棉花黄萎病拮抗内生贝莱斯芽胞杆菌在土壤-植物系统中定殖特征及强化抗病机制研究

It is a promising new biocontrol strategy to control plant diseases using antagonistic endophytic bacteria. Whether the introduced biocontrol bacteria can colonize in rhizosphere soil and roots is the key to the success and failure of biocontrol. Previously, we found that cotton endophytic bacteria Bacillus velezenensis BHZ-29 could survive in tissue and the rhizosphere of cotton plants for a long time and the colonization time was significantly correlated with the control effects. But it is not clear that colonization of B. velezensis BHZ-29 in Soil-Plant system and its mechanism of enhancement of disease resistance. Therefore, the strain BHZ-29 was tagged with green fluorescent protein (gfp), and the effects of cotton root exudates on the growth and membrane formation of antagonistic bacteria were examined. The colonization characteristics of antagonistic bacteria in cotton rhizosphere were assessed. The researches revealed the interaction between cotton and biocontrol bacteria mediated by root exudates, and the colonization of biocontrol bacteria in the rhizosphere of cotton. Colonization rule of antagonistic strain BHZ-29 in cotton was assayed by tracking monitoring the adhesion of infection, colonization sites, colonization of transmission, distribution and proliferation behavior in cotton. By analyzing the changes of the activities of resistance-related enzymes, the content of other disease-related substances and the gene expression of defense enzymes in cotton induced by antagonistic bacteria, the mechanism of antagonistic endophytic bacteria to enhance cotton resistance was revealed, in order to improve the colonization ability and to stabilize and improve the control effect of biocontrol bacteria.

利用植物拮抗内生菌控制植物病害是极具研发前景的生防新策略，而拮抗菌能否在植物根际或体内定殖是决定生防成败的关键。申请人前期研究发现棉花内生贝莱斯芽胞杆菌BHZ-29能在棉花根际与体内存活较长时间且与防病效果显著相关，但不清楚BHZ-29在土壤-植物系统中的定殖特征及强化抗病机制。因此，本研究拟利用绿色荧光蛋白标记拮抗菌，通过研究棉花根系分泌物对拮抗菌生长和成膜的影响与拮抗菌在棉花植株根际的定殖特征，揭示根系分泌物介导的"棉花-生防细菌"互作关系以及生防细菌在棉花根际的定殖动态；通过跟踪检测拮抗菌在棉花体内的粘附侵染，定殖位点，定殖传导，分布增殖等行为，明确其在棉花体内的定殖规律；通过分析拮抗菌诱导棉花抗性相关酶类活性、其他抗病相关物质含量及防御酶基因表达量的变化，揭示拮抗内生细菌强化棉花抗病性的诱导机制，为提高生防菌的定殖能力、稳定和提高防效奠定基础。

棉花黄萎病(Verticillium dahlia Kleb.)是新疆棉花生产上最重要的病害之一，严重阻碍着新疆棉花产业的发展。棉花内生贝莱斯芽胞杆菌BHZ-29能在棉花根际与体内存活较长时间且与防病效果显著相关，由于对BHZ-29在土壤-植物系统中的定殖特征缺乏深入认识，对其强化抗病机制知之甚少，因而，解析BHZ-29在土壤-植物系统中的定殖特征及强化抗病机制成为亟待解决的关键问题。课题组前期获得了1株有较强抑菌活性的内生贝莱斯芽胞杆菌BHZ-29。本课题利用抗生素标记、PMA-qPCR检测、挑战接种、实时荧光定量PCR等技术，首先获得了Rifampicin标记的贝莱斯芽胞杆菌BHZ-29，明确了拮抗细菌和大丽轮枝菌在棉花根际土壤中的定殖规律，揭示了根系分泌物介导的"棉花-生防细菌"互作关系：不同棉花品种根系分泌物提高了BHZ-29成膜能力，其中葡聚糖、精氨酸、缬氨酸、丙氨酸显著提高菌株BHZ-29成膜能力。BHZ-29在不同棉花品种中的定殖能力均表现出根部＞茎部＞叶部，定殖规律呈先增加后减少变化趋势。BHZ-29具有明显的棉花根粘附性能，能通过棉花根、茎、叶部伤口和自然孔口入侵棉花。BHZ-29在棉花根、茎、叶部的定殖位点与侵染部位相关，根、茎和叶侵染，定殖量分别为根部＞叶部＞茎部，叶部＞茎部＞根部，茎部＞叶部＞根部，且灌根法接种定殖量高于其它接种方法。qPCR揭示了菌株BHZ–29由根际，经根、茎向叶部传导的数量递减规律。明确了BHZ–29定殖顺序根际＞根部＞叶部＞茎部分布与增殖特征。BHZ-29提高了不同抗性品种棉花过氧化物酶（POD）、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、多酚氧化酶（PPO）、苯丙氨酸解氨酶（PAL）活性和Vc、丙二醛（MDA）含量。BHZ–29诱导了3条主酶带，1条副酶带的棉叶过氧化物酶同工酶酶谱；诱导了3条主酶带的棉叶超氧化物歧化酶同工酶酶谱；接种初期诱导了1条主酶带，中后期诱导主副酶带活性增强区，同时诱导酯酶同工酶活升高，副带增多的棉叶酯酶同工酶酶谱。BHZ-29诱导pal、pod和cat基因相对表达量呈显著性先降低后升高变化趋势。