的发展<em> Metarhizium anisopliae</em>作为防腐剂：从隔离到现场表现
Summary
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。
Abstract
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％。
Introduction
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“> 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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Agar | Hi-Media | RM666 | Reagent |
| Ammonium sulphate | Thomas Baker | 11645 | Reagent |
| DNA analyzer | Applied biosystem | ABI prism 3730 | Instrument |
| DNA islation kit | Qiagen | 69104 | Reagent |
| Dodine | Sigma | 45466 | Reagent |
| Gel extraction kit | Qiagen | 28604 | Reagent |
| Glucose | Hi-Media | GRM077 | Reagent |
| Knapsac sparyer | Kaypee | HY-16L (1004) | Instrument |
| Peptone | Hi-Media | RM006-500G | Reagent |
| Polypropylene vials | Laxbro | SV-50 | Plasticware |
| Potato dextrose agar (PDA) | Hi-Media | M096-500G | Reagent |
| Tween-80 | SRL | 28940 | Reagent |
| Ultra low volume sparyer | Matabi | INSECDISK | Instrument |
| Unicorn-bags | Unicorn | UP-140024-SMB | Autoclavalbe bag for SSF |
| Yeast extract | Hi-Media | RM027-500G | Reagent |
| Chromas 2.1 | software |
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