| Mycoparasitism,a lifestyle where one fungus is parasitic on another fungus,has special relevance when the prey is a plant pathogen,providing a strategy for biological control of pathogen fungi for plant protection.Banana wilt disease or Panama disease is caused by Fusarium oxysporum f.sp.cubense 4(Foc4)in later stage in plantation and is one of the most economically important and harmful diseases of banana(Musa spp.).The possible preventing methods include the application of chemical and biological fungicides to eliminate the soil-borne pathogen.Chemical fungicides,often result in environmental and food quality problems.Primary biological fungicides,together with the integrated preventing and controlling technique will be of great importance.Biological fungicides mainly consist of bacteria,fungi and antibiotics.Rhizosphere-competent Trichoderma spp.strains that can colonize the surfaces of roots have been shown to have direct effects on plants,promoting plant growth and yields,enhancing nutrient uptake,improving seed germination and stimulating the induced systemic resistance(ISR)to diseases is most widely applied in production of agriculture.Rhizosphere-competent Trichoderma spp.strains that can colonize the surfaces of roots have been shown to have direct effects on plants,promoting plant growth and yields,enhancing nutrient uptake,improving seed germination and stimulating the induced systemic resistance(ISR)to diseases is most widely applied in production of agriculture.Trichoderma spp.strains also have strong mycoparasitism against plant pathogen fungi and reduce the disease incidence invaded by pathogen fungi.The species of Trichoderma spp.in production include Trichoderma harzianum,Trichoderma virens,and Trichoderma atroviride.However,the difficult in the technology of tranformation limited research of the molecular mechanism of mycoparasitism of Trichoderma sp..This study started from characterization of a new Trichoderma sp.strain,and was mainly focused on the molecular mechanism of mycoparasitism against Foc4.Phylogenetic analysis classified the NJAU 4742 in the Trichoderma sp.Its survival in soil and organic fertilizers and colonization on plant root was verified after NJAU 4742 labeled with gfp by the optimized transformation method with Agrobacterium tumefaciens.Then,mutants library constructed by the transformation with Agrobacteria were used to screen mutant lost strong mycoparasitism.The inserting sites of T-DNA was analyzed for the research of gene function.At last,The primary factor of mycoparasitism of NJAU 4742 against plant pathogen fungi was identify from deleting and over-expressing the related gene and comparing the antifungal ability of different components excreted by NJAU 4742.The main results obtained are listed as follows.1.A Trichoderma sp.strain,having strong mycoparasitism against pathogen fungi Foc4,Botrytis cinerea,Alternaria alternata,Sclerotinia sclerotiorum,Rhizoctonia solani and Sclerotium rolfsii,was screened,purified and named as NJAU 4742.NJAU 4742 hyphae can coil or attach along Foc4 hyphae observed under Scanning Electronic Microscopy(SEM).The metabolites released by NJAU 4742 mycelia coiling Foc4 damaged the integrity of Foc4 hyphae,which was stained with blue by Trypan Blue.Water-soluble metabolites of NJAU 4742 completely inhibited the growth of Botrytis cinerea,Alternaria alternata,Sclerotinia sclerotiorum andRhizoctonia solani,but showed low inhibition to Foc4.GC-MS analysed the component of volatiles released by NJAU 4742.In sealed plates,3-Octanone,Naphthalene,1-methylnaphthalene and 1-Octen-3-one can inhibit the growth of Foc4 mycelia.After measurement of extracellular enzymes,chitinases,trypsin-like proteases and chymotrypsin-like proteases were induced by Foc4 mycelia.NJAU 4742 is clarified as a new clade of Trichoderma sp.,based on Bayesian phylogenetic tree according to gene call,chil8-5 and tefl.It has stronger mycoparasitism against pathogen fungi than the type strain Trichoderma harzianum CBS 226.95(CBS),which can’t cover the Foc4 colonies in darkness and showed low