ANALYSIS OF DROUGHT AND RUST DISEASES TOLERANCE CONFERRED BY ENDOPHYTES IN

WHEAT CROP

By

HAFIZ ARSLAN ANWAAR

M.Sc. (Hons.) Plant Pathology2005-ag-1757

A thesis submitted in partial fulfilment of the requirements for the degree

of

DOCTOR OF PHILOSOPHY

INPLANT PATHOLOGY

Department of Plant PathologyFaculty of Agriculture

University of Agriculture, Faisalabad

2018

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DEDICATED TO

Our national political leader

IMRAN KHAN

A SYNONYMOUS OF CHANGE

MAN OF COMMITMENT, STRUGGLING AGAINST CORRUPT VULTURES.

A LONG AWAITED RAY OF HOPE FOR PITIABLE PAKISTANI NATION GRANTED AFTER GREAT QUID M.A JINNAH.

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ACKNOWLEDGEMENTSAll the praises and thanks for ALMIGHTY ALLAH, who bestowed me with the

opportunity and ability to contribute a little material to the existing knowledge. Trembling

lips and wets eyes praises for Holy prophet, MUHAMMAD (PBUH) who is the thriving

city of knowledge.

Words don’t come easy for me to articulate the feelings of unassuming admiration

to the great scholar of this era SYED ABU ALA MODOODI (R.H). He behaved a torch

of guidance and knowledge for me and impart that “Islam a complete code of life” is not

a utopia and get rid from sectarianism.

I would like to thanks to my supervisors Dr. Safdar Ali and Prof. Dr. Shahbaz

Talib Sahi for their valuable comments, guidance and continuous support during my

Ph.D. studies and research work. I also express my deep gratitude to Dr. Ye Xia, Ohio

State University, Columbus, USA for sharing valuable ideas. No acknowledgement

could ever express my obligation to my affectionate PARENTS whose endless efforts

and encouragements sustained me all stages of my life and whose hands always rose in

prayers for my success.

I shall be missing something if I don’t extend my admiration and appreciation to

my fellows and friends Dr Sahibzada Rizwan and Dr Hafiz Sajid for their support, lot

of smiles and enjoyable moments throughout my Ph.D. study period. Special thanks to

Zubair Safdar for making some arrangement of financial assistance as well as my

colleague Dr Safdar Hussain for assistance in writing of this manuscript.

Hafiz Arslan Anwaar

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List of contentsCHAPTER TITLE PAGE

1 INTRODUCTION 12 REVIEW OF LITERATURE 5

2.1 Origin and geographical significance of wheat 52.2 Economic importance and taxonomic status of the wheat 52.3 Significance of wheat in Pakistan 62.4 Wheat productivity constraints 62.5 Impact of drought on wheat genotypes 72.6 Effects of terminal drought 7

2.6.1 Leaf Senescence 72.6.2 Light harvesting and fixation of carbon 72.6.3 Grain development 82.7 Tolerance to terminal drought 8

2.7.1 Drought escape 92.7.2 Solute accumulation and osmotic adjustment 92.7.3 Antioxidant defence 92.7.4 Stay green 102.7.5 Root system architecture 102.7.6 Reserve translocation 102.7.7 Hormonal regulations 112.8 Role of rusts in wheat yield reduction 112.9 Historic epidemics and yield losses 122.10 Classification and life Cycle of Puccinia 132.11 Symbiosis 142.12 Plant Symbionts 152.13 Endophytism 152.14 Endophytism VS Pathogenism 162.15 Classification of endophytes 172.16 Fungal endophytes 172.17 Fungal endophytes diversity 17

2.17.1 Class 1 endophytes 182.17.2 Class 2 endophytes 182.17.3 Class 3 endophytes 192.17.4 Class 4 endophytes 19

2.18Mechanisms of endophytic fungi confer tolerance against abiotic stresses 19

2.19Mechanisms of endophytic fungi confer tolerance against biotic stresses 20

2.20Role of fungal endophytes in conferring plant tolerance against different abiotic and biotic stresses 21

2.20.1 Piriformospora indica 212.20.2 Trichoderma 24

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2.20.3 Colletotrichum 263 MATERIALS AND METHODS 28

3.1 Experimental site and conditions 283.2 Collection of wheat germplasm 283.3 Screening of wheat germplasm against drought 293.4 Screening of wheat germplasm against rust diseases 303.5 Rust inoculation Procedures 30

3.5.1 Collection of inoculums 303.5.2 Storage of inoculums 313.5.3 Methods of inoculation 31

3.5.2.1 Hypodermic needle injection method 313.5.2.2 Spraying method 323.5.2.3 Dusting method 32

3.6 Data recording of leaf and stripe rust 333.7 Rusts screening of wheat genotypes 333.8 Yield and yield components 34

3.8.1 Number of tillers m-2 343.8.2 Number of productive tillers 343.8.3 Number of grains per spike 343.8.4 Thousand grains weight (g) 348.8.5 Biological yield (gm-2) 343.8.6 Grains yield (gm-2) 343.8.7 Harvest index (%) 358.8.8 Percent yield reduction (%) 353.8.9 Percent yield increased (%) 353.8.10 Area under disease progress curve (AUDPC) 353.8.11 Coefficient of infection (CI) 35

3.9Optimization of efficient and compatible endophytes with wheat seed 35

3.9.1 Collection of samples for endophytes 353.9.2 Morphological identification and mass culturing of endophytes 363.9.3 In-vitro evaluation of endophytes 36

3.10Field appraisal of selected efficient and compatible endophytes against drought and rust diseases 36

3.11 Statistical analysis 374 RESULTS AND DISCUSSIONS 38

4.1 Response of different wheat genotypes against drought stress. 384.1.1 Number of grains per spike 384.1.2 1000-grains weight (g) 394.1.3 Number of productive tillers 394.1.4 Harvest index (%) 504.1.5 Biological yield (gm-2) 504.1.6 Grain yield (gm-2) 524.1.7 Drought sensitivity indices 54

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4.2 Response of different wheat genotypes against Leaf rust 574.2.1 Disease severity (%) 574.2.2 Area under disease progress curve (AUDPC) 594.2.3 Coefficient of infection (CI) 614.3 Response of different wheat genotypes against yellow rust 63

4.3.1 Disease severity (%) 634.3.2 Area under disease progress curve (AUDPC) 664.3.3 Coefficient of infection (CI) 68

4.4Symbiotic effect of fungal endophytes on two drought sensitive wheat genotypes in drought conditions 70

4.4.1 1000-grain weight (g) 704.4.2 Number of productive tillers 714.4.3 Biological yield (gm-2) 724.4.4 Grain yield (gm-2) 734.4.5 Percent yield increased 75

4.5Symbiotic effect of fungal endophytes on two leaf rust susceptible wheat genotypes in disease conditions 76

4.5.1 Disease severity (%) 764.5.2 Area under disease progress curve (AUDPC) 774.5.3 1000- grains weight (g) 784.5.4 Grain yield (gm-2) 794.5.5 Percent yield increased (%) 80

4.6Symbiotic effect of fungal endophytes on two yellow rust susceptible wheat genotypes in disease conditions 82

4.6.1 Disease severity (%) 824.6.2 Area under disease progress curve (AUDPC) 834.6.3 1000- grain weight (g) 844.6.4 Grain yield (gm-2) 854.6.5 Percent yield increased (%) 864.7 Discussion 875 SUMMARY 93

CONCLUSION 95RECOMMENDATIONS 95

6 LITERATURE CITED 96

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List of TablesTable 3.1 Wheat genotypes used in the screening experiment 29Table 4.1 Influences of drought stress on the yield related traits of different

wheat genotypesTable 4.2 Analysis of variance for morphological traits of wheat in normal

and drought stress conditions43

Table 4.3 Influence of drought stress on the yield related traits of biological yield (gm-2) and Harvest Index %

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Table 4.4 Influences of drought stress on the yield related traits of GY, YR%, TOL, MP and SSI

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Table 4.5 Impacts of wheat leaf rust on FDS, AUDPC and CI in field conditions

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Table 4.6 Analysis of variance for FDS, AUDPC and CI under field conditions

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Table 4.7 Impacts of yellow rust on traits of FDS, AUDPC and CI in field conditions

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Table 4.8 Analysis of variance for FDS, AUDPC and CI under field conditions

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Table 4.9 Symbiotic effects of endophytes for 1000-grain weight in tolerance of drought stress

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Table 4.10 Symbiotic effects of endophytes for Number of Productive Tillers in tolerance of drought stress

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Table 4.11 Symbiotic effects of endophytes for biological yield in tolerance of drought stress

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Table 4.12 Symbiotic effects of endophytes for grain yield in tolerance of drought stress

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Table 4.13 Analysis of variance for TGW, PT, BY, GY and YI% under field conditions

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Table 4.14 Symbiotic effects of endophytes for percent yield increased in tolerance of drought stress

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Table 4.15 Antagonistic effects of endophytes for disease severity in tolerance of disease (leaf rust) conditions

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Table 4.16 Antagonistic effects of endophytes for AUDPC in tolerance of disease (leaf rust) conditions

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Table 4.17 Analysis of variance for FDS, AUDPC, TGW, GY and YI% under field conditions

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Table 4.18 Symbiotic effects of endophytes for 1000-grain weight in tolerance of disease (leaf rust) conditions

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Table 4.19 Symbiotic effects of endophytes for grain yield in tolerance of disease (leaf rust) conditions

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Table 4.20 Symbiotic effects of endophytes for percent grain yield increase in tolerance of disease (leaf rust) conditions

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Table 4.21 Antagonistic effects of endophytes for disease severity in tolerance of disease (yellow rust) conditions

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Table 4.22 Antagonistic effects of endophytes for AUDPC in tolerance of disease (yellow rust) conditions

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Table 4.23 Analysis of variance for FDS, AUDPC, TGW, GY and YI% 84

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under field conditionsTable 4.24 Symbiotic effects of endophytes for 1000-grain weight in

tolerance of disease (yellow rust) conditions85

Table 4.25 Symbiotic effects of endophytes for grain yield in tolerance of disease (yellow rust) conditions

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Table 4.26 Symbiotic effects of endophytes for percent grain yield increase in tolerance of disease (yellow rust) conditions

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List of FiguresFig 2.1 Life cycle of Rust 13Fig 2.2 Life cycle of Puccinia 14

Fig 3.1 Modified diagrammatic Cobb Scale described by Peterson et al., 1948 31Fig 3.2 Artificial inoculums of leaf rust (left) and yellow rust (right) 32Fig 3.3 Field view of Leaf rust (left) and Yellow rust (right) 34Fig 4.1 Comparison of 50 wheat genotypes for No. of grains per spike

under normal and drought conditions44

Fig 4.2 Comparison of 50 wheat genotypes for No. of productive tillers under normal and drought conditions

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Fig 4.3 Comparison of 50 wheat genotypes for 1000-grains weight under normal and drought conditions

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Fig 4.4 Comparison of 50 wheat genotypes for Biological yield (gm-2) under normal and drought conditions

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Fig 4.5 Comparison of 50 wheat genotypes for Grain yield (gm-2) under normal and drought conditions

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Fig 4.6 Comparison of 50 wheat genotypes for Harvest index (%) under normal and drought conditions

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LIST OF ABBREVIATIONS

ABBREVIATIONS Meanings

AARI Ayub Agricultural Research Institute

ABA Abscisic Acid

AMF Arbuscular mycorrhiza fungi

ANOVA Analysis of Variance

APX Ascorbate peroxidase

AUDPC Area under disease progress curve

BY Biological yield

CAT Catalase

CEs Clavicipitaceous endophytes

CI Coefficient of Infection

CIMMYT International maize and Wheat improvement centre

CRD Completely Randomized Design

DON Deoxynivalenol

DSE Darkly melanized septa

FAO Food and Agriculture Organization

FDS Final Disease Severity

FHB Fusarium head blight

GY Grain yield

HI Harvest Index

IAA Indole acetic acid

ISR Induced systemic resistance

LSD Least Significant Difference

MHB Mycorrhization helper bacteria

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MP Mean Productivity

NCEs Non-clavicepataceous endophytes

NUE Nitrogen used efficiency

P. indica Piriformospora indica

PGPB Plant growth promoting bacteria

PGPR Plant growth promoting rhizobacteria

POX Peroxidase

PT Productive Tillers

RCBD Randomized complete block design

ROS Reactive Oxygen Specie

RuBP Ribulose bisphosphate

SOD Superoxide dismutase

SSI Stress Susceptibility Index

TGW Thousand grain weight

TOL Tolerance Index

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ABSTRACT

Excessive use of pesticides has caused agricultural and environmental hazards. Microbial inoculation is an alternate to pesticides for confronting pathogens and is an environmental friendly approach. Endophytes are beneficial microbes and biologically safe for inducing tolerance in plants. Hence, the current research was conducted to evaluate endophytes potential in wheat against drought stress and rust diseases. Fifty genotypes of wheat were sown under Randomized Complete Block Design (RCBD) in field conditions for evaluation against rust diseases and drought conditions. The drought stress was given to the wheat genotypes by skipping the irrigation at flowering and grain filling stage for drought resistance whereas all genotypes were also inoculated against rust diseases through natural and artificial method at tillering and heading stage. Data regarding drought stress were recorded on the basis of different growth parameters viz. number of grains per spike, 1000-grain weight (g), number of productive tillers m2, biological yield (gm-2), grain yield (gm-2), harvest index % and percent yield reduction, drought tolerance indices like mean productivity (MP), tolerance index (TOL) and stress susceptibility index (SSI). Data regarding final disease severity percentage, area under disease progress curve and coefficient of infection were recorded for rust diseases. Kohsar-95 and Parwaz-94 exhibited most drought sensitive genotypes while two leaf rust (Faisalabad-85, Aas-02) and two yellow rust genotypes viz. Fareed-06 and Shafaq-06 expressed susceptible response. Plant samples for endophytes were collected from desert areas (Cholistan, Thar and Rohi) of Pakistan. The endophytes were isolated, identified and purified in Plant Mycology Lab. on sterilized Potato Dextrose Agar media (PDA). The in-vitro efficacy of endophytes was evaluated in test tubes containing 0.3% agar concentration. The wheat seed was sown in test tube containing distilled water along with fungal spore suspensions (1×106/ml) and incubated at 28±2oC. The root and shoot length was measured after 4 days of interval. The four endophytes expressed significant results were used for further studies. Spore suspensions of these endophytes were prepared and their concentrations were observed through haemocytometer. Seeds of disease and drought susceptible wheat genotypes were inoculated by dipping in spore suspension and sown in field under factorial RCBD. Inoculated susceptible wheat genotypes exhibited the tolerance against drought and rust diseases. The endophyte Piriformospora indica showed significant increase in grain yield 15.4% of drought sensitive genotypes followed by Colletotrichum lindemuthianum, Trichoderma viride, and Acremonium lolii 11.3 %, 8.1 % and 7.5 % respectively. Similarly, for leaf and yellow rust diseases P. indica also exhibited statistically significant increase in grain yield 17.5% and 12.3%, respectively followed by Trichoderma spp. (13.7 % and 10.6 %). Colletotrichum spp. and Acremonium spp. showed (7.1%, 6.2%) as well as (8.2%, 4.2%) under leaf rust and yellow rust conditions respectively.

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CHAPTER 1 INTRODUCTION

Pakistan is an agricultural country. A huge section of the population is dependent

directly or indirectly on agriculture. The trade of Pakistan totally depends on agriculture.

The food security is highly focused on obtaining more food to fulfil the needs of

burgeoning population which can only be accomplished when the production of cereal

crops is increased globally. Wheat (Triticum aestivum L.) belongs to family Poaceae and

is extensively cultivated in the majority of the world regions. In Pakistan, wheat as main

staple food, is cultivated on the area of 9.23 million hectare with the estimated production

of 25.3 million metric tons with 2.74 metric tons/ha grain yield (USDA, 2017; Pakistan

Bureau of Statistics, 2017)

The yield of cereal crops is very low in Pakistan as compared to developed

countries due to many abiotic and biotic constraints. Biotic (diseases and insect pest) and

abiotic stresses (drought, temperature, salinity and water logging) for crop are highly

concerned in this regard (Hussain et al., 2015). Faced with scarcity of water resources,

only a single factor of drought is utmost uncertain hazard to food security of world. It has

been the catalyst of the past’s worse famines (Loewenberg, 2014). The drought severity

is unpredictable as it is influenced by rainfall, evaporation and soils’ moisture storing

capacity (Muscolo et al., 2015; Ramirez et al., 2015).

The drought incidence and severity will certainly increase in coming future as a

result of global warming that will direct towards a rigorous decline in overall production

of food. Predicted temperature rise of 1.5-5.8 °C by 2100 will lead ruthless troubles for

agricultural production (IPCC, 2012). At the same time progressively increasing human

population that might achieve to nine billion in 2050, requires a surge in food supplies.

Since desertification swells further by reason of constant trouncing of arable area the

condition will be shattering and distressing more in the upcoming days (IPCC, 2007;

2012).

Drought conditions impact an influential demerit of low wheat productivity.

Drought reduces the number of fertile tillers which are main contributors of grain yield

(Al- Ajlouni et al., 2016). Drought deters cell enlargement and cell division and also

reduces respiration, photosynthesis, translocation, nutrient metabolism, ion uptake and

carbohydrates (Farooq et al., 2008). Severe water stress upsets plant water relations and

reduces water-use efficiency which ultimately causes decline of photosynthesis decline,

metabolism disorder and thus results in plant death (Jaleel et al., 2009; Cattivelli et al.,

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2008; Sikuku et al., 2012). All the wheat growth phonological stages may affected by

drought and the stages of reproductive and grain-filling are the most responsive (Pradhan

et al., 2012; Nawaz et al., 2013).

Terminal drought causes significant yield reductions in wheat because of

oxidative damage to photo-assimilatory machinery (Farooq et al., 2009), accelerated leaf

senescence (Yang et al., 2007), pollen sterility (Dorion et al., 1996; Cattivelli et al.,

2008), decreased rates of fixation of carbon and translocation of assimilates (Asada,

2006), decreased set of grains and their development (Ahmadi and Baker, 2001; Nawaz

et al., 2013) and condensed sink capacity (Liang et al., 2001). Drought at post-anthesis

lessen the wheat yields by 1-30%, mild stress at heading stage losses 57%, whereas

persistent terminal drought shrinks the grain yields by 58–92% (Dias de Oliveria et al.,

2013).

Massive losses in wheat yield are attributed to various diseases in which rusts

have caused huge yield losses in the recent years. New races of rusts are evolving

unremittingly day by day which have infected the resistant varieties (Brian, 2006). Leaf

or brown rust, stem or black rust and yellow or stripe rusts are generally observed in

wheat. Leaf rust of wheat caused by the fungus Puccinia recondita infects the leaf blades,

leaf sheaths and glumes in vulnerable genotypes (Huerta-Espino et al., 2011). As a result

decline the number of grains per spike and grain weight (Kolmer et al., 2005; Marasas et

al., 2004). Early infection of leaf rust in wheat usually reasons high yield losses; infection

of 60–70% on the flag leaf at emergence of spike may cause more than 30% yield losses.

Stem or black rust caused by Puccinia graminis is the most destructive disease of wheat.

In the presence of favourable conditions, it causes up to 100 % yield losses in susceptible

wheat genotypes (Roelfs, 1985; Leonard and Szabo, 2005). Stripe rust caused by

Puccinia striformis is the most significant favoured mild winter, cool summer and long

cool and wet spring. The yield losses of 10-70% on wheat depend on susceptibility of the

genotypes, initial infections, inoculum density and inoculum multiplication rate (Chen,

2005).

Plants combat different abiotic stresses by their own natural defence and in

cooperation with different soil microorganisms (Marulanda et al., 2006). Activities of

microorganisms facilitate in continuance of biological equilibrium and soil’s

sustainability in stresses (Sieber et al., 2005; Alexander, 2005; Kavamura et al., 2013).

Endophytes are fungal or bacterial microbes that colonize healthy plant tissue

intracellularly and intercellularly without causing any obvious disease symptoms

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(Bandara et al., 2006; Castillo et al., 2006; Azevedo et al., 2000). They are ubiquitous,

colonize the plants which are isolated from almost all species of plants examined to date

(Mohali et al., 2005; Zhang et al., 2006). Their symbiotic and mutualistic association can

be obligate or facultative and causes no harm to the host plants. Endophytic

microorganisms induce defence mechanisms in host plants to counteract pathogen attack

and others generated antibiotic substances that hamper growth of pathogen, competition

for host resources and space may also take place between incoming pathogens and

already existent endophytes (Arnold et al., 2007; Wang et al., 2007).

Endophytes are also playing their imperative role for mutual interaction with their

hosts for better adaptability and systemic resistance, augmenting nutrient uptake, stress

tolerance, balancing minerals concentration and composition, bolstering abiotic and

pathogenic tolerance or resistance (Redman et al., 2011; Kavamura et al., 2013). The

constructive and valuable characteristics of endophytic fungi have observed in host plants

against numerous stresses (Waller et al., 2005; Rodriguez et al., 2008; Hamilton et al.,

2010). The endophytes also play a considerable function in food safety, bioremediation

and sustainable crop production (Ganely et al., 2006; Singh et al., 2011).

So, to enhance farmers' earnings and productivity of wheat, appropriate strategies

might be adopted to minimize severe yield losses by different abiotic stresses and

pathogenic diseases. Numerous scientists and researchers are discovering sustainable

alternative approaches to pesticides and chemical fertilizers. Natural resources are

decreasing with the passage of time due to spontaneously increasing world population. It

is need of the hour to find out an alternative approach for growing more food in such a

manner that can reduce detrimental environmental impacts of intensive farming,

fungicide resistance and environmental pollution.

The beneficial effects of endophytes on plants against diseases have increased the

interest of researchers and farmers for enhancing agricultural production. This bio-control

strategy for combating different a biotic stresses and pathogenic diseases have altered the

attention of farmers for better crop production. It is tried to discover different efficient

and compatible fungal endophytes for wheat tolerance against drought and rust diseases.

The present research was conducted to achieve the following objectives:

To screen out resistant and sensitive / susceptible genotypes of wheat against

drought and rust diseases

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To find out efficient and compatible endophytes for wheat tolerance against

drought and rust diseases

To meet the above stated objectives the following line of work was adopted

a) Seed collection of different wheat genotypes

b) Screening of resistant and sensitive / susceptible genotype of wheat against

drought and rust diseases

c) Collection and isolation of fungal endophytes

d) In-vitro evaluation of efficient and compatible endophytes for wheat

e) Preparation of endophytic fungal media for inoculating wheat seeds

f) Soaking of wheat seeds for inoculation of selected endophytes

g) Re-isolation and identification of fungal endophytes from inoculated wheat

plants for confirmation of Koch’s postulate

h) Data collection regarding growth parameters

i) Statistical analysis

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CHAPTER 2 REVIEW OF LITERATURE

2.1 Origin and geographical significance of wheat

Wheat (Triticum aestivum L.) is very important crop for the huge population of

the world. It has been remained as first domesticated and essential staple food crop for the

key civilizations of North Africa, West Asia and Europe for last 10,000 years. The history

of wheat and history of human culture have co-evolved and co-existed, in a close

reciprocal relationship. Fertile Crescent is the part of the world (near and middle east) of

its earliest evolution and then its culture extended to all directions, from the Tigris-

Euphrates drainage basin (known as centre of wheat diversity) to entire world (Marcussen

et al., 2014).

Wheat is adapted to climatic conditions from latitudes of 30° and 60°N to 27° and

40°S; but cultivation in wide climate range commencing the Arctic Circle to the higher

elevations close to the equator can occur (Nutteson, 1957). CIMMYT reported that

production of wheat in warmer regions is technologically feasible. In altitude, it can be

cultivated from sea level to 3,000 m. The optimum cultivating temperature is 25°C, with

maximum and minimum growth temperatures of 30° to 32°C and 3° to 4°C, respectively.

It adapted to wide ranges of wet environment and might be cultivated in the major

localities having 250 to 1750 mm of precipitation range. Categorization into spring and

winter wheat is general and conventionally consider the season of the crop cultivation. In

winter wheat, stage of emergence of spike is delayed until a period of cold winter

temperature (0° to 5°C) experiences by the plant and the Spring wheat, the same as name

implies, is generally cultivated during spring (during autumn season in countries i.e.

Pakistan experience the mild winter season) and matures in summer (Marcussen et al.,

2014).

2.2 Economic importance and taxonomic status of the wheat

The grass family Poaceae (Gramineae) comprises most important crops for

example wheat (Triticum aestivum L.), rice (Oryza sativa L.), maize (Zea mays L.),

barley (Hordeum vulgare L.), oat (Avena sativa L.), and rye (Secalecereale L.). Triticeae

is the tribe including 300 species and more than 15 genera. Diploid and tetraploid were

previously cultured wheat forms and succeeding evolutionary adaptation and constant

research formed hexaploid wheat that is presently extensively cultivated at 96% area of

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world wheat (Dubcovsky and Dvorak, 2007). Chromosome number sets (genomes) for

each generally known type of wheat are firstly reported by Sakamura in 1918.

