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STUDIES ON Choanephora TWIG

BLIGHT (Choanephora cucurbitarum

(Berk & Rav.) Thaxt. OF CHILLI

(Capsicum frutescens L.) AND ITS

MANAGEMENT

J. CHANDRAKALA

B.Sc. (Ag.)

MASTER OF SCIENCE IN AGRICULTURE (DEPARTMENT OF PLANT PATHOLOGY)

2016

STUDIES ON Choanephora TWIG BLIGHT

(Choanephora cucurbitarum (Berk & Rav.)

Thaxt. OF CHILLI (Capsicum frutescens L.)

AND ITS MANAGEMENT

BY

J. CHANDRAKALA

B.Sc. (Ag.)

THESIS SUBMITTED TO THE

PROFESSOR JAYASHANKAR TELANGANA STATE

AGRICULTURAL UNIVERSITY

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF THE DEGREE OF

MASTER OF SCIENCE IN AGRICULTURE (PLANT PATHOLOGY)

CHAIRPERSON: Dr. B. VIDYASAGAR

DEPARTMENT OF PLANT PATHOLOGY

COLLEGE OF AGRICULTURE

RAJENDRANAGAR, HYDERABAD - 500 030

PROFESSOR JAYASHANKAR TELANGANA STATE

AGRICULTURE UNIVERSITY

2016

CERTIFICATE

Ms. J. Chandrakala has satisfactorily prosecuted the course of research and that

thesis entitled “Studies on Choanephora twig blight (Choanephora cucurbitarum

(Berk & Rav.) Thaxt. of Chilli (Capsicum frutescens L.) and its management”

submitted is the result of original research work and is of sufficiently high standard to

warrant its presentation to the examination. I also certify that neither the thesis nor its

part thereof has been previously submitted by her for a degree of any university.

(Dr. B. VIDYA SAGAR)

Date: Chairperson

CERTIFICATE

This is to certify that the thesis entitled “Studies on Choanephora twig blight

(Choanephora cucurbitarum (Berk & Rav.) Thaxt. of Chilli (Capsicum frutescens

L.) and its management” submitted in partial fulfilment of the requirements for the

degree of ‘Master of Science in Agriculture’ of the Professor Jayashankar Telangana

State Agricultural University, Hyderabad, is a record of the bonafide original research

work carried out by Ms. J. Chandrakala under our guidance and supervision. The

subject of the thesis has been approved by the Student's Advisory Committee.

No part of the thesis has been submitted by the student for any other degree or

diploma. The published part and all assistance received during the course of the

investigation have been duly acknowledged by the author of the thesis.

Thesis approved by the Student Advisory Committee

Chairperson Dr. B. VIDYA SAGAR

Associate Professor

Department of Plant Pathology

College of Agriculture, PJTSAU

Rajendranagar, Hyderabad-500 030.

Member Dr. R. Jagdeeshwar

Principal scientist

Agricultural Research Institute, Rice section


Member Dr. P. Rajanikanth

Assistant professor

Department of Entomology

College of Agriculture, PJTSAU


Date of final viva-voce:

LIST OF CONTENTS

Chapter No. Title Page No.

I INTRODUCTION

II REVIEW OF LITERATURE

III MATERIAL AND METHODS

IV RESULTS AND DISCUSSION

V SUMMARY AND CONCLUSIONS

LITERATURE CITED

DECLARATION

I, J. Chandrakala, hereby declare that the thesis entitled “Studies on

Choanephora twig blight (Choanephora cucurbitarum (Berk and Rav.) Thaxt. of

Chilli (Capsicum frutescens) and its management” submitted to the Professor

Jayashankar Telangana State Agricultural University for the degree of Master of

Science in Agriculture is the result of original research work done by me. I also

declare that any material contained in the thesis has not been published earlier in any

manner.

Place: Hyderabad (J. CHANDRAKALA)

Date: I. D. No. RAM/13-55

ACKNOWLEDGEMENTS

I thank the almighty GOD for his love and blessings, without which I would not

have been able to complete my studies hitherto and present this piece of work. No

creation in this world is a solo effort, neither is this thesis. I would like to take this

opportunity to acknowledge all, who supported me directly or indirectly, bringing in

this thesis to a really good shape

It gives me great pleasure to humbly place on record my profound sense of

gratitude, indebtedness and heartfelt thanks to my major advisor and chairperson of

my advisory committee, Dr. B. Vidya sagar, Associate professor, Dept. Of Plant

Pathology, College of Agriculture, Rajendranagar, Hyderabad for his initiative,

benevolence, constant encouragement, warm affection, caring nature and ready help

which enabled me to overcome several stumbling blocks during the period of my

investigation and also in the preparation of this thesis. I will be always indebt for all

the help provided by my chairperson which cannot be valued in any terms and a

great, heartfelt thanks to my chairperson without his support and his motivation, I

could not have completed my thesis and come to this position.

I deem it my privilege to record my sincere thanks and deep sense of

gratitude to luminous educationalist and esteemed Co-chairman of my advisory

committee, Dr. R. Jagdeeswar, Principal Scientist, ARI, Rice section, Hyderabad for

his scholastic guidance, unceasing interest, technical guidance, scientific view,

cherishable counselling, moral support and also his help in bringing out this thesis.

It gives me great pleasure to humbly express my profound gratitude and

heartfelt thanks to Dr. G. Uma devi, Professor and University Head, Department of

Plant Pathology, College of Agriculture, Rajendranagar, Hyderabad for her

valuable suggestions, careful and reasoned criticism and meticulous attention to the

details and also for her constant encouragement which has led to the present

investigation to the final shape.

I express my sincere gratitude and thanks to my caring professors Dr. Bharati

bhatt, Dr. Kishore varma, Dr. P. Narayan reddy, Dr. M. Rajeswari, Dr. G. Samuel,

Dr. Krishna Mohan, Dr. Ch. Sreenivas Rao of College of Agriculture, Rajendranagar,

Hyderabad.

I am in dearth of words to express my sense of gratitude to my beloved

parents Sri. J. Chenchanna and Smt. J. Subba Lakshmi for giving me this

wonderful life, my caring and loving sisters Priyanthi, Krishna priya and loving

brothers Krishna kumar, Harish, Mani and Srinivas who have always strived for

my well being and their unforgettable efforts in bringing me to this stage, without

whom i could not have achieved all my success in my life.

Least but not last my friends Mamatha, Ajitha, Anitha, Navya, Ramya,

Krishna, and Madhukar for their love and affection towards me in making my

career successful.

I use this opportunity to sincerely thank my classmates Raviteja, Yashaswini,

Yamuna and Suman Raj meena for their cooperation and help during these two years

of study.

No scholar can complete the work on his own. He or she has to get a little help

from their friends for one or another item of works, so I owe my gratitude towards my

friends Rakesh, Ramesh, Bhimu, Roja, Ragini, Surya, Gouthami, Sravanthi and

Srilatha for the great support they gave me. I extended warmest thanks to my seniors

Rajendra Prasad, Yellagoud, Gopika, Suresh, Sumalatha, Padmaja, Rajesh Khanna,

Swetha Singh, Divya Rani, Jadesha, M. Suresh, Bindu Madhavi, Chandramani,

Ramesh, Srinivas, Ramprasad and Premalatha and juniors Manjunaath, Srikanth,

Chiranjeevi, Kalpna, Rani, Ramakrishna and Deepak for all their help.

Words could not help me when I need to thank to non-teaching staff of the

department Andalu, Chandrakala, Ramkoti, Asiabi, Ramakoti and Mariyamma their

help during my post-graduation. I felt elated to express my thanks to those who directly

or indirectly helped me in successful completion of thesis work.

I am grateful to PJTSAU and Government of Telangana for the financial

help in the form of stipend during my study period which cannot be forgettable.

Date :

Place : Hyderabad (J. Chandrakala)

LIST OF ABBREVIATIONS

% : per cent

- ve : Negative

+ ve : Positive

μg/ml : Micrograms per millilitre

a.i. : Active ingredient

BOD : Biological Oxygen Demand

CA : Carrot agar

cfu : Colony forming units

CD : Critical Difference at 5 per cent level

cm : Centimeter

CRD : Complete Randomized Design

cv. : Cultivar

DAS : Days after sowing

DAI : Days after inoculation

et al. : and other

etc. : and so on

Fig. : Figure

g : gram

gl-1 : Grams per litre

g-1 : Per gram

μg : Microgram (s)

h : hours

ha : hectare

H2O2 : Hydrogen peroxide

HgCl2 : Mercuric Chloride

i.e. : that is

kg : Kilogram

kg acre-1

: Kilogram per acre

kg ha-1

: Kilogram per hectare

KMNO4 : Potassium permanganate

l : litre

m : Metre

M : Molar

m2 : Meter square

mg : milligram

ml : milliliter

mm : millimeter

NA : Nutrient agar

No. : Number

oC : Degree Centigrade

PDA : Potato Dextrose Agar

PDI : Per cent Disease Incidence

psi : pounds per square inch

PH : Hydrogen ion concentration

SEm : Standard error of mean

Spp : Species

t : Tonne

viz., : namely

Author : J. CHANDRAKALA

Title of the thesis : “Studies on Choanephora twig blight (Berk and

Rav.) Thax. on chilli (Capsicum frutescens L.)

and its management”

Degree : MASTER OF SCIENCE IN AGRICULTURE

Faculty : AGRICULTURE

Major Field : PLANT PATHOLOGY

Chairperson : Dr. B. VIDYA SAGAR

University : PROFESSOR JAYASHANKAR TELANGANA STATE

AGRICULTURAL UNIVERSITY

Year of Submission : 2016

ABSTRACT

Choanephora cucurbitarum is a facultative saprobe that belongs to the Sub

division Zygomycotina, Order: Mucorales and Family Choanephoraceae and it is

responsible for causing twig blight in chilli. It is a fungal plant pathogen and has a wide

host range (Abel Motaal et al., 2010). The disease was first reported by Dastur (1920) in

India and it became endemic in certain monocropping areas. A little is known about

chilli twig blight, hence the present investigation has been initiated to study the

symptoms and management of disease.

A preliminary survey was conducted in 6 major chilli growing districts of

Telangana district representing 13 mandals and 38 villages during kharif 2013-14 for

the prevalence of Choanephora twig blight diseases in chilli.

Khammam recorded maximum disease incidence varying from 20 to 32% in all

five villages surveyed where as in Karimnagar and Warangal district the disease

incidence was 18 and 15% respectively. There was no incidence of twig blight disease

in chilli in Medak, Ranga Reddy and Nizambad districts.

The pathogen was isolated from the leaf and proved for its pathogenecity. The

drooping multisporous sporangia were subglobose in shape and 90.15 - 100 μm in size.

The sporangia were non columellate and dehisce into two half releasing the spores.

Sporangiospores were elliptic, fusiform or ovoid in shape, light brown or dark brown in

color and are characterized by longitudinal striatures on the surface of wall and 12-19 x

7.23-9.5 μm by size. The sporangiophore (conidiophore) from which the monosporous

sporangiola arose was long slender, branched at the apex with primary vesicle from

which secondary vesicles were produced on the stalks which bears sporangiospores

(conidia). Monosporous sporangiola were elliptical, fusiform or ovoid, striate and

measured 11-19 × 4-12 μm. Zygospores of C. cucurbitarum appear not to have been

observed prior to the present study. The mycelial and morphological characters were

similar to that described by Wolf, 1917. On the basis of morphological and cultural

characters, growth habitat and temperature relations, the fungus was identified as

Choanephora cucurbitarum.

The pathogen restricts its attack on young green twigs as well as petiole, fruit

stalk and flower bases. The affected areas on twig first develop as water soaked, brown

coloured lesion which enlarge rapidly encircling the twig. The portion of twig above the

affected region becomes wilted, blighted and dried up. The fungus develops stiff thread

like sporangiophores with black headed asexual reproductive structures on diseased

lesions.

Prominent hairy growth of Choanephora cucurbitarum on the infected tissue

was found in the morning hours. This is visible with a hand lens. Usually whitish

mycelia and monosporous sporangial were produced on the lesions. The disease

gradually spreads to more and more branches even to the stem causing severe damage

under warm and humid conditions that favor the disease development.

Studies on the effect of different pH, media and temperature on the radial

growth were carried out. The suitable pH for mycelial growth of C. cucurbitarum was at

pH 4.5. Among the eight media tesed, PDA was found more suitable for growth of C.

cucurbitarum. The temperature of 25oC was found favourable for the radial growth of

C. cucurbitarum.

Four isolates of Trichoderma spp and 8 fungal antagonists isolates were isolated

from rhizosphere and phylloplane of chilli respectively and tested the efficacy against

test pathogen Choanephora cucurbitarum by dual culture technique. Among these,

Trichoderma viride isolate-1 was found most effective in inhibiting the test pathogen

followed Fusarium spp.

Seven fungicides viz., azoxystrobin, carbendazim, captan, copper oxychloride,

chlorothalonil, hexaconazole and mancozeb were tested at six concentrations i.e. 0.05,

0.1, 0.15, 0.2, 0.25 and 0.3% respectively under in vitro conditions. Among them captan

at 0.15% at least concentration inhibited the mycelia growth of Choanephora

cucurbitarum.

Ten varieties were screened against the test pathogen C. cucurbitarum and out of

them LCA-334 and G-4 showed disease severity index with 27 and 24 per cent

respectively.

The potential biocontrol agent and effective fungicide were screened under

greenhouse conditions against twig blight of chilli caused by Choanephora

cucurbitarum with different combinations. The treatment, T2 (captan @ 0.15%)

recorded was found to most effective in reducing the disease incidence by 66.47%

followed by T7 (Trichoderma viride + captan + Neem oil) 28.9% when compared to

control (13.95%).

Chapter I

INTRODUCTION

Chilli (Capsicum frutescens) is a self-pollinated crop bearing a pod like

fruit (berry) and belongs to family Solanaceae. It originated from South and Central

America where it is still under cultivation (Pickersgill, 1997). It is a tropical and

subtropical crop mainly grown in India, Japan, Mexico, Turkey, United States of

America and African countries. India is the largest producer of chillies in the world,

accounting for over 45% of the total area under cultivation from almost the sea level up

to an altitude of 2000 meters with an annual rainfall of 60-150 cm (Anonymous, 2008).

Chilli is an annual sub-shrub constitutes one of the most important spices

cultivated all over the world except in colder parts. India is the leading country in the

production of chillies contributing 41.11% of the world’s production. India stands first

in production and consumption of chilli globally. India is well known as the land of

spices the world over. In India, they occupied about ninety thousand hectares with a

production of ninety thousand tonnes of chillies. Andhra Pradesh, Maharashtra,

Karnataka, Orissa and Tamil Nadu alone account for about 75% of the total area as well

as production. Almost all varieties of low and medium pungency that are cultivated on

field scale in India belong to Capsicum spp. Export of chillies during 1999-2000 was

64776 metric tonnes valued at Rupees 250.66 crores. However, chilli is the second

commodity in our export basket earning nearly 13% of foreign exchange from spices

(Subramaniam, 2001).

The area under Chilli was estimated at 792.1 thousand hectare during 2010-11

with the production of 1223.4 thousand tones green chilli. During this year production

of green chilli was recorded 1.5 MT per hectare (Anonymous, 2011).

Capsicum frutescens is cultivated either for pungent fruited genotypes called

chilli (synonyms: hot pepper, American pepper, chile, azi, cayenne, paprika etc.) or

non-pungent fruited genotypes called sweet pepper (synonyms: Capsicum, paprika, bell

pepper, Shimla mirch). Chilli has many culinary advantages. It comprises numerous

chemicals including steam-volatile oils, fatty oils, capsaicinoids, carotenoids, vitamins,

proteins, fibers and mineral elements (Bosland and Votava, 2000).

Chilli is commercially important for two qualities, i.e., its red colour is due to

the pigment capsanthin and its biting pungency is due to capsaicin. Capsaicin also

known as hinders cholesterol. Chilli production suffers from many diseases caused by

fungi, bacteria, viruses, nematodes and also by abiotic stresses. Chilli suffers from

several seed borne fungal fruit rot diseases.

Choanephora cucurbitarum is a plant pathogenic fungus causing fruit rots,

flower rot and leaf blights on a variety of plants including squash, pumpkin, pepper, pea

and bean. This fungus is known to attack several other crops which include cereals such

as millet, rice and sorghum. The fungus also causes pod blight known as wet rot,

blossom blight and whisker rot (Kacharek et al., 2003). This disease is also common on

squash and southern pea but occurs on the floral parts of many types of plants (Afolabi,

1994). It causes blossom blight, die back, wet rot and soft rot of stems or side shoots of

chilli plants (Maeda et al., 2010).

