| Fungal insecticides developed with entomopathogenic fungi for soil pest control hasshowed potential, but propagales tolerant to drought, with long shelf life, are still neededfor massproduction. Highly resistant microsclerotia (MS, hereafters) have been discoveredto be able to form in submerged culture, showing potential against soil pests. However, thestate and art for MS production are still not mature and need to be optimized. A seriesoptimization experiments were run to improve culture condition and provide reliableparameters.Nitrogen sort was determined to be significant affecting factors of MS by change ofnutrition conditions of shake culture for Beauveria bassiana, Beuveria brongniarti andMetarhizium anisopliae,3isolates each. Different isolates had different optimal nitrogenresource; absence of Tween through a series single factor tests, a highly productive isolateof each species was chosen: Bb2729for B. bassiana, Bbr17for B. brongniartii, and Ma55for M. anisopliae, with MS production at3.87×103ml-1,6.80×103ml-1å'Œ4.07×103ml-1,respectively.Based on a single factor test, proportion of carbon and nitrogen resources wasrescreened, with a result that MS production and biomass both were affected by carbonconcentration and carbon/nitrogen ratio. On the basis of the single factor test, anotheroptimization was further made by a two-level fractional factor test on non nutritionconditions, with carbon concentration, C/N, rotation speed, amount of conidial suspensioninoculum, and load of liquid medium as5influencing factors and with MS production andbiomass as response to screen significant factors. The result indicated that the significantinfluential factors were all carbon concentration, C/N and inoculum amount. Then asteepest ascent test determined the central point of the response surface: for Bb2729,carbon concentration was at27g/L, C/N ratio at8:1and inoculum amount at0.9%; forBbr17: carbon concentration at30g/L, C/N at7:1and inoculum amount at1.0%, for Ma55:carbon concentration at30g/L, C/N at35:1, and inoculum amount at0.85%. Finally,theoretical maximal productions of the MS for the3isolates were determined by aBox-Benhnken central composite test:8.24×103MS/mL for Bbr17, with carbonconcentration at30.66, inoculum amount at0.84%, and C/N at7.59:1;5.57x103forBb2729, with carbon concentration at27.07, inoculum amount at1.03%, and C/N at7.29:1;4.63×103for Ma55, with carbon concentration at30.29, inoculum amount at0.84%, andC/N at34.66:1. Under the respective theoretical parameters,3batch of verification tests were made for each isolate, resulting in errors of MS production all within1%, proving thecredibility of the results of the response surface test.In a drought tolerance test, the fermentation products were added with diatomite,vacuum-filtrated, freeze-dried, and milled into MS-DE powder. Its revival rate kept above90%at4℃for4months. Comparatively, MS derived in a low carbon and high C/Nrevived up to100%.A short-term storage tests indicated that the temperature is an important factor onstorage of MS. Under4℃, MS-DE revival rate of Bb2729declined slowly and their revialrate kept over90%after60days. Under31℃, the rate of MS-DE tended to significantlydecline significantly, but still kept over87%. Comparatively, MS-DE of Bbr17was moretough for storage, with revival rate up to91.93%after6-days. MS revival was affectedslightly at early stage of formularization, but some protective effect was showed after thatperiod. After long room temperature storage, the revival rate of the unformulated MS-DEdeclined dramatically from the4th month and declined down to40.22%after10months.The rate of formulated MS-DE declined obviously after10months and kept at55.19%after10months. |
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