chapter-viii bioassay of antagonists on disease...

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CHAPTER-VIII

Bioassay of antagonists on disease suppression and plant growth under

green house condition.

8.1 Background

Since the pathogens that cause root disease of tea are soil borne, it becomes essential

to study the effect of soil microbes for their antagonistic/ hyperparasitic behaviour against the

test pathogens U. zonata and F. lamaoensis, under study .The diseases transmitted from the

infected plant to the healthy plant through the infected root.

Various soil microbes which act as effective biocontrol agents against soil borne

pathogens are well-known for their beneficial effect as plant growth promoter (Barka et al .,

2000 ; Chakraborty et al.,2005). In the present study, an attempt was made to study the effect

of selected antagonistic microbial strains isolated from tea soil and was found effective in in-

vitro antagonistic test. Bioassay of highly antagonistic microbial strains screened in-vitro is

very essential to determine their potentiality under natural condition, therefore a nursery trial

was conducted for assessing the effect of different treatments on growth parameters and plant

health. Treatments were applied in the root zone as there are evidence that actinomycetes are

quantitatively and qualitatively important in the rhizosphere (Barakate et al., 2002; Crawford

et al.,1993; Doumbou et al., 2001; Miller et al., 1989, 1990), where they may influence plant

growth and protect plant roots from invasion by root pathogenic fungi (Lechevalier,1988).

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8.2 Materials and Methods

8.2.1 Evaluation of screened antagonist strains under nursery conditions

The nursery trial was conducted in a glasshouse at Tocklai Experimental Station,

Jorhat (Latitude 26043

/ 46.38

// N; Longitude 94

013

/ 43.44

//E), with tea seedlings for a period of

18 months (fig 8.1). The isolates used in this trial were selected on the basis of their efficacy

to inhibit the growth of two test pathogens under in- vitro conditions (antagonistic test

conducted in chapter VII)

8.2.2 Soil used

The soil used in the nursery trial for filling up the polythene sleeves (34cmx19cm)

was collected from the tea plantation area under Tocklai Experimental Station. Black

polythene sleeves (34cm×19cm) were filled with 6 kg of uniformly broken soil. The soil was

of sandy loam texture with moisture content % 17.5 and PH

of 4.5-5.3. The microbial load of

the soil sample was estimated before filling up of the sleeves (Table 8.1) to have an idea of

the biological status of the soil.

8.2.3 Planting Material

Cuttings of tea plants of clone TV1 cultivars of Assam variety, (Camellia sinensis

var. assamica) were procured from nursery of Borbheta Experimental plot (Jorhat) having

latitude 26.71o, longitude 94.20

o and elevation 322ft. The cuttings were transported to glass

house and set in the campus of Tocklai Experimental Station where they were raised in

nursery bed for the study (fig 8.2). After the formation of callus, the tea plantlets were

transferred to the polythene sleeves and maintained well through regular monitoring.

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8.2.3 .1 Description of selected clone

TV 1 Type : Assam China hybrid

Frame : Compact and dense

Shoot size (2L+B) : Medium

Weight

Fresh (gm) : 0.59

Dry (gm) : 0.12

Leaf type (size) : medium

Rooting : very good

Leaf shape : broadly lanceolate

Pubescence : medium

Drought resistance : good

Susceptible to disease: High to moderate depending on pathogen

Involved.

8.2.4 Experimental Treatments

Nursery trial was conducted to assess biocontrol potential of selected PSB and

actinomycetes isolates against two primary root disease causing pathogens of tea by dual

inoculation of the pathogens (U.zonata or F.lamaoensis) and the antagonistic isolates of PSB

(MM/PH/BST and MM/PH/KMP) and actinomycetes (MM/PH/AC-02 and MM/PH/AC-09)

following Ganesan and Gnanamanickam, (1987). The experiment was conducted with

thirteen treatments consisting 4 isolates and two reference strains of Trichoderma viride, one

absolute control (no pathogen and no biocontrol agent) and another two control with only

pathogens (ordinary control).The experiment was performed in the plastic sleeves with tea

151

plantlets under greenhouse condition with 13 treatments and 5 replications following the

Completely Randomized Block Design (CRBD).The layout of the nursery trial plot is shown

in fig 8.3. The treatments (antagonistic isolates) used for nursery evaluation of the selected

PSB and actinomycetes isolates in controlling the root disease causing pathogens as well as in

enhancing growth of tea plants are given below:

