不同温度下两株球孢白僵菌侵染西花蓟马的生长动力学及其毒力

doi:10.3864/j.issn.0578-1752.2018.08.006

摘要/Abstract

摘要： 【目的】通过比较两株球孢白僵菌（Beauveria bassiana）在不同温度条件下侵染西花蓟马（Frankliniella occidentalis）的生长动力学及其毒力，探讨球孢白僵菌菌体增殖和杀虫毒力的关系，为提高球孢白僵菌对西花蓟马的杀虫效率提供理论支持。【方法】首先，在20、25和30℃ 3个温度条件下，连续记录第1—8天西花蓟马被两株球孢白僵菌菌株（SCWJ-2和GZGY-1-3）侵染后的死亡率，并以未被真菌感染的西花蓟马为对照，计算其累积校正死亡率，选择第3天的数据（对照死亡率为2%—5%）比较两个菌株的杀虫效率。其次，在20、25和30℃ 3个温度条件下，连续记录第1—8天内平板培养的上述两个菌株的菌落直径，选取第3天两个菌株的菌落生长直径数据进行比较分析。最后，提取20、25和30℃温度下，西花蓟马分别被两株球孢白僵菌侵染第1、2和3天的混合DNA，通过实时荧光定量PCR（qRT-PCR）技术定量球孢白僵菌在每个宿主样品体内的拷贝数，选取第3天两个菌株的基因拷贝数进行比较分析。【结果】生物测定结果显示，在检测温度（20—30℃）范围内，球孢白僵菌菌株GZGY-1-3和SCWJ-2对西花蓟马成虫均具有较高致病性，无论任何温度和何种菌株，西花蓟马从处理后第2天开始有死亡个体出现，第8天时，SCWJ-2和GZGY-1-3造成的校正死亡率分别为83%—91%和79%—90%。以第3天的校正死亡率为指标（对照死亡率为2%—5%），30℃下菌株SCWJ-2毒力显著高于GZGY-1-3（P＜0.05），25℃和20℃下两个菌株毒力无显著差异（P＞0.05）。平板培养试验表明，在检测温度（20—30℃）范围内，两个菌株菌落直径随着时间推移而增加，第8天时，菌株SCWJ-2和GZGY-1-3的菌落直径分别为31—36、28—32 mm。选取第3天菌落生长直径数据进行比较，菌株SCWJ-2在3个温度下的菌落直径均显著大于菌株GZGY-1-3（P＜0.05）。实时荧光定量结果显示，除30℃下的菌株SCWJ-2之外，两个菌株在西花蓟马体内的基因拷贝数都在第1天时下降，2 d后又逐渐上升。使用第3天西花蓟马体内的基因拷贝数进行分析，在30℃下菌株SCWJ-2真菌拷贝数显著大于GZGY-1-3（P＜0.05），25℃和20℃下两个菌株基因拷贝数无显著差异（P＞0.05）。【结论】真菌在被侵染虫体内的基因拷贝数受到菌株和温度的影响，这与生物测定结果相一致。与菌株GZGY-1-3相比，SCWJ-2更适合高温条件下对西花蓟马的防治。

