investigation on biocontrol potential of endophytic yeasts...

INVESTIGATION ON BIOCONTROL POTENTIAL

OF ENDOPHYTIC YEASTS ASSOCIATED WITH

HEALTHY PLANTS

AYESHA FAREED

DEPARTMENT OF BOTANY,

UNIVERSITY OF KARACHI,

PAKISTAN

2018

INVESTIGATION ON BIOCONTROL POTENTIAL

OF ENDOPHYTIC YEASTS ASSOCIATED WITH

HEALTHY PLANTS

Thesis Submitted for the Partial Fulfilment of the Degree of

Doctor of Philosophy in Botany

By

AYESHA FAREED

TO THE FACULTY OF SCIENCE

UNIVERSITY OF KARACHI

PAKISTAN

2018

INVESTIGATION ON BIOCONTROL POTENTIAL OF

ENDOPHYTIC YEASTS ASSOCIATED WITH HEALTHY PLANTS

AYESHA FAREED

Ph.D. THESIS

APPROVED

THESIS ADVISOR EXTERNAL EXAMINER

PROF. DR. SYED EHTESHAMUL-HAQUE

DEPARTMENT OF BOTANY

UNIVERSITY OF KARACHI

DEDICATED TO

MY LOVING HUSBAND

FAREED M. KHAN

AFRIDI

TABLE OF CONTENTS PAGES

LIST OF FIGURES i

LIST OF TABLES iii

KHULASA vi

SUMMARY viii

1. INTRODUCTION 1

2. MATERIALS AND METHODS 11

2.1. Collection of healthy plant samples for the isolation of indigenous

yeasts 11

2.2. Isolation of indigenous endophytic yeasts from different parts

of healthy plants 11

2.3. Purification and maintenance of yeast isolates for further studies 11

2.4. Morphological/physiological tests for the identification of yeasts 12

2.4.1. Microscopic appearance of non-filamentous vegetative cells 12

2.4.2. Microscopical Examination for Filamentous Growth (Dalmau

Plate Culture) 12

2.4.3. Microscopical examination for ballistoconidia 12

2.4.4. Microscopical examination for ascospores 13

2.4.5. Germ Tube Formation 13

2.4.6. Ability to grow at different temperatures 13

2.4.7. Tolerance of 1% acetic acid 14

2.5. Biochemical tests for the identification of yeasts 14

2.5.1. Carbon assimilation test 14

2.5.2. Assessing the ability to use certain sugars anaerobically 15

2.5.3. Urease test 15

2.6. Molecular identification of selected yeast strains 15

2.7. Determination of indole acetic acid production (In vitro) by

endophytic yeasts 16

2.8. Phosphate solubilization capacity of yeasts 16

2.9. Isolation of root infecting fungi from soil 17

2.9.1. Isolation of Fusarium spp. through Soil Dilution Method 17

2.9.2. Isolation of Rhizoctonia solani through Baiting Procedure 17

2.9.3. Isolation of Macrophomina phaseolina through Wet Sieving and

Dilution Technique 17

2.10. TEST FOR ANTIFUNGAL ACTIVITY TOWARDS ROOT

ROTTING FUNGI 18

2.10.1. Dual Culture Plate Assay for In Vitro Testing against Root

Rotting Fungi 18

2.11. Cell free culture filtrates of yeast isolates 18

2.12. Nematicidal activity of yeasts 18

2.12.1. In Vitro Testing for Juvenile Mortality 18

2.13. Population of yeast bioagents (Antagonists) 18

2.13.1. Colony forming unit (cfu per mL) in suspension 18

2.14. Determination of phosphorus from leaf samples 19

2.14.1. Sample (wet) digestion 19

2.14.2. Reagents 19

2.14.3. Procedure for the preparation of test, standards and blank 19

2.14.4. Calculation 20

2.15. Estimation of polyphenols 20

2.16. Estimation of antioxidants 21

2.17. Estimation of salicylic acid 21

2.18. Infection Percentage

2.19. Analysis of data

21

21

3. EXPERIMENTAL RESULTS 22

3.1. Isolation of endophytic yeasts from different parts of healthy plants 22

3.2. Screening of the endophytic yeasts against root knot nematodes

and root infecting fungi 22

3.2.1. In vitro nematicidal activity of cell free culture filtrates of


3.2.2. In vitro antifungal activity of endophytic yeasts against root rotting

fungi by dual culture plate assay 30

3.3. Morphological and physiological identification of endophytic

yeasts isolated from different sources 41

3.4. Molecular identification of endophytic yeasts 41

3.5. In vitro indole acetic acid (IAA) production by potential isolates of


3.6. Phosphate solubilization activity by potential yeast isolates 46

3.7. In vivo studies

49 3.8. Effect of endophytic yeast as soil drench on the growth of

sunflower in sterilized soil 49

3.8.1. Effect of endophytic yeasts applied as soil drench on the growth of

sunflower in sterilized soil (EXPERIMENT 1) 49

3.8.2. Vegetative growth parameters 50

3.8.3. Effect of endophytic yeasts applied as soil drench on the growth of

sunflower in sterilized soil: (EXPERIMENT 2) 52


3.9. Biocontrol screen house experiments 56

3.9.1. Use of endophytic yeasts as soil drench treatment for the

regulation of root fungal pathogens in screen house

experiment on sunflower

56


3.9.3. Root infection 57

3.10. Use of endophytic yeasts as soil drench treatment for the

regulation of root fungal pathogens in screen house experiment on

sunflower

60



3.11. Application of selected endophytic yeasts using soil drench

method for the regulation of root fungal pathogens in screen

house experiment on mungbean

62



3.12. Application of selected endophytic yeasts using soil drench

method for the regulation of root fungal pathogens in screen

house experiment on tomato

65



3.13. Effect of neem cake as organic amendment on the efficiency of 71

endophytic yeasts application in soil drench as biocontrol agents

against root rotting fungi

3.13.1. Effect of neem cake as organic amendment on the efficiency of

endophytic yeasts applied as soil drench in controlling root

rotting fungi of sunflower

71



3.14. Effect of neem cake as organic amendment on the efficiency of


rotting fungi of mungbean

77



3.15. Effect of neem cake as organic amendment on the efficiency of


rotting fungi of tomato

81



3.16. Effect of chemical fertilizer on the biocontrol and growth

promoting efficacy of endophytic yeasts 85

3.17. Effect of chemical fertilizers on the efficacy of biocontrol and

plant growth promoting ability of endophytic yeasts on sunflower 85



3.17.3. Phosphorus content 86


plant growth promoting ability of endophytic yeasts on

mungbean

89

3.18.1. Growth parameters 89




plant growth promoting ability of endophytic yeasts on tomato 92




FIELD EXPERIMENTS 96 3.20. Application of yeasts as soil drench in order to control root

rotting fungi of sunflower (Helianthus annuus L.) under field

conditions

96



3.21. Estimation of biochemical parameters of leaf 98


3.21.2. Total phenolic contents 98

3.21.3. Antioxidant activity 98

3.21.4. Salicylic acid 98

3.22. Application of yeasts as soil drench in order to control root

rotting fungi of sunflower (Helianthus annuus L.) in soil amended

with neem cake under field conditions

104





3.23.2. Total phenolic contents 109



3.24. Application of yeasts as soil drench in order to control root

rotting fungi of tomato (Lycopersicon esculentum Mill.) in soil

amended with neem cake under field conditions

112


3.24.2. Root Infection 112



3.25.2. Total Phenolic contents 118



4. DISCUSSION

121

5. CONCLUSION 133

6. REFERENCES 135

7. AKNOWLEDGEMENTS 167

8. PUBLICATIONS 168

LIST OF FIGURES

FIGURE

NO. TITLE

PAGE

NO.

1. Restriction of mycelial growth of root rotting fungi by

endophytic yeast KUAY-5 in dual culture method 35











7. Molecular identification of endophytic yeast. 42

8. Fermentation of Glucose by various endophytic yeasts 44

9. Urea Hydrolysis by some endophytic yeasts. 45

10. Effect of soil drenching with endophytic yeasts on the growth of

sunflower plants. 54


sunflower plants. 55


tomato plants. 68

13. Effect of soil drenching with endophytic yeasts on the growth

of tomato plants. 69


of sunflower plants in neem cake amended soil. 74


of sunflower plants in neem cake amended soil. 75


of mungbean plants in neem cake amended soil. 79

17. Effect of endophytic yeasts applied as soil drench on the

nodulation in mungbean roots in neem cake amended soil. 80

18. Effect of NPK soil amended with endophytic yeasts on

phosphorus uptake (mg/g) in sunflower plants. 88


phosphorus uptake (mg/g) in mungbean plants. 91

20. Effect of chemical fertilizer with endophytic yeasts on the

growth of tomato plants. 94


phosphorus uptake (mg/g) in tomato plants. 95

22. Fruit Formation in sunflower plants treated with endophytic

yeasts. 99

23. Fruit Formation in sunflower plants treated with endophytic

yeasts. 100

24. Effect of endophytic yeasts on the overall growth of tomato

plants in neem cake amended soil under field conditions. 115

25. Fruit formation in tomato plants grown in soil amended with

endophytic yeasts and neem cake in field plots 116

LIST OF TABLES

TABLE

NO. TITLE

PAGE

NO.