The fungus is more successful under humid conditions and thrives best at a

temperature of 25°C and relative humidity of about 100%. A temperature of about 31°C

stimulates the production of large sporangia but unfavorable for conidia formation

(Umana and Ikotun, 2000).

The crop is suffering from various diseases of which the fungal disease,

Choanephora blight in chilli caused by Choanephora cucurbitarum has become one of

the constraints in chilli growing areas resulting in poor yields, besides reducing quality.

Hence the present research work is planned with the following objectives.

Objectives of investigation

1. Survey for the occurrence of Choanephora twig blight in major chilli growing areas

of

Telangana districts and isolation and characterization of Choanephora cucurbitarum.

2. Screening of fungicides and biocontrol agents against Choanephora cucurbitarum in

in vitro.

3. Screening of popular chilli varieties against Choanephora cucurbitarum.

4. Management of twig blight disease with effective fungicide and potential biocontrol

agents

against Choanephora cucurbitarum under green house conditions.

Chapter II

REVIEW OF LITERATURE

A brief review of relevant literature for the present investigation is presented

under the following headings.

2.1 The disease

2.1.1 Geographical distribution

2.1.2 Host range

2.1.3 Economic importance

2.1.4 Symptomatology

2.2 Survey for twig blight incidence

2.3 The pathogen

2.3.1 Morphological characterization of Choanephora cucurbitarum

2.3.2 Isolation, purification, identification and pathogenicity

2.4 Physiological studies

2.4.1 Effect of media

2.4.2 Effect of pH

2.4.3 Effect of temperature

2.5 Evaluation of fungicides against Choanephora under in vitro conditions

2.6 Isolation and evaluation of phylloplane and rhizosphere mycoflora against

Choanephora cucurbitarum

2.7 Screening of popular varieties against Choanephora cucurbitarum

2.8 Screening of effective fungicide and potential biocontrol agent against twig

blight caused by Choanephora cucurbitarum under greenhouse conditions

2.1 The disease

2.1.1 Distribution of the Disease

The genus Choanephora was first described by Currey (1873) from

fructifications on flowers of Hibiscus rosaesinensis furnished by D. D. Cunningham

from Calcutta, India. Currey described fungus as Cunninghamia but later changed the

generic name to Choanephora since the name Cunninghamia had already been used for

a conifer.

Berkeley (1875) reported the occurrence of fungus in United States and

described as Rhopalomyces cucurbitarum from decaying squashes collected in South

Carolina by Ravenel. He also observed the similar fungus isolated from squashes under

the name Aspergillus cucurbiteus. Peck (1890) listed the same species on squashes from

New York under the name Rhopalomyces cucurbitarum. Similarly Rick (1906) reported

Choanephora infundibulifera from Brazil and Sydow et al. (1907) reported

Choanephora from India.

Clinton (1903) reported the occurrence of the disease in Connecticut and

included photographic illustrations of the fungus on squash flowers. This species has

also been reported in Brazil on the petals of a species of Hibiscus and on certain other

plants.

Thaxter (1903) named the species Choanephora cucurbitarum and recorded its

occurrence on squash in Massachusetts and on Hibiscus, a wild malvaceous plant in

Florida. Moller (1901) also recorded the same species under another name on Hibiscus

from Brazil.

Wolf (1917) reported C. cucurbitarum from squash, flowers of cucumber,

Hibiscus syriacus, H. coccineus, okra and cotton. He also described the zygosporic

stage of this species. Furthermore, he showed that certain insects transmit the disease

from one flower to another and that infection in squashes occurs through the flower end.

The occurrence of C. cucurbitarum on chillies (Capsicum sp.) was reported by Dastur

(1920) in India and Choanephora blight on cowpea was recorded in North Carolina

Wolf and Lehman (1920).

Palm and Jochems (1924) reported C. cucurbitarum from Amaranthus blitum and

A. spinosus. Dade (1929) reported that C. cucurbitarum occurred on papaya leaves,

cacao husks and withered blossoms of Hibiscus and Zephyranthes collected in the Gold

Coast region of Africa. Blakeslee et al. (1921) stated that C. cucurbitarum is commonly

found on withered flowers of pumpkin and squash from gardens near Cold Spring

Harbor.

Three species of Choanephora, C. cucurbitarum on leaves, stems and flowers of

Capsicum spp.; C. simsoni on Ipomoea and on flowers of Zinnia elegans and C.

infundibulijera on flowers of Hibiscus rosaesinensis and on inflorescence of

Tabernaemontana coronaria were reported by Butler and Bisby (1931) in their

monograph of fungi in India. C. cucurbitarum was also reported from Florida which is

causing blossom blight on pepper (Weber, 1932). Similarly, C. cucurbitarum was

isolated from red beans in Puerto Rico (Echavez, 1990).

The widely distributed pod rot of cowpea induced by Choanephora cucurbitarum

(Berk and Rav.) Thaxt. was reported in the province of Cordoba, Colombia (Munoz and

Tamayo, 1994).

Choanephora blight of potato caused by Choanephora cucurbitarum (Berk and

Rav.) Thaxt. was reported in Peru (Turkensteen, 1979).

Yasmin and Mirza (1988) reported that the disease symptom was observed on

maize cv. Shaheen in Islamabad, Pakistan, after monsoon rains in July-September, 1983

in the form of leaf blight. The causal organism was isolated and identified as

C. cucurbitarum and its pathogenicity was confirmed. Maize appears to be a new host

of C. cucurbitarum in Pakistan.

Top rot of Amaranthus hypochondriacus was first reported in India in August

1986. Infection starts at the top of the shoot as a brownish discoloration that spreads

downwards to the stem and leaves. Infected shoot droops, rots and finally plant dies.

C. cucurbitarum was identified from affected parts and its pathogenicity was confirmed.

It was established that infection occurs primarily from soil borne inoculum; seed

transmission was 0.5%. This is the first report of C. cucurbitarum on this host in India

(Roy and Deka, 1989).

Outbreak of Choanephora blight (Choanephora cucurbitarum) on green bean

(Phaseolus vulgaris) and bell pepper plants (Capsicum annuum) was reported in

Florida, USA. Analysis of outbreak showed that it is influenced by environmental

conditions such as long periods of high rainfall, humidity and temperature. Crops like

bean and pepper that was not normally affected by the disease may be infected (Roberts

et al., 2003).

Kwon and Jee (2005) reported and described the soft rot disease of eggplant

caused by C. cucurbitarum in the year 2002-2003 in Korea.

Choanephora wet rot of okra fruits was recorded in Malaysia (Siddiqui, 2006 and

Siddiqui et al., 2008).

https://ovidsp.tx.ovid.com/sp-3.14.0b/ovidweb.cgi?&S=AFGKFPKBLBDDBBGBNCLKPFJCPOILAA00&Search+Link=%22Yasmin+Ahmad%22.au.

https://ovidsp.tx.ovid.com/sp-3.14.0b/ovidweb.cgi?&S=AFGKFPKBLBDDBBGBNCLKPFJCPOILAA00&Search+Link=%22Roy%2c+A+K%22.au.

https://ovidsp.tx.ovid.com/sp-3.14.0b/ovidweb.cgi?&S=AFGKFPKBLBDDBBGBNCLKPFJCPOILAA00&Search+Link=%22Deka%2c+K+C%22.au.

The Choanephora blight has been reported to occur in Bangladesh, China,

Cambodia, Japan and India etc. In India the disease was noticed in Andhra Pradesh,

Assam, Bihar, Chhattisgarh, Delhi, Gujarat, Karnataka, Kerala, Madhya Pradesh,

Punjab, Rajasthan and Uttar Pradesh states (Anonymous, 2008).

Five fungi were recorded during the cropping year on Brinjal viz., Fusarium

solani, Helminthosporium spiciferum, Choanephora cucurbitarum, Curvularia lunata,

Trichothecium roseum. The growth of these fungi was found luxurious especially from

Oct to Nov and Feb to April. Fruit spoilage was maximum from Aug to Jan due to high

humidity, high temperature and less rainfall (Pandey, 2010).

Hussein and Ziedan (2013) reported the wet rot of okra caused by Choanephora

cucurbitarum in Egypt and observed white aerial mycelial strands on affected branches

which later turns into light yellow colour.

2.1.2 Host range

The disease was found on several varieties of Cucurbita pepo being most

destructive on the “pattypan” types of summer squashes, commonly known as

cymlings. The fungus has also been found on the fading flowers of cucumber (Cucumis

sativus), althea (Hibiscus syriacus), scarlet hibiscus (Hibiscus coccineus), okra

(Hibiscus esculentus) and cotton (Gossypium herbaceum). It appears to be parasitic,

however, only upon the squash (Wolf, 1917).

In Sarwak, it was reported that C. cucurbitarum infects 21 host species which

consist of 14 herbaceous species and 7 aborescent species (Turkensteen, 1979).

The pathogen (Choanephora cucurbitarum) has been reported to infect cotton

(Sapkal et al., 1975), cowpea, cucumber, tobacco (Sinclair, 1982), amaranthus (Afolabi,

1994), Lucerne (Randhawa et al.,1985) and cowpea (Bashir et al., 1985).

The genus Choanephora, a member of Zygomycetes was known to attack

withering floral parts of many plants after fertilization and invades the fruits, causing a

soft rot of primarily summer squash or pumpkin, pepper and okra (Agrios, 1997).

C. cucurbitarum reported to cause fruit rots, flower and leaf blights on a variety

of plants including okra, squash, pumpkin, pepper, pea, bean and cucumber (Yu and

Ko, 1997).

https://ovidsp.tx.ovid.com/sp-3.14.0b/ovidweb.cgi?&S=AFGKFPKBLBDDBBGBNCLKPFJCPOILAA00&Search+Link=%22Ziedan%2c+E%22.au.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774878/#B1

Recently, several authors reported various diseases caused by Choanephora

cucurbitarum such as blossom blight of petunia (Kwon et al., 2001), pod rot of cowpea

(Kwon et al., 2001) and flower rot on cotton rose (Kwon and Park, 2002).

Choanephora cucurbitarum found to be a plant pathogenic fungus causing fruit

rots, flower rot and leaf blights on a variety of plants including squash, pumpkin,

pepper, pea and bean. The fungus is known to attack several other crops which include

cereals such as millet, rice and sorghum. The fungus also causes pod blight known as

wet rot, blossom blight and whisker rot (Kacharek et al., 2003).This disease is also

common on squash and southern pea but occurs on the floral parts of many types of

plants (Afolabi, 1994).

Diseases caused by the Choanephora have been recorded on pea, four o’clock,

cabbage, cucumber, white clover, sugar beet, garden petunia, spinach,

Mesembryanthemum spp., pumpkin and eggplant (National Institute of Agrobiological

Sciences, 2010).

2.1.3 Economic importance

The disease caused of appreciable damage to squashes annually, but appears in

epidemic form only under certain meteorological conditions. Conditions of high

humidity and excessive rainfall were generally prevalent in the Southern States during

the past summer, and are believed to be correlated with epidemics of the squash disease

(Wolf, 1917).


The symptoms of the disease on flowers and fruits of squashes are quite

characteristic. The fungus covers the affected parts with a luxuriant crop of

conidiophores, with no evidence of any vegetative mycelium. The fructifications of two

other fungi, Rhizopus nigricans and Botrytis vulgaris may appear on the surface of

decaying squash fruits but neither of them possesses the metallic luster which

characterizes the fructifying filaments of C. cucurbitarum (Wolf, 1917).

The symptoms of Choanephora disease on red gram was reported by Misra and

Mehra, 1969. They observed symptoms initially on the tender unfolding leaves, later

spreads to older leaves and tender shoots. The infected portions partly loose the green

colour in the beginning and later show tendency of wet rot.




Black et al. (1975) reported the occurrence of wet rot on pepper. Initial symptoms

were often associated with flowers, flower buds, or apical growing points of the plant.

Infected tissue turns brown to black and the fungus grows rapidly downward, killing off

portions of the upper plant. A wet rot was associated with infected tissue which often

appears somewhat silvery to gray because of the spore producing stalks which are

visible to the naked eye.

Chahal and Grove (1974) reported the occurrence of soft rot on chilli. Initial

symptoms were often associated with flowers, flower buds or apical growing points of

the plant. Flowers first turn brown and then black and begins to rot. The rot then rapidly

extends downwards attacking buds and tender leaves. The infected part of the stem

shows a very marked wet green colour and the bark easily peels off in shreds as the

disease spreads downwards the whole plant, in a severe case of attack entire plant dies

off.

C. cucurbitarum mostly attacks tissues that have been damaged by insects or

mechanical means or crops that were poorly adapted to a hot humid climate. The

general appearance of Choanephora blight is similar to that of diseases caused by other

Mucorales of the genera Mucor and Rhizopus. Infected tissues have a hairy appearance

resulting from the tall sporangiophores that produce a cluster of brown sporangiola

(often referred to as conidia) at their tips (Turkensteen, 1979).

Gupta and Madan (1982) reported several parasitic fungi on Solanaceous plants

from North India. Choanephora cucurbitarium was observed on the leaves, flower buds

and fruits. Leaves and fruits were equally affected by black color sporangia on their

surfaces. It was abundantly present during October-November and first week of

December, while in the rest period it was not recorded either from the field or market.

Sinclair (1982) described symptoms of leaf blight caused by C. cucurbitarum

on soybean. The infected leaves first develop a grayish color similar to hot water scald

or chemical injury. The infected portion dries up and curls. Sporangiophores and

sporangia develop on the damaged portion. If relative humidity is high, severe

defoliation occurs. If humidity is low, only the areas showing symptoms drop off,

leaving the unaffected portion intact.

The pathogen Choanephora infundibulifera Sacc. was isolated from the infected

plants of soybean on potato dextrose and water agar. The fungus produced sporangioles

after two days in culture. This was the first report of Choanephora blight in Louisiana

(Subbarao, 1990).

Black (2001) first described symptoms of Choanephora rot on cucurbit which

first attacks the blossoms and progresses into the developing fruit causing a wet rot at

the blossom end. Sporulation of the fungus appears as spines with dark heads on the

surface of infected tissues.

Kwon et al. (2001) found blossom blight of petunia caused by Choanephora

cucurbitarum in greenhouses around Jinju area and reported that the disease begins with

water-soaked lesions on the flower which rapidly withered and rotted.

Typical symptoms of the soft rot on eggplant fruit appeared as water-soaking and

dark-green lesions. After infection the diseased tissues were rotten rapidly under

favorable environmental conditions. The pathogen penetrated mainly through wounds

on the fruit and rotted rapidly. Usually whitish mycelia and monosporous sporangial

were produced on the lesions. The disease was severe under a high temperature and

humid conditions that favor the disease development. It was often observed in the fields

that the fruit surface was covered by the fungal hyphae with abundant sporangia and

sporangiospores. The symptoms were similar to the soft rot caused by Rhizopus sp and

Mucor sp (Kwon and Jee, 2005).

Choanephora cucurbitarum fungus is a weak parasite; it colonizes on dead or

dying tissue before it actively invades living pepper tissue. Pepper plants were

susceptible from seedling to early flowering stage. Fruit infection was observed

predominantly around the calyx. Most of the time, it starts in senescing flower petals.

Once established, entire flowers are overgrown, resulting in a brown to black mass of

soft tissue. Flower stalks, buds and leaves may subsequently be invaded. In the field, the

disease diagnosed based on the appearance of a stiff silvery mass of whisker-like or

hairy strands of the fungus growing out of the affected pepper tissue, topped with a

black ball made of great numbers of spores and is visible with a hand lens. Infected

young fruit may abort. Individual branches of a plant get infected and die back. Stems

that are infected appear wet and green and the bark peels off easily in shreds (Kalb,

2004).

Sikora (2004) observed Choanephora wet rot on summer squash. Fungal mold

appears on the infected area, and fruit rot rapidly. The fruit resembles a pin cushion

with numerous small, black-headed pins stuck in it. Initially, the heads are white to

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brown but turn purplish black within a few days. Affected flowers, pedicels (flower

stalks) and immature fruit become water-soaked and a soft wet rot develops. An entire

fruit can rot in a 24 to 48 hour period. Symptoms usually begin on the blossom end of

the fruit.

Twig blight caused by Choanephora cucurbitarum (Berk and Rav.) Thaxt. was

responsible for severe damage of chilli and bell pepper. The disease was more on young

growing stages. Water soaked lesion was observed at a point of an individual branch

above which the ultimate branches were produced. The lesion encircles the branch

causing death of young branch above the lesion. Prominent hairy growth of C.

cucurbitarum on the infected tissue was found in the morning hours. The disease

gradually spreads to more and more branches, even to the stem causing severe crop

damage under warm and humid condition (Jana et al., 2011).

