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
Summary
昆虫致病真菌作为农业害虫的生物防治剂越来越重要。本研究利用农粮产品成功大规模生产了足够数量的南非 梅塔根菌 和 山 茱萸分离株的弹性感染性繁殖体，用于商业应用。
Abstract
茴香属复合物的昆虫致病真菌作为农业害虫的生物防治剂而变得越来越重要。害虫对化学杀虫剂的抗药性增加，对杀虫剂对人类健康的负面影响的日益关注，以及农药对环境的污染，导致全球努力寻找新的可持续作物保护和害虫控制战略。以前，已经尝试过大规模培养诸如Beauveria bassiana之类的昆虫致病真菌（EPF）物种。然而，只有有限的尝试进行了大规模的培养Metarhizium robertsii和M. pinghaense用于对抗害虫。本研究旨在大规模生产足够数量的南非罗拔沙分枝杆菌和平海氏菌的弹性感染性繁殖体，用于商业应用。三种农产品，片状燕麦，片状大麦和大米，被用作EPF固体发酵基质。采用两种接种方法，分生孢子悬浮液和胚芽孢子的液体真菌培养来接种固体底物。观察到使用分生孢子悬浮液的接种相对不那么有效，因为相对于使用囊孢子接种方法，在固体底物上观察到污染水平增加。发现片状燕麦不是M. robertsii和M. pinghaense的合适生长基质，因为没有从基质中收获干燥的分生孢子。发现片状大麦有利于分生孢子菌的产生，而不是平海分生孢子菌的生产，平均1.83克±1.47克干分生孢子菌，从基质中收获零克分生孢子分生孢子。 发现水稻籽粒有利于分生孢子大规模生产M. pinghaense和M. robertsii分离株，平均从基质中收获平均8.2g±4.38 g和6 g±2 g。
Introduction
昆虫致病真菌（EPF）作为作物保护剂，在重要农业害虫的生物防治中具有重要意义1，2。在土壤中自然发生的昆虫病原体在各种害虫物种的种群中引起动物流行病3。EPF的物种是宿主特异性的，在攻击非靶向物种方面构成的风险相对较小，并且对环境无毒4。EPF具有入侵宿主的独特机制，以及在其直接环境中繁殖和持续存在1。它们主要通过无性孢子攻击宿主，这些孢子附着并穿透宿主角质层，以侵入宿主血细胞并增殖。宿主最终由于血淋巴营养物质的消耗或真菌释放的有毒代谢物引起的毒血症而死亡。死亡后，在理想的环境条件下，真菌出现在宿主尸体5，6的外表面（明显的真菌病）。
人们越来越关注化学残留物对人类健康、环境污染和虫害抗药性的发展的负面影响，导致全球努力减少化学杀虫剂的投入，并寻找替代、新颖和可持续的作物保护和虫害防治战略6，7，8.这为开发用于病虫害综合治理（IPM）计划的微生物杀虫剂提供了机会，这些计划比传统的化学控制更具生态优势3，8。
为了开发成功的农业害虫微生物控制剂，必须首先分离，表征，鉴定合适的生物体并确认其对目标害虫的致病性。然而，需要一种简单、具有成本效益的大规模生产微生物剂的方法，以生产用于生物控制方案的可行产品9、10、11、12、13。大量高质量昆虫病原体的大规模生产取决于微生物菌株，环境，目标害虫，配方，市场，应用策略和所需的最终产品14，15，16。EPF可以大规模生产，使用液体底物发酵产生囊孢子或固体底物发酵过程产生地上分生孢子6，17，18。然而，昆虫病原体的大规模生产和配制过程直接影响最终产品的毒力，成本，保质期和现场功效。为了在IPM中成功使用，昆虫病原体的生产过程必须易于运行，需要最少的劳动力，产生高产浓度的毒力，活体和持久性繁殖体，并且成本低4，13，14，16。
了解昆虫病原体的营养需求对于所有培养方法的大规模培养非常重要4，12.生产培养基的营养成分对所得繁殖体的属性有显著影响，包括生物防治功效、产量、干燥耐受性和持久性8、19、20、21。生产程序的优化旨在解决这些因素22.对于EPF，真菌分生孢子的良好生长，孢子形成和大规模生产的主要要求是充足的水分，最佳生长温度，pH值，CO 2和O2 的气体交换以及 营养，包括良好的磷，碳水化合物，碳和氮源18。
Jaronski和Jackson18 将固体底物发酵方法描述为相对于液体底物发酵方法而言，与EPF生产自然过程最有效和最接近的方法，因为在自然条件下，真菌分生孢子存在于固体直立结构上，就像昆虫尸体的表面一样。含有淀粉的农产品和副产品主要用于下胰腺真菌的大规模生产，因为真菌很容易通过从其菌丝尖端分泌高度浓缩的水解酶来分解淀粉，以渗透固体物质，并获得物质11，17，18，23中存在的营养物质.谷物产品也为健康的生物质生产提供了要求，因为当它们被水合和灭菌时，基质可以从任何液体培养基16，18，24中吸收更多的营养物质。
此前，有几项研究试图大规模培养EPF物种，如 Beauveria bassiana （Bals）。Vuil.， 冬虫夏草 （Wize） Kelper B. Shrestha & Spatafora， Verticillium lecanii （Zimm.）维加斯和一些 Metarhizium anisopliae （Metschn.）Sorokin物种复合物在各种基质上分离16，23，24。这种大规模生产和商业化开发的分离物包括Green Muscle® （菌株IMI 330189），由 M. anisopliae var Metarhizium acridum （Driver&Milner）J.F. Bisch，Rehner&Humber，Metarhizium 69（Meta 69菌株ICIPE69）和Real Metarhizium 69（L9281），由 M. anisopliae开发，宽带® （菌株PPRI 5339）和Eco-Bb®，由 B. bassiana25，26开发。.然而，对大众文化 Metarhizium robertsii J.F. Bisch.，S.A. Rehner&Humber和 Metarhizium pinghaense Chen&Guo的尝试有限。这两种分离株在之前的一项研究中被选为对控制粉虱最有效的， Pseudococcus viburni Signoret（半翅目：Pseudococcidae）27。因此，目前的研究旨在配制和大规模生产足够数量的当地 田上梅和平 海氏菌的弹性感染性繁殖体 ， 用于商业应用以对抗害虫。采用固体底物发酵法对两种EPF分离株进行菌源分生孢子的大规模生产。使用两种EPF接种方法，使用分生孢子悬浮液和芽孢子的液体真菌培养来接种固体底物。
