STUDIES ON LEAF BLIGHT OF BLACK GRAM (Vigna mungo (L ... · GRAM (Vigna mungo (L.) Hepper) OF CHHATTISGARH PLAINS M. Sc. (Ag.) Thesis by Manish Kumar Sahu DEPARTMENT OF PLANT PATHOLOGY

STUDIES ON LEAF BLIGHT OF BLACK

GRAM (Vigna mungo (L.) Hepper) OF

CHHATTISGARH PLAINS

M. Sc. (Ag.) Thesis

by

Manish Kumar Sahu

DEPARTMENT OF PLANT PATHOLOGY

COLLEGE OF AGRICULTURE, RAIPUR

FACULTY OF AGRICULTURE

INDIRA GANDHI KRISHI VISHWAVIDYALAYA,

RAIPUR (Chhattisgarh)

2018

STUDIES ON LEAF BLIGHT OF BLACK

GRAM (Vigna mungo (L.) Hepper) OF

CHHATTISGARH PLAINS

Thesis

Submitted to the

Indira Gandhi Krishi Vishwavidyalaya, Raipur

by

Manish Kumar Sahu

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

Master of Science in

Agriculture

(Plant Pathology)

U.E. IDNo.20161724933 ID No.120116177

JULY, 2018

ACKNOWLEDGEMENT

“Education plays vital role in personal and social development and

teacher plays a fundamental role in imparting education. Teachers have crucial

role in shaping young people not only to face the future with confidence but also

to build up it with aim and responsibility. There is no substitute for teacher pupil

relationship”. I take this golden opportunity to express my heartful humble and

deepest sense of gratitude to those who helped me to complete my research

possible. These words are small acknowledgement but never fully recompensed for

their great help and co-operation.

I start in the name of God-who has bestowed upon me all the physical

and mental attributes that I posses and skill to cut through and heal a fellow

human.

It is my privilege to study and conduct my research under Dr. N. Khare

Principal Scientist, Department of Plant Pathology, College of Agriculture,

Raipur (C.G.), Chairman of my advisory committee, who provided me the research

insight, illuminating and meticulous guidance, calm endurance, continuous and

unfailing encouragement, scholarly suggestions, unique supervision, constructive

criticisms, sympathetic attitude, plausible appreciation and sustained support

during the entire course of investigation and preparation of manuscript. I am

highly indebted to him for his invaluable painstaking efforts taken towards my

study while devoting his precious time. His scientific approach and generosity

without any reservation greatly helped me to work under his supervision,

knowledge and enthusiastic interest, which he provided me throughout my post

graduation and research investigation despite his busy schedule of work.

I emphatically and gratefully acknowledge extend my loyal and venerable

thanks to members of my Advisory Committee, Dr. A.S. Kotasthane, Professor

and Head (Department of Plant Pathology), Dr. P. L. Johanson, Senior Scientist,

(Department of Genetices and plant Breeding), Dr. N. Lakpale, Associate

Professor, Dr. R. K. Dantre, Professor, (Department of Plant Pathology) and Dr.

(Smt.) G. Chandrakar, Professor, (Department of Agril. Statistics and Social

Sciences, Language) for providing proper guidance, critical comments, valuable

suggestions and deligent support lead to timely completion of this work.

I am also highly obliged to Hon’ble Vice Chancellor, Dr. S. K. Patil, Dr.

S. S. Rao, Director Research Services, Dr. O. P. Kashyap, Dean, College of

Agriculture, Raipur, Dr.M.P. Thakur, Director of Instructions andDr.(major)

TABLE OF CONTENTS

Chapter Title Page ACKNOWLEDGEMENT

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF PLATES

LIST OF ABBREVIATIONS

ABSTRACT

I INTRODUCTION

II REVIEW OF LITERATURE

2.1General

2.2 Collection, Isolation, Purification and

identification of pathogen Macrophomina

phaseolina

2.3 Screening of black gram entries for their

reaction to Macrophomina blight in-vivo

2.4 In-vitro evaluation of Pseudomonas

fluorescence on the radial mycelial growth

of Macrophomina phaseolina

2.5 In-vitro evaluation of fungicides on the

radialmycelial growth of Macrophomina

phaseolina

iii-vi

iv-v

vi

vii

viii

ix

x-xiii

1-4

5-21

5-6

7-8

8-10

11-15

16-21

III MATERIALS AND METHODS 22-27

3.1 Collection, Isolation, Purification and 22

identification of pathogen Macrophomina

Phaseolina

3.1.1 Cleaning and Sterilization of materials 22

3.1.2 Collection of disease sample 23

3.1.3 Isolation 23

3.1.4 Purification and maintenance of culture and 23

identification of pathogen

3.1.4.1 Media used 23

3.1.5 Identification of the fungus 24

3.2 Screening of black gram entries against 24

Macrophomina phaseolina

Chapter Title Page 3.3 In-vitro evaluation of Pseudomonas

fluorescence on the radial mycelial growth

of Macrophomina phaseolina

3.4 In vitro evaluation of fungicide on the

radialmycelial growth of Macrophomina

phaseolina

IV RESULTS AND DISCUSSION

4.1 Collection of disease sample

4.1.1 Isolation

4.1.2 Identification of the fungus 4.2 Screening of black gram entries for their reaction

to Macrophomina blight 4.3 In-vitro evaluation of Pseudomonas fluorescence

on the radial mycelial growth of Macrophomina phaseolina

4.4 In vitro evaluation of fungicides on the mycelialgrowth of Macrophomina phaseolina

V SUMMARY ANDCONCLUSION

REFERENCES

RESUME

25

26-27

28-47

28

29

29-34

35-37

38-42

43-47

48-49

50-58

59

LIST OF TABLES

Table No. Title Page No.

3.1 Categorization of entries on the basis of per cent disease 24

incidence and 1-5 scale (IIPR, Kanpur, 1996)

3.2 List and Concentration of fungicides used in in vitro 27

Evaluation

4.1 Pathogen isolated from infected leaf and stem samples of 28

different entries of black gram

4.2 Screening of blackgram entries for their reaction to 36

Macrophomina blight

4.3 Efficacy of differentPseudomonas isolateson the 39

growth of M. phaseolina in vitro

4.4 Effect of different fungicides on growth of 44


LIST OF FIGURES

Figure No. Title Page No.

4.4 In-vitro evaluation of Pseudomonas fluorescence on 41

the radial mycelial growth of Macrophomina phaseolina

4.5 Per cent reduction in mycelial growth over control 42

4.6 Effect of different fungicides on growth of 46

Macrophomina phaseolina in vitro

4.7 Per cent reduction in mycelial growth over control 47

LIST OF PLATES

Plates Title Page

4.1 Disease symptoms in field 31

4.2 Symptoms used for isolation of pathogen 32

4.3 Pure culture of Macrophomina phaseolina on PDA 33

4.4 Pure culture of Macrophomina phaseolina on water agar 33

4.2 Sclerotia of Macrophomina phaseolina 34

4.3 Screening of black gram entries 37

4.4 Effect of different Pseudomonas fluorescenceisolates on 40

growth of Macrophomina phaseolina in vitro

4.5 Effect of different fungicides on growth of Macrophomina 44

phaseolina in vitro

LIST OFABBREVIATIONS

Abbreviations Description

% : Per cent

0C : Degree Celsius

Cm : Centimetres

Fig. : Figure

Gm : Gram

ha-1 : Per hectare

i.e. : That is

Kg : Kilogram

m-2 : Per Meter square

Mm : Milometer

S. No. : Serial number

Viz : That is to say / in other words

q ha-1 : quintal per hectare

CD : critical difference

et al. : et alia (and others)

PDI : Percent disease Incidence

ppm : Part per million

LSI : Location Severity Index

SEm± : Standard error of means

Out of Forty entries screened, none were resistant or moderately resistant.

Only 4 entries i.e. KPU 17- 1, KPU 17- 2, KPU 17- 3 and KPU 17- 4 were

moderately susceptible. Rests of the varieties were susceptible and highly

susceptible against Macrophomina phaseolina.

Ten different isolates of Pseudomonas fluorescence were tested for their

antagonistic potential against Macrophomina phaseolina, the result revealed that

among all isolates isolate 1 exhibited in vitro maximum antagonistic potential and

significantly reduced mycelia growth (36mm) and percent inhibition (60%) of M.

phaseolina, followed by P3.

All the fungicides except propineb significantly reduced the mycelia

growth of Macrophomina phaseolina. Carboxine+Thiram, Hexaconazole+Zineb

and Tricyclazole completelyinhibited the mycelial growth. Thiram and

Carbendazim were found to be least and Propineb is not effective in reducing the

growth of the test fungus.

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CHAPTER - I

INTRODUCTION

Pulses in India have long been considered as the poor man’s only source of

dietary protein. Besides being a rich source of protein, they maintain soil fertility

through biological nitrogen fixation in soil and thus play a vital role in sustainable

agriculture (Kannaiyan, 1999). In India, pulses have been cultivated since time

under rainfed situations which is characterized by poor soil fertility and moisture

stress. These crops are energy rich but cultivated largely under energy starving

situations. Unlike in cereals, varietal breakthrough in pulses has not been taken

place. Pulses occupies 25.25 m ha area and contributes 16.46 m tonnes production

with an average productivity of 652 kg ha-1 (Anonymous, 2015-16a). During the

last four decades, the total area under pulses remained virtually stagnant (22 to 24

million ha) with almost stable production (12 to 14 million tonnes), even though

the population has been increased. As a result, per capita availability of pulses has

been declined from 64 g per day in 1951-56 to less than 40 g per day as against

WHO’s recommendation of 80 g per day (Asthana and Chaturvedi, 1999). This

situation led to the severe shortage of pulses in India, which has aggravated the

problem of malnutrition in large section of vegetarian population of our country.

