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Control and Management of Soil Borne Fungal Pathogens ofBetelvine
Control of Soil Borne Fungi of Betelvine by Botanicals
The control of fungal plant diseases was mainly depends on the
fungicides, the regular use of fungicides has to be limited due to its costs,
adverse environmental hazards, besides development of resistance in pathogens
(Anandaraj and Leela, 1996). Disease resistance in some plants is due to the
presence certain chemical substances in the host tissues are know to show the
antifungal activity (Shekhawat and Prasada, 1971). In the present study the
plant extracts of Azadirachta indica and Allium cepa were tested for their
antifungal activity against the isolated soil borne fungi of betelvine by poison
food technique (Dhingra and Sancliar, 1993f).
Materials and methods
The different concentrations of plant extract like 10%, 30%, 60%, 80%
and 100% were prepared by diluting with sterile distilled water. Two ml of
poison (plant extract) was pipetted into the autoclaved petriplates of size 9cm
and for the control only 2ml of distilled water was added. Later it was then
mixed aseptically with 20ml of sterile, melted and cooled (40 °C) PDA, which
was supplemented with antibacterial. Then PDA was poured equally to the
autoclave-sterilized petriplates (four replications were maintained for each
concentration).
After solidification the plates were inoculated with 5mm mycelial discs
of test fungi at the center of the petriplate that was taken from the actively
growing seven day old culture growing on the PDA with the cork borer and
then the inoculated petiplates were incubated at 28 ± 2°C temperature under day
light tubes with arrangement for alternate periods of 12 hours light and 12 hours
darkness (Shukla et al, 1972; Dhingra and Sancliar, 1993b; Dipak et al., 1999).
The colony diameter was measured after 48 h of incubation (Aneja, 1996c).
80
Control and Management of Soil Borne Fungal Pathogens of Betelvine
The radial growth of mycelium was measured at two points along the
diameter of the plate and the mean of these two readings was taken as the
diameter of the colony (Anandaraj and Leela, 1996). The inhibition percentage
of the fungal colony due to different concentrations of poison was calculated by
using the formula C-T/C x 100 (Vincent, 1947). Where ' C is the colony
diameter in the control and 'T' is the colony diameter in treated.
Preparation of Garlic extract
Cold water extract of garlic was prepared (Sarvamangala,ef ai,
1993;Monica and Gupta, 2003) 50g of garlic bulb (Allium cepa) was taken in
100ml of distilled water, it is macerated in a blender and it is filtered in double
layered muslin cloth. The filtrate is the stock solution, the stock solution itself is
100%(Chendrasekaran and Rajappan, 2002). It was diluted with distilled water
to get the different dilutions like 10, 30, 60, 80 % respectively.
The obtained extracts were stored in the refrigator at 4°C , but not for
more than 48 hours ( Dubey and Dwivedi, 1991). The in-vitro evaluation of its
antifungal activity was done against the test fungus by employing poison food
technique (Vincent, 1947; Singh and Thapliyal, 1986 and Dipak et al., 1999).
Preparation of Neem extract
Cold water extract of neem was prepared (Monica and Gupta, 2003)
200g of neem leaves (Azadirachta indicia L.) was taken, it was first washed
under tap water and then in distilled water, it is macerated in a blender and
filtered in double layered muslin cloth (Jayashree et a/., 1999). The filtrate is the
stock solution, the stock solution itself is 100%. The different dilutions like 10,
30, 60, 80 % , were prepared from the stock respectively (Chendrasekaran and
Rajappan, 2002).
81
Control and Management of Soil Borne Fungal Pathogens ofBetelvine
The in-vitro evaluation of its antifungal activity was done against the test
fungus by employing poison food technique (Vincent, 1947; Dipak et ah,
1999).
Results
The plant extracts of Allium sativum (Garlic) and Azadirachta indica
(Neem) were showed antifungal activity against the isolated soil borne fungi of
betelvine like Phytophthora parasitica, Pythium vexans, Fusarium oxysporum,
Rhizoctonia solani and Sclerotium rolfsii was given in Tables 5.1 to 5.5 and
Figures 4.67 to 4.76.
At 10%, 30%, 60%, 80% and 100% concentrations Allium sativum
showed inhibition of Phytophthora parasitica colony by 20.56%, 25.81%,
40.32%, 47.58% and 53.63% respectively. Azadirachta indica at 10%, 30%,
60%, 80% and 100% concentrations showed inhibition percentage of
Phytophthora parasitica colony by 21.37%, 26.61%, 28.23%, 28.63% and
34.68% respectively (Table 5.1)(Fig 4.67, 4.68 and 4.77).
Allium sativum at 10%, 30%, 60%, 80% and 100%) concentrations
showed inhibition percentage of Pythium vexans colony by 12.50%, 25.00%,
49.57%o, 52.59% and 100% respectively. Azadirachta indica at 10%, 30%,
60%, 80% and 100% concentrations showed inhibition percentage of Pythium
vexans colony by 0.86%, 2.59%, 15.95%, 18.53%, and 48.71% respectively
(Table 5.2)(Fig 4.69, 4.70 and 4.78).
Allium sativum at 10%, 30%, 60%, 80% and 100% concentrations
showed inhibition percentage of Fusarium oxysporum colony by 34.72%,
62.96%, 100%, 100% and 100% respectively.
82
Control and Management of Soil Borne Fungal Pathogens of Betelvine
Azadirachta indica at 10%, 30%, 60%, 80% and 100% concentrations
showed inhibition percentage of Fusarium oxysporum colony by 3.70%, A.(i7>%,
10.19%, 15.28% and 30.09% respectively (Table 5.3) (Fig 4.71, 4.72 and 4.79).
