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Biocontrol of Cereal Pathogens

Shivaditya Gautam

DISSERTATION.COM

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Biocontrol of Cereal Pathogens

Copyright © 2009 Shivaditya Gautam

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Dissertation.com Boca Raton, Florida

USA • 2010

ISBN-10: 1-59942-347-2 ISBN-13: 978-1-59942-347-0

Biocontrol Of Cereal Pathogens

ABSTRACT

Septoria leaf blotch has been the major disease of wheat in Britain and much of the

rest of Europe. It was reported that the disease cause serious yield losses to range

from 31 to 53%. Mycosphaerella graminicola (anamorph: Septoria tritici) is the

pathogen which causes Septoria leaf blotch. The disease can be controlled by various

methods such as cultural practices, chemical control, using resistant varieties and

biological control. In plant pathology, the term biological control leads to the

introduction of microbial antagonists or host specific pathogens to suppress diseases

and populations of one or more plant pathogens. This study investigated the microbial

community on and within wheat leaves which can suppress Septoria leaf blotch by

reducing the inoculum level of the causative pathogen M. graminicola. Plate count

and DGGE analysis techniques were used to assess the microbial community. The

changes in the microbial populations of healthy, senescent and M. graminicola

infected i.e. diseased wheat leaves were investigated in vitro on agar plates using plate

colony count method. It was found that method was successful in assessing both

bacterial and fungal population sizes showing distinct colonies. DGGE is a rapid

method which can analyze large number of samples simultaneously. The bands

appearing in DGGE profile represent different species present in the microbial

population. The DGGE technique was successfully used in this study to assess

bacterial community. Bioinformatics tools have also played a vital role in this study

for identifying bacterial and fungal species. Total five bacterial species and five

fungal species were identified by bioinformatics tools. The antagonistic abilities of

identified bacterial and fungal species were tested against a Septoria isolate in vitro on

dual culture PDA plates. Four microorganisms (Fusarium sp., Verticillium sp.,

Penicillium sp., Sporobolomyces sp. and Microbotryum sp.) in the dual cultures vary

in their colony length, width, ratio of length and width and distance bwtween them.

But after satatistical analysis all the values are insignificant observations of dual

culture plates indicated that the microorganisms might inhibit Septoria by competing


for space. These microorganisms after testing might be good candidates for further in

vivo testing of Septoria inhibition. A thorough knowledge of wheat leaf microbial

communities is required for further use of biocontrol in the future.


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TABLE OF CONTENTS

LIST OF FIGURES……………………………………………………………….…4

LIST OF TABLES…………………………………………………………………...7

CHAPTER 1 INTRODUCTION & LITERATURE REVIEW…………….…….8

1.1. Septoria leaf blotch……………………………………………………………....8

1.1.1. Pathogen………………………………………………………………………8

1.1.2. Hosts………….…………………………………………………….…………9

1.1.3. Symptoms…………………….……………………………………...……......9

1.1.4. Life cycle………………………………………………………………..…...11

1.2. Today’s challenge, Septoria leaf blotch…………………………………….....12

1.3. Septoria tritici epidemiology…………………………………………..………12

1.4. Control……………………………………………………………………….…13

1.4.1. Resistant Varieties……………………………………………….………….13

1.4.2. Cultural Practices…………………………………………………….……...13

1.4.3. Chemical Control……………………………………………………………14

1.5. Biological control of plant diseases…………………………...……………….14

1.5.1. Mutualism…………………………………………………….…………….15

1.5.2. Protocooperation…………………………………………………….......….15

1.5.3. Commensalism……………………………………………………………...16

1.5.4. Neutralism…………………………………………………………………..16

1.5.5. Competition………………………………………………………………....16

1.5.6. Parasitism …………………………………………………………………..17

1.5.7. Predation………………………………………...………………………….17

1.6. Biocontrol of wheat pathogens…………………...……………………………18

1.6.1. Fungal organisms as biocontrol agent against wheat pathogens…….………18

1.6.2. Yeast species as biocontrol agent against wheat pathogens……...…….....…19

1.6.3. Bacterial organisms as biocontrol agent against wheat pathogens…………..19

1.7. Focus of the current project…………..………………………………………..20

1.7.1. Assessing microbial communities……………………..……………………..20


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1.7.2. Screening microorganisms from microbial communities for disease control…...21

