外源添加L-脯氨酸对棉花黄萎病发生及其根际土壤微生物群落的影响

Effects of Exogenous Addition of L-Proline on the Occurrence of Cotton Verticillium Wilt and Its Soil Microbial Community in Rhizosphere

【目的】根系分泌物是植物与土壤微生物进行互作的信号媒介,对于植物病害发生和植株生长均具有重要调控功能。论文旨在明确棉花根系分泌物L-脯氨酸抵御棉花黄萎病发生的微生态机制,揭示L-脯氨酸介导的根际微生物与棉花黄萎病发生的互作关系,为构建生物防治土传病害的有益菌群提供新视角。【方法】通过温室盆栽试验,以外源添加不同浓度的L-脯氨酸(0、50、100、200和400 mmol·L^(-1))为试验处理,采用实时荧光定量PCR(real-time qPCR)和宏基因组测序技术分别测定不同处理的土壤中大丽轮枝菌(Verticillium dahliae)DNA拷贝数量和土壤微生物群落结构及功能,采用主成分分析比较不同处理的根际土壤微生物群落结构,利用冗余分析研究土壤养分因子与微生物群落结构的相关性,利用斯皮尔曼相关分析研究微生物群落结构功能代谢途径的相关性。【结果】与空白对照相比,低浓度(50 mmol·L^(-1))L-脯氨酸处理不能够减轻棉花黄萎病的发生,高浓度(100、200和400 mmol·L^(-1))L-脯氨酸处理的病情指数分别下降22.51%、60.23%和64.23%。qPCR结果表明,L-脯氨酸处理不能够显著降低土壤中大丽轮枝菌拷贝数量。宏基因组测序分析表明,L-脯氨酸处理后细菌多样性Shannon指数显著增加,真菌多样性Shannon指数呈下降趋势。在属水平上,L-脯氨酸处理后类诺卡氏菌属(Nocardioides)、溶杆菌属(Lysobacter)、节杆菌属(Arthrobacter)、Phycicoccus、假单胞菌属(Pseudomonas)和毛霉菌属(Mucor)的相对丰度呈上升趋势。线性判别分析表明,除浓度为100 mmol·L^(-1)的L-脯氨酸外,外源添加L-脯氨酸处理后根际土壤微生物KEGG通路的富集情况发生改变。冗余分析表明,细菌群落组成受pH、电导率、硝态氮、铵态氮和有机质显著影响,而真菌群落组成与铵态氮存在显著相关性。Spearman相关分析表明,细菌的KEGG代谢通路与pH、有机质、铵态氮含量呈负相关关系,而与电导率和硝态氮呈正相关关系;真菌的大部分KEGG代谢通路与土壤养分的相关性较差。【结论】外源添加适量L-脯氨酸通过改变土壤细菌群落结构和功能,增加有益微生物的相对丰度,从而影响棉花黄萎病的发生,但其不能够改变病原菌数量。同时,根际细菌群落组成和功能均与土壤养分存在相关性。

【Objective】Root exudates are signal mediators for the interaction between plants and soil microorganisms,which have important regulatory functions for plant disease occurrence and plant growth.The objective of this study is to clarify the microecological mechanism of L-proline in root exudates of cotton against the occurrence of Verticillium wilt,reveal the relationship of L-proline-mediated interaction between rhizosphere microorganisms and the occurrence of cotton Verticillium wilt,and to provide a new perspective for the construction of beneficial bacterial communities for the biological control of soil-borne diseases.【Method】Through greenhouse pot experiments,different concentrations of L-proline(0,50,100,200,and 400 mmol·L^(-1))were set as experimental treatments,and real-time fluorescence quantitative PCR and metagenomic sequencing techniques were used to determine the DNA copy numbers of Verticillium dahliae and microbial community structure and function of soil samples treated with L-proline.The microbial community structures of rhizosphere soils under different concentrations of L-proline were compared using principal component analysis.Redundancy analysis was used to study the correlations between soil nutrient factors and microbial community structure,and Spearman correlation analysis was focused on the relationships between microbial community structure and functional metabolic pathways.【Result】Compared with the blank control,the occurrence of cotton Verticillium wilt could not reduce under low concentration(50 mmol·L^(-1))L-proline treatment,while the disease indexes under high-concentration(100,200,and 400 mmol·L^(-1))L-proline treatments were decreased by 22.51%,60.23%,and 64.23%,respectively.The results of qPCR showed that L-proline treatment did not significantly decrease the copy numbers of V.dahliae in soil.The metagenomic sequencing analysis showed that the Shannon index of bacterial diversity significantly increased after L-proline treatment,while the Shannon index of fungal diversity showed a downward trend.At the genus level,the relative abundances of Nocardioides,Lysobacter,Arthrobacter,Phycicoccus,Pseudomonas,and Mucor were increased after treatment with L-proline.Linear discriminant analysis(LDA)showed that the enrichment of microbial KEGG pathways in rhizosphere soil changed after exogenous addition of L-proline,except for L-proline at the concentration of 100 mmol·L^(-1).Redundancy analysis showed that bacterial community composition was significantly influenced by pH,conductivity,nitrate nitrogen,ammonium nitrogen,and organic matter(OM),while fungal community composition was significantly correlated with ammonium nitrogen.Spearman correlation analysis showed that the bacterial KEGG pathways were negatively correlated with pH,OM and ammonium nitrogen,while positively correlated with conductivity and nitrate nitrogen.Most of the KEGG pathways of fungi had poor correlation with soil nutrients.【Conclusion】The exogenous addition of an appropriate amount of L-proline can influence the occurrence of cotton Verticillium wilt through altering the structure and function of soil bacterial communities,increasing the relative abundance of beneficial microorganisms,but it cannot change the DNA copy numbers of pathogen.Meanwhile,the composition and function of bacterial community are related to soil nutrients.