玉米根际土壤中镰孢菌的构成与定量分析

doi:10.3864/j.issn.0578-1752.2018.19.006

摘要/Abstract

摘要： 【目的】了解我国玉米根际土壤镰孢菌（Fusarium spp.）的种群结构及相对含量，为相关土传病害的发生提供早期预警。【方法】从全国17个省（自治区、直辖市）采集的47份玉米田间土样中，采用稀释涂布平板法共分离到58个镰孢菌分离物，通过形态学、特异性引物及ITS、TEF-1α基因测序等方法进行鉴定，从GenBank以及镰孢菌数据库Fusarium MLST中下载标准参照菌株，利用MEGA 6.0软件以邻接法构建多基因位点系统发育树；挑选代表菌株利用玉米自交系黄早四分别进行幼苗、种子以及离体叶片的致病性测定；基于已建立的实时荧光定量PCR（RT-qPCR）检测体系，提取土壤总DNA对玉米土样中镰孢菌属以及部分种的含量进行检测，针对不同玉米产区进行划分统计。【结果】共鉴定出9种镰孢菌，其中拟轮枝镰孢（F. verticillioides）（22.41%）、木贼镰孢（F. quisete）（20.69%）、禾谷镰孢复合种（F. graminearum species complex）（18.97%）分离频率较高，其余为尖镰孢复合种（F. oxysporum species complex）、层出镰孢（F. proliferatum）、单隔镰孢（F. dimerum）、锐顶镰孢（F. acuminarum）、茄镰孢（F. solani）和居群镰孢（F. commune），分离频率分别为12.07%、8.62%、5.17%、5.17%、3.40%和3.40%，相同镰孢菌与其参照菌株处于同一分支，不同区域的镰孢菌存在多样性；致病性测定结果显示除单隔镰孢外，其余镰孢菌均具有致病性，但致病力各不相同，同一镰孢种的不同菌株间致病性也不尽相同；其中居群镰孢为首次在玉米根际土壤中发现，并首次证实其对玉米具有致病性。RT-qPCR检测结果表明，玉米根际土壤中镰孢菌属、拟轮枝镰孢与禾谷镰孢复合种的含量分别为2.91—169.90、0.08—5.34和0.76—78.37 pg·g^-1，各玉米产区的镰孢菌含量不同，其中禾谷镰孢含量均大于拟轮枝镰孢。【结论】玉米根际土壤中镰孢菌种类众多，其中拟轮枝镰孢、木贼镰孢和禾谷镰孢复合种为优势镰孢种，各地区的镰孢菌含量均不相同，其中西南山地玉米区的镰孢菌含量最多，黄淮海玉米区的禾谷镰孢含量高于其他区域。

关键词: 玉米, 根际土壤, 镰孢菌, 实时荧光定量PCR

Abstract: 【Objective】The objective of this study is to understand the population structure and relative content of Fusarium spp. in maize rhizosphere soil in China, and to provide an early warning of the occurrence of soil-borne maize diseases.【Method】Using dilution plate method, a total of 58 Fusarium isolates were obtained from 47 maize soil samples of 17 provinces (autonomous region, municipality). Based on morphological characteristic, specific PCR amplification, and ITS and TEF-1α gene sequences analysis, these Fusarium spp. were further determined. The reference strain sequences were downloaded from GenBank and Fusarium MLST. Multi-gene loci phylogenetic tree was constructed by neighbor-joining method (NJ) using MEGA 6.0 software. Some representative strains were selected for pathogenicity test by maize seedlings, seeds and in vitro leaves infection. The content of Fusarium spp. and some species in maize rhizosphere soil samples was detected using RT-qPCR determination system. Statistical analysis was carried out for different maize producing areas.【Result】 A total of 9 Fusarium species were identified. Among them, F. verticillioides (22.41%), F. quisete (20.69%) and F. graminearum species complex (18.97%) were isolated more frequently. The rest of 6 species were F. oxysporum species complex, F. proliferatum, F. dimerum, F. acuminarum, F. solani and F. commune, with the isolation frequency of 12.07%, 8.62%, 5.17%, 5.17%, 3.40%, and 3.40%, respectively. The results showed that the same Fusarium spp. and the reference strain were in the same branch, and the diversity of Fusarium spp. existed in different regions. All Fusarium species were pathogenic except F. dimerum. F. commune was isolated from maize rhizosphere soil and confirmed to be pathogenic to maize seed, seedling, and detached leaf for the first time. The pathogenicity of different strains of the same Fusarium species was not the same. Using RT-qPCR detection system, the total of Fusarium, F. verticillioides and F. graminearum species complex in maize soil samples were quantitatively assayed, with the content range of 2.91-169.90, 0.08-5.34 and 0.76-78.37 pg·g^-1, respectively. The content of Fusarium species in different maize producing areas was different, and the content of F. graminearum was higher than that of F. verticillioides. 【Conclusion】There are multiple species of Fusarium in maize rhizosphere soil and F. verticillioides, F. equiseti, and F. graminearum species complex are dominant species. The content of Fusarium spp. differs in different areas. Among them, the content of Fusarium spp. is the highest in Southwest mountainous area of maize, and the F. graminearum content in the Huang-huai-hai summer sowing maize area is higher than that in other regions.

Key words: maize;rhizosphere soil, Fusarium spp., RT-qPCR

葛波，王宝宝，郭成，孙素丽，陈国康，王晓鸣，朱振东，段灿星. 玉米根际土壤中镰孢菌的构成与定量分析[J]. 中国农业科学, 2018, 51(19): 3683-3693.

GE Bo, WANG BaoBao, GUO Cheng, SUN SuLi, CHEN GuoKang, WANG XiaoMing, ZHU ZhenDong, DUAN CanXing. Composition and quantitative analysis of Fusarium species in maize rhizosphere soil [J]. Scientia Agricultura Sinica, 2018, 51(19): 3683-3693.

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