Allium sativum at 10%, 30%, 60%, 80% and 100% concentrations
showed inhibition percentage of Rhizoctonia solani colony by 31.57%, 82.53%,
88.16%, 100% and 100% respectively. Azadirachta indica at 10%, 30%, 60%,
80% and 100% concentrations showed inhibition percentage of Rhizoctonia
solani colony by 1.69%, 2.59%, 7.22%, 10.71% and 20.41% respectively
(Table 5.4) (Fig 4.73, 4.74 and 4.80).
Allium sativum at 10%), 30%, 60%, 80%) and 100% concentrations
showed inhibition percentage of Sclerotium rolfsii colony by 56.80%, 63.89%,
100%, 100% and 100% respectively. Azadirachta indica at 10%), 30%, 60%,
80% and 100% concentrations showed inhibition percentage of Sclerotium
rolfsii colony by 15.42%, 32.05%, 35.70%, 45.84% and 65.92% respectively
(Table 5.5)(Fig 4.75, 4.76 and 4.81). Both the plant extracts of Allium sativum
and Azadirachta indica were showed antifungal activity at the said
concentrations (Tables 5.1 to 5.5).
Discussion
The plant extracts of Allium sativum (Garlic) and Azadirachta indica
(Neem) were showed more antifungal activity against the isolated soil borne
fungi of betelvine like Phytophthora parasitica, Pythium vexans, Fusarium
oxysporum, Rhizoctonia solani and Sclerotium rolfsii was given in Tables 5.1 to
5.5 and Figures 4.67 to 4.76. At Five percent level of stastical significance the
concentrations of Allium sativum and Azadirachta indica which were showed
significant inhibition percentage on the colony growth of pathogenic fungal
were discussed.
83
Control and Management of Soil Borne Fungal Pathogens ofBetelvine
At 30%, 60%, 80% and 100% concentrations. Allium sativum inhibited
the Phytophthora parasitica colony by 25.81%, 40.32%, 47.58% and 53.63%
respectively. Whereas Azadirachta indica at 30%, 60%, 80% and 100%
concentrations showed inhibition percentage of Phytophthora parasitica colony
by 26.61%, 28.23%, 28.63% and 34.68% respectively (Table 5.1)(Fig 4.67,
4.68 and 4.77).
At 10%, 30%, 60%, 80% and 100% concentrations, Allium sativum
inhibited Pythium vexans colony by 12.50%, 25.00%, 49.57%, 52.59% and
100% respectively. Azadirachta indica at 60%, 80% and 100% concentrations
showed inhibition percentage of Pythium vexans colony by 15.95%, 18.53%,
and 48.71% respectively (Table 5.2)(Fig 4.69, 4.70 and 4.78).
Allium sativum at 10%o, 30%, 60%o, 80%) and 100%; concentrations
showed inhibition percentage of Fusarium oxysporum colony by 34.72%,
62.96%, 100%, 100% and 100% respectively. Azadirachta indica at 80% and
100% concentrations showed inhibition percentage of Fusarium oxysporum
colony by 15.28% and 30.09% respectively (Table 5.3) (Fig 4.71, 4.72 and
4.79).
At 10%, 30%, 60%, 80% and 100% concentrations Allium sativum
inhibited Rhizoctonia solani colony by 31.57%, 82.53%o, 88.16%, 100% and
100% respectively. Azadirachta indica at 80% and 100% concentrations
showed inhibition percentage of Rhizoctonia solani colony by 10.71% and
20.41% respectively (Table 5.4) (Fig 4.73, 4.74 and 4.80). Allium sativum at
10%, 30%, 60%, 80% and 100% concentrations showed inhibition percentage
of Sclerotium rolfsii colony by 56.80%, 63.89%, 100%, 100% and 100%
respectively. Azadirachta indica at 30%, 60%, 80% and 100% concentrations
showed inhibition percentage of Sclerotium rolfsii colony by 32.05%, 35.70%,
45.84% and 65.92% respectively (Table 5.5)(Fig 4.75, 4.76 and 4.81).
84
Control and Management of Soil Borne Fungal Pathogens ofBetelvine
Both the plant extracts of Allium sativum and Azadirachta indica were
showed antifungal activity at the said concentrations (Tables 5.1 to 5.5), but the
overall performance of Allium sativum was more than Azadirachta indica under
in-vitro condition .The extracts of A. indica which was showed lesser activity
(Figures 4.67 to 4.81) the similar observations was made by Anandaraj and
Leela (1996); Gohil and Vala (1996);Chattopadhyay et al., (2002) Qais et al,
(2004).
In-vltro Control of Soil Borne Fungal pathogens of betelvine by Dual culture technique
The soil borne fungal pathogens of betelvine were capable of surviving
on the 'non-host' plant residues and showed continued activity throughout the
season (Table 3.4) (Fig 4.7 and 4.8) and was very difficult to control these soil
borne fungal pathogens of betelvine.
The disease control can be achieved by chemical, biological and
agronomical practices. Though the control of diseases by systemic fungicides
are quite promising (Sinclair, 1971 and Backman, 1978), but the frequent and
indiscriminate use of fungicides often leads to atmospheric pollution and
development of fungicide resistance in pathogens (Gattani, 1951 and Thind et
al, 2001). In this context, biological control is coming up as an alternative
strategy for disease management, which is also environment friendly
(Manoranjitham, et al, 2001) and the biological control of plant pathogens by
the addition of antagonists like Trichoderma offers an alternative method to
combat the ravages of soil borne diseases (Abraham and Gupta, 1998 &
Thirumala and Sitaramaiah, 2000).
