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i

EVALUATION OF PURPUREOCILLIUM LILACINUM AND

GLOMUS ON PLANT GROWTH AND CONTROL OF

MELOIDOGYNE INCOGNITA OF EGGPLANT

IN ARSENIC CONTAMINATED SOIL

KHALID HASAN

DEPARTMENT OF PLANT PATHOLOGY

SHER-E-BANGLA AGRICULTURAL UNIVERSITY

DHAKA-1207

JUNE, 2015

ii





BY

KHALID HASAN

REG. NO. 09-03598

A Thesis

Submitted to

The Department of Plant Pathology, Faculty of Agriculture,

Sher-e-Bangla Agricultural University, Dhaka,

in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

IN

PLANT PATHOLOGY

SEMESTER: JAN-JUN, 2015

APPROVED BY:

(Dr. F. M. Aminuzzaman)

Professor

Department of Plant Pathology

Sher-e-Bangla Agricultural University

Supervisor

(Dr. M. Salahuddin M. Chowdhury)

Professor



Co-Supervisor

(Dr. Md. Belal Hossain)

Associate professor

Chairman

Examination Committee


Sher-e-Bangla Agricultural University, Dhaka

iii

CERTIFICATE

This is to certify that the thesis entitled, “EVALUATION OF

PURPUREOCILLIUM LILACINUM AND GLOMUS ON PLANT GROWTH

AND CONTROL OF MELOIDOGYNE INCOGNITA OF EGGPLANT IN

ARSENIC CONTAMINATED SOIL” submitted to the Department of Plant

Pathology, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka,

in the partial fulfillment of the requirements for the degree of MASTER OF

SCIENCE (M. S.) IN PLANT PATHOLOGY, embodies the result of a piece of

bonafide research work carried out by KHALID HASAN bearing Registration No.

09-03598 under my supervision and guidance. No part of the thesis has been

submitted for any other degree or diploma.

I further certify that such help or source of information, as has been availed of during

the course of this investigation has duly been acknowledged.

Dated:

Place: Dhaka, Bangladesh

(Dr. F. M. Aminuzzaman)

Professor



Supervisor

Department of Plant Pathology Fax: +88029112649

Sher - e - Bangla Agricultural Universit y Web site: www.sau.edu.bd

Dhaka - 1207 , Bangladesh

iv

LIST OF ABBREVIATED TERMS

ABBREVIATION FULL WORD

AMF Arbuscular Micorrhizal Fungi

As Arsenic

et al. And others

BARI Bangladesh Agricultural Research

Institute

Cm3 Centimeter cube

Cm2 Centimeter square

Cm Centimeter

µgcm-2 Microgram/cm2

CV. Cultivar

oC Degree centigrade

Etc. Etcetera

Ed. Edited

Eds. Edition

G Gram

J. Journal

No. Number

PDA Potato Dextrose Agar

LSD Least Significant Difference

DMRT Duncan’s New Multiple Range Test

% Percent

RCBD Randomized Completely Block Design

Res. Research

SAU Sher-e-Bangla Agricultural University

Viz. Namely

ppb Parts per billion

v

ACKNOWLEDGEMENTS

First of all, I would like to submit my gratitude to Almighty Allah, the most

merciful and compassionate. The most Gracious and beneficent to whom every

admire is due and to his Prophet Muhammad (SM) who is perpetually a set on

fire of knowledge and leadership for humanity as a whole with whom delighting

the present endeavor has been beautiful.

Now I would like to give inexpressible gratefulness to my commendable supervisor

Dr. F. M. Aminuzzaman, Professor, Department of Plant Pathology, Sher-e-

Bangla Agricultural University, Dhaka. I am obliged to his ever inspirational

direction, studious comments, constructive suggestions and well-mannered

behavior right through the course of my study.

I express my especial thanks to my esteemed Co- Supervisor, Dr. M. Salahuddin

M. Chowdhury, Professor, Department of Plant Pathology, Sher-e-Bangla

Agricultural University, Dhaka, for his correct direction, inspirational

collaboration and support during the research work and preparation of thesis.

I am decidedly express my thanks to my honorable teachers Professor Mrs. Nasim

Akhtar, Dr. Md. Rafiqul Islam, Dr. Nazneen Sultana, Assoc. Prof. Khadija

Akhter, Dr. Nazmoon Naher Tonu, Dr. Md. Belal Hossain, Abu Noman Faruq

Ahmmed, Asstt. Prof. Shukti Rani Chowdhury, Md. Ziaur Rahman Bhuiya,

Department of Plant Pathology and Professor Dr. Md. Razzab Ali, Department of

Entomology, Faculty of Agriculture, Sher-e-Bangla Agricultural University, for

their valuable teaching, direct and indirect suggestion and encouragement and

support during the whole study period.

vi

I am impressed to thank all stuffs and employees of Plant Pathology Department

and all farm labors of Sher-e-Bangla Agricultural University, Dhaka for their

valuable and sincere help in carrying out the research work.

I also express my especial thanks to my well-wishers and friends Amit Kumar,

Md. Abdullah-Al-Mamun, Md. Mahabub Elahi, Md. Mostaqur Rahman, Babul

Akhter, Afrin Akter Faria, Kollol, Shimul, Sanjida, Nitu and Shammi for their

help and support during my work.

I found no words to thanks my parents, brother, brother-in-law and my sister for

their unquantifiable love and constant support, their sacrifice never ending

affection, immense strength and untiring efforts for bringing my dream to proper

shape. They were constant source of inspiration, zeal and enthusiasm in the

critical moment of my studies.

The Author

vii

CONTENTS

CHAPTER TITLE PAGE

LIST OF ABBREVIATED TERMS iv

ACKNOWLEDGEMENTS v- vi

LIST OF CONTENTS vii-xi

LIST OF TABLES xii

LIST OF PHOTOGRAPHS Xiii

LIST OF FIGURES xiv-xvi

LIST OF PLATES xvii

ABSTRACT xviii

1. INTRODUCTION 1-3

2. REVIEW OF LITERATURE 4-34

2.1 Role of AMF in different crops 4-11

2.2 Interaction of arsenic and AMF 13-18

2.3 Interaction of AMF and nematode 19-27

2.4 Interaction of P. lilacinum and M. incognita 28-34

3. MATERIALS AND METHODS 35-42

3.1. Experimental site and experimental period 35

3.2. Environment of experiments 35

3.3. Pot Experiment 35

3.3.1. Crop variety used 35

3.3.2. Collection of seeds 35

viii

CONTENTS (cont’d)

CHAPTER TITLE PAGE

3.3.3. Soil collection and sterilization 35

3.4. Raising of seedling 36

3.5. Preparation of pots 37

3.6. Treatments and design of the experiment 38

3.6.1. Treatments 38

3.6.2. Design of the experiment 38

3.7. Isolation, identification and culturing of AMF 39

3.8. Fungal Isolate 43

3.8.1. Culture, mass production and harvesting of

Purpureocillium lilacinum

43

3.9. Nematode inoculum preparation 45

3.10. Preparation of arsenic solution 47

3.11. General inoculation procedure for the experiment 47

3.12. Intercultural operations 47

3.13. Harvesting and data recording 48

3.14. Data recorded 49

3.14.1. Plant data 49

3.15. Counting of nematode egg masses/root system 51

3.16 Slide preparation and counting of eggs/egg mass 53

3.17 Extraction of nematode from soil and counting of

juveniles

53

ix

CONTENTS (cont’d)

CHAPTER TITLE PAGE

3.18. Gall index 56

3.19. Egg masses colonization (%) by Purpureocillium lilacinum 57

3.20. Soil colonization by Purpureocillium lilacinum (CFUg-1 soil) 58

3.21. Observation of roots for mycorrhizal infection 59

3.22. Study of spore population in soil 60

3.23. Chemical analysis of plant sample 61

3.23.1. Nutrient analysis 61

3.23.2. Preparation of plant sample 61

3.23.3. Digestion of plant samples with nitric-perchloric acid mixture 61

3.23.4. Phosphorus 61

3.23.5. Potassium 61

3.23.6. Nitrogen 62

3.23.7. Arsenic 62

3.24. Analysis of data 62

4. RESULTS AND DISCUSSION 63-106

4.1. For all treatment combination 63

4.1.1. Shoot length 63

4.1.2. Root length 64

4.1.3. Shoot fresh weight 67

4.1.4. Root fresh weight 67

x

CONTENTS (cont’d)

CHAPTER TITLE PAGE

4.1.5. Shoot dry weight 67

4.1.6. Root dry weight 68

4.1.7. Leaf area 68

4.1.8. Chlorophyll content 69

4.2. For Glomus sp. involved treatment combination 73



4.2.3. Leaf area 76






4.2.9. Number of spore/10 g soil 83

4.2.10. Root infection 84

4.3. For Purpureocillium lilacinum treatments 85



4.3.3. Leaf area 87


xi

CONTENTS (cont’d)

CHAPTER TITLE PAGE





4.3.9. CFU/g soil 93

4.4.1. Gall index 94

4.4.2. Number of eggmass/ root 95

4.4.3. Number of egg/ eggmass 96

4.4.4. Eggmass colonization 97

4.4.5. Reproduction factor 98

4.5. For Meloidogyne incognita involved treatments 99

4.5.1. Number of eggmass/root 99

4.5.2. Gall index 100

4.5.3. Number of egg/ eggmass 101

4.5.4. Reproduction factor 102

4.5.5. Nutrient uptake 103-105

4.5.6. Arsenic uptake 106

5. SUMMARY AND CONCLUSION 107-109

REFERENCES 110-129

xii

LIST OF TABLES

SL.

NO.

TITLE PAGE

1. Physicochemical characteristics of pot soil

37

2. Influence of Purpureocillium lilacinum in combination

with Glomus sp. on shoot length, root length, shoot and

root fresh weight of eggplant in arsenic amended soil

challenged with Meloidogyne incognita

66


with Glomus sp. on dry weight of shoot and root, leaf area

and chlorophyll content of eggplant in arsenic amended

soil challenged with Meloidogyne incognita

69


with Glomus sp. on phosphorus, potassium and Sulphur

percentage of shoot of eggplant in arsenic amended soil


104

xiii

LIST OF PHOTOGRAPHS

SL.

NO.

TITLE PAGE

1 Raising of eggplant seedling 36

2 Identification of Glomus sp. 40

3 Confirmation of colonization observing vesicle inside

the cell of Cassia tora

40

4 Inoculation of Glomus spore on maize seed 41

5 Inoculation of Glomus spore in maize root 41

6 Mass culture of Glomus sp. with trap plant maize 42

7 Pure culture of P. lilacinum on PDA media 44

8 Mass culture of P. lilacinum on chick pea 44

9 M. incognita inoculum production in association with eggplant root 46

10 M. incognita infected root showing eggmass and gall 46

11 Leaf area measurement by CI-202 Portable Laser Leaf Area Meter 50

12 Measurement of chlorophyll content of eggplant

leaf by SPAD 502 Plus Chlorophyll Meter

50

13 Heavily galled root treated with Phloxine-B solution 52

14 Phloxine-B treated root for counting of eggmass/ root 52

15 Counting the number of egg/ eggmass 54

16 Extraction of nematode by Bangladeshi plate method (modified

White Head and Heaming method, 1965)

54

17 Second stage juveniles of Meloidogyne incognita 55

18 Determination of CFUg-1 soil using the soil dilution plate method 58

19 Different growth pattern of eggplant in different treatment

combination during two months of growing

71

http://www.cid-inc.com/products/leaf-area-lai/portable-laser-leaf-area-meter

http://www.specmeters.com/nutrient-management/chlorophyll-meters/chlorophyll/spad502p/

xiv

LIST OF FIGURES

SL.

NO.

TITLE PAGE

1 Shoot length of eggplant influenced by the eight mycorrhizal

treatments in different combination of P. lilacinum, M. incognita

and arsenic

72

2 Represents the root length of eggplant influenced by the eight

mycorrhizal treatments with different combination of P. lilacinum,

M. incognita and arsenic

73

3 The role of Glomus sp. on leaf area of eggplant in combination of

P. lilacinum in arsenic amended soil challenged with Meloidogyne

incognita

74

4 Chlorophyll content of eggplant influenced by Glomus sp in

combination of P. lilacinum in arsenic amended soil challenged

with Meloidogyne incognita

75

5 Variation of shoot fresh weight of eggplant due to eight

mycorrhizal treatments of eggplant influenced by Glomus sp. in



76

6 Shoot dry weight of eggplant influenced by Glomus sp. in



77

7 Root fresh weight of eggplant influenced by Glomus sp. in



78

8 Shoot dry weight of eggplant influenced by Glomus sp. in



79

9 Number of spore/ 10 g soil influenced by Glomus sp in



80

10 Root infection (%) influenced by Glomus sp in combination of P.

lilacinum in arsenic amended soil challenged with Meloidogyne

incognita

81

xv

LIST OF FIGURES (cont’d)

SL.

NO.

TITLE PAGE

11 Shoot length of eggplant influenced by the eight P. lilacinum

involved treatments in different combination with Glomus sp., M.

incognita and arsenic

82

12 Root length of eggplant influenced by the eight P. lilacinum

treatments with different combination of Glomus sp., M. incognita

and arsenic

83

13 The role of P. lilacinum involved treatments on leaf area of

eggplant in combination of Glomus sp in arsenic amended soil


84

14 Chlorophyll content of eggplant influenced by the role of eight P.

lilacinum treatments in combination of Glomus sp. in arsenic

amended soil challenged with Meloidogyne incognita

85

15 Variation of shoot fresh weight of eggplant due to eight P.

lilacinum treatments of eggplant influenced by the role of P.

lilacinum in combination of Glomus sp. in arsenic amended soil


86

16 Shoot dry weight of eggplant influenced by P. lilacinum involved

treatments in combination of Glomus sp. in arsenic amended soil


87

17 Root fresh weight of eggplant influenced by P. lilacinum involved

treatments in combination of Glomus sp in arsenic amended soil


88

18 Root dry weight of eggplant influenced by P. lilacinum involved

treatments in combination of Glomus sp. in arsenic amended soil


89

19 Influence of P. lilacinum in different combination with Glomus sp.

in arsenic amended soil challenged with Meloidogyne incognita

on CFU/ g soil

90

20 Gall index of eggplant influenced by P. lilacinum 91

21 Role of P. lilacinum on the number of eggmass/ root 92

22 Effect of P. lilacinum on the number of M. incognita egg/ eggmass 93

xvi

LIST OF FIGURES (cont’d)

SL.

NO.

TITLE PAGE

23 Role of P. lilacinum on of eggmass colonization by bioagent 94

24 Role of P. lilacinum on of reproduction factor of M. incognita 95

25 Number of eggmass/ root of M. incognita influenced by Glomus

sp. in combination of P. lilacinum in arsenic amended soil

96

26 Gall index of eggplant influenced by P. lilacinum and Glomus sp.


97

27 Number of egg/ eggmass of M. incognita on different combination

with P. lilacinum and Glomus sp. in arsenic amended soil


98

28 Reproduction factor of M. incognita influenced by Glomus sp in

combination of P. lilacinum in arsenic amended soil

99

29 Arsenic uptake by shoot of eggplant influenced by Glomus sp in



104

xvii

LIST OF PLATES

SL.

NO.

TITLE PAGE

1 Egg colonization of M. incognita by P. lilacinum 57

2 Observation of eggplant roots for mycorrhizal infection 60

3 Eggplant root at different treatments combination 70

xviii





BY

KHALID HASAN

ABSTRACT

Root-knot nematode Meloidogyne incognita remarkably reduces eggplant growth

and yield in Bangladesh. Nematophagous fungus Purpureocillium lilacinum has

profound role on suppression of M. incognita. Again, contamination of groundwater

by arsenic and plant uptake from soil contaminated by groundwater or irrigation

water. AM fungi have significant effect on plant growth reducing arsenic

contamination to plant. This study determined the role of AMF (Glomus sp.)

combined with the P. lilacinum on growth of eggplant and nematode control in

arsenic amended soil challenged with M. incognita. AMF colonized root fragments

of maize seedlings and rhizosphere soil (100 g) containing spore were used for AMF

treatment in combined with P. lilacinum maintaining CFU (5x106) of P. lilacinum/g

soil mixed with 50 ppm arsenic. Eggs of M. incognita was adjusted to 10000

eggs/pots for inoculation. All growth characteristics was higher in combined

treatment of AMF (Glomus sp.) and P. lilacinum (G+Pl) inoculated plants in

comparison to the other treatments, and decreased significantly with the M.

incognita (Mi) and arsenic (As) involved treatment combination. But, M. incognita

(Mi) and arsenic (As) combined with (G), (Pl) and (G+Pl) gave better results rather

than their individual treatments. The findings of this research revealed less M.

incognita infection and arsenic content, highest leaf area, higher chlorophyll content

and nutrient uptake in (G+Pl) inoculated plants.

1

INTRODUCTION

A large area being covered with vegetable crops resulting in increased yield and thus

created a better socio-economic status for farmers in Bangladesh. The challenge now

is to grow more food of high nutritive quality. The solution lies in developing and

adopting hi-tech agriculture to improve the productivity in eco-friendly manner.

Eggplant belonging to Solanaceae family is chosen for the study, as this is a commonly

consumed vegetable in our country. Commercially it is less expensive and

economically more important. It is widely grown in Bangladesh, China, India,

Pakistan and Philippines. It is also popular in others countries like Balkan area,

France, Indonesia, Italy, Japan, Mediterranean, Turkey and United states (Bose and

Som, 1986). It is well-known as “Begoon” (Eggplant) in Bangladesh, is a very

common and favorite vegetable. It is grown in an area of about 1,15,424 acres

producing about 341262 Mt of fruits where 44,377 acres in Kharif season and 71,047

acres in Rabi season of the year with total annual production of 3,41,262 M. Tons and

the average yield is 5.86 t/ha in 2009-2010 (BBS, 2011). The yield potential of

eggplant is low in Bangladesh compared to other countries. Incidence of insects, pests

and diseases generally hampered the production of eggplant. This crop suffers from

the various diseases; about 13 different diseases so far recorded in Bangladesh (Das

et al. 2000; Rashid, 2000). Among those diseases, root knot of eggplant has been

treated as one of the major constraints in eggplant cultivation in Bangladesh. Root

knot nematodes are plant parasitic organism of the genus Meloidogyne spp. About

2000 plants are susceptible to infection by root knot nematodes Meloidogyne spp.

