Construction Of A T-DNA Insertional Library Of Purureocillium Lilacinum And Function Analysis Of Infection Related Genes

Purpureocillium lilacinum(Thom) Samson, a soil-inhabitant and egg-parasitic fungus, is one of the most effective parasites of nematode eggs and juveniles. P. lilacinum is not only a bio-pesticide, but also aplant growth promotor. Extensive work has been conducted on P. lilacinum focusing on biological characteristics, biocontrol applications, physiological metabolites, etc. However, at the molecular level, few papers were reported about pathogenic genes and pathogenesis. The present research work is mainly focusing on exploring the pathogenic mechanism when P.lilacinum parasitizes nematode eggs and kill juveniles through the phenotypic changes of transformed strain by Agrobacterium tumefaciens-mediated transformation(ATMT). The main results are listed as follows:1. The system of A. tumefaciens-mediated transformation of the filamentous fungus P.lilacinum was successfully constructed and and optimized with the following conditions: constructed vectoe containing bar gene encoding glufosinate resistance and CBX gene encoding carboxin resistance, spore concentration of 106 spores/m L, A. tumefaciens AGL-1 OD600 value of 0.2, co-culture temperature of 25 °C, co-culture period of 54 hours, acetosyringone AS concentration of 250 μg/m L and shaking speed 120 rpm / min. Screening of 359 strains for their virulence against nematodes was performed.2. New amino acid substitution sites in β-tubulin gene have been analyzed for effective carbendazim-resistance. Amino acid substitutions caused by site-direct changes at particular target codons(145,185 and 200) were manifested to be the cause of fungicide resistance by loss or reduction of the binding affinity to benzimidazole associated with the amino acid changes in β-tubulin.The carbendazim resistance of P.lilacinum has been increased to a concentration of 100μg/m L. The change of β-tubulin gene did not affect either the morphology or virulence of P.lilacinum.3. Leucinostatins is an indicator of nematicidal activity. They could kill nematodes with no effect on egg parasitism. The result showed that the Zn(II)2Cys6 transcription factor genes ‘rol P’ encodes production of leucinostatins A, and showed a negative effect on leucinostatins B. The highest mortality rate and the relative expression level of rol P gene were achieved when lactose as a carbon source and L-Alanine as a source of nitrogen. Acidic environment was more suitable for the P. lilacinum strain to produce leucinostatins and high m RNA level expression of rol P gene.4. phospholipase D can cleave the phospholipids of nematodes eggs and surface. This study showed that pld gene encodes phospholipase D with two PLD phosphodiesterase domains(HKD), Infection trial provided evidence that the the presence of nematodes or their metabolites may stimulate the expression of pld. Maltose and L-alanine were found to increase the expression of pld gene. An acidic environment and moderate temperatures(27 oC to 29 oC) are favorable for pld expression in P. lilacinum.5. Leucinostatins D does not only kill nematodes, but also has a strong antagonistic inhibition of Sclerotinia sclerotiorum. Furthermore, the glycoside hydrolase gene PGH31 of P. lilacinum was found to have a negative effect on production of leucinostatins D. P. lilacinum is a novel and promising biocontrol agent against oilseed rape Sclerotinia stem rot as confirmed through the laboratory and field trials.