1.8. Aims of this study………………………………………………………….……….22

CHAPTER 2 MATERIAL & METHODS…………………………………………....23

2.1. Sampling of wheat plants………………………………………………………….23

2.2 Colony counts…………………………………………………………………...…..24

2.2.1.Recovery of leaf-associated microorganisms………………………………….....24

2.3. DNA Extraction……………………………………………………………..……...25

2.3.1. CTAB Method (Using Liquid Nitrogen)………………………………………...25

2.3.2. CTAB method for Soil samples (Using Glass Beads)………….………………..26

2.3.3. Sub cultured plates…………………..…………………………………………....27

2.3.4. Whole Leaf Extracts………….……….………………………………………….27

2.4. PCR Amplification………………………………………………………………...28

2.4.1. Bacterial 16S rDNA………..…………………………………………………...28

2.4.2. Fungal 18S rDNA…………...…………………………………………………..28

2.5. Analysis of PCR products………………………………………………………...29

2.6. Sequencing…………………………………………………………………………30

2.7. Analyzing bacterial community………………………………………………….30

2.7.1. DGGE Analysis………………………………………………………………...30

2.7.2. Sequencing of DGGE gene fragments………………………………………….31

2.8. Identification of microorganism using Bioinformatics tools…………………...31

2.8.1. 16S Bacterial samples………………………………………………………….31

2.8.2. 18S Fungal samples……………………………………………………………32

2.9. Antagonism Tests………………………………………………………………...32

2.9.1. Septoria growth inhibition trial………………………………………………..32

2.9.2. Statistical Analysis…………………………………………………………….33

CHAPTER 3 RESULTS……………………………………………………………...34

3.1 Colony counts……………………………………………………………………..34

3.2. DNA Extraction…………………………………………………………………..37

3.2.1. CTAB Method………………………………………………………………...37


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3.2.2. Sub cultured plates………………………………………………………………37

3.2.3. Whole Leaf Extracts……………………………………………………………..43

3.3. PCR Amplification…………………………………………………………………44

3.3.1. Bacterial 16S rDNA…….……………………………………………………….44

3.3.2. Fungal 18S r DNA……….……………………………………………………...46

3.4. Analyzing bacterial community………………………………………………..…47

3.4.1. DGGE Analysis…………………………………………………………………47

3.4.2. Sequencing of DGGE gene fragments…………………………………………..49

3.5. Identification of microorganism using Bioinformatics tools…………………….51

3.5.1. 16S Bacterial samples…………………………………………………………...51

3.5.2. 18S Fungal sample………………………………………………………………55

3.6. Antagonism Tests………………………………………………………………….57

3.6.1. Septoria growth inhibition trial………………...…………………………...…..57

3.6.2. Statistical Analysis……………………………………………………………...63

CHAPTER 4 DISCUSSION…………………………………………………………..64

4.1. DNA Extraction…………………………………………………………………...64

4.2. Identification of microorganisms………………………………………………...65

4.3. DGGE analysis…………………………………………………………………….68

4.4. Antagonism test…………………………………………………………………...69

CHAPTER 5 CONCLUSION……………………………………………...…………71

ACKNOWLEDGEMENTS…………………………………………………………...72

REFERENCES………………………………………………………………………...73

APPENDICES…………………………………………………………………………85

Appendix 1. Reagent and gel preparation……………………………………………85

Appendix 2. TSA Bacterial Plates…………………………………………………….88

Appendix 3. PDA Fungal Plates………………………………………………………89

Appendix 4. Mycosphaerella graminicola isolate plate.……..…………………..…..91

Appendix 5. Analysis of variance (ANOVA) tables from statistical analysis of

antagonist plates……………………………………………………………………….92


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LIST OF FIGURES

Main Document

Figure 1. Images of Septoria leaf blotch on wheat leaves…………...……..…..…..10

Figure 2. Life cycle of Septoria tritici……..…………………………...…..……….11

Figure 3. Wheat planting plan……………………………………………..………..23

Figure 4. Dual culture antagonism test…………….………………….………..…...33

Figure 5. TSA plates with subcultured bacteria..….………………...…..…………..38

Figure 6. Agarose gel analysis of bacterial DNA……..….……………………….…39

Figure 7. PDA plates with subcultured fungi……………………………………..…41

Figure 8. Agarose gel analysis of fungal DNA…………………………………..….42

Figure 9. Agarose gel analysis of whole leaf extract samples…..………….....……..43

Figure 10. Agarose gel analysis of bacterial 16S rDNA gene fragments obtained after

