从土壤样品中分离昆虫致病真菌的方法比较
Summary
使用 Tenebrio 诱饵， Galleria 诱饵以及选择性人造培养基（即富含氯霉素，噻苯达唑和环己酰亚胺（CTC培养基）的酵母提取物的马铃薯葡萄糖琼脂从热带土壤样品中分离出昆虫致病真菌菌落。
Abstract
本研究的目的是比较使用昆虫诱饵与人工选择性培养基从土壤样品中分离昆虫致病真菌（EPF）的有效性。土壤是微生物的丰富栖息地，包括EPF，特别是属于Metarhizium和Beauveria属，它们可以调节节肢动物害虫。市场上有基于真菌的生物制品，主要用于农业节肢动物害虫防治。然而，尽管地方性生物多样性很高，但全世界只有少数菌株用于商业生物制品。本研究在富含氯霉素、噻苯达唑和环己酰亚胺（CTC培养基）的酵母提取物的马铃薯葡萄糖琼脂上培养了524个土壤样品。观察真菌菌落的生长3周。所有Metarhizium和Beauveria EPF都在属水平上进行了形态学鉴定。此外，一些分离株在物种水平上被分子鉴定。在这524个土壤样本中，有24个也使用昆虫诱饵法（Mellonella和Tenebrio molitor）调查了EPF的发生率。从524个土壤样品中共分离出51个EPF菌株（41个Metarhizium spp.和10个Beauveria spp.）。所有真菌菌株都从农田或草原中分离出来。在选取比较的24个样本中，91.7%使用Glaeria诱饵的EPF阳性，62.5%使用Tenebrio诱饵，41.7%使用CTC。我们的结果表明，使用昆虫诱饵从土壤中分离出EPF比使用CTC培养基更有效。除了EPF的识别和保护之外，分离方法的比较还对生物多样性知识产生了积极影响。EPF收集的改进支持科学发展和技术创新。
Introduction
土壤是几种微生物的来源，包括昆虫致病真菌（EPF）。这种特殊的真菌群通过它们定殖并经常杀死节肢动物宿主的能力而得到认可，特别是昆虫1。经过分离、表征、杀伤性菌株的选择和注册后，EPF被大量生产用于节肢动物害虫控制，这支持了它们的经济相关性2。因此，分离EPF被认为是开发生物农药的第一步。 Beauveria spp.（Hypocreales：Cordycipitaceae）和 Metarhizium spp.（Hypocreales：Clavicipitaceae）是用于节肢动物害虫控制的最常见真菌3。EPF已成功从土壤，具有可见真菌病的节肢动物，定植植物和植物根际4，5中分离出来。
分离EPF也可用于研究该特定组的多样性，分布和生态学。最近的文献报道说，EPF的使用被低估了，引用了EPF的几个非常规应用，例如它们改善植物生长的能力4，从土壤中去除有毒污染物，以及用于医学6。本研究旨在比较使用昆虫诱饵从土壤中分离EPF与人工培养基的效率7，8，9。在EPF分离的背景下，使用 Galleria mellonella L.（鳞翅目：Phyralidae）作为昆虫诱饵已被广泛接受。这些幼虫被全世界科学界用作研究宿主 – 病原体相互作用的实验模型10，11。 Tenebrio molitor L.（鞘翅目：Tenebrionidae）幼虫被认为是涉及毒力和分离EPF的研究的另一种昆虫模型，因为这种昆虫在实验室中很容易以低成本稀有7，12。
与培养无关的方法，例如使用各种PCR技术，可用于检测和定量其基质上的EPF，包括土壤13，14。然而，为了正确分离这些真菌菌落，应将其基质培养到选择性人工培养基9上，或者可以使用敏感昆虫15对样品中存在的真菌进行诱饵。一方面，CTC是一种不含多定的人造培养基，由富含酵母提取物的马铃薯葡萄糖琼脂组成，并辅以氯霉素，噻苯达唑和环己酰亚胺。这种培养基是由费尔南德斯等人开发的。9 .以最大限度地从土壤中恢复天然存在的 Beauveria spp.和 Metarhizium spp.。另一方面， G. mellonella 和 T. molitor 幼虫也可以成功地用作诱饵，以从土壤中获得EPF分离物。然而，根据Sharma等人15的说法，报告伴随使用和比较这两种诱饵昆虫的研究较少。葡萄牙葡萄园土壤对 Metarhizium robertsii（Metscn .）进行了显着恢复。索罗金使用 T。 与 G. mellonella 幼虫相比的molitor幼虫;相比之下， Beauveria bassiana （Bals. -Criv.）Vuill分离与使用 G. mellonella 诱饵15有关。因此，应根据研究目标和实验室基础设施来决定使用哪种EPF分离方法（即 ，G. mellonella-bait， T. molitor-bait或CTC培养基）。本研究的目的是比较使用昆虫诱饵与人工选择性培养基从土壤样品中分离EPF的有效性。
Protocol
Representative Results
Discussion
自然和农业土壤生境是EPF22 的典型环境，也是一个极好的天然水库。在本研究中，讨论了使用昆虫诱饵与选择性培养基分离的两种EPF方法。分离的第一步是收集土壤样本。正确储存和鉴定土壤样品至关重要。关于纬度、经度、土壤类型和生物群落的信息对于涉及流行病学、建模和地理空间主题的研究至关重要23，24.收集后，建议尽快处…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究部分由巴西的Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior（CAPES），财务代码001，Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Rio de Janeiro（FAPERJ）（项目编号E-26/010.001993/2015）和巴西国家电子和技术委员会（CNPq）资助。
Materials
| Autoclave | Phoenix Luferco | 9451 | |
| Biosafety cabinet | Airstream ESCO | AC2-4E3 | |
| Chloramphenicol | Sigma-Aldrich | C0378 | |
| Climate chambers | Eletrolab | EL212/3 | |
| Coverslip | RBR | 3871 | |
| Cycloheximide | Sigma-Aldrich | C7698 | |
| Drigalski spatula | Marienfeld | 1800024 | |
| GPS app | Geolocation app | 2.1.2005 | |
| Lactophenol blue solution | Sigma-Aldrich | 61335 | |
| Microscope | Zeiss Axio star plus | 1169 149 | |
| Microscope camera | Zeiss Axiocam 105 color | 426555-0000-000 | |
| Microscope softwere | Zen lite Zeiss 3.0 | ||
| Microscope slide | Olen | k5-7105-1 | |
| Microtube | BRAND | Z336769-1PAK | |
| Petri plates | Kasvi | K30-6015 | |
| Pipette tip | Vatten | VT-230-200C/VT-230-1000C | |
| Pippette | HTL – Labmatepro | LMP 200 / LMP 1000 | |
| Plastic pots | Prafesta descartáveis | 8314 | |
| Polypropylene bags | Extrusa | 38034273/5561 | |
| Potato dextrose agar | Kasvi | K25-1022 | |
| Prism software 9.1.2 | Graph Pad | ||
| Shovel | Tramontina | 77907009 | |
| Tenebrio mollitor | Safari | QP98DLZ36 | |
| Thiabendazole | Sigma-Aldrich | T8904 | |
| Tween 80 | Vetec | 60REAVET003662 | |
| Vortex | Biomixer | QL-901 | |
| Yeast extract | Kasvi | K25-1702 |
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