Wheat owes much of its popularity to its high gluten content and bread making

quality, an important attribute, which assured its continuing role in the development of

human society. It is the vital and essential staple food of 2 billion human populations

(36% world’s population). Universally, it gives 20% food calories and 55% carbohydrates

consumed globally (Detterbeck et al., 2016).Thus being the most popular staple food for

the huge share of the world population it has become the major food commodity of

international trade. It offers well nutrition profile with 12.1 percent protein, 70 % total

carbohydrates, 59.2 % starch, 1.8 % ash, 1.8 % lipids, 2.0 % reducing sugars, 6.7 %

pentosans and provides 314K cal/100 g of food (USDA, 2017).

2.3 Significance of wheat in PakistanIn terms of yield per hectare, total production and wheat cultivation area, Pakistan

ranks in 10 key wheat producing countries in the world. In Pakistan, 125 kg is the

average consumption per capita, hence has a key location in agricultural policies of the

government. It contributes 2.7% to GDP and 11.2% to agriculture value addition. Wheat

production has been amplified from 3.35 to 25.3 million tonnes (617%) in 1948 to 2017

while raise in the area was from 3.9 to 9.23 million hectare throughout this period

(129%). Grain yield has been amplified from 848 Kg ha-1(1948) to 2845Kg ha-1 (2017)

with 325 % increase (Pakistan Bureau of Statistics, 2017).

Historically, production of wheat might be categorized into three distinctive

periods: release of semi dwarf wheat during 1947-65, the “Green Revolution” period

during 1966-76when adaptation of high yielding varieties along with chemical fertilizers

and post green revolution period 1977 to date in which high yielding varieties along with

disease resistance sustained to cover the majority wheat area through coordination efforts

at national level (Pakistan Economic Survey 2016-17).

2.4 Wheat productivity constraints

Wheat production in the country, due to lower yields, remained variable and

potentially below. The main grounds for instability and low productivity comprises: biotic

stresses such as diseases (emergence of new Pathogen races) and pests, abiotic stresses

like drought, heat, salinity, frost and different cultural practices like shortage of irrigation

water, delayed harvesting of kharif crops like sugarcane, rice and cotton, and resultantly

late wheat sowing, ineffective use of fertilizer, soil degradation, infestation of weeds and

weak extension services system (USDA, 2017).

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2.5 Impact of drought on wheat genotypes

The total irrigated area has been enlarged in Pakistan from 15.73 to 18.32 million

hectares since 1982 to 2017 whereas the wheat irrigated area enlarged from 5.959to 7.11

million hectare from 1985-86 to 2016-17. The major portion of irrigation water is not

used by the crops and the collective consequence of seepage wastage and leakage

amounts to loss of 40%. Wheat requires water for the whole growth period, but some

stages are more vulnerable to drought and serious yield loses may result if any water

shortage occur during that stage. Drought conditions at crown root initiation, grain filling,

booting and reproductive stage results in major yield losses (Pakistan Economic Survey

2016-17).

2.6 Effects of terminal drought

Drought at flowering and grain filling stage causes extensive decrease in yield

which are mostly down to increased leaf senescence (Yang et al., 2007), decreased rates

of assimilate translocation and carbon fixation (Asada, 2006), oxidative damage to photo-

assimilatory machinery (Farooq et al., 2009), pollen sterility (Cattivelli et al., 2008),

decreased set of grains and their development (Nawaz et al., 2013) and decreased sink

capacity (Liang et al., 2001).

2.6.1 Leaf Senescence

Gradual change in colour and functions of leaf due to breakdown of chlorophyll

and membrane that leads to a decline in photosynthesis with reduced water content with

the age is known as leaf senescence (Gregersen and Holm, 2007). Drought at

reproduction speed up the rate of leaf senescence, principally accountable for wheat yield

reductions (Nawaz et al., 2013). Moreover, due to senescence early on, drought at grain

filling stage shortens the duration of grain filling. It excites senescence of entire wheat

plant that amplifies remobilization of stored carbohydrates from the leaves and stem and

to developing grains, ultimately initiated grain yield losses (Ji et al., 2011).

2.6.2 Light harvesting and fixation of carbon

Drought influences fixation of carbon via stomatal closure, declines CO2 influx

into mesophyll cells (Flexas et al., 2004). It affects on metabolism which leads to

decreased ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco) content, ribulose

bisphosphate (RuBP) regeneration and impaired ATP synthesis (Bota et al., 2004; Farooq

et al., 2009). At the initial onset of drought, reduced photosynthesis is primarily caused

by reduced stomatal conductance. However, drought at later stages causes’ tissue drought

directing to metabolic impairment (Farooq et al., 2009).Under terminal drought, Rubisco

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activity is not primarily affected; however, drought occurs at anthesis decreases its

activity in flag leaves leading to reduced chlorophyll contents and soluble protein in

wheat. In prolonged drought, stomatal closure and exposure to constant extreme light

reduce the CO2 influx which direct electrons toward molecular oxygen and causes the

production of ROS and superoxide ions in photosystem I (PSI) which injure the

photosynthetic machinery leading to a large decline in carbon fixation (Asada, 2006).

2.6.3 Grain development

Photosynthesis and stored assimilates redistribution from vegetative tissues are

responsible of filling of grains in wheat (Farooq et al., 2011). In drought conditions, the

rate of grain filling declines because of decreased photosynthesis, increased senescence

and sink limitations (Wei et al., 2010: Madani et al., 2010).

Terminal drought shortens life cycle and period of grain filling having more

impact on number of grains, instead of size of grain, principally responsible for the

decrease in wheat yield (Dolferus et al., 2011). Reduced grain number is recognized to be

unsuccessful egg fertilization, therefore an undeveloped ovule. Drought happens at

microspore stage of pollen development causes pollen sterile, thus reducing grain number

(Ji et al., 2010). Anthesis and meiosis are very drought susceptible, and their lack directly

distress number of grains, so leading significant reduction of grain yield (Cattivelli et al.,

2008).

After anthesis, drought impose any impact on number of grains (Ji et al., 2011)

and slight impact on grain filling rate but period of grain filling is reduced, causes

significant reduction in dry weight of grain (Plaut et al., 2004 ; Altenbach et al., 2003).

2.7 Tolerance to terminal drought

Strategies and mechanisms of drought resistance divided into 2 main types:

drought avoidance and drought tolerance (Levitt, 1972). Physiological and morphological

features (e.g. deposition of epicuticular waxes, deep roots, early flowering and osmotic

adjustment) that facilitate the whole plant or plants parts to maintain drought are

categorized in drought avoidance. On the contrary, features for example water soluble

carbohydrates remobilization and accretion of molecular protectants which permit to the

plant to sustain in proper function in severe drought conditions are classified in drought

tolerance.

Plants show numerous mechanisms for tolerance against drought. They stay away

from the adversities of drought by early flowering, declining duration of growth,

8

improved and extra efficient uptake of water through preserve available water via

shortening canopy size and adjusting stomatal openings (Wolf et al., 2016). Osmotic

adjustment (Tambussi et al., 2007), stay-green character (Christopher et al., 2016),

antioxidant defense (Jones, 2004), hormonal regulations (Guoth ´ et al., 2009) and reserve

translocation (Wardlaw and Willenbrink, 2000) and also contribute to terminal drought

tolerance. A concise synopsis of mechanisms for terminal drought tolerance is discussed

for wheat here:

2.7.1 Drought escape

Plants’ ability to finish their life cycle earlier than inception of dry spell or

drought is termed as drought escape (Basu et al., 2016).Advance of plasticity and

remobilise assimilates of pre-anthesis to developing grains refer the vital traits of drought

escape (Ehdaie et al.,2008). Also time to flowering is extremely crucial in this regard.

2.7.2 Solute accumulation and osmotic adjustment

Drought impacts plants’ growth and yield with decreasing status of turgor and

water of plants tissues. Thus, in drought conditions, plants of wheat are constrained to

shrink their internal water potential to facilitate dehydration avoidance and to endorse

balance the water potential. To accomplish that, plants’ cells accumulate osmolytes that

contribute to phenomenon of osmotic adjustment, therefore, assist plant cells for water

balance maintenance (Tekle and Alemu, 2016). So the accretion of osmolytes, like

polyols, glycinebetaine, polyamines proline, and ions (i.e., potassium) considers a

significant element of the drought tolerance mechanism (Farooq et al., 2009).

2.7.3 Antioxidant defence

Drought encourages oxidative stress because of over generation of ROS and anti-

oxidative defence system, containing enzymatic or non-enzymatic components, assists in

reactive oxygen species scavenging in drought (Huseynova, 2012). Catalase (CAT),

superoxide dismutase (SOD), glutathione reductase (GR), ascorbate peroxidase (APX)

and peroxidase (POX) are designate the enzymatic antioxidants and ascorbic acid, β-

carotene, glutathione and α-tocopherol are designate as non-enzymatic antioxidants ,thus,

gathers in plants in drought stress to evade oxidative damage (Farooq et al., 2009;

Scandalios, 2005).

2.7.4 Stay green

Plants have the ability to stay photosynthetically active as a result of delayed

senescence which is called stay green character (Thomas and Howarth, 2000). The flag

leaf’s duration of the stay green makes positive correlation with water use efficiency at

9

the stage of development of grain (Gorny and Garczynski, 2002). Cultivars maintaining

flag leaf photosynthesis give improved yields like 35-55% of the photosynthates essential

in grain filling are supplied by photosynthesis of flag leaf (Larbi and Mekliche, 2004).

Hence, character of stay green in flag leaves of wheat performs a major function in

deciding tolerance to drought.

2.7.5 Root system architecture

It may extremely essential contributor for drought tolerance. A root system of

better development permits better extraction of soil water, thus constant and better yields

in drought stress (Dodd et al., 2011). Deep root system enhances density of root by

surface towards depth along with better radial hydraulic conductivity on depth direct to

greater production during drought stressed conditions (Wasson et al., 2012).

2.7.6 Reserve translocation

Photosynthesis and redistribution of stored assimilates from vegetative organs and

afterwards transportation towards emergent grains and assimilates accumulate in the ears

might responsible of grains fillings (Ehdaie et al., 2006). A positive relationship between

grain yield and stem reserves in both irrigated and drought conditions. On account of leaf

desiccation, current photosynthesis is reduced as a result of reduced stomatal aperture and

CO2 influx (Wardlaw and Willenbrink, 2000). In general, a decline at present

photosynthesis at terminal drought encourages mobility of stem reserve to growing grains

(Yang et al., 2004). Thus, re-translocation of a huge quantity of stem reserves to growing

grains in water stress to alleviate the impacts of accelerated leaf senescence (Ehdaie et al.,

2006). Consequently grain filling and yield may extremely reliant on reserve

carbohydrates’ translocation in leaves and stems in terminal drought (Plaut et al., 2004).

2.7.7 Hormonal regulations

A plenty of hormones modulate plant responses to water stress (Kar et al., 2011);

for example, ABA adjusts water content of tissue via stomatal oscillations which manage

cellular drought tolerance (Basu et al., 2016). It also adjusts growth of plant by regulating

leaf elongation and expansion and growth enhancement of roots in stress (Sah et al.,

2017). During drought ABA produces inside xylem tissues, that is next shifted to

reproductive organs there it impacts grain filling through regulating the genes expression

concerned to cell division and carbohydrate metabolism (Liu et al., 2005). Under mild

drought conditions, ABA as well as cytokinins is the central player to enhance the rate of

grain filling by the regulation of sink activity by key enzymes modulation in the starch

conversion from sucrose. However in severe stress, high concentrations of ABA, ACC

10

and ethylene occur, decreasing the filling of grain rate in wheat (Vishwakarma et al.,

2017). A balance between ethylene and ABA in growing grains modulates rate of grain

filling and a boost in the ratio of ABA to ethylene enhances rate of grain filling

(Maevskaya and Nikolaeva, 2013).

2.8 Role of rusts in wheat yield reduction

The wheat crop is often low as contrast to its actual capacity in terms of

production and yield because it hit by many of the abiotic and biotic maladies (Jellis,

2009). Rusts, smuts, bunts and aphids are some of the main biotic constraints and

drought, terminal heat, salinity fogs, hailstorms, winds and extreme cloudy weather are

the key abiotic constrains to wheat production (Hussain et al., 2006). The wheat rusts

have historically been key biotic constraints in Asia as well as other world. Out of 3000

rust species in the world, three are pathogenic on wheat (Laudon, 1973). Brown or leaf

rust by Puccinia reconddita, stem or black rust by Puccinia graminis and stripe or yellow

rust by Puccinia striiformis are well known diseases of rust (Hussain et al., 2011).

The happenings of cereals rust diseases have drastically influenced the human

civilization development (Large, 1940). Puccinia graminis urediospores taken from

Israeli mines were dated by 1300 B.C and biblical accounts pointed that Patriarch Jacob’s

family forced to look for refuge in Egypt due to rust epidemics at about 1870 B.C (Roelf

et al., 1992) and curse of rust is evidence in the Roman and Greek literature, whereas at

about 500 B.C. the ritual ceremonies point out liturgies to appease Robigus, the Corn

God, with the purpose of avoid the crop losses (Kislev, 1982).

According to Chester (1946), Felice Fontana was the first one who recognizes in

1767 that a fungal parasite is the casual agent of the rust. However, a difference was made

between the smuts and rusts into the 19th century by de Candolle that explained pathogens

of brown rust as Uredo rubigo-vera and thus distinct it from Puccinia graminis (Chester

1946). In the 1860s De Bary presented evidence of heteroecism of Puccinia graminis on

cereals and reported that Eriksson (1896) was the person who defined formae specials to

depict “special forms” (f.sp.) of stripe and black rust pathogens with theirs specialization

on different hosts (Walker, 1950). Thus it was generally accepted in the early 20 th century

that several fungus rusts, also expressed as rust or explosion in a number of publications,

compare with host range. Enormous efforts were made to control of cereal rust and

reduce their losses since 1880s in account of the advancement in basic science and

practical research. Large (1940) asserted that “the greatest sole undertaking in plant

pathology history was to be the rusts’ attack on cereals”.

11

2.9 Historic epidemics and yield losses

The social and economic turmoil as a consequence of losses in epidemics of wheat

rust have been leading impacts in advisory and research activities (McIntosh et al., 1995).

Even though cereal rust were evidently of great historical significance, assess of actual

yield losses got interest in the twentieth century because of well awareness of the disease’

biology and a deem require to economically justify the monetary investment in control

programs. A state wise overview compiled by Roelfs et al., (1978) on losses in United

State of America from 1918 to 1976, mentioning the reduction was more than 55% in

years of epidemic down to leaf and black rust and up to 70% in commercial fields due to

stripe rust. During the 1960s rust predictably reduced wheat yields more than one million

tonnes (2%) yearly in North America (Wise, 1977).

In Pakistan, an approximate loss of US $ 86 million as a result of severe rust

epidemic was thrashed in 1978 (Hussain et al., 1999). Egypt experienced 50% losses

because of brown rust (Abdel-Hak et al., 1980). A significant yield loss in Ethiopia,

Yemen and Iran in 1993 due to yellow rust epidemic in choice of wheat line generates by

international maize and Wheat improvement centre (CIMMYT) (McIntosh et al., 1995).

Losses by cereal rust are principally related stripe and leaf rust in Europe. A decline

occurred in the value of stem rust due to the irregular incidence of favourable

temperatures and the early 20th century’s effective barberry eradication campaign.

Economic evaluation of winter wheat in UK by Priestly and Bayles (1988) reported an

approximate loss of $83 million caused by yellow and brown rust. In china winter wheat

is influenced by persistent epidemics of yellow rust (Stubbs, 1985).

In Australia, crop losses 30-55% in wheat susceptible to both brown and black

rust (Rees and Platz, 1975). In 1992, a wide spread leaf rust epidemic in susceptible

cultivars resulted 37% loss occurred in Western Australia (McIntosh et al., 1995). Yellow

rust supposed more significance in the Southern Africa, China, Northern Europe, West

Asia and South America. Huge losses from leaf rust have been recorded in South East

Asia, Asia, South America and North Africa.

Yellow and leaf rust are presently the most imperative wheat diseases worldwide

and threaten the food security globally (Hovmoller et al., 2010, 2011). Frequent severe

epidemics of yellow rust are affecting world’s main wheat growing areas since 2000,

when two high temperature tolerant and highly aggressive Pst strain appeared

(Hovomoller et al., 2008).

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2.10 Classification and life Cycle of Puccinia

Puccinia is a genus of fungi and comprises 4000 species. Most of the species of

this genus are obligate pathogens of plant.

Kingdom : Fungi

Division : Basidiomycota

Class : Pucciniomycetes

Order : Pucciniales

Family : Pucciniaceae

Genus : Puccinia

Fig 2.1 Life cycle of Rust

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Fig 2.2 Life cycle of Puccinia2.11 Symbiosis

The living together of diverse organisms is recognized since de Bary (1879)

defined as symbiosis. The term symbiosis (Greek: Syn "with" and Biosis "living")

exploited for interspecies interaction and is concerned for a variety of associations. These

are classified into mutualistic symbiosis (benefits for both partners), commensalistic

symbiosis (benefits for one partner and neutral effect for another) and antagonistic or

pathogenic (beneficial for one but detrimental for the other partner) (Paszkowski, 2006;

Smith and Read, 2008). For the construction, evolution of well organized life and recent

ecological relationships, symbiosis played a vital role (Shtark et al., 2010).

The oldest known fossils demonstrating the first bryophyte-like land plants had

symbiotic associations with fungi (having similar properties to arbuscular mycorrhiza

fungi (AMF) that were collected from dolomite rocks in Wisconsin and are estimated to

be 460 million years old (Brundrett, 2002).This confirms evolution of plants from aquatic

environments to terrestrial strictly concurrent to plant microbes interactions. In natural

ecosystems, majority of plants show symbiotic relationship with microbes and recognized

14

potential hosts for a broad range of microbes living on the surface of host plants called as

epiphytes and colonize of the plant tissues called as endophytes. Scientists, owing to

significance of symbiosis of plant and microbes, suggested the term “plant symbiont”

(Harman, 2011).

2.12 Plant Symbionts

Microbes that produce symbioses with plant without cause apparent symptoms,

and offer boosting impact on plant growth, tolerance to abiotic stresses and pathogenic

diseases, recognize as plant symbionts. They are present in vast diversity and classified

into two groups: bacterial and fungal. Fungal symbionts can be classified into three

categories; Mycorrhizal fungi (ectomycorrhizal fungi and arbuscular mycorrhizal fungi),

Special root endophytes (Piriformospora indica as well as different dark septate

endophytes), Endophytic fungi (Type I and Type II endophytes are included). In the case

of bacterial symbionts the classification is more complex due to overlapping in terms.

Frequently used terms in the literature, such as ‘rhizobia’, ‘mycorrhization helper

bacteria’ (MHB), ‘plant growth promoting bacteria’ (PGPB) /‘plant growth promoting

rhizobacteria’ (PGPR) and ‘N-fixing bacteria’, and all refer to bacterial symbionts. The

PGPB and PGPR groups cover the most of plant bacterial symbionts. Five groups can be

recognized within PGPR/PGPB, as follows: non-legume rhizobia, MHB, legume

rhizobia, N-fixing bacteria and different plant related functional bacteria. Rhizobia are

able to give promotional impact on growth of plant, so as classified to deal rhizobia as a

distinct, individual type of bacterial symbiont, excluded from PGPB (Wang et al., 2012).

2.13 EndophytismThe term endophyte takes from the Greek endo “within” and phyton “plant”

which refers a wide range of plant endosymbionts. Endophytes are characteristically

metabolically active microbes (fungi, bacteria or virus) that colonize healthy plant tissue

intercellularly and intracellularly without apparent symptoms (Stone et al., 2000; Schulz

and Boyle, 2006; Reinhold-Hurek and Hurek, 2011; Compant et al., 2011). They are

ubiquitous, colonize in all plants, grow internally in tissue of living plants for at least part

of their life and have been isolated from almost all plants examined to date. They exhibit

complex interactions and a variety of symbiotic lifestyle with their hosts ranging from

parasitism to mutualism (Schulz and Boyle, 2005; Redman et al., 2001) and microscopic,

culturing, or molecular methods are used to detect their presence in host plant tissues

(Lebeis, 2014; Hardoim et al., 2015).

15

Endophytic microbes exhibit to augment the host plant’s fitness, competitiveness,

growth and show a strong effect on their growth, survival and ecology (Brundrett, 2006;

Rudgers and Clay, 2008; Jani et al., 2010; Beltran-Garcia et al., 2014). In several cases,

endophytic microbes produce bioactive organic compounds, secondary and antimicrobial

metabolites that resist plant pathogens (Clay and Holah, 1999, 2002; Clarke et al., 2006;

Ambrose and Belanger, 2012; Gond et al., 2014). Some endophytic microbes show to

improve tolerance of host plants to abiotic stresses (Redman et al., 2002; Malinowski and

Belesky, 2000; Kuldau and Bacon, 2008).

Endophytic microbes are horizontally transmitted to their hosts by means of

airborne spores. On the contrary, some are perhaps transmitted vertically to the

subsequent generations of plants through seeds (Tadych et al., 2012). Once they enter in

host tissue, they believe in dormant state either for the entire lifetime of host or for a time

period when environmental circumstances become favourable for the them or for some

host development state initiate for the benefit of the them (Sieber, 2007). Plants sternly

avert the growth of endophytes but they exploit numerous mechanisms to gradually

acclimatize to their living environments. So as to sustain stable symbiosis, endophytes

form several volatile and novel compounds that endorse growth of plants and help them

adjust better to the environment.

2.14 Endophytism VS Pathogenism

A question is raised that how plants distinguish the presence of beneficial (or

harmless) endophytes from pathogens and how a pathogen in some host becomes

endophyte in other host. For that it is very important to focus on endophytic phase rather

that endophyte defining all aspects of the organism. If an endophytic microbe enhances

stress tolerance of hosts or imparts some other benefit to hosts during the growth and

reproductive phase of the host but causes disease when plants become old, the microbe is

merely a pathogen? What is it that keeps a microbe in the endophytic non-pathogenic

phase rather than a pathogenic phase? What causes the transition from one phase to the

next? The pathogen is beneficial endophyte or pathogen depending on the circumstances.

A lot of times, when isolated endophytes from healthy plants inoculate to other plants

they can cause disease. It occurs when the circumstances of the fungus in the endophytic

phase are not known. By understanding the factors that make an endophyte to a pathogen

there might be able to control microbes in plants to express only endophytic phases

(Alvarez-Loayza et al., 2011)

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2.15 Classification of Endophytes

Endophytes are categorized into 2 broader groups, systemic/true and non-

systemic/transient endophytes, based on their biology, functional diversity, taxonomy and

mode of transmission. The idea of true/systemic endophytes was set forth by Mostert et

al., (2000). Systemic endophytes described the microbes that express symbiotic

relationship without causing any visible disease symptoms at any stage of host plants.

Conversely, the general established definition of Petrini (1991) with a slight amendment

is acknowledged for the non-systemic endophytes, the microbes that exist in the tissues of

hosts as a minimum part of their life cycle without showing any visible symptoms in

hosts in normal environments but in stressed or resource-limited conditions transform

symbiotic to pathogenic.

Systemic endophytes are cocladogenetic that means a host plant comprise

phylogentically similar endophytes in diverse environmental conditions, whereas

transient endophytes differ both in abundance and diversity through changed environment

(Higgins et al., 2014; Botella and Díez, 2011). Systemic endophytes are naturally inhabit

in different parts of plants oblegately and are transmitted to next generation vertically

(through seeds or vegetative propagules) (Schardl et al., 2004; Selosse and Schardl, 2007;

Tadych et al., 2014) whereas the transient endophytes turn into systemic in plants not

obligatory endophytic and usually they put up extremely close association to pathogens

and soil microbes (Johnston-Monje and Raizada, 2011; Compant et al., 2010). These

endophytes are in general horizontally transmitted, by means of spores. In some cases

they can be transmitted by seeds but vectored on seed surfaces instead of they enter into

embryos (Moricca and Ragazzi, 2008).

2.16 Fungal endophytes

Fungal endophytes comprise a broad spectrum from latent pathogens to mutualistic which

grow internally in tissue of living plants for at least part of their life without causing

symptoms of disease (Schulz and Boyle, 2005). They show a promotional effect on

growth of plant and their survival and upsurge plant ecology and fitness (Leofort et al.,

2016), and manifest strong influence on symbiotic microorganisms (e.g. nematodes,

bacteria and insects) (Gundelet al., 2011; Tadych et al., 2015).

