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BOTANICAL RESEARCH AND PRACTICES









SUNFLOWERS

GROWTH AND DEVELOPMENT,
ENVIRONMENTAL INFLUENCES
AND PESTS/DISEASES
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SUNFLOWERS

GROWTH AND DEVELOPMENT,
ENVIRONMENTAL INFLUENCES
AND PESTS/DISEASES







JUAN IGNACIO ARRIBAS
EDITOR












New York

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Library of Congress Cataloging-in-Publication Data
Sunflowers : growth and development, environmental influences and pests/diseases / editor: Juan
Ignacio Arribas (Electrical Engineering Department, Univ. Valladolid, Spain).
pages cm
Includes index.
1. Sunflowers. I. Arribas, Juan Ignacio.
QK495.C74S87 2014
583'.99--dc23
2014003599



Published by Nova Science Publishers, Inc. † New York

ISBN: (eBook)
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CONTENTS


Preface vii
Chapter 1 An Introduction to the Sunflower Crop 1
Fabián Fernández-Luqueño, Fernando López-Valdez,
Mariana Miranda-Arámbula, Minerva Rosas-Morales,
Nicolaza Pariona and Roberto Espinoza-Zapata
Chapter 2 Floral Biology of Sunflowers: A Histological
and Physiological Analysis 19
Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
Chapter 3 Development of Female Reproductive Structures and Apomixis
in Sunflowers 43
Olga N. Voronova
Chapter 4 Genetics and Genomics Applied to Sunflower Breeding 61
Carla Filippi, Jeremías Zubrzycki, Verónica Lía,
Ruth A. Heinz, Norma B. Paniego and H. Esteban Hopp
Chapter 5 Sunflower Genetic Resources: Interspecific Hybridization
and Cytogenetics in Prebreeding 95
Jovanka Atlagić and Sreten Terzić
Chapter 6 Functional Genomics and Transgenesis Applied to
Sunflower Breeding 131
Sebastian Moschen, Laura M. Radonic,
Guillermo F. Ehrenbolger, Paula Fernández,
Verónica Lía, Norma B. Paniego, Marisa López Bilbao,
Ruth A. Heinz and H. Esteban Hopp
Chapter 7 Disease Management in Sunflowers 165
Regina M. V. B. C. Leite
Chapter 8 Recent Advances for Developing Resistance against
Plasmopara halstedii in Sunflowers 187
Osman Radwan

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Contents vi
Chapter 9 Effects of Crop Management on the Incidence and Severity
of Fungal Diseases in Sunflowers 201
P. Debaeke, E. Mestries, M. Desanlis and C. Seassau
Chapter 10 Insect Pests of Sunflowers in Africa 227
Hannalene du Plessis
Chapter 11 Soil Amendments and Their Effects on Sunflower Growth 239
Fernando López-Valdez, Fabián Fernández-Luqueño,
Perla Xóchitl Hernández-Rodríguez, Minerva Rosas-Morales
and Silvia Luna-Suárez
Chapter 12 Nutrition and Fertilization of Sunflowers in Brazilian Cerrado 257
C. de Castro, F. A. Oliveira, A. Oliveira Junior
and N. P. Ramos
Chapter 13 Environmental Issues in the Sunflower Crop of Midwestern Brazil:
Diversification and Complementarities in the Biodiesel Chain 281
N. P. Ramos, A. M. M. Pires, C. C. A. Buschinelli,
H. B. Vieira, C. de Castro and G. S. Rodrigues
Chapter 14 Micro and Macro-Morphological Variation of Cosmos bipinnatus
and Cosmos bipinnatus var. Albiflorus in Sympatric Zones in
Central Mexico 297
M. Paniagua-Ibañez, A. Zepeda-Rodríguez, P. Mussali-Galante,
R. Ramírez-Rodríguez and E. Tovar-Sánchez
Index 309




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To Juan Ignacio, Jr., Elena, Jr. and Elena



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PREFACE


We are all well aware that the importance of the sunflower (Helianthus Annuus) as a crop
has increased significantly in recent years, not only in the food industry but also as a natural
energy resource in oil production. I am, thus, very pleased to be able to present this
comprehensive monograph on a wide range of important issues regarding sunflowers, with an
emphasis on environmental influences, pests and diseases in order to maximise production
whilst minimising costs.
Contributors where selected based on their proven experience in the field of sunflowers.
Contributors submitted an extended abstract that was assessed for relevance. They were then
invited to contribute draft chapters. Each chapter underwent a stringent and thorough peer
review process by other experts in the field, with final approval by the editor who, thus, was
able to balance the topics from all contributors.
The book contains important original results. Each chapter deals with a different topic,
and draws, where appropriate, from studies and results previously published by the authors.
Authors were encouraged to complement their writing with original and high quality graphs,
charts, tables, figures, pictures and photographs.
It‘s my honour and pleasure to acknowledge the rigorous work carried out by all authors
in this book, and at the same time I am very grateful to them for trusting me in leading this
project in the role of the editor of their work. My thanks also go to the anonymous reviewers
who contributed their time so generously to this book, and without whom it would not exist.
I am also very grateful to Nova Science Publishers for inviting me to lead this book, and
thank them for the help and coverage provided during the whole time that this project lasted.
I really do hope that you find this book of interest and wish you enjoy its reading as much
as I have done through the whole editing process and as much I am sure all authors have done
while writing it.
The book is structured as follows: Chapter 1 introduces sunflowers. Chapters 2 and 3
detail the biology of a sunflower. Chapters 4, 5 and 6 explore the important topics of
sunflower production: genetics, genomics and the breeding of sunflowers. Chapters 7, 8, 9
and 10 address with other important aspects of sunflower production, pests and diseases.
Chapters 11 and 12 deal with sunflower nutrition and growth. Finally, Chapter 13 presents
environmental sunflower crop issues and Chapter 14 studies sunflower morphological
Cosmos variations.

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Juan Ignacio Arribas x
In Chapter 1, entitled An Introduction to the Sunflower Crop, Dr. Luqueno and colleagues
from the Natural Resources and Energy Group, Cinvestav-Saltillo, Mexico, present an
enjoyable historical perspective on sunflowers. Sunflower (Helianthus annuus L.) belongs to
the family Asteraceae. The sunflower plant originated in eastern North America. It is thought
to have been domesticated around 3000 B.C. by Native Americans. In the late 1800s the
sunflower was introduced in the Russian Federation where it became a food crop and Russian
farmers made significant improvements in the way that the sunflower was cultivated. Since
3000 B.C. a wide range of uses of sunflower have been reported throughout the world.
Sunflower is well known by its phytoremediation potential and by its seed oil content.
Because the sunflower has several potential markets, it is a good choice for growers on both
small and large scales. However, it has to be remembered that scientific, technical or
agricultural projects linked with sunflower have to include side effects elsewhere in order to
shape a sustainable future.
In Chapter 2, entitled Floral Biology of Sunflower - A Histological and Physiological
Analysis, Dr. Bhatla and colleagues from the Laboratory of Plant Physiology and
Biochemistry, Department of Botany, University of Delhi, India, introduce a meticulous
approach to the development of sunflower inflorescence as considered under three phases
listed next: inflorescence initiation, floret development and anther formation. Anthesis of disc
florets is a phytochrome-mediated response and is also modulated by phytohormones, such as
auxins and gibberellic acid. Dr. Bhatla and colleagues focus on the role of various
biomolecules, like glycoproteins, calcium, nitric oxide, reactive oxygen species, and
associated scavenging enzymes in relation to stigma maturation. Specific expression of
lignoceric acid (24:0) in the pollen coat and localization of lipase in pollen and stigma are
likely to have possible roles during pollen-stigma interaction. The phenomenon of self-
incompatibility and pseudo self-compatibility in sunflower has been discussed. The initial
processes accompanying pollen-stigma interaction and their regulation, especially the
adhesion of pollen on the stigma surface, hydration, formation of an "attachment foot" and
pollen tube germination in sunflower with respect to self-and cross-pollinated situations, has
also been dealt with in detail.
In Chapter 3, entitled Development of Female Reproductive Structures and Apomixis in
Sunflowers, Dr. Voronona from the Department of Embryology and reproductive biology,
Komarov Botanical Institute of RAS, Saint-Petersburg, Russia, presents an scrupulous visual
analysis of archesporial cells which are formed and gave rise to megaspore mother cells. The
meiotic divisions produced a linear tetrad of haploid megaspores and from one chalazal
megaspore a Polygonum-type embryo sac is formed. Under natural conditions the apomixis
phenomenon was hardly observed in genus Helianthus L. In addition, author shows how on
plants of CMS-lines a number of anomalies in development of female reproductive system
were detected, including such phenomena as total absence of embryo sac, apospory and
integumentary embryony. Lack of the main embryo sac and formation of additional
aposporous embryo sacs could be observed in the same ovule. Finally, investigation of the
early stages of ovule development showed that aposporous embryo sacs originated from the
same ovule subepidermal cells as a normal embryo sac.
In Chapter 4, entitled Genetics and Genomics Applied to Sunflower Breeding, Dr. Hopp
and colleagues from the Instituto de Biotecnologia, Centro de Investigaciones Veterinarias y
Agronomicas, Instituto Nacional de Tecnologia Agropecuaria, Hurlingham, Argentina,
present a thoughtful study about new breeding strategies based on molecular markers, like
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Preface xi
quantitative trait loci mapping, association mapping and genomic selection that are currently
being developed for commercial crop improvement. The need to increase efficiency and
precision, and save time, resources and efforts, has motivated the application of Marker
Assisted Selection (MAS) in sunflower breeding programs. Furthermore, nowadays, the focus
is on tolerance improvement to biotic and abiotic stresses and oil quality and yield increasing,
in order to reduce the gap between potential and actual sunflower production.
In Chapter 5, entitled Sunflower Genetic Resources – Interspecific Hybridization and
Cytogenetics in Prebreeding, Drs. Atlagic and Terzic present a rigorous description of the
genus Helianthus by reviewing genetic resources, cytogenetic research and application in
breeding. Besides the review of available literature, research results of the Institute of Field
and Vegetable Crops are presented in detail since the establishment of its collection in 1980.
Experience collected during this period indicates the difficulties in collection maintenance,
interspecific crosses and isolation of desired genes. Nevertheless, genus Helianthus proved to
be a good source of material for the constant improvement of cultivated sunflower.
In Chapter 6, entitled Functional Genomics and Transgenesis Applied to Sunflower
Breeding, Dr. Hopp and colleagues from the Instituto de Biotecnologia, Centro de
Investigaciones Veterinarias y Agronómicas, Instituto Nacional de Tecnologia Agropecuaria,
Hurlingham, Argentina, introduce an interesting chapter where they analyze different
strategies which have been developed in the last decade from functional genomics and post
genomics disciplines to contribute to the elucidation of gene regulation and identification of
key metabolic pathways involved in the response to biotic and abiotic stresses in sunflower.
The state of the art of strategies for gene function, studies in silico and in planta, by stable
gene transfer or agroinfiltration in sunflower as well as in the model system for Asteraceae
species, lettuce, are discussed within the frame of their application in sunflower breeding.
In Chapter 7, entitled Disease Management in Sunflowers, Dr. Leite from Embrapa
Soybean, Brazil, presents an interesting approach regarding the most important sunflower
diseases and strategies for disease management. Sunflower can be affected by the presence of
diseases, which may, depending on climatic conditions that favor the occurrence of pathogens
and the infective process, lead to a significant reduction on yield and quality of product.
Disease management should be based on an integrated program, in order to give support for
the sustainability and competitiveness of the sunflower crop.
In Chapter 8, entitled Recent Advances for Developing Resistance against Plasmopara
Halstedii in Sunflowers, Dr. Radwan from the Department of Natural Resources and
Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA,
presents an interesting visual approach to Downy Mildew disease, as one of the most
important diseases of sunflower, which leads to an economic yield loss. In last two decades,
different approaches of genetics and genomics have significantly contributed to better
understand sunflower-Plasmopara halstedii. This progress directed to development of
sunflower lines carrying resistance to different races of this pathogen.
In Chapter 9, entitled Effects of Crop Management on the Incidence and Severity of
Fungal Diseases in Sunflowers, Dr. Debaeke and colleagues from the Institut National de la
Recherche Agronomique (INRA), Toulouse, France, dissected the effects of crop
management on the incidence and severity of major fungal diseases in sunflower, including
downy mildew, phoma, phomopsis and sclerotinia. They deeply reviewed and discussed the
influence of sowing date, plant population, N fertilization, and irrigation on sunflower
diseases from numerous experiments conducted in France during the last twenty years. They
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Juan Ignacio Arribas xii
proposed indicators of canopy development and nutritional status that could be useful when
developing crop management systems with reduced chemical applications.
In Chapter 10, entitled Insect Pests of Sunflowers in Africa, Dr. du Plessis from the Unit
for Environmental Sciences and Management, North-West University, Potchefstroom, South
Africa, present a complete study regarding a number of insect species which have adapted to
cultivated sunflower as a source of food and have consequently become economically
important pests. However, although many insect species is associated with sunflower in
Africa, only few are considered to be of potential economic importance. Insects most
commonly reported as injurious to this crop, occur sporadically, but usually in high numbers.
The families Noctuidae, Tenebrionidae, Curculionidae, Pentatomidae and Orsillidae are the
most important. Various types of damage are caused to sunflower seedlings, but the damage
symptoms are specific to a particular pest species. Dr. du PLessis argues that these seedling
pests mainly constitute dusty surface beetles (Gonocephalum simplex), greater false wire
worms (Somaticus spp.), cutworms (Agrotis spp.) and ground weevils (Protostrophus spp.).
Total defoliation can be incurred by the Plusia looper, Trichoplusia orichalcea. Hemipterans
and the African bollworm, Helicoverpa armigera are the most important insect pests of
sunflower during the heading stages of crop development. Intensive feeding by hemipterans
during this development stage results in deformed heads, which delay flower opening. The
occurrence of the false chinch bug, Nysius natalensis on sunflower during the heading stage
onwards in South Africa, is similar to that of N. stali in Nigeria. Since high summer
temperatures prevail throughout the sunflower production area of South Africa and the most
favourable temperature range for N. natalensis development is between 26°C and 38 °C, the
potential for rapid population build-up by this pest during the sunflower production season is
good. It is likely that N. natalensis can become important in sunflower production in other
African countries with similar weather conditions too. The insect is polyphagous and a variety
of wild host plants, mainly weed species as well as crops such as grain sorghum play an
important role in sustaining its populations. Sunflower is not the preferred host but N.
natalensis lays its eggs on sunflower when its preferred host plants are removed or dead. This
behaviour explains the insects‘ injuriousness to late-planted sunflower because weed species
have already senesced before the sunflower. This period often coincides with seed fill of late-
planted sunflower, providing an alternative for the insect for moisture, as well as seeds that
are necessary for reproduction of the pest. Weeding in and around sunflower during seed fill
of the crop, therefore results in destruction of the preferred host plants of N. natalensis, and
they consequently move to sunflower where they feed and cause damage. When considering
application of insecticides for control of this pest, it should take into consideration that N.
natalensis is highly mobile and continuous re-infestations could occur. Timing of insecticide
application is therefore important. African bollworm (H. armigera) is frequently present
during the reproductive stage of cultivated sunflower in Africa. The attractiveness of
sunflower to this pest is demonstrated by the trap‘s use as a trap crop in and around organic
cotton fields in Tanzania. Larvae occur from the budding stage onwards. Levels of infestation
vary between localities and seasons, sporadically reaching epidemic proportions. Sunflower
has, however, the ability to compensate for head damage and along with the fact that
preferential feeding sites of H. armigera are not the achenes, a significant number of larvae
could be tolerated without any significant effect on yield. Actual damage is, therefore, the
only criterion that could be used in the determination of economic injury levels for control of
African bollworm on sunflower crop.
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Preface xiii
In Chapter 11, entitled Soil Amendments and Their Effects on Sunflower Growth, Dr.
Lopez and colleagues from the Centro de Investigacion en Biotecnologia Aplicada, Instituto
Politecnico Nacional, Tlaxcala, Mexico, present a novel approach to several forms of amend
or fertilize sunflowers as alternative to improves this important cultivar. In particular authors
are interested in the study of organic materials that could be applied to soil in order to
improve their properties and plant growth, keeping in mind that we must recycle or reuse this
kind of materials. Finally, the organic amendments could be a beneficial disposal approach
that must be considered.
In Chapter 12, entitled Nutrition and Fertilization of Sunflowers in Brazilian Cerrado,
Dr. Castro and colleagues present an interesting chapter centered in a particular region in
Brazil, which authors argue that is recognized as a major global food producer and despite the
fact that its agriculture occupies only less than 5 % of the national territory, the estimated
grain production for 2013/2014 growing season is 195 million tons. The Brazilian Cerrado is
the main agriculture expansion region in the country, driven by soybean cultivation in an area
of 13 million ha. In this tropical agricultural region, sunflower has great potential for
expansion and consolidation as an important component of sustainable crop rotation
production systems. This chapter addresses the major limiting soil fertility factors which
hinder the crop development and discusses fertilization management practices related to the
main limiting nutrients, like the macronutrients nitrogen, phosphorus and potassium and
micronutrients such as boron and molybdenum. Adequate management of soil acidity and
fertilization has been proved as a powerful tool to improve the natural conditions of acidic
and chemically poor arable tropical soils. In addition to the soil fertility assessment, leaf
analysis is essential for the proper interpretation of plant nutritional status, thereby enabling
better refinement of the crop nutritional management.
In Chapter 13, entitled Environmental Issues in the Sunflower Crop of Midwestern Brazil
– Diversification And Complementarities in the Biodiesel Chain, Dr. Ramos and colleagues
from Embrapa Environment, Brazil, present an interesting study regarding the increase in
global demand for renewable energy, the production of oilseeds, including sunflower, as
feedstock for biodiesel. The increase in global demand for renewable energy has encouraged,
both directly and indirectly, the production of oilseeds, including sunflower, as feedstock for
biodiesel. In this scenario, authors argue that Brazil stands out for its excellent agronomic and
climatic conditions for growing these crops throughout its territory. Sunflower is considered
an interesting option and your production in the mid-western region of Brazil has done
valuable contributions as a second, and especially the production practices adopted by the
reference farmers of the country, rendering complementarities and diversification to both the
food and the bioenergy sectors.
In Chapter 14, entitled Micro and Macro-Morphological Variation of Cosmos Bipinnatus
and Cosmos Bipinnatus Var. Albiflorus in Sympatric Zones in Central Mexico, Dr. Paniagua
and colleagues from the Centro de Investigacion en Biodiversidad y Conservacion, Univ.
Autonoma Estado de Morelos, Morelos, Mexico, present a concise but at the same time
precise analysis of the morphological variations in various sunflower species in central
Mexico area. Mexico is considered one of the centers of diversification of the Asteraceae
family, which contains the greatest richness of flowering plants. Particularly, the Trans-
Mexican Volcanic Belt (TMVB) is a heterogeneous mountain belt, located in the central part
of the country in an east–west direction, which has been considered a diversification site for
many genera due to the vast number of species that it contains, as well as its high degree of
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Juan Ignacio Arribas xiv
endemism. Cosmos bipinnatus is present in various types of vegetation along the TMVB
which is favored by disturbances. Taxonomic studies have documented that this sunflower
species presents white to lilac ligules. However, horticulturists have considered the white
variety as C. bipinnatus var. albiflorus. Still, there is no scientific evidence to support their
observations. Therefore, the goal of this study was to compare the micro and macro
morphology characters between C. bipinnatus individuals with white and lilac ligules to
determine a possible morphological differentiation between both phenotypes in sympatric
zones in the central Mexico region. Principal Component Analysis and Non-Metric
Multidimensional Scaling showed a clear morphological differentiation between both groups;
this pattern was consistent even when the ligule‘s color was not considered for the statistical
analyses. Dr. Paniagua and colleagues results sign a possible speciation between these
phenotypes and support a taxonomic shift for the Mexican sunflower with white ligules to C.
bipinnatus var. albiflorus.


Juan Ignacio Arribas, PhD
Associate Professor of Electrical Engineering
University Valladoild, Spain
Valladolid, November 2013
Tel: +34 983423000
Fax: +34 983423667
jarribas@tel.uva.es



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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 1



AN INTRODUCTION TO THE SUNFLOWER CROP


Fabián Fernández-Luqueño
1,
, Fernando López-Valdez
2
,
Mariana Miranda-Arámbula
2
, Minerva Rosas-Morales
2
,
Nicolaza Pariona
1
and Roberto Espinoza-Zapata
3

1
Natural Resources and Energy Group, Cinvestav-Saltillo, Coahuila, México
2
CIBA - Instituto Politécnico Nacional, Tepetitla de Lardizábal, Tlaxcala, México

3
Crop Breeding Department, UAAAN, Saltillo, Coahuila, México


ABSTRACT

Sunflower (Helianthus annuus L.) belongs to the family Asteraceae. The Helianthus
genus contains 65 different species of which 14 are annual plants. The sunflower plant
originated in eastern North America. It is thought to have been domesticated around 3000
B.C. by Native Americans. In the late 1800s the sunflower was introduced in the Russian
Federation where it became a food crop and Russian farmers made significant
improvements in the way that the sunflower was cultivated. Since 3000 B.C. a wide
range of uses of sunflower have been reported throughout the world such as ornamental
plant, medicinal, alimentary, feedstock, fodder, dyes for textile industry, body painting,
decorations, and so on. Sunflower species are allelopathic in nature and this crop appears
to have a bright future, especially if the scientists can translate the cutting-edge research
into technologies that will reduce the reliance on synthetic herbicides, pesticides, and
crop protection chemicals. On the one hand sunflower is well known by its
phytoremediation potential, thus it can be speculated that the good tolerance of sunflower
towards pollutants coupled with an increased accumulation/degradation capacity might
contribute to an efficient removal of pollutants from soil and water; on the other hand
sunflower possesses the potential to develop bioenergy systems that allow for synergies
between food and energy production. Because the sunflower has several potential
markets, it is a good choice for growers on both small and large scales. However, it has to
be remembered that scientific, technical or agricultural projects linked with sunflower
have to include side effects elsewhere in order to shape a sustainable future.

*
Corresponding author: F. Fernández-Luqueño, Natural Resources and Energy Group, Cinvestav-Saltillo, Coahuila.
C. P. 25900, México Tel.: +52 844 4389625; Fax: +52 844 4389610. E-mail address: cinves.cp.cha.luqueno@
gmail.com.
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F. Fernández-Luqueño, F. López-Valdez, M. Miranda-Arámbula et al. 2
Keywords: Allelopathy, biodiesel, phytoremediation, renewable energy, sustainable
development, symbiosis


1. INTRODUCTION

Sunflower (Helianthus annuus L.) belongs to the family Asteraceae. Helianthus genus
contains 65 different species (Andrew et al., 2013). The name Helianthus, being derived from
helios (the sun) and anthos (a flower), has the same meaning as the English name Sunflower,
which has been given these flowers from a supposition that they follow the sun by day,
always turning towards its direct rays. The sunflower that most people refer to is H. annuus,
an annual sunflower. In general, it is an annual plant which possesses a large inflorescence
(flowering head), and its name is derived from the flower's shape and image, which is often
used to depict the sun. The plant has a rough, hairy stem, broad, coarsely toothed, rough
leaves and circular heads of flowers (Khaleghizadeh, 2011). The heads consist of many
individual flowers which mature into seeds on a receptacle base (Seghatoleslami et al., 2012).
Sunflower is the world‘s fourth largest oil-seed crop and its seeds are used as food and its
dried stalk as fuel. It is already been used as ornamental plant and was used in ancient
ceremonies (Harter et al., 2004; Muller et al., 2011). Additionally, medical uses for
pulmonary afflictions have been reported. In addition, parts of this plant are used in making
dyes for the textile industry, body painting, and other decorations. Sunflower oil is used in
salad dressings, for cooking and in the manufacturing of margarine and shortening
(Kunduraci et al., 2010). Sunflower is used in industry for making paints and cosmetics. A
coffee type could be made with the roasted seeds. In some countries the seed cake that is left
after the oil extraction is used as livestock feed. In the Soviet Union the hulls are used for
manufacturing ethyl alcohol, in lining for plywood and growing yeast. The dried stems have
also been used for fuel. The stems contain phosphorous and potassium which can be
composted and returned to soil as fertilizer. Sunflower meal is a potential source of protein
for human consumption due to its high nutritional value and lack of anti-nutritional factors
(Fozia et al., 2008).
Sunflower was a common crop among American Indian tribes throughout North
America. Evidence suggests that the plant was cultivated by natives in present-day Arizona
and New Mexico about 3000 B.C. Some archaeologists suggest that sunflower may have been
domesticated before corn (NSA, 2013). Although the scientific consensus had long been that
sunflower was domesticated once in eastern North America, the discovery of pre-Columbian
sunflower remains at archaeological sites in Mexico led to the proposal of a second
domestication center in southern Mexico. However, evidences from multiple evolutionary
important loci and from neutral markets support a single domestication event for extant
cultivated sunflower in eastern North America (Blackman et al., 2011).
The objective of this chapter is to present and discuss a summary about the huge amount
of information in which the sunflower is the main subject. The chapter aims to assist people
involved in all aspects of sunflower management, including conservation, agriculture, mining,
energy, food production, health and other industries, to obtain a broad knowledge of
sunflower and of its ecosystem services.

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An Introduction to the Sunflower Crop 3
2. BOTANICAL AND MORPHOLOGICAL DESCRIPTION

Sunflowers are botanically classified as Helianthus annuus L. (Table 1). They are large
plant and are grown throughout the world because of their relatively short growing season.
Sunflower is an annual herb, with a rough, hairy stem, 3 to 12 feet high, broad, coarsely
toothed, rough leaves, 3 to 12 inches long and circular heads of flowers, 3 to 6 inches wide in
wild specimens and often a foot or more in cultivation. The flower-heads are composed of
many small tubular flowers arranged compactly on a flattish disk: those in the outer row have
long strap-shaped corollas, forming the rays of the composite flower. Each sunflower head, or
inflorescence, is actually composed of two types of flowers. What appears to be yellow petals
around the edge of the head are actually individual ray flowers. The face of the head is
comprised of hundreds of disk flowers, which each form into a seed (achene).
The basic chromosome number for the Helianthus genus is 17. Diploid, tetraploid and
hexaploid species are known. There are only 14 annual species of Helianthus. Plant breeders
have made interspecific crosses within the genus and have transferred such useful characters
as higher oil percentage, cytoplasmic male sterility for use in production of hybrids, and
disease and insect resistance to commercial sunflower.

Table 1. Scientific classification of H. annuus L.; this genus counts 65 different species

Taxa
Kingdom Plantae
Subkingdom Viridaeplantae
Infrakingdom Streptophyta
Division Tracheophyta
Subdivision Spermatophytina
Infradivision Angiospermae
Class Magnoliopsida
Superorder Asteranae
Order Asterales
Family Asteraceae
Subfamily Helianthoideae
Tribe Heliantheae
Genus Helianthus
Specie annuus
The taxonomic classification has been in place since 1753.


3. PRODUCTION

In recent years, the sunflower cultivated area has been steadily increasing due to the
breeding of dwarf high yielding hybrids that also facilitate mechanization and the emphasis
given to polyunsaturated acids for human consumption. Global production grew steadily in
last 25 years (PSD-USDA, 2011), and FAO expect a total world output close to 60 million
tons towards 2050. The four largest producers (Russia, Ukraine, European Union and
Argentina) account for 70% of global volume, with an exponential growth of production in
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the last ten years in the Black Sea region, with increased acreage an higher yields achieved by
the replacing old varieties by hybrid seeds.
According to data from FAOSTAT (FAOSTAT, 2011) Russia Federation ranked first
producing ca. 9.7 millions of tons of sunflower seeds or 26% of the world total. Ukraine and
Argentina ranked second and third place with 8.6 and 3.6 tons of sunflower seeds,
respectively. France, Romania, China, Bulgaria, Hungary, Turkey, and Spain produced
between 1.0 and 1.9 millions of tons of sunflower seeds (Table 2). The United States
produced ca. 1.0 millions of tons of sunflower seeds, or 5% of the world‘s total production.
That is enough to make the United States rank eleventh in that category. South Africa ranked
twelfth producing ca. 0.9 millions of tons of sunflower seeds.

Table 2. The highest twelve sunflower seed producing countries
in the world during 2011

Place Countries Production (tons)
1 Russia Federation 9,696,450
2 Ukraine 8,670,500
3 Argentina 3,671,750
4 France 1,882,450
5 Romania 1,789,330
6 China 1,700,000
7 Bulgaria 1,439,700
8 Hungary 1,374,780
9 Turkey 1,335,000
10 Spain 1,084,300
11 United States of America 924,550
12 South Africa 860,000
Russia followed by Ukraine are harvesting almost half of the world sunflower seed production. The
total sunflower seed production is reaching ca. 35 millions of tons
Data source: data obtained from FAOSTAT (2011).

According to FAO (FAO, 2010), there are some key production parameters which have
to be known by farmers throughout the world:

 Sunflowers are grown in warm to moderate semi-arid climatic regions of the world
from Argentina to Canada and from central Africa to the Commonwealth of
Independent States (Esmaeli et al., 2012; Onemli, 2012).
 Frost will damage sunflowers at all stages of growth. The plant grows well within a
temperature range of 20-25°C; temperatures above 25°C reduce yields and oil
content of the seeds (Thomaz et al., 2012).
 Plants are drought-resistant, but yield and oil content are reduced if they are exposed
to drought stress during the main growing and flowering periods. Sunflowers will
produce moderate yields with as little as 300 mm of rain per year, while 500-750 mm
are required for better yields (Gholamhoseini et al., 2013; Ghaffari et al., 2012).
 Sunflowers adapt to a wide variety of soil, but perform best on good soils suitable for
maize or wheat production (Radanielson et al., 2012).
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An Introduction to the Sunflower Crop 5
 Sunflower plant density of 5-8 plants per m
2
is required to form the optimum leaf
area for plant photosynthesis. Kernel weight (40-80 g per 1000 kernels) and the
average number of kernels in a sunflower head (1200-1500) are the other most
important yield component (Seassau et al., 2012; Emami-Bistghani et al., 2012).
 Sunflower growth depends more on nitrogen than any other nutrient. Due to its deep
rooting system, sunflower is able to use nitrogen from soil layers that are inaccessible
to wheat, corn or other field crops. The plant requires a maximum of 150 kg of
nitrogen per hectare to produce a three tons ha
-1
yield. Over fertilization may lead to
sunflower lodging. Phosphorous, potassium, boron, magnesium and molybdenum are
also needed to achieve the best yields (Jabeen and Ahmad, 2012; Babaeian et al.,
2011).
 The average fatty acid composition of oil from temperate sunflower crops is 55-75%
linoleic acid and 15-25% oleic acid. Protein content is 15-20% (Aznar-Moreno et al.,
2013; Ali and Ullah, 2012).
 Planting in the Western Balkan countries, Eastern Europe and countries of the
Former Soviet Union takes place during March and April (Zheljazkov et al., 2012;
Saleem et al., 2008).
 Sunflower has one of the shortest growing seasons of the major economically
important crops of the world. Early maturing varieties are ready for harvesting 90 to
120 days after planting, and late maturing varieties 120 to 160 days after planting.
Delayed harvesting causes unwelcome changes in oil quality, with an increase in free
fatty acid content. The seeds are ready to harvest when the heads turn black or brown
and the seed moisture content reaches 10-12%. Grain combines are fairly easily
adapted for the harvesting of sunflower by the addition of a head snatcher (Borbely et
al., 2008).
 Depending on climatic and cultivation conditions, yields can vary from as much as
600 to 3000 kg ha
-1
; irrigation is a key factor for obtaining high yields (Chigeza et
al., 2013; Khan et al., 2013; Akhtar et al., 2012).

Table 3 shows the oil yields in gallons per acre of oil producing crops, the yields will
vary in different agroclimatic zones. Sunflower produces 98 Gal oil acre
-1
.

That is enough to
make the sunflower rank twenty-third in that category. Additionally, higher-yielding oil crops
like safflower, mustards and sunflower have significant rotational benefits. For example, deep
safflower and sunflower roots help break up hardpan and improve soil tilth.


4. GROWTH AND DEVELOPMENT

Sunflower is a broadleaf plant that emerges from the soil with two large cotyledons
(Rawat et al., 2010). The emergence will take four to five days when planted an inch deep in
warm soil, but will take a few days longer in cooler soils or when planted deeper. Soil
crusting can make it difficult for the large seedlings to push out of the soil. Sunflowers grow
rapidly, producing large and rough leaves. Current sunflower varieties reach an average
height of six feet, varying between five and seven feet depending on planting date and soil
conditions (Saensee et al., 2012). After reaching their full height and blooming, heads on
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F. Fernández-Luqueño, F. López-Valdez, M. Miranda-Arámbula et al. 6
commercial cultivars turn downwards, designed to make it harder for birds to eat the seed.
Commercial sunflowers have flowers that are self-compatible for pollination, meaning they
do not require a pollinating insect, although some studies have shown bee pollinators
providing a slight yield boost (de Carvalho and de Toledo, 2008). Some farmers prefer
sowing their rows from north to south so that the capitula can lean into the row space, rather
than bumping against an adjacent plant, causing some seed to fall (Olowe and Adeyemo,
2009).

Table 3. Oil producing crops

Number Crop Scientific name Yield (Gal oil acre
-1
)
1 Oil palm Elaeis guineensis Jacq. 610
2 Macauba palm Acrocomia aculeata Jacq. 461
3 Pequi Caryocar brasiliense Camb. 383
4 Buriti palm Mauritia flexuosa L. 335
5 Oiticia Licania rigida Benth 307
6 Coconut Cocos nucifera L. 276
7 Avocado Persea americana Mill. 270
8 Brazil nut Bertholletia excelsa Humb & Bonpl. 245
9 Macadamia nut Macadamia ternifolia F.V. Muell. 230
10 Jatrofa Jatropha curcas L. 194
11 Babassu palm Orbignya martiana Mart. 188
12 Jojoba Simmondsia chinensis Link 186
13 Pecan Carya illinoensis Wangenh. 183
14 Bacuri Platonia insignis Mart. 146
15 Castor bean Ricinus communis L. 145
16 Ghoper plant Euphorbia lathyris L. 137
17 Pissava Attalea funifera Mart. 136
18 Olive tree Olea europea L. 124
19 Rapessed Brassica napus L. 122
20 Opium poppy Papaver somniferum L. 119
21 Peanut Arachis hypogea L. 109
22 Cocoa Theobroma cacao L. 105
23 Sunflower Helianthus annuus L. 98
24 Tung oil tree Aleurites fordii Hemsl. 96
Yields of common energy crops are associated with biodiesel production. This is not related to ethanol
production, which relies on starch, sugar, and cellulose content instead of oil yields.

Experiments have been carried out to improve the growth and development of sunflower
under natural or stress conditions (Gerardo et al., 2013; Nasim et al., 2011; Da Silva et al.,
2012). Naz and Bano (2013) reported that the adverse effects of salt stress on sunflower
growth could be alleviated by foliar application of salicylic acid alone or in combination with
Azospirillum and Pseudomonas inoculations (Table 4). Gholamhoseini et al. (2013) shown
that the application of Glomus musseae and Glomus hoi could be critical in the cultivation of
sunflowers under arid and semi-arid conditions, where water is the most important factor in
determining plant growth and yield. Additionally, Akbari et al. (2011) reported that
inoculating the sunflower seeds with plant-growth promoting rhizobacteria increased the
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An Introduction to the Sunflower Crop 7
qualitative and quantitative properties of sunflower significantly, as compared to the control
treatment.

Table 4. Recent uses of the sunflower during the last years; main or alternative uses
make evident the diversity of sunflower

Area Description References
Food
Blends of high linoleic sunflower oil with
selected cold pressed soils.
(Ramadan, 2013)
Production of florets of sunflower. (Liang et al., 2013)
Tocopherols and phytosterols for the human
food market.
(Fernández-Cuesta et al.,
2012)
Sunflower flour as a rich source of high
quality proteins.
(Levic et al., 2012)
Protein hydrolysis using proteases. (Tavano, 2013)
Animal Feed
Sunflower products fed to finishing pigs. (González-Vega and Stein,
2012)
Ingestive behavior and physiological
responses of goats fed with sunflower cake.
(Agy et al., 2013)
Nutritional value of sunflower meal on broiler
chickens.
(Moghaddam et al., 2012)
Potential nutritive value as source of feed for
ruminants in Kenya.
(Osuga et al., 2012)
Energy
Methane production. (Fernández-Cegrí et al., 2013;
Todorovic et al., 2013)
Biodiesel production. (Iriarte and Villalobos, 2013;
Iglesias et al., 2012)
Bioenergy: biotechnology progress and
emerging possibilities.
(González-Rosas et al., 2013)
Anaerobic digestion of sunflower oil cake. (De la Rubia et al., 2013)
Oil production. (Spinelli et al., 2012)
Sustainability
Of sunflower cultivation within the EU
Renewable Energy Directive.
(Spugnoli et al., 2012)
Sustainable sunflower processing. (Weisz et al., 2013)
Economic sustainability of sunflower
production.
(Keskin and Dellal, 2011)
Symbiosis and Plant-Growth Promoting Rhizobacteria
Effect of arbuscular mycorrhizal inoculation
on sunflower.
(Naz and Bano, 2013; Audet
and Charest, 2013;
Gholamhoseini et al., 2013)



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F. Fernández-Luqueño, F. López-Valdez, M. Miranda-Arámbula et al. 8
Table 4. (Continued)

Area Description References
Symbiosis and Plant-Growth Promoting Rhizobacteria
Bacterial inoculation speeds zinc release from
ground tire rubber.
(Khoshgoftarmanesh et al.,
2012)
A strain of Bacillus subtilis stimulates
sunflower growth.
(López-Valdez et al., 2011)
Remediation
Biodegradation of PAHs. (Tejeda-Agredano et al.,
2013)
Plant response to lead. (Doncheva et al., 2013)
Metal accumulation on sunflower. (Mahmood et al., 2013; Hao et
al., 2012)
Fertilization, pesticides and environment
Foliar fertilization with molybdenum. (Skarpa et al., 2013)
Fertilization affects the agronomic traits of
high oleic sunflower hybrid.
(Mohammadi et al., 2013)
Gas exchange in sunflower plants. (Da Silva et al., 2013)
Effect of different nitrogen level on yield
components.
(Rafiei et al., 2012)
Biological control
Encrusting offers protection against
phytotoxic chemicals.
(Szemruch and Ferrari, 2013)
Biological control of Macrophomina
phaseolina on sunflower.
(Ullah, 2010)
Allelopathic effects
On growth of rice and subsequent wheat crop. (Bashir et al., 2012)
On seed germination and seedling growth of
Trianthema portulacastrum.
(Rawat et al., 2012)
Health
In vivo evaluation of an oral health toothpaste
with sunflower oil.
(Schafer et al., 2007)
Health benefits of the sunflower kernel. (Holliday and Phillips, 2001)


5. SUNFLOWER ALLELOPATHY

Sunflower species are allelopathic in nature; as well cultivated sunflower has great
allelopathic potential and inhibits weed-seedling growth of velvet leaf, thorn apple, morning
glory, wild mustard and other weeds (Macías et al., 1998a). Two members of the genus
Helianthus contain a great quantity of allelopathic compounds. H. annuus is well known for
its allelopathic compounds, including sesquiterpene lactones, heliespirones A, annoionones,
helibis-abonols and heliannols (Macías et al., 1998b). Heliannols A, D and E have special
relevance due to high phytotoxic activity (Macías et al., 1999).

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An Introduction to the Sunflower Crop 9

Figure 1. Some molecular structures of allelopathic compounds presents in sunflower cultivars. A)
Annuithrin (sesquiterpene lactone) or Niveusin C, a growth inhibitor. B) Furanoheliangoline, a
biologically active molecule. C) Germacranolide, a toxic sesquiterpene lactone (a potent feeding
deterrents).
Helianthus tuberosus contains helian-gine and H. annuus contains a sesquiterpene
lactone; a heliangolide [Annuithrin or Niveusin C (Figure 1A)] (a growth inhibitor);
furanoheliangolide [(Figure 1B) a biologically active]; three additional sesquiterpene
lactones: the known compound niveusin B, a germacranolide (Figure 1C) (the tifruticin-type);
a 3-ethoxy-niveusin B; an ethoxyheliangolide (Spring et al., 1982) and coumarins (only
accumulate in healthy sunflower plants as a response to the variation in environmental
conditions that affect field-grown plants). In sunflower, it was reported that the concentrations
of scopolin exceeded those in both infected and uninfected plants (Gutiérrez-Mellado et al.,
1996).
Scopoletin have been described as phytoalexins and allelopathic compounds, being
accumulated in response to fungal and parasitic plant infection, insect attack, mechanical
injury and treatment with abiotic elicitors such as sucrose and CuCl
2
, and plant hormones;
besides scopoletin has also been shown to have a physiological activity, including the
promotion of stomatal closure in sunflower and inhibition of bud growth in pea at very low
concentrations (Gutiérrez-Mellado et al., 1996).
Annuithrin was tested using a bioassay with Avena straight growth test. The addition of a
concentration range from 50 to 180 μM resulted in a linear reduction of growth between 10
and 90%. In fact, annuithrin was shown to have antibacterial qualities. However, fungi and
yeast were either less inhibited or not inhibited (minimal inhibitory concentration, MIC 45 μg
mL
-1
on Bacillus brevis; MIC 90 μg mL
-1
on Proteus vulgaris; MIC 90 μg mL
-1
on
Eremothecium ashbyi; Macías et al., 1996). In addition, in vivo DNA and RNA synthesis in
cells of the ascitic form of Ehrlich carcinoma was drastically reduced by annuithrin (at an
annuithrin concentration of 20 μg mL
-1
about 50% inhibition of DNA synthesis and about
75% inhibition of RNA synthesis) (Spring et al., 1981).
It is well known that there are examples of allelopathic cover crops being used for weed
management in other crops, as well as other cultural methods to employ allelopathy (Duke,
2010). However, there are still no cultivars of crops being sold with allelopathic properties as
a selling point (Cheema and Khaliq, 2000; Tesio and Ferrero, 2010). Enhancement or
impartation of allelopathy in crops through the use of transgenes could eventually be used to
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F. Fernández-Luqueño, F. López-Valdez, M. Miranda-Arámbula et al. 10
produce such a cultivar. The study of allelopathic crops appears to have a bright future,
especially if the scientists can translate the cutting-edge research into technologies that will
reduce the reliance on synthetic herbicides, pesticides, and crop protection chemicals. Tesio
and Ferrero (2010) reported that the use of allelopathic traits from crops or cultivars with
important weed inhibition qualities, together with common weed control strategies, can play
an important role in the establishment of sustainable agriculture. It has to be noted that
allelopathy may also be another component of desired improved weed management. It will
not solve all weed problems in any field, but may help considerably to reduce the population
of weeds in the fields (Labrada, 2008).


6. PHYTOREMEDIATION WITH SUNFLOWER

Phytoremediation consists of mitigating pollutant concentrations in contaminated soils,
water, or air, with plants able to contain, degrade, or eliminate contaminants and its
derivatives (Malaviya and Singh, 2012). H. annuus is a plant with not only food and energy
values, but also with phytoremediation potential (Seth et al., 2011; Mukhtar et al., 2010). It is
one of the most widely studied plants for heavy metal phytoremediation (Kara et al., 2013).
However, it is well known that sunflower is able to contain, degrade or eliminate metals
(Chen et al., 2012; Ker and Charest, 2010; Lee and Yang, 2010), polycyclic aromatic
hydrocarbons (Tejeda-Agredano et al., 2013; Gan et al., 2009) and polychlorinated biphenyls
(Fiebig et al., 1997) from soil or water. Investigations with H. annuus have revealed that
several heavy metals, including lead, cadmium, copper, zinc and cobalt, accumulate at high
concentrations in shoots as well as in roots. Heavy metal uptake is minor in seeds than in
roots and shoots. However, few attempts have been made to use plant-growth promoting
rhizobacteria to facilitate phytoextraction and cadmium uptake in H. annuus planted in
cadmium-contaminated soil (Prapagdee et al., 2013). Sunflower is a documented metal
accumulator and its growth on contaminated soil for simultaneous remediation and further
energy production has been studied (Marques et al., 2013; Madejon et al., 2003). The good
tolerance of sunflower toward pollutants coupled with an increased accumulation/degradation
capacity might contribute to an efficient removal of pollutants from soil and water. Clearly it
is not an easy job, thus scientists of multidisciplinary areas have to work hard. Additionally,
there is a lack of knowledge concerning the pollutants accumulation and antioxidant
responses during the growth and development of sunflowers.


7. SUNFLOWER AS A RENEWABLE ENERGY SOURCE

Thousands of years ago, people in many regions throughout the world began to process
vegetable oils, utilizing whatever food stuffs they had on hand to obtain oils for a variety of
cooking purposes. The Chinese and Japanese produced soy bean oil as early as 2000 B.C.,
while southern Europeans had begun to produce olive oil by 3000 B.C. In Mexico and North
America, sunflower seeds were roasted and beaten into a paste before being boiled in water;
the oil that rose to the surface was skimmed off (FAO, 2010). During the last decade, an
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An Introduction to the Sunflower Crop 11
increased attention would be observed being paid on the use of sunflower as renewable
energy source.
Oilseed sunflower is quickly gaining popularity as a feedstock crop for biodiesel because
it shares several positive agronomic features with other common oil crops such as canola and
soybean; yields well in a variety of conditions, and can be grown easily and profitably at both
small farm and large field scales. It is well known that a number of crops can be used for both
food and bioenergy production such as sunflower (Kibazohi et al., 2012). Under some
circumstances, the potential exist to develop bioenergy systems that allow for synergies
between food and energy production. Integrated food and energy systems could produce food
crops while simultaneously addressing energy needs (Bogdanski et al., 2010).
There is a trend world-wide to grow crops in short rotation or in monoculture (such as
sunflower), particularly in conventional agriculture (Bennett et al., 2012). This practice is
becoming more prevalent due to a range of factors including economic market trends,
technological advances, government incentives, and retailer and consumer demands. Land-
use intensity will have to increase further in future in order to meet the demands of growing
crops for both bioenergy and food production, and long rotations may not be considered
viable or practical. Notwithstanding, evidence indicates that crops grown in short rotations or
monoculture often suffer from yield decline compared to those grown in longer rotations or
for the first time (Zambrano-Navea et al., 2012). Numerous factors have been hypothesized as
contributing to yield decline, including biotic factors such as plant pathogens, deleterious
rhizosphere microorganisms, mycorrhizas acting as pathogens, and allelopathy or autotoxicity
of the crop, as well as abiotic factors such as land management practices and nutrient
availability (Sun et al., 2011). This section identifies gaps in our understanding about the
energy production of biomass and the interaction of the ecosystems. Additionally, it has to be
remembered that each bioenergy development projects have to include side effects elsewhere
in order to shape a sustainable future.


CONCLUSION

Sunflower was domesticated in eastern North America and since 3000 B.C. this crop was
bred by natives. Thenceforth a wide range of uses of sunflower have been reported
throughout the world. Sunflowers are a permanent source of food, oilseed and biofuels
because they are well adapted to a variety of conditions and often require fewer agricultural
inputs than other more common crops, while under some circumstances, the potential exist to
develop bioenergy systems that allow for synergies between food and energy production.
Because the sunflower has several potential markets, it is a good choice for growers in both
small and large scales. However, scientific, technical or agricultural projects linked with
sunflower have to include environmental side effects such as pollution, greenhouse gases
emissions, salinization, or energy consumption elsewhere in order to shape a sustainable
future.



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F. Fernández-Luqueño, F. López-Valdez, M. Miranda-Arámbula et al. 12
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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 2



FLORAL BIOLOGY OF SUNFLOWERS:
A HISTOLOGICAL AND PHYSIOLOGICAL ANALYSIS


Basudha Sharma, Rashmi Shakya and Satish C. Bhatla
*

Laboratory of Plant Physiology and Biochemistry, Department of Botany,
University of Delhi, Delhi, India


ABSTRACT

The development of sunflower inflorescence can be considered under three phases,
namely inflorescence initiation, floret development and anther formation. Floret
primordia appear at the rim of the receptacle where ray or disc florets are generated. Disc
florets are arranged in Fibonacci series whereby a spiral pattern emerges as new florets
arise in rows of bumps consisting of a bract and a floret. Floral morphogenesis in
sunflower occurs according to the ABC model, whereby genes of the MADS box are
activated. Anthesis of disc florets is a phytochrome-mediated response and is also
modulated by plant hormones, such as auxins. The disc florets are hermaphrodite and
protandrous in nature, whereas the ray florets are sterile, incomplete and have an
attractive, fused and flag-like corolla. Stigma in sunflower is semi-dry in nature,
producing lipid rich exudates in the crevices of the adjacent papillae. Stigma undergoes
physiological maturity with the passage of development from bud, staminate and, finally
to the pistillate stage. The production of extracellular lipid rich secretions is initiated at
the staminate stage of stigma development and increases at the receptive stage through
the availability of elaioplasts and endoplasmic reticulum network in the basal regions of
the papillae. Transfer cells, earlier identified only in the wet type of stigma, are also
present in the transmitting tissue of sunflower stigma. Neutral esters and triacylglycerols
(TAGs) are the major lipidic constituents in pollen grains and stigma, respectively.
Lignoceric acid (24:0) and cis-11-eicosenoic acid (20:1) are specifically expressed only
in the pollen coat. Similar long-chain fatty acids have earlier been demonstrated to play a
significant role during the initial signalling mechanism leading to hydration of pollen
grains on the stigma surface. Lipase activity is expressed both in the pollen grains and
stigma papillae. Stigma exhibits a better expression of acyl-ester hydrolase activity the
pollen grains. Specific expression of lignoceric acid (24:0) in the pollen coat and

*
Corresponding author: Professor S.C.Bhatla; E mail: bhatlasc@gmail.com.
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Basudha Sharma, Rashmi Shakya and Satish C. Bhatla 20
localization of lipase in pollen and stigma are likely to have possible roles during pollen-
stigma interaction. During the course of stigma development in sunflower, a correlation is
evident in the accumulation of reactive oxygen species (ROS), nitric oxide (NO) and the
activities of ROS scavenging enzymes [superoxide dismutase (SOD) and peroxidase
(POD)]. Mn-SOD (mitochondria localized) and Cu/Zn-SOD (cytoplasmic) exhibit
differential expression during the staminate stage of stigma development. An increase in
total SOD activity at the staminate stage is followed by a peak of POD activity during the
pistillate stage of stigma development, indicating the sequential action of the two
enzymes in scavenging ROS in maturing stigma. The number of POD isoforms increases
with the passage of stigma development and two POD isoforms are unique to pistillate
stage. This highlights their role in ROS scavenging mechanism. ROS and NO
accumulation exhibit reverse trends during pollen-stigma interaction. All these recent
findings indicate the modulation of floral development in sunflower by an array of
biomolecular signalling components which influence development through a series of
cross-talk mechanisms.

Keywords: Lipids, nitric oxide, non-specific esterase, pollen, peroxidase, reactive oxygen
species, superoxide dismutase, stigma, pollen-stigma interaction


INTRODUCTION

A large diversity of floral structures of varying complexities are evident in plants for the
attraction of pollinators. Cross-pollinated plants exhibit a kind of synchrony among
themselves and also with their pollinators in order to bring about optimal seed set. It is
necessary that plants must flower at the correct time of the year for optimal reproductive
fitness. Such a strong correlation with the environment for the onset of flowering poses many
questions about the mechanisms of sensing of the environmental signals by the plants and
also about the sequence of biochemical events which ultimately bring about flowering in
response to the environmental signals. A vegetative shoot bud exhibits noteworthy differences
when compared with a floral bud, in terms of the constituent forms and types of cells. A
change in the fate of cells at the shoot apex is governed by the expression of a set of genes,
leading to various biochemical events in the shoot apex which bring about floral evocation, i.e
the ability of the apical meristem to produce flowers. Floral evocation is regulated by
endogenous factors, such as circadian rhythms and hormones, and exogenous factors such as
photoperiod and temperature. The initiation of four types of floral organs from the floral
meristem is observed in whorls around the flanks of the meristem. The floral primordial start
as small bumps of cells and their further development into reproductive structures is governed
by the environmental signals, various metabolic events and also by the activation of specific
genetic programs. Broadly, an attempt has been made in the following chapter to understand
the histological, physiological and biochemical changes taking place in the shoot apex of
sunflower during this process of phase change, i.e., transition from adult vegetative phase to
adult reproductive phase. The initiation of whorls of disc florets in the inner core and
development of peripheral ray florets during capitulum development in sunflower is a gradual
process, showing various stages of development of florets in a capitulum. The present chapter
provides detailed information on the ultrastructural changes associated with stigma
development in sunflower. These features have been analyzed in relation with the
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Floral Biology of Sunflowers: A Histological and Physiological Analysis 21
biochemical events accompanying stigma maturation. Likewise, a detailed structural analysis
of pollen (intact and germinating) has been discussed. Finally, the mechanism of pollen-
stigma interaction has been analyzed under natural and experimental conditions.


PATTERN OF FLORAL DEVELOPMENT AND ITS MODULATION
BY LIGHT, HORMONES AND GENETIC FACTORS

Members of Asteraceae maximize their reproductive output by condensing inflorescence
and forming a capitulum. Vegetative apex is indeterminate, domed and densely meristematic
whereas reproductive apex broadens, flattens and becomes determinate (Teeri et al., 2006).
Sunflower inflorescence is a disc-shaped capitulum located at the shoot tip and its shape and
size vary according to the cultivar, season and agricultural conditions (Weiss, 2000). The
capitulum is surrounded by three rows of ovate to ovate-laceolate involucral bracts or
phyllaries which function as sepals and protect the capitulum during its development (Figure
1). Various phases of reproductive development in sunflower have earlier been categorized
under nine stages (Schneiter and Miller, 1981). Beginning from the initiation of floral bud
(R1) to the attainment of physiological maturity (R9), R5 marks the beginning of flowering.
This stage (R5) is further subdivided from R5.1 to R5.2 and so on, representing the percent of
disc florets which have completed or are flowering. The florets in the capitulum are arranged
in a spiral and geometric pattern (Hernández and Green, 1993). Floret primodia appear at the
rim of the receptacle where ray or disc florets are generated. Disc florets are arranged in
Fibonacci series leading to the emergence of a spiral pattern as new florets arise in rows of
bumps consisting of a bract and a floret (Hernández, 1997). Ray florets (outer) are sterile,
incomplete and have an attractive, fused and flag-like corolla whereas disc florets (inner) are
complete and exhibit centripetal maturation pattern. Each disc floret consists of an inferior
ovary, two pappus scales (modified sepals) and a tubular corolla, which is fused, except at the
tip (Figure 2). Flowering begins with the unfolding of the ray florets in the capitulum. Disc
florets gradually open in whorls towards the centre of the head, as a consequence exhibiting
different stages of floret maturation in a single capitulum. Such a pattern of development also
increases flowering time of a capitulum, thereby attracting insects for pollination. The
maturation stages of disc florets are referred as bud, staminate, transitional and pistillate
(Figure 2). The disc florets are protandrus and are cross-pollinated by insects, particularly
bees. At the bud stage, disc florets exhibit the development of corolla, androecium and
gynoecium. Stigma is clasped and the pollen grains inside the anther lobes have a well
developed exine. Anthesis begins in the morning at the staminate stage when staminal
filaments elongate and black syngenesious stamens are exposed through the tubular corolla.
Protandry in sunflower is induced by photoperiod and corolla has been suggested to be the
site of light perception, stimulating the growth of anther filament (Lobello et al., 2000).
Elongation of the antheridial filaments is initiated after the dark period and occurs for about
2-6h. The pollen grains are then released inside the anther tube as three-celled structures. At
the transitional stage, the stigma elongates through the anther lobe and hairy pseudopapaillae
are observed at the apex of the anther tube. Members of Asteraceae exhibit the phenomenon
of secondary pollen presentation whereby pollen grains are relocated from the anther to
another floral organ, which then presents pollen for pollination. The pollen grains left in the
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Basudha Sharma, Rashmi Shakya and Satish C. Bhatla 22
anther tube adhere to the sweeping hair (pseudopapillae) of the stigma and they are
mechanically forced out and exposed to the pollinators (Hong et al., 2008). Stigma exhibits
biphasic growth kinetics at the pistillate stage during which it first elongates, detaches along
the median, and the tip curls outward (Sammataro et al., 1985).


Figure 1. Stages of the development of capitulum in sunflower.

Figure 2. Various stages of disc floret development in sunflower. A: Young bud, B: Mature bud,
C: Staminate stage, D: Transitional stage, E: Pistillate stage.
Floret maturation is reported to be under the control of phytochrome and plant hormones
(Baroncelli et al., 1990; Koning, 1983). The development of capitulum from R2 to R4 is
affected by photoperiod (Rezadoust et al., 2010). Although sunflower is considered to be a
day neutral plant, the development of floral buds and their maturation is known to be affected
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Floral Biology of Sunflowers: A Histological and Physiological Analysis 23
by daylength. Light leads to an enhancement of photosynthesis, causing growth of the tissue
for the formation of floral bud. Short days are known to modulate anthesis by promoting post-
initiation development of the floral buds (Marc and Palmer, 1981). Depending on the
influence of photoperiod in green house plantations (from the period of emergence to floral
bud development), different sunflower genotypes have been classified as long-day, short-day
or day-neutral plants. In addition, some genotypes have been observed to be
ambiphotoperiodic, a condition in which the floral buds can develop in long or short day
conditions but their further development is delayed in intermediate day length (Goyne and
Schneiter, 1987).
The young capitula (heads) exhibit heliotropism which is marked by the eastward
movement of head in the morning and its westward turning along the direction of sun. As the
capitulum matures, the opened heads are locked in the eastward direction (Weiss, 2000). An
eastwardly direction of the capitulum dries the night dew in the morning hours and decreases
the possibility of fungal attack. It also prevents overheating of the developing stigmas and
preserves pollen viability, consequently enhancing the efficiency of fertilization. It has been
proposed that the heliotropic movement of the young capitulum is related to auxin
distribution in the actively growing parts of the plants (Weiss, 2000). Growing regions of
plants contain relatively higher concentration of IAA than as compared to the fully developed
plant parts resulting in the accumulation of assimilated substances (Duca, 2006). The content
of gibberellic acid also increases, particularly in the cytoplasmic male sterile lines.
Gibberellic acid is involved in floral induction and the process of sexual differentiation (Duca
et al., 2003; Duca, 2006). The role of phytochrome in favouring protandry, and hence cross
pollination, has also been established (Baroncelli et al., 1990; Lobello et al., 2000). Variations
in photoperiod and relatively higher concentrations of gibberellic acid are known to cause a
deviation in floral development (Blackman et al., 2011). The elongation of antheridial
filaments is stimulated by auxins (IAA and NAA) or light. Auxins are known to be involved
in the light-regulated expansion of cells. In vitro experiments have confirmed that auxins can
reduce the inhibitory action of red or dichromatic treatment (far red + red light) on the
elongation of antheridal filaments. Filament elongation caused by light and dark cycles or
auxins, is also known to be dependent on the critical phase of growth of the florets (Lobello et
al., 2000). High concentrations of gibberellic acid (GA
3
) are known to inhibit filament and
style elongation in favourable photoperiodic conditions (Lobello et al., 2000). Light, thus,
plays an important role in altering the availability of gibberellic acid which is essential for
cell expansion.
The development of floral organs in each whorl is regulated by the differential activities
of various genes encoding MADS-box transcription factors (Dezar et al., 2003). The
identification of the genes responsible for sunflower morphogenesis has highlighted two types
of floral differentiation. Reproductive meristem follows the ABC model of flower
development which refers to the class of genes that are required for the development of
whorls of sepals, petals, stamens or carpels. The genes corresponding to ABC encode the
MADS-box transcription factors which are conserved motifs controlling transition from
vegetative to reproductive growth, thus determining the identity of floral meristem and
organs. Floral homeotic mutants of Arabidopsis thaliana, Antirrhinum majus and Petunia
hybrida have extensively been used to understand the controlling factors for the initiation of
floral buds by the ABC model (Coen and Meyerowitz, 1991; Angenent et al., 1995). Seven
full length cDNAs of HAM genes (Helianthus annuus MADS) have been isolated and their
control in the development of pistil, stamen and petals has been established in sunflower
(Shulga et al., 2008). Blot hybridization from different parts of sunflower has further revealed
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Basudha Sharma, Rashmi Shakya and Satish C. Bhatla 24
that HAM 75 and HAM 92 genes are expressed in petals, seed coat HAM 45 is expressed in
the ovule and HAM 59 is expressed in the ovules, stamens and pistil. HAM 59 is expressed
essentially in the disc florets and is absent in ray florets, causing sterility in the ray florets.
HAM 59 expression during ray floret initiation seems to be important for the structural and
functional differences in the developing inflorescence. A correlation between the structure
and function of these proteins is evident. Since HAM genes code for proteins that belong to
different subfamilies of MADS-box, a duplication of the sunflower genome during evolution
has been proposed. Antimicrobial proteins are known to be produced by plants which
contribute to resistance. Among the antimicrobial peptides, cysteine-rich thionine, lipid-
transfer proteins, defensins and snakin have been described. In sunflower, defensins have
been reported to accumulate as florets mature. The defensins are known to be localized
mainly in cell wall or vacuoles (Urdangarín et al., 2000).


