EFFECT OF ALLELOPATHIC BACTERIA ON THE

GROWTH AND YIELD OF WHEAT (Triticum aestivum L.)

AND ITS ASSOCIATED WEEDS

By
Tasawar Abbas
M. Sc. (Hons.) Agriculture Soil Science
(Regd. # 2007-ag-2296)

A thesis submitted as partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY
IN
SOIL SCIENCE

FACULTY OF AGRICULTURE,
UNIVERSITY OF AGRICULTURE, FAISALABAD.
PAKISTAN.
2017

i
 Abstract
Weeds growth in crops causes more economic losses to crops than any other pest. The
conventional control methods have given rise to serious issues of environment and human
health. The importance of development of alternative techniques based on biological
approaches has increased. In the present study, allelopathic bacteria were evaluated for
suppression of weeds associated with wheat. A large collection of rhizobacteria was
obtained from the rhizosphere of wheat and its associated weeds (wild oat, little seed
canary grass, broad leaved dock, common lambs’ quarter and field bindweed) which were
in turn sampled from chronically infested wheat fields. These rhizobacterial strains were
screened through in vitro bioassays based on production of phytotoxic metabolites i.e.,
HCN production, E. coli antimetabolite assay and lettuce seedling bioassay. Eighty nine
of 393 rhizobacterial strains were found to be cyanogenic. Nineteen of the 89 cyanogenic
strains inhibited the growth of sensitive E. coli strain K12 due to antibiosis. These 19
strains were applied to lettuce in agar bioassay on Petri plates. Growth of lettuce
seedlings was inhibited by 6 strains, 5 significantly increased while 8 strains did not
affect the growth of lettuce seedlings. These 19 strains were applied to wheat and 4 weeds
(wild oat, little seed canary grass, broad leaved dock and common lambs’ quarter) in
similar agar bioassay. Results indicated non-selective inhibition of all the weeds and
wheat due to inoculation with 2 strains. Three strains selectively inhibited the germination
and growth of weeds but also suppressed wheat. Three strains selectively inhibited weeds
and remained non-inhibitory to wheat. However, 9 strains selectively inhibited weeds and
promoted the growth of wheat. Ten strains from the later 2 groups were re-tested on 3
weeds and wheat under axenic conditions in growth room. Twenty five days old plants
were measured for different growth parameters. The applied strains caused inhibition of
germination and dry matter of wild oat from 15.2 to 63.3 and 12.4 to 65%, little seed
canary grass from 18.5 to 58.7 and 22.8 to 81.4% and broad leaved dock from 18.4 to
60.5 and 21.7 to 71.3% than their controls, respectively. Four of these strains improved
the growth of wheat while others remained non-inhibitory.

Five strains from the previous study (T42, L9, 7O₀, O₀10 and W9) were selected
to study effects of allelopathic bacteria on 3 weeds (wild oat, little seed canary grass and
broad leaved dock) grown in wheat and infested wheat in pot and field trials. Infestation
of wild oat caused reduction in grain yield of wheat up to 60.8% than weed free control.
Suppression of wild oat by strain T42, L9, 7O₀, O₀10 and W9 controlled the loss in grain

xiii
yield of infested wheat up to 60.0, 73.6, 35.8, 22.0 and 49.7%, respectively. Infestation of
little seed canary grass caused loss in grain yield of infested wheat up to 59.9% than weed
free control. Suppression of this weed by strain T42, L9, 7O₀ and O₀10 recovered the loss
in grain yield of infested wheat up to 20.1, 55.0, 66.9 and 59.0%, respectively. Infestation
of broad leaved dock caused loss in grain yield of infested wheat up to 55.8% than weed
free control. Suppression of this weed by strain T42, L9, 7O₀ and W9 controlled the loss
in grain yield of infested wheat up to 45.2, 53.9, 46.3 and 68.0%, respectively. These
effects of allelopathic bacteria were also evident from other growth, yield and
physiological parameters of weeds and infested wheat. Infestation of weeds caused loss in
grain yield of infested wheat up to 54.1, 53.9 and 56.3% than weed free control,
respectively. In field trial I, suppression of broad leaved dock and common lambs’ quarter
controlled the loss of grain yield of infested wheat up to 38.3, 64.0, 51.0 and 62.9% due
to inoculation with strain T42, L9, 7O₀ and W9, respectively. Suppression of little seed
canary grass in field trial II controlled the loss in grain yield of infested wheat up to 34.3,
55.1, 64.3 and 57.2% due to inoculation with strain T42, L9, 7O₀ and O₀10, respectively.
In field trial III, suppression of wild oat and little seed canary grass controlled the loss in
grain yield of infested wheat up to 47.9, 60.7, 53.7, 29.0 and 36.6% due to inoculation
with strain T42, L9, 7O₀, O₀10 and W9, respectively. These effects of allelopathic
bacteria were also evident from other growth, yield and physiological parameters of
weeds and infested wheat. Under weed free conditions, these strains remained non-
inhibitory to wheat. Instead, inoculation with strain L9 and 7O₀ significantly improved
the growth and yield of wheat. Microbiological and biochemical characterization of these
strains also revealed the possession of molecular characteristics of weed suppression and
plant growth promotion, and identified as Pseudomonads. This research suggests
utilization of allelopathic bacteria to control weed infestations in wheat and avoid harmful
effects of other weed control techniques on human health and environment.

xiv
CHAPTER-I INTRODUCTION
Destruction of natural ecosystems for agricultural practices and other
anthropogenic activities has destroyed the habitats of wild organisms. It has caused the
shift of wild organisms from their habitats to crops and they became pests. They
disastrously reduce the yields of crops highlighting the need for adoption of wise
management practices. So, pest control is an important component of agriculture since its
inception. The major pests of crops include weeds, insects, pathogens and rodents.
Among these pests, weeds cause more losses to crops than any other pest worldwide
(Oerke, 2006). The wild plants which interfere with the growth of crop plants and
compete for its resources to cause yield and quality loss of crops are called weeds (Ashiq
and Aslam, 2014). They reduce 18-30% yield of wheat, 39-89% yield of vegetables and
24-47% yield of maize in Pakistan (Anonymous, 2011-12). Worldwide weeds cause ca.
10% losses in crops whose recovery may be sufficient to feed one million people (Froud-
Williams, 2002; Berca, 2004). Conventionally weeds are controlled by using manual,
mechanical and chemical methods. Manual weeding involves pulling, plucking and
hoeing of weeds using hand driven equipments by labor. It is the most primitive and
efficient method of weed control with minimum side effects (Naylor, 2002). The
successful use of this method depends upon the availability of labor. Rapid industrial
growth throughout world uses more labor with high wages. It has caused the shift of labor
from farm to industry. However, manual weeding is still adopted in countries backward in
industrial development. Mechanical and chemical weed control methods are most popular
now.
Mechanical weeding is performed with the help of tillage implements driven by
traction or draught power. Weeds are controlled by uprooting and burying plants grown
between crop rows. It is applicable in crops with wider row spacing and suitable sowing
methods (Hakansson, 2003). Also, weeds grown within crop rows are not controlled.
Apart from causing root injury to crops, partially uprooted weeds may restore their vigour
through regeneration. Aftereffects of tillage may also stimulate germination and growth
of weeds due to suitable tilth. Therefore, mechanical weeding has a limited scope in crop
production (Hakansson, 2003). On the other hand, mechanical weeding has adverse
environmental effects which include expedition of soil erosion leading to eutrophication
of waterbodies, increased leaching of nutrients, formation of photo-oxidants and global
warming (due to high energy consumption) (Hakansson, 2003; Ahlgren, 2004). Narrow

1
wheel cover area is used for weeding which has more impact on soil compaction than
other tillage practices (Ahlgren, 2004).
Due to several drawbacks of these weed control methods, chemical control has
now gained popularity worldwide. This is accomplished by application of chemical
herbicides. Herbicides account for 44% of total pesticides worldwide (Quimby et al.,
2002). Global consumption of herbicides increased from US $ 170 million in 1960 to US
$ 14.97 billion in 2005 (Zhang et al., 2011). Application of herbicides increased the yields
of different crops from 10 to 50% (Ashiq and Aslam, 2014). Gianessi and Sankula (2003)
reported that yields of different crops would have decreased from 5 to 67% without use of
herbicides. Use of herbicides has reduced the need for tillage from 50 to 80% (Ashiq and
Aslam, 2014). Lesser requirement of labor and energy makes the weed control easier
(Ghorbani et al., 2005). Therefore, global demand for herbicides keeps rising due to more
reliance on chemical control of weeds and their associated benefits. The side effects of
chemical herbicides were ignored in the past which increased their magnitude. The
passage of time has given rise to serious limitations of use of herbicides. Instead of
minimizing the use of herbicides with time, continuous application of herbicides resulted
into shift of weed flora. The reduction in abundance of one weed species due to
herbicides gave opportunity to invasions of other weed species which were less abundant
and less susceptible to the applied herbicides (Zimdhal, 1994). Solaimalai et al. (2004)
reported the appearance of more troublesome grassy weeds after few years of application
of 2,4-D to control broadleaved weeds. Continuous application of such herbicides has
also induced intraspecific selection of weed species leading to the development of
herbicide resistant biotypes of weeds (Bradley and Hagood, 2001; de Prado et al., 2004).
More than 300 examples of herbicide resistance have been reported against 15 families of
chemical herbicides (Quimby et al., 2002; Chhokar et al., 2006; Heap, 2013). It demands
the continuous replacement of existing compounds with new compounds having new
mode of action (Powles and Yu, 2010; Kao-Kniffin et al., 2013). The discovery of new
compounds has drastically reduced during the current years (Duke, 2012). Another issue
in chemical weed control is the exposure and effects of herbicides on susceptible non-
target organisms including wildlife (Geiger et al., 2010). Some herbicides like triazines
and sulphonyl urea may persist in soil long enough to affect subsequent susceptible crops
(Zimdhal, 2007). Herbicides, although do not change the population of microorganisms in
soil but it may induce intraspecific and interspecific selection leading to disturbance in
microbial biodiversity and useful soil biological processes (Hakansson, 2003). Drainage
2
of contaminated water exposes aquatic life to herbicides. Herbicides absorbed by crop
plants accumulate in different plant parts including edible portions (Solaimalai et al.,
2004). Feeding animals on contaminated plant materials causes poisoning, diseases and
death of animals (Blakley et al., 1999). Ultimately human beings are exposed to
herbicides through contaminated water, food and air (Blair et al., 2015). Herbicides have
been reported to cause life threatening diseases in human beings including cancer of
various types, embryonic malformations, neurological, liver and reproductive disorders
(Schreinemachers, 2003; Hakansson, 2003; Alavanja et al., 2004). In short, multiple
issues regarding efficiency in weed control, human health, environment and biodiversity
are faced at the cost of chemical weed control. The costs incurred on remediating
environment, saving biodiversity and curing human diseases demand that alternative
weed control methods must be developed to reduce yield losses of crops caused by
weeds.
Another approach for weed control is biological weed control. It refers to the
application of natural antagonists of weeds or their products to reduce their vigor, density,
growth, reproductive capacity and impact on crops (Quimby and Birdsall, 1995). It is
divided into two categories; inoculative biocontrol and inundative biocontrol approach.
Inoculative biocontrol refers to the introduction and naturalization of a weed antagonist
into a new area (Scheepens et al., 2001). The biocontrol agents maintain its population
balance with host populations in nature. The organism once released can not be recalled.
Therefore, great care is required in testing an inoculative agents before its release.
Tsukamoto et al. (1997) introduced a moth Cactoblastis cactorum from North America in
Australia to control prickly-pear cactus (Opuntia spp.) after successful laboratory
experiments in mid 1920s. It reduced the dense vegetation of cactus to minor patches. The
hazards of such biocontrol agents may appear if they change their host in the environment
and emerge as a new pest of the area (Scheepens et al., 2001). Inoculative biocontrol
approach meets success when the target weed species is quite unrelated to other plant
species of the area because host range of inoculative agents is usually wide (Ghorbani et
al., 2005). Since weed flora is commonly similar to crop plants, the threats of emergence
of new pest continue even after careful testing. Therefore, research on development of
weed biocontrol agents based on this approach is limited. Inundative biocontrol refers to
the application of high populations of a natural antagonist of weed which already inhabits
that environment (Hakansson, 2003). Such biocontrol agents are also called
bioherbicides. Plant pathogenic fungi and bacteria have been applied in the past as
3
bioherbicides. The characteristics of such pathogens included high virulence against
target plants, establishment of high populations on the host, long viability and safety of
non-target species (Agrios, 1997; Auld et al., 2003; Zhang et al., 2003). Several
bioherbicides containing plant pathogenic bacteria and fungi have been commercialized.
Infection and disease development in weeds by such biocontrol agents showed
dependence on suitable environmental conditions which favor the survival and infection
of these pathogens (Leiss, 2001; Charudattan, 2001; Ghorbani et al., 2005). The
susceptibility of hosts to pathogens also varies with environmental and growth conditions
and growth stages of plants (Agrios, 1997; Ghorbani et al., 2002; Dudycha and Roach,
2003; Kremer and Li, 2003). Environmental conditions in the field keep fluctuating and
scarcely are suitable to disease development by biocontrol agents (Ghorbani et al., 2005).
Therefore, scope of plant pathogens for weed biocontrol is limited.
The knowledge of allelopathy has also been exploited for biocontrol of weeds. It
refers to the interactions between one plant species and others or microorganisms through
the release of chemical substances (secondary metabolites) called allelochemicals (Torres
et al., 1996). Extracts and residues of plants containing phytotoxic allelochemicals against
certain weeds have been applied to control them (Farooq et al., 2011; Farooq et al., 2013).
This technique has been applied to control several weeds grown in wheat, cotton,
mungbean and rice at various levels (Cheema and Khaliq, 2000). Production of
allelochemicals by donor plants and their phytotoxic effects on receiver plants are
influenced by growth conditions and stages of both as well as environmental conditions
(Wu et al., 1999; Tongma et al., 2001; Suzuki et al., 2001; Iqbal et al., 2002). Another
important consideration is the involvement of soil factors in the bioavailability and
phytotoxicity of allelochemicals applied through plant extracts and residues (Kobayashi,
2004). The effects of allelochemicals on target plants are reduced by adsorption on clay,
mobility of allelochemicals within soil to target plants, hydrodynamics of soil and
degradation of allelochemicals by soil microorganisms (Kobayashi, 2004; Olver-Velona
et al, 2008). This drastically reduces the phytotoxic activity and bioavailability of
allelochemicals to weeds (Mandal, 2001; Belz et al., 2009). These factors may be
overcome if allelochemicals are produced at root surfaces in the rhizosphere of weeds by
allelopathic bacteria and immediately absorbed. Therefore, it is suggested that application
of allelopathic bacteria may suppress weeds more efficiently than plant extracts and
residues. The rhizobacteria which produce phytotoxic secondary metabolites
(allelochemicals) and suppress the germination and growth of certain weeds are called
4
allelopathic bacteria (AB) (Sturz and Christie, 2003; Kremer, 2006). They survive in the
rhizosphere of weeds using their root exudates for energy and precursors of secondary
metabolites. They keep producing phytotoxins in the rhizosphere as long as plant survives
thereby reducing its competitive ability, vigor and impact on crops (Kulmatiski et al.,
2004). On the other hand, plant extracts and residues contain only specific amount of
allelochemicals. The future of allelopathy and weed biocontrol research therefore needs
focus on allelopathic bacteria which is suggested to be more effective than plant
extracts/residues and plant pathogens under field conditions. Other benefits of using
allelopathic bacteria include narrow host range (Kennedy et al., 1991; 2001). Host
specificity of allelopathic bacteria has been attributed to selective phytotoxicity of
allelochemicals for different plant species, partial/suboptimal colonization of non-host
rhizosphere, modified availability of substrates in the root exudates in the rhizosphere of
non-host plants or a combination of these factors (Begonia and Kremer, 1994;
Piotrowska-Seget, 1995; Owen and Zdor, 2001; Zeller et al., 2007). The outcome of such
interactions of allelopathic bacteria with crop plants may be beneficial, harmful or neutral
(Kennedy et al., 2001; Weissmann et al., 2003; Zdor et al., 2005). Plant growth promotion
effects of allelopathic bacteria usually observed may be due to production of plant growth
promoting metabolites by AB (Zahir et al., 2004). These characteristics of allelopathic
bacteria offer an opportunity to be screened from the rhizosphere of plants as biocontrol
agents of weeds. Therefore, this study has been conducted with the following objectives:

 Isolation and screening of rhizobacteria from wheat and its associated weeds in
laboratory bioassays.
 Characterization of allelopathic bacteria for suppression of one or more weeds
associated with wheat and non-inhibitory effects on wheat.
 Evaluation of bioherbicidal activity of allelopathic bacteria on majors weeds
associated with wheat and non-inhibitory effects on wheat under axenic conditions
 Evaluation of effects of allelopathic bacteria on wheat and its associated weeds
under natural conditions in pot and field trials.
 Microbiological and biochemical characterization of allelopathic bacteria, and
their identification through 16S rRNA gene sequencing technique.

5
CHAPTER-II REVIEW OF LITERATURE
Introduction of agro-chemicals, irrigation, improved cultural practices and high
yielding varieties after 1950 paved the way for green revolution. It substantially increased
food production worldwide and helped to cope with ever increasing hunger of human
beings worldwide. Wheat yields in Pakistan increased from 4.55 million tons in 1966
(Anonymous, 1965-66) to 25.48 million tons in 2014 (Anonymous, 2014-15).
Domestication of wild plants triggered manipulation of nature for agricultural practices.
Apart from benefits gained from these practices, they have also given rise to unexpected
outcomes bearing threats to sustainability of such practices as well as survival of human
beings and other life forms on Earth. Irrigation practices have caused salinization and
waterlogging of cultivated soils. High yielding cultivars of crops depleted the soils of
nutrients and precious organic matter. Tillage deteriorated soil structure, oxidized organic
matter and produced compaction layers in the subsoil. Many wild species of plants,
animals and microorganisms emerged as pests of crop plants due to destruction of their
natural habitats. No species can be eliminated from nature and continuous disturbance of
its habitat through agricultural practices increased the emergence and diversity of pests.
They pose the biggest challenge to food production worldwide since the start of
agriculture. Therefore, pest management is an important component of crop production
since its inception (Oerke, 2006). Although, it involves centuries old practices but major
breakthrough in pest control happened due to application of pesticides. Pesticides are the
chemical substances applied to control population of pests. The amount of pesticides used
worldwide for pest control increased by 15-20 folds in the last 50 years (Oerke, 2006).
Apart from controlling pest populations, they have posed new challenges in pest control.
The side effects of pesticides include killing of susceptible non-target species and
disturbance of biodiversity. The number of pests developing resistance to pesticides is
growing. Contaminated food, air, soil and waterbodies have exposed the life on Earth to
hazardous agrochemicals. Every individual on earth has pesticide residues in his body
which have caused chronic diseases leading to death in human beings (Annett et al.,
2014). The challenge of food security still continues and demands the adoption of new
approaches to ensure safe and sufficient food for everyone.

Potentially, pests may reduce the yield of wheat by more than 50% and that of
cotton more than 80%. However, due to pest management practices, the yield reductions

6
of wheat, maize, rice, soybean and potatoes account for 26, 31, 37, 29 and 40%,
respectively. The crop pests include weeds, insects, pathogens and rodents. Among these
pests, weeds contribute maximum losses in crop production which may potentially reduce
34% production of crops followed by animal pests (18%) and pathogens (16%). Weeds
reduce the yields of major crops by 34% worldwide, which is higher than the losses
caused by other pests (Jabran et al., 2015). Weeds are the plant species which compete
with crop plants for resources and reduce their growth, yield and quality (Ashiq and
Aslam, 2014). This competition deprives the crop plants of space, nutrients, moisture,
light and other growth requirements. The severity of competition brings higher losses to
crop yields (Rajcan and Swanton, 2001). They have been reported to decrease the yield of
cotton, rice, maize, soybean and potatoes by 36, 37, 40, 37 and 30%, respectively (Oerke,
2006). Other than yield losses, they may also harbor other pests and pathogens and
enhance their incidents on crops (Booth et al., 2003; Jabran et al., 2015). Certain weeds
have also been reported to produce allelochemicals which suppress root and shoot growth
of crop plants. Root hairs of Chenopodium murale L. released allelochemicals in a case
study by Dmitrovic et al. (2014). These allelochemicals disturbed cell cycle and caused
oxidative damage to wheat and Arabidopsis thaliana seedlings. Similarly, allelopathic
water extracts from Malva Parviflora L. and Chenopodium murale L. suppressed growth
and photosynthetic activity of barley (Al-Johani et al., 2012). Weeds reduce 18-30% yield
of wheat in Pakistan per annum (Anonymous, 2011-12). The worth of these losses
amounts to Rs. 146 billion (Razzaq et al., 2012). Weeds may potentially reduce the yield
of wheat up to 23% (18-29%) worldwide but due to weed management practices actual
losses account for 7.7% (3-13%) (Oerke, 2006). Globally, yield losses to weeds have
reduced with time because of improvements in weed control practices and improved weed
management.

Weed management in arable lands involves direct and indirect methods. Indirect
methods involve utilizing such cultural practices and preventive measures which reduce
germination, growth, vigor and reproductive capacity of weeds. Direct methods employ
all the techniques primarily aimed at weed control (Shad, 2015). These techniques include
manual, mechanical, chemical and biological. Conventionally, weeds are controlled using
manual, mechanical and chemical methods (Chauvel et al., 2012). These methods have
given rise to serious limitations and their place in sustainable modern agricultural systems
is jeopardized. Therefore, search for new non-conventional weed control methods

7
continues which may be aimed at utilization of biological weed control approaches.
Threats posed by conventional weed control methods to environment, food quality and
human health have been discussed below followed by feasibility of developing a most
efficient biological weed control approach.

2.1. Manual weeding

This method involves the use of labor for uprooting, plucking and hoeing with or
without hand driven equipments for weeding and is being followed since ancient times
(Young et al., 2014). It is the most efficient weed control method and is used in areas
where labor is cheaper and easily available. It can be adopted for weed control in all
crops, sowing methods and growth conditions. However, in-time availability of labor
before the weeds cause economic losses to crops is a critical factor in the success of this
method (Shad, 2015). Rapid expansion of industries has caused shift of labor from farm
to industry and simultaneous need for labor for weeding of whole cropped area has
restricted manual weeding to areas backward in industrial development (Naylor, 2002).
These factors have caused lesser availability as well as high cost of labor for manual
weeding (Carballido et al., 2013; Gianessi, 2013). Sometimes, farmers may be forced to
follow manual weeding due to lack of technical know-how and uncertain market
conditions of herbicides regarding cost and availability (Shad, 2015). Repeated hand
weedings and involvement of intense labor makes this method usually inconvenient,
uneconomical and unfeasible (Rao et al., 2007). Weed control on large scale usually
becomes impossible if manual control method is adopted (Donald, 2000). Akbar et al.
(2011) compared the efficiency of manual weeding with other conventional weed control
methods in direct-seeded rice cultivation. They reported that manual weeding was more
efficient than mechanical weeding and both were better than chemical control. Grain
yield of direct seeded rice was improved by 30% where manual weeding was done, 25%
where mechanical weeding was done and 7-19% where recommended doses of different
herbicides were applied. However, manual weeding is still in operation where labor is
cheaper, easily available and landholdings are small.

8
2.2. Mechanical weeding

It is usually more economical to use mechanical than manual weeding. It involves

the use of tillage implements like harrows, weeders and cultivators driven by animals or
engine. These implements rely on burying and uprooting weeds grown between crop rows
which are wide enough to facilitate movement of the implements without significant
injury to crops. Therefore, this method is applicable only in specific crops sown in
straight rows and having suitable row to row spacing. Weeds grown within crop rows and
closer to crop plants escape the control (Shad, 2015). Weeds grown within crop rows
incur much higher losses to crops than those grown between crop rows (Melander et al.,
2012). Partially uprooted weeds may regain vigor through regeneration and root injury to
crops may also occur (Hakansson, 2003). It requires repeated operations for effective
weed control using mechanical methods (Jabran et al., 2015). It reduces the efficiency of
mechanical weeding over other conventional methods (chemical and manual control)
(Bond and Grundy, 2001; Akbar et al., 2011). Narrow cover area of wheel tracks is used
for mechanical weeding which has more effect on soil compaction than other tillage
practices (Hakansson, 2003; Smith et al., 2011). Other adverse environmental effects of
using tillage for mechanical weeding include expedited soil erosion, leaching of plant
nutrients, formation of photo-oxidants, global warming and eutrophication (Ahlgren,
2004). Mechanical control utilizes high energy and contributes to global warming. It also
increases decomposition and oxidation of organic matter in soil causing its depletion and
loss of soil fertility. Apart from deteriorating soil structure, it also aggravates compaction
of subsoil. Other disadvantages include destruction of natural habitats and wildlife
(Culliney, 2005). Any effort of reducing tillage puts more pressure on use of other
methods of weed control especially herbicides (Hakansson, 2003). Mechanical weeding is
being used inspite of its disadvantages due to non-availability of safer techniques, lack of
awareness among farmers and lack of environmental concerns on the part of farmers.

2.3. Chemical weed control

Chemical control of weeds is accomplished with the application of herbicides.

Chemicals for weed control were used in the start of 20th century which included copper
salts and sulphuric acid (Hamill et al., 2004). During World War II, defoliating agents
were used for destruction purposes. Later on these chemicals were familiarized as
herbicides. Discovery of selective herbicides lead to the application of herbicides in

9
arable lands. Introduction of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-
chlorophenoxyacetic acid (MCPA) in 1940s revolutionized weed control in cereals
(Zimdahl, 1999). They were deemed to control weeds more efficiently than all other
techniques due to several advantages of herbicides. These included less requirement of
labor, low cost of application, reduced soil erosion and energy saving (Ghorbani et al.,
2005). Global consumption of herbicides increased from US$ 170 million in 1960 to
US$14.97 billion in 2005 (Zhang et al., 2011). They include chemical substances which
control weeds by inhibiting photosynthesis, animo acids biosynthesis, lipid biosynthesis,
respiration and other mechanisms. They also include some auxin mimics. Chemical
control of weeds has improved the yields of different crops from 10 to 50% (Ashiq and
Aslam, 2014). Gianessi and Sankula (2003) reported that yields of different crops would
have decreased from 5 to 67% without the use of herbicides. They have reduced the need
for tillage from 50 to 80% in different crops (Ashiq and Aslam, 2014). Application of
herbicides has been necessitated in certain crops and sowing methods e.g. direct-seeded
rice cultivation. Rice is mainly grown in flooded puddling conditions because they reduce
emergence of several weeds (Chauhan and Johnson, 2009). This is no more a sustainable
approach due to rising water scarcity situation worldwide (Rosegrant et al., 2002;
Bhushan et al., 2007). The research on sustainable rice production is now focused at
success of direct-seeded rice cultivation through improved weed management which may
save precious water from 20 to 35% (Weerakoon et al., 2011). Weed infestations under
direct seeded rice cultivation may reduce the yield upto 85% leading to complete failure
of the crop (Phuong et al., 2005; Rao et al., 2007). Narrow row spacing makes mechanical
weed impractical whereas manual weeding becomes impossible due to need of more
frequent weeding and shortage of labor on large scale production of rice. Pre- and post-
emergence techniques of herbicide applications have shown their effectiveness to
suppress weeds grown in direct seeded rice (Gitsopoulos and Froud-Williams, 2004).
These and other factors have caused the current popularity of herbicides for weed control
worldwide.

However, ever increasing reliance on herbicides has given rise to serious

limitations. Zimdahl (1994) studied the shift in weed flora of US rice-maize-soybean
cropping system. The original weed flora comprised of grasses (60%), sedges (25%) and
broadleaved weeds (15%). Chemical control was administered for these weeds
continuously up to six years. After this period, weed flora comprised of 80% grasses, 7%

10
sedges and 13% broadleaved weeds. He suggested that shift in weed flora occurs due to
interspecific selection of weed species which was induced by herbicides application
having similar mode of action and selectivity pattern. The chemical control of new flora
becomes even more difficult with herbicides (Hakansson, 2003). 2,4-D is widely used to
control broadleaved weeds. Solaimalai et al. (2004) reported that after few years of 2,4-D
application more troublesome growth of grassy weeds appeared. The continuous
application of such herbicides also leads to intraspecific selection of weeds and caused
the development of herbicide resistant biotypes of weeds (Bradley and Hagood, 2001; de
Prado et al., 2004). More than 300 examples of herbicide resistance have been reported
against 15 families of chemical herbicides (Chhokar et al., 2006; Heap, 2013). Stuart
(2003) reported 273 weed species resistant to different herbicides used for their control.
Heap (2005) reported 177 weed species which had evolved resistance to 18 classes of
different herbicides. It requires additional applications as well as high doses of
compounds for control of these weeds which further increases the magnitude of
resistance. To overcome this situation, several researchers have suggested that current
herbicidal compounds should be continuously replaced with others having new mode of
action (Powles and Yu, 2010; Kao-Kniffin et al., 2013). The discovery of new
compounds is a concern in chemical weed control but it has drastically reduced during the
current years (Hakansson, 2003; Duke, 2012). It has widened the gap between increasing
number of weed species to be controlled and available herbicidal compounds effective
against those weeds.

Herbicides also cause losses of crops and crop products through toxicity caused by
drift and residual effects. Residues of herbicides are accumulated in different plant parts
and may enter the food chain. Solaimalai et al. (2004) reported the presence of residues of
atrazine in grains of sorghum and that of butachlor in rice grains and straw. Similarly,
they also reported the presence of residues of pendimethalin, metolachlor and pretilachlor
in edible portions of several food crops. A major portion of herbicides applied under field
conditions falls on non-target species and soil (Crone et al., 2009). Some herbicides like
triazines and sulphonyl ureas may persist in soil long enough to affect the growth of
subsequent sensitive crops (Hakansson, 2003; Zimdahl, 2007). This phenomenon has
been reported in US corn-soybean rotations. When atrazine or sceptor herbicides were
applied to corn, they persisted in soil and reduced germination and growth of soybean
grown later on as subsequent crop (Pimentel, 2005). Pimentel (1994) demonstrated

11
increased susceptibility of some crops to insects and diseases following application of
2,4-D for weed control. Herbicides applied at inappropriate soil and weather conditions
may cause yield reductions of crops from 2 to 50% (Pimentel et al., 1993). Herbicides
may be transported to nearby non-target crops through drift. It may reduce the growth and
yield of non-target sensitive crops upto several miles downwind (Hakansson, 2003).

Herbicide drift is also responsible for irreparable damages to wildlife and

mammals by causing death, growth reduction, poisoning and loss of fertility (Pimentel,
2005). The abundance and distribution of wild plants in a native region is regulated by
populations of their natural enemies. Herbicides may also kill beneficial natural enemies
(predators and parasites) of crop pests which may induce the pest attack more severe as
well as emergence of new pests (Hoddle, 2004; Pimentel, 2005). This may require
additional and more expensive control treatments to sustain existing crop yields.
Herbicides in soil although may not reduce population of soil microflora and microfauna
but may induce intraspecific and interspecific selection (Hakansson, 2003).
Microorganisms and invertebrates present in soil are crucial to the functioning of
ecosystems. They are useful in recycling of plant nutrients, decomposition of organic
wastes, biological nitrogen fixation, formation of soils, and regulation of plant nutrients.
Herbicides have also shown toxicity to these organisms leading to disturbances in
biologically driven processes of soil microorganisms and other invertebrates. Apart from
these, herbicides have also been identified as the sole chemical threat to 33 endangered
species of which 27 are angiosperms (Smith et al., 2000). Dinitropheonls (DNOC and
Dinoseb) disturb respiration processes of many organisms (Hakansson, 2003). Herbicides
have also caused toxicity and diseases to exposed animals (Hakansson, 2003).

Herbicides may also be transported to waterbodies through leaching and runoff

especially water soluble compounds. Cornell (2003) found the presence of atrazine,
alachlor and aldicarb in ground and well water. One half of human population uses
ground water for drinking purpose worldwide (Pimentel, 2005). He suggested that
degradation of these compounds in ground water is extremely slow due to presence of
very few microorganisms and <1% ground water recharge per year. Monitoring,
surveying and cleaning water of pesticide residues is much costly operation to make the
process impossible. Contamination of waterbodies has drastic effects on aquatic life. This
water affects fish directly by killing them and indirectly by eliminating food of fish like
insects and algae etc. These fish also produce contaminated meat reducing its quality and

12
market price (Pimentel, 2005). Fabricius (2005) found photophysiological stress on corals
after short term exposure to lower concentrations (<1 ug L-1) of atrazine and diuron.
Herbicides in agricultural runoff were implicated to cause the elimination of macro-phyte
populations from Ebo River delta of Spain (Manosa et al., 2001). Later on, it reduced the
population of waterfowl which used to feed on these macro-phytes.

As human beings are located at the top of food chain, they are ultimately exposed
to herbicides through contaminated water, food, soil and air. Twenty six million people
are reported to suffer from non-fatal poisoning of pesticides every year (Richter, 2002).
Of these cases about 3 million people are hospitalized; ca. 0.22 million deaths occur, and
ca. 0.75 million people face chronic illnesses (Hart and Pimentel, 2002). Blair et al.
(2015) reported that 1 million people suffer from chronic diseases and death due to
herbicide exposures annually. Herbicides cause acute poisoning to human beings in short
term effects. The long term effects of herbicides are held responsible for neurological
disorders, embryonic malformations, reproductive disorders, cancer of various types, loss
of immunity, and liver and kidney disorders (Hart and Pimentel, 2002; Schreinemachers,
2003; Hakansson, 2003; Alavanja et al., 2004; Watson et al., 2004). Chronic exposures to
herbicides have been linked with neurological anomalies such as Parkinsonism (Alavanja
et al., 2004). Laboratory studies have also impaired the functioning of immune system
due to herbicide exposure (Blakley et al., 1999). It also includes some irreversible nerve
damages like organophosphate induced delayed poly-neuropathy (OPIDP) (Pimentel,
2005). Kim et al. (2003) associated exposures to defoliating agents (2,4-D and 2,4,5-T) in
Vietnam War veterans to various diseases like eczema, radiculopathy, diabetes, peripheral
neuropathy, hypertension, heart disease and retinopathy. Pregnant women exposed to
chlorophenoxy compounds face increased incidents of birth defects in their children
(Schreinemachers, 2003). Most popular herbicides of the world glyphosate, 2,4-D,
bromoxynil and triazines are also reported to be carcinogenic (Guyton et al., 2015).
Studies have shown higher risks of certain types of cancer in farm workers and others
engaged in herbicide business (Pimentel and Hart, 2001). They have been shown to cause
disturbances in estrogen levels in women which may result in breast cancer in women.
McCarthy (1993) reported that pesticides which induce changes in levels of endocrine
hormone may result in reproductive, immunological and developmental disorders.
Although, effects on endocrine levels due to these compounds rarely result in acute
poisoning but their effects on reproduction and embryonic development have far reaching

13
consequences (Colborn et al., 1996). These effects are more pronounced in children due
to their high metabolic rates (which detoxifies, activates and excretes more compounds
than adults), more use of food items and less consciousness of eating and drinking
(Pimentel, 2005). The proportion of brain to body is more than five times higher in
children than adults. It further increases the vitality of cholinestrases in children. The
children exposed to agricultural field conditions have been shown to produce lesser
quantity of cholinestrases which is a strong indication of poisoning due to carbamates and
organophosphates (Repetto and Baliga, 1996). The risks of cancer are also ten times
higher in children than adults (Hebert, 2003).

The magnitude of problems posed by herbicides is still bigger than the benefits
gained from herbicides. Therefore, many scientists across the globe have recognized the
need for development of alternative techniques to chemical herbicides for sustainable
crop production.

2.4. Biological weed control

The above mentioned weed control methods become even more inappropriate
where weed infestations are widespread, land value is small and access to land is
inconvenient. Biological weed control is an approach of using living organisms or their
products to reduce population, growth, reproduction and impact of weeds on crops with
no harmful effects on crops (TeBeest, 1996; Charudattan, 2005). Biological control of
weeds has several advantages over other weed control methods. The biocontrol agents
establish self-perpetuating populations on weeds and distribute themselves throughout the
target weed’s range. The biocontrol approach can be used in areas not easily accessible
and where other approaches can not be used. Biocontrol agents are non-polluting to
environment, energy efficient, lesser expensive, biodegradable and leave no toxic
residues (Goeden and Andres, 1999). Natural enemies may reduce the populations of
weeds in invaded habitats to levels found in natural home range. This may lead to
restoration of ecological balance and biodiversity (Bellows, 2001). Biocontrol agents
either directly control weeds by interfering the physiology of plants and destroying their
vital parts or indirectly by making plants more susceptible to diseases and other pests
(Bellows and Headrick, 1999). Biological control of weeds is divided into inoculative
(classical) biocontrol and inundative biocontrol.

14
2.4.1. Classical or inoculative biological control

Classical or inoculative approach refers to the introduction and naturalization of a

natural antagonistic organism into a new area to control an exotic plant species
(Scheepens et al., 2001). The biocontrol agent is searched from the dynamically balanced
population in the native region of the weed and is applied in the newly invaded area
(Charudattan and Dinoor, 2000). The control using this technique is permanent but slower
due to gradual spread of initial population applied (El-Sayed, 2005). Classical control
agents once released into the environment maintain their population balance with the host
population. The expenses on weed control are incurred only once in contrast to other
control methods. The organisms are evaluated before and after release which improves
understanding the ecology of invasive plants (Goeden and Andres, 1999). The organism
once released can not be recalled. So, great care and testing is required before its release
to ensure safety of non-target species. The control agent may also change its host after
release into a new environment and emerge as a new pest (Scheepens et al., 2001).
Phytophagous insects have been principally used in classical biocontrol of weeds. Goeden
(1988) reported the first successful case using this approach in India for the control of a
cactus (Opuntia vulgaris Miller) in 1836. Cochineal insect (Dactylopius ceylonicus
(Green) (Homoptera : Dactylopiidae)), native to Brazil, was introduced for this purpose.
Other important example of classical biocontrol is of prickly-pear cactus (Opuntia spp.)
by a moth Cactoblastis cactorum which was introduced from North America to Australia
in the mid of 1920s (Tsukamoto et al., 1997). Three million eggs were released onto weed
flora after very careful laboratory testing. It reduced the dense vegetation of cactus to
minor patches. Towards the end of 19th century, vedalia beetle (Rodolia cardinalis) was
introduced into California from Australia to control cottony cushion scale (Icerya
Purchasi) (Waage and Greathead, 1988).

Host range of classical biocontrol agents is usually wide. Therefore, classical

biocontrol meets success when agents are introduced to control a plant species which is
quite unrelated to valued plants of the area. Populations of such biocontrol agents can be
protected and improved in nature by conservation biocontrol practices which involve
favoring environmental and growth conditions of biocontrol agent (Culliney, 2005;
Ghorbani et al., 2005). Rust and smut fungi have also been applied to control rush
skeleton (Chondrilla juncea), musk thistle (Caduus thoermeri), Ageratina riperia, Acacia
saligna and weedy species of Rubus in different parts of the world (Charudattan and

15
Dinoor, 2000; Leiss, 2001). Classical biocontrol programs require many years of research
and huge funds. These programs typically involve foreign exploration for biocontrol
agents, pre-release testing, post-release monitoring and evaluation which may take 10 to
20 years as well as cost multi-million dollars (McFadyen, 2000). Inoculative biocontrol
agents are also kept in quarantine for exhaustive host range testing. It is essential to
ensure the safety of nontarget plant species. For example, two curculinoid weevils
(Acythopeus spp.) were introduced in Hawaii for control of invasive cucurbit (Coccinia
grandis L. (Voigt)). The agents remained under quarantine for seven years before their
release for authorization of their use in natural environment (Culliney et al., 2003). Weed
flora is commonly related to crop plants in physiology and phenology which increases the
probability of nontarget effects of biocontrol agents. European weevil (Rhinocyllus
conicus (Frolich) (Coleoptera : Curculionidae)) was introduced in the continental United
States for control of exotic thistles (Carduus spp.) in 1969 (Julien and Griffiths, 1998).
Louda et al. (1997) found that European weevil also significantly reduced seed
production of Cirsium spp. and jeopardized the existence of some of the rarer species.
Non-target effects of insect biocontrol agents have been oftenly reported (Culliney,
2005). Funasaki et al. (1988) reported four cases of insect biocontrol agents introduced
into Hawaii which attacked non-target native or beneficial plants. Similarly, McFadyen
(1998) described eight cases of non-target impacts of insect biocontrol agents worldwide.
Furthermore, success of this approach is also dependent on favorable conditions which
increase the establishment and high populations of biocontrol agents on target plants
(Dagno et al., 2012). Therefore, this approach is limited to low value lands having broad
area of weed infestation and has never been employed on arable lands. It needs to be very
cautious when introducing a new species into an area which is generally discouraged.

2.4.2. Inundative or augmentative biological control

It involves relatively lesser risks to apply indigenous natural enemies than

introduction of exotic species into a new habitat (Charudattan, 2005). Therefore, research
for development of weed biocontrol is focused on inundative or augmentative biocontrol.
It refers to the application of natural antagonists of weeds at high populations periodically
which are already present in the area (Hakansson, 2003). These biocontrol agents are also
called bioherbicides. Fungi have been mostly exploited for weed biocontrol which may
also be called mycoherbicides. They are supposed to immediately suppress target weeds
and are fast acting than classical biocontrol agents (Dagno et al., 2012). The process

16
begins with the search for active biocontrol agents followed by efficient mass production,
delivery system using suitable formulations, strategies for use, registeration and
commercialization of product (Bateman, 2001). The important characteristics of these
biocontrol agents include; high virulence against target plants (Agrios, 1997),
establishment of high populations in host plants, easy to produce and store, suitable
formulation and safety of non-target species (Auld et al., 2003; Zhang et al., 2003).
Kenny (1986) reported that first commercial bioherbicide was introduced in 1980s called
DeVine. This contained a fungus Phytophthora palmivora and was used to control
Moronea odorata grown in citrus plantations of Florida. Many plant pathogenic fungi and
bacteria have been developed as bioherbicides. Dagno et al. (2012) have described sixteen
bioherbicides registered for biocontrol of different weeds grown in different countries.
Some of these containing plant pathogenic fungi include Collego, BioMal, Stumpout and
Dr. BioSedge. They contained Colletotricum gloeosporioides f.sp. Aeschynomene,
Colletotrichum gloeosporioides f.sp. Malvae, Puccinia canaliculata and Cylindrobasidum
leave which were supposed to control Aeschynomene virginica in rice and soybean,
Malva pusilla, Cyperus esculantus and resprouting of cut trees, respectively (Charudattan
and Dinoor, 2000; Charudattan, 2001; Ghorbani et al., 2005). The commercialized
bioherbicide based on phytopathogenic bacteria is CAMPERICO. It contained
Xanthomonas campestris pv Poae which induced wilt in Poa annua grown in golf
courses (Charudattan and Dinoor, 2000; Charudattan, 2001).

The success of pathogenic microorganisms is based on the presence of suitable

number of biocontrol agent on susceptible host population and appropriate environmental
conditions required for infection and disease development (Leiss, 2001). The
management techniques of weed biocontrol using pathogens are opposite to management
of pathogenic diseases in crop plants. The environmental conditions which favor disease
development in crops are controlled whereas those conditions are promoted for disease
development in weeds using almost similar methods in reverse (Charudattan and Dinoor,
2000). These environmental conditions include optimum temperature range, moisture
contents and humidity, dew period, wind and light (Charudattan, 2001; Ghorbani et al.,
2005). Dew period and temperature are determinant factors of primary infection by
biocontrol agents which vary from field to field and year to year. These variations and
their effects were observed during experiments with Colletotrichum gloeosporioides f.sp.
Malvae (BioMal, Philom Bios) and Alternaria alternata (Babu et al., 2003). Collego also

17
showed dependence on free moisture for causing primary infection. Different biocontrol
agents take variable time period for disease development after application.
Helminthosporium sp. took 36 days after application to defoliate 73% of wild poinsettia
plants grown in soybean in Brazil (Dagno et al., 2012). The requirement of time period
after application for disease development in water hyacinth by Alternaria alternata was
two months (El-Morsy, 2004). Disease development in target plants also depends on the
susceptibility of hosts to the attack of such pathogens. Susceptibility of host to infection
of pathogens varies with growth conditions of target plants and crop-weed competition
(Agrios, 1997; Ghorbani et al., 2002; Kremer and Li, 2003). Susceptibility to infection of
pathogens is different at different growth stages of weed species (Dudycha and Roach,
2003). Therefore, proper timing to manipulate environmental conditions and application
and establishment of biocontrol agents on host plants is a critical factor in the success of
this approach. It requires a thorough understanding of interactions of biocontrol agents
with host plants in nature and role of environmental factors on their performance. Other
limitations associated with this biocontrol approach include difficulties in mass
production of pathogens, assurance of efficacy, loss of virulence, inadequate shelf life of
inoculum and lack of delivery system which have often caused the failure of promising
biocontrol agents (Charudattan and Dinoor, 2000; Dagno et al., 2012). Limited success of
bioherbicides has also been linked with rapid desiccation of control agents in the field due
to their high humidity requirement for infection and damages done by UV light (El-
Sayed, 2005). Suitable formulation not only helps efficient application but also improves
the efficiency of biocontrol agents (El-Morsy et al., 2006). Suitable formulation of plant
pathogens is a challenge which should impart long shelf life, viability of high population
of control agents, ease in application and improved efficiency of control agents,
compatibility with end users equipments and practices, and effectiveness at suitable rates
(Auld et al., 2003; Zhang et al., 2003). Environmental conditions in the field keep
fluctuating and scarcely are suitable to disease development by biocontrol agents
(Ghorbani et al., 2005). After overcoming all these limitations, some promising
biocontrol agents have shown their effectiveness against a single weed species or very
closely related species (Rosskopf et al., 1999). More economic returns are expected when
multiple weed species are targeted because reduction in one weed species invites more
infestations by other weeds. The scope of inundative biocontrol has been suggested to be
enhanced by adopting multiple pathogens strategy. This strategy is aimed at combining
very host specific multiple pathogens in a single application (Chandramohan et al., 2000).
18
They used three fungal pathogens, Drechslera gigantea, Exserohilum longirostratum and
E. rostratum in an emulsion. This multiple pathogens strategy was used to control seven
grassy weed species in greenhouse and field trials.

