DNA Barcoding And Molecular Systematics Of Pythium

Pythium species are widely distributed throughout the world. Most species are saprobic in soil, water and the remains of dead plants and animals. Some species are important pathogens of plants and animals. China contains rich Pythium resources since it has a vast area and the various ecological environment. In this study, Pythium strains were isolated from rhizosphere soil samples of various plants or collected from the Agricultural Culture Collection of China (ACCC). A study on taxonomy, DNA barcoding and molecular systematics of Pythium and the composition and distribution of Pythium species in lawn soils in Guangxi were earned out. The main results were summarized as follows:1. The collected525Pythium strains were identified as51taxa based on the combination of morphological characteristics and sequence data of COI (mitochondrial cytochrome oxidase I) and ITS. Of the51taxa, five new species were described, i.e. Pythium agreste, P. baisense, P. breve, P. guangxiense, and P. wuhanense, twelve new records in China were found, i.e. P. attrantheridium, P. campanulatum, P. folliculosum, P. longisporangium, P. oopapillum, P. pleroticum, P. plurisporium, P. recalcitrans, P. rhizo-oryzae, P. takayamanum, P. terrestris, and P. zingiberis and ten unpublished taxa were delimited as putative new species.2. Barcodes of Pythium species were screened from four candidate DNA segments, i.e. COI, COII (mitochondrial cytochrome oxidase Ⅱ), ITS and β-tub (Beta-tubulin gene). The availability of the four candidate DNA segments from high to low were COII (100%)> COI (97.1%)> ITS (83.1%)> β-tub (68.7%) based on the success rate of PCR and sequencing. Intra-specific similarity of COI (average intra-specific similarity, for AS=0.993) was close to that of COII (AS-0.994) and ITS (AS=0.993), but lower than that of β-tub (AS=0.996). Inter-specific similarity of COI (average inter-specific similarity, for ASi-0.921) was close to that of COII (ASi=0.912), but higher than that of ITS (ASi= 0.627) and6-tub (ASi=0.881). There was a relatively clear boundary between intra-and inter-specific similarity in COI and COII, while intra-and inter-specific similarity in ITS and B-tub was overlapped. Among the51Pythium taxa collected in this study, two couple, i.e P. folliculosum/P. torulosum and P. guangxienselP. periilum, could not be differentiated with these four candidate DNA segments. In addition, P. myriotylum and P. zingiberis could not be differentiated with COI and B-tub, P. diclinum and P. dissotocum could not be differentiated with COII and ITS, P. oligandrum and Pythium sp. nov.4could not be differentiated with ITS and β-tub, a group of P. folliculosum, P. torulosum and P. rhizo-oryzae and a couple of P, spnosum and Pythium sp. nov.7could not be differentiated with ITS. A couple of P. spinosum and Pythium sp. nov.6and a group of P. sylvaticum, Pythium sp. nov.9and P. terrestris could not be differentiated with β-tub. COI and COII provided the highest species resolution (88.2%), followed by ITS (78.4%) and B-tub (74.5%). The preliminary results indicated that both COI and COII were more discriminative than ITS and B-tub.,It was proposed that COI could be used as a main barcode and COII as a supplementary barcode because publicly available COII database of reference species was limited and COI database of reference species was relative plentiful.3. The results of phylogenetic analysis of Pythium based on single and multiple DNA fragments of COI, COII, ITS and B-tub showed that the relationship of Pythium species was revealed well in the phylogenetic analyses of only ITS, the combination of ITS and COI and the combination of the four DNA fragments, respectively. By comparison, there was better correlationship between morphological characters and the major clades of phylogenetic tree generated with combination of four DNA fragments because the phylogenetic tree was divided into three major clades corresponding with three types of sporangial shape, i.e. the first type was the filamentous sporangium or complex of filamentous and (sub)globose sporangium, the second type was the catenulate globose sporangium or complex of swollen filamentous and (sub)globose sporangium, and the third type was the single (sub)globose sporangium. Among the three types of sporangium, the type of catenulate globose or complexes of swollen filamentous and (sub)globose sporangium appeared to be an intermediate form between the type of filamentous or complexes of filamentous and (sub)globose sporangium and the type of single (sub)globose sporangium. Morphological characters of sexual organs, as the most important characters in traditional taxonomic key of Pythium genus, were not correlated with the major clades of phylogenetic tree.4. The122Pythium strains isolated from lawn soil in Guangxi were identified as24taxa based on the main barcode COI. The result of two-way ANOVAs showed that species richness (F=8.945, P=0.000) and Shannon’s diversity index (F=4.599, P=0.004) were significantly influenced by seasons. Species richness in winter (1.10±0.18) was significantly higher than in summer (0.43±0.12) and autumn (0.20±0.10), as well as in spring (0.80±0.15)than in autumn (0.20±0.10). Shannon’s diversity index was significantly higher in winter (0.26±0.07) than in autumn (0.04±0.03) and summer (0.03±0.03). Species richness was also significantly influenced by geographic location (F=3.087, P=0.013). Species richness was significantly higher in Guilin (1.00±0.22) than in Beihai (0.15±0.08). The result of single regression analysis showed that species richness was negatively correlated with seasonal mean temperature (R2=0.4259, P>0.001). The result of non-metric multidimensional scaling (NMDS) indicated that the community composition of Pythium was strongly structured by sites (R2=0.257, P-0.004), and the community composition of Pythium in Beihai was significantly different from the other sites.