controcancf management ofsoic^bome tunga[(patfiogens of...

ControCancf Management ofSoiC^Bome Tunga[(Patfiogens of (BeteCvine

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


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


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


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


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


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



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


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


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


(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


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


sorghum grains were used as substrate for preparation of inoculum seeds

(Upadhyay and Mukhopadhyay, 1986).


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


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


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



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


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).


Paddy straw - sorghum mixture, showed the growth of 16.7, 14.9, 19.0, and


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,



Saw dust- sorghum mixture, showed the growth of 13.8, 13.4, 14.1 and 14.0 cm

respectively (Table 5.10).


Paddy straw - sorghum mixture, showed the growth of 16.7, 14.9, 19.0, and


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


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


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


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


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


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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