1. List of yeasts isolated from different parts of healthy plants. 23

2. Effect of cell free culture filtrates of different isolates of yeasts on

juvenile mortality of Meloidogyne javanica, the root knot nematode. 27

3. In vitro inhibition of Fusarium solani, F. oxysporum, Macrophomina

phaseolina and Rhizoctonia solani by yeast isolates. 32

4. RFLP analysis of ITS amplicons of endophytic yeast isolates. 41

5. Morphological and biochemical/physiological characteristics of

selected endophytic yeasts. 43

6. Production of Indole Acetic Acid (IAA) by some potential yeast

isolates during in vitro assay 47

7 In vitro phosphate solubilization activity by some potential yeast

isolates 48

8. Effect of different yeast isolates applied as soil drench on vegetative

growth of sunflower (Helianthus anuus L.) in sterilized soil 51


growth of sunflower (Helianthus anuus L.) in sterilized soil 53


growth of sunflower (Helianthus anuus L.) in naturally infested soil 58

11.

Effect of different yeast isolates on root infection by Fusarium solani,

F. oxysporum, Macrophomina phaseolina and Rhizoctonia solanion

sunflower (Helianthus annuus L.) roots.

59

12.

Effect of different yeast isolates applied as soil drench on

vegetative growth of sunflower (Helianthus anuus L.) in naturally

infested soil

61

13.

Effect of different yeast isolates on root infection by Fusarium solani,

F. oxysporum, Macrophomina phaseolina and Rhizoctonia solanion

sunflower (Helianthus annuus L.) roots.

62


growth of mungbean (Vigna radiata L.) in naturally infested soil 64

15.

Effect of different yeast isolates on root infection by Fusarium

solani, F. oxysporum, Macrophomina phaseolina and Rhizoctonia

solani on mungbean (Vigna radiata L.) roots.

65

16.

Effect of different yeast isolates applied as soil drench on vegetative

growth tomato (Lycopersicon esculentum Mill.) in naturally infested

soil

67

17.

Effect of different yeast isolates on root infection by Fusarium

solani, F. oxysporum, Macrophomina phaseolina and Rhizoctonia

solani on tomato roots.

70

18. Effect of neem cake amendment along with different yeast isolates

applied as soil drench on vegetative growth of sunflower plants 73

19.

Effect of different yeast isolates on infection by Fusarium solani, F.

oxysporum, Macrophomina phaseolina and Rhizoctonia solani on

sunflower roots in neem cake amended soil.

76


applied as soil drench on vegetative growth of mung bean plants 78

21.

Effect of different yeast isolates on infection by Fusarium solani,

F. oxysporum, Macrophomina phaseolina and Rhizoctonia solani on

mungbean roots in neem cake amended soil.

81


applied as soil drench on vegetative growth of tomato plants 83

23.



tomato roots in neem cake amended soil.

84

24. Combined effect of chemical fertilizer and yeast isolates on

vegetative growth of sunflower plants under screen house. 87

25.

Effect of chemical fertilizer with yeast isolates used as soil drench

on infection by Fusarium solani, F. oxysporum, Macrophomina

phaseolina and Rhizoctonia solani on roots of sunflower plants.

88

26. Combined effect of chemical fertilizer and yeast isolates on the

growth of mungbean plants under screen house 90

27.

Combined effect of chemical fertilizer and yeast isolates on infection

by Fusarium solani, F. oxysporum, Macrophomina phaseolina and

Rhizoctonia solani on roots of mungbean plants.

91

28. Combined effect of chemical fertilizer and yeast isolates on the

vegetative growth of tomato plants under screen house 93

29.

Combined effect of chemical fertilizer and yeast isolates on infection

by Fusarium solani, F. oxysporum, Macrophomina phaseolina and

Rhizoctonia solani on roots of tomato plants.

95

30. Effect of different yeast isolates on the growth parameters of

sunflower plants under field conditions 98

31.



sunflower roots in field plot experiment

101

32. Effect of selected yeast isolates on the polyphenols and antioxidant

activity of sunflower plants under field conditions 103

33. Effect of selected yeast isolates on Phosphorus uptake and Salicylic

acid production by sunflower plants under field conditions 104

34. Effect of neem cake soil amended with yeast isolates on growth of

sunflower plants under field conditions. 107

35. Effect of selected yeast isolates on infection by Fusarium solani, 108


sunflower roots in neem cake amended soil under field conditions.

36.

Effect of neem cake soil amended with yeast isolates on the

polyphenols and antioxidant activity of sunflower plants under field

conditions

110

37.

Effect of neem cake soil amended with yeast isolates on Salicylic

acid and Phosphorus uptake by sunflower plants under field

conditions

111

38. Effect of neem cake amended soil drenched with selected yeast

isolates on the growth of tomato plants under field conditions 114

39.



tomato roots in neem cake amended soil under field conditions.

117

40.

Effect of different yeast isolates on the polyphenols and antioxidant

activity of tomato plants in soil amended with neem cake under field

conditions.

119

41. Effect of neem cake soil amended with yeast isolates on Salicylic

acid and Phosphorus uptake by tomato plants under field conditions. 120

SUMMARY

The exposure of crops to a variety of fungal and bacterial diseases leads to their huge

losses both in quantity and quality. Different strategies are being adopted to control of

these diseases among which the use of chemical pesticides is a common one. But the health

risk due to chemical exposure is being associated with this common method. The

application of biological control antagonists (BCAs) can provide a positive plus promising

alternative to chemicals. Many mycelial fungi and bacteria have proved to be good

antagonists against several plant pathogens, but less literature is available, in contrast, for

the use of yeasts as biological antagonists. This study was an effort to evaluate the

biocontrol efficiency of indigenous endophytic yeasts associated with healthy plants

against different root rot pathogens. Hundred yeast strains were isolated from different

vegetative parts of healthy plants belonging to seven genera. Out of hundred yeasts tested,

sixty-nine isolates showed inhibitory effect during in vitro assay against Fusarium solani,

F. oxysporum, and Macrophomina phaseolina, to varying degrees. Maximum inhibitory

effect against root rotting fungi was observed by yeast strains KUAY-17, KUAY-34,

KUAY-62. Out of hundred yeast isolates, sixty-nine isolates repressed the mycelial growth

of the three tested fungi viz., Fusarium solani, F. oxysporum and Macrophomina

phaseolina. No yeast was effective against Rhizoctonia solani during in vitro testing.

KUAY-34, 62 and 66 caused maximum inhibition of F. solani while F. oxysporum was

greatly suppressed by KUAY-9, 25, 34, 38 and 62. Mycelial growth of M. phaseolina was

restricted greatly by KUAY-5, 9, 34 38 and 62. Eleven yeast isolates showed 100%

nematicidal activity against second stage juveniles of root knot nematodes after 48 hours,

while other strains killed the juveniles to varying degrees. Out of 15 isolates tested for

Indole Acetic Acid production, 2 yeast isolates produced maximum concentration of IAA

in vitro, whereas, all the tested yeast isolates exhibited the phosphate solubilization

property.

The effective yeast strains were subjected to testing for their biocontrol activity in

screen house experiments using Sunflower (Helianthus annuus L.), Mungbean (Vigna

radiata (L.) Wilczek) and Tomato (Lycopersicon esculentum Mill.) as test crops. The

yeasts were able to inhabit the roots of test crops when applied externally to plants in

sterilized soil. They significantly enhanced the growth of tested plants. In soil, having

naturally infestation of root rotting fungi, the application of endophytic yeasts not only

reduced the incidence of pathogens but also promoted the plant growth. Out of 17 tested

yeast isolates, 4 isolates, viz., KUAY-5, KUAY-17, KUAY-34 and KUAY-62 exhibited

great potential of reducing fungal pathogens along with the promotion of plant growth both

in screen house experiments as well as under field conditions.

The combined treatment of yeasts and neem cake had positive impact on plants growth

and on the suppression of fungal pathogens. The application of yeast isolate KUAY-62

also increased the nodulation in the roots of mungbean plants.

A notable increase was observed in the phosphorus uptake and antioxidant activity in

the plants treated with endophytic yeasts. Induction of Systemic Acquired Resistance

(SAR) in the plants have an imperative role in fighting with pathogens. In the current

study, the application of endophytic yeasts improved the status of phenolic compounds

and salicylic acid in the test crops which contributed towards the obliteration of pathogens

and promotion of plant growth under field conditions.

1. INTRODUCTION

Nature has provided us with all kinds of vegetable and fruit crops that can be grown

in different seasons of the year in different regions of the world. Thanks to agriculture we

can enjoy a bounty of food crops. These vegetables and fruits are a basic source of our

nutrition for having valuable carbohydrates, proteins, minerals and vitamins. In medicine

industry, chemicals from certain vegetables and fruits help in the cure of many diseases.

Besides food source, the economy of a country also depends largely on cultivation of food

crops. Due to increasing human population, the global food demand has also been driven

up. Food demand is expected to increase anywhere between 59% to 98% by 2050 (Maarten

& Florian, 2016). To sustain a large population, agriculture is playing its vital role. The

world’s food supply relies upon on about 150 plant species. Of these 150, just 120 offer 3-

quarters of the world’s food (FAO). Greater than 50% of the world’s food power comes

from restricted varieties of three ‘mega-vegetations’: rice, wheat and maize. To meet with

the global food demand, agricultural industry has to increase the crop production on yearly

basis.

Unfortunately, the food production faces a prime threat from pathogenic

microorganism which disturbs the steadiness of world ecosystem (Compant et al., 2005).

