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Agriculture Issues and Policies Series


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Agriculture Issues and Policies Series

Agriculture Issues & Policies, Volume I

Alexander Berk (Editor)
2001. ISBN 1-56072-947-3

Agricultural Conservation
Anthony G. Hargis (Editor)
2009. ISBN 978-1-60692-273-6

Hired Farmworkers: Profile and Labor Issues

Rea S. Berube (Editor)
2009. ISBN 978-1-60741-232-8

Environmental Services and Agriculture

Karl T. Poston (Editor)
2009 ISBN: 978-1-60741-053-9

Weeds: Management, Economic Impacts and Biology

Rudolph V. Kingely (Editor)
2009 ISBN 978-1-60741-010-2

Effects of Liberalizing World Agricultural Trade

Henrik J. Ehrstrom (Editor)
2009 ISBN: 978-1-60741-198-7

Economic Impacts of Foreign-Source Animal Disease

Jace R. Corder (Editor)
2009 ISBN: 978-1-60741-601-2

Agricultural Wastes
Geoffrey S. Ashworth and Pablo Azevedo (Editors)
2009 ISBN: 978-1-60741-305-9

Soybean and Wheat Crops: Growth, Fertilization, and Yield

Samuel Davies and George Evans
2009 ISBN: 978-1-60741-173-4

Corn Crop Production: Growth, Fertilization and Yield

Arn T. Danforth (Editor)
2009 ISBN: 978-1-60741-955-6


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Danforth, Arn T.
Corn crop production growth, fertilization and yield / Arn T. Danforth.
p. cm.
Includes index.
ISBN 978-1-60876-860-8 (E-Book)
1. Corn--Growth. 2. Corn--Fertilizers. 3. Corn--Yields. I. Title.
SB191.M2D27 2009

Published by Nova Science Publishers, Inc. Ô New York


Preface vii
Chapter 1 Corn Crop Production: Growth, Fertilization and Yield 1
K. D. Subedi and B. L. Ma
Chapter 2 Responses of Agronomically Important Crops to Inoculation
With Plant-Associated Beneficial Bacteria in Crop-Farming
Systems – A Review 85
M. Madhaiyan, S. Poonguzhali, M. Senthilkumar and P.
Chapter 3 Effect of Abiotic Stresses on Growth, Metabolic Alterations
and Tolerance Mechanisms in Rice Crop 111
Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey
Chapter 4 Domestication and Conservation Genetics of the Lima Bean
(Phaseolus Lunatus L.) in Its Mesoamerican Diversity Center 187
Jaime Martínez-Castillo, Patricia Colunga-GarcíaMarín, Daniel
Zizumbo-Villarreal, Filogonio May-Pat and Julián Coello-Coello
Chapter 5 Potential Impact of Biological Nitrogen Fixation
and Organic Fertilization on Corn Growth and Yield in Low
External Input Systems 227
Márcia do Vale Barreto Figueiredo, Mario de Andrade Lira Junior,
Arminda Saconi Messias and Rômulo Simões Cezar Menezes
Chapter 6 Distribution and Risk Assessment of PAHs in Soils and Corns
Around Zhongyuan Oil Field, China 257
Shaoping Kuang and Hong Chen
Chapter 7 Low Temperature Effects on the Early Development
of Corn Seedlings 291
Ricardo Aroca
vi Contents

Chapter 8 Soil Water Balance and Yield of Dryland Maize Using

the CropSyst Model 307
M. G. Abraha and M. J. Savage
Chapter 9 Wrongful Exploitation and Terminator Technology 319
Keith Bustos
Chapter 10 Maize Doubled Haploids Via Anther and Microspore Culture 333
Bohuš Obert, Ľubica Uváčková and Anna Preťová
Chapter 11 Modelling of Maize Production and the Impact of Climate Change
on Maize Yields in Croatia 345
Višnja Vučetić
Index 353

Corn or maize is a crop that originated in Mexico and has spread all over the world as a
major food crop. Sustainable production of a corn field crop as grain corn for feed, food and
biofuels, as well as sweet corn for fresh market or processing, and as silage for high energy
sources, requires scientific management of nutrients along with several other crop
management practices such as proper plant population density, timely seeding and harvesting,
soil water, weeds and pests control. Corn has become the major item in the diet of many
tropical peoples, the main grain used for animal feed in temperate regions, as well as new
stocks for many other purposes including recently used as feedstock for biofuels. Rapid
expansion of grain based ethanol production in North America, has already caused concern
about future food and feed supplies. This important book gathers the latest research from
around the world in this dynamic field.
Chapter 1 - Sustainable production of a corn (Zea mays L.) crop as grain corn for feed,
food and biofuels, as sweet corn for fresh market or processing, and as silage of high energy
source, requires scientific management of nutrients along with several other crop management
practices such as proper plant population density (PPD), timely seeding and harvesting, soil
water, weeds and pests management. This chapter reviews the recent advances on corn
nutrients management in relation to crop development, yield formation, economic
consideration and environmental sustainability of corn production. Corn types, physiological
basis of corn yield, nutrients uptake and partitioning by different types of corn will be briefly
reviewed. Critical timing of nutrients requirements by a corn plant, factors affecting nutrients
uptake, removal and utilization efficiencies are discussed under different crop rotations,
cropping systems, and growing environments. Recent approaches of determining/predicting
corn nutrients requirements such as crop-based indicators, site-specific nutrients management
and variable rates application for the sustainable nutrients management are discussed along
with the conventional soil test and plant tissue test approaches. Impacts of manures and
fertilizers and methods, timing, and rates of their applications on the crop yield and
environment such as nitrate (NO3--N) leaching, ammonia (NH3) volatilization and greenhouse
gas emissions such as nitrous oxide (N2O) from corn fields are also outlined. The concept,
importance and practical approaches of integrated plant nutrients management (IPNM) for
corn production are also discussed. Finally, the importance of corn residue for biofuel
(ethanol) production is discussed in relation to its impact on soil fertility.
Chapter 2 - Root colonizing bacteria (rhizobacteria) that exert beneficial effects on plant
development via direct or indirect mechanisms have been defined as plant growth promoting
viii Arn T. Danforth

rhizobacteria (PGPR). The search for PGPR and investigation of their modes of action are
increasing at a rapid pace as efforts are made to exploit them commercially as biofertilizers.
This review focuses on different kinds of PGPR, their mode of action as biofertilizers and
also development of microbial consortia is mentioned. These mode of action include fixing
N2, increasing the availability of nutrients in the rhizosphere, positively influencing root
growth and morphology. Although significant control of plant pathogens or direct
enhancement of plant development has been demonstrated by PGPR in the laboratory and in
the greenhouse, results in the field have been less consistent. Because of these and other
challenges in screening, formulation, and application, PGPR have yet to fulfill their promise
and potential as commercial inoculants. Recent progress in our understanding of their
diversity, colonization ability, mechanism of action, formulation, and application should
facilitate their development as reliable components in the management of sustainable
agricultural systems. Obtaining maximum benefits on farms from plant growth promoting
biofertilizers will require a systematic strategy designed to fully utilize all these beneficial
factors, allowing crop yields to be maintained or even increased with reduced fertilizer
Chapter 3 - Rice is a staple food crop for the majority of world population. Abiotic
stressful conditions of the environment such as salinity, drought, heat, chilling, anaerobiosis,
metal toxicity impose limitations on productivity of rice in the regions which are prone to
such constraints. The manifestations of these stresses include non-expression of full genetic
potential, differential transcription of many genes, induction of stress responsive genes
leading to cellular metabolic changes, alteration in activity behaviours of many enzymes,
overproduction of several compatible metabolites like amino acids, sugars, polyamines,
phytochelatins, organic acids, increased synthesis of many enzymes and stress specific
proteins. Salinity and drought are prime stressful conditions for rice crop in arid and semi arid
regions of the world. Changes in temperature rhythm impose heat or chilling injury. Soil
flooding or submergence causes oxygen deprivation leading to anaerobic stress. Metal ions
such as Pb, Cd, Hg, As, Ni are key pollutants of the soil, whereas Al toxicity is a problem in
acid upland soils. Most of the abiotic stresses cause overproduction of reactive oxygen
species (ROS) within the cell which cause oxidative damage to membranes and biomolecules.
Increased accumulation of compatible solutes, overproduction of antioxidative enzymes,
overexpression of transcription factors have been shown to confer tolerance in rice plants to a
wide range of stresses like salinity, drought and low temperature. Stress induced gene
products those involved in stress tolerance and those involved in signal transduction or as
transcription regulators have served as basis to engineer stress tolerant plants. To contribute to
food security and sustainability in rice production, it is essential to produce stress tolerant rice
plants suitable for cultivation in stress prone areas. This needs a detail understanding of
physiological and molecular mechanisms associated with stress tolerance more specially gene
products involved in stress tolerance and signal transduction. Transcriptome profiling of rice
seedlings has helped in great way in understanding how rice plants respond to abiotic stresses.
Successful attempts have been made to produce transgenic rice plants tolerant to different
abiotic stresses. However, with the rapid progress in the areas of functional genomics,
proteomics and metabolomics a more improved understanding of novel stress responsive
genes and their expression under various stresses is anticipated which will provide the basis
of new strategies to produce genetically engineered rice plants tolerant to a single or multiple
of abiotic stresses.
Preface ix

Chapter 4 - The lima bean (Phaseolus lunatus L.) is the second major cultivated species
of the genus Phaseolus. It possesses high levels of genetic diversity and its primary gene pool
includes both wild and domesticated forms grouped into two main gene pools: Mesoamerican
and Andean. In the Yucatan peninsula, it is integrated into the traditional agricultural system
focused on corn cultivation, known as milpa, where it is planted as an associated crop. This
region possesses the largest diversity of domesticated forms in Mexico, a diversity that is
possibly being generated and maintained, in part, by their sympatric growth with wild
populations. However, the repercussions of human population growth and socio-economic
changes occurring in this region during the last 50 years have resulted in major modifications
to the milpa. One of the most evident consequences of these changes has been a decreased
planting of the crops associated with corn and a loss of the vegetational areas next to the
milpa where wild relatives grow. This review chapter shows the results of eight years of
research on the conservation genetics of P. lunatus in the Yucatan peninsula, México, using
ethnobotanical, ecological and genetic evidence. The results indicate that: 1) the genetic
diversity of P. lunatus from the Yucatan peninsula is higher in comparison to other
Mesoamerican regions; 2) wild populations show higher values of diversity than the
domesticated ones, probably due to a founder effect or recent processes of genetic erosion in
the domesticated forms; 3) the three most abundant landraces (70% of the planted area) had
the lowest values of genetic diversity—in contrast, 12 landraces with high levels of genetic
diversity were planted only by few farmers, a situation that shows the high risk of genetic
erosion in the domesticated gene pool; 4) wild and domesticated gene pools show a strong
genetic differentiation due to distance isolation and low levels of gene flow; 5) the wild-
domesticated gene flow is low, but it is three times higher than the domesticated to the wild
populations than in the opposite direction. This situation may lead to genetic assimilation of
the wild lima bean by its domesticated counterpart and may lead to the possible escape of
transgenes in this center of diversity. In situ programs urgently need to be established in this
important Mesoamerican region to conserve the milpa system, including the lima bean
landraces and its wild populations.
Chapter 5 - Maize productivity in tropical low external input systems is usually limited
by low soil fertility because crop uptake leads to a gradual depletion of soil nutrient stocks.
Since the use of chemical fertilizers is infeasible or undesired, the management of the fertility
of these soils depends primarily on low-cost processes based on nutrient recycling. The main
processes that may contribute to this are 1) biological nitrogen fixation (BNF), 2) nutrient
recycling through organic fertilization using plant residues or animal manures, and 3) where
feasible, the use of industrial and/or urban waste. BNF may contribute to maize growth and
yield by direct fixation in corn, or through the use of legume plants either as green manure or
as crops in rotation or intercropped with corn. Either way, BNF can usually be considered
sustainable long term, and usually would be one of the preferred nitrogen sources for low
external input corn production systems. Since almost all soil nitrogen is derived from the
atmosphere, in the absence of substantial use of nitrogen fertilizer most of the remaining
nitrogen pool is a product of BNF, either recent or past. The main difference between on-field
BNF and use of plant residues and animal manures is that nitrogen is previously fixed or
obtained from the soil pool on another field and later taken to the corn field. At the same time,
nutrient recycling through organic fertilization is usually limited due to the low amounts of
organic matter available for this use, especially considering the concurrent demands for this
material. Therefore, the efficient use of the different types of organic matter used as fertilizer
x Arn T. Danforth

requires knowledge about its quality and patterns of decomposition in order to guarantee
synchronization between nutrient supply and crop demand. Finally, the third approach in
these systems centers on the use of urban waste, most usually compost or sewage sludge, or
industrial by-products. Some of these may be quite rich in several nutrients at the same time,
but usually require careful investigation into possible negative effects of items such as heavy
metals and pathogens. Information is reviewed regarding BNF directly on corn, in green
manure or crop rotations involving this culture; strategies to improve the amount and quality
of organic fertilizers produced in low input systems; and some possible alternatives of urban
or industrial by-products, describing the current rationale to supply nutrients to maize crops at
a low cost using the resources available within the agroecosystems.
Chapter 6 - The purpose of this study was to determine the degree of contamination with
polycyclic aromatic hydrocarbons (PAHs) in samples of soils in both winter and summer,
corn roots and corn leaves around three different oily sludges in Zhongyuan Oil Field. The
contents of PAHs in samples were determined with HPLC. According to these data and the
ratio of Fla/Pyr, PAHs in oily sludge samples were mainly from petrogenic sources, and the
soil samples in both winter and summer were divided into petrogenic soil samples and both
petrogenic and pyrogenic soil samples by the source of PAHs. The PAHs contents of soil
samples in both winter and summer around three different oily sludges from high to low
present that the Third Wenming Plant of the oily sludge (3W)>the Third Mazhai plant of the
oily sludge (3M)>the Fourth Wener Plant of the oily sludge (4W), and the contents of PAHs
in soil samples in summer were lower than those in winter, and 2-4 rings were major
pollutants of PAHs in oily sludge. The contents of PAHs in soil samples around oily sludges
varied widely from 434.5 to 408.8 ng·g-1. Naphthalene, acenaphthene, fluorine, phenanthrene
and pyrene were major pollutants of PAHs in petrogenic soil samples, and the two
predominant PAHs in both petrogenic and pyrogenic soil samples were naphthalene and
phenanthrene. The PAHs contents of corn root samples from soils around three different oily
sludges from high to low were 3W > 3M> 4W, which was consistent with the order of the
contents of PAHs of soil samples in three plants. And 2-4 rings PAHs were still the most
predominant components of PAHs in corn root samples. The PAHs contents of corn leaf
samples from soils around three different oily sludges from high to low were 3W> 4W > 3M,
which was not consistent with the order of the contents of PAHs of soil samples and corn root
samples in three plants. And 5-6 rings PAHs were the most predominant components of
PAHs in corn leaf samples. Based on Nemero index P, the result of classification evaluation
showed soils around oily sludge were heavily polluted, and also present that the PAHs
pollution level in soil samples in winter was much higher than that in summer. According to
the pollution characters of PAHs in soil samples in winter and summer, and the contents
characters of PAHs in corn samples in summer, this article also determined the health risk
assessment and ecological risk assessment in soils around oily sludge in Zhongyuan Oil Field,
and the security of the corn as a crop.
Chapter 7 - Corn farming is extended all around the world, from temperate regions to
tropical ones. Moreover, corn crop represents an essential component of the global food
security. In temperate regions, corn growth is reduced by low temperatures at the early stage
of development (from germination to fourth leaf fully developed stage). At these stages, corn
seedlings are very sensitive to low temperatures as can be determined by several
physiological processes. These physiological processes include among others water transport,
respiration, photosynthesis and oxygen metabolism. On the other hand, there is a degree on
Preface xi

the sensitivity to low temperature among corn genotypes cultivated worldwide. Corn
seedlings suffer a decrease in their leaf water content upon exposure to low temperatures.
This water deficit is caused by the lack of stomatal response and the diminution of the root
water uptake. Together with this water deficit, corn seedlings diminish their respiration and
their CO2 fixation. However, although CO2 fixation decreases, the amount of light that the
leaf receives remains constant. The excess of light non used to fix CO2 causes an excess of
energy in the photosystems, that ultimately is captured by the oxygen molecules, forming the
so called reactive oxygen species (ROS). These ROS are highly harmful when they exceed
the capacity of the leaves to remove them. Thus the differences on low temperature sensitivity
among corn genotypes is partially linked to better water status maintenance, keeping higher
rates of photosynthesis, and having more effective ROS removing mechanisms. Here, we will
review all these physiological aspects involved on corn seedlings tolerance to low
Chapter 8 - Crop growth models are increasingly being used as decision support tools to
help optimize crop and soil management strategies. Crop growth models need to be calibrated
and validated for the site and crop variety of interest. This study is undertaken to evaluate the
ability of CropSyst, a cropping systems simulation model, to simulate the yield and soil water
balance of dryland maize (Zea mays L. cv. PAN 6568) at Cedara, KwaZulu-Natal, South
Africa. Soil, plant, weather and management data were used as inputs for calibration and
validation of the CropSyst model. Model crop parameters were calibrated using past
experiments or obtained from model documentation, with slight modification to account for
varietal differences. Validation of crop parameters was carried out using an independent
dataset not previously used for calibration. The model-predicted phenology and grain yield of
maize with reasonable accuracy, but consistently under-estimated the soil water content of the
deeper depths with more pronounced under-estimation about 80 days after start of simulation.
The CropSyst model appears to be an adequately suitable tool for crop management
applications, climate change studies and research applications. For more accurate and reliable
results, the CropSyst model should be validated for the site and crop under study with more
observed data.
Chapter 9 - In an effort to restrict seed piracy, Monsanto intends to implement some
variation of genetic use restriction technology (GURT). Regarding such intentions, many
activist groups throughout the world (mainly in the US, Canada, and the UK) adamantly
contend that Monsanto and possibly other multinational agrochemical corporations (MACs)
will be acting immorally if GURTs, such as Terminator Technology (TT), are implemented in
the global agricultural industry. These activists argue that the potential implementation of TT
is immoral because it will grant Monsanto the power to wrongfully exploit resource-poor
farmers (RPFs) by reducing RPFs to mere means of production.
I contend that Monsanto will not necessarily be wrongfully exploiting RPFs through the
implementation of TT. More specifically, as long as Monsanto allows these RPFs to make an
autonomous choice to use terminator seeds and sponsors public plant breeding initiatives
(PPBIs), then Monsanto cannot be accurately considered to be wrongfully exploiting these
There are three main parts to this essay. In the first part, I explain what exploitation is and
the conditions that must obtain for it to be immoral from a Kantian perspective. In the second
part, I briefly describe a few of the major objections that some activist groups have made
regarding the potential implementation of TT. In the third part of this essay, I apply the
xii Arn T. Danforth

conception of wrongful exploitation developed below to the current debate concerning the
potential implementation of TT in the global agricultural industry.
Chapter 10 - Within the past years, great progress has been made in development of
technologies for improvement of cereal crops of economic importance including maize.
Induction of embryogenesis from gametic and somatic cells and tissue culture are the main
techniques necessary for practical application of advanced biotechnological tools for targeted
improvement of plant. There is a requirement for haploid and doubled haploid material and
homozygous lines for cell culture studies and breeding in maize. Anther culture is currently
the most successful method producing doubled haploid lines in maize, but microspore culture
was also described as a good source of doubled haploids.
This review focuses on tissue and plant regeneration using anther culture, and cultivation
of isolated microspores. The effect of genotype, physiological status of donor plants, donor
material pre-treatment, cultivation conditions for maize anthers and microspores as well as
ploidy level of regenerated tissue and plants, and use of colchicine during early stages of
androgenesis induction for chromosome doubling are discussed here. Processes connected
with developmental switch towards embryogenic development of microspores and process of
plant regeneration from anther- and microspore-derived calli are also in the focus of this
Chapter 11 - The effect of climate change on maize growth and productivity in the central
part of Croatia has been researched using the crop CERES-Maize model. The Zagreb
Maksimir meteorological data during the period 1949–2004 and pedological, physiological
and genetic data obtained in the field maize experiment in Zagreb 1999 have been used. In
order to estimate the intensity of the regional impact of climate change on maize production, a
synthetic meteorological series was created by the stochastic weather generator MetandRoll
for different climate change scenarios. The CERES-Maize model was run with
meteorological series representing the present climate and synthetic meteorological series
representing the changed climate. All climate change scenarios during the 21st century,
including only the climate change effect, projected a shorter growing season of 34-44 days
and a reduction in maize yields of 8-15%.
In: Corn Crop Production Growth, Fertilization and Yield ISBN 978-1-60741-955-6
Editor: Arn T. Danforth © 2009 Nova Science Publishers, Inc.

Chapter 1



K. D. Subedi and B. L. Ma1

Eastern Cereal and Oilseed Research Centre (ECORC), Agriculture and Agri-Food
Canada (AAFC), Central Experimental Farm, 960 Carling Avenue,
Ottawa, ON, K1A, 0C6, Canada

Sustainable production of a corn (Zea mays L.) crop as grain corn for feed, food and
biofuels, as sweet corn for fresh market or processing, and as silage of high energy source,
requires scientific management of nutrients along with several other crop management
practices such as proper plant population density (PPD), timely seeding and harvesting,
soil water, weeds and pests management. This chapter reviews the recent advances on corn
nutrients management in relation to crop development, yield formation, economic
consideration and environmental sustainability of corn production. Corn types,
physiological basis of corn yield, nutrients uptake and partitioning by different types of
corn will be briefly reviewed. Critical timing of nutrients requirements by a corn plant,
factors affecting nutrients uptake, removal and utilization efficiencies are discussed under
different crop rotations, cropping systems, and growing environments. Recent approaches
of determining/predicting corn nutrients requirements such as crop-based indicators, site-
specific nutrients management and variable rates application for the sustainable nutrients
management are discussed along with the conventional soil test and plant tissue test
approaches. Impacts of manures and fertilizers and methods, timing, and rates of their
applications on the crop yield and environment such as nitrate (NO3--N) leaching,
ammonia (NH3) volatilization and greenhouse gas emissions such as nitrous oxide (N2O)
from corn fields are also outlined. The concept, importance and practical approaches of
integrated plant nutrients management (IPNM) for corn production are also discussed.
Finally, the importance of corn residue for biofuel (ethanol) production is discussed in
relation to its impact on soil fertility.

1 or AAFC-ECORC Contribution No. 08-953.
2 K. D. Subedi and B. L. Ma

BMP best management practice;
CHU Crop Heat Unit;
DM dry matter;
GDD growing degree days
HI harvest index;
IPNM integrated plant nutrient management;
LAI leaf area index;
LRS leafy reduced-stature;
NDVI normalized difference vegetation index;
NLRS non-leafy reduced stature;
NUE nitrogen use efficiency;
OM organic matter;
PAR photosynthetically active radiation;
PPD plant population density;
PM physiological maturity;
PPNT pre-plant nitrate test
PSNT pre-sidedress nitrate test;
SOC soil organic matter;
SSNP site specific nutrients management;
VRA variable rate application.

Corn or maize (Zea mays L.), a crop originated in Mexico from where, it spreads to all
over the world as a major food crop. In the late 15th and the early 16th centuries when the
Europeans came to North and South America, they brought corn back home and spread it
throughout the world during the rest of their conquer ( Corn is now one of the
most widely grown crops, and it is cultivated from the equator to the approximately 50 ° north
and south, and altitude from sea level to 3000 m above sea level (Morris, 2002). Although corn
is cosmopolitan in nature, it is the major staple food of several countries of Latin America,
Eastern Africa, Central America and South-east Asia including China. In the North America,
corn is grown as a food, feed and industrial crop. In the USA, corn production uses over 25%
of the nations’ cropland and more than 40% of the commercial fertilizer applied (Christensen,
2002). Unlike the other major cereal crops, corn is a C4 grass, efficient in utilizing water,
nutrients and CO2 to produce carbohydrates which are stored in the leaves and stalks and
harvested as grain starch. It is one of the most efficient field crops in producing superior
amount of dry matter (DM) per unit area (Oktem, 2005). Consequently, corn becomes the
major item in the diet of many tropical people, the main grain used for animal feed in
temperate region, as well as new stocks for many other purposes including recently used as
feedstock for biofuels. Rapid expansion of grain based ethanol production in North America
has already caused concern about future food and feed supplies. Crop improvement strategies
Corn Crop Production: Growth, Fertilization and Yield 3

for increased biomass and yield and site-specific nutrient best management practices (BMPs)
should be developed to adapt to the changes and meet the new demands.
Nutrients management is a vast topic which involves cropping systems management, corn
types, BMPs suitable for improved nutrients use efficiency, maintain soil fertility levels for
sustainable production while protecting the environment. Maintenance of soil fertility to a
desired level is the most important production challenge for corn worldwide. Continuous corn
cultivation and declining soil organic matter (SOM) levels are the limiting factors for corn
production, especially in the smallholders in the tropical and subtropical corn production areas.
Efforts have been made to collect information on best management technologies of soil,
manure and fertilizers management on corn crops. In this chapter, acquisition and roles of
essential nutrients on corn development, nutrients requirements, and yield are discussed. Major
corn-based cropping systems, essential nutrients and their deficiencies, diagnostic tools for
nutrients deficiencies, integrated nutrients management approaches, and environmental
impacts of corn nutrients management are discussed.
Since corn is cultivated in a vast area, diverse cropping systems and agro-ecologies from
tropics to cool temperate environments, and from small-holding subsistence farmings to highly
mechanized and precision farmings such as of North America and Europe, it is not possible to
give detailed accounts of specific system in a single chapter. Therefore, attempts have been
made to provide a general overview of nutrients management for sustainable corn production.
This chapter describes the current understanding and advances on nutrients management for
corn production. Corn production practices in relation to its nutrients management are
discussed based on the peer-reviewed journal publications and non-reviewed sources globally.
Efforts have been made to give a global perspective of corn nutrients management, whereas
majority of the published literature are available from the North America. Although, this
chapter deals mainly on the nutrients management aspects of corn production, some of the
terminology and background information such as corn types, growth stages, corn-production
systems, are also briefly discussed.


Botanically, corn is a monoceious species, with a male flower (tassel) located at the top of
the stem and female inflorescence (ear) located in the middle of the stem nodes of the same
plant. The spatial arrangement of the flowers facilitates both selfing and crossing (Morris,
2002). At flowering (anthesis), numerous pollens shed from the tassels, which are then trapped
by the receptive stigmas (i.e. the silks). The male inflorescence (tassel) of corn can produce
considerably more pollen grains than are required for pollination of a single plant (Schoper et
al., 1987). Goss (1968) estimated that as many as 2 to 5 million pollen grains are produced by
a typical corn plant. Pollen shed can begin before tassels have completely emerged from the
whorl and can continue over a week or longer (Ritchie et al., 1993).
There are many forms or types of corn, and they are classified based on botanical
description, utilization, growing environments, maturity types and so on. Some of the common
classifications of corn are briefly described below:
4 K. D. Subedi and B. L. Ma

2.1. Classification Based on Plant Taxonomy

• Flour corn — Zea mays var. amylacea

• Popcorn — Zea mays var. everta
• Dent corn — Zea mays var. indentata
• Flint corn — Zea mays var. indurata
• Sweet corn — Zea mays var. saccharata and Zea mays var. rugosa
• Waxy corn — Zea mays var. ceratina

2.2. Classification Based on Growing Environments or Adaptation

Although no universally accepted system exists for classifying corn production

environments, the International Maize and Wheat Research Center (CIMMYT), recognizes the
following four Mega Environments for corn production:

• Low land tropical

• Sub-tropical and mid-altitude transition
• Tropical highlands, and
• Temperate

Tropical and sub-tropical corn varieties are adapted to the more tropical environments.
They are characterized by tall plants, large tassels and have substantial capacity to store
photosynthates as simple sugars in the stem at crop maturity (Hay and Gilbert, 2001).
Tropical highlands and temperate varieties are developed such that they have reduced
tassel size with shorter plant height and retain less sugar in the stem at crop maturity.
Temperate varieties are the ones that are adapted to cooler, temperate regions of the world
such as North America and Europe.

2.3. Classification Based on Plant Stature or Leaf Types

Leafy corn: The Leafy corns are those with extra number of leaves above the ear node
(Shaver, 1983). For most temperate corn hybrids (varieties), the mature plant has about 5
leaves above the primary ear node. The gene Lfy causes the plant to have extra number of
leaves above the ear (> 6 and up to 11) compared to normal hybrids of the same maturity.
Leafy hybrids have recently gained popularity as silage corn (Roth, 2003), probably due to
potential longer window for silage harvest and greater silage dry matter (Ma et al., 2006b).
These hybrids have greater total number of leaves and leaf area than their conventional
counterparts (Shaver, 1983; Subedi and Ma, 2005a, 2005b). Because of the heavy foliage and
higher biomass (Andrews et al., 2000; Subedi and Ma, 2004), N nutrition requirement of Leafy
hybrids may be greater than the conventional hybrids.
Erect-leaf corn: Certain phenotypes of corn are developed with more erect leaf orientation
than the conventional corn types. Erect types are believed to be more efficient in canopy light
Corn Crop Production: Growth, Fertilization and Yield 5

Brown mid-rib (BMR) corn: Brown midrib (bmr) plants are characterized by a brown or
reddish-brown pigmentation in the leaf midrib, rind and pith. Such corn contains a gene (bm3)
that results in lower lignin content being produced during the plant development. As a
consequence, BMR silage corn contains fibre that is more digestible than conventional silage
corn (Gehman et al., 2008).
Stay-green (SG) corn: Expression of the stay green (SG) trait has been reported in corn
(Tollenaar and Daynard, 1978; Ma and Dwyer, 1998; Rajcan and Tollenaar, 1999a, 199b).
Such phenotypes exhibit delayed senescence and have higher water and chlorophyll content in
the leaves at maturity than the conventional corn phenotypes. There is a limited understanding
of the physiological processes underlying this trait. Rajcan and Tollenaar (1999b) proposed
that leaf senescence in a recent corn hybrid was delayed because of an improvement in the
ratio of assimilate supply (i.e. source) to assimilate demand (i.e. sink) during kernel filling
period. They also found that total N uptake in aboveground portions were 10 and 18% greater
in the SG hybrid than an older hybrid under low and high soil N conditions, respectively.
Reduced stature (rht) corn: Reduced stature (Rht) corns are genetically modified for
reduced plant height. The total stalk height substantially reduced through reduced internode’s
length. These types may be with leafy or normal leaf numbers.

2.4. Classification Based on Uses

Grain corn: grain corns refer to flour, dent and flint corns that are mainly used for human
consumption, animal feed and industrial uses such as corn flour, starch, ethanol and others.
Pop corn: pop corns have ears with small kernells that pop while roasting. Pop corns are
used as one of the most common snacks.
Sweet corn: sweet corns are planted for fresh market or processing (e.g., canning) uses.
Sweet corn grains contain higher concentration of sugars than other corns. Some varieties of
sweet corn are more sugary which are also called se (sugary enhanced) and supersweet types
depending on the types of genes on them. Sweet corn consumption has increased considerably
worldwide. Fresh consumption of sweet corn is more beneficial compared to other derivatives
of corn due to its soft kernels, thin shells, high concentration of sugar and tastefulness. Dough
made from dry kernels of sweet corn is used for baby food, chips, dough products, pasta and
cakes. Sweet corn can be an excellent source of some minerals.
Baby corn: cobs harvested early, while the ears are very small and immature from the
specialized corn plants. Baby corn ears are usually processed and preserved prior to the
Silage corn: corns that are harvested for fodder or silage before the plant reaches
physiological maturity. Corn silage production is an integral component of many dairy and
beef operations. In Canada, silage hybrids make up approximately 20% of corn acreage with
concentrated production in Ontario and Quebec, supporting the dairy industry (Dwyer et al.,
1998). Principally, any types of corn can be used as silage corn, but certain varieties have more
desirable qualities than others such as Leafy and the brown-midrib (BRM) types are more
silage specific hybrids. Traditionally, grain corn hybrids have been used dual-purposely for
silage production. However, selection criteria such as hard, high density kernels, strong stalks
and rapid kernel dry-down, which favour grain production, may be undesirable for silage
harvest, fermentation and digestibility (Ma et al., 2006b). Silage varieties should have the
6 K. D. Subedi and B. L. Ma

characteristics of slow maturing, softer starch kernels, and slow dry-down of stalks and lower
neutral detergent fibre (NDF) with high NDF digestibility (Dwyer et al., 1998).

2.5. Classification Based on Growing Duration (Maturity Classes)

• Early maturing varieties (<110 d)

• Intermediate (medium) duration varieties (110-120 d)
• Late maturing varieties (>120 d).

The rate of development of corn from planting to maturity is dependent mainly on

temperature (accumulated heat), although other factors such as photoperiod (day length),
altitude, and agronomic management practices such as water and nutrition management also
influence maturity to some extent. Therefore, air temperature is used to quantify response to
corn growth. The more realistic estimation of crop maturity would be based on the amount of
heat accumulated by a variety or hybrid. Rating of hybrid corn maturity and zonation of
production areas in North America employ several systems, including Growing Degree Days
(GDD; Wang, 1960), Crop Heat Unit (CHU; Brown and Bootsma, 1993), and Minnesota
Relative Maturity Rating (MRMR; Peterson and Hicks, 1973). The accumulated heat is
calculated according to the following formulae:

(i) Growing Degree Days (GDD; Wang, 1960; Dwyer et al., 1999a):

GDD = ∑ (TA – TB)∆t (1)

where TA is the average of daily maximum (Tmax) and minimum (Tmin) air temperatures, TB is a
base temperature below which development is assumed to cease and is usually set at 10 oC for
corn, and ∆t is a time step in days (Dwyer et al., 1999a). In addition, temperatures below 10 oC
and above 30 oC are assumed to be ineffective for development and Tmax values > 30 oC are set
to 30 oC and Tmin values < 10 oC are set to 10 oC.
(ii) Minnesota Relative Maturity Rating (MRMR) provides the relative ranking of the
number of days corn hybrid requires to reach maturity in relation to the time require
by previously ranked hybrid (Peterson and Hicks, 1979).
(iii) Crop Heat Unit (CHU; Brown and Bootsma, 1993) is similar to GDD, but it considers
that response of development to temperature differs between the day and the night,
therefore, is more commonly used in northern states of USA and Canada. The
maximum or daytime relationship uses 10°C (50°F) as the base temperature and 30°C
(86°F) as the optimum because warm-season crops do not develop at all when daytime
temperatures fall below 10°C, and develop fastest at about 30°C. The minimum or
night time relationship uses 4.4°C (40°F). The CHU is calculated according to eq. [2].

CHU = (Ymin + Ymax)/2 (2)

Corn Crop Production: Growth, Fertilization and Yield 7

Ymin = 9/5 (Tmin - 4.4) (3)

and Ymax = 3.33 (Tmax - 10.0) - 0.084(Tmax - 10.0)² (4)

Recently, another index, named General Thermal Index (GTI) was developed (Dwyer et
al., 1999). This system takes into consideration of the temperature functions separately for
vegetative and reproductive phases of corn (Stewart et al., 1998) as

FT(veg) = 0.0432 TA² - 0.000894TA3 (5)

FT(fill) = 5.358 + 0.01128TA² (6)

and GTI is expressed as


where, FT is the Temperature Response Function fitted to the vegetative (veg; before silking)
or grain filling period (fill; after silking) phase, n is the number of days in a period and ∆t is
time step in days.


Cropping system refers the sequence of crops in a piece of land or farm unit, grown in a
fixed period of time. There are diverse corn-based cropping systems in practice throughout the
world. Describing all systems is beyond the scope of this chapter. Only brief account of the
key corn-based cropping systems are discussed.

3.1. Monoculture

Monoculture refers growing a single crop over growing seasons in the same field. In the
temperate corn production systems such as Northern Corn-Belts of USA and Canada, and
some European Union countries, historically growing sole corn crop is a common practice.
However, corn and soybean (Glycine max L.) in biannual rotation is gaining more popularity.
8 K. D. Subedi and B. L. Ma

3.2. Intercropping

In the more tropical or sub-tropical environments, corn is grown in a subsistence label by

the small holders, and mostly under non-irrigated (rainfed) conditions. Corn-based mixed
cropping, such as in the subsistence farming of the Latin America, Asia and Africa where
usually legumes are intercropped (mixed, row or relay intercropping) with corn. Legumes such
as soybean, cowpea (Vigna unguiculata L.), field bean (Phaseolus vulgaris L.), mungbean (V.
radiate L.), groundnut (Arachis hypogeae), velvet bean (Mucuna pruriens L.) and others are
intercropped. There are also some cropping systems where corn is intercropped with cereal
crops such as finger millet (Elecusine corocana L.) (Subedi, 2002) or upland rice (Oryza
sativa L.) (Subedi et. al., 1993) or cotton (Gossypium hirsutum L.).

3.3. Corn Based Crop-Rotations

Different crop species can be rotated in the same piece of land one after another in the
same year or in different years. The common corn-based rotations in different parts of the
world are as follows:

• Corn followed by a legume crop (e.g. corn-soybean, corn-alfalfa (Mdicago sativa L.).
This is the dominant corn-based cropping rotation in US Corn Belt and Canada.
Christensen (2002) estimated that corn-legume rotation was used on almost 60% of
US corn acreage in 1996.
• Corn-small grain cereal-legume (e.g. corn-wheat (Triticum aestivum L.)-soybean,
corn-oat (Avena stiva L.)-soybean, corn-rice-lentils (Lens culinaris), etc.
• Corn- small grain cereal- small grain cereal or corn- small grain cereal (annual
system): In the tropical and sub-tropical regions such as parts of India and China,
Nepal and other countries, corn is rotated with other cereals such as wheat and rice in
a rice-wheat-corn or rice-fallow-corn cropping system.

3.4. Based on Water Availability

• Unirrigated or rainfed (most of tropical and sub-tropical) corn production: Where crop
production is totally dependent on the seasonal precipitation.
• Irrigated corn: Where corn production is supplemented with irrigation water. This
system is dominant in the plains states of USA, most of Australia, and in certain areas
of tropical and sub-tropical Asia such as India, China and Thailand.

3.5. Based on Tillage Practices

Tillage systems have a profound effect on soil fertility and nutrients management in corn.
Therefore, a brief account of various tillage systems in corn cultivation is introduced here.
Nutrients management in relation to tillage systems is discussed in the respective sections.
Corn Crop Production: Growth, Fertilization and Yield 9

3.5.1. Conventional Tillage

In a conventional tillage system, corn is planted with mouldboard plough or any
conventional land preparation practices including ploughing, harrowing, planking, etc. This
practice was historically dominated in the US Corn Belt, and continues to follow in the
majority of small-holders, subsistence farmers of the tropics and sub-tropics. This system is
labour and/or machinery intensive and prone to extensive soil loss due to erosion. Almost all
of the crop residues are removed from the soil. Even in the highly mechanized crop production
system of USA, still about 30% of the corn acreage is under this system (Christensen, 2002).

3.5.2. Reduced-Tillage
This is a system in which soil disturbance is minimal and leaves at least 15-30% of the soil
surface covered by crop residue at planting. This system excludes the use of mouldboard
plough, and the intensity of tillage is reduced. Weed control is accomplished with herbicides
and/or cultivation. The reduced tillage practices were used on about 30% of corn cultivation in
1996 in USA (Christensen, 2002).

3.5.3. Conservation Tillage

Conservation tillage (CT) refers to any system that uses some tillage, but less than the
conventional techniques of seedbed preparation. Any tillage system that maintains at least 30%
of the soil surface covered by crop residue after planting is considered as conservation tillage.
Conservation cultivation is considered as one of the most successful agricultural inventions in
terms of soil management. In the USA, conservation tillage practices were estimated to be in
32% of the land in corn production in 1996, and there is a higher percentage of no-till/ridge-
till conservation tillage with the irrigated corn production system (Christensen, 2002).
Generally, the advantages of CT system are to conserve soil and moisture, reduce the costs
of fuel, machinery, and labour (Halvorson et al., 2006). No-till seedbed conditions pose
challenges for nutrient retention, fertilizer amendments, application methods and timing of
operations. There are also some disadvantages of NT system. In some situations, mulches
(crop residues) under the conservation tillage system acts as an insulating layer over the soil
surface, which can contribute to lower soil temperatures in the upper soil profile (Wolfe and
Eckert, 1999; Niehues et al., 2004) and shelter for insects. The decreased soil temperature
thereby lowers early season soil NO3--N released from organic matter mineralization (Andraski
and Bundy, 2008), may lead to the increased N-immobilization and denitrification (Fox and
Bandel, 1986). Reduced plant population densities, slow early growth and delayed tasselling
(Halvorson et al., 2006), and reduced grain yield (Dwyer et al., 1995b) has also been reported
as a result of cooler spring soil temperatures in the NT systems. The following two CT are
more common in practice:
No-till (NT) is defined as planting crops in unprepared soil with at least 30% mulch cover
(Triplett and Dick, 2008). In most no-till systems, no land preparation or cultivation is done
during production. The soil is left undisturbed so as to minimize the disturbance and to
maximize retention of crop residue on the surface. Seeds are planted in narrow seedbeds by
coulters or disk opener or row chisels. Normally, fertilizer or granular soil insecticides are
applied at the time of planting. Knockdown herbicides are generally applied before planting.
With better planters, herbicides and accumulated experiences, NT has gradually become a
more adopted practice in USA, Canada, and Australia since 1980. Basic idea of no-till planting
10 K. D. Subedi and B. L. Ma

is that the crop residue will provide benefits, including (1) conserving moisture; (2) reducing
runoff and erosion; (3) increased sequestration of soil organic carbon (SOC), and (4) reducing
weeds pressure through shading out. Higher level of SOC and soil organic N can be attained
by increasing cropping intensity under no-till management (Ortega et al., 2002).
Ridge-tillage is where soil is undisturbed from harvest to planting except for nutrients and
seeds injection. Seeds are planted on seedbeds prepared on ridges with sweeps, disc openers or
row cleaners.
Tillage systems have a significant effect on SOM. For example, after 8 years of no-till,
chisel plough and mouldboard plough, the chisel plough and mouldboard ploughs had less
total organic C than no-till plots in the 0-5 cm depth (Hussain et al., 1999). In the fine-textured
clay soils, no-till system often resulted in 15% or more reduction in dry matter and grain yields
of corn than the conventional mouldboard plough system in the cool and humid northeastern
Canada (Dwyer et al., 1995b).


4.1. Growth Stages of Corn

Corn plant undergoes different distinct developmental stages to complete its life cycle.
Although various scales of growth measurement are used, the most practical and commonly
used scale is the one developed by Ritchie et al. (1993). It describes corn growth in two
distinct growth phases, i.e. “Vegetative” and “Reproductive”. Within each phase, different
growth stages are designated with different scales (Table 1).

Table 1. Vegetative and reproductive stages of a corn plant (Ritchie et al., 1993)

Growth stage Morphological characterization

Vegetative Stages
VE Seedling emergence
V1 First leaf unfolded and fully expanded
V2 Second leaf unfolded and fully expanded
V3 Third leaf eaf unfolded and fully expanded
V(n) nth leaf eaf unfolded and fully expanded
VT Tasseling
Reproductive Stages
R1 Silking
R2 Blister
R3 Milk
R4 Dough
R5 Dent
R6 Physiological maturity
Corn Crop Production: Growth, Fertilization and Yield 11

4.2. Yield Components and Yield Determinants

Yield of any crop refers to the total amount of the part of a crop harvested on a given area
of land for economic uses. In corn, yield may refer to grain yield (grain corn), fodder yield
(forage or silage crop), marketable cob yield (sweet corn) and cobs weight (baby corn) and so
on. Yield components are the portions, each of which affects the yield as a whole. Yield
components in corn can be as follows:

a) Grain corn:

Grain yield (Mg ha-1) = Plants or ears ha-1 x grains ear-1 x mean grain weight (8)
Grain yield is usually expressed on a 155 g kg-1 water basis.

b) Silage corn:

Silage yield (Mg dry matter ha-1) = Plants ha-1 x weight of individual plant (9)

Silage corn is usually harvested when the whole plant moisture is within the range of 62 to
70%. Silage yield is often reported on a 650 g kg-1 water basis.

c) Sweet corn yield is usually reported as the number or weight of marketable ears per
ha, which is the product of number of plants per unit area by the number of marketable
ears per plant. Marketable ears refer to those ears with over 80% filled kernels and a
minimum length of 12 cm (Ma et al., 2007).

Clearly, plant population density (PPD; the number of plants per unit area) is the key
determinant of yield for all types of corn. Plant population density ultimately affects yield
through altering all the yield components. At high PPD, ear and kernel abortion occur because
of interplant competition for assimilates during the flowering period (Tollenaar, 1977), which
reduces the size of ear and ultimately the number of grains per ear and the size of individual
kernels. Andrade et al. (1999) suggested that PPD has also an important effect on partitioning
of dry matter (DM) between vegetative and reproductive sinks, and kernel set responded to the
amounts of resources available for each individual plant. Grain yield per unit area increases
with PPD until the increase in yield attributable to plants is not greater than the decline in
mean yield per plant (Tollenaar and Wu, 1999). At supra-optimal PPD, the number of grains
per ear, mean grain weight and cob-length were reduced (Bavec and Bavec, 2002). High PPD
coupled with low N supply often leads to high rates of kernel abortions and results in more
barren plants (Subedi et al., 2006). On the other hand, lower than optimum PPD delays the
time of canopy closure and thus reduces the interception of seasonal incident solar radiation
(Westgate et al., 1997), leading to larger number of grains per plant, but lower grain yield per
unit area (Andrade et al., 1999; Subedi and Ma, 2009). The PPD affects the post-flowering
source-sink ratio through its effects on plant leaf area, the amount of light intercepted per plant
and kernel number per plant (Borrás et al., 2003). Generally, higher PPD would enable the
crop to capture more PAR initially, but crowding increases after canopy closure.
The number of grains (or often refer to kernels) per ear is another important component for
grain corn. In corn, grain yield is correlated with kernel number, but uncertainty exists about
12 K. D. Subedi and B. L. Ma

the extension of the critical period of kernel set (Otegui and Bonhommer, 1998). Kernel
number is closely related to intercept photosynthetically active radiation (IPAR) during the
critical period. Pre-silking environment appears to define the potential number of kernels that
will be set as well as sub apical fertility, but the effectively fixed kernel number is dependent
upon post-silking conditions and hybrid characteristics. Pearson and Jacob (1987) observed no
evidence that shoot size per se controlled grain number or rate of grain growth; rather fertilizer
management during spikelet differentiation had most effect on yield. Number of kernels per
unit area is the most critical determinant of corn grain yield (Ritchie et al., 1988). Stresses that
enlarge anthesis-silking-interval beyond 5 d reduced grain yield per ear drastically (Ellings et
al., 1998). Monneveux et al. (2005) also observed that in tropical corn, grain yield was
negatively correlated with kernel abortion rate under low N stress.
Plant nutrition has also a significant effect on yield components. Grain yield was affected
by both N supplied before and after anthesis. For example, soil and foliar applied N around
silking can increase grain yield and nitrogen use efficiency by up to 15% (Ma et al., 2004).
The concentration of grain N remained declined rapidly during the first 20 d of grain filling
and remained constant thereafter (Ma et al., 2001). Subedi et al. (2006) observed that under
high PPD (90, 000 plants ha-1), as high as 15% of the plants were barren (plants without fully
developed ears), especially when the supply of N was limited. The effect of N stress on kernel
number occurs through its indirect effect on photosynthesis, silking dates and anthesis-silking-
interval (Singh and Wilkens, 2002).

4.3. Dry Matter Production and Partitioning

Corn is a C4 grass, which means, during the process of CO2 assimilation, the first stable
product of carbon reduction is a 4-C molecule. On a leaf surface and per unit time basis, C4
plants are more efficient in utilizing water, nutrients and CO2 to produce photoassimilates than
C3 plants such as wheat, barley (Hordeum vulgare L.) and rice (Oryza sativa L.). Unlike the
most small grain cereals, in which grain yield improvement during the past 60 years was
associated with the better partitioning of photoassimilates into the grains, resulting in the
significant improvement in harvest index (HI), corn yield improvement is attributable to its
general improvement in tolerance to abiotic (crowding, lodging, extreme temperatures, water,
nutrients, etc.) and biotic (insects, diseases, weeds) stresses (Tollenaar and Wu, 1999).
Partitioning of total biomass to the harvestable grains in tropical (Hay and Gilbert, 2001) and
temperate (Tollenaar and Wu, 1999) corn hybrids (varieties) has largely unchanged.
Harvest index refers to the proportion a crop that is of economic use. The HI in grain corn
is calculated as:

Grain yield
HI = x 100 (10)
Total biomass ( grain + stover )

Harvest Index is used as an indicator of the efficacy with which assimilates are partitioned
into the economically useful component of the crop. Generally, HI for corn without severe
stress ranges from 0.48 to 0.52, i.e. at maturity, around 50% of plant dry matter is partitioned
into kernels.
Corn Crop Production: Growth, Fertilization and Yield 13

5.1. Essential Plant Nutrients

Higher plants require at least 17 nutrient elements for their growth and completion of life
cycle. These elements are also called essential nutrients. Arnon and Stout (1939) first proposed
the term. For an element to be considered as an essential, it must meet the following three

(i) The plant cannot complete its life cycle in the absence of this element,
(ii) The function of an essential element cannot be replaced or compensated by another
element, and
(iii) The element is directly involved in the plants’ growth and reproduction.

5.2. Classification of Essential Plant Nutrients

The essential nutrients of higher plants, their sources and typical concentrations in plant
tissues are summarized in Table 2. Carbon (C), hydrogen (H) and oxygen (O) are considered
as non-mineral elements and are derived from air and water (Jones and Jacobson, 2005a). The
remaining 13 nutrients are classified either as macronutrients and micronutrients based on
their relative amounts of requirements by the plants. Within the macronutrients, nitrogen (N),
phosphorus (P) and potassium (K) are considered as “Primary Nutrients” while calcium (Ca),
magnesium (Mg) and sulphur (S) are called as “Secondary Nutrients”. The micronutrients
include boron (B), chlorine (Cl), copper (Cu), iron (Fe), manganese (Mn), molybdenum (MO),
and zinc (Zn). Nickel (Ni) is recently included among the micronutrients.

Table 2. Essential plant nutrients, their source, roles in the plant,

and typical concentrations in plant tissues

Element Origin Ionic forms absorbed by Role in plant Typical concentration

plants on dry tissue
Backbone of all organic matter;
Carbon (C) Air
necessary for photosynthesis
Important for osmotic balance,
Hydrogen (H) Water biochemical reactions and
constituent of carbohydrate
Constitution of carbohydrate,
Oxygen (O) Air/ Water
necessary for respiration
NO3- Constituent of proteins, chlorophyll 1.0-5.0%
Nitrogen (N) Air /soil
NH4+ and nucleic acids
Constituent of coenzymes, nucleic 0.1-0.5%
acids (DNA) and metabolic
substrates; storage of energy (ATP)
Phosphorus (P) Soil and important in energy transfer;
transportation of nutrients across
the cell wall and synthesis of
nucleic acid and proteins
14 K. D. Subedi and B. L. Ma

Table 2. (Continued)

Element Origin Ionic forms absorbed by Role in plant Typical concentration

plants on dry tissue
Involved with photosynthesis, 0.5-0.8%
+ carbohydrate translocation, protein
Potassium (K) Soil K
synthesis, disease resistance and
drought tolerance
A component of cell walls; plays a 0.2-1.0%
Calcium (Ca) Soil Ca+2 role in the structure and
permeability of membranes
Component of chlorophyll 0.1-0.4%
Magnesium +2 molecule, acts as an enzyme
Soil Mg
(Mg) activator, involves in carbohydrate
Important component of plant 0.1-0.4%
Sulphur (S) Soil SO4-2 proteins (amino acid synthesis) and
Important in sugar translocation, 6-60 mg kg-1
Boron (B) Soil carbohydrate metabolism, N and P
metabolism, pollination
Cl- Involves with oxygen production in 0.1-1.0%
Chlorine (Cl) Soil photosynthesis, water use, disease
A catalyst for respiration; a 2-20 mg kg-1
component of various enzymes,
Copper (Cu) Soil Cu+2
protein synthesis and chlorophyll
formation, N metabolism
Involves with chlorophyll synthesis 50-250 mg kg-1
+2 +3
Iron (Fe) Soil Fe , Fe and in enzymes for electron
Controls several oxidation- 20-200 mg kg-1
+2 reduction systems, essential for
Manganese (Mn) Soil Mn
chlorophyll manufacturing and thus
Molybdenum MoO4-2 Involves with N fixation, protein 0.05-0.2 mg kg-1
(Mo) synthesis, N metabolism
Involves with enzyme systems that 25-150 mg kg-1
regulate various metabolic
Zinc (Zn) Soil Zn+2
activities, including protein
synthesis and root development
Adapted from Dr. C.E. Swift (1993). Colorado State University, Extension, Tri River Area Agent
(Horticulture); W.F. Bennett (editor). Nutrient Deficiencies and Toxicities in Crop Plants, APS
Press, St. Paul, Minnesota.

All essential nutrients move from roots to the other parts of the plant, but they differ in
their pattern such that some nutrients move or are translocated from the older leaves to the
newer leaves when the supply of these nutrients to the growth point is limited. This
phenomenon is also referred as “mobility” of nutrients. Based on the mobility of nutrient
elements within the plant, plant nutrients are classified as mobile and immobile as presented in
Table 3.
Corn Crop Production: Growth, Fertilization and Yield 15

Table 3. Mobility of essential plant nutrients within the plant

Mobile Nutrients Immobile Nutrients

Nitrogen Boron
Phosphorus Calcium
Potassium Copper
Magnesium Iron
Molybdenum Sulphur
Chlorine Zinc

Within the mobile category, nutrients also vary greatly. For example, nitrate (NO3-) is
more mobile than phosphate (HPO4). The general rule is that deficiency symptoms are first
shown in the lower (older) leaves for the mobile nutrients whereas shortage of immobile
nutrients shows first symptom in the new leaves or terminal growth. In the soil system,
nutrients are also mobile such as NO3- is highly mobile while NH4+ is less mobile. Mobile
nutrient forms in the soil are easier to be taken up by the plant than non-mobile forms.

5.3. Deficiency Symptoms of the Essential Plant Nutrients

All essential nutrients have their specific and unique roles in plant growth. Although the
deficiency symptoms of some of the nutrients are identical in certain ways, they can be
distinguished each other. The typical deficiency symptoms on plant and soil-water system that
favors the deficiency are summarized in Table 4.

Table 4. Typical deficiency symptoms of various plant nutrients and favourable

conditions that enhance deficiency of different plant nutrients in corn

Nutrient Deficiency symptoms Favourable conditions

V-shaped yellow coloration from the margins in Low supply of N fertilizers
new leaves. Yellowing progresses from the Low mineralization in soil
Nitrogen (N) lower to the upper leaves and plants appear pale Water logging
green to yellow. Leaching of NO3--N and
N loss in gaseous forms (NH3 or N2O)

Purple margins or entire leaves especially Low test-P containing soils

during the seedling stage. Low fertilizer-P applied
Cooler and wetter weather during planting which
Phosphorus (P)
reduces the mobility of P and its uptake by the
Low soil pH
Yellowing to brown (necrosis) of the outer leaf Low test- K containing soils
margins. These symptoms begin at the leaf tip Under applied fertilizer K
and progress down the margin toward the leaf Cooler and wetter environments may induce K-
Potassium (K)
base. Plants become weak and may lodge. deficiency
Soil compaction
Excess N supply can also lower K availability
Failure of the leaf tips to separate from the Low Ca-containing soils
Calcium (Ca) whorl. This is often called "laddering". Calcium is often limited in acidic soils that
receive abundant rainfall to leach Ca
16 K. D. Subedi and B. L. Ma

Table 4. (Continued)

Nutrient Deficiency symptoms Favourable conditions

Leaves have light green to yellow strips that run More common in acidic and sandy soils that are
Magnesium parallel with the blade. Chlorotic leaves prone to leaching
(Mg) generally turn reddish and develop spotted Mock or organic soils
necrotic areas. Cool, wet soils
Yellowing of the younger leaves of the corn Sulphur is a mobile nutrient and is water soluble,
plant. Sulphur deficiency symptoms show up as high rainfall during corn planting can cause more
interveinal chlorosis of the leaves emerging S leaching.
Sulphur (S) from the whorl. As the plant ages and the Poor root development of corn
deficiency become more pronounced, the entire Low soil OM or reduced mineralization of
leaf turns yellow with slightly greener veins. organic-S in the no-till systems.
Generally S-deficient plants are stunted.
Severe B deficiency results in small, misshapen Sandy soil, leached soils and calcareous soils are
cobs or do not produce ears or ears with deficient in B
Boron (B) missing kernels (barren cobs). Under extreme B Soils low in OM are deficient in B
deficiency, the leaves also may have small
white dead spots and be curled and brittle.
Wilting and restricted, highly branched root Chlorine deficiencies can occur on sandy soils in
Chlorine (Cl) systems are the main chloride-deficiency high rainfall areas or those derived from low-
symptoms. chloride parent materials.
Yellowing of leaves, stunted growth and pale Copper deficiencies are mainly reported on peat
green leaves that wither easily. (muck) soils, sandy soils which are low in OM.
Copper (Cu) Copper uptake decreases as soil pH increases.
Increased P and Fe availability in soils decreases
copper uptake by plants.
Leaf yellowing first appears on the younger Iron deficiencies are found mainly on high pH
upper leaves in interveinal tissues due to low soils, sandy soils low in OM.
levels of chlorophyll. Severe Fe deficiencies Cool, wet weather enhances iron deficiencies,
cause leaves to turn completely yellow or especially on soils with marginal levels of
Iron (Fe) almost white interveinal chlorosis and then available Fe.
brown as leaves die. Poorly aerated or compacted soils also reduce iron
uptake by plants.
Uptake of Fe is also adversely affected by high
levels of available P, Mn and Zn in soils.
Inter-veinal chlorosis with white/grey spots of Manganese deficiencies mainly occur on organic
the upper, new leaves of corn, resulting in soils, high-pH soils, sandy soils low in OM, and
premature leaf drop. Delayed maturity is on over-limed soils. Soil Mn may be less available
another deficiency symptom and is also a sign in dry, well-aerated soils.
of manganese deficiency.
Pale - green to yellow leaves and marginal Mo deficiency occurs under acidic conditions,
chlorosis along side and tip of blade and thick sandy soils and soils low in OM.
cupped leaves.
Zinc deficiency is the most widely occurring Soils deficient in Zn
among the micronutrients. Zinc deficiency Calcareous soils with pH >7.5
symptoms begin at the leaf base of the upper
leaves and expand toward the leaf tip as
Zinc (Zn)
interveinal chlorosis or a band of chlorotic
tissue between the leaf edge and the midrib.
Zinc deficient plants also exhibit delayed
Sources: Jones and Jacobson (2005a)
Corn Crop Production: Growth, Fertilization and Yield 17

5.4. Nutrients Composition in Corn Plants

Generally, typical values of nutrient composition are given based on repeated observations
and from unstressed plants. The typical nutrient concentrations in different parts of a matured
corn plant are presented in Table 5. Composition of essential nutrients in any plant depends on
growing conditions such as amount of nutrients supplied in the growing medium/soil, growing
environments (unstressed crop), crop type, variety, growth stage and several other factors.
Therefore, typical concentration is a vague definition and cautions should always be taken to
interpret such data. For example, chemical constituent of corn plant was dependent on amount
of nutrients supplied and frequency of irrigation (Ibrahim and Kandil, 2007).

Table 5. Dry matter and nutrient composition by corn plant part at maturity
(after Hanway, 1962)

Component Dry Matter Nitrogen Phosphorus Potassium

% of total %N % P2O5 % K2O
Grain 48 1.44 0.69 0.50
Stalks 22 0.43 0.14 0.90
Leaves 10.6 1.80 0.69 2.05
Sheaths 5.3 0.64 0.37 1.74
Husks 4.3 0.36 0.21 1.32
Shanks 1.5 0.50 0.18 1.68
Cobs 7.5 0.33 0.11 0.62
Tassels 0.5 0.97 0.50 1.70
Lower ears 0.5 2.04 0.87 3.00
Silks 0.2 3.50 0.87 2.57
Total 100 - - -

The concentrations reported above are not universal. The tissue nutrients composition
varies with the supply of nutrients (soil plus applied nutrients), genotypic ability to take up and
partition nutrients to different components, growing environment of the crop (water supply,
stress-free growing period, etc.) and stage of the crop at harvest. For example, tropical corn
varieties are reported to contain 1.46% N, 0.33% P, and 0.39% K in the grain (Feil et al.,

5.5. Sources of Essential Plant Nutrients

Plant nutrients that are to be supplemented for plant growth (i.e. in addition to from soil
and water systems) are available in many forms and through different sources. Broadly, the
nutrients for corn are supplied through two major sources:
18 K. D. Subedi and B. L. Ma

(i) Organic form: Organic sources are those originated from living organisms after their
decomposition. These include crop residues, green manures, biologically fixed N,
farm yard manures (FYM), liquid or solid compost or uncomposted animal manures,
processing waste, municipal waste, etc.
(ii) Inorganic form: Plant nutrients (macro and micro) and soil amendments (e.g. dolomite
lime) are supplied in artificially manufactured chemical medium called fertilizers and

The typical nutrient concentrations in various organic sources are presented in Tables 6, 7
and 8, and macronutrient fertilizers and micronutrient supplements are summarized in Tables 9
and 10.

Table 6. Nutrient content of organic materials

Percentage by Weight
Organic Material
N P2O5 K2O Ca Mg S Cl
Blood (dried) 12 to 15 3.0 — 0.3 — — 0.6
Bone meal (raw) 3.5 22.0 — 22.0 0.6 0.2 0.2
Bone meal (steamed) 2.0 28.0 0.2 23.0 0.3 0.1 —
Cotton waste from factory 1.3 0.4 0.4 — — — —
Cottonseed meal 6 to 7 2.5 1.5 0.4 0.9 0.2 —
Cowpea forage (green manure) 0.4 0.1 0.4
Legume 3.0 1.0 2.4 1.2 0.2 0.3 —
Grass 1.5 0.5 1.9 0.8 0.2 0.2 —
Peanut hull meal 1.2 0.5 0.8 — — — —
Peanut meal 7.2 1.5 1.2 0.4 0.3 0.6 0.1
Peat/muck 2.7 — — 0.7 0.3 1.0 0.1
Pine needles 0.5 0.1 — — — — —
Poultry processing: DAF sludge 8.0 1.8 0.3 — — — —
Sawdust 0.2 — 0.2 — — — —
Seaweed (dried) 0.7 0.8 5.0 — — — —
Sewage sludge (municipal) 2.6 3.7 0.2 1.3 0.2 — —
Soybean meal 7.0 1.2 1.5 0.4 0.3 0.2 —
Adapted from Zublena et al. (1991).
Table 7 Average nutrient contents of livestock manures. Data from manure samples collected between 1992 and 2004
and analyzed by different Ontario Labs

Manure Type Dry Total N NH4+-N P K (%) Ca Mg Zn (mg kg-1) Cu (mg kg-1) Mn (mg kg-1)
(number of samples) Matter (%) (mg kg-1) (%) (%) (%)
Dairy liquid (860) 8.5 0.36 1,527 0.09 0.24 0.49 0.14 48 17 40
solid (150) 24.2 0.61 1,278 0.17 0.50 1.54 0.36 95 29 107
Swine liquid (924) 3.8 0.40 2,648 0.13 0.17 0.12 0.06 85 30 22
solid (54) 29.8 0.90 2,582 0.47 0.56 -- --- 172 103 --
Poultry liquid (137) 10.6 0.83 5,581 0.3 0.3 1.6 0.08 70 11 64
solid (623) 52.6 2.37 5,495 1.11 1.17 4.6 0.28 238 33 204
Beef liquid (81) 7.95 0.52 1,794 0.13 0.43 0.7 0.3 57 14 61
solid (176) 28.6 0.73 1,011 0.23 0.57 1.5 0.41 129 36 112
Sheep solid (54) 31.3 0.76 1,862 0.27 0.70 1.5 0.38 170 20 140
Horse solid (32) 33.41 0.42 684 0.13 0.36 1.7 0.56 73 23 113
Source (Brown, 2005).
20 K. D. Subedi and B. L. Ma

These concentrations are typical nutrient ranges found in the highly nutritive animal
feeding systems. The nutrient concentration depends on protein content in the feed stuff, types
of animal, age of animal, manure management system (liquid versus stock piled, open versus
shaded piles and liquid versus solid and so on). This table gives a general guideline, but more
accurate data are required from determinations by chemical analysis locally and frequently.
The actual concentrations of nutrients in the small-scale subsistence farming vary greatly. For
example, Lupwayi et al. (2000) reported in highland Ethiopia, that manure samples taken from
experimental stations contain more N, P, K, Mg, Cu and Zn than those from smallholder
farms, probably due to differences in feed availability and quality. Stored manures usually
contain slightly higher N concentration than the same of fresh manures, probably because of
loss of some carbon.

