作者承认来自瑞士生物技术部印度 - 瑞士合作生物技术合作社（ISCB）计划的贡献，以及瑞士伯尔尼瑞士发展与合作署。公认的项目学员和工作人员参与发展杀丝胶药剂的贡献包括Vandana Ghormade，Pallavi Nahar，Priya Yadav，Shuklangi Kulkarni，Manisha Kapoor，Santosh Chavan，Ravindra Vidhate，Shamala Mane和Abhijeet Lande。 EKP和SGT分别感谢印度大学教育资助委员会和印度科学与工业研究理事会（CSIR）的研究奖学金。 MVD承认新德里工业和科学研究理事会对名誉科学计划的支持。作者非常感谢印度新德里生物技术系在ISCB和SBIRI项目下的财政支持。我们很感激评论者的投入。

昆虫病原真菌的分离<ol><li> 土壤稀释法 <ol><li>收集来自不同作物田间的土壤样品和霉菌昆虫（ 表1 ）。使用土壤稀释电镀方法从土壤样品中分离昆虫病原真菌8 。 注意:本研究中从Pune（18°31&#39;13&#39;&#39;N; 73°51&#39;24&#39;&#39;E）和Buldhana（19°58&#39;36&#39;&#39;N 76°30&#39;30&#39;&#39;E ）地区，马哈拉施特拉邦，印度。 </li><li>称取10g每个土壤样品，并分别加入到90 mL无菌0.1％（w / v）Tween-80中。 </li><li>使用磁力搅拌器彻底混合样品60分钟，以释放附着在土壤颗粒上的孢子。 </li><li>混合后，将100至200μL等分试样从每个样品扩散至含有（g / L）:蛋白胨10的选择培养基上。葡萄糖20琼脂18链霉素0.6;四环素，0.05; CYC氯己酰胺，0.05;和dodine，0.1 mL; pH 7.0 9 。将板在28℃孵育3-7天。 </li><li>在同一培养基上选择和传代培养单个孢子菌落以得到纯培养物。 </li></ol></li><li> 从微妙的昆虫 <ol><li>从野外收集微生物的昆虫。 注意:一个硬的幼虫体很可能会被昆虫病原体感染。由于细菌或病毒感染，死亡的昆虫体很软。 </li><li>收集异常行为的活昆虫，协调不协调，动作不畅。 </li><li>保持昆虫直到死亡，然后将其转移到潮湿室，以进一步的真菌病和孢子形成（如果有的话），在28°C和70-80％RH。 </li><li>在上述选择性培养基上从孢子形成的尸体上分离出孢子，通过在同一培养基上进一步传代2-3次获得纯培养物。 </li></ol></li><li> 诱饵法 <ol><li>在一个含有60g土壤样品的小瓶（3.85×6.0cm），加入4只稻蛾幼虫（ Corcyra cephalonica ），并将小瓶保持在25±2℃，持续14天。 </li><li>每天将小瓶倒置。 14天后，筛选土壤样品中存在迷迭香米蛾幼虫。通过在选择性培养基上从孢子形成的尸体上划出孢子来分离昆虫病原真菌。 </li><li>在获得纯培养物后，将分离物转移到马铃薯葡萄糖琼脂（PDA）斜面上，并在28℃和70-80％RH下孵育7天以允许孢子形成。孢子形成后，将母体培养物保持在8°C直至使用。 </li></ol></li></ol>昆虫病原真菌鉴定<ol><li>通过观察形态特征（ 即无性孢子大小和形状以及孢子对分生孢子的排列）来鉴定昆虫病原真菌;分离3个主要属， Metarhizium ， Beauv<html> 呃和Nomuraea ，可以确定。 </li><li>对于Metarhizium菌株的分子鉴定，使用DNA分离试剂盒从菌丝体生物量提取基因组DNA;按照制造商的说明（参见材料表 ）。通过在0.8％琼脂糖凝胶上进行电泳检查基因组DNA的质量。 <ol><li> PCR扩增ITS1-5.8S-ITS2和26S rDNA区。使用基因组DNA作为模板，使用ITS1正向（TCCGTAGGTGAACCTGCGG）和ITS4反向（TCCTCCGCTTATTGATATGC）引物10 。 </li><li>凝胶洗脱并使用凝胶提取试剂盒纯化预期大小的扩增子;按照制造商的说明（参见材料表 ）。量化纯化的扩增子和序列。 </li><li>使用软件读取和编辑序列，并对NCBI GenBank数据库中的核苷酸序列进行BLAST检索，以分析其密切同源性="xref"&gt; 11。 </li><li>将确定的致病原分离株的序列存储到NCBI GenBank数据库中以检索登录号。 </li></ol></li></ol> 3. 绿僵菌的筛选分离株抗棉铃虫 <ol><li> 昆虫饲养 <ol><li>通过从野外收集昆虫的健康幼虫和蛹，建立棉铃虫的初始培养。 </li><li>对于饲养，将幼虫分别生长在含有0.5％（v / v）次氯酸钠消毒的秋葵粉碎的无菌聚丙烯小瓶（3.85×6.0cm，50mL容量）中10分钟12 。 </li><li>收集饲养期间铺设的昆虫卵，并用0.5％（v / v）次氯酸钠进行表面消毒。 </li><li>保持幼虫在25±2℃和65±5％RH。 </li></ol></li><li> 昆虫生物测定 注意:对于昆虫生物测定，生产孢子，和田间性能研究，除非另有说明，否则使用来自mycosed H. armigera幼虫的Metarhizium菌株的第一次亚文化。 <ol><li>对于昆虫生物测定，使用棉铃虫的 III龄幼虫。 </li><li>将一组30只幼虫一式三份分别浸入10分钟的Metarhizium分离株的孢子悬浮液中（1×10 7孢子/ mL，除非另有说明;活力&gt; 90％）5秒。 </li><li>治疗后，将每只幼虫单独转移到一个单独的无菌小瓶中，以避免同类相食。向每个小瓶中加入潮湿的Whatmann滤纸1号和一片消毒的秋葵作为饲料。更换纸张和替代天馈送。 </li><li>将幼虫保持在25±2℃，65±5％相对湿度和16:8光线:暗处14天或直至死亡。 </li><li>将死亡的幼虫转移到含有湿棉签的无菌培养皿中，并将其保持在28℃和70-80％相对湿度下3-7天，以允许菌丝体和孢子在尸体上形成。 </li><li>对于对照，用无菌蒸馏水中的0.1％（w / v）Tween-80处理一组30只幼虫一式三份。 </li><li>使用新鲜制备的孢子悬浮液进行一式三份的所有实验。收集和汇集三个实验中百分比死亡率的数据，以获得平均值。使用雅培公式13计算校正百分比死亡率。 </li><li>使用随机完整块设计（RCBD）布局进行实验，每次处理包含一组30只幼虫，一式三份。基于对棉铃实夜蛾的百分比死亡率，选择Metarhizium分离株进一步筛选用于商业生产的最佳分离物。 </li><li>选择对棉铃虫 III型幼虫显示&gt; 90％死亡率的分离株。 注意:这里选择了12个分离株。 </li><li>确定这些分离株的LT 50 ，并选择分离株评估最快的杀戮（在更短的时间内）。 注意:在这里，从12个最有效的分离株中选出5个分离物。 </li><li>使用孢子悬浮液的四种不同浓度（即1×10 3，1 ×10 5，1 ×10 7和1×10 9孢子/ mL）确定所选分离物的LC 50值。 </li><li>确定Metalhizium分离株对棉铃实夜蛾三龄幼虫的LC 50 ，以提高识别具有高死亡率的分离株的毒力差异的可能性，如果仅使用单次剂量，则可能未检出。 </li></ol></li></ol> 4.生产用于田间表现研究的Metarhizium Isolate孢子<ol><li> 通过SSF生产Metarhizium孢子 <ol><li>对于SSF，通过添加2×10 7个 Metarhizium分离株的孢子来准备接种物200mL YPG（0.3％酵母提取物，0.5％蛋白胨，1.0％葡萄糖）培养基。将烧瓶在28℃振荡（180rpm）孵育48小时。 </li><li>对于SSF大量生产孢子，除非另有说明，否则以米为基质。 </li><li>对于SSF，用2公斤的大米将高压釜袋（型号/ 14，用0.5μm的单个滤膜; 2kg容量; 64×36cm）填充。在袋中加入1000 mL蒸馏水，浸泡过夜14 。用浸泡的米饭在121℃高压灭菌45分钟15 。 </li><li>用48小时的菌丝体接种物（10％接种物，200mL的2kg的大米）接种袋子，并在28℃和70-80％的相对湿度条件下温育14天。 </li><li>使用0.1％Tween-80通过液体萃取收获孢子。为此，将袋子的内容物加入到0.1％吐温-80（每1kg大米3L）中，充分混合，通过离心分离孢子与液体，并在37℃下干燥2天。<html> / LI&gt; <li>或者，将含有孢子的袋子和一些菌丝体在37℃下干燥2天以降低含水量（&lt;20％）。用收割机或振动筛收获孢子。 </li></ol></li><li> 生存能力研究 <ol><li>决定采用不同的方法15收获孢子的存活百分比。为此，在0.1％（w / v）Tween-80中制备孢子悬浮液，并将其计数为1×10 3个孢子/ mL。 </li><li>将孢子悬浮液（0.1mL）一式三份扩散到PDA平板上，并在28℃和70-80％RH下孵育72小时。 </li><li>手动计数分离的菌落并确定相应样品的总计数。 </li></ol></li><li> 孢子沉降率 <ol><li>对于均匀剂量，需要均匀的孢子悬浮液;确定如下所述的Metarhizium分离株的孢子沉降速率"xref"&gt; 16。检查0.2M硫酸铵和0.1％Tween-80中孢子的沉降速率。 </li><li>将孢子悬浮液的计数调至〜7×10 7孢子/ mL，得到540nm初始吸光度为0.6。允许比色皿不受干扰6小时使孢子沉降。 </li><li>记录吸光度达6小时。以百分比表示沉降速率，计算50％沉降所需的时间（ST 50 ）。使用新鲜制备的孢子悬浮液重复实验三次。 </li></ol></li></ol> 5.选择M. anisopliae分离株控制豌豆H. the的能力现场表现研究<ol><li> 可湿性粉末制剂M. anisopliae M 34412孢子 <ol><li>通过将孢子与滑石混合来制备2.5-5％的可湿性粉剂。 </li><li>将最终可行计数（TVC）调整为1×10 12个 spores每kg配方。 </li></ol></li><li> M. anisopliae M 34412孢子的田间性能研究 <ol><li>对于所选择的绿僵菌的能力字段性能研究隔离以控制棉铃实夜蛾侵扰在木豆，使用RCBD，4个重复。 注意:在这里，在圣雄法师Krus Vidyapeeth（MPKV），Rahuri（19.3927°N，74.6488°E）执行。 </li><li>使用两种不同的喷雾配方，一种油配方的孢子（5×10 12个孢子/ 3升柴油:向日葵油，7:3）和吐温-80（0.1％）中的水性制剂。用背包式喷雾器（5×10 12个孢子，500L / ha）用超低体积（ULV）喷雾器（70分钟; 3L / ha）喷洒油制剂。 注意:这里，在喷雾前一天记录幼虫种群，并在每次喷雾施用5次随机选择的植物后3和7天。总人口为t转换为n + 1的平方根进行统计分析。 <ol><li>根据豌豆作物的农业实践，在产蛋后10至15天进行第一次喷洒，间隔14天进行2次。在16:00至18:00 HST之间进行喷雾。监测风向，如有必要，使用布帘避免孢子漂移到邻近地块。 </li></ol></li><li>为了比较，用手压缩背包喷雾器喷洒化学杀虫剂。 </li><li>通过在喷雾后0,3,5,7和14天收集棉铃虫幼虫来确定现场接种物的持久性。 </li><li>将这些幼虫保持观察14天，死亡后，将其转移到含有湿滤纸的塑料小瓶中。在25±2℃和70±10％RH下孵育以观察真菌病。 </li><li>根据第p页确定接种物对幼虫种群的持续性<html>喷雾后从田间收集的幼虫的死亡率数据。 </li><li>基于效率百分比评估现场研究17 ，荚果损失百分比和产量百分比18 。 注:在这里，在一次试验中记录了温度，湿度，风速（km / h），阳光（h），降雨量（mm），雨天和蒸发量（mm）等参数的数据农业大学（Mahatma Phule Krushi Vidyapeeth，Rahuri，19.3927°N，74.6488°E）。 </li></ol></li><li> 农民参与方案 <ol><li>选择示范试验的农民人数。向农民供应豌豆种子（BSMR - 736）和肥料。 注:本研究涉及20名农民。村庄:Deolali Pravara，（19.473°N 74.6°E）。 </li><li>使用与步骤5.2相同的喷雾配方和喷雾数量。 </li></ol></li></ol>tle"&gt; 6。对非目标生物的影响<ol><li>观察豌豆叶上的防腐剂喷雾的效果（如果有的话）。 </li><li>收集土栖节肢动物和叶栖昆虫在未处理地块的每个处理后1天，并与绿僵菌处理的地块。 </li><li>处理后24 h内收集土壤节肢动物，并在处理后的早晨（ 即治疗后约15-18 h）收集带有扫蚊网的叶栖昆虫。 </li><li>将它们分别保存在直径为3.5厘米，高4.0厘米的圆柱形塑料盒中。每天检查昆虫感染，并喂食适当的食物。 </li><li>记录M. anisopliae的存在，如果有的话，并分离真菌。 </li></ol> 7.质量控制参数的识别<ol><li>通过测量PDA上的孢子萌发28°检查孢子的活力;C。 </li><li>测量角质层降解酶活性，如几丁质酶，几丁质脱乙酰酶，壳聚糖酶，蛋白酶和脂肪酶，如前所述15生产的YPG和几丁质培养基。 </li><li>确定实验室生物测定中棉铃实夜蛾的百分比死亡率15 。 </li><li>使用分子标记，如几丁质酶的PCR-RFLP图谱（ 捷 1，2，和4）和蛋白酶（ 的PR1a）基因，毒力为绿僵菌属性。 <ol><li>使用DNA分离试剂盒15从菌丝体生物质中提取基因组DNA。使用基因组DNA作为模板，使用引物对Chit1F / Chit1R（CTCTGCAGGCCACTCTCGGT / AGCCATCTGCTTCCTCATAT）和Chit2F / Chit2R（GACAAGCACCCGGAGCGC / GCCTTGCTTGACACATTGGTAA）进行PCR扩增Chit1和Chit2基因片段。对于Chit4 ，使用引物对Chit4F / Chit4R（ATCCGGCAGCACGGCTAC / CTTGGATC CGTCCCAGTTG）。 </li><li>对于Pr1A基因的扩增，使用METPR2和METPR5引物对（AGGTAGGCAGCCAGACCGGC / TGCCACTATTGGCCGGCGCG）。 </li><li>用BsaJI ， BstUI和ScrFI进行Chit1基因的限制性消化19 ;的Chit2基因与AluI ， HpyCH4IV和HpyCH4V ，和Chit4基因与BstUI ， HaeIII和MboI 。为了消化Pr1A基因，使用RsaI ， DdeI和MspI 20 。 </li><li>通过电泳对大多数毒性菌株（&gt; 90％死亡率）的每个基因进行电泳观察1.5％琼脂糖凝胶上的限制性片段长度多态性（RFLP）模式;这可以用作选择五角茴香的毒力标记。