The Effect On Biocontrol Of Paecilomyces Lilacinus E2-4 And The Genetic Transformation Mediated By Agrobacterium Tumefaciens

The excessive application of chemical pesticides has left the agro-ecosystem collapse, environmental contamination and human health concerns. So the biological strategy for biological control is receving much attention worldwide. Among the biocontrol microorganism, pathogenic fungi have very important contribution to biological control, especially for plant etiological agent. Compared with chemical pesticide, pathogenic fungi were no toxic to environment, human and animals. Therefore , mycoinsecticides are being considered as alternative and supplement to chemical pesticides. Paecilomyces lilacinus is well known not only one of nematodes egg-pathogenic fungi, but also a kind of entomopathogenic fungi ,which currently receiving attention for their potential in biological control agent. However, the acceptance of fungal products for pest control is very limited .Because the fungal insecticides are not as fast acting as chemical products and sensitive to adverse environment and lose their effectiveness more rapidly. So it is necessary to improvement P.lilacinus strain .With the basis of gene engineering ,chitinase gene from Metarhizium anisopliae was cloned and transformated to P.lilacinus E2-4 by Agrobacterium tumefaciens-mediated procedure.Transformants of P.lilacinus E2-4 were obtained and the biocontrol ability of transformants were significant enhanced.The main results are as following:1) Firstly ,in order to sure the biocontrol potential of P.lilacinus E2-4 which was screened and stored by our lab, some virulence test were done . The effection of P.lilacinus E2-4 sporium suspension to the hatching of nematode egg masses showed that the number of nematode hatching from nematode egg masses which were treated by low concentration of sporium suspension(10⁴conidia/mL)were more exceded than control treated by water.When the concentration of sporium suspension was increased to 10⁵conidia/mL,numbers of nematode were significantly reduced and the fractional inhibition of 10⁵, 10⁶,10⁷,10⁸coidia/mL to nematode egg masses were 21.72%,42.21%, 49.19 % ,54.95 % respectively.The effection of sporium suspension10⁶,10⁷ and 10⁸conidia/mL to the hatching of nematode egg masses existed significant deviation between control . The nematode egg masses treated by sporium suspension were observed by Olympus stereomicroscopy, found that many mycelial of P.lilacinus on the surface of egg masses and wrapped around it.P.lilacinus E2-4 solid granula were made by liquid-solid diphasic fermentation. Soybean meals were used as the substrate of liquid fermentation and the substrate of solid fermentation were grains of rice .Studied on the effect of inducer on conidia yield.When chitin and chitosan were added to grains of rice as the inducer, the conidia yield of P.lilacinus E2-4 added to 4.15×10⁹ and 2.37×10⁹ conidia/mL.A pot experiment was conducted to test the effect of P.lilacinus E2-4 solid granula on tomato root-knot nematodes.The results proved that the occurrence of tomato root-knot nematodes was inhibited and plant growth was promoted by P.lilacinus E2-4 solid granula. And CK which were not treated by P.lilacinus E2-4 solid granula occurred diseases ,for example the leaves become brown or yellow and even to withered , the root had many root knots. The disease symptoms of tomato stalks,leaves and root were less by P.lilacinus E2-4 compared with CK meanwhile.A experiment was conducted to test the effection of P.lilacinus E2-4 sporium suspension to the death rating of chinese cabbage aphids.The result showed that the death rating of aphids treated with different concentration of sporium suspension have some difference.Treated with 10⁴ conidia/mL sporium suspension,the virulence to aphids was not distinct difference compared with control which treated by water. And then the death rating of aphids were increased obviously,when the concentration of sporium suspension was increased to 10⁵,10⁶,10⁷ and 10⁸conidia/mL.The mycelial threads could be observed on the surface of aphids by microscope.2)According to the above experiments proved that P.lilacinus E2-4 have the biocontrol ability to root-knot nematodes and aphids.Therefore ,in order to improve the virulence of E2-4 strain for more effective biocontrol application, a simple,highly efficient and reliable transformation system of P.lilacinus mediated by Agrobacterium tumefaciens was developmented ,cloning of chitinase gene from M.anisopliae and then enhancing virulence of P.lilacinus by genetic transformation.First of all , a expression vector including chi gene and gfp gene was constructed. Using pCAMBIA1302 ,a commonly used vector for plant transformation,as the backbone,the binary vector pTBAR was constructed .According to the fact that P.lilacinus was not sensitive to hygromycin,the herbicide resistance gene bar which was PCR amplified from vector pBHt2 was used as selectable marker gene for P.lilacinus transformation and bar gene was under the promoter Trpc which was amplified from vector pC30RG in pTBAR. And then a expression element trpc-chi was constructed, through cloning chi gene from M.anisopliae and connecting with Trpc promoter by invaginate PCR.At last , vector pTBAR was inserted trpc-chi expression element by BamH ? and HindШsitus, and expression vector pTBTCHI was constructed successfully. 3) Mediated by A.tumefaciens strain GV310³,T-DNA of pTBTCHI was successfully integrated into P.lilacinus genome. After being perfected transformation steps were described below. The transformation efficiency of P.lilacinus was highest when the co-cultivation condition was A.tumefaciens OD₆₀₀=0.2-0.3, P.lilacinus spore 10⁶conidia/mL and 200μg/mL AS. When the A.tumefaciens OD₆₀₀ was low to 0.1 or high over 0.3, the transformation efficincy was low. For the concentration of spore , the resistant transformants obtained from transformation were very few when the concentration of spore was 10⁴conidia/mL,10⁵conidia/mL, 10⁷conidia/mL and 10⁸conidia/mL,except10⁶conidia/mL.Under the condition of A.tumefaciens OD₆₀₀=0.2, the concentration of spore was 10⁶conidia/mL, AS was added to broth IM ,the transformation efficincy were stable ,and could obtain about 40 transformants/dish. When no AS added to IM co-culture ,no resistant transformants obtained.A total of 53 resistant transformants were obtained , the most of their morphology were similar with E2-4 strain ,and only few have different. In order to test if the transformants were positive transformants, 11 resistant transformants were random selected and extracted genomic DNA which were used to amplified bar gene by PCR. Six resistant transformants were amplified the bar gene.Because of the T-DNA which integrated into P.lilacinus including gfp gene, the positive transformations could be observed green fluorescence by fluorescence microscope. And Southern blotting analysis on the transformants randomly chosen from resistant transformants revealed that the T-DNA have insertioned to P.lilacinus.4)To determine if expression of chi can enhance the virulence of P.lilacinus. Four positive transformants were applied to the chitinase expression analysis in the exuxiae of the cicada medium.The result of Duncan multiple range test fo variable showed that the chitinase activity of tchi001,tchi002,tchi007,tchi009,tchi021 and tchi030 compared with E2-4 existed significant deviation. The protein level produced by six transformants were also very different with E2-4.Virulence test were conducted by two positive transformants tchi001 and tchi007 ,which have higher chitinase activity .The result showed that both of the two transformants and E2-4 strain had inhibitory action to the haching of nematode egg masses. But the inhibitory action speed of tchi001 was obviously faster than E2-4 and tchi007, the ability to inhibit hatching of nematode egg was also higher than E2-4 and tchi007. The death rate test to nematodes revealed that all of the transformations and wild type E2-4 strain had evident virulence to tomato root-knot nematodes. As seen from the analysis of SAS , against E2-4, the correct lethality rate to tomato root-knot nematodes of tchi001 and tchi007 were all over 60%, The result suggesting that expression chitinase can enhance the virulence of P.lilacinus.