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EVALUATION OF SELECTED ECO-FRIENDLY
COMPONENTS AGAINST PHOMOPSIS FRUIT ROT
(Phomopsis vexans) OF EGGPLANT
SUMAIYA KHANAM
DEPARTMENT OF PLANT PATHOLOGY
SHER-E-BANGLA AGRICULTURAL UNIVERSITY
DHAKA-1207
JUNE, 2015
EVALUATION OF SELECTED ECO-FRIENDLY
COMPONENTS AGAINST PHOMOPSIS FRUIT ROT
(Phomopsis vexans) OF EGGPLANT
SUMAIYA KHANAM
REGISTRATION NO. 09-03292
A Thesis
Submitted to the Faculty of Agriculture, Dept. of Plant Pathology
Sher-e-Bangla Agricultural University, Dhaka,
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
IN
PLANT PATHOLOGY
SEMESTER: JANUARY - JUNE, 2015
Approved by:
Department of Plant Pathology
Supervisor
__________________________________
Dr. Md. Belal Hossain
Associate Professor
Chairman
Department of Plant Pathology
Sher-e-Bangla Agricultural University
Dr. Md. Rafiqul Islam
Professor
Supervisor
Dr. Nazneen Sultana
Professor
Department of Plant Pathology
Co-supervisor
Dedicated to My
Beloved Parents
CERTIFICATE
This is to certify that the thesis entitled, “EVALUATION OF
SELECTED ECO-FRIENDLY COMPONENTS AGAINST
PHOMOPSIS FRUIT ROT (Phomopsis vexans) OF
EGGPLANT” submitted to the Department of Plant Pathology, Faculty of
Agriculture, Sher-e-Bangla Agricultural University, Dhaka, in the partial
fulfillment of the requirements for the degree of MASTER OF SCIENCE
(M.S.) IN PLANT PATHOLOGY, embodies the result of a piece of bona
fide research work carried out by SUMAIYA KHANAM bearing
Registration No. 09-03292 under my supervision and guidance. No part of
the thesis has been submitted for any other degree or diploma.
I further certify that such help or source of information, as has been availed
of during the course of this investigation has duly been acknowledged.
Dated: 26 May, 2016 Prof. Dr. Md. Rafiqul Islam
Place: Dhaka, Bangladesh Department of Plant Pathology
Sher-e-Bangla Agricultural University
Supervisor
Department of Plant Pathology Fax: +88029112649
Sher - e - Bangla Agricultural Universit y Web site: www.sau.edu.bd
Dhaka - 1207 , Bangladesh
ACKNOWLEDGEMENTS
All admiration and praises are solely to “Almighty Allah” Whose mercy
absolutely enabled the author to pursue the higher education in Agriculture
discipline and successful completion of M.S. courses and research work for
the degree of M.S. in Plant Pathology.
The author expresses her immense respect and deepest sense of gratitude and
heartfelt thanks to her most reverend teacher and research Supervisor,
Professor Dr. Md. Rafiqul Islam, Department of Plant Pathology, Sher-e-
Bangla Agricultural University, Dhaka for his inestimable help, dynamic
and cordial guidance, planning, ingenious and valuable suggestion,
continuous encouragement, constructive criticism and scholastic supervision
throughout the research work and preparation of the thesis.
The author also expresses her gratitude, profound respect and immense
indebtedness to her honorable teacher and research Co-supervisor, Professor
Dr. Nazneen Sultana, Department of Plant Pathology, Sher-e-Bangla
Agricultural University, Dhaka for her sincere guidance, keen interest,
valuable advices, helpful suggestions and sympathetic co-operation regarding
this research work.
The author also wishes to pay her deep respect to Dr. Md. Belal Hossain,
Chairman, Department of Plant Pathology, Sher-e-Bangla Agricultural
University, Dhaka for his valuable advice and continuous effort throughout
the study and research period.
The author also wishes to express her sincere gratitude to all other respected
teachers of the Department of Plant Pathology, Sher-e-Bangla Agricultural
University, Dhaka for their valuable suggestion, kind cooperation and help
during the entire study and research period.
Cordial appreciation and thanks are extended to the staff of the laboratory
of Department of Plant Pathology and Horticulture Farm, Sher-e-Bangla
Agricultural University, Dhaka for their responsible help during the
experimental work.
Finally, the author expresses her immense gratefulness to her beloved
parents, husband, little daughter and well wishers whose inspiration,
sacrifice, advice, continuous encouragement and moral support opened the
gate and paved the way to higher studies.
The Author
EVALUATION OF SELECTED ECO-FRIENDLY COMPONENTS AGAINST
PHOMOPSIS FRUIT ROT (Phomopsis vexans) OF EGGPLANT
ABSTRACT
An eco-friendly bioagent Trichoderma harzianum, four plant extracts viz. garlic clove
extract, allamonda leaf extract, neem leaf extract and lemon grass extract, a fungicide
Bavistin 50WP and two soil amendment tools saw dust and poultry waste were evaluated
against Phomopsis vexans for the management of phomopsis fruit rot disease of eggplant.
The experiment was carried out in the horticultural farm of Sher-e-Bangla Agricultural
University during Rabi season (October, 2014 to March, 2015). Trichoderma harzianum
and Bavistin 50WP showed promising performance in controlling the disease. The lowest
disease incidence was 9.54, 14.77 and 16.52, respectively at 105, 114 and 123 DAT and
the disease severity was 3.73, 6.09 and 4.83, respectively at 105, 114 and 123 DAT in
Trichoderma harzianum treated plot. The fungicide Bavistin 50WP treated plot showed
disease incidence 10.77, 16.53 and 18.43, respectively at 105, 114 and 123 DAT. The
disease severity was also found 5.09, 9.64 and 10.01 in Bavistin 50WP treated plot at 105,
114 and 123 DAT, respectively. The plant extracts also showed significant effects in
controlling the disease reducing the disease incidence and disease severity. Among the
plant extracts, allamonda leaf (1:2) and neem leaf (1:2) extract were found very effective
against Phomopsis vexans. Allamonda leaf extract showed disease incidence (14.16, 17.96
and 18.60) and disease severity (7.50, 10.01 and 12.73) at 105, 114 and 123 DAT,
respectively. Disease incidence recorded in neem leaf extract treated plot was 16.66, 19.65
and 24.16 at 105, 114 and 123 DAT, respectively and the disease severity was 9.98, 10.46
and 15.14 at 105, 114 and 123 DAT, respectively. The effect of other plant extracts also
found moderate in reducing the disease incidence and severity. Saw dust and poultry
waste showed poor performances against the disease.
