Identification Of Mutants Of Agrobacterium Tumefaciens-mediated Transformation Of Paecilomyces Lilacinus And Analyses Of Their Enzyme Activity

Root knot nematode disease is one of plant root diseases caused by root-knot nematodes,which is widely distributed,and root-knot nematodes cause huge losses in agriculture and forestry.Paecilomyces lilacinus is considered as an efficient bio-control fungus that can control the root-knot nematodes.But the pathogenic mechanism of P.lilacinus is unknown.In order to take full advantage of P.lilacinus and improve the effects of biological control,P.lilacinus mutant bank was conducted,mutants whose phenotypes had changed were screened and enzyme activities were analyzed,those researches may contribute to understand the pathogenic mechanism of P.lilacinus and clone genes related to pathogenicity.All of 14 mutants generated by T-DNA insertion were tested with PCR for their insertional sequence.Phenotypes of mutants were also analyzed about morphology,colony growing rate,spore production,conidia germination rate,dry weight of mycelium and pathogenicity against Meloidogyne incognita by comparing with wild-type strain 20-7.The results demonstrated that the genomes of those mutants contain the sequence of beta-tubulin gene and T-DNA had been integrated into wild-type genome of P.lilacinus.Compared with wild-type strain 20-7,these colony morphologies were changed at different degrees.85.71% strains slowed down colony growing rate obviously,but only strain BN-11 speeded up its colony growing rate distinctly.14.29% strains increased the spore production,which were BN-11 and BN-12.78.57% mutants changed their conidia germination rate significantly.The rates of mutants that dry weight of mycelium was reduced and increased were respectively 21.43% and 21.43%.1 mutant whose pathogenicity was increased and 11 mutants whose pathogenicity was decreased enormously were obtained.Chitinase and protease activity is an important indicator which evaluates the biological control potential of P.lilacinus,chitinase activity and protease activity of the mutants were analyzed,the results revealed chitinase and protease activity were affected at different levels by T-DNA insert into genomes.The chitinase activity of wild-type strain 20-7 reached a maximum value 2.2944 U/m L after 5 days of culture,and the chitinase activity of wild-type strain 20-7 was larger compared with enzyme activity of every mutant strain in each day.Chitinase enzyme activity peak of each mutant strain appeared sooner or later.On the other hand,the protease activity of wild-type strain 20-7 reached a maximum value 2.6453 U/m L after 6 days of culture.In all strains,protease activity of mutant BN-7 was at lowest value compared with enzyme activity of other mutant strains in each day.