Root knot is widely distributed important diseases in the country (Talukdar, 1974;

Ahmed and Hossain, 1985). In Bangladesh root knot may cause as much 27.2% loss

in fruit yield of eggplant (BARI, 2001). Eggplant cultivation in Bangladesh is severely

impaired by three important wilt causing pathogens viz. Ralstonia solanacearum,

Fusarium oxysporum and Meloidogyne spp. the causal agent of Bacterial wilt,

2

Fusarium wilt and Nemic wilt, respectively and caused considerable damage of

eggplant (Talukdar, 1974; Ahmed and Hossain, 1985; Ali et al., 1994). These are also

the major limiting factors for eggplant production throughout the world (Hinata,

1986). Infection of roots by nematodes alter uptake of water and nutrients and

interferes with the translocation of minerals and photosynthesis (Williamson and

Hussey, 1996). Such alterations change the shoot and root ratio (Anwar and Van

Gundy, 1989) and expose the plant to other pathogens. For example, nematode root

infection increases the incidence and severity of Fusarium wilt diseases on a variety

of crops (Martin et al., 1994), which can negatively influence yield (Orr and Robison,

1984). Vegetable yield reductions have reached as high as 30% for susceptible

genotypes in the presence of plant parasitic nematodes in some production areas

(Anwar et al., 2009a). The control of plant parasitic nematode is a difficult task has

mainly depended on chemical nematicide for remarkable reduction of nematode

population has been achieved (Jatala,1985). Biocontrol seems to be the most relevant

and practically damaging approach for the control of root knot nematode is an

excellent biocontrol agent in tropical and subtropical agricultural soils.

Purpureocillium lilacinum has been reported to reduce nematode population densities

and is considered as one of the most promising practiciable biocontrol agent for the

management of plant parasitic nematodes (Jatala., 1986; Siddiki et al., 2000; Eapen et

al., 2005; Atkins et al., 2005; Kiewnick et al., 2011). A pre-planting soil treatment

reduced root galling by 66% egg masses by 74% and the final nematode population

in the roots by 71% compared to the inoculated control (Kiewnick et al., 2006). It is

now recognized that AM fungi can be harnessed in order to improve productivity in

agriculture, fruit culture, and forestry by reducing the input of fertilizers and/or by

enhancing plant survival, thus offsetting ecological and environmental concerns. For

this reason, studies on mycorrhizae gained importance due to its practical use as a low

input technology for managing soil fertility and plant nutrition. Cofcewicz et al.

(2001) under greenhouse conditions studied the interaction of arbuscular mycorrhizal

fungi Glomus etunicatum and Gigaspora margarita and root knot nematode,

3

Meloidogyne javanica and their effects on the growth and mineral nutrition of tomato.

The result pointed out that the shoot dry matter yield was reduced by nematode

infection and this reduction was less pronounced in plants colonized with G.

etunicatum than plant colonized with G. margarita and non-mycorrhizal plants.

Arbuscular mycorrhizae (AM) are the most common mycorrhizal form and formed

arbuscules (Agrios, 1988). Arbuscular mycorrhizal fungi (AMF), as an important

group of soil fungi, can form symbiotic associations with more than 80% of the land

plant families (Schwarzott et al., 2001). AMF can essentially improve plant mineral

nutrition and plant water relations and enhance plant resistance to heavy metal

contaminations (Hildebrandt et al., 2007). Recent studies show that the arbuscular

mycorrhizas naturally occur in As-contaminated soils (Smith et al., 2010) and

mycorrhizal inoculation can improve the As tolerance of tomato (Liu et al., 2005b),

maize (Bai et al., 2008). For the better efficacy in eco-friendly management of

nematode and mitigating arsenic problem soil during vegetable production in arsenic

polluted area of Bangladesh, evaluation of Purpureocillium lilacinum in combination

with Arbuscular Mycorrhizal Fungus (Glomus sp.) on plant growth and suppression

of Meloidogyne incognita on eggplant in arsenic amended soil is an endeavor to find

out the following objectives.

Objectives:

1. To study the effect of Purpureocillium lilacinum either alone or in combination

with Mycorrhizal fungus (Glomus sp.) on plant growth and suppression of root knot

nematode Meloidogyne incognita on eggplant in arsenic amended soil.

2. To determine the effect of fungal antagonist P. lilacinum either alone or in

combination with Arbuscular Mycorrhizal fungus on nutrient uptake and reducing

arsenic toxicity of eggplant in arsenic amended soil.

4

REVIEW OF LITERATURE

2.1. Role of AMF in different crops

Priyadharsini et al. (2015) proposed that most of the agricultural and

horticultural crops are associated with common soil fungi, the arbuscular mycorrhizal

(AM) fungi. These fungi are crucial for plant health and fitness as they increase the

efficiencies of the plant root systems. The hyphae of these fungi originating from roots

grow into the soil and absorb nutrients especially phosphorus and deliver it to the

roots. They also play a crucial role in imparting tolerance to plants against various

stresses as well as modifying soil structure.

Caporale et al. (2014) investigated the effect of arbuscular mycorrhizal fungi

(AMF - Glomus spp.) on the growth of the vetiver grass (Chrysopogon zizanioides L.)

and its As uptake from contaminated hydroponic and soil systems. An ameliorative

effect of the AMF inoculation in enhancing plants growth was found, mainly by

stimulating the development of their root system. In addition, AMF-inoculated plants

also took up more As from both contaminated systems compared to non-inoculated

plants, although the differences were not always statistically significant (p < 0.05).

Kelkar et al. (2013) carried out this experiment where soils with different

concentrations of arsenic with and without mycorrhizal inoculums were tested in

Trigonella foenumgraceum. The response of mycorrhiza in T. foenumgraceum was

determined in terms of percentage germination of seeds, sustainability of seedlings,

fresh weight and dry weight of plants etc. It was observed that in the pot soil

contaminated with arsenic and no mycorrhizal inoculum, performance was very bad

in terms of all aspects of growth, whereas in the pot where mycorrhizal inoculum was

added along with contaminated soil, the performance of the plant was better.

5

Beltrano et al. (2013) conducted a study in a greenhouse, to investigate the

effects of arbuscular mycorrhizal fungi (Glomus intraradices), soil salinity and P

availability on growth (leaf area and dry weight), nutrient absorption and ion leakage,

chlorophyll, soluble sugar and proline content and alkaline phosphatase activity of

pepper plants (Capsicum annuum L.). Plants were grown at four levels of salinity (0,

50, 100 and 200 mM NaCl) and two P levels (10 and 40 mg kg-1). Colonization was

80 to 51% in non-stressed and high salt-stressed plants, respectively. The mycorrhizal

dependency was high and only reduced at the higher salinity level. Mycorrhizal plants

maintained greater root and shoot biomass at all salinity levels compared to non-

mycorrhizal plants, regardless the P level. Interactions between salinity, phosphorous

and mycorrhizae were significant for leaf area, root and shoot dry mass. The results

indicate that the mycorrhizal inoculation is capable of alleviating the damage caused

by salt stress conditions on pepper plants, to maintaining the membranes stability and

plant growth, and this could be related to P nutrition.

Abdullahi et al. (2013) conducted a field experiment to determine the effect of

arbuscular mycorrhizal fungi (AMF) in reducing the excessive amount of chemical

fertilizer used in cultivation of onion. Inoculated and un-inoculated onion plant were

grown with varying levels of N and P fertilizer (00-00, 40-20, 60-30, 80-40, 100-50

and 120-60 kg ha-1 N and P, respectively), K was constant at 50 kg ha-1 laid out in

randomized complete block design with 3 replications. Mycorrhizal colonization (%),

plant height (cm), number of leaves per plant, fresh and dry shoot biomass (g), and N,

P, and K concentrations in plant were determined. The results showed no significant

difference in plant height and number of leaves per plant between inoculated and un-

inoculated plants at 4 weeks after transplant (WAT) for all treatments. Inoculated

plants with 60-30-50 kg ha-1 NPK produced plants with highest growth parameters

(38.63 cm, 13.66, 27.80g and 3.74 g) for plant height, number of leaves, fresh shoot

and dry biomass, respectively as compared to un-inoculated plants with high dosages

6

(120-60-50 kg ha-1 NPK) of fertilizer. From this study, it can be concluded that using

AMF could reduce the amount of excessive chemical fertilizer needed to produce

onion.

Gomes et al. (2012) found that Anadenanthera peregrina is a Brazilian

savanna tree species that occurs naturally in arsenic (As)-contaminated areas, and it’s

As resistance has been associated with arbuscular mycorrhizal fungi (AMF)

symbiosis. A plant’s ability to survive in stressful environments is correlated with its

nutrition status, which can be affected by As uptake. The present study evaluated the

influence of As on the concentrations and distribution of nutrients in the roots and

shoots of A. peregrina grown in the absence of AMF. These plants were grown in

substrates spiked with 0, 10, 50, and 100 mg As kg-1 for 25 d under greenhouse

conditions, and the concentrations of essential macro- (P, K, Ca, Mg, N, and S) and

micro- (Fe, Mn, Cu, Zn, B, and Mo) nutrients in the roots and shoots were then

determined. Enhanced As levels increased the concentrations of P, S, and N and

decreased Ca, Mg, and Fe.

Bücking et al. (2012) investigated approximately 80 % of all known land plant

species form mycorrhizal interactions with ubiquitous soil fungi. The majority of

these mycorrhizal interactions is mutually beneficial for both partners and is

characterized by a bidirectional exchange of resources across the mycorrhizal

interface. The mycorrhizal fungus provides the host plant with nutrients, such as

phosphate and nitrogen, and increases the abiotic (drought, salinity, heavy metals) and

biotic (root pathogens) stress resistance of the host. In return for their beneficial effect

on nutrient uptake, the host plant transfers between 4 and 20% of its

photosynthetically fixed carbon to the mycorrhizal fungus.

Smith et al. (2011) reviewed new findings about the roles of the arbuscular

mycorrhizas (mycorrhiza = fungus plus root) in plant growth and phosphorus (P)

7

nutrition. They focus particularly on the function of arbuscular mycorrhizal (AM)

symbioses with different outcomes for plant growth (from positive to negative) and

especially on the interplay between direct P uptake via root epidermis (including root

hairs when present) and uptake via the AM fungal pathway. The results are highly

relevant to many aspects of AM symbiosis, ranging from signaling involved in the

development of colonization and the regulation of P acquisition to the roles of AM

fungi in determining the composition of natural plant assemblages in ecological

settings and their changes with time.

Irfan et al. (2011) conducted an experiment on Solanum melongena L. a

medicinally and economically important crop plants were grown in polythene bags.

The effect of mycorrhizal inoculation (Glomus mosseae) and increasing phosphate

levels on the expression of the photosynthetic activity in terms of chlorophyll content.

Antioxidant enzymes like peroxidase, polyphenol oxidase, root acid and alkaline

phosphatase activity of Solanum melongena were evaluated. The experimental design

was entirely at CRBD with eight treatments with three levels of phosphate

(50,100,150 mg kg-1 of soil). Root colonization ranged from 50.33 to 67.33%. The

activity of the studied antioxidant enzymes was found to be increased in arbuscular

mycorrhizal (AM) Solanum plants. This work suggests that the mycorrhiza helps

Solanum plants to perform better in low and high phosphate level by enhancing

antioxidant enzyme activity, acid and alkaline phosphatase activity and total

chlorophyll content.

Aggarwal et al. (2011) reported that mycorrhizal symbiosis is a highly evolved

mutually beneficial relationship. This symbiosis confers benefits directly to the host

plant’s growth and development through the acquisition of Phosphorus (P) and other

mineral nutrients from the soil by the AMF. In addition, their function ranges from

stress alleviation to bioremediation in soils polluted with heavy metals. They may also

enhance the protection of plants against pathogens and increases the plant diversity.

8

This is achieved by the growth of AMF mycelium within the host root (intra radical)

and out into the soil (extra radical) beyond. Proper management of Arbuscular

Mycorrhizal fungi has the potential to improve the profitability and sustainability of

agricultural systems.

Ortas (2010) evaluated the effects of different arbuscular mycorrhizal fungi

(AMF) under field conditions for cucumber production. The parameters measured

were seedling survival, plant growth and yield, and root colonization. In 1998 and

1999, Glomus mosseae and Glomus etunicatum inoculated cucumber seedlings were

treated with and without P (100 kg P2O5 ha-1) application. A second experiment was

set up to evaluate the response of cucumber to the inoculation with a consortia of

indigenous mycorrhizae, G. mosseae, G. etunicatum, Glomus clarum, Glomus

caledonium and a mixture of these four species. Inoculated and control non inoculated

cucumber seedlings were established under field conditions in 1998, 2001, 2002 and

2004. Seedling quality, seedling survival under field conditions and yield response to

mycorrhiza were tested. The field experiment results showed that mycorrhiza

inoculation significantly increased cucumber seedling survival, fruit yield, P and Zn

shoot concentrations. The most relevant result for growers was the increased survival

of seedlings.

Akond et al. (2008) carried out an investigation for fifteen plant species,

cultivated widely as vegetable crops in mycorrhizal colonization in their root tissues

with a range of 7 to 98% variations in root infections and spore densities were found

statistically significant. Plant species had a significant role in root tissue colonization

by mycorrhizal fungi.

Ali (2008) reported that AMF has great influence on growth of some

agricultural crops like brinjal, tomato, okra, danta and chili. Mycorrhiza enhanced

9

disease reduction in all the selected agricultural crops and also significantly influenced

the nutrient uptake capacity of crops over control.

Van et al. (2008) explored the various roles that mycorrhizal fungi play in

sustainable farming systems with special emphasis on their contribution to crop

productivity and ecosystem functioning. A number of mechanisms and processes by

which mycorrhizal fungi can contribute to crop productivity and ecosystem

sustainability. Results showed that the significance of mycorrhizal fungi for

sustainable farming systems.

Nogueira et al. (2006) evaluated the response of Rangpur lime (Citrus limonia)

to arbuscular mycorrhiza (Glomus intraradices), under P levels ranging from low to

excessive. Plants were grown in three levels of soluble P (25, 200 and 1,000 mg kg-

1), either inoculated with Glomus intraradices or left noninoculated, evaluated at 30,

60, 90, 120 and 150 days after transplanting (DAT). Total dry weight, shoot P

concentration and specific P uptake by roots increased in mycorrhizal plants with the

doses of 25 and 200 mg kg-1 P at 90 DAT. With 1,000 mg kg-1 P, mycorrhizal plants

had a transient growth depression at 90 and 120 DAT, and non-mycorrhizal effects on

P uptake at any harvesting period. Root colonization and total external mycelium

correlated positively with shoot P concentration and total dry weight at the two lowest

P levels. Although the highest P level decreased root colonization, it did not affect

total external mycelium to the same extent.

Islam (2006) carried out an experiment on the role of arbuscular mycorrhizal

(AM) fungi on growth and nutrient uptake of some legumes. He observed growth

response was positive to AMF in all the selected legumes. The seedlings emergence,

plant height, shoot length and root length of inoculated legumes were comparatively

higher than that of uninoculated legumes.

10

Giri et al. (2005) assessed the effect of two arbuscular mycorrhizal (AM) fungi,

Glomus fasciculatum and G. macrocarpum on shoot and root dry weights and nutrient

content of Cassia tora in a semi-arid wasteland soil. Under nursery conditions

mycorrhizal inoculation improved growth of seedlings. Root and shoot dry weights

were higher in mycorrhizal than non-mycorrhizal plants. The concentration of P, K,

Cu, Zn and Na was significantly higher in AM inoculated seedlings than non-

inoculated seedlings. On transplantation to the field, the survival rate of mycorrhizal

seedlings (75-90%) was higher than that of non-mycorrhzal seedlings (40%).

Combination of AMF and Pseudomonus proved to be better. Present findings

indicated that microbial gene pool especially the key helpers for the maintenance of

soil health residing in the vicinity if roots were positively affected by using

Pseudomonus and AMF.

Karagiannidis et al. (2002) studied the effect of the arbuscular mycorrhizal

fungus (AMF) Glomus mossae and the soil borne Verticillium dahliae and their

interaction on root colonization, plant growth and nutrient uptake in eggplant and

tomato seedlings grown in pots. Root colonization by the AMF as well as spore

formation was higher (34.6% and 30.5%, respectively) in the eggplant than in tomato.

The mycrrohizal treatments increased fresh and dry weight and mean plant height in

tomato by 96, 114, and 21% compared to control.

Mridha and Xu (2001) studied the genus diversity of AMF fungi in some

vegetable crops in Bangladesh. They identified Acaulospora, Entrophosphora and

Glomus abundantly. But Gigaspora and Sclerocystis were poor in number.

George et al. (2000) reported that arbuscular mycorrhizal fungi (AMF) can

greatly affect the plant uptake of mineral nutrients. It may also protect plants from

harmful elements in soil. The contribution of AM fungi to plant nutrient uptake is

mainly due to the acquisition of nutrients by the extra-radical mycorrhizal hyphae.

Many mycorrhizal fungi can transport nitrogen, phosphorus, zinc and copper to the

11

host plant, but other nutrients can also be taken up and translocated by the hyphae.

Among the nutrients, phosphorus is often the key element for increased growth or

fitness of mycorrhizal plants because phosphorus is transported in hyphae in large

amounts compared to the plant phosphorus demand.

Dodd et al. (2000) reported AMF are primarily responsible for nutrient transfer

from soil to plant but have other roles such as soil aggregation, protection of plant

against drought stress and soil pathogens and increasing plant diversity. This is

achieved by the growth of their fungal mycelium within a host root and out into the

soil beyond. In agro- and natural ecosystems AMF are pivotal in closing nutrient

cycles and have a proven multi-functional role in soil-plant interactions.

Mridha et al. (1999) studied AM colonization in some crops of Bangladesh.

They observed high levels of colonization in the numbers of Leguminosae family and

no colonization in Amaranthaceae, Chenopodiaceae and Cruciferae.

12

2.2. Interaction of arsenic and AMF

Zhang et al. (2015) conducted two pot experiments where wild type and a non-

mycorrhizal mutant (TR25:3-1) of Medicago truncatula were grown in arsenic (As)-

contaminated soil to investigate the influences of arbuscular mycorrhizal fungi (AMF)

on As accumulation and speciation in host plants. The results indicated that the plant

biomass of M. truncatula was dramatically increased by AM symbiosis. Mycorrhizal

colonization significantly increased phosphorus concentrations and decreased As

concentrations in plants. Moreover, mycorrhizal colonization generally increased the

percentage of arsenite in total As both in shoots and roots, while dimethyl arsenic acid

(DMA) was only detected in shoots of mycorrhizal plants. The results suggested that

AMF are most likely to get involved in the methylating of inorganic As into less toxic

organic DMA and also in the reduction of arsenate to arsenite. The study allowed a

deeper insight into the As detoxification mechanisms in AM associations.

González et al. (2014) conducted research to identify the in situ localization

and speciation of arsenic (As) in the AM fungus Rhizophagus intraradices [formerly

named Glomus intraradices] exposed to arsenate [As(V)]. By using a two-

compartment in vitro fungal cultures of R. intraradices-transformed carrot roots,

micro-spectroscopic X-ray fluorescence (m-XRF), and micro spectroscopic X-ray

absorption near edge structure (m-XANES). It was observed that As(V) is absorbed

after 1 h in the hyphae of AMF. Three hours after exposure a decrease in the

concentration of As was noticed and after 24 and 72 h no detectable As concentrations

were perceived suggesting that As taken up was pumped out from the hyphae. No As

was detected within the roots or hyphae in the root compartment zone three or 45 h

after exposure. This suggests a dual protective mechanism to the plant by rapidly

excluding As from the fungus and preventing As translocation to the plant root. m-

XANES data showed that gradual As(V) reduction occurred in the AM hyphae

between 1 and 3 h after arsenic exposure and was completed after 6 h.