Table 8. Nutrients concentration (g kg-1 dry weight basis) of cattle manure collected
from small-scale farms and experimental stations in Ethiopian highlands

Nutrient Range Mean± SD

Nitrogen (N) 11.7-27.4 18.3±4.6
Phosphorus (P) 2.2-7.0 4.5±1.5
Potassium (K) 10.6-54.4 21.3±11.2
Calcium (Ca) 10.1-24.6 16.4±3.9
Magnesium (Mg) 3.2-12.4 5.6±2.3
Iron (Fe) 3.7-22.4 10.8±0.5
Manganese (Mn) 0.27-1.90 0.78±0.39
Copper (Cu) 0.008-0.086 0.024±0.015
Zinc (Zn) 0.049-0.0217 0.092±0.036
Adapted from Lupwayi et al. (2000).

Table 9. Common chemical fertilizers and their nutrients composition

Nutrients concentration (% by weight)

Material Chemical formula
N P2 0 5 K20 Ca Mg S

Ammonium nitrate NH4NO3 30-33 0 0 0 0 0

Ammonium nitrate sulphate NH4NO3+(NH4)2SO4 26 0 0 0 0 15
Ammonium sulphate 21 0 0 0.3 0 24
Ammonium thiosulfate (NH4)2S2O3 12 0 0 0 0 26
Anhydrous ammonia 82 0 0 0 0 0
Aqua ammonia NH4OH 16 -25 0 0 0 0 0
Ca(NO3)2 4H2O
Calcium nitrate 15 0 0 19 1.5 —

Calcium nitrate/urea Ca(NO3)2+4CO(NH2)2

34 0 0 10 0 0

Potassium nitrate KNO3

13 0 44 0.6 0.4 0.2

Urea CO(NH2)2
46 0 0 0 0 0
Corn Crop Production: Growth, Fertilization and Yield 21

Nutrients concentration (% by weight)

Material Chemical formula
N P2 0 5 K20 Ca Mg S
Urea (sulphur coated) CO(NH2)2+S
36 -38 0 0 0 0 13-16
Urea sulphate CO(NH2)2.H2SO4 17 — — — — 20
Diammonium phosphate (DAP) 18 46 0 0 0 0

Monoammonium phosphate (MAP) NH4H2PO4

11 48 0.2 1 0.3 2.2
Ammonium phosphate nitrate NH4H2PO4 NH4NO3
27 15 0 0 0 0

Ammonium phosphate sulphate 4NH4H2PO4+(NH4)2SO4

13-16 20-39 0.2 0.3 0.1 15

Ammonium polyphosphate (APP) (NH4)3HP2O7

10 34 0 0 0 0
. .
Basic slag 5CaO P2O5 SiO2 0 2-17 0 3 -3 3 —
Concentrated superphosphate Ca(H2PO4)2 H2O
0 42-50 0.4 14 0.3 1.4
Ordinary superphosphate Ca(H2PO4)2.H2O+CaSO4 0 18-20 0.2 20 0.2 12

Nitric phosphate
14-22 10-22 0 8-10 0.1 0.3

Phosphate rock 0 2-35 0 — — 0

Urea ammonium phosphate (UAP) CO(NH2)2.NH4H2PO4

25 35 0 0 0 0
Potassium chloride KCI
(Muriate of potash) 0 0 60-62 0.1 0.1 0

Potassium nitrate KNO3

13 0 44 0.6 0.4 0.2

Potassium sulphate K2SO4

0 0 50 0.7 1 18

Calcium chloride CaCl2

0 0 0 36 0 0

Calcitic limestone CaCO3

0 0 0.3 32 3 0.1

Dolomitic limeston CaCO3+MgCO3

0 0 0 21-30 6-12 0.3
Gypsum CaSO4 2H2O
0 0 0.5 22 0.4 17

Hydrated lime (Slaked lime) Ca(OH)2

0 0 0 50 0 0

Magnesium oxide (Magnesia) MgO

0 0 0 0 45 0
Magnesium sulfate MgSO4 7H2O
0 0 0 2 10 14

Ammonium sulphate (NH4)2SO4

21 0 0 0.3 0 24

Elemental sulphur/ Wettable S S

0 0 0 0 0 90-100

Elemental sulphur (S): Flowable S S

0 0 0 0 0 52-70
Compiled from Zublena et al. (1991).
22 K. D. Subedi and B. L. Ma

Table 10 . Types and the approximate nutrient concentrations of different micronutrients

Nutrient Material Chemical Formula Concentration (%)

Boron (B) Borax (sodium tetraborate 11

decahydrate) Na2B4O7.10H2O

Boric acid 17
Clorine (Cl) Ammonium chloride 66

Calcium chloride 74

Magnesium chloride 65

Potassium chloride 47

Sodium chloride 60
Copper (Cu) Copper chelates 13
Copper sulfate CuSO4.H2O 35

Cupric ammonium phosphate Cu(NH4)PO4.H2O 32

Iron (Fe) Ferric sulphate 20


Ferrous ammonium phosphate 29


Ferrous ammonium sulphate 14


Ferrous oxalate 30

Ferrous sulphate 20
Iron chelates (Fe EDTA) 9 to 12
Manganese Manganese ammonium phosphate 28
Manganese chelate 12

Manganese sulphate 24

Manganous oxide 41 to 68
Molybdenum Sodium molybdate 38 to 46
Ammonium molybdat (NH4)6Mo7O24.4H2O up to 54
Zinc (Zn) Zinc chelate Na2Zn EDTA 9 to 14

Zinc oxide 78 to 80

Zinc sulphate ZnSO4.H2O 22 to 36

Compiled from Zublena et al. (1991).


Nutrient uptake by a crop refers to the total amount of the nutrient as a fraction of the plant
DM at harvest. The amount of nutrients removed by a corn plant at harvest depends on the
Corn Crop Production: Growth, Fertilization and Yield 23

availability of the nutrients in the soil, soil moisture, corn hybrids, and growing conditions that
determine the crop growth rate. Table 11 shows the typical nutrient concentrations found in a
corn crop producing 18.7 Mg ha-1 DM. These values give a general idea but the actual
concentrations vary considerably with different growing conditions, varieties and nutrients
supplying capacity of the soils.

Table 11. Typical concentrations of 13 essential plant nutrients in a corn crop yielding
18.7 Mg ha-1 dry matter yield

Primary Nutrients Content Micro-nutrient Content

kg ha-1 kg ha-1
Nitrogen (N) 240 Chlorine (Cl) 110
Phosphorus (P) 44 Iron (Fe) 3
Potassium (K) 200 Manganese (Mn) 0.6
Secondary Nutrients Zinc (Zn) 0.6
Sulphur (S) 34 Copper (Cu) 0.2
Calcium (Ca) 45 Boron (B) 0.1
Magnesium (Mg) 56 Molybdenum (Mo) >0.1
Adapted from Johnston and Dowbenko (2004).

One should not be confused with the “nutrients uptake” with “nutrients removal”. The
nutrient removal is the amount of nutrients that are removed in the harvested portions of the
crop such as grain, silage or forage (Ma et al., 2006a). In the case of corn, generally grain
(about 50% DM and 60 to 70% N) is harvested while 50% DM and about 30% N in the residue
DM (leaves, stalk, cobs etc.) are left on the same field if only grains are harvested. Therefore,
at least 1/3 of the total N and other nutrients in much higher proportions are remained and
recycled in the same field. In the small holder subsistence farming systems, corn residues are
often considered value for livestock feed, or as materials for heating, fencing and staking etc.,
corn stovers are partially or entirely removed from the corn-fields, leading to the land
vulnerable to erosion and much less nutrients available for the following crops. In such
systems, replenishment of plant nutrients is difficult to achieve and there is always a negative
balance of SOM and nutrients unless large amounts of manure and fertilizers are added each
To determine if a nutrient element is critical for plant development and yield formation,
the concept of critical nutrient concentration is often referred. For example, the concept of
critical N concentration (Ncrit) assumes at any time a minimum shoot N concentration
necessary for maximum biomass production (Herrmann and Taube, 2005). A quadratic-plateau
model is used to derive Ncrit values. The relationship of Ncrit (g N kg-1 DM) and biomass
production is described by a mononomial function: W (Mg DM ha-1): Ncrit=34.12 x W-0.391.
Uptake rates of N, P and K nutrients can often be expressed as a linear relationship between
nutrients uptake rate and transpiration rate of corn canopy (Novak and Vidovic, 2003).


Nutrients required by a corn crop can be determined in a variety of ways. Some of the
methods are quantitative while others give subjective judgement of whether the crop is
24 K. D. Subedi and B. L. Ma

deficient in particular nutrient(s). The common methods in practice to determine or assess the
nutrients requirement of corn crops are as follows:

6.1. Visual Observation

Corn plants deficient on a particular nutrient can be detected visually based on the
symptom they develop (See Table 2). This is a simple and inexpensive method but needs skills
of detection and knowledge of crop growth environment. Sometimes symptoms of more than
one nutrient can be confusion. For example, the deficiency symptoms of N and Mg are similar
unless they are carefully diagnosed. Colour pictures of deficiency symptoms are helpful for
such detections. The disadvantage of this approach is that (i) normally it will be already too
late to follow corrective actions, and (ii) this approach does not quantify how much nutrients
are to be added if application is needed for correction.

6.2. Soil –Based Indicators

Soil analysis to determine the nutrient availability in the soil is one of the most common
methods for determining nutrients requirement in any crop. This is a traditional method;
although newer methods and approaches are being developed for the determination of nutrient
status and for recommendations of the optimum fertilizer rates. Soil tests both pre-season and
pre-sidedress can help farmers to predict optimum fertilizer rates (Binford and Peterson, 1998;
Ma and Wu, 2008). Soil test methods require considerable time, efforts and cost for sampling,
processing and analysis (Bausch and Duke, 1996).

6.2.1. Pre-Plant Soil Test (PPNT)

This test quantifies the amount of soil residual NO3-- and NH4+-N concentrations (PPNT
soil test) and other nutrients such as P and K present in the crop rooting zone so that farmers
can adjust their fertilizer rates accordingly. Soil samples are collected before planting in
spring, generally from a soil depth of up to 60 cm and concentrations of available N (NO3--
and NH4+-N), P, K and any other nutrients of interest are determined in a chemical laboratory.
Fertilizers are recommended based on the concentrations of soil available nutrients. In the
humid environments, such as northeastern USA and Canada, response of corn grain yields to N
amendments is often poorly correlated with soil mineral N at pre-plant or pre-sidedress,
because they do not address the spatial and temporal variability of soil N (Ma and Dwyer,
1999). Similarly, Stevens et al. (2005) concluded that the PPNT performed less than
satisfactorily in many cases when compared with actual N responses from 75 site-year data.
Khan et al. (2001) proposed an approach of predicting soil organic N contributions to the
plant-available N supply through the analysis of NH4+ and hydrolysable amino sugar. This test
is known as Illinois Soil Nitrogen Test (ISNT). Osterhaus et al. (2008) evaluated this test and
concluded that ISNT and the soil organic fractions studied are not reliable predictions of corn
N response. The limitation with the PPNT is that soil samples are taken before corn planting
which does not account for growing season mineralization and denitrification, which
determine the amount of NO3--N available for corn.
Corn Crop Production: Growth, Fertilization and Yield 25

6.2.2. Pre-Sidedress Nitrate Test (PSNT)

Magdoff et al. (1984) proposed the pre-sidedress soil nitrate test (PSNT), in which
recommendations of N fertilizer sidedress rates are based on the soil test prior to the time of
application, usually in late June or early July, during the crop growing season. This is also
called as late-spring soil nitrate test. This approach involves (i) time (when corn is 20-30 cm
tall) and (ii) soil depth (top 30 cm) of sampling. This test quantifies the amount of soil NO3--N
present in the crop rooting zone so that farmers can adjust the fertilizer rates accordingly (Ma
and Wu, 2008). A critical value of PSNT was defined to be 20 to 30 µg NO3--N g-1 soils
(Magdoff et al., 1984).
Unlike PPNT, PSNT is partially accountable for soil mineralization and preplant
application of N fertilizers and manures. Recommendations based on PSNT can trim down the
extra amount of fertilizer N used by farmers to guard against N-deficient corn (Magdoff, 1991;
Heckman et al., 1995). This approach has the greatest potential for soils with high
mineralization potentials (e.g. soils with high OM, or with manure history). Therefore, the
PSNT has shown promise as a means of quantifying and improving N management for corn
production (Magdoff et al., 1984; Binford et al., 1992). Andraski and Bundy (2002) concluded
that adjusting N application rates for corn using PSNT is more profitable than not making such
adjustment. Andraski and Bundy (2002) also reported that the accuracy of PSNT was highest
for sites with above-average May-June air temperatures and high yield potentials. Use of
PSNT has also been successfully extended to sweet corn and other vegetables (Heckman et al.,
2002). In Ontario, working with sweet corn, Ma et al. (2007) observed that the PSNT NO3--N
increased linearly with the fertilizer N rates, and there were significant positive correlations
between PSNT at V4 to V6 growth stages and the number of marketable ears. The drawback of
this test is that (i) it can be costly as it involves several samples per ha to be taken and
analysed, (ii) laboratory analysis of soil samples requires more time before critical stage of
corn N requirement elapses, and (iii) PSNT may not precisely address the spatial variability of
soil N.

6.2.3. Post-Harvest Nitrate Test (PHNT)

The post harvest nitrate test (PHNT) is an approach in which soil samples are collected
after the corn is harvested and analyzed for the residual soil NO3--N and other nutrients of
interest such as P and K. This test is not as common as PSNT or PPNT. However, PHNT
appears to be valuable to identify N sufficient and deficient sites (Schröder et al., 2000), and
justify environment assessment (Ma et al., 2006a). This test also indicates the potential for
ground water pollution since it measures the NO3--N not used by the crop (Sullivan and
Cogger, 2004). Elevated post-harvest soil NO3--N is an indicator of excess amount of N
fertilizer application in the previous crop (Gehl et al., 2006). Slight differences in site
characteristics (e.g. textural boundaries), can greatly influence conclusions derived post-
harvest soil sampling regarding the risk of NO3--N leaching (Gehl et al., 2006). The weather
patterns such as precipitation and temperatures affect the interpretation as they influence
mineralization of SOM and also leaching. This approach can help to decide on the preplant
fertilizer recommendations where PPNT is not possible. However, this is not a tool for in-
season N corrections.
26 K. D. Subedi and B. L. Ma

6.3. Plant Tissue Analysis (Destructive Method)

Destructive plant sampling is one of the commonly used indicators to assess crop N status
during the crop growing season. Plant analysis has proven useful in confirming nutrient
deficiencies, toxicities or imbalances, identifying “hidden hunger”, evaluating fertilizer
programs, determining the availability of elements not tested for by other methods, and
studying interactions among nutrients (Schulte and Kelling, 1999). Therefore, plant analysis is
considered as a tool for troubleshooting crop problems.
There are some disadvantages of tissue analysis other than labour and cost, such as (i)
contamination of plant samples with soil particles or pesticides residue can lead to erroneously
high results for iron, Al, Mn or Cu, (ii) decomposition of plant samples before it reaches to the
laboratory can result in a loss of carbon through respiration thereby increases the concentration
of other nutrients, (iii) measurement of N uptake by plants does not necessarily indicate the N
requirement of the plant as several studies have indicated that N concentration in shoot can be
greater than the minimum plant requirements for maximum growth (Dharmakeerthi et al.,
2006), (iv) As N supply decreases, N uptake, translocation and remobilization are also
affected. Therefore, it might not give a true picture of the N status.
Nevertheless, tissue tests give an overall picture of the nutrient level within the plant at the
time of sample taken. Generally, good relationships can be developed between soil nutrient
supplies, nutrient levels in the plant, and crop yield for a given location in a year. However,
differences in locations, variety, time and management often cause variations in these
relationships and make them difficult to interpret (Schulte and Kelling, 1999). For most
diagnostic purpose, plant analysis is interpreted on the basis of “critical levels” for each
nutrient. The critical level has been defined as the concentration that below which yields
decrease or deficiency symptoms appear (Schulte and Kelling, 1999). The nutrient
concentration of the crop changes as the plant matures and with the portion of plant sampled;
therefore, the critical levels are defined for a specific plant part at a specific stage of growth.
Tissue test can be whole plant analysis or a particular plant part such as individual leaf, plant
sap content or shoots. The commonly used tissue tests that are used in corn are as follows:

6.3.1. Whole Plant Tissue Test

Generally, corn seedlings (V6-V8 stage) are sampled and analysed for the nutrients
concentration, especially for total or NO3--N. This test is employed to diagnose the nutrients
status at specific stage and make recommendation for corrections. Iversen et al. (1985) found
that stalk N concentration at approximately 30 d after emergence appeared to be correlated
with relative grain yield and N uptake. Binford et al. (1992), however, suggested that tissue
test based on the concentrations of N in young plants would not be reliable indicators of N
availability in corn fields. Although, the relationships between plant N concentration and grain
yield were established, results varied with season, soil types and stages of measurement.
Similarly, Schröder et al. (2000) concluded that tissue tests are less value for the support of
decisions on N supplementation than indicators that are directly related to the soil or to the
measurement of leaf and canopy greenness. Tissue tests are unable to quantify excessive
availability of N at early crop stages as opposed to soil related indicators. Driskell and Richer
(1952) observed a strong correlation between tissue N concentration and visual deficiency
symptoms, however, no correlation was found between tissue test and potential nitrification.
Strong correlation was observed between tissue tests of P and K with deficiency symptoms.
Corn Crop Production: Growth, Fertilization and Yield 27

In general, the greatest risk of high nitrate levels has been in drought-stunted fields that
have received excessive manure or N fertilizer. The risk of high nitrate levels is highest
immediately following a drought-ending rain. Nitrates accumulate in the lower portion of the
plant, so harvesting higher position of the plant under these conditions can help avoid high
nitrate concentrations. Plant analysis requires considerable efforts for sample collection and
processing. Repeated sampling throughout the growing season can be very laborious, time
consuming and costly.

6.3.2. Leaf Tissue Test

Concentration of earleaf at silking (R1) has been used as a tool for evaluating the N status
of crop by comparing the observed concentration of earleaf N with published critical values.
For corn, the earleaf from tasselling to silking is commonly used for analysis (Schulte and
Kelling, 1999). The typical concentration of nutrients in corn earleaf at silking stage is
presented in Table 12. Cerrato and Blackmer (1991) assessed the reliability of leaf N
concentration as an indicator of the N status of corn. They found that leaf N concentration
tended to increase with increase in rates of N application and with increase in grain yields.
Critical N concentration in the earleaf between tasselling and silking ranged from 2.6 to 3.6%
on DM basis (Roberts and Rhee, 1993). Cerrato and Blackmer (1991) concluded that for grain
yield, the critical N concentration in the leaf opposite or below the ear is not a sensitive
indicator of N status. More importantly, in most cases, this is too late to correct the deficiency
through in-season nutrients application.

Table 12. Typical Composition of Plant Nutrients in the Corn Leaf (Mid-Third of the
Earleaf Opposite The Ear) At Silking Stage

Nutrient Units Critical concentration1 Maximum normal concentration2

Nitrogen % 2.50 3.50
Phosphorus % 0.28 0.50
Potassium % 1.20 2.50
Calcium % 1.50
Magnesium % 0.10
Sulphur % 0.14 0.60
Boron ppm 2.0 25.0
Copper ppm 2.0 20.0
Manganese ppm 15.0 150.0
Zinc ppm 20.0 70.0
1 Maximum yield loss due to nutrients deficiency is expected with nutrient concentrations at or below
the "critical" concentration.
2 Maximum normal concentrations are more than adequate but do not necessarily cause toxicities.
Adapted from OMAFRA (2002).

6.3.3. Post-Harvest (End-of–Season) Stalk Nitrate Test

The end-of-season NO3--N test or post-harvest stalk nitrate test was proposed by Binford
et al. (1990), as a post-mortem to determine if excessive or insufficient N was available to the
corn crop during the later part of the development. This indicator gives valuable hints on N
fertilizer requirements for the subsequent corn crops. The 20 cm portion of corn stalk, 15-35
cm above the ground are analysed for NO3- concentration immediately after grain harvest. A
concentration of between 700 to 2000 mg NO3- kg-1 indicates adequate N supplied and over
28 K. D. Subedi and B. L. Ma

2000 mg kg-1 indicates that excess N was supplied or present in the soil during the growing
season (Binford et al., 1992). This test helps in N management in the coming season but not
for in-season correction for N - deficiency. Wilhelm et al. (2004) observed that NO3-
concentration of the individual sample varied greatly from <100 to >8000 mg NO3--N kg-1
DM, and increased downwards the stalk from ear level to the aboveground level. Moreover,
the range of NO3--N concentration for grain corn will not be applicable for sweet corn or silage
corn because these crops are harvested earlier than the grain crop. Therefore, stalk N
concentration will be higher prior to maturity. Similarly, N-dynamics in the soil such as
mineralization, denitrification and leaching are not taken into account. Thus stalk NO3--N
concentrations are also soil and climate dependent.

6.4. Crop-Based Indicators (Non-Destructive Methods)

Because soil and plant analysis require considerable efforts, time and cost to collect and
analyse samples, alternative technologies that reflect plant nutrients status can be useful. In the
recent past, many types of instantaneous diagnostic techniques have been developed to monitor
the crop nutrients status. These are called remote sensing or crop sensing devices.
Chlorophylls, xanthophylls and carotenes absorb solar radiation in the visible part of the
spectrum and thus reflect a small portion in these ranges. Reflectance in the visible range (λ =
550-675 nm) has been used to estimate leaf chlorophyll (Benedict and Swilder, 1961; Slafer
and Andrade, 1991), and carotenoid (Filella et al., 1995; Thomas and Gausman, 1977) levels,
and by extension the photosynthetic capacity of the crop (Ma et al., 1995). In the near infrared
(NIR) range, green vegetation strongly reflects incident radiation. The magnitude of the NIR
reflectance is governed by the scattering of light by plant tissues at different levels in the
canopy (Knipling, 1970) and is proportional to the vegetation biomass (Gutierrez-Rodriguez et
al., 2004). This distinct contrast in spectral behaviour between visible absorbance and NIR
reflectance formed the background and principle of terrestrial remote sensing for the past three
decades (Gitelson, 2004). Many remote sensing devices operate in the green, red and NIR
regions of the electromagnetic spectrum, which discriminate radiation absorption and
reflection from the surface of green vegetation. Such devices are used not only to detect crop
nutrient deficiencies but also crop stresses induced moisture deficiency (drought), disease, and
pests. The stresses are indicated generally by decrease in NIR reflectance.

6.4.1. Leaf Chlorophyll Meter (SPAD)

The leaf chlorophyll meter, which is commonly known as SPAD (Soil Plant Analysis
Development; SPAD–502 Minolta Camera Co. Ltd. Japan), is an easy to use device which
measures the intensity of light transmitted through the leaf at the 650 and 940 nm wavelengths.
The obtained SPAD values are linearly correlated with leaf chlorophyll content determined
with destructive measurements (e.g. Marquard and Tipton, 1987; Schaper and Chacko, 1991).
Wood et al. (1992) evaluated its field performance and found to be good predictor of grain
yield. Blackmer and Schepers (1995), Bausch and Duke (1996), and Waskom et al. (1996) also
found that chlorophyll meter is a useful method for rapid monitoring of in-season crop N status
and grain yield potential. The ability of the SPAD to accurately identify N deficiencies is
improved when normalizing the chlorophyll meter reading to an adequate or non-N limiting
Corn Crop Production: Growth, Fertilization and Yield 29

reference plot within the same field (Schepers et al., 1992; Bausch and Duke, 1996; Ruiz Diaz
et al., 2008).
It is well established that chlorophyll meter readings are highly correlated with N
concentration in corn leaf tissue (Schepers et al., 1992; Wood et al., 1992). However, at the V6
stage, there is a narrow range of leaf chlorophyll, which made difficult to separate N-deficient
from N-sufficient field (Dwyer et al., 1991) and a large number of observations are required
(Costa et al., 2001). Although Schröder et al. (2000) concluded that leaf greenness and tissue
tests both are unable to quantify excessive availability of N at early stages as opposed to soil
related indicators, Ruiz Diaz et al. (2008) stated that sensing of crop to determine in-season N
addition seems to be a cost effective strategy with the reduced sidedress N rate.
The chlorophyll meter is a quick, easy to use, and results are instantaneous for in-season N
application decisions. However, there are some drawbacks of the SPAD system such that (i) N
sufficiency is not represented by a unique value as the SPAD value of sufficient N increases
with crop age (Blackmer and Schepers, 1995), (ii) corn hybrids differ substantially in
chlorophyll meter readings within a given N rates (Subedi and Ma, 2005a; Subedi et al., 2006),
(iii) position of leaf, and the readings taken at the early stage (before V6 growth stage) are less
effective, and the later season diagnosis of N deficiency (i.e. after V8 growth stage) is
generally too late to correct the deficiency, and (iv) the initial investment is high for small
scale farmers.

6.4.2. Canopy Reflectance Measurements

The use of remote sensing techniques such as canopy light reflectance could help
eliminate the need for extensive field samplings (Gilabert et al., 1996; Ma et al., 1996).
Canopy reflectance is defined as the ratio of the amount of radiation reflected by an individual
leaf or canopy to the amount of incident radiation (Schröder et al., 2000). Leaves absorb
mainly blue (450 nm) and red (660 nm) wavelengths and reflect mainly green (550 nm)
wavelengths. Reflectance measurements at these wavelengths therefore, give a good indication
of leaf greenness (Schröder et al., 2000). Several researchers have used the multi-spectral
canopy reflectance to indicate plant N status and predict yield potential in many crops,
including corn (Ma et al., 1996, 2005; Bausch and Duke, 1996; Osborne et al., 2002b), rice
(Casanova et. al., 1998), soybean (Ma et al., 2001), cotton (Gossypium hersutum L.) (Bronson
et al., 2003) and wheat (Aparicio et al., 2000; Flowers et al., 2003). Similarly, Osborne et al.
(2002a) used the spectral radiance to detect the P-deficiency in corn. On-the-go sensing
devices based on canopy reflectance have now been developed and tested for variable rate
application of N fertilizer according to site-specific field conditions (Raun et al., 2002).
A hand-held multi-spectral radiometer (Crop Scan, CropScan Inc., Rochester, MN), which
records percent light reflectance in 11 wavelength bands (460, 507, 559, 613, 661, 706, 850,
900 and 950 nm), approximately at 50 nm intervals is used for measuring nutrient status, weed
infestation and foliar disease intensity in corn. The data are processed through a minicomputer
connected to the sensor. The sensor readings are used to derive different vegetation indices.
The normalized difference vegetation index (NDVI) is one of the common indices (Ma et al.,
2005), and is derived as follows:

( R813 − R613)
NDVI= (13)
( R813 + R613)
30 K. D. Subedi and B. L. Ma

Ma et al. (2005 and 2007) evaluated NDVI data in comparison with soil tests (PSNT),
tissue test and chlorophyll meter readings all taken simultaneously at the V6 to V8 growth
stages of corn. They observed that PSNT, tissue N concentration at V6, SPAD and NDVI all
differentiated corn N response similarly (Figure 1), and these measurements were highly
correlated with one another. However, they concluded that none of the indicators tested at the
V6 to V8 growth stage was able to predict corn yield at harvest sufficiently, indicating that
environmental factors after N sidedress may have played dominant roles in their studies.
Recently, another ground-based commercial canopy device, GreenSeeker (NTech
Industries Inc., Ukiah, CA) optical sensor, is developed to measure canopy reflectance using
an active light source. The unit emits red (656 nm) and NIR (774 nm) light and measures how
much is reflected back to the sensor from the canopy. An NDVI is calculated with the same
assumptions as the Crop Scan. This technology has been extensively tested in recent years on
corn as a tool for variable rate application of N fertilizers. Teal et al. (2006) reported a poor
exponential relationship between NDVI of early season measurement (V6-V7) and grain yield.
But, they found a strong relationship (R² = 0.77) by V8 growth stage. In their study, the sensor
failed to distinguish variations in green biomass among fertilizer levels at the later stages (V8-
V9), likely due to canopy closure. They concluded that yield potentials in corn could be
accurately predicted in season with NDVI measured with GreenSeeker.
Such crop-based indicators are quicker (on-the-go) and require less labour, and can be
used as alternatives in predicting N requirement for corn production. For the N reflectance
index to be a practical, usable technique, it must represent plant N status as early as the V6
growth stage (Busch and Duke, 1996). It appears that, however, the N-stress sensing is more
accurate and successful later in the growing season (Ma et al., 2005, 2007; Ruiz Doaz et al.,
2008). Another drawback of canopy reflectance sensors is that soil background interferes with
the NDVI data before the corn canopy closure. The general consensus is that crop sensors are
useful tools in determining the N status of corn plants, it is important to take measurements at
the appropriate stage because NDVI values change with growth stages, the relationship
between chlorophyll concentration and soil N status is not linear – there is a maximum
concentration of chlorophyll that a plant can pack into a leaf, and results in the NDVI
saturation, the technology is not yet plug-and-play, and more work is needed to improve the
algorithms used for on-the-go application to adjust fertilizer rates based on NDVI in corn.
However, variable-rate fertilizer application based on crop sensors “seeing” and responding to
plants requirements will become reality in the near future.


Sustainable nutrients management refers to the use of various available sources of plant
nutrients and agronomic management practices that optimizes the crop yield while maintaining
soil health and environment in a longer run. Although sustainable agriculture is an often
discussed subject, the application of its principles in practices is insufficient. In this section,
best management practices (BMP) in nutrients management for sustainable corn production are
Corn Crop Production: Growth, Fertilization and Yield 31

Figure 1. Leaf chlorophyll content (SPAD), canopy reflectance (NDVI), plant N uptake (kg ha-1) and soil
NO3- - N as affected by preplant N application. All measurements were taken at the V6 stage of grain
corn. The bars followed by different letters indicate significant differences (P < 0.05). Derived from Ma
et al. (2005).

7.1. Nitrogen

Nitrogen is one of the most important plant nutrients as it is required for the production of
proteins and chlorophyll, maintenance of photosynthetic efficiency, leaf area development, and
ultimately DM production (Muchow, 1998). It is also the most important yield limiting
nutrients all over the world. Limitation of N is more severe in tropical and sub-tropical farming
systems where cropping systems are intensive and degradation of soil fertility is alarming. In
Sub-Saharan Africa, for example, low soil fertility especially low N is among the major abiotic
stress limiting corn yield (Worku et al., 2007).
The component of soil N include SOM, residual organic and inorganic N from previous N
application, atmospheric N fixed by legumes and free-living bacteria and atmospheric
deposition (Legg and Meisinger, 1982). The availability of soil mineral N (SMN; i.e. NH4+ and
NO3-) affects the rate of leaf initiation and expansion, final leaf size and foliar senescence rate
(Schröder et al., 2000). In agricultural soils, SMN usually accounts for < 2% of the total N
(Keeney and Nelson, 1982). The SMN concentration does not necessarily reflect the crop’s N
32 K. D. Subedi and B. L. Ma

status but a positive relationship can be found between SMN and the NO3- concentration in
plants (Schröder et al., 2000).
Nitrogen management is one of the most extensively researched topics in agriculture
(Subedi et al., 2006). The use of N fertilizer has been identified as the most energy-consuming
component of corn production (Ma and Dwyer, 1998; Ruiz Diaz et al., 2008). Nitrogen use is
an issue of great concern in corn production because higher N rates are used by corn growers
as “insurance” which may have an adverse effect on the environment (Schröder et al., 2000).
Efficient use of N fertilization is becoming increasingly important in modern corn production
due to raising cost of N fertilizer and growing concerns about NO3- contamination in ground
and surface waters (Stevens et al., 2005), and gaseous N emissions to the atmosphere as a
major source of GHGs and air pollutions. Nitrogen uptake and partitioning, critical timing of N
requirement by corn plant, genotypic differences in N requirement, NUE and considerations
for N fertilizer recommendation will be discussed in this section.

7.1.1. Nitrogen Uptake and Partitioning in Corn

Soon after corn seedling emergence, the roots start to take up N from the soil solution. As
plant growth progresses, the rate of N uptake increases linearly (Ma and Dwyer, 1998). How
long a corn plant keeps on taking N from the soil is not very clear. In general, the rate of N
uptake by corn is relatively slow before entering the period of rapid growth at about the V6
growth stage, and great N accumulation occurs during the mid to late vegetative growth stages
(Ritchie et al., 1993). By silking, up to 70% or more (depending on hybrids, yield potentials
and weather conditions after silking) of the total plant N has been taken. After silking, rate of
N uptake becomes slow and eventually ceases prior to physiological maturity. Ziadi et al.
(2008) defined the minimum N concentration required to achieve the maximum growth as the
critical N concentration (NCrit). During the first three to six weeks after emergence, corn plants
take up soil mineral N at a rate of < 0.5 kg ha-1 d-1; during which period, soil net mineralization
rate can vary from 0.25 to 1.5 kg N ha-1 d-1 (Ziadi et al., 2008; Wu et al., 2008). Subedi and Ma
(2005a) observed that restriction of N supply until V8, and from V8 to physiological maturity
caused irreversible effects on grain yield and N-uptake in three contrasting corn hybrids. They
concluded that the timing of N application and level of N-deficiency in plant significantly
influenced N uptake, remobilization and N-dynamics in corn.
Partitioning of total N taken up by a corn plant at maturity may be dependent on the
hybrids, growing duration, level of stresses suffered by the crop and adequacy of soil N supply
during the growing season. Subedi and Ma (2005b) observed that the more severe the plants
were deficient in N, the greater was the recovery of applied 15N fertilizer. Under an adequate N
supplied situation, according to Subedi and Ma (2007), about 47% of the applied 15N was
recovered at harvest, of which 74% was partitioned in the kernels, followed by 14% in the
leaves, 10% in the stalk and only 3% in the roots (Figure 2). During grain filling period, there
are two sources of N for kernel development: (i) absorbed N from the soil, and (ii) re-
mobilized N from vegetative tissues (Ta and Weiland, 1992). With later application, 15N-
labeled N fertilizer was predominately partitioned to the ear, and stalk played an important role
in providing N to the grain during grain filling period. Subedi and Ma (2005b) observed that
the demand for N by grains from leaves and stalk was small when plants received adequate N
supply from current uptake. If the absorbed N from the soil is not adequate, great proportions
of N stored in the leaves and stalks are remobilized for kernel development. On the contrary,
when there is adequate N supply, corn plants continue to take up N from the soil until later
Corn Crop Production: Growth, Fertilization and Yield 33

grain filling period. For example, Subedi and Ma (2005a) reported that corn plant continued to
take up N beyond 3 wk after silking, and the later N was applied, the higher proportion of it
was partitioned to the grain.
The sources of N for uptake are (i) residual soil mineral N, (ii) current-season mineralized
N, and (iii) N applied through fertilizers. Fertilizer N accounted for an increasing proportion of
crop N uptake as the N rate was increased, but N uptake from the soil source was always more
extensive, accounting for 54-83% of total plant N uptake (Ma et al., 1999a; Stevensen et al.,

Figure 2. Distribution (%) of dry matter, N content, 15N content, and N use efficiency (NUE) among
roots, stalks, leaves and kernels of a corn plant labelled with 5%15N2-NH4NO3, averaged over two
growing seasons. Adapted from Subedi and Ma (2007).

7.1.2. Critical Timing of Nitrogen Requirement by Corn

The critical time here refers to the stage of corn plant at which lack of N supply can cause
adverse effect on its growth and yield. As a general rule, any stage of corn development should
not experience an N-stress, although the demand of N varies considerably over the growing
period. The generalized trend of N uptake and response of crop yield to N supply is presented
in Figure 3 derived from Brown (1970). This figure shows that at very low soil N levels, there
is a clear evidence of N deficiency, and grain yield of corn increases rapidly with N fertilizer.
At very high soil N levels, grain yield declines, while plant N concentration continues to
increase. There is a window when plant tissue N concentration is low, and the crop suffers
from “hidden hunger”. It is important to know when N nutrition increases to a certain level,
although plant tissue N concentration continues to increase, grain yield does not respond to
increased soil N supply or yield stags. Further increases of soil N supply, grain yield may
suffer due to imbalanced source-sink ratios (Rajcan and Tollenaar, 1999a, 1999b).
Nitrogen supplied before anthesis has two main effects on yield as (i) plant size, and (ii)
grain number. High N from the onset of floral initiation directly increased the number of grains
34 K. D. Subedi and B. L. Ma

per plant or per unit area, presumably by increasing the rate of differentiation of spikelets
(Pearson and Jacob, 1987). Knowledge of both soil factors and crop N requirement is a pre-
requisite to the development of management strategies to maximize the yield response to
fertiliser N (Muchow, 1998). The critical timing of N requirement is important to know
because N amendment decisions can be made so that irreversible yield loss can be avoided.
There are, however, inconsistent reports about the critical timing of N requirement and N
uptake by a corn plant. In the US Corn Belt, it was observed (Scharf et al., 2002) that there was
no significant yield loss when N application was delayed until V11 to V16 growth stages;
although full yield was not achieved when N applications were postponed until silking, corn
was still responsive to N supply at the silking stage. In a controlled greenhouse study, Subedi
and Ma (2005a) reported that withholding N supply fromV8 to maturity reduced kernel yield
by 22% and N uptake by 53%. In the same experiment, when N supply was restricted until V8
stage, there was an irreversible effect on the size of ear and kernel number although overall
size of the plant (leaf number and shoot DM) was unaffected. They concluded that N supply
was more critical prior to silking than after silking as limiting N supply reduced ear size,
kernel yield and N uptake. Rendig and Crawford Jr. (1985) reported that post-anthesis N
nutrition affected the composition of the vegetative growth, but had no effect on yields or N
accumulation in the grain. Under low N conditions, however, Worku et al. (2007) observed
that post flowering N-uptake and utilization contributed to the improved performance in a set
of tropical corn varieties whereas N uptake before anthesis was of little relevance.

Figure 3. Relationship between nutrient supply, corn yield and nutrient concentrations in earleaf tissue.
Adapted from Brown (1970).

The uptake of N can be described by a linear relationship between the specific ion uptake
velocity from the soil and the rate of respiration (Novak and Vidovic, 2003). Therefore, for an
efficient uptake of N, the transpiration rate should be unaffected. For this, water supply plays
an important role. Maho et al. (2007) suggested that the amount of retained soil NO3- - N was
positively correlated with transpiration by corn (r = 0.943, P < 0.01, n = 12). Therefore, NO3-
leaching from a granitic regosol during the rainy season could be reduced by the increasing of
Corn Crop Production: Growth, Fertilization and Yield 35

planting density due to the increase of N uptake by the plants and the increase of retained N in
soil derived from the increasing of plant transpiration.

7.1.3. Genotypic Differences in Nitrogen Uptake and Requirements

Nutrient requirements, uptake and utilization by different corn types (e.g. grain corn,
silage corn, sweet corn, leafy corn, conventional, and transgenic corn) may vary considerably.
Genotypic variation on N uptake and partitioning has been widely reported in conventional
corn hybrids (Beauchamp et al., 1976; Chevalier and Scharder, 1977; Moll et al., 1982;
Weiland and Ta, 1992; McCullough et al., 1994; Ma and Dwyer, 1998; Bertin and Gallais,
2000). For example, previous field studies show that the SG types taken up greater amounts of
N than the conventional hybrids (Ma and Dwyer, 1998; Rajcan and Tollenaar, 1999b; Borrell
et al., 2001). Costa et al. (2002) found no difference in different types of leafy, non-leafy,
reduced or not reduced stature corn hybrids. Similarly, Subedi and Ma (2005a, 2005b), in
greenhouse studies, reported there was no difference in total N acquisition, partitioning of 15N
and NUE among three contrasting (i.e. Leafy, stay-green and conventional) corn hybrids
(Table 13; adapted from Subedi and Ma, 2005a). Although Bruns and Abel (2003) reported an
increased N concentration and σ-endotoxin with increased supply of N in the whole plant of a
Bt corn hybrid at the V5 growth stage, Subedi and Ma (2007) found no such difference in Bt
and non-Bt conventional hybrids when compared the N uptake and partitioning patterns until
crop maturity.

Table 13. Total dry matter (DM, g plant-1), N concentration (NC, %) and N content (g
plant-1) in different plant parts or in the whole plant of conventional (Pioneer 3905), stay
green (Pioneer 39F06 Bt) and Leafy (Maizex LF 850 RR) corn hybrids, averaged over
five N treatments

Plant Parts Parameters Hybrid

Pioneer 3905 Pioneer 39F06 Bt Maizex LF850 RR
Root DM (g) 33.5b† 37.9b 54.3a
NC % 0.68ab 0.71a 0.59b
N content (g) 0.23b 0.25ab 0.29a

Stalk DM (g) 55.7b 60.0b 70.3a

NC (%) 0.47a 0.51a 0.44a
N content (g) 0.26a 0.30a 0.30a

Leaves DM (g) 35.3b 36.0b 45.9a

NC (%) 1.10a 0.98ab 0.85b
N content (g) 0.39a 0.35a 0.39a

Kernel DM (g) 100.4a 101.9a 102.9a

NC (%) 1.76a 1.59b 1.74ab
N content (g) 1.76a 1.61a 1.79a

Entire Plant DM (g) 224.9b 235.9b 273.2a

NC (%) 1.17a 1.06a 1.01a
N content (g) 2.64a 2.51a 2.77a
† Values followed by the same letter within each row are not statistically significant at P ≤ 0.05.
(After Subedi and Ma, 2005a).
36 K. D. Subedi and B. L. Ma

Subedi et al. (2006) observed that the Leafy corn was more sensitive to high PPD, especially
under low N supply conditions than a conventional corn hybrid, although the hybrids did not
differ in N acquisition and partitioning. In silage corn, Sheaffer et al. (2006) reported that
brown midrib and Leafy hybrids did not differ in N response.

7.1.4. Nitrogen Use Efficiency (NUE) in Corn Production

There are several definitations of NUE in the scientific literature. In general, soil scientists
and agronomists define NUE as the N uptake by a crop expressed as a fraction (or percent) of
total N fertilizer applied. Crop physiologists refer NUE as the dry matter produced per unit of
N taken up (i.e. g DM g-1 N) or the ratio of net photosynthetic rate to leaf N content. In order
to evaluate hybrid differences in plant N uptake and N utilization efficiencies, crop
physiologists also use the following formulae to calculate NUE and its components, N uptake
efficiency (NUptE) and N utilization efficiency (NUtiE) according to Moll et al. (1982) on a
kg ha-1 basis (Ma et al., 2003).

NUE = GDM / SN (14)

NuptE = PTN / SN (15)

UtiE = GDM / PTN (16)

where GDM refers to total grain dry matter (kg ha-1), PTN is plant total N at final harvest (kg ha-
) excluding roots, and SN is soil available N at planting (kg ha-1).
In this review, we use the terminology accepted by both soil and crop scientists: the plant
total N uptake as a percentage of applied N fertilizer. Using this method, the worldwide
estimated NUE of cereals including corn is approximately 33% (Raun and Johnson, 1999).
Clearly, improving NUE for cereal crops including corn production becomes more and more
important, both for economic benefit to producers and environment to the general public. The
use of best agronomic practices that help ensure the development of vigorous healthy crop will
increase the efficiency of applied N fertilizers. Of course, there are hybrids and varieties that
are more efficient in N utilization than others. Nitrogen use efficiency is measured using
various methods including the difference method and 15N-labeling techniques. The following
equation (Liang and Mackenzie, 1994) is commonly used to calculate NUE:

∑ [W × N (
i =1
i i
N i1 −15 N i 0 × 100
NUE (%) = (17)
f ( a − b)

where Wi and Ni are the ith component of plant dry weight (g plant-1 or kg ha-1) and total N
concentration (fraction), respectively, 15Ni1 and 15Ni0 are the 15N% a.e. in the ith component of
the 15N-labelled and non-labelled plants, f is the total amount of N applied (g pot-1 or kg ha-1)
through the labelled source, and a and b are the 15N% a.e. in the fertilizer and background,
The NUE is an important criterion to assess crop management systems. Nitrogen use
efficiency varies from one situation to another due to variability in several factors such as crop
Corn Crop Production: Growth, Fertilization and Yield 37

health (plant stress), weather factors influencing soil temperature and moisture availability
(Westerman et al., 1999), PPD (Al-Kaisi and Yin, 2003), soil types (texture and SOM), years
and locations (Lory and Scharf, 2003), application timing, method of application/incorporation
of the fertilizer in the soil, and the nutrient responses of the cultivated variety (Nagy, 1997;
Nielsen, 2006). Improving NUE is therefore, an art of addressing the factors appropriately in
corn production. Similarly, methods of application, crop rotation and tillage practices also
affect NUE.
Selecting appropriate source of fertilizer or method and timing of application certainly
influences NUE. In order to increase fertilizer NUE and reduce N loading to the environment,
reliable methods that quantify crop N requirements must be developed and N fertilizers should
be applied precisely and timely. Matching supply of N from soil with the crop demand for the
nutrients is one of the nutrients management challenges of crop production (Heckman, 2002).
Precision agricultural practices attempt to allow timely and precise application of N fertilizer
to meet plant needs as they vary across the landscape (Raun and Johnson, 1999). For example,
sidedress can reduce NH3 volatilization, denitrification and NO3- leaching losses and increase
the availability of mineral N to the crop. Studies have shown that sidedress N applied during
early growth stages (i.e. close to the time of the crop’s greatest need) are used more efficiently
than preplant application (Magdoff et al., 1984; Magdoff, 1991; Ma et al., 2005). There is less
time for leaching or denitrification losses when N is applied after plant emergence (Vetsch and
Randall, 2004). Sainz Rozas et al. (2004) stated that higher NUE with economically
competitive grain yields can be obtained when N is applied at the V6 stage because gaseous N
losses are low and NO3--N leaching would be reduced. Split application of N fertilizers is
generally found to be beneficial than a single application. Corn plants can be responsive to
applied N until silking stage and later (Subedi and Ma., 2005a). The use of a sidedress
application strategy remains one of the easiest and least expensive ways to maximize NUE.
Other application methods and timings need to be matched wisely with N fertilizer source to
minimize the risk of N loss prior to plant uptake.
Applying N fertilizers without information about N-supplying capacity of the soil can
contribute to NO3- leaching and polluting ground and surface waters or not supplying enough
for economic yield (Heckman et al., 1995). Soil N supply is expected to vary among year and
location. Residual soil nitrogen (RSN) is the amount of inorganic N that remains in the soil at
the end of growing season after crops have been harvested. Adjustment of N rates to the
amounts of RSN present shortly before planting can contribute to efficient N-use (Schroder et
al., 2000). Soil RSN is estimated as the difference between all N-inputs (fertilizer N, manure-
N, biological fixed-N and atmospheric N deposition) and all N-output (N removed in crop
harvest, N losses through NH3 volatilization and denitrification), assuming that mineralization
and immobilization are generally balanced (Durby et al., 2005). A history of excessive N
application may decrease response of subsequent crops to fertilizer N due to greater release
from non-available N forms, most likely as a result of increased mineralization of crop
residues and recently formed SOM (Stevens et al., 2005).
Cropping systems, tillage practices and water availability all affect the timing and amount
of N fertilization for greater NUE. In the irrigated corn production systems, for example,
irrigation inputs needs to be optimized to prevent nutrients leaching from root zone, especially
on sandy soils (Raun and Johnson, 1999). Similarly, N application timing and rates should be
different for no-till or conservation tillage system than for conventional tillage system.
38 K. D. Subedi and B. L. Ma

Sources of N also affect NUE. For example, NH4+-containing fertilizers are less subject to
leaching or denitrification loss than fertilizers containing NO3--N. Ma et al. (1999a) observed a
greater NUE when manure was applied at the lower rate presumably because the slow release
of N from manure and increased uptake of N during exponential phase of plant growth. There
was a difference in timing of availability of manure N from fertilizer and manure treatment.
Mechanisms, such as leaching, volatilization, and denitrification significantly affect N
losses (Webster et al., 1992). Therefore, heavy N fertilization should be avoided and the most
effective N-fertilizer recommendation should be determined. Placing N where it can easily be
absorbed by the plant and using site-specific or variable rate of application techniques
improves NUE. Similarly, selecting an appropriate N fertilizer source is important for corn.
Selection of fertilizer depends on availability in the market, relative cost, soil pH, and
application equipments, etc. For example, Bacon and Thompson (1984) reported that urea was
superior to aqua ammonia because it minimized mineral N retention near the soil surface and it
was not susceptible to volatilization losses. Techniques that provide rapid assessment of soil
and plant N status on a frequent basis will be useful for the in-season N amendment (Bausch
and Duke, 1996).
Appropriate fertilizer N rate for corn crops is important in order to meet the crop critical N
demands during the rapid growing period, to minimize the wasteful application and to increase
NUE and economic benefit to the producers. Recommendation of N fertilization should
primarily focus on application (i) rates, (ii) timing, (iii) method, and (iv) choice of manure and
fertilizers. The economically optimum N rate (EONR) required for corn may vary spatially due
to variations in soil characteristics and temporally due to the interactions of environmental
factors (Schmitt and Randall, 1994; Miao et al., 2006; Mamo et al., 2003; Katsvario et al.,
2003; Scharf et al., 2005). Generally, the total amount of N utilized by a corn crop will
increase with yield level. However, recommendations of fertilizer rates based on yield goals
are often poorly correlated with actual EONR (Doerge, 2002). The variation in corn N
response has been attributed to differences in soil N supply, corn N needs for a given yield
level, and hybrid’s NUE potential. For example, EORN should be reduced in soils containing
high organic N concentration at the start of growing season. Therefore, corn N
recommendation typically includes a system of N credits from conditions that increase the
quantity of soil N available to the crop (Lory and Scharf, 2003). As a general rule, synchrony
of nutrients supply with crop demand is essential in order to ensure optimum crop yield, high
NUE, while reducing negative environmental effects (Ma et al., 2005).

7.2. Phosphorus

Although P is not present as large quantities as N in the plant tissues, it is involved in

many crucial metabolic functions that occur in plant cell (Johnston and Dowbenko, 2004). Soil
P is generally less mobile and is often not in shortage or excess for crop growth in most soils
of fine texture or with manure history. From production point of view, management of P is,
therefore, not as urgent and critical as N. However, there is a growing response of grain yield
of corn to P fertilizer, especially at high yield potentials. On the other hand, P leaching to the
water bodies has become one of the serious environmental problems.
Phosphorus deficiency usually appears when corn plants are young, when solution P
concentrations are either inadequate to meet the high P requirement of the faster growing
Corn Crop Production: Growth, Fertilization and Yield 39

shoots, or the juvenile corn plants have difficulty absorbing sufficient amount of P, especially
in the cold spring conditions (Johnston and Dowbenko, 2004; Bittman et al., 2004).
Soil test P level is an important factor for understanding corn grain yield responses to
various P placements and tillage practices (Randall et al., 2001). Soil test before planting is the
best way to detect P requirement for corn, based on which recommendation of fertilizer P can
be made. The total soil P is usually much higher than the available P index because it includes
both the mineral and organic P pools. Soil P availability is usually evaluated with soil test
Bray-P concentrations, the amount of P that is likely available for uptake by the plant.
Phosphorus fixes easily with many compounds in the soil, and in a fixed form, it is less easily
available for the plants. When soil has a low pH, free Fe and Al ions bind with P, thereby
making P less available for plant roots. In the calcarious soils (high pH), P availability also
reduces as it binds with Ca to form an insoluble compound. Available soil P was correlated
positively with grain P content, and there was a considerable variability in grain P content for
any given soil test level (Lithourgidis et al., 2007).
The sources of P fertilizer can be organic (manures and supplements) and inorganic such
as rock phosphate and P containing chemical fertilizers. The inorganic sources of P fertilizer
are applied either broadcast before seeding or as a starter banded with corn planting. The use
of a starter fertilizer at planting is certainly one option for conditions where available P is low
in the soil or temperature is low during the early growth stage of corn. There is a growing
interest of "pop-up" fertilization (placement of small amounts of fertilizer in direct contact
with corn seed) in recent years. There are certain advantages of each of broadcast and banding
application, and suitability of each method varies with soil type, climate, crop rotation, tillage
systems and equipment availability and so on. The banded P can be placed either directly
below the seed or to the side of and below the seed. The most common method of supplying P
fertilizer is to apply a band about 5 cm depth to the side of the seed furrow (Bittman et al.,
2004). Banded application has been found to be more effective in the ridge-till and no-till
systems. The negative effect of fertilizer placed too close to the seed on germination is a
concern. However, the early research of Garg and Welch (1967) showed that percent seed
emergence did not differ among the placement methods. Yields of forage, percent P, and yield
of P were greater when P was placed in contact with seeds than when it was either mixed or
banded. Sanchez et al. (1991) compared the broadcast P as surface applied and disked into the
soil before planting with banded P as applied about 3 cm below the corn seeds. Band
placement reduced the amount of P required for specified sweet corn yield and also appeared
to result in higher total yield. The relative efficacy of banded to broadcast P depended on soil
test P level. They concluded that banded P was a reliable strategy of P used for sweet corn
production in Histosols. Placement is less of a consideration when soil tests are high. However,
when soil tests are low, substantial yield increases may be seen when P is applied either
broadcast or banded (Randall et al., 2001).
The relative efficacy of broadcast P was dependent on soil test-P. Yost et al. (1979) found
that broadcast treatments gave greater yields than banded treatments at the same rates for the
first crop, at the end of four crops. However, total yields of P uptake were very similar for
broadcast and banded treatment in which the same total amount of P had been applied. Borges
and Mallarino (2001) reported that there was a similar response of corn yield to P application
as broadcast or deep band (15-20 cm). Heckman et al. (2006) conducted a study in 12 northern
States of USA at 51 experimental sites, of which 17-47% of the sites testing below the critical
40 K. D. Subedi and B. L. Ma

level of soil P exhibited a yield increase to broadcast P. Marked residual effects were observed
with the higher rates of broadcast P.
In conservation tillage systems, surface application of P fertilizers and annual return of
corn residue to the soil surface can result in the P and K stratification (Mackay et al., 1987).
Phosphorus stratification occurs under long-term no-till conditions. This means that a build-up
of P occurs on or near the soil surface over time when the soil is managed in a no-till system.
This stratification is caused by normal crop demands on the nutrients deeper in the soil profile,
and the absence of incorporation of the surface applied P. However, unless an excessive
amount of P is applied, the build-up of P is always low. Vertical stratification due to deep-band
fertilization of P was evident for all treatments, but was more with band application, and
especially so on no-till cultivation (Mallarino and Borges, 2006).

7.3. Potassium

Potassium is required for photosynthesis, carbohydrate translocation, protein synthesis,

and for disease resistance and drought tolerance in plants. Optimum K fertilization is also
believed to increase N concentration in the grain as well as enhanced N-use by the crop. Not
all of the measured K is available to plants because the positive (+ve) cations are attracted by
the negatively charged ions in the soil humus and clay particles; also known as the cation
exchange capacity (CEC) of the soil. Only a small fraction of total K (<2%) is available in soil
solution as “soluble K”, which is the form available for plant uptake. Exchangeable soil K is
highly correlated with tissue test and deficiency symptoms (Driskell and Richer, 1952). In a
region or a corn field, the amount of K needed is site-specific and its application is therefore
dependent on three principal factors (i) soil test K, (ii) yield goal, and (iii) soil CEC. Potassium
uptake by plants can be affected by high salinity and N concentrations in the soil solution.
Heckman et al. (2003) reported that concentrations of K in the grains of corn were
positively associated with yield levels. Although K-deficiency is not always visually evident
and can be masked by other crop stress symptoms (Johnston and Dowbenko, 2004), K must be
available to corn plant from early growth stage for optimum corn development since over 70%
of the total K requirement is taken up by silking (Johnston and Dowbenko, 2004). Application
of K fertilizer is based on soil test recommendations. When soil test indicated shortage of
available K, up to 13% reduction in corn yield occurred in a humid temperate environment of
Canada (Subedi and Ma, 2009).
Tissue test and soil test (exchangeable K) are used to determine the requirement of K by a
corn crop. In northern climates, due to cold soils and slow plant growth rates early in the
growing season, responses of plant vigor and grain yield to starter fertilizers are often
anticipated. Heckman and Kamprath (1992) reported that corn yield increased linearly with
application of K up to 112 kg K ha-1. Similarly, Bundy and Andraski (1999) reported that it is
more likely for a positive response of corn yield to starter fertilizers on soils with soil test K
levels below 140 mg kg-1. Starter K fertilizer appears to be particularly important in reduced
tillage, since responses to applied K can occur even at high soil test levels.
Unlike N fertilizer, many farmers in the US Corn Belt apply a single dose of broadcast K
fertilizer before corn planting. In practice, responses of corn yields to preplant K fertilizer vary
considerably over locations, cropping systems and growing seasons. In conventional tillage
systems, the preferred method of K application is through land preparation and K is
Corn Crop Production: Growth, Fertilization and Yield 41

broadcasted and incorporated into the soil. While in no-till systems, K fertilizers can be either
broadcasted on the surface or preferably banded. Bardoli and Mallarino (1998) observed that
no-till corn responded to deep banded K at some sites with high soil K levels. Vyn and
Janocicek (2001) showed that corn yield responses to starter K were larger with no-till than
tillage. Maximized corn yields are often obtained in the no-till system with high rate of starter
K, when no K fertilizer was applied in the previous fall. In furrow cultivation, starter K
fertilization for corn is, however, not an effective practice. Borges and Mallarino (2001)
observed a deep placement (15-20 cm) of K fertilizer superior to broadcast application. They
also observed that broadcast K fertilization leads to stratification of K in the ridge-till system,
which may reduce fertilizer use efficiency. To overcome K stratification, it is suggested that at
least some of the K fertilizer should be applied in a band with or near the seed row.

7.4. Calcium

Although considerable amount of Ca is required in plants as an integral part of plant

structure (i.e. cell wall), deficiency in Ca is not that common in agricultural soils. While most
neutral and alkaline soils contain adequate Ca, deficiency in Ca is sometime observed in acidic
soils. Correction for low soil pH with lime usually brings the soil Ca to the adequate levels for
corn crops. Similarly, maintenance of proper SOM through application of farm manures and
crop residues to the soil helps maintain Ca and other nutrient levels.

7.5. Magnesium

Magnesium is the constituent of chlorophyll molecule, it acts as an enzyme activator and

involves in carbohydrate metabolism. Deficiency in Mg is generally greater than Ca, but Mg is
often not a major yield limiting nutrient in most soils. Driskell and Richer (1952) reported
significant correlations between exchangeable soil Mg and visual deficiency symptoms of Mg
or tissue test in corn. Dolomite limestone contains large amount of Mg. Soils originated from
parent materials containing dolomitic limestone will not require Mg fertilization. Similarly, if
soil amendment is made with dolomitic limestone, there will be no additional requirement of
Mg fertilizers. If Mg deficiency is detected by visual symptoms or tissue or soil test, corrective
measures can be taken by soil or foliar application of Mg containing fertilizers such as
Chelated Mg or Magnesium oxide.

7.6. Sulphur

Sulphur is a secondary element, whose role on plant is vital as it is the component of plant
amino acids (precursor of proteins) and involved in chlorophyll formation. Sulphur plays the
key role in balanced nutrient application for top yields and superior quality produce. Both
organic and inorganic forms of S exist, with organic S as the predominant form in most soils
(Kowalenko, 2004). Organic S is unavailable to plants unless it is mineralized to inorganic
oxidized sulphate (SO4-). Rainfall and animal manure amendment supply soils with significant
amounts of S. Sulphur is attaining importance in all regions of the world because of frequent
42 K. D. Subedi and B. L. Ma

S-deficiencies in time and space (Khan et al., 2006). However, excess soluble S in the soil will
enhance the uptake of toxic Cd element and harm the normal growth of corn (Cui and Wang,
Several factors contribute to S deficiencies, including the increased use of S-free high
analysis fertilizers, intensive cropping, removal of crop residues and soil erosion. S-deficiency
symptoms are more often observed in crops at early growth stages since S can be easily
leached by precipitation and often accumulate in subsoil layers (Hitsuda et al., 2005). Critical
shoot S concentration of corn at early stages is reported at 0.8 g kg-1 (Hitsuda et al., 2005).
Reports of significant yield improvement with S fertilization are available, although responses
varied considerably over locations. Application of S up to 30 kg ha-1 enhanced average grain
yield of corn by 22% over unapplied control treatments (Dwivedi et al., 2002). Niehues et al.
(2004) showed enhanced early season DM production, and increased grain yield and nutrient
uptake when 11 kg S ha-1 was applied as a subsurface starter fertilizer. Khan et al. (2006)
reported that application of 60 kg S ha-1 significantly increased corn yield components and
grain yield, tissue S concentration as well as residual S in the soil after crop harvest in

7.7. Micronutrients

Deficiencies in micronutrients have been frequently observed due to intensive cropping

practices and adoption of high yielding cultivars. Micronutrient deficiencies are particularly
problematic on sandy coastal plain soils due to low CEC, and organic soils due to low mineral
contents. Deficiencies of micronutrients are also widespread in many Asian countries due to
calcareous nature of soils, high soil pH, low SOM, soil salinity, continuous drought, high
bicarbonate content in irrigation water, and imbalanced application of fertilizers (Malakouti,
2008). Micronutrients are also often deficient in volcanic soils (Lisuma et al., 2006).
The availability of most micronutrients is influenced by soil pH. In general, the higher the
soil pH, the worst the problem becomes for many of the micronutrients. For example, alkaline
soils depress the availability of soil Fe, Mn, Zn, and increased the ratios of Na/Zn and P/Zn in
plant tissues (Mehrotra et al., 1986). Excessive application of N and P fertilizers can induce
Among the micronutrients, Zn followed by B is the element, which occurs most often in
deficiencies for corn production worldwide. Zinc deficiency is a very important nutrient
problem in the world’s soil as available Zn in most soils is in deficient level (Adiloglu and
Adiloglu, 2006). It was observed reduction in corn DM yield with B application, and B
accumulation and toxicity in plant roots especially in Zn-deficient soils (Adiloglu and
Adiloglu, 2006). Zinc deficiency is greatly correlated with band or broadcast application of Zn.
Application of small amount of Zn with seed could be used as a procedure for correcting Zn-
deficient corn. Pumbhrey et al. (1963) reported that ZnSO4 broadcast application followed by
incorporation before planting increased early growth and grain yield of corn. Higher
application rate increased Zn concentration in young and nearly mature corn plants. They also
showed that small amount of N banded with ZnSO4 enhanced the effectiveness of Zn fertilizer.
Dwivedi et al. (2002) reported that application of Zn up to 5 kg ha-1 increased corn yield by
19% over the control. The optimum dose of Zn was estimated to be 7.1 kg ha-1. Tariq et al.
(2002) in Pakistan reported that grain yield, yield components and uptake of Zn by corn
Corn Crop Production: Growth, Fertilization and Yield 43

responded to Zn application up to 20 kg Zn ha-1. Hosseini et al. (2007) reported a significant B

× Zn interaction on plant growth and tissue nutrient concentration which were rate dependent.
In general, the effect of B × Zn interaction was antagonistic on nutrient concentration and
synergistic on growth. It is recommended that adequate Zn should be supplied when corn is
grown in high B soils, especially when availability of Zn is low. Serchan et al. (2004) in an
acid soil in Nepal found that corn yield decreased in the absence of B while Zn in the corn leaf
was deficient. Hensler et al. (1970) found that concentrations of Ca, Mg, P, S, Fe and Mo in
the plant tissue were usually higher from the limed soils than not limed soils but reverse was
true with Zn and Mn.
Boron deficiencies are mainly found in sandy soils, calcarious soils, soils with low SOM,
and in the regions with high rainfall or under irrigation, since borate ions are mobile in soils
and leach out quickly. Responses of crop yields to micronutrients have been evident in small
farms of developing countries. Kayode and Agboola (1983) reported a significant yield
increase of corn when N, P, and K fertilizers were applied in combination with Fe, Cu and Zn.
In tropical corn, application of N fertilizer reduced the concentration of Zn and Ca and
increased the concentration of Mn in grains (Feil et al., 2005). Lamond and Leikam (2002)
reported a positive response of applied Cl in corn, and application based on soil tests and plant
analyses has proven useful in identifying potential deficiencies of chloride.



