二月兰叶斑病病原甘蓝链格孢的分离鉴定及生物学特性研究

2017年10月在河北省黄骅市发现二月兰的一种叶部病害,感病叶片初生黑褐色、形状不规则的小病斑,后逐步发展成为具有明显同心轮纹状病斑,平均病株率和病叶率分别为96.2%和87.6%。为了明确该病害的致病菌,以组织分离法进行病原物的分离培养,对分离得到的菌落进行纯化和单孢分离后,选取代表性菌株GS1-1、HH2-1和HH3-1按照柯赫氏法则进行致病性测定,均能引起二月兰叶斑病。并对这3个菌株进行rDNA-ITS和EF-1α(tef)序列分析,结果表明,菌株GS1-1、HH2-1和HH3-1的ITS序列均与甘蓝链格孢(MN173824、MN173825、MN173823、MF462311)相似性达99%以上,其中与甘蓝链格孢(MF462311)相似性达100%;GS1-1、HH2-1和HH3-1的EF-1α基因序列与甘蓝链格孢(JX213350、KF889266、KT895946、KC584642、LC480212和KT895946)的亲缘关系最近,同源性达99%,且和以上各自对应的菌株在系统发育树上聚为一类,与形态学鉴定结果相一致。选取菌株GS1-1进行生物学特性研究,结果表明该菌株最适营养生长和产孢的培养基为马铃薯蔗糖琼脂培养基,最适营养生长的碳源和氮源分别为麦芽糖和蛋白胨,最适产孢的碳源和氮源分别为葡萄糖和酵母膏;最适菌丝生长温度为25 ℃(在5~35 ℃均可生长),最佳产孢温度为28 ℃;12 h光暗交替条件促进菌丝营养生长和产孢。

Isolation,identification and biological characteristics of Alternaria brassicicola leaf spot on Orychophragums violaceus

A leaf spot disease was found on Orychophragums violaceus in Huanghua City, Hebei Province, during October of 2017. The infected leaves were initially black and brown with irregular small disease spots, which gradually developed into necrotic spots with concentric circles, affecting 96.2% of plants observed and 87.6% of leaves on each plant. In order to identify the pathogen, samples were isolated from infected leaves by tissue separation and cultured. After purification, three isolates were obtained (GS1-1, HH2-1, and HH3-1), and their pathogenicity tested according to Koch’s rule. Test results confirmed that the three isolates reproduced the original symptoms and were therefore the causative agent of the observed O. violaceus leaf spot disease. For the three isolates, DNA was extracted, amplified and sequenced by rDNA-ITS, and the results showed the three isolates had a very close relationship to Alternaria brassicicola (MN173824, MN173825, MN173823 and MF462311), and 100% similarity with A. brassicicola (MF462311). The results also showed a very close relationship of the three isolates to certain other A. brassicicola isolates (JX213350, KF889266, KT895946, KC584642, LC480212 and KT895946) through EF-1α gene sequencing, which was consistent with the results of morphological identification. The vegetative growth and sporulation conditions of isolate GS1-1 were studied by biological methods. It was found that the optimal medium for vegetative growth and spore production of isolated GS1-1 was PSA, and the optimal carbon and nitrogen sources for vegetative growth were maltose and peptone, respectively. The optimal carbon and nitrogen sources for sporulation were glucose and yeast extract, respectively. The optimal colony growth temperature was 25 ℃, with the temperature range over which colony growth occurred being 5-35 ℃, and the optimal spore production temperature was 28 ℃. Alternate 12 h light and dark periods promoted colony growth and sporulation.