Blackgram is one of the important kharif pulse crop grown throughout the India,

next to green gram. It is consumed in form of ‘dal’ (whole or split, husked or

unhusked) or parched. It is chief constituent of ‘papad’ and also of ‘bari’ (spiced

balls) which make a delicious curry. Urdbean differs from other pulses in its

peculiarity of attaining when mixed with water a somewhat mucilaginous pasty

character, giving additional body to the mass. In the south, the husked dal is

ground into a fine paste and allowed to ferment and is mixed with equal quantity of

rice flour to make ‘dosa’ and ‘idli’. It is also fried to serve as savoury dish. Urd dal

is also used in the preparation of ‘halwa’ and ‘imarti’.

Blackgram (Vigna mungo. L.) also known as urdbean or mashbean belongs

to popular plant family Papillionaceae and is among the most important pulse

crops of the world. It has been cultivated all over the world, mainly in south

western Asia, Egypt, Europe, India, Pakistan, Nepal and china. It has great value as

food, fodder and green manure. In addition to improving the soil fertility it is a

cheap source of protein for direct human consumption. The economic product of

black gram is seed grain, which is a good source of dietary protein. Urdbean

contains approximately 25-28% protein, 1.0-1.5% oil, 3.5 - 4.5% fibre, 4.5-5.5%

ash and 62-65% carbohydrates on dry weight basis. High values of lysine make

urdbean an excellent complement to rice in terms of balanced human nutrition.

Being short duration and photo, thermo insensitive, black gram is considered as an

excellentfor crop intensification and diversification.

Urdbean is one of the most important pulse crops of India cultivated over a

wide range of agro-climatic situations. Urdbean is mainly grown in tropical and

sub-tropical climate. Urdbean is a native of India and originated from wild plant

i.e. Phaseolus sublobatus. It occupies sizable area in India, Bangladesh, Pakistan,

Myanmar, Sri Lanka, and West indies. It is grown all over country in kharif and

summer seasons also, while in south India, it is raised mainly in rabi season.The

major urdbean growing states of the country are Maharashtra, Andhra Pradesh,

Madhya Pradesh, Uttar Pradesh, Tamilnadu, Karnataka and Rajasthan.

Development of short duration, photo, thermo-insensitive and disease resistant

varieties has led to its cultivation as a sole or intercrop during spring season in

North India and as a sole / relay crop during rabi season in the rice fallows of the

coastal peninsula. It is occupying an area of 3.06 million ha and total production of

1.70 million tonnes with an average productivity of 555 kg ha-1in India

(Anonymous 2014-15.c). In Chhattisgarh it occupies an area of 154.53 thousand ha

and total production of 128.00 thousand tonnes with average productivity 470 kg

ha-1 (Anonymous, 2014 – 15.c).

Throughout the India, the black gram is used for different purposes. The

major portion is utilized in making dal, curries, soup, sweets and snacks. With

sprouting there is an increase in the thiamine, niacin and ascorbic acid, thus

mungbean sprouts are increasingly becoming popular in certain vegetarian diets.

Moreover, its food values lie in high and easily digestible protein. Amino acid

analysis indicates that it is an excellent complement to rice for balanced human

nutrition.

The major fungal diseases which infect the crop are leaf blight

[Macrophomina phaseolina (Tassi) Goid], powdery mildew (Erysiphe polygoni

DC), web blight(Thanatephorus cucumeris (Fr.) Donk (=Rhizoctonia solani

Kuhn), Cercospora leaf spots (Cercospora canescens Ellis and Martin, C. cruenta

Sacc., C. dolichi Ellis and Everlast, C. kikuchi Matsumoto & Tomoyasu and

Anthracnose (Colletotrichumdematium and C. lindemuthianum (Philip et al., 1969,

Dwivedi and Saksena, 1974.,and Grewal,1988).

Macrophomina phaseolina (Tassi) Goid is one of the most damaging seed

andsoil borne pathogen, infecting about 500 plant species in more than 100

families throughout the world [(Kunwar et al, 1986, Mihail and Taylor 1995)].

Under favorable conditions the fungus causes many diseases like leaf blight,

damping off, seedling blight, collar rot, stem rot, charcoal rot and root rot in

various economically important crops. Mung bean was observed severely affected

by leaf blight caused by Macrophomina phaseolina (Tassi.) Goid. In Kharif as well

as during summer season.It was first reported from Jabalpur (M.P.) India (Philip et

al., 1969).

The pathogen attacks on all parts of plant i.e. root, stem, branches, petioles,

leaves, pods and seeds. Moreover, seed infection of Rhizoctonia bataticola (M.

phaseolina) ranges from 2.2-15.7% which causes 10.8% losses in grain yield and

12.3% inprotein content of seed in black gram (Kaushik et al.1987). The infected

seeds act as an important source of primary inoculum for new areas. Soil and seed

borne nature of the disease possesses problems for an effective disease

management. Therefore, an attempt has been made to integrate management of leaf

blight disease on black gram incited by Macrophomina phaseolina (Tassi.) Goid

which have become a serious problem in hampering the production of the black

gram in all growing areas of India.

Considering the severity of the disease in Chhattisgarh state and crop losses in

the farmer's field the present investigations entitled, “STUDIES ON LEAF

BLIGHT OF BLACK GRAM (Vignamungo (L.) Hepper) OF

CHHATTISGARH PLAIN”were undertaken with following objectives:

http://www.mycologia.org/content/99/6/797.full#ref-12



Objectives of investigation:

1. Collection, Isolation, Purification and identification of pathogenMacrophomina

phaseolina

2. Screening of black gram entries for their reaction to Macrophomina blight in-vivo

3. In-vitro evaluation of Pseudomonas fluorescence on the radial mycelial growth of


4. In-vitro evaluation of fungicides on the radial mycelial growth of Macrophomina

phaseolina

CHAPTER - II

REVIEW OF LITERATURE

In this chapter the literature on Macrophomina phaseolina has be reviewed

and cited below under different heading. Macrophomina phaseolina

(Rhizoctoniabataticola) is widely distributed throughout the tropical part of the

world. It is highlydestructive in nature and brings about severe losses in economic

crop plants.

2.1 General

Macrophomina phaseolina (Tassi) Goid(Tiarosporella phaseolina (Tassi)

Van der Aa) is a soil borne plant pathogenic fungus. It belongs to the anamorphic

Ascomycetes and is characterized by the production of both pycnidia and sclerotia

in host tissues and culture media. The pycnidial state was initially named

Macrophoma phaseolina by Tassi in 1901 and Macrophoma phaseoli by Maublanc

in 1905. In 1927, Ashby maintained the name Macrophomina phaseoli, while

Goidanich (1947) proposed Macrophomina phaseolina. Tiarosporella phaseolina

(Tassi) Van der Aa was used in 1981 by Van der Aa to designate the species. The

sclerotial state was described for the first time by Halsted as Rhizoctonia bataticola

(Taub.) Butler on Ipomoea batatas in 1890. Singh (1981) has given a brief review

on the origin and interrelationship of Vigna mungo and V. radiata. The symptoms,

mode of transmission, and host range ofimportant diseases, namely mungbean

yellow mosaic virus, leaf crinkle virus, leaf curl virus, mosaic mottle virus, and

diseases caused by Cercospora canescens, Erysiphepolygoni, Rhizoctonia

bataticola [Macrophomina phaseolina], R. solani, Xanthomonas phaseoli and

Pseudomonas phaseolicola are given. Screening forresistance, sources of

resistance, including interspecific hybridization, and induced mutations as well as

the genetics of resistance are treated along with suggestions for future breeding

strategies, symptoms, hosts and transmission of some virus, fungal and bacterial

diseases of Vigna radiata and V. mungo are outlined and information on breeding

and the genetics of resistance is reviewed. Sharma and Singh (2000) reported

that24 per cent of mungbean (Vignaradiata) seed samples collected from 11

districts of Rajasthan in India, during 1996-97, showed 0.5-38 % infection with

Macrophomina phaseolina (Rhizoctoniabataticola).

Rauf (2000) detected 24 seed born fungi belonging to different genera

using blotter paper method, from 145 seed samples of major legume crops in

Pakistan. Among these Macrophomina phaseolina, Alternaria alternata,

Ascochyta sp., Colletotrichum sp., Fusarium sp. andMacrophomina phaseolina

were the mostfrequent and known as common pathogenic fungi.

Khan et al. (2008) identified sources of genetic resistance in mung bean

against charcoal rot caused by Macrophomina phaseolina, 29 germplasm

accessions were evaluated by paper towel technique under laboratory conditions.

Two genotypes (NCM 252-10 and 40536) were highly resistant, whereas 5

cultivars (40504, NCM 257-5, 40457, NCM 251-4 and 6368-64-72) were resistant

and 6 were moderately resistant. Three genotypes were tolerant whereas the rest of

the accessions were susceptible or highly susceptible. The paper towel technique

proved to be quick and efficient for identification of resistance in mung bean for

charcoal rot disease.

Singh et al. (2013) had undertaken a study to screen for percent incidence

of seed borne mycoflora of two mung bean varieties viz. HUM-4 and HUM-7

They were screened by standard blotter paper and agar plate methods. Agar plate

method was found to be suitable as even under lesser incubation there was higher

observed percent incidence of seed mycoflora.