85
Control and Management of Soil Borne Fungal Pathogens ofBetelvine
In the present study, the control and management of soil borne fungal
pathogens was concentrated on biological control, the chemical control was
eliminated because of the residual effects, that persist in leaves (Dixit, et al,
1994), since our betelvine crop yields leaves and these leaves are consumed as
raw, the chemical control of diseases cannot be recommended, so the control
and management of soil borne fungal pathogens was concentrated on biological
control. Control of disease with the help of the activity of some organisms or
organism is known as biological control (Saksena, 1969b).
Betelvine is an important commercial crop of Karnataka this plant is
very sensitive to temperature, soil moisture, soil condition, nutrient level and
agronomic practices like manuring, pruning, lowering, trailing and layering, it
is also sensitive to slight fluctuations on climatic factors the higher heat of
scorching sun will makes the leaf to dry (Fig 4.82 and 4.83). Betelvine is also
sensitive to disease, many serious plant diseases are associated with soil borne
plant pathogens, which cause root rot, crown or collar rot, damping- off,
blights, wilts in field and horticultural crops. Efficient management of soil-
borne diseases is possible only through good knowledge of crop husbandry,
physical, chemical and biological properties of soil, ecology and epidemiology
of root diseases.
Among the various diseases of betelvine the soil borne fungal diseases
were very important, they were causing foot rot, collar rot, and wilt. Regarding
the soil borne fungal diseases the visual symptoms appears only when the root
system and the vascular system has been completely damaged, at that time it
was too late to do any thing, to take any remedial measures for the recovery of
diseased plant. The diseases due to soil borne fungi is one of the major
problems facing in many parts of the world.
86
Control and Management of Soil Borne Fungal Pathogens ofBetelvine
Materials and methods
The soil samples were collected from the rhizosphere soil of the healthy
betelvines, the Trichoderma species were isolated from these collected soils by
following soil serial dilution method. The Trichoderma species were identified
by its morphological characters like colony characters, conidial morphology
and philiades. The ability of native isolates to inhibit the growth of S.rolfsii
under in-vitro conditions was determined through dual culture technique
(Morton and Stroube, 1955).
The antagonistic activity of each isolate was measured by using a
modified Bell's Scale (Bell et al, 1982). Five millimeter-diameter discs of
Trichoderma isolates were removed from the edge of colonies of 7-day-old
PDA cultures and placed on one side of a petri dishes containing PDA medium.
Similar dishes of Sclerotium rolfsii grown in the same manner were placed on
the opposite side of petri dishes. Each treatment was replicated for four times.
Cultures were observed daily and recorded for colony growth, and
antagonism of Trichoderma spp, against S. rolfsii. This was done because
among the isolated soil borne fungal pathogens of betelvine, Sclerotium rolfsii
showed strong resistance against the Trichoderma isolates compared with other
soil fungal pathogens like Phytophthora, Pythium, Fusarium and Rhizoctonia
(Fig 4.88, 4.89 and 4.90). Among the native isolates of Trichoderma spp, the
isolate Trichoderma harzianum was selected for the further work as it has
performed well against the Sclerotium rolfsii than the rest of the isolates (Table
5.6) and (Fig 4.92 and 4.93).
Later the Trichoderma harzianum isolate was tested for its ability to
inhibit the growth of soil borne fungal pathogens of betelvine like Phytophthora
parasitica, Pythium vexans, Fusarium oxysporum, and Rhizoctonia solani,
87
Control and Management of Soil Borne Fungal Pathogens of Betelvine
under in-vitro conditions. The seven day old cultures of Phytophthora
parasitica, Pythium vexans, Fusarium oxysporum, Rhizoctonia solani ,
Sclerotium rolfsii and Trichoderma harzianum which were growing on PDA
were taken. With the help of sterilized cork borer, 5mm mycelial discs were
taken from the periphery of the actively growing seven day old colony.
The T. harzianum was allowed to grow along with the above said
pathogens separately. The 5mm mycelial disc of T.harzianum and 5mm
mycelial disc of P. parasitica were placed on PDA taken in petriplates. The
5cm distance was maintained between the mycelial discs, the same was done
for the rest of the pathogens.
For the control the 5mm mycelial discs of pathogens were inoculated
separately on PDA and allowed to grow without T. harzianum. Four
replications were maintained for each pathogen. The inoculated cultures were
incubated at 28±2°C. After 72 h of incubation the colony diameter was
measured in centimeters. The inhibition percentage of fungal colony of
Phytophthora parasitica, Pythium vexans, Fusarium oxysporum, Rhizoctonia
solani and Sclerotium rolfsii due to T. harzianum was calculated by the formula
C-T/C X 100 (Vincent, 1947). Where ' C is the colony diameter of pathogens in
the control and 'T' is the colony diameter of pathogens when grown with T.
harzianum as dual culture.
Results
Among the isolates Trichoderma viride 1 (TVl), Trichoderma viride 2
(TV2) and Trichoderma harzianum (TH) were tested against the antagonistic
activity against Sclerotium rolfsii. Among the Trichoderma isolates
Trichoderma harzianum showed more antagonistic activity then the rest of the
isolates (Table 5.6) (Fig 4.90).