first amplification…………………………………...………………………………..44

Figure 11. Agarose gel analysis of bacterial 16S rDNA gene fragments obtained

after second amplification using GC clamp VFC/VR primers…….………….……..45

Figure 12. Agarose gel analysis of fungal 18S rDNA gene fragments obtained after

amplification…………...…………………………………………...….……………..46

Figure 13. DGGE analysis of bacterial community from wheat leaves……….….....48

Figure 14. Part of Band D1 (from DGGE gel, figure 13, section 3.4.1) DNA sequence

viewed in FinchTV...…………………………………………..………………..……49


viewed in FinchTV…...………………………………………………………..……..49


viewed in FinchTV……...……………………………………………………..……..50


viewed in FinchTV……...……………………………….……………………..…….50


viewed in FinchTV...………………………………………….………………..…….50



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viewed in FinchTV………………….………………………………………………..51

Figure 20. The bootstrap neighbour joining tree constructed from forward primer

sequences of B1, B2, B3, B4 and B5 bacterial 16S rDNA samples using

ClustalX...……………………………………………………………….….………..53

Figure 21. The bootstrap neighbour joining tree constructed from reverse

complement primer sequences of B1, B2, B3, B4 and B5 bacterial 16S rDNA samples

using ClustalX…………...……………………………………………….…..……....54

Figure 22. The bootstrap neighbour joining tree constructed from consensus

sequences of F1, F2, F3, F4, F6 and F7 fungal 18S rDNA samples using ClustalX...56

Figure 23. Length and width of different antagonist colony………………………...57

Figure 24. Length and width of different Septoria colony..........................................58

Figure 25. Ratio of length and width of fungal antagonist colony……………….….59

Figure 26. Ratio of length and width of fungal Septoria colony………………...…..60

Figure 27. Distance between antagonist and Septoria colony………………….....…61

Figure 28. Septoria (SP1, SP2, SP3, SP4, SP6 and SP7) growth inhibition trial……62

Appendices

Figure 1. R1, gd, 10-3 ……………………………………………………………….88

Figure 2. R1, gh, 10-1……………………………………………….……………….88

Figure 3. R1, s, 10-3 ………………………………………………...……………….88

Figure 4. R2, gd, 10-3…………………………………………….………………….88

Figure 5. R2, gh, 10-3………………………………………….…………………….88

Figure 6. R2, s, 10-3…………………………………………………………………88

Figure 7. R1, gd, 10-3, 17/06/09…………….……………………………………….89

Figure 8. R1, gh, 10-3, 17/06/09…………….……………………………………….89

Figure 9. R1, s, 10-3, 17/06/09……………….……………………………...……….89

Figure 10. R2, gd, 10-3, 18/06/09……………………………………………………89

Figure 11. R2, gh, 10-3, 18/06/09………………………………………...………….89

Figure 12. R2, s, 10-3, 18/06/09……………………………………………………...89


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Figure 13. R1, gd, 10-3, 19/06/09……………………………………………………90

Figure 14. R1, gh, 10-3, 19/06/09…………………………………………………....90

Figure 15. R1, s, 10-3, 19/06/09…………………………………………………..….90

Figure 16. R2, gd, 10-3, 19/06/09…...……………………………………………….90

Figure 17. R2, gh, 10-3, 19/06/09………………………………………………...….90

Figure 18. R2, s, 10-3, 19/06/09……………………………………………………...90

Figure 19. Control plate of Septoria isolate (CBS 100332)…………………………91


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LIST OF TABLES

Main Document

Table 1. Leaf sampling categories………….………...……………...……………….24

Table 2. PCR primer sequences and PCR conditions for the amplification of 16S

bacterial and 18S fungal rDNA..………………………………….……………....….29

Table 3. Colony Count on TSA Bacterial Plates……….……………………………35

Table 4. Colony Count on PDA Fungal Plates………….…………………………...36

Table 5. Bacterial isolates used for DNA extraction………………………..………37

Table 6. Fungal isolates used for DNA extraction……….....………………………40

Table 7. Bacterial species identified from BLAST search at

http://bioinfo.unice.fr/blast/.…..............................................………………………..53

