大豆高隆象致病球孢白僵菌菌株BEdy1的鉴定及毒力测定

doi:10.3864/j.issn.0578-1752.2020.14.020

摘要/Abstract

摘要：

【目的】大豆高隆象（Ergania doriae yunnanus）是我国南方大豆田间主要害虫之一。本研究旨在明确一株高隆象自然致病真菌的种类及其生物学特性,并测定该菌株对大豆高隆象成虫的毒力,为大豆高隆象的生物防治提供新材料。【方法】对采集的真菌进行分离、纯化培养,形态学观察;提取真菌DNA进而扩增rDNA-ITS,利用BLAST比对和系统进化树构建,鉴定田间致高隆象自然死亡的真菌种类;设置不同培养温度,通过十字交叉法和血球计数板方法测量计算真菌生长速率和产孢量;配置不同浓度的球孢白僵菌（Beauveria bassiana）BEdy1孢子悬浮液,采用浸虫处理法测定对大豆高隆象的毒力,与商品化球孢白僵菌产品LH毒力进行对比;大豆叶片喷施BEdy1孢子悬浮液,评价高隆象取食后的致死效果。【结果】致使高隆象田间自然发病死亡的真菌为球孢白僵菌,菌株命名为BEdy1。该菌株在22—28℃有较高的生长速率,26℃下生长速率最高,达4.34 mm·d^-1;在20—26℃有较高的产孢量,22℃下产孢量最大,为4.63×10⁶孢子/mm²。浓度为1.0×10⁵、1.0×10⁶、1.0×10⁷和1.0×10⁸孢子/mL的BEdy1孢子悬浮液处理高隆象成虫10 d,死亡率分别为49.33%、77.33%、88.67%和98.00%,对照死亡率仅为10%。1×10⁸孢子/mL孢子悬浮液处理下,LT₅₀为（6.79±0.13）d,僵虫率为74.67%,发病严重度最高。10 d的LC₅₀和LC₉₅分别为4.80×10⁴和3.57×10⁷孢子/mL。浓度为1×10⁸孢子/mL的商品化球孢白僵菌产品LH处理高隆象成虫,10 d死亡率为58.00%,僵虫率为4.67%,极显著低于同浓度的BEdy1处理。大豆叶片喷施BEdy1孢子悬浮液后喂食高隆象,10 d死亡率为100%,僵虫率为63.33%,LT₅₀为（5.27±0.35）d。【结论】适当环境条件下,球孢白僵菌菌株BEdy1生长速度快,产孢量高,对大豆高隆象成虫有很高的致死率,具有较大的开发应用潜力。

关键词: 大豆高隆象, 球孢白僵菌, 生物学特性, 毒力, 生物防治

Abstract:

【Objective】Ergania doriae yunnanus has become one of the main pests in soybean fields in southern China. The objective of this study is to investigate the species and biological characteristics of an entomopathogenic fungus that naturally caused the death of E. d. yunnanus, determine the virulence of this fungal strain to E. d. yunnanus adults, and to provides a new way for biological control of E. d. yunnanus.【Method】The fungus from E. d. yunnanus was isolated and purified, and the fungal DNA was extracted for amplification of rDNA-ITS. Subsequently, BLAST alignment and phylogenetic tree construction were used to identify fungal species that caused the natural death of E. d. yunnanus. The growth rate and sporulation of the fungus were measured by cross method and hemocytometer method at different culture temperatures. The virulence of fungus BEdy1 to E. d. yunnanus was assessed on the lethality of the pests by treatment with different concentrations of spore suspension and estimated by comparison with the commercial fungal agent LH. Finally, the soybean leaves sprayed with BEdy1 spore suspension were used to evaluate the lethal effect after E. d. yunnanus feeding.【Result】The fungus caused the death of E. d. yunnanus under natural conditions was identified as Beauveria bassiana, named BEdy1. BEdy1 had a high growth rate at 22-28℃, and the highest growth rate was 4.34 mm·d^-1 at 26℃. There was a high sporulation at 20-26℃, and the highest sporulation was 4.63×10⁶ spores/mm²at 22℃. The mortality at 10th day of E. d. yunnanus adults treated with 1.0×10⁵, 1.0×10⁶, 1.0×10⁷ and 1.0×10⁸ spores/mL of B. bassiana BEdy1 spore suspension was 49.33%, 77.33%, 88.67% and 98.00%, respectively. The mortality of the control treatment was only 10%. The effect was best under the treatment of 1×10⁸ spores/mL, with the LT₅₀ of (6.79±0.13) d and the cadaver rate of 74.67%. The LC₅₀ and LC₉₅ at 10th day were 4.80×10⁴ and 3.57×10⁷ spores/mL, respectively. Treatment with 1×10⁸ spores/mL of a commercial B. bassiana agent LH, the mortality and cadaver rate at 10th day were only 58.00% and 4.67%, which were significantly lower than those of the same concentration of BEdy1 treatment. After 10 days of E. d. yunnanus feeding on BEdy1 treated soybean leaves, the mortality and cadaver rate were 100% and 63.33%, respectively, and the LT₅₀ was (5.27±0.35) d. 【Conclusion】Under appropriate conditions, the B. bassiana strain BEdy1 has fast growth rate, high sporulation, and high lethality rate to E. d. yunnanus adults, which has great potential for development and application in the future.