Protocol
Representative Results
Discussion
在农业生态系统中成功整合微生物制剂以生物控制重要的农业害虫取决于昆虫病原的成功和容易程度，这是实验室条件下的第一步。EPF的大规模生产对于使用生物防治的IPM计划的EPF产品的大规模应用和可用性非常重要9，10，11，12，13。不同EPF分离物的大规模生产的成功受生?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要感谢Hort Pome，Hort Stone以及技术和人力资源行业计划（THRIP：TP14062571871）为该项目提供资金。
ORCID：
莱托迪·马图 http://orcid.org/0000-0002-5118-3578
安托瓦内特·马兰 http://orcid.org/0000-0002-9257-0312
诺玛克霍尔瓦·斯托克韦 http://orcid.org/0000-0003-2869-5652
Materials
| 0.05% Tween 20 | Lasec | Added to conidial suspensions to allow fungal spores to mix with water | |
| 20 mL McCartney bottles | Lasec | Used to make conidial suspensions | |
| Aluminium foil | Used as a cover of the cotton wool plugs on 250-mL flask | ||
| Autoclave | Used to sterilize materials and ingredients used for the conidia production process | ||
| Autoclave bags | Lasec | Fermentation bags or solid substrate containers | |
| Autoclave tape | Lasec | To secure PVC pipes on the fermentation bags | |
| Brown Kraft paper bags | Used to dry conidia cultures on agricultural grains | ||
| Bunsen burnner | Labnet (Labnet International, Inc.) | Used to flame equipment (surgical blades,inoculating loops and rims of flasks) | |
| Clear edge test sieve | Used to separate fungal conidia from agricultural grain substrates | ||
| Corn steep liquor | SIGMA | 66071-94-1 | Ingredient of the blastospore liquid medium |
| Cotton Wool | Lasec | Used as plug of the neck for fermentation bags | |
| Duran laboratory bottles | Neolab | Used to autoclave SDA medium and distilled water | |
| Electrical tape | Used to tape and seal the sieve joints to prevent the escape of conidial dust | ||
| ENDECOTTS test sieve | Used to separate fungal conidia from agricultural grain substrates | ||
| Erlenmeyer Flasks, Narrow neck,250-mL flask | Lasec | Carrier of the blastospore liquid medium | |
| Ethanol (99%) | Lasec | Used to sterilize surgical blades and inoculating loops | |
| Flaked barley | Health Connection Wholefoods | Agricultural grain used as a solid substrate growth medium for conidia of both M. pinghaense and M. robertsii | |
| Flaked oats | Tiger brands | Agricultural grain used as a solid substrate growth medium for conidia of both M. pinghaense and M. robertsii | |
| Glucose | Merck | Ingredient of the blastospore liquid medium | |
| Growth Chamber/ incubators | For growing fungal conidia culture | ||
| Haemocytometer | Used to determine conidial concentrations | ||
| Inoculating loops | Lasec | For harvesting spores to innoculate liquid medium for blastospores growth | |