关键词: 球孢白僵菌, 西花蓟马, 真菌生长动力学, 实时荧光定量PCR

Abstract: 【Objective】The objective of this study is to compare the growth kinetics and virulence of two strains of Beauveria bassiana infected Western flower thrips (Frankliniella occidentalis) under different temperatures, analyze the relationship between the proliferation and insecticidal virulence of B. bassiana, and to provide a theoretical support for improving the efficiency of B. bassiana against F. occidentalis.【Method】Firstly, from 1st to 8th day, the mortality of the infected F. occidentalis with two strains of B. bassiana (SCWJ-2 and GZGY-1-3) under 20, 25, and 30℃ was recorded, and cumulative corrected mortality was calculated while uninfected F. occidentalis was used as control. The mortality of F. occidentalis infected with the two strains at the 3rd day (the mortality of control was 2%-5%) was compared. Secondly, under 20, 25 and 30℃, the colony diameter of the two strains was recorded continuously from 1st to 8th day. The colony diameter of the two strains at the 3rd day was compared. Finally, the DNA mixture of F. occidentalis infected with fungi from 1st to 3rd day under 20, 25 and 30℃ was extracted, respectively. The real-time fluorescence quantitative PCR (qRT-PCR) was used to quantify the copy number of fungi within each insect host sample. The gene copy of two strains under the same temperature at the 3rd day was compared. 【Result】The bioassay results of B. bassiana against F. occidentalis showed that within the tested temperature range (20-30℃), both strains GZGY-1-3 and SCWJ-2 were highly lethal to F. occidentalis adults. No matter what the temperature or the strain, the dead individuals F. occidentalis appeared from the 2nd day after treatment. On the 8th day, the corrected mortality of F. occidentalis infected by strain SCWJ-2 and GZGY- 1-3 was 83%-91% and 79%-90%, respectively. The corrected mortality of F. occidentalis infected by the two strains on the 3rd day was compared (the morality of control were 2%-5%). the virulence of strain SCWJ-2 was significantly higher than that of GZGY-1-3 under 30℃ (P＜0.05), but there was no significant difference between them under 25 and 20℃ (P＞0.05). Within the tested temperature range from 20 to 30℃, the colony diameter of two strains increased with time. On the 8th day, the colony diameter of the strains SCWJ-2 and GZGY-1-3 was 31-36 and 28-32 mm, respectively. On the 3rd day, the colony diameter of strain SCWJ-2 was significantly larger than that of strain GZGY-1-3 under all test temperatures (P＜0.05). The results of qRT-PCR showed that, with the exception of strain SCWJ-2 at 30℃, the gene copy number of both strains in F. occidentalis decreased on the 1st day and gradually increased after 2 days at all the three temperatures. The gene copy number of strain SCWJ-2 within F. occidentalis was significantly higher than that of strain GZGY-1-3 under 30℃ (P＜0.05), but there was no significant difference between them under 25 and 20℃ (P＞0.05). 【Conclusion】 The number of fungal gene copies within insect host was affected by strains and temperatures, which is in accordance with the result of bioassay. Compared with B. bassiana strain GZGY-1-3, strain SCWJ-2 is more suitable for controlling F. occidentalis under high temperatures.

Key words: Beauveria bassiana, Frankliniella occidentalis, fungal growth kinetics, real-time fluorescence quantitative PCR (qRT-PCR)

刘晓晨，吴圣勇，雷仲仁，王海鸿. 不同温度下两株球孢白僵菌侵染西花蓟马的生长动力学及其毒力[J]. 中国农业科学, 2018, 51(8): 1484-1492.

LIU XiaoChen, WU ShengYong, LEI ZhongRen, WANG HaiHong. Growth Kinetics and Virulence of Two Beauveria bassiana Strains in Frankliniella occidentalis Under Different Temperatures[J]. Scientia Agricultura Sinica, 2018, 51(8): 1484-1492.

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参考文献

[1] REITZ S R. Biology and ecology of the western flower thrips (Thysanoptera: Thripidae): The making of a pest. Florida Entomologist, 2009, 92(1): 7-13.

[2] REITZ S R, Gao Y L, LEI Z R. Thrips: pests of concern to China and the United States. Journal of Integrative Agriculture, 2011, 10(6): 867-892.

[3] 雷仲仁, 问锦曾, 王音. 危险性外来入侵害虫——西花蓟马的鉴别、危害及防治. 植物保护, 2004, 30(3): 63-66.

LEI Z R, WEN J Z, WANG Y. The identification, damage and control of an invasive western flower thrips (Frankliniella occidentalis). Plant protection, 2004, 30(3): 63-66. (in Chinese)

[4] Ullman D E, Meideros R, Campbell L R, Whitfield A E, Sherwood J L, German T L. Thrips as vectors of tospoviruses. Advances in Botanical Research, 2002, 36: 113-140.

[5] 张友军, 吴青君, 徐宝云, 朱国仁. 危险性外来入侵生物——西花蓟马在北京发生危害. 植物保护, 2003, 29(4): 58-59.