2.4.3. Allelopathy
Non-target impacts of biocontrol agents and dependence on suitable
environmental conditions by pathogenic microorganisms for infection and disease
development limit the practical use of such biocontrol agents (Ghorbani et al., 2005). In
this scenario, the importance of allelopathy has increased for the sake of biological
control of weeds. It is related to inhibition of growth of one plant through release of
phytotoxic substances by other plants and microorganisms into the environment (Torres
et al., 1996). Higher plants and microorganisms produce and release phytotoxic
substances in their surroundings which play role in defense and competition of these
organisms with other co-existent species (Mattner, 2006). These substances are
commonly termed as allelochemicals. Past efforts on allelopathy research were
concentrated on the use of allelopathic plants which showed inhibitory effects on weeds
grown in different cropping systems (Farooq et al., 2011, 2013). Allelochemicals
produced by these allelopathic plants have been shown to disrupt physiology of other
plants like respiration, photosynthesis, enzymes activity, water and hormonal balance,
growth and germination processes (Kruse et al., 2000; Farooq et al., 2013).
Allelochemicals have diverse chemical structures and act at multiple sites of plants. This
reduces specificity of these substances which is evident in herbicides (Nimbal et al.,
1996). Unlike herbicides, allelochemicals active to suppress plants are generally
comprised of mixtures of several compounds and have no residual effects (Inderjit et al.,
2005; Bhadoria, 2011).
Important crop plants which release allelochemicals are rye, sorghum, brassica,
sunflower, tobacco and wheat (Cheema and Khaliq, 2000; Jabran et al., 2015). Schulz et
al. (2013) listed 16 allelochemicals produced by rye which inhibited other crops and
weeds. Sorghum is most studied allelopathic plant which produces allelochemicals in its
different parts and has been shown to inhibit many weeds. The concentration and
phytotoxicity of these allelochemicals varies with cultivars, environmental conditions and
growth stages of the crop (Weston et al., 2013). Plants from Brassicacae family have been
reported to produce glucosinolates in different plant parts (Fahey et al., 2001). These
glucosinolates are converted to isothiocyanates after release. Isothiocyanates target

19
respiration process to suppress weeds and crops upon absorption by these plants (Petersen
et al., 2001). Sunflower allelopathy has also been investigated for suppression of several
weeds. The allelopathic potential of these and other plants has been manipulated for
biocontrol of weeds. Synergistic effects of mixtures of various sources of allelochemicals
have also been used in several studies to control multiple weeds grown under field
conditions (Awan et al., 2012). Alsaadawi et al. (2012) evaluated the effect of
allelochemicals from eight cultivars of sunflower to suppress weeds grown in wheat.
They applied residues of the cultivars to crop and weeds grown. All the cultivars of
sunflower varied in reducing weed density (24-75%) and weed biomass (12-67%) due to
variable allelopathic potential and resultantly, increased grain yield and yield components
of wheat over non-treated control. As allelopathic potential of crop cultivars is controlled
genetically, they can be improved for more releases of allelochemicals to control weeds
inexpensively, easily and environment friendly (Kong et al., 2011). Artemisinin is a
phytotoxin released by annual wormwood (Artemisia annua) which is inhibitory to
Amaranthus retroflexus, Ipomoea lacunose, Portulaca oleracea and A. annua. Lydon et
al. (1997) described that weed suppression was reduced when soil was amended with pure
artemisinin than that of leaf extract of Artemisia annua. Marigold, eucalyptus and many
other plants are also known for their allelopathic potential which may be investigated for
biocontrol of weeds.
The major input of allelochemicals from sources to the environment comes from
root exudates but other modes of release are also prominent in different cases (Inderjit
and Weston, 2003). The timing of release by allelopathic plants is also a critical factor in
the defense of plants against weeds. Release of higher amounts at early seedling stage
plays more effective role in plant’s life cycle than later stages (Inderjit et al., 2005).
Allelochemicals are also released by decomposing dead plant tissues, leaching and
volatilization from plant parts (Jabran et al., 2015). Therefore, benefits of allelopathic
potential in weed suppression may be gained through applying residues of allelopathic
plants or cultivating allelopathic plants in crop rotation (Tabaglio et al., 2008; Farooq et
al., 2011). Allelopathic plants may be grown as an intercrop and benefits gained from
their production and weed suppressing ability simultaneously (Makoi and Ndakidemi,
2012). Some cover crops having allelopathic potential like brassica may be preferred over
others to get dual benefits of weed control as well as cover crop (Altieri et al., 2011).
Bernstein et al. (2014) suggested that soybean may be planted in rye cover crop with no
apparent damage to soybean crop while maintaining rye cover crop for longer period to
20
effectively control weeds. Residues of plants containing allelochemicals may be applied
as mulches in different crops for weed suppression as well as water saving. Dhima et al.
(2006) applied barley residues in maize crop as mulch which reduced the emergence of
Setarria verticillata (0-67%) and Echinochloa crussgalli (27-80%) and improved yield of
maize by 45% compared with non-mulched treatment. Introduction of allelopathic crops
in rotation may also lower weed infestations. Allelopathic crops in rotation add
allelochemicals in soil to suppress weeds in next crops of the rotation (Dmitrovic et al.,
2014; Garrison et al., 2014). Growing allelopathic crops for weed suppression needs a
change in existing cropping pattern. Cropping patterns are primarily determined by agro-
climatic conditions and economic benefits of growing a crop which makes it difficult to
introduce allelopathic crops in rotation. Therefore, it is more reliable option to apply plant
residues and extracts containing allelochemicals to suppress weeds. Allelochemicals can
be extracted from potentially allelopathic plant parts in water and applied to weeds for
their suppression. This technique has been suggested to be more efficient than others for
significant weed suppression (Razzaq et al., 2010). Rice (1995) suggested that application
of extracts containing allelochemicals is more advantageous than raw biomass because of
slower release from biomass and lack of concentration build up adequate to suppress
weeds. Slower release from raw biomass is also accompanied by continual degradation
and other processes on allelochemicals which further reduce their impact on weed
control. The allelopathic effects on target plants are concentration dependent which may
be stimulatory at lower concentrations and inhibitory at higher concentrations (Belz et al.,
2005). Hence, the required amount of allelopathic residues of plants is impracticably
higher to get adequate concentration of allelochemicals under field conditions (Rice,
1984).
The phytotoxic effects of allelopathic plants are affected by concentration, age of
target plants, flux rate, metabolic state of plant and climatic and environmental conditions
(Singh et al., 1999). Biotic and abiotic stresses to allelopathic plants increase the
production of allelochemicals (Einhellig, 1996). Cheema and Khaliq (2000) and Irshad
and Cheema (2005) determined the dose, time and number of applications of sorghum
extracts (also called Sorgaab) for weed control in various crops. They found that 10%
sorghum extract applied twice at intervals of 25 days can provide economic returns in
soybean, wheat, rice and cotton. Maximum gains were accrued from rice where biomass
of barnyard grass (Echinochloa crussgalli L.) was reduced by 40% and yield of rice was
increased by 18%. Similarly, extract of different plant parts of sunflower (also known as
21
Sunfaag) has been evaluated for suppression of weeds grown in wheat in various studies
(Anjum et al., 2007; Naseem et al., 2009). It was established from these studies that three
applications of different extracts of sunflower at 7 days interval starting from 3-4 weeks
after emergence may effectively control two common weeds, lambsquarters
(Chenopodium album L.) and toothed dock (Rumex dentatus L.) by 70 and 97%,
respectively. In these cases, the yield of wheat was obtained equivalent to weed free
control. Zuo et al. (2014) reported that microbial activity in the rhizosphere of crop plants
increases upon application of allelopathic materials. It helped to increase nutrient
availability to crop and out-compete weeds.
As allelochemicals have to be transported from donor to receiver plants through
environment usually soil, the soil reactions essentially influence these substances
(Kobayashi, 2004). It is evident from the observation that many chemicals have only
shown phytotoxic effects without soil in laboratory bioassays (Inderjit et al., 2005; Kaur
et al., 2005). The fate of allelochemicals after release into soil is determined by physical,
chemical and biological processes in the soil carried out by organic and inorganic
constituents of soil and soil organisms. These processes modify bioavailability and
phytotoxicity of allelochemicals in soil thereby modifying their impact on target plants.
Physical processes which affect allelochemicals include sorption, mobility and
hydrodynamics of soils. Chemical reactions of allelochemicals with soil constituents
affect allelochemicals in soil. Microbial and enzymatic reactions on allelochemicals in
soil also modify bioavailability and phytotoxicity of allelochemicals to target plants.
Processes of biogeochemical cycles of plant nutrients in soils like immobilization and
mineralization have also been found to act on allelochemicals in soil and modify their
bioavailability and phytotoxicity to target plants (Inderjit, 2006). Metabolic reactions of
microbes on allelochemicals in soil may inactivate and degrade them to lower or no
phytotoxicity (Mandal, 2001; Belz et al., 2009). The large diversity in structure of
allelochemicals invites diverse group of microorganisms and a variety of reactions for
their biodegradation (Kobayashi, 2002).
Growth conditions of donor and receiver plants also influence the amount and
impact of allelochemicals which are in turn dependent on environmental conditions
(Kobayashi, 2004). The concentration of allelochemicals released differs with growth
stages, plant organs, crop cultivar and distance from the donor plants (Wu et al., 1999;
Tongma et al., 2001). The susceptibility of target plants to allelochemicals also varies
with species type, plant organs and growth stages (Suzuki et al., 2001; Iqbal et al., 2002).
22
The principal mode of absorption of allelochemicals is through roots (Kobayashi, 2004).
Absorption of certain forms of allelochemicals by target plants and their transport in soil
is more efficient than others like water solubility (Kobayashi, 2004). It shows that only a
portion of diverse forms of allelochemicals showing activity under laboratory conditions
may be effective in weed suppression under natural conditions (Kobayashi, 2004). A
variety of soil, environment, growth conditions/stages of donor and receiver plants, plant
species and cultivar factors have complicated the use of allelopathy in weed control and
benefits gained from this environment friendly weed control approach.
2.4.4. Allelopathic bacteria
Now research efforts are being diverted towards microbial allelopathy due to
involvement of multiple factors affecting the efficiency of allelopathic plants and
limitations of plant allelopathy for its practical use in weed biocontrol. It has been
realized that microbial allelopathy has several advantages over plant allelopathy and its
role in biocontrol of weeds may be more prominent than plant allelopathy. Microbial
allelopathy involves the application of such microorganisms to weeds which colonize
rhizosphere of plants, release phytotoxic metabolites and directly or indirectly suppress
growth, density, reproductive capacity and impact of weeds on crops (Kremer, 2013). The
effects of allelopathic microorganisms are reproduced by transferring soil from their
origin into an open field (Barazani and Friedman, 1999). These rhizosphere inhabiting
microorganisms include allelopathic bacteria and fungi. Allelopathic bacteria are being
reviewed and investigated for their potential benefits in overcoming these limitations and
a promising efficiency for biocontrol of weeds. These privileges of allelopathic bacteria
are appreciated because production and absorption of allelochemicals takes place almost
at the same site (rhizosphere of weeds) and time. It reduces the role of soil and
environmental factors in decreasing the bioavailability and phytotoxicity of bacterial
phytotoxins. The rhizobacteria which release phytotoxic secondary metabolites
(allelochemicals) in the rhizosphere of certain plants, and inhibit their germination and
growth are called allelopathic bacteria (AB) (Barazani and Friedman, 1999; Sturz and
Christie, 2003). As microbial phytotoxins produced and released in the rhizosphere do not
have to move long distance from source to sink, it rules out the possibility of reduction in
concentration and phytotoxicity of bacterial allelochemicals due to soil reactions. The
amount produced is absorbed by target plants roots nearby. It keeps the production,
release and flow between source and sink. The allelopathic bacteria continue their
activities in the rhizosphere as long as they survive there and suppress the growth and
23
development of target plants (Kremer, 2006). Therefore, the impact of phytotoxic
metabolites of rhizobacteria is suggested to be higher than plant allelochemicals applied
to soil. The future of alleopathy research therefore needs to focus on development of
allelopathic bacteria which is suggested to be more effective under field conditions.
Rhizosphere inhabiting Pseudomonads are well known for their biocontrol activity
(Weller, 2007), plant growth promotion and competitive root colonization (Rosas et al.,
2009). Specific effects of these bacteria on plants appear as reduced germination, growth
inhibition, reduced elongation of root, root deformation and enhanced infection of
pathogens (Li and Kremer, 2006). Although several phytotoxic substances have been
determined from rhizobacteria in different cases but cyanide production has been
suggested to be a common substance among phytotoxins producing rhizobacteria
(Kremer, 2006). Fluorescent pseudomonads have been extensively studied for
cyanogenesis by using glycine, methionine, plant glucosinolates and other simple
substances (Gallagher and Manoil, 2001). A membrane bounded three sub-unit enzyme
encoded by hcnABC produces cyanide through oxidative decarboxylation of glycine
(Blumer and Haas, 2000). Cyanide is also produced by induction of 1-
aminocyclopropane-1-carboxylic acid (ACC) synthase activity for ethylene production. It
further enhances the phytotoxic effects of cyanide (Grossmann, 2004). Cyanide has been
reported to disturb electron transport chain of aerobic respiration which reduces
respiration rates and causes cell death (Siedow and Umbach, 2000). It inhibits electron
transport from cytochrome c oxidase (COX) to O2 which impairs proton motive force and
ATP synthesis (Umbach et al., 2006). Cyanide is also a potential inhibitor of enzymes
involved in CO2 and nitrate metabolism. It may also bind with plastocyanin and block
electron transport chain of photosynthesis (Grossman, 1996). Additionally cyanide has
also been reported to inhibit auxin biosynthesis and transport. Rudrappa et al. (2008)
described that cyanogenesis inhibited primary root growth of Arabidopsis thaliana,
lettuce, barnyard grass and green foxtail by suppression of auxin signaling mechanism.
Kremer and Souissi (2001) characterized over 2000 isolates for cyanogenesis, out of
which ca. 32% produced cyanide which varied from trace concentrations to >30 nmols
per mg cellular protein. Eight cyanide producing strains significantly reduced root length
of lettuce (35-84% by different strains) and barnyard grass (15-57% by different strains)
seedlings in agar bioassay. Two non-cyanogenic strains also inhibited root lengths of
lettuce (35 and 56%) and barnyard grass (44 and 17%) in this study, which were
suggested to be due to phytotoxins other than cyanide. Sarwar and Kremer (1995) isolated
24
a high auxins producing isolate of Enterobacter taylorae (72 mg L-1 IAA equivalents)
which suppressed root growth of field bindweed by 90.5% when L-TRP was amended
compared to non-treated control. They suggested that providing L-TRP with selected
auxins producing rhizobacteria increases their phytotoxic effects and may be used as a
biological weed control strategy. Similarly, higher concentrations of other phytohormones
have also been suggested to inhibit plant growth (Nehl et al., 1997). Gurusiddaiah and
Gealy (1994) purified phytotoxin from D7 strain (the strain which inhibited downy brome
in Kennedy et al., 1991). This phytotoxin was a complex which contained at least two
polypeptides, one chromophore, fatty acid esters and a lipopolysaccharide matrix. Further
purification of this compound lost its phytotoxicity which may be due to damage to the
chemical structure of the compound. Patrick et al. (1993) studied the mechanism of D7
for root growth inhibition. They suggested that the effects observed due to application of
D7 were shown due to disruption of fatty acid biosynthesis and cell membrane integrity.
Banowetz et al. (2008) studied phytotoxic effects of five strains of Pseudomonas
fluorescens WH6 on germination of several grassy weed species. They found that these
rhizobacteria secrete a compound or compounds which stop germination process after
emergence of coleorhiza and plumule. They named this compound as germination arrest
factor (GAF). This compound arrested germination of grassy weeds, wheat and barley but
not corn. The diluted concentration of this compound did not inhibit germination of eight
dicot plant species. Banowetz et al. (2009) described physical and chemical properties of
GAF. It showed insolubility in all organic solvents but moderate solubility in methanol
indicating that the compound has non-aromatic structure. Studies based on gel filtration
and ion exchange chromatography indicated that the compound must contain an acid
group and its molecular weight to be lesser than 1000. Pseudomonas fluorescens BRG100
has also been reported to suppress green foxtail. Quail et al. (2002) attributed this growth
reduction of green foxtail to the production of extracellular metabolites including
pseudophomins A and B, and cyclic lipodepsipeptides. Various un-identified phytotoxins
produced by rhizobacteria have also been associated with growth inhibition of weeds
(Nehl et al., 1997). Phytotoxic metabolites in the rhizosphere may also enhance the
release of root exudates, increase permeability of plasmalemma and damage
plasmalemma protein function in the root (Barazani and Friedman, 1999). Other
phytotoxic substances released by such bacteria include lytic agents and enzymes (Glick
et al., 1998), extracellular polysaccharides (Fett et al., 1989), siderophores (Marschener
and Crowley, 1997) and antibiotics (Bender et al., 1999). Plant growth may also be
25
suppressed due to decrease in root weight induced by reduction in root sugar contents
(Brinkman et al., 1999). Kremer et al. (2006) reported the production of cell wall and cell
membrane degrading enzymes by plant growth inhibitory rhizobacteria. The presence of
allelopathic microorganisms in rhizosphere of plants and their phytotoxic metabolites
may increase the susceptibility of plants to other pathogens and thereby inhibit their
growth and development indirectly (Frederickson and Elliott, 1985). Suppression of plant
symbionts by biocontrol agents may also suppress the growth of these plants indirectly by
depriving them of benefits gained through symbiosis. Rhizobium leguminosarum forms
symbiotic association with two important weeds, white clover and common lambs’
quarter. These weeds get benefits from this association by gaining nitrogen for growth.
Application of antagonistic microorganisms of R. leguminosarum may suppress their
growth. Omer et al. (2010) identified several such isolates of lactic acid bacteria (LAB)
which suppressed this symbiotic association and indirectly growth of white clover and
common lambs’ quarter. They reported that field application of such antagonistic
microbes may reduce the growth of these weeds up to 80% using this approach. Barazani
and Friedman (1999) reported two allelochemicals, bialaphos and phosphinothricin
extracted from Streptomyces hygroscopicus and Streptomyces viridochromogenes,
respectively, which were developed and commercialized as natural herbicides. Two other
allelochemicals have also been isolated from S. hygroscopicus called geldanamycin and
nigericin which suppressed Lapidium sativum by 50% in petri plate assay (Heisey and
Putnam, 1986). Additionally hydantocidin was also isolated from the same species which
inhibited growth of several annuals and perennials in soil when applied in foliar spray
(Nakajima et al., 1991). All the rhizosphere inhabiting bacteria which produce few or
multiple phytotoxic substances in a species specific manner can be used for development
of bioherbicides in different cropping systems (Boyetchko, 1997).
Downy brome is an annual grass weed which infests millions of hectares of land
planted to winter wheat in western USA. Kennedy et al. (1991) isolated over 1000
isolates from the rhizosphere of downy brome (Bromus tectorum) and winter wheat. They
characterized these rhizobacteria for growth suppression of downy brome and lack of
inhibition of winter wheat for their potential as biocontrol agents of this weed. Out of
total, 81 of the isolates suppressed downy brome without inhibiting winter wheat in agar
bioassay. Six of these isolates consistently suppressed downy brome but not winter wheat
in growth chamber studies conducted in potted soil. Then they applied these isolates in
nursery field trials in which the weed density was reduced up to 30% and biomass upto
26
42% by different isolates. They also conducted three field trials in which 3 isolates of
brome suppressing bacteria were applied to naturally grown downy brome populations.
Two out of three isolates suppressed downy brome population and resultantly, growth up
to 31 and 53%, respectively, over un-inoculated control. The yield of winter wheat was
improved by 18 to 35% at two sites.
O’Hara and Vargas (2005) isolated 442 strains from soil and roots of three weeds
wild radish, annual ryegrass and capeweed and characterized them for biocontrol
potential of these weeds. Seventy four of these strains inhibited weeds in laboratory
bioassay which were later applied on these weeds, grapevines and a cover crop
subterranean clover. In this study, 19 strains suppressed wild radish and/or annual
ryegrass. Ten of these weed suppressive strains also promoted growth of subterranean
clover whereas no deleterious effects were observed on grapevines. These biocontrol
agents were identified using biochemical and molecular techniques as Pseudomonas
fluorescens and Alcaligenes xylosoxidans.
Striga hermonthica (Del.) Benth. being an obligate root parasite causes severe
crop losses in cereals in sub-Saharan Africa. The germination and later growth of S.
hermonthica seeds causes economic losses to crops. The control methods adopted usually
target emerged seeds. Biocontrol approach aimed at germination inhibition of S.
hermonthica seeds may not only be most effective technique but cheaper also in resource
poor African countries. Ahonsi et al. (2002) isolated 460 strains of fluorescent
pseudomonads from S. hermonthica seeds taken from naturally suppressive soil. These
isolates were screened for germination inhibition of S. hermonthica seeds in an in vitro
bioassay using glass-fiber disks. Fifteen isolates significantly inhibited germination of
target weed in this bioassay which belonged to Pseudomonas fluorescens and P. putida.
These 15 strains were used in an in vivo screening assay by inoculation of maize seeds in
pot conditions using steam-sterilized soil. The results showed that 6 strains reduced
germination of S. hermonthica seeds and increased biomass of maize compared to un-
inoculated control. These biocontrol effects were not retained in subsequent maize crop
which necessitated periodic application of these agents for sustained biocontrol of S.
hermonthica. The results obtained were suggested to be due to production of phytotoxic
metabolites, and plant species and even cultivar specificity of biocontrol agents. These
observations were synonymous to Kennedy et al. (1991) and Gurusiddaiah et al. (1994).
Leafy spurge (Euphorbia esula-virgata) infests millions of hectares across
Northern America. This weed resists most of weed control methods due to regeneration
27
from root and shoot buds and deep root system. Kremer and Souissi (2013) tested over
500 isolates of rhizobacteria which were isolated from the rhizosphere of leafy spurge for
its biological control. These rhizobacteria were first characterized for production of
phytotoxins in sensitive seedlings (lettuce) bioassay. Twenty strains were selected based
on data from previous step for application to target weed cuttings. One third of these
strains significantly suppressed leafy spurge in this study. Half of these leafy spurge
inhibiting bacteria only showed effects when intact cells were also attached to plants
rather than culture filtrates only. They suggested that such strains may also be relying on
formation of biofilms along with other mechanisms to show biocontrol activity.
Previously root damaging insects were successfully applied as insect biocontrol agents of
leafy spurge (Caesar, 2000). Kremer et al. (2006) investigated the synergistic
relationships between these insect biocontrol agents and plant associated rhizobacteria for
biocontrol of this weed. They isolated rhizobacteria from the root of leafy spurge
damaged by biocontrol insects. These isolates were screened based on production of
indole-3-acetic acid, HCN, siderophores and extracellular polysaccharides. The potential
candidates for biocontrol of leafy spurge were selected based on this characterization and
applied to callus tissue of the weed in tissue culture bioassay. Thirty microlitre culture of
each strain was applied on callus piece and incubated in dark at 27°C for 48 hours. After
that, tissue injury symptoms were visually rated from 0 (no injury evident) to 4 (severe
growth reduction and color change). All of the isolates suppressed callus growth in the
bioassay to various extents. However, two isolates in their study suppressed callus growth
of leafy spurge but did not possess the biochemical characteristics tested in the laboratory.
They suggested that these effects may be due to production of some other unknown
phytotoxins. The results of this study showed potential of these isolates to be integrated
with root damaging insects for biocontrol of leafy spurge.
Jointed goatgrass (Aegilops cylindrica Host.) is also a notorious grassy weed
which infests millions of hectare land cultivated to wheat annually. It annually causes
losses of $ 145 million to crop yield and quality (25-50%) (Ogg, 1999). Physiology and
morphology of this weed are quite similar to wheat which limits herbicide use and
application of biocontrol agents may prove very effective to reduce economic losses
(Anderson et al., 2004). Kennedy and Stubbs (2007) isolated 2450 strains from
rhizosphere of downy brome, jointed goatgrass, winter wheat, and from soil. All of these
isolates were applied to winter wheat and jointed goatgrass in seedling bioassay using
water agar in Petri plates. Data regarding germination and root length of seedlings was
28
recorded after 6 days. The results showed that half of the applied strains inhibited root
growth of jointed goatgrass. Seventy six out of these strains inhibited jointed goatgrass by
more than 50% and they had no inhibitory effects on winter wheat. These 76 strains were
evaluated in growth chamber studies in which seeds were at the surface of silt loam filled
containers and culture of respective strains applied (@108 colony forming units mL-1 for
each container). The results showed that only seven strains inhibited jointed goatgrass but
not winter wheat in this study. The response for root growth suppression ranged from
stimulatory (82%) to inhibitory (71%) as compared to control. The response for shoot
growth ranged from a stimulatory of 87% to inhibitory of 71% as compared to control.
These seven strains were then applied under field conditions for weed suppression over a
period of three years. Growth of jointed goatgrass was reduced by more than 20% in the
1st year. In 2nd year growth reduction ranged from 20 to 74% by different strains. In 3rd
year growth of jointed goatgrass was suppressed from 27 to 47% as compared to control
by different strains. However, no reduction in plant number of jointed goatgrass was
observed during these years.
Omer and Balah (2011) isolated rhizosphere inhabiting bacteria and fungi from
three important weeds Polyogon monspiliensis, Convolvulus arvensis and Phalaris
paradoxa. These isolates were applied to 7-day old seedlings of these weeds obtained in
sterile filter papers after surface-sterilization of seeds. After 5 days, seedlings were
removed from tubes containing MS basal medium and their biomass was recorded. Two
isolates of rhizobacteria (identified as Pseudomonas syringae st.1 and P. syringae st.2)
and one fungal isolate (identified as Colletotrichum sp.) significantly suppressed biomass
of these weeds. The secondary metabolites were extracted from these three strains using
ethyl acetate and applied to surface sterilized seeds in pre and post emergence bioassays
to determine their effects on germination and biomass reduction. These isolates reduced
the seedling biomass of P. monspiliensis (upto 47.5%), C. arvensis (upto 22.8%) and P.
paradoxa (upto 51.3%) as compared to respective un-inoculated controls. The reductions
in root length, shoot length and germination were observed up to 79, 35 and 31% for P.
monspiliensis, 61, 38 and 43% for C. arvensis, and 96, 82 and 73% for P. paradoxa,
respectively. The metabolites of these isolates were then applied to these weeds grown in
faba bean under greenhouse conditions in potted sand. The results showed that biomass of
P. monspiliensis, C. arvensis and P. paradoxa was reduced up to 40, 32.6 and 46.4%,
respectively, over respective un-inoculated controls.

29
 Similarly, Pseudomonas fluorescens LS102 and LS174 have been found to
suppress leafy spurge (Brinkman et al., 1999). Boyetchko (1997) reported several isolates
of Pseudomonas fluorescens for their herbicidal activity against green foxtail (Setaria
viridis) and wild oat (Avena fatua). Caldwell et al. (2012) applied Pseudomonas
fluorescens BRG100 to green foxtail in growth pouch bioassay which suppressed its root
length from 73 to 79% over un-inoculated control. A large collection of rhizobacteria was
isolated by Li and Kremer (2000) from several weed species and screened for the
suppression of their germination and growth in laboratory bioassays. The competent
strains from their study were selected for evaluation under greenhouse conditions using
natural soil containing indigenous soil microorganisms (Li and Kremer, 2006). The
response due to inoculation of these potential weed inhibitory isolates varied from
stimulatory to inhibitory. The results showed that rhizobacteria of giant foxtail reduced
root and shoot lengths of green foxtail up to 77 and 79%, that of barn yard grass up to 67
and 73%, that of field bindweed up to 27 and 60%, and that of ivyleaf morningglory up to
79 and 28%, respectively, by different isolates. The rhizobacteria isolated from
morningglory and field bindweed inhibited root and shoot lengths of green foxtail up to
70 and 51%, that of barnyard grass upto 45 and 34%, that of field bindweed up to 19.5
and 55%, and that of ivyleaf morningglory up to 43 and 27%, respectively, by different
isolates. Isolates from Amarathus sp. and common cocklebur showed that root and shoot
lengths of green foxtail may reduce up to 54 and 66%, that of redroot pigweed up to 74
and 59%, and that of common cocklebur up to 49.5 and 40%, respectively, by different
isolates. All these isolates were also applied to soybean and wheat in a similar manner.
Majority of the isolates significantly increased root and shoot lengths of wheat and
soybean, some caused non-significant impact and few also suppressed the crops. Four
isolates significantly reduced root length and one significantly reduced shoot length of
soybean. Three of the isolates also significantly reduced root length of wheat. It was
concluded from this study that host specific weed inhibiting rhizobacteria may suppress
these weeds under natural conditions ensuring the safety of non-target plants.
To avoid laborious plate and seedling bioassays for screening of rhizobacteria as
potential biocontrol agents of weeds, Souissi and Kremer (1998) devised a rapid
microplate callus assay. In this assay, callus pieces of target weed (leafy spurge) were
obtained by culturing tissue of the plant in Gamborg’s B5 medium. Pieces of the callus
were made weighing 0.5 grams and placed in microtiter plates supplemented with
Gamborg’s B5 medium. Thirty microliters culture of rhizobacterial strains was applied in
30
respective well and placed in dark at 27°C for 48 hours. After that the rapidly growing
callus was rated for tissue injury symptoms from 0 to 4. The results obtained from this
study were synonymously effective as in seedling bioassay. It was also found from this
study that as much as 30% of rhizobacteria obtained from leafy spurge grown in Europe
and North America were inhibitory to this weed.
Differential effects are usually observed on crop plants due to application of
allelopathic bacteria which ensures the safety of non-target plants and selectivity of
bioherbicides. Host specificity is related to differential colonization and other interaction
mechanisms in the rhizosphere of different plant species (Kennedy et al., 2001; Wieland
et al., 2001; Weissman, 2002). Another possible reason of host-specificity may be
selective toxicity of phytotoxic metabolites of allelopathic bacteria for different plant
species (Piotrowska-Seget, 1995; Owen and Zdor, 2001). This trait of selective toxicity is
attributed to partial/suboptimal colonization of AB in non-host rhizosphere, change in the
functional bahaviour of AB in different plant spp., differential toxicity of phytotoxic
metabolites for different plant species (even cultivars) or a combination of these factors
(Owen and Zdor, 2001; Zeller et al., 2007). Functional characteristics of bacteria may
change in the rhizosphere of non-host species due to difference in the nature of root
exudates of different plant species. This may change the available amount of
substrates/precursors required for specific activities of AB (Begonia and Kremer, 1994;
Zeller et al., 2007). The outcome of such interactions of allelopathic bacteria with crop
plants may be beneficial, harmful or neutral for these plants or even cultivars (Kennedy et
al., 2001; Weissmann et al., 2003; Zdor et al., 2005). Gurley and Zdor (2005) applied two
strains of cyanogenic rhizobacteria (Pseudomonas putida ATH2-1RI/9 and Acidovorax
delafieldii ATH2-2RS/1) to suppress velvetleaf (Abutilon theophrasti) grown in corn
under field conditions using pesta formulation. Both of these strains colonized
rhizosphere of both plant species to the same extent but cyanide production was much
lesser in the rhizosphere of corn (1 and 14 mM/g root) than velvetleaf (53 and 68 mM/g
root). Resultantly, these two strains suppressed biomass production and lengths of roots
and shoots of velvetleaf but not corn. Previously, Owen and Zdor (2001) applied these
two strains to velvetleaf and corn under gnotobiotic conditions using autoclaved soil.
They observed that HCN produced in the rhizosphere of corn was nearly twice the
concentration produced in the rhizosphere of velvetleaf. The rhizospheric population of
these strains was also higher in corn than velvetleaf. The results of this study showed that
velvetleaf growth was suppressed from 40 to 80% over control but no growth reduction of
31
corn was observed. These studies reflect the differential sensitivity of plants to cyanide
production by rhizobacteria which was also supported by Piotrowska-seget (1995) and
Astrom (1991). Zeller et al. (2007) also reported strong host plant selectivity to
phytotoxins produced by rhizobacteria. They isolated eight cyanogenic strains of
rhizobacteria from four weed species and applied them to these weeds and two crops. The
interactions of these strains varied with bacterial strains in which strong negative effects
were observed on Echinochloa crus-galli. Plant growth promotion effects of allelopathic
bacteria usually observed may be due to solubilization of soil minerals, production of
plant growth promoting metabolites like auxins, siderophores, chitinases, increasing stress
tolerance in plants by ACC-deaminase activity, and inducing systemic disease resistance
(Zahir et al., 2004). In this case allelopathic bacteria may also be called plant growth
promoting rhizobacteria (PGPR). These characteristics of allelopathic bacteria offer an
opportunity to be screened out from the rhizosphere of plants as biocontrol agents of
weeds.

2.5. Integrated weed management

It is a technique of using multiple weed control approaches in an integrated

manner with an objective of avoiding crop losses and negative effects of different weed
control methods. The sole reliance on any of conventional weed control methods would
enhance its negative impacts on crop production and environment which have been
discussed above. So, the best approach for weed control is integrated approach which
does not let the side effects of any control method exceed above a permissible limit while
sustaining productivity of crops. Therefore, biological weed control techniques should be
integrated with existing weed control methods in almost all cropping patterns for gaining
their potential benefits in weed control and safety of environment. Inoculative biocontrol
approach is a time consuming, lesser host specific and relies on suitable environmental
and growth conditions of plants. These characteristics have restricted the use of this
technique in non-cropped lands only (Culliney, 2005). Use of inundative biocontrol
approach on the other hand is synonymous to herbicide use which provides seasonal
effects, may be applied along with other crop inputs, possess narrow host range ensuring
the safety of non-target species, and selective phytotoxicity (Owen and Zdor, 2001;
Hakansson, 2003; Kremer, 2005). This suggests that inundative biocontrol agents may be
conveniently applied in cropped areas to most effectively suppress weeds with reduced

32
reliance on existing destructive control methods. The adjustment of inundative biocontrol
agents can be made with reduced doses of herbicides initially and gradually minimizing
herbicide usage. Peng and Byer (2005) applied 25% of the recommended dose of
herbicide along with a pathogenic biocontrol agent of green foxtail. Complete control of
this weed was attained due to this synergy. They may also be used alongwith allelopathic
plants to increase the magnitude of biocontrol of weeds using their synergistic
associations. Multiple biocontrol agents may also be applied simultaneously to increase
the number of weed species to be controlled biologically (Charudattan, 2001). Different
biocontrol strategies (insects, pathogens and rhizobacteria) may also be integrated to
achieve successful biocontrol of weeds (Caesar, 2003). Cultural practices may also be
modified to enhance the survival and activity of natural antagonistic species of weeds
(Hatcher and Melander, 2003; Kremer, 2006). A small reduction in vigor of weedy plants
due to any control method may also increase the susceptibility of these plants to
biocontrol agents (Kremer, 2006). Therefore, it is suggested that biocontrol agents should
be used as an essential component of integrated weed management.

The plant species which release phytotoxic allelochemicals against certain weed
species can also be used to biologically control weeds. The residues of such allelopathic
plants may be incorporated into soil, these plants may be grown purposefully and the
phytotoxic allelochemicals may be extracted from the parts of such plants and applied to
weeds to suppress their growth (Jabran et al., 2015). The ultimate goal of weed control
techniques should not be to eliminate weedy plants but to avoid economic losses to crops,
and other harmful effects of plant pests on animals, man and his environment.

Conclusion

The ultimate goal of weed control techniques should not be to eliminate weedy
plants but to avoid economic losses to crops, and other harmful effects of plant pests on
animals, man and his environment. Biological weed control techniques should be adopted
in weed management to minimize the challenges posed by conventional weed control
methods to sustainability of agricultural resources, safety of environment and human
health. The biological approaches which may be investigated for weed control include
inoculative and inundative approaches and allelopathy. It is suggested that inoculative
approaches should be used for control of weeds grown in non-cropped areas where they
may be the single most effective technique due to large magnitude of problem weed, low

33
value of land and non-availability of other cost-intensive techniques. The successful
biocontrol agents from inundative biocontrol may be most effectively used in arable lands
when integrated with other control methods and cultural practices. It may be the single
most effective option when weeds grown in crops are resistant to applied herbicides, labor
for manual weeding is not available and cultural practice can not be applied due to certain
reasons e.g., narrower crop rows and topography of land etc. The plant species which
release phytotoxic allelochemicals against certain weed species can also be used to
biologically control weeds. The residues of such allelopathic plants may be incorporated
into soil, these plants may be grown purposefully and the phytotoxic allelochemicals may
be extracted from the parts of such plants and applied to weeds to suppress their growth.
The integration of these biocontrol approaches may increase the spectrum of weed control
which may further be integrated with reduced administration of conventional weed
control methods.

34
CHAPTER-III MATERIALS AND METHODS
Limitations of existing weed control methods necessitate the development of
alternative control methods using biological approaches. Allelopathic bacteria are
suggested to establish in the rhizosphere of weed species, produce phytotoxins and
suppress their germination and growth. It recognizes the role of these bacteria in
biocontrol of weeds. Therefore, allelopathic bacteria were isolated, screened and
characterized from the rhizosphere of wheat (Triticum aestivum L.) and notorious weeds
grown in wheat. Plant samples for this purpose were collected from fields chronically
infested with wild oat (Avena fatua L.), little seed canary grass (Phalaris minor), field
bindweed (Convolvulus arvensis), broad leaved dock (Rumex dentatus L.) and common
lambs’ quarters (Chenopodium album). Rhizobacteria from these plants were screened
and characterized for production of phytotoxins, inhibition of sensitive seedlings of
lettuce (Lectuca sativa L.), inhibition of major weeds of wheat and non-inhibitory effects
on wheat. The promising strains from these studies were applied to weeds grown in wheat
in potted soil under greenhouse conditions and naturally grown weeds in wheat under
field conditions. Data regarding de 0 14 50 0 1 441 44 559.75 Tm[(L )]24)ed74 70n tate the 4140 0 hizo

35
obtained by dipping roots in 100 mL volumetric Erlenmeyer flasks containing sterilized
distilled water and shaking for half an hour in orbital shaker (100 revolutions per minute).
Serial dilutions of this suspension were prepared in test tubes containing 9 mL sterilized
distilled water. Hundred microliters from each dilution were added onto Petri plates
containing King’s B agar media (King et al., 1954) using spreader aseptically. These Petri
plates were incubated at 28 ± 1 ºC for 48 hours. Fast growing colonies from these plates
were purified by repeated streaking on freshly prepared King’s B agar plates. This
procedure was repeated for all the plant species sampled for isolation of rhizobacteria and
393 strains were isolated and purified. All these strains were encoded and preserved
cryogenically in 40% glycerol at -20 ± 1 ºC.

3.2. Screening of rhizobacteria by cyanide production assay:

All the strains isolated from the rhizosphere of selected weed species and wheat
were tested for cyanide production following Bakker and Schipper (1987). Filter paper
pieces were cut to the size of underside of lid of Petri plates and sterilized. These filter
papers were soaked in 1% picric acid solution for overnight and dried on the bench of
laminar flow next day. Alongside quarter-strength King’s B agar media amended with
glycine at 5 g L-1 was prepared and poured in Petri plates. The dried filter papers were
fastened on the underside of the lid of Petri plates using 10% Na2CO3 solution. All the
strains were streaked on prepared plates of glycine amended KB agar media. Petri plate
lids containing filter paper pieces were placed on plates containing freshly streaked
strains of rhizobacteria. These plates were sealed with parafilm to avoid leakage of gas
and incubated at 28°C. Change in color of filter paper was visually observed after 4, 24
and 48 hours of incubation and compared with control plates containing no culture. The
intensity of color change from yellow to light brown, brown or reddish brown reflects
increasing concentration of cyanide produced by the rhizobacterial strains. Triplicate
plates were set up for each strain. Rhizobacterial strains were rated as non-cyanogenic,
and low, medium, high and very high cyanide producing ability based on no color change
of filer paper, and change in color of filter paper after 48, 36, 24 and 12 hours of
incubation, respectively.

3.3. Screening of rhizobacteria by E. coli antimetabolite assay:

The rhizobacterial strains which showed positive cyanide activity (89 of 393
strains) were selected for this test. This is an indicator technique for antimetabolite toxic

36
reaction towards E. coli by rhizobacterial isolates (Gasson, 1980). Non-pathogenic E. coli
strain K12 stock culture was obtained from Centre for Agricultural Biochemistry and
Biotechnology, University of Agriculture, Faisalabad. Fresh culture of E. coli was
obtained by streaking stock culture on LB agar media and incubating at 28°C for 48 hours
to conduct this test. Culture of E. coli was suspended by adding 0.01M MgSO4 and
rubbing the surface gently. This suspended culture was pipetted out into test tubes and
cell density was maintained at 108 cells mL-1 by measuring optical density (OD) at 600
nm (0.55-0.6 value) and adding 0.01M MgSO4 buffer. These cells were added on molten
King’s B agar media in Petri plates at the ratio of 1: 10 and temperature <40°C.
Cyanogenic strains of rhizobacteria were later spot inoculated at three equidistant points
on E. coli overlain Petri plates and incubated at 28°C for 48 hours. Triplicate plates were
set up for each strain. Presumed allelopathic bacteria produced clear zones devoid of E.
coli growth around the point of inoculation due to production of antimetabolites. The
reaction of test was recorded as plus or minus and diameter of clear zone was measured
for minus reactions using a ruler. The strains showing negative association with E. coli
and forming halo zones were tested again for consistency of results. The diameter of halo
zone produced by each strain was statistically analyzed following completely randomized
design (CRD).

3.4. Screening of rhizobacteria by lettuce (Lectuca sativa L.) seedling

bioassay:
The cyanogenic strains of rhizobacteria which showed antimetabolite activity
against E. coli (19 out of 89 strains) were further tested for production of phytotoxins and
consistency of results in phytotoxins sensitive plant species (lettuce) seedling bioassay.
Fresh culture of each selected strain was obtained by streaking on King’s B agar plates
incubated at 28°C for 48 hours. The culture of each strain was suspended in 0.01M
MgSO4 buffer by gently shaking and poured in test tubes. The cell density of culture of
all strains was maintained at 106 cells mL-1 by measuring optical density (OD) using
spectrophotometer (0.33 value). Lettuce seeds were surface sterilized by momentarily
dipping in 70% ethanol followed by treatment with 5% NaClO for 3 minutes and rinsing
with sterilized distilled water for 4-5 times (Abd-Alla et al., 2012). Water agar (1:100)
was prepared, autoclaved and poured in large Petri plates (15 cm diameter). Twenty pre-
germinated surface sterilized seeds of lettuce were placed on surface of water agar
equidistantly. Culture suspension of each strain was dispensed on each seed at the rate of

37
thirty microliters for each lettuce seed. Three replicate plates were set up for each isolate.
Control plates were prepared by adding 0.01M MgSO4 on seeds. These test plates were
placed in dark at ambient temperature for growth. After 4 days, seedlings were removed
from water agar and blotted with filter paper. Data regarding root and shoot lengths, and
biomass was recorded and statistically analyzed for significant differences as described
by Kremer (2013).

3.5. Screening of presumed allelopathic bacteria on target plants

seedlings:

Presumed allelopathic bacteria were obtained after screening in the above

described laboratory studies. All the rhizobacterial strains tested in lettuce seedling
bioassay (19 strains) were selected for screening on wheat (cultivar Punjab-2011) and
four economically important weeds of wheat (wild oat, little seed canary grass, broad
leaved dock and common lambs’ quarter) using almost similar experimental arrangement.
Culture of each selected strain was prepared by inoculating in sterile King’s B broth
media and incubating at 28°C in shaking incubator at 100 rpm for 48 hours. Bacterial
population of each strain was determined by measuring optical density (600 nm) and
maintained at 108 cells mL-1. The culture of each strain was mixed into molten sterile
water agar (1% agar in water) at the ratio 1:100 aseptically and placed on the bench of
laminar flow for solidification in Petri plates (15 cm diameter). Sterile King’s B media
was added into molten sterile water agar to prepare control plates. Mature seeds of each
selected weed species (wild oat, little seed canary grass, broad leaved dock and common
lambs’ quarter) were collected from the field and their viability was checked by placing
seeds within folds of moistened filter paper. The seeds of weeds and wheat were surface
sterilized using method as described earlier for lettuce seedling bioassay. Twenty surface
sterilized seeds of each weed species and wheat were placed on surface of inoculated
water agar using sterilized forceps. Thirty microliters culture of each strain was dispensed
to each seed in the respective plates and the same amount of sterile growth medium was
added onto seeds placed in control plates. Four replicate plates were set up for each
treatment and placed in dark at ambient temperature. The experiment was conducted at
separate times for convenience. Seedlings of weeds were removed from water agar after 7
days and blotted with filter paper for drying. Seedlings of wheat were removed from
water agar after 5 days and blotted for drying. Data regarding germination and seedling

38
growth of weed species and wheat were recorded and statistically analyzed for significant
differences.

3.6. Growth room studies:

Growth room studies were conducted to evaluate in vivo bioherbicidal activity of

promising strains of allelopathic bacteria under gnotobiotic conditions. Ten strains were
selected for growth room studies based on maximum inhibition of germination or growth
of one or more weed species without showing any harmful effects on wheat in agar
bioassay. Fresh culture of these strains was prepared in King’s B broth by incubating at
28 ⁰C in shaking incubator at 100 rpm for 48 hours. Bacterial population in culture media
was determined by measuring optical density (600 nm) and population was maintained at
108 cells mL-1. Three weed species were selected for growth room studies based on
response of isolated strains on suppression of these weed species and non-inhibitory
effects on wheat in agar bioassays. No promising strain was found in agar bioassay of
common lambs’ quarter for its inhibition in more rigorous conditions. Seeds of these
selected weed species (wild oat, little seed canary grass and broad leaved dock) and wheat
(cultivar Punjab-2011) were surface sterilized by using method as described earlier for
lettuce seedling bioassay. One hundred and two glass jars (13 × 6 cm each) were filled
with sand (350 g each) and autoclaved twice at 121⁰C for 20 minutes. Seeds of each
selected weed species were inoculated with the respective strains by dipping seeds in
culture for 20 minutes. These inoculated seeds were sown in the sand filled glass jars.
One milliliter culture of each strain was applied to its respective weed species and wheat
on sand surface in glass jars. The surface of glass jars was then covered with a thin layer
of sand to avoid evaporation from culture. For control, seeds were sown after dipping in
sterile growth medium and 1 mL of sterile growth medium was applied on sand surface
followed by thin layering of sand on surface. Three replicates were set up for each strain
and un-inoculated control. All the glass jars were placed in growth room having
fluorescent and incandescent lamps according to completely randomized design. Light
and dark periods were adjusted at 10 and 14 hours with temperature 21°C and 16°C,
respectively. Sterilized Hoagland’s nutrient solution (half strength) was applied to
growing seedlings for nutrients and water requirements of plants (Hoagland and Arnon,
1950). Seedlings of each plant species were gently removed from glass jars by adding
water after 25 days and blotted with filter paper. Data regarding germination and growth

39
parameters of weeds and wheat were recorded and statistically analyzed for significant
differences.

The entire screening from cyanide production assay to growth room studies was
repeated on these 10 strains to assess consistence and stability in their characteristics.
This was done for further selection of these strains to conduct pot and field trials.