A severe disease of Amaranthus was observed in Nigeria during 1985 and 1986. It

was characterized by extensive blighting of the inflorescence, often accompanied by

inflorescence dieback and breaking of the floral head. Seed production was severely

affected. C. cucurbitarum was isolated from various parts of infected inflorescences

(Adebanjo, 1988).

Rotting and shrivelling flowers of cotton rose (Hibiscus mutabilis) were found in

the flower beds. Water soaked and dark green lesions on the petals and then whole

flower was rotted rapidly. Whitish mycelia and monosporous sporangiophore with

monosporous sporangiola caused by C. cucurbitarum were formed abundantly on the

lesions (Kwon and Park, 2002).

Saroj et al. (2012) reported the disease symptoms of Choanephora wet rot of

Withania somnifera in India and worldwide. The initial symptoms appeared as water

soaked lesions on leaves and stems that developed into a wet rot. Mature lesions

harbored black fructifications of the pathogen. The fungus was identified

as Choanephora cucurbitarum based on cultural and morphological characteristics and

nucleotide sequence data. This is thought to be the first report of wet rot of caused by

Choanephora cucurbitarum.

Water soaked lesions on Capsicum annuum appear on the leaves and the margins,

leaf tips gets blighted. The disease causes severe destruction in rainy season in tropical

climates. Initial symptoms were associated with flowers, buds or apical growing points

and later fungus grows downward killing the plant. Wet rot caused by C. cucurbitarum

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develops on affected parts. Infected parts of the stem appear wet and green. The bark

easily peels off in shreds (Abhishek and Pooja, 2014).

2.2. Survey for twig blight incidence

A survey on incidence of Choanephora cucurbitarum on Amaranthus sp was

conducted during 1984-1985 in Nigeria. It was found that the disease was recorded

throughout the year at varying levels. Stem lesion and leaf blight incidences was higher

during Oct-Dec season (Adebanjo, 1988).

The severity of Choanephora blight on bean is estimated up to 40 per cent under

normal to favorable condition (Roy, 1993).

An extensive survey on post-harvest fungal diseases of fleshy chilli fruits was

conducted in various markets of Warangal district. In this survey, a total of 20 diseases

caused by 24 fungal species were recorded in different varieties of chilli (Prabhavathy

and Reddy, 1995).

Data from reports on crop losses due to disease during 1983-88 are reviewed.

Eight diseases were reported during survey for watermelon (Citrullus lanatus). Blossom

end rot (Choanephora cucurbitarum) recorded the highest incidence of all pathogens

for all years followed by damping off, gummy stem blight (Didymella bryoniae) and

root knot nematodes (Meloidogyne spp.) (Shetty and Wehner, 1999).

During the disease survey on eggplant from 2002 to 2003, a severe fruit soft rot

caused by Choanephora cucurbitarum on the plant was observed in the experimental

field of Gyeongsangnam-do Agricultural Research and Extension Services in Korea.

Infection rate of the diseased fruit was about 2.6% in two fields surveyed (Kwon and

Jee, 2005).

A survey at Herbal Park in Jaipur, India, plants of Boerhavvia diffusa were found

infected with twig blight caused by Choanephora cucurbitarum. The disease begins as

foliar infections which then spread and kill twig tissues (Sikora, 2004).

Choanephora cucurbitarum was recorded in the months of July to Dec. Its

frequency was almost equal in all the months of its occurrence. As per climatic data it

may be concluded that C. cucurbitarum favors most humid condition and temperature

ranging around 30oC (Mishra and Vinit, 2012).

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It has been reported that in Bareilly city (U.P.) humid and cloudy weather

favoured in development of fungal diseases on brinjal and five fungal species were

encountered viz., Fusarium solani, Helminthosporium speciferum, Choanephora

cucurbitarum, Curvularia lunata and Trichothecium roseum (Dilip et al., 2013).

2.3 The pathogen

2.3.1 Morphological characterization of Choanephora cucurbitarum

Kuo et al. (1999) isolated Choanephora cucurbitarum on lima bean and

described the morphology of the fungus. The colonies of the Choanephora

cucurbitarum grown on potato dextrose agar at 25oC are white at the early stage of

growth with abundant aerial mycelium, but at later growth stages turn pale yellow.

Sporangia and sporangiola are readily observed on the tip of upright sporangiophores,

which are numerous on aerial mycelium. Sporangia spherical in shape, 25-124 µm in

diameter, white at first but turning dark brown at maturity. Sporangiola, containing only

one spore, indehiscent, ellipsoid to broadly ellipsoid. 11-13 x 13-20 µm in size

subtended by a short cylindrical pedicel. Sporangiospores, ellipsoid to broadly ellipsoid

in shape, brown to pale brown, 8-12 x 20-24 µm in size and are characterized by

longitudinal striatures on the wall surface and several fine hyaline appendages at both

poles.

Kwon et al. (2001) described the morphology of Choanephora cucurbitarum. The

fungus produces whitish mycelia and monosporous sporangia on the lesions. The

fungus isolated from the lesions produce white to pale yellowish brown mycelia with

scattered monosporous sporangia on potato dextrose agar (PDA) plates. The size of the

sporangium was 37.2-135.8 μm. Monosporous sporangia were elliptic, fusiform or

ovoid and brown in color and their size was 10.4-22.4 x 7.4-12.9 μm. Sporangiospores

were elliptic, fusiform or ovoid in shape, dark brown or brown in color and were 13.7-

23.5 x 8.7-13.8 μm in size and had appendaged appressorium of 3 or more. Zygospores

were black and were 40.8-61.5 μm in size.

Kwon and Park (2002) isolated C. cucurbitarum causing flower rot of cotton

rose (Hibiscus mutabilis) and described morphology. Colony appeared as white to pale

yellowish brown mycelia on potato dextrose agar medium (PDA). Monosporous

sporangiophore was long slender and branched at the apex, each branch bearing a head

of sporangiospores. Sporangium was subglobose in shape and was 42.6-114.2 μm in

size. Monosporous sporangiola were elliptic, fusiform or ovoid and brown in color and

12.3-21.6 x 8.3-11.6 μm in size. Sporangiospores were elliptic, fusiform or ovoid in

shape, dark brown or brown in color and 16.3-23.8 x 8.2-13.6 μm in size and they had

three or more appendages at bipolar end. Zygospores were mostly globose, dark black

colored and sized was 42.6-78.4 μm in diameter.

The fungal colonies on PDA were white to pale yellowish brown abundant

monosporous sporangiola were radially formed on the medium. Sporangia were

subglobose in shape and 40~130 µm in size. Monosporous sporangiophore was long

slender and branched at the apex and each branch beared a head of sporangiospores.

Monosporous sporangiola were elliptic, fusiform or ovoid, pediculate, striate and

measured 12~20 × 6~14 µm. Sporangiospores attached three or more appendages.


color and sized 14~22 × 7~10 µm. Zygospores were not observed in this study (Kwon

and Jee, 2005).

Abel Motaal et al. (2010) described the morphology of Choanephora

cucurbitarum the fungal colony was colorless as it aged, it became white to yellowish

brown and had aseptate hyphae with irregular branching. Monosporous sporangiophores

were long, slender and branched at the apex with monosporous sporangiola on each

branch. The monosporous sporangiola were subspherical to ovate in shape and 13.1–

25.3 x 8.2–15.0 μm in size. The few spored sporangia were subglobose in shape and

43.4 –121.2 μm in size. The dark brown sporangiospores were fusiform or elliptical to

ovoid and 8–10 x 12–16 μm in size with hair-like appendages on both ends. Four day

old cultured vegetative cells of two isolates, HM16-N and HM16-V were streaked 1 cm

apart on a new PDA plate and incubated at 25oC for 15–20 days to test mating.

Heterothallic, hemispherical zygospores were black, 45.3–85.6 μm in diameter and

formed between the tips of entwining branches. Chlamydospores were found in chains

in old cultures.

White aerial mycelia that later turned pale yellow was consistently isolated

from infected plant parts. Mycelia were hyaline and nonseptate. Sporangiophores

bearing sporangiola were erect, hyaline, unbranched, apically dilated to form a clavate

vesicle from which arose dichotomously branched distally clavate secondary vesicles.

Sporangiola were indehiscent, ellipsoid, brown to dark brown with distinct longitudinal

striations and measured 12 to 20 × 6 to 12 μm. Sporangia were multispored, spherical;

initially white to yellow and pale brown to dark brown at maturity and measured 40 to

160 μm. Sporangiospores from sporangia were ellipsoid to broadly ellipsoid, brown to

http://www.ncbi.nlm.nih.gov/pubmed/?term=Jee%20HJ%5Bauth%5D

dark brown, indistinctly striate with fine hyaline polar appendages and measured 16 to

20 x 8 to 12 μm (Saroj et al., 2012).

The fungal colonies produced cottony, white mycelium and black spore masses

were consistently isolated from diseased tissue. The mycelium was non-septate,

unbranched and hyaline. Secondary vesicles borne conidia that were smooth walled;

light brown, about 2.4 μm in diameter and typically obovate. Sporangiophores were

non-septate 5-13 mm high, straight but not swollen and roughened at the base.

Sporangia were numerous in culture measuring 20-45 x 150-200 μm in diameter,

bearing few to many sporangiospores, that were ellipsoid in shape and brown to pale

brown in colour, measuring 15-21 x 10-13 μm (Singh et al., 2012).

2.3.2 Isolation, Purification, Identification and Pathogenicity

The pathogenicity of the C. cucurbitarum was conducted by detaching healthy

pods of cowpea and surface sterilized with 0.5% sodium hypochloride for 1.5 min and

washed several times in sterilized water. The washed pods were inoculated by dipping

them into a conidial suspension of C. cucurbitarum (3.5 x104

conidia/ml) and were then

placed in sterilized culture test tubes along with control at room temperature ranging

from 24 to 30oC. After one week identical symptoms were observed on the inoculated

pods and the same organism was reisolated consistently from the diseased pods (Bashir

et al., 1985).

Yasmin Ahmad et al. (1988) isolated and identified C. cucurbitarum on maize and

confirmed its pathogenicity. Pathogenicity tests consisted of spray inoculation (5,000

spores per ml) of 6-week-old plants each with and without wounding made by lightly

scratching the leaf surface with a needle. Plants were placed in the greenhouse with

temperatures ranging from 21 to 26°C and symptom development was observed as

early as 3 days after inoculation.

The causal agent of fruit rot of bhendi (okra) was identified as C. cucurbitarum in

Kerala, India, in 1990 and pathogenicity was confirmed (Naseema and Wilson, 1993).

Daughtory (1995) performed pathogenicity test by misting a mixture of

sporangiola and sporangiospores (25,000 to 70,000/ml of water taken from 7 to 10 days

old culture grown on PDA). Inoculated plant and uninoculated control plant (2 to 4 of

each treatment in each test) were held in a dew chamber 28oC for 48 hr and then moved

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to a greenhouse within 48 hr. After inoculation, plant developed water soaked lesion on

flower, leaves and stems then wilted and rotted. Koch postulates were completed by re

isolation of pathogen from disease inoculated plants. C. cucurbitarum and C.

infundibulifera have been reported to cause flower blight of petunia in U.S.

Isolation and Koch's postulates were fulfilled by re-isolating the fungal pathogen,

which was identified as Choanephora cucurbitarum causing wet rot on Capsicum

annuum has been reported in India (Prabhavathy and Reddy, 1995).

Balogun and Babatola (1999) studied the effect of plant age and injury on the

pathogenicity of Choanephora cucurbitarum in okra. Inoculation at planting, seedling

emergence or 10 days after planting caused the leaf infection significantly earlier than

inoculations made at later stages of growth.

Fruits of eggplant showing soft rot symptom caused by C. cucurbitarum were

collected and fruits were cut into pieces disinfected with 0.1% mercuric chloride

solution for 30 sec, placed on potato dextrose agar and left for 48 hrs at 25o ± 2

oC. The

fungal tip growing out from the tissues were transferred to potato dextrose agar (Kwon

and Jee, 2005).

A monosporous sporangiola suspension (2 x 104 sporangiola/ml sterilized water)

was applied on newly opened flowers. The control flowers received the same amount of

sterilized water. The plants were covered with plastic bags to increase the moisture

content. Three days later, the inoculated flowers were wilting and symptoms had begun

to appear. The rotting extended to the remaining parts of the flower within 8 days and

reached the stem and leaves after 10 days; eventually the floral tops wilted and died. C.

cucurbitarum was reisolated from the diseased plants (Abel-Motaal et al., 2010).

Samples of rotted blossoms and young pods of okra infected by C. cucurbitarum

were cut into bits and surface sterilized using 1% sodium hypochlorite 1 minute then

washing with sterilized distilled water several times then cultured on potato dextrose

agar medium (PDA) and incubated at 25ºC for 7days. Colony margins were then

transferred onto (PDA) as part of the culture purification process by single spore culture

technique (Hussein and Ziedan, 2012).

Saroj et al. (2012) isolated, identified and confirmed pathogenicity of the

Choanephora cucurbitarum by artificial inoculation on healthy plants with spray of an

aqueous spore suspension containing 106 spores/ml. Plants sprayed with sterile distilled

water were used as control. Both inoculated and control plants were kept in a humidity

chamber (96%) for 3 days and there after placed in the glasshouse at 28 ± 2°C. Initial

symptoms developed in 2 to 3 days while typical disease symptoms appeared on all the

inoculated plants after 7 to 10 days. Control plants were free from infection.

Infected tissues of Amaranthus cruentus were cut into small pieces and

superficially sterilized for 3 min by immersion in a 75% ethanol solution and then

thoroughly rinsed three times with sterile distilled water. The tissues were incubated in

a moist chamber (autoclaved Petri plates containing wet Whatman No. 1 filter paper)

for five days at 27°C temperature dark/light. The developed fungal colonies were

transferred on Petri dishes containing potato dextrose agar (PDA) and incubated 3 to 5

days at 27 ± 2°C for spore production. Pathogenecity tests were performed by spraying

20 ml of spore suspension. The tissues of the plant were exposed to the inoculum by

slightly pressing with sand paper. After spraying, plants were covered with polythene

foil for 72 h to create an environment conducive for C. cucurbitarum growth. Seedlings

treated with sterile water were used as an untreated control (Awurum and Uchegbu,

2013).

Akwaji et al. (2014) performed pathogenecity test by spraying the spore

suspension, approximately 5 x 105

spores /ml were inoculated on the abaxial surface of

the wet healthy host leaves, stems and soil by spraying to run off level. The leaves,

stems and soil was covered with transparent polyethylene bags and allowed to stay for

24 hours. Spores measurement was done with a haemocytometer. The control

experiment was carried out with sterile distilled water without spores.

2.4. PHYSIOLOGICAL STUDIES

2.4.1 Effect of Media

Ikediugwu (1981) reported that sterile distilled water, 1% glucose and diluted leaf

extract of Amaranthus hybridus supported the germination of the spores of C.

cucurbitarum but noted that high percentage germination was attained in the presence

of potato dextrose medium.

Saini and Sidhu (1983) tested 28 carbon compounds and reported that C.

cucurbitarum grew best on glucose and C. infundibulifera on maltose.

Bandopadhyay & Som (1984) reported that Choanephora cucurbitarum grews

very fast on PDA at 28oC.

Iroakazi (1984) evaluated the different culture media for the growth of C.

cucurbitarum in laboratory and found that PDA was the best for the growth of fungus

and the yam dextrose medium was the best medium for sporulation and germination of

the fungus.

Awuah (1989) reported Choanephora cucurbitarum grew and sporulated well on

potato dextrose agar media.

Kuo et al. (1999) tested 12 media for Choanephora wet rot of lima bean

(Phaseolus limensis [P. lunatus]) caused by Choanephora cucurbitarum and found the

potato dextrose agar and 10% V8 medium were the most favorable, followed by

czapek-dox yeast extract agar for mycelial growth of the fungus. Maximum sporangia

formation was observed on 10% V8 medium and carrot agar, while sporangiola

formation was observed on czapek-dox yeast extract agar.

Kwon and Park (2002) reported that fungus grows well on PDA media and the

optimum temperature was 30oC.

2.4.2 Effect of PH levels

Arya and Prasad (1952) observed pH 3 to pH 8.5 range is suitable for the growth

of Alternaria spp.

Almedia (1977) and Sinclair (1982) found that maximum radial growth and

sporulation Choanephora cucurbitarum at pH 6.5 to 7.5.

Holcomb (1998) isolated Choanephora cucurbitarum from flower spots by

plating necrotic tissue on acidified potato dextrose agar.

Ojionuka (1988) also reported that maximum radial growth of Choanephora

cucurbitarum at pH 5.0. Growth at pH 5.0 was significantly greater than growth at other

pH levels except 4.5, 5.5, 6.0 and 6.5.