ZHANG Y J, WU Q J, XU B Y, ZHU G R. Dangerous invasive alien species - Western flower thrips in Beijing. Plant protection, 2003, 29(4): 58-59. (in Chinese)

[6] 王海鸿, 雷仲仁, 李雪, 代安国, 陈翰秋. 西藏发现重要外来入侵害虫——西花蓟马. 植物保护, 2013, 39(1): 181-183.

WANG H H, LEI Z R, LI X, DAI A G, CHEN H Q. An important invasive pest, Frankliniella occidentalis, inspected in Tibet. Plant protection, 2013, 39(1): 181-183. (in Chinese)

[7] 张慧, 吴圣勇, 李娟, 张璐璐, 张林雅, 雷仲仁. 不同培养基继代培养球孢白僵菌对西花蓟马毒力和产孢量的影响. 中国农业科学, 2016, 49(15): 2977-2987.

ZHANG H, WU S Y, LI J, ZHANG L L, ZHANG L Y, LEI Z R. Influence of subculture on virulence to Frankliniella occidentalis and conidial production of the entomopathogenic fungus Beauveria bassiana. Scientia Agricultura Sinica, 2016, 49(15): 2977-2987. (in Chinese)

[8] LEE S J, KIM S, KIM J C, LEE M R, HOSSAIN M S, SHIN T S, KIM T H, KIM J S. Entomopathogenic Beauveria bassiana granules to control soil-dwelling stage of western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae). Biocontrol, 2017, 62(5): 639-648.

[9] WU S Y, GAO Y L, ZHANG Y P, WANG E D, XU X N, LEI Z R. An entomopathogenic strain of Beauveria bassiana against Frankliniella occidentalis with no detrimental effect on the predatory mite Neoseiulus barkeri: Evidence from laboratory bioassay and scanning electron microscopic observation. PLoS One, 2014, 9(1): e84732.

[10] 李银平, 雷仲仁, 王海鸿. 对西花蓟马高效的球孢白僵菌菌株筛选及产孢特性研究. 中国生物防治学报, 2013, 29(2): 219-226.

LI Y P, LEI Z R, WANG H H. Selection of Beauveria bassiana strains against Frankliniella occidentalis and their conidial production characteristics. Chinese Journal of Biological Control, 2013, 29(2): 219-226. (in Chinese)

[11] 袁盛勇, 张宏瑞, 孔琼, 李正跃, 骆弟乾, 王学丽, 董丽玲. 球孢白僵菌MZ060812菌株对西花蓟马的致病性研究. 西北农林科技大学(自然科学版), 2010, 38(3): 145-149.

YUAN S Y, ZHANG H R, KONG Q, LI Z Y, LUO D Q, WANG X L, DONG L L. Detection of pathogenicity of Beauveryia bassiana MZ060812 against Frankliniella occidentalis. Journal of Northwest A & F University (Natural Science Edition), 2010, 38(3): 145-149. (in Chinese)

[12] ZHANG T, REITZ S R, WANG H H, LEI Z R. Sublethal effects of Beauveria bassiana (Ascomycota: Hypocreales) on life table parameters of Frankliniella occidentalis (Thysanoptera: Thripidae). Journal of Economic Entomology, 2015, 108(3): 975-985.

[13] 张烨, 雷仲仁, 王海鸿, 吉青战. 球孢白僵菌HsbA蛋白的原核表达及免疫定位. 中国农业科学, 2013, 46(21): 4534-4541.

ZHANG Y, LEI Z R, WANG H H, JI Q Z. Prokaryocyte expression and immune localization of HsbA in Beauveria bassiana. Scientia Agricultura Sinica, 2013, 46(21): 4534-4541. (in Chinese)

[14] HAJEK A E, LEGET R J S. Interactions between fungal pathogens and insect hosts. Annual Review of Entomology, 1994, 39: 293-322.

[15] Clarkson J M, CHAMLEY A K. New insights into the mechanisms of fungal pathogenesis in insects. Trends in Microbiology, 1996, 4(5): 197-203.