Suradkar S. et al. (2015) tested Twenty-seven varieties/ germplasms of

Black gram grown at Pulse Research Station, N.A.U. Navsari in Kharif season,

2008. The reactions of varieties/ germplasms against the disease were recorded. In

which TPU-4 (0.95 %) recording minimum per cent disease index was resistant

while eight germplasms were rated as moderately resistant. GU-1 (52.00 %), UB-8

(55.00%), UB-15 (56.00 %), UB-16 (60.00 %), showed susceptible reaction while

the germplasms UB-19 (78.00%) and UB-22 (76.33 %) showed highly susceptible

reactions. Other varieties showed moderately susceptible reactions.

2.2 Collection, Isolation, Purification and identification of pathogen


Philip et al. (1969) reported that Rhizoctonia bataticola infects the root's

hypocotyl region and leaf of the moong and urd crop.

Saxena et al. (1970) reported that same strains of Macrophomina

phaseolina (Rhizoctonia bataticola) caused blight and dieback of black and green

gram. It is alsoreported to cause leaf spot and blight in pigeon pea in North-

India.Sakuja (1974) recorded infection of mung and urdbean causing leaf blight

disease.

Jain et al. (1973) studied isolates of R. bataticola obtained from urd plant

parts (root, stem, leaf, pod and seeds) and soil. The isolates from various plant

parts and soil showed differences in virulence. The soil isolate was most

pathogenic. The isolates differed in their growth pattern and sclerotial size. The

leaf isolate developed the largest sclerotia and the seed and soil isolates developed

the smallest sclerotia. When grown on different media the isolates varied in growth

pattern and growth rate. The soil isolate showed the least amount of growth in

almost all media.

Hooda and Grover (1982) reported that isolates of Macrophomina

phaseolina obtained from different plant species and plant parts of the same host

differed in their morphological and cultural characteristics. There was no

correlation between these characteristics and their pathogenicity on Vigna radiata.

Young inoculum (3-5 days old) was more infective than old (7-34days) and with

increase in inoculum density disease intensity also increased. Macrophomina

phaseolina causing leaf spot of Vignaradiata crop was reported in Queensland,

Australia. The inoculum sources wasconsidered to be microsclerotia of the fungus

in soil splashed on the leaves (Fuhlbohm et al., 1986).

Devi and Singh (1998) revealed that total of 56 Macrophomina phaseolina

isolates were obtained form black gram and green gram crops. The isolates were

categorized as highly virulent (MP-2 and MP-3) moderately virulent (MP-1, MP-4,

MP-6) and weakly virulent (MP-5) on the basis of disease incidence and intensity

on black gram and green gram.

Kale (1999) isolated Rhizoctonia bataticola from infected leaf samples by

using tissue isolation method and pathogenicity was proved on three weeks old

mungbean plants.

Ahmadi et al. (2010) revealed that Macrophomina phaseolina is one of the

most important plant pathogenic fungi species that causes charcoal rot in many

plants. In September of 2009, during a study of epidemics of this disease in

Soybean fields of Golestan province, symptoms of charcoal rot on a number of

weeds was observed and after isolation and purification, the causal fungus was

identified as Macrophomina phaseolina. The weeds species includes Stinging

nettle (Urtica dioica), Black LaceElderberry (Sambucus nigra), wild mustard

(Sinapis arvensis) and camelthorn (Alhagimaurorum). All of four hosts are

introduced as new hosts for this fungus in Iran.

Iqbal and Mukhtar (2014) studied the morphological and pathogenic

variability among the 65 isolates of Macrophomina phaseolina from different agro

ecological regions of Punjab and Khyber Pakhtunkhwa provinces of Pakistan.

Characters taken for study were radial growth, sclerotial size, weight and

pathogenicity. Sixteen isolates were rated as fast growing, 11 as slow growing, and

the rest of the isolates as medium growing. Nine isolates were classified as large

sized, 26 as small sized, and the remaining 30 as medium sized. Ten fungal isolates

appeared to be least virulent, whereas eight isolates of diverse origin proved to be

highly virulent against mungbean cultivars. The remaining isolates were regarded

as moderately virulent. No relationship was found among the morphological

characters and pathogenicity of the isolates.

2.3 Screeningof black gram entries for their reaction to Macrophomina blight

in-vivo

Deshkar et al. (1974) screened 163 varieties of mungbean for their

resistance to Rhizoctonia bataticola (Macrophomina phaseolina) and reported that

none of the variety evaluated was resistant or moderately resistant. Only one

variety 11160 (a) was moderately susceptible and rests were susceptible.

In field trials with 19 tolerant lines of Phaseolus aureus (Vigna radiata)

only two showed high resistance to Macrophomina phaseolina (Vidhyasekaran et

al., 1977).

Sivaprakasam et al. (1983) screened 20 Vigna radiata genotypes for

reaction to Macrophomina phaseolina under artificial conditions, PIMS2, PIMS3,

PIMS4 andBGG2 proved to be resistant.

Zote et al. (1983) screened mung cultivars and found none of the cultivar to

be completely free from Macrophomina blight, however ML-5, ML-26, ML-65

and ML-62 were found to be moderately susceptible to Macrophomina blight. J-

781 was highly susceptible.

Shirshikar et al. (1991) conducted field trials over 3 years with 30

Vignaradiata cultivars exposed to artificial infection by M. phaseolina, only BCG-

1 wasconsistently highly resistant, 4 others being moderately resistant. BCG-1 may

be used in a breeding programme to develop high yielding resistant lines.

Suresh et al. (1992) observed that a pure line selection from local

Vignaradiata cv Kaveripattinam was identified as a high yielder and designated as

DPI-703.It matures in 85-90 days, withstands drought during early growth has an

erect, compact habit and broad green leaves. Resistance was shown to

Macrophomina phaseolina and Erysiphe polygoni.

Burman et al. (1998) observed that Vigna radiata genotypes PDM-86-199

and K-851 were planted in a loamysand soil with a 10.4% moisture holding

capacity in Jodhpur, Rajasthan in India. Genotype K-851 was more susceptible to

Macrophomina phaseolina infection than PDM-86-199.

Dhutraj et al. (2005) screened eighteen urd bean genotypes, along with two

controls (pusa vishal and barkha), for their resistance to powdery mildew (Erysiphe

polygoni) and Macrophomina blight (Macrophomina phaseolina) during kharif

2004 in Badnapur, Maharashtra, India. All genotypes were resistant to both

powdery mildew and Macrophomina blight. Other resistant sources can be utilized

in breeding programmes for developing disease resistance in urd bean.

Choudhary et al. (2010) conducted an experiment of screening during 2006

and 2007 to evaluate 25 greengram [Vigna radiata (L.) Wilczek] genotypes for

resistance to root rot caused by Macrophomina phaseolina (Tassi.) Goid. Complete

resistance to root rot could not be found, however, 'MSJ 118' genotype exhibited

highest suppression of dry root rot, followed by the genotype 'KM 4-59' and

appeared as moderately resistant genotypes.

Deepthi et al. (2014) revealed that Charcoal rot is one of the most

destructive disease which causes heavy loss in sesame. Macrophomina phaseolina

was identified as pathogen of charcoal rot disease on the basis of morphological

studies and pathogenicity. Among the evaluated sesame cultivars, none of the

cultivars showed complete resistance against M. phaseolina. Only one entry

PKDS-91 was found as moderately resistant to charcoal rot. Three entries (OSC-

366-I, SSD-2-I and OSC-79) were recorded as moderately susceptible.

Tak et al. (2015) screened Mungbean varieties for associated mycoflora

and its management. Three mungbean cultivars SML 668, ML 818 and PAU 911

showed association of mainly six fungi viz. Macrophomina phaseolina (52.66%),

Aspergillusflavus (39.32%), Aspergillus niger (15.33%), Aspergillus versicolor

(9.11%), Aspergillus clavatus (8.89%) and Penicillium sp. (6.00%). The maximum

mycofloraincidence was observed in mungbean cv. SML 668, where highest

incidence of M.phaseolina (38.44%) and Aspergillus flavus (28.44) was observed.

Suradkar S. et al. (2015) tested Twenty-seven varieties/genotypes of

Blackgram grown at Pulse Research Station, N.A.U. Navsari in Kharif season,

2008. The reactions of varieties/ germplasms against the disease were recorded. In

which TPU-4 (0.95 %) recording minimum per cent disease index was resistant

while eight germplasms were rated as moderately resistant. GU-1 (52.00 %), UB-8

(55.00%), UB-15 (56.00 %), UB-16 (60.00 %), showed susceptible reaction while

the germplasms UB-19 (78.00%) and UB-22 (76.33 %) showed highly susceptible

reactions. Other varieties showed moderately susceptible reactions.

2.4 In-vitro evaluation of Pseudomonas fluorescence on the radial mycelial

growth of Macrophomina phaseolina

Pande and Chaube (2003) reported that In vitro antibiosis of 6 isolates of

P.fluorescence resulted in reduction of mycelial growth of R. solani and the

inhibitionzone ranged from 1.3 to 22.5 mm in different isolates on Kings-B

medium.

Ahmadzadeh et al. (2006) evaluated biological control activity of 47

Pseudomonas fluorescence spp., against certain soil-borne phytopathogenic fungi

such as, M. phaseolina, R. solani, P. nicotianae var. parasitica, Pythium sp. and

Fusarium sp. Theresults indicated that 66%, 40.42%, 63.82%, 48.94% and 27.65%

of strains revealed antagonistic activity against R. solani, M. phaseolina, Pythium

sp., P. nicotianae and Fusarium sp., respectively.

