The Genetic Diversity Of Phytophthora Nicotianae In Yunnan Province

Tobacco as the main economic income for Yunnan province, while black shank disease caused by Phytophthora nicotianae seriously affects the yield and quality of tobacco leaves. Which has becomed an important factor of restricting tobacco agriculture production. The study mainly researched P.nicotianae phenotypic traits (growth rate on OA、spore-bearing rate、 pathogenicity test) in different tobbaco planting regions of Yunnan province.In addition, two different mt-IGS sequences was analysised to study genetic polymorphism of P.nicotianae.1) 26 P.nicotianae isolates from different counties of Yunnan were collected,which were inoculated on OA medium recording the growth rates. As a result, majorities of the isolates’ growth rate differences weren’t statistically significant. The differences of zoospores-bearing from 26 isolates of P. nicotianae was researched by artificial induction. The results show that significantly difference were found between isolates.2) Phaogenicity test is key traits for studying closely related strains of P.nicotianae.Pumpkin tissues and tobacco stems were inoculated by placing on each wound a OA agar containing growing mycelium respectively, evaluating differences of phaogenicity for 26 strains.Lesion lengths of 26 isolates on pumpkin slices were analysised, which illustrated all isolates can affected pumpkin tissuess. Lesion spreaded from the vaccination site with rot. In addition, pumpkin slices will cover with a layer of mycelium.Significant differences about lesions lengths were founded among sort of isolates at 0.05 level. Clustering analysis showed that isolates were divided into 5 clusters.Lesion lengths of 26 isolates on tobbaco stems were analysised,which indicated that all isolates can affected tobacco. Lesion spreaded from the vaccination site with water damage of dark green to black brown. Significant differences about lesions lengths were observed among some isolates. Clustering analysis according lesions lengths among all isolates, all isolates were divided into 5 clusters. Comparing to pumkin inoculation, some isolates have strong pathogenicity on pumpkin,while have weak pathogenicity on tobacco.The same phenomenon was found on tobacco stems inoculation is that no correction betweening phaogenicity with geographic origin.3) Two mitochondrial intergenic regions (mt-IGS) were characterized by intraspecific variation in DNA sequences, which were applied for studing of the p.nicotianae from Yunnan.It turned out that trnY/rns sequences has A/T bias. Pi, Hd, variation sites were 0.00101、 0.053,6 respectively.6 haplotypes were detected in 26 sequences, H5 is advantage haplotypes including 21 isolates. Another five haplotype with only one isolate individual. The average genetic distance between two sequences is 0.001, The average genetic distance between two haplotypes is 0.004. Molecular phylogenetic tree based on haplotypes explainning that all haplotypes were divided into 2 clusters. H4 can be solely clustered into one clade, another five haplotypes be clustered into another clade.The same as trnY/rns sequence, trnW/cox2 sequences has A/T bias.8 variation sites were found by sequence alignment. Haplotype diversity index (Hd) and mean nucleotide diversity index (Pi) were 0.415 and 0.00189 respectively.7 haplotypes were detected in 26 sequences,H1 is advantage haplotypes including 20 isolates. Another six haplotypes with only one isolate individual. The average genetic distance between two sequences is 0.002, The average genetic distance between two haplotypes is 0.004. Molecular phylogenetic tree based on haplotypes explainning that all haplotypes were divided into 2 clusters. H4 (Hongta) can be solely clustered into one clade, another six haplotypes be clustered into another clade.The results of analysising two mt-IGS sequences and pathogenicity tests illustrated that the genetic diversity of P.nicotianae from Yunnan at a lower level as a whole. Combining the results on phenotypic traits, there is no correlations between phaogenicity diversity, growth rate, geographic origin or DNA sequence analysis for mt-IGS. Meanwhile, the differences of pathogenicity tests were not relevant to geographic origin.