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

doi:10.3864/j.issn.0578-1752.2024.11.008

中国农业科学 ›› 2024, Vol. 57 ›› Issue (11): 2143-2160.doi: 10.3864/j.issn.0578-1752.2024.11.008

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

赵卫松(), 郭庆港, 崔钠淇, 鹿秀云, 李社增, 马平()

河北省农林科学院植物保护研究所/农业农村部华北北部作物有害生物综合治理重点实验室/河北省农业有害生物综合防治技术创新中心/河北省作物有害生物综合防治国际科技联合研究中心，河北保定 071000

收稿日期:2024-01-04 接受日期:2024-02-16 出版日期:2024-06-01 发布日期:2024-06-07
通信作者:
马平，E-mail：pingma88@126.com
联系方式: 赵卫松，E-mail：zhaoweisong1985@163.com。
基金资助:
河北省自然科学基金(C2022301036); 国家现代农业产业技术体系(CARS-15-19); 河北省重点研发计划(22326509D)

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

ZHAO WeiSong(), GUO QingGang, CUI NaQi, LU XiuYun, LI SheZeng, MA Ping()

Plant Protection Institute, Hebei Academy of Agriculture and Forestry Sciences/Key Laboratory of Integrated Pest Management on Crops in Northern Region of North China, Ministry of Agriculture and Rural Affairs/IPM Innovation Center of Hebei Province/ International Science and Technology Joint Research Center on IPM of Hebei Province, Baoding 071000, Hebei

Received:2024-01-04 Accepted:2024-02-16 Published:2024-06-01 Online:2024-06-07

摘要/Abstract

摘要：

【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。

关键词: L-脯氨酸, 根际微生物, 棉花黄萎病, 大丽轮枝菌, 土壤养分

Abstract:

【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.

Key words: L-proline, rhizospheric microorganism, cotton Verticillium wilt, Verticillium dahliae, soil nutrient

赵卫松, 郭庆港, 崔钠淇, 鹿秀云, 李社增, 马平. 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[J]. 中国农业科学, 2024, 57(11): 2143-2160.

ZHAO WeiSong, GUO QingGang, CUI NaQi, LU XiuYun, LI SheZeng, MA Ping. Effects of Exogenous Addition of L-Proline on the Occurrence of Cotton Verticillium Wilt and Its Soil Microbial Community in Rhizosphere[J]. Scientia Agricultura Sinica, 2024, 57(11): 2143-2160.

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处理 Treatment	指标Index
处理 Treatment	株高 Plant height (cm)	地径 Ground diameter (cm)	地上鲜重 Shoot fresh weight (g)	地上干重 Shoot dry weight (g)	地下鲜重 Root fresh weight (g)	地下干重 Root dry weight (g)
CK	53.48±0.99a	3.04±0.21a	8.73±0.73a	3.12±0.34a	1.02±0.15a	0.40±0.06a
L50	56.73±6.14ab	3.01±0.20a	10.03±1.99a	2.82±0.23a	0.90±0.23a	0.52±0.29a
L100	63.34±6.82b	2.97±0.17a	9.79±0.63a	2.83±0.29a	1.05±0.45a	0.39±0.21a
L200	59.85±6.80ab	3.04±0.21a	10.35±1.57a	2.94±0.61a	0.82±0.26a	0.35±0.09a
L400	55.53±5.08ab	2.80±0.09a	9.73±0.95a	2.83±0.22a	0.85±0.20a	0.26±0.02b

指标 Index	处理Treatment
指标 Index	CK	L50	L100	L200	L400
速效钾含量AK content (mg·kg^-1)	157.40±1.27a	155.99±5.75a	154.45±2.45a	157.96±6.58a	140.38±3.19b
速效磷含量AP content (μmol·g^-1)	1.39±0.12a	1.44±0.02a	1.58±0.11a	1.44±0.15a	1.40±0.22a
铵态氮含量NH₄⁺-N content (mg·kg^-1)	23.85±2.79a	21.33±1.98a	24.26±0.58a	18.17±0.35b	17.27±2.33b
硝态氮含量NO₃^--N content (μg·g^-1)	11.87±2.86b	4.06±0.32d	6.75±0.79c	12.80±2.55b	375.76±7.31a
有机质含量OM content (%)	4.66±0.02b	4.71±0.11b	4.98±0.07a	4.93±0.04a	4.99±0.03a
pH	7.89±0.01ab	7.90±0.02a	7.93±0.01a	7.85±0.04b	7.75±0.02c
电导率Conductivity (μm·cm^-1)	215.67±31.94b	197.37±4.38b	191.20±3.08b	212.67±3.06b	316.00±1.00a

处理 Treatment	细菌Bacteria		真菌Fungi
处理 Treatment	Shannon	ACE	Shannon	ACE
CK	4.57±0.01c	2806.70±16.17a	4.60±0.11a	388.67±12.67a
L50	4.59±0.01ab	2791.00±17.78a	4.55±0.04a	367.00±7.00a
L100	4.60±0.01a	2808.00±14.53a	4.52±0.10a	382.50±9.19a
L200	4.60±0.01a	2796.00±13.53a	4.46±0.11ab	369.00±54.56a
L400	4.59±0.01ab	2794.00±8.66a	4.32±0.11b	366.00±7.55a

微生物 Microorganism	指标 Index	RDA1	RDA2	决定系数 Coefficients of determination (R²)	P值 P value
细菌Bacteria	pH	0.9613	-0.2756	0.7921	0.001
	电导率Conductivity	-0.9959	0.0907	0.7810	0.004
	硝态氮含量NO₃^--N content	-0.9977	0.0685	0.8673	0.005
	铵态氮含量NH₄⁺-N content	0.8755	-0.4833	0.7556	0.001
	速效磷含量AP content	0.6975	-0.7166	0.0443	0.760
	速效钾含量AK content	0.9999	0.0116	0.6554	0.002
	有机质含量OM content	0.3713	-0.9285	0.5679	0.005
真菌Fungi	pH	-0.8198	-0.5723	0.0988	0.532
	电导率Conductivity	0.4810	0.8767	0.0553	0.729
	硝态氮含量NO₃^--N content	0.3452	0.9385	0.0345	0.827
	铵态氮含量NH₄⁺-N content	-0.8095	-0.5872	0.5188	0.015
	速效磷含量AP content	-0.6515	0.7587	0.0989	0.543
	速效钾含量AK content	-0.5847	-0.8113	0.0306	0.850
	有机质含量OM content	-0.9769	0.2136	0.3668	0.064

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

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