88
Control and Management of Soil Borne Fungal Pathogens of Betelvine
The inhibition percentage of Phytophthora parasitica due to T.
harzianum was 30.67% and the colony diameter ratio of Phytophthora
parasitica : T. harzianum was 2:5. The inhibition percentage of P. vexans due
to T. harzianum was 22.69% and the colony diameter ratio of P. vexans : T.
harzianum was 1:2. The inhibition percentage of F.oxysporum due to T.
harzianum was 44.32% and the colony diameter ratio of F.oxysporum: T.
harzianum was 3:8.
The inhibition percentage of R. solani by T. harzianum was 9.21% and
the colony diameter ratio of R. solani: T. harzianum was 9:5. The inhibition
percentage of S. rolfsii due to T. harzianum was 41.91% and the colony
diameter ratio of 5. rolfsii: T. harzianum was 4:5.
Discussions
The biocontrol approach was adopted since the chemical method will
damage the native microflora and soil properties (Manoranjitham et al, 2001).
The native isolate of Trichoderma spp was isolated from the soils of the
betelvine garden by serial dilution agar plating technique and identified (Rifai,
1969). T. harzianum isolate(TH) grew rapidly at room temperature(Fig 4.90),
cultures at first were white and cottony then turned to bright green, finally they
became dark green. Single phialides arose laterally on the conidiophores and in
clusters of 2-4 terminally; they were short, averaged 11.88 x 3.94 \im.
Phialospores arose by budding from the tips of phialides: they were smooth-
walled, short obovoid, averaged 1.98 x 3.96 fxm (Fig 4.84).
Trichoderma viride Pers. Ex Fries, the vegetative hyphae of
Trichoderma viride isolates (TVl and TV2) grew fairly rapidly at room
temperature, mycelium fluffy, cultures were white, gradually become whitish
green, ultimately appeared dark green (TVl) (Fig 4.88) and pale green (TV2)
89
Control and Management of Soil Borne Fungal Pathogens of Betelvine
(Fig 4.89). Phialides were long, nin-pin-shaped (Narrow at base and more so
above), arise singly or opposite (TV2) or in groups of two or three (TVl), the
averaged length of the isolate TVl has 11.98 x 5.94 [Am and TV2 has 7.92 x
3.96 ^m. Each have the head of containing about ten to thirty or more spores
held together. Conidia globoid averaged size 4.59 jxm (TVl) and 4.04|xm
(TVl). One celled, smooth walled with inconspicuous roughenings.
Among the isolates Trichoderma viride 1, Trichoderma viride 2 and
Trichoderma harzianum were tested against the antagonistic activity against
Sclerotium rolfsii. Among the Trichoderma isolates Trichoderma harzianum
showed more antagonistic activity then the rest of the isolates (Table 5.6). This
Trichoderma harzianum is believed to be a potential bio-control agent as it can
parasitise many soil borne fungal pathogens (Homer, 1993).
However, even the fast growing like Rhizoctonia solani was later
parasitised by Trichoderma harzianum (Fig 4.93) (Bakshi and Singh, 1956).
The native isolate Trichoderma harzianum was found successful potential
biocontrol agent, as it has parasitised all the isolates of soil borne fungi of
betelvine like Phytophthora parasitica, Pythium vexans (Shanmugam and
Sukurana, 1999), Fusarium oxysporum, Rhizoctonia solani and Sclerotium
rolfsii (Weindling,1932 and 1934; Deb and Dutta, 1992; Mehrothra et al,1993 ;
Hoitink et al, 1997; Pandey et al, 1999a & 1999b; Suseelendra and Schlosser,
1999 & Yogendra singh, 2002) (Fig 4.92 and 4.93). The inhibition percentage
on the colony growth of Phytophthora parasitica, lithium vexans (Shanmugam
and Sukurana, 1999), Fusarium oxysporum, Rhizoctonia solani and Sclerotium
rolfsii due to the presence of T. harzianum was given in the Table (5.7) and Fig
(4.93).
90
Control and Management of Soil Borne Fungal Pathogens of Betelvine
The inhibition percentage of Phytophthora parasitica due to T.
harzianum was 30.67% and the colony diameter ratio of Phytophthora
parasitica : T. harzianum was 2:5. The inhibition percentage of P. vexans due
to T. harzianum was 22.69% and the colony diameter ratio of P. vexans : T.
harzianum was 1:2.
The inhibition percentage of F. oxysporum due to T. harzianum was
44.32% and the colony diameter ratio oiF. oxysporum: T. harzianum was 3:8.
The inhibition percentage of R. solani due to T. harzianum was 9.21%
and the colony diameter ratio oiR. solani: T. harzianum was 9:5.
The inhibition percentage of S. rolfsii due to T. harzianum was 41.91%
and the colony diameter ratio of 5. rolfsii: T. harzianum was 4:5.
In all the cases the colony ratio with the pathogens is higher except in
case of R. solani (Table 5.7). Since R. solani is one of the fast growing fungi
(Dhingra, and Sinclair, 1993g), as it occupies majority of the available space in
the petriplate (Fig 4.92), but it was later parasitised by T. harzianum (Fig 4.93).
In the present study the native isolate T. harzianum was found to be a
potential biocontrol agent under in-vitro condition. The success of Trichoderma
harzianum lies in its rapid colonization, mycoparasitism and exceptionally high
growth rate (Saksena, 1969b)(Table 5.7)(Fig 4.91, 4.92 and 4.93).
Mass multiplication of Trichoderma harzianum
The performance of the native isolate Trichoderma harzianum under in-
vitro condition was quite considerable. The mass production of T. harzianum
inoculum was carried out by using locally available substrates like paddy straw
(Sangeetha and Jeyarajan, 1993), saw dust (Bunker and Kusum, 2001) and
91
Control and Management of Soil Borne Fungal Pathogens of Betelvine
sorghum grains were used as substrate for preparation of inoculum seeds
(Upadhyay and Mukhopadhyay, 1986).