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+and+direct+sequencing+of+fungal+ribosomal+RNA+genes+for+phylogenetics.">Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.</a> PCR-Protocols: A guide to methods and applications. , 315-322 (1990).</li><li>Ignoffo, C. M., Futtler, B., Marston, N. L., Hostetter, D. L., Dickerson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+incidence+of+the+entomopathogenic+fungus+Spicaria+rileyi+associated+with+noctuid+pests+of+soybeans.">Seasonal incidence of the entomopathogenic fungus Spicaria rileyi associated with noctuid pests of soybeans.</a> J Invertebr Pathol. 25 (1), 135-137 (1975).</li><li>Abbott, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+computing+the+effectiveness+of+an+insecticide.">A method for computing the effectiveness of an insecticide.</a> J Econ Entomol. 18 (2), 265-267 (1925).</li><li>Nahar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development of biocontrol agents for the control of pests in agriculture using chitin metabolism as target. , 137 (2004).</li><li>Kulkarni, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Metarhizium+isolates+for+biocontrol+of+Helicoverpa+armigera+(Lepidoptera:+Noctuidae)+in+chickpea.">Comparison of Metarhizium isolates for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) in chickpea.</a> Biocontrol Sci Tech. 18 (8), 809-828 (2008).</li><li>Jeffs, L. B., Khachatourians, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+spore+hydrophobicity+for+members+of+the+genera+Beauveria,+Metarhizium,+and+Tolypocladium+by+salt-mediated+aggregation+and+sedimentation.">Estimation of spore hydrophobicity for members of the genera Beauveria, Metarhizium, and Tolypocladium by salt-mediated aggregation and sedimentation.</a> Can J Microbiol. 43 (1), 23-28 (1997).</li><li>Henderson, C. F., Tilton, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tests+with+acaricides+against+the+brow+wheat+mite.">Tests with acaricides against the brow wheat mite.</a> J Econ Entomol. 48 (2), 157-161 (1955).</li><li>Hassani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development and proving of biocontrol methods based on Bacillus thuringiensis and entamopathogenic fungi against the cotton pests Spodoptera littoralis, Helicoverpa armigera (Lepidoptera: Noctuidae) and Aphis gossypii (Homoptera: Aphididae). , (2000).