iv
CONTENTS
CHAPTER TITLE PAGE
ACKNOWLEDGEMENTS i
ABSTRACT iii
CONTENTS iv
LIST OF TABLES vii
LIST OF FIGURES viii
LIST OF APPENDICES ix
1 INTRODUCTION 1-3
2 REVIEW OF LITERATURE 4-12
2.1 Symptom of phomopsis blight and fruit rot of eggplant 4
2.2 Incidence and severity of phomopsis blight and fruit rot
of eggplant 6
2.3 Morphology of Phomopsis vexans, the causal agent of
phomopsis fruit rot of eggplant 7
2.4 Seed infection and its impact 8
2.5 Management of phomopsis fruit rot through plant
extracts and biological control agent 10
3 MATERIALS AND METHODS 13-28
3.1 In vitro Experiment 13
3.1.1 Experimental site 13
3.1.2 Experimental period 13
3.1.3 Preparation of culture media 13
v
3.1.4 Isolation and maintenance of culture of
Phomopsis vexans 14
3.1.5 Preparation of spore suspension of Phomopsis
vexans 14
3.2 Field Experiment 15
3.2.1 Experimental site 15
3.2.2 Experimental period 15
3.2.3 Experimental design and layout 15
3.2.4 Test material (cultivar) 15
3.2.5 Raising of seedlings 15
3.2.6 Land preparation and fertilizer application 18
3.2.7 Transplanting of of seedlings to the main field 18
3.2.8 Intercultural operation 21
3.2.9 Plant extracts used in the experiment 21
3.2.10 Preparation of plant extracts 21
3.2.11 Spraying of plant extracts 23
3.2.12 Spraying of bioagent as spore suspension 23
3.2.13 Spraying of fungicide 25
3.2.14 Inoculation of eggplant by Phomopsis vexans 25
3.2.15 Data collection 27
3.2.16 Seed extraction 27
3.2.17 Analysis of data 30
vi
4 RESULTS 31-47
4.1 Laboratory experiment 31
4.1.1 Isolation and identification of causal agent 31
4.2 FIELD EXPERIMENT 33
4.2.1
Development of symptoms in brinjal plants following inoculation
with Phomopsis vexans 33
4.2.2 Evaluation of different treatments against disease incidence of
eggplant by Phomopsis vexans 35
4.2.3 Evaluation of different treatments against disease severity of
eggplant by Phomopsis vexans 38
4.2.4 Effect of different treatments on plant growth characteristics 42
4.2.4.1 Effect of different treatments on height of plant 42
4.2.4.2 Effect of different treatments on number of branch of eggplant 42
4.2.4.3
Effect of different treatments on number of leaf per branch of
eggplant 43
4.2.5 Effect of different treatments on fruit yield and seed yield of
eggplant 45
5 DISCUSSION 48-52
6 SUMMARY AND CONCLUSION 53-54
7 REFERENCES 55-64
8 APPENDICES 65-67
vii
LIST OF TABLES
Table Title Page
1. Effect of different treatments on disease incidence of fruit rot of
Eggplant at different days after transplanting (DAT) 37
2. Effect of different treatments on disease severity of fruit rot of
Eggplant at different days after transplanting (DAT) 41
3. Effect of different treatments on growth characteristics of eggplant 44
4. Effect of different treatments on fruit and seed yield of eggplant 47
viii
LIST OF PLATES
Plate Title Page
1 Eggplant seedlings (Variety-BARI Begun 8) raised in plastic tray 17
2 Different growth stages of eggplant showing in the experimental
field 19
3 A view of experimental field in Horticultural Farm of SAU 20
4 Preparation of different plant extracts. 22
5 Pure culture of Trichoderma harzianum 24
6 Trichoderma harzianum under compound microscope 24
7 Inoculation of eggplant by Phomopsis vexans 26
8 Harvesting of fruit for seed production 28
9 Extraction of seed from fruit 29
10 Pure culture of Phomopsis vexans 32
11 Alpha conidia of Phomopsis vexans under compound microscope 32
12 Infected plant showing phomopsis fruit rot symptom 34
ix
LIST OF APPENDICES
APPENDIX TITLE PAGE
NO.
I Map showing the experimental site under study 65
II Physiochemical properties of soil of the experimental
site 66
III
Monthly average relative humidity, maximum and
minimum temperature, rainfall and sunshine hour of
the experimental period (October 2014 to March 2015)
67
1
Introduction
The eggplant (Solanum melongena) belonging to the family Solanaceae originates
from India and is now generally grown as a vegetable throughout the tropical, sub-
tropical and warm temperate areas of the world. It is a popular, nutritious and widely
grown vegetable in Bangladesh as well as in the world and has got multifarious use as
a dish item (Rashid, 1976; Bose and Som, 1986).About 8 million farm families are
involved in eggplant cultivation. Its position in terms of acreage production is second
in vegetable crops in Bangladesh (BBS, 2003).The total cultivable area of eggplant is
44,377 acres with total annual production of 3,68,000 metric tons (BBS, 2013). A
large number of cultivars are grown in Bangladesh, which is showing a wide range of
variation in yield performance. This gives small, marginal and landless farmers a
continuous source of income and provides employment facilities for the rural people.
For most of the time, except peak production period, market price of eggplant
compared to other vegetables remains high which is in favor of the farmer’s solvency.
Therefore, it plays a vital role to boost up our national economy. It is grown round the
year especially during the lean period when the seasonal vegetables are in a scarcity
in the market. Eggplant, thus regarded as a cash crop to the farmers which provides
them continuous harvesting and financial assistance.
Such a potential crop is known to suffer from 12 diseases and among them phomopsis
fruit rot caused by Phomopsis vexans Harter has been treated as one of the major
constraints in eggplant cultivation in the country (Khan, et al., 2002; Das, 1998). The
disease was first reported from Gujrat in 1914 and since then from many parts of
India. Fakir (1983) and Ahmad (1987) have reported occurrence of this disease in
Bangladesh. The infected fruit develops pale and sunken spots that progress covering
the entire surface and cause dropping. Affected portion is mummified producing huge
2
pycnidia on the surface and becomes non-edible (Singh, 1992; Ashrafuzzaman,
1986).
Phomopsis vexans is viable for about 14 months in soil debries and in the seed from
infected fruits. The pathogen is reported both externally and internally seed borne
(Islam, 2005). It has become a major constraint for intensive cultivation of eggplant.
The disease affects the crops from seedling to maturity (Singh, 1992). Crop losses
due to this disease are evident, loss ranges from 15-20% in general but 30-50% in
severe case (Das, 1998).
Efforts for controlling the disease through different measures have been made by
many researchers (Khan, 1999; Hawlader, 2003; Nazimuddin, 2004 and Rummana,
2004). Plant extracts might be a substantial alternative of chemical pesticides in
controlling phomopsis blight and fruit rot of eggplant. Bavistin is reported to be an
effective fungicide against P. vexans (Khan et al., 2002; Meah et al., 1998; Mohanty
et al., 1994). Chemicals, though seriously discouraged in present plant protection
system especially for horticultural crops by Montreal Protocol, it might be
incorporated in the disease management program as the last option in absence of any
alternative.
Seed is the infection source of P. vexans and may serve as a substrate for pathogen
survival (Pan et al., 1995). The pathogens remain on the seed coat and the cotyledons
of eggplant seeds causing various degrees of seed discoloration and minute black
pycnidial bodies which is distinctly observed on the surface of the dry seed (Karuna
et al., 1994). Therefore, by the management of phomopsis fruit rot we can control
seed infection caused by P. vexans and thus seed yield is higher. Many plants
extracts, bioagents and hot water are reported to be antimicrobials against plant
pathogenic fungi (Bowers et al., 2000; Lawson et al., 1998; Hossain, 2004; Hassan,
3
2000; Cook, 1993; Harman, 1989). Those components may address against P. vexans.
Besides, seed dressing fungicide Bavistin may be assayed against the pathogen
Phomopsis vexans.
Under such scenario, developing a biologically based and environmentally safe eco-
friendly approach for management of phomopsis fruit rot is essential. To screen out
the effective components for management of phomopsis blight and fruit rot of
eggplant, the present experiment was undertaken with the following objectives:
1. To isolate and identify the causal organism of phomopsis fruit rot of eggplant.
2. To evaluate eco-friendly components for the management of phomopsis fruit rot of
eggplant for seed production.
4
REVIEW OF LITERATURE
Eggplant (Solanum melongena L.) is a popular Solanaceous vegetable crop of
Bangladesh. It is cultivated throughout the year and used by people of all strata of the
country (Rashid, 1993). Brinjal suffers from many diseases caused by fungi, bacteria,
virus, nematode and mycoplasma. Of them, phomopsis blight and fruit rot of eggplant
caused by the fungus Phomopsis vexans (Sacc and Syd) is a serious disease which
attacks all above ground parts of plant. It is mentionably damaging to the crop and is
a threat particularly in kharif season and late crop in winter season.
Phoma solani Hals. was first described as the organism of fruit rot of eggplant by
Halsted in 1982 in the United States of America (Harter, 1914). Since the name had
been used for another fungus it was changed to Phoma vexans by Saccardo and
Sydow in 1899 (Harter, 1914). Harter (1914) and Punithalingam and Holliday (1972)
have reported that brinjal (Solanum melongena L.) is the only economic host of
Phomopsis vexans. Evidences of research work regarding phomopsis blight and fruit
rot of eggplant is very dearth. However, it is known from literatures that emphasis
have been given to control the disease through biological control agents and plant
extracts. Literatures on the symptom, incidence and severity of phomopsis fruit rot,
its seed borne nature and control by eco-friendly components are given below.
2.1 Symptom of phomopsis blight and fruit rot of eggplant
Phomopsis blight occurs on fruit and occasionally on leaves or stems. Infection of
foliage and stem is less important than fruit infection.
5
Harter (1914) reported that on the fruits the symptoms appear first as minute sunken
greyish spots with a brownish halo, which later enlarge and coalesce, producing
concentric rings of yellow and brown zones. These spots increase in size and form
large rotten areas on which conidiomata often develop concentrically, covering most
of the rotten fruit surface. Pycnidia on fruit are larger than those on stems and leaves.