3.7. Pot trials:

Five efficient strains of allelopathic bacteria were selected based on performance

in growth room studies for pot and field trials. The efficiency of these strains was
depicted by suppression of one or more weeds and non-inhibitory effects on wheat in
previous studies. Pot trials were conducted on three weed species (broad leaved dock,
wild oat and little seed canary grass) grown in wheat and wheat under weed free
conditions. Fresh culture of these strains was prepared in King’s B broth by incubating in
orbital shaking incubator at 28°C for 48 hours. Bacterial population in these cultures was
maintained at 108 cells mL-1 by measuring optical density at 600 nm using
spectrophotometer. Pots having diameter of 30 cm were filled with air dried and sieved
soil at 8 kg per pot. This soil was taken from the plough layer of field at Research area of
Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad,
Pakistan. The soil used in pots was characterized for physico-chemical characteristics i.e.,
texture, organic matter, pHs, electrical conductivity (ECe), saturation percentage (SP),
cation exchange capacity (CEC), total nitrogen, available phosphorus and extractable
potassium. The soil was classified as Typic Haplocambids according to Soil Survey Staff
(1999). Pots were fertilized with recommended doses of NPK at the rate of 120: 90: 60 kg
ha-1 using urea, diammonium phosphate and sulphate of potash fertilizers, respectively.
Viability of weed seeds was checked by germination test within folds of moistened filter
paper pieces. Forty viable seeds of each selected weed species were dipped in the culture
of each selected strain for 20 minutes and sown slightly below soil surface in their
respective pots. Eight seeds of wheat crop were also dipped in culture of respective
strains and sown in pots containing weed seeds. Thirty milliliters fresh culture of selected
bacterial strains was added onto soil surface of respective pots containing weed and crop
seeds which were later covered with a thin layer of sand. For control, seeds of weeds and
test crop were dipped in sterile King’s B broth and sown in potted soil. Later on, thirty
milliliters sterile King’s B broth was added onto soil surface and soil surface was covered
with thin layer of sand to avoid evaporation in control pots. A similar set up of pots was
40
arranged in which only crop was sown following the same inoculation method under
weed free conditions to study the effect of inoculation of allelopathic bacteria on growth
and yield of wheat under weed free conditions. Three sets of all these pots were arranged
to record data at three different growth stages of the crop (tillering, booting and
harvesting). All these pots were placed in the wire house of Institute of Soil and
Environmental Sciences, University of Agriculture, Faisalabad (31.438976° N and
73.069029° E). Good quality irrigation water was used to irrigate the pots as and when
required. At each growth stage, soil of pots was removed from roots by washing to get
intact plants having roots. The plants were then cut at the junction of root and shoot. Data
regarding density and growth parameters (lengths of roots and shoots, and biomass of
roots and shoots) of weeds, infested wheat and wheat grown under weed free conditions
was collected at the three growth stages of the crop to evaluate the potential benefits of
applying allelopathic bacteria as bioherbicide for weed control in wheat.

3.8. Field trials:

Three field trials were conducted at different sites to evaluate bioherbicidal

activity of allelopathic bacteria under natural conditions. The same five strains were
selected for field trials as in pot trials. Fields were selected based on their previous history
of weed infestations. Field trial-1 was conducted at Post-graduate Agriculture Research
Station, University of Agriculture, Faisalabad (31.391533° N and 73.014199° E). Field
trial-2 was conducted at Chak No. 75 RB Lohkay, Khurrianwala, District Faisalabad
(Photo 1) (31.483213° N and 73.329242° E). The third field trial was conducted at
research area of Institute of Soil and Environmental Sciences, University of Agriculture,
Faisalabad (31.438738° N and 73.070080° E). The soils of these fields were classified as
Typic Haplocambids according to Soil Survey Staff (1999). These trials were conducted
during Rabi 2014-15. The field area in all the experiments was divided into two halves;
one to study biocontrol of weeds grown in wheat and other to study non-inhibitory effects
on wheat under weed free conditions. Twenty one sub-plots were made (4 × 5 m each) in
the first half of field for seven treatments (weedy control, weed free control, strain 7O₀,
O₀10, T42, L9 and W9) in three blocks. Weeds were not allowed to grow in weed free
un-inoculated control treatment by manually pulling germinated weed seedlings every
week. For un-weeded un-inoculated control no weed seeds were added into these plots.
The other half of field area was divided into 18 sub-plots to apply these five strains in
three blocks according to randomized complete block design. Recommended doses of

41
Photo 1. Glimpses from field trial II at Chak No. 75 RB Lohkay, Faisalabad at tillering
stage (50 days after sowing).

42
NPK fertilizers at the rate of 120-90-60 kg ha-1 were applied using urea, diammonium
phosphate and sulphate of potash, respectively besides other cultural practices. Fresh
culture of selected strains was prepared in King’s B broth and population was maintained
at 108 cells mL-1. Culture of these strains was applied at 5 mL m-2 (at 5 × 108 cells m-2) in
sterilized water as carrier in the respective plots while sterile growth medium was applied
with water in control plots. Seeds of two weeds (wild oat and little seed canary grass)
were mixed in equal proportions at 60 seeds m-2 for each and manually broadcasted in
first half of field in field trial-3. Representative soil samples were collected from all the
fields by composite soil sampling technique. These samples were processed by air drying,
grinding and sieving for analyses of physico-chemical characteristics. Wheat was sown in
lines 20 cm apart in all the plots. Canal water was used to irrigate the crop as required.
Field trial-1 was naturally infested with broad leaved dock and common lambs’ quarter
while the density of other weeds was too low to be studied. Field trial-2 was naturally
infested with little seed canary grass while the density of other weeds was too low to be
studied. Data regarding density, growth and yield parameters of weeds and wheat was
collected at tillering, booting and harvesting of the crop. Measurements of weeds, infested
wheat and wheat under weed free conditions were taken from a quadrat (1 m2 area). Data
regarding physiological parameters of wheat and its associated weeds was collected at 60
DAS. Grain and straw samples of wheat were analyzed for NPK contents.

3.9. Physico-chemical characterization of soil:

Soil samples were taken from potted soil and field trials. These samples were
analyzed for various physical and chemical properties following standard protocols as
described below:

3.9.1. Determination of textural class

Soil textural class was determined following the method described by Moodie et
al. (1959). Forty gram soil was put in a 600 mL plastic beaker and 40 mL 2% sodium
hexametaphosphate (dispersing agent) was added. The mixture was left overnight and
then suspension was stirred by mechanical stirrer. The suspension was poured into 1 L
cylinder and volume was made up to mark with deionized water. After stirring the
suspension with plunger, Buoyoucos hydrometer readings were noted after intervals for
sand, silt and clay contents. Textural class was calculated using USDA online textural
class calculator (USDA, 2011).

43
Table 3.1. Physico-chemical characteristics of the soil used in pots

Characteristic Unit Value

Textural class -- Sandy clay loam

Saturation percentage % 30.2

ECe dS m-1 1.44

pHs -- 7.5

Organic matter % 0.89

Total nitrogen % 0.04

Available phosphorus mg kg-1 7.8

Extractable potassium mg kg-1 132

Table 3.2. Physico-chemical characteristics of the soil selected for field trials

Characteristic Unit Value

Field trial-1 Field trial-2 Field trial-3

Textural class -- Sandy clay Sandy Clay Sandy clay

loam Loam loam

Saturation % 32 35.4 30.7

percentage

ECe dS m-1 1.82 1.2 1.76

pHs -- 7.4 7.2 7.5

Organic matter % 0.65 0.9 0.85

Total nitrogen % 0.05 0.07 0.05

Available mg kg-1 7.6 8.3 7.5

phosphorus

Extractable mg kg-1 116 154 138

potassium

44
3.9.2. Saturation percentage

Two hundred gram air dried soil was saturated with deionized (DI) water and
weighed in a tarred china dish. The saturated paste was oven dried at 105°C till constant
weight. The saturation percentage was calculated following the formula (Method 27a,
U.S. Salinity Laboratory Staff, 1954).

3.9.3. Electrical conductivity of soil extract (ECe)

Water was extracted from saturated paste of soil through a ceramic filter using
vacuum pump. Electrical conductivity of the extract was determined by digital
conductivity meter (Jenway Conductivity meter Model 4070) (Method 3a and 4b, U.S.
Salinity Laboratory Staff, 1954).

3.9.4. pH of the saturated soil paste (pHs)

The pH of saturated soil paste was determined by using pH meter (Kent Eil 7015,
England) following Method 21a, U.S. Salinity Laboratory Staff (1954).

3.9.5. Cation exchange capacity (CEC)

Five grams of each soil sample were saturated with 1 N CH3COONa (pH 8.2),
washed thrice with ethanol and extracted with 1 N CH3COONH4. The concentration of
sodium was determined by using flame photometer (Jenway PFP-7, England) using Na+
filter in place. The instrument was first standardized with a series of standards of NaCl (0,
2, 4, 6, 8 and 10 mg L-1). The standard curve was used to determine sodium
concentration. The CEC was calculated by using the following formula;

Where,

3.9.6. Organic matter content

Soil organic matter was determined following Moodie et al. (1959). For that, one gram
soil was mixed with 10 mL potassium dichromate (1 N) solution in 500 mL conical flask.
Then 20 mL concentrated sulphuric acid was added, agitated and left for 30 minutes. One

45
hundred and fifty milliliters DI water and 25 mL freshly prepared 0.5 N ferrous sulphate
were added. The mixture was titrated against potassium permanganate (0.1 N) solution till
pink end point. Same procedure was followed to run a blank (without soil).

3.9.7. Total nitrogen

Soil was wet digested using concentrated sulphuric acid (Gunning and Hibbard’s
method) and ammonia was distilled into boric acid (4%) using Kjeldhal’s apparatus. The
receiver was back titrated against 0.01 N sulphuric acid.

3.9.8. Available phosphorus

Five grams of air dried soil was agitated with 100 mL sodium bicarbonate solution
(0.5 M) on a horizontal shaker for 30 minutes in 250 mL conical flask and filtered
through Whattman No. 42 filter paper. Five milliliters of filtrate and ascorbic acid (color
developing reagent) were mixed and volume was made with DI water. Absorbance was
recorded at 880 nm wavelength on spectrophotometer (ANA-720W, Tokyo Photo-electric
Company Limited, Japan). The concentration of P was determined by using standard
curve (Watanabe and Olsen, 1965).

3.9.9. Extractable potassium

Soil was extracted with neutral 1 N ammonium acetate and potassium was
measured by flame photometer (Jenway PFP-7, England). Standard solutions of KCl with
different concentrations were prepared. Readings of standard solutions were used to make
standard curve. The concentration of potassium from samples was derived by using the
standard curve (Method 11a, U.S. Salinity Laboratory Staff, 1954).

3.10. Plant analyses

At maturity of crop, shoot and grain samples of wheat were taken from pot and
field trials. These samples were ground, digested and analyzed for NPK contents.

3.10.1. Digestion

Ground samples (0.1 g) were transferred in 25 mL conical flasks. Two milliliters

concentrated sulphuric acid was added to these flasks and left overnight at room
temperature. One milliliter hydrogen peroxide (35%) was added in the flask and placed
on the hot plate at 350°C for 20 minutes. After that, the flasks were cooled, further 1 mL
hydrogen peroxide added and again placed on hot plate for 20 minutes. The procedure

46
was repeated until the material in the flask became colorless. After digestion, the
colorless material was diluted with DI water, filtered into 50 mL volumetric flask, volume
was made upto mark and stored for NPK determination (Wolf, 1982).

3.10.2. Nitrogen determination

Five milliliters of the digested sample solution was taken into a digestion tube and
fitted on the Kjeldhal ammonium distillation apparatus. Ten milliliters of NaOH (40%)
was added in the tube and started distillation. The distillate (ammonium) got absorbed by
the receiver (100 mL conical flask) containing boric acid (2%) and mixed indicator
(methyl red and bromocresol green mixed in ethanol). Distillation was stopped when
volume of receiver reached upto 50 mL. Then the receiver was removed from the
Kjeldhal apparatus and cooled for few minutes. After cooling, the contents were back
titrated with 0.01 N H2SO4 till the light pink end point.

3.10.3 Phosphorus determination

Total phosphorus in plant was determined by adding 10 mL Barton’s reagent in 5

mL sample aliquot and making the volume upto 50 mL with DI water. The mixture was
left for 30 minutes and the absorbance was measured at 410 nm wavelength on
spectrophotometer (ANA-720W, Tokyo Photo-electric Company Limited, Japan). Actual
concentration of P was derived following standard curve.

Barton’s reagent was prepared following Ashraf et al. (1992). Solution A was
made by dissolving 25 g ammonium molybdate in 400 mL DI water. Solution B was
developed by dissolving 1.25 g ammonium metavenadate in 300 mL boiling water and
after cooling, 250 mL HNO3 (conc.) was added. Solution A and B were mixed in 1 L
volumetric flask and volume was made upto the mark with DI water.

3.10.4. Potassium determination

Digested sample aliquot was fed to the flame photometer (Jenway PFP-7,
England) and its concentration was calculated by using a calibration curve developed by
known K concentrations ranging from 0 to 100 mg L-1.

3.11. Photosynthetic measurements of plants

Photosynthetic parameters of flag leaves of wheat and its associated weeds were
measured using CIRAS-3 portable photosynthesis system (PP Systems, USA) during 9:00
to 12:00 a.m. (at 1200-1400 µmol m-2 s-1 photon flux density) (Ben-Asher et al., 2006).

47
Data of physiological parameters e.g. assimilation rate (A), evapo-transpiration rate (E),
stomatal conductance (gs) and sub-stomatal conductance (Ci) were collected.

3.12. Formulae for calculation of loss factors

Percentage loss incurred in any growth, yield and physiological parameter of

wheat due to weed infestation in any treatment was calculated by using the following
formula;

Percentage recovery in loss of any growth, yield and physiological parameter of

infested wheat due to any treatment was calculated by using the following formula;

3.13. Characterization and identification of allelopathic bacteria

The selected strains of allelopathic bacteria were further characterized for various
plant growth promoting traits and finally identified following standard protocols as
described below:

3.13.1. Root colonization assay

Microbial root establishment capability was determined by modifying the method

from Simon et al. (1996). Seeds of wheat (cultivar Punjab-2011) and selected weed
species (wild oat, little seed canary grass and broad leaved dock) were surface sterilized
by momentarily dipping in 70% ethanol, followed by treatment with 5% sodium
hypochlorite for 3 minutes and subsequent washing with sterile distilled water 4-5 times
(Abd-Alla et al., 2012). Fresh culture of five efficient strains of allelopathic bacteria (7O₀,
O₀10, T42, L9 and W9) was prepared in King’s B broth media for 48 hours and bacterial
population was maintained at 108 cells mL-1. Surface sterilized seeds of weeds and wheat
were dipped in the culture of these strains for 20 minutes and sown in double autoclaved
sand filled (350 g) glass jars (13 × 6 cm). These jars were placed in growth room for 15
days and sterilized half strength Hoagland solution was added for water and nutrients
requirements of plants. At harvest, four roots from each treatment were selected and 1 cm
root tip was cut and transferred to test tube containing 0.9% sterilized saline buffer. The
tubes were shaken on the mechanical shaker for 30 minutes. Then sterilized King’s B
agar plates were inoculated with different dilutions of root extract with three repeats and

48
incubated at 28°C. After 48 hours, colonies were counted on digital colony counter and
expressed in cfu cm-1 of root.

3.13.2. Auxin production assay

Ability of selected strains of allelopathic bacteria to produce auxins in the

presence and absence of substrate (L-TRP) was determined using spectrophotometer
following the procedure described by Sarwar et al. (1992). Hundred milliliters conical
flasks containing 20 mL sterilized King’s B broth was taken and 5 mL of filter sterilized
(Whattman membrane filter of 0.2 µm) L-TRP (5%) solution was added to reach 1 g L-1
concentration. Fresh culture of each strain was prepared in conical flasks containing 60
mL King’s B broth and incubated at 28°C for 48 hours and 100 rpm in orbital shaking
incubator. Bacterial cells were harvested by centrifugation at 4°C and 4000g for 15
minutes. Culture of 0.5 OD was developed in sterilized DI water with harvested cells
using OD meter. Flasks containing broth media with or without L-TRP were inoculated
with 1 mL of inoculum. The flasks were incubated in orbital shaking incubator at 28°C
and 100 rpm for 48 hours. Un-inoculated control was also kept for comparison. After
incubation, the media was filtered through Whattman No. 2 filter paper. Three milliliters
of the filtrate were poured into test tubes and Salkowski reagent (2 mL) was added as
color developing reagent. After adding color developing reagent, tubes were left to stand
for 30 minutes at room temperature. The absorbance of the contents was measured at 535
nm on spectrophotometer. Auxins were measured in terms of IAA equivalents by using
the standard curve developed from known concentrations of IAA.

3.13.3 Phosphate solubilization assay

Solubilization of inorganic phosphate by selected strains was evaluated on

National Botanical Research Institute Phosphate Bromophenol Blue (NBRI-PBB) media
(Mehta and Nautiyal, 2001). Loopful culture of each strain was inoculated at four
equidistant points in the Petri plates. Three plates were set up for each strain. The
inoculated plates were incubated at 28°C for seven days. After incubation, colonies
showing clearing zone around them were considered positive for phosphate
solubilization.

3.13.4. Siderophores production assay

Petri plates containing chrome azurol S (CAS) agar media were prepared and four
wells were dug in each plate by using cork borer. Thirty microliters of each inoculum was

49
poured into the wells with three repeats. Control plates were also prepared by pouring
sterilized broth for comparison. After two days incubation at 28°C, change in medium
color was considered positive sign for siderophore production activity (Schwyn and
Neilands, 1987).

3.13.5. Exo-polysaccharides production assay

Fresh culture of each strain was prepared in King’s B broth in orbital shaking
incubator at 28°C and 100 rpm for 48 hours. After maintaining an OD of 0.5 by using OD
meter, loopful of inocula was placed at four points on Petri plates containing RCV-
glucose media (Ashraf et al., 2004). After 96 hours of incubation, plates were examined
for mucoid growth of bacterial colonies. Colonies showing mucoid growth were taken as
positive for exo-polysaccharide production activity.

3.13.6. Chitinase activity

The ability of isolates to produce chitinase was examined by the addition of chitin
in King’s B agar media (Chernin et al., 1998). Loopful of inocula having an OD of 0.5
was placed on four places of Petri plates containing King’s B agar amended with chitin.
After 96 hours of incubation at 28°C, halos around the colonies were observed. The
presence of halos was considered positive sign for chitinase activity.

3.13.7. Catalase activity

Presence of catalase was identified in bacteria following the method described by

MacFaddin (1980). Loopful of culture of each strain was spread on microscopic glass
slide and one drop of 35% hydrogen peroxide was dropped on the culture with a dropper.
Bubbling in response to hydrogen peroxide introduction was an indication for the
presence of catalase in the culture (Diane Hartman, Baylor University, Waco, TX).

3.13.8. Motility

Motility of allelopathic bacteria was tested following the method given by Prescot
et al. (1993). Triphenyltetrazolium chloride (TTC) was added into freshly prepared
semisolid King’s B agar media (0.4% agar was used) and autoclaved at 121°C for 20
minutes in test tubes. Allelopathic bacteria were inoculated in these test tubes by stabbing
in a line. These test tubes were incubated at 28°C for 48 hours. Spreading of growth
around the line of inoculation was taken as positive sign for motility of allelopathic

50
bacteria. The spreading of growth is evident due to formation of red colored pigment
(formazan) from the reduction of TTC by bacteria.

3.13.9. Urease activity

Allelopathic bacterial strains were tested for the presence of urease activity
following the method given by MacFaddin (2000). Fresh culture of the selected strains of
allelopathic bacteria was prepared in King’s B media. Urea broth was prepared by adding
filter sterilized urea in autoclaved ingredients of composition containing yeast extract, pH
buffers and phenol red. The bacterial strains were inoculated into urea broth and
incubated at 28°C for 24 hours. The urease activity was indicated by change in color of
media from orange yellow to pink color.

3.13.10. Oxidase activity

Oxidase was examined in freshly cultured isolates on King’s B agar plates (24
hours old). Loopful of bacterial culture of each strain was rubbed on filter papers
containing 1% Kovacs reagent (dried after dipping in the reagent). Change in paper color
from light blue to purple within 90 seconds was considered as positive for the oxidase
activity (Steel, 1961).

3.13.11. Identification of selected strains of allelopathic bacteria

Five efficient strains of allelopathic bacteria (7O₀, O₀10, T42, L9 and W9) were
identified through 16S rRNA gene sequencing technique described by Yanagi and
Yamasato (1993). DNA of bacteria were isolated and sent to Macrogen® for sequencing
based on 16S rRNA gene. The sequence of each isolate was then analyzed on
http://www.ncbi.nlm.nih.gov.

3.14. Statistical analyses

Standard statistical procedures were applied to analyze the data recorded from all
the studies (Steel et al. 1997). The completely randomized design (CRD) was followed
for all the laboratory studies and pot trials. Randomized complete block design (RCBD)
was adopted for field trials. Means of different parameters were compared to compute
significant differences using Duncan’s multiple range test (DMR) (Duncan, 1955).
Software used for the analyses was Statistix 8.1 (Analytical Software, USA).

51
CHAPTER-IV RESULTS

Growth of weedy plants in crops poses major challenge to crop production than
any other pest. It necessitates the use of available weed control methods to grow crops.
Conventionally weeds are controlled using manual, mechanical and chemical methods.
These control methods apart from being unsustainable have also given rise to serious
limitations which includes degradation of natural resources, and damages to wildlife,
animals and human health. Introduction of some non-conventional control method has
become the need of the day at least to be used along with conventional methods based on
biological approaches. This study was therefore, planned to evaluate rhizobacteria for
their ability to colonize weedy plants, produce phytotoxic metabolites, suppress their
germination and growth, and differential effects on wheat crop. Such rhizobacteria were
isolated from the rhizosphere of five economically important weeds grown in wheat
namely wild oat (Avena fatua L.), broad leaved dock (Rumex dentatus L.), common
lambs’ quarter (Chenopodium album), field bindweed (Convolvulus arvensis) and little
seed canary grass (Phalaris minor), and wheat (Triticum aestivum L.). A large collection
of rhizobacterial strains from these plant species was characterized for production of
phytotoxins, inhibition of sensitive bacterial and plant species, inhibition of major weeds
of wheat and non-inhibition of wheat in various laboratory studies. The promising strains
from these studies were evaluated for bioherbicidal activity in pot and field trials. The
results of all these studies are described below;
4.1. Cyanide production by rhizobacteria
All the isolated strains from the rhizosphere of weeds and wheat (393) were tested
for cyanide production (Photo 2). The results shown in Table 4.1 indicated that 77.3%
strains (304 out of 393) were characterized as non-cyanogenic, 8.4% strains (33 out of
393) showed low cyanide activity, 6.4% strains (25 out of 393) showed medium cyanide
activity, 5.09% strains (20 out of 393) showed high cyanide activity while 2.8% strains
(11 out of 393) showed very high cyanide production activity (Photo 3). These results
were partitioned into rhizosphere of origin (wheat, wild oat, little seed canary grass,
common lambs’ quarter, broad leaved dock and field bindweed). Out of 72 strains of
rhizobacteria of wheat, 54, 8, 6, 2 and 2 strains were non-cyanogenic, low cyanide
producing, medium cyanide producing, high cyanide producing and very high cyanide
producing which accounted for 75, 11.1, 8.33, 2.78 and 2.78% of total isolates,

52
respectively (Table 4.2). A total of 78 strains were isolated from the rhizosphere of broad
leaved dock among which 46, 3, 12, 14 and 3 strains showed non-cyanogenesis, low
cyanide activity, medium cyanide activity, high cyanide activity and very high cyanide
activity which accounted for 59, 3.8, 15.4, 18 and 3.8% of rhizobacteria of broad leaved
dock, respectively (Table 4.3). Eighty one strains of rhizobacteria were isolated from wild
oat among which 65, 8, 3, 0 and 5 strains were non-cyanogenic, low cyanide producing,
medium cyanide producing, high cyanide producing and very high cyanide producing
strains, respectively. The abundance of these rhizobacteria was recorded to be 80.2, 9.9,
3.7, 0 and 6.2% of rhizobacteria of wild oat, respectively (Table 4.4). Out of seventy eight
strains of rhizobacteria of little seed canarygrass, 72, 5, 0, 1 and 0 strains were non-
cyanogenic, low, medium, high and very high cyanide producing, and their abundance
was 92.3, 6.4, 0, 1.3 and 0% in the rhizosphere of little seed canary grass (Table 4.5).
Thirty eight strains were isolated from the rhizosphere of common lambs’ quarter among
which 29, 6, 2, 1 and 0 strains showed no cyanide activity, low, medium, high and very
high cyanide activity, respectively. The abundance of these groups of rhizobacteria was
76.3, 15.8, 5.3, 2.6 and 0% in the rhizosphere of common lambs’ quarter, respectively
(Table 4.6). The rhizosphere of field bindweed was processed for isolation of 46 strains of
rhizobacteria. The results showed that 38, 3, 2, 2 and 1 strains were non-cyanogenic, low,
medium, high and very high cyanide producing strains which accounted for 82.6, 6.5, 4.4,
4.4 and 2.2% in the rhizosphere of field bindweed, respectively (Table 4.7).
4.2. E. coli antimetabolite assay
All the cyanogenic strains of rhizobacteria (89) from wheat and its associated
weeds were subjected to E. coli antimetabolite assay. Both mutualistic (70 strains) and
antagonistic associations (19 strains) of selected rhizobacteria were observed when grown
on sensitive E. coli strain K12 overlain plates (Table 4.8). The share of these strains
accounted for 78.7 and 21.3% of cyanogenic rhizobacteria, respectively. The growth
inhibition of E. coli was evident from the formation of halo zone around spot of
inoculation of rhizobacterial strain. The diameter of halo zones was measured which
varied among different strains. Maximum diameter was recorded due to inoculation with
strain 7O₀ (13 mm) followed by strain W9, O₀10, T42, W28, T12, T23 and L9 up to 12.3,
12.1, 10.1, 9.6, 8.8, 8.2 and 7.2 mm, respectively (Photo 4).

53
Table 4.1. Summary of cyanide production by rhizobacteria of wheat and its
associated weeds
Rhizosphere of origin
Little
Broad Common Total
Category Wild seed Field
Wheat leaved lambs’ strain
oat canary bindweed
dock quarter s
grass
Non-
cyanogenic 54 46 65 72 38 29 304
strains (-)
Low
cyanide
8 3 8 5 3 6 33
activity
strains (+)
Medium
cyanide
6 12 3 0 2 2 25
activity
strains (++)
High
cyanide
activity 2 14 0 1 2 1 20
strains
(+++)
Very high
cyanide
activity 2 3 5 0 1 0 11
strains
(++++)
Total strains 72 78 81 78 46 38 393
- sign shows no change in color of filter paper placed underside the lid of petri plates in
cyanide test while +, ++, +++ and ++++ signs indicate change in the color of filter papers
placed underside the lid of petri plates observed after 48, 36, 24 and 12 hours of
incubation, respectively.

54
Table 4.2. Summary of cyanide production by rhizobacteria of wheat
Total
Category Strains
strains
T51, T54, T55, T59, T60, T61, T62, T63, T65,
T66, T67, T68, T69, T71, T72, T73, T74, T76,
T77, T78, T79, T80, T81, T82, T83, T85, W1,
Non-cyanogenic strains (-) W2, W3, W5, W6, W7, W8, W10, W11, W12, 54
W13, W14, W15, W16, W17, W19, W21, W22,
W23, W24, W25, W26, W30, W31, W33, W34,
W35, W36
Low cyanide activity (+) T64, T70, T84, T86, W4, W18, W27, W32 8
Medium cyanide activity
T52, T56, T57, T58, W28, W29 6
(++)
High cyanide activity (+++) T53, W20 2
Very high cyanide activity
T75, W9 2
(++++)
Total strains 72 72
- sign shows no change in color of filter paper placed underside the lid of petri plates in
cyanide test while +, ++, +++ and ++++ signs indicate change in the color of filter papers
placed underside the lid of petri plates observed after 48, 36, 24 and 12 hours of
incubation, respectively.

Photo 2. Pictorial view of cyanide production by rhizobacteria

55
Table 4.3. Summary of cyanide production by rhizobacteria of broad leaved dock
Total
Category Strains
strains
T4, T5, T7, T8, T9, T10, T17, T21, T25, T27,
T28, T29, T30, T32, T36, T37, T39, T41, T45,
Non-cyanogenic strains (-) T47, T48, T49, T50, P1, P2, P3, P4, P7, P8, P9, 46
P10, P11, P12, P13, P14, P15, P17, P18, P20,
P22, P23, P24, P25, P26, P27, P28
Low cyanide activity (+) P5, P16, P21 3
Medium cyanide activity T2, T3, T15, T16, T20, T33, T34, T35, T40,
12
(++) T44, P6, P19
T1, T6, T11, T12, T13, T14, T19, T22, T23,
High cyanide activity (+++) 14
T24, T26, T31, T43, T46
Very high cyanide activity
T18, T38, T42 3
(++++)
Total strains 78 78
- sign shows no change in color of filter paper placed underside the lid of petri plates in
cyanide test while +, ++, +++ and ++++ signs indicate change in the color of filter papers
placed underside the lid of petri plates observed after 48, 36, 24 and 12 hours of
incubation, respectively.

56
Table 4.4. Summary of cyanide production by rhizobacteria of wild oat
Total
Category Strains
strains
ERO-1, ERO-2, ERO-3, ERO-4, ERO-5, ERO-
6, ERO-7, ERO-10, 1O₀, 3O₀, 4O₀, 5O₀, 6O₀,
8O₀, 9O₀, 10O₀, 11O₀, 12O₀, 13O₀, 14O₀,
16O₀, 17O₀, 19O₀, 20O₀, 21O₀, 22O₀, 23O₀,
24O₀, O₀1, O₀2, O₀3, O₀4, O₀5, O₀6, O₀7, O₀8,
Non-cyanogenic strains (-) 65
O₀9, O₀11, O₀12, O₀13, O₀14, O₀15, O₀16,
O₀18, O₀20, O₀21, O₀22, O₀23, O₀24, O₀25,
O₀26, O₀27, O₀28, O₀29, O₀30, ESO-3, ESO-6,
ESO-7, ESO-9, ESO-10, ESO-12, ESO-13,
ESO-14, ESO-16, ESO-17
ERO-8, ERO-9, 15O₀, 18O₀, O₀17, O₀19, ESO-
Low cyanide activity (+) 8
5, ESO-15
Medium cyanide activity
ESO-1, ESO-2, ESO-4 3
(++)
High cyanide activity (+++) -- 0
Very high cyanide activity
2O₀, 7O₀, O₀10, ESO-8, ESO-11 5
(++++)
Total strains 81 81
- sign shows no change in color of filter paper placed underside the lid of petri plates in
cyanide test while +, ++, +++ and ++++ signs indicate change in the color of filter papers
placed underside the lid of petri plates observed after 48, 36, 24 and 12 hours of
incubation, respectively.

57
Table 4.5. Summary of cyanide production by rhizobacteria of little seed canary
grass
Total
Category Strains
strains
DK1, DK2, DK3, DK4, DK5, DK6, DK7, DK8,
DK9, DK10, DK11, DK12, DK13, DK14,
DK15, DK16, DK17, DK18, DK19, DK20,
DK21, DK22, DK23, DK24, DK25, DK26,
DK27, DK28, DK29, DK30, DK31, DK32,
Non-cyanogenic strains (-) 72
RD1, RD3, RD4, RD5, RD6, RD8, RD9, RD10,
RD11, D1, D2, D3, D4, D5, D6, D8, D9, D10,
D11, D13, D14, D15, D16, D17, D18, D19,
D20, D21, D24, D25, D26, D27, D28, D29,
D30, D31, D32, D33, D34, D35
Low cyanide activity (+) RD2, D7, D12, D22, D23 5
Medium cyanide activity
-- 0
(++)
High cyanide activity (+++) RD7 1
Very high cyanide activity
-- 0
(++++)
Total strains 78 78
- sign shows no change in color of filter paper placed underside the lid of petri plates in
cyanide test while +, ++, +++ and ++++ signs indicate change in the color of filter papers
placed underside the lid of petri plates observed after 48, 36, 24 and 12 hours of
incubation, respectively.

58
Table 4.6. Summary of cyanide production by rhizobacteria of common lambs’
quarter
Total
Category Strains
strains
B1, B3, B5, B6, B7, B8, B9, B12, B13, B14,
B16, B17, B21, B22, B23, B24, B25, B26, B27,
Non-cyanogenic strains (-) 29
B28, B29, B30, B31, B32, B33, B34, B35, B36,
B37
Low cyanide activity (+) B2, B4, B10, B15, B18, B20 6
Medium cyanide activity
B19, B38 2
(++)
High cyanide activity (+++) B11 1
Very high cyanide activity
-- 0
(++++)
Total strains 38 38
- sign shows no change in color of filter paper placed underside the lid of petri plates in
cyanide test while +, ++, +++ and ++++ signs indicate change in the color of filter papers
placed underside the lid of petri plates observed after 48, 36, 24 and 12 hours of
incubation, respectively.

Photo 3. Pictorial view of cyanide production at various levels by rhizobacteria

59
Table 4.7. Summary of cyanide production by rhizobacteria of field bindweed
Total
Category Strains
strains
LDM1, LDM3, LDM4, LDM5, LDM6, LDM7,
LDM8, LDM9, LDM10, LDM11, LDM12,
Non-cyanogenic strains LDM14, LDM15, LDM16, LDM17, L1, L2,
38
(-) L3, L5, L7, L10, L11, L12, L14, L15, L16,
L17, L18, L19, L20, L21, L22, L23, L24, L26,
L27, L28, L29
Low cyanide activity (+) LDM2, LDM13, L4 3
Medium cyanide activity
L8, L13 2
(++)
High cyanide activity
L6, L25 2
(+++)
Very high cyanide
L9 1
activity (++++)
Total strains 46 46
- sign shows no change in color of filter paper placed underside the lid of petri plates in
cyanide test while +, ++, +++ and ++++ signs indicate change in the color of filter papers
placed underside the lid of petri plates observed after 48, 36, 24 and 12 hours of
incubation, respectively.

60
Table 4.8. Inhibition of sensitive E. coli strain K12 by cyanogenic rhizobacteria in E.
coli antimetabolite assay
Rhizosphere of Mutualistic strains of Antagonistic strains of Total
origin cyanogenic rhizobacteria cyanogenic rhizobacteria strains
T52, T53, T56, T57, T58,
T75 (-4.7 h-j), W9 (-12.3
T64, T70, T84, T86, W4,
Wheat ab), 18
W18, W20, W27, W29,
W28 (-9.6 cd)
W32
T12 (-8.8 de), T18 (-5.5
T1, T2, T3, T6, T11, T13,
g),
T14, T15, T16, T20, T22,
T19 (-4 j), T23 (-8.2 e),
Broad leaved dock T26, T33, T34, T35, T40, 32
T24 (-5.3 gh), T31 (-2.5
T43, T44, T46, P5, P6, P16,
k),
P19, P21
T38 (-5.6 g), T42 (-10.1 c)
15O₀, 18O₀, O₀17, O₀19,
2O₀ (-4.4 ij), 7O₀ (-13 a),
ERO-8, ERO-9, ESO-1,
Wild oat O₀10 (-12.1 b), ESO-8 (-5 16
ESO-2, ESO-4, ESO-5,
g-i), ESO-11 (-5.1 gh),
ESO-15
Little seed D7, D12, D22, D23, RD2,
6
canarygrass RD7,
Common lambs’ B2, B4, B10, B15, B18,
B11 (-3.3 jk) 9
quarter B19, B20, B38
LDM2, LDM13, L4, L8,
Field bindweed L6 (-5 g-i), L9 (-7.2 f) 8
L13, L25,
Total strains 70 19 89
Values written within parenthesis indicate the average diameter (in mm) of halo zone
around the spot of inoculation of rhizobacterial cyanogenic strains. The values of
diameter sharing same letter(s) are statistically non-significant with each other.

61
 T6 ESO-11 W9

T75 L9 7O₀
Photo 4. Pictorial view of E. coli antimetabolite assay

62
4.3. Lettuce seedling bioassay
Nineteen strains of cyanogenic bacteria which inhibited growth of E. coli were
selected for lettuce seedling bioassay on water agar plates. Data in Table 4.9 showed
effect of inoculation of selected strains on seedling growth of lettuce in which variable
response was observed due to different strains in different parameters of lettuce viz root
length, shoot length, fresh biomass and dry matter. Eight strains (T12, T18, T31, T38,
O₀10, L6, W9 and W28) significantly reduced root length of lettuce over un-inoculated
control. Maximum reduction in root length of lettuce was caused by strain W28 (36%
lesser than un-inoculated control) followed by strain W9, O₀10, T18, T38, T12, T31 and
L6 up to 34.3, 33.1, 25.4, 19.9, 19.7, 14.1 and 11.8%, respectively. Inoculation with strain
T75, WSO-8, ESO-11 and B11 did not differ significantly from un-inoculated control
regarding increase or decrease in root length of lettuce. Inoculation of lettuce with seven
strains (7O₀, L9, T19, T42, 2O₀, T23 and T24) significantly increased root length of
lettuce up to 61.4, 57.7, 39.9, 25.0, 22.0, 19.8 and 16.7% as compared to un-inoculated
control, respectively. Shoot length of lettuce was significantly reduced due to inoculation
with strain W28, W9, O₀10, T12, T18 and T38 up to 24.3, 22.6, 20.7, 20.5, 17.3 and
16.0% over un-inoculated control, respectively. However, inoculation of lettuce with
strain 7O₀, L9, T19, T23, 2O, T24, T42, ESO-8 and B11 significantly increased the shoot
length of lettuce up to 43.4, 42.7, 34.2, 29.8, 28.5, 26.2, 15.2, 10.4 and 9.0% over un-
inoculated control, respectively. Slight increase or decrease in shoot length was caused by
inoculation with strain T31, T75, ESO-11 and L6 which did not differ significantly from
un-inoculated control. Data regarding fresh biomass showed that inoculation of lettuce
with six strains (W9, W28, O₀10, T12, T18 and T38) significantly reduced the fresh
biomass of lettuce seedlings up to 25.7, 24.3, 23, 23, 18.9 and 17.6% as compared to un-
inoculated control, respectively. Inoculation of lettuce with strain T19, T23, T24, 2O₀,
7O₀ and L9 significantly increased the fresh biomass of lettuce seedlings up to 29.7, 28.4,
25.7, 27, 41.9 and 43.2% over un-inoculated control, respectively. Fresh biomass of
lettuce seedlings inoculated with strain T42, ESO-8, ESO-11, B11, T31, T75 and L6
remained statistically non-significant with un-inoculated control. Dry matter yield of
lettuce seedlings was significantly reduced due to inoculation with strain W9, W28, T12,
O₀10, T18, T38 and T31 up to 30.1, 26, 24.4, 22.7, 18.8, 18.3 and 14.2% as compared to
un-inoculated control, respectively. Inoculation of lettuce with strain L6, ESO-8, ESO-11,
B11 and T75 caused a non-significant

63
Table 4.9. Effect of cyanogenic E. coli inhibiting rhizobacteria on lettuce seedlings in
water agar bioassay
Root length Shoot Length Fresh Dry matter
Treatments
(cm) (cm) biomass (g) (mg)
Un-inoculated 51.49 fg
5.08 de 4.01 fg 0.25 de
control
38.94 ij
T12 4.07 gh 3.19 i 0.19 fg

T18 3.79 hi 3.32 i 0.2 fg 41.81 h-j

T19 7.1 b 5.39 bc 0.32 ab 67.13 ab

T23 6.08 c 5.22 c 0.32 a-c 61.7 b-d

59.57 c-e
T24 5.92 c 5.07 c 0.31 a-c

T31 4.36 fg 3.68 gh 0.22 d-g 44.2 hi

T38 4.06 gh 3.37 hi 0.203 fg 42.07 h-j

T42 6.35 c 4.63 d 0.27 b-d 58.42 de

50.7 fg
T75 4.8 ef 4.02 f 0.24 d-f

6.19 c 5.16 c 0.31 a-c 65.26 bc

2O₀

8.19 a 5.76 a 0.35 a 72.19 a

7O₀

3.4 ij 3.18 i 0.19 fg 39.78 h-j

O₀10
55.0 ef
ESO-8 5.37 d 4.43 d 0.27 b-d

ESO-11 5.03 de 4.06 ef 0.25 de 52.0 fg

L6 4.48 fg 3.79 fg 0.22 d-g 45.79 gh

L9 8.01 a 5.73 ab 0.35 a 72.88 a

54.53 ef
B11 5.26 de 4.38 de 0.26 cd

W9 3.34 ij 3.11 i 0.18 g 35.99 j

W28 3.231 j 3.04 i 0.19 fg 38.08 ij

LSD 0.517 0.345 0.047 6.42

Table presents data of 4 days old seedlings of lettuce. Values sharing same letter(s) do not
differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test.

64
increase or decrease in dry matter of lettuce as compared to un-inoculated control.
However, dry matter of lettuce seedlings was significantly increased due to inoculation
with strain L9, 7O₀, T19, 2O₀, T23, T24 and T42 up to 41.5, 40.2, 30.4, 26.7, 19.8, 15.7
and 13.5% over un-inoculated control, respectively.
4.4. Effects of presumed allelopathic bacteria on weed seedlings in agar bioassays

All the strains tested in lettuce seedling bioassay were selected to study their
effects on inhibition of weed seedlings in agar plate bioassays. These strains were applied
to four weed species (broad leaved dock, wild oat, little seed canarygrass and common
lambs’ quarter) and their results are described below;

4.4.1. Agar bioassay on broad leaved dock (Rumex dentatus L.)

Data regarding germination and seedling growth parameters of broad leaved dock
in agar plate bioassay is presented in Table 4.10.
4.4.1.1. Germination (%)
The results showed that inoculation of broad leaved dock with 10 strains of
presumed allelopathic bacteria (T19, L6, T38, O₀10, ESO-11, W28, 7O₀, L9, T42 and
W9) significantly reduced germination of seeds over un-inoculated control. Maximum
inhibition of germination of broad leaved dock seeds was caused by inoculation with
strain W9 (64.4% lesser than un-inoculated control) followed by strain T42, L9, 7O₀,
W28, ESO-11, O₀10, T38, L6 and T19 (55.6, 44.4, 44.4, 42.2, 35.6, 31.1, 26.7, 24.4 and
15.6% lesser than un-inoculated control, respectively). The reduction or improvement in
germination of broad leaved dock due to inoculation with strain T23, T75, T24, ESO-8,
T18, B11, 2O₀ and T12 did not differ significantly from un-inoculated control. However,
inoculation of broad leaved dock seeds with T31 significantly increased its germination
over un-inoculated control which was 15.6% higher than un-inoculated control.
4.4.1.2. Root length (cm)
Data regarding root length showed that inoculation of broad leaved dock with 10
of the selected strains (W9, W28, T42, L9, 7O₀, O₀10, ESO-11, T38, L6 and T19
significantly inhibited its root length up to 61.8, 59.9, 50.9, 46.7, 44.3, 38.4, 28.2, 24,
21.4 and 15.4% as compared to un-inoculated control, respectively. Inoculation with
strain B11, 2O, T75, T23, ESO-8, T24, T12 and T18 caused a small non-significant
change in root length of broad leaved dock over un-inoculated control. However,

65
Table 4.10. Effect of presumed allelopathic bacteria on germination and seedling
growth of broad leaved dock in agar bioassay
Germination Root length Shoot length Fresh Dry
Treatment biomass
(%) (cm) (cm) matter (g)
(g)
Un-inoculated
75.0 bc 3.52 b 6.06 bc 1.5 bc 0.31 a-c
control
T12 73.4 bc 3.48 b 5.76 cd 1.37 cd 0.29 a-d
T18 80.0 ab 3.5 b 5.81 b-d 1.49 bc 0.303 a-c
T19 63.4 de 2.98 cd 5.04 e 1.26 de 0.25 c-e
T23 68.4 cd 3.33 bc 5.54 d 1.37 cd 0.28 b-d
T24 71.6 b-d 3.46 b 6.08 bc 1.46 bc 0.297 a-d
T31 86.6 a 4.1 a 6.69 a 1.75 a 0.35 a
T38 55.0 ef 2.68 de 4.45 f 0.99 fg 0.23 d-f
T42 33.4 ij 1.73 hi 3.12 i 0.6 hi 0.13 hi
T75 70.0 cd 3.33 bc 5.8 b-d 1.35 cd 0.28 b-d
2O₀ 73.4 bc 3.61 b 6.14 b 1.5 bc 0.303 a-c
7O₀ 41.6 hi 1.96 gh 3.66 gh 0.79 gh 0.17 f-h
O₀10 51.6 fg 2.17 fg 3.94 g 1.03 f 0.20 e-g
ESO-8 73.4 bc 3.41 b 6.06 bc 1.4 cd 0.29 a-d
ESO-11 48.4 f-h 2.53 ef 5.67 d 1.05 f 0.20 e-g
L6 56.6 ef 2.77 de 4.75 ef 1.12 ef 0.24 c-e
L9 41.6 hi 1.88 gh 3.47 hi 0.75 h 0.16 g-i
B11 76.6 bc 3.65 b 6.07 bc 1.63 ab 0.32 ab
W9 26.6 j 1.34 i 2.67 j 0.44 i 0.10 i
W28 43.4 gh 1.41 i 3.45 hi 0.63 hi 0.14 g-i
LSD 9.82 0.429 0.359 0.205 0.068
Table presents data of 7 days old seedlings. Values sharing same letter(s) do not differ
significantly from each other at p <0.05 according to Duncan’s Multiple Range Test.

66
inoculation of broad leaved dock with strain T31 significantly increased its root length
(16.5%) over un-inoculated control.
4.4.1.3. Shoot length (cm)
Results indicated that shoot length of broad leaved dock was significantly reduced
due to inoculation with strain ESO-11, T23, T19, L6, T38, O₀10, 7O₀, L9, W28, T42 and
W9 which varied from 6.4 to 55.9% lesser than un-inoculated control. Maximum
reduction in shoot length was caused by inoculation with strain W9 (55.9% lesser than
un-inoculated control) followed by strain T42, W28, L9, 7O₀, O₀10, T38, L6, T19, T23
and ESO-11 (48.5, 43, 42.8, 39.6, 35, 26.5, 21.5, 16.9, 8.5 and 6.4% lesser than un-
inoculated control, respectively). Slight increase and decrease in shoot length was
observed due to inoculation with strain strain T12, T75, T18, ESO-8, B11, T24 and 2O₀
which did not differ significantly from un-inoculated control. However, inoculation of
broad leaved dock with strain T31 significantly increased shoot length over un-inoculated
control.
4.4.1.4. Fresh biomass (g)
Inoculation of broad leaved dock with strain W9, T42, W28, L9, 7O₀, T38, O₀10,
ESO-11, L6 and T19 significantly reduced its fresh biomass which varied from 16.2 to
70.7% lesser than un-inoculated control. Maximum inhibition of biomass was caused due
to inoculation with strain W9 (70.7% lesser than un-inoculated control) followed by strain
T42, W28, L9, 7O₀, T38, O₀10, ESO-11, L6 and T19. Inoculation of dock with strain
T75, T12, T23, ESO-8, T24, T18, 2O₀ and B11 remained statistically non-significant with
un-inoculated control regarding their effect on fresh biomass of broad leaved dock.
However, fresh biomass of broad leaved dock was significantly increased up to 16.4%
due to inoculation with strain T31 over un-inoculated control.
4.4.1.5. Dry matter (g)
Inoculation with strain T38, T42, 7O₀, O10, ESO-11, L9, W9 and W28
significantly reduced the dry matter from 23.1 to 68.1% than un-inoculated control,
respectively. Maximum inhibition of dry matter was caused by strain W9 followed by
strain T42, W28, L9, 7O₀, ESO-11, O₀10 and T38. Inoculation with other strains (L6,
T19, T23, T75, ESO-8, T12, T24, T18, 2O₀, B11 and T31) caused non-significant effect
on dry matter than control.