Every year crops are exposed to a variety of fungal and bacterial diseases which not only

reduce the aesthetic value of harvestable yield but also affect the storage life of agricultural

crops. This exposure of crops to diseases leads to huge economical losses. The major

agricultural pests which are more likely to damage food, ornamental and fiber crops,

include soil-borne pathogens, root knot nematodes and root rotting fungi. These

pathogenic organisms are responsible for disorders like crown and root rots, wilt disease,

root knot disease and damping off (Ehteshamul-Haque et al., 2007a, b). Treating fruit and

vegetable crops with synthetic chemicals provides prime technique for regulating pre-

harvest and post-harvest diseases. The usage of fungicides to preserve the good value of

harvest also generates a hazard for humans to these chemical compounds. Currently, the

health hazard related to fungicide acquaintance is a question of prodigious controversy and

discussion. As a direct consequence of this escalating alarm, substantial care has been

positioned on evaluating the possibility of the use of microbial antagonists as a viable

substitute to the artificial fungicides.

Amongst the root decomposing fungi, Fusarium spp. Rhizoctonia and

Macrophomina are the major threat to food crops. Fusarium spp. are responsible for the

cause of wilt, rot diseases on a broad range of valuable plants. More than 2000 plant

species are vulnerable to the attack of Fusarium (Brown and Proctor, 2013). The viability

of Fusarium Chlamydospores for several years in soil and their invasion to the root surface

reduces the disease management by crop protection (Haware et al., 1996). Eighty

notorious strains of only F. oxysporum cause the vascular wilt diseases in particular crops.

Mirza and Qureshi (1978) found that most of root fungal pathogens, including Fusarium

spp. are known to attack many cultivated plants and parasitized 36 host in Pakistan

including Cotton, Rape seed, Mustard, Groundnut, Linseed, Sunflower, Gram, Mungbean,

Tomato, Pea, Guar, Chilies, Citrus etc.

Another disease i.e Charcoal rot caused by Macrophomina phaseolina is also of

serious status in tumbling crop yield exclusively in arid region of the world (Hoes, 1985).

This pathogen spreads actively in varied climatic situations from arid to tropical regions

and has a wide host choice (Cottingham 1981; Abawi and Partor Carrales, 1990). Greater

than 500 hosts including legume and cereal plants are exposed to this fungus (Dhingra and

Chagas 1981; Sinclair 1982). Charcoal rot has been reported as a huge threat for sunflower

yield production (Ijaz et al., 2013, Iqbal and Mukhtar, 2014, Iqbal et al., 2014). Attack of

Rhizoctonia solani on a wide range of hosts especially herbaceous plants causes serious

diseases like Collar Rot, root rot, wire stem and damping off to a wide range of hosts

especially herbaceous plants. Various environmental conditions put the plant at higher risk

of contamination due to this pathogen. Hotter and moist climates favor its infection and

development. The seedlings are more vulnerable to disease in their early stages.

Use of synthetic fungicides seem to be the best-known technique for the control of

soil-pathogens. The application of pesticides, no doubt, results in the suppression of

diseases and crop yield and quality is also enhanced. However, the regular use of these

chemicals, may produce collateral problems. One of the major threat is the contamination

of environment with toxic elements which directly or indirectly affects the human and

animal health. Continuous application of fungicides to the crops also increases the

pathogen resistance to such chemicals in particular soil environments (Sparks 2013; Tupe

et al., 2014). These treatments are mostly non-specific i.e not only affect target pathogens

but also other beneficial microorganisms (Ranganathswamy et al., 2013).

Biological antagonists have now emerged as a practical substitute to the artificial

fungicides. Biological Control Agents (BCAs) are used to suppress the survival or activity

of a pathogen resulting in precising the incidence of diseases. In Biocontrol system, usually

fungi and bacteria are involved as BCAs. Indeed, natural microbial antagonists had proven

to control numerous rot pathogens on assorted commodities. A variety of successful cases

of biological control have been published for pre- or postharvest control: of Fusarium rot

in sorghum using rhizobacteria (Idris et al., 2007), application of Trichoderma in compost

soil for control of strawberry roots (Leandro et al., 2007), post-harvest diseases of citrus

fruit by preharvest applications of Pantoea agglomerans CPA-2 (Cañamás et al., 2008) or

application of Torulaspora globosa to control Colletotrichum sublineolum in sorghum

(Rosa et al., 2010), etc. Various other microorganisms such as Bacillus subtilis, B.

megaterium, B. mycoids, Pseudomonas putida, P. fluorescence, Enterococcus spp.

Azospirillum spp. and Trichoderma spp. are also reported as effective antagonists against

phytopathogens in many countries (Nguyen et al., 2011; Abd El-Kader et al., 2012;

Dawoud et al., 2012). The microorganisms that are used as BCAs either live naturally as

epiphytes on plant surface or as endophytes within plant tissues (Arnold et al., 2000; Inacio

et al., 2002; Lindow and Brandl, 2003; Yadav et al., 2004, 2005; Stapleton and Simons,

2006). Adopting of these organisms as antagonists for crop disease management is a

difficult task. Mode of action of the organism, its persistence and spread, and latent

harmfulness to non-target species is mainly considered during the selection (Harman,

2000., Lumsden, 1996., Mathre, et al., 1999). One specific feature is not accountable for

antagonistic effect of the organism. Considering their mode of action, the most essential

capabilities that have appeared from scientific inquiries include: 1) production of antibiotic

compounds 2) secretion of hydrolytic enzymes and 3) competition for nutrition.

Along with other microbial antagonists, yeasts are recently gaining interest by

scientists for use as a biocontrol agent against various phytopathogenic fungi. Yeast

represents a unique group of eukaryotic microorganisms among the Kingdom Fungi. They

are single-celled, despite the fact that some yeast species may have multicellular forms

due to the presences of pseudo hyphae (Kurtzman and Fell, 2005). This group of fungi

offers a substantial role in human affairs., the yeast species. The conversion

of carbohydrates to carbon dioxide and alcohols via fermentation by Saccharomyces

cerevisiae, has been utilized for thousands of years in baking and alcohol industries

http://en.wikipedia.org/wiki/Carbohydratehttp://en.wikipedia.org/wiki/Carbon_dioxidehttp://en.wikipedia.org/wiki/Ethanolhttp://en.wikipedia.org/wiki/Fermentation_(food)http://en.wikipedia.org/wiki/Baking

(Legras et al., 2007). Many other yeasts have the ability to convert byproducts of low

protein value into single cell protein which is then used for animal feeding (Alexopoulos

et al., 1996). This has been recorded in the process of cheese making, potato processing,

brewing and paper production. Apart from profitable benefits, yeasts are also encountered

as plant pathogens and mycoparasites, in the contamination and spoilage of food items,

and as human pathogens.

In nature, fermentable sugar rich liquids, extracts and plant exudates, provide

suitable niches for the development of yeasts (Phaff et al., 1964, Miller et al., 1962).

Ecology of yeasts is greatly affected by some physiochemical factors among which, the

most important appear to be the energy sources, nutrients, temperature, pH value and water

(Rose and Harrison, 1969). The single cell habit allows yeast to attain a much wider

ecological distribution than mycelial forms. Due to lack of photosynthetic power, the

yeasts strictly depend on the presence of organic carbon as an energy and carbon source.

Soil may offer true ecological niche for yeasts. Although found in lower numbers as

compared to other microorganisms in the soil, yeasts are able to build up significant

populations in a highly competitive environment (Miller and Webb, 1954). Different soil

quality, organic composition, moisture, soil pH at diverse geographical places and varied

climatic conditions affect the population of soil yeasts (Rose and Harrison, 1969).

Struggle for nutrition is possibly the single topmost issue in yeast ecology. Cuticle,

epidermis, lesions, senescing host tissues or openings like stomata and lenticels are

commonly nutrition-rich due to secretion of sugars and amino acids. These areas actually

serve as the entry points for the fungal pathogens into the plant body. Biocontrol agents

like yeast can strive their best to reside in these spots. They use the nutrients and

successfully displace the pathogen by averting germination of propagules or infection

(Filonow, 1998). Yeasts also act as antagonists of other organisms e.g. bacteria and fungi,

through the induction of environmental pH change (Rose and Harrison, 1969). The change

in pH is known to occur by the production of killer toxins, a unique character of some

yeasts (Bevan and Makower, 1963; Bussey, 1972; Rosini, 1983). These toxins have lethal

effects on strictly associated strains but the killer yeast itself is resistant to its toxin

production (Woods et al., 1968). They may also produce hydrolytic enzymes responsible

for degrading the cell walls of phytopathogenic fungi (Masih and Paul, 2002; Urquhart

and Punja, 2002), and poisonous volatile compounds (Bruce et al., 2004). Natural yeast

isolates are potential opponents (Ghaouth et al., 2003; Droby et al., 2002; Paster et al.,

1993), because they are biodegradable, cost effective, non-toxic to humans or other

animals. Like any other effective microbial antagonist, the biocontrol activity of those

yeasts encompasses various approaches like struggle for nutrition and space (Ghaouth et

al., 1998), cell-wall degrading hydrolytic enzymes production (Bar-Shimon et al., 2004),

physical contact with the pathogen (Chan and Tian, 2005), induction of host-resistance

(Yao and Tian, 2005) etc. Species of Pseudozyma were testified to hinder the development

of target organism by limiting the permeability of their cell membranes through some

secondary metabolites with antimicrobial activity (Avis and Be´langer, 2001).