</li><li>Enkerli, J., Ghormade, V., Oulevey, C., Widmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR-RFLP+analysis+of+chitinase+genes+enable+efficient+genotyping+of+Metarhizium+anisopliae+var.+anisopliae.">PCR-RFLP analysis of chitinase genes enable efficient genotyping of Metarhizium anisopliae var. anisopliae.</a> J Invert Pathol. 102 (2), 185-188 (2009).</li><li>Bidochka, M. J., Melzer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+polymorphism+in+three+subtilisin-like+protease+isoforms+(Pr1A,+Pr1B+and+Pr1C)+from+Metarhizium+strains.">Genetic polymorphism in three subtilisin-like protease isoforms (Pr1A, Pr1B and Pr1C) from Metarhizium strains.</a> Can. J. Microbiol. 46 (12), 1138-1144 (2000).</li><li>McCoy, C. W., Samson, R. A., Boucias, D. G., Ignoffo, C. M., Mandava, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomogenous+fungi.">Entomogenous fungi.</a> Handbook of natural pesticides, Microbial insecticides, Part A. Entomogenous protozoa and fungi. , 151-236 (1988).</li><li>Nahar, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+repeated+in+vitro+sub-culturing+on+the+virulence+of+Metarhizium+anisopliae+against+Helicoverpa+armigera+(Lepidoptera+Noctuidae).">Effect of repeated in vitro sub-culturing on the virulence of Metarhizium anisopliae against Helicoverpa armigera (Lepidoptera Noctuidae).</a> Biocontrol Sci Tech. 18 (4), 337-355 (2008).</li><li>Kapoor, M., Deshpande, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+mycoinsecticide+for+the+control+of+insect+pests:+Issues+and+challenges+in+transfer+of+technology+from+laboratory+to+field.">Development of mycoinsecticide for the control of insect pests: Issues and challenges in transfer of technology from laboratory to field.</a> Kavaka. 40, 45-56 (2013).</li><li>Deshpande, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycopesticide+Production+by+Fermentation:+Potential+and+Challenges.">Mycopesticide Production by Fermentation: Potential and Challenges.</a> Crit Rev Microbiol. 25 (3), 229-243 (1999).</li></ol>