Materials and methods
Paddy straw was cut into size of 3-5 cm bits, lOOg of bits were taken and
soaked overnight in water, after that excess water was decanted and mixed with
lOOg sorghum grains and were autoclaved. To the autoclaved paddy straw -
sorghum mixture 10mm mycelial discs of freshly grown seven day old culture
of Trichoderma harzianum was inoculated at 1:10 ratio (fungus : substrate),
i.e. for every lOgrams of substrate one 10mm mycelial disc was inoculated.
Later it was incubated in an illuminated chamber at 28±2 °C. After
fifteen days the substrate was observed for the colonization of Trichoderma
harzianum over the substrate (Fig 3.23).
Another substrate saw dust was weighed to 200grams and was soaked in
water overnight. The excess of water was decanted and autoclaved,
Trichoderma harzianum was inoculated to the substrate at 1:10 ratio (fungus :
substrate), i.e. for every 10 grams of substrate one 10 mm mycelial discs was
inoculated. Later it was incubated in an illuminated chamber at 28±2C. After
fifteen days the substrate was observed for the colonization of Trichoderma
harzianum over the substrate (Fig 3.24).
The same procedure was followed to grow T. harzianum on sorghum
grains. After fifteen days of incubation the sorghum substrate was observed for
the colonization of Trichoderma harzianum (Fig 3.25). each was replicated for
four times. Later lOgrams of substrate colonized by Trichoderma harzianum
was sampled and diluted in 100 ml distilled water the spore count reading for
each substrate was taken by using Haemocytometer.
92
Control and Management of Soil Borne Fungal Pathogens ofBetelvine
Results
The substrate sorghum grain yield more cfu ml"̂ 24 x 10̂ followed by Paddy
straw 12 x 10̂ and Saw dust 8 x lO'̂ (Table 5.8).
Discussion
The mass production of Trichoderma inoculum was carried out by using
locally available substrates which are cost effective, the substrates includes,
paddy straw (Sangeetha and Jeyarajan, 1992), saw dust (Bunker and Kusum,
2001) and sorghum grains were used as substrate for preparation of inoculum
seeds(Upadhyay and Mukhopadhyay, 1986).
1 7
The substrate sorghum grain yield more cfu ml"' 24.719 x 10' followed
by Paddy straw 12.844 x lO' and Saw dust 8.188 x lOl The results obtained
were similar to the results obtained by Upadhyay and Mukhopadhyay (1986),
Kousalya and Jeyarajan (1990), Sangeetha and Jeyarajan (1992), Biswas
(1999), Bunker and Kusum, (2001).
Control of Soil Borne Disease of Betelvine Under Field Conditions
The main objective of integrated crop protection (ICP) is the co
ordination of all cultural, biological, ecological and chemical methods in such a
way as to obtain the maximum total benefit and to minimize harmful side
effects which is due to the excessive usage of pesticides and fungicides in
agriculture. There are four major non-living components of soil, mineral
particles, organic matter, water and air. The solid matter composing the soil
consists of 50% by volume, the rest constitutes the pores and spaces which are
filled with air and water. The organic matter in the soil is the major energy
source on which the majority of microorganisms depends for life.
93
Control and Management of Soil Borne Fungal Pathogens of Betelvine
The organic matter is added to the soil mainly in the form of leaves and
branches from above ground and dead roots and root exudates below the
ground. Each species of microorganisms has its own complex of enzymes,
which enables it to act in a particular way. There is a competition between
organisms which have similar type of physiology and derive their food from the
same type of substrate. As the organic matter gets depleted the activity of micro
organisms declines and these in general pass into dormant and resting stages.
This gives in a broad outline the nature of the soil environment which a root
disease causing fungus has to encounter when it is not inside the host. For the
plants to grow normally light and soil are very important physical factors. Soil
is one of the important natural nutrient media for the plants as it provides,
nutrients, aeration and moisture.
Conserving the soil nutrient status and making disease free is one of the
important challenges to the agricultural scientists. The disease control and
management can be chemical, biological and agronomical practices. Though
the control of diseases by systemic fungicides are quite promising (Sinclair,
1971 and Backman, 1978), but the frequent and indiscriminate use of
fungicides often leads to atmospheric pollution and development of fungicide
resistance in pathogens (Gattani, 1951 and Thind etal, 2001).
In this context, biological control is coming up as an alternative strategy
for disease management, which is also environment friendly (Manoranjitham, et
al, 2001) and the biological control of plant pathogens by the addition of
antagonists like Trichoderma offers an alternative method to combat the
ravages of soil borne diseases (Abraham & Gupta, 1998 and Thirumala &
Sitaramaiah 2000).
94
Control and Management of Soil Borne Fungal Pathogens ofBetelvine
Materials and methods
The soil amendment was done to know the effect of the Trichoderma
harzianum inoculum on the growth and recovery of betelvine under field
condition. The T. harzianum was mass multiplied in the two substrates like
Paddy straw - sorghum and Sawdust - sorghum mixtures.
The experiment was conducted during pre-monsoon season of the year
2003. The four blocks were selected in betelvine gardens of Tarikere tauk, in
each block the diseased betelvine plants which were in the grade 3 were
selected (Fig 3.3).
