Isolation And Identification Of Nematicidal Compound And Efficient Construction Of Engineered Strains Of Gymnoascus Reesii

One fungus strain za-130was obtained with high toxin to root-knotnematode by systematic screening method as root-knot nematode for the target andone novel nematicidal compound was purified from filtrates of the strain za-130. Itstaxonomy, nematicidal activity, fermentation conditions, separation and purificationprogress, control effect and evaluation of safety to mice were studied systematically.The random insertion transformants library of G. ressii was established byrestriction enzyme mediated technique. Twenty mutants were obtained with1.5timeshigher nematicidal activity than the original strain’ and candidate gene sequencefragments, which might be related to the way of producing nematicidal compound,were also isolated successfully. Results showed as follows:1. A total of1319fungal strains collected from different regions China wereisolated from618soil samples. The nematicidal activities of filtrates of these fungalstrains were tested with root-knot nematode by the high-effective screening systemfor the nematicidal materials established by ourselves. The results showed that therewere29fungus strains which could knock down J2of Meloidogyne incogonita. Onestrain designed za-130with good repeatability was obtained from above29strainsafter tests conducted by re-screening. The strain za-130was Gymnoascus reessiidefinited by traditional classification. The results of greenhouse pot experimentsshowed that the control effects on tomato root-knot nematode were both over80%after45and80days by its5times concentrated filtrates. The safety evaluation test oforal toxicity to mouse showed the fermentation filter of za-130belong to“micro-toxicity” by LD50. The strain za-130has potential research and developmentprospects based on the above results.2．Pure nematicidal compound whose purity was98.296%was extracted fromfermentation, which pre-treated by5×ethanol, and condensed by decompression,separated and purified by macroporous adsorption resin chromatography, silica gelchromatography, Sephadex LH-20chromatography and recycling preparative liquidchromatography. It showed that pure nematicidal compund was light yellow powdery,whose molecular weight was326, UV absorption spectrum was275nm. Its IR absorption spectrum also showed it contained methylene, hydroxyl and carbon carbondouble bond. Ultimate analysis showed that it contained carbon, hydrogen and oxygen.The pure compound was identified as（3E,5E）-2,5-dihydroxy-2,7-dihydrooxepine-3-carboxylic anhydride according to its1H、13C、dept135、COSY、HMQC、HMBC spectral data. It possessed light, thermal,acid and base stability. Therefore, za-130was a biocontrol strain which had greatprospect to control plant parasitic nematode.3. The fermentation conditions of za-130were researched by single factor anddesigns of orthogonal experiments, results were as follows: the optimum carbon andnitrogen resource was cornstarch and fish peptone, respectively; the liquid medium offermentation constitutes by cornstarch （2.0%）, fish peptone （0.4%）, potassiumhydrogen phosphate anhydrous （0.1%）, magnesium sulfate（0.03%）, and Manganouschloride （0.1mmol/L）; the optimum fermentation condition included25℃～28℃ofculture, the initial pH6.5, liquid seed cultured48hours,10%of the inoculationamount, the volume of medium in shaking flasks60mL～100mL/500mL flask,150r·min^-1～200r·min^-1of rotation speed,120hours of culture time.4. To characterize the molecular function of G. reesii, an effective transformationsystem of G. reesii needs to be established. Firstly, the factors which can affect theprotoplast preparation and regeneration were optimized. The optimal conditions forprotoplast preparation and generation are as follows: mycelia growth time in CMmedium of42h, driselase as the cell wall-degrading enzyme, a ratio of1.5mLenzyme solution （20mg/mL driselase） per gram mycelia, a treatment time of4h at28°C, and0.7M NaCl as the osmotic stabilizer buffer. Under the conditions above,the yield of G. reesii protoplasts could reach1.84×107protoplasts per mL and theregeneration rate reached up to21.17%on the SR medium. Subsequently, a constructof Rp27::GFP was transformed into the genome of the za-130. The resultingtransformants were confirmed by PCR analysis and fluorescence detection. Datasuggested the PEG mediated transformation system we established was suitable for G.reesii.5. To obtain the root-knot nematode resistant strain, a novel mutant library of G.reesii containing1182transfomants was constructed by the restriction enzyme mediated transformation （REMI）. Using the high-effective screening system for thenematicidal materials,20transformants with1.5times higher nematicidal activitythan the original strain za-130’s were obtained. Finally, in order to isolate thecandidate gene reasonable for the nematicidal compound, the flanking sequences ofinsertion fragment were obtained by plasmid rescue technique for further analysis.