作者没有什么可以披露的。

<html>在19世纪80年代，第一个尝试使用绿僵控制金龟子，Anisoplia austriaca和甜菜象鼻虫，Cleonis punctiventris 21。在该方案中，先决条件之一是从土壤中或从感染的昆虫中分离出毒性菌株。事实上，其它参数，诸如LC 50，LT 50和ST 50，显著到产品22，23的成本效益作出了贡献。为了优化孢子生产，孢?...

<html>从在印度上世纪40年代引进有机氯农药，农药的使用增加了许多倍，1，与作物病虫害仍然花费数十亿卢比，每年2的农业生产产量损失的条款。广泛和不合理地使用合成农药是对环境和人类健康的持续威胁1 。不分青红皂白地使用农药会导致土壤中的残留物和天敌害虫的消耗。它也是改变有害生物种群遗传构成的强大选择压力，导致抗性发展1 。尽管绿色革命带来了巨大的好处，这种革命需要高投入，如肥料和农药，但害虫仍然是一个主要的生物限制。印度和全世界录得的年度作物损失的一般估计为120亿美元ef"&gt; 2和2000亿美元3 。 当化学农药在用于防治害虫时具有不利影响，寻找生态健康，可靠，经济，可持续的替代方法成为当务之急。生物防治提供了合适的替代方案，包括使用寄生虫，捕食者和微生物病原体4 。例如，真菌感染各种各样的害虫，包括鳞翅目，鳞翅目，鞘翅目和dip子，经常导致天然流行病。此外，与其他细菌和病毒昆虫控制剂不同，昆虫致病真菌的作用方式是通过接触5 。这些真菌包含超过100属的异源组，其中不同昆虫中报告了大约750种。重要的真菌病原体是: Metarhizium sp。， Beauveria sp。， Nomuraea rileyi ， Lecanicillium lecanii和Hirsutella sp。，仅举几例6 。 M. anisopliae （Metchnikoff）Sorokin是生物控制中第二大广泛应用的昆虫病原真菌。已知攻击200多种昆虫7 。 在这项研究中，涉及到使用绿僵一个mycopesticide的知识为基础的发展不同阶段呈现。这包括:1）确定有毒昆虫病原体的来源（ 即土壤或微生物昆虫），2）昆虫病原体鉴定和选择，3）在实验室生物测定和野外维持其毒性和有效性的策略4 ）传染性繁殖体的成本有效的制剂，5）开发用于有毒制剂的独特的质量控制参数，以及6）生物勘探和增值。