The number of available plants in the grade 3 were equally divided into
three sets. One set is used for control and another set is used to treat the plants
with Trichoderma inoculum which was mass multiplied in saw dust - Sorghum
mixture and the remaining set was used to treat the plants with Trichoderma
inoculum which was mass multiplied in Paddy straw - Sorghum mixture. The
diseased plants that were in the grade 3 were selected, these plants were
lowered, diseased portion of the plants and rhizosphere soil were removed. The
infected soil and plant parts were dumped into 'sanitation pit'. (The sanitation
pit was dug at the corner of the garden, containing decaying plant material and
Trichoderma inoculum).
The betelvine is twined into circular fashion (Fig 3.26), planted in 2.0 x
2.0 x 0.5 feet pit and supplemented with 20 kg of new soil, 2 kg of Farm Yard
Manure (FYM) since it acts as substrate as well as it increases the population of
Trichoderma (Indu and Sawant, 1996; Hoitink et al., 1997) and 200g of
Trichoderma harzianum inoculum that was mass multiplied in the substrates
like Sorghum - Saw dust mixture and Sorghum - Paddy straw mixture in the
ratio 1:1 V / V, each substrate containing Trichoderma inoculum was added
95
Control and Management of Soil Borne Fungal Pathogens of Betelvine
separately to know its effect on the growth of plant (Upadhyay and
Mukhopadhyay, 1986; Kousalya and Jeyarajan, 1990; Ram et al, 1999;
McPherson and Hunt, 1995 and John, 2000).
For the control, the plants were lowered, the diseased portion of the
plants were removed, the vine is twined into circular fashion, planted in 2.0 x
2.0 X 0.5 feet pit. The treated plants were numbered and marked, they were
regularly watered and monitored. The treated plants were examined for their
recovery. After forty five days the shoot length was recorded. The treated plants
that had grown more than 10 cm were regarded as the recovered plants (Fig
3.27). The recovery percentage of the treated plant were calculated by the
following formula:
Total Number of plants recovered X 100 Recovery percentage =
Total Number of plants treated
Results
The shoot length was recorded after forty five days of treatment, in the
block I, II, III & IV, the average shoot length of vine in control is 5.7, 5.9, 7.0,
5.5 cm respectively (Table 5.10).
The plants treated with Trichoderma harzianum which was multiplied in
Saw dust- sorghum mixture, showed the growth of 13.8, 13.4, 14.1 and 14.0 cm
respectively (Table 5.10).
The plants treated with Trichoderma harzianum which was multiplied in
Paddy straw - sorghum mixture, showed the growth of 16.7, 14.9, 19.0, and
17.0 cm respectively (Table 5.10).
The recovery percentage of treated plants in all the four blocks were
ranged from 48.49% to 58.33% (Table 5.12) (Fig 3.27).
96
Control and Management of Soil Borne Fungal Pathogens of Betetvine
Discussions
The soil was amended with Trichoderma harzianum inoculum, which
was mass multiplied in Paddy straw - sorghum and Saw dust - sorghum
substrates.
The shoot length was recorded after forty five days of treatment, in the
block I, II, III & IV, the average shoot length of vine in control is 5.7, 5.9, 7.0,
5.5 cm respectively (Table 5.10).
The plants treated with Trichoderma harzianum which was multiplied in
Saw dust- sorghum mixture, showed the growth of 13.8, 13.4, 14.1 and 14.0 cm
respectively (Table 5.10).
The plants treated with Trichoderma harzianum which was multiplied in
Paddy straw - sorghum mixture, showed the growth of 16.7, 14.9, 19.0, and
17.0 cm respectively (Table 5.10).
The recovery percentage of treated plants in all the four blocks were
ranged from 48.48% to 58.33% (Table 5.12) (Fig 3.27).
The stastical analysis ANOVA was done for the treatments and for
blocks (ANOVA Table 5.11). The variation between the blocks remained
nonsignificant, where as in treatments the plants which were treated with
Trichoderma harzianum that was mass multiplied in Paddy straw - sorghum
mixture was found to be significant at (P = 0.05).
The significant growth was observed in the plants treated with the
Trichoderma inoculum prepared in the substrate paddy straw - sorghum
mixture. The cellulose based substrates like paddy straw and sorghum,
availability of air pores within the culms, might have resulted in the building of
97
Control and Management of Soil Borne Fungal Pathogens ofBetelvine
increased conidial concentration of Trichoderma in the soil. The change in
routine organic cultural practices, that is by amending soil with the addition of
farm yard manure, new soil and the Trichoderma harzianum inoculum with
cellulose based substrates played a significant role in the overall growth and
recovery of the plant.
The decomposition of organic matter in the soil was known to increase
microorganism activity and suppressing root infecting pathogens (Khanna and
Singh, 1974). This is especially important in connection with root diseases
because in these cases besides the interaction between host and the parasite, one
factor of soil environment is the soil microbial Population. The rhizoplane and
rhizosphere population intimately reacts with the disease causing fungus before
it enters the root tissues. If there were any antagonists present in these regions
they will actively influence against the disease causing fungus. Weindling
(1932 and 1934) recorded the parasitism of Trichoderma on other species of
soil fungi like Phytophthora, Pythium, Sclerotium and including Rhizoctonia
solani a common soil pathogens.
Trichoderma harzianum occurs widely in nature in soil substrate and this
is being commercialization because of its ability to compete with
phytopathogenie fungi and produce toxins. This fungus has been recommended
for the control of soil-inhabiting pathogenic fungi like Fusarium, Rhizoctonia,
Sclerotium Phytophthora and Pythium (Hoitink et al., 1997 and Pandey et ai,
1999a & 1999b). This fungus competes in the soil for nutrients and rhizosphere
dominance with phytopathogenic fungi. In presence of sufficient organic carbon
it produces enzymes having lytic effect on target fungi and in contrast in
adverse conditions it produces toxins which are equally harmful.