Sl. No. Treatments

1. MM1- MM/PH/AC-02+ Ustulina zonata (U.zonata)

2. MM2- MM/PH/AC-09+ U.zonata

3. MM3- MM/PH/KMP + U.zonata

4. MM4- MM/PH/BST + U.zonata

5. MM5- MM/PH/AC-02+ Fomes lamaoensis (F. lamaoensis )

6. MM6- MM/PH/AC-09+ F. lamaoensis

7. MM7- MM/PH/KMP+ F. lamaoensis

8. MM8- MM/PH/BST+ F. lamaoensis

9. MM9 –Trichoderma viride + U.zonata

10. MM10- T. viride + F. lamaoensis

11. MM11- Control (only U.zonata )

12. MM12- Control (only F. lamaoensis )

13. MM13- Absolute control (No pathogen and no antagonists)

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The plants were kept under optimum shade condition throughout the experimental

period. The plants were watered regularly and plant protection measures were taken as and

when necessary to protect the plants against insect pest damage.

8.2.4.1 Preparation of inocula for treatment

Tea bushes infected with charcoal stump rot were collected from root disease infested

area of Kakojan Tea Estate (28, Deberapara div.). For brown root rot disease, infected tea

bushes were taken from Tyroon Tea Estate, (Kheremia section No.10). Infected tea roots

were initially washed properly with tap water to remove all the soil particles, and again rinsed

with distilled water. The roots were made into small rectangular blocks (4cm x 2cm x1cm),

and incubated at 25 ± 2o C in conical flasks under moist condition. After 15-20 days the

infested wooden blocks were found to be covered by thick subfelty mycelia with colour

ranging from brownish white to brown in case of F. lamaoensis (Brown root rot) and thick

charcoal like encrustation and black lining in all the surfaces of the wooden blocks in case of

U. zonata (Charcoal stump rot).

8.2.4.2 Pure culture of applicable strains

Pure cultures of four selected strains (MM/PH/BST,MM/PH/KMP, MM/PH/AC-02

and MM/PH/AC-09) were selected on the basis of in-vitro antagonistic screening, and

prepared in broth of Pikovskaya‘s medium (MM/PH/BST, MM/PH/KMP) and Kenknights

medium (MM/PH/AC-02, MM/PH/AC-09) under laboratory conditions. The cultures were

incubated at their optimum temperatures till the appearance of full growth of the strains.

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8.2.4.3 Treatment application

The incubated tea root bits were soaked in the pure culture broth of the test strains and

kept overnight before application of treatments. One year tea plants raised in the polythene

sleeves for nursery trial were inoculated artificially in the root zone with pure inocula

cultured on fresh infested tea root bits under laboratory condition. The freshly infested tea

root bits with and without implication of pure culture of antagonistic strains that are to be

applied as treatments were introduced in the soil by making four holes around the plants so

that the bits touches the root of the target tea plants. The treatment application was conducted

following the method of Barthakur (1997) with some modification. Thirty target plants were

taken for each treatment and equal number of plants were not subjected to any treatment were

considered as control.

8.2.6 Plant growth measurements

The experiments were maintained for eighteen months. Stem girth, root length, fresh

weight and dry weight of biomass per plant were recorded by non-destructive methods on the

day of inoculation and at quarterly intervals (after every three months) after application of the

treatment.

8.2.5.1 Root length

On expiry of the harvesting schedule, plants were carefully uprooted and washed

gently the root system to get them free of soil. The root length was recorded by measuring

from base of the stem to tip of the longest root and expressed in centimetres.

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8.2.5.2 Stem girth

Stem girth was determined by measuring the diameter of the stems at collar region

using a vernier calliper applying the formula 2πr and expressed in centimetres.

8.2.5.3 Total Biomass content

Both the fresh and dry weight of the uprooted plants was recorded. Uprooted plants

were first air dried and then oven dried at 600C to a constant weight for recording the dry

biomass and were expressed in g per plant.

8.2.6 Periodic observation of plant health and disease incidence

Visual observation of the plant health was recorded after every harvesting by

recording the number of healthy plants, plants with stunted growth, plants started wilting,

plants with acute wilting and dead. On the basis of plant health, disease occurrence and

incidence of mortality, a score chart was prepared for recording the periodic observation.

Score Chart.

Score 1: Healthy

Score 2: Stunted growth

Score 3: Initial wilting

Score 4: Progressive wilting

Score 5: Dead

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8.2.6.1 Determination of disease incidence

The tea plants exhibiting stunted growth, initial wilting, progressive wilting and

death were examined thoroughly by uprooting the plants. The uprooted tea plants after each

harvesting were washed properly with tap water and the disease incidence was determined

from external appearance, according to characteristic symptoms mentioned in I.T.A.