67
4.4.2. Agar bioassay on wild oat (Avena fatua L.)

The selected strains of presumed allelopathic bacteria were applied to wild oat in
agar plate bioassay to evaluate their effects on its germination and seedling growth. The
results of various parameters of this study are described below;
4.4.2.1. Germination (%)
Data regarding germination of wild oat is presented in Table 4.11. Inoculation
with 12 of the selected strains (L9, T42, W9, W28, O₀10, 7O₀, ESO-11, 2O₀, T18, ESO-
8, T75 and T12) significantly reduced germination of wild oat up to 70.9, 56.4, 43.6, 40,
38.2, 32.7, 25.4, 25.4, 20, 18.2, 18.2 and 14.5% over un-inoculated control, respectively.
Inoculation of wild oat with strain T23, T24, B11, T31 and T38 showed slight decrease
and increase in germination that was statistically non-significant with un-inoculated
control. However, inoculation of wild oat with L6 and T19 significantly increased the
germination of wild oat up to 12.7 and 14.5%, respectively over un-inoculated control.
4.4.2.2. Root length (cm)
Effect of selected strains on root length of wild oat is shown in Table 4.11. The
results showed that root length was significantly reduced due to inoculation with strain
T24, ESO-8, 2O₀, W28, 7O₀, O₀10, W9, T42 and L9 over un-inoculated control.
Maximum reduction in root length was recorded due to inoculation with strain L9 which
was 60.2% lesser than un-inoculated control. Inoculation of wild oat with strain T42, W9,
O₀10, 7O₀, W28, 2O₀, ESO-8 and T24 reduced root length up to 54.3, 43, 40, 37.2, 28.3,
24, 19.4 and 11.9% as compared to un-inoculated control, respectively. Inoculation of
T19 significantly increased root length of wild oat up to 13.3% as compared to un-
inoculated control while other strains remained statistically at par with un-inoculated
control.
4.4.2.3. Shoot length (cm)
Data regarding shoot length of wild oat as influenced by inoculation with selected
strains is presented in Table 4.11. The data indicated that inoculation with strain L9, T42,
W9, O₀10, 7O₀, W28, 2O₀ and ESO-8 significantly reduced the shoot length of wild oat
up to 47, 42.9, 35.1, 34.2, 28.6, 22.1, 19.9 and 13.7% as compared to un-inoculated
control, respectively. Inoculation with strain T24, T75, T38, T12, T23, T31, T18, B11, L6
and ESO-11 remained statistically at par with un-inoculated control regarding their effect
on shoot length of wild oat. Inoculation of wild oat with strain T19 significantly increased
shoot length of wild oat which was 11.8% higher than un-inoculated control.

68
Table 4.11. Effect of presumed allelopathic bacteria on germination and seedling
growth of wild oat
Germination Root length Shoot Fresh Dry
Treatment
(%) (cm) length (cm) biomass (g) matter (g)
Control 73.33 bc 6.00 b-d 7.49 bc 1.98 bc 0.32 b-d
T12 62.70 d-f 5.60 c-e 7.55 a-c 1.82 c-e 0.29 b-d
T18 58.70 fg 5.52 de 7.27 b-d 1.72 de 0.277 cd
T19 84.00 a 6.79 a 8.38 a 2.20 a 0.41 a
T23 66.73 c-e 6.16 b 7.59 a-c 1.93 b-d 0.30 b-d
T24 68.02 cd 5.28 ef 6.76 c-e 1.85 c-e 0.29 b-d
T31 73.33 bc 5.75 b-e 7.21 b-d 2.08 ab 0.32 b-d
T38 77.33 ab 5.96 b-d 7.55 a-c 2.01 a-c 0.33 bc
T42 32.00 k 2.74 j 4.28 ij 0.48 h 0.11 hi
T75 60.00 e-g 5.88 b-d 7.17 b-d 1.74 de 0.28 b-d
2O₀ 54.70 gh 4.55 g 6.00 e-g 1.43 f 0.20 ef
7O₀ 49.32 hi 3.77 hi 5.35 gh 1.07 g 0.16 f-h
O₀10 45.33 ij 3.6 i 4.93 hi 1.02 g 0.15 f-h
ESO-8 60.00 e-g 4.83 fg 6.46 d-f 1.48 f 0.20 fg
ESO-11 54.70 gh 5.56 de 7.31 b-d 1.65 ef 0.26 de
L6 82.70 a 6.13 bc 7.62 ab 2.13 ab 0.35 ab
L9 21.3 l 2.39 j 3.97 j 0.27 i 0.06 i
B11 70.70 bc 6.00 b-d 7.61 a-c 1.98 bc 0.32 b-d
W9 41.33 j 3.42 i 4.86 hi 0.90 g 0.13 gh
W28 44.00 ij 4.33 gh 5.83 fg 0.99 g 0.17 f-h
LSD 7.32 0.545 0.857 0.214 0.064
Table presents data of 7 days old seedlings. Values sharing same letter(s) do not differ
significantly from each other at p <0.05 according to Duncan’s Multiple Range Test

69
4.4.2.4. Fresh biomass (g)
Fresh biomass of wild oat was significantly reduced due to inoculation with 11 strains
(L9, T75, T18, ESO-11, ESO-8, 2O, 7O₀, O₀10, W28, W9 and T42) up to 86.6, 12, 13.3,
17, 25.2, 27.7, 45.9, 48.7, 50.3 and 54.6% over un-inoculated control, respectively. A
non-significant increase or decrease in fresh biomass was observed due to inoculation
with strain L6, T38, T12, T24, T23, B11 and T31 over un-inoculated control. Fresh
biomass of wild oat was significantly increased by 10.9% over un-inoculated control due
to inoculation with T19.
4.4.2.5. Dry matter (g)
Inoculation with strain 2O₀, 7O₀, O₀10, ESO-8, L9, W9 and W28 significantly
reduced the dry matter of wild oat up to 37.5, 49.0, 52.1, 38.5, 80.2, 58.3, 58.3 and 47.9%
than un-inoculated control, respectively. Inoculation with strain T19 significantly
increased the dry matter up to 28.1% than un-inoculated control. Inoculation with other
strains (T12, T18, T23, T24, T31, T38, T75, B11, L6 and ESO-11) caused non-significant
effect on dry matter than control.

4.4.3. Agar bioassay on little seed canary grass (Phalaris minor)

Presumed allelopathic bacteria were applied to little seed canary grass to evaluate
their impact on its germination and seedling growth in agar plate bioassay (Photo 5). Data
regarding different parameters of germination and seedling growth are presented in Table
4.12 and are described below;
4.4.3.1. Germination (%)
Data given in Table 4.12 showed that inoculation of little seed canary grass with
10 strains (O₀10, 7O₀, ESO-11, W28, L9, T75, T42, T12, W9 and T18) significantly
reduced its germination up to 52.7, 41.8, 34.5, 32.7, 32.7, 27.3, 25.4, 25.4, 20 and 18.2%
over un-inoculated control, respectively. The effectiveness of other strains (2O, ESO-8,
T23, T24, B11, T38, T31, L6 and T19) in suppression of germination of little seed canary
grass varied from -12.7% to +10.9% as compared to un-inoculated control but they all did
not differ significantly from un-inoculated control.
4.4.3.2. Root length (cm)
Results indicated that root length of little seed canary grass was significantly reduced up
to 63.6, 60.9, 49.1, 48.3, 44.8, 37, 28.9, 24.2, 20, 10.8 and 10.5% as compared to un-
inoculated control due to inoculation with strain W28, of O₀10, T75, 7O₀, L9, T42, W9,

70
Table 4.12. Effect of presumed allelopathic bacteria on germination and seedling
growth of little seed canary grass
Germination Root length Shoot length Fresh Dry
Treatments
(%) (cm) (cm) biomass (g) matter (g)
Control 73.3 ab 4.59 bc 5.62 b 1.6 a-c 0.283 bc
T12 54.7 d-g 4.11 de 5.61 b 1.593 a-c 0.287 bc
T18 60.0 c-f 3.68 ef 5.64 b 1.573 a-c 0.267 b-d
T19 81.3 a 5.07 a 6.16 a 1.763 a 0.35 a
T23 66.7 bc 4.64 ab 5.6 bc 1.593 a-c 0.26 b-d
T24 68.0 bc 4.28 b-d 5.07 d 1.487 a-c 0.237 c-e
T31 74.7 ab 4.32 b-d 5.46 b-d 1.67 a-c 0.287 bc
T38 74.7 ab 4.58 bc 5.49 b-d 1.603 a-c 0.277 bc
T42 54.7 d-g 2.89 gh 4.03 g 0.88 e-g 0.153 g-i
T75 53.3 e-h 2.34 i 4.1 fg 0.953 ef 0.175 f-h
2O₀ 64.0 b-e 4.1 de 5.05 de 1.383 b-d 0.223 d-f
7O₀ 42.7 hi 2.37 i 3.42 h 0.583 gh 0.100 j
O₀10 34.7 i 1.8 j 2.8 i 0.34 h 0.087 j
ESO-8 65.3 b-d 4.16 cd 5.1 cd 1.427 b-d 0.237 c-e
ESO-11 48.0 gh 3.48 f 5.0 de 1.347 cd 0.237 c-e
L6 80.0 a 4.57 bc 5.75 ab 1.69 ab 0.293 b
L9 49.3 f-h 2.54 hi 3.61 gh 0.737 fg 0.123 ij
B11 74.7 ab 4.52 b-d 5.79 ab 1.627 a-c 0.257 b-d
W9 58.7 c-g 3.26 fg 4.54 ef 1.15 de 0.197 e-g
W28 49.3 f-h 1.67 j 3.14 hi 0.737 fg 0.137 h-j
LSD 11.432 0.445 0.512 0.323 0.0507
Table presents data of 7 days old seedlings. Values sharing same letter(s) do not differ
significantly from each other at p <0.05 according to Duncan’s Multiple Range Test

71
 W28 7O₀

T42 Control

Photo 5. Pictorial view of agar bioassay on little seed canary grass

72
ESO-11, T18, 2O₀ and T12, respectively (Table 4.12). Strains ESO-8, T24, T31, B11, L6
and T38 also reduced root length as compared to un-inoculated control but they were
statistically at par with the control. Strain T23 increased root length that differed non-
significantly from un-inoculated control. However, inoculation of little seed canary grass
with strain T19 significantly increased root length of little seed canary grass up to 10.4%
over un-inoculated control.
4.4.3.3. Shoot length (cm)
The effect of selected strains of presumed allelopathic bacteria on shoot length of
little seed canary grass is shown in Table 4.12. Data indicated that inoculation of little
seed canary grass with 11 of the selected strains (O₀10, W28, 7O₀, T31, T42, T75, W9,
ESO-11, 2O, T24, and ESO-8) significantly reduced its shoot length up to 50.2, 44, 39.1,
27, 28.2, 27, 19.1, 11, 10, and 9.3% over un-inoculated control, respectively. A non-
significant reduction in shoot length due to inoculation with strain T31, T38, T23 and T12
was observed over un-inoculated control. Inoculation with strain T18, L6 and B11 caused
non-significant increase in shoot length over un-inoculated control. However, inoculation
of little seed canary grass with strain T19 significantly increased shoot length up to 9.6%
over un-inoculated control.
4.4.3.4. Fresh biomass (g)
Inoculation with strain O₀10, W9, T75, T42, W28, L9 and 7O₀ significantly
reduced fresh biomass of seedlings up to 78.8, 28.1, 40.4, 45.2, 54, 54 and 63.5% as
compared to un-inoculated control, respectively (Table 4.12). Fresh biomass of little seed
canary grass was also reduced non-significantly due to inoculation with strain ESO-11,
2O, ESO-8, T24, T18, T23 and T12 over un-inoculated control. On the other hand, strain
T38, B11, T31, L6 and T19 increased fresh biomass of seedlings that did not differ
significantly from un-inoculated control.
4.4.3.5. Dry matter (g)
Inoculation with strain T42, T75, 2O₀, 7O₀, O₀10, L9, W9 and W28 significantly
decreased the dry matter up to 45.9, 38.2, 21.2, 64.7, 69.4, 56.5, 30.6 and 51.8% than un-
inoculated control, respectively. Inoculation with strain T19 significantly increased the
dry matter up to 23.5% than un-inoculated control. Inoculation with other strains (T12,
T18, T23, T24, T31, T38, L6, B11, ESO-8 and ESO-11) caused non-significant effect on
dry matter as compared to un-inoculated control.

73
4.4.4. Agar plate bioassay on common lambs’ quarter (Chenopodium album)

The selected strains of presumed allelopathic bacteria were evaluated for their
effects on germination and seedling growth of common lambs’ quarter in agar bioassay.
The results of this study are summarized in table 4.13 and are described below;
4.4.4.1. Germination (%)
Inoculation with strain W28, ESO-11, B11 and T75 significantly reduced the
germination rate of common lambs’ quarter up to 24.6, 21, 22.8 and 22.8% as compared
to un-inoculated control, respectively (Table 4.13). Inoculation of common lambs’ quarter
with strain L6 and 2O, however, significantly increased germination of common lambs’
quarter seeds by 14.8 and 19.3% over un-inoculated control, respectively. A non-
significant increase or decrease in seed germination was observed due to inoculation with
other strains as compared to un-inoculated control.
4.4.4.2. Root length (cm)
The results showed that root length of common lambs’ quarter seedlings was
significantly reduced up to 55, 20.3, 27.3, 29 and 41% over un-inoculated control due to
inoculation with strain T18, B11, T75, ESO-11 and W28, respectively (Table 4.13). A
significant increase in root length was recorded due to inoculation with strain 2O₀ which
was 22.2% higher than un-inoculated control while other strains showed non-significant
increase over control.
4.4.4.3. Shoot length (cm)
Shoot length of common lambs’ quarter was influenced due to inoculation with 6 of the
selected strains (B11, W28, T18, T75 and ESO-11) (Table 4.13). They produced 11.5,
41.8, 15.3, 22.2 and 28% lesser shoot length of common lambs’ quarter than un-
inoculated control, respectively. Inoculation of common lambs’ quarter with strain 2O₀
significantly increased shoot length by 15.24% as compared to un-inoculated control.
However, non-significant increase or decrease in shoot length was observed due to
inoculation with other strains.
4.4.4.4. Fresh biomass (g)
Inoculation with strain W28, B11, T75 and ESO-11 significantly reduced the fresh
biomass of common lambs’ quarter up to 47, 21, 25 and 35.3% as compared to un-
inoculated control, respectively (Table 4.13). Inoculation of common lambs’ quarter with
strain 2O₀ significantly increased fresh biomass of seedlings which was 27.9% higher

74
Table 4.13. Effect of presumed allelopathic bacteria on germination and seedling
growth of common lambs’ quarter
Germination Root Shoot Fresh Dry matter
Treatments
(%) Length (cm) Length (cm) Biomass (g) (g)
Control 63.33 c-e 2.87 b-e 3.15 b-f 1.17 b-e 0.27 a-c

T12 61.0 c-e 2.57 d-g 2.83 f-h 1.11 c-g 0.25 b-d

T18 61.55 c-e 2.29 f-h 2.36 hi 1.04 d-g 0.243 b-d

T19 63.2 c-e 2.5 e-h 3.45 bc 1.25 a-d 0.27 a-c

T23 58.67 c-e 2.73 c-f 2.98 d-g 1.14 c-f 0.25 b-d

T24 66.7 bc 3.14 a-c 3.32 b-e 1.31 a-c 0.29 ab

T31 57.7 d-f 2.9 b-e 3.28 b-f 1.21 b-e 0.28 ab

T38 57.24 d-f 2.61 d-f 2.92 e-g 1.1 c-g 0.24 b-d

T42 65.7 b-d 3.04 a-d 3.47 bc 1.13 c-f 0.24 b-d

T75 47.67 g 2.04 hi 2.01 ij 0.88 gh 0.19 d-f

2O₀ 75.62 a 3.51 a 3.94 a 1.5 a 0.32 a

7O₀ 55.67 e-g 2.9 b-e 3.05 c-f 1.1 c-g 0.24 b-e

O₀10 63.3 c-e 2.93 b-e 3.39 b-d 1.0 e-g 0.243 b-d

ESO-8 64.32 b-d 2.93 b-e 3.44 b-d 1.25 b-d 0.277 a-c

ESO-11 49.0 g 1.69 ij 1.71 j 0.76 hi 0.17 ef

L6 72.33 ab 3.32 ab 3.56 ab 1.39 ab 0.30 ab

L9 63.34 c-e 2.69 c-f 3.19 b-f 1.27 a-d 0.273 a-c

B11 49.0 g 2.09 g-i 2.56 gh 0.92 f-h 0.21 c-e

W9 59.0 c-e 2.98 b-e 2.85 e-g 1.17 b-e 0.257 a-d

W28 50.0 fg 1.29 j 1.0 k 0.62 i 0.133 f

LSD 8.127 0.524 0.47 0.244 0.073

Table presents data of 7 days old seedlings. Values sharing same letter(s) do not differ
significantly from each other at p <0.05 according to Duncan’s Multiple Range Test

75
than un-inoculated control. However, inoculation with other strains caused non-
significant impact on fresh biomass being statistically at par with un-inoculated control.
4.4.4.5. Dry matter (g)
Inoculation with strain T75, ESO-11 and W28 significantly reduced the dry matter
of common lambs’ quarter up to 27.5, 36.3 and 50.5% as compared to un-inoculated
control, respectively. Inoculation with other strains (T12, T18, T19, T23, T24, T31, T38,
T42, L6, L9, B11, 2O₀, 7O₀, O₀10, ESO-8 and W9) caused non-significant effect on dry
matter than un-inoculated control.
4.4.5. Agar plate bioassay on wheat (Triticum aestivum L.)
All the strains used in agar bioassays of four weed species (wild oat, little seed
canary grass, broad leaved dock and common lambs’ quarter) were tested for their
possible effects on wheat in agar plate bioassay using the same experimental arrangement
(Photo 6). The results regarding germination and different growth parameters of wheat
obtained from this study are described below;
4.4.5.1. Germination rate (%)
Data regarding germination rate of wheat revealed that inoculation of wheat with strain
L9, T19, 2O, 7O₀, T23, T24 and T42 significantly increased its germination rate up to
18.8, 16.7, 16.7, 16.7, 12.5, 12.5 and 8.33% over un-inoculated control, respectively
(Table 4.14). A non-significant increase or decrease in germination rate was recorded due
to inoculation with strain O₀10, L6, B11, ESO-8, T12, T75, T31, T38 and W9 over un-
inoculated control. However, inoculation of wheat with strain T18, ESO-11 and W28
significantly decreased its germination rate over un-inoculated control which was 8.3, 8.3
and 10.4% lesser than un-inoculated control, respectively.
4.4.5.2. Root length (cm)
The results indicated that inoculation of wheat with strain L9, 7O₀, 2O, T24, T23 and T19
significantly increased its root length up to 37.7, 37.6, 19.4, 20, 23 and 24.3% as
compared to un-inoculated control, respectively (Table 4.14). A non-significant increase
in root length was observed due to inoculation with strain O₀10, T42, W9 and T38 over
un-inoculated control. Strains B11, L6, T31 and ESO-8 caused non-significant reduction
in root length as compared to control. However, inoculation with strain T12, T18, ESO-
11, T75 and W28 significantly decreased root length up to 24.8, 27.2, 48.8, 50 and 52.8%
as compared to un-inoculated control, respectively.

76
Table 4.14. Effect of presumed allelopathic bacteria on germination and seedling
growth of wheat
Germination Root Shoot Fresh Dry matter
Treatments
(%) Length (cm) Length (cm) Biomass (g) (g)
Control 80.0 de 6.60 c-e 8.58 cd 1.55 e 0.313 fg

T12 75.0 ef 4.97 f 6.96 e 1.29 f 0.233 h

T18 73.35 f 4.81 f 6.78 e 1.32 f 0.240 h

T19 93.4 a 8.22 b 9.83 b 1.78 b-d 0.407 c

T23 90.0 ab 8.13 b 10.13 b 1.75 c-e 0.390 cd

T24 90.0 ab 7.92 b 10.05 b 1.74 de 0.400 c

T31 80.0 de 6.47 de 10.19 b 1.57 e 0.300 g

T38 80.0 de 6.67 c-e 10.21 b 1.57 e 0.310 fg

T42 86.5 bc 7.15 c 9.097 c 1.63 de 0.343 ef

T75 75.0 ef 3.3 g 5.53 f 1.25 f 0.230 h

2O₀ 93.4 a 7.89 b 10.03 b 1.96 a-c 0.413 bc

7O₀ 93.4 a 9.09 a 11.13 a 2.1 a 0.450 ab

O₀10 85 b-d 7.17 c 8.69 cd 1.63 de 0.353 de

ESO-8 81.5 cd 6.53 c-e 8.52 cd 1.58 de 0.347 ef

ESO-11 73.4 f 3.38 g 5.56 f 1.25 f 0.220 h

L6 83.4 cd 6.41 e 8.6 cd 1.98 ab 0.357 de

L9 95 a 9.09 a 11.13 a 2.16 a 0.463 a

B11 83.4 cd 6.32 e 8.42 d 1.96 ab 0.400 c

W9 81.7 cd 7.14 cd 8.62 cd 1.68 de 0.337 e-g

W28 71.6 f 3.12 g 5.28 f 1.18 f 0.207 h

LSD 6.565 0.682 0.673 0.208 0.0376

Table presents data of 7 days old seedlings. Values sharing same letter(s) do not differ
significantly from each other at p <0.05 according to Duncan’s Multiple Range Test

77
 Control L9

T19 W28

Photo 6. Pictorial view of agar bioassay on wheat

78
4.4.5.3. Shoot length (cm)
The results shown in Table 4.14 indicated that inoculation with strain T19, 2O,
T24, T23, T31, T38, L9 and 7O₀ significantly increased the length of wheat up to 14.6,
16.8, 17, 18, 18.7, 18.9, 29.6 and 29.7% as compared to un-inoculated control,
respectively. Inoculation with strain T42, O₀10, W9, L6, B11 and ESO-8 caused a non-
significant increase or decrease in shoot length of wheat over un-inoculated control.
Shoot length of wheat was significantly reduced due to inoculation with strain W28, T75,
ESO-11, T18 and T12 that were 38.5, 35.5, 35.3, 21 and 18.9% lesser than un-inoculated,
respectively.
4.4.5.4. Fresh biomass (g)
Inoculation with strain T19, 2O₀, B11, L6, 7O₀ and L9 significantly increased the
fresh biomass of wheat up to 14.8, 26, 26, 27.2, 35.4 and 38.7% over un-inoculated
control, respectively (Table 4.14). Increase in fresh biomass due to inoculation with strain
T23, T24, W9, T42, O₀10, ESO-8, T31 and T38 remained statistically non-significant
with un-inoculated control. However, inoculation of wheat with strain W28, T75, ESO-
11, T12 and T18 significantly decreased its fresh biomass up to 24.3, 19.6, 19.4, 17 and
15% as compared to un-inoculated control, respectively.
4.4.5.5. Dry matter (g)
Inoculation with strain T12, T18, T75, ESO-11 and W28 significantly reduced the
dry matter up to 25.5, 23.4, 26.6, 29.8 and 34.0% as compared to un-inoculated control,
respectively (Table 4.14). Inoculation with strain T19, T23, T24, L6, L9, B11, 2O, 7O
and O10 significantly increased the dry matter up to 29.8, 24.5, 27.7, 13.8, 47.9, 27.7,
31.9, 43.6 and 12.8% as compared to un-inoculated control, respectively. However,
inoculation with strain T31, T38, W9, T42 and ESO-8 caused non-significant effect on
dry matter of wheat as compared to un-inoculated control.
4.5. Effect of allelopathic bacteria on germination and growth of wheat
and its associated weeds under axenic conditions

Ten strains were selected to study their impact on wheat and 3 weeds (wild oat,
little seed canary grass and broad leaved dock) based on suppression of one or more
weeds and non-inhibition effects on wheat in agar plate bioassays. These strains were
applied to these plants under axenic conditions in growth chamber. The results of these
studies are described below;

79
4.5.1. Effect of allelopathic bacteria on germination and growth of wild oat
4.5.1.1. Germination (%)
The data in Table 4.15 revealed that germination of wild oat was significantly
reduced due to inoculation with all the selected strains as compared to un-inoculated
control. Maximum reduction in germination (63.3%) was observed due to inoculation
with strain L9 followed by T42 (53.2%) which were significantly less than un-inoculated
control and other strains. Inoculation with strains W9, O₀10, 7O₀, 2O₀ and ESO-8
significantly reduced germination by 41.8, 43.2, 31.6, 21.5 and 15.2% over un-inoculated
control, respectively (Photo 7).
4.5.1.2. Root length (cm)
The results in Table 4.15 showed that inoculation of wild oat with all of the
applied strains significantly reduced root length as compared to un-inoculated control.
Inoculation strain L9 maximally reduced root length (65.8%) followed by T42 (54%)
which were significantly less than un-inoculated control and other strains T42, W9, O₀10,
7O₀, 2O₀ and ESO-8. Inoculation of wild oat with strain W9, O₀10, 7O₀, 2O₀ and ESO-8
reduced root length by 44.8, 29.7, 23.6, 17.8 and 15%% which were significantly from
un-inoculated control, respectively.
4.5.1.3. Shoot length (cm)
The data in Table 4.15 depicted that shoot length of wild oat was significantly
reduced due to inoculation with all of the selected strains except ESO-8 over un-
inoculated control. Shoot length of wild oat was maximally reduced due to inoculation
with strain L9 which was 25.7% lesser than un-inoculated control followed by inoculation
with strain W9, O₀10, 7O₀, 2O₀ and ESO-8. Inoculation with strain T42 decreased shoot
length of wild oat by 21.2% which was significantly different from un-inoculated control
and other strains. Similarly, inoculation of wild oat with strain W9, O₀10, 7O₀ and 2O₀
reduced the shoot length by 18.9 and 16.3, 13.1 and 8.2% over un-inoculated control,
respectively. Minimum reduction in shoot length was observed by strain ESO-8.
4.5.1.4. Root dry weight (g)
Data indicated that inoculation with strain T42, L9, 7O₀, O₀10 and W9
significantly reduced the root dry weight of wild oat up to 60.2, 72.1, 26.4, 32.3 and
48.3% than un-inoculated control, respectively (Table 4.15). Maximum reduction in root
dry weight was observed due to inoculation with strain L9 which was significantly less
than strain 2O₀, 7O₀, O₀10, ESO-8 and W9. Strain T42 caused significant reduction in

80
Table 4.15. Effect of allelopathic bacteria on germination and growth of wild oat
under axenic conditions
Root Shoot
Germination Root dry Shoot dry
Treatments Length Length
(%) weight (g) weight (g)
(cm) (cm)
Control 75.31 a 13.64 a 18.83 a 0.67 a 1.34 a
T42 35.27 e 6.28 e 14.84 ef 0.27 de 0.56 e
L9 27.65 f 4.67 f 13.99 f 0.19 e 0.38 f
2O₀ 59.11 b 11.20 b 17.29 bc 0.55 ab 1.07 b
7O₀ 51.48 c 10.41 bc 16.35 cd 0.49 b 0.92 c
O₀10 49.57 cd 9.59 c 15.76 de 0.45 bc 0.80 d
ESO-8 63.87 b 11.59 b 17.78 ab 0.57 ab 1.12 b
W9 43.85 d 7.53 d 15.27 de 0.35 cd 0.66 e
LSD 6.627 1.233 1.104 0.125 0.122
Table presents data of 25 days old plants. Values sharing same letter(s) do not differ
significantly from each other at p <0.05 according to Duncan’s Multiple Range Test

Photo 7. Pictorial view of effect of allelopathic bacteria on 25 days old plants of wild oat
under axenic conditions

81
root dry weight than strain 2O₀, 7O₀, O₀10 and ESO-8. Strain W9 also significantly
reduced the root dry weight than strain 2O₀, 7O₀ and ESO-8. Strain 2O₀, 7O₀, O₀10 and
ESO-8 remained non-significant with each other in affecting the root dry weight of wild
oat.
4.5.1.5. Shoot dry weight (g)
Inoculation with strain T42, L9, 2O₀, 7O₀, O₀10, ESO-8 and W9 significantly
reduced the shoot dry weight up to 58.0, 71.9, 20.1, 31.3, 40.5, 16.2 and 51.0% than un-
inoculated control, respectively (Table 4.15). Strain L9 also significantly reduced the
shoot dry weight than all other strains. Strain T42 and W9 also significantly reduced the
shoot dry weight than strain O₀10 also significantly reduced the shoot dry weight than
strain 2O₀, 7O₀ and ESO-8. Inoculation with strain 7O₀ also significantly reduced the
shoot dry weight than strain 2O₀ and ESO-8. Strain 2O₀ and ESO-8 remained non-
significant with each other in affecting the shoot dry weight of wild oat.

4.5.2. Effect of allelopathic bacteria on germination and growth of little seed canary
grass

4.5.2.1. Germination (%)

Data indicated that inoculation with strain O₀10, 7O₀, L9, T42 and W9
significanty reduced the germination rate of little seed canary grass up to 58.7, 35.7, 37,
27.2 and 18.5% as compared to un-inoculated control, respectively (table 4.16).
Inoculation with strain O₀10 performed significantly better than other strains in reducing
the germination rate of little seed canary grass over un-inoculated control. Inoculation of
little seed canary grass with strain 7O₀ and L9 also performed significantly better than
W9 in reducing the germination rate over un-inoculated control (Photo 8).
4.5.2.2. Root length (cm)
Table 4.16 indicated that root length of little seed canary grass was significantly
reduced due to inoculation with O₀10, 7O₀, L9, T42 and W9 up to 68, 62.5, 54.9, 46.7
and 27.8% as compared to un-inoculated control, respectively. Inoculation with strain
O₀10 and 7O₀ performed significantly better than strain T42, L9 and W9 in decreasing
the root length over un-inoculated control. Inoculation with strain L9 performed
significantly better than strain T42 and W9 in decreasing the root length over un-

82
Table 4.16. Effect of allelopathic bacteria on germination and growth of little seed
canary grass under axenic conditions
Root Shoot Root dry Shoot dry
Germination
Treatments Length Length weight weight
(%)
(cm) (cm) (g) (g)
Control 87.7 a 10.09 a 10.89 a 0.097 a 0.20 a

T42 63.9 bc 5.37 c 8.24 c 0.057 bc 0.12 bc

L9 55.3 cd 4.55 d 7.84 c 0.042 cd 0.09 cd

7O₀ 47.7 d 3.76 e 6.93 d 0.034 d 0.07 de

O₀10 36.2 e 3.23 e 6.09 e 0.023 d 0.05 e

W9 71.5 b 7.28 b 9.18 b 0.063 b 0.15 b

LSD 8.975 0.631 0.845 0.0238 0.042

Table presents data of 25 days old plants. Values sharing same letter(s) do not differ
significantly from each other at p <0.05 according to Duncan’s Multiple Range Test

Photo 8. Pictorial view of effect of allelopathic bacteria on 25 days old plants of little
seed canary grass under axenic conditions

83
inoculated control. The reduction in root length of little seed canary grass due to
inoculation with strain T42 was also significantly less than strain W9.
4.5.2.3. Shoot length (cm)
Shoot length of little seed canary grass was significantly reduced due to
inoculation with O₀10, 7O₀, L9, T42 and W9 up to 44, 36.3, 28, 24.3 and 15.7% as
compared to un-inoculated control, respectively (Table 4.16). Inoculation with strain
O₀10 performed significantly better than strain 7O₀, L9, T42 and W9 in reducing the
shoot length over un-inoculated control. Inoculation of little seed canary grass with strain
7O₀ produced significantly less shoot length than strain L9, T42 and W9. Inoculation
with strain L9 and T42 also performed significantly better than W9 in reducing the shoot
length of little seed canary grass.
4.5.2.4. Root dry weight (g)
Inoculation with strain T42, L9, 7O₀, O₀10 and W9 significantly reduced the root
dry weight of little seed canary grass up to 41.4, 58.6, 69.0, 79.3 and 34.5% as compared
to un-inoculated control, respectively (Table 4.16). Inoculation with strain O₀10 and 7O₀
performed significantly better than strain W9 and T42 in reducing the root dry weight
than un-inoculated control. Inoculation with strain L9 also significantly reduced the root
dry weight than strain W9.
4.5.2.5. Shoot dry weight (g)
Table 4.16 indicated that inoculation with strain T42, L9, 7O₀, O₀10 and W9
significantly reduced the shoot dry weight of little seed canary grass up to 41.0, 54.1,
67.2, 77.0 and 27.9% as compared to un-inoculated control, respectively. Inoculation with
strain O₀10 also significantly reduced the shoot dry weight than strain T42, L9 and W9.
Inoculation with strain 7O₀ also caused significant reduction in shoot dry weight than
strain T42 and W9. Shoot dry weight produced by strain L9 was also significantly less
than that produced by strain W9.
4.5.3. Effect of allelopathic bacteria on germination and growth of broad leaved
dock
4.5.3.1. Germination (%)
Data shown in Table 4.17 indicated that inoculation with strain W9, T42, L9, 7O₀,
O₀10, T38, L6 and T19 significantly reduced the germination rate of broad leaved dock
up to 60.5, 55.3, 44.7, 36.8, 28.9, 23.7, 21 and 18.4% over un-inoculated control,
respectively. Inoculation with W9 performed significantly better than strain L9, 7O₀,

84
Table 4.17. Effect of allelopathic bacteria on germination and growth of broad
leaved dock under axenic conditions
Shoot
Germination Root Length Root dry Shoot dry
Treatments Length
(%) (cm) weight (g) weight (g)
(cm)
Control 63.35 a 9.16 a 10.81 a 0.095 a 0.39 a
T19 51.65 b 7.50 b 8.60 b 0.077 ab 0.29 b
T38 48.34 b 6.78 bc 7.19 c 0.063 bc 0.23 c
T42 28.33 ef 3.10 f 4.41 f 0.033 ef 0.15 f
L6 50.0 b 7.22 b 7.53 c 0.074 b 0.27 b
L9 35.0 de 4.19 e 4.81 ef 0.043 de 0.17 ef
7O₀ 40.0 cd 5.32 d 5.06 de 0.047 de 0.19 de
O₀10 45.0 bc 6.03 cd 5.61 d 0.053 cd 0.20 d
W9 25.0 f 2.32 g 3.45 g 0.024 f 0.13 g
LSD 8.25 0.78 0.608 0.0141 0.02
Table presents data of 25 days old plants. Values sharing same letter(s) do not differ
significantly from each other at p <0.05 according to Duncan’s Multiple Range Test

85
O₀10, T38, L6 and T19 in reducing the germination rate over un-inoculated control.
Reduction in germination rate due to inoculation with strain L9 and T42 was also
significantly less than strain O₀10, T38, L6 and T19. Inoculation with strain 7O₀ also
performed significantly better than strain T38, L6 and T19 in reducing the germination
rate of broad leaved dock over un-inoculated control.
4.5.3.2. Root length (cm)
Results indicated that inoculation with strain W9, T42, L9, 7O₀, O₀10, T38, L6
and T19 significantly reduced the root length of broad leaved dock up to 74.7, 66, 54.2,
41.9, 34.1, 25.9, 21.1 and 18% as compared to un-inoculated control, respectively (Table
4.17). Inoculation with strain W9 performed significantly better than other strains in
reducing the root length over un-inoculated control. Inoculation with strain T42 decreased
the root length significantly less than that caused by strain L9, 7O₀, O₀10, T38, L6 and
T19. Reduction in root length due to strain L9 was also significantly less than that caused
by inoculation with strain 7O₀, O₀10, T38, L6 and T19. Inoculation with strain 7O₀ and
O₀10 performed significantly better than strain L6 and T19 in reducing the root length
over un-inoculated control.
4.5.3.3. Shoot length (cm)
Shoot length of broad leaved dock was significantly reduced due to inoculation
with strain W9, T42, L9, 7O₀, O₀10, T38, L6 and T19 up to 68.1, 59.2, 55.5, 53.2, 48.1,
33.5, 30.4 and 20.4% as compared to un-inoculated control, respectively (Table 4.17).
Inoculation with strain W9 performed significantly better than other strains. Reduction in
shoot length caused by inoculation of broad leaved dock with strain T42 and L9 remained
significantly less than that caused by strain O₀10, T38, L6 and T19. Inoculation with
strain 7O₀ and O₀10 performed significantly better than strain T38, L6 and T19 in
reducing the shoot length over un-inoculated control. Reduction inn shoot length caused
by strain T38 and L6 was also significantly less than that caused by inoculation with
strain T19.
4.5.3.4. Root dry weight (g)
Results showed that root dry weight of broad leaved dock was significantly
decreased due to inoculation with strain T38, T42, T42, L6, L9, 7O₀, O₀10 and W9 up to
29.6, 63.0, 22.2, 51.8, 48.1, 40.7 and 74.1% as compared to un-inoculated control,
respectively (Table 4.17). Maximum reduction in root dry weight was observed due to
inoculation with strain W9 which was aso significantly less than strain L9, 7O₀, O₀10,

86
T38, L6 and T19. Inoculation with strain T42 also significantly reduced the root dry
weight than strain T19, T38, L6 and O₀10. Inoculation with strain L9 and 7O₀ also
performed significantly better than strain T19, T38 and L6 in reducing the root dry
weight. Inoculation with strain O₀10 also significantly reduced the root dry weight than
strain L6. Reduction in root dry weight due to inoculation with strain T19, T38 and L6
remained statistically at par with each other.
4.5.3.5. Shoot dry weight (g)
Shoot dry weight of broad leaved dock was significantly reduced due to
inoculation with strain T19, T38, T42, L6, L9, 7O₀, O₀10 and W9 up to 24.1, 39.7, 60.3,
29.3, 55.2, 50.9, 47.4 and 67.2% as compared to un-inoculated control, respectively
(Table 4.17). Inoculation with strain W9 caused maximum reduction in shoot dry weight
which was also significantly less than all other strains. Strain T42 also significantly
reduced the shoot dry weight than strain T19, T38, L6, 7O₀ and O₀10. Inoculation with
strain L9 also performed significantly better than strain T38, L6, O₀10 and T19 in
reducing the shoot dry weight. Inoculation with strain 7O₀ and O₀10 also significantly
reduced the shoot dry weight than strain T19, T38 and L6. Inoculation with strain T38
also significantly reduced the shoot dry weight than strain T19 and L6.
4.5.4. Effect of allelopathic bacteria on germination and growth of wheat under
axenic conditions
4.5.4.1. Germination (%)
Results showed that none of the strains caused any significant reduction in
germination rate of wheat over un-inoculated control (Table 4.18). Inoculation of wheat
with strain 7O₀, T19 and L9 significantly increased germination rate up to 14.8, 14.8 and
18.5% over un-inoculated control, respectively. Maximum increase in germination rate
was observed due to inoculation with strain L9 which was also significantly higher than
strain ESO-8. Inoculation with other strains (T42, 2O, O₀10, W9, ESO-8, T38 and L6)
caused a non-significant increase or decrease in germination rate of wheat (Photo 9).
4.5.4.2. Root length (cm)
Data indicated that root length of wheat was not significantly reduced due to
inoculation with the selected strains. Inoculation of wheat with strain 2O₀, T19, 7O₀ and
L9 significantly increased its root length up to 21.6, 24.7, 40.8 and 43.7% as compared to
un-inoculated control, respectively (Table 4.18). Inoculation of wheat with strain T42,
O₀10, W9, ESO-8, T38 and L6caused a non-significant increase or decrease in root

87
Table 4.18. Effect of allelopathic bacteria on germination and growth of wheat
under axenic conditions
Shoot
Germination Root Root dry Shoot dry
Treatments Length
(%) Length (cm) weight (g) weight (g)
(cm)
Control 75.0 cd 9.20 d 20.69 cd 0.10 c 0.22 cd
T19 86.1 ab 11.48 b 25.20 ab 0.13 b 0.27 b
T38 74.9 cd 9.77 d 21.61 cd 0.11 c 0.24 c
T42 83.3 a-c 10.11 cd 22.08 c 0.11 c 0.24 c
L6 75.0 cd 9.28 d 21.08 cd 0.097 c 0.23 cd
L9 88.9 a 13.22 a 26.54 a 0.16 a 0.32 a
2O₀ 80.6 a-c 11.19 bc 24.91 b 0.14 b 0.27 b
7O₀ 86.1 ab 12.96 a 26.43 a 0.15 ab 0.31 a
O₀10 77.8 bc 10.15 cd 22.03 c 0.11 c 0.237 cd
ESO-8 66.7 d 9.15 d 20.52 d 0.09 c 0.21 d
W9 77.8 bc 9.70 d 21.42 cd 0.103 c 0.227 cd
LSD 10.42 1.204 1.5 0.02 0.028
Table presents data of 25 days old plants. Values sharing same letter(s) do not differ
significantly from each other at p <0.05 according to Duncan’s Multiple Range Test

Photo 9. Pictorial view of effect of allelopathic bacteria on 25 days old plants of wheat
under axenic conditions

88
length over un-inoculated control.
4.5.4.3. Shoot length (cm)
Data in Table 4.18 showed the effect of allelopathic bacteria on shoot length of
wheat. Results showed that shoot length of wheat was not significantly reduced due to
inoculation with any of the selected strains. Inoculation with strain 2O₀, T19, 7O₀ and L9
significantly increased shoot length of wheat up to 20.4, 21.8, 27.7 and 28.2% as
compared to un-inoculated control, respectively. Inoculation of wheat with strain T42,
O₀10, T38, W9, L6 and ESO-8 caused a non-significant increase or decrease in shoot
length over un-inoculated control.
4.5.4.4. Root dry weight (g)
Data indicated that none of the strains caused significant reduction in root dry
weight of wheat than un-inoculated control (Table 4.18). Inoculation with strain T38,
T42, L6, O₀10 and W9 caused non-significant effect on root dry weight than un-
inoculated control. Inoculation with strain T19, L9, 2O₀ and 7O₀ significantly increased
the root dry weight up to 33.3, 60, 36.7 and 53.3% than un-inoculated control,
respectively. Inoculation with strain L9 caused maximum increase in root dry weight
which was also significantly higher than increase in root dry weight caused by strain T19
and 2O₀. The increase in root dry weight due to strain 2O₀, 7O₀ and T19 remained
statistically at par with each other.
4.5.4.5. Shoot dry weight (g)
Results showed that shoot dry weight of wheat was not significantly reduced due
to inoculation with any of the selected strains (Table 4.18). Inoculation of wheat with
strain O₀10, T42, T38, L6, W9 and ESO-8 caused non-significant effect on shoot dry
weight than un-inoculated control. Inoculation with strain T19, L9, 2O₀ and 7O₀
significantly increased the shoot dry weight up to 24.2, 45.4, 22.7 and 40.9% than un-
inoculated control, respectively. Maximum increase in shoot dry weight was recorded due
to inoculation with strain L9 and 7O₀ which was significantly higher than strain 2O₀ and
T19. Inoculation with strain T19 and 2O₀ remained statistically at par with each other in
improving the shoot dry weight.
Pot Experiments

Four pot trials were conducted to study effect of allelopathic bacteria on; wild oat
grown in wheat, little seed canary grass grown in wheat, broad leaved dock grown in
wheat and wheat under weed free conditions.

