Identification and Culture Optimization of Effective Biocontrol Agent on Thosea senensis for Tea Plantations
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摘要:
目的 筛选适宜生态有机茶园防治扁刺蛾的生防菌。 方法 采用温度筛选法从自然罹病死亡的扁刺蛾肠道体内初筛生防菌,浸液饲喂法测定生防菌对扁刺蛾幼虫的室内毒力;综合菌株的形态特征、分子生物学鉴定以及构建的系统发育树分析进行菌株的鉴定;通过单因素和正交试验对生防菌的发酵参数条件进行优化。 结果 筛选到1株对扁刺蛾幼虫具有较高防效的内生生防菌X-11,扁刺蛾3龄幼虫取食经2.56×108个·mL−1 X-11菌液处理的茶枝条7 d后扁刺蛾累计校正死亡率达到90.5%,致死中时(LT50)仅为2.88 d。菌株X-11经16s rDNA分子生物学鉴定以及构建的系统发育树分析确定为偶氮还原拟杆菌(Paenibacillus azoreducens)。发酵条件优化试验表明,菌株X-11的适宜发酵条件为:菌株接种量2%,在发酵温度36℃条件下发酵培养60 h,菌株含量最高达1.34×109个·mL−1。 结论 筛选到1株对扁刺蛾幼虫具有较高防效的内生生防菌x-11,可作为防治扁刺蛾幼虫的生物防治菌剂使用。 Abstract:Objective A biocontrol microbe on Thosea senensis Walker suitable for organic tea agriculture was identified and optimal culture conditions studied. Method From the intestines of T. senensis died from disease in nature, microbes were cultured and screened by means of varying the temperature. Indoor virulence of the selected microbes on T. senensis was determined by immersion feeding and further identified by morphological, molecular biological, and phylogenetic tree analyses. Culture of the isolated strain was optimized by univariate and orthogonal experiments. Result The endophytic strain X-11 with high efficacy in controlling the pest was isolated. The 3rd instar larvae of T. senensis fed with a 2.56×108 conidia·mL−1 X-11 solution displayed a cumulative corrected mortality rate of 90.5% with a median lethal time LT50 of 2.88 d on the 7th day after treatment. Based on the 16S rDNA molecular identification and phylogenetic analysis, X-11 was determined to be Paenibacillus azoreducens. The optimized culture conditions for the bacterium included the use of 2% inoculum to culture for 60 h at 36℃ with a final count approaching 1.34×109 conidia·mL−1. Conclusion The endophytic strain with high control efficacy on T. senensis larvae was identified as P. azoreducens. It could be cultured to be used as a bioagent for the pest control at tea plantations. -
Key words:
- Thosea senensis Walker /
- strain screening /
- bacteroidetes /
- culture conditions /
- orthogonal test
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表 1 正交试验优化因素水平
Table 1. Factors and levels of orthogonal optimization test
水平
Level接种量A
Inoculation
amount A/%发酵温度B
Fermentation
temperature B/℃发酵时间C
Fermentation
time C/h1 1.5 36 55 2 2 39 60 3 2.5 42 65 表 2 生防菌毒力测定
Table 2. Determination of virulence of biocontrol bacteria
菌株
Strain死亡率 Mortality /% 累计死亡率
Cumulative mortality /%累计校正死亡率
Cumulative adjusted mortality /%致死中时LT50
Lethal time
LT50/d3 d 5 d 7 d X-2 58.5±2.36 a 78.3±8.49 a 88.3±8.49 ab 88.3±8.49 ab 86.8±8.49 ab 2.86±0.23 a X-7 25.0±4.08 b 36.7±2.36 b 48.3±6.24 b 48.3±6.24 b 41.5±6.24 b — X-9 26.7±6.24 b 53.3±8.49 ab 76.7±6.24 ab 76.7±6.24 ab 73.6±6.24 ab 4.74±0.34 b X-11 58.3±8.49 a 73.3±10.27 a 91.6±2.36 a 91.6±2.36 a 90.5±2.36 a 2.88±0.32 a X-12 28.3±6.24 b 60.0±7.07 ab 66.7±6.24 b 66.7±6.24 b 62.3±6.24 b 4.56±0.48 b Bt 53.3±6.24 a 66.7±4.71 a 86.7±8.49 ab 86.7±8.49 ab 84.9±8.49 ab 2.93±0.32 a CK 5.0±4.08 c 6.67±2.36 c 11.6±2.36 c 11.6±2.36 c — — 表中数据为平均值±标准差;同列数据后不同的小写字母表示 0.05 水平上差异显著。
Datas are mean±SD; those with different lowercase letters on same column indicate significant difference at 0.05 level.表 3 X-11菌发酵条件优化正交试验
Table 3. Orthogonal test for X-11 culture optimization
序号
Serial接种量
Inoculation amount发酵温度
Fermentation temperature发酵时间
Fermentation time菌株生物量
Strain biomass/(mg·L−1)1 3 3 1 322.9 2 1 2 3 390.5 3 3 1 3 455.4 4 1 3 2 357.9 5 2 3 3 339.2 6 3 2 2 410.6 7 2 2 1 357.9 8 2 1 2 534.9 9 1 1 1 429.8 K1 392.7 473.4 370.2 K2 410.7 386.4 434.6 K3 396.4 340 395.1 R 14.3 133.4 39.5 -
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