辽宁西瓜和甜瓜细菌性果斑病的病原鉴定

Identification of the Pathogen Causing Bacterial Fruit Blotch of Watermelon and Melon in Liaoning

辽宁省锦州市凌海市西瓜、甜瓜果实上发生疑似细菌性果斑病,从发病西瓜果实上分离获得了菌株JZ17,从发病甜瓜果实上分离纯化获得了菌株JZX2和JZT8。柯赫氏法则测定表明,接种的西瓜和甜瓜上均表现出与田间病害相同的症状,且从接种的发病植株上又重新分离到相同的菌株。采用西瓜噬酸菌检测试剂盒进行DAS-ELISA鉴定,表明3个菌株均呈阳性反应。设计特异性引物YH1/YH2,对3个菌株的16S-23S rDNA ITS序列进行扩增,3个菌株的ITS序列与西瓜噬酸菌各菌株ITS序列的同源性均高达99%以上。综上可知,菌株JZ17,JZX2和JZT8可鉴定为西瓜噬酸菌(Acidovorax citrulli)。利用特异性引物PL1/PL2对3个菌株进行亚群鉴定,结果表明菌株JZ17和对照菌株AAC00-1(亚群Ⅱ)均可扩增出大小为332 bp的片段,而菌株JZX2、JZT8和对照菌株pslbtw8(亚群Ⅰ)未扩增出此条带,因此可将菌株JZ17鉴定为西瓜噬酸菌亚群Ⅱ型菌株,菌株JZX2和JZT8鉴定为I型菌株。为比较3个菌株的亲缘关系,采用通用引物27F/1492R对3个菌株的16S rDNA序列进行扩增,获得了目的片段,在GenBank中进行同源性比对的结果表明,菌株JZ17,JZX2,JZT8与A.citrulli各菌株16S rDNA的相似性均达99%以上。利用NeighborJoining法构建的系统发育进化树结果表明,菌株JZ17与江西地区菌株JXZS6处于同一分支,菌株JZX2,JZT8与广东地区菌株XJ-6,pslb-25亲缘关系较近。首次报道辽宁省凌海市发生的西瓜和甜瓜细菌性果斑病,并鉴定其病原菌,为该病害在辽宁省的检疫和防控奠定了基础。

The suspected bacterial fruit blotch disease in watermelon and melon happened in Linghai City,Liaoning Province.We isolated the strain JZ17 from diseased watermelon fruit and JZX2 and JZT8 from diseased melon fruit.The pathogenicity test of Koch’s postulate showed the same symptoms on the inoculated leaves as that in the field,and the same strainwas re-isolated from the inoculated plants.The DAS-ELISA serological identification of the three strains was carried out using Acidovorax citrulli test kit.The results indicated that all of these three strains showed positive reaction.A pair of specific and sensitive primers(YH1/YH2)were designed to amplify the 16 S-23 S rDNA ITS sequences of the three strains,the results showed these three strains shared more than 99%similarity with that of Acidovorax citrulli.In conclusion,the strains JZ17,JZX2 and JZT8 were clearly identified as Acidovorax citrulli.The primers PL1/PL2 were used to identify the groups.The results showed that 332 bp bands were generated in the strain JZ17 and the control strain AAC00-1(groupⅡ),respectively;however,the strains JZX2,JZT8 and the control strain pslbtw8(groupⅠ)showed negative result.Therefore,the strain JZ17 could be identified as Acidovorax citrulli groupⅡwhile the strain JZX2 and JZT8 were identified as group I.In order to analyze the phylogenetic relationship of the three strains,16 S rDNA sequences were amplified by PCR with universal primers 27 F/1492 R.Homology analysis of the obtained sequences was carried out in GenBankand the results showed that the 16 S rDNA sequences of the strain JZ17,JZX2 and JZT8 shared more than 99%similarity with that of A.citrulli.Phylogenetic tree analysis based on Neighbor-Joining method showed that the strain JZ17 was clustered with the strain JXZS6,and the strain JZX2 and JZT8 were closed to the strain XJ-6 and pslb-25.This is the first report on the bacterial fruit blotch of watermelon and melon in Linghai city of Liaoning province and identification of its pathogen,laying the foundation for the quarantine inspection and prevention of bacterial fruit blotch disease in Liaoning province.