89
4.6. Effect of allelopathic bacteria on wild oat grown in wheat in pot
experiment

Wild oat was grown in wheat in pots and fresh culture of 5 strains of allelopathic
bacteria was applied in the respective pots. Three sets of pots were arranged to study
weed suppression effects of allelopathic bacteria and resultant improvement in growth
and yield of infested wheat at three growth stages (tillering, booting and harvesting). Data
regarding growth and yield parameters of wild oat and infested wheat were recorded at
these growth stages and compared with weedy and weed free control. The results of this
experiment are described below;
4.6.1. Effect of allelopathic bacteria on growth of wild oat and infested wheat at
tillering in pot experiment
4.6.1.1. Number of tillers per pot
Data given in Table 4.19 indicated that growth of wild oat significantly reduced
the number of tillers of infested wheat up to 60.3% as compared to weed free un-
inoculated control. Inoculation with strain L9, T42, W9, 7O₀ and O₀10 significantly
reduced the number of tillers of wild oat up to 42.2, 30.4, 22.2, 17 and 11.9% over weedy
control, respectively. Reduction in number of tillers due to inoculation with strain L9 was
also significantly less than strain T42, W9, 7O₀ and O₀10. Inoculation with strain T42
performed significantly better than strain 7O₀ and O₀10 in reducing the number of tillers
of wild oat over weedy control. Inoculation with strain W9 also significantly reduced
number of tillers of wild oat over inoculation with strain O₀10. Resultantly, inoculation
with strain L9, T42, W9, 7O₀ and O₀10 recovered the loss in number of tillers of infested
wheat up to 65.8, 55.3, 42.1, 31.6 and 13.2%, respectively.
4.6.1.2. Root length (cm)
Results showed that root length of wheat was significantly reduced up to 61% due
to growth of wild oat (Table 4.19). Inoculation with strain L9, T42, W9, 7O₀ and O₀10
significantly reduced the root length of wild oat up to 44, 30.1, 25.2, 21.1 and 16.6% as
compared to weedy control, respectively. Inoculation with strain L9 also signidicantly
decreased the root length of wild oat as compared to inoculation with strain T42, W9, 7O₀
and O₀10. Inoculation with strain T42, W9, 7O₀ and O₀10 remained statistically non-
significant with each other in reducing the root length of wild oat over weedy control.
Resultantly, the loss in root length of infested wheat was recovered up to 61.0, 53.9, 43.1,
34.9 and 17.9% due to inoculation with strain L9, T42, W9, 7O₀ and O₀10, respectively.
90
Table 4.19. Effect of allelopathic bacteria on growth of wild oat and infested wheat at tillering in pot experiment
Wild oat Infested wheat
Treatments No. of Root Shoot Root fresh Shoot fresh No. of Root Shoot Root fresh Shoot fresh
tillers Length length weight weight tillers Length length weight weight
pot-1 (cm) (cm) (g pot-1) (g pot-1) pot-1 (cm) (cm) (g pot-1) (g pot-1)
Weed free
-- -- -- -- -- 21.0 a 19.2 a 34.5 a 35.6 a 45.0 a
Control
Weedy
45.0 a 22.2 a 41.7 a 23.0 a 34.3 a 8.3 f 7.5 e 26.9 d 12.1 f 16.5 f
Control
T42 31.3 d 15.5 bc 33.3 c 12.4 d 20.9 d 15.3 bc 13.8 b 32.0 a-c 25.7 c 32.6 bc
L9 26.0 e 12.4 c 27.0 d 9.2 e 17.1 e 16.7 b 14.7 b 33.3 ab 28.3 b 35.8 b
7O₀ 37.3 bc 17.5 b 35.3 bc 16.1 bc 26.9 c 12.3 de 11.6 cd 30.3 b-d 21.2 d 26.5 d
O₀10 39.7 b 18.5 b 37.5 b 18.7 b 29.9 b 10.0 ef 9.6 de 28.6 cd 16.2 e 20.7 e
W9 35.0 cd 16.6 b 34.0 bc 13.8 cd 24.7 c 13.7 cd 12.6 bc 31.3 a-c 23.8 c 30.8 c
LSD 4.04 3.46 3.47 2.84 2.96 2.86 2.14 4.09 2.47 4.04
These measurements were taken at 50 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

91
4.6.1.3. Shoot length (cm)
Infestation of wild oat significantly reduced the shoot length of wheat up to 22%
over weed free control (Table 4.19). Inoculation with strain L9, T42, W9, 7O₀ and O₀10
significantly reduced the shoot length of wild oat up to 35.2, 20.2, 18.4, 15.3 and 10% as
compared to un-inoculated control, respectively. Inoculation with strain L9 also
significantly reduced the shoot length over strain T42, W9, 7O₀ and O₀10. Inoculation
with strain T42 performed significantly better than strain O₀10 in reducing shoot length
over weedy control. Resultantly, the loss in shoot length of infested wheat was recovered
up to 84.1, 66.2, 57.0, 43.8 and 21.9% due to inoculation with strain L9, T42, W9, 7O₀
and O₀10, respectively.
4.6.1.4. Root fresh weight (g pot-1)
Root fresh weight of wheat was significantly increased due to weed suppression
effects of applied strains (Table 4.19). Inoculation with strain L9, T42, W9, 7O₀ and
O₀10 significantly reduced the root fresh weight of wild oat up to 60, 46, 40, 30 and
18.7% as compared to weedy control, respectively. Inoculation with L9 also performed
significantly better than strain T42, W9, 7O₀ and O₀10 in reducing root fresh weight over
weedy control. Inoculation with strain T42 also significantly reduced root fresh weight as
compared strain 7O₀ and O₀10. Root fresh weight produced by strain W9 was also
significantly less than strain O₀10. Resultantly, the loss in root fresh weight of infested
wheat was recovered up to 68.6, 57.7, 49.5, 38.5 and 17.4% due to inoculation with strain
L9, T42, W9, 7O₀ and O₀10, respectively.
4.6.1.5. Shoot fresh weight (g pot-1)
Results showed that shoot fresh weight of wheat was significantly reduced up to
63.2% due to infestation of wild oat (Table 4.19). Inoculation with strain L9, T42, W9,
7O₀ and O₀10 significantly reduced the shoot fresh weight of wild oat up to 50, 39, 28,
21.7 and 13% as compared to weedy control, respectively. Inoculation with strain L9 also
significantly reduced shoot fresh weight as compared to strain T42, W9, 7O₀ and O₀10.
Inoculation with strain T42 also remained significantly less than strain W9, 7O₀ and O₀10
in reducing shoot fresh weight. Inoculation with strain W9 and 7O₀ also significantly
reduced shoot fresh weight over strain O₀10. Resultantly, the loss in shoot fresh weight of
infested wheat was recovered up to 67.6, 56.6, 50.3, 35.1 and 14.5% due to inoculation
with strain L9, T42, W9, 7O₀ and O₀10, respectively.

92
4.6.2. Effect of allelopathic bacteria on growth of wild oat and infested wheat at
booting in pot experiment
4.6.2.1. Number of tillers per pot
Infestation of wild oat significantly reduced the number of tillers of wheat up to
67.2% over weed free control (Table 4.20). Inoculation with strain L9, T42, W9, 7O₀ and
O₀10 significantly reduced number of tillers of wild oat up to 41.7, 29.5, 22, 18.9 and
14.4% as compared to weedy control, respectively. Inoculation with strain L9 performed
significantly better than strain T42, W9, 7O₀ and O₀10 in reducing the number of tillers
over weedy control. Inoculation with strain T42 also significantly reduced number of
tillers over inoculation with strain W9 and O₀10. Inoculation with strain 7O₀, W9 and
O₀10 remained statistically similar with each other in reducing number of tillers over
weedy control. Resultantly, the loss in number of tillers of infested wheat was recovered
up to 53.3, 46.7, 40.0, 28.9 and 15.6% due to inoculation with strain L9, T42, W9, 7O₀
and O₀10, respectively (Photo 10).
4.6.2.2. Root length (cm)
Infestation of wild oat significantly reduced the root length of wheat up to 70.4%
as compared to weed free control (Table 4.20). Data indicated that root length of wild oat
was significantly reduced due to inoculation with strain L9, T42, W9, 7O₀ and O₀10 up to
46.1, 41, 36.4, 28.9 and 17.3% as compared to weedy control, respectively (Table 4.21).
Inoculation with strain L9 also significantly reduced root length over inoculation with
strain 7O₀ and O₀10. Inoculation with strain T42 and W9 also significantly reduced root
length as compared to weedy control. Resultantly, the loss in root length of infested wheat
was recovered up to 47.0, 39.8, 36.5, 29.0 and 19.4% due to inoculation with strain L9,
T42, W9, 7O₀ and O₀10, respectively.
4.6.2.3. Shoot length (cm)
Infestation of wild oat significantly reduced the shoot length of wheat up to 19.8%
as compared to weed free control (Table 4.20). Inoculation with strain L9, T42, W9 and
7O₀ significantly reduced the shoot length of wild oat up to 30.4, 22.1, 19.6 and 16.2% as
compared to weedy control, respectively. Inoculation with strain L9 also significantly
reduced shoot length as compared to inoculation with strain O₀10. Inoculation with strain
O₀10 caused a non-significant decrease in shoot length of wild oat. Resultantly, the loss
in shoot length of infested wheat was recovered up to 82.6, 68.0, 56.6, 44.8 and 27.0%
due to inoculation with strain L9, T42, W9, 7O₀ and O₀10, respectively.

93
Table 4.20. Effect of allelopathic bacteria on growth of wild oat and infested wheat at booting in pot experiment
Wild oat Infested wheat
Treatments No. of Root Shoot Root fresh Shoot fresh No. of Root Shoot Root fresh Shoot fresh
tillers Length length weight weight tillers length length weight weight
pot-1 (cm) (cm) (g pot-1) (g pot-1) pot-1 (cm) (cm) (g pot-1) (g pot-1)
Weed free
-- -- -- -- -- 22.3 a 30.2 a 49.1 a 51.0 a 79.7 a
Control
Weedy
44.0 a 28.6 a 62.3 a 39.4 a 95.6 a 7.3 e 8.9 e 39.4 e 16.1 f 28.5 f
Control
T42 31.0 c 16.9 cd 48.5 bc 18.4 d 59.9 c 14.3 bc 17.4 bc 46.0 a-c 35.7 c 56.2 c

L9 25.7 d 15.4 d 43.4 c 14.2 e 47.7 d 15.0 b 18.9 b 47.4 ab 41.3 b 60.4 b

7O₀ 34.3 bc 20.4 bc 52.2 bc 26.0 c 76.4 b 11.7 cd 15.1 cd 43.8 cd 27.5 de 45.7 d

O₀10 37.7 b 23.7 b 56.5 ab 31.2 b 80.6 b 9.7 de 13.1 d 42.0 de 24.0 e 35.1 e

W9 35.7 b 18.2 cd 50.1 bc 22.4 cd 71.8 b 13.3 bc 16.7 bc 44.9 b-d 31.8 cd 53.6 c

LSD 3.73 3.6 9.21 4 11.9 2.91 3.41 3.52 4.54 4.14
These measurements were taken at 100 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

94
 wheat wheat

Wild oat

Wild oat

Weed free Weedy

L9
Control Control

Photo 10. Pictorial view of effect of allelopathic bacteria on wild oat grown in wheat at
booting (100 days after sowing)

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4.6.2.4. Root fresh weight (g pot-1)
Infestation of wild oat significantly reduced the root fresh weight of wheat up to
68.4% as compared to weed free control (Table 4.20). Inoculation with strain L9, T42,
W9, 7O₀ and O₀10 significantly reduced root fresh weight of wild oat up to 64, 53.2, 43,
34 and 20.7% a as compared to un-inoculated control, respectively. Inoculation with
strain L9 also significantly significantly reduced the root fresh weight over inoculation
with strain T42, W9, 7O₀ and O₀10. Inoculation of wild oat with strain T42 also
significantly decreased its root fresh weight over inoculation with strain 7O₀ and O₀10.
Inoculation of wild oat with strain W9 and 7O₀ also significantly reduced its root fresh
weight over inoculation with strain O₀10. Resultantly, inoculation with strain L9, T42,
W9, 7O₀ and O₀10 recovered the loss in root fresh weight of infested wheat up to 72.1,
56.0, 44.7, 32.5 and 22.7%, respectively.
4.6.2.5. Shoot fresh weight (g pot-1)
Shoot fresh weight of wheat was significantly reduced up to 64.3% than weed free
control due to infestation of wild oat (Table 4.20). Inoculation with strain L9, T42, W9,
7O₀ and O₀10 significantly reduced the shoot fresh weight of wild oat up to 50.2, 37.4,
24.9, 20 and 15.7% as compared to weedy control, respectively. Inoculation with strain
L9 also significantly reduced shoot fresh weight over inoculation with strain T42, W9,
7O₀ and O₀10. Inoculation with strain T42 produced significantly less shoot fresh weight
than inoculation with strain W9, 7O₀ and O₀10. Inoculation with strain W9, 7O₀ and
O₀10 remained statistically non-significant with each other in reducing shoot fresh weight
over weedy control. Consequently, the loss in shoot fresh weight of infested wheat was
recovered up to 62.3, 54.0, 49.0, 33.5 and 13.0% due to inoculation with strain L9, T42,
W9, 7O₀ and O₀10, respectively.
4.6.3. Effect of allelopathic bacteria on growth and yield of wild oat and infested
wheat at harvesting
4.6.3.1. Number of tillers per pot
Data indicated that infestation of wild oat significantly reduced the number of
tillers of wheat up to 67.1% over weed free control (Table 4.21). Inoculation with strain
L9, T42, W9, 7O₀ and O₀10 significantly reduced the number of tillers of wild oat up to
45, 32.6, 25, 22.5 and 17% as compared to weedy control, respectively. Inoculation with
strain L9 also significantly reduced number of tillers over inoculation with strain T42,

96
Table 4.21. Effect of allelopathic bacteria on growth and yield of wild oat and infested wheat at harvesting in pot experiment
Wild oat Infested wheat
Treatments No. of Shoot Root fresh Straw Grain No. of Shoot Root fresh Grain Straw
tillers length weight yield yield tillers Length weight Yield yield
pot-1 (cm) (g pot-1) (g pot-1) (g pot-1) pot-1 (cm) (g pot-1) (g pot-1) (g pot-1)
Weed free
-- -- -- -- -- 23.3 a 54.3 a 25.5 a 21.1 a 31.6 a
Control
Weedy
43.0 a 62.3 a 22.0 a 32.3 a 9.2 a 7.7 f 43.9 d 8.4 f 8.3 f 10.7 f
Control
T42 29.0 c 46.7 cd 10.8 d 19.2 d 4.9 d 16.7 bc 51.1 a-c 17.6 bc 16.0 bc 21.9 bc

L9 23.6 d 41.0 d 9.6 d 15.5 e 3.7 e 17.7 b 53.0 ab 20.6 b 17.7 b 24.8 b

7O₀ 32.7 bc 50.7 bc 15.2 bc 26.1 bc 6.5 c 13.3 de 48.8 c 13.8 de 12.8 de 16.9 de

O₀10 35.7 b 55.3 b 17.4 b 27.7 b 7.1 b 11.0 e 47.8 c 12.5 e 11.1 e 14.4 e

W9 33.3 bc 49.0 bc 13.4 c 24.4 c 6.4 c 14.7 cd 49.6 bc 16.0 cd 14.6 cd 20.1 cd

LSD 4.61 6.39 2.38 3.16 0.5 2.81 3.73 3.07 2.59 3.62
These measurements were taken at 150 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

97
W9, 7O₀ and O₀10. Inoculation with strain T42 produced number of tillers significantly
less than strain O₀10. Inoculation of wild oat with strain 7O₀ and W9 remained
statistically at par with each other and O₀10 in reducing number of tillers of wild oat.
Weed suppression effects of strain L9, T42, W9, 7O₀ and O₀10 resultantly recovered the
number of tillers of infested wheat up to 51.1, 46.8, 38.3, 31.9 and 17.0%, respectively.
4.6.3.2. Shoot length (cm)
Results showed that infestation of wild oat significantly reduced the shoot length
of wheat up to 19.2% as compared to weed free control (Table 4.21). Inoculation with
strain L9, T42, W9, 7O₀ and O₀10 significantly reduced the shoot length of wild oat up to
34.2, 25.1, 21.4, 18.7 and 11.2% as compared to weedy control, respectively. Inoculation
with strain L9 produced significantly less shoot length than strain W9, 7O₀ and O₀10.
Inoculation with T42 also significantly reduced the shoot length as compared to strain
O₀10. Inoculation with strain W9, 7O₀ and O₀10 remained statistically non-significant
with each other in reducing shoot length over weedy control. Resultantly, the loss in shoot
length of infested wheat was recovered up to 87.2, 69.1, 54.9, 47.4 and 36.9% due to
inoculation with strain L9, T42, W9, 7O₀ and O₀10, respectively.
4.6.3.3. Root fresh weight (g pot-1)
Infestation of wild oat significantly decreased the root fresh weight of wheat up to
67.1% over weed free control (Table 4.21). Root fresh weight of wild oat was
significantly reduced due to inoculation with strain L9, T42, W9, 7O₀ and O₀10 up to
56.3, 51, 39.4, 31.1 and 21.1% as compared to weedy control, respectively. Inoculation
with strain L9 and T42 maximally reduced root fresh weight of wild oat produced
significantly less root fresh weight than strain W9, 7O₀ and O₀10. Inoculation of wild oat
with strain W9 remained significantly better than strain O₀10 in reducing the root fresh
weight of wild oat. Resultantly, the loss in root fresh weight of infested wheat was
recovered up to 71.4, 53.5, 44.2, 31.5 and 23.7% due to inoculation with strain L9, T42,
W9, 7O₀ and O₀10, respectively.
4.6.3.4. Straw yield (g pot-1)
Infestation of wild oat significantly decreased the straw yield of infested wheat up
to 66.1% over weed free control (Table 4.21). Results showed that straw yield of wild oat
was significantly reduced due to its inoculation with strain L9, T42, W9, 7O₀ and O₀10
up to 52.1, 40.4, 24.4, 19.2 and 14.3% as compared to weedy control, respectively.
Inoculation with strain L9 produced significantly less straw fresh weight than strain T42,

98
W9, 7O₀ and O₀10. Inoculation with strain T42 also significantly reduced the straw fresh
weight over strain W9, 7O₀ and O₀10. Inoculation with strain W9 produced significantly
better than strain O₀10 in reducing straw fresh weight over weedy control. Weed
suppression effects of strain L9, T42, W9, 7O₀ and O₀10 resultantly recovered the loss in
straw yield of infested wheat up to 67.7, 53.9, 45.2, 29.8 and 18.0%, respectively.
4.6.3.5. Grain yield (g pot-1)
Data indicated that grain yield of wheat was significantly reduced up to 60.8% due
to infestation of wild oat (Table 4.21). Inoculation with strain L9, T42, W9, 7O₀ and
O₀10 significantly reduced the grain yield of wild oat up to 60, 46.4, 30.4, 29 and 22.3%
as compared to weedy control, respectively. Inoculation with strain L9 also significantly
reduced the grain yield of wild oat over inoculation with strain W9, 7O₀ and O₀10.
Inoculation with strain W9 and 7O₀ also significantly reduced the grain yield as
compared to inoculation with strain O₀10. Weed suppression effects of strain L9, T42,
W9, 7O₀ and O₀10 resultantly controlled the loss in grain yield of infested wheat up to
73.6, 60.0, 49.7, 35.8 and 22.0%, respectively.
4.6.4. Effect of allelopathic bacteria on physiology of wild oat grown in wheat and
infested wheat
4.6.4.1. Assimilation rate (A)
The results showed that infestation of wild oat significantly reduced the
assimilation rate of wheat up to 28.4% than weed free control (Table 4.22). Inoculation
with strain L9, T42, W9 and 7O₀ significantly reduced the assimilation rate of wild oat up
to 45, 34.2, 21.1 and 17.2% over weedy control, respectively. Inoculation with strain L9
and T42 significantly reduced the assimilation rate over strain W9, 7O₀ and O₀10.
Inoculation of wild oat with strain W9 performed significantly better than strain O₀10 in
reducing assimilation rate over weedy control. Weed suppression effects of strain L9,
T42, W9, 7O₀ and O₀10 recovered the loss in assimilation rate of infested wheat up to
96.0, 82.1, 69.1, 49.4 and 32.1%, respectively.
4.6.4.2. Stomatal conductance (gs)
Data indicated that infestation of wild oat significantly decreased stomatal conductance of
infested wheat up to 27.6% over weed free control (Table 4.22). Stomatal conductance of
wild oat was significantly reduced due to inoculation with strain L9, T42, W9, 7O₀ and
O₀10 up to 58.5, 46.2, 32, 26.3 and 14.4% over weedy control, respectively. Inoculation
of wild oat with strain L9 also significantly reduced the stomatal conductance over strain

99
Table 4.22. Effect of allelopathic bacteria on physiology of wild oat grown in wheat
and infested wheat in pot experiment
Wild oat Infested wheat
Treatments Assimilation Stomatal Assimilation Stomatal
rate (A) conductance (gs) rate (A) conductance (gs)
Weed free
-- -- 12.05 a 246.0 a
Control
Weedy
13.23 a 213.0 a 8.63 e 178.0 d
Control
T42 8.7 d 114.7 d 11.44 ab 232.3 ab

L9 7.27 d 88.3 e 11.92 a 246.0 a

7O₀ 10.97 bc 157.0 bc 10.32 cd 201.3 c

O₀10 12.07 ab 182.3 b 9.73 d 195.3 c

W9 10.43 c 144.7 c 11.0 bc 224.3 b

LSD 1.47 26.07 0.84 16.76

These measurements were taken at 60 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

100
T42, W9, 7O₀ and O₀10. Inoculation with strain T42 also significantly reduced the
stomatal conductance over W9, 7O₀ and O₀10. Inoculation of wild oat with strain W9
remained statistically similar with strain 7O₀ and significantly better than strain O₀10 in
reducing the stomatal conductance of wild oat. Weed suppression effects of strain L9,
T42, W9, 7O₀ and O₀10 recovered the loss in stomatal conductance of infested wheat up
to 100, 79.9, 68.1, 34.3 and 25.5%, respectively.
4.7. Effect of allelopathic bacteria on little seed canary grass grown in
wheat in pot conditions
Little seed canary grass was grown in wheat in pot conditions in three sets to be
harvested at three growth stages of the crop (tillering, booting and at harvesting). Data
regarding reduction in growth and yield parameters of little seed canary grass and
resultant improvement in growth and yield of infested wheat was recorded at these
growth stages. The results of this study are described below;
4.7.1. Effect of allelopathic bacteria on little seed canary grass grown in wheat and
infested wheat at tillering
4.7.1.1. Number of plants per pot
Infestation of little seed canary grass significantly reduced the number of tillers of
wheat up to 49.4% than weed free control (Table 4.23). Inoculation with strain L9, T42,
7O₀ and O₀10 significantly decreased its number of plants up to 35.5, 27.4, 24.4 and
15.6% over weedy control, respectively. Inoculation with strain 7O₀ produced
significantly less number of plants than strain L9, T42 and W9. Inoculation with strain
O₀10 and L9 also significantly reduced number of plants over strain T42 and W9. Weed
suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in number of
tillers of infested wheat up to 38.5, 84.6, 100.0, 89.7 and 7.7%, respectively (Photo 11).
4.7.1.2. Root length (cm)
Results showed that infestation of little seed canary grass significantly reduced the
root length of wheat up to 49.7% than weed free control (Table 4.23). Inoculation with
strain 7O₀, O₀10, L9 and T42 significantly decreased the root length of little seed canary
grass up to 40.2, 37.2, 32.9 and 19.2% over weedy control, respectively. Inoculation with
strain 7O₀ and O₀10 also significantly reduced the root length over strain T42 and W9.
Inoculation with strain L9 and T42 also produced root length of little seed canary grass
significantly less than strain W9. Resultantly, the loss in root length of infested wheat was
recovered up to 44.0, 84.5, 98.4, 91.6 and 4.9% due to inoculation with strain T42, L9,

101
Table 4.23. Effect of allelopathic bacteria on growth of little seed canary grass grown in wheat and infested wheat at tillering in pot
conditions
Little seed canary grass Infested wheat
Treatments No. of Root Shoot Root fresh Shoot fresh No. of Root Shoot Root fresh Shoot fresh
tillers Length Length weight weight tillers Length Length weight weight
pot-1 (cm) (cm) (g pot-1) (g pot-1) pot-1 (cm) (cm) (g pot-1) (g pot-1)
Weed free
-- -- -- -- -- 26.3 a 20.7 a 38.8 a 25.0 a 46.6 a
Control
Weedy
45.0 a 14.6 a 23.1 a 22.1 a 48.9 a 13.3 c 10.4 c 31.4 c 10.8 d 22.0 c
Control
T42 38.0 b 11.8 b 19.4 b 15.4 b 37.4 b 18.3 b 15.0 b 35.3 ab 17.1 c 28.0 b

L9 34.0 c 9.8 bc 15.4 c 12.6 bc 27.1 c 24.3 a 19.1 a 36.9 a 21.8 b 42.6 a

7O₀ 29.0 d 8.7 c 13.8 c 8.4 d 21.3 d 26.0 a 20.6 a 38.7 a 23.0 ab 45.3 a

O₀10 32.7 cd 9.2 c 15.0 c 11.0 cd 25.5 cd 25.0 a 19.9 a 37.3 a 22.3 ab 43.4 a

W9 41.3 ab 14.2 a 21.8 ab 19.8 a 45.5 a 14.3 c 10.9 c 33.0 bc 11.9 d 23.3 c

LSD 3.96 2.19 2.79 2.85 4.54 3.625 2.98 3.6 2.97 4.23
These measurements were taken at 50 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

102
 Wheat

Wheat
Little seed
canary grass

Weed free
Control Weedy Control 7O₀

Photo 11. Pictorial view of effect of allelopathic bacteria on little seed canary grass
grown in wheat at tillering (50 days after sowing)

103
7O₀, O₀10 and W9, respectively.
4.7.1.3. Shoot length (cm)
Infestation of little seed canary grass significantly reduced the shoot length of
wheat up to 19% than weed free control (Table 4.23). Shoot length of little seed canary
grass was significantly decreased due to its inoculation with strain T42, L9, O₀10 and
7O₀ up to 40, 35.1, 33.4 and 15.8% over weedy control, respectively. Inoculation with
strain 7O₀, O₀10 and L9 also significantly decreased shoot length over inoculation with
strain T42 and W9 while being statistically at par with each other. Weed suppression
effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in shoot length of infested
wheat up to 52.9, 75.1, 98.6, 80.1 and 22.2%, respectively.
4.7.1.4. Root fresh weight (g pot-1)
Results showed that growth of little seed canary grass in wheat significantly
reduced the root fresh weight of infested wheat up to 56.9% (Table 4.23). Root fresh
weight of little seed canary grass was significantly decreased due to its inoculation with
strain 7O₀, O₀10, L9 and T42 up to 62, 50.1, 43 and 30.3% as compared to weedy
control, respectively. Inoculation with strain 7O₀ also significantly reduced the root fresh
weight over inoculation with strain L9, T42 and W9. Inoculation of little seed canary
grass with strain O₀10 also significantly decreased its root fresh weight over strain T42
and W9. Inoculation with strain L9 and T42 remained statistically similar with each other
and significantly better than strain W9 in reducing the root fresh weight over weedy
control. Resultantly, the loss in root fresh weight of infested wheat was controlled up to
44.3, 77.3, 86.2, 81.3 and 8.0% due to inoculation with strain T42, L9, 7O₀, O₀10 and
W9, respectively.
4.7.1.5. Shoot fresh weight (g pot-1)
Infestation of little seed canary grass significantly decreased shoot fresh weight of
infested wheat up to 52.8% over weed free control (Table 4.23). Shoot fresh weight of
little seed canary grass was significantly reduced due to its inoculation with strain 7O₀,
O₀10, L9 and T42 up to 56.5, 47.9, 44.5 and 23.6% as compared to weedy control,
respectively. Inoculation with strain 7O₀ also significantly reduced shoot fresh weight
over inoculation with strain L9, T42 and W9. Inoculation with strain O₀10 and L9
produced significantly less shoot fresh weight as compared to strain T42 and W9.
Inoculation with strain T42 also significantly reduced the shoot fresh weight as compared
to inoculation with strain W9. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and

104
W9 recovered the loss in shoot fresh weight of infested wheat up to 24.5, 83.6, 94.7, 86.9
and 5.3%, respectively.
4.7.2. Effect of allelopathic bacteria on little seed canary grass grown in wheat and
infested wheat at booting in pot conditions
4.7.2.1. Number of tillers per pot
Infestation of little seed canary grass significantly reduced the number of tillers of
wheat up to 56% than weed free control (Table 4.24). Number of tillers of little seed
canary grass were significantly reduced up to 34.9, 28.6, 23.8 and 15% over weedy
control due to its inoculation with strain 7O₀, O₀10, L9 and T42, respectively. Inoculation
with strain 7O₀ produced significantly less number of plants than strain L9, T42 and W9.
Inoculation with strain O₀10 significantly decreased its number of plants over strain T42
and W9. Inoculation with strain L9 and T42 also performed significantly better than strain
W9 in reducing number of plants over weedy control while being statistically non-
significant with each other. Inoculation with strain W9 caused a non-significant decrease
in its number of plants as compared to un-inoculated control. Weed suppression effects of
strain T42, L9, 7O₀, O₀10 and W9 recovered the number of tillers of infested wheat up to
26.2, 64.3, 78.6, 71.4 and 2.4%, respectively.
4.7.2.2. Root length (cm)
Growth of little seed canary grass significantly reduced the root length of infested wheat
up to 56.3% over weed free control (Table 4.24). Inoculation with strain 7O₀, O₀10 and
L9 significantly decreased its root length up to 48.1, 43.2 and 40.1% as compared to un-
inoculated control, respectively. These three strains were statistically similar with each
other and significantly better than strain T42 and W9 in decreasing the root length of little
seed canary grass over un-inoculated control. Inoculation of little seed canary grass with
strain T42 significantly decreased its root length up to 20% over un-inoculated control
which was significantly less than strain W9. Root length of little seed canary grass was
also reduced up to 5% as compared to un-inoculated control but it was statistically similar
with un-inoculated control. Resultantly, the loss in root length of infested wheat was
recovered up to 33.3, 64.4, 75.0, 70.2 and 3.0% due to inoculation with strain T42, L9,
7O₀, O₀10 and W9, respectively.
4.7.2.3. Shoot length (cm)
Results showed that shoot length of wheat was significantly reduced up to 19.9%
due to infestation of little seed canary grass (Table 4.24). Shoot length of little seed

105
Table 4.24. Effect of allelopathic bacteria on growth of little seed canary grass grown in wheat at booting in pot conditions
Little seed canary grass Infested wheat
Treatments No. of Root Shoot Root fresh Shoot fresh No. of Root Shoot Root fresh Shoot fresh
tillers Length Length weight weight tillers Length Length weight weight
pot-1 (cm) (cm) (g pot-1) (g pot-1) pot-1 (cm) (cm) (g pot-1) (g pot-1)
Weed free
-- -- -- -- -- 25.0 a 31.8 a 51.3 a 44.6 a 68.3 a
control
Weedy
42.0 a 19.1 a 30.3 a 26.1 a 69.7 a 11.0 d 13.9 d 41.1 e 16.5 e 27.0 d
control
T42 35.7 b 15.3 b 25.7 b 18.3 b 52.9 c 14.7 c 19.9 c 44.9 cd 25.7 d 34.8 c

L9 32.0 bc 11.5 c 18.8 c 14.3 c 38.3 d 20.0 b 25.4 b 47.0 bc 32.9 c 52.0 b

7O₀ 27.3 d 9.9 c 17.0 c 9.9 d 30.6 e 22.0 ab 27.3 b 49.7 ab 38.2 b 55.4 b

O₀10 30.0 cd 10.9 c 18.1 c 13.0 c 36.1 d 21.0 b 26.5 b 48.1 a-c 33.4 c 53.4 b

W9 39.7 a 18.2 a 28.9 ab 23.4 a 63.6 b 11.3 d 14.4 d 43.0 de 18.1 e 27.5 d

LSD 3.9 1.95 3.33 2.7 5.32 3 2.87 3.47 3.17 4.87
These measurements were taken at 100 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

106
canary grass was significantly reduced due to its inoculation with strain 7O₀, O₀10, L9
and T42 up to 44, 40.2, 38.1 and 15.3% over weedy control, respectively. Inoculation
with strain 7O₀, O₀10 and L9 also produced significantly less shoot length over strain
T42 and W9 while being statistically at par with each other. Weed suppression effects of
strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in shoot length of infested wheat up
to 37.9, 58.5, 85.0, 69.0 and 18.9%, respectively.
4.7.2.4. Root fresh weight (g pot-1)
Infestation of little seed canary grass significantly decreased root fresh weight of
infested wheat up to 62.9% (Table 4.24). Inoculation with strain 7O₀, O₀10, L9 and T42
significantly decreased its root fresh weight up to 62.2, 50.1, 45.2 and 30% over weedy
control, respectively. Inoculation with strain 7O₀ also produced significantly less root
fresh weight over strain O₀10, L9, T42 and W9. Inoculation with strain O₀10 and L9
remained significantly better than strain T42 and W9 in reducing root fresh weight while
being statistically non-significant with each other. Inoculation with strain T42 also
significantly reduced root fresh weight as compared to strain W9. Weed suppression
effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in root fresh weight of
infested wheat up to 32.5, 58.4, 77.1, 60.1 and 5.6%, respectively.
4.7.2.5. Shoot fresh weight (g pot-1)
Results showed that shoot fresh weight of wheat was significantly reduced up to
60.45% due to infestation with little seed canary grass (Table 4.24). Shoot fresh weight of
little seed canary grass was significantly reduced due to its inoculation with strain 7O₀,
O₀10, L9, T42 and W9 up to 56.1, 48.2, 45.1, 24.2 and 8.7% as compared to weedy
control, respectively. Inoculation with strain 7O₀ also produced significantly less shoot
fresh weight over inoculation with strain O₀10, L9, T42 and W9. Inoculation with strain
O₀10 and L9 also significantly decreased shoot fresh weight over strain T42 and W9
while being statistically similar with each other. Inoculation with strain T42 also
significantly decreased the shoot fresh weight over strain W9. Weed suppression effects
of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in shoot fresh weight of infested
wheat up to 18.8, 66.7, 68.7, 64.0 and 1.1%, respectively.

107
4.7.3. Effect of allelopathic bacteria on little seed canary grass grown in wheat and
infested wheat at harvesting in pot conditions
4.7.3.1. Number of tillers per pot
Results showed that infestation of little seed canary grass significantly reduced
number of tillers of infested wheat up to 60.8% over weed free control (Table 4.25).
inoculation with strain T42, L9, 7O₀ and O₀10 significantly reduced the number of tillers
of little seed canary grass up to 40, 36, 34.4 and 18.4% as compared to weedy control,
respectively. Inoculation with strain 7O₀, O₀10 and L9 also significantly decreased
number of plants over strain T42 and W9 while being statistically non-significant with
each other. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the
loss in number of tillers of infested wheat up to 25.0, 56.3, 68.8, 60.4 and 2.1%,
respectively.
4.7.3.2. Shoot length (cm)
Data given in Table 4.25 indicated that shoot length of wheat was significantly
reduced up to 31.1% due to infestation of little seed canary grass. Inoculation with strain
T42, L9, 7O₀ and O₀10 significantly reduced the shoot length of little seed canary grass
up to 14.3, 38.2, 43.1 and 40.0% than weed control, respectively. Inoculation with strain
L9, 7O₀ and O₀10 also significantly reduced the shoot length of little seed canary grass
than strain T42 and W9. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9
recovered the loss in shoot length of infested wheat up to 20.1, 31.5, 44.4, 36.2 and 3.1%,
respectively.
4.7.3.3. Root fresh weight (g pot-1)
Infestation of little seed canary grass significantly decreased the root fresh weight of
infested wheat up to 62.8% over weed free control (Table 4.25). Inoculation with strain
7O₀, O₀10, L9 and T42 significantly decreased its root fresh weight up to 56, 48.1, 45.1
and 25.1% over weedy control, respectively. Inoculation with strain 7O₀ and O₀10 also
significantly reduced root fresh weight over strain T42 and W9 while statistically similar
with each other. Inoculation with strain L9 significantly reduced root fresh weight over
strain W9. Resultantly, the loss in root fresh weight of infsted wheat was controlled up to
32.7, 57.0, 73.5, 62.3 and 3.8% due to inoculation with strain T42, L9, 7O₀, O₀10 and
W9, respectively.

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Table 4.25. Effect of allelopathic bacteria on growth of little seed canary grass grown in wheat and infested wheat at harvesting in pot
conditions
Little seed canary grass Infested wheat
Treatments No. of Shoot Root fresh Straw Grain No. of Shoot Root fresh Grain Straw
tillers length weight yield yield tillers length weight yield yield
pot-1 (cm) (g pot-1) (g pot-1) (g pot-1) pot-1 (cm) (g pot-1) (g pot-1) (g pot-1)
Weed free
-- -- -- -- -- 26.33 a 59.2 a 24.7 a 21.2 a 33.4 a
control
Weedy
41.7 a 36.0 a 15.6 a 27.0 a 5.1 a 10.3 d 40.8 e 9.2 d 8.5 d 12.5 d
Control
T42 34.0 b 30.9 b 11.7 bc 20.7 b 4.2 b 14.33 c 44.5 cd 14.3 c 11.0 c 17.0 c

L9 27.3 c 22.2 c 8.6 cd 15.2 c 3.8 bc 19.3 b 46.6 bc 18.0 b 15.4 b 23.6 b

7O₀ 25.0 c 20.5 c 6.9 d 12.9 c 3.1 c 21.3 b 49.0 b 20.6 b 17.0 b 25.7 b

O₀10 26.7 c 21.6 c 8.1 d 14.3 c 3.5 bc 20.0 b 47.5 bc 18.9 b 16.0 b 24.2 b

W9 38.3 ab 34.4 ab 14.5 ab 25.0 a 5.2 a 10.7 d 41.4 de 9.8 d 8.9 cd 13.4 d

LSD 5.87 4.03 3.24 4.29 0.82 2.96 3.47 3.05 2.36 2.65
These measurements were taken at 150 days afte sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

109
4.7.3.3. Straw yield (g pot-1)
Data presented in Table 4.25 indicated that infestation of wheat with little seed
canary grass significantly reduced its straw yield up to 62.6% over weed free control.
Inoculation with strain 7O₀, O₀10 and L9 significantly decreased straw yield of little seed
canary grass up to 52.2, 46.9 and 43.8% as compared to weedy control, respectively.
These three strains were statistically similar with each other and significantly better than
strain T42 and W9 in reducing the straw yield of little seed canary grass. Inoculation of
little seed canary grass with strain T42 also significantly decreased its straw yield as
compared to un-inoculated control which was also significantly less than strain W9.
Straw yield of little seed canary grass was decreased by 7.4% due to inoculation with
strain W9 which was statistically at par with un-inoculated control. Weed suppression
effects of strain T42, L9, 7O₀, O₀10 and W9 reocovered the loss in straw yield of infested
wheat up to 21.5, 53.1, 63.2, 56.1 and 4.3%, respectively.
4.7.4.4. Grain yield (g pot-1)
Grain yield of wheat was significantly reduced up to 59.9% due to infestation of
little seed canary grass (Table 4.25). Grain yield of little seed canary grass was
significantly reduced due to inoculation with strain 7O₀, O₀10, L9 and T42 up to 40, 31.3,
25.7 and 17.3% over weedy control, respectively. Inoculation with strain 7O₀ also
produced significantly less reproductive capacity over strain T42 and W9, and remained
statistically non-significant as compared to strain O₀10 and L9. Inoculation with strain
O₀10, L9 and T42 performed significantly better than strain W9 in reducing the grain
yield over weedy control, while being non-significant with each other. Weed suppression
effects of strain T42, L9, 7O₀, O₀10 and W9 resultantly recovered the loss in grain yield
of infested wheat up to 20.1, 55.0, 66.9, 59.0 and 3.4%, respectively.
4.7.5. Effect of allelopathic bacteria on physiology of little seed canary grass grown
in wheat and infested wheat in pot conditions
4.7.5.1. Assimilation rate (A)
Results indicated that assimilation rate of wheat was significantly decreased up to
27.1% due to infestation of little seed canary grass (Table 4.26). Assimilation rate of little
seed canary grass was significantly decreased due to inoculation with strain 7O₀, O₀10,
L9 and T42 up to 25.6, 21.1, 20.5 and 14.7% as compared to un-inoculated control,
respectively. These three strains remained statistically similar with each other and
significantly better than strain T42 and W9 in reducing the assimilation rate as compared

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Table 4.26. Effect of allelopathic bacteria on physiology of little seed canary grass
grown in wheat and infested wheat in pot conditions
Little seed canary grass Infested wheat
Treatments Stomatal Stomatal
Assimilation Assimilation
conductance conductance
rate (A) rate (A)
(gs) (gs)
Weed free
-- -- 11.64 a 233.0 a
Control
Weedy
5.2 a 151.3 a 8.73 c 181.3 e
Control
T42 4.43 bc 135.0 a 10.33 b 202.3 d

L9 4.13 c 108.0 b 11.55 a 216.0 c

7O₀ 3.87 c 87.67 c 11.98 a 228.7 ab

O₀10 4.1 c 104.7 bc 11.63 a 218.0 bc

W9 4.83 ab 146.0 a 9.78 b 192.0 de

LSD 0.58 18.6 0.551 12.63

These measurements were taken at 60 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

111
to weedy control. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9
recovered the loss in assimilation rate of infested wheat up to 49.2, 86.8, 89.6, 89.2 and
32.3%, respectively.
4.7.5.2. Stomatal conductance (gs)
Results showed that infestation of little seed canary grass significantly reduced the
stomatal conductance of infested wheat up to 22.2% over weed free control (Table 4.26).
Inoculation with strain 7O₀, O₀10 and L9 significantly reduced its stomatal conductance
up to 42, 30.8 and 28.6% as compared to weedy control, respectively. Inoculation with
these 3 strains performed ignificantly better than strain T42 and W9 in reducing stomatal
conductance over weedy control, and remained non-significant with each other. Weed
suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in
assimilation rate of infested wheat up to 40.6, 67.1, 91.6, 71.0 and 20.6%, respectively.
4.8. Effect of allelopathic bacteria on broad leaved dock grown in wheat
in pot conditions
Five efficient strains of allelopathic bacteria were selected to conduct pot
experiment on suppression of broad leaved dock grown in wheat and resultant
improvement in growth and yield of infested wheat. Three sets of pots were arranged to
collect data at three growth stages of the crop (tillering, booting and at harvesting). The
results of these studies are described below;

4.8.1. Effect of allelopathic bacteria on broad leaved dock grown in wheat at tillering
in pot conditions

4.8.1.1. Number of plants per pot

Data indicated that infestation of broad leaved dock significantly decreased the
number of tillers of infested wheat up to 45.4% over weed free control (Table 4.27).
Inoculation with strain W9, T42, L9, 7O₀ and O₀10 significantly reduced the number of
plants of broad leaved dock up to 35.8, 32.8, 29.8, 22.4 and 13.4% as compared to weedy
control, respectively. Inoculation with strain W9 also significantly reduced number of
plants as compared to strain 7O₀ and O₀10. Inoculation with strain L9 and T42 also
performed significantly better than strain O₀10 in reducing number of plants over weedy
control. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the
loss in number of tillers of infested wheat up to 37.1, 54.3, 45.7, 17.1 and 80.0%,
respectively.