Key words: Ergania doriae yunnanus, Beauveria bassiana, biological characteristics, virulence, biological control

张磊,贾琦,巫蔚,赵路评,薛冰,刘欢欢,尚静,雍太文,李庆,杨文钰. 大豆高隆象致病球孢白僵菌菌株BEdy1的鉴定及毒力测定[J]. 中国农业科学, 2020, 53(14): 2974-2982.

ZHANG Lei,JIA Qi,WU Wei,ZHAO LuPing,XUE Bing,LIU HuanHuan,SHANG Jing,YONG TaiWen,LI Qing,YANG WenYu. Species Identification and Virulence Determination of Beauveria bassiana Strain BEdy1 from Ergania doriae yunnanus[J]. Scientia Agricultura Sinica, 2020, 53(14): 2974-2982.

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参考文献 27

[1]	景河铭. 高隆象生活习性初步观察及其防治. 四川林业科技, 1986,7(4):63-66.
	JING H M. Observation on the living habits of Ergania doriae yunnanus Heller and its prevention. Journal of Sichuan Forestry Science and Technology, 1986,7(4):63-66. (in Chinese)
[2]	RICE M E, PILCHER C D. Imported longhorned weevil,Calomycterus setarius, defoliation and damage to soybean in Iowa. Journal of the Kansas Entomological Society, 1997,70(4):272-280.
[3]	RICE M E. An odd soybean pest: Imported longhorned weevil. Integrated Crop Management News, 2000:2068. https://lib.dr.iastate. edu/cropnews/2068/.
[4]	MUTCHLER A J. A Japanese weevil,Calomycterus setarius Roelofs, which may become a pest in the United Stetes. American Museum Novitates, 1930,418:1-3.
[5]	HUNT T E, HIGLEY L G, HAILE F J. Imported longhorned weevil (Coleoptera: Curculionidae) injury to soybean: Physiological response and injury guild-level economic injury levels. Journal of Economic Entomology, 2003,96(4):1168-1173. pmid: 14503588
[6]	SOSA-GOMEZ D R, CORONEL N, BINNECK E, ZUCCHI M I, ROSADO-NETO G. RAPD and mitochondrial DNA analysis of the soybean stalk weevil, Sternechus subsignatus(Coleoptera: Curculionidae). Bulletin of Entomological Research, 2008,98(5):475-481. doi: 10.1017/S0007485308005798
[7]	AMBROGI B G, ZARBIN P H G. Aggregation pheromone inSternechus subsignatus(Coleoptera: Curculionidae): Olfactory behaviour and temporal pattern of emission. Journal of Applied Entomology, 2008,132(1):54-58. doi: 10.1111/j.1439-0418.2007.01240.x
[8]	LÓPEZ-GUILLÉN G, TERAN-VARGAS A P, RUIZ J G, LARA J S, ROSADO-NETO G H, O’BRIEN C W, CRUZ-LÓPEZ L, RODRÍGUEZ-DEL-BOSQUE L A, ALATORRE-ROSAS R. First record of Rhyssomatus nigerrimus(Curculionidae: Molytinae: Cleogonini) infestations in soybeans in Mexico. Florida Entomologist, 2012,95(2):524-528.
[9]	DU J B, HAN T F, GAI J Y, YONG T W, SUN X, WANG X C, YANG F, LIU J, SHU K, LIU W G, YANG W Y. Maize-soybean strip intercropping: Achieved a balance between high productivity and sustainability. Journal of Integrative Agriculture, 2018,17(4):747-754.
[10]	尚静, 肖任果, 汤忠琴, 常小丽, 杨文钰. 两种防治大豆高隆象药剂防效及其对大豆的氧化胁迫效应. 大豆科学, 2017,36(5):768-773.
	SHANG J, XIAO R G, TANG Z Q, CHANG X L, YANG W Y. The control of two kinds of insecticideson to Ergania doriae yunnannus and their oxidative stress on soybean. Soybean Science, 2017,36(5):768-773. (in Chinese)
[11]	YASIN M, WAKIL W, GHAZANFAR M U, QAYYUM M A, TAHIR M, BEDFORD G O. Virulence of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against red palm weevil, Rhynchophorus ferrugineus(Olivier). Entomological Research, 2019,49(1):3-12. doi: 10.1111/enr.2019.49.issue-1
[12]	MBATA G N, IVEY C, SHAPIRO-ILAN D. The potential for using entomopathogenic nematodes and fungi in the management of the maize weevil,Sitophilus zeamais(Motschulsky)(Coleoptera: Curculionidae). Biological Control, 2018,125:39-43.
[13]	王定锋, 李良德, 李慧玲, 张辉, 王庆森, 吴光远. 一株茶大灰象甲寄生真菌的分子鉴定及其毒力测定. 茶叶科学, 2018,38(3):313-318.
	WANG D F, LI L D, LI H L, ZHANG H, WANG Q S, WU G Y. Molecular identification and virulence evaluation of an entomopathogenic fungus isolated from the cadaver of Sympiezomias citri. Journal of Tea Science, 2018,38(3):313-318. (in Chinese)