According to Walker (1952) the first phase of phomopsis blight is a blight of young
seedling. The stem is girdled slightly above the soil line. The seedlings topple over
and die. The stem lesion is dark brown, becoming gray in the center as the pycnidia
develop. Round or oval brown spots develop on the leaf, becoming irregular in shape
as they enlarge upto 3 cm in diameter. The centers become gray and contain pycnidia.
The fruit is attacked while on the plant. The decay being first soft and watery and
later the fruits become black and mummified as the pycnidia develop abundantly over
the surface.
Pawar and Patel(1957) reported that the symptoms of phomopsis blight range from
poor germination and seedling blight to fruit rot. If the infection enters the fruits
through the calyx, the whole fruit may become mummified due to dry rot. Post-
emergence damping-off of seedlings results from infection of the stem just above the
soil surface. The symptoms on leaves are more prominent during the early stages of
plant growth. The lesions first are small, more or less circular, and buff to olive, later
becoming cinnamon buff, with an irregular blackish margin. Irregular spots result
from coalescence. After transplanting, leaves coming in contact with the soil may
become infected directly or develop leaf spot due to infection by conidia. Lesions on
the petiole or the lower part of the midrib can result in death of the entire leaf.
6
Affected leaves may drop prematurely, and the blighted areas become covered with
numerous black pycnidia.
Ashrafuzzaman (1986) stated that due to this disease damping off takes place at
seedling stage. Leaf may be attacked at any time. Generally the first symptom appears
on the lower leaves. Spots are clear, circular and grayish. Numerous pycnidia are
formed on aged spots. Infected leaves become yellowish and eventually may die.
Cankers observed at the base of the stem, bark is cracked and woody portion opened.
Light coloured, sunken spots are observed on fruit. Black pycnidia are formed in
spots and the fruits become mummified.
Kumar et al. (1986) described that Phomopsis vexans caused fruit rot of eggplant
which appeared as minute, circular, water soaked, sunken, grayish spots with
brownish halo and have a light coloured center which later enlarged to produce
concentric rings and brownish zones. Spots increased in size and formed larger rotten
areas on which pycnidia developed causing blackening of the affected area.
Ken et al (2009) described that on older plants, round or oval spots develop on the
leaf and stem, which enlarge and become more irregular. Fruits are infected while on
the plant and exhibit spots which appear as pale sunken areas which may cover the
entire fruit if not treated. The small black pycnidia are present in abundance in the
fruit spots.
If conditions become dry, infected fruit become shrivelled, dry, and form black
mummies (Howard et al, 2009).
7
2.2 Incidence and severity of phomopsis blight and fruit rot of eggplant
Meah et al., (2002) recorded up to 100% disease incidence and 9.8% disease severity
in cultivar Dohazari. Meah et al., (2002) also reported a loss equivalent to Taka 808
million (US$ 134 million) per annum due to phomopsis blight and fruit rot of
eggplant in Bangladesh. Khan (1999) reported that phomopsis blight and fruit rot of
eggplant causes about 21% fruit rot and 7% seed rot in eggplant.
Meah (2003) did an epidemiological survey on the incidence and severity of
phomopsis blight and fruit rot of eggplant in 18 major growing areas of Bangladesh
and reported that sandy loam soil, soil moisture in the range of 50-60%, moderate air
temperature (20- C) and high atmospheric moisture (65-78 % RH) significantly
influenced the prevalence of the disease. The prevalence was higher in the southern
part of Bangladesh with moderate weather than the middle and northern part of
Bangladesh with severe weather.
2.3 Morphology of Phomopsis vexans, the causal agent of phomopsis fruit rot of
eggplant
According to Edgerton and Moreland, 1921, pycnidia of Phomopsis vexans is
subepidermal, erumpent, dark, thick-walled and flattened to globose shaped, varying
in size, often 100-300 µm in diameter, with or without a beak. Phialides are hyaline,
simple or branched, sometimes septate, 10-16 µm long and arising from the
innermost layer of cells lining the cavity. Alpha conidia are hyaline, aseptate, sub-
cylindrical and 5-8 × 2-3 µm in size. Beta conidia are filiform, curved, hyaline,
septate, 18-32 x 0.5-2.0 µm in size and non- germinating. Hyphae are hyaline, septate
and 2.5-4 µm diameter.
8
Gratz (1942) stated that, perithecia in culture of Phomopsis vexans are usually in
clusters, 130-350 µm diameter and beaked. Beaks are sinuous, carbonaceous,
irregular and 80-500 µm long. Asci clavate, sessile, 24-44 × 5-12 µm, eight-spored.
Ascospores are biseriate, hyaline, narrowly ellipsoid to bluntly fusoid, one-septate,
constricted at the septum and 9-12 × 3-4.5 µm in size.
Punithalingum and Holliday (1972) reported that, pycnidia of Phomopsis vexans on
stems, leaves and fruits are solitary or gregarious, initially immersed, becoming
erumpent, black, globose to irregular, up to 350µm wide having opening by 20-50
wide ostioles. Conidiophores (phialides) are hyaline, simple or branched, sometimes
septate,10-16µm long, arising from the innermost layers of cells lining the cavity.
Conidia are fusoid to ellipsoidal, hyaline, filiform, curved, rarely erect and 20-30 ×
0.5-1µm in size.
2.4 Seed infection and its impact
The fungus P. vexans is seed borne at significant levels (Edgerton and Moreland,
1921; Toole et al., 1941)
Infection in seed adversely affects the seed quality, causing seed discoloration,
reduced seed weight and density, poor germinability and reduced viability (Toole et
al., 1941; Porter, 1943)
Phomopsis vexans is an important fungus isolated from surface sterilized eggplant
seeds and stated to be seed borne by Walker (1952) in USA. Panwar (1957) reported
that the causal organism of the disease remained viable for about 14 months in soil
debris and in the seeds from infected fruits, which were poor in germination.
9
Infected seeds contain profuse branched septate mycelium aggregated in the seed
coat, between the seed coat and endosperm and in the embryo region of the seeds.
Pycnidia are produced in the seed coat, between the seed coat and endosperm, and in
the endosperm tissue (Vishunavat and Kumar, 1994).
Karuna et al. (1994) studied on seed borne inocula of P. vexans and the effect of
infection on seed quality in eggplant and found that seed borne infection of varieties
PBR -7, PBR-5, MHB-1 and Pant rituraj were 29%, 36%, 35% and 56% respectively.
Seed infection caused various degrees of seed discoloration. On the surface of dry
seed, the fungus was observed as black pycnidial bodies. Pre-treatment of seeds with
0.1% HgCl2 solution reduced seed infection. P. vexans infection adversely affected
seed quality.
Karuna et al. (1994) also studied locations of infection of P. vexans in brinjal seeds
and observed that component plating of seed of the arburgine cultivar PBR-5 yielded
22% and 12% infection in seed coats and cotyledons, respectively. Pycnidia and
mycelium were seen on seed coats and in the embryo.
Pan et al. (1995) studied the seed borne nature of P. vexans and reported that P.
vexans was present on seed coat and on the cotyledons of arburgine seeds collected
from disease fruit in West Bengal, India. It was suggested that the seeds were an
infection source and might serve as a substrate for pathogen survival.
Gangadharaswamy et al. (1997) studied the impact of P. vexans on seed quality of
brinjal and reported that 13% seed infection was found in variety Erenagere collected
from farmers and no seed infection was found in variety Erenagere collected from
farmers and no seed infection was found in variety “Pusa purple” collected from
10
National Seed Corporation. P. vexans even at low incidence caused failure of
emergence of root, ultimately rotting of seeds.
Khan et al. (2002) analyzed 22 seed samples collected from farmers of different
eggplant growing areas of Bangladesh and recorded 0.5-7.0% infection of P. vexans.
2.5 Management of phomopsis fruit rot through plant extracts and biological
control agent
Mohanty et al. (1995) investigated the allelopathic control of Phomopsis vexans,
causal agent of phomopsis fruit rot of brinjal by aqueous leaf extracts of five plants.
Fungal growth was inhibited to a maximum by leaf extracts of Allamonda cathertica
(93.75%) followed by Aegle mermelos (85.38%). Leaf extracts of Catheranus roseus,
Polyalthia longifolia and Azadirachta indica were equally effective, but that of
Octimum sanctum was the least effective causing 52.23% growth inhibition.
Khaleduzzaman (1996) reported that out of four plant extracts, garlic bulb extract was
found best in reducing seed-borne prevalence and increasing germination percentage
of seed followed by ginger, neem and bishkatali extracts. The medicinal plant extracts
reduced seed borne prevalence of all the fungi. The reduction of different fungi
differed significantly from the control. However, among the 6 extracts, ginger extract
performed better in reducing seed-borne prevalence of all the major fungi and
increasing germination followed by followed by garlic, Bishkatali and ghagra extract.