8.1. The Concept of Integrated Plant Nutrients Management

Nutrients depletion through harvested crop components and residue removal or by

leaching, surface runoff and soil erosion accentuates the often very low inherent fertility of
many soils in the tropics (Syers, 1997). Sustainability of cropping systems requires that
nutrients removed from the soil be balanced by nutrients replacement so that soil fertilities are
maintained or improved (Ma et al., 2006a). Chemical fertilizer alone cannot sustain long-term
productivity on many soils and organic materials inputs are required to restore SOM levels and
crop productivity (Syers, 1997). Enhancing sustainable food production will therefore require
integrated strategies for the use of various sources of plant nutrients in conjunction with
improved soil, water and crop management practices (Keerthisinghe et al., 2003). For corn
production, farmers’ nutrient management decisions influence the amount and form of
nutrients used, the timing and method of fertilizer application, which in turn influences on how
much of a nutrient is used by the corn crop, how much is stored as a residual in the soil, and
how much becomes available as a potential water and air pollutant (Christensen, 2002).
Integrated plant nutrient management (IPNM) refers to the approach of integrating
available sources of plant nutrients to meet the crop’s nutrients requirement to achieve an
optimum yield while maintaining desired soil fertility (FAO, 1998). The IPNM attempts to
integrate all available means of soil and crop management so as to achieve locally optimum
land productivity under sustainable soil and fertility management (Subedi and Sapkota, 2002).
All sources of plant nutrients should be considered to optimize crop yield and quality, while
minimizing the impact of these nutrient sources on environment. Therefore, the main aim of
44 K. D. Subedi and B. L. Ma

IPNM is to increase and sustain soil fertility to provide a sound basis for flexible food
production systems that within the constraints of soil and climate can grow a wide range of
crops to meet changing needs (FAO, 1998). The role of IPNM seems to be very important in
the subsistence farming systems because there is always a limited nutrients supply, and soil
degradation is a major production and environmental threat. The following key steps are to be
considered while designing and implementing location-specific nutrient management for a
given domain or cropping system:

(i) Assessment of soil status: Information on soil parameters such as SOM, pH, nutrients
availability, texture, extent of nutrient leaching and erosion are to be gathered through
site visit and laboratory analysis.
(ii) Setting of yield target: based on the availability of farm resources, expected yield
levels for the crop under a particular production system are set.
(iii) Calculation of nutrient balance: based on the two estimates above, a nutrient balance
(i.e. input-output) can be calculated which indicates how much and which nutrients
are to be added. Emphasis is to be given for the balance of SOM.
(iv) Listing available nutrient sources: all available internal (farm level) and external
(purchased) nutrients sources are to be considered.
(v) Integrating all possible nutrient sources, and
(vi) Determine the amount, timing and methods of manure and fertilizers application for a
given crop, cropping system and land type.
(vii) Follow-up and monitoring: once manure and fertilizers are applied based on the
above assessments, periodic monitoring of corn fields is important in order to address
the in-season deficiencies if any induced by climatic variability such as excess rainfall
or drought and trouble shooting for such deficiencies.

In the IPNM system, soil, crop and nutrients are judiciously managed based on the
existing decision environments such as cropping systems, soil properties, labour availability,
market forces and social equity (Subedi and Sapkota, 2002). Several factors influence crop
yield and nutrients requirement by a corn crop. Therefore, soil properties (e.g. texture, SOM,
residual nutrients), climatic factors (e.g. precipitation, temperatures), cropping systems (e.g.
previous crops, tillage systems), crop variety grown (yield potential), and economic and
market considerations (e.g. prices of grain and fertilizers) should be taken into account when
making recommendations for manure and fertilizer application.
While deciding the amounts of chemical fertilizers, organic, inorganic and other sources of
plant nutrients are integrated along with soil and crop management options. It is a strategy that
incorporates both organic and inorganic plant nutrients to attain higher crop productivity,
prevent soil degradation, and thereby help meet future food supply needs. Therefore, IPNM
relies on a number of factors, including appropriate nutrient application and conservation and
the transfer of knowledge (Gruhn et al., 2000). This approach emphasizes applying nutrients as
and when needed. Similar to any other crop or cropping system, the key components of IPNM
for corn production are as follows:
Corn Crop Production: Growth, Fertilization and Yield 45

8.2. Legumes in Rotations

Integration of various food, forage or vegetable legumes either in the crop rotations or as
intercropping with corn crop is a common practice everywhere, although the frequency of
rotations varies across locations and cropping systems. The advantages of legume
incorporation in a cropping system is well understood even in the subsistence farming systems
in terms of improved soil fertility (biologically fixed N), diversity in food values, nutrition and
farm income. Most of the legume species have a symbiotic relationship with certain bacteria
and fix N from the atmosphere. Corn crop can also benefit from the residual N from legumes
as well as the in-season N fixed by the companion legumes when grown in the intercropping
system. Numerous rotation studies have shown the benefit of extended rotations with legumes,
including interrupted insect populations (Cook, 1988), improved soil physical properties
(Raimbault and Vyn, 1991), a better balance of plant nutritional factors, increased root activity
(Copeland et al., 1993), a shift in soil mycorrhizal populations (Johnson et al., 1992; Jawson et
al., 1993), reduced disease severity (Reid et al., 2001), and enhanced seasonal N mineralization
(Vanotti and Bundy, 1995; Ding et al., 1998), reduced N inputs and increased corn yield (Ma
et al., 2003b). For example, corn in annual rotation with legume crops could increase corn
yields by as much as 20% and reduce the amount of chemical fertilizer N by as much as 180
kg N ha-1 (Ma et al., 2003b). The average increase of corn yield following soybean ranges
from 10 to 15% in the Midwestern USA (Griffith et al., 1988). Wolfe and Eckert (1999)
observed that in a no-till system, corn following soybean produced greater yield than corn
following corn system, indicating greater contribution of previous legume crop in the rotation
than the amount of crop residue on soil. Lawrence et al. (2008) found that following legume
forage turn over, only a small quantity of starter N was sufficient for optimum of silage corn
yield. In the subsistence farming systems, inclusion of legumes will play an important role in
diversifying crops and ensuring improved household food security.

8.3. Green Manures and Cover Crops

Green manures are the plants grown for the purpose of incorporation into the soil while
they are green. Green manuring involves the soil incorporation of any field or forage crop
while green or soon after flowering, for the purpose of soil improvement (Sullivan, 2003).
Literarily any plant species can be used as a green manure crop, however; species vary
considerably in their adaptation, green manure values and nutrients composition. Generally
leguminous species, plants with succulent tissues, fast growing and plant parts not suitable for
other uses (e.g. forage) are the desirable traits for green manure crops. Green manure crops can
be used as in-situ green manure (i.e. grown in the same field and incorporated in-situ) or green
leaf manure (i.e. lopping from plants or trees from the same field or from outside and then
incorporating them into the soil). Several species of plants, preferably legumes such as
Sesbania, sunhemp (Crotalaria juncea), Pillipesara sp., rice bean (Vigna umbellate L.), velvet
bean (Mucana deeringina), jackbean (Canavalia ensiformis), lablab bean (Dolichos lablab),
red clover (Trifolium pratense), cowpea (Vigna unguiculata) and other leguminous species are
considered good for green manuring, although certain non-legume species as Adhatoda vasica,
Artemisia vulgaris have also superior green manuring properties (Subedi, 1997). All green
manures have a positive effect on soil biological properties, plant nutrition and crop yield
46 K. D. Subedi and B. L. Ma

parameters (Tejada et al., 2008). In addition, the other benefits of green manures are reported
to be weed suppression, moisture conservation, and protection of soil from erosion (Fischler
and Wortmann, 1999). It is estimated that the portion of the N available to a following crop is
usually about 40 to 60% the total amount contained in the legume (Sullivan, 2003).
Cherr et al. (2006) used sun hemp green manures as supplement N sources for sweet corn
in a reduced tillage system. Sun hemp residue and living winter legumes together contained
120 to 125 kg N ha-1. Shehu et al. (1997) reported that a corn crop grown with the green
manure of pigeon pea (Cajanus cajan) produced yield equivalent to application of chemical
fertilizers at 120 kg N ha-1, 30 kg P ha-1 and 30 kg K ha-1. Similarly, Fischler and Wortmann
(1999) observed that grain yield of corn following a one-season fallow with velvet bean and
lablab bean that were 60 and 50% higher, respectively than corn following corn. These studies
demonstrated that green manures have the potential of substituting most of the N requirements
of succeeding corn crop.
Cover crops refer the growing of any plant species for the purpose of covering soil surface
to protect the soil from erosion and to trap nutrients losses without affecting the main crop.
Similar to green manures, cover crops can be annual, biennial, or perennial herbaceous plants
grown in a pure or mixed stand during all or part of the year (Sullivan, 2003). Cover crop can
be grown during the off-season (i.e. when there is no crop in the field) or underneath the crop.
When cover crops are planted to reduce nutrient leaching following a main crop, they are often
termed as "catch crops" (Sullivan, 2003). Cover crops can be legumes such as hairy vetch
(Vicia villosa Roth.), alfalfa, crimson clover (Trifolium incaratum L.); peas (Pisum sativum L.)
and others, and non-legume plant species such as wheat, oat, rye (Secale cereale) and barley.
Cover crops are incorporated into the soil as green or killed with herbicides.
The key benefits of cover crops are (i) soil conservation by reduced runoff, (ii) trapping of
soil NO3--N that is prone to leaching, (iii) weeds suppression, (iv) soil moisture conservation,
(v) contribution to nutrients pool thereby reduces the amount of fertilizer N requirement, and
(vi) addition of valuable SOM (Decker et al., 1994; Vaughan and Evanylo, 1998; Griffin et al.,
2000; Kuo and Jellum, 2002; Bittman et al., 2004; Snapp et al., 2005; Andraski and Bundy,
2005). For example, cover crop amendments increased soil aggregate stability and the
percentage of water stable 2-6 mm aggregates (Liu et al., 2005). Oyer and Touchton (1990)
reported that reseeding of crimson clover in combination with soybean-corn rotation,
constantly produced the highest yields of the system studied and provided an equivalent of 68
to 159 kg N ha-1 to corn. Griffin et al. (2000) reported that legume cover crops of alfalfa and
hairy vetch were able to replace all or nearly all of the N fertilizer required by a subsequent
sweet corn crop with a fertilizer replacement value of 58 to 156 kg N ha-1. Nevertheless, a
cover crop of cereal rye recovered more fertilizer N (48 kg ha-1) applied in the preceding corn
crop than by legume crops such as vetch and crimson clover (8 kg ha-1). Roy and Ball-Coelho
(2004) also reported that in southern Ontario, winter cereal such as rye appeared to be the best
cover crop for capturing residual N after corn harvest. Rye establishes and grows vigorously in
the fall taking up significant amounts of soil N. Relay crop of cover crops (i.e. planting or
establishing cover crops before corn harvest) can also be practised so as to capture maximum
amount of residual soil N and provide ground cover against erosion and runoff (Bittman et al.,
2004). Relay planting of different crops such as soybean, cowpeas, velvet beans and finger
millet (Eleusine coracana) under corn crop are traditional practices in the subsistence farmings
of tropical and sub-tropical regions, which also help conserve soil and trap N loss.
Corn Crop Production: Growth, Fertilization and Yield 47

8.4. Organic Manures

Organic manures are the natural renewable sources of soil organic matter, containing all
the essential plant nutrient elements. Literarily, when any living things die and decay, the
resultant product is organic manure. Until the artificially synthesized chemical fertilizers were
developed, manures and organic supplements were the major sources of plant nutrients and
SOM. There are different types of organic manures. Farm yard manure (FYM), both solid and
liquid, represents the major source of organic manure, although poultry manure, hog manure
and other animal faeces, compost prepared from crop residues and other farm wastes,
vermicompost, oil cakes, and biological wastes such as - animal bones, slaughter house refuse,
etc. all represent organic manure. Most farmers in the developing countries with low input for
corn production are dependent upon organic sources of plant nutrients. Even in the developed
countries, organic manures constitute significant sources of nutrients for corn crop. For
example, in the poultry and dairy production states of USA, almost 40% of the acreage use
manures as nutrient sources for corn production (Christensen, 2002).
Decomposition and mineralization are the means by which plant nutriments held in SOM
are released into the soil as inorganic forms (Jarvis et al., 1996). Mineralization is the process
of transformation of OM into NH4+ - and NO3- - forms of inorganic N and several other
elements by soil microorganisms. Manure from livestock is an important source of N for crop
production in many areas, but efficient management of manure is critical to improve the
economics of manure use and to minimize the impact on water quality (Jokela, 2004). Most of
the readily available N in solid manure from pigs and cattle is in NH3 form while that in
poultry manure is in uric acid form (Misselbrook, 2004). The proportions of readily available
N as NH3 after applications on the soil are typically greater for solid manure than for slurries.
For solid manures, the only practical measure to reduce the ammonia losses after spreading is
to incorporate the manure into the soil (Misselbrook, 2004; Jokela, 2004). The amount of N
that will be available to a crop after manuring depends on the time and method of application.
For the fall (autumn) applications, the majority of available N will be lost through NH3
volatilization soon after application and by NO3- leaching over the winter and spring
(Misselbrook, 2004). It is important to apply manure as later as possible in the fall to minimize
mineralization before soil frozen (Ma et al., 1999b).
Compost is the organic material derived from aerobic decomposition of recycled plant
waste, manures, crop residues, biosolids, and animal shed waste such as left-over fodder.
Nutrients in the compost must be released by soil microorganisms through a decomposition
process called mineralization. This biological process is affected by variations in moisture,
temperature, and the microbial species and populations present in the soil. Composting manure
is a useful method of producing stabilized product that can be stored or spread with little odour
or fly-breeding potential (Fronning et al., 2008).
Organic manures have several advantages such as (i) they serve as the carrier of plant
nutrient and provide several of the essential nutrients, (ii) increase soil OM, (iii) enable soil to
hold more water and also help improve the drainage in clay soils, (iv) they provide organic
acids that help dissolve soil nutrients and make them available for the plants, (v) nutrients from
the organic manures release slowly in soil and supply nutrients over seasons, and (vi) they
enhance the physical, chemical and biological properties of soils.
Nutrients concentrations in the organic manure vary greatly depending on animal species,
the size and age of the animal, type of ration fed to the animals, bedding materials, storage,
48 K. D. Subedi and B. L. Ma

processing, and the season of the year. For example, manures from dairy cattle contain higher
N than from beef cattle, broiler chicken litter contain higher N than layer hens and so on.
Rieck-Hinz et al. (1996) reported that nutrient values of dairy feedlot manure were higher in
summer and fall than in spring and winter. The approximate nutrient composition of various
solid manures, including some composted manures, is presented previously (Tables 6 and 7).
While these tables provide a general guide of manure or compost’s nutrient concentrations, it
is strongly recommended that routine sampling and analysis needs to be carried out for the
determination of more precise application rates because as stated earlier, nutrients
concentrations vary greatly over time, location and sources.
There have been some systematic studies on the advantages of FYM and composted
manure applications on soil properties, environment and crop nutrient values. In eastern
Ontario, Ma et al. (1999a) reported greater increase in corn yield by the application of either
uncomposted or partially composted manure, in comparison with inorganic fertilizer N.
Manure application also enhanced soil N release from seasonal mineralization (Ma et al.,
1999b). Eghball and Power (1999a) found there was no effect of tillage practice, and manure
and compost applied plots resulted in similar corn grain yields. They noted that the first year N
availability was approximately 8% for manure and 20% for compost in both no-till and tillage
systems. Apparent NUE was 17% for manure and 12% for compost as compared to 45% in the
chemical fertilizer. In another study (Eghball and Power, 1999b), P-based manure and compost
application resulted in similar grain yield to those N-based treatments, but had significantly
less soil available P level after 4-yr of application. There was no yield difference between the
biannual and annual manure and compost application. They concluded that when application
rate is based on correct N or P availability, manure and compost can produce corn grain yields
that are equal to or greater than that for fertilizer application. Annual P-based manure or
compost application is the most effective method of using these resources when soil P build-up
is a concern. Similarly, when studied N-mineralization from beef-cattle feedlot manure and
compost, Eghball (2000) reported about 11% of the composted manure and 21% of the non-
composted manure released through mineralization during the succeeding growing season.
Eghball et al. (2004) also observed that residual effects of manure and compost applications on
corn grain yield and N uptake lasted for at least over growing seasons while effects on soil
properties lasted longer. Eghball et al. (2005) recommended that in P-deficient soils, a P
availability of 70% should be used. There is a serious lacking of studies on the residual effects
of manure and compost on soil and environment in the developing countries.
The rate of manure or compost applied to fields depends on the crop being grown, soil test
levels and nutrient composition of the manure or compost. There are some studies that
evaluated the efficacy of manure and composts on corn yield in comparison with chemical
fertilizers. Xie and MacKenzie (1986) reported that hog manure resulted in more NO3--N
compared with fresh and composted cow manures. Annual P-based manure or compost
application is the most effective method using these resources when soil P buildup is a concern
(Eghball et al., 2005). One to 5 kg manure-N was found to be equivalent to 1 kg of urea-N in
terms of increasing soil NO3--N levels at the end of growing season. Laboski and Lamb (2003)
reported that P from liquid swine manure was more available than fertilizer P. It is postulated
that decomposition of manure resulted in concentrations of organic acids that effectively
reduced P sorption to the soil and increased P availability.
Corn Crop Production: Growth, Fertilization and Yield 49

In a corn-soybean-corn rotation study conducted in Michigan, USA, Fronning et al. (2008)

found that total SOM increased in the 2 to 25 cm soil profile by 41 and 25% for the compost
and manure treatments, respectively and decreased by 3% in the untreated control plots.
Nitrogen uptake increased with increases in N application rates and was higher with hog
manure than with cow manures.
With an expected yield of 9.4 Mg ha-1, Dormaar and Chang (1995) reported that corn
yields of annual or biennial beef cattle manure or compost application based on N or P
requirements of the crop were similar to those for fertilizer application. Ginting et al. (2003)
reported that effects of compost and manure resulted in 20 to 40% higher soil microbial
biomass C, 42 to 74% higher potentially mineralizable N, and 0.5 unit higher pH, as compared
with the fertilizer treatment. For silage corn production, Butler et al. (2008) reported that three
years after compost application, soil P and K concentrations were greater in plots receiving 70
and 150 Mg DM ha-1 manure. Soil OM increased in all treatments receiving >35 Mg DM ha-1
after first season application.
In the smallholder farmers in the tropics and sub-tropical regions, organic manures are the
main sources of plant nutrients for corn. Both quality and quantity of the manures are the
concerns and the environmental effects of manures are less perceived. For example, in a
subsistence farming system of Kenya, Jama et al. (1997) reported that application of 10 kg P
ha-1 as organic, inorganc and organic + inorganic sources significantly increased corn yield.
Sensitivity ananlysis suggested that organic materials was most suitable for use as P source
and low in cost of production. In a similar study conducted in Zimbabwe, supplement manure
with varying levels of mineral fertilizers resulted in corn yields that were still below the
potentials due to inadequate amounts, poor quality of organic materials and inefficient
combinations (Murwira and Palm, 1998).
Nutrients losses from the barn, storage and field are a big concern over manure
management. Burger and Venterea (2008) reported that estimates of first-season available N
from manure would be improved by measuring manure NH4+. In contrast, in soil amended with
solid manure, which had the lowest initial NH4+ content, 22% of organic N was mineralized.
Gaseous N losses were <1% of the added N in all treatments. Oenema et al. (2007) studied the
magnitude of nutrient losses in manure management systems in 27 European Union member
states, and found about 65% of the N excreted in barns was collected in barn and stored for
some time prior to application to agricultural land. Almost 30% of the N excreted in barns was
lost during storage; approximately 19% via NH3 volatilization, 7% via emission of NO, N2O
and N2, and 4% via leaching and run-off. Low-protein animal feeding is an effective measure
to reduce gaseous emissions of NH3, NO and N2, and NO3-- leaching from animal manure. Al-
Kaisi and Kwaw-Mensah (2008) reported that tillage and N rates beyond 85 kg ha-1 had no
effect on corn yield regardless of whether supplied from fertilizer or manure source. Tillage
and N rate had a significant effect on plant N and P uptake. Recovery of percentage of applied
N across all tillage systems and N rates was 40% and 27% from manure and fertilizer sources,
respectively at V12 of corn.
The challenges with use of organic manures are (i) large volume of materials which needs
more application cost, (ii) conservation of nutrients from organic manure, (iii) environmental
issues such as ground and surface water contaminations (Eghball and Power, 1999b), (iv)
odours, and (iv) buildup of P with continuous application and others. If organic materials are
over applied, it may lead to contamination of surface and/or groundwater by excess nutrients
such as NO3--N. Manure application in excess of crop needs can cause a significant buildup of
50 K. D. Subedi and B. L. Ma

soil P, N and other ions and salts (Dormaar and Chang, 1995; Eghball and Power, 1999b). The
environmental concerns of manure and compost application are discussed in detail in Section

8.5. Mulching

Mulching is a practice of covering soil surface with some organic (e.g. organic reasidues
or live mulch) or inorganic materials. Living mulches grow for a long time with the main crops
and legumes used as mulches also provide N fixation thereby reducing the need for fertilizer.
This is a traditional practice in the subsistence farming systems of tropical and sub-tropical
regions and it has also gained popularity recently in the conservation agriculture. The key
benefits of using mulch are to (i) conserve soil moisture, (ii) prevent weeds growth, (iii)
protect soil from erosion, (iv) lower soil temperature, and (iv) add OM and nutrients into the
The value of mulching is very important in sloppy lands and with conventional tillage
practices where soil erosion is a major challenge. Atreya et al. (2008) in the mountains of
Nepal, estimated that up to 60 to 90% of annual nutrients losses occurred during the pre-
monsoon period (May). They reported that mulching reduced annual SOM loss by 52%,
annual total N loss by 46%, annual available P2O5 by 32%, and annual exchangeable K2O by
53% in a corn – mustard (Brassica campestris L.) cropping system. Similarly, intercropping
corn with soybean reduced the annual loss of these nutrients by 58, 49, 26 and 60%,

8.6. Hedge Rows

Hedge row intercropping is a system of growing mainly leguminous hedges between the
crop rows and incorporation of pruned biomass on the soil. This system has gained some
attention in subsistence farming systems of tropics, e.g. Africa and Latin America, especially
in the sloppy lands. The benefits of hedge-row system are to (i) conserve soil from runoff, (ii)
add SOM and plant nutrients, and (iii) lift nutrients from deep soil to the surface. Swınkels and
Franzel (1997) evaluated the hedge row intercropping in western Kenya. Although about half
the farmers claimed that hedges improved crop yields, after three years of experimentation
only about one-fifth planted additional hedges and only 14% did so to improve soil fertility.
Agus et al. (1998) reported that Gliricidia in a contour hedgerow increases food crop yield on
strongly acid Oxisols by recycling nutrients and partially supplementing the N demand by the
food crops. It appeared that the potential for its adoption as a soil fertility practice is low,
mainly because of high labour requirements.

8.7. Chemical Fertilizers

Chemical fertilizers are the artificially manufactured sources of plant nutrients. The role of
chemical fertilizers in the modern day’s agriculture are paramount. The agricultural production
Corn Crop Production: Growth, Fertilization and Yield 51

in the absence of fertilizers would not have met the growing food demand by the alarmingly
increasing population. Since the 1960s, additional nutrients applied through fertilizers have
been responsible for 55% of the yield increase in developing countries (FAO, 1998). There
should not be an argument whether fertilizers are needed, and corn production in the absence
of chemical fertilizer would not be possible only with rest of other sources. Therefore fertilizer
is one of the key components of IPNM. In an ideal situation, in addition to the nutrients
supplied by the components discussed above, remaining nutrients should be complemented
with chemical fertilizers. Consequently, a judicious use of chemical fertilizer to complement
the nutrients supplied through various other sources is a component of IPNM. The concept of
IPNM therefore, emphasizes the balanced amount of fertilizers to complement the nutrients
insufficient from other sources. However, excessive and unwarranted use of chemical
fertilizers has huge negative impacts on production efficiency, producers’ net returns, regional
and global environment, human health, and cost of production.
Changing practices from conventional to conservation tillage makes chemical fertilizer
management more crucial importance and challenge. Adequate fertility must be maintained
when producers switched to no-till corn production. It is because, for the no-till system, proper
fertilization is imperative to optimize production and maintain SOM (Campbell et al., 1998).
Fertilizer N addition, legume incorporation and tillage systems had significant positive effects
on N-uptake (Dharmakreethi and Beauchamp, 2006). Total N required for producing 1 Mg
grain yield of corn was slightly greater (20 kg Mg-1 grain) in the NT than conventional tillage
(19 kg Mg-1 grain). Thus, Halvorson et al. (2006) stated that current N fertilization
recommendations for conventional tillage corn may need to be modified for NT to account for
the lower yield potential and slightly higher N requirements.
Use of fertilizers is limited in the subsistence farmings of the developing countries because
of (i) unavailability in required time, (ii) lack of money to purchase by the resource poor
farmers, and (iii) lack of technical know-how of the judicious use of fertilizers.

8.7.1. Basis of Fertilizers Recommendation

As stated earlier in the concept of IPNM, the amount of chemical fertilizers required for a
corn crop depends on several factors such as target yield, available nutrients in soil,
mineralization potential of SOM, and nutrients supply from other sources such as FYM or
compost, green manures and others, tillage practices (e.g. no-till or conventional tillage), and
soil water availability (irrigated or non-irrigated). This is a decision making process and needs
some sort of exercise of nutrients budgeting. Current N management practices based on yield
goals have been found to be poorly correlated with optimum N rates (Vonotti and Bundy,
1994). Proper crediting of N from animal manures, legumes in rotation and SOM
mineralization has to be made although it is difficult to estimate the exact value of inputs from
these sources. The nutrients from chemical fertilizers can be supplied through one or more than
one fertilizers (e.g. N, P, K, and micronutrients sources). A decision of which fertilizers to be
applied depends on (i) availability of fertilizers, (ii) relative cost of fertilizers, (iii) soil texture,
(iv) soil pH, and (iv) type and availability of equipments, etc.
Management of N is one of the most important aspects of IPNM. The rate of fertilizer N to
corn depends on several crop management aspects such as hybrid/variety types, purpose of
crop production (grain or silage or sweet corn), yield goal, residual SMN, soil texture, SOM
content, manure history, preceding crops (e.g. legumes or cereals), PPD, and economic
considerations (costs of fertilizer, fuel and produce, etc.), and amount of other sources for N
52 K. D. Subedi and B. L. Ma

such as organic manure available. Therefore, determination of a precise N fertilizer rate has
been a complicated topic. It is because the best rate of N for corn crops varies greatly by
complex interactions of soil, climatic, biological, management practices and location
(Blackmer, 1997; Winhold and Halvorson, 1999; Doerge, 2002). Even if SMN is known,
in the humid environments such as eastern Canada, corn yield response to N amendments was
poorly correlated with soil mineral N content prior to planting because of greater spatial and
temporal variability (Ma and Dwyer, 1999). The cumulative effects of past N management
practices, crops in rotations, tillage systems, and excessive use of manure and chemical
fertilizers makes the recommendation further complicated. Therefore, no single rate or a
blanket recommendation is desirable for diverse production systems. Any recommendation
should take into consideration of these variabilities. Recommendation of other nutrients such
as P, K, S and micronutrients should be based on the soil tests, and consideration should be
made for the P and K buildup in soil and micronutrients toxicities due to over-application or
improper application practices.

8.7.2. Timing of Fertilizer Application in Corn

Once the amount of nutrient element required is calculated and the type of fertilizer is
decided, next step is to determine the timing of fertilizer application. Fertilizers can be applied
as (i) before corn planting (preplant), (ii) at planting (starter), or (iii) after seedling emergence
(sidedress or topdress) depending on various circumstances. The key factors that determine the
apropriate timing of fertilizer application to corn could be (i) soil residual nutrients, (ii)
previous crop in the rotation, (iii) amount of manure and fertilizers applied on the previous
crops, (iv) soil texture and (v) other factors such as availability of fertilizer, labour, credit, etc.
For example, if the soil contains adequate P and K, it would unlikely need to add these
nutrients. On the other hand, if the soil test shows lack of these nutrients and other sources are
inadequate, these nutrients should be supplied based on the soil-test recommendations during
land preparation.
Timing of N fertilizer application is critical, especially for corn production. Nitrogenous
fertilisers are generally applied to corn as preplant, starter (at planting), and as sidedress at the
V6 to V8 growth stage (in-season). One approach to better matching N application with crop
need is a split application, focussing the split component at mid-to-late vegetative stages (Ruiz
Diaz et al., 2008). The goal is to make sure the crop not experience any shortage of N during
the critical periods of requirement. If organic manure such as FMY is to be applied, they
should be incorporated well before corn planting.
The rate of N uptake by corn is relatively slow before entering the period of rapid growth
at about the V6 growth stage. The highest rate of N mineralization tends to occur before the
highest rates of N uptake by corn (Wu et al., 2008) at which, soil NO3--N that accumulates
during the early part of the growing season, usually still presents in the root zone during the
peak demand period (Magdoff, 1991). Tillage system has a role on the timing of N
requirement. Schepers et al. (1995) reported that 50 to 80% of the N fertilizer use in corn is
applied prior to planting; however this varies with location. Less than 20% of the total N
uptake by corn occurs prior to sidedress (Schepers et al., 1995). Soil temperature rather than N
immobilization by residue and/or N supplied from residue, were primary factors affecting net
N mineralization in high surface residue corn systems (Andraski and Bundy, 2008).
Starter fertilizer is an efficient way of stimulating early growth and improving yield of
corn (Niehues et al., 2004). Starter fertilizers, regardless of placement, often increased early
Corn Crop Production: Growth, Fertilization and Yield 53

season dry matter (DM) production and significantly increase grain yields (Vetsch and
Randall, 2002; Niehues et al., 2004). Restriction of starter N supply until V8 stage (Ritchie et
al., 1993) caused an irreversible effect on grain yield of corn even though adequate N was
supplied thereafter (Subedi and Ma, 2005a). Plant competition between the vegetative stage
and anthesis had a large effect on grain yield reduction, which ranged from 8 to 21% (Hashemi
et al., 2005). Subedi and Ma (2009) reported that while lack of preplant N application (100 kg
ha-1) reduced yield by up to 10 to 22%, there was no yield increment due to additional
sidedress of 50 kg N ha-1 in a medium textured loamy soil. The disadvantage of a single
preplant application is that excess N applied early can be lost on coarse-textured sandy soils or
with irrigated corn due to leaching, and on fine-textured soils due to runoff. While starter
fertilizers accelerate early plant growth under these conditions, yield increases do not always
occur. Bullock et al. (1993) reported that starter fertilizers on soils of high testing mineral N
increased plant growth and development rates, but the increase in early growth often did not
result in a yield increase. In general, use of starter fertilizers for corn may be more important in
no-till or reduced tillage systems than in conventional tillage because it may help overcome the
effects of slow early growth and soil compaction. In Wisconsin, a comparison of starter
fertilizer in no-till and conventional tillage showed that planting date had a major influence on
the role of starter fertilizer with the largest yield response in the no-till system when corn was
planted late (Bundy and Widen, 1991). No-till soils are often cooler and less aerated than tilled
soils. These conditions decrease N mineralization so that less N is available to the crop early in
the season. Failing to apply sufficient N fertilizer at planting time when the remainder of N
fertilizer is to be sidedress may limit yield of no-till corn. In the no-till or mulch covered fields,
incorporating or knifing-in the UAN improves efficiency slightly over dribble applications.
Delaying of some or all N fertilizer until after seedling emergence may allow for precise
diagnosis of N needs by either in-season soil testing, tissue tests, sensing crop colour or
estimating weather effect on soil N availability (Magdoff et al., 1984; Blackmer et al., 1989;
Scharf et al., 2002). Post-emergence applications are practised so as to avoid frequent wet
seasons and to minimise the in-season N loss in wet years (Scharf et al., 2002). However,
delaying N application may lead to irreversible yield loss (Subedi and Ma, 2005a). Binder et
al. (2000) reported a nearly 12% reduction in maximum grain yield when delaying in N
application until V6 growth stage. Scharf et al. (2002) found that yield was still responsive to
N application until silking, but full yield was not achieved when applications were delayed till
then. Corn responded more to sidedress N for total N uptake than for preplant application, and
it is very likely that grain yield is affected to a larger extent by the conditions after sidedress
(Ma et al., 2005). Split application of N can help reduce the N loss and also meet the high
demand of corn during its peak uptake stage. Split-application also provides farmers with the
opportunity for fine-tuning of N application. Russell et al. (1998) indicated that N recovery by
corn may be greater when part of the N is applied as a delayed split rather than a single
application at planting.
Studies have shown that N applied as sidedress during the early growth stage (i.e. close to
the time of rapid uptake of N by the crop) has been used more efficiently (Magdoff et al.,
1984; Magdoff, 1991; Ma et al., 2005). There is less time for leaching or denitrification loss
when N is applied after plant emergence (Vetch and Randall, 2004). This is especially critical
on soils with a high potential for leaching or denitrification. Although corn plants can be
responsive to applied N until later stages and it is easy to detect N-deficiency at the later
stages, application after V8 growth stages has a practical problem of the crop being damaged.
54 K. D. Subedi and B. L. Ma

Broadcasting fertilizers such as urea on corn plants can cause leaf injury; therefore this is not a
feasible practice for corn.
In the humid temperate regions of USA and Canada, where there is usually a single crop in
a year, fertilizer N can also be applied either during the fall (after crop harvest) or during the
spring (prior to corn planting). Smicklas and Moore (2008) compared fall and spring
applications in terms of grain yield of corn and NO3--N recovered in the drainage water.
Application of full dose of anhydrous ammonia in the spring produced equivalent yields to that
of fall-applied N treatments and it decreased NO3--N release into the drainage water.

8.7.3. Methods of Fertilizer Application

Chemical fertilizers can be applied to corn as surface broadcast or banded with planting or
injected in furrows at sidedress. Appropriate application method also depends on fertilizer
type, tillage system or land preparation, and stage of crop at the time of fertilizer application.
For instance, N fertilizer is often applied in no-till systems using surface broadcast of granular
urea or N solutions containing urea (UAN). If it is anhydrous ammonia, it has to be soil
incorporated (knifed) to 15 to 20 cm deep. Fertilizers such as ammonium nitrate, ammonium
sulfate, and the ammoniated phosphates are less prone to volatilization losses, therefore can be
surface-applied to soils with neutral or low pH, while application of urea as surface broadcast
would lead to significant losses. Surface Broadcast Application

Generally, fertilizers and manures are applied as broadcast on the soil surface mainly in
the non-mechanized farmings. Even in the mechanized farming, fertilizer or manure is spread
over the soil surface such as solid manure or preplant application of fertilizer. There are some
possible adverse effects of the surface applied manure or fertilizers on soil and environment
such as surface-applied fertilizers and manures are prone to runoff and, surface-applied or
shallow banding of P and K could lead to stratification of these nutrients. Incorporation of
surface applied manure or fertilizer into the soil immediately after application is the
recommended best nutrient management practice. Banding
Banding of fertilizers along the seed row allows combining the seeder with the fertilizer
distributor in one operation and saves timing and fuel cost in field operation. The consensus to
date is that P and K applications via strip tillage in the fall may be too deep to provide starter
fertilizer benefits to the following corn crop. Fall banding operations need to be particularly
concerned with depth of placement and perhaps with the orientation of previous bands as well. Foliar Application

Foliar application of nutrients to corn has not been very effective although micronutrients
can be foliar-applied. Foliar application can sometimes cause foliar injury (phytotoxicity) of
the crop if proper sprayer calibrations are not carried out or if sprayers are not properly cleaned
after herbicide application. Foliar application is more useful for micronutrients that require
very small concentration. However, for the macronutrients such as N, the amount of total
requirements by high yield crops cannot be applied at once; split applications with foliar
supplement may useful to avoid plant toxicities.
Corn Crop Production: Growth, Fertilization and Yield 55

8.7.4. Variable Rate-Application (VRA)/Site-Specific Nutrient Management (SSNM)

Yield variation in a corn field may be caused by many factors, including spatial variability
in landscape position, soil structure and texture, crop production and field operation history,
soil physical and chemical properties and nutrients availability (Wibawa et al., 1993; Penny et
al., 1996). Site-specific nutrients management (SSNM) is a concept developed in Asia for rice
(IRRI, 2007). It emphasizes on ‘feeding’ nutrients as and when the crop needed. The SSNM
strives to enable farmers to dynamically adjust fertilizer use to optimally and timely fill the
deficit between the nutrient needs of a high-yielding crop and the nutrient supply from
naturally occurring indigenous sources such as soil, organic amendments, crop residues,
manures, and irrigation water (IRRI, 2007). Site-specific N management based on an in-season
assessment of crop N status may offer producers with increased grain yield, profitability, and
NUE. To address the spatial variability of nutrients in corn fields, precision agriculture
practices such as SSNM will benefit growers by increasing the efficiency of fertilizers, reduce
the total amount of N application, improving the productivity and thereby reducing
environmental impacts. In Kenya, Tabu et al. (2006) reported that SSNM was found to be
important in the small holder farmer fields. A survey of the fertilizer use, however, shows that
farmers use sub-optimal levels probably because of the poor resources level and non-specific
recommendations that aim at optimizing crop yield but not necessarily NUE.
Variable rate application (VRA) is a similar concept as SSNM but it is based on more
precision agricultural practices than in the SSNM system. The strategies used in the VRA are
assessing site characteristics that affect soil nutrients dynamics. Fertilizer rates, application
timing and methods are determined based on extensive soil tests to determine pre- and post
season NO3--N and other nutrients of interest using intensive grid soil sampling or according to
crop need-based indicators (Ma et al., 2005). Crop-based indicators (e.g. remote sensing,
SPAD), and real-time site specific yield monitors are gaining popularity in recent years.
Variable responses to the amount of N fertiliser application in the same region or field have
been more common than other nutrients in corn (Magdoff et al., 1984; Scharf et al., 2002;
Andraski and Bundy, 2002). The first step in site-specific N management is to examine the
field history, in particular its effect on N mineralization, and to take account of anticipated
effects of texture, drainage and precipitation (Schröder et al., 2000). Site-specific N
management requires indicators of the N status of the soil-crop system. Various indicators
have been developed and evaluated during the last decade (Schröder et al., 2000). An ideal
indicator has a reproducible relationship with the N status of the soil-crop system and must be
able to detect or predict both deficiency and excess of N.
Heiniger (1998) reported that VRA increased yields only in the strips testing low in P and
K, but an increased economic profit of VRA where variability existed in the fields. Ferguson et
al. (2002) realised no significant difference between the uniform application and VRA while
VRA reduced soil residual NO3--N. Schmidt et al. (2002) stated that VRA based only on SOM
are too simplistic to reflect variability in soil N availability within a field. Other workers have
shown the potential of VRA to improve fertilizer use efficiency (Raun et al., 2002), increase
yield (Wang et al., 2003), economic returns (Wang et al, 2003; Yang et al., 2001; Koch et al.,
2004), and reduced environmental impacts (Raun et al., 2002; Roberts et al., 2002). Wittry and
Mallarino (2004) reported that VRA resulted in better P fertilizer management because it
applied 12 to 14% less fertilizer and reduced soil test P variability compared with the
traditional uniform fertilization method. Inman et al. (2005) used site specific management
zones (SSMZ) for N management and such zones were found to be less spatially variable than
56 K. D. Subedi and B. L. Ma

the whole field. The SSMZs accurately characterized variability in N uptake as well as grain
yield response to applied N. They concluded that variation in N uptake and grain yield can
potentially be managed using SSMZs. Nevertheless, adoption of this practice by corn
producers in the North America is rather low because of the recommended N fertilizers based
on yield goal are often poorly correlated with actual economically optimum N rates (Doerge,
2002). The challenges are that N response patterns are often field- and season-specific and can
vary within the same fields (Doerge, 2002). The availability of N to crop plant is affected by
complex set of interacting soil, biological, climatic and management factors (Blackmer et al.,
1992; Degree, 2002; Ma and Dwyer, 1999). Economically optimum rate of N (EORN) was
very different between fields and was also highly variable within fields (Scharf et al., 2005).
Scharf et al. (2005) concluded that the average level of within field variability in EORN is high
enough than the potential of VRA to produce economic and environmental benefits. The total
amount of N utilized by corn crop will increase with yield level. However, the predicted,
potential, average or actual yields are poorly correlated with EORN (Blackmer et al., 1992;
Doerge, 2002). As EORN was poorly correlated with grain yield, Lory and Scharf (2003)
suggested that the Delta Yield (i.e. the grain yield at optimum N rate minus grain yield at
control) may be a better predictor of EORN, and farmers should be encouraged to monitor
Delta Yield as a more effective indicator of EORN than the actual yield.
There are no consistent advantages for variable or uniform rate of N applications, whole-
field N rates were similar for both strategies and post-season soil NO3--N levels were not
appreciably reduced when using VRA (Wu and Ma, 2008). Doerge (2002) reviewed the
prospect of VRA and concluded that there is a need for new diagnostic tools that provide a
better prediction of EORN in sub-regions of a field. Adoption of this technology has been
hampered due to the difficulty of classifying fields into management units, the high cost of
sampling soils on a grid basis, and the variability of soil and plant properties in the landscape.
Moreover, intensive grid soil sampling approaches involving analysis of soils is also another
drawback for VRA to be a viable technology. Further research on tools that detect the nutrients
variability will make this technology more economical and practical for adoption by growers.


Corn production consumes probably the largest amounts of fertilizers worldwide. Even
with the most efficient ways of nutrient management practices, the efficacy of applied
nutrients is always low (< 50%). The low efficiency of applied nutrients especially N leads to
significant economic and environmental consequences. Therefore, excessive and improper
application of manure and fertilizers on corn production has raised public concern about
surface and ground water contamination, and gaseous N emissions to pollute the environment
(Christensen, 2002; Jarvis et al., 1996). While there are various pathways of N loss from corn
fields, the major routes of N losses occur through NH3 volatilization, NO3- leaching, and N2O
emissions (especially due to denitrification). Plant N losses as NH3 could be up to 52 to 73%
of labeled 15N fertilizer in a corn field (Francis et al., 1993). In a tillage study, gaseous N
losses due to denitrification from applied N fertilizer are up to 10% for conventionally-tilled
and 22% for no-till corn (Hilton et al., 1994). The leaching loss of NO3--N is dependent on the
Corn Crop Production: Growth, Fertilization and Yield 57

amount of residual NO3- in the soil, applied manures and N fertilizers, rate of SOM
mineralization which is dependent on soil temperatures and seasonal rainfall and/or irrigation
water. Nitrate leaching is the most serious problem on well drained and sandy soils, but it also
can occur on well-drained upland limestone and shale soils. In Ontario, Drury et al. (1996)
estimated that up to 26 kg ha-1 yr-1 of NO3--N is lost through tile drainage. Denitrification is
most serious on soils rated as somewhat poorly drained but it also can occur on any soil that is
saturated with water. In Germany, Herrmann et al. (2005) concluded that forage corn
production is characterised by a significant excess of N supply, and there leaves ample
opportunity for reduction in N use without risk of yield loss. Therefore, manures and fertilizers
must be used judiciously to maximize profits, optimize crop quality, save energy, and protect
the environment (Schröder et al., 2000). In this section, the different ways of nutrients loss
from corn fields are discussed in relation to their impacts on environment.

9.1. Issues with the Use of Manure and Biosolids

Composted or liquid manure from farm animals is one of the major sources of plant
nutrients. Crop production in the small-holder farms is more dependent on manures as a source
of plant nutrients. Odour, runoff and leaching of nutrients such as NO3--N are some of the
important key environmental problems associated with manure and biosolids use in corn field.
Over-application of fertilizer or manure P can negatively impact water quality. Nitrate leaching
occurs most commonly after the corn harvest and in the following spring, and the risk of NO3--
N movement in groundwater is lower in loamy than in sandy soil (Ball-Coehlo and Roy,
2004). There has been substantial research conducted on this topic in Europe and North
America, but there is a lack of information in the developing countries, where manure is the
major source of nutrients for crop production.
In general, animal manure contains more P than needed by the crop when the amount of
manure application is based on its N content. Therefore, continuous manure application to
meet crop N requirements will result in higher soil test P, water soluble P and higher potential
runoff P (Zhang et al., 2007). Proper management of P sources and P transport over the
landscape can reduce negative impacts of P on water quality. Where P buildup in soil is a
concern, Zvomuya et al. (2006) suggest that accurate prediction of P availability and plant P
recovery may help tailor manure and compost applications to plant needs and minimize the
buildup of bioavailable P, which can contribute to eutrophication of sensitive aquatic systems.
Accumulation and redistribution of NO3--N within the soil varies due to management
practices, soil characteristics and growing season precipitation. Hensler et al. (1970) concluded
that for soils near the neutral points, nutrients in the manure, even at the very high rates of
application can be utilized in crop production and soil improvement with relatively little
danger of plant toxicity. Lithuurgidis et al. (2006) reported that concentrations of NO3--N in
the soil profile of manure-plots were higher than control but similar to inorganic fertilizer
treatments. They concluded that soil applied liquid cattle manure at a rate equivalent to the
recommended inorganic fertilization can produce corn yield and maintain soil fertility at
desired level.
When raw or treated liquid swine manure (LSM) was used on corn as sidedress followed
by immediate incorporation, Chantigny et al. (2008) found that the raw and treated LSM have
fertilizer value of N uptake and corn yields similar to fertilizer N; the risk of post-harvest NO3-
58 K. D. Subedi and B. L. Ma

accumulation with the raw and treated LSM was similar to mineral fertilizer on the loam soil
and lower on clay soils. Application of organic manures affects the rate of mineralization. Net
mineralization in the historically amended soil was twice that in the historically non-amended
soil, mostly due to difference in soil total N stock (Mallory and Griffin, 2007).
Research has shown that 40% of the total N in beef feedlot manure and 15% in composted
beef feedlot manure is available to the crop plant in the first year it is applied and incorporated
(Eghball et al., 1999a). Apparent NUE was 17% for manure, 12% for compost, and 45% for
the fertilizer treatment across 4 years. When beef feedlot manure is applied and not
incorporated in a no-till system, research has shown first-year availability of 38% of total N for
manure and 20% for compost (Eghball et al., 1999b). In their study, surface application did not
show significant N loss because the N in both manure and compost were in very stable forms.
A significant portion of the N can be lost from manure before it is applied to the land.
Much of this is caused by the gaseous loss of ammonia (NH3). This process occurs in the
feedlot pens and during storage. Ammonia loss will also continue after manure is applied to
crop land. Research has shown that incorporation of manure into the soil within the first 48 h
of application minimizes the gaseous loss of ammonia to about 15%. Delaying incorporation
will cause greater losses. Injection of liquid manure can also greatly reduce gaseous loss and
retain more N for crop use. Equipment should be calibrated based on the application method so
the correct amount is used.

9.2. Nitrous Oxide (N2O) Emission

Nitrous oxide (N2O) is one of the main greenhouse gases (GHG) generated from
agricultural sources. It has a global warming potential of 298 times greater than that of carbon
dioxide (CO2) and 25 times than that of methane (CH4) over a 100-year time horizon (IPCC,
2007). It accounts for approximately 5% of atmospheric GHG effect globally (Hutchinson et
al., 2007). The dramatic increase of atmospheric N2O is due to the human alterations of the
global N cycle, with 24% of annual emissions produced by agricultural soils and the
application of N fertilizer (Bouwman, 1996; Mosier et al., 1996; Mosier, 2001; Bouwman and
Boumans, 2002; IPCC, 2007). In groundwater under agricultural fields receiving N
applications, or in riparian zones receiving groundwater or runoff water, excessive NO3- may
be transformed to N2O through the process of denitrification (Mosier et al., 1998). Nitrous
oxide emissions account for almost 60% of on-farm GHG emissions in Canada (Desjardins et
al., 2005). Hutchinson et al. (2007) estimated that the direct N2O from manured corn field
ranged from 12.9 to 17.3 Tg with an average of 15.1 Tg equivalents during 1981-2001. The
greatest source of emissions estimated was from N fertilizer followed by crop residue (4.24
Tg). During a corn growing season (mid-May to mid-September), N2O emission from an
irrigated corn field was totaled only 2.5 kg N ha-1and about 30% of the N2O lost from the corn
field was emitted during the 2 wk following fertilization (Mosier and Hutchinson, 1981). The
amount of N2O produced is determined by the rate of nitrification and denitrification in
agricultural systems, and soil moisture plays an important role. The flux of N2O was not
significantly correlated with soil NO3--N concentration but was strongly correlated with soil
water content and N2O concnetration in the soil atmosphere (Mosier and Hutchinson, 1981).
Similarly, Lessard et al. (1996) observed that fluxes of N2O occurred in episodes and the high
fluxes coincided with periods when NO3--N levels and water content were relatively high.
Corn Crop Production: Growth, Fertilization and Yield 59

Under a simulated rainfall condition, Whalen (2000) observed pulsed N2O emission from
denitrification of accumulated NO3--N, indicating that further emissions will occur with an
increase in soil moisture. Agricultural activities influence N2O emissions primarily by
changing the magnitude and pattern of N-cycle in the soil-plant system (Hutchinson et al.,
2007). However, Ginting et al. (2003) concluded that residual effects of manure and compost
on CO2, N2O, and CH4 emissions were minimal. Kim et al. (2009) also concluded that the flux
of dissolved N2O from the cropped field was negligible in comparison to soil N2O emission in
the crop fields. Fronning et al. (2008) observed that compost and manure amendments resulted
in a net global warming potential (GWP) of equivalent to 1811 and 1160 g CO2 m-2 yr-1,
respectively, compared to 12 g CO2 m-2 yr-1 for unapplied control treatment.

9.3. Nitrate (NO3--N) Leaching and Water Quality

Excessive rates and inappropriate method of application or inefficient use of N fertilizer

may have adverse effects on ground water through leaching of soil NO3--N. High NO3- levels
in ground water can cause adverse health concerns, especially for infants, and it also cause
excess plant and bacterial growth, which upon death and decay can deplete much oxygen in
river and lake water. The primary inorganic N component in the soil profile was NO3--N, and
the zone of maximum accumulation was between 2 and 2.5 m. Excessive levels of NO3--N in
subsurface drainage from row crops, especially corn are well documented (Owens, 2008).
Such accumulation is subjected to loss during winter and spring periods through leaching
and/or denitrification, especially in the humid environments (Drury et al., 2007).
A considerable amount of research has been conducted on NO3--N leaching and
redistribution in soil. Timmons and Dylla (1981) estimated that annual average NO3--N
leaching loss ranged from about 29 (non-fertilized, non-irrigated) to 112 kg ha-1 (high N with
irrigation), the combination of variable rainfall, soil NO3--N content and low soil water holding
capacity cause great variation within and among years. De Jong et al. (2007) estimated that N
loss via leaching and concentration of NO3--N in the leached water in Canada ranged from 5.1
kg N ha-1 in 1991 to 6.4 kg ha-1 in 2001. The actual concentration of NO3--N in the ground
water is affected by the amount of fertilizers applied in the previous crops, soil texture,
seasonal rainfall (percolation), tillage operations, crop rotations and so on. Elevated post-
harvest soil NO3--N usually provides evidence that N was applied in excess for corn uptake
(Ferguson et al., 1991; Andraski et al., 2003). Nitrate remaining in the post-harvest soil profile
representing a potential risk for leaching during the fallow period has been shown to be closely
related to N fertilization rate, seasonal precipitation and soil texture (Gehl et al., 2006). Year-
to-year variations in loads of NO3--N occur as a result of variations in weather and crop yields.
There are different ways which can minimize the NO3- leaching in the ground water. As N
is lost from cropping systems in a number of pathways, a single solution of N management is
unlikely (Binder et al., 2000). Precise matching of application rates with crop needs could
reduce residual soil NO3--N available for leaching (Bausch and Duke, 1996; Vyn et al., 1999;
Andraski and Bundy, 2002). For this, in-season, site-specific or variable-rate N management
based on remote sensing tools may reduce N losses to groundwater while maintaining or
increasing yield and NUE. Similarly, periodic application of liquid N through the irrigation
system may reduce average annual NO3--N leaching by about 12 kg ha-1 at the 5 cm irrigation
level. When N application rate was higher than the optimum, little fertilizer derived N leached
60 K. D. Subedi and B. L. Ma

from the profile during the first growing season, but losses did occur during the off-season and
subsequent growing season (Stevens et al., 2005). Yang et al. (2007) found that improved N
fertilization practices reduced residual soil nitrate (RSN) by 13%. In China, Fang et al. (2008)
estimated that there was a potential of saving more than 30% of the current N application rates
per crop from 300 to 200 kg N ha-1, which could reduce about 60% of the N leaching without
compromising yields. Timing of N fertilizer can have a major effect on NO3- leaching.
Smiciklas and Moore (2008) observed that spring applied fertilizer produced equivalent corn
yield with decreased NO3--N in the drainage water, in comparison with that of fall-applied N
treatments. Similarly, maintaining healthy and dense corn plants may help to reduce NO3-
leaching. Herron et al. (1971) proposed that use of a nitrification inhibitor with an NH carrier
can help in preserving mineral N in irrigated fine-textured soils. Maho et al. (2007) suggested
that NO3--N leaching from a granitic regosol during the rainy season could be reduced by the
increasing of planting density of corn due to the increase of N uptake by the plants.
The amount of NO3- leaching also depends on the soil type, water management practice
and cropping system. For example, no-till crop production increases the amount of soil
macropores and allows for greater water infiltration, which could lead to more NO3- leaching
in groundwater (Izaurralde et al., 1995). However, more recent studies (Halvorson, et al.,
2001; Gupta et al., 2004) showed no difference in N leaching between tillage types. Soil type,
rainfall, crop rotation and other external factors will influence the amount and rate of the
macropore flow. Therefore, proper N fertilization management is important to prevent
producers from applying too much crop-usable N and increasing the risk of N leaching in
macropore flow. Weather events can lead to substantial leaching, runoff, or denitrification of
residual N. As a general rule, the lower end of the recommended N rate range is more
appropriate under conditions when residual N is expected. Planting of cover crops and
avoiding of late season sidedress can minimize the post harvest NO3--N concentration in soil,
which is liable for leaching.

9.4. Phosphate in Surface Water

Transport of P from field is influenced by rainfall, soil erosion, surface runoff, and wind
(Lemunyon, 1993). The rate, timing, form, and method of application, along with the site
location on the landscape affect the likelihood of P movement and environmental impact
(Lemunyon, 1993). The transport of manure nutrients off-site in runoff is a major source of
surface water contamination. Phosphorus and N in surface runoff are the major contributors to
the impairment of lakes and ponds through the process of eutrophication. Eutrophication is the
result of excessive bacteria and algae growth in surface waters due to nutrient enrichment,
usually of N and phosphates. Applying poultry manure according to the N needs of corn
typically applies more P than is recommended for two crop-years in a corn-soybean rotation,
which increases the risk for P losses to surface waters (Mallarino et al., 2002; Kaiser et al.,
Phosphorus from liquid swine manure was more available than fertilizer P. It was
postulated that the decomposition of manure resulted in concentrations of organic acids that
effectively reduced P sorption to the soil and increased P availability. Incorporating swine
manure when the probability of immediate rainfall is high reduces the risk of P loss in surface
runoff; however, this benefit sharply decreases with time (Allen and Mallarino, 2008). Grande
Corn Crop Production: Growth, Fertilization and Yield 61

et al. (2005a, 2005b) observed that high residue levels combined with spring-applied manure
led to enrichment in the clay-sized fraction of runoff sediment. After 4-yr of last application of
manure and compost in a corn field, P leaching to a soil depth of 45 to 60 cm was observed
with N-based manure application (Eghball et al., 2004).
Recently applied manure and higher residue levels achieved by high-cutting silage can
substantially lower sediment losses in spring runoff when soil is most susceptible to erosion.
Kleinman et al. (2005) reported that water-extractable P (WEP) ranged widely (0.2 -16.8 g ha-
), with swine manure having the highest average concentrations (9.2 g ha-1), followed by
turkey (6.3 g ha-1), layer chickens (4.9 g ha-1), dairy cattle (4.0 g ha-1), broiler chickens (3.2 g
ha-1), and beef cattle manure (2.3 g ha-1). Phosphorus leaching from manure applications on
loamy sand soils does not pose environmental concerns as long as soil P levels remain below
the saturation level (van Es et al., 2004). Phosphorus leaching can be extreme and represents a
great concern in many coarse-textured soils with low P-sorption capacities (Alleoni et al.,
2008). In a simulated rainfall study in Alberta, Canada, runoff of total P, soil test P and
dissolved reactive P concentrations increased with manure rate for both fresh and residual
manure (Volf et al., 2007). Band placement of P fertilizer can be considered an environmental
best management practice because the fertilizer banded below the soil surface is much less
susceptible to loss via surface runoff (Jokela, 2004). Recent manure additions were most
influential in enriching transported sediments with P (Grande et al., 2005b).
There was no relationship between soil test P levels and runoff P concentrations or loads in
no-till systems. Long-term manure P applications in excess of P removal by corn in
conventional tillage systems ultimately increased the potential for greater dissolved and
bioavailable P losses in runoff by increasing soil P levels (Andraski et al., 2003). They suggest
that maintaining high surface residue cover such as those found in long-term NT corn
production systems can mitigate this risk in addition to reducing sediment and particulate P



Residues are necessary to protect soil from erosion and to contribute SOM levels. Crop
residues protect the soil from wind and water erosion, provide inputs to form soil organic
matter (a critical component determining soil quality) and play a key role in nutrient cycling
(Johnson et al., 2003). In the developing countries, corn stover is removed mainly for animal
feed, while in the mechanized and developed countries such as in the USA, corn residue
(stover) has been increasingly considered as a source of cellulosic biomass for biofuel
production to supplement fossil fuel. Ethanol derived from corn is considered as a clean source
of energy that is in use to power transportation vehicles. Use of corn residue for production of
biofuel has the advantage of reducing dependence on the imported fossil fuel and developing
renewal energy source (Wilhelm et al., 2004). Because of the growing demand of corn based
ethanol, corn growers in the USA are getting good profits from corn crops. Its use is increasing
and there is a growing demand of biofuel although this technology is generally still in its
infancy. Under conservation tillage systems, the corn residues are left over after the grain
harvest, which can be a major source of nutrients cycling and SOM. Removing corn stover as a
62 K. D. Subedi and B. L. Ma

feed stock for biofuel production may decrease the amount of carbon stored in soil, and lead to
an adverse impact on overall soil fertility.
There are no consistent conclusions on the impacts of residue removal on soil
characteristics and crop yield. In a short-term test, stover removal resulted in increased soil
crust strength and reduced soil water content (Blanco-Canquia et al., 2006). Over a three-year
period, where crop residues were completely removed after harvest, yield of corn was reduced
by 22% than where residues were not removed (Dorna et al., 1984). Yield reduction of corn
was primarily from decreased soil water storage and excessive surface soil temperatures where
residue was completely removed. There is a potential of reduction in grain yields as well as
greater potential for nutrients removal (Dorna et al., 1984). Blanco-Canqui and Lal (2008)
reported that removal of stover at rates ≥ 50% reduced sub-critical water repellence by 2 to 10
times in all soils and concluded that stover removal adversely affects both macro- and micro-
scale soil properties. In the contrary, Wilhelm et al. (2004) stated that within limits, corn stover
can be harvested for biofuel production. Johnson et al. (2003) concluded that the range of crop
and soil responses to crop residue removal was attributed to interactions with climate,
management and soil type. Crop residues also impact radiation balance and energy fluxes and
reduce evaporation. The impact of crop residue removal on soil quality and crop productivity
must be assessed before prudent decision and policy can be developed (Wilhelm et al., 2004).
Other potential problems with using corn stover for bio-energy include competition with
livestock feed, competition in land for other food grains production, and more importantly, the
negative impact on soil fertility and quality due to increasing continuous corn monoculture.
The removal of corn stover will significantly reduce the amount of OM returned in the soil,
which in time will reduce the SOM and plant nutrients. Alternative practices such as green
maturing, cover cropping and using animal manures should be practices for the lands where
corn stovers are removed for biofuel production. Similarly, height of cutting of stover will
minimize the total OM removal from the soil. In general, the benefits of using crop residues as
fuel, which removes crop residues from the field, must be balanced against negative
environmental impacts (e.g. soil erosion), maintaining SOM levels, and preserving or
enhancing productivity.


