昆虫病原真菌对孤雌生殖昆虫、芥菜蚜虫、 Lipaphis erysimi （Kalt.） 的蚜虫作用评估
Summary
该协议提出了一种优化的分离叶生物测定系统，用于评估昆虫病原真菌（EPF）对孤雌生殖昆虫芥菜蚜虫（Lipaphis erysimi （Kalt.））的有效性。该方法概述了培养皿实验期间的数据收集过程，使研究人员能够始终如一地测量EPF对芥菜蚜虫和其他孤雌生殖昆虫的毒力。
Abstract
芥菜蚜虫（L. erysimi）是一种害虫，会侵扰各种十字花科作物并传播植物病毒。为了实现环保的害虫管理，昆虫病原真菌（EPF）是控制这种害虫的潜在微生物控制剂。因此，在现场应用之前，有必要在培养皿条件下对EPF分离株进行毒力筛选。然而，芥菜蚜虫是一种孤雌生殖昆虫，因此在培养皿实验中很难记录数据。为了解决这个问题，开发了一种改进的分离叶生物测定系统，使用微型喷雾器将分生孢子接种到蚜虫上，并通过促进孢子悬浮后的空气干燥来防止溺水。在整个观察期间，该系统保持较高的相对湿度，叶盘保持新鲜十天以上，使蚜虫得以孤雌生殖繁殖。为了防止后代堆积，实施了每天使用画笔去除的过程。该方案展示了一种稳定的系统，用于评估EPF分离株对芥菜蚜虫或其他蚜虫的毒力，从而能够选择潜在的分离物进行蚜虫控制。
Introduction
芥菜蚜虫（L. erysimi）是一种臭名昭著的害虫，它侵扰各种十字花科作物，造成重大经济损失1。虽然已经推荐了几种系统性杀虫剂来对抗蚜虫侵扰，但这些杀虫剂的频繁使用引起了人们对杀虫剂耐药性的担忧2,3。因此，在环境友好型害虫管理方面，昆虫病原真菌（EPF）可以作为一种合适的替代防治策略。EPF 是一种昆虫病原体，能够通过穿透宿主的角质层来感染宿主，使其成为控制蚜虫和其他吸食植物的昆虫的有效药物4.此外，EPF 已被证明是一种可行且可持续的害虫管理技术，具有植物病原体拮抗和促进植物生长等益处5.
EPF可以通过昆虫土壤诱饵获得，也可以从野外的昆虫尸体中分离出来6,7。然而，在进一步使用真菌分离株之前，有必要进行致病性筛查。已经对 EPF 对蚜虫的有效性进行了几项研究，蚜虫是可造成严重损害的重要作物害虫 8,9。芥菜蚜虫，在各种蚜虫中，已经测试了对几种菌株的易感性，包括Beauveria spp.、Metarhizium spp.、Lecanicillium spp.、Paecilomyces spp.，甚至链格孢菌，它主要被称为腐生和植物病原真菌，但对芥菜蚜虫显示出一些致命的作用10,11,12。
为了评估EPF在实验室条件下对蚜虫的有效性，生物测定可分为两个主要部分：接种室和真菌接种。目前的方案描述了接种室的构造，其中蚜虫可以使用各种方法进行维护，例如用湿棉花包裹叶柄的切除叶片，带有衬有湿滤纸的培养皿的切除叶盘，直接维护盆栽植物，或切除的叶盘嵌入培养皿或容器10中的水琼脂中，11,13.常见的真菌接种方法包括分生孢子喷洒、蚜虫浸泡到分生孢子悬浮液中、叶片浸入分生孢子悬浮液中以及植物内生菌接种11、14、15、16。虽然存在各种接种方法，但生物测定应模拟现场应用条件。例如，在叶浸法12,17的情况下，可以评估EPF的效率，但由于蚜虫侵扰了装载真菌的叶子，因此蚜虫的背侧是一个优先的渗透部位，通常不会暴露于真菌。
为了评估EPF在实验室条件下的杀蚜作用，该方案建议使用Yokomi和Gottwald18描述的分离叶方法进行一些修改，然后使用微型喷雾器接种分生孢子。该方法在生物测定室中保持约100%的湿度至少七天，而无需额外补充水18,19。此外，将蚜虫限制在一个表面上可确保它们暴露于分生孢子喷洒并促进观察20.然而，蚜虫在接种室内移动时可能会卡在暴露的琼脂表面。此外，在培养皿实验中记录芥菜蚜虫的数据，芥菜蚜虫是孤雌生殖昆虫，由于它们的快速发育和繁殖，可能具有挑战性。如果不移除，很难区分接种的成虫及其后代。如何进行这一步的细节很少提及，一些不一致的因素，如叶片消耗面积，需要优化。
该方案展示了一种稳定的系统，用于筛选EPF分离株对芥菜蚜虫的毒力，从而能够从广泛的EPF库中选择针对各种蚜虫物种的潜在分离物。可以鉴定现场收集的蚜虫，并且可以建立足够的芥菜蚜虫实验室种群，以使用简单可行的方法评估各种真菌分离株的杀蚜效果，并具有一致的结果。蚜虫在应对农业生态系统中强烈和反复的人为压力时发展出多种进化机制，对粮食安全构成挑战9.因此，所描述的方法可以扩展到评估针对各种蚜虫物种的潜在EPF分离株。
Protocol
注意： 完整的流程图如图 1 所示。 1.芥菜蚜虫的收集与维护 芥菜蚜虫的收集翻转叶子，目视检查田间十字花科作物上是否有芥菜蚜虫侵扰。 记录采样地点信息（即GPS）和寄主植物，并与农民确认使用杀虫剂的历史。 使用昆虫吸痰器或精细画笔（见 材料表）从田间十字花科作物中收集约50个…
Representative Results