Memorandum No.8 (Tunstall, 1940)

To determine the incidence of fungi on roots, the modified technique of the method of

Short et al., (1980) was used where root from each uprooted plants after each harvesting were

cut into one cm long root pieces, surface sterilized with 1% Ca(OCl)2 for 3 min and

transferred onto potato dextrose agar plates containing streptomycin (0.2gm/litre). After

incubation for 5 to 7 days at 280C, incidence of root infecting fungi and percent disease

reduction (DIR) were recorded using the formula as given by Shameem, (2006).

Total number of plants infected by the pathogen

Disease incidence % = x 100

Total number of plants

.

No. of plants infected in control – No. of plants infected in treatment

% DIR= ———————————————————————————— x 100

No. of plants infected in control

8.2.7 Experimental design and statistical analysis

Nursery experiment was arranged in a complete randomized block design with four

replicates for each treatment as well as control. Statistical analysis of the obtained data was

subjected to standard analysis of variance procedure. The values of LSD were calculated at

5% level according to the method described by Gomez and Gomez (1984).

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8.3 Results

8.3.1 Effect of PSB and actinomycetes isolates on growth parameters of tea plants

After the 1st harvest, growth parameters like stem girth, root length, fresh weight and

dry weight of tea plants was found to be increased as a result of treatments in MM5

(MM/PH/AC-02 and F.lamaoensis) and in MM9 (T.viride and U.zonata) (Table 8.2 and Fig.

8.a). Except MM3 (MM/PH/KMP and U.zonata), MM8 (MM/PH/BST and F.lamaoensis)

and MM11 (only U.zonata), all other treatments showed better stem girth.

More root length was observed in the plants treated with MM5 (MM/PH/AC-02 and

F.lamaoensis), MM7 (MM/PH/KMP and F.lamaoensis), MM9 T.viride and U.zonata ) and

MM10 (T.viride and F.lamoensis ) than untreated plants.

Fresh weight of the plants under all the treatments were found higher except in

treatment MM3 (MM/PH/KMP and U.zonata) and MM11 (only U.zonata) which was at par

with the untreated control. Dry weight of the plants with treatment MM1, MM5, MM7 and

MM9 were found significantly higher than untreated control. All the findings were found to

be statistically significant.

The data recorded after 2nd

harvest (Table 8.3 and Fig. 8.b) revealed that the plants

treated with MM9 (T.viride and U.zonata) showed maximum growth in stem girth, root

length and fresh weight over untreated plants. But, in case of dry weight, plants treated with

MM8 (MM/PH/BST and F.lamaoensis) showed the highest value. All the plants treated with

combination of antagonist microbes and pathogen showed better growth in stem girth, root

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length, fresh weight and dry weight of the plants than the untreated plants, while the plants

grown with the pathogen only did not showed any significant difference with the untreated

plants.

The data recorded after 3rd

harvesting is presented in Table 8.4. and Fig. 8.c Plants

treated with MM4 (MM/PH/BST and U.zonata) showed maximum stem girth followed by

treatment MM8 (MM/PH/BST and F.lamaoensis). In case of root length, fresh and dry

weight biomass, MM8 recorded the highest. All the treatments of antagonist isolates in

combination with the test pathogen (treatments MM1, MM2, MM3, MM4, MM5, MM6,

MM7, MM8) were found to increase all the growth parameters significantly,(fig 8.4). But the

plants treated with only pathogens (treatments MM11 and MM12) did not show any

significant variation with the untreated plants (treatment MM13).

After the 4th harvest, the growth parameters like stem girth, root length and fresh

weights were found to be increased, with treatments MM2, MM4, MM6 and MM8 than the

untreated plants. Among all the treatments, MM2 (MM/PH/AC-09 and U. zonata) was found

to be the best followed by treatment MM8 (MM/PH/BST and F. lamaoensis). Plants treated

with only pathogens (treatments MM11 and MM12) showed significantly lesser growth in all

the growth parameters, however the results were at par with the untreated plants in case of

dry weight(fig 8.5&fig 8.6)

The records of the 5th harvest (Table 8.6 and Fig. 8.e) clearly states the maximum

effect of treatment MM2 (MM/PH/AC-09 and U.zonata), MM4 (MM/PH/BST and

U.zonata), MM6 (MM/PH/AC-09 and F.lamaoensis) and MM8 (MM/PH/BST and F.

lamoensis) on all the growth parameters. All the treatments, except MM11 (only U. zonata)

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and MM12 (only F. lamaoensis) showed significant (P<0.05) increase in growth parameters

over untreated plants.