Figure 3. Structural analysis of receptive stigma and pollen in sunflower. A: Scanning electron
micrograph of receptive stigma surface (65X); B: Transverse section of mature stigma showing the
presence of papillae (P), vascular strand (VS) and secretory canal (SC) (400X); C: Localization of
proteins in the papillae and transmitting tissue (TT) after staining with mercuric bromophenol solution
(400X); D: Electron micrograph from the basal region of the papillae showing the accumulation of
extracellular secretions (1,150X); E: Transmission electron micrograph showing cluster of
mitochondria at the base of papillae at the staminate stage of stigma development (8000X); F:
Transmission electron micrograph showing transfer cells in the transmitting tissue below the papillae in
the receptive stigma; G: Transmission electron micrograph of cells of transmitting tissue showing
plasmodesmatal connections (1,150X); H and I: Transmission electron micrograph of the pollen wall
showing spinular region (4,600X) and the inter-spinular region (8,400X). Abbreviations: P, Papillae;
PP, Pseudopapillae; E, Extracellular secretions; PD, Plasmodesmata; N, Nucleus; V,Vacuole; M,
Mitochondria; WI, Wall Ingrowths; TT, Transmitting tissue; VS, Vascular strand.
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Floral Biology of Sunflowers: A Histological and Physiological Analysis 25
STRUCTURAL ANALYSIS OF DEVELOPING STIGMA
AND TRANSMITTING TISSUE

Mature stigma in sunflower is forked, bifid and consists of two parts- the peripheral
brush-like pseudopapillae and the inner, thin, finger-like papillae, which are raised and
densely arranged outgrowths of the peripheral cells (Figure 3A). The papillate surface of
stigma increases the pollen capturing area and ensures the proper interaction of pollen with
stigma surface (Heslop-Harrison and Shivanna, 1977). A transverse section of stigma shows
the presence of a four-layered transmitting tissue immediately below the papillae where the
cells are surrounded by an intercellular matrix. Beneath the transmitting tissue is the ground
tissue, in the centre of which is a vascular canal and a large secretory canal (Figure 3B).
Papillae and the transmitting tissue are abundant in proteins (Figure 3C). In order to attain
receptivity, stigma undergoes many structural and physiological changes which allows it to
become competent for the directional growth of pollen tubes (Kandasamy et al., 1994; Yi et
al., 2006). Transmission electron microscopic analysis has revealed that the papillae in the
bud stage of developing stigma are densely cytoplasmic as compared to the ones in mature
stigma, in which they are elongated and vacuolated. The papillae have a large nucleus with a
prominent nucleolus and abundant mitochondria. Nature of stigma surface in sunflower has
remained controversial over the years. It has earlier been described by some investigators as
dry and lacking secretions on the surface (Heslop-Harrison and Shivanna, 1977; Vithanage
and Knox, 1977; Gotelli et al., 2010). Recently, it has also been described as semi-dry in
sunflower (Shakya and Bhatla, 2010), and in some other members of Asteraceae, namely
Senecio squalidus (Hiscock et al., 2002a), Lessingianthus grandiflorus and Lucilia
lycopodioides (Teixeira et al., 2011). Semi-dry stigma possesses a surface cuticle on the
papillae similar to the dry stigma which, however, is not continuous at the base of the
papillae. Like the wet stigma, mature semi-dry stigma possesses a small amount of
extracellular secretion at the base of the papillae (Allen et al., 2010). The initiation of
secretory activity in Helianthus annuus is observed at the staminate stage which leads to an
accumulation of lipid-rich extracellular secretion at the base of the papillae during the
pistillate stage of stigma development (Figure 3D; Sharma, 2012). The secretory activity
coincides with the presence of endoplasmic reticulum and elaioplasts in the basal region of
the papillae. These secretions accumulate in the intercellular and subcuticular gaps, causing a
disruption of the cuticle and release of exudates on the stigma surface. Dry stigma in Brassica
rapa, Arabidopsis thaliana and Raphanus sp. do not show extracellular secretions and the
cuticle extends to the base of the papillae (Hiscock et al., 2002a). Lipids are known to be the
major components of the exudates in some wet stigmas and are responsible for pollen
hydration (Cresti et al., 1986). During the course of evolution, the function of hydration has
been taken over by the pollen coat in dry stigmas (Sage et al., 2009). Probably, the pollen coat
and lipids on the stigma surface of semi-dry stigma aid during the processes of adhesion and
hydration.
The cells of transmitting tissue are loosely arranged below the papillae. The secretory
products of the transmitting tissue in sunflower are rich in pectins and other polysaccharides,
as has also been observed in Tibouchina sp. (Ciampolini et al., 1995), Vitis vinifera
(Ciampolini et al., 1996), Passiflora edulis (Souza et al., 2006). The extracelluar secretions of
the transmitting tissue increase as stigma attains maturity, showing that it acts as a source of
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Basudha Sharma, Rashmi Shakya and Satish C. Bhatla 26
nutrition for the growing pollen tube. Cells of the transmitting tissue are polyhedral to
spherical, with a prominent nucleus, plastids and endoplasmic reticulum. Cells of the
transmitting tissue can be differentiated into three types. Type I cells are vacuolated with
parietal cytoplasm and few mitochondria. Type II cells have a large nucleus, several
mitochondria and a vacuole smaller than that in Type I cells. Type III cells have a dense
cytoplasm (Figure 3G). Some cells of the transmitting tissue show internal ramification
(Figure 3F). The finger-like projections into the cytoplasm formed by secondary wall
ingrowths increase the surface-volume ratio for enhanced metabolic activities across the cells.
The involvement of these cells in secretory activity has also been reported in the members of
Rosaceae (Heslop-Harrison and Shivanna, 1977) and watermelon (Sedgley, 1981). Cells of
the transmitting tissue possess numerous plasmodesmic connections which are involved in the
transfer of metabolic signals from the ovary (Figure 3G). Some plasmodesmic connections
are also present between the basal region of papillae and cells of the transmitting tissue,
indicating their involvement in the symplastic pathway for the transport of metabolites from
the transmitting tissue to the papillae (Figure 3F).


ACCUMULATION OF INTRACELLULAR AND EXTRACELLULAR
LIPIDS AND ASSOCIATED ENZYMES IN RELATION WITH STIGMA
MATURATION

During pollen-stigma interaction, lipids are known to play a role in pollen hydration,
germination and pollen tube penetration into the style (Wolters-Arts et al., 1998). Lipids
prevent evaporation of stigmatic tissue in dry stigma and prevent desiccation of exudates in
wet stigma (Shivanna, 2003). Some lipids in the exudates serve as attractants and are of
nutritional value for the pollinators (Lord and Webster, 1979). In wet stigmas, lipids are
present in the stigmatic exudates, as observed in Phaseolus vulgaris (Lord and Webster,
1979), Nicotiana tabacum (Cresti et al., 1986), Olea europaea (Serrano et al., 2008), while in
the dry stigmas, lipids are present as a continuous layer of cuticle beneath the pellicle, as
demonstrated in Zephyranthus sp. (Ghosh and Shivanna, 1984). In semi-dry stigmas, such as
those in Helianthus annuus and Senecio squalidus, a small amount of lipid-rich extracellular
secretion is evident in the crevices of the papillae, and cuticle is not continuous (Shakya and
Bhatla, 2010; Allen et al., 2010; Sharma, 2012). Lipid content in sunflower stigma increases
with the attainment of stigma receptivity, as observed in wet stigmas of Forsythia intermedia
and Nicotiana tabacum (Matsuzaki et al., 1985; Dumas, 1977). Triacylglycerol (TAGs)
content decreases from bud to staminate stage in sunflower and shows an increase at the
pistillate stage. It is probable that TAGs at the bud stage are degraded during the growth of
the stigma and are synthesized during the pistillate stage of stigma development. In addition,
terpenes have also been detected at the pistillate stage of stigma development. Cis-unsaturated
fatty acids have been reported to be essential components of stigma secretions among wet
stigmas and are required for restoring stigma fertility (Wolters-Arts et al., 2002). Fatty acid
composition in Helianthus annuus shows the abundance of some saturated and unsaturated
fatty acids, as has also been observed in Nicotiana tabacum and Forstythia intermedia
(Shakya and Bhatla, 2010; Matsuzaki et al., 1983; Dumas, 1977). Palmitic acid (16:0) is the
major saturated fatty acid at all the stages of stigma development and its content increases as
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Floral Biology of Sunflowers: A Histological and Physiological Analysis 27
the stigma attains maturity (Figure 4A; Shakya and Bhatla, 2010; Sharma, 2012). Stearic acid
(18:0) content, however, decreases with stigma maturation. Linoleic acid is the major
unsaturated fatty acid in the lipids of sunflower stigma (Figure 4B). Its content decreases on
maturity, indicating its role in stigma maturation. In contrast, linolenic acid increases as the
stigma attains maturity. Linolenic acid is required as a substrate for octadecanoid pathway
which results in the production of signalling molecules, such as jasmonic acid (McConn and
Browse, 1996).


Figure 4. Relative content of major saturated (A) and unsaturated (B) free fatty acids from stigma at
different maturation stages as resolved by gas liquid chromatography. Each value is a mean of three
independent values (±standard error). Zymographic detection of fatty acyl esterase isoforms in different
fractions of pollen (C) and developmental stages of stigma (D) following treatment with α-napthyl
acetate and fast blue B.
The cuticle on the surface of stigmatic papillae in sunflower has an outer proteinaceous
pellicle (Figure 3C). Cytochemical studies have indicated the presence of glycoproteins and
some enzymes, predominantly esterases, peroxidases and acid phosphatase, as the major
components of the pellicle (Vithanage and Knox, 1977; Shakya and Bhatla, 2010). Non-
specific esterase activity has earlier been implicated in the attainment of stigma receptivity
and has a role to play in the metabolism of fatty acids and in host-pathogen interaction
(Bilková et al., 2009; Shivanna and Rangaswamy, 1992). Non-specific esterases include
carboxylesterase (EC 3.1.1.1), arylesterase (EC 2.1.1.2) and acetyl esterase (EC 3.1.1.6). The
activity of these enzymes has been detected in dry stigmas [Linum grandiflorum (Ghosh and
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Basudha Sharma, Rashmi Shakya and Satish C. Bhatla 28
Shivanna, 1980), Pennisetum americanum (Reger, 1989) and Brassica (Mattsson et al.,
1974)], wet stigmas [Impatiens sp. (Kulloli et al., 2010), Moringa oleifera (Bhattacharya and
Mandal, 2004) and Nicotiana sylvestris (Kandasamy and Kristen, 1987)] and semi-dry
stigmas, as in Helianthus annuus and Senecio squalidus (Shakya and Bhatla, 2010; Hiscock et
al., 2002a). Non-specific esterase activity is evident at all stages of stigma development in
sunflower and it increases as stigma attains maturity. As in Helianthus annuus, esterase
activity has also been detected in the early stages of stigma development in Nicotiana
sylvestris (Kandasamy and Kristen, 1987) and Linum grandiflorum (Ghosh and Shivanna,
1980), which can be correlated with the initiation of cell differentiation (Bílková et al., 1999).
Qualitative analysis of stigma proteins has revealed a change in the isoform pattern of
esterases as stigma approaches maturity (Figure 4D). An increase in the number of isoforms
indicates their correlation with stigma maturation (Bhattacharya and Mandal, 2004). Non-
specific esterases present on the stigma surface have been reported to be involved in forming
a cutinase complex (Knox et al., 1976; Hiscock et al., 2002b). The cutinase complex interacts
with esterases from pollen grain wall and pellicle which allows the breakdown of cuticle,
facilitating the penetration of pollen tube into the stigmatic tissue. Removal of stigma surface
components by chemical treatment or using an inhibitor of serine esterases, reduces the ability
of pollen tubes to penetrate the stigma (Knox et al., 1976). This suggests that serine esterases
are associated with cutinase complex formation needed for pollen tube penetration in dry
stigmas (Hiscock et al., 2002b). Non-specific esterases have the ability to hydrolyze cross-
bonds of cell wall polysaccharides and are, therefore, important in the establishment and
reorganization of cell wall. Lipase- (Triacylglycerol acyl hydrolase; EC 3.1.1.3) like proteins
have also been reported in mature stigma papillae of sunflower (Shakya and Bhatla, 2010),
Petunia and Nicotiana (Beisson et al., 2003). Lipases act on the triacylglycerols (TAG) and
might be involved in altering the lipidic composition of the stigmatic surface and have been
located on the stigmatic papillae and pseudopapillae of the receptive stigma (Shakya and
Bhatla, 2010).


REACTIVE OXYGEN SPECIES ACCUMULATION DURING STIGMA
DEVELOPMENT AND ASSOCIATED SCAVENGING MECHANISMS

Recent reports have suggested that during stigma receptivity, angiosperms exhibit an
accumulation of high levels of reactive oxygen species (ROS), principally H
2
O
2
(McInnis et
al., 2006a,b; Allen et al., 2011; Losada and Herrero, 2012). ROS are the byproducts of
aerobic metabolism which are removed by enzymes and antioxidants. In recent years ROS
have been shown to act as signalling molecules in various phases of growth and development,
response to environmental stress and during pollen germination and tube growth (McInnis et
al., 2006a; Hiscock et al., 2007; Zafra et al., 2010). ROS-mediated signalling is controlled by
a fine balance between the production and scavenging of ROS. The main sites of ROS
production are located in the plasma membrane, NADPH-oxidases, plastids and peroxisomes
(Karuppananpandian et al., 2011; Apel and Hirt, 2004). An increase in ROS accumulation is
observed in sunflower stigma accompanying the attainment of stigma receptivity (Figure 5 I;
Sharma and Bhatla, 2013). Stigmas are a source of nutrients for the pollen grains and may be
prone to microbial attack. It is suggested that ROS play a role in defence mechanisms since
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Floral Biology of Sunflowers: A Histological and Physiological Analysis 29
high levels of ROS have been detected in the floral nectar which never experience microbial
attack (Carter and Thornburg, 2004). ROS may directly be toxic to the pathogen or may
trigger hypersensitive reaction and programmed cell death at the site of pathogen attack (De
Rafael et al., 2001). It has also been demonstrated that ROS is required for the polarized
growth of the pollen tube (Potocký et al., 2007).
Superoxide dismutases (SOD; EC 1.5.1.1) form the first line of defence against the
accumulation of superoxide anion (O
2
-
) and lead to its conversion into H
2
O
2
and dioxygen.
SOD activity is associated with redox cycle in the nectar in Nicotiana (Carter and Thonburg,
2004; Carter et al., 2007). The SOD in stigma activity is mainly due to Mn-SOD
(mitochondria localized) which may be associated with enhanced nectar secretion at the base
of florets during pollen producing stage and also when stigma is receptive (Figure 5 III;
Tripathi and Singh, 2008; Sharma and Bhatla, 2013). Coinciding with the increased SOD
activity, a cluster of mitochondria is evident at the basal region of papillae cell cytoplasm,
showing the increased metabolic activity accompanying stigma elongation and dehiscence of
anther (Figure 3E; Sharma, 2012). A slight reduction in SOD activity at the pistillate stage of
stigma development leads to a reduction in O
2
-
form of ROS and an increase in H
2
O
2
in the
stigmatic papillae. Peroxidases (POD; EC 1.11.7) are heme-containing glycoproteins and
their activity has been reported both in wet and dry types of stigmas (McInnis et al., 2005).
Peroxidase activity in the stigmatic tissue increases as the floret development reaches its
maximum at receptivity. In Arabidopsis thaliana, Petunia hybrida and Senecio squalidus,
POD activity increases as stigma attain receptivity (Dafni and Maués, 1998; McInnis et al.,
2006b). Younger stages of developing stigma show reduced POD activity in Peduclaris
canadensis, Clintonia borealis and Helianthus annuus which has been correlated with poor
pollen adhesion and germination (Figure 5 IV; Galen and Plowright, 1987; Sharma and
Bhatla, 2013). Recent investigations have reported peroxidase isoforms specific to stigma.
Expression of stigma-specific peroxidases is developmentally regulated, their activity being
maximally observed during stigma receptivity (McInnis et al., 2005). Among the stigma-
specific peroxidases, three isoforms have earlier been identified in Arabidopsis, one in
Senecio squalidus, and one in hazelnut (Beltramo et al., 2012). Stigma-specific expression of
POD indicates the key role of peroxidases in the loosening of cell wall components of stigma
to allow pollen tube growth into the stigma. They might be involved (through H
2
O
2

metabolism) in signalling network, mediating species-specific pollen recognition (McInnis et
al., 2005). Some peroxidases are also known to be induced/upregulated in association with
hypersensitive response or stress, thereby indicating their role in defense mechanism of
stigma (McInnis et al., 2005; Beltramo et al., 2012).
Nitric oxide (NO) is a gaseous signalling molecule known to be involved in different
plant processes related to growth and development and in responses to stress. Recent
investigations have also revealed that NO is likely to be involved in plant reproductive
processes (Hiscock et al., 2007; Seligman et al., 2008; Yadav et al., 2013). In sunflower, NO
accumulation increases as stigma attains maturity (Figure 5 II; Sharma and Bhatla, 2013).
Similar increase in NO has been reported in the developing stigma of olive and Arabidopsis
(Zafra et al., 2010; Seligman et al., 2008). NO is important in imparting immunity to the
receptive stigma and it also increases thermotolerance by activating ROS scavenging
enzymes, thereby playing a role in ROS-mediated signalling processes (Piterková et al., 2013;
Sharma and Bhatla, 2013). Upon pollination, a crosstalk between pollen-localized NO and
stigmatic ROS has been proposed which may have a role in pollen recognition and signalling
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Basudha Sharma, Rashmi Shakya and Satish C. Bhatla 30
between the stigmatic papillae and pollen grains (Hiscock et al., 2007; Sharma and Bhatla,
2013).


Figure 5. Accumulation of reactive oxygen species (ROS) and expression of associated scavenging
enzymes during stigma development. I: Localization of ROS on the surface of developing stigma after
treatment with fluorescent probe-dichlorodihydrofluorescein diacetate (DCFH-DA). Magnification:
100X. Inset shows intense ROS accumulation in the papillae. II: Localization of nitric oxide (NO) on
surface of developing stigma after treatment with MNIP-Cu {Copper derivative of (4-methoxy-2-(1H-
napthol [2,3-d] imidazol-2-yl) phenol)}. Magnification: 200X. III: Zymographic detection of
superoxide dismutase (SOD) isoforms in developing stigma, after treatment with nitro blue tetrazolium.
IV: Zymographic detection of peroxidase (POD) isoforms in developing stigma following benzidine
treatment.

LIPIDS IN POLLEN COAT AND THEIR ROLE IN FOOT FORMATION

Mature pollen grains are released in a highly desiccated condition and are metabolically
inactive. Pollen grains are suboblate, echinate, tectate and tricolporate (Gotelli et al., 2008).
The innermost layer of pollen wall (intine) is thin as compared to the outer layer (exine)
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Floral Biology of Sunflowers: A Histological and Physiological Analysis 31
which is spinulate and has pollen coat substances embedded on it. The spines of pollen are
conical and have spinular microperforations, indicating their entomophilous nature (Figure
3H; Harry et al., 1978; Shakya, 2005; Coutinho and Dinis, 2007). The exine is differentiated
into ektexine and endexine, both of which are separated by a space designated as cavus 2.
Ektexine is formed of spinules, tectum, internal foramina (openings), columella, large internal
spaces (cavus 1) and foot layer (Figure 3I). Proteins originating from tapetal cytoplasm attach
to the cavae and internal foramina of exine, which are known to act as allergens and
recognition substances for interspecific compatibility (Horner and Pearson, 1978). The exine
pattern is of ‗Helianthoid type‘, referring to abundant internal foramina in the columella and
tectum, equal length of columella having basally fused regions and presence of cavae and a
thin foot layer (Skvarla and Turner, 1966). Connected basal region of columella, internal
formina in columella and enlarged cavae in sunflower (as in other members of Asteraceae,
such as Pallenis maritiama, Jasonia tuberose and Astericus aquaticus) allows easy
communication between pollen surface and cavae, facilitating the exchange of water and
physiologically active substances between them (Coutinho and Dinis, 2007). Pollen coat is
rich in lipids which originate from the tapetal cytoplasm (Horner and Pearson, 1978). Pollen
capture is exine-dependent and at a later stage it involves the formation of ―attachment foot‖
at the point of its contact with the stigmatic papillae. Pollen coat contains essential
components required for adhesion and cell to cell interaction between the stigmatic cells and
pollen. The pollen coat material flows out from between the columellae of exine to form an
adhesive foot at the surface of the papillae (Wheeler et al., 2001). Lipidic constituents in the
pollen grains of sunflower belong to two different domains- the external tryphine (pollen
coat) and the internal cytoplasmic (internal pollen). The lipidic content of pollen coat is more
than that of the internal pollen (Shakya and Bhatla, 2010). Among the total neutral lipids,
neutral esters (wax esters) and free fatty acids are the major components of the two pollen
fractions. Gas chromatographic profile of free fatty acids has revealed an abundance of
saturated and unsaturated free fatty acids in the internal pollen and the pollen coat. As in
stigma, the major saturated fatty acids are palmitic (16:0) and stearic (18:0) acids both in
pollen coat and internal pollen, thus indicating a functional similarity in the lipidic
constituents of stigmatic exudates and pollen (Piffanelli et al., 1997; Shakya and Bhatla,
2010). Lignoceric acid (24:0), which is expressed more in the pollen coat than in the internal
pollen fraction, is specifically expressed only in pollen grains, pointing to its role in the
involvement of long fatty acids in the signalling mechanism for hydration of pollen on the
stigma surface. Among the unsaturated fatty acids, oleic (18:1), linoleic (18:2), linolenic
(18:3) and cis–eicosenoic (20:1) acids have been detected in the pollen grains. Linolenic acid
(18:3) is the major unsaturated fatty acid in the pollen coat and internal pollen in sunflower,
and Brassica napus (Evans et al., 1987; Shakya and Bhatla, 2010). Cis-eicosenoic acid (20:1)
is the major component of intact pollen grains in sunflower (Schulz et al., 2000). Pollen coat
in sunflower exhibits lipase activity, as has also been reported in Arabidopsis thaliana
(Mayfield et al., 2001; Shakya and Bhatla, 2010). Lipid profile of the pollen fractions shows
an absence of triacylglycerides in the pollen coat fraction. It is likely that lipases in pollen
coat are activated when pollen coat makes a contact with the stigmatic tissue. Pollen coat
lipases may be involved in the degradation of the lipids, such as cuticle present in the
stigmatic tissue, and they may also participate in various signalling activities (Murphy, 2006).
Several esterase isoforms have been detected in the two fractions of pollen (internal
pollen and pollen coat). Four and three isoforms have been detected in the pollen coat and
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Basudha Sharma, Rashmi Shakya and Satish C. Bhatla 32
internal pollen, respectively (Figure 4C; Shakya and Bhatla, 2010). Esterase activity has
earlier been localized in pollen intine suggesting its role in cuticle degradation during the
entry of pollen tube (Vithanage and Knox, 1979). It has been proposed that esterases from the
pollen and stigma cause a species-specific recognition event resulting in the formation of a
‗cutinase complex‘ which digests the cuticle for the successful penetration of pollen tube into
the stigma (Knox et al., 1976; Hiscock and Allen, 2008). Only one peroxidase isoforms is
present in the intact pollen of sunflower (Shakya, 2008). Three isoforms have been detected
in the internal pollen whereas pollen coat does not show peroxidase activity. Pollen
peroxidases are known to degrade phenolic compounds (such as chlorogenic acid, caffeic acid
and cinnamic acid) present in the stigmatic exudates (Bredemeijer, 1984, 1982). Phenolics
present on the stigmatic exudates or stigma surface seem to be involved in the stimulation or
inhibition of IAA oxidase activity which influences growth activity in stigma (Shakya, 2008;
Bredemeijer and Blaas, 1975).


OTHER BIOCHEMICAL FEATURES OF POLLEN GRAINS
AND RECEPTIVE STIGMA

Glycoproteins believed to have important role/s in pollen-stigma interaction, are known
to be present in the pollen grains (Suraez-Carvera et al., 2005; Kimura et al., 2002; Aelst and
Went, 1992; Shakya, 2008). In the pollen grains of sunflower, four glycoproteins have been
detected. As in the pollen grain of Elaeis guineensis, an isoforms of 31kDa has been detected
in sunflower as well. The other three glycoproteins correspond to the presence of xylanases
which help in the hydrolysis of xylan in the cell wall of stigma (Shakya, 2008). Some
glycoproteins in stigma are known to be correlated with the expression of S gene proteins,
namely SLG (S locus glycoprotein) and SLR-1 (S locus related glycoprotein-1; Luu et al.,
1997b). SLG is a polymorphic protein known to have a role in self-incompatibility and it is
secreted into the cell wall of the stigmatic papillae (Kandasamy et al., 1989; Umbach et al.,
1990; Doughty et al., 1993). The cytosolic fraction of receptive stigma of sunflower contains
a single glycoprotein of 31 kDa. The expression of SLR1 gene is reduced at the later stages of
pollen-stigma interaction, showing its involvement in pollen-stigma cross-linking (Luu et al.,
1997b). These glycoproteins on the papillae surface interact with pollen coat proteins (PCP)
and facilitate in the process of adhesion. SLG has been demonstrated to bind to PCP-A1 and
about ten PCP-like proteins, pointing towards the involvement of SLG in many other
processes in addition to pollen-stigma adhesion (Swanson et al., 2004). Various enzymes,
including proteases, are required for the proteolytic digestion of proteinaceous pellicle during
the initial stages of pollen-stigma interaction (Luu et al., 1997a; Graff de et al., 2001;
Swanson et al., 2004). Protease activity may also result in the damage of proteins and
enzymes on the stigma surface, which leads to the activation of pollen cutinase necessary for
the degradation of cuticle (Radlowski, 2005). A protease 54kDa protease has been reported in
the cytosolic fraction of sunflower at the receptive stage, in contrast with Cynara cardunculus
where two proteases have been reported in the storage vacuoles of stigmatic papillae and
transmitting tissue of mature stigma (Verissimo et al., 1996; Shakya, 2008). Calcium is an
important constituent of in vitro germinating pollen and serves as a chemoattractant for
guiding the growth of pollen tube. Membrane-bound calcium appears to be generally
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Floral Biology of Sunflowers: A Histological and Physiological Analysis 33
distributed in the papillae at all the stages of development. Young buds, however, show lesser
accumulation of bound calcium in the stigmatic papillae. At the staminate stage, calcium
content increases, reaching a maximum at the pistillate stage of stigma development. Bound
calcium has been detected in the pellicle and upper region corresponding to the cytoplasmic
organelles in the papillae at the pistillate stage of stigma development. The tip region of
stigma also shows an increase in bound calcium content. Investigations using
45
Ca
2+
have
revealed calcium uptake by the germinating pollen from the stigma tissue (Bednarska and
Butowt, 1995). De-esterified pectins in the apoplast are capable of binding with calcium ions.
Upon pollination, bound calcium is liberated due to the enzymatic lysis of de-esterified
pectin, leading to an increase in the levels of free calcium (Bednarska, 1989; Lenartowska et
al., 2001). In the growing pollen tubes, callose synthesis is also a calcium-dependent process
(Bednarska, 1989).


THE PROCESS OF POLLEN ADHESION, HYDRATION
AND GERMINATION ON THE RECEPTIVE STIGMA

Pollination involves the transfer of viable pollen onto the receptive stigmatic surface.
Adhesion is initiated due to non-specific van der Waal forces between the rough surface of
stigma bearing papillae and the spikes of pollen grains (Ferrari et al., 1985; Thio et al., 2009).
The process of pollen adhesion is rapid, maximal at the receptive stage of stigma and initiates
in a similar manner both in self- and cross-pollinated conditions. As has also been reported in
Brassica oleracea, the papillae undergo physiological changes with the attainment of stigma
maturity thereby affecting their ability to interact with pollen grains (Heizmann et al., 2000;
Sharma, 2012). The process of adhesion involves the interaction of proteins present in the
pellicle (arabinogalactan proteins) and the pollen wall (Hiscock and Allen, 2008; Losada and
Herrero, 2012). As in Brassica sp., the initial stages of adhesion are not dependent on Sterlity
locus (S). Therefore, recognition or rejection of compatible or incompatible pollen does not
occur at this stage. At a later stage, however, with the involvement of S locus glycoproteins,
adhesion between incompatible pollen grains does not increase (Heizmann et al., 2000). The
pollen coat and the stigmatic pellicle of the papillae have an important role in the process of
adhesion. Upon removal of the pollen coat, a reduction in the degree of adhesion between the
internal pollen and stigmatic papillae is evident. Adhesion of few decoated pollen and
stigmatic papillae has, however, revealed that pollen coat proteins and carbohydrate-based
molecules associated with exine are involved in the process of adhesion (Zinkl et al., 1999;
Sage et al., 2009). The importance of pellicle in the process of adhesion is further evident by
the treatment of pellicle with acetone which leads to a significant decrease in pollen adhesion
suggesting that glycolipids and lipids located at the base of the papillae play a role in the
process of adhesion. The lipids of the pollen coat and cuticle and extracellular lipids in the
basal region of papillae are likely to form hydrophobic bonds, leading to adhesion between
the pollen grains and the stigmatic papillae. After adhering to the stigmatic papillae, the
pollen grains hydrate. The exact mechanism by which water, nutrients and other essential
molecules are taken up by the pollen grains from the stigmatic papillae, is not yet fully
understood (Frion et al., 2012). Various proteins from pollen coat, including glycine-rich
proteins, calcium-binding proteins, lipases and cutinases aid in the process of hydration by
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Basudha Sharma, Rashmi Shakya and Satish C. Bhatla 34
causing the breakdown of lipidic constituents at the pollen-stigma interface (Mayfield and
Preuss, 2000; Hiscock et al., 2002b; Updegraff et al., 2009; Shakya and Bhatla, 2010). Pollen
grains hydrate leading to an increase in their volume, both in self- and cross-pollinated
conditions (Vithanage and Knox, 1977). After making a contact with stigma, the pollen coat
material flows out from the columella of the exine to the surface of papillae (Ellemen et al.,
1992; Allen et al., 2011). This leads to the formation of ‗attachment foot‘ where an interaction
between pollen and stigma-derived biomolecules takes place. Lipids create conditions and
govern the movement of water for guidance cue for the development of the pollen tube which
emerges from the pore in the middle of colpus area to grow through the attachment foot
towards the basal region of the papillae (Ellemen et al., 1992; Hiscocok et al., 2002a). The
acceptance or rejection of pollen grains on the stigmatic surface seems to be dependent on the
interaction between the pollen and stigma-derived attachment foot (Hiscock, 2000; Sharma,
2012). Some of the incompatible pollen grains are not able to germinate while others pass
perpendicularly towards the basal region of the papillae. Some pollen grains grow parallel to
the papillate surface of stigma thereby showing some incompatible rejection reactions
(Figure 6).


Figure. 6. Transverse section through germinating pollen grain and the associated stigma following
cross (A) and self (B) pollination. Magnification: 400X.
In sunflower, cross pollination is favoured due to the presence of self-incompatibility, as
also observed in the members of Brassicaceae. Members of both the families (Asteraceae and
Brassicaceae) have some similarities in pollen-stigma interaction. The common features
include the presence of dry stigma surface (Vithanage and Knox, 1977), three-celled pollen
grains (Hiscock and Allen, 2008), release of pollen coat on contact with papillae (Elleman et
al., 1992) and arrest of incompatible pollen soon after germination accompanying the
deposition of callose on the pollen tube and papillae (Allen et al., 2011; Vithanage and Knox,
1977). Habura (1957) was first to report sporophytic self-incompatibility in sunflower and it
was later confirmed by various other plant scientists (Luciano et al., 1965; Asthana, 1973).
Recent reports have, however, pointed out flexibility in sporophytic self incompatibility in
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Floral Biology of Sunflowers: A Histological and Physiological Analysis 35
other members of Asteraceae (Hiscock, 2000; Ortiz et al., 2006). As in Senecio squalidus,
sunflower shows germination of self-incompatible pollen. Some of the incompatible pollen
grains are not able to germinate, while others germinate and grow between the papillae
(Vithanage and Knox, 1977). It has been suggested that the degree of self-incompatibility and
self-fertility depends on genetic control, environmental factors and the morphology of the
inflorescence (Miller and Fick, 1997). Self-incompatibility is compensated in situations when
the reproductive opportunity of the pistil is affected. Such cases exhibit pseudo self-
compatibility which involves a delayed acceptance of the otherwise incompatible pollen
(Brennan et al., 2011). The modification of self-incompatibility may also be brought about by
G locus, which is a second gametophytic ancestral locus, remnant of ancestral gametophytic
self incompatibility in Brassicaceae, which permits compatible cross between the individuals
with the same S alleles, which are otherwise incompatible (Lewis et al., 1988; Hiscock, 2000;
Brennan et al., 2011). Recent molecular analysis has also pointed out that sporophytic self-
incompatibility in Asteraceae operates by a mechanism different from the SRK/SCR
molecular mechanism operative in Brassicaceae (Hiscock et al., 2003; Tabah et al., 2004;
Allen et al., 2011). Further investigations related to seed set and the molecular mechanisms in
sunflower are required to understand the physiological nature of self-incompatibility.
To sum up, the present work highlights the complex interaction between floral
development and environmental factors, which seem to operate through a critical balance of
auxin and gibberellic acid distribution at the bud primordium. Subsequently, in order to
facilitate an effective pollen and stigma interaction, both the reproductive structure undergo
biomolecular changes required for ‗foot‘ formation on the surface of receptive stigma
papillae. The knowledge gained so far in this direction highlights the role of reactive oxygen
species and associated scavenging enzymes, glycoproteins, calcium and many more
biomolecules. Alterations in the availability of these biomolecules correlate with distinct
subcellular structural changes both in pollen and stigma. Although a lot is known by now
about these structural and biochemical events accompanying floral development in sunflower,
further investigations on their physiological, biochemical and genetic aspects will provide
further information on the crosstalk mechanisms regulating floret development and pollen-
stigma interaction.


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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 3



DEVELOPMENT OF FEMALE REPRODUCTIVE
STRUCTURES AND APOMIXIS IN SUNFLOWERS


Olga N. Voronova
*

Department of Embryology and Reproductive Biology,
Komarov Botanical Institute of RAS, Saint Petersburg, Russia


ABSTRACT

The apomixis phenomenon was hardly observed in the genus Helianthus L. under
natural conditions. In this study, a series of experiments on interspecific hybridization
was carried out on plants of Cytoplasmic Male Sterility lines. A number of anomalies in
the development of female reproductive system were uncovered, including an apospory
and integumentary embryony. A surprising phenomenon was noted, such as total absence
of the embryo sac in some ovules. Lack of the main embryo sac and formation of
additional aposporous embryo sacs could be observed in the same ovule at the same time.
Investigation of the early stages of ovule development showed that aposporous
embryo sacs originated from the same ovule subepidermal cells as a normal embryo sac.
Aposporous embryo sac included the same elements, as the main one: the egg cell, the
synergids, the central cell with polar nuclei or secondary nucleus, and antipodes.
Asynchrony in development of the main and aposporous embryo sacs was noted.
Integumentary embryos were observed in unfertilized embryo sac (7-9 day after
flowering). These embryos were formed from the group of initial cells in the chalazal
region of the integumentary tapetum; their apical poles were turned to the micropyle. The
integumentary embryos consisted of 1-2 suspensor cells and 7-8 embryonic cells and by
structure corresponded to Asterad-type, as a typical zygotic embryo.
Apospory embryo sac and initial cells of integumentary embryo are formed at the
fixed stage of ovule development, the first – when female gametophyte forms, the second
– when the development of sexual embryo should begin.

Keywords: Helianthus, CMS, apospory, integumentary embryony, interspecific hybridization


*
Department of Embryology and Reproductive Biology, Komarov Botanical Institute, RAS, 2 Professor Popov St.,
Saint-Petersburg, Russia 197376. tel./ fax: +7(812) 3725441. Email: o_voronova@list.ru.
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Olga N. Voronova 44
LIST OF ABBREVIATIONS

ac archesporial cell
bc1 first basal cell
bc2 second basal cell
bn basal area of nucellus
cln cells of lateral area of nucellus
hc hypostases cells
ic initial cell
in integument
it integumentary tapetum
msc megasporocyte
sec subepidermal cells


INTRODUCTION

The early embryological studies of cultivated sunflower were carried out by Goldflus
(1899) and Nawashin (1900a,b) who described different aspects of formation and
development of its reproductive system. Many studies of sunflower reported that the ovule is
tenuinucellar, unitegmic, anatropous with integumentary tapetum, Polygonum-type
development of embryo sac, and Asterad-type (Senecio-variation) embryo formation
(Newcomb, 1973a,b; Toderich, 1988; Yan et al., 1991; Gotelli et al, 2008, 2010). Under
natural conditions, only cross-pollination is typical for sunflower and the apomixis
phenomenon was hardly observed in genus Helianthus L. (Noyes, 2007).
Formation of several aposporous embryo sacs under unfavorable weather conditions,
such as low temperature and excessive humidity, is described by Ustinova (1955, 1964, 1970)
in sunflower cultivars Zhdanovskiy and Saratovskiy. She suggested that aposporous embryo
sacs arise from the integument cells because the nucellus cells are lysed early. Dzyubenko
(1959) noted some cases of apospory in ornamental double-flower form of sunflower. She
pointed out the presence of a group of cells near the chalazal end of the normal embryo sac
that could be initial aposporous embryo sacs. She named these cells the sporogenous cells.
Additionally, Toderich (1988) mentioned several examples of the formation of two embryo
sacs in the same ovule in different wild sunflowers (H. rigidus, H. petiolaris, H. occidentalis),
but she suggested that they originated from two different archesporial cells, which were
formed within one ovule. Pleten et al. (2011) reported hemigamy in sunflower lines APS-11.
The majority of the earlier studies of sunflower were carried out on its cultivars
(Ustinova, 1955, 1964, 1970; Dzyubenko, 1959; Newcomb, 1973a, b; Toderich, 1988; Yan et
al., 1991). Cytoplasmic male sterility (CMS) lines are widely used in sunflower breeding
because it allows a breeder to avoid the difficult process of manual sterilization of the flower
in order to create a genetic variety by hybridization. Unfortunately, the CMS sunflower lines
were not studied comprehensively. Previous studies have paid attention mainly to male
reproductive structures (Horner, 1977; Simonenko and Karpovich, 1978; Balk and Leaver,
2001) and only in recent years the researchers began to study the female reproductive
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Development of Female Reproductive Structures and Apomixis in Sunflowers 45
structure of CMS lines using cyto-embryological analysis (Voronova, 2006, 2008a, 2010;
Voronova and Gavrilova, 2007; Gotelli et al., 2008).
In this study, a series of experiments on interspecific hybridization was carried out on
plants of CMS-lines followed by a cyto-embryological research of ovule development. As a
result, a number of anomalies in development of female reproductive system were uncovered,
including such phenomena as an apospory and integumentary embryony.


METHODS

The main material of the study were the sunflower plants of Helianthus annuus cv.
Peredovik and lines VIR 114 form A, VIR 116 form A and their fertile analogs VIR 114 form
B and VIR 116 form B. Plants were grown in the Kuban experimental station of N.I. Vavilov
Research Institute of Plant Industry (VIR) in Krasnodar region (45°12′54.95″N,
40°47′37.12″E).
Perdovik plants were cross-pollinated. Whole heads and separated tubular flower of
plants of H. annuus cv. Peredovik (standard) were collected and preserved at different stages
of flower development, starting from ovule initiation stage (about 1cm heads diameter ) to
torpedo embryo stage (over 20 cm heads diameter).
Flower heads of CMS plants were protected from foreign pollen by bagging. When the
second circle on a head began to flower, they were pollinated with the fresh pollen of wild
sunflower species or lines B. The pollen was collected immediately prior to pollination. The
following perennial species were used as pollinators: H. ciliaris, H. decapetalis,
H. divaricatus, H. gigantheus, H. hirsutus, H. maximiliani, H. mollis, H. nuttalli,
H. occidentalis, H. strumosus (all from collections of VIR). Also, the plants of CMS lines
form A were pollinated by their fertile analog of the form B.
The material for cyto-embryological investigations was preserved beginning from 1 hour
and until 9 days after pollination, that is 1, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48 hours after
pollination and 3, 4, 5, 6, 7, 8, 9 days after pollination. The whole flower heads or separate
tubular flowers taken by pincers from a head were preserved in FAA solution (formalin,
acetic acid and 70% ethanol in 7:7:100 ratio). The pieces were then dehydrated in an ethanol
series, infiltrated in ethanol-chloroform mixtures and embedded in Histomix®. Permanent
preparations for light microscopy were stained with Heidenhain's hematoxylin with alcian
blue (for more details see Zhinkina and Voronova, 2000). The method of the total cleared-
ovule by methyl-benzoate was used for accelerated analysis of the material (Crane, 2001;
Voronova, 2008b). The sections were observed and photographed using a Zeiss Axioplan 2
Imaging microscope. Cleared ovaries were examined with Nomarscki interference contrast
optics.


RESULTS AND DISCUSSION

A sunflower ovule is initiated in the base of an ovary, about 7-10 days before flowering.
The ovule primordium is formed by periclinal divisions of cell in the subepidermal placental
layer (Figure 1a, b, c).
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Olga N. Voronova 46

First stages of ovule development in the sunflower.
Figure 1a, b, c – tubular flower at the stage of ovule primordium initiation.
Figure 2a, b – the ovule primordium with initial cell in subepidermal layer.
Figure 3a, b – the initial cell elongates and starts mitotic division.
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Development of Female Reproductive Structures and Apomixis in Sunflowers 47

First stages of ovule development in the sunflower (Continued).
Figure 4a, b, c – the initial cell divided into archesporial cell and basal cell, integument initiates in epidermal
layer of ovule, the ovule starts being curved.
Figure 5a, b – the archesporial cell elongates, the integument actively develops, the vascular band initiates in
ovule.
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Olga N. Voronova 48


First stages of ovule development in sunflower (Continued).

Figure 6a, b, c – tubular flower at the stage of megasporocyte formation, the ovule curves in anatropous
position.
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Development of Female Reproductive Structures and Apomixis in Sunflowers 49


First stages of ovule development in sunflower (Continued).
Figure 7a, b – megasporocyte before meiosis: the first stage of integumentary tapetum formation – two
cell in epidermal layer start flatten.
Figure 8 – megasporocyte before meiosis, integumentary tapetum is formed around megasporocyte.
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Olga N. Voronova 50

Embryo sac formation and anomalies in ovule development in sunflower. 9 – ovule with chalazal megaspore,
three micropilar megaspore degenerated, walls of integumentary tapetum cell are thickened, 10 – mature
embryo sac, 11 – mature embryo sac and aposporous embryo sac with egg apparatus, antipodal cells and two
polar nuclei, 12 –ovule with a cell complex instead of a embryo sac, 13 – ovary with mature main embryo sac
and aposporous embryo sac, 14 – mature embryo sac, integumentary tapetum forms derivates around the
embryo sac, 15– ovule without main embryo, 16 – ovule without main embryo sac and with aposporous
embryo sac, 17a,b,c – ovule with integumentary embryo in degenerated embryo sac: a,b – neighboring
section of one ovule, c - integumentary embryo, 18 – ovule with embryo and endosperm.
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Development of Female Reproductive Structures and Apomixis in Sunflowers 51


1-4 – CMS line VIR 114 A, 5-8, 18 – cv. Peredovik, 9-17 – CMS line VIR 116 A;
1-13, 17-18 – longitudinal slides were stained with Heidenhain's hematoxylin and alcyan blue, 14-16 – total
ovule (phase contrast).
ac – archesporial cell, aes – aposporous embryo sac, anc – antipodal cell, an – anther, bc – basal cell, bn –
basal area of nucellus, cc – cell complex, ccn – central cell nucleus, ch - chalaza, cln – cell of lateral area
of nucellus, dea - degenerating egg apparatus, ec – egg cell, em – embryo, en – endosperm, es – embryo
sac, ic - initial cell, ie – integumentary embryo, ii – initial of integument, hc – hypostase cell, in –
integument, it – integumentary tapetum, izi – inner zone of integument, mc – micropile, msc –
microsporocyte, ms – microspore, ozi – outer zone of integument, ov – ovary, pn – polar nuclei, pt –
pistil, sec – subepidermal cell, tc – table-like cell, vb – vascular bands.
Scale bars: 1b, 2a, 3a, 4b, 5a, 6b, 7b, 8, 9, 10, 11, 12, 17a,b,c, – 30 mkm, 1a, 4a, 6a – 150 mkm, 13 – 300
mkm.
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Olga N. Voronova 52
Previous studies suggested that usually 1-2 (for wild species up to 3-4) archesporial cells
were formed in subepidermal layer of the primordium ovule, which then develops into
megasporocyte directly without any mitotic divisions (Ustinova, 1964; Dzyubenko, 1959;
Newcomb, 1973a; Toderich, 1988, etc.). In this study, a different order of structures
formation were observed. One to two (rarely 3) subepidermal cells (sec) divided mitotically
and gave rise to initial (ic) and first basal cells (bc1) located under initial cell in the ovule
primordium of cultivated sunflower. That results in couples of cells located around future
long axis of an ovule (Figure 2a,b). The ic cell of the central couple divided once again
periclinally and formed an archesporial cell (ac) on the top and second basal cell (bc2) on the
bottom (Figure 3a,b, 4a,b,c). Both basal cells (bc1+ bc2) became a part of basal area of
nucellus (bn). Other pair of cells (analog ic+bc1 cells) located around the archesporial cell
formed a lateral area of nucellus (cln) (Figure 4c, 5b, 6c, 8, 9). The archesporial cell
represented an apical area of nucellus.
Thus, the apical, lateral, and basal areas of nucellus were identified on the early stage of
sunflower ovule development. About the division the nucellus onto different areas see
Shamrov, 1998, 2008. Therefore, the ovule of a sunflower can be considered not as
tenuinucellar, but rather as medionucellar, with sindermalny variation and single-layer sub-
variation, according to Shamrov's classification (2008). According to Endress‘s classification
(2011), it would be an incompletely tenuinucellar ovule, that is without a hypodermal cell
layer between the megasporocyte and nucellus apex, but with hypodermal tissue at the
nucellus flanks and below megasporocyte.
The single integument (in) was developed in epidermal placental layer at the same time
as the formation of an archesporial cell (Figure 4b,c, 5a,b). The layer of hypostases cells (hc)
developed at the level of initial cells of integument and on top of them, the basal cells (bc1)
differentiated. An axial row was formed as ac+bc2+bc1+hc (Figure 4c). The basal cells bc1
increased in size and became strongly vacuolated, with enlarged and poorly stained nucleus.
Because two to three such cells were present in an ovule, they formed a peculiar light zone
transparent in the center of the ovule. The basal cell located under the archesporial cell was
larger than the latter.
Archesporial cell (ac) elongated and gave rise to megasporocyte (msc). At the same time
the internal epidermal cells of integument located nearby also underwent changes. They
started differentiating into integumentary tapetum (it) which was well expressed already at the
stage of mononuclear embryo sac and consisted of one layer of dark-stained, 1-2-nuclear,
flattened cell of table-like form with thickened walls (Figure 7a,b, 8, 9). Later, the
integumentary tapetum became a two to three layers deep and formed derivates around the
mature embryo sac (Figure 10, 11, 14), as typical for Asteraceae (Musial et al., 2012, 2013;
Bencivenga et al., 2011).
During differentiation of archesporial cell to megasporocyte, the cell of basal area of the
nucellus underwent one more mitotic division and formed two cells of table-like form (tc1
and tc2). An axial row was formed as msc+tc1+tc2+bc1+hc (Figure 6c). The table-like cells
and basal cells together formed the basal area of nucellus. The cells of the basal area of
nucellus remained for a long time and formed a pathway from an archesporial cell (or
megasporocytes and megaspore) to hypostasis. Formation of rows of table-like cells, the cells
of hypostases, in a broad sense, under a megasporocyte is a characteristic for a number of
floral plants, in particular for cereals, such as wheat and corn (Batygina, 1974, 1987,
Voronova et al., 2003). It was noted that the archesporial cell of the wheat is terminating an
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Development of Female Reproductive Structures and Apomixis in Sunflowers 53
already existing number of the cells located along a polar axis of nucellus (Batygina, 1974).
As one can see, in sunflower the differentiation of archesporial cell begins with the formation
of a special cell which will further form a basal area of nucellus. But unlike cereals, where
rows of table-like cells remained unchanged up to seed maturation, in sunflower only two
table-like cells got formed. Soon these cells got elongated and formed a narrow row together
with the underlying cells of basal area of nucellus (Figure 9, 10).
Formation of apospory embryo sac took place within the row (Figure 11, 13). Apospory
embryo sacs, as a rule, had the improper shape and was formed from the integumentary
tapetum behind the main embryo sac, but was not adjoined to it.
Initial cells of aposporous embryo sac were positioned near the basal area of nucellus
and, probably, originated from the same subepidermal cell (sec) of ovule primordium, like an
archesporial cell (Table 1). Thus, the initial cells of aposporous embryo sacs arose from the
cells of the nucellus, and not from the integument as proposed by Ustinova (1970). And,
probably, they can be related to the cells that Dzyubenko (1959) called sporogenous cells
without considering their origin.
Cells of lateral area of nucellus (cln = ic+bc1) exist only at early stages of ovule
development. Initially located around an archesporial cell, they are forced out downwards
during the process of growth (Figure 4c, 5b, 6c, 7a). These cells continued to put pressure on
the cells of the lateral area of nucellus and shifted them down towards the chalaza with the
development of megasporocyte and then megaspore. The cells of the lateral area of nucellus
were compressed, but remained distinct up to the stage of mononuclear embryo sac
(Figure 8, 9).
The young ovule of sunflower is ortotropuos (Figure 1a,b, 2a, 3a), but one side of the
integument got curved because of the more intensive growth (Figure 4a, 5a, 6a,b). The
mature ovule of sunflower is anatropous (Figure 13), unitegmic, with integumentary tapetum
or endothelium, like in other Asteraceae.

Table 1. The sequence of divisions at an early stage of sunflower ovule development


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Olga N. Voronova 54
The ovule showed a zonal differentiation, that is particularly visible in the central part.
The cells of the outer zones were elongated with thin cell wall, whereas the cells of the inner
zone were disintegrated and had thick cell wall intensively stained with alcian blue (Figure 9,
12, 13, 17a,b, 18). The vascular bundle passed through chalaza and penetrated integument
almost reaching the micropyle (Figure 5b, 6c, 7a), like in some other Asteraceae species
(Musial et al., 2013).
The megasporocyte developed directly from archesporial cell without any mitotical
division (Figure 5a,b, 6b,c, 7a,b, 8). The meiotic divisions of megasporocyte produced a
linear tetrad of haploid megaspores. Three of them degenerated and Polygonum-type embryo
sac was formed from one chalazal megaspore (Figure 9, 10).
The embryo sac was completely developed by the time of pollination. The mature
embryo sac consisted of the three-cellular egg apparatus, the central cell with big, fused,
secondary nucleus, and antipodal cells (Figure 10). Egg apparatus consisted of three pear-
shaped cells: two synergids and the egg. The egg cell nucleus was rather large, with obvious
nucleolus, and positioned in the apical part of the cell. The synergid nuclei were barely
distinguishable and positioned in the center of cells. A filiform apparatus with hamiform
evagination was found in the basal part of synergids. The bulk of cytoplasm of the central cell
was located near the egg apparatus and thin bundles between large vacuoles. The large central
cell nucleus was located near the apical end of the egg cell. Antipodes were linear. Antipodal
cells were strongly vacuolated, with the large, polyploid nuclei. Usually, there are only two
antipodes in the mature embryo sac. Antipodal cell were adjoined to the central cell, usually
larger, underlying, and can increase 2-3 times in size. The remains of the third antipode were
sometimes observed in the chalazal end of embryo sac. The antipodal complex as a whole
took half of the general linear size of the embryo sac.
An additional embryo sacs were found in the ovule of the plants pollinated by perennial
wild species. They were found at different times after pollination, from two hours to nine
days, and in different combination of pollinations (see Table 2).
The relative position of additional and main embryo sacs confirmed that the additional
embryo sac was aposporous. Besides, the groups of big megaspore-like cells were found
around chalazal end of main embryo sacs at earlier stages of development (Voronova,
Gavrilova, 2007). These are the cells of the basal area of the nucellus that have originated
from the subepidermal cells of the ovule (see Table 1).
The main embryo sac in all cases had a normal structure that is characteristic for
sunflower. Aposporous embryo sac included the same elements as the main one: the egg cell,
the synergids, the central cell with polar nuclei or secondary nucleus, and antipodes. All
elements of additional embryo sac were similar to those of the main, but differed in sizes and
by the level of differentiation. There were more than three antipodal cells (Figure 11).
Aposporous embryo sacs lied below the level of integumentary tapetum. Absence of
integumentary tapetum around an additional embryo sac leaded to a variety of shapes and
sizes of this embryo sac. Apparently, the integumentary tapetum plays a role of a bearing
element and gives definite form to the whole structure of the main embryo sac.
The asynchrony in development of the main and additional embryo sac was noted. The
additional embryo sacs, as a rule, delayed in development. For example, the main embryo sac
could be fully formed while the polar nuclei of additional embryo sac did not fuse yet
(Figure 11).

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Development of Female Reproductive Structures and Apomixis in Sunflowers 55
Table 2. Anomalies in ovule development after pollination of CMS
lines by wild perennial sunflower

Pollination combination
Ovules,
in total
Ovules without any
embryo sac, in %
Ovules with apospory
embryo sac, in %
VIR 114 A х H. ciliaris 35 2,9 51,4
VIR 116 A х H. nuttalli 55 0 14,5
VIR 116 A х H. occidentalis 43 20,9 14,0
VIR 114 A х H. occidentalis 85 1,2 11,8
VIR 114 A х H. gigantheus 69 0 10,0
VIR 114 A х H. nuttalli 35 34,3 8,6
VIR 116 A х H. divaricatus 35 14,3 5,7
VIR 114 A х H. hirsutus 89 16,9 4,5
VIR 116 A х H. gigantheus 45 0 2,2
VIR 114 A х VIR 114 B 119 3,4 1,7
VIR 116 A х VIR 116 B 49 0 0
VIR 116 A х H. maximiliani 48 0 0
VIR 116 A х H. ciliaris 46 0 0
VIR 116 A х H. decapetalis 45 0 0
VIR 116 A х H. mollis 44 0 0
VIR 116 A х H. strumosus 40 0 0
cv. Peredovik (free pollination) 126 0 0

A surprising phenomenon, the total absence of embryo sac, was recorded in different
ovules of some CMS lines (VIR 114, VIR 116) (Figure 12, 15, 16). Previously, the total
absence of the embryo sac was described for tetraploid form of sunflower only (Efremov,
1967; Pustovoit et al., 1976). The authors suggested that the embryo sac underdevelopment
was caused by disorders in the preceding meiotic divisions (Pustovoit et al, 1976). Also they
noted that in some tetraploid sunflowers the ovule is underdeveloped in general and lacking
the embryo sac (Efremov, 1967). In this investigation, all ovule structures were formed
normally and phases of their development and relative positioning of elements corresponded
to the stages of flower development. Two small and 1-3 large cells with light, strongly
vacuolated cytoplasm and small nuclei were found in a cavity surrounding integumentary
tapetum, instead of an embryo sac (Figure 12). Possibly, these cells are derivatives of
nucellus. Usually, nucellus cells appear squeezed by the developing megasporocyte and
degenerate by the time of formation of an embryo sac. It is possible to assume that the
transport of nutrients in this zone remains in the absence of embryo sac, and, nucellus cells
remain viable for a long time in the absence of effect from outside megasporocyte.
Lack of the main embryo sac and formation of additional embryo sacs could be observed
at the same time in the same ovule (Figure 16). In case of formation of seeds from similar
ovule, the embryo in them could be formed by apomixes only.
Integumentary embryos were formed in some ovules of CMS line VIR 116 (Figure
17a,b,c). after parent plants were pollinated by H. occidentalis pollen. Though pollination
was carried out, fertilization was not observed even 7-9 days after the pollination. Normally,
both, the embryo and endosperm were observed in the ovule at that time. But in some plants
of CMS line VIR 116 A, the embryo sac still remained in an 8-shaped form at this stage. It
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Olga N. Voronova 56
was still possible to discern the egg apparatus, the central cell with the secondary nucleus, and
the antipodes (Figure 17b). Sometimes the egg cell was significantly increased in size, while
synergids were compressed and darkened. The egg nucleus could be larger than the secondary
nucleus of the central cell. The cytoplasm of the central cell occupied wall position with a
huge vacuole in the center. The secondary nucleus was located near the egg cell.
The integumentary tapetum became multilayered and folded, and its cells were
transparent with small and poorly visible nuclei. The integumentary tissues near the chalazal
pole of the embryo sac were destroyed, the cells were disintegrated and cavities were formed.
Integumentary embryos were located in the chalazal region of the integumentary tapetum
with their apical poles turned to the micropyle, which is distinct from the sexual embryo
(Figure 17a,c). The embryos consisted of 1-2 suspensor cells and 7-8 embryonic cells. Cells
were dark-colored and had large nuclei. The lighter nucleoli with dark-colored center were
visible in the nucleus (Figure 17a). These embryos corresponded to Asterad-type Senecio –
variation (Voronova, 2010).
In control pollination of VIR 116 A line with the pollen of the fertile analogue of VIR
116 B and of cv. Peredovik the formed embryos and endosperm at the similar stage of ovule
development were observed (Figure 18); later the seeds were fully formed. Unfortunately, the
development of fully formed seeds after pollination of the CMS line with pollen of wild
sunflower species were not observed. The seeds formed in the head did not contain embryos.
It is known that interspecific hybridization in sunflower is possible, but the number of seeds
formed is very insignificant, such as 1-10 for a head (Gavrilova and Anisimova, 2003).


CONCLUSION

Three types of deviations in the reproductive system of CMS lines were found during this
study: absence of an embryo sac; apospory; and integumentary embryony. The last two
represent different forms of apomixis.
It was noted that apomixes phenomenon is connected with hybridization in different
systematic groups (Carman, 1995). CMS lines obtained a sterile cytoplasm from wild
sunflower species (Leclerq, 1969) and, therefore, have an interspecific hybridization in their
pedigrees (Voronova, Gavrilova, 2007). It is possible, that the reproductive system of CMS
lines is less stable than in cultivated species and cultivars. Therefore, the alternative ways of
development, such as apospory and integumentary embryony, are rare for the cultivated
sunflower but typical for other representatives of the Asteraceae family (Noyes, 2007) and
work under stress conditions, for example pollination with an alien pollen, delay or the full
absence of the fertilization.
Based on the earlier publications (Molchan, 1973; Petrov, 1988; Bencivenga et al, 2011),,
it could be assumed that the pollination of cultivated sunflower with the pollen of wild
Helianthus species and the absence of normal fertilization can stimulate appearance of
different anomalies in the morphogenesis of ovule structures. The disturbance in fertilization
can lead to interruption of normal ―cross talk between the sporophyte and megagametophyte‖
(Bencivenga et al, 2011) resulting in the alternative way of development of reproductive
system in sunflower lines.
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Development of Female Reproductive Structures and Apomixis in Sunflowers 57
The interspecific hybridization method was used during the breeding work on sunflower
and the pollination process was changed repeatedly. At first, there was a search of self-fertile
forms and then, among them, the sterile lines were located. CMS lines were maintained using
so called "B lines" or fertile analogs. For production of heterosis industrial hybrids, the
process of pollination was changed again and CMS lines were cross-pollinated. The balance
existing in reproductive system of stable cross-pollinated cultivars was apparently disrupted
in lines and hybrids.
Integumentary embryony, as one of apomixes forms, represented indubitable theoretical
and practical interest because it implies embryo formation from cells of somatic tissue of
ovule. By origin, integumentary embryos represent clones of a parent organism; their
formation can result in genetic heterogeneity of seed (Batygina, Vinogradova, 2007). This
means that in the progeny of a single plant some of the seeds may have a paternal heredity
and part of the maternal. Furthermore, both parents can participate in the endosperm
formation. For this reason, possibility of integumentary embryos formation in cultural
sunflower perhaps may explain the deviations from expected result of crossings, so-called
partial hybridization and lack of splitting in hybrids. These were noted by different authors
(Lyashchenko 1940, 1948; Gavrilova et al., 1997; Faure et al., 2002; Gavrilova and
Anisimova, 2003). When sunflower was pollinated by wild species under interspecies
hybridization, part of progeny tended to resemble a cultivated sunflower, whereas when wild
species was pollinated by sunflower, the progeny looked like wild species. It is possible that
genes of the endosperm inherited from both parents would be included in the work at the
early stages and affected the development of the embryo.
It was observed in other plants that the initial cells of integumentary embryos are formed
at a certain stage of ovule development, corresponding to the time when the normal embryo
should start to develop (Naumova, 1993, 2008). In this study, a similar picture was observed.
The alternative (aposporic) embryo sacs were formed if the violation in the development of
basic reproductive structures occurred at an earlier stage. The integumentary (somatic)
embryos were developed if the violation has occurred at a stage when zygotic embryo must
be formed. Apospory embryo sac and integumentary embryo were formed at the determined
stage of ovule development, the first during the formation of female gametophyte, and the
second during the development of zygotic embryo. Formation of the alternative and
additional structures depends on general stage of the ovule development and on the time that
passed after pollination.


