玉米穗腐病样品中黄色镰孢的分离鉴定及rDNA-ITS序列分析

为了明确甘肃玉米穗腐病的病原菌,于2009年9月在甘肃省四大生态区采集玉米穗腐病样品,以组织分离法进行病原物的分离培养,对分离得到的镰孢菌菌落进行纯化和单孢分离后,以形态学为基础,参照Leisle分类系统进行鉴定。结果表明,分离到271株镰孢菌菌株中有105株经形态学鉴定为黄色镰孢,占分离镰孢菌的38.7%。分析发现黄色镰孢种群数量随采样区而异,陇东和陇南地区采集的样品中分离频率分别为52.0%,55.4%,本研究充分证明了黄色镰孢是甘肃省陇东和陇南地区玉米穗腐病的优势病原菌。按照柯赫氏法则用混合菌株接种法对沈单16和金穗96832进行致病性测定,证实了黄色镰孢对玉米果穗的致病性。随机选取3株黄色镰孢菌株进行rDNA-ITS基因序列分析,将PCR产物回收测序后在GenBank 上比对,菌株3-1-1与GenBank 中登记的黄色镰孢菌株K1004和K216、6-4-1和21-2-1与黄色镰孢菌株CBS122.73亲缘关系最近,同源性达99%。利用DNAStar 软件绘制其系统发育树状图,结果表明,菌株3-1-1与K1004和K216、菌株6-4-1和21-2-1与CBS122.73位于系统发育树的同一分支,聚为一类,与形态学的鉴定结果一致。

Isolation, identification and analysis of rDNA-ITS gene sequence of Fusarium culmorum from maize ear rot samples

To identify pathogens of maize ear rot in Gansu province, samples were collected from 4 ecological zones in September, 2009 and the pathogens were isolated from cankered maize ear onto a standard medium. Fusarium colonies obtained were identified according to the Leisle taxonomic system after subculture and single-spore isolation. Of 271 isolates, 105 were identified as F. culmorum, contributing about 38.7% of the Fusarium isolates. The populations differed in different ecological areas, for example, F. culmorum was the dominant pathogen in Longdong and Longnan ecological areas, where its isolation frequencies were 52.0% and 55.4% respectively. The pathogenicity of F. culmorum was tested according to Koch’s Postulate using mixed isolates on maize varieties Shendan 16 and Jinsui 96832. F. culmorum was verified as pathogenic to maize ears. Three F. culmorum isolates were taken at random for sequences analysis of the rDNA-ITS gene. The PCR products of the three isolates were collected, purified, and sequenced. The sequences were aligned in GenBank. There was a very close relationship of isolates 3-1-1 with K1004 and K216, 6-4-1 and 21-2-1 with CBS122.73 downloaded from GenBank. Their maximum similarity was 99% with F. culmorum. Microsoft DNAStar was used to draw the phylogenetic tree of isolates 3-1-1, 6-4-1 and 21-2-1. This tree showed that 3-1-1 was in the same cluster as the K1004 while K216 isolates, 6-4-1 and 21-2-1 were in the same cluster as the CBS122.73 isolate, results that were consistent with morphological identification.