这项研究得到了科学技术部 （MOST） 的 109-2313-B-005 -048 -MY3 的支持。

注意： 完整的流程图如图 1 所示。
1.芥菜蚜虫的收集与维护
<ol><li>芥菜蚜虫的收集<ol><li>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。</li>
		<li>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。</li>
		<li>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个芥菜蚜虫到50mL离心管中，并在3小时内将样品带到实验室。</li>
		<li>准备一个临时维护培养皿，底部有切除的十字花科叶子和渗水的滤纸。</li>
		<li>将五只蚜虫置于室温（25±2°C）下，相对湿度为70%，12：12小时（光：暗）光周期为14天，以确认蚜虫没有天敌，然后再进一步饲养。 注意：为确保田间收集的蚜虫没有寄生蜂或昆虫病原真菌等天敌，在进行进一步饲养之前确认不存在这些生物至关重要。</li>
	</ol></li>
	<li>芥菜蚜虫的维护 注意：要保持芥菜蚜虫，请使用不含农药（包括生物农药）的十字花科叶子。<ol><li>提供稳定和充足的十字花科叶子供应（约 25 cm2）。 注意：从市场上获得十字花科的叶子或种植植物。在长期大规模饲养芥菜蚜虫之前，可以测试几种不同种类的十字花科植物，以找到合适的十字花科植物。一旦十字花科叶子的种类固定，就不要改变它。在这项研究中，使用了6-7叶期的小松（Brassica rapa var. perviridis）（见 材料表）。此外，处理掉 2-3 片最老的叶子。</li>
		<li>在底部的滤纸完全干燥之前加水。</li>
		<li>每天观察芥菜蚜虫的种群密度。7 天后，人口应增长到 2-3 倍。</li>
		<li>当蚜虫数量增加时，将原来用芥菜蚜虫切除的叶子切成4-6块小块，并将每片带有芥菜蚜虫的小叶片放入一个培养皿中，培养皿中装有新鲜切除的叶子和浸水的滤纸。</li>
		<li>将培养皿置于25°C的培养箱中，具有12：12小时（光：暗）的光周期，并每天观察芥菜蚜虫的种群密度。</li>
		<li>在原始叶子腐烂之前，大多数蚜虫迁移到新鲜的切除叶子后，去除原始切除的叶子。</li>
	</ol></li>
</ol>2.芥菜蚜虫的分子鉴定
注：为了确认田间采集的芥菜蚜虫的种类，使用两种分子标记进行分子鉴定：Lu等人设计的基于扩增区（SCAR）的A05Le的序列表征21，以及芥菜蚜虫的细胞色素氧化酶亚基1（COI）区域。
<ol><li>芥菜蚜虫基因组DNA的提取<ol><li>在 1.5 mL 离心管中收集约 50 只蚜虫。 注意：用于分子鉴定的蚜虫应为单个蚜虫的后代，并且各个阶段也可以汇集在同一管中进行DNA提取。</li>
		<li>用沉淀杵匀浆蚜虫，并按照制造商的说明使用 Gene-Spin 基因组 DNA 分离试剂盒提取 DNA（参见 材料表）。</li>
		<li>用 50 μL 预热的无核酸酶水洗脱基因组 DNA。</li>
	</ol></li>
	<li>PCR扩增和DNA测序<ol><li>使用PCR预混液（2x）扩增蚜虫基因组DNA中的靶DNA序列，引物对A05LeF / A05LeR和LeCO1F / LeCO1R（表1）具有不同的PCR程序21。 注：总体积为 20 μL 的 PCR 反应由 2 μL 蚜虫基因组 DNA 模板、1 μL 正向和 1 μL 反向引物、10 μL PCR 预混液（参见 材料表）和 6 μL ddH2O 组成。</li>
		<li>使用以下程序在热循环仪中进行PCR（参见 材料表）：94°C5分钟，94°C循环25次，持续45秒，61°C（对于A05LeF / A05LeR）和58°C（对于LeCO1F / LeCO1R）45秒，72°C持续1分钟，然后最终延长72°C5分钟。</li>
		<li>通过1%琼脂糖凝胶电泳分析PCR产物，以确认芥菜蚜虫的身份（图2）。 注：如果引物对 A05LeF/A05LeR 成功扩增了 DNA 片段的正确大小，则它已令人信服地将现场收集的蚜虫鉴定为芥菜蚜虫21。引物对列于 表2中。</li>
		<li>按照制造商的说明（参见 材料表），使用市售的凝胶/PCR试剂盒纯化芥菜蚜虫COI扩增子的PCR产物，并使用商业测序服务对PCR产物进行测序。</li>