2.17 Fungal endophytes diversity

Fungal endophytes are broadly categorized in 2 key groups. “Clavicipitaceous

endophytes (CEs)” (Clavicipitaceae, Ascomycota) colonise rhizomes and shoots of a

17

narrow host range of warm and cool seasonal grasses (Poaceae), whereas “non

clavicepataceous endophytes (NCEs)” are phylogenetically diverse in which mostly

belonging to Ascomycota, isolated from roots and shoots (Saikkonen et al., 1998;

Petrini, 1996). CEs and NCEs can be characterized into 4 discrete functional classes or

groups according to biodiversity, host range, inhabited in host tissue, transmission,

biodiversity, colonization pattern and fitness benefits (Rodriguez et al., 2009).

2.17.1 Class 1 endophytes

Class 1 endophytes characterize of clavicipitaceous species which are

predominantly transmitted vertically to offspring via seed infections and confined to

some warm and cool season grasses (Saikkonen et al., 1999). Distinctively whole life

cycle of class 1 endophytes inside the aerial part of the plant host, producing systemic,

nonpathogenic, and frequently intercellular associations (Kuldau and Bacon, 2008).These

endophytic associations are reliant on host genotypes, host species and environmental

aspects (Faeth and Sullivan, 2003; Saikkonen et al., 1999; Faeth et al., 2006). These

increase plant biomass, augment the ecophysiology of host plants, confer the ability of

drought tolerance and generate numerous toxic substances, to animals and decreases

herbivory (Arnold et al., 2008; Clay, 2002; Malinowski and Belesky, 2000).

2.17.2 Class 2 endophytes

Non-clavicipitaceous or Class 2 endophytes are recognized the largest group of

fungal symbionts that colonize roots, stems and leaves. They can culturable on artificial

media and have broad host range. They also accomplished to form extensive infections in

plants and transmitted by way of rhizomes and seed coats (Cope-Selby et al., 2016;

Hastings et al., 2017; Rodriguez et al., 2008). This class related to a small number of

members of the Agaricomycotina (Basidiomycota), Pucciniomycotina and

Pezizomycotina (Ascomycota) for example Arthrobotrys spp., Phoma spp., Fusarium

culmorum, Curvularia protuberate and Colletrichum spp. (Rodriguez et al., 2009). These

endophytes improve the biomass of shoot and roots of plant (Mucciarelli et al., 2003;

Gasoni and de Gurfinkel, 1997; Ernst et al., 2003) and supports plats to confront biotic

stress as diseases (Narisawa et al., 2002; Campanile et al., 2007), and abiotic stresses as

desiccation, drought, salinity and heat (Márquez et al., 2007; Redman et al., 2001, 2002;

Rodriguez et al., 2008).

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2.17.3 Class 3 endophytes

Class 3 endophytes isolated from tropical forest to boreal and Arctic plant

populations (Higgins et al., 2007; Arnold and Lutzoni, 2007) belong to Ascomycota

(Sordariomycetes, Dothidiomycetes, Pezizomycetes, Leotiomycetes and Eurotiomycetes)

and Basidiomycota (Agaricomycotina, Pucciniomycotina and Ustilaginomycotina)

(Rodriguez et al., 2009). They are transmitted horizontally by rain, wind, and insects

(Feldman et al., 2008; Arnold, 2008) and show a localized colonization limited to shoots.

Their assistance to the host plant is non habitat adapted as well as complex (Vega et al.,

2010; Rodriguez et al., 2009).

2.17.4 Class 4 endophytes

Class 4 make melanised structures like that microsclerotia, interellular and

intracellular hyphae and colonize only in roots. They might not host specific or not

mostly habitat; thus, represent a broad host range from diverse phylogenetic groups

amongst Ascomycota and amongst non-mycorrhizal members in Sebacinales order

(Basidiomycota) (Addy et al., 2005; Weiss et al., 2004; Xie et al., 2016). They colonize

roots of conifer trees and boreal shrubs as well as temperate zones, lowlands, coastal

plains and tropical ecosystems. Based on limitation to host roots and existence of darkly

melanized septa (DSE) these are distinctive with other endophytes and recognized as a

separate group.

2.18 Mechanisms of endophytic fungi confer tolerance against abiotic stresses

Tolerance against abiotic Stresses in host plants could be attained by symbiotic

association of fungi that engages 2 mechanisms. Initiation of host stress responsive

systems might be the first mechanisms when exposed plants to stress that directed host

plants to either stay away from that stress or tackle it. Synthesis of anti-stress bio-

chemicals by the endophytes inside the hosts might be the second mechanisms that assist

in alleviating the stress circumstances (Singh et al., 2011).

Some of the evidences during symbiotic association of fungal endophytes with

plants to adopt tolerance against abiotic stress conditions by osmotic adjustment, water

relations, increasing photosynthetic rates, accumulation of drought protective osmolytes

or amendments in the concentrations of osmolytes in host plant and by dehydrins

production, an unstructured proteins group abundant in late embryogenesis (Carson et al.,

2004). Endophytes also promote drought tolerance by means of high produce of loline

alkaloids through protection the denaturation of macromolecules or reduction the number

of reactive oxygen species (Schardl et al 2004).

19

Endophytes amplified water-use efficiency in plants to adopt tolerance against

water deficit conditions via mechanisms of stomatal regulation, osmotic adjustment by

improved levels of biomass, reduced consumption of water and efficient usage of water

(Carroll, 1988; Wu and Xia, 2006; Schafer et al., 2009). Reduced water consumption and

improved water-use efficiency supply an exclusive symbiotic mechanism for alleviation

of drought tolerance (Rodriguez et al., 2008).

Endophyte infected grasses display a rise in length and rate of growth of root

which performs a part in drought tolerance and nutrient acquirement (Richardson et al.,

1990, 1993). Fungi formed cytokinin itself and a key role in drought and salinity

tolerance in host plants in the course of its cross talk to abscisic acid (Crafts and Miller,

1974; Nishiyama et al., 2011). Endophytic plants modified their biochemical mechanism

in stress condition, consequently mineral and water uptake may rise for enhanced

structure, function and growth of plant cell (Kohler et al., 2008; Adesemoye et al., 2008).

Also fungal endophytes recognized competent of generating GA3 and IAA to stimulate

plant growth and development (Waqas and Khan, 2012; Cicatelli et al., 2010).

Accumulation of ROS in all abiotic and biotic stresses is a common plant

biochemical process. ROS cause oxidative damage to lipids, DNA, and proteins,

extremely toxic to biological cells. Conversely, they are signal molecules to stresses and

their formation is an early incident in host response to stress. In abiotic stress, endophytes

either prevent ROS production or scavenge ROS themselves or induce plants to extra

scavenge ROS efficiently (Apel and Hirt, 2004). In stress conditions, the reactive oxygen

species ROS may degrade in roots of endophyte infected wheat because of the activities

of antioxidants (peroxidise; ascorbate peroxidase, APX; peroxidase, POD; catalase, CAT;

superoxide-dismutase, SOD) (Viterbo et al., 2010; He et al., 2007).

Fungal endophytes stimulate the development of lateral roots and extended

surface area of roots by means of producing gibberellins and auxins that support the host

to uptake extra minerals and water in stress and thus better presence and generate is

obtained (Schafer et al., 2009).Endophytes produce high level of auxin, thus, increase the

mobilization and translocation of reserve carbohydrate. They are osmotically active

carbohydrates (glucose and fructose), needed by plants in drought stress when leaf area

becomes insufficient to produce the required energy (Arachevaleta et al., 1989).

2.19 Mechanisms of endophytic fungi confer tolerance against biotic stresses

Various mechanisms through they may confront the development of pathogen were

observed. Endophytic species act as mycoparasites (Sánchez Márquez et al., 2007; Rivera

20

Varas et al., 2007) or produce antibiotic substances which inhibit pathogen growth

(Strobel, 2006; Wang et al., 2007; Schulz and Boyle, 2005) or synthesis of secondary

metabolites or modify host plant biochemistry that induce defence mechanisms against

pathogens which counteract their attack or direct competition for resources and space

may also happen between incoming pathogens and resident endophytes (Arnold et al.,

2007) or produce toxic fungal alkaloids which lower the vector reproduction in case of

viruses (Lehtonen et al., 2006). For example, most tissue vacant for infection already

occupied, or endophytes make inhibition zones checking the entrance of other fungi.

2.20 Role of fungal endophytes in tolerance against abiotic and biotic stresses

The fungal endophytes acquire substantial growth promoting activity in a broad

host range and their competency in supporting host tolerance to several abiotic and biotic

stresses is incredible.

2.20.1 Piriformospora indica

Piriformospora indica is a basidiomycete arbuscular mycorrhizal like fungus with

an endophytic life style that can be grow easily on pure culture or synthetic media, where

it produces typical pear-shaped chlamydospores. P. indica is versatile and multifunctional

root endophytic fungus of the family Sebacinaceae colonises the roots of a large diversity

of dicotyledonous and monocotyledonous plants and also the model plants Arabidopsis

and barley (Peškan-Berghöfer et al., 2004; Oelmu¨ller et al., 2005; Waller et al., 2005;

Shahollari et al., 2007; Lee et al., 2011; Qiang et al., 2012; Sherameti et al., 2008;

Glaeser et al., 2015). That root colonizing mutualist was initially discovered in

association with woody shrubs from northwest Rajasthan in the Indian Thar desert in

1997 and were widely studied for its beneficial effects when they interact with plants. It

is extensively reported that inoculation of spores and culture filtrates of Piriformospora

indica directs to improvement of the growth, increase in root and aboveground biomass

in its mutualistic relationship with a broad diversity of plants (Achatz et al., 2010; Zarea

et al., 2012; Jogawat et al., 2013; Bakshi et al., 2014) increase in grain yield, better

phosphate and nitrate uptake (Yadav et al., 2010; Cruz et al., 2013; Shrivastava and

Varma, 2014) and improved tolerance to major biotic and abiotic stresses in both field

and glasshouse conditions (Ghahfarokhy et al., 2011; Alikhani et al., 2013; Ghabooli et

al., 2015; Varma et al., 2015; Harrach et al., 2015; Johnson et al., 2015; Prasad et al.,

2015; Gill et al., 2016; Trivedi et al., 2016). It is reported that P. indica experiences the

21

successful interaction with its hosts was attained by an active suppression of defence

responses of host (Waller et al., 2005; Jacobs et al., 2011).

In an experiment the performance of Piriformospora indica evaluated in many

substrata in greenhouse and field conditions by colonizing winter wheat roots with that

hypothesised biocontrol endophyte against different root, stem and leaf pathogens. In

greenhouse conditions, severity of typical root (Fusarium culmorum), stem

(Pseudocercosporella herpotrichoides) and leaf (Blumeria graminis f. sp. tritici)

pathogens were reduced significantly. However, in field conditions, leaf pathogen

symptoms were not different in Piriformospora indica colonized compared with control

plants. But the stem pathogen disease severity of Pseudocercosporella herpotrichoides

was much reduced in endophytic colonized plants. Enlarged concentrations of hydrogen

peroxide and numbers of sheath layers after B. graminis attack were detected in

endophytic colonized plants that means induction of systemic resistance was done in

plants (Serfling et al., 2007).

The frequently observed quality of improved stress tolerance in colonised hosts is

reflected in barley and Arabidopsis by an increased salt tolerance and systemic resistance

induction against the fungi of powdery mildew Golovinomyces orontii and Blumeria

graminis f.sp. hordei (Waller et al., 2005). The mutual interaction of P. indica with

Arabidopsis or barley and the phenomenon of induced systematic resistance present an

important combination to unravel processes used for symbiotic association and beneficial

effects. Additionally, this symbiotic association was encouraging in exploration of the

genetic basis of programmed cell death due to microbes’ invasion. Interestingly, P. indica

can colonize great root surface areas without aggravating tissue narcotisation although

sporulation and root colonisation occur together and depend strongly on death of root

cells (Deshmukh et al., 2006).

The symbiosis association of Piriformospora indica and barley was also

associated with an improved antioxidative status presented by cycle of ascorbate–

glutathione. P. indica colonized barley displayed an enlarged gathering of ascorbate in

roots where as glutathione contents considerably high in leaves in contrast to non-infested

hosts (Waller et al., 2005). Infested hosts showed an improved drought and salt stress

tolerance (Eltayeb et al., 2007).

Piriformospora indica confront fungal root pathogens such as Fusarium spp.,

Rhizoctonia solani and Cochliobolus sativus and protect barley plants from their

deleterious effects (Waller et al., 2005). Additionally, protection of systemic type to

22

foliar pathogens is achieved which is a beneficial effect connected to the mutualistic

interaction. Generally, systemic resistance show a defence approach of the plants to

check microbes’ invasions to sites of initial infection, thus, to defend yet uninfected

organs of plants. However, in case of barley, P. indica activates respective host defence

responses that ultimately direct a systemic resistance against the leaf pathogen Blumeria

graminis f.sp. hordei (Waller et al., 2005).

P. indica was reported helpful in biological control of Fusarium wheat diseases.

Assessment was done for the biocontrol consequence of P. indica on the disease of

fusarium head blight (FHB) of spring and winter wheat resulting by the contamination of

the mycotoxin deoxynivalenol (DON) in UK weather conditions. Application of P. indica

reduced 70% of disease incidence and severity and reduced 70 and 80% concentration of

mycotoxin DON in winter and spring wheat respectively. P. indica also improved

aboveground biomass, 1000-grain weight and grain yield (Rabiey and Shaw, 2015).

In another experiment the performance of Piriformospora indica was estimated in

control of Fusarium crown rot of wheat which can decrease grain quality and yield and

straw production. Wheat Seedlings were inoculated with P. indica and F. culmorum at

the time of sowing growth of 7 days, under glasshouse condition. Seedling without the

inoculation of P. indica were badly damaged by F. culmorum pathogen but root seedlings

inoculated with P. indica and F. culmorum were not damaged and also free of visible

symptoms. DNA quantification reflected that decrease of the amount of F.culmorum

DNA in the end. These results proposed that P. indica can defend seedlings of wheat

from damage of Fusarium crown rot and decrease inoculums return towards the soil

(Rabiey et al., 2013). Also P. indica showed an extensive effect on soil and root

microflora and having altered relation of wheat with native weeds as well as decreased

the competitiveness of the weeds with wheat in UK weather and soil conditions (Rabiey

et al., 2015).

Among microbe–plant interactions, symbiotic association P. indica with plants is

well recognized to improve plant growth by lessening the effects of stress and resultantly

improve the plant fitness. Colonization of P. indica generally increases the ROS

metabolism by maintaining ROS homeostasis, and thus prevents high level of ROS

accumulation in cellular processes of plants and survival and growth of plants in stressful

environments (Nath et al., 2016). A distinct balance between scavenging and generation

of ROS is vital to exploit ROS as an adaptive defence response of plants in biotic and

abiotic stress conditions. Adverse conditions of abiotic and biotic stresses can extensively

23

accelerate the ROS generation (Gill and Tuteja, 2010; Rasool et al., 2013). P. indica

could enhance the plant fitness through altering the chemical plasticity via changing

generation-scavenging of ROS under biotic and abiotic stresses (Goh et al., 2013;

Beneventi et al., 2013; Mo et al., 2016; Hashem et al., 2016).

The root endophyte P. indica be a leading candidate to increase plants’ growth

and yield and performs as a bioprotector and growth promoter, in addition fighting

against environmental stresses to a variety of plant species. In a greenhouse experiment,

effects of culture filtrate of Piriformospora indica reported on Helianthus annus

(Japanese) and Helianthus annus (Sun gold). Overall plants growth and seed production

were promoted as well as increased 50 to 70% oil content of the seeds in the culture

filtrate of Piriformospora indica inoculated plants (Bagde et al., 2011).

In a field experiment, influence of interaction of P. indica on C. forskohliia

medicinal plant was studied. The observed results were of overall improvement in aerial

biomass, phosphorus acquisition and chlorophyll contents, as well as promoted

inflorescence development, resultantly an increase in the amount of p-cymene in the

inflorescence. However, endophytes treated plants were become fibrous and developed

more lateral roots consequently root thickness was reduced. So the smaller root biomass,

thus a decrease in content of forskolin. Symbiotic association of P. indica and C.

forskohlii promoted aerial biomass production as well as flower development. Thus,

exploit of root endophyte Piriformospora indica in sustainable agriculture increased the

production of medicinally significant chemicals (Das et al., 2012).

2.20.2 Trichoderma

Apart from P.indica, several fungal microbes also colonize plant roots from the

genus of Trichoderma. It has also been recognized as biocontrol fungi for many of the

decades; however, a few strains are plant symbionts with endophytic lifestyle and induce

systemic resistance in their host plants. Mycoparasitism and antibiosis were the prime

biocontrol mechanisms previously, but currently induced systemic resistance (ISR) is

believed to be imperative mechanism. A few strains also colonize stems and twigs too,

however, as root endophytes; they modify plant physiology and ascertain chemical

communication of plant, results in reprogramming of gene expression of host (Mastouri

et al., 2010). They can also promote growth, improve photosynthesis and respiratory rates

and nitrogen used efficiency (NUE) and it induce resistance to diversity of biotic and

abiotic stresses including drought, temperature, salt and osmotic stress (Dingle and

McGee, 2003; Harman and Mastouri, 2009). It was reported about 30% decrease in

24

nitrogen use for selected crops without losing any yield (Mastouri et al., 2010). Some

Trichoderma strains were reported having direct positive impacts on host plants by

escalating nutrient uptake, seed germination rate and percentage, growth potential,

fertilizer use efficiency, and induction of host defences against damage of abiotic and

biotic stress (Shoresh et al., 2010; Mastouri et al., 2010).

Isolates of Trichoderma viride were reported as endophytes on roots as well as

aboveground tissues of Theobroma cacao (Holmes et al., 2004; Bailey et al., 2009).

Earlier in cacao, Trichoderma viride were considered for their ability to manage diseases

(Bailey et al., 2009; Mastouri and Harman, 2009), but after modifications in gene

expression patterns, revealed by Characterization of cacao/Trichoderma, that abiotic

stresses including drought could be conferred by Trichoderma viride in cacao (Bailey et

al,. 2006; Harman, 2011). Endophyte Trichoderma viride colonized the seedlings of

cacao might be cause the delay in different aspects of drought response. Hence drought

tolerance in cacao was proposed to mediate by improved water status through promoted

root growth (Bae et al., 2009).

In another experiment, improved seedling vigour and physiological protection

against oxidative damage in plants were studied when seeds treated with T. harzianum

strain T22. Under numerous biotic stress (Pythium ultimum diseases of seeds and

seedlings), abiotic stress (salt, osmotic and temperature) and physiological stress (reduced

seed quality result of seed aging) consistently faster and uniform germination of treated

seeds were reported than those untreated seeds (Rodriguez et al. 2010). Accumulation of

ROS was a general feature that negatively influenced plants in these stresses. The

application of endophyte showed a positive effect on seed germination as well as treated

seeds presented a decrease in damages by accumulation of ROS and accumulation of

peroxides in seedlings of cacao in aged seeds or during osmotic stress (Miller et al.,

2007; Mastouri et al., 2010).

The status of antioxidant defence by colonization of T. harzianum T22 with

tomato seedlings in drought was investigated.T22 alters the expression of genes encoding

antioxidant enzymes, thus, colonized plants retained their redox state higher than non

colonized plants. The higher redox state of colonized plants could be revealed by their

enhanced activity of glutathione and ascorbate-recycling enzymes, enhanced activity of

ascorbate peroxidase, catalase and superoxide dismutase (Yildirim et al., 2006) As an

expected mechanism to improve tolerance to biotic and abiotic stresses, increased

tolerance in colonized hosts in drought was reported as a result of advance ability to

25

scavenge ROS and recycle oxidized glutathione and ascorbate (Rizhsky et al., 2004;

Mastouri et al., 2012).

The capacity of different Trichoderma to alleviate severe environmental

conditions facilitates their existence in different geographical locations (Hermosa et al.,

2004). It is no surprise on account of their ubiquitous nature and fast substrate

colonization; they have been usually utilized as biocontrol microbes in agriculture

(Montero-Barrientos et al., 2010). These genes isolation and transfer to plant genome

might outcome in a considerable enhancement in tolerance to abiotic and biotic stresses

(Dana et al., 2006) and these genes symbolize a significant resource in exploitation of

agri-biotechnology and development of soil resources (Montero-Barrientos et al.,2010).

Trichoderma as biocontrol agents catch the attention for managing diversity of

soil borne fungi of Sclerotiorum cepivorum, Botrytis allii and Aspergillus niger which are

the causal organisms of neck rot black mould and white rot disease of onion, respectively

(Clarkson et al., 2004; Metcalf et al., 2004; McLean et al., 2005). A combination of

numerous mechanisms such as induced resistance, moderate the enzymes of pathogen

and competition are liable for biocontrol. Trichoderma mode of action for managing

foliar pathogens was credited to induce tolerance against them (Elad, 2000; El Hassni et

al., 2007). Induced accumulation of antifungal phenolic compounds was related to the

level of tolerance to black mould and basal rot in different onion growth stages (O ¨ zer et

al., 1999, 2003, 2004). It is valuable in protecting Arachis hypogaea, Cucumis sativus

and a number of other crops from destruction by several pathogens (Ha, 2010). It has also

been broadly practice in agricultural management practices specifically in control of

weeds (Daisog et al., 2012).

2.20.3 Colletotrichum

Colletotrichum spp. categorized as virulent pathogens but a number of species

express mutualistic and symbiotic lifestyles in host plants. Symbiotic benefits conferred

by Colletotrichum spp. are drought tolerance, growth improvement and disease resistance

(Redman et al., 2001). Slight variations in host plant genomes show strong impact on the

result of symbiotic interactions (Rodriguez et al., 2008). For instance, some genetic

variations among varieties of tomato (Solanum lycopersicum) with the colonization of

Colletotrichum magna caused different types of lifestyles of fungus from either parasitic,

commensal or mutualistic lifestyles (Tanksley, 2004; Brewer et al., 2007) like that

Colletotrichum gloeosporioides was known the pathogen on strawberry but designated a

commensal for tomato as it expressed no disease defence (Redman et al., 2001).

26

However, it conferred drought tolerance in tomato and also enhanced host biomass and

was therefore categorized a mutualist (Rodriguez et al., 2008). So it confirmed that

several microbes contain pathogenic or symbiotic lifestyles dependent on the host plants.

Some of the fungal endophytes fungi were evidence for single host specificity at host

species level (Higgins et al., 2007; Arnold, 2007; Hoffman and Arnold, 2008).

Conversely, many of the endophytes including Colletotrichum members showed a broad

range of host as they colonized various taxonomically unrelated hosts (Murali et al.,

2006; Sieber, 2007) signifying that they attained different types of adaptations to

overcome a number of host defences (Suryanarayanan et al., 2009).

Colletotrichum spp. showed symbiotic lifestyle devoid of inducing defence

systems in watermelon (Citrullus lanatus) when colonized its roots, stems and leaves,

However, attack of a virulent pathogen activated the defence systems in those

Colletotrichum colonized plants to that high level which was not achieved by

noncolonized plants (Redman et al., 1999). A number of research studies reported the

confrontation and alleviation of pathogenic diseases and different abiotic stresses

including drought stress through the infestation of plants with fungal endophytes such as

Acremonium lolii (Poling et al., 2008) and Colletotrichum spp.( Mejı´a et al., 2008; Lee

et al., 2009).

27

CHAPTER 3 MATERIALS AND METHODS

The current study was carried out to consider the beneficial role of fungal

endophytes in enhancing drought and rust diseases tolerance in wheat genotypes through

field and lab experiments. The field experiments were carried out during 2014-15 and

2015-16 (spring wheat growing season in Pakistan) to find out drought sensitive and rust

diseases susceptible genotypes as well as investigate the role of symbiotic relationship of

fungal endophytes with drought sensitive and rust susceptible genotypes under drought

and rust prone environments. The lab experiment was conducted in 2015 to evaluate

different efficient and compatible endophytes for wheat plant growth. Materials and

methods employing during these studies are briefed as:

3.1 Experimental site and conditions

The study was carried out in the mycology lab and field conditions at Department

of Plant Pathology research area, University of Agriculture Faisalabad. A series of field

and lab experiments were performed for this study. The lab experiments were carried out

in test tubes and Petri plates containing agar and potato dextrose agar (PDA).

Randomized complete block design (RCBD) with factorial arrangements having three

replications in sandy clay loam soil were used for field experiments.

3.2 Collection of wheat germplasm

The germplasm of 50 local genotypes/lines of wheat were collected from Gene

Pool of Plant Breeding and Genetics (PBG) department and Wheat Section, Ayub

Agricultural Research Institute (AARI) Faisalabad. The fifty local genotypes/lines of

wheat were evaluated (Table 3.1).