98
Control and Management of Soil Borne Fungal Pathogens ofBetelvine
Trichoderma harzianum is a widely distributed member of the soil
microflora and exerts its effect by competing for nutrients and producing toxins
against phytopathogenic species.
The Trichoderma harzianum was effectively used against several soil
borne fungi like Rhizoctonia solani, Sclerotium rolfsii, and Pythium
aphanidermatum (Hadar et al, 1979; Elad et al, 1980; Chet et al, 1981; Elad
et al, 1982; Deb and Dutta, 1992; Mehrothra et a/., 1993; Suseelendra and
Schlosser, 1999). The main objective of integrated crop protection (ICP) is the
co-ordination of all cultural, biological, ecological and chemical methods in
such a way as to obtain the maximum total benefit and to minimize harmful
side effects which is due to the excessive usage of pesticides and fungicides in
agriculture (Charles, 1997).
Compost acts as suitable substrate for many microorganisms, which
includes biocontrol agents like Trichoderma which suppresses the broad
spectrum of soil borne fungal pathogens (Hoitink et al., 1997).
The decomposition of organic matter helps in alternation of physical,
chemical and biological conditions of the soil and the altered conditions may be
reducing the inoculum potential of soil- borne pathogens including Rhizoctonia
solani and Sclerotium rolfsii (Singh, 1983 and Sachin et a/.,2002).
It also improves soil structure, which promots root growth of the host
plant, various biochemicals like antibiotics and phenols are released in
decomposition, which induces resistance in root system and increase overall
growth of the plant (Sachin et al, 2002).
99
Effect of Garlic extract on growth of Phytophthora parasitica
120
100
80 u a. c o 60
H 40
20
0 10 30 60 80 100 Cotxaitrations
Concentration %- Inhibition"/
Fig 4.67 Effect of Garlic extract on the mycelial growth o^ Phytophthora parasitica
Effect of Neem extract on growth of Phytophthora parasitica
120 1
100 y 80
so
1 60 u
4-^
/ _ _
1 20i /y^ 0 ^ — 0 ' ' ' ' '
0 10 30 60 80 100 Concentratjon
• Cuiiceiilraliuii % • ixuiibiliou%
Fig 4.68 Effect of Neem extract on the mycelial growth of Phytophthora parasitica
Effect of Garlic extract on growth of Pythium vexans
120
0 10 30 60 80 100
Concentration
Concentration °/<r-»— Inhibition"/^
Fig 4.69 Effect of Garlic extract on the mycelial growth o^ Pythium vexans
Effect of Neem extract on growth of Pythium vexans
120
so a *.» c u u u a. c o
100
10 30 60 80 100
Concentration
Concentration %-•^— Inhibition"/:
Fig 4.70 Effect of Neem extract on the mycelial growth of Pythium vexans
Effect of Garlic extract on growth of Fusarium oxysporum
120
10 30 60 80 100
Concentration
-•— Concentration %-»— Inhibition"/^
Fig 4.71 Effect of Garlic extract on the mycelial growth of Fusarium oxysporum
Effect of Neem extract on growth of Fusarium oxysporum
120 1
100 y a, 80 B c
i 60^ a. c
:l 40i JO
^ 20
/ ^
0 _̂-̂::̂i—̂"̂— 0 0 10 30 60 80 100
—•— concentration %"•— inhiDitionT^
Fig 4.72 Effect of Neem extract on the mycelial growth of Fusarium oxysporum
Effect of Garlic extract on the growth of Rhizoctonia solani
120
100
10 30 60 80 100
Concentration
-•— Concentration % • Inhibition"/:
Fig 4.73 Effect of Garlic extract on the mycelial growth oi Rhizoctonia solani
Effect of Neem extract on the growth o{ Rhizoctonia solani
120
B c u u o. c o
100
10 30 60 80 100
Concentration
Concentration % • Inhibition"/1
Fig 4.74 Effect of Neem extract on the mycelial growth of Rhizoctonia solani
Effect of Garlic extract on growth of Sclerotium rolfsii
120
a 100 2 c u o w u o. c _o
IS 'JE c
10 30 60 80 100
Concentration
Concentration %-•— Inhibition"/!