	</ol></li>
	<li>序列分析 注：根据Lu等人21，A05Le的DNA弯曲是芥菜蚜虫特异性DNA片段;因此，A05Le PCR产物不需要进一步测序。<ol><li>获得LeCO1序列后，通过Chromas软件（见 材料表）检查读取质量。</li>
		<li>通过打开 fasta（或 txt）文件并直接删除低质量读取来修剪上游和下游低质量读取。</li>
		<li>使用默认参数将修剪后的序列提交到 NCBI web BLAST （https://blast.ncbi.nlm.nih.gov/Blast.cgi）。 注：如果 A05Le 和 LeCO1 引物组的扩增子大小正确，理论上，针对 NCBI 标准数据库的序列原始搜索必须与芥菜蚜虫匹配。</li>
	</ol></li>
</ol>3.昆虫病原真菌的制备
注：本研究中使用的EPF列于 表1中。
<ol><li>从真菌库中回收EPF<ol><li>从-80°C冰箱中解冻保存的真菌原液（悬浮在30%甘油中的分生孢子）。</li>
		<li>将少量（约10μL）分生孢子悬浮液转移到6cm 1/4 Sabouraud葡萄糖琼脂（SDA）平板（0.75g Sabouraud葡萄糖肉汤，每100mL ddH2O含1.5g琼脂）中，并在层流罩中使用细胞扩散器均匀分布。</li>
		<li>用石蜡膜密封培养皿，并在25°C的黑暗中孵育10-14天。</li>
		<li>通过在1/4SDA平板上划线真菌团并将真菌在25°C的黑暗中孵育10-14天，在收获分生孢子22之前重新培养真菌分离物。 注意：真菌培养物应在接种蚜虫前约10天使用。不建议将超过 14 天的 EPF 培养用于毒力试验。</li>
	</ol></li>
	<li>分生孢子悬浮液的制备<ol><li>加入2-3mL 0.03%吐温80后，通过接种环从10-14天龄的真菌培养物上刮除10-14天龄真菌培养物的分生孢子（参见 材料表）。</li>
		<li>将真菌悬浮液收集到离心管中。</li>
		<li>以最高速度涡旋溶液，在光学显微镜下使用血细胞计数器计数分生孢子的数量，并将分生孢子悬浮液的浓度调节至108 分生孢子/ mL。</li>
		<li>将分生孢子悬浮液转移到紫外线灭菌的微型喷雾器中。 注意：在转移分生孢子悬浮液之前再次涡旋分生孢子悬浮液，因为分生孢子容易沉淀。使用前，微型喷雾器应在层流罩中暴露于紫外线辐射 30 分钟。</li>
	</ol></li>
</ol>4. 针对芥菜蚜虫的毒力筛查
<ol><li>新成虫的准备<ol><li>将有翅（无翅）和有翅（有翅）的成虫从培养皿中移到新鲜切除的叶子上，以繁殖同龄的新后代。24小时后取出成虫以避免过度拥挤，并尽量减少后代的产生23,24。在进一步的实验中，只有适配的成虫才会被使用。</li>
		<li>将后代培养到成虫阶段，并在48小时后去除未发育到成虫阶段的后代（图3），以防止蚜虫之间的年龄差异进行实验。此外，删除有缺陷的成虫。</li>
	</ol></li>
	<li>接种室的准备 注意：建议先准备直径为9厘米的十字花科叶子，以便在凝固前将叶盘嵌入水琼脂中。<ol><li>用自来水清洁十字花科的叶子，去除污垢和残留物，以及任何其他节肢动物（如果有）。</li>
		<li>用纸巾擦去多余的水，并使用立体显微镜检查十字花科叶子表面是否有任何其他节肢动物。移除任何节肢动物（如果存在）。</li>
		<li>通过将底部培养皿直接压在叶子上作为模具或使用剪刀来切割直径为9厘米的叶盘。 注意：如果叶子直径小于 9 厘米，请合并几片切除的叶子。</li>
		<li>通过使用微波将450mg琼脂（参见 材料表）加热并溶解在30mL ddH2O中，为每个培养皿制备1.5%水琼脂。 注意：1.5%水琼脂的量可以根据实验规模进行调整。</li>
		<li>在层流罩中固化之前，将30mL的1.5%水琼脂倒入9cm培养皿中，然后等待琼脂冷却至~40°C，直到琼脂表面半固化。 注意：通过轻轻水平摇动培养皿来检查表面是否半凝固。</li>
		<li>将叶盘，轴面朝上，放在水琼脂上，将叶盘嵌入琼脂中。 注意：尽量减少琼脂表面的暴露。</li>
		<li>水琼脂完全凝固后，关闭培养皿的盖子。 注意： 如果盖子上有大量液滴凝结，请立即打开盖子进行水汽化。</li>
		<li>将 20 只适配的成虫放在叶盘上。 注意： 建议在立体显微镜下操作，以防止损坏其口器。</li>