[14]	张建华, 查玉平, 陈京元, 王义勋, 蔡三山, 洪承昊, 赵东容. 一株白僵菌菌株的分离鉴定及对紫薇梨象成虫致病力的初步研究. 湖北林业科技, 2016,45(2):37-38, 42.
	ZHANG J H, ZHA Y P, CHEN J Y, WANG Y X, CAI S S, HONG C H, ZHAO D R. Isolation and identification of a strain of Beauveria bassiana and its biological activity to Pseudorobitis gibbus. Hubei Forestry Science and Technology, 2016,45(2):37-38, 42. (in Chinese)
[15]	HATTING J L, MOORE S D, MALAN A P. Microbial control of phytophagous invertebrate pests in South Africa: Current status and future prospects. Journal of Invertebrate Pathology, 2019,165:54-66. pmid: 29427636
[16]	吴进才. 农药诱导害虫再猖獗机制. 应用昆虫学报, 2011,48(4):799-803.
	WU J C. Mechanisms on pesticide-induced resurgence of pests. Chinese Journal of Applied Entomology, 2011,48(4):799-803. (in Chinese)
[17]	雷仲仁, 吴圣勇, 王海鸿. 我国蔬菜害虫生物防治研究进展. 植物保护, 2016,42(1):1-6, 25.
	LEI Z R, WU S Y, WANG H H. Progresses in biological control of vegetable insect pests in China. Plant Protection, 2016,42(1):1-6, 25. (in Chinese)
[18]	REHNER S A, BUCKLEY E. A Beauveria phylogeny inferred from nuclear ITS and EF1-α sequences: Evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia, 2005,97(1):84-98. doi: 10.3852/mycologia.97.1.84 pmid: 16389960
[19]	SEID A M, FREDENSBORG B L, STEINWENDER B M, MEYLING N V. Temperature-dependent germination, growth and co-infection of Beauveria spp. isolates from different climatic regions. Biocontrol Science and Technology, 2019,29(5):411-426. doi: 10.1080/09583157.2018.1564812
[20]	周仙红, 范晓杰, 曹雨, 张思聪, 庄乾营, 于毅. 低温对球孢白僵菌生物学特性及其对韭蛆毒力的影响. 植物保护, 2018,44(2):158-161.
	ZHOU X H, FAN X J, CAO Y, ZHANG S C, ZHUANG Q Y, YU Y. Effects of low temperature on the biological characteristics of Beauveria bassiana and its toxicity against Bradysia odoriphaga. Plant Protection, 2018,44(2):158-161. (in Chinese)
[21]	邝灼彬, 吕利华, 冯夏, 陈焕瑜, 武亚敬, 何余容. 温度及常见农药对球孢白僵菌生物学特性的影响. 华南农业大学学报, 2005,26(3):26-29.
	KUANG Z B, LÜ L H, FENG X, CHEN H Y, WU Y J, HE Y R. Effect of temperature and chemical pesticides on the biological characteristics of Beauveria bassiana. Journal of South China Agricultural University, 2005,26(3):26-29. (in Chinese)
[22]	刘召, 徐丽, 雷仲仁. 温度和光照对白僵菌加拿大1号菌株菌落及产孢量的影响. 西南大学学报 (自然科学版), 2014,36(5):1-6.
	LIU Z, XU L, LEI Z R. The effects of temperature and photoperiod on sporulation and colony development of Beauveria bassiana strain Canada 1. Journal of Southwest University (Natural Science Edition), 2014,36(5):1-6. (in Chinese)
[23]	王定锋, 黎健龙, 王庆森, 李慧玲, 吴光远. 柑橘灰象甲一株高毒力白僵菌菌株的筛选鉴定及培养特性. 中国生物防治学报, 2014,30(6):750-758.
	WANG D F, LI J L, WANG Q S, LI H L, WU G Y. Selection, identification and culture characteristics of a highly virulent strain of Beauveria towards Sympiezomias citri. Chinese Journal of Biological Control, 2014,30(6):750-758. (in Chinese)
[24]	宋晓兵, 彭埃天, 程保平, 凌金锋, 陈霞, 张炼辉. 一株侵染柑橘木虱的球孢白僵菌的分离及鉴定. 植物保护, 2017,43(4):139-144.
	SONG X B, PENG A T, CHENG B P, LING J F, CHEN X, ZHANG L H. Isolation and identification of a Beauveria bassiana strain infecting Diaphorina citri. Plant Protection, 2017,43(4):139-144. (in Chinese)
[25]	RONDOT Y, REINEKE A. Endophytic Beauveria bassiana in grapevine Vitis vinifera(L.) reduces infestation with piercing-sucking insects. Biological Control, 2018,116:82-89.
[26]	MAHMOOD Z, STEENBERG T, MAHMOOD K, LABOURIAU R, KRISTENSEN M. Endophytic Beauveria bassiana in maize affects survival and fecundity of the aphid Sitobion avenae. Biological Control, 2019,137:104017.
[27]	RUSSO M L, PELIZZA S A, VIANNA M F, ALLEGRUCCI N, CABELLO M N, TOLEDO A V, MOURELOS C, SCORSETTI A C. Effect of endophytic entomopathogenic fungi on soybean Glycine max(L.) Merr. growth and yield. Journal of King Saud University - Science, 2019,31(4):728-736.