Fig 4.75 Effect of Garlic extract on the mycelial growth of Sclerotium rolfsii
Effect of Neem extract on growth of Sclerotium rolfsii
120
c
c o
'.E
100
0 10 30 60 80 100
Concentration
Concentration % Inhibition"/
Fig 4.76 Effect of Neem extract on the mycelial growth of Sclerotium rolfsii
Tab e 5.1 Effect o f plant extracts on the mycel ial g rowth of Phytophthora parasitica
Percentage of plant extracts Average colony diameter in cms
Percentage of inhibition
Control (C J_ 2.48
10% 1.97 20.56 30% 1.84 25.81*
Allium sativum 60% 1.48* 40.32* 80% 1.30* 47.58*
100% 1.15* 53.63*
10% 1.95 21.37
30% 1.82 26.61* Azadirachta indica 60% 1.78 28.23*
80% 1.73 28.63*
100% 1.62* 34.68*
CD at 5% 0.8 4.24
Average of four replications *significant value
Table 5.2 Effect o f plant extracts on the mycel ia l growth oiPythium vexans
Percentage of plant extracts Average colony diameter Percentage of inhibition in cms
Control (C ) 2.32
10% 2.03 12.50*
30% 1.74 25.00* Allium sativum 60% 1.17* 49.57*
80% 1.10* 52.59*
100% 0.00* 100.00*
10% 2.3 0.86
30% 2.26 2.59
Azadirachta indica 60% 1.95 15.95* 80% 1.89 18.53*
100% 1.19* 48.71*
CD at 5% 0.84 7.01
Average of four replications *significant value
Tab e 5.3 Effect of plant extracts on the mycelial growth oi Fusarium oxysporum
Percentage of plant extracts Average colony diameter Percentage of inhibition In Cms
Control (C) 2.16
10% 1.41* 34.72* 30% 0.80* 62.96*
Allium sativum 60% 0.00* 100.00* 80% 0.00* 100.00* 100% 0.00* 100.00*
10% 2.08 3.70 30% 2.06 4.63
Azadirachta indica 60% 1.94 10.19 80% 1.83 15.28* 100% 1.51* 30.09*
CD at 5% 0.61 8.13
Average of four replications *significant value
Table 5.4 Effect of plant extracts on the mycelial growth oi Rhizoctonia solum
Percentage of plant extracts Average colony diameter Percentage of inhibition in Cms
Control (C) 8.87
10% 6.07* 31.57* 30% 1.55* 82.53*
Allium sativum 60% 1.05* 88.16* 80% 0.00* 100.00* 100% 0.00* 100.00* 10% 8.72 1.69 30% 8.64 2.59
Azadirachta indica 60% 8.23 7.22 80% 7.92 10.71* 100% 7.06* 20.41*
CD at 5% 1.11 8.3
Average of four replications •significant value
Table 5.5 Effect of plant extracts on the mycelial growth of Sclerotium rolfsii
Percentage of plant extracts Average colony diameter Percentage of inhibition in cms
Control (C) 4.93 10% 2.13* 56.80* 30% 1.78* 63.89*
Allium sativum 60% 0.00* 100.00* 80% 0.00* 100.00* 100% 0.00* 100.00* 10% 4.17 15.42 30% 3.35* 32.05*
Azadirachta indica 60% 3.17* 35.70* 80% 2.67* 45.84* 100% 1.68* 65.92*
CD at 5% 1.35 7.02
Average of four replications *significant value
Ta 3le 5.6 Antagonistic activity of Trichoderma isolates on Sclerotium rolfsii Antagonistic activity of Trichoderma isolates on Sclerotium rolfsii
Isolate After 7 days After 11 days After 14 days
Trichoderma viride 1 + + + + + +
Trichoderma viride 2 + + + + + +
Trichoderma harzianum + + + + + + + +
+++ Trichoderma spp. inhibit and overgrow S. rolfsii .(Fig 4.90) ++ Trichoderma spp. inhibit growth of 5. rolfsii but they stop at the inhibiting line (Fig 4.88) + Trichoderma spp. inhibit S. rolfsii but they are overgrown by S. rolfsii.
Trichoderma spp. do not inhibit S. rolfsii but are overgrown by S. rolfsii.
Table 5.7 Inhibition percentage of Soil borne fungal pathogens of betel vine by Trichoderma harzianum in dual culture.
Test Fungi
Colony diameter of test fungi in control
Colony diameter of test fungi when grown with Trichoderma
Colony diameter of Trichoderma in dual culture
Colony diameter ratio of Test fungi: Trichoderma
Inhibition percentage of test fungi due to Trichoderma in dual culture
Phytophihura parasitica 3.75 2.6 6.56 2:5 30.67
Pyihium vejcans 4.01 3.1 6.84 1:2 22.69 Fusarium oxysporum 4.31 2.4 6.28 3:8 44.32
Rhizocioniu sutani 9.01 8.18 4.57 9:5 9.21
Sclerotium rolfsii 8.47 4.92 6.16 4:5 41.91
Table 5.8 Growth of Trichoderma harzianum on different substrates
Substrate Trichoderma Spores mi' Sorghum 24 X 10̂ Paddy straw - sorghum 12 X 10' Sawdust-sorghum 8.0x10'
Table 5.10 Average shoot length of Betelvine in cm due to Trichoderma harzianum amendment
Blocks Control Sawdust-sorghum Paddystraw - sorghum* 1 5.7 13.8 16.7 II 5.9 13.4 14.9 III 7.0 14.1 19.0 IV 5.5 14.0 17.0
* significant at (5%) CD = 1.02 ** Treatment is soil amended with Trichoderma harzianum mass multiplied on Sawdust-sorghum
and Paddy straw - sorghum mixtures.
Table 5.11 ANOVA for average shoot length of Betelvine in cm due to Trichoderma harzianum*
Source of variation DF SS MS F observed F5% Treatments 2 251.4 126 193.38* 5.14 Blocks 3 6.17 2.1 3.2 4.75 Error 6 3.91 0.7
Total 11 * Treatment is soil amended with Trichoderma harzianum mass multiplied on Sawdust-sorghum
and Paddy straw - sorghum mixtures, is significant at F = 5%.
Table 5.12 Recovery of diseased plants due to Trichoderma harzianum
Control Saw-sorg Pady-sorg Total treated Dead plants Recovered % recovery
Block 1 14 15 16 45 8 23 51.12 Block 2 8 7 9 24 2 14 58.33 Block 3 11 8 10 29 3 15 51.73 Block 4 12 9 12 33 5 16 48.49
Saw-sorg = Trichoderma harzianum mass multiplied on Sawdust-Sorghum mixture. Pady-sorg= Trichoderma harzianum mass multiplied on Paddystraw-Sorghum mixture.
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