This chapter covered a wide prospectus of sustainable nutrients management in corn crops.
Corn types, corn-based cropping systems, corn growth stages and maturity group, essential
nutrients and their classification, nutrients-deficiencies and sources of nutrients for corn crops
are the introductory sections. Methods of detecting nutrients requirements, and integrated
management of different sources of nutrients as well as strategies to achieve optimum corn
yield without causing adverse effects on soil health and environment have been the other
aspects of this chapter. Emphasis has been given on the nutrients use efficiencies especially for
N and impacts of different nutrients management practices on environment, such as on soil
NO3--N leaching to ground water, P in surface runoff and gaseous N emissions. The effects of
using organic manures and biosolids on corn fields and removal of corn stover for bioenergy
production are also briefly discussed. The information provided is based on the advances in
contemporary research outcomes suitable to the topic. Although we have tried to provide a
Corn Crop Production: Growth, Fertilization and Yield 63

global perspective of the topic, it is obvious that majority of the research works that have been
reviewed are from the North Americas.
Low efficiency of applied manures and fertilizers, especially N fertilizer has been found to
be one of the most important nutrients management challenges in corn production.
Consequently, the impacts of chemical fertilizers and animal manures on soil, water and
eventually on the environment are significant. Tillage practices such as conservation tillage has
positive impacts on nutrients use efficiency, and soil conservation, and environment although
it demands more nutrients initially. Nutrients application timing (such as preplant or
sidedress), rate and methods have also been influenced by the tillage systems. The IPNM,
which is a nutrients management approach of integrating soil, crop and nutrients sources for
sustainable crop production seems to be the key nutrients management approach for all corn
production systems. Components of IPNM have been discussed in relation with the
environmental protection and sustainability of the corn production systems.
Nutrients management on corn is one of the most researched areas, while nutrients losses
from the corn fields are noteworthy. Still there are several areas of research that needed to be
concentrating for the sustainable nutrients management in corn crops in order to maximize the
nutrients use efficiencies while reducing the impacts on environment. Based on the review of
published literatures on this topic, information gaps in corn nutrients management seem to be
in the following key areas:

• There are several researches on an individual component of the overall nutrients

management for corn, such as N, P or micronutrients management. However, research
on a complete package is lacking. For example, integrating soil, crop and nutrients
management on a holistic system basis would provide more useful results on a system
perspective. Therefore, research projects focus on IPNM approach of nutrients
management, i.e. integrating nutrients, soils, crop and environment would be desirable
for the sustainable corn production.
• It has been observed that the use of N fertilizer on corn production is far more than
what is actually required. There is always a lower NUE, usually < 40% and excessive
N are beyond recovery in the soil and water systems. Therefore, efforts towards
reducing the use of unwarranted N fertilizers or manure in corn fields are important.
Large-scale on-farm research and demonstration projects with the participation of corn
producers will be useful and perhaps the best technology transfer avenues to raise
producers awareness and acceptance of any new innovative technologies.
• There is lack of suitable and agronomic and economically feasible controlled-release
fertilizers that would provide the crop with timely nutrient supply while minimizing or
eliminating NO3- and gaseous N emission losses. Integrating experts in the areas of
soil, plant, and engineering, including nanotechnology to develop intelligent smart
fertilizers and matched nutrient management systems will certainly improve the
system efficiency.
• There is a serious lack of research on crop site-specific nutrients management,
especially in the developing countries. Generalizing the research results from
geographically distinct regions is not appropriate. Therefore, more research based on
the local cropping systems, soil and environment should be encouraged. Nutrients
recommendations without complete set of research information can be misleading in
64 K. D. Subedi and B. L. Ma

terms of environmental and human health issues while increase the cost of manure or
• Manures from animals are the natural sources of plant nutrients and undoubtedly they
have several benefits to soil and crops. However, improper storage, application and
uses can have severe impacts on environment and human health. There is limited
research on the use of organic manures, especially in the developed countries, while
research in the developing countries is negligible. This component of plant nutrients
management should be given much attention.
• The conservation tillage system has been proven to be beneficial for soil, water and
nutrients conservation and reducing energy and cost of production. The nutrients
management practices for conservation agriculture systems may be different than the
conventional systems. Therefore, more research on appropriate nutrient management
for the conservation agriculture system will be required for the wide-scale promotion
of this system.
• Cover crops have been shown importance in preserving soil NO3- after corn harvest,
providing nutrients to the following crops and preventing wind and water erosions.
Research on suitable crops and their cultivation and technologies for the
implementation of cover crops are urgently needed.
• Although, site-specific nutrient management is considered to be a potential approach
of reducing nutrients waste thereby protecting environment and providing
economically profitable system, there are limitations on the adaptation of this
approach. There has been considerable interest in the development of crop need-based
indicators using remote sensing technology. Up-to-date research on field level remote
sensing has shown that i) optical sensing at certain wavelength bands or spectral
indices is able to detect crop reaction to biotic (diseases) and abiotic (nutrient, water)
stresses; (ii) at early stages of crop development, measuring leaf chlorophyll or
canopy reflectance can predict biomass and yield potential in certain crops and at
specific growth stages/conditions; (iii) the ability of optical sensing to detect crop
health status varies with environmental and cultural conditions; (iv) under best
scenarios in research plots (shortage of plant available nutrients in the soil, e.g. N,
weed-free, no severe drought after fertilizer sidedress, low N supplying power fields –
sandy loam soil, no manure or legume preceding crop), optical sensor guided N
application can increase yield and NUE; (v) algorithms for converting optical sensor
signals into fertilizer N rates may vary across environments; (vi) optical sensor
readings will be saturated when available N above certain levels and/or crop reaches
advanced stages. Clearly, more focused research on the development of on-the-go
uniform or variable rate application of N to corn is of paramount importance to
improve nutrient and water use efficiencies and reducing N and agrochemical loading
of the environment for efficient food and biomass production.
Corn Crop Production: Growth, Fertilization and Yield 65

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Agus, F.; Garrity, D.P.; Cassel, D.K. and Mercado, A. (1998). Grain crop response to contour
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Chapter 2



M. Madhaiyana,c, S. Poonguzhalia,c, M. Senthilkumarb

and P. Santhanakrishnanc
Department of Agricultural Chemistry, Chungbuk National University,
Cheongju 361-763, Republic of Korea
Department of Agricultural Microbiology, Horticultural College and Research Institute,
TamilNadu Agricultural University, Periyakulam 625 604, TamilNadu, India
Department of Agricultural Microbiology, Tamilnadu Agricultural University,
Coimbatore 641 003, Tamilnadu, India

Root colonizing bacteria (rhizobacteria) that exert beneficial effects on plant
development via direct or indirect mechanisms have been defined as plant growth
promoting rhizobacteria (PGPR). The search for PGPR and investigation of their modes of
action are increasing at a rapid pace as efforts are made to exploit them commercially as
biofertilizers. This review focuses on different kinds of PGPR, their mode of action as
biofertilizers and also development of microbial consortia is mentioned. These mode of
action include fixing N2, increasing the availability of nutrients in the rhizosphere,
positively influencing root growth and morphology. Although significant control of plant
pathogens or direct enhancement of plant development has been demonstrated by PGPR in
the laboratory and in the greenhouse, results in the field have been less consistent. Because
of these and other challenges in screening, formulation, and application, PGPR have yet to
fulfill their promise and potential as commercial inoculants. Recent progress in our
understanding of their diversity, colonization ability, mechanism of action, formulation,
and application should facilitate their development as reliable components in the
management of sustainable agricultural systems. Obtaining maximum benefits on farms
from plant growth promoting biofertilizers will require a systematic strategy designed to
86 M. Madhaiyan, S. Poonguzhali, M. Senthilkumar et al.

fully utilize all these beneficial factors, allowing crop yields to be maintained or even
increased with reduced fertilizer applications.

Keywords: PGPR, rhizosphere, growth-promotion, biocontrol, root morphology

World’s population has been growing faster than cereal production since the early 1980s
with decrease in global per-capita cereal yield and output. The strong implication is that this
decline is caused by increasing various environmental production constraints. In addition to
these problems, between now and the year 2025, the human population is expected to rise from
about 6 billion to 8 billion (Tim Dyson, 1999). Can the world’s farmers produce 3 billion tons
for 8 billion people in 2025? Probably yes, but at an accelerated impact on sustainability and
environmental quality (Trewavas, 2001). As defined by Golley et al. (1992), sustainable
agriculture is that which is managed towards greater resource efficiency and conservation
while maintaining an environment favorable for the evolution of all species. More simply,
sustainability is meeting today’s needs without compromising the needs of the future. By
either of these definitions, crop production is compromising the global future use of fertilizers
and chemicals sustainability. It is only relatively recently that microbial ecologists have begun
to recognize the importance of those complex interactions between plants and the microbial
components in soil that sustain them. While the physical and biological benefits of manures
and composts have been quantified (Brady, 1974; Hoitink and Boehm, 1999), the microbial
mechanisms that underpin their effectiveness are still little understood. Though it is often
considered that soil microbes play a key role in soil quality and health (Vessey, 2003), a
general dearth of information exists when it comes to categorizing the assembly of species that
assure soil fertility, the critical forces that govern their community structure and function. It is
now clearly indicates that root zone microflora and plants interaction involves a continuum of
colonization events that are initiated at seed germination and traverse from rhizosphere to the
rhizoplane, to the endoroot via the root epidermis and apoplast, directly to the shoots (Petersen
et al., 1981; Kloepper et al.,1992a; Van Bruggen et al., 2002). Consequently, phyto-microbial
relationships can become extremely intimate, extending over the life-cycle and growth habit of
the plant, and involving both exo- and endo plant environments (Wardle, 2002).

Functionally there are three broad interacting components were recognized in the
rhizosphere namely - rhizosphere (soil), the rhizoplane, and the root itself. The rhizosphere is
the zone of soil influenced by roots through the release of substrates that affect microbial
activity. The rhizoplane is the root surface, including the strongly adhering soil particles. The
root itself is a part of the system, because certain microorganisms, the endophytes, are able to
colonize root tissues (Kennedy, 1998 and Bowen and Rovira, 1999). Microbial colonization of
the rhizoplane and/or root tissues is known as root colonization, whereas the colonization of
the adjacent volume of soil under the influence of the root is known as rhizosphere
Responses of Agronomically Important Crops to Inoculation… 87

colonization (Kloepper et al., 1991; Kloepper, 1994). The use of molecular techniques to
identify microorganisms (O’Gara et al., 1994) is currently a key tool to study rhizosphere
ecology (Puhler et al., 2004).
Bacterial inoculants which help in plant growth are generally considered to be of two
types a) symbiotic and b) free-living (Kloepper et al., 1988). One group of microorganisms
which are beneficial to crops is bacteria that colonize roots or rhizosphere soil of crop plants.
These bacteria are referred to as plant growth promoting rhizobacteria (PGPR). Beneficial
free-living bacteria referred to as PGPR are found in the rhizosphere of the roots of many
different plants (Kloepper et al., 1989). PGPR are free-living, soil-borne bacteria, isolated from
the rhizosphere, which, when applied to seeds or crops, enhance the growth of the plant or
reduce the damage from soil-borne plant pathogens (Kloepper et al., 1980a). Breakthrough
research in the field of PGPR occurred in the mid 1970s with studies demonstrating the ability
of Pseudomonas strains capable of controlling soil-borne pathogens to indirectly enhance plant
growth and increase the yield of potato and radish plants (Kloepper and Schroth, 1981a; Howie
and Echandi, 1983). The rhizosphere is the soil found around the root and under the influence
of the root. It is a site with complex interactions between the root and associated
microorganisms. PGPR enhance plant growth either by direct or indirect mechanisms (Glick,
1995). The direct growth promoting mechanisms includes nitrogen fixation, phosphorus
solubilization and mobilization, siderophores production, production of phytohormones such
as auxins, cytokinins, gibberellins and lowering of ethylene concentration (Kloepper et al.,
1989; Glick, 1995; Glick et al., 1999). The indirect mechanisms of plant growth promotion by
PGPR include antibiotic production, depletion of iron from the rhizosphere, synthesis of
antifungal metabolites, production of fungal cell wall lysing enzymes, competition for sites on
roots and induced systemic resistance (Kloepper et al., 1988; Liu et al., 1995; Glick et al.,
1999). The thin layer of soil (about 1 to 2mm thick) surrounding crop roots and the volume of
soil occupied by roots are known as the rhizosphere. Hiltner (1904) introduced the term
rhizosphere, which is derived from the Greek word ‘rhiza’, meaning root, and ‘sphere’,
meaning field of influence. He defined the rhizosphere as the zone of soil immediately
adjacent to legume roots that supports high levels of bacterial activity. Now the term has been
broadened to include both the volume of soil influenced by the root and the root tissues
colonized by microorganisms (reviewed in Pinton et al., 2001). The shear extent of crop roots
in soil dictates that a significant portion of soil is actually within the rhizosphere (about 5 to
40% of soil in the rooting zone depending upon crop root architecture). This zone is where the
majority of soil microorganism (bacteria and fungi) reside. They reside in the rhizosphere to
utilize compounds and materials released from crop roots providing microorganisms with
energy. Rhizobacteria that establish inside plant roots, forming more intimate associations, are
endophytes. These include a wide range of soil bacteria forming less formal associations than
the rhizobia–legume symbiosis; endophytes may stimulate plant growth, directly or indirectly
(Kobayashi et al., 1995) and include the rhizobia. Given the semantic overlap, the following
definition for endophytic bacteria has been proposed: “those bacteria that can be isolated from
surface-disinfected plant tissue or extracted from within the plant, and that do not visibly harm
the plant” (Hallmann et al., 1997). In general, a greater proportion of endophytes are PGPR
than in the case for bacteria inhabiting the rhizoplane or rhizosphere (Nowak, 1998).
Nodulating (rhizobia) and other N2-fixing rhizobacteria are also endophytes (Lodewyckx et al.,
2002), living in specially developed root organs; given their ability to promote plant growth
through N fixation.
88 M. Madhaiyan, S. Poonguzhali, M. Senthilkumar et al.


Beneficial, root colonizing, rhizosphere bacteria, the PGPR, are defined by three intrinsic
characteristics: (i) they must be able to colonize the root, (ii) they must survive and multiply in
microhabitats associated with the root surface, in competition with other microbiota, at least
for the time needed to express their plant promotion/protection activities, and (iii) they must
promote plant growth (Barea et al., 2005). The PGPR are known to participate in many
important ecosystem processes, such as the biological control of plant pathogens, nutrient
cycling, and/or seedling growth (Persello-Cartieaux et al., 2003; Barea et al., 2004; Zahir et al.,
2003). The rhizosphere supports diverse bacteria that can stimulate growth of plants. As
rhizosphere processes result from the activities of diverse groups of microorganisms (Table 1),
determining the significance of changes to community structure presents a major challenge for
the future. As the effect of PGPR on plants was demonstrated, the concept of PGPR began to
gain importance and a large number of bacterial strains have been isolated, screened and
evaluated for plant growth promotion.

Table 1. Examples of plant-growth promoting bacteria

Bacterial species Reference

Azotobacter chroococcum Kennedy and Tchan (1992)
Clostridium spp. Kennedy and Tchan (1992)
Azospirillum spp. Reinhold and Hurek (1988)
Azoarcus sp. Hurek et al. (1994)
Burkholderia vietnamiensis Baldani et al. (1997)
H. seropedicae Baldani et al. (1986)
Rhizobium leguminosarum bv. trifolii Yanni et al. (1997)
Rhizobium etli bv. phaseoli Gutiérrez-Zamora and Martı´nez-Romero (2001)
Azorhizobium caulinodans Matthews et al. (2001)
Acetobacter (Gluconacetobacter) diazotrophicus Baldani et al. (1997)
Actinobacter sp., Enterobacter agglomerans,
Tanii et al. (1990)
Flavobacterium spp
Aeromonas caviae Inbar and Chet (1991)
Agrobacterium radiobacter Ryder and Jones (1990)
Alcaligenes sp. Yuen et al. (1985)
Bacillus cereus Handelsman et al. (1990); Ryder et al. (1999)
Bacillus circulans Berge et al. (1990)
Bacillus firmus, Bacillus licheniformis Chen et al. (1996)
Bacillus subtilis Turner and Blackman (1991)
Enterobacter cloacae, Erwinia herbicola Nelson (1998)
Phyllobacterium sp. Lambert et al. (1990)
Pseudomonas aureofaciens Kluepfel and McInnis (1991)
Pseudomonas cepacia, Pseudomonas putida De Freitas and Germida (1991)
Pseudomonas fluorescens Voisard et al. (1989)
Serratia fonticola Chanway et al. (1991)
Serratia liquefaciens Zhang and Smith (1996)
Serratia macescens Ordentlich et al. (1988)
Serratia proteamaculans Chanway et al. (1991) and Bai et al. (2002a)
Bacillus thuringiensis Bai et al. (2002b)
Responses of Agronomically Important Crops to Inoculation… 89

The soil is a living environment that supports extremely diverse communities of micro and
macro organisms and is often considered a ‘black box’. The plant absorb all the minerals from
the soil by the solubilization action of the microbes in the rhizosphere region which indicates a
series of generalized and specific plant – microbe associations exists to perform this function.
The release of carbon compounds from plants into the soil results in greater microbial
populations in the rhizosphere relative to the bulk soil, and increased microbial biomass and
activity (Bending, 2003). When considering the rhizosphere effect in general, the
rhizosphere/bulk soil (r/s) ratios for bacteria, actinomycetes, and fungi are usually in the ranges
2–20, 5–10, and 10–20, respectively. In young plant roots it is thought that the rhizosphere
bacterial communities are dominated by r-strategists, which are species with fast growth rates
and capacities to utilize simple substrates (Andrews and Harris, 1986). As the roots mature,
there is a shift in dominance to bacterial communities with relatively slow growth rates and the
capacity to degrade more complex substrates (k-strategists). As a rule, although a general
increase in microorganisms in the rhizosphere is always noted, the community structure and
functional consequences of this increase are less well understood.
The rhizosphere environment is relatively rich in nutrients released by the roots and its
microbial communities differ from those outside the influence of the roots. Since roots act as a
source of organic carbon, the population density of microorganisms, especially bacteria, is
considerably higher in the rhizosphere than in the bulk soil. Root colonization is the initial
mechanism by which PGPR survive when inoculated onto seeds or into the soil and proliferate
in response to seed or root exudates rich in carbohydrates and amino acids (Kloepper et al.,
1985). In the root zone the rhizobacteria are efficient microbial competitors, can displace
native root colonizing microorganisms (Kloepper and Schroth, 1981b). The bacteria are
distributed in the rhizosphere in a log normal pattern with a sharp increase as the root surface
is approached (Loper et al., 1984) and are sporadically located along roots (Bahme and
Schroth, 1987).
Colonization of the roots is a complex phenomenon in which one of the first steps is the
migration of microorganisms towards the roots. The microorganisms present in the rhizosphere
colonize the host plant through chemotaxis movement with root exudates act as an important
source of nutrients. Chemotactic response towards amino acids, sugars, or organic acids is
fundamental for bacterial behavior both in vitro and in situ (Bashan and Holguin, 1994) and
represents, very probably, the first step in root colonization (Zheng and Sinclair, 1996). Once
bacteria are in the vicinity of the root, attachment to target cells on the plant surface can be
inoculation of wheat seedlings with A. brasilense increased the number and length of lateral
roots. Both grain and straw yields of wheat infected with R. leguminosarium bv. trifolii are
greater than uninfected controls. Yields of wheat plants are even higher when commercial
fertilizer is applied (Biswas et al., 2000). Inoculation with Azotobacter replaced up to 50% of
the urea-N for wheat in greenhouse trials under aseptic (gnotobiotic) conditions (Soliman et
al., 1995). Inoculation with A. brasilense can increase wheat grain yield by up to 30% and
other yield components significantly in field conditions (Okon and Labandera-Gonzalez,
1994), but only at lower rates of fertiliser-N (50–60 kg N ha-1). Beneficial effects of
inoculation with Azospirillum on wheat yields in both greenhouse and field conditions have
been reported (Ganguly et al., 1999). Azorhizobium caulinodans increased the dry weight and
90 M. Madhaiyan, S. Poonguzhali, M. Senthilkumar et al.

N content of wheat plants in a greenhouse experiment (Matthews et al., 2001). Its inoculation
saved up to 50% of the recommended rate of urea N in greenhouse trials under gnotobiotic (or
sterile) conditions (Saleh et al., 2001).

Common diazotrophs found in the rhizosphere of maize are Enterobacter spp., Rahnella
aquatilis, Paenibacillus azotofixans, Azospirillum spp., H. seropedicae, Bacillus circulans and
Klebsiella sp. (Chelius and Triplett, 2000). Corn (Zea mays L.) plants under water stress
during flowering (Casanovas et al., 2003) exhibited better growth, enhanced grain yield and
quality. A. lipoferum (N7), A. brasilense (N8), and P. putida CQ179 strains isolated from corn
rhizosphere and isolates G. azotocaptans DS1 from corn rhizosphere,G. diazotrophicus Wt
from sugarcane when inoclulated into corn plant under green house condition in sterile and
unsterilie condition significantly increased the root and shoot growth (Mehnaz and Lazarovits,
2006). Javed et al., (1998) selected 11 isolates of plant growth promoting rhizobacteria and
reported that four of these improved the growth of maize and could be used as biofertilizers.
However, results of these bacteria used as biofertilizers are variable in the field. Fallik and
Okon, (1989) showed that when maize seedlings were inoculated with 107 cfu of A. brasilense,
root surface area was significantly increased two weeks after sowing compared to that of the
control. The increase in root surface area was attributed to the synthesis of IAA, identified in
culture medium using GLC and GC-MS. The ability of Pseudomonas fluorescens M.3.1 to
produce auxins in the presence of maize root exudates in the culture medium indicated that the
strain can convert exudates into secondary metabolites which can play an important role in
plant development and yield (Benizri et al., 1998). Dobbelaere et al. (2001) found A.
brasilense increased grain yield of maize by 0.7–1.0 t ha-1 (50–95% increase) depending on
soil conditions when N was applied at low to medium (18–46 kg ha-1) rates. Other species of
Azospirillum capable of increasing the yield of maize are A. lipoferum and A. indigens, and
Azorhizobium caulinodans was also capable of giving such beneficial effects (Riggs et al.,
2001). In addition, they concluded from the results of extensive greenhouse and field
experiments using non sterilised soils that there were beneficial effects of maize seed
inoculation H. seropedicae, with increased yield in greenhouse conditions by 49–82% with
applied fertilizer N compared to an increase of only 16% without fertilizer N. In field
experiments, the increases in yields due to H. seropedicae inoculation were up to 19.5%.
Rhizobium etli bv. phaseoli can colonize maize roots, and increase plant dry weight
(Gutie´rrez-Zamora and Martı´nez- Romero, 2001). Riggs et al., (2001) had shown that
inoculation of R. leguminosarum bv. trifolii increased maize yields by 34 and 11% in the
greenhouse and field conditions, respectively. Sinorhizobium sp. can increase maize yields by
35–43% depending on the maize genotype (Riggs et al., 2001). Burkholderia spp. application
in the field trials was able to increase maize yield by 5.9–6.3% (Riggs et al., 2001).
Application of Bacillus sp. has been found to substantially control seedling blight, root
rots and stalk rots of maize caused by Fusarium graminearum, when used as seed inoculant
(Kommedahl and Chang, 1975). Trichoderma viride and Pseudomonas species were also
capable of controlling stalk rots of maize (Chen et al., 1999). Application of root-associated
Pseudomonas cepacea as seed coating biocontrol agent could reduce the Fusarium
Responses of Agronomically Important Crops to Inoculation… 91

moniliforme-induced infection of maize root by 23–80%. Pseudomonas cepacea was also

found to inhibit a range of soil-borne fungal pathogens including Fusarium graminearum,
Fusarium moniliforme and Macrophomina phaseolina (Hebbar et al., 1992). Burkholderia
cepacea was also found potent in controlling Fusarium moniliforme besides plant growth
promotion of maize (Bevivino et al., 1998). Raju et al., (1999) reported the reduction of
Fusarium moniliforme-induced diseases of maize by application of Trichoderma harzianum,
Pseudomonas fluorescens and Chaetomium globosum.

The diazotrophs commonly present in sugarcane plants are Acetobactor diazotrophicus,
Azospirillum brasilense, A. lipoferum, A. amazonense, B. brasilensis, B. tropicalis, H.
seropedicae and H. rubrisubalbicans (Reis et al., 2000). Asymbiotic nitrogen fixing bacteria
replaced 60% of the nitrogen requirements of sugarcane amounting to 200 kg N/ha (Urquiaga
et al., 1992). Gluconacetobacter diazotrophicus, a species found in high numbers in roots and
stems of sugarcane in Brazil and Australia (Stephan et al., 1991), was the only known
nitrogen-fixing species of this genus until Jimenez-Salgado et al., (1997) isolated two other
acetic-acid-producing, nitrogen-fixing bacteria (Gluconacetobacter azotocaptans and
Gluconacetobacter johannae) from the rhizosphere of coffee plants (Fuentes-Ramirez et al.,
2001). G. diazotrophicus has numerous properties associated with enhanced plant growth
including nitrogen fixation at low pH (5 or less), which is only partially inhibited by ammonia,
production of growth hormones, phosphate solubilization, and antagonistic potential against
plant pathogens (Muthukumarasamy et al., 2002). In sugarcane, inoculation with a mixture of
diazotrophic bacteria and mycorrhizal fungi was shown to be equivalent to half the dose of
recommended fertilisers. Muthukumarasamy et al., (1999) claimed that combination of G.
diazotrophicus, Herbaspirillum spp. and Azospirillum lipoferum with AM fungi helps in
reducing the input of nitrogen fertilizers. Also, investigations on the co-inoculation of G.
diazotrophicus with other diazotrophs resulted in improved growth of Brazilian sugarcanes
(Oliveira et al., 2002). Soil applications of Azospirillum can significantly increase cane yield in
both plant and ratoon crops in the field (Shankariah and Hunsigi, 2001). They reported that the
mean cane yield increases were 9 and 5 t ha-1 in plant and ratoon crops, respectively.


Strains of Pseudomonas putida and Pseudomonas fluorescens were particularly effective
in increasing root and shoot elongation in canola, lettuce, and tomato and yield of potato,
radish, sugar beet, tomato, lettuce, apple, citrus, bean and ornamental plants (Rodriguez and
Fraga, 1999). Methylobacterium strains also contributed to the flavor of strawberries and have
been localized as endo symbionts within cells of Scotch pine buds (Zabetakis, 1997). Increase
in potato growth occurs following infection with Pseudomonas (Kloepper et al., 1980b),
increasing total plant weight up to 100% when compared to the control. Microbial inoculation
of tissue culture plants showed enhanced growth characters like pseudo stem height and girth
in nursery stage. Similarly, improved plant growth after microbial inoculation was reported
92 M. Madhaiyan, S. Poonguzhali, M. Senthilkumar et al.

earlier in fruit crops like apple (Utkhede and Smith, 1992), strawberry (Kokalis-Burelle, 2003)
and Prunus spp. (Bonaterra et al., 2003). Similar influence of growth and development of fruit
crops were observed by Mello et al., (2000) and Jaizme- Vega et al., (2004) in pineapple and
banana respectively. The use of PGPR in the management of viral diseases in the field
condition is of very recent trend as reported from Tomato mottle virus in tomato (Murphy et
al., 2000), Cucumber mosaic virus in cucumber (Zehnder et al., 2000), Tomato spotted wilt
virus in tomato (Kandan et al., 2005). Pseudomonas strains 84 and 4B when introduced to
banana roots of tissue-cultured plants at de-flasking stage significantly improved plant growth
and reduced infection of Fusarium oxysporum f. sp. cubense in the rhizome under green house
conditions (Smith et al., 2003). Recently, Jaizme-Vega et al., (2004) found that bacterization of
micro propagated banana plants cv. Grand Nain and ITC-1297 with Bacillus spp. significantly
improved plant growth and foliar mineral contents. Similarly, strawberry cv. Camarosa
transplant plugs amended with PGPR strain LS 213 showed enhanced growth and yield under
field conditions (Kokalis-Burelle, 2003).



The majority of interactions considered so far concern with single PGPR strains either as a
growth promoter or as a biocontrol agent in the rhizosphere. However, one way of improving
the efficiency of PGPR in the rhizosphere may be to add mixtures or combinations of bacterial
genera, particularly if they exhibit different or complementary modes of action or abilities to
colonize root microsites. Such multiple interactions are the normal situation in the rhizosphere.
However the major obstacle to the successful application of PGPR strains in soil has been the
lack of stable effectiveness of the inoculant. This was always due to ineffective colonization of
the plant as well as poor survival and/or low activity of the introduced population.
Coinoculation of plant growth promoting rhizobacteria PGPR with B. japonicum has been
shown to increase soybean nodulation, nitrogen fixation, growth and physiological activity at
suboptimal root zone temperatures. Coinoculation of PGPR and B. japonicum increases
soybean root and shoot weight, grain yield, plant vigour, nodulation and nitrogen fixation (Li
and Alexander, 1988) and yield. Azospirillum brasilense strain Az39 and Brayrhizobium
japonicum strain E109 when inoculated singly or in combination, showed the capacity to
promote seed germination, nodule formation, and early development of corn and soybean
seedlings (Cassan et al., 2008).
Compared to the use of individual PGPR strains, mixtures of several strains may result in
a more stable rhizosphere community, provide several mechanisms of biological control, and
may suppress a broader range of pathogens. Compatible mixtures of certain biocontrol strains
with antagonism as the main mechanism of action have provided greater disease suppression
than that used individually. The seed application of a combination of three PGPR, Bacillus
pumilus Meyer & Gotheil, Bacillus subtilis (Ehrenberg) Cohn and Curtobacterium
flaccumfaciens (Hedges) Collins & Jones provided greater control of several pathogens on
cucumber (Cucumis sativa L.) than when any were inoculated singly (Raupach and Kloepper,
1998), combinations of Paenibacillus sp. and a Streptomyces sp. suppressed Fusarium wilt of
cucumber better than when either was used alone (Singh et al., 1999) and a combination of
Responses of Agronomically Important Crops to Inoculation… 93

Pseudomonas fluorescens and Stenotrophomonas maltophila improved protection of sugar beet

against Pythium-mediated damping-off in comparison with either applied individually (Dunne
et al., 1998). Jetiyanon and Kloepper, (2002) suggest that mixtures of PGPR and individual
strains of PGPR can elicit induced systemic resistance to fungal, bacterial, and viral diseases in
tomato, pepper, cucumber and green kuang futsoi tested. It has been reported that mixtures of
PGPR strains either in a two-way or three-way combination gave a greater protection,
compared to single-strain treatments, of cucumber angular leaf spot disease caused by
Pseudomonas syringae pv. lachrymans under field conditions (Raupach and Kloepper, 2000).

Plants growing in field soil cannot be viewed as single organisms. The science of PGPR is
thus relatively young in comparison to nitrogen fixing bacteria and momentarily applications
to crop production are limited. The possible role of PGPR signals in plant growth stimulation
presents exciting possibilities and research opportunities. As already outlined, PGPR increase
plant growth and the mechanisms underlying this are poorly understood. However, where they
are understood they could be exploited to increase plant growth. Agronomically, PGPR effects
are of particular interest and play roles in crop production (reviewed in Broughton et al.,
2003). The research required to fully understand them will require work at all levels, from
ecology to proteomics and metabolomics. What is needed for the future is a clear definition of
what bacterial traits are useful and necessary for different environmental conditions and plants,
so that optimal bacterial strains can either be selected or constructed. Also, it would be very
useful to have a better understanding of how different bacterial strains work together for the
synergistic promotion of plant growth (Lucy et al., 2004). Additional studies need to be
conducted on the effectiveness of different and novel inoculant delivery systems such as
alginate encapsulation. In addition, a better understanding of the factors that facilitate the
environmental persistence of the PGPR strains would be very useful. Finally inoculant strains
should be labelled e.g., with lux or gfp genes, so they can be readily detected in the
environment after their release. In this regard, finding new ways of establishing stable
associations between plants and beneficial organisms and understanding the molecular and
biochemical mechanisms of signal recognition and transduction that occur in plant–microbial
interactions under different environments are most challenging study elements.

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Editor: Arn T. Danforth © 2009 Nova Science Publishers, Inc.

Chapter 3



Pallavi Sharma, bAmbuj Bhushan Jha and R. S. Dubey1
Department of Biochemistry, Faculty of Science
Banaras Hindu University
Varanasi-221005, India

Rice is a staple food crop for the majority of world population. Abiotic stressful
conditions of the environment such as salinity, drought, heat, chilling, anaerobiosis, metal
toxicity impose limitations on productivity of rice in the regions which are prone to such
constraints. The manifestations of these stresses include non-expression of full genetic
potential, differential transcription of many genes, induction of stress responsive genes
leading to cellular metabolic changes, alteration in activity behaviours of many enzymes,
overproduction of several compatible metabolites like amino acids, sugars, polyamines,
phytochelatins, organic acids, increased synthesis of many enzymes and stress specific
proteins. Salinity and drought are prime stressful conditions for rice crop in arid and semi
arid regions of the world. Changes in temperature rhythm impose heat or chilling injury.
Soil flooding or submergence causes oxygen deprivation leading to anaerobic stress. Metal
ions such as Pb, Cd, Hg, As, Ni are key pollutants of the soil, whereas Al toxicity is a
problem in acid upland soils. Most of the abiotic stresses cause overproduction of reactive
oxygen species (ROS) within the cell which cause oxidative damage to membranes and
biomolecules. Increased accumulation of compatible solutes, overproduction of
antioxidative enzymes, overexpression of transcription factors have been shown to confer

1 Corresponding author: Dr. R. S. Dubey, Department of Biochemistry, Faculty of Science, Banaras Hindu
University, Varanasi-221005, India, E-mail:, Tel.: +91-542-2317190; Fax: +91-542-
aPresent Address: Department of Plant Sciences, College of Agriculture and Bioresources, University of
Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
bPresent Address: Department of Food and Bioproduct Sciences, College of Agriculture and Bioresources, University of
Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
112 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

tolerance in rice plants to a wide range of stresses like salinity, drought and low
temperature. Stress induced gene products those involved in stress tolerance and those
involved in signal transduction or as transcription regulators have served as basis to
engineer stress tolerant plants. To contribute to food security and sustainability in rice
production, it is essential to produce stress tolerant rice plants suitable for cultivation in
stress prone areas. This needs a detail understanding of physiological and molecular
mechanisms associated with stress tolerance more specially gene products involved in
stress tolerance and signal transduction. Transcriptome profiling of rice seedlings has
helped in great way in understanding how rice plants respond to abiotic stresses.
Successful attempts have been made to produce transgenic rice plants tolerant to different
abiotic stresses. However, with the rapid progress in the areas of functional genomics,
proteomics and metabolomics a more improved understanding of novel stress responsive
genes and their expression under various stresses is anticipated which will provide the
basis of new strategies to produce genetically engineered rice plants tolerant to a single or
multiple of abiotic stresses.

Rice, a staple crop for over half of the world population, is sensitive to a variety of abiotic
stresses (Lafitte et al., 2004a; Gao et al., 2007). Rice-growing areas span over the tropics,
subtropics, semi-arid tropics, and temperate regions of the world (Gorantla et al., 2007). These
areas are often threatened by severe abiotic stresses, including drought, salinity, extremes of
temperature, anaerobiosis, excessive levels of heavy metals, gaseous pollutants, irradiation,
etc. (Figure 1). Abiotic stresses (singly or in combination) cause both general and specific
detrimental effects on plant growth and development, and finally lead to reduced crop yield. At
the whole plant level, the effect of stress is usually perceived as a decline in photosynthesis
and growth associated with alteration in carbon and nitrogen metabolism (Dubey and Singh,
1999; Kumar et al., 2000; Feng et al., 2003; Chen et al., 2004; Jha and Dubey, 2004a, b;
Sharma and Dubey, 2005a; Moradi and Ismail, 2007; Mishra and Dubey, 2008a). One
characteristic cellular feature evident under abiotic stresses is the high production of reactive
oxygen species (ROS) in the chloroplasts, mitochondria and peroxisomes, which cause
irreversible cellular and tissue damages. Rice plants respond and adapt to variable
environmental conditions with a wide range of cellular and metabolic alterations that are
triggered by signaling and regulatory pathways (Shah and Dubey, 1998a; Shah et al., 2001;
Sharma and Dubey, 2004, 2005a, b, 2007; Rohila and Yang, 2007; Cho et al., 2008; Kumar et
al., 2008). Plants perceive the signals from environment and transmit them in a specific
manner to the genetic machinery in the nucleus to regulate the response to abiotic stresses.
Post-translational modifications of proteins play a key role in different signaling cascades. The
phosphorylation/dephosphorylation of proteins by specific protein kinases/phosphatases
modulate the activities of specific signaling molecules, resulting in signal amplification (Luan,
2003; Mishra et al., 2006; Kumar et al., 2008). Signal transduction cascade regulate gene
expression in a temporal and spatial pattern, leading to changes at the metabolic, physiological
and biochemical levels. These changes lead to adaption of rice plants to these stresses, thus
enabling the plants to survive.
Effect of Abiotic Stresses on Growth… 113

Figure 1. Aboitic stresses such as drought, salinity, heat, chilling, metal toxicity, gaseous pollutants, UV-
B radiation are major constraints to rice production. Rice cultivars that are tolerant to aboitic stresses
tolerate abiotic stresses whereas sensitive rice cultivars suffer from productivity loss.

Significant number of genes, gene products, and pathways associated with abiotic
response and adaptation have been identified in rice using a variety of experimental
approaches (Shah and Dubey, 1998a; Sharma and Dubey, 2004, 2007; Kumar et al., 2008;
Kawakami et al., 2008; Wang et al., 2009).
Most common responses to abiotic stresses in rice plants include induction of stress-
responsive gene expression (Gorantla et al., 2007), anatomical and morphological changes
(Caldwell et al. 1998; Matsui and Omasa, 2002; Feng et al., 2003), cellular metabolic changes
(Shah et al., 1995, 1997a, 1998a; Shah et al., 2001; Jha and Dubey, 2004a, b, c, 2005; Sharma
and Dubey, 2005a, b, 2007), overproduction of several compatible organic solutes termed
osmoprotectants or osmolytes and synthesis of stress-specific proteins (Dubey and Rani, 1989;
Shah and Dubey, 1998a, b, d; Sharma et al., 2005a; Lee et al., 2007; Kawakami et al., 2008;
Wang et al., 2009). Figure 2 shows the most common responses to abiotic stress in rice plants.
In our quest to achieve sustainable food production, abiotic stresses seem to present major
challenge. Generally plants respond to low and moderate levels of abiotic stresses, but when
the intensity of stress increases, physiological mechanisms imparting tolerance to plants start
breaking down which ultimately may result into plant death (Tayal et al., 2004). Development
of crop plants tolerant to environmental stresses appears to be a promising approach to help
satisfy growing food demands of the developing and under-developed nations where abiotic
stresses are severe constraints to crop productivity. Decades of breeding and selection have
resulted in limited improvements of stress tolerance in rice and other crops largely due to the
physiological and genetic complexities involved (Zhang et al., 2006). The advances in
physiology, genetics, and molecular biology have greatly improved our understanding of the
mechanisms of responses to abiotic stresses in rice.
114 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

Figure 2. Some of the common responses of rice plants to abiotic stresses.

Though considerable progress has been made in the improvement of transgenic rice for
abiotic stress tolerance yet the achievements are not promising. Progress is now anticipated
through comparative genomics studies of an evolutionarily diverse set of model organisms,
and through the use of techniques such as high-throughput analysis of expressed sequence
tags, large scale parallel analysis of gene expression, targeted or random mutagenesis, and
gain-of-function or mutant complementation. The identification of novel genes in rice,
determination of their expression patterns in response to the stresses, and an improved
understanding of their functions in stress adaptation (obtained by the use of functional
genomics) will provide us the basis of effective engineering strategies to generate rice plants
with enhanced tolerance to a single or multiple abiotic stresses in order to enhance the yield of
this crop (Rabbani et al., 2003).
The present review focuses on our current status of knowledge related to the effect of
various abiotic stresses on growth and metabolism of rice plants and components associated
with abiotic stress tolerance. Further the strategies adopted for improving stress tolerance in
rice plants have also been discussed.



Abiotic stresses such as drought, salinity, heat, chilling, excess levels of heavy metals,
gaseous pollutants, frequently limit growth and productivity of rice crop (Saini and Westgate,
2000; Yan et al., 2006; Yang et al., 2008). These conditions can delay growth and
development, reduce yield and, in extreme cases, can inflict lethal injuries to the rice plants.
Adverse effects of environmental stresses have been noted both during vegetative and
reproductive growth stages in rice plants (Sarvestani et al., 2008). Process leading to seedling
emergence, growth of the seedling, flower development and quality of seeds are critically
affected by these stresses. The cellular organelles like plasma membrane, mitochondria and
endoplasmic reticulum are known to be severely affected in response to adverse environmental
conditions (Pareek et al., 1997). The major events of plant response to abiotic stresses are
perception and transduction of the stress signals through signaling components resulting in the
Effect of Abiotic Stresses on Growth… 115

activation of a large number of stress related genes and synthesis of diverse functional proteins
that finally lead to various physiological and metabolic responses. The responses to a specific
stress may vary with the genotype; nevertheless, some general reactions occur in all genotypes.
Some of the most common responses which are triggered in rice plants subjected to different
abiotic stresses include alteration in gene expression, anatomical and morphological changes,
decreased efficiency of photosynthesis, reduced N assimilation capacity, alterations in plasma
membrane characteristics, cellular metabolic changes, overproduction of several metabolites,
altered activities of several key enzymes, increased synthesis of stress induced novel proteins,
etc. In the following sections common abiotic stresses and their effects on growth and
metabolism in rice plants has been described.

a) Drought

Drought is an imminent threat to food security. Water deficit occurs when land plants
absorb less water from their roots compared to that transpired (evaporated) from their leaves
resulting in reduction of relative water content, cell volume and cell turgor (Lawlor and
Cornic, 2002). The frequency of such phenomena is likely to increase in the future even
outside today’s arid/semi-arid regions (Chaves et al., 2002). Water deficit adversely affects a
range of key metabolic processes in plants like normal synthesis of proteins and nucleic acids,
photosynthesis, respiration, nitrogen assimilation, etc. which may ultimately result in poor
growth of plants and reduction in yield (Dubey, 1994). Plant responses to water deficit are
complex, involving deleterious and/or adaptive changes, and under field conditions these
responses can be synergistically or antagonistically modified by the superimposition of other
stresses (Chaves et al., 2002).
Rice productivity is severely affected due to recurrent droughts in almost all
agroecological zones of the world (Gorantla et al., 2007). Growth and development of rice
plants can be detrimentally impaired by water deficit at anytime during its life cycle. However,
the damage from water deficit is particularly exacerbated if the stress occurs during the
meiosis stage of male gametophyte development in the anthers (Saini and Westgate, 2000).
The effects of water deficit on pollen formation and related events in rice anthers were
investigated by Giao and coworkers (2007). A reduction of grain set and decline in
concentration of the adenosine-5'-triphosphate (ATP) pool in rice anthers in water stressed rice
plants compared to the controls was recorded. Decline in the concentration of the ATP pool
could possibly cause programmed cell-death (PCD) and adverse effects on ATP dependent
processes in the cells which could be the physiological causes for the loss of viable pollens.
Water stress detrimentally affects flower induction, pollen production and subsequently leads
to failure of fertilization and hence grain set (Sheoran and Saini, 1996). Water stress at
vegetative stage of rice plants was found to significantly reduce plant height due to decrease in
photosynthesis rate and dry matter accumulation (Sarvestani et al., 2008) whereas at flowering
stage imposition of greater water stress caused a reduction in grain yield due to reduction in
fertile panicle and filled grain percentage. Total biomass, harvest index, plant height, filled
grain, unfilled grain and 1000 grain weight were reduced under water stress in all rice cultivars
studied (Sarvestani et al., 2008). Plant root is the most important organ for uptake of water,
therefore it plays important role in tolerance to osmotic and drought stresses (Zombori et al.,
2008). Rice reacted to drought stress with changes in root dry matter and rooting depth. Rice
116 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

roots grew deeper under drought stress (Asch et al., 2005). Under drought, aerobic genotypes
were more tolerant than the irrigated lowland genotypes due to their higher ability to maintain
nodal root production, elongation, and branching, thus, less reduction in dry matter production.
Aerenchyma was also formed in droughted roots regardless of genotypes. The inability of
roots to acclimate to drought stress results in reduced growth and function thereby reduced dry
matter production (Suralta and Yamauchi, 2008).
Plants act in response to drought stresses through perception and transduction of the stress
signals through signaling components, leading to activation of a large number of stress related
genes and synthesis of diverse functional proteins that finally lead to various physiological and
metabolic responses (Shinozaki et al., 2003; Seki et al., 2003). Osmotic stress activates several
protein kinases including mitogen-activated kinases, which may mediate osmotic homeostasis
and/or detoxification responses. A number of phospholipid systems are activated by osmotic
stress, generating a diverse array of messenger molecules, some of which may function
upstream of the osmotic stress–activated protein kinases (Zhu, 2002). Abscisic acid (ABA) is
thought to be involved in the signal transduction. Evidence exists demonstrating the presence
of both ABA-independent and ABA-dependent regulatory systems governing drought-
inducible gene expression (Yamaguchi-Shinozaki and Shinozaki, 2005). Rice roots are known
to produce signals in response to progressive drought, which regulate leaf stomatal
conductance (Gs) and transpiration (Tr) and shoot growth (Siopongco et al., 2008). The
increase in leaf ABA concentration under field drought, and its strong association with soil
moisture tension and Gs, suggested its involvement in mediating stomatal responses during
early drought in rice (Siopongco et al., 2008). Water stress leads to following sequence of
responses of the rice leaves (i) rise in ABA content, (ii) closure of stomata, (iii) initiation of
leaf rolling (Henson, 1982). Leaf diffusive resistance and degree of leaf rolling were found to
be linearly related to leaf water potential. Stomatal resistance was found to increase more on
the abaxial than the adaxial leaf surface in rice cultivars. At the grain filling stage of early rice,
the Gs, Tr and Gs/Tr ratio fluctuated insignificantly under mild and moderate water stress,
while photosynthetic rate and leaf water use efficiency increased significantly, with an increase
in grain yield under mild water stress (Chen et al., 2005). Cabuslay and coworkers (2002)
demonstrated the greater sensitivity of leaf enlargement to water stress than dry matter
accumulation. Rice cultivars tolerant to mild water stress were found to have relatively high
rate of transpiration, low initial leaf area, high carbon isotope discrimination in the leaf, and
low specific leaf weight. These factors enabled tolerant cultivars to maintain high moisture in
the leaf and to have high values of leaf area, shoot dry matter and sugar as well as starch in the
tissues of stressed plants relative to the controls. Mild water deficit increased water use
efficiency in stressed plants and caused more degradation of starch than sugar in the leaf blade,
and resulted in more accumulation of these carbohydrates in the leaf sheath (Cabuslay et al.,
Activities of photosynthetic O2 evolution, Hill reaction, photophosphorylation and Ca2+-
ATPase and ATP content were significantly reduced in chloroplasts from flag leaves of rice
plants subjected to water stress (Chen et al., 2004). The membrane lipid content especially
sulfoquinovosyl-diacylglycerol and phosphatidylglycerol also declined. The changes in the
ultrastructure of chloroplasts included mainly a decrease in number of grana and increase in
number of osmiophilic granules. Yamane and coworkers (2004) found that the ultrastructural
changes of chloroplasts in bundle sheath cells were more prominent than those in mesophyll
cells of rice plants under drought stress treatments. Contents of ribulose-1,5-bisphosphate
Effect of Abiotic Stresses on Growth… 117

carboxylase (RUBISCO) reduced more dramatically in bundle sheath chloroplasts than in

mesophyll chloroplasts under drought stress. Thylakoids were less affected by drought stress
than chloroplast envelope (Yamane et al., 2004). Impairment of enzymes of sugar metabolism
and starch synthesis were suggested to be among the potential causes of pollen sterility and
inhibition of starch accumulation in water-stressed rice plants (Sheoran and Saini, 1996). Fast
hydrolysis of starch and increased carbon remobilization observed in rice under water stress
was attributed to the enhanced α-amylase activity and the high activation state of sucrose
phosphate synthase (SPS) (Yang et al., 2001).
The rate of dark respiration in rice plants increased in a normal light and dark cycle with
mild water stress whereas overall conversion efficiency (the ratio of grain dry-matter against
the gross carbohydrate input to the construction and maintenance processes) decreased. With
increasing water stress a decrease in respiration rate was observed (Kobata and Takami et al.,
1986). Photorespiration is associated with metabolism through the glycolate pathway (Tolbert,
1971). It was observed that increasing the duration of water-deficit-stress produced a
proportional decrease in relative reduced glycolate content and catalase (CAT) activity, but
increased glycolate oxidase activity, hydrogen peroxide (H2O2) and glyoxylate contents in the
leaves of rice plants (Goyal, 1986). Rice seedlings subjected to severe water stress showed
adverse effects on nitrogen assimilation. A marked decline in the levels of both NRact (Nitrate
reductase activity in the presence of Mg2+ representing the non-phosphorylated NR state) and
NRmax (Nitrate reductase activity in the presence of EDTA representing maximum NR
activity) and almost unaltered NR activation were observed by Sharma and Dubey (2005a) in
water stress seedlings. The accumulation of ammonium in detached rice leaves under water
stress has been observed which is attributed to a decrease in glutamine synthetase (GS)
activity. Ammonium accumulation in detached rice leaves, induced by water stress, was
accompanied by an increase in tissue sensitivity to ethylene which, in turn, accelerated leaf
senescence (Lin and Kao, 2000).
Amplified fragment length polymorphism (AFLP) analysis of cDNA identified 103
transcript-derived fragments corresponding to differentially induced genes in a tolerant rice
variety upon water-deficit stress. Most of the transcripts identified genes were related to
metabolism, energy, protein biosynthesis, cell defense, signal transduction and transport
(Rodriguez et al., 2006). Gorantla and coworkers (2007) identified genes associated with
water-stress response in rice using expressed sequence tags (ESTs) generated from a
normalized cDNA library, constructed from drought-stressed leaf tissue of an indica cultivar
Nagina 22. Comparison of leaf stress responsive genes (SRGs) to expression profiles for a
drought-stressed rice panicle library identified some common sequences. A total of 125 genes
were found to be expressed under drought stress in both tissues. The functional classification
of these 125 genes showed that the majority of them were associated with cellular metabolism,
signal transduction and transcriptional regulation (Gorantla et al., 2007). With the advent of
molecular and genomic technologies, emphasis is now being placed on understanding the
mechanisms of genetic control of the drought-stress response. To elucidate genome-level
responses to drought in rice, a 70 mer oligomer microarray covering 36,926 unique genes or
gene models was used to profile genome expression changes in rice shoot, flag leaf and panicle
under drought conditions. Drought stress appears to alter the expression patterns of a
significant number of genes involved in transcription and cell signaling in a largely organ-
specific manner. The promoter regions of genes induced by drought stress possess relative
118 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

enrichment of two cis-elements (ABRE core and DRE core) known to be associated with water
stress (Zhou et al., 2007).
An experiment was conducted to compare the mRNA expression difference in rice leaves
and roots under drought stress and normal conditions using fluorescent differential display
method. NADPH oxidoreductase gene expression level was found to be higher under drought
stress than that under normal conditions in rice leaves and roots (Chen et al., 2006a). Rice
seedlings subjected to drought showed increased concentration of superoxide anion (O2·−),
increased level of lipid peroxidation showing oxidative stress, chlorophyll bleaching, loss of
antioxidants [ascorbate (AsA), glutathione (GSH), α-tocopherol and carotenoids], total soluble
protein and thiols (Boo and Jung, 1999; Sharma and Dubey, 2005b; Bai et al., 2006). Various
studies related to the identification of components of water stress tolerance in rice suggest that
tolerance to water stress is associated with a co-ordination of physiological and biochemical
alterations at the cellular and molecular level such as the accumulation of various osmolytes,
polyamines and induced synthesis of stress specific proteins specially late embryogenesis
abundant proteins (LEAs) coupled with an efficient antioxidative defense system.

b) Salinity

Salinity is a widespread soil problem limiting productivity of cereal crops worldwide. The
United Nations Environment Program estimates that approximately 20% of agricultural land in
the world is salt-stressed (Flowers and Yeo, 1995). In saline soils predominant salts include
chlorides and sulphates of sodium, magnesium and calcium. NaCl is the more prevalent salt in
saline soils due to its exceptionally high solubility in water. High salinity causes hyperosmotic
stress and ion disequilibrium that produce secondary effects (Hasegawa et al., 2000; Zhu,
2001). The chemical potential of the saline solution initially establishes a water potential
imbalance between the apoplast and symplast that leads to turgor decrease, which if severe
enough can cause growth reduction (Bohnert et al., 1995). Processes such as seed germination,
seedling growth and vigour, vegetative growth, flowering and fruit set are adversely affected
by high salt concentration, ultimately causing diminished economic yield and also quality of
produce. In general, electrical conductivity of saturated soil extract (ECe) of 4.0 deciSiemens
per meter (dS-1m) is considered as a lower limit to define saline soils. Rice and most grain
crops are glycophytes that show stress symptoms and reduced yield even when the ECe is
lower than 4.0 dS-1m (~40 mmol/L NaCl). The salinity threshold for rice is 3.0 dS-1 m with a
12% reduction in yield per dS-1 m beyond this threshold (Maas, 1990). Sensitivity of rice to
salinity stress varies with the growth stage. Rice is considered to be relatively more salt
tolerant at germination (Heenan et al., 1988; Khan et al., 1997). Seed germination is not
significantly affected up to salinity level of 16.3 dS m-1 (Heenan et al., 1988). Rice is
particularly sensitive to salinity during the seedling stage, with consequent poor crop
establishment, as well as during reproduction stage, where salinity can severely disrupt grain
formation and yield (Moradi and Ismail, 2007). From an agronomic point of view, tiller
number determined at vegetative stage and numbers of spikelets per panicle determined at
panicle initiation stage have been regarded as the most salinity-sensitive yield components in
rice genotypes (Zeng and Shannon, 2000). Salinity imposes both ionic and osmotic stresses on
plants (Munns et al., 2006). Yamane and coworkers (2003) suggested that in salt-treated rice
plants, the ionic effects induced swelling of thylakoids and the osmotic effects caused the
Effect of Abiotic Stresses on Growth… 119

destruction of chloroplast envelope. According to these workers salt-induced injury in

chloroplasts is dependent on light and that increased production of H2O2 and ·OH are
responsible for the deleterious effects of salt stress on chlorophyll content and chloroplast
Pareek and coworkers (1997) analyzed short-term salt stress-induced subcellular
alterations in undifferentiated leaf cells of rice seedlings. The subcellular changes evoked by
salinity stresses after 4 h were plasmolysis, lysis of the cytoplasm, accumulation of electron-
dense granules in the cytoplasm, distension in the endoplasmic reticulum membranes,
enhanced association of ribosomes with the endoplasmic reticulum, reduction in the number of
mitochondrial cristae, increased cytoplasmic vesiculation as well as disorganization of cell wall
fibrillar material (Pareek et al., 1997). Salt can induce programmed cell death (PCD) in rice
root tip cells (Li et al., 2006). NaCl treatment could lead to specific features of PCD in root
tips, such as DNA ladder formation nuclear condensation and deformation and transferase
mediated dUTP nick end labeling positive reaction, which were initiated at 4 h of treatment
and progressed thereafter. Cytochrome c release from mitochondria into cytoplasm was also
observed, which occurred at 2 h and was prior to the above nuclear events. In very early phase
of PCD, an immediate burst in H2O2 and O2·− production rate was accompanied by two-phase
changes of superoxide dismutases (SOD) and ascorbate peroxidase (APX) activities. A short
period of increase in the activity was followed by prolonged impairment (Li et al., 2006).
Salinity leads to decreased efficiency of photosynthesis. Net photosynthesis was inversely
correlated with the sodium concentration in the rice leaf tissues. There was no evidence of a
threshold effect; net photosynthesis declined linearly with increasing leaf sodium concentration
(Yeo et al., 1985). Dionisio-Sese and Tobita (2000) suggested that the decline in net
photosynthetic rate, measured in terms of CO2 assimilation of the youngest fully expanded leaf
of four rice varieties, might be due to a direct effect of salt on stomatal resistance via a
reduction in guard cell turgor. The relationship between transpiration rate and leaf sodium
concentration paralleled closely that for photosynthesis. Photosynthesis was reduced by half at
a sodium concentration in the leaf which did not reduce the concentration of chlorophyll.
Moradi and Ismail (2007) observed decreased photosynthetic CO2 fixation, Gs and
transpiration rate in rice plants due to salt stress, with greater reduction in the sensitive cultivar
IR29 than in the tolerant lines IR651 and IR632. The tolerant lines IR651 and IR632 had more
responsive stomata that tended to close faster during the first few hours of stress, followed by
partial recovery after a brief period of acclimation. However, in the sensitive line, Gs continued
to decrease for longer duration and with no recovery afterwards. Chlorophyll fluorescence
measurements revealed that non-photochemical quenching increased, whereas the electron
transport rate decreased under salt stress (Moradi and Ismail, 2007). The sucrose metabolizing
enzymes SPS and acid invertase show varying behaviours in activity in crop cultivars differing
in salt tolerance when raised in saline medium (Dubey and Singh, 1999). Nitrate assimilation
is reduced in plants growing in saline medium, however salt tolerant plants tend to maintain
higher N assimilation efficiency than the sensitive plants. Among the enzymes of nitrate and
ammonia assimilation NR and GS are more prone to inhibition under salinity, more inhibitory
effect is observed in salt sensitive cultivars than in tolerant ones when raised under similar
salinity levels (Katiyar and Dubey, 1992; Richharia et al., 1997; Kumar and Dubey, 1999).
Increased rate of proteolysis in salt-stressed rice seedlings and an association of salt-tolerance
ability with higher protease and aminopeptidase activities and lower carboxypeptidase activity
under salinisation were suggested by Dubey and Rani (1990). Varying behaviour of the
120 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

dehydrogenases malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH) is

observed when two sets of rice cultivars differing in salt tolerance are raised in saline medium
(Kumar et al., 2000). Inhibition in the activities of dehydrogenases, NR and GS as well as
reduced photosynthetic activity in salt sensitive rice cultivars due to salinity appear to be key
contributory factors for the decreased growth of plants under saline conditions (Katiyar and
Dubey, 1992; Dubey and Singh, 1999; Kumar et al., 2000).
In lamina of rice 32 proteins were observed to be significantly regulated by salinity and so
far 11 of these proteins were identified by tandem mass spectrometry. An increase in the level
of eight proteins, including RUBISCO activase and ferritin, occurred by 24 h of exposure to
NaCl (50 mM) and it continued to increase during the following 6 d. Only one protein, a
putative phosphoglycerate kinase, was found to increase in expression within 24 h and it did
not increase over a longer period of exposure to salt. There were also proteins that showed no
change after 24 h exposure to salt. In such cases an increased SOD or decreased S-adenosyl-L-
methionine synthetase was observed after 7 d salt treatment (Parker et al., 2006). As rice is
most sensitive to salinity during the reproductive stage, proteomic approach was employed to
further investigate the mechanism of plant responses to salinity at an early reproductive stage.
The expression pattern of 13 proteins significantly changed in all panicle sizes in response to
stress. These proteins were involved in several salt responsive mechanisms which may
increase plant adaptation to salt stress including higher constitutive expression level and up-
regulation of antioxidants, up-regulation of proteins involved in translation, transcription,
signal transduction, and ATP generation (Dooki et al., 2006). The signaling processes in plants
that initiate cellular responses to biotic and abiotic factors are believed to be located in the
plasma membrane (PM). A better understanding of the PM proteome response to
environmental stresses might lead to new strategies for improving stress-tolerant crops.
Comparative two-dimensional electrophoresis revealed that 24 proteins were differentially
expressed in response to salt stress. These include protein related to regulation of PM pumps
and channels, membrane structure, oxidative stress defense, signal transduction, protein
folding, etc. (Sahar et al., 2007). Studies have suggested a critical role of protein
phosphorylation in salt stress response in plants. However, the phosphoproteome in rice,
particularly under salinity stress, has not been well studied. Chitteti and Peng (2007) studied
rice phosphoproteome differential expression under salt stress. Seventeen differentially
upregulated and 11 differentially downregulated putative phosphoproteins were identified.
Further analyses indicated that 10 of the 17 upregulated proteins are probably upregulated at
post-translational level instead of the protein concentration. While eight of them are known
salt stress responsive proteins, many of them have not been reported earlier in the literature
(Chitteti and Peng, 2007).
Results suggest that in response to high salinity stress in rice, various genes get
upregulated, the products of which are involved either directly or indirectly in plant protection.
Some of the genes encoding enzymes of antioxidant defense system, osmolytes, polyamines,
ion channels and receptors are able to confer salinity-tolerance phenotypes when transferred to
sensitive plants. Overall, the sensitivity or tolerance to high salinity in plants is a coordinated
action of multiple stress responsive genes, which also cross talk with other components of
stress signal transduction pathways. High salinity exerts its negative impact mainly by
disrupting the ionic and osmotic equilibrium of the cell. Mechanisms of salinity tolerance
involve sequestration of Na+ and Cl− in vacuoles of the cells, blocking of Na+ entry into the
cell, Na+ exclusion from the transpiration stream, and some other mechanisms that help in
Effect of Abiotic Stresses on Growth… 121

salinity tolerance. Understanding these mechanisms of stress tolerance, along with a plethora
of genes involved in the stress signaling network, is important to improve high salinity stress
tolerance in crops plants (Tuteja, 2007).

c) Chilling

Chilling and freezing temperatures are major constraint that limits the productivity and the
geographical distribution of many important crops species including rice (Yan et al., 2006).
Poor seedling vigor and fertility, and consequent reductions in yield have been major problems
provoked by cold conditions. In rice, chilling sensitivity is one of the main features of the
genotypic diversity among varieties, and is one of the major factors limiting its production at
high altitude and in temperate or subtropical regions. Rice can perceive chilling stress signals
by putative sensors and can transmit them to the cellular machinery by signal transduction to
regulate gene expression. Through regulation of transcription, RNA processing, translation,
and protein processing, changes in the abundance and activities of functional proteins involved
in redox homeostasis, photosynthesis, photorespiration, and metabolisms of carbon, nitrogen,
sulfur and energy occur in rice plants. Redox homeostasis can also act as signals. These
processes might work cooperatively to establish a new cellular homeostasis under chilling
stress (Yan et al., 2006). Cold tolerance during germination is important for ensuring fast and
uniform establishment of rice crop early in the season (da Cruz et al., 2006). For rice,
temperatures lower than 20°C decrease both the speed and percentage of germination
(Yoshida, 1981) and result in lower crop stands and higher production costs. The first visible
symptom of rice seedling damage caused by chilling is wilting, and prolonged low
temperatures result in complete dehydration of the leaves. It is thought that transpiration
exceeds the rate of water absorption through the root system under low temperature conditions,
causing a loss of turgor in leaf tissues (Kabaki and Tajima, 1981; Kawakami et al., 2008).
Chilling during male gametophyte development in rice inhibits development of microspores,
causing male sterility. Changes in cellular ultrastructure observed under mild chilling include
microspores with poor pollen wall formation, abnormal vacuolation and hypertrophy of the
tapetum and unusual starch accumulation in the plastids of the endothecium in post-meiotic
anthers. Anthers observed during tetrad release also show callose (1,3-β-glucan) wall
abnormalities as shown by immunocytochemical labelling. Expression of rice anther specific
monosaccharide transporter (OsMST8) is greatly affected by chilling treatment (Mamun et al.,
Rolling of leaves, increased relative electrolyte leakage and decreased net photosynthetic
rate are observed in rice plants subjected to chilling treatment (Yan et al., 2006). Chilling
induced higher electrolyte leakage in rice leaves was also reported by Bertin and coworkers
(1996). Chilling temperatures resulted in light-dependent loss of photosynthetic electron
transport in chilling-sensitive rice varieties. Analysis of in vivo chlorophyll fluorescence in
chilling-sensitive rice suggests that low temperatures cause an increased reduction of the
plastoquinone pool that could result in photoinhibitory damage to the photosystem II reaction
centers. Chilling temperature inhibited protein phosphorylation in rice (Moll et al., 1987).
Jeong and coworkers (2002) observed that when rice leaves were exposed to various low
temperatures with illumination (150 μmol m-2 s-1) for 6 h, the leaf photosynthesis of chilling
122 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

sensititve rice cultivar decreased faster than that of chilling tolerant rice cultivar. The light-
chilling induced differential photoinhibition of photosynthesis between the two cultivars was
suggested to be caused by the photon-activation of PSII but not of PSI, since the potential
quantum yield of PSII followed a similar trend to the changes in photosynthetic rates. Among
various genes associated with sucrose metabolism the transcript levels of SPS significantly
decreased in sensitive rice cultivar by light-chilling stress compared to tolerant cultivar. Based
on these results, Jeong and coworkers (2002) proposed that PSII, not PSI, is the sensitive site
for light-chilling stress in chilling-sensitive rice. Several proteins show enhanced degradation
during chilling stress. The large subunit of photosynthetic enzyme protein RUBISCO showed
19 fragments due to chilling stress (Yan et al., 2006).
Low temperatures affect protein and RNA metabolism in leaves of rice seedlings. Hahn
and Walbot (1989) found that while de novo accumulation of several abundant proteins was
suppressed, some polypeptides were consistently found to be cold-induced. Synthesis of
RUBISCO was drastically reduced after 7 days of cold treatment. Using immunoprecipitation
of RUBISCO, evidence was obtained that the suppression was greater for the small subunit
(over 90%) than for the large subunit (80%), indicating a partial loss of coordination in their
synthesis. Cold-sensitive rice cultivars responded with drastic changes in the protein synthesis
pattern when compared to cold-tolerant varieties under cold stress. Chloroplast functions are
disturbed during cold stress. The levels of chloroplast-encoded mRNAs, especially
photosystem I reaction center polypeptide (psaB), and of the nuclear encoded chlorophyll a/b
binding protein decreased drastically in the cold. One nuclear gene known to be induced by
water stress and ABA (Rab21) was also found to be induced by cold treatment (Hahn and
Walbolt, 1989). Rice plants exposed to low temperatures showed a decrease in CO2
assimilation rate without photoinhibition, and increases in the fraction of thermal dissipation in
PSII and in the electron flux through the water-water cycle (WWC). Hirotsu and coworkers
(2004) concluded that although low temperature led to a decrease in CO2 assimilation, rice
potentially could cope up with the excess light energy by increasing the thermal dissipation
and the electron flux of WWC under low temperature irrespective of leaf development and
genotypes (Hirotsu et al., 2004).
To investigate the responses of rice to cold stress, changes in protein expression were
analyzed using a proteomic approach. Slight changes in stress responsive proteins were clearly
displayed, and four proteins were newly detected after cold stress. From identified proteins, it
was concluded that proteins related to energy metabolism were up-regulated and defense-
related proteins were down-regulated in leaf blades by cold stress (Hashimoto and Komatsu,
2007). A few days of cold treatment (< 20°C) at the young microspore stage induced severe
pollen sterility and large grain yield reductions. In an experiment using cold-sensitive cultivar
Doongara and relatively cold-tolerant cultivar HSC55 it was observed that the abundance of 37
anther proteins changed more than 2-fold after 1, 2, and 4 days of cold treatment in cv.
Doongara. Among these, one protein was newly induced, 32 protein spots were up-regulated,
and four protein spots were down-regulated. Of these 37 protein spots, two were identified as
anther-specific proteins (putative lipid transfer protein and Osg6B) and a calreticulin that were
down-regulated and a cystine synthase, a β-6 subunit of the 20 S proteasome, an H protein of
the glycine cleavage system, cytochrome c oxidase subunit VB, an osmotin protein
homologue, a putative 6-phosphogluconolactonase, a putative adenylate kinase, a putative
cysteine proteinase inhibitor, ribosomal protein S12E, a caffeoyl-CoA O-methyltransferase and
a monodehydroascorbate reductase (MDHAR) that were up-regulated (Imin et al., 2006).
Effect of Abiotic Stresses on Growth… 123

Rice is more chilling sensitive at the onset of microspore release. Chilling treatment at this
stage causes male sterility. The gene expression profile during the microspore development
process under chilling stress revealed that as many as 160 cDNAs were up- or down-regulated
due to chilling during the microspore release stage. RT-PCR analysis of 5 genes OPDAR (12-
oxo-phytodienoic acid reductase), SAMDS (s-adenosyl methionone decarboxylase), Radc (rice
anther down-regulated by chilling), OsSalT (salt tolerant protein) and Act1 (Rice actin 1)
confirmed the microarray results (Yamaguchi et al., 2004). Roots are regarded as highly
sensitive organs to abiotic stresses in plants. To gain a better knowledge of the chilling stress
responses of plants, it is imperative to analyze the tissue-specific proteome patterns under
chilling stress. Proteomic analysis of the rice roots is an important step towards
characterization of differentially expressed proteins and elucidation of the mechanisms
underlying the stress effects (Hashimoto and Komatsu, 2007). Lee and coworkers (2009)
identified a number of chilling stress-responsive proteins such as aconitate hydratase, glycine
dehydrogenase, heat shock protein (HSP-70), calreticulin precursor, oxalyl-co-A
decarboxylase, ubiquitin conjugation enzyme, enolase, cysteine synthase, and cytoplasmic
MDH, which had also been previously identified in the proteomic analysis of rice or other
plant species in response to cold stress. A group of novel proteins were also identified
including acetyl transferase, phosphogluconate dehydrogenase, NADP-specific isocitrate
dehydrogenase, fructokinase, putative α-soluble N-ethylmaleimide-sensitive factor (NSF)
attachment protein, and glyoxalase. These proteins are involved in several cellular processes,
including energy production and metabolism, vesicular trafficking and detoxification etc. (Lee
et al., 2009).
Plants respond to low temperature through an intricately coordinated transcriptional
network. C-repeat binding factors (CBFs) also known as dehydration-responsive element
binding proteins (DREB) are transcription factors in plants involved in response to low
temperature. The CBF/DREB-regulated network of genes has been shown to play a prominent
role in freezing tolerance through the process of cold acclimation (CA). DREB1/CBF cold-
responsive pathway is conserved in rice and the DREB1-type genes are quite useful for
improvement of stress tolerance to low temperature in transgenic rice plants (Ito et al., 2006).
Cheng and coworkers (2007) used rice cultivars with wide contrast in chilling sensitivity
between indica and japonica rice as model to identify other regulatory clusters by integrative
analysis of promoter architecture (ab initio) and gene expression profiles. A hypothetical
model of an ROS-mediated regulon (ROS-bZIP-as1/ocs) triggered by chilling stress was
assembled in rice. Based on these results, it appears that this regulon is independent of ABA
and CBF/DREB, and that its activation has an important contribution in configuring the rapid
responses of rice seedlings to chilling stress.
Various genes encoding osmolytes, polyamine, regulatory proteins, including mitogen-
activated protein kinases, calcium-dependent protein kinases, and 14-3-3 proteins have been
shown to be up-regulated in response to low temperature. In addition, families of transcription
factors, the CBF/DREB1 proteins, have been identified in rice that control the expression of a
regulon of cold-induced genes that increase plant freezing tolerance.
124 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

d) Heat

As sessile organisms, plants are constantly exposed to changes in temperature rhythm.