13

Bhalerao et al. (2013) found that arbuscular mycorrhizal (AM) Fungi provide

an attractive system to advance plant based environmental clean-up. AM associations

beingintegral functioning parts of plant roots and are widely recognized as enhancing

plant growth on severely disturbed sites, including those contaminated with heavy

metals. They are reported to play an important role in metal tolerance and

accumulation. Isolation of the indigenous and presumably stress-adapted AM fungi

can be a potential biotechnological tool for inoculation of plants for successful

restoration of degraded ecosystems.

Orłowska et al. (2012) assessed the role of indigenous and non-indigenous

arbuscular mycorrhizal fungi (AMF) on As uptake by Plantago lanceolata L. growing

on substrate originating from mine waste rich in As in a pot experiment. P. lanceolata

inoculated with AMF had higher shoot and root biomass and lower concentrations of

As in roots than the non-inoculated plants. There were significant differences in As

concentration and uptake between different AMF isolates. Inoculation with the

indigenous isolate resulted in increased transfer of As from roots to shoots; AMF from

non-polluted area apparently restricted plants from absorbing As to the tissue. The

mycorrhizal colonization affected also the concentration of Cd and Zn in roots and Pb

concentration, both in shoots and roots.

Karim et al. (2011) found that arbuscular mycorrhizal fungi (AMF) present on

the roots of plants growing on heavy metals contaminated soils and play an important

role in metal tolerance and accumulation enhancing plant growth on severely

disturbed sites, including those contaminated with heavy metals (HMs). Isolation of

the indigenous and presumably stress-adapted AMF can be a potential

biotechnological tool for inoculation of plants for successful restoration of degraded

ecosystems. Plants grown in metal contaminated sites harbor unique metal tolerant

and resistant microbial communities in their rhizosphere. These rhizomicroflora

14

secrete plant growth-promoting substances, siderophores, phytochelators to alleviate

metal toxicity, enhance the bioavailability of metals (phytoremediation) and

complexation of metals (phytostabilisation).

González et al. (2011) utilized the two-compartment system to study the effect

of arsenic (As) on the expression of the Glomus intraradices high-affinity phosphate

transporter GiPT, and the GiArsA gene, a novel protein with a possible putative role

as part of an arsenite efflux pump and similar to ArsA ATPase. Results showed that

induction of GiPT expression correlates with As (V) uptake in the extra-radical

mycelium of G. intraradices. It also showed a time-concerted induction of transcript

levels first of GiPT, followed by GiArsA, as well as the location of gene expression

using laser microdissection of these two genes not only in the extra-radical mycelium

but also in arbuscules. This work represents the first report showing the dissection of

the molecular players involved in arbuscular mycorrhizal fungus (AMF)-mediated As

tolerance in plants, and suggests that tolerance mediated by AMF may be caused by

an As exclusion mechanism, where fungal structures such as the extra-radical

mycelium and arbuscules may be playing an important role.

Elahi et al. (2010) carried out an experiment to determine the influence of AMF

inoculation on growth, nutrient uptake, arsenic toxicity and chlorophyll content of

eggplant grown in arsenic amended pot soil. Three levels of arsenic concentrations

(10ppm, 100ppm and 500ppm) were used in pot soil and eggplant was grown in

arsenic amended soils with or without mycorrhizal inoculation. Root length, shoot

height, root fresh weight, shoot fresh weight, root dry weight and shoot dry weight

were higher in AMF inoculated plants in comparison to their respective treatments

and decreased significantly with the increase of rate of arsenic concentrations. Less

arsenic content and higher chlorophyll and nutrient uptake were recorded in

mycorrhiza inoculated plants in compare to noninoculated plants. The findings of the

15

study indicated that AMF inoculation not only reduces arsenic toxicity but also can

increase growth and nutrient uptake of eggplant shoot.

Saha (2008) conducted three experiments to justify the effect of mycorrhiza on

seed germination in soil amended with different concentrations of arsenic solution.

He conducted the experiments in plastic tray, blotter plate and poly bags. Seedling

emergence, shoot height, root length, fresh and dry weight of shoot, fresh and dry

weight of root and also nutrient uptake by shoot of plants is increased in mycorrhiza

inoculated plants than that of inoculated plants.

Akhter (2008) conducted an experiment an experiment on the effect of

mycorrhizal fungi on growth and nutrient uptake by few crops in arsenic amended

soil. She found that 10 ppm arsenic solution + mycorrhiza treatment showed the

lowest performance in all the selected crops. The experiment exposed that with the

increase of arsenic concentration plants show the decrease growth performance.

Chen et al. (2007) observed that mycorrhizal fungi may play an important role

in protecting plants against arsenic contamination. They compartmented pot

cultivation system to investigate the roles of Glomus mosseae in plant P and As

acquisition by Medicago sativa and P-As interactions. The results indicate that fungal

colonization increased plant dry weight and also substantially increased both plant P

and As contents. The decreased shoot As concentrations were largely due to “dilution

effects” that resulted from simulated growth of AM plants and reduced As partitioning

to shoots.

Xia et. al. (2007) examined the effects of arbuscular mycorrhizal fungus

(Glomus mosseae) and P addition on As uptake by maize plants from an As-

contaminated soil. The results indicated that addition of P inhibited root colonization,

shoot and root biomass and development of extraradical mycelium. Root length. Dry

16

weight and shoot and root As contaminations both increased with mycorrhizal

colonization under the zero-P treatments. AM fungal inoculation decreased shoot As

concentrations when no P was added. AM colonization therefore appeared to enhance

plant tolerance to As in low P soil, and have some potential for the phytostabilization

of As-contaminated soil.

Ultra et al. (2007) set up an experiment to find out the effects of arbuscular

mycorrizal (AM) and phosphorus application on arsenic toxicity in As-contaminated

soil. The treatments consisted of a combination of two levels of AM (Glomus

aggregatum) inoculation and two levels of P application. AM inoculation as well as

P application reduced As toxicity symptoms and increased plant growth. Shoot As

concentrations were reduced by AM inoculation but enhanced by P application.

Dong et al. (2007) reported that, in a compartmented cultivation system white

clover and ryegrass were grown together in a As contaminated soil. The influence of

AM inoculation on plant growth, As uptake, phosphorus nutrition, and plant

competitions were investigated. Results showed that both plant species highly

depended on mycorrhiza for surviving the As contamination.

Trotta et al. (2006) studied the effects of arbuscular mycorrhizae on growth

and As hyperacumulation in the Chinese brake fern Pteris vittata. The As treatment

produced a dramatic increase of As concentration in pinnae and a much lower increase

in roots of both mycorrhizal and control plants. Mycorrhization increased pinnae dry

weight and leaf area, strongly reduced root As concentration and increased the As

translocation factor. The concentration of P in pinnae and roots was enhanced by

mycorrhizal fungi.

Kim et al. (2006) were investigated the effects of arbuscular mycorrhizal fungi

(Glomus mosseae) inoculation on arsenic and phosphorus uptake by Trifolium refresin

and Oentothera odorata. These results indicate that inoculation of AM fungi to host

17

plants obtained high yield and increased arsenic resistance to its toxicity and has a

potential applicability to enhance the efficiency of phyto-stabilization in soils highly

contaminated with arsenic.

Leung et al. (2006) conducted the greenhouse trial to investigate the role of

arbuscular mycorrhiza in aiding arsenic uptake and tolerance by Pteris vittata and

Cynodon dactylon. The infectious percentage of mycorrhizae and the average biomass

of shoots in infected P. vittata increased according to the increase of As levels when

compared to control. The indigenous mycorrhizas enhanced As accumulation in the

As mine populations of P. vittata and also sustained its growth by aiding P absorption.

For C. dactylon, As was mainly accumulated in mycorrhizal roots and translocation

to shoots was inhibited.

Ahmed et al. (2006) reported that Arsenic contamination of irrigation water

represents a major constraint to Bangladesh agriculture. This study examined the

effects of As and inoculation with an AM fungus, Glomus mosseae, on lentil. Plant

height, leaf/pod number, plant biomass and shoot and root P concentration/uptake

increased significantly due to mycorrhizal infection. Plant height, leaf /pod number,

plant biomass and shoot and root P concentration/uptake decreased significantly with

increasing As concentration. However mycorrhizal inoculation reduced As

concentration in roots and shoots. This study shows that growing lentil with

compatible AM inoculum can minimize As toxicity and increase growth and P uptake.

Agely et al. (2005) said that Chinese brake fern (Pteris vittata L.) is a

hyperaccumulator and mycorrhizal symbiosis may be involved in As uptake by this

fern. This is because arbuscular mycorrhizal (AM) fungi have a well-documented role

in increasing plant phosphorus (P) uptake and ferns are known to be colonized by AM

fungi. They found that the AM fungi not only tolerated As amendment, but their

presence increased found dry mass at the highest As application rate. These data

18

indicate that AM fungi have an important role in arsenic accumulation by Chinese

brake fern.

Liu et al. (2005) conducted a glasshouse experiment to sudy the effect of

arbuscular mycorrhizal (AM) colonization by Glomus mosseae on the yield and

arsenate uptake of tomato plants in soil experimentally contaminated with five As

levels. Mycorrhizal colonization was little affected by As application and declined

only in soil amended with 150 mg Askg-1. Shoot As concentration increased with

increasing As addition up to 50mgkg-1 but decreased with mycorrhizal colonization.

Mycorrhizal plants had higher shoot and root P/As ratios at higher As application rates

than did non-mycorrhizal controls. Mycorrhizal colonization may have increased

plant resistance to potential As toxicity at the highest level of As contamination.

Gonzalez et al. (2002) studied the role of arbuscular mycorrhizal fungi (AMF)

in arsenate resistance in arbuscular mycorrhizal associations for two Glomus spp.

isolated from the arsenate-resistant grass Holcus lanatus. Glomus mosseae and

Glomus caledonium were isolated from H. lanatus growing on an arsenic-

contaminated mine-spoil soil. The arsenate resistance of spores was compared with

nonmine isolates using a germination assay. Short-term arsenate influx into roots and

long-term plant accumulation of arsenic by plants were also investigated in uninfected

arsenate resistant and nonresistant plants and in plants infected with mine and

nonmine AMF. Mine AMF isolates were arsenate resistant compared with nonmine

isolates. Resistant and nonresistant G. mosseae both suppressed high-affinity

arsenate/phosphate transport into the roots of both resistant and nonresistant H.

lanatus. Resistant AMF colonization of resistant H. lanatus growing in contaminated

mine spoil reduced arsenate uptake by the host. AMF evolved arsenate resistance, and

conferred enhanced resistance on H. lanatus.

19

2.3. Interaction of AMF and nematode

Yang et al. (2014) found that AMF enhanced plant tolerance to herbivores,

nematodes, and fungal pathogens and also found reciprocal inhibition between AMF

and nematodes as well as fungal pathogens, but unidirectional inhibition for AMF on

herbivores. Negative effects of AMF on biotic stressors of plants depended on

herbivore feeding sites and actioning modes of fungal pathogens. More performance

was reduced in root-feeding than in shoot-feeding herbivores and in rotting- than in

wilt-fungal pathogens. However, no difference was found for AMF negative effects

between migratory and sedentary nematodes.

Hayder et al. (2014) A 60 days greenhouse experiment was conducted to

evaluate the efficacy of certain rhizobacteria (P. fluorescens, B. subtilis, Azotobacter

spp.), mycorrhizal fungi (Glomus fasciculatum) alone and in combination on the

multiplying on Meloidogyne incognita and growth of brinjal. The experiment

consisted of eighteen treatments with four replicates arranged in RBD. The plants

treated with the combinations of certain rhizobacteria and mycorrhizal fungus

significantly suppressed number of galls per root system, second stage juveniles J2

and improved plant growth over control, single treatments of rhizobacteria,

Mycorrhizal fungus and Carbofuran 3G (chemical check). P. fluorescens, B. subtilis,

G. fasciculatum when used in combination showed intermediary effects on both

nematode reproduction and plant growth, while Azotobacter sp. was found to be least

effective.

Banuelos et al. (2014) studied on biocontrol traits of arbuscular mycorrhizal

fungi (AMF), in terms of single and mixed species inoculum, against the root knot

nematode Meloidogyne incongita in Impatiens balsamina L., with and without

mineral fertilization in a greenhouse pot experiment. At harvest, 60 days after sowing,

general plant growth parameters and plant defense response in terms of antioxidant

20

activity and content of phenolic compounds in roots and leaves were measured. Also

AMF root colonization and abundance of nematode root-knots were determined.

Mineral fertilization increased all plant growth parameters measured, which coincided

with an increased disease development caused by M. incognita. Inoculation with AMF

mitigated the observed plant growth reduction caused by M. incognita though, higher

abundance of M. incognita root knots was found in mycorrhizal plants.

Marro et al. (2014) found that a possible biological control alternative to reduce

the damage caused by this species may be the use of arbuscular mycorrhizal fungi

(AMF). In the present work, the effect of Glomus intraradices on tomato plants

inoculated with the nematode at transplanting and three weeks later was tested. At 60

days, the following parameters were estimated: percentage of AMF colonization, root

and aerial dry weight, number of galls and egg masses, and reproduction factor

(RF=final population/initial population) of N. aberrans. AMF colonization was higher

in the presence of the nematode. The use of AMF favoured tomato biomass and

reduced the number of galls and RF on the plants inoculated with the nematode at

transplanting.

Udo et al. (2013) carried out an experiment to investigate the single and

combined effects of different arbuscular mycorrhizal fungi (AMF) and bio formulated

Paecilomyces lilacinus against M. incognita race 1 on tomato. Dysteric Cambisol soil

was used. The experiment took place in Calabar, Cross River State, Nigeria. The

experiment was laid out as a 3x6 factorial in a completely randomized design (CRD)

with three replications. Three applications of the bionematicide were combined with

five species of AMF plus an uninoculated control. The results indicated that AMF

species differed significantly (p < 0.05) in their efficacy of gall and egg mass

inhibition, tomato root colonization rate as well as growth and fresh fruit yield

enhancement. Glomus etunicatum and G. deserticola were the most efficient species.

Two applications of the bionematicide more significantly (p < 0.05) reduced galling

21

and egg production than a single application. Individual combinations of two AMF

(G. etunicatum and G. deserticola) with a double application of the bionematicide,

resulted in the greatest gall and egg mass inhibition and consequently the greatest

growth and fresh fruit yield enhancement.

Aggangan et al. (2013) conducted a study to determine the potential of

arbuscular mycorrhizal fungi (AMF) and nitrogen fixing bacteria (NFB) bio fertilizers

as growth promoters and biological control agents against nematodes in tissue-

cultured banana var. Lakatan under screen house conditions. Meriplants were

inoculated with AMF (MykovamTM) and NFB (Bio-NTM) during planting in

individual plastic bags filled with sterile soil sand mixture. Plant parasitic nematodes,

Radopholus similis and Meloidogyne incognita suspension were poured into the soil,

two months after inoculation with biofertilizers at concentrations of 1,000 and 5,000

larvae or eggs per seedling, respectively. Plant height, pseudostem diameter and leaf

area were taken every 2 weeks. At fourth month, results showed that AMF and

AMF+NFB inoculated seedlings grew better than the control plants. AMF treated

plants were taller, had bigger pseudostem diameter, larger leaf area, highest fine,

coarse root and total plant dry weights than the control and the other treatments. AMF

reduced root galls by 33% relative to those inoculated with M. incognita.

Hajra et al. (2013) conducted an experiment to evaluate the efficacy of

arbuscular mycorrhizal (AM) fungi (Glomus spp. and Gigaspora spp.) as bio-

protectant against root-knot nematode Meloidogyne incognita in sponge gourd (Luffa

cylindrica (L.) Roem.), mycorrhizal plant of family Cucurbitaceae. All parameters

were estimated in roots, shoot and leaves of mycorrhizal and nonmycorrhizal plants.

Physical/biochemical and carbon profile were taken into account. Comparative study

clearly indicates the significant variations in all parameters. Leaf area and plant height

were increased in mycorrhizal plants than non-mycorrhizal, while it showed a sharp

22

decrease in nematode infected plants, same plants also showed less water content due

to xylem vessels damage.

Vos et al. (2012) investigated the effects of AMF and mycorrhizal root

exudates on the initial steps of Meloidogyne incognita infection, namely movement

towards and penetration of tomato roots. M. incognita soil migration and root

penetration were evaluated in a twin-chamber set-up consisting of a control and

mycorrhizal (Glomus mosseae) plant compartment (Solanum lycopersicum cv.

Marmande) connected by a bridge. Penetration into control and mycorrhizal roots was

also assessed when non-mycorrhizal or mycorrhizal root exudates were applied and

nematode motility in the presence of the root exudates was tested in vitro. Results M.

incognita penetration was significantly reduced in mycorrhizal roots compared to

control roots. In the twin-chamber set-up, equal numbers of nematodes moved to both

compartments, but the majority accumulated in the soil of the mycorrhizal plant

compartment, while for the control plants the majority penetrated the roots.

Application of mycorrhizal root exudates further reduced nematode penetration in

mycorrhizal plants and temporarily paralyzed nematodes, compared with application

of water or non-mycorrhizal root exudates.

Herman et al. (2012) studied the effects of inoculation of sweet passion fruit

plants with the arbuscular mycorrhizal (AM) fungus Scutellospora heterogama on the

symptoms produced by Meloidogyne incognita race 1 and its reproduction were

evaluated in two greenhouse experiments. In the 1st, the M. incognita (5000

eggs/plant) and S. heterogama (200 spores/plant) inoculations were simultaneous; in

the 2nd, the nematodes were inoculated 120 days after the fungal inoculation. In both

the experiments, 220 days after AM fungal inoculation, plant growth was stimulated

by the fungus. In disinfested soil, control seedlings (without S. heterogama) were

intolerant to parasitism of M. incognita, while the growth of mycorrhized seedlings

was not affected. Sporulation of S. heterogama was negatively affected by the

23

nematodes that did not impair the colonization. M. incognita did not affect

mycorrhizal seedling growth.

Adriano et al. (2011) conducted an experiment where banana plants (Musa spp.

L.) cv 'Great dwarf' were amended with free-living N2 fixing bacteria (FLNFB) and

arbuscular mycorrhizal fungi (AMF) and the presence of the burrowing nematode

Radopholus similis was monitored in the field. Five treatments were applied by

inoculating banana roots with four strains of FLNFB, that is C1, C2, C3 and C4

isolated from the rhizoplane of the same banana cultivar, or by keeping them

uninoculated. The largest number of nematodes was found in the untreated roots and

the lowest in the roots inoculated with C4. The largest percent of mycorrhizal

colonization was found when banana roots were inoculated with C1 and the lowest in

roots that were not inoculated. The number of R. similis decreased with increased

colonization with AMF.