Another key factor in the yeast antagonism is the production of hydrolytic enzymes

which may vary from specie to specie. Candida has proven to produce sufficient levels of

these enzymes for the Botrytis control (Saligkarias et al., 2002) as compared to Tilletiopsis

with insignificant enzymatic action towards powdery mildews (Urquhart and Punja, 2002).

The efficacy of these cell wall degrading enzymes is largely enhanced with the physical

contact of yeasts to fungal hyphae (Cook et al., 1997). In a study, more than 250 yeast

isolates showed positive attachment capability to Botrytis spores (Allen et al., 2004).

Amongst those, numerous yeast species were proved to be bioagents which include

Rhodotorula glutinis, Cryptococcus laurentii, and Pichia guillermondii for several post-

harvest pathogenic fungi (Zhang et al., 2011, Lahlali et al., 2014). Hyphal disintegration

of Monilinia fructicola, Penecillium expansum and Rhizopus stolonifer by the attachment

of P. membranefaciens and C. albidus was also reported in another study by Chan and

Tian (2005). A sturdy attachment of Rhodotorula glutinis to Botrytis spores is also recently

reported with increased biocontrol activity (Boqiang et al., 2016). These bioagents may

cause mycelial disruption of the pathogen by secreting certain enzymes like chitinase,

protease and glucanase (Chan and Tian, 2005, Zhang et al., 2011, Banani et al., 2014).

In the recent past, there has been a lot of studies accomplished on the evaluation of

yeast as biocontrol agent against fungal pathogens. Rhizosphere as well as phyllosphere

both provide suitable niches for the establishment of microorganisms. Owing to the cell

single cell habit and rapid cell growth, yeasts have advantage over other mycelial

organisms to inhabit these environments more rapidly. Many of the yeasts isolates from

these regions were investigated for their biocontrol action towards several serious

phytopathogens, both under screenhouse and field environments. Research is going on

both epiphytic and endophytic yeasts for their use as promising bioagents. Isolation of

active microorganisms from different plant species and their utilization against post-

harvest diseases have been previously reported (Janisiewicz and Roitman 1988, Gullino et

al., 1991). Blue mold (Penecillium expansum) and Grey mold (Botrytis cinerea) are the

two major diseases of apples leading to significant postharvest losses. Epiphytic yeasts,

Cryptococcus laurentii and Candida ciferrii, secluded from the surface of healthy apples

showed their biocontrol activity against blue mold caused by P. expansum (Vero et al.,

2002).

Numerous yeast strains have been recognized to inhibit plant diseases. Pichia

membranefaciens is reported to be positive inhibitor of Rhizopus stolonifer on nectarine

fruit (Fan and Tian, 2000), while Cryptococcus albidus acted a successful bioagent against

Penecillium expansum in apples and pears (Tian et al., 2002).

Yeasts strains of Cryptococcus laurentii have also been used as the postharvest

bioagent against grey mold rot of apples (Roberts, 1990), grey and blue mold rot of pears

and diseases of other fruits including kiwi fruits, strawberries, and table grapes (Lima, DE

Curtis 1998). Saccharomyces cerevisiae is also suggested to be a powerful biocontrol

agent towards apples blue mold (Jalal et al., 2009). Another research indicates that plant

growth enhancing, and biocontrol ability of S. cerevisiae makes it a strong competitor of

Fusarium oxysporum that causes damping-off symptoms in sugar beet seedlings (Shalaby

and El-Nady, 2008). Species of Fusarium are nectrotrophic pathogens of winter wheat that

contaminate crops through their mycotoxins (Mankeviciene et al., 2011, Baliukoniene et

al., 2011). Yeasts of genera Cryptococcus, Rhodotorula and Saccharomyces are

pronounced to have deleterious effects on Fusarium sporotrichioides to some satisfactory

degrees (Wachowska et al., 2013). Rhodotorula rubra strain TG-1 is an endophytic yeast

which has previously shown significant inhibitory effects against several fungal and

bacterial plant pathogens including Fusarium and Xanthomonas malvacearum strains

(Akhtyamova and Sattarova, 2013).

A unique phenomenon found in most of the yeast strains is the production of some

toxins which might contribute to the antagonistic activity of yeasts. These toxins termed

as ‘killer toxins’ were first reported in Saccharomyces cerevisiae by Bevan and Makower

in 1963. Later several other genera like Candida, Cryptococcus, Hanseniaspora,

Kluyveromyces, Pichia, Torulopsis, Ustilago, Williopsis and Zygosaccharomyces were

also identified as killer yeasts (Starmer et al., 1987). The killer toxins are proteinaceous in

nature which have fatal effects on other sensitive yeast strains. One killer strain may also

be sensitive to the toxin produced by other strong killer yeast strain (Woods et al., 1974).

Based on the killer character, the yeast phenotypes are characterized as killer, neutral and

sensitive. Generally, activity of these toxins is greatly enhanced at low pH optima but

elevated temperatures (35 oC) normally inactivate them. Analysis of toxin production and

immunity response of killer yeasts are extremely reliant on the use of sensitive strain and

suitable conditions for toxin activity (Tipper and Bostain, 1984; Mohamudha Parveen and

Ayesha Begum, 2010).

Killer yeasts have been classified into at least 11 groups (K1 to K11) based on

cross reactions (Rogers and Bevan, 1978; Young and Yagiu, 1978; Wickner, 1979).

Among these, only three groups viz K1, K2 and K3 have thoroughly been investigated.

There is a genetic variation for the expression of different killer phenotypes. For instance,

extensively studied K1 killer toxin in Saccharomyces cerevisiae is found dependent on the

existence of encapsulated double stranded RNA (dsRNA) molecules located in the

cytoplasm (Bussey, 1981; Tipper and Bostian, 1984). On the other hand, this killer

phenotype in Kluyveromyces and Pichia is encoded by linear dsDNA plasmid (Schaffrath

and Meinhardt, 2005) and chromosomally encoded in Williopsis sp. (Theisen et al., 2000;

Suzuki, 2005). K1 is a non-glycosylated extracellular protein having an estimated

molecular weight of approx. 11500 (Palfree and Bussey, 1979). It is a heat labile, being

stable around 22 oC and active only with a narrow pH range (4.2-4.8). The cell wall has an

imperative character in the interaction of toxin and host cell. This is a receptor mediated

process involving three main steps: 1) passive binding to a toxin primary receptor (β-1,6-

D-glucan) within the cell wall of a sensitive target cell, 2) energy-dependent transmission

of toxin to the plasma membrane to interact with a secondary receptor, 3) disruption of

membrane function by establishing cation-selective ion channels or blocking synthesis of

DNA and capture cells in primary S phase of the cell cycle. Thus, the toxin acts on the

plasma membrane by changing its permeability properties (Bussey and Sherman, 1973;

Skipper and Bussey, 1997; Schmitt and Radler, 1988; Santos et al., 2000). The known

resistance mechanisms for the killer yeasts to their own killer toxins are at the cell wall

receptor level (Brown et al., 1993). Production of these killer toxins ensures the

interference competition from one yeast excluding the other in a particular niche. For

example, strains of Torulaspora delbrueckii produce dsRNA encoded toxin that have the

ability to destroy the earlier recognized S. cerevisiae killer strains, along with other non-

Saccharomyces yeasts (Ramirez et al., 2015).

Many of the killer yeast strains have also proved to be promising biological

antagonists of plant pathogenic fungi. For example, Pichia membranefaciens is once

reported as an effective biocontrol agent against Grey mold (Botrytis cinerea) (Santos and

Marquina, 2004). In a research, Lopes et al., 2015 assessed the positive antifungal and

lethal effects of some killer isolates of S. cerevisiae against Colletotrichum acutatum,

causal agent of post bloom fruit drop.

Aim of the application of any BCA is to not only suppress the disease but also to

get high crop yield with good quality. A good and effective biocontrol agent has both the

inhibitory effect on the pathogen as well as plant growth promoting ability. Many of the

bioagents have been reported earlier as effective antagonists with positive impacts on plant

growth. In recent few years, PGPR has gained large attention of scientists as promising

biofertilizers. Many of the bacterial genera including Pseudomonas, Azotobacter,

Arthrobacter, Klebsiella have been recognized as plant growth promoters (George et al.,

2012; Beneduzi et al., 2013). PGPR can stimulate plant growth through various

mechanisms like by interacting with the phytopathogen, producing certain

phytohormones, inducing plant resistance or solubilizing phosphate or minerals in the soil

solution (Prasher et al., 2014; Myresiotis et al., 2014; Ahemad and Kibret; 2014).

Although, yeasts are comparatively present in low numbers in rhizospheric regions

than bacteria and filamentous fungi, they have the capability to elevate growth in plants

just like any other useful organism. A variety of yeast are known to produce

phytohormones (Nassar et al., 2005). Most common plant auxin, Indole Acetic Acid which

is directly involved in the promotion of several features of plant growth (Teale et al., 2006;

Spaepen et al., 2007), has been reported by many workers to be secreted by various yeasts

(Xin et al., 2009; Fu et al., 2016).

Capability of many microorganisms is known for the solubilization of the insoluble

phosphorus in soil and making it accessible to plants has extensively been studied (Rao,

1982; Halder et al., 1991; Abdalla, 1994; Whitelaw, 2000). Phosphorus deficiencies are

amongst the restraining issues for crop production. Gizaw et al., (2017) reported certain

yeast species including Pichia norvegensis, Rhodotorula aurantiaca, Cryptococcus

albidus var albidus, etc, as active phosphate solubilizer.