Temperatures high and hot enough for sufficient time cause irreversible
damage to plant function and development and lead to extensive agricultural
losses (Mittler, 2006). With the predicted increase in global air temperature induced by the
greenhouse effect, plant responses to increasing temperatures have become a major area of
concern (Gunderson et al., 2000; Rustad et al., 2001). Heat stress disturbs cellular homeostasis
and can lead to severe retardation in growth and development and even death. The heat stress
response is characterized by inhibition of normal transcription and translation, higher
expression of heat shock proteins (HSPs) and induction of thermotolerance. If stress is too
severe, signaling pathways leading to apoptotic cell death are also activated (Krishna, 2003).
Many physiological factors could be involved in heat stress injury. For example, heat stress
inhibits photosynthesis, limits partitioning of carbohydrates and can damage cell membranes
leading to cell death (Abernethy et al., 1989; Liu and Huang, 2000; Tang et al., 2007).
High temperature stress adversely affects growth and development of rice plants. Izumi
and coworkers (2007) observed decline in rice yield when mean air-temperature exceeded
28°C (Izumi et al., 2007). A variable degree of increase in germination percentage, seedling
growth in terms of root and shoot lengths, water uptake, respiration and activities of hydrolytic
enzymes viz. α-amylase, adenosine triphosphatase (ATPase) and phytase and a positive
correlation between seedling vigour and hydrolase activities were observed in rice seedlings
treated for 30 min at 50°C compared to control. The treatment for 30 min at 60°C, on the other
hand, elicited a retarding influence on these characters. As regards the enzyme activities, the
damaging effect of 60°C could, however, be visualized only after 72 h of germination which
was preceded by an enhancement during the early hours (Mukherjee et al., 1971). The
subcellular changes in rice plant provoked by heat stress after 4 h of treatment were lysis of the
cytoplasm, accumulation of electron-dense granules in the cytoplasm, distension in the ER
membranes, enhanced association of ribosomes with the endoplasmic reticulum, reduction in
the number of mitochondrial cristae, as well as disorganization of cell wall fibrillar material.
Discontinuity in the plasma membrane with close association of the osmiophilic granules were
observed in response to high temperature (Pareek et al., 1997). Flowering (anthesis and
fertilization) and to a lesser extent booting (microsporogenesis) are more sensitive stages in the
life cycle of rice plants to high temperatures (Farrell et al., 2006). Rice is grown mainly in
tropical and subtropical zones and a high temperature at flowering can induce floret sterility
and limit grain yield (Matsushima et al., 1982). The effect of high temperature at anthesis on
spikelet fertility was studied on IR64 (lowland indica) and Azucena (upland japonica) rice
varieties at different tissue temperatures. In IR64, high temperature increased the number of
spikelets reaching anthesis, whereas in Azucena numbers were reduced. In both genotypes ≤ 1
h exposure to ≥ 33.7°C at anthesis caused sterility (Jagadish et al., 2007). Percentage fertility is
negatively correlated with the number of cell layers that separated the anther locule from the
lacuna that formed between the septum and the stomium. Anther dehiscence requires the
rupture of the cell layers. Matsui and Omasa (2002) concluded that the tight closure of the
locules by the cell layers delayed locule opening and decreased fertility at high temperatures in
rice (Matsui and Omasa, 2002). Tang and coworkers (2008) observed a marked decrease in
pollen activity, pollen germination and floret fertility in rice due to high temperature treatment,
however, the high temperature tolerant rice cv. Shanyou 63 showed a much slower rate of
Effect of Abiotic Stresses on Growth… 125

decrease than the high temperature sensitive cv. Teyou 559. A possible correlation between
pollen viability/floret sterility and high temperature-caused changes in indole-3-acetic acid
(IAA), gibberellic acids (GAs), ABA, free proline and soluble protein contents was observed
by Tang and coworkers (2008). High temperature during the grain-filling stage causes
deleterious effects on the yield and quality of crop products (Peng et al., 2004). Temperature
above certain growth-optimal temperatures impairs dry matter production, generally
decreasing grain size in all major cereal crops, such as rice. Such small grains result in not only
decreased yield but also low milling quality. For japonica cultivars of rice, temperatures higher
than 26°C render chalky grain appearance as well as reduction of grain weight (Yamakawa et
al., 2007). To elucidate the effect of high temperature on grain-filling metabolism, Yamakawa
and coworkers (2007) exposed developing rice caryopses to high temperature (33°C/28°C) or
control temperature (25°C/20°C) during the milky stage. Comprehensive gene screening
revealed that several starch synthesis-related genes, such as granule-bound starch synthase I
(GBSSI) and branching enzymes, especially BEIIb, and a cytosolic pyruvate orthophosphate
dikinase gene were down-regulated by high temperature, whereas those for starch-consuming
α-amylases and HSPs were up-regulated. Biochemical analyses of starch showed that the high
temperature-ripened grains contained decreased levels of amylose and long chain-enriched
amylopectin, which might be attributed to the repressed expression of GBSSI and BEIIb,
respectively. SDS-PAGE and immunoblot analysis of storage proteins revealed decreased
accumulation of 13-kD prolamin, which is consistent with the diminished expression of
prolamin genes under elevated temperature (Yamakawa et al., 2007).
Stomatal diffusive conductances of 35°C treated rice plants were found to be significantly
higher than 25°C treated plants (Yang and Heilman, 1991). Relationship between
photosynthetic responses to high temperature at the whole-plant level and sensitivity of light
reactions, the most labile systems, to brief heat treatment was determined by Al-Khatib and
Paulsen (1999). Photosynthetic rates of protoplasts and chloroplasts from all rice species
decreased after being treated in vitro with high temperature. Photosystem II (PSII) activity
declined steadily in protoplasts, chloroplasts, and thylakoids of rice when treated from 22 to
42°C. The results suggest that differences in photosynthetic responses to high temperature are
associated with light reactions. Several components of the photosynthetic apparatus and
associated metabolic processes are heat labile. At a temperature of 40°C, specific activity of
RUBISCO and the titre of RUBISCO holoenzyme are increased or remained unaffected, while
at 45°C, the specific activity and holoenzyme level were more stable in the tolerant cultivar
than in the sensitive one. In both cultivars, a decline in activity and holoenzyme level with
time was pronounced at 50°C (Bose and Ghosh, 1995). Photosystem PSI and PSII mediated
photoreactions of thylakoids isolated from the seedlings exposed to high temperature did not
differ significantly from the thylakoids isolated from control seedlings (25°C). The high
irradiance induced loss in PSII activity and increased lipid peroxidation measured in terms of
malondialdehyde production was more rapid in thylakoids isolated from stressed seedlings as
compared to that of control seedlings. Thus the thylakoids isolated from the stressed seedlings
were more prone to photodamage than those from the controls (Vani et al., 1996).
Effects of high temperature on amylose concentration and amylopectin fine structure in
endosperm were found to depend on the genotypes of rice studied. High temperature caused a
reduction in amylose concentration and an increase in the short chain (CL< 22) proportion of
amylopectin for cv. Jia 935 (showing low amylose concentration); while opposite was true for
cv. Jia 353 (showing high amylose concentration). Accumulation rate of amylose was
126 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

significantly and positively correlated with glycine betaine accumulation for Jia 935, but not
for Jia 353. Amylose accumulation was also significantly and positively correlated with the
activities of starch-debranching enzyme (SDBE), starch branching enzyme (SBE), ADP-
glucose pyrophosphorylase and sucrose synthase (SS) for both varieties. Rice grain quality is
also affected with the environmental temperature it experiences. At higher temperature the
decreased activity of starch branching enzyme reduces the branching frequency of the
branches of amylopectin, which results in the increased amount of long chains of amylopectin
of endosperm in rice grain at high temperature (Jiang et al., 2003).
Proteins are essential to rice caryopsis development and quality formation. High
temperature is an important environmental factor, which may decrease grain quality. Rice
caryopsis proteins profiled by two-dimensional polyacrylamide gel electrophoresis showed 70
proteins that were differentially expressed. Total of 54 proteins were identified with known
functions. Of these, 21 were involved with carbohydrate metabolism, 14 with protein synthesis
and sorting, and 9 with stress responses. High temperature decreased the expression of waxy
(Wx) proteins, allergen-like proteins, and elongation factor 1β, but increased the expression of
small heat shock proteins (sHSPs), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and
prolamin. sHSPs formation was positively correlated with the appearance of chalky kernels.
During development, glutelins were phosphorylated and glycosylated, indicating that these
molecules were post-translationally modified (Lin et al., 2005). Rice leaf proteome in response
to heat stress was investigated by Lee et al. (2007). Increased relative ion leakage and lipid
peroxidation was observed which suggested that oxidative stress was frequently generated in
rice leaves exposed to high temperature. Among 73 differentially regulated proteins under heat
treatment, total of 48 proteins were identified. These proteins were categorized into classes
related to HSPs, energy and metabolism, redox homeostasis, and regulatory proteins. A group
of low molecular weight HSPs was newly induced by heat stress (Lee et al., 2007). Four of the
differentially accumulated proteins that corresponded to antioxidant enzymes were analyzed at
the mRNA level and it was confirmed that increased gene expression occurred for these
proteins. Expression of a group of proteins called heat shock proteins HSPs is increased when
the plants are exposed to elevated temperatures. These HSPs have diverse range of molecular
weights ranging from 20-30 kDa to high 120 kDa.
In order to cope up with heat stress, plants tend to develop various mechanisms, such as
maintenance of membrane stability, scavenging of ROS, production of antioxidants,
accumulation of compatible solutes, induction of mitogen-activated protein kinase (MAPK)
and calcium-dependent protein kinase (CDPK) cascades, as well as most importantly
chaperone signaling and transcriptional activation. All these mechanisms, which are regulated
at the molecular level, enable plants to thrive under heat stress (Wahid et al., 2007). Enhanced
production and accumulation of free and conjugated polyamines as well as increased activities
of their biosynthetic enzymes in rice plants have been associated with heat stress (Roy and
Ghosh, 1996).

e) Metal Toxicity

Metal contamination is a major global environmental problem, threatening the health of

wildlife, humans and plants (Salt et al., 1998; Taiz and Zeiger, 1998; Shah and Dubey, 1998a;
Sharma and Dubey, 2007). Metal concentrations in soil range from less than 1 mg/kg (ppm) to
Effect of Abiotic Stresses on Growth… 127

as high as 100,000 mg/kg (Blaylock and Huang, 2000). Rice plants exposed to high levels of
metals in the soil environment typically accumulate these metals in their tissues (both in roots
and shoots), often to toxic levels that decrease growth and alter metabolic functions (Shah and
Dubey, 1995, 1998b; Verma and Dubey, 2003; Jha and Dubey, 2004a; Liu et al., 2007;
Maheshwari and Dubey, 2007; Sharma and Dubey, 2007). Absorbed metal has been shown to
be distributed in an organ specific manner with its localization greater in roots than in shoots
indicating that the roots are the primary sites of metal accumulation and only small quantities
of metals are transported or accumulated in the shoots (Shah and Dubey, 1995, 1998b; Verma
and Dubey, 2003; Jha and Dubey, 2004a; Maheshwari and Dubey, 2007; Sharma and Dubey,
2007). Metals like Cd, Cu, Pb, As, Al and Ni have been reported to decrease the germination
percentage of rice seeds and reduce the length of root as well as shoot of rice seedlings (Shah
and Dubey, 1995, 1998b; Verma and Dubey, 2003; Jha and Dubey, 2004a; Ahsan et al., 2007;
Maheshwari and Dubey, 2007; Sharma and Dubey, 2007). Muramoto (1989) suggested the
order of metal toxicity to rice plants is CdO>ZnO>PbO. Concentration of 10,000 ppm Cd in
the form of CdO is the critical one towards rice plant. By contrast, such damage was not
observed at even higher levels of ZnO and PbO and hence these metals were considered to
have low toxicity toward rice plant (Muramoto, 1989).
A wide array of metabolic alterations is seen in metal stressed plants. Rice plants grown in
nutrient medium containing metals show a significant decrease in water content as a
consequence of the stress (Ahsan et al., 2007; Llamas et al., 2008). The plasma membrane of
root cells constitutes the first barrier between cytoplasm and metal in the soil and therefore
plasma membrane gets rapidly affected by metals. Addition of Ni2+ to the solution bathing the
roots induced a concentration-dependent PM depolarization but the activity of the PM-H+-
ATPase was not inhibited by the presence of Ni2+. In the long term (days) a drastic loss of K+
was observed in roots and shoots, which could be responsible for the changes in the water
content measured, since stomatal conductance and the transpiration rate remained unaffected
by Ni2+ treatment. The effects induced by Ni2+ were not permanent and could be reverted, at
least in part, by transferring the plants to a medium without Ni2+ (Llamas et al., 2008). Rice
plants grown in presence of Cd, Pb, Al show increased generation of ROS and oxidative stress
marked by increased lipid peroxidation and protein oxidation (Shah et al., 2001; Verma and
Dubey, 2003; Sharma and Dubey, 2007; Ahsan et al., 2008).
Metals perturb carbohydrate metabolism and impair their partitioning in growing rice
plants. The effect of increasing concentrations of Cd, As and Al in situ on the content of
starch, sugars and activity behaviour of enzymes related to their metabolism were studied in
rice seedlings (Verma and Dubey, 2001; Jha and Dubey, 2004b; Mishra and Dubey, 2008a).
All three metals in the growth medium caused an increase in the contents of starch, total sugars
as well as reducing sugars in roots as well as shoots of the rice seedlings. The activities of the
enzymes of starch hydrolysis α-amylase, β-amylase declined, whereas the activities of sucrose
hydrolyzing enzymes sucrose synthase (SS) and acid invertase increased in the rice seedlings
due to these metals. The enzyme of sucrose synthesis, SPS showed decreased activity in Cd,
As as well as Al treated seedlings compared to controls. Enhanced activity of starch
phosphorylase enzyme was observed in As stressed rice seedlings (Jha and Dubey, 2004b).
Results indicated that these metals in rice seedlings caused perturbations in carbohydrate
metabolism leading to the accumulation of soluble sugars by altering enzyme activities in the
rice seedlings. Under metal toxicity sucrose synthase possibly appears to play a positive role in
synthesis of sucrose (Jha and Dubey, 2004b). A marked decline in nitrogen assimilation due to
128 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

inhibition in the activities of the nitrate assimilatory enzymes nitrate reductase (NR), nitrite
reductase (NiR) and glutamine synthethase (GS) is observed in rice seedlings subjected to As
toxicity, whereas an increase in the transamination reactions marked by elevated activities of
alanine and aspartate aminotransferases is observed under such conditions (Jha and Dubey,
2004a). Inhibition in the activities of N assimilatory enzymes accompanied with decreased
affinity of the enzymes towards their substrates is suggested to eventually lead to a marked
suppression of N assimilation and impaired growth of rice seedlings in As polluted
environment (Jha and Dubey, 2004a). Shah and Dubey (1995) observed increase in RNase
activity in rice seedlings due to moderate Cd treatment level of 100 μM, whereas higher Cd
level of 500 μM was inhibitory to the enzyme. Maheshwari and Dubey (2007) suggested that
nickel toxicity in rice seedlings suppresses the hydrolysis of RNA and proteins by inhibiting
the activity of ribonuclease (RNase) and protease, respectively. Suppression of proteolytic
activity marked by decreased activities of protease and peptidase was observed due to Cd
treatments in germinating rice seeds leading to altered levels of protein and amino acids (Shah
and Dubey, 1998b). An increase in the level of RNA, proteins and proline accompanied with a
decline in the level of free amino acid pool was observed in rice seedlings under As
supplementation compared to controls (Mishra and Dubey, 2006). Arsenic caused marked
decline in activities of RNase, protease and leucine aminopeptidase (LAP) whereas the activity
level of carboxypeptidase was enhanced (Mishra and Dubey, 2006). Cd impairs phosphorus
metabolism in plants (Shah and Dubey, 1998c). The increased concentration of As led to the
decrease in phosphate content in plant organs (Milivojevic et al., 2003; Mishra and Dubey,
2008b). Mishra and Dubey (2008b) suggested that exposure of rice plants to arsenite leads to
lowering of the phosphate pool and alteration in the activities of key phosphohydrolytic
enzymes which might contribute to metabolic perturbations and decreased growth of rice
plants in an As (III) polluted environment. Tissue specific inhibition of the activities of
phosphatases both under in situ and in vitro conditions has been observed due to Cd in
growing plants (Shah and Dubey, 1998c). Decrease in activity as well as synthesis of acid
phosphatase isoforms in embryoaxes of Cd-treated germinating rice seeds limit the energy
need of germinating seeds thereby decreasing the vigour of establishing seedlings (Shah and
Dubey, 1997b). A decline in the level of total phosphate pool along with inhibition in the
activities of phosphorolytic enzymes acid phosphatase, alkaline phosphatase and inorganic
pyrophosphatase appear to be one of the key reasons for decreased metabolic activity and
inhibited growth of rice plants grown under high Cd levels (Shah and Dubey, 1998c).
Drastic changes in high-resolution two-dimensional electrophoresis protein patterns of rice
leaf after treatment with Cu, Cd and Hg over control were found, including changes in the
morphology of the leaf segments. Changes in the major leaf photosynthetic protein, RUBISCO
(both suppression and fragmentation), and induction of synthesis of some of the proteins are
reported under metal toxicity. Most of the proteins showed homology to RUBISCO protein,
and some to defense/stress-related proteins, like the pathogenesis related class 5 protein
(OsPR5), the probenazole-inducible protein (referred to as the OsPR10), SOD, and the oxygen
evolving protein. Results indicated a highly specific action of some of these metals in
disturbing the photosynthetic machinery, as evidenced by prominent reductions/fragmentation
of the major photosynthetic protein RUBISCO (Hajduch et al., 2001).
Seed germination is a complex physiological process in plants that can be affected
severely by excessive level of metals. Protein profile alternations during the germination stage
following exposure to Cd has been studied. Seeds exposed to wide range of Cd concentration
Effect of Abiotic Stresses on Growth… 129

revealed increased Cd accumulation in seeds and increased lipid peroxide content, whereas
germination rate, shoot elongation, biomass and water content decreased significantly. When
temporal changes of the total proteins were investigated by two-dimensional electrophoresis,
21 proteins including proteins involved in defense and detoxification, antioxidant, protein
biosynthesis and germination processes were found to be upregulated at least 1.5-fold in
response to Cd stress (Ahsan et al., 2007). Very little information is available regarding As
stress-elicited changes in plants at the proteome level. Arsenic treatment to rice plants resulted
in increases of As accumulation, lipid peroxidation, and in vivo H2O2 contents in roots. A total
of 23 As-regulated proteins including predicted and novel ones were identified. Results
suggested that S-adenosylmethionine synthetase (SAMS), cysteine synthase (CS), glutathione
S transferases (GSTs) and glutathione reductase (GR) presumably work synchronously
wherein GSH plays a central role in protecting cells against As stress (Ahsan et al., 2008).
Gene expression in response to Cu stress in rice leaves was quantified using DNA microarray
and real-time PCR technology. Microarray analysis flagged 305 Cu-responsive genes, and
their expression profile showed that a large proportion of general and defense related genes
were up-regulated under excess Cu conditions, whereas photosynthesis and transport-related
genes were down-regulated. Results indicated that defense-related genes involved in
phytoalexin and lignin biosynthesis were the most sensitive to Cu and that plant management
of abiotic and pathogen stresses has overlapping components, possibly including signal
transduction (Sudo et al., 2008). Rice plants possess a range of potential cellular mechanisms
that may be involved in the detoxification of heavy metals and thus tolerance to metal stress.
These include constitutively high levels or increased synthesis of antioxidant enzymes to cope
up with the oxidative stress caused due to excessive level of metals, release of extracellular
exudates reduced uptake or efflux pumping of metals at the plasma membrane; synthesis of
peptides such as phytochelatins or metallotheionin for chelation of metals in the cytosol, repair
of stress-damaged proteins compartmentation of metals in the vacuole by tonoplast-located
transporters, etc.

f) Gaseous Pollutants

Plants react to changes in the composition of the atmosphere. It is estimated that the levels
of several gaseous components like SO2, NO2, ozone (O3) will increase within the biosphere in
the near future. Plants are much more susceptible for gaseous air pollutants than humans and
animals. Air pollutants penetrate in plants mainly through skin pores. Through the skin pores
gases can penetrate cell walls and get absorbed by the cell fluids. Some of the gaseous air
pollutants directly damage plant leaves when they penetrate into plant cells. This can cause the
leaves of plants and trees to lose their colour, or even leaves die off. It can also cause plant
growth to stagnate. The leaves and stems may slack and curl up. Ozone is one of the major
gaseous pollutants detrimental to crop growth and metabolism (Yang et al., 2008). Ozone
enrichment causes visible injury symptoms and affects photosynthesis, water relationship,
phenology, dry matter production and allocation, leaf membrane protective system and grain
yield as well as its components in rice seedlings (Yang et al., 2008). When adverse effects of
phytotoxic levels of ambient ozone on growth and yield of two rice cultivars were studied a
clear difference in the sensitivity of the two cultivars was found. A yield reduction of 6.3%
was observed in a cultivar MR 185 (p< 0.01) which was largely due to an increase in grain
130 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

sterility, whilst the yield reduction in cultivar MR 84 was not statistically significant (Ishii et
al., 2004). Ozone leads to decrease in dry matter of rice plants. Changes in dry matter can be
accounted due to a decrease or increase in the relative growth rate (RGR). The changes in the
RGR caused by ozone could be mainly attributed to the effect of ozone on the net assimilation
rate. Root/shoot ratio of rice plants was lowered by ozone treatment throughout the exposure
period. Time-course changes in NH4-N root uptake rate were similar to that of the root/shoot
ratio on ozone exposure (Nouchi et al., 1991).
Effects of ozone on rice growth processes were addressed in terms of light use of plants
exposed to ozone in field exposure chambers (Kobayashi and Okada, 1995). While no effects
of ozone on light absorption were found, the light use efficiency was decreased due to ozone
exposure. The effect of ozone on light use efficiency was much greater in the reproductive
than in the vegetative stage (Kobayashi and Okada, 1995). Integrated transcriptomics,
proteomics and metabolomics approaches were applied to investigate the molecular responses
of ozone in the leaves of 2 week old rice seedlings. A total of 1535 nonredundant genes
showed altered expression of more than 5-fold over the control, representing 8 main functional
categories. Genes involved in information storage and processing (10%) and cellular
processing and signaling categories (24%) were highly represented within 1 h of ozone
treatment (Cho et al., 2008). Proteomics analyses identified 23 differentially expressed protein
spots (21 nonredundant proteins) in rice leaves exposed to ozone for 24 h compared to
respective control. Identified proteins were found to be involved in cellular processing and
signaling (32%), photosynthesis (19%), and defense (14%). Metabolomic profiling revealed
accumulation of amino acids, gamma-aminobutyric acid, and GSH in ozone exposed leaves
until 24 h over control. This systematic survey showed that ozone triggers a chain reaction of
altered gene, protein and metabolite expressions involved in multiple cellular processes in rice
plants (Cho et al., 2008). Ozone induced synthesis of defense/stress-related proteins in the
leaves of two-week-old rice seedlings, as evidenced by high-resolution two-dimensional
electrophoresis. These inductions by ozone were preceded by very early (within minutes) and
specific changes in the phosphorylation status of proteins, including the appearance of new
phosphoproteins, over the unchanged filtered pollution free air control. Furthermore, a protein
of approximately 66-kDa in leaf extracts showed strong and specific cross-reaction with an
anti-MAPKinase (ERK1) antibody, and whose levels increased within 5 min of ozone
exposure, over its decrease in control, which suggests possible involvement of ERK-type
MAPKs in the ozone-elicited self-defense response pathways in rice (Agrawal, 2002).
Moreover, in-gel kinase assay revealed rapid activation of a 48-kDa myelin basic protein-
phosphorylating activity by ozone in seedling leaves over control. It is concluded that the
activation of kinase-signaling cascades downstream of ozone perception in rice seedlings
might be involved in as self defense responses (Agrawal, 2002).
Exposure of rice plants to low concentrations of O3 and SO2 singly and in combination
showed foliar injury of different levels. The maximum leaf injury was noted in case of O3+SO2
treated plants and the minimum in O3 treated ones. Also the reductions in chlorophyll a,b and
total chlorophyll and carotenoid contents in leaves exposed to O3+SO2 mixtures were higher
than the reduction noted in case of each individual pollutant. Thus the results suggest existence
of a synergism between O3 and SO2 related to plant injury, especially with respect to
chlorophyll and carotenoid contents of rice (Agrawal et al., 1982). Highly damaging effect of
air pollutant SO2 and SO2 triggered multiple events linked with defense/stress response in the
leaves of rice seedlings (Rakwal et al., 2003). Distinctive reddish-brown necrotic spots and
Effect of Abiotic Stresses on Growth… 131

interveinal browning were observed on the leaf surface after exposure to SO2, over control,
partly reminiscent of the hypersensitive reaction lesions. Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and immunoblotting analysis revealed strong induction of
ascorbate peroxidase (APX) and changes in cysteine proteinase inhibitors (‘phytocystatins’)-
like proteins. Employing classical two-dimensional electrophoresis followed by amino acid
sequencing, induced accumulation of a pathogenesis-related (PR) class 5 (OsPR5) protein,
three PR 10 class proteins (OsPR10s), a novel ATP-dependent CLP protease and an unknown
protein was observed. Subsequent northern analysis showed accumulation of OsPR5 and
OsPR10 transcripts in leaves. Finally, mass spectrometry analysis revealed a strong production
of phytoalexins, sakuranetin and momilactone A in SO2 stressed leaves (Rakwal et al., 2003).
Various mechanisms of tolerance to ozone have been suggested. Stomatal regulation is
important in controlling gas influx into the leaf mesophyll and can help to exclude ozone from
entering the leaves (Fiscus et al., 2005). Moreover, several ROS defense systems exist in
plants, which can counteract oxidative damage caused by ozone. Among these defense systems
are the ROS scavenging enzymes SOD, CAT and peroxidases, as well as a network of low
molecular mass antioxidant compounds, such as AsA, GSH, phenolic compounds, and
tocopherols (Blokhina et al., 2003). Activities of the enzymes SOD, APX, GR and POD were
significantly higher in a sensitive rice cultivar TN 1 than in the tolerant cultivar TNG 67
subjected to ozone treatment (Lin et al., 2001). It is suggested that the genotypic variation for
these tolerance mechanisms can be exploited for breeding ozone tolerant crop varieties (Frei et
al., 2008).

g) Anaerobiosis

Plants depend on the supply of molecular oxygen from their environment in order to
support respiration and various other life-sustaining oxidations reactions (Vartapetian and
Jackson, 1997). Exposure to oxygen deficits is more widespread in biological systems than it is
commonly believed. Since excessively wet soils are common in large areas of the world, poor
soil aeration is an important practical problem facing both agriculture and forestry. Soil
flooding, or more complete submergence, is the most common environmental cause of oxygen
deprivation for vascular plants. Flooding is a recurrent phenomenon in several lowland rice-
growing areas in India and elsewhere. Rice is the only major crop plant that can grow well in
flooded conditions. However, rice plants are severely injured when submerged totally in water
for several days (Ram et al., 2002; Jackson and Ram, 2003). Nearly 25% of the world’s rice
(i.e. 38 million hectares) is cultivated in the rainfed lowland ecosystem. However, the produce
from rainfed lowland ecosystem accounts for only 17% of the global rice supplies (Mohanty et
al., 2000). Growth and survival during submergence of rice is affected by partial (hypoxia) or
complete loss (anoxia) of O2. Reduced supply of O2 and CO2 as well as reduced C2H4 diffusion
limit respiratory activities, photosynthesis and have a negative impact on elongation and
growth of rice plants. Submergence of plants inhibits aerobic respiration and photosynthesis
and stimulates a variety of responses that can enhance survival, such as a switch from aerobic
to anaerobic respiration. Rice seeds can germinate in the complete absence of oxygen. Under
anoxia, the rice coleoptile elongates, reaching a length greater than that of the aerobic one.
Two cultivars (‘FR13A’ and ‘Kurkaruppan’) already known to tolerate submergence, differed
little from submergence-intolerant ‘IR42’ in their relative growth rate and soluble carbohydrate
132 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

concentration during submergence (Jackson et al., 1987). Complete submergence almost

stopped the accumulation of dry matter, depressed soluble carbohydrate concentration by over
75% and promoted chlorosis in fully expanded leaves. Increase in fresh weight by the shoots
was not impaired. Extension by the youngest visible leaf was stimulated but extension by the
next leaf to appear was retarded by submergence. However, underwater leaf extension of
cultivar ‘FR13A’ and ‘Kurkaruppan’ was less than in ‘IR42’. Greater leaf extension and
chlorosis of submerged plants could be attributable to accumulated ethylene (Jackson et al.,
Compared with coleoptiles from air-grown seedlings, anaerobically grown coleoptiles had
depressed cytochrome oxidase activity and a much lower capacity for respiratory oxygen
uptake. Since a high crista density develops in rice coleoptile mitochondria with a very much
depressed cytochrome oxidase activity, there is no obligate correlation between crista density
and cytochrome oxidase activity in this tissue (Opik, 1973). Mitochondrial structures appear to
proliferate in rice seedlings even when plants are grown under anoxic conditions from dry
seed. Fox and Kennedy (1990) found that the activities of the tricarboxylic acid cycle enzymes
succinyl-CoA synthase and citrate synthase were similar in aerobically and anaerobically
grown seedlings, whereas the activities of 2-oxoglutarate dehydrogenase complex, aconitase,
isocitrate dehydrogenase, and fumarase were reduced in anaerobic seedlings. The rate of
succinate oxidation and succinate dehydrogenase activity have been reported to be lower in
mitochondria from rice seedlings grown submerged but increased during adaptation to air.
When in rice seedlings the function of mitochondria under 6 days of anoxia following 1 day of
air adaptation were compared with mitochondria isolated from 7-day aerobically grown
seedlings, it was observed that the mitochondria isolated from anoxia grown seedlings respired
very slowly compared to air-adapted and air-grown seedlings. Activity analysis showed that
respiratory oxidases markedly increased in activity during the air adaptation of seedlings. The
abundance of cytochrome c1 oxidase complex, assembled b/c complex, total heme content,
cytochrome absorbance spectra, and the electron carrier cytochrome c increased markedly on
air adaptation. These results likely reflect limited heme synthesis for cytochrome assembly in
the absence of oxygen and represent a discrete and reversible blockage of full mitochondrial
biogenesis in the anoxia-tolerant rice species (Millar et al., 2004).
Irrespective of tolerance class, decreased soluble protein concentrations was observed
under submergence condition at all sampling times. Pyruvate decarboxylase (PDC) activity
was slightly higher in submergence intolerant lines, compared with tolerant lines, under both
dark submergence and anoxia. Such differences in PDC activity between the two groups of rice
lines were not observed when they were submerged under the natural diurnal cycle. Increased
PDC activity in roots at night demonstrated a probable incidence of tissue hypoxia or anoxia
during submergence during each dark period (Mohanty and Ong, 2003). Although most cereal
roots cannot elongate under anoxic conditions, primary roots of three-day-old rice seedlings
are able to elongate during anoxia. Kato-Noguchi (2004) showed that rice roots are able to
utilize the set of enzymes (SS, glucokinase, fructokinase, PDC, alcohol dehydrogenase)
involved in the metabolism of soluble sugars under anoxia. The ability to maintain an active
fermentative metabolism for production of ATP by fueling the glycolytic pathway with
fermentable carbohydrate is probably greater in hypoxic-pretreated (H-PT) than in non-
pretreated (N-PT) roots (Kato-Noguchi, 2004). Results indicate that sucrose synthase is a
typical anaerobic protein in rice (Ricard et al., 1991). Sucrose synthase activity increased in 2
day old rice seedlings subjected to anaerobic stress. Significantly higher steady-state levels of
Effect of Abiotic Stresses on Growth… 133

SS mRNA, as determined by northern blots and the ability of total RNA to direct in vitro
synthesis of SS, were induced by anaerobic treatment. Analysis of run-on transcripts showed
increased transcription of SS genes as early as 60 minutes after initiation of anaerobic stress.
Anaerobic condition prevents aerobic respiration so that plant survival becomes dependent
on fermentative glycolysis which replaces the Krebs cycle as the main source of energy
(Crawford and Braendle, 1996; Vartapetian and Jackson, 1997). Fermentative glycolysis is
accompanied by the accumulation of a number of metabolites. The major fermentative
metabolites are ethanol, lactate and alanine, all derived from pyruvate, the end-product of
glycolysis (Ricard et al., 1994; Drew, 1997; Tadege et al., 1998). Although there is still much
to learn about the biochemical and molecular basis of anaerobic rice germination, the ability of
rice to maintain an active fermentative metabolism (i.e. by fuelling the glycolytic pathway with
readily fermentable carbohydrates) is certainly crucial (Magneschi and Perata, 2009). Relative
importance of ethanolic, lactate and alanine fermentation pathways was determined in
coleoptiles of rice seedlings subjected to anoxic stress by Kato-Noguchi (2006). The in vitro
activities of alcohol dehydrogenase, pyruvate decarboxylase and alanine aminotransferase in
the coleoptiles increased due to anoxia, whereas no significant increase was observed in lactate
dehydrogenase activity. Kato-Noguchi (2006) suggested that potential carbon flux from
pyruvate to ethanol may be much greater than the potential flux from pyruvate to lactate and
alanine in rice coleoptiles during anoxia (Kato-Noguchi, 2006). The ethanol concentration in
the coleoptiles was correlated with anoxia tolerance with respect to the ATP concentration and
coleoptile elongation. These results suggest that the ability to increase ethanolic fermentation
may be one of the determinants in anoxia tolerance of rice coleoptiles (Kato-Noguchi and
Morokuma, 2007).
Effects of anoxia on the levels of free-amino acids were investigated in the coleoptiles of
rice seedlings. Rice coleoptiles are able to grow in extremely low oxygen conditions. Anoxic
stress increased the concentration of total free-amino acids in the coleoptiles (Kato-Noguchi
and Ohasi, 2006). Alanine (Ala) and γ-aminobutyric acid (GABA) were the main amino acids
which accumulated. Since Ala and GABA are bio-compatible solutes and their accumulation is
known to stabilize osmotic potential and/or cytoplasmic pH in plant cells, these stress-induced
amino acids may allow rice coleoptiles to make biochemical adjustment that enable them to
cope with the stress conditions. Therefore, the ability to increase the concentrations of Ala and
GABA may be important for anoxic and submergence stress tolerance of rice seedlings (Kato-
Noguchi and Ohashi, 2006).
When seven day old rice seedlings were subjected to anaerobic stress, only minor changes
in the pattern of proteins were observed in the shoots, whereas disappearance of many protein
bands was observed in the roots. Three anaerobic stress proteins (ANPs; 36-, 40- and 87-kD
protein) were selectively induced in both roots and shoots of the seedlings, and 36-kD ANP
was identified as the glycolytic enzyme, GAPDH by limited N-terminal amino acid
sequencing. Activities of GAPDH in the shoots and roots increased due to stress of over 24 h
and were 3.4- and 6.2-fold greater than those in non-stressed seedlings at 24 h. These results
suggest that anaerobiosis induces the production of ANPs including GAPDH in the seedlings,
which may allow the seedlings to survive under stress condition (Kato-Noguchi, 2000).
Sugars appear to play a signaling role under anoxia, with several genes indirectly up-
regulated by anoxia-driven sugar starvation. Analysis of the effects of anoxia on the expansin
gene families revealed that EXPA7 and EXPB12 are likely to be involved in rice coleoptile
elongation under anoxia. Genes coding for ethylene response factors and HSPs are among the
134 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

genes modulated by anoxia in rice. Genes coding for some enzymes requiring oxygen for their
activity were dramatically down-regulated under anoxia, suggesting the existence of an energy
saving strategy in the regulation of gene expression (Lasanthi-Kudahettige et al., 2007). Gene
expression profiles of submergence tolerant rice cv. FR13A and sensitive IR39595-503-2-1-2,
when compared after submergence stress using differential display reverse transcriptase-
polymerase chain reaction (DDRT-PCR), 42 differential display bands were revealed from the
submergence tolerant variety, four of them showed high homology with genes related to a
water stress response: genes encoding ATP-binding protein, isocitrate dehydrogenase, NADH
dehydrogenase and terminal acetyltransferase, respectively. The remaining three fragments
were novel cDNA fragments (Chen et al., 2007).
Deep-water rice cultivars can diminish flooding stress by rapid elongation of their
submerged tissues to keep up with the rising waters. Aerenchyma and aerenchymatous
adventitious roots are formed that facilitate oxygen diffusion to prevent anaerobic conditions in
the submerged tissues. Aerobic as well as irrigated lowland rice genotypes grown under well-
watered (control) and waterlogged soil conditions for 30 days were found to be tolerant to
waterlogging because of their comparable abilities to enhance aerenchyma that effectively
facilitated O2 diffusion to the roots for maintaining root growth and dry matter production
(Suralta and Yamauchi, 2008). The ability of roots to resist waterlogging stresses might have
strong implications for the adaptation of rice growing in environments with fluctuating soil
water regimes (Suralta and Yamauchi, 2008). Two important factors influencing rice plant
survival during submergence are limitations to gas diffusion under water, and reduced
irradiance that impair photosynthesis and efficient utilization of carbohydrates. Thus, survival
during submergence may largely depend on accumulation of high carbohydrate concentrations
prior to submergence and a capacity for maintaining energy production through rapid alcoholic
fermentation under oxygen shortage. During flash flooding, a third factor thought to affect
survival is the aerobic shock during the post-submergence period when floodwaters recede.
Changes in the level of antioxidants and enzymes such as SOD suggest that tolerant rice
cultivars develop protective systems to air after exposure to hypoxic or anoxic environments.
In rice the two main strategies are to elongate and escape, or not to elongate and conserve
resources. For rainfed lowland rice exposed to flash flooding, elongation growth during
complete submergence has major adverse effects on survival, since this competes with
maintenance processes which require carbohydrates and energy (Ram et al., 2002).

h) UV-B Radiation

Ultraviolet-B (UV-B, 280-320 nm) radiation on plants is a major concern to plant

biologists due to its threat to productivity in global agriculture. The depletion in stratospheric
ozone has prompted renewed efforts in assessing the potential damage to plant and animal life
due to enhanced levels of solar UV-B radiation (Caldwell, 1998; Casati and Walbot, 2004;
Yannarelli et al., 2006). The enhanced UV-B radiation is detrimental to growth, development,
yield and quality of some crop plants including rice (Teramura and Ziska, 1996; Vass, 1997).
Enhanced UV-B radiation can affect sensitive species or cultivars by inhibition of
photosynthesis, DNA damage, decrease of pollen germination and tube growth, changes in
morphology, phenology, and biomass accumulation and partitioning (Caldwell et al., 1998;
Feng et al., 2003).
Effect of Abiotic Stresses on Growth… 135

Plant height, leaf area, dry weight, net assimilation rate (NAR), and relative growth rate
(RGR) are significantly affected due to UV-B treatment in rice cultivars. Changes in plant
height and leaf area induced by UV-B can alter the rice plant canopy structure (Dai et al.,
1992). Tiller number, dry mass, panicle number, grain yield and grain size of rice was found to
decrease significantly under elevated UV-B radiation. Among grain storage proteins, glutelin
content significantly increased but albumin-globulin and prolamin contents did not. It was thus
evident that not only grain size but also grain storage protein of rice was markedly influenced
due to elevated UV-B radiation (Hidema et al., 2005). Rice plants exposed to UV-B exhibited
significantly reduced dry matter production (total plant and shoot), shoot height, leaf blade
length and total leaf area, increased number of tillers, and greater weight fractions in leaf
blades and roots. For most cultivars, the relative effects of UV-B on shoot morphology were
greater than the effects on biomass production (Barnes et al., 1993).
Supplementation of visible radiation with UV-B radiation resulted in reduced amounts of
total leaf nitrogen, chlorophyll, soluble protein and RUBISCO in rice leaves (Hidema et al.,
1996). It has been shown that under realistic UV-B conditions, reduction in the levels of
RUBISCO and other enzymes of the Calvin cycle as well as photoinhibition are the primary
cause for the decline in photosynthetic rate in higher plants as well as in aquatic photosynthetic
organisms (Baker et al., 1997; Sinha and Häder, 2002; Abdullaev et al., 2005). Compared with
control plants, the content of soluble protein and RUBISCO protein decreased significantly
after the UV-B treatment in rice leaves (He et al., 1993). UV-B radiation inactivates
photosystem II (PS II) (Biswal et al., 2006), oxygen evolving complex (OEC) (Hideg et al.,
1993), quinone component (Melis et al., 1992) and D1-D2 reaction centre protein complex
(Friso et al., 1994a, b). Marked decrease occurred in the ratios of variable to maximum
chlorophyll fluorescence yield and in the quantum yield of photosynthetic oxygen evolution in
rice leaves (He et al., 1993).When the effects of supplementary UV-B radiation on the changes
in synthesis and degradation of RUBISCO and light-harvesting chlorophyll a/b binding protein
of PSII (LHCII) as well as mRNA levels for small and large subunits of RUBISCO (rbcS and
rbcL, respectively) and LHCII (cab) were examined with leaf age in UV-sensitive (Norin 1)
and UV-resistant rice (Sasanishiki) varieties, synthesis of RUBISCO but not LHCII was
significantly suppressed by UV-B in Norin 1 (Takeuchi et al., 2002). The degradation of
RUBISCO was enhanced by UV-B around the time of leaf maturation in both the rice
cultivars. The levels of rbcS and rbcL were reduced by UV-B at the early stages after leaf
emergence in both cultivars. Cab transcripts were first present at high levels in the two
cultivars, but drastically decreased due to UV-B treatment immediately after leaf emergence in
Norin 1. RUBISCO synthesis was significantly suppressed by supplementary UV-B radiation
at the transcription step during the early leaf stages. It is suggested that the difference in UV-B
sensitivity in RUBISCO synthesis between the two rice cultivars is due to specific suppression
both transcriptionally and post-transcriptionally (Takeuchi et al., 2002).
Free radicals with a long lifetime were observed in the leaves of two rice cultivars
Sasanishiki (UV-B resistant) and Norin-1 (UV-B sensitive), by electron spin resonance (ESR)
spectroscopy. The leaves of both cultivars grown with visible light showed very similar ESR
spectra composed of radical 1 (R1) and radical 2 (R2), which could be attributed to the
formation of P700 cation radicals in the reaction center of photosystem I and tyrosine cation
radicals in the reaction center of photosystem II, respectively. The ESR spectrum composed of
R1 and R2 radicals in the leaves of Sasanishiki grown under visible light with supplemental
UV-B was similar to that in the plant grown without supplemental UV-B. On the other hand,
136 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

the amount of R2 radicals in the leaves of Norin-1 grown under visible light with supplemental
UV-B was significantly smaller than that in the plant grown without supplemental UV-B. It is
suggested that the loss of R2 radicals in Norin-1 upon UVB irradiation is related to the
instability of the plant (Kumagai et al., 1999). Effect of UV-B radiation on stomatal density
and opening was determined to test whether the stomatal response to UV-B was associated
with different sensitivity of growth to UV-B in four cultivars. Ten-day-old seedlings of IR 45
and IR 74 (UV-B sensitive), and IR 64 and IR 30 (UV-B less sensitive), were subjected to UV-
B radiation. Under 4-week UV-B exposure, leaf area and plant dry mass of IR 45 and IR 74
were significantly reduced. Stomatal density decreased in all cultivars, except in IR 64. Greater
reduction of stomata on the adaxial surface than on the abaxial surface under 3 and 4 weeks of
UV-B exposure suggested a direct effect of UV-B radiation on stomata. Difference in plant dry
mass between UV-B treated and control plants could be significantly correlated with the
reductions in stomatal opening and density on adaxial surface under UV-B treatment (Dai et
al., 1995).
The impact of elevated UV-B radiation on membrane systems and lipid peroxidation
indicated a significant increase in O2·−) generation, H2O2 content, malondialdehyde (MDA)
concentration and relative electrolyte conductivity (EC) in the two rice cultivars IR 74 and
Dular due to effective UV-B (UV-BBE) treatment (Dai et al., 1997).This indicates disruption of
membrane systems as an eventual reason for UV-B induced injury in rice plants. There was a
positive correlation between O2·−) generation and increases in EC or MDA in leaves. Activities
of CAT and SOD (but not APX) and concentrations of ascorbic acid and GSH were enhanced
by UV-BBE after 14 days of UV-BBE exposure. Further, exposure to 28 days of UV-BBE was
associated with a decline in enzyme activities and ascorbic acid, but not GSH. It is suggested
that UV-BBE-induced injury may be associated with disturbance of active oxygen metabolism
through the destruction and alteration of both enzymatic and nonenzymatic defense systems in
rice (Dai et al., 1997).
Plants have evolved defense mechanisms against UV radiation. There are two basic
strategies. One is the accumulation of UV absorbing compounds (Caldwell et al., 1983; Bharti
and Khurana, 1997) and the other is the development of an efficient DNA repair mechanism
(Britt, 1999). It is suggested that the difference between cultivars in the resistance to UV-B
radiation might be due to the differences in the levels of RUBISCO and in UV-absorbing
compounds that are induced by UV-B radiation (Hidema et al., 1996). Although UV absorbing
compounds are effective in reducing cyclobutane pyrimidine dimer (CPD) induction in plants
exposed to a challenge exposure to UV-B, certain levels of CPD are maintained constitutively
in light conditions containing UV-B, regardless of the quantity or presence of visible light.
These findings imply that the systems for repairing DNA damage and scavenging ROS are
essential for plants to grow in light conditions containing UV-B. CPD photolyase activity is a
crucial factor determining the differences in UV-B sensitivity between rice cultivars. Teranishi
and coworkers (2004) examined the correlation between UV-B sensitivity and CPD photolyase
activity in 17 rice cultivars of progenitors and relatives in breeding of UV-resistant Sasanishiki
and UV-sensitive Norin 1. Results suggested that cultivars more resistant to UV-B exhibited
higher photolyase activities than less resistant cultivars and that single amino acid alteration
from glutamine to arginine leads to a deficit of CPD photolyase activity (Teranishi et al.,
2004). These findings open up the possibility of increasing the resistance of rice to UV-B
radiation, by selective breeding or bioengineering of the genes encoding CPD photolyase
(Hidema and Kumagai, 2006).
Effect of Abiotic Stresses on Growth… 137



Rice feeds about one half of the world’s population, mainly in Asia, Africa, and South
America. Increases in annual rice production are no longer keeping pace with the growth in the
number of consumers (Sasaki and Burr, 2000). Rice production in the next fifty years faces
even greater challenges, so the development of new cultivars with enhanced abiotic stress
tolerance will undoubtley have an important effect on the global food production. Abiotic
stresses induce a wide range of physiological and metabolic changes in rice plants. These
changes apparently adaptive, include a host of biochemical pathways associated with signal
perception, transduction and regulation of gene expression in a temporal and spatial pattern.
The advances in physiology, genetics and molecular biology have greatly improved our
understanding of the responses of rice plants to these stresses. In rice plants these adaptations
are dependent on the activation of cascades of molecular networks involved in stress
perception, signal transduction and the expression of stress related genes and metabolites.
Figure 3 shows the components associated with abiotic stress tolerance in rice plants. Abiotic
stresses induces expression of stress responsive genes which lead to accumulation of stress
related proteins and metabolites like osmolytes, polyamines, organic acids as well as increased
activities of certain enzymes in rice plants. These components which have been discussed in
detail in the following sections can be successfully used as attractive targets to produce abiotic
stress tolerant rice plants using biotechnological approaches (Table 1).

Table 1. Transgenic rice plants produced using some of the components associated
with stress tolerance

Components Gene Tolerance References

cod A
Osmolytes cox Kawakami et al., 2008
Proline, otsA Su et al., 2006
Glycine betaine, otsB Su and Wu, 2004
Trehalose, OsTPP1 Low temperature Sakomoto et al., 1998
Sorbitol, mtlD Salinity Mohanty et al., 2002
Mannitol, gutD Drought Garg et al., 2002
Fructans wft1 Wang et al., 2000
wft2 Ge et al., 2008

Putrescine, Capell et al., 2004
adc Drought
Spermine, Roy and Wu, 2001
SAMDC Salinity
Spermidine Roy and Wu, 2002

Organic acids
PEPC Aluminum Osaki et al., 2001
138 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

Table 1. (Continued)

Components Gene Tolerance References

Antioxidants CAT Low temperature Matsumura et al., 2002

Catalase, MnSOD Drought Wang et al., 2005a
Mn-SOD katE Salinity Moriwaki et al., 2008
Xu et al., 1996,
Late embryogenesis abundant PMA80 Drought
Cheng et al., 2002,
proteins PMA1959 Salt stress
Xiao et al., 2007

Katiyar-Agarwal et al., 2003

Hsp101 Heat
Murakami et al., 2004
Heat shock proteins sHSP17.7 Drought
Sato and Yokoya, 2008

nhaA Salinity Wu et al., 2005a

Transport proteins
OsNHX1 Drought Fukuda et al., 2004

OsMAPK44 Salinity Jeong et al., 2006

OsCIPK genes Multiple stresses Xiang et al., 2007
Signaling pathways
OsCDPK7 Cold Ma et al., 2005
calcineurin gene Drought Saijo et al., 2000

ATAF Drought Wang et al., 2008b
CUC (NAC) Salt stress Hu et al., 2006
Transcription factors OsDREB1F Heat Wu et al., 2009
OsWRKY11 Low temperature Wang et al., 2007
OsWRKY89 UV-B Xu et al., 2006

Figure 3. Components associated with abiotic stress tolerance in rice plants. Abiotic stresses lead to
induction of stress responsive genes expression and accumulation of stress specific proteins and
metabolites like polyamines, osmolytes, organic acids in rice plants. These components can be used to
produce abiotic stress tolerant rice plants using plant breeding or genetic transformation using
biotechnological approaches.
Effect of Abiotic Stresses on Growth… 139

a) Osmolytes

The acclimation of plants to a constantly changing environment involves the accumulation

of certain organic compounds of low molecular mass collectively known as compatible
cytoplasmic solutes or osmolytes. These osmolytes fall into three major groups: amino acids
(e.g. proline), quaternary ammonium compounds (e.g. glycine betaine, β-alanine betaine,
dimethylsulfoniopropionate, etc.) and polyols/sugars (e.g. mannitol, trehalose). Recent studies
indicate that compatible solutes besides serving as osmoprotectants also act as free-radical
scavengers or chemical chaperones by directly stabilizing membrane phospholopids, proteins
and enzymes (Rudolph et al., 1986; Diamant et al., 2003; Sharma and Dubey, 2004; Mishra
and Dubey, 2006). Unlike perturbing solutes (such as inorganic ions), which readily enter the
hydration sphere of proteins favouring unfolding, compatible solutes tend to exclude these ions
from the hydration sphere of proteins and stabilize folded protein structures (Low, 1985).
Proposed roles of osmolytes in abiotic stress tolerance in rice plants have been shown in figure
4. Osmolytes can act as osmoprotectant, chemical chaperon, antioxidant, storage compound,
metal chelator, protein stabilizer, enzymes and hence protect DNA, lipid, protein, membrane
etc. against the denaturing effect of abiotic stresses. As rice is not capable of natural
production or accumulation of all of these compounds in response to stresses, extensive
research has been conducted examining various approaches to enhance increased production of
these compounds in rice plants. Engineering increased osmolyte content in transgenic plants is
a promising strategy for protecting plants against various abiotic stresses. Transgenic rice
plants engineered to accumulate proline, mannitol, fructans, trehalose, glycine betaine exhibit
marked improvements in salt and/or drought tolerance (Wang et al., 2000; Su and Wu, 2004;
Su et al., 2006; Ge et al., 2008; Kawakami et al., 2008). Figure 5 shows the biosynthetic
pathway of some of the common osmolytes that have been used to increase abiotic stress
tolerance in rice plants.

Amino Acids
Increase in the content of amino acids during environmental stresses has been correlated
with tolerance to many abiotic stresses in plants. Dubey and Rani (1989) reported significant
increase in the levels of free amino acids with a substantial elevated level of proline in rice
seedlings subjected to salinity treatment of 7 and 14 dS m-1. NaCl induced increase in proline
content in rice leaves was related to proteolysis, an increase in ornithine-δ-aminotransferase
activity, a decrease in proline dehydrogenase activity, a decrease in proline utilisation and an
increase in the content of the precursors of proline biosynthesis, ornithine and arginine (Lin et
al., 2002). Proline accumulation caused by NaCl in detached rice leaves was also associated
with ammonium ion accumulation (Lin et al., 2002). Proline content was observed to increase
in rice seedlings subjected to water stress also. Both drought resistant and sensitive rice
genotypes showed increased proline content when subjected to water stress. The increase was
higher in tolerant genotypes due to increased Δ1-pyrroline-5-carboxylate synthetase (P5CS)
activity (Choudhary et al., 2005). Metal toxicity also leads to increased proline accumulation
in rice plants. With Al treatment level of 160 μM in situ about 180-276% increase in proline
content was observed in roots and shoots of 15 day grown rice seedlings (Sharma and Dubey,
2005a). In rice leaves CuSO4 and CuCl2 were equally effective in inducing proline
accumulation, indicating that proline accumulation is induced by Cu (Chen et al., 2001).
140 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

Figure 4. Proposed roles of osmolytes in abiotic stress tolerance in rice plants. Osmolytes can act as
osmoprotectants, enzyme protectants, antioxidants, storage compounds, metal chelators, protein
stabilizer, chemical chaperons and hence protect lipids, proteins, DNA, membranes etc against the
damaging effects of abiotic stresses.

Figure 5. Biosynthetic pathways of some of the osmolytes which accumulate in rice plants under
stresses. Enzymes used in attempts to increase abiotic stress tolerance in rice plants includes enzymes
involved in the biosynthesis of osmolytes proline, glycine betaine, trehalose, mannitol, sorbitol and
fructan as Δ1- pyrroline-5-carboxylase reductase (P5CR), Δ1- pyrroline-5-carboxylase synthase (P5CS),
glutamate-5-phospho transferase (G5P), betaine aldehyde dehydrogenase (BADH), choline
monooxygenase (CMO), choline dehydrogenase (CDH), trehalose-6-P phosphatase (T6PP), trehalose-6-
P synthetase (T6PS), non specific phosphatase (NPT), mannitol-1-P dehydrogenase (MPDH), sorbitol-6-
P phosphatase (S6PP), aldose-6-P reductase (A6PR), sorbitol-6-P dehydrogenase (S6PD), fructosyl
transferase (FT).
Effect of Abiotic Stresses on Growth… 141

Sulfate salts of Mg, Mn, and Fe were ineffective in inducing proline accumulation in
detached rice leaves. Excess Cu had no effect on relative water content of detached rice leaves,
suggesting that Cu-induced proline accumulation is unlikely due to water deficit. Proline
accumulation induced by excess Cu was related to proteolysis and an increase in Δ1-pyrroline-
5-carboxylate reductase (P5CR) or ornithine-δ-amino-transferase activity and could not be
explained by proline utilization or stress-induced modifications in proline dehydrogenase
(ProDH) or Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities. The content of
glutamic acid was decreased by excess Cu. The increase in arginine but not ornithine was
found to be associated with the increase in proline content in Cu-stressed detached rice leaves
(Chen et al., 2001). There has been little agreement regarding the mechanism by which proline
reduces heavy metal toxicity. Proline supplement to Cu-treated rice seedlings not only reduced
the Cu absorption in the roots but also increased Cu exclusion, suggesting that supplement of
proline accompanied by Cu exposure induces a barrier of Cu influx and efflux in rice roots
(Chen et al., 2001). Wang and coworkers (2009) suggested a protective effect of proline on
Hg2+ toxicity through detoxifying reactive oxygen species, rather than chelating metal ions or
maintaining the water balance under Hg2+ stress (Wang et al., 2009). In vitro experiments
using enzymes extracted from rice leaves have suggested that proline protects enzyme activity
under various stressful conditions. Activity of NR enzyme from rice seedlings appeared to be
sensitive to H2O2, PEG-6000, NaCl and various metal salts. Addition of proline in the enzyme
assay medium caused a considerable protection to the enzyme against the damaging effects of
these stressful components (Sharma and Dubey, 2005a). Similar protective effect of proline on
GS isoforms and RNase extracted from rice has been shown under water stress and metal
toxicity (Kumar and Dubey, 1999; Mishra and Dubey, 2006; Maheshwari and Dubey, 2007).
In plants, proline is synthesized from L-Glutamic acid (L-Glu) by two enzymes, P5CS and
P5CR. L-proline is metabolized to L-Glu by two enzymes, proline dehydrogenase (ProDH)
and P5CDH (Figure 5). It has been reported that P5CS and ProDH are rate-limiting enzymes in
proline synthesis and metabolism of plants under water stress, respectively. Therefore, it is
expected that genetically engineered plants produced by overexpression of P5CS gene or
suppression of ProDH gene overproduce proline and acquire osmotolerance, namely, the
ability to tolerate environmental stresses such as high salinity and drought. Chisako and
coworkers (1999) investigated whether or not transgenic plants with a ProDH antisense cDNA
accumulate proline of high level. Transgenic rice plants were generated with a ProDH
antisense cDNA from Arabidopsis thaliana by Agrobacterium method. Several transgenics
accumulated proline at a significantly higher level than wild type plants under normal growth
conditions. Iyer and Caplan (1998) found that intermediates in proline biosynthesis and
catabolism such as glutamine and Δ1-pyrroline-5-carboxylic acid (P5C) can increase the
expression of several osmotically regulated genes in rice, including salT and dhn4. One
millimolar P5C or its analog 3,4-dehydroproline, produced a greater effect on gene expression
than 1 mM proline or 75 mM NaCl. P5C- and 3,4-dehydroproline-treated plants were found to
consume less O2, had reduced NADPH and increased NADH levels and accumulated many
osmolytes. These experiments indicate that osmotically induced increases in the concentrations
of one or more intermediates in proline metabolism could be influencing some of the
characteristic responses related to osmotic stress tolerance (Iyer and Caplan, 1998).
Su and Wu (2004) compared the growth rate of transgenic rice plants in which the
expression of a mothbean p5cs cDNA was driven separately with a constitutive and a stress-
inducible promoter. They found that both constitutive expression and stress-inducible
142 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

expression of the p5cs cDNA in transgenic rice led to the accumulation of p5cs mRNA and
proline. Third-generation (R2) transgenic rice seedlings showed significantly higher tolerance
to stress produced by high levels of NaCl or water deficiency as judged by faster growth of
shoots and roots in comparison with non-transformed plants. However, stress-inducible
expression of the P5CS transgene showed significant advantages over the constitutive
expression in increasing the biomass production of transgenic rice grown in soil under stress
conditions (Su and Wu, 2004).