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Table 4.27. Effect of allelopathic bacteria on broad leaved dock grown in wheat and infested wheat at tillering in pot conditions
Broad leaved dock Infested wheat
Treatments No. of Root Shoot Root fresh Shoot fresh No. of Root Shoot Root fresh Shoot fresh
plants length length weight weight tillers length length weight weight
pot-1 (cm) (cm) (g pot-1) (g pot-1) pot-1 (cm) (cm) (g pot-1) (g pot-1)
Weed free
-- -- -- -- -- 25.7 a 19.5 a 35.3 a 27.3 a 37.9 a
Control
Weedy
22.3 a 17.7 a 21.3 a 10.0 a 35.9 a 14.0 e 11.0 d 26.3 c 12.4 c 18.0 c
Control
T42 15.7 cd 11.9 bc 17.2 ab 6.7 bc 25.7 bc 18.3 cd 14.8 bc 31.6 a-c 20.8 b 29.2 b
L9 15.0 cd 9.4 cd 16.3 ab 5.2 cd 21.5 cd 20.3 bc 16.7 ab 32.9 ab 23.6 ab 30.6 b
7O₀ 17.3 bc 10.8 cd 17.5 ab 6.0 c 23.6 b-d 19.3 b-d 15.6 bc 32.2 ab 21.7 b 29.7 b
O₀10 19.3 b 14.1 b 19.3 ab 7.9 b 30.5 ab 16.0 de 13.7 cd 28.3 bc 15.7 c 21.8 c
W9 14.3 d 7.9 d 14.9 b 4.1 d 17.9 d 23.3 ab 17.3 ab 34.2 a 25.9 a 35.5 a
LSD 2.68 3.029 5.42 1.7 7.32 4.06 2.95 5.42 4.12 4.56
These measurements were taken at 50 days afte sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

113
4.8.1.2. Root length (cm)
Results showed that root length of wheat was significantly reduced up to 43.8%
due to infestation of broad leaved dock (Table 4.27). Root length of broad leaved dock
was significantly reduced due to inoculation with strain W9, L9, 7O₀, T42 and O₀10 up to
55.3, 47, 38.9, 32.9 and 20.3% as compared to weedy control, respectively. Inoculation
with strain W9 also significantly reduced root length as compared to inoculation with
strain T42 and O₀10. Inoculation with strain L9 and 7O₀ produced significantly less root
length as compared to inoculation with strain O₀10. Inoculation with strain T42 and O₀10
remained statistically similar with each other in reducing root length over weedy control.
Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in
number of tillers of infested wheat up to 45.3, 67.2, 53.9, 32.4 and 74.6%, respectively.
4.8.1.3. Shoot length (cm)
Data given in Table 4.27 indicated that infestation of broad leaved dock
significantly decreased the shoot length of infested wheat up to 25.5% over weed free
control. Shoot length of broad leaved dock was significantly decreased due to inoculation
with strain W9 up to 30% as compared to weedy control. Inoculation with strain W9
remained statistically non-significant with strain L9, T42, 7O₀ and O₀10. Inoculation with
these 4 strains remained statistically non-significant with each other and weedy control.
Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in
number of tillers of infested wheat up to 58.5, 73.3, 64.8, 21.8 and 87.8%, respectively.
4.8.1.4. Root fresh weight (g pot-1)
Root fresh weight of wheat was significantly reduced up to 54.6% due to
infestation of broad leaved dock (Table 4.27). Inoculation with strain W9, L9, 7O₀, T42
and O₀10 significantly decreased the root fresh weight of broad leaved dock up to 59.1,
48.2, 40.2, 33.6 and 21.3% as compared to weedy control, respectively. Inoculation with
strain W9 also significantly reduced the root fresh weight over strain 7O₀, T42 and O₀10.
Inoculation with strain L9 and 7O₀ performed significantly better than strain O₀10 in
decreasing the root fresh weight over weedy control. Resultantly, the loss in root fresh
weight of infested wheat was recovered up to 56.5, 74.8, 62.3, 22.1 and 90.2% due to
inoculation with strain T42, L9, 7O₀, O₀10 and W9, respectively.
4.8.1.5. Shoot fresh weight (g pot-1)
Shoot fresh weight of infested wheat was significantly reduced up to 52.5% over
weed free control due to infestation of broad leaved dock (Table 4.27). Shoot fresh weight

114
of broad leaved dock was significantly reduced due to inoculation with strain W9, L9,
7O₀ and T42 up to 50.1, 40, 34.3 and 28.4% as compared to weedy control, respectively.
Inoculation with strain W9 also significantly decreased shoot fresh weight over strain T42
and O₀10. Inoculation with strain L9 produced significantly less shoot fresh weight as
compared to strain O₀10. Inoculation with strain O₀10 caused non-significant decrease of
14.9% as compared to weedy control. Weed suppression effects of strain T42, L9, 7O₀,
O₀10 and W9 recovered the loss in number of tillers of infested wheat up to 56.2, 63.6,
59.1, 19.1 and 87.9%, respectively.
4.8.2. Effect of allelopathic bacteria on broad leaved dock grown in wheat and
infested wheat at booting in pot conditions
4.8.2.1. Number of plants per pot
Data given in Table 4.28 indicated that growth of broad leaved dock in wheat
significantly decreased its number of tillers up to 52.5%. Inoculation with strain W9, L9,
7O₀ and T42 significantly decreased its number of plants up to 30, 26.7, 11.7 and 21.7%
as compared to weedy control, respectively. Inoculation with strain W9, L9 and T42 also
significantly reduced number of plants over inoculation with strain O₀10. Weed
suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in number of
tillers of infested wheat up to 33.3, 47.6, 38.1, 11.9 and 54.8%, respectively (Photo 12).
4.8.2.2. Root length (cm)
Results indicated that infestation of broad leaved dock significantly reduced the
root length of wheat up to 50.4% than weed free control (Table 4.28). Root length of
broad leaved dock was significantly inhibited due to inoculation with strain W9, L9, 7O₀,
T42 and O₀10 up to 42.4, 35, 31.3, 27.2 and 19.5% as compared to weedy control,
respectively. Inoculation with strain W9 also significantly reduced root length over strain
7O₀, T42 and O₀10. Inoculation with strain L9 and 7O₀ produced significantly less root
length than strain O₀10. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9
recovered the loss in root length of infested wheat up to 47.4, 62.4, 55.4, 17.6 and 71.0%,
respectively.
4.8.2.3. Shoot length (cm)
Results indicated that shoot length of wheat was significantly decreased up to
22.7% due to infestation of broad leaved dock (Table 4.28). Inoculation with strain W9,
L9, 7O₀, T42 and O₀10 significantly decreased its shoot length up to 28.8, 25, 20, 18.2
and 12.5% as compared to weedy control, respectively. Inoculation with strain W9 and

115
Table 4.28. Effect of allelopathic bacteria on broad leaved dock grown in wheat at booting in pot conditions
Broad leaved dock Infested wheat
Root
Treatments No. of Root Shoot
fresh
Shoot fresh No. of Root Shoot Root fresh Shoot fresh
plants length length weight tillers length length weight weight
weight
pot-1 (cm) (cm) (g pot-1
) pot -1
(cm) (cm) (g pot-1) (g pot-1)
(g pot-1)
Weed free
-- -- -- -- -- 26.7 a 30.8 a 52.4 a 43.77 a 68.4 a
Control
Weedy
20.0 a 22.2 a 27.2 a 14.1 a 50.7 a 12.7 d 15.3 d 40.5 d 16.6 d 25.7 d
Control
T42 15.0 c 16.1 bc 22.2 bc 9.3 bc 35.4 c 17.3 bc 22.7 c 47.4 bc 27.77 c 42.5 c
L9 14.7 c 14.4 cd 20.4 c 7.7 c 32.4 cd 19.3 b 25.0 bc 48.6 ab 29.9 bc 44.3 c
7O₀ 15.7 bc 15.2 c 21.7 bc 8.9 c 34.4 c 18.0 b 23.9 bc 47.8 ab 28.43 c 43.3 c
O₀10 17.7 ab 17.8 b 23.8 b 10.6 b 42.7 b 14.3 cd 18.0 d 42.9 cd 19.5 d 28.3 d
W9 14.0 c 12.8 d 19.3 c 6.0 d 27.4 d 20.3 b 26.3 b 50.7 ab 33.2 b 48.6 b
LSD 2.62 2.38 3.12 1.69 6.53 3.5 3.62 4.72 3.61 3.51
These measurements were taken at 100 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

116
 Wheat
Wheat

Broad
leaved dock

Broad
leaved dock

Weed free Control W9 Weedy Control

Photo 12. Pictorial view of effect of allelopathic bacteria on broad leaved dock grown in
wheat at booting (100 days after sowing)

117
L9 also significantly reduced shoot length over inoculation with O₀10. Inoculation with
strain 7O₀, T42 and O₀10 remained statistically non-significant with each other in
reducing shoot length over weedy control. Weed suppression effects of strain T42, L9,
7O₀, O₀10 and W9 recovered the loss in shoot length of infested wheat up to 58.3, 68.3,
61.6, 20.4 and 85.4%, respectively.
4.8.2.4. Root fresh weight (g pot-1)
Root fresh weight of wheat was significantly decreased up to 62% due to
infestation of broad leaved dock (Table 4.28). Inoculation strain W9, L9, 7O₀, T42 and
O₀10 significantly reduced the root fresh weight up to 57.2, 45.1, 37.1, 34 and 25.1% as
compared to weedy control, respectively (Table 4.36). Inoculation with strain W9 also
significantly reduced root fresh weight over inoculation with strain L9, 7O₀, T42 and
O₀10. Inoculation with strain L9 and 7O₀ also performed significantly better than strain
O₀10 in reducing root fresh weight over weedy control. Weed suppression effects of
strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in root fresh weight of infested
wheat up to 41.1, 49.0, 43.6, 10.7 and 61.1%, respectively.
4.8.2.5. Shoot fresh weight (g pot-1)
Results shown in Table 4.28 indicated that infestation of wheat with broad leaved
dock caused significant reduction in its shoot fresh weight up to 62.4%. Shoot fresh
weight of broad leaved dock was significantly reduced up to 46, 36, 32.1, 30.1 and 15.7%
over weedy control due to its inoculation with strain W9, L9, 7O₀, T42 and O₀10,
respectively. Inoculation with W9 also significantly reduced shoot fresh weight over
inoculation with strain 7O₀, T42 and O₀10. Inoculation with strain L9, 7O₀ and T42
remained statistically non-significant with each other and significantly better than strain
O₀10 in reducing shoot fresh weight over weedy control. Weed suppression effects of
strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in shoot fresh weight of infested
wheat up to 39.3, 43.6, 41.2, 6.2 and 53.7%, respectively.
4.8.3. Effect of allelopathic bacteria on broad leaved dock grown in wheat and
infested wheat at harvesting in pot conditions
4.8.3.1. Number of tillers per pot
Data given in Table 4.29 indicated that number of tillers of wheat were
significantly decreased up to 52.9% due to infestation of broad leaved dock. Number of
plants of broad leaved dock were significantly decreased up to 39, 35.6, 33.9, 27.1 and
15.2% as compared to weedy control due to inoculation with strain W9, L9, 7O₀, T42 and

118
Table 4.29. Effect of allelopathic bacteria on broad leaved dock grown in wheat and infested wheat at harvesting in pot conditions
Broad leaved dock Infested wheat
Treatments No. of Shoot Root fresh Grain Straw No. of Shoot Root fresh Grain
Straw yield
plants length weight yield yield tillers length weight yield
(g pot-1)
pot-1 (cm) (g pot-1) (g pot-1) (g pot-1) pot-1 (cm) (g pot-1) (g pot-1)
Weed free
-- -- -- -- -- 23.3 a 62.8 a 25.43 a 22.71 a 32.82 a
Control
Weedy
19.7 a 42.5 a 9.4 a 6.5 a 19.8 a 11.0 c 45.7 c 10.47 c 10.03 d 14.86 d
Control
T42 13.0 c 32.5 bc 6.1 c 4.0 c 13.5 bc 17.7 b 54.1 b 17.67 b 15.75 c 24.56 c

L9 12.7 c 30.9 c 5.6 c 3.6 c 12.7 c 18.7 b 55.5 b 18.88 b 16.85 bc 25.59 bc

7O₀ 14.3 bc 32.2 bc 6.0 c 3.8 c 13.1 c 18.0 b 54.6 b 18.12 b 15.89 c 24.78 c

O₀10 16.7 b 36.1 b 7.6 b 5.2 b 16.2 b 12.7 c 48.7 c 13.22 c 11.49 d 17.21 d

W9 12.0 c 29.7 c 5.1 c 3.1 c 11.1 c 20.0 ab 57.4 b 20.33 b 18.65 b 27.88 b

LSD 2.44 4.23 1.53 1.21 2.9 3.46 3.3 2.88 2.38 2.64
These measurements were taken at 150 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

119
O₀10, respectively. Inoculation with strain W9, L9 and T42 performed significantly better
than strain O₀10 in reducing the number of plants over weedy control. Weed suppression
effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in number of tillers of
infested wheat up to 29.7, 40.5, 35.1, 8.1 and 51.4%, respectively.
4.8.3.2. Shoot length (cm)
Results showed that shoot length of infested wheat was significantly decreased up
to 27.2% due to infestation of broad leaved dock (Table 4.29). Inoculation of broad
leaved dock with strain W9, L9, 7O₀, T42 and O₀10 significantly decreased its shoot
length up to 30.2, 27.2, 24.3, 23.5 and 15% as compared to weedy control, respectively.
Inoculation with strain W9 and L9 also significantly reduced shoot length over strain
O₀10. Inoculation with strain 7O₀, T42 and O₀10 remained statistically non-significant
with each other in reducing shoot length over weedy control. Weed suppression effects of
strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in shoot length of infested wheat up
to 49.1, 57.1, 51.7, 17.1 and 68.2%, respectively.
4.8.3.3. Root fresh weight (g pot-1)
Infestation of broad leaved dock significantly decreased the root fresh weight of
infested wheat up to 58.8% over weed free control (Table 4.29). Inoculation with strain
W9, L9, 7O₀ and T42 significantly decreased its root fresh weight up to 45.8, 40.1, 36.5
and 35.1% as compared to un-inoculated control, respectively. These four strains were
statistically at par with each other and significantly less than strain O₀10 regarding
production of root fresh weight of broad leaved dock. Root fresh weight of broad leaved
dock was also significantly reduced due to inoculation with strain O₀10 up to 18.7% as
compared to un-inoculated control. Resultantly, the loss in root fresh weight of infested
wheat was recovered up to 48.2, 56.3, 51.0, 18.0 and 66.0% due to inoculation with strain
T42, L9, 7O₀, O₀10 and W9, respectively.
4.8.3.4. Grain yield (g pot-1)
Infestation of broad leaved dock significantly decreased the grain yield of infested
wheat up to 86% as compared to weed free control (Table 4.29). Grain yield of broad
leaved dock was significantly reduced due to inoculation with strain W9, L9, 7O₀, T42
and O₀10 up to 51.3, 44.8, 41.6, 38.9 and 19.6% as compared to weedy control,
respectively. Inoculation with strain W9, L9, 7O₀ and T42 performed significantly better
than strain O₀10 in decreasing the grain yield of broad leaved dock while being
statistically at par with each other. Weed suppression effects of strain T42, L9, 7O₀, O₀10

120
and W9 recovered the loss in grain yield of infested wheat up to 45.2, 53.9, 46.3, 11.6 and
68.0%, respectively.
4.8.3.5. Straw yield (g pot-1)
Data given in Table 4.29 indicated that infestation of broad leaved dock
significantly decreased the straw yield of infested wheat up to 54.7% as compared to
weed free control. Straw yield of broad leaved dock was significantly reduced up to 44,
35.5, 33.7, 31.6 and 17.9% over weedy control due to its inoculation with strain W9, L9,
7O₀, T42 and O₀10. Inoculation with strain W9, L9 and 7O₀ performed significantly
better than strain O₀10 in reducing the straw yield over weedy control. Weed suppression
effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in number of tillers of
infested wheat up to 54.0, 59.8, 55.6, 13.0 and 72.6%, respectively.
4.8.7. Effect of allelopathic bacteria on physiology of broad leaved dock grown in
wheat and infested wheat in pot conditions
4.8.7.1. Assimilation rate (A)
Data given in Table 4.30 indicated that assimilation rate of wheat was
significantly reduced up to 28.6% due to infestation of broad leaved dock. Assimilation
rate of broad leaved dock was significantly reduced up to 40.2, 35.4, 31.8, 29.9 and
17.5% over weedy control due to inoculation with strain W9, L9, 7O₀, T42 and O₀10,
respectively. Inoculation with strain W9 also significantly reduced assimilation rate over
strain 7O₀, T42 and O₀10. Inoculation with strain L9, 7O₀ and T42 remained statistically
similar with each other, and significantly less than strain O₀10 in reducing assimilation
rate over weedy control. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9
recovered the loss in assimilation rate of infested wheat up to 71.9, 84.6, 73.8, 40.0 and
99.5%, respectively.
4.8.7.2. Stomatal conductance (gs)
Infestation of broad leaved dock significantly reduced the stomatal conductance of
wheat up to 28% than weed free control (Table 4.30). Stomatal conductance of broad
leaved dock was significantly decreased up to 45, 36.1, 32, 30.3 and 20.1% over weedy
control due to inoculation with strain W9, L9, 7O₀, T42 and O₀10, respectively (Table
4.40). Inoculation with strain W9 significantly reduced the stomatal conductance of broad
leaved dock over other strains. Inoculation with strain L9, 7O₀ and T42 performed
significantly better than strain O₀10 in reducing stomatal conductance over weedy control
which remained statistically similar with each other. Weed suppression effects of strain

121
Table 4.30. Effect of allelopathic bacteria on physiology of broad leaved dock grown
in wheat and infested wheat in pot conditions
Broad leaved dock Infested wheat
Treatments Assimilation Stomatal Assimilation Stomatal
rate conductance rate conductance
(A) (gs) (A) (gs)
Weed free
-- -- 11.85 a 241.3 a
Control
Weedy
15.83 a 394.7 a 8.46 e 173.7 e
Control
T42 11.1 c 275.0 c 10.9 c 213.3 c

L9 10.23 cd 252.3 c 11.33 b 225.3 bc

7O₀ 10.8 c 268.3 c 11.0 bc 215.7 c

O₀10 13.07 b 315.3 b 9.8 d 187.7 d

W9 9.47 d 217.0 d 11.83 a 234.7 ab

LSD 0.945 23.4 0.381 12.14

These measurements were taken at 60 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

122
T42, L9, 7O₀, O₀10 and W9 recovered the loss in stomatal conductance of infested wheat
up to 58.6, 76.4, 62.1, 20.7 and 90.1%, respectively.
4.9. Effect of allelopathic bacteria on the growth and yield of wheat
grown under weed free conditions in pot experiment
The selected strains of allelopathic bacteria for above discussed experiments (T42,
7O₀, O₀10, L9 and W9) were applied to wheat under weed free conditions to examine
their effects on growth and yield of wheat in the absence of weeds. Data regarding growth
and yield parameters of the crop were recorded at three growth stages (tillering, booting
and at harvesting). The results of this experiment are explained below;
4.9.1. Effect of allelopathic bacteria on growth of wheat in the absence of weeds at
tillering in pot conditions
Results indicated that none of the strains caused significant reduction in number of
tillers, root length, shoot length, root fresh weight and shoot fresh weight of wheat grown
under weed free conditions at tillering stage (Table 4.31). However, inoculation with
strain L9 and 7O₀ significantly increased the number of tillers of wheat up to 21.1 and
16.9% than control, respectively. These strains also caused significant increase in root
length up to 28 and 22%, shoot length up to 21.3 and 18.1%, root fresh weight up to 45.3
and 36.3%, and shoot fresh weight up to 38 and 32.1% as compared to un-inoculated
control, respectively.
4.9.2. Effect of allelopathic bacteria on growth of wheat in the absence of weeds at
booting under pot conditions
Data given in Table 4.32 indicated that none of the strains of allelopathic bacteria caused
any significant decrease in number of tillers, root length, shoot length, root fresh weight
and shoot fresh weight of wheat grown under weed free conditions at booting stage.
However, inoculation with strain L9 and 7O₀ significantly increased the number of tillers
of wheat up to 18.2 and 15.6% than un-inoculated control. Inoculation with these strains
also caused significant increase in root length up to 30.2 and 24.1%, shoot length up to
25.8 and 19.8%, root fresh weight up to 38.5 and 32.5%, and shoot fresh weight up to 43
and 35.3% as compared to un-inoculated control, respectively.

123
Table 4.31. Effect of allelopathic bacteria on growth of wheat in the absence of
weeds at tillering in pot conditions
Root Shoot Root fresh Shoot fresh
No. of
Treatments Length length weight weight
tillers pot-1
(cm) (cm) (g pot-1) (g pot-1)
Control 23.7 c 18.07 b 31.63 b 20.1 b 36.47 b

T42 25.0 bc 19.13 b 33.07 b 22.1 b 38.03 b

L9 28.7 a 23.13 a 38.36 a 29.2 a 50.33 a

7O₀ 27.7 ab 22.07 a 37.36 a 27.4 a 48.17 a

O₀10 25.0 bc 18.9 b 32.5 b 21.63 b 38.4 b

W9 24.3 c 18.43 b 31.9 b 20.43 b 37.6 b

LSD 3.27 2.74 3.84 4.09 4.48

These measurements were taken at 50 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

Table 4.32. Effect of allelopathic bacteria on growth of wheat in the absence of

weeds at booting in pot conditions
Shoot
No. of Root Shoot Root fresh
fresh
Treatments tillers per Length length weight
weight
pot (cm) (cm) (g pot-1)
(g pot-1)
Control 25.67 c 29.16 b 48.5 b 44.94 b 83.36 b

T42 27.33 a-c 30.76 b 51.37 b 48.17 b 90.71 b

L9 30.33 a 37.97 a 61.03 a 62.23 a 119.21 a

7O₀ 29.67 ab 36.2 a 58.1 a 59.56 a 112.77 a

O₀10 27.0 a-c 30.6 b 50.1 b 48.61 b 88.43 b

W9 26.33 bc 31.43 b 49.67 b 47.9 b 88.55 b

LSD 3.38 4.32 5.49 5.89 13.31

These measurements were taken at 100 days after sowing. Values sharing same letter(s)
do not differ significantly from each other at p <0.05 according to Duncan’s Multiple
Range Test

124
4.9.3. Effect of allelopathic bacteria on growth and yield of wheat grown in the
absence of weeds at harvesting in pot conditions
Data indicated that inoculation with the selected strains of allelopathic bacteria did
not cause any significant reduction in growth and yield of wheat grown under weed free
conditions at harvesting (Table 4.33). However, inoculation with strain L9 and 7O₀
significantly increased the number of tillers up to 17.8 and 16.4%, shoot length up to 31.2
and 23.5%, root fresh weight up to 42 and 34.2%, grain yield up to 40.3 and 34.3%, and
straw yield up to 37.1 and 32.1% as compared to un-inoculated control, respectively
(Photo 13).
4.9.4. Effect of allelopathic bacteria on physiology of wheat grown in the absence of
weeds in pot conditions
Allelopathic bacteria did not cause any significant reduction reduction in the
physiological parameters (assimilation rate, stomatal conductance and evapo-transpiration
rate) of wheat grown under weed free conditions (Table 4.34). However, inoculation with
strain L9 and 7O₀ significantly increased the assimilation rate of wheat up to 21.5 and
15.3%, stomatal conductance up to 43.2 and 30.1%, and evapo-transpiration rate up to
23.8 and 18.2% than un-inoculated control, respectively.

125
Table 4.33. Effect of allelopathic bacteria on growth and yield of wheat in the
absence of weeds at harvesting in pot conditions
Shoot Root fresh Grain Straw
No. of
Treatments length weight yield (g yield
tillers pot-1
(cm) (g pot-1) pot-1) (g pot-1)
Control 24.33 b 56.67 b 25.83 b 22.33 b 31.23 b

T42 26.0 ab 61.66 b 27.78 b 24.0 b 33.1 b

L9 28.67 a 74.33 a 36.69 a 31.33 a 42.83 a

7O₀ 28.33 a 70.0 a 34.68 a 30.0 a 41.26 a

O₀10 25.33 b 61.0 b 27.2 b 23.66 b 32.63 b

W9 25.0 b 59.66 b 26.0 b 22.66 b 32.26 b

LSD 2.75 6.51 4.49 3.43 4.14

These measurements were taken at 150 days after sowing. Values sharing same letter(s)
do not differ significantly from each other at p <0.05 according to Duncan’s Multiple
Range Test

Table 4.34. Effect of allelopathic bacteria on physiology of wheat grown in the

absence of weeds in pot conditions
Evapo-
Assimilation rate Stomatal
Treatments transpiration rate
(A) conductance (gs)
(E)
Control 9.6 b 185.0 b 3.35 b

T42 9.6 b 191.0 b 3.33 b

L9 11.67 a 265.0 a 4.15 a

7O₀ 11.07 a 240.7 a 3.96 a

O₀10 9.67 b 184.7 b 3.37 b

W9 9.63 b 188.7 b 3.35 b

LSD 1.4 26.86 0.32

These measurements were taken at 60 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

126
Photo 13. Pictorial view of effect of allelopathic bacteria on wheat grown under weed
free conditions at harvesting (150 days after sowing)

127
Field Experiments
Three field trials were conducted to evaluate the bioherbicidal activity of
allelopathic bacteria under natural conditions at different locations. Field trial-I was
conducted at Post-graduate Agriculture Research Station (PARS), University of
Agriculture, Faisalabad. Field trial-II was conducted at farmer’s field at Chak No. 75 RB
Lohkay, Faisalabad. Third field trial was conducted at Research Area of Institute of Soil
and Environmental Sciences, University of Agriculture, Faisalabad. Field trial-I was
infested with broad leaved dock and common lambs’ quarter, Field trial-II was infested
with little seed canary grass while Field trial-III was infested with wild oat and little seed
canary grass. The presence of other weeds was also noted in these trials but the effects of
allelopathic bacteria could not be studied on these weeds due to their low density.
4.10. Effect of allelopathic bacteria on wheat and its associated weeds in
field trial-I
The field area was divided into two halves; one to study suppression of weeds
grown in wheat and resultant improvement in growth and yield of wheat while other to
study effect of allelopathic bacteria on growth and yield of wheat under weed free
conditions. Data regarding growth and yield parameters of wheat and its associated weeds
was collected at three different stages (tillering, booting and harvesting). The results of
these studies are explained below;
4.10.1. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at
tillering
4.10.1.1. Density (number of plants m-2)
Infestation of weeds significantly reduced the number of tillers of infested wheat up to
50.8% than weed free control (Table 4.35). Inoculation with strain T42, 7O₀, L9 and W9
significantly decreased density of broad leaved dock up to 24.2, 22.3, 30.1 and 31.1% as
compared to weedy control, respectively. Inoculation with strain L9 and W9 also
produced significantly less density of broad leaved dock than strain O₀10. Inoculation
with strain O₀10 caused a non-significant decrease in density of broad leaved dock up to
10.1% over weedy control. None of the selected strains caused any significant reduction
in density of common lambs’ quarter than weedy control. Weed suppression effects of
strain T42, L9, 7O₀, O₀10 and W9 resultantly recovered the loss in number of tillers of
infested wheat up to 25.7, 44.8, 35.8, 12.0 and 58.3%, respectively.

128
Table 4.35. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at tillering

Broad leaved dock Common lambs’ quarter Infested wheat

Treatments Density Fresh Shoot Density Fresh Shoot No. of Fresh Shoot
(no. of biomass length (no. of biomass length tillers biomass length
plants m-2) (t ha-1) (cm) plants m-2) (t ha-1) (cm) m-2 (t ha-1) (cm)
Weed free
-- -- -- -- -- -- 278.3 a 14.5 a 42.2 a
Control
Weedy
178.0 a 2.25 a 18.0 a 48.0 a 1.47 a 21.7 a 137.0 f 5.6 f 34.1 d
Control
T42 135.0 bc 1.62 c 14.9 bc 46.67 a 1.48 a 21.6 a 173.3 de 8.52 d 36.5 b-d

L9 124.3 c 1.37 cd 14.0 c 42.66 a 1.29 a 19.5 a 200.3 bc 10.1 b 39.5 ab

7O₀ 138.3 bc 1.54 c 14.3 bc 44.66 a 1.36 a 20.1 a 187.7 cd 9.33 c 38.5 a-c

O₀10 160.0 ab 1.91 b 16.9 ab 48.0 a 1.51 a 21.4 a 154.0 ef 6.73 e 35.66 cd

W9 122.7 c 1.24 d 13.9 c 48.0 a 1.52 a 21.7 a 219.3 b 10.54 b 40.9 a

LSD 32.62 0.274 2.769 14.64 0.292 2.9 24.16 0.502 3.75
These measurements were taken at 50 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

129
4.10.1.2. Fresh biomass (t ha-1)
Infestation of weeds significantly reduced the fresh biomass of wheat up to 61.4%
than weed free control (Table 4.35). Inoculation with strain W9, L9, 7O₀, T42 and O₀10
significantly decreased fresh biomass of broad leaved dock up to 45.1, 39, 31.7, 28.3 and
15.2% as compared to weedy control, respectively. Inoculation with strain W9 also
significantly reduced fresh biomass over strain 7O₀, T42 and O₀10. Inoculation with
strain L9, 7O₀ and T42 performed significantly better than strain O₀10 in reducing the
fresh biomass over weedy control while being non-significant with each other.
Inoculation with strain L9 and 7O₀ caused non-significant decrease in fresh biomass of
common lambs’ quarter up to 12.2 and 7.5% than weedy control, respectively. Weed
suppression effects of strain T42, L9, 7O₀, O₀10 and W9 resultantly recovered the loss in
fresh biomass of infested wheat up to 32.8, 50.6, 41.9, 12.7 and 55.5%, respectively.
4.10.1.3. Shoot length (cm)
Weed infestation significantly reduced the shoot length of infested wheat up to
19.2% than weed free control (Table 4.35). Shoot length of broad leaved dock was
significantly reduced due to inoculation with strain W9, L9, 7O₀ and T42 up to 23.1,
22.2, 20.5 and 17.2% as compared to weedy control, respectively. Inoculation with strain
W9 and L9 also significantly reduced shoot length than inoculation with strain O₀10.
None of the strains caused significant reduction in shoot length of common lambs’
quarter than weedy control. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and
W9 resultantly recovered the loss in shoot length of infested wheat up to 28.8, 66.3, 54.3,
18.9 and 83.9%, respectively.
4.10.4. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at
booting
4.10.4.1. Density (number of plants m-2)
Infestation of weeds significantly reduced the number of tillers of infested wheat
up to 47% over weed free control (Table 4.36). Inoculation with strain T42, 7O₀, L9 and
W9 significantly decreased the density of broad leaved dock up to 24.1, 25.1, 34.2 and
31.2% as compared to weedy control, respectively. These four strains were statistically at
par with each other and significantly better than strain O₀10 in decreasing the density of
broad leaved dock grown in wheat. Inoculation with strain L9 and 7O₀ caused a non-
significant decrease in density of common lambs’ quarter up to 14.4 and 9.6% than weedy
control, respectively. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9

130
Table 4.36. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at booting

Broad leaved dock Common lambs’ quarter Infested wheat

Treatments Density Fresh Shoot Density Fresh Shoot No. of Fresh Shoot
(no. of biomass length (no. of biomass length tillers biomass length
plants m-2) (t ha-1) (cm) plants m-2) (t ha-1) (cm) m-2 (t ha-1) (cm)
Weed free
-- -- -- -- -- -- 270.3 a 19.88 a 63.83 a
Control
Weedy
163.3 a 3.38 a 33.0 a 34.7 a 2.36 ab 34.7 a 143.3 f 8.42 f 50.57 c
Control
T42 124.0 b 2.26 c 27.0 bc 35.0 a 2.40 a 34.8 a 184.3 de 13.22 d 59.5 ab
L9 107.3 b 1.71 d 25.1 c 29.7 a 1.99 c 30.2 b 212.3 bc 15.59 b 63.73 a
7O₀ 122.3 b 2.02 c 25.7 c 31.3 a 2.11 bc 31.4 ab 200.7 cd 14.19 c 61.37 ab
O₀10 150.0 a 2.75 b 30.4 ab 36.3 a 2.35 ab 34.9 a 162.7 ef 10.12 e 54.73 bc
W9 112.3 b 1.69 d 25.1 c 36.0 a 2.35 ab 33.8 ab 227.0 b 15.75 b 64.73 a
LSD 21.37 0.257 4.38 11.6 0.2613 4.37 22.19 0.746 7.13
These measurements were taken at 100 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

131
resultantly recovered the loss in number of tillers of infested wheat up to 32.3, 54.3, 45.1,
15.2 and 65.9%, respectively.
4.10.4.2. Fresh biomass (t ha-1)
Growth of weeds caused reduction in fresh biomass of wheat up to 57.7% than
weed free control (Table 4.36). Inoculation with strain W9, L9, 7O₀, T42 and O₀10
significantly reduced fresh biomass of broad leaved dock up to 50, 49.2, 40.1, 33.2 and
18.1% as compared to weedy control, respectively. Inoculation with strain W9 and L9
proved significantly better than strain 7O₀, T42 and O₀10 in decreasing the fresh biomass
over weedy control. Inoculation with strain 7O₀ and T42 performed significantly better
than strain O₀10 in reducing the fresh biomass over weedy control. Fresh biomass of
common lambs’ quarter was significantly reduced up to 15.4 and 10.5% than weedy
control due to inoculation with strain L9 and 7O₀, respectively, while other strains caused
non-significant effects. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9
recovered the loss in fresh biomass of infested wheat up to 41.9, 62.5, 50.4, 14.9 and
64.0%, respectively.
4.10.4.3. Shoot length (cm)
Data indicated that infestation of weeds significantly reduced the shoot length of
infested wheat up to 20.8% than weed free control (Table 4.36). Inoculation with strain
W9, L9, 7O₀ and T42 significantly decreased shoot length of broad leaved dock up to
24.1, 24, 22.2 and 18.3% as compared to weedy control, respectively. Inoculation with
strain W9, L9 and 7O₀ remained statistically similar with each other and strain T42, and
significantly better than strain O₀10 in reducing the shoot length of broad leaved dock.
Shoot length of common lambs’ quarter was significantly reduced up to 13% than weedy
control due to inoculation with strain L9 while other strains caused non-significant
effects. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss
in shoot length of infested wheat up to 63.1, 92.9, 76.2, 29.4 and 93.6%, respectively.
4.10.7. Effect of allelopathic bacteria on broad leaved dock grown in wheat and
infested wheat at harvesting
4.10.7.1. Density (number of plants m-2)
Data indicated that number of tillers of wheat were significantly reduced up to
57% due to infestation of weeds (Table 4.37). Density of broad leaved dock was
significantly reduced up to 35.1, 38.2, 30 and 28.1% over weedy control due to
inoculation with strain W9, L9, 7O₀ and T42, respectively. Inoculation with strain O₀10

132
Table 4.37. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at harvesting

Broad leaved dock Common lambs’ quarter Infested wheat

Treatments Density Shoot Grain Straw Density Shoot Grain Straw No. of Shoot Grain Straw
(no. of length yield yield (no. of length yield yield tillers length yield yield
plants m-2) (cm) (t ha-1) (t ha-1) plants m-2) (cm) (t ha-1) (t ha-1) m-2 (cm) (t ha-1) (t ha-1)
Weed free
-- -- -- -- -- -- -- -- 302.7 a 83.3 a 3.34 a 5.57 a
Control
Weedy
172.0 a 33.0 a 124.6 a 400.5 a 41.0 a 48.5 a 1.51 a 4.53 a 130.0 d 62.5 d 1.54 e 2.86 e
Control
27.0
T42 123.7 b 80.8 c 250.0 c 42.3 a 48.7 a 1.44 ab 4.44 a 174.3 c 72.2 bc 2.23 c 4.25 c
bc
L9 106.3 b 25.1 c 52.9 d 167.9 d 36.7 a 40.8 b 1.13 c 3.59 b 198.3 bc 78.9 ab 2.69 b 4.75 b
7O₀ 120.3 b 25.7 c 73.4 c 234.2 c 38.0 a 43.2 b 1.21 bc 3.84 b 184.0 bc 74.8 b 2.46 bc 4.54 bc
30.4
O₀10 155.7 a 110.7 b 311.8 b 42.3 a 48.9 a 1.48 ab 4.49 a 146.3 d 66.8 cd 1.85 d 3.47 d
ab
W9 111.7 b 25.1 c 56.0 d 176.8 d 41.7 a 48.5 a 1.50 ab 4.54 a 201.7 b 78.7 ab 2.67 b 4.79 b
LSD 27.66 4.38 12.23 25.55 14 5.08 0.2997 0.4123 26.82 6.97 0.262 0.421
These measurements were taken at 150 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

133
decreased density of broad leaved dock non-significantly over weedy control. None of the
strains caused significant reduction in density of common lambs’ quarter. Weed
suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in number of
tillers of infested wheat up to 25.7, 39.6, 31.3, 9.5 and 41.5%, respectively.
4.10.7.2. Shoot length (cm)
Infestation of weeds significantly reduced the shoot length of wheat up to 24.9%
than weed free control (Table 4.37). Inoculation with strain W9, L9, 7O₀ and T42
significantly reduced the shoot length of broad leavd dock up to 24.1, 24.0, 22.2 and
18.3% than weedy control, respectively. Inoculation with strain W9, L9 and 7O₀ also
significantly reduced the shoot length of broad leaved dock than strain O₀10. Inoculation
with strain L9 and 7O₀ significantly reduced the shoot length of common lambs’ quarter
up to 15.7 and 10.9% than weedy control, respectively. Weed suppression effects of strain
T42, L9, 7O₀, O₀10 and W9 recovered the loss in shoot length of infested wheat up to
46.5, 78.7, 59.2, 20.4 and 77.8%, respectively.
4.10.7.3. Grain yield (t ha-1)
Data given in Table 4.37 indicated that Grain yield of wheat was significantly
decreased up to 54.1% due to infestation of weeds. Grain yield of broad leaved dock was
significantly reduced up to 55, 57.5, 41.1, 35.1 and 11.2% over weedy control due to
inoculation with strain W9, L9, 7O₀, T42 and O₀10, respectively. Inoculation with strain
W9 and L9 also performed significantly better than strain 7O₀, T42 and O₀10 in reducing
the reproductive capacity over weedy control. Inoculation with strain 7O₀ and T42 also
significantly reduced reproductive capacity over strain O₀10. Grain yield of common
lambs’ quarter was significantly decreased up to 25.3 and 20.1% than weedy control due
to inoculation with strain L9 and 7O₀, respectively. Weed suppression effects of strain
T42, L9, 7O₀, O₀10 and W9 resultantly controlled the losses in grain yield of infested
wheat up to 38.4, 64.0, 51.0, 17.3 and 62.9%, respectively.
4.10.7.4. Straw yield (t ha-1)
Data indicated that Straw yield of wheat was significantly reduced up to 48.7%
due to infestation of weeds (Table 4.37). Straw yield of broad leaved dock was
significantly reduced due to inoculation with strain W9, L9, 7O₀, T42 and O₀10 up to
55.9, 58, 41.5, 37.6 and 22.1% as compared to weedy control, respectively. Inoculation
with strain W9 and L9 performed significantly better than strain 7O₀, T42 and O₀10 in
reducing the shoot fresh weight over weedy control. Inoculation with strain 7O₀ and T42

134
produced significantly less shoot fresh weight over inoculation with strain O₀10.
Inoculation with strain L9 and 7O₀ significantly reduced the straw yield of common
lambs’ quarter up to 20.8 and 15.2% than weedy control, respectively. Weed suppression
effects of strain T42, L9, 7O₀, O₀10 and W9 resultantly controlled the losses in straw
yield of infested wheat up to 51.4, 69.5, 61.9, 22.4 and 71.2%, respectively.
4.10.10. Effect of allelopathic bacteria on physiology of weeds grown in wheat and
infested wheat
4.10.10.1. Assimilation rate (A)
Data given in Table 4.38 showed that assimilation rate of wheat was significantly
reduced up to 26.7% than weed free control due to infestation of weeds. Inoculation with
strain W9, L9, 7O₀, T42 and O₀10 significantly reduced the assimilation rate of broad
leaved dock up to 17.8, 18.6, 14.5, 13.1 and 9.7% as compared to weedy control,
respectively. Inoculation with strain L9 produced significantly less assimilation rate than
strain T42 and O₀10. Inoculation with strain W9 also performed significantly better than
strain O₀10 in reducing assimilation rate over weedy control. Inoculation with strain L9
and 7O₀ significantly reduced the assimilation rate of common lambs’ quarter up to 13
and 11.7% than weedy control, respectively. Other strains caused non-significant effects
on assimilation rate of common lambs’ quarter. Weed suppression resultantly caused
recovery in loss of assimilation rate of infested wheat up to 46.7, 90.0, 74.5, 27.8 and
91.1% due to inoculation with strain T42, L9, 7O₀, O₀10 and W9, respectively.
4.10.10.2. Stomatal conductance (gs)
Results showed that stomatal conductance of wheat was significantly decreased up
to 28.8% than weed free control due to infestation of weeds (Table 4.38). Stomatal
conductance of broad leaved dock was significantly reduced due to inoculation with strain
W9, L9, 7O₀, T42 and O₀10 up to 24.3, 25.2, 20, 17.9 and 10% as compared to weedy
control, respectively. Inoculation with strain L9 produced significantly less stomatal
conductance than strain 7O₀, T42 and O₀10. Inoculation with strain W9 also significantly
reduced stomatal conductance over inoculation with strain T42 and O₀10. Inoculation
with strain 7O₀ and T42 also performed significantly better than strain O₀10 in reducing
stomatal conductance over weedy control. Stomatal conductance of common lambs’
quarter was significantly reduced up to 12.1 and 9.7% than weedy control due to
inoculation with strain L9 and 7O₀, respectively. Other strains caused non-significant
effects on stomatal conductance of common lambs’ quarter. Weed suppression resultantly

135
Table 4.38. Effect of allelopathic bacteria on physiology of broad leaved dock grown in wheat and infested wheat
Broad leaved dock Common lambs’ quarter Infested wheat
Treatments Assimilation Stomatal Assimilation Stomatal Assimilation Stomatal
rate (A) conductance (gs) rate (A) conductance (gs) rate (A) conductance (gs)
Weed free
-- -- -- -- 11.2 a 297.7 a
Control
Weedy Control 21.9 a 415.7 a 22.3 ab 427.3 a 8.2 d 212.0 d

T42 19.0 bc 341.3 c 22.5 a 423.0 a 9.6 bc 249.3 c

L9 17.8 d 311.0 e 19.4 c 375.7 b 10.9 a 294.7 a

7O₀ 18.7 b-d 332.3 cd 19.7 c 386.0 b 10.5 ab 273.7 b

O₀10 19.8 b 374.0 b 21.6 b 430.0 a 9.1 cd 236.3 c

W9 18.0 cd 314.7 de 21.6 b 429.0 a 11.0 a 288.3 ab

LSD 1.11 21.12 0.81 20.9 1.18 19.69

Physiological parameters of plants were measured at 60 days after sowing. Values sharing same letter(s) do not differ significantly from each
other at p <0.05 according to Duncan’s Multiple Range Test

136
caused recovery in loss of assimilation rate of infested wheat up to 43.6, 96.5, 72.0, 28.4
and 89.1% due to inoculation with strain T42, L9, 7O₀, O₀10 and W9, respectively.
4.11. Effect of allelopathic bacteria on growth and yield of wheat grown
under weed free conditions in field trial I
Allelopathic bacteria were applied to wheat under weed free conditions to study
their impact on its growth and yield in the same field where they were tested for their
weed suppression effects. Weeds grown in field were manually controlled from time to
time. Data regarding growth and yield of wheat was collected at different stages of the
crop (tillering, booting and at harvesting). The results of this study are explained below;
4.11.1. Effect of allelopathic bacteria on wheat grown under weed free conditions at
tillering
Data indicated that none of the strains caused significant reduction in any of the
measured growth parameters of wehat than un-inoculated control at tillering stage (Table
4.39). However, inoculation with strain L9 and 7O₀ significantly increased the number of
tillers up to 19.2 and 14.8%, fresh biomass up to 26.1 and 20.2% and shoot length up to
15.4 and 9.8% as compared to un-inoculated control, respectively.
4.11.2. Effect of allelopathic bacteria on wheat grown under weed free conditions at
booting
Results showed that none of the selected strains caused significant reduction in
number of tillers, fresh biomass and shoot length than un-inoculated control at booting
stage (Table 4.40). However, inoculation with strain L9 and 7O₀ significantly increased
the number of tillers of wheat up to 20.9 and 17.2%, fresh biomass up to 27.2 and 22.4%
and shoot length up to 15.4 and 12.3% as compared to un-inoculated control,
respectively.
4.11.3. Effect of allelopathic bacteria on growth and yield of wheat grown under
weed free conditions at harvesting
Data indicated that inoculation with the selected strains did not cause in
significant reduction in the measured growth and yield parameters than control (Table
4.41). However, inoculation with strain L9 and 7O₀ significantly increased the number of
tillers of wheat up to 20.1 and 15.1%, fresh biomass up to 25.4 and 21.1%, shoot length
up to 14.1 and 11.2%, grain yield up to 23.2 and 18.1% and straw yield up to 28.2 and
23.3% than un-inoculated control, respectively.

137
Table 4.39. Effect of allelopathic bacteria on wheat grown under weed free
conditions at tillering
Fresh biomass Shoot length
Treatments No. of tillers m-2 -1
(t ha ) (cm)
Control 285.0 c 14.76 c 36.567 b

T42 296.0 bc 15.34 c 37.8 b

L9 339.67 a 18.62 a 42.2 a

7O₀ 327.33 ab 17.75 b 40.167 ab

O₀10 304.67 a-c 15.41 c 37.033 b

W9 296.0 bc 15.36 c 37.933 b

LSD 42.23 0.83 3.85

These measurements were taken at 50 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

Table 4.40. Effect of allelopathic bacteria on wheat grown under weed free
conditions at booting
Fresh biomass Shoot length
Treatments No. of tillers m-2 -1
(t ha ) (cm)
Control 295.33 b 22.11 c 52.1 c

T42 299.33 b 22.32 c 53.0 c

L9 357.0 a 28.12 a 60.1 a

7O₀ 346.0 a 27.06 b 58.5 ab

O₀10 301.0 b 22.25 c 54.333 bc

W9 304.33 b 22.43 c 54.233 bc

LSD 31.89 0.845 4.75

These measurements were taken at 100 days after sowing. Values sharing same letter(s)
do not differ significantly from each other at p <0.05 according to Duncan’s Multiple
Range Test

138
Table 4.41. Effect of allelopathic bacteria on growth and yield of wheat grown under
weed free conditions at harvesting
Fresh Shoot Grain Straw
No. of
Treatments -2 biomass length yield yield
tillers m
(t ha-1) (cm) (t ha-1) (t ha-1)
Control 308.33 b 8.2 b 74.2 b 3.28 b 4.95 b

T42 311.33 b 8.29 b 74.467 b 3.32 b 4.99 b

L9 370.33 a 10.28 a 84.667 a 4.04 a 6.35 a

7O₀ 355.0 a 9.93 a 82.533 a 3.87 a 6.11 a

O₀10 308.67 b 8.25 b 75.5 b 3.32 b 5.07 b

W9 310.0 b 8.32 b 75.4 b 3.33 b 5.06 b

LSD 28.51 0.75 6.71 0.26 0.47

These measurements were taken at 150 days after sowing. Values sharing same letter(s)
do not differ significantly from each other at p <0.05 according to Duncan’s Multiple
Range Test

139
4.11.4. Effect of allelopathic bacteria on physiology of wheat grown under weed free
conditions
Results showed that none of the selected strains caused significant reduction in
assimilation rate, stomatal conductance and evapo-transpiration rate of wheat than un-
inoculated control (Table 4.42). However, inoculation with strain L9 and 7O₀
significantly increased the assimilation rate up to 20.1 and 15.2%, stomatal conductance
up to 19.0 and 16.8% and evapo-transpiration rate up to 16.2 and 11.1% than un-
inoculated control, respectively.
4.12. Effect of allelopathic bacteria on wheat and its associated weeds in
field trial II
Bioherbicidal activity of allelopathic bacteria on weed flora of wheat was
evaluated in field trial II. The field area was divided into two halves; one to study weed
suppression effects of allelopathic bacteria and resultant improvement in growth and yield
of infested wheat while other to study effects of allelopathic bacteria on wheat under
weed free conditions. This field was severely infested with little seed canary grass and
data regarding effect of allelopathic bacteria on other weeds could not be collected due to
their low population. Data regarding growth and yield parameters of little seed canary
grass and wheat was taken at different growth stages of the crop (tillering, booting and
harvesting). The results of these studies are explained below;
4.12.1. Effect of allelopathic bacteria on little seed canary grass grown in wheat and
infested wheat at tillering
4.12.1.1. Density (number of plants per sq. meter)
Data indicated that infestation of little seed canary grass significantly reduced the
number of tillers of wheat up to 50.5% than weed free control (Table 4.43). Inoculation
with strain O₀10, L9 and 7O₀ significantly reduced the density of little seed canary grass
up to 28.4, 30.3 and 40.3% as compared to weedy control, respectively. Inoculation with
strain 7O₀ also significantly reduced the density than inoculation with strain W9 and T42.
Inoculation with strain L9 and O₀10 proved significantly better than strain W9 in
reducing the density than weedy control. Suppression of little seed canary grass
resultantly recovered the loss in number of tillers of infested wheat up to 36.4, 60.9, 72.6,
59.0 and 3.6% due to inoculation with strain T42, L9, 7O₀, O₀10 and W9, respectively.

