Identification And RDNA-ITS Sequence Analysis Of Phytophthora Sojae In Huaiyuan Of Anhui

Phytophthora root rot caused by Phytophthora sojae is a kind of worldwide soybean disease and also the plant diseases in quarantine in China.In this thesis,the isolation,identification and pathotypes of Phytophthora sojae in the Huaiyuan area of Anhui Province were studied.The main research results are as follows:1.Thirty-four soil samples in soybean fields were collected from Huaiyuan area.Using the method of soybean leaf-disc baiting,7 isolates in Phytophthora were obtained from the soil samples with the detection frequency of20.59%.Three representative strains(HY11,HY15,HY17)from the seven strains of Phytophthora in Huaiyuan soybean field were selected for morphological identification.The results show that the isolates HY11,HY15,HY17 grow slow on Lima bean agar medium(LBA),and the hypae were dense,with hyphal swellings and nearly right-angled branches,measuring diameter in 2.5-?m.Terminal sporangia were obpyriform,and did not fall off,with the internal proliferation,measuring 20.00 ~91.00 ? 14.1 ~ 38.8 ?m(avg.52.57 ? 31.98 ?m),with the ratio of length / breadth(L/B)of 1.64.The strains were homothallic and signle isolate produced abundent sexual organs on LBA.Oogonia were spherical,with diameters from 25.0 to 41.3?m.Antheridia were mostly paragynous,occasionally amphigynous,According to the main Phytophthora classification system,these isolates could be identified as Phytophthora sojae.Pathogenicity test showed that all strains were pathogenic to Hefeng 35 soybean plants.The wound inoculated soybean plants showed the same symptoms as the field soybean plant did.According to Koch's rule,the tested isolates could be confirmed as Phytophthora sojae.2.The 7 strains of Phytophthora sojae from soybean field soil samples in Huaiyuan area were inoculated on the 15 of soybean varieties with different resistance genes by the hypocotyl inoculation.The experimental results show that 7 strains belong to 7 different pathotypes: 1d,3a,3b,3c,4,5,6 1d,2,3a,3b,3c,4,5,6,7,1d,3a,3b,3c,4,5,6,7,4,5,6,7,2,3c,4,5,6,7,3c,4,6,7 and 4,6,7.There was 1 strain that can overcome the 11 disease-resistant genes,suggesting that the highly virulent strain of soybean diseases exists in Huaiyuan area.The rate of the strains that can overcome Rps3 c,Rps4,Rps5,and Rps6 and Rps7 was more than 70%,and these resistant genes had little value in breeding.The rate of the strains that can overcome Rps1 d and Rps3 b,Rps3a was more than 40%.The rate of the strains could overcome Rps2 was28.57%.These results provide a experimental evidence for the soybean breeding and rational layout of resistance varieties in Huaiyuan area.3.The molecular identification was carried out by applying the analysis of the internal transcribed spacer regions of the ribosomal DNA(ITS).Genetic polymerase chain reaction(PCR)products from the internal transcribed spacer regions of the ribosomal DNA of three Phytophthora isolates(HY11,HY15,HY17)were amplified.The results showed that a single band of PCR product about300 bp was amplified from above three tested isolates.At the meanwhile,the ITS regions of the ribosomal DNA gene from above three Phytophthora isolates were amplified using PCR by a couple of specific primers for P.sojae and sequenced.Sequences from the above isolates were compared with the published sequences of P.sojae,P.medicagini,P.trifolii and so on in GenBank database and phylogenetic trees were produced based on ITS sequence data by unweighted pairgroup method with arithmetic means mehtods.The results indicated that all of the three base sequences of ITS were equal length,and the nucleotide sequence was identical each other.The homology of three Phytophthora isolates tested ITS sequence and he sequences of P.sojae isolates,AY256844,AB688408,GU993914 and GU259351,published in GenBank was 100%.The clustering analysis result showed that the tested Phytophthora strains HY11,HY15,HY17 and the P.sojae isolates,AY256844,AY590274,AB688408,GU993914 and GU259351 in the GenBank were clustere into one group,with the highly genetic similarity.But the 3isolates from Huaiyuan were distant to the other isolates in Phytophthora in genetic relationship.The molecular identification indicated that the isolates in Phytophthora from soybean field soil samples in Huaiyuan were Phytophthora sojae.