ACKNOWLEDGMENTS

I am grateful to Dr. V. A. Gavrilova (N.I.Vavilov Research Institute of Plant industry)
and Dr. V. T. Rozhkova and Dr. T. T. Tolstaja (the Kuban experimental station) for their help
in obtaining the experimental material and Dr. T. Lobova for her help in editing the
manuscript.
This work was performed under the project № 01201255606 and supported by the
Russian Foundation for Basic Research (grant № 11-04-01466).


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Olga N. Voronova 58
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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 4



GENETICS AND GENOMICS APPLIED
TO SUNFLOWER BREEDING


Carla Filippi
1,2
, J eremías Zubrzycki
1,2
, Verónica Lía
1,2,3
,
Ruth A. Heinz
1,2,3
, Norma B. Paniego
1,2
and H. Esteban Hopp
*
1,3

1
Instituto de Biotecnología, Centro de Investigaciones Veterinarias y Agronómicas,
Instituto Nacional de Tecnología Agropecuaria (INTA Castelar),
Repetto y Los Reseros, Hurlingham, Provincia de Buenos Aires, Argentina
2
Consejo Nacional de Investigaciones Científicas y Técnicas,
Ciudad Autónoma de Buenos Aires, Argentina
3
Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina


ABSTRACT

Since sunflower domestication by pre-hispanic American cultures at least 3000 BC,
the use of empiric and scientifically based genetics led to an amazing genetic
diversification of the crop going from sophisticated nutraceutical applications up to
ornamental purposes, including the traditional confectionary and oilseed production.
Commercial sunflower breeding based on genetics started in the first half of the twentieth
century and genomics at its endings, with breeding efforts being directed towards the
most economically important traits such as increasing seed and oil yield, improving
quality traits and conferring resistance or tolerance to biotic and abiotic stresses. In the
last few years, advancements in genotyping and sequencing technologies allowed the
development of increasingly dense genetic and physical maps, enabling the development
of new breeding strategies based on molecular markers, like QTL mapping, association
mapping and genomic selection. The need to increase efficiency and precision has
motivated the application of marker assisted selection (MAS) in sunflower breeding
programs. This chapter will review the different genomic breeding approaches that are
currently used to improve sunflower tolerance to biotic and abiotic stresses, increase oil
quality and enhance agronomic yield associated traits in order to reduce the gap between
potential and actual sunflower production in the present cultivated sunflower area and

*
Corresponding author: Professor H. Esteban Hopp; E-mail: ehopp@cnia.inta.gov.ar
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Carla Filippi, Jeremías Zubrzycki, Verónica Lía et al. 62
under global weather changing conditions that negatively impact on it. An overview of
the state of the art on sunflower genomics is presented and the potential of high
throughput sequencing and genotyping technologies for crop breeding is discussed.

Keywords: Sunflower, marker assisted selection, QTL mapping, association mapping,
cytogenetyc mapping, linkage mapping


INTRODUCTION

The Compositae (Asteraceae) is the largest plant family on earth, with over 24000
described species, representing almost 10% of all flowering plant species (Stevens 2010).
Compositae species include economically important crops, rare and beautiful wildflowers,
invasive weeds and several species harboring common allergens and valuable medical
molecules (Dempewolf et al., 2008). They are referred to as Composites because what looks
like a single large flower is actually a composite of many, maybe thousands tiny flowers. It
includes sunflowers, lettuce, artichokes, dandelions, thistles, daisies, ragweed, goldenrod and
chicory (Kane et al., 2011).
The genus Helianthus contains about 50 species of annual and perennial herbs (Heiser
and Schilling 1981) native to America. It includes diploids, tetraploids and hexaploids, all
with the basic chromosome number of 17 (Rieseberg 1991).
Asteraceae and its related families Goodeniaceae and Calyceraceae do not have an
extended fossil record. In 2010, Barreda et al. described a fossil capitulum and pollen grains
from the Eocene that were found in Patagonia, Southern Argentina. This finding evidenced
that the family evolved from an ancestor originated putatively in South America about 50
million years ago (Barreda et al., 2010).
Sunflower (Helianthus annuus L. var. macrocarpus) was originally domesticated in the
east-central part of North America circa 3000-4000 years ago (Crites 1993; Harter et al.,
2004; Smith and Yarnell 2009; Bowers et al., 2012) by pre-Columbian civilizations that used
it as a source of edible seeds and for other applications (as a source of natural dyes and for
ceremonial purposes) (Mandel et al., 2013). Since then, sunflower has been grown with many
purposes: as oil crop (its main use), for beauty (ornamental sunflower) and for direct
consumption of the seeds (confectionary sunflower). Furthermore, there is an increasing
interest in the use of sunflower proteins in human nutrition. The content of protein remaining
in cakes and extraction residues after seed oil extraction, accounts for 30 to 50% (Dorrell and
Vick 1997). The properties of sunflower proteins are comparable with those of soy and other
leguminous proteins (González-Pérez et al., 2005; Zilic et al., 2010). Regarding its main use,
sunflower is currently the world‘s fourth largest source of vegetable oil
(http://www.fas.usda.gov/). Sunflower oil is considered premium due to its high unsaturated
fatty acid composition and low content of linolenic acid. Nowadays, it is cultivated on over
23 million hectares worldwide (http://www.fao.org/), with an annual production of 32 million
metric tons, mainly concentrated in the Russian Federation, Ukraine, India, and Argentina
(Bowers et al., 2012).
Conventional breeding has been successful in raising sunflower yield potential and its
stability, as well as in controlling resistance to some fungal diseases, pests and parasitic
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Genetics and Genomics Applied to Sunflower Breeding 63
weeds (Sala et al., 2012). The advent and development of molecular markers and genetic
maps have facilitated understanding the genetic basis of different agronomic traits.
In this chapter, we present and discuss the different genetic and genomic breeding
approaches that are currently used to improve sunflower yield and its tolerance to diseases,
focusing on the study of pathogen resistance responses and reviewing the state of the art of
sunflower genomics.


1. SUNFLOWER BREEDING

Commercial sunflower breeding started in most of the producing countries (Eastern and
Western Europe, North and South America) between 1920 and 1950 by phenotypic selection,
a method of selecting desirable plants from a population on the basis of phenotypic traits.
The introduction of heterosis, first described in 1966 (Leclercq 1966), the incorporation of
cytoplasmic male sterility after interspecific crossing with H. petiolaris Nutt (Leclercq 1969),
and the development of fertility restorer lines by Kinman in 1970 (Miller and Fick 1997),
allowed the development of sunflower hybrids. This process is based on a single cytoplasmic
male sterility (CMS) source, the PET1. The CMS was associated with the expression of a 16
kDa protein encoded by orfH522 in the PET1 cytoplasm, which is anther-specific reduced in
fertility restored hybrids (Moneger et al., 1994). This protein is co-transcribed with the atpA
mitochondrial gene in the male sterile lines.
The first sunflower hybrids were produced in 1972 and reached 80% of production in five
years (Miller and Fick 1997), due to their higher yield and quality potential, high
homogeneity, maturing time synchronicity and better adaptation to cultural applications.
Sunflower breeding efforts were directed towards the most economically important traits,
including: increased seed and oil yield (number of seeds per plant, test weight, 1000-seed
weight, low husk content and high oil concentration in the seed), increased harvest index
(plant height, head size and shape, angle of the head, leaf area and leaf canopy, early
maturation, short stem and uniform height), improved quality traits (oil quality, protein
concentration and composition), and resistance to biotic and abiotic stresses (Škorić 1992).
Despite the optimism for continued improvement by conventional breeding, new
technologies are needed to significantly increase efficiency and precision, and to save time,
resources and efforts. Agriculturally important traits, such as yield, quality and some forms of
disease resistance are controlled by many genes and known as quantitative traits. The regions
within genomes that contain those genes associated with a particular quantitative trait are
known as quantitative trait loci (QTL). Owing to genetic linkage, DNA markers can be used
to detect the presence of allelic variation for major genes or QTL underlying the traits of
interest.
The first molecular markers described in sunflower were restriction fragment length
polymorphism (RFLP) (Berry et al., 1995, 1996, 1997; Gentzbittel et al., 1995, 1999; Jan et
al., 1998), random

amplified polymorphic DNA (RAPD) (Rieseberg et al., 1993; Rieseberg
1998) and amplified fragment length polymorphisms

(AFLPs) (Peerbolte and Paleman 1998;
Flores Berrios et al., 2000; Gedil et al., 2001; Al-Chaarani et al., 2004).

However, RFLPs are
technically laborious for routine use as molecular markers and while RAPD and AFLP

markers have many advantages, they are mostly dominant, abundant but often non-specific
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Carla Filippi, Jeremías Zubrzycki, Verónica Lía et al. 64
and not very useful for comparison of a genome-wide synteny of molecular markers for

cross
referencing genetic linkage maps (Paniego et al., 2007). Nowadays, the most popular
molecular markers are microsatellite markers (also called single sequence repeats, SSR) and
single nucleotide polymorphisms (SNP). While SSR markers are multiallelic, SNPs are
generally assumed as biallelic markers. However, this disadvantage is compensated by the
relative abundance of SNPs (Oraguzie et al., 2007) and emerging high throughput profiling
technologies.
Marker-assisted selection (MAS) is a method whereby a phenotype is selected using
molecular markers linked to the trait genetic determinants. The advantages of MAS include:
time saving by substitution of field trials with molecular tests; selection carried out at seedling
stage; the possibility of combining multiple gene selection; avoid the transfer of undesirable
genes by background selection; selection of traits with low heritability; selection of single
plants (Collard et al., 2005).
One of the most important tools in MAS strategy is having a high-resolution map,
composed of polymorphic molecular markers covering all chromosomes, in order to identify
markers flanking those QTL that control traits of interest. To avoid losing the selected trait
due to recombination events between the marker locus and the QTL, they need to be in strong
linkage disequilibrium (at least <5 cM but ideally <1 cM away from the gene). However, not
any marker associated with a QTL is directly useful in MAS, and its reliability for predicting
phenotypes has to be tested (Collard et al., 2008). Advancements in genotyping technologies,
and its direct consequences, lower genotyping costs and denser genome coverage, allowed the
development of new breeding strategies like association mapping and genomic selection
(GS). Association mapping, or linkage disequilibrium (LD) mapping, has emerged as a tool to
resolve complex trait variation, because the historical recombination and natural genetic
diversity are exploited for high resolution mapping (Risch and Merikangas 1996; Nordborg et
al., 2002; Zhu et al., 2008). Meanwhile, GS uses a genome-wide panel of dense markers as
predictors of performance, calculating genomic estimated breeding values for complex traits.
Phenotypes and high-density marker scores are analyzed in the population of study, and the
breeding values of any line can be predicted with a high level of accuracy (in simulations, the
correlation between true and estimated breeding values reached a level of 0.85 (Heffner,
Sorrells, and Jannink 2009). This selection method promises to accelerate the breeding times,
while changing the role of phenotyping only to corroborate and update prediction models.
However, due to the lack of a reference genome, this method cannot be applied in sunflower
until the establishment of a much larger number of SNPs markers validated for GS.


2. SUNFLOWER LINKAGE MAP

Over the past decades, several genetic linkage maps differing in length, density and type
of molecular markers, were developed for cultivated sunflower (n=17 chromosomes).
Gentzbittel et al (1994, 1995) have shown that the RFLP technique can be used to identify
sunflower genotypes and that there is sufficient variability to develop an RFLP framework
map. Thus, first descriptions of a consensus sunflower map, based on segregation of RFLP
markers, were published by Gentzbittel et al., (1995) and Berry et al., (1995). Since then,
denser linkage maps were developed, based on RFLP (Berry et al., 1996, 1997; Jan et al.,
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Genetics and Genomics Applied to Sunflower Breeding 65
1998; Gentzbittel et al., 1999), RAPD (Rieseberg et al., 1993; Rieseberg 1998), AFLPs
(Peerbolte and Paleman 1998; Flores Berrios et al., 2000; Gedil et al., 2001; Al-Chaarani et
al., 2002), direct amplification of length polymorphism markers (DALPs) (Langar et al.,
2003), and target region amplification polymorphisms (TRAPs), that use EST database
information to generate polymorphic markers around targeted candidate gene sequences (Hu
and Vick 2003).
In sunflower, RAPDs have been used primarily for tagging phenotypic loci (Lawson et
al., 1998; Lu et al., 2000), while the RFLP maps have been used as tools for mapping
phenotypic and quantitative trait loci (Leon et al., 1995, 2000; Berry et al., 1996; Lu et al.,
1999; Bert et al., 2001; Lee et al., 2001; Al-Chaarani et al., 2002; Pérez-Vich et al., 2002).
The TRAP technique has been employed to construct a linkage map (Hu and Vick 2003), to
define the telomeric regions of sunflower linkage groups through the use of TRAP markers
based on Arabidopsis-type telomere repeat sequences (Hu 2006), to map some resistance loci
(Hu et al., 2004; Yue et al., 2008); and to assess germplasm relationships (Sala et al., 2012).
The development of the first microsatellite markers in sunflower supplied the critical
mass of DNA markers needed to create a public reference map and unify independently
developed genetic linkage maps. They rapidly became the markers of choice for linkage
analysis due to the fact that they are highly polymorphic, usually inherited in a codominant
manner and, in most cases, chromosome-specific.
In the last years, development of microsatellites by South American researchers at INTA
in Argentina (HAx) together with those of European (CARTISOL; CRS) and North American
(ORSx) origin summed up to 2040 markers (Dehmer and Friedt 1998; Paniego et al., 2002;
Tang et al., 2002; Yu et al., 2002).

These markers were used for developing reference maps
using F2 and recombinant inbred line (RIL) populations, which are highly homozygous lines
derived from crosses between highly contrasting sunflower inbred lines. The recombinant
inbred line parental crosses used were: PAC-2 x RHA266, RHA280 x RHA801 and PHA x
PHAB (Flores Berrios et al., 2000; Tang et al., 2002, 2003; Yu et al., 2003; Al-Chaarani et
al., 2004). Furthermore, several reference genetic maps that integrate the independently
developed linkage maps have been reported (Yu et al., 2003; Paniego et al., 2007). Yu et al.,
(2003) integrated 120 SSR loci from the public SSR map and 28 RFLP markers from Jan et
al., (1998) map with 80 RFLP loci of the genetic map of Berry et al., (1997). Lai et al.,
(2005a) produced the first SNP map by integrating 243 loci in the RHA280 x·RHA801
reference map (Tang et al., 2002; Yu et al., 2003). Kiani et al (2007b) established a high-
density genetic map with a mean density of one marker per 3.7 cM, by integrating 191 SSR
marker loci (Paniego et al., 2002; Tang et al., 2003) into a previous map based on 304 AFLP
markers (Al-Chaarani et al., 2004). All the maps reported above, plus unpublished linkage
maps, can be accessed through the Sunflower CMap Database (http://www.sunflower.
uga.edu/cmap/).
These SSR-based maps have been used to identify quantitative trait loci (QTL)
underlying numerous traits of agronomic importance (Shunxue Tang et al., 2006; Wills and
Burke 2007, Kianni et al 2007, 2008, 2009, Haddadi et al., 2010, Haddadi et al 2011a, 2011b)
and to investigate variation in genome structure between sunflower and other Helianthus
species (Burke et al., 2002; Heesacker et al., 2008).
Nowadays, next-generation sequencing technologies and high-throughput genotyping
platforms make it possible to simultaneously interrogate thousands of SNPs throughout the
genome (Gupta et al., 2008), enabling the development of increasingly dense genetic maps.
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Carla Filippi, Jeremías Zubrzycki, Verónica Lía et al. 66
Zubrzycki et al (2012a) reported the construction of an Oligonucleotide Probe Assay
(Illumina) including 384 SNPs representing putative candidate genes for stress resistance
responses. In parallel, a 10K Illumina Infinium SNP genotyping array was developed
(Bachlava et al., 2012) and used in different sunflower mapping populations to produce a
consensus linkage map of the sunflower genome, that constitutes the longest map reported in
the literature until now (Bowers et al., 2012). In this study, four different sunflower mapping
populations were used to produce four separate high-density genetic maps containing 3500–
5500 loci each. Although the individual maps were highly colinear, localized variation in
recombination rates in several genomic regions and gaps up to 26 cM in length were
observed. Because these regions differed by cross between the different populations, the
consensus map of 10,080 loci contained no such gaps, demonstrating the value of
simultaneously analyzing multiple mapping populations. This high density genetic map has
the potential to facilitate the assembly of the forthcoming sunflower genome (Kane et al.,
2011), enabling the efficient and detailed genotypic characterization of germplasm
collections, and providing an important tool for genome wide association mapping (GWAS)
and GS studies.
Bowers et al., (2012) described the genotypic characterization of a subset of 69 lines of
the RHA280 x RHA801 mapping population using a custom Affymetrix GeneChip that
contains 2389915 features, targeting 86023 unigenes from sunflower and related species. The
previously generated genetic map (Bowers et al., 2012), was used to filter out low quality data
and place 67486 features, corresponding to 22481 unigenes, on the sunflower genetic map.
The resulting map contains a substantial fraction of all sunflower genes, and will facilitate
genome assembly and the identification of candidate genes underlying QTL or traits of
interest.


3. QTL MAPPING

3.1. Disease Resistance

Biotic stress is considered one of the most important factors affecting sunflower all over
the world. Cultivated sunflower is susceptible to several fungal and bacterial diseases, being
the infection severity greatly dependent on environmental conditions. Diseases affecting most
of the sunflower growing areas include wilt, middle stalk rot, and head rot (mainly caused by
Sclerotinia sclerotiorum), downy mildew (Plasmopara halstedii), stem canker (Phomopsis
helianthi), rust (Puccinia helianthi) and Verticillium wilt (Verticillium dahliae). Other
diseases such as head rots (Rhizopus arrhizus, R. stolonifera, Botrytis cinerea), phomopsis
black stem (Phoma macdonaldii), Alternaria leaf and stem spot (Alternaria helianthi or A.
zinniae), Septoria leaf spot (Septoria helianthi), charcoal rot (Macrophominia phasiolina),
bacterial infections (Pseudomonas syringae pv. Tagetis), powdery mildew (Erysiphe
cichoracearum) have local impacts in some productive areas (Seiler 1992; Pereyra and
Escande 1994; Schneiter 1997).
Disease control by applying fungicides is not always possible due to the vast marginal
cultivated areas of sunflower crop. Therefore, the most efficient and sustainable strategy to
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Genetics and Genomics Applied to Sunflower Breeding 67
control the disease and pest impact is to strengthen the crop by the generation of inbred lines
with genetic disease tolerance (Sackston 1962).
A relevant contribution to the study of sunflower disease resistance factors is the
comprehensive work carried out by Radwan et al., (2008) mapping unique resistance gene
candidates, encoding nucleotide binding site-leucine rich repeat sequences (NBS-LRR), to
167 loci throughout the 17 sunflower linkage groups. NBS-LRR loci were mainly distributed
in cluster or singletons at the upper and lower end of most linkage groups. They described
two large clusters on linkage groups 8 and 13, whereas smaller clusters were mapped on
linkage groups 1, 4, 9, and 15. Interestingly, most of the resistance loci mapped in sunflower
until now, lie in the same linkage groups (LG, see below).
A compendium of disease resistance genes mapped in sunflower genome is summarized
in Table 1.

Table 1. Disease resistance and oil related genes mapped in sunflower

Trait Gene Location Literature source
Downy Mildew
Resistance Pl1 LG8
Zimmer and Fick, 1974; Gedil et al., 2001; Bertero de
Romano et al., 2010

Pl2 LG8 Zimmer and Fick, 1974; Bertero de Romano et al., 2010

Pl4 ? Sackston, 1990

Pl5 LG13 Bert et al., 2001; Radwan et al., 2004

Pl6 LG8
Miller and Gulya,1991; Bouzidi et al., 2002; Vear et al.,
2003; Panković et al., 2007

Pl7 LG8 Miller and Gulya, 1991; Bert et al., 2001;

Pl8 LG13
Miller and Gulya,1991; Radwan et al., 2004; Bachlava
et al., 2011

Pl9 ? Sackston, 1990; Molinero-Ruiz et al., 2003

Pl13 LG1 Mulpuri et al., 2009; Bertero de Romano et al., 2010

Pl14 LG1 Bachlava et al., 2011

Pl15 LG8 Bertero de Romano et al., 2010

PlArg LG1
Dussle et al., 2004; Wieckhorst et al., 2010; Imerovski
et al., 2011
Black Rust
Resistance R1 LG8 Lawson et al., 1996

Radv LG13 Lawson et al., 1998; Bachlava et al., 2009

R4 LG13 Qi et al., 2011; Miller et al., 1988

R2 LG9 Lawson et al., 2010

R3 ? Goulter 1990

R5 LG2 Qi et al., 2011

Pu6 ? Yang 1989

R11 LG13 Qi et al., 2012

Verticillium Wilt
R12
V1
?
?
Gong et al., 2013
Gulya, 2007
Sclerotinia Head
Rot Resistance HaRIC_B LG11 Fusari et al., 2012
Oil Yield Related B LG10 Tang et al., 2006

Hyp LG16 Tang et al., 2006
P LG17 Tang et al., 2006
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Carla Filippi, Jeremías Zubrzycki, Verónica Lía et al. 68
Downy Mildew

Genetic analysis of downy mildew resistance includes the characterization and
inheritance of both complete and partial resistance (Jocic et al., 2012). Complete, monogenic
or qualitative resistance is given by Pl genes (Vranceanu et al., 1981; Miller and Gulya 1991).
Until today, a total of 18 disease resistance genes have been identified from wild and
cultivated sunflower (Pl
1
to Pl
16
, P
21
, Pl
Arg
). The Pl
5
gene from H. tuberosus, a perennial
hexaploid sunflower, protects against race 3 (virulence phenotype 700) (Pustovoit 1966;
Leclercq et al., 1970). Resistance to races 1, 2, 3 and 4 (virulence phenotype 730) was
introgressed into cultivated sunflower from three sunflower species: H. annuus ssp. annuus
(Pl
6
), H. praecox ssp. runyonii (Pl
7
) and H. argophyllus (Pl
8
) (Miller and Gulya 1988, 1991).
Also from H. argophyllus was isolated Pl
Arg
, a gene which conferred resistance to at least four
tested races (virulence phenotypes 300, 700, 730, 770). Recently, new sources of resistance
were described, one derived from the inbred line HAR5 which was designated as Pl
13

(Mulpuri et al., 2009), while the second was designated as Pl
14
(Bachlava et al., 2011). The
effectiveness of the major resistance genes Pl
6
and Pl
7
has been overcome by new races in
France (Delmotte et al., 2008) and in USA; however, there are no records of races
overcoming other broad spectrum (Pl
8
, and Pl
Arg
) genes (Gulya et al., 2010). The gene Pl
15

found in RNID, a restorer inbred line derived from an open pollinated population, shows
resistance to the four predominant races of downy mildew in all sunflower producing
countries (300, 700, 730, and 770 (Gulya 2007), and also to the less prevalent races, including
those recently described in USA (714 and 734) and in France (304) which overcome Pl
6

(Vear 2004; Gulya et al., 2010). The Pl
1
(Mouzeyar et al., 1995), Pl
2
(Vear et al., 1997), Pl
6
(Roeckel-drevet et al., 1996), Pl
7
(Bert et al., 2001), Pl
15
(Bertero de Romano et al., 2010)
cluster on LG 8. Finally, Pl
5
(Bert et al., 2001), Pl
8
(Radwan et al., 2003) and Pl
21
(Vincourt
et al., 2012) were mapped to LG 13.
Partial or quantitative resistance is usually non race-specific and assumed to be polygenic
(Jocic et al., 2012). Al-Chaarani et al., (2002) described three major QTL located on LG 1, 9
and 17, and explained 54.9% of the total phenotypic variance. Vear et al., (2008) reported two
QTL located on LG 10 and LG 8. Vincourt et al., (2012) confirmed and refined the QTL
described by Vear et al (2008) and mapped a third QTL in LG 7. QTL on LG 7 and LG 10 are
not located within the major resistance gene clusters (LG 8, LG 13, LG 1), suggesting that the
genes underlying these QTL may involve other biological processes than those directly
involved in plant pathogen interaction. Moreover, QTL mapped in the same region as a gene
involved in stomatal opening and root growth, which was suggested as a possible candidate to
explain the control of mildew resistance (Vincourt et al., 2012).
The use of partial resistances does not appear to have an effect as strong as the
monogenic resistance on pathogen populations. By combining the partial resistance provided
by minor genes with specific resistance genes, durable resistance could be achieved (Vear et
al., 2008).


Black Rust

Sunflower rust, occasioned by Puccinia helianthi can result in significant losses in both
yield and seed quality. The rapid changes that occur in its virulence represent a continuous
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Genetics and Genomics Applied to Sunflower Breeding 69
threat to the effectiveness of existing rust-resistance inbred lines and hybrids. Several genes
conferring resistance to rust have been identified in sunflower including R
1
, R
2
, R
3
, R
4
, R
5
,
Pu
6
, R
adv
, R
11
and R
12
(Putt and Sackston 1957; Miah and Sackston 1970; Miller and Gulya
1988; Yang 1989; Lawson et al., 1998; Gong et al., 2013), but only a few of them have been
genetically characterized, mapped and linked to molecular markers. In addition, several
inbred lines and interspecific germplasm lines were reported to have resistance to different
rust races (Quresh et al., 1993; Bulos et al., 2012).
Rust resistance R
1
gene was found in the restorer inbred line RHA279 (Lawson et al.,
1996) and was mapped on LG 8. Three resistance genes (R
adv
, from an Australian hybrid
(Lawson et al., 1998), R
adv
from the public line RHA340 (Bachlava et al., 2009) and R
4
from
HAR3 (Qi et al., 2011) were located on LG 13. R
1
, and both R
adv
and R
4
are close to the
largest clusters of NBS-LRR resistance candidate genes currently described in sunflower
genetic maps (Lawson et al., 1998, 2010; Radwan et al., 2008; Qi et al., 2011). R
2
from MC29
was located using SSR markers in LG 9 (Lawson et al., 2010). In each case, it was
demonstrated that rust resistance is controlled by a single factor.
Inheritance and mapping of rust resistance genes from different sources were recently
reported (Bulos et al., 2012). Bulos and coworkers (2013) identified 8 markers linked to the
rust resistance gene from the line HAR6, located between molecular markers ZVG61 and
ORS581, at 0.7 and 1.5 cM, respectively. Currently, research is in progress in order to
elucidate the allelic relationships among all these sources, and to generate allele specific
markers for all of them. A pre-breeding strategy to eliminate linkage drag from the wild donor
species is also in progress (Paniego et al., 2012).


Verticillium Wilt

Verticillium dahliae (Kleb) is a soil-borne pathogen widely distributed which causes the
premature death and stem breakage of sunflower. Resistance to this pathogen was described
as qualitative or quantitative, dominant or recessive, depending on the host species considered
(Pegg and Brady 2002; Fradin and Thomma 2006). In sunflower genetic resistance, based on
a single, dominant gene from wild H. annuus (V1), was previously identified and
incorporated into released inbreds (Gulya 2007). Resistant oilseed hybrids were produced
containing this gene. However, a race of V. dahliae not controlled by the V1 gene was
previously identified in Argentina (Bertero and Vasquez 1982).
Galella et al., (2004), reported two new races isolated from Argentina which were called
Varg1 and Varg2. A new strain of V. dahliae, identified in the U.S., as well as these new
reported Argentinean races, are able to overcome V1 resistance gene (from HA89 inbred line)
(Galella et al., 2004; Gulya 2007). A major Varg1 resistance QTL, mapped and incorporated
by MAS to hybrids of different backgrounds, gave effective protection under natural infection
at four locations, independently of the background of the parental lines (Creus et al., 2007). A
second QTL contributing to Varg2 resistance was mapped in a different linkage group. Both
QTL were transferred to the same susceptible line conferring resistance to the two most
abundant pathogenic races present in Argentina, under controlled independent inoculations.
Nevertheless, few individual plants presented typical symptoms under natural infection
conditions indicating the presence of minor Verticillium races (Galella et al., 2012). These
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Carla Filippi, Jeremías Zubrzycki, Verónica Lía et al. 70
results indicate that it is possible to combine resistance QTL to different races in order to
obtain a more durable resistance to Verticillium wilt.


Alternaria Leaf Blight

Alternaria helianthi is a worldwide distributed pathogen associated to important yield
reductions. A. helianthi attacks leaves, stems petioles, bracts and heads of sunflower (Kong et
al., 1995) causing premature foliar senescence and defoliation due to spot coalescence or
petiole necrosis. The nature of resistance is polygenic and limited information about
resistance QTL location has been reported (Virányi 2008). The lack of good sources for
resistance is a major limiting issue in sunflower breeding (Reddy et al., 2006).
In order to detect resistance sources, 370 lines were evaluated under natural infection in
the Northern crop region of Argentina. The lesion density and the percentage of totally
necrotic leaves were measured. Three major QTL in LG 4, 10 and 17 showing additive effect
were detected (Galella et al., 2010). The QTL were introgressed by backcross from the
resistance source to L97 and L03 susceptible lines using MAS. The converted lines and their
respective counterparts were evaluated under very severe natural infection conditions. The
percentage of total necrotic foliage was reduced from 100 to 50 % in L03 and from 70 to 40
% in L97 backgrounds, respectively.


Sclerotinia Rots

Sunflower production is seriously affected by Sclerotinia sclerotiorum infection with
very variable attack levels, according to temperature and rainfall at particular periods of crop
growth and the presence of other susceptible crops in the rotation or in neighbouring fields.
This pathogen is a highly polyphagous and necrotrophic fungus that infects different plant
parts, including roots, stem bases (stalk rot), leaves, terminal buds and capitula (head rot)
producing great losses. Disease control on S. sclerotiorum is difficult, since the fungus
persists in soils for long periods and at high inoculum levels (Huang and Dueck 1980; Troglia
2003).
Currently, no complete resistance to S. sclerotiorum is available in cultivated sunflower,
even if differences in susceptibility exist (Tourvieille de Labrouhe et al., 1996). The nature of
resistance has been described as partial quantitative disease resistance (QDR) and highly
dependent on environmental conditions (Castaño et al., 1993) involving several loci with
different effects, most of them additives (Vear and Tourvieille de Labrouhe 1988; Gulya et
al., 1997).
Conventional breeding programs involve field resistance tests on segregating progenies
and inbred lines and field trials on potential hybrid varieties. Regarding this point, Mestries et
al., (1998) identified four QTL for leaf resistance and two for head rot resistance based on a
genetic map with RFLP and isozyme markers. One of these zones appears to be involved in
resistance to both types of S. sclerotiorum attack, while the others appear specific for
resistance in one part of the plant. Individual QTL explained between 9% and 48% of the
phenotypic variability, confirming the polygenic basis of this trait. Overall, the quantitative
trait loci explained 60% of the genetic variation for leaf resistance and 38% for capitulum
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Genetics and Genomics Applied to Sunflower Breeding 71
resistance to S. sclerotiorum. Simultaneously, Gentzbittel et al., (1998) reported several QTL
for resistance to penetration or extension of the S. sclerotiorum mycelium in different tissues.
The latter authors also mapped a serine-threonine protein kinase-like (PK) marker that co-
segregates with a QTL apparently specific to an inbred line, PAC1. It explained 50% of
variation in a cross with a very susceptible line and 15% in a cross with a different highly
resistant line. However, this QTL has not been found in other populations, even when the
resistance source came from recurrent selection of a population from which PAC1 was
originated and when the parents showed polymorphism for the PK gene.
Later, Bert et al., (2002) detected 15 QTL for Sclerotinia reaction across several linkage
groups, whereas Bert et al., (2004) described seven QTL for resistance to S. sclerotiorum
terminal bud attack, each explaining less than 10% of the phenotypic variance. For capitulum
attack, there were 4 QTL, each explaining up to 20% of variation. All QTL found involved
six different genetic crosses between cultivated inbred lines, being reported QTL distributed
in at least 14 linkage groups.
Regarding S. sclerotiorum head rot, relative QTL effect levels vary between different
years and locations considered. Thus, QTL effects studied during a specific year and location
differ within a range of 23% (Bert et al., 2004) to 44% (Mestries et al., 1998), and also differ
from those studied over two or more years in different locations for which the variation
ranged from 13% to 16% (Gentzbittel et al., 1998; Bert 2002).
Micic et al., (2004) identified nine QTL for leaf lesion, eight for stem lesion, and six for
speed of fungal growth. For stem lesion two genomic regions explained 26.5% of the
genotypic variance (Micic et al., 2005b). Three to four putative QTL were detected for stem
lesion, leaf lesion and speed of fungal growth explaining between 40.8% and 72.7% of the
genotypic variance. Two QTL for stem lesion showed large genetic effects and corroborated
earlier findings from the cross NDBLOSsel (resistant) x CM625 (susceptible) (Micic et al.,
2005a).
Rönicke et al., (2005) identified five QTL for lesion length and head rot explaining 10.6
to 17.1% of the total phenotypic variance. The most frequent QTL being that (or those) linked
to the recessive branching gene b1 on Linkage Group 10.
Yue et al., (2008) identified nine and seven QTL for disease incidence and disease
severity, respectively. Each QTL explained 8.4 to 34.5% of phenotypic variance, suggesting
the polygenic basis of the resistance to head rot. Five of these QTL were identified in more
than one experiment, and each QTL explained more than 12.9% of phenotypic variance.
Although a positive correlation existed between the two disease indices, most of the
respective QTL were located in different chromosomal regions, suggesting a different genetic
basis for the two phenomena indices.
Davar et al., (2010) detected 7 QTL for percentage necrotic area, localized on 7 linkage
groups whose effects were small to moderate.
Zubrzycki et al (2012b) identified 20 QTL, accounting for incidence, severity and the
area under the disease progress curve (AUDPC), located mostly in linkage groups 10, 13 and
14. Each encountered QTL explained less than 10% of the observed phenotypic variance. In
the upper end of LG 10, two reproducible QTL associated with lower incidence were found
on this population and on the population derived from the cross of HA89 X RHA801
analyzed by Maringolo (2007).
Association studies of transcript expression of candidate genes with disease resistance
represent an alternative strategy to find new sources of resistance to Sclerotinia. The
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Carla Filippi, Jeremías Zubrzycki, Verónica Lía et al. 72
development of a large EST public database for sunflower (http://www.ncbi.nlm.nih.gov/
dbEST/dbEST_summary.html) including entries from sequenced differential cDNA libraries
with highly represented defense genes (Fernandez et al., 2003, 2012) enables the utilization of
the candidate gene approach for searching new QTL determinants.
The QTL studies for S. sclerotiorum resistance have shown that the main QTL detected
from different crosses appear to be different, thus suggesting that pyramiding QTL for
resistance is possible. Furthermore, resistance to this disease is governed by multiple QTL,
located on almost all the sunflower linkage groups. However, like any other quantitative trait,
it is necessary to confirm the position of the QTL and to carry out fine-scale mapping before
MAS can be undertaken.
The possibility of increasing the level of resistance of elite lines by introgression of
several QTL with the help of MAS and embryo culture tools, without affecting yield
components, constitutes a relative simple way to reduce the impact of this complex disease.


3.2. Mapping Yield-and Quality Related Traits

Sunflower yield is a complex trait regulated by a number of factors that can be studied as
component traits. Oil yield per unit area is determined by the product of seed yield per unit
area and oil percentage in the seed. Then, one of the most important goals of sunflower
breeding programs is to develop high oil yield hybrids considering both components.
Several QTL accounting for yield-related phenotypic variation across environments
ranging from 5 to 90.4%, were detected on different LGs (Leon et al., 1995, 2000; Mokrani et
al., 2002).
Tang et al., (2006) identified 40 QTL for yield-related traits in a low- x high-oil RIL
mapping population (RHA280 x RHA801) segregating for apical branching (B, located on
LG 10), phytomelanin pigment (Hyp, located on LG 16), and hypodermal pigment (P,
Located on LG 17) loci. Twenty-four of the QTL were tightly linked to B, P, and Hyp and
may have been partly or completely caused by the pleiotropic effects of B, P, and Hyp. Five
QTL controlling seed weight were reported explaining 72.8% of the phenotypic variation.
However, the contribution of the pleiotropic effect of the apical branching gene B accounted
again for the bulk (52.5%) of this variation (Tang et al., 2006). Interestingly, the same
relationship between apical branching and seed oil content was observed in other mapping
populations (Mestries et al., 1998; Bert et al., 2003). The linked, pleiotropically acting, and
epistatically interacting QTL identified for seed traits were presumably targeted by selection
in the transition from large-seeded, low-oil to small-seeded, high-oil cultivars in sunflower.
Putative QTL for 1000-grain weight were detected explaining from 5.4% to 37% of total
phenotypic variance (Mokrani et al., 2002; Al-Chaarani et al., 2004). Some of the QTL
controlling seed weight overlapped with that mentioned above. The QTL on the same LG as
the branching gene b1, also reported by Mestries et al., (1998) for another cross, was almost
certainly linked to capitulum size. The second QTL for seed weight was close to the one for
flowering date.
By evaluation of the same mapping population under different conditions it is possible to
discriminate constitutive QTL effects from adaptive ones. These studies allow investigating
the underlying genetic basis of trait association by looking for colocation of corresponding
QTL for yield-related traits on the genetic map under stress and nonstress conditions.
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In this context, several studies have conducted QTL mapping for different traits to
highlight the existence of adaptive QTL detected only under specific environmental
conditions or QTL modifying their expression with the level of an environmental factor. In
2008, Ebrahimi et al (2008) mapped QTL controlling seed quality traits under well-watered
and water-stressed conditions. Significant variation among RILs was observed even though
that there were no significant differences for oil content in the seed between both parents.
Two to 15 QTL explaining from 5% to 31% of phenotypic variation were found depending on
trait and growth conditions. Several QTL for oil content overlapped with QTL for palmitic
and stearic acid contents in all four conditions. An overlapping region on linkage group 3 was
linked to an SSR marker (ORS657). Furthermore, the latter author also identified genomic
regions controlling other yield-related traits (seed protein content, kernel and hull weights,
and seed density) in the same water-stressed conditions. Several specific and nonspecific
QTL were detected for all traits under these conditions. A QTL for protein content accounting
for 17% of phenotypic variance under water-stress conditions was detected. Overlapping QTL
for protein content and seed density were identified in LG 15. This region probably has a
pleiotropic effect on protein content and seed density. QTL for protein content co-localizing
with grain weight traits were also identified. Three markers linked to protein content
(ORS671_2 and HA2714) and kernel weight (E35M60_4) were identified (Ebrahimi et al.,
2009). QTL for grain yield per plant (GYP) under four water treatments were identified on
different linkage groups, among which two were specific to a single treatment (Kiani et al.,
2009). The identified QTL explained 4% to 40% of phenotypic variance. Three QTL for GYP
overlapped with several QTL for drought-adaptive traits detected in a previous study (Kiani et
al., 2007a).
In 2010, Haddadi et al., mapped QTL associated with percentage of seed protein, oil and
fatty acids content under well-, partial-irrigated and late-sowing conditions. A specific QTL
of palmitic acid content on LG 6 was detected under late-sowing conditions. Common
chromosomic regions were observed for percentage of seed oil and stearic acid content on
linkage group 10 and 15. Seven QTL associated with palmitic, stearic, oleic and linoleic acids
content were identified on LG 14. Additionally, overlapping occured for QTL of oleic and
linoleic acids content on linkage groups 10, 11 and 16.
Haddadi et al., (2011) mapped QTL associated with agronomic traits such as yield, plant
height, days from sowing to flowering, and leaf-related traits. The study was carried out over
well- and partial-irrigated conditions. Three to seven QTL, explaining from 4 to 49% of
phenotypic variance, were found for each studied trait in both conditions. Three to six QTL
were found for each yield-related trait in both conditions. These QTL were located on LG 2,
9, 10, 11, 12, 13 and 14. Likewise, Hadaddi et al (2011) conducted seven experiments in
different environments to identify genomic regions underlying the genetic control of
tocopherol, a non-enzymatic antioxidant known as lipid-soluble vitamin E, and phytosterol,
molecules with interesting properties, which can result in decreased risk of chronic diseases in
humans and with several beneficial effects in plants. Five QTL for total tocopherol content
accounting for 45% of phenotypic variation were located on LG 1, 8, 10 and 14, whereas four
QTL for total phytosterol content on linkage groups 1, 2, 16 and 17 explained 27% of the
phenotypic variation. Moreover, four candidate genes co-located with QTL for total
phytosterol content (GST, PAT2, SFH3 and POD) and four candidate genes co-localized with
QTL for total tocopherol content (VTE4, HPPD, GST and Droug1). Moreover, the candidate
gene SMT2 also mapped on linkage group 17 near the QTL of total phytosterol content.
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Evidence of co-localization of ESTs and QTL in sunflower had already been provided by
Lai el al. (2005a). They reported different candidate genes that map coincident with QTL
controlling physiological, morphologic and developmental traits. On the other hand, a high
level of linkage disequilibrium has been reported in cultivated sunflower (Kolkman et al.,
2007; Fusari et al., 2008). Thus, it has been suggested that association-based approaches will
provide a high degree of resolution for the mapping of functional variations in sunflower (Liu
and Burke 2006). Interspecific QTL mapping has been reported for the wild annual species H.
annuus and H. petiolaris (Lexer et al., 2005) and for three hybrid sunflower species derived
from them: H. anomalus, H. deserticola, and H. paradoxus (Lai et al., 2005b). QTL analysis
in these studies indicated that karyotypic differences among species contributed to
reproductive isolation and evaluated inter- and intraspecific QTL magnitudes.
Studies of co-localization of developmental and agronomic traits with resistance to
pathogens were also reported. A QTL for percentage of plants attacked by S. sclerotiorum
was detected in a LG close to one for flowering date, with one of the parental alleles that
reduced the days to flowering being linked with increases in resistance to this pathogen (Bert
et al., 2003). Other co-localization was reported for QTL for resistance to S. sclerotiorum and
QTL for seed weight and oil content (Mestries et al., 1998; Bert et al., 2003). Furthermore,
some morphology-related traits such as branching regulated by the gene b1 were studied for
their association with S. sclerotiorum resistance and agronomic characters (Bert et al., 2003).
A review of oil related genes in sunflower is summarized in Table 1.


4. ASSOCIATION MAPPING AND GENOME SELECTION

Association genetics via Linkage Disequilibrium (LD) mapping is an emerging field of
genetic mapping that has the potential for resolution to the level of individual genes (alleles)
underlying quantitative traits (Oraguzie et al., 2007). In contrast to classical biparental
population mapping, AM is a method that detects relationships between phenotypic variation
and gene polymorphisms in existing germplasm, without development of mapping
populations. This method incorporates the effects of recombination occurring in many past
generations into a single analysis and thus, it is complementary to the classical biparental
approach (Jorde 2000; Fusari et al., 2012).
The first association mapping work reported in sunflower focused on the study of
Sclerotinia head rot resistance using a candidate gene AM based approach on a population of
94 inbred lines belonging to the ―Sunflower Breeding Program‖ of INTA (Fusari et al., 2012).
Disease incidence was measured using assisted inoculation with the fungal pathogen during
two campaigns in replicated field trials. Given that no biological mechanisms or biochemical
pathways have been positively identified for head rot resistance, selection of candidate genes
was based on previous transcript profiling studies in sunflower (Giacomelli et al., 2010;
Peluffo 2010; Fernandez et al., 2003) and B. napus (Zhao et al., 2007) infected with S.
sclerotiorum. From 43 previously selected genes, 30 candidate genes were amplified in 10
sunflower inbred lines selected as Core Set (CS) for polymorphism discovery, and 16
candidate genes were finally genotyped in the association mapping population. Associations
among SHR incidence and haplotype polymorphisms in those 16 candidate genes were tested
using Mixed Linear Models (MLM) that account for population structure and kinship
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relationships. A significant association between the candidate gene HaRIC_B and SHR
incidence was found, accounting for a SHR incidence reduction of about 20 % (Fusari et al.,
2012).
A study combining association and linkage mapping was carried out by Cadic et al.,
(2013) to identify QTL and causative mutations involved in the control of flowering time in
cultivated sunflower. Flowering time is a major event in the plant life cycle that is controlled
by both genetic and environmental stimuli. A panel of 384 inbred lines and a recombinant
inbred line (RIL) population comprising 273 lines were phenotyped, and genotyped with a set
of 5,923 SNPs markers. A large similarity between association peaks detected in both the
QTL mapped in a RIL population and the linkage disequilibrium mapping approach was
shown (Cadic et al., 2013).
In a recent study carried out by Mandel et al (2013), a total of 271 lines capturing nearly
90% of the allelic diversity present within the cultivated sunflower gene pool was genotyped
on an Illumina Infinium 10 k SNP array (Bachlava et al., 2012). These data along with
phenotypic data gathered from multi-environmental trials, were used to characterize overall
patterns of genomic diversity and to characterize associations underlying agronomic and
evolutionary important traits (Mandel et al., 2013). Briefly, the number of days to flowering
(DTF, calculated from the planting date) and the total number of branches per plant were
measured in the field at the r-5 and R-9 reproductive stage of the Schneider & Miller scale,
respectively. Four plants per accession were scored for replicates at three locations. Three
different association mapping models were run for each trait including a mixed linear model
(MLM) accounting for kinship, and two MLMs using kinship and population structure as
estimated via either principle component analysis (PCA) or the program STRUCTURE
(Pritchard et al., 2000). Significant positive correlations were found for both branching and
flowering time across locations, whereas there was an overall significant negative correlation
between branching and DTF. The authors described substantial variation in levels of linkage
disequilibrium (LD) across the genome, with islands of elevated LD generally corresponding
to genomic regions underlying traits that have been targeted by selection, and large region
where LD decays relatively rapidly across the genome.
Genomic selection (GS) strategy was implemented for the first time in sunflower
breeding research as a method to predict the performance of untested hybrids based
phenotypic and genotypic data (Reif et al., 2013). The work was based on the study of 15
female and 8 male elite sunflower lines adapted to Central Europe, genotyped with 572 AFLP
markers (Reif et al., 2013). Experimental data from sunflower hybrids and their parental lines
were used to examine the accuracy to predict performance for grain yield, oil yield and oil
content, based on phenotypic and genomic data. Among the different biometrical approaches
to implement GS, in this study ridge regression-best linear unbiased prediction (RR-BLUP;
(Whittaker et al., 1999; Meuwissen et al., 2001) was the method of choice, because it
appeared to be well suited for regular plant breeding trials (Albrecht et al., 2011). Prediction
accuracy of hybrid performance based on general combining ability effects (GCA) was high.
In those cases where no information on GCA effects of parental lines was available, hybrid
prediction based on GS methods was accurate for groups of related lines. For unrelated lines,
a strong decrease in the prediction accuracy was observed, suggesting that prediction of
hybrid performance for crosses based on genetically distant parents is still a challenge.
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5. PHYSICAL MAPPING

5.1. Molecular Cytogenetic Mapping

As described above, as a consequence of the increasing availability of various molecular
markers and high-throughput genotyping assays sunflower linkage maps are becoming more
saturated. In spite of that advance, the association between linkage groups (LGs) and
chromosomes has not been established yet. Since the 70s, sunflower cytogenetic studies were
focused on karyotypic analyses using conventional techniques as Feulgen‘s staining and C-
banding (Raicu et al., 1976; Al-Allaf and Godward 1979; Kulshreshtha and Gupta 1981;
Cuellar et al., 1996; Feng et al., 2013). In recent years, development of molecular cytogenetic
techniques such as genomic in situ hybridization (GISH), fluorescence in situ hybridization
(FISH) and primed in situ labeling (PRINS) provided key tools for association of physical
and genetic maps, physical mapping of interesting genomic regions, as well as diversity and
evolutionary studies between species of the genus Helianthus (Rocco 2002; Rocco et al.,
2003). The GISH technique using genomic DNA as probes on cytological preparations has
been successfully used in different plant species to evaluate introgression of wild species in
cultivated crops (Benabdelmouna et al., 2003; Shigyo et al., 2003; Wei et al., 2003) as well as
in evolutionary studies using wild polyploids (Bennett 1995; Poggio et al., 1999; Paniego et
al., 2007); FISH has been the method of choice for mapping repetitive sequences, such as
rRNA gene, and thus, enabling the characterization of the chromosome complement and the
karyotypic analysis in different species (Talia et al., 2011; Minelli, Maggini, and Gelati 2000;
Ceccarelli et al., 2007; Cuellar et al., 1996; De Paula et al., 2005); PRINS technique (Koch et
al., 1989) represents an alternative to the use of FISH for localization of DNA sequences in
plant chromosomes. This strategy was found to be particularly useful for detection of high-
copy tandem repeats. However, for the detection of low-copy repeats, a more sensitive variant
of PRINS named cycling PRINS (C-PRINS), which involves serial thermal cycles analogous
to polymerase chain reaction (Gosden et al., 1991; Kubaláková et al., 2001), has been
recommended (Talia et al., 2011). The combination of bacterial artificial chromosome -
fluorescence in situ hybridization (BAC-FISH) and C-PRINS techniques were evaluated for
their use in the integration of physical and genetic maps of sunflower ( Talia et al., 2011).
Classical cytogenetic studies have been important for the analysis of interspecific
hybridization in the genus. Comparative genetic linkage maps and co-linearity are powerful
tools for the study of karyotypic evolution. The evolution of genome structure between two
annual sunflower species, H. annuus and H. petiolaris was examined using a SSR/RAPD
genetic linkage map of the H. petiolaris genome and an integrated SSR genetic linkage map
derived from four independent H. annuus mapping populations (Burke et al., 2004). The
results of this work indicate the presence of 11 rearrangements (8 translocation and 3
inversions). In spite of that fact, these two species hybridize extensively throughout their
shared range, have high rates of complex backcross production (Rieseberg et al., 1999), and
have long-term rates of gene flow (Strasburg and Rieseberg 2008; Strasburg et al., 2009).
Patterns of genetic diversity and divergence in rearranged versus co-linear regions in H.
annuus and H. petiolaris were examined using sequence data from 77 loci distributed
throughout the genomes of the two species, finding weak evidence for increased genetic
divergence near chromosomal break points but not within rearranged regions overall. No
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Genetics and Genomics Applied to Sunflower Breeding 77
evidence for increased rates of adaptive divergence on rearranged chromosomes was found;
suggesting a limited role for chromosomal rearrangements in genetic divergence (Strasburg et
al., 2009).
Transposable elements are the major drivers of genome size increases in plants. Thus,
understanding the diversity and evolutionary dynamics of transposable elements in sunflower
is of considerable importance for understanding the evolutionary history of this species. FISH
studies using repetitive sequences derived from retroelements from H. annuus allowed the
detection of sequences of two families of retroelements dispersed along the length of all
chromosomes in all species studied. However, the Ty3 /gypsy-like sequences were localized
preferentially at the centromeric regions in most of the studied species, whereas Ty1/ copia-
like sequences were less represented or absent around the centromeres and plentiful at the
chromosome ends only in H. annuus (Santini et al., 2002; Natali et al., 2006; Paniego et al.,
2007), suggesting that these two sequence families played a role in Helianthus genome
evolution and species divergence, evolved independently in the same genomic backgrounds
and in annual or perennial species, and acquired different possible functions in the host
genomes. The analysis of approximately 25% of the sunflower genome from random
sequence reads and assembled bacterial artificial chromosome (BAC) clones, shows that it is
composed of over 81% transposable elements, 77% of which are long terminal repeat (LTR)
retrotransposons, predominantly chromodomain-containing Gypsy LTR retrotransposons
(related to chromoviruses). It was shown that the vast majority of observed LTR
retrotransposon insertions have likely occurred since the origin of this species, providing
evidence that LTR retrotransposon activity has played a major role in shaping the chromatin
and DNA landscape of the sunflower genome (Staton et al., 2012).
The first GISH procedure applied in sunflower was developed for identifying
chromosomes or chromosome segments of wild species in the background of cultivated
sunflower, by examining interspecific hybrids involving four wild perennial species, H.
californicus, H. angustifolius, H. nuttallii and H. maximiliani. With different blocking/probe
ratios and washing stringencies, chromosomes or segments of the four wild species were
clearly identified, demonstrating that the procedure is a practical tool for introgression studies
in the genus Helianthus (Liu et al., 2009).
The use of bacterial artificial chromosomes (BACs) containing large genomic DNA
inserts in physical mapping by FISH technology enabled studies of genome diversity,
evolutionary pathways as well as chromosomal location of specific genes or genes families in
different species (Nagaki et al., 2003; Wei et al., 2003; Ji et al., 2004; Zhang et al., 2004;
Paniego et al., 2007). Recently, a bacterial artificial chromosome (BAC) clone containing
repetitive sequences with similarity to retrotransposons and a homologous rDNA sequence
isolated from the sunflower genome were used for characterize the complete chromosome
complement of sunflower. The BAC clone containing highly represented retroelements
hybridized with all the chromosome complement in FISH, and used together with the rDNA
probe allowed the discrimination of all chromosome pairs of sunflower. The karyotype could
be subdivided into 3 clear-cut groups of 12 metacentric pairs, 1 submetacentric pair, and 4
subtelocentric pairs ( Talia et al., 2010).
In 2013, Feng et al., tried to assign sunflower linkage groups to individual chromosomes,
and integrate the genetic map with the cytogenetic map, by using a previously developed
RFLP linkage map with 232 cDNA probes on 20 linkage groups (some small linkage groups
were not integrated with the others (Jan et al., 1998) and two BAC and binary bacterial
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Carla Filippi, Jeremías Zubrzycki, Verónica Lía et al. 78
artificial chromosome (BIBAC) libraries from the cultivated cv. HA89 (Feng et al., 2006). A
set of linkage group-specific BAC/BIBAC clones (~100 kb in size) was identified from the
libraries using the mapped cDNA derived RFLP markers, and were used as probes to in situ
hybridize to HA89 mitotic chromosomes at metaphase using the BAC-FISH technique. All
sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific
FISH signals (Feng et al., 2013). As chromosome-specific cytogenetic markers, the selected
BAC/BIBAC clones that encompass the 17 linkage groups will be valuable cytogenetic
markers for molecular cytogenetic and genomic research in sunflower, helping to improve our
knowledge regarding localization of agronomically interesting characters.


6. WHOLE GENOME SEQUENCING

Despite the economic importance of various species of the Asteraceae, there is no
reference genome sequence available for the family, which impedes research and
improvement efforts. Because the sunflower genome is really large (3.5 billion bases long
(Baack et al., 2005), and has many repetitive sequences, for several years it has been
considered costly and impractical to sequence. However, the advances in DNA sequencing
technologies are making it more practical and much less expensive to sequence and assemble
(Mardis 2008; Schatz et al., 2010; Kane et al., 2011).
Kane et al (2011) reported a strategy to sequence the whole sunflower genome,
combining whole-genome shotgun sequencing using the Solexa and 454 platforms with the
generation of high-density genetic and physical maps that serve as scaffolds for the linear
assembly of whole-genome shotgun sequences. This hybrid approach combined the benefits
of the two technologies: Solexa brings great depth, but cannot bridge regions of low
complexity, whereas 454 reads can span longer repeats, but their higher cost makes deep
coverage expensive (Dalloul et al., 2010; Schatz et al., 2010). The performance of this
approach was enhanced by the construction of a sequence-based physical map that provides
unique sequence-based markers every 5–6 kb across the genome.
Preliminary analyses indicated that almost 80% of the sunflower genome consists of
repetitive sequences, thus making difficult the ensemble of the reads. However, 76% of
contigs longer than 5 kb could be assigned to either the physical or genetic map or to both,
suggesting that the approach is likely to deliver a highly accurate and contiguous reference
genome for sunflower (Kane et al., 2011).


CONCLUSION

Sunflower is one of the most important oilseed crops, with an annual production
estimated at 32 million metric tons. However, yield is often below the maximum potential due
to economics, management and many biological factors. Moreover, over the last decade
cultivation has been shifting to marginal areas (with lower-fertility soils, drought and drastic
temperature changes), so the genetic progress for oil yield may have been masked by the
decline in agronomic quality of the growing region. The rapid changes that occur in pathogen
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Genetics and Genomics Applied to Sunflower Breeding 79
virulence, their high persistence in soil and their distribution all around the sunflower growing
areas are also contributing to yield losses.
The challenge now is to breed sunflowers adaptable to these marginal environments,
improve resistance to pathogens and, at the same time, increase seed and oil yield.
Significant advances have been made in this area, including the identification of useful
germplasm for many agronomic traits, the discovery of a large number of loci affecting yield
under stress conditions and several genes and QTL implicated in disease resistance.
Furthermore, more integrated and saturated linkage and physical maps are becoming
available. Marker-assisted selection could improve selection methods, but to date, the impact
on variety development has been minimal, due principally to insufficient linkage between
marker and gene/ QTL locus, limited number of markers and polymorphisms among breeding
materials, the interaction between QTL and environmental effects and the high cost of
marker-assisted selection. The emergence of high-throughput genotyping technologies based
on DNA-chips and ultimately on new generation sequencing technologies, along with modern
breeding techniques based on population and quantitative genetics, combined with the
possibility of obtaining the complete sunflower sequenced genome, should allow MAS to
become more widely applicable for sunflower breeding programs. The further step will be to
combine the best conventional and modern molecular approaches to improve sunflower
germplasm, in order to maintain it as an economically important global crop.


ACKNOWLEDGMENTS

This research was supported by ANPCyT/FONCYT, PICT 2011-1365, PID 2007 00073,
INTA-PRR AEBIO 241001 and 245001. CF and JZ are PhD students from CONICET, VL,
RAH and NBP are career members of the Consejo Nacional de Investigaciones Científicas y
Técnicas (CONICET, Argentina) and INTA Researchers, HEH and RAH are INTA
Researchers and Professors at the Faculty of Sciences, University of Buenos Aires (FCEN-
UBA).


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Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 5



SUNFLOWER GENETIC RESOURCES:
INTERSPECIFIC HYBRIDIZATION AND
CYTOGENETICS IN PREBREEDING


Jovanka Atlagić
*
and Sreten Terzić


Institute of Field and Vegetable Crops, (IFVCNS), Novi Sad, Serbia


ABSTRACT

Sunflower (Helianthus annuus L.) belongs to the Helianthus genus which is
composed of 51 species (14 annual and 37 perennial). Lowered genetic variability and
sensitivity towards large number of pathogens on cultivated sunflower, point to wild
relatives as useful breeding material. Practically from the early breeding efforts in Russia
in 1930s, first uses of wild relatives were registered, and it was intensified with
introduction of hybrids since 1970s.
Increased usage led to formation of several major collections of Helianthus species,
starting from the collection in USA. They were since enlarged with new accessions in
collecting expeditions and exchange with other gene banks. The collection at Novi Sad,
Serbia was formed in 1980 with 11 annual and 32 perennial species (over 1000
accessions) and considered as one of the largest collections worldwide. Wild species are
usually kept as seeds in cold chambers, while perennial species can also be kept as living
collections in the field.
Wild species are mostly used in sunflower breeding as a source of desirable genes
for resistance to pathogens, to find Cytoplasmic Male Sterility and Restoring fertility
genes, specific oil quality, traits for new ideotypes and herbicide resistance. The
characterization data significantly increased usability and value of collections by
facilitating their use in breeding programs thus justifying the effort of collection and
maintenance.
The divergence and heterogeneity of the genus cause considerable difficulties, such
as cross-incompatibility, embryo abortiveness, sterility and reduced fertility in
interspecific hybrids. All annual species and a large number of perennial species have

*
Corresponding author: Jovanka Atlagić, Institute of Field and Vegetable Crops, (IFVCNS), Maksima Gorkog 30,
21000 Novi Sad, Serbia, jovanka.atlagic@nsseme.com

sreten.terzic@nsseme.com
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Jovanka Atlagić and Sreten Terzić 96
been crossed with the cultivated sunflower using the conventional hybridization method.
Other methods like somatic hybridization, "in vitro" embryo culture and chromosome
doubling are less often used but can be helpful for more difficult cross combinations. An
example is the interspecific program at IFVCNS which resulted in crosses with 7 annual
species (F
1
and BC
1
F
1
- BC
4
F
1
) and 14 perennial species (F
1
- BC
2
F
1
), some of which
were included in the breeding program. The success of interspecific hybridization is
evaluated based on morphological observations, cytogenetic analysis, as well as
molecular markers.
Cytogenetic studies are used for determinations of chromosome number and
structure and analysis of meiosis-microsporogenesis and pollen viability. Such studies
made it possible to establish phylogenetic relations between wild sunflower species and
the cultivated sunflower and enabled the use of the former in sunflower breeding.
The experience gathered over such a long period of sunflower prebreeding point to
difficulties in wild sunflower collection maintenance, interspecific hybridization and
isolation of desirable genes. None the less Helianthus genus has become a model genus
for studding speciation and evolution while still being a constant source of material for
improvement of cultivated sunflower.

