贝莱斯芽孢杆菌Pm9生物防治潜力及全基因组分析

Biological control potential and complete genome analysis of Bacillus velezensis Pm9

为探究贝莱斯芽孢杆菌(Bacillus velezensis)Pm9对植物病害的生防潜力,明确其抑菌广谱性、无菌发酵滤液抑菌活性的稳定性、抑菌机制和抑菌物质遗传基础,并为生防制剂开发利用提供依据,采用平板对峙法测定菌株Pm9对小麦赤霉病菌(Fusarium graminearum)、小麦全蚀病菌(Gaeumannomyces graminis)、小麦纹枯病菌(Rizoctonia cerealis)、君子兰茎基腐病菌(Fusarium proliferatum)、番茄灰霉病菌(Botrytis cinerea)和南天竹炭疽病菌(Colletotrichum karstii)6种供试植物病原菌的抑制作用。以葡萄座腔菌(Botryosphaeria dothidea)为供试病原菌,测定菌株Pm9无菌发酵液抑菌物质稳定性,并考察无菌发酵滤液对葡萄座腔菌孢子萌发和菌丝生长的影响。利用3代Nanopore测序仪测定菌株Pm9的全基因序列,通过BLAST比对分析明确抑菌作用的遗传基础。结果表明,菌株Pm9对6种植物病原菌均具有抑制作用,抑制率在55.5%~87.5%之间。菌株Pm9无菌发酵滤液在pH 4~10之间抑菌活性稳定,耐受酸碱范围广,同时对紫外线照射和蛋白酶K不敏感,80℃以内具有较强的热稳定性。菌株Pm9可使葡萄座腔菌菌丝膨大畸形,抑制菌丝生长和孢子萌发。菌株Pm9含有合成地非西丁(difficidin)、丰原素(fengycin)、bacillibactin、表面活性素(surfactin)等多种肽聚糖和聚酮糖类抗性化合物的基因簇,以及能够降解病原菌细胞壁的β-1,3-葡聚糖酶和几丁质酶相关的基因。贝莱斯芽孢杆菌Pm9可抑制多种植物病原菌的生长,抑菌物质稳定性高,具有产生多种肽聚糖和聚酮糖类抗性化合物和细胞壁水解酶的能力,本研究为菌株Pm9进一步开发应用奠定了基础。

To explore the biocontrol potential of endophytic Bacillus velezensis Pm9 isolated from Malus sieversii,the antagonistic effect of strain Pm9 against Botryosphaeria dothidea and various plant pathogens,the inhibitory stability of its metabolites,and the molecular mechanism underlying the biocontrol processes were studied.The antagonistic effect of strain Pm9 against six pathogenic fungi was evaluated by the confrontation culture method,including Fusarium graminearum,Gaeumannomyces graminis,Rizoctonia cerealis,Fusarium proliferatum,Botrytis cinerea and Colletotrichum karstii.The stability of bacteriostatic substances in sterile fermentation broth of strain Pm9 was determined using B.dothidea as the test pathogens.The effect of sterile filtrate on the spore germination and mycelial growth of B.dothidea was studied.The potential biocontrol molecular mechanism of strain Pm9 was analyzed by the complete genome sequence,and BLAST comparative analysis was used to confirm the genetic basis of the bacteriostatic action.Strain Pm9 exhibited antagonistic abilities to six plant pathogens,and the inhibition rate was between 55.5%and 87.5%.In addition,it still maintained the antifungal activity after the treatment of acidic,alkaline,heat(<80℃),proteinase K and ultraviolet radiation.Strain Pm9 could cause the abnormal growth of mycelium and inhabit the growth of myceliu and the germination of the spore.Complete genome sequence analysis showed that strain Pm9 contained gene clusters related to the biosynthesis of a variety of polypeptide and polyketide compounds,such as difficidin,fengycin,bacillibactin and surfactin,and the genes encoding enzymes likeβ-1,3-glucase and chitinase,that could hydrolyze the pathogen cell wall.Strain Pm9 had strong inhibitory activity against fungal pathogens in fruit trees,wheat,vegetable,and so on.And the inhibitory activity was stable.With the ability to produce a variety of peptidoglycan and polyketosaccharide resistant compounds and cell wall hydrolases,strain Pm9 can be exploited as bio-controlling strains with high potential value for further study.