[16] 王海鸿, 王登杰, 张桃, 李雪, 雷仲仁. 一种球孢白僵菌菌株GZGY-1-3及其应用: ZL 2014 1 0449467.4[P]. (2017-04-19)[2017- 11-21].

WANG H H, WANG D J, ZHANG T, LI X, LEI Z R. A strain of Beauveria bassiana GZGY-1-3 and its application: ZL 2014 1 0449467.4[P]. (2017-04-19)[2017-11-21]. (in Chinese)

[17] 王海鸿, 王登杰, 张桃, 雷仲仁. 一种球孢白僵菌菌株SCWJ-2及其应用: ZL 2014 1 0449469.3[P]. (2017-05-24)[2017-11-21].

WANG H H, WANG D J, ZHANG T, LEI Z R. A strain of Beauveria bassiana SCWJ-2 and its application: ZL 2014 1 0449469.3[P]. (2017-05-24) [2017-11-21]. (in Chinese)

[18] KERSHAW M J, MOORHOUSE E R, BATEMAN R, REYNOLDS S E, CHAMLEY A K. The role of destruxins in the pathogenicity of Metarhizium anisopliae for three species of insect. Journal of Invertebrate Pathology, 1999, 74(3): 213-223.

[19] MWAMBURI L A, LAING M D, MILLER R M. Effect of surfactants and temperature on germination and vegetative growth of Beauveria bassiana. Brazilian Journal of Microbiology, 2015, 46(1): 67-74.

[20] EKESI S, MANIANIA N K, AMPONG-NYARKO K. Effect of temperature on germination, radial growth and virulence of Metarhizium anisopliaeand and Beauveria bassiana on Megalurothrips sjostedti. Biocontrol Science and Technology, 1999, 9(2): 177-185.

[21] Yeo H, Pell J K, Alderson P G, Clark S J, Pye B J. Laboratory evaluation of temperature effects on the germination and growth of entomopathogenic fungi and on their pathogenicity to two aphid species. Pest Management Science, 2003, 59(2): 156-165.

[22] SHIMAZU M. Effects of temperature on growth of Beauveria bassiana F-263, a strain highly virulent to the Japanese pine sawyer, Monochamus alternatus, especially tolerance to high temperatures. Applied Entomology and Zoology, 2004, 39(3): 469-475.

[23] TEFERA T, PRINGLE K. Germination, radial growth, and sporulation of Beauveria bassiana and Metarhizium anisopliae isolates and their virulence to Chilo partellus (Lepidoptera: Pyralidae) at different temperatures. Biocontrol Science and Technology, 2003, 13(7): 699-704.

[24] Adamo S A, Lovett M M. Some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket Gryllus texensis. The Journal of Experimental Biology, 2011, 214(12): 1997-2004.

[25] Hunt V L,Zhong W,McClure C D,Mlynski D T, DuxburyE M,Charnley A K, Priest N K. Cold-seeking behaviour mitigates reproductive losses from fungal infection in Drosophila. The Journal of Animal Ecology, 2016, 85(1): 178-186.

[26] OUEDRAOGO R M, CUSSON M, GOETTEL M S, BRODEUR J. Inhibition of fungal growth in thermoregulating locusts, Locusta migratoria, infected by the fungus Metarhizium anisopliae var. acridum. Journal of Invertebrate Pathology, 2003, 82(2): 103-109.

[27] BELL A S, BLANFORD S, JENKINS N, THOMAS M B, READ A F. Real-time quantitative PCR for analysis of candidate fungal biopesticides against malaria: technique validation and first applications. Journal of Invertebrate Pathology, 2009, 100(3): 160-168.

[28] ANDERSON R D, BELL A S, BLANFORD S, PAAIJMANS K P, THOMAS M B. Comparative growth kinetics and virulence of four different isolates of entomopathogenic fungi in the house fly (Musca domestica L.). Journal of Invertebrate Pathology, 2011, 107(3): 179-184.