Key words: Cultivated sunflower, wild species, desirable traits, hybridization, cytogenetics


INTRODUCTION

Sunflower breeding has reached a plateau for a number of important agronomic traits.
The major limiting factor for further improvements of the genetic potentials for seed yield and
oil quality is the susceptibility of the sunflower to a large number of pathogens. Studies in the
field of population genetics have shown that the genetic variability of the cultivated sunflower
has been drastically narrowed.
Molecular data on the diversity of the cultivated sunflower is indicating a narrow genetic
base, and thus a limited possibility for further evolution of this economically important crop
(Rieseberg and Seiler, 1990). On the other hand, molecular studies have indicated the
presence of large variability, i.e., "primitive polymorphism", in both wild species and local
populations of sunflower.
Sunflower germplasm resources can be categorized as in situ resources (i.e., wild
population and landraces) or ex situ resources (accessions preserved in gene banks).
The term 'interspecific hybridization' implies the crossing between different species of the
same genus. This method is frequently used in plant breeding, especially when variability of a
cultivated form (primary gene pool) has been exhausted and it became necessary to search for
desirable genes in its wild relatives (secondary and tertiary gene pools). This has been the
case with the cultivated sunflower (Helianthus annuus var. macrocarpus (DC.) Ckll.) which
has been crossed with wild sunflowers (Helianthus spp.). The classical hybridization method
is typically used for that purpose, while "in vitro" embryo culture and somatic hybridization
are less frequent. Interspecific hybridization is typically used for transferring resistance to
disease agents, soil salinity and acidity, and drought as well as for finding new sources of
cytoplasmic male-sterility (CMS) and fertility restoration (Rf) genes and the development of
new sunflower ideotypes.
The development and application of sunflower cytogenetics have been associated with
utilization of the Helianthus germplasm for improvement of the cultivated sunflower genome
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Sunflower Genetic Resources 97
and the use of Helianthus genus as a model for evolutionary studies. It has progressed from
cytology, via cytotaxonomy and classical cytogenetics to molecular cytogenetic studies.
This chapter reviews systematics and taxonomy of the genus Helianthus, its genomic
structure and the usefulness of wild Helianthus species as a source of desirable genes.
Cytogenetic studies on sunflower are reviewed through the analyses of chromosome number
and appearance (karyotype), meiosis (microsporogenesis), pollen viability and CMS.
The experience gathered over a long period of sunflower pre breeding point to difficulties
in wild sunflower collection maintenance, followed by cross incompatibility, lowered fertility
or complete sterility of interspecific hybrids, difficult isolation of unwanted genes and loss of
desirable genes through backcrossing.


GENETIC RESOURCES

Sunflower Origin and Dispersion

It is believed that sunflower was the first cultivated plant in North America where Native
Americans used its seeds for food. Sunflower can adapt to different habitats, including
variable vegetation length of more than seven months in Mexico, to four months in Canada.
The first written document about sunflower in Europe comes from 1567. Initially used as a
decorative plant that impressed by its size, sunflower quickly spread across Europe, it was
recognized as an oilseed crop in Russia only. Since 1880s Russian varieties could be found on
the American market. In a short period, sunflower rose from tenth (1930) to second (1970) on
the list of oil crops, behind soybeans. The percentage of oil content from about 28% (1920)
was raised to nearly 50% (1955), but the disease problem persisted (Heiser, 1976).
The first recorded attempt of interspecific hybridization between cultivated sunflower and
wild species to increase disease resistance was made in 1915. Because of the observed
interspecies compatibility in the genus Helianthus and sources of resistance to pathogens in
wild species, the use of interspecific hybridization is considered the most effective method to
compete with the new races of pathogens and maintenance of sunflower as an important oil
crop.


Systematics and Taxonomy of the Genus Helianthus

The sunflower belongs to a large and polymorphic genus Helianthus, Asteraceae family.
In the course of the 18th and 19th centuries, a number of authors had described more than 200
species from this genus. Sunflower systematics and taxonomy have been subject to continual
changes and amendments. Heiser et al. (1969) described 66 species, 48 from North America
and 18 from South America. The former group comprises 11 annual and 37 perennial species
classified into 3 sections and 7 series. Robinson (1979) reclassified the latter group into a new
genus named Helianthopsis. The North American group of the genus Helianthus (Heiser et
al., 1969) has been reconstructed following analyses of 42 morphological traits (Schilling and
Heiser, 1981). Using the biosystematics and cluster methods, 49 species were classified into 4
sections and 6 series. Section Helianthus covers 11 annual species including the cultivated
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Jovanka Atlagić and Sreten Terzić 98
sunflower. Section Agrestis includes one annual species. Section Ciliares includes two series,
Ciliares and Pumili, each containing three perennial species from North America. Section
Corona solis, Microcephali, Atrorubens and Angustifolius (Schilling and Heiser, 1981).
The classification of Schilling and Heiser (1981) is presented herein with six
modifications as described by Seiler and Gulya (2004). First, the sectional name Atrorubens
used by Anashchenko (1974) has taxonomic priority, thus the section Divaricati E. Schilling
and Heiser is replaced by section Atrorubens Anashchenko. Second, H. exilis is recognized as
a species, as opposed to an ecotype of H. bolanderi due to information which has shown it to
be morphologically and genetically distinct. Third, the species name H. pauciflorus has
priority over H. rigidus and is treated accordingly herein. Fourth, Viguiera porteri has been
transferred to H. porteri. Fifth, H. verticillatus has been rediscovered and redescribed and is
now recognized as a species. Sixth, H. niveus ssp. canescens has been transferred to H.
petiolaris ssp. canescens. This brings the number of species to 51, with 14 annual and 37
perennial (Tables 1 and 2).

Table 1. Infrageneric classification of annual Helianthus species

Section* Species
Common
Name
Chromosome
Number (n)
Helianthus H. annuus L. Prairie 17
H. anomalus Blake Anomalous 17
H. argophyllus T.& G. Silver-leaf 17
H. bolanderi A. Gray Bolander‘s, Serpentine 17
H. debilis
ssp. debilis Nutt. Beach 17
ssp. cucumerifolius (T. & G.) Heiser Cucumber leaf 17
ssp. silvestris Heiser Forest 17
ssp. tardiflorus Heiser Slow-Flowering 17
ssp. vestitus (Watson) Heiser Clothed 17
H. deserticola Heiser Desert 17
H. exilis A. Gray Serpentine 17
H. neglectus Heiser Neglected 17
H. niveus
ssp. niveus (Benth.) Brandegee Snowy 17
ssp. tephrodes (Gray) Heiser Ash-Colored, Dune 17
H. paradoxus Heiser Pecos, Puzzle, Paradox 17
H. petiolaris
ssp. canescens (A. Gray) Gray 17
E. E.Schilling
ssp. fallax Heiser Deceptive 17
ssp. petiolaris Prairie 17
H. praecox
ssp. hirtus Heiser Texas 17
ssp. praecox Englm. & A.Gray Texas 17
ssp. runyonii Heiser Runyon‘s 17
Agrestes H. agrestis Pollard Rural, Southeastern 17
Porteri H. porteri (A. Gray) J. F. Pruski Confederate Daisy, Porter‘s 17
(*Seiler and Gulya, 2004).


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Sunflower Genetic Resources 99
Table 2. Infrageneric classification of perennial Helianthus species

Section* Series Species
Common
Name
Chromosome
Number (n)
Ciliares Ciliares H. arizonensis R. Jackson Arizona 17
H. ciliaris DC. Texas blueweed 34, 51
H. laciniatus A. Gray Alkali 17
Ciliares Pumili H. cusickii A. Gray Cusick‘s 17
H. gracilentus A. Gray Slender 17
H. pumilus Nutt. Dwarfish 17
Atrorubens Corona-solis H. californicus DC. California 51
H. decapetalus L. Ten-petal 17, 34
H. divaricatus L. Divergent 17
H. eggertii Small Eggert‘s 51
H. giganteus L. Giant 17
H. grosseserratus Martens Sawtooth 17
H. hirsutus Raf. Hairy 34
H. maximiliani Schrader Maximilian‘s 17
H. mollis Lam. Soft, Ashy 17
H. nuttallii
ssp. nuttallii T. and G. Nuttall‘s 17
H. nuttallii
ssp. rydbergii (Brit.) Long Rydberg‘s 17
H. resinosus Small Resinous 51
H. salicifolius Dietr. Willow-leaf 17
H. schweinitzii T. and G. Schweinitz‘s 51
H. strumosus L. Swollen, Woodland 34, 51
H. tuberosus L. Jerusalem artichoke 51
Atrorubens Microcephali H. glaucophyllus Smith White-leaf 17
H. laevigatus T. and G. Smooth 34
H. microcephalus T. and G. Small-headed 17
H. smithii Heiser Smith 17, 34
Atrorubens Atrorubentes H. atrorubens L. Purple-disk 17
H. occidentalis
ssp. occidentalis Riddell Few-leaf, Western 17
H. occidentalis
ssp.plantagineus (T. & G.) Heiser Few-leaf, Western 17
H. pauciflorus
ssp. pauciflorus Stiff 51
H. pauciflorus ssp.
subrhomboides (Rydb.) O. Spring Stiff 51
H. silphioides Nutt. Odorous 17
Atrorubens Angustifolii H. angustifolius L. Narrow-leaf, swamp 17
H. carnosus Small Fleshy 17
H. floridanus A. Gray ex Chapman Florida 17
H. heterophyllus Nutt. Variable-leaf 17
H. longifolius Pursh Long-leaf 17
H. radula (Pursh) T. and G. Scraper, Rayless 17
H. simulans E. E. Wats. Muck, Imitative 17
H. verticillatus Small Whorled 17
(*Seiler and Gulya, 2004).

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The taxonomic complexity of the genus Helianthus stems from many different factors.
Natural hybridization and introgression between many of the species resulted in
morphological intergradations between otherwise distinct forms. Polyploidy in the perennial
species also contributed to the complexity of species classification in Helianthus (Jan and
Seiler, 2007).
Detailed descriptions of the species (plant habit, site, geographic distribution, period of
flowering, ploidy level, etc.) were provided by Heiser et al. (1969) and Rogers et al. (1982).
The extent of variability in the genus Helianthus has not been sufficiently studied. Heiser
et al. (1969) reported that subspecies, varieties and forms existed in some Helianthus species.
The taxonomy of Schilling and Heiser (1981) retained subspecies only for some Helianthus
species. This taxonomy is simpler to use but many researchers are baffled by the intraspecific
variability occurring in their collections.
Studies of Reiseberg and Burke (2001), Rieseberg et al. (2002, 2003) and Mandel et al.
(2013) made special contributions to the knowledge of origin and speciation of Helianthus
species. Seiler (1992) concluded that changes in plant habit and distribution of species occur
as consequences of natural adaptation and natural selection in the Helianthus genus.
Gentzbittel et al. (1992) compared their molecular classification of the genus Helianthus with
morphological taxonomy of Schilling and Heiser (1981) and concluded that they are in
agreement.
Study of Miljanović et al. (2000), and Saftić-Panković et al. (2005) showed a large
variability for some taxonomically stable traits in the perennial species H. giganteus and H.
maximiliani, which could even justify the recognition of new infraspecific forms.
Nonetheless, possible existence of natural hybrids and growing the accessions in a common
environment vs. their natural environment must also be considered.


Germplasm Resources Collecting

Having the wild species of Helianthus within the boundaries of the USA has facilitated
collecting sunflower germplasm. The explorations for wild sunflowers in almost 40 years
have resulted in the assemblage of a USDA-ARS collection that is the most complete
collection in the world (Marek et al., 2004). It is presently located at the National Plant
Germplasm System, Plant Introduction Station at Ames, Iowa. Currently, the wild Helianthus
collection contains 2163 accessions, about two-thirds of which are annual species. Continual
collecting of wild sunflower germplasm for preservation in gene banks is critical so that
germplasm may be readily available for research and the breeding community (Seiler and
Gulya, 2004).
Dr. Charles Heiser, Indiana University was one of the early collectors of Helianthus
germplasm (Heiser, 1947). His focus was primarily taxonomy, systematics, and evolution and
speciation of the genus. His early work formed the basis of the current knowledge and
understanding of the Helianthus genus. Later explorations were undertaken in Texas and
Oklahoma in 1963 (Seiler, 1988) where the most represented wild species was H. annuus.
During the 1970s, wild sunflowers were collected throughout the southwestern USA,
resulting with approximately 200 accessions. Most of those were annual species, represented
with wild H. annuus. An exploration for sources of rust resistance was undertaken in 1972 in
the North Central Great Plains and resulted with additional 100 accessions of mostly wild
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Sunflower Genetic Resources 101
annual species. The USDA-ARS formally established a wild Helianthus collection at
Bushland, TX, in 1976. Creation of a permanent collection greatly increased the number of
collecting expeditions for wild Helianthus. During 1976, 200 accessions were collected in
Texas and New Mexico, while in 1978, 175 accessions were added from western,
southwestern, and southeastern USA. Several short explorations were made throughout the
USA in 1979 (Seiler, 1988).
Several expeditions for collecting wild sunflower were undertaken in 1980s and one in
1991 in cooperation of USDA-ARS stations in Bushland, TX and Fargo, ND with Institute of
Field and Vegetable Crops (IFVC), Novi Sad, Yugoslavia (Table 3). In a 1984 exploration to
southern Texas, Gerald Seiler collected 32 accessions of annual H. argophyllus, H. debilis
and H. praecox.
The first collecting expedition outside of the USA was in 1994 in several provinces of
Canada. Sixty-three accessions of wild sunflower were collected. Thirty-one accessions were
annual, while 32 were perennial. Almost 40% of the accessions were H. annuus (Seiler and
Brothers, 1996).

Table 3. Wild sunflower species collecting expeditions including IFVCNS staff
(1980-1991)

Collected by Year No. of state
No. of
species
No. of
accessions
Seiler, G. (USDA-ARS)
Ćuk, L. (YUGIFVC)
1980. 21 (USA) 37 384
Marinković, R. (YUGIFVC)
Miller, J. (USDA-ARS)
1984. 1 (Canada) 7 88
Škorić, D. (YUGIFVC)
Seiler, G. (USDA-ARS)
Rooth, (USDA-ARS)
1985. 12 (USA) 13 88
Seiler, G. (USDA-ARS)
Pomeroy, J. (USDA-ARS)
Marinković, R. (YUGIFVC)
1987. 6 (USA) 7 52
Dozet, B. (YUGIFVC)
Seiler, G. (USDA-ARS)
Pomeroy, (USDA-ARS)
Gavrilova, V. (SUNWIR)
1989. 6 (USA) 12 84
Dozet, B. (YUGIFVC)
Marinković, R. (YUGIFVC)
1990. 1(Montenegro) 1 81
Marinković, R. (YUGIFVC)
Seiler, G. (USDA-ARS)
Stauffer, C. (USDA-ARS)
Duhoon, S. (NBPGR)
1991. 7 (USA) 9 140

Starting from 2000, collecting expeditions were more oriented to specific
underrepresented species and filling the gaps in the current wild species collection. An
exploration to southwestern USA in 2000 was undertaken for annual species H. anomalus and
H. deserticola. Annual serpentine sunflower, H. exilis, was collected in California in 2002. In
September 2003, an exploration was undertaken to California to collect the endemic perennial
species H. californicus, while perennial species H. eggertii, H. schweinitizii, H. verticillatus
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and annual H. porteri were collected in 2003 in the southeastern USA (Seiler and Gulya,
2004).
The collection efforts of the USDA-ARS were supported by US, European and
International organizations involved with plant genetic resources. The obtained support also
provided resources for maintenance so that the germplasm can be distributed. These
accessions have become the basis of wild species research programs in Argentina, Russia,
India, China, Mexico and many European countries. Besides USDA collection at Ames, IA,
notable collections exist at the N.I. Vavilov Research Institute of Plant Industry, St.
Petersburg, Russia, (Gavrilova and Anisimova, 2003) Institute of Field and Vegetable Crops,
Novi Sad, Serbia, (Cuk and Seiler, 1985), Dobroudja Agricultural Institute (DAI) at General
Toshevo, Bulgaria, (Christov et al., 2001) and INRA, Montpellier, France (Serieys, 1992).


The Collection of Wild Species in Novi Sad

The collection of wild sunflower species in Novi Sad was made through 7 collecting
expeditions in the period from 1980-1991., when 917 accessions were collected. The trips
varied in the number of collected species (1-37) and accessions (52-384) (Table 3).
Of the 49 sunflower species in the genus Helianthus, the collection included 43 species.
Section Helianthus was complete, and 6 species were missing from the group of perennials.
Regrettably, 14 species have been lost in the previous period, 4 annuals and 10 perennials.
The reasons for this were in the first place different conditions for growing between the
collection and the original natural habitat (winterkill, long growing season), as well as low
self-fertility and poor viability of seed of the wild species. At present, the collection
comprises 21 perennial and 7 annual species. The total number of accessions in the collection
is 447. Reserve seeds of the accessions of annual species range from several scores to several
thousands (Table 4).

Table 4. The IFVCNS collection of annual wild sunflower species

Species
No. of accessions
No. of seeds
in gene bank date base
with seed
reserves
H.annuus 108 70 10-2539
H.petiolaris 33 25 80-9130
H.neglectus 4 4 1564-4838
H.debilis 21 13 33-5490
H.praecox 15 14 335-10710
H.argophyllus 7 7 1616-10396
H.niveus 4 3 259-5910

The situation is similar with the perennial species, where the discrepancy in the number
of accessions registered in the gene bank and the number of available reserve seed lots
indicates that a certain number of accessions have been lost. From the initial 41 accessions of
H. tuberosus, only 16 remained. In the case of H. grosserratus, 9 accessions remained from
the original 29. The number of perennial accessions maintained in the field exceeds for most
part the number of accessions having seeds in storage (Table 5). It should be mentioned that
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the number of reserve seeds of the perennials varies from one seed to several hundred,
exceptionally several thousands. Compared with the reserve seeds of the annuals, the number
of reserve seeds of the perennials is considerably smaller.
Detailed information about the collected accessions including passport data can be
accessed online on the IFVCNS site: http://www.nsseme.com/about/inc/oilcrops/wild.php

Table 5. The IFVCNS collection of perennial wild sunflower species

Species
No. of accessions
No. of seeds
No. of
accessions in
the field
In gene bank
data base
With seed
reserves
H.tuberosus 41 16 1-650 112
H.rigidus (H.pauciflorus) 13 12 1-400 11
H.mollis 7 5 3-1162 6
H.maximiliani 36 32 1-8630 60
H.divaricatus 10 10 8-680 8
H.decapetalus 8 7 17-622 7
H.grosseserratus 29 9 2-335 31
H.nuttallii 23 22 1-399 15
H.strumosus 20 12 2-354 20
H.laevigatus 7 7 7-91 8
H.glaucophyllus 1 1 38 1
H.giganteus 16 15 1-1800 19
H.eggertii 2 2 1-9 1
H.hirsutus 4 3 3-280 2
H.californicus 1 1 1 1
H.resinosus 2 2 125, 4858 2
H.silphioides 1 1 13 1
H.atrorubens 1 1 2 1
H.microcephalus 2 2 28, 300 2
H.smithii 2 2 13, 90 2
H.salicifolius 1 1 10 1

Low auto fertility in wild species makes the collection maintenance difficult. The method
of inflorescence isolation with paper bags was used at the beginning for seed production, than
the method of transferring pollen from one inflorescence to another (pollen mix), while in the
recent period a method of isolation with cages and the use of solitary bees is applied. Low
seed viability is a problem in collection maintenance, especially in perennial wild sunflower
species. Different methods for germination enhancement were tested and the results showed
that the most efficient was by removing the seed hull and seed coat (Atlagić et al., 2006).
The species in the collection have been measured and observed according to IBPGR
descriptors. The obtained results indicated that extremely high variability for a number of
characteristics existed not only between the species but also among accession within a single
species (Dozet et al., 1993; Atlagić et al., 1999; Miljanović et al., 2000). The morphological
variability found by Miljanović et al. (2000) was confirmed by molecular analysis in the two
taxonomically close species, H. maximiliani and H. giganteus (Saftić - Panković et al., 2005),
which were frequently used in breeding programs. The importance of this high variability was
demonstrated through cross compatibility when crossing accessions of the same species with
cultivated sunflower. Despite the problems in maintaining the collection, the fact that all
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Jovanka Atlagić and Sreten Terzić 104
annual and 14 perennial species have been crossed with cultivated sunflower by the
conventional hybridization method (Atlagić, 2004) should be considered as success. Several
thousand crossings were made as a part of the Novi Sad breeding program, resulting in
successful transfer of desirable characteristics into cultivated sunflower.
According to Atlagić et al. (2012a) F
1
interspecific hybrids derived from hybridization
between perennial species and cultivated sunflower can be classified as genetic resources.
Perennial species reproduce vegetatively (rhizomes, tubers) so that it is relatively easy to
maintain them in ex situ collections in the field. Hybrid plants between perennial species
(H.tuberosus, H.rigidus, H.eggerttii, H.resinosus, H.strumosus, H.laevigatus, H.decapetalus,
H.divericatus and H.salicifolius) and cultivated sunflower keep the ability of vegetative
reproduction in F
1
generation. Feasibility of maintaining and using F
1
interspecific hybrids
was investigated in the collection of wild sunflower species in Novi Sad. Interspecific hybrids
were obtained using conventional crossing method in the period from 1987 to 2005 and are
grown in a quarantine field under the same conditions as wild perennial sunflower species.
The obtained results indicate that it is possible to maintain interspecific hybrids in F
1
generation in the field for 6-24 years after hybridization under the same growing conditions
(clone maintenance) as wild perennial sunflower species.
Considering difficulties in obtaining hybrids between perennial species and cultivated
sunflower, it is very useful to keep the existing hybrids and use them for obtaining further
crossing generations (BC
1
F
1
, BC
2
F
1
...).


Germplasm Evaluation and Use

Application of interspecific hybridization in cultivated sunflower breeding has an
important role in the creation of resistant hybrids to economically important diseases, which
are at present one of the main problems limiting sunflower yield. Sources of resistance are
most often found in perennial wild species. Traits that are usually associated with wild
species are resistance to pathogens and insects, but also quality and quantity of oil and
proteins (Jan and Seiler, 2007).
Considering resistance genes for sunflower pathogens, early reports date from 1970s and
describe the use of annual wild species H. annuus, H. petiolaris and H. praecox as a source of
resistance to Verticilium wilt (Verticilium dahliae Kleb.) (Hoes et al., 1973). Wild perennial
species H. resinosus, H. tuberosus, H. decapetalus, H. grosseserratus, H. nuttallii and H.
pauciflorus were found to be the source of resistance to Sclerotinia head rot (Pustovoit and
Gubin, 1974). Tolerance to Sclerotinia head rot (Sclerotinia sclerotiorum (Bk) De Bary) was
investigated in inbred lines of sunflower created within the program of interspecific
hybridization (Dedić et al., 2008), and crosses were carried out by conventional methods and
in laboratory (Vasić et al., 2002). Sclerotinia mid-stalk rot and root rot was investigated by
Škorić (1987) who found H. resinosus and H. tuberosus to have tolerance.
Annual wild species H. annuus, H. petiolaris and H. praecox are also a source of
resistance genes for downy mildew (Plasmopara halstedii (Farl.) Berl and DeToni) and rust
(Puccinia helianthi Schwein) (Tan et al., 1992; Quresh et al., 1993). We tested the resistance
of wild species to downy mildew and started hybridization program for transferring genes
into cultivated sunflower lines (Terzić et al., 2007).
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One factor that hampers the cultivation of sunflower is a root parasite broomrape
(Orobanche cumana Wallr). Resistance to broomrape has been found in most of the wild
perennial species (Fernandez-Martinez et al., 2000). Species of the Helianthus genus were
tested for resistance to the parasitic flowering and the possibility to transfer resistance genes
into cultivated sunflower (Terzić et al., 2010). Material testing also led to improved methods
of testing (Dedić et al., 2011).
Phoma black stem (Phoma macdonaldii Boerma) resistance was found in perennial
species H. decapetalus, H. eggertii, H. hirsutus, H. resinosus and H. tuberosus (Škorić,
1985). When working on the transfer of resistance to the phomopsis stem canker (Phomopsis
helianthi Munt-Cvet. et al.) embryo rescue technique was used in order to obtain interspecific
hybrids (Dozet et al., 1996) while resistance was found in perennials H. maximiliani, H.
pauciflorus, H. hirsutus, H. resinosus, H. mollis, and H. tuberosus (Škorić, 1985; Dozet,
1990). H. tuberosus was examined for resistance to the Phomopsis stem canker as a trait of
interest for its cultivation, but also as a source of resistance in breeding of cultivated
sunflower (Terzić et al., 2011).
Alternaria leaf spot (Alternaria helianthi (Hansf.) Tubaki and Nishihara) resistance was
observed in perennials H. hirsutus, H. pauciflorus, and H. tuberosus (Morris et al., 1983).
Rhizopus head rot (Rhizopus arrhizus Fischer) resistance was observed in perennials H.
divaricatus, H. hirsutus, H. resinosus, and H. × laetiflorus (Yang et al., 1980). Powdery
mildew (Erysiphe cichoracearum DC. ex. Meret) resistance was observed in annuals H.
debilis subsp. debilis, H. bolanderi, and H. praecox (Saliman et al., 1982; Jan and Chandler,
1985) and perennials H. decapetalus, H.divaricatus, and H. laevigatus (Dedić et al., 2012).
Investigations on insect resistance genes were mostly performed in the US where insect
related yield losses are most abundant. Tolerance to sunflower moth (Homoeosoma electellum
(Hulst)) (Rogers et al., 1984), stem weevil [Cylindrocopturus adspersus (LeConte)] (Rogers
and Seiler, 1985) and sunflower beetle [Zygogramma exclamationis (Fabricius)] (Rogers and
Thompson, 1980) were mostly found in perennial species like H. tuberosus.
Oil content is higher in cultivated sunflower than in the wild species but wild species can
be used for changing the fatty acid composition affecting the oil quality (Seiler, 1998). As
reported by Pustovoit and Krasnokutskaya (1975) protein concentrations ranged from 290 to
350 g/kg in a survey of 39 wild Helianthus species which implies that there is sufficient
variability in the genus for breeding on protein concentration increase.
To reduce the cost of cultivation of commercial hybrids, it is essential to have resistance
to total herbicides such as imazethapyr and imazamox (Miller and Al-Khatib, 2002) or
sulfonylurea (tribenuron) (Miller and Al-Khatib, 2004; Terzić and Atlagić, 2008).
The accumulation of evaluation data increases the value of germplasm collections and
makes it possible for researchers and breeders to work more efficiently by selecting the right
material for their program. One example of providing efficient means for identifying useful
genetic traits is the assembly of core collections (Brothers and Miller, 1999). They are made
based on the evaluation and passport data and enable the researchers to sample the available
diversity without testing the whole collection.



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Jovanka Atlagić and Sreten Terzić 106
CYTOGENETICS

Cytogenetic studies in sunflower have a century long tradition. The first study was related
to determination of the number of chromosomes in cultivated sunflower. Karyotype of
species in the genus Helianthus was then made. The number and characteristics of
chromosomes were used to classify species and study phylogenetic relationships in the genus
Helianthus. The use of interspecific hybridization and gene transfer from wild species into
cultivated sunflower required the use of cytogenetic methods. The development of hybrid
sunflower during seventies was accompanied by studies of CMS and Rf whose application
was of great importance in the new breeding programs. Cytogenetic studies followed the
application of in vitro methods of cultivation, especially anther culture, then the study of
fertilization process in terms of separating prior- and post-zygotic incompatibility in
interspecific hybridization and determining cross-compatibility in selected hybrid parental
pairs. In recent years, cytogenetic studies are often combined with the application of
molecular markers.


The Sunflower Genome

Chromosome number in somatic cells of the cultivated sunflower (2n=34) was
determined by Tahara (1915) and confirmed by Wagner (1932), Ševčenko (1936), and
Kostoff (1939). Studying the chromosome number in different Helianthus species, Geisler
(1931) found species with n=17, 34 and 51 chromosomes. This finding was later on
corroborated by Heiser and Smith (1955) and Georgieva-Todorova (1976). The basic
chromosome number in the genus Helianthus is n=17, while the genus is a polyploidy
complex composed of diploid (2n=2x=34), tetraploid (2n=4x=68) and hexaploid
(2n=6x=102) species (Figure 1).


Figure 1. Polyploidy in the genus Helianthus: a) H. annuus, n=34, b) H. hirsutus, n=68 and c) H.
rigidus, n=102. (Atlagić, 2004)
All 14 annual species are diploid; the 37 perennials include 25 diploid, 3 tetraploid, 6
hexaploid and 3 "mixoploid" species. H. ciliaris and H. strumosus occur in the tetraploid and
hexaploid forms, H. decapetalus in diploid and tetraploid forms (Schilling and Heiser, 1981).
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Atlagić et al. (1992) found that the diploid species H. smithii occurs also in the hexaploid
form, while the species H. strumosus occurs in diploid, tetraploid and hexaploid forms.
Most authors used to think that the basic chromosome number (n=17) comprised the
sunflower genome. Hypotheses have been made on the origin of polyploidy, whether it was
auto- or allopolyploidy. Some authors reported finding aneuploids following hybridization.
Although the annual diploids and the perennial diploids have the same chromosome number,
they are either difficult to cross or cannot be crossed at all. Heiser and Smith (1964)
concluded that these two groups of species had different genomes. Georgieva-Todorova
(1976) arrived at a similar conclusion on the basis of an analysis of interspecific hybrids. The
genome of the annual wild species evidently differs from that of the cultivated sunflower.
Analyzing the meiosis in a group of annual diploids and their interspecific hybrids, Chandler
et al. (1986) concluded that the basic chromosome number is not a single genome, i.e., that
the 17 chromosomes do not have the same origin. Thus they confirmed the finding of
Kulshreshtha and Gupta (1979) who proposed that the basic number of chromosomes in the
genus Helianthus had developed secondarily, by hybridization. Which are the original species
that had hybridized in order to give rise to the diploid sunflower species?
This question could not be answered because of the impossibility to mutually cross the
species of the genus Helianthus (cross incompatibility) and the flaws in the cytogenetic
methods (analysis of meiosis, C-bending and ―in situ” hybridization). Great hopes have been
invested in the method of molecular markers. Using the RAPD technique, Sossey-Alaoui et
al. (1998) analyzed 40 Helianthus taxa, 36 identified and 4 non-classified. The analysis
showed that there existed the following genomes:

1 C-genome, common for all species from the three analyzed sections,
2 H-genome, specific for section Helianthus,
3 P-genome, common for perennial species (sections Atrorubens and Ciliares), and
4 A-genome, specific for section Atrorubens.

The genomic constitution was therefore HC for section Helianthus, CPA for section
Atrorubens and CP for section Ciliares. The question remains if it is possible or not to find
RAPD fragments which define the genome. It would also be important, when identifying the
different genomes, to find an effective method of comparison of RAPDs against other
molecular markers.


DNA Content

Because of the different ploidy levels in the different Helianthus species, it was
considered worthwhile to determine their total DNA contents. A study of DNA content in 22
Helianthus species and subspecies indicated that 2C DNA increased continuously from 6.4 pg
in H. neglectus to 12.02 pg in H. angustifolius (Sims and Price, 1985). The highest DNA
contents were found in H. divaricatus and H. agrestis, 16.90 pg and 25.91 pg, respectively.
Such differences are difficult to explain without considering the polyploidization. It has
been noticed that DNA content was more similar among close species than among distant
ones. This was an indication of small intraspecific variation. It was also an indication that
DNA content depended on chromosome size. So, diploid perennials have a higher DNA
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Jovanka Atlagić and Sreten Terzić 108
content than diploid annuals or some diploid species have a higher DNA content than
polyploid ones. It means that the origin of polyploid levels in the Helianthus species cannot
be proved experimentally using DNA content.


Karyotype

Karyotype describes the haploid chromosome set of an organism, i.e., the form and
length of chromosomes, length index of chromosome arms, position of the centromere and
secondary constrictions and the size and position of heterochromatic knots. The analysis of
karyotype is most frequently performed on mitotic metaphase chromosomes. Using the
classical Feulgen technique Klimočkina (1940) performed a detailed karyological analysis of
H. annuus. Based on the position of the centromere, she divided chromosomes into four
groups according to their morphology. Numerous authors have conducted the analysis of
karyotype in sunflower. The nomenclature used is based on the relations between
chromosome arms. This classification distinguishes metacentric, submetacentric,
subtelocentric and telocentric chromosomes in which the ratios longer vs. shorter arm are 1.0-
1.7, 1.7-3.0, 3.0-7.0 and 7.0-?, respectively.
The karyotype of the species H. mollis was analyzed by Georgieva-Todorova et al.
(1974), H. annuus and H. debilis by Raicu et al.(1976), cultivated H. annuus by Al-Allaf and
Godward (1977), H. salicifolius by Georgieva-Todorova and Lakova (1978), H. hirsutus and
H. decapetalus by Georgieva-Todorova and Bohorova (1979), the hybrid H. annuus × H.
hirsutus by Georgieva-Todorova and Bohorova (1980). Finally, Kulshreshtha and Gupta
(1981) analyzed the karyotypes of 12 H. species.
Raicu et al. (1976) found that the total length of the haploid chromosome set of the
cultivated sunflower (the cultivar Record) was 73.82 μm. The lengths of the individual
chromosomes varied from 3.76 to 5.15 μm. The karyotype consisted of 10 metacentrics, 3
submetacentrics and 4 subtelocentrics. Three chromosomes had secondary constrictions and a
large variation of arm ratio, from 1.08 to 5.34. In H. debilis, the length of the haploid
chromosome set was 110.19 μm and the lengths of the individual chromosomes varied from
5.69 to 7.91 μm. Regarding their morphology, two of them were satellite chromosomes, 6
were meta centrics, 7 were submetacentrics and 2 were subtelocentrics (Georgieva-Todorova,
1976). The karyotype of H. mollis differed from those of H. annuus and H. debilis
(Georgieva-Todorova et al., 1974). The chromosomes were short, from 3.16 to 4.50 μm, and
the karyotype formula was 2SAT + 11SM + 4ST. H. salicifolius was similar to H. mollis
(Georgieva-Todorova and Lakova, 1978).
The closely related tetraploid species, H. decapetalus and H. hirsutus, had similar
karyotypes, but the former had somewhat longer chromosomes. Both species had 4 SAT
chromosomes. Georgieva-Todorova and Bohorova (1980) presented the karyotype and
ideogram of the F
1
interspecific hybrid H. annuus (2n=34) × H. hirsutus (2n=68). The
somatic cells of the hybrid contained 51 chromosomes, the karyotype formula was 3
SAT+8M+11SM+4ST, and one chromosome was incomplete. The authors compared the
karyotypes of the parent species with the karyotype of the F
1
hybrid. The total length of the
chromosome set in the hybrid was 131.68 μm, while H. annuus and H. hirsutus had the
lengths of 104.68 μm and 172.03 μm, respectively. It was difficult to identify chromosomes
of the parent species on the basis of the karyotype of the F
1
hybrid. Kulshreshtha and Gupta
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(1981) constructed the karyotypes of 12 Helianthus species. They identified only one SAT
chromosome in each of the diploid H. tuberosus species and three SAT chromosomes in the
hexaploid H. tuberosus.
Karyotypic characteristics of individual Helianthus species are useful for the study of
interspecific relations as well as for the study of evolutionary changes. The relatively small
size and the large number of chromosomes in Helianthus species make it difficult to
distinguish similar chromosomes on the basis of mitotic metaphase alone. Karyotype may be
studied on the basis of meiotic pachytene chromosomes using C- or N- bending techniques.
These techniques have allowed the identification of trisomics in many plant species (corn,
tomato, rice, barley, and others).
Because of the limited possibilities to study karyotype exclusively by cytogenetic
methods, a new direction of study has evolved, called molecular cytogenetic. Such studies
allow us to understand the genomic organization of species with both large and small
genomes. "In situ" hybridization, one of the methods employed in the cytogenetic-molecular
studies, contributes the identification of chromosomes and their arrangement which are
indicators of the evolutionary history of the genome (Heslop - Harrison, 1995). Chromosomal
variability in H. annuus var. macrocarpus was determined on the basis of heterochromatin
distribution, number and position of NORs (Nuclear Organizer Regions) and the number and
location of rDNA sequences using the method of Feulgen staining, C-bending,
fluorochromium staining, silver staining and "in situ" hybridization (Cuellar et al., 1996).
Such complex studies using chromosome markers permit:

 determining whether a species is diploid, tetraploid or hexaploid, i.e., is the basic
chromosome number n=17 maybe of polyploid origin (Chandler et al., 1986);
 determining whether the different Helianthus races are or are not chromosomally
different, i.e., whether they had been obtained from crosses between perennials and
annuals or between diploids and polyploids (Kulshreshtha and Gupta, 1979), and
 gaining cytogenetic information from the analysis of meiotic configurations in
interspecific hybrids. Such information is exceedingly important in interspecific
hybridization.


Meiosis - Reduction Division

Meiosis or reduction division is a process of micro- and macrosporogenesis taking place
in plant stamens and ovaries, respectively. Microsporogenesis involves the development of
pollen grains or male gametes in the anther. Macrosporogenesis involves the development of
embryos or female gametes in the embryo sac.

Microsporogenesis
In Helianthus species, microsporogenesis is studied in immature anthers, most frequently
by the acetocarmine method (Georgieva-Todorova, 1976). Analyzing the meiosis in different
Helianthus species, Atlagić (1989), and Atlagić et al. (2010) made the following
observations:

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1 Leptonema and zygonema, early stages of prophase I, cannot be detected. Although
pachynema occurs frequently in preparations, it is not suitable for analysis because
sunflower chromosomes are thin and long. In diplonema, the chromosomes are short.
Diakinesis is the most suitable stage within prophase I for determination of the
numbers of bivalents, univalents, multivalents as well as chiasmata. Chromosome
configurations may also be determined in metaphase I, when chromosomes are
aligned along the equatorial plane. Anaphase I and telophase I occur frequently in
preparations. Meiosis II, metaphase II and anaphase II can seldom be detected in the
cultivated sunflower and almost never in the wild species, while telophase II is
frequent in both. After telophase II, tetrads are most frequently formed, although
diads, triads and pentads can be seen. Microspores are further divided mitotically,
giving rise to pollen grains (Figure 2).


Figure 2. Phases of meiosis (normal) a) Pachyten; b) Diakinesis; c) Metaphase I; d) Anaphase I;
e) Telophase I; f) Metephase II; g) Telophase II; h) Tetrads; i) Mononuclear microspores; j,k)
Microspores, l) Pollen grains. (Atlagić et al., 2010)
2 Each sunflower head contains a large number of disk flowers which differ in age
from the periphery to the center of the head. This is why a bud contains all phases of
meiosis - microsporogenesis. Each disk flower has 5 anthers, which allows one to
find all meiotic phases in a single preparation. In sunflower, the meiotic division is
asynchronous, i.e., karyokines is not followed by cytokinesis.

Georgieva-Todorova (1976) made a detailed analysis of meiosis in the cultivated
sunflower. At diakinesis, she observed 17 bivalents. The numbers of chiasmata per cell and
per bivalent were 23.88 and 14, respectively. The number of ring bivalents ranged between 3
and 10. Meiotic abnormalities were observed even in normal fertile plants, but in less than 5%
of meiocytes. These occurred as a result of spontaneous interruptions and changes.
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The analyses of meiosis – microsporogenesis in interspecific hybrids (F
1
, BC
1
) are
important for determination of phylogenetic relations among Helianthus species (Figure 3).
The analysis of meiosis provides valuable data on the following:

 chromosome homology and translocations (configurations at diakinesis);
 changes in genetic material (number of chiasmata);
 unpaired chromosomes (univalents);
 non-included chromosomes (fast and lagging chromosomes);
 inversions (chromosome bridges and fragments).


Figure 3. Phases of meiosis (irregular) a) Diakinesis with quadrivalent and hexavalent.; b) Metaphase I
with fast chromosomes; c) Anaphase I with lagging chromosomes; d) Anaphase I with chromosome
bridge; e) Telophase II with lagging chromosomes; f) Telophase II with chromosome bridges. (Atlagić
et al., 2010)
Pollen Viability
Mature pollen grains are yellow-orange, spherical, covered with spines (echinate), and
have three apertures. Whelan (1978) screened sunflower pollen grains by electron
microscopy. In polar view, the grains show three equidistant colpi in the wall. The equatorial
view shows that each colpus extends almost from pole to pole, with an aperture near the
middle. The germinating pollen tube emerges from one of these apertures. The diameter of
the body of the pollen grain, without the spines, varies from 33 to 39 μ. The abortive pollen
grain is smaller and it has an increased number of spines. Gundaev (1971) reported that the
diameter of sunflower pollen grains varied from 30 to 45 μ.
Preparations that are used for pollen viability determination can also be used to analyze
other pollen grain traits (size–length, width; pore number) using light microscope Axiovert
40C (20x/0.30Ph1) Carl Zeiss Jena. Microphotographs were obtained using a digital camera
Canon Power shot A80, and the image analysis software AxioVision LE; Rel.4.3. Pollen
grain size of cultivated sunflower was in the range of 29-34µm and 25-34µm in wild
sunflower species (Atlagić et al., 2012b).
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Pollen viability is an important biological trait. It is typically assessed by staining
methods (Georgieva-Todorova, 1976; Alexander, 1969; Atlagić et al., 2012b) (Figure 4).


Figure 4. Sunflower pollen grain staining using Alexander method (Fertile-red and sterile-green pollen).
(Atlagić, 2004)
Pollen grain germination and pollen tube growth in sunflower are analyzed by fluorescent
microscopy (Xanthopoulos, 1991; Atlagić et al., 2012b) (Figure 5).


Figure 5. a. Pollen grain germination on stigma (light microscope); b. Pollen germination on stigma
(fluorescent microscope); c. Pollen tube growth through the style (fluorescent microscope). (Atlagić et
al., 2012b)

Male Sterility

The sunflower is known to possess two types of male sterility, nuclear (NMS) and
cytoplasmic (CMS). Generally, nuclear male sterility results from the action of individual
recessive gene pairs. The number of genes determining this trait differs (ms1 to ms5,
Vranceanu, 1970; ms6 to ms9, Jan, 1992). The cytology of NMS lines has not been described
in too much detail. Paun (1974) examined the NMS lines AS-110 and AS-116, which
contained the ms1 gene and which behaved similarly. He found that 2-4 univalents occur in
diplonema-diakinesis as a consequence to asynapsis or desynapsis. The author noted unequal
segregation, equatorial division and the elimination of univalents in subsequent phases.
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Chromosome clumping and agglutination were observed together with chromosome bridges
and fragments. The tetrad stage was mostly abnormal and it contained micronuclei. During
flowering, 4-6% of the pollen grains were stained with acetocarmine, but only 75% of these
had normal size and they lacked the spiny exine characteristic for Helianthus species. A
question arises as to the viability of these 75% pollen grains. Numerous authors (Nakashima
and Hosokawa, 1974; Pirev, 1968; Georgieva-Todorova, 1974) that have studied NMS found
that meiosis was normal until the tetrad stage, degenerations starting to occur at the stages of
microspores or pollen grains.
Leclercq (1969) discovered the first source of CMS in H. petiolaris ssp. petiolaris. This
discovery, together with the discovery of fertility restoration genes (Enns et al., 1970;
Kinman, 1970; Vranceanu and Stoenescu, 1971), made possible the development of hybrid
sunflower. Numerous CMS sources have been discovered in programs of crossing between
wild Helianthus species and the cultivated sunflower. Initially, the FAO list had registered 26
CMS sources (Serieys, 1991). Jan (1997) reviewed 38 CMS sources, which had been
mentioned in publications released in the period 1972-1994. These sources typically belonged
to the annual species H. annuus, H. petiolaris and H. argophyllus. Serieys (2002) reported
that the most recent FAO list included 70 CMS sources. The list specified the origin,
collection number of donor and author of the source. Of these 70 CMS sources, 62 were
derived from annual species in the section Helianthus (38 from H. annuus, 24 from other
annual species), and 8 were derived from perennial species in the section Atrorubens.
Restorer genes have been found for most of these CMS sources. CMS in the sunflower is
most frequently alloplasmatic. This term was coined by Pearson (1981) who used it to
describe male sterility resulting from interspecific and intergeneric crosses. He believed that
alloplasmatic CMS was a result of incompatibility between the nucleus and cytoplasm.
Considering the origin of CMS and the expression of this trait after incorporation into
different sunflower genotypes, cytogenetic studies in this field are both interesting and
valuable for breeding. Paun (1974) studied Leclercq's CMS. He analyzed meiosis in 4 sterile
lines and their fertile analogues. Meiosis was normal in the fertile lines, while degeneration of
sporogenous tissue occurred in the sterile lines. Degeneration occurred in pre-meiotic stages
in two sterile lines and after tetrad stage in the other two lines. The author hypothesized that
the degeneration was due to enzymatic reactions which inactivated the mechanisms for pollen
development. Studying microsporogenesis in sunflower male sterile lines, Rjabota (1969)
registered the occurrence of chromosome bridges in anaphase I and degenerative changes in
the post-meiotic cycle, i.e., in uninuclear microspore, binuclear microspore and pollen grain
stages.
Whelan and Dedio (1980) substituted H. annuus nuclei into H. petiolaris cytoplasm.
Applying a series of backcrossing, they obtained progenies whose anthers were either empty
or contained non-functional pollen. "In vitro" testing of pollen fertility in 23 BC
5
plants
showed that complete male sterility existed in 14 plants. Female fertility remained intact, as
demonstrated by normal seed set. Meiosis was normal in the BC
5
plants. Meiotic
abnormalities observed in the F
1
interspecific hybrids (univalents, chromosome bridges and
fragments) were eliminated by backcrossing, resulting in the BC
5
generation being
cytoplasmic male sterile.
Similar cytogenetic results were found when substituting H. annuus into H. maximiliani
cytoplasm (Whelan and Dorell, 1980). Meiotic abnormalities (multivalents and chromosome
bridges) were observed in early generations of crossing. The F
1
generation was observed to
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Jovanka Atlagić and Sreten Terzić 114
contain aneuploids (trisomic plants) which, together with the plants with modified anthers,
were the most frequent CMS sources. A high percentage of abnormalities in the interspecific
hybrid H. giganteus × H. annuus suggested that the parents differed in genomic structure,
although they had the same number of chromosomes (Whelan, 1978).
Based on the analysis of meiosis in this F
1
interspecific hybrid, Whelan concluded that H.
giganteus differed from H. annuus in three translocations and one paracentric inversion.
Whelan concluded that the consequence of these changes in chromosome structure was the
occurrence of sterile plants. Backcrossing eliminated abnormalities, but sterility remained.
Whelan (1980) inferred that nuclear and cytoplasmatic factors are intermingled in early
generations of interspecific crossing. Since meiotic abnormalities (nuclear factors) are
eliminated by backcrossing, the male sterility remaining after BC
4
is inevitably cytoplasmic
which produces normal fertile progeny when crossed with restorers.
Using light and electron microscopy, Horner (1977) compared microsporogenesis in a
fertile line HA232 to its sterile analogue which contained CMS-PET-1. He analyzed anthers,
sporogenous tissue and chromosomes. He divided microsporogenesis into 11 stages, stages 1
to 4 ranging from the first sporogenous tissue to the initiation of tetrads, and stages 5 to 11
from late tetrad to mature pollen. Sterile and fertile analogues did not differ in
microsporogenesis until stage 5. The elongation and degeneration of tapetal cells at the end of
stage 5 caused degeneration of microspores in the tetrads, which ultimately resulted in
sterility. Similar results were obtained by Vlčkova and Kovačik (1981) and Szabo et al.
(1984). Atlagić et al. (1996) studied the stability of 5 CMS sources (PET-1, PET-2, MAX-1,
GIG-1, ANN-6) during substitution into the inbred line HA89, and the stability of 5 CMS
sources (PET-1, PET-2, ANN-5, ANN-44, ANN-164) during substitution into the inbreeds L-
1, L-98, L-74 and L-22. It was found that the anthers differed in development pattern from
normal to rudimentary. Microsporogenesis developed normally until the tetrad stage in most
of the cases. Some anthers contained deformed and sterile pollen grains. The authors
concluded that the sources GIG-1 and PET-2 were unstable - their pollen viability was
10.42% and 1% to 63.43%, respectively). Atlagić and Marinković (1998) conducted a
cytogenetic study on potential CMS sources (interspecific hybrids with 6 H. annuus
accessions and one H. petiolaris accession). All plants in the BC
1
F
1
generation were male
sterile. Differences existed in the stage of anther development and related meiotic phases.


APPLICATION OF WILD SPECIES IN SUNFLOWER BREEDING

Wild species were first crossed with cultivated sunflower in a classical fashion by
moving pollen from male to female parent, until the embryo culture method (Chandler and
Beard, 1983) was described which facilitated obtaining more difficult cross combinations
besides other techniques like chromosome doubling (Jan and Chandler, 1989). Wild H.
annuus was also used as an intermediate parent to produce hybrids with H. giganteus and H.
maximiliani by Whelan (1978).



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Diploid Species

The annual wild species were studied in considerable detail by Chandler et al. (1986).
They found that all annuals were crossable both mutually and with the cultivated sunflower.
However, they frequently had to resort to embryo culture. The analyses of meiosis and pollen
viability, which included all annuals and their F
1
interspecific hybrids, showed that the
annuals differed in 0 to 6 translocations and 0 to 8 paracentric inversions. This was a further
proof that the basic chromosome number (n=17) is not a single genome.
Annual species are phylogenetically closer to cultivated sunflower so that they can be
used in interspecific programs without larger effort. Species H.annuus, H.argophyllus,
H.petiolaris, H.praecox (Figure 6), H.debilis, H.neglectus and H.niveus were successfully
crossed with the cultivated sunflower lines (Atlagić, 1986, 1990; Škorić et al.; 1988, Terzić,
2006). The obtained interspecific hybrids of different cross generations (F
1
, BC
1
F
1
– BC
4
F
1
)
were most often used as sources of CMS and Rf genes.


Figure 6. Wild diploid sunflower species H.praecox (2n=2x=34) and their F
1
interspecific hybrid with
cultivated sunflower. (Atlagić, 1986)
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Meiotic abnormalities and reduced pollen viability in F
1
hybrids between wild annual
species and the cultivated sunflower have been reported by Heiser (1947, 1961), Heiser et al.
(1969), Georgieva-Todorova (1976, 1990), Whelan (1979), Atlagić (1988, 1990), and Terzić
(2006). One of the truly useful interspecific hybrids was made by Leclercq (1969) between H.
petiolaris and the cultivated sunflower. This was the first stable source of CMS (PET1) which
is still used exclusively for the development of commercial hybrids. Study of wild annuals
has lagged in recent years. Wild annuals are seldom used in inferspecific hybridization
programs because they are as sensitive to major diseases as the cultivated sunflower.


Figure 7. Wild diploid sunflower species H.salicifolius (2n=2x=34) and their F
1
interspecific hybrid
with cultivated sunflower. (Atlagić, 2004)
Wild perennial sunflowers of various ploidy levels have been mutually crossed mostly for
the purpose of cytotaxonomy (Heiser and Smith, 1955; Jackson, 1963; Heiser et al., 1969;
etc.). Wild perennials were also crossed with the cultivated sunflower (Christov, 1991), but
these interspecific hybrids were seldom subject to cytogenetic analysis. Diploid perennials are
interesting for breeders as potential sources of resistance to diseases (H. giganteus, H.
maximiliani), high oil content in seed (H. salicifolius) (Figure 7) or development of a new
ideotype (H. mollis). Crossability between diploid perennials and the cultivated sunflower is
poor, as demonstrated in studies of Georgieva-Todorova (1976, 1990), Jan (1987), Atlagić
(1994a), Atlagić et al. (1995), etc.
Using the embryo culture method, Kräuter et al. (1991) succeeded in obtaining hybrids
between H. mollis and H. maximiliani on one hand and the cultivated sunflower on the other.
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Sunflower Genetic Resources 117
Cytogenetic analyses of F
1
interspecific hybrids (Georgieva-Todorova, 1967, 1976, 1990;
Whelan, 1978; Atlagić et al., 1995) detected numerous meiotic abnormalities (uni- and
quadrivalents in diakinesis, dislocated chromosomes in meta-, ana- and telophases, and
chromosome bridges in anaphase I). Also, these authors found complete sterility or reduced
pollen viability in F
1
hybrids. These results indicated that the genomes of diploid perennials
and diploid annuals differ (Heiser and Smith, 1964), which limits the use of the former in
breeding programs.


Tetraploid Species

The analyses of meiosis and pollen viability in the tetraploid species has raised the
question of the origin of polyploidy in the sunflower. Georgieva-Todorova and Bohorova
(1979) and Georgieva-Todorova (1990) reported that meiotic abnormalities and reduced
pollen viability in tetraploid species H. hirsutus, H. decapetalus, H. strumosus and H.
scaberimus reflect their allopolyploid nature. On the other hand, Atlagić (1991) reported that
these species had regular meiosis, which suggests autopolyploidy.
Hybridization between tetraploids and the cultivated sunflower was observed only in a
few cases, by Heiser et al. (1962), Georgieva-Todorova et al. (1979), Pustovoit (1975),
Christov (1991) and Atlagić (1994b). Georgieva-Todorova et al. (1979), Georgieva-Todorova
(1984), Atlagić (1994b), and Terzić (2006) have successfully crossed the tetraploid species H.
hirsutus (Figure 8), H. decapetalus, H. laevigatus and H. strumosus with the cultivated
sunflower and conducted cytogenetic analyses of the hybrids.


Figure 8. Wild tetraploid sunflower species H.hirsutus (2n=4x=68) and their F
1
interspecific hybrid
with cultivated sunflower. (Atlagić, 2004)
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Jovanka Atlagić and Sreten Terzić 118
The results of these analyses showed an exceedingly high percentage of meiotic
abnormalities and a frequent occurrence of complete sterility. Obviously, it is difficult to
transfer desirable genes from these species into the cultivated sunflower. In order to make
these crosses, conventional hybridization methods have to be combined with the embryo
rescue method (Kräuter et al., 1991) and chromosome doubling in the F
1
and BC
1

interspecific hybrids (Jan, 1988).


Hexaploid Species

The species from the hexaploid group most frequently used in sunflower breeding are H.
tuberosus (a source of resistance genes to Phomopsis stem canker, Alternaria leaf spot and
downy mildew), H. pauciflorus (H. rigidus) (resistance to disease agents and high protein
content in seed) and H. resinosus (resistance to disease agents and high content of oleic acid
in seed). These species have undergone extensive cytogenetic studies by a number of
researchers. Kostoff (1934) was the first to conduct a detailed analysis of H. tuberosus and he
established two hypotheses on the genomic structure of the species. First, H. tuberosus is
autohexaploid (AAAAAA); and second, H. tuberosus is amphiploid (AABBCC) made from a
cross of an autotetraploid and a diploid form. In 1939, the same author conducted a study of a
hybrid between H. tuberosus and the cultivated sunflower, which confirmed his hypothesis on
the different genomes in these two species.
Clevenger and Heiser (1963) claimed on the basis of cytogenetic analyses that H.
tuberosus is a natural hybrid but the results of Georgieva-Todorova (1990) and Atlagić et al.
(1993) indicated that it is an original species. Many researchers studied the meiosis and pollen
viability in F
1
hybrids between H. tuberosus and the cultivated sunflower (Kostoff, 1939;
Heiser and Smith, 1964; Cauderon, 1965; Heiser et al., 1969; Pustovoit, 1969; Georgieva-
Todorova, 1990; Atlagić et al., 1993; Espinasse et al., 1995; Terzić, 2006). Their results
invariably showed that the complete sterility and reduced fertility in these interspecific
hybrids were due to a large number of meiotic abnormalities occurring as a consequence of
the differences in chromosome number and structure between the parent species.


Figure 9. Wild hexaploid sunflower species H.rigidus (2n=6x=102) and their F
1
, F
1
BC
1
interspecific
hybrids with cultivated sunflower. (Atlagić, 2004)
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Sunflower Genetic Resources 119
Table 6. The potential for interspecific hybridization of wild and cultivated sunflower

Species
Chrom
No.
1981 – 1991 year. 1992 - 2006 year.
Accession No. F
1
gen. Accession No. F
1
gen.
Polli-
nated
Crossed
No.
of plants
Polli-
nated
Crossed
No. of
plants
H.annuus 17 24 17 123 28 11 169
H.petiolaris 17 22 20 35 23 3 7
H.argophyllus 17 8 6 24 9 2 30
H.neglectus 17 2 2 14 4 1 12
H.debilis 17 12 9 32 13 1 3
H.praecox 17 11 9 24 19 4 0
H.niveus 17 2 1 8 3 0 0
H.mollis 17 4 3 10 7 2 0
H.salicifolius 17 2 1 7 7 2 0
H.maximiliani 17 7 3 10 31 5 0
H.occidentalis 17 3 1 8 0 0 0
H.nuttallii 17 3 1 9 24 1 0
H.smithii 17 2 2 27 0 0 0
H.decapetalus 17, 34 5 1 28 10 1 0
H.hirsutus 34 3 2 66 3 1 0
H.strumosus 34, 51 8 1 13 14 5 24
H.laevigatus 34 5 3 51 3 1 4
H.tuberosus 51 17 9 90 11 4 27
H.rigidus 51 7 3 105 11 4 0
H.eggertii 51 1 1 5 1 1 0
H.resinosus 51 2 2 89 3 1 0
H.divaricatus 17 6 1 1 13 2 3
H.giganteus 17 6 1 0 12 0 0
H.grosseserratus 17 3 0 0 16 0 0
H.microcephalus 17 2 0 0 1 0 0

H. pauciflorus (H. rigidus) is another wild species extensively used in sunflower breeding
programs (Figure 9). The species itself has not been extensively studied (Atlagić, 1996a), but
F
1
interspecific hybrids between H. rigidus and the cultivated sunflower were studied by
Whelan (1978), Georgieva-Todorova (1990) and Atlagić (1996a). All of these studies showed
irregularities in chromosome pairing and diakinesis. Of all hexaploid species, Georgieva-
Todorova (1990) found H. resinosus to be most similar to the cultivated sunflower. Within
the scope of cytogenetic analyses of interspecific hybrids, the analyses of meiosis and pollen
viability were studied by the largest number of researchers. In addition to crossability,
sterility and reduced fertility in interspecific hybrids are most indicative of the applicability of
these hybrids in sunflower breeding programs. Based on her long-term studies, Georgieva-
Todorova (1984, 1990) concluded that pollen viability is invariably associated with meiosis
as well as that it is genetically controlled. Conversely, Chandler et al. (1986) came to a
conclusion that pollen viability is invariably affected by the number and type of meiotic
abnormalities, but these effects do not necessarily have to be direct. Cytogenetic studies have
mostly been done on F
1
interspecific hybrids. However, analyses of BC
1
F
1
hybrids showed
even larger percentages of meiotic abnormalities, as well as the occurrence of aneuploids,
plants with different chromosome numbers, reduced pollen viability, etc. (Whelan, 1979;
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Jovanka Atlagić and Sreten Terzić 120
Whelan and Dorrell, 1980; Atlagić, 1996b; Atlagić and Škorić, 1999). Although Whelan
(1979) and Whelan and Dorrell (1980) claimed that backcrossing eliminates meiotic
abnormalities observed in F
1
interspecific hybrids, cytogenetic analyses of BC hybrids have
shown that the elimination takes place in later generations of backcrossing. Defining
problems associated with the use of wild Helianthus species in sunflower breeding programs,
Atlagić and Škorić (2000) pointed out that phylogenetic differences among species are as
important if not more important than differences in ploidy level.
Recent studies of interspecific hybridization in sunflower have included various aspects
of occurrence of partial hybrids in wide crosses between sunflower (H. annuus) and perennial
species (H. mollis and H. orgyalis) (Faure et al., 2002a, 2002b, 2002c).
The hybridization potential presented in table 6. shows that majority of species apropos
accessions, was crossed in the period between 1981 and 1991. That is a result of more
intensive work on interspecific program, because larger number of crosses was made than in
the following period. Similar to the Novi Sad program, large number of crosses between wild
species and the cultivated sunflower was made and the potential of interspecific hybridization
usage in sunflower breeding was described by Georgieva-Todorova, 1990, Christov, 1991,
Jan, 1997.