According to the data recorded after 6th harvest (Table 8.7 and Fig. 8.f) treatment

MM2 (MM/PH/AC-09 and U.zonata) was found to be highly effective in increasing stem

girth, root length and dry weight in comparison to all other treatments, effect of treatment

MM11 (only U. zonata) and MM12 (only F. lamaoensis) was found to be the lowest among

all the other treatments.

8.3.2 Periodic observation of plant health and disease incidence

Observation on the plant health and development of morphological disease symptoms

was taken after every three months from the date of treatment applications. Some of the

plants were found healthy throughout the observation, while some showed disease incidence

according to the type of treatment.

Records of the periodic observation on plant health and disease occurrence of treated

tea plants under greenhouse trial were presented in Table 8.8.The data reveal that all the

antagonist isolates (MM1, MM2, MM3, MM4, MM5, MM6, MM7, MM8) effectively check

disease occurrence. Plants treated with antagonist exhibited good health at the time of 1st

harvesting irrespective of treatments. Initial wilting in one plant and stunted growth in two

plants was noted at the time of 2nd

harvest, in plants treated with U. zonata (Treatment

MM11) while rest of the plants were healthy. At the time of 3rd

harvesting all other plants

except the treatment MM11 showed healthy growth. In the treatment MM11, progressive

wilting was noticed in one plant along with initial wilting in two and stunted growth in other

two. Stunted growth (1plant) and initial wilting (2 plants) were also observed in plants treated

with only F. lamaoensis (Treatment MM12). At the time of 4th harvesting, four plants from

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MM11 and three plants from MM12 treated plants showed progressive wilting. Initial wilting

was recorded in plants with treatment MM1 (3 plants), MM2 (1plant), MM3 (2 plants), MM5

(1 plant) and MM10 (1 plant). At the time of 5th

and 6th

harvesting all the plants taken for

observation in MM11 and MM12, were found dead with withered leaves (fig 8.7). Uprooting

of dead plants recorded in MM11 treated plants showed symptoms of charcoal stump rot

infection (fig 8.8) and that in MM12 treated plants showed initiation of brown root rot

symptoms.

8.3.2.1 Determination of disease incidence

Appearance of small white, turning black cushions were evidenced in root of

progressive wilting and dead plants under treatment MM11 (U.zonata) (fig 8.9). Infected root

when peeled of carefully, white delicate fans of mycelium were observed. When the root was

cut off longitudinally black lines were detected when they were observed minutely which

shows their resemblance with the characteristics of charcoal stump rot. After uprooting of

progressive wilting and dead plants of MM12 (F.lamaoensis), the roots were found to be

rough with soil even after washing, cream to brown mycelium were also found encrusted to it

which were considered to be infected by brown root rot ( fig 8.10).

Overall percentage of disease incidence was recorded to be optimum (50%) in

plants treated with MM11 (only U.zonata) followed by 43% in plants treated with MM12

(only F.lamaoensis) (Table 8.7 and Fig. 8.f). Disease incidence was found to be less (6.67%)

in the plants treated with treatment MM2 (MM/PH/AC-09+ U.zonata) and MM6

(MM/PH/AC-09+ F. lamaoensis) in each of the cases.

The percentage of disease reduction by the inoculated antagonist PSB and

actinomycetes varied from 61.54 to 86.67% (Table 8.7 and fig 8.g). Isolate MM/PH/AC-09

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showed maximum control (86.67%) of U. zonata, followed by the same isolate against F.

lamaoensis, with 84.62 % reduction in disease over control (treated with only pathogens).

8.4 Discussion

Periodic observation on plant health and disease incidence of the plants under

greenhouse trial indicated that, all the four antagonists were effective in checking the disease

occurrence as compared to the plants treated with only pathogen (control). Among the

thirteen treatments including untreated plants (Absolute control), the progress of disease

occurrence was less in MM/PH/AC-09 and MM/PH/BST treated plants with both the test

pathogens. The severity of diseases substantially increased in plants treated with only

pathogens. From the investigation it was found that percent of disease incidence was 50 and

43 in plants treated with only U.zonata and F.lamaoensis respectively. 100 percent disease

incidence was not recorded in any of the treatment because due to time unavailability our

nursery trial was confined to 18 months, which was not enough for both the pathogens to

attack the tea plants completely. According to the previous record it was known that charcoal

stump rot (U.zonata) requires a period of six months to four years to attack plant completely

and Brown root rot (F.lamaoensis ) attacks tea plants completely above three year but young

tea plants are affected within few months.