Dhoke and Kurundkar(2006) tested the efficacy of isolates of P.

fluorescence against M.phaseolila on potato dextrose agar medium by dual culture

technique. In generalmycelia growth of bacterial isolates decreased and formed

zone of inhibition indicating their antagonistic nature. The most promising isolates

found were FP1 and FP2.

Rini and Sulochana (2007) isolated twenty-six isolates of Trichoderma spp.

and 56 isolates of Pseudomonas fluorescence from Kerala were evaluated for their

antagonistic activity against R. solani under in vitro conditions. Different isolates

showed varying degrees of antagonism. The two most antagonistic isolates against

R.solani were T. pseudokoningii TR17 and T. harzianum TR20 of the

Pseudomonas fluorescence, P. fluorescence isolates P28 and P51 showed the

greatest inhibition (26.6%) and (27.5%) against R. solani.

Goud and Muralikrishnan (2009) tested antifungal activity of P.

fluorescence against P. oryzae, P. ultimim and M. phaseolina. All three pathogenic

fungi were inhibited by P. fluorescence with inhibitory activities ranging from

50% to 80%.

Fifteen rhizobacterial Pseudomonas fluorescence isolates obtained from

rice in the region of Andhra Pradesh, India. In all 10 strains of Pseudomonas

fluorescence were selected based on preliminary screening of all these isolates for

antifungal activity against rice fungal pathogens (P. oryzae and R. solani) inhibited

the growth of rice fungal pathogens in Fe deficient King‟s B medium varied from

(3 to 58% inhibition). Among these Pf 003 strain completely inhibited the mycelial

growth of two rice pathogens both in presence and absence of FeCl3 which

indicated the siderophore mediation along with antifungal metabolites. (Battu and

Reddy, 2009).

Afsharmanesh et al. (2010) reported that Pseudomonas fluorescence able to

produce secondary antifungal metabolites can inhibit soil-borne plant pathogens.

For this reason, the antagonistic activity of P. fluorescence UTPF5 against R.

solani AG-4 was assessed in bean under in-vivo and in-vitro conditions. Production

of some secondary and nonvolatile metabolites on growth of the fungus were

observed in UTPF5 and their impact on mycelial growth of R. solani was also

studied. The results showed that UTPF5 could inhibit the growth of R. solani both

in-vitro and in-vivo, and suppress the disease by 33.34% and 14.29% by soil

drenching and seed treatment, respectively. Production of HCN, siderophore and

protease and involvement of siderophore, volatile and nonvolatile metabolites on

growth of the fungus were observed in UTPF5.

Dev and Dawande (2010) evaluated the antagonistic property of

Trichodermaspp. and P. fluorescence against R. solani and found that the

mycolytic enzymesproduced by the antagonists suppressed the growth of R. solani.

Two hundred isolates from cotton rhizosphere isolated by Fallahzadeh and

Ahmadzadeh (2010), out of which 39% pertained to Pseudomonas fluorescence.

Dual culture assays were conducted against R. solani AG4 for these isolates to

evaluate the ability of antibiotic production. In greenhouse studies, all of the

isolates significantly suppressed the disease on plant.

Pan and Josh (2010) studied that the inhibition in growth of Trichoderma

was 8.59% when Trichoderma was used first and 59.44% when P. fluorescence

was used first in a dual culture test. Highest growth inhibition (59.44%) of

Trichoderma (Th3) was recorded when P. fluorescence was first inoculated in

King's medium B agar (KMB) 24h prior to inoculation of T. harzianum.

Malhotra et al. (2011) evaluated thirteen bio-control fungi and 4 bacterial

strains against R. solani using dual culture technique. The results showed that

among 12 the fungal species Gliocladium virens and T. harzianum (T8) were the

most effective isolates and inhibited R. solani mycelial growth by 74.82% and

73.33% respectively. Among the bacterial strains maximum growth inhibition was

caused by P. fluorescence P.f.1 (73.33%) followed by P. fluorescence P.f.2

(62.22%).

Rajeswari and Kannabiran (2011) reported that T. viride, T. harzianum, and

P. fluorescence were evaluated for their antagonistic activity against F. oxysporum

in-vitro. Among them, highest percent inhibition of conidial germination was

brought out by T. viride (89.4%) followed by T. harzianum (85.7%) and P.

fluorescence (83.15%) and inhibition of radial mycelial growth were (86.6%),

(84.0%), (60.0%) respectively.

Singh (2011) selected antagonists isolates i. eTrichoderma (Th38 and

Tv34), Pseudomonas fluorescence (Pf5 and Pf7), Bacillus subtilis (Bs5), non-

pathogenic Fusarium (Fo52) and Epicoccum purpurescens (Ep5) as potential bio-

control agents. The selected antagonists are highly effective against R. solani,

Sclerotinia sclerotiorum, M. phaseolina and F. oxysporum causing diseases in

vegetable crops.

Gull and Hafeez (2012) examined 28 Pseudomonas bacterial strains,

among the 28 strains tested, 14 were found to be siderophore producers. These

strains were evaluated for their bio-control potential against R. solani using various

dual culture assays. The role of siderophores in the inhibition of R. solani was

confirmed by iron chloride (FeCl3) experiment. Data demonstrated that bacterial

strain Mst 8.2 produces more than one antifungal agent but the siderophore

production is the key mechanism involved in the antagonism. Bacterial strains MS-

3y, Mst 8.2 and Mst 7.4 were the most effective with more than 70% disease

reduction in wheat.

Kapoor et al. (2012) determined the efficacy of different microbes for their

antagonistic ability in-vitro against F. oxysporum f. sp pisi by dual culture method.

The antagonists inhibited the mycelial growth of F. oxysporum f. sp. Pisi.,

T. viride (51.55), P. fluorescence-1 (49.62) and P. fluorescence -2 (50.08) showed

maximum inhibitionantagonistic activity in vitro. In-vivo T. viride and P.

fluorescence resulted in maximum reduction of seed rot root and wilt (F.

oxysporum) of pea.

Ten isolates of bacteria, designated as PGB1, PGB2, PGB3, PGB4, PGB5,

PGT1, PGT2, PGT3, PGG1 and PGG2, were successfully isolated and

characterized by Manivannan et al. (2012). Subsequently, to investigate the PGPR

isolates for their antagonistic activity against phytopathogenic fungi such as F.

oxysporum, R. solani and Sclerotium rolfsii. Furthermore, most of the PGPR

isolates shown antifungal activityagainst F. oxysporum, R.solani, and Sclerotium

rolfsii.

Adhikari et al. (2013) evaluated seventy isolates, antagonistic twenty-one

representing bio-vars of P. fluorescence (bio-vars I, II, III, and V) were collected

from the rhizosphere of okra, chilli, ground nut, brinjal, cabbage and tomato from

different agro-ecological regions of West Bengal and were subjected to evaluate

for their antifungal activity under in vitro condition against R. solani, the most

important soil-borne plant pathogen. Two isolates, PF-8 and PF-7 effectively

inhibited the mycelial growth of R. solani (72.05 and 68.25%, respectively) in dual

culture method.

Arumugam et al. (2013) isolated P. fluorescence and T. viride from the

rhizosphere of rice fields by the serial dilution method and tested against rice

pathogens for R. solani, Helminthosporium oryzae and S. oryzae in dual culture

method. The test result revealed that P. fluorescence actively inhibited,

Heminthosporium oryzae 75.6%, R. solani 47.8%, S. oryzae 68.9%.

Ravindran and Shaike (2013) isolated R. solani from naturally infected

vanilla plants and attempted to minimize the damage caused by the pathogen using

bio-control agents T. harzianum and P. fluorescence isolated from soil. The

combined inoculation of T. harzianum with P. fluorescence treatment showed

maximum disease suppressionfollowed by the single inoculation of P.

fluorescence, T. harzianum, P. putida and T.virens respectively in decreasing

order.

Saravanan et al. (2013) focused on the antagonistic potential of

Pseudomonas fluorescence in vitro and its inoculation effect on growth

performance of Lycopersicon esculentum in R. solani infested soil. Isolates Pf5 and

Pf6 wereantagonistic against 14 bacterial species, and two pathogenic fungi (F.

oxysporum and R. solani).

Thirty isolates were screened for PGP attributes and isolates showing PGP

properties were further screened for in vitro antagonism against soil borne phyto-

pathogens via, R. solani, Sclerotium rolfsii and Fusarium spp. Results revealed that

50% (15 out of 30 isolates) reacted positively for one or more PGP properties. A

high prevalence of antagonists was found against the three fungal pathogens.

Majority of the bacterial isolates (33%) displayed antagonism through the

production of siderophores or HCN. (Sarvani and Reddy, 2013)

Akter et al. (2014) isolated 325 bacteria and 14 isolates were found to be

antagonistic against the pathogen, and in the dual culture test selected bacterial

isolates KMB25, TMB33, PMB38, UMB20 and BMB42 showed 68.44%, 60.89%,

60.22%, 50.00% and 48.22% fungal growth inhibition, respectively. These

bacterial isolates were identified as Pseudomonas fluorescence by morphological

and biochemical characterization.

Four bacterial strains of P. fluorescence were isolated from tomato field

soil by Mezeal (2014). The antagonistic microorganisms against the pathogens

were observed by dual culture technique. P. fluorescence5 isolate was found to

show 81.3% and 77.4% of growth inhibition against R. solani and F. oxysporum

respectively.

