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| Microbiological Control of Rust Disease of Wheat Crop | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Paper Id :
16166 Submission Date :
01/06/2022 Acceptance Date :
21/06/2022 Publication Date :
25/06/2022
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| Abstract | Effect of spores and metabolites of dominant phylloplane fungi on germination and growth of germ tubes of uredospores of Puccinia recondita tritici, causal agent of rust disease of wheat crop was studied in vitro. In general, the percentage of inhibition of uredospores germination and growth of germ tube were in an increasing magnitude with the increase in the concentrations of spores of phylloplane micro fungi in the uredospore suspension. Addition of extra cellular products of phylloplane micro fungi also caused inhibition of spore germination and germ tube growth of uredospores and growth and development of uredosori under in vitro and in vivo conditions respectively. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Keywords | Phylloplane, micorfungi, Rust disease, Metabolites. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Introduction |
Despite growing awareness about environmental pollution, pesticides still dominate the measures of plant disease control. As a result of their indiscriminate use, a variety of agro – chemical is being introduced into the aerial and soil environment of plants. There is evidence that pesticides applied to leaves affect mineralization of elements, loss dry weight of liter and composition of fungal communities appearing in succession of decaying litter (Macauley, 1997; Campbell, 1985). Effects of pesticides on such natural processes should, therefore, affect the soil fertility, Substantial money of the world is being spent annually on the manufacture of agrochemicals to be used as pesticides and fertilizers. |
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| Aim of study | This study is an attempt has been taken to assess the possibility of controlling wheat rust disease by non-target phylloplane microfungi of wheat plant. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Review of Literature |
The use of pesticides in agriculture could provide little success in disease control. It is well known that some pathogens have acquired resistance to these chemicals. There are reports that application of chemicals, instead of providing any control, resulted in an increase in disease severity of some plants. thought such information are mostly on root diseases (Dekker, 1972; Fokkema et al, 1975), there is evidence for leaf disease also is evidence that delicate balance of composition, number and activities of phylloplane micro – organisms is considerably modified by the application of pesticides to leaf surface (Hislop, 1971; Dickinson, 1973; Sainger, 1982). Bollen and Scholten (1971) have shown that occurrence of benomy1 resistant Botrytis cinerea on sprayed cyclamen plants may be a consequence of the reduction of antagonistic Penicillium sp. Keeping in view the above, |
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| Main Text | Isolation
of spores of Non – target Phylloplane Fungi Spores
of non – target phyllloplane fungi were scrapped form the 10 – day – old
culture, grown on sterile PDA slants. These spores 10- days old culture, grown
on sterile PDA slants. These
spores along with mycelial fragment were mixed in sterile water. The mycelial
fragments were removed by filtering the spore suspension through there layered
cheesecloth. The spore concentration (2 x 105 spores cm-3) was measured
with the help of heamocytometer. This known concentration of spore suspension
was taken as stock sample. Spore suspension of desired concentration was
obtained by mixing the stock spore suspension and sterile water. Formula
for Preparation of Known concentration of Spores S1V1 = S2V2 where, S1 = concentration
of spore suspension to e prepared, V1 = Volume
of spore suspension to be prepared, S2 = concentration
of stock spore suspension, V2 = volume
to be taken from stock spore suspension, V1-V2 = volume
of sterile water mixed with volume of V2. Isolation
of Metabolites of Non- target Phylloplane Fungi. Teen
species of fungi as said earlier were grown individually in liquid czapek,
medium pH-6. Each 250 ml Erlenmeyer flask, containing 100 ml of the medium, was
inoculated with 10- day-old disc of the test fungus grown on PDA. The fungus
was allowed to grow for one week at 25+10C and incubated at room
temperature for 5 weeks. During this time the flasks were shaken at regular
intervals. The contents of each flask were filtered through what man No. 44
filter paper. The filtrate was centrifuged at 3000 rpm for 15 min. The supernatant
contain fungal metabolites was collected in sterile test tubes for further
experiments. Isolation
of Uredospores The
diseased leaf was suspended in Petri dishes containing sterile water. The leaf
surface was scrached with the help of forcep and needle so as to collect the
uredospores in the sterile water. The uredospores suspension was centrifuged at
3000 rpm for 15 min. at 150C. The centrifugation process was carried by
Remi-refrigerator centrifuge. The supernatant was discarded and 5ml of fresh
sterile water was added to the pellet containing uredospores. By the help of
Vortex mixer, homogenous spore suspension was obtained. The concentration or
uredospores was fixed at 104 uredospores cm-3 with the help of heamocytometer. The
study was made under sterile condition. Interactions
of Non-target Phylloplane Fungi and Pucinia recondita tritici (in
vitro) Germination
of rust uredospores of Puccinia recondita tritici was
studies in vitro by hanging drop method. A sterile
cavity slide was taken having petroleum jelly smear around the cavity. During,
petroleum jelly smearing process care was taken to keep the cavity free from
petroleum jelly contamination. A sterile cover slip with a drop of uredospore