28

Table 3.1: Wheat genotypes used in the screening experimentsGenotypes Genotypes GenotypesPunjab-2011 Pbw-222 Pict-62Gomal-2008 MH-97 Wafaq-2001Iqbal-2000 Fareed-2006 AARI-11Pak-81 Shaheen-94 NARC-08Watan-92 Bathoor-2008 Chenab-2000Saher-06 Pirsabak-2004 Abadghar-939495 Faisalabad-85 9725Punjab-85 Shafaq-2006 SH-95LU-26 Kohenoor-83 Millat-2011Moomal-2002 Manthar-2003 Hashim-2010Kohistan-97 Lasani-2006Faisalabad-83 9610As-1011 SH-02Kohsar-95 Parsab-08Faisalabad -2008 Anmol-91Uqab-2000 As-2002GA-02 Parwaz-94Glaxy-2013 9444Chakwal-50 Inqalab-91Bhakhar-2002 Potohar-73

3.3 Screening of wheat germplasm against drought

In the screening experiment two plots with each 50 wheat genotypes were sown

under randomized complete block design (RCBD) in field with three replications to find

out the drought sensitive wheat genotypes during 2014-15. Drought conditions were

provided by skipping the irrigation at reproductive and grain filling stage of wheat in one

plot in comparison of normal plot where no irrigation was skipped. The following wheat

growth parameters for screening terminal drought were:

i. Number of grains per spike

ii. 1000-grain weight (g)

iii. Number of productive tillers m2

iv. Biological Yield (gm-2)

v. Grain yield (gm-2)

vi. Harvest Index %

vii. Percent Yield Reduction %

viii. Stress Susceptibility Index (SSI)

ix. Mean Productivity (MP)

29

x. Tolerance Index (TOL)

3.4 Screening of wheat germplasm against rust diseases

In the other two screening experiments 50 wheat genotypes were sown in two

separate plots under randomized complete block design (RCBD) in field with three

replications to evaluate the leaf and yellow rusts susceptible genotypes during 2014-15.

The rust conditions were produced by providing artificial inoculation of yellow and leaf

rust at tillering and heading stage on wheat genotypes by means of various methods like

dusting with talcum powder, spraying with distilled water and needle injection methods.

Disease pressure was offered by maintaining high intensities of fungal spores of rusts by

rows of highly susceptible Morocco as spreader around the fields. The following wheat

growth parameters for screening leaf and yellow rust susceptibility were:

i. Area under disease progress curve (AUDPC)

ii. Final disease severity % (FDS)

iii. Coefficient of infection (CI)

3.5 Rust inoculation Procedures

3.5.1 Collection of inoculums

For making the artificial inoculation of leaf and yellow rusts, the inoculums of

leaf and yellow rusts Puccinia recondita and Puccinia striiformis were taken from Wheat

department, Ayub Agricultural Research Institute (AARI) Faisalabad. Those inoculums

were collected, preserved and stored by Wheat department during earlier wheat crop

seasons from the Wheat department, different fields of Faisalabad area, Kaghan, Murree

and various farmers field of the Punjab for their different ongoing projects. The idea of

collection the inoculum from different sources was to use diversified inoculums to create

the rust infection.

30

Fig 3.1: Modified diagrammatic Cobb Scale described by Peterson et al., 1948

3.5.2 Storage of inoculums Collection of inoculums of yellow and leaf rusts was done by a vacuum rust

collector and spores of rusts were desiccate in calcium sulphate (CaSO4) desiccators for

5-6 days. Storage of inoculums for long term was occurred in glass vials, gelatine

capsules, aluminum foil pouches and plastic bags at -800C freezer and liquid nitrogen at

WRI, for further study and different ongoing projects of wheat rusts. The fungi which can

survive in freezing, cooling and subsequent thawing can be stored for an indefinite period

in liquid nitrogen (Dhingra, 1993). In that process, dispensing of the organism aseptically

into ampoules, sealing and dipping into liquid nitrogen (N2).

3.5.3 Methods of inoculation

For making artificial inoculation 3 methods were done, given below:

3.5.2.1 Hypodermic injection method

Highly susceptible genotype Morocco in the spreader rows were inoculated by

means of hypodermic injection method. In that method hypodermic needle injection with

an aqueous suspension of uredinospores at the rate of 106/ml of water to elongating stem

(Rao et al., 1989) two times weekly until the development of heavy inoculums (Roelfs et

31

al., 1992). Those inoculums of brown and yellow rusts comprised of mixture of brown

rust races (TKTPR, TKTRN, PGRTB, KSR/JS, FHTTL and PHTTL) and stripe rust races

(80E85) collected from Kaghan, Faisalabad and Murree. That diversification was done by

dusting and spraying of rust inoculums to spreader rows, made 3 times in the month of

January and February 2015.

3.5.2.2 Spraying method

Rust suspension prepared comprising 250 mg uredospores per liter in purified

water along with 2 drops of Tween-20. Inoculums applied by means of a hand sprayer

with a pressure of 1.1 kg/cm to experimental plots (Tarvit and Cassell, 1951; Roelfs et al.,

1992). For ensuring the successful infection, rust spore suspension was sprayed in the

morning time. For increasing humidity, inoculated plants sprayed with pure water. That

method directed towards the development of plentiful rust spores and also repeated until

anthesis stage. For that reason, there was no chance of disease escape by susceptible

genotypes.

3.5.2.3 Dusting method

Rusts inoculums were mixed with talcum powder (Travit and Cassell, 1951) and

dusted in morning time on the experimental plots during the month of January and

February, 2015, making sure each plot was dusted properly. That was done to stay away

from any failure during other methods in the development of artificial rust infection.

Fig 3.2: Artificial inoculums of leaf rust (left) and yellow rust (right)

32

3.6 Data recording of leaf and stripe rust

In the screening experiments, during 2014-15 data of brown and stripe rusts

recorded at intervals of ten days. Severity of rusts in percentage was assessed according

to modified Cobb’s scale described by Peterson et al., (1948). These Severity ratings

were based on visual observations assessed at ten days intervals and field response as

susceptible, moderately susceptible, moderately resistant and resistant by the values of

coefficient of infection (CI) were calculated by the equation described by Pathan and

Park, 2006. Four observations about rusts severity were assessed before the physical

maturity of the crop.

3.7 Rusts screening of wheat genotypes

In the leaf and yellow rusts screening experiments, during 2014-15 selected 50

genotypes were planted by hand drill in RCBD design along with Morocco as a rust

spreader during 1st week of December, 2014 to evaluate their resistance and susceptibility

against leaf and yellow rusts. The experimental plots were surrounded by planting three

rows of highly susceptible genotype Morocco for developing rust infection. The spreader

plants Morocco were inoculated artificially by means of hypodermic injection method

(Rao et al., 1989) over and above dusting and spraying methods, two times in the months

of January and February for the development of a heavy rust infection (Roelfs et al.,

1992; Tarvit and Cassell (1951). Severities of rusts in percentage were recorded in four

observations at 10 days intervals when Morocco showed 40-50% rust severity. The rust

severities were calculated with modified Cobb’s diagrammatic scale as mentioned earlier.

33

Fig 3.3: Field view of Leaf rust (left) and Yellow rust (right). Photographs were taken at 10th April 2015 showing 50 % disease severity

3.8 Yield and yield components3.8.1 Number of total tillers (m2)

For counting tillers per meter square (m2), a quadrant of 1 meter square was used.

From each plot numbers of tillers were recorded at random at harvest and then were take

average.

3.8.2 Number of productive tillers (m2)

For counting, the productive tillers from each plot selected at random by

deducting the non productive tillers from totals.

3.8.3 Number of grains per spike

For counting number of grains per spike, 5 spikes from each quadrate selected at

random after harvesting and manually threshing, grains counted and taken the average.

3.8.4 1000-grains weight (g)

The 1000 grains randomly selected from each quadrate were measured through an

electric balance.

3.8.5 Biological yield (gm-2)

Biological yield per meter square was measured through an electric balance by

weighing plants.

3.8.6 Grains yield (gm-2)

34

The grain yield per meter square was measured after harvesting the crop and

threshing manually and through an electric balance by weighing grains.

3.8.7 Harvest index (%)

Harvest index calculated for each quadrate by employing the formula:

HI= [(Economic yield / Biological yield)] x 100Where,

Biological yield = grains + straw Economic yield = grain yield

3.8.8 Percent yield reduction (%)

Percentage of yield reduction = [1 – (Grain yield in drought/Grain yield in normal water)]

x 100

3.8.9 Percent Yield Increased (%)

Percentage of yield increased = [1 + (Grain yield in drought/Grain yield in normal

water)] x 100

3.8.10 Area under Disease Progress Curve

AUDPC estimated for each genotype according to the equation adopted by Pandy et al., (1989).

AUDPC = d [1/2 (y1 + yk) + (y2 + y3 + - - - - - + yk-1)]Where,

d= days between two consecutive records (time intervals)y1 + yk = Sum of the first and last disease recordsy2 + y3 + - - - - - + yk-1= Sum of all in between disease scores3.8.11 Coefficient of Infection (CI)

Calculated the coefficient of infection (CI) by multiplying value of severity by 0.2, 0.4,

0.6, 0.8, 0.9 or 1.00 for host response ratings of resistant (R), moderately resistant (MR),

moderately susceptible (MS) or susceptible (S) respectively (Pathan and Park, 2006) to

categorize wheat genotypes into different groups.

3.9 Optimization of efficient and compatible endophytes with wheat seed

3.9.1 Collection of samples for endophytes

The samples of healthy leaves, roots and stems were taken from the desert plants

from various locations of Cholistan, Thar and Rohi Deserts. Samples were taken at

random from 3 to 5 healthy plants per site. The zip-lock bags were used for samples and

after that stored in ice buckets and were shifted to the lab, stored in refrigerator and were

used for isolation of endophytes within 72 hours. Samples were cleaned and washed until

yet soil removed, and then their surfaces were sterilized in 1 % (v/v) sodium hypochlorite

solution for 3 times and with distilled water.

35

By means of aseptic technique, 2-3 cm pieces of leaves, roots, and stems placed

on 10% PDA in petri plates and after that incubated at 28°C for 6-8 days to let the

emergence of fungi. Pure culture was obtained by the sub culturing of isolated fungi.

3.9.2 Morphological identification and mass culturing of endophytes

Fungal identification methods were based on the morphological characteristics of

their colonies (Najjar, 2007). The shape and size of conidia and phailides were calculated

and also compared the micro and macro morphological features to the identification key

(Hanlin, 1990; Barnett and Hunter, 1998; Pitt and Hocking, 2009) and after that pure

culture were multiplied.

3.9.3 In-vitro evaluation of endophytes

Spores of endophytic fungi were harvested in distilled water by rubbing the

surface of a sporulating pure culture with a sterile bent glass rod. Spore densities were

estimated using a hemocytometer and compound microscope (200x total magnification).

The spore suspensions were diluted in distilled water to prepare 105-106 spore mL-1.

Germinating wheat seed were kept in test tubes containing 0.3% agar concentration in

distilled water with fungal spore suspension of known concentration and were incubated.

After suitable intervals root and shoot length were measured and the efficacy of

endophytes were tested.

3.10 Field appraisal of selected efficient and compatible endophytes against drought and rust diseases

Seeds of two selected (from screening experiments) drought sensitive, leaf and

yellow rust susceptible genotypes of wheat each were soak in different selected (from lab

experiments) efficient and compatible endophytic spore suspensions for 24 hours and

then were sown in field with three replications and measured the antagonistic effect of

endophytes for enhancing the yield by reducing the disease severity as well as symbiotic

effect for tolerance of terminal drought. The parameters of Final disease severity (FDS),

AUDPC value, 1000-grain weight, Grain yield and Percent yield increased were

measured in case of leaf and yellow rust whereas 1000-grain weight, Number of

productive tillers, Biological yield, Grain yield and Percent yield increased were

measured in case of drought for assessing the role of fungal endophytes in stressed

environments. The endophytes were re-isolated from the inoculated plants to confirm the

colonization of the fungus in plant tissues. Re-isolation and identification of fungal

36

endophytes from inoculated wheat plants were done for the confirmation of their

existence.

3.11 Statistical analysis

Data were analysed using analysis of variance (ANOVA) and Dunkun’s New

Multiple Range Test (DNMRT), Tukey’s test at 5% probability level in screening

experiment and Least Significant Difference (LSD) test for other experiment (Steel et al.,

1997).

37

CHAPTER 4 RESULTS AND DISCUSSIONS

4.1: Response of different wheat genotypes against drought stress

4.1.1 Number of grains per spike:

While assessing the growth behaviour of wheat genotypes a decline was seen in

all growth parameters with escalating the drought conditions. Number of grains per spike

is an imperative yield interposing attribute for achieving high yield. Greater number of

grains per spike confirm high final yield. Data analysis of number of grains per spike is

the evidence for a considerable effect of imposed drought at the flowering and grain

filling stage on the number of grains per spike of different wheat genotypes during the

screening of 50 genotypes for assessing and screening of most effected genotypes by the

drought. Results showed highly significant (P≤0.001) effects among investigated wheat

genotypes for number of grains per spike in drought and normal conditions (Table 4.2).

The data concerning the number of grains per spike during 2014-15 year revealed

significant impact of drought stress on plants for this yield related parameter. In normal

water conditions, plants produced maximum grains per spike, while the imposed drought

stress significantly reduced the number of grains per spike.

A perusal of the table 4.1 showed the range of the number of grains per spike

among 50 wheat genotypes from 36 (Punjab-2011, Hashim-2010) to 49 (Millat-11, MH-

97) under normal conditions while under drought conditions the range for number of

grains was observed as 32.4 (Punjab-2011, Hashim-2010) to 46.6 (MH-97).

Under both normal and stress conditions, Hashim-10, Punjab-11, Watan-92,

Faisalabad-85 and Shafaq-06 were the genotypes which showed low number of grains per

spike while the genotypes Faisalabad-08, Uqab-00, MH-97, 9444, Pirsabak-04,

Faisalabad-83 Kohistan-97, Chakwal-50, Fareed-06 and Punjab-85 showed high number

of grains. Genotypes Punjab-85, 9495, Pothohar-73, Kohsar-95, Kohenoor-83 and

Parwaz-94 showed high number of grains in normal condition but produced low number

of grains per spike in drought condition, thus higher loss of number of grains was

depicted (Table 4.1).

Similar results were found by Bayoumi et al. (2008) who reported drought stress

resulted reductions in number of grains per spike and other yield components. Such

observations reported previously by Chandler and Singh (2008) and later by Dorostkar et

38

al. (2015). Differences in results may be because of the difference in circumstances in

which experiments were carried out.

4.1.2 1000-grains weight (g):It is one of the imperative yield contributing attribute in getting greater yield and

contains a pivotal character in restrictive yield potential of a genotype. 1000-grain weight

depends upon seed size. Greater the size of grain, higher will be the grain weight. The

analysed data of TGW of wheat exhibited a significant impact of terminal drought on

grain weight of different wheat genotypes during the screening of genotypes for assessing

and evaluating of most effected genotypes under drought. Results showed highly

significant (P≤0.001) effects among investigated wheat varieties for grains weight in

normal and drought conditions (Table 4.2). In normal water conditions, plants depicted

higher 1000 grain weight, while the drought stress significantly reduced it.

Results for 1000 grains weight during 2014–2015, showed that the highest grains

weight belonged to the genotypes Kohenoor-83 (44.79g) and the lowest related to

Hashim-2010 (35.25g) in normal conditions. In drought conditions, Millat-11 (42.1g) and

Hashim-2010 (28.2g) showed highest and lowest grain weight respectively (Table 4.1).

Under both normal and stress conditions, Hashim-10, Punjab-11, Watan-92,

Faisalabad-85 and Shafaq-06 were the genotypes which showed low grain weight while

the genotypes MH-97, 9444, Pirsabak-04, Faisalabad-83 and Punjab-85 showed high

grain weight. Genotypes Kohenoor-83, Kohsar-95 and Parwaz-94 showed 1000 grain

weight under normal conditions but produced low grain yield in stress condition, thus

higher loss in grains weight was observed (Table 4.1).

Similar results were found by Bayoumi et al. (2008) who reported drought stress

resulted reductions in 1000-grain weight and other yield components. Such observations

reported previously by Chandler and Singh (2008) and later by Dorostkar et al. (2015).

4.1.3 Number of productive tillers:Mean comparison of number of productive tillers m2 showed considerable

difference in normal and drought stress conditions. A significant decline in the number of

productive tillers m2 of wheat was observed while was increasing the drought conditions.

Number of productive tillers is an imperative yield contributing attribute for achieving

higher yield. Greater number of productive tillers validate higher final yield. Data

analysis of number of productive tillers m2 of wheat is the evidence for a considerable

effect of imposed drought at the flowering and grain filling stage on the number of

productive tillers of different wheat genotypes during the screening of 50 genotypes for

39

assessing and screening of most effected genotypes by the drought. Results showed

highly significant (P≤0.001) effects among investigated wheat genotypes for number of

productive tillers under normal and drought conditions (Table 4.2). In normal water

conditions, plants formed more productive tillers, while terminal drought stress drastically

reduced this trait.

In present study, results for the number of productive tillers described that highest

number of productive tillers observed in the genotypes MH-97 (454) and lowest observed

in Punjab-11 (305) under normal conditions. While the genotypes of MH-97 (436) and

Punjab-11(290) had the highest and lowest number of productive tillers under stress

conditions respectively (Table 4.1).

Under both conditions, Shafaq-06, Faisalabad-85 Hashim-10, Punjab-11 and

Watan-92 were the genotypes which showed low number of productive tillers while the

genotypes MH-97, 9444, Pirsabak-04, Faisalabad-83 and Punjab-85 showed high number

of productive tillers. Genotypes Parwaz-94, Kohsar-95 and Kohenoor-83 showed number

of productive tillers under non stress conditions but produced low number of productive

tillers in stress conditions, thus higher decrease in the number of productive tillers was

observed (Table 4.1).

Described results are in harmony with the observations of Bayoumi et al. (2008)

and Dorostkar et al., (2015).Difference in results may be because of the difference in

circumstances in which experiments were carried out.

40

Table 4.1: Influences of drought stress on the yield related traits of different wheat genotypes

Genotypes

Number of grains per Spike

1000-Grains weight (g)

Number of productive tillers m2

NormalDrough

t Normal Drought Normal DroughtPb-11 36.0 32.4 34.69 31.2 305 290Gomal-08 39.0 35.1 37.03 34.1 334 317Iqbal-00 39.0 35.1 37.20 35.7 345 328Pak 81 40.0 34.8 38.92 36.2 360 342Watan-92 37.3 32.5 35.85 33.3 311 295Saher-06 43.0 37.4 40.44 37.6 374 3569495 45.0 34.7 41.81 32.2 401 361Pb-85 45.0 38.3 41.73 37.1 391 367LU-26 39.3 33.4 37.80 33.6 337 317Moomal-02 44.3 37.7 41.59 37.0 386 363Kohistan-97 45.3 38.5 42.31 38.9 405 381Fsd-83 45.0 38.3 42.00 38.6 405 380Ass-11 40.7 34.2 38.92 35.8 344 323Kohsar-95 43.0 33.1 41.13 31.3 371 330Fsd-08 43.0 40.4 40.47 36.0 373 362Uqab-00 45.0 42.3 41.57 37.0 391 380GA-02 39.7 37.3 37.69 33.5 340 330Glaxy-13 40.3 37.9 38.69 34.4 353 343Chakwal-50 45.3 42.6 42.29 39.8 410 397Bhakhar-02 44.3 42.1 41.81 39.3 401 385Pbw-222 46.0 43.7 43.16 40.6 425 408MH-97 49.0 46.6 44.58 41.9 454 436Fareed-06 46.0 43.7 42.21 38.8 410 394Shaheen-94 45.0 42.8 42.55 39.1 406 390Bathoor-08 43.3 41.2 40.83 37.6 383 368Pirsabak-04 48.3 45.9 44.52 41.0 448 430Fsd-85 37.0 35.2 36.00 33.1 320 307Shafaq-06 37.0 35.2 36.20 33.3 319 306Kohenoor-83 48.3 37.2 44.79 33.6 447 393Manthar-03 43.3 37.3 41.13 35.8 388 361Lasani-06 43.0 37.0 41.30 35.9 385 3589610 40.3 34.7 38.30 33.3 351 326SH-02 45.3 39.0 41.46 36.1 409 381Parsab-08 43.0 37.0 40.27 32.6 383 357Anmol-91 43.3 39.0 41.27 33.4 386 359Aas-02 41.3 37.2 38.26 31.0 348 324Parwaz-94 44.0 34.3 41.18 30.9 380 3309444 48.3 45.4 44.67 39.3 445 419Inqalab-91 47.7 44.8 43.70 38.5 421 396Potohar-73 45.7 42.9 42.25 37.2 397 373Pict-62 45.0 42.3 42.18 36.7 392 369Wafaq-01 40.7 38.2 38.47 33.5 350 329

41

Genotypes

Number of grains per Spike

1000-Grains weight (g)

Number of productive tillers m2

NormalDrough

t Normal Drought Normal DroughtAARI-11 45.0 42.3 42.19 36.7 401 377NARC-08 43.0 40.4 40.11 34.9 376 353Chenab-00 48.3 37.7 44.62 34.4 447 398Abadghar-93 45.0 41.9 42.03 39.5 392 3729725 48.7 45.3 44.61 41.9 441 419SH-95 41.7 38.8 38.50 36.2 350 333Millat-11 49.0 45.6 44.76 42.1 444 422Hashim-10 36.0 32.4 35.27 28.2 314 298

42

Table 4.2: Analysis of variance for morphological traits of wheat in normal and drought stress conditionsSource of variation

Df Number of grains per spike Number of productive tillers m2

1000-grain weight Biological yield Grain yield Harvest index Yield reduction

Normal Drought Normal Drought Normal Drought Normal Drought Normal Drought Normal Drought

Replication 2 7.2600 0.3618 768.74 255.37 1.1062 0.2495 111364 86467.7 9393.4 8798.5 2.8219 4.2705 14.3950

Genotype 49 33.8781** 48.5330** 6084.26** 4063.88** 20.3494** 30.8956** 139026** 98891.0** 11674.3** 11674.3** 12.3979** 21.4768** 98.4144**

Error 98 2.6001 0.9142 353.98 107.65 0.7452 0.1525 9965 7727.0 898.4 893.9 2.5193 2.7423 8.5063

*= Significant (P<0.05)

**= Highly Significant (P<0.01)

N.S= Non-significant

43

44

Pb 11

Iqbal 00

Watan 92

9495LU

26

Kohistan

97Ass

11Fsd

08GA 02

Chakwal-

50

Pbw 222

Fareed 06

Bathoor 0

8Fsd

85

Kohenoor 83

Lasan

iSH

02

Anmol 91

Parwaz

94

Inqalab 91

Pict 62

AARI11

Chenab 00

9725

Millat 1

10.0

10.0

20.0

30.0

40.0

50.0

60.0

Non Stressed

Drought

Wheat GenotypesFig 4. Comparison of 50 wheat genotypes for No. of grains per spike under normal and drought conditions.

Num

ber

of G

rain

s per

Spi

ke

Pb 11

Iqbal 00

Watan 92

9495LU

26

Kohistan

97Ass

11Fsd

08GA 02

Chakwal-

50

Pbw 222

Fareed 06

Bathoor 0

8Fsd

85

Kohenoor 83

Lasan

iSH

02

Anmol 91

Parwaz

94

Inqalab 91

Pict 62

AARI11

Chenab 00

9725

Millat 1

10.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

400.0

450.0

500.0

Non Stressed

Drought

Wheat GenotypesFig 4.2 Comparison of 50 wheat genotypes for No. of productive tillers under normal and drought conditions.

Num

ber

of P

rodu

ctiv

e T

iller

s m-2

45

Pb 11

Iqbal 00

Watan 92

9495LU

26

Kohistan

97Ass

11Fsd

08GA 02

Chakwal-

50

Pbw 222

Fareed 06

Bathoor 0

8Fsd

85

Kohenoor 83

Lasan

iSH

02

Anmol 91

Parwaz

94

Inqalab 91

Pict 62

AARI11

Chenab 00

9725

Millat 1

10.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

Non Stressed

Drought

Wheat GenotypesFig 4.3 Comparison of 50 wheat genotypes for 1000-grains weight(g) under normal and drought conditions.