Crude extracts and dilution extracts (1:1v/v) of the selected plant species were used
for treating the seeds. Neem bark, garlic clove, bishkatali gave better results. Further
investigation in controlling seed borne fungi in wheat seed using alcoholic extracts of
neem bark and garlic clove also gave best results.
11
Panda et al. (1996) tested the efficacy of leaf extracts from Polyalthia longifolia,
Aegle mermelos, Azadirachta indica, Catheramus roseus, Octimum sanctum and
Allamonda cathertica for control of phomopsis blight (caused by P. vexans). Leaf
extracts of Allamanda cathertica had excellent potential as a fungicide.
Khan (1999) studied the effect of plant extracts (Allamanda, Bael, and Neem) for the
management of phomopsis blight and fruit rot of eggplant in field condition. Among
three plant extracts, allamanda spray was found most effective. He also found that the
incidence and severity of phomopsis fruit rot varied from one plant extract to another
when aqueous leaf extract of neem, allamanda and bael applied at flowering, fruiting
and peak fruiting stages in different doses (S, S/10 and S/100).
Meah (2003) reported that garlic bulbs extract (1:1) and allamonda leaves extract
efficiently controlled P. vexans in the laboratory, nursery house and in the field
reducing severity of leaf blight and fruit rot by 71-75%.
Hawlader (2003) reported that seed treatment with allamanda leaf extracts (1:1)
effectively increased germination of eggplant seeds and considerably decreased
nursery diseases.
Islam (2004) found garlic bulbs and allamonda leaves extract cause 76-100%
inhibition of mycelial growth of Phomopsis vexans. Diethyl ether, Dichloromethane
and water acted as effective solvents. TCL studies showed the presence of a number
of compounds having very low polarity in garlic bulbs and allamanda leaf extracts
acting against the pathogen.
12
Antagonistic Pseudomonas fluorescens and Trichoderma harzianum seed treatment
and spray treatment were found to be effective against P. vexans (Srinivas et al.,
2005)
Alam (2005) tested 11 botanicals to control phomopsis blight and fruit rot of
eggplant. Out of 11 botanicals, garlic (Allium sativum L.) bulb and allamanda
(Allamanda cathertica L.) leaf extracts were found promising arresting mycelial
growth and inhibiting spore germination of Phomopsis vexans in in vitro condition
and controlled phomopsis blight and fruit rot of eggplant in the field significantly. He
also found that combinations of apparently healthy seed, seed treated with garlic bulb
extract and soil treated with Trichoderma harzianum completely controlled damping
off, tip over and seedling blight in the nursery bed in the net house and increased
germination by 48.83% over control.
13
Materials and methods
3.1 In vitro EXPERIMENT
3.1.1 Experimental site
The In vitro experiments were conducted at M.S. Laboratory of the Department of
Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka.
3.1.2 Experimental period
The experiments were conducted in August-September, 2014.
3.1.3 Preparation of culture media
Composition of Potato Dextrose Agar (PDA) media are as follows:
Potato (Peeled and sliced) 200g
Dextrose 20g
Agar 20g
Water 1000ml
Peeled and sliced potato was boiled to collect the extract by sieving with a fine piece
of cloth. Afterwards, the other ingredients were mixed up and then heated gently for
few minutes. After preparation, the contents were poured into 250ml Erlenmeyer
flasks, plugged with cotton and were sterilized in autoclave at C under 15 PSI
for 20 minutes. This medium was acidified with 30 drops of 50% lactic acid per 250
ml medium to inhibit the growth of bacteria. These 20 ml of the medium was poured
in to each petri-plate (9cm dia.) and allowed to solidify.
14
3.1.4 Isolation and maintenance of culture of Phomopsis vexans
The pathogen, Phomopsis vexans was isolated by tissue planting method from
naturally infected brinjal fruit collected from the Horticulture Garden of Sher-e-
Bangla Agricultural University, Dhaka (Islam et al.). The sample showed typical
phomopsis fruit rot symptom having pale, sunken spots on the mummified fruit (CMI
Description no.338). The infected fruit was repeatedly washed in fresh water and
surface sterilized with 10% Clorox for 1 minute followed by three times washing in
distilled water to eliminate traces of mercury. Infected tissues were cut and placed in
PDA media and incubated at 22±2 for 10 days. After incubation, it was observed that
white mycelia and pycnidia were formed. Several slides were prepared and examined
under compound microscope. The fungus was identified according to CMI
description (No. 338). The pathogen was purified and multiplied subsequently
through block culture on PDA medium and culture was stocked in PDA slant for
future use.
3.1.5 Preparation of spore suspension of Phomopsis vexans
Phomopsis vexans was grown on PDA medium in petridish at C temperature.
After formation of pycnidia (in about 15-20 days), 5ml/plate sterile water was added
and the spore masses was scraped away with sterile needle/scalpel. The conidial
suspension was made with additional 45ml water and blended in Moulinex blender
for 2 minutes in medium speed and filtered through sterile cheesecloth. The
suspension was adjusted to 105conidia/ml solution and stored at C for future use.
15
3.2 FIELD EXPERIMENT
3.2.1 Experimental site
The experiments were conducted in the experimental field of Department of
Horticulture, Sher-e-Bangla Agricultural University, Dhaka.
3.2.2 Experimental period
The experiments were conducted during the winter season (October, 2014 to March,
2015).
3.2.3 Experimental design and layout
The experiment was laid out in a Randomized Block Design (RCBD) with three
replications where unit plot size was 3.5 sq.m. (3.5m x 1m). Plot to plot distance was
maintained 1m and row to row distance was1.5m. Plant to plant distance 75cm. Soil
in between two plots were taken over to make it raised, thereby facilitating free
movement for nursing and free drainage of excess water during rain. There were three
replications in the experiment. Four plant extracts, one biological control agent, one
fungicide, manuring with saw dust, poultry waste constituted the treatments.
3.2.4 Test material (Cultivar)
Eggplant cultivar “BARI Begun 8” was used for the experiment. The seeds were
collected from BARI (Bangladesh Agricultural Research Institute), Gazipur.
16
3.2.5 Raising of seedlings
Seedlings were raised in plastic trays with proper care and management. Trays were
prepared by mixing soil, sand and well decomposed cowdung in proportion of 2:1:1.
The mixture were sterilized in autoclave. Then the trays were filled with sterilized
soil and the seeds were sown. Shade was provided to save the young and delicate
seedlings from heavy showering and scorching sunlight. Seedlings were observed
regularly and watering was done as per necessary up to transplanting in the main
field.
17
Plate 1. Eggplant seedlings (Variety-BARI Begun 8) raised in plastic tray
18
3.2.6 Land preparation and fertilizer application
A piece of medium high land with well drainage system was selected and prepared by
ploughing, followed by laddering. The soil was well pulverized for good tilth
condition. Weeds and stubbles were removed. During field preparation, application of
fertilizers and manuring was done with recommended doses (Anonymous, 1997). The
experimental plot was partitioned into the unit plots in accordance with the
experimental design.
Fertilizers and manures used in experimental field
Fertilizer Rate (Kg/ha)
Urea 130
TSP 125
MP 100
Cowdung 10000
Oilcake 500
Whole amount of well decomposed cowdung, TSP and half amount of MP were
applied during land preparation. Urea and remaining half of MP were applied in three
installments as top dressing.
3.2.7 Transplanting of seedlings to the main field
After proper growth of the seedlings they were transplanted in the field followed by
watering. Five seedlings were planted in each subplot maintaining plant-to-plant
distance 75 cm and line-to-line distance 0.5m.
19
a) 20 DAT b) 30 DAT
c) 45 DAT d) 60 DAT
e) 80 DAT f) 90 DAT
Plate 2(a-f).Different growth stages of eggplant showing in the experimental field
20
Plate 3. A view of experimental field in Horticultural farm of SAU
21
3.2.8 Intercultural operation
Various intercultural operations were accomplished for better growth and
development of the seedlings. Weeding, manuring and irrigation were done time to
time according to plants’ need. The plants were also kept free from insect pest attack
by spraying insecticides as required intervals.