CONCLUSION

Based on literature data and the results of our own studies, it became clear that the
method of interspecific hybridization is extensively used in sunflower breeding programs.
Obstacles that precede interspecific hybridization are also in the forming and
maintenance of a wild species collection and making accessions available to breeders. The
most frequent barriers for interspecific hybridization application are: cross incompatibility
(prezygotic and postzygotic - embryo abortivity), lowered fertility or complete sterility of F
1

and other early generations of interspecific hybridization. Differences in ploidy, phylogenetic
origin and taxonomic belonging of the wild species in comparison to the cultivated sunflower
are the cause of the mentioned barriers.
Interspecific hybridization brings not only desirable, but also a large number of
undesirable traits (branching, small head diameter, low oil content, etc.) and that is why it is
necessary to back cross F
1
interspecific hybrids with cultivated sunflower. Cytogenetic
analysis of BCF
1
hybrids have shown large percentage of irregularities in meiosis, aneuploids,
plants with different chromosome number, lowered pollen viability. On the other hand,
desirable genes are lost after several back crosses with cultivated sunflower. That is why it is
necessary to analyze not only on cytogenetic, but also on molecular level for the presence of
wild species genome in comparison to the cultivated sunflower genome in interspecific
hybrids.
Application of wild species in cultivated sunflower breeding is the most significant in
creating resistant hybrids to economically important diseases like Phomopsis stem canker and
Sclerotinia rot and root parasite broomrape. Perennial wild species were most often the source
of resistance. Nevertheless, annual species like H.annuus and H.petiolaris provided fertility
restoration genes for CMS (PET-1) and some new sources of CMS.
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Sunflower Genetic Resources 121
Despite the difficulties in wild sunflower collection maintenance, interspecific
hybridization and isolation of desirable genes, Helianthus genus remained a constant source
of material for improvement of cultivated sunflower. Greater efficiency in wild sunflower
germplasm usage depends on successful application of modern techniques like marker-
assisted selection and a growing database on gene function and location, while having secured
germplasm collections.


REMARKS

The authors note that in the writing of the chapter they used most of the text from a
review paper ―Roles of interspecific hybridization and cytogenetic studies in sunflower
breeding‖ by Atlagić J. published in the Helia journal in 2004.


ACKNOWLEDGMENTS

We are grateful to the Ministry of Education, Science and Technological Development of
Republic of Serbia, which has funded projects in which the research was conducted and
produced results partly described in this chapter, including the current project TR31025. The
authors thank the Institute of Field and Vegetable Crops.
The technical assistance of Jasminka Pilipović and Đuro Kovačević is gratefully
acknowledged for the help and enthusiasm to work on wild species maintenance, interspecific
hybridization, cytogenetic analysis and technical assistance with writing.


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Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 6



FUNCTIONAL GENOMICS AND TRANSGENESIS
APPLIED TO SUNFLOWER BREEDING


Sebastian Moschen
1,2,
, Laura M. Radonic
1,*
,
Guillermo F. Ehrenbolger
1,2
, Paula Fernández
1,2,3
,
Verónica Lía
1,2,4
, Norma B. Paniego
1,2
,
Marisa López Bilbao
1
, Ruth A. Heinz
1,2,4
and H. Esteban Hopp
†,1,4

1
Instituto de Biotecnología, Centro de Investigaciones en Ciencias
Agronómicas y Veterinarias, Instituto Nacional de Tecnología
Agropecuaria - INTA, Hurlingham, Buenos Aires, Argentina
2
Consejo Nacional de Investigaciones Científicas y Técnicas,
Ciudad Autónoma de Buenos Aires, Argentina
3
Escuela de Ciencia y Tecnología, Universidad Nacional
de San Martín, San Martín, Buenos Aires, Argentina
4
Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina


ABSTRACT

The advances in genomics and post genomics in the last decade allowed the
discovery and functional characterization of many genes simultaneously on a genome-
wide scale. However, the sunflower genome was not systematically sequenced until the
recent advent of next-generation sequencing technologies and is still in progress. In
parallel, comprehensive EST datasets were developed and used to design oligo-
nucleotide-based microarrays focusing both in genotyping and expression analysis
purposes, which in turn, help to study the transcriptomics response to different growing
conditions as water deficit, senescence or response to pathogens. In addition, first
metabolomics analyses of tolerance to diseases started few years ago. This chapter
reviews the functional genomics analysis of several important sunflower characters
including the development and application of bioinformatic approaches to explore


SM and LMR are equal co-senior authors.

Corresponding author: Professor H. Esteban Hopp; E-mail: ehopp@cnia.inta.gov.ar
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Sebastian Moschen, Laura M. Radonic, Guillermo F. Ehrenbolger et al. 132
massive data derived from high throughput sequencing technologies to elucidate complex
traits and identify candidate genes playing a key role in metabolic pathways with special
emphasis in the generation of new breeding tools. In addition, functional strategies for
candidate gene validation either by TILLING approach, and/or by overexpression in
model and sunflower plants are also reviewed and discussed.
In vitro tissue culture techniques, like immature embryo rescue, were incorporated in
sunflower breeding before the existence of the functional genomics disciplines allowing
the development of four inbreeding generations per year and helping breeders to sort
sterility or incompatibility barriers in wide interspecies crossings. Even so, sunflower was
considered a recalcitrant species for genetic transformation due to difficulties in plant
regeneration procedure. Sunflower transformation protocols have improved in the last
years, but they are still time consuming and produce low numbers of transgenic
regenerants per assay. For some applications, like complementation studies, these
difficulties were solved by transient transformation and the stable transformation of
lettuce, as model system. Still, the introduction of agronomical useful genes successfully
delivered transgenic lines with herbicide tolerance, insect resistance, disease resistance
and improved oil composition. They were developed by seed companies and nearly
reached commercial stage. However, since pollen from cultivated sunflower can spread
to adjacent wild populations, any intended release of transgenic sunflowers requires a
previous analysis of the population biology of wild relatives to assess the potential added
fitness or detrimental effects that agronomic traits might have on the ecosystem. This has
impeded the intended release of commercial transgenic sunflowers until now. However,
as a consequence of the appearance of the non-transgenic imidazolinone resistant
sunflower and its global marketing, many of the concerns about the release of transgenic
sunflowers are rapidly decreasing.

Keywords: Sunflower, transcriptomics, metabolomics, biotic and abiotic stresses,
transgenesis, transient expression, in vitro culture


INTRODUCTION

During the last decade, sunflower crop production has been gradually driven to marginal
areas due to the rapid change of agricultural practices in crops such as soybean and maize,
which have greatly increased their cultivated areas as a consequence of favourable
commodity prices and because farmers found more profitable to sow transgenic crops with
resistance to herbicides and insects.
Difficulties in obtaining transgenic events (Davey and Jan, 2010) as well as the concerns
raised about the environmental risk after the release of these events (Cantamutto and
Poverene, 2007) prevented the appearance of genetically modified sunflowers on the global
marketplace. This situation, together with global climate change and the increase in the
frequency and severity of abiotic stresses that take place in the actual sunflower cultivated
areas, has led researchers to redirect the objectives and widen the tools currently used in
sunflower breeding (Sala et al., 2012). This adverse situation has been partially overcome in
sunflower by contributing to yield maintenance through breeding for herbicide resistance and
tolerance to biotic and abiotic stresses (Paniego et al., 2007, Sala et al., 2012). Different
abiotic stresses such as nitrogen starvation, drought, cold, heat, salinity and high temperature
can reduce crop yields and the genetic potential to increase the production is not reached,
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Functional Genomics and Transgenesis Applied to Sunflower Breeding 133
indicating that the development of cultivars with an increased adaptation to environmental
changing conditions should be undertaken (Boyer, 1982).
Sunflower is described as normally susceptible to low temperatures and salinity (Kratsch
and Wise, 2000, Huang et al., 2005, Maas and Hoffman, 1977), but with a relative tolerance
to drought stress due to its highly explorative root system (Connor and Jones, 1985, Sadras et
al., 1991, Connor et al., 1985). However, available information on gene expression in
response to abiotic stresses in sunflower is still limited to few works.
Biotic stress is considered one of the most important limiting factors affecting sunflower
yield production worldwide. In the last ten years, several attempts have been made through
conventional breeding and molecular biology studies to dissect the bases for fungal resistance
to allow biotechnology assisted selection. Considering that sunflower is cultivated in vast
marginal areas and therefore, the application of fungicides is not always feasible and
unfriendly to the environment, breeding for generation of inbred lines with genetic tolerance
to most common pathogens represents a main goal. Downy mildew (Plasmopara halstedii),
Sclerotinia stalk and head rot (Sclerotinia sclerotiorum), black rust (Puccinia helianthi),
Verticillium wilt (Verticillium dahliae), Alternaria leaf blight (Alternaria helianthi) among
others, appear as the highest impact diseases, since they seriously reduce yield in many
sunflower crop regions (Viranyi, 2008). Therefore, breeding for disease resistance has been,
and will continue to be, essential to strengthen this crop. There exist several sources of
resistance genes in sunflower germplasm, mainly present in the numerous wild sunflower
species, landraces and old open pollinated varieties, some of which have been introgressed
into cultivated elite inbred lines (Seiler, 2010, Gulya et al., 1999, Moreno, 2013) but many
others certainly remain to be discovered, genetically characterized and incorporated in the
high yielding cultivated genetic pool.
The advances in genomics in the last decade allowed the discovery and functional
characterization of many genes in a simultaneous way by use of a genome-wide scale
approach. Genomics tools can improve the rate of genetic gain in breeding programs by either
extending the amount or nature of variation available for selection, or by accelerating the
selection process to produce varieties more rapidly (Langridge and Fleury, 2011). In the case
of sunflower, genomics approaches have already started both in the public and private sectors,
which resulted in the generation of several tools that are likely to change some paradigms of
sunflower breeding (Seiler and Jan, 2010, Kane et al., 2011, Fernandez et al., 2012a, Paniego
et al., 2012). In this chapter we review and discuss different strategies that have been
developed in the last decade from functional genomics and post genomics disciplines to
contribute to the elucidation of gene regulation and identification of key metabolic pathways
involved in the response to biotic and abiotic stresses in sunflower. The state of the art of
strategies for gene function, studies in silico and in planta, induction of genetic diversity and
gene transfer are discussed within the frame of their application in sunflower breeding.





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Sebastian Moschen, Laura M. Radonic, Guillermo F. Ehrenbolger et al. 134
FUNCTIONAL GENOMICS FOR ABIOTIC AND BIOTIC STRESS
RESPONSES IN SUNFLOWER

cDNA Libraries, Differential Display and Candidate Gene Approaches

The first functional genomics studies applied to abiotic stress in sunflower came from
cDNA libraries and differential display analysis.
Ouvrard et al. in 1996 assessed two drought contrasting sunflower lines and identified six
sunflower drought induced genes (sdi) through subtractive hybridization. These genes were
up-regulated by drought in the tolerant as well as in the sensitive line and included non-
specific lipid transfer proteins (nsLTP), early light-induced proteins (ELIP), RAB proteins
and ACC oxidases or dehydrins.
For three of these genes, putative ELIP, RAB -16B and the ACC oxidase protein, the
level of transcripts accumulated in response to drought was higher in the tolerant genotype,
indicating that these proteins might represent potential candidates for being involved in
drought tolerance (Ouvrard et al., 1996).
Dehydrin proteins are usually expressed following any environmental stimulus involving
dehydration, such as drought or cold stress and salinity (Close et al., 1993, Godoy et al., 1990,
Neven et al., 1993). In sunflower, the increase of two dehydrins, HaDhn1 and HaDhn2 was
correlated with the decrease of midday leaf water potential during progressive stress and
mainly up-regulated in a drought tolerant line (Cellier et al., 1998). The influence of day/night
cycle on the expression profile of these two dehydrin genes, showed an oscillation of HaDhn1
in a diurnal way with a peak of messenger RNA (mRNA) at midday. In contrast the increase
of HaDhn2 transcript was independent of day/night periods (Cellier et al., 2000).
Changes in the dehydrin transcript levels were also studied in two sunflower mutants for
ABA synthesis and accumulation during embryo and plantlet development and drought stress:
nd-1 (an albino, non-dormant and lethal mutant with a very low ABA content and no ABA
accumulation in response to stress) and w-1 (a wilty mutant, with reduced ABA
accumulation). These results showed the existence of two regulation pathways of dehydrin
transcript accumulation, an ABA-dependent manner and an ABA-independent one, which
may have cumulative effects (Giordani et al., 1999).
Expression profiles of four water-stress associated genes, aquaporin, dehydrin, leafy
cotyledon1-like protein and fructose-1,6 bisphosphatase were assessed in four RILs and
parental lines showing contrasting responses to dehydration and rehydration (Kiani et al.,
2007). The expression level of aquaporin and fructose-1,6 bisphosphatase genes in leaves was
down regulated by water stress and was associated with relative water content. The level of
dehydrin transcripts increased in leaves of all studied RILs in response to water stress.
Transcript accumulations of dehydrin and leafy cotyledon1-like genes, likely involved in
protective tolerance processes, were not directly correlated with plant water status. Down-
regulation of fructose-1,6 bisphosphatase was observed under water stress. Net
photosynthesis rate and the fructose-1,6 bisphosphatase gene expression levels were
associated mainly after rehydration. This phenomenon indicates an association between
physiological response to water stress and differential expression of water-stress related genes
(Kiani et al., 2007).
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Functional Genomics and Transgenesis Applied to Sunflower Breeding 135
Gene expression profiles between drought and salinity stressed plants in sunflower, two
of the most important environmental stresses that alter plant water status and severely limit
plant growth, development and crop productivity, were assessed through differential display
polymerase chain reaction, (Liu and Baird, 2003). Five drought regulated cDNAs and 12
salinity regulated cDNAs were cloned and sequenced. Out of them, 13 genes were analysed in
leaves of drought stressed plants, and in roots and shoots of drought and salinity stressed seed
lines. These results showed that certain genes respond to both stresses, evidencing a cross-talk
and complex regulation, while others are uniquely regulated either in terms of the stress
stimulus or the plant tissue.
Sequence analysis of these cloned genes identified five with homology to known genes,
guanylate kinase (signal transduction), lytB (antibiotic/drug resistance), selenium binding
protein (heavy metal stress), polyprotein (reverse transcriptase), and AC-like transposable
element (Liu and Baird, 2003). In addition, SOS2 and PMP3 genes, deeply involved in Na
+

efflux mechanisms in glycophytes, were reported in sunflower as expressed in root-cap as a
way to exploit plant adaptation to saline environments. PMP3, a small hydrophobic peptide
first described in yeast and involved in preventing Na
+
entry, was found in the root cap of a
monocotyledonous halophyte but not identified in dicots (Inada et al., 2005), whereas SOS2
was found to be expressed gradually in seedlings of tolerant sunflower lines (Saadia et al.,
2013). Recently Jabeen and Ahmad (2013) reported chitosan as an effective biostimulator to
enhance safflower and sunflower seedlings growth and plant tolerance under salinity
conditions. Chitosan is a natural biopolymer formed by alkaline deacetylation of chitin which
has recently attracted additional attention because of its antioxidant activities, being
previously reported in rice and other crops (Chibu and Shibayama, 2001, Ruan and Xue,
2002).
During 2003 it was reported for the first time in sunflower, the isolation and
characterization of expressed sequence tags (ESTs) from organ-specific cDNA libraries
constructed by suppressed subtractive hybridization (Diatchenko, 1996) as an alternative to
identify differentially expressed sunflower transcripts. They presented the differential level of
representation for functional EST groups based on Gene Ontology annotation (Ashburner et
al., 2000), as well as a comprehensive description of the individual non-redundant sequences
generated (Fernandez et al., 2003).
Within a large number of genes that are activated by stress conditions, the study of
transcription factor genes (TFs) is essential to understand different stress mechanisms. Stress
responses are regulated by multiple signalling pathways (Knight and Knight, 2001, Singh et
al., 2002, Chen and Zhu, 2004); several abiotic stress pathways share common elements that
are potential ‗nodes‘ for cross-talk (Knight and Knight, 2001) and TFs can regulate various
stress inducible genes controlling these gene networks.
HAHB4 transcription factor belongs to the sunflower subfamily I of HD-Zip proteins and
is transcriptionally regulated by water availability, abscisic acid and ethylene signalling. The
overexpression of HAHB4 cDNA in sunflower, together with knockdown by iRNA, showed
that this TF helps to prevent the accumulation of gene transcripts related to photosynthesis
(Manavella et al., 2006, Manavella et al., 2008a).
Moreover, Arabidopsis transgenic plants expressing this gene showed an up-regulation of
HAHB4 induced by different external factors such as drought, extreme temperatures, osmotic
stress, and light conditions, evidencing a complex cross-talk network (Manavella et al.,
2008b).
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Sebastian Moschen, Laura M. Radonic, Guillermo F. Ehrenbolger et al. 136
NAC transcription factors (TFs) represent a large family of the senescence-regulated
genes in many plant species (Guo et al., 2004, Buchanan-Wollaston et al., 2005, Lin and Wu,
2004, Balazadeh et al., 2008, Gregersen and Holm, 2007). ORE1, a NAC-TF, was first
reported to act downstream of ethylene and auxin signalling pathways. It is involved in salt
stress response and lateral root development (He et al., 2005), and several of its downstream
genes are also affected by salt stress leading to leaf senescence (Balazadeh et al., 2010). Leaf
senescence is a complex and highly coordinated process, controlled by multiple variables,
either from genetic and environmental origin that has a strong impact on crop yield (Noodén
et al., 1997).
In sunflower, the senescence process takes place abruptly, coinciding with adverse
environmental conditions and the incidence of foliar diseases (Dosio and Quiroz, 2010).
HaORE1 transcription factor, appears as a potential molecular functional marker, showing an
early activation in relation to physiological and biochemical events, with an early expression
pattern, followed by an increase of expression prior to anthesis and the first symptoms of
senescence, when the critical period of grain filling has already begun, which makes this gene
a potential indicator of the triggering of the process, (Fernandez et al., 2012b, Moschen et al.,
2012). Senescence is tightly linked to nutritional stress and N starvation (Nasser, 2002,
Cabello et al., 2006, Agüera et al., 2010) and CO
2
environmental level (Larios et al., 2004, de
la Mata et al., 2012). Recently, different light intensity studies were performed in H. annuus
L. where these results showed that high photon flux densities caused early senescence in
primary leaves by altering not only the CO
2
fixation rate and sugar levels but also the activity
of key enzymes of nitrogen metabolism (de la Mata et al., 2013).
Plants growing in adverse environmental conditions change their protein concentrations,
by both transcriptional and/or post-transcriptional regulatory mechanisms. At a post-
transcriptional level, an important aspect to understand plant signalling pathways related to
different stresses is the study of microRNAs (miRNAs). MicroRNAs are noncoding,
endogenous, small RNAs about 20–24 nucleotides long, conserved in plants and animals
(Bonnet et al., 2004, Huang et al., 2010). They have an important role in post transcriptional
gene regulation (Bartel, 2004, Carrington and Ambros, 2003). miRNAs negatively regulate
gene expression either by inhibiting translation elongation or by triggering mRNA destruction
(Aukerman and Sakai, 2003, Tang et al., 2003). In sunflower, precursors (pre-miRNAs), from
known Helianthus expressed sequence tags (ESTs) were found by a systematic search
approach (Barozai et al., 2012). The study resulted in 61 novel miRNAs belonging to 34
families from Helianthus ESTs database. Out of these 61 new miRNAs, 20 are from H.
tuberosus, 17 miRNAs belong to H. annuus, 8 are from H. ciliaris, 5 from H. exilis, 4 from
H. argophyllus, H. petiolaris each and 3 are from H. paradoxus. Targets of these miRNAs
consist of growth and development related genes, transcription factors, signalling pathway
kinases, stress resistant proteins and transport related proteins (Barozai et al., 2012).
The sunflower HaWRKY6 is a particularly divergent WRKY transcription factor gene,
unique to plants and identified as mediating varied stress responses (Ulker and Somssich,
2004, Eulgem and Somssich, 2007, Rushton et al., 2010). HaWRKY6 exhibit a putative target
site for the miR396. Arabidopsis plants expressing a miRNA396-resistant version of
HaWRKY6, confirmed the dependency of HaWRKY6 silencing by miRNA (Giacomelli et
al., 2012). Sunflower plants exposed to high temperatures or salicylic acid presented opposite
expression of HaWRKY6 and miR396. Experiments using the wild type and miRNA-
resistant versions of HaWRKY6 showed altered stress responses. These results showed a role
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Functional Genomics and Transgenesis Applied to Sunflower Breeding 137
of the recently evolved miR396 in the regulation of HaWRKY6 during early responses to
high temperature (Giacomelli et al., 2012). Candidate gene approaches based on sequence
similarity to previously characterized genes has greatly contributed to identifying and
characterizing orthologous genes in sunflower both for biotic and abiotic stress response. The
isolation and characterization of transcription factors mentioned above is an example of the
successful application of this strategy. Another example is the identification and functional
characterization of members of the Germin-like Protein (GLP) genes in sunflower
(Ehrenbolger, 2012). Over the last years, evidence has accumulated regarding the
involvement of GLPs in defence against pathogens (Davidson, 2009).
In a previous work overexpression of the wheat oxalate oxidase (gf2.8), a member of the
Germin family, showed enhanced tolerance to the oxalic acid producing pathogen Sclerotinia
sclerotiorum (Hu et al., 2003). Germins are included within the Cupin Superfamily, along
with the more diverse Germin-like proteins (GLPs), which are present in a wide range of
species and are characterized by containing two highly conserved motifs. Although the
biochemical properties of many of the GLPs described to date are still unknown, superoxide
dismutase activity has been reported in different species such as barley, grape, and tobacco
(Carter and Thornburg, 2000, Zimmermann, 2006, Godfrey, 2007). Given that most GLPs
exhibit glycosylation and cell wall localization signals, they have been related to cell wall
strengthening and papillae formation (Wei, 1998). In addition, they have been proposed to
play a role in reactive oxygen species detoxification and to function as signalling molecules
inducing a range of defence responses in a direct or indirect manner (e.g., Lane 1994; Zhou et
al. 1998). The expression of a GLP from Beta vulgaris (BvGLP-1) in Arabidopsis has proved
to elevate H
2
O
2
content and confer significant resistance to Verticillium longisporum and
Rhizoctonia solani (Knecht, 2010). Recently, Ehrenbolger et al. (2012) described the
evolution, diversification, and function of sunflower GLPs (HaGLPs) with the aim of
identifying new candidate genes for crop improvement. They identified nine putative
subfamilies within HaGLPs and found that these genes are divergent in terms of their primary
sequence, the size of their encoded proteins and the length of the introns. Expression studies
showed that most HaGLPs were transcribed in major plant organs, albeit to varying degrees
in different sunflower tissues (root, leaf, stem, flower, receptacle, and seed). Transgenic
Arabidopsis lines overexpressing HaGLP1 showed enhanced tolerance to Rhizoctonia solani.


LARGE SCALE TRANSCRIPTOMICS ANALYSIS

The first breakthrough technology for transcriptome analysis was the development of
microarrays. Microarrays have proven to be a powerful tool for the discovery of many
stressed-induced genes involved in stress response and tolerance. Macro and microarray
studies of stress responses in Arabidopsis and Oryza sativa allowed the identification of genes
involving both functional and regulatory proteins (Seki et al., 2001, Sahi et al., 2006,
Kanesaki et al., 2002).
Although analysis of gene expression using the microarray approach has already
provided significant insights into the signalling processes involved in defence responses in
model plants (Dowd et al., 2004, Maleck et al., 2000, Schenk et al., 2000, Scheideler et al.,
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Sebastian Moschen, Laura M. Radonic, Guillermo F. Ehrenbolger et al. 138
2002), there are few reports describing the global changes in transcription activities in
response to pathogen infection in sunflower.
Microarrays can be classified into two groups based on the nature of the DNA fixed to
solid support; oligonucleotide microarrays and cDNA microarrays (Aharoni and Vorst, 2002).
For oligonucleotide (shortened as ―oligo‖) microarrays, also called ‗gene chips‘, synthetic
oligos are arrayed on a glass substrate through direct synthesis or spotting. Oligo arrays have
the advantage of greater gene density and more uniform hybridization because of similar
sequence lengths, and more specific hybridization, which enables differentiation between
duplicated or differentially spliced genes. The oligo approach also obviates concerns about
potential mismatches between cDNA clones and EST sequences (Knight, 2001).
However, synthetic oligo arrays are expensive to produce and less sensitive than cDNA
arrays, particularly in hybridizations involving distantly related species (Lee and Roy, 2004).
The first transcriptional studies for cultivated sunflower, involved the development of
cDNA macro and microarrays to evaluate sunflower responses to biotic (Alignan et al., 2006)
and abiotic stresses (Hewezi et al., 2006a, Roche et al., 2007, Fernandez et al., 2008).
Alignan et al. (2006) developed and used a 1000-element cDNA nylon microarray (also
known as low density array). They chose genes putatively involved in primary metabolic
pathways, signal transduction and response to biotic stress, identified on the basis of their
homology to A. thaliana genes. The low density array allowed investigating transcriptional
changes that occur during the activation of multi-genic resistance in partially resistant and
susceptible sunflower lines inoculated with P. macdonaldii. They proposed a model in which
negative regulation of a dual-specificity MAPK phosphatase could be implicated in sunflower
defence mechanisms against the pathogen. The resulting activation of the MAP kinase
cascade could subsequently trigger defence responses under the control of transcription
factors belonging to MYB and WRKY families. It was also proposed that the activation of
protein phosphatase 2A (PP2A), which is implicated in cell death inhibition, could limit
pathogen development (Alignan et al., 2006).
Nylon microarrays containing >8000 putative unigenes were developed to assess two
contrasting sunflower flowering genotypes under low temperature treatment (Hewezi et al.,
2006a). A total of 108 cDNA clones were differentially expressed between plants grown at
low temperature (15°C and 7°C) and their corresponding controls across the two genotypes.
Almost 90% of the genes whose products are involved in low temperature tolerance such as
LTPs, aquaporins, chaperones, and sucrose metabolism related genes were down-regulated. It
was postulated that the down-regulation of genes having important functions under low
temperature tolerance might be responsible for the sensitivity of sunflower plants to low
temperature.
Drought tolerant and drought sensitive genotypes subjected to soil water deficit stress
under field conditions were assessed through sunflower cDNA microarrays containing about
800 clones, representing high sequence similarity with known or predicted Arabidopsis genes
(Roche et al., 2007). The tolerant genotype showed up-regulation of genes involved in cell
detoxification (aldehyde deshydrogenase, amino transferases and tocopherol enzymes) and
down-regulation of genes involved in the cell division control and cellular differentiation
(proteins kinases, transcription factors and receptors), suggesting a specific adapted
mechanism in response to water stress. Embryos and leaves showed different expression
patterns. Genes related to amino acid and carbohydrate metabolisms, and signal transductions
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Functional Genomics and Transgenesis Applied to Sunflower Breeding 139
were up-regulated in embryos and down-regulated in leaves, suggesting a differential
response to water stress of vegetative and reproductive organs.
Fluorescence cDNA microarray assays based on sunflower organ-specific unigenes was
first developed to study gene expression in early responses to chilling and salinity (Fernandez
et al., 2008). The aim of this work was to detect candidate genes associated to regulatory and
stress response pathways common to both stresses and to identify those genes exclusively
expressed in response to each kind of stress condition. A total of 80 candidate genes for either
salinity and/or chilling stresses were detected. Fifty of them were up or down regulated under
both stresses, evidencing a cross-talk of regulatory mechanisms in the responses to chilling
and salinity. Only 15 and 12 sequences were up regulated or down regulated specifically in
one stress but not in the other, respectively.
Interestingly, 39 genes detected in this study correspond to genes with unknown
predicted function. Microarray profiling revealed dynamic changes in transcript abundance,
including transcription factors, defence/stress related proteins, and effectors of homeostasis,
all of which highlight the complexity of both stress responses.
The development of genomics projects during the last decade generated an exponential
increase in the number of sequences available in public databases involving complete genome
sequencing for some species and Expressed sequences (ESTs), in the case of species with
large genomes, such as sunflower.
The availability of large number of EST sequences in public databases for cultivated
sunflower and wild Helianthus species, jointly with the development in the last years of high-
scale transcriptional analysis techniques (through development of oligonucleotide
microarrays) allowed the implementation of expression studies across different stages of
development in many species, organs and/or growth conditions, including biotic and abiotic
stress. In the case of sunflower different oligonucleotide based microarray platforms were
developed. Affymetrix and NimbleGen technologies have been applied to the development of
chips for the Helianthus genus and weeds of the Asteraceae family, respectively (Lai et al.,
2012, Bazin et al., 2011). The Affymetrix GeneChip was designed based on wild and
cultivated sunflower raw ESTs available in public databases (Bazin et al., 2011). The
NimbleGen platform comprises one 4-plex microarray developed from the assembly of
Sanger ESTs from several H. annuus L. cultivars deposited in GenBank up to the year 2007,
and one 12-plex array based on the 454 Titanium platform transcriptome assembly from one
weedy H. annuus L. genotype (Lai et al., 2012). Using the same public Helianthus EST data
set, plus 454 sequences from the HA89 inbred line transcriptome, a Helianthus gene reference
assembly was built to conduct SNP discovery and to design an Illumina Infinium BeadChip
for genotyping (Bachlava et al., 2012). More recently, an oligonucleotide based chip on
Agilent technology has been developed for stress-induced gene expression studies (Fernandez
et al., 2012c). The use of a longer probe format represents an advantage of the Agilent
oligonucleotide microarrays over other technologies, because the longer oligonucleotides
provide higher hybridization stability for sequence mismatches; being thus more suitable for
the analysis of highly polymorphic regions (Hardiman, 2004). This chip consisted of
approximately 41,000 unigenes representative of the sequences available for sunflower within
public ESTs and non-redundant sequences (Fernandez et al., 2012c). A repository of the
sunflower unigene collection, plus the probes associated to each unigene and functional
annotation information is available at the URL atgc-sur.inta.gob.ar (Figure 1). The microarray
was experimentally validated for a pilot senescence experiment and the performance of
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different statistical and bioinformatics strategies for data analysis were explored to identify
candidate genes and key metabolic pathways associated to the outbreak of the leaf senescence
process and response to biotic stress in sunflower (Fernandez et al., 2012c, Fernandez et al.,
2012a).
The advent of next-generation sequencing methods with reduced costs and higher
throughput has encouraged the generation of more comprehensive and in-depth studies for a
wider range of organisms and transcriptomes. RNA-Seq technology allows the generation of
valuable information for species with high economic interest but limited genomics resources.
Recently, the interaction between sunflower and Plasmopara halstedii (downy mildew)
has been studied using the 454 FLX pyrosequencing of cDNAs from H. annuus seedlings
infected by the oomycete.
As a result, sequences expressed by either organism were identified in the frame of their
interaction. This study represents a substantial improvement of existing knowledge regarding
P. halstedii sequences that are expressed during the interaction of this species with sunflower
because twenty putative cytoplasmic effectors were characterized, including RXLR and CRN
effectors that are responsible for crinkling and necrosis phenotypes in the leaves of the
infected plants. Additionally, 22 SNPs were detected, providing new information on pathogen
polymorphisms (As-sadi et al., 2011).


OTHER POST GENOMICS FUNCTIONAL STRATEGIES:
METABOLOMICS

Multiparallel analyses of mRNA, metabolite and protein profiles are central to today's
functional genomics initiatives. The use of metabolite profiling as a tool for a comparative
display of gene function has the potential to provide deeper insight into complex regulatory
processes (Fiehn et al., 2000).
Studies of soluble metabolite exchange between sunflower and Sclerotinia have been
performed by Jobic et al. (2007). This investigation reported a metabolic study of the
necrotrophic interaction between S. sclerotiorum and cotyledonary leaves of sunflower based
on NMR-spectroscopy (nuclear magnetic resonance) (Roberts and Jardetzky, 1981, Shachar-
Hill, 1996, Ratcliffe and Shachar-Hill, 2001). In order to analyse metabolic processes that
promote fungal development in plant tissues, they established the profiles of soluble
metabolites for each partner and followed the quantitative modifications of the metabolites
during the course of infection. The results indicated a progressive exhaustion of plant
carbohydrate stores in favour of the accumulation of glycerol of fungal origin. Expression of
fungal hexose transporters in plants was also described (Jobic et al., 2007).
Other metabolic studies adopted a gas chromatography–mass spectrometry (GC–MS)-
based metabolite profiling approach capable of identifying a broad spectrum of metabolites
including major and minor sugars and sugar alcohols, organic acids, amino acids, fatty acids
and few soluble secondary metabolites in the sunflower capitulum (Peluffo et al., 2010). As
plant–host interactions represent one of the most complex systems at the biochemical level,
the aim of this work was to develop a metabolomics approach to shed light onto the
metabolism of sunflower florets by a comparative analysis of two well characterized

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Functional Genomics and Transgenesis Applied to Sunflower Breeding 141

Figure 1. Sunflower Unigene Repository (SUR) for curated sunflower unigenes. (http://atgc-sur.
inta.gob.ar).

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genotypes with contrasting tolerance levels to S. sclerotiorum. The metabolite data obtained
in the work of Peluffo et al. (2010), showed that, under field conditions, primary metabolism
is differentially synchronized in the different genotypes.
As a result, the analysis allowed detecting differential metabolic regulation between
susceptible and resistant genotypes in the sunflower-Sclerotinia system. Primary metabolic
events in floret cells are differentially synchronized in genotypes with contrasting behaviours
tested under field conditions. GC-MS approach has proven to be one of the key tools in
metabolomics per se and by extension in plant functional genomics (Sumner et al., 2003,
Fernie, 2007).
Thus, NMR and GC–MS approaches have been proposed as complementary technologies
for metabolic profiling, especially in the context of biomarker discovery (Fernie et al., 2004,
Meyer et al., 2007).


REVERSE GENETICS BY TILLING STRATEGY

Non-conventional sunflower breeding has advanced through a variety of approaches
including Molecular Marker Assisted Selection (see chapter Genetics and Genomics Applied
to Sunflower Breeding in this book), genetic engineering (Lucas, 2000), in vitro techniques
(somaclonal variation, protoplast fusion and in vitro embryo rescue) and conventional
mutagenesis (Encheva, 2008) to improve yield, oil quality and disease-, salt- and pest-
resistance. TILLING (Targeting Induced Local Lesions in Genomes) is one of the latest
additions. It is a molecular biology-based method that allows directed identification of
mutations in a specific previously characterized gene. TILLING was first reported in 2000,
using the model plant Arabidopsis thaliana and has been adapted since then as a method in
other plant species (McCallum et al., 2000a, McCallum et al., 2000b, Prina et al., 2010).
TILLING provides a reverse genetic technique that is suitable for most plants (McCallum
et al., 2000b) in order to select new genetic variability for useful alleles to more precisely
design new traits. Thanks to the generation of random point mutations at high density by this
technique, allelic series of missense mutations can be discovered; as well as short insertion/
deletions (INDELs) (Greene et al., 2003). An advantage is that even working with only a
small population, multiple alleles of a specific gene may be obtained regardless of the gene
size (Till, 2007).
Due to the complexity and cost of releasing transgenic traits, TILLING became the
method of choice when the desired trait is related to the silencing of a given gene. It
significantly reduces costs and time for product development when compared to transgenic
crop plants.
The first sunflower TILLING population was developed by Sabetta et al. (2011). They
were able to generate point mutations like nucleotide substitutions by conventional ethyl
methanesulfonate (EMS) mutagenesis and optimized the procedure for an efficient sunflower
SNP detection system.
As result, a sunflower TILLING population of 3,651 independent mutant lines was
developed, providing an important tool for the identification of interesting phenotypes; 37 of
the mutant lines showed a single altered trait, while 138 lines displayed multiple mutant traits.
This mutagenic strategy was successfully applied to assay 1,152 sunflower M2 lines. Four
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Functional Genomics and Transgenesis Applied to Sunflower Breeding 143
genes have been subjected to the reverse genetics screening: the kasII and kasIII genes
(codifying the isoforms II and III of the β-keto-acyl-ACP-synthetase); the fad2-1 gene,
encoding the enzyme responsible for the converting reaction of oleic acid to linoleic acid; the
AY490791 gene, involved in P. halstedii resistance. The authors focused on some key
enzymes of the fatty acid pathway, because of the interest in increasing the nutritional value
of sunflower oil by the reduction of the ratio of saturated to unsaturated fatty acids. Moreover,
P. halstedii is one of the most dangerous pathogens that affect sunflower cultivation in the
Mediterranean area (Radwan et al., 2005). Therefore the availability of a stable and effective
system, as genetic resistance, for the pest-control results of prime importance (Sabetta et al.,
2011).
This reverse genetics tool has also been used in sunflower by Kumar and co-workers with
the objective of screening mutations in two genes, FatA and SAD, which are involved in the
accumulation of short and medium chain fatty acids. As a result they obtained 26 induced
mutations using an EMS mutant population under controlled conditions.
They focused in the production of more stable oils for biolubrication and the increase of
healthy substitutes for food industry (Kumar et al., 2013).
Currently, the shortage of well characterized candidate genes underlying agronomic
important traits represents one of the main drawbacks in sunflower molecular breeding. In
this context, functional tools which allow concerted transcriptional studies, such as high
density oligonucleotide microarrays, TILLING populations and RNA-seq, strongly support
the discovery and characterization of novel genes.


IN VITRO CULTURE AND GENETIC TRANSFORMATION

In vitro tissue culture techniques, as immature embryo rescue, were incorporated in
sunflower breeding before the existence of the functional genomics and post genomics
disciplines allowing the development of four to six generations per year and therefore,
notably accelerating inbreeding speed. Embryo rescue and protoplast fusion also helped to
sort sterility or incompatibility barriers in wide interspecies crossings. Examples of protoplast
fusion techniques applied to rescue of wide crossings are those between H. annuus and H.
petiolaris or with H. debilis (Alibert et al., 1994). The usefulness of these techniques is
currently valuable as they were recently applied in the transference of architectural traits from
H. mollis Lam. to H. annuus L. (Breton et al., 2012) and for the development of drought and
broomrape resistant sunflower germplasm utilizing H. argophyllus and H. maximiliani (Petcu
and Pâcureanu, 2011).
Transgenic breeding has clearly demonstrated its ability to make significant
improvements to agriculture, being maize and soybean the best examples of the advantages
that have been achieved using transgenics. The establishment of an efficient and reproducible
in vitro regeneration system is the first, and maybe the most important, step to develop a plant
transformation protocol.
Sunflower plant regeneration was obtained by direct or indirect organogenesis
(depending on the induction and passage through an undifferentiated callus stage), somatic
embryogenesis, and vegetative multiplication. Indirect organogenesis and embryogenesis
through callus stages were soon discarded because of seldom regenerated viable shoots or
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embryos (Greco et al., 1984, Paterson and Everett, 1985, Wilcox McCann et al., 1988). While
somatic embryogenesis was mainly obtained from immature embryos, the most successful
approach seems to be direct organogenesis, which was reported to occur from different
explant sources. Thus, several tissues have been used as a source of explants in an important
number of publications, plant regeneration have been essayed from cotyledons (Power, 1987;
Knittel et al., 1991; Pugliesi et al., 1993; Ceriani et al., 1992; Chraibi et al., 1992; Fiore et al.,
1997; Deglene et al., 1997; Alibert et al., 1999; Baker et al., 1999; Flores Berrios et al.,
1999a; Flores Berrios et al., 1999b; Dhaka and Kothari, 2002; Molinier et al., 2002; Mayor et
al., 2003; Lewi et al., 2006; Radonic et al., 2006; Radonic et al., 2008; Dağüstü et al., 2008;
Pradeep et al., 2012; Sujatha et al., 2012b), immature embryos (Power, 1987; Finer, 1987;
Freyssinet and Freyssinet, 1988; Wilcox McCann et al., 1988; Jeannin and Hahne, 1991;
Bronner et al., 1994; Jeannin et al., 1995; Witrzens et al., 1988; Espinasse et al., 1989; Lucas
et al., 2000; Sujatha and Prabakaran, 2001; Hewezi et al., 2002), shoot-tips (Knittel et al.,
1994; Laparra et al., 1995; Grayburn and Vick, 1995; Burrus et al., 1996; Gürel and Kazan,
1999; Mohamed et al., 2004), protoplasts (Burrus et al., 1991; Fischer et al., 1992;
Krasnyanski et al., 1992; Krasnyanski and Menczel, 1993; Laparra et al., 1995; Moyne et al.,
1988; Müller et al., 2001), hypocotyls (Everett et al., 1987; Pelissier et al., 1990; Escandón
and Hahne, 1991; Müller et al., 2001; Dağüstü et al., 2008), leaves (Konov et al., 1998;
Yordanov et al., 2002) and two days old seedlings (Rao and Rohini, 1999).
Regarding production of transgenic plants, different approaches were achieved.
Polyethylene glycol (PEG)-induced uptake of vectors was used for the transformation of
protoplasts (Moyne et al., 1988), but because of the labour required to isolate and culture
protoplasts, it was necessary to develop other transformation procedures. Some attempts were
made to transform sunflower via microprojectile bombardment with DNA coated particles
(Hunold et al., 1995; Laparra et al., 1995) or electroporation (Kirches et al., 1991) but no
transgenic plants were obtained. A combination of microprojectile bombardment with
uncoated particles and Agrobacterium tumefaciens was also used obtaining transgenic plants
(Bidney et al., 1992; Malone-Schoneberg et al., 1994; Knittel et al., 1994; Lucas et al., 2000;
Müller et al., 2001; Mohamed et al., 2006). Despite of these attempts, most published
transformation systems for sunflower are based on Agrobacterium-mediated transformation
(Everett et al., 1987; Schrammeijer et al., 1990; Escandón and Hahne, 1991; Grayburn and
Vick, 1995; Burrus et al., 1996; Alibert et al., 1999; Müller et al., 2001; Molinier et al., 2002;
Mohamed et al., 2004; Ikeda et al., 2005; Lewi et al., 2006; Radonic et al., 2006; Dağüstü et
al., 2008; Radonic et al., 2008; Neskorodov et al., 2010; Pradeep et al., 2012; Sujatha et al.,
2012b) and these protocols are modifications of the following scheme: Imbibition of seeds,
excision of embryonic axes, co-culture with Agrobacterium tumefaciens, induction of shoots,
recovery of transformed shoots, selection, shoot elongation, transfer to greenhouse and
acclimatization.
All these published protocols suffer from overall low transformation efficiencies. The
critical steps in plant transformation procedure are the transfer of the T-DNA into as many
cells as possible with good regeneration potential, and the regeneration of the transformed
cells into shoots. However, the induction efficiency of adventitious shoots in sunflower is low
due to the fact that the regeneration process is in almost every case by direct organogenesis,
without a callus phase that might serve to amplify the response. This regeneration event is
often of multicellular origin, so regenerated shoots and plants are frequently chimeric
(Schrammeijer et al., 1990) and the transgenic sectors may or may not lead to the recovery of
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Functional Genomics and Transgenesis Applied to Sunflower Breeding 145
transgenic progeny (Burrus et al., 1996). Some efforts were made to increase the regeneration
potential in the meristematic area, like bombardment with coated particles with a construct
containing the ipt gene, which codes for an isopentenyl transferase involved in the
biosynthesis of cytokinins in plants, to induce transient expression of this growth regulator
and consequently an increase in the number of shoots per explant (Molinier et al., 2002).
Furthermore, to stimulate shoot regeneration from hypocotyl explants, Koopmann and
Kutschera (2005) reported a novel system by immersing the explants in a suspension of
Metylobacterium prior to culture on a shoot-regeneration medium.
Other important reported improvement in plant regeneration was the addition of silver
nitrate in all regeneration media to diminish hyperhydricity of primordia and shoots (Mayor et
al., 2003). Besides, rooting was improved by transferring the regenerated shoots to a medium
containing ancymidol as gibberellin inhibitor (Fiore et al., 1997) and naphthalene-acetic acid
as the only auxin (Baker et al., 1999).
More recently, Sujatha et al. (2012a) used 2-isopentenyladenine in the shoot induction
and elongation media to prevent precocious flowering.
Some of these transformation protocols rely on one of several treatments in order to
stimulate Agrobacterium virulence genes, such as adding phenolic compounds as
acetosyringone to the co-culture medium (Mohamed et al., 2004), tissue wounding by either
bombardment with naked particles (Bidney et al., 1992; Müller et al., 2001; Sawahel and
Hagran, 2006), or glass beads (Grayburn and Vick, 1995), or by macerating with enzymes
(Alibert et al., 1999; Weber et al., 2003), sonication (Weber et al., 2003), vacuum (Hewezi et
al., 2003), DMSO treatment (Sujatha et al., 2012b), explant dehydration for several minutes
before co-culture (Hewezi et al., 2002) or combination of some of these treatments (Weber et
al., 2003; Radonic et al., 2006; Radonic et al., 2008; Sujatha et al., 2012b).
Efficient selection of transgenic plants is an important aspect of any transformation
protocol. Kanamycin is the most widely used selection agent for sunflower transformation,
although it is known to be detrimental to organogenic potential (Everett et al., 1987; Müller et
al., 2001).
It has been used in different concentrations (from 1 to 200 mg/L) and with different
levels of success, ranging from the inability of regenerating shoots to a 79% of escapes
(Knittel et al., 1994; Hunold et al., 1995; Gürel and Kazan, 1999; Rao and Rohini, 1999;
Lucas et al., 2000; Hewezi et al., 2002; Rousselin et al., 2002; Ikeda et al., 2005; Lewi et al.,
2006; Radonic et al., 2006; Sawahel and Hagran, 2006; Hewezi et al., 2006b, Radonic et al.,
2008; Pradeep et al., 2012; Sujatha et al., 2012b; Escandón and Hahne, 1991; Malone-
Schoneberg et al., 1994; Grayburn and Vick, 1995; Burrus et al., 1996).
Most of these works used shoot bleaching as selection criteria, except Radonic et al.
(2006, 2008) that showed that in vitro root development in presence of kanamycin was the
most effective parameter to discriminate between transformed and non-transformed shoots
with no escapes (Figure 2).
In some cases where kanamycin selection could not be applied, GFP (Müller et al., 2001;
Weber et al., 2003; Mohamed et al., 2006) and GUS (Mohamed et al., 2004) were used as
vitals marker to select transgenic shoots. Escandón and Hahne (1991) tried selection in
phosphinotricin as they found that kanamycin was not suitable for selection because it
reduced regeneration potential in sunflower. More recently, Neskorodov et al. (2010)
demonstrated that bar is an efficient selective marker for sunflower genetic transformation as
false transformants did not regenerate.
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TRANSIENT TRANSFORMATION AND USE OF LETTUCE AS MODEL
SYSTEM FOR SUNFLOWER FUNCTIONAL GENOMICS STUDIES

From all the transformation attempts mentioned in the previous paragraphs there are two
remarkable conclusions: neither there is a rapid protocol nor it is possible to obtain a large
number of transgenic regenerants per assay.
On the other hand, the availability of an Agrobacterium-mediated transient transfor-
mation system represents a powerful tool to study both biological processes and the role
played by selected candidate genes derived from genomics approaches on agriculture
important traits.

Figure 2. Kanamycin selection of transgenic plants using rooting as selection criterion. Non-
transformed plants develop a normal root in media without kanamycin (A). In kanamycin containing
media, non-transformed plants do not root (B) whereas transformed plants develop a normal root (C).

Figure 3. Standard agroinfiltration system. Sunflower greenhouse plant in V2 stage used to perform
agroinfiltration (A). GUS staining of a leaf showing expression in different agroinfiltration areas (B).
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Functional Genomics and Transgenesis Applied to Sunflower Breeding 147
Agroinfiltration of sunflower is not as easy as in Nicotiana benthamiana or other species,
and it was first reported by Manavella and Chan (2009) who developed a system where leaf
discs in vitro cultured for 3 days could be transiently transformed.
More recently, a standard agroinfiltration system was developed by establishing basically
the Agrobacterium strain and the developmental stages of the greenhouse grown plants used
(Radonic, 2010) (Figure 3).
However, transient expression systems only allow studies for up to 3 or 4 days and when
a follow up experiment for a longer time is required, for example to assess the effect of a gene
or a promoter on developmental stages, the stable transformation of plants is completely
necessary.

Figure 4. Use of lettuce as a model system for the Asteraceae family, in this case, to evaluate tissue
specificity of sunflower HaAP10 promoter. GUS staining results from non-transformed (A, B) and
transformed (C, D) Arabidopsis seeds showing expression only in transformed seeds. The same result
was obtained for non-transformed (E) and transformed (F, G) lettuce seeds.
In this context, in a meeting of the Compositae Genome Project (http://compge-nomics.
ucdavis.edu/cwp) it was concluded that lettuce is the best model system as both species
belong to the Asteraceae family. Lettuce is friendly to in vitro culture producing a high
number of regenerants that later adapt easily to greenhouse conditions. This system was used
to evaluate tissue specificity of the sunflower HaAP10 promoter transiently directing GUS
expression only in lettuce seeds (Zavallo et al., 2011), in the same way as it was shown in
Arabidopsis seeds (Zavallo et al., 2010) (Figure 4).
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Sebastian Moschen, Laura M. Radonic, Guillermo F. Ehrenbolger et al. 148
MOST IMPORTANT AGRONOMICAL TRAITS ENDORSED BY GENETIC
ENGINEERING OF SUNFLOWER

The majority of the transgenic plants obtained in the previously mentioned publications
were developed to establish the transformation methodology with selective markers and
reporter genes, like kanamycin resistance gene nptII, phosphinotricin resistance gene bar,
glucuronidase gene uidA and green fluorescent protein gene egfp. Nevertheless, there are
some studies centred upon the introduction of agronomically useful genes into sunflower.
Three agronomically important transgenic traits were incorporated in sunflower, basically
by seed companies: herbicide tolerance, insect resistance and disease (fungal) resistance.
Regarding insect resistance, transgenic expression of the Cry1F (a Bacillus thuringiensis
or ―Bt‖ gene) in sunflower confers significant control of Rachiplusia nu and Spilosoma
virginica, two important insect pests that impact on sunflower production in various
producing countries. A feeding assay was performed using transgenic leaf discs of Cry1F Bt
plants and both, larvae at seedling and preflowering stages, clearly exhibited enhanced insect
resistance compared to the control. Also, two field bioassays were carried out in Argentina
where two Cry1F-transgenic sunflower events exhibited complete resistance to R. nu (Pozzi
et al., 2000). Another Bt gene, Cry1Ac, when introduced in a transgenic sunflower line also
showed insect resistance. When this transgenic event was crossed with a wild H. annuus
accession, the hybrid progeny showed significant enhanced resistance to Lepidopteran
damage, which was not a surprising result. However, the transgenic × wild sunflower hybrid
produced 55% more seeds than the control hybrids lacking the Cry1Ac gene (Snow et al.,
2003). This study confirmed the effectiveness of Bt genes in controlling insect pests in
sunflower, and at the same time raised a concern about ―gene flow‖ to wild species that has
the potential to enhance the fitness of weeds in the environment.
For fungal diseases like downy mildew and rust that can be controlled by single dominant
resistance genes, breeders have been very successful in identifying new resistance alleles in
wild species and integrating them into elite germplasm (Gulya et al., 1997). However, for
complex fungal diseases like Sclerotinia head rot which is the most damaging disease, the
success of conventional breeding was only partial and many breeders feel that the only
possible solution is to develop transgenic resistant plants. This approach was carried out by
introducing the wheat germin gf2.8 OXO gene (Lane et al., 1991) to generate the so-called
OXO-transgenic sunflower plants (Lu et al., 2000, Scelonge et al., 2000). Bioassays of these
plants were carried out at second generation (T2) and at more advanced progeny generations
indicating the stable inheritance of the transgene. Lesion sizes in the transgenic leaves were
inversely related to the endogenous levels of OXO activity and Sclerotinia spread to the
capitulum tissue only in control plants while the lesions were confined to the main stem on
transgenic plants, proving that OXO can confer significant Sclerotinia resistance in transgenic
sunflower (Hu et al., 2003). Moreover hybrids from the crossing of transgenic lines with
natural Sclerotinia rot-tolerant lines that carried the OXO transgene were more resistant to
Sclerotinia than the corresponding non-transgenic isogenic hybrids, so the combination of
OXO with natural tolerance genetic background provides a greater level of resistance than the
one observed in the commercial hybrids (Bazzalo et al., 2000). This was the first example of a
successful transgene-mediated fungal resistance mechanism in plants. However, the OXO
enzyme has been found to persist in simulated gastric fluid digestion studies, raising concerns
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Functional Genomics and Transgenesis Applied to Sunflower Breeding 149
about whether the protein may become a human allergen (Jensen-Jarolim et al., 2002).
Regarding the environmental concerns, Burke and Rieseberg (2003) suggest that genetically
modified wild plants will not be a worse weed because they do not produce more seeds than
the other wild plants, so the OXO transgene will diffuse neutrally after its escape in the
absence of selection pressure for Sclerotinia tolerance. Chapman and Burke (2006) studied
the escape of transgenes from crop × wild hybridization and showed that natural selection is
the main factor governing the spread of favourable transgene alleles, and not the gene flow.
Genetically transformed crops with herbicide resistance genes give farmers greater
flexibility for the control of weeds. Among the herbicides used in combination with herbicide
tolerant transgenic crops, phosphinothricin and glyphosate have demonstrated effective
control of weeds, low toxicity and low impact on the environment. In the case of sunflower,
this is also the case for a non-transgenic trait that was introgressed from a wild accession. An
imidazolinone (IMI) genetic resistance was identified in a wild population of H. annuus in
Kansas by Miller and Al-Khatib (2002).
The basis of this resistance is the expression of two natural alleles containing mutations
in the gene that encodes for ALS (acetolactate synthase) that produce a conformational
change in the structure of the enzyme which avoids herbicide binding. IMI resistance was
incorporated into elite germplasm through conventional breeding and is currently sold
worldwide under the Clearfield
TM
trademark owned by BASF.
Although this resistance to IMI herbicides was incorporated through conventional
breeding methods, some concerns have raised on gene flow of the IMI-resistance trait to wild
species. Massinga et al. (2003, 2005) created hybrids of domesticated sunflower with both
common sunflower and prairie sunflower with and without the IMI-resistance trait, and
measured the growth of IMI-R and IMI-S hybrids under non-competitive conditions,
concluding that hybrids of both sunflower species were equally competitive. These results
show the safety on environmental impact of IMI technology and besides that the ALS mutant
genes can be used for engineering sunflower to produce transgenic sunflower for this or other
herbicide resistance genes.
However, this door is not completely open yet, since transgenic tolerance to glyphosate
and phosphinotricin have already been introduced in sunflower showing efficacy in field
trials, but they did not reach the market because of the concerns regarding the environmental
consequences of the transfer of these genes to sexually compatible weedy relatives.
Other transgenic approaches are directed to modify oil quality. Sunflower oil is one of the
four major sources of edible oils worldwide and is well known as healthy oil. Besides it is
very stable against lipid peroxidation and is used for salad dressings, cooking and frying.
High oleic acid sunflower, Pervenets lines, were originally developed by mutagenesis
(Soldatov, 1976). The cross of a Pervenets female with the inbred line HA89 created the first
mid oleic or NuSun
TM
sunflower hybrid (Miller et al., 1987). The existence and the rapid
adoption of these lines encourage the improving intentions for oil profile by transgenesis.
The coding sequence of D9-stearoyl-(acyl carrier protein) desaturase from Ricinus
communis was introduced into sunflower, under the control of seed-specific promoter and
terminator sequences of the late embryogenesis abundant gene from sunflower, Hads10. Fatty
acid composition of the seed oil was followed over five generations under greenhouse and in
open field conditions.
Some of the transgenic lines produced oil with a significantly reduced stearic acid content
compared with non-transformed plants under greenhouse and field conditions (Rousselin et
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Sebastian Moschen, Laura M. Radonic, Guillermo F. Ehrenbolger et al. 150
al., 2002). Dağüstü et al. (2008) introduced into sunflower the Erwinia uredovora phytoene
desaturase (crtl) and hydroxymethylglutaryl-CoA (Hmgr-CoA) genes, which have the
potential to increase oil quality.
In summary, current scenarios in sunflower transgenesis shows contrasting facts. As
mentioned previously, Snow and colleagues (2003) showed some environmental concerns
regarding Bt transgenic sunflower release, while Burke and Rieseberg reported lack of such
risk after gene flow to weeds for the OXO event.
Besides, as detailed by Cantamutto and Poverene (2007) each new release should be
analysed separately as it must be taken into account whether the release would take place in
the centre of origin (North America) or in other geographical areas (South America, Europe
or Africa) and also that it must be considered the adaptive nature of the introduced character.
For example, it is foreseen that the adaptive impact of a change in oil quality will give no
selective advantage to weeds, compared to the case of an herbicide tolerance under the
selective pressure of the agroecosystem.
However, even in this extreme example of adaptive advantage it may be argued that it
rapidly disappears in the absence of the selective agent (the herbicide) in natural environment
outside the agroecosystem.
Moreover, it would be interesting for environmental sustainability to count with the
variety of plant tolerant to different herbicides in order to alternate their sowing with other
herbicide tolerant species, like soybean or even with IMI sunflowers, thereby preventing the
emergence of resistant weeds to one of these herbicides or helping to control them.
As a consequence of the appearance of Clearfield sunflower and its global marketing
since 2010, many of the concerns about the release of transgenic sunflowers are rapidly
decreasing. In addition, the constant efforts in improving sunflower transformation protocols
will strength the emergence of some transgenic varieties of this oilseed in the near future.


CONCLUSION

The advances and emergence of new technologies in functional genomics, post genomics
and transformation were described in this chapter.
Although sunflower is still an orphan crop, remains as a crop without reference genome
available; the development of knowledge at transcriptomics, metablomics and proteomics
level along with statistical and bioinformatics procedures for the analyses and data mining of
the massive information derived from high-throughput processing methodologies greatly
contributes to support plant breeding.
The application of these strategies allows not only the elucidation of complex
agronomical pathways but also the identification of candidate robust genes playing key roles
in determining these traits.
In addition, the possibility to analyse those candidate sequences as genes or promoters in
a transient or stable expression system both in sunflower plants as in model species like
Arabidopsis or lettuce assures their functional validation.
These genomics approaches are nowadays being used both in the public and private
sectors, and will impact positively on sunflower breeding bringing this crop to a competitive
place in the international marketplace.
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Functional Genomics and Transgenesis Applied to Sunflower Breeding 151
ACKNOWLEDGMENTS

This research was supported by ANPCyT/FONCYT, PICT 2011-1365, PME 2007-024,
PICT 2007-019, PICT 15-32905, PICT 2007-727, CONICET PIP 5788, INTA-AERG
233261, INTA PE1131024, INTA PE1131023, INTA-PRR AEBIO 241001 and 245001.
SM and GFE are PhD students from CONICET; PF, VL, NBP and RAH are career
members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET,
Argentina) and INTA Researchers; LR and MLB are INTA researchers, HEH is INTA
Researcher and Professor at the University of Buenos Aires.


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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 7



DISEASE MANAGEMENT IN SUNFLOWERS


Regina M. V. B. C. Leite
*

Embrapa Soybean, Londrina, Brazil


ABSTRACT

The expansion of sunflower can be affected, among other factors, by the presence of
diseases, since it hosts over 30 phytopathogenic microorganisms, mostly fungi, which
may, depending on climatic conditions that favor the occurrence of pathogens and the
infective process, lead to a significant reduction on yield and quality of product.
Alternaria leaf spot and Sclerotinia wilt and head rot are the most severe diseases in
Brazil. Other important diseases occurring worldwide, like downy mildew, powdery
mildew, Phomopsis stem canker and sunflower rust, are likely to affect sunflower, under
very diverse climatic conditions. Once installed on the crop, sunflower diseases are hard
to control. Therefore, measures of disease management are mostly preventive and should
not be used alone. Effective control is based on an integrated program, which includes
zoning for climate risk and diverse cultural practices. The present chapter aims to discuss
about the most important sunflower diseases and strategies for disease management, in
order to give support for the sustainability and competitiveness of the sunflower crop.

Keywords: Alternariaster helianthi, Sclerotinia sclerotiorum, Plasmopara halstedii, Erysiphe
cichoracearum, Phomopsis helianthi, Puccinia helianthi


INTRODUCTION

Sunflower (Helianthus annuus L.) cropped area is increasing in Brazil, with 74 thousand
hectares in the last growing season (Conab, 2013). In the world, sunflower production in
2012/2013 was 36 million tons of oilseeds, 14 million tons of meal and 13 million tons of oil.
The largest producers were: Ukraine (24.7%), Russia (21.8%), European Union (19.1%),
Argentina (8.8%) and Turkey (2.8%) (United States, 2013).

