1株抗番茄灰霉病的生物有机肥菌种的筛选、发酵条件优化及其应用潜力

Screening,fermentation conditions and application potential of a bio-organic fertilizer strain against tomato gray mold

为了筛选有效的番茄灰霉病拮抗菌,研究其固体发酵条件,制备生物有机肥并研究其对番茄灰霉病的生防效果。采用稀释涂平板及平板划线法分离,平板对峙法筛选,对所筛选的菌株进行菌株形态鉴定和ITS区序列解析鉴定;通过单因素和正交试验探索了其发酵条件对该菌产孢量的影响;将拮抗菌与已腐熟有机肥发酵后应用于盆栽试验。经分离、初筛和复筛,最终得到1株编号为Z47的菌株,该菌株对番茄灰霉病病原菌的抑制效果最好,抑制率高达74.80%,鉴定为草酸青霉(Penicillium oxalicum);该菌最优发酵条件为固料瓶装量15g/250-mL三角瓶,稻壳粉:麸皮按8∶2混合,外加麦芽糖、硫酸铵和磷酸氢二钾的质量分数分别为1.5%,2%,0.15%,料水比为1∶0.6(g/mL),接种量为12.5%(mL/g),全黑暗培养,25—20—28℃下依次发酵2d,持续发酵12d;盆栽试验表明,施用富含拮抗菌草酸青霉菌株Z47的生物有机肥对番茄灰霉病的防治效果达到73.67%,显著高于拮抗菌Z47菌液处理。筛选所得的草酸青霉菌株通过优化固体发酵条件后,可获得高达5×109 CFU/g的产孢量,该菌发酵的生物有机肥对防治番茄灰霉病有较好的生物防治潜力。

Summary Tomato gray mold is a common disease which is caused by Botrytis cinerea and does harm to tomato seriously.Chemical control of tomato gray mold is easy to produce negative effects,such as drug resistance, pesticide residues and ecological imbalance.Biological control has advantages of high efficiency,no pollution,no drug resistance and combining prevention with treatment.Bio-organic fertilizer has both superiorities of bio-fertilizer and organic fertilizer,which can not only increase production,improve product quality,but also regulate and improve the composition of soil microorganisms,and then reduce the danger of crop diseases to some extent. Screening effective antagonistic strains of tomato gray mold and then preparing antagonistic agent and bio-organic fertilizer are important biotechnology ways to prevent and control tomato gray mold. The studies on solid fermentation of antagonistic strains are less involved at home and abroad. This study aimed at screening an effective antagonistic strain of tomato gray mold,exploring the solid fermentation conditions,preparing biological organic fertilizer and studying its bio-control effect on tomato gray mold. The strains of soil samples were isolated by the method of dilution coated tablet andstreak plate,and then screened by tablet confrontation.Furthermore,they were identified by morphological and ITS sequence analysis.The influence factors on the conidial concentration of this strain were investigated by single factor and orthogonal experiments,including solid medium,external carbon,external nitrogen,external phosphate,the ratio of solid material to water,amount of bottled solid material,inoculums percentage,illumination,temperature and time of fermentation.The antagonistic strain was added to the rotten organic fertilizer to ferment,and then it was applied to pot experiment. After isolating,primary screening and rescreening,a strain of number Z47 was obtained eventually,which had the best inhibition effect and its inhibition rate reached 74.80% on B.cinerea .The strain was identified as Penicillium oxalicum .Fermentation condition optimization results for the P.oxalicum Z47 were as follows.The orthogonal optimization results for powdered rice hulls ∶ bran,maltose,ammonium sulfate,and dipotassium hydrogen phosphate in medium formula were 8 ∶ 2, 1.5%,2% and 0.1 5%,respectively.The orthogonal optimization results for the ratio of solid material to water,amount of bottled solid material,inoculums percentage, illumination conditions and fermentation temperature in medium formula were 1∶0.6 （g/mL）,1 5 g,12.5%（mL/g）,dark totally and fermentation at a fluctuant temperature of 25—20—28 ℃ for 2 days,respectively.P. oxalicum Z47 had the highest spore number when cultured for 12 days under the optimal solid medium and culture conditions.The result of the pot experiment showed that the control effect of tomato gray mold by applying biological organic fertilizer which was rich in antagonistic strain Z47 was up to 73.67%,which was significantly higher than the treatment of antagonistic strain Z47 liquid. In sum,under the optimization of solid fermentation,the spore numbers of P.oxalicum Z47 were up to 5 ＆#215; 10 9 CFU/g.The fermentation of biological organic fertilizer has good potential for prevention of tomato gray mold.