3.2.9 Plant extracts used in the experiment
Local name Sceintific name Plant parts used Concentration (w/v)
Garlic Allium sativum Clove 1:2
Allamanda Allamanda cathertica Leaf 1:2
Neem Azadirachta indica A. Juss. Leaf 1:2
Lemon grass Cymbopogon flexuosus Leaf 1:2
3.2.10 Preparation of plant extract
For preparation of plant extracts, required amount of respective parts of each plant
was taken, washed in tap water, crushed in a mortar and pestle. The crushed materials
were blended in an electric blender adding equal amount of sterile water for 1:1
solution. The blend was filtered through sterile cheesecloth. The supernatant was
diluted in equal amount of sterile water for 1:2 solutions.
22
Plate 4. Preparation of different plant extracts. A. Blender B. Garlic clove
C. Garlic clove extract. D. Allamonda leaf E. Allamonda leaf extract F. Neem
leaf G. Neem leaf extract H. Lemon grass I. Lemon grass extract
A
B C
D E
F G
H I H
23
3.2.11 Spraying of plant extracts
The extracts were sprayed at fruiting stage as pre-inoculation spray (before 24 hr. of
inoculation) and post inoculation spray (after 24 hr. of inoculation). The spraying was
done by a hand sprayer to cover whole surface of plant leaf, flower and fruits. An
amount of 50ml solution was sprayed in one plant. Precautions were taken to avoid
drifting of spray materials to neighbouring plants with polythene barrier.
3.2.12 Spraying of bioagent as spore suspension
Spraying of eggplants with spore suspension (5 x 106 spore /ml) of Trichoderma sp.
was done at fruiting stages of the crop through spraying conidial suspension onto all
leaves and fruits of a plant @ 100ml/plant.
24
Plate 5. Pure culture of Trichoderma harzianum
Plate 6. Trichoderma harzianum under compound microscope
25
3.2.13 Spraying of fungicide
The fungicide (Bavistin 50WP) was sprayed at fruiting stage of the crop by a hand
sprayer. Fungicide solution was prepared by taking requisite amount of fungicide
(2gm/L) for optimum concentration.
3.2.14 Inoculation of eggplant by Phomopsis vexans
All leaves and fruits of an eggplant were inoculated with Phomopsis vexans before
and after 24 hr. of treatment with plant extracts. Spore suspension of Phomopsis
vexans (107spores/ml) was sprayed with an atomizer @ 70 ml/plant. The inoculated
plants were kept covered with moist polythene sheet for 24 hr. (Plate. 5)
26
Plate 7. Inoculation of eggplant by Phomopsis vexans
27
3.2.15 Data collection
After inoculation and spraying of fungicide and plant extracts, data were taken at
every 10 days intervals for the following parameters:
1. % Fruit infection
2. % FAD (Fruit area diseased)
Percent FAD (Fruit Area Diseased) was measured by eye estimation. Area of single
fruit was considered as 100%. Deducting the healthy area, the diseased area was
estimated. Average of %FAD was then calculated by dividing the total diseased areas
by the total number of investigated fruits.
Total diseased areas
% FAD = ----------------------------------------------
Total number of investigated fruits
Seed weight of individual plot was also measured after ripening of the fruit.
3.2.16 Seed extraction
The harvested fruits were stored for three to four days until they become soft. This
allows the seed to mature fully. Seeds were extracted by cutting, crushing and
macerating with a mechanical extractor. After extraction, seeds were washed and
cleaned with extra water in a container. Seed drying was done by spreading the wet
seeds in the sun. Seeds were stired with hands at least 2-3 times a day, turning them
over to dry uniformly. The seed should be completely dry to about 8% moisture
content. Amount of seed production was counted in case of each plot.
28
Plate 8. Harvesting of fruit for seed production
29
Plate 9. Extraction of seed from fruit
30
3.2.17 Analysis of data
All collected data were tabulated and analyzed following statistical computer program
(MSTAT-C). Treatment means were compared with Duncan’s Multiple Range Test
(DMRT).
31
RESULTS
4.1 LABORATORY EXPERIMENT
4.1.1 Isolation and identification of causal agent
The causal fungus was isolated from infected fruits and studied in the laboratory. The
fungus was purified and identified as Phomopsis vexans (Fig. 8.). In PDA, the fungus
grew with whitish mycelia which later developed dark gray colony. Conidia of
Phomopsis vexans were hyaline and single celled. There are two types of conidia
(Alpha and Beta) of Phomopsis vexans. Alpha conidia was globose and fusoid and
Beta conidia was hyaline and filiform shaped.
32
Plate 10. Pure culture of Phomopsis vexans
`
Plate 11. Alpha conidia of Phomopsis vexans under compound microscope
33
4.2 FIELD EXPERIMENT
4.2.1 Development of symptoms in brinjal plants following inoculation with
Phomopsis vexans
Seven days after inoculation of the plants by inoculum, leaves showed clearly
defined, circular gray to brown spots with rough coloured centre. The spots coalesced
and affected leaves turned yellow and eventually died. The old spots showed
numerous black pycnidia.
Most of the flowers dropped after 5 days of inoculation. Fruit rot appeared as minute,
globose, water soaked spots with brownish colour, which later enlarged to produce
concentric rings. The outer most ring got separated from the healthy fruit surface.
Spots increased in size and formed large rotten area which produced blackish
pycnidia distributed throughout the rotten fruit. Fruits were mummified. Some
affected fruits dropped off.
Biological control agent Trichoderma harzianum, plant extracts viz. garlic clove
extract, allamonda leaf extract, neem leaf extract, lemon grass extract and fungicide
Bavistin 50WP were applied on cultivated high yielding variety of BARI Begun 8 at
fruiting stage after inoculation of Phomopsis vexans. Saw dust and poultry waste
were also applied in the soil before seedling transplanting to evaluate their
effectiveness against Phomopsis vexans.
34
Plate 12. Infected plant showing phomopsis fruit rot symptom
35
4.2.2 Evaluation ofdifferent treatments against disease incidence of eggplant by
Phomopsis vexans
The effect of Biological control agent Trichoderma harzianum, plant extracts,
Bavistin 50WP, saw dust and poultry waste on disease incidence (percent fruit
infection) at 105 DAT was summarized in table 3. Trichoderma harzianum and
Bavistin 50WP showed significant effect in respect of percent fruit infection in
comparison to control. The highest percent fruit infection was observed in control
plot (26.86) and the lowest percent fruit infection was observed in Trichoderma
harzianum treated plot (9.54). Bavistin 50WP treated plot showed 10.77% fruit
infection. The plant extracts (garlic clove extract, allamonda leaf extract, neem leaf
extract and lemon grass extract) also showed significant effect in reducing percent
fruit infection in comparison to control. However, the effects were statistically similar
among themselves. Among the plant extracts, the lowest fruit infection was observed
in allamonda leaf extract treated plot (14.16%) followed by neem leaf extract
(16.66%), garlic clove extract (20.78%) and lemon grass extract (21.51%).Saw dust
and poultry waste treated plot showed 14.79% and 15.54% fruit infection
respectively.
The effect of biological control agent Trichoderma harzianum, plant extracts,
Bavistin 50WP, saw dust and poultry waste on disease incidence at 114 DAT was
summarized in table 3.Application of fungicide (Bavistin 50WP) and Trichoderma
harzianum significantly reduced percent fruit infection. The lowest percent fruit
infection was observed in Trichoderma harzianum treated plot (14.77) followed by
Bavistin 50WP treated plot (16.53). Among the plant extracts, the lowest fruit
infection was observed in Allamonda leaf extract treated plot (17.96%) followed by
Garlic clove extract (18.29%), Neem leaf extract (19.65%) and Lemon grass extract
36
(24.82%).Saw dust and poultry waste also showed significant effect in reducing
percent fruit infection where20.91% fruit infection was recorded in saw dust treated
plot and 21.46% fruit infection was found in poultry waste treated plot. The control
plot showed 23.64% fruit infection.
The biological control agent Trichoderma harzianum, plant extracts, Bavistin 50WP,
saw dust and poultry waste showed significant effect in terms of disease incidence
(i.e.; percent fruit infection) at 123 DAT in comparison to control. The lowest percent
fruit infection (%FI) was observed in Trichoderma harzianum treated plot (16.52).
Bavistin 50WP treated plot showed 18.43% fruit infection. The plant extracts (garlic
clove extract, allamonda leaf extract, neem leaf extract and lemon grass extract)
showed significant effect in reducing percent fruit infection compared to control. But
effects were statistically similar among themselves. Among the plant extracts, the
lowest fruit infection was found in allamonda leaf extract treated plot (18.60%)
followed by lemon grass extract (20.24%), neem leaf extract (24.16%) and garlic
clove extract (25.31%). Saw dust and poultry waste also showed significant effect in
reducing percent fruit infection (%FI) and percent fruit area diseased (%FAD).