Quaternary Ammonium Compounds

These are amino acid derivatives in which the nitrogen atom is fully methylated. In plants,
glycine betaine is a representative member of this group of osmolytes. Glycine betaine not
only acts as an osmoregulator, but also stabilizes the structures and activities of enzymes and
protein complexes, and maintains the integrity of membranes against the damaging effects of
excessive salt, cold, heat and freezing (Gorham, 1995; Sakamoto and Murata, 2002). In vitro
experiments using enzymes extracted from rice leaves suggest that glycine betaine protects
activity of many enzymes under various abiotic stresses. Glycine betaine under in vitro
conditions protects activity of enzyme NR against NaCl, water stress and H2O2 in rice.
Activities of isoforms of GS, namely GS1 and GS2 were also reported to be protected by the
osmolytes proline, glycine betaine and sucrose. Among these three osmolytes, glycine betaine
appeared to be the most suitable protectant for GS (Kumar and Dubey, 1999).
Glycine betaine is synthesized in the chloroplast from choline by a two-step process. The
first step (choline to betaine aldehyde) is mediated by choline monooxygenase (CMO), which
can be induced by drought and salinity (Russell et al., 1998). The second step (betaine
aldehyde to glycine betaine) is catalyzed by the enzyme betaine aldehyde dehydrogenase
(BADH) a NAD+-dependent dehydrogenase (Figure 5). Rice which has two genes for BADH,
does not accumulate glycine betaine because it lacks a functional gene for CMO. Rice plants
accumulate glycine betaine in the presence of exogenously applied betaine aldehyde, which
leads to the development of a significant tolerance to salt, cold and heat stress. Kishitani and
coworkers (2000) reported the transgenic rice plants constitutively expressing precise barley
BADH1 that converted high levels of exogenously applied betaine aldehyde to glycine betaine
more efficiently than did wild-type plants. The lower conversion efficiency in the wild-type
plants probably results from the limitation of precise native BADH proteins found in this
study. Shirasawa and coworkers (2006) reported transgenic rice plants harboring a single copy
of expressed spinach CMO which accumulated detectable glycine betaine and had enhanced
tolerance to salt and temperature stresses. Because CMO alone only converts choline into
betaine aldehyde, these transgenic plants still need native functional BADH proteins for
conversion of betaine aldehyde into glycine betaine. Transgenic lines of indica rice were
generated by Agrobacterium-mediated transformation with the choline oxidase (codA) gene
from Arthrobacter globiformis. A significant amount of choline oxidase product, i.e. glycine
betaine, accumulated in R0 as well as R1 plants. Challenge studies performed with R1 plants
by exposure to NaCl stress (0.15 M) for 1 week, followed by a recovery period, revealed that
in some cases more than 50% of the transgenic plants could survive salt stress and set seed
whereas wild-type plants failed to recover (Mohanty et al., 2002). Several glycine betaine-
producing transgenic rice lines were generated in which the Arthrobacter pascens choline
oxidase (COX) gene, fused to a chloroplast targeting sequence (TP) was expressed under the
control of an ABA-inducible promoter (SIP; stress-inducible promoter) or a ubiquitin (UBI)
Effect of Abiotic Stresses on Growth… 143

gene promoter. Saline growth conditions enhanced glycine betaine accumulation by up to 89%
in the SIP lines, whereas up to 44% increase was seen in the UBI line. In all these cases the
glycine betaine levels were many-fold below the range reported for plant species that produce
glycine betaine naturally. In spite of lower glycine betaine concentrations, statistically greater
levels of stress tolerance were found in SIP lines than in UBI lines, suggesting that the stress
protection observed in SIP plants cannot be totally explained by the increase in the glycine
betaine content (Su et al., 2006).

The accumulation of sugar alcohols may protect plants against environmental stresses. The
accumulation of polyols have been proposed to have dual functions: facilitating osmotic
adjustment and supporting redox control. A significant increase in sucrose content in roots and
shoots of water stressed as well as Al3+ stressed rice seedlings compared to the level in control
seedlings was reported by Sharma and Dubey (2005a). In vitro studies using enzymes
extracted from rice seedlings suggest that sucrose can act as enzyme protectant. In vitro water
stress of -0.5MPa or -2.0 MPa caused a loss in activity of the GS isoforms with an increase in
enzyme Km values. Sucrose provided considerable protection to the enzyme against the
deleterious effect of water stress induced by polyethylene glycol. Similarly activity of NR
enzyme extracted from rice seedlings appeared to be sensitive to H2O2, PEG-6000, NaCl and
various metal salts in vitro. Addition of 1 mol/L sucrose in the enzyme assay medium caused a
considerable protection to the enzyme against the damaging effects of stressful components
(Sharma and Dubey, 2005a).
Trehalose serves as a stress protectant and/or reserve carbohydrate in a variety of
organisms including bacteria, yeast and invertebrates. With the notable exception of the
desiccation-tolerant resurrection plants, trehalose is not thought to accumulate to detectable
levels in most plants. A systematic search of rice databases discovered a large TPS/TPP gene
family in the rice genome, which is similar to that found in Arabidopsis thaliana, especially in
the gene family structure. Expression analysis demonstrated that OsTPP1 was initially and
transiently up-regulated after salt, osmotic and ABA treatments but slowly up-regulated under
cold stress. OsTPP1 overexpression in rice enhanced tolerance to salt and cold stress (Ge et
al., 2008). Regulated overexpression of Escherichia coli trehalose biosynthetic genes (otsA
and otsB) as a fusion gene for manipulating abiotic stress tolerance in rice was reported by
Garg and coworkers (2002). Compared with non-transgenic rice, several independent
transgenic lines exhibited sustained plant growth, less photo-oxidative damage, and more
favorable mineral balance under salt, drought, and low temperature stress conditions.
Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3-10
times that of the non-transgenic controls. The observation that peak trehalose levels remain
well below 1 mg g-1 fresh weight indicates that the primary role of trehalose is not as a
compatible solute. Rather, increased trehalose accumulation correlates with higher soluble
carbohydrate levels and an elevated capacity for photosynthesis under both stress and
nonstress conditions, consistent with a suggested role in modulating sugar sensing and
carbohydrate metabolism. These findings demonstrate the feasibility of engineering rice for
increased tolerance to abiotic stress and enhanced productivity through tissue-specific or
stress-dependent overproduction of trehalose (Garg et al., 2002).
Fructans are water-soluble fructose oligomers and polymers and have been implicated in
protecting plants against water stress. Rice is highly sensitive to chilling temperatures and is
144 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

not able to synthesize fructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose 1-

fructosyltransferase, encoded by wft2, or sucrose:fructan 6-fructosyltransferase, encoded by
wft1, were introduced into rice plants, and rice transformants that accumulate fructans were
successfully obtained. Transgenic rice lines expressing wheat-derived fructosyltransferase
genes accumulated large amounts of fructans in mature leaf blades and exhibited enhanced
chilling tolerance at the seedling stage. This is the first report owing that fructan accumulation
enhanced tolerance to non-freezing low temperatures in rice (Kawakami et al., 2008).
Role of mannitol and sorbitol as stress protectant was investigated. Agrobacterium
tumefaciens mediated integration of mtlD gene (encoding mannitol-1-phosphate
dehydrogenase) and gutD gene (encoding glucitol-6-phosphate dehydrogenase) was done in
the rice genome. Analysis of sugar alcohol showed that transgenic rice plants could produce
and accumulate mannitol and sorbitol. The salt tolerance of transgenic plants was much higher
than that of their controls (Wang et al., 2000). It is suggested that transgenic rice produces and
accumulates mannitol and sorbitol so as to enable itself to absorb water and grow normally
under high osmotic pressure.

b) Polyamines

Polyamines have been shown to be involved in plant stress responses. However, the
precise role(s) of polyamine metabolism in these processes remains ill-defined. Transgenic
approaches demonstrate that polyamines play essential roles in stress tolerance and open up the
possibility to exploit this strategy to improve plant tolerance to multiple environmental stresses
(Alcazar et al., 2006). The diamine putrescine and the polyamines spermidine and spermine
are ubiquitous in nature. Polyamines are abundant polycationic compounds involved in many
plant physiological processes such as cell division, dormancy breaking, plant morphogenesis
and in response to environmental stresses (Garufi et al., 2007). Figure 6 shows the biosynthetic
pathway of some of the common polyamines. The starting point for polyamine biosynthesis is
the basic amino acids ornithine and arginine, which are decarboxylated by ornithine
decarboxylase (ODC) and arginine decarboxylase (ADC), respectively, to yield putrescine.
Putrescine then serves as the substrate for the biosynthesis of spermidine and spermine via the
activities of S-adenosylmethionine decarboxylase and spermidine as well as spermine
synthases (Walters, 2000). The polycationic nature of polyamines, positively charged at
physiological pH, has attracted the attention of researchers and has led to the hypothesis that
polyamines could affect physiological systems by binding to anionic sites, such as those
associated with nucleic acids and membrane phospholipids (Groppa and Benavides, 2008).
Polyamines have been reported to be involved in protein phosphorylation (Ye et al., 1994),
post transcriptional modifications (Mehta et al., 1994), and conformational transition of DNA.
The physiological role of putrescine in abiotic stress responses is a matter of controversy. It
has been very difficult to establish a direct cause-and-effect relationship between increased
putrescine levels in plants and abiotic stress (Capell et al., 2004). Elevated amount of
putrescine might be the cause of stress-induced injury or alternatively, a protective response
resulting from stress (Reggiani et al., 1993).
Rice has a large capacity to enhance polyamines biosynthesis in leaves in response to
stresses. The role of polyamines in plant defense to stresses varies with polyamines forms and
stress stages.
Effect of Abiotic Stresses on Growth… 145

Figure 6. Biosynthetic pathways of diamine putrescine and the polyamines spermidine and spermine.
The enzymes involved in the biosynthesis of putrescine, ornithine decarboxylase (ODC); spermidine, S-
Adenosyl-methionine decarbosxylase (ADC) and spermidine synthase; and spermine, spermine synthase;
have been used in attempts to increase abiotic stress tolerance by their overexpression in rice plants.

In rice germinating under saline condition, increased levels of polyamines spermine and
spermidine was observed (Katiyar and Dubey, 1990). Krishnamurthy and Bhagwat (1989)
compared polyamine accumulation of nine rice cultivars with different salt sensitivity, they
found that the salt-tolerant cultivars accumulated high concentrations of spermidine and
spermine, while the salt-sensitive ones accumulated excessive putrescine and low levels of
spermidine and spermine. However, marked increase in spermine as well as spermidine level
was also observed in seedlings of sensitive rice cultivars under higher level of salinity. In all
cultivars salt stressed seedlings had higher agmatine level compared to non-stressed. Salinity
led to greater accumulation of certain unidentified polyamines in seedlings of sensitive
cultivars. Increased levels of total polyamines, putrescine, spermidine and unknown
polyamines in rice seedlings under salinization suggest their possible role in combating the
adverse effects of salinity stress (Katiyar and Dubey, 1990).
A correlation was found between polyamine and salt stress-induced responses in rice
genotypes when physiological responses of various rice genotypes were studied in relation to
NaCl stress (Basu and Ghosh, 1991). Cultivars CSR-1 and Dular germinated well in different
NaCl regimes compared to cvs. Rupsail, Assam Getu and M-1-48. Cultivars CSR-1 and Dular
were relatively effective in maintaining high concentrations of polyamines as well as arginine
decarboxylase activity in coleoptiles and roots in a non-stressed condition. The activities of
two biodegradative enzymes, diamine oxidase and polyamine oxidase were lowest in cv. CSR-
1. The polyamine content was not significantly altered when seedlings of cv. CSR-1 were
exposed to 100 mM NaCl. However, in cv. M-1-48 enhancement of arginine decarboxylase
activity with concomitant accumulation of polyamines was observed suggesting a correlation
between polyamine and salt stress-induced responses in rice genotypes (Basu and Ghosh,
Yang and coworkers (2007) suggested that the physiological traits like higher levels of
free spermidine/free spermine and insoluble-conjugated putrescine, as well as early
accumulation of free polyamines, could be important for rice crop in adapting to water stress.
146 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

The activities of arginine decarboxylase, S-adenosyl-L-methionine decarboxylase and

spermidine synthase were significantly enhanced in rice leaves under water stress, in good
agreement with the increase in putrescine, spermidine and spermine contents. The
increased contents of free spermidine, free spermine, and insoluble-conjugated putrescine
under water stress were significantly correlated with the yield maintenance ratio (the ratio of
grain yield under water-stressed conditions to grain yield under well-watered conditions) of the
cultivars. Free polyamines showed significant accumulation when leaf water potentials reached
-0.51 MPa to -0.62 MPa for the drought-resistant cultivars and -0.70 MPa to -0.84 MPa for the
drought-susceptible ones (Yang et al., 2007). Enhanced production and accumulation of free
and conjugated polyamines as well as increased activities of their biosynthetic enzymes in rice
plants have also been associated with heat stress. Perchloric acid-soluble free, as well as
conjugated polyamines, and their metabolic enzymes were studied under 45°C heat stress in
callus raised from heat-tolerant and -sensitive rice cultivars. The levels of free and conjugated
polyamines, as well as arginine decarboxylase and polyamine oxidase activities were higher in
tolerant than in sensitive rice callus under non-stressed conditions (Roy and Ghosh, 1996).
Heat stress caused greater accumulation of free and conjugated polyamines and increased
activities of arginine decarboxylase and polyamine oxidase in callus of the heat-tolerant
cultivar (N 22) than in that of the heat-sensitive cultivar (IR 8). In particular, the uncommon
polyamines norspermidine and norspermine were detected in cv. N 22, which increased
appreciably during stress, but these were not detected in callus of cv. IR 8. Increased levels of
transglutaminase activity indicated the high titre of conjugated polyamines (Roy and Ghosh,
1996). Akiyama and Jin (2007) presented the first direct evidence supporting essentially
chilling-specific regulation of a rice ADC gene that also potentially influences putrescine
accumulation, a phenomenon previously noted in cold-stressed rice seedlings. RNA gel blot
analysis revealed markedly increased OsADC1 mRNA levels in rice seedling leaves subjected
to chilling stress. Interestingly, this treatment induced a concomitant increase in free putrescine
levels in these samples, coincident with the observed elevated OsADC1 mRNA levels.
At physiological concentrations spermine and spermidine are found to significantly
prevent the leakage of electrolytes and amino acids from roots and shoots induced by salinity
stress in rice seedlings. To different degrees they also prevent chlorophyll loss, inhibition of
photochemical reactions of photosynthesis as well as downregulation of chloroplast-encoded
genes like psbA, psbB, psbE and rbcL, indicating a positive correlation between salt tolerance
and accumulation of higher PAs in rice. The inhibitory effect of salinity stress and its reversal
by exogenous PAs are more pronounced in the salt-sensitive M-1-48 rice plants than in the
tolerant Pokkali plants (Chattopadhayay et al., 2002). In the case of salt stress, a beneficial
effect of an exogenous polyamine may also be related to the improvement of the ion balance in
salt-treated cells due to its cationic nature (Ndayiragije and Lutts, 2006). Exogenous putrescine
at 1 mM clearly decreased both Na+ and Cl- accumulation of rice calli exposed to salt. Roy and
coworkers (2005) determined the extent to which polyamine mediated restoration of activities
of plasma membrane (PM)-bound enzymes occurs and differs within salt-sensitive and salt-
tolerant rice cultivars. Results showed that nine-fold higher level of H+-ATPase (100%
vanadium sensitive) was detected from PM of roots of salt-tolerant cultivar (Nonabokra) in
comparison to roots of salt sensitive cultivar (M-1-48). Salinity stress alone to the seedlings
significantly reduces the activity of PM-bound H+-ATPase. The activity of H+-ATPase was
restored to some extent in the roots treated with NaCl stress in presence of 1 mM spermidine.
Analysis of PM-bound polyamine from untreated control roots showed only putrescine from
Effect of Abiotic Stresses on Growth… 147

salt sensitive cultivars, whereas roots of salt-tolerant plants had only spermidine and spermine.
Western blot using polyclonal antibody raised against H+-ATPase (PM-bound) of Arabidopsis
thaliana showed NaCl stress-induced decrease and spermidine-induced recovery of 100 kDa
polypeptide (known MW of 100 kDa H+-ATPase from rice). Roy and coworkers (2005)
suggested that accumulation of Na+, loss of K+ ion, salinity stress-induced sharp inhibition of
PM-bound H+-ATPase activity, could be overcome by supplying spermidine exogenously.
Spermidine and spermine, but not putrescine, were found to be effective in reducing CdCl2-
induced toxicity in rice leaves (Hsu and Kao, 2007). Spermidine and spermine were shown to
protect Cd-induced oxidative damage and this protection is most likely related to the avoidance
of H2O2 generation and the reduction of Cd uptake. Spermidine and spermine prevented
CdCl2-induced increase in the contents of H2O2 and malondialdehyde (MDA), decrease in the
contents of ASC and GSH, and increase in the activities of antioxidative enzymes (Hsu and
Kao, 2007).
Arginine decarboxylase (ADC) cDNA from oat (Avena sativa L.) was introduced into rice
genome by an Agrobacterium-mediated transformation method. Expression of the ADC
transgene under the control of an ABA-inducible promoter led to stress-induced upregulation
of ADC activity and polyamine accumulation in transgenic rice plants. Second-generation (Rl)
transgenic rice plants showed an increase in biomass under salinity stress conditions, as
compared to the non-transformed control plants (Roy and Wu, 2001). Capell and coworkers
(2004) demonstrated that the manipulation of polyamine biosynthesis in plants can produce
drought-tolerant germplasm in rice. Transgenic plants expressing Datura adc produced much
higher levels of putrescine under stress, promoting spermidine and spermine synthesis and
ultimately protecting the plants from drought. Further these workers generated transgenic rice
plants expressing the Datura stramonium adc gene and investigated their response to drought
stress. Wild-type plants responded to the onset of drought stress by increasing endogenous
putrescine levels, but this was insufficient to trigger the conversion of putrescine into
spermidine and spermine (the agents that are believed to protect plants under stress). In
contrast, transgenic plants expressing Datura adc produced much higher levels of putrescine
under stress, promoting spermidine and spermine synthesis and ultimately protecting the plants
from drought (Capell et al., 2004). Transgenic plants exhibited less chlorophyll loss and leaf
curling than the wild type (Capell et al., 2004). Roy and Wu (2002) reported that the
introduction of the Tritordeum SAMDC gene into rice resulted in a three to four-fold increase
in spermidine and spermine levels in the transformed plants. These transgenic rice plants
showed normal growth and development even under NaCl stress, which indicated that the
transformants were more stress tolerant than the wild type.

c) Organic Acids

Organic acids are key intermediates in carbon metabolism but can also serve as key
components to cope with metal tolerance. One of the most interesting strategies to ameliorate
the effects of metal toxicity involves formation of complexes with organic acid molecules to
decrease the availability of the metal within the plant cell, thus limiting the toxic effects
(Peterson and Oliver, 2006). Organic acid anions such as citrate, malate, oxalate are potential
ligands for metals and so could play an important role in metal tolerance and their
detoxification (Kochian et al., 2004; Yang et al., 2006). Organic acids have been shown to
148 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

play a key role in Al exclusion via their exudation from the root apex on exposure to Al, and
internal detoxification of symplastic Al by complexation, where organic acids chelate Al and
reduce its toxic effects at the cellular level (Kochian et al., 2004). Hue and coworkers (1986)
suggested a list of acids in the decreasing order of the ability to precipitate Al: oxalic acid >
citric acid > malic acid > succinic acid. Higher levels of citrate effectively alleviate Al-induced
toxicity in Indica rice (Meriga et al., 2003). Seedlings of two Indian rice cultivars (Suraksha
and Vikas) differing in Al sensitivity when grown in Yoshida’s culture solution containing 80
μmol Al in absence and presence of citrate, showed that citrate at a concentration of 200 mM
alleviate the toxic effects of Al in both cultivars of rice, mostly by chelating with the metal.
Phosphoenolpyruvate carboxylase (PEPC) transgenic rice plants, in which the gene of
PEPC of maize was introduced, were tested for Al resistance (Osaki et al., 2001). When the
intact gene of maize PEPC was introduced into the C3 crop rice, PEPC activity in leaves of
transgenic rice plants were 2 to 3 times higher than those in maize. 14C distribution to organic
acids after 5 minutes of 14CO2 assimilation was almost two times higher in transgenic rice
plants than in control plants. Relative growth of control rice plants decreased with increasing
Al application. However, that of transgenic rice plants increased with increase of Al
application. Organic acid-anion exudation from roots of control plants persisted remained
regardless of Al application. However, Al treatment enhanced particularly oxalate exudation
from roots of the transgenic plants. These results are in agreement with the better growth of the
transgenic plants compared to the control in presence of Al. Osaki and coworkers (2001)
suggested that the higher Al resistance of the PEPC transgenic plants is due to enhanced
synthesis of organic acids in the leaves, transport to the roots and exudation from the roots.

d) Antioxidants

Under most of the abiotic stressful conditions such as drought, salinity, heat, chilling,
metal toxicity, increased gaseous pollutants, radiations, etc., many plant species overproduce
ROS. Under such conditions the scavenging system for ROS may lose its function and the
balance between producing and quenching ROS can get disturbed, resulting in oxidative
damage to plant system (Bowler et al., 1992; Halliwell and Gutteridge, 2006; Sharma and
Dubey, 2007). If stress-induced production of reactive oxygen species is not adequately
counter balanced by cellular antioxidnts, oxidative damage of lipids, proteins and nucleic acids
ensues (Halliwell and Gutteridge, 1989; Duval et al., 2002; Sharma and Dubey, 2007).
Antioxidants serve to keep down the levels of free radicals, permitting them to perform
useful biological functions without too much damage to cellular biomolecules and organelles
(Halliwell and Gutteridge, 2006). Enhancement of antioxidant defense in plants can thus
increase tolerance to different stresses. Antioxidants (ROS scavengers) include enzymes such
as CAT, peroxidase (POD), SOD, APX, MDHAR, DHAR and GR as well as non-enzyme
molecules such as ascorbic acid (AsA), glutathione (GSH), carotenoids and anthocyanins.
Table 2 shows the reactions catalyzed by enzymes involved in antioxidative defense system in
rice plants. Efficient scavenging/destruction of reactive oxygen species generated during
abiotic stresses require the action of several antioxidant enzymes. The ascorbate-glutathione
cycle present in at least four different subcellular locations including the cytosol, chloroplast,
mitochondria and peroxisomes, scavenges H2O2.
Effect of Abiotic Stresses on Growth… 149

Table 2. Reactions catalyzed by enzymes involved in antioxidative defense system in rice

plants. Efficient scavenging/destruction of reactive oxygen species generated during
abiotic stresses requires the action of several antioxidant enzymes

Antioxidant Enzymes Reaction catalyzed

Catalase 2H2O2 2H2O + O2

Peroxidase H2O2 + AH2 H2O + A

Ascorbate peroxidase H2O2 + Ascorbate H2O + Monodehydroascobate

Monodehydroascorbate MDHAR
Monodehydroascorbate + NAD(P)H Ascorbate + NAD(P)+

Dehydroascorbate DHAR
Dehydroascorbate + 2GSH Ascorbate + GSSG

Glutathione reductase GSSG + NAD(P)H GR 2GSH + NAD(P)+

Superoxide dismutase O2·− + O2·− + 2H+ H2O2 + O2

In this cycle, AsA and GSH are not consumed, using NADPH as reducing power H2O2 is
reduced to H2O. APX reduces H2O2 to H2O using AsA, which generates
monodehydroascorbate. Monodehydroascorbate radical can be reduced to AsA by MDHAR. If
not reduced rapidly, monodehydroascorbate is disproportionated into AsA and
dehydroascorbate. Dehydroascorbate is reduced to AsA by dehydroascorbate reductase
(DHAR) using GSH as the reducing agent. Oxidized glutathione (GSSG) is in-turn reduced by
GR using NADPH (Figure 7). Additional compounds, such as osmolytes, proteins (e.g.
peroxiredoxin) and amphiphilic molecules (e.g. tocopherol) can also function as ROS
scavengers (Bowler et al., 1992; Noctor and Foyer, 1998).
Increased concentration of O2·−, increased level of lipid peroxidation showing oxidative
stress, chlorophyll bleaching, loss of antioxidants (AsA, GSH, α-tocopherol and carotenoids),
decline in total soluble proteins and thiols were observed in rice seedlings subjected to water
stress (Boo and Jung, 1999; Sharma and Dubey, 2005b; Bai et al., 2006). Increase in the
capacity of AsA regeneration system in rice plants by de novo synthesis of MDHAR, DHAR
and GR is one of the primary responses of plants to water deficit so as to mitigate oxidative
stress (Boo and Jung, 1999; Sharma and Dubey, 2005b). APX serves as an important
component of antioxidative defense system under water stress (Sharma and Dubey, 2005b).
150 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

Figure 7. Components of ascorbate-glutathione cycle play an important role in hydrogen peroxide

decomposition in rice plants under abiotic stresses. This cycle maintains a high level of ascorbate in the
stroma. Monodehydroascorbate (MDHA) is formed by oxidation of ascorbate and is reconverted to
ascorbate via protonation by MDHA reductase (MDHAR) and NAD(P)H. This disproportionation also
forms dehydroascorbate (DHA). DHA is reduced to ascorbate by DHA reductase (DHAR) while reduced
glutathione (GSH) gets converted to oxidized glutathione (GSSG).

In the leaves of rice plants, salt stress preferentially enhanced the content of H2O2 as well
as the activities of SOD, APX, and peroxidase specific to guaiacol, whereas it decreased CAT
activity (Lee et al., 2001)On the other hand, salt stress had little effect on the activity levels of
GR. Expression of Cu/Zn-1, -2, and Mn-SOD-2 isoforms was preferentially enhanced by salt
stress. Out of seven APX isoforms the intensities of APX-4 to -7 were enhanced by salt stress,
whereas those of APX-1 to -3 remained almost uncharged. Lee and coworkers (2001)
suggested that SOD leads to the overproduction of H2O2 in the leaves of rice plants subjected
to salt stress and that the overproduction of H2O2 functions as the signal of salt stress, which
induces the induction of specific APX isoforms but not specific GR isoforms under CAT
deactivation (Lee et al., 2001). In 11-day-old rice seedlings, subjected to salt stress APX,
CatB, GR, SodCc1, and SodCc2 were found to be up-regulated, while CatA, CatC, and
guaiacol peroxidase (GPX) remained unaltered. In 6-week-old plants, higher mRNA levels
were observed for CatB, GR, and SodCc2. Salt had no significant effect on APX, GPX, and
SodCc1. CatA accumulation was significantly impaired. Salt stress was suggested to trigger a
differential modulation of antioxidant transcripts, possibly due to disruption of cell redox
homeostasis (Menezes-Benavente et al., 2004). Anand and coworkers (2006) suggested that
(a) peroxidase enzymes detoxify H2Ο2 under high temperature (b) CAT enzyme scavenges
H2Ο2 when the plant shifts from vegetative to reproductive stage (Anand et al., 2006). Under
chilling stress activities of enzymes SOD, CAT, APX and GR and content of ascorbic acid
increased in seedlings of rice cv. Xiangnuo-1 (chilling tolerant), while in cv. IR-50 (chilling
sensitive) decline was noticed. The results indicated that higher activities of defense enzymes
Effect of Abiotic Stresses on Growth… 151

and higher content of antioxidant under stress were associated with tolerance to chilling
(Huang and Guo, 2005).
Metals like Cd, Pb and Al have been shown to induce oxidative stress in rice plants which
is evident from the increased content of lipid peroxides in the seedlings treated with these
metals (Shah et al., 2001; Verma and Dubey, 2003; Sharma and Dubey, 2007). A concomitant
increase in the activities of the enzymes SOD, GPX, APX and GR was observed in rice
seedlings with increasing concentration of Cd, Pb and Al treatment. APX activity was reported
to increase several fold in response to Cd and Al toxicity. Results suggest that those metals
induces oxidative stress in rice plants and that SOD, POX, GR and APX could serve as
important components of antioxidative defense mechanism against metal induced oxidative
injury (Shah et al., 2001; Verma and Dubey, 2003; Sharma and Dubey, 2007).
The potential role of SOD in the protection against salt stress was examined using
transgenic rice plants (Tanaka et al., 1999). The coding region of the yeast mitochondrial Mn-
SOD gene was introduced into rice protoplasts. The activities of overexpressed Mn-SOD and
cytosolic Cu/Zn-SOD did not change upon salt stress in either the transgenic or control plants,
whereas the chloroplastic Cu/Zn-SOD activity in control rice decreased significantly. At high
salinity, the APX activity of the transformant was about 1.5-fold higher than that in the
control. These results suggest that increased levels of APX and high levels of chloroplastic
SOD in the transformant are important factors for salt resistance in rice (Tanaka et al., 1999).
When the role of Mn-SOD in the drought tolerance of rice was examined by introducing Mn-
SOD from pea (Pisum sativum) into chloroplasts of rice, it was observed that under drought
stress transgenic leaf slices showed reduced electrolyte leakage compared to wild type (WT)
leaf slices, suggesting that transgenic plants were more resistant to PEG-induced oxidative
stress (Wang et al., 2005a). Transgenic plants also exhibited less injury, measured by net
photosynthetic rate, when treated with PEG. Wang and coworkers (2005a) suggested that SOD
is a critical component of the ROS scavenging system in chloroplasts and that the expression
of Mn-SOD can improve drought tolerance in rice. Moriwaki and coworkers (2008) showed
that the simple genetic modification of rice to express E. coli derived CAT can efficiently
increase its tolerance against salt stress. The expression of E. coli CAT gene, katE in BR5
plants caused 150% higher CAT activity than in nontransgenic plants and such transgenic rice
plants exhibited high tolerance to salt stress compared with nontransgenic plants (Moriwaki et
al., 2008). Similarly the expression of wheat CAT cDNA in transgenic rice was shown to
enhance tolerance against low temperature injury (Matsumura et al., 2002).
Non-enzymatic antioxidants include the major cellular redox buffers AsA and GSH, as
well as carotenoids and tocopherol. Alleviation of oxidative injury by the use of antioxidants
can enhance plant tolerance to abiotic stresses. Rice roots were fed with AsA and its putative
precursors to observe AsA and oxalate concentrations and the tolerance of rice plants to
chilling, water stress, and Al toxicity. Ascorbic acid concentration was significantly enhanced
in both shoots and roots of rice seedlings by feeding with D-glucose or L-galactono-γ-lactone
(Guo et al., 2005). Ascorbic acid or L-galactono-γ-lactone treatment increased accumulation of
oxalate mainly in soluble form, while these treatments decreased electrolyte leakage from root
cells, H2O2 and lipid peroxidation level in rice seedlings subjected to chilling, water stress and
Al toxicity. Antioxidant AsA also alleviated the inhibition of root growth by Al. These results
indicate that AsA and its immediate precursors protect plants against the oxidative damages
induced by various stresses. Enhanced Al resistance caused by AsA and L-galactono-γ-lactone
152 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

could possibly be arise from increased level of oxalate, which acts as metal chelator. Thus it is
proposed that manipulation of AsA and oxalate biosynthesis through enhancement of L-
galactono-γ-lactone level in plants could be a strategy for improving abiotic stress tolerance
(Guo et al., 2005). During the antioxidation process, AsA itself is oxidized to
dehydroascorbate; DHAR re-reduces the oxidized ascorbate. A high ratio of reduced to
oxidized ascorbate is important for ROS-scavenging efficiency. Ushimaru and coworkers
(2006) reported that overexpression of rice DHAR1 in Arabidopsis increases AsA levels,
which leads to increased salt tolerance.

e) Stress Induced Peptides and Proteins

Many abiotic stressful conditions lead to transcriptional activation of a large set of plant
genes, which in turn causes accumulation of certain novel proteins or increased synthesis of
pre-existing protein (Skriver and Mundy, 1990; Xu et al., 1996; Sharma and Dubey, 2007). It
is generally assumed that stress-induced proteins could play a role in stress tolerance (Xu et
al., 1996). Heat shock proteins (HSPs), which are synthesized in plants on exposure to high
temperatures, protect plants from damage caused due to heat stress or help repair the damage
caused by stress. All organisms produce HSPs in response to elevation in temperature and
certain other stresses (Lindquist and Craig, 1988). HSPs, many of them also called chaperones
are responsible for protein folding, assembly, translocation and degradation in many normal
cellular processes, stabilize proteins and membranes, contribute to cellular homeostasis and
can assist in protein refolding under stress conditions (Wang et al., 2004). Plant HSPs consist
of a few high-molecular-weight classes and a complex group of low-molecular-weight proteins
with molecular sizes ranging from 15 to 30 kDa (Vierling, 1991). Rice is sensitive to high-
temperature stress at almost all the stages of its growth and development. Rice seedlings
accumulate stainable amounts of the 104 and 90 kDa proteins which accumulate to different
extents on exposure to salinity, water stress, low-temperature treatment and exogenous ABA
application (Pareek et al., 1995). The in vitro polysome translation-products from heat shocked
rice seedlings showed twelve HSPs (Dai et al., 1996). Two full-length cDNA clones, pTS1 and
pTS3, specific for heat-shock proteins were isolated from a rice cDNA library. Both
encoded 16 to 20 kDa class I heat-shock proteins. Heavy-metal stress, in addition to heat stress,
increased the levels of the corresponding mRNAs (Tseng et al., 1993).
Considering the crucial role of HSP-101 in imparting thermotolerance to cells, Katiyar-
Agarwal and coworkers (2003) introduced A. thaliana HSP101 (AtHSP101) cDNA into Pusa
basmati 1 cultivar of rice. The transgenic rice lines showed significantly better growth
performance in the recovery phase following the stress. This thermotolerance advantage
appeared to be solely due to over-expression of HSP101 as neither the expression of low
molecular weight HSPs nor of other proteins was altered in the transgenic rice (Katiyar-
Agarwal et al., 2003).
Small HSPs (sHSPs) represent the major family of HSPs induced by heat stress in plants
(Waters et al., 1996). Rice seedlings subjected to high temperature showed 73 differentially
expressed proteins. A total of 48 proteins were identified. The results showed that a group of
low molecular small sHSPs were newly induced by heat stress. Among these sHSPs, a low
molecular weight mitochondrial (Mt) sHSP was validated further by western blot analysis (Lee
et al., 2007). Small heat shock protein sHSP17.7 was isolated from heated rice seedlings, and
Effect of Abiotic Stresses on Growth… 153

the influence of rice sHSP17.7 expression on the viability of E. coli under heat-shock
conditions was assessed (Murakami et al., 2004). After heating, the survival rate of sHSP17.7
cells was 2-fold higher than that of the control cells. The molecular chaperone activity of
sHSP17.7 was investigated using CAT as a substrate. Recombinant sHSP17.7 cells had heat-
stable chaperone properties that were capable of protecting stressed CAT from precipitation.
Transgenic rice plants with increased levels of sHSP17.7 protein exhibited significantly
increased thermotolerance compared to untransformed control plants. The transgenic rice plant
with the highest constitutive expression of sHSP17.7 had significantly greater resistance to
UV-B stress than untransformed control plants. Increase in the degree of resistance of
transgenic plants to UV-B was accompanied by an increase in production of sHSP17.7 protein
(Murakami et al., 2004). Overproduction of sHSP17.7 could increase drought tolerance in
transgenic rice seedlings (Sato and Yokoya, 2008). At the end of drought treatments, only
transgenic seedlings with higher expression levels of sHSP17.7 protein were found to regrow
after rewatering.
Plants have a number of defense mechanisms for combating the toxicities of many metals.
One such mechanism involves the production of cysteine-rich peptides, such as phytochelatins
(PCs) and metallothioneins (MTs), for detoxification or homeostasis of heavy metals (Cobbett
and Goldsbrough, 2002). PCs are derived from GSH and related thiols in a γ-glutamylcysteinyl
transpeptidation reaction catalyzed by phytochelatin synthases (PC synthases) (Grill et al.,
1989; Rea et al., 2004) and have the general structure (γ-Glu-Cys)n-glycine, where n can vary
from 2 to 11 (Rea et al., 2004). PCs form stable complexes with heavy metals in the cytosol,
and these metal-PC complexes are subsequently sequestered into the vacuole (Zenk, 1996;
Cobbett, 2000). Overexpressing the PC synthase gene in transgenic plants appears to be a
promising approach for developing metal tolerance in plants. From 3 week old rice seedlings,
treated with 50 μM copper sulfate, PC was extracted and purified. Cd was the most effective
stimulator, followed by Pb, Cu, Ag, Co and other divalent cations (Yan et al., 2000). Exposure
of several species of the family Poaceae to cadmium results in the formation of metal-induced
peptides of the general structure (γ-Glu-Cys)n-Ser (n = 2-4) (Klapheck et al., 1994). These
peptides are presumably formed from hydroxymethyl-glutathione (γ-Glu-Cys-Ser) and are
termed hydroxymethyl-phytochelatins (hm-PCs). The hm-PCs were isolated from the roots of
Cd-exposed rice plants. The hm-PCs probably play a significant role in heavy metal
detoxication in rice. In addition to this new form of γ-Glu-Cys (γ-EC) peptide, PCs and γ-EC
peptides without C-terminal Ser or Gly are found. All γ-EC peptides are synthesized in roots as
well as in shoots of rice plants without delay after incubation of plants in 100 μM CdCl2
(Klapheck et al., 1994). To reduce the accumulation of Cd in rice seeds, the expression of
phytochelatin synthase (PCS) gene OsPCS1 was suppressed by RNA interference (RNAi). Cd
accumulation was reduced by about half in the seeds of RNAi rice whereas no apparent
difference of growth appeared between RNAi and wild-type plants. The results suggest that
this new approach can be used to control heavy metal accumulation in seeds of rice crop. Shah
and Dubey (1998a) isolated and characterized a 18 kDa cadmium inducible protein complex
from rice seedlings and suggested that Cd2+ binds possibly with the help of 4-SH groups to this
peptide in mercaptide bonds. This complex, which has a comparatively higher molecular
weight (18 kDa) than conventional phytochelatins, may help in sequestration of excess Cd2+
ions in rice plants.
154 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

MTs are cysteine-rich polypeptides encoded by a family of genes. MTs are low molecular
weight (6-7 kDa), 60-65 amino acid residue long, cysteine (20 molecules)-rich metal-binding
(through mercaptide bonds) proteins (Liu et al., 2000). A novel rice genomic sequence
encoding coding segments homologous to other metallothionein-like genes was isolated from
rice genomic library. This sequence, designated as rgMT (rice genomic metallothionein-like
gene) was found to be differentially expressed in rice plants under different stress conditions.
Excess heavy metals and heat shock, lead to elevated expression of rgMT (Hsieh et al., 1995).
In many eukaryotic and prokaryotic organisms under salinity, water deficit, high
osmolarity and low temperature a group of glycine-rich, hydrophilic proteins known as
hydrophilins or late embryogenesis abundant (LEA) proteins are synthesized in increased
amounts. A number of mechanisms have been proposed to justify the roles of these proteins in
freezing tolerance, salinity tolerance, desiccation tolerance, water replacement, ion
sequestering and membrane stabilization (Close, 1996; Cuming, 1999; Dubey, 1999; Tompa et
al., 2006; Abu-Abied et al., 2006; Tolleter et al., 2007). It has been hypothesized, based on the
correlation of LEA gene expression with physiological and environmental stresses, that LEA
proteins may play a protective role in plant cells under various stress conditions as a protective
measure for the survival of the plant (Chandler and Robertson, 1994).
Cold-regulated gene called Wcs19 encodes a protein of unknown function (WCS19) in
rice, which shares identity with three different groups of LEA proteins LEA3-L1, LEA3-L2
and LEA3-L3 (NDong et al., 2002). Arabidopsis plants transformed with the wcs19 gene
showed a significant increase in freezing tolerance (NDong et al., 2002). Tolleter and
coworkers (2007) observed that a mitochondrial LEA protein (LEAM) when expressed in
seeds interacted with membranes in the dry state and protected liposomes as well as inner
mitochondrial membranes during desiccation (Tolleter et al., 2007). Transgenic rice expressing
wheat LEA genes PMA80 and PMA1959 showed enhanced drought and salt tolerance in
glasshouse tests (Cheng et al., 2002). When a LEA protein gene, HVA1, from barley (Hordeum
vulgare L.) was introduced into rice suspension cells, second generation transgenic rice plants
showed significantly increased tolerance to water deficit and salinity. Xu and coworkers
(1996) found that the extent of increased stress tolerance could be correlated with the level of
the HVA1 protein accumulated in the transgenic rice plants. Thus, LEA genes hold
considerable potential for use as molecular tools for genetic crop improvement toward stress
tolerance. A LEA protein gene OsLEA3-1 was over-expressed in rice to test the drought
resistance of transgenic lines under the field conditions. OsLEA3-1 is induced by drought and
salt but not by cold stress. Drought resistance pre-screening of T1 families at anthesis stage
revealed that the over-expressing families with OsLEA3-S and OsLEA3-H constructs had
significantly higher relative yield (yield under drought stress treatment/yield under normal
growth conditions) than the wild type under drought stress conditions, although a yield penalty
existed in T1 families under normal growth conditions. These results indicate that transgenic
rice with significantly enhanced drought resistance and without yield penalty can be generated
by over-expressing OsLEA3-1 gene with appropriate promoters (Xiao et al., 2007).
Effect of Abiotic Stresses on Growth… 155

f) Other Components (Signaling pathway, Transport proteins, Transcription


Alteration in components associated with signaling pathway may improve stress tolerance
of plants. The generalized scheme of signal transduction implies that the extracellular signal
binds to a transmembrane receptor, which in turn activates GTP-binding proteins. The GTP-
binding protein either regulates a cascade of kinases (MAPKKK, MAPKK, and MAPK;
MAPK stands for mitogen-activated protein kinase) or a G-protein effector, leading to a
change in the level of intracellular signals called second messengers (such as cAMP, cGMP,
protein kinase C, Ca2+-dependent kinases and calmodulin-dependent kinases). Mitogen
activated protein kinase cascade plays a crucial role in various biotic and abiotic stresses.
Identification and characterization of upstream kinases and other regulatory components is
necessary to understand the mechanism of MAPK activation by different external stimuli
(Rohila and Yang, 2007). MAP kinase kinase performs an important function of integrating
upstream signals to mitogen activated protein kinase for further appropriate cellular responses.
Kumar and coworkers (2008) suggested involvement of specific MAP kinase kinase in
different abiotic stress signaling and also possible cross talks that could exist during the
signaling processes. Agrawal and coworkers (2003) characterized a jasmonic acid-inducible
MAPK gene. The weak constitutive mRNA expression of OsBWMK1 (blast and wound
inducible MAPK gene of Oryza sativa L.) was potently enhanced and transiently regulated
within 15-30 min of heavy metals, drought, high salt, high temperature and environmental
pollutants such as ozone and sulfur treatment, suggesting that OsBWMK1 converges diverse
stress signals in rice (Agrawal et al., 2003). These results strongly suggest the physiological
importance of OsBWMK1 in mediating multiple extrinsic signals in rice. Jeong and coworkers
(2006) isolated and characterized a putative rice MAPK gene (designated OsMAPK44).
OsMAPK44 gene. expression in rice cv. Pokkali (salt resistant) was slightly up-regulated
within 30 min and then disappeared rapidly, while cv. IR64 (salt sensitive) maintained its
expression for 1 h following down-regulation. Under salinity stress, OsMAPK44
overexpression in transgenic rice plants showed less damage and greater ratio of potassium and
sodium than OsMAPK44 suppressed transgenic lines did, suggesting that OsMAPK44 may
have a role to prevent damages due to salinity (Jeong et al., 2006).
Cyclin-dependent protein kinases (CDKs) form a conserved superfamily of eukaryotic
serine/threonine protein kinases which require binding to a regulatory cyclin for activity.
Huang and coworkers (2008) proposed that CDKs may be involved in the salt stress signaling
in rice. The rice CDKC1 transcript was shown to be induced by salt stress. OsSIPK gene
(salicylic acid-induced protein kinase gene of Oryza sativa L.) can be implicated in salt
responsive signaling cascades and transcription of certain genes (Lee et al., 2008). A time
course (30 to 120 min) experiment using salt stress revealed that the OsSIPK mRNA is
strongly induced by sodium chloride. OsSIPK protein was found to be localized in the nucleus.
Calcium-dependent protein kinases (CDPKs) belong to the family of serine/threonine kinases.
In situ detection of the transcript and immunolocalization of rice Ca2+-dependent protein
kinase, OsCDPK7 revealed that OsCDPK7 was expressed predominantly in vascular tissues of
crowns and roots, vascular bundles and central cylinder, respectively, where water stress
occurs most severely. Similar localization patterns with stronger signals were observed in
stress-tolerant OsCDPK7 over-expressing transformants. Over-expression of OsCDPK7
enhanced induction of some stress-responsive genes in response to salinity/drought, but not to
156 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought
tolerance are different from each other. It seems likely that at least two distinct pathways
commonly use a single CDPK, maintaining the signalling specificity through unknown post-
translational regulation mechanisms. These results demonstrate that simple manipulation of
CDPK activity has great potential with regard to plant improvement (Saijo et al., 2000). The
transcript of a putative target gene of the OsCDPK7 signaling pathway, rab16A, was also
detected essentially in the same tissues upon salt stress, suggesting that the OsCDPK7 pathway
operates predominantly in these regions (Saijo et al., 2001). OsCDPK7 gene was transferred
into rice via Agrobacterium-mediated method. T2 transgenic seeds could germinate in 0.2
mol/L NaCl medium, and T2 transgenic young plants could rejuvenate after treatment with 0.4
M NaCl for 10 days, while the control plants could not germinate and died in salt stress. This
finding proved that the regulation factor of the plant signal transduction could enhance the salt
tolerance of transgenic plants (Wang et al., 2008a).
Calcineurin is a Ca2+- and calmodulin-dependent serine/threonine phosphatase and has
multiple functions in animal cells including regulating ion homeostasis. The differentially
induced expression of OsCIPK genes by different stresses and the examples of improved stress
tolerance of the OsCIPK transgenic rice plants suggest that rice CIPK genes have diverse roles
in different stress responses and some of them may possess potential usefulness in stress-
tolerance improvement of rice (Xiang et al., 2007). OsCIPK23, a member of CBL (Calcineurin
B-Like) Interacting Protein Kinase (CIPK) family, was found to be upregulated by various
abiotic stresses. RNA interference-mediated suppression of OsCIPK23 expression conferred a
hypersensitive response to drought stress, indicating its possible role in drought stress. In
consistent, overexpression of OsCIPK23 induced the expression of several drought tolerance
related genes (Yang et al., 2008). Ma and coworkers (2005) generated transgenic rice plants
that not only expressed a truncated form of the catalytic subunit of mouse calcineurin, but also
were able to grow and fertilize normally in the field. Expression of the mouse calcineurin
protein functionally improved the salt stress tolerance of rice partly by limiting Na+
accumulation in the roots (Ma et al., 2005).
14-3-3 proteins function as major regulators of primary metabolism and cellular signal
transduction in plants. However, their involvement in plant defense and stress responses is
largely unknown. Chen and coworkers (2006b) examined the rice GF14 family that comprises
eight numbers. The phylogenetic comparison with the Arabidopsis 14-3-3 family revealed that
the majority of rice GF14s might have evolved as an independent branch. At least four rice
GF14 genes, GF14b, GF14c, GF14e and Gf14f were differentially regulated by salinity and
drought (Chen et al., 2006b).
Rice plants are relatively sensitive to soil salinity, and NaCl is a major salt in saline soils
(Flowers, 2004). Sodium enters plant roots by two ways: the symplastic pathway mediated by
cation channels/transporters and the apoplastic pathway in which Na+ enters the transpiration
stream. In rice it has been shown that HKT (High-affinity K+ Transporter) transporters are
involved in root Na+ uptake (Garciadeblás et al., 2003; Platten et al., 2006). In conditions of
high Na+ concentrations, Na+ may be taken up ectopically by K+ and other cation transporters.
SKC1, a HKT family member (OsHKT8/OsHKT1;5), is a Na+ selective transporter identified
in a salt-tolerant indica variety, Nona Bokra. The Na+ transport activity of NSKC1 (from Nona
Bokra) was higher than that of KSKC1 (from a salt susceptible japonica variety, Koshihikari).
Under salt stress, rice seedlings carrying NSKC1 exhibited more tolerance to salinity than
those carrying KSKC1, resulting from greater Na+ extraction from xylem sap by NSKC1. The
Effect of Abiotic Stresses on Growth… 157

xylem sap and shoot Na+ content in NSKC1 seedlings was lower than that noted in KSKC1
seedlings, whereas the reverse was true for K+ content (Lin et al., 2004).
Vacuolar sequestration of Na+ is catalyzed by OsNHX1 gene (Na+/H+ antiporter gene of
Oryza sativa L.) in rice (Fukuda et al., 2004). The expression of the NHX1 gene was up-
regulated by salinity in rice (Fukuda et al., 1999). The overexpression of OsNHX1 in rice
improved salt tolerance of the transgenic plants, without adverse effects on their Na+ and K+
contents and plant growth (Fukuda et al., 2004). This may be useful for genetic improvement
of salt tolerance in rice. E. coli nhaA gene encodes a Na+/H+ antiporter, which plays a critical
role in ion homeostasis. Wu and coworkers (2005a) transferred a bacterial nhaA gene into rice
and detected high expression in the transgenic rice. The germination rate, growth and average
yield per plant of the transgenic lines were better than those of control lines under salt or
drought stress. Moreover, the sodium and proline content of the transgenic lines under salt or
drought stress was also higher than in control lines, implying that nhaA over-expression
enhances osmoregulation by activating the biosynthesis of proline (Wu et al., 2005a).
Multiple transcription factors, including ICE (inducer of CBF expression), CBFs/DREBs,
AREB/ABF/ABI/bZip, MYC/ MYB and NACs, have been well characterized (Chinnusamy et
al., 2004, 2006). Induction of stress tolerance through engineering for over-expression of
transcription factor genes is emerging as an attractive proposition in recent years. A cis-acting
promoter element DRE (drought responsive element) plays an important role in regulating
gene expression in response to abiotic stresses (salt, drought and cold stresses). DREB
transcription factors play key roles in plant stress signaling transduction pathway, they can
specifically bind to DRE/CRT element (G/ACCGAC) and activate the expression of many
stress inducible genes. Dubouzet and coworkers (2003) identified five DREB cDNAs in rice:
OsDREB1B were induced by cold, while OsDREB2A was regulated by salt and drought stress.
Chen and coworkers (2008) isolated three novel rice DREB genes, OsDREB1E, OsDREB1G
and OsDREB2B, which are homologous to Arabidopsis DREB genes. The yeast one-hybrid
assay indicated that OsDREB1E, OsDREB1G, and OsDREB2B can specifically bind to the C-
repeat/DRE element. To elucidate the function of respective OsDREB genes, these were
introduced into rice by Agrobacterium-mediated transformation. Transgenic rice plants
analysis revealed that over-expression of OsDREB1G and OsDREB2B in rice significantly
improved their tolerance to water deficit stress, while over-expression of OsDREB1E could
only slightly improve the tolerance to water deficit stress, suggesting that the OsDREBs might
participate in the stress response pathway in different manners (Chen et al., 2008). Gutha and
Reddy (2008) reported the functional characterization of a DREB transcription factor,
DREB1B gene from rice. The OsDREB1B gene was differentially regulated at the
transcriptional level by osmotic stress, oxidative stress and cold. The data obtained provided
strong in vivo evidence that OsDREB1B is involved in both abiotic and biotic stress responses,
and confers broad-spectrum stress tolerance to transgenic plants (Gutha and Reddy, 2008). A
novel rice DREB transcription factor, OsDREB1F, was cloned and characterised via
subtractive suppression hybridisation (SSH) from upland rice. Expression analysis revealed
that OsDREB1F gene was induced by salt, drought, and cold stresses. Subcellular localization
results indicated that OsDREB1F localizes in nucleus. Transgenic plants harbouring
OsDREB1F gene showed enhance tolerance to salt, drought and low temperature in rice
(Wang et al., 2008b).
158 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

WRKY transcription factors contain one or two conserved WRKY domains, about 60
amino acid residues with the WRKYGQK sequence followed by a C2H2 or C2HC zinc finger
motif. Evidence is accumulating to suggest that the WRKY proteins play significant roles in
responses to biotic and abiotic stresses, and in development in plants (Wu et al., 2005b). An
OsWRKY11 gene, which encodes a transcription factor with the WRKY domain, was identified
as one of the genes that were induced by both heat shock and drought stresses in seedlings of
rice. To determine if overexpression of OsWRKY11 confers heat and drought tolerance,
OsWRKY11 cDNA was fused to the promoter of HSP101 of rice and introduced into a rice
cultivar Sasanishiki. Overexpression of OsWRKY11 was induced by heat treatment. After heat
pretreatment, the transgenic lines showed significant heat and drought tolerance, as indicated
by the slower leaf-wilting and less-impaired survival rate of green parts of plants (Wu et al.,
2009). Wang and coworkers (2007) reported functional characterization of a rice WRKY gene,
OsWRKY89. RNA gel blot analysis indicated that OsWRKY89 was strongly induced by UV-B
radiation treatment. Overexpression of the OsWRKY89 gene enhanced tolerance to UV-B
irradiation (Wang et al., 2007). Ramamoorthy and coworkers (2008) analyzed the publicly
available rice genome sequence databases and predicted 103 genes encoding WRKY
transcription factors. Their expression profiles under normal and abiotic stress were examined.
Under normal growth conditions, 65 WRKY genes were expressed differentially either in their
transcript abundance or in their expression patterns. Under abiotic (cold, drought and salinity)
stresses and various phytohormone treatments, 54 WRKY genes exhibited significant
differences in their transcript abundance; among them three genes were expressed only in
stressed conditions. Among the stress-inducible genes, 13 genes were regulated only by abiotic
stresses, another set of 13 genes were responsive to only phytohormone treatments and the
remaining 28 genes were regulated by both factors, suggesting an interaction between abiotic
stress and hormone signaling (Ramamoorthy et al., 2008).
It has been established that ethylene response factor (ERF) proteins play important
regulatory roles in plant response to abiotic and biotic stresses. Compared with the wild-type
plants, overexpression of TERF1 (encoding a tomato ERF protein) resulted in an increased
tolerance to drought and high-salt in transgenic rice and such enhanced tolerance was
associated with the accumulation of proline and the decrease of water loss (Gao et al., 2008).
Furthermore, TERF1 can effectively regulate the expression of stress-related functional genes
Lip5, Wcor413-l, OsPrx and OsABA2, as well as regulatory genes OsCDPK7, OsCDPK13 and
OsCDPK19 under normal growth conditions. Analyses of cis-acting elements showed the
existence of DRE/CRT and/or GCC-box in TERF1 targeted gene promoters. Gao and
coworkers (2008) suggested that ectopic expression of TERF1 in transgenic rice caused a
series of molecular and physiological alterations with enhanced tolerance to abiotic stress,
indicating that TERF1 might have similar regulatory roles in response to abiotic stress in rice
(Gao et al., 2008). Most rice cultivars die within a week of complete submergence. A few
cultivars, such as the cultivar FR13A, are highly tolerant and survive up to two weeks of
complete submergence owing to a major quantitative trait locus designated Submergence 1
(Sub1) near the centromere of chromosome 9. Overexpression of Sub1A-1 in a submergence-
intolerant O. sativa sp. japonica conferred enhanced submergence tolerance to the plants,
downregulation of Sub1C and upregulation of alcohol dehydrogenase 1 (Adh1), indicating that
Sub1A-1 is a primary determinant of submergence tolerance (Xu et al., 2006).
NAM, ATAF and CUC (NAC) transcription factors comprise a large plant-specific gene
family and a few members of this family have been characterized for their roles in plant
Effect of Abiotic Stresses on Growth… 159

growth, development, and stress tolerance. Fang and coworkers (2008) performed systematic
sequence analysis and found 140 putative NAC or NAC-like genes (ONAC) in rice. The
expression levels of stress-responsive NAC (SNAC) genes family were checked under various
abiotic stresses including drought, salinity and low temperature. Based on microarray data,
Fang and coworkers (2008) found that more than 40 genes of this family were responsive to
drought and salt stresses (Fang et al., 2008). Chao and coworkers (2005) found that multiple
rice transcription factors, including a NAC gene, were induced in the early stage of salt stress.
OsNAC6, a member of ATAF subfamily, was also induced by cold, salt and drought (Ohnishi
et al., 2005).
Hu and coworkers (2008) showed that overexpression of stress responsive gene SNAC1
significantly enhances drought resistance and salt tolerance at the vegetative stage as well as
reproductive stage while showing no phenotypic changes or yield penalty. SNAC1 is induced
predominantly in guard cells by drought and encodes a NAM, ATAF, and CUC (NAC)
transcription factor with transactivation activity. DNA chip analysis revealed that a large
number of stress-related genes were up-regulated in the SNAC1-overexpressing rice plants.
Therefore Hu and coworkers (2008) suggested that SNAC1 holds promising utility in
improving drought and salinity tolerance in rice.



Abiotic stresses are major constraints in cultivation of rice crop throughout the world. In a
world where population growth exceeds food supply, it is imperative that scientists should
focus efforts to find solutions that may help plants overcome the increasing challenging
environmental conditions (Alcazar et al., 2006). Rice is a major crop in the world and provides
the staple food for over half of the world’s population. From thousands of years of cultivation
and breeding to recent genomics, rice has been the focus of agriculture and plant research. Rice
domestication, breeding and genetics have led a great foundation for modern rice research (Ma
et al., 2007). Rice has the smallest genome among the cultivated cereals, and it conserves
much of the gene content and, to some extent, gene order present in other species (Gale and
Devos, 1998). The full rice genome has now been sequenced (Goff et al., 2002), allowing the
identification and localization of genes related to stress tolerance. Due to recent advances in
the understanding of the rice genome, substantial progress has been made in the development
of rice varieties with tolerance to various abiotic stress factors, including drought, salinity and
flooding (Wang et al., 2005a; Xu et al., 2006; Gao et al., 2007, 2008; Jena and Mackill, 2008).
Comparative genomics and techniques such as high-throughput analysis of expressed sequence
tags, large scale parallel analysis of gene expression, targeted or random mutagenesis, and
gain-of-function or mutant complementation, discovery of novel genes, determination of their
expression patterns in response to abiotic stress, and an improved understanding of their roles
in stress adaptation (obtained by the use of functional genomics) will provide the basis of
effective engineering strategies leading to greater stress tolerance (Cushman and Bohnert,
2000). Currently, a number of genes related to abiotic stress tolerance are available and the
technology to incorporate these traits to rice varieties is practicable. However, further advances
in understanding the requirements for transgene regulation, expression, and refinement in the
160 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

gene transfer techniques will strengthen the process for the development of rice cultivars
tolerant to various abiotic stresses (Giri and Laxmi, 2000).
Two basic genetical approaches currently being utilized to improve stress tolerance
include: (1) exploitation of natural genetic variations, either through direct selection in
stressful environments or through the mapping of quantitative trait loci (QTL) and subsequent
marker-assisted selection and (2) generation of transgenic plants to introduce novel genes or
alter expression levels of the existing genes to affect the degree of abiotic stress tolerance
(Blumwald et al., 2004). If rice breeding and genetic endeavors have generated genetic
materials that paved the way for recent advances in studying specific genes that are important
for many developmental and physiological traits, then the sequencing of the rice genome and
the subsequent functional genomics and proteomics efforts have yielded great volumes of
global molecular and biochemical information on many thousands of genes and proteins (Ma
et al., 2007). Such information has already greatly benefited rice research and allows
researchers to investigate specific processes or pathways with a global perspective of the
genome and great comprehensiveness hitherto not possible (Ma et al., 2007).
Genetic improvement of rice through conventional breeding methods is an effective
strategy for developing high yielding varieties. This has been accomplished by transferring
genes from the secondary gene pool of the wild relatives to the cultivated species of rice
through distant hybridization (Giri and Laxmi, 2000). Early attempts to evaluate the genetic
basis of stress tolerance in plants were restricted to simple genetic models (Blumwald et al.,
2004). In recent years, improved understanding of how rice responds to abiotic stresses and the
basis of varietal differences in tolerance has been applied in marker-aided selection. Marker-
assisted selection of progeny from crosses between tolerant, low-yielding cultivars and
susceptible, high yield-potential lines theoretically allows for much greater efficiency in a
breeding program, because extensive unreliable phenotypic screening can be eliminated, and
linkage drag can be effectively reduced (Lafitte et al., 2004b). Through expression profiling of
many genes simultaneously, it has been possible to identify three types of stress-responsive
gene networks: early signaling pathways, adaptive responses and genes that reflect
downstream results of damage. For crop improvement, the identification of useful allelic
variation for genes related to adaptive responses may be the most promising approach. Once
such genes or gene combinations are identified, either molecular approaches or trait-specific
physiological screens can be used to search for these superior alleles. Marker-assisted
backcrossing can then be applied to incorporate these alleles into agronomically superior
germplasm (Lafitte et al., 2004a).
With the development of molecular markers, evaluating the inheritance of abiotic stress
tolerance became a more tractable problem since specific QTL could be identified. Costly and
time-consuming process of fine-mapping may be circumvented by considering the stress-
responsive candidate genes that underlie a given QTL (Wayne and McIntyre, 2002; Ishimaru
et al., 2004). A strong putative candidate region can be used directly in breeding, however,
even if final gene identity is not known, as long as its position is confirmed through association
with phenotype in mapping populations (Thorup et al., 2000; Ramalingam et al., 2003).
Moreover, combining the transgenic approaches with traditional breeding methods will be an
effective approach to develop abiotic stress-tolerant rice cultivars. Several studies have been
done to identify QTL conferring tolerance to abiotic stresses, and to relate QTLs to
physiological tolerance mechanisms (Wang et al., 2005b; Ren et al., 2005; Wan et al., 2005;
Yue et al., 2008; Frei et al., 2008). Further map-based cloning and/or marker aided selection
Effect of Abiotic Stresses on Growth… 161

will help to map, isolate, and clone more genes of agronomic importance for its transfer to rice
plants (Martin et al., 1993; McCouch et al., 1997).
Significant advances have been made in the genetic engineering of rice since the first
transgenic rice plant production in the late 1980s. Selection and use of appropriate promoters,
selectable markers and reporter genes have been helpful for development of efficient protocols
for production of transgenic rice in a number of cultivars (Giri and Laxmi, 2000). To address
some of the problems in transferring desirable traits, however, would require either the
modification of the gene or engineering of transformation vector with multiple genes and their
exploitation in developing transgenic rice. Particle bombardment method has been used
extensively for the development of gene transfer protocols in rice to overcome genotypic
barriers however gene silencing has been found predominantly in transgenic rice plant
produced via particle bombardment (Kohli et al., 1999). This has facilitated multiple copy
integration of transgenes into the genome of rice triggering gene silencing. Recent
developments in gene transfer involving Agrobacterium appear to be beneficial to address the
problem of gene silencing (Khanna and Raina, 1999; Tyagi et al., 1999; Yokoi and Toriyama,
2000). Apprehension of environmental risks from commercial transgenic crops in general and
rice in particular cannot be ruled out. The hunch is that, the transgenics can convert the crop
plants into weeds in the cultivated area or the transgene escaping into natural habitats, and this
would affect public acceptance (Bhatia and Mitra, 1998; Ferber, 1999).
Though considerable progress has been made in the production of transgenic rice for
abiotic stress tolerance yet the achievements are not satisfactory. Some of the main problems
which have been encountered in producing stress tolerant rice plants include (1) tolerance to
abiotic stressis due to a complex trait influenced by the coordination and differential
expression of a network of genes, (2) while T-DNA integrates essentially everywhere in the
Arabidopsis genome, integration is detected only in the gene space, namely in the gene-rich,
transcriptionally active, regions of the rice genome (Barakat, 2000), (3) absence of transgene
expression in T0 plants, sterility of T0 plants, non-transmission of intact transgenes to some or
all progenies, silencing of transgene expression in progeny plants. Transgene stability was
significantly related to differences in transgene structure and expression levels (Vain et al.,
2002), (4) the marker gene used in transformation may affect the food safety and biosafety and
thus limit the application of transgenic rice in agricultural production (Giri and Laxmi, 2000).
One major limitation to progress is the lack of knowledge of the functions and interactions of
tolerance-related genes. While many genes have been identified with great potential for
abiotic-stress engineering, most of them, more or less, affect rice morphology when they are
constitutively overexpressed. Utilization of some of these genetic characteristics, therefore,
while producing desirable stress-related results, may have concomitant negative impacts.
Quantifying the advantages and disadvantages of breeding may take some time. However, it is
desirable to generate transgenic plants with gene expression driven by a controllable promoter
so that the gene products are not produced unless plants are subjected to stress.
With the study of the functional genomics of plants, considerably more information about
the mechanisms by which plants perceive and transduce these stress signals to initiate adaptive
responses will be obtained and with the improvement of the transgenic approach, marker free
transgenic rice will be produced (Breitler et al., 2004; Cao et al., 2006). The increase of the
productivity of rice and other crops is the main purpose of transgenic studies. Thereby,
different strategies need to be tested and adapted experimentally to genetically improve the
abiotic stress tolerance in rice. Ultimately, the different strategies must be integrated, and the
162 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

genes representing distinctive approaches must be combined to substantially increase abiotic

stress tolerance in rice. Our limited knowledge related to stress-associated metabolic
alterations remains a major gap in our understanding; therefore, comprehensive profiling of
stress-associated metabolites is most relevant to the successful molecular breeding of stress-
tolerant rice plants. Unraveling additional stress-associated gene resources, from both rice
plants and highly salt and drought tolerant model plants, will enable future molecular
dissection of salt-tolerance mechanisms in rice plants (Vinocur and Altman, 2005).
Abiotic stress tolerant transgenic rice plants have been produced using a host of different
genes and transcript profiling by micro- and macroarray-based methods which has opened the
gates for the discovery of novel salt stress mechanisms in rice and comparative genomics is
turning out to be a critical input in this respect. From the data on comprehensive transcript
expression profiling of clones representing abiotic stress-associated genes of rice, it is shown
that transcriptional and translational machineries are important determinants in controlling
abiotic stress response and gene expression response in tolerant and susceptible rice plants
differs mainly in quantitative terms. Interconnected signal transduction pathways that lead to
multiple responses to abiotic stresses have been difficult to study using traditional approaches
because of their complexity and the large number of genes and gene products involved in the
various defensive and developmental responses of the plants. New insights into signaling
networks involved in abiotic stress adaptation have now been gained by transcriptome analyses
that suggest the existence of both specific signaling and of crosstalk between signal
transduction pathways in response to environmental changes (Baier et al., 2006). During the
past couple of years, proteomics has been a leading technology for the high-throughput
analysis of proteins on a genome-wide scale. Considerable research effort has been made to
analyze rice proteome and remarkable progress has been achieved in the systematic and
functional characterization of proteins in various tissues and organelles of rice. As part of this
research, a system for direct differential display using 2-dimensional electrophoresis (O’Farell,
1975) has been developed for the identification of rice proteins that vary in expression under
different physiological conditions and among different tissues. The information obtained from
proteomic technologies regarding proteins modification, protein-protein interaction and the
development of new methods for differential proteomics will aid in deciphering more precisely
the functions of known and/or unknown proteins in rice (O’Farell, 1975).
Various studies have demonstrated that common genes are activated by such diverse
stresses as wounding, pathogen attack, salt stress and high temperature, etc in rice. Rabbani
and coworkers (2003) using a rice cDNA microarray including about 1,700 independent
cDNAs derived from cDNA libraries prepared from drought, cold, and high salinity treated rice
plant identified a total of 73 genes as stress inducible including 58 novel unreported genes in
rice. Among them, 36, 62, 57 and 43 genes were induced by cold, drought, high salinity and
ABA, respectively. These workers have observed strong association in the expression of
stress-responsive genes and found 15 genes that responded to all four treatments. The rice
genome database search enabled them not only to identify possible known cis-acting elements
in the promoter regions of several stress-inducible genes but also to expect the existence of
novel cis-acting elements involved in stress-responsive gene expression in rice stress-inducible
promoters. The complexities of abiotic stress responses essentially preclude the precise
experimental dissection of individual abiotic stresses, and suggest that further studies of
individual stresses might not be the best approach. Genomics, proteomics and metabolomics,
coupled with a strong bioinformatics capability, now enable a ‘broad’ approach to be taken in
Effect of Abiotic Stresses on Growth… 163

the study of plant responses to abiotic stresses. Thus, the entire system of networks of
signalling pathways and key interconnecting processes that lead to the multiple defensive
responses can be described in detail. The identification and analysis of genes exhibiting large
expression responses to several different types of stress may provide insights into the
functional basis of multiple stress tolerance in plant species including rice (Swindell, 2006).

