布氏白僵菌菌株JG-17的鉴定及对3种金龟子的毒力测定

doi:10.3864/j.issn.0578-1752.2024.12.008

中国农业科学 ›› 2024, Vol. 57 ›› Issue (12): 2364-2377.doi: 10.3864/j.issn.0578-1752.2024.12.008

布氏白僵菌菌株JG-17的鉴定及对3种金龟子的毒力测定

曹伟平¹(), 丰硕¹, 程佳旭¹, 陈丹¹, 吴青君², 宋健¹()

¹ 河北省农林科学院植物保护研究所/农业农村部华北北部作物有害生物综合治理重点实验室/河北省农业有害生物综合防治技术创新中心/河北省作物有害生物综合防治国际科技联合研究中心，河北保定 071000
² 中国农业科学院蔬菜花卉研究所/蔬菜生物育种全国重点实验室，北京 100081

收稿日期:2024-01-08 接受日期:2024-02-29 出版日期:2024-06-25 发布日期:2024-06-25
通信作者:
宋健，E-mail：sj3250@163.com
联系方式: 曹伟平，E-mail：cwplx751209@163.com。
基金资助:
国家特色蔬菜产业技术体系(CARS-24-C-02); 河北省农林科学院科技创新专项课题(2022KJCXZX-ZBS-6); 河北省重点研发计划(21326505D)

Species Identification of Beauveria brongniartii Strain JG-17 and Virulence Against Three Scarabaeoidae Pests

CAO WeiPing¹(), FENG Shuo¹, CHENG JiaXu¹, CHEN Dan¹, WU QingJun², SONG Jian¹()

¹ Plant Protection Institute, Hebei Academy of Agriculture and Forestry Sciences/Key Laboratory of Integrated Pest Management on Crops in Northern Region of North China, Ministry of Agriculture and Rural Affairs/IPM Innovation Center of Hebei Province/ International Science and Technology Joint Research Center on IPM of Hebei Province, Baoding 071000, Hebei
² Institute of Vegetable and Flowers, Chinese Academy of Agricultural Sciences/State Key Laboratory of Vegetable Biobreeding, Beijing 100081

Received:2024-01-08 Accepted:2024-02-29 Published:2024-06-25 Online:2024-06-25

摘要/Abstract

摘要：

【目的】基于金龟子类害虫种类繁多、危害严重的现状以及农业对具有生态友好、可持续治理特点的生物防治的迫切需求，研究布氏白僵菌（Beauveria brongniartii）菌株JG-17对我国3种重要金龟子幼虫和成虫的毒力，为金龟子生防制剂的研发提供优良的菌种资源。【方法】采用稀释挑取法和病原菌回接法，在感病金龟子成虫虫尸上分离获得高毒力病原真菌；通过形态学与rDNA-ITS基因序列分析确定菌株JG-17的分类地位；采用十字交叉测量法和血球计数板法测定不同温度下菌株JG-17的生长速率和产孢量；采用浸渍法测定菌株JG-17对华北大黑鳃金龟（Holotrichia oblita）、暗黑鳃金龟（Holotrichia parallela）和铜绿丽金龟（Anomala corpulenta）成虫和幼虫的毒力；通过盆栽试验测定菌株JG-17对暗黑鳃金龟2龄幼虫的防治效果。【结果】分离获得一株对3种金龟子成虫和幼虫同时具有较高毒力的白僵菌菌株JG-17，结合形态学和rDNA分子鉴定结果，确定菌株JG-17为布氏白僵菌。25—29 ℃为JG-17的适宜生长温度，生长速率均在4 mm·d^-1以上，27 ℃下，JG-17的生长速率和产孢量最高。JG-17 1×10⁸个孢子/mL处理浓度下，华北大黑鳃金龟、铜绿丽金龟和暗黑鳃金龟成虫13 d累计死亡率分别为78.0%、100.0%和86.0%，LT₅₀分别为9.458、6.911和8.391 d。相同处理浓度下，JG-17对暗黑鳃金龟幼虫的毒力高于铜绿丽金龟和华北大黑鳃金龟，1×10⁸个孢子/mL浓度下，华北大黑鳃金龟、铜绿丽金龟和暗黑鳃金龟1龄幼虫13 d的累计死亡率分别为76.7%、96.7%和100.0%，2龄幼虫13 d的累计死亡率分别为66.7%、36.7%和100.0%；不同孢子浓度下，对暗黑鳃金龟1龄和2龄幼虫的LT₅₀均明显低于铜绿丽金龟和华北大黑鳃金龟。盆栽试验中，相同施用剂量下，中耕期淋灌处理的暗黑鳃金龟幼虫死亡率与沟施处理的幼虫死亡率差异不显著，播种前撒施方式下取得相近的防治效果需要10倍以上的孢子量。【结论】布氏白僵菌JG-17对华北大黑鳃金龟、暗黑鳃金龟和铜绿丽金龟成虫和幼虫均具有较高致死率，对防治金龟子类地下害虫具有较大的开发应用潜力。