In the present study, all the antagonistic isolates utilized as treatment were able to

improve the plant growth leading to increase in stem girth, root length, Fresh and dry weight

biomass over the pathogen treated as well as plants without any treatments, under nursery

condition which correlates with the work of Phukan et al. (2011),where they reported several

indigenous species of Bacillus, Pseudomonas, Azotobacter, Azospirillum isolated from the tea

soil showing plant growth promoting as well as antagonistic effect against Black rot and

blister blight disease of tea in both nursery and commercial trial and also with the work of

Mishra (2006). Previous work of many workers explains that growth promotion occurs as a

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result of direct stimulation of plant growth, induction of host plant systemic resistance or

suppression of plant pathogens (Dwivedi and Johri, 2003; Lugtenberg et al., 2002; Fakhouri

et al., 2001; Van Loon et al., 1998). Isolates MM/PH/AC-09 and MM/PH/BST was found to

be highly effective in increasing the growth parameters of the plants significantly (P<0.05) as

well as in decreasing disease occurrence which justify the fact that antagonism is one of the

important trait of Plant growth promoting microbes, therefore Such microorganism have been

applied to a wide range of plants for the purpose of plant growth enhancement and disease

control (Barka et al., 2000 and Chakraborty et al., 2005). The increment in growth

parameters in response to microbial antagonists inoculation endorsed the fact that the test

strains were having one or more growth promoting mechanism including mobilization and

efficient uptake of nutrients ( Biswas et al.,2000), solubilization of insoluble phosphates

(Alikhani et al., 2006), and inhibition of fungal growth (Nautiyal, 1997).

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Table 8.1: Microbial load of the soil used for nursery trial.

Type of microbes Population

density

Bacteria

39.6 x10-5

cfu/g

Fungi 12.0 x10-4

cfu/g

Actinomycetes 2.3 x10-4

cfu/g

Azotobacter 1.3 x10-5

cfu/g

Azospirillum ND

Phosphate solubilizer 1.0 x10-5

cfu/g

ND- not detected, cfu- colony forming units.

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Table 8.2: Effect of different treatments on stem girth, root length and

biomass of the plant (1st harvesting, Dec 09-Mar 10 )

Treatments Stem girth (cm) Root length

(cm)

Fresh weight of

plant

(gm)

Dry weight of

plant

(gm)

MM1 1.48ab 48.60ab 117.80abcd 59.22a

MM2 1.35cd 41.40e 115.44de 55.74bc

MM3 1.27de 38.00f 113.34e 53.24c

MM4 1.40bc 44.28cde 118.40abc 56.50b

MM5 1.50a 49.52a 120.56a 61.41a

MM6 1.46ab 41.68de 115.94cde 55.16bc

MM7 1.39bc 46.00abc 119.26ab 60.80a

MM8 1.25e 44.88bcde 115.14de 56.48b

MM9 1.55a 49.66a 120.40a 61.82a

MM10 1.31cde 46.24abc 115.80cde 54.04bc

MM11 1.25e 44.16cde 113.34e 55.50bc

MM12 1.33cde 46.32abc 116.82bcd 56.50b

MM13 1.30de 45.28bcd 113.58e 54.18bc

S.Ed () 0.04 1.66 1.24 1.23

CD(0.05) 0.08 3.37 2.51 2.49

The figures followed by same letter(s) do not differ significantly

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Table 8.3: Effect of different treatments on stem girth, root length and

biomass of the plant (2nd

harvesting, Mar10-june-10)

Treatments Stem girth (cm) Root length (cm) Fresh weight of

plant

(gm)

Dry weight of

plant

(gm)