Table 8. One-way analysis of variance of the colony growth of Septoria tritici

isolates……………………………………..…………………………………..…….63

Appendices

Table 1. Phosphate buffer reagents…...………………………….……………….….85

Table 2. CTAB buffer and 25% PEG reagents…………...…………..…………...…86

Table 3. Reagents for a 10% (wt/v) polyacrylamide gel with a 30-60% denaturing

gradients…..…………………..……………………………………………………...87


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CHAPTER-1

INTRODUCTION & LITERATURE REVIEW

1.1. Septoria leaf blotch

Septoria leaf blotch, also known as speckled leaf blotch, is caused by the fungus Septoria

tritici (Wolf, 2008). The disease is widely spread in all wheat-growing areas of the world

and is also a major problem in many regions (Van Ginkel et al. 1999). Septoria leaf

blotch has been the major disease of wheat in Britain and much of the rest of Europe for

nearly two decades (Pillinger et al. 2004). Septoria leaf blotch has the potential to cause

serious losses if the environmental conditions are favourable for its spread during late

May and June (Lipps et al. 2002). It was reported that the disease cause serious yield

losses to range from 31 to 53 % (Eyal, 1981; Babadoost and Herbert, 1984; Polley and

Thomas, 1991). This disease is common on wheat in the tillering stages but after ear

emergance the disease becomes quite severe on the upper leaves (Jenkins et al.1969).

1.1.1. Pathogen

Mycosphaerella graminicola (anamorph: Septoria tritici) is the pathogen which causes

Septoria leaf blotch (Eyal, 1987; Eyal, 1999). The pathogen is only found on foliar parts

of infected plants. It is a haploid, hemibiotrophic ascomycete having both filamentous

and yeast-like growth phases i.e. yeast-like growth at 15°C and mycelial growth at 25°C

(Joint Genome Institute 2008). M. graminicola can be easily cultured on both liquid and

solid media. Kema et al. (1996) showed that the crosses on wheat leaves were

successfully achieved in their study and found the bipolar heterothallic mating system in

M. graminicola. The population genetics of M. graminicola is better understood than any

other fungus due to natural occurrence of sexual recombination several times per year

(Joint Genome Institute, 2008).


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Classification:

(Source: Index of Fungorum)

Kingdom: Fungi

Phylum: Ascomycota

Class: Dothideomycetes

Subclass: Dothideomycetidae

Order: Capnodiales

Family: Mycosphaerellaceae

Genus: Mycosphaerella

Species: Graminicola

1.1.2. Hosts

Major host is wheat, but also occurs on rye, triticale and some grass species (HGCA,

2007).

1.1.3 Symptoms

Symptoms of Septoria leaf blotch are commonly visible in the beginning of growing

season whereas lesions of Septoria tritici usually appear on young autumn-sown wheat.

These lesions are elongate ovals which runs parallel to leaf veins. By early December

grey water-soaked patches are apparent on the lowest leaves which quickly turns brown

and necrotic (Murray et al. 1998). These patches have visible black pycnidia which are

mainly characteristic feature of M. graminicola (HGCA, 2007). The presence of pycnidia

is a good diagnostic characteristic (Nyvall, 1999). Pycnidia are most frequent on dead

overwintering leaves of winter wheat where they are embedded in lesions, which have

ashen centres (Maloy & Inglis, 1993). On mature plants, these lesions are brown and can

be restricted by veins giving rectangular appearance which may combine leading to large


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areas of necrotic brown tissue (HGCA, 2007). Severely diseased leaves become yellow

and die prematurely. Sometimes the entire plant may be killed.

A B

C D

Figure 1. Images of Septoria leaf blotch on wheat leaves. (A). Symptoms of Septoria

tritici. (B). Brown necrotic lesions on young wheat plant showing black pycnidia. (C).

Lesions consisting of brown necrotic tissue. (D). Coalesced lesions giving large areas

of necrotic brown tissue.

(Source: Cereal Disease Encyclopedia, HGCA, 2007)


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1.1.4.Life cycle

The life cycle of M. graminicola is favoured by optimum temperatures (15-20°C), and

to cause infection it requires long periods of high humidity (HGCA, 2007). The

Septoria leaf blotch has two conspicuous phases. First during the winter on the basal

leaves of autumn sown crops which normally infected by long distance spread of air-

borne ascospores, and during the summer on upper leaves of host plants (Nyvall,

1999). Initial infections are caused from wind-borne ascospores which are released

from pseudothecia and asexually produced water splash-dispersed pycnidiospores

produced from pycnidia (Murray et al. 1998). Infection requires at least 6 hours or

more hours of leaf wetness, for maximum infection. After occurrence of infection, the

fungus develops black fruiting bodies in 21 to 28 days and produces a new generation

of spores. The spores produced in these fruiting bodies are discharged in sticky

masses and through rain these spores are splashed onto the upper leaves and heads

(Wolf, 2008). Septoria leaf blotch can become a serious disease during environmental

conditions that are conducive for disease development (Nyvall, 1999).

