Identification Of Alternaria Section Undifilum Gansuense And Detection Of LAAO,KS Genes In Alternaria Section Undifilum Spp

Locoweeds are some toxic plants of the genera Oxytropis and Astragalus,which contain the alkaloid swainsonine(SW)and be distributed widely in China,the United States,Canada.After,Locoism can be induced by overfeeding on locoweeds and causes significant economic losses.Alternaria Sect.Undifilum spp is responsible for the biosynthesis of SW in locoweed and closely related to toxicity of locoweed.The pathway of SW biosynthesis is not illuminated in the endophytic fungi yet but that is important to reduce or eliminate the toxicity of locoweed from the perspective of biological control.Nowadays,it is found that only saccharopine reductase gene could be responsible for regulation of SW biosynthesis.The pathway of SW biosynthesis might be similar to that of Rhizoctonia leguminicola and Metarhizium anisopliae Astragalus adsurgens Pall is regarded as a kind of pasture that grown widely in China.Phytopathogenic fungus,Alternaria gansuense,is similar and closely related to Alternaria Sect.Undifilum spp,the endophytic fungi from locoweed of the in morphology and phylogeny.However,it has not been known whether fungus can produce SW.The first objective is to isolate fungi from Astragalus adsurgens Pall and screen the fungi producing SW.LAAO and KS genes could involve in the regulation of L-lysine degradation and SW biosynthesis.But function of LAAO and KS genes is known well in fungal enophytes of locoweeds.The second objective is to screen and analyze LAAO and KS genes of swainsonine-producing fungi from Astragalus adsurgens Pall and four species of locoweeds.The third objective is screen the mutants that swainsonine-producing is significantly different from control strain from isolates treated by UV EMS and NTG mutagenesis.It will offer mutant for differentially expressed genes(DEGs)of fungi that involved in SW biosynthesis.The main findings are as follows:1.The fungi were isolated from plant tissues of Astragalus adsurgens Pall.All of isolates were screened to determine which isolates producing swainsonine.The sequences of ITS,GPD and KS of SW-producing strains were sequenced and analyzed by genetic evolution.The results showed that 13 of 43 isolates from A.adsurgens Pall can produce SW.However,the SW content of the fungi was only 004 ±0.046 mg.g-1 and significantly lower than SW-producing endophytic fungi from locoweeds.The 13 of fungi are most closely related to reported pathogenic fungus of A.adsurgens Pall,Alternaria gansuense,and Alternaria Section Undifilum sp from locoweeds.They are based on morphological characteristics and phylogenetic analysis.The 13 of fungi producing swainsonine from A.adsurgens Pall were classified into the Alternaria Section Undifilum sp and named as Alternaria Section Undifilum gansuense.2.LAAO and KS genes of 13 SW-producing isolates from Astragalus adsurgens Pall,Astragalus variabills,Astragalus,strictus,Oxytropis.glacialis and Oxytropis.ochrocephala were tested,sequenced and compared.The results showed that all of fungal LAAO fragments were 798 bp and KS fragments were 690 bp or 691 bp.LAAO sequences of the 12 endophytic fungi of locoweeds were abosolutely same.However,there were 6 nucleotide differences between Alternaria Section U.gansuense and other fungi from locoweeds.Comparing with Alternaria Section U.oxytropis(KY365741),there were 0 to 11 nucleotide differences among KS genes sequences of 13 SW-producing isolates that caused 0 to 9 amino acid residue differences.However,all of mutation sites in Alternaria Section U.gansuense from A.adsurgens Pall were not present in other 12 fungi from locoweeds.This study hypothesized that LAAO gene could involvein the synthesis of L-lysine or the regulation of the synthesis pathway from L-lysine to L-pipecolic acid.KS could involve in the synthesis of from L-pipecolic acid to SW.The variation of the nucleotide sequence of KS gene in different isolates could be related to expression of KS gene and its regulation of SW synthesis in fungi.3.Candidate mutants of Alternaria Section U.oxytropis treated by UV,EMS and NTG mutagenesis were successively subcultured for 3 generations.The SW yield of each mutant was tested and statistically analyzed to pick out the mutant that SW yield differ significantly from control but remain stable.The results showed that three mutants,E-02,E-25 and E-23,are obtained.Three mutants with significant differences(p<0.05)in the yields of SW and control strains.SW yield of E-02 was significantly different from the control group(p<0.01)after continuously cultured.This study can provide strains for further transcriptome analysis of locoweed endophytic fungi.