MM1 1.64bc 54.20c 140.60cd 67.50bc

MM2 1.55fg 47.46f 132.60f 60.40e

MM3 1.58ef 46.40f 126.68gf 58.10f

MM4 1.62cd 52.60cd 135.80e 61.60de

MM5 1.66b 57.48b 142.52c 69.48ab

MM6 1.64bc 50.50de 131.42f 56.66fg

MM7 1.60de 52.40cd 139.60d 63.42d

MM8 1.67b 58.46b 145.38b 71.60a

MM9 1.75a 61.66a 148.50a 66.42c

MM10 1.54gh 53.50cd 127.50g 61.26de

MM11 1.51h 47.74ef 125.42gh 55.22gh

MM12 1.52gh 46.48f 124.60h 53.48h

MM13 1.54gh 47.26f 126.50gh 57.30fg

S.Ed () 0.02 1.39 1.24 1.05

CD(0.05) 0.03 2.81 2.50 2.13


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Table 8.4: Effect of different treatments on stem girth, root length and biomass

of the plant (3rd

harvesting June10-sep-10)


plant

(gm)

Dry weight of

plant

(gm)

MM1 1.65f 51.50de 145.10c 70.18bc

MM2 1.69cd 59.20a 150.10b 75.32a

MM3 1.71bc 51.60de 142.50d 65.06d

MM4 1.75a 56.40b 148.71b 72.20b

MM5 1.68de 54.20c 139.20e 62.30e

MM6 1.69cd 59.20a 155.60a 75.60a

MM7 1.65f 53.50cd 135.51f 61.60e

MM8 1.72b 58.60a 157.48a 76.56a

MM9 1.71bcd 51.30de 141.71d 68.20c

MM10 1.66ef 51.00e 141.56d 62.40e

MM11 1.56h 48.16f 129.40g 58.30f

MM12 1.62g 49.28ef 130.20g 60.20ef

MM13 1.66ef 50.20f 133.31f 60.26ef

S.Ed () 0.01 1.03 1.15 1.04

CD(0.05) 0.02 2.08 2.33 2.11


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of the plant (4th

harvesting, Dec10-Mar-11)


plant

(gm)

Dry weight of

plant

(gm)

MM1 1.75d 53.08cd 150.82de 72.60de

MM2 1.85a 58.50a 160.06a 78.42a

MM3 1.75d 53.30cd 149.20e 72.28de

MM4 1.82b 58.50a 156.48bc 76.60abc

MM5 1.75d 55.50bc 148.78ef 72.52de

MM6 1.81b 57.50ab 158.50ab 77.64ab

MM7 1.74d 52.50d 152.12d 74.84cd

MM8 1.78c 56.70ab 155.22c 75.28bc

MM9 1.74d 52.10d 152.40d 70.10ef

MM10 1.75d 53.64cd 148.78ef 72.20de

MM11 1.65g 47.60e 145.80g 68.10f

MM12 1.68f 48.50e 146.24fg 59.26f

MM13 1.71e 52.54d 148.78ef 68.88f

S.Ed () 0.01 1.16 1.25 1.23

CD(0.05) 0.03 2.35 2.53 2.48


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Table 8.6 : Effect of different treatments on stem girth, root length and biomass

of the plant (5th

harvesting, Sep10-Dec-10)

The figures followed by same letter(s) do not differ significant


plant

(gm)

Dry weight of

plant

(gm)

MM1 1.87bc 59.20cd 188.40bc 81.34c

MM2 2.12a 64.26a 195.48a 88.56b

MM3 1.84cd 57.16de 182.68d 87.22

MM4 2.11a 62.38ab 196.44a 82.60

MM5 1.90bc 60.10bc 183.52d 76.28

MM6 1.93b 61.32bc 195.48a 82.28

MM7 1.78d 57.16de 184.58cd 88.56

MM8 1.91bc 60.10bc 190.12b 77.22

MM9 1.88bc 59.24cd 185.50cd 82.60

MM10 1.88bc 57.34de 183.52d 88.12

MM11 1.53e 41.30f 172.16e 68.46

MM12 1.43f 42.38f 171.56e 65.42

MM13 1.87bc 56.36e 181.62d 75.91

S.Ed () 0.04 1.08 2.04 1.26

CD(0.05) 0.07 2.19 4.13 2.56

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of the plant (6st harvesting, Mar11-june-11)



plant

(gm)

Dry weight of

plant

(gm)

MM1 2.02bc 59.24c 235.48fg 100.36ef

MM2 2.20a 64.84a 242.46b 110.20a

MM3 1.96c 58.66c 238.36de 100.96e

MM4 2.14ab 64.60a 244.82a 111.62d

MM5 2.00bc 61.36bc 236.62ef 99.40ef

MM6 2.11ab 62.72ab 242.46b 107.38b

MM7 2.00bc 58.66c 234.52g 100.40ef

MM8 2.08abc 61.46bc 241.72b 107.12bc

MM9 2.06bc 59.24c 239.60cd 104.56cd

MM10 2.08abc 58.68c 240.50bc 102.32de

MM11 1.69d 42.56d 161.72i 97.70f

MM12 1.75d 43.52d 172.56h 100.96e

MM13 1.96c 58.68c 234.52g 100.64ef

S.Ed () 0.06 1.26 0.91 1.32

CD(0.05) 0.12 2.55 1.85 2.66

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Table 8.8 Periodic observation on plant health and disease incidence