Figure 2. Life cycle of Septoria tritici

(Source: Cereal Disease Encyclopedia, HGCA, 2007).


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1.2.Today’s challenge, Septoria Leaf Blotch

In recent years, Septoria leaf blotch disease is widely spread in winter wheat crops and it

is considered as the major factor of losses in the wheat crops in many countries (Lucas,

1998). In epiphytotic years crop yield losses due to this disease may be up to 20-30% of

the total. It also worsens grain quality. For wheat breeders Septoria leaf blotch has

emerged as a challenging disease (Zadoks, 2004). A history of Septoria leaf blotch shows

that from the last century it is a serious problem about every 25 years, once per human

generation (Zadoks, 2004). Zadoks (2004) showed that before 1900 severe epidemics of

Septoria tritici occurred in some parts of europe but during 1970 Septoria tritici had a

world wide attack and it is still a frightening fungus. Lucas (1998) deduced that there are

several factors associated with the increased severity of Septoria leaf blotch such as

earlier sowing of the crop, direct drilling into crop residues which may carry inoculum,

and the use of dwarf varieties which are more likely to get infection.

1.3. Septoria tritici epidemiology

The primary sources of inoculum in the epidemics of Septoria leaf blotch are airborne

ascospores and rain splash distributed asexual pycnidiospores from infected plant debris

and possibly seed-borne infections (Eyal, 1999). Rain-splashed pycnidiospores are

transported vertically upward from the base of the crop to the upper leaves where they

germinate and penetrate through stomata and causes infection (Hunter et al 1999).

Ascospore discharge depends on various environmental factors such as rain duration,

intensity, wind and temperature. The teleomorph stage (Mycosphaerella graminicola) is

also known to play an important role in the disease cycle. It was first described in the UK

by Scott et al. (1988). M. graminicola ascospores contribute to the genetic diversity of the

fungal population (Hunter et al. 1999). In comparison to other wheat foliar pathogens M.

graminicola has relatively long latent period i.e. ranging between 11-42 days. The period

between infection and the first appearance of sporulating structures is generally known as


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latent period (Shearer & Zadoks, 1972). The symptoms are usually developed under

strong influence of environmental conditions such as moisture, optimum temperature in

the range 18-25°C and light (Lovell et al. 2004). During a growing season S. tritici is able

to produce several sexual cycles and ascospore generations (Kema et al. 1996).

1.4 Control

1.4.1. Resistant Varieties

Resistant varieties are the best control for Septoria tritici blotch in winter wheat but only a

moderate degree of resistance is exhibited in the field and several popular varieties in the

Europe have partial resistance (Murray et al. 1998). Several genes regulate resistance to

the leaf blotch phases of the disease. Although by providing resistance to the foliar phase

serious yield losses can be prevented (Wolf, 2008). The varieties which are very prone to

this disease have to be avoided as they will build up inoculum levels and cause yield loss

in that variety and also in close susceptible wheat crops (DPI, 2008).

1.4.2. Cultural Practices

Cultural control of Septoria leaf blotch includes crop rotation or the disposal of

contaminated wheat crop debris by burning or ploughing will reduce the number of

ascospores which infect the new season's crop (Murray et al. 1998) Though burning of

crop debris is illegal in U.K. but still an alternative to control disease. In order to reduce

the amount of inoculum crops are rotated out of cereals and grasses for 3-4 years, wheat

stubble are deeply ploughed and volunteer hosts are destroyed. Even disease risk are

higher with minimum tillage practices. Septoria diseases are predominant when wheat is

heavily fertilized and have dense foliage (Maloy & Inglis, 1993). By crop rotation or

destruction of residues of earlier wheat crops Septoria tritici blotch cannot be fully

controlled as it partially effect the severity of the disease (Wolf, 2008). Cultural practices

will have more effect if practiced on a district basis. In order to prevent erosion cultural

practices are not used in light soil areas where stubble is stored (DPI, 2008).


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1.4.3. Chemical Control

Chemical control of the disease is widely practiced in Europe. The application of azole

based fungicides such as triazoles, usually to the flag leaf of wheat, can be highly

effective in reducing Septoria tritici blotch (Murray et al. 1998). Fungicidal treatment on

seed can also reduce disease severity (Maloy & Inglis, 1993). Before spraying fungicide it

is important to correctly identify Septoria tritici blotch as nutritional disorders such as

aluminium toxicity can be confused with Septoria tritici blotch (DPI, 2008). Moreover

before application of foliar fungicide, the important thing to keep in mind is to protect the

last two leaves because these leaves supply most of the energy which is required in

production of grain. In order to protect of these leaves the best way is to apply the

fungicide between emergence of the flag leaf and the beginning of flower (Wolf, 2008).

