月季炭疽病内生拮抗细菌的筛选鉴定及抑菌作用

Screening, identification and bacteriostasic activity of antagonistic endophytic bacteria against Rosa chinensis Jacp.anthracnose

  • 摘要: 【目的】发掘月季内生拮抗细菌资源,为月季炭疽病生物防治提供高效而稳定的拮抗菌株资源。【方法】以20个健康无病害蔷薇属植物的叶片为试验材料,采用组织匀浆法分离叶片内生细菌,采用平板对峙法筛选对月季炭疽病病原菌(Colletotrichum fructicola)YJY-20具有拮抗作用的内生细菌菌株,结合菌落形态、生理生化和16S rRNA序列分析对拮抗菌进行种类鉴定。利用平板对扣法测定拮抗菌挥发性有机化合物的拮抗效果;利用牛津杯扩散法测定拮抗菌无菌发酵液对炭疽病病原菌及菌丝的影响;通过PCR扩增脂肽类抑菌物质合成基因;采用琼脂孔扩散法测定拮抗菌粗蛋白和脂肽类抑菌物质的抑菌活性;利用平板检测法测定拮抗菌产胞外酶活性;通过平板对峙法测定拮抗菌对12种植物病原真菌的抑制效果;采用离体叶片试验测定拮抗菌发酵液对月季炭疽病的防治效果。【结果】从蔷薇属植物叶片中分离筛选出2株对菌株YJY-20具有较强拮抗效果的内生细菌DMGSMG4和QZM1,经鉴定分别为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和贝莱斯芽孢杆菌(B. velezensis)。菌株DMGSMG4和QZM1的挥发性有机化合物对菌株YJY-20的抑制率分别为11.72%和30.71%,同时能减少病原菌黑色素生成;其无菌发酵液对菌株YJY-20的抑制率分别为60.25%和61.24%,并能造成病原菌菌丝断裂扭曲。菌株DMGSMG4和QZM1粗蛋白对菌株YJY-20的抑制率分别为59.76%和50.63%,且热稳定性较好;其脂肽类抑菌物质对菌株YJY-20的抑制率分别为50.07%和45.03%。2株拮抗菌均能扩增出bmyBfenDsrfAAsrfAB脂肽类抑菌物质合成相关基因。2株拮抗菌可产纤维素酶和蛋白酶,其中菌株DMSGMG4还可产有机酸。菌株DMGSMG4和QZM1对12种植物病原真菌的抑制率均大于50.00%,其对月季炭疽病离体叶片防治率分别为59.35%和20.58%。【结论】筛选获得的菌株DMGSMG4和QZM1对炭疽病病原菌具有较强的拮抗效果,具有作为月季炭疽病生防菌株的潜力。

     

    Abstract: 【Objective】To explore the resources of endophytic antagonistic bacteria of Rosa chinensis Jacp., and provide efficient and stable antagonistic strain resources for biocontrol of R. chinensis anthracnose. 【Method】The leaves of 20 healthy and disease-free Rosa species were used as test materials, the endophytic bacteria were isolated from the leaves by tissue homogenization method, the endophytic bacteria strains with antagonistic effect against Colletotrichum fructicola YJY-20 were screened by plate confrontation method. The antagonistic bacteria were identified according to colony morphology, physiological and biochemical characteristics and 16S rRNA sequence analysis. The antagonistic effect against C. fructicola YJY-20 of volatile organic compounds from antagonistic bacteria were determined by plate interlocking method. The inhibitory activity of sterile fermentation broth of the antagonistic bacteria on C. fructicola YJY-20 and its hyphae were determined by the Oxford cup diffusion method. The PCR was used to amplify lipopeptide biocontrol functional genes. The inhibitory activity of the crude protein and lipopeptide antibacterial substance from antagonistic bacteria against C. fructicola YJY-20 were determined by agar well diffusion method. The extracellular enzyme activity of the antagonistic bacteria were detected by plate assay method. The inhibitory activity of antagonistic bacteria against 12 different plant pathogenic fungi were determined by plate confrontation method. The control effects of fermentation broth of antagonistic bacteria against R. chinensis anthracnose caused by C. fructicola were detected by in vitro leaves experiment. 【Result】Two endophytic bacteria strains DMGSMG4 and QZM1 with strong activity against C. fructicola YJY-20 were isolated and screened from the leaves of Rosa species, and they were identified as Bacillus amyloliquefaciens and B. velezensis respectively. The inhibition rates against C. fructicola YJY-20 of strains DMGSMG4 and QZM1 volatile organic compounds were 11.72% and 30.71% respectively, and they both could reduce melanin production of C. fructicola YJY-20.The inhibition rates against C. fructicola YJY-20 of strains DMGSMG4 and QZM1 sterile fermentation broth were 60.25%and 61.24% respectively, and they could cause hyphae of C. fructicola YJY-20 breakage and distortion. The inhibition rates against C. fructicola YJY-20 of strains DMGSMG4 and QZM1 crude protein were 59.76% and 50.63% respectively, and their thermal stability was good. The inhibition rates of the strains DMGSMG4 and QZM1 lipopeptide antibacterial compounds on C. fructicola YJY-20 were 50.07% and 45.03% respectively. The two strains both could amplify lipopeptide antibacterial substance synthetic related genes such as srfAA, srfAB, fenD and bmyB. The two strains both could produce cellulase and protease, and strain DMSGMG4 could produce organic acids. The inhibition rates of two strains DMGSMG4 and QZM1 were more than 50.00% against 12 different plant pathogenic fungi, and control rates on the in vitro leaves with anthracnose were 59.35% and 20.58% respectively. 【Conclusion】The screened strains DMGSMG4 and QZM1 have strong antagonistic effect against C. fructicola, and they show potential to be biocontrol strains for R. chinensis anthracnose.

     

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