	</ol></li>
	<li>EPF接种和观察<ol><li>打开接种室的盖子，将0.3mL分生孢子悬浮液（来自步骤3.2）直接喷洒到叶盘上方约15cm处的蚜虫和叶盘上。 注意：喷洒前再次涡旋分生孢子悬浮液。应在实验前测试喷涂区域，因为它们可能因不同的喷雾器而异。在这种情况下，建议使用 0.3 mL，距离为 15 cm。喷洒区域应覆盖整个叶盘，叶盘应覆盖一层薄薄的分生孢子悬浮液。如果液滴在叶子上汇聚成积水，则应减少接种量。接种前和使用不同分离株之间用70%乙醇清洁桌子。</li>
		<li>等待分生孢子悬浮液干燥，然后盖上盖子，防止芥菜蚜淹死。 注意：在实验过程中，蚜虫很少四处游荡;然而，确保蚜虫不会逃脱仍然是必要的。</li>
		<li>用石蜡膜密封接种室以保持高相对湿度，并将接种室置于25°C的培养箱中，光周期为12：12小时（光：暗）。</li>
		<li>打开接种室的盖子，每12小时在立体显微镜下计算死亡率，持续5天。</li>
		<li>用纸巾擦去粘附在盖子上的蜜露，用细画笔去除新出现的蚜虫。每 24 小时用自来水清洁盖子。 注意：根据成虫的寿命，不同种类的蚜虫的观察期可能会有所不同。</li>
		<li>将尸体放在叶盘上进行真菌病，以确认接种成功。</li>
	</ol></li>
	<li>分生孢子萌发率的确认 注意：此步骤是可选的。在进行毒力筛选时，要确认分生孢子的萌发率，以确保分生孢子的活性。<ol><li>将一滴 5 μL 分生孢子悬浮液滴在 1/4 SDA 上，重复 3 次。</li>
		<li>18小时后，用光学显微镜计算发芽率。在单个液滴中随机计数至少 100 个孢子，以确认真菌萌发率。 注：通常，实验中使用的分离株的发芽率应高于90%。</li>
	</ol></li>
</ol>5. 选定的EPF分离株的生物测定
注：从步骤4中选出显示出高毒力的EPF分离株，使用四种浓度的分生孢子悬浮液（范围为104 至107 分生孢子/ mL）对芥菜蚜虫进行生物测定。
<ol><li>分生孢子悬浮液的制备<ol><li>重复步骤 3.1 以恢复选定的 EPF 分离株。</li>
		<li>重复步骤3.2制备四种浓度的分生孢子悬浮液，包括104、105、106 和107 分生孢子/ mL。</li>
	</ol></li>
	<li>新成虫的准备<ol><li>重复步骤 4.1 以准备新出现的成虫。</li>
	</ol></li>
	<li>接种室的准备<ol><li>重复步骤 4.1。准备接种室。</li>
	</ol></li>
	<li>EPF接种和观察<ol><li>重复步骤 4.3。分别用四种浓度的分生孢子悬浮液进行EPF接种和观察。</li>
	</ol></li>
</ol>6. 统计分析
<ol><li>校正死亡率的计算<ol><li>使用 Abbott 公式25 计算校正死亡率，如下所示： 校正死亡率 （%） = [（MT− M C） × 100%] / （100− MC） MT 代表治疗组的死亡率，MC 代表对照组的死亡率。 注意：对照组的死亡率不应高于20%。</li>
		<li>打开SPSS软件平台（见 材料表），创建包括“治疗”和“cor_mor”（代表校正死亡率）在内的变量，并输入同一时间点以相同接种浓度接种不同菌株的计算结果。</li>
		<li>在 SPSS 中选择分析、 比较均值和比例、独立样本 t 检验以进行独立 t 检验 分析。</li>
		<li>在 SPSS 中，在“分组变量”框中输入 处理 ，然后在“测试变量”中 cor_mor 。按不同的真菌分离株定义组。按 OK 进行分析。</li>
	</ol></li>
	<li>中位致死时间（LT 50）和中位致死浓度（LC50）的计算<ol><li>打开 SPSS，创建变量“total”、“response”和“duration”/“concentration”，并将记录的结果输入电子表格。</li>
		<li>在 SPSS 中选择分析、回归、概率以计算 LT 50 和/或 LC50。 注意：如果累积死亡率最终不超过 50%，则无法估计 LT 50 和/或 LC50。</li>
		<li>在“观察到的总量”框中输入 总 值，在“响应频率”框中输入 响应，在 “协变量”框中输入 持续时间/浓度 。按 OK 进行分析。 注意：通常，按“选项”并将 “ 使用异质性因子的显著性水平”设置为 0.05。</li>
	</ol></li>
</ol>

