148 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 i n- 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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148
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).
149
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.
150
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)
152
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.
153
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.
154
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).
156
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
157
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)
158
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