Amongst other plant growth promoting traits, siderophore production and NH3-

releasing ability is also noteworthy. Certain microorganisms are able to produce organic

compounds with low molecular masses known as Siderophores. These are the iron-

chelating compounds that bind and transport iron in microorganisms in Fe-stress

environments. Yeasts have also been documented for siderophore productions (Vero et al.,

2013; Spadaro et al., 2011). The yeast genus Rhodotorula is known to produce

‘Rhodotorulic acid’, a hydroxamate-type siderophore. Calvente et al., (1999) described the

antagonistic activity of these siderophores from Rhodotorula glutinis against Penecillium

expansum.

During a study, Fu et al., (2016) observed the production of NH3 and polyamines

by some yeast species including Pseudozyma, Aureobasidium, Dothideomyces,

Galactomyces candidum. NH3-releasing yeast become beneficial to plants because

nitrogen is a vital nutrient and the most frequently the limiting factor in crop yield.

Polyamines are the bioactive organic compounds occurring abundantly in cells of

microorganisms. Although these compounds are not plant hormones, but they are engaged

in numerous aspects of plant growth and their development (Evans and Malmberg, 1989;

Baron and Stasolla, 2008).

The present research describes the effort of isolating indigenous endophytic yeasts

from healthy plants and determining their biocontrol potential against the survival of root

rotting fungal pathogens accompanied with their plant growth promotion. The report also

describes the role of endophytic yeasts in enhancing the nutrient uptake and increase in

antioxidant, salicylic acid and phenolic status in plants.

2. MATERIALS AND METHODS

2.1. COLLECTION OF HEALTHY PLANT SAMPLES FOR THE ISOLATION

OF INDIGENOUS YEASTS

In the present study, 70 healthy plant samples belonging to 17 plant species viz.,

Cucumis sativus, Chenopodium album, Rubus idaeus, Solanum lycopersicum,

Abelmoschus esculantus (L.) Moench, Achyranthus aspera L., Azadirachta indica A.

Juss., Calendula officinales, Capsicum annuum L., Carica papaya L., Corchorus ridens

L., Cucurbita pepo L., Helianthus annuus L., Lycopersicon esculentum Mill., Momordica

charantia L., Solanum melongena L., and Tribulus terristeris were collected from

experimental fields of University of Karachi, Karachi. These plants were carefully taken

to the laboratory in sterile, and sealed plastic bags for further investigations.

2.2. ISOLATION OF INDIGENOUS ENDOPHYTIC YEASTS FROM

DIFFERENT PARTS OF HEALTHY PLANTS

Stems roots and leaves were separately cut into 2-3 cm long sections. The adhered

debris and epiphytic microorganisms were removed by washing the cuttings with sterile

water. They were then imperiled to consecutive washes for 1 minute with solutions

containing 1% sodium hypochlorite, 70% ethanol and sterile distilled water. Surface-

disinfected tissue was aseptically deliquesced with homogenizers. Serial dilution was

made up to 10-6 by adding 1 ml of well-shaken suspension and to 9 ml water blank tubes.

0.5 ml from last two dilutions was transfered on YM medium plates which were then

incubated for 5-7 days at 25 oC + 1 oC.

2.3. PURIFICATION AND MAINTENANCE OF YEAST ISOLATES FOR

FURTHER STUDIES

Three isolates from morphologically similar looking growing colonies of yeast

from each plate were carefully chosen as representatives of the yeast flora. These cultures

were preserved on yeast-morphology agar medium having pH 4.5. pure yeast cultures were

further preserved on anhydrous silica gel (Trollope, 1975). Yeast cells from 3-5 days old

cultures were mixed with sterilized double distilled water formerly chilled at 4 oC. Same

amount of chilled skim milk (15 % w/v, sterilized at 121 oC for 10 min.) was supplemented

to produce yeast suspension. The silica gel (8-18 mesh) was sterilized (180 oC for 15-24

min.) in Pyrex glass tubes. 0.5 ml of pre-cooled cell suspension was evenly distributed on

the gel granules in each chilled tube. These tubes were kept in close fitting boxes with self-

indicating silica gel either at room temperature or at 4 oC.

2.4. MORPHOLOGICAL TESTS FOR THE IDENTIFICATION OF YEASTS

2.4.1. Microscopic appearance of non-filamentous vegetative cells

The vegetative yeast cells were examined microscopically to check the type of

reproduction whether budding, splitting or both, location of buds in case of budding

whether multilateral or polar on the mother cells and shape and size of the vegetative cells.

Yeasts from a young culture (say, 1 day old) was inoculated into 30 ml. Of sterile

liquid malt extract medium in a 100 ml conical (erlenmeyer) flask. The cultures were

examined microscopically after 2-3 days incubation period at 25 oC ± 2 oC.

2.4.2. Microscopical examination for filamentous growth (Dalmau Plate Culture

Technique)

This examination ensures the ability of a yeast to produce filaments, kind of

filaments (pseudo or true hyphae), and kind of cells (if any e.g. Ballistoconidia,

arthroconidia etc.) Grown from the filaments (beech, 1972).

Sterilized petri plates were poured sterilized corn meal agar medium. The plates

were dried for 2 days at room temperature (beech, 1972). Five yeast cultures were streaked

per plate a small portion of each streak was covered with a sterile cover slip. The aerobic

portions of the streaks were observed for the filamentous growth of yeast cells after 3-5

days of incubation period.

2.4.3. Microscopical examination for ballistoconidia

Finding ballistoconidia gives decisive identification of a yeast as a member of the

family Sporobolomycetaceae (Bensingtonia, Bullera, Fibulobasidium, Sporobolomyces)

or the genus Sporidiobolus.

Agar medium 10 to 15 ml of malt extract or potato glucose was poured into a Petri

plate and dried for 2 days. Petri dish with the medium was inoculated with the test yeast

with 2 lines at right angles across its diameter. This plate was inverted onto another Petri

plate having agar medium with a sterile slide on it. The two dishes were taped together all

around the circumference. The setup was incubated for three weeks at 20 oC. Colonies

were formed on the agar of the lower plate by the discharged ballistoconidia. The spores

are also collected on the slide which can be examined microscopically (Barnett et al.,

1990).

2.4.4. Microscopical examination for ascospores

This examination was carried out to check if a yeast forms ascospores and if so,

the region of the ascospores whether they are produced from ordinary vegetative cells

without immediate previous conjugation or after conjugation between a mother cell as well

as its bud and the shapes and sizes of the ascospores.

A young culture, actively growing overnight, or for up to 2 days, at 25 oC on malt

yeast glucose peptone was used to inoculate ascosporulation medium i.e. acetate agar or

malt yeast glucose peptone agar. These inoculated media were incubated for 3 days at 25

oC and examined microscopically.

2.4.5. Germ Tube Formation

The formation of germ tube is a consistent means of identifying Candida albicans

and closely related species. Germ tube is defined as a thin continuous outgrowth of

filament without a constriction at the point of its origin. The germ tube formation can be

induced by suspending yeast cells from a 24-hour old culture in either normal blood serum

or egg albumin. Germ tubes, if formed by yeast, can be examined microscopically after 1-

3 h incubation time at 37 oC.

2.4.6. Ability to grow at different temperatures

It is of interest taxonomically to determine if a yeast will grow at various

temperatures especially at 25 oC, 30 oC, 35 oC, 37 oC, and 40 oC because possible

inferences regarding ability to grow in association with warm blooded animals may be

obtained. A tube with normal glucose assimilation medium, inoculated with test yeast and

incubated at the above said temperatures. Yeast growth was observed after 7 and 14 days.

The results were noted in the similar way as in the test for carbon compound assimilation.

2.4.7. Tolerance of 1% acetic acid (Yarrow, 1984d)

Acetic acid (1%) agar medium was prepared in 100 mL of double distilled water

and sterilized at 121 oC for 15 min. After cooling the medium to 50 oC, glacial acetic acid

(1 ml) was added to it, mixed rapidly and then poured into plates. The yeast suspension

was streaked on agar plates of test medium. The plates were incubated at 25 oC and

examined after 3 and 6 days for the development of colonies.

2.5. BIOCHEMICAL TESTS FOR THE IDENTIFICATION OF YEASTS

2.5.1. Carbon assimilation test

Many yeast species are easily distinguished by their differing ability to utilize

certain organic compounds each as a chief source of carbon for aerobic growth. The

medium used in carbon assimilation test were similar to that used for morphological

studies i.e., yeast morphology agar except that it contained no glucose, asparagine and

agar. Five gram of ammonium sulphate per liter was used as a nitrogen source. A ten-fold

concentrated stock medium was prepared along with 50 mM of appropriate carbon source.

The pH was adjusted to 4.5 or 5.2 if the carbon source was an acid. The solution was filter-

sterilized and kept in the refrigerator until needed. Half ml of the liquid medium was

transferred into clear 16 mm tubes filled with 4.5 ml of sterile distilled water.

An inoculation medium was prepared having the standard concentration of other

constituents with only 0.1 % of glucose. Cells from 24-48 h slants of the yeast culture were

deferred in 1 mL of prepared medium and were incubated at 25 oC for 48 h. The inoculum

was then diluted with the base medium which contained no glucose. Each tube in the set

of test medium received 0.1 ml of the diluted inoculum.