2.4.3 Effect of Temperature

Temperature is the most important environmental factor that influences the

growth and sporulation of Choanephora cucurbitarum.

Lilly and Barnett (1950) reported that the temperature was the only factor which

greatly influenced the formation of sporangia of C. cucurbitarum. A temperature of

31oC during sporulation was reported unfavorable for conidia production. While

maximum mycelia growth was obtained at 34oC.

Tiwary and Yadav (1974) recorded vegetative growth of C. cucurbitarum greatest

in violet and green light whereas sporulation was best in red and yellow light in both

strains of growth and sporulation was minimum in continuous dark and continuous

artificial light.

Naito and Sugmito (1989) observed maximum mycelial growth of Choanephora

cucurbitarum on nutrient agar at 30oC, while hyphal growth was slight at 10 and 40

oC.

Artificial inoculation at the flowering stage with a spore suspension of sporangiospores

of Choanephora sp. isolated from the lesions caused flower stalk rot in sugar beet’s.

Kang et al. (1993) studied the effect of temperature on Choanephora

cucurbitarum and found optimum temperature for mycelial growth between 25-30oC,

while conidial germination and appresorium formation was best at 30oC.

Kuo et al. (1999) recorded the optimum temperature for mycelial growth and

germination of sporangiospores of C. cucurbitarum was between 25 to 35oC.

The C. cucurbitarum grews well under humid conditions and thrives best at a

temperature of 25°C and relative humidity of about 100%. A temperature of about 31°C

stimulates the production of large sporangia but unfavorable for conidia formation

(Umana and Ikotun, 2000).

Kwon et al. (2001) also studied the effect of temperature on the blossom blight of

petunia caused by C. cucurbitarum in Korea and reported that the fungus grew well on

PDA at 20-40oC and optimum temperature was 30

oC.

The production of the asexual structures of the two strains of C. cucurbitarum on

L. cylindrica and Hibiscus esculentus (Abelmoschus esculentus) is influenced by such

environmental conditions as temperature, relatively lower temperatures (between 20-

30oC) favors early formation of sporangia. However they found that temperature did not

have any effect on the formation of conidia (Yadav and Tiwary, 2001).

Wolf (1917) observed alternate periods of dark and light or light and dark encouraged

the formation of sporangia. While continuous bulb light produced the poorest effect, a one-

hour exposure to sunlight proved better for sporangial formation of C. cucurbitarum.

2.5 Evaluation of fungicides against Choanephora cucurbitarum under

in vitro conditions

Oladiran (1980) found that chloroneb, thiram, thiram + BHC, fentin acetate,

captafol, fentin hydroxide and captan as inhibitory fungicides of mycelial growth of C.

cucurbitarum. Thiram, thiram + BHC and captan inhibited conidial germination of

fungus.

Raju et al. (1982) recommended captan as the effective fungicide against

Choanephora cucurbitarum disease due to its compatibility with pesticides like

quinalphos, dimathoate, phosolone and carbaryl. Spraying with paushamycin at 200

ppm along with 0.3% Blitox, 0.25% Dithane M-45 controlled the incidence of C.

capsici and Xanthomonas.

Oikawa et al. (1986) tested nine compounds under in vitro against Choanephora

Cucurbitarum among which mancozeb, triazine and triflumizole showed little control

but was ineffective under in vivo. This may be due to their poor adhesion.

Hammouda (1988) reported that mancozeb, dinocap and thiabendazole as

effective fungicides in reducing the infection of Choanephora.

Ridomil inhibited mycelial growth at all the concentrations used except at the

lowest concentration of 0.5 g/litre, while Bordeaux mixture and benlate inhibited

mycelial growth correspondingly at higher concentrations. Dithane M-45 allowed the

highest mycelial growth of all the fungicides tested (Fasemo, 1997).

Panja (1999) reported that captan 50% (2.0 g/litre), ziram 27% (1.5 ml/litre) and

copper oxychloride with 50% copper (3.0 g/litre) completely inhibited the mycelial

growth and sporulation of C. cucurbitarum and improved yields of Capsicum annum

during 1995-1996.

2.6 Isolation and evaluation of phylloplane and rhizosphere mycoflora

against Choanephora cucurbitarum

Singh and Sinha (1962) reported that Alternaria, Aspergillus, Cladosporium,

Curvularia, Helminthosporium, Heterosporium, Mucor, Penicillium, Rhizopus and

Trichoderma were isolated from blighted chilli leaves.

Individual and combined application of Pseudomonas aeruginosa and

Paecilomyces lilacinus reduced the infection of root infecting fungi Fusarium

oxysporum and Fusarium solani in chilli (Perveen et al., 1998).

Kamlesh et al. (2002) reported that fungal biocontrol agents Trichoderma viride,

Trichoderma harzianum and two isolates of Fusarium solani isolated from rhizosphere

of chilli significantly inhibited the mycelia growth of Rhizoctonia solani to an extent of

68.6 and 61.0 mm, respectively and reduced sclerotial productions of Rhizoctonia solani

causing stem rot of chilli.

Okigbo and Ikediugwu. (2001) isolated from the tuber surface of cultivar D.

alata. The fungus Aspergillus niger, Botryodiplodia theobromae, Fusarium solani,

Choanephora cucurbitarum, Penicillium oxalicum, Rhizopus spp, Trichoderma spp and

B. theobromae was most frequently isolated. However A. niger, Trichoderma spp and

Rhizopus spp isolated throughout the year except in D. alata on which it was relatively

sparse. P. oxalicum was recorded only during the early months of storage. The surface

mycoflora of D. alata was particularly high in Rhizopus spp, Trichoderma spp, was

consistently isolated from all cultivars throughout storage; however, as most of the

other fungi except A. niger and B. theobromae, its frequency decreased with storage

period.

2.7 Screening of popular chilli varieties against Choanephora

cucurbitarum

Pino (1980) screened several cultivars of cassava against C. cucurbitarum in

Cuba. Out of 212 cassava cultivars, 12 varieties escaped defoliation and recorded

resistance.

In Ibadan, Nigeria, nine cultivars of okra were screened for resistance to

premature fruit abortion caused by C. cucurbitarum during two seasons over 2 years. A

range of resistance/susceptibility was reported from 24 to 73% abortion. The most

resistant cultivar was NHAc 621 (Adebanjo and Dede, 1985).

Punjab Agricultural University, Ludhiana has developed 18 multiple disease

resistant lines in chillies crop (Singh and Kaur, 1990). Among these, ‘Punjab Lal’ (S-

118-2) has been released as multiple resistant chilli variety in 1985 for general

cultivation in the State. The other important multiple resistant lines are ‘Perennial’,

‘BG-1’, ‘Loral’, ‘Tiwari’, ‘Indonesian Selection’, etc. These are not only found resistant

to various fungal and viral diseases in India but also in France, Hungary, Spain,

Malaysia, Korea, USA and Taiwan (Thakur et al., 1987).

Psophocarpus scandens was resistant to leaf spot (Pseudocercospora

psophocarpi) and leaf curl and less susceptible to yellow mosaic, necrotic mosaic

and Choanephora flower blight (C. cucurbitarum) when compared with P.

tetragonolobus accessions. Leaf spot disease was positively correlated with plant

age. P. scandens yielded the highest number of pods and seeds/plant. Among P.

tetragonolobus accessions UPS 99, SLS 7, TPT 1 and UPS 45 produced comparatively

good pod yields (Gunasekera et al., 1990).

The influence of the environment on incidence of dieback, stem and leaf blights in

the Amaranthus cruentus cultivars NHAc33, NHAc30 and NHAc100 was investigated for 6

seasons during 1987-88 in Nigeria against Choanephora cucurbitarum. The lowest

incidences (0%) of dieback and stem blight (8%) were recorded during the 1st season

for NHAc33 and NHAc30, while NHAc100 had 4% leaf blight in the 3rd season (Dec-Feb).

The lowest mean percentage of leaf blight was recorded for all cultivars by the 3rd

season. Conversely, the highest incidence of symptoms caused by Choanephora

cucurbitarum in cultivars occurred in the 2nd season (Aug-Oct). The health of A.

cruentus seeds was influenced both by the seasons of harvest, environmental conditions

and seed variety. Precipitation, high temperature and humidity that accompanied

harvests of the 1st and 2nd seasons greatly reduced seed quality. High quality seeds

were obtained from NHAc100 and NHAc33 (Adebanjo, 1988).

The occurrence of C. cucurbitarum on cowpea was recorded from the province of

Cordoba, Colombia. The incidence of the disease was studied on 2 cowpea varieties

(ICA Calamari and ICA Betanci) and 10 lines (Munoz and Tamayo, 1994).

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Naimuddin et al. (1999) screened 148 genotypes of pigeonpea. Out of these 103

genotypes were disease free and 45 were infected. Disease incidence in these 45

genotypes varied between 3.8 and 15.4%. During disease surveys it was observed that

in almost all fields of pigeonpea, there was an association between leaf folder insect

pests and C. cucurbitarum. It is suggested that as C. cucurbitarum is generally a

facultative parasite, the mechanical injury due to feeding of the insect provides avenues

for parasitization by the fungus in addition to increasing humidity in the folded leaves.

Adegbite and Amusa (2008) screened 71 cowpea lines against the disease

Choanephora pod rot of lima bean and found that 80% lines were susceptible to the

disease.

A total of thirty varieties of chilli were screened for evaluation of resistant

varieties. Among thirty varieties screened, only two varieties CO-4 and DLC-352 were

found 100% resistant, while five varieties, viz., Ajeet-3, DLC-524, F-112-5-83, KCS-

2013, Hot pepper Nun-2060 were moderately resistant (83.34%) and Sel 11 was most

susceptible variety against wilt of chilli induced by pathogenic F. oxysporum (Mamta

Joshi et al., 2012 ).

2.8 Management of twig blight of chilli caused by Choanephora

cucurbitarum by using effective fungicides and potential biocontrol

agents against under greenhouse conditions

McMillan (1972) obtained effective control of C. cucurbitarum unreported

disease of pole bean (Phaseolus vulgaris). Effective control was obtained with DCNA

75WP at 2 lb/100 gal water applied as a protective spray.

Chahal and Grover (1974) reported zineb, mancozeb, ziram and thiram as the

most effective fungicides used as pre-inoculation sprays on ripe chilli fruits for

controlling C. cucurbitarum.

Iroakazi (1984) reported that the isolates of B. subtilis from soil and ogili (a local

food condiment prepared from watermelon seeds) controlled Choanephora shoot

disease of A. hybridus in the greenhouse. Disease developed on plants inoculated

simultaneously with C. cucurbitarum and the ogili isolate, but not the soil isolate of B.

subtilis. Application of either bacterial strain to plants one day before challenging

with C. cucurbitarum prevented disease development. A single application of either

strain prevented disease development from a subsequent challenge with the pathogen at

any time over a 30 day period.

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Gunasekera et al. (1990) tested eight systemic fungicides for in-vitro activity

against C. cucurbitarum causing blight of winged bean (Psophocarpus tetragonolobus)

flowers. Triadimenol, Vinclozolin and Bitertanol were most effective in inhibiting spore

germination and mycelial growth. Monthly applications of these fungicides in a field

trial reduced the blight severity.

Farzana Banu et al. (1990) reported that captan and thiram were able to reduce

the incidence of Colletotrichum dematium and increase the germination ability of chilli

seed. They also reported that seed treatment with biocontrol agents, Trichoderma

harzianum and T. viride and hot water treatment at 50oC also reduce seed borne

incidence of C. dematium.

Field evaluation of effective plant extracts and antagonists and fungicide

revealed that spraying with T. viride (2%) showed a maximum disease reduction of

61.41% followed by P. fluorescens (58.10%). However, the fungicide ziram (0.25%)

recorded 80.84% maximum disease reduction (Balogun and Babatola, 1999).

The efficacy of leaf, fruit, bark and root extracts of Azadirachta indica (Neem) in

controlling growth and sporulation of Choanephora cucurbitarum, which causes wet rot

disease of Amaranthus was evaluated both in vitro and in vivo. While the effects of leaf

and root extracts on mycelial growth were not significantly different between

themselves and the control, they had significantly less inhibitory effect than fruit and

bark extracts. The % inhibition of mycelial growth was 13.85, 75.38, 100.00 and

13.27% for leaf, fruit, bark and root, respectively. The inhibition rate of mycelial

growth was increased with the increase in plant extract concentration. Sporulation also

followed the same trend as mycelial growth. However, the results showed that the

fungicidal attributes are concentrated more in the fruit and bark than in leaf and root

extracts since, at 8.0 mg/ml, percentage spore production inhibition was 82.50, 100, 100

and 66.67% for leaf, fruit, bark and root extracts, respectively (Olufolaji, 1999).

Okigbo and Ikediugwu (2001) inoculated yam cv. Iyawo tubers by spraying with

conidiospore suspension of Trichoderma viride in potato dextrose broth showed a

drastic reduction in the range and number of mycoflora, including pathogen

Choanephora spp on the tuber surface during five months of storage in a traditional

yam barn.

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Siddiqui et al. (2008) studied the potential of water extract from rice straws (RST)

and empty fruit bunch of oil palm (EFB) composts fortified with Trichoderma

harzianum for the control of Choanephora cucurbitarum on okra under field condition.

The disease severity was lowest in plant treated with Trichoderma fortified (RST)

extract. There was a reduction of 85.04% in Choanephora cucurbitarum wet rot

severity when treated with Trichoderma-fortified RST extracts during 12 weeks of

assessment in the field, which was comparable to the conventional fungicide dithane M-

45, suggesting that application of extracts produced from well-matured compost

fortified with biocontrol agents could be an alternative control strategy.

The results indicated that the highest infection rate of the disease was obtained in

the first six weeks of inoculation of the organism, with plants in the control pots having

the highest disease severity (9.8). There was a significant (P < 0.05) difference in the

growth and yield of plants treated with Benomyl and plant extracts, compared with

those of the control. The least performance was observed from plants treated with sterile

water, which acted as the control (Awurum and Uchegbu, 2013).

The extracted essential oils were assessed for their antifungal potential through

poison food technique against two phytopathogens, Rhizoctonia solani and

Choanephora cucurbitarum, which cause root and wet rot diseases in various crops.

Ocimum tenuiflorum, O. gratissimum and O. kilimandscharicum exhibited complete

growth inhibition against R. solani and C. cucurbitarum after 24 and 48 h of

treatment. O. basilicum chemotypes showed variable levels of growth inhibition

(63.0%-100%) against these two phytopathogens (Padalia et al., 2014).

Chapter III

MATERIALS AND METHODS

This chapter includes all the materials used and methods adopted in the

investigation and the techniques are detailed under respective headings.

3.1 Location of the work

The present experiments were carried out in the Department of Plant Pathology,

College of Agriculture, Professor Jayashankar Telangana State Agricultural University,

Rajendranagar, Hyderabad, Ranga Reddy District, Telangana.

3.2 Laboratory techniques

The general laboratory techniques followed for the present study were those

described by Nene and Thapliyal (1993), Dhingra and Sinclair (1995) and Aneja (2001)

for preparation of media, sterilization, isolation and maintenance of fungal and bacterial

cultures with slight modifications wherever necessary.

3.3 Glassware

Glassware made of Borosil was used throughout the present investigation. The

glassware used in the study were Petri plates (90 mm diameter), conical flasks (250,

500, 1000 ml), measuring cylinders (25, 250 and 500 ml), test tubes, pipettes (0.1, 1.0,

2.0, 5.0 ml and 10 ml) etc.

3.3.1 Cleaning of Glassware

The glassware was first cleaned with a detergent followed by thorough cleaning

with tap water before placing them in cleaning solution for 24 hours and finally rinsed

with distilled water for 3-4 times. Then they were air dried before use.

Potassium dichromate (K2Cr2O7) : 60.0 gm

Concentrated sulphuric acid (H2SO4) : 60.0 ml

Distilled water : 1000.0 ml

3.3.2 Chemicals

Chemicals of analytical reagent (AR) and guaranteed reagent (GR) grade of

standard make were used. The pH of the media was adjusted using either 0.1 N HCl or

0.1 N NaOH. Streptomycin sulphate was added to the medium to avoid bacterial

contamination. Formaldehyde 10% solution was used to fumigate the laminar air flow

chamber and commercial formulations of fungicides and insecticides were used in all

experiments. Fungicides and insecticides were stored at 25oC in the dark to maintain

and preserve their biocidal activity.

3.3.3 Sterilization of glassware

Glassware like Petri plates, test tubes, pipettes etc., were wrapped in butter paper

and sterilized in hot air oven at 180oC for one hour. Different media and water used in

the study were sterilized at 15 lb psi (121 oC) for 20 minutes in an autoclave.