Abiotic stresses adversely affect the productivity of rice in various parts of the world. The
abiotic stresses such as drought, salinity, extremes of temperature, anerobiosis, heavy metals,
gaseous pollutants cause considerable loss in rice yield every year. These stresses lead to
disruption of homeostasis in the rice plant. Abiotic stresses lead to a series of physiological,
biochemical and molecular changes within the rice plants. The abiotic stresses adversely affect
key metabolic processes in plants like synthesis of proteins and nucleic acids, photosynthesis,
respiration, nitrogen assimilation, etc. which may ultimately result in poor growth of plants
and reduction in yield. Plants respond to these stresses by displaying complex, quantitative
traits that involve the functions of many genes. Abiotic stresses lead to accumulation of low-
molecular weight organic compounds, compatible solutes or osmolytes, stress specific
proteins, late-embryogenesis-abundant proteins, heat shock proteins, phytochelatins,
metallothioneins, and lead to activation of several detoxification and antioxidative enzymes.
Although, different rice cultivars have variable thresholds for stress tolerance, and some of
them can successfully tolerate severe stresses and still complete their life cycles, most cultivars
are highly sensitive and either die or suffer from productivity loss after being exposed to long
periods of stress. Development of crop plants tolerant to environmental stresses appears to be a
promising approach to help satisfy growing food demands of the developing and under-
developed nations where abiotic stresses are severe constraints to crop productivity. Decades
of breeding and selection have resulted in limited improvements of stress tolerance in rice
plants largely due to the physiological and genetic complexities involved. More knowledge
about the genetics and molecular basis of abiotic stress related traits will be helpful in this
direction. In conjunction with these efforts, characterization of the genetic and functional
interactions of more abiotic stress-related genes is necessary. Genomics, proteomics and
metabolomics, coupled with bioinformatics provide better approach to study entire system of
networks of signalling pathways, key interconnecting processes, genes and gene products
associated with multiple defensive responses in rice plants which will help in deep
understanding of the functional basis of multiple stress tolerance in plants. Identification and
characterization of stress inducible proteins/enzymes involving proteomics approach,
discovery of novel and responsive genes determination of their expression patterns in response
to abiotic stresses, improved understanding of their roles in stress adaptation will provide the
basis of new strategies to improve stress tolerance in rice plants.
164 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

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Chapter 4



Jaime Martínez-Castillo1, Patricia Colunga-GarcíaMarín,

Daniel Zizumbo-Villarreal, Filogonio May-Pat
and Julián Coello-Coello
Centro de Investigación Científica de Yucatán (CICY), Calle 43 No. 130,
Col. Chuburná de Hidalgo, Mérida, Yucatán 97200, México

The lima bean (Phaseolus lunatus L.) is the second major cultivated species of the
genus Phaseolus. It possesses high levels of genetic diversity and its primary gene pool
includes both wild and domesticated forms grouped into two main gene pools:
Mesoamerican and Andean. In the Yucatan peninsula, it is integrated into the traditional
agricultural system focused on corn cultivation, known as milpa, where it is planted as an
associated crop. This region possesses the largest diversity of domesticated forms in
Mexico, a diversity that is possibly being generated and maintained, in part, by their
sympatric growth with wild populations. However, the repercussions of human population
growth and socio-economic changes occurring in this region during the last 50 years have
resulted in major modifications to the milpa. One of the most evident consequences of
these changes has been a decreased planting of the crops associated with corn and a loss of
the vegetational areas next to the milpa where wild relatives grow. This review chapter
shows the results of eight years of research on the conservation genetics of P. lunatus in
the Yucatan peninsula, México, using ethnobotanical, ecological and genetic evidence.
The results indicate that: 1) the genetic diversity of P. lunatus from the Yucatan peninsula
is higher in comparison to other Mesoamerican regions; 2) wild populations show higher
values of diversity than the domesticated ones, probably due to a founder effect or recent
processes of genetic erosion in the domesticated forms; 3) the three most abundant

188 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

landraces (70% of the planted area) had the lowest values of genetic diversity—in contrast,
12 landraces with high levels of genetic diversity were planted only by few farmers, a
situation that shows the high risk of genetic erosion in the domesticated gene pool; 4) wild
and domesticated gene pools show a strong genetic differentiation due to distance isolation
and low levels of gene flow; 5) the wild-domesticated gene flow is low, but it is three
times higher than the domesticated to the wild populations than in the opposite direction.
This situation may lead to genetic assimilation of the wild lima bean by its domesticated
counterpart and may lead to the possible escape of transgenes in this center of diversity. In
situ programs urgently need to be established in this important Mesoamerican region to
conserve the milpa system, including the lima bean landraces and its wild populations.

According to the Rural Advancement Foundation International (RAFI), agriculture
worldwide has lost three-quarters of the genetic diversity in major food crops, and this erosion
continues at an annual rate of 1–2% (Mazhar, 1997). Genetic erosion is the loss or reduction of
genetic diversity between and within populations of the same species over time (Jarvis et al.,
2000), and most often results from agricultural, economic and social changes (FAO, 1996). In
cultivated species, this phenomenon has been evaluated at the landrace level (Tsegaye and
Berg, 2006; Hammer and Laghetti, 2005), principally as this is the primary available genetic
pool for hybridization and genetic improvement programs (Harlan and De Wet, 1971).
However, genetic erosion is an important phenomenon in the wild relatives, too. Nowadays
there has been renewed interest in studying the wild ancestors of domesticated species as a
plant genetic resource, given their potential value as reservoirs of genetic variation for crop
improvement (Degreef et al., 2002). Genetic erosion is a significant issue in crop
domestication areas since a) they concentrate the highest genetic diversity; b) traditional
producers conserve ancestral landraces, along with the knowledge and cultural practices that
created this diversity; and c) there exist inter-reproductive wild-weedy-domesticated
complexes, favoring wild-domesticated gene flow (Bellon and Taylor, 1993; Brush, 1991).
Mexico forms part of the Mesoamerican center of domestication (Vavilov, 1926). The
ecological, productive and cultural conditions of traditional agroecosystems in Mexico have
helped to conserve a large number of domesticated species. These conditions have also
maintained these species as part of a dynamic scenario for development of new crops and
species evolution, both of which are processes that favor high levels of variation and genetic
contact with wild relatives (Hernández-Xolocotzi, 1973). In this Mesoamerican region,
Phaseolus beans are a very important crop. After corn, beans are the second most important
crop of the milpa production system, an ancestral Mesoamerican dry land farming system
based on human energy in which vegetation is cyclically slashed and burned in order to plant a
group of basic crops, such as corn (Zea mays L.) and squash (Cucurbita moschata [Duch]
Duch ex Poir; C. argyrosperma Huber). Alongside these basic crops, other secondary species
are cultivated, such as chilis (Capsicum spp.), sweet potatoes (Ipomoea batatas L.), cassava
(Manihot esculenta Crantz) and tomatoes (Solanum esculentum L.). After two to four years of
cultivation (depending on soil fertility), the land is allowed to rest for a period of five to 15
years before a new cycle is begun (Hernández-Xolocotzi, 1992; Pérez-Toro, 1945). The
conservation of patches of vegetation that are cyclically cultivated is, in turn, the mainstay of
the milpa’s productivity, as it assures the recovery of soil fertility and maintains the habitat for
Domestication and Conservation Genetics of the Lima Bean… 189

a large part of the plant genetic resources integrated into the milpa agroforestry production
system (Colunga-GarcíaMarín and May-Pat, 1992; Hernández-Xolocotzi, 1992; Zizumbo-
Villarreal, 1992).
Phaseolus beans are the world’s most important grain legumes for direct human
consumption. Dry beans (P. vulgaris L.) were grown on 27.7 million ha in 148 countries in
2004 and total production was 18.7 million metric tons (MT) (
default.aspx; 2006). The main production areas of beans are Latin America (with Brazil and
Mexico as the most important producers) and eastern Africa (Broughton et al., 2003). From an
economic perspective, dry bean exports generate US $812 million for developing countries,
whereas developed countries receive US $460 million for their exports. However, in many of
the countries in the world, especially in developing countries, beans are consumed as an
important part of the diet and not exported. The importance of beans to diets in the developing
world is reflected in the fact that for developing countries only 13% of production is exported.
This contrasts with developed countries, which export 31% of their production. Beans are
nutritionally rich, especially in protein and iron, along with being a good source of dietary
fiber and complex carbohydrates. Given their nutritional quality and high consumption levels,
beans make an important contribution to human nutrition, especially for poor consumers. In
addition to high quality protein, a single serving (1 cup) of beans provides at least half of the
USDA-recommended daily allowance of folic acid and 25–30 percent of the daily
recommended iron levels. Similarly, the same serving of beans provides 25 percent of the daily
requirements of magnesium and copper, and 15 percent of potassium and zinc (http://www.
Beans (Phaseolus spp.) are one of the many other groups of domesticated species that are
at risk of genetic erosion in their domestication and diversity centers. This genus has a
complex taxonomic and nomenclatural history, but recent phylogenetic investigations support
a monophyletic Phaseolus with an origin strictly in the New World, concentrated in tropical
and warm temperate America. The exact number of species within the genus is still being
debated (Delgado-Salinas et al., 1999, 2006; Freytag and Debouck, 2002). However, there are
probably around 50 species in the genus, of which five are domesticated: the common bean (P.
vulgaris L.), scarlet runner bean (P. coccineus L.), tepary bean (P. acutifolius A. Gray) and
year bean (P. polyanthus Greenm.), and lima bean (P. lunatus L.) (Baudoin et al., 2004).
After the common bean, the lima bean (Phaseolus lunatus L.) is the second most
important cultivated species of the genus Phaseolus in the world. It is an important source of
protein for rural populations in America and Africa and an important commercial species for
countries such as the United States, the world largest producer of the lima bean, followed by
Malagasy and Peru (Akande and Balogun, 2007). It is an annual or short-lived perennial
species, with a mixed mating system, that is predominantly autogamous but with outcrossing
levels up to 48% (Baudoin et al., 1998). It is characterized by high levels of genetic diversity
and its primary gene pool includes both wild and domesticated forms (Baudet, 1977). Analyses
of genetic variation and relationships among cultivars, landraces, and wild populations have
indicated the existence of two major gene pools for P. lunatus: Mesoamerican and Andean,
and a minor group of intermediate genotypes (Caicedo et al., 1999; Fofana et al., 1997; Fofana
et al., 2001; Lioi and Galasso 2002). Three cultigroups (cv-gr) are recognized in the cultivated
forms (Baudet 1977): 1) Potato, with small and round seeds; 2) Sieva, with medium-sized and
kidney-shaped seeds; and 3) Big Lima, with large and flat seeds. The Potato and Sieva
cultigroups represent the Mesoamerican gene pool and the Big Lima represents the Andean
190 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

one. Two main independent domestication events have been hypothesized for the P. lunatus
cultigroups on the basis of studies supported by archaeological (Kaplan and Lynch 1999),
biochemical (Debouck et al., 1987; Gutiérrez Salgado et al., 1995; Maquet et al., 1997), and
molecular data (Fofana et al., 1997; Fofana et al., 1999; Lioi et al., 1998; Lioi and Galasso,
2002): Big Lima cv-gr. was domesticated in South America, in the west valleys from Ecuador
and Peru, site where the Andean wild populations grown. In relation to the Sieva and Potato
cultigroups, it is considered that these were domesticated in Mesoamerica. However, the
Mesoamerican wild populations exists from north of Mexico to north of Argentina and the
cultivated forms from southeast of USA to the east coast of Brazil (Debouck et al., 1987). This
made difficult to find the domestication site, however, studies using alloenzyme markers
indicated that the region situated between southeastern of Mexico and northern of Guatemala
could be the putative domestication area of this gene pool (Gutiérrez-Salgado et al., 1995).
Recently, molecular analyses indicated that the Tehuantepec Itsmus in the southeastern of
Mexico is the putative site of domestication of this gene pool (D. Debouck, personal
communication, 2008).
Studies about the diversity and genetic structure of P. lunatus considering accessions from
all its natural geographic distribution are few. Maquet et al. (1997), using alloenzyme markers,
reported a Nei’s gene diversity (He = 0.26) and a total genetic diversity (HT = 0.317) for the P.
lunatus base collection of the Germplasm Bank of CIAT. These authors stated that the He
value found is a significant level and higher than reported for other plants that, like P. lunatus,
are mixed-mating or short-lived perennial species (He = 0.12) (Hamrick et al., 1991). This high
diversity of P. lunatus could be a result of its wide geographic distribution. According to
Hamrick et al. (1991), geographic range accounts for the largest proportion of the explained
variation in genetic diversity at the species level. Maquet et al. (1997), analyzing at the gene
pool level, found that the Andean accessions had a higher genetic diversity (He = 0.24; HT =
0.33) than the Mesoamerican accessions (He = 0.16, HT = 0.23). Also, these authors reported
that the wild accessions had higher genetic diversity than cultivated ones, HT = 0.352, 0.331,
respectively. Fofana et al. (1997) using DNA-RAPDs markers and HOMOVA two-way nested
AMOVA statistics, reported that the genetic variance within the Mesoamerican gene pool (σ2 =
0.0079) was significantly higher than the Andean gene pool (σ2 = 0.0051). Also, these authors
found that the variation within landraces was significantly higher than within wild forms.
Nienhuis et al. (1995), analyzing only cultivars and landraces accessions with RAPD markers,
found that the Mesoamerican group had more genetic diversity (He = 0.110) than the Andean
group (He = 0.097). In relation to the genetic structure, Maquet et al. (1997), found that on
average, lima bean showed 76% (GST = 0.755) and 24% of the total diversity, respectively,
among and within accessions. The mating system and life form of the species are both usually
highly associated with differences in GST values (Hamrick et al., 1991). These results indicate
that species with a limited potential for gene flow show more differentiation among
populations than do species with a greater potential for gene flow.
Considering only the Mesoamerican gene pool of P. lunatus, few studies of its diversity
and genetic structure exist. Castiñeiras et al. (2007), using AFLP DNA-markers, analyzed the
genetic diversity of Potato and Sieva landraces planted in the Cuban home gardens and they
reported a HT = 0.119. These authors, considering three collect regions, found a low genetic
differentiation (GST = 0.08). In relation to the wild populations of P. lunatus, the most
important studies have been made in the Central Valley of Costa Rica. Ouédraogo et al.
(2005), using alloenzymes markers, reported a He = 0.08 and a HT = 0.12; and using
Domestication and Conservation Genetics of the Lima Bean… 191

microsatellite markers they found a He = 0.143 and a HT = 0.22. In relation to the genetic
differentiation, these authors found a GST = 0.303 with both type of markers. Zoro-Bi et al.
(2003), using enzyme loci, reported a He = 0.03 and a HT = 0.193. Also, they reported a GST =
0.519 and low levels of gene flow (Nm = 0.39). Maquet et al. (2001), using isozyme markers,
reported a GST = 0.575 and an Nm = 0.18. In general, all these studies suggest: 1) low levels of
genetic diversity, 2) high levels of genetic differentiation and, 3) a restricted gene flow among
In the Mesoamerican part of Mexico, the lima bean is cultivated on lowland slopes
adjacent to the Pacific Ocean and the Gulf of Mexico. It is a common cultivar in traditional
cropping systems and is cultivated by diverse ethnic groups (Ballesteros, 1999). In the Yucatan
Peninsula, domesticated forms receive the Mayan name of ib and they are the fourth most
important crop of the milpa. As reported by Ballesteros (1999), the Yucatan Peninsula is the
most important center of diversity in relation to cultivated forms for Mexico. In spite of this
and of its importance as a crop for the Mayan farmers of this part of Mexico, studies about the
genetic diversity of P. lunatus in the Yucatan Peninsula were few until year 2000, and these
were based in morphological evidence, principally (Debouck et al., 1979; Hernández and
Delgado, 1992; Nahal, 1993; Ballesteros, 1999). In short, all these studies reported a high
diversity in cultivated forms with Potato, Sieva and intermediate variants grouped in 25 local
names, and the existence of wild and weedy populations. This high number of landraces is
possibly being generated and maintained, in part, by its sympatric growth with wild
populations in the milpa. However, during the last few decades, this traditional agricultural
system has undergone a series of transformations associated, in part, with the growth of the
rural population, which has doubled in the last 30 years (Cuanalo and Arias, 1997). Among the
most notable changes are (i) a shortening of the fallow period, (ii) an increased use of and
dependency on agrochemicals, (iii) a greater integration of the Mayan farmers to an external
marketing system, (iv) a reduction in the diversity of cultivated species, and (v) a reduction of
the areas of vegetation bordering the milpas, where the wild populations usually grow (Reyes
and Aguilar, 1992; Lazos, 1995; Ku-Naal, 1995; Remmers and Ucán, 1996). Nowadays, with
the development of transgenic crops, the environmental risk of transgenics escaping via
hybridization between crops and wild relatives in centers of origin has emerged as another
potential problem (Hails, 2000; Snow, 2002).
In spite of the changes that have occurred in the milpa, it continues to be the most
important agricultural system in the Yucatan Peninsula of Mexico. In this region, the milpa has
survived over the centuries due to, in part, the rocky and shallow soils that have limited the
introduction of agricultural machinery, other cereals, and broadcast sowing. Another signifi-
cant factor contributing to the persistence of the milpa system has been the cultural resistance
offered by the Mayan people who have maintained this agricultural system as the material
basis of their culture for many generations (Zizumbo-Villarreal, 1992). At present, there are
four geographic areas in the Mexican part of the Yucatan Peninsula where the milpa continues
to be the most important economic activity. The location of these areas and their agroeco-
logical characteristics are shown in Figure 1 and Table 1, respectively. These areas correspond
to four of the 13 cultural-geographic zones established by Adams and Culbert (1977) for the
origin of the Maya lowland civilization. These four areas have their own particular physio-
graphic, vegetational, and agroecological features (Duch-Gary, 1991), and have followed their
own specific cultural and economic trajectories since the arrival of the Europeans. They
include: 1) northeastern Campeche, in “Los Chenes” zone. In this area, a series of low-to-tall
192 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

hills alternate with interspersed plains, including floodplains. Vegetation includes savanna,
seasonally flooded low tropical deciduous forest, and medium tropical semi-evergreen forest;
2) southern Yucatan, in the “Puuc” zone. This area is characterized by hillock formations,
continuous stretches of long, elevated hills and variable-sized plains between them, favorable
for intensive agriculture. In terms of vegetation, this region is noted for low tropical deciduous
forest and medium tropical sub-deciduous forest. This is the area where the influence of
commercial agriculture has most recently been the greatest; 3) southeastern Yucatan, located
within the “Northern Plains” zone. Undulating plains with hillocks and shallow bottomlands
characterize the physiography of this region. Vegetation consists of medium tropical sub-
deciduous forest. The most traditional Mayan communities in the Yucatan state are
encountered here; and 4) central eastern Quintana Roo, within the “Río Bec” zone. This area
corresponds to the region currently known as the “Mayan Resistance Area” (Bartolomé and
Barabas, 1977). It is comprised of human populations that emigrated from the southeastern
part of the Yucatan state during the Caste Wars (Guerra de Castas) in the nineteenth century.
These populations remained independent until the beginning of the twentieth century. The
dominant vegetation in this area is the medium tropical semi-evergreen forest.

Figure 1. Agricultural areas where the ethnobotanical research was made and plant material was
collected. SYUC, Southern Yucatan; NECAMP, Northeastern Campeche; SEYUC, Southeastern
Yucatan; CEQROO, Central eastern Quintana Roo. Taken from Martínez-Castillo et al., 2008.
Domestication and Conservation Genetics of the Lima Bean… 193

Table 1. Environmental characteristics of the four studied areas in the Yucatan

Peninsula, Mexico. Modified from Martínez-Castillo et al. (2004).

Southern Northeastern Southeastern Central eastern

Yucatan Campeche Yucatan Quintana Roo
Temperature (o C) 26.4 26.3 25.3 26.1
Average annual rainfall (cm)
Vegetation types1 1-3 2-4-5 3- 4
Soil types 2 r-cl r r-l l-r
Average years of 0 0 8 15
Fallow period
Agricultural m (70%) m-lb (80%) pc pc
management 3, 4 m-lb (30%) pc (20%).
Use of herbicide 100 % 100 % 70 % 70 %
and fertilizer (%)
1= low tropical deciduous forest, 2 = seasonally- flooded low tropical deciduous forest, 3 = medium
tropical subdeciduous forest, 4 = medium tropical semievergreen forest, 5 = savanna.
r= rendzine, l = litosol, cl = cromic luvisol.
m = monoculture of maize, m-lb = maize with lima bean; pc = maize with lima bean and other species.
Percentages are on respect to 160 peasants interviewed.

Establishing guidelines for conserving native germplasm in the Mayan area of the Yucatan
Peninsula is of great relevance when this region is one of the Mesoamerican sub-areas with the
greatest cultural continuity and historical persistence of their traditional farming practices. The
objective of this chapter is to give a resume of the most important results of eight years of
research (2000-2008) about the domestication and conservation genetics of P. lunatus in the
Yucatan Peninsula, México. The basic questions to ask in this chapter are: 1) what is the
intraspecific diversity and in situ conservation problematic of P. lunatus wild and domesticated
gene pools in the Yucatan Peninsula, México?; 2) what are the structure and genetic diversity,
and levels of gene flow in these gene pools?; 3) what is the magnitude and direction of the
wild-domesticated gene flow?; and 4) what is the risk of genetic erosion of the landraces
cultivated by the Mayan farmers?. The last three questions are approached using molecular
data and the results analyzed with the support of ethnobotanical and ecological information.
With this, we expect contribute with basic information to generate in situ conservation
programs to P. lunatus as well as to all the milpa system of the Yucatan Peninsula, México.


Ethnobotanical Research

An ethnobotanical study in the four most important geographic areas where the milpa
continues to be the most important economic activity was made to know and characterize the
in situ conservation problematic of lima bean in the Yucatan Peninsula, Mexico. The areas
were: 1) Northeastern Campeche, 2) Southern Yucatan, 3) Southeastern Yucatan, and 4)
194 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

Central Eastern Quintana Roo (Figure 1). The study was undertaken during 2000 and 2001 in
eight two-week field trips, two trips for each region studied (Martínez-Castillo et al., 2004):
three agricultural communities were selected from each one of the four study areas. In each
community, 13 to 14 farmers (40 per study area) were randomly chosen from a list provided by
local civil authorities and a semi-structured interview was applied to each farmer which
focused on the following information associated with wild, weedy, and cultivated variants of
P. lunatus: Mayan name, morphological and phenological characters used for traditional
classification, selection criteria, agricultural management practices, production purpose, and
percentage of area cultivated of each variant in relation to the total area cultivated of P.
lunatus. Data obtained through the semi-structured interviews were complemented with data
obtained through participative observation during the agricultural works at the milpa done by
We calculated relative abundance of the landraces as the number of hectares cultivated
with each landrace in relation to the total cultivated hectares for the species in the four study
areas, according to reports from the farmers; diversity using Shannon’s index (Shannon and
Weaver, 1949: H’ = - Σ pi ln pi), and dominance utilizing Simpson’s index (Simpson, 1949: D
= Σ pi2). In calculating the Shannon and Simpson indices, pi represented the percentage of the
area cultivated per landrace. These indices were calculated using the Biodiversity Professional
software Release 2.0 (McAleece, 1997).
Samples of all landraces recognized as different by each farmer were collected from
existing seed stock in the farmer’s barns and directly from the milpas, too. For the collection of
in situ germplasm from the wild and weedy populations, botanical explorations were
conducted in the agricultural areas of each community visited, with the participation of peasant
farmers. From each wild and weedy population, samples were collected that included an
average of 10 pods from 20 to 30 individuals. Botanical vouchers were collected with the help
of the interviewed farmers, and deposited in the CICY herbarium. In total, 149 cultivated
accessions obtained from the farmer barns and 24 populations (11 wild, 1 weedy, 12
domesticate) obtained in situ were collected.

Molecular Research

Samples of seed of all landraces recognized by the Mayan farmers, and seeds of the wild,
weedy and domesticated populations collected in situ were planted in a greenhouse in the
Centro de Investigación Científica de Yucatán, in Merida, Mexico. For all molecular analyses,
genomic DNA was obtained from young leaves following the CTAB method (Doyle and
Doyle, 1987).
Diversity, genetic structure and gene flow in the wild and domesticated gene pools. To
make these analyses, the microsatellite (Simple Sequence Repeat or SSR) technique was used.
SSRs are loci that consist of repeating units of 1-6 base pairs in length. Nine pair primers
reported as polymorphic for Gaitán-Solis et al. (2002) was used (Table 2). To details of PCR
conditions see Martínez-Castillo et al. (2006, 2007).
Diversity, genetic structure and gene flow were analyzed in wild and domesticated gene
pools, separately. 11 wild populations and 12 domesticated ones were used, respectively
(Table 3). The genetic diversity was estimated considering three levels of analysis:
populations, agricultural regions and the Yucatan Peninsula. The estimates were the number of
Domestication and Conservation Genetics of the Lima Bean… 195

alleles (A), the evenness of the allelic frequencies (Ae/A), the observed heterozygosity (Ho),
and the Nei´s genetic diversity index (H). All these indices were estimated using POPGENE
1.31 (Yeh and Boyle, 1999). Using the GLM procedure of the SAS ver. 6.12 (SAS, 1997)
program, a one-way analysis of variance (ANOVA) and Duncan’s multiple comparison of
means tests (α = 0.05) were conducted to compare the values of allelic richness and diversity
obtained at the regional level.

Table 2. Characteristics of the nine microsatellite (SSR) loci used in the analysis
of the diversity, genetic structure and gene flow in the wild and domesticated gene pools

Code SSR sequence 5’ to 3’ Primer sequence Tm1 NoA2 RF 3

BM160 (GA)15(GAA)5 Left CGTGCTTGGCGAATAGCTTTG 52 4 178-188
BM164 (GT)9(GA)21 Left CCACCACAAGGAGAAGCAAC 52 5 135-143
BM211 (CT)16 Left ATACCCACATGCACAAGTTTGG 52 16 194-219
Annealing temperature in ºC (Tm). 2 Number of alleles per locus (NoA). 3 Range of fragment size
found in base pairs (RF).

Wright’s inbreeding coefficient [FIS = 1-(Ho/He)] (Wright, 1978) was obtained as an

indicator of excess or deficit of heterozygotes for each locus/population using POPGENE 1.31.
We estimated if these values were different from zero (α = 0.05) with a chi-squared test, X2 =
N (r-1) FIS2 with r (r-1)/2 degrees freedom, where N is sample size and r is the number of
alleles at the locus (Li and Horvitz, 1953). The FIS were averaged across polymorphic loci for
each population using a jackknife procedure. To estimate if these values were significantly
different from zero (α = 0.05) we used a two-tailed Student t test based on jackknife-generated
standard error values (Sokal and Rohlf, 1995).
Genetic structure was analyzed using two statistical procedures: (i) the GST statistic was
estimated by POPGENE 1.31, and (ii) analysis of molecular variance (AMOVA) using
ARLEQUIN ver 2.0 (Schneider et al., 2000). To evaluate the hypothesis of isolation by
distance, a Mantel test was performed using the matrixes of genetic and geographic distances
of the populations using ARLEQUIN. The genetic relationships among populations were
inferred with the construction of an UPGMA based on the standard genetic distance of Nei for
various loci (Nei, 1972), using the TFPGA program. Robustness of the topology was evaluated
by selecting the bootstrapping option with 1000 random resamplings with replacement over
loci (Felsenstein, 1985). Long term gene flow was indirectly estimated for both regional and
the Peninsula levels from Nm [Nm = 0.25 (1 - GST)/GST] using POPGENE.
196 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

Table 3. Twenty-four wild, weedy and domesticated P. lunatus populations

from four agricultural areas on the Yucatan Peninsula, Mexico

Agricultural region Population 1

Southeastern Yucatan (SEYUC) San Fernando (w) (i)
Marcos (we) (i)
Boje (w) (i)
San Fernando (d)
Marcos (d)
X-Uilub (d)
Central eastern Quintana Roo (CEQROO) Nohca (w)
Kik (w)
Holpat (w)
Celestino (d)
Domingo (d) (i)
Julián (d)
Southern Yucatan (SYUC) Xohuayán-1 (w)
Nohcacab (w)
Xohuayán-2 (w)
Xohuayán-1 (d)
Xohuayán-2 (d)
Rubén (d)
Northeastern Campeche (NECAMP) Itzinté (w) (i)
Bolonchén (w) (i)
Chunchintok (w)
Bolonchén (d) (i)
Elias (d)
Pascual (d)
Domesticated (d), weedy (we), wild (w); populations with morphological or molecular evidence of
introgression (i).

Finally, the genetic structure of all wild and domesticated populations of P. lunatus was
analyzed in conjunction, using the GST statistic estimated by POPGENE 1.31, and AMOVA
using ARLEQUIN ver 2.0.
Wild-domesticated gene flow. Data obtained previously with the microsatellite technique
on the 11 wild and 12 domesticated populations, plus molecular data obtained of one weedy
population, were considered in this section (Table 3). To analyze the magnitude and direction
of wild–domesticate gene flow, we used two different approaches: genotype assignment
methods to analyze recent gene flow and frequency methods to analyze long-term gene flow.
The two approaches are complementary, providing information about gene flow on different
timescales. Differences between both methods are showed in Manel et al. (2005).
We used Bayesian genotype assignment methods implemented in the Structure 2.1
program (Pritchard et al., 2000). This program uses a Bayesian clustering approach with
Monte Carlo Markov Chain (MCMC) methods and assigns individual genotypes to a
predefined number of populations (K) in a given sample (X) to achieve Hardy-Weinberg and
linkage equilibriums. This method assumes a model with K populations, each characterized by
a set of allele frequencies at each locus. Individuals in the sample are assigned probabilistically
Domestication and Conservation Genetics of the Lima Bean… 197

to populations, or jointly to two populations if their genotypes indicate they are admixed. Gene
flow magnitude and direction were based on the proportion of estimated ancestry of each
individual (q) and each population (Q) as calculated by Structure. Individuals were classified
into two categories according to their biological status: wild or domesticated. Weedy
individuals were classified as wild. The analysis was done on what were called the Peninsula,
interregional, intraregional, and parcel levels. The Peninsula level included a simultaneous
analysis of all studied individuals on the Yucatan Peninsula and grouping of them to calculate
Q for the different populations where they were collected. The interregional level included
simultaneous analysis of all studied populations and grouping of them to calculate Q for eight
gene pools by biological status and region. The intraregional level involved separate analyses
of populations in the same agricultural region and grouping to calculate Q in two gene pools
per region according to biological status. The parcel level involved separate analyses of the
populations in the Marcos parcel, where both weedy and domesticate populations grew, and
grouping to calculate Q for the two gene pools by biological status. For the Yucatan Peninsula,
intraregional and parcel levels, populations were assigned to K = 2 gene pools (i.e., wild and
domesticate). Populations in the interregional level were assigned to K = 8 gene pools, that is,
one wild and one domesticate per region. The model was applied using the previous data on
the population option; these data were their geographic location to determine which
individuals in the sample were immigrants or had recent immigrant ancestors. Burn-in length
was 104 and run length was 105 to allow the Markov chain to reach stationarity.
Two frequency methods were used: (1) Estimation of Nm at the Peninsula and
intraregional levels was done using the POPGENE 1.31 program (Yeh and Boyle, 1999). (2)
Estimation of mY, which is based on the average coalescence time of genes obtained from
within and between parental and admixed populations. This estimator was initially described in
Bertorelle and Excoffier (1998) and extended to any number of parental populations by
Dupanloup and Bertorelle (2001). The analysis was performed using Admix 2.0 software
developed by Dupanloup and Bertorelle genetica/Isabelle/admix2
_0.html. The admixture model used was based on Papa and Gepts (2003). It considers that
both wild and domesticate populations consist of two subpopulations: “true” wild (PW) and
domesticated (PD) types, without introgression from their domesticated or wild counterparts;
and hybrid wild (PhyW) and domesticated (PhyD) populations, with introgression. Each hybrid
population consists of N (1-μ) loci randomly obtained from a parent population and Nμ loci
from the other population: PhyW= μ2PD + (1- μ2)PW and PhyD = μ1PW = (1 + μ2)PD, where μ is
the parent population’s contribution to the hybrid population. This allows comparison of μ1
(contribution of PW to PhyD) and μ2 (contribution of PD to PhyW). Selection of the individuals
to create the PhyD and PhyW populations was done based on the Bayesian clustering results,
the classification of the seeds by Mayan farmers, and the morphological data collected in situ.
Genetic erosion in landraces. To analyze the genetic erosion in the domesticated gene
pool of P. lunatus, 21 landraces collected and recognized for Martínez-Castillo et al. (2004)
were chosen. For each landrace a number of accessions ranging from 1 to 5 was analyzed
(Table 4). When possible, the seeds were taken from accessions collected in the four
agricultural regions considered for this study to provide greater genetic representativity. The
21 landraces were listed on the basis of the percentage of cultivated area that a sample of 160
farmers used for each landrace. This produced three groups (Table 4): (a) abundant landraces,
consisting of three landraces, each grown on more than 16% of the cultivated area and planted
198 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

by 10–33 producers in the four agricultural zones; (b) common landraces, including six
landraces, each grown on 3–5% of the area and by 5–14 producers (this group included
Chakpetch and Balche, with low percentages but planted by nine and five producers,
respectively); and (c) rare landraces, consisting of 12 landraces, each planted on less than 2%
of the area and grown by one to four farmers.
The Inter-Simple Sequence Repeat (ISSR) technique was used. It allows detection of
polymorphism without previous knowledge of DNA sequences. It involves polymerase chain
reaction (PCR) amplification of DNA using a single primer composed of a SSR sequence,
anchored at the 3 or 5 end by two to four arbitrary, often degenerate, nucleotides (Zietkiewicz
et al., 1994). Each ISSR band was considered as an independent locus and polymorphic bands
were scored as absent (0) or present (1) for all samples. Only clearly reproducible bands were
scored and differences in band intensity were not considered. Four primers were used:
(GACA)3RG, YR(GACA)3, (GACAG)3AG and (CACAG)3RG. To details of PCR conditions
see Martínez-Castillo et al. (2008).

Table 4. Plant material used in the analysis of the genetic erosion of the 21 lima bean
landraces of the Yucatan Peninsula, Mexico. Taken from Martínez-Castillo et al. (2008).

Local name Culti-group Number of accesions Relative abundance % of cultivated area Agricultural regions1

Mulición Potato 5 Abundant 29.61 All regions

Sac Potato 5 Abundant 25.13 All regions
Putsica-sutsuy Potato 5 Abundant 16.5 All regions
Bacalar Sieva 5 Common 5.82 CEQROO
Nuk Sieva 5 Common 4.12 SYUC
Chak- saac Sieva 5 Common 4.1 CEQROO, SEYUC
Mejen Sieva 5 Common 3.00 SYUC
Chak- petch Sieva 5 Common 1.79 CEQROO, SEYUC
Balche Sieva 5 Common 0.92 CEQROO
Box-petch Sieva 1 Rare 1.85 CEQROO,
Balam-pach Potato 1 Rare 1.1 SEYUC
Tsisibal Potato 2 Rare 1.1 SEYUC
Kan Potato 1 Rare 1.01 SEYUC
Chak-mejen Sieva 2 Rare 0.32 NECAMP
Madza-kitam Sieva 1 Rare 0.31 SEYUC
Pool-santo Potato 1 Rare 0.26 CEQROO, SEYUC
Tabaco Sieva 1 Rare 0.16 CEQROO
Box-uolis Potato 1 Rare 0.08 CEQROO
Chak-uolis Potato 4 Rare 0.06 CEQROO, SEYUC
Chak-chi Sieva 1 Rare 0.02 SEYUC
Chocolate Sieva 1 Rare 0.02 CEQROO
Agricultural regions: SEYUC, southeastern Yucatan; CEQROO, central eastern Quintana Roo; SYUC,
southern Yucatan; NECAMP, northeastern Campeche.

Genetic diversity was estimated at two levels: (a) total domesticated gene pool, and (b)
landraces groups. To avoid common problems associated with the analysis of dominant data
(Culley and Wolfe, 2001; Lynch and Milligan, 1994), analyses did not involve Hardy–
Weinberg equilibrium (HWE). It was considered due to domesticated populations of P. lunatus
from the Yucatan Peninsula are known to deviate from Hardy–Weinberg equilibrium with co-
Domestication and Conservation Genetics of the Lima Bean… 199

dominant microsatellite markers (Martínez-Castillo, 2005). It was assumed that there was no
co-migration of alleles from different loci, alleles shared by two individuals descend from a
common ancestor and each locus consisted of only two alleles that segregate in Mendelian
The parameters used were: (1) polymorphic loci percentage (% P), calculated directly
from the data; (2) the Shannon-Weaver diversity index (I) (Shannon and Weaver, 1949)
obtained with the POPGENE ver. 1.31 program (Yeh and Boyle, 1999); (3) Nei genetic
diversity (H) considering the Taylor expansion (Lynch and Milligan, 1994) and obtained with
the TFPGA program (Miller, 1997). Although in this study was considered that the data are not
in Hardy–Weinberg equilibrium, based in the results reported by Kremer et al. (2005) we
decided to evaluate average heterozygosity (HB) using the Bayesian approach proposed by
Zhivotovsky (1999). This estimator was obtained with AFLP-SURV version 1.0 program
(Vekemans, 2002). Paired Student t tests were done to compare I, H and HB values between
pairs of landrace groups (α = 0.05) using the Statistica ver. 6.0 program (Statsoft, Tel Aviv,

1. Intraspecific diversity and in situ conservation of P. lunatus
in theYucatan Peninsula, Mexico

Wild gene pool

Wild populations were found in habitats generated by human activities where there was a
high, but variable incidence of disturbance. Both wild and weedy populations were called in
Mayan ib-cho (ib rat) because according to farmers, rats consumed the seeds of these plants.
Both types of populations showed forms with obvious differences in size, shape, and color
(Figure 2), however, in spite of these differences, farmers did not give them different names.
For these populations, no selection criteria were detected, basically because they are not given
any use. Some farmers reported that a few people have consumed these populations because of
their considerable pod production and similarity to the cultivated material, but they stopped
doing so when they subsequently became ill. The wild and weedy seeds of P. lunatus have a
high HCN content which makes them unfit for consumption (Maquet, 1991). Recently one
Mayan farmer reported that a weedy variant is planted and eaten by himself and his family (F.
Dzul, personal communication, 2008).
The wild populations were located in the four study areas, but were most abundant in
CEQROO. This situation appears to be correlated with: 1) longest fallow periods, 2) lowest
herbicide use, and 3) best soil fertility conditions (Table 1). Some farmers tolerate the presence
of wild populations within the milpas when their population density does not affect the correct
development of their crops. They were kept under control by: 1) hand weeding, when the
population density was low (<15 plants). It exist two kinds of hand weeding: haranchak and
lochepak. The first is more effective as the weeds are eliminated along with their roots.
Lochepak, on the other hand, eliminates only the aerial part of the plant, allowing a subsequent
200 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

Figure 2. Seed morphological variation of wild, weedy and cultivated variants, recognized by traditional
farmers in the Peninsula of Yucatan, Mexico. Lane 1 = Sieva variants, lane 2 = Potato variants, lane 3 =
wild and weedy populations. Traditional variants not recognized with morphological characters alone:
chakpetch-chakmejen(cp-cm), xmejen-nuk (me-nu), mulición-sacmejen (mu-sm). Wild (wi) and weedy
(we) variants. Taken from Martínez-Castillo et al., 2004.

With this kind of weeding, the farmers allow the weed population to reach the stage of
seed production along with their crops. 2) Use of herbicides. When the population density was
higher (>50 plants), as in some populations which manage to grow within the corn
monoculture, the peasants limited the density with the application of herbicides, but only until
the maize plants had reached a point in their development that assured the production of ears.
Once the harvest was secured, the peasants allowed these populations to grow freely. In
several wild populations we found seeds with colors, forms, and sizes similar to the cultivated
variants, as a consequence, perhaps, of past events of wild-domesticated gene flow. Only in the
southeast of Yucatan did the farmers report the elimination of wild populations in order to
prevent them from ‘‘mixing’’ with the landraces of this species.
The weedy populations were found in CEQROO, SEYUC and NECAMP at very low
population densities. It could be due to the differential management of the agricultural areas by
the farmers. The areas where wild populations grow are preferred for corn monoculture treated
with herbicides, since hand picking is not effective in eliminating high-density wild
populations. This agricultural management does not permit the existence of species associated
with corn as P. lunatus, thus the generation of P. lunatus weedy populations by gene flow is
not possible. So, weedy populations of P. lunatus were specifically associated with long fallow
periods that allowed the existence of vegetational patches and with traditional weeding
practices as lochepak.

Domesticate gene pool

All landraces of lima bean were named ib; however, each one had its own name. Twenty-
five landraces were recognized by farmers (Table 5), who used two morphological characters
and one phenological character to differentiate them: 1) Seed shape. Landraces pertaining to
the cv-gr. Potatoes were called x-uolis ib (ib ball) or mulición (ib birdshot). Landraces
pertaining to the cv-gr. Sieva were called petch (flat). 2) Seed color pattern. Landraces with
seeds of only one color, such as chak-ib (ib red), box-ib (ib black), and sac-ib (ib white) were
named by their color. Landraces whose seeds showed combinations of colors often received
names related to the things, plants, or animals that they resembled, such as madza-kitam (ib
Domestication and Conservation Genetics of the Lima Bean… 201

wild boar eyebrows) and pool-santo (ib saint’s head) landraces. The variation found in these
two morphological characters is shown in Figure 2. 3) Production cycle. Landraces also
received names related to the duration of the plant’s production cycle, such as mejen-ib (ib
short cycle; a landrace with a three to four months production cycle) and nuk-ib (ib long cycle;
a landrace with a seven to eight months production cycle). The combination of these three
characteristics is used to distinguish among landraces. Synonyms for landraces’ names were
encountered on many occasions. One landrace might receive more than one name, depending
on its different attributes. Sometimes different landraces received the same name if the farmers
used only one classification criterion.
In relation to the cultivated area by landrace, the ones of greatest importance in the region
were mulición (29.61%), sac (25.12%), and putsicasutsuy (16.5%). Many of the other
landraces did not reach the 1%, being planted by just a farmer (0.02%) (Table 5). Central
eastern Quintana Roo was the most diverse area (H’ = 0.8), followed by southeastern Yucatan
(H’ = 0.76). Tied for third place were NECAMP and the SYUC region (both with H′ = 0.71).
The lowest richness of landraces observed in the SYUC area appears to have been correlated
with a high degree of agricultural intensification and with the incorporation of smallscale
farmers into markets. In this area, we encountered almost half the landraces occurring in
southeastern Yucatan. Deep soils in the SYUC area have allowed the introduction of irrigation
and farm machinery into geographically tiny areas. This has favored monocultures and
agricultural intensification due to the planting of short cycle landraces (xmejen) with high
market values.
The abundance of each landrace and the diversity of each agricultural area are a result of
the selection criteria applied for the Mayan farmers nowadays. Farmers indicated taste
(27.39%) and color (20.75%) as the main criteria, following for cooking time (12.45%),
production purpose (11.2%), and productive cycle (5.39%), among others. The landraces
identified as having the best taste were mulición, sac, xmejen, nuk, sacmejen, and
putsicasutsuy, the first five with white testa. White as primary color (5 variants) was the most
represented, followed by the red (three variants). The landraces with white testa were also
identified as the fastest cooking. In relation to the production purpose, Mayan farmers plant ib
with auto-consumption and commercial purposes. Although no variant appeared to be selected
exclusively for a commercial interest, when the production was directed mainly to the market,
the white testa variants were preferred, occupying 66.66% of the total cultivated area reported
by the farmers. Xmejen landrace reaches the highest prices because of its white testa, larger
seeds, and shorter productive cycle, allowing seed production in months when there is no
production of other landraces. Indeed, xmejen was the only one that was eaten as immature
seed. It is planted principally in SYUC and it does not represent a high percentage of cultivated
area (only 3.03%) as its production depends on mechanized and, in several cases, irrigation
systems. The production of P. lunatus also included landraces that were cultivated only for
auto-consumption as these were not popular in the market, apparently because of the dark
color and semi-bitter taste of the seed. This component of auto-consumption was reflected in
the low percentage of cultivated area for this class of landraces. In relation to the productive
cycle, 21 of the 25 variants were reported as long cycle variants and only four as short cycle
variants (Table 5). The preference of a large number of long cycle landraces is probably
related to their better adaptation to dry land farming. A phenological analysis showed that
xmejen has the most short cycle among all landraces (Martínez-Castillo et al., 2004).
202 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

Table 5. Traditional landraces recognized by Mayan farmers from the Yucatan

Peninsula, Mexico. Percentage of cultivated area (A); First and second color of seed (B);
Time to the harvest (C); Agricultural management (D); Production purpose (E);
Taste (F); Cooking time (G); SEYUC (H); NECAMP (I); SYUC (J); CEQROO (K).
Modified from Martínez-Castillo et al. (2004).

Name A3 B4 C5 D6 E7 F8 G9 H I J K
cv-gr. Sieva
Bacalar 4.92 br-b l a m-s s i 0 0 0 14
Balche 0.92 br-b l a m-s s i 0 0 0 5
Batun 0.74 c l a S s i 0 0 1 0
Boxpetch 1.85 b l a S sb i 0 0 0 1
Chakchí 0.02 r-w s a S s i 0 0 1 0
Chakmejen 0.32 r s a s-m s f 0 1 1 0
Chakpetch 1.79 r l a s-m s i 0 1 4 4
Chaksaac 4.21 r-b l a s-m s i 0 2 1 0
Chocolate 0.02 br l a S sb s 0 0 0 1
Madzakitam 0.31 b-w l a S sb s 1 0 0 0
Mejen 3.03 w s m m-s s f 6 1 0 0
Nuk 4.12 w l a m-s s f 2 0 0 0
Tabaco 0.16 br l a S sb i 0 0 0 1
cv-gr. Papa
Balampach2 0.10 r-g l a m-s s i 0 0 0 1
Boxuolis 0.08 b l a S sb s 0 0 0 1
Chakuolis 0.06 r l a S sb s 0 0 0 4
Kan 1.01 y l a S s i 0 0 1 0
Munición 29.61 w l a m-s s f 9 7 2 15
Poolsanto 0.26 g-b l a S sb s 0 0 1 0
Putsicasutsuy 15.90 r-g l a m-s s i 1 2 5 2
Sac 25.12 w l a m-s s f 0 2 24 2
Sacmejen 4.78 w s a m-s s f 0 0 1 1
Tsisibal2 0.30 r-g l a m-s s i 0 0 2 0
Tuchasutsuy2 0.10 r-g l a m-s s i 0 0 1 0
Xananmucuy2 0.10 r-g l a m-s s i 0 0 0 1
Synonymous of Bacalar landrace. 2 Synonymous of Putsicasutsuy landrace. 3: % cultivated area in 160
farmers interviewed. 4: w = white, b = black, br = brown, r = red, g = gray, y = yellow, c = cream. 5:
l = 7 to 8 months, s = 3 to 4 months. 6: a = polyculture, m = monoculture. 7: m-s = subsistence and
market, s-m = principally subsistence
but also market, s = just subsistence. 10: s = sweet, sb = semibitter, b = bitter. 11: f = fast, i = intermediate,
s = slow.

The number of landraces cultivated by the farmers ranged from one to seven, with an
average of two. With the exception of xmejen, all the landraces were cultivated in polyculture
under the milpa system, with maize as the main crop and tutor plant for P. lunatus. Xmejen
was planted with sticks as tutors, in small, mechanized, irrigated areas. When the seed was
sufficient and the production had a commercial component, the farmers planted each landrace
separately in different sections of the milpa or in different milpas in order to maintain the
purity of their germplasm and obtain a better price on the market. When the quantities of seed
from each landrace were small and the production was destined to auto-consumption, they
planted their germplasm in the form of mixed seeds (the planting of seeds from different
Domestication and Conservation Genetics of the Lima Bean… 203

landraces mixed together in the same part of the milpa), regardless of the loss of the particular
characteristics of each landrace. This practice of planting a large number of variants (up to 7)
in one milpa, either in rows or mixed, favors the possibility of gene flow between one landrace
and another and subsequently the generation of variation which eventually could have been
selected by the farmers. Indeed, this practice favors the possibility of wild-domesticated
introgression, because the management of mixed seeds could to hide the generation and
existence of weedy forms.

2. Structure, genetic diversity, and gene flow in P. Lunatus

Wild gene pool

Fifty-nine alleles were found in all wild populations. At the Yucatan Peninsula level, we
found high levels of genetic diversity: A = 7.38, Ho = 0.67 and H = 0.69. Even though this
study considered a smaller number of populations, the obtained values were greater than those
reported for Central Valley of Costa Rica using isozymes: A = 1.1, Ho = 0.006 (Zoro Bi et al.,
2003); Ho = 0.012 (Ouédraogo and Baudoin, 2002), and SSRs: Ho = 0.031 (Ouédraogo and
Baudoin, 2002). These differences can be explained considering different factors: a) the
greater sensitivity of the SSR markers in the detection of polymorphisms. b) Different
outcrossing rates. In the Central Valley of Costa Rica, the outcrossing rate was estimated
between 0.027 and 0.268 (Zoro Bi, 1999). Recently, Chimal-Chan (2008) reported higher
outcrossing rates of up to 74% to CEQROO populations favored, possibly, for the high
abundance and diversity of local pollinating insects. c) The differential size of the populations
studied. The Yucatan Peninsula populations presented in their majority more than 100
individuals in the reproductive stage, whereas the Central Valley of Costa Rica populations
were much smaller, as 66% of populations were no larger than 30 individuals (Maquet et al.,
2001). A positive correlation between intra-population genetic variation and the size of the
population has been reported for Zoro Bi et al. (2003) in the Central Valley of Costa Rica for
P. lunatus, it suggesting endogamy to be the most plausible cause. d) Founder effect and
bottlenecks. Populations of the Central Valley of Costa Rica are subject to intensive
commercial agricultural management and to encroachment from urban areas. (Zoro Bi et al.,
2003). In contrast, the Yucatan Peninsula agriculture is still essentially traditional. Although
there have been important changes, fallow periods from three to eighteen years are conserved.
It had permitted that of the lima bean populations exist and they have not undergone episodes
of extinction and re-colonization or that these episodes have not been as frequent as those in
the Central Valley of Costa Rica.
At the population level, Xohuayán-2 exhibited the highest genetic diversity (Ho= 0.90)
(Table 6). The agricultural management of this region may explain this. Xohuayán-2 is located
in SYUC, a region where the farmers plant xmejen landrace, which has a high market value.
For marketing reasons, the farmers take great care in maintaining purity of this landrace by
ensuring that it do not crossbreed with the wild populations. Thus, they plant this germplasm in
sites where wild populations are not found or, if found, they eliminate them with herbicides. In
this way, by protecting the purity of this germplasm, the farmers are indirectly maintaining the
identity and diversity of the wild populations still existing in this area. In regard to allelic
richness, the Holpat, Kik, and Nohca populations presented the highest values (Table 6), which
could be a result, in the case of the two first populations, of their larger population size. In
204 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

contrast, Bolonchén presented the lowest value, a consequence of its small population size and
recent events of extinction–recolonization, which would have caused a process of genetic drift
in this population. Theoretical works on the effects of genetic drift suggest that allelic
frequencies fluctuating in small populations will produce a reduction in Ho (Wright, 1931;
Kimura, 1955; quoted by Cole, 1998). It has also been noted that genetic drift must have a
more immediate effect on the loss of rare alleles, thus causing a reduction of A (Cole, 1998).
Apart from the Bolonchén population, the San Fernando and Boje populations showed the
lowest allelic richness of all wild populations analyzed. These populations had some plants
with morphological characteristics of flowers, pods, and seeds very similar to those of the
domesticated germplasm, which is suggestive of past introgression events with domesticated
germplasm, causing a reduction in their genetic diversity.

Table 6. Estimators of the genetic diversity of 11 wild populations of P. lunatus

from the Yucatan Peninsula, Mexico. Taken from Martínez-Castillo et al. (2006).

Agricultural region Population Name N A Ae / A Ho H

CEQROO Holpat 20 3.38 0.70 0.46 0.54
Kik 20 3.38 0.64 0.60 0.48
Nohcá 14 3.38 0.67 0.52 0.51
Mean 44.7 3.38 0.67 0.53 0.51
SEYUC Boje 20 2.87 0.72 0.51 0.47
San Fernando 20 2.38 0.70 0.49 0.35
Mean 20 2.63 0.71 0.50 0.41
NECAMP Bolonchén 20 2.37 0.81 0.67 0.41
Chunchintok 19 3.13 0.83 0.67 0.57
Itzinté 20 3.25 0.85 0.82 0.59
Mean 19.7 2.92 0.83 0.72 0.52
SYUC Nohcacab 20 3.13 0.73 0.87 0.53
Xohuayán-1 20 3.25 0.77 0.82 0.55
Xohuayán-2 20 3.01 0.81 0.90 0.57
Mean 20 3.13 0.77 0.86 0.55
CEQROO, central eastern Quintana Roo; SEYUC, southeast Yucatan; NECAMP, northeast Campeche;
SYUC, south of Yucatan; Number of plants (n); Average number of alleles (A); Allelic frequencies
eveness ( Ae / A ); Observed heterozygosity (Ho); Nei’s index of diversity (H).

A comparative analysis among agricultural areas indicated significantly higher Ho values

for SYUC and NECAMP than those of CEQROO and SEYUC (Table 7). The SEYUC had the
lowest A value, but only significantly lower than CEQROO, which had the highest value.

The high A and low Ho values for CEQROO are explained by the low evenness of allelic
frequencies evaluated by Ae/A (Table 6). The SEYUC, in addition to the lowest A value, had a
low evenness coefficient. A possible explanation for the low diversity of CEQROO and
SEYUC may be the existence of gene flow from the domesticated to the wild populations.
Martínez-Castillo et al. (2004) found weedy plants growing within two domesticated
populations of P. lunatus in CEQROO.
Domestication and Conservation Genetics of the Lima Bean… 205

Table 7. Duncan’s test for comparison of means for the values of observed
heterozygosity (Ho) and average number of observed alleles (A) found
in 11 wild populations of P. lunatus from four agricultural regions
in the Yucatan Peninsula, Mexico. Taken from Martínez-Castillo et al. (2006).

Ho A
Agricultural region Mean Duncan test Mean Duncan test
SYUC 0.86 A 3.13 AB
NECAMP 0.72 B 2.92 AB
CEQROO 0.53 C 3.38 A
SEYUC 0.50 C 2.63 B
SYUC, south of Yucatan; NECAMP, northeast Campeche; CEQROO, central east Quintana Roo;
SEYUC, southeast Yucatan. Regions with the same letter are not different significantly (α= 0.05).

These weedy plants could be hybrid forms generated by gene flow events between
domesticated and wild populations. These authors also found a weedy population growing
beside a domesticated population in SEYUC. In this weedy population, plants were found with
wild-type seeds and others with domesticated-type seeds. Also, they found two wild
populations in SEYUC with wild–domesticated introgression characteristics (Table 3). Several
studies have indicated that the gene flow from the domesticated populations can diminish the
genetic diversity of the wild populations through the displacement of wild alleles (Gepts et al.,
The FIS tests indicated that 40.5% of the locus–population analyzed have an excess of
heterozygotes, 11.9% a deficit, and 47.6% are in Hardy-Weinberg equilibrium (Data not
showed). When the average values of FIS are obtained per population for all the loci studied,
the tests show that the 11 populations are in Hardy-Weinberg equilibrium, even though
Xohuayán-1, Nohcacab, and Xohuayan-2 had a high number of loci with heterozygote excess
(each with five). At the level of loci for the entire peninsula, the tests indicated that four of the
eight loci studied presented an excess of heterozygotes (AG1, BM140, BM156, and BM160),
one locus showed a deficit (GATS91), and three loci were in Hardy-Weinberg equilibrium
(BM164, BM183, and BM211). These results at the locus–population and the loci–Peninsula
levels show evidence of a tendency toward an excess of heterozygotes in the wild pool of P.
lunatus, an excess, perhaps, as a consequence of natural selection favoring heterozygosity
and/or of a Wahlund effect inside the populations. This result is in contrast with that reported
by Zoro Bi et al. (2003) for the Central Valley of Costa Rica. These authors found that the
populations of that region also deviated from the HWE; however, they found a deficit of
heterozygosity in those populations. These differences between both regions appear to
correspond to the difference in the size of the populations and the levels of endogamy.
In the Yucatan Peninsula, 27 % of the total variation was found among populations (GST =
0.27). This result was supported by an AMOVA, which showed that 27 % of the total variation
was found among populations and 73% within populations. These results can be explained by
a low level of long-term gene flow found (Nm = 0.66). Our results were lower than those
reported in the Central Valley of Costa Rica (GST = 0.56) (Ouédraogo and Baudoin, 2002).
This could be a consequence of a lower level of long-term gene flow present in that region
(Nm = 0.17) than in the Yucatan Peninsula. Low levels of gene flow in the wild populations of
206 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

P. lunatus in the Central Valley of Costa Rica were also reported by Hardy et al. (1977) and
Baudoin et al. (1998).
Figure 3 shows the UPGMA of the 11 wild populations analyzed. The topology indicates a
grouping in accordance with the geographical location of the populations, with the exception
of the group comprising the populations of San Fernando and Boje, located in SEYUC, and
Chunchintok, located in NECAMP. A possible explanation of the clustering of Chunchintok
with the populations of SEYUC could be the accidental transportation of seed by Campeche
farmers, who mentioned that they transport their agricultural products for sale in Valladolid,
the principal town of SEYUC. The clustering patterns of the populations agreed with the
results obtained from the Mantel test (data no showed), which indicates the existence of a
geographical isolation among these wild populations.

Domesticated gene pool

Thirty-one alleles were found in the domesticated populations. Considering the entire
Yucatan Peninsula, we found the following values of allelic richness and genetic diversity: A =
4.0, Ho = 0.62 and H = 0.51. These values were lower than those found in the wild populations
of this region. These differences can be explained for a founder effect occurring during the
domestication process, which has been reported for P. lunatus (Gutiérrez-Salgado et al., 1995)
and other cultivated species (Ladizinsky, 1985), and/or for a genetic erosion effect in the
domesticated gene pool due to changes associated with intensification of traditional agriculture
during recent decades, as it has been reported for common bean (P. vulgaris L.) in central
Mexico (Payro de la Cruz et al., 2005; Zizumbo-Villarreal et al., 2005).
At the population level, San Fernando exhibited the highest diversity (Ho and H) (Table
8). In regard to allelic richness, San Fernando, Marcos and Pascual populations presented the
highest values, the two first located in SEYUC and the other one in NECAMP. This could be a
result, in the case of the two first populations, due to they presented the biggest number of
landraces planted and they grew near to wild populations favoring the introgression of wild
alleles into the domesticated populations.
A comparative analysis among agricultural areas for genetic diversity and allelic richness
indicated significantly higher Ho values for SEYUC, NECAMP and SYUC than those of
CEQROO (Table 9). In relation to the allelic richness, the SEYUC had again the highest A
value, following for NECAMP. SYUC and CEQROO had significantly lower values.
Martínez-Castillo et al., (2004) found a weedy population growing beside a domesticated
population in SEYUC. It could be that this situation increased the genetic diversity and allelic
richness of this area due to the entrance of wild alleles to its domesticated gene pool. In the
case of SYUC, a possible explanation for the low genetic diversity and allelic richness could
be the high dominance of xmejen in this area.
This landrace has replaced to the other ones due to its highest economic value and with
this the replacement of rare alleles present in the other landraces. In the case of CEQROO, its
low genetic diversity and allelic richness could be a result of the high interchange of seeds
among Mayan farmers from the agricultural towns where the germplam analyzed was
collected. Many of the farmers of these towns are relatives or friends
When the farmers lost its seed, they recover it from the seed lot of their relatives or
friends. This situation may increase the endogamy phenomenon and with this to decrease the
genetic diversity.
Domestication and Conservation Genetics of the Lima Bean… 207

Table 8. Estimators of the genetic diversity of 12 domesticated

and one weedy population of P. lunatus from the Yucatan Peninsula, Mexico

Agricultural region Population Name N A Ae/A Ho H

CEQROO Celestino 19 1.87 0.83 0.50 0.30
Domingo 18 1.63 0.95 0.53 0.28
Julián 20 1.63 0.97 0.56 0.30
SEYUC Marcos 20 2.13 0.82 0.63 0.34
San Fernando 20 2.13 0.92 0.73 0.41
X-Uilub 20 2.01 0.84 0.67 0.35
NECAMP Bolonchén-3 20 1.75 0.93 0.63 0.32
Elías 20 1.75 0.92 0.63 0.32
Pascual 20 2.13 0.79 0.64 0.35
SYUC Rubén 20 1.63 1.00 0.63 0.31
Xohuayán-2 20 1.75 0.93 0.62 0.32
Xohuayán-3 20 1.63 1.00 0.63 0.31
CEQROO, central eastern Quintana Roo; SEYUC, southeast Yucatan; NECAMP, northeast Campeche;
SYUC, south of Yucatan; Number of plants (n); Average number of alleles (A); Allelic frequencies
eveness ( Ae / A ); Observed heterozygosity (Ho); Nei’s index of diversity (H).