关键词: 布氏白僵菌, 金龟子, 幼虫, 成虫, 分离鉴定, 毒力

Abstract:

【Objective】 Based on the current situation of the wide variety and serious damage of scarabs, and the urgent demand of agriculture for biological control with eco-friendly and sustainable management, the virulence of Beauveria brongniartii strain JG-17 to the larvae and adults of three important scarabs in China was studied in order to provide excellent microbial resources for the research and development of scarabs biological control agents.【Method】A high virulence fungus strain was isolated from the infected adult scarab cadavers by spore dilution method and re-inoculation experiment. The taxonomic status of strain JG-17 was determined by morphology and rDNA-ITS gene sequence analysis. The growth rate and sporulation of strain JG-17 at different culture temperatures were measured by cross method and hemocytometer method. The virulence of strain JG-17 to the adults and larvae of Holotrichia oblita, Holotrichia parallela and Anomala corpulenta was determined by immersion method and the control efficacy against the 2nd instar larvae of H. parallela was determined by peanut pot experiment.【Result】A strain of Beauveria JG-17, which caused high virulence against adults and larvae of three scarabs, was isolated from the naturally infected scarabs adult. Strain JG-17 was identified as B. brongniartii combined with the morphological and molecular identification results. 25-29 ℃ is the optimal growth temperature for strain JG-17, and the growth rate is greater than 4 mm·d^-1. The growth rate and sporulation of strain JG-17 are the highest at 27 ℃. The cumulative mortality at 13 d of the adults of H. oblita, A. corpulenta and H. parallela treated with 1×10⁸ spores/mL of strain JG-17 spore suspension was 78.0%, 100.0% and 86.0% with the LT₅₀ values of 9.458, 6.911 and 8.391 d, respectively. At the same treatment concentration, the virulence of JG-17 to the larvae of H. parallela was higher than that of A. corpulenta and H. oblita. At the 1×10⁸ spores/mL of JG-17 concentration treatment, the cumulative mortality at 13 d of the 1st instar larvae of H. oblita, A. corpulenta and H. parallela was 76.7%, 96.7% and 100.0%, respectively, and that of the 2nd instar larvae was 66.7%, 36.7% and 100.0%, respectively. Under different spore concentrations of JG-17 treatment, the LT₅₀ of JG-17 against the 1st and 2nd instar larvae of H. parallela was significantly lower than that of A. corpulenta and H. oblita. In the pot experiment, there was no significant difference in larvae mortality between spraying evenly around the root and the furrow application with same spore dosage during the inter-tillage period, and more than 10 times of spore were needed to achieve similar control efficacy with spreading application before sowing.【Conclusion】B. brongniartii JG-17 has a high lethality rate to the adults and larvae of H. oblita, H. parallela and A. corpulenta. It is a biocontrol microbial resource with great potential for development and application to control underground scarab pests.

Key words: Beauveria brongniartii, scarab beetle, larva, adult, isolation and identification, virulence

曹伟平, 丰硕, 程佳旭, 陈丹, 吴青君, 宋健. 布氏白僵菌菌株JG-17的鉴定及对3种金龟子的毒力测定[J]. 中国农业科学, 2024, 57(12): 2364-2377.

CAO WeiPing, FENG Shuo, CHENG JiaXu, CHEN Dan, WU QingJun, SONG Jian. Species Identification of Beauveria brongniartii Strain JG-17 and Virulence Against Three Scarabaeoidae Pests[J]. Scientia Agricultura Sinica, 2024, 57(12): 2364-2377.

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菌株 Isolate	采集地 Collecting site	菌落形态 Colony texture	菌落颜色 Colony color	生长速率 Growth rate (mm·d^-1)	产孢量 Sporulation (×10⁶spores/cm²)	侵染率 Infection rate (%)
JG-4	河北保定Baoding, Hebei	毡状Felt-like	白色White	3.65±0.16d	4.83±0.93c	32.0±4.5cd
JG-5	河北博野Boye, Hebei	绒状Villous-like	白色White	5.39±0.15a	7.65±0.76a	18.0±8.4e
JG-9	河北沧州Cangzhou, Hebei	绒状Villous-like	黄色Yellow	2.64±0.13f	3.24±0.50d	20.0±7.1e
JG-12	河北高阳Gaoyang, Hebei	毡状Felt-like	淡黄色Light yellow	4.07±0.04c	4.98±0.56c	26.0±5.5de
JG-17	河北清苑Qingyuan, Hebei	绒状Villous-like	乳白略黄Yellowish white	4.23±0.07b	7.29±0.65a	92.0±8.4a
JG-18	河北满城Mancheng, Hebei	绒状Villous-like	淡黄色Light yellow	3.31±0.04e	3.43±0.60d	46.0±8.9b
JG-21	河北乐亭Laoting, Hebei	绒状Villous-like	乳白色Cream white	4.02±0.10c	6.31±0.48b	40.0±10.0bc

