作 者 ：刘艳霞,李想,蔡刘体,张恒,石俊雄

期 刊 ：植物营养与肥料学报 2016年 22卷 2期 页码：418-428

关键词：烟草根系分泌物；病原菌；拮抗菌；根际微生物；土壤微生物群落功能多样性；

Keywords:tobacco root exudates, pathogen, antagonist, rhizosphere microorganism, soil microbial functional diversity,

摘 要 ：【目的】烟草连作已导致土传病害发生、 烟株生长受抑制、 产量下降和品质恶化等问题。烟株对自身及土壤微生物产生的化感作用,是烟草产生连作障碍的一个重要原因,其中化感物质中的根系分泌物是烟株与土壤微生物间相互作用的重要物质,探索烟草根系分泌物对根际微生物生长的影响是生物防控烟草青枯病的理论依据。【方法】本文利用超高效液相色谱串联四级杆飞行时间质谱(UPLC-Q-TOF/MS)技术,分离、 鉴定烟草根系分泌物中主要酚酸类物质的种类和含量; 通过添加外源酚酸类物质,研究在液体培养基中烟草根系分泌物中的主要酚酸类物质对病原菌及拮抗菌的影响,并在土壤中添加鉴定出的主要酚酸类物质; 通过土壤培育试验,研究其对土壤微生物多样性和数量变化,特别是对烟草青枯病菌及其拮抗菌生长的影响。【结果】1)烟草根系分泌物粗提物对病原菌(茄科劳尔氏菌)生长的促进率为16.8％,对拮抗菌(短短芽孢杆菌)的生长抑制率达到29.4％; 2)UPLC-Q-TOF/MS检测根系分泌物中主要酚酸类物质为苯甲酸和3-苯丙酸,含量分别为0.25 μg/g干根重和1.15 μg/g干根重; 3)液体培养外源添加低浓度的苯甲酸(≤ 2 μg/L)和3-苯丙酸(≤ 3 μg/L)促进病原菌和拮抗菌的生长; 4 μg/L的苯甲酸对病原菌生长抑制作用不显著,对拮抗菌生长的抑制率达到90.2％,6 μg/L的 3-苯丙酸对病原菌的生长具有促进作用,对拮抗菌的生长抑制率达到81.1％,外源高浓度苯甲酸(≥4 μg/L)和3-苯丙酸(≥ 7 μg/L)抑制病原菌与拮抗菌的成长; 4)土壤中添加3 μg/kg土的苯甲酸时,土壤中病原菌的数量增加12.3％,而拮抗菌的数量减少21.0％,土壤细菌、 放线菌和真菌的数量分别降低37.5％,41.9％和55.6％; 3-苯丙酸浓度达到8 μg/kg土时,拮抗菌生长量减少14.5％,对病原菌没有显著影响,土壤细菌、 放线菌和真菌的数量分别降低69.9％,57.2％和80.7％; 5)土壤添加4 μg/kg的苯甲酸和7 μg/kg的3-苯丙酸后,土壤微生物Shannon指数、 Simpson指数、 McIntosh指数显著下降,分别仅为对照的57.7％、 94.1％、 88.1％和97.6％ 73.3％、 80.0％。【结论】烟草根系分泌物粗提物促进病原菌生长抑制拮抗菌生长,根系分泌物中酚酸类物质主要为苯甲酸和3-苯丙酸,液体培养中4 μg/L的苯甲酸或6 μg/L的 3-苯丙酸浓度是对病原菌生长抑制不明显但显著抑制拮抗菌生长的分界点,土壤外源添加3 μg/kg的苯甲酸或8 μg/kg的3-苯丙酸时,是土壤增加病原菌减少拮抗菌数量的分界点,同时土壤微生物功能多样性显著下降,病原菌对根系分泌物中苯甲酸和3-苯丙酸的利用优于拮抗菌,这也是烟草长期连作引起青枯病暴发流行的机理之一。

Abstract:【Objectives】 Tobacco continuous mono-cropping has caused very serious problems, including soil-borne disease outbreak, tobacco growth suppression, yield reduction and quality decline. Allelopathy comes from tobacco and soil microorganisms may be one of the most important reasons leading to mono-cropping obstacle. Tobacco root exudates (TRE) play a key role in the plant-microorganism interactions in the rhizosphere. It is of great importance to explore effect of tobacco root exudates on rhizosphere microorganisms. 【Methods】 In this study, the main phenolic acids were screened and identified by the UPLC-Q-TOF/MS method, and their contents in tobacco root exudates were evaluated. Effect of identified phenolic acids from TRE in liqiud medium on pathogen and its antagonist was investigated by applying exogenous phenolic acids. Besides, the soil was added with the identified phenolic acids and cultured for 3 d. After that, the rhizosphere microbial diversities and counts, especially the population of pathogen Ralstonia solanacearum and its antagonist Brevibacillus brevis in the soil were measured. 【Results】 1)The tobacco root exudates promote the growth of pathogen by 16.8％ and suppress the growth of antagonist by 29.4％. 2)Two phenolic acids are screened and identified by UPLC-Q-TOF/MS with the concentrations of 0.25 μg/g and 1.15 μg/g dry root, respectively. 3)When the exogenous phenolic acids were added to the culture media, low concentrations of benzoic acid (≤2 μg/L) and 3-phenylpropanoic acid (≤3 μg/L) promote the growth of the pathogen and antagonist, and the 4 μg/L benzoic acid does not significantly affect R. solanacearum, whereas the population of antagonist is decreased by 90.2％. The 6 μg/L 3-phenylpropanoic acid promotes the growth of the pathogen while inhibits the growth of antagonist by 81.1％. High concentrations of benzoic acid (≥ 4 μg/L) and 3-phenylpropanoic acid (≥ 7 μg/L) significantly suppress the pathogen and antagonist. 4)When the soil is applied with the 3 μg/kg benzoic acid, the pathogen count was increased by 12.3％ while the antagonist population is decreased by 21.0％. Besides, the populations of soil bacteria, actinomycetes and fungi are declined by 37.5％,41.9％ and 55.6％, respectively. The 8 μg/kg 3-phenylpropanoic acid significantly suppresses the antagonist growth by 14.5％, and does not significantly affect the pathogen. Soil bacteria, actinomycetes and fungi are decreased by 69.9％, 57.2％ and 80.7％, respectively under this concentration of 3-phenylpropanoic acid. 5)After the 4 μg/kg benzoic acid or 7 μg/kg 3-phenylpropanoic acid application to the soil, the Shannon index, Simpson index and McIntosh index are significantly declined to 57.7％, 88.1％, 73.3％ and 94.1％, 97.6％, 80.0％ of CK. 【Conclusions】 The tobacco root exudates promote the pathogen growth whereas suppress the antagonist growth. The main phenolic acids in tobacco root exudates are benzoic acid and 3-phenylpropanoic acid. The 4 μg/L exogenous benzoic acid or 6 μg/L exogenous 3-phenylpropanoic acid are the boundary concentrations, which do not significantly suppress the growth of pathogen but inhibits the antagonist. When benzoic acid and 3-phenylpropanoic acid are applied to the soil, the growth of pathogen is promoted, while the antagonist population and the rhizo-microbial functional diversity are significantly reduced under the 3 μg/kg benzoic acid or 8 μg/kg 3-phenylpropanoic acid. Pathogen could survive under the condition of tobacco root exudates, rather than antagonist. This may be an explanation of tobacco bacterial wilt outbreak resulted from mono-cropping obstacle.