There is a widespread resistance to the benzimidazole (MBC) group of fungicides in UK

populations of Septoria tritici (Murray et al. 1998). There are various foliar fungicides

which are effective against Septoria leaf blotch such as Bumper, Headline, Proline,

Propimax, Quilt, Quadris, Stratego, and Tilt (DPI, 2008). These fungicides belong to

strobilurins and triazoles chemical group. Early applications of foliar fungicides and

fungicide seed treatments can only provide partial protection from Septoria leaf blotch

during early season, as these applications fails to protect the upper canopy during grain

fill when the plants are more prone to disease (Wolf, 2008).

1.5. Biological control of plant diseases

Any living organism that can be manipulated by man to suppress the pest or pathogen is

known as the biological control agent or BCA (Baker & Cook, 1974). In plant pathology,

the term biological control leads to the introduction of microbial antagonists or host

specific pathogens to suppress diseases and populations of one or more plant pathogens

(Pal & Gardener, 2006). There are various factors implied in biological control such as

host, pathogen or parasite, physical environment and antagonists. Biological control also

involves the use of microbial inoculants in suppression of single type of plant disease.


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Although diseases can be suppressed by managing soils in order to raise the combined

activities of native soil and plant associated organisms (Pal & Gardener, 2006).

Moreover to understand the mechanisms of biological control, it is very useful to know

the different ways through which organisms interact. All plants and pathogens throughout

their life cycle interact with a wide variety of organisms by direct or indirect contact.

These interactions have drastic affect on plant health (Pal & Gardener, 2006). The

interactions of two populations are defined by the outcomes for each (Odum et al. 1953).

There are various types of interactions such as mutualism, protocooperation,

commensalism, neutralism, competition, parasitism, and predation.

1.5.1. Mutualism

Mutualism is a biological interaction between two organisms that benefits both

individuals, such as increased survival. Sometimes, it also becomes a lifelong interaction,

which involves close physical and biochemical contact for example mutuality between

plants and mycorrhizal fungi. (Pal & Gardener, 2006). Mycorrhizas are symbiotic

relationship between rhizosphere fungi and the roots of plants. Many studies have shown

that exchange of mineral and other organic resources between plant and fungal symbionts

has benefited both individuals (Johnson et al. 1997). However, they are generally

facultative and opportunistic. These types of mutualistic association led to biological

control, by strengthening the plant with improved nutrition or by stimulating host

defenses.

1.5.2. Protocooperation

Protocooperation is a form of mutualism in which the organisms involved is not fully

dependant on each other for survival. Examples are protocooperation between soil

bacteria or fungi, and the plants that occur growing in the soil. Soil bacteria and fungi

interrelate with each other, producing nutrients essential to the plant’s survival. Plants

benefit by getting essential mineral nutrients and carbon dioxide from root nodules and

decomposing organic substances. Fowler et al. (1989) showed protocooperation between


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soil inhabiting mole crickets and nematophagous fungi in South America. This

protocooperation depends on the parasitic nematode of mole crickets, which has proven

to be a promising biological control agent against pest insects. A lot of isolated

microorganisms which are classified as biological control agents can be considered as

mutualists involved in protocooperation, because their survival does not depends on any

specific host and disease suppression also varies with the prevailing environmental

conditions (Pal & Gardener, 2006).

1.5.3. Commensalism

It is a symbiotic interaction between two living organisms, where one individual benefits

and the other is unaffected. Mostly microorganisms which are plant-associated are

supposed to be commensals in relation to the host plant (Pal & Gardener, 2006). Peterson

et al. (2006) showed that peptidoglycan from Bacillus cereus mediates commensalism

with rhizosphere bacteria from the Cytophaga-Flavobacterium group and also enhances

Cytophaga-Flavobacterium rhizosphere isolates growth in root exudate medium.

1.5.4. Neutralism

It is the biological interaction when the organisms occupy different and non-overlapping

niches in the same microhabitat together without affecting each other (Campbell, 1989).

Although both species are not affected by the population density of each other. In terms

of biological control, an inability to associate the population dynamics of a pathogen with

that of another organism shows neutralism (Pal & Gardener, 2006). In contrast,

antagonism between organisms results in a negative outcome for one or both.