The tubes with various carbon sources were then incubated at 25 oC. results were

recorded at 7 days and again at 20 to 24 days. The presence of pellicles and visible amount

of riboflavin (practically the only soluble yellow compound produced by yeasts) was

noted. After shaking the tubes vigorously, they were placed against white card having 3/4

mm wide lines drawn with black ink. The growth was recorded as 3+ if it completely

obliterated the lines; 2+, if the lines were seemed as diffused bands, and the growth was

recorded as 1+, where the lines were clear as such but had indistinct edges. The absence

of growth was noted as negative. A 3+ or 2+ reaction at 7 days was considered as positive,

a 1+ reaction as weak (W), after 7 days as delayed (D), variable (V) response (i.e. either +

or -) and ? (response not known).

2.5.2. Assessing the ability to use certain sugars anaerobically

Just over the half of the known yeast species ferment at least D-glucose semi-

anaerobically. The ability of the yeast to use sugars anaerobically is assessed by looking

for the formation of gas, CO2. For this purpose, test tubes, 150 mm x 16 mm, with insert

Durham tubes of 50 mm x 10 mm were used. 10 to 15 ml of yeast extract medium [0.5 %

(w/v) of a commercially formed dehydrated yeast extract] was drawn into the tubes with

0.5% / 50 mm separately filter sterilized test sugars. Tubes without sugar served as

negative control (Barnett et al., 1990).

2.5.3. Hydrolysis of urea

Ability to hydrolyze urea distinguish ascogenous yeast from basidiomycetous one.

This property is mainly vague in ascogenous forms but found in basidiomycetous genera

such as Rhodotorula and Cryptococcus (Abadie 1967, Hagler and Ahearn 1981).

Test was done on Christensen’s Urea Agar medium (Christensen, 1946). 1 g peptone, 5 g

sodium chloride, 2 g dihydrogen phosphate and 12 µg phenol red dissolved in 800 mL of

distilled water with pH adjusted to 6.8. The solution was filter sterilized. 20 g of agar was

dispensed into 200 mL of water and autoclaved at 121 oC for 15 min. the agar was cooled

down to 50 %. Then urea base solution was added to agar medium. 5 ml of medium was

dispensed into screw capped glass tubes and positioned into slants.

The agar medium was inoculated with fresh cultures of yeast isolates and incubated

at 25 oC. The positive result is designated by the appearance of deep pink color on the

slant.

2.6. MOLECULAR IDENTIFICATION OF SELECTED YEAST STRAINS

The selected yeast isolates (KUAY-34, KUAY-38, KUAY-62 and KUAY-67)

were further identified by polymerase chain reaction (PCR) and restriction endonuclease

analysis (RFLP) of internal transcribed spacer region (ITS1-5.8S-ITS2) of ribosomal DNA

(rDNA) as significant molecular marker (Esteve-Zarzoso et al., 1999; Mohammadi et al.,

2013). Two reference yeast species, Saccharomyces boulardii (Enflor, probiotic, Hilton

Pharma) and Saccharomyces cerevisiae (Rossmor, Baker's yeast) were also included as

positive control. Yeast isolates were grown for48 hours in YPD broth at 37 ºC, the cell

pallets were prepared by centrifugation at 10,000 g (Hanil, Korea) for 10 minutes.

Genomic DNA was extracted by fungi/yeast genomic DNA isolation kit (Norgen biotek,

Canada) as per vender instruction. Purity and quantity of DNA preps were assessed by

Nano-drop (Nano-Drop 200, Thermo Scientific, USA). The contiguous ITS1-5.8S-ITS2

region was amplified with the primers set ITS1 (5´-TCCGTAGGTGAACCTGCGG-3´)

and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) as pronounced by Karimi et al., (2015).

PCR reactions were achieved in 20 μL volume containing; 1.5 μL genomic DNA (90

ng/μL), 0.8 μL of 16 μM each primer, 6.9 μL nuclease free water and 10 μL of 2x dream

taq master mix (Thermo Scientific, USA). For RFLP analysis of ITS1-5.8S-ITS2, the

restriction endonuclease selection was aided by in-silico digest of ITS regions in

Saccharomyces boulardii (GenBank: AY428861.1) and Saccharomyces cerevisiae

(GenBank: AY247400.1) using Snap Gene Viewer 2.2.2. The reconstructive

phylogenetics was performed after digesting 10 µL of ITS amplicon by Hae-III

(Fermentas, USA). The restriction patterns were analyzed by Gel Cluster software. The

UPGMA-dice coefficient method was used to construct phylogenetic tree on the distance

matrix.

2.7. DETERMINATION OF INDOLE ACETIC ACID PRODUCTION (IN VITRO)

BY ENDOPHYTIC YEASTS BY YEASTS

The yeasts were grown in liquid medium containing 0.1% (w/v) l-tryptophan and

then incubated in the dark for five days. Supernatants were collected after centrifuging the

tubes at 4000 rpm for 15 min. 1 ml of supernatant was mixed with 2 ml salkowski’s

reagent. The mixture could stand for 30 min for color development (red). Color intensity

was measured at a wavelength of 530 nm using a spectrophotometer (Gordon and Weber,

1951). IAA production was calculated against the calibration curve using authentic IAA.

2.8. PHOSPHATE SOLUBILIZATION CAPACITY OF YEASTS

The ability of yeast to solubilize inorganic phosphorus was determined in vitro by

using Pikovskaya’s agar medium (Pikovskaya, 1948). The fresh cultures of yeast isolates

were point inoculated on plates containing solid medium and left to incubation for 5 days

at 25 oC. The solubilization of phosphorus was specified by the production of distinct halos

around yeast colonies.

2.9. ISOLATION OF ROOT INFECTING FUNGI FROM SOIL

2.9.1 Isolation of Fusarium spp. through Soil Dilutions

1 g of sample soil was added to sterilized test tubes containing 10 ml of 0.1% agar

suspension. From 10 ml mixture, more dilutions were made and 1 ml from last dilution

was evenly spread over PCNB agar medium surface (Nash and Synder, 1962). Fusarium

species were identified from the plates after 5 days of incubation at 28 oC (Brown and

Proctor, 2013).

2.9.2 Isolation of Rhizoctonia solani through Baiting Procedure

For isolating R. solani, sterilized sorghum grains were used as bait. These grains

were placed on the surface of moist soil in sterilized Petri plate. The seeds were removed

after 24 hours, washed and then shifted to PDA medium for fungal growth (Wilhelm,

1955). Population percentage in soil was determined by counting seeds colonized by R.

solani.

2.9.3 Isolation of Macrophomina phaseolina through Wet Sieving and Dilution

Technique

Soil sample (20 g) was passed over 100 mesh (150 µm) and 300 mesh (53 µm)

sieves. After washing the residues retained on 53 µm under tap water for 1 minute, they

were transferred to a beaker containing 0.5 % Ca(OCl)2 solution with 1:5 dilution and

made up to 100 ml volume. The suspension containing sclerotia of M. phaseolina was

stirred over magnetic stirrer. 1 ml of aliquot was spread over PDA medium supplemented

with penicillin (1000000 units/L), streptomycin (0.2 g/L), rose bangal (0.1 g/L) and

domsan (0.3 g/L). After the incubation period of 5 days at 28 oC, grayish to black colonies

produced by M. phaseolina were recognized as explained by Shaikh and Ghaffar (1975).

2.10. TEST FOR ANTIFUNGAL ACTIVITY TOWARDS ROOT ROTTING

FUNGI

2.10.1 Dual Culture Plate Assay for In Vitro Testing against Root Rotting Fungi

The antagonistic activity of yeasts against root rotting fungal pathogens was

determined by dual culture plate assay. The Petri plates were poured with sterilized PDA

medium. After solidification, the medium was streaked with yeast isolates on one side. A

disc (5 mm) of fungi viz., Fusarium solani, F. oxysporum, Rhizoctonia solani and

Macrophomina phaseolina was placed on the side of plates opposite to each yeast streak.

The plates were incubated at 28 oC for 3-7 days. The inhibitory effect of yeasts was

assessed by measuring zones of inhibition produced by yeasts against test fungi.

2.11. CELL FREE CULTURE FILTRATES OF YEASTS

Yeast cultures were inoculated in yma liquid medium and incubated at 25 oc for 5

days. After incubation period, the broths were centrifuged for 10 min at 4000 rpm. The

supernatants were shifted to separate tubes whereas pellets were discarded.

2.12. NEMATICIDAL ACTIVITY OF YEASTS

2.12.1 In Vitro Testing for Juvenile Mortality

1 mL suspension of freshly hatched second juvenile (20 juveniles) were shifted in

cavity blocks with 1 mL of a yeast culture filtrate. Each isolate was kept with three

replicates. The setup was left at 26 ±5 oC for 48 hours after which mortality of second

stage juveniles was determined. The nematodes were considered dead if didn’t move when

probed with needle (Cayrol et al., 1989).

2.13. POPULATION OF YEAST BIOAGENTS (ANTAGONISTS)

2.13.1 Colony Forming Unit (cfu/mL) in Suspension

Number of yeast cells were determined with the help of Dilution plate method. 0.1

mL yeast suspension from last dilution was dispensed on YMA medium and incubated for

three days at 25 oC. Plates with yeast colonies were counted and multiplied by the dilution

factor that gave cfu/mL of yeast.

