Establishment Of The Molecular Identifying System Of Curvualria And Application In Difficult Species

Curvularia is one of the important plant pathogens, most species are born in Gramineae plants. Accurate and rapid identification of Curvularia species is the necessary for the diagnosis of plant diseases and the base of Curvularia system evolution research. Usually it has a certain arbitrariness to identify some Curvularia speciese relying on morphological characteristics, because of the morphological characteristics of Curvularia species are changeful in the different culture conditions. This study screened out ideal DNA barcodes suitable for Curvularia classification and preliminarily established the Curvualria molecular identifying system after analysed the consecutive and variability of seven genes from intra- and inter-specific of Curvularia based on the morphological identification. Meanwhile, we identified accurately the diffcult species of Curvularia by combination of morphological and molecular characteristics.During the investigations of Curvularia Boedijn in China from 2008 to 2011, more than 1000 specimens were collected from several provinces of China. All the specimens were cultured by keeping damp incubation and tissure isolation. 150 strains of Curvularia were isolated from those specimens, plus another 200 isolates from our lab, domestic and foreign research institutions, 350 isolates of Curvularia were collected in this study. All the isolates were rechecked via morphological characteristics under the standard culture condition, at last 23 species and 3 unidentified difficult species of Curvularia were identified.Based on the morphological characteristics, 40 strains of 14 morphological stability representative species were selected to study molecular identifying system of Curvularia. PCR amplification, purification, sequencing of seven candidate genes (5.8S/ITS,Gpd,Brn1,large subunit rDNA D1/D2,ACT,CAL and EF-1α) were conducted. After analysed the consecutive and variability of seven genes from intra- and inter-specific of Curvularia combined the success rates of PCR amplification and sequencing which were considered as important criteria to estimate the candidate genes, 5 ideal DNA barcodes for the Curvularia fungi were screened out.A total of 255 sequences of the candidate genes were analyzed in this study. rDNA5.8S/ITS,rDNA D1/D2,Gpd and ACT genes had a high PCR and sequencing success rate(100%).The consecutive and variability of Gpd and ACT gene from intra- and inter-specific of Curvualria is steadily, they are suitable for species-level molecular identification for the Curvularia species. rDNA5.8S/ITS gene are more consecutive than Gpd and ACT gene, it can distinguish most species except few similar species, as the main molecular classification basis of fungi with most sequences in GenBank has been recognized by most of experts and scholars, it can be the preferred gene in the the molecular identifying system of Curvualria.CAL and EF-1αgenes can also distinguish thedifferent Curvularia species, however, the PCR and sequencing success rate is less than the Gpd and ACT genes, they can as complement genes in the the molecular identifying system of Curvualria, increase genetic information of the Curvularia species. Brn1 gene has low PCR and sequencing success rates, merely 55% of the toal species. Large subunit rRNA D1/D2 gene has highly conserved sequence, can not distinguish the most Curvularia species. So Brn1 and large subunit rRNA D1/D2 genes were unsuited to use in the the molecular identifying system of Curvualria.3 unidentified taxa of Curvularia were discovered in this study, they were confirmed to be three new species by the morphological and molecular identification including 5.8S/ITS, Gpd, ACT and CAL genes, they were named as ZMJ23:Curvularia trifolicola Meng Zhang, T. Tsukiboshi & C.L. Wang, sp. nov.; ZM10088: Curvularia fatsia Meng Zhang & C.L. Wang, sp. nov.; ZM10255-1:Curvularia fujianensis Meng Zhang & C.L. Wang, sp. nov.This study established the the molecular identifying system of curvularia which contain the preferred genes 5.8S/ITS,Gpd or ACT, and the complemental genes CAL or EF-1α. This study provided a reliable technology platform for accurate and rapid Curvularia species identification, which can solve the identification problem of Curvularia difficult species, also laid the foundation for the Curvularia DNA barcode project.. The specimens examined, semi-permanent slides, living cultures and the type specimens of three new species are deposited in the Herbarium of Henan Agricultural University: Fungi (HHAUF).