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

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作者声明这项工作不涉及利益冲突。

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#956;L Inoculating Loop</td>
			<td>NEST Scientific</td>
			<td>718201</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp DNA Ladder III</td>
			<td>Geneaid</td>
			<td>DL007</td>
			<td></td>
		</tr>
		<tr>
			<td>2x SuperRed PCR Master Mix</td>
			<td>Biotools</td>
			<td>TE-SR01</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Bioman Scientific</td>
			<td>ET5050-12</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm Petri dish</td>
			<td>Alpha Plus Scientific</td>
			<td>16021</td>
			<td></td>
		</tr>
		<tr>
			<td>6 mm insect aspirator</td>
			<td>MegaView Science</td>
			<td>BA6001</td>
			<td></td>
		</tr>
		<tr>
			<td>70 mm filter paper NO.1</td>
			<td>Toyo Roshi Kaisha</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>9 cm Petri dish</td>
			<td>Alpha Plus Scientific</td>
			<td>16001</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Bioman Scientific</td>
			<td>AGR001.1</td>
			<td>Microbiology grade</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioman Scientific</td>
			<td>PB1200</td>
			<td></td>
		</tr>
		<tr>
			<td>BioGreen Safe DNA Gel Buffer</td>
			<td>Bioman Scientific</td>
			<td>SDB001T</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromas</td>
			<td>Technelysium</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GeneDoc</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GenepHlow Gel/PCR Kit</td>
			<td>Geneaid</td>
			<td>DFH300</td>
			<td>https://www.geneaid.com/data/files/1605861013102532959.pdf</td>
		</tr>
		<tr>
			<td>Gene-Spin Genomic DNA Isolation Kit</td>
			<td>Protech Technology</td>
			<td>PT-GD112-V3</td>
			<td>http://www.protech-bio.com/UserFiles/file/Gene-Spin%20Genomic%20DNA%20Kit.pdf</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Paul Marienfeld</td>
			<td>640030</td>
			<td></td>
		</tr>
		<tr>
			<td>Komatsuna leaves (Brassica rapa var. perviridis)</td>
			<td>Tai Cheng Farm</td>
			<td>1-010-300410</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsprayer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MiniAmp Thermal Cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>A37834</td>
			<td></td>
		</tr>
		<tr>
			<td>Mustard aphid (Lipaphis erysimi)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Painting brush</td>
			<td>Tian Cheng brush company</td>
			<td>4716608400352</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet pestle</td>
			<td>Bioman Scientific</td>
			<td>GT100R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sabouraud Dextrose Broth</td>
			<td>HiMedia</td>
			<td>MH033-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS Statistics</td>
			<td>IBM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer 50x</td>
			<td>Bioman Scientific</td>
			<td>TAE501000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>PanReac AppliChem</td>
			<td>142050.1661</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of aphidicidal effect of entomopathogenic fungi against parthenogenetic insect, mustard aphid, lipaphis erysimi (kalt.)

