| Cereal cyst nematode(CCN, Heterodera spp.) is a kind of worldwide important pathogenic nematodes which parasitize the wheat(Triticum aestivum), barley(Hordeum vulgare), oat(Avena sativa) and other cereal crops. In addition to harm cereal crops, CCN also parasitize some perennial gramineous plants as the intermediate host or the bridging host. According to the distribution features of farming-pastoral region of Tianzhu steppe, a systematical study on occurrence, ecological distribution, species of cyst nematode and dominant population of CCN in tianzhu alpine meadow steppe was made in this research.Surveys on cereal cyst nematode in main pastoral area of Tianzhu alpine meadow steppe, Gansu province were made in 2013-2014. 248 sample were collected in the surveys covered the main pastoral areas and 13 townships of farming-pastoral region using random sampling method. The samples were taken to the laboratory and checked out using the simple floating separation method. The results showed that 56 samples with cyst nematodes were detected, accounted for 22.6% of the total number of samples. And samples in 7 towns among the 11 townships were found cyst nematodes. The population desity of cyst nematodes in the town of zhuaxixiulong was highest, up to 54/100 g soil samples. The population desity of sampling sites in other towns are relatively low. The soil samples where the cyst nematodes have been found were collected by stratified sampling according to the vertical sampling standard of 0 ~ 10 cm, 10 ~ 20 cm, 20 ~ 30 cm, 30 ~ 40 cm to research the vertical distribution of CCN in tianzhu steppe. The results showed that the cyst nematodes mainly distributed in 0 ~ 10 cm and 10 ~ 20 cm soil layer, and cyst nematodes below the 30 cm soil samples was not detected. In addition, the density of cyst nematodes of all soil samples in 0 ~ 10 cm was greater than the corresponding soil samples in 10 ~ 20 cm.Morphological identification and molecular biology identification were adopted in this study. Identification results showed that the populations of cyst nematodes in Tianzhu Alpine meadow steppe were Heterodera avenae. The cysts were light brown, large lemon-shaped, vulva cones with bifenestrate and obvious bullae, but without under-bridge of vulva. Ribosomal DNA-ITS PCR amplifications were made using general primers AB28 and TW81. The fragments of ITS region were 1045 bp after PCR product purification, cloning and sequencing. The sequences have no insertions and deletions, but have base substitutions. By alignment on Genbank and Blasting, sib-species sequences were downloaded and a phylogenetic tree was constructed using UPGMA method in MEGA5.2. The results showed that the similarities between these groups and H. avenae were 99% ~ 100% by alignment on Genbank and Blasting. ITS constructing phylogenetic tree showed that these groups, cereal cyst nematode of China, Kentucky bluegrass cyst nematode(H. pratensis) of Russia and Kentucky bluegrass cyst nematodes(H. pratensis) of Germany groups into a small branch at the same time, which revealed the higher genetic relationship between them. These molacular results confirmed that the cyst nematodes parasitizing on pastures of Tianzhu alpine meadow steppe in Gansu province were H. avenae.In order to evaluate the pathogenic ability of cereal cyst nematode populations(H. avenae) of Tianzhu pasture on wheat using the indoor artificial inoculation method. Wheat cultivar "Aikang58" were inoculated with 2 populations of from CCN with 3000 eggs per plant. The number of cysts of wheat rhizosphere and rhizosphere soil were observed and counted after inoculation 75 d. The results showed that the cereal cyst nematode groups of Tianzhu pasture can infect wheat, and treat wheat as the host. |
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