3.3.4 Sterilization of other instruments

Surface of Laminar Air Flow Chamber was sterilized by wiping with cotton swab

dipped in alcohol. Inoculation needles, forceps, cork borers and blades by passing

through flame after dipping in alcohol.

3.3.5 Plastic ware

Polythene bags (LDPE) of 20 guage polythene bags were used to collect twig

blight samples and also to cover the whole pot along with the plant to maintain

humidity.

3.3.6 Equipment

Compound microscope (10x, 40x magnifications) was used for observing the

fungi. Hot air oven and autoclave were used for sterilization of glassware and media

respectively. Incubators were used for incubating test materials at different

temperatures. The cultures were stored in a refrigerator. Weighments were done on a

single pan electronic balance with a sensitivity of 0.001 g. Other tools which were used

in the present investigation for various purposes include camel brush, inoculation

needle, pots etc.

3.4.1 Preparation of media

3.4.1.1 Potato Dextrose Agar (PDA) Medium

PDA medium was prepared using the following components for culturing of fungi

in the laboratory.

Potato : 200 g

Dextrose : 20 g

Agar : 20 g

Distilled water : 1000 ml

Peeled potato pieces were boiled in 500 ml of distilled water in a 1000 ml beaker

till the pieces got softened. The extract was filtered through a double layered muslin

cloth. To another 500 ml of distilled water in another 1000 ml beaker, 20g of agar was

added and melted till it got dissolved. Both the solutions were mixed in another 1000 ml

beaker into which 20 g of dextrose was added. The final volume of the medium was

made up to 1000 ml by addition of distilled water. The pH of the medium was adjusted

to 6.8 with 1 N NaOH or 1 N HCl as the case may be with the pH meter. The medium

was distributed to culture tubes and conical flasks at 8.0 ml and 100 ml each,

respectively. The medium was sterilized in an autoclave at 15 psi (121oC) for 15

minutes.

3.4.1.2 Preparation of PDA slants

PDA slants were prepared by transferring 8.0 ml of the medium to culture tubes.

The tubes were plugged with non-absorbent cotton and sterilized in an autoclave. After

sterilization, the tubes were removed from the autoclave when they were still in hot

condition (i.e. approximately 40°C) and kept in a slanting position for the medium to

solidify. After solidification, the slants were kept in refrigerator for further use.

3.4.1.3 Preparation of carrot agar slants

Carrot agar slants were prepared by transferring 8.0 ml of the medium to culture

tubes. The tubes were plugged with non-absorbent cotton and sterilized in an autoclave.

After sterilization the tubes were removed from the autoclave when they were still in

hot condition (i.e. approximately 500C) and kept in a slanting position for the medium

to solidify. After solidification the slants were kept in refrigerator for further use.

3.4.1.4 Potato Dextrose Broth

Potato Dextrose Broth was prepared using the following components for culturing

the fungi in the laboratory.

Potato : 200 g

Dextrose : 20 g

Distilled water : 1000 ml

Peeled potato pieces were boiled in 500 ml of distilled water in a 1000 ml beaker

till the pieces got softened. The extract was filtered through a double layered muslin

cloth. To another 500 ml of distilled water in another 1000ml beaker, both the solutions

were mixed in another 1000 ml beaker into which 20 g of dextrose was added. The final

volume of the medium was made up to 1000 ml by addition of sterile distilled water.

The pH of the medium was adjusted to 6.8 with 0.1 N NaOH or 0.1 N HC1 as the case

may be with the pH meter. About 100 ml of the medium was dispensed into each

conical flask. The medium was sterilized in an autoclave at 15 psi for 15 minutes.

3.5 General procedure followed

For each set of treatment different replication were used in all in vitro studies. In

general, in each Petri dish about 15-20 ml of potato dextrose agar medium was poured,

supplemented with streptomycin in order to check the undesirable bacterial

contamination.

Wherever growth studies were conducted seven mm disc of pure culture of C.

cucurbitarum by the help of cork borer, was used for inoculation of medium in Petri

dish. The inoculated plates were incubated in the 25oC ± 20

oC for three days.

Observation for the growth was recorded daily after inoculation 1st day to 3rd days.

3.6 Survey

Survey was conducted in major chilli growing districts of Karimnagar,

Warangal, Nizambad, Medak, Khammam and Ranga Reddy during kharif 2014. One

square meter area was marked in the center of field and four corners leaving the border

rows. The naturally infected leaf of chilli crop with the typical Choanephora blight

symptoms i.e. irregular spot, leaf area & leaf tip and twigs were collected (Plate 3.1).

The number of twig blight infected plants was counted and the per cent disease

incidence was calculated. Soil samples were also collected from the rhizosphere of the

infected plants. Six to eight samples were collected from each field, made into a

composite sample of about 1 kg and placed in a polythene bag. The bags were labeled

and information as per the data sheet mentioned below was collected (Table 3.1).

The Per cent Disease Incidence (PDI) was calculated as per the following

formula (Kotle and Vishunavat, 2005):

No. of infected plants

PDI = _____________________________________

x 100

Total No. of plants observed

Table 3.1 Specimen copy of the data sheet used during survey of Choanephora

twig blight in chilli.

Survey of Choanephora twig blight in chilli

Data Sheet

1 Sample No. : 1

2 Date of collection : 22-12-2014

3 Name of the farmer : Mr.Bhaskar rao

4 Locality

a) Village

b) Mandal

: Gudimalla

Khammam rural

5 Name of the variety : Super teja

6 Rainfed /Irrigated : Irrigated (vegetable or for red chilli)

7 Total area : 5 acres

8 Time of sowing : July 3rd

week

9 Purpose of variety

(vegetable or for

red chilli)

: For red chilli

10 Stage of the crop : Harvesting, 2nd

picking

11 Condition of the

crop

:

a. Twig blight

incidence

: 32.0 %

12 Previous crop : Maize

13 Type of soil : Sandy clay loam soil

3.7 Isolation, purification and identification

3.7.1 Collection of diseased sample

The naturally infected leaf of chilli crop with the typical Choanephora blight

symptoms i.e. irregular spot, leaf area & leaf tip were collected from the field during

survey. Such samples were brought to the laboratory for critical examination, isolation

and the identification of causal organism under compound microscope.

3.7.2 Isolation of the pathogen

The entire work of isolation and purification was done in isolation chamber and

laminar air flow, which was sterilized by alcohol or formaldehyde and UV Tubes, prior

to use. Chilli plants showing typical disease symptoms were collected from the farmers’

fields during survey and used for isolation of the test pathogen.

The infected twigs and leaves were thoroughly washed and cut into small pieces

of 1 to 2 cm long. The cut pieces were surface sterilized with 1% Sodium hypo chlorite

for one minute followed by three washings with sterile distilled water before placing on

PDA (Plate 3.2).

The plates were incubated in BOD incubator at 25o ± 2

oC. Fungal growth

emerging from diseased host tissue was directly transferred to the PDA medium under

aseptic conditions and incubated at 25o ± 2

oC.

Mycelial mats were then sub-cultured, purified by hyphal tip method and further

pure culture of pathogenic fungi was maintained on PDA by periodic sub-culturing and

preserved the test pathogen on PDA slant. All the growth characters were recorded and

compared with the standard descriptions of pathogen for confirmation.

3.7.3 Identification of the pathogen

The fungus associated with the disease was brought into pure culture on Potato

Dextrose Agar medium and identified based on the descriptions given by Wolf, F.A

(1917).

3.7.4 Microscopic examination

Temporary aqueous mounts were prepared from the pure culture of the fungus

and observed under the microscope. Measurements of sporangia and sporangiophore of

the fungus were taken with the help of ocular micrometer after calibration.

3.7.5 Pathogenicity test

Pathogenicity test was conducted in pots under glass house conditions. The

susceptible chilli cv. Madhuri of 120-135 DAS susceptible to Choanephora

cucurbitarum twig blight disease was raised in pots and 3 day old culture grown on

potato dextrose agar media was inoculated to chilli plant with hand sprayer and covered

with polythene covers.

3.7.6 Preparation of Inoculum

Inoculum of the pathogen was prepared by using Three day old culture of

Choanephora cucurbitarum grown on potato dextrose agar medium. Spores of the

pathogen were harvested by flooding sporulating cultures with sterile distilled water and

gently scraping the surface with a sterile needle. The resultant suspension was filtered

through a muslin cloth and the spore suspension was standardized to 5 x 105 spores/ml

through Haemocytometer.

3.7.7 Inoculation

Chilli plants of 135 days old were spray inoculated with pathogen spore

suspension (5 x 105

spores/ml) added with 0.025% tween twenty to provide uniform

spreading of spores was spray inoculated on leaves and twigs of chilli by using hand

sprayer.

3.7.8 Incubation

The inoculated plants were placed in a growth chamber at 25 ± 2oC temperature

and above 90% humidity to affect the infection process and observed for development

of typical symptoms of the disease.

4. Physiological studies

4.1 Effect of pH

The effect of different pH ranging from 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,

8.5 and 9.0 studied on potato dextrose agar medium. The pH of the medium was

adjusted before autoclaving with help of digital pH meter by addition of 0.1 N HCl and

0.1 N NaOH into the PDA medium after autoclaving, 20 ml of medium was poured in

the sterilized Petri plates. The Petri plates were inoculated with 3 day old culture of 7

mm disc of inoculums with the help of cork borer. Petri plates were incubated in BOD

incubator at 25 oC ± 2

oC. The three replications were maintained for each treatment and

observation for radial growth was recorded at 1st day and 2nd days of inoculation.

4.2 Effect of Temperature

The experiment was conducted to find out, the most suitable temperature for

radial growth of C. cucurbitarum. The sterilized poured Petri plate with potato dextrose

media were inoculated with 7 mm disc of the test pathogen of three day old culture. The

Petri plates were incubated at 0oC, 10

oC, 15

oC, 20

oC, 25

oC, 30

oC, 35

oC and 40

oC

temperature. Four replications were maintained for each treatment and observation for

radial growth was recorded at 1st day, 2nd day after inoculation. 3-day-old culture was

placed aseptically at the centre of each Petri dish (90 mm) containing 20 ml PDA

medium and incubated at given temperatures for 2 days. The colony diameter was

recorded after first and second day after inoculation.

4.3 Effect of media

To know the effect of different media viz. Oat meal agar, Carrot agar, Dextrose

nitrate agar, Corn meal agar, V8 juice agar, Water agar, Czapek dox agar and Potato

dextrose agar media were used to find out the best medium for the growth and

sporulation of Choanephora cucurbitarum. Twenty ml of media was poured in Petri

plate, 7mm disc of three day old culture of test fungus was placed in the center of

medium.

4.3.1 Morphological characterization of the pathogen associated with

twig blight of chilli

4.3.2 Morphological identification

The fungus causing twig blight of chilli was isolated on potato dextrose agar

medium and was examined periodically and identified based on morphological

characteristics, viz., colour, growth pattern and morphology of spores and

sporangiophores structure. Different media were used for cultural characterization of

pathogen and the compositions of the media are mentioned in 3.4.

5. Isolation of mycoflora from phylloplane and rhizosphere and testing

their antagonistic activity on Choanephora cucurbitarum under

in vitro conditions

5.1 Isolation of phylloplane mycoflora

The mycoflora associated with chilli leaves was isolated following modified leaf

washing technique (Deb et al., 1999). Fresh leaves from healthy and diseased plants

were collected randomly during survey in a polythene bag and brought to the laboratory.

Five discs were cut from each leaf with the help of 5 mm sterile cork borer. As

such 50 leaf discs were cut and transferred to a conical flask containing 100 ml sterile

water blank. The leaf discs were thoroughly agitated for 20 minutes using a Neolab

Vortex mixer. Serial dilutions were prepared in the standard way and the dilutions (10-3

and 10-4

) were plated in Petri dishes containing potato dextrose agar and the inoculated

Petri dishes were incubated in a BOD incubator at 25 + 2oC. Four replicates were

maintained for all the dilutions tested. The Petri plates were observed daily and the

fungal colonies obtained were transferred to PDA slants for further use.

5.2 Isolation of rhizosphere mycoflora

Diseased and healthy plant samples with rhizosphere soil of chilli plants was

collected in polythene bags from each survey plots and isolated immediately for

antagonistic organisms by following serial dilution method. One gram of soil from each

sample is taken in a 250 ml conical flask with 99 ml of sterile distilled water and

agitated for 5 minutes. 10-4

ml suspension was poured in sterilized Petri plates

containing PDA and nutrient agar media, respectively and incubated at room

temperature (25oC). Three replicates were maintained for each sample. Isolated fungal

antagonists from rhizosphere soil were transferred to culture tubes containing

appropriate media for further examination.

5.3 Maintenance of cultures

The fungi isolated from leaves were further purified by single spore isolation.

Pure cultures were maintained in culture tubes containing the PDA for further

examination and identification.

5.4 Identification

The isolated fungi were identified up to the level of genus or species by

illustrations given by Raper and Fennel (1965), Raper and Thom (1949), Subramanian

(1971) and Rifai (1969).

5.5 Evaluation of phylloplane and rhizosphere mycoflora against

Choanephora cucurbitarum under laboratory conditions

The fungi isolated from phylloplane and rhizosphere of chilli were screened for

antagonism against Choanephora cucurbitarum under in vitro conditions on potato

dextrose agar medium by following dual culture technique (Mortan and Sproube, 1995;

Mukherjee et al., 1995). Fifteen millimeter of sterilized luke warm potato dextrose agar

medium was aseptically poured into 90 mm diameter sterilized Petri plates.

Five mm discs of various fungal cultures and the test pathogen was cut with a

sterilized cork borer from the edge of three day old cultures and was placed on the

solidified medium opposite to each other. Three replicates were maintained for each of

the treatment. Suitable control was maintained by placing only the pathogen on culture

medium. The plates were incubated at 23 + 1oC. Petri plates were observed daily for

recording antagonistic interactions between the pathogen and biocontrol agent. The Per

cent growth reduction (I) of the test pathogen was calculated when the growth of the

pathogen was full in the control plates by using the formula as given below.

I = C – T x 100

C

Where,

I = Per cent inhibition of mycelial growth

C = Radial growth of pathogen in control (mm)

T = Radial growth of pathogen in treatment (mm)

6. Evaluation of different fungicides against Choanephora

cucurbitarum in vitro

Seven fungicides viz. Copper oxychloride, Mancozeb, Chlorothalonil,

Azoxystrobin, Captan, Hexaconazole and Carbendazim were evaluated against

Choanephora cucurbitarum by poisoned food technique (Nene and Thapliyal, 1993) at

6 different concentrations i.e., 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3 and the details of the

fungicides are presented in Table 3.2

The required quantity of fungicide were weighed and mixed in the potato

dextrose agar medium by thorough shaking for uniform mixing of the fungicide before

pouring into Petri dishes so as to get the desired concentration of active ingredient of

each fungicide. Six concentrations of each treatment were used. Three replications were

maintained for each fungicide for each of its concentration in CRD. To avoid bacterial

contamination a little amount of streptomycin was added in each flask before plating;

seven mm disc was cut with the help of sterilized cork borer from three day old culture

of the test fungus and was placed in the center of the medium in the reversed position to

maintain continuous contact of the pathogen with poisoned medium. PDA plates

without fungicide served as control.

The radial growth of the colony was measured when the growth in control plates

reached the rim of the Petri plates; per cent growth inhibition under the influence of

different fungicides was calculated on the basis of the control. Per cent inhibition of

mycelial growth was calculated using the formula (Vincent, 1927)

I = (C-T/C) x 100

Where,

I = Per cent inhibition of mycelial growth

C = Colony diameter in control (mm)

T = Colony diameter in treatment (mm)

7 Screening of popular chilli varieties against Choanephora

cucurbitarum

Screening of popular varieties (Table 3.2) against twig blight under green house

conditions were done using susceptible check (Madhuri). The ten varieties were

procured from LAM farm, Guntur. Ten replications were maintained for each treatment.

The respective cultivars were maintained in separate polythene bags in glass house and

screened for resistance to Choanephora twig blight.

Table 3.3 List of the popular chilli cultivars

The cultivars were inoculated with the spore suspension of pathogen at a

concentration of 5 x 105 spores/ ml and disease severity was calculated for each cultivar.

The disease severity was recorded by following 0 - 5 scale.