Shreenivasa et al. (2011) conducted an experiment where Tomato

Lycopersicon esculentum Cv. Pusa ruby inoculated with indigenous isolates of

Glomus fasciculatum and Meloidogyne incognita individually and in combination

were analyzed for sequential biochemical variations in roots with respect to total

proteins, phenols and polyphenol oxidase activity. There was a post inflectional

increase in the concentration of total proteins in G. fasciculatum, M. incognita + G.

fasciculatum and M. incognita inoculated roots, respectively. Concentration of

phenols and polyphenol oxidase activity was higher in inoculated roots as a result the

nematode infection reduced in Glomus infected root.

Manandhar et al. (2011) explored the biocontrol effect of different species of

arbuscular mycorrhizal fungi (AMF) (Glomus intraradices and G. mossae) was tested

against Meloidogyne graminicola in rice (Oryza sativa) cultivars Azucena and

UPLRi5 under greenhouse conditions. Seed - based inoculum and root-based

24

inoculum were used for G. intraradices in Azucena, while in vivo cultured G. mossae

was used for UPLRi5. Calcium alginate beads were used as inoculant carrier for the

entrapment of G. intraradices propagules in rice seeds. Low percentage of

colonisation was observed for G. intraradices while percentage of G. mossae

colonisation varied between experiments. G. intraradices did not show biocontrol,

however, despite lower colonisation, G. mossae exhibited suppression of the

nematode multiplication. Variations in the control efficiency of different species of

Glomus in different rice cultivars indicate the host-AMF specificity to achieve control.

Further study is needed in order to optimize AMF colonisation in rice and to determine

the biocontrol potential of AMF against M. graminicola which is a major problem in

all rice growing areas.

Ambo et al. (2010) conducted a glass house experiment for the effectiveness

of vermicompositing and rhizotrophic micro-organisms (arbuscular mycorrhizal

fungus (AMF) Glomus aggregatum and mycorrhiza helper bacterium (MHB) Bacillus

coagulans) for the management of Meloidogyne incognita on tomato cv Pusa Ruby.

Among the different treatments evaluated, vermicompost and G. aggregatum alone

and in combination with B. coagulans recorded the maximum growth, biomass and

nutrients of tomato cv Pusa Ruby with decreased root- knot nematode population and

root- knot index. But amending the soil with application of vermicomposting + B.

coagulans + G. aggregatum in tomato was significantly increased the plant growth,

biomass and nutrients of tomato cv Pusa Ruby. Similarly, reduction in root- knot

nematode population, root- knot index (RKI), nematode reproduction rate (NRR)

number of galls and egg masses per plant were recorded in the above treatment.

Highest mycorrhizal colonization of 92.5% and minimum nematode population of

145.0/ 250cc soil was observed in the same treatment.

Akhtar et al. (2009) studied on the effects of Phosphate solubilizing

microorganisms (Glomus intraradices, Pseudomonas putida, P. alcaligenes, P.

25

aeruginosa (Pa28), A. awamori) and Rhizobium sp. on the growth, nodulation yield

and root-rot disease complex of chickpea under field condition. Inoculation of

Rhizobium sp. caused a greater increase in growth and yield than P. putida, P.

aeruginosa or G. intraradices. The number of nodules per root system was

significantly higher in plants inoculated with Rhizobium sp. compared to plants

without Rhizobium sp. Inoculation of P. putida caused highest reduction in galling

followed by P. aeruginosa, P. alcaligenes, G. intraradices and A. awamori while

Rhizobium sp. caused almost similar reduction in galling as caused by P. putida.

Sankaranarayanan et al. (2009) conducted an experiment under glasshouse

conditions to study the reciprocal influence of the arbuscular mycorrhizal fungus

(AMF) Glomus fasciculatum and the root-knot nematode Meloidogyne incognita and

their interaction effects on the growth of blackgram. Prior inoculation of AMF

increased significantly shoot and root growth and pod yield of blackgram, especially

when applied 20 days before nematode inoculation, and suppressed root gall index

and the nematode population in the soil, with earlier application of AMF resulting in

greater suppression of the nematode. Inoculation of the nematode prior to AMF

affected negatively root mycorrhizal colonization and spores in the soil with the

suppressing effects being more pronounced when nematodes were inoculated 20 days

prior to AMF. AMF treatments increased phosphorus content of shoots and roots of

blackgram.

Jefwa et al. (2008) found up to 20 AMF species to be associated with banana

plantations (Musa spp.) in East and Central Africa. Spore abundance, the inoculum

reservoir that determines colonization, is largely influenced by management practices.

The data generated to date increasingly illustrates the importance of AMF in banana

systems and its sensitivity to crop and soil management practices, effects on banana

growth, nutrient uptake and control of root damage by nematodes.

26

Siddiqui et al. (2008) carried out an experiment on the effects of Glomus

intraradices and Pseudomonas putida which were observed alone and in combination

with fertilizers (composted cow manure and urea) on the growth of tomato and on the

reproduction of Meloidogyne incognita. Inoculation of P. putida caused a greater

increase in the tomato growth than G. intraradices and inoculation of both together

caused a greater increase than by either of them. The maximum reduction in galling

and nematode multiplication was observed when P. putida was used with G.

intraradices together with composted manure.

Siddiqui et al. (2007) investigated the effects on chickpea (Cicer arietinum) of

the phosphate-solubilizing microorganisms Aspergillus awamori, Pseudomonas

aeruginosa (isolate Pa28) and Glomus intraradices in terms of growth, and content of

chlorophyll, nitrogen, phosphorus and potassium and on the root-rot disease complex

of chickpea caused by Meloidogyne incognita and Macrophomina phaseolina.

Application of these phosphate-solubilizing microorganisms alone and in

combination increased plant growth, pod number, and chlorophyll, nitrogen,

phosphorus and potassium contents, and reduced galling, nematode multiplication and

root-rot index of chickpea. Pseudomonas aeruginosa reduced galling and nematode

multiplication the most followed by A. awamori and G. intraradices.

Jaizme-Vega et al. (2006) aimed of study to determine whether the combined

inoculation of two AMF species and a Bacillus consortium based on three strains

previously described as PGPR in other crops were able to reduce nematode infection

and damage on papaya. Papaya seedlings were inoculated with two AMF isolates

(Glomus mosseae or G. manihotis) at the beginning of the nursery phase. Results in

terms of plant development and nutrition, benefits due to AMF inoculation persisted

in the presence of PGPR. However, the effect of dual inoculation was different,

depending on the Glomus species. This positive effect was also evident in plants with

nematode. Meloidogyne infection was significantly reduced in mycorrhizal plants.

27

However, the addition of PGPR does not seem to improve the results of AMF single

treatments in terms of nematode infection.

Masadeh et al. (2004) studied the effects of the combination of the arbuscular

mycorrhizal fungus (AMF) Glomus intraradices and the biological control fungus

Trichoderma viride on the control of the root-knot (RK) nematode, Meloidogyne

hapla, were investigated in greenhouse experiments on the tomato cultivars ‘Hildares’

(very suitable as host for RK) and ‘Tiptop’ (less suitable as host for RK) showing

retarded development of the giant cell system, retarded growth of the nematode, and

consequently reduced production of egg-sacs. There was no evidence of negative

interactions between the two beneficials with regard to AMF root colonization or

population development of T. viride in the rhizosphere.

28

2.4. Interaction of P. lilacinum and M. incognita

Aminuzzaman et al. (2013) used alginate pellets of Paecilomyces lilacinus

YES-2 and Pochonia chlamydosporia HDZ-9 for controlling of M. incognita on

tomato in a greenhouse by adding them into a soil with sand mixture at rates of 0.2,

0.4, 0.8 and 1.6% (w/w). P. lilacinus pellets at the highest rate (1.6%) reduced root

galling by 66.7%. P. chlamydosporia pellets at the highest rate reduced the final

nematode density by 90%. The results indicate that P. lilacinus and P.

chlamydosporia as pellet formulation can effectively control root-knot nematodes.

Usman et al. (2012) observed the effect of some fungal strains for the

management of root-knot nematode (Meloidogyne incognita) on eggplant (Solanum

melongena. They used two biocontrol fungal strains of Trichoderma harzianum and

Paecilomyces lilacinus at 1g/pot and 2g/pot. Inoculation of fungus was done

simultaneously along with 1000 second stage juveniles (J2) of M. incognita. Strains

of T. harzianum were found to be most effective when treated at 2g/pot. P. lilacinus

also gave almost similar results and enhanced all plant growth characters with the

reduction in the root knot infestation.

Kiewnick et al. (2011) evaluated the fungal bio-control agent, P. lilacinus

strain 251 for its potential to control the root-knot nematode Meloidogyne incognita

on tomato at varying application rates and inoculums densities. He demonstrated that

a pre-planting soil treatment with the lowest dose of commercially formulated PL251

(2×105 CFU/g soil) was already sufficient to reduce root galling by 45% and number

of egg masses by 69% when averaged over inoculums densities of 100 to 1600 eggs

and infective juveniles per 100 cm3 of soil. A real time PCR revealed a significant

relationship between egg mass colonization by PL 251 and the dose of product applied

29

to soil but no correlation was found between fungal density and biocontrol efficacy or

nematode inoculums level. These results demonstrate that rhizosphere competence is

not the key mode of action for PL 251 in controlling M. incognita on tomato.

Aminuzzaman and Liu (2011) first reported the isolation and evaluation of

biocontrol fungus Paecilomyces lilacinus recorded in Bangladesh. The results under

pot experiment and field experiment demonstrated significant variation among the

treatment. They mentioned that the fungus showed more than 80% egg parasitism and

52% juvenile mortality of Meloidogyne spp. They also reported that the fungus

increased shoot height, fresh shoot weight, root length and fresh root weight and also

reduced root galling up to 30% and number of eggmass per root system up to 40%

when compared to control treatment. P. lilacinus enhanced plant growth and reduced

galling index and nematode population which was supported by Aminuzzaman et al.

(2011). They also reported that root galling index and final nematode population

decreased up to 40.7 and 73.8% respectively for brinjal of application of the biocontrol

fungus. They also mentioned that P. lilacinus enhanced plant growth and reduced

galling index with its increased doses.

Kalele et al. (2010) worked with antagonistic fungus P. lilacinus strain 251 in

controlling root knot nematodes in tomato and cucumber. He applied P. lilacinus

inoculums at different rates and different times. He found that pre-planting soil

treatment reduced final nematode populations by 69% and 73% in the roots and soil,

respectively compared to the non-inoculated control in tomato. However, soil

treatment at planting recorded reduction level of 54 and 74% in the roots and soil

respectively he described that PL251 was a promising potential that could be exploited

in the management of Meloidogyne spp. in vegetable production systems.

Aminuzzaman (2009) used fungal pellet containing spores of nematophagous

fungus P. lilacinus YES-2 in green house condition to assess its biocontrol potency

30

against root knot of tomato and observed P. lilacinus significantly reduced the number

of nematode population in soil and root and increased 20.75% tomato yield over

untreated control.

Bhat et al. (2009) observed the interaction of fungus P. lilacinus and in

Meloidogyne incognita in bitter gourd at different time intervals. They found that

Meloidogyne incognita induced large sized galls on the plants. The xylem and the

phloem exhibited abnormalities in structure near the giant cells. Abnormal vessel

elements were occupying larger area near giant cells. The plants that were treated with

fungus either one week before nematode inoculation or simultaneously produced

significantly (P+0.01) small sized galls in comparison to untreated plants.

Lopez-llorca et al. (2008) observed the mode of action and interactions of

nematophagous fungi and discussed types of recondition phenomena (e.g. chemotaxis

and adhesion), signaling and differentiation, penetration of the nematode

cuticle/eggshell using mechanical as well as enzymatic (protease and chitinase)

means. They observed that P. lilacinus is an egg and female parasitic fungus and it

infects nematode by its appressoria. It produced chitinases enzymes and damage the

eggshell and destroyed nematode.

Singh (2007) examined root galls of rice caused by Meloidogyne graminicola

for natural colonization by nematophagous fungi and observed that application of

inocula of A. dactyloides and D. brachophaga in soil infested with Meloidogyne

graminicola respectively reduced the number of root galls by 86% and females by

94% and the eggs and juveniles by 94%. The application of these fungi to soil

increased plant growth.

Anastasiadis et al. (2007) evaluated a formulated product (BioAct) is made up

of the spores of the naturally occurring fungus P. lilacinus, strain 251, against root

31

knot nematodes in pot and green house experiments. They observed that application

of P. lilacinus and the bacteria Bacillus fitmus, significantly or together in pot

experiments, provided effective control of second stage juveniles, eggs or egg masses

of root knot nematodes.

Esphahani and Pour (2006) observed that P. lilacinus was effective in

controlling root knot nematode on tomato and suppressing its population growth and

effectively promoted the growth of plant.

Khan et al. (2006) described the mode and severity of infection of nematodes

by a soil saprophyte P. lilacinus. Infection of stationary stages of nematodes by P.

lilacinus was studied with three plant parasitic nematodes M. javanica, Heterodera

avenae and radopholus similis.P. lilacinus infected eggs, juveniles and females of M.

javanica by direct hyphal penetration. The early developed eggs were more

susceptible than the eggs containing fully developed juveniles. P. lilacinus also

infected immature cyst of H. avenae including eggs in the cysts and the eggs of R.

similis and the fungus was shown to infect mobile stages of all the plant-parasitic

nematodes.

Kiewnick and Sikora (2006) mentioned that successful biocontrol of RKN

depends on initial low nematode density in the soil. They used fungal biocontrol agent,

P. lilacinus strain 251 (PL251), and evaluated for its potential to control the root knot

nematode Meloidogyne incognita on tomato. In growth chamber experiment, a pre-

planting soil treatment reduced root galling by 66% number of egg masses by 74%

and the final nematode population in the roots by 71% compared to the inoculated

control. They also mentioned that a single pre-plant application at a concentration of

1× 106 CFU/g is needed for sufficient biocontrol of Meloidogyne incognita by PL251.

32

Kiewnick and Sikora (2004) conducted a greenhouse experiments to control

root knot nematodes Meloidogyne incognita and M. hapla on tomato using P. lilacinus

251. All single or in combination treatments tested decreased the gall index and the

number of egg masses compared to the untreated control 12 weeks after planting.

However, the combination of the seedling treatment with a pre or at planting

application of P. lilacinus was necessary to achieve higher levels of control. They

found that the above mentioned combination of pre-planting application plus the

seedling and one post plant drench gave the best control and resulted in a significant

fruit yield increase in concurrence with a decrease in number of galls per roots.

Oduor-owino (2003) used agrochemicals, organic matter and the antagonistic

fungus P. lilacnus in controlling root knot nematode in natural field condition. He

found that the smallest galling index, number of galls and nematode population were

in soil treated with aldicarb in combination with P. lilacinus.

Rao and Reddy (2001) used Glomus mossae in combination with P. lilacinus

and neem cake to control root knot nematode of eggplant. The parasitization of eggs

of root knot nematode was significantly increased by P. lilacinus and the transplants

yielded significantly more fruit. Neem cake amendment in the nursery beds played a

positive role in increasing the colonization of endomycorrhiza and the biocontrol

fungus on the roots of transplants before and after transplanting. The combined effect

of these three components facilitated the sustainable management of M. incognita on

eggplants under field condition.

Siddiqui et al. (2000) studied the efficacy of Pseudomonas aeruginosa alone

or in combination with P. lilacinus on controlling of root knot nematode and root

infecting fungi under laboratory and field conditions. Ethyl acetate extract (1mg/ml)

of P. lilacinus and P. aeruginosa, respectively, caused 100 and 64% mortality of

Meloidogyne javanica larvae after 24h. In field experiments, biocontrol fungus and

33

bacterium significantly suppressed soil bore root infecting fungi including

Macrophomina phaseolina, Fusarium oxysporum, Fusarium solani, Rhizoctonia

solani and the root knot nematode Meloidogyne javanica, P. lilacinus parasitized eggs

and female of Meloidogyne javanica.

Oduor and Waudo (1996) evaluated P. lilacinus, Phoma herbarum and three

isolates of Fusarium oxysporum in controlling root knot (M. javanica) in eggplant. P.

lilacinus and Fusarium oxysporum-1 significantly (p<0.05) parasitized more than

70% eggs and female while Fusarium oxysporum-3 parasitized less than 20% control

of Meloidogyne incognita in eggplant.

Mittal et, al. (1995) evaluated P. lilacinus, a rhizosphere inhabiting

nematophagous fungus, along with chitin in sterilized soil for the suppression of

Melooidogyne incognita, causal agent of root knot disease in Solanum melongena,

Lycopersicon esculentum and Cicer arietinum. The plant growth after 30, 60 and 90

days was assessed in terms of shoot and root length, shoot and root fresh and dry

weight and number of galls/gm root fresh weight. Combination of fungus wth chitin

enhanced suppression of Meloidogyne incognita more than using them alone.

Cabanillas et al. (1989) isolated of 13 P. lilacinus isolates from various

geographic regions as biocontrol agents against Meloidogyne incognita. The best

control of M. incognita was provided by an isolate from Peru or a mixture of isolated

of P. lilacinus. As soil temperatures increased from 16OC to 28OC, both root knot

damage caused by M. incognita and percentage of egg masses infected by P. lilacinus

increased. The greatest residual P. lilacinus activity on M. incognita was attained with

a mixture of fungal isolates. These isolates effected lower root galling and necrosis,

egg development, and enhanced shoot growth compared with plants inoculated with

M. incognita alone.

34

Cabanillas and Barker (1989) conducted a microplot experiment to evaluate

the inoculam level and time of application of P. lilacinus on the protection of tomato

against M. incognita. They observed that P. lilacinus applied into soil 10 days before

planting increased yield with the improvement of plants compared with the nematode

alone plots.

35

MATERIALS AND METHODS

3.1. Experimental site and experimental period

The present investigation was carried out during the period from September 2014 to

July 2015 in the shade house and in the laboratory of the Department of Plant

Pathology, Sher-e-Bangla Agricultural University, Sher-e Bangla Nagar, Dhaka -

1207.

3.2. Environment of experiments

All the experimental plants were kept in the shade house where the temperature was

28 ± 2º C during the “day” and 23 ± 2º C during “night” with an average temperature

of 26± 2º C.

3.3. Pot Experiment

3.3.1. Crop variety used

Eggplant variety BARI Begun-10 was used as selected crop in this experiment.

3.3.2. Collection of seeds

Seeds of BARI Begun-10 was collected from Bangladesh Agricultural Research

Institute (BARI), Joydebpur, Gazipur.

3.3.3. Soil collection and sterilization

Required soils were collected from agricultural farm of Sher-e-Bangla Agricultural

University. Sand, decomposed cow dung and compost were also collected with soil.

Then soil, sand, cow dung and compost mixed properly in a ratio of 6:3:1. For raising

seedlings in plastic trays and for final experiment set up the mixture was autoclaved

at 121ºC, 15 psi for 15 minutes on two successive days. The sterilized soil was allowed

to cool to room temperature and was later used to fill the plastic trays for raising

seedlings and pot for seedling transplanting.