所呈现的流程图说明了芥菜蚜虫从田间采集到毒力筛选的稳定状态。通过田间采集对蚜虫的维护确保了蚜虫群落的稳定增加和充足的食物供应。通过使用分子标记（包括PCR扩增子大小和LeCO1测序）将现场收集的蚜虫确认为芥菜蚜虫。使用分离叶法进行的毒力筛查显示，芥菜蚜虫的存活率一致，对照组的存活率为85%（图4）。 在毒力筛选过程中，Cc-NCHU-213表…
Discussion
十字花科植物是一组蔬菜，经常被多种蚜虫侵染，包括芥菜蚜（L. erysimi）和卷心菜蚜虫（Brevicoryne brassicae）26。这两个物种在台湾已有报道 27，它们有可能在采集地点共存。为了区分密切相关的蚜虫种类，本研究采用多重引物组21的分子鉴定技术。通过从芥菜蚜虫COI基因片段中设计分子标记，我们成功地鉴定了芥菜蚜虫，从而证?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了科学技术部 （MOST） 的 109-2313-B-005 -048 -MY3 的支持。
Materials
| 10 μL Inoculating Loop | NEST Scientific | 718201 | |
| 100 bp DNA Ladder III | Geneaid | DL007 | |
| 2x SuperRed PCR Master Mix | Biotools | TE-SR01 | |
| 50 mL centrifuge tube | Bioman Scientific | ET5050-12 | |
| 6 cm Petri dish | Alpha Plus Scientific | 16021 | |
| 6 mm insect aspirator | MegaView Science | BA6001 | |
| 70 mm filter paper NO.1 | Toyo Roshi Kaisha | ||
| 70% ethanol | |||
| 9 cm Petri dish | Alpha Plus Scientific | 16001 | |
| Agar | Bioman Scientific | AGR001.1 | Microbiology grade |
| Agarose | Bioman Scientific | PB1200 | |
| BioGreen Safe DNA Gel Buffer | Bioman Scientific | SDB001T | |
| Chromas | Technelysium | ||
| GeneDoc | |||
| GenepHlow Gel/PCR Kit | Geneaid | DFH300 | https://www.geneaid.com/data/files/1605861013102532959.pdf |
| Gene-Spin Genomic DNA Isolation Kit | Protech Technology | PT-GD112-V3 | http://www.protech-bio.com/UserFiles/file/Gene-Spin%20Genomic%20DNA%20Kit.pdf |
| Hemocytometer | Paul Marienfeld | 640030 | |
| Komatsuna leaves (Brassica rapa var. perviridis) | Tai Cheng Farm | 1-010-300410 | |
| Microsprayer | |||
| MiniAmp Thermal Cycler | Thermo Fisher Scientific | A37834 | |
| Mustard aphid (Lipaphis erysimi) | |||
| Painting brush | Tian Cheng brush company | 4716608400352 | |
| Parafilm M | Bemis | PM-996 | |
| Pellet pestle | Bioman Scientific | GT100R | |
| Sabouraud Dextrose Broth | HiMedia | MH033-500G | |
| SPSS Statistics | IBM | ||
| TAE buffer 50x | Bioman Scientific | TAE501000 | |
| Tween 80 | PanReac AppliChem | 142050.1661 |
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Cite This Article
Yang, C., Nai, Y. Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.). J. Vis. Exp. (197), e65312, doi:10.3791/65312 (2023).