S.No Name of the

variety

1 LCA 206

2 LCA 334

3 LCA 625

4 G 4

5 LCA 353

6 G 36

7 LCA 235

8 G 3

9 CA 960

Control Madhuri

Numerical rating Disease intensity

0 No incidence

1 1-5% of mature leaves with necrotic and chlorotic symptoms

2 6-15% of mature leaves with necrotic and chlorotic symptoms

3 16-50% young shoots and stem with water soaked lesions

4 51-95% water soaked lesions with abundant mycelia growth

and fructification

5 Dead plant

Disease severity in each treatment was calculated and disease severity index was

recorded. Disease severity index was calculated using the formula:

Disease severity index = Sum of all disease ratings x 100

Total no. of ratings x maximum disease grade

The disease progress in various treatments was assessed using the disease severity scale

given by Tarig et al., (1989).

8. Screening of effective fungicide and potential biocontrol agent against

Choanephora cucurbitarum under green house conditions

The effective fungicide and potential biocontrol agent were screened for their

biocontrol potential against C. cucurbitarum in a closed polythene humid chamber.

Chilli plants were arranged in glasshouse in a randomized complete block design

(RCBD) in three replication. T1 treatment was sprayed with spore suspension (107

conidia ml-1

) of Trichoderma spp. and inoculum (5 x105

spores/ml) of C. cucurbitarum

was sprayed 1week after pathogen inoculation. Chilli plants sprayed with water alone

and chilli plants inoculated with spore suspension (5 x105

spores/ml) of C. cucurbitarum

alone served as healthy and inoculated controls. Treated plants were transferred to

polythene humid chamber with fogging devices in which temperature and humidity

were maintained at 22 + 2oC.

Similarly based on poisoned food technique studies effective fungicides which

made cent per cent mycelial inhibition were selected and sprayed on the chilli plants. In

T3 treatment Neem oil was sprayed at the rate of 3 ml/l and combinations of the above

treatments data is recorded in the following table.

Table 3.4 Details of the treatment

9. Statistical Analysis

The data obtained in different experiments was transferred using angular

transformations wherever necessary and was statistically analyzed using Completely

Randomized Block Design (CRD) as per the procedures suggested by Panse and

Sukhatme (1978).

S.No. Treatments Combinations

1 T1 Effective bio agent/ antagonist

2 T2 Effective Fungicide

3 T3 Neem oil (3 ml/l )

4 T4 T1+ T3 ( Effective bio agent/ antagonist +

Neem oil)

5 T5 T2 + T3 ( Effective Fungicide + Neem oil)

6 T6 T1+ T2 ( Effective bio agent/ antagonist +

Effective Fungicide)

7 T7 T1 +T2 + T3 ( Effective bio agent/

antagonist + Effective Fungicide +

Neem oil)

8 T8 Control

Table 3.2: Details of fungicides employed in the present investigation

Sl.

No.

Trade Name Common name Chemical name Formulation Source of

supply

1 Amistar Azoxystrobin Methyl (2E)-2-(2-{[6-(2-cyanophenoxy)pyrimidin-4-

yl]oxy}phenyl)-3-methoxyacrylate

23% SC Syngenta

2 Captaf Captan (3aR,7aS)-2-[(trichloromethyl)sulfanyl]-3a,4,7,7a-tetrahydro-

1H-isoindole-1,3(2H)-dione

50% WP Tata Rallis

3 Bavistin Carbendazim Methyl-2-benimidazole carbamate 75% WDG BASF

4 Dithane M-45 Mancozeb Manganese(2+) zinc 1,2-ethanediyldicarbamodithioate 80% WP Dow Agro

sciences

5 Blitox Copper oxy

chloride

Dicopper chloride trihydroxide 50% WDG BASF

6 Kavach Chlorothalonil 2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile 75% WDG Syngenta

7 Contaf plus Hexaconazole 2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)hexan-2-ol 5% EC Tata Rallis

3.3 Media and their composition

The following media were used in the present investigation

1. Potato Dextrose Agar (PDA)

Peeled and sliced potatoes 200.0 g

Dextrose 20.0 g

Agar 20.0 g

Distilled water 1000.0 ml

2. Dextrose nitrate medium

Dextrose 1.0 g

Potassium dihydrogen phosphate 0.1 g

Potassium chloride 0.10 g

Magnesium sulphate 0.10 g

Agar 1.5 g


4. V8 juice agar medium

V8 juice extract 8.3 g

L-aspargine 10.0 g

Calcium carbonate 2.0 g

Glucose 2.0 g

Yeast extract 2.0 g

Agar 20.0g


5. Corn meal agar

Corn meal 50.0 g

Agar 20.0 g


6. Oat meal agar

Oat meal 72.0 g

Agar 20.0 g


7. Carrot agar

Carrot agar 200.0 g 200 g

Dextrose 20.0 g

Agar 20 g


8. Water agar

Agar 20.0 g


9. Czapex dox agar

Sodium Nitrate 2.0 g

Potassium dihydrogen phosphate 1.0 g

Magnesium sulphate 0.5 g

Potassium chloride 0.5 g


Chapter IV

RESULTS AND DISCUSSION

The experimental results and discussion of various studies conducted on

Choanephora cucurbitarum (Berk and Rav.) Thaxt. of chilli (Capsicum frutescens L.)

crop are as follows:

4.1 SURVEY FOR CHILLI TWIG BLIGHT INCIDENCE

Survey was conducted in six major chilli growing districts of Telangana state

representing thirteen mandals and 38 villages during kharif 2014 for the prevalence of

Choanephora twig blight diseases in chilli and the results are presented in Table 4.1 and

Plate 4.1. For field survey, necessary data was collected from the farmers spread over in

Ranga Reddy, Karimnagar, Nizambad, Medak, Khammam and Warangal districts of

Telangana. During the survey, out of six districts of Telangana twig blight disease was

observed only in three districts viz., Warangal, Karimnagar and Khammam.

Khammam recorded maximum disease incidence varying from 20 to 32% in all

five villages of surveyed where as in other district the disease was observed only in one

village with 18 and 15% disease incidence of each out of 9 and 10 villages surveyed in

Karimnagar and Warangal districts respectively.

However there was no incidence of twig blight in Medak, Nizambad and Ranga

Reddy districts of Telangana during surveying.

Among the three districts villages in Khammam recorded more than 20% of

incidence where as in Warangal and Karimnagar disease incidence was less than 15%

and 18% respectively. The survey results indicated that the disease of economic

importance and has the potential to cause heavy yield losses.

The disease samples of twig blight of chilli were collected during survey the

infected leaves developed a grayish color similar to hot water scald or chemical injury.

The infected portion dries up and curls. Host tissues have a hairy appearance resulting

from the tall sporangiophores that produce a cluster of brown sporangiola.

During the survey the maximum incidence of twig blight was recorded in

Gudimalla village of Khammam rural mandal (32%), while least incidence was

recorded in Palathodu village of Mandapet mandal (15%) and no incidence was

recorded in the remaining districts of Telangana. It may be concluded from the above

discussion that various biotic and abiotic factors influence the incidence of twig blight.

From the above districts surveyed in Telangana the incidence of twig blight caused by

Choanephora cucurbitarum was maximum in Khammam followed by Karimnagar as

the pathogen finds the environment more conducive for survival and hence induce

disease in the host plant. The frequency of oocurence of Choanephora twig blight was

regular in Khammam which may be due to continuous raising of chilli year after year

from a long time hence it is concluded that disease was prevalent at Khammam districts.

Prabhavathy and Reddy (1995) conducted an extensive survey on post-harvest

fungal diseases of fleshy chilli fruits in various markets of Warangal district. During the

survey a total of 20 diseases caused by 24 fungal species were recorded in different

varieties of chilli in which one of the fungal pathogen recorded was Choanephora

cucurbitarum.

4.1.2 Isolation

The test pathogen Choanephora cucurbitarum was isolated from chilli twigs

which were collected from the farmers’ field during survey in Khammam district with

maximum disease incidence in the field. Twig blight lesions were surface sterilised (1%

Sodium hypo chlorite) for one minute, followed by the sterile water wash and kept for

incubation. After 48 hours fungal colonies developed and fresh mycelium transferred to

Petri plate containing PDA medium. Pathogen was purified by single spore techniques

and pathogen culture was multiplied and maintained on PDA at 25 ± 2oC in BOD

incubator.

4.1.3 Identification of Choanephora cucurbitarum

The test fungus was isolated from infected twigs and leaves on potato dextrose

agar medium and was identified as C. cucurbitarum with the help of descriptions given

by Wolf (1917). The fungal colony appeared white to pale yellow on PDA plates. The

white coloured mycelium on maturity produced black pin heads indicating onset of

sporulation. The cultural characters were in accordance with the descriptions given by

Saroj et al. (2012).

The mycelia was hyaline, unbranched and without any septations.

Sporangiophores were non-septate, hyaline and smooth walled. Two types of asexual

structures were produced drooping sporangia and monosporous sporangiola. The

drooping multisporous sporangia were subglobose in shape and 90.15 - 100 μm in size.

The sporangia were non columellate and dehisce into two half releasing the spores.


color and are characterized by longitudinal striatures on the wall surface and 12-19 x

7.23-9.5 μm by size. The sporangiophore (conidiophore) from which the monosporous

sporangiola arose was long slender, branched at the apex with primary vesicle from

which secondary vesicles were produced on the stalks which bears sporangiospores

(conidia). Monosporous sporangiola were elliptical, fusiform or ovoid, striate and

measured 11-19 × 4-12 μm. Zygospores of C. cucurbitarum appear not to have been

observed prior to the present study. The mycelial and morphological characters were

similar to that described by Wolf (1917). The fungus grew well between 25oC to 30

oC

temperatures. On the basis of microscopic examination of the fungus, the morphology

was identical to that of Choanephora cucurbitarum (Berk and Rav.) Thaxt. (Agrios,

(1997), Abel-Motaal et al. (2010), Singh et al. (2012) and Kwon et al. (2001).

Similar observations were made by Kuo et al. (1999) in C. cucurbitarum. The

colonies of the causal fungus grown on potato dextrose agar at 25oC are white at the

early stage of growth with abundant aerial mycelium, but at later growth stages turn

pale yellow. Sporangia and sporangiola are readily observed on the tip of upright

sporangiophores, which are numerous on aerial mycelium. Sporangia spherical in shape,

25-124 µm in diameter, white at first but turning dark brown at maturity. Sporangiola,

containing only one spore, indehiscent, ellipsoid to broadly ellipsoid, 11-13 x 13-20 µm

in size, subtended by a short cylindrical pedicel. Sporangiospores, ellipsoid to broadly

ellipsoid in shape, brown to pale brown, 8-12 x 20-24 µm in size are characterized by

longitudinal striatures on the wall surface and several fine hyaline appendages at both

poles.

Similar results were also reported by Kwon and Jee (2005) the fungal colonies on

PDA were white to pale yellowish brown abundant monosporous sporangiola were

radially formed on the medium. Sporangia were subglobose in shape and 40~130 µm in

size. Monosporous sporangiophore was long slender and branched at the apex and each

branch beared a head of sporangiospores. Monosporous sporangiola were elliptical,

fusiform or ovoid, pediculate, striate and measured 12~20 × 6~14 µm. Sporangiospores

attached three or more appendages. Sporangiospores were elliptic, fusiform or ovoid in

shape, light brown or dark brown in color and sized 14~22 × 7~10 µm. Zygospores

were not observed in this study.

4.1.4 Pathogenicity

Pathogenicity test was conducted selecting suspectible chilli cv. Madhuri. The

test plants were grown in pots. The spore suspension of the isolate was prepared in

sterile water from 3 days old culture grown on PDA. The spore suspension was diluted

https://ovidsp.tx.ovid.com/sp-3.14.0b/ovidweb.cgi?&S=AFGKFPKBLBDDBBGBNCLKPFJCPOILAA00&Search+Link=%22Kuo%2c+C+H%22.au.

with sterile distilled water in such a way that it contains 5 × 105

spores/ml. The chilli

plants of 120-135 DAS were used for inoculation. The plants were covered with

suspension was sprayed with the hand-sprayer on the plants. Control plants were

sprayed with the same volume of sterile distilled water. All the plants were immediately

covered with polythene bags to maintain high humidity and kept undisturbed as such for

24 hr. Periodical observations on disease development were made. Seven days after

inoculation, the plants were observed for the disease development.

The initial symptoms produced in the form of water soaked or scald lesions on

the leaf surface later these lesions appeared grayish or gray to brown color and after 5

days leaves become totally blighted and curled.

On upper surface of infected leaf tissue creamy white spine like fungus mycelia

and black spore masses was consistently present. The same pathogen of C.

cucurbitarum was re-isolated from the infected leaf. No symptoms were observed in

control. The Koch postulates was proved and pathogenicity of C .cucurbitarum on chilli

was confirmed.

Yasmin Ahmad et al. (1988) similarly performed pathogenicity tests by spray

inoculation (5,000 spores per ml) on plants each with and without wounding made by

lightly scratching the leaf surface with a needle. Plants were placed in the greenhouse

with temperatures ranging from 21 to 26oC and symptom development was observed as

early as 3 days after inoculation.

In the present study, the fungus was inoculated into leaves and stem of the test

plants and the symptoms were observed on the both the plant parts.


The initial symptom of disease was appeared in leaves after 120 days of sowing

(at flowering stage) at distal halves of infected leaves. Symptoms developed as a grayish

color similar to hot water scald or chemical injury.

The infected portion become dried and curled upward. Host tissues have a hairy

appearance resulting from the tall sporangiophores that produce a cluster of brown

sporangiola at their tips.

Water soaked lesions appear on the leaves and the margins, leaf tips gets

blighted. The appearance of a stiff silvery mass of whisker-like or hairy strands of the

fungus growing out of the affected pepper tissue, topped with a black ball made of great

numbers of spores.

Sporangiophores and sporangia observed on the infected portion of the leaves.

Under high relative humidity (>90%) conditions cause a heavy or severe defoliation,

while under low relative humidity condition infected portion dropped off without

sporulations leaving the uninfected portion intact.

Similarly Sinclair (1984) reported that the distal halves of infected leaves first

develop a grayish color similar to hot water scald or chemical injury. The infected

portion dries up and curls. Sporangiophores and sporangia develop on the damaged

portion. If relative humidity is high, severe defoliation occurs. If humidity is low, only

the areas showing symptoms drop off, leaving the unaffected portion intact. When

defoliation is severe, plants and seeds are smaller than normal.

Similar observations were also made by Abhishek and Pooja (2014) water

soaked lesions appear on the leaves and the margins, leaf tips get blighted. The disease

causes severe destruction in rainy season in tropical climates. Initial symptoms were

associated with flowers, buds or apical growing points and later fungus grows

downward killing the plant. Wet rot develops on the stem which appeared as wet and

green. The bark easily peels off in shreds.

Black (2001) first observed symptoms of Choanephora rot on cucurbit which

first attacks the blossoms and progresses into the developing fruit causing a wet rot at

the blossom end. Sporulation by the fungus appears as spines with dark heads on the

surface of infected tissues.

4.2 Physiological studies

4.2.1 Effect of different media on radial growth of Choanephora cucurbitarum

The data presented in Table 4.2 and Fig 4.1 indicates that the radial growth of

Choanephora cucurbitarum differed significantly with different types of media used.

The cultural studies of Choanephora cucurbitarum were carried on eight different

media. Among them maximum radial growth (90 mm) of fungus was observed on both

PDA and carrot agar media followed by V8 juice agar media and czapex dox agar

which recorded almost similar radial growth of 87.67 mm and 87.59 mm respectively,

whereas, minimum radial growth was observed on oat meal agar medium with 33.50

mm. On the other hand, water agar, dextrose nitrate agar and corn meal agar medium

recorded radial growth of 82.67 mm, 78.17 mm and 75.17 mm respectively at two days

after incubation (Plate 4.4).

Our results showed that PDA and carrot agar media were most suitable media

for the growth of Choanephora cucurbitarum. Among these two media PDA was found

highly suitable with maximum radial growth of 58.83 mm followed by carrot agar

media (54.67 mm) after 24 hrs of incubation. The present finding is in agreement with

of Bandopadhya and Som (1984) who reported that Choanephora cucurbitarum grows

very fast on PDA at 28oC then other media.

Our findings are similar to the reports of Iroakazi (1984), Ikediugwi (1981),

Awuah (1989) and Kwon and Park (2002) reported that PDA was most suitable medium

for sporulation and germination of C. cucurbitarum.

Kuo et al. (1999) also reported that potato dextrose medium and 10% V8

medium was most favourable medium for C. cucurbitarum causing wet rot of lima

bean. In the present investigation PDA and carrot agar recorded maximum per cent of

radial growth after 2 DAI followed by V8 juice agar medium.