antifungal ability of water-soluble metabolites.With the analysis of phenotype of NJAU 4742 and CBS by Biolog,NJAU 4742 belongs to Trichoderma guizhouense.2.The method of transformation with Agrobacterium tumefaciens were optimized in NJAU 4742 and suitable to T.aggressivum CPK 375,T.pleuroticola CPK 2104,T.pleurotum CPK 2094 and Foc4,with high transformation efficiency.NJAU 4742 labeled with GFPcan survive in soil and organic fertilizers,observed by tracking green fluorescence.The CFU was measured by plate counting with hygromycin B.NJAU 4742 colonizing the roots of banana plants had a synergistic effect in promoting banana growth and the biocontrol against Foc4 in a greenhouse.3.To identify genes involved in mycoparasitism,T-DNA insertional mutagenesis was applied with transformation with Agrobacterium.A mutant out of 80 purified mutants was unable to overgrow mycelia of Foc4,Botrytis cinerea and Alternaria alternata,and reduced antifungal ability of its water-soluble metabolites.With HiTAIL-PCR,The T-DNA inserting site in the genome was detected in the terminator of a neutral metalloprotease gene(Encoding a MEROPS family M35 protease),which was named nmpl.The antifungal activity of the mutant was rescued by retransformationwith wild-type nmpl gene.NMP1(overexpressed in P.pastoris),18.7 kDa contain a unique aspzincin motif in which two zinc-binding in an HEXXH motif.In addition,further third zinc ligand is formed by a GTXDXXYG motif.The NMP1 did not inhibit the growth and germination of other fungi in vitro.However,NMP1,partly recovered the antifungal activity of the mutant strain against Sclerotium rolfsii and strengthened the antifungal activity of wild type.The expression of nmpl was induced by the presence of other fungi and by dead fungal biomass4.The interaction between Foc4 and NJAU 4742 was investigated.Chitinases,proteases and H2O2 were detected and measured in the yellowish exudate that was collected from the NJAU 4742 hyphae surface that covered Foc4 mycelia.GC-MS analysis identified almitic and stearic acids,which is constituent parts of cytomembrane.H2O2 suppressed Foc4 growth in the Oxford Cup experiment,compared with crude enzymes(induced by Foc4 mycelia)and n-butanol extracts(dissolving NJAU4742 metabolites).H2O2 accumulated when NJAU 4742 mycelia physically contacted Foc4 mycelia by the method of dye.Under confrontation test,Fe-sod,Mn-sod and Cu/Zn-sod,which encode the SOD enzymes that convert O2·-to H2O2,were up-regulated by Quantitative Real-Time PCR analysis.The plasma membrane of Foc4 cells was damaged when treated with H2O2 by the propidium iodide(PI)staining.5 M H2O2 can inhibit the growth of Rhizoctonia solani and Sclerotium rolfsiiand changed the morphology of Alternaria alternata,Sclerotinia sclerotiorum and Foc4 on PDA plates.PEG-CaCl2 mediated transformation of protoplasts was used after constructing a plasmid of deleting nox1(EncodingNADPH oxidase 1,Noxl).A positive mutant out of 410 mutants was screened by PCR.Deleting nox1 madethe mutant lost strong mycoparasitism against Foc4,Alternaria alternata,and Sclerotium rolfsii in darkness.The Anoxl mutant also showed weak hurt response when its mycelia was cut.Over-expressing noxl in the Anoxl strain can recovery the mycoparasitism against Sclerotium rolfsii.Foc4 and Sclerotinia sclerotiorum in darkness.Over-expressing noxl in wild type NJAU 4742 completely changed the mycoparasitism and had the ability to cover Foc4,Sclerotinia sclerotiorum,Sclerotium rolfsii,Botrytis cinerea completely in light.Conclusion:NJAU 4742 with strong mycoparasitism against several pathogen fungi can survive in soil.Fermentation of NJAU 4742 in organic fertilizers is useful to increase the orders of magnitude of strains.After the series analysis of different component of metabolites,H2O2 served as primary antifungal chemical is regulated by Nox1.More research should be focused on the function of reactive oxygen species.Moreover we show that lytic enzymes are essential for reusing the Foc4 dead biomass in mycotrophy. |
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