| Crown rot of wheat is a soil-borne disease which has newly discovered in China recent years, and has showed the trend of increasing year by year. In order to clarify the occurrence and distribution of this disease, meanwhile, to determine the pathogen species in Huanghuai wheat growing region which is the main producing area of China, we collected the field samples extensively throughout Huanghuai wheat growing region in this study and obtained isolates by tissue isolation and purification technics, then recognized the species of these isolates by process of morphological comparison, molecular identification, and pathogenicity test, thereby determined the predominating pathogens species. Then, NJ, UPGMA and ML method were used by MEGA5.0 to construct phylogenetic tree based on rDNA-ITS and EF-1α gene sequence, and had clarified the phylogenetic relationships among isolates obtained from various geographic locations of Huanghuai wheat growing region. The main results were as follows:1. Samples were collected from wheat plants at maturity displaying crown rot symptoms from 45 places in 25 cities of 7 provinces, and sections removed from infected crown tissues to be used for isolation (504 isolates) and identification (147 isolates) of pathogens. The results showed that Fusarium pseudograminearum was the most common in North Henan, South Shanxi, North Shandong and the southeast of Hebei province in Huanghuai wheat growing region, Bipolaris sorokiniana taked the second place. While in the North Centre of Anhui province, North Jiangsu and the west area of Shandong province, over 70% of isolates from this area was F. graminearum, and B. sorokiniana (about 10%) taked second place. F. pseudograminearum and F. graminearum were both situated high isolation rate in the East Centre of Henan province. Overall, in all of isolated pathogens, F. pseudograminearum accounted for 29.2%, F. graminearum 39.4%,B. sorokiniana 19.4%. In addition, we also obtained some other pathogens, such as F. tricinctum, F. equiseti and F. proliferatum, but the proportions of them were lower than 5%.2. Phylogenetic analysis of isolates from Huanghuai winter wheat growing region was carried out by constructing phylogenetic trees used methods of NJ, UPGMA and ML, which based on the rDNA-ITS and the translation elongation factor-1 alpha (EF-1α) gene sequence data. The cladograms showed that, when the genetic distance was 0.01 (UPGMA),147 strains of isolates were divided into 6 groups. There were closer phylogenetic relationships among a majority of isolates in same geographic locations in F. pseudograminearum and F. graminearum subtree, especially on the cladograms based on EF-1α gene sequence data. However, there were some variation existing in almost every subtree. Therefore, this suggested the existence of genetic diversity to some extent among pathogens of crown rot in various geographic locations across Huanghuai wheat growing region.3. The representative 6 kinds of isolates were inoculated into the colonized millet medium for pathogenicity test in greenhouse trial and field experiment, then validated by finishing Koch’s Rule. The pathogenicity test results showed that, the pathogenicity of F. pseudograminearum was strongest, the average disease severity index were 44.3 and 38.2 respectively in greenhouse and field trial, as well as the difference was significant compared with treatments inoculated other pathogenic isolates. B. sorokiniana and F. graminearum, whose severity index are 29.8/16.8 and 29.6/12.3(greenhouse/field), taked the second place. Besides, the other 3 Fusarium isolates had partly pathogenicity, but displayed the lower disease severity index. Combining with the results of isolation frequency and pathogenicity test in this study, the predominant pathogens in Huanghuai wheat growing region of China were F. pseudograminearum and F. graminearum, B. sorokiniana was also one of the main pathogens causing the symptom of brown discoloration on subcrown, crown, and lower stem tissues. |
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