Maurya et al. (2014) found that total eight micro flora resembling

P.fluorescence were isolated and three isolates were confirmed as P. fluorescence

(strainP.f.01, strain P.f.05 and strain P.f.07). P. fluorescence strains P.f 07 were

found most effective with the highest antagonistic activity against three fungal

pathogen and show maximum inhibition of mycelial growth of R. solani (68.23%),

F. moniliforme (65.45%) and Alternaria alternate (48.13%)

Solanki et al. (2014) were screened 220 bacteria isolated from tomato

rhizosphere for in vitro antagonistic activity against R. solani AG-4. Five potent

antagonistic strains viz., Pseudomonas spp. (M10A and MB65), P. aeruginosa

(MPF14 and MB123) and P. fluorescence (MPF47) were identified.

2.5 In-vitro evaluation of fungicides on the radial mycelial growth of


Jhooty and Bains (1972) evaluated systemic and non-systemic

fungitoxicants against Rhizoctonia solani in-vitro. They found that Dithane M-45

at 10 µg/ml and 44 µg/ml inhibited the growth of fungus completely and 50%

respectively.

Kapoor and Chohan (1974) concluded that Thiram when mixed with Agar

medium and incubated at 30°C inhibited growth of Macrophomina phaseolina

completely.

Goel and Mehrotra (1981) tested nine fungicides in-vitro against

Rhizoctoniabataticola (Macrophomina phaseolina) of gram and found that bavistin

could inhibitthe fungal growth completely. Among 39 fungicides tested in the

laboratory studies carbendazim, benomyl, guazatine, dichlozoline and iprodione

were highly toxic to mycelial growth of Rhizoctonia bataticola (Macrophomina

phaseolina) in Czapek's medium (Hooda and Grover, 1983).

Martin et al. (1984) tested chlorothalonil at 0.1% against 16 isolates of

Rhizocatonia solani and reported that they were sensitive to chlorothalonil. Among

the non-systemic fungicides tested, mancozeb followed by copper oxychloride

were most effective in inhibiting the growth of Rhizoctonia bataticola

(Macrophomina phaseolina) (Patil and Wangikar, 1984).

Gautam and Narain (1986) tested efficacy of seven fungicides against

Macrophomina phaseolina causing blight of cowpea and found that bavistin,

thiramand topsin-M inhibited growth of fungus completely.

Basu et al. (1988) tested fourteen fungicides and found that Dithane-M-45

increased inhibition of fungus with increase in concentration.

Giri and Peshney (1993) tested a fungicides in-vitro and found that

carbendazim (0.1%) and mancozeb (0.25%) inhibited the spore germination and

mycelial growth of Colletotrichum graminicola and Rhizoctonia bataticola

(Macrophomina phaseolina) causal agent of leaf spot of mungbean.

Carbendazim and Thiophanate methyl at 0.2% were most effective growth

inhibitors of Macrophomina phaseolina causing seedling mortality of black gram

(Devi and Singh, 1997).

Kanakamahalakshmi et al. (1998) determined the effect of root rot caused

by M. phaseolina at different stages of castor bean growth. In addition, the

economiclosses due to the disease and its in vitro management using Carbendazim,

Thiophanate-methyl, Captan and Thiram. All the growth yield parameters were

adversely affected by M. phaseolina. Incidence of root rot at early stages of crop

growth drastically reduced plant height, spike formation, yield and oil contents.

Thiophanate-methyl, Carbendazim, Thiram and Captan were equally and highly

effective at 1500 ppm inhibiting the growth of M. phaseolina completely. The

fungicides were effective against M. phaseolina in vitro using poisoned food

technique were further evaluated in the laboratory to test the efficacy on seed

germination and seedling vigour index. Both systemic and non-systemic fungicides

tested were effective in increasing seed germination, hypocotyl length and root

length thereby reducing abnormal seedling production including browning and

blackening.

Lokesha and Benagi (2004) tested seven fungicides (Benomyl,

carbendazim, Carboxin, Thiophanate-methyl, Captan, Thiram and Mancozeb) at 3

different concentrations (250, 500 and 1000 ppm) for their efficacy against

Macrophomina phaseolina using the poison food technique in vitro. At 250 ppm,

benomyl recordedthe highest percentage of mycelial growth inhibition (98.14%),

followed by carbendazim (84.07%). At 500 ppm, both benomyl and carbendazim

fully inhibited the growth of the fungus (100%), followed by mancozeb (97.40%)

and thiram (97.03%). Benomyl, carbendazim, thiram and mancozeb showed 100%

growth inhibition at 1000 ppm. All fungicides were tested in pot culture

experiments conducted in 2002 in Raichur, Karnataka, India. Pigeon pea (ICP-

8863) seeds were treated with individual fungicides at 2 gkg-1

and sown (8 seeds

per pot) in earthen potscontaining sterilized soil mixed with 10% pathogen

inoculum. Maximum plant survival at 130 days after sowing was obtained with

both carbendazim and benomyl (both 86.70%), followed by mancozeb (82.02%).

Raut and Patil (2005) conducted a field experiment in Thane, Maharashtra,

India, during the 2002/ 03 rabi season on tomato cv. Pusa Ruby to evaluate the

efficacy of fungicides, botanicals and Trichoderma viride against Fusarium

oxysporum and Rhizoctonia bataticola [Macrophomina phaseolina]. The

treatments comprised 0.1%carbendazim, 0.1% Bordeaux mixture, 2.5 g T. viride

per plant, 0.1% Reviver, 0.1% Chetana and 0.1% garlic extract. Bordeaux mixture

gave the lowest disease incidence (22.22%) and highest tomato yield (17.12 tha-1

).

Khan and Khan (2006) determined the efficacy of several fungicides

against Macrophomina leaf spot (Macrophomina phaseolina) of mung beans in

vitro and inthe field (Uttar Pradesh, India). For the in vitro tests, treatments

comprised: carbendazim, Topsin M [thiophanate-methyl], benomyl, captafol,

mancozeb and thiram all at either 0.10 or 0.25% concentration and an untreated

control. For the field tests, treatments comprised: 0.10% carbendazim + seed

treatment with thiram (ST; 3 g/kg); 0.10% Topsin M + ST; 0.10% benomyl + ST;

0.25% captafol + ST; 0.25% mancozeb + ST; and an untreated control.

Carbendazim, Topsin M and benomyl werehighly effective against the pathogen in

vitro and recorded 100% pathogen radial growth inhibition at both concentrations.

At 0.10 and 0.25% concentrations, thiram recorded 89.7 and 95.5% pathogen

radial growth inhibition, captafol recorded 85.1 and 93.8% growth inhibition and

mancozeb recorded 78.7 and 83.1% growth inhibition, respectively.

Konde et al. (2008) reported that Soybean (Glycine max (L.) Merill) is

important oil seed crop in India. Rhizoctonia bataticola (Pycnidial stage -

Macrophomina phaseolina) is the important soil borne pathogen causes

rootrot/charcoal rot disease in soybean. In vitro studies of fungicides and bioagents

efficacy against Rhizoctonia bataticola, revealed carbendazim+thiram (0.1+0.2%),

penconazole (0.1%), thiophanate-M (0.1%) completely inhibited (100%) the

growth of pathogen. Among bioagents, Trichoderma viride inhibited the growth of

Rhizoctoniabataticola to the extent of 96.39 per cent in pot culture studies,

maximum germination(84.45%) and per cent disease reduction (52.39%) was

recorded in the seed treatment with thiram+carbendazim (2±1 g kg-1

).

Khalikar et al. (2011) observed that chemical control is one of the measures

to manage the disease and avoid the losses. The evaluation study was therefore

conducted in vitro. Seven fungicides were tested against the pathogen i.e.

Macrophomina phaseolina in vitro. The highest inhibition (100%) of M.

phaseolina was observed due to carbendazim (500 ppm), chlorothalonil (500 ppm),

hexaconazole (500 ppm) and captan (2500 ppm) followed by mancozeb (2500

ppm) (94.39%) and benomyl (1000 ppm) 93.4% and rest of the treatments

significantly inhibited colony growth over control. The significantly highest

inhibition (100%) of scleortial production was recorded due to carbendazim (500

ppm), chlorothalonil (800 ppm), hexaconazol (500 ppm) and captan (2500 ppm)

followed by mancozeb (2500 ppm) 96.59% and benomyl (1000) 96.59%.

Dhingani et al. (2012) tested eleven fungicides of four different categories

viz., six systemic, two non-systemic and three mixed formulations and six

herbicides at their three different concentrations in vitro by poisoned food

technique for evaluatintheir efficacy against M. phaseolina causing root rot of

chickpea. Among all concentrations tested, the higher concentrations of each of

fungicides and herbicides produced maximum growth inhibition of the pathogen.

From tested fungicides, carbendazim (Bavistin 50 WP), tricyclazole (Beam 75

WP), propiconazole (Tilt 25 EC), Quintal 50%WP (carbendazim 25%+iprodine

27%) and Sixer 75WP (carbendazim 12%+mancozeb 63%) at all three

concentrations completely inhibited growth of the pathogen and proved to be

highly toxic to the pathogen. Among all herbicides tested, pendimethalin (Stomp

30 EC), oxyflourfan (Galagan 23.5 EC) and alachlor (Laso 50 EC) were proved to

be effective in inhibiting the mycelial growth of the pathogen.