36

3.4. Raising of seedlings

Several plastic trays were filled with sterilized and fertile soil. Seeds of BARI Begun-

10 was soaked in water for one night and treated with NaOCl for one minute and

washed with distilled water for three times. After that the seeds were sown in plastic

trays and covered with a thin layer of soil and watered. Then the trays were covered

with polythene sheet and kept in sunlight for raising seedlings (Photograph 1.).

Seedlings were observed regularly and watering was done as per necessity up to

hardening the seedling in plastic pot.

Photograph 1. Raising of eggplant seedlings

37

3.5. Preparation of pots

Plastic pots of 1000 cm3 were cleaned, washed and dried up. Sterilized and fertile soil

was filled in required amount into each pot. Each pot contained 600 g soil. Then the

pots were arranged according to selected experimental design. Detailed of soil

properties presented in Table1.

Table 1. Physicochemical characteristics of pot soil

PH Organic

matter

Total

N

K Ca Mg P S B Zn

% Meq/100 gm µg/gm(ppm)

6.6 2.29 0.114 0.16 8.22 1.71 4.84 9.99 0.14 5.35

38

3.6. Treatments and design of the experiment

3.6.1. Treatments

There were sixteen integrated treatment combination: 2 (1 Glomus sp. + 1control) x 2

(1 P. lilacinum + 1 control) x 2 (1 Arsenic + 1 control) x 2 (1 M. incognita + 1 control).

1. C

2. C+G

3. C+Pl

4. C+Mi

5. C+As

6. G+As

7. Pl+As

8. Mi+As

9. G+Mi+As

10. Pl+Mi+As

11. G+Pl+As

12. G+Pl+As+Mi

13. G+Mi

14. G+Pl

15. Pl+Mi

16. G+Pl+Mi

C= Control, G= Glomus sp., Pl= Purpureocilium lilacinum, Mi= Meloidogyne incognita, As=

Arsenic

3.6.2. Design of the experiment

The experimental design was CRD with 5 replicated pots per treatment. Plants were

disposed in the shadehouse

39

3.7. Isolation, identification and culturing of AMF

Spores of AMF in soil was collected by the wet sieving and decanting method

(Gerdeman and Nicolson, 1963) followed by sucrose density gradient centrifugation

technique described by Daniel and Skipper (1982). After collection of roots and

rhizosphere soil of Cassia tora, approximately 100 g of root and rhizosphere soil was

separately suspended into a 2-liter container and 1.5 liter distilled water was added.

Vigorous mixing of the suspension was done to make free of spore from soil

(photograph 2.) and roots. For root sample blending the sample for 1 min in 300 ml

of distilled water was essential to free the spore from roots. Heavier particles in

suspension was allowed to settle for 15 to 45s and the supernatant was decanted

through standard sieves. A 500 um pore sieve over a 50 µm pore size sieves was used.

Suspension was transferred to 50 ml centrifuge tubes with a fine stream of water and

was centrifuge at 1200 to 1300x for 3 min. the suspension was removed carefully. Soil

or root particle (pellet) was suspended in chilled 1.17 M sucrose, mixing the content

and centrifuge again at 1200 to 1300x for 1.5 min. The supernatant was poured

through small mesh sieve and rinse carefully with distilled water and wash the spores

sufficiently. Spores were sterilized following Budi et al. (1999) by immersed for 10 s

in 96% ethanol and washed using a 25 um sieve. Spores will then be immersed for 10

min in a solution of .02% streptomycin, 2% chloramines T and a drop to Tween 20

followed by subsequent washing in 25 um sieve. A final emersion was done in 6%

bleach for 1 min and subsequent washing in distilled water. The AMF was sterilized

with 1% sodium hypochlorite for 2 min, rinsed three times in distilled water and sown

in 50 multi-pot trays containing soil of lower P content which was previously

inoculated by sufficient spore of AMF inoculation was done following (Jaizmevega

et al. 2005). Maize seedlings were allowed for sufficient root and soil colonization.

The multi-pot trayswere kept in shade house and irrigated regularly. After 8 weeks,

AMF colonized root fragments (photograph 3) of seedlings and rhizosphere soil

containing spore were used for AMF treatment.

40

Photograph 2. Identification of Glomus sp. (40x)

Photograph 3. Confirmation of colonization observing vesicle inside

the cell of Cassia tora(40x)

Spore

Vesicle

41

Photograph 4. Inoculation of Glomus spore on maize seed (40x)

Photograph 5. Inoculation of Glomus spore in maize root (40x)

Spore

Maize seed

Maize root

Spore

42

Photograph 6. Mass culture of Glomus sp. with trap plant maize

43

3.8. Fungal Isolate

The isolate of Purpureocillium lilacinum isolated from Mymensingh, Bangladesh,

previously shown to have high biocontrol ability against root knot nematode was used

in this study (Aminuzzaman and Liu, 2011).

3.8.1. Culture, mass production and harvesting of Purpureocillium lilacinum

Purpureocillium lilacinum was grown on Potato Dextrose Agar (PDA) medium for 8-

10 days (Photograph 7) (Aminuzzaman and Liu, 2011). Within 8-10 days the fungus

was transferred on chick pea for mass production (Photograph 8). For mass production

one hundred grams of chickpea seed free of any pesticide treatment was placed in 250-

ml conical flasks and soaked in lukewarm water for 3-4 hours. Then the water was

drained off, and each flask was closed with a cotton plug and covered with brown

paper in two layer of paper. Then flasks were placed in an autoclave for 15 minutes at

121OC temperature at 15 psi. After the flasks and contents cooled, P. lilacinum as a

mycelial mat growing on PDA was added aseptically to one flask and shaken for better

distribute of the fungus; the other flask served as an un-inoculated control. The flasks

were incubated at 25-30OC for 20 days. After incubation the sterile water was added

into the conical flask and the spore masses scraped away with sterile brush within

laminar air flow chamber. The harvested spores were filtered through sterilized

cheesecloth. The spore was harvested from each conical flask and spore was counted

with a hemocytometer.

44

Photograph 7. Pure culture of P. lilacinum on PDA media

Photograph 8. Mass culture of P. lilacinum on chick pea

45

3.9. Nematode inoculum preparation

The RKN was maintained on eggplant (Photograph 9) in shade house for two months

and the eggs was extracted from the roots following sucrose floatation method as

described by Liu and Chen (2001). The fresh roots were collected from the shade

house into plastic bag and nematode extraction was carried out within two days. The

collected roots (Photograph 10) were washed in running tap water and cut into 1.5

small pieces. The cut pieces were crushed in 500 ml sterile water with a mini sample

blender for 1min at high speed. The suspensions were treated with 1% sodium

hypochlorite (NaOCl) for 1 min to dissolve eggs was collected on 25 µm aperture

sieve. The eggs were washed three times with sterile distilled water to remove residual

NaOCl and collected in a 50 ml plastic tube. The eggs were separated from debris by

centrifugation in 37.5% (w/v) sucrose solution for 5 min and then was rinsed with 1%

NaOCl for 1 min and washed three times with sterile distilled water to remove residual

NaOCl and collected in 50 ml plastic tube. Eggs was adjusted to 500 eggs/100 µl

suspension.

46

Photograph 9. M. incognita inoculum production in association with eggplant root

Photograph 10. M. incognita infected root showing eggmass and gall

47

3.10. Preparation of arsenic solution

Arsenic solution was prepared following Elahi et al. (2010). For preparation of 1000

ppm 1L arsenic solution at first 4g of sodium hydroxide was taken in a 100ml

measuring cylinder. Sodium hydroxide was diluted with distilled water and the

volume of the cylinder rose up to the 100 ml mark. Then 1.32g arsenic powder was

taken in another 1000 ml measuring cylinder and dilute with that diluted sodium

hydroxide. 10% HCl was taken in a beaker. Then HCl was added into the 1000ml

measuring cylinder to make it acidic. Finally, the volume of the flask rose up to the

1000ml mark.

3.11. General inoculation procedure for the experiment

Each plastic pot (600cm3) was filled with the 500 cm3 soil. Fungal biocontrol agent

was added into soil and mixed thoroughly before 7 days of transplantation. To

determine specific CFU (5x106) of P. lilacinum/g soil, a separate experiment was

carried out where fungal inocula were added in 100 g soil in different rate and mixed

thoroughly with three replicates. Soil moisture to be maintained to field capacity.

After seven days of inoculation, CFU of P. lilacinus/g soil were determined following

soil dilution plate method and relationship between amount of P. lilacinum pellet

applied to the soil and CFU of P. lilacinum/g soil. Arsenic suspension was prepared

by dissolving arsenic powder in NaOH. Additional HCl were added to make it acidify.

As solution was mixed thoroughly with the pot soil at 50 ppm. As concentration was

confirmed by subsequent soil analysis following Beer’s law. Twenty-five gram AMF

colonized maize root and colonized rhizosphere soil was used for each pot soil.

Seedling of 30 days (BARI Eggplant 10 var.) was transplanted into each pot.

Nematode eggs at the rate of 10000 eggs/ pots was inoculated into the central area of

each pot through four 2cm depth holes. Each pot will have a dish underneath to

eliminate cross contamination and each treatment was replicated five times. Then,

pots were transferred to the shade house. Seedlings was irrigated with tap water daily.

48

3.12. Intercultural operations

After transplantation of seedling and final experiment set up weeding and irrigation

were regularly done as per necessity. General sanitation was maintained throughout

the growing period.

3.13. Harvesting and data recording

After two months of transplanting, plants were harvested and data was recorded.

The following parameters were considered

Shoot length (cm)

Root length (cm)

Shoot fresh and dry weight (g)

Root fresh and dry weight (g)

Leaf area (cm2)

Chlorophyll content (μg cm−2)

Mycorrhizal root infection (%)

Mycorrhizal spore (number/10gm soil)

Gall index (0-10 scale)

Number of egg masses per root

Number of eggs per egg mass

Number of eggs per root system

Number of juveniles per 800 g soil

Total number of nematode population/plant (J2+ eggs)

Reproduction factor (RF)

% Egg masses colonized by P. lilacinum

Soil colonization by P. lilacinum (CFUg-1 soil)

49

3.14. Data recorded

3.14.1. Plant data

Shoot and root length were measured before harvest. The shoot height (cm) was

measured from the base of the plant to the growing point of the youngest leaf with a

measuring scale. Then the roots are harvested by cutting with an anti-cutter. Roots are

carefully separated from soil, cleaned gently with water and collected in different

polybag that were leveled according to different treatments. Finally, the root length

(cm) was taken. The length of root was measured from the growing point of root to

the longest available lateral root apex. For fresh weight (g) of root and shoot was

blotted dry and the weight was recorded. For dry weight (g), the shoot and root were

sun dried for three days and then kept in drier machine for 4-6 hours at 40º C

temperatures. And after complete drying the weight was recorded. Leaf area (cm2)

was measured by leaf area meter (Photograph11) and chlorophyll content (µgcm-2) of

eggplant leaves were measured by chlorophyll meter (Photograph12).

50

Photograph 11. Leaf area measurement by CI-202 Portable Laser Leaf Area Meter

Photograph 12. Measurement of chlorophyll content of eggplant

leaf by SPAD 502 Plus Chlorophyll Meter

http://www.cid-inc.com/products/leaf-area-lai/portable-laser-leaf-area-meter

http://www.specmeters.com/nutrient-management/chlorophyll-meters/chlorophyll/spad502p/

51

3.15. Counting of nematode egg masses/root system

Number of egg masses/root system was counted following Holbrook et al. (1983).

The roots were soaked in Phloxine-B (2mg/l) for 15 minutes (Photograph 13)

(Hartman and Sasser, 1985). The roots were observed and egg masses/root was

counted with a magnifying glass. Then egg masses were picked with forceps treated

with NaOCl for three minutes to dissolve gelatinous materials. After subsequent

washing with water eggs were counted under compound microscope (Photograph 14).

52

Photograph 13. Heavily galled root treated with Phloxine-B solution

Photograph 14. Phloxine-B treated root for counting of eggmass/ root

53

3.16. Slide preparation and counting of eggs/egg mass

Heavily galled roots were collected and properly washed with water. Care was taken

so that an egg mass does not washed with water. Then the roots were soaked in

Phloxine-B (2mg/l) solution for 15 minutes (Hartman and Sasser, 1985). Then water

was soaked by placing the root in tissue paper for one minute. A clean slide was

prepared. Three drops of glycerin were placed on the slide. Then egg masses were

collected from the root with the help of fine forceps and placed on the slide and also

crashed with the help of bottom side of needle. Then after placing cover slip the slide

was examined under microscope and counting the eggs/egg mass (Photograph 15).

3.17. Extraction of nematode from soil and counting of juveniles

The extraction of nematodes from soil was done by using a Whitehead and Hemming

tray method (1965) as follows: Pot soil was mixed thoroughly and different samples

of 100 g soil was weighted and put it on the sieve that was on a bowl filled with water.

The upper portion of sieve was lined with three layers of kitchen tissue paper. After 5

days the nematode suspension was collected in a beaker and left for a day, excess

water was discarded leaving 100 ml suspension and 5 ml sub sample was taken and

put into a counting dish. Juveniles counting were done by using a compound

microscope (Photograph 16 and Photograph 17).

54

Photograph 15. Counting the number of egg/ eggmass (40x)

Photograph 16. Extraction of nematode by Bangladeshi plate method (modified

White Head and Heaming method, 1965)

55

Photograph 17. Second stage juveniles of Meloidogyne incognita(40x)

56

3.18. Gall index

Root galls were indexed on a 0-10 scale of Bridge and Page (1980), which were as

follows

Scales Specification

0 No galls

1 Few small gall, difficult to find

2 Small gall only, clearly visible, main root clean

3 Some larger galls visible, main root clean

4 Larger galls predominant but main root clean

5 50% of the roots infected, galling on some main roots, reduced root

system

6 Galling on main roots

7 Majority of the main roots galled

8 All main roots including tap roots galled, few clean roots visible

9 All roots severely galled, plants usually dying

10 All roots severely galled, no root system

57

3.19. Egg masses colonization (%) by Purpureocillium lilacinum

Egg masses were collected as per treatment from the eggplant plant roots, washed

with water and disinfected with a solution of 10% Clorox, rinsed with sterile water

and put on a Potato Dextrose Agar (PDA) media in petridish. Randomly ten

eggmasses/root were collected so that 80 egg masses per treatments were collected.

The number of colonized egg masses was determined after 5 days of incubation. The

presence of P. lilacinum with egg mass of M. incognita was confirmed by preparation

of slides from the culture grown on PDA (Plate 1).

A B

Plate 1. Egg colonization of M. incognita by P. lilacinum (A and B)

58

3.20. Soil colonization by Purpureocillium lilacinum (CFUg-1 soil)

Samples of 1g soil from each treatment were collected after harvest of the crop around

the root zone. The number of colony forming unit (CFUg-1 soil) per gram soil was

determined using the soil dilution plate method (Photograph 18).

Photograph 18. Determination of CFUg-1 soil using the soil dilution plate method

59

3.21. Observation of roots for mycorrhizal infection

Following Phillips and Hayman (1970) roots was treated with 10 % KOH solution for

30 min to 1-2 hours in a hot bath. Treated roots was washed with water and treated

with 2 % HCl solution. Acidified root samples were stained with 0.05 % acid fuchsin

in lactic acid for 10-15 min in a hot bath. The roots were destained with lactic acid or

lacto-glycerol. Then the destained root segments were mounted in acetic glycerol on

slides and the cover slips was placed and slightly pressed. The roots were observed

under the microscope. The presence or absence of infection of AMF in the root

segments was recorded and the percent infection was calculated using the following

formula:

Number of AMF positive segments

% Root infection = ×100

Total number of segments recorded

A root segments was considered to be infected if it showed mycelium, vesicle and

arbuscules or any other combination of AM fungi.

3.22. Study of spore population of in soil

After confirming Mycorrhizal association in the root system, we identify the spore

population in soils were isolated and inoculated. Identification was done in the Central

Laboratory of Department of Plant Pathology, SAU, Dhaka.

60

Plate 2. Observation of eggplant roots for mycorrhizal infection.

(A and B): vesicle. (C and D): Mycelium

vesicle

Mycelium

A

D

B

C

61

3.23. Chemical analysis of plant sample

3.23.1. Nutrient analysis

Chemical analysis of the plant samples was done in the department of Soil Science

and in the Department of Agricultural Chemistry, SAU, Dhaka.

3.23.2. Preparation of plant sample

Plant (shoot) samples were dried in oven at 700C for 70 hours and then ground the

samples and sufficient amount of sample for each treatment was kept in desiccators

for chemical analysis.

3.23.3. Digestion of plant samples with nitric-perchloric acid mixture

An amount of 0.5g of sub-sample was taken into a dry clean 100ml Kjeldahl flask,

10ml of di-acid mixture (HNO3, HClO4 in the ratio of 2:1) was added and kept for few

minutes. Then, the flask was heated at a temperature rising slowly to 2000C. Heating

was instantly stopped as soon as the dense white fumes of HClO4 occurred and after

cooling, 6ml of 6N HCl were added to it. The content of the flask was boiled until

they became clear and colorless. This digest was used for determining P, K and S.

3.23.4. Phosphorus

Phosphorus in the digest was determined by ascorbic acid blue color method (Murphy

and Riley, 1962) with the help of a Spectrophotometer (LKB Novaspec, 4049).

3.23.5. Potassium

Potassium content in the digested plant sample was determined by flame photometer

Sulphur content in the digest was determined by turbidimetric method as described by

Hunt (1980) using a Spectrophotometer (LKB Novaspec,4049).

62

3.23.6. Nitrogen

Plant samples were digested with 30% H2O2, conc. H2PO4 and a catalyst mixture

(K2SO4: CuSO4.5H2O: Selenium powder in the ratio of 100: 10: 1, respectively) for

the determination of total nitrogen by Micro-Kjeldahl method. Nitrogen in the digest

was determined by distillation with 40% NaOH followed by titration of the distillate

absorbed in H3BO3 with 0.01N H2SO4 (Bremner and Mulvaney, 1982).

3.23.7. Arsenic

Analysis of arsenic was conducted by following steps:

10% HCl was prepared as a carrier liquid by dilution of 100 ml conc. HCl into

1000 ml volumetric flask. Then the flask was volume upto the mark of 1000

ml.

Potassium borohydride solution was prepared by taking 7.5 g of potassium

borohydride and 0.3% sodium hydroxide in a 500 ml volumetric flask. Then

the volume of 500 ml made by adding water.

Standard arsenic solution of 0, 2.5, 7.5, 10 and 12.5 µg/L were prepared from

As2O5.

Detection of arsenic was done with Hydride Generation Atomic Absorption

Spectrophotometer (Analytik jena), an arsenic detection equipment in soil

science laboratory of Sher-e-Bangla Agricultural University. After preparation

of samples and all necessary chemicals, the equipment was operated

maintaining manufacturer’s guideline.

This equipment is a computer based, so the result was displayed on the monitor

through the respective software and the reading was taken as ppb.

3.24. Analysis of data

The data were statistically analyzed using analysis of variance to find out the variation

of results from experimental treatments. Treatment means were compared by

Duncan’s New Multiple Range Test (DMRT) according to Gomez and Gomes,

(1984).

