Molecular Diagnosis Of Diseased Plant Tissues By Radopholus Similis

The burrowing nematode, Radopholus similis (Cobb) Thorne, is an important quarantine pest in China. It causes serious losses to the agricultural production nearly all over the world. We took work on the researches about early detection for Radopholus similis in order to preventing its spread into China.In this study we obtained gene sequences from the D2 and D3 expansion segments of 28S rDNA from populations of Radopholus similis and populations of the genus Pratylenchus Filipjev. Lengh of the D2-D3 expansion segments varied from 758 bp (J01.pra) - 785 bp (TW.pra).Aligment were constructed using the software DNAMAN, and it confirmed that the high similarity (96.23%) among the 5 populations of Radopholus similis, and 84.72% between Radopholus similis and Pratylenchus Filipjev. Phylogenetic analysis of D2D3 sequences using UPGMA method showed that: Radopholus similis and Pratylenchus Filipjev.groups were clusted to each monophyletic group; and in the group of Radopholus similis, XH1 and XH2 populations were clusted to one group, and other populations as another group.The fragment of rDNA - ITS varied in Radopholus similis from 705 bp to 708 bp, and in Pratylenchus Filipjev from 704 bp to 1248 bp. Multiple sequence aligments showed that the populations of Radopholus similis shared high similarity (92.90%); and showed low similarity (47.02%) between Radopholus similis and Pratylenchus Filipjev. Phylogentic analysis of ITS sequence showed that Radopholus similis and Pratylenchus Filipjev had their own monophyletic group; Radopholus similis was also divided into two geographic populations, HN5 population was clusted to a single group.The amplification of the coding region and intergenic space (IGS) with the primers 5S F1 / R1 of one population of Radopholus similis and one population of Pratylenchus coffeae yielded two different fragments(462 bp,164 bp). The IGS sequence of Radopholus similis was significantly different from the Pratylenchus coffeae.For the design of the specific primer, nucleotide sequences of the ITSregion from several populations of Radopholus similis before in my lab. Duplex PCR with two pair of primers D2A / D3B, RsF1 / RsR1 was developed to detect seven populations of R. similis and other species of nematodes. All Radopholus similis populations yieled distinct fragments (271 bp and 780 bp), while all other species produced only one (780 bp). The sensitivity of this method is high of 1 / 256 times of the DNA trace of the single female adult Radopholus similis. It would be detected as long as there is a minimal part of Radopholus similis.This method is rapid, specific, sensitive, simple, economical, etc., and fits for quarantine department.According to the sequence of mtDNA-COII of Radopholus similis and other nematodes in NCBI, one pair of specific primers CF1 / CR1 was designed. Single Radopholus similis could be detected specifically using this pair of primers, too.Based on the above experiments, three methods of DNA isolation from plant directly were devised. Through the modified CTAB method, the total genomic DNA of the diseased plant tissues by R. similis was extracted. We invented"A method of early molecular detection for Radopholus similis"using the species specific primers RsF1 / RsR1 and universal primers D2A / D3B, and applied for national patent (application number: 201010113930.X). Radopholus similis could be detected specifically and directly from the diseased plant without separating the nematodes from soil or plants through this method. The actual samples could also be detected when there were only 4 Radopholus similis in 700 mg Anthurium roots. The results showed that this method was widely applicable, accurate and reliable, easy and fit for ports and quarantine departments.