烤烟连作障碍因子的发生及生物修复机制研究

Difficulties in establishing continuous cropping are seriously limiting the sustainable development of tobacco industry in Yunnan and China. The current project investigates the effects of allelochemicals on the secondary metabolism of flue-cured tobacco that restrict the continuous cropping of flue-cured tobacco. The allelopathic effects are caused by phenolic-acid derived compounds produced by decomposing organic matter originating from crop residues. Experiments of continuous cropping were conducted in potted plants and in the field in various locations to isolate and determine the primary phenolic-acid derived allelopathic toxins which prevent the continuous cropping of flue-cured tobacco. The aim of the research was to establish the relationship between phenolic-acid derived allelochemicals excreted by flue-cured tobacco and the secondary metabolism of this crop. A particular focus was on the exudation of allelochemicals into the rhizosphere and their accumulation in the products of secondary metabolism of flue-cured tobacco leading to the autotoxicity of tobacco during continuous cropping. Furthermore, attention was given to the composition of the soil microflora that may influence the allelopathic effects on the flue-cured tobacco. The research was looking at the response mechanisms by screening the microbial strains in the rhizosphere that are showing high-resistance and are capable of decomposing the phenolic-acid derived allelochemicals affecting flue-cured tobacco base on the screening technique of microbial acclimation and microbial variation caused by artificial phenolic-acid allelopathic poison, and gram-negative bacteria exploitation to overcome continuous cropping barrier of flue-cured tobacco.

连作障碍已成为制约云南乃至全国烟草种植业的关键因素之一。本项目依据烤烟次生代谢分泌、根茬腐解产生酚酸类化感毒物诱发烤烟连作障碍的原理，采用实验模拟、盆栽重茬试验结合大田多点定位连作试验方法，研究分离鉴定诱发烤烟连作障碍的主要酚酸类化感毒物，深入探究烤烟分泌酚酸化感毒物与烤烟代谢、尤其是次生代谢的关系；探明烤烟主要酚酸致毒物质对土壤微生物群落结构演化的诱导机制及其对烤烟病菌侵染的影响，并探讨主要病原（根黑腐病菌、青枯病菌、黑胫病菌）侵染诱导烤烟次生代谢导致毒素积累、分泌及其对连作烤烟自毒的响应，揭示烤烟连障碍因子发生的机理。在此基础上，通过连作驯化、酚酸毒物诱导变异的方法，筛选出对烤烟主要酚酸致毒化感物质有高抗性、高降解特性的微生物菌株和菌剂，用该菌株和菌剂的接种土培试验探讨生物修复克服烤烟连作致害的机制和方法。本研究将明确烤烟连作障碍因子的发生、生物修复机制及其在克服烤烟连作障碍中的意义。

连作障碍已成为制约云南乃至全国烟草种植业发展的主要因素之一。本项目依据烤烟次生代谢分泌、根茬腐解产生酚酸类化感毒物诱发烤烟连作障碍的原理，采用盆栽重茬结合大田限根栽培根际模拟试验、大田连续定位连作试验方法，① 研究分离鉴定出诱发烤烟连作障碍的主要酚酸类化感毒物，包括对羟基苯甲酸、肉桂酸、4-羟基苯甲酸、香草酸、棕榈酸，同时采用这些酚酸致毒物为唯一碳源的选择培养基，逐步提高有关酚酸物质浓度的筛选方法，从重茬连作植烟土壤中成功筛选出能有效降解对羟基苯甲酸的菌株；② 研究得出烤烟连续重茬会导致土壤养分残留增多，盐分富集，土壤碳氮代谢相关酶活性降低，且由于植烟根际酚酸化感毒物积累，导致少数真菌类群富集，多数真菌、细菌、放线菌类群数量减少，种群变化明显呈单一化趋势，结果导致土壤微生态结构失衡，连作病害爆发，烤烟枯萎死亡，造成恶性循环；③系统研究表明深耕、大塘均匀施肥结合增施生物炭、增炭减肥和配套实施烤烟-万寿菊套作的系列烤烟重茬连作生物、生态修复技术，在完全不打农药条件下，试验示范尺度上达到90%以上的烤烟重茬烟株连作病害免疫，且有减少烤烟重茬连作的土壤肥料养分残留和盐分积累，促进肥料养分的吸收利用，显著提升烤烟质量的重要作用；④ 应用高通量测序技术，系统研究探讨了接种病原诱导、增施生物炭及烤烟-万寿菊套作的连作障碍微生物生态修复机制，结果表明接种黑胫病菌、根结线虫病原物的诱导作用并配合烤烟-套作万寿菊套作措施，能显著提高烤烟重茬连作的植烟根际土壤各微生物群落种群多样性和微生物丰度，且同步显著提高烟草内生细菌多样性，适量施用生物炭以及生物炭结合烤烟-万寿菊套作的措施也有相似作用效果。这表明相关生物、生态修复是从内生、根际层面同步提高了微生物群落种群多样性，从而在烟株根际和植物体层面构造了“双重生物多样性防御圈”，从而起到有效控制和减轻烤烟重茬连作病害的重要作用。