4.2.3 Effect of different pH on radial growth of Choanephora cucurbitarum

The effect of different pH levels on radial growth of C. cucurbitarum was

studied by using PDA medium and the data are presented in Table 4.4 and Fig 4.2. The

radial growth of C. cucurbitarum was recorded at 24hrs and 48hrs after incubation to

observe the effect of different pH levels. Among the different pH levels maximum

radial growth (90 mm) was recorded by seven pH levels varying from pH 4.7 (74.3 mm)

to pH 7.5 (64.4 mm) after 48 hrs of incubation followed by pH 8.5 which recorded

radial growth of about 81.5 mm and is onpar with pH 4.0 with a radial growth of 80.9

mm where as pH 8.0 recorded radial growth of 79.5. However low radial growth of 29.9

mm was recorded by pH 9.0 after 48hrs (Plate 4.5).

Though the maximum radial growth recorded from pH 4.5 to pH 7.5. However the

mean radial growth varied at different pH levels. In the present study it was observed

that the test fungus C. cucurbitarum was adapted to wide range of pH levels.

The maximum radial growth of C. cucurbitarum was found to be pH 4.5 followed

by pH 5.0 while minimum radial growth was recorded at pH 9.0.

Our results are highly supported with Holcomb (1998) who reported maximum

growth in acidified potato dextrose agar medium. Similarly Ojionuka (1988) also found

that maximum radial growth occurred at pH level 5.0. The radial growth at pH 5.0 was

significantly greater when compared to pH levels of 4.5, 5.5, 6.0 and 6.5.

4.2.4 Effect of different temperature on radial growth of Choanephora

cucurbitarum

The effect of temperature on growth of C. cucurbitarum was tested on PDA

medium under in vitro conditions. The data presented in Table 4.5 and Fig 4.3 indicated

that the radial growth of C. cucurbitarum varied significantly when it was grown at

different temperatures. The radial growth was significantly higher by 90 mm at 20oC

and 25oC followed 73.9 mm at 30

oC after 2 DAI respectively. Mycelial growth was not

observed at 0, 10 and 15oC and slowed at 40

oC. In the present study, the temperature of

20oC to 25

oC was most suitable for growth of C. cucurbitarum (Plate 4.6).

Similarly Kang et al. (1993) studied the effect of temperature on Choanephora

cucurbitarum and found optimum temperature for mycelial growth between 25-30oC,

while conidial germination and appresorium formation were best at 30oC. In the present

investigation the growth of C. cucurbitarum was maximum at 20oC and 25

oC and the

growth was also high at 30 oC when compared to other treatments.

Kwon and Park (2002) reported that the maximum mycelial growth was on PDA

at 28oC. Similarly Naito et al. (1989) also observed maximum mycelial growth of

Choanephora cucurbitarum on nutrient agar at 30oC.

4.3 Identification of fungal bio control agents from rhizosphere

The antagonistic activity of isolated microorganisms from rhizosphere of chilli

leaves was tested against Choanephora cucurbitarum by dual culture technique. Four

isolates of fungal bio control agent particularly Trichoderma spp., were isolated from

the rhizosphere of chilli and the morphological characters were studied and the isolates

were identified based on the key characteristics provided by Rifai (1969) (Plate 4.7).

The four isolates of Trichoderma spp were used to test antagonistic performance

in dual culture with a test pathogen C. cucurbitarum. The data on the inhibition of

S.No. Trichoderma isolate Characteristics of the isolate

1 Trichoderma viride isolate- 1 Plain green colony with distinct regular

growth and profuse sporulation

2 Trichoderma viride isolate- 2 Slightly fluffy growth, light green colony

with profuse sporulation.

3 Trichoderma harzianum isolate-1 Fluffy growth, colour greenish yellow, later

turns yellowish green.

4 Trichoderma harzianum isolate-2 Fluffy with sparsely rhythmic growth, yellow

colony, profuse sporulation.

growth of C. cucurbitarum by Trichoderma isolates are presented in Table 4.6 and Fig

4.4.

The data on dual culture test by Trichoderma spp., revealed that all the

Trichoderma isolates inhibited radial growth of Choanephora cucurbitarum, but there

was a variation in their inhibition. All isolates exhibited more than 40.11% inhibition of

mycelial radial growth of Choanepora cucurbitarum. However, Trichoderma viride

isolate-1 (62.44%) showed maximum per cent inhibition of test pathogen followed by

Trichoderma viride isolate-2 (62.22%), Trichoderma harzianum isolate-2 (55.33%) and

Trichoderma harzianum isolate-1 (40.11%).

Kamlesh et al. (2002) reported that fungal biocontrol agents Trichoderma viride,

Trichoderma harzianum and two isolates of Fusarium solani isolated from rhizosphere

of chilli significantly inhibited the mycelia growth of Rhizoctonia solani to an extent of

68.6 and 61.0 mm, respectively and reduced sclerotial productions of Rhizoctonia solani

causing stem rot of chilli.

4.3.1 Identification of fungal biocontrol agents from phylloplane

The antagonistic activity of isolated microorganisms from phylloplane of chilli

was tested against C. cucurbitarum by dual culture technique. About seven fungi were

isolated from the phylloplane. The data on the inhibition of growth of C. cucurbitarum

by fungal biocontrol agents isolates are presented in Table 4.7 and Fig 4.5.

The data revealed that all the fungal isolates inhibited radial growth of C.

cucurbitarum, but varied in their efficacy. The fungal isolates isolated from phylloplane

of chilli leaves significantly inhibited radial growth of C. cucurbitarum vary from

57.77% (Fusarium sp) to 47.00% (Alternaria sp) (Plate 4.8a and Plate 4.8b). The fungal

isolates of Aspergillus genera such as Aspergillus niger and Aspergillus flavus showed

more than 56.00% of inhibition where as Mucor, Rhizopus which belongs to same

taxonomic position of Choanephora showed nearly similar per cent inhibition (53.3,

53.66) respectively. Penicillium sp and Paecilomyces sp recorded similar per cent of

inhibition (51.88, 52.2).

Singh and Sinha (1962) reported that Alternaria, Aspergillus, Cladosporium,

Curvularia, Helminthosporium, Heterosporium, Mucor, Penicillium, Rhizopus and

Trichoderma were isolated from blighted chilli leaves.

4.3.3 Evaluation of fungicides at different concentration on inhibition of radial

growth of Choanephora cucurbitarum

The sensitivity of different fungicides against C. cucurbitarum was tested in-

vitro by poisoned food technique at different concentrations (0.05, 0.1, 0.15, 0.2, 0.25

and 0.3%) and the data are presented in Table 4.8.

Most of the tested fungicides significantly inhibited the mycelial growth of C.

cucurbitarum over control.

The present results revealed that none of the test fungicide showed 100%

inhibition at all the tested concentrations but with the increasing in concentration of

fungicides there was a decrease in radial growth of C. cucurbitarum and per cent

inhibition over control was increased.

Out of seven fungicides tested five fungicides significantly recorded inhibition

over control. However the fungicides azoxystrobin and captan recorded maximum

(100%) inhibiton at 0.15% concentration followed by chlorothalonil (96.2%),

hexaconazole (90.2%), mancozeb (86.8%) and copper oxychloride with 86.6 per cent at

0.15 per cent concentration. The fungicide chlorothalonil, hexaconazole and mancozeb

recorded maximum (100%) inhibition at 0.2% concentration while copper oxychloride

at 0.3%. The fungicide carbendazim did not record any inhibition at all concentrations

tested (Plate 4.9).

Captan was found most effective in inhibiting germination of the test

fungus. Oladiran (1980) reported that captan was most effective fungicide against C.

cucurbitarum causing pod blight of cowpea. In the present study the captan recorded

maximum inhibition of test fungus. Panja (1999) also reported that captan 50% (2.0 g/l),

ziram 27% (1.5 ml/l) and copper oxy chloride 50% (3 gm/l) was most effective in

inhibition of radial growth and sporulation of C. cucurbitarum at different

concentrations tested. The present findings report that captan was effective against C.

cucurbitarum. Jana et al. (2011) reported that copper oxychloride + metalaxyl

formulation was most effective in controlling the twig blight of chilli and bell pepper

followed by copper oxychloride and ziram, while carbendazim was not effective against

C. cucurbitarum. In the present investigation, carbendazim was found least effective in

inhibiting the test fungus.

4.4 Varietal screening against Choanephora cucurbitarum

Resistant varieties offer one of the best and safe strategies for managing the

diseases. Identification of resistant sources and inheritance are considered important

factors in determining the breeding methodology to be adopted for incorporating

resistance. Thus varietal screening plays a significant important role in integrated

disease management. Popular cultivars of chilli were screened against test pathogen

under glass house conditions using susceptible check (Madhuri). The following varieties

were procured from Guntur LAM, LCA-206, LCA-334, LCA-353, LCA-235, LCA-625,

CA-960, G36, G4 and G3. The respective cultivars were maintained in separate

polythene bags in glass house and screened for resistance to Choanephora twig blight.

The cultivars were inoculated with the spore suspension of pathogen at a concentration

of 5 x 105 spores/ml and disease severity was recorded after 15 DAI and per cent

disease index was calculated for each cultivar and the data is presented in the Table 4.9

and Fig 4.6.

Disease severity index was used as an indicator of the varietal screening against

the test pathogen. Low disease severity indicated the presence of disease suppression.

Disease severity was significantly higher in the control plants, Madhuri (85.3%). A

varied level of resistance was observed against the test pathogen C. cucurbitarum.

Screening of nine varieties with C. cucurbitarum showed the per cent disease index

ranging from 24% to 75%. Among the ten varieties screened, the variety LCA-235

recorded highest per cent disease severity index of 76 followed by G 36 (72), LCA-206

(50), LCA-625 (50), LCA-353 (48), G-3 (46) and CA-960 (32) while low per cent severity

index was recorded in LCA- 334 (27) and G-4 (24) (Plate 4.10). As per the data it is

concluded that G-4 and LCA-334 are resistant to twig blight disease. There is no

information on varietal resistance for chilli twig blight disease. However, varied level of

resistance was recorded for C. cucurbitarum on other crops.

In Ibadan, Nigeria, nine cultivars of okra were screened for resistance to

premature fruit abortion caused by C. cucurbitarum during two seasons over 2 years. A

range of resistance/susceptibility was reported from 24 to 73% abortion. The most

resistant cultivar was NHAe 621 (Adebanjo and Dede, 1985).

Mamta Joshi et al. (2012) screened thirty varities of chilli for evaluation of

resistant varieties. Among thirty varieties screened, only two varieties CO-4 and DLC-

352 were found 100% resistant, while five varieties, viz., Ajeet-3, DLC-524, F-112-5-

83, KCS-2013, Hot pepper Nun-2060 were moderately resistant (83.34%) and Sel 11

was most susceptible variety against wilt of chilli induced by pathogenic F. oxysporum.

Adegite and Amusa (2008) screened 71 cowpea lines against the disease

Choanephora pod rot of lima bean and found that 80% lines were susceptible to the

disease. Similarly in the present investigation most of the varieties recorded more than

30% disease index.

4.5 Management of twig blight disease with effective fungicide and potential

biocontrol agent under greenhouse conditions

The most potential biocontrol agent and effective fungicide from the earlier

experiment were further screened against twig blight disease under glass house

conditions along with neem oil and total seven treatments were used with different

combinations. Chilli plants were arranged on a greenhouse bench in a randomized

complete block design (RCBD) and replicated thrice. The effective fungicide captan (2

gm/lit), spore suspension (107

conidia ml-1

) of Trichoderma spp. and inoculum (5 x105

spores/ml) of C. cucurbitarum was sprayed 1week after pathogen inoculation.

Chilli plants spray inoculated with spore suspension of C. cucurbitarum alone

served as inoculated controls. Treated plants were transferred to polythene humid

chamber with fogging devices in which temperature and humidity were maintained at

22 + 2oC and relative humidity at 90%. Disease severity index was calculated for each

treatment and indicator of the effectiveness of the fungicide and potential biocontrol

agent and data on disease index was recorded and presented in Table 4.10 and Fig 4.7.

The data revealed that all the treatments were significant in reducing the per cent

disease index over control. Treatment T2 (Effective fungicide captan @ 0.15%) was

highly significant in reducing the disease incidence by 66.47 per cent disease over

control (85.3) while least at T3 i.e. Neem oil with a 13.5 per cent disease over control.

Among the individual Treatments captan was found effective when compared to

control (85.3%). Among the integrated Treatments, T7 (T. viride isolate 1+ captan +

Neem oil) recorded low disease severity index of 28.9 followed by T6 (T. viride isolate

1+ captan) and T5 (captan + Neem oil) with 28.9, 29.1 and 30.1 respectively (Plate

4.11).

In the present experiment T.viride isolate 1 recorded the disease index of 39.7 at

per cent disease over control of 53.45 while other integrated treatments (T4 , T6 and T7 )

recorde per cent disease of control 26.26, 65.88 and 66.11 respectively. It was observed

that, captan alone or integrated with other treatments viz., Trichoderma viride isolate 1

and neem oil was found effective.

Farzana Banu et al. (1990) reported that captan and thiram were able to reduce

the incidence of Colletotrichum dematium and increase the germination ability of chilli

seed. They also reported that seed treatment with biocontrol agents, Trichoderma

harzianum and T. viride and hot water treatment at 50oC also reduce seed borne

incidence of C. dematium.

Raju et al.(1982) found that captan was highly effective against C.

cucurbitarum. Panja (1999) also reported that captan (2.0 gm/lit) was most effective in

inhibiting the twig blight pathogen in chilli.

Krishna Reddy (2001) reported that Choanephora twig blight can be managed

by collection and destruction of infected plant parts. He also reported that spraying

copper oxy chloride @ 3 gm + 0.1 gm streptocyclin per litre of water with NSKE @ 5%

was effective.

Balogun and Babatola (1999) studied the effect of plant age and injury on the

pathogenicity of Choanephora cucurbitarum in okra. Inoculation at planting, seedling

emergence or 10 days after planting caused the leaf infection significantly earlier than

inoculations made at later stages of growth. Field evaluation of effective plant extracts

and antagonists and fungicide, revealed that spraying with T. viride (2%) showed a

maximum disease reduction of 61.41% followed by P. fluorescens (58.10%). However,

the fungicide ziram (0.25%) with 80.84% disease reduction ranked first. In the present

study, fungal biocontrol agents was not effective when compared to captan. The

Trichoderma isolate 1 recorded low (39.7%) per cent disease when compared to

fungicide. This may be due to variation in isolate.

Table 4.1 Survey of Choanephora twig blight in chilli

Name of the district Name of the

Mandal Name of the village

Sample

number

Twig blight

incidence (%)

Ranga Reddy Chevella

Urella

Shankarpally

Deverierra Pally

Antharam

Hasthepur

1

2

3

4

5

0.0

0.0

0.0

0.0

0.0

Vikarabad

Kamareddiguda

Kompalle

Pathur

Phulmaddi

Pendlimadgu

6

7

8

9

10

0.0

0.0

0.0

0.0

0.0

Medak Zahirabad

Shekarpur

Hothidi

11

12

0.0

0.0

Karimnagar Malharrao

Nacharam

Mathkupalle

Dubbapeta

13

14

15

0.0

0.0

0.0

Mahadevpur

Edapalle

Elkeswaram

Kamanpalle

16

17

18

0.0

0.0

0.0

Manthini

Eklaspur

Maidipalle

Mallaram

19

20

21

18.0

0.0

0.0

Warangal Mahabubabad

Laxmipur

Ammangal

Anantharam

Pendyal

Nandipalle

22

23

24

25

26

0.0

0.0

0.0

0.0

0.0

Mandapet

Palathodu

Yeditha

Ippanapadu

27

28

29

15.0

0.0

0.0

Kuravi

Nerada

Narayanpur

30

31

0.0

0.0

Nizambad Sirikonda

Gadkole

32

0.0

Nandipet

Ailapur

33

0.0

Khammam Garla

Chinnakistappu

Siripuram

34

35

23.0

20.0

Khammam rural Gudimalla

Gollapadu

Chintapalli

36

37

38

32.0

25.0

25.0

Table 4.2: Radial growth of Choanephora cucurbitarum on different media

Sl.No.

Medium

Radial growth (mm)

1 DAI 2 DAI

1 Oat meal agar 1.20 33.50

2 Dextrose nitrate agar 16.97 78.17

3 V8 Juice agar 55.67 87.67

4 Carrot agar 54.67 90.0

5 Water agar 33.67 82.67

6 Corn meal agar 49.33 75.17

7 Czapek dox agar 47.67 87.59

8 PDA 58.83 90.0

CD (P=0.05) 1.71 2.41

SE (m) 0.56 0.79

DAI: Days after inoculation

Table 4.7: Evaluation of phylloplane in inhibiting the radial growth of Choanephora

cucurbitarum in vitro


Sl. No. Treatment Radial growth of C.

cucurbitarum (mm)

Per cent inhibition

of mycelia growth

over control (%)

1 Paecilomyces sp 43.0 52.22

2 Aspergillus niger 39.5 56.11

3 Penicillium sp 43.3 51.88

4 Aspergillus flavus 39.6 56.00

5 Fusarium sp 38.0 57.77

6 Alternaria sp 47.7 47.00

7 Mucor sp 42.0 53.33

8 Rhizopus sp 41.7 53.66

9 Control 90.0 0.00

CD (P=0.05) 1.41

SE (m) 0.57

Table 4.6: Effect of bioagents on radial growth of Choanephora cucurbitarum in vitro

Sl. No.