De R. K. (2014) tested new fungicides for management of stem rot of jute

(Corchorus olitorius L. and C. capsularis L.), caused by Macrophomina

phaseolina, revealed that pre-sowing seed treatment followed by foliar spraying of

tebuconazole one month after sowing resulted in lowest stem rot incidence of 25%

and it was statistically at par with carbendazim (28.5%) and hexaconazole (28.6%)

compared to 45.2% in check. These were best fungicides against jute stem rot

pathogen as also observed in in vitro tests. These were followed by Tricyclazole,

Copper oxychloride and Mancozeb, respectively, with 33.4, 33.5 and 35.9% of

stem rot incidence. Among the fungicides tested, Thiophanate methyl was least

effective against stem rot of jute with 38.7% disease. The progress of stem rot was

slowest in Tebuconazole and Carbendazim in all the dates of crop growth from 30

to 90 days after sowing. Chemicals and fungicides causing complete inhibition of

M. phaseolina under in vitro tests were Propiconazole 25% EC (10 µg/ml),

turmeric oil (10 µg/ml), Carbendazim 50 WP (25 µg/ml), Copper oxychloride 50

WP (50 µg/ml), Tebuconazole 25.9% EC (50 µg/ml), Hexaconazole 5% EC (100

µg/ml), curcumin mixture (100 µg/ml) and tricyclazole 75% WP (10000 µg/ml).

Deepthi et al. (2014) revealed that out of different fungicides evaluated

under in vitro conditions against M. phaseolina; vitavax power and penflufen gave

100%inhibition at 500 ppm, while tricyclazole gave 100% inhibition at 1000 ppm

and otherfungicides were less effective. The field evaluation of different fungicides

indicated that vitavax power gave highest seed germination and less pre and post

emergence mortality, and yield loss, whereas the sesame seeds treated with vitavax

power alongwith one foliar application of carbendazim was found most effective

for enhancing seed germination and reducing pre, post emergence mortality and

losses in yield of sesame.

Pawar et al. (2014) evaluated efficacy of the recommended pesticides viz.,

fungicides, and botanicals on the growth and sporulation of Rhizoctonia bataticola

causing (charcoal rot), of soybean under in vitro condition was. Using poisoned

foodtechnique, radial growth and visual observations were taken. Pyraclostrobin,

Picoxystrobin, Azcoxystrobin are highly effective in reducing myecial growth and

sclerotial formation of Rhizoctonia bataticola. Out of eight fungicides tested only

(carboxin, copper oxychloride, mancozeb, thiram, carbendazim) were effective in

reducing the growth of Rhizoctonia bataticola. Nilgiri, neem and ashoka were

moderately effective in reducing the mycelial growth and sclerotial formation of

Rhizoctonia bataticola.

Sangappa and Mallesh (2016) reported that Blackgram (Vigna mungo L.) is

an important pulse crop grown throughout India. A new disease of blackgram i.e.

aerial blight and dry root rot caused by Rhizoctonia bataticola is primarily a soil

inhabitant. Fungicides like contact, systemic and combi products were also tested

against the aforementioned pathogen. Among five contact fungicides captan,

propineb and zineb recorded cent per cent inhibition (100%) of mycelial growth at

all the concentrations (i.e., 0.1, 0.2 and 0.3% respectively). Among seven systemic

fungicides and combi fungicides tested, benomyl, carbendazim, hexaconazole,

thiophanate methyl and tridemefon showed 100 per cent mycelia inhibition and

also in carbendazim 12% + mancozeb 63%, cymoxanil 8% + mancozeb 64%,

captan70% + hexaconazole 5%, tricyclozole 18% + mancozeb 62% and mancozeb

(64%) + metalaxyl (4%) showed cent per cent (100%) inhibition at all the

concentrations (0.05, 0.10 and 0.2%), respectively.

CHAPTER- III

MATERIALS AND METHODS

The materials used in the present investigation and methods followed are given

below.

All the in-vitro studies on Macrophomina phaseolina (Tassi) Goid. of

Black gram, were conducted in the Department of Plant Pathology, Indira Gandhi

Krishi Vishwavidhyalaya, Raipur (C. G.). The field studies were conducted in the

Pulse Pathology Field, Block Number-23 of I.G.K.V., Research Farm of the

University. Glassware's were of Borosil Grade and chemicals of standard trade

markes (BDH, Qualigens and Merck, etc.) were used during the course of the

investigations. The following instruments were used in the present studies:

1. Autoclave for sterilization.

2. BOD incubator for incubation.

3. Hot air oven for glassware sterilization

4. Laminar flow for isolation.

5. Anamid electronic digital balance for weighing

3.1 Collection, Isolation, Purification and Identification of

pathogen Macrophomina phaseolina 3.1.1 Cleaning and Sterilization of materials

Prior to use glasswares were cleaned with detergent powder and finally

rinsed with tap water as per requirement of the experiment. The dried glassware’s

were sterilized in hot air oven at 180°C for two-to-three hours. The forceps and

other metallic instruments were sterilized by dipping them in alcohol and heating

over the flame before using them, sterilization of the media was done by

autoclaving at 1.02 kg/cm2 pressure for 20 minutes. The plastic plates were

sterilized with alcohol and air dried before use.

3.1.2 Collection of disease sample

The disease samples of Black gram having dark brown irregular lesions

were collected from the field of Pulse Pathology Research Farm, I.G.K.V., Raipur.

3.1.3 Isolation

The isolation of pathogen was made from the disease-infected leaf and stem

collected from the field of Pulse Pathology Research Farm. The usual tissue

isolation method was followed for the isolation of the fungus from leaves, infected

branches and stem. Infected leaf bits of black gram were first washed with tap

water and then with distilled water. The bits were then surface sterilized by dipping

in 0.1% Mercuric Chloride solution for a minute and again washed by giving three

successive changes of sterilized distilled water to remove the traces of Mercuric

Chlorides. The isolation work was carried out by using laminar airflow. The leaf

and stem bits were then placed on sterilized potato dextrose agar in Petri plates.

Plates were then incubated at room temperature (28±2°C). As soon as the growth

of fungus was observed in plates, small portion of mycelial growth was transferred

on potato dextrose agar slants. Number of slants were prepared for further

investigation.

3.1.4 Purification and maintenance of culture and identification of pathogen

3.1.4.1 Media used

Two per cent Water Agar and Potato Dextrose Agar was used for isolation,

purification, maintenance and morpho-cultural studies of isolates.

Water Agar

Agar agar : 20 gm

Distilled water : 1 liter

For preparing two percent water agar, 20 gm of agar-agar is added to one

literof water and mixed thoroughly. The medium was sterilized in an autoclave at

1.02 kg/cm2 (121 ºC) for 20 minutes.

Potato dextrose agar

Potato (peeled, washed and sliced) : 200g

Dextrose : 20g

Agar agar : 20g

Distilled water : 1000ml

For preparing PDA medium, 200g Potato washed, peeled off the skin and

sliced them into small pieces. The sliced potato was boiled in 500 ml water in a

vessel for 20 minutes. Simultaneously 20g of agar was mixed in 500 ml of water

and boiled for 30 minutes. The potato extracts was collected by filtering through

muslin cloth. Twenty gm of dextrose was added to the potato extract, mixed

thoroughly with potato-agar mixture and volume was made upto 1 liter with

distilled water. The pH of medium was checked by using pH metre, adjusted the

pH by adding 1 N HCl or 1 N NaOH as the case may be. The medium was

sterilized in an autoclave at 1.02 kg/cm2 (121 ºC) for 20 minutes.

King’s B medium,

A selective one (Kings et al., 1954) was used for the isolation and

multiplication of P. fluorescence. The composition of the medium was as

follows:

Ingredients Quantity

Protease peptone 20.0 gm

Dipotassium hydrogen phosphate 1.5 gm

Magnesium sulphate 1.5 gm

Glycerol 10.0 ml

Agar-Agar 20.0 gm

Distilled water 1000.0 ml

3.1.5 Identification of the fungus

Colony colour of Macrophomina phaseolina varies in culture from black to

brown or gray and becomes dark in color with age. Abundant aerial mycelium

isproduced in the culture plate with sclerotia imbedded within the hyphae

orengrossed in the agar or on the agar surface with smooth precincts. Hyphae are

septate, initially hyaline turning to a honey or black color. Numerous dark

brownsto black colored sclerotia can be seen on the reverse side of the culture

plate. Thevegetative mycelium is characterized by the formation of monilid or

barrel-shapedcells and the formation of septum near the branching of the

mycelium. Branchingoccurs at right angle to parent hyphae, but branching at acute

angles is also common (Dhingra and Sinclair, 1977).Isolates were given to name

on the basis of genus and species name of M.phaseolina i.e. MP.

The fungus was purified by repeated isolation from the culture plates. The

pathogen was identified on the basis of character of the mycelium and sclerotia.

The characters were compared with the standard description of Macrophomina

phaseolina from literature (Singh, 1998).

3.2 Screening of black gram entries against Macrophomina

phaseolina

Forty entries of black gram were sown with two replications. Each of the

mungbean entries were sown by dibbling at a distance of 10cm x 30cm. The

observation on incidence of disease was recorded at flowering and pod initiation

stage. Per cent incidence was recorded on the basis of visual observation. The

entries were categorized as follows.