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。

The mustard aphid (L. erysimi) is a notorious pest that infests a variety of cruciferous crops, causing significant economic losses1. While several systematic insecticides have been recommended to combat aphid infestations, the frequent use of these insecticides raises concerns about pesticide resistance2,3. Therefore, in terms of eco-friendly pest management, entomopathogenic fungi (EPF) could serve as a suitable alternative control strategy. EPF is an insect pathogen with the ability to infect hosts by penetrating their cuticles, making it a potent agent for controlling aphids and other plant-sucking insects4. Furthermore, EPF has proven to be a feasible and sustainable pest management technique, offering benefits such as plant pathogen antagonism and plant growth promotion5.
EPF can be obtained through insect-soil baiting or isolated from insect cadavers in the field6,7. However, before further use of fungal isolates, pathogenicity screening is necessary. Several studies have been conducted on the effectiveness of EPF against aphids, which are significant crop pests that can cause severe damage8,9. Mustard aphids, among various species of aphids, have been tested for susceptibility to several strains of Beauveria spp., Metarhizium spp., Lecanicillium spp., Paecilomyces spp., and even Alternaria, which is primarily known as a saprophytic and plant pathogenic fungus but has shown some lethal effects against mustard aphids10,11,12.
To evaluate the effectiveness of EPF against aphids under laboratory conditions, bioassays can be divided into two main parts: the inoculation chamber and fungal inoculation. The current protocol describes the construction of an inoculation chamber, where aphids can be maintained using various methods such as an excised leaf with a petiole wrapped in moist cotton, an excised leaf disc with a Petri dish lined with damped filter paper, direct maintenance on pot plants, or an excised leaf disc embedded in water agar within a Petri dish or container10,11,13. Common methods for fungal inoculation include conidia spraying, aphid immersion into a conidia suspension, leaf dipping into a conidia suspension, and plant endophyte inoculation11,14,15,16. While various inoculation methods exist, the bioassays should simulate field application conditions. For example, in the case of the leaf dipped method12,17, the efficiency of EPF can be evaluated, but since the aphids infest the fungus-loaded leaves, the dorsal side of the aphid, which is a preferential penetration site, does not usually get exposed to the fungus.
To evaluate the aphidicidal effect of EPF under laboratory conditions, this protocol suggests using the detached-leaf method described by Yokomi and Gottwald18 with some modifications, followed by conidia inoculation using a micro-sprayer. This method maintains approximately 100% humidity in the bioassay chamber for at least seven days without requiring additional replenishment of water18,19. Additionally, confining aphids to one surface ensures their exposure to conidia spraying and facilitates observations20. However, aphids may become stuck in the exposed agar surface while moving within the inoculation chamber. Furthermore, recording data in the Petri dish experiment with mustard aphids, which are parthenogenetic insects, can be challenging due to their rapid development and reproduction. It is difficult to distinguish between inoculated adults and their progeny without removal. The details of how to proceed with this step are seldom mentioned, and some inconsistent factors, such as leaf consumption area, need to be optimized.
This protocol demonstrates a stable system for screening the virulence of EPF isolates against mustard aphids, enabling the selection of potential isolates against various aphid species from an extensive EPF library. Field-collected aphids can be identified, and a sufficient laboratory population of mustard aphids can be established to evaluate the aphidicidal effect of various fungal isolates using an easy and feasible methodology with consistent outcomes. Aphids have developed multiple evolutionary mechanisms in response to intense and repeated anthropogenic pressures in agroecosystems, posing challenges to food security9. Therefore, this described method could be extended to evaluate potential EPF isolates against various aphid species.

Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

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

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...

Watch this Scientific Journal Video about Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids at JoVE.com

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。

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, ...

如今，环保害虫管理已广受青睐。然而，在实施之前，昆虫病原真菌必须经过彻底的测试。我们的研究展示了昆虫病原真菌库在对抗芥菜蚜虫方面的有效性，这是其潜力的一个典型例子。降低昆虫病原真菌预处理的高死亡率和预防孢子悬浮液具有挑战性。我们已经成功地开发了一个稳定的系统，产生了预期的结果。这涉及使用流行的系统或生物测定室，以在测试过程中有效地维护这种微小的害虫。该协议成功地展示了一种无需测量琼脂温度即可设置分离叶方法的可行方法，从而简化了实验室的建立。此外，我们提出了尽量减少琼脂表面暴露的重要性，以防止蚜虫在腔室内漫游时卡住。我们对芥菜蚜虫在蚜虫死亡之前面对昆虫病原真菌时的免疫反应很感兴趣。通过研究基因表达数据，我们可以结合昆虫病原真菌应用，确定可以作为基因敲低潜在靶标的关键基因。

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Unit 1

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.)

Biostatistics: Introduction

SCIENTISTS IN ACTION

1.2K 次观看.  National Chung Hsing University. 如今，环保害虫管理已广受青睐。然而，在实施之前，昆虫病原真菌必须经过彻底的测试。我们的研究展示了昆虫病原真菌库在对抗芥菜蚜虫方面的有效性，这是其潜力的一个典型例子。降低昆虫病原真菌预处理的高死亡率和预防孢子悬浮液具有挑战性。我们已经成功地开发了一个稳定的系统，产生了预期的结果。这涉及使用流行的系统或生物测定室，以在测试过程中?...

视频: Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.

This protocol presents an optimized detached-leaf bioassay system for evaluating the effectiveness of entomopathogenic fungi (EPF) against the mustard aphid (Lipaphis erysimi (Kalt.)), a parthenogenetic insect. The method outlines the data collection process during Petri dish experiments, enabling researchers to consistently measure the virulence of EPF against mustard aphids and other parthenogenetic insects.

科研

生物学

Collection, Maintenance, and Molecular Identification of Mustard Aphids

Start by preparing a temporary maintenance Petri dish with excised cruciferous leaves and water infiltrated filter paper at the bottom. Next place five aphids on the prepared Petri dish. When the aphids increase in number, cut the originally excised leaves into four to six smaller pieces.Transfer each small leaf piece with the aphids into separate Petri dishes containing fresh leaves and wet filter paper. To collect the aphid genomic DNA, first homogenize the aphids using a pellet pestle. Next, extract the DNA using a Gene-Spin genomic DNA isolation kit as per the manufacturer's guidelines.Finish the extraction process by eluting the genomic DNA with 50 microliters of preheated nuclease-free water. Perform PCR amplification and DNA sequencing by adding the DNA sample to the PCR master mix with primer pairs. Analyze the PCR product using 1%agarose gel electrophoresis to confirm the identity of the mustard aphid.The field collected aphids were confirmed as mustard aphids using molecular markers, including the PCR amplicon size and Lipaphis erysimi CO1 sequencing.