*
Embrapa Soybean, C.P. 231, CEP 86001-970, Londrina, PR, Brazil. Email: regina.leite@embrapa.br.
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Regina M. V. B. C. Leite 166
However, there is an enormous potential for expansion if sunflower is planted as a second
summer crop, in succession to soybean, which occupies an area of around 28 million ha in
Brazil (Conab, 2013). The potential for area expansion is also driven by a crescent demand
for special oils for human consumption, for example high oleic oil, and the Brazilian
government demand for biofuel.
The expansion of sunflower can be affected, among other factors, by the presence of
diseases caused by viruses, bacteria, fungi and nematodes. Sunflower hosts over 30
phytopathogenic microorganisms, mostly fungi, which may, depending on climatic conditions
that favor the occurrence of pathogens and the infective process, lead to a significant
reduction on yield and quality of product (Zimmer & Hoes, 1978; Gulya et al., 1997).
It is estimated that diseases are responsible for an average annual loss of 12% of the
world production of sunflower (Zimmer & Hoes, 1978), this being the most limiting factor
for crop production in most regions. In Brazil, there is no accurate data on yield losses caused
by disease, but it is known that can reach 100%, depending on climate conditions. In the state
of Paraná, for example, diseases were considered one of the main factors responsible for the
decline in the production of sunflower in the early 1980s, with the reduction of cultivated area
of approximately 80,000 ha in 1981 to about 5,000 ha in 1984. The yield, which reached 1800
kg per hectare in the previous year, was reduced to 480 kg per hectare, caused by excessive
moisture and low temperatures in the end of the cycle, which favored the occurrence of
Sclerotinia sclerotiorum (Yorinori et al., 1985).
At least 19 diseases have already been reported affecting sunflower in Brazil (Table 1).

Table 1. Diseases affecting sunflower previously reported in Brazil

Disease Pathogen References
Alternaria leaf spot Alternariaster helianthi, Alternaria
zinniae and Alternaria alternata
Embrapa (1983), Yorinori et al. (1985)
Bacterial blight Pseudomonas cichorii Embrapa (1983), Yorinori et al. (1985)
Bacterial leaf spot Pseudomonas syringae pv. helianthi Kimura et al. (1974)
Bacterial stalk rot and head rot Pectobacterium carotovorum subsp.
carotovorum
Embrapa (1983), Yorinori et al. (1985)
Charcoal rot Macrophomina phaseolina Embrapa (1983), Yorinori et al. (1985)
Downy mildew Plasmopara halstedii Embrapa (1983), Yorinori et al. (1985)
Gray mold Botrytis cinerea Embrapa (1983), Yorinori et al. (1985)
Phoma black stem Phoma oleracea var. helianthi-
tuberosi
Embrapa (1983), Yorinori et al. (1985)
Phomopsis stem canker Phomopsis helianthi Embrapa (1983), Yorinori et al. (1985)
Powdery mildew Erysiphe cichoracearum Embrapa (1983), Yorinori et al. (1985)
Rhizoctonia root rot Rhizoctonia solani Embrapa (1983), Yorinori et al. (1985)
Rosellinia root rot Rosellinia sp. Embrapa (1983), Yorinori et al. (1985)
Rust Puccinia helianthi Embrapa (1983), Yorinori et al. (1985)
Sclerotinia wilt and head rot Sclerotinia sclerotiorum Embrapa (1983), Yorinori et al. (1985)
Sclerotium wilt and damping
off
Sclerotium rolfsii Embrapa (1983), Yorinori et al. (1985)
Septoria leaf spot Septoria helianthi Maldaner et al. (2009)
Sunflower mosaic virus Bidens mosaic virus (BiMV) Embrapa (1983), Yorinori et al. (1985)
Verticillium wilt Verticillium dahliae Almeida et al. (1981)
White rust Albugo tragopogonis Almeida et al. (1981)

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Disease Management in Sunflower 167
Some diseases have significant importance, like Alternaria leaf spot and Sclerotinia wilt
and head rot, which are the most severe (Embrapa, 1983). Alternaria disease seems to be
prevalent in all sowing dates, in different growing regions (Leite et al., 2006). Sclerotinia
head rot is one of the main diseases occurring worldwide and occurs mainly under conditions
of low temperature and high humidity, making it almost impossible to cultivate sunflower as
a commercial crop during the autumn in southern Brazil (Leite et al., 2000). Furthermore, this
pathogen can leave resistance structures (sclerotia) in the soil, which may remain viable for
many years and constitutes a source of inoculum for other host crops, like soybean, canola,
and beans, which may be affected in the next growing seasons. Otherwise, other important
diseases in the world, like downy mildew, powdery mildew, Phomopsis stem canker and
sunflower rust, are likely to affect sunflower in our country, since it is grown from latitude
31ºS to latitude 2ºN, under very different climatic conditions that can favor diseases.
The present chapter aims to discuss about the most important sunflower diseases and
strategies for disease management, in order to give support for the sustainability and
competitiveness of the sunflower crop.


SUNFLOWER DISEASES

Alternaria Leaf Spot (Alternariaster helianthi)

In areas of tropical and subtropical climates, that are prevalent in Brazil, Alternaria leaf
spot is a major disease, occurring in virtually all regions and in all sowing dates. The damage
caused by the disease can be attributed to a great reduction in photosynthetic area of the plant
(Leite et al., 2006). Severely attacked plants show early maturation, with reduced production
and seed weight. Besides Brazil (Aquino et al., 1971, Ribeiro et al., 1974), the disease occurs
in countries of North America and Africa, Argentina, India, Japan, Australia, Serbia and
Montenegro (former Yugoslavia), Romania and France (Anahosur, 1978; Zimmer & Hoes,
1978; Davet et al., 1991; Pereyra & Escande, 1994).
Typical initial symptoms on leaves are small necrotic lesions with 3 to 5 mm diameter,
variable color from brown to black, and round to angular chlorotic halo (Figure 1).
Characteristic lesions exhibit concentric circles, similar to a target. These lesions may
coalesce, forming large areas of necrotic tissue, causing premature defoliation of plants. The
symptoms occur primarily in the lower leaves, appearing later in the whole plant. However,
there may be generalized infection of the leaves, regardless of their position on the plant. On
stem and petiole, lesions start as small dots or stripes, and when numerous can form large
necrotic areas. Severe attack can result in blight and finally plant death. The fungus also
colonizes floral bracts and receptacle and may even cause head rot (Anahosur, 1978; Almeida
et al., 1981; Davet et al., 1991).
Various fungi species cause similar symptoms in sunflower plants. Three species of the
fungus are reported as pathogenic to sunflower in Brazil: Alternariaster helianthi (Hansf.)
E.G. Simmons (syn. Alternaria helianthi (Hansf.) Tubaki & Nishihara and Helminthosporium
helianthi Hansf.), Alternaria zinniae H. Pape ex M.B. Ellis and Alternaria alternata (Fr.)
Keissler (syn. Alternaria tenuis Nees). The first one is the most commonly found.
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Regina M. V. B. C. Leite 168

Figure 1. Alternaria leaf spot in sunflower.
The conidia of A. helianthi are cylindrical to ellipsoidal, colorful and dimension of 74 x
19 µm. They have five transverse septa, no tail, and are formed singly in cylindrical
conidiophores. The mycelium is olive-brown, septate, branched and colonizes the
intercellular spaces of the mesophyll cells. The fungus grows slowly and sporulates well on
medium potato-dextrose-agar, forming grayish colonies. A. helianthi is also pathogenic to
chrysanthemum and all annual and perennial species of Helianthus. No physiological
specialization is reported (Anahosur, 1978; Gulya et al., 1997; Simmons, 2008).
The fungus can be spread by contaminated seeds, due to its presence internally or in plant
fragments mixed in the lot, which can remain viable for many years (Godoy & Fernandes,
1985a). However, the major source of primary inoculum consists in plant debris infected with
the fungus (Davet et al., 1991). Under favorable conditions, the fungus produces a large
amount of conidia that are spread by wind and rain, and reach other parts of the plant or other
plants. The optimum conditions for conidia germination are high relative humidity and
temperature between 25°C to 30°C. The germ tube penetrates directly through the epidermis
and the cuticle (Davet et al., 1991). The fungus is highly pathogenic under favorable
conditions. The optimum conditions for infection by A. helianthi are leaf wetness for 24 h at
25°C (Leite & Amorim, 2002). The sunflower plants are susceptible at all stages of
development, with a phase of increased susceptibility from the flowering to the beginning of
grain filling (Anahosur, 1978; Davet et al., 1991). The relationship between Alternaria
severity and yield in the R3 (second phase of inflorescence elongation) growth stage proved
that plants with disease severity higher than 10% had yield lower than 500 kg per hectare,
regardless of the sowing date (Leite et al., 2006). The disease progresses rapidly from the
lower leaves to upper leaves. More severe infections occur in later stages of development,
after flowering (Allen et al., 1983; Godoy and Fernandes, 1985b; Pereyra & Escande, 1994).
Genetic resistance is quantitative, based on the intensity of infection. Studies under
condition of natural infection showed that all tested genotypes were susceptible to A.
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Disease Management in Sunflower 169
helianthi, with different levels of resistance (Leite et al., 1999; Leite & Oliveira, 2012).
Sunflower breeding program should be directed to higher disease resistance. Studies
developed in India indicate that it is possible to synthesize hybrids with reasonable degree of
tolerance even involving susceptible parents (Keertht et al., 2012). Certain species of
Helianthus as wild H. annuus, H. hirsutus, H. rigidus and H. tuberosus, show resistance to A.
helianthi (Lipps & Herr, 1986; Davet et al., 1991). The interspecific hybridization may allow
the incorporation of resistance genes in cultivated genotypes (Davet et al., 1991).
Survival of the fungi species affecting sunflower crop debris indicates prophylactic
control measures. Sunflower should be included within a system of crop rotation, returning in
the same area only after at least four years. The destruction or incorporation of infected crop
debris is recommended to limit sporulation and the amount of primary inoculum (Davet et al.,
1991).
A key measure to minimize the severity of Alternaria leaf spot is the choice of sowing
date. Adjustment of sowing dates is an obviously good strategy to minimize the disease and
obtain better yields (Gadhave et al., 2011). Late sowing is favorable to high Alternaria disease
severity in sunflower crop in Rio Grande do Sul, Brazil (Loose et al., 2012). Sowing should
be done at a time which satisfies the requirements of the plant at different stages of
development, and not favorable to this disease. To minimize the occurrence of disease, it
should avoid when the flowering coincides with periods of heavy rain.
Chemical control with fungicides can be difficult due to the inability of the conventional
input machines enter the crop, because of high plant height. However, fungicides such as
benomyl, imazalil, iprodione, mancozeb + iprodione, procymidone, propiconazole and
vinclozolin were effective in controlling the disease in other countries, with substantial
increases in yield, achene weight and oil content (Pereyra & Escande, 1994; Gulya et al.,
1997; Karuna et al., 2012). In Brazil, fungicides azoxistrobin + ciproconazole and
difenoconazole are registered for control Alternaria leaf spot in sunflower (Ministério, 2012),
but there are no available data that indicates efficiency on disease control until now.


Sclerotinia Wilt and Head Rot (Sclerotinia sclerotiorum)

This fungus is considered the most important pathogen for sunflower in the world and is
distributed in all regions, under temperate, tropical or subtropical climates (Gulya et al.,
1997).
The losses caused by Sclerotinia sclerotiorum depend on the part of the plant affected by
the fungus, which can infect roots, stem or sunflower head. The losses attributed to basal rot
depend on plant age at the infection. S. sclerotiorum quickly kills the infected plants at
seedling stage, resulting in stand failures. Losses associated with head rot directly affect yield,
reducing the number of seeds per chapter, seed weight and oil content. The oil quality is
lower due to the increased concentration of free fatty acids on seeds infected by the fungus.
Seeds of infected head can fall, resulting in total loss of production (Zimmer & Hoes, 1978;
Davet et al., 1991; Masirevic & Gulya, 1992; Pereyra & Escande, 1994). In the state of
Paraná, Brazil, incidence of stem and head disease was high (17.6% to 100.0%), when
sunflower was cultivated after the harvest of summer crops in 1996-1998 growing seasons
(Leite et al., 2000). Indirect losses occur due to contamination of seeds with sclerotia, often of
the same size, shape and specific weight of these, being impossible their removal during
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Regina M. V. B. C. Leite 170
cleaning operation. In addition to these losses, the fungus persists in the soil for many years,
representing a potential danger for sunflower and other hosts (Zimmer & Hoes, 1978; Davet
et al., 1991; Masirevic & Gulya, 1992; Pereyra & Escande, 1994).
S. sclerotiorum can cause different symptoms in sunflower (Zimmer & Hoes, 1978;
Almeida et al., 1981; Davet et al., 1991; Masirevic & Gulya, 1992; Pereyra & Escande, 1994;
Gulya et al., 1997).
The basal rot may occur from the seedling stage to maturity. In seedlings, infection is less
frequent, as plants die quickly and the process does not result in spread to other plants.
Infection is mostly observed near flowering. Diseased plants appear as a group of two or more
plants on the line. Rot starts when the mycelium of the fungus, originating from sclerotia in
the soil, comes in contact with the lateral roots. The first symptom observed is a sudden
wilting of the plant. The infected plant can recover turgidity at night or after a rainfall, but
within a few days, this symptom becomes irreversible, and the disease is named Sclerotinia
wilt. A light brown and soft injury appears at ground level and typically surrounds the rod. If
there is high humidity, the lesion may be covered by white mycelium. The fungus develops
internally and destroys the internal tissues of the stem. Many sclerotia are found within the
colonized portion of the stem, but few are found in the root and in the outdoor area. Diseased
plants can lodge easily.
The rot in the middle portion of the stem occurs in plants from the end of the vegetative
stage to maturity. Infection occurs on leaves wounds and proceeds toward the petiole, ending
the stem. The appearance of the lesion is similar to those of the basal rot. It is most noticeable
in mature stems, because the affected tissue appears lighter than the normal brown coloring
physiological maturity. A white mycelium can cover the lesion, and sclerotia are observed
within the stem. Plants can break at the lesion site.


Figure 2. Sclerotinia head rot in sunflower.
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Disease Management in Sunflower 171
Symptoms in sunflower heads occur at the end of flowering or later. The infection may
start in any part of the receptacle. The initial symptoms are characterized by light brown and
soft lesions on the dorsal side of the head covered by portions of white mycelium (Figure 2).
Eventually, the fungus destroys the interior of the head, leaving intact only the vascular
elements. Sclerotia in large numbers and irregular shape are found within the head. Finally,
there is the complete disintegration of the head, which remains with the exposed fibrous
vascular elements, like a broom. A mass of achenes and sclerotia falls into the base of the
plant.
Sclerotinia sclerotiorum (syn. Sclerotinia libertiana Fuckel and Whetzelinia sclerotiorum
(Lib.) Korf & Dumont) form mycelium and sclerotia in the asexual phase and asci with
ascospores in the sexual phase. Microconidia are produced in senescent cultures in the
laboratory, can be functional for asexual reproduction, but their role in the biology of the
pathogen is not known. The mycelium is composed of hyaline multicellular hyphae with 6.5
to 7 µm in diameter (Mordue & Holliday, 1976).
Sclerotia is formed from the anastomosis of a large number of hyphae in a hard body with
variable format, and may reach several centimeters long. Sclerotium germination occurs in
two ways: a myceliogenic way forming only hyphae and another carpogenic way producing
apothecia (Masirevic & Gulya, 1992; Gulya et al., 1997).
The apothecium is a flat or cup-shaped structure that produces sexual spores of S.
sclerotiorum. Many apothecia may be formed from a single sclerotium. The apothecia are
pale brown in color and have 4 to 10 mm in diameter. Moist soil for a long period and light
are essential for the formation of apothecia. The upper layer contains many paraphyses and
asci. The asci are cylindrical and extended to the apex and range 130-163 µm long and 8 to 10
µm wide (Mordue & Holliday, 1976; Zimmer & Hoes, 1978; Davet et al., 1991; Masirevic &
Gulya, 1992; Gulya et al., 1997).
S. sclerotiorum is a polyphagous fungus, having as host plants of 75 families, 278 genera,
408 species and 42 subspecies or varieties. Except one species of the phylum Pteridophyta, all
hosts of S. sclerotiorum belong to phyla Gymnospermae and Angiospermae (Boland & Hall,
1994). No reports of physiological specialization of the fungus are available (Mordue &
Holliday, 1976; Gulya et al., 1997).
The sclerotium begins and ends the life cycle of S. sclerotiorum (Zimmer & Hoes, 1978).
Miceliogenic germination of sclerotia causes infection on tissues of the base of the plant to
produce root rot, stem rot and wilting (Davet et al., 1991). The hyphae penetrate through
tissue injuries or stomata by the cuticle, invading the intercellular spaces, and finally reach the
interior of cells. The fungus causes lesions visible at the base of the stem and wilting of
shoots due to obstruction of the conducting vessels (Pereyra & Escande, 1994). Secondary
contamination is possible through direct contact of the diseased tissue with healthy tissue
from neighboring plants (Davet et al., 1991). Carpogenic germination of sclerotia generates
the apothecia, which emerge on the soil surface and release the ascospores. In conditions of
high relative humidity above 70% a mature apothecium can produce up to 2 x 10
8
ascospores
over a period of several weeks. The ascospores are released at temperatures of 3ºC to 22ºC,
with greater intensity between 19ºC and 20ºC. Temperatures above 25ºC and relative
humidity below 35% are limiting for ascospore survival. The ascospores germinate in
favorable conditions and infect the host, causing mainly head and stem rot. The susceptibility
to infection of the sunflower head is higher in the period between the initial flowering and up
to two weeks after flowering. After a latent period of 15 to 40 days, the fungus invades the
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Regina M. V. B. C. Leite 172
parenchyma and causes tissue rotting. The optimum temperature for development of the
mycelium is between 18ºC and 25ºC. The sclerotia produced within and on the surface of the
colonized tissuse return to the soil and crop debris are responsible for the conservation of the
fungus (Zimmer & Hoes, 1978; Davet et al., 1991; Gulya et al., 1997). The sclerotia can
remain in the soil for many years, keeping intact their pathogenic power (Pereyra & Escande,
1994). Seeds are important vehicles for the dissemination of S. sclerotiorum, as sclerotia
mixed with the seeds or fungus mycelium colonizing the internal tissues (Mordue & Holliday,
1976; Zimmer & Hoes, 1978).
Sclerotinia wilt and head rot control is difficult due to the persistence of sclerotia viable
for a long time in the soil, to the fact that the fungus produces ascospores responsible for
aerial infection until long distances, the lack of effective chemical control and high
susceptibility of sunflower genotypes (Pereyra & Escande, 1994; Gulya et al., 1997). Thus,
the most effective control is based on an integrated program of measures, which include
several cultural practices.
Exclusion measures were adopted, starting in 1984, to prevent the introduction of the
fungus through contaminated seed from other countries. An ordinance of the Brazilian
Ministry of Agriculture, Livestock and Supply established the importation of seeds only from
sunflower production areas free of S. sclerotiorum.
Genetic resistance to Sclerotinia wilt and head rot has been studied in several countries.
Efforts have been made in breeding programs around the world aimed at finding resistance to
the pathogen, but little progress has been made (Zimmer & Hoes, 1978; Gulya et al., 1997).
All studies indicate a lack of immunity in the sunflower cultivated and other wild species,
similar to what is observed in all species of plants are affected by S. sclerotiorum (Gulya et
al., 1997). The resistance of the sunflower to S. sclerotiorum is partial and controlled by
multiple genes. The reaction of the same genotype may vary depending on the mode of
fungus attack, or one genotype can display a high level of resistance to basal rot and show to
be very sensitive to head rot. Furthermore, genes that are expressed in a stage of plant
development may be ineffective in another stage (Davet et al., 1991). Wild species of
Helianthus, such as H. resinosus, H. debilis, H. lenticularis and H. petiolaris, have high
resistance genes (Zimmer & Hoes, 1978; Davet et al., 1991). There are reports of variation
among cultivars for incidence of head rot, but apparently these differences are related to
greater plant height, which would provide less favorable conditions for fungus infection
(Zimmer & Hoes, 1978). Restorers and maintainers oilseed sunflower germplasms with
improved resistance to head rot have already been developed and released (Miller & Gulya,
2006; Miller et al., 2006), but until now there are no hybrids or commercial varieties that have
resistance level suitable for cultivation of sunflower in areas where this disease is endemic.
Methods including mapping of Quantitative Trait Locus (QTL), as they detect relationships
between phenotypic variation and gene polymorphisms in existing germplasm, can be useful
in dissecting complex traits in sunflower, like resistance to Sclerotinia diseases, thus
providing a valuable tool to assist in crop breeding (Fusari et al., 2012).
Crop rotation is a suitable practice to help management of S. sclerotiorum. The use of
crops resistant to this fungus, like grasses, serves to allow time for natural degradation of
sclerotia by their natural enemies. Due to susceptibility to S. sclerotiorum, sunflower
cultivation after soybean, canola, peas, beans, alfalfa, tobacco, tomatoes and potatoes, among
other crops, should be avoided. Rotation with non-host crops during three to five years
reduces the number of sclerotia in the soil and minimizes the impact of the sunflower root
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infection (Gulya et al., 1997). Weeds should be well controlled, because they may be
alternative hosts for S. sclerotiorum. An obvious recommendation, but very important, is to
avoid the use of seed mixed with sclerotia (Pereyra & Escande, 1994).
A key measure to prevent the occurrence of Sclerotinia wilt and head rot is the choice of
sowing date, in order to reduce periods of high humidity and low temperature during the
cycle, especially at flowering. In southern Brazil, for cultivation of sunflower after the harvest
of summer crops, sowing should be performed until mid-March and early-maturity genotypes
(100 days between emergence and harvest) should be used, considering studies of zoning for
climate risk, to prevent low temperature at the end of the cycle (Leite et al., 2000).
Other cultural practices are important to minimize the problems caused by S.
sclerotiorum. Spatial isolation is a measure effective in reducing the incidence of infection by
ascospores. Generally, it is recommended to choose areas at least 1 km away from crops
infected with S. sclerotiorum in the previous year (Masirevic & Gulya, 1992). It is convenient
to choose lower seeding densities and larger spacing so as to enable adequate plant aeration
and decrease the chances of contact with diseased adjacent plants (Zimmer & Hoes, 1978).
Chemical control has not proven to be effective for several reasons. For sunflower, there
are no products with systemic efficiency (Davet et al., 1991). The duration of flowering and
consequently the susceptibility to head infection requires at least two or three preventive
sprays with fungicides. Furthermore, the penetration of products in floral organs is very
difficult (Davet et al., 1991) and the fungicide needs to be applied to the head face to be
effective (Masirevic & Gulya, 1992). In Brazil, fluazinan is registered for control Sclerotinia
head rot in sunflower (Ministério, 2012), but there are no available data that indicates
efficiency on disease control until now.


Downy Mildew (Plasmopara halstedii)

Downy mildew is a major disease of sunflower in the world, to be potentially very
destructive. Although originating in North America, with the movement of sunflower around
the world, the pathogen is currently endemic in all places where sunflower is grown (Gulya et
al., 1997). Most countries have specific regulations to prevent the introduction or spread of
the pathogen (Pereyra & Escande, 1994), including Brazil, where it is considered quarantine
pest category "A". In Brazil, it was found for the first time in 1982, in the municipalities of
Santo Augusto and Veranopolis, Rio Grande do Sul, and later in 1983 in Londrina, Paraná.
The disease was found in experimental plots and diseased plants were eradicated and
immediately burned (Ferreira et al., 1983; Henning & França Neto, 1985). Plants with downy
mildew were observed again under experimental conditions in Londrina, in the late-1990s,
and were again eradicated (Leite et al., 2007). The damage caused by downy mildew may
result from premature death of plants, decrease of head size, decrease in oil content of
contaminated seeds or finally, complete yield loss (Pereyra & Escande, 1994; Gulya et al.,
1997).
This disease can show different types of symptoms, depending on the amount of
inoculum, plant age, reaction of the genotype and conditions of humidity and temperature
(Zimmer & Hoes, 1978; Almeida et al., 1981; Davet et al., 1991; Pereyra & Escande, 1994;
Gulya et al., 1997; Tourvieille-de-Labrouhe et al., 2000).
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Damping-off results from infection of the root system of the plants in the early stages of
development, under conditions of low temperature and high humidity. This symptom is
manifested by the presence of primary inoculum in the soil, which may affect the seedlings
before or after emergence, resulting in reduction of stand.
Plants with systemic infection have slow growth or stunting, chlorotic leaves with
abnormally thick, brittle stems with head erect and usually sterile (Figure 3). The initial
symptoms are yellowing of the first pair of true leaves, usually at the base of the leaves or
along the midrib. During flowering, plants infected systemically present height from 0.1 to
1.0m and do not follow the movement of the sun, while healthy plants have 1.5 to 1.8 m and
show heliotropism. In conditions of high humidity and low temperature, grayish-white
structures, composed of conidiophores and conidia, can be seen on the underside of chlorotic
leaves.
The causal agent of downy mildew is Plasmopara halstedii (Farl.) Berl. & de Toni. It is
an systemic obligate parasite, which produces intercellular mycelium with globular haustoria
and sporangia that arise through the stomata. The sporangia are thin and branched
monopodially to form zoosporangia at the ends of the branches. The zoosporangia break up,
release biflagellate zoospores (Zimmer & Hoes, 1978). The structures of the pathogen are
found in all tissues of the seedling and adult plant, but never in contact with the
undifferentiated cells of meristematic tissues, nor in conducting vessels (Davet et al., 1991).


Figure 3. Downy mildew in sunflower.
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Disease Management in Sunflower 175
P. halstedii causes disease in at least 80 species of 35 genera belonging to the subfamily
Asteroidae and Cichorioidea (Gulya et al., 1997). Besides H. annuus, other species of
Helianthus, such as H. argophyllus, H. debilis, H. divaricatus, H. grosseserratus and, as well
as other genera of the family Asteraceae (including Ambrosia, Artemisia, Bidens, Centaurea,
Gerbera, Solidago, Vernonia and Xanthium) are susceptible to downy mildew pathogen
(Davet et al., 1991; Gulya et al., 1997).
The life cycle of P. halstedii starts with oospores, which are resistance structures with
thin wall that are produced sexually (Gulya et al., 1997). The oospores occur in contaminated
debris from the previous sunflower crop, as well as within the seeds from systemically
infected plants. After the winter, the oospores germinate, especially in moist conditions of
spring. Some oospores, however, remain dormant for up to 14 years (Zimmer & Hoes, 1978).
The percentage of diseased plants drops considerably from the sixth year (Davet et al., 1991).
The oospores germinate producing thin-wall zoosporangia which produce the biflagellate
zoospores. In contact with the host tissue, especially primary roots and hypocotyls of newly
emerged seedlings, the zoospore encysts and emits haustoria into the host cell (Zimmer &
Hoes, 1978). It sporulates on the surface of the invaded tissues by producing zoosporangia
that are responsible for secondary infections. With the advancement of the crop cycle, the
male (antheridia) and female (oogonium) organs of the pathogen are formed in the
intercellular spaces of the roots, stem, and often seeds. Fertilization occurs, giving rise to a
thin wall oospore. Finally, the oospore returns to the soil, completing the life cycle of P.
halstedii (Zimmer & Hoes, 1978).
Disease incidence, type and severity of downy mildew symptoms are determined by the
nature and quantity of the inoculum, by plant age at the time of infection and by
environmental conditions (Zimmer & Hoes, 1978; Pereyra & Escande, 1994). The disease is
favored by high rainfall conditions (relative humidity higher than 95%) and temperature
between 15ºC to 18ºC (Davet et al., 1991).
The nomenclature used to describe the races of downy mildew was very ambiguous,
since races were called by the name of the region of origin (Race Red River, European race),
numbers (races 1-9 of American nomenclature) or letters (races A to D of French
nomenclature). To standardize the nomenclature, a set of nine publicly available lines was
established to be used as differential lines named D1 to D9 regrouped into three sets of three
lines (triplets). To encode the race, a score is assigned to each triplet, based on the reaction of
susceptibility or resistance of each differential line. If the first line of a set of three is
susceptible, it imparts a value of 1; if the second line is susceptible, it imparts a value of 2;
and if the third line is susceptible, it imparts a value of 3. In the case of resistance genotype it
imparts a value of zero. The score is the sum of the coefficients assigned to triplet. The final
race name will be a 3-digit code, one digit from each of the three sets of lines (Gulya et al.,
1998).
In the world, at least eleven physiological races of P. halstedii have been reported
affecting sunflower (Tourvieille-de-Labrouhe et al., 2000). In Brazil, there was, in 1982, the
occurrence of race 2 American (race 300 in the current nomenclature) (Henning & França
Neto, 1985). In recent reports, in Londrina, race 330 or former American race 7 was
identified (Leite et al., 2007). Former American race 7 is also prevalent in Argentina (Castaño
et al., 1998).
Exclusion measures have been adopted, since 1984, to prevent the entrance of downy
mildew in Brazil. An ordinance of the Brazilian Ministry of Agriculture, Livestock and
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Supply banned the importation of sunflower seeds and other common species of the genus
Helianthus and tubers of H. tuberosus, when coming from the following countries: Argentina,
Canada, Chile, Spain, United States, France, Hungary, Iran, Israel, Serbia and Montenegro
(former Yugoslavia), Japan, Jordan, Pakistan, Dominican Republic, Romania, Russia, Czech
Republic (former Czechoslovakia) and Uruguay, and other countries where the pathogen is
found. Imports from other countries are restricted to seeds produced in areas free of downy
mildew. Since 1995, following the harmonization of quarantine procedures for Mercosur
countries, seeds can be imported from Argentina, Paraguay and Uruguay (Leite & Oliveira,
1998). However, there is a continuous threat of reintroduction of P. halstedii by infected
seeds, especially from Argentina, where the disease is important (Pereyra & Escande, 1994).
The use of resistant cultivars is the most secure method to prevent the disease (Pereyra &
Escande, 1994). The resistant genotypes produce defense reactions against the pathogen,
forming a barrier to disease progression, and the disease does not become systemic (Davet et
al., 1991; Gulya et al., 1997). The resistance to downy mildew is oligogenic dominant and
controlled by several Pl genes. At least nine genes for resistance to downy mildew (Pl1 the
PL9) are the most widely used in breeding programs (Davet et al., 1991). The lines of
germplasm from the USDA (United States) HA-335 to HA-339 and RHA 340 have resistance
to all known races of P. halstedii (Gulya et al., 1997; Gulya et al,. 1998). In France, cultivars
resistant to races 710 and 703, that are prevalent in the country, were used in at least 80% of
the crops, in 2000 growing season (Tourvieille-de-Labrouhe et al., 2000). Also, genotypes from
Argentina have incorporated downy mildew resistant genes (Pereyra & Escande, 1994).
Research on more durable resistance has been undertaken, including examples of host-
parasite interactions to integrate the pathosystem P. halstedii x H. annuus (Sakr, 2012).
Sunflower show a large variability for resistance to downy mildew that is independent of
major Pl genes giving race specific resistance in breeding programs. This quantitative
resistance could be used in breeding programs using a simple method to phenotype progenies.
Infection of the first pair of true leaves and observation of the proportion of plants showing
systemic downy mildew symptoms appear as a promising technique (Tourvieille-de-Labrouhe
et al., 2008).
Cultural measures are also important in the prevention of downy mildew. These include
crop rotation for four years and the destruction of volunteer plants that emerge after
harvesting sunflower (Davet et al., 1991; Gulya et al., 1997).
Chemical control through foliar sprays with fungicides is not recommended in France,
since this treatment can lead to selection pressure on the population of P. hasltedii, which can
answer manifesting resistance to fungicides (Davet et al., 1991; Tourvieille-de-Labrouhe et al.,
2000). Resistant races have been detected in this country since 1994 (Tourvieille-de-Labrouhe
et al., 2000).
Seed treatment with specific fungicides for P. halstedii, such as metalaxyl, is mandatory
in some countries, such as France and Argentina, in susceptible open pollinated varieties or
hybrids. Thanks to its systemic property, metalaxyl allows controlling primary contamination
and ensures good protection in the early stages of crop development (Davet et al., 1991;
Pereyra & Escande, 1994).



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Disease Management in Sunflower 177
Powdery Mildew - Erysiphe cichoracearum

Powdery mildew is a disease distributed worldwide, but occurs in greater intensity in
tropical areas where occasionally cause plant senescence in the flowering stage or later. In
temperate areas, powdery mildew is usually not observed until flowering and rarely has
economic importance (Zimmer & Hoes, 1978; Gulya et al., 1997).
The disease is characterized by the appearance of velvety white or gray structures on the
aerial parts of the plant, especially lower leaves, but occasionally on the stem and bracts
(Figure 4). The lesions may grow and coalesce, covering most of the plant surface. With the
evolution of the cycle, black dots can be observed randomly distributed in velvety areas
(Zimmer & Hoes, 1978; Almeida et al., 1981).
Powdery mildew is caused by the fungus Erysiphe cichoracearum (DC) ex Meret (syn.
Golovinomyces cichoracearum (DC) Heluta), which is an obligate parasite. The velvety
structures are mycelia, conidiophores and conidia of the fungus. The mycelium is usually well
developed. Spores are formed in long chains, have ellipsoid shape and size ranging from 25-
45 µm x 14-26 µm. At the end of the cycle, the fungus produces cleistotecia, which are black
structures responsible for survival of the pathogen, containing asci with two ascospores
(Kapoor, 1967).
E. cichoracearum is restricted to the Asteraceae family, causing powdery mildew in 230
species belonging to 50 genera. There are reports of at least 13 formae speciales of the fungus
(Kapoor, 1967).
Transmission is primarily through cleistotecia that survive from one growing season to
another. In some cases the conidia can also survive (Kapoor, 1967). Conidia are disseminated
primarily by wind, which can reach long distances. The optimum conditions for infection are
temperatures around 25ºC and relative humidity of 95%. The conidia do not germinate when
there is a water film on the leaf surface. The disease is favored by hot and dry periods
(Kapoor, 1967; Zimmer & Hoes, 1978).


Figure 4. Powdery mildew in sunflower.
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Although there are specific fungicides for powdery mildew, such as sulfur, chemical
control is not performed due to the low prevalence of the disease (Kapoor, 1967; Zimmer &
Hoes, 1978). In Brazil, fungicides azoxistrobin + ciproconazole and difenoconazole are
registered for control powdery mildew in sunflower (Ministério, 2012), but there are no
available data that indicates efficiency on disease control until now.
Few efforts have been made in the development of cultivars resistant to powdery mildew.
However, there seems to be wide differences in the response of different cultivars to the
pathogen (Zimmer & Hoes, 1978).


Phomopsis Stem Canker - Phomopsis helianthi

Phomopsis stem canker was first reported in Serbia and Montenegro (former Yugoslavia)
in 1980, and has been highly destructive in the countries of Eastern Europe and in France
(Davet et al., 1991; Masirevic & Gulya, 1992; Pereyra & Escande, 1994). The damage is
caused due to breakage and lodging of attacked plants, seriously damaging the crop.
Depending on climatic conditions, the degree of incidence can reach 50% to 80% of the
plants (Masirevic & Gulya, 1992; Pereyra & Escande, 1994; Gulya et al., 1997).
The first symptoms of the disease often occur in foci, usually after flowering. About 10 to
15 days after infection, small necrotic spots surrounded by a yellow halo appear at the margin
of lower or median leaves and evolve towards the midrib of the leaf. Infected leaves wither
and die quickly (Davet et al., 1991; Masirevic & Gulya, 1992; Pereyra & Escande, 1994;
Gulya et al., 1997).
The fungus grows toward the stem, where the most characteristic symptoms appear
(Figure 5). The lesions on the stem, always initiated in the axils of leaves, begin as small
brown spots and grow rapidly, becoming round or ellipsoidal, usually girdling the stem. The
fungus destroys the internal tissues of the lesions and the stem breaks easily, rendering the
plants subject to lodging (Figure 5). Small and dark pycnidia can be observed in diseased
tissues. The final symptom is the complete blight (Davet et al., 1991; Masirevic & Gulya,
1992; Pereyra & Escande, 1994; Gulya et al., 1997).


Figure 5. Phomopsis stem canker in sunflower.
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Disease Management in Sunflower 179
This disease is caused by Phomopsis helianthi Munt.-Cvet. et al., which teleomorph is
Diaporthe helianthi Munt.-Cvet. et al. (Davet et al., 1991; Masirevic & Gulya, 1992; Pereyra
& Escande, 1994). Another unidentified species of Phomopsis can also cause disease in
sunflower, but P. helianthi seems to be prevalent (Carriere & Petrov, 1990). Three new
species (P. gulyae, P. kochmanii and P. kongii) are associated with stem canker on sunflower
in Australia (Thompson et al., 2011).
Under natural infection, pycnidia begin to be formed soon after the lesions appear on the
stem. They can be found in leaves, but always fewer in number than the stem. The pycnidia
are globular, dark brown,with 120-190 µm in diameter, and often immersed in host tissue.
Hyaline beta-conidia, with 17 to 42 µm in length by 0.5 to 2 µm in width, are formed. The
developing perithecia can be found in sunflower residues in cortical tissues, individually or in
groups. Numerous globular, cylindrical asci, with 60 to 76.5 µm in length by 8.7 to 12.5 µm
in width, develop in the perithecia. After maturation, eight ascospores per ascus are released
(Masirevic & Gulya, 1992).
Excepted species of the genus Helianthus, no reports of other host plants of P. helianthi
are confirmed. However, a species of Phomopsis isolated from Xanthium italicum is reported
as pathogenic to sunflower (Carriere & Petrov, 1990).
The optimum temperature for fungal growth is around 25ºC. The spores infect plants in
conditions of high humidity for 12 to 15 consecutive hours. Frequent and abundant rains
result in increased infection. The fungus persists in crop debris as mycelium. Under
conditions of temperature between 18ºC and 20ºC and high humidity, perithecia are produced
and they release ascospores, which are then disseminated by wind and rainwater (Pereyra &
Escande, 1994). The fungus can also be found in sunflower seeds (Masirevic & Gulya, 1992).
The ascospores germinate in the insertion of the leaf and initiate infection through the
invasion of the petiole, eventually reaching the stem. The characteristic lesion of the disease
appears 25 to 30 days after initial infection of the leaf.
Increasing plant density favor increased disease incidence and severity, resulting in a
greater proportion of girdling stem lesions, detrimental to yield, because of earlier infection
under dense canopies. The number of girdling lesions per plant also increases with high N
fertilization (Debaeke & Moinard, 2010).
A large number of accessions of wild species of Helianthus have a satisfactory level of
resistance to Phomopsis stem canker: H. tuberosus, H. resinosus, H. decapetalus, H.
divaricatus, H. eggertii, H. giganteus, H. grosserratus, H. hirsutus, H. mollis, H. salicifolius,
H. nuttallii and H. radula. Interspecific crosses between cultivated sunflower and H.
argophyllus or H. tuberosus resulted in lines used for the development of commercial hybrids
with high disease resistance (Masirevic & Gulya, 1992; Gulya et al., 1997). The resistance is
controlled by many genes, with additive effects. Resistance to P. helianthi is connected
positively to resistance to Macrophomina phaseolina, Phoma oleracea var. helianthi-tuberosi
and drought, possibly attributed to linked genes (Masirevic & Gulya, 1992; Pereyra &
Escande, 1994; Deglène et al., 1999).
Cultural control practices such as the use of plant populations below 50,000 plants per
hectare, adequate nitrogen fertilization and crop rotation are necessary to reduce the disease
incidence. Moreover, incorporation or removal of contaminated crop debris helps to reduce
fungus inoculum (Davet et al., 1991; Masirevic & Gulya, 1992; Gulya et al., 1997; Debaeke
& Moinard, 2010).
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Benzimidazole-based fungicides have been used to control the disease in France and
Argentina (Davet et al., 1991; Pereyra & Escande, 1994). Chemical control with two aerial
applications of fungicides, the first in the vegetative stage V8 to V10 and the second during
flowering is recommended by Masirevic & Gulya (1992). Seed treatment has also been
proven effective (Davet et al., 1991). Despite minimizing yield losses, the use of fungicides is
not as efficient as the use of genotypes with resistance genes (Masirevic & Gulya, 1992).


Rust - Puccinia helianthi

Sunflower rust is an important disease in many regions of the world. Severe losses have
been attributed to this disease, which causes premature defoliation. Disease severity is greater
in areas of humid (Zimmer & Hoes, 1978; Pereyra & Escande, 1994; Gulya et al., 1997).
The high incidence of rust in São Paulo state, Brazil in the mid-1960s, was the main
factor responsible for the discouragement of sunflower cultivation in the northwest region of
the state at that time. This occurred due to the high susceptibility of cultivars used by
producers (Lasca, 1993).
Typical symptoms of sunflower rust are small round powdery pustules, with 1 to 2 mm
diameter, pale orange to black, distributed randomly over the entire surface of the plant
(Figure 6). They are more common in lower leaves, progressing to the top. Normally, the
pustules are surrounded by small yellow halos. At high levels of infection, stem, petiole and
floral parts may exhibit symptoms. The coalescence of pustules may occupy almost the entire
leaf surface, causing premature leaf senescence, which causes reduced yield and low seed
quality (Zimmer & Hoes, 1978; Almeida et al., 1981; Pereyra & Escande, 1994, Gulya et al.,
1997).
Rust is caused by Puccinia helianthi Schwein. The fungus develops the whole cycle in a
single host and produces two types of spores: uredospores and teliospores. The uredospores
are the orange powdery mass, which is characteristic of the disease, and are produced in
uredia, during favorable climate conditions for the pathogen. The uredia are formed on the
lower surface of the leaves and have a diameter of 1 mm. The uredospores are ellipsoidal or
sometimes cylindrical, with size ranging from 25-32 x 19-25 µm. Teliospores with 40-60 x
18-30 µm are produced for survival (Laundon & Waterson, 1965; Zimmer & Hoes, 1978;
Pereyra & Escande, 1994).
Puccinia helianthi is a specific pathogen of the genus Helianthus, affecting more than 35
annual and perennial species (Zimmer & Hoes, 1978; Pereyra & Escande, 1994). There are
several known races of the pathogen: nine races have already been reported in Canada, seven
in Australia and 10 in Argentina, and 20 virulence patterns have been detected by using a
series of nine differential lines in the United States (Laundon & Waterson 1965; Zimmer &
Hoes, 1978; Pereyra & Escande, 1994; Gulya et al., 1997).
The pathogen can be perpetuated in plants of the genus Helianthus, where uredospores
are produced. This is possibly the usual way of perpetuating the fungus in regions where
winter is not severe (Pereyra & Escande, 1994). The uredospores are transmitted to other
plants from contaminated crops, crop debris or volunteer plants. Air currents at high altitudes
may contribute to the spread of uredospores over long distances. The infection occurs shortly
after flowering, when uredospores are deposited on leaves and germinate under conditions of
high relative humidity (Pereyra & Escande, 1994).
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Disease Management in Sunflower 181

Figure 6. Sunflower rust.
The disease severity varies with age of the plant, environmental conditions and genotype
resistance. The pathogen is favored by temperatures of 18ºC to 22ºC and high relative
humidity and, under these conditions, can cause epidemics (Pereyra & Escande, 1994).
Measures to reduce the initial inoculum and reduce the risk of severe losses caused by
rust are recommended, as the elimination of sunflower volunteer plants, crop rotation for at
least three years and destruction of crop debris (Laundon & Waterson, 1965; Zimmer & Hoes,
1978; Pereyra & Escande, 1994).
Sulfur or copper-based fungicides, in spite of controlling this fungi, have not been used in
commercial crops (Laundon & Waterson, 1965).
The method of rust control that is universally used is based on resistant cultivars.
Selections and cultivars resistant to this fungus have been developed in countries like Russia,
Peru, Chile, Serbia and Montenegro (former Yugoslavia), United States and Argentina
(Laundon & Waterson 1965; Zimmer & Hoes, 1978; Pereyra & Escande, 1994; Gulya et al.,
1997). Recently, 107 genotypes were screened against sunflower rust under field conditions
in India but none of them were found to be immune to rust (Nargund et al., 2011). The rust
resistance is dominant and inherited by a single gene. Many sources of rust resistance are
known. Collections of wild sunflower, including H. annuus and H. petiolaris, represent a
reservoir of resistance genes that can be used in breeding. R1 and R2 genes have been widely
used for development of resistant cultivars (Zimmer & Hoes, 1978). Many cultivars have
developed resistance to race 1, more frequent and distributed worldwide. However, the use of
resistant cultivars may be limited due to the existence of races of the fungus or the selection
of races that can overcome this resistance (Pereyra & Escande, 1994).


CONCLUSION

Once installed on the crop, sunflower diseases are hard to control, due to the difficulties
of chemical application, because of the dynamics of plant growth, hindering or even
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preventing the entry of machinery in the field and lack of efficient application of fungicides,
requiring in some situations aerial application of fungicides.
Therefore, measures of disease management are mostly preventive and should not be
used alone. Thus, effective control is based on an integrated program, which includes zoning
for climate risk and diverse cultural practices.
Genetic resistance to diseases is highly desirable because it does not increase directly the
cost of production and many times can dispense other control measures. Studies on the
reaction of genotypes and breeding aiming resistance have been carried out for different
diseases and should be done continuously.
A key measure to minimize the occurrence and severity of diseases is the choice of
sowing date. Considering the different diseases and plant requirements, the date suitable for
sowing sunflower varies with different soil and climatic regions. It should be noted that the
indication of sowing date should be guided by studies of zoning for climate risk in order to
establish the date that will satisfy the requirements of the plant at different stages of
development, and that disfavor diseases.
Another important aspect is to use crop population around of 40,000 to 45,000 plants per
hectare. Sunflower requires deep soils with good nutrient levels and proper pH. Liming and
fertilization should always be made based on soil analysis and technical and economic
criteria.
As many sunflower pathogens are transmitted by seed, it is imperative to use healthy
seeds from known origin. With respect to chemical control, several fungicides are available
and can be used as tool for the farmers.
In addition to these measures, it is noted that sunflower should be included within a
system of crop rotation, returning in the same area only after at least four years.


REFERENCES

Allen, S. J., Brown, J. F., Kochman, J. K. (1983). Effects of temperatures, dew period, and
light on the growth and development of Alternaria helianthi. Phytopathology 73, 893-
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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 8



RECENT ADVANCES FOR DEVELOPING RESISTANCE
AGAINST PLASMOPARA HALSTEDI I IN SUNFLOWERS


Osman Radwan
1,2,
*

1
Department of Natural Resources and Environmental Sciences,
University of Illinois at Urbana-Champaign, Urbana, IL, US
2
Department of Plant Production, College of Technology and Development,
Zagazig University, Zagazig, Sharkia, Egypt


ABSTRACT

Downy mildew caused by the Oomycete Plasmopara halstedii is one of the most
important diseases of sunflower (Helianthus annuus L.). During the last two decades, a
significant progress has been reported in different research areas of plant pathology,
genetics and genomics. For example, molecular and cellular approaches have been
employed to characterize two types of resistance and the results enhanced our
understanding of sunflower-P. halstedii interactions. In addition, a comparative genomics
approach has been used to identify and isolate resistance gene candidates (RGCs)
belonging to NBS-LRR class. These RGCs were mapped to different linkage groups and
associated with the resistance to several important diseases. In particular, molecular
markers derived from RGCs were used for mapping and positional cloning of genes
conferring resistance to downy mildew disease. This progress in genetic resistance will
certainly help breeders to develop new sunflower lines with unique and durable resistance
to P. halstedii.

Keywords: Sunflower, downy mildew, Plasmopara halstedii, resistance, mapping






*
Corresponding Author address. Email: oradwan@illinois.edu.
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Osman Radwan 188
INTRODUCTION

Sunflower (Helianthus annuus L.) is one of the important seed oil crops in the world and
it grows on all of the continents. In 2012, Ukraine was the major producer followed by
Russian Federation, Argentina, Romania and France (Figure 1; www.FAO.org).


Figure 1. Top nine producers of sunflower in 2012. Yield production was estimated by metric tons
(MT).
Sunflower oil is the second most important vegetable crop oils and it is comparable to
corn and safflower oils in unsaturated fatty acids with a high level of linoleic fatty acid. Many
diseases attack sunflower causing a significant yield loss and poor quality of the produced oil.
Among these diseases is downy mildew, which is caused by the Oomycete P. halstedii. In
this book chapter, I will review the recent research findings focused on downy mildew: i) the
causal agent of downy mildew, ii) genetic resistance of sunflower to downy mildew disease,
iii) two types of sunflower resistance to downy mildew, and iv) gene discovery and molecular
mapping.


The Causal Agent of Downy Mildew

Downy mildew disease is caused by Plasmopara halstedii (Farlow) Berlese and De
Tony, a member of the Oomycota, which includes several important plant diseases such as the
late blight disease of potato and tomato that is caused by Phytophthora infestans. This
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Recent Advances for Developing Resistance … 189
pathogen was discovered in North America in 1922 (Young and Morris, 1927) whereas it was
reported in France 44 year later (Delanoë, 1971).


Figure 2. Plasmopara halstedii disease cycle (modified from Tourvieille de Labrouhe et al., 2000).
Like other downy mildew pathogens, it is an obligate parasite and can be dispersed by
wind via infected seeds, but is mostly soil-borne pathogen. Mobile zoospores in the soil infect
seedlings through the roots and the systemic infection follows. Normally, downy mildew
infection leads to the death of the sunflower host, however some plants can survive after
infection with severe dwarfism, yellowing leaves and a white mass of mycelium on the
undersides of their leaves. This pathogen is diploid, homothallic, and it reproduces both
sexually and asexually phases (Figure 2).
The pathogen uses the sexual phase (oospores) as overwintering propagates, while it uses
the asexual phase (zoospores) for the secondary infection during the growing season of
sunflower from spring to autumn.


Genetic Resistance of Sunflower to Downy Mildew Disease

P. halstedii is an obligate parasite with several physiological strains that can be
distinguished by their differential virulence on sunflower genotypes (Tosi and Beccari, 2007;
Gulya et al., 1991; Rashid, 1993). Control of this disease is achieved mainly by genetic
resistance that is conditioned by major genes denoted Pl (for Plasmopara; Vranceanu and
Stoenescu, 1971). These Pl genes provide complete, dominant and race-specific resistance.
Several loci, involved in resistance, to P. halstedii have been mapped in different linkage
groups of sunflower. These loci include: (I) the Pl1/Pl6 locus on linkage group (LG) 8
(Bouzidi et al., 2002; Gedil et al., 2001); (II) the Pl5/Pl8 and Pl21 loci on LG13 (Vincourt et
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Osman Radwan 190
al., 2012; Radwan et al, 2003; 2004); (III) the PlArg locus on LG1 (Dußle et al., 2004); (IV)
the Pl13 and Pl16 loci on LG1 (Liu et al., 2012; Mulpuri et al., 2009) and (V) a newly
mapped locus (Pl14) on LG1, which is located independently from the PlArg locus (Bachlava
et al., 2011; Dußle et al., 2004; Wieckhorst et al., 2010).
These genes form a cluster, however it is not clear if each cluster contains a single gene
that confers resistance to a particular race of the pathogen or each cluster comprises several
genes and each one confers resistance to a particular race of the pathogen. There is a variation
of resistance level from one cluster to another, for example some clusters, such as Pl6, have
been overcome by the new fungal races while others, such as Pl8 confer resistant to several
races of the pathogen. It is interesting to report that these clusters contain resistance to other
important diseases such as rust.
In a recent work, Franchel et al. (2013) explored the previous work related to Pl6 locus
(Bouzidi et al., 2002) and resistance gene candidates of sunflower (Radwan et al., 2008) for
positional cloning of Pl6 locus. They employed 13 RGCs markers (Bouzidi et al., 2002)
linked to the Pl6 locus to develop two codominant STS markers (NBS8 and NBS1-3)
flanking the Pl6 locus. These two codominant markers were used to identify recombinant
plants among 3,072 F2 individuals. Using this strategy, they have identified 160 F2
recombinant individuals harboring Pl6 locus. Each family of F3 and/or F4 from recombinant
individuals was tested using 300, 703 and 710 races of P. halstedii. In the Pl6 locus, a
segregation of downy mildew resistance to each race has been reported, suggesting that this
cluster contains different sub-clusters and each one confers resistance to a particular race of
the pathogen (Franchel et al., 2013).


Two Types of Sunflower Resistance to Downy Mildew

Depending on the host-pathotype combination, two types of sunflower-P. halstedii
incompatibility reactions have previously been identified. Type I resistance can restrict the
growth of the pathogen in the basal region of the hypocotyls, whereas Type II cannot, thus
allowing the pathogen to reach the cotyledons. In a microscopic study, Mouzeyar and his
colleagues (Mouzeyar et al., 1993) demonstrated that both susceptible and resistant sunflower
lines could be invaded by P. halstedii. Five days after root infection, a hypersensitive reaction
(HR) develops within the hypocotyls of both types I and II of resistant plants, but the fate of
the infection depends on both the resistance gene in the host and the avirulence (avr) gene in
P. halstedii. In plants with Type I resistance, the pathogen is restricted to the basal area of the
hypocotyls, whereas in plants with Type II resistance, the hypocotyls could be completely
invaded and the cotyledons are reached by the pathogen, although in most cases the pathogen
does not reach the true leaves (Mouzeyar et al., 1993; 1994). This phenomenon was first
described by Gulya et al. (1991) and Sackston (1992) and so-called Cotyledon Limited
Infection (CLI), which characterizes the Type II resistance.
To explore the molecular and cellular characterization of both types of resistance,
Radwan et al. (2011) have used five sunflower lines that are different in their responses to P.
halstedii Pathotypes. HA89 is a susceptible line. HA335 and RHA419 confers Type I
resistance to Pathotype 300 (P 300). RHA340 and 29004 confer Type II resistance to P 300.
Macroscopic observations from susceptible plants (HA89) showed normal downy mildew
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Recent Advances for Developing Resistance … 191
symptoms such as stunting, leaf chlorosis and fungal sporulation in both cotyledons and
leaves (Figure 3A, Radwan et al., 2011).


Figure 3. Morphological, cellular and molecular (Radwan et al., 2011) differences between cotyledons
of sunflower lines HA89 (susceptible), HA335 (Type I) and RHA340 (Type II) after 15 days of root
infection by P. halstedii. Pathogen sporulation and HR can be noted on cotyledons in case of
susceptible (HA89) and resistant Type II (RHA340) interactions, which is confirmed by the
microscopic examination of cotyledon sections at 12 (B) and 15 dpi (C). The arrows point to haustoria
of P. halstedii in the compatible reaction and the HR in incompatible combination. Black bars=20 µm.
D, Semi-quantitative PCR detected the Ph-tef1 gene of P. halstedii (higher band, 331 bp) in cotyledons
of susceptible and type I and II resistant plants. Actin (lower band, 129 bp) was used as an internal
reference to quantify sunflower cDNA. M=DNA ladder (Invitrogen, Carlsbad, CA), C=control and 1.5
to 15=days post infection. The microscopic observation and semi-quantitative PCR are derived from
one representative experiment of three independent biological replications.
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Osman Radwan 192

Figure 4. Morphological, cellular and molecular (Radwan et al., 2011) differences between hypocotyls of the
susceptible line (HA89), and Type I (HA335) and II (RHA340) resistant plants of sunflower to P. halstedii.
A, HR is extended on hypocotyls of RHA340 (Type II) while restricted to the basal part of hypocotyls in the
case of Type I, no such reaction was observed in susceptible hypocotyls. The boxed B, C and D parts are
corresponding to basal, middle and apex parts of hypocotyls, respectively. Sections were microscopically
examined from the basal region of hypocotyls at 9 dpi (B) the middle part (C) at 12 dpi and the apex part of
hypocotyls at 15 dpi (D). The arrows point to P. halstedii haustoria in a compatible reaction and the HR in an
incompatible reaction. All size bars are 20 µm except for the susceptible B and C (column 3), which are 10
µm E, Semi-quantitative PCR was used to detect the Ph-tef1 gene of P. halstedii (higher band, 331 bp) in
hypocotyls of susceptible and type I and II resistant plants. Actin (lower band, 129 bp) was used as an
internal reference to quantify sunflower cDNA. M= DNA ladder (Invitrogen, Carlsbad, CA), C=control and
1.5 to 15=days post infection. The microscopic observation and semi-quantitative PCR are derived from one
representative experiment of three independent biological replications.

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Recent Advances for Developing Resistance … 193
In both types of resistance, the infected plants showed neither stunting nor chlorosis, and
they appear virtually healthy. In Type II resistance, the plants showed an initiation of necrosis
with slightly sporulation on some cotyledons (Figure 3A, column 3, Radwan et al., 2011)
while in Type I resistance neither sporulation nor necrosis were observed (Figure 3A, column
2, Radwan et al., 2011). The susceptible plants (HA89) and both types of resistant plants were
all invaded by P. halstedii, but about 5-6 days post infection (dpi), an HR developed within
the basal region of the hypocotyls in both types of resistance. While the HR can extend
halfway along the hypocotyls in Type II resistance, the susceptible response is characterized
by the absence of such a reaction (Figure 4A, column 1, Radwan et al., 2011).
From both the susceptible and the resistant plants, microscopic sections from the basal
region of hypocotyls were examined at 9 dpi. In the compatible reaction, intercellular hyphal
growth with abundant haustoria and an absence of necrotic parenchyma cells was apparent in
the hypocotyls (Figure 4A, column 1, Radwan et al., 2011), whereas in the incompatible
combination of both types of resistance, the parenchyma cells penetrated by haustoria had
undergone cell death (Figure 4A and B, columns 2 and 3, Radwan et al., 2011). To follow the
progress of the pathogen towards the cotyledons, microscopic sections from middle and upper
parts of hypocotyls from susceptible and resistant lines were examined at 12 and 15 dpi,
respectively. Microscopic observations showed the presence and absence of the pathogen in
susceptible and Type I resistant plants, respectively. On the other hand, Type II resistance is
characterized by a collapse of some cells, indicative of an HR (Figure 4C and D, column 3;
Radwan et al., 2011). Semi-quantitative PCR detected P. halstedii in hypocotyls of
susceptible and Type I and II resistant plants (Figure 4E, columns 1, 2 and 3; Radwan et al.,
2011). The pathogen was detected in the hypocotyls of susceptible and Type II-resistant
plants from 1.5 until 15 dpi, whereas in Type I resistance the pathogen was detected from 1.5
dpi until 9 dpi. Semi-quantitative PCR (Figure 4E; Radwan et al., 2011) reflected the
abundant growth of the pathogen in the susceptible line and substantially less growth of the
pathogen in the Type II-resistant lines.
Morphological observations of cotyledons (Figure 3A; Radwan et al., 2011), microscopic
examination (Figure 3B and C; Radwan et al., 2011), and semi-quantitative PCR (Figure 3D;
Radwan et al., 2011) provided evidence that the pathogen reaches cotyledons of both
susceptible and Type II-resistant plants. Hyphal growth with abundant haustoria and an
absence of necrotic parenchyma cells was apparent in the cotyledons of susceptible plants at
12 and 15 dpi (Figure 3B and C, column 1; Radwan et al., 2011). Microscopic examination of
sections from cotyledons of Type I-and II-resistant plants, at 12 and 15 dpi, confirmed a HR
in the case of Type II resistance, whereas the cells appeared normal in the case of Type I
resistance (Figure 3B and C, column 2; Radwan et al., 2011). In Type II resistance, cotyledon
cells close to the hyphae showed a collapse indicating a HR (Figure 3B and C, column 3;
Radwan et al., 2011). By using specific primers from the elongation factor gene (Ph-tef1) of
P. halstedii (Figure 3D; Radwan et al., 2011) PCR products confirmed the presence of the
pathogen in the cotyledons of susceptible and Type II-resistant plants. The pathogen reached
the cotyledons of susceptible plants early (6 dpi), and then its growth increased gradually
until 15 dpi. On the other hand, the pathogen did not reach the cotyledons of Type II-resistant
plants until 12 dpi and then its growth decreased by 15 dpi. These results (Radwan et al.,
2011) suggested that pathogen growth differs according to the host-pathogen combination.


