| As an important soil-borne plant pathogenic fungus,Fusarium oxysporum can produce virulence factors such as cell wall degrading enzymes,effector proteins and secondary metabolic toxins during the infection of plants.The secondary metabolic toxins mainly include fusaric acid,beauvericin and enniatins,the secondary metabolic toxin biosynthesis genes have been identified in some forma specialis of Fusarium oxysporum,but there are few studies on the secondary metabolic toxins produced by Fusarium oxysporum f.sp.medicaginis and the expression rules of its metabolic toxin biosynthesis genes.In this study,FUB genes of Fusarium oxysporum f.sp.medicaginis strains for tests were identified and phylogenetic analysis were carried out according to fusaric acid biosynthesis gene(FUB)sequences of different forma specialis,and FUB genes expression level in tested strains during different stages of in vitro culture and infection of alfalfa seedlings were studied.Finally,the yield of fusaric acid of tested strains during in vitro culture and the toxic effects of different fusaric acid content crude toxin on alfalfa were analyzed,This study is expected to provide theoretical basis for elucidation of the pathogenic mechanism of F.oxysporum f.sp.medicaginis and the control of alfalfa fusarium wilt.The main findings are as follows:1.The identification results of FUB genes in the tested strains showed that FUB1,FUB4 and FUB10 gene existed in all tested strains(T6～T9),FUB3 and FUB8 gene only existed in T6～T8,and FUB6 gene only existed in T9.Phylogenetic analysis of FUB1,FUB4 and FUB10 gene showed that the sequences of FUB gene of strain T6～T8 were highly similar to each other,while the sequences of FUB gene of strain T9 were quite different from those of other strains.Phylogenetic analysis of FUB genes can distinguish forma specialis as well as strong and weak virulence strains(T9and T6).2.The expression levels of FUB1,FUB4 and FUB10 gene of strong virulence strain T9 were higher than those of weak virulence strain T6 at 21 d and 28 d after in vitro culture.The expression levels of FUB1 and FUB4 gene of strong virulence strain T9 were also higher than those of weak virulence strain T6 at 21 d and 28 d after inoculation of alfalfa seedlings,indicating that the transcription level of FUB genes affected the virulence of the tested strains to a certain extent.3.The fusaric acid production of the strong virulence strain T9 was higher than that of the weak virulence strain T6 at 14 days and 21 days after in vitro culture.The crude toxin containing fusaric acid 2670.5μg/ml could completely inhibit the germination of alfalfa seeds and cause the dieback of alfalfa seedlings.The crude toxin containing fusaric acid 267μg/ml could significantly inhibit the growth of radicle and plantule of alfalfa seeds and significantly increase the disease index of alfalfa seedlings(P<0.05).The crude toxin with fusaric acid content <26.7μg/ml had slight inhibitory effect on alfalfa seed germination. |
