Diversity Of Yeast-like Symbiotes (YLS) In Brown Planthopper And Its Variation In Different Nymph Periods And On Different Resistant Rice

Brown planthopper (BPH), feed on the phloem sap of rice plants, harborendosymbiont. Yeast-like symbiotes (YLS) are endosymbionts that are intimatelyassociated with the growth, development, reproduction of their host, the brownplanthopper, Nilaparvata lugens (Hemiptera: Delphacidae). It has been demonstratedthat there are not only one species of YLS in planthoppers and the number of YLSchanged with the change of the host’s living environment. In this study, to analyzethe YLS of BPH, a novel nested PCR-denaturing gradient gel electrophoresis (DGGE)strategy was developed in this paper. And with this method, the variation of YLS inthe BPH was analyzed, which was in different nymph period or feed on differentresistant rice.1. A novel nested PCR-denaturing gradient gel electrophoresis (DGGE) strategywas developed in this paper to analyze the YLS of BPH, using a nested PCR protocolthat involved the18S rDNA gene and the5.8S-ITS gene using fungal universalprimers. The nested PCR protocol was developed as follows: firstly, the18S rDNAgene and5.8S-ITS gene were amplifed using fungal universal primers. Subsequently,these products were used as a template in a second PCR with primers ITS1GC-ITS2,ITS1FGC-ITS2and NFGC-NR, which was suitable for DGGE. Using this highlyspecific molecular approach, we found several previously detected fungi: Noda,Pichia guilliermondii, Candida sp., and some previously undetected fungi, such asSaccharomycetales sp., Debaryomyces hansenii and some uncultured fungi. Inconclusion, the nested PCR system developed in this study, coupled with DGGEfingerprinting, offered a new tool for uncovering fungal endosymbiont diversitywithin planthoppers.2. The brown planthopper, Nilaparvata lugens, harbors YLS. The number ofYLS varies with nymphal period. To study the variation of the three YLS (Noda,Pichia guilliermondii and Candida quercitrusa) in BPH from different nymphal period respectively, PCR-DGGE combined with Real-time quantitive PCR was firstintroduced in this paper. The result of DGGE showed that there was not significantvariation of band’s intensity of this three YLS in different nymph stage, but thedetection signal of Noda and Pichia guilliermondii was stronger than that of Candidain same nymph stage. With the Real-time quantitive PCR, the results indicated thatthe number of the three YLS increased gradually in accordance with the increasing ofnymphal period but decreased in the5th instar nymph and increased in female adultremarkably. In addition, the number of Noda was more than the other species. It hasbeen confirmed the truth that the Noda was the dominant endosymbiont in the YLS.3. Variation of the three YLS in the first eight generations was monitored in theBPH which fed on different resistant rice varieties. The results elucidated that thenumber of the three YLS in BPH that was fed on resistant rice varieties were alwayslower significantly than values of susceptible rice variety TN1. The number of thethree YLS reached the minimum at the second generation and increased graduallyfrom the third generation. The change of three YLS in BPH from different resistantrice in the same generation was different: there was no significant difference ofPichia guilliermondii and Candida. In the case of Noda strain, which is sensitive tothe resistant rice Mudgo, the amounts of Noda were much lower than those of othersresistant rice varieties. In addition, the results also indicated that the amount of Nodawas more than the other species.