芥菜蚜虫的采集、维护和分子鉴定

Entomopathogenic Fungi (EPF) Preparation and Virulence Screening Against Mustard Aphids

For the EPF recovery from the fungal library, start by smearing the fungal isolates on a quarter strength SDA plate. Incubate it in darkness at 25 degrees Celsius for 10-14 days before conidia harvest. Following this, pipette 2-3 mL of 0.03%Tween 80 onto a cultured plate.Then use an inoculation loop to scrape the conidia by. Next, transfer the fungal suspension to a centrifuge tube. Vigorously vortex the suspension at the highest speed, then pipette out a sample into a hemocytometer to count the number of conidia.Dilute the conidia within the suspension using 0.03%Tween 80 til desired concentration is obtained. To prepare the inoculation chamber, first cut a 9 centimeter diameter leaf disk using the bottom Petri dish as a mold or with scissors. Next, prepare 1.5%water agar for each dish using a microwave.Pour 30 mL of the cooled water agar into the Petri dish and let it solidify in a laminar flow hood. When the agar surface is semisolid, place the leaf disk with its abaxial surface facing up, then press it slightly to imbed it in the agar. Now, place 20 apterous adults on the leaf disk.Open the cover of the inoculation chamber to spray 0.3 mL of conidia suspension directly onto the aphids and the leaf disk. Once the suspension has dried, close the cover to prevent the drowning of the mustard aphids. Subsequently, seal the inoculation chamber using paraffin film to maintain a high mount of humidity.Place the chamber in an incubator set at a temperature of 25 degrees Celsius with a light and dark photoperiod of 12 hours each. Use a stereomicroscope to count the mortality every 12 hours for 5 days. The virulence screening revealed a consistent survival rate for mustard aphids with the control group exhibiting an 85%survival rate.Cordyceps cateninnulata demonstrated the fastest aphid-killing ability, resulting in 50%and 90%moralities at 3 days and 4.5 days post-inoculation, or DPI. Beauveria strains 141, 143, and 153 showed slow aphid-killing abilities with only 5%mortality at 3 DPI. Apart from Purpureocillium, most EPF isolates exhibited corrected mortality rates higher than 70%within 5 DPI.Beauveria bassiana demonstrated the highest mortality rate of 100%among EPF isolates. EPF mycosis was observed on cadavers of mustard aphids infected with Metarhizium species, Beauveria species, Purpureocillium lilacinum, and Cordyceps cateniannulata during the virulence screening.

昆虫病原真菌（EPF）制备及芥菜蚜虫毒力筛选

Aphicidal Assessment of Entomopathogenic Fungi by the Statistical Analysis of Corrected Mortality in Mustard Aphids

Begin by launching the SPSS software platform. Create variables termed treatment and corrected mortality. Then input the calculated results for different isolates inoculated at the same time point with the same concentration.Navigate to Analyze, Compare Means and Proportions, then Independent Samples T Test in SPSS for independent T-test analysis. Next, input treatment into the grouping variable box and corrected mortality into the Test Variables box. Classify groups according to different fungal isolates and press OK to begin the analysis.To calculate the median lethal time and median lethal concentration, create the variables defined as Total, Response, and either Duration or Concentration. Next, enter the recorded result into the spreadsheet. Finally, choose Analyze, followed by Regression, and then Probit to compute the median lethal time and median lethal concentration.Inoculations of 10 to the seven conidia per milliliter resulted in significantly different corrected mortalities for Metarhizium strain 197 and Cordyceps strain 213 at three and four DPI. The Cordyceps strain 213 exhibited a significantly shorter lethal duration compared to other treatments. It also showed a lower lethal dose value relative to the Metarhizium 213 strain.