Table 3.1 Categorization of entries on the basis of per cent disease incidence and 1-5 scale (IIPR, Kanpur, 1996)

S. No. Score PDI Category

1 1 0 Resistant

2 2 0.1-10.0 Moderately resistant

3 3 10.1-25.0 Moderately susceptible

4 4 25.1-50.0 Susceptible

5 5 Above 50.0 Highly susceptible

3.3 In-vitro evaluation ofPseudomonas fluorescence on the radial

mycelial growth of Macrophomina phaseolina

In-vitro evaluation of Pseudomonas fluorescence on the radial mycelial

growth of Macrophomina phaseolina was done by dual culture method to test

the efficacy as bio-control agent against M. phaseolina. Pseudomonas

fluorescence isolates were multiplied on King's B broth and incubated for 2 days

at 280C till the fluorescent pigment appeared in the broth. Equal volume of

sterilized potato dextrose agar (PDA) and King's B medium were mixed in

sterilized Perti plates. The edge of a glass funnel was sterilized by dipping in

alcohol followed by flaming. Broth containing young growing cell of

Pseudomonas fluorescence was dispensed in sterile Petri plate and picked at

the edge of the funnel by dipping. Care was taken to remove the excess

inoculum by gently shaking the funnel. Inoculation was done by gently

touching the edge of the funnel (containing Pseudomonas fluorescence) which

encircled the pre-inoculated with M. phaseolina on was placed in centre of the

plate. Control was also maintained to see the difference. Observation was taken

after three days of inoculation.

In vitro antagonistic potential of different isolates of Pseudomonas

fluorescence was assessed by calculating percentage inhibition growth of

pathogen (M. phaseolina) over control in presence of Pseudomonas fluorescence

using the formula of Vincent (1947):

C-T

Per cent inhibition = I = --------- X 100

C

Where,

I = Per cent inhibition

C = Radial growth of pathogen in control

T= Radial growth of pathogen in presence of Pseudomonas fluorescence

3.4 In vitro evaluation of fungicide on the radial mycelial growth of


Six fungicides were tested in vitro against M. phaseolina i.e. Thiram,

Carbendazim, Carboxin+Thiram, Tricyclazole, Propineb, Hexaconazole+Zineb.

Potato dextrose agar medium was sterilized in conical flask of 250 ml capacity.

Prior to pouring, fungicides Thiram(1%), Carbendazim (1%), Carboxin+Thiram

(2%), Tricyclazole (1%), Propineb(2%)and Hexaconazole+Zineb(1%) of active

ingredient were added to 100 ml of PDA seperately. The medium was then poured

in sterilized petriplates. Five mm discs of the test pathogen cut from the margin of

seven days old culture were placed centrally in each of the petriplate. The disc was

kept inverted to allow the contact of the fungus with the medium. The inoculated

petriplates without fungicides served as control. The inoculated plates were kept in

incubator at 28±2°C. Colony diameter of the pathogen was measured after 4, 6 and

8 days of inoculation with the help of scale and sclerotial formation was recorded

after 8 days of inoculation. Three replications of each treatment were maintained.

The Petri plates were incubated at 28±2 °C temperature till the complete coverage

in control plate. The per cent growth inhibition (PGI) of the pathogen was worked

out by using formula given by Vincent (1947).

C-T

Growth inhibition = I = --------- X 100 C

Where,

I= Percent inhibition

C = Mycelial growth in control plate

T = Mycelial growth in treatment plate

Table 3.2 List and Concentration of fungicides used in in vitro evaluation

S. No. Fungicides

Concentration in %

1. Thiram 2%

2. Propineb 2%

3. Carbendazim 1%

4. Tricyclazole 1%

5. Carboxin+Thiram 2%

6. Hexaconazole+Zineb 1%

7. Control -

CHAPTER-IV

RESULTS AND DISCUSSION

Present investigation on Macrophomina blight of blackgram was carried

out during2017-18 in laboratory and experimental field of Dept. of PlantPathology,

College of Agriculture Indira Gandhi Krishi Vishwavidyalaya, Raipur and the

results of the experiment are presented and discussed in the light of findings of

earlier researches in this chapter.

4.1 Collection of diseased sample

The Macrophomina blight disease samples of leaf and stem were collected

from thirteen different entries and subjected to isolation of the causal organism.

The details of the pathogen isolations were made from different entries and parts of

the plant are given in table 4.1.

Table 4.1: Pathogen isolated from infected leaf and stem samples of different

entries of Blackgram

S. No. Entries

Plant Parts

stem and leaf

No. of samples

Collected

M. phaseolina

Isolated

1. KPU 17- 6 Stem 2 Nil

Leaf 2 2


Leaf 2 1

3. KPU 17- 14 Stem 2 1

Leaf 2 2


Leaf 2 2

5. KPU 17-19 Stem 2 1

Leaf 2 2


Leaf 2 1


Leaf 2 2


Leaf 2 2


Leaf 2 1

10. KPU 17- 32 Stem 2 1 Leaf 2 2


Leaf 2 2


Leaf 2 2


Leaf 2 1

Total no. of stem samples 26 3

Total no. of leaf samples 26 22

Two disease samples from stem and leaf of each variety were collected.

Macrophomina phaseolina could only be isolated from leaf samples of all the

varieties because the pathogen was observed to be more pre-dominant on leaf as

compared to the stem. Therefore, the pathogen isolated from leaf was used for

further investigation.

4.1.1 Isolation

Isolation of the fungus was done in Petri dishes using Potato Dextrose Agar

medium. The surface sterilized diseased bits yielded the fungus after 48 hours of

incubation at 28 ±2°C temperature. The uniform colonies originating from diseased

bits were separated, purified and were maintained on potato dextrose agar slants

for further use during entire course of investigation.

Purification

Purification of the fungus was done in Petri dishes using Water Agar

medium. The uniform colonies originating from diseased bits were separated, and

allowed to grow on Water Agar plates for single sclerotia isolation and purification

of pathogen and were maintained on potato dextrose agar slants for further use

during entire course of investigation.

4.1.2 Identification of the fungus

The pathogen was identified on the basis of character of the mycelium and

sclerotia. The mycelium was septate and hyaline, the sclerotia were brown to black

in colour, rounded or oblong in shape. Numerous black sclerotia were produced

within 5 to 6 days on Potato Dextrose Agar medium. Pathogenicity test was

performed by Koch’s Postulates and fungus isolated was confirmed as

Macrophomina phaseolina.

The isolate of Macrophomina phaseolina produced dark black to brown,

raised colony on potato dextrose agar media. Mycelium was well developed,

hyaline and septate. The fungus produced numerous sclerotia on host and also in

culture medium (PDA). The sclerotia were more or less round with an exception of

oval to irregular in shape. The present finding corroborates with the morphological

characters reported by Singh et al. (1998), such as black, smooth, hard round to

oblong or irregular shape of sclerotia. They measure about 100 microns to 1 mm in

diameter (in culture 50 to 300 microns). However, size is highly variable within an

isolate.

Plate 4.1: Disease symptoms in field

Plate 4.2: Symptoms used for isolation of pathogen

Plates 4.4 Pure culture of Macrophomina phaseolinaon water agar

Plates 4.3 Pure culture of Macrophomina phaseolinaon PDA

Plate 4.5 Sclerotia of Macrophomina phaseolina

4.2 Screening of black gram entries for their reaction to

Macrophomina blight

Forty entries with susceptible check; Barabanki local of black gram

screened for Macrophomina blight under natural field conditions, none of them

were found resistant and moderately resistant. Four entries i. e. KPU 17-1, KPU

17-2, KPU 17-3, KPU 17-4 were found moderately succeptible, nine varieties i. e.

KPU 17-7, KPU 17-9, KPU 17-11, KPU 17-13, KPU 17-25, KPU 17-33, KPU 17-

35, KPU 17-37 and KPU 17-40 were found to be succeptible and twenty seven

entries i. e. KPU 17-5, KPU 17-6, KPU 17-8, KPU 17-10,KPU 17-12, KPU 17-14,

KPU 17-15,KPU 17-16, KPU 17-17,KPU 17-18, KPU 17-19, KPU 17-20,KPU 17-

21,KPU 17-22, KPU 17-23, KPU 17-24, KPU 17-26, KPU 17-27, KPU 17-28,

KPU 17-29, KPU 17-30, KPU 17-31, KPU 17-32, KPU 17-34, KPU 17-36, KPU

17-38 and KPU 17-39 were found to be highly succeptible.

It was cleared that; no entries were found resistant or moderately resistant.

Only 4 entries were moderately succeptible. Rests of the entries were susceptible

and highly susceptible against Macrophomina phaseolina. Deepthi et al. (2014)

revealed that only one entry PKDS-91 was found as moderately resistant to

Macrophomina leaf blight of mungbean. Three entries (OSC-366-I, SSD-2-I and

OSC-79) were recorded as moderately susceptible.

Pandurang (2002) found that screening of thirty-eight black gram entries

was done for testing their resistance to Macrophomina blight. Eleven entries were

found to be moderately resistant to Macrophmina blight. The moderately resistant

entries were K-3, T-9, PU-19, PH-25, LBG-625, PU-30, LAMBG-296, LAMBG-

17, KU-309, KU-314 and KU-303. Rest of the entries were moderately susceptible,

susceptible and highly susceptible against Macrophomina blight.

Zote et al. (1983) also found that out of 19 cultivars screened, none was

completely free of Macrophomina blight however 4 lines were moderately

susceptible.

Table: 4.2 Screening of black gram entries for their reaction to Macrophomina

blight

S. No. Score Reaction Frequency Entries

Distribution

1. 1 Resistant 0 Nil

2. 2 Moderately 0 Nil

Resistant

3. 3 Moderately 4 KPU 17-1, KPU 17-2, KPU 17-3,

susceptible KPU 17-4

4. 4 Susceptible 9 KPU 17-7, KPU 17-9, KPU 17-

11, KPU 17-13, KPU 17-25, KPU

17-33, KPU 17-35, KPU 17-37,

KPU 17-40

5. 5 Highly 27 KPU 17-5, KPU 17-6, KPU 17-

susceptible 8, KPU 17-10, KPU 17-12, KPU

17-14, KPU 17-15, KPU 17-16, KPU 17-17, KPU 17-18, KPU




17-38, KPU 17-39

Total entries – 40

LSI – 4.57

Plate 4.6 Field view of screening trial at different crop stages

4.3 In-vitro evaluation of the Pseudomonas fluorescence isolates for

antagonistic potential against Macrophomina phaseolina.