63

RESULTS AND DISCUSSION

Arbuscular mycorrhizal fungus has the remarkable influence on plant growth, nutrient

uptake and arsenic toxicity. In arsenic contaminated soil AMF reduce the plant arsenic

uptake in contrast to uninoculated plant. This fungus triggers several channel to

bypass the arsenic during nutrient uptake thus reduce arsenic toxicity. When

artificially AMF is inoculated in arsenic amended soil, it strengthens plant to face the

adversity as a result it can tolerate the adverse situation and can grow as a healthy

plant. Due to this mechanism plant growth and nutrient uptake is increased. In this

experiment, arsenic and the nematode M. incognita challenged soil was prepared to

know the combined effect of AMF and P. lilacinum. It is well known that P. lilacinum

is a nematophagus fungi and on the other hand AMF has also some reported positive

influence on nematode control. Here sixteen treatment combinations were evaluated

all the treatments of AMF, P. lilacinum, M. incognita and arsenic to determine the

combined and individual effect of this treatments on plant growth, root-knot

development, plant nutrition and As toxicity of eggplant.

4.1. For all treatment combination

4.1.1. Shoot length

The influence of Purpureocillium lilacinum in combination with Glomus sp. on shoot

length of eggplant in arsenic amended soil challenged with Meloidogyne incognita are

presented in Table 2. The shoot length was different significantly among various

treatments (Photograph 21). The highest shoot length was recorded from treatment (G

+ Pl) followed by (C+G) and (C+Pl) which were statistically similar. The highest

shoot length for treatment (G+Pl) was 26.40 cm and for treatment (C+G) and (C+Pl)

it was 20.80 cm and 20.04 cm, respectively. Shoot length with treatment (G+As) was

18.26 cm followed by treatment (G + Pl+ Mi), (G + Pl+ As + Mi), (C) and (Pl+ Mi)

where shoot lengths were 16.06 cm, 15.74 cm, 15.10 cm and 14.50 cm, respectively.

64

The lowest shoot length 9.64 cm found from treatment (Mi + As) that was statistically

similar with treatment (C+Mi) where the shoot lengths were 9.64 cm and 9.10 cm

respectively followed by treatments with (C+As), (Pl+Mi+As), (Pl+As) and

(G+As+Mi) for shoot lengths 12.88 cm, 12.90 cm, 13.18 cm and 13.30 cm

respectively. Rao et al. (1998) reported the same result that plant height was

significantly greater in case of eggplant treated with both G. mosseae and P. lilacinus.

Mycorrhizal inoculation significantly enhanced shoot height. This was probably due

to uptake of more nutrients, which increased vegetative growth. Present findings are

in agreement with Matsubara et al. (1994). Reduction in root galling and nematode

reproduction by P. lilacinus, and induction of systemic resistance/tolerance through

an improved host nutrition or modification of mycorrhizosphere by AMF, could

possibly account for the growth and yield enhancement. In the findings of Tushar et

al. (2012), it was observed that in the pot with soil contaminated with arsenic and no

mycorrhizal inoculum, performance was very bad in terms of all aspects of growth,

whereas in the pot where mycorrhizal inoculum was added along with contaminated

soil, the performance of the plant was better.

4.1.2. Root length

The influence of Purpureocillium lilacinum in combination with Glomus sp. on root

length of eggplant in arsenic amended soil challenged with Meloidogyne incognita are

presented in Table 2. The root length was different significantly among various

treatments (Photograph 22). The highest root length (cm) was recorded from treatment

(G + Pl) followed by (C+G) which were significantly distinct from other treatments

followed by (C+Pl) which was statistically similar with (G + Pl+ Mi) and (G+As).

The highest root length for treatment (G+Pl) was 20.44 cm and for treatment (C+G)

root length was 18.44 cm. Rao et al. (1998) reported the same result that root length

was significantly greater in case of egg plants treated with both G. mosseae and P.

lilacinus. The root length with treatment (C+Pl) was 15.14 cm followed by 14.70 cm

and 14.10 cm for treatment (G + Pl + Mi) and (G+As) respectively. The lowest root

65

length 7.16 cm was found from treatment (Mi + As) that was statistically similar with

treatment (C+Mi) of 8.34 cm root length followed by 9.10 cm for treatment

(Pl+Mi+As). Mycorrhizal inoculation significantly enhanced root length in

comparison to noninoculated control. This was probably due to uptake of more

nutrients, which increased vegetative growth. Present findings are in agreement with

Matsubara et al. (1994). The findings of Xia et al. (2007) is similar with the findings

of present study. They conducted an experiment under glasshouse condition in an As-

contaminated soil and they reported arbuscular mycorrhizal (AM) fungus (Glomus

mosseae) increased root length markedly under the zero-P treatments. This validates

the synergistic interaction between the two biocontrol agents as reported earlier by

some authors (Al-Raddad, 1995; Trivedi, 1997; Rao et al. 1998; Bhat and Mahmood

2000; Sharma).

4.1.3. Shoot fresh weight

The role of Purpureocillium lilacinum in combination with Glomus sp. on shoot fresh

weight of eggplant in arsenic amended soil challenged with Meloidogyne incognita

are presented in Table 2. The result revealed that among all the treatments, the highest

fresh weight of shoot 15.51g was recorded in treatment (G + Pl) which was

remarkably different from other treatments followed by (C+G) and (C+Pl) of which

weight of shoot was 13.29g and 12.95g, respectively which were statistically similar.

The lowest fresh weight of shoot 6.34g found from treatment (C+Mi) followed by (Mi

+ As) and (Pl+ Mi + As) for 6.34g and 6.26g, respectively that was statistically similar.

Rao et al. (1998) reported the same result that shoot fresh weight was significantly

greater in case of eggplants treated with both G. mosseae and P. lilacinus. Tarafdar

and Parveen, (1996) reported that shoot biomass was significantly improved in

mycorrhiza inoculated plants.

66

Table 2. Influence of Purpureocillium lilacinum in combination with Glomus sp. on

shoot length, root length, shoot and root fresh weight of eggplant in arsenic


Treatments Shoot length

(cm)

Root length

(cm)

Shoot fresh

weight (g)

Root fresh

weight (g)

C 15.10 d 12.32 ef 8.06 ghi 5.48 g

C + G 20.80 b 18.44 b 13.29 b 9.32 b

C + Pl 20.04 b 15.14 c 12.95 bc 8.18 c

C + Mi 9.640 g 8.340 ij 6.34 j 4.82 g

C + As 12.88 f 11.00 fg 7.09 ij 5.34 g

G + As 18.26 c 14.10 cd 11.70 cd 7.48 cd

Pl + As 13.18 ef 10.00 gh 8.66 fgh 5.30 g

Mi + As 9.100 g 7.160 j 6.24 j 5.19 g

G + As + Mi 13.30 ef 11.04 fg 10.06 ef 5.80 fg

Pl + Mi + As 12.90 f 9.100 hi 6.26 j 5.26 g

G + Pl + As 14.64 de 12.94 de 11.98 bcd 6.80 de

G + Pl + As +

Mi

15.74 d 12.40 ef 11.24 de 6.48 ef

G + Mi 13.44 ef 12.24 ef 9.680 f 6.80 de

G + Pl 26.50 a 20.44 a 15.51 a 11.06 a

Pl + Mi 14.50 de 11.58 ef 7.58 hij 7.36 cde

G + Pl + Mi 16.06 d 14.70 c 9.04 fg 7.56 cd

Lsd (0.05) 1.42 1.37 1.333 0.88

CV (%) 7.35 8.69 10.84 10.31

C = Control, G = Glomus, Pl = P. lilacinum, Mi = M. incognita, As = Arsenic

67

4.1.4. Root fresh weight

Table 2 shows that the results of the effect of Purpureocillium lilacinum in

combination with Glomus sp. on root fresh weight of eggplant in arsenic amended soil

challenged with Meloidogyne incognita. The result revealed that among all the

treatments, the highest fresh root weight (11.06 g) was recorded in treatment (G + Pl)

which was statistically significant from other treatments followed by treatment (C+G)

gave root weight of 9.32 g that was statistically different from treatment (C+Pl) which

gave 8.18 g root weight. The lowest fresh root weight 5.48 g was found from treatment

(C) followed by (C+Mi), (Pl+ As), (Mi + As) and (Mi + Pl+ As) which gave 4.82,

5.30, 5.19 and 5.26 g of root weight, respectively that were statistically similar. The

results of the present study coroborates with the findings of Carling and Brown (1980)

who reported that root colonization by most of the Glomus isolates significantly

increased plant root fresh weight in low fertility soil.

4.1.5. Shoot dry weight

Table 3 shows that the results of the effect of Purpureocillium lilacinum in

combination with Glomus sp. on shoot dry weight of eggplant in arsenic amended

soil challenged with Meloidogyne incognita. The result revealed that among the 16

treatments, the highest dry weight of shoot (1.91 g) was recorded in treatment (G +

Pl) that was statistically similar to treatment (C+G) which gave shoot weight of 1.73

g followed by weight of shoot 1.63 g with treatment (C+Pl) which was statistically

similar to (C+G) but different from (G+Pl) treatment. The lowest dry weight of shoot

0.94 g was recorded from treatment (Mi+As) that was statistically similar to (C+As)

that provide the dry weight of shoot of 1.10 g. Shoot dry weights were higher in

mycorrhizal than nonmycorrhizal plants is reported by Giri et al., 2005. The findings

of the present study are in accordance with the findings of Xia et al. (2007) who

reported that both of dry weight and root biomass of maize plants increased markedly

when inoculated with arbuscular mycorrhizal (AM) fungus (Glomus mosseae) under

glasshouse condition in an arsenic amended soil.

68

4.1.6. Root dry weight

Sixteen treatments were taken to evaluate the effect of Purpureocillium lilacinum in

combination with Glomus sp. on root dry weight of eggplant in arsenic amended soil

challenged with Meloidogyne incognita (Table 3). The highest dry root weight 1.76 g

was recorded in treatment (G + Pl) which was statistically similar with treatments

(C+G) which gave 1.71 g dry root weight followed by (C+Pl) from which dry root

weight was obtained 1.38 g. The results of treatment (G+Pl) and (C+G) were

statistically different from all others treatments. The lowest dry root weight (0.27 g)

found from treatments (Mi+As) followed by treatments (Pl+As), (Mi+Pl+As) and

(C+As) which provided results of 0.31 g, 0.40 g and 0.35 g for dry root weight

respectively that were statistically similar. Root dry weights were higher in

mycorrhizal than nonmycorrhizal plants is reported by Giri et al., 2005. The findings

of the present study are in accordance with the findings of Xia et al. (2007) who

reported that both of dry weight and root biomass of maize plants increased markedly

when inoculated with arbuscular mycorrhizal (AM) fungus (Glomus mosseae) under

glasshouse condition in an arsenic amended soil.

4.1.7. Leaf area

Leaf area influencd by the Purpureocillium lilacinum in combination with Glomus sp.

of eggplant in arsenic amended soil challenged with Meloidogyne incognita presented

in Table 3. The result revealed that among the 16 treatments, the highest leaf area

(18.18 cm2) was obtained from treatment (G+Pl) that was statistically different from

all other treatments followed by (C+G) that was statistically different from (C+Pl).

The leaf area found 37.64 cm2 and 34.92 cm2 from treatments (C+G) and (C+Pl)

respectively. The lowest leaf area 15.07 cm2 found from treatment (C+Mi) that was

statistically different from all other treatments followed by (Mi+As) and (C+As)

which gave leaf area of 22.27 cm2 and 24.46 cm2 respectively. As plant height, shoot

weight, root weight and root length/g of root were significantly greater so the leaf area

69

also increased accordingly due to the combined and individual effect of Glomus sp.

and P. lilacinum.

4.1.8. Chlorophyll content

Chlorophyll content influencd by the Purpureocillium lilacinum in combination with

Glomus sp. of eggplant in arsenic amended soil challenged with Meloidogyne

incognita presented in Table 3. Among the 16 treatments, the highest chlorophyll

content 42.18 μg cm−2 was recorded in treatment (G + Pl) which was significantly

different from all other treatments followed by treatments (C+G) and (C+Pl) which

provided the chlorophyll content of 39.12 μg cm−2 and 34.20 μg cm−2 respectively.

The result of treatment (C+G) and (C+Pl) was statistically different from each other

and from all other treatments. The lowest chlorophyll content 22.32μg cm−2 was

recorded from treatment (Mi + As) followed by treatment (C+As) from which 23.28

μg cm−2 chlorophyll content was obtained. All other treatments provided

significantly different results according the Table 3. Mycorrhizal infection

ameliorated chlorophyll content of lettuce reported by Zuccarini (2007). Again the

findings of Elahi et al. (2010) stated that AMF has a positive influence on chlorophyll

content. Cabanillas et al. (1989), Kiewnick and Sikora (2004) and Esfahani and Pour

(2006) reported that plant growth parameters and yield promoted by using the

bioagent P. lilacinum compared with plants treated with this nematode alone.

70


dry weight of shoot and root, leaf area and chlorophyll content of eggplant in

arsenic amended soil challenged with Meloidogyne incognita

Treatments Shoot dry

weight (g)

Root dry

weight (g)

Leaf area

(cm2)

Chlorophyll

content(μgcm−2)

C 1.42 cdef 0.47 f 32.54 cde 30.72 d

C + G 1.73 ab 1.71 a 37.64 b 39.12 b

C + Pl 1.63 bc 1.38 b 34.92 c 34.20 c

C + Mi 1.17 gh 0.62 e 15.07 k 25.10 f

C + As 1.10 hi 0.35 fg 24.46 ij 23.28 g

G + As 1.41 cdefg 1.38 b 29.80 fg 28.52 e

Pl + As 1.46 cde 0.31 g 29.96 efg 27.78 e

Mi + As 0.94 i 0.27 g 22.27 j 22.32 g

G + As + Mi 1.28 efgh 0.47 f 28.34 gh 31.28 d

Pl + Mi + As 1.08 hi 0.40 fg 26.23 hi 27.86 e

G + Pl + As 1.50 cde 0.79 d 30.95 efg 34.10 c

G + Pl + As +

Mi

1.39 cdefg 0.67 de 29.25 fg 28.10 e

G + Mi 1.55 bcd 1.36 b 30.75 efg 31.10 d

G + Pl 1.91 a 1.76 a 48.18 a 42.18 a

Pl + Mi 1.19 fgh 1.08 c 31.70 def 27.80 e

G + Pl + Mi 1.36 defg 1.29 b 34.11 cd 31.02 d

Lsd (0.05) 0.21 0.11 2.40 1.12

CV (%) 12.16 10.44 6.27 2.93


71

Photograph 19. Growth pattern of eggplant in different treatment combination

during two months of growing

72

Plate 3. Eggplant root at different treatments combination; A = C, B = C+G, C = C+Pl,

D = C+Mi, E = C+As, F = G+As, G = Pl+As, H = Mi+As, I = G+As+Mi, J = Pl+Mi+As,

K = G+Pl+As, L = G+Pl+As+Mi, M = G+Mi, N = G+Pl, O = Pl+Mi, P = G+Pl+Mi

(C= Control, G = Glomus, Pl = P. lilacinum, Mi = M. incognita, As = Arsenic)

I J L K M N P

A B C D F G H

O

E

73

4.2. For Glomus sp. involved treatment combination

4.2.1. Shoot length

Shoot length of eggplant influenced by the eight mycorrhizal treatments in different

combination of P. lilacinum, M. incognita and arsenic is shown in Fig. 1. It was found

that Glomus sp. in combination of P. lilacinum treatment (G + Pl) gave the highest

shoot length 26.50 cm which was significantly different from all other treatments

followed by (C+G) provided the shoot length of 20.80 cm. The lowest shoot length

13.30 cm was revealed from treatment (G + As + Mi) followed by treatment (G + Mi)

which gave shoot length of 13.44 cm. Udo et al. (2013) reported same findings

investigating the single and combined effects of different arbuscular mycorrhizal

fungi (AMF) and bio formulated Paecilomyces lilacinus against M. incognita race 1

on tomato. Venkatesan et al. (2013) investigated that As uptake was increased up to

three times due to the nematode infection of roots, the increase being more at the

higher inoculum level. So, the treatment (G+As+Mi) gave the lowest shoot length.

Mycorrhizal inoculation significantly enhanced shoot height and root length. This was

probably due to uptake of more nutrients, which increased vegetative growth. Present

findings are in agreement with Matsubara et al. (1998).

74

C= Control, G= Glomus, Pl= P. lilacinum, Mi= M. incognita, As= Arsenic

Fig. 1. Shoot length of eggplant influenced by the eight mycorrhizal treatments in

different combination of P. lilacinum, M. incognita and arsenic

b

c

cde

d

e

a

d

0

5

10

15

20

25

30Sh

oo

t le

ngt

h (

cm)

Treatments

75

4.2.2. Root length

Fig. 2 represents that the root length of eggplant influenced by the eight mycorrhizal

treatments with different combination of P. lilacinum, M. incognita and arsenic.

Among eight mycorrhizal treatments, it was found that Glomus sp. in combination of

P. lilacinum (G+Pl) treatment provided the highest root length 20.44 cm that was

significantly different to other treatment combination followed by treatment (C+G)

which gave 18.44 cm root length. The lowest root length 11.04 cm was revealed from

treatment (G+As+Mi) that was statistically similar to treatment (G+Mi) which gave

12.24 cm root length. Arsenic uptake was increased up to three times due to the

nematode infection of roots reported by Venkatesan, et al. (2013). So, the treatment

(G+As+Mi) gave the lowest shoot length. Mycorrhizal inoculation significantly

enhanced shoot height and root length in comparison to noninoculated control. This

was probably due to uptake of more nutrients, which increased vegetative growth are

in agreement with Matsubara et al. (1998).

76


Fig. 2. Root length of eggplant influenced by the eight mycorrhizal treatments with

different combination of P. lilacinum, M. incognita and arsenic

b

cdf

de e ef

a

c

0

5

10

15

20

25

Ro

ot

len

gth

(cm

)

treatments

77

4.2.3. Leaf area

The role of Glomus sp on leaf area of eggplant in combination of P. lilacinum in

arsenic amended soil challenged with Meloidogyne incognita is presented in Figure

3. Results revealed that treatment (G+Pl) gave highest leaf area 48.18 cm2 which

statistically significant and different from all other treatments followed by treatment

(C+G), (G+Pl+Mi) for leaf area 37.64 and 34.11 cm2, respectively. The lowest leaf

area 28.34 cm2 was revealed from treatment (G+As+Mi) followed by treatment

(G+Pl+As+Mi), (G+As), (G+Mi), and (G+Pl+As) from which leaf area obtained

29.25, 29.80, 30.75, 30.75 and 30.95 cm2, respectively where all of these treatments

results were statistically similar. Tarafdar and Parveen, (1996) reported that shoot

biomass was significantly improved in mycorrhiza inoculated plants. Again, P.

lilacinum has synergistic effect on growth parameters with AMF, so the combined

and individual presence of Glomus and P. lilacinum increased leaf area.