Cfu/mL = Number of yeast colonies on plate x Dilution factor

2.14. DETERMINATION OF PHOSPHORUS FROM LEAVE SAMPLES

Both wet digestion or dry ashing procedures can be used for the estimation of total

phosphorus content in plant material but later one is simpler, easier, non-hazardous and

economical. The dry ashing method described by Rayan et al., (2001) was used with a few

modifications.

2.14.1 Wet Digestion of Sample:

1 g oven dried leave sample (dried at 120 oC for 24 hrs) was crushed and liquified

in 2N-HCl. The extract was digested for 60 min and then filtered (What man No.1 filter

paper).

2.14.2. Reagents:

• Barton reagent (Ammonium vanadate-molybdate)

For the preparation of Barton’s reagent, two solutions were needed:

Solution A: 25 g ammonium molybdate was dissolved in 400 ml distilled water.

Solution B: 1.25 g ammonium metavanadate was dissolved in 300 ml of boiling

water, cooled and then 250 ml concentrated HNO3 was added to it.

Then solution A and B were mixed, and volume made up to one liter.

• Standard stock solution of phosphorus

50 ppm phosphorus stock solution was prepared by dissolving 0.2197 g of anhydrous

KH2PO4 in 1000 mL of distilled water.

2.14.3. Procedures for preparation of tests, standards and blank

• Test:

Digested filtrate (10 ml) and freshly prepared Barton reagent (10 mL) were

transferred to a 100-mL volumetric flask. Final volume of the mixture was made up to 100

mL with distilled water. Mixture was kept at normal temperature for 30 min. Within

incubation time, yellow phosphorus vando-molybdate complex appeared. The absorbance

was recorded against blank at 420 nm spectrophotometrically.

• Standards:

Concentrations from 1-10 ppm phosphorus/ml were arranged to make various

standards and headed as test.

• Blank:

Mixture containing 10 mL of Barton reagent in 100 mL of distilled water served as

blank.

2.14.4. Calculation

Concentration of phosphorus was calculated using the formula

% P = ppm P (from calibration curve) x Wt x 10000

Where:

R = Ratio of total volume of the digest and volume of the digest used for

measurement

Wt = Weight of the dry plant (g)

2.15. ESTIMATION OF POLYPHENOLS

The extraction of oven dried leaves was done with ethanol (96 % v/v). After

centrifuging the extracts at 3000 rpm for 20 min, supernatants were used for analyzing

polyphenols and antioxidants.

The Folin-Ciocalteu assay described by Chandini et al., (2008) was applied for the

quantification of total phenols from plant leaves. 2 mL of 2% Na2CO3 was added to 100

mL of extract (10 mg/mL) and left untouched for 2 min at room temperature. After adding

100 µL Folin-Ciocalteu reagent (50%), the mixture was incubated in dark place for 30

min. The test samples were subjected to spectrophotometry for determining their

absorbances at 720 nm. Standard curve obtained from Gallic acid was used for the

estimation of total phenol contents in the samples.

2.16. ESTIMATION OF ANTIOXIDANT ACTIVITY

DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay (Zubia et al., 2007) was employed

for determining antioxidant activity in sample leaves.

An aliquot of 200 µL of sample extract was mixed with 800 µL of 10 Mm Tris-

HCl buffer (pH 7.4). 30 µM DPPH (dissolved in DMSO) was added to the mixture and

vortex. The mixture was left to stand at room temperature in dark. Mixture of 1 mL ethanol

and 1 mL DPPH was served as control. The absorbance was measured at 517 nm

spectrophotometrically against blank after 1 minute and 30 minutes of incubation. The

DPPH radical scavenging ability was calculated using the following equation:

% of inhibition = Acontrol - Asample x 100

Acontrol

Where, Acontrol is the absorbance of the control (DPPH solution without sample)

Asample is the absorbance of the test sample (DPPH solution plus test

sample)

2.17. ESTIMATION OF SALICYLIC ACID

Amount of salicylic acid (SA) in sample leaves was determined by

spectrophotometric method as described by Warrier et al., (2013). 0.1 mL of chilled

ethanolic leaf extracts was assorted with 3 mL of 0.1 % freshly prepared ferric chloride.

The absorbance of the reaction complex (violet) was noted at 540 nm by

spectrophotometry. The quantity of SA (μg mL-1) was calculated and expressed in mg/gm-

1 dried sample by using standard curve, where 100 mg of SA was dissolved in 100 mL of

ethanol.

2.18. INFECTION PERCENTAGE:

Infection percentage of root pathogens was calculated as follows:

Infection % = No. of plants infected by a pathogen x 100

Total no. of plants

2.19. DATA ANALYSIS:

Two-way ANOVA was used for fungal infection in order to compare the means

among the treatments and also among different fungal pathogens. The follow up of

ANOVA included least significant difference (LSD) at (p

3. EXPERIMENTAL RESULTS

3.1 ISOLATION OF ENDOPHYTIC YEASTS FROM DIFFERENT PARTS

OF HEALTHY PLANTS

100 endophytic yeasts were isolated from different parts i.e. leaves, stems and roots

of the plant samples belonging to 7 species viz; Azadirachta indica A. Juss., Calendula

officinales, Carica papaya L., Chenopodium sp., Lycopersicon esculentum Mill., Rubus

idaeus, and Cucumis sativus. (Table 1). These yeasts isolates were purified and preserved

on Yeast Morphology Agar medium at 4 oC for further investigations.

3.2 SCREENING OF THE ENDOPHYTIC YEASTS AGAINST ROOT KNOT

NEMATODE AND ROOT INFECTING FUNGI

3.2.1 In Vitro Nematicidal Activity of Cell Free Culture Filtrates of Endophytic

Yeasts:

Nematicidal activity of isolated yeasts was determined by coinoculating freshly

hatched second stage nematode juveniles with cell free yeast culture filtrates. The juvenile

mortality was observed after 24 hrs. and 48 hrs. of incubation period. The experiment was

set up with three replicates for each yeast isolate.

Culture filtrates of different yeast isolates showed nematicidal activity against root

knot nematode juveniles at varying degrees. 100 % juvenile’s mortality was exhibited by

yeast isolates viz; KUAY-5, KUAY-19, KUAY-29, KUAY-30, KUAY34, and KUAY-66

within 24 hrs. of incubation while yeast isolates KUAY-10, KUAY-21, KUAY-31,

KUAY-65, and KUAY-67 killed the juveniles completely (100 %) after 48 hrs. Yeast

cultures KUAY-18, KUAY-28, KUAY-32, KUAY-35, KUAY-37, KUAY-38, KUAY-40,

KUAY-41, KUAY-43, KUAY-44, KUAY-45, KUAY-50, KUAY-51, KUAY-56,



KUAY-83, KUAY-84, KUAY-86, KUAY-87, KUAY-88, and KUAY-96 showed 72%,

80%, 80%, 95%, 75%, 70%, 86.5%, 89%, 75%, 88%, 88%, 88%, 78%, 80%, 77%, 90%,

89%, 76%, 78%, 88%, 89%, 89%, 76%, 86%, 91%, 91%, 80%, 89%, 79%, 87%, 84%,

79%, 89%, and 72% mortality respectively (Table 2).

Table 1. List of yeasts isolated from different parts of healthy plants.

Yeast isolates Source Locality

KUAY-1 Calendula officinalis University of Karachi

KUAY-2 " University of Karachi






KUAY-8 Azadirachta indica University of Karachi




KUAY-12 Cucumis sativus University of Karachi














KUAY-26 Chenopodium album University of Karachi






KUAY-62 Carica papaya University of Karachi


















KUAY-80 Rubus idaeus University of Karachi










KUAY-90 Solanum lycopersicum University of Karachi

KUAY-91

University of Karachi


KUAY-92 Solanum lycopersicum University of Karachi








Table 2. Effect of cell free culture filtrates of different isolates of yeasts on

juvenile mortality of Meloidogyne javanica, the root knot nematode.

Yeast isolates Nematode mortality (%)