<table><tbody><tr><td>Agar</td><td>Hi-Media</td><td>RM666</td><td>Reagent</td></tr><tr><td>Ammonium sulphate&amp;nbsp;</td><td>Thomas Baker</td><td>11645</td><td>Reagent</td></tr><tr><td>DNA analyzer&amp;nbsp;</td><td>Applied biosystem</td><td>ABI prism 3730&amp;nbsp;&amp;nbsp;</td><td>Instrument</td></tr><tr><td>DNA islation kit</td><td>Qiagen</td><td>69104</td><td>Reagent</td></tr><tr><td>Dodine</td><td>Sigma</td><td>45466</td><td>Reagent</td></tr><tr><td>Gel extraction kit</td><td>Qiagen</td><td>28604</td><td>Reagent</td></tr><tr><td>Glucose</td><td>Hi-Media</td><td>GRM077</td><td>Reagent</td></tr><tr><td>Knapsac sparyer</td><td>Kaypee</td><td>HY-16L (1004)</td><td>Instrument</td></tr><tr><td>Peptone</td><td>Hi-Media</td><td>RM006-500G</td><td>Reagent</td></tr><tr><td>Polypropylene vials&amp;nbsp;</td><td>Laxbro</td><td>SV-50</td><td>Plasticware</td></tr><tr><td>Potato dextrose agar (PDA)&amp;nbsp;</td><td>Hi-Media</td><td>M096-500G</td><td>Reagent</td></tr><tr><td>Tween-80</td><td>SRL</td><td>28940</td><td>Reagent</td></tr><tr><td>Ultra low volume sparyer</td><td>Matabi</td><td>INSECDISK</td><td>Instrument</td></tr><tr><td>Unicorn-bags&amp;nbsp;</td><td>Unicorn</td><td>UP-140024-SMB</td><td>Autoclavalbe bag for SSF</td></tr><tr><td>Yeast extract</td><td>Hi-Media</td><td>RM027-500G</td><td>Reagent</td></tr><tr><td>Chromas 2.1</td><td></td><td></td><td>software</td></tr></tbody></table>

development of metarhizium anisopliae as a mycoinsecticide: from isolation to field performance

与化学杀虫剂相比，开发商用防霉杀菌剂的主要问题是杀死速度。因此，选择快速，高毒力昆虫病原真菌的分离筛选是重要的步骤。昆虫病原真菌如Metarhizium，Beauveria和Nomurea通过接触作用，比苏云金芽孢杆菌或核多角体病毒（NPV）更适合，必须被害虫摄入。在目前的工作中，我们使用土壤稀释和诱饵方法从感染的昆虫中分离出68株Metarhizium菌株。通过ITS1-5.8S-ITS2和26S rDNA区域的扩增和测序鉴定了分离株。基于针对棉铃实夜蛾三龄幼虫的昆虫生物测定中获得的中值致死浓度（LC 50 ）和时间（LT 50 ）选择最具毒性的Metarhizium anisopliae菌株。通过使用稻作为底物的固相发酵（SSF）进行14天的选择菌株的大量生产孢子。使用0.1％吐温-80从孢子生物质中提取孢子，并制备不同的孢子制剂。通过随机区块设计进行了用于控制棉蚜棉铃虫侵染的配方的现场试验。用油和水性制剂（分别为78.0％和70.9％）获得的侵染控制水平优于用化学农药获得的63.4％。

在调查期间， 绿僵菌，白僵菌 ，和野村的不同菌株通过各种分离方法（数据未示出） 如图6所示 ，分离14作为绿僵菌的菌株被发现是在控制棉铃虫 ，在脉冲6可怕的害虫，14更有效目的是进一步分离来自不同作物田和昆虫的Metarhizium菌株（ 表1 ）。通过文化和形态...

