The Function Of NRPS Gene VMG10 In Valsa Mali

Apple valsa canker,caused by Valsa mali,has greatly impaired the healthy development of the apple industry in China.Due to its wide spread and serious damage,it is very difficult to control,as called"cancer"of apple trees.At present,the research on apple valsa canker mainly focuses on cell wall degrading enzymes,effector proteins and secondary metabolites.The whole genome sequencing of Valsa Mali indicated a large number of NRPS genes present in the genome,in which 13 NRPS genes were up-regulated at the early stage of infected branches,suggesting that NRPS genes played an important role in the pathogenesis.In this study,five NRPS genes were first knocked out,followed by virulence screening.Finally,we focused on the growth and development of NRPS gene VmG10 and its pathogenic mechanism.The mainly results are as follows:1.Quantitative qRT-PCR was used to analyze the expression of VmG10 in the Valsa mali infected branches at different time points.The results showed that the expression of NRPS gene VmG10 was up-regulated by 7.12 times at 6 hpi compared with control.Then one knockout mutant and six over-expressed mutants were obtained by PEG-mediated genetic transformation,and 4 revertant mutants were obtained by one-step cloning and final primer detection;2.By observing the growth rate and morphology of respective mutants cultured on PDA,it was found that the deletion of VmG10 affected its growth morphology and did not affect the growth rate,the mycelial edge ofΔVmG10-19 produced more bifurcations than that of03-8.However,the conidia number ofΔVmG10-19 decreased by about 50%compared with03-8.Pathogenicity analysis showed that the virulence ofΔVmG10-19 decreased by 72.17%in leaves and loss the virulence on twigs compared with 03-8.The pathogenicity of over-expressed mutants increased,among which Q15 increased by 21.73%and Q16increased by 12.50%.The melanin phenotype observation and the quantitative analysis of the six genes related to melanin transcriptional regulation showed that the melanin content inΔVmG10-19 was higher than that of 03-8,and the six genes related to melanin transcriptional regulation inΔVmG10-19 were in mycelial growth 2,3,4,5 d the expression levels were higher than those in 03-8;3.Under abiotic stresses of 0.5 M KCl,18 mM H₂O₂,200 mg/L CR and 0.01%SDS,mutantΔVmG10-19 was more inhibited than 03-8.Especially under 18 mM H₂O₂,the inhibiting rate was 44.44%higher than 03-8.The confrontation experiment between mutant03-8 andΔVmG10-19 with other four saprophytic fungi on apple tree showed thatΔVmG10-19 was antagonistic to 03-8.Resistance to apple wheel pattern and apple anthrax was weaker,especially when confronted with apple rings and anthrax.The results of secondary metabolite separation showed that compared with the necrotic spots produced by the tobacco leaves treated with the 03-8 fermentation broth,the necrotic spots produced by theΔVmG10-19 fermentation broth were reduced by 32.8%,and the fermentation broth was diluted 100 times,the results were consistent;The results of free amino acid content showed that the aspartic acid content in the fermentation broth of 03-8 was 4 times that ofΔVmG10-19 fermentation broth,and glutamic acid and arginine were twice as much;It was found that the knockout of VmG10 did affect the synthesis of some secondary metabolites of the V.mali,but not necessarily the gramicidin;The fermentation broth treated the lettuce seeds,and the results showed that the compound treated with less than 1 KD was processed.The germination rate of lettuce seeds was significantly different.The germination rate of lettuce seeds treated withΔVmG10-19 fermentation broth was 63.3%,the germination rate of lettuce seeds treated with 03-8 fermentation broth was 23.7%,and the germination rate of ddH₂O treatment was 86.7%.