140
Table 4.42. Effect of allelopathic bacteria on physiology of wheat grown under weed
free conditions
Assimilation rate Stomatal Evapo-
Treatments
(A) conductance (gs) transpiration (E)
Control 9.63 b 215.7 b 3.26 b

T42 9.9 b 222.0 b 3.2 b

L9 11.57 a 256.7 a 3.78 a

7O₀ 11.1 a 252.0 a 3.62 a

O₀10 9.5 b 210.7 b 3.24 b

W9 9.43 b 217.0 b 3.24 b

LSD 1.07 23.1 0.17

These measurements were taken at 60 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

Table 4.43. Effect of allelopathic bacteria on little seed canary grass grown in wheat
and infested wheat at tillering
Little seed canary grass Infested wheat
Treatments Density Fresh Shoot Fresh Shoot
No. of
(no. of biomass length biomass length
tillers m-2
tillers m-2) (t ha-1) (cm) (t ha-1) (cm)
Weed free
-- -- -- 368.3 a 19.85 a 43.2 a
Control
Weedy
123.3 a 3.21 a 27.6 a 182.3 d 8.65 e 33.6 d
Control
T42 108.3 ab 2.45 b 23.4 bc 250.0 c 12.56 d 38.3 bc

L9 86.0 bc 1.79 c 19.8 cd 295.7 b 16.46 c 40.2 ab

7O₀ 73.7 c 1.41 d 17.9 d 317.3 b 17.58 b 42.0 ab

O₀10 88.3 bc 1.88 c 20.4 cd 292.0 bc 16.03 c 40.1 ab

W9 121.7 a 3.08 a 26.8 ab 189.0 d 8.91 e 35.0 cd

LSD 28.9 0.367 3.68 43.27 0.48 4.64

These measurements were taken at 50 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

141
4.12.1.2. Fresh biomass (t ha-1)
Data indicated that infestation of little seed canary grass significantly reduced the
fresh biomass of wheat up to 56.4% than weed free control (Table 4.43). Fresh biomass of
little seed canary grass was significantly reduced up to 56.1, 44.2, 41.3 and 23.7% than
weedy control due to its inoculation with strain 7O₀, L9, O₀10 and T42, respectively.
Inoculation with strain 7O₀ also produced significantly less fresh biomass than other
strains (L9, O₀10, T42 and W9). Inoculation with strain L9 and O₀10 performed
significantly better than strain T42 and W9 in reducing the fresh biomass than weedy
control. Inoculation with strain T42 also significantly reduced fresh biomass than strain
W9. Suppression of little seed canary grass resultantly recovered the loss in fresh biomass
of infested wheat up to 34.9, 69.8, 79.7, 65.9 and 2.3% due to inoculation with strain T42,
L9, 7O₀, O₀10 and W9, respectively.
4.12.1.3. Shoot length (cm)
Infestation of little seed canary grass caused the loss in shoot length of wheat up
to 22.1% than weed free control (Table 4.43). Shoot length of little seed canary grass was
significantly reduced up to 35.1, 28.4, 26.1 and 15.1% over weedy control due to
inoculation with strain 7O₀, L9, O₀10 and T42, respectively. Inoculation with strain 7O₀
provided significantly less shoot length than strain T42 and W9. Inoculation with strain
L9 and O₀10 also performed significantly better than strain W9 in reducing the shoot
length over weedy control. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and
W9 resultantly controlled the loss in shoot length of infested wheat up to 49.3, 68.5, 87.4,
68.2 and 14.0%, respectively.
4.12.3. Effect of allelopathic bacteria on little seed canary grass grown in wheat and
infested wheat at booting
4.12.3.1. Density (number of plants m-2)
Data indicated that infestation of little seed canary grass significantly reduced the
number of tillers of wheat up to 53.1% than weed free control (Table 4.44). Density of
little seed canary grass was significantly reduced up to 43.1, 33.1, 29.1 and 15.9% than
weedy control due to inoculation with strain 7O₀, L9 and O₀10, respectively. Inoculation
with strain 7O₀ also significantly reduced the density than strain T42 and W9. Inoculation
with strain L9 and O₀10 also performed significantly better than strain W9 in reducing
the density than weedy control. Suppression of little seed canary grass resultantly
recovered the loss in number of tillers of infested wheat up to 32.8, 56.1, 67.5, 54.2 and

142
Table 4.44. Effect of allelopathic bacteria on little seed canary grass grown in wheat
and infested wheat at booting
Little seed canary grass Infested wheat
Treatments Density Fresh Shoot No. of Fresh Shoot
(no. of biomass length tillers biomass length
tillers m-2) (t ha-1) (cm) m-2 (t ha-1) (cm)
Weed free
-- -- -- 358.0 a 29.21 a 72.1 a
control
Weedy
106.7 a 4.81 a 44.2 a 168.0 e 12.61 f 56.5 d
Control
T42 89.7 ab 3.66 b 36.7 b 230.3 d 18.05 e 64.4 c

L9 71.3 bc 2.58 c 30.9 c 274.7 c 24.23 c 69.1 ab

7O₀ 60.7 c 1.97 d 27.8 c 296.3 b 25.58 b 71.3 ab

O₀10 75.7 bc 2.69 c 31.5 c 271.0 c 23.48 d 67.6 bc

W9 106.7 a 4.78 a 43.77 a 174.0 e 12.95 f 58.1 d

LSD 26.55 0.337 4.2 20.51 0.641 4.23

These measurements were taken at 100 days after sowing. Values sharing same letter(s)
do not differ significantly from each other at p <0.05 according to Duncan’s Multiple
Range Test

143
3.2% due to inoculation with strain T42, L9, 7O₀, O₀10 and W9, respectively.
4.12.3.2. Fresh biomass (t ha-1)
Infestation of little seed canary grass caused loss in fresh biomass of infested
wheat up to 56.8% than weed free control (Table 4.44). Fresh biomass of little seed
canary grass was significantly reduced up to 59.1, 46.4, 44 and 24% than weedy control
due to inoculation with strain 7O₀, L9, O₀10 and T42, respectively. Inoculation with
strain 7O₀ produced significantly less fresh biomass than strain L9, O₀10, T42 and W9.
Inoculation with strain L9 and O₀10 performed significantly better than strain T42 and
W9 in reducing fresh biomass over weedy control. Fresh biomass produced by
inoculation with strain T42 was also significantly less than that produced by W9. Weed
suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in fresh
biomass of infested wheat up to 32.8, 70, 78.1, 65.5 and 2.0%, respectively.
4.12.3.3. Shoot length (cm)
Results showed that infestation of little seed canary grass significantly reduced the
shoot length of wheat up to 21.6% than weed free control (Table 4.44). Shoot length of
little seed canary grass was significantly reduced due to inoculation with strain 7O₀, L9,
O₀10 and T42 up to 37.1, 30.1, 28.7 and 17% as compared to weedy control, respectively.
Inoculation with strain 7O₀, L9 and O₀10 performed significantly better than strain T42
and W9 in decreasing the shoot length of little seed canary grass over weedy control.
Inoculation with strain T42 decreased the shoot length significantly less than strain W9.
Inoculation with strain W9 caused a non-significant decrease in shoot length over weedy
control. Suppression of little seed canary grass by strain T42, L9, 7O₀, O₀10 and W9
controlled the loss in shoot length of infested wheat up to 50.3, 80.5, 94.9, 70.9 and
10.0%, respectively.
4.12.5. Effect of allelopathic bacteria on little seed canary grass grown in wheat and
infested wheat at harvesting
4.12.5.1. Density (number of plants m-2)
Infestation of little seed canary grass significantly reduced the number of tillers of
wheat up to 52.5% than weed free control (Table 4.45). Density of little seed canary grass
was significantly reduced up to 45.3, 36.2, 30.2 and 16.1% as compared to weedy control
due to inoculation with strain 7O₀, L9, O₀10 and T42, respectively. Inoculation with
strain 7O₀ produced significantly lesser density than strain O₀10, T42 and W9.
Inoculation with strain L9 and O₀10 also performed significantly better than strain T42

144
Table 4.45. Effect of allelopathic bacteria on little seed canary grass grown in wheat and infested wheat at harvesting
Little seed canary grass Infested wheat
Treatments Density Shoot Grain Straw Shoot Grain Straw
No. of
(no. of length yield yield length yield yield
tillers m-2
tillers m-2) (cm) (t ha-1) (t ha-1) (cm) (t ha-1) (t ha-1)
Weed free
-- -- -- -- 396.0 a 104.9 a 3.86 a 6.60 a
Control
Weedy Control 99.3 a 106.4 a 0.72 a 1.49 a 188.0 d 83.2 c 1.78 d 3.01 e

T42 83.3 b 83.3 b 0.62 ab 1.14 b 259.7 c 92.1 b 2.49 c 4.35 d

L9 63.3 cd 76.2 bc 0.48 bc 0.75 c 308.7 b 102.1 a 2.92 b 5.38 bc

7O₀ 54.3 d 69.0 c 0.43 c 0.58 c 324.0 b 103.6 a 3.11 b 5.51 b

O₀10 69.3 c 80.2 b 0.51 bc 0.81 c 300.3 b 99.9 a 2.97 b 5.05 c

W9 98.3 a 103.0 a 0.71 a 1.53 a 190.7 d 84.0 c 1.80 d 3.13 e

LSD 13.14 8.35 0.156 0.234 28.2 5.61 0.24 0.42

These measurements were taken at 150 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

145
and W9 in reducing the density over weedy control. Suppression of little seed canary
grass by strain T42, L9, 7O₀, O₀10 and W9 controlled the loss in number of tillers of
infested wheat up to 34.4, 58.0, 65.4, 54.0 and 1.3%, respectively.
4.12.5.2. Shoot length (cm)
Infestation of little seed canary grass significantly reduced the shoot length of
wheat up to 20.7% than weed free control (Table 4.45). Inoculation with strain T42, L9,
7O₀ and O₀10 significantly reduced the shoot length of little seed canary grass up to 21.8,
28.4, 35.1 and 24.6% than weedy control, respectively. Inoculation with strain 7O₀ also
significantly reduced the shoot length than strain T42 and O₀10. Weed suppression
effects of strain T42, L9, 7O₀, O₀10 and W9 resultantly recovered the loss in shoot length
of infested wheat up to 41.0, 87.4, 94.2, 77.1 and 4.0%, respectively.
4.12.5.3. Grain yield (t ha-1)
Results showed that infestation of little seed canary grass caused loss in grain
yield of infested wheat up to 53.9% than weed free control (Table 4.45). Inoculation with
strain 7O₀, L9 and O₀10 significantly reduced the grain yield of little seed canary grass
up to 40.1, 33.3 and 29.8% as compared to weedy control, respectively. Inoculation with
strain 7O₀ produced significantly less grain yield than strain T42 and W9. Inoculation
with strain L9 and O₀10 significantly decreased the grain yield than strain W9.
Suppression of little seed canary grass resultantly recovered the loss in grain yield of
infested wheat up to 34.3, 55.1, 64.3, 57.2 and 1.3% due to inoculation with strain T42,
L9, 7O₀, O₀10 and W9, respectively.
4.12.5.4. Straw yield (t ha-1)
Suppression of little seed canary grass by allelopathic bacteria resultantly
improved the straw yield of infested wheat which is shown in Table 4.45. Results showed
that straw yield of wheat was significantly reduced up to 54.4% due to infestation of little
seed canary grass. Inoculation with strain 7O₀, L9, O₀10 and T42 significantly reduced
the straw yield of little seed canary grass up to 60.6, 49.6, 45.5 and 23.1% than weedy
control, respectively. Inoculation with strain 7O₀, L9 and O₀10 performed significantly
better than strain T42 and W9 in reducing the shoot fresh weight over weedy control.
Inoculation with strain T42 significantly decreased the shoot fresh weight over
inoculation with strain W9. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and
W9 recovered the loss in straw yield of infested wheat up to 37.2, 65.8, 69.4, 56.7 and
3.3%, respectively.

146
4.12.7. Effect of allelopathic bacteria on physiology of little seed canary grass grown
in wheat and infested wheat
4.12.7.1. Assimilation rate (A)
Infestation of little seed canary grass significantly reduced the assimilation rate of
infested wheat up to 23.4% over weed free control (Table 4.46). Assimilation rate of little
seed canary grass was significantly reduced up to 33.3, 25, 22.3 and 13.2% over weedy
control due to inoculation with strain 7O₀, L9, O₀10 and T42, respectively. Inoculation
with strain 7O₀ produced significantly less assimilation rate than strain O₀10, T42 and
W9. Inoculation with strain L9 significantly decreased assimilation rate over inoculation
with strain T42 and W9. Inoculation with strain O₀10 and T42 performed significantly
better than strain W9 in reducing the assimilation rate over weedy control. Weed
suppression effects of strain T42, L9, 7O₀, L9, O₀10 and W9 recovered the loss in
assimilation rate of infested wheat up to 51.2, 81.7, 97.6, 70.7 and 4.9%, respectively.
4.12.7.2. Stomatal conductance (gs)
Results indicated that infestation of little seed canary grass caused loss in stomatal
conductance of wheat up to 21.8% than weed free control (Table 4.46). Stomatal
conductance of little seed canary grass was significantly reduced up to 34.3, 27.5, 24.5
and 13.3% over weedy control due to inoculation with strain 7O₀, L9, O₀10 and T42,
respectively. Inoculation with strain 7O₀ produced significantly less stomatal
conductance than strain L9, O₀10, T42 and W9. Inoculation with strain L9 and O₀10
performed significantly better than strain T42 and W9 in reducing the stomatal
conductance over weedy control. Inoculation with strain T42 also significantly decreased
the stomatal conductance over inoculation with strain W9. Weed suppression effects of
strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in stomatal conductance of infested
wheat up to 51.0, 84.0, 88.8, 77.2 and 2.9%, respectively.
4.12.7.3. Evapo-transpiration rate (E)
Infestation of little seed canary grass significantly reduced the evapo-transpiration
rate of infested wheat up to 18.7% than weed free control (Table 4.46). Inoculation with
strain 7O₀, L9, O₀10 and T42 significantly reduced the evapo-transpiration of little seed
canary grass up to 33.9, 26.7, 24.5 and 15.8% as compared to weedy control, respectively.
Inoculation with strain 7O₀ also significantly reduced evapo-transpiration than strain L9,
O₀10, T42 and W9. Inoculation with strain L9 and O₀10 performed significantly better
than strain T42 and W9 in reducing the evapo-transpiration over weedy control.

147
Table 4.46. Effect of allelopathic bacteria on physiology of little seed canary grass grown in wheat and infested wheat
Little seed canary grass Infested wheat
Treatments Evapo- Evapo-
Assimilation rate Stomatal Assimilation Stomatal
transpiration transpiration
(A) conductance (gs) rate (A) conductance (gs)
rate (E) rate (E)
Weed free
-- -- -- 11.13 a 287.7 a 3.99 a
Control
Weedy Control 8.8 a 340.0 a 3.26 a 8.4 c 219.0 d 3.24 d
T42 7.63 b 294.7 b 2.75 b 9.8 b 254.0 c 3.59 c
L9 6.6 cd 246.3 c 2.39 c 10.63 ab 276.7 ab 3.8 b
7O₀ 5.87 d 223.3 d 2.16 d 11.07 a 280.0 ab 3.92 ab
O₀10 6.83 bc 256.7 c 2.46 c 10.33 ab 272.0 b 3.76 bc
W9 8.77 a 348.7 a 3.25 a 8.53 c 221.0 d 3.34 d
LSD 0.959 16.87 0.159 0.98 15.16 0.174
Physiological parameters of plants were measured at 60 days after sowing. Values sharing same letter(s) do not differ significantly from each
other at p <0.05 according to Duncan’s Multiple Range Test

148
Inoculation with strain T42 also significantly decreased the evapo-transpiration rate than
strain W9. Suppression of little seed canary grass resultantly recovered the loss in evapo-
transpiration rate of infested wheat up to 47.3, 75.4, 91.5, 69.6 and 13.8% due to
inoculation with strain T42, L9, 7O₀, O₀10 and W9, respectively.
4.13. Effect of allelopathic bacteria on growth and yield of wheat under
weed free conditions in field trial II
4.13.1. Effect of allelopathic bacteria on wheat grown under weed free conditions at
tillering
Results indicated that none of selected strains caused significant reduction in the
measured growth parameters of wheat grown under weed free conditions at tillering stage
(Table 4.47). However, inoculation with strain L9 and 7O₀ significantly increased the
number of tillers of wheat up to 21.1 and 16.2%, fresh biomass up to 24 and 18.1% and
shoot length up to 17.4 and 11.8% than un-inoculated control, respectively.
4.13.2. Effect of allelopathic bacteria on wheat grown under weed free conditions at
booting
Data indicated that effect of selected strains of allelopathic bacteria on the
measured growth parameters of wheat remained non-inhibitory at booting stage (Table
4.48). Inoculation with strain L9 and 7O₀ significantly increased the number of tillers of
wheat up to 18.4 and 14.3%, fresh biomass up to 24.3 and 19.2% and shoot length up to
18.0 and 13.1% as compared to un-inoculated control, respectively.
4.13.3. Effect of allelopathic bacteria on wheat grown under weed free conditions at
harvesting
Inoculation with the selected strains of allelopathic bacteria did not cause significant
reduction in the measured growth and yield parameters of wheat at harvesting (Table
4.49). However, inoculation with strain L9 and 7O₀ significantly increased the number of
tillers of wheat up to 19.8 and 14.83%, shoot length up to 17.1 and 11.8%, grain yield up
to 30.1 and 23% and straw yield up to 27.2 and 19.8% as compared to un-inoculated
control, respectively.
4.13.4. Effect of allelopathic bacteria on physiology of wheat grown under weed free
conditions
Results indicated that none of the selected strains caused significant reduction in
assimilation rate, stomatal conductance and evapo-transpiration rate of wheat grown

149
Table 4.47. Effect of allelopathic bacteria on wheat grown under weed free
conditions at tillering
Fresh biomass Shoot length
Treatments No. of tillers m-2 -1
(t ha ) (cm)
Control 381.7 b 19.35 c 38.57 b

T42 387.0 b 19.61 c 38.6 b

L9 462.3 a 24.0 a 45.27 a

7O₀ 443.3 a 22.86 b 43.1 a

O₀10 384.3 b 19.67 c 38.73 b

W9 388.0 b 19.41 c 38.6 b

LSD 36.55 0.59 3.88

These measurements were taken at 50 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

Table 4.48. Effect of allelopathic bacteria on wheat grown under weed free
conditions at booting
Fresh biomass Shoot length
Treatments No. of tillers m-2 -1
(t ha ) (cm)
Control 370.0 b 28.9 c 65.7 b
T42 380.3 b 29.1 c 66.23 b
L9 438.7 a 36.05 a 77.57 a
7O₀ 423.3 a 34.56 b 74.3 a
O₀10 379.3 b 29.1 c 66.2 b
W9 371.7 b 29.25 c 65.97 b
LSD 28.39 0.68 3.785
These measurements were taken at 100 days after sowing. Values sharing same letter(s)
do not differ significantly from each other at p <0.05 according to Duncan’s Multiple
Range Test

150
Table 4.49. Effect of allelopathic bacteria on wheat grown under weed free
conditions at harvesting
No. of tillers Shoot length Grain yield Straw yield
Treatments
m-2 (cm) (t ha-1) (t ha-1)
Control 395.67 b 98.8 b 3.73 c 6.43 c

T42 401.67 b 99.1 b 3.74 c 6.52 c

L9 474.0 a 115.7 a 4.85 a 8.18 a

7O₀ 454.33 a 110.5 a 4.59 b 7.71 b

O₀10 397.0 b 99.3 b 3.73 c 6.56 c

W9 401.67 b 99.1 b 3.74 c 6.54 c

LSD 30 5.67 0.23 0.332

These measurements were taken at 150 days after sowing. Values sharing same letter(s)
do not differ significantly from each other at p <0.05 according to Duncan’s Multiple
Range Test

Table 4.50. Effect of allelopathic bacteria on physiology of wheat grown under weed
free conditions
Evapo-
Assimilation rate Stomatal
Treatments transpiration
(A) conductance (gs)
rate (E)
Control 9.64 b 235.0 b 3.3 b

T42 9.67 b 234.0 b 3.27 b

L9 11.27 a 276.3 a 3.76 a

7O₀ 10.8 a 265.7 a 3.63 a

O₀10 9.4 b 239.7 b 3.33 b

W9 9.74 b 235.3 b 3.26 b

LSD 0.902 12.15 0.191

Physiological parameters of plants were measured at 60 days after sowing. Values sharing
same letter(s) do not differ significantly from each other at p <0.05 according to Duncan’s
Multiple Range Test

151
under weed free conditions at 60 days after sowing (Table 4.49). However, inoculation
with strain L9 and 7O₀ significantly increased the assimilation rate of wheat up to 16.8
and 12.0%, stomatal conductance up to 17.6 and 13.1% and evapo-transpiration rate up to
14 and 10.2% than un-inoculated control, respectively.
4.14. Effect of allelopathic bacteria on wheat and its associated weeds in
field trial III
Field trial III was conducted at Research Area of Institute of Soil and
Environmental Sciences, University of Agriculture, Faisalabad. Effects of allelopathic
bacteria were observed on wild oat and little seed canary grass. The effects of allelopathic
bacteria on other flora could not be studied due to their low population. The same field
area was used to investigate weed suppression effects of allelopathic bacteria on weeds
and their effects on wheat under weed free conditions by dividing the field into two equal
halves. The weeds grown in one half were periodically controlled using manual labor to
keep it free of weeds. Data regarding growth and yield parameters of weeds, infested
wheat and wheat grown under weed free conditions was collected at three growth stages
of the crop (tillering, booting and at harvesting). The results of this field trial are
described below;
4.14.1. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at
tillering
4.14.1.1. Density (number of plants m-2)
Infestation of weeds caused loss in number of tillers of infested wheat up to 51.8%
than weed free control (Table 4.51). Results showed that density of wild oat was
significantly reduced up to 34.2, 46.1, 18 and 19% over weedy control due to inoculation
with strain T42, L9, 7O₀ and W9, respectively (Table 4.74). Inoculation with strain L9
and T42 also significantly reduced the density over strain 7O₀, O₀10 and W9. Inoculation
with strain 7O₀ and W9 also significantly reduced the density over strain O₀10.
Inoculation with strain O₀10 remained non-significant with weedy control in reducing the
density of wild oat over weedy control. Density of little seed canary grass was
significantly reduced up to 35.2, 45.4 and 30.6% than weedy control due to inoculation
with strain L9, 7O₀ and O₀10, respectively. Inoculation with strain 7O₀ also significantly
reduced the density than strain T42 and W9. Inoculation with strain L9 and O₀10 also
significantly reduced the density than strain W9. Suppression of weeds by strain T42, L9,

152
7O₀, O₀10 and W9 caused recovery in loss of number of tillers of infested wheat up to
49.2, 78.1, 48.4, 27.9 and 33.5%, respectively.
4.14.1.2. Fresh biomass (t ha-1)
Infestation of weeds caused loss in fresh biomass of infested wheat up to 54.5%
than weed free control (Table 4.51). Inoculation with strain T42, L9, 7O₀ and W9
significantly decreased the fresh biomass of wild oat up to 40.1, 53.2, 22.5 and 21.6% as
compared to weedy control, respectively. Inoculation with strain L9 also significantly
reduced the fresh biomass over other strains (T42, 7O₀, O₀10 and W9). Inoculation with
strain T42 also significantly decreased the fresh biomass over strain 7O₀, O₀10 and W9.
Fresh biomass produced by inoculation with strain 7O₀ and W9 was also significantly
less than that produced by strain O₀10. Inoculation with O₀10 remained non-significant
with weedy control in reducing the fresh biomass. Fresh biomass of little seed canary
grass was significantly reduced up to 14.7, 42.4, 52.3 and 37.3% than weedy control due
to inoculation with strain T42, L9, 7O₀ and O₀10, respectively. Inoculation with strain
7O₀ also significantly reduced the fresh biomass than strain T42, O₀10 and W9.
Inoculation with strain L9 and O₀10 also significantly reduced the fresh biomass than
strain T42 and W9. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9
caused recovery in loss of fresh biomass of infested wheat up to 57.0, 79.5, 54.7, 37.7 and
43.5%, respectively.
4.14.1.3. Shoot length (cm)
Shoot length of wheat was significantly reduced up to 26.9% than weed free control due
to infestation of weeds (Table 4.51). Inoculation with strain T42, L9, 7O₀ and W9
significantly reduced the shoot length of wild oat up to 20.2, 34.5, 16 and 15.2% than
weedy control, respectively. Inoculation with strain L9 also significantly reduced the
shoot length than other strains (T42, 7O₀, O₀10 and W9). Inoculation with strain T42,
7O₀ and W9 remained non-significant with each other and significantly better than strain
O₀10 in reducing the shoot length of wild oat than weedy control. Shoot length of little
seed canary grass was significantly reduced up to 15.3, 25.2, 30.5 and 22.8% than weedy
control due to inoculation with strain T42, L9, 7O₀ and O10, respectively. Inoculation
with strain 7O₀ also significantly reduced the shoot length over strain T42 and W9.
Inoculation with strain L9, T42 and O₀10 remained non-significant with each other in
reducing the shoot length over weedy control. Weed suppression effects of strain T42, L9,

153
Table 4.51. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at tillering
Wild oat Little seed canary grass Infested wheat
Treatments Density Fresh Shoot Density Fresh Shoot Fresh Shoot
number of
(number of biomass length (number of biomass length biomass length
tillers m-2
tillers m-2) (t ha-1) (cm) tillers m-2) (t ha-1) (cm) (t ha-1) (cm)
Weed free
-- -- -- -- -- -- 320.0 a 12.85 a 40.87 a
Control
Weedy
140.3 a 3.37 a 37.6 a 65.2 a 1.77 a 29.0 a 154.0 e 5.85 e 29.87 e
Control
T42 92.3 c 2.02 c 30.0 b 58.6 ab 1.51 b 24.6 b 235.7 c 9.84 c 37.93 bc

L9 75.6 c 1.58 d 24.6 c 42.3 bc 1.02 cd 21.7 bc 283.7 b 11.42 b 40.3 ab

7O₀ 115.0 b 2.61 b 31.6 b 35.6 c 0.84 d 20.2 c 234.3 c 9.68 c 36.87 cd

O₀10 138.6 a 3.34 a 37.5 a 45.3 bc 1.11 c 22.4 bc 200.3 d 8.49 d 34.5 d

W9 113.7 b 2.64 b 31.9 b 67.0 a 1.72 ab 28.6 a 209.7 d 8.9 d 35.27 cd

LSD 20.75 0.255 4.62 17.34 0.248 3.24 19.57 0.458 2.91
These measurements were taken at 50 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

154
7O₀, O₀10 and W9 caused recovery in loss of shoot length of infested wheat up to 73.3,
95.2, 63.6, 42.1 and 49.1%, respectively.
4.14.4. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at
booting
4.14.4.1. Density (number of plants m-2)
Infestation of weeds significantly reduced the number of tillers of infested wheat
up to 48.3% than weed free control (Table 4.52). Inoculation with strain T42, L9, 7O₀
and W9 significantly decreased the density of wild oat up to 36, 48.4, 21.1 and 20.3%
than weedy control, respectively. Inoculation with strain L9 also significantly reduced the
density than strain 7O₀, O₀10 and W9. Inoculation with strain T42, 7O₀ and W9 also
significantly reduced the density than strain O₀10 while being non-significant with each
other. Inoculation with strain L9, 7O₀ and O₀10 significantly reduced the density of little
seed canary grass up to 37.2, 48.4 and 32.6% as compared to weedy control, respectively.
These strains also significantly reduced the density than strain T42 and W9 while being
non-significant with each other. Suppression of weeds by strain T42, L9, 7O₀, O₀10 and
W9 caused recovery in loss of number of tillers of infested wheat up to 59.0, 94.4, 60.2,
34.4 and 40.8%, respectively.
4.14.4.2. Fresh biomass (t ha-1)
Infestation of weeds caused loss in fresh biomass of infested wheat up to 51.9%
than weed free control (Table 4.52). Inoculation with strain T42, L9, 7O₀ and W9
significantly reduced the fresh biomass of wild oat up to 42.3, 56.1, 26.2 and 24% as
compared to weedy control, respectively. Inoculation with strain L9 also significantly
reduced the fresh biomass over strain T42, 7O₀, O₀10 and W9. Inoculation with strain
T42 also significantly reduced the fresh biomass over strain 7O₀, O₀10 and W9.
Inoculation with strain 7O₀ and W9 caused a significant reduction in fresh biomass over
strain O₀10. Inoculation with strain O₀10 caused a non-significant reduction in fresh
biomass over weedy control. Fresh biomass of little seed canary grass was significantly
reduced up to 11.7, 44.1, 55 and 39% as compared to weedy control due to inoculation
with strain T42, L9, 7O₀ and O10, respectively. Inoculation with strain 7O₀ also
significantly reduced the fresh biomass than other strains (T42, L9, O₀10 and W9).
Inoculation with strain L9 and O₀10 also significantly reduced the fresh biomass than T42
and W9. Inoculation with strain T42 also performed significantly better than strain W9 in
reducing the fresh biomass than weedy control. Weed suppression effects of strain T42,
155
Table 4.52. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at booting
Wild oat Little seed canary grass Infested wheat
Treatments Density Fresh Shoot Density Fresh Shoot Number Fresh Shoot
(number of biomass length (number of biomass length of tillers biomass length
tillers m-2) (t ha-1) (cm) tillers m-2) (t ha-1) (cm) m-2 (t ha-1) (cm)
Weed free
-- -- -- -- -- -- 334.7 a 27.41 a 80.1 a
Control
Weedy
134.3 a 5.47 a 56.3 a 71.67 a 2.71 a 52.1 a 173.0 d 13.18 f 55.3 d
Control
T42 86.0 bc 3.16 c 44.2 b 64.0 a 2.40 b 43.7 ab 268.3 b 22.19 c 71.1 bc
L9 69.3 c 2.40 d 35.5 c 45.0 b 1.52 c 37.7 b 325.7 a 26.31 b 77.7 ab
7O₀ 106.0 b 4.04 b 46.5 b 37.0 b 1.22 d 36.5 b 270.3 b 22.25 c 71.4 bc
O₀10 132.0 a 5.35 a 56.2 a 48.33 b 1.65 c 40.0 b 228.7 c 19.41 e 65.5 c
W9 107.0 b 4.16 b 47.3 b 74.0 a 2.68 a 51.2 a 239.0 c 20.46 d 67.0 c
LSD 22.11 0.31 4.2 15.4 0.244 8.46 23.8 0.566 7.65
These measurements were taken at 100 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

156
L9, 7O₀, O₀10 and W9 caused recovery in loss of fresh biomass of infested wheat up to
63.3, 92.2, 63.8, 43.8 and 51.2%, respectively.
4.14.4.3. Shoot length (cm)
Results showed that shoot length of wheat was significantly reduced up to 30.9%
than weed free control due to infestation of weeds (Table 4.52). shoot length of wild oat
was significantly reduced up to 21.5, 37, 17.4 and 16% over weedy control due to
inoculation with strain T42, L9, 7O₀ and W9, respectively. Inoculation with strain L9
caused a significant reduction in shoot length than other strains (T42, 7O₀, O₀10 and
W9). Inoculation with strain T42, 7O₀ and W9 also significantly reduced the shoot length
than strain O₀10 while being non-significant with each other. Inoculation with strain
O₀10 caused a non-significant reduction in shoot length than weedy control. Inoculation
with strain L9, 7O₀ and O₀10 significantly reduced the shoot length of little seed canary
grass up to 27.6, 30 and 23.2% as compared to weedy control, respectively. Inoculation
with strain L9, 7O₀ and O₀10 also significantly reduced the shoot length than strain W9
while being non-significant with each other and strain T42. Weed suppression effects of
strain T42, L9, 7O₀, O₀10 and W9 caused recovery in loss of shoot length of infested
wheat up to 63.6, 90.2, 64.9, 41.2 and 47.1%, respectively.
4.14.7. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at
harvesting
4.14.7.1. Density (number of plants m-2)
Infestation of weeds significantly reduced the number of tillers of infested wheat
up to 48.9% than weed free control (Table 4.53). Inoculation with strain T42, L9, 7O₀
and W9 significantly reduced the density of wild oat up to 38.2, 50.1, 23.4 and 21.3% as
compared to weedy control, respectively. Inoculation with strain L9 and T42 also
significantly reduced the density than strain 7O₀, O₀10 and W9. Inoculation with strain
7O₀ and W9 performed significantly better than strain O₀10 in reducing the density over
weedy control. Inoculation with strain O₀10 caused a non-significant decrease in density
of wild oat than weedy control. Density of little seed canary grass was significantly
reduced up to 50.5, 39.4 and 34.1% than weedy control due to inoculation with strain
7O₀, L9 and O₀10, respectively. These three strains remained statistically at par with each
other and significantly better than strain T42 and W9 in reducing the density of little seed
canary grass. Suppression of weeds by strain T42, L9, 7O₀, O₀10 and W9 caused
recovery in loss of number of tillers of infested wheat up to 58.9, 75.0, 60.9, 34.8 and

157
Table 4.53. Effect of allelopathic bacteria on weeds grown in wheat and infested wheat at harvesting

Wild oat Little seed canary grass Infested wheat

Density Density
Treatments Shoot Grain Straw Shoot Grain Straw Number Shoot Grain Straw
(number (number
length yield yield length yield yield of tillers length yield yield
of tillers of tillers
(cm) (t ha-1) (t ha-1) (cm) (t ha-1) (t ha-1) (m-2) (cm) (t ha-1) (t ha-1)
m-2) m-2)
Weed free
-- -- -- -- -- -- -- -- 365.0 a 112.1 a 3.78 a 6.00 a
Control
Weedy
148.3 a 95.6 a 0.91 a 2.36 a 69.3 a 92.6 a 0.35 a 0.755 a 186.7 e 78.0 d 1.65 e 2.58 d
Control
T42 91.6 c 73.1 c 0.64 bc 1.30 c 62.3 a 84.4 b 0.33 a 0.647 a 291.7 c 100.0 bc 2.67 c 4.32 b

L9 74.0 c 57.3 d 0.51 c 0.89 d 42.0 b 64.7 d 0.25 bc 0.390 b 320.3 b 103.7 ab 2.94 b 4.66 b

7O₀ 113.6 b 76.4 b 0.71 b 1.61 b 34.3 b 61.1 d 0.23 c 0.313 b 295.3 c 100.1 bc 2.79 bc 4.50 b

O₀10 147.3 a 95.4 a 0.91 a 2.33 a 45.7 b 70.3 c 0.27 b 0.419 b 248.7 d 92.3 c 2.27 d 3.86 c

W9 116.7 b 79.1 b 0.75 ab 1.70 b 71.3 a 92.1 a 0.35 a 0.743 a 256.7 d 95.2 bc 2.43 d 3.83 c

LSD 21.77 3.02 0.172 0.226 14.85 3.845 0.022 0.128 23.48 8.803 0.193 0.343
These measurements were taken at 150 days after sowing. Values sharing same letter(s) do not differ significantly from each other at p <0.05
according to Duncan’s Multiple Range Test

158
39.2%, respectively.
4.14.7.2. Shoot length (cm)
Shoot length of wheat was significantly reduced up to 30.4% than weed free
control due to infestation of weeds (Table 4.53). Inoculation with strain T42, L9, 7O₀ and
W9 significantly decreased the shoot length of wild oat up to 23.6, 40.1, 20.0 and 17.2%
than weedy control, respectively. Inoculation with strain L9 also significantly reduced the
shoot length than other strains. Inoculation with strain T42 also significantly reduced the
shoot length than strain 7O₀, O₀10 and W9. Inoculation with strain T42, L9, 7O₀ and
O₀10 significantly reduced the shoot length of little seed canary grass up to 8.8, 30.1,
34.0 and 24.0% than weedy control, respectively. Inoculation with strain L9 and 7O₀ also
significantly reduced the shoot length than strain T42, O₀10 and W9. Inoculation with
strain O₀10 also significantly reduced the shoot length than strain T42 and W9. Weed
suppression effects of strain T42, L9, 7O₀, O₀10 and W9 caused recovery in loss of shoot
length of infested wheat up to 64.4, 75.2, 64.7, 41.8 and 50.3%, respectively.
4.14.7.3. Grain yield (t ha-1)
Grain yield of wheat was significantly reduced up to 56.3% than weed free control
due to infestation of weeds (Table 4.53). Inoculation with strain T42, L9 and 7O₀
significantly reduced the grain yield of wild oat up to 29.6, 43.9 and 21.3% as compared
to weedy control, respectively. Inoculation with strain L9 caused a significant reduction
in grain yield than strain 7O₀, O₀10 and W9. Inoculation with strain T42 and 7O₀ also
significantly decreased the grain yield of wild oat over strain O₀10 while being non-
significant with each other and strain W9. Inoculation with strain O₀10 remained
statistically at par with weedy control in reducing the grain yield of wild oat. Grain yield
of little seed canary grass was significantly reduced up to 28.6, 34.1 and 23.4% as
compared to weedy control due to inoculation with L9, 7O₀ and O10, respectively.
Inoculation with strain 7O₀ also significantly reduced the grain yield than strain O10, T42
and W9. Inoculation with strain L9 and O₀10 significantly decreased the grain yield of
little seed canary grass than strain T42 and W9. Suppression of weeds by strain T42, L9,
7O₀, O₀10 and W9 resultantly recovered the loss in grain yield of infested wheat up to
47.9, 60.7, 53.7, 29.0 and 36.6%, respectively.
4.14.7.4. Straw yield (t ha-1)
Straw yield of wheat was significantly reduced up to 57.0% than weed free control
due to infestation of weeds (Table 4.53). Straw yield of wild oat was significantly reduced

159
up to 45.1, 62.2, 32 and 28.1% as compared to weedy control due to inoculation with
strain T42, L9, 7O₀ and W9, respectively. Inoculation with strain L9 also significantly
reduced the straw yield than other strains (T42, 7O₀, O₀10 and W9). Inoculation with
strain T42 caused a significant reduction in shoot fresh weight than strain 7O₀, O₀10 and
W9. Inoculation with strain 7O₀ and W9 also significantly reduced the straw yield than
strain O10. Inoculation with strain O₀10 caused a non-significant reduction in straw yield
than weedy control. Inoculation with strain 7O₀, L9 and O₀10 significantly reduced the
straw yield of little seed canary grass up to 58.6, 48.3 and 44.5% than weedy control,
respectively. These three strains remained statistically at par with each other and
significantly better than strain T42 and W9 in reducing the straw yield than weedy
control. Weed suppression effects of strain T42, L9, 7O₀, O₀10 and W9 caused recovery
in loss of straw yield of infested wheat up to 51.1, 61.0, 56.1, 37.6 and 36.6%,
respectively.
4.14.10. Effect of allelopathic bacteria on physiology of wild oat grown in wheat and
infested wheat
4.14.10.1. Assimilation rate (A)
Infestation of weeds caused significant reduction in assimilation rate of wild oat up to
21.0% than weed free control (Table 4.54). Assimilation rate of wild oat was significantly
reduced due to inoculation with T42, L9, 7O₀ and W9 up to 37.6, 43.8, 30.3 and 24.9%
than weedy control, respectively. Inoculation with strain L9 and T42 also caused a
significant reduction in assimilation rate than strain 7O₀, O₀10 and W9. Inoculation with
strain 7O₀ and W9 also significantly reduced the assimilation rate than strain O₀10.
Inoculation with strain 7O₀, L9 and O₀10 significantly reduced the assimilation rate of
little seed canary grass up to 32, 25.1 and 22% as compared to weedy control,
respectively. Inoculation with strain L9, 7O₀ and O₀10 also significantly reduced the
chlorophyll contents over strain T42 and W9. Weed suppression effects of strain T42, L9,
7O₀, O₀10 and W9 caused recovery in assimilation rate of infested whet up to 73.6, 90.3,
80.6, 44.4 and 54.2%, respectively.
4.14.10.2. Stomatal conductance (gs)
Results indicated that infestation of weeds caused significant reduction in stomatal
conductance of infested wheat up to 31.2% than weed free control (Table 4.54).
Inoculation with strain T42, L9, 7O₀ and W9 significantly reduced the stomatal
conductance of wild oat up to 29.3, 33.9, 23.3 and 21.5% as compared to weedy control,
160
Table 4.54. Effect of allelopathic bacteria on physiology of weeds grown in wheat and infested wheat
Wild oat Little seed canary grass Infested wheat
Treatments
Assimilation rate Stomatal Assimilation rate Stomatal Assimilation rate Stomatal
(A) conductance (gs) (A) conductance (gs) (A) conductance (gs)
Weed free
-- -- -- -- 11.43 a 308.0 a
Control
Control 12.87 a 267.7 a 8.63 a 347.7 a 9.03 d 212.0 e

T42 8.03 c 189.3 cd 7.9 a 312.3 b 10.8 a-c 276.3 c

L9 7.23 c 177.0 d 6.47 b 238.3 c 11.2 ab 296.3 ab

7O₀ 8.97 b 205.3 bc 5.87 b 205.7 d 10.97 a-c 281.7 bc

O₀10 12.07 a 262.3 a 6.73 b 244.3 c 10.1 c 239.7 d

W9 9.67 b 210.0 b 8.33 a 336.3 a 10.33 bc 246.0 d

LSD 0.9 16.55 1.02 17.87 1.05 16.5

Physiological parameters of plants were measured at 60 days after sowing. Values sharing same letter(s) do not differ significantly from each
other at p <0.05 according to Duncan’s Multiple Range Test

161
respectively. Inoculation with strain L9 also significantly reduced the stomatal
conductance than strain 7O₀, O₀10 and W9. Inoculation with strain T42 also caused a
significant reduction in stomatal conductance than strain O₀10 and W9. Inoculation with
strain 7O₀ and W9 also significantly reduced the stomatal conductance than strain O₀10.
Stomatal conductance of little seed canary grass was significantly reduced up to 10.2,
31.4, 40.8 and 29.7% than weedy control due to inoculation with strain 7O₀, L9, O₀10
and T42, respectively. Inoculation with strain 7O₀ also significantly reduced the stomatal
conductance over other strains (T42, L9, O₀10 and W9). Inoculation with strain L9 and
O₀10 also significantly reduced the stomatal conductance over strain T42 and W9.
Inoculation with strain T42 also caused a significant reduction in stomatal conductance
than Suppression of weeds by strain T42, L9, 7O₀, O₀10 and W9 caused recovery in
stomatal conductance of infested whet up to 67.0, 87.8, 72.6, 28.8 and 35.4%,
respectively.
4.15. Effect of allelopathic bacteria on the growth and yield of wheat
grown under weed free conditions
4.15.1. Effect of allelopathic bacteria on wheat grown under weed free conditions at
tillering
Results indicated that none of the strains caused significant reduction in the measured
growth parameters of wheat grown under weed free conditions at tillering stage (Table
4.55). However, inoculation with strain L9 and 7O₀ significantly increased the number of
tillers of wheat up to 17.3 and 12.3%, fresh biomass up to 21 and 16.3% and shoot length
up to 14.1 and 9.7% than un-inoculated control, respectively.
4.15.2. Effect of allelopathic bacteria on wheat grown under weed free conditions at
booting
Inoculation with the selected strains of allelopathic bacteria remained non-inhibitory to
wheat at booting stage (Table 4.56). Results indicated that inoculation with strain T42,
O₀10 and W9 caused non-significant effects on number of tillers, fresh biomass and shoot
length of wheat than un-inoculated control. However, inoculation with strain L9 and 7O₀,
significantly increased the number of tillers of wheat up to 19.1 and 15%, fresh biomass
up to 24 and 18.2% and shoot length up to 16 and 13.2% than un-inoculated control,
respectively.

162
Table 4.55. Effect of allelopathic bacteria on wheat grown under weed free
conditions at tillering
Number of tillers Fresh biomass Shoot length
Treatments
m-2 (t ha-1) (cm)
Control 329.0 b 13.16 c 37.3 b

T42 330.0 b 13.2 c 37.5 b

L9 386.0 a 15.92 a 42.6 a

7O₀ 369.3 a 15.3 b 40.9 a

O₀10 330.3 b 13.34 c 37.4 b

W9 334.3 b 13.33 c 37.4 b

LSD 28.85 0.45 2.93

These measurements were taken at 50 days after sowing. Values sharing same letter(s) do
not differ significantly from each other at p <0.05 according to Duncan’s Multiple Range
Test

Table 4.56. Effect of allelopathic bacteria on wheat grown under weed free
conditions at booting
number of tillers Fresh biomass Shoot length
Treatments
m-2 (t ha-1) (cm)
Control 345.6 b 24.88 c 63.7 b

T42 345.3 b 24.92 c 63.9 b

L9 411.7 a 30.86 a 73.9 a

7O₀ 396.6 a 29.4 b 72.1 a

O₀10 345.6 b 24.96 c 63.9 b

W9 349.0 b 25.0 c 63.9 b

LSD 25.52 0.59 3.11

These measurements were taken at 100 days after sowing. Values sharing same letter(s)
do not differ significantly from each other at p <0.05 according to Duncan’s Multiple
Range Test

163
4.15.3. Effect of allelopathic bacteria on wheat grown under weed free conditions at
harvesting
Data indicated that inoculation with the selected strains of allelopathic bacteria did
not cause significant reduction in the measured growth and yield parameters of wheat at
harvesting (Table 4.57). Inoculation with strain T42, W9 and O₀10 caused non-significant
effects on the growth and yield of wheat. However, inoculation with strain L9 and 7O₀
significantly increased the number of tillers of wheat up to 20.2 and 16.1%, fresh biomass
up to 27 and 21%, shoot length up to 14.2 and 10.2%, grain yield up to 22.1 and 18.2%
and straw yield up to 28.2 and 21.1% than un-inoculated control, respectively.
4.15.4. Effect of allelopathic bacteria on physiology of wheat grown under weed free
conditions
Results indicated that the selected strains of allelopathic bacteria remained non-inhibitory
to physiological parameters of wheat (Table 4.58). Inoculation with strain T42, W9 and
O₀10 caused non-significant effects on assimilation rate, stomatal conductance and
evapo-transpiration rate of wheat than un-inoculated control. Inoculation with strain L9
and 7O₀ caused significant increase in assimilation rate of wheat up to 21.1 and 14.9%,
somatal conductance up to 15.8 and 12.1% and evapo-transpiration rate up to 21.2 and
17.4% than un-inoculated control, respectively.
4.16. Microbiological characterization of allelopathic bacteria
The efficient strains of allelopathic bacteria (T42, L9, 7O₀, O₀10 and W9) were
used to conduct pot and field trials after their further characterization for microbiological
characteristics. These characteristics included gram staining, motility, catalase, oxidase,
urease, chitinase, exo-polysaccharides production, siderophores production, phosphate
solubilization, auxins production and root colonization ability. The results of these tests
are shown in Table 4.59. The selected strains showed negative gram reaction. All the
strains were motile in nature and positive for catalase, oxidase, urease and chitinase
activity. Three of the selected strains (L9, T42 and 7O₀) showed exo-polysaccharides
production activity whereas strain O₀10 and W9 did not show this activity. The selected
strains also possessed siderophores production activity. Inorganic phosphate
solubilization activity was observed in strain T42 and L9 only. Auxins production ability
of allelopathic bacteria was determined in the presence and absence of precursor, L-
Tryptophan. The results showed high auxins production ability of strain L9 and 7O₀ in
the presence of L- tryptophan (96.02 and 74.56 ug/mL, respectively) and in the absence
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Table 4.57. Effect of allelopathic bacteria on wheat grown under weed free
conditions at harvesting
Shoot Fresh
Number of Grain yield Straw yield
Treatments length biomass
tillers m-2 -1 (t ha-1) (t ha-1)
(cm) (t ha )
Control 335.0 b 94.4 b 8.45 c 3.64 b 4.82 b

T42 335.0 b 94.6 b 8.48 c 3.65 b 4.84 b

L9 402.7 a 107.8 a 10.74 a 4.44 a 6.18 a

7O₀ 389.0 a 104.0 a 10.23 b 4.3 a 5.84 a

O₀10 335.3 b 95.5 b 8.47 c 3.64 b 4.83 b

W9 337.3 b 94.6 b 8.46 c 3.66 b 4.84 b

LSD 23.4 4.00 0.415 0.162 0.37

These measurements were taken at 150 days after sowing. Values sharing same letter(s)
do not differ significantly from each other at p <0.05 according to Duncan’s Multiple
Range Test

Table 4.58. Effect of allelopathic bacteria on physiology of wheat grown under weed
free conditions
Assimilation rate Stomatal Evapo-
Treatments
(A) conductance (gs) transpiration (E)
Control 9.17 b 228.0 b 3.23 b

T42 9.2 b 227.3 b 3.25 b

L9 11.1 a 264.0 a 3.92 a

7O₀ 10.53 a 255.7 a 3.79 a

O₀10 9.13 b 228.7 b 3.23 b

W9 9.17 b 228.0 b 3.24 b

LSD 1.2 12.4 0.168

Physiological parameters of plants were measured at 60 days after sowing. Values sharing
same letter(s) do not differ significantly from each other at p <0.05 according to Duncan’s
Multiple Range Test

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Table 4.59. Microbiological characterization of allelopathic bacteria
Characteristics T42 L9 7O₀ O₀10 W9
Gram staining - - - - -
Motility + + + + +
Catalase + + + + +
Oxidase + + + + +
Urease + + + + +
Exo-polysaccharides - + + - -
Siderophores + + + + +
P solubilization - + + - -
Chitinase + + + + +
IAA (ug/mL) with L-
18.32 96.02 74.56 7.63 4.82
Tryptophan
IAA (ug/mL) without L-
1.26 4.68 3.45 0.26 0.21
Tryptophan
+ denotes presence of a characteristic while – denotes absence of a characteristic in the
strain.