Treatment Radial growth

(mm)

Per cent inhibition

of mycelial growth

over control

1 Trichoderma viride isolate 1 33.8

62.44

2 Trichoderma viride isolate 2 34.0 62.22

3 Trichoderma harzianum isolate 1 53.9 40.11

4 Trichoderma harzianum isolate 2 40.2 55.33

5 Control 90.0 0.00

CD (P=0.05) 1.47

SE (m) 0.47


Table 4.9: Screening of popular chilli varieties against twig blight caused by



S.No. Name of the variety Disease severity

Index (%)

1 LCA 206 50

2 LCA 334 27

3 G4 24

4 LCA 625 50

5 G3 46

6. CA 960 32

7 LCA 353 48

8 G 36 72

9 LCA 235 76

Control Madhuri 85.3

CD (P=0.05) 3.28

SE (m) 1.34

Table 4.10: Management of twig blight of chilli caused by Choanephora

cucurbitarum by using effective fungicide, potential biocontrol agent

and neem oil

S.No. Treatment Combinations

Per cent

disease

index

Per cent

disease

control

1 T1 Trichoderma viride isolate 1 39.7 53.45

2 T2 Captan 28.6 66.47

3 T3 Neem oil 73.4 13.95

4 T4 T1 +T3 (Trichoderma viride

isolate 1+ Neem oil) 62.9 26.26

5 T5 T2+T3 (Captan + Neem oil) 30.1 64.71

6 T6 T1+ T2 (Trichoderma viride

isolate 1+ captan) 29.1 65.88

7 T7

T1 +T2 + T3 (Trichoderma

viride isolate 1 + captan +

Neem oil)

28.9 66.11

8 T8 Control 85.3 0

CD (P=0.05) 3.27

SE (m) 1.28


Table 4.5: Effect of temperature on radial growth of Choanephora

cucurbitarum


S.N0. Temperature ( o

C ) Radial growth (mm)

1 DAI

2 DAI

1 0 o

C 0 0

2 10 o

C 0 0

3 15 o

C 0 0

4 20 o

C 50.1 90.0

5 25 o

C 58.9 90.0

6 30 o

C 69.3 83.9

7 35 o

C 29.8 67.8

8 40 o

C 19.8 27.3

CD (P=0.05) 1.66 1.53

SE (m) 0.55 0.35

Table 4.4: Effect of different pH on radial growth of Choanephora

cucurbitarum


S.NO pH

Radial growth (mm)

1 DAI 2 DAI

1 4.0 35.5 80.9

2 4.5 63.8 90.0

3 5.0 68.5 90.0

4 5.5 69.8 90.0

5 6.0 71.3 90.0

6 6.5 74.3 90.0

7 7.0 60.9 90.0

8 7.5 64.4 90.0

9 8.0 52.8 79.5

10 8.5 35.6 81.5

11 9.0 13.9 29.9

CD (P=0.05) 2.24 1.70

SE (m) 0.75 0.57

Table 4.3: Morphological growth characteristics of C. cucurbitarum on different culture media

S. No.

Medium

Colony characters

Colony colour

Growth pattern

1

Oat meal agar White Irregular growth with pluffy mycelium at inoculation

point without sporulation at colony edges

2

Dextrose nitrate agar White Sparse mycelial growth at the center with irregular

margins without sporulation

3

V8 Juice agar Initially white to dirty white

in colour and later turned to

cream colour

Pluffy mycelium at the center with sporulation

4

Carrot agar

Whitish at the center and

creamish yellow at the

periphery

Pluffy mycelium at the inoculation point with sparse

growth at the periphery without sporulation

5

Water agar Whitish at the center and

creamish yellow at the

periphery

Round pluffy mycelium at the inoculation point with

sparse growth at the periphery without sporulation

6

Corn meal agar White Sparse mycelial growth at the center without

sporulation

7 Czapek dox agar Initially white to dirty white

in colour and later turned to

cream colour

Pluffy mycelium at the center with sporulation

8 PDA White Round pluffy mycelium at the inoculation point and at

periphery with sporulation

Table 4.8: Evaluation of fungicides at different concentrations in inhibiting the mycelia growth of Choanephora cucurbitarum in vitro

Sl.No Fungicide 0.05 0.1 0.15 0.2 0.25 0.3

Radial

growth

(mm)

Inhib

ition

(%)

Radial

growth

(mm)

Inhib

ition

%

Radial

growth

(mm)

Inhib

ition

%

Radial

growth

(mm)

Inhib

ition

%

Radial

growth

(mm)

Inhib

ition

(%)

Radial

growth

(mm)

Inhib

ition

(%)

1 Azoxystrobin 18.3 79.6 11.4 87.3 0.0 100 0.0 100 0.0 100 0.0 100

2 Carbendazim 90.0 0.0 90.0 0.0 90.0 0.0 90.0 0.0 90.0 0.0 90.0 0.0

3 Captan 20.6 77.1 18.5 79.4 0.0 100 0.0 100 0.0 100 0.0 100

4 Copper oxychloride 18.7 79.2 15.9 82.3 12.0 86.6 10.3 88.5 9.7 80.3 0.0 100

5 Chlorothalonil 15.6 82.6 11.0 82.6 3.4 96.2 0.0 100 0.0 100 0.0 100

6 Hexaconazole 17.2 80.8 14.7 83.6 8.8 90.2 4.2 95.3 0.0 100 0.0 100

7 Mancozeb 15.7 82.5 13.6 84.8 11.8 86.8 2.9 96.7 0.0 100 0.0 100

8 Control 90.0 0.0 90.0 0.0 90.0 0.0 90.0 0.0 90.0 0.0 90.0 0.0

CD (P=0.05) 0.521 0.085 0.511 0.276 0.428 0.232

SE(m) 0.188 0.027 0.164 0.994 0.148 0.107

Fig 4.1: Radial growth of Choanephora cucurbitarum on different media

T1 - Oat meal agar

T2 - Dextrose nitrate agar

T3- V8 Juice agar

T4 - Carrot agar

T5 - Water agar

T6 - Corn meal agar

T7 - Czapek dox agar

T8 - PDA

0

10

20

30

40

50

60

70

80

90

T1 T2 T3 T4 T5 T6 T7 T8

Per

cen

t ra

dia

l gro

wth

Different media

Fig 4.6: Screening of different popular varieties of chilli against twig blight of caused by


T1 - LCA 206

T2 - LCA 334

T3 - G4

T4 - LCA 625

T5 - G3

T6 - CA 960

T7 - LCA 353

T8 - G 36

T9 - LCA 235

Control - Madhuri

0

10

20

30

40

50

60

70

80

90

LCA 206 LCA 334 G4 LCA 625 G3 CA 960 LCA 353 G 36 LCA 235 Madhuri

Dis

ease

sev

erit

y i

nd

ex (

%)

Treatments

Fig 4.7: Evaluation of effective fungicide and potential biocontrol agent in reducing twig

blight of chilli caused by Choanephora cucurbitarum

T1 - Trichoderma viride isolate 1

T2 - Captan

T3 - Neem oil

T4 - T1 +T3 (Trichoderma viride isolate 1+ Neem oil)

T5 - T2+T3 (Captan + Neem oil)

T6 - T1+ T2 (Trichoderma viride isolate 1+ captan)

T7 - T1 +T2 + T3 (Trichoderma viride isolate 1+ captan + Neem oil)

T8 – Control (Madhuri)

0

10

20

30

40

50

60

70

80

90

T1 T2 T3 T4 T5 T6 T7 T8

Per

cen

t D

isea

se I

nd

ex

Treatments

Fig 4.5: Effect of bioagents on radial growth of Choanephora cucurbitarum in vitro



T3 - Trichoderma harzianum isolate 1

T4 - Trichoderma harzianum isolate 2

T5 - Control

0

10

20

30

40

50

60

70

T1 T2 T3 T4 T5

Per

cen

t in

hib

itio

n (

%)

Treatments

Fig 4.2: Effect of different pH on radial growth of Choanephora cucurbitarum

0

10

20

30

40

50

60

70

80

90

4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9

Ra

dia

l g

row

th (

mm

)

Different pH

Fig 4.5: In vitro evaluation of phylloplane in inhibiting the radial growth of Choanephora

cucurbitarum

T1 - Paecilomyces sp

T2 - Aspergillus niger

T3 - Penicillium sp

T4 - Aspergillus flavus

T5 - Fusarium sp

T6 - Alternaria sp

T7 - Mucor sp

T8 - Rhizopus sp

T9 - Control

0

10

20

30

40

50

60

T1 T2 T3 T4 T5 T6 T7 T8 T9

Per

cen

t in

hib

itio

n (

%)

Treatments

Fig 4.3: Effect of temperature on radial growth of Choanephora cucurbitarum

0

10

20

30

40

50

60

70

80

90

0 10 15 20 25 30 35 40

Per

cen

t in

hib

itio

n (

%)

Temperature

Medak

Plate 4.1 Map showing different districts of Telangana to know the occurrence

of twig blight disease on chilli

Plate 3.1: (a) Typical symptoms of twig blight disease on chilli leaves and twigs

during survey

(b) Chilli twigs with sporangia and sporangiophore of C. cucurbitarum

Plate 3.2: (a) Symptoms of twig blight disease on chilli leaves, twigs and fruits

(b) Isolation of Choanephora cucurbitarum on PDA medium

Plate 4.2: Pure culture of Choanephora cucurbitarum on potato dextrose medium

Plate 4.3a: Photomicrograph showing sporangia and sporangiophores of

Choanephora cucurbitarum (10X)

Plate 4.3b: Photomicrograph showing sporangial hyphae of Choanephora

cucurbitarum (40X)

Plate 4.3c: Photomicrograph showing fertile heads of Choanephora cucurbitarum

(40X)

Plate 4.3d: Photomicrograph showing sporangiospores of Choanephora

cucurbitarum (40X)

Longitudinal

striations

Plate 4.5: Effect of different pH on Choanephora cucurbitarum

Plate 4.4: Growth of Choanephora cucurbitarum on different media

1 Oat meal agar

2 Dextrose nitrate agar

3 V8 Juice agar

4 Carrot agar

5 Water agar

6 Corn meal agar

7 Czapek dox agar

8 PDA

Plate 4.6: Effect of different temperature on radial growth of Choanephora cucurbitarum

Plate 4.9: Effect of different fungicides on the radial growth of Choanephora cucurbitarum

at different concentrations

1 Azoxystrobin

2 Carbendazim

3 Captan

4 Copper oxychloride

5 Chlorothalonil

6 Hexaconazole

7 Mancozeb

8 Control

Plate 4.7: Pure culture of different Trichoderma spp.

1 Trichoderma viride isolate 1


3 Trichoderma harzianum isolate 1


Plate 4.7a: Antagonistic activity of Trichoderma spp. against Choanephora

cucurbitarum





Plate 4.7b: Photomicrograph showing Trichoderma viride isolate-1

Plate 4.7c: Photomicrograph showing Trichoderma harzianum isolate-2

Plate 4.8a: Photomicrograph of Fusarium spp. isolated from chilli leaves

Plate 4.8b: Photomicrograph of Aspergillus niger isolated from chilli leaves

Plate 4.10: Different degrees of twig blight severity chilli leaves by isolates of Choanephora

cucurbitarum showing white mycelium

Treatment T2 Treatment T1

Plate 4.11: Screening of effective fungicide and biocontrol agent against Choanephora

cucurbitarum

CHAPTER V

SUMMARY AND CONCLUSION

The findings of the investigation on “Studies on Choanephora twig blight

(Choanephora cucurbitarum (Berk and Rav.) Thaxt. of Chilli (Capsicum

frutescens) and its management” are summarized below:

Survey was conducted in six major chilli growing districts of Telangana state

representing thirteen mandals and 38 villages during kharif 2014 for the prevalence of

Choanephora twig blight disease in chilli. During the survey, out of six districts of

Telangana maximum twig blight disease incidence was recorded in Khammam

followed by Karimnagar and Warangal where as no disease incidence was recorded in

Ranga Reddy, Medak and Nizambad.

The disease incidence of twig blight in chilli ranged from 15.0 to 32.0%. Highest

incidence of twig blight disease was recorded in Gudimalla village (32.0%) of

Khammam district while the lowest was observed in Palathodu (15.0%) village of

Warangal district.

The symptoms of Choanephora twig blight of chilli were observed under natural

field condition on leaves, stem, petioles and fruits during survey. Isolation was done

from the plant parts and showed presence of Choanephora cucurbitarum which was

identified on the basis of its morphological characters.

To identify the pathogen, its cultural and morphological characters were studied

on different media. The mycelium was irregularly branched, coenocytic when young

and filamentous with full granular protoplasm. In older cultures, the mycelium was

filamentous, hyaline and aseptate and was found to produce globose sporangia.

Twig blight disease caused by the pathogen Choanephora cucurbitarum was

identified based on morphological and cultural characters. The pathogenicity of fungus

was tested and Koch’s postulates were confirmed. Symptoms first appeared as water

soaked lesion on leaves and stems. The infected portion dries and curls up and rapidly

extends downward, attacking buds and tender leaves. Prominent hairy growth of

Choanephora cucurbitarum on the infected tissue was found in the morning hours. This

is visible with a hand lens. Usually whitish mycelia and monosporous sporangial were

produced on the lesions. The disease gradually spreads to more and more branches even

to the stem causing severe damage under warm and humid conditions that favor the

disease development.

The diversity in cultural characters of C. cucurbitarum was studied on eight

different media.

The radial growth of C. cucurbitarum was high on potato dextrose agar medium

(90 mm) and carrot agar medium (90 mm) followed by v8 juice agar medium (87.67

mm) while least on oat meal agar medium (33.50 mm).

The effect of different pH on radial growth of C. cucurbitarum was studied on

PDA medium at different pH levels ranging from 4.0 to 9.0. Among the different pH

level tested the maximum growth was observed in the range from 4.5. The temperature

of about 20oC to 25

oC was found most suitable for growth of C. cucurbitarum.

The phylloplane and rhizosphere mycoflora of chilli were isolated by serial

dilution method and identified as Aspergillus niger, Aspergillus flavus, Penicillium

spp., Paecilomyces spp., Fusarium spp., Alternaria spp., Mucor spp and Rhizopus spp.

Among the fungal isolates, Fusarium spp recorded significantly highest inhibition

(57.77%) of test fungus compared to all other treatments followed by Penicillium spp

with 51.88 per cent inhibition. Alternaria spp recorded least (47.00%) inhibition of

mycelial growth of Choanephora cucurbitarum compared to all other treatments.

The antagonistic activity of Four Trichoderma isolates was evaluated against

Choanephora cucurbitarum under in vitro conditions. Among all Trichoderma

isolates, Trichoderma viride isolate 1, Trichoderma harzianum isolate 2, Trichoderma

viride isolate 2 and Trichoderma harzianum isolate 1(62.44, 55.33, 62.22 and 40.11

per cent, respectively) recorded significantly highest per cent inhibition of mycelial

growth of C. cucurbitarum.

Bio efficacy of several fungicides was tested against Choanephora cucurbitarum

by poison food technique at six concentrations i.e 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3%

respectively under in vitro conditions. Among them captan at 0.15% recorded

maximum inhibition of mycelia growth of Choanephora cucurbitarum.

Disease severity index was used as an indicator of the varietal screening against

the test pathogen. The following varieties were procured from Guntur LAM, LCA-

206, LCA-334, LCA-353, LCA-235, LCA625, CA960, G36, G4 and G3. Out of ten

varieties screened G 4 and LCA 334 showed disease severity index of about 24% and

27% respectively. CA 960 and G3 showed disease severity index about 32% and 46%

followed by LCA-353 (48%), LCA-625 (50%), LCA-206 (50%), G 36 (72%) and

LCA-235 (76%).

The most effective fungicide (captan) and potential biocontrol agent

(Trichoderma viride isolate 1) were evaluated against twig blight incidence of chilli

caused by Choanephora cucurbitarum under glass house conditions along with neem

oil. These treatments were used alone and in various combinations. Among the various

treatments the application of captan (T2) was recorded maximum (66.47%) per cent

disease control while least was recorded in T3 (Neem oil) 13.95%. The fungicide

captan alone and their combination were proved effective in the disease management.

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