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Osman Radwan 194
Relationship between Resistance Type, Sequence and Transcript

It is interesting to note that Type II resistance is effective against a large number of
pathotypes. For example, the RHA340 line, which develops sporulation on cotyledons, is
resistant to all known pathotypes (Radwan et al., 2003). It seems that Type II resistance takes
some time to be effective in the plant, but is quite sufficient in the field (Vear et al., 2003).
Radwan et al. (2011) investigated the possible relationships between types of resistance,
sequence types and the transcript activation in response to infection. They employed qRT-
PCR to quantify the transcript accumulation of four sunflower RGCs. PU3 and HA-NTIR11
were previously described and mapped to LG6 and 13, respectively (Bouzidi et al., 2002;
Radwan et al, 2004). RGC203 and RGC151 associated with Pl14 (Bachlava et al., 2011) and
Pl
Arg
(Dußle et al., 2004; Wieckhorst et al., 2010) genes, respectively and were mapped to
different locations of LG1 of sunflower (Radwan et al., 2008). An increase of the transcript
expression of RGC203 and HA-NTIR11 was detected in infected hypocotyls of 29004 and
RHA340 lines infected by P 300. In contrast, no such increase of RGC151 and PU3
transcripts was shown (Radwan et al., 2011). The products of resistance gene appear to
interact directly or indirectly with products of pathogen avirulence (Avr) genes (Hammond-
Kosack and Parker, 2003; Jones and Dangl, 2006). While the pathogen proteins provide the
ligands, R proteins employed as receptors and their genes are usually constitutively expressed
at low levels to guarantee the presence of these receptors in the absence of pathogens, in order
to detect the pathogen rapidly (Jones and Dangl, 2006).
Both 203 and Ha-NTR11 RGCs (Radwan et al., 2011; 2004) are constitutively expressed
in mock-infected plants, with dramatically enhanced expression upon P. halstedii infection,
suggesting that the amount of the R-gene product may be important for Type II resistance
mediated by such genes. In Type II-resistant plants, the pathogen grows through the entire
hypocotyl and substantially more tissue is subjected to the infection and HR compared with
Type I resistance. Gene expression of HA-NTIR11 and RGC203 during the defense response,
probably leads to an HR similar to that observed with the RPW8.1 and RPW8.2 genes, where
their enhanced transcription via salicylic acid feedback is necessary to induce the HR in
Arabidopsis plants infected by Erysiphe cichoracearum (Xiao et al., 2003). The induction of
an HR in the upper part of plant hypocotyls (resistant Type II) was demonstrated by
microscopic examination and by the accumulation of hsr203j transcript as a marker of HR
induction. The hsr203J gene is specifically activated during an HR (Pontier et al., 1994) and
occurs in various incompatible combinations involving tobacco and sunflower (Pontier et al.,
1994, Radwan et al., 2005A; 2005B). In Type I resistance, it seems that the constitutive
expression of PU3 and RGC151 is enough to guarantee recognition (either directly or
indirectly) of the products of pathogen avirulence (Avr) genes and to induce the HR, which is
localized only in the basal part of the hypocotyls. In this case the HR is strong enough to
restrict the spread of the pathogen towards the upper part of the hypocotyls. On the other
hand, in Type II resistance both HR and systemic acquired resistance (SAR) are employed
together to halt the pathogen growth. HR is initiated as a consequence of direct or indirect
recognition between the pathogen avr protein and host R protein (Hammond-Kosack and
Parker, 2003), while SAR is initiated by the transmitted signal from the infected area of
hypocotyls (Figure 5; Radwan et al., 2005B).
Amino acid sequence analysis of RGC203 and RGC151 revealed that these proteins
belong to the CC-NBS-LRR and TIR-NBS-LRR classes of plant resistance genes,
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Recent Advances for Developing Resistance … 195
respectively, and have a high degree of similarity to other plant R-proteins. In sunflower,
sequences from the Pl6 and Pl
Arg
genes belong to the TIR-NBS-LRR class (Bouzidi et al.,
2002; Radwan et al., 2011), whereas those from the Pl8 and Pl14 genes belong to the CC-
NBS-LRR class (Radwan et al., 2004; 2011). Both the Pl6 and Pl
Arg
(TIR-NBS-LRR), and
the Pl8 and Pl14 (CC-NBS-LRR) clusters harbor resistance genes to sunflower downy
mildew P 300.


Figure 5. (Radwan et al., 2005B) Model describing the events leading to the resistance of sunflower to
the oomycete Plasmopara halstedii after root infection. The parasite penetrates the host roots where no
hypersensitive reaction (HR) is observed. When it reaches the lower part of the hypocotyl, an HR takes
place which may restrict oomycete growth without killing it. In addition, the SAR, which is initiated in
the upper parts of the seedlings, contributes to the weakening of the parasite as it progresses upwards.
In this model, sequential action of both the HR and the SAR are required to enable recovery of the
seedling from the disease.
The four sequences of sunflower that were mapped in different genomic locations may
confer resistance to P 300 of the downy mildew pathogen. The complexity and independence
of the four clusters facilitate selection for resistance to multiple pathotypes and the
development of resistant lines to multiple pathotypes (Radwan et al., 2008).
As the HA335 and RHA419 lines carry Pl sequences belonging to the TIR-NBS-LRR
class of R proteins (Bouzidi et al., 2002; Radwan et al., 2011) and the RHA340 and 29004
lines carry Pl sequences belonging to the CC-NBS-LRR class (Radwan et al., 2004; 2011),
genetic analysis is needed to verify if there is a relationship between the sequence types and
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Osman Radwan 196
sunflower resistance types. Because the N termini of sequences related to the Pl6 and Pl
Arg

genes on one hand, and to the Pl8 and Pl14 genes on another hand are different, it is possible
that one or more effector proteins from P 300 of P. halstedii are recognized by two different
R-proteins of sunflower. Other different mechanisms may be suggested [e.g., different guard
proteins (Hammond-Kosack and Parker, 2003)], depending on the N-terminus of the gene.


Gene Discovery and Molecular Mapping

Isolation and identification of Resistance Gene Candidates (RGCs) is considered as one
of the most important works in sunflower research program. To achieve this goal, Radwan et
al. (2008) targeted nucleotide binding site (NBS) leucine rich repeat (LRR) proteins as three-
fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants
encode NBS-LRR genes. Two strategies have been pursued to identify new RGCs, by
targeting the conserved sequences of NBS-LRR genes and by mining ESTs in the Compositae
Genome Program Database (CGPdb; http://cgpdb.ucdavis.edu/ cgpdb2/).
The comparative genomics approach has been employed (Radwan et al., 2008) to isolate
sunflower RGCs by designing two pairs of degenerate oligonucleotide primers
complementary to highly conserved NBS sequence motifs (Meyers et al., 1999; He et al.
2004). To enrich the sources of RGCs, they (Radwan et al., 2008) used genomic DNA
extracted from both cultivated and wild sunflower (H. annuus, H. argophyllus, H. deserticola,
H. paradoxus and H. tuberosus). The designed primers were predicted to amplify about 500
bp fragments from both TIR and non-TIR-NBS-LRR encoding genes. Using this strategy,
they (Radwan et al., 2008) cloned and sequenced 1,248 amplicons with 84.3% homologous to
NBS-LRR encoding genes from sunflower or other plant species. Among those, 784
sequences had uninterrupted ORFs from the P-loop to the GLPLAL motif, 542 were unique
and lacked stop codons or frame shift mutations, and 242 harbored stop codons or frame shift
mutations and were suspected to be pseudogenes. Sequencing errors should have been
minimal because each clone was bidirectionally sequenced. The 542 unique NBS sequences
ranged in length from 164 to 173 amino acids. Three-fourths of the newly isolated NBS
sequences without stop codons belong to TIR class (Meyers et al., 1999, 2003; Pan et al.,
2000). TIR-NBS-LRR class represented 53.6% and 93.9% of NBS-LRR RGCS isolated from
H. annuus H. tuberosus, respectively. On the other hand TIR-NBS-LRR class represented
95.3% of the NBS sequences harboring stop codons.
By mining 284,745 Helianthus ESTs in the Compositae Genome Program Database,
Radwan et al. (2008) have identified 88 unigenes homologous to NBS-LRR encoding R-
genes. The ESTs spanned pre-NBS, NBS, LRR-, or post-LRR domains or combinations
thereof. ESTs spanning NBS domains were aligned with previously and newly isolated NBS
domain sequences (Gedil et al., 2001; Radwan et al., 2003; Plocik et al., 2004). The sequence
alignment showed that nucleotide identities ranged from 28 to 100%, while amino acid
similarities ranged from 16 to 100%.
The sunflower NBS sequences (Radwan et al., 2008) clustered into 14 groups with strong
bootstrap support, eight solely comprised of TIR-NBS and six solely comprised of non-TIR-
NBS sequences. To map these RGCs to sunflower genetic map, Radwan et al. (2008)
developed SSCP and INDEL markers for 196 RGCs. The parents of the RHA280 x RHA801
(Tang et al., 2002) and NMS373 x ANN1811 (Gandhi et al., 2005) mapping populations were
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Recent Advances for Developing Resistance … 197
screened for presence-absence polymorphisms (dominant INDELs), SSCPs, or both.
Collectively, 167 NBS-LRR loci were mapped, nine by genotyping INDEL markers and 158
by genotyping SSCP markers. Markers developed from NBS-LRR RGCs were integrated into
a framework of previously mapped SSR and INDEL markers distributed throughout the
sunflower genome. RGCs markers were used for mapping and positional cloning of
resistance to important diseases of sunflower such as downy mildew, rust and oribanche
(Bachlava et al., 2011, Franchel et al., 2013; Wieckhorst et al., 2010; Radwan et al., 2009;
Vincourt et al., 2012; Mulpuri et al., 2009; Liu et al., 2012; Gong et al., 2013).
In conclusion, sunflower is not only an important oil crop, but also a promising biodiesel
crop demanding more future research to reduce the yield loss from diseases. Advances of
molecular breeding and molecular biology research areas focused on genetic mapping and
understanding resistance mechanisms of sunflower to P. halestdii opened the door for more
perspective research projects to improve sunflower resistance to downy mildew and other
important diseases.


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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 9



EFFECTS OF CROP MANAGEMENT
ON THE INCIDENCE AND SEVERITY
OF FUNGAL DISEASES IN SUNFLOWERS


P. Debaeke
1,4
*
, E. Mestries
2
, M. Desanlis
1,4
and C. Seassau
3,4

1
INRA, UMR AGIR, CS 52627, 31326 Castanet-Tolosan, France
2
CETIOM C/O ENSAT, Auzeville, Tolosane, France
3
Ecole d‘Ingénieurs de Purpan, Toulouse, France
4
Université de Toulouse, INPT, UMR AGIR, Toulouse, France


ABSTRACT

The main fungal diseases of sunflower are black stem disease (Phoma macdonaldii),
downy mildew (Plasmopara halstedii), Phomopsis stem canker (Phomopsis helianthi)
and white mold (Sclerotinia sclerotiorum). Thanks to genetic improvement, the main
method of disease control is the use of varieties with good tolerance or resistance. Crop
management includes a set of measures to reduce the risk of fungal attacks and to limit
the impact of these attacks on the crop. Cultural control is based on prevention (e.g., by
crop rotation), escape (e.g., by adjusting the sowing date) or the promotion of
microclimatic conditions unfavorable to pathogens (through control of vegetative
growth), all of which have been shown to be effective. A suitably timed combination,
organized at the regional level, of cultural, genetic, chemical and biological control
methods is the key to an effective, integrated and sustainable control of diseases in
sunflower.

Keywords: Sunflower, fungal diseases, crop management, Phomopsis helianthi, Phoma
macdonaldii




*
Corresponding author: Email: debaeke@toulouse.inra.fr.
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P. Debaeke, E. Mestries, M. Desanlis et al. 202
INTRODUCTION

Sunflowers were grown in France on 680 000 ha in 2012 and the grain yield averaged 2.3
t.ha
-1
, while potential yield is about 4 t.ha
-1
in experimental plots where most limiting factors
are controlled. Water stress and fungal diseases are the two most frequent and detrimental
factors limiting crop yield (Jouffret et al., 2011). A dozen phytopathogenic fungi are currently
reported as potential problems for this oilseed crop, but only four diseases cause significant
yield losses nationally: downy mildew (Plasmopara halstedii), phoma (Phoma macdonaldii),
phomopsis (Phomopsis helianthi) and sclerotinia (Sclerotinia sclerotiorum) (Cetiom, 2002).
Their main biological and ecological characteristics are reported in Table 1.
The incidence and severity of a disease result from complex interactions between a
pathogen, a host plant and their common biophysical and biological environment defined by
soil, weather and crop management.
The biological cycle of sunflower diseases conforms with the general scheme proposed
by Lucas (2007) in Figure 1.
Reducing the harmfulness of disease attacks in a field in a given year is the main
objective of crop protection. But reducing injury (sporulating lesions), even in the absence of
detrimental effects on annual crop yield, is a way to reduce the production of primary
inoculum and thus future epidemics. For example, in Argentina, where verticillium is a
serious problem on sunflower, the response of varieties is evaluated from the symptoms they
express, but also on their ability to multiply the fungal inoculum (by assessing the amount of
microsclerotia in the stem pith). Hence the planning of disease control methods must consider
both temporal and spatial dimensions as soon as the fungus is able to spread widely.
To avoid (or at least limit) the injury (symptoms) and the damage (loss of yield and
quality of the crop), farmers have three possible control strategies to trigger (Delos et al.,
2004; Attoumani-Ronceux et al., 2010):

 prevent the disease risk (prophylactic methods)
 avoid the contamination when inoculum is still present
 mitigate crop injury and damage after infection

There are four ways to control fungal diseases: genetic (through the choice of resistant or
tolerant varieties), chemical (fungicidal), biological (e.g., Coniothyrium minitans against
sclerotinia) and agronomic (or cultural) (Aubertot et al., 2005).
During the last two decades there has been a lot of progress in controlling sunflower
diseases by genetic methods (Vincourt et al., 2011; Vear and Muller, 2011); nowadays it is
surely the most efficient, practical and repeatable method to control most of the diseases.
Nevertheless, the other methods should not be neglected, especially cultural control
(Sackston, 1992). Most of these effects are presented in Table 2.
In order to reduce the application of pesticides for environmental and public health
concerns, and to preserve the sustainability of genetic resistance, agronomic control should
also be part of crop protection programs. For this, the interactions between the pathogen, its
host plant, the biophysical and biological environment and crop management must be
dissected and modeled in a comprehensive system approach (Desanlis et al., 2013).

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Effects of Crop Management on the Incidence and Severity of Fungal … 203
Table 1. Characteristics of four main sunflower diseases in France

Phoma Downy mildew Phomopsis Sclerotinia




Type of fungus necrotrophous
widespread
biotrophous local
attacks

necrotrophous
widespread
necrotrophous
widespread
(except for stem
base attacks)
Most detrimental
symptoms
Stem base necrosis and
premature ripening
Dwarf plants (for
primary systemic
attacks)
Girdling spots
on stem
Stem base and
head rot
Potential yield loss 30 to 50 % 50 % (if primary
attacks)
0.1 to 0.3
t.ha
-1
for 10 %
of plants with
girdling lesions
> 50 %
Attacks responsible
for the spread of
inoculum
Stem Sporulating
dwarf plants
Stem (girdling
and non-
girdling spots)
Stem, collar,
head
Inoculum
survival
Form Mycelium Oospores,
mycelium
Mycelium Sclerotia
Support Crop residues, seeds Crop residues,
seeds, soil
Crop residues Crop residues,
soil
Life span > 3 years 10 years 1 year 7 to 10 years
Key period for
disease
development
Attack at stem base early
enough to provoke a
stem base sheath at
anthesis
Synchronism
between plant
emergence and
presence of free
water in the soil
Time to reach
the stem from
an attack at leaf
margin
Anthesis
coincides with
spore emission
Favorable
conditions for the
establishment of the
fungus
Strong inoculum
pressure, environmental
conditions moderately
limiting
50 mm of
precipitation
required within
10 days around
sowing time, soil
temperature >
10°C
Relative
humidity > 90%
during 36h,
optimal
temperature
range 20-24°C
Free water
during 42h at
floret surface
Time for symptoms
to appear after
contamination
8 to 15 days 10 days to 7
weeks (primary
infection)
20-25 days (leaf
spots) then 20
more days
before stem
lesions
3-8 weeks on
heads





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P. Debaeke, E. Mestries, M. Desanlis et al. 204
Table 1. (Continued)

Phoma Downy mildew Phomopsis Sclerotinia
Distribution
(France)





(from CETIOM, 2002; Delos et al., 2000; Delos et al., 2004; Gulya et al., 1997; Moinard et al., 2009).

Table 2. Efficacy of control methods (from Aubertot et al., 2005)

Downy mildew Phoma Phomopsis Sclerotinia
Genetic control +++ + +++ ++
Chemical control +++ + +++ -
Biological control - - - +
Cultural control + + + ++
(+) efficacy level of the method, (-) no method available.


Figure 1. Simplified life cycle of a fungus on a plant, within a crop, or at landscape scale (Lucas, 2007).
When genetic control is based on the use of specific resistance (e.g., mildew), agronomic
control is necessary for maintaining the effectiveness of this resistance over time.
The reduction of the initial inoculum is the first step in the disease control strategy. The
main method is lengthening the crop rotation, i.e., delaying the return of the same crop on the
same field but also the ‗previous effect‘ of each crop. The rotation could be diversified by
alternating host and non-host species, together with soil tillage methods, in order to break the
reproductive cycle of soil-borne diseases. At regional level, an optimal landscape mosaic
should be arranged to prevent unwanted spore dissemination from sources of primary
multiplication
dissemination
survival
contamination
crop damage
injury
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Effects of Crop Management on the Incidence and Severity of Fungal … 205
inoculum to host plants. The second lever is the management of infected crop residues which
are the main source of primary inoculum: crushing and burying the crop residues reduces the
inoculum‘s life span and its ability to continue its cycle. The sanitary quality of seeds is also
an important criterion.
Avoidance strategies consist of reducing the risk of synchronism between the period of
maximal crop receptivity and the phase of emission of contaminating spores. The main lever
is the choice of the sowing date, associated with the choice of suitable varieties (when
earliness is possible).
Mitigating the impact of diseases in crops consists of reducing the magnitude of disease
injury and damage when contamination occurs. Usually the management objective is to
manipulate the canopy vigor through various cultural practices (sowing date, seeding rate,
nitrogen fertilization, irrigation). Variety choice is also involved, through its level of genetic
resistance to the initial infection but also other characters such as earliness, plant architecture
and tolerance (i.e., ability to limit the damage caused by the pathogen injury).
Studies on the impact of crop management on the expression of sunflower diseases began
in the mid-80s, but are still limited as compared to other crops (for instance wheat). The
purpose of this chapter is to review the results of these studies with a special focus on two
major fungal diseases (phomopsis and phoma) which have been intensively studied in the last
decade by agronomists and pathologists because they are significantly influenced by cultural
practices.


Figure 2. A schematic diagram representing the strategies for controlling Phomopsis helianthi and the
technical solutions than can be applied: 1. prophylactic methods; 2. tolerance (genetic control);
3 escape; 4. avoidance; 5 attenuation (chemical control). Cultural control is 1 (cropping system level,
before sunflower crop or on adjacent fields), 3 and 4 (crop management system, intra-field).
Sunflower
diseases (e.g
Phomopsis) are
the main limiting
factors in wet
years
2. Phomopsis tolerant
genotypes
3. Susceptible stages
escaping the main
periods of infection
4. Canopy conditions
unfavourable to
infection and injury
5.Prevent or stop the
leaf attacks
Varieties
TPS & R
Late sowing date
Low plant
density, low N,
low irrigation
Fungicides
1. Low primary inoculum
Crop residue
management
C
r
o
p
M
a
n
a
g
e
m
e
n
t

S
y
s
t
e
m
C
r
o
p
p
i
n
g
S
y
s
t
e
m
Longer
rotations
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P. Debaeke, E. Mestries, M. Desanlis et al. 206
REDUCE PRIMARY INOCULUM

The inoculum of major fungal diseases persists in various forms, either as spores
produced by asexual or sexual reproduction (oospores, ascospores) or by mycelium and
sclerotia, on different organs of the plant (vegetative parts, seeds) or diffuse in the soil. Its
survival time is variable (1-10 years) depending on environmental conditions and agricultural
practices. Knowing this duration is critical to evaluate the effectiveness of the methods
applied for inoculum reduction.


Longer Rotations: A More Effective Practice When the Inoculum Is Not
Widespread at Regional Level

In general, short rotations or landscapes with a high concentration of sunflower fields
both increase the risk of disease. They contribute to the frequent soil enrichment in
contaminated residues (mildew, phomopsis, phoma) or various forms of storage (sclerotia of
Sclerotinia, oospores of mildew, microsclerotia of Macrophomina or Verticillium), which
constitute different sources of inoculum for the following sunflower crop. Spreading the
sunflower crop on the largest possible number of plots to delay the return of sunflower on the
same plot usually helps reduce risk. Whatever the cropping system, crop rotation is always an
effective practice to prevent the risk of disease. Two successive sunflower crops are banned
in France to prevent the spread of mildew (Delos et al., 2004). Masirevic and Gulya (1992)
recommended a minimum period of three years between two successive sunflower crops.
In the case of mildew, although weather conditions around sowing are critical for
infection, the risk is particularly great in areas where rotations are very short, because the
level of infestation of a field depends on the presence of plants with mildew symptoms in
previous years. A survey of 225 fields monitored between 2007 and 2008 in southwestern
France revealed a significant effect of crop rotation duration on the percentage of infected
plants and on the infectious potential of the field, these two effects being closely associated
with soil type: the risk was higher in calcareous clay soils and for short rotations (one
sunflower every two years) compared to rotations where sunflower returned not less than one
year in three (Moinard et al., 2009).
In the case of sclerotinia, the risk of attack increases with the amount of sclerotia in the
soil. The return frequency of susceptible crops (e.g., oil or protein crops) in the rotation
potentially increases the risk of contamination of the field. Lengthening the crop rotation is a
good way of reducing the bank of sclerotia in the soil, part of which disappears naturally each
year due to insufficient melanisation or because of mycoparasites (Huang and Scott Erickson,
2008). This measure will be all the more effective if it is applied as soon as the first attacks
occur (Heffer-Link and Johnson, 2007), and if other susceptible crops are considered (such as
oilseed rape, pea, soybean) (Delos et al., 2004). Gulya et al. (1997) consider that a minimum
of 5 years without a susceptible crop in the rotation is necessary to reduce the infectivity
potential of a plot.
In the case of phomopsis and phoma, the short life span of the inoculum on crop residues
and the long distance dissemination of ascospores both reduce the effect of the return period
of sunflower cropping (Jouffret, 2005). When the next crop (often a cereal) is established
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Effects of Crop Management on the Incidence and Severity of Fungal … 207
after simplified tillage, the sunflower-infected debris remains on the surface in wheat plots,
thus constituting a source of inoculum for the surrounding sunflower plots. In the case of
phoma, this was verified in recent years: in the Poitou-Charentes region (center-west of
France): despite sunflower returning less often than in the southwest for many years (10 to
15% of plots with less than one sunflower crop every 3 years against 40 to 50% in the
southwest, Wagner and Lieven, 2010), significant attacks of phoma and premature ripening
syndrome were still observed in 2010.


Management of Primary Inoculum: At Field Level, but Not Only ....

Assessing the amount of primary inoculum is critical to predict the risk of disease; this
amount is dependent both on the severity of attack in previous years and how infected crop
residues are managed after harvest.
Crop residues infected by Phomopsis and Phoma are a source of inoculum for attacks in
sunflower fields in subsequent years. These two fungi continue their cycle by producing
fruiting bodies (perithecia and/or pycnidia) on residues, which constitute new sources of
contaminating spores. Thus, the fate of these residues after harvest determines the level of
inoculum that can infect sunflowers grown in the following year. Crushing sunflower stalks
also accelerates the decomposition of stalks and limits the trophic support for different fungi
which are sources of inoculum (Delos et al., 2004). In addition, burying residues disrupts the
cycle of these fungi (Jinga et al., 1992), the formation of fruiting bodies being determined by
light.
Given the ability of the inoculum of Phoma and Phomopsis to be released into the air,
measures promoting the rapid degradation of infected residues will be more effective if they
are applied in all the fields in a region (Delos et al., 1994). In the case of phomopsis, the
dissemination of contaminating spores generally follows the direction of prevailing winds
(Masirevic and Gulya, 1992); for phoma, ascospores may be dispersed over long distances by
rain and wind (Maric et al., 1998) and were identified in former Yugoslavia as the source of
attacks on sunflower in places where it had never previously been grown.
Regarding sclerotinia, soil tillage influences the survival of sclerotia and their ability to
germinate in the mycelial form (responsible for the stem base attacks) or to form apothecia,
sources for airborne contamination by ascospores. However, some results on soybean in the
United States appear to be contradictory: a deep tillage can bury sclerotia, avoiding the
formation of apothecia, but may encourage attacks at the stem base. Maintaining sclerotia less
than 5 cm deep with a shallow tillage would promote their germination but also would expose
them to degradation by mycoparasites present in this layer (Duncan et al., 2005; Heffer,
2007).
According to Quiroz et al. (2009), the combination of no-tillage and highly resistant
cultivars promises to be an interesting tool to manage V. dahliae and Verticillium wilt in
sunflower. Doran and Linn (1994) found that conservation tillage (no tillage) increased the
level of microbial populations from 0.5 to 2.7 times in the top 15 cm of soil. Increases in soil
microbial activity would provide a highly competitive environment, leading to competition
effects between soil microbes and encouraging disease suppression (Chen et al., 1988).

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P. Debaeke, E. Mestries, M. Desanlis et al. 208
Biological Control against Sclerotia of Sclerotinia

Among mycoparasites, Coniothyrium minitans is a fungus that can destroy hyphae and
sclerotia of Sclerotinia sclerotiorum. Mycelium penetrates into sclerotia through their
pigmented bark or by altering the surface, and fully colonizes them (Huang and Kokko,
1987). Under optimal conditions, sclerotia can be destroyed in 15 days. Today, an organic
product based on spores of this fungus is available on the market: Contans ® WG. Studies on
the effectiveness of this control method against sclerotinia in oilseed rape suggest that
parasitized sclerotia cease to produce apothecia, resulting in a reduction in crop attacks of 20
to 30% (Penaud and Michi, 2009). Moreover, this biological control is more effective when it
is applied to each crop in the rotation (Penaud et al., 2003), because not all sclerotia are fully
destroyed after a single application.


The Sanitary Quality of Seeds

The sanitary quality of seeds is an important method of control for all fungi that can be
transmitted through this way. Mildew is the disease for which the transmission of the disease
by seeds played a crucial role in the spread of the 703 and 710 races in France. Sunflower
seeds can harbour the mildew in the form of oospores and mycelium. In case of a severe
attack, the proportion of infected seeds can reach 100% (CETIOM, 2000). The use of
certified seeds is imperative to avoid the intake of inoculum from outside.
Today, phoma is a fungus whose ability to be transmitted by seed (especially in unclean
seed lots) has been clearly shown, which explains its sudden onset in countries where
sunflower was not grown till now (Luo et al., 2011). This would not be the case for
phomopsis.


REDUCE THE RISK OF DISEASE BY ESCAPING INFECTION

The success of an infectious event and its expression (injury) depends on the crop growth
stage when it occurs.
The choice of sowing date should be based on the notion of an escape strategy, which
consists of optimizing the delay between the phase of crop receptivity and the usual periods
of contaminating events, as derived from historical records, i.e., the date of ripening of
perithecia for Phomopsis and Phoma, and rainy periods favoring the emission of Sclerotinia
spores or the germination of mildew oospores in soil.


Adjusting Sowing Date According to Weather Conditions: A Key Factor in
Mildew Control

Primary mildew attacks are the most serious: they are responsible for dwarf plants and
head sterility. Rainfall plays a major role in the success of infections: these occur at the time
of crop germination, as contaminating oospores (arising from the dormant form of the fungus)
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Effects of Crop Management on the Incidence and Severity of Fungal … 209
need free water in the soil to come into contact with sunflower rootlets. The cumulative
rainfall between 10 and 20 days around sowing time is thus a risk factor, especially if it
exceeds 50 mm in 10 days around sowing. Soil temperature is also involved, the success of
infections being greatly reduced for temperatures less than 10°C (Delos et al., 2000).


Phomopsis: Higher Risk for Early Sowing

The period of crop receptivity to Phomopsis ranges between flower bud stages E1 (star
bud stage) and E5 (pre-anthesis). According to several studies, early sowings often result in
severe attacks while delaying planting can shorten the overlap between the period of crop
receptivity and the time when the risk of attack is at its maximum (spring rainy events).
In 1992, Jinga et al. were the first to demonstrate the effect of sowing date on phomopsis
incidence: a sowing made 20 days later than usual (late April instead of early April), reduced
the fraction of injured plants by nearly 30%.
From two experiments done in Toulouse in 1996 and 2000 it was evident that delayed
sowing significantly reduced the fraction of plants infected: delaying sowing by 12 days
(2000: from April 22 to May 3) or 35 days (1996: 10 April to 15 May) together with low
input management, resulted in 3% attacks in 1996 (against 73% in high input, early sown
management) and 42% in 2000 (against 97%) (Debaeke et al., 2003). However, a delay such
as that tested in 1996 has a major drawback, as potential yield was affected too.
The escape effect can be attributed to the phenological stage of the crop during the period
highly conducive to infection: although infections are possible as long as green leaves are
present, the highest proportion of infected stems results from attacks that occur in the early
stages of flower bud development. Late planting can usually limit the number of infections
due to less favorable weather conditions (less frequent rainfall events and higher frequency of
days with lethal temperatures for fungus) coinciding with a shorter duration of the crop
receptivity phase and of the time of canopy closure (Debaeke et al., 2003).
These experimental approaches have been confirmed by the use of the Asphodel model,
an epidemiological model which was applied to a range of annual weather patterns and
sowing dates to simulate the risk of infection (Debaeke et al., 2001).


Phoma: The Random Effect of Sowing Date

Sackston (1950) first described a wilt and stalk rot of unknown etiology as ‗‗premature
ripening‘‘, and earlier evidence showed that stem base girdling canker caused by P.
macdonaldii was its primary cause. Crops show loss of vigor from mid- to late summer;
leaves become wilted and necrotic, the stalk turns dark brown to black, and this is followed
by senescence and plant death a few weeks before physiological maturity (Donald et al.,
1987).
Regarding phoma, the periods of crop receptivity and emissions of contaminating
ascospores are both very broad: attacks can take place throughout the growing season.
Consequences of delaying planting appear less clear than with phomopsis: according to
several authors (Fayazalla and Maric, 1981; Delos et al, 1998), the risk of attack on stems and
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P. Debaeke, E. Mestries, M. Desanlis et al. 210
its severity would be lower with late plantings. Indeed, late plantings tend to reduce the
infection rate because of lower rainfall before flowering (escape strategy). On deep soils,
avoiding too early planting (late March) can reduce Phoma attacks which are generally
frequent on these high potential situations (Taverne, 2005). However, deferring planting to
late spring is not always effective, especially if June and July are wet: in three experiments
carried out in different regions of France (1999 and 2000), it was observed that the attacks
increased by an average of 23% on stems and 12% at stem base when delaying sowing by
three to four weeks (Debaeke and Peres, 2003).
Besides, although early plantings (early April) are often more exposed to attacks on stems
(including phoma), they can escape terminal water stress more easily, with limited risk of
premature ripening.
The optimum planting date is thus a balance between sacrificing potential yield (which
needs a long season), escaping water stress (through early sowing) and mitigating/escaping
disease attacks by delaying sowing and thus avoiding risky periods for disease infection.
Other examples of sowing date effects on disease incidence were given for Alternaria
helianthi in Brazil where sunflower is planted as a second crop after soybean (Leite et al.,
2008).


MITIGATE DISEASE IMPACT

Several elements of crop management can reduce disease development and the final
proportion of diseased plants. This is the case for phomopsis and phoma at stem level, phoma
and sclerotinia at stem base level, and sclerotinia on heads.
The severity of symptoms can also be reduced: in the case of phomopsis, harmful
symptoms are girdling lesions; for phoma, crop management can have a major influence on
premature ripening due to attacks at stem base level (Seassau et al., 2010).


Practices to Reduce the Proportion of Infected Plants

Phomopsis
In the case of phomopsis, plant density has a significant effect on the fraction of plants
with lesions on leaves and stems.
From nine experiments done at INRA Toulouse between 1994 and 2001, Debaeke et al.
(2003) observed that increasing plant density from 5 to 7.5 plants.m
-2
resulted in an increase
of 22% in infected stems.
Similarly, when early infections and rainfall are conducive to the onset of symptoms on
stems, crop irrigation around anthesis favors and secures these attacks: from 1997 to 2000, on
41 experimental situations, irrigation has contributed to a 22% increase in the percentage of
attack on stems (Debaeke et al., 2003).
Regarding the effect of nitrogen fertilization under conditions of low inoculum pressure,
the proportion of infected stems increased by 36% when increasing N fertilization from 60 to
120 kg N.ha
-1
(Debaeke et al., 2003).
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Effects of Crop Management on the Incidence and Severity of Fungal … 211
When weather conditions are such that the inoculum is not limiting, relative humidity
conditions within the canopy are favorable to the success of infections even if the field is
sparsely covered. Under these extreme conditions, the dissemination of contaminating spores
would be favored within a more open canopy, causing more spots on leaves; the reduced plant
growth (smaller leaf size and petiole length due to nitrogen deficiency in these conditions)
would facilitate the leaf-to-stem passage of the fungus (Desanlis et al., 2013). Disease
severity was thus accentuated by the smaller stem diameter due to high plant density
(Debaeke et al., 2003; Delos et al., 2004).
Hence, on the same soil, with the same weather and for the same inoculum pressure,
changing plant density, nitrogen fertilization and irrigation can vary the percentage of
infected stems for a susceptible genotype from 0 to 100% (Debaeke et al., 2003).

Phoma
The impact of crop management on Phoma attacks at stem and collar level have been
reported in several studies (Formento and Velasquez, 2000; Debaeke and Peres, 2003;
Seassau et al., 2010).
The influence of N fertilization on phoma incidence was illustrated by the 2005
experiment in Toulouse (Debaeke and Taverne, 2006). The number of Phoma spots per stem
was evaluated for 2 cultivars and 6 nitrogen treatments differing in N amount and splitting.
Irrigation was fully supplied. The number of Phoma spots per stem increased dramatically
with excessive N fertilization (120-160 kg N.ha
-1
), cv.Melody being always less infected than
cv.Heliasol (Figure 3).
Fifteen field experiments between 1996 and 2000, in four French regions, with different
combinations of sowing date, sowing density, nitrogen fertilization (0 to 120 kg N.ha
-1
) and
irrigation (rainfed or different dates and amounts of irrigation) were subjected to natural
Phoma attacks (Debaeke and Peres, 2003).
The fraction of plants with stem spots was higher under wetter conditions in June (heavy
rainfall or irrigation): 94% cf. 77%. This percentage and the number of spots per stem were
both closely related to the N available to the crop: on average, 5.9 stem spots with 120 kg
N.ha
-1
and 4.2 with 60 kg N.ha
-1
. The higher the number of spots per plant, the higher the
inoculum potential for future sunflower crops in the area (Debaeke and Peres, 2003).
At a given plant density, the proportion of infected plants increased steadily with N
fertilization rate. An interaction between the level of N fertilization and plant density was also
shown: when the crop was N-limited, the number of Phoma spots on the stem increased with
plant density. Conversely, in non-limiting N conditions, the highest number of spots was
observed in low-density conditions (Debaeke and Peres, 2003). This effect can be attributed
to the fact that the troughs at the insertion points of petioles on the stem, which act as
receptacles for free water, are deeper in low density and high N conditions and that the
frequent bursting of petiole tissue at groove level may favor the penetration of the fungus
within the plant (Delos et al., 2000).
Regarding stem base attacks of Phoma, a positive correlation was found between the
fraction of plants with a girdling lesion at stem base and leaf area index at flowering
(Figure 4).

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P. Debaeke, E. Mestries, M. Desanlis et al. 212

Figure 3. Effect of N management on phoma incidence for 2 cultivars (Toulouse, France - 2005) – LSD
at 5%.


(a)

(b)
Figure 4. Relationship between the proportion of plants with harmful lesions of Phoma macdonaldii:
(a) on stems (class 4); (b) on stem base (class 2), and leaf area index at flowering. Field trial in Saint-
Florent (central France), year 2000 (Debaeke and Peres, 2003).
Thus, below a leaf area index of 2.5 at flowering, the risk of potentially harmful attacks
of Phoma at stem base remains low (below 30%). This synthetic index (LAI) being the result
of complex interactions between each of the factors of crop management (sowing date, plant
density, water and nitrogen availability before flowering), probably reveals the underlying
existence of an impact of any or all of these factors on the extent of collar attacks, even if
each individual relationship was not demonstrated in this series of experiments.

Phomopsis and Phoma: the extent of canopy development for predicting the attacks
on stem
The extent of canopy development is commonly expressed by leaf area index, which is a
synthetic indicator of crop growth and development. The fraction of photosynthetically active
radiation intercepted by the canopy (fPAR
i
) is closely related to LAI and thus to the intensity
of management before flowering (nitrogen fertilization, plant density, irrigation) and partly to
the variety.


0
1
2
3
4
5
6
7
8
9
0 N 40 N 80 N early 120 N 80 N late 80 N early
+ 80N late
P
h
o
m
a

s
p
o
t
s


/

s
t
e
m
cv.Heliasol cv.Melody
c
c
c
c
c
a
b
bc
c
c b
a

y = 19,6 x - 18,4
R
2
= 0,83
0
10
20
30
40
50
60
70
0 1 2 3 4 5
Leaf Area Index at flowering (F2)
%

s
t
e
m
s

w
i
t
h

P
h
o
m
a

c
l

4

y = 14,7 x - 11,5
R2 = 0,66
0
10
20
30
40
50
60
0 1 2 3 4 5
Leaf Area Index at flowering (F2)
%

c
o
l
l
a
r
s

w
i
t
h

P
h
o
m
a

c
l
2


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Effects of Crop Management on the Incidence and Severity of Fungal … 213
Concerning phomopsis, in experiments where the weather conditions and crop
management (early sowing, pre-flowering irrigation) favored natural attacks, the percentage
of infected stems increased exponentially from 0 to 100% with the fraction of radiation
intercepted by the canopy (55 to 95%), whatever the susceptibility level of the varieties
(Susceptible, Moderately Susceptible, Tolerant). Beyond fPAR
i
of 85% at flowering (i.e., LAI
> 2.5), the proportion of stems with infected lesions increased rapidly; below this threshold,
the attack rate remained below 20% (Figure 5). Therefore, obtaining a high leaf area index by
increasing plant density and nitrogen fertilization increased the fraction of infected stems,
regardless of the intrinsic susceptibility of the varieties (Debaeke and Estragnat, 2003).
This relationship is also valuable in the case of Phoma attacks on stems (Debaeke and
Peres, 2003): within a range of variation of fPAR
i
from 65% to 95% (corresponding to a LAI
range of 1.5 to 5 at flowering), the proportion of infected stems increased with the value of
fPAR
i
. The number of spots on stems increased significantly above a fPAR
i
of 85% (up to 12
spots per plant) while it remained below 7 per stem below this fPAR
i
threshold (Figure 6).


Figure 5. Relationship between the proportion of stems infected by Phomopsis and the fraction of PAR
intercepted by the sunflower crop at anthesis for weather conditions promoting leaf and stem infection
(n = 129) – Genotypes were susceptible, moderately susceptible or tolerant (Debaeke et al., 2003).

Figure 6. Relationship between the proportion of stems with simple Phoma spots (class 2) and the
fraction of PAR intercepted by sunflower (cv. Select) at early anthesis for different crop management
systems (INRA, 1994). D1 = 5.2 plants.m
-2
, D2 = 6.8 plants.m
-2
, N30 and N60: N fertilization at sowing
time (kg N.ha
-1
), irr = one irrigation before anthesis, sprayed =one application of Punch CS 0.8 L.ha
-1
,
water stress: plots where senescence was observed before anthesis as a result of water stress (Debaeke
and Peres, 2003).

y = 0.00003.e
0.158x
R
2
= 0.668***
0
10
20
30
40
50
60
70
80
90
100
50 60 70 80 90 100
f PARi at anthesis (%)
I
n
f
e
c
t
e
d

s
t
e
m
s

(
%
)
0
10
20
30
40
50
60
70
60 70 80 90 100
f PARi at early anthesis (%)
S
t
e
m
s

i
n
f
e
c
t
e
d

b
y

P
h
o
m
a

(
%
)
D1-N30
D2-N60
D2-N60-irr
Sprayed
Water stress
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P. Debaeke, E. Mestries, M. Desanlis et al. 214
For Phomopsis, this close relation can be explained by the effect of crop management on
the dynamics of canopy closure: with a high population density (> 6.5 plants.m
-2
), canopy
closure is faster, creating suitable microclimatic conditions (notably relative humidity) for
leaf infection (Debaeke and Moinard, 2010; Desanlis et al., 2013); these conditions give rise
to early attacks, causing the most severe injury to yield and oil concentration. In addition,
dense stands are characterized by smaller leaves and thinner stems, which are more quickly
destroyed by Phomopsis (Desanlis et al., 2013). N concentration of plant tissues does not
appear to affect the fungus‘s growth within the tissue, and thus the leaf-to-stem passage
(Berault, 2000; Desanlis et al., 2013). Changes in the rate of spread of symptoms are related
to the susceptibility of the variety and the microclimatic conditions (temperature and relative
humidity), irrigation causing an acceleration of the fungus‘s progression, which on a
susceptible variety may vary from 1.5 to 4 cm.day
-1
, depending on weather conditions.
For phoma, unlike phomopsis, successful infections at the collar seem more dependent on
the weather than on microclimatic conditions. Changes in microclimate within the canopy
appear more critical for attacks on leaves than at the stem base, where soil moisture
concentrated near the collar generally provides favorable conditions for the infection to
succeed. This "lesser requirement" may explain the high incidence of collar attacks under
natural conditions in areas where the inoculum is abundant (Bordat et al., 2011). Sometimes,
however, low levels of attack are observed when weather conditions in June and July are
particularly hot and dry. Regarding attacks on the stem, they are widespread in all areas of
sunflower cultivation. Unlike Phomopsis, where the fungus necessarily infects the leaves
before infecting the stem, Phoma attacks on the stem can occur at its insertion point with the
petiole. The trough that forms at this level (generally from the 10-12 leaf stage) is conducive
to water storage. This creates a microenvironment extremely favorable for the germination of
Phoma spores as soon as free water is maintained.
Intensifying crop management in sunflower (nitrogen fertilization, plant density, and
irrigation) thus increases the fraction of plants infected by Phomopsis and Phoma.

Sclerotinia at stem base: An underground transmission of the disease
Germination of sclerotia and mycelial growth of Sclerotinia affect the root system of
sunflower plants. The disease is more common in soils rich in organic matter (deep silt soils,
marshland). Reducing plant density (plants spaced at least 40 cm apart) reduces plant-to-plant
contamination. Similarly, the distribution of sclerotia down the soil profile affects the attack
rate: it decreases from 50% to about 20% when sclerotia are buried at 20 cm, rather than
10cm, depth (Huang and Hoes, 1980).

Sclerotinia on heads: Avoid synchronizing flowering and irrigation
The period of sunflower susceptibility to Sclerotinia attacks on the capituluum starts from
the beginning of flowering and lasts until mid-flowering: ascospores from apothecia settle at
floret level on one side of the head. Conditions of high humidity are necessary for spore
germination and early colonization of the inflorescence: the presence of continuous free water
for at least 39 hours determines the success of the infection phase (Pauvert and Lamarque,
1981). Attacks take place during rainy periods in July. In case of dry weather, the main
element of crop management that affects the percentage of infected heads is irrigation at
flowering: in low inoculum pressure conditions, for the same total amount of irrigation (100
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Effects of Crop Management on the Incidence and Severity of Fungal … 215
mm), infection rates can increase from 1% with two applications to 27% with 5 applications
around flowering (Peres and Allard, 2000). Stand structure, through inter-row spacing, also
appears to have a significant effect on the percentage of heads with Sclerotinia: for the same
population density (60,000 plants.ha
-1
), a row spacing of 50 cm increases the attack level on
heads (17% of injured plants) compared to an inter-row of 80 cm (10% of injured plants)
(Peres et al., 1992; Peres and Allard, 2000).


Practices to Reduce the Severity of Symptoms

Phomopsis – The harm caused by Phomopsis to yield and oil content is related to the fate
of spots on stems, the most detrimental symptom being at the girdling lesion, where the pith
is completely destroyed, leading more or less rapidly to a wilted plant.
Delaying sowing till May helps to reduce the extent of attacks on stems and also has a
significant effect on the fraction of plants with at least one girdling spot: depending on the
delay (between 12 and 35 days), late planting associated with low-input management results
in reduced vegetative growth that can limit the infection rate to between 2% and 31%, as
against 73% to 85% with high-input management (Debaeke et al., 2003). In addition, the hot
dry periods most common after late infections are unfavorable to fungal growth on plants.
Debaeke et al. (2003) also showed in nine experiments that increasing plant density from
5 to 7.5 plants.m
-2
increased the proportion of plants developing girdling spots by up to 82%.
The effect of plant density on disease severity is the same regardless of the varietal
susceptibility (Debaeke and Moinard, 2010).
Irrigation around flowering is the third crop management factor which may affect the
severity of attacks: with reinforced semi-natural infection, the fraction of plants with at least
one girdling spot increased by 48 % (from 27 to 40%) with two water applications of 35 mm
around flowering (Debaeke and Moinard, 2010). Irrigation at flowering increases leaf area
duration and thus offers more chances for the fungus to progress on the leaves and develop
girdling lesions on stems.
From 1994 to 2005, several field experiments were done in south-west France by INRA
and CETIOM to quantify the influence of crop management systems on disease occurrence
and yield loss in sunflower (Debaeke et al., 2003).
To illustrate the main results on phomopsis, the 2002 experiment is presented here: 8
cultivars (from tolerant to very susceptible) were submitted to 4 management systems
differing in plant density (5 vs 7 plants.m
-2
), N fertilization (25, 70, 25+80 kg N.ha
-1
), and
irrigation (0 vs 40 mm at anthesis) (Debaeke and Taverne, 2006). The proportion of stems
with necrotic lesions was observed on early August. The treatments had a big effect on the
proportion of stems severely infected by Phomopsis (5 - 95 %) confirming previous results
(Debaeke et al., 2003). Disease incidence increased with plant density, N rate and irrigation,
irrespective of genotypic tolerance (Figure 7). Splitting nitrogen at sowing and star bud stage
appeared to be a good strategy to limit phomopsis occurrence, but an interaction with
genotype susceptibility was observed: increasing total N rate was detrimental to susceptible
cultivars in spite of splitting. In 10 plots out of 32, yield loss was below the injury threshold
(15 % of stems with girdling lesions) and chemical treatment could have been replaced by
cultivar tolerance and canopy management in these situations.
Complimentary Contributor Copy
P. Debaeke, E. Mestries, M. Desanlis et al. 216

Figure 7. Effect of crop management (N fertilization and irrigation) on phomopsis incidence for 8
cultivars differing in their susceptibility to this disease: (T) tolerant, (S) susceptible, (VS) very
susceptible, (-) moderately S or moderately T (Toulouse, 2002) – Comparison of means by LSD at P <
0.05 (Debaeke and Taverne, 2006).
A relationship between fPAR
i
at flowering and the fraction of plants with at least one
girdling lesion due to Phomopsis (PHO) was suggested for susceptible to moderately
susceptible varieties (Debaeke and Estragnat, 2009):

PHO (%) = 1.10 fPAR
i
(%) - 38.3 (r² = 0.661, P <0.0001)

On two experiments done in 2000 and 2001 in Auzeville (31), the fraction of plants
bearing at least one girdling lesion of Phomopsis was closely related to relative humidity
(RH) during the infection period (Figure 8). RH was monitored by placing thermo-
hygrometers at 40 cm above the soil. Intensifying crop management through plant density and
nitrogen fertilization resulted in higher values of LAI, fPAR
i
and RH, with a greater risk of
disease development in sunflower (Debaeke and Moinard, 2010).
The observation of the onset of symptoms on a susceptible variety studied in 2000 in
Toulouse showed that the earliest attacks on leaves, affecting the bottom layers (leaf nodes 1
to 9), have a lower chance of reaching the stem than those infecting the plant later at upper
nodes (leaf nodes 10 to 16). There are two reasons for this: physiological leaf senescence,
which begins at the bottom of the plant due to leaf age and shading, and the attacks of Phoma
on the stem, which begin at the bottom of the plant and prevent the passage of Phomopsis
from leaf to stem (Debaeke and Moinard, 2010).
The effect of high-input management on Phomopsis is therefore obvious on both its
incidence and severity. Although under favorable weather conditions associated with
abundant inoculum the effects of crop management, however, remain limited (Delos et al.,
1994), tests of crop management systems have shown that a given level of Phomopsis attack
could be achieved either by conventional management based on fungicide or by an integrated
management without fungicide, provided crop management is adapted to disease risk; i.e., by
reducing plant density and N fertilization and delaying sowing date in a coordinated way
(Debaeke and Estragnat, 2003).

0
10
20
30
40
50
60
70
80
90
100
ALB HEL LG5660 MEL PRI (S) PRO SAN (T) VID (VS)
s
t
e
m
s

w
i
t
h

g
i
r
d
l
i
n
g

s
p
o
t
s

(
%
)
25 N
70 N
70 N + irr
105 N + irr
b ab a a
b ab a ab
c bc ab a
NS
c bc a ab
c b b a
b b a ab
b b a b
crop
management
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Effects of Crop Management on the Incidence and Severity of Fungal … 217

Figure 8. Relationship between the percentage of plants with girdling spots on the stem and the average
relative humidity (RH) in the canopy during the infection period. The figures indicate the number of
days suitable for infection as simulated by the Asphodel model in 2000 and 2001 (Debaeke and
Moinard, 2010).
Table 3 gives an overview of the effects of irrigation, N fertilization, plant density and
variety on the three disease components of phomopsis: number of leaf symptoms, leaf-to-
stem passage (%) and fraction of girdling symptoms (%) which all contribute to the final
number of girdling symptoms of Phomopsis per plant (Desanlis et al., 2013).

Table 3. Effects of crop management on the three components of phomopsis injury

Leaf symptoms (number)
Leaf-to-Stem passage
(% leaf symptoms)
Girdling symptoms
(% stem symptoms)
High plant
density
early symptoms 
microclimate
late symptoms 
green leaf area
+

-
small leaves
early senescence

+
-
thin stems +
High
Nitrogen
Microclimate
Green leaf area
+
+
big leaves
more phoma
high N tissue content
-
-
+
thick stems -
Irrigation Microclimate
green leaf area
+
+
delayed senescence +
Susceptible
genotype
low tissue resistance (leaf) + low tissue resistance
(stem)
+ low tissue resistance
(stem)
+
(+: factor promoting the disease; -: factor impeding the disease).

Phoma and premature ripening
Regarding premature ripening caused by phoma at the stem base, which is the most
harmful form of the disease, the idea emerged in 1984 that a combination of biotic stress
(related to the pathogen: nature, quantity, aggressiveness) and abiotic stress (related to water
status and crop nitrogen status) could be the cause of this syndrome (Gulya et al., 1984).

0
10
20
30
40
50
60
70
80
90
100
70 75 80 85
Mean RH (%)
S
t
e
m
s

w
i
t
h

g
i
r
d
l
i
n
g

l
e
s
i
o
n
s

(
%
)
2000
2001
7
15
13
16
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P. Debaeke, E. Mestries, M. Desanlis et al. 218
Once Phoma has infected the plant collar, the onset and progression of premature
ripening are strongly influenced by crop water and nitrogen status, resulting from the
combined actions of soil, weather and management (N fertilizer, irrigation, plant density).
Four experiments were carried out at INRA Toulouse between 2006 and 2009 to study
the effects of crop management on this syndrome (Seassau et al., 2010 ; Seassau et al., 2012).
The tests were conducted on two varieties with different susceptibility to phoma (cv. Melody
vs cv. Heliasol), during very contrasting growing seasons, combining:

 two water regimes (rainfed vs irrigated after flowering)
 three levels of nitrogen fertilization (0, 50/75 and 150 kg N.ha
-1
)
 three densities, 4, 6.5 and 9 plants.m
-2


The effects of these factors were characterized after natural or artificial infection of plants
at stem base by mycelium of a single conidium culture of Phoma macdonaldii selected for its
aggressiveness.
The analysis of the percentage of prematurely ripened plants (i.e., ripened about 2-3
weeks before plants in disease-free conditions) observed from 2006 to 2009 was used to
estimate the proportion of variability explained by each crop management factor (water
regime, nitrogen availability, variety, population density) and by the method of contamination
in order to rank their own contribution to the pathological syndrome (Table 4).
Of the factors studied, N availability contributed the most to the explanation of the PR
fraction. Its contribution ranged from 14% (2006: a dry year) to 84% (2008: a wet year) to the
variance of PR.
Varietal susceptibility appears as the second most explanatory factor. Water regime may
have a strong effect on the disease, but this factor is highly variable depending on
precipitation in spring and summer. In 2006 and 2009, when post-flowering stress was
pronounced, water regime had a bigger effect on the disease.
In 2007 and 2008, the effect of water regime was concealed by the importance of the
nitrogen effect, which alone accounted for between 60 and 84% of PR expression.
Population density contributed little to the variability of PR expression (4-8 %).

Table 4. Contribution of five agronomic factors to the total variance of prematurely
ripened plants (PR, %) – about 1500 plots observed - the factor was not studied
(Seassau, 2010)

Factor 2006 2007 2008 2009
Variety 26,5% 23,4% - -
N availability 13,7% 60,2% 84,1% 72,5%
Post-flowering water regime 31,4% 0 0 7,9%
Plant density - - 3,9% 7,8%
Contamination method 13,3% 3,9% - -

Crop management appears to be sufficient by itself to modify disease expression and to
increase the proportion of PR plants: a variety susceptible to PR, under conditions of natural
contamination, highly fertilized and not irrigated can sustain 100% of PR plants when N
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Effects of Crop Management on the Incidence and Severity of Fungal … 219
deficiency and water satisfaction may result in totally healthy plants. This should be
considered in trials used for testing the susceptibility of inbreds and commercial hybrids.
From these experimental results, a simple linear model was built to relate PR plants (from
artificial and natural contamination) to crop indicators of N and water status. NNI, the
Nitrogen Nutrition Index was selected to represent the crop N status at flowering and the ratio
of actual evapotranspiration (ETa) to maximum evapotranspiration (ETo) was used to
estimate the plant water status during the post-contamination period (Seassau et al., 2010):

PR (%) = 27.8+ 118.3 NNI – 102.1 (ETa /ETo) (n = 35 plots, r ² = 0.787)

This relationship emphasizes how the adjustment of nitrogen fertilization and satisfaction
of water requirements both affect the control of premature ripening.
Plant density is a management factor that would constitute a further control lever of PR.
Increasing plant density (D) increased the proportion of PR plants in 83 % (2008) and 100%
(2009) of the paired plots (low vs high density) (Seassau et al., 2012).
For this reason, in spite of the slight quantitative effect of plant density, Seassau et al.
(2012) proposed a second regression model with 4 variables describing leaf area (LAI), stem
diameter growth (SBD, mm), N shoot content (Nm, %) and water satisfaction rate (ETa/ETo,
%) explaining 73 % of the variability of PR:

PR (%) = 105.79 + 45.3 Nm – 103.0 (ETa /ETo) – 5.0 (SBD) + 12.4 LAI
(n= 36 plots, r² = 0.731)

PR increased with LAI and Nm (indicators of plant growth and N status) and decreased
with SBD and ETa/ETo, indicators of plant density and water status
In these experiments, the average stem diameter at collar level for plants grown at
different densities (4-9 plants.m
-2
) can differ from 8 mm between extreme densities. Thus, a
population density greater than 7 plants.m
-2
, by reducing the stem diameter, accelerates the
establishment of PR and the resulting damage. By contrast, highly fertilized plants are more
susceptible to PR in spite of their thicker stems. It could be a dominant effect of nitrogen:
plant susceptibility to disease is independent of collar diameter, but due to a trophic effect of
nitrogen which stimulates the development of fungus inside the plant (Seassau, 2010).
Unlike Phomopsis, the severity of Phoma attacks at collar level appeared less related to
the canopy microclimate (Seassau et al., 2012).
From the canopy characterization by agronomic indicators related to N shoot status, plant
growth and architecture, stem base morphology, plant water satisfaction rate and
microclimate, Seassau et al. (2012) proposed a conceptual framework of the effect of plant
density on the expression of the disease (Figure 9). The microclimate apparently has a
moderate effect on disease epidemiology and PR, unlike the other variables that directly
affect PR. High leaf area index (due to high N fertilization and high plant density) accelerates
soil water exhaustion and a drop in transpiration after anthesis. Avoiding excessive N
fertilization by using the soil N balance method could significantly reduce disease severity.
Also, manipulating the stem diameter, mainly through planting density and N supply, could
be exploited more, instead of resorting to fungicide use. This is probably a morphological trait
that breeders could exploit in the future. Indeed, genetic tolerance to PR should be evaluated
in order to achieve complete non-chemical control (Bordat et al., 2011). Promising cultivars,
Complimentary Contributor Copy
P. Debaeke, E. Mestries, M. Desanlis et al. 220
with thick stems, should therefore be tested at high density and N supply under water-limited
conditions, a procedure which could be used in resistance tests during breeding programs for
an effective control of sunflower PR.

Sclerotinia
Regarding white mold attacks on heads, the severity of symptoms depends on the level of
genotype resistance and on weather conditions. The head symptoms appear within 3 to 8
weeks after infection at flowering. In areas at risk, the choice of sowing date and the use of
early maturing varieties allow wet weather at maturity and hence poor drying conditions for
the heads (after mid-September) to be avoided, and may limit the losses at harvest provided
that sowing is not too late in spring (CETIOM, 2002). Plant nutrition has a substantial
influence on the predisposition of plants to be attacked or affected by diseases. Miladinovic et
al. (2008) found a high positive correlation between N content of infected sunflower plants
and resistance, which indicates the important role of this nutrient in sunflower defense from
Sclerotinia attack.


CONCLUSION

Cultural control must be seen as a combination of practices which can exert some control
over the development and expression of fungal diseases (Table 5). Crop management,
especially through techniques that control canopy establishment and its duration (plant
density, row spacing, nitrogen fertilization, irrigation), can significantly modify the
expression of sunflower diseases which develop during the vegetative period.
In the case of phomopsis, manipulating plant density, nitrogen fertilization and irrigation
can vary the percentage of stems from 0 to 100% under the same conditions of soil, weather
and inoculum pressure. However, in high-risk conditions and with abundant rains in late
spring, agronomic measures are not sufficient alone to maintain a level of infection below the
threshold of economic profitability of fungicide when susceptible varieties are grown
(Debaeke et al., 2003).
In the case of phoma and premature ripening, the range of variation is similar to
phomopsis and efficient levers are available (Seassau et al., 2010 ; Seassau et al., 2012).
These practices are most effective when they are combined with other practices to help reduce
primary inoculum and disease impact on the crop, varietal choice being one of the main
levers.
However, although significant genetic progress has been made by breeders to increase the
level of resistance / tolerance of sunflower varieties to major fungi, varietal choice may not be
the only method to control disease.
In the case of phomopsis, in very favorable conditions for disease, from the observation
of 125 varieties over 10 years of experimentation in south-western France, it was concluded
that even varieties with good levels of tolerance may result in 10 to more than 30% of plants
with girdling spots which is more that the economic injury threshold (Debaeke and Estragnat,
2009).
Complimentary Contributor Copy
Effects of Crop Management on the Incidence and Severity of Fungal … 221

Figure 9. Potential effect of crop management (water deficit, high nitrogen supply and plant density) on
crop canopy development (architecture, morphology) inducing favorable conditions for the spread of
P. macdonaldii in the plant (xylem) leading to premature ripening (from Seassau et al., 2012).
Table 5. Assessment of the effects of agronomic control and other control methods
on sunflower diseases

Disease
Cultural control
G
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(
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Mildew - 0 - 0 0 0 - - Ø
Phomopsis Leaf spots 0 - + + + + - - Ø
Non girdling spots (stem) 0 - + + 0 + - 0 Ø
Girdling spots (stem) 0 - + + + + - 0 Ø
Phoma Stem spots 0 - 0 + + + - - Ø
Stem base spots 0 - 0 + + 0 - - Ø
Premature ripening 0 - - + + - - 0/- Ø
Sclerotina Stem base - - 0 + + 0 - Ø -
Capituluum - - - - + + - Ø -
+ Favorable to the disease, 0: no effect or random effect, - unfavorable to the disease, Ø: not applicable.

Complimentary Contributor Copy
P. Debaeke, E. Mestries, M. Desanlis et al. 222
In the case of sclerotinia diseases, for which breeding is particularly difficult given the
highly quantitative genetic determinism of plant-pathogen interactions, all the varieties
identified with a good response could be 100% infected when faced with highly conducive
weather conditions, or could bear sclerotia before harvest with harmful consequences for
following crops and penalties for the harvest.
Finally, with mildew, as the sustainability of specific resistance is uncertain, the choice of
varieties with different types of resistance is a crucial decision to be made from year to year
(Moinard et al., 2009).
In addition, cultural practices can contribute to the decision of whether to use chemical
control as in the case of phomopsis: for instance, LAI or plant height values at star bud stage
can be assessed to estimate the risk of disease and influence the spraying decision (Debaeke
and Estragnat, 2009).
It is therefore important to enter a virtuous circle, where cultural control has its own place
and will be more effective when the disease pressure is lower. The combination of all control
methods with partial effects, by their additive or complementary effects, makes for the
appropriate overall strategy (Lucas, 2007). In the case of sunflower, current surveys on
farming practices in France provide opportunities for progress (judging nitrogen fertilization,
suitable sowing time, longer rotations) (Wagner and Lieven, 2010; Jouffret et al., 2011). In
addition, the implementation of appropriate farming practices can help to reduce the number
of sprays (2.1 treatments; Butault et al., 2010) which is one of the main agronomic advantages
of this low input and drought-tolerant oilseed crop
The sound association, in time and at regional level, of cultural, genetic, chemical and
biological control methods is the key to an effective, integrated and sustainable control of
sunflower diseases


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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 10



INSECT PESTS OF SUNFLOWERS IN AFRICA


Hannalene du Plessis


Unit for Environmental Sciences and Management,
North-West University, Potchefstroom, South Africa


ABSTRACT

Numerous insect species attack sunflower in Africa. While only a few species have
high pest status, the majority are of little importance or occur sporadically. Sunflower is
subject to insect damage from planting onwards to drying of seeds on the heads. The
pests that attack sunflower are largely polyphagous and attack a variety of crops and wild
host plants.
Pests can be categorised into seedling pests, leaf pests and pests of sunflower heads.
Seed and seedlings are mainly damaged by soil insects, while mainly Lepidoptera larvae
and Hemiptera species cause damage to sunflower heads. The most important are the
semi-looper, Trichoplusia orichalcea, a highly sporadic pest that attacks leaves of older
plants and when outbreaks occur, total defoliation may result and the false chinch bug,
Nysius natalensis which damages developing seed, resulting in a reduction in yield, oil
content and germination of damaged seeds.
The African bollworm, Helicoverpa armigera is a common pest which infests
sunflower heads during the budding stage and cause damage to achenes from anthesis
onwards. Actual damage and not number of larvae per head is the only criterion which
can be used in the determination of economic injury levels for control of African
bollworm on sunflower.
With the exception of the African bollworm, no economic threshold levels for
chemical control of sunflower pests have been developed in southern Africa. This often
leads to unnecessary application of insecticides. The management of N. natalensis and H.
armigera on sunflower is discussed in this chapter.

