Screening And Identification Of Antagonistic Bacteria Against Sclerotinia Sclerotiorum From Sunflower Rhizosphere, Preliminary Study On The Mechanism

Six samples of sunflowers rhizpsphere were gathered from siol in the farm of Inner Mongolia. 640 bacteria isolates were abtained by the way of gradient dilution and plate pairing culture. Take the Sclerotinia sclerotiorum as target bacteria , 18 isolates which have the antagonistic properties were got by the plate pairing culture, of which XRK1, XRK2, XRK4, XRK5, XRK9 have the most strongest antagonistic properties, the most stable and good application potential on biological defense.The XRK1, XRK2, XRK4, XRK5 and the XRK9 was identified as Bacillus subtilis, Streptomyces sp, Bacillus licheniformis, Lysobacter capsici, Pseudomonas brassicacearum respectively through morphologic observation, physiological, phenotypic characterization and 16S rDNA Sequencing.The activity detect ofβ-1,3-1,4-glucanase shows that XRK4 produces efficient, stable glucanase. XRK4 can also degradate the cell walls of Sclerotinia sclerotiorum. Based on the reported conserved sequence of Bacillus glucanase gene, primer PJTM3(5'-CCGGATTATGGCAAAAAGC-3'), PJTM4 (5'-GAGCCAGTCATCGACACCT-3') were designed to amplify the conserved sequence of theβ-1,3-1,4-glucanase gene of XRK4. Through PCR amplification, DNA segments of 580bp were obtained. By referring to the highly homology sequence on NCBI, the primer PJTM5,PJTM6, PJTM7 and PJTM8 were designed to amplifythe open reading frame ofβ-1,3-1,4 glucanase gene. Through PCR amplification, DNA segments of 732bp were obtained. It revealed that theβ-1,3-1,4 glucanase gene open reading frame had the highest similarity level to theβ-1,3-1,4-glucanase bglA(AY365256),which is up to 99%. Analysis of the active sites showed that:β-1,3-1,4 glucanase gene of XRK4 had 4 highly conserved amino acid sequences, which included Tryptophan, Glutamic acid, Aspartic acid and Glutamine.Tested by the plate of nutrition agar with antibiotics, Pseudomonas brassicacearum XRK9 were sensitive to kanamycin, but could tolerate 20μg/mL chloromycetin. The Tn5-1063a was used to mutagenize XRK9. Designing primers to amplify the special seqeunce of XRK9 genome, it shows that the mutant strain is from the original XRK9. Designing primers according to luxA sequences from Tn5-1063a, the DNA of wild strains, pRL1063a and the mutants as the templates, the sequences of luxA was obtained by PCR. The luxA of the mutants is identical to that of Tn5-1063a at 100% level, it indicates that Tn5-1063a was inserted directly into the genome of XRK9.After fermentation, salting-out, dialysis and plate-confrontation, it suggests that the extracellular proteins of XRK9 have antagonistic ability to Sclerotinia sclerotiorum of Sunflower. The molecular weight of the separated protein was detected through SDS-PAGE and Native-PAGE. Physiological and biochemical determination of the antagonistic protein indicates that the protein have high activeness under 60% of ammonium sulfate concentration, it is a kind of hydrophilic protein, it can resist high temperatures above 120℃, and it is not sensitive to proteinase K, ultraviolet rays and Chloroform.