20.37% fruit infection was recorded in saw dust treated plot and 27.13% fruit
infection was found in poultry waste treated plot. The highest percent fruit infection
(%FI) was observed in control plot (29.73%) (Table 3)
37
Table 1. Effect of different treatments on disease incidence of fruit rot of
eggplant at different days after transplanting (DAT)
Treatment Disease Incidence
105 DAT 114 DAT 123 DAT
T1 9.54b 14.77 b 16.52 c
T2 20.78 ab 18.29 ab 25.31 abc
T3 14.16 b 17.96 ab 18.60 bc
T4 16.66 ab 19.65 ab 24.16 abc
T5 21.51 ab 24.82 a 20.24 bc
T6 10.77 b 16.53 ab 18.43 bc
T7 14.79 ab 20.91 ab 20.37 bc
T8 15.54 ab 21.46 ab 27.13 ab
T9 26.86 a 23.64 ab 29.73 a
CV (%) 27.90 30.66 21.28
Here,
T1= Trichoderma harzianum
T2= Garlic clove extract
T3= Allamonda leaf extract
T4= Neem leaf extract
T5= Lemon grass extract
T6= Bavistin 50WP
T7= Saw dust
T8= Poultry waste
T9=Control
38
4.2.3 Evaluation of different treatments against disease severity of eggplant by
Phomopsis vexans
The effect of Biological control agent Trichoderma harzianum, plant extracts,
Bavistin 50WP, saw dust and poultry waste on disease severity (percent fruit area
diseased) at 105 DAT was summarized in table 4. The biological control agent
Trichoderma harzianum and fungicide Bavistin 50WP showed significant effect in
terms of percent fruit area diseased in comparison to control. The highest percent fruit
area diseased (%FAD)was observed in control plot (14.69) and the lowest percent
fruit area diseased (%FAD) was observed in Trichoderma harzianum treated plot
(3.73). Bavistin 50WP treated plot showed 5.09% fruit area diseased (%FAD).The
plant extracts (garlic clove extract, allamonda leaf extract, neem leaf extract and
lemon grass extract) showed significant effect in reducing percent fruit area diseased
(%FAD) in comparison to control. Among the plant extracts, the lowest fruit area
diseased (%FAD) was recorded in allamonda leaf extract treated plot (7.50%)
followed by neem leaf extract (9.98%), garlic clove extract (10.77%) and lemon grass
extract (12.18%). Saw dust and poultry waste also showed significant effect in
reducing percent fruit area diseased in comparison to control. But the effects were
statistically similar between themselves. 8.03% fruit area diseased was observed in
poultry waste treated plot and 8.37% fruit area diseased was observed in saw dust
treated plot. The highest fruit area diseased 14.69% was recorded in control plot.
The effect of biological control agent Trichoderma harzianum, plant extracts,
Bavistin 50WP, saw dust and poultry waste on disease severity (percent fruit area
diseased) at 114 DAT was summarized in table 4. The biological control agent
Trichoderma harzianum and fungicide Bavistin 50WP showed significant effect in
terms of percent fruit area diseased in comparison to control. The lowest percent fruit
39
area diseased (%FAD) was observed in Trichoderma harzianum treated plot (6.09).
Bavistin 50WP treated plot showed 9.64% fruit area diseased (%FAD). Among the
plant extracts, the lowest fruit area diseased (%FAD) was recorded in allamonda leaf
extract treated plot (10.01%) followed by neem leaf extract (10.46%), garlic clove
extract (10.77%) and lemon grass extract (16.99%). Saw dust and poultry waste also
showed significant effect in reducing percent fruit area diseased in comparison to
control. But the effects were statistically similar between themselves. 12.28% fruit
area diseased was observed in poultry waste treated plot and 12.31% fruit area
diseased was observed in saw dust treated plot. 12.71% fruit area diseased was
recorded in control plot.
The effect of Biological control agent Trichoderma harzianum, plant extracts,
Bavistin 50WP, saw dust and poultry waste on disease severity (i.e.; percent fruit area
diseased) at 123 DAT was summarized in table 4. The biological control agent
Trichoderma harzianum and fungicide Bavistin 50WP showed significant effect in
terms of percent fruit area diseased in comparison to control. The highest percent fruit
area diseased (%FAD) was observed in control plot (18.95) and the lowest percent
fruit area diseased (%FAD) was observed in Trichoderma harzianum treated plot
(4.83). 10.01% fruit area diseased (%FAD) was recorded in Bavistin 50WP treated
plot. The plant extracts (garlic clove extract, allamonda leaf extract, neem leaf extract
and lemon grass extract) showed significant effect in reducing percent fruit area
diseased (%FAD) in comparison to control. Among the plant extracts, the lowest fruit
area diseased (%FAD) was recorded in lemon grass extract treated plot (10.48)
followed by allamonda leaf extract (12.73), neem leaf extract (15.14) and Garlic
clove extract (15.55). Saw dust and poultry waste also showed significant effect in
reducing percent fruit area diseased in comparison to control. However, the effects
40
were statistically similar between themselves. The disease severity (%FAD) were
13.94 and 17.45 in saw dust and poultry waste treated plot respectively.
41
Table 2. Effect of different treatments on disease severity of fruit rot of
eggplant at different days after transplanting (DAT)
Treatment Disease Severity (%FAD)
105 DAT 114 DAT 123 DAT
T1 3.737 c 6.090 c 4.830c
T2 10.77 abc 12.74 ab 15.55 ab
T3 7.507 bc 10.01 bc 12.73 ab
T4 9.983 abc 10.46 bc 15.14 ab
T5 12.18 ab 16.99 a 10.48 bc
T6 5.090 bc 9.647 bc 10.01 bc
T7 8.373 abc 12.31 ab 13.94 ab
T8 8.037 abc 12.28 ab 17.45 a
T9 14.69 a 12.71 ab 18.95 a
CV (%) 41.12 29.43 18.60
Here,
T1= Trichoderma harzianum
T2= Garlic clove extract
T3= Allamonda leaf extract
T4= Neem leaf extract
T5= Lemon grass extract
T6= Bavistin 50WP
T7= Saw dust
T8= Poultry waste
T9=Control
42
4.2.4 Effect of different treatments on plant growth characteristics
4.2.4.1 Effect of different treatments on height of plant
The effect of fungicide Bavistin 50WP and Biological control agent Trichoderma
harzianum on height of eggplant was summarized in table 1. Application of
fungicide (Bavistin 50WP) and Trichoderma harzianum significantly influenced
plant height. The highest (89.33cm) height was observed in Trichoderma harzianum
treated plot followed by Bavistin 50WP treated plot (84.33 cm). The effect of plant
extracts on plant height differed significantly in comparison to control. Among the
plant extracts, the highest height was observed in case of Garlic clove extract treated
plot (80 cm), preceded by neem leaf extract (78.67cm), lemon grass extract
(77.33cm) and allamonda leaf extract (76 cm). The effect of saw dust and poultry
waste in terms of plant height was statistically similar compared to control plot. The
lowest plant height was observed in control plot (69 cm).
4.2.4.2 Effect of different treatments on number of branch of eggplant
The effect of fungicide Bavistin 50WPand Biological control agent Trichoderma
harzianum on number of branch per plant was presented in table 1. The highest
number of branch per plant was observed in Trichoderma harzianum treated plot (9)
followed by Bavistin 50WP treated plot (8). The effect of plant extracts on number of
branch per plant were statistically identical among themselves but significantly
different in comparison to control. Among the plant extracts, the highest number of
branch per plant (7.33) was observed in garlic clove extract and neem leaf extract
treated plot preceded by lemon grass extract (6.66) and allamonda leaf extract (6.33).
The effect of saw dust and poultry waste in terms of number of branch per plant was
statistically similar to control. The number of branch per plant of poultry waste and
43
saw dust treated plots was consequently 5.66 and 5.33. The number of branch per
plant in control plot was 5.33.