温度 Temperature (℃)	生长速率 Growth rate (mm·d^-1)	产孢量Sporulation (×10⁶ spores/cm²)
18	1.64±0.05e	2.41±0.34f
21	2.74±0.05c	3.50±0.71e
23	3.70±0.12b	5.17±0.68d
25	4.12±0.13a	6.33±0.49bc
27	4.23±0.07a	7.66±0.73a
29	4.16±0.11a	6.96±0.63ab
31	2.28±0.08d	5.78±0.63cd
33	1.38±0.08f	2.90±0.84ef
35	0g	0g

金龟子种类 Scarab species	僵虫率Cadaver rate (%)
金龟子种类 Scarab species	1×10⁸ spores/mL	5×10⁷spores/mL	2×10⁷ spores/mL	5×10⁶ spores/mL	2×10⁶ spores/mL
华北大黑鳃金龟H. oblita	72.0±4.5c	56.0±5.5c	48.0±8.4c	28.0±8.4c	12.0±4.5c
铜绿丽金龟A. corpulenta	94.0±5.5a	86.0±5.5a	78.0±4.5a	66.0±5.5a	58.0±4.5a
暗黑鳃金龟H. parallela	82.0±4.5b	70.0±7.1b	62.0±4.5b	52.0±4.5b	30.0±7.1b

金龟子种类 Scarab species	孢子浓度 Spore concentration (spores/mL)	回归方程 Regression equation (y=)	LT₅₀(d)，95%置信限 Confidence limit	χ²	P
华北大黑鳃金龟 H. oblita	1×10⁸	-2.666+0.282x	9.458 (8.834-10.107)	6.075	1.000
华北大黑鳃金龟 H. oblita	5×10⁷	-2.751+0.249x	11.033 (10.311-11.968)	5.790	1.000
铜绿丽金龟 A. corpulenta	1×10⁸	-2.783+0.403x	6.911 (6.330-7.410)	6.732	1.000
铜绿丽金龟 A. corpulenta	5×10⁷	-2.431+0.314x	7.846 (7.192-8.426)	11.861	0.973
暗黑鳃金龟 H. parallela	1×10⁸	-2.066+0.246x	8.391 (7.638-9.084)	6.034	1.000
暗黑鳃金龟 H. parallela	5×10⁷	-2.241+0.227x	9.875 (9.134-10.715)	7.490	0.999

金龟子种类 Scarab species	龄期 Stage	累计死亡率Cumulative mortality (%)
金龟子种类 Scarab species	龄期 Stage	1×10⁸ spores/mL	5×10⁷ spores/mL	2×10⁷ spores/mL	5×10⁶ spores/mL	2×10⁶ spores/mL	CK
华北大黑鳃金龟 H. oblita	1龄1st instar	76.7±11.5	63.3±5.8	50.0±10.0	23.3±15.3	6.7±5.8	0
华北大黑鳃金龟 H. oblita	2龄2nd instar	66.7±5.8	53.3±5.8	33.3±6.7	13.3±3.3	3.3±5.8	0
铜绿丽金龟 A. corpulenta	1龄1st instar	96.7±5.8	90.0±10.0	76.7±5.8	40.0±10.0	26.7±5.8	6.7±5.8
铜绿丽金龟 A. corpulenta	2龄2nd instar	36.7±5.8	23.3±5.8	13.3±5.8	6.7±5.8	0	0
暗黑鳃金龟 H. parallela	1龄1st instar	100.0±0	100.0±0	96.7±5.8	86.7±5.8	60.0±0	0
暗黑鳃金龟 H. parallela	2龄2nd instar	100.0±0	93.3±5.8	76.7±5.8	53.3±5.8	36.7±5.8	0

布氏白僵菌菌株JG-17的鉴定及对3种金龟子的毒力测定

Species Identification of Beauveria brongniartii Strain JG-17 and Virulence Against Three Scarabaeoidae Pests

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处理Treatment	死亡率Mortality (%)	僵虫率Cadaver rate (%)
撒施Spreading application (500×10⁸spores/pot)	50.00±0e	41.67±7.22c
撒施Spreading application (1000×10⁸spores/pot)	66.67±7.22cd	58.33±7.22ab
撒施Spreading application (2000×10⁸spores/pot)	83.33±7.22ab	70.83±7.22a
淋灌Spraying around peanut root (20×10⁸spores/pot)	45.83±7.22e	37.50±12.50c
淋灌Spraying around peanut root (40×10⁸spores/pot)	58.33±7.22de	50.00±0bc
淋灌Spraying around peanut root (80×10⁸spores/pot)	70.83±7.22bcd	66.67±7.22a
沟施Furrow application (40×10⁸spores/pot)	50.00±0e	41.67±7.22c
沟施Furrow application (80×10⁸spores/pot)	66.67±7.22cd	62.50±0ab
沟施Furrow application (160×10⁸spores/pot)	75.00±12.50abc	70.83±7.22a
45%毒死蜱Chlorpyrifos (2000×)	87.50±12.50a	0d
CK	8.33±7.22f	0d

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