Tho

usan

d G

rain

Wei

ght (

g)

46

Pb 11

Iqbal 00

Watan 92

9495LU

26

Kohistan

97Ass

11Fsd

08GA 02

Chakwal-

50

Pbw 222

Fareed 06

Bathoor 0

8Fsd

85

Kohenoor 83

Lasan

iSH

02

Anmol 91

Parwaz

94

Inqalab 91

Pict 62

AARI11

Chenab 00

9725

Millat 1

10.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0

Non Stressed

Drought

Wheat GenotypesFig 4.4 Comparison of 50 wheat genotypes for Biological yield (gm-2) under normal and drought conditions.

Bio

logi

cal Y

ield

(gm

-2)

47

Pb 11

Iqbal 00

Watan 92

9495LU

26

Kohistan

97Ass

11Fsd

08GA 02

Chakwal-

50

Pbw 222

Fareed 06

Bathoor 0

8Fsd

85

Kohenoor 83

Lasan

iSH

02

Anmol 91

Parwaz

94

Inqalab 91

Pict 62

AARI11

Chenab 00

9725

Millat 1

10.0

50.0

100.0

150.0

200.0

250.0

300.0

350.0

400.0

450.0

500.0

Non Stressed

Drought

Wheat GenotypesFig 4.5 Comparison of 50 wheat genotypes for Grain yield (gm-2) under normal and drought conditions.

Gra

in Y

ield

(gm

-2)

48

Pb 11

Iqbal 00

Watan 92

9495LU

26

Kohistan

97Ass

11Fsd

08GA 02

Chakwal-

50

Pbw 222

Fareed 06

Bathoor 0

8Fsd

85

Kohenoor 83

Lasan

iSH

02

Anmol 91

Parwaz

94

Inqalab 91

Pict 62

AARI11

Chenab 00

9725

Millat 1

10.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

Non Stressed

Drought

Wheat GenotypesFig 4.6 Comparison of 50 wheat genotypes for Harvest index (%) under normal and drought conditions.

Harv

est i

ndex

(%)

49

4.1.4 Harvest index (%):Harvest Index is the competence of a genotype for translocation of assimilates in

economically imperative part of crop. It is the ratio of grain yield to biological yield or total

biomass. The analysed data of harvest index of wheat exhibited a significant impact of

terminal drought on biological yield and grain yield of different wheat varieties during the

screening of genotypes for assessing of the most effected genotypes by drought. Results

showed highly significant (P≤0.001) effects among different under studied wheat genotypes

for harvest index in drought and normal conditions (Table 4.2).The data concerning the grain

yield and biological yield during 2014-15 year revealed significant impact of drought stress

on plants for this index.

Under normal conditions, minimum value of harvest index (28.6) was observed by the

Parsab-08 and As-02 and maximum value (37.8) was found in LU-26, while in stress

conditions, minimum and maximum values were observed in Kohsar-95 (24.1) and MH-97

(36.20) respectively. Harvest index decreased under drought conditions since both biological

and grain yields decreased at different rates. Decrease in the value of harvest index possibly

because of impact on grain yield and higher enhance in biological yield comparatively in

grain yield (Table 4.3).

Described results are consistent with the results of Bayoumi et al. (2008) and

Dorostkar et al. (2015). Difference in results may be because of the difference in

circumstances in which experiments were carried out.

4.1.5 Biological yield (gm-2):Biological yield is the sum of grains and yield of straw. It is the role of crop’s genetic

characteristics, soil’s nutrient conditions and the ecological pattern within neighbouring of

the crop. During the screening of wheat genotypes for estimation of the most affected

genotypes by drought, the analysed data of biological yield of wheat depicted a significant

impact of terminal drought on the biological yield of different wheat genotypes. Results

showed highly significant (P≤0.001) effects among investigated genotypes for biological

yield in normal and drought stress conditions (Table 4.2). The data concerning the biological

yield during 2014-15 revealed significant impact of drought stress on plants for this growth

parameter. In normal water conditions, plants exhibited higher biological yield, while the

imposed drought stress significantly reduced the biological yield.

Results for biological yield in 2014–2015 represented that highest biological yield

related to the genotypes Millat-11 (1413.3 gm-2) and lowest related to Punjab-11 (657.0 gm-2)

50

in normal conditions. Under drought conditions, 9444 (1229.7 gm-2) and Punjab-2011 (591.3

gm-2) showed highest and lowest biological yield, respectively (Table 4.3).

Under both normal and stress conditions, Hashim-10, Punjab-11, Watan-92,

Faisalabad-85 and Shafaq-06 were the genotypes which showed low yield while the

genotypes MH-97, 9444, Pirsabak-04, Faisalabad-83 and Punjab-85 showed high biological

yield. Genotypes Pothohar-73, Kohsar-95 and Parwaz-94 showed high yield under normal

conditions but produced low grain yield in drought conditions, thus higher biological yield

loss was observed (Table 4.3).

The biological yield losses were presumably directed to ripening of photosynthetic

portions prematurely which hampered photosynthesis and resultantly less grain yield. Same

findings found by Pierivatolum et al. (2010) who considered the impact of soil water stress

on biological yield of 4 wheat genotypes and depicted that resistant genotypes showed greater

biological and grain yield.

Table 4.3: Influence of drought stress on the yield related traits of biological yield (gm-2) and Harvest Index %

GenotypesBiological yield (gm-2) Harvest index %

Normal Drought Normal DroughtPb-11 657.0 591.3 32.2 31.9Gomal-08 817.3 735.6 33.7 33.4Iqbal-00 850.3 765.3 32.8 28.2Pak 81 894.3 804.9 32.7 32.3Watan-92 689.3 620.4 32.3 30.0Saher-06 1177.0 1059.3 30.1 29.99495 1235.7 1112.1 31.6 27.8Pb-85 1280.7 1152.6 30.0 28.4LU-26 812.7 731.4 37.8 36.0Moomal-02 1220.0 1098.0 30.4 29.1Kohistan-97 1272.0 1144.8 32.0 31.5Fsd-83 1340.0 1206.0 32.1 32.0Ass-11 973.7 876.3 32.3 30.5Kohsar-95 1134.3 1020.9 31.3 24.1Fsd-08 1211.0 1089.9 29.0 25.6Uqab-00 1251.7 1063.9 32.1 31.5GA-02 919.0 781.2 30.5 30.4Glaxy-13 1034.3 879.2 29.3 29.2Chakwal-50 1168.7 993.4 35.2 35.0Bhakhar-02 1271.3 1080.6 30.8 29.4Pbw-222 1309.7 1113.2 32.4 31.9MH-97 1306.7 1110.7 36.3 36.2Fareed-06 1280.7 1088.6 35.4 35.3Shaheen-94 1272.7 1081.8 31.7 30.3Bathoor-08 1186.7 1008.7 30.5 30.0Pirsabak-04 1340.0 1139.0 34.8 34.7

51

GenotypesBiological yield (gm-2) Harvest index %

Normal Drought Normal DroughtFsd-85 700.7 595.6 34.3 32.8Shafaq-06 697.3 592.7 34.5 32.5Kohenoor-83 1373.3 1167.3 34.3 28.1Manthar-03 1230.0 1045.5 32.9 32.0Lasani-06 1223.3 1039.8 32.9 31.99610 1020.3 867.3 29.8 29.3SH-02 1273.0 1082.1 31.7 28.1Parsab-08 1216.7 1034.2 28.6 27.4Anmol-91 1208.7 1027.4 32.2 31.9As-02 1035.7 828.5 28.6 28.0Parwaz-94 1186.0 948.8 31.2 26.99444 1343.3 1229.7 35.0 34.3Inqalab-91 1324.3 1059.5 34.4 34.1Potohar-73 1289.7 1031.7 31.3 30.9Pict-62 1268.7 1014.9 32.7 32.8Wafaq-01 1020.3 816.3 31.1 30.5AARI-11 1281.0 1024.8 31.3 28.9NARC-08 1190.0 952.0 29.7 27.0Chenab-00 1386.7 1109.3 33.7 29.2Abadghar-93 1277.3 1021.9 31.3 28.09725 1393.3 1184.3 33.8 32.5SH-95 1026.3 872.4 29.9 29.4Millat-11 1413.3 1201.3 33.6 32.5Hashim-10 661.7 529.3 34.0 30.5

4.1.6 Grain yield (gm-2):Final grain yield is interplay of cumulative effects of different growth and yield

contributing components which are number of grain per spike, number of productive tillers,

and average grain weight. These yield contributing components may be affected by many of

abiotic, pathogenic diseases and different agronomic practices. The computed data of grain

yield of wheat exhibited a significant impact of terminal drought on this trait of different

wheat genotypes during the screening of genotypes for assessing and evaluating of the most

effected genotypes by drought. Results showed highly significant (P≤0.001) effects among

investigated wheat varieties for grain yield in normal and drought conditions (Table 4.2). The

data concerning the grain yield during 2014-15 year revealed significant impact of drought

stress on plants for this growth parameter. In normal water conditions, plants exhibited higher

grain yield, while the imposed drought stress significantly reduced this parameter.

A perusal of the Table 4.4 showed the results for grain yield during the 2014–2015

represented that the highest and lowest grain yield belonged to the genotypes Millat-11

52

(475.3 gm-2) and Punjab-11 (211.7gm-2) under normal conditions. Genotype 9725 also gave

high yield having no significance different from Millat-11. Under stress conditions, 9444

(419.4gm-2) and Hashim-10 (161.2gm-2) showed highest and lowest grain yield respectively

(Table 4.4). Genotypes Hashim-10 and Punjab-11 showed low yield and MH-97 and 9444

showed high yield in both drought and normal conditions in the 2014–2015. Genotypes

Kohsar-95 and Parwaz-94 showed high yield under normal conditions but produced low grain

yield in drought conditions.

Based on following grain yield mean comparisons (Table 4.4), genotypes were

categorized into 4 groups.

I. The genotypes viz. 9725, Millat-11, Inqalab-91, 9444, Lasani-06, Manthar-03,

Pirsabak-04, MH-97, Kohistan-97 and Faisalabad-83 expressed less grain yield losses

or in other words high yield in both drought and normal conditions.

II. The genotypes of Hashim-10 and Punjab-11, Watan-92, GA-02, Faisalabad-85,

Shafaq-06 and Aas-02 showed minimum grain yield as compared to first group in

both drought and normal conditions.

III. Chenab-00, Kohsar-95, Parwaz-94 and Kohenoor-83 genotypes of wheat expressed

high grain yield under normal conditions and low grain yield under drought

conditions. Genotypes of this group confirmed high grain yield loss due to drought

stress. Similarly, it was also observed that genotypes belong to this groups are most

sensitive to drought.

IV. Likewise, the rest of the genotypes are included in the fourth group.

Differences in grain yield of genotypes pointed out differential tolerance and

sensitivity to drought which can be detailed by grain yield loss as an index. Chenab-00,

Kohsar-95, Parwaz-94 and Kohenoor-83showed highest grain yield loss (31.3%, 30.4 %,

30.6% and 30.7% yield losses) that means these were highly sensitive to drought, while the

lowest loss (less sensitive or resistant) belonged to 9444, Faisalabad-83 (9.9% and 10.5%

yield losses), respectively (Table4.4).

These results are in harmony with the results of Bayoumi et al. (2008), Dorostkar et

al. (2015), Khakwani et al. (2012), Richards et al., 2002, Lopes & Reynolds, 2010). While

evaluating and screening of 36 bread wheat genotypes for drought tolerance and

susceptibility under normal and different stress regimes, the same type of four groups or

clusters of investigated genotypes were also reported by Dorostkar et al. (2015). Difference

in results may be because of the difference in circumstances in which experiments were

53

carried out. It is in agreement that grain yield of wheat is highly influenced by drought stress,

thus, decreasing its value by it.

4.1.7 Drought sensitivity indices:

The highest values of tolerance index (TOL) belonged to genotypes Chenab-00,

Kohinoor-08, Parwaz-94, Abadghar-93, Kohsar-95 and AARI-11 having the values of 143.8,

143.2, 115.4, 113.4, 107.8, 104.2 respectively and the highest values of stress susceptibility

index (SSI) belonged to genotypes Parwaz-94, Kohsar-95, Kohinoor-08 and Chenab-00

having the values of 1.7, 1.6, 1.6 and 1.6 respectively (Table 4.4). These genotypes had high

grain yield in normal conditions and low yield in drought conditions and thus were identified

as sensitive ones. Although these genotypes produced high yields, but are not suitable for

cultivating over wide areas on account of their high losses of grain yield. So the selection on

the basis of high values of TOL and SSI would outcome low yielding and sensitive genotypes

in drought conditions.

The lowest value of TOL belonged to genotypes Punjab-11 and Gomal-08 having

value of 23.1gm-2 and 30.0 gm-2. Dorostkar et al., 2015; Khakwani et al., 2011; Aghaei-

Sarbarze et al., 2009 and Rosielle and Hamblin (1981) recommended that low values of TOL

and SSI are related to low sensitivity to drought and selection solely based on these indices

leads to high yielding genotypes in drought conditions. As TOL index shows the difference

between the grain yields in normal and drought conditions, low value of grain yield in stress

conditions or high value of grain yield in normal conditions leads to an increase in TOL

value, and thus, genotypes with high TOL value have higher sensitivity to drought stress.

The MP (mean productivity) index reported about genotypes of low yielding in

drought conditions and high yielding in normal conditions. The genotypes are having the

values of SSI lower than one and produced high yields in both drought and normal conditions

indicating their genetic potential in drought condition. Guttieri et al., (2001) depicted that SSI

higher than one shows above-average susceptibility, whereas value of SSI less than one

shows below-average susceptibility to drought stress. Hence, for selecting genotypes in

drought conditions stress susceptibility index (SSI) is a better index along with mean

productivity (MP) and tolerance index (TOL).

54

Table 4.4: Influences of drought stress on the yield related traits of GY, YR%, TOL, MP and SSI

GenotypesGrain Yield (gm-2) Yield

Reduction %TOL(gm-2) MP SSINormal Drought

Pb-11 211.7 188.6 11.0 23.1 200.1 0.6Gomal-08 275.7 245.7 10.9 30.0 260.7 0.6Iqbal-00 278.0 216.7 22.5 61.3 247.3 1.2Pak 81 292.3 260.0 11.1 32.3 276.2 0.6Watan-92 223.0 186.0 16.6 37.0 204.5 0.9Saher-06 355.0 318.0 10.7 37.0 336.5 0.69495 390.3 309.3 21.0 81.0 349.8 1.1Pb-85 385.3 328.2 14.8 57.1 356.8 0.8LU-26 306.6 262.6 14.4 44.0 284.6 0.8Moomal-02 372.3 321.8 13.8 50.5 347.1 0.7Kohistan-97 408.2 362.0 11.4 46.2 385.1 0.6Fsd-83 425.5 385.4 9.9 40.1 405.5 0.5As-11 314.1 267.1 15.0 47.0 290.6 0.8Kohsar-95 354.6 246.8 30.7 107.8 300.7 1.6Fsd-08 346.6 276.3 20.4 70.3 311.5 1.1Uqab-00 402.5 335.1 16.7 67.3 368.8 0.9GA-02 280.7 237.4 15.4 43.2 259.1 0.8Glaxy-13 302.7 256.2 15.4 46.4 279.5 0.8Chakwal-50 406.0 344.0 15.3 62.0 375.0 0.8Bhakhar-02 391.7 317.9 18.9 73.8 354.8 1.0Pbw-222 425.1 355.2 16.2 69.9 390.2 0.9MH-97 468.9 398.1 15.2 70.8 433.5 0.8Fareed-06 453.7 383.9 15.4 69.8 418.8 0.8Shaheen-94 403.3 328.1 18.7 75.3 365.7 1.0Bathoor-08 363.7 302.7 16.5 60.9 333.2 0.9Pirsabak-04 465.2 395.6 15.1 69.6 430.4 0.8Fsd-85 240.7 195.1 18.9 45.6 217.9 1.0Shafaq-06 240.7 192.8 19.9 47.8 216.8 1.1Kohinoor-83 471.2 328.1 30.4 143.2 399.7 1.6Manthar-03 403.2 334.7 17.2 68.4 369.0 0.9Lasani-06 401.8 332.8 17.5 69.0 367.3 0.99610 304.0 254.7 16.3 49.3 279.3 0.9SH-02 403.3 304.2 24.6 99.1 353.8 1.3Parsab-08 348.0 283.4 18.6 64.6 315.7 1.0Anmol-91 387.7 328.2 15.4 59.5 358.0 0.8As-02 296.0 231.3 21.9 64.7 263.7 1.2Parwaz-94 371.2 255.9 31.3 115.4 313.6 1.79444 468.1 419.4 10.5 48.7 443.7 0.6Inquilab-91 454.1 360.4 20.6 93.6 407.3 1.1Potohar-73 402.7 318.1 21.0 84.6 360.4 1.1Pict-62 414.7 332.9 19.7 81.8 373.8 1.1Wafaq-01 317.0 249.0 21.4 68.0 283.0 1.1AARI-11 400.7 296.5 26.0 104.2 348.6 1.4NARC-08 354.3 257.6 27.4 96.8 306.0 1.5

55

GenotypesGrain Yield (gm-2) Yield

Reduction %TOL(gm-2) MP SSINormal Drought

Chenab-00 467.9 324.1 30.6 143.8 396.0 1.6Abadghar-93 399.3 285.9 28.4 113.4 342.6 1.59725 471.2 384.3 18.3 86.9 427.8 1.0SH-95 307.3 256.7 16.5 50.6 282.0 0.9Millat-11 475.3 390.3 17.8 85.0 432.8 1.0Hashim-10 224.7 161.2 28.2 63.5 192.9 1.5

56

4.2: Response of different wheat genotypes against Leaf rust4.2.1 Disease Severity (%):

Disease damage or lesions covered on host tissues or organ described in percentage is

called disease severity. Severity results from the number and size of the lesions. Data analysis

and mean comparison indicated that 50 wheat genotypes were significantly different based on

the final disease severity (FDS) parameter (Table 4.5). Results showed highly significant

(P≤0.001) effects among investigated wheat genotypes for disease severity under field

conditions. In screening experiment, the results of the response of different commercial wheat

genotypes to leaf rust under field conditions are shown in Table 4.6. In 2014-15 growing

season, all the investigated wheat genotypes exhibited different disease severity ranged from

30-80%. The genotypes of Punjab-11, Faisalabad-85 and Aas-02 showed the highest final

rust severity of 80% followed by Sehar-06 and Wafaq-01which had the value of 70% rust

severity. While the minimum final disease severity value was 30 % showed by Manthar-03,

Parwaz-94, Bathoor-08 and some other cultivars, thus, were more resistant to the pathogen of

the leaf rust puccinia recondita.

Current results are in a harmony with results reported by Taye et al. (2015); Hasan et

al. (2012); Macharia and Wanyera, (2012); El-Shamy et al. (2011); Salman et al. (2006);

Singh et al(2004); Sayre et al. (1998). A study by Hasan et al. (2012) was aimed to estimate

of losses of grain yield because of brown rust disease on the five local commercial

susceptible wheat cultivars in field conditions during 2011-2012 growing seasons at

Gemmeiza Agriculture Research Station, Egypt. The investigated cultivars exhibited 5-80%

disease severity. And was depicted a relationship between rust severity and yield components

which resultantly converted to financial losses. Their results were also endorsed by El-Shamy

et al. (2011) who observed a significant link among percentage losses of 1000 grain weight

and grain yield and final rust severity. This trend is also in synchronization with earlier

results by Shaner et al. (1978) against brown rust disease of wheat.

Singh et al. (2004) reported that highly significant negative correlation of rust severity

and AUDPC with grain weight and yield while evaluating 5,107 advanced lines in field trials

at Mexico in 2001, only 2.4% lines were highly resistant, 28% were moderately resistant (up

to 30% rust severity) and the rest of the lines showed 40 to 100% rust severity. Macharia and

Wanyera, (2012) investigated fifteen wheat cultivars including six advanced lines from

CIMMTY germplasm and others were commercially grown cultivars in Kenya and depicted

that disease severity and area under disease progress curve were highly positively correlated

with yield losses thus, were negatively correlated with grain yield and yield contributing

57

components. These results are positively correlated and agreed with the outcomes of Taye et

al. (2015); Safavi et al. (2012); Shah et al. (2010); Ali et al. (2007); Sandoval-Islas et al.

(2007); Allen et al. (1963).

Based on the disease parameters of FDS and AUDPC, Macharia and Wanyera, (2012)

reported infection responses of the wheat cultivars and classified them into four discrete

categories: a) S-susceptible) MS-moderately susceptible c) MR-moderately resistant d) R-

resistant which was previously reported by Roelfs (1992).

Table 4.5: Impacts of wheat leaf rust on FDS, AUDPC and CI under field conditionsGenotypes FDS AUDPC CI IRPb-11 80 1600 76 SGomal-08 30 300 15 RIqbal-00 40 625 24 MRPak 81 30 475 19 RWatan-92 30 450 19 RSaher-06 70 1300 64 S9495 50 615 38 MSPb-85 40 525 24 MRLU-26 30 465 19 RMoomal-02 50 875 37 MSKohistan-97 30 300 15 RFsd-83 60 725 50 SAss-11 30 475 15 RKohsar-95 30 450 19 RFsd-08 60 950 50 SUqab-00 30 450 15 RGA-02 50 800 37 MSGlaxy-13 40 565 24 MRChakwal-50 40 525 28 MRBhakhar-02 30 465 19 RPbw-222 50 875 37 MSMH-97 30 475 19 RFareed-06 30 450 15 RShaheen-94 40 750 29 MRBathoor-08 30 450 17 RPirsabak-04 40 750 29 MRFsd-85 80 1350 78 SShafaq-06 30 300 12 RKohenoor-83 40 625 24 MRManthar-03 30 475 15 RLasani-06 30 450 19 R9610 40 750 25 MRSH-02 50 615 37 MSParsab-08 60 825 51 SAnmol-91 30 465 15 RAs-02 80 1550 78 S

58

Parwaz-94 30 475 17 R9444 30 450 15 RInqalab-91 50 800 38 MSPotohar-73 40 750 27 MRPict-62 50 615 39 MSWafaq-01 70 875 63 SAARI-11 30 465 17 RNARC-08 50 875 37 MSChenab-00 30 475 15 RAbadghar-93 30 450 17 R9725 40 750 25 MRSH-95 30 465 15 RMillat-11 50 875 39 MSHashim-10 40 750 24 MR

4.2.2 Area under disease progress curve (AUDPC):

Data analysis and mean comparison indicated that leaf rust infection was well

established apparently in all the tested wheat cultivars screened for the disease. Results

showed highly significant (P≤0.001) effects among investigated wheat genotypes for area

under disease progress curve under field conditions (Table 4.6). In screening experiment, the

results of the values of area under disease progress curve of different wheat varieties to leaf

rust are shown in Table 4.5. The genotypes Punjab-11, As-02, Faisalabad-85 and Sehar-06

were ranked as susceptible to area under disease progress curve (AUDPC) with the values of

1600, 1550, 1350 and 1300 respectively, whereas Shafaq-06, Kohistan-97 and Gomal-08

were ranked as resistant having the minimum value 300 of area under disease progress curve.

The rest of the cultivars had the range of value from 450 to 850 were ranked as moderately

resistant to moderately susceptible.

Current results are in a harmony with observations reported by Taye et al. (2015);

Hasan et al. (2012); Macharia and Wanyera, (2012); El-Shamy et al. (2011); Salman et al.

(2006); Singh et al (2004); Sayre et al. (1998). Results of current study showed that AUDPC

run in a parallel manner to rust severity. It is obvious from the previous findings by Taye et

al. (2015); Safavi et al. (2012); Shah et al. (2010); Sandoval-Islas et al. (2007); Ali et al.

(2007) that a very strong positive relation found between AUDPC and final rust severity that

means severely infected cultivar showed higher AUDPC values. And the second thing was

disease progress rates and AUDPC values were positively correlated and highly significant.

Taye et al. (2015) and Salman et al. (2006) reported that area under disease progress

curve (AUDPC) and the final disease severity (FDS) were negatively correlated with final

grain yield as well as yield contributing components, and very positively correlated with

59

grain yield loss. Thus, it demonstrated a negative relation when there is an increase in these

disease parameters there will be a decline in yield related parameters and vice versa.

Macharia and Wanyera, (2012) investigated fifteen wheat cultivars including six

advanced lines from CIMMYT germplasm and others were commercially grown cultivars in

Kenya and depicted that disease severity and area under disease progress curve were highly

positively correlated with yield losses thus, were negatively correlated with grain yield and

yield contributing components.

Based on the FDS and AUDPC, Macharia and Wanyera, (2012) reported infection

responses of the wheat cultivars and classified them into four discrete categories: I) S-

susceptible; II) MS-moderately susceptible; III) MR-moderately resistant and IV) R-resistant

which was previously reported by Roelfs (1992).