Treatment

Status of plant health and disease incidence after treatments

3 months 6 months 9 months 12 months 15 months 18 months

MM1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 3 3 1 1 4 3 4 2 4 3 4 5

MM2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 3 1 1 1 1 3 3 5 1 2 3 4

MM3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 1 1 2 4 5 1 4 1 4 1

MM4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 4 1 1 5 3 4

MM5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 3 1 4 5 2 4 1

MM6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 3 1 1 1 1 1 4 4 3

MM7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 4 1 4 5 4 3 3 4

MM8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 4 4

MM9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 2 4 1 4 1 2 4 5 5

MM10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1 1 2 3 4 5 5 4 4 4

MM11 1 1 1 1 1 1 1 2 2 3 2 2 3 3 4 1 4 4 4 4 5 5 4 4 4 4 5 5 5 5

MM12 1 1 1 1 1 1 1 1 1 1 1 1 3 3 2 1 4 4 4 2 5 4 4 4 5 5 5 5 5 4

MM13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 2 1 3 3

170

Table 8.9 Details of the plant health and disease incidence at the completion

of study.

Treatments Total numbers

of plants

Numbers of

diseased plants

%Disease reduction

over control

MM1 30 5 66.67

MM2 30 2 86.67

MM3 30 4 73.33

MM4 30 3 80.00

MM5 30 4 69.23

MM6 30 2 84.62

MM7 30 5 61.54

MM8 30 3 76.92

MM9 30 5 66.67

MM10 30 6 53.85

MM11(control) 30 15 00.00

MM12(control) 30 13 00.00

MM13(Absolute control)

30 1 00.00

N.B: Control denotes the plants treated with only pathogen and Absolute

control denotes the plants without any treatments

171

Table 8.10 Effect of antagonists on charcoal stump rot

Treatments % disease incidence

MM/PH/AC-02 + U.zonata 16.67

MM/PH/AC-09 + U.zonata 6.67

MM/PH/KMP + U.zonata 13.33

MM/PH/BST + U.zonata 10.00

T.viride (reference ) +U.zonata 16.67

U.zonata (control) 50.00

S.Ed (±) 0.8755

CD(0.05) 2.145

Table 8.11 Effect of antagonists on Brown root rot

Treatments % disease incidence

MM/PH/AC-02 + F.lamaoensis 13.33

MM/PH/AC-09 + F.lamaoensis 6.67

MM/PH/KMP + F.lamaoensis 16.67

MM/PH/BST + F.lamaoensis 10.00

T.viride (reference ) + F.lamaoensis 20.00

F.lamaoensis (control) 43.33

S.Ed (±) 0.8944

CD(0.05) 2.191

172

Fig 8.a. Effect of different treatments on stem girth, root length and

biomass of the plant (1st harvesting, Dec 09-Mar 10)

Fig. 8.b. Effect of different treatments on stem girth, root length and biomass of

the plant (2nd


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0

20

40

60

80

100

120

140

MM1 MM2 MM3 MM4 MM5 MM6 MM7 MM8 MM9 MM10MM11MM12MM13

Ste

m g

irth

(cm

)

Ro

ot le

ng

th (

cm

), F

resh

weig

ht

of

pla

nt

(gm

), D

ry w

eig

ht

of

pla

nt

(gm

)

Treatment

RL (cm) Fwt

Dwt Stem girth (cm)

1.35

1.4

1.45

1.5

1.55

1.6

1.65

1.7

1.75

1.8

0

20

40

60

80

100

120

140

160


Ste

m g

irth

(cm

)

Ro

ot le

ng

th (

cm

), F

resh

weig

ht

of

pla

nt

(gm

), D

ry w

eig

ht

of

pla

nt

(gm

)

Treatment

RL (cm) Fwt

Dwt Stem girth (cm)

173

Fig. 8.c. Effect of different treatments on stem girth, root length and biomass of the

plant (3rd

harvesting June10-sep-10)