suspension (0.01 ml) was carefully placed on the cavity. The purpose of
smearing the jelly was to keep the free space and cavity slide tight and also
to check evaporation form spore suspension. Including control two sets of
experiment were maintained during the trials. One set of experiment was made
from the spores/metabolites and in second set, the uredospore suspension was
mixed in liquia metabolites/spore suspension of individual non-target
phylloplane fungi. The slides were incubated at 20+10C for 24 hr
and then examined under high power 40x compound light microscope for
germination of uredospores. Length of germ-tube equal to the diameter of
uredospore was taken as criteria of germination. Germination
of Uredospores in Spore Suspension of Non-target Phylloplane Fungi. Know
concentration of uredospores (104 uredospores cm-3) and spores of
non-target fungi suspension (2 x 105 spores cm-3) were taken (v/v) and
mixed as per the formula stated earlier so as to get final spore concentration
of uredospores and individual phylloplane fungi in the ratio of 1:2, 1:5 and
1:2. The sutdy of uredospores germination was made as started above. Germination
of Uredospores in Presence of Metabolites The
germination of uredospores was studied in presence of heat-treated (550C temp.) and unheated
metabolites of individual phylloplane fungi. Measurement of germ tube length of
uredospores was also made. Sterile liquid Czapek, medium was taken as control. The
percentage of inhibition of germination and the length of germ tube was
calculated as the formula given below: Interactions
of Non-target phylloplane Fungi and Puccinia recondita tritici
(in vivo) Wheat
seedlings were grown in controlled laboratory conditions. The 11 days old
seedlings were transferred to field for further experiments. Excluding the
control ten experimental plots (2 m x 2 m) were taken for seedlings plantation.
Around 10-45 seedlings were planted in each plot and left for further growth.
After the three weeks of seedlings plantation, the dilute detergent (10%)
Triton 100 x was sprayed on the wheat plants. The purpose of dilution detergent
spray was to make leaf surface free from dust particles if any. The plants were
sprayed with uredospores suspension and metabolites of individual fungus. The
plants on control plot were sprayed only with sterile water. Sufficient care
was taken so as to make free the treatment plots from undesirable spore
suspension contamination. This practice was carried out simply by covering the
wheat plants with polythene bags while giving specific treatment to be a
desired experimental plot. Studies
on Uredosori Development in Presence of Metabolites under Field Conditions
Desirable concentration (8 x 103 cm-3) of uredospores suspension in fungal metabolite was prepared by using the formula cited earlier. After 45 hrs of the treatment, the polythene bags were removed from leaves to expose them to natural condition for 20 days. After 20 days the leaves were removed from the wheat plant and the number of uredosori was examined and calculated cm-2 leaf surface. The percentage inhibition of rust uredosori was calculated as follows:
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| Methodology | Isolation of Non – target Phylloplane Fungi
For the present studies 10 most common phylloplance fungi i.e. Alternaria alternata, Aspergillus flavus, A. luchuensis, A. niger, chaetomium globossum, Cladosporium cladosporioides, Drechslera australiensis, Fusarium moniliforme, Nigrospora sphaerica and Penicillium chrysogenum were selected to study their antagonistic nature with reference to rust diseases of wheat caused by Puccinia recondita tritici. |
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| Result and Discussion |
Data
on effect of different concentrations of spores of phylloplane fungi on
germination of uredospores of Puccinia recondita tritici has been computerized
in Table – 1. Fusarium moniliforme showed
more or less same order of impact on germination of uredospores as given in
Table- 1 However, metabolite of Aspergillus luchensis, was
more innibitory as compared to its spores (Table-1). This could have been due
to the effect of specific extra-cellular metabolite of Aspergillus luchuensis produced
in the vegetative hyphae the late phase of the growth and development of
mycelium (Whipps, 1987). A. luchuensis, Cladosporium cladosporioides, Drechslera australiensis, Alternaria alternata and Nigrospora sphaerica. In general, the order of inhibitory effect of metabolites under in vivo condition was different from that of in vitro condition. Table
– 1
Table – 2 Effect of extra-cellular product (metabolites) of some phylloplane fungi grown in liquid Czapek’s medium on germination of uredospore of Puccinia recondita tritici.
Table – 3 Effect of metabolite on germ tube growth of uredospores ofPuccinia recondita tritici.
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| Conclusion | Numerous antagonistic microorganisms with potential for biocontrol of diseases of wheat (Morgan, 1963; Steward and Hill, 1965 and other cultivated plants (Sinha, 1965; Henis and chet, 1975; Rai and Singh, 1980; Cook and Baker, 1983; Sharma, 1985; Paulitz et al, 1987) have been indentified during the recent past either in the laboratory or in greenhouse trials. This antagonistic nature of microorganisms due to direct or indirect effect of metabolites either in the form of specific inhibitor or as enzymes (Dennis and Webster, 1971; Elad et al, 1982,) is responsible for the lysis of the vegetative structure of pathogens (Elad et al, 1982;) Generally the enzymes are very much susceptible to temperature approaching boiling point of water. In this connection date on increase in inhibitory effect of heat treated metabolites rule out the possibility of any extraellular enzymatic effect on germination of uredospores as described earlier. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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