Hasan et al. (2012) assessed grain yield losses due to brown rust disease on the five

local commercial susceptible wheat cultivars under field conditions during 2011-2012

growing seasons and was depicted a relationship between rust severity and yield components

which resultantly converted to financial losses. These results endorsed by El-Shamy et al.

(2011) who found a significant relation among percentage losses of 1000 grain weight, grain

yield and final rust severity. This trend is also in synchronization with earlier results by

Shaner et al. (1978) against brown rust disease of wheat.

Ochoa and Parlevliet (2007) described that yield losses were strongly correlated with

area under disease progress curve. Singh et al. (2004) reported highly significant (P < 0.01)

negative correlation of rust severity and AUDPC with grain weight and yield while

evaluating 5,107 advanced breeding lines in field trials at Mexico in 2001, only 2.4% lines

were highly resistant, 28% were moderately resistant and the rest of the lines were

moderately susceptible to susceptible.

60

Table 4.6: Analysis of variance for FDS, AUDPC and CI under field conditionsMean squares

Source of variation Df Final rust severity

Area under disease progress

curve

Coefficient of Infection

Replications 2 6794.00 2421640 9168.75

Genotypes 49 652.53** 256313** 930.93**

Error 98 149.10 16743 211.26

*= Significant (P<0.05) **= Highly Significant (P<0.01) NS= Non-significant

4.2.3 Coefficient of infection (CI):

Data analysis and mean comparison indicated that wheat cultivars were considerably

different based on the coefficient of infection values (Table 4.5). Results showed highly

significant (P≤0.001) effects among investigated wheat genotypes for coefficient of infection

under field conditions (Table 4.6).The results of the response of those commercial wheat

genotypes to leaf rust under field conditions during screening experiment are shown in Table

4.5. In 2014-15 growing season, all investigated wheat genotypes showed the significant

values of coefficient of infection. The values 10-20, 21-30, 31-45 and 46-80 were categorized

as high, moderate, low and very low levels of resistant respectively.

Coefficient of infection has been frequently used parameter for the purpose of partial

resistance. Positive correlation exists between coefficient of infection and area under disease

progress curve (AUDPC) and final disease severity (FDS) was found in present study. During

this study, an effort was also done to elucidate the association between described parameters.

Among the different cultivars in screening experiment, Aas-02 and Faisalabad-85 and

Punjab-11 showed highest values of coefficient of infection 77.6, 77.6 and 76.0 followed by

Sehar-06 and Wafaq-01 having values of 63.6 and 63.0 respectively, thus, these were ranked

as susceptible. However, Shafaq-06 showed the minimum value of coefficient of infection

12.0 and followed by Manthar-03, Anmol-91, 9444, Gomal-08, Khistan-97, Fareed-06,

Uqab-00 and SH-95having values of 14.6, so were ranked as resistant to leaf rust pathogen

(Table 4.5).

The described results are in harmony with outcomes of Ali et al. (2008); Pathan and

Park, (2006) and Shah et al. (2014). Ali et al. (2008) investigated 20 wheat genotypes and

61

‘Morocco’ as susceptible check at NIFA for describing variability for field based partial

resistance to rust and lines with coefficient of infection values of 0-20, 21-40 and 41-60 were

considered as possessing better, moderate and low levels of partial resistance, respectively.

Only Morocco (a susceptible check) was possessing 100 CI value. Earlier, Pathan and Park

(2006) assessed a kind of partial resistance called adult plant resistance (APR) to brown rust

in European lines and reported various levels of APR among the investigated wheat lines by

means of average coefficient of infection (ACI) by estimating average of CI across different

locations.

62

4.3: Response of different wheat genotypes against yellow rust4.3.1 Disease severity (%):

As described earlier Disease damage or lesions covered on host tissues or organ

described in percentage is called disease severity. Severity results from the number and size

of the lesions. Mean comparison indicated that different wheat genotypes were considerably

different based on the final disease severity parameter (Table 4.7). Results showed highly

significant (P≤0.001) effects among studied wheat genotypes for disease severity under field

conditions (Table 4.8). In screening experiment, the results of the response of 50 commercial

wheat genotypes to yellow rust under field conditions are shown in (Table 4.7).

In 2014-15, all investigated wheat genotypes exhibited different disease severity

ranged from 20-80%. The cultivars of Fareed-06, MH-97, Shafaq-06, Inqalab-91 and Aas-02

showed the highest 80% final rust severity followed by Punjab-11, Watan-92, Kohistan-97,

Shaheen-94 and SH-02 showed 70% while Moomal-02, Faisalabad-83, Parsab-08 and

Hashim-10 had 60% rust severity, so these were ranked as susceptible cultivars. Genotypes

Punjab-85, LU-26, Kohenoor-83, 9610, Millat-11, Pict-62 and 9610, were Categorized as

moderately susceptible to yellow rust under field conditions with the value of 50% rust

severity. Whereas the minimum final disease severity value was 20 % showed by Bhakhar-

02, Fsd-08, thus may be more resistant to the pathogen of the yellow rust Puccinia

striiformis.

Results are in a harmony with earlier outcomes reported by Safavi (2015); Gupta et

al. (2013); Shah et al. (2014); Ali et al. (2009); Qamar et al. (2014); Tabassum et al. (2010);

Afzal et al. (2010); Kazi et al. (2012); Afzal et al. (2007) and Qamar et al. (2012).

Shah et al. 2014 studied 50 commercially grown wheat genotypes of Pakistan to

identify rust resistance against stripe rust caused by Puccinia striiformis by doing the

seedling tests at Thiverval-Grignon, INRA, France under glasshouse conditions whereas field

trials using epidemiological variables at Peshawar, Pakistan during 2005-07. The investigated

cultivars exhibited 0-87% disease severity. Twenty nine cultivars were susceptible, 11 were

resistant whereas, 10 were moderately resistant to moderately susceptible at seedling stage.

Ali et al. (2009) assessed partial resistance level of 37 wheat genotypes introduced

from Oklahoma State University, with local check at Hazara Agricultural Research Station

(HARS), Abbottabad. 6 genotypes were showed more than 70% final disease severity with a

maximum value of 95% while remaining genotypes showed less than 70% along with local

check. In the same way, based on rust severity, the studied genotypes were categorized into

three groups of partial resistance. These showed disease severity range of 1-30%, 31-50%

63

and 50-70% was ranked as high, moderate and low levels of resistant respectively. Earlier,

Ali et al. (2007) also evaluated wheat cultivars from Pakistan to estimate their partial

resistance in terms of slow rusting behaviour. Similarly, Broers et al. (1996) also conducted

quantitative resistance assessment in field for stripe rust for ranking of wheat genotypes. In

keeping with the resistance level based on final rust severity together with other disease

parameters, they depicted that resistance levels ranged from very low to very high among the

studied wheat genotypes.

Afzal et al. (2010) reported a survey of field based screening of fifty seven Pakistani

commercial genotypes to assess their potential against yellow rust was carried out during the

years of 2005-06 and 2006-07. The commercial varieties including GA-2002 and Iqbal-2000

ranked the resistant and MH-97, Inquilab-91, Faisalabad-85, SH-02, Abadgar-93, Watan-92

and Moomal-02 displayed susceptible reactions as the findings of present experiment

described. The results are second by Qamar et al. (2012) who performed an experiment to

endow with information on the level of resistance in 113 wheat genotypes against stripe rust

from 2007 to 2011 at NARC. The results are in agreement of Kazi et al. (2012) who

performed seedling tests to screen 95 wheat cultivars against prevalent races of yellow rust in

Pakistan in glasshouse conditions. These results are also endorsed by Tabassum et al. (2010)

and Qamar et al. (2014).

A Study was conducted to estimate wheat yield losses caused by Puccinia striiformis.

Outcomes revealed that a direct connection between disease level and the yield loss in

Pakistani commercially adopted wheat genotypes. The yield was negatively correlated with

the effected leaf area by yellow rust. A changing resistance level was showed among various

wheat genotypes. The widely cultivated cultivars Wafaq-2001and Inquilab-91 were observed

the most resistant with minimum yield loss. On the bases of those results Inquilab-91 and

Wafaq-2001 were recommended to sow for avoiding serious yield losses inflicted by the

yellow rust which is contradicted to my study where Inquilab-91 and Wafaq-2001 show low

level of resistance and thus ranked as susceptible varieties (Afzal et al. 2007).

A survey of the arbitrarily selected wheat fields situated in Jammu and Kashmir to

estimate the distribution and prevalence of wheat stripe rust was carried out repeatedly for 3

years of 2009 to 2012. The growing season of 2010-2011 recorded highest disease

prevalence, possibly as a result of favourable environmental conditions joined to monoculture

wheat varieties and outbreak of virulent races of Puccinia striiformis (Gupta et al., 2013).

64

In the study by Safavi (2015) the efficiency of different categories of resistance was

evaluated at Ardabil Agricultural Research Station (Iran) in field plots in 2011-2013. Yield

and yield contributing components in conjunction with disease parameters of area under

disease progress curve (AUDPC), final disease severity (FDS) and coefficient of infection

(CI) were evaluated for 16 wheat genotypes. Five wheat genotypes were observed race-

specific resistant, one susceptible and remaining 10 genotypes were found with different

levels of slow rusting resistance.

Table 4.7: Impacts of yellow rust on traits of FDS, AUDPC and CI in field conditionsGenotypes FDS AUDPC CI IRPb-11 70 1042 63 SGomal-08 40 450 25 MRIqbal-00 30 400 19 RPak 81 40 500 24 MRWatan-92 70 945 58 SSaher-06 30 400 19 R9495 40 465 27 MRPb-85 50 675 38 MSLU-26 50 765 37 MSMoomal-02 60 825 50 SKohistan-97 70 875 64 SFsd-83 60 825 51 SAs-11 30 475 19 RKohsar-95 40 542 25 MRFsd-08 20 200 8.0 RUqab-00 30 300 19 RGA-02 40 502 25 MRGlaxy-13 40 465 25 MRChakwal-50 40 625 25 MRBhakhar-02 20 265 8 RPbw-222 40 725 24 MRMH-97 80 1413 76 SFareed-06 80 1463 76 SShaheen-94 70 983 64 SBathoor-08 30 450 15 RPirsabak-04 40 750 24 MRFsd-85 30 450 19 RShafaq-06 80 1400 76 SKohenoor-83 50 900 40 MSManthar-03 30 450 19 RLasani-06 30 450 19 R9610 50 800 37 MSSH-02 70 875 64 SParsab-08 60 825 50 SAnmol-91 40 725 25 MRAs-02 80 1283 78 S

65

Parwaz-94 40 525 28 MR9444 30 465 15 RInqalab-91 80 1367 76 SPotohar-73 40 725 25 MRPict-62 50 615 37 MSWafaq-01 30 300 19 RAARI-11 40 515 28 MRNARC-08 40 515 28 MRChenab-00 80 1283 76 SAbadghar-93 30 300 15 R9725 40 725 25 MRSH-95 30 475 15 RMillat-11 50 775 38 MSHashim-10 60 925 51 S

4.3.2 Area under disease progress curve (AUDPC):

In screening experiment, the results of the values of area under disease progress curve

of 50 commercial wheat genotypes to yellow rust in field conditions are shown in Table 4.7.

Mean comparison indicated that yellow rust infection was well established apparently in all

the tested wheat cultivars screened for the disease. Results showed highly significant

(P≤0.001) effects among studied wheat genotypes for area under disease progress curve

under field conditions (Table 4.8). The cultivars Fareed-06, MH-97, Shafaq-06, Inqalab-91,

Chenab-00, Aas-02 and Punjab-11 were ranked as susceptible with area under disease

progress curve (AUDPC) of 1463, 1413, 1400, 1367, 1283,1283 and 1042 respectively,

whereas Fsd-08 and Bhakhar-02 had the minimum value of area under disease progress curve

of 200 and 265 followed by Abadghar-93, Wafaq-01 and Uqab-00 contained the value of

300, thus, were ranked as resistant. The rest of the cultivars had the range of value from 400

to 983 were ranked from moderately resistant to moderately susceptible.

The findings of present study showed that AUDPC run in a parallel manner to rust

severity. It is clear from the results by Taye et al. (2015); Safavi et al. (2012); Shah et al.

(2010); Ali et al. (2007); Sandoval-Islas et al. (2007); that severely infected genotypes

showed higher AUDPC values which means a positive correlation was observed between

AUDPC and FDS. Moreover, disease progress rates and AUDPC values were positively

correlated and highly significant.

Taye et al. (2015) and Salman et al. (2006) reported that area under disease progress

curve (AUDPC) and the final disease severity (FDS) were negatively correlated with final

grain yield as well as yield contributing components, and very positively correlated with

grain yield loss. Thus, it demonstrated a negative relation when there is an increase in these

66

disease parameters there will be a decline in yield related parameters and vice versa. These

results are agree with Hailu and Fininsa (2009) and other researchers (Safavi et al., 2012;

Ahmad et al., 2010; Herrera-Fossel et al., 2006). They also concluded that resistant cultivars

of wheat had low reduction in yield components against stripe rust. Fifteen wheat cultivars

including 6 advanced lines from CIMMYT germplasm and others were commercially grown

cultivars in Kenya were reported by Macharia and Wanyera, (2012) and revealed that disease

severity and area under disease progress curve were highly positively correlated with yield

losses, so negatively correlated with grain yield and yield contributing components.

Ali et al. (2009) assessed partial resistance level of 37 wheat genotypes introduced

from Oklahoma State University, with local check at Hazara Agricultural Research Station

(HARS), Abbottabad. Seventeen genotypes showed higher AUDPC values while remaining

21 genotypes showed different range of AUDPC values. Based on their AUDPC values, the

studied genotypes were categorized into three distinct groups for partial resistance.

Genotypes exhibiting AUDPC values up to 500 were considered as high level of resistant,

while those exhibited AUDPC values from 500 to 800 were considered as moderately

resistant genotypes. Similarly, genotypes having AUDPC ranging from 800 to 1100 were

grouped as low level of partial resistant.

Shah et al. (2014) investigated 50 widely grown wheat genotypes of Pakistan during

2005-07 to assess resistance against stripe rust caused by Puccinia striiformis by doing the

seedling tests at INRA, France under glasshouse conditions while field trials using

epidemiological variables at Peshawar, Pakistan. The investigated cultivars depicted different

range of AUDPC. Based on AUDPC and disease severity 11 genotypes were resistant, 29

were susceptible, whereas, 10 were moderately resistant to moderately susceptible. Shaheen-

94, Inqilab-91, Watan-92 and Kohistan-97 were included in susceptible verities which are

also in the susceptible category in my study.

Gupta et al. (2013) survey prevalence and distribution of wheat stripe rust by the

randomly selected wheat fields situated in Jammu and Kashmir for consecutively three years

of 2009 to 2012. The growing season of 2010-2011 recorded highest disease prevalence,

possibly as a result of favourable environmental conditions joined to monoculture wheat

varieties and outbreak of virulent races of Puccinia striiformis. The results depicted that all

the studied wheat growing areas were found infested by the disease with maximum 2422 and

minimum AUDPC values 351, respectively.

Yield and yield contributing components in combination with area under disease

progress curve (AUDPC) and some other disease parameters were evaluated for 16 wheat

67

genotypes at Ardabil Agricultural Research Station (Iran) in 2011-2013 to study the

efficiency of different categories of resistance by Safavi (2015) Five wheat genotypes were

observed race-specific resistant, one susceptible and remaining 10 genotypes were found with

different levels of slow rusting resistance.

Table 4.8: Analysis of variance for FDS, AUDPC and CI under field conditionsMeans Squares

Source of variation

Df Final disease severity

Area under disease progress

curve

Coefficient of infection

Replications 2 7736.00 1007775 10874.1

Genotypes 49 676.53** 265715** 939.9**

Error 98 58.45 3681 92.0

Total 149

**= Highly Significant (P<0.01) *= Significant (P<0.05) N.S= Non-significant

4.3.3 Coefficient of infection (CI):

Mean comparison indicated that different wheat cultivars were significantly different

based on the Coefficient of infection value (Table 4.7). Results showed highly significant

(P≤0.001) effects among studied wheat genotypes for Coefficient of infection under field

conditions (Table 4.8). In 2014-15, all of the tested wheat genotypes exhibited the significant

values of Coefficient of infection. 10-20, 21-30, 31-45 and 46-80 were categorized as having

high, moderate, low and very low levels of resistance respectively.

Among the different cultivars under screening, Aas-02 showed highest values of

Coefficient of infection 77.7 followed by Fareed-06, MH-97, Shafaq-06, Inqalab-91, Chenab-

00 having value of 76.0, thus, these were ranked as susceptible. However, Bhakhar-02 and

Faisalabad-08 showed the minimum value of Coefficient of infection 8.0 and it statistically at

par with Bathoor-08, Abagghar-93,9444 and SH-95 contained the value of 14.7, followed by

Wafaq-01, Manthar-03, Lasani-06, Uqab-00, Iqbal-00 and Aas-11 Faisalabad-85 and Sehar-

06 having values of 18.7, so were all ranked as resistant to leaf rust pathogen (Table 4.7).

A Positive correlation of coefficient of infection between final disease severity (FDS)

and area under disease progress curve (AUDPC) was found in present study. Same results

observed by Ali et al. (2008); Pathan and Park, (2006) and Shah et al. (2014). Strong

68

association reported among coefficient of infection (CI), AURPC and FDS and by Ali et al.

(2008) when they studied 20 wheat genotypes for describing variability for field based partial

resistance to stripe rust at NIFA. Genotypes with coefficient of infection values of 0-20, 21-

40 and 41-60 were considered as possessing better, moderate and low levels of partial

resistance, respectively.

Those results are endorsed by another study, when Ali et al (2009) assessed partial

resistance level of 37 wheat genotypes introduced from Oklahoma State University, with

local check at Hazara Agricultural Research Station (HARS), Abbottabad. Based on the

coefficient of infection, the tested genotypes were grouped into high (27 genotypes),

moderate (9 genotypes) and low levels of resistance (one genotype).

Pathan and Park (2006) assessed a kind of partial resistance called adult plant

resistance (APR) to rust in European lines and reported various levels of APR among the

investigated wheat lines by means of average coefficient of infection (ACI) by estimating

average of CI across different locations.

To assess resistance against stripe rust, 50 extensively grown wheat genotypes of

Pakistan were taken under study by Shah et al. (2014) under glasshouse conditions. They

performed seedling tests at INRA, France whereas field trials using epidemiological variables

at Peshawar, Pakistan. The genotypes under study showed different range of coefficient of

infection (CI) from 0 to 53. Based on CI, area under disease progress curve and disease

severity 11 genotypes were resistant, 29 were susceptible, whereas, 10 were moderately

resistant to moderately susceptible.

Based on Coefficient of infection and some other disease parameters along with yield

and yield contributing components, 16 wheat genotypes were evaluated to study the

efficiency of different categories of resistance at Ardabil Agricultural Research Station (Iran)

by Safavi et al. (2015). Five wheat genotypes were observed resistant, one susceptible and

remaining 10 genotypes were found with different levels of resistance.

69

4.4: Symbiotic effect of fungal endophytes on two drought sensitive wheat genotypes in drought conditions4.4.1 1000-grain weight (g):

Enhanced grain weight is the valid option for yield maximization followed by

enhanced number of grains. Thousand grain weights is one of the imperative yield

contributing attribute in getting greater yield and contains a prominent character in restrictive

yield potential of a cultivar. 1000-grain weight is directly depended on seed size. Greater the

size of grain, higher will be the grain weight (TGW). Results revealed that fungal endophytes

and genotypes significantly affected grain weight. Similarly, interaction of endophytes and

genotypes also affected grain weight under the drought conditions imposed on the wheat

genotypes at the flowering and grain filling stage during 2015-16.

Among the four fungal endophytes, maximum 1000-grain weight (39.2 g) was

observed in the case Piriformospora indica and it was statistically at par with Colletotrichum

lindemuthianum (38.4 g) followed by Acremonium lolii and Trichoderma viride (36.4 g) and

(35.9 g) respectively. While minimum grain weight (34.0 g) was observed in control

condition where no endophyte was applied to the drought sensitive genotypes in drought

condition while all other agronomic requirements were provided equally. Among the

genotypes, maximum grain weight (37.3 g) was observed by the Parwaz-94 (Table 4.9).

In case of interaction, endophyte Piriformospora indica interacted comparatively

better for grain weight (39.6 g) with drought sensitive genotype Parwaz-94 and it was found

similar symbiotic interaction of Piriformospora indica with genotype Kohsar-95 having

grain weight (38.8 g). Colletotrichum lindemuthianum also showed better performance to

combat the drought conditions while interacting with the sensitive wheat genotypes (38.6 g)

and (38.1 g) (Table 4.9).

The rest of the endophytes Acremonium lolii and Trichoderma viride were also

showed moderate performance viz (37.4 g), (35.5 g), (36.7 g) and (35.1 g) respectively, under

drought condition. Synergetic and symbiotic effect of endophytes significantly contribute for

stabilizing wheat plant under drought conditions through enhancing 1000- grain weight, the

prime attribute in harvesting greater yield. The performance of drought sensitive genotypes

viz Parwaz-94 and Kohsar-95 was at par in terms of fungal endophytes.

70

Table 4.9: Symbiotic effects of endophytes for 1000-grain weight in tolerance of drought stress

Fungal endophytes Wheat genotypes Mean(g)Parwaz-94

(g)Kohsar-95

(g)Control 34.127 33.920 34.023CColletotrichum lindemuthianum

38.660 38.187 38.423A

Acremonium lolii 37.427 35.527 36.477BTrichoderma viride 36.740 35.160 35.950BPiriformospora indica

39.553 38.847 39.200A

Mean 37.301A 36.328BLSD at 5% E=0.9255,V=0.5853 E*V=1.3088

4.4.2 Number of productive tillers:Increased number of productive tillers is the valid option for yield maximization

followed by enhanced number of grains and grain weight. Number of productive tillers is a

very important yield contributing parameter for achieving higher yield. Greater number of

productive tillers validate higher final yield. Results revealed that fungal endophytes and

genotypes significantly affected the number of productive tillers per unit area; likewise,

interaction of endophytes and genotypes also affected the number of productive tillers in the

drought conditions imposed on the drought sensitive wheat genotypes at the flowering and

grain filling stage.

Among the four endophytes, maximum number of productive tillers (361.3) was

observed in case of Piriformospora indica and it was statistically at par with Colletotrichum

lindemuthianum (354.6) followed by Trichoderma viride and Acremonium lolii (345.3) and

(343.3) respectively. While minimum number of productive tillers (336.5) were observed in

control condition where no endophyte was applied to the drought sensitive genotypes in

drought condition while all other agronomic requirements were provided equally. Among the

genotypes, maximum productive tillers (355.3) were observed by the Kohsar-95 (Table 4.10).

In the case of interaction, endophyte Piriformospora indica showed interacted

comparatively better for productive tillers (362.6) with drought sensitive genotype Kohsar-

95and it was found a similar symbiotic interaction of Piriformospora indica with genotype

Parwaz-94 having productive tillers (360.0), respectively (Table 4.10). Colletotrichum

lindemuthianum also showed better performance to combat the drought conditions while

interacting with the sensitive wheat genotypes (350.0) and (359.3) (Table 4.10).

71

The rest of the endophytes Trichoderma viride and Acremonium lolii also showed

moderate performance viz (354.6), (336.0), (334.0) and (352.6) respectively, under drought

conditions. Symbiotic effect of endophytes significantly contribute for stabilizing wheat plant

under drought conditions through increasing the number of productive tillers, the prime

attribute in harvesting greater yield. The performance of drought sensitive genotypes viz

Parwaz-94 and Kohsar-95 was at par in terms of fungal endophytes.

Table 4.10: Symbiotic effects of endophytes for Number of Productive Tillers in tolerance of drought stress

Fungal endophytes Wheat genotypes MeanParwaz-94 Kohsar-95

Control 325.67 347.33 336.50BColletotrichum lindemuthianum

350.00 359.33 354.67A

Acremonium lolii 334.00 352.67 343.33BTrichoderma viride 336.00 354.67 345.33BPiriformospora indica

360.00 362.67 361.33A

Mean 341.13B 355.33ALSD at 5% E=8.9296,V=5.6476 E*V=12.628

4.4.3 Biological Yield (gm-2):Biological yield is the sum of grains and yield of straw. It is the role of crop’s genetic

characteristics, soil’s nutrient conditions and the ecological pattern within neighbouring of

the crop. Results revealed that fungal endophytes and genotypes significantly affected the

biological yield; however, interaction of endophytes and genotypes also affected the

biological yield in the drought conditions imposed on the drought sensitive wheat genotypes

at the flowering and grain filling stage during the growth season of the crop.