166
of L-tryptophan (4.68 and 3.45 ug/mL). Other three strains (T42, O₀10 and W9) showed
poor auxins producing ability. They produced auxins in the presence of L-tryptophan up
to 18.32, 7.63 and 4.82 ug/mL, respectively, and in the absence of L-tryptophan, auxins
producing ability of these strains was observed to be 1.26, 0.26 and 0.21 ug/mL,
respectively.
4.16.1. Root colonization assay
Allelopathic bacteria were evaluated for their root colonization ability in wheat and its
associated weeds. Five strains of allelopathic bacteria (T42, L9, 7O₀, O₀10 and W9) were
tested on wheat and three weeds of wheat (wild oat, little seed canary grass and broad
leaved dock) in root colonization assay. Root colonization ability of allelopathic bacteria
in wheat is shown in Figure 4.1. The depicted that root colonization ability of allelopathic
bacteria in wheat was variable. Inoculation with strain L9, T42 and 7O₀ optimally
colonized the root of wheat while being statistically similar with each other. Root
colonization ability of these strains was significantly higher than strain W9 and O₀10.
Data regarding root colonization ability of allelopathic bacteria on wild oat is shown in
Figure 4.2. The data indicated that high root colonization ability in wild oat was observed
due to inoculation with strain L9 which was statistically at par with strain T42 and
significantly higher than strain W9, O₀10 and 7O₀. Inoculation with strain T42 optimally
colonized the root of wild oat which was significantly higher than strain 7O₀ and non-
significant with strain O₀10 and W9. However, root colonization ability of strain 7O₀,
O₀10 and W9 in wild oat was statistically at par with each other. Data regarding root
colonization ability of allelopathic bacteria in little seed canary grass is shown in Figure
4.3. The results showed that the selected strains optimally colonized the root of little seed
canary grass while being statistically similar with each other. Figure 4.4 contains data
regarding root colonization ability of allelopathic bacteria in broad leaved dock. The
results showed that maximum root colonization ability was observed due to inoculation
with strain W9 which was significantly higher than strain 7O₀ and O₀10 while being
statistically non-significant with strain T42 and L9. Inoculation with strain T42, L9, 7O₀
and O₀10 also optimally colonized the roots of broad leaved dock while being statistically
at par with each other.

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 7 a
×105 cfu g-1 of dry root

6 a
a
5
4
3
b b
2
1
0
T42 L9 7O₀ O₀10 W9

Figure 4.1. Root colonization ability of allelopathic bacteria in wheat

7
×105 cfu g-1 of dry root

a
6 ab
5 bc bc
c
4
3
2
1
0
T42 L9 7O₀ O₀10 W9

Figure 4.2. Root colonization ability of allelopathic bacteria in wild oat

168
 6.5
×105 cfu g-1 of dry root

a
6
a
a
5.5
a a
5

4.5
T42 L9 7O₀ O₀10 W9

Figure 4.3. Root colonization ability of allelopathic bacteria in little seed canary grass

6
×105 cfu g-1 of dry root

a
5 ab
ab
b b
4
3
2
1
0
T42 L9 7O₀ O₀10 W9

Figure 4.4. Root colonization ability of allelopathic bacteria in broad leaved dock

169
4.16.2. Identification of allelopathic bacteria
Five efficient strains of allelopathic bacteria (T42, L9, 7O₀, O₀10 and W9) were
identified through 16S rRNA gene sequencing technique. The results indicated that the
selected strains of allelopathic bacteria belonged to pseudomonads. Strain T42 was
identified to be Pseudomonas putida KT2440, strain L9 was identified to be
Pseudomonas fluorescens F113, strain 7O₀ was identified as Pseudomonas fluorescens
SBW25, strain O₀10 was identified as Pseudomonas aeruginosa PAO1 and strain W9
was identified to be Pseudomonas alcaligenes NBRIC14159. The phylogenetic
relationship between these strains has been shown in Figure 4.5.

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 Pseudomonas stutzeri
Bradyrhizobium japonicum
Bacillus megaterium
Bacillus subtilis
Pseudomonas alcaligenes NBRC 14159 (W9)
Pseudomonas gessardii
Pseudomonas fluorescens F113 (L9)
Pseudomonas fluorescens SBW25 (7O0)
Pseudomonas putida KT2440 (T42)
Pseudomonas aeruginosa PAO1 (O010)
Pseudomonas azotoformans
Bacillus thuringiensis
Bacillus cereus
Bacillus anthracis
Pseudomonas trivialis
Rhizobium leguminosarum
Rhizobium etli

Figure 4.5. Phylogenetic relationship between different strains of allelopathic bacteria.

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CHAPTER-V DISCUSSION

Weeds invasion in crops causes huge economic losses. To avoid these economic
losses, different weed control practices are adopted which are based on chemical, manual
and mechanical approaches. Use of chemical and mechanical control methods has given
rise to several issues of environmental degradation, biodiversity and human health.
Manual weeding is considered to be environment and user friendly technique but shortage
and high cost of labor for large cropped area has reduced its adoption. The side effects of
these control methods have accumulated over time which has necessitated the
development of some other non-conventional control methods based on biological
approaches. Therefore, this research work was conducted to investigate the role of
allelopathic bacteria in the biological control of weeds associated with wheat. The use of
natural antagonists of weeds to reduce their growth, density, reproductive capacity or
impact on crops is called biological weed control (Quimby and Birdsall, 1995). The
rhizosphere inhabiting bacteria which produce phytotoxic metabolites and hence suppress
the germination and growth of certain plant species are called allelopathic bacteria (Sturz
and Christie, 2003). Allelopathic bacteria were isolated from the rhizosphere of wheat and
its associated weeds which were in turn sampled from chronically infested fields.
Schippers et al. (1987) reported that plant growth inhibitory rhizobacteria are abundantly
found in fields under continuous growth of single plant species year after year.

A large collection of rhizobacteria was made from the rhizosphere of wheat and
its associated weeds in the Soil Microbiology and Biochemistry Laboratory of Institute of
Soil and Environmental Sciences, University of Agriculture, Faisalabad. These
rhizobacteria were subjected to several bioassays based on their ability to produce
phytotoxic metabolites. Rhizobacteria were first evaluated for cyanide production on
plate assay following Bakker and Schippers (1987). Kremer and Souissi (2001) reported
that cyanide produced by some rhizobacteria is the major plant growth inhibitory
substance in affecting some plant species.

This study showed that 22.6% strains (89 out of 393) of rhizobacteria from wheat
and its associated weeds produced cyanide at various levels and their distribution in
different rhizospheres was variable. Twenty five percent of the rhizobacteria of wheat
produced cyanide to various degrees. The proportion of cyanogenic rhizobacteria in the

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rhizosphere of broad leaved dock, wild oat, little seed canary grass common lambs’
quarter and field bindweed accounted for 41, 19.8, 7.7, 23.7 and 17.4% among their
isolates, respectively. The cyanogenic rhizobacteria were divided into low, medium, high
and very high cyanide producing rhizobacteria which accounted for 8.4, 6.4, 5.1 and 2.8%
of the rhizobacteria from wheat and its associated weeds, respectively. These results are
in line with Kremer and Souissi (2001) in which cyanogenic rhizobacteria accounted for
32% of the rhizobacteria which produced cyanide at various levels. Cyanogenic
rhizobacteria were selected for further bioassays based on production of phytotoxic
metabolites by rhizobacteria in this study. However, non-cyanogenic rhizobacteria have
also been reported to suppress root length of barnyard grass and lettuce by Kremer and
Souissi (2001). Zeller et al. (2007) found that the sensitivity of different plant species to
cyanide was variable. They tested the effect of cyanide application on six plant species
(Echinochloa crus-galli, Galium mollugo, Daucus carota, Triticum aestivum, Hordeum
murinum and Centaurea jacea). The results indicated that E. crus-galli, G. mollugo and
D. carota were more sensitive to cyanide than T. aestivum, H. murinum and C. jacea.
Hence, they suggested that cyanide producing rhizobacteria could be selectively used to
suppress H. murinum, C. jacea and G. mollugo grown in wheat.

The cyanogenic rhizobacteria were then tested in E. coli anti-metabolite assay

which was also used in screening process of rhizobacteria for potential phytotoxicity by
Kremer et al. (1990). The selected strains were spot inoculated on plates overlain with
growth of sensitive E. coli strain K12. Nineteen of the cyanogenic strains of rhizobacteria
inhibited growth of sensitive E. coli strain K12 in this assay which was evident due to
formation of growth inhibition zone around the spot of inoculation of cyanogenic
rhizobacteria.

These 19 strains of rhizobacteria were selected for further screening in lettuce

seedling bioassay. Variable effects of these strains were observed on growth of lettuce
seedlings. The results of this study indicated that Root length of lettuce seedlings was
significantly reduced due to inoculation with 7 of the strains (36.8% of the isolates) as
compared to un-inoculated control. Seven strains significantly increased the root length of
lettuce seedlings than un-inoculated control. Other 5 strains remained non-significant
with un-inoculated control regarding their effect on root length of lettuce seedlings. Shoot
length of lettuce seedlings was significantly reduced, promoted and not affected by 6, 9

173
and 4 of the selected strains than control, respectively. Biomass of lettuce seedlings was
significantly reduced, enhanced and not affected by 5, 6 and 8 of the selected strains than
control, respectively. All the E. coli inhibiting strains did not inhibit the growth of lettuce
seedlings in the present study which is in agreement with Kremer et al. (1990). This study
showed contrasting effects of cyanogenic rhizobacteria on growth of lettuce seedlings
which have also been reported by Alstrom and Burns (1989). They also reported the
promotion of plant growth due to the application of cyanogenic rhizobacteria. Similar
results were also achieved by Zermane et al. (2007). The sensitivity of lettuce to
phytotoxic metabolites of rhizobacteria has been reported in some studies (Alstrom, 1987;
Kremer et al., 1996). Therefore, use of this bioassay to screen rhizobacteria for biocontrol
of weeds was suggested by Li and Kremer (2000) and Kremer (2013). Alstrom and Burns
(1989) found that cyanogenic rhizobacteria may promote or reduce the growth of lettuce
seedlings. They screened rhizobacteria using lettuce seedling bioassay for biocontrol of
weeds. They reported that 11% of the 337 isolates significantly inhibited the root growth
of lettuce seedlings, 5% of the isolates significantly increased the root growth of lettuce
seedlings while 84% of the isolates had non-significant effect on root growth of lettuce
seedlings. Ahonsi et al. (2002) also conducted lettuce seedling bioassay using water agar
in which the proportion of rhizobacteria suppressive to the growth of lettuce seedlings
was 3.3%. However, the proportion of rhizobacteria suppressive to root growth of lettuce
was greater in this study because only cyanogenic and E. coli growth inhibiting
rhizobacteria were selected for this bioassay in the present study. This study also reflects
stability in the characteristics of our strains which could further be explored for growth
inhibition of weeds. The strains showing non-significant effects on growth of lettuce
seedlings might be due to non-host interactions or lack of stability in their characteristics
(Weiland et al., 2001).

The strains used in lettuce seedling bioassay were selected for agar bioassays on
wheat and its associated weeds. The results of this study indicated that all the E. coli
inhibiting rhizobacteria did not reduce the growth of lettuce seedlings which is in
agreement with Kremer et al. (1990). It may be due to different nature of interaction of E.
coli inhibiting rhizobacteria with lettuce, weeds and E. coli which may alter the
production of phytotoxic metabolites and their effects. However, these results are
inconsistent with Fredrickson and Elliott (1985). The results of this study showed that the

174
rhizobacteria which do not suppress lettuce seedlings may suppress weeds and vice versa.
This observation was also reported by Souissi and Kremer (2010) in which no correlation
was found between the results of lettuce seedling bioassay and that of seedling bioassays
on wheat and its associated weeds. Even though the lettuce seedlings assay is often used
in preliminary screening foe evaluation of phytotoxicity, the lack of a good correlation
with agar bioassays on weeds and crops suggest that the inhibitory activity of the majority
of rhizobacteria is host specific (Alstrom, 1987). Previous work has also shown that the
inhibitory activity of rhizobacteria originating from different plant species depended on
particular hosts only (Dotyel et al., 1994). Therefore, the rhizobacteria may also be
directly screened on target plants but it requires uniformity in germination and growth of
seeds of target plant species. However, Kremer and Souissi (1998) suggested that lettuce
seedlings respond to microbially produced phytotoxins almost similarly as leafy spurge.
They suggested that lettuce seedling bioassay may be used to screen phytotoxins
producing rhizobacteria. They found high correlation (r2 = 0.8) between effect of
deleterious rhizobacteria on lettuce seedlings and leafy spurge. Therefore, Kremer et al.
(1990) suggested that rhizobacteria should be directly screened on weeds rather than
indicator bioassays due to inconsistency in results. Li and Kremer (2000) also suggested
that it is more accurate to test rhizobacteria on weed species rather than lettuce seedlings.
However, lettuce seedling bioassay is conventionally used in studies on biological weed
control due to its sensitivity to several phytotoxins. Kremer and Souissi (2013) also
screened rhizobacteria for potential phytotoxicity to leafy spurge through lettuce seedling
bioassay.

These 19 strains were, hence, selected for agar bioassay on target plants seedling
bioassay following Kennedy et al. (1991). These strains were tested on 4 weed species
(wild oat, little seed canary grass, broad leaved dock and common lambs’ quarter) and
wheat. Inoculated seeds of weeds were allowed to grow on water agar plates for a week
and that of wheat for 5 days. Inoculation with strain ESO-11 and W28 inhibited the
germination and seedling growth of all the tested plant species being non-selective (non-
host specific). Inoculation with strain T12, T18 and T75 suppressed the germination and
seedling one or more weeds and wheat. The strain T38, ESO-8 and W9 inhibited the
germination and seedling growth of one or more weeds but remained non-inhibitory to
wheat being selective (host specific). However, inoculation with 9 strains inhibited one or

175
weeds and promoted the germination and growth of wheat. Selective response of
allelopathic bacteria in different plant species may be due to tolerance of some plants to
the phytotoxic substances produced by allelopathic bacteria (Owen and Zdor, 2001).
Selective effects of these strains may also be due to non-production of phytotoxic
metabolites in interaction with rhizosphere of non-host plants (Kennedy et al., 2001).
There may be differences in root colonization of strains in different plants giving rise to
selectivity (Zeller et al., 2007). Production of phytotoxic metabolites by allelopathic
bacteria also depends on nature of substrates available in the root exudates of a plant
species. As the composition and nature of substances in the root exudates are also specific
to plant species. This can also give rise to selectivity in rhizobacteria (Begonia and
Kremer, 1994). The results of this study are in agreement with Kennedy et al. (1991).
They tested over 1000 isolates on wheat and downy brome in agar bioassays and found
81 strains inhibitory to downy brome and non-inhibitory to wheat. Harris and Stahlman
(1996) isolated over 800 bacteria from soil and plant roots and screened through agar
bioassay on jointed goat grass, downy brome, Japanese brome and winter wheat. They got
162, 202 and 129 strains inhibitory to downy brome, Japanese brome and jointed goat
grass. Over 350 strains from their collection remained non-inhibitory to winter wheat.
Vargas and O’Hara (2006) applied 125 strains of rhizobacteria on annual ryegrass and
wild radish. The results indicated that 74 strains significantly reduced root growth of wild
radish and/or annual ryegrass. Li and Kremer (2000) obtained 43 isolates inhibitory to
green foxtail, redroot pigweed and/or common cocklebur among a large collection of
thizobacteria isolated from several weed hosts. Kennedy and Stubbs (2007) isolated 2450
rhizobacterial strains from winter wheat, jointed goat grass and downy brome. They
screened these rhizobacteria for inhibition of jointed goat grass and non-inhibitory effects
on winter wheat using agar bioassay. They obtained 1236 isolates inhibitory to jointed
goat grass and among these isolates only 76 did not inhibit seedling growth of winter
wheat. Hence, the strains of rhizobacteria inhibitory to one or more weeds with non-
inhibitory effects on crops may be used for biological control of these weeds in crops.
Lakshmi et al. (2015) isolated Pseudomonas aeruginosa KC1 from the rhizosphere of
castor plants and tested on spiny amaranth, pigweed and wheat in agar bioassay. Results
indicated that inoculation with Pseudomonas aeruginosa KC1 significantly reduced the
growth of weeds and wheat.

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 The strains found inhibitory to germination and growth of common lambs’ quarter
were also found to be inhibitory to wheat growth in the present study. Therefore, growth
room studies were conducted on 3 weeds (wild oat, little seed canary grass and broad
leaved dock) and wheat using 10 strains (which suppressed one or more weeds and not
wheat in agar bioassays). These strains showed variable potential of inhibition of
germination and growth of weeds. Germination of little seed canary grass, wild oat and
broad leaved dock was reduced from 18.5 to 58.7, 15.2 to 63.3 and 18.4 to 60.5% than
un-inoculated control, respectively. Similar reductions in different growth parameters of
weedy plants were also observed. Results showed that none of the selected strains caused
any significant reduction in any growth parameter of wheat. Inoculation with strain T19,
L9 and 7O₀ significantly increased the germination of wheat than un-inoculated control.
Inoculation with strain T19, L9, 2O₀ and 7O₀ significantly improved the growth of wheat
plants than un-inoculated control. The results of these growth room studies are in
agreement with the previous studies. Kennedy and Stubbs (2007) applied 76 strains of
rhizobacteria in growth chamber suppressive to jointed goat grass and not winter wheat.
They obtained 7 strains of rhizobacteria in this study which were inhibitory to the growth
of jointed goat grass but not winter wheat under growth chamber conditions. The
proportion of rhizobacteria suppressive to the weed was lesser in their study than the
present study because they used non-sterile soil as growth medium where the availability,
concentration build up and movement of phytotoxins remains low. Kennedy et al. (1991)
screened a large collection of rhizobacteria for selective suppression of downy brome
grown in winter wheat in laboratory bioassays. They got 81 isolates from this screening
and applied them in potted non-sterile soil on downy brome grown in wheat in growth
chamber studies. Six strains consistently reduced the growth of downy brome but not
wheat in their experiment. Harris and Stahlman (1996) obtained 40 strains after
laboratory screening and applied to downy brome, jointed goat grass and winter wheat in
potted non-sterilized soil:sand (4:1) mixture in growth chamber. Nearly 25% isolates
inhibited root and shoot biomass of jointed goat grass or downy brome but not winter
wheat in this experiment. Caldwell et al. (2012) applied Pseudomonas fluorescens
BRG100 on green foxtail in growth chamber conditions which caused 74% reduction in
its root growth. Rakian et al. (2015) isolated 5 strains of rhizobacteria from weeds and
tested for germination inhibition and relative growth rate of Paspalum conjugatum,
Ageratum conyzoides, Amaranthus spinosus and Chromolaena odorata. Germination

177
capacity was reduced up to 44, 2.67, 13.3 and 2.67% of P. conjugatum, A. conyzoides, A.
spinosus and C. odorata, respectively. Relative growth rate of P. conjugatum, A.
conyzoides, A. spinous and C. odorata were reduced up to 25.5, 1.4, 8.11 and 1.48% over
un-inoculated control, respectively.

The present study showed selectivity in effects of rhizobacteria on weeds and

wheat. Inoculation with strain L9 and 7O₀, instead increased the growth of wheat. Similar
results were also obtained by Mejri et al. (2010). They tested biological weed control
agent Pseudomonas trivialis X33d on downy brome, durum wheat, barley, oat, chickpea,
pea, mung bean and faba bean in potted sterile soil mixture under growth chamber
conditions. The results showed 41 and 20.5% reduction in root and shoot biomass of
downy brome over control due to inoculation with Pseudomonas trivialis X33d. Root
biomass of durum wheat, barley, oat, pea chickpea, mung bean and faba bean were
increased up to 59, 61, 15, 25, 2, -1 and -17.5% over control, respectively. Inoculation
with Pseudomonas trivialis X33d, however, increased the shoot biomass of durum wheat,
barley, oat, pea, chickpea, mung bean and faba bean up to 20.5, 35.5, 4, 1.5, 1.5 and 7.5%
over control, respectively. Pesudomonas fluorescens D7 was previously reported to
inhibit the growth of downy brome by Kennedy et al. (1991). Kennedy et al. (2001)
reported growth promotion of oilseed rape due to inoculation with this strain.
Pesudomonas fluorescens strain G2-11 was found to suppress barnyard grass, field
bindweed, ivyleaf morningglory and green foxtail. Li and Kremer (2006) reported growth
promotion of wheat and soybean due to application with this strain. Miche et al. (2000)
also reported contrasting effects of applying Azospirillum brasilence strain L4 on
inhibition of weed (Striga hermonthica) and growth promotion of crop (Sorghum
bicolor).

Five strains of allelopathic bacteria (T42, L9, 7O₀, O₀10 and W9) were selected
for further evaluation of their bioherbicidal activity under more rigorous conditions in pot
trials. These strains were applied on 3 weed species (wild oat, little seed canary grass and
broad leaved dock). These weeds were grown along with wheat to test the weed
suppression effects of allelopathic bacteria and resultant improvement in growth and yield
of infested wheat. Data regarding growth and yield parameters of weeds and wheat was
collected at 3 growth stages of wheat (tillering, booting and harvesting). The wild oat
suppression effects of strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in grain yield

178
of infested wheat up to 60.0, 73.6, 35.8, 22.0 and 49.7%, respectively. The suppression of
little seed canary grass by these strains caused recovery in loss of grain yield of infested
wheat up to 20.1, 55.0, 66.9, 59.0 and 3.4%, respectively. Suppression of broad leaved
dock recovered the loss in grain yield of infested wheat up to 45.2, 53.9, 46.3, 11.6 and
68.0%, respectively. These strains remained non-deleterious to wheat grown under weed
free conditions. However, inoculation with strain L9 and 7O₀ significantly increased the
growth and yield of wheat i.e., grain yield up to 40.3 and 34.3% and straw yield up to
37.1 and 32.1% than control, respectively. These effects of allelopathic bacteria were also
evident from other growth, yield and physiological parameters of wheat and its associated
weeds.

Inoculation with strain L9 most effectively suppressed the growth of wild oat
followed by strain T42 and W9. The magnitude of growth suppression of wild was lesser
due to inoculation with strain 7O₀. Inoculation with strain O₀10 remained non-significant
with weedy control in affecting the growth of wild oat in most of growth parameters at
different stages. Inoculation with strain 7O₀ proved most effective in suppressing the
growth of little seed canary grass at all growth stages. The strains L9, O₀10 and T42
proved next effective strains for biocontrol of little seed canary grass. However,
inoculation with W9 remained non-significant with weedy control in most of growth
parameters at all growth stages. The strain W9 was the most effective strain in
suppressing broad leaved dock followed by L9, T42, 7O₀ and O₀10. Results obtained in
these pot trials are in agreement with the previous studies on biological control of weeds
by rhizobacteria (Bakker and Schippers, 1987; Begonia and Kremer, 1994; Gealy et al.,
1996; Owen and Zdor, 2001). Flores-Vargas and O’Hara (2006) isolated 442 strains of
rhizobacteria from wild radish, annual rye grass and capeweed and screened in laboratory
bioassay. They applied 74 strains of potential biocontrol agents of annual rye grass and
wild radish to these weeds, grapevine and legume cover crop sub-terranean clover in a
glasshouse experiment. The results indicated that 19 strains significantly reduced root and
shoot lengths and dry weights of weeds. Fourteen strains inhibited growth of both weeds
while five strains inhibited only wild radish. Three promising strains among these were
applied to sub-terranean clover and grapevine in which none of the strains caused any
significant deleterious effects on sub-terranean clover or grapevine. The increase in
growth and yield of wheat has been reported in the present study due to application of

179
allelopathic bacteria. This increase in plant growth by these bacteria may be due to
production of plant growth promoting metabolites by these rhizobacteria as reported by
Raju et al. (1999). Different nature of interaction of rhizobacteria in different plant hosts
has also been reported by Bouillant et al. (1997) and Miche et al. (2000). They reported
that Azospirillum brasilence strain L4 prevented the germination of the parasitic weed
Striga hermonthica and promoted the growth of Sorghum bicolor. Pseudomonas
fluorescens D7 aggressively reduces the growth of downy brome under field conditions
(Kennedy et al., 1991). Kennedy et al. (2001) reported that Pseudomonas fluorescens D7
stimulates the growth of oilseed rape. Boyetchko (1997) reported growth suppression of
green foxtail by rhizobacteria which also stimulated growth of spring wheat giving
competitive advantage to spring wheat to overcome weed invasion. The present study
indicated that allelopathic bacteria did not completely suppress weeds but significantly
reduced early growth of weeds and help the crop plants to effectively compete with
weakened weed plants. This is in agreement in studies reported by Kremer and Kennedy
(1996). Lakshmi et al. (2015) studied effect of Pseudomonas aeruginosa KC1 on spiny
amaranth, pigweed and wheat in a pot trial. Results indicated that root growth of spiny
amaranth, pigweed and wheat were reduced up to 42.5, 51.6 and 12.7% over un-
inoculated control, respectively. Li and Kremer (2006) applied 43 isolates to their host
weed, wheat, soybean, one non-host monocotyledonous and one non-host dicotyledonous
weeds (green foxtail, barn yard grass, field bindweed, ivyleaf morningglory, redroot
pigweed and common cocklebur) in potted soil under greenhouse conditions. These
isolates were obtained through laboratory agar bioassays on respective weeds and crops.
Forty three strains were applied to green foxtail among which 29 significantly suppressed
its root and shoot growth. Twenty five strains were applied to barn yard grass among
which 8 significantly inhibited its root and shoot growth. Inoculation with 8 of the
selected strains (25) significantly reduced the root and shoot growth of field bindweed.
Nine strains significantly reduced the root and shoot growth of ivyleaf morningglory out
of 25 applied strains. Eleven strains were applied to redroot pigweed out of which 3
significantly reduced its root and shoot growth. These 11 strains were also applied to
common cocklebur out of which 2 significantly inhibited its root and shoot growth.
Twenty seven strains were applied to wheat and soybean among which 5 strains
significantly reduced their root growth. Sixteen strains significantly increased the root
growth of soybean, 10 significantly increased the shoot growth of soybean while other

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strains remained statistically non-significant with un-inoculated control. Eighteen strains
significantly increased the root growth of wheat, 13 significantly increased the shoot
growth of wheat while other strains remained statistically at par with un-inoculated
control in affecting the growth of wheat. Allelopathic bacteria showed host specificity in
the present study in its response which varied with the plant species which also agrees
with Kennedy et al. (2001). Therefore, Li and Kremer (2006) suggested that due to
possession of dual abilities of plant growth promotion and weed suppression by
rhizobacteria, they should be screened to get dual benefits in different cropping systems.
Inoculation of infested wheat with two strains (L9 and 7O₀) showed more bioherbicidal
activity than their potential in the present study. This happened because they also carried
plant growth promoting characteristics for wheat. Dual benefits of weed suppression and
promotion of growth of infested wheat were recorded in the present study which
increased the competitive ability of wheat and overcome weed infestation. This is in
agreement with Harris and stahlman (1993). They described that competition from wheat,
when present might reduce weed growth even further. Possession of dual characteristics
in some strains may provide competitive advantage to crop thereby further suppressing
the growth of weeds and losses caused to crop. The potential of weed suppression of the
applied strains was lesser in the present study than previously studied under laboratory
conditions which agrees with Harris and Stahlman (1996). This might bee due to
competition of applied strains with indigenous microbial communities of soil and stress of
environmental and growth conditions. These conditions may influence the population and
production of metabolites of allelopathic bacteria resulting into their weed biocontrol
activity.

Allelopathic bacteria were also applied to weeds grown in wheat and wheat grown
under weed free conditions under natural conditions in 3 field trials. The selected strains
of allelopathic bacteria (T42, L9, 7O₀, O₀10 and W9) were applied at concentration (108
to 1012 colony forming units m-2) which is considered adequate to achieve biocontrol of
weeds under field conditions (Kennedy et al., 1991). In field trial I, suppression of broad
leaved dock due to inoculation with strain T42, L9, 7O₀, O₀10 and W9 resulted into
recovery in loss of grain yield of infested wheat up to 38.4, 64.0, 51.0, 17.3 and 62.9%
and straw yield up to 51.4, 69.5, 61.9, 22.4 and 71.2%, respectively. In field trial II,
suppression of little seed canary grass resulted into recovery of loss of grain yield of

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infested wheat up to 34.3, 55.1, 64.3, 57.2 and 1.3%, and straw yield up to 37.2, 65.8,
69.4, 56.7 and 3.2%, respectively. In field trial III, suppression of wild oat and little seed
canary grass caused recovery in loss of grain yield of infested wheat up to 47.9, 60.7,
53.7, 28.9 and 36.6%, and straw yield up to 51.1, 61.0, 56.1, 37.6 and 36.6%,
respectively. These effects were also evident from other growth, yield and physiological
parameters of wheat and its associated weeds. These strains of allelopathic bacteria
remained non-inhibitory to wheat grown under weed free conditions. However,
inoculation with strain L9 and 7O₀ significantly improved the growth and yield of wheat
grown under weed free conditions. Previous research work on biological control of weeds
with rhizobacteria was limited to laboratory studies and only scanty information is
available on biological control potential of rhizobacteria under field conditions. Kennedy
et al. (1991) conducted three field trials to evaluate bioherbicidal activity of 3 isolates of
downy brome inhibitory bacteria in fields naturally infested with downy brome. Two
isolates reduced the growth of downy brome up to 31 and 53% over control, respectively.
Reproductive capacity of downy brome was reduced up to 64% in this field study.
Resultantly, the yield of infested wheat was increased from 18 to 35% over weedy control
at 2 sites. Harris and Stahlman (1996) also applied downy brome inhibitory isolates in the
field. In this study, inoculation with 2 strains increased the yield of infested wheat by 13
and 10%, respectively. Weissmann et al. (2003) evaluated bioherbicidal activity of
Serratia plymuthica strain A153, strain A131 and their combination in spring wheat,
spring barley and potatoes. This bacterium was found effective against broad leaf weeds
in previous studies (Weissmann and Gerhardson, 2001). The treatments significantly
reduced the growth of weeds but the yield of infested wheat was not improved due to
severe drought stress. Inoculation with A153 significantly reduced number and growth of
chickweed grown in spring barley. However, combination of A153 and A131
significantly reduced most of weeds in spring barley. No improvement in grain yield of
infested barley crop was observed in this experiment which also showed some
phytotoxicity symptoms. Foliar application of biocontrol agents, although produced some
necrotic lesions on weeds but no reduction in their vigor was observed in this study and
no significant reduction or improvement in yield of potatoes was noted. These results are
inconsistent with the findings of our study in which yield of infested crop was improved
due to suppression of weeds. This may be due to non-selectivity of bacteria applied in
barley and foliar application of biocontrol agent in their study. Kennedy and Stubbs

182
(2007) used 6 strains of rhizobacteria inhibitory to jointed goat grass which were screened
after laboratory and growth chamber studies. The field trial was conducted consecutively
up to 3 years. Above ground growth of jointed goat grass was reduced by >20% in the
first year by 2 strains. The growth suppression of jointed goat grass was recorded from 20
to 74% in the second year. However, in the third year, selected strains reduced the growth
of jointed goat grass from 27 to 47%. These results are consistent with findings of the
present study.

These five strains of allelopathic bacteria were characterized for microbiological

and biochemical characteristics. They were gram negative, motile and urease, oxidase,
catalase and chitinase carrying bacteria. Flores-Vargas and O’Hara (2006) also reported
positive results for catalase, oxidase, motility, HCN production and negative gram
reaction of rhizobacteria with potential for biocontrol of wild radish and annual rye grass.
Kremer et al. (1990) also reported similar characteristics of rhizobacteria associated with
weeds. Catalase activity in bacteria improves the resilience of plants against
environmental stresses (Rathaur et al., 2012). Chitinase activity may degrade the cell wall
of plant pathogenic fungi (Recep et al., 2009). These strains also carried siderophores
producing ability. Siderophores are considered to chelate iron compounds which become
more available to plants and lesser to plant pathogens (Robin et al., 2008). Production of
antibiotics by these strains was tested in E. coli antimetabolite assay. The production of
antibiotics in the rhizosphere may suppress the population of plant pathogenic
microorganisms (Haas and Keel, 2003). These characteristics help to improve growth of
plants by reducing the population and activity of plant pathogens in their rhizosphere.
Strain L9 and 7O₀ also had the ability to solubilize inorganic phosphate and produce exo-
polysaccharides. Aggregation and structure of soil adhering to plants is improved due to
production of extra-cellular polysaccharides. It improves uptake of water and minerals
thereby causing promotion of plant growth (Dobbelaere et al., 2003). Strain L9 and 7O₀
also produced higher amount of IAA equivalents in the presence and absence of L-
tryptophan, T42 was intermediate, and strain O₀10 and W9 produced lower amounts of
IAA equivalents. The level of IAA produced by rhizobacteria affects plant growth based
on its concentration and nature of plant species (Peck and Kende, 1995; Spaepen et al.,
2007). However, elevated levels of IAA produced in the rhizosphere of plants suppress its
growth including weeds (Sarwar and Kremer, 1995). Overall impact of IAA on plants

183
depends on sum of endogenous and exogenous productions. At lower levels, it increases
lateral and adventitious root growth and hence other growth and yield components of
plants (Lambrecht et al., 2000). Ability of solubilization of inorganic phosphorus was
observed in strain L9 and 7O₀ which improved its availability to plants resulting into
promotion of plant growth (Yao, 2004). Promotion of growth of wheat by allelopathic
bacteria has been attributed to the possession of these characteristics.

Kremer et al. (1990) reported that the rhizobacteria associated with weeds possess
characteristics which are inconsistent with the accepted definitions of plant growth
promoting and deleterious rhizobacteria. Therefore, the term of allelopathic bacteria is
suggested to be more convenient for such rhizobacteria. Allelopathic bacteria were also
assayed for root colonization ability in wheat and associated weeds. Results showed that
inoculation with strain T42, L9 and 7O₀ optimally colonized the roots of wheat while
inoculation with strain O₀10 and W9 showed poor root colonization. Inoculation with
strain L9 and T42 optimally colonized the roots of wild oat followed by strain 7O₀, O₀10
and W9. Inoculation with strain L9, T42, 7O₀, O₀10 and W9 optimally colonized the
roots of little seed canary grass. Inoculation with strain W9 optimally colonized the roots
of broad leaved dock followed by inoculation with T42 and L9. However, inoculation
with strain 7O₀ and O₀10 caused intermediate root colonization of broad leaved dock.
Root colonization pattern of plant growth promoting and biocontrol rhizobacteria has also
been studied by Gamalero et al. (2005) and Rosas et al. (2009). The difference in
colonization ability of different strains of allelopathic bacteria was observed in the present
study. This characteristic has been attributed to the selectivity of biocontrol agents in the
rhizosphere of different plants by Gurley and Zdor (2005).

Allelopathic bacteria were identified through 16S rRNA gene sequencing

technique. Results showed that T42 is Pseudomonas putida KT2440, L9 is Pseudomonas
fluorescens, 7O₀is Pseudomonas fluorescens SBW25, O₀10 is Pseudomonas aeruginosa
and W9 is Pseudomonas alcaligenes NBRC14159. Pseudomonads have been reported for
their potential role in biological control of weeds (Kennedy et al., 1991; Kremer and
Kennedy, 1996; Flores-Vargas and O’Hara, 2006) and plant pathogens (Compant et al.,
2005).

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CHAPTER-VI SUMMARY

Indiscriminate use of chemical herbicides has given rise to multiple issues of

human health and environment. Weeds still pose challenges in crop production and
continuous use of herbicides has piled up their side effects. Therefore, importance of
alternative control methods based on biological approaches has increased with time which
may at least be integrated with the existing weed control practices. The biocontrol agents
which were investigated in past included insects and pathogens. Wide host range, change
of host in the environment and excessive reliance on suitable environmental conditions
for action reduced the value of such biocontrol agents. Allelopathy has also been
investigated widely for weed control. Extracts and residues of plants containing
allelochemicals have been applied for weed control. The rhizobacteria which release
phytotoxic metabolites in the rhizosphere of certain weed species also have the potential
to suppress their growth and reproduction. These rhizobacteria may be called allelopathic
bacteria. They remained ignored in the past for their possible role in biocontrol of weeds.
The current scenario has increased the importance of such bacteria. Therefore, this study
was conducted to investigate the role of allelopathic bacteria in the biocontrol of weeds
associated with wheat and their impact on the growth and yield of both infested and weed
free wheat.

Plants samples of wheat and its associated weeds were collected from chronically
infested fields across the district Faisalabad, Pakistan (30.903042° to 31.732165° N and
72.818847° to 73.491648° E). Rhizobacteria (393 strains) were isolated from these
samples using dilution plating technique. These strains were screened through several
bioassays based on in vitro production of phytotoxic metabolites and in vivo effects on
different plant species (phytotoxins sensitive lettuce seedlings, wheat and associated
weeds). All the strains were tested for cyanide production in a plate assay having filter
papers soaked with alkaline picrate. Eighty nine of the strains were found to be
cyanogenic and their abundance in the rhizospheres of individual plants was found to be
variable. All the cyanogenic rhizobacteria were tested in antimetabolite assay against
sensitive E. coli strain K12. Growth of E. coli was obtained on Luria Bertani (LB) Agar
media plates. The growth of E. coli was overlain on King’s B Agar media. The
rhizobacterial strains were spot inoculated on E. coli overlain plates and incubated. Halo
zones around the spot of inoculation were observed after 48 hours. The halo zones

185
indicated antibiosis activity due to inhibition of growth of E. coli by cyanogenic
rhizobacteria. Nineteen of the 89 strains suppressed the growth of E. coli in this assay
which produced the variable diameter of halo zone from 2.5 to 12.3 mm. The E. coli
inhibiting cyanogenic strains were applied to phytotoxins sensitive plant, lettuce, in agar
bioassay at 106 cells mL-1. Pre-germinated seeds of lettuce were placed on sterilized water
agar (100:1) and placed in dark for 96 hours. Triplicate plates were set up for each strain.
Results indicated that 6 strains significantly reduced the root and shoot lengths of lettuce
seedlings while 5 strains significantly reduced the dry matter of lettuce seedlings at p
<0.05. Five strains promoted the seedling growth of lettuce while others caused non-
significant effects. Host specific effects of rhizobacteria may cause inhibition of weeds
but not lettuce. Therefore, these 19 strains were selected for agar bioassays on wheat ans
its associated weeds. These strains were applied to wheat and 4 weeds (wild oat, little
seed canary grass, broad leaved dock and common lambs’ quarter) in similar bioassays as
for lettuce. The strains were applied to surface sterilized seeds of wheat and weeds at cell
density of 106 cells mL-1. Data regarding germination and seedling growth parameters
was collected after 7 days for weeds and after 5 days for wheat. This study reported
inhibition of all the tested plants non-selectively due to inoculation with 2 strains. Three
strains selectively inhibited one or more weeds but also remained inhibitory to wheat.
Three strains selectively inhibited one or more weeds and caused non-significant effects
on wheat. However, 9 strains were characterized as selectively suppressive to 1 or more
weeds and promotory to wheat (Figure 6.1). Ten strains were selected from the later 2
groups based on maximum inhibition of weeds to conduct growth room studies. These
strains were applied to 3 weeds and wheat which were sown in sand jars (13 × 6 cm and
filled with 350 g sand) under axenic conditions. These strains showed variable potential
of inhibition of germination and growth of weeds. Four of these strains significantly
increased the growth parameters of wheat. Five strains most inhibitory to weeds in this
study (T42, L9, 7O₀, O₀10 and W9) were selected to conduct pot and field trials. Three
pot trials were conducted on 3 weeds, each co-seeded with wheat. One pot trial was
conducted to evaluate effect of allelopathic bacteria on wheat grown under weed free
conditions.

Infestation of wild oat, little seed canary grass and broad leaved dock caused loss
in grain yield of infested wheat up to 60.8, 59.9 and 55.8% than weed free control,

186
 Isolation of rhizobacteria from wheat and its
associated weeds grown in chronically infested fields

Cyanogenic Rhizobacteria Non-cyanogenic

Rhizobacteria
Low Medium High Very High

Cyanogenic and inhibitory to Cyanogenic and non-

growth of sensitive E. coli inhibitory to sensitive E. coli

Inhibitory to Neutral effects on Promotory to

growth of lettuce growth of lettuce growth of lettuce
seedlings seedlings seedlings

1. Selective inhibition of weeds but 1. Non-Selective 1. Selective

not wheat inhibition of inhibition of
2. Selective inhibition of weeds and weeds and weeds but not
promotion of wheat promotion of wheat
3. Selective inhibition of weeds and wheat 2. Selective
wheat 2. Selective inhibition of
4. Non-selective inhibition of weeds inhibition of weeds and
and wheat weeds and also promotion of
wheat wheat

Selection of strains (which were inhibitory to weeds and non-

inhibitory (neutral or promotory effects) to wheat) and re-
testing on 3 weeds and wheat under axenic conditions

Pot and field trials on application of allelopathic bacteria to

control infestation of wild oat, little seed canary grass and
broad leaved dock in wheat

Determination of biochemical and microbiological characteristics of

allelopathic bacteria including plant growth promotion characteristics.
Identification of allelopathic bacteria using 16S rRNA sequencing

Figure 6.1. Flow chart diagram of isolation, screening, characterization and field testing
of allelopathic bacteria to control weeds associated with wheat

187
respectively. Inoculation with strain T42, L9, 7O₀, O₀10 and W9 significantly inhibited
the germination and growth of wild oat and recovered the loss in grain yield of infested
wheat up to 60.0, 73.6, 35.8, 22.0 and 49.7%, respectively. Inoculation with strain T42,
L9, 7O₀ and O₀10 significantly inhibited the germination and growth of little seed canary
grass causing recovery in loss of grain yield of infested wheat up to 20.1, 55.0, 66.9 and
59.0%, respectively. Significant inhibition of germination and growth of broad leaved
dock by strain T42, L9, 7O₀, O₀10 and W9 recovered the loss in grain yield of infested
wheat up to 45.2, 53.9, 46.3, 11.6 and 68.0%, respectively. These effects of allelopathic
bacteria were evident from other growth, yield and physiological parameters of wheat and
its associated weeds. These strains remained non-inhibitory to wheat grown under weed
free conditions. However, inoculation with strain L9 and 7O₀ significantly improved the
growth, yield and physiological parameters of wheat under weed free conditions.

These 5 strains of allelopathic bacteria were conducted to evaluate their weed

suppression effects at 3 sites of chronic weed infestation under field conditions during
Rabi 2014-15. Each field was divided into 2 halves; one to study weed suppression
effects while other to study effects on wheat grown under weed free conditions. Culture
of these strains having 108 cfu mL-1 was prepared and applied to soil at 5 ml m-2 by
mixing in water. Sterilized King’s B broth was mixed in water and applied in weedy
control and weed free control. Wheat was sown in lines 20 cm apart. Infestation of weeds
in field trial I, II and III caused loss in grain yield of infested wheat up to 54.1, 53.9 and
56.3% than weed free control, respectively. Inoculation with strain T42, L9, 7O₀ and W9
significantly inhibited the germination and growth of broad leaved dock in field trial I
resulting into recovery in loss of grain yield of infested wheat up to 38.3, 64.0, 51.0 and
62.9%, respectively. Suppression of little seed canary grass in field trial II due to
inoculation with strain T42, L9, 7O₀ and O₀10 caused recovery in loss of grain yield of
infested wheat up to 34.3, 55.1, 64.3 and 57.2%, respectively. Suppression of wild oat and
little seed canary grass by strain T42, L9, 7O₀, O₀10 and W9 in field trial III caused
recovery in loss of grain yield of infested wheat up to 47.9, 60.7, 53.7, 29.0 and 36.6%,
respectively. These effects of allelopathic bacteria were also evident from other growth,
yield and physiological parameters of infested wheat and weeds. The tested strains were
found to be non-inhibitory to common lambs’ quarter in laboratory studies in field trial I.
However, inoculation with strain L9 and 7O₀ significantly reduced the growth and grain

188
yield of common lambs’ quarter in this trial. This is suggested to be due to growth
promotion effects of these strains on wheat. Under weed free conditions, inoculation with
strain L9 and 7O₀ significantly improved the growth, yield and physiology of wheat in
these field trials.

These effects of allelopathic bacteria on weeds, infested wheat and wheat were
further confirmed by determination of biochemical and microbiological characteristics of
these strains. These five strains (T42, L9, 7O₀, O₀10 and W9) showed negative gram
reaction. They were motile, gram negative, siderophores producing, and positive for
urease, oxidase, catalase and chitinase activity. Two strains (L9 and 7O₀) had the ability
to solubilize inorganic phosphate and produce exo-polysaccharides. Auxins producing
ability of these strains was variable. Strain L9 and 7O₀ indicated high auxin producing
ability both in the presence and absence of precursor L-tryptophan. However, strain W9,
O₀10 and T42 showed lower auxin producing ability. These strains were characterized for
root colonization ability in wheat and its associated weeds. Selectivity in effects of these
strains was also reflected from the study of their root colonization ability in different
plants. These 5 strains of allelopathic bacteria were identified using 16S rRNA gene
sequencing technique. The strain T42 was identified as Pseudomonas putida KT2440, L9
as Pseudomonas fluorescens F113, 7O₀ as Pseudomonas fluorescens SBW25, O₀10 as
Pseudomonas aeruginosa and W9 as Pseudomonas alcaligenes NBRIC14159.

It is concluded from this research work that allelopathic bacteria exist in the
rhizosphere of wheat and its associated weeds in areas of their chronic infestation. The
presence of selectivity in these bacteria suggests that they may be used in crops to
biologically control weed infestations. Apart from weed suppression, two strains (L9 and
7O₀) were found to promote the growth and yield of wheat under weed free conditions.
Such strains also improve the competitive ability of wheat against weeds and broaden the
magnitude of their control using their dual nature. Complete control of weeds was not
achieved in the present study using allelopathic bacteria but it caused significant
improvement in growth and yield of infested wheat and economic losses were highly
reduced. It is suggested that the potential of allelopathic bacteria may be used to control
weeds in crops. It may reduce the economic losses significantly but the complete loss
may not be recovered. The benefits of using allelopathic bacteria to environment and

189
human health should be gained by reducing the high input of chemicals and other
hazardous approaches.

Future Research Areas:

In future, research may be focused on increasing the scope of control of weeds by

using allelopathic bacteria through adoption of different techniques. These may be

 Phytotoxic substances may be extracted from culture and applied to

evaluate its weed suppression effects.
 Allelopathic bacteria may be used in an integrated approach along with
other biocontrol agents and plant allelochemicals.
 Integrated use of allelopathic bacteria with reduced control by using
conventional control methods (manual, chemical and mechanical).
 Allelopathic bacteria which also promote the growth of crop may increase
the competitive ability of crop over weeds and help to suppress their
effects on crops.
 Multiple biocontrol agents strategy may be adopted to broaden the scope
of allelopathic bacteria under diverse soil and climatic conditions.

190
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