4.2.4.3 Effect of different treatments on number of leaf per branch of eggplant
The effect of fungicide Bavistin 50WP and Trichoderma harzianum on number of
leaf per branch of eggplant was summarized in table 1. The highest number of leaf
per branch was observed in case of Trichoderma harzianum treated plot (27) and the
second highest (25) was observed in Bavistin 50WP treated plot. The effect of plant
extracts on number of leaf per branch differed significantly in comparison to control
but the effects between them was statistically similar. Among the plant extracts, the
highest number of leaf per branch was observed in garlic clove extract treated plot
(22) preceded by neem leaf extract (21.67), lemon grass extract (20.67) and
allamonda leaf extract (20). The effect of saw dust and poultry waste in terms of
number of leaf per branch differed significantly compared to control. The number of
leaf per branch of saw dust and poultry waste treated plots were respectively 17 and
16. The number of leaf per branch in control plot was 13.
44
Table 3. Effect of different treatments on growth characteristics of eggplant
Treatment Plant
height
(cm)
Number of branch
per plant
Number of leaf
per branch
T1 89.33 a 9.000 a 27.00 a
T2 80.00 c 7.333 abc 22.00 b
T3 76.00 d 6.333 bcd 20.00 b
T4 78.67 cd 7.333 bc 21.67 b
T5 77.33 cd 6.667 bcd 20.67 b
T6 84.33 b 8.000 ab 25.00 a
T7 71.33 e 5.333 d 17.00 c
T8 71.00 e 5.667 cd 16.00 c
T9 69.00 e 5.333 d 13.00 d
CV (%) 1.55 9.84 4.96
Here,
T1= Trichoderma harzianum
T2= Garlic clove extract
T3= Allamonda leaf extract
T4= Neem leaf extract
T5= Lemon grass extract
T6= Bavistin 50WP
T7= Saw dust
T8= Poultry waste
T9=Control
45
4.2.5 Effect of different treatments on fruit and seed yield of eggplant
The effect of fungicide Bavistin 50WP and biological control agent Trichoderma
harzianum on fruit yield was summarized in table 5. Application of fungicide
(Bavistin 50WP) and Trichoderma harzianum significantly increased fruit yield in
comparison to control. The highest (36.38 ton/ha) fruit yield was observed in
Trichoderma harzianum treated plot where the second highest (35.01 ton/ha) was
observed in Bavistin 50WP treated plot. The effect of plant extracts on fruit yield
differed significantly in comparison to control but the effects were statistically
similar. Among the plant extracts, the highest fruit yield was observed in lemon grass
extract treated plot (27.99 ton/ha) preceded by neem leaf extract (27.28 ton/ha), garlic
clove extract (27.11 ton/ha) and allamonda leaf extract (26.27 ton/ha).The effect of
saw dust and poultry waste in terms of fruit yield was statistically identical. The
plants of poultry waste and saw dust treated plots consequently produced 18.39 ton/ha
and 16.15 ton/ha fruit. 14.67 ton/ha fruit yield was found in control plot.
The effect of fungicide Bavistin 50WP and biological control agent Trichoderma
harzianum on seed yield was summarized in table 5. Application of fungicide
(Bavistin 50WP) and Trichoderma harzianum significantly increased seed yield. The
highest (35.67 gm/plot) seed yield was observed in Trichoderma harzianum treated
plot and the second highest (32.48 gm/plot) was observed in Bavistin 50WP treated
plot. The effect of plant extracts in terms of increasing seed yield differed
significantly in comparison to control. Among the plant extracts, the highest seed
yield was observed in garlic clove extract treated plot (28.51 gm/plot), preceded by
lemon grass extract (28.07 gm/plot), neem leaf extract (24.85 gm/plot) and allamonda
leaf extract (13.95 gm/plot). The effect of saw dust and poultry waste in terms of seed
yield was statistically similar compared to control plot. The plants of saw dust and
46
poultry waste treated plots consequently produced 9.88 gm/plot and 15.05 gm/plot
seed. Seed production found in control plot was 10.77 gm/plot.
47
Table 4. Effect of different treatments on fruit and seed yield of eggplant
Treatment Fruit yield (tonha-1
) Seed Yield (gm/plot)
T1 36.38a 35.67a
T2 27.11b 28.51bc
T3 26.27b 13.95d
T4 27.28b 24.85c
T5 27.99b 28.07bc
T6 35.01a 32.48ab
T7 16.15c 9.880d
T8 18.39c 15.05d
T9 14.67c 10.77d
CV (%) 9.32 9.48
Here,
T1= Trichoderma harzianum
T2= Garlic clove extract
T3= Allamonda leaf extract
T4= Neem leaf extract
T5= Lemon grass extract
T6= Bavistin 50WP
T7= Saw dust
T8= Poultry waste
T9=Control
48
DISCUSSION
The eggplant belongs to the family Solanaceae, is a popular, nutritious and widely
grown vegetable in Bangladesh as well as in the world. About 8 million farm families
are involved in eggplant cultivation in Bangladesh. Its position in terms of acreage
production is second in vegetable crops and the total acreage of eggplant is 44,377
acres with total annual production of 3,68,000 metric tons in the country (BBS,
2013). A large number of cultivars are grown in Bangladesh, which is showing a wide
range of variation in yield performance. It is a continuous source of income for the
rural farmers and provides employment facilities for the rural people. Therefore, it
plays a vital role to boost our national economy. It is grown round the year especially
during the lean period when the seasonal vegetables are in a scarcity in the market.
Eggplant, thus regarded as a cash crop to the farmers which provides them continuous
harvesting and financial assistance.
Such a potential crop is known to suffer from 12 diseases and among them phomopsis
fruit rot caused by Phomopsis vexans has been treated as one of the major constraints
in eggplant cultivation in the country (Khan, et al., 2002; Das, 1998).The infected
fruit develops pale and sunken spots that progress covering the entire surface and
cause dropping. Affected portion is mummified producing huge pycnidia on the
surface and becomes non-edible (Singh, 1992; Ashrafuzzaman, 1986).
The organism isolated from diseased fruits collected from the field and experimental
plot was resembled with those described by Punithalingum and Holliday (1972),
Islam (2005) and CMI description No. 338.
49
Effect of Trichoderma harzianum and plant extracts against phomopsis fruit rot
of eggplant caused by Phomopsis vexans
From the field study it was evident that the pathogen Phomopsis vexans was
effectively controlled by Trichoderma harzianum. Spraying of spore suspension of
Trichoderma harzianum reduced disease incidence to 9.54, 14.77 and 16.52 at 105,
114 and 123 DAT, respectively. Disease severity was reduced to 3.73, 6.09 and 4.83
at 105, 114 and 123 DAT, respectively. The findings of the present investigation are
well supported by the findings of Meah and Howlader (2003). Howlader (2003)
reported that spraying of spore suspension of T. harzianum CP yielded good result
against phomopsis blight and fruit rot of eggplant in the field.
Evidence of using Trichoderma spp. against plant pathogens especially against fungal
pathogens are available in the literature (Shamsuzzaman et al; 2003a; Bari et al; 2000;
Prassad and Rangeshwarn, 2000; Kaur and Mukhapaddhyay, 1992; Kumar and
Khare, 1990; Harman et al., 1989; Sivan, et al., 1984; Agrawal et al., 1977) however
research with Trichoderma spp. against P. vexans are few. Meah (2003) found that T.
harzianum CP significantly controlled the nursery diseases caused by Phomopsis
vexans and increased seed germination by 49% over control. He also reported that
spraying of spore suspension of T. harzianum CP was effective in controlling
phomopsis blight and fruit rot of eggplant in the field.
Different strains of T. harzianum act as strong competitors to other pathogenic
microorganisms in the rhizosphere colonizing the root zone. (Harman et al. 1989).
The account for disease reduction by Trichoderma strains was explained as the
competition for space and nutrients with the pathogenic fungi (Alabouvette et al.
1992) and the production of antibiotics by the antagonists (Larkin et al. 1996). Elad et
50
al., (1982) reported the lylic activity of extra cellular enzymes of T. harzianum that
aided to antagonize the pathogen.
The plant extracts also effectively controlled the disease reducing fruit infection
significantly. Garlic clove extract reduced disease incidence to 20.78, 18.29 and 25.31
at 105, 114 and 123 DAT respectively. In garlic clove extract treated plot disease
severity was reduced to 10.77, 12.74 and 15.55 at 105,114 and 123 DAT,
respectively. The efficacy of garlic clove extract in controlling P. vexans has been
reported by other workers. Inhibition of pathogenic fungi by garlic bulb extract might
be due to the presence of antimicrobial compounds in the extract. It has been reported
that the antibiotic substance present in garlic is the allyl compound of allyl
thiosulphate (Cavallito et al.1944). It is also reported that garlic contains an amino
acid alliin which on crush transferred into allicin by the action of allicinase enzymes
and this allicin is toxic to microorganism (http//:www.gourmetgarlicgardens.com).
