假苍白杆菌A4对空间舱内小麦镰刀菌根腐病的生防机制研究

Plants with an important life-support functions in the space cabin, face the threat of fungal diseases. Plants, crew and equipment coexist in the closed and confined environment of space life support system.The control of plant diseases must be taken into account in crop growth, crew health and equipment safety in space carbin. Based on this requirment, we proposed the idea of screening Low-temperature-adapted biocontrol bacteria from the cave to control plant fungal diseases of space carbin. We have previously isolated a novel antagonist isolate, Pseudochrobactrum kiredjianiae A4, which has been found to colonize in plant roots, be able to secrete antimicrobial substances, and display a remarkably broad spectrum of biocontrol activity against plant pathogens. However, the biocontrol mechanism of Pseudochrobactrum kiredjianiae A4 is unclear because that bacteria of Pseudochrobactrum genus remain unreported as biocontrol bacteria. In this project, we plan to clarify the biocontrol mechanisms of strain A4 using Fusarium graminearumas as target pathogen, which have ability to cause wheat root rot.First，we will analyze colonization capability of strain A4 under Earth's gravity and simulated microgravity in a close carbin using antibiotic resistance labeling or green fluorescent protein labelling method. Second, we will analyze the inhibition of the strain A for target pathogen in the plant root at tissue and cell levels; Third, we will characterize the active antifungal compounds from the strain A4 by chromatographic techniques and spectroscopic approaches. Last, we will interprete the molecular mechanism of antifungal compounds of the strain A4 agaist the target pathogen using proteomics technique. The study will not only provide theoretical basis for the use of strain A4 as biological control agent in space life support system, but also lay a foundation for the development of biological pesticides using cave bacteria.

发挥着重要生命保障功能的植物，在空间舱面临着真菌病害的威胁。空间舱的封闭环境要求植物病害防控必须兼顾作物生长、人员健康、设备安全三个方面。前期申请人提出了筛选低温生防细菌防治空间舱内植物真菌病害的设想，已从地下洞穴分离出可在植物根部定殖、分泌抗菌物质、广谱抗病原真菌的低温适生菌株A4，其具备在空间舱内应用的潜力。进一步研究发现A4属于假苍白杆菌，生防机制尚不清楚。本项目拟以小麦根腐镰刀菌为靶标菌，开展A4在模拟空间环境下的生防机制研究。首先采用抗性标记或荧光蛋白标记技术分析密闭舱中不同重力下A4在小麦根部的定殖规律；随后以显微技术分析定殖后A4对靶标菌侵染根系的抑制作用；最后运用层析分离方法和波谱手段鉴定A4分泌的抗菌活性物质，并采用差异蛋白质组学揭示活性物质对靶标菌抑制的分子机理。本研究不仅可为A4菌株在空间舱中的实际应用提供理论依据，同时也为洞穴菌作为生物农药的开发奠定一定基础。

植物栽培在载人航天生命保障系统中发挥着重要作用。在长时间、远距离的受控生态生命保障系统中， 植物能够再生氧气和食物，而且能够消耗人类生存产生的二氧化碳和废水。在近地轨道航天器的物理化学式生命保障系统中，植物满足乘员的饮食多样性和新鲜维他命、粗纤维的需要。空间舱内的温湿度适宜，适合微生物的生长和繁殖。此外，重力的改变也能够刺激病原微生物的生长繁殖，而且增加其致病力和对农业化学药品的抵抗力。同时，密闭且重力改变的环境会导致植物对病原真菌的抗性减弱。因此，植物病害防治已经变成空间植物栽培亟待解决的关键问题之一。考虑到载人航天器集人、植物、设备于一个密闭有限空间的特殊性，我们提出了低温细菌更适合作为航天器生命保障系统中植物病原菌生物防治的微生物源。在本项目中，针对分离得到一种新型生防菌细菌——Pseudochrobactrum kiredjianiae A4，进行了模拟空间舱环境下的生防机制研究。我们研究了A4菌在正常重力（1G）和模拟微重力（μG）下对小麦幼苗生长发育和抗病能力的影响，发现在1G正常重力下A4菌能够促进小麦幼苗生长，然而这种促进作用在模拟微重力下减弱， A4菌通过诱导抗氧化酶活性和植物抗性相关激素来缓解模拟微重力和病原真菌侵染对植物造成的胁迫。我们进一步发现了模拟微重力下A4菌株在小麦根际定殖数量相对于常规重力下减少；同时发现A4菌通过改变小麦细胞壁、叶绿体形态结构，进而影响小麦镰刀菌Fusarium graminearum对小麦的侵染；模拟微重力下A4菌对小麦组织细胞形态结构影响发生显著变化。鉴定出其发酵液成分中含有戊二酰亚胺类物质为主要的抗菌活性物质。A4的抗F. graminearum的生防分子机制表现为上调小麦幼苗的过氧化物酶、谷胱甘肽转移酶、苯丙氨酸解氨酶、血红素结合蛋白转录水平，增强小麦抗病抗逆途径中苯丙氨酸代谢途径、木质素合成途径。这些结果不仅能够拓展我们关于模拟微重力对植物-微生物相互作用影响的理解，而且能够为太空植物栽培和作物保护提供有价值的途径方法。