一株拮抗黄单胞菌的贝莱斯芽孢杆菌的分离和鉴定

Isolation and identification of a Bacillus velezensis strain against plant pathogenic Xanthomonas spp.

摘要:

[目的]为了筛选防治水稻条斑病（bacterial leaf streak，BLS）的生防细菌。[方法]以水稻条斑病菌（Xanthomonas oryzae pv.oryzicola，Xoc）的模式菌株RS105为靶标菌，采用平板稀释和抑菌圈法，从空心菜根际土壤中筛选到一株对RS105具有拮抗作用的细菌菌株504。通过形态学、生理生化特征以及16S rDNA和gyrA序列分析对菌株504进行了鉴定。利用牛津杯法测定504对植物病原黄单胞菌的拮抗活性及其无菌发酵液拮抗活性的稳定性。通过PCR扩增预测504编码合成脂肽类和聚酮类化合物的合成相关基因。采用苗期水稻注射接菌法来评价水稻组织中504对Xoc的拮抗活性。[结果]菌株鉴定结果表明504为贝莱斯芽孢杆菌，命名为Bacillus velezensis 504。抑菌实验显示，B.velezensis 504对黄单胞菌属的细菌具有较好的抑菌活性，对水稻白叶枯病菌（X.oryzae pv.oryzae，Xoo）的拮抗效果最显著。基因预测结果显示，B.velezensis 504含有fenA、dhbA、sfrA、bmyA、beaS、dfnA及bacA等编码脂肽类和聚酮糖类抑菌化合物的基因簇。其无菌发酵液的活性物质耐高温和蛋白酶降解，但不耐强酸、强碱，在pH值为5.5-8.9时仍具有稳定的拮抗活性。在高感水稻品种原丰早上，B.velezensis 504对Xoc在水稻叶片中引起的水渍症状具有显著的抑制作用。[结论]B.velezensis 504能够特异性拮抗黄单胞菌，在黄单胞菌引起的细菌性病害的生物防治中将具有较大的应用潜力。

Abstract:

[Objective] To obtain biocontrol bacteria to control bacterial leaf streak (BLS) of rice caused by Xanthomonas oryzae pv. oryzicola (Xoc). [Methods] A bacterial strain 504 with antibacterial activity was screened from water spinach rhizosphere soil by agar disk dilution and inhibition zone method using an Xoc model strain RS105 as target. Strain 504 was identified through morphological, physiological and biochemical characteristics in combination with 16S rDNA and gyrA sequence analysis. Antagonistic activity of 504 against some plant pathogenic Xanthomonas spp. was measured and stability of its sterile fermentation broth against Xoc was analyzed by Oxford Cup method. We predicted the genes encoding the synthesis of multiple lipopeptide and polyketide compounds by PCR amplification. Antibacterial activities of 504 against Xoc in the leaves of rice seedlings were evaluated by injection inoculation method. [Results] Strain 504 was identified as Bacillus velezensis, named B. velezensis 504. B. velezensis 504 showed specific inhibitory effects on some plant pathogenic Xanthomonas, especially on X. oryzae pv. oryzae. The secondary metabolites prediction showed that B. velezensis 504 contains genes such as fenA, dhbA, sfrA, bmyA, beaS, dfnA and bacA encoding the biosynthesis of various peptidoglycans and polyketose antibacterial compounds. The cell-free supernatant showed stable inhibitory effects against Xoc under high temperature and by protease treatment, however, there is no inhibitory activities when the pH value of supernatant is beyond 5.5 to 8.9. B. velezensis 504 exhibits significantly inhibitory effect on water-soaked lesions caused by Xoc in the leaves of the high susceptible rice cultivar Yuanfengzao. [Conclusion] B. velezensis 504 exhibits the broad-spectrum antagonistic activity against Xanthomonas spp., and has the potential as an effective biocontrol agent on diseases caused by plant pathogenic Xanthomonas spp..