SCREENING OF ACETAMIPRID TOLERANT PLANT GROWTH PROMOTING MICROORGANISM
ABSTARCT:
Increased demands for food have also increased the usage of pesticides. A pesticide not only kills the pests and pathogens but it also kills microorganisms that are beneficial to plant. So plant growth promoting microorganisms that are tolerant to pesticides would help to enrich soil microorganisms thus protecting the environment. In the present work four soil samples were collected from agricultural fields were pesticide usage was a regular practice. Acetamiprid tolerant microbes were isolated by using acetamiprid amended minimal agar medium.
Streptomyces AT (acetamiprid tolerant) isolate was isolated and its PGPR properties were evaluated. The isolate was found to posses properties like IAA secretion, phosphate solubilisation and siderophore production. Coriandrum sativum (coriander) was used as a model plant for evaluation of plant growth promoting properties of promising isolate, Streptomyces AT. The coriander seedlings were grown in soil inoculated with the AT1 isolate, Control plants were maintained by growing in un-inoculated soil. Root length, shoot length and number of plants were measured after 10 days of sowing.
Out of 60 coriander seeds sown in each pot, 25 and 50 seeds got germinated in uninoculated and inoculated soils respectively. The average root length of plants grown in uninoculated and inoculated soils was recorded as 37mm and 59mm respectively. The average shoot length of plants grown in uninoculated and inoculated soils was recorded as 34mm and 64mm respectively.
KEYWORDS: Acetamiprid, Streptomyces AT, coriandrum sativum, tolerance, plant growth promoting.
INTRODUCTION:
In developing countries like India agriculture is the major sector.
In agriculture pesticides play an important role for control of the pests which is important for better yield and disease free crops. Pesticides are the chemical substances intended to eradicate the pests. The use of pesticides in agriculture is unavoidable to most farmers as pesticides keep the crops free from infestation. Apart from the advantages of the pesticides in pest control they pose a high risk to the environment. Due to the repeated use of pesticides they get accumulated in the soil, water, turf and contaminate the environment. It is evident that pesticides posses potential risk to humans and other forms of with unwanted side effects. Microorganisms present in the soil due to the continuous and life excessive use of the pesticides either knowingly or unknowingly make the organisms tolerant to the pesticides.
The pesticide use in the present study was Acetamiprid (C10H11CIN14) , the organism tolerant to Acetamiprid was isolated invitro and tested for the plant growth promoting activites on the plant coriandrum sativum . The increase in the number of plants along with increased root and shoot length made the organism efficient in plant growth promotion.
MATERIALS AND METHODS
The experiment mentioned in the research include the isolation of pesticide tolerant microorganism and screening for PGPR activity IAA production, siderophore activity, HCN production, phosphate solubilisation and determining the most responsive plant by inoculating the responsive culture in soil for high yield of crops.
Isolation of the microbes from soil:
The soil samples were isolated from the local fields of Jalalpur (Yadadri) districts of Telangana, India; where agriculture and the use of pesticides was a regular practice. Latitude and Longitudinal of those areas are 17.340959, 78.535664 and 17.584976, 78.727894 respectively. The surface soil and the rhizosphere soil are collected respectively.
Method
0.05gms of each soil sample was weighed and inoculated into 50ml of minimal media containing 0.005gms of Acetamprid (1%), and incubated for 5days 370c in orbital shaking incubator. The soil samples were then plated on minimal agar plates amended with 1% acetamprid and incubated at 350c +/- 20c for 48 hours. Pickup the isolated colonies and re streak on minimal agar plates containing 1% of Acetamprid.
Procedure
- Day1:0.5gms of soil is weighed and mixed along with 1% of acetamprid in 20ml of minimal media in sterile Borosil/Corning make flask (100ml) and incubated for 5days in orbital shaking incubator.
- Day6:The soil samples are plated on minimal media plates containing 1% acetamiprid. The isolate was identified as Streptomyces AT.
- Day7: The isolates are re streaked on minimal media containing 1% acetampridDay8: The isolate was tested for plant growth promoting activities.
- Day17:The culture is growth in 100ml of minimal broth.
- Day21:The Streptomyces culture media was subjected to pots for the plant growth promoting activity for coriander seedlings.
RESULTS AND DISCUSSION
The isolate which is tolerant to 1% Acetamiprid grown on minimal salt medium was gram stained and identified as Streptomyces AT. The isolate was then tested for its plant growth promoting properties
In-vitro screening of Isolate for Plant Growth Promoting (PGP) Activities:
Siderophore Production:
The isolate was inoculated into nutrient broth and incubated 0.5m of ferric chloride is added for the detection of siderophore, if produced the colour change in the medium was observed indicating positive for Streptomyces . (1)
Phosphate Solubilizing Activity of Bacteria:
The isolate was screened for phosphate solubilisation on a selective media i.e. pikovaskaya’s agar medium (PAM).After incubation a clear zones develops around the colonies showing phosphate solubilization activity positive for Streptomyces.
Production of IAA (Indole acetic Acid):
The isolate was inoculated into Luria broth and incubated .After incubation freshly prepared salkowasky reagent was added to the medium. The change in the colour of medium from yellow to red indicates its positive reaction.(2)
Catalase test:
The Culture is placed on a clean glass slide with the help of a loop and H2O2 was added upon the culture the rapid evolution of oxygen within 5-10 seconds indicates positive.(3)
Amylase test:
Cultures of Streptomyces AT was placed on starch agar plate and incubated at 370C for 48hrs. Add 2-3 drops 10% iodine solution directly onto the edge of colony and wait for 10-15minutes and record the results. The medium turns dark and the area around the colony appears clear indicating that starch has been hydrolyzed.
Cellulase test:
The culture is grown on carboxy methyl cellulose agar for 48hrs at 370C after incubation the plate is flooded with Congo red. Wait for 15minutes if no clear zone is observed the organism is negative for cellulose production
Protease activity:
The culture was placed on sterile casein agar plates along with BCG and incubated for 48hrs at 370C after incubation a zone of proteolysis was detected on casein agar plates.(4)
In vivo study:
15 days after sowing coriander seeds plants were harvested and measured for different plant growth variables like root and shoot length and number of rootlets.(5)
CONCLUSION
From the agricultural soil, Streptomyces AT1 isolate was collected which is tolerant to acetamprid. The organism is potential as plant growth promoting bacteria due its ability to produce indole acetic acid (IAA), siderophore, phosphate solubilisation.The organism also possesses enzyme activities such as catalase, amylase, cellulase and protease activities. Coriandrum sativum (coriander) was used as a model plant for evaluation of plant growth promoting properties of promising isolate, Streptomyces AT. The coriander seedlings were grown in soil inoculated with the AT1 isolate, Control plants were maintained by growing in un-inoculated soil. Root length, shoot length and number of plants were measured after 10 days of sowing. Out of 60 coriander seeds sown in each pot, 25 and 50 seeds got germinated in uninoculated and inoculated soils respectively. The average root length of plants grown in uninoculated and inoculated soils was recorded as 37mm and 59mm respectively. The average shoot length of plants grown in uninoculated and inoculated soils was recorded as 34mm and 64mm respectively.
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