Cultivation, identification and quantification of one species of yeast-like symbiotes, Candida, in the rice brown planthopper, Nilaparvata lugens

The brown planthopper （BPH）, Nilaparvata lugens Stal, which is one of the most destructive pests of rice, has been confirmed to harbor yeast-like symbiotes （YLS） in the fat body. Several morphologically different YLS have been previously isolated and cultured in vitro from BPH, but direct evidence is lacking to further clarify whether the cultured YLS were from BPH. In this study, one species of YLS was successfully cultured in vitro and simultaneously verified to exist in the fat body of BPH by sequence analysis and nested polymerase chain reaction （PCR）. The cultured YLS isolate in vitro was identified as a member of the genus Candida on the basis of 18S rDNA （ribosomal DNA） and 5.8S-ITS （internal transcribed spacer） rDNA sequence and a phylogenetic analysis of ITS sequences from yeast. Therefore, this yeast isolate was named as Candida-like symbiotes. Candida-like symbiotes was found to exist in fat bodies, ovaries and newly laid eggs of the BPH, but not in the heads, thoraxes and mid-guts. In addition, the number of Candida- like symbiotes in 1×106 of purified YLS from BPH fat bodies was speculated to be （5.32±0.22）×104 on the basis of a quantitative PCR analysis.

三种稻飞虱体内类酵母共生菌18S rDNA和ITS-5.8S rDNA序列克隆及进化分析

为研究水稻3种主要害虫灰飞虱Laodelphax striatellus、褐飞虱Nilaparvata lugens和白背飞虱Sogatella furcifera体内类酵母共生菌(yeast-like symbiotes,YLS)的种属地位及与寄主的进化关系,测定了其体内YLS的18S rDNA及ITS-5.8Sr DNA的全长序列。基于3种稻飞虱体内YLS的18SrDNA序列比对表明,褐飞虱YLS和白背飞虱YLS的一致性比其与灰飞虱YLS的高(褐飞虱YLS和白背飞虱YLS为98.91%,灰飞虱YLS和褐飞虱YLS为95.74%,灰飞虱YLS和白背飞虱YLS为96.02%),而基于ITS-5.8SrDNA序列比对,灰飞虱YLS和白背飞虱YLS的一致性比其与褐飞虱YLS的要高(白背飞虱YLS和灰飞虱YLS为99.57%,灰飞虱YLS和褐飞虱YLS为91.91%,白背飞虱YLS和褐飞虱YLS为90.46%)。基于真菌18SrDNA和ITS-5.8SrDNA的系统发育树均表明,3种稻飞虱体内YLS与其他已知真菌进化关系较远。本研究证实了昆虫真菌类共生菌与寄主形成了长期的进化关系,从而形成了不同于已知真菌的分类地位。