温度 Temperature (℃)	生长速率 Growth rate (mm·d^-1)	产孢量 Sporulation (×10⁶孢子/mm²)
16	2.09±0.10d	0.93±0.22cd
20	3.32±0.04c	2.95±0.35b
22	4.01±0.14b	4.63±0.63a
25	4.02±0.04b	3.63±0.30b
26	4.34±0.12a	3.55±0.35b
28	4.21±0.04ab	1.47±0.12c
30	2.01±0.02d	0.33±0.09de
35	0e	0e

时间 Time (d)	毒力回归方程 Toxicity regression equation (y=)	LC₅₀(孢子/mL)	LC₉₅(孢子/mL)	相关系数 Correlation coefficient
7	0.1287x-0.5047	6.43×10⁷	2.02×10¹¹	0.9470
8	0.2120x-0.8513	2.37×10⁶	3.14×10⁸	0.9879
9	0.1760x-0.4473	2.41×10⁵	8.70×10⁷	0.9560
10	0.1567x-0.2333	4.80×10⁴	3.57×10⁷	0.9196

时间 Time (d)	死亡率 Mortality (%)	僵虫率 Cadaver rate (%)	LT₅₀(d)
8	38.67±4.81 ***	0 ***	-
9	50.67±6.36 ***	2.00±1.15 ***	-
10	58.00±3.06 ***	4.67±1.76 ***	9.00±0.52 **