Population Genetic Diversity Of Phytophthora Infestans In Fujian Province And Sequence Analysis Of AVR3a Gene In Phytophthora Infestans

Potato and tomato late blight is caused by Phytophthora infestans, and the disease is the most serious one for the production of potato and tomato. This study focuses on the isolating、physio- logical race、chemical resistance、SRAP genotypic diversity and sequence analysis of AVR3a of the fungus in Fujian provice. The results are as following.The methods of routine isolation,dew chamber and leaf chaping with potato slices were tested based on semi-composed medium of Rye for different disease samples, Isolates were purified through cutting the top of mycelium of pathogen.The results showed that the method of the disease leaf claping with potato slices is the best method for isolating P.infestans from different disease samples.67 isolates of P.infestans from Fujian province were tested for physiological races with different cultivars of 11 dominant mono-genes, R1,R3,R3,R4,R5,R6,R7,R8,R9,R10 and R11。The results showed that there were 37defferent races among 67isolates.Race 3.7 and race 3.4.7 were the most common with the frequency of 11.94% respectively, followed by race 3.10、3.7.11、3.6.7.8 and 3.4.6.7.8 with frequency 4.47%. The results also indicated that virulence composition of P.infestans population was complex in Fujian.There was great virulent difference among different areas, different fields, even from same area and same varieties. The most of known major resistant genes have lost their resistance to P.infestans in Fujian and new resistant resources or breeding strategies need to be deployed.Cross-resistance of metaxyl-sensitive and metaxyl-resistant P.inestans strains to 5 fungicides was tested by measuring radial growth on agar medium amended with fungicides,and compared with the floating-leaf-disk method.The results showed that the strains had cross-resistance to Metaxyl and Cymoxanil each other, while to Dimethomorph,Flumorph,Azoxystrobin, Pyraclostrobin,they did not have cross-resistance. The results could give the basic information for chemical control of potato and tomato late blight.Sequence-related amplified polymorphism (SRAP) analysis was used to detect the genetic variation in populations of P.infestans in Fujian. Among110 combinations of primer pairs screened, 10 that generated consistent polymorphic bands with DNA of 10 isolates were got.They were further applied to amplify the genomic DNA of 90 isolates collected from different areas and hosts in Fujian. In total, 92 SRAP markers were obtained. Among the bands, 90 were polymorphic,which ocuupying 97.8%. Based on the genetic distance, a dendrogram was constructed with the sofeware NTSYSpc Version 2.1. At linkage distance of 0.71, 90 isolates were divided into 9 SRAP groups. There was no correlation found between SRAP group and the geographic origin or their hosts. The analysis showed that some strains from one area closely related to that from another area. Nonetheless, rich genetic variation existed among the tested strains. Migration and A2 type mating of the late blight fungus might have been the cause of the diversity.According to the AVR3a DNA sequence of Phytophthora infestans in GenBank (accession No.AJ893357) and its primers(Pex147F,Pex147R) reported in references, PCR was carried out with the genomics DNA of P.infestans as template and the specific fragment was purified and cloned. The results showed that the fragment was 451bp and analysis of sequence indicated that the coding region was 441bp and encoded 147 amino acids. The specific fragment sequence of P.infestans shared homology of 99.34% with AVR3a gene sequence. Alignments of amino acids showed that there were three amino acids were different from that of AVR3a,Their locations were absent at position 19、80 and 103,which could explain variation in virulence of AVR3a in different isolates of P.infestans.The research laid a sound foundation for studying the regulation and control in variation mechanism in avirulence gene AVR3a at the molecular level in the future...