Fig. 3. The role of Glomus sp on leaf area of eggplant in combination of P. lilacinum


b

d dd

dd

a

c

0

10

20

30

40

50

60

leaf

are

a (c

m2

)

Treatments

78


Chlorophyll content of eggplant influenced by the role of Glomus sp in combination

of P. lilacinum in arsenic amended soil challenged with Meloidogyne incognita is

presented in Fig. 4. Among eight mycorrhizal treatments, it was found that treatment

(G+Pl) gave highest chlorophyll content 42.18 μg cm-2 of eggplant leaves which was

statistically different from all other treatments followed by treatment (C+G) provided

39.12 μg cm-2 chlorophyll content. The lowest chlorophyll content 28.10 μg cm-2 was

revealed from treatment (G+Pl+As+Mi) that was statistically similar to treatment

(G+As) that gave leaf chlorophyll content 28.52 μg cm-2. The chlorophyll is the

essential component for photosynthesis and it increases with mycorrhizal colonization

(Colla et al., 2008). AM symbiosis enhanced the chlorophyll content of Solanum

leaves which was in agreement with the results of other studies (Elahi et al., 2010;

Kapoor and Bhatnagar, 2007).


Fig. 4. Chlorophyll content of eggplant influenced by Glomus sp in combination of P.

lilacinum in arsenic amended soil challenged with Meloidogyne incognita

b

ed

c

e

d

a

d

0

5

10

15

20

25

30

35

40

45

Ch

loro

ph

yll c

on

ten

t (u

gcm

-2)

Treatments

79


Variation of shoot fresh weight of eggplant due to eight mycorrhizal treatments of

eggplant influenced by the role of Glomus sp in combination of P. lilacinum in arsenic

amended soil challenged with Meloidogyne incognita is shown in Figure 5. Among

these treatments, it was found that treatment (G+Pl) gave highest fresh shoot weight

15.51 g that was significantly different from all other treatments followed by treatment

(C+G) which gave 13.29 g fresh shoot weight. The lowest fresh shoot weight 9.04 g

was revealed from treatment (G+Pl+Mi) which was statistically similar to treatment

(G+Mi), (G+As+Mi) for 9.68 and 10.06 g fresh shoot weight, respectively. Since the

eggplant was grown with two reported ecofriendly organisms so their combined effect

invaded pathogen and mediated arsenic contamination. So, shoot weight increased

when they were combindly inoculated rather than uninoculated and individual

treatments combination.


Fig. 5. Variation of shoot fresh weight of eggplant due to eight mycorrhizal treatments

of eggplant influenced by Glomus sp in combination of P. lilacinum in arsenic


bbc

debc cd

de

a

e

0

2

4

6

8

10

12

14

16

18

Aer

ial f

resh

wei

ght

(g)

Treatments

80


Shoot dry weight of eggplant influenced by the role of Glomus sp. in combination of

P. lilacinum in arsenic amended soil challenged with Meloidogyne incognita is

presented in Fig. 6. Among eight mycorrhizal treatments, it was found that treatment

(G+Pl) gave the highest dry shoot weight 1.91g that was statistically similar to

treatment (C+G) from which 1.73 g shoot dry weight obtained. The lowest dry shoot

weight, 1.28 g was obtained from treatment (G+As+Mi) followed by (G+Pl+As+Mi)

and (G+As) which provided 1.39 and 1.41 g shoot dry weight respectively. Present

findings validate the findings of Xia et al. (2007). They found that both of dry weight

and root biomass of maize plants increased markedly when inoculated with arbuscular

mycorrhizal (AM) fungus (Glomus mosseae) under glasshouse condition in an arsenic

amended soil. The present findings are also in accordance with the findings of Ahmed

et al. (2003).


Fig. 6. Shoot dry weight of eggplant influenced by Glomus sp in combination of P.


ab

cdd

bcd cdbc

a

cd

0

0.5

1

1.5

2

2.5

Aer

ial d

ry w

eigh

t (g

)

treatments

81


Influence of Glomus sp. involved treatments combination on root fresh weight is

shown in Fig. 7. Results revealed that treatment (G + Pl) gave highest fresh root

weight 11.06 g that was statistically different from all other treatments followed by

treatment (C+G) from which 9.32 g root fresh weight was obtained. The lowest fresh

root weight 5.80g among eight mycorrhizal treatments was found from treatment

(G+As+Mi) which was statistically similar to (G+Pl+Mi+As), (G+Pl+As) and

(G+Mi) where 6.48, 6.80 and 6.80 g root fresh weight, respectively were obtained.

Results of the experiment confirmed various reports on enhanced plant growth due to

AM inoculation to medicinal plants (Nisha and Rajeshkumar, 2010) and forest trees

species (Rajan et al., 2000).


Fig. 7. Root fresh weight of eggplant influenced by Glomus sp in combination of P.


b

c

d

cd cd cd

a

c

0

2

4

6

8

10

12

14

Fres

h r

oo

t w

eigh

t (g

)

Treatments

82


Root dry weight of eggplant influenced by the role of Glomus sp in combination of P.

lilacinum in arsenic amended soil challenged with Meloidogyne incognita is presented

in Figure 8. Among 8 mycorrhizal treatments, it was found that treatment (G+Pl)

provided the highest result of root dry weight 1.76 g that was statistically similar with

the effect of treatment of (C+G) which gave 1.71 g dry root weight. The lowest dry

root weight 0.47 g was revealed from treatment (G+As+Mi) which was significantly

different from all other treatments. Present findings are in accordance with Xia et al.

(2007) who reported that both of dry weight and root biomass of maize plants

increased markedly while inoculated with arbuscular mycorrhizal (AM) fungus

(Glomus mosseae) under glasshouse condition in an arsenic amended soil.


Fig. 8. Shoot dry weight of eggplant influenced by Glomus sp in combination of P.


ab

e

cd

ba

b

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Dry

ro

ot

wei

ght

(g)

Treatments

83

4.2.9. Number of spore of Glomus sp. /10 g soil

Influence of Glomus sp involved treatments combination on of spore of Glomus sp.

/10 g soil is shown in Fig. 9. Among 8 mycorrhizal treatments, it was found that

treatment (G+Pl) gave the highest result of 35.60 number of spore of Glomus sp. /10

g soil that was statistically similar to the result of 30.40 spore/10 g soil for treatment

(C+G). The result confirms the findings of Rao et al. (1998). The lowest result of

15.80 number of spore of Glomus sp. /10 g soil was found from treatment (G+As+Mi).


Figure 9. Number of spore of Glomus sp. /10 g soil influenced by the role of Glomus

sp in combination of P. lilacinum in arsenic amended soil challenged with

Meloidogyne incognita

ab

cd

e

bcd bc

d

a

b

0

5

10

15

20

25

30

35

40

No

. of

spo

re/1

0 g

so

il

Treatments

84

4.2.10. Root infection by Glomus sp.

Root infection by Glomus sp. in different level of combination with P. lilacinum, M.

incognita and arsenic is represented in Fig. 10. Among eight mycorrhizal treatments,

it was found that treatment (G+Pl) gave the highest root infection of 58.30% which

was significantly different from all other treatments followed by 54.80% root

infection by the treatment (C+G). The lowest root infection 34% was found from

treatment (G+As+Mi) which was statistically dissimilar to all other treatments. The

result of the present study corroborating with the findings of Rao et al. (1998) of 64

and 62% infection in eggplant due to the combined inoculation of Glomus sp. and P.

lilacinum and individual inoculation of Glomus sp., respectively.


Fig. 10. Root infection (%) influenced by the role of Glomus sp in combination of P.


be

f

d ce

ade

0

10

20

30

40

50

60

70

Ro

ot

infe

ctio

n (

%)

Treatments

85

4.3. For Purpureocillium lilacinum treatments

4.3.1. Shoot length

Shoot length of eggplant influenced by the eight P. lilacinum involved treatments in

different combination with Glomus sp., M. incognita and arsenic is shown in Fig. 11.

Among eight Purpureocillium lilacinum treatments, it was found that treatment

(G+Pl) gave the highest result for shoot length 26.50 cm which was statistically

different from all other treatments followed by (C+Pl) from which 20.04 cm shoot

length was obtained. The lowest shoot length 12.90 cm was found from treatment

(Pl+Mi+As) statistically similar to the treatment (Pl+As) (Pl+Mi) and (G+Pl+As) for

shoot length of 13.18, 14.50 and 14.64 cm, respectively. Improved plant growth

characters by application of P. lilacinus in controlling root-knot nematodes was also

reported earlier by Walia et al. (1999), and Khan and Goswami (2000). So, the

Glomus effect on arsenic mediation and nematode controlling effect of P. lilacinum

marked well in aspect of shoot length.


Fig. 11. Shoot length of eggplant influenced by the eight P. lilacinum involved

treatments in different combination with Glomus sp., M. incognita and arsenic

b

dd cd c

a

cd c

0

5

10

15

20

25

30

Sho

ot

len

gth

(cm

)

Treatments

86

4.3.2. Root length

Fig. 12 represents that the root length of eggplant influenced by the eight P. lilacinum

treatments with different combination of Glomus sp., M. incognita and arsenic. It was

found that treatment (G+Pl) gave the highest root length 20.44 cm that was

remarkably different from all other treatments followed by treatment (C+Pl) gave the

root length of 15.14 cm. The lowest root length 11.58 cm was found from treatment

(Pl+Mi) statistically similar to (G+ Pl+Mi+As), (G+Pl+As) for 12.40 cm and 12.94

cm root length. This is validated by Hasan (2004), who narrated the better effect of P.

lilacinus on growth parameters.


Fig. 12. Root length of eggplant influenced by the eight P. lilacinum treatments with

different combination of Glomus sp., M. incognita and arsenic

b

dd

c c

a

c

b

0

5

10

15

20

25

Ro

ot

len

gth

(cm

)

Treatments

87

4.3.3. Leaf area

The role of P. lilacinum involved treatments on leaf area of eggplant in combination

of Glomus sp in arsenic amended soil challenged with Meloidogyne incognita is

presented in Fig. 13. Of eight P. lilacinum treatments, it was found that treatment

(G+Pl) gave the highest leaf area 48.18 cm2 that was statistically significant from all

other treatments followed by (C+Pl) from which 34.92 cm2 leaf area found. The

lowest leaf area 26.23 cm2 was found from treatment (Pl+Mi+As) statistically similar

to (G+Pl+As+Mi) for leaf area 29.25 cm2. Probably individual and combined effect

of this two bio-agent P. lilacinum and Glomus sp. contributed on healthy growth of

eggplant which triggers better leaf area.


Fig. 13. The role of P. lilacinum involved treatments on leaf area of eggplant in

combination of Glomus sp in arsenic amended soil challenged with Meloidogyne

incognita

bbcd

dbcd de

a

bc bc

0

10

20

30

40

50

60

Folia

r su

rfac

e (c

m2

)

Treatments

88


Chlorophyll content of eggplant influenced by eight P. lilacinum treatments in

combination of Glomus sp. in arsenic amended soil challenged with Meloidogyne

incognita is shown in Fig. 14. Of eight P. lilacinum treatments, it was found that

treatment (G+Pl) gave the highest chlorophyll content 42.80 μg cm-2 that was

statistically different from all other treatments followed by (G+Pl+As) and (C+Pl) for

34.10 μg cm-2 and 34.20 μg cm-2 chlorophyll content. The lowest chlorophyll content

27.78 μg cm-2 was found from treatment (Pl+As) statistically similar to (Pl+Mi),

(Pl+Mi+As), (G+Pl+Mi+As) for 27.80, 27.86 and 28.10 μg cm-2 of chlorophyll

content, respectively. As P. lilacinum has been reported as soil ameliorates and which

keep the rhizosphere area safe for plant root growth as well as has synergistic relation

with Glomus sp. so the chlorophyll content increased due to their influence.


Figure 14. Chlorophyll content of eggplant influenced by the role of eight P. lilacinum

treatments in combination of Glomus sp. in arsenic amended soil challenged with

Meloidogyne incognita

b

d d

b

d

a

dc

0

5

10

15

20

25

30

35

40

45

Ch

loro

ph

yll c

on

ten

t (u

g/cm

-2)

Treatments

89


Variation of shoot fresh weight of eggplant due to eight P. lilacinum treatments of

eggplant in combination of Glomus sp. in arsenic amended soil challenged with

Meloidogyne incognita is shown in Fig. 15. Among eight P. lilacinum treatments, it

was found that treatment (G+Pl) gave the highest fresh shoot weight 15.51 g that was

statistically different from other treatments followed by (C+Pl) from which 12.95 g

fresh shoot weight was obtained. The lowest fresh shoot weight 6.26 g was found from

treatment (Pl+Mi+As) that was statistically similar to the treatment of (Pl+Mi) for

shoot fresh weight 7.58 g. Though the presence of M. incognita and arsenic

contamination decreased the shoot weight but when P. lilacinum present in the

treatment combination with M. incognita, due to its nematophagous properties,

increased the normal plant growth in combination with Glomus sp.


Fig. 15. Variation of shoot fresh weight of eggplant due to eight P. lilacinum

treatments of eggplant influenced by the role of P. lilacinum in combination of

Glomus sp in arsenic amended soil challenged with Meloidogyne incognita

b

de

f

bc c

a

ef

d

0

2

4

6

8

10

12

14

16

18

Aer

ial f

resh

wei

ght

(g)

Treatments

90


Shoot dry weight of eggplant influenced by P. lilacinum related treatments in


incognita is presented in Fig. 16. Among eight P. lilacinum treatments, the highest

result for dry shoot weight 1.91 g was found from the treatment (G+Pl) which was

significantly different from all other treatments followed by (C+Pl) that gave 1.63 g

dry shoot weight. The lowest dry shoot weight 1.08 g was found from treatment

(Pl+Mi+As) that was statistically similar to the treatment of (Pl+Mi) for 1.19 g shoot

dry weight. P. lilacinum gave better plant growth in the experiment of Davide et al.

(1987) which confirms our study viz. when P. lilacinum was in combination of

Glomus the growth parameters projected better results.


Fig. 16. Shoot dry weight of eggplant influenced by P. lilacinum related treatments in


incognita

ab

cdd

bcd cdbc

a

cd

0

0.5

1

1.5

2

2.5

Aer

ial d

ry w

eigh

t (g

)

treatments

91


Influence of P. lilacinum involved treatments combination on root fresh weightis

shown in Fig. 17. Results revealed that treatment (G+Pl) gave the highest fresh root

weight 11.06 g that was statistically different from all other treatments followed by

the treatment (C+Pl) gave result of 8.18 g. The lowest fresh root weight 5.26 g was

found from treatment (Pl+Mi+As) that was statistically similar to (Pl+ As) for 5.30 g

root fresh weight. P. lilacinum significantly increased root weights and other growth

characteristics in line with Gomathi et al. (2006) in brinjal. P. lilacinum provided

positive impact in combination with the Glomus sp.


Fig. 17. Root fresh weight of eggplant influenced by P. lilacinum related treatments

in combination of Glomus sp. in arsenic amended soil challenged with Meloidogyne

incognita

b

d dc c

a

bc bc

0

2

4

6

8

10

12

14

Fres

h r

oo

t w

eigh

t (g

)

Treatments

92


Root dry weight of eggplant influenced by eight P. lilacinum involved treatments in


incognita is presented in Fig. 18. Of all the P. lilacinum treatments, treatment (G+Pl)

gave the highest dry root weight 1.76g compared to other treatment that was

statistically different from all other treatments followed by the treatments (C+Pl) for

1.38 g root dry weight. The lowest dry root weight 0.31 g was found from treatment

(Pl+As) that was statistically similar to the treatment (Pl+Mi+As) for root dry weight

0.40 g. This result is in support to the reports of Pandey and Dwivedi (2001) and

Dhawan et al. (2004) who recorded maximum root weight in P. lilacinus applied

treatment. Since both of two bio-agents have synergistic effect on each other so they

provided better dry weight in different individual and combined treatment.


Fig. 18. Root dry weight of eggplant influenced by P. lilacinum related treatments in


incognita

b

ec

d d

a

c

b

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Dry

ro

ot

wei

ght

(g)

Treatments

93

4.3.9. Soil colonization by P. lilacinum (CFU/g soil)

Influence of P. lilacinum in different combination with Glomus sp. in arsenic amended

soil challenged with Meloidogyne incognita on CFU/ g soil is shown in Fig. 19.

Among this P. lilacinum treatments, the highest obtained CFU 40.20 per gram soil

was from the treatment (G+Pl) that was statistically similar to the result of the

treatment (C+Pl) for 37.80 CFU/g soil. The lowest result was found from treatment

(Pl+Mi+As) with 14.80 CFU/ g soil. Lowest result from treatment (Pl+Mi+As) was

found may be due to the presence of M. incognita and arsenic combindly which

probably deteriorated the effect of P. lilacinum.


Fig. 19. Influence of P. lilacinum in different combination with Glomus sp. in arsenic

amended soil challenged with Meloidogyne incognita on CFU/ g soil in dilution of

(1:1000)

a

cd

dcd

bc

a

bb

0

5

10

15

20

25

30

35

40

45

CFU

(1

0 4

) /g

so

il

Treatments

94

4.4. For P. lilacinum and M. incognita parallel treatments

4.4.1. Gall index

Gall index of eggplant influenced by P. lilacinum is shown in Fig. 20. Effect of P.

lilacinum on gall index was found highest in treatment (Pl+Mi+As) of 3.26 that was

statistically distinct from other treatments. The lowest gall index was found from the

treatment (G+Pl+Mi) that was statistically similar to (Pl+Mi) and (G+Pl+Mi+As) for

1.04, 1.50 and 1.50 gall index, respectively. The findings of the present study were

supported by the findings of Kiewnick et al. (2011). They reported the fungal bio-

control agent, P. lilacinus strain with the lowest dose of 2×105 CFU/g soil was already

sufficient to reduce root galling by 45%. Application of bioformulated P. lilacinus

significantly reduced root galling. This result confirms the report of Oclarit and

Cumagun (2009) and Khalil et al. (2012) that P. lilacinus is an effective biocontrol

agent of M. incognita against tomato.


Figure 20. Gall index of eggplant influenced by P. lilacinum

a

b

b

b

0

0.5

1

1.5

2

2.5

3

3.5

4

Gal

l in

dex

treatments

95

4.4.2. Number of eggmass/ root

Role of P. lilacinum on the number of eggmass/ root was represented in Fig. 21.

The lowest number of eggmass/ root was 5.80 at the treatment of (G+Pl+Mi) that

was statistically different from all other treatments. The highest number of eggmass/

root 13.6 was found from the treatment (Pl+Mi+As) that was statistically distinct

from all other combination of the P. lilacinum and M. incognita combined

treatment. The findings of the present study were supported by the findings of

Kiewnick et al. (2011). They reported the fungal bio-control agent, P. lilacinus

strain with the lowest dose of 2×105 CFU/g soil was already sufficient to reduce

number of egg masses by 69%. Application of bioformulated P. lilacinus

significantly reduced root galling and egg production by the nematode species. This

result validates the report of Oclarit and Cumagun (2009), and Khalil et al. (2012)

that P. lilacinus is an effective biocontrol agent of M. incognita against tomato.


