进境美国柚子中冬生疫霉的分离鉴定

Isolation and identification of Phytophthora hibernalis fromthe Californian Citrus grandis fruit

从美国加州进境柚子表现褐腐症状的组织中分离到1个疫霉菌株9099,对其进行形态学特征观察、PCR检测、序列分析和致病性测定。试验结果表明,该菌株在V8培养基上菌落平贴,呈花瓣状;在PDA培养基上菌落边缘不规则,白色。孢子囊卵形或椭圆形,大小为(28.4~46.8)μm×(16.3~23.8)μm(平均37.5μm×19.4μm),长宽比为1.7~2.2∶1(平均1.9∶1)。菌株为同宗配合,卵孢子黄橙色,球形,满器,大小26.3~39.3μm(平均32.7μm),壁厚1~3μm。利用冬生疫霉(Phytophthora hibernalis)特异引物对PHIB1/PHIB2和751F/752R扩增9099的基因组DNA分别得到407 bp和616 bp的预期目标条带。菌株9099的ITS序列与Gen Bank中P.hibernalis的序列相似性为99.89%~100%。基于ITS序列构建的系统进化树显示菌株9099与P.hibernalis聚在同一个分支,亲缘关系最近。创伤接种柚子果实,接种处组织出现典型褐腐症状。根据形态学特征及ITS序列分析等,将菌株9099鉴定为P.hibernalis。

A Phytophthora isolate 9099 from the diseased Californian Citrus grandis fruit was identified in this study. The observation of morphological characteristics of the isolate, pathogenicity assay, PCR detection based on ITS sequence and sequence analysis were conducted. The colony of the isolate is flat, velvet-like on V8 medium, and white-colored with irregular margin on PDA medium. The sporangia are ovate or elliptic in shape, and the size is 28.4-46.8 μm long × 16.3 - 23.8 μm wide （ average 37.51μm × 19.4 μm）, with a length/width ratio of 1.7-2.2 ： 1. The isolate is homothallic, and its oospores are yellow-orange, plerotic and 26.3-39.3（ average 32.7）μm in diameter with a cell wall thickness of 1-3 μm. The expected bands of 407 bp and 616 bp were amplified from the isolate by P. hibernalis specific primer pairs PHIB1/PHIB2 and 751F/ 752R, respectively. ITS sequence of the isolate showed 99.89%-100% identity with P. hibernalis ITS sequences （Accession No. EU686391, GU258886 and AY369370） deposited in GenBank. Phylogenetic tree based on the ITS sequences showed the isolate 9099 was clustered closely with P. hibernalis. Typical symptom of brown rot was observed on the grapefruit 7 days after wound inoculation with isolate 9099. Taken together, the isolate 9099 was identified as P. hibernalis.