Fig. 8.d. Effect of different treatments on stem girth, root length and biomass of the

plant (4th

harvesting, Dec10-Mar-11)

1.45

1.5

1.55

1.6

1.65

1.7

1.75

1.8

0

20

40

60

80

100

120

140

160

180


Ste

m g

irth

(cm

)

Ro

ot le

ng

th (

cm

), F

resh

weig

ht

of

pla

nt

(gm

), D

ry w

eig

ht

of

pla

nt

(gm

)

Treatment

RL (cm) Fwt

Dwt Stem girth (cm)

1.55

1.6

1.65

1.7

1.75

1.8

1.85

1.9

0

20

40

60

80

100

120

140

160

180


Ste

m g

irth

(cm

)

Ro

ot le

ng

th (

cm

), F

resh

weig

ht

of

pla

nt

(gm

), D

ry w

eig

ht

of

pla

nt

(gm

)

Treatment

RL (cm) Fwt

Dwt Stem girth (cm)

174

Fig. 8.e. Effect of different treatments on stem girth, root length and biomass of the

plant (5th

harvesting, Sep10-Dec-10)

Fig. 8.f. Effect of different treatments on stem girth, root length and biomass of the

plant (6th


0

0.5

1

1.5

2

2.5

0

50

100

150

200

250


Ste

m g

irth

(cm

)

Ro

ot le

ng

th (

cm

), F

resh

weig

ht

of

pla

nt (g

m),

Dry

weig

ht o

f p

lan

t (g

m)

Treatment

RL (cm) Fwt

Dwt Stem girth (cm)

0

0.5

1

1.5

2

2.5

0

50

100

150

200

250

300


Ste

m g

irth

(cm

)

Ro

ot le

ng

th (

cm

), F

resh

weig

ht

of

pla

nt

(gm

), D

ry w

eig

ht

of

pla

nt

(gm

)

Treatment

RL (cm) Fwt

Dwt Stem girth (cm)

175

Fig 8.g: Graphical representation of percent disease incidence and disease

reduction over control.

0

10

20

30

40

50

60

70

80

90

100

Treatments %Disease incidence %Disease reduction over control

Treatments

176

M 10 M 10 M 9 M95 4 5 4 5 4 5 4

M 4 M 3 M 2 M15 3 5 3 5 3 5 3

M 4 M 3 M 2 M15 5 5 5 5 5 5 5

M 11 M 11 M 11 M115 2 5 2 5 2 5 2

M 8 M 7 M 6 M55 5 5 5 5 5 5 5

M 8 M 7 M 6 M55 3 5 3 5 3 5 3

M 13 M 13 M 13 M135 2 5 2 5 2 5 2

M 12 M 12 M 12 M125 2 5 2 5 2 5 2

ENTRANCE

E

S

W

N

Fig 8.3 Layout of nursery trial plots

MM1- MM/PH/AC-02+ U.zonata

MM2- MM/PH/AC-09+ U.zonata

MM3- MM/PH/KMP + U.zonata

MM4- MM/PH/BST + U.zonata

MM5- MM/PH/AC-02+ F . lamoensis

MM6- MM/PH/AC-09+ F . lamoensis

MM7- MM/PH/KMP+ F . lamoensis

MM8- MM/PH/BST+ F . lamoensis

MM9 –T..viride + U.zonata

MM10- T..viride + F . Lamoensis

MM11- U.zonata

MM12- F . Lamoensis

MM13- Without Treatment.

177

fig 8.1: Site of Nursery trial

fig 8.2:

Cutting of TV1

cultivars raised in

nursery bed

178

fig 8.4: Observation of treated plants at the time of third harvesting

A- Treated with only pathogens; B- Treated with pathogen and antagonist.

fig 8.5: Observation of treated plants at the time of fourth harvesting

A-Treated with (MM/PH/AC-09+U.zonata ;

B-Treated with (MM/PH/BST + F.lamaoensis)

A

B

A

B

C

D

179

fig 8.6(a): Treated with pathogens +antagonists

fig 8.6(b): Treated with only pathogens

fig 8.6(c): untreated plants

180

Fig8.7: Observation of plants treated with only pathogens

at the time of sixth harvesting.

fig 8.8: Observation of root in uprooted plants after sixth harvesting

A- Healthy plant B- Diseased plant

A B

B

181

Fig 8.9: Root infected with U.zonata after 12 months

Of treatment application

Fig 8.10: Root infected with F.lamaoensis after 18 months

o f treatment application

chapter-viii bioassay of antagonists on disease...

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