1.5.5. Competition

Competition is defined as an interaction between organisms in which one organism is

harmed by the presence of another and also reduces the activity, growth rate and fertility

of the interacting organisms (Begon et al. 1996). Biological control occurs when non-

pathogens compete with pathogens for nutrients in and around the host plant. The


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understanding of biological control is also affected by the direct interactions that benefit

one population at the expense of another (Pal & Gardener, 2006).

1.5.6. Parasitism

It is a type of symbiotic correlation between two unrelated organisms where one organism

(the parasite) benefits or obtains its food from the host, and both organisms coexist over a

prolonged period of time (Campbell, 1989). In this type of interaction parasite benefits

from host for growth and reproduction. It also emphasizes that excessive harm done to a

host by parasite makes host less competitive and also endangers the survival of the

parasite species. The activities of various hyperparasites that parasitize plant pathogens

can result in biocontrol (Pal & Gardener, 2006).

1.5.7. Predation

Predation is an interaction between organisms in which biomass is captured by one

organism from another for consumption and sustenance (Begon et al. 1996). Predators are

generally animals that feed at higher trophic levels in the macroscopic world such as

microarthropods and fungal feeding nematodes that consume pathogen biomass for

sustenance (Pal & Gardener, 2006).

From all of these types of interactions, biological control results in varying degrees

depending on the environment within which they occur. Biological control commonly

arises from manipulating antagonisms between microorganisms and pathogens or from

manipulating mutualisms between microorganisms and their plant hosts (Pal & Gardener,

2006).


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1.6. Biocontrol of wheat pathogens

1.6.1. Fungal organisms as biocontrol agents against wheat pathogens

Most of the colonizers on leaves of cereals are capable of antagonism (Fokkema, 1978).

Trichoderma harzianum is an effective biocontrol agent against several fungal soilborne

plant pathogens (Green et al. 1999). It is an antagonistic fungus that is widely recognized

as a potential biocontrol agent against several soilborne plant pathogens (Jensen et al.

1995 & Papavizas, 1985). Trichoderma harzianum isolates have shown potential to

reduce levels of fusarium head blight of wheat by reducing perithecial and ascospore

production of Gibberella zeae on wheat straw (Inch & Gilbert, 2007). One Trichoderma

isolate T-22, is registered as a biocontrol agent in the USA. In their study it was found

that when T-22 strain was inoculated with G. zeae it reduced perithecial formation by

70%. Application of biocontrol agents to wheat residues has proven to be useful for

controlling Septoria leaf blotch (Perelló et al. 2009). Trichoderma harzianum, T.

aureovide and T. koningii isolates have potential to inhibit mycelial growth of the

necrotrophic pathogen Pyrenophora tritici-ripentis, causing tan spot of wheat (Perelló et

al. 2003).

Trichoderma spp. is a most successful antagonistic agent used for biocontrol of pathogens

on plant surfaces of cruciferous, solanaceous and gramineous plants (Perelló et al. 2006).

The earlier studies of Perelló et al. (1997, 2003) have shown biocontrol potential of

Trichoderma isolates to suppress P. tritici-repentis and M. graminicola on wheat under

laboratory experiments and greenhouse conditions. Most recently Perelló et al. (2006)

found that on application of Trichoderma isolates in some trial pots it gave disease

control comparable to that achieved with conventional fungicides. It is believed that T.

harzianum acts as an effective antagonist against Septoria leaf blotch by causing a

biochemical induced response in wheat plants (Cordo et al. 2007).


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1.6.2. Yeast species as biocontrol agent against wheat pathogens

Some commonly found yeast species have also proven to be good biocontrol agents

against wheat pathogens. These include Aureobasidium sp., Sporobolomyces sp., and

Cryptococcus sp. which has shown antagonistic, effects towards foliar pathogens

(Fokemma et al. 1976). Fokemma et al. (1976) showed that these yeast species have

reduced the superficial mycelial growth of Septoria nodorum and the infection of wheat

leaves by 50%. The studies have also revealed that strain J121 of the yeast Pichia

anomala reduces Penicillium roqueforti growth on wheat cereal grains (Druvefors &

Schnurer, 2005).

1.6.3. Bacterial organisms as biocontrol agent against wheat pathogens

Besides fungal and yeast bacterial species have also played a vital role in suppression of

wheat diseases as biocontrol agents. A Bacillus licheniformis strain when inoculated with

Pyrenophora trichostoma reduces tan spot disease of wheat (Ghazanfari & Gough, 1981).

