Screening And Identification Of Antagonistic Bacteria From The Rhizosphere Of Pepper, Cloning Of Antagonistic Gene Of Paenibacillus Polymyxa SC2

With Fusarium solani as indicator, 5 antagonistic bacteria were screened from the rhizosphere of pepper in Guizhou and were named as SC2, SC2-4, SC2-4-1, SC2-4-2, and SC2-4X, respectively. They have a broad-spectrum antimicrobial activity and can antagonize Fusarium oxysporum f. sp. cucumerinum, Pseudoperonospora cubensis, Botrytis cinerea Pers and Botrytis cinerea. Isolates SC2, SC2-4-2, and SC2-4X were identified as Paenibacillus polymyxa and isolates SC2-4 and SC2-4-1 were identified as Bacillus subtilis by morphological test, physiological and biochemical tests and 16S rDNA sequence analysis. The isolate SC2 probably produce antimicrobial protein or peptides.Detection ofβ-1, 4-xylanase andβ-1, 3-1, 4-glucanase activity revealed that Paenibacillus polymyxa SC2 could produceβ-1, 4-xylanase andβ-1, 3-1, 4- glucanase. A 4807bp DNA fragment was cloned by Thermal asymmetric interlaced PCR with genomic DNA of P. polymyxa SC2 as template. DNAMAN analysis of the DNA fragment revealed that the sequence contained two open reading frames (ORF1, ORF2), ORF1 of 1908bp encoded a protein of 635 amino acids with a molecular mass of 67.8 kDa, ORF2 of 714bp encoded a protein of 237 amino acids with a molecular mass of 26.8 kDa. According to the result of BLAST analysis, ORF1 was identical to xynD of P. polymyxa ATCC 842 at the level of 94%, ORF2 was identical to gluB of P. polymyxa WY110 at the level of 99%. Furthermore, based on the phylogenetic analysis, it was shown that the ORF1 and ORF2 could be xynD encodingβ-1, 4-xylanase and gluB encodingβ-1, 3-1, 4-glucanase, respectively.We obtained a 497bp conserved DNA fragment of NRPSs gene, with the primers TGD and LGG detecting non-ribosomal peptide synthetases concerved domain, with genomic DNA of P. polymyxa SC2 as template. According to the conserved DNA sequence, we designed specific primers LNRP1, LNRP2, LNRP3 and RNRP1, RNRP2, RNRP3 of TAIL-PCR. We amplified the DNA sequences flanking on the conserved DNA fragment by the method of TAIL-PCR with genomic DNA of P. polymyxa SC2 as template. Subsequently, the TAIL-PCR of upstream or downstream DNA sequences of the last amplified DNA fragments were continually carried out for several times. Alternatively, depending on the identity of the NRPS gene to bamC, mycC, ituC sequences of Bacillus subtilis strains and bmyC of Bacillus amyloliquefaciens FZB42, degenerate primers were designed to amplify the aimed DNA fragments. Finally, we obtained a resulting DNA sequence with the length of 19040bp by assembling all amplified DNA fragments.DNAMAN analysis revealed this DNA sequence includes a 5'-incomplete ORF of 17502bp. BLAST analysis revealed the ORF is identical to the homologous fusA sequence of Paenibacillus polymyxa E681 at the level of 90%, and similarity of the deduced amino acid sequences to FusA of the strain E681 is 92%. Based on the results of domain prediction of P. polymyxa SC2 FusA, it was shown that the deduced amino acid sequence (partial FusA) contain four modules typical of NRPSs. The domain organization of these four modules is C-A-T, C-A-T-E, C-A-T-E and C-A-T, respectively. The predicted substrate specificities of the four A domains within FusA were consistent with the last four amino acids, L-Tyr, D-Thr, D-Asn and Ala, in the peptide moiety of fusaricidin C. Similarity of the four A domains to the corresponding domain A3, A4, A5 and A6 of FusA of P. polymyxa E681 is 86%, 94%, 98% and 96%, respectively. Accordingly, identity of their DNA sequences is 85%, 89%, 94% and 92%. We deduce that fusA is a key gene involved in biosynthesis of nonribosomal peptide fusaricidin C from P. polymyxa SC2.