Among the four fungal endophytes, maximum biological yield (1125.0 gm-2) was

observed in case of Piriformospora indica and it was statistically at par with Colletotrichum

lindemuthianum (1072.3 gm-2) followed by Trichoderma viride and Acremonium lolii (1068.0

gm-2) and (1060.3 gm-2) respectively. While minimum biological yield (1012.1 gm-2) was

observed in control where no endophyte was applied to the drought sensitive genotypes in

drought condition. Among the genotypes, maximum biological yield (1099.5 gm-2) was

observed by the Parwaz-94 (Table 4.11).

In the case of interaction, endophyte Piriformospora indica interacted comparatively

better for biological yield (1148.0 gm-2) with drought sensitive genotype Parwaz-94 and it

was found a similar symbiotic interaction of Piriformospora indica with genotype Kohsar-95

having biological yield (1102.0 gm-2), respectively (Table 4.11). Colletotrichum

72

lindemuthianum also showed better performance to combat the drought conditions while

interacting with the sensitive wheat genotypes (1109.3 gm-2) and (1035.3 gm-2) [Table 4.11].

The rest of the endophytes Trichoderma viride and Acremonium lolii were also

showed moderate performance in drought conditions viz (1108.7gm-2), (1027.3gm-2),

(1089.3gm-2) and (1031.3gm-2) respectively, under drought condition. Symbiotic effect of

endophytes significantly contributed for stabilizing wheat plant under drought conditions

through increasing total aboveground mass or biological yield. The performance of drought

sensitive genotypes viz Parwaz-94 and Kohsar-95 was at par in terms of fungal endophytes.

Table 4.11: Symbiotic effects of endophytes for biological yield in tolerance of drought stress

Fungal endophytes Wheat genotypes Mean(gm-2)Parwaz-94

(gm-2)Kohsar-95

(gm-2)Control 1042.1 982.0 1012.1CColletotrichum lindemuthianum

1109.3 1035.3 1072.3B

Acremonium lolii 1089.3 1031.3 1060.3BTrichoderma viride 1108.7 1027.3 1068.0BPiriformospora indica

1148.0 1102.0 1125.0A

Mean 1099.5A 1035.6BLSD at 5% E=39.506,V=24.986 E*V=55.870

4.4.4 Grain yield (gm-2):Results revealed that fungal endophytes and genotypes affected significantly the grain

yield; likewise, interaction of endophytes and genotypes also affected the grain yield in the

drought conditions imposed on the wheat genotypes during the 2015-16 growth season of the

crop. Among the four fungal endophytes, maximum grain yield (315.9 gm-2) was observed in

case of Piriformospora indica and it was statistically at par with Colletotrichum

lindemuthianum (301.3 gm-2) followed by the endophytes Trichoderma viride and

Acremonium lolii (290.6 gm-2) and (288.7 gm-2) respectively. While minimum grain yield

(267.3 gm-2) was observed in control condition where no endophyte was applied to the

drought sensitive genotypes in drought condition. Among the genotypes, maximum grain

yield (293.1gm-2) was observed by the Parwaz-94 (Table 4.12).

Among the interaction, endophyte Piriformospora indica interacted comparatively

better for grain yield (316.6 gm-2) with drought sensitive genotype Kohsar 95 and it was

found a similar symbiotic interaction of Piriformospora indica with genotypeParwaz-94

73

having grain yield (315.2 gm-2), respectively (Table 4.12). Colletotrichum lindemuthianum

also showed better performance to combat the drought conditions while interacting with the

sensitive wheat genotypes (300.4 gm-2) and (302.2 gm-2) (Table 4.12).

The rest of the endophytes Trichoderma viride and Acremonium lolii were also

showed moderate performance in drought condition viz (290.3 gm-2), (291.0 gm-2), (291.7 gm-

2) and (285.7 gm-2) respectively, under drought condition. Symbiotic effect of endophytes

significantly contributed for stabilizing wheat plant under drought conditions through

increasing the grain yield. The performance of drought sensitive genotypes viz Parwaz-94 and

Kohsar-95 was at par in terms of fungal endophytes.

Table 4.12: Symbiotic effects of endophytes for grain yield in tolerance of drought stressFungal endophytes Wheat genotypes Mean

(gm-2)Parwaz-94(gm-2)

Kohsar-95(gm-2)

Control 268.2 266.4 267.3CColletotrichum lindemuthianum

300.4 302.2 301.3AB

Acremonium lolii 291.7 285.7 288.7BTrichoderma viride 290.2 291.0 290.6BPiriformospora indica

315.2 316.6 315.9A

Mean 293.1A 292.4ALSD at 5% E=15.591,V=9.8604 E*V=22.049

Table 4.13: Analysis of variance for TGW, PT, BY, GY and YI% under field conditions

Mean Squares

Source of variation

Df TGW PT BY GY YI%

Replications 2 1.08 337.23 5270.80 2587.81 16.12

Endophytes 4 25.40** 574.63** 9681.10** 1916.00** 194.57**

Genotypes 1 7.11** 1512.30** 30617.70** 4.49 NS 0.16 NS

E*G 4 0.80 NS 94.63 NS 290.80NS 15.78 NS 1.55 NS

Error 18 0.58 54.20 1060.80 165.21 11.28

Total 29

**= Highly Significant (P<0.01) *= Significant (P<0.05 NS= Non-significant

74

4.4.5 Percent Yield Increased:Results revealed that fungal endophytes significantly affected the grain yield and

became the reason in increase; likewise, interaction of endophytes and genotypes also

affected positively the grain yield under drought conditions imposed on drought sensitive

wheat genotypes at the flowering and grain filling stage. Among the four selected fungal

endophytes, maximum increase in yield 15.4 % was observed in case of Piriformospora

indica and followed by Colletotrichum lindemuthianum 11.3 %, Trichoderma viride 8.1 %

and Acremonium lolii 7.5 %. Among the genotypes, maximum grain yield increased 10.7%

was observed by the Kohsar-95 (Table 4.14).

Among the interaction, endophyte Piriformospora indica interacted comparatively

better for grain yield (15.8 %) with drought sensitive genotype Kohsar-95 and it was found a

similar symbiotic interaction of Piriformospora indica with genotype Parwaz-94 showing

increase in grain yield (15.1 %) respectively (Table 4.14). Colletotrichum lindemuthianum

also showed better performance to combat the drought conditions while interacting with the

sensitive wheat genotypes 10.8 % and 11.7 % (Table 4.14).

The rest of the endophytes Trichoderma viride and Acremonium lolii were also

showed moderate performance in drought condition viz (7.8 %), (8.4 %),(8.3 %) and(6.7 %)

respectively, under drought condition. Symbiotic effect of endophytes significantly

contributed for stabilizing wheat plant under drought conditions through increasing the grain

yield. The performance of drought sensitive genotypes viz Parwaz-94 and Kohsar-95 was at

par in terms of fungal endophytes.

Table 4.14: Symbiotic effects of endophytes for percent yield increased in tolerance of drought stress

Fungal endophytes Wheat genotypes Mean(%)Parwaz-94

(%)Kohsar-95

(%)Contol 0.00 0.00 0.00C

Colletotrichum lindemuthianum

10.86 11.73 11.30B

Acremonium lolii 8.30 6.73 7.51B

Trichoderma viride 7.83 8.46 8.15B

Piriformospora indica

15.06 15.86 15.46A

Mean 10.51A 10.69A

LSD at 5% E=4.0742,V=2.5768 E*V=5.7618

75

4.5: Symbiotic effect of fungal endophytes on two leaf rust susceptible wheat genotypes in disease conditions

4.5.1 Disease Severity (%):

Results revealed that fungal endophytes significantly affected the disease severity and

became the reason in its decrease; however, interaction of endophytes and genotypes also

affected disease severity under the leaf rust conditions inoculated artificially on the wheat

genotypes at tillering and heading stage. Among the four fungal endophytes, minimum

disease severity was observed in Piriformospora indica and Trichoderma viride 45 %

followed by Acremonium lolii and Colletotrichum spp. 55 % and 60 % respectively. While

maximum disease severity 80 % was observed in control where no endophyte was applied to

leaf rust susceptible genotypes whereas all other agronomic requirements were provided

equally.

Among the genotypes, Aas-02 showed least disease severity than that of Fsd-85.

Among the interaction, endophyte Piriformospora indica interacted comparatively better for

least disease severity (40 %) with genotype Aas-02 and it was found statistically at par with

the interaction of Trichoderma viride with genotype Aas-02 having the same value of disease

severity 40 % (Table 4.15).

Fifty percent severity was observed in cases of interaction of genotype Fsd-85 with

both Piriformospora indica and Trichoderma viride and Aas-02 with Acremonium lolii while

rest of the cases showed moderate performances. Symbiotic effects of fungal endophytes with

susceptible genotypes and antagonistic effect for leaf rust fungal pathogen Puccinia recondita

were contributed significantly for stabilizing wheat plant against leaf rust through confronting

the attack of disease and decreasing disease severity.

Table 4.15: Antagonistic effects of endophytes for disease severity in tolerance of disease (leaf rust) conditions

Fungal endophytes Wheat Genotypes Mean(%)Fsd-85

(%)Aas-02

(%)Control 80.0 80.0 80.0AColletotrichum lindemuthianum

60.0 60.0 60.0B

Acremonium lolii 60.0 50.0 55.0BCTrichoderma viride 50.0 50.0 50.0CPiriformospora indica

40.0 40.0 40.0D

Mean 58.0A 56.0ALSD at 5% E=8.6718,V=5.4845 E*V=12.264

76

4.5.2 Area under disease progress curve (AUDPC):

Results revealed that endophytes significantly affected the AUDPC value and became

the reason in decrease; likewise, interaction of endophytes and genotypes also affected

AUDPC value under the leaf rust conditions inoculated artificially on the wheat genotypes at

tillering and heading stage. Among the different endophytes, minimum AUDPC value was

observed in Piriformospora indica 525 followed by Trichoderma spp. 738.3 Acremonium

lolii 875 and Colletotrichum lindemuthianum 908.3, respectively. While maximum AUDPC

value 1400 was observed in control condition where no endophyte was applied to the leaf rust

susceptible genotypes while all other agronomic requirements were provided equally.

Among the interaction, endophyte Piriformospora indica interacted comparatively

better for least AUDPC value 525 with both genotypes Fsd-85 and Aas-02 Trichoderma

viride also showed better performance to combat the leaf rust disease while interacting with

the susceptible wheat genotypes Fsd-85 and Aas-02 indicating AUDPC values 733.3 and

743.3 respectively (Table 4.16).

The rest of the endophytes Acremonium lolii and Colletotrichum lindemuthianum

were also showed moderate performance viz (900.0), (850), (925) and (891) respectively.

Synergetic and symbiotic effects of fungal endophytes with susceptible genotypes and

antagonistic effect for leaf rust fungal pathogen Puccinia recondita were contributed

significantly for stabilizing wheat plant against leaf rust through confronting the attack of

disease and decreasing disease severity resultantly decreasing AUDPC values.

Table 4.16: Antagonistic effects of endophytes for AUDPC in tolerance of disease (leaf rust) conditions

Fungal endophytes Wheat Genotypes MeanFsd-85 Aas-02

Control 1350.0 1450.0 1400.0AColletotrichum lindemuthianum

925.0 891.7 908.3B

Acremonium lolii 900.0 850.0 875.0BTrichoderma viride 733.3 743.3 738.3BPiriformospora indica

525.0 525.0 525.0C

Mean 886.67A 892.00ALSD at 5% E=208.18,V=131.67 E*V=294.41

77

Table 4.17: Analysis of variance for FDS, AUDPC, TGW, GY and YI% under field conditions

SOV Df FDS AUDPC TGW GY YI%

Replications 2 640.00 269341 3.1343 112.53 3.561

Genotypes 1 30.00NS 213NS 24.1203** 710.53** 4.408NS

Endophytes 4 1320.00** 625330** 19.3938** 2319.12** 270.229**

G*E 4 30.00 NS 5088 NS 0.0628NS 130.12* 11.691NS

Error 18 51.11 29457 1.1543 41.72 4.714

Total 29

*= Significant (P<0.05) **= Highly Significant (P<0.01) NS= Non-significant

4.5.3 1000-grains weight (g):Enhanced grain weight is the suitable option for maximization of yield in both normal

and disease condition. Thousand grain weights is one of the imperative yield contributing

attribute in getting greater yield and contains a prominent character in restrictive yield

potential of a genotype. 1000 grain weight is directly depended on seed size. Greater the size

of grain, higher will be grain weight. Results revealed that fungal endophytes and genotypes

affected significantly the 1000-grain weight. Similarly, interaction of endophytes and leaf

rust susceptible genotypes also affected grain weight under the leaf rust conditions inoculated

artificially on the wheat genotypes at tillering and heading stage during the growth season of

the crop.

Among the four fungal endophytes, maximum 1000-grain weight 40.3 g was observed

in the case Piriformospora indica and it was statistically at par with Trichoderma viride 39.7

g followed by Colletotrichum lindemuthianum 38.0 g and Acremonium lolii 37.9 g

respectively. While minimum 1000-grain weight 35.7 g was observed in control condition

where no endophyte was applied to the leaf rust susceptible genotypes in disease provided

condition while all other agronomic requirements were provided equally. Among the

genotypes, maximum 1000-grain weight 39.2 g was observed by the Fsd-85 (Table 4.18).

In case of interaction, endophyte Piriformospora indica interacted comparatively

better for 1000-grain weight 41.2 g with leaf rust susceptible genotype As-02 and it was

found similar symbiotic interaction of Piriformospora indica with genotype Fsd-85 having

1000- grain weight 39.5 g. Trichoderma viride also showed better performance to combat the

leaf rust while interacting with the leaf rust susceptible wheat genotypes 40.5 g and 38.8 g

(Table 4.18).

78

The rest of the endophytes Acremonium lolii and Colletotrichum lindemuthianum

were also showed moderate performance viz (37.2 g), (38.8 g), (37.0 g) and (38.8 g)

respectively, under disease condition. Synergetic and symbiotic effects of fungal endophytes

with susceptible genotypes and antagonistic effect for leaf rust fungal pathogen Puccinia

recondita were contributed significantly for stabilizing wheat plant against leaf rust through

confronting the attack of disease and decreasing disease severity resultantly enhancing 1000-

grain weight, the prime attribute in harvesting greater yield.

Table 4.18: Symbiotic effects of endophytes for 1000-grain weight in tolerance of disease (leaf rust) conditions

Fungal endophytes Wheat Genotypes Mean(g)Fsd-85

(g)Aas-02

(g)Control 34.7 36.8 35.7CColletotrichum lindemuthianum

37.2 38.8 38.0B

Acremonium lolii 37.0 38.8 37.9BTrichoderma viride 38.8 40.5 39.7APiriformospora indica

39.5 41.2 40.3A

Mean 39.2A 37.4BLSD at 5% E=1.3032,V=0.8242 E*V= 1.8430

4.5.4 Grain Yield (gm-2):Results revealed that fungal endophytes and genotypes significantly affected the grain

yield; likewise, interaction of endophytes and genotypes also affected the grain yield in the

leaf rust conditions inoculated artificially on the wheat genotypes at tillering and heading

stage. Among the four fungal endophytes, maximum grain yield 290.8 gm-2 was observed in

case of Piriformospora indica and it was statistically at par with Trichoderma viride 278.2

gm-2 followed by the endophytes Acremonium lolii and Colletotrichum lindemuthianum 261.0

gm-2 and 256.0 gm-2 respectively. While minimum grains yield 239.6 gm-2 was observed in

control condition where no endophyte was applied to the leaf rust susceptible genotypes in

disease condition. Among the genotypes, maximum grain yield 270.4gm-2 was observed by

the Parwaz-94 (Table 4.19).

Among the interaction, endophyte Piriformospora indica interacted comparatively

better for grain yield 301.3 gm-2 with rust susceptible genotype As-02followed by

Trichoderma viride with genotype Aas-02 having grain yield 287.3 gm-2. There was found

similar results where interaction of Piriformospora indica and Trichoderma viride were done

with genotype Fsd-85 having grain yield 280.3 gm-2 and 269.0 gm-2 respectively.

79

The rest of the endophytes Acremonium lolii and Colletotrichum lindemuthianum

were also showed moderate performance in disease condition viz (259.7gm-2), (262.3 gm-2),

(257.7 gm-2) and (258.3 gm-2) respectively. Symbiotic effects of fungal endophytes with

susceptible genotypes and antagonistic effect for leaf rust fungal pathogen Puccinia recondita

were contributed significantly for stabilizing wheat plant against leaf rust through confronting

the attack of disease and decreasing disease severity resultantly greater grain yield. The

performance of disease susceptible genotypes viz Aas-02 and Fsd-85 was at par in terms of

fungal endophytes.

Table 4.19: Symbiotic effects of endophytes for grain yield in tolerance of disease (leaf rust) conditions

Fungal endophytes Wheat Genotypes Mean(gm-2)Fsd-85

(gm-2)Aas-02(gm-2)

Contol 236.67 242.67 239.67DColletotrichum lindemuthianum

257.67 258.33 258.00C

Acremonium lolii 259.67 262.33 261.00CTrichoderma viride 269.00 287.33 278.17BPiriformospora indica

280.33 301.33 290.83A

Mean 260.67B 270.40ALSD at 5% E=7.8345,V=4.9550 E*V= 11.080

4.5.5 Percent Yield Increased (%):Results revealed that fungal endophytes significantly affected the grain yield and

became the reason in increase; likewise, interaction of endophytes and genotypes also

affected positively the grain yield in the leaf rust conditions inoculated artificially on the

wheat genotypes at tillering and heading stage. Among the four selected fungal endophytes,

maximum increase in yield 17.5 % was observed in case of Piriformospora indica followed

by Trichoderma viride 13.7 %, Acremonium lolii 8.2 % and Colletotrichum lindemuthianum

7.1 %. Among the genotypes, maximum grain yield increased 12.1% was observed by the

Aas-02 (Table 4.20).

Among the interaction, endophyte Piriformospora indica interacted comparatively

better for grain yield 19.4 % with drought sensitive genotype Aas-02 and it was found a

similar symbiotic interaction of Piriformospora indica with genotype Fsd-85 and

Trichoderma viride with genotype As-02 showing increase in grain yield 15.5 %. Also

statistically similar results in increasing grain yield 15.5 % when Trichoderma viride

interacted with genotype Fsd-85 (Table 4.20).

80

The rest of the endophytes Acremonium lolii and Colletotrichum lindemuthianum

were also showed moderate performance in disease condition viz (8.8 %), (7.4 %),(8.1 %)

and(5.9 %) respectively. Symbiotic effects of fungal endophytes with susceptible genotypes

and antagonistic effect for leaf rust fungal pathogen Puccinia recondita were contributed

significantly for stabilizing wheat plant against leaf rust through confronting the attack of

disease and decreasing disease severity resultantly greater grain yield. The performance of

disease susceptible genotypes viz Aas-02 and Fsd-85 was at par in terms of fungal

endophytes.

Table 4.20: Symbiotic effects of endophytes for percent grain yield increase in tolerance of disease (leaf rust) conditions

Fungal endophytes Wheat Genotypes Mean(%)Fsd-85

(%)Aas-02

(%)Control 0.0 0.0 0.0DColletotrichum lindemuthianum

8.1 5.9 7.1C

Acremonium lolii 8.8 7.4 8.2CTrichoderma viride 12.0 15.5 13.7BPiriformospora indica

15.5 19.4 17.5A

Mean 11.1A 12.1ALSD at 5% E=7.8345,V=4.9550 E*V= 11.080

81

4.6: Symbiotic effect of fungal endophytes on two yellow rust susceptible wheat genotypes in disease conditions

4.6.1 Disease Severity (%):Results revealed that fungal endophytes significantly affected the disease severity and

became the reason in its decrease; however, interaction of endophytes and genotypes also

affected disease severity under the yellow rust conditions inoculated artificially on the wheat

genotypes at tillering and heading stage. Among the four fungal endophytes, minimum

disease severity was observed in Piriformospora indica (40 %) and Trichoderma viride (50

%) followed by Colletotrichum lindemuthianum and Acremonium lolii (55 %) and (60 %)

respectively. While maximum disease severity (80 %) was observed in control condition

where no endophyte was applied to the yellow rust susceptible genotypes whereas all other

agronomic requirements were provided equally (Table 4.21).

Among the genotypes, Fareed-06 showed least disease severity than that of Shafaq-

06. Among the interaction, endophyte Piriformospora indica interacted comparatively better

for least disease severity 40 % with genotypes Fareed-06 and Shafaq-06. The interaction of

Trichoderma viride with genotypes Fareed-06 and Shafaq-06 showed value of disease

severity 50 % (Table 4.21).

Sixty percent severity was observed in cases of interaction of genotype Shafaq-06

with both Colletotrichum lindemuthianum and Acremonium lolii and Fareed-06 with

Acremonium lolii while Fareed-06 showed 50% severity when interact with Colletotrichum

lindemuthianum Symbiotic effects of fungal endophytes with susceptible genotypes and

antagonistic effect for yellow rust fungal pathogen Puccinia striformis were contributed

significantly for stabilizing wheat plant against yellow rust through confronting the attack of

disease and decreasing disease severity.

Table 4.21: Antagonistic effects of endophytes for disease severity in tolerance of disease (yellow rust) conditions

Fungal endophytes Wheat Genotypes Mean(%)Fareed-06

(%)Shafaq-06

(%)Control 80.0 80.0 80.0AColletotrichum lindemuthianum

50.0 60.0 55.0BC

Acremonium lolii 60.0 60.0 60.0BTrichoderma viride 50.0 50.0 50.0BCPiriformospora indica

40.0 40.0 40.0C

Mean 56.0A 58.0ALSD at 5% E=18.921,V=11.967 E*V= 26.759

82

4.6.2 Area under disease progress curve (AUDPC):Results revealed that endophytes significantly affected the AUDPC value and became

the reason in decrease; likewise, interaction of endophytes and genotypes also affected

AUDPC value under the leaf rust conditions inoculated artificially on the wheat genotypes at

tillering and heading stage. Among the different endophytes, minimum AUDPC value was

observed in Piriformospora indica (625) followed by Trichoderma viride (800),

Colletotrichum lindemuthianum (933.3) and Acremonium lolii (1104) respectively. While

maximum AUDPC value (1325) was observed in control condition where no endophyte was

applied to the yellow rust susceptible genotypes while all other agronomic requirements were

provided equally (Table 4.22).

Among the interaction, endophyte Piriformospora indica interacted comparatively

better for least AUDPC value 625 with both genotypes Fareed-06 and Shafaq-06.

Trichoderma viride also showed better performance to combat the yellow rust disease while

interacting with the susceptible wheat genotypes Fareed-06 and Shafaq-06 indicating

AUDPC value 800 (Table 4.22).

The rest of the endophytes Colletotrichum lindemuthianum and Acremonium lolii

were also showed moderate performance viz (900), (966), (1083) and (1125) respectively.

Synergetic and symbiotic effects of fungal endophytes with susceptible genotypes and

antagonistic effect for yellow rust fungal pathogen Puccinia striformis were contributed

significantly for stabilizing wheat plant against yellow rust through confronting the attack of

disease and decreasing disease severity resultantly decreasing AUDPC values.

Table 4.22: Antagonistic effects of endophytes for AUDPC in tolerance of disease (yellow rust) conditions

Fungal endophytes Wheat Genotypes MeanFareed-06 Shafaq-06

Control 1400.0 1250.0 1325.0AColletotrichum lindemuthianum

900.0 966.7 933.3BC

Acremonium lolii 1083.3 1125.0 1104.2BTrichoderma viride 800.0 800.0 800.0CPiriformospora indica

625.0 625.0 625.0D

Mean 961.6A 953.3ALSD at 5% E=173.9,V=109.9 E*V= 245.9

83

Table 4.23: Analysis of variance for FDS, AUDPC, TGW, GY and YI% under field conditions

SOV Df FDS AUDPC TGW GY YI%

Replications 2 1510.00 349230 0.3163 147.73 4.819

Genotypes 1 30.00NS 521 NS 28.6163** 2842.13** 23.941**

Endophytes 4 1320.00** 438771** 29.5270** 1201.28** 146.476**

G*E 4 30.00NS 10625 NS 0.5963* 56.72NS 4.550**

Error 18 243.33 20557 0.1926 21.66 0.974

Total 29

**= Highly Significant (P<0.01) *= Significant (P<0.05) NS= Non-significant

4.6.3 1000-grain weight (g):Enhanced grain weight is the suitable option for maximization of yield in both normal

and disease condition. Thousand grain weights is one of the imperative yield contributing

attribute in getting greater yield and contains a prominent character in restrictive yield

potential of a genotype. 1000 grain weight is directly depended on seed size. Greater the size

of grain, higher will be the grain weight. Results revealed that fungal endophytes and

genotypes affected significantly the 1000-grain weight. Similarly, interaction of endophytes

and yellow rust susceptible genotypes also affected the grain weight under the leaf rust

conditions inoculated artificially on the wheat genotypes at tillering and heading stage.

