| Locoweed is general name of some Astragalus and Oxytropis species toxic to livestock, which is mainly poisonous plant in China and USA. Locoweed caused large amount of livestock be poisoned or death, and severe loss every year. The toxicity of locoweed is associated with endophyte Alternaria spp. set. Undifilum. Up to data,20 locoweed species contained endophyte in the world. However, only 3 species of such endophyte were described. It seems that lots of locoweed share with a same species endophyte. In order determine the unknown mechanism that endophyte how choose its hosts, and subsequently manage the locoweed, the co-evolution relationship between locoweed and their endophyte was studied with American and Chinese locoweed in this thesis. The main results as follows:1. O. falcata from Qilian and Gonghe County, Qinghai Province was the 8th locoweed contained endophyte in China.2. The results of phylogenetic tree analyzed with 18s, ITS and KGCB regions after DNA extraction, PCR, and sequenced with 7 species of Chinese locoweed indicated that KGCB is better than 18s or ITS for locoweed identification. Chinese locoweeds were clearly different from American locoweeds no matter in Astragalus or Oxytropis. However, Chinese locoweed O. falcata is closer to American locoweed O. lambertii and O. sericea than other 4 species Chinese locoweed. Furthermore, O. kansuensis was close to O. ochrocephala and not be separated with all 3 primers.3. After cultured on PDA, WA, V8, and WHDA media at 22℃ for 3 weeks, some differences of 11 Chinese strains exhibited on the colony color, construct and growth rate, in which 8 strains produce conidia, length-width ratio of 6 strains were significantly different from each other. This implied that differences existed in Chinese locoweed endophyte.4. Phylogenetic trees based on ITS, gpd, calmodulin, actin, chitin synthesis genes of 11 Chinese endophyte strains indicated ITS was better than other primers for locoweed endophyte identification. Three species in Alternaria sect. Undifilum(A. cinereum, A. bornmuelleri, A. fulvum) were separated obviously using ITS sequences. Nevertheless, the fourth species A. oxytropis were divided into 2 groups (group 1: America endophyte of O. sericea and O, lambertii, group 2:Chinese endophyte of O. kansuensis). Actin, gpd and chitin synthesis sequences supported that all Chinese endophytes were a same species, but ITS showed it should not include endophyte from O. kansuensis. From calmodulin sequences Chinese endophytes include 3 groups:group 1:endophytes from O. sericopetala, group 2:endophytes from O. kansuensis and O. ochrocephala, group 3:endophytes from O. falcata, O. glabra, A. strictus and A. variabilis. In conclusion, it is possible there is new species in Chinese locoweed endophyte, or they may contained several subspecies.5. The relationship between locoweed and their endophytes by the method of the topology consistence analysis implied that American locoweed endophyte had strong co-evolution relationship with their hosts, while Chinese locoweed endophyte was not (the consistence rate was 42.8%). There were 6 groups of locoweed endophyte and 4 groups of their hosts. Conidia of endophyte produced from O. ochrocephala,O. kansuensis, O. glabra and O. falcata plant surface. It means endophyte likely infect plant in field by conidia. This suggests some Chinese locoweed possibly share the same endophyte species by horizontal transmission with conidia. This also may explain why only weak co-evolution relationship between Chinese locoweed endophytes and their hosts. |
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