Keywords: Sunflower, damage, pests, Helicoverpa armigera, Nysius spp.




hannalene.duplessis@nwu.ac.za.
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Hannalene du Plessis 228
INTRODUCTION

Sunflower is utilized by many insect species, both as food plant and as a source of pollen
and nectar. A number of insect species have adapted to cultivated sunflower and have become
economically important pests (Charlet et al., 1997). Rajamohan (1976) reported 255 different
species of insects and mites attacking sunflower globally, while Misari (1990) recorded 172
species belonging to 77 families and 9 orders in Nigeria. Eighty insect species were reported
from sunflower roots, stems, leaves and flower-heads in Kenya (Khaemba & Mutinga, 1982).
Only those species regarded as of economic importance will be discussed in more detail.


PEST OF ECONOMIC IMPORTANCE

Although many insect species occur on sunflowers in southern Africa, only a few are
considered to be of potential economic importance. These are: the common cutworm [Agrotis
segetum (Denn. & Schiff) (Lepidoptera: Noctuidae)], dusty surface beetles, Gonocephalum
simplex Fab. (Coleoptera: Tenebrionidae), greater false wire worms (Somaticus spp.)
(Coleoptera: Tenebrionidae), ground weevils, Protostrophus spp. (Coleoptera:
Curculionidae), the African bollworm, Helicoverpa armigera (Hübner) (Lepidoptera:
Noctuidae), plusia looper, Trichoplusia orichalcea (Fabricius) (Lepidoptera: Noctuidae) and
the false chinch bug, Nysius natalensis Evans (Hemiptera: Orsillidae). These insects occur
sporadically in high numbers and are then injurious to sunflower crops. The pest complex
regarded as ‗major‘ pests in Kenya are Agrotis spp., Plusia orichalcea Fab, H. armigera and
Nezara viridula L. (Khaemba & Mutinga, 1982), which is similar to southern Africa with the
exception of N. viridula. Nysius natalensis is the most important hemipteran pest in South
Africa.


SEEDLING PESTS

Although various types of damage are caused to sunflower seedlings, the damage
symptoms are characteristic of particular pest species. Damage caused to seedlings vary from
cutting through the stems at or just below the soil surface by dusty surface beetles
(G. simplex), chewing into the stem of seedlings just below the soil surface by greater false
wire worms (Somaticus spp.) and above-ground feeding on seedlings, mainly on leaves by
ground weevils (Protostrophus spp.). These seedlings can be destroyed during severe
infestations (Du Plessis, 1999).
Cutworms (Agrotis spp.) damage seedlings by cutting through the stems at or just below
the soil surface. Severe cutworm infestations can destroy a crop and necessitate replanting.
Strategies to manage cutworms include the application of insecticides and cultural control
strategies. In areas with very cold winters, such as in southern Africa, cultural control by
means of winter cultivation which destroys winter weeds and bring the larvae and pupae to
the soil surface where they are taken by birds or the pupae killed by frost, can reduce
infestation levels present in fields. The destruction of winter weeds deprives cutworm moths
of oviposition substrates and larvae of a food source. Application of these practices over a
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Insect Pests of Sunflowers in Africa 229
wide area, results in decreased moth numbers by the beginning of spring (Drinkwater, 1997).
Preplant weed control is the best method of cutworm control. If there are no volunteer plants
on fields during spring, the large numbers of cutworm moths that start flying during early
spring cannot lay their eggs in fields. Where practically possible, all fields must be cultivated
35 days before planting, or even earlier, to keep them free from weeds until planting time.
This will also cause small cutworm larvae that may already be present in fields at that stage to
die of starvation (Drinkwater, 1997).


PEST OF SUNFLOWER ROOTS

Khaemba and Mutinga (1982) considered root-feeding pests in Kenya to be of little
economic importance since their attacks are sporadic. Termites were, however, reported by
Evans (1951) to cause considerable losses in sunflower by damaging the roots that resulted in
lodging of the plants in Tanzania. Riekert & Van den Berg (2003) evaluated 13 sunflower
cultivars in South Africa and found no plants of any of the cultivars to lodge as a result of
termite damage indicating resistance of these cultivars to termites in contrast to maize plants
that lodge under similar infestation pressures. The planting of sunflower did, however, not
suppress termite activity in the subsequent seasons (Riekert & Van den Berg, 2003).


PEST OF LEAVES

Plusia sp. was reported to visit flowering sunflower plants in South Africa (Cockerell,
1916). Trichoplusia orichalcea larvae can cause devastating damage to sunflower. If
outbreaks occur and large numbers are present, plants may be totally defoliated, stems and
flower buds are damaged, often resulting in total crop loss. Damage usually does not occur on
the edges of fields and is therefore not visible from outside the field (Du Plessis, 1999).


PESTS DURING THE PRE-HEADING AND HEADING STAGES

The most important pests of sunflower during the heading stages of crop development are
hemipterans and H. armigera. Although hemipterans have been reported in abundant numbers
on sunflower in many regions in Africa, none of them, except for Nysius natalensis, have
been studied in detail. Because of their small size, hemipterans are often inconspicuous on
sunflower heads and the qualitative damage they may incur is therefore largely
underestimated. Due to its omnipresence and because H. armigera may attain pest status
wherever sunflower is grown it has been studied on sunflower.
Hemipterans have been reported as the most abundant insects occurring on sunflower
plants during the pre-heading growth stages in Nigeria (Misari, 1990). They suck sap from
stems and leaves resulting in stunted growth of plants. Macrosteles spp. (Cicadellidae)
comprised over 90% of all these hemipterans reported in Nigeria (Misari, 1990). It has,
however, not been documented as such for other African countries. Calidea dregii Germar
(Hemiptera: Pentatomidae), C. bohemia (Stål) (Hemiptera: Pentatomidae), and Nezara
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Hannalene du Plessis 230
viridula L. (Hemiptera: Pentatomidae), are regarded as pests of immature sunflower seeds in
east Africa (Hill, 1975; Khaemba & Mutinga, 1982). Very high populations of C. dregii
(140 000/ha) was reported by Evans (1951) on sunflower in Urambo, Tanzania and their
feeding resulted in reduced seed weight, oil content and subsequent seed viability.
The intensive feeding by hemipterans during the heading stage results in deformed heads
which delay flower opening (Misari, 1990), a phenomenon which is also observed under
heavy infestation of N. viridula in South Africa (H. du Plessis, personal observation).
The most common hemipteran family on sunflower in South Africa is, however, the
Lygaeidae (seed bugs) (Du Toit & Holm, 1992). The genus Nysius was previously classified
under the family Lygaeidae. Most species of the Lygaeidae, both ground-living and plant-
living, subsist upon seeds of many plant species (Sweet, 1960). The name ―seed bugs‖ was
therefore suggested for the Lygaeidae by Sweet (1964). The family Lygaeidae was, however,
divided into 11 families (Henry, 1997), with the family Orsillidae (also containing the genus
Nysius) now considered to be a distinct family from the Lygaeidae (Sweet, 2000). Misari
(1990) recorded Nysius stali Evans to occur from the heading stage to the grain stage on
sunflower in Nigeria. The occurrence of the false chinch bug, Nysius natalensis Evans
(Hemiptera: Orsillidae) is similar on sunflower in South Africa and sunflower crops are
usually damaged in late summer (Du Plessis et al., 2007). Damage by N. natalensis to seeds
results in a reduction in yield, oil content and germination of damaged seeds.
Sunflower in South Africa is grown mainly between the 24°S and 30°S latitudes, where
the climate is semi-arid, characterized by erratic and relatively low rainfall, low humidity and
high radiation intensity during summer (Nel, 1998). Nysius occurs throughout the South
African sunflower production area but only attains pest status in certain areas (Du Plessis et
al., 2007). In a study to determine the effect of temperature on N. natalensis development and
survival, Du Plessis et al. (2011) found that the females lay eggs sooner at higher
temperatures (shorter pre-oviposition period), but that the optimum temperature for
oviposition (most eggs laid) is between 26 and 28°C and it is also the temperature range
where the female lifespan is the longest (19 – 28°C) (Table 1). The lower threshold
temperature for N. natalensis eggs and nymphs differed by only 1.25°C. Eggs will not hatch
at temperatures too low for nymphal development, which is 14.0°C for eggs and 15.2°C for
nymphs (Du Plessis et al., 2011). Since high summer temperatures prevail throughout the
sunflower production area of South Africa and the most favourable temperature range for N.
natalensis development was determined as 26 to 38°C, the potential for rapid population
build-up exists during the sunflower production season. Potential therefore also exist that this
pest can become important on sunflower in other African countries too.
Wild host plants and crops such as grain sorghum play an important role in sustaining
populations of N. natalensis (Kruger et al., 2008; Du Plessis et al., 2007). To determine
whether a plant is a suitable host of a particular insect, the insect must be able to complete its
life cycle on the host plant and replace itself, or have an intrinsic rate of population increase
equal to or greater than zero (Zalucki et al., 1986). Shorter development times and higher
rates of reproduction with low mortality rates on a host indicate greater suitability of a
particular host plant (Awmack & Leather, 2002). Eggs, nymphs, and adults of N. natalensis
have been collected from 26 species of host plants in eight families, as well as from cultivated
sunflower plants in South Africa (Du Plessis et al., 2007).

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Table 1. Mean fecundity and longevity (+ S.E.) of Nysius natalensis females at constant temperatures and 14L:10D photoperiod.
On the day of their final moult, single male-female pairs were confined to Petri dishes (90 mm diameter)
and life history parameters recorded until females died

Temperature
(± 1 °C)
n Pre-
oviposition
period (days)
Post-
oviposition
period (days)
No. of days
during which
no eggs were
laid
Longevity of
adult female
(days)
Total no.
of eggs laid
Mean no. of
eggs laid per
day
Max. no. of
eggs laid per
day
Infertile
eggs (%)
19 28 13.6 + 1.3 a 0.9 + 0.2 ab 31.3 + 3.2 a 45.0 + 2.9 a 122.0 + 13.6 b 3.9 + 0.3 a 10.9 + 0.7 a 1.2 bc
26 35 5.9 + 0.4 b 1.2 + 0.3 ab 3.3 + 1.1 b 32.4 + 2.2 b 245.8 + 19.6 a 10.6 + 0.7 b 21.3 + 1.1 b 2.8 a
28 28 6.3 + 0.7 b 1.5 + 0.5 a 2.5 + 0.7 b 28.9 + 1.8 b 275.8 + 28.8 a 12.2 + 0.8 bc 23.5 + 1.5 b 0.1 c
31 37 4.3 + 0.2 c 0.6 + 0.2 ab 0.9 + 0.3 b 13.9 + 1.0 c 107.1 + 12.4 b 11.7 + 0.8 bc 22.3 + 1.3 b 4.9 ab
36 30 2.3 + 0.1 c 0.2 + 0.1 b 0.3 + 0.1 b 8.8 + 0.7 c 95.7 + 12.3 b 15.0 + 1.5 c 25.9 + 2.1 b 1.7 c
38 31 3.6 + 0.2 c 0.3 + 0.2 b 0.4 + 0.2 b 9.2 + 0.5 c 81.6 + 8.8 b 14.6 + 1.0 c 23.5 + 1.6 b 0.6 c
Means within the same column followed by the same letter do not differ significantly at P = 0.05 (Tukey‘s HSD). (From: Du Plessis et al., 2011).
(Reproduced with permission of the Entomological Society of Southern Africa).



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Hannalene du Plessis 232
Table 2. Plant species recorded as host plants of Nysius natalensis in South Africa.
Sampling of Nysius natalensis and host plants was conducted through a roadside
survey in each quarter-degree grid in the area in the sunflower production
area of South Africa. Nysius natalensis was reared for one generation
in the laboratory on seed of each wild host plant to confirm the host status
of the respective plants (Du Plessis et al., 2007).

Family Plant species Common name
Amaranthaceae
Amaranthus hybridus L. subsp. hybridus var.
hybridus
Cape pigweed
A. spinosus L. Thorny pigweed
A. thunbergii Moq. Red pigweed
Asteraceae Conyza aegyptiaca (L.) Aiton Fleabane
C. albida Spreng. Tall fleabane
C. bonariensis (L.) Cronq. Flax-leaf fleabane
C. canadensis (L.) Cronq. Horseweed fleabane
C. podocephala DC. Conyza
Felicia muricata (Thunb.) Nees subsp. muricata -
Helichrysum atgyrosphaerum DC. -
H. nudifolium (L.) Less -
H. rugulosum Less -
Nidorella resedifolia DC. subsp. resedifolia -

Pseudognaphalium luteo-album (L.) Hilliard &
Burtt
Jersey cudweed
P. oligandrum (DC.) Hilliard & Burtt -
Senecio consanguineus DC. Ragwort
S. inornatus DC. -
Campanulaceae Wahlenbergia undulata (L.f.) A.DC. Pale bluebell
Chenopodiaceae Chenopodium album L. White goosefoot
C. carinatum R. Br. Green goosefoot
C. cristatum F. Muell. Crested goosefoot
Poaceae
Melinis repens (Willd.) Zizka subsp. grandiflora
(Hochst.) Zizka

(Hochst.) Zizka
Natal red top
Portulacaceae Portulaca oleracea L. Pigweed
P. quadrifida L. Wild purslane
Solanaceae Physalis viscose L. Sticky gooseberry
Verbenaceae Verbena bonariensis L. Wild verbena
Reproduced with permission of the Entomological Society of Southern Africa

Fewer eggs are laid by females when they fed on stems of plants, in spite of the seed of
these plants being a suitable food source for their survival. Seeds were therefore found to be
essential for N. natalensis reproduction. The insect is polyphagous and a variety of wild host
plants as well as sunflower are suitable for feeding and reproduction. As N. natalensis adults
are good fliers, it is likely that nymph-to-adult host switching is a common phenomenon. It is
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Insect Pests of Sunflowers in Africa 233
also likely that the insects switch hosts between generations when adults migrate and lay eggs
on plant species different from those on which they developed. The effects of within-
generation host plant switching were studied by Du Plessis et al. (2012), by providing various
combinations of foods to nymphs and adults. Nymphs were reared on crushed seed of five
plant species, namely A. hybridus, P. oleracea, C. album, C. albida and H. annuus. After
completion of the nymphal stage, emerging adults of each group were either provided with
the same diet, or were transferred to different seeds as adult food. The duration of the pre-
oviposition period is affected by the nymphal food source, but fecundity is determined by
food consumed during the adult stage (Du Plessis et al., 2012). The number of eggs laid by N.
natalensis is affected by adult food as well as by the interaction between nymphal and adult
food sources while adult longevity is influenced by the host plant species consumed by both
nymphs and adults (Du Plessis et al., 2012).
The wild host plants, A. hybridus, P. oleracea, C. album, and C. albida were also
evaluated as paired combinations in two-choice experiments to determine the comparative
attractiveness of sunflower and wild host plants for oviposition by N. natalensis. The control
was a piece of pipe cleaner (Du Plessis et al., 2012). When given a choice, N. natalensis
females prefer to lay their eggs on wild host plants (weeds), compared with sunflower (Figure
1). Sunflower is therefore not the preferred host, but N. natalensis lay its eggs on sunflower if
its preferred host plants are removed or dead (Du Plessis et al., 2012). These results explain
host plant switching by N. natalensis from its wild host plants to sunflower as a result of
restricted temporal availability of wild host plants. It also explains their injuriousness to late-
planted sunflower when senescence of weed species occurs during autumn. This period often
coincides with seed fill of late-planted sunflower, providing host plants for the insect with a
high moisture content, as well as seeds necessary for reproduction of the pest. Weeding of the
headlands of sunflower during seed fill of the crop results in destruction of the preferred host
plants of N. natalensis. Insect consequently move to sunflower where they feed and cause
damage (Du Plessis et al., 2007).
The risk of short-range dispersal from host weed species should be limited to protect
sunflower crops, especially late-planted crops, from N. natalensis damage. Headlands and
adjacent fields should be kept weed free from the beginning of the season to limit pest
population build up. Furthermore, weedy headlands and adjacent fields on which weedy host
plants occur should not be hoed during seed-fill period of sunflower crops because these
activities result in destruction of the wild host plants, causing subsequent invasion of
sunflower fields (Du Plessis et al., 2007). Late plantings should therefore be avoided where
possible and particular attention should be paid to informed control of annual weeds in the
vicinity of the crop (Du Plessis et al., 2012).
The decision to apply insecticides for control of this pest should take the following into
consideration. It is a polyphagous pest that is highly mobile and continuous re-infestations
can occur. Timing of insecticide application is therefore important. Most damage to sunflower
seeds inflicted by N. natalensis is done at the distal end of seeds (Du Plessis et al., 2005). It
takes the cotyledons approximately 10 to 12 days to develop and reach the distal end of the
husk, depending on the cultivar. If damage is the only concern, it is not necessary to spray
during anthesis. Keeping in mind that anthesis commences at the periphery of the sunflower
head and progresses inward, it can be equal to the period that heads of certain cultivars take to
complete anthesis (Du Plessis et al., 2005). The period that the heads take to turn downward
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Hannalene du Plessis 234
after completion of anthesis will, however, determine the period of application of insecticides
against N. natalensis during seed fill (Du Plessis et al., 2005).


Source: Entomologia Experimentalis et Applicata © 2012 The Netherlands Entomological Society
Figure 1. Mean number (± SD) of eggs laid by Nysius natalensis on various host plants (Helianthus
annuus, Amaranthus hybridus, Chenopodium album, Conyza albida, and Portulaca oleracea) in paired-
choice tests. Means of a pair capped by the same letter do not differ significantly (Tukey‘s HSD:
P>0.05). (Du Plessis et al., 2012).
The African bollworm, H. armigera, is an important pest of many crops in many parts of
the world (Zalucki et al., 1986; Sharma, 2001). It is also regularly present during the
reproductive stage of cultivated sunflower in Africa. Its attractiveness to sunflower is
demonstrated by its use as a trap crop in and around organic cotton fields in Tanzania (Cherry
et al. 2003). Larvae occur from the budding stage onwards (Von Maltitz, 1993). Levels of
infestation vary between localities and seasons, sporadically reaching epidemic proportions.
First and second instar larvae are mainly found on sunflower buds, with a feeding preference
for involucral bracts. Later instar larvae feed more extensively than younger larvae,
consequently doing more damage. One mature larvae feeding inside a bud may completely
destroy the immature florets. However, plants infested at the budding stage escape serious
feeding damage as the majority of larvae mature during anthesis (Von Maltitz, 1993; Du
Plessis, 1997). Larvae of all instars also burrow under and between bracts into the receptacles
(Von Maltiz, 1993), and are, therefore often not exposed to insecticides. Insecticides for H.
armigera control on sunflower in South Africa are applied aerially and the question often
arises whether effective insecticide application is limited by the downward inclination of
sunflower heads and by the fact that the heads are often turned in the opposite direction to
aerial application. Du Plessis (1997) found aerial application of insecticides for bollworm
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Insect Pests of Sunflowers in Africa 235
control to be effective during the budding as well as the anthesis and pollination stages when
later instars, which are more damaging, occurred on heads.
Simulated bollworm damage was done to florets (R-5.1stage) and young achenes in the
milky stage (R-6.0) of sunflower in a field trial in South Africa (Du Plessis, 1997). R-5.1 is
the reproductive stage at which 10 % of the head area (disk flowers) has completed or is in
flower. R-6.0 is the reproductive stage in which anthesis and pollination are completed and
the ray flowers are wilting (Schneiter and Miller, 1981). The mean mass of randomly chosen
achenes in these heads increased at increased levels of damage (Figure 2).
Compensation for lost florets and achenes, therefore, occurred over the entire head.
Compared with undamaged heads, the mean mass of achenes was significantly higher at
levels of 15, 20 and 30 % damage at both reproductive stages (Figure 2). The total mass of
fertile achenes per head did not differ between damaged and undamaged heads, up to a 30 %
level of damage (although lower at 5 and 30 % damage levels) (Figure 3) for both
reproductive stages evaluated. It showed that sunflower has the ability to compensate for
damage (Du Plessis, 1997). Sunflower variety and climatic conditions may provide results
which differ from those found by Du Plessis (1997).


Figure 2. Mean mass of 15 achenes surrounding simulated bollworm damage area(s) and chosen at
random in sunflower heads at different levels of damage done to florets in the R-5.1 reproductive stage
and to young achenes in the milky stage (R-6.0). │= LSD
T
(P = 0.05). (From: Du Plessis, 1997).
(Source: African Crop Science Journal)

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Hannalene du Plessis 236

Figure 3. Mean mass of fertile achenes per sunflower head at different levels of simulated bollworm
damage (n=24) done to florets in the R-5.1reproductive stage and to young achenes in the milky stage
(R-6.0). │= LSD
T
(P = 0.05). (From: Du Plessis, 1997). (Source: African Crop Science Journal)
Based on the results of this study by Du Plessis (1997), it is proposed that insecticides be
applied when at least 20% damage per head occurs, in order to promote timely control
measures. Taking into account that the preferential feeding sites of H. armigera are not the
achenes, as well as the ability of plants to compensate for damage to florets and achenes, a
significant number of larvae can, however, be tolerated without any significant effect on
yield. Actual damage is therefore the only criterion which can be used in the determination of
economic injury levels for control of African bollworm on sunflower (Du Plessis, 1997). The
large volumes of insecticides applied for H. armigera control is therefore not justifiable, since
damage levels greater than 20% seldom occur and natural enemies are abundant.
Approximately 170 parasitoid species and a large number of predators of H. armigera have
been reported from southern and east Africa (Cherry et al., 2003). Furthermore, a nuclear
polyhedrosis virus that occurs freely in nature, contribute significantly in controlling H.
armigera populations in southern Africa.


CONCLUSION

Although many insect species occur on sunflower in Africa, only a few of these obtain
pest status. Insecticide application for control of pest species should therefore be done with
care since economic injury levels of all, except for H. armigera, have not been determined
and natural enemies are plentiful. Further research on the hemipteran pest complex of this
crop is needed since the presence of these pests may lead to qualitative crop losses.


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Insect Pests of Sunflowers in Africa 237
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In: Sunflowers ISBN: 978-1-63117-347-9
Editor: Juan Ignacio Arribas © 2014 Nova Science Publishers, Inc.






Chapter 11



SOIL AMENDMENTS AND THEIR EFFECTS
ON SUNFLOWER GROWTH


Fernando López-Valdez
1
*
, Fabián Fernández-Luqueño
2
,
Perla Xóchitl Hernández-Rodríguez
1
, Minerva Rosas-Morales
1

and Silvia Luna-Suárez
1

1
Centro de Investigación en Biotecnología Aplicada, Instituto Politécnico Nacional,
Tepetitla de Lardizábal, Tlaxcala, México
2
Natural Resources and Energy Group, Cinvestav-Saltillo,
Coahuila, México


ABSTRACT

An interesting topic in agriculture is the search for forms of fertilisation that have a
low impact on soil, plants, humans, and the environment. Wastewater treatment plants
almost always separate the organic matter, called wastewater sludge or sewage sludge.
This sludge is rich in mineral macronutrients such as nitrogen (ammonium or nitrate),
phosphorous (phosphate), potassium, and micronutrients. The sewage sludge might
provide the majority of necessary nutriments for growing plants, and also provide many
beneficial effects when it is applied to soils, resulting in an improvement in the chemical,
physical, and biological characteristics. We know that wastewater sludge and soil
microorganisms play an important synergetic role on the released and available nitrogen.
Although urea is the most accepted type of fertiliser used worldwide, it does have some
inconvenient drawbacks such as pH changes, microflora modifications from the soil, and
others; these issues must be considered in order to avoid N losses. An interesting
alternative might be the use of Plant Growth-Promoting Rhizobacteria (PGPR, soil
bacteria that colonize the roots of plants by inoculation onto seeds or roots and that allow
enhanced plant growth) as helpers. Examples such as Pseudomonas, Azotobacter,
Burkholderia, Klebsiella, and Bacillus, among other genera, have been reported. In this

*
Corresponding author: F. López-Valdez, Centre for Research in Applied Biotechnology (CIBA) - Instituto
Politécnico Nacional. Carr. Est. Sta. Inés Tecuexcomac – Tepetitla km. 1.5 s/n, Tepetitla de Lardizábal,
Tlaxcala, C.P. 90700, México. Tel.: +52 55 5729 6000 ext. 87805; Fax: +52 248 487 0762. E-mail address:
flopez2072@yahoo.com.
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F. López-Valdez, F. Fernández-Luqueño, P. X. Hernández-Rodríguez et al. 240
chapter, we focus on specific types of fertilisation or soil amendments and their effects on
this important crop. In particular, Bacillus subtilis has showed promising results. It is
known that these bacteria might improve plant growth by several direct mechanisms as
well as indirect mechanisms, such as controlling phytopathogen organisms, or a
combination of both. A regular strain of Bacillus subtilis was co-inoculated with urea in
sunflower roots, and it was found that the strain temporarily stimulated sunflower cultivar
growth. We found that B. subtilis improves the sunflower establishment, particularly its
strength and vigour during the early stages, resulting in an increase of the mass and
length of roots compared with the control treatment. Application of either organic or
mineral fertiliser improved the crop yield under various conditions, but fertilising the
crops with sewage sludge might be more environmentally friendly than using mineral
fertiliser. Wastewater sludge has a high NH
4
+
content that is slowly oxidized to NO
3
-

while being absorbed by sunflower cultivars. In both cases (organic amendment or
bacterial inoculation with urea), the NO
3
-
is not lixiviated or leaked during experiments.
Accordingly, the wastewater sludge and B. subtilis might be potential methods of
fertilisation for sunflowers or any ornamental cultivars.

Keywords: Bacillus subtilis, fertilisers, sunflower, urea, wastewater sludge


INTRODUCTION

Agriculture is an essential discipline that concerns the possible cultivation of domestic
plants in order to improve food or biomass productions. Enhancing food security and a
sustainable production of foods (agricultural sustainability) are modern global topics.
Therefore, fertilisation is a critical topic, particularly aspects such as the reasonable
application of fertilisers, reutilization of materials (wastewater sludge, manure, tannery
sludge, whey, biochar, inter alia), and application of beneficial microorganisms (PGPRs,
mycorrhizal fungi, among other microorganisms) as soil amendments. These could all be
strategies that lead to a sustainable agriculture. In this chapter, we explore some of the
strategies that we have used to experiment on an important crop, the sunflower.


SUNFLOWER

The sunflower (Helianthus annuus L.) is a significant crop and an attractive ornamental
plant known throughout the world. This crop is a considerable source of edible oil, protein
(Nesterenko et al., 2013; Lin et al., 1974), oil for diesel biosynthesis, phytoextraction, and
even animal feed. It shows a well-developed and deeply penetrating root system, allowing a
good establishment, which is why it is considered a drought-tolerant plant (Stone et al.,
2002). Therefore, the sunflower is easily cultivated, exhibiting growth with minimal or no
fertilisation (Ruiz and Maddonni, 2006), and even exhibiting growth under rain-fed
conditions. Mexico might be the origin of domestication of this crop. There is evidence of
some early remains of H. annuus discovered at the San Andrés site in the Gulf Coast region
of Tabasco, Mexico, constituting the earliest record of the domesticated sunflower (Lentz et
al., 2001; Wills and Burke, 2006).
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Soil Amendments and Their Effects on Sunflower Growth 241
CHEMICAL FERTILISERS

It is known that nitrogen (N) is the most important mineral nutrient for non-leguminous
plants. This mineral has several chemical forms within the soil, such as ammonium (NH
4
+
-N),
nitrite (NO
2
-
-N), and nitrate (NO
3
-
-N); ammonium and nitrate are both valuable ions for
plants. There are several synthetic N fertilisers, such as ammonia, urea, ammonium nitrate,
calcium ammonium nitrate, ammonium sulphate, and urea ammonium nitrate, among others.
The world's production of ammonia (NH
3
) exceeded 134.2 x 10
9
kg y
-1
in 2011 (IFA, 2013).
With the highest N content (82%), it is feedstock for N fertilisers and other products.
However, when ammonia is applied as aqua ammonia (dissolving ammonia in water from
20% to 24% N solution) as fertiliser, special care must be taken: it must not be placed in close
proximity to seeds, and it must be handled carefully for safety purposes.
Urea is the most common N fertiliser used worldwide, constituting over 71.1 x 10
9
kg y
-1

in 2011 (IFA, 2013). The production of ammonia and urea is increasing annually; in fact, the
ratio of world urea production is about 2.1 x 10
6
kg y
-1
(the result was estimated from data
from 2002 to 2011). Urea has several advantages over others fertilisers, such as a high N
content (46.7%) and solubility (20 °C) at 1,080 g L
-1
. In addition, it is easier to handle than
NH
4
NO
3
or ammonia, easier to store, less corrosive to machinery, and less likely to explode
or burn. However, some problems have been reported, including damage to seeds, seedlings
or young plants; NO
2
-
toxicity; phytotoxicity of foliar-applied urea; and volatilization of urea-
N as NH
3
(Bremner, 1995).


WASTEWATER SLUDGE

Wastewater sludge or sewage sludge is an unavoidable by-product from wastewater
treatment plants. The wastewater sludge is rich in organic matter as well as macro- and
micronutrients for plants (N, P, S, and minerals). It can even provide C and energy sources for
the growth of soil microorganisms (López-Valdez et al., 2010, 2011b). Also, it has the
potential to improve the physical, chemical, and biological properties of the soil. However,
sewage sludge can also contain some pathogens—bacteria, virus, protozoa cysts, or helminth
ova (Jiménez-Cisneros et al., 2001; Jiménez, 2007)—which are heavy metals and organic
pollutants that must be eliminated. According to USEPA, sewage sludge can be classified as
Class A or Class B with regard to pathogens (2013). Class A refers to sludge that might be
amended to agricultural soils with edible cultivars. In contrast, Class B refers to sewage
sludge that might to applied to non-agricultural soils or even soils where ornamental plants
will be cultivated.
Approximately 37% of the sewage sludge produced in the world was used in agriculture
and 12% was used in forestry (Fytili and Zabaniotou, 2008). The remainder of the sludge was
not used, commonly resulting in its being confined or burned. Application of wastewater
sludge in agriculture is an alternative disposal approach that, relative to the burn-up approach,
offers the opportunity to recycle nutrients that enhance plant growth, return organic matter to
the soil (López-Valdez et al., 2011b), and avoid CO
2
-C return to the atmosphere. It has been
reported that sludge significantly increases the yield in apple trees (Malus pumila Mill.)
(Bozkurt et al., 2010), commun beans (Phaseolus vulgaris L.) (Fernández-Luqueño et al.,
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F. López-Valdez, F. Fernández-Luqueño, P. X. Hernández-Rodríguez et al. 242
2010), cotton (Gossypium hirsutum L.) (Samaras et al., 2008), mung beans (Vigna radiata L.
cv. Malviya janpriya) (Singh and Agrawal, 2010b), rice (Oryza sativa L. cv. Pusa sugandha)
(Singh and Agrawal, 2010a), soybeans (Glycine max (L.) Merrill) (Souza et al., 2009), and
sunflowers (Helianthus annuus L.) (Lavado, 2006).
The goal of sewage sludge application into the soil is to provide an alternative, to restore
degraded or N-depleted soils and simultaneously enhance plant growth (Fernández-Luqueño
et al., 2009; López-Valdez et al., 2010, 2011b).


BACTERIA AS HELPERS (PGPR)

Another compelling alternative is the application of microorganisms such as bacteria and
filamentous fungi (as mycorrhizal fungi). Bacteria are the most abundant microorganisms in
soil (up to 6 x 10
8
cells g
-1
of soil and a weight of approximately 10,000 kg ha
-1
) (Kilian et al.,
2000). The number of bacteria found in soil depends on the season, the type of soil, the
cultivar used, the moisture content, the oxygen supply in the soil, the tillage and fertilisation
of the soil, the penetration of the soil by plant roots, and the depth from which the soil
samples were taken (Kilian et al., 2000). Pseudomonas, Arthrobacter, Achromobacter,
Clostridium, Micrococcus, Flavobacterium, Azospirillum, Azotobacter, and Bacillus are the
most representative genera of bacteria from soil and other environments (López-Valdez et al.,
2011a). Bacteria have notorious advantages over fungi such as faster growth, a ubiquitous
presence, and the ability to be cultured in vitro. Bacteria might even compete for an
ecological niche; bacteria produces metabolites or enzymes that enhance or stimulate the
growth of plants through direct or indirect mechanisms. The Bacillus genus shows a wide
spectrum of mechanisms that might (a) stimulate plant growth as fungistatic or bactericidal
compounds (Singh and Deverall, 1984; Ongena et al., 2005; Forchetti et al., 2007; Sang et al.,
2008; Swain and Ray, 2009; Alvindia, 2013), (b) produce phytohormones involved in root or
shoot growth (Araújo et al., 2005; Yao et al., 2006; Karadeniz et al., 2006; Forchetti et al.,
2007; Swain and Ray, 2009; Ahmed and Hasnain, 2010), (c) induce systemic resistance
(Gupta et al., 2000) by volatile organic compounds (Ryu et al., 2003; Ping and Boland, 2004),
(d) colonize plant roots (Dijkstra et al., 1987) by remaining very close to the root tip by
passive displacement, and (e) produce extracellular enzymes. Another interesting result is the
reduction of ethylene production, as described by Penrose and Glick (2003). Also, the
Bacillus genus is known to affect the fruit ripening, leaf senescence, flower abscission,
germination, cell elongation and proliferation, nodulation, and response to plant pathogen
attack. Hence, bacteria that can stimulate or promote plant growth are called PGPR.


SAMPLING SITE AND PROPERTIES OF SOIL

We used several soils from different land uses. The first sampling site was located in the
former lake of Texcoco (Mexican valley, Mexico; 19°30‘ N, 98°53‘ W) at an altitude of
2,250 m above sea level. This soil is alkaline saline, classified as Typic Ustifluvent with a pH
between 8.5 and 11, electrolytic conductivity (EC) between 4 and 170 dS m
-1
, and water
holding capacity (WHC) of 684 g kg
-1
soil. The former lake bed is not cultivated, but some
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Soil Amendments and Their Effects on Sunflower Growth 243
grasses and small trees can grow. More details are available in López-Valdez et al. (2010).
The second soil samples were obtained from Otumba (Estado de México, Mexico; 19°42‘ N,
98°49‘ W), near the former lake Texcoco. The soil, classified as Typic Fragiudepts, was
characterized as sandy loam, pH 7.6, EC of 1.1 dS m
-1
, WHC of 530 g kg
-1
soil, and an
organic C content of 7.2 g C kg
-1
soil. This soil was cultivated with maize, receiving a
minimum amount of mineral fertiliser without being irrigated. More details are available in
Fernández-Luqueño et al. (2009). The third sampling site is located in Alcholoya, an Acatlán
village (Hidalgo, Mexico; 2,120 m above sea level and 20°09‘ N, 98°26‘ W). The soil was
classified as Typic Fragiudepts with pH 6.5, EC 0.7 dS m
−1
, a WHC of 846 g kg
−1
soil, an
organic C content of 11.1 g kg
−1
soil, and a total N content of 1.0 g kg
−1
soil. This soil
received organic fertiliser (cow excreta) occasionally. More information is available in
López-Valdez et al. (2011b). All soils were sampled by augering (0 to 15 cm depth) from
three plots equivalent to 0.5 ha. Soils from each plot were pooled and sieved. In total, three
soil samples were obtained.


CHARACTERISTICS OF THE WASTEWATER SLUDGE

Reciclagua (Sistema Ecológico de Regeneración de Aguas Residuales Ind., S.A. de C.V.)
in Lerma (Estado de México, Mexico) treats wastewater from various sources, mainly
alimentary industries and households. In the primary treatment, the wastewater is mixed with
a flocculant, and the sludge obtained is passed through a belt filter in order to reduce the
water content. Although the concentration of heavy metals and toxic organic compounds has
been low, (Franco-Hernández et al., 2003) this sludge can be classified as Class B due to its
pathogen content. The characteristics of sludge were pH 8.1, CE 7.9 dS m
-1
, water content
847 g kg
−1
, organic C content 288 g kg
−1
, and total N content 41.8 g kg
−1
. The mineral N was
NH
4
+
-N 13 g kg
-1
, NO
2
-
-N 8.3 mg kg
-1
, and NO
3
-
-N 122 mg kg
-1
on a dry matter.
These characteristics show that sewage sludge is a strong candidate for an organic
fertiliser, since the C/N ratio is over 14, meaning that sludge has a high N content. Almost
30% is ammonium, indicating that the mineral N is immediately available for plants; the
remaining 70% is organic N (such as proteins, amino acids, and nucleic acids, among others),
available for microorganisms that mineralize organic N up to mineral nitrogen. This mineral
N is released slowly and remains available for plants.


BACI LLUS SUBTI LI S

We studied a regular strain of Bacillus subtilis that was isolated from the potato
rhizosphere (identified by 16S ribosomal RNA, rRNA, gene sequencing). The strain was
provided by Dr. Olalde-Portugal (Laboratory of Microbial Ecology, Department of
Biotechnology and Biochemistry, Cinvestav, Gto., Mexico). This strain was tested and
selected due to its ability to provide positive results in some in vitro tests, thus demonstrating
its antagonistic activity against some phytopathogenic fungi (Fusarium oxysporum and
Rhizoctonia solani AG1). Also, this strain embodies many characteristics, such as the ability
to solubilize phosphate as described by Burr et al. (1984), the 1-aminocyclopropane-1-
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F. López-Valdez, F. Fernández-Luqueño, P. X. Hernández-Rodríguez et al. 244
carboxilate deaminase enzymatic activity as reported in Penrose and Glick (2003), the indole-
3-acetic acid production by the colorimetric method (Azcón et al., 2009), and the root
colonization on maize and sunflower in a Petri dish using Phytagel (Sigma Co.) as a medium
(Dijkstra et al., 1987). This strain belongs to a collection that is being explored as PGPR. In
our opinion, this is a regular or common strain. More information can be found in López-
Valdez et al. (2011a).


METHODS

The sunflower seeds were provided by the Departamento de Fitotecnia, Universidad
Autónoma de Chapingo, Texcoco, Estado de México, Mexico [Department of Plant Science,
Autonomous University of Chapingo, Texcoco, Estado de México, Mexico]. The non-treated
seeds were received for amendments with sewage sludge, urea, or unamended soil. However,
according to López-Valdez et al. (2011a), the seeds were disinfected for inoculation with B.
subtilis and were incubated in an agar-agar plate under aseptic conditions in order to
determine whether microorganisms grow after one day of incubation. Afterward, these seeds
were dressed with a suspension of B. subtilis at 10
7
CFU mL
-1
in 1% carboxymethylcellulose.
One hundred and eight sub-samples were prepared as follows: each sub-sample contained
6.5 kg soil in a cylindrical pot with the purpose of obtaining five treatments, three plots, three
replicates, and three sampling times (at 37, 60, and 95 days after sowing) during the
experiment (López-Valdez et al., 2011b). At the onset of the experiment, 0.5 g urea was
added to nine pots from each of the three sampled plots; twelve days after emergence, the
nine plantlets were amended with another 0.5 g urea (equivalent to 150 kg N ha
-1
, the UREA
treatment). Nine others pots were amended with 30 g sludge (150 kg N ha
-1
, the SLUDGE
treatment), nine were left unamended (the PLANT treatment), and nine were left unamended
and unsown (the CONTROL treatment). The last treatment was amended with B. subtilis (the
BACTERIA treatment); the seeds were disinfected, dried, and dressed with the B. subtilis
suspension (as described above). In this treatment, the plants were fertilised with 0.5 g urea
(75 kg N ha
-1
). Each pot was sown with three sunflower seeds. Eight days after emergence,
two of the three plantlets in each pot were discarded. The tap water was analysed, 19 kg
mineral-N ha
-1
was additionally added to each treatment during the whole experiment, and no
water was leached out from the pots (López-Valdez et al., 2011b).
At the onset of the experiment, approximately every two days up to 30 days after sowing,
the columns were airtight and the atmosphere was analysed for CO
2
and N
2
O at 0, 3, 15, and
30 minute intervals. On every sample day (37, 60, and 95 days), nine pots were selected at
random from each treatment. Soil samples were collected at depths of 0-15 cm and 16-30 cm.
The roots were separated from the shoots. The variables measured in the plants were shoot
height, root length, fresh shoot weight, fresh root weight, dry shoot weight, dry root weight,
seed weight per plant, number of seeds per plant, and total N content; in the soil, the variables
measured were pH, EC, NH
4
+
-N, NO
2
-
-N, and NO
3
-
-N. Also, the gas production was
measured as CO
2
-C and N
2
O-N. Further details can be found in López-Valdez et al. (2011b).
Significant differences between plant and soil characteristics as a result of the various
treatments were determined by analysis of variance (ANOVA) and based on the least
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Soil Amendments and Their Effects on Sunflower Growth 245
significant difference test, using the general linear model procedure (PROC GLM; SAS
Institute Inc., 1989).


UREA NOTES

Sunflower Characteristics (Alcholoya Soil)

According to our results, the UREA treatment was not significantly different in shoot
height and dry root weight compared with the other treatments at one, two, and three months.
After two months, the root length and fresh root weight were similar to the BACTERIA and
PLANT treatments. Finally, the effect of urea was significantly different in the dry shoot
weight compared with the PLANT treatment. Compared with the unfertilised plants, the urea
enhanced the dry shoot weight, causing a significant increase of total N content per kg of
dried plant (P<0.05). As we know, the urea has a disadvantage: it decomposes up to NO
3
-
,
which could form leaching, and it continues decomposing and losing N
2
O through the
nitrification or denitrification processes.


Soil Characteristics of Alcholoya

The pH did not significantly differ by treatment in either soil depth (0-15 and 16-30 cm).
At the end of the experiments, EC was not significantly different at a depth of 0-15 cm, but a
slight increase (not a significant difference) was noted at a depth of 16-30 cm. Fernández-
Luqueño et al. (2009) showed that soil amended with urea and cultivated with common bean
did not result in a change of pH. Mineral N, NO
2
-
-N, and NH
4
+
-N did not significantly differ
by treatment in either soil depth. However, the treatments with urea (the UREA and
BACTERIA treatments) showed significant NO
3
-
-N differences compared with the
unfertilised plants. The urea-N was transformed to NO
3
-
-N almost totally, and remained in the
soil at the given depth (16-30 cm). As reported by Casado-Vela et al. (2006), tap water
provides mineral N at the end of the experiments. An analysis by principal components
(PCA) showed that the PLANT treatment is opposite to the UREA and BACTERIA
treatments (quadrant +,+) in the PCA plot (Figure 1); these results indicate that the treatments
have positive effects on sunflower plants, in contrast to sunflower plants without some kind
of fertilisation. The UREA and BACTERIA treatments have effects on the variables
measured on soil, i.e., increasing in naturally the N
2
O and CO
2
productions, EC, and NO
3
-
(in
both depths) as consequent of urea application and its decomposition (to CO
2
and NH
3
). Also
minor effects have on pH, NH
4
+
, NO
2
-
due to nitrification process (where NH
4
+
was
transformed to NO
2
-
and NO
2
-
was transformed to NO
3
-
). The urea addition had little effect on
the pH of the soil in these experiments, but is well known that long-term applications of urea
decrease the pH of soil. In the long-term, ammonium decreases soil pH by oxidation to NO
3
-
,
generating a proton (Enwall et al., 2007). The sunflowers fertilised with urea did not affect
the production of CO
2
; however, the production rate of N
2
O was more than double that of the
mean produced by unamended soil.

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F. López-Valdez, F. Fernández-Luqueño, P. X. Hernández-Rodríguez et al. 246
WASTEWATER SLUDGE NOTES

Otumba and Texcoco Soils

When sludge or sterile sludge was applied to an alkaline saline soil (Texcoco) or a
depleted nitrogen soil (agricultural soil from Otumba), dynamic profiles of CO
2
-C in both
soils showed that the microorganisms of sludge were affected by the alkalinity and salinity of
Texcoco soil as opposed to agricultural soil. On the other hand, the ammonium dynamics
showed that microorganisms immobilise the ammonium from several treatments of sludge,
sterile sludge, or ammonium sulphate, on the third day of the experiment. After that, the
ammonium was released, and later, the ammonium concentration was consumed. The NH
4
+
-N
dynamics showed that soil microorganisms immobilise N, release it, and finally, transform it
to NO
3

N; in other words, the N became available for assimilation by the plants. The final
concentration of NO
3
-
-N was higher in amended soil with sludge and sterile sludge than with
ammonium sulphate (López-Valdez et al., 2010). Both soils were considered as nitrogen
deficient: NH
4
+
-N concentration was 10 mg N kg
-1
dry soil in both soils, and NO
3
-
-N
concentration was 30 and 10 mg N kg
-1
dry soil for Otumba and Texcoco soils, respectively.
These results suggest that wastewater sludge could be a useful organic fertiliser. Nevertheless,
the pathogenic microorganisms in the sludge must be reduced.


Sunflower Characteristics (Alcholoya Soil)

The amended soil with sludge showed no significant difference in shoot height compared
with the other treatments at any sampled time. There was a significant difference between the
fresh and dry shoot weights of the PLANT and UREA treatments during the first two months.
Fresh and dry root weights were similar to the UREA treatment, but they were significantly
different to the PLANT treatment (P<0.05). The sludge was not different in the number of
seeds per plant and total N content mean compared with the UREA and PLANT treatments,
but the seed weight per plant was significantly different in the SLUDGE treatment. It has
been reported (Singh and Agrawal, 2010b) that sludge could increase the shoot length in
crops such as wheat (Triticum sp.), mung bean (Vigna radiate L. cv. Malviya janpriya), and
kenaf (Hibiscus cannabinus L.), but in this work it was not observed. One explanation could
be that the three experiments were begun at different seasons; it is well known that the
weather and amount of sunlight have strong effects on the growth of crops. Christodoulakis
and Margaris (1996) reported that sewage sludge amendment improved the maize growth in
comparison to commercial fertiliser, increasing plant height by 77%, whereas commercial
fertiliser increased plant height by 25%, compared with the control treatment. Liu et al.
(2009) reported an incremental increase of plant height from soil amended with 10% sludge
(doses from 0% to 30% sludge); additionally, they found that fresh weight, chlorophyll
content, activities of peroxides (POD) and catalase (CAT), or decreasing contents of
malondialdehyde (MDA) and membrane permeability (MP) increased the sewage sludge ratio
from 0% to 10% sludge. This sludge had 2% N kg
-1
, and it was disinfected by chlorination.
The PCA revealed that the SLUDGE treatment could actually surpass the UREA and PLANT
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Soil Amendments and Their Effects on Sunflower Growth 247
treatments because sludge has a positive effect on the total N content of plants as well as the
weight and height of shoots (Figure 2).


Soil Characteristics of Alcholoya

At the end of the experiment, the EC was similar to the UREA and PLANT treatments in
both depths, but the SLUDGE treatment was significantly different in soil pH, reaching a
final value of 7.4 and 7.1 in depths of 0-15 cm and 16-30 cm, respectively. The soil pH of
Alcholoya had an average of 6.5. Additionally, at the end of the experiments, the NO
2
-

concentration was significantly different in the SLUDGE treatment at a depth of 0-15 cm, but
it was not different at a depth of 16-30 cm. Final concentrations ranged from 0.33 to 0.43 mg
NO
2
-
-N kg
-1
dry soil for a depth of 0-15 cm, and 0.23 to 0.26 mg NO
2
-
-N kg
-1
dry soil in a
depth of 16-30 cm. The concentrations were low because this ion is an intermediate for NO
3
-

production. Interestingly, the dynamic of the NO
3
-
concentration resulted in a higher
concentration during the first month and a lower concentration at the end of the experiments
in both depths; in addition, non-leaching was found. Furthermore, we found that the NO
3
-

concentration was significantly different for the UREA treatment compared with the other
treatments in both soil depths (P<0.05) (Table 1).
The lowest residual concentration of NO
3
-
-N was 35%, found in the SLUDGE treatment
at a depth of 16-30 cm. These results could suggest that sludge decomposes slowly. The NO
3
-
-N concentration was similar to unfertilised plants.


Figure 1. Principal components analysis of unfertilised and fertilised sunflower plants. The PLANT
treatment refers to unfertilised sunflowers, the UREA treatment refers to fertilised sunflowers with 1 g
urea (150 kg N ha
-1
), and the BACTERIA treatment refers to sunflower seeds inoculated with B. subtilis
and fertilised with urea at 75 kg N ha
-1
. The experiments were carried out under greenhouse conditions.
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F. López-Valdez, F. Fernández-Luqueño, P. X. Hernández-Rodríguez et al. 248

Figure 2. Principal components analysis of unfertilised and fertilised sunflower plants. The PLANT
treatment refers to unfertilised sunflowers, the UREA treatment refers to fertilised sunflowers with 1 g
urea (150 kg N ha
-1
), and the SLUDGE treatment refers to amended sunflowers with sewage sludge at
150 kg N ha
-1
.
The dynamic of ammonium concentration at a depth of 0-15 cm was significantly higher
in the PLANT treatment (unfertilised plants) and was significantly different with regard to the
SLUDGE treatment, but it was not different with regard to the UREA treatment. This increase
in ammonium concentration could be due to an inlet of N from tap water; there was no N
supply from another source. On the soil depth of 16-30 cm, the dynamic was similar, but the
NH
4
+
-N concentration showed a significant decrease in the SLUDGE treatment: from 8.0 to
6.4 mg N kg
-1
soil (P<0.05) (Table 2).

Table 1. Dynamics of NO
3
-
-N (mg N kg
-1
soil) concentration by treatment in both
Alcholoya soil depths. The PLANT treatment refers to unfertilised sunflowers,
the UREA treatment refers to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
,
and the SLUDGE treatment refers to amended sunflowers with sewage sludge
at 150 kg N ha
-1
. The experiments were carried on under greenhouse conditions

Months PLANT UREA SLUDGE
0 – 15 cm depth
1
st
11.8 B
a
c
b
33.1 A a 22.4 A b
2
nd
10.7 B c 36.1 A a 22.5 A b
3
rd
14.9 A b 19.4 B a 14.6 A b
16 – 30 cm depth
1
st
17.0 A c 49.8 A a 34.0 A b
2
nd
12.7 B b 35.0 B a 18.4 B b
3
rd
11.7 B b 29.3 B a 12.2 B b
The significant difference between treatments was determined by analysis of variance, using LSD test.
a
Values with the same capital letter are not significantly different over time.
b
Values with the same letter are not significantly different between treatments.
SLUDGE
PLANT
UREA
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
PC2: 28%
PC1: 41%
Shoot dry weight
Root fresh weight
Number of seeds
Seed weight
Shoot length
Root dry weight
Root length
Shoot fresh weight
Total N content
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Soil Amendments and Their Effects on Sunflower Growth 249
Table 2. Dynamics of NH
4
+
-N (mg N kg
-1
soil) concentration by treatment from
Alcholoya soils. The PLANT treatment refers to unfertilised sunflowers, the UREA
treatment refers to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
,
and the SLUDGE treatment refers to amended sunflowers with sewage sludge
at 150 kg N ha
-1
. The experiments were carried on under greenhouse conditions

Months PLANT UREA SLUDGE
0 – 15 cm depth
1
st
7.0 B
a
b
b
9.2 A a 8.8 A ab
2
nd
4.3 B b 14.1 A a 6.2 B b
3
rd
11.7 A a 9.6 A ab 8.1 A b
16 – 30 cm depth
1
st
6.7 B b 8.7 A a 8.0 A a
2
nd
4.0 B b 6.2 A a 4.9 C ab
3
rd
10.4 A a 8.9 A ab 6.4 B b
The significant difference between treatments was determined by analysis of variance, using LSD test.
a
Values with the same capital letter are not significantly different over time.
b
Values with the same letter are not significantly different between treatments.

In the SLUDGE treatment, the rate of CO
2
production increased to 86% (1.4 mg C kg
-1

day
-1
) with regard to the uncultivated and unamended soil (CONTROL treatment). The
SLUDGE treatment had a significant difference in the rate of N
2
O production compared with
all other treatments: the mean production was 2.31 µg N kg
-1
day
-1
, which was 12 times that
of the CONTROL treatment. The wastewater sludge refers to organic matter—matter that
involves a high production of greenhouse gasses by decomposing microorganisms—but there
is no such comparison when sludge is burned. The sewage sludge used in these experiments
had an organic C content of 28.8%, meaning about 18,000 mg CO
2
-C kg
-1
dry sludge (on a
dry matter basis). PCA showed that the SLUDGE and UREA treatments had a major effect
over all soil parameters. The SLUDGE treatment affected the ammonium and nitrate in both
soil depths and the EC at a depth of 0-15 cm; the UREA treatment affected the pH in both soil
depths, principally (Figure 3).


Recent Work

Recently, we have studied wastewater sludge in order to remove or reduce the amount of
pathogen microorganisms such as Salmonella sp., faecal coliforms, coliphage, protozoa cysts,
and helminth ova. The sewage sludge was characterised in terms of the initial concentrations
of helminth ova that were determined. Viability was determined by continuous washing,
combined with several filtration steps: suspension, concentration, and incubation in H
2
SO
4

0.1 N at 26 °C for 30 days (USEPA, 2013). One kg of wastewater sludge was placed in
plastic containers of 0.30 × 0.25 × 0.12 m dimensions, and 100 g CaO was added. The sludge
was exposed by 15-day increments from day 0. Viable ova quantification was performed at 0,
5, and 15 days after the start of treatment.

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Figure 3. Principal components analysis of unfertilised and fertilised sunflower plants and their effects
on soil properties. The PLANT treatment refers to unfertilised sunflowers, the UREA treatment refers
to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
, and the SLUDGE treatment refers to amended
sunflowers with sewage sludge at 150 kg N ha
-1
.
Table 3. Viable ova count by treatment and by sampling day (g
-1
total solids).
The experiments were carried out under two different conditions
(cold and warm seasons)

Day
Alkaline Non-Alkaline
MSD
c

Ova g
-1
TS
0 3053 A
a
a
b
3470 A a 2077
5 1387 A a 1805 A b 2960
15 0 B a 1943 A b 773
MSD 3110 1207
a
Values with the same capital letter are not significantly different between treatments.
b
Values with the same letter are not significantly different over time.
c
MSD: Minimal significant difference (P<0.05).

We found that the lime used significantly decreased embryonic development of helminth
ova from Day 15, when we did not find viable ova. The alkaline treatment increased the
temperature from 15°C to 33°C on the first day. Furthermore, the lime dose increased the pH
of the sewage sludge to 12 units upon contact; the pH remained above this value for 72 hours
(Table 3). Finally, treatment with the lime at 10% allowed for the inactivation of helminth
ova. However, future studies should be conducted in order to confirm the inactivation of
helminth ova in limed sewage sludge treated under large scale or field conditions. We are
interested in researching other treatments that reduce pathogen microorganisms, in order to
reach a Class A sewage sludge that can amend agricultural soils.

SLUDGE
PLANT
UREA
-1
-0.75
-0.5
-0.25
0
0.25
0.5
0.75
1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
PC2: 17%
PC1: 22%
pH, 15-30 cm
pH, 0-15 cm
EC, 0-15 cm
NO
2
-
,
0-15 cm
Production of CO
2
EC, 15-30 cm
NO
2
-
, 15-30 cm
NO
3
-
, 0-15 cm
NO
3
-
, 15-30 cm
NH
4
+
, 0-15 cm
NH
4
+
, 15-30 cm
Production of N
2
O
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Soil Amendments and Their Effects on Sunflower Growth 251
BACI LLUS SUBTI LI S NOTES

Sunflower Characteristics (Alcholoya Soil)

The shoot height and dry root weight characteristics were not significantly different
between treatments (P<0.05). However, at the end of the experiment, the fresh and dry shoot
weight characteristics were significantly different compared with the UREA and PLANT
treatments. The root length and fresh root weight parameters showed a significant difference
with regard to the UREA and PLANT treatments at the start of the experiment, but not at the
end. Meanwhile, parameters such as weight of seeds, number of seeds, and total N content of
plants did not differ significantly throughout the entire experiment (P<0.05). As mentioned
above, this bacterium is a regular microorganism. As such, the tested strain produces IAA,
solubilizes phosphorus, and tests positive for ACC deaminase, but it was not an outstanding
PGPR. The application of B. subtilis to plants cultivated in soil amended with urea increased
root growth—namely, the root length and root weight—as opposed to plants only fertilised
with urea at the first month. We found that the B. subtilis strain had a temporary stimulation
effect on the sunflower crop at the first month; similar results were reported by Swain and
Ray (2009).


Figure 4. Principal components analysis of unfertilised and fertilised sunflower plants and their effects
on soil properties. The PLANT treatment refers to unfertilised sunflowers, the UREA treatment refers
to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
, and the BACTERIA treatment refers to
sunflower seeds inoculated with B. subtilis and fertilised with urea at 75 kg N ha
-1
.

BACTERIA
PLANT
UREA
-1
-0.75
-0.5
-0.25
0
0.25
0.5
0.75
1
-1.25 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25
PC2: 21%
PC1: 33%
pH, 15-30 cm
pH, 0-15 cm
EC, 0-15 cm
NO
2
-
,
0-15 cm
Emission of CO
2
EC, 15-30 cm
NO
2
-
,
15-30 cm
NO
3
-
, 0-15 cm
NO
3
-
, 15-30 cm
NH
4
+
,
0-15 cm
NH
4
+
,
15-30 cm
Emission of N
2
O
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The PCA showed that the BACTERIA treatment had a positive effect on the length and
weight of shoots; meanwhile, the UREA treatment had a positive effect on the length and
weight of roots (Figure 4). In contrast, the unfertilised plants had negative PC1 and PC2,
meaning less development of roots and shoots as well as fewer and lighter seeds. On the other
hand, the BACTERIA treatment was established with half of the N amount with regard to the
UREA treatment. Also, unfertilised plants showed chlorotic leaves with regard to the UREA
and BACTERIA treatments.


Soil Characteristics of Alcholoya

Treatments had no effect on the pH in either soil depth. However, the soil pH increased
significantly at the end of the experiments. In the case of EC, the BACTERIA, UREA, and
PLANT treatments showed no significant difference at a depth of 0-15 cm by the end of the
experiment (P<0.05). For the soil depth of 16-30 cm, the first two months were significantly
higher for the UREA and BACTERIA treatments, but by the end of the experiment, the
BACTERIA treatment showed a significant difference with regard to unamended plants. Urea
application seem to affect the EC. Otherwise, these increments could be due to the extra
minerals supplied by tap water (high contents of Ca
+2
, Mg
+2
, and Na
+
) that were increasing
over time (Lavado, 2006). The NO
2
-
concentration was similar between treatments in both
soil depths throughout the entire experiment. Regarding NO
3
-
-N, we found no significant
difference between the UREA and BACTERIA treatments, but we did find a difference with
regard to unamended plants (P<0.05). Also, the analysis shows that the NO
3
-
concentration
decreased significantly over time (Table 4).

Table 4. NO
3
-
-N concentration by treatment in both Alcholoya soil depths.
The PLANT treatment refers to unfertilised sunflowers, the UREA treatment
refers to fertilised sunflowers with 1 g urea at 150 kg N ha
-1
, and the BACTERIA
treatment refers to sunflowers inoculated with B. subtillis plus urea at 75 kg N ha
-1


NO
3
-
-N (mg N kg
-1
soil)
Months PLANT UREA BACTERIA
0 – 15 cm depth
1
st
11.8 B
a
c
b
33.1 A a 43.1 A a
2
nd
10.7 B c 36.1 A a 27.2 B a
3
rd
14.9 A b 19.4 B a 19.8 B a
16 – 30 cm depth
1
st
17.0 A c 49.9 A a 49.9 A a
2
nd
12.7 B b 35.0 B a 28.7 B a
3
rd
11.7 B b 29.3 B a 32.3 B a
The significant difference between treatments was determined by analysis of variance, using LSD test.
a
Values with the same capital letter are not signifi