Allamonda leaf extract also reduced disease incidence to 14.16%, 17.96% and 18.6%
at 105, 114 and 123 DAT, respectively and disease severity to 7.50%, 10.01% and
12.73% at 105, 114 and 123 DAT, respectively. The findings of the present
investigation are well supported by the findings of Khan et al.(2002), Kuprasvile
(1996), Panda et al.(1996) and Mohanty et al., (1995) who reported that allamonda
leaf extract inhibited the growth of P. vexans significantly. Tiwari et al., (2002)
reported that Allamonda cathertica acted as antidermatophitic agent against fungi.
Reports on the toxic compounds of allamonda leaf contains some substances toxic to
the micro-organisms (Hwang, 2001).Some unidentified compounds extracted from
Allamanda cathertica using organic solvents prevented growth of P. vexans in culture
at unspecified concentrations (Masuduzzaman et al., 2008). In the present
investigation, neem leaf extract also reduced disease incidence and disease severity
51
effectively. Disease incidence was reduced to 9.54, 14.77 and 16.52 at 105, 114 and
123 DAT, respectively and disease severity to 9.98, 10.46 and 15.14 at 105, 114 and
123 DAT, respectively. The present results of effectiveness of neem leaf extract
corroborate with the report of other workers. Neem and its products have been widely
reported to control plant fungal diseases (Vir and Sharma, 1985; Amadioha, 2000;
Dubey et al., 2009).The bio-efficacy of neem extract over pathogens can be attributed
to the fact that neem has active compounds such as azadirachtin, nimbin, nimbidin,
nimbinene and azadirone which are antifungal, antibactrial and anti-insecticidal in
nature (Bohra et al., 2006). From the field study it was evident that the pathogen
Phomopsis vexans was effectively controlled by lemon grass extract. Lemon grass
extract reduced upto 21.51, 24.82 and 20.24disease incidence at105, 114 and 123
DAT, respectively. It also reduced disease severity to 12.18, 16.99 and 10.48 at 105,
114 and 123 DAT, respectively. The findings of the present investigation are well
supported by the findings of Ogunlana et al. (1987) who described antimicrobial
effects of lemon grass, including activity against both gram-positive and gram-
negative bacterial pathogens and fungi.
Effect of Bavistin 50WP, saw dust and poultry waste against Phomopsis vexans
Bavistin 50WP was found promising against P. vexans in the field. Bavistin reduced
disease incidence to 10.77, 16.53 and 18.43 at 105,114 and 123 DAT respectively.
Disease severity was also reduced to 5.09, 9.64 and 10.01 at 105, 114 and 123 DAT,
respectively. The present findings are in agreement with the Meah (2003), Meah et al.
(1998), Islam and Sitansu (1992) and Islam et al.(1990). Meah (2003) reported that
Bavistin 50WP (0.1%) significantly controlled phomopsis blight and fruit rot of
eggplant in the nursery and as well as in the field condition. Meah et al. (1998) also
stated that Bavistin 50WP @ 0.2% applied both before and after inoculation of P.
52
vexans on the surface rough fruit showed effective performance in reducing percent
fruit infection and percent fruit area diseased producing the least sized lesion. Islam
and Sitansu (1992) reported that in a field trial against Phomopsis vexans, Bavistin
50WP (0.1%) provided nearly complete control of leaf blight and fruit rot of eegplant
with 3 sprays at 15 days interval. Islam et al. (1990) found in field trial following in
vitro screening of different fungicides, Bavistin 50WP @ 0.1% gave the best control
against phomopsis blight and fruit rot of eggplant. In the present experiment, saw dust
and poultry waste were also found effective against P. vexans. Saw dust showed
lower disease incidence than poultry waste. Saw dust treated plot showed 14.79,
20.91 and 20.37 disease incidence at105, 114 and 123 DAT, respectively. Poultry
waste treated plot showed 15.54, 21.46 and 27.13 disease incidence at 105, 114 and
123 DAT, respectively. In case of disease severity, it was found that saw dust treated
plot showed 8.37, 12.31 and 13.94 disease severity at 105,114 and123 DAT,
respectively. Poultry waste treated plot showed 8.03, 12.28 and 17.45 disease severity
at 105,114 and123 DAT, respectively. Saw dust and poultry waste are now being
considered as environment friendly approach that make the soil suppressive
improving the antagonistic activities of the soil microorganisms. The findings of the
present study corroborate with the findings of Bhuyan (2010) and Dataram (1988).
Bhuyan (2010) reported that poultry waste was found potential for controlling fungus
diseases like rhizome rot of ginger. Dataram (1988) reported that the incidence of
rhizome rot was low when Trichoderma viride was applied to soil along with wood
saw dust.
53
SUMMARY AND CONCLUSION
Phomopsis fruit rot is one of the most important biotic disease that significantly
reduce eggplant production. The fungus Phomopsis vexans is seed borne and can
survive in crop debris in the absence of the host. The fungus can survive for more
than a year in fields where a diseased crop was grown and is mainly favoured by
warm, wet weather. People often manage the disease with plant protection chemicals.
But it is environmentally hazardous and hampers our ecosystem. Thus, finding out the
alternatives of chemical fungicides with eco-friendly components the present
investigation has been undertaken to evaluate the effect of bio-agent, plant extracts
and soil amendments against the disease and find out the suitable management
components for controlling phomopsis fruit rot in our country. The present
experiments were conducted during Rabi season (October, 2014 to March, 2015) in
the Horticultural farm of Sher-e-Bangla Agricultural University. A biological control
agent (Trichoderma harzianum), four plant extracts viz. garlic clove extract,
allamonda leaf extract, neem leaf extract and lemon grass extract, a fungicide
(Bavistin 50WP), saw dust and poultry waste were evaluated against Phomopsis
vexans causing phomopsis fruit rot.
Trichoderma harzianum showed promising performance in reducing percent fruit
infection, percent fruit area diseased and seed infection. Fruit and seed yield was
found the highest in case of Trichoderma harzianum treated plot. Fruit yield and seed
yield was 36.38 ton/ha and 35.67 gm/plot in Trichoderma harzianum treated plot,
respectively. Fruit and seed yield were also found promising in Bavistin 50WP
treated plot where fruit yield and seed yield were 35.01 ton/ha and 32.48gm,
respectively.
54
Among the four plant extracts, allamonda leaf (1:2) extract and neem leaf (1:2)
extract found to be effective against Phomopsis vexans reducing percent fruit
infection and percent fruit area diseased. The fruit yield and seed yield were more or
less similar among the plant extracts treated plot and higher than the control plot. Saw
dust and poultry waste were also found effective in increasing fruit and seed yield.
Considering the performances of bio-agent, plant extracts, fungicide and soil
amendments evaluated in the experiment it is suggested that Trichoderma harzianum,
Bavistin 50WP, allamonda leaf extract and neem leaf extract at 1:2 concentration
could be used for successful management of phomopsis fruit rot of eggplant.
55
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65
APPENDICES
Appendix I. Map showing the experimental site under study
Source: https://www.google.com.bd/maps/place/horticulture-farm
66
Appendix II. Physiochemical properties of soilof the experimental site
Source: Soil Resources Development Institute (SRDI), Dhaka-1207. (Year-
2014)
Characteristics Value
Particle size analysis
% Sand
% Silt
% Clay
Textural class
pH
Organic carbon (%)
Organic matter (%)
Total N (%)
Phosphorus(µg/g soil)
Exchangeable K (me/100 g soil)
Sulphur (µg/g soil)
Boron (µg/g soil)
Zinc (µg/g soil)
25.68
53.85
20.47
Silty-loam
5.8-7.1
0.31
0.54
0.027
23.66
0.60
28.43
0.05
2.31
67
Appendix III. Monthly average relative humidity, maximum and minimum
temperature, rainfall and sunshine hour of the experimental period (October 2014
to March 2015)
Source: Bangladesh Meteorological Department (Climate division), Agargaon, Dhaka
-1207.
Month Average RH
(%)
Average Temperature (ºC) Total
Rainfall
(mm)
Average
Sunshine
hours Min. Max.
March 64 20.4 32.5 65.8 5.2
April 69 23.6 33.7 165.3 4.9
May 81 24.5 32.9 339.4 4.7
June 84 25.4 33.7 415.6 4.8
July 89 27.5 34.8 512.4 4.7