In vitro antagonistic potential of different isolates of P. fluorescence

wasstudied (Table 4.3 and fig. 4.4) on the mycelial growth of M. phaseolina of

black gram following dual culture method. The mycelial growth was assessed after

7 days of inoculation. Mycelial growth of M. phaseolinaof all the treatments was

significantly less than that of control. Minimum mycelial growth (36 mm) (%

inhibition 60%) of test fungus was observed with isolate P1 followed by P3 growth

(38 mm) and % inhibition (60%), P2 growth (42 mm) and % inhibition (53.6%),

P10 growth (43 mm) and % inhibition (52.3%), P9 growth (44 mm) and %

inhibition (51.2%), P5 growth (45 mm) and % inhibition (50%), P7 growth (45

mm) and % inhibition (50%), P8 growth (48 mm) and % inhibition (46.7%), P4

growth (50 mm) and % inhibition (44.5%) and with highest mycelia growth isolate

P6 growth (65 mm) and % inhibition (27.8%).

Mycelial growth of treatment P1 was at par with P3 and treatment P3 was

at par with P2 and P10.

The work carried out by Pande and Chaube (2003) reported that in vitro

evaluation of Pseudomonas fluorescence isolates resulted in reduction of mycelial

growth of M. phaseolina and the inhibition zone ranged from 1.3 to 22.5 mm in

different isolates on King’s B medium. Similarly work carried out by

Krishnamurthy and Gnanamanickam (1998) reported that Pseudomonas

fluorescence strains recorded antagonistic to P. grisea, R. solani and S. oryzae

were isolated from rice rhizosphere.

Maurya et al. (2014) also found that Pseudomonas fluorescence strain P.f

07 was found most effective with the highest antagonistic activity against three

fungal pathogen and show maximum inhibition of mycelial growth of

Rhizoctoniasolani (68.23%), Fusarium moniliforme (65.45%) and Alternaria

alternata (48.13%).

Table 4.3: Efficacy of Pseudomonas fluorescence isolates againstM. phaseolina

*Average of three replications

Isolates Ave. mycelial growth

(mm)

% growth inhibition

over control

P1 36* 60.0 P2 42 53.6 P3 38 57.8 P4 50 44.5 P5 45 50.0 P6 65 27.8 P7 45 50.0 P8 48 46.7 P9 44 51.2 P10 43 52.3

Control 90.00 -

SE(m)± 1.842

C.D. (5%) 5.438

4.4 In vitroevaluation of fungicides on the mycelial growth of


Six fungicides were tested for their efficacy on Macrophomina phaseolina

in-vitro condition. The chemicals used were Thiram, Propineb,Carbendazim,

Tricyclazole, Carboxin+Thiram, Hexaconazole+Zineb. Incorporated in Potato

Dextrose Agar medium.

The data presented in table 4.4 revealed that all treatments except propineb

were significantly superior over control in checking the mycelial growth of the test

fungus. Tricyclazole, Carboxin+Thiram and Hexaconazole+Zineb completely

inhibited the mycelial growth (0 mm) and % inhibition (100%) followed by

Thiram (60.0 mm) % inhibition (33.4%)), Carbendazim (12.00 mm) % inhibition

(86.66%), and Propineb (90.0 mm)% inhibition (0%).

Mycelial growth of treatment T4 was at par with treatment T5 and T6.

The findings of present study agreed with the findings of other researchers.

Deepthi et al. (2014) revealed that out of different fungicides evaluated under in

vitro conditions against M. phaseolina; vitavax power and penflufen gave

100%inhibition at 500 ppm, while tricyclazole gave 100% inhibition at 1000 ppm

and otherfungicides were less effective. The field evaluation of different fungicides

indicated that vitavax power gave highest seed germination and less pre and post

emergence mortality and yield loss, whereas the sesame seeds treated with vitavax

power alongwith one foliar application of carbendazim was found most effective

for enhancing seed germination and reducing pre and post emergence mortality and

losses in yield of sesame.

Pawar et al. (2014) also revealed that out of eight fungicides tested

Carboxin, Copper oxychloride, Mancozeb, Thiram and Carbendazim were

effective in reducing the growth of M. phaseolina. Sangappa and Mallesh (2016)

revealed that among seven systemic fungicides tested, Benomyl, Hexaconazole,

Thiophanate methyl and Triademefon showed 100 per cent mycelial growth

inhibition.

Table 4.4: Effect of different fungicides on mycelia growth of Macrophomina

phaseolina

Treatment Fungicides

Concentration

in %

Ave. colony

diameter (mm)

Growth reduction

over control (%)

T1. Thiram 1% 60* 33.4

T2. Propineb 2% 90 0

T3. Carbendazim 1% 12 86.66

T4. Tricyclazole 1% 0 100

T5. Carboxin+

Thiram 2% 0 100

T6. Hexaconazole+

Zineb 1% 0 100

T7. Control - 90 -

Se(m)± 0.218

CD at 5% 0.668

*Average of three replications

CHAPTER-V

SUMMARY AND CONCLUSION

The present investigation entitled “STUDIES ON LEAF BLIGHT OF

BLACK GRAM (Vigna mungo (L.) Hepper) OF CHHATTISGARH

PLAINS”were carried out in the laboratories and fields of Departmentof Plant

Pathology, College of Agriculture, Indira Gandhi Krishi Vishwavidyalaya, Raipur

(C.G.). Various aspects statistically analized and results obtained are summarized

below.

Fungal organism was isolated from black gram plants showing typical

symptoms of leaf blight caused by Macrophomina phaseolina. This organism was

purified, identified and pathogenicity test revealed that fungal pathogen was

virulent and produced typical symptoms of leaf blight on black gram.

Out of forty entries screened for resistance against Macrophomina blight,

among all entries except 4 showed susceptible and highly susceptible. Only 4

entries i e. KPU 17- 1, KPU 17- 2, KPU 17- 3, KPU 17- 4 were moderately

succeptible against Macrophomina blight.

In vitro antagonistic potential of different isolates of P. fluorescence

following dual culture method. The mycelial growth was assessed after 7 days of

inoculation. Mycelial growth of M. phaseolina of all the treatments was

significantly inhibit as compare to control. Least mycelial growth (36 mm) term of

inhibition % (60%) of test fungus was observed with isolate P1 followed by P3

growth (38 mm) and % inhibition (60%), P2 growth (42 mm) and % inhibition

(53.6%), P10 growth (43 mm) and % inhibition (52.3%), P9 growth (44 mm) and

% inhibition (51.2%), P5 growth (45 mm) and % inhibition (50%), P7 growth (45

mm) and % inhibition (50%), P8 growth (48 mm) and % inhibition (46.7%), P4

growth (50 mm) and % inhibition (44.5%) and with highest mycelia growth isolate

P6 growth (65 mm) and % inhibition (27.8%).

The data revealed that all treatments except propineb were significantly

superior over control in checking the mycelial growth of the test fungus.

Tricyclazole, Carboxin+Thiram and Hexaconazole+Zineb completely inhibited the

mycelial growth (0 mm) term of inhibition % (100%) followed by Thiram (60.0

mm) % inhibition (33.4%)), Carbendazim (12.00 mm) % inhibition (86.66%), and

Propineb (90.0 mm)% inhibition (0%).

CONCLUSION

The fungus isolated was purified by repeated isolation from the culture plates. The

pathogen was identified on the basis of character of the mycelium and sclerotia.

The mycelium was septate and hyaline, the sclerotia were brown to black in colour,

rounded or oblong in shape.

4 entries i.e. KPU 17- 1, KPU 17- 2, KPU 17- 3, KPU 17- 4 were moderately

succeptible and 9 were shown as susceptible and 27 were shown as highly

susceptible.

All the P. fluorescenceisolates significantly reduced the mycelial growth of

Macrophomina phaseolina over untreated (control) treatment. All the 10 isolates of

P. fluorescenceshowed potential forreduction in mycelial growth. Percent

inhibitions of M. phaseolina ranged from 60 to 27.8 percent.

All the fungicides except propineb were significantly effective in reducing the

radial mycelial growth of Macrophomina phaseolina. Tricyclazole,

Carboxin+Thiram and Hexaconazole+Zineb completely controlled the mycelial

growth and Thiram and Carbendazim was also control the growth of fungus.

SUGGETIONS FOR FUTURE RESEARCH WORK

1) Regular survey works are need to be conducted to assess the severity at regular

intervals. This will help in devising management practices.

2) Need to be development of resistant cultivar against pathogen.

3) Variations within the pathogen also need to be identified for effective resistant

breeding programme.

4) The biological management’s aspects need to be thoroughly investigated.

5) Need based application of chemical fungicides i.e. Tricyclazole, Carboxin+Thiram

and Hexaconazole+Zineb may reduce the diesese when applied at timely interval.

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STUDIES ON LEAF BLIGHT OF BLACK GRAM (Vigna mungo (L ... · GRAM (Vigna mungo (L.) Hepper) OF CHHATTISGARH PLAINS M. Sc. (Ag.) Thesis by Manish Kumar Sahu DEPARTMENT OF PLANT PATHOLOGY