海南岛柑橘肾形线虫病的发生及病原鉴定

Occurrence and Pathogen Identification of Citrus Reniform Nematode Disease in Hainan Island

据调查发现海南岛柑橘根部寄生线虫危害严重,其根部主要寄生线虫为肾形线虫(Rotylenchulus spp.),且分布广泛,侵染根部对其造成严重影响。从海南岛柑橘园中采集表现为黄化、小叶等症状的发病植株根部土样,用改良贝曼漏斗法分离土壤线虫,通过形态学观察与测量、28S rDNA D2-D3区序列扩增、测序、比对及进行系统发育分析。结果表明,在60份土样中该线虫检出率为66.7%,虫口密度最高达513条/100 mL土;由形态学鉴定为肾形线虫。对肾形线虫28S rDNA D2-D3区PCR扩增条带大小为780 bp左右,与目标基因大小一致。测序结果与GenBank中登录的数据进行Blast比对,与肾状肾形线虫(Rotylenchulus reniformis)数据同源性高达99%以上。根据比对结果构建系统发育树,结果与已报道的肾状肾形线虫聚为一类。结合形态学与分子生物学技术,将该线虫鉴定为肾状肾形线虫。本研究对柑橘根部土壤肾形线虫的调查和鉴定研究,为该病害的诊断和防治提供理论参考。

Accord ing to the survey,the parasitic nematodes in the citrus roots of Hainan Island were seriously endangered.The main parasitic nematodes in the roots were Rotylenchulus spp.which were widely distributed in Hainan Island.The nematodes parasite plants roots and made a severe influence on them.Root soil samples were collected from the diseased plants showing yellowing and lobular of the citrus orchards in Hainan island,and the soil nematodes were isolated by modified Beman funnel method.Morphological observation and measurement,28 S rDNA D2-D3 region amplification and sequencing were performed,then alignment and phylogenetic analysis were conducted.The results showed that the detection rate of the nematode was 66.7%in 60 soil samples,andthe highest population density was 513/100 mL soil;it was identified by morphology as Rotylenchulus spp.The size of the PCR amplification band of the 28 S rDNA D2-D3 region of the Rotylenchulus spp.was about 780 bp,which was consistent with the target gene size.The sequencing results were compared with the data registered in GenBank for Blast,and the homology with the Rotylenchulus reniformis was over 99%.The phylogenetic tree was constructed based on the alignment results,and the results were clustered with the reported nephrons.The nematode was identified as Rotylenchulus reniformis in combination with morphology and mole cular biology techniques.This paper investigated and identified Rotylenchulus spp.in the citrus roots,providing a theoretical reference for the diagnosis and prevention of the disease.