Among the four fungal endophytes, maximum 1000-grain weight (40.0 g) was

observed in the case Piriformospora indica and it was statistically at par with Trichoderma

viride (38.8 g) followed by Colletotrichum lindemuthianum (36.6 g) and Acremonium lolii

(35.9 g) respectively. While minimum 1000-grain weight (34.5 g) was observed in control

condition where no endophyte was applied to the yellow rust susceptible genotypes in disease

provided condition while all other agronomic requirements were provided equally. Among

the genotypes, maximum 1000-grain weight (38.1 g) was observed by the Shafaq-06 (Table

4.24).

In case of interaction, endophyte Piriformospora indica interacted comparatively

better for grain weight 40.8 g with yellow rust susceptible genotype Shafaq-06 and it was

found similar symbiotic interaction of Piriformospora indica with genotype Fareed-06 having

grain weight 39.2 g. Trichoderma viride also showed better performance to combat the

yellow rust while interacting with the yellow rust susceptible wheat genotypes 39.5 g and

38.2 g (Table 4.24).

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The rest of the endophytes Colletotrichum lindemuthianum and Acremonium lolii also

showed moderate performance viz (35.2 g), (37.9 g), (34.6 g) and (37.2 g) respectively, under

disease conditions. Synergetic and symbiotic effects of fungal endophytes with susceptible

genotypes and antagonistic effect for yellow rust fungal pathogen Puccinia striformis were

contributed significantly for stabilizing wheat plant against yellow rust through confronting

the attack of disease and decreasing disease severity resultantly enhancing 1000-grain weight,

the prime attribute in harvesting greater yield.

Table 4.24: Symbiotic effects of endophytes for 1000-grain weight in tolerance of disease (yellow rust) conditions

Fungal endophytes Wheat Genotypes Mean(g)Fareed-06

(g)Shafaq-06

(g)Control 33.7 35.4 34.5EColletotrichum lindemuthianum

35.2 37.9 36.6C

Acremonium lolii 34.6 37.2 35.9DTrichoderma viride 38.2 39.5 38.8BPiriformospora indica

39.2 40.8 40.0A

Mean 36.2B 38.1ALSD at 5% E=0.5324,V=0.3367 E*V= 0.7529

4.6.4 Grain yield (gm-2):Results revealed that fungal endophytes and genotypes significantly affected the grain

yield; likewise, interaction of endophytes and genotypes also affected the grain yield in the

yellow rust conditions inoculated artificially on the wheat genotypes at tillering and heading

stage during the growth season of the crop. Among the four fungal endophytes, maximum

grain yield (283.0 gm-2) was observed in case of Piriformospora indica and it was statistically

at par with Trichoderma viride (278.0 gm-2) followed by the endophytes Colletotrichum

lindemuthianum (264.5 gm-2)and Acremonium lolii (259.0 gm-2) respectively. While

minimum grains yield 248.1 gm-2 was observed in control condition where no endophyte was

applied to the leaf rust susceptible genotypes in disease condition. Among the genotypes,

maximum grain yield (276.2gm-2) was observed by the Shafaq-06 (Table 4.25).

Among the interaction, endophyte Piriformospora indica interacted comparatively

better for grain yield (294.0 gm-2) with rust susceptible genotype Shafaq-06followed by

Trichoderma spp. with genotypeShafaq-06 having grain yield (292.3 gm-2). There was found

similar results where interaction of Piriformospora indica and Trichoderma viride were done

with genotype Fareed-06 having grain yield (272.0 gm-2) and (263.6 gm-2) respectively.

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The rest of the endophytes Colletotrichum lindemuthianum and Acremonium lolii

were also showed moderate performance in disease condition viz (255.3gm-2), (273.6 gm-2),

(251.3 gm-2) and (266.6 gm-2) respectively. Synergetic and symbiotic effects of fungal

endophytes with susceptible genotypes and antagonistic effect for yellow rust fungal

pathogen Puccinia striformis were contributed significantly for stabilizing wheat plant

against yellow rust through confronting the attack of disease and decreasing disease severity

resultantly greater grain yield. The performance of disease susceptible genotypes viz Shafaq-

06 and Fareed-06 was at par in terms of fungal endophytes.

Table 4.25: Symbiotic effects of endophytes for grain yield in tolerance of disease (yellow rust) conditions

Fungal endophytes Wheat Genotypes Mean(gm-2)Fareed-06

(gm-2)Shafaq-06

(gm-2)Control 241.6 254.6 248.1CColletotrichum lindemuthianum

255.3 273.6 264.5B

Acremonium lolii 251.3 266.6 259.0BTrichoderma viride 263.6 292.3 278.0APiriformospora indica

272.0 294.0 283.0A

Mean 256.8B 276.2ALSD at 5% E=5.6451,V=3.5703 E*V= 7.9834

4.6.5 Percent Yield Increased (%):Results revealed that fungal endophytes significantly affected the grain yield and

became the reason in increase; likewise, interaction of endophytes and genotypes also

affected positively the grain yield in the yellow rust conditions inoculated artificially on the

wheat genotypes at tillering and heading stage. Among the four selected fungal endophytes,

maximum increase in yield (12.2 %) was observed in case of Piriformospora indica followed

by Trichoderma viride (10.61 %), Colletotrichum lindemuthianum (6.15 %) and Acremonium

lolii (4.18 %). Among the genotypes, maximum grain yield increased (9.41 %) was observed

by Shafaq-06.

Among the interaction, endophyte Piriformospora indica interacted comparatively

better for grain yield (13.36 %) with rust susceptible genotype Aas-02 and it was found a

similar symbiotic interaction of Piriformospora indica with genotype Shafaq-06 and

Trichoderma viride with genotype Shafaq-06 showing increase in grain yield (12.86 %). Also

statistically similar results in increasing grain yield (11.13 %) and (8.36 %) when

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Piriformospora indica and Trichoderma viride interacted with genotype Fareed-06

respectively (Table 4.26).

The rest of the endophytes Colletotrichum lindemuthianum and Acremonium lolii

were also showed moderate performance in disease condition viz (5.36 %), (6.93 %), (3.86

%) and (4.50 %) respectively. Symbiotic effects of fungal endophytes with susceptible

genotypes and antagonistic effect for yellow rust fungal pathogen Puccinia striformis were

contributed significantly for stabilizing wheat plant against yellow rust through confronting

the attack of disease and decreasing disease severity resultantly greater grain yield. The

performance of disease susceptible genotypes viz Shafaq-06 and Fareed-06 was at par in

terms of fungal endophytes.

Table 4.26: Symbiotic effects of endophytes for percent grain yield increase in tolerance of disease (yellow rust) conditions

Fungal endophytes Wheat Genotypes Mean(%)Fareed-06

(%)Shafaq-06

(%)Control 0.00 0.00 0.00EColletotrichum spp. 5.36 6.93 6.15CAcremonium lolii 3.86 4.50 4.18DTrichoderma viride 8.36 12.86 10.61BPiriformospora indica

11.13 13.36 12.25A

Mean 7.17B 9.41ALSD at 5% E=1.1970,V=0.7570 E*V= 1.6928

4.7 Discussion:Wheat crop production and yield is lower than its actual capacity because its

physiology is interrupted by many abiotic and biotic factors (Jellis, 2009). Among biotic

factors, diseases badly affect wheat yield in which rusts have caused huge yield losses in

recent years. Wheat rusts are prevalent throughout the world and its new races are evolving

day by day and infecting resistant varieties (Brian, 2006). Yellow and leaf rusts are presently

the imperative wheat disease worldwide, which threaten the global food security. There are

many reports about social and economic losses due to wheat rust epidemics (Hovmoller et al.,

2010).

Drought is one of the foremost abiotic stresses (Ramachandra et al., 2004; Boubacar,

2012) which cause low wheat productivity. Drought reduces the number of fertile tillers

which are main contributors of grain yield (Pfeiffer et al., 2005). It deters cell enlargement

and cell division and also reduces nutrient metabolism, respiration, photosynthesis and

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translocation (Farooq et al., 2008). Water stress disturbs plant water relations, minimizes

photosynthesis and plant energy results in death of the plant (Jaleel et al., 2007; Cattivelli et

al., 2008; Sikuku et al., 2012). Drought affects all of the wheat phonological stages, the

grain-filling and the reproductive wheat stages are the most sensitive (Pradhan et al., 2012;

Nawaz et al., 2013). Drought at post-anthesis reduces the wheat yield from 1-30%, mild

stress at heading stage losses 57% yield, whereas persistent terminal drought shrinks the grain

yield by 58–92% (Wei et al., 2010; Madani et al., 2010; Dias de Oliveria et al., 2013).

The productivity of wheat is enhanced by adopting appropriate strategies which

consequently minimizes severe yield losses caused by different abiotic stresses and

pathogenic diseases. Numerous scientists and researchers are discovering sustainable

alternative approaches to pesticides and chemical fertilizers. Natural resources are decreasing

with the passage of time due to spontaneously increasing world population. It is need of the

hour to find out an alternative approach for growing more food in such a manner that can

reduce detrimental environmental impacts of intensive farming, fungicide resistance and

environmental pollution.

The present research was conducted on endophytes, drought and rust diseases

tolerance in wheat. The endophytic beneficial effects on plants against diseases increased the

attention of researchers and farmers for enhancing agricultural production. This bio-control

strategy for combating different a biotic stresses and pathogenic diseases have altered the

attention of farmers for better crop production. It is tried to discover different efficient and

compatible fungal endophytes for wheat tolerance against drought and rust diseases.

Endophytes are metabolically active microbes (fungi, bacteria or virus) that colonise healthy

plant tissue intracellularly and intercellularly without causing any obvious disease symptoms

(Schulz and Boyle, 2006; Compant et al., 2008; Reinhold-Hurek and Hurek, 2011). They

exhibit complex interactions and a variety of symbiotic lifestyles with their hosts ranging

from parasitism to mutualism (Schulz and Boyle, 2005; Redman et al., 2001).

Endophytic microorganisms induce defence mechanisms in host plants against

pathogen attack. Plants combat different abiotic stresses by their own natural defence and in

cooperation with different microbes (Marulanda et al., 2006; Hardoim et al., 2015). In several

cases endophytic microbes produce bioactive organic compounds, secondary and

antimicrobial metabolites that resist pathogens (Clarke et al., 2006; Ambrose and Belanger,

2012; Gond et al., 2014). Several endophytic microbes also create tolerance in host plants

against abiotic stresses (Redman et al., 2002; Kuldau and Bacon, 2008). Endophytes are also

playing their imperative role for mutual interaction with their hosts for better adaptability and

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systemic resistance, augmenting nutrient uptake, stress tolerance and pathogenic tolerance or

resistance. The constructive and valuable characteristics of endophytic fungi have also

observed in host plants against numerous stresses (Hamilton et al., 2010; Kavamura et al.,

2013).

During the cropping season 2015-16 studies regarding symbiotic and synergetic

relationship of four selected fungal endophytes with two drought sensitive genotypes were

conducted on the wheat at the flowering and grain-filling stage. Results revealed that fungal

endophytes and genotypes significantly affected the grain yield and other yield related

parameters. Symbiotic effect of endophytes considerably contributed for alleviating drought

conditions through enhancing number of productive tillers, 1000-grains weight, biological

yield and finally grain yield of the crop. Piriformospora indica exhibited comparatively

significant response among the four fungal endophytes. Colletotrichum lindemuthianum also

showed better performance to combat the terminal drought conditions while interacting with

the sensitive wheat genotypes of Parwaz-94 and Kohsar-95 and increased 11.3% of final

grain yield after P. indica which gave 15.4% grain yield increase as compare to control

conditions while all other agronomic requirements were provided equally. Trichoderma

viride and Acremonium lolii also showed moderate response and 8.1% and 7.5% increase in

grain yield, respectively. The results of current research are also in line with the findings of

Shahabivand et al., (2012) and Yaghoubian et al., (2014) who reported that Piriformospora

indica enhanced wheat growth and its inoculation augmented the defence mechanisms in

wheat, conferred drought tolerance and increased yield and productivity of wheat plants,

suggesting P. indica application was playing its beneficial role by establishing interaction

with its host. Results are also consistent with Rodriguez et al., (2008) and Suryanarayanan et

al., (2009) who reported symbiotic and synergetic benefits conferred by Colletotrichum

lindemuthianum in tomato plants by improved drought tolerance, and also enhanced growth

and biomass of host.

In the same cropping season 2015-16, studies were also carried out related to the

exploring the symbiotic and synergetic relationship of four selected fungal endophytes with

two leaf rust susceptible genotypes Faisalabad-85 and Aas-02 and two yellow rust susceptible

genotypes namely Fareed-06 and Shafaq-06 for stabilizing wheat plant under artificial and

natural inoculation environments. The artificial inoculation of yellow and leaf rusts was

performed on wheat genotypes by means of different methods like rubbing, dusting through

talcum powder, spraying with distilled water and needle injection methods. The same studies

concerning artificial inoculation conducted by Hussain et al., 2015; Rao et al., 1989 using

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same method of hypodermic injection through an aqueous spore’s suspension. Dusting and

spraying methods used by Roelfs et al., 1992 who mixed fresh spores in talcum powder and

distilled water and followed by applied to wheat plants.

It is evident from the results that symbiotic effect of fungal endophytes significantly

contributed for alleviating pathogenic leaf and yellow rust conditions through minimizing

both of the final rust severities (FDS) and AUDPC values and via enhancing 1000-grain

weight and wheat grain yield by comparing with control while all other agronomic

requirements were provided equally. Piriformospora indica and Trichoderma viride were

performed significant among the four endophytes to confront the disease conditions while

interacting with the leaf and yellow rust susceptible wheat genotypes. Both reduced the leaf

rust severities by 40% and 30% and also yellow rust severities 40% and 30%. Thus, final

grain yield were increased 17.5% and 13.7% against leaf rust whereas 12% and 10% against

yellow rusts. Colletotrichum lindemuthianum and Acremonium lolii showed moderate

performance.

The findings of present research are similar with the results of Rabiey and Shaw

(2015) who reported that P. indica was helpful in biological control of Fusarium wheat

diseases. Assessment was done for the antagonistic and biocontrol effect of P. indica on

fusarium head blight (FHB) disease of spring and winter wheat. Fusarium head blight caused

by the contamination of the mycotoxin deoxynivalenol (DON). Application of P. indica

reduced 70% of disease incidence and severity and reduced 70 and 80% concentration of

mycotoxin deoxynivalenol in winter and spring wheat respectively. P. indica also increased

1000-grains weight, biological and grain yield. The average increase of biological and grain

yield were reported 24.2 % and 17.3 %.

The results are also in consistent in case of antagonistic effect of P. indica by Rabiey

et al., (2015) for control of air-borne diseases of spring and winter wheat, including yellow

rust, powdery mildew and septoria leaf blotch in outdoor conditions. The host genotypes

were inoculated naturally and artificially with corresponding pathogens of air-borne diseases.

Application of P. indica at sowing time reduced the disease severities of yellow rust,

powdery mildew and septoria leaf blotch by 29, 63 and 65% respectively. Consequently, it

also increased 1000-grain weight, biological and grain yield by 25, 48 and 27 % respectively.

The performance of Piriformospora indica was also evaluated by Rabiey et al.,

(2015) for control of Fusarium crown rot of wheat which caused decrease in straw production

as well as grain quality and grain yield. Wheat seedlings were inoculated with P. indica and

pathogenic Fusarium culmorum at sowing time and growth of 7 days were checked under

90

glasshouse conditions. Seedlings without inoculation of P. indica were badly damaged by F.

culmorum pathogen but root seedlings inoculated with P. indica and F. culmorum were not

damaged.

Serfling et al., (2007) reported the effect of P. indica in different substrata under field

and greenhouse conditions by colonizing winter wheat roots with this biocontrol endophyte

against different root, stem and leaf pathogens. In greenhouse conditions, severity of typical

root (Fusarium culmorum), stem (Pseudocercosporella herpotrichoides) and leaf (Blumeria

graminis f. sp. tritici) pathogens were reduced significantly. However, in field conditions,

symptoms of leaf pathogen were not significant in Piriformospora indica colonized

compared with control. But the stem pathogen disease severity of Pseudocercosporella

herpotrichoides was much reduced in endophytic colonized plants. Enlarged concentrations

of hydrogen peroxide and numbers of sheath layers after B. graminis attack were detected in

endophytic colonized plants that means systemic resistance induction was done in plants.

Results are also consistent with Montero-Barrientos et al., (2010), Shoresh et al.,

(2010) and Mastouri et al., (2010) who reported antagonistic effect conferred by Trichoderma

viride in many of the host plants. For that reason it considered as bio-control fungi for their

ability to manage diseases. Trichoderma as biocontrol agents catch the attention for

managing diversity of soil borne fungi of Sclerotiorum cepivorum, Botrytis allii and

Aspergillus niger which are the causal organisms of neck rot black mould and white rot

disease of onion, respectively (Metcalf et al., 2004; Clarkson et al., 2004; McLean et al.,

2005). It is reported valuable in protecting Arachis hypogaea, Cucumis sativus and a number

of other crops from destruction caused by several pathogens (Ha, 2010).

P. indica confront fungal root pathogens such as Fusarium spp., Rhizoctonia solani

and Cochliobolus sativus and protect barley plants from their deleterious effects (Waller et

al., 2005). Additionally, protection of systemic type against foliar pathogens is achieved

which is a beneficial effect connected to the mutualistic interaction. Generally, systemic

resistance show a defence approach by hosts to check microbes’ invasions to infection sites,

thus, to defend yet uninfected organs of host. However, in case of barley, Piriformospora

indica activates plant defence responses that ultimately direct a systemic resistance against

Blumeria graminis f.sp. hordei (Waller et al., 2005).

The frequently observed quality of improved stress tolerance in colonized host plants

of P. indica was also reflected in barley and Arabidopsis by an increased salt tolerance and

the systemic resistance induction against the powdery mildew fungi Golovinomyces orontii

and Blumeria graminis f.sp. hordei (Waller et al.,2005). It is extensively reported that

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infestation by P. indica spores and culture filtrates leads to improvement of the growth,

increase in root and biological yield in its mutualistic relationship with a broad range of hosts

(Achatz et al., 2010; Zarea et al., 2012; Jogawat et al. ,2013; Bakshi et al., 2014) increase in

grain yield, enhanced nitrate and phosphate uptake (Yadav et al., 2010; Cruz et al., 2013;

Shrivastava and Varma, 2014) and enhanced tolerance to major biotic and abiotic stresses

under glasshouse and field conditions (Ghahfarokhy et al., 2011; Alikhani et al., 2014;

Ghabooli et al., 2014; Varma et al., 2014; Harrach et al., 2014; Prasad et al., 2014; Johnson

et al., 2014; Trivedi et al., 2016; Gill et al., 2016).

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CHAPTER 5 SUMMARY

Wheat production and yield is much low in Pakistan due to numerous abiotic and

biotic factors in which drought, leaf and stripe rusts are very important. Leaf rust/brown rust

is caused by the Puccinia recondita yellow rust/stripe rust is caused by Puccinia striformis.

These pathogens spread through air as well as these are persistent threat for sustainable wheat

productivity.

To combat these threats for food security, it is necessary to discover alternative

sustainable and environment friendly approaches. Therefore, during 2014-15 and 2015-16,

the research was conducted to confirm the hypothesis that endophytes confer drought and rust

diseases tolerance in wheat genotypes. During 2014-15, fifty genotypes were screened to find

out drought sensitive, leaf and yellow rusts susceptible genotypes in 3 separate screening

experiments. The rust conditions were produced by providing artificial inoculation of yellow

and leaf rust at tillering and heading stage on wheat genotypes by means of various methods

like dusting with talcum powder, spraying with distilled water and needle injection. Drought

conditions were provided by skipping the irrigation at reproductive and grain filling stage.

In screening experiment, the mean comparisons of yield parameters and drought stress

related indices such as tolerance index, mean productivity and stress susceptibility index of

wheat exhibited a significant impact of terminal drought on the number of grains per spike,

1000-grain weight, number of productive tillers, harvest index, biological yield and finally

grain yield. Based on mean comparisons, the genotypes were categorized into four groups;

i. The genotypes of 9725, Millat-11, Inqalab-91, 9444, Lasani-06, Manthar-03,

Pirsabak-04, MH-97, Kohistan-97 and Faisalabad-83 expressed less grain yield losses

as well as high yield in both drought and normal conditions.

ii. The genotypes of Hashim-10 and Punjab-11, Watan-92, GA-02, Faisalabad-85,

Shafaq-06 and Aas-02 showed minimum grain yield than first group in both drought

and normal conditions.

iii. Chenab-00, Kohsar-95, Parwaz-94 and Kohenoor-83 genotypes of wheat expressed

high grain yield under normal condition and low grain yield under drought condition.

Genotypes of this group confirmed higher loss of grain yield due to drought stress.

Similarly, it was also observed that genotypes belong to this group are most sensitive

to drought.

iv. Likewise, the rest of the genotypes are included in the fourth group.

93

In the second screening experiment, the mean comparisons of different disease

parameters of final disease severity % (FDS), AUDPC and coefficients of infection (CI)

exhibited a significant impact of leaf rust on 50 wheat genotypes. On the basis of above

mentioned disease parameters, the genotypes were classified into four categories: a) S-

susceptible b) MS-moderately susceptible c) MR-moderately resistant d) R-resistant. The

genotypes namely Punjab-11, Faisalabad-85, Aas-02, Sehar-06 and Wafaq-01 expressed

susceptible response whereas Manthar-03, Parwaz-94, Bathoor-08 and few among the rest

genotypes showed resistant response.

Similarly, in further screening trial Fareed-06, MH-97, Shafaq-06, Inqalab-91 and

Aas-02 exhibited susceptible response whereas Bhakhar-02 and Fsd-08expressed resistant

response. In in-vitro evaluation endophytes were conducted by keeping the seeds in test tubes

containing 0.3% agar concentration in distilled water with endophytic fungal spore

suspension of known concentration. The efficacy of endophytes was tested by measuring the

root and shoot length of wheat seedlings. Four efficient endophytes namely Piriformospora

indica, Colletotrichum lindemuthianum, Trichoderma viride and Acremonium lolii were

selected among them for further studies.

During 2015-16, three experiments were conducted to investigate the symbiotic

relationship of four selected endophytes for stabilizing wheat plant under drought, leaf and

yellow rust conditions respectively. The drought sensitive genotypes like Kohsar-95 and

Parwaz-94 showed significant results along with endophytes. Endophytes enhanced the

number of productive tillers, 1000-grains weight, grain and biological yield of endophytic

inoculated drought sensitive genotypes in drought stress conditions as compare to control.

Piriformospora indica showed significant performance by enhancing 15.4% final grain yield

followed by Colletotrichum lindemuthianum (11.3 %), Trichoderma viride (8.1 %) and

Acremonium lolii (7.5 %) respectively.

In case of leaf and yellow rust conditions, the leaf rust susceptible genotypes namely

Faisalabad-85 and Aas-02 and the yellow rust susceptible genotypes namely Fareed-06 and

Shafaq-06 showed significant results with fungal endophytes. Endophytes enhanced 1000-

grains weight and grain yield by reducing the disease severity and AUDPC values of those

endophytic inoculated rust susceptible genotypes in disease conditions as compare to control.

Piriformospora indica also showed significant performance in cases of leaf and yellow rust

diseases by enhancing 17.5% and 12.3% final grain yield followed by Trichoderma viride

which showed 13.7 % and 10.6 % in leaf and yellow rust conditions respectively. The rest of

94

endophytes Colletotrichum lindemuthianum and Acremonium lolii showed 7.1% and 6.2%,

8.2% and 4.2% in leaf and yellow rust conditions respectively.

CONCLUSION: Environmental factors strengthen the wheat by confront abiotic and biotic stresses in

combination with symbiosis.

Endophytes confer the drought and rust diseases tolerance in wheat genotypes.

Endophytes can protect wheat from damage caused by Puccinia recondita and Puccinia

striiformis by reducing the disease severity and consequently enhance the grain yield

under field conditions.

Piriformospora indica showed best performance against drought and rust diseases.

Trichoderma viride exhibited outstanding performance against rust diseases under field

conditions.

Colletotrichum lindemuthianum showed significant performance against drought stress

under field trials.

RECOMMENDATIONS: It is strongly recommended that endophytes should utilize against drought and rust

diseases under favorable environmental conditions.

Wheat seeds should be treated with Piriformospora indica to get maximum yield under

rust favorable and water deficient conditions.

Wheat seeds treated with Colletotrichum lindemuthianum is most suitable to get good

yield from deficient water environment.

Maximum wheat yield was obtained when seeds were treated with Trichoderma viride

against rust diseases.

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