| Locoweed is a general term for some plant genera contained sowninsonine and caused the typical neurological and pathological changes in herbivores.The most famous locoweed species belongs to Oxytropis and Astragalus,which mainly distributes in China and United States,and caused economic losses every year.The content of swainsonine in locoweed is positively correlated to the amount of endophyte(Alternaria spp.sect.undifilum).Up to data,it is clear that the endophytic fungus lives inside of locoweed plants and seeds.However,life cycle of such fungus is not completely determined such as what is roles of conidia in life cycle.The object of this dissertation is to study life cycle of locoweed endophyte using the plants and seeds of O.glabra,O.ochrocephala and O.kansuensis respectively collected from Alashan County of Inner Mongolia,Haiyuan County of Ningxia,Tianzhu County of Gansu.The main results are as follows:1.Lifestyle of endophyte during locoweed overwintering period.Endophyte was detected from 40% and 80% roots,and 50% and 90% young stems among 10 plants after regreening of O.ochrocephala respectively by specific primers OmtssuF/OmtssuR and OR1/ITS5.there was a plant(different plants)contained endophyte in root but was free from young stem in each primers.Detection percentage of plants with endophyt in the second pair of primer(90%)was higher than the first pair primers(50%).However,there were many unexpected bands on the electrophoretogram with second primer but not with the first primer.the bands at roots were lighter than at stems.It is means that the amount of endophyte hypha in roots was higher than that in stems,and spread to shoots from roots after regreening.2.Endophyte during the plant growing stages: In the period of shooting,flowering and maturity of O.ochrocephala,O.falcata and O.kansuensis,conidia of endophyte produced from surface of plants,in which mostly from white receptacles,then from died leaves,and less from stems.This indicate endophyte conidia produced during the whole growing period only from died or senile tissues.3.Endophyte lives inside and outside of seeds: endophyte conidia present at the surface of O.ochrocephala seeds at 32.78 conidia per seed after washig seeds and accounted.Endophyte mycelia reside in 80.00% of O.glabra seeds,85.83% of O.ochrocephala seeds,and 8.75% of O.kansuensis seeds.Hypha of endophyte distributed between seedcoats and embyos in aleurone layer,and free on seedcoats and embyoes.4.Locoweed endophyte from seeds to seedlings: endophyte was detected from seedlings grown from whole seeds by PCR with specific primers OmtssuF/OmtssuR,while not prsented from seedlings grown from embyoes.This shown that endophyte entered into seedlings by hypha during seed germinating or seedling grwoth.5.Conidia of locoweed endophyte infect to seedlings: when conidia were inoculated onto detached O.ochrocephala leaves and observed under microscope,conidia germinated,and germtubes entered into leaves.6.Similarities and differences of endophyte from 3 species of locoweeds: endophyte from 3 species of locoweed(LYZ0091,LYZ0093 and LYZ0109)were Alternaria oxytropis based phylogenetic tree with ITS sequences.The optium temperature for colonies growth were 20?,25? and 25?.All strains grew at 30? except LYZ0109.All strains grew at pH 4 to pH11.Differences were found for morphology of hypha,in which hypha of LYZ0091 and LYZ0109 were waved and rare chlamydospores,while hypha of LYZ093 were straight or plentiful chlamydospores ranged as tree-liked construction from media bottom to surface.Colonies of diffrent strains grew separated at early 4 weeks when cultured in pair on PDA.However hypha did not fused,and no sexual construction produced at later cultures.No conidia were found on all cultures on different media,pH and temperature. |
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