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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
Rajendranagar, Hyderabad-500 030.
Member Dr. P. Rajanikanth
Assistant professor
Department of Entomology
College of Agriculture, PJTSAU
Rajendranagar, Hyderabad-500 030.
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).
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).
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).
2.1.4 Symptomatology
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
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
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).
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.
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 (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
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
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).
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.
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.
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
Distilled water 1000.0 ml
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
Distilled water 1000.0 ml
5. Corn meal agar
Corn meal 50.0 g
Agar 20.0 g
Distilled water 1000.0 ml
6. Oat meal agar
Oat meal 72.0 g
Agar 20.0 g
Distilled water 1000.0 ml
7. Carrot agar
Carrot agar 200.0 g 200 g
Dextrose 20.0 g
Agar 20 g
Distilled water 1000.0 ml
8. Water agar
Agar 20.0 g
Distilled water 1000.0 ml
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
Distilled water 1000.0 ml
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.
Sporangiospores were elliptic, fusiform or ovoid in shape, light brown or dark brown in
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
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.
4.1.5 Symptomatology
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
DAI: Days after inoculation
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
DAI: Days after inoculation
Table 4.9: Screening of popular chilli varieties against twig blight caused by
Choanephora cucurbitarum
DAI: Days after inoculation
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
DAI: Days after inoculation
Table 4.5: Effect of temperature on radial growth of Choanephora
cucurbitarum
DAI: Days after inoculation
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
DAI: Days after inoculation
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
Choanephora cucurbitarum
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
T1 - Trichoderma viride isolate 1
T2 - Trichoderma viride isolate 2
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
2 Trichoderma viride isolate 2
3 Trichoderma harzianum isolate 1
4 Trichoderma harzianum isolate 2
Plate 4.7a: Antagonistic activity of Trichoderma spp. against Choanephora
cucurbitarum
1 Trichoderma viride isolate 1
2 Trichoderma viride isolate 2
3 Trichoderma harzianum isolate 1
4 Trichoderma harzianum isolate 2
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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