Study On Submerged Fermentation Conditions And Formulations For Paecilomyces Lilacinus

The fungal biocontrol agent,Paecilomyces lilacinus,as evaluated as one of the most potential nematophagous fungi to control the root-knot nematode.However,the research was just started about the mass production of conidia,preparation of conidia powder and formulations and the shelf life of formulations of P.lilacinus,which was restricted to apply to practice. Therefore,it is absolutely necessary to carry out these studies on mass production and preparation of formulations of P.lilacinus.The optimization of medium ingredient and the fermentation conditions were got by flask fermenting and submerged culturing in the small fermenting pot. Some methods of drying the production of fermentation were compared. Some synergists and the carrier and adjuvants of formulations production which were compatible with P.lilacinus20-7were screened by the method of investigating wether mycelial growth and conidial germination were influenced on the PDA containing synergists or carrier or adjuvants. The shelf life of formulations of P.lilacinus20-7under different tempereture was campared. The results were as folows:(1) The optimal C/N (weigh ratio) and concentration of C was determined as8:1and160g/L.Fermentation cycles was shortened and the spore production was increased by adding potato sauce or egg yolk.Using CaCO3as buffer of fermentation broth can improve spore production.The composition of medium improved (g/L):glucose160, NaNO310,(NH4)2SO45, soybean powder5, KH2PO41.5, MgSO4·7H2O1.5,1%egg yolk or200g/L potato sauce, CaCO32.(2) The optimum technological conditions of submerged culture were as follows: tank pressure was0.06-0.08Mpa,temperature27℃,the best intial pH value6which was not controled or kept by adding some CaCO3as buffer,inoculum volume3%-5%.The optimal conditions of strring rate and aeration rate were controled as follows:The strring rate was100r/min between0-48h,150r/min between48-84h;200r/min between84h-108h,150r/min after108h till finally.The aeration rate was200L/h between0-60h,250L/h between60-108h,200L/h after108h till finally.The optimum fermentation period was84-96h. (3) The optimum conditions of recovering conidium by centrifugal method were as follows:the flocculant was KAl (SO4)2that its concentration was2.0g/100ml, relative centrifugal force4000g and centrifugation time30min.(4) The drying methods had an important influence on the survival rate and moisture content of condium powder. The optimal temperature of drying condium powder in the oven was comfirmed as35℃.Different drying methods can be adopted in the different situations. Several sectional methods which were studied can be adopted.(5) Three carbohydrate cryprotectants,two polymers cryoprotectants,an amino acid cryoprotectant and a protein cryoprotectant were screened which were10%lactose,10%xylose and10%maltose,5%glycerol and1%CMCNa,10%sodium glutamate and4%peptone.Their survival rate after vacuum freezedrying were separately more than80%,about60%,more than70%and about60%.(6) Some synergists which were compatible with P.lilacinus20-7were screened as follows:0-1%Nemathorin,0.1-100mg/L IAA,500-1000mg/ml2,4-D10-100mg/L oxalic acid,1%NH4NO3and NH4Cl.(7) The optimal composition of conidia germination stimulus as an additive to P.lilacinus20-7formulation were as follows:0.1%peptone,0.1%vitamin B1,0.1%L-alanine,0.15%sucrose.The parasitism rate of P.lilacinus20-7to eggs of Meloidogyne spp.was more than80%and the hatch rate of eggs of Meloidogyne spp. was almost0after the action of P.lilacinus20-7and Meloidogyne spp for5days.(8) Diatomite and kaolin were screened as the carrier of P.lilacinus20-7and diatomite was much better.D1002,NNO,lignin sufonic natrium and lignin sufonic calcium as dispersant agents,Tween-20,PEG and PVA as wetting agents,PEG and oxalic acid as stabilizers,riboflavin and sodium fluorescein as protective agent of UV were screened.(9) The composition of wettable powder of P.lilacinus20-7were as follows: condium powder:50%,dispersant agent2-5%; anti-UV protective agent0.5-2%,stabilizer0.5-1%,wetting agent1-2%,phytohormone0.1-1000mg/L,inorganic salts synergists or conidia germination stimulus:0.5-2%,finally make up to100%with carrier.(10) The shelf life of formulations of P.lilacinus20-7was related to the type of formulation and storage temperature.The shelf life when stored under4℃was longer than that when stored under room temperature.The shelf life of microencapsulation agents was longer than granules ans powder. The shelf life of formulations was more than6months at4℃but only microencapsulation agents could be kept for6months at room temperature and others only could be saved for3months and microencapsulation agents could be both kept alive for more than12months under room temperature and4℃.