茶园扁刺蛾生防菌筛选、鉴定及其发酵条件优化

贺望兴; 谢小群; 杨普香; 李文金; 李延升; 江新凤; 毛平生

doi:10.19303/j.issn.1008-0384.2022.011.012

茶园扁刺蛾生防菌筛选、鉴定及其发酵条件优化

doi: 10.19303/j.issn.1008-0384.2022.011.012

贺望兴^{1, 2,},
谢小群^{1, 2},
杨普香^{1, 2, ,},
李文金^{1, 2},
李延升^{1, 2},
江新凤^{1, 2},
毛平生^{1, 2}

1.
江西省蚕桑茶叶研究所，江西　南昌　330203
2.
江西省经济作物研究所，江西　南昌　330203

基金项目: 江西省重点研发项目（20201BBF61006）；江西省茶叶质量与安全控制重点实验室项目（20192BCD40007）；江西省星火计划项目（20161BBF61011）

详细信息

作者简介:
贺望兴（1989−），男，硕士研究生，助理研究员，研究方向：微生物学（E-mail：545221036@qq.com）

通讯作者:
杨普香（1965−），女，研究员，研究方向：茶学（E-mail：jxypx@163.com）

中图分类号: S 435
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- 文章访问数: 289
- HTML全文浏览量: 128
- PDF下载量: 38
- 被引次数: 0
出版历程
- 收稿日期: 2022-04-12
- 修回日期: 2022-06-17
- 网络出版日期: 2022-12-28
- 刊出日期: 2022-11-28

Identification and Culture Optimization of Effective Biocontrol Agent on Thosea senensis for Tea Plantations

HE Wangxing^{1, 2
,},
XIE Xiaoqun^{1, 2},
YANG Puxiang^{1, 2
, ,},
LI Wenjin^{1, 2},
LI Yansheng^{1, 2},
JIANG Xinfeng^{1, 2},
MAO Pingsheng^{1, 2}

1.
Jiangxi Sericulture and Tea Research Institute, Nanchang, Jiangxi　330203, China
2.
Industrial Crops Institute of Jiangxi, Nanchang, Jiangxi　330203, China

摘要

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。
- 扁刺蛾 /
- 菌株筛选 /
- 拟杆菌 /
- 发酵条件 /
- 正交试验
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 3^rd 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 7^th 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.
- Thosea senensis Walker /
- strain screening /
- bacteroidetes /
- culture conditions /
- orthogonal test

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图 1 生防菌侵染死亡的扁刺蛾幼虫虫体

Figure 1. Dead T. senensis larvae infected by biocontrol bacteria

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图 2 x-11菌在培养基上的菌落形态

Figure 2. Colony morphology of X-11 on culture medium

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图 3 x-11菌株 16S rDNA基因扩增电泳图

1：LB-1；2：空白对照；M：DL10000。

Figure 3. Electrophoresis of amplified X-11 16S rDNA gene

1: LB-1; 2: blank control; M: DL10000.

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图 4 基于16S rDNA 基于序列构建X-11菌株系统发育树

Figure 4. Phylogenetic tree of X-11 based on 16S rDNA sequence

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图 5 接种量对X-11菌株活性的影响

不同的小写字母表示 0.05 水平上差异显著。图6~7同。

Figure 5. Effect of inoculation amount on activity of X-11

Those with different lowercase letters indicate significant difference at 0.05 level. Same for Fig.6-7.

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图 6 发酵时间对X-11菌株活性的影响

Figure 6. Effect of fermentation time on activity of X-11

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图 7 温度对X-11菌株活性的影响

Figure 7. Effect of temperature on activity of X-11

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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/h
1	1.5	36	55
2	2	39	60
3	2.5	42	65

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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/d
菌株 Strain	3 d	5 d	7 d	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/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.

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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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