Fig. 21. Role of P. lilacinum on the number of eggmass/ root

a

bb

c

0

2

4

6

8

10

12

14

16

Eggm

ass/

roo

t

Treatments

96

4.4.3. Number of egg/ eggmass

Figure 22 shows that the effect of P. lilacinum on the number of M. incognita egg/

eggmass. The effect of P. lilacinum on the highest number of egg/ eggmass 297.4 was

found highest in the treatment of (Pl+Mi+As) that was statistically different from all

other treatments. The lowest number of egg/ eggmass 164.0 was found from the

treatment of (G+Pl+Mi) which was statistically different from all other treatments.


Fig. 22. Effect of P. lilacinum on the number of M. incognita egg/ eggmass

a

b bc

0

50

100

150

200

250

300

350

Egg

No

./eg

gmas

s

Treatments

97

4.4.4. Eggmass colonization

Role of P. lilacinum on the number of eggmass colonization was represented in Fig.

23. Effect of P. lilacinum on eggmass colonization was found the highest (37.80%) in

the treatment of (G+Pl+Mi) that were differed significantly from all other treatments.

The lowest eggmass colonization was 22.70% found from the treatment (Pl+Mi+As)

that was also statistically significant from all other treatments. Aminuzzaman and Liu

(2011) reported more than 80% egg parasitism and 52% juvenile mortality of

Meloidogyne spp. by Paecilomyces lilacinus. Our results are in agreement with earlier

findings of Santos et al. (1992) who observed the variations of P. lilacinus for egg

parasitism on M. incognita.


Fig. 23. Role of P. lilacinum on the number of eggmass colonization

d

c b

a

0

5

10

15

20

25

30

35

40

45

Eggm

ass

colo

niz

atio

n

Treatments

98

4.4.5. Reproduction factor

Of all the P. lilacinum treatments, four treatments were in combination of M. incognita

and the influence of P. lilacinum on the reproduction factor for nematode is shown in

Fig. 24. P. lilacinum on reproduction factor of M. incognita was found the lowest in

the treatment of (G+Pl+Mi), where the reproduction factor was lowest 1.85 which was

statistically different from all other treatments. The highest reproduction factor 5.39

was found from the treatment of (Pl+Mi+As). This result was in agreement with the

findings of Udo et al. (2013). Park et al. (2004) reported the production of

leucinotoxin and other nematicidal compounds by P. lilacinus. The overall effect was

the decrease in population and pathogenicity of the nematode species.


Fig. 24. Role of P. lilacinum on reproduction factor of M. incognita

a

b b

c

0

1

2

3

4

5

6

Rep

rod

uct

ion

fac

tor

Treatments

99

4.5. For Meloidogyne incognita involved treatments

4.5.1. Number of eggmass/root

Number of eggmass/ root of M. incognita influenced by the effect of Glomus sp in

combination of P. lilacinum in arsenic amended soil is presented in Fig. 25. Among

eight M. incognita involved treatments, the highest eggmass/ root 65.50 was found

from the treatment (C+Mi) that was significantly different from all other treatments

followed by (Mi+As) which gave 30.80 eggmass/ root. The lowest number of

eggmass/ root 5.80 was obtained from treatment (G+Pl+Mi) that was also

significantly different from all other treatments followed by the treatments (Pl+Mi)

and (G+Pl+As+Mi) for 8.80 and 9.80 eggmass/ root respectively. This result validates

the report of Oclarit and Cumagun (2009) and Khalil et al. (2012) that P. lilacinus is

an effective biocontrol agent of M. incognita against tomato.


Fig. 25. Number of eggmass/ root of M. incognita influenced by the effect of Glomus

sp. in combination of P. lilacinum in arsenic amended soil

a

b

c

ef

d

fg

0

10

20

30

40

50

60

70

Eggm

ass/

ro

ot

Treatments

100

4.5.2. Gall index

Gall index of M. incognita influenced by the role of P. lilacinum and Glomus sp. in

arsenic amended soil challenged with Meloidogyne incognita is shown in Fig. 26.

Among eight Meloidogyne incognita involved treatments, the highest gall index 6.52

was found from the treatment (C+Mi) that was significantly different from all other

treatments followed by gall index for treatment of (Mi+As). The lowest gall index

1.04 was obtained from treatment (G+Pl+Mi), statistically similar to the gall index

1.50 and 1.50 for the treatment of (Pl+Mi) and (G+Pl+Mi+As), respectively. Nicolás,

et al. (2014) worked on the effect of Glomus intraradices on tomato plants inoculated

with the nematode at transplanting. He found the use of AMF favored tomato biomass

and reduced the number of galls and reproduction factor on the plants inoculated with

the nematode at transplanting.


Fig. 26. Gall index of M. incognita influenced by the role of P. lilacinum and Glomus

sp. in arsenic amended soil challenged with Meloidogyne incognita

a

b

ccd

e

d

e

e

0

1

2

3

4

5

6

7

8

Gal

l in

dex

Treatments

101

4.5.3. Number of egg/ eggmass

Fig. 27 shows that the results of the number of egg/ eggmass of M. inconita on

different combination with P. lilacinum and Glomus sp. in arsenic amended soil

challenged with Meloidogyne incognita. Of all M. incognita involved treatments, the

highest number of egg/ eggmass (480.6) was found from the treatment (C+Mi) that

was statistically different from all other treatments followed by the number of egg/

eggmass (330.2) from the treatment of (G+Mi). The lowest number of egg/ eggmass

164.0 was revealed from the treatment (G+Pl+Mi) that was statistically different from

all other treatments followed by the number of egg/ eggmass 190.4 for the treatment

(Pl+Mi+As). The other treatments also gave significantly different number of egg/

eggmass for different combination of the treatments. Since both the bio-agents have

the antagonistic effect against M. incognita, might be their combined effect

synergistically boosted the reduction of Number of egg/eggmass.


Fig. 27. Number of egg/ eggmass of M. incognita on different combination with P.

lilacinum and Glomus sp. in arsenic amended soil challenged with Meloidogyne

incognita

a

ce d

f

b

fg

0

100

200

300

400

500

600

No

. of

egg/

eggm

ass

Treatments

102

4.5.4. Reproduction factor

Reproduction factor of M. incognita influenced by the effect of Glomus sp. in

combination of P. lilacinum in arsenic amended soil is presented in figure28. Among

eight M. incognita involved treatments, the highest reproduction factor 14.4 was

found from the treatment (C+Mi) that was significantly different from all other

treatments followed by (Pl+Mi+As) which gave 5.39 reproduction factor. The lowest

reproduction factor 1.85 was obtained from treatment (G+Pl+Mi) that was also

significantly different from all other treatments followed by the treatments (Pl+Mi),

(G+Pl+As+Mi) and (Mi+As) for 3.09, 3.14 and 3.42 reproduction factor, respectively.

Nicolás, et al. (2014) worked on the effect of Glomus intraradices on tomato plants

inoculated with the nematode at transplanting. He found the use of AMF favored

tomato biomass and reduced the number of galls and reproduction factor on the plants

inoculated with the nematode at transplanting.


Fig. 28. Reproduction factor of M. incognita influenced by Glomus sp. in combination

of P. lilacinum in arsenic amended soil

a

dc

b

d

c

d

e

0

2

4

6

8

10

12

14

16

Rep

rod

uct

ion

fac

tor

Treatments

103

4.5.5. Nutrient uptake

Nutrient uptake influencd by the Purpureocillium lilacinum in combination with

Glomus sp. on shoot of eggplant in arsenic amended soil challenged with Meloidogyne

incognita presented in Table 4. The result revealed that among the 16 treatments, the

highest nutrient uptake was recorded in treatment (G + Pl) and those were 0.48 ppm

P, 0.62 ppm K, 3.10 ppm S in shoot which was significantly different from all other

treatments. It was conspicuously observed that with the arsenic toxicity, nutrient

uptake decreases. The lowest P uptake was found from the treatment (Pl + Mi + As)

that was statistically similar with (Pl + As) and (Mi + As). The lowest K uptake was

found from treatment (C + Mi) that was statistically similar to the result of treatment

(Mi + As). Again, the lowest S uptake was recorded in treatment in (C + As) which

was statistically similar with the treatment (Mi + As). Findings indicate that the

treatment combination with the arsenic and M. incognita decreases the nutrient

uptake. It was observed from the analysis, nutrient uptake (N, P, K, S) was increased

in the treatment combination of arbuscular mycorrhizal fungus (Glomus sp.).

Mycorrhizae inoculated treatment increased the nutrient uptake significantly in

contrast to other treatments. The best treatment combination was found (G+Pl) where

when Glomus sp. and P. lilacinum was combinedly inoculated to the soil. Moreover,

the treatment combination with the Glomus sp. increased the nutrient uptake rather

than the without-Glomus sp. treatment combination. The present results have the

similarity with the findings of Al-Amri (2013) who reported that AM inoculated

Broadbean plants had higher shoot and root, and N, P, K content than non-AM plants.

In his experiment, broad bean plants grown in wastewater contaminated soil. AM

broad bean plants had higher shoot and root P, N and K contents than nonAM plants.

104


phosphorus, potassium and Sulphur percentage of shoot of eggplant in arsenic


Treatments Shoot

N (%) P (%) K (%) S (%)

C 1.00 g 0.25 de 0.17 gh 0.61 h

C + G 1.63 b 0.40 b 0.55 b 2.39 b

C + Pl 1.24 de 0.31 c 0.50 c 1.90 c

C + Mi 0.86 h 0.21 ef 0.12 i 0.43 i

C + As 0.80 hij 0.20 f 0.17 gh 0.33 i

G + As 1.52 c 0.38 b 0.37 e 1.55 d

Pl + As 0.76 ij 0.19 f 0.19 g 0.63 h

Mi + As 0.84 hi 0.21 ef 0.15 hi 0.42 i

G + As + Mi 1.29 d 0.32 c 0.50 c 1.02 f

Pl + Mi + As 0.74 j 0.18 f 0.24 f 0.45 i

G + Pl + As 1.58 bc 0.39 b 0.45 d 1.22 e

G + Pl + As + Mi 1.12 f 0.28 cd 0.35 e 0.92 fg

G + Mi 1.24 de 0.31 c 0.21 fg 0.88 g

G + Pl 1.92 a 0.48 a 0.62 a 3.10 a

Pl + Mi 0.77 ij 0.19 f 0.36 e 0.58 h

G + Pl + Mi 1.19 ef 0.29 c 0.24 f 0.67 h

CV (%) 5.35 5.35 7.92 3.75


105

Giri et al. (2005) assessed the effect of two arbuscular mycorrhizal (AM) fungi,

Glomus fasciculatum and G. macrocarpum on shoot and root dry weights and nutrient

content of Cassia tora in a semi-arid wasteland soil. The concentration of P, K, Cu,

Zn and Na was significantly higher in AM inoculated seedlings than non-inoculated

seedlings.

Mycorrhizal inoculation reduced the arsenic (As) concentration in shoots. This study

shows that among the eight arsenic involved treatment combinations when Glomus

sp. and P. lilacinum combinedly present in the treatment of (G+Pl+As) the nutrient

uptake was better than their individual combination with arsenic. This findings

supported by the experiment of Elahi et al. (2010) who evaluated the influence of

AMF inoculation on growth, nutrient uptake, arsenic toxicity and chlorophyll content

of eggplant grown in arsenic amended soil. The findings of the study indicated that

AMF inoculation not only reduces arsenic toxicity but also can increase growth and

nutrient uptake of eggplant shoot. Less arsenic content and higher chlorophyll and

nutrient uptake were recorded in mycorrhiza inoculated plants in compare to non-

inoculated plants. The findings emphasized that AMF inoculation reduced arsenic

translocation from soil to plant and increase growth and nutrient uptake and

chlorophyll content of eggplant.

The treatment combination of arsenic (As) and M. incognita reduced the nutrient

uptake of roots and shoots. This higher nutrient uptake in mycorrhizal plants might

be attributed to the contribution of fungal external mycelia which explore a large

volume of soil and thus absorb more nutrients (Gupta and Janardhanan, 1991).

Previous report showed that arbuscular mycorrhizal fungi increase plant uptake of

phosphate (Bolan, 1991).

106

4.5.6. Arsenic uptake

Arsenic uptake by shoot of eggplant influenced by Glomus sp in combination of P.

lilacinum in arsenic amended soil challenged with Meloidogyne incognita is presented

in Fig. 4. Among eight arsenic involved treatments, it was found that treatment

(G+Pl+As) gave lowest amount arsenic uptake 29.30 ppb of eggplant which was

statistically different from all other treatments followed by treatment (C+G). The

highest amount of arsenic uptake was recorded from treatment (C+As) ie. 60.70 ppb

which was statistically different from all other treatments. AMF can essentially

improve plant mineral nutrition and plant water relations (Li et al., 2014), and enhance

plant resistance to heavy metal contaminations (Hildebrandt et al., 2007). Recent

studies show that the arbuscular mycorrhizas naturally occur in As-contaminated soils

(Smith et al., 2010) and mycorrhizal inoculation can improve the As tolerance of

tomato (Liu et al., 2005b), maize (Bai et al., 2008) which corroborated to the present

findings.

Figure 29. Arsenic uptake by shoot of eggplant influenced by Glomus sp in

combination of P. lilacinum in arsenic amended soil challenged with Meloidogyne

incognita

a

fe

c c

b

g

d

0

10

20

30

40

50

60

70

Co

nce

ntr

atio

n (

pp

b)

Treatments

107

SUMMARY AND CONCLUSION

The serious arsenic contamination of groundwater in Bangladesh has come out

recently as the biggest natural calamity in the world. The people in 59 out of 64

districts comprising 126,134 km2 of Bangladesh are suffering due to the arsenic

contamination in drinking water. From a major review of studies conducted in

Bangladesh, and elsewhere in Asia, the report concludes that people may be exposed

to arsenic not only through drinking water, but indirectly though food crops irrigated

by contaminated groundwater. A number of studies have also reported a correlation

between arsenic in soil and reduction in crop yield.

This experiment was conducted to determine the influence of Glomus sp. and P.

lilacinum on growth of eggplant in arsenic amended soil challenged with M.

incognita. The investigation of results showed significant difference among

treatments in response to several combination of treatment. Of all treatments, the

highest shoot length, root length, fresh weight of shoot and root was found from

treatment (G+Pl) that was 26.50, 20.44, 15.51 and 11.06 cm, respectively. The lowest

shoot length (9.64 cm) and root length (7.16 cm) found from treatment (Mi + As) that

was statistically similar to treatment (C+Mi). Fresh weight of shoot found lowest

6.34g from treatment (C+Mi) followed by (Mi + As). The lowest fresh root weight

5.48 g was found from treatment (C) followed by (C+Mi), (Pl+ As) and (Mi + As)

which gave 4.82, 5.30g and 5.19g, respectively. Dry weight of shoot (0.94 g) and root

(0.27 g) that was recorded lowest from treatment (Mi+As) that was statistically similar

to the treatment (C+As) and (Mi+As), respectively. The highest dry weight of shoot

and root, leaf area and chlorophyll content of eggplant was revealed from treatment

(G+Pl) that was 1.91 g, 1.76 g, 48.18 cm2 and 42.18 μgcm-2, respectively. The lowest

leaf area 15.07 cm2 found from treatment (C+Mi) that was statistically different from

all other treatments followed by (Mi+As) and (C+As) which gave leaf area of 22.27

108

cm2 and 24.46 cm2, respectively. And the chlorophyll content 22.32 μg cm-2 was

recorded lowest from treatment (Mi + As).

Influence of Glomus sp. involved treatments combination on number of Glomus

spore/10 g soil and root infection shown that treatment (G+Pl) gave highest result. Of

eight AMF involved treatments 35.60 number of Glomus spore/10g soil and 58.30%

of root infection was recorded highest from treatment (G+Pl). The lowest root

infection and number of spore/10g soil found 34% and 15.80, respectively from

treatment (G+As+Mi).

Influence of P. lilacinum in different combination with Glomus sp. in arsenic amended

soil challenged with Meloidogyne incognita on CFU/ g soil is obtained highest 40.20

per gram soil from treatment (G+Pl) that was statistically similar with the result of the

treatment (C+Pl) for 37.80 CFU/g soil. The lowest CFU/ g soil was found from

treatment (Pl+Mi+As) with 14.80 CFU/ g soil.

Gall index, number of eggmass/ root, number of egg/ eggmass and reproduction factor

of M. incognita influenced by the role of P. lilacinum found lowest in treatment

(G+Pl+Mi) that was 1.04, 5.80, 164.0 and 1.85 respectively. On the other hand, the

highest gall index (6.52), number of eggmass/ root (65.60), number of egg/ eggmass

(480.6) and reproduction factor (14.4) was revealed from treatment (C+Mi). Eggmass

colonization of M. incognita found highest, 37.80% by the influence of Glomus sp.

and P. lilacinum involved treatment combination (G+Pl+Mi). The lowest eggmass

colonization was 22.70% found from the treatment (Pl+Mi+As) that was also

statistically significant from all other treatments.

109

The result revealed that among the 16 treatments, the highest nutrient uptake was

recorded in treatment (G + Pl) and those were 0.48% P, 0.62% K, 3.10% S in shoot

which was significantly different from all other treatments. It was observed that with

the arsenic toxicity, nutrient uptake decreases. The lowest P uptake 0.18% was found

from the treatment (Pl + Mi + As) that was statistically similar to (Pl + As) and (Mi +

As) for 0.19 and 0.21 % respectively. The lowest K uptake 0.12% was found from

treatment (C + Mi) that was statistically similar to the result of treatment (Mi + As)

for 0.15%. For S uptake, the lowest amount of uptake was 0.33% found from treatment

(C+As) that was statistically similar to (Pl+Mi+As) and (Mi+As).

Among eight arsenic involved treatments, it was found that treatment (G+Pl+As) gave

lowest amount arsenic uptake 29.30 ppb of eggplant which was statistically different

from all other treatments followed by treatment (C+G). The highest amount of arsenic

uptake was recorded from treatment (C+As) ie. 60.70 ppb which was statistically

different from all other treatments.

It is now recognized that AM fungi and Purpureocillium lilacinum can be harnessed

in order to improve productivity in agriculture by reducing the input of fertilizers

and/or by enhancing plant survival, thus offsetting ecological and environmental

concerns. AMF helps plant in nutrient uptake, reducing arsenic toxicity as well as

reducing plant diseases and insect attack through induced resistance. On the other

hand, P. lilacinum and AMF both have the management effect on M. incognita in

eggplant. (G+Pl) treatment can be recommended in the M. incognita infested and

arsenic contaminated soil of Bangladesh. In future the different species of Glomus

need to be considered in combination with different doses of P. lilacinum for

specification of treatment. As a result, this research will be a platform which will

minimize the farmers concern of expenditure for pesticide and fertilizer and also will

ensure the sustainable agriculture.

110

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