青皮核桃采后病害生防菌贝莱斯芽胞杆菌XRD006全基因组分析及防治效果研究

Bacillus velezensis XRD006: genomic characteristics and biocontrol effects on diseases of postharvest green walnuts

摘要:

【目的】探究生防菌贝莱斯芽胞杆菌(Bacillus velezensis) XRD006对青皮核桃采后病害的生防能力及其贮藏保鲜效果，解析菌株的基因特性和次级代谢产物，了解菌株的抑菌机制。【方法】通过抑菌试验确定XRD006对青皮核桃采后病原菌的抑制能力。利用活体抑菌及贮藏试验探究生防菌对青皮核桃采后病原菌的抑制能力及对青皮核桃贮藏品质的影响。以全基因组测序了解菌株XRD006的基因组特征及潜在抑菌相关基因；利用antiSMASH软件预测XRD006的次级代谢产物；结合比较基因组学分析XRD006和贝莱斯芽胞杆菌标准株FZB42、SQR9之间的共线性关系和次级代谢产物基因簇差异。利用高效液相色谱(high performance liquid chromatography, HPLC)和质谱鉴定XRD006次级代谢产物并通过牛津杯法测定其抑菌能力。【结果】抑菌试验表明菌株XRD006对青皮核桃采后病原菌隐秘刺盘孢(Colletotrichum aenigma)、暹罗炭疽菌(Colletotrichum siamense)、葡萄座腔菌(Botryosphaeria dothidea)和藤仓镰刀菌(Fusarium fujikuroi)的抑菌率分别为49.22%、50.61%、53.83%和58.71%。活体抑菌试验表明，XRD006在果实上能有效抑制病原菌的侵染与生长。贮藏试验显示，XRD006发酵上清液可以显著延缓果实的失重、减少微生物的生长，降低过氧化物酶(peroxidase, POD)和多酚氧化酶(polyphenol oxidase, PPO)酶活变化的同时，保持核仁品质。全基因组测序显示XRD006基因组长4 371 975 bp，GC含量为46.07%，含蛋白编码基因4 362个，具有胞外水解酶、生物膜等抑菌促生相关基因；antiSMASH预测显示XRD006分别含有9种已知和5种未知的次级代谢产物的编码基因簇。菌株XRD006与FZB42和SQR9亲缘关系密切，三者有8种相同的次级代谢产物基因簇，但是基因簇的位置和编码基因有差异。次级代谢产物鉴定显示，XRD006可以产生伊枯草菌素(iturin)和丰原素(fengycin)两种脂肽家族；抑菌试验表明，相比于C13-iturin、C14-iturin、C15-iturin和C17-fengycin C，fengycin家族的C16-fengycin B对暹罗炭疽菌(C. siamense) HT12的抑菌效果最好。【结论】贝莱斯芽胞杆菌XRD006对青皮核桃采后病害具有良好生防能力，具备实际应用潜力。

Abstract:

[Objective] To investigate Bacillus velezensis XRD006 in terms of the inhibitory effects on diseases of postharvest green walnuts, the fresh-keeping effect on green walnuts, genetic characteristics, secondary metabolites, and antifungal mechanism. [Methods] The activities of XRD006 against pathogens of postharvest green walnuts were determined by the inhibition experiments. In vivo fungal inhibition and storage quality experiments were conducted to investigate the inhibitory effects of the strain on pathogens and the effect of the strain on the storage quality of postharvest green walnuts. The genomic characteristics and potential antifungal genes of XRD006 were investigated by whole genome sequencing. The secondary metabolites of XRD006 were predicted by antiSMASH, and the collinearity and differences of the secondary metabolite gene clusters between strains XRD006, FZB42, and SQR9 were analyzed by comparative genomics. The secondary metabolites of XRD006 were identified by high performance liquid chromatography (HPLC) and mass spectrometry. The antagonistic ability of the strain was tested by the oxford cup method. [Results] The inhibition rates of XRD006 against Colletotrichum aenigma, Colletotrichum siamense, Botryosphaeria dothidea, and Fusarium fujikuroi of postharvest green walnuts were 49.22%, 50.61%, 53.83%, and 58.71%, respectively. In vivo antifungal experiments showed that XRD006 effectively inhibited the infection and growth of pathogenic fungi on the fruits. The fermentation supernatant of XRD006 significantly retarded the weight loss, inhibited the microbial growth, and reduced changes in peroxidase (POD) and polyphenol oxidase (PPO) activities while maintaining the kernel quality. The whole genome sequencing showed that the genome of XRD006 was 4 371 975 bp in length, containing 46.07% GC and 4 362 protein-coding genes (including antifungal and plant growth-promoting genes such as extracellular hydrolase and biofilm genes). The antiSMASH predicted that XRD006 had nine known and five unknown gene clusters of secondary metabolites. XRD006 was closely related to FZB42 and SQR9, and they shared eight secondary metabolite gene clusters, which showed varied location and coding genes. XRD006 produced two families of lipopeptides: iturin and fengycin. Compared with C13-iturin, C14-iturin, C15-iturin, and C17-fengycin C, C16-fengycin B of the fengycin family had the strongest inhibitory effect on C. siamense HT12. [Conclusion] B. velezensis XRD006 has good biocontrol effects on diseases of postharvest green walnuts and the potential for application in production.