Biological Characterization And Fermentation Conditions Of Lecanicillium Attenuatum TN002 Isolates

With far-ranging geographical distribution and wide host range,Lecanicillium attenuatum is considered to be one of the most important entomopathogenic fungus with magnificent application prospect in plant protection.Ever since 1980 s,the commercial products of Lecanicillium was already used as an alternative control agent for aphids and whiteflies abroad.However,in China,the research on this fungi is still at its initial stage.In this thesis,the biological characteristics and the conditions for liquid-solid biphasic fermentation of the L.attenuatum TN002 strain isolated from whitefly body in Chongqing were studied.The results provide more theoretic support for the massive commercial production of TN002 strain.The works and results can be listed as follows:(1)Biological characterization of L.attenuatum TN002 isolatesThe biological characterization of TN002 isolates was studied by using single factor experiment.The colony morphology and spore production were selected as the evaluation index.The results showed that,the colony diameter TN002 isolates was 32.33 mm in 10 days on PDA,high,circular,white,radial,and reverse yellowish-white.The conidia was cylindrical,monospore and transparent.Solitary or whorled phialides produced on prostrate hyphae.The conidia of this strain could germinate and grow quickly and sporulate highly.Besides,the optimal culture medium and pH values for TN002 strain were also evaluated.The results indicated that PJA and SDAY were the optimal growth medium for TN002,while PDA was optimum for spore production.TN002 could survive from 10 to 35?.But the optimal temperature for growth and spore production was 25?.The influences of pH value of medium on colonial growth rate were not significant.However,the highest value of conidial production was found at pH being 5.5.Different carbon and nitrogen sources had different impacts on the colony growth and sporulation quantity.D-maltose and yeast extract powder were considered to be the best carbon and nitrogen source for its growth,while sucrose,glucose,galactose and peptone were optimum for conidial production,respectively.In addition,the affects of trace elements and vitamins on the growth and yield of conidia were also estimated.The data indicated that Cu,Mn,Zn(concentration: 50 ?g/mL)had negative influence on the production of conidia on the medium,but the vitamins,thiamine hydrochloride(VB1),riboflavin(VB2),folic acid(VM),pyridoxine hydrochloride(VB6)(concentration: 10 ?g/mL)had positive influence on conidia,folic acid is most conducive to its growth.In general,the TN002 strain was relatively sensitive to UV-B radiation,but was tolerant to high temperature treatment.The conidia germination remained to 70% after treated with 40? for 1 h.Furthermore,the condidial suspension showed virulent to Panonychus citri McGregor,with the indicators of median lethal time(LT50),6.13 days(8.20×105 spores·mL-1)and median lethal concentration(LC50),1×107 spores·mL-1.(2)Culture conditions for liquid fermentation of L.attenuatum TN002 isolatesThe liquid culture conditions of the TN002 isolates was selected by using single factor experiment and orthogonal design.Under shake flask culture condition,glucose yeast extract liquid medium was optimum for spore production.In addition,the highest spores production and mycelial dry weight were observed at 60 h after shaker cultivation for 24 h~84 h in the glucose yeast extract medium(50mL/250 mL dish)with 1% inoculation.The influence of the different pH values on the mycelial dry weight were not significant,and the maximal conidial production was found at initial pH being 5.5.The influence of volume of culture medium,initial inoculum size and rotation speed on the spore production was estimated with orthogonal test.The results showed that all of these three factors were important for spore production,and the optimal conditions for first stage seed production were 4% for inoculum size in 100 mL medium at 190 r/min.For the mass production of L.attenuatum TN002 isolates,different carbon and nutrition sources were screened for the optimal conditions for spore production.As demonstrated,sucrose,white sugar,soybean meal and wheat bran were the best carbon and nitrogen sources,respectively.In consideration of the costs,the medium with wheat bran and white sugar was selected as the optimal medium combation for the massive fermentation.The orthogonal test for wheat bran,white sugar and initial inoculation further indicated that,No.10 treament(the medium with 5% wheat bran,5% sugar)was best for spore production.The optimal combination for batch fermentation was 5% wheat bran,5% sugar with 2% initial inoculation.(3)Culture conditions for solid fermentation of L.attenuatum TN002 isolatesThe medium components and culture medium were screened for the mass production in diphasic liquid-solid fermentation using canned bottle.The yield of spores and quality of conidial viability were chosen as the evaluation index.The results showed that the conidia production reach its maximum level in the wheat bran or soybean meal solid-state medium after cultivating.After evaluation,the medium with 30% rice hull,35% wheat bran and 35% soybean meal was finally selected as the optimal condition for solid-state fermentation.The optimal conditions for conidia production on the 20 g medium were 25?,initial conidia concentration being 1×106 conidia/g dry substrate and the initial ratio for material-water being 1.00:1.It was suitable to harvest after cultivating for 5 to 7 days.Compared with the original strain,the virulence of L.attenuatum TN002 isolates generated from diphasic solid-state fermention was relatively stable,with the indicators of LT50(5.06 days,at 2×107 spores·mL-1)and LC50(7.43×106 spores·mL-1).