Figure 3. UPGMA based on Nei’s genetic distance (1972) of 11 wild populations of P. lunatus studied in
four agricultural areas of the Yucatan Peninsula, Mexico. The numbers above the lines are the proportion
of similar replicates supporting each node. Taken from Martínez-Castillo et al., 2006.
208 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

The FIS tests indicated that 96.96% of the locus–population analyzed has a deficit of
heterozygotes and 3.4% an excess (Data not showed). When the average values of FIS are
obtained per population for all the loci studied, the tests show that the 12 populations are not in
Hardy-Weinberg equilibrium. At the level of loci for the entire peninsula, the tests indicated
that the nine loci studied presented a deficit of heterozygotes. These results at the locus–
population and the loci–Peninsula levels show evidence of a deficit of heterozygotes in the
domesticated gene pool of P. lunatus, a deficit, perhaps, as a consequence of an interchange of
domesticated seeds among farmers into the same areas favoring homozygosity in this gene

Table 9. Duncan’s test for comparison of means for the values of observed heterozygosity
(Ho) and average number of observed alleles (A) found in 12 domesticated populations of
P. lunatus from four agricultural areas in the Yucatan Peninsula, Mexico

Ho A
Agricultural region Mean Duncan test Mean Duncan test
SEYUC 0.68 A 2.09 A
NECAMP 0.63 A 1.88 A-B
SYUC 0.63 A 1.67 B
CEQROO 0.53 B 1.71 B
SYUC, south of Yucatan; NECAMP, northeast Campeche; CEQROO, central east Quintana Roo;
SEYUC, southeast Yucatan. Regions with the same letter are not different significantly (α = 0.05).

In the Yucatan Peninsula, 36 % of the total variation was found among populations (GST =
0.36). This result was supported by an AMOVA, which showed that 35.5 % of the total
variation was found among populations and 64.5 % within populations. The AMOVA showed
that the highest differentiation was among populations from different areas, too. These results
can be explained by a low level of long-term gene flow found at the Peninsula level (Nm =
0.44) in comparison with the highest values found at the area level, where CEQROO have the
highest value (Nm = 18.1) and SYUC the lowest (Nm = 2.9).
Figure 4 shows the topology generated with a UPGMA of the 12 domesticated populations
analyzed. This topology indicates a grouping in accordance with the geographical location of
the populations. The clustering patterns of the populations agreed with the results obtained
from the Mantel test (data no showed), which indicates the existence of a geographical
isolation among the domesticated populations of P. lunatus.
Analyzing in conjunction the 24 wild, weedy and domesticated populations, at the
Peninsula level the GST value was of 0.47.
This result was supported by an AMOVA which showed that 45.89% of the total variation
was among populations (24.10% among gene pools and 21.79% among populations into each
gene pool). At the intraregional level, the GST values were of 0.33 to NECAMP, 0.37 to
CEQROO, and 0.45 to SEYUC and SYUC.
These results were supported by an AMOVA which showed that in NECAMP the 37.72%
of the total variation was among populations (27.3% among gene pools and 10.42% among
populations into each gene pool); in CEQROO was of 46.54% (41.66% among gene pools and
4.54% among populations into each gene pool); in SEYUC was of 49.17% (43.8% among
gene pools and 5.37% among populations into each gene pool); and in SYUC was of 51.49%
Domestication and Conservation Genetics of the Lima Bean… 209

(44.7% among gene pools and 6.79% among populations into each gene pool). At the parcel
level, the GST value was of 0.29. It was supported by an AMOVA which showed that 38.33%
of the total variation was among weedy and domesticated populations.

Genetic structure of P. lunatus.

Figure 4. UPGMA based on Nei’s genetic distance (1978) of 12 domesticated populations of P. lunatus
studied in four agricultural areas of the Yucatan Peninsula, Mexico. The numbers above the lines are the
proportion of similar replicates supporting each node.

These results show that the genetic differentiation in the wild–weedy–domesticated

complex of P. lunatus was high at the different levels analyzed, even at the parcel level where
the weedy and domesticated plants grew very close to each other (inclusive on the same maize
plant). It could be a result of the low levels of gene flow between the wild and domesticate
gene pools, as it was indicated for the AMOVA analyses that showed higher levels of
differentiation among gene pools than among populations from the same gene pools.

3. Wild-domesticated gene flow

Recent gene flow

At the Peninsula level, Bayesian clustering analysis showed that most of the wild
populations were subjected to gene flow from the domesticate gene pool (Figure 5, Table 8),
with the highest Q values in the Bolonchén (0.513) and Itzinté (0.167) wild populations in
NECAMP (Table 8). Neither of these populations had morphological evidence of introgression
but they did grow a short distance from domesticated populations. The Chunchintok (0.035)
and Boje (0.027) populations had midlevel Q values (Table 8), the latter included two plants
with morphological seed characteristics indicating introgression from the domesticate gene
pool. After the Bolonchén wild population, the weedy population had the second highest Q
value (0.370) (Table 8). This population was found growing together with a domesticated
210 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

population in SEYUC and two types of seeds were collected from it: those with wild-type
characteristics and others with domesticate-type characteristics.

Figure 5. Coefficients of estimated ancestry per individual (q), grouped by population, biological status
and agricultural region. Each individual is represented by a single vertical line broken into two colored
segments, with lengths proportional to the individual’s estimated ancestry fraction from each of the two
biological statuses: Wild (gray) and domesticate (black). Populations: San Fernando-1 (1), Weedy (2),
Boje (3), San Fernando-2 (4), Marcos (5), X-Uilub (6), Celestino (7), Domingo (8), Julián (9), Nohca
(10), Kik (11), Holpat (12), Xohuayán-1 (13), Nohcacab (14), Xohuayán-2 (15), Xohuayán-3 (16),
Xohuayá n-4 (17), Rubén (18), Itzinté (19), Bolonchén-1 (20), Chunchintok (21), Bolonchén-2 (22),
Elías (23), and Pascual (24). Taken from Martínez-Castillo et al., 2007.

In contrast, most of the domesticated populations had very low gene flow levels from the
wild gene pool (Figure 5, Table 8). The highest Q values were in Pascual (0.063) and
Bolonchén (0.018), located in NECAMP, and Celestino (0.029) in CEQROO (Table 8). No
morphological evidence of introgression was noted in the Pascual population and there were
no wild populations nearby. Bolonchén was one of the domesticated populations growing next
to the Itzinté wild population, but it did not manifest any morphological evidence of
introgression. The farmer cultivating the Celestino population reported that wild plants had
grown there in the last 3 yr, and one weedy plant with wild-type seeds similar to those in the
weedy population was collected there.
The interregional analysis showed that gene flow does exist between agricultural areas,
although at low levels (Table 9). The SEYUC wild pool had a higher reception of domesticate
genes from other agricultural regions, with the CEQROO (Q = 0.004) and NECAMP (Q =
0.003) domesticate gene pools being those contributing the most domesticate alleles. One
individual from the weedy population in SEYUC had a q-probability = 0.072 of having a
domesticated parent from CEQROO, while four others had a q-probability = 0.057 of the
same. In CEQROO, one wild individual showed a q-probability = 0.012 of having a
domesticated grandparent from NECAMP, and in SYUC another individual showed a q-
probability = 0.032 for the same. The region with the highest levels of wild gene infiltration
toward the domesticate pool was CEQROO, where one domesticated individual had a q-
probability = 0.022 of belonging to the SEYUC wild pool. In NECAMP, one domesticated
individual from the Bolonchén population showed a q-probability = 0.072 of having a SEYUC
wild grandparent and a q-probability = 0.059 of having a CEQROO wild grandparent.
Domestication and Conservation Genetics of the Lima Bean… 211

Table 8. Proportion of estimated ancestry (Q) of 24 wild and domesticated P. lunatus

populations from four agricultural regions on the Yucatan Peninsula, Mexico.
Taken from Martínez-Castillo et al. (2007).

Agricultural region Population Q domesticate pool Q wild pool

SEYUC San Fernando (w) 0.006 0.994
Marcos (we) 0.370 0.630
Boje (w) 0.027 0.973
San Fernando (d) 0.992 0.008
Marcos (d) 0.995 0.005
X-Uilub (d) 0.994 0.006
CEQROO Nohca (w) 0.028 0.972
Kik (w) 0.007 0.993
Holpat (w) 0.007 0.993
Celestino (d) 0.971 0.029
Domingo (d) 0.993 0.007
Julián (d) 0.993 0.007
SYUC Xohuayán-1 (w) 0.006 0.994
Nohcacab (w) 0.012 0.988
Xohuayán-2 (w) 0.011 0.989
Xohuayán-1 (d) 0.995 0.005
Xohuayán-2 (d) 0.994 0.006
Rubén (d) 0.995 0.005
NECAMP Itzinté (w) 0.167 0.833
Bolonchén (w) 0.513 0.487
Chunchintok (w) 0.035 0.965
Bolonchén (d) 0.982 0.018
Elias (d) 0.987 0.013
Pascual (d) 0.937 0.063
SEYUC, southeastern Yucatan; CEQROO, central eastern Quintana Roo; SYUC, southern Yucatan;
NECAMP, northeastern Campeche. Domesticated (d), weedy (we), wild (w).

The intraregional analysis showed gene flow levels even lower than at the Peninsula level
(data not shown). An appreciable gene flow from the domesticate gene pool toward the wild
gene pool was observed in SEYUC. In the weedy population one individual showed a q-
probability = 0.184 of having a domesticated parent and four individuals showed a q-
probability = 0.094 of having a domesticated grandparent. Wild individuals from other areas
showed q-probabilities from 0.000 to 0.04 of having a domesticated grandparent. Gene flow
was even lower from the wild gene pool toward the domesticate gene pool. In CEQROO, just
one individual showed a q-probability = 0.720 of being a wild immigrant, and in NECAMP
only two domesticated individuals showed q-probabilities between 0.170 and 0.199 of having
a wild grandparent. In SEYUC, one domesticated individual showed a q-probability = 0.147 of
having a wild grandparent, while the remaining individuals had q-probabilities between 0.001
and 0.004. All domesticated individuals in SYUC had q-probabilities = 0.001 of having a wild
212 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

Table 9. Proportion of estimated ancestry (Q) of eight wild and domesticate P. lunatus
gene pools from four agricultural areas on the Yucatan Peninsula, Mexico.
Taken from Martínez-Castillo et al. (2007).

Q of agricultural region / biological status gene pool

Agricultural Biological SEYUC CEQROO SYUC NECAMP
region Status W D W D W D W D
SEYUC W 0.988 0.001 0.001 0.004 0.000 0.002 0.000 0.003
D 0.000 0.998 0.000 0.000 0.000 0.001 0.000 0.001
CEQROO W 0.000 0.000 0.998 0.000 0.000 0.000 0.000 0.000
D 0.001 0.000 0.005 0.994 0.000 0.000 0.000 0.000
SYUC W 0.001 0.000 0.001 0.000 0.997 0.000 0.000 0.000
D 0.000 0.001 0.000 0.000 0.000 0.998 0.000 0.000
NECAMP W 0.002 0.000 0.001 0.000 0.001 0.000 0.995 0.000
D 0.001 0.000 0.001 0.001 0.000 0.000 0.000 0.997
SEYUC, southeastern Yucatan; CEQROO, central eastern Quintana Roo; SYUC, southern Yucatan;
NECAMP, northeastern Campeche. Domesticate (d), wild (w).

The parcel level analysis also showed low gene flow levels between wild and domesticate
gene pools. Just one individual manifested a q-probability = 0.006 for having a domesticate
parent, while the remaining individuals showed q-probabilities ranging from 0.003 to 0.021 for
having a domesticate grandparent. Domesticate individuals generally showed lower q-
probabilities (from 0.004 to 0.005) for having a wild grandparent, though one did show a q-
probability = 0.001 for having a wild parent and a q-probability = 0.064 for having a wild
The low observed recent gene flow levels at the interregional, intraregional, and parcel
levels were likely due to the limited outcrossing potential of P. lunatus. This is correlated with
its short life cycle, the predominance of self-pollination and its limited ability for pollen and
seed dispersal (Maquet et al., 1997). Though crossing rates of up to 48% have been reported,
the synchronized ripening of pollen grains and the stigma, as well as their proximity in the
bud, favor autogamy in P. lunatus (Baudoin et al., 1998). These authors reported that
horizontal pollen and seed transference did not exceed 6 m. and the neighborhood size was 1.6
m. in wild populations in the Central Valley of Costa Rica.

Long-term gene flow

The Nm estimator showed relatively low gene flow values at the Peninsula level (Nm =
0.28), as well as at the intraregional level: NECAMP (Nm = 0.51), CEQROO (Nm = 0.42),
SEYUC (Nm = 0.31), and SYUC (Nm = 0.31). These results coincide with data reported for
wild populations in Costa Rica (Hardy et al., 1997; Maquet et al., 2001; Ouédraogo and
Baudoin, 2002). This may be explained by the joint action of limited recent gene flow and
continuous selective pressure exercised by Mayan farmers against wild progenitor hybrids and
The admixture analysis showed the estimated contribution of Pw to PhyD (mWD = 0.12 ±
0.02) to have been less than the estimated contribution of PD to PhyW (mDW = 0.34 ± 0.04).
These values generate a ratio of mDW/mWD = 2.83, meaning there was an asymmetrical gene
flow almost three times greater from the domesticate pool toward the wild pool. The highly
asymmetrical gene flow can be explained for the migratory-recurrent nature of the swidden
system. This characteristic, combined with the existence of wild P. lunatus seed banks in the
Domestication and Conservation Genetics of the Lima Bean… 213

soil, can favor or limit genetic contact between wild and domesticated populations. Mayan
farmers on the Peninsula cultivate their plots for 1 to 3 yr and then leave them fallow for 5 to
15 yr. If wild P. lunatus seeds are in the soil, they will germinate when farmers cut and burn
the vegetation for a new agricultural cycle, leading to sympatric growth with domesticated
populations and thus increasing the possibility of introgression between the two gene pools.
Papa and Gepts (2003) found similar results in P. vulgaris from Central Mexico and
suggested two factors to explain the asymmetrical gene flow: (i) the smaller size or lower
density of wild populations compared to domesticated populations; and (ii) the role of farmers
in seed selection. Though hand weeding is still common on the Yucatan Peninsula, increasing
use of herbicides is leading to drastic reductions in the density of wild populations, a greater
pollen production by the domesticate pool relative to that of the wild population, and
consequently a higher pollen flow toward the wild pool. Seed selection also clearly favors the
domesticate pool. In cultivated environments, farmers easily recognize and select against F1
domesticate X wild hybrids because their seeds have generally an intermediate size between
those of the parents and a different color from the domesticated maternal parent. Natural F1
wild X domesticate hybrids, in contrast, can be better adapted due to their hybrid vigor and the
overall dominance of wild-type traits favoring later recombination and introgression of
domesticate alleles into the wild pool (Singh et al., 1995; Papa et al., 2005). In addition to this,
Mayan farmers can also distinguish and select against hybrids based on seed flavor. Wild seeds
contain high concentrations of linamarine, a cyanogenic compound that makes them inedible
(Maquet, 1991). In cases of introgression, hybrid seeds acquire a bitter taste that is easily
detected, leading farmers to dispose of the harvest. Another factor that may be further limiting
the entrance of wild genes into the domesticate gene pool is selection for external markets.
Regional markets on the Yucatan Peninsula currently prefer white-seed landraces, favoring
elimination of hybrid seeds of different colors. This may be the case in SYUC, where a
dominant selection criterion is focused on production of seed for sale. Farmers in this region
report the intentional elimination of wild populations with herbicides to avoid mixing with
their landraces and attain a better price (Martínez-Castillo et al., 2004). This may explain why
SYUC domesticated populations have a lower degree of genetic infiltration.
Certain aspects of the Mayan traditional agriculture in the region, however, favor the
entrance of wild genes into domesticated populations (Martínez-Castillo et al., 2004): (a)
lochepak allows wild and weedy plants to reach the flowering stage at the same time as
domesticated populations because it eliminates only the aerial part of the plant, allowing the
subsequent recovery: (b) wild populations growing near domesticated ones are tolerated when
they do not affect the correct development of their crops, as was the case in NECAMP and; (c)
cultivation for subsistence purposes includes up to seven different types of landraces that, after
hybridizing, create a wide variety of seed shapes, sizes, and colors that can hide the presence
of weed seeds; (d) women and children, who may not readily recognize weedy P. lunatus
seeds, sometimes participate in agricultural activities such as harvest. All of these aspects can
explain why reports for P. vulgaris in Costa Rica contrast with the present results in that gene
flow appears to move from the wild toward the domesticate gene pool in that country, as was
found by González-Torres (2004) using chloroplast DNA markers. Another explication about
the findings of González-Torres (2004) in contrast with ours could be that chloroplast
introgression occurs predominantly from wild to domesticate gene pool, whereas the
introgression of nuclear genes, as our microsatellite data, is predominantly from the
domesticate to wild gene pool (Papa and Gepts, 2003; Chacón et al., 2005).
214 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

4. Genetic erosion in the domesticated gene pool

A total of 90 loci were analyzed of which 71 were polymorphic and 19 monomorphic. At

the total domesticated gene pool level, genetic diversity was high, and the three estimators
showed similar values (I = 0.33, H = 0.28, HB = 0.31) (Table 10). Using alloenzymes, Maquet
et al. (1997) reported an H = 0.26 for the P. lunatus base collection of the Germplasm Bank of
the International Center for Tropical Agriculture (CIAT-Colombia). These authors stated that
this is a significant level and higher than reported for other plants that, like P. lunatus, are
mixed-mating or short-lived perennial species (H = 0.12) (Hamrick et al., 1991). The values
obtained in this study are higher to those found for Nienhuis et al. (1995) using RAPDs on the
domesticated Mesoamerican material (H = 0.11) and to those found for Castiñeiras et al.
(2007), using AFLP molecular markers on the Potato-Sieva landraces planted in the Cuban
home gardens (H = 0.119). Compared with these studies, our results could be reflecting the
high genetic diversity maintained by Mayan farmers in the milpa of the Yucatan Peninsula,
Mexico. However, the studies cited here were made using different methodologies to collect
the plant material (size of samples, sampling methods, different origin of samples-field or
genebank-), and cautions should be taken.
Compared to the wild gene pool, the domesticated lima bean gene pool from the Yucatan
Peninsula had lower genetic diversity values. Using SSR loci, Martínez-Castillo et al. (2006)
reported an H = 0.69 for wild lima beans, which is almost three times higher than found for the
domesticated gene pool (H = 0.28). A number of factors may explain these differences: (a) a
founder effect occurring during the domestication process, which has been reported for P.
lunatus (Gutiérrez-Salgado et al., 1995) and other cultivated species (Ladizinsky, 1985); (b) a
genetic erosion effect in the domesticated gene pool due to changes associated with
intensification of traditional agriculture during recent decades, as it has been reported for
common bean (P. vulgaris) in central Mexico (Payro de la Cruz et al., 2005; Zizumbo-
Villarreal et al., 2005); and (c) the type of data generated by the different markers used in this
analysis (ISSR-dominant markers) vs. Martínez-Castillo et al. (2006) analysis (SSR-
codominant markers).
At the landrace groups level, the common landraces had the highest genetic diversity
values (except for % P), although the differences between the three groups were not
statistically significant.
The rare landraces group had genetic diversity values (H and I) slightly lower than the
common landraces group, but higher for % P (Table 10). It is probably due to the fact that nine
of the 12 rare landraces were represented only by a single accession, whereas all the common
landraces were represented by at least five accessions.
The rare landraces’ minimal abundance is the main factor that most increases the risk of
genetic erosion since it can lead to their local extinction. During a germplasm collection in
2007, a farmer from SEYUC reported that he had lost his seed of Pool-santo and Chak-chí
landraces in the 2006 agricultural cycle due to a lack of rain.
In another case, a farmer from CEQROO stopped planting the Chocolate and Tabaco
landraces in 2005 because he became sick that year and did not cultivate his milpa.
Unfortunately, when we made the germplasm collection, this farmer was the only one who had
these landraces. At present these two rare landraces have not been collected again.
Domestication and Conservation Genetics of the Lima Bean… 215

Table 10. Estimators of genetic diversity of lima bean landraces groups

from the Yucatan Peninsula, Mexico, using 90 ISSR loci.
Taken from Martínez-Castillo et al. (2008).

Percentage of Shannon´s Nei´s gene Average

polymorphic diversity diversity (H) heterozygosity (HB)
loci (% P) index (I)
Total domesticated gene pool 78.9 0.33 0.29 0.31
Groups of landraces
Dominant landraces 26.7 0.17 a 0.13 a 0.27 a
Common landraces 58.9 0.33 a 0.26 a 0.37 a
Rare landraces 66.7 0.27 a 0.24 a 0.28 a
Groups with the same letter are not different significantly (α= 0.05).

Two factors that could reduce the risk of genetic erosion in some of the rare landraces are
dark seed color and their mixed management by Mayan farmers. Both aspects favor the
entrance of wild alleles through formation of wild-weedy-domesticated complexes and the
generation of weedy forms (Martínez-Castillo et al., 2004). Two special cases in the use of
seed mixtures are the Bacalar and Balche landraces. These have become a kind of ‘‘genetic
dump’’ as they contain seeds similar to many different landraces, such as Box-petch, Putsica-
sutsuy, Chak-petch, Chaksaac, Pool-santo, among others. Indeed, in 2007 weedy forms were
observed among the seeds harvested of Bacalar in CEQROO.
The abundant landraces group had the lowest values of genetic diversity among the three
groups for all estimators, except for H that showed the same value showed for the rare landrace
group (Table 10). These low values could be reflecting a germplasm selection influenced by
external market demands. Martínez-Castillo et al. (2004) reported that one of the main
selection criteria applied to the three most abundant landraces is seed production for sale. As a
result of this, Mayan farmers currently tend to plant white seeded landraces. This leads to a
selection against weedy forms produced from crosses between landraces and the wild
populations surrounding milpas, consequently limiting introgression of wild alleles and
increasing the risk of genetic erosion. In relation to dominant lima bean landraces, Debouck
(1979) collected at least 10 different landraces in the Mayan community of Nohalal,
Campeche, but currently only three have been observed and these are dominated by Mulición
and Sac (direct observation). Informal interviews with Mayan producers in Nohalal suggest
that this loss of landraces is associated with the introduction of mechanized agriculture and
monoculture of improved varieties of corn. Recent field observations indicate that even the
planting of abundant lima bean landraces is decreasing in response to low prices. A similar
case is happening in SYUC, where the xmejen has been replacing the other landraces with
color of seed different from white (Martínez-Castillo et al., 2004). Recently, it has decreased in
cultivated area as a result of a low in demand markets. Even though in this study xmejen is
considered as a landrace, there are evidences that it could be a improved variety introduced
approximately 25 years ago: (1) it was not found by Debouck in 1979, (2) it is a variety
planted as a monocrop (an aspect non common in the traditional Mayan agriculture) and it is
no associated with maize as all the other landraces, and (3) it is a variety with a very short
productive cycle that depends on a lot of water, a limited resource in the Yucatan Peninsula.
This decrease in the number and density of planted populations may mean that a new genetic
bottleneck is soon to come for the abundant landraces.
216 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

One little-studied factor in the genetic erosion of crops is change in the food preferences of
the rural populations. For lima bean in the Yucatan Peninsula this currently takes three forms:
(a) young adults and children do not eat it; (b) only the elderly plant many of the rare landraces
for their own use; and (c) cowpea (Vigna unguiculata (L.) Walpers, locally known as x-pelon),
introduced to the region from Africa in the 20th century, has been replacing P. lunatus. In fact,
lima bean is progressively being replaced in some regions of Latin America by other food
legumes (Maquet et al., 1997). As it is a long process to re-introduce a crop plant, in a study
conducted in Cuba, Esquivel and Hammer (1988) proposed to maintain lima bean landraces as
part of the traditional horticultural system. In several Mayan towns of the Yucatan Peninsula,
some landraces are planted into the home gardens. However, it is not a common agricultural
practice. On the other hand, loss of landraces is also apparently linked to the different
generations in human populations. Reports document that the Mayan farmers planting a large
variety of rare landraces are elderly and their death almost surely means the loss of these
landraces (direct observation).

The greatest intra-specific diversity of P. lunatus found in the Yucatan Peninsula by
Ballesteros (1999), when compared to the rest of Mexico, could be explained by the results
presented in this chapter. The variation in P. lunatus observed suggested the presence of
complex gene pools in the Maya lowlands area that may have resulted from sympatric contact
between wild, weedy, and cultivated populations. Such a situation is similar to those described
by Beebe et al. (1997) for P. vulgaris in the Andean center and by Papa and Gepts (2003) and
Zizumbo-Villarreal et al. (2005) for Mesoamerica. In both cases, gene flow between
components of the complexes has been considered an important mechanism for generating
genetic variability (Beebe et al. 1997; Harlan 1992). Ethnobotanical data suggested that Mayan
farmers favored the formation and maintenance of these complex gene pools through: 1)
traditional weeding practices that did not totally eliminate weedy plants; 2) side-by-side
planting of different landraces; and, 3) sowing mixtures of landraces when seeds were scarce.
On the other hand, this great diversity could lie in the profound and continuous history of
selection pressures and traditional management practices exercised by Mayan farmers over
thousands of years and/or to be a result of the complexity of human migrations in the area and
the history of germplasm exchange that has been maintained with other subcultural areas in
Mesoamerica since the establishment of the first agricultural communities (Colunga-
GarcíaMarín et al., 2003).
Molecular data indicated that the wild and domesticated gene pools of P. lunatus have a
high intrapopulation genetic diversity and a structure that results from processes of geographic
isolation and low levels of gene flow. The wild gene pool has a higher genetic diversity than
the domesticated one due to, possibly, a founder effect occurring during their domestication
process. In the case of the wild populations, molecular data suggested a positive correlation
between agricultural intensification and increase in diversity, as greater values of Ho were
recorded in the areas with greater intensification. These results suggest that wild populations of
P. lunatus are actually favored by the intensification of disturbance in situations involving at
least 3 yr of fallow. However, the opposite could be true at higher levels of intensification, as
Domestication and Conservation Genetics of the Lima Bean… 217

can be observed in the Central Valley of Costa Rica, where the diversity is diminishing. Two
aspects could explain these results: (i) long fallow periods, combined with the existence of a
soil seed bank, favors the existence of gene flow from domesticated toward the wild pool,
diminishing with this its genetic diversity, as it could be the case of the SEYUC (fallow of 10
yr): and (ii) longer fallow periods determine stronger bottlenecks and genetic drift on the soil
seed bank, diminishing with this also the genetic diversity of the wild populations. In relation
to the domesticated gene pool, although no clear relationship between genetic diversity and
intensification was observed using molecular data, the area showing the highest levels of
genetic diversity was SEYUC, which presented the lowest levels of agricultural intensification
and in which wild-weedy-domesticated complexes were found. In this way, the higher genetic
diversity found in one of the areas with lower agricultural intensification and with the presence
of weedy populations (SEYUC), in contrast with the lower allelic richness found in the
domesticated gene pool of the area with the highest agricultural intensification (SYUC),
suggested that the existence of wild-weedy-domesticated complexes and the agricultural
intensification are key factors in designing in situ conservation programs. When the genetic
diversity of the lima bean was analyzed at the landrace level, we found that it remains high in
comparison with other studies. However, it is important to say that the rare landraces from the
Yucatan Peninsula are in a higher risk of genetic erosion because, with few individuals living
per landrace and with moderate genetic diversity, it represents the greatest loss of unique
alleles if these landraces go to local extinction. On the other hand, the abundant landraces have
the lowest genetic diversity levels and are thus at great risk of genetic erosion due to selection
criteria imposed by an external market, too.
If data about relative abundance reported by Martínez-Castillo et al. (2004) reflect the
current condition of the domesticated lima bean pool in the Yucatan Peninsula, then this
species is at very high risk of genetic erosion since this region is one of its main centers of
genetic diversity in Mesoamerica. If current trends continue in the region, many lima bean
landraces may cease to be grown into the milpa in two to three generations. When compared
with other Mesoamerican regions, wild and domesticated gene pools of P. lunatus presents in
the Yucatan Peninsula showed genetic diversity values higher than those reported for Cuba and
Costa Rica, and similar genetic diversity when compared with all species. Our results could be
reflecting the high genetic diversity of this species maintained in the traditional agricultural
systems of the Yucatan Peninsula, Mexico.
On the other hand, the findings reported in this chapter are very important for the
conservation and biosafety of domesticated and wild P. lunatus populations within this
Mesoamerican center of diversity. Even with the low levels of gene flow reported in this
chapter, the asymmetrical gene flow from the domesticated to the wild gene pool may create a
drastic reduction in the genetic diversity of wild populations and even lead to local extinctions.
This in turn could affect the genetic diversity of the domesticate gene pool and the availability
of agriculturally interesting genes for plant breeders. In addition, many of the characteristics
incorporated into domesticated plants using traditional improvement methods (e.g., lack of
seed latency, dwarfing, and dependence on nutrient-rich soils) are maladaptive for wild plants
(Ellstrand and Hoffman, 1990), meaning hybrids between domesticated forms and their wild
parents may be poorly adapted to uncultivated environments, thus diminishing or even
preventing transference of domesticate genes within natural populations (Doebley, 1992;
National Research Council, 1989). However, the characteristics genetically transferred by
genetic engineering (e.g., herbicide, pest and disease resistance) may provide an adaptive
218 Pallavi Sharma, Ambuj Bhushan Jha and R. S. Dubey

advantage to wild plants (Gasser and Fraley, 1989). If these characteristics are introduced into
this crop by genetic engineering, domesticate–weedy hybrids could threaten the current host–
pest balance (Ellstrand and Hoffman, 1990). The problem becomes even more complex taking
into account gene flow between pools from different agricultural regions through deliberate or
accidental seed movement.
The results presented here indicate the importance of initiating in situ and ex situ
conservation programs for P. lunatus in the Yucatan Peninsula, especially given the
accelerated pace of agricultural intensification in the region. To do this, areas need to be
selected that favor in situ conservation while considering the natural, economic, social and
cultural factors that contribute to this conservation. Two areas that must be considered are
CEQROO and SEYUC, areas with low agricultural intensification and a high number of rare
landraces. Unexpected conditions such as the randomness of rainfall events, relatively few
peasants planting ‘‘rare’’ varieties, and the older age of farmers could lead to significant losses
of germplasm even within the span of a single human generation. In situ conservation
programs must include significant cultural reinforcement programs, since the relevant
objective is not the conservation of available germplasm by farmers, but that farmers continue
to play a dynamic role in the generation of new germplasm. In situ conservation programs are
needed that address: (a) an emergency collecting effort to save all landraces ex situ as a backup
for the in situ conservation activities, (b) in situ conservation of landraces and the alleles they
consist of; (c) generation of wild-weedy-domesticated complexes that allow introgression of
wild alleles into landraces; and (d) reintroduction of rare landraces and programs to promote
their planting and acceptance among young Mayan producers and their families.

This chapter is a result of the first author’s doctoral dissertation at the Centro de
Investigación Científica de Yucatán, A. C. The authors thank P. Gepts, Hugo Perales and P.
Delgado for their academic advice. The first author thanks the Consejo Nacional de Ciencia y
Tecnología-Mexico for the scholarship for his postgraduate studies, UCMEXUS-CONACYT
and SINAREFI-SAGARPA for the economic support to carry out this research.

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In: Corn Crop Production Growth, Fertilization and Yield ISBN 978-1-60741-955-6
Editor: Arn T. Danforth © 2009 Nova Science Publishers, Inc.

Chapter 5



Márcia do Vale Barreto Figueiredo1, Mario de Andrade Lira Junior2,

Arminda Saconi Messias3 and Rômulo Simões Cezar Menezes4

Maize productivity in tropical low external input systems is usually limited by low
soil fertility because crop uptake leads to a gradual depletion of soil nutrient stocks. Since
the use of chemical fertilizers is infeasible or undesired, the management of the fertility of
these soils depends primarily on low-cost processes based on nutrient recycling. The main
processes that may contribute to this are 1) biological nitrogen fixation (BNF), 2) nutrient
recycling through organic fertilization using plant residues or animal manures, and 3)
where feasible, the use of industrial and/or urban waste. BNF may contribute to maize
growth and yield by direct fixation in corn, or through the use of legume plants either as
green manure or as crops in rotation or intercropped with corn. Either way, BNF can
usually be considered sustainable long term, and usually would be one of the preferred
nitrogen sources for low external input corn production systems. Since almost all soil
nitrogen is derived from the atmosphere, in the absence of substantial use of nitrogen

Biologist, PhD. Research Fellow of National Research and Technological Development, Brazil. Agronomical
Institute of Pernambuco IPA/CARH, 1371, Gen. San Martin Avenue, Recife, PE, Brazil, 50761-000. E-mail:
Agronomist, PhD. Research Fellow of National Research and Technological Development, Brazil. Federal
Agricultural University of Pernambuco, Agronomy Department. UFRPE/DEPA, s/n D. Manoel de Medeiros St,
Recife, PE, Brazil. 52171-900. E-mail:
Chemical engineer, PhD. Pèrnambuco Catholic University. UNICAP, 526 Principe st, Recife, PE, Brazil, 50050-
900. E-mail:
Agronomist, PhD. Research Fellow of National Research and Technological Development, Brazil. Federal
University of Pernambuco, Nuclear Energy Department. UFPE/DEN, 1000 Professor Luis Freire Av., Recife,
PE, Brazil, 50740-540. E-mail:
228 M. do Vale Barreto Figueiredo, M. de Andrade Lira, A. Saconi Messias et al.

fertilizer most of the remaining nitrogen pool is a product of BNF, either recent or past.
The main difference between on-field BNF and use of plant residues and animal manures
is that nitrogen is previously fixed or obtained from the soil pool on another field and later
taken to the corn field. At the same time, nutrient recycling through organic fertilization is
usually limited due to the low amounts of organic matter available for this use, especially
considering the concurrent demands for this material. Therefore, the efficient use of the
different types of organic matter used as fertilizer requires knowledge about its quality and
patterns of decomposition in order to guarantee synchronization between nutrient supply
and crop demand. Finally, the third approach in these systems centers on the use of urban
waste, most usually compost or sewage sludge, or industrial by-products. Some of these
may be quite rich in several nutrients at the same time, but usually require careful
investigation into possible negative effects of items such as heavy metals and pathogens.
We review information regarding BNF directly on corn, in green manure or crop rotations
involving this culture; strategies to improve the amount and quality of organic fertilizers
produced in low input systems; and some possible alternatives of urban or industrial by-
products, describing the current rationale to supply nutrients to maize crops at a low cost
using the resources available within the agroecosystems.

Keywords: BNF, legume, green manure, sludge, compost

Corn (Zea mays) is a major user of synthetic nitrogen fertilizer; therefore if Biological
Nitrogen Fixation (BNF) in corn is successful, there could be far-reaching economic
consequences (Halbrendt and Blase, 1989). With the current cost of fertilizer approaching half
the total variable cost of producing corn, the potential savings could be substantial if BNF is
developed and adopted (Mendonça et al., 2006). By definition, BNF is synonymous with
sustainability. This process offers an economically attractive and ecologically sound means of
reducing external nitrogen input and improving the quality and quantity of internal resources
(Saikia and Jain, 2007). Clearly, it is not realistic to consider sustainable agriculture on a broad
scale in the absence of BNF.
Some cereal crops of commercial importance like corn, rice, wheat, and millets are found
to have association with microorganisms that are capable of assimilating atmospheric nitrogen
(Döbereiner and Boddey, 1981; Okon and Kapulnik, 1986; Baldani et al., 1986; Urquiaga et
al., 1992; Chelius and Triplett, 2001; Riggs et al., 2001; Boddey et al., 2003; Tejera et al.,
2006; Barassi et al., 2007; Herridge et al., 2008). Corn yields have also risen steadily, largely
because of use of the hybrids and increased input of fertilizer nitrogen. To accommodate the
world’s expanding population, which is projected to double by 2050, an ever-increasing
production of food crops will be necessary. This must be achieved primarily by increasing the
productivity of currently farmed areas, since suitable new land is very limited. An obvious
goal of BNF research is to find ways to enable the major cereal crops to utilize BNF directly as
a partial or major source of their nitrogen needs (Raymond et al., 2004).
Potential Impact of Biological Nitrogen Fixation and Organic Fertilization… 229


BNF is the process by which the bulk of the atmospheric nitrogen has been incorporated
into living matter throughout the evolution of our planet. Even today, this process is the main
pathway of nitrogen incorporation to the ecosystem, which is constantly recycled into the
atmosphere primarily by the action of organisms’ decomposition of soil organic matter.
Therefore, the action of microorganism nitrogen fixers and denitrification warrants an
inexhaustible reservoir of nitrogen in the atmosphere. In addition to ensuring an ecosystem in
balance, a reduction in the application of excessive doses of nitrogen compounds such as
nitrate, which contaminates water and plants consumed by humans, enables the development
of less aggressive agriculture in the environment. The estimate is that the contribution of
biologically fixed nitrogen ranges from 139 to 170.106 tons of nitrogen a year, at least double
the chemical fixation (Peoples and Craswell, 1992).
The microorganisms that promote the BFN have great importance, since this element is an
essential component of proteins, nucleic acids and other nitrogen compounds, and therefore of
life for all living beings (Döbereiner, 1997). Under optimal conditions in an ecosystem the
microbiota are in balance in the soil, maintaining its biodiversity and sustainability, but this
balance can easily be broken by humans or by natural phenomena (Döbereiner, 1992). Even
though the greatest contributions of BNF has been detected in oceans and leguminous plants,
some plants of the family Gramineae have shown a very significant potential in obtaining
nitrogen by the action of nitrogen fixing bacteria (Baldani et al., 2002; Alves et al., 2006).
These plants have a fascicule root system, taking advantage of the leguminous’ pivoting
system to extract water and soil nutrients, and because they are widely used as food by
humans. Therefore, even if only part of N could be provided by association with fixing
bacteria, the economy in nitrogen fertilizers would be equal to or higher than that observed
with leguminous plants that can be self-sufficient in nitrogen (Döbereiner, 1992; Boddey et al.,
Undoubtedly, after carbon, oxygen and hydrogen, nitrogen is quantitatively the most
important element required by plants and animals for growth both in water and on land,
reaching about 1.5% of the dry weight of innumerable agricultural crops (Van Loon and
Duffy, 2001). So, this was one of the nutrients that most contributed to the so-called Green
Revolution. Its indiscriminate use has led to environmental problems (Bouchard et al., 1992).
Some of the adverse environmental effects of excessive use of nitrogenous fertilizers are the
following: (i) metheamoglobinemia in infants due to NO3 and NO2 in water and food; (ii)
cancer due to secondary amines; (iii) respiratory illness due to NO3, aerosols, NO2 and HNO3;
(iv) eutrophication due to N in surface water; (v) material and ecosystem damage due to HNO3
in rainwater; (vi) plant toxicity due to high levels of NO2 and NH4 in soils; and (vii) excessive
plant growth due to more available N and depletion of stratospheric ozone due to NO and N2O
(Saikia and Jain, 2007).
In tropical countries, 40% of the costs of maize cultivation, for instance, are committed to
the purchase of mineral nitrogen (Majerowicz et al., 2002). Overall, cereal cultivation
consumes 60% of the total nitrogen fertilizer used in the world. However, on average, only
33% of all N applied is recovered in the grains, promoting the loss of $15.9 billion in 1999
(Raun et al., 2002). Questions like these encourage the creation of technologies that reduce the
excessive amount of fertilizer applied. The study of efficiency in the use of N allows multiple
230 M. do Vale Barreto Figueiredo, M. de Andrade Lira, A. Saconi Messias et al.

methods from the simplest, based on the mere reduction of doses of fertilizers, to productive
levels, even those based on genetic improvement able to set up productive plants in poor N
soils. On the physiological—i.e., molecular—level, the study of the acquisition and use of N
should be linked to the understanding of absorption, assimilation and redistribution of this
nutrient in cell, along with its balance between storage and use in cellular and whole plant
biology (Majerowicz et al., 2002).
Even now, new methods for its use are intensely studied. The quest for greater efficiency
in its use, through recognition of biochemical and molecular pathways of absorption and
assimilation in plants, as well as the agroecological methods, such as the BNF, are proposals to
allow the sustainable use of this material without production loss (Traore and Maranville,
1999; Pradella et al., 2001).
A number of reviews of plant-associated N2 fixation have clearly highlighted the many
methodological problems and inconsistencies in the published studies (Boddey, 1987; Chalk,
1991; Giller, 2001; Giller and Merckx, 2003). One of the key problems is distinguishing
between inputs of N by free-living and associative agents and other external sources of N
contributing to agricultural soils, e.g., N in rainfall and dry deposition (Herridge et al., 2008).
Such inputs can represent 3–50 kg N/ha/year (McNeill and Unkovich 2007). Roper and Ladha
(1995) concluded that the free-living, heterotrophic bacteria may fix significant amounts of N
in agricultural systems, using crop residues as an energy source.

2.1. Diazotrophic Bacteria

The diazotrophic bacteria occupy separate niches, and may be free-living, symbiotic or
associative. BNF was first described in diazotrophic bacteria from the rhizosphere and
rizoplane of a wide variety of non-leguminous plants (Döbereiner,1992; Boddey et al., 2003).
Common diazotrophs found in the rhizosphere of maize are Enterobacter spp., Rahnella
aquatilis, Paenibacillus azotofixans, Azospirillum spp., Herbaspirillum seropedicae, Bacillus
circulans and Klebsiella sp. (Chelius and Triplett, 2001).
The positive effects of Azospirillum on maize growth are mainly derived from
physiological changes of the inoculated plant roots, which enhance water and mineral nutrient
uptake (Okon and Kapulnik, 1986; Barassi et al., 2007). Both A. brasiliense and A. irakense
are used as inoculant biofertilizers for maize. Others species of Azospirillum capable of
increasing the yield of maize are A. lipoferum and A. indigens, and Azorhizobium caulinodans
was also capable of giving such beneficial effects (Riggs et al., 2001). The magnitude of this
increase varie with the Azospirillum strain and maize cultivar and depending on soil
Cereals of economic importance, such as corn, sugar cane, rice, wheat, sorghum, and some
fodder were identified with hosts of different species of endophytic diazotrophic bacteria.
Among endophytic diazotrophic are: Gluconacetobacter diazotrophicus (Cavalcante and
Döbereiner, 1988; Boddey et al., 2003), Azoarcus spp. (Reinhold-Hurek et al., 1993),
Herbaspirillum seropedicae (Baldani et al., 1986; James, 2000), Herbaspirillum
rubrisubalbicans (Baldani, 1996; Gillis et al., 1991), Burkholderia spp. (Baldani et al., 1997)
and H. lusitanum (Valverde et al., 2003).
Initially the endophytic microorganisms were considered harmless to plants, but from the
1970s its importance to the plants began to be observed (Azevedo et al., 2002). There are
Potential Impact of Biological Nitrogen Fixation and Organic Fertilization… 231

several positive effects attributed to endophytic bacteria, such as the promotion of plant growth
(Okon and Labandera- Gonzalez, 1994; Okon and Itz1gsohn, 1995; Raja et al., 2006),
biological control of pests and diseases in plants (Mariano et al., 2004), biological nitrogen
fixation (Döbereiner and Boddey, 1981; Downing et al., 2000; Verma et al., 2004; Reis Junior
et al., 2008), induction of systemic resistance (Hallmann et al., 1997), production of
siderophores (Burd et al., 1998; Wenbo et al., 2001) and production of antibiotics (Strobel and
Daisy, 2003).
The promotion of plant growth occurs mainly by the production of phytohormones as
auxins, cytokinins, gibberellins, abscise acid and ethylene by the endophytic bacteria. The
production of these phytohormones has been reported in bacteria as Gluconoacetobacter,
Azospirillum, Herbaspirillum, Erwinia, Pseudomonas and Pantoea (Kuklinsky-Sobral et al.,
2004). The indoleacetic acid is a naturally occurring important auxin that causes physiological
effects on the plant, such as increased growth (Lambrecht et al., 2000; Nefedieve, 2003;
Figueiredo et al., 2008).
The association of cereals and grasses with endophytic diazotrophic bacteria may
represent one of the most promising alternatives for the promotion of plant growth, soil
management and environmental quality since bacteria are able to promote growth, increase
disease resistance, through biological fixation of nitrogen or the phytohormones production
(Thuler et al., 2003; Bashan et al., 2004). In addition, diazotrophic endophytic may present
advantages in relation to diazotrophic associated with roots once they are better located to
explore the carbon sources released by plants and these bacteria have been isolated from
several grasses species (Riggs et al., 2001; Tejera et al., 2006).
There is a great interest in characterizing the diversity of these microorganisms in order to
use their potential in different cultures, especially the corn, where a wide diversity of
diazotrophic has been found colonizing this plant (Baldani et al., 1997; Chelius and Triplett,
2001; Pitnner et al., 2007). In general, studies on diversity are based on cultivation techniques
and subsequent characterization of isolates. However, the cultivation of microorganisms
provides limited information about diversity since most existing organisms is not easily
isolated by conventional cultivation techniques. Techniques of Random Amplified
Polimorphic RAPD-DNA, Polymerase chain reaction-BOX-PCR, Amplified Fragment Length
Polymorphism-AFLP and Amplified ribosomal DNA Restriction Analysis-ARDRA are
applied in assessing the diversity of microbial community cultured. The application of
independent cultivation techniques such as Denaturing Gradient Gel Electrophoresis-DGGE,
the construction and analysis of clone libraries and qPCR real time quantitative are applied to
the study of microbial communities (Andreote et al., 2008). For evaluation of BFN diversity in
different ecosystems, universal primers have been used to amplify the gene nifH through
techniques of independent cultivation (Bashan et al., 2004). Such techniques make possible to
obtain a more complete characterization of the diazotrophic community than dependent
techniques of cultivation (Roesch et al., 2007).
232 M. do Vale Barreto Figueiredo, M. de Andrade Lira, A. Saconi Messias et al.

2.2. Plant Growth-Promoting Rhizobacteria (PGPR) Increase

Crop Performance

The mechanisms by which PGPR increase crop performance is not well understood. There
are several inoculants currently commercialized that seem to promote growth through at least
one mechanism; suppression of plant disease (termed Bioprotectants), improved nutrient
acquisition (termed Biofertilizers), or phytohormone production (termed Biostimulants).
Inoculant development has been most successful to deliver biological control agents of plant
disease, that is organisms capable of killing other organisms pathogenic or disease causing to
crops (Tenuta, 2003).

Table 1. Biology, and potential role of some diazotrophs promoting crop production
(adapted by Kennedy et al., 2004)

Condition Mechanism
Diazotrophs Habitat Energy source References
for BNF of effect

Aerobic Rhizozphere Organics in soil BNF Kennedy and Tchan (1992)

Clostridium spp. Anaerobic Soil saprophyte Organics in soil BNF Kennedy and Tchan (1992)

mildly Organics in soil, Reinhold and Hurek (1998)
Azospirillum spp. Microaerobic endophytic in root exudates and BNF, PGP Mirza et al. (2000)
roots, stems plant tissue Okon and Kapulnik (1986)
and leaves
Herbaspirillum Endophytic,
Microaerobic Root exudates BNF, PGP Baldani et al. (1986, 2000)
seropedicae Rhizozphere
Hurek et al. (1994)
Azoarcus sp. Microaerobic Endophytic Root exudates BNF
Reinhold-Hurek et al. (1993)
Burkholderia Rhizozphere, Organics in soil
— BNF, PGP Baldani et al., (1997, 2000)
vietnamiensis Endophytic, and root exudates
Endophytic in Yanni et al. (1997)
leguminosarum bv — Root exudates PGP
roots Biswas et al. (2000)
Rhizobium etli bv Endophytic in Guitiérrez-Zamora (2001)
— Root exudates PGP
phaseoli roots Martínez-Romero (2001)
Endophytic in
A. caulinodans Microaerobic Root exudates PGP Matthews et al. (2001)
Endophytic in
Glucanoacetobacter Root exudates Baldani et al., (1997)
Microaerobic roots, stems BNF
diazotrophicus and plant tissue Boddey et al., (1991)
and leaves
BFN, Biological nitrogen fixation; PGP, plant growth promoting.

BNF by associative diazotrophic bacteria is a spontaneous process where soil N is limited

and adequate C sources are available. Yet the ability of these bacteria to contribute to yields in
crops is only partly a result of BNF. A range of diazotrophic plant growth-promoting
rhizobacteria participate in interactions with C3 and C4 crop plants (e.g. rice, wheat, maize,
sugarcane and cotton), significantly increasing their vegetative growth and grain yield. The
mechanisms involved have a significant plant growth-promoting potential, retaining more soil
Potential Impact of Biological Nitrogen Fixation and Organic Fertilization… 233

organic-N and other nutrients in the plant–soil system, thus reducing the need for fertilizer N
and P. Table 1 suggests that the diversity of habitat and effectiveness might logically require
more than one bacterial strain to obtain the maximum biological effects on plant growth are
summarized indicating the proposed mechanisms of PGP (plant growth promoting ) effects
(Kennedy, et al., 2004).
According to Kennedy et al. (2004), this diversity will need to be carefully considered in
the future design of the most efficient inoculant biofertilisers. For example, an important
question is whether inoculants should be restricted to a single strain of bacterium, such as
Azospirillum, or not. If all of the PGP mechanisms can be well expressed in a single strain of
bacterium this would simplify the design of inoculant products. However, it would be unlikely
that a single strain of bacterium would be capable of optimal activity.


Another approach to biological nitrogen fixation used with corn is the inclusion of
legumes as part of the cropping system, most commonly as grain crops or green manures.
The widespread knowledge, and even to a large degree “faith” , that legumes always
improve soil nutritional status has historically led most farmers to include legume in the
cropping systems, either directly or indirectly through fallow periods (Van Kessel and
Roskoski, 1988; Gathumbi et al., 2002; P hoomthaisong et al., 2003; Sanginga, 2003; Sanginga
et al., 2003; Okito et al., 2004).

3.1. Crop Rotations

Legume crops are a key component of most traditional tropical cropping systems (Van
Kessel and Roskoski, 1988; Peoples et al., 1995; Sanginga, 2003), either cultivated at the same
time as the main commercial crop, which would most frequently be corn, sorghum or millet
(Anthofer, 2005; Fan et al., 2006; Okogun et al., 2007), depending on the region, or in
sequential cropping on the same field, which is usually the preferred solution for current
commercial cropping due to easier management (Goss et al., 2002; Smith et al., 2008).
Some of the results achieved with the use of a legume crop in the production system can
be shown as examples. For instance Fan et al. (2006) have found that when corn and faba bean
(Vicia faba) were intercropped in Northern China, corn yield was not significantly different
from the achieved by corn single-cropped which did not receive nitrogen fertilizer, and slightly
lower than what corn would yield if nitrogen fertilizer was used (12.04 t.ha-1 for the intercrop,
and 13.31 t.ha-1 for single corn), but this difference was not significant. In another paper,
yields in plots previously sown to soybean were significantly larger than yields in the fallow
plots, with averages of about 3 t.ha-1 and 0.5 t.ha-1, respectively (Osunde et al., 2003).
Under either of the approaches, although common sense indicates that there is always a
gain in soil fertility due to the inclusion of the legume crop, this frequently is not the case with
modern cultivars (Singh et al., 2003). This happens because these cultivars may achieve higher
nitrogen harvest indexes than the total nitrogen fixation. This possible negative nitrogen
balance may be the main responsible for the huge variation in results from the several papers
234 M. do Vale Barreto Figueiredo, M. de Andrade Lira, A. Saconi Messias et al.

dealing with the effect of inclusion of legume crops in the cropping system (Gentry et al.,
2001; Tilman et al., 2002; Fortuna et al., 2003; Rosolem et al., 2004; Robertson et al., 2005;
Smith et al., 2008).
Besides leaving crop residues on the field, which would usually be the recommended
practice for reasons besides nitrogen balance, such as weed control, reduction of soil loss
through erosion and of superficial soil temperature (Oliveira et al., 2002; Cabezas, 2004;
Balkcom and Reeves, 2005; Silva, 2006), other manageable aspects of the legume crop that
allow a positive nitrogen yield for the following crop would be selection of legume cultivars
with longer growth period, lower nitrogen harvest indexes or higher nitrogen fixation potential.
Increasing growing period for the legume in the system will usually allow larger biomass
accumulation with all of the above-mentioned advantages, and may be applied both for cover
and grain legume crops (Anthofer, 2005; Fan et al., 2006; Chikoye et al., 2008). On the other
hand, it may be interesting when the pulse crop is grown on alternate years on the same plot
with corn, as is usually the case on the corn-soybean rotation, very common on some of the
main soybean growing regions of the world, such as Brazil and the United States. The corn-
soybean rotation is probably the most important cereal-legume rotation in large scale intensive
agriculture, since both crops are major commodities. Since both are also important potential
sources for biofuels, respectively ethanol under current American practice, and biodiesel an
increase in area under cultivation is predicted for both (Salvagiotti et al., 2008).
The importance of the possibly negative nitrogen balance in legume crops such as soybean
may be observed in Salvagiotti et al. (2008). These authors have examined 637 data sets (site-
year-treatment combinations) from field studies that had examined nitrogen balance data and
had been published in refereed journals from 1966 to 2006. In most situations they found that
the amount of N fixed was not sufficient to replace N export from the field in harvested seed.
The partial N balance (fixed N in aboveground biomass - N in seeds) was negative in 80% of
all data sets, with a mean net soil N mining of -40 kg N.ha-1. However, when an average
estimated belowground N contribution of 24% of total plant N was included, the average N
balance was close to neutral (-4 kg N.ha-1). This gap between crop N uptake and N supplied by
BNF tended to increase at higher seed yields for which the associated crop N demand is higher
On the other hand, as long as NHI is lower than the nitrogen obtained from biological
nitrogen fixation, nitrogen export through seeds would be lower than nitrogen fixation, and
there would be a net increase of available soil nitrogen (Phoomthaisong et al., 2003;.Singh et
al., 2003; Alves et al., 2006). This increase in nitrogen fixation is one of the main aims of most
soil microbiologists currently working hand in hand with the legume inoculants’ industry
(Date, 2000; Graham and Vance, 2000; Catroux et al., 2001; Hardarson and Atkins, 2003;
Deaker et al., 2005; McInnes et al., 2005). There are strong indications for some of the crops
commonly used in rotation with corn that another feasible approach is cultivar selection for
higher nitrogen fixation potential (Tsai et al., 1998; Hardarson and Atkins, 2003; Bouton,
While the results for inclusion of legume green manures on the nitrogen balance are more
positively consistent than those for legume crops, this practice has not achieved the same
degree of grower adoption as the former (Chikowo et al., 2004; Anthofer, 2005; Crews and
Peoples, 2005; Musiyiwa et al., 2005; Shelton et al., 2005; Rufino et al., 2006; Ojiem et al.,
A recent paper (Tonitto et al., 2006) discusses 36 papers on the effect of the inclusion of
legumes as green manure in North American cropping systems, of which 28 had corn as the
Potential Impact of Biological Nitrogen Fixation and Organic Fertilization… 235

main crop. The authors conclude that in half of the 228 individual experiments the legume
green manure resulted in a net input of 50 to 150 kg of N.ha-1. They also conclude that with
that level of biological nitrogen input there was usually no significant difference on yield
between conventionally managed systems and legume based ones. They also conclude that if
fixed nitrogen was higher than 180 kg of N.ha-1 there was usually a 5% gain on crop yield,
while if input was below 110 kg of N.ha-1 the expected result would be loss of yield. These
results were achieved with loss of a growing season, since all experiments included only short
season green manure crops.
In another paper, a field experiment was conducted in Michigan, USA, studying how corn
was affected by inclusion of crop rotation with soybean and/or short season legume green
manures (Smith et al., 2008). The experimental design was unique in that no fertilizer or
pesticides were used, and the only management variable manipulated was number of species in
the rotation, thus providing a strong comparison to grassland diversity-ecosystem function
experiments. Corn grain yield increased linearly in response to the number of crops in the
rotation, with yields in the treatment with corn, soybean and winter wheat as crops and red or
crimson clovers and rye as short season cover crops were over 100% higher than in continuous
monoculture. Most importantly, the yields were not significantly different from the county
average for each of the 3 years despite the absence of chemical inputs.
An important point concerning use of green manures is that it is highly knowledge
dependent, since it must be highly environmentally adapted, or it won’t achieve the expected
result. Although low formal education levels are widespread among developing world farmers,
usually they have high level knowledge of their farming environment. Both of these aspects
indicate that green manure research should ideally be conducted under field conditions,
preferably with farmer management at least on final research stages (Jensen and Hauggaard-
Nielsen, 2003; Chikowo et al., 2004;Crews and Peoples, 2005; Mapfumo et al., 2005).


Sewage sludge (SS), or pie, is waste of urban and/or industrial origin which results from
the treatment of effluents, presenting highly variable composition. The differences vary with
the type of process employed (primary, raw sludge produced in primary decanters; activated
sludge, produced in biological reactors and, digested sludge, process of biological
stabilization), with the physiographic location of Wastewater Treatment of Sewage (WTS)
(which reflects the dietary habits of the population), with the balance of nutrients from food
consumed, with a time of year and with the waste discharge (Saito, 2007; Tsutiya, 2000;
PROSAB, 1999; Vidor, 1999). According to Bettiol and Camargo (2000) depending on the
origin and the process of obtainment used, the sewage sludge presents quite variable
composition, being rich in organic matter (40 to 60%), nitrogen and some micronutrients such
as iron, copper, zinc and manganese. Typical sewage sludge contains 40 % organic matter, 4 %
of nitrogen, 2% phosphorus and 0.4% potassium.
When there is possibility of sterilization with sulfate / calcium carbonate in the process of
WTS, the resulting product becomes the biosolids. Therefore, biosolids is the name given to
the sludge resulting from the sewage treatment, with features that allow recycling in rational
and environmentally safe way. The term biosolids was created and disseminated throughout
236 M. do Vale Barreto Figueiredo, M. de Andrade Lira, A. Saconi Messias et al.

the world to encourage the use of sewage as fertilizer and soil-conditioners (Oliveira et al.,
2005; Smith, 2005; USEPA, 1999). The urban sludge, when treated, may eliminate the
pathogenicity of viruses, bacteria, fungi, protozoa and helminthes (Barbosa et al., 2007).
However, the possible presence of pollutants such as pathogens agents and potentially toxic
elements - heavy metals are factors that may cause negative impacts, so its application requires
special care to be avoided damage to the population and the environment.
The application to soil is one of the oldest practices of final destination for sanitary sewer.
The "sewage farms", became known as the first experiments in England at the beginning of the
nineteenth century, quickly spread through Europe and the United States (Bastos, 2003). The
best known information is those from China. In the West, particularly in Prussia, irrigation
with sewage effluent was practiced since 1560. In England, around 1800, many projects were
developed for agricultural use of sewage effluent, especially due to combat cholera’s
epidemics. The adoption of the practice of using soil as means of sewage or sludge disposal
has been frequent in many countries (Nascimento et al., 2004).
The use of sewage sludge as organic fertilizer has been mentioned as an alternative to the
final destination of the waste, mainly by the predominant concentration of organic matter and
source of nutrients (Messias, 1993; Messias and Moraes, 1992; Pires, 2005; Andreoli et al.,
2004; Gadioli and Fortes Neto, 2004; Faria, 2007).
Besides the environmental and economical point of view, the use of sludge in agriculture
is advantageous once it promotes greater soil’s water holding capacity, porosity (aeration of
the roots) and aggregate stability. Also, greater resistance to erosion, residual effect usable for
subsequent crops, and possibly induce the suppression of soil to phytopatohgens (Bettiol and
Fernandes, 2004; Silva et al., 2002; Berton et al., 1997; Melo et al. 1994). The criteria for
application of SS should be based as well in soil attributes and not only in their total levels of
metals. The knowledge of how these attributes influence metals behavior is then able to show
the amount of waste that can receive the soil (Borges and Coutinho, 2004). Some inventories
were created for monitoring and managing the sludge disposal in space, including surveys of
environmental data (soil, water, geology, geomorphology and vegetation), current use of rural
and urban soil and institutional context. Gomes et al. (2001) proposed an inventory, followed
by the location of areas potentially suitable for the sludge recycling, by eliminating areas
incompatible with the necessary environmental attributes and the legislation requirements.
Therefore, it was considered the distance of water resources, urban spot, flooded areas and
land slope, among other factors. Soils’ agricultural ability was assessed from the current levels
of fertility, its ability to recover physically and chemically by organic addition, beyond the risk
of erosion of them.
Most studies have aimed to verify the effect of organic fertilizers on the yield, compared
to, in general, with complete (NPK) or incomplete mineral fertilization including corrective
soil. With rare exceptions, most studies shows effects of fertilization, both organic and
mineral, in relation to the control, but the differences between organic and mineral fertilizers
are variable, depending on the soil characteristics, doses of fertilizers, crop and study area.
According to studies performed, results expected with organic and mineral fertilization,
isolate, may be represented by a curve of quadratic type, in which production rises relative to
the dose (Malavolta, 1981). The application of organic compounds in a continuous manner
raises the nitrogen level to the point of making dispensable its application in the form of
chemical fertilizer. In poor soils, the amount of organic matter may increase considerably its
potential productivity. The efficiency of organic fertilizers to improve soil productivity
Potential Impact of Biological Nitrogen Fixation and Organic Fertilization… 237

depends on several factors that should be considered: (a) quality and quantity of application,
(b) ages and conditions of use; (c) methods of implementation; (d) the adequacy to farming
systems prevailing in the region, (e) and, especially, the relative cost of their use.
Daros et al. (1993) verified residual effect of N and P in soil sludge-fertilized in the
production of millet with subsequent associated cultivation of oats and vetch. Mazzarino et al.
(1998) state that the release of P by sludge depends on the soil type and origin of the residue.
Silva et al. (2002) corroborated that the sludge used presented 25 % more efficiency than the
triple superphosphate as phosphorus source for corn. The application of increasing doses of
sewage sludge promoted decrease in pH and increase in levels of organic matter, total N, P, K,
Na, Ca and Mg in soils with crops of maize and beans, however dry material of both crops was
lower than that obtained by complete mineral fertilization (Nascimento et al., 2004). Galdo et
al. (2004) and Tsadilas et al. (1995) observed higher grains yield in maize cultivation, with
application of sewage sludge, as well as Cripps et al. (1992) found grains yield 47 % higher
with application of sewage sludge in comparison with conventional fertilization. Silva et al.
(2002) and Biscay and Miranda (1996) reported higher grain yield in relation to the control and
the NPK fertilizer for three years, after sludge application, demonstrating its residual effect.
Some authors showed an increase in levels of Cd and Cu (Logan et al., 1997; Favaretto, 1997;
Pierrisnard, 1996; Al-Jaloud et al., 1995; Reddy et al., 1989, Ritter and Eastburn, 1978), Cr, Ni
and Zn in corn, beans and sorghum, with increase in the doses of sewage sludge application
from 40.5 t ha-1 (Boaretto et al., 1992; Oliveira, 1995; Angels and Mattiazzo, 2000).
The absorption of large quantities of Zn by plants in treatments with sewage sludge
considered above the adequate range for the cultivation of corn, according to Malavolta et al.
(1989), may have caused lower productivity of this treatment. The application of increasing
doses (10, 20, 30, 40 and 60 t.ha-1) of sewage sludge promoted decrease in pH and increase in
levels of organic matter, N, P, K, Na, Ca and Mg in crops of maize and beans, however dry
matter of both cultures was lower than that obtained by mineral fertilization (Nascimento et al.,
2004). Maize’s dry matter production increased with the dose of sludge in the presence or
absence of potassium (Simonete et al., 2003; Simonete and Kiehl, 2002; Simonete, 2001;
Berton et al., 1989). Gomes et al., (2007), to evaluate the chemical changes in soil caused by
the addition of sewage sludge, installed an experiment with corn in the field conditions, in
Yellow Argisol, which consisted of six treatments (0; 7.7; 15.4; 29.7; 45.1 and 60.5 t. ha-1).
The production of grains increased depending on the doses of sewage sludge up to the
application of 26 t. ha-1 which provided the maximum agronomic efficiency for the corn
production. The sl