Ghazanfari & Gough (1981) showed that Bacillus strains have great potential to suppress

wheat foliar diseases. Other bacterial species such as Pseudomonas sp. isolated from soil

environment also have ability to reduce disease levels of two foliar wheat pathogens (Levy et

al. 1989). Levy et al. (1989) concluded that a fluorescent Pseudomonas sp. strain when

inoculated onto wheat seedlings has reduced Septoria tritici disease by 88% and Puccinia

recondita by 98%.

It is clear from the work discussed that phyllosphere microorganisms have potential for

biocontrol of foliar diseases. It will also be a promising strategy in managing Septoria leaf

blotch in the fields which has been the major disease of wheat in Britain and much of the

rest of Europe for nearly two decades (Perelló et al. 2009).


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1.7. Focus of the current project

An increasing volume of literature on plant-associated microorganisms as biocontrol

agents shows that these agents have great potential to control plant diseases. But due to

multiple interactions between plant pathogens and biocontrol agents it is an intricate

process to identify and assess biocontrol agents which can successfully control plant

disease. So the focus of the current project is to study the microbial community on and

within wheat leaves which could suppress Septoria leaf blotch by reducing the inoculum

level of the causative pathogen M. graminicola. To study plant microbial community

initial step is to assess the potential of resident microflora for biocontrol of plant diseases.

1.7.1. Assessing microbial communities

Researchers have used several methods for analyzing structure and diversity of microbial

communities Øvreås et al. (1997). These methods were categorized into two groups,

biochemical-based techniques for example plate count and molecular-based techniques

for example DGGE analysis (Kirk et al. 2004). Kirk et al. (2004) showed that these

techniques used for assessing microbial communities are not very accurate and reliable

due to their limitations. There are several advantages and disadvantages associated with

both methods such as that the plate count method is fast and cheap method but cannot

detect unculturable microorganisms (Kirk et al. 2004), on the other hand DGGE is a rapid

method which can analyze large number of samples simultaneously but can only detect

dominant species and each band can represent more than one species (Kirk et al. 2004).

Legard et al. (1994) investigated seasonal changes in the microbial populations of spring

wheat and found that both bacterial and fungal population sizes could be assessed

successfully using plate colony count method whereas Øvreås et al. (1997) showed that

plate count method is an incomplete method for assessing biodiversity, as less than 1% of

the bacterial population was cultivable on the selected media.


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Moreover, in case of molecular techniques denaturing gradient gel electrophoresis

(DGGE) is used for comparing rDNA gene fragments of microbial communities (Saito et

al. 2007). To generate the rDNA gene fragments for DGGE PCR amplification was done

with total extracted community DNA samples using universal bacterial and fungal

primers (Øvreås et al. 1997; Lane 1991; Muyzer et al. 1993; Borneman & Hartin 2000).

DGGE separates gene fragments of the same length but with different base-pair sequence

on the basis of their melting properties which are sequence specific (Muyzer et al. 1993).

Muyzer et al. (1993) showed that all the bands appearing in DGGE profile represent

different species present in the microbial population. They also proved by their study that

it is possible to identify a species that represents only 1% of the total population. Thus

DGGE is a useful method for studying bacterial and fungal communities.

Although limitations were associated with both methods used for assessing these

microbial communities, but still both the biochemical technique i.e. plate count and

molecular techniques i.e. DGGE were successfully used to assess microbial communities

in terms of size and diversity (Nakatsu et al. 2000). Therefore in this project also plate

count and DGGE technique is used for assessing microbial community.

1.7.2. Screening microorganisms from microbial communities for disease control

For screening the antagonistic activity of microorganisms towards plant pathogens, a co-

culture of the test pathogen and the antagonist was established in vitro on agar plates

(Gachomo et al. 2008). It is a simple and inexpensive method mostly used as a prelude to

more complex in vivo studies of antagonism. For assessment of antagonistic ability of

microorganisms against pathogen the time needed for colony overgrowth is considered as

an important parameter (Begum et al. 2008). Innocenti et al. (2003) showed that success

of biocontrol measures depends on the characteristics of both pathogen and antagonist

microorganisms. Agar plate dual cultures have been widely used in many studies to

observe the specific effects of particular pathogen-antagonist pairings on pathogen

growth and spore production (Andrews et al. 1983; Begum et al. 2008; Gachomo et al.

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