[29] 王登杰, 王海鸿, 张桃, 雷仲仁. 三株球孢白僵菌侵染烟粉虱的比较生长动力学及其毒力. 菌物学报, 2016, 35(5): 577-585.

WANG D J, WANG H H, ZHANG T, LEI Z R. Comparative growth kinetics and virulence of three isolates of Beauveria bassiana in the whiteflies Bemisia tabaci. Mycosystema, 2016, 35(5): 577-585. (in Chinese)

[30] 俞佳. 球孢白僵菌不同来源菌株分生孢子耐热性及其对桃蚜的毒力评价[D]. 杭州: 浙江大学, 2006.

YU J. Variation in conidial thermotolerance and virulence of originally different Beauveria bassiana strains against Myzus persicae[D].Hangzhou: zhejiang university, 2006. (in Chinese)

[31] VIDAL C, FARGUES J, Lacey L A. Intraspecific variability of Paecilomyees fumosorosezts: Effect of temperature on vegetative growth. Journal of Invertebrate Pathology, 1997, 70(1): 8-26.

[32] 袁盛勇, 张宏瑞, 孔琼, 王平, 孙士卿, 李正跃, 肖春. 球孢白僵菌MZ041016菌株对西花蓟马的毒力测定. 华中农业大学学报, 2011, 30(2): 197-199.

YUAN S Y, ZHANG H R, KONG Q, WANG P, SUN S Q, LI Z Y, XIAO C. Detection on the virulence of Beauveryia bassiana MZ041016 against Frankliniella occidentalis (Pergande). Journal of huazhong agricultural university, 2011, 30(2): 197-199. (in Chinese)

[33] 王雅卉, 郑长英, 王俊平. 球孢白僵菌对西花蓟马成虫的毒力及体表侵染的扫描电镜观察. 中国生物防治学报, 2011, 27(3): 324-330.

WANG Y H, ZHENG C Y, WANG J P. Virulence of Beauveria bassiana to Frankliniella occidentalis adults and scanning electron microscopic observation on its infection process. Chinese journal of biological control, 2011, 27(3): 324-330. (in Chinese)

[34] 任洁, 韩雪梅, 刘昭, 雷仲仁. 烟粉虱血细胞对白僵菌的防御反应. 中国蔬菜, 2013(12): 61-65.

REN J, HAN X M, LIU Z, LEI Z R. Defense response of hemolymph in Bemisia tabaci to Beauveria bassiana. China Vegetables, 2013(12): 61-65. (in Chinese)

[35] 张慧, 吴圣勇, 王晓青, 雷仲仁. 球孢白僵菌对葱蝇成虫血淋巴蛋白质及游离氨基酸的影响. 中国农业科学, 2017, 50(3): 591-598.

ZHANG H, WU S Y, WANG X Q, LEI Z R. Changes in the contents of proteins and free amino acid in haemolymph of Delia antique adult infected by Beauveria bassiana. Scientia Agricultura Sinica, 2017, 50(3): 591-598. (in Chinese)

[36] Ferguson L V, Heinrichs D E, Sinclair B J. Paradoxical acclimation responses in the thermal performance of insect immunity. Oecologia, 2016, 181(1): 77-85.

[37] 张永军, 王中康, 殷幼平, 裴炎. 球孢白僵菌的生物学特性及对小麦蚜虫的毒力. 西南农业大学学报, 2001, 23(2): 144-146.

ZHANG Y J, WANG Z K, YIN Y P, PEI Y. Biological characteristics of three isolates of Beauveria bassiana and their virulence against wheat aphids. Journal of southwest agricultural university, 2001, 23(2): 144-146. (in Chinese)

[38] 魏灵燕, 宋健, 曹伟平, 冯书亮, 王金耀, 朱宝成, 杜立新. 球孢白僵菌不同分离株的生物学特性的研究. 中国生物防治学报, 2012, 28(4): 502-507.

WEI L Y, SONG J, CAO W P, FENG S L, WANG J Y, ZHU B C, DU L X. Study on biological characteristics of different Beauveria bassiana strains. Chinese Journal of Biological Control, 2012, 28(4): 502-507. (in Chinese)