The Functional Research Of Extracellular Proteas Es Related To Nematode Egg Parasitism In Pochonia Chlamydosporia

Root-knot nematodes （Meloidogyne spp.） are sedentary endoparasites that cause significant economic and yield losses of a wide range of agricultural crops worldwide, mainly in tropical and sub-tropical regions.Up to now, many methods have been used to control nematodes, such as chemical control, breeding for disease resistance, biological control methods and so on. But chemicals are toxic to human and environment and the resistance-breeding is lack of nematode-resistant genes and with longer cycles. Lacking alternative controls has prompted researchers to pay more attention to biological control methods for nematodes. The egg-pathogenic fungus Pochonia chlamydosporia is one of the most widely studied and tested soil hyphomycetes for the biological control of plant-parasitic nematodes. In the past, the investigators mainly focus on the study on the biocontrol efficacy of P.chlamydospora as biological control agents and the progress of P. chlamydosporia infecting nematodes. There are few reports on the molecular mechanism of the fungus P. chlamydosporia infecting nematodes for lacking effective means of gene knockout. In this study we have developed a effective P. chlamydosporia gene knockout systerm with neomycin-resistant gene neo as selectable marker. And we also did some research on the functions of protease and chitinase relating to M. incognita eggs parasitic. All of this will provide a basis for the research of the molecular mechanism. The main contents are listed as following:1. The infection rates of P. chlamydosporia for M. incognita eggs in dissociated state or in the egg masses were measured. Results are 71.6±2.2 and 74.6±3.6（at 7 days after infection）, respectively. Under pot experiment, the control efficiency of strain PC-170 combined with abamectin to M. incognita was measured. The results indicated that PC-170 is a potential biological control agent M. incognita and with the control efficiency maintained, the use of PC-170 could reduce abuse of avermectin.2. A stable green fluorescent protein （GFP）-expressing transformant （PC-170gfp） was constructed, make the infection process of the fungus PC-170gfp in M. incognita eggs in the eggs masses and in cucumber roots visible with confocal laser microscopy.3. Two protease genes （VFPPC₁0088 and VFPPC₀6535） and one chitinase gene （VFPPC₀1099） was selected as candidate genes based on the analysis of P. chlamydosporia genomic and transcriptomic data.4. Developed an effective P. chlamydosporia gene knockout systerm with neomycin-resistant gene neo as a selectable marke using fusion PCR-based split maker gene disruption method and PEG-mediated protoplast transformation method. Two protease genes （VFPPC₁0088 and VFPPC₀6535） and one chitinase gene （VFPPC 01099） were respectively deleted in strain PC-170 by the gene knockout systerm. And the parasitic ability of wild type strain PC-170 and mutants were tested. The results showed that all the mutants but PC-170-△VFPPC₁0088 had a significant reduction （P<0.05） in the ability to parasitize M. incognita eggs compared to the wild strain, but they still had an ability to parasitize M. incognita eggs. This suggested that the three genes might have a partial role in the process of P. chlamydosporia infecting M. incognita eggs.5. The genomic data of PC-170 and other 17 fungi was analysed together for grouping orthologous protein sequences by OrthoMCL. In PC-170, there are two genes belong to oxyzin precursor group, VFPPC₀3048 （coding VCP1 protein） and VFPPC₁0088. Here, both genes were successfully deleted respectively via homologous recombination in PC-170 and the M. incognita eggs parasitic ability of mutants and wild type strain were tested. Results showed that the parasitic ability between the two mutants had no significant difference and both had significant reduction in the ability to parasitize M. incognita eggs in eggs masses compared to the wild strain （P<0.05）, but still had an ability to parasitize M. incognita eggs.This indicated that the three genes might have a partial role in the process of P. chlamydosporia infecting M. incognita eggs.