Genetic diversity in biocontrol - Pseudomonas species in coastal agri-ecosystem of Southern India, using RFLP and SSCP techniques
S, Bharathkumar; Paul, Diby and Nair, Sudha (2004) Genetic diversity in biocontrol - Pseudomonas species in coastal agri-ecosystem of Southern India, using RFLP and SSCP techniques. In: 13th Southern Regional Conference on Microbial Inoculants, 3-5 Dec 2004, Bijapur, India.
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Rhizospheric Pseudomonas species were isolated from crops grown in coastal agri-ecosystem. These strains were tested against the pathogens, Cercospora personata (Tikka disease) in groundnut and Xanthomonas campestris pv. vesicatoria (Leaf spot) in chilly and tomato. The major antagonistic principles in these strains were proved to be 2, 4- diacetylphloroglucinol (2,4 – DAPG), Pyrrolnitrin (PRN) and Pyoluteorin (PLT) which are broad-spectrum antibiotics. The present study deals with the genetic diversity among these antibiotic-producing strains of Pseudomonas spp. using Restriction Fragment Length Polymorphism (RFLP) and Single strand Conformation Polymorphism (SSCP) of DNA techniques. Gene-specific primers were developed from conserved sequences and 2,4-DAPG, PrnD and PltC fragments were amplified from 10 different species of Pseudomonas that produced either two or three of these antibiotics. Polymorphisms within the genes were analyzed by RFLP method using a number of restriction enzymes. Single-strand-conformation polymorphism (SSCP) of DNA, a more efficient method, widely used in mutation analysis was adapted for the analysis of differentiation among amplified genes coding for the antibiotic compounds. The antibiotic genes were amplified with respective primers and the phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with MDE polyacrylamide gel. The polymorphisms among the antibiotic coding genes revealed great level of diversity among the Pseudomonas spp. in the coastal agri-eco system in the southern coast of India.
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