Watch this Scientific Journal Video about Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance at JoVE.com

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

在这里，我们报告了有效的杀丝粒菌知识开发中涉及到的不同阶段，包括分离，鉴定，筛选和选择"最适合"昆虫病原真菌（ Metarhizium anisopliae ）来控制农业中的害虫。

的发展<em&gt; Metarhizium anisopliae</em&gt;作为防腐剂:从隔离到现场表现

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

development-metarhizium-anisopliae-as-mycoinsecticide-from-isolation

Universidad San Sebastian

Research

JoVE Journal

Environment

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

20.6K 次观看.  CSIR-National Chemical Laboratory. 在这里，我们报告了有效的杀丝粒菌知识开发中涉及到的不同阶段，包括分离，鉴定，筛选和选择"最适合"昆虫病原真菌（ Metarhizium anisopliae ）来控制农业中的害虫。 

视频: Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

实验室确定的磷通量湖泊沉积物作为内部磷载入一个测量

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

科研

环境科学

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

实验协议生产生物柴油与饱和酮的分离从商业余积<em&gt;等鞭金藻</em&gt;藻类生物量

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

修改和叶鼓风机-VAC的节肢动物领域采样中的应用

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

生物学

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. The increase in pest resistance to chemical insecticides, the growing concerns regarding the negative effects of insecticides on human health, and the environmental pollution from pesticides have led to a global drive to find novel sustainable strategies for crop protection and pest control. Previously, attempts to mass culture such entomopathogenic fungi (EPF) species as Beauveria bassiana have been conducted. However, only limited attempts have been conducted to mass culture Metarhizium robertsii and M. pinghaense for use against insect pests. This study aimed to mass-produce a sufficient number of resilient infective propagules of South African isolates of M. robertsii and M. pinghaense for commercial application. Three agricultural grain products, flaked oats, flaked barley, and rice, were used as the EPF solid fermentation substrates. Two inoculation methods, conidial suspensions and the liquid fungal culture of blastospores were used to inoculate the solid substrates. Inoculation using conidial suspensions was observed to be relatively less effective, as increased levels of contamination were observed on the solid substrates relative to when using the blastospore inoculation method. Flaked oats were found not to be a suitable growth substrate for both M. robertsii and M. pinghaense, as no dry conidia were harvested from the substrate. Flaked barley was found to favor the production of M. robertsii conidia over that of M. pinghaense, and an average of 1.83 g &plusmn; 1.47 g of dry M. robertsii conidia and zero grams of M. pinghaense conidia was harvested from the substrate. Rice grains were found to favor the conidial mass production of both M. pinghaense and M. robertsii isolates, with an average of 8.2 g &plusmn; 4.38 g and 6 g &plusmn; 2 g harvested from the substrate, respectively.

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi have gained importance as the biological control agents of agricultural insect pests. In this study, the mass production of a sufficient number of resilient infective propagules of South African isolates of both Metarhizium robertsii and M. pinghaense for commercial application against insect pests was successfully conducted using agricultural grain products.

大规模生产昆虫致病真菌、 罗伯氏菌 和 异根真菌，用于昆虫害虫的商业应用

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

从土壤样品中分离昆虫致病真菌的方法比较

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

针对芥菜蚜虫的昆虫病原真菌的优化生物测定

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

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.

作者聚焦:使用昆虫病原真菌对抗芥菜蚜虫的环保害虫管理 

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.

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

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

的发展

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此视频中的章节

实验室确定的磷通量湖泊沉积物作为内部磷载入一个测量

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

实验协议生产生物柴油与饱和酮的分离从商业余积

修改和叶鼓风机-VAC的节肢动物领域采样中的应用

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

大规模生产昆虫致病真菌、罗伯氏菌和异根真菌，用于昆虫害虫的商业应用

从土壤样品中分离昆虫致病真菌的方法比较

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

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

的发展

摘要

探索更多视频

此视频中的章节

实验室确定的磷通量湖泊沉积物作为内部磷载入一个测量

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

实验协议生产生物柴油与饱和酮的分离从商业余积

修改和叶鼓风机-VAC的节肢动物领域采样中的应用

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

大规模生产昆虫致病真菌、 罗伯氏菌 和 异根真菌，用于昆虫害虫的商业应用

从土壤样品中分离昆虫致病真菌的方法比较

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

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

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

大规模生产昆虫致病真菌、罗伯氏菌和异根真菌，用于昆虫害虫的商业应用

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

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