BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF BACILLUS SPP. ISOLATED FROM RHIZOSPHERIC SOILS OF FIELD CROPS AND THEIR BIOCONTROL ACTIVITY AGAINST FUSARIUM OXYSPORUM F. SP. CUMINI
Loading...
Files
Date
2013-08
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
jau,junagadh
Abstract
Fusarium wilt caused by F. oxysporum f. sp. cumini (FOCu) is the major limiting factor for cumin productivity. Keeping this in view, 27 Bacillus spp. were isolated from 11 crop rhizospheres and screened for their in vitro antagonistic activity. Out of 27 isolates, 11 B. subtilis and 5 B. cereus were subjected to further antagonism which evaluated B. subtilis JND-KHCa-10A as best antagonist with 63.33 % growth inhibition of FOCu at 10 DAI followed by B. subtilis JND-KHCo-11A (56.67%), B. subtilis KSD-RHCu-1A (53.33%). However, B. cereus JND-RHCh-9C gave 35.83% maximum growth inhibition of test pathogen among five B. cereus isolates. The production of defence related substances by antagonists were assayed in synthetic medium containing cell wall of pathogen at 4 DAI. The percent growth inhibition was positively correlated with antibiotics siderophore and lytic enzymes chitinases and β-1,3-glucanase. However, cellulase and IAA production were negatively correlated with percent growth inhibition of test pathogen.
Total 16 antagonists and one test pathogen were characterized by whole cell protein profiles using Native and SDS-PAGE. A total 7 bands were generated in range during Native PAGE without any unique bands while SDS-PAGE of whole cell protein extract revealed 17 bands with 94.1% polymorphism and also distinguished B. subtilis from B. cereus by 118.83, 97.58 and 83.64 kDa marker bands.
The RAPD profiles of genomic DNA of antagonists and test pathogen demonstrated high level of polymorphism. The calculated PIC values for RAPD markers ranged from 0.152 to o.886 and RAPD primer index (RPI) differed from 0.30 to 12.37 which revealed that OPL-12 is the most inforamtive primer among 20 random primres used. Total 17 primers gave 32 specific unique amplicon for B. subtilis JND-KHCa-10A isolate associated with best antagonism against FOCu. The phylogenetic tree constructed by UPGMA method generated two main clusters and similarity coefficient was ranged from 9.8 to 96 % and it also discriminated
best antagonist (B. subtilis JND-KHCa-10A) from other Bacillus spp. with minimum 21.7 % similarity.
The discriminative pattern among the Bacillus isolates was validated by the 16s rDNA & 23s ITS analysis which produced the single amplicon of size more than 1kb with a few exceptions. The similarity coefficient was found ranging from 0.433 to 1.000. The Bsub5F & Bsub3R primers showed presence of single amplicon at 713 bp for 11 B. subtilis while it was not found for 5 B. cereus indicating species level identification.
On the basis of biochemical as well as molecular results, the isolate B. subtilis JND-KHCa-10A was found to be best biocontrol agent inhibiting the test pathogen FOCu. Biochemical markers chitinase, β-1,3-glucanase, protease, siderophore and salicylic acid are positively correlated with inhibition of test parhogen during in vitro antagonism. Moreover, the molecular characterization of antagonists produced 17 RAPD markers which discriminate best antagonist to control FOCu.
Key words: Bacillus spp., Fusarium wilt, Antagonism, Protein profiling, RAPD markers, 16s rDNA amplification
Description
Keywords
BIOTECHNOLOGY