24 hrs 48 hrs

Control

0

20

KUAY-1 38.3 53.3

KUAY-2 28 33.3

KUAY-3 25 30

KUAY-4 64 66.6

KUAY-5 100 100

KUAY-6 30 63.3

KUAY-7 30 42.5

KUAY-8 26.5 38.3

KUAY-9 25.5 31.66

KUAY-10 60 100

KUAY-11 50 59.3

KUAY-12 56 62

KUAY-13 0 12

KUAY-14 53 60

KUAY-15 50 50

KUAY-16 42.6 60

KUAY-17 62 68

KUAY-18 68 72

KUAY-19 100 100

KUAY-20 11 17.3

KUAY-21 66.6 100,

KUAY-22 28.77 42.33

KUAY-23 41.5 60

KUAY-24 56 68

KUAY-25 11 18

KUAY-26 29 37.6

KUAY-27 50.6 54

KUAY-28 68 80

KUAY-29 100 100

KUAY-30 100 100

KUAY-31 50 100

KUAY-32 68 80

KUAY-33 56 66

KUAY-34 100 100

KUAY-35 78 95

KUAY-36 40.5 43


24 hrs 48 hrs

KUAY-37

58

75

KUAY-38 64.8 70

KUAY-39 64 69

KUAY-40 70 86.5

KUAY-41 80 89

KUAY-42 66 75

KUAY-43 75 88

KUAY-44 64 88

KUAY-45 87 88

KUAY-46 58 66

KUAY-47 55 59

KUAY-48 57.5 63

KUAY-49 67 69

KUAY-50 66 78

KUAY-51 58 80

KUAY-52 38 43.8

KUAY-53 56 67

KUAY-54 45 66

KUAY-55 48 65

KUAY-56 57 80

KUAY-57 55 65

KUAY-58 23 30

KUAY-59 56 77

KUAY-60 89 90

KUAY-61 65 68

KUAY-62 48 54

KUAY-63 78 89

KUAY-64 56 76

KUAY-65 92 100

KUAY-66 100 100

KUAY-67 98 100

KUAY-68 50 55.7

KUAY-69 58 78

KUAY-70 58 67

KUAY-71 49 60

KUAY-72 48.6 59.7

KUAY-73 59 65

KUAY-74 52 65

KUAY-75 66 89

KUAY-76 58 76

KUAY-77 79 86


24 hrs 48 hrs

KUAY-78

48

56

KUAY-79 68 91

KUAY-80 78 91

KUAY-81 56 80

KUAY-82 76 89

KUAY-83 68 79

KUAY-84 66 87

KUAY-85 46 67

KUAY-86 66 84

KUAY-87 67 79

KUAY-88 74 89

KUAY-89 30 35

KUAY-90 26 40

KUAY-91 39 51

KUAY-92 45 58

KUAY-93 29.5 48

KUAY-94 25 32

KUAY-95 55.5 67

KUAY-96 58 72

KUAY-97 41.5 52

KUAY-98 58 66

KUAY-99 38 51.5

KUAY-100 50 63.5

3.2.2 In Vitro Antifungal Activity of Endophytic Yeasts Against Root Rotting

Fungi:

The inhibitory effect of yeasts towards root decaying fungal pathogens viz;

Fusarium solani, F. oxysporum, Macrophomina phaseolina and Rhizoctonia solani was

assessed in vitro by Dual Culture Plate method. The inhibitory effect was determined by

observing varying zones of inhibition produced by yeast cultures after 3-7 days of

incubation at 28 oC

One hundred yeast isolates, tested for their antifungal activity, showed varying

degrees of inhibition against test fungi. Sixty-nine isolates inhibited the mycelial progress

of three test fungi viz; F. solani, F. oxysporum and M. phaseolina. No yeast isolate showed

inhibition of R. solani. Amongst these isolates, some produced significant zones of

inhibition around fungal growth. KUAY-1 isolate produced zones of 17, 20 and 18 mm

against F. solani, F. oxysporum and M. phaseolina respectively. KUAY-5 formed 10 mm

inhibitory zone around F. solani growth, 15 mm against F. oxysporum, whereas 25 mm

zone towards M. phaseolina. KUAY-9 showed 13, 25 and 22 mm zones of inhibition with

Fusarium solani, F. oxysporum and M. phaseolina respectively. Zones of 14, 9 and 11 mm

were produced by KUAY-10 isolate for F. solani, F. oxysporum and M. phaseolina

respectively. KUAY-17 represented inhibition of F. solani by forming 16 mm clear zones,

whereas 27 and 15 mm zones against mycelial progress of F. oxysporum and M.

phaseolina respectively. KUAY-34 produced 22, 28 and 24 mm whereas KUAY-38

represented inhibitory zones of 14, 22 and 25 mm for the mycelial growth of F. solani, F.

oxysporum and M. phaseolina respectively. KUAY-52 suppressed the mycelial progress

of F. solani, F. oxysporum and M. phaseolina by producing inhibitory zones of 15, 3 and

20 mm respectively. KUAY-54 restricted the mycelial growth of the three fungi by

producing inhibition zones of 18 mm, 21 mm and 22 mm respectively. KUAY-62

suppressed F. solani, F. oxysporum and M. phaseolina by producing 28 mm, 24 mm and

26 mm zones respectively. KUAY-66 produced 22 mm, 19 mm and 20 mm inhibitory

zones against Fusarium solani, F. oxysporum and M. phaseolina respectively. KUAY-70

represented the restriction of F. solani, F. oxysporum and M. phaseolina by producing

zones of 10 mm, 15 mm and 19 mm respectively. In the same way, zones of 11, 20 and 14

mm were produced to stop the growth of F. solani, F. oxysporum and M. phaseolina

respectively by the yeast isolate KUAY-72. Lysis of fungal hyphae was also caused by

some yeast isolates for example in case of KUAY-37, 39, 41, 42, 43, 44, 45, 46, 47, 48,

49, 51, 53, 64, 69, 74, 77, 78, 80, 82, 85, 86, 87, 88, 89, 91, 92, 93, 95 and KUAY-96

(Table 3). Maximum inhibition of F. solani growth was observed by the yeast culture

KUAY-34, KUAY-62, and KUAY-66. Yeast isolates KUAY-9, KUAY-25, KUAY-34,

KUAY-38, and KUAY-62 produced maximum growth inhibition of F. oxysporum,

whereas proliferation of Macrophomina phaseolina was greatly affected by isolates

KUAY-5, KUAY-9, KUAY-34, KUAY-38, and KUAY-62.

Table 3. In vitro inhibition of Fusarium solani, F. oxysporum,

Macrophomina phaseolina and Rhizoctonia solani by yeast isolates.

Yeast isolates Zone of inhibition (mm)

F. solani F. oxysporum M. phaseolina R. solani

KUAY-1

17

20

18

0

KUAY-2 4 11 15 0

KUAY-3 7 8 12 0

KUAY-4 7 5 15 0

KUAY-5 10 15 25 0

KUAY-6 15 10 12 0

KUAY-7 14 8 11 0

KUAY-8 10 15 7 0

KUAY-9 13 25 22 0

KUAY-10 14 9 11 0

KUAY-11 11 11 10 0

KUAY-12 11 10 20 0

KUAY-13 12 7 15 0

KUAY-14 7 14 11 0

KUAY-15 6 6 5 0

KUAY-16 0 2 8 0

KUAY-17 16 27 15 0

KUAY-18 0 ** 11 0

KUAY-19 10 11 15 0

KUAY-20 15 18 21 0

KUAY-21 9 18 12 0

KUAY-22 5 10 14 0

KUAY-23 11 6 9 0

KUAY-24 7 13 7 0

KUAY-25 9 7 12 0

KUAY-26 5 9 15 0

KUAY-27 4 11 12 0

KUAY-28 11 7 5 0

KUAY-29 12 6 5 0

KUAY-30 5 10 4 0

KUAY-31 11 14 9 0

KUAY-32 10 ** 7 0

KUAY-33 15 ** 11 0

KUAY-34 22 28 24 0

KUAY-35 17 19 12.5 0

KUAY-36 5 11 12 0

KUAY-37 10 ** ** 0

KUAY-38 14 22 25 0

KUAY-39 ** ** 11 0



KUAY-40

11

10

7

0

KUAY-41 20 ** ** 0

KUAY-42 11 ** ** 0

KUAY-43 ** 5 7 0

KUAY-44 ** 7 5 0

KUAY-45 ** ** 2 0

KUAY-46 ** 2 ** 0

KUAY-47 ** ** 5 0

KUAY-48 ** ** 7 0

KUAY-49 ** ** ** 0

KUAY-50 10 2 50 0

KUAY-51 11 ** ** 0

KUAY-52 15 3 20 0

KUAY-53 11 ** 10 0

KUAY-54 18 21 22 0

KUAY-55 15 18 18 0

KUAY-56 12 6 7 0

KUAY-57 12 5 8 0

KUAY-58 11 10 15 0

KUAY-59 11 8 12 0

KUAY-60 15 18 20 0

KUAY-61 18 20 15 0

KUAY-62 28 24 26 0

KUAY-63 25 18 15 0

KUAY-64 18 15 ** 0

KUAY-65 20 12 15 0

KUAY-66 22 19 20 0

KUAY-67 15 11 12 0

KUAY-68 18 9 5 0

KUAY-69 11 ** ** 0

KUAY-70 10 15 19 0

KUAY-71 5 11 10 0

KUAY-72 11 20 14 0

KUAY-73 7 15 11 0

KUAY-74 6 ** 9 0

KUAY-75 5 15 5 0

KUAY-76 7 11 3 0

KUAY-77 7 7 ** 0

KUAY-78 6 8 ** 0

KUAY-79 9 10 11 0

KUAY-80 11 11 ** 0



KUAY-81

10

9

5

0

KUAY-82 5 ** ** 0

KUAY-83 4 6 5 0

KUAY-84 11 3 7 0

KUAY-85 10 9 ** 0

KUAY-86 9 11 ** 0

KUAY-87 8 ** ** 0

KUAY-88 14 17 ** 0

KUAY-89 15 ** 11 0

KUAY-90 11 5 7 0

KUAY-91 7 ** 8 0

KUAY-92 5 5 ** 0

KUAY-93 6 9 ** 0

KUAY-94 13 5 11 0

KUAY-95 12 ** 4 0

KUAY-96 5 4 ** 0

KUAY-97 18 5 7 0

KUAY-98 11 7 6 0

KUAY-99 4 9 11 0

KUAY-100 10 11 8 0

** = Lysis of mycelium

Fig. 1. Restriction of mycelial growth of root rotting fungi by Endophytic yeast

KUAY-5 in dual culture method


KUAY-9 in dual culture method

investigation on biocontrol potential of endophytic yeasts...

Documents