| Kitchen rolling pin | Used to manipulate the solid grain substrate bed | ||
| Laminar flow Cabinet | ESCO Laminar Flow Cabinet | Provide as sterile environment during substrate inoculation | |
| Metarhizium pinghaense conidia | Stellenbosch University | 5HEID | Cultures used to mass culture conidia of Metarhizium pinghaense |
| Metarhizium robertsii conidia | Stellenbosch University | 6EIKEN | Cultures used to mass culture conidia of Metarhizium robertsii |
| Microscope | ZEIZZ (Scope. A1) | Used to determine conidial concentrations and conidial viability | |
| Orbital shaker | IncoShake- LABOTEC | Used for the blastospore production process | |
| Parboiled rice | Spekko | Agricultural grain used as a solid substrate growth medium for conidia of both M. pinghaense and M. robertsii | |
| Penicillin-Streptomycin | SIGMA | Added to the SDA medium to prevent bacterial contamination | |
| Petri-dishes | Lasec | Containers for the SDA medium | |
| Pipettes and pipette tips | Labnet (BioPette PLUS) | Used to measure liquids ingredients | |
| Polyvinylchloride Marley waste pipe | Used to create a neck for the fermentation bag | ||
| Potassium phosphate dibasic (K2HPO4) | SIGMA-ALDRICH | Ingredient of the blastospore liquid medium | |
| Rubber band | Used to secure the secure the surgical paper over the fermentation bag PVC pipe necks | ||
| Sabaroud dextrose agar (SDA) | NEOGEN Culture Media | Medium used to culture spores of both Metarhizium pinghaense and Metarhizium robertsii | |
| Sterile distilled water | To hydrate agricultural grains, to make conidial suspensions | ||
| Sticky pad | Used to secure the seives on the vibratory shaker | ||
| Surgical blade | Lasec | Used to scrape off spores from fungal cultures | |
| Surgical paper | Lasec | Used to cover the PVC necks and cotton wool plugs of the fermentation bag | |
| Vibratory shaker | Used to shake conidia off the agricultural grain substrates | ||
| Vortex mixer | Labnet (Labnet International, Inc.) | Used to mix conidial suspensions in Mc Cartney bottles | |
| Yeast extract | Biolab | Added to the SDA medium to improve spore germination and growth | |
| Zipper-lock bags | GLAD | Used to to store harvested fungal conidia |
Referências
- Shah, P. A., Pell, J. K. Entomopathogenic fungi as biological control agents. Applied Microbiology and Biotechnology. 61 (5), 413-423 (2003).
- Mathulwe, L. L., Malan, A. P., Stokwe, N. F. A review of the biology and control of the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae), with special reference to biological control using entomopathogenic fungi and nematodes. African Entomology. 29 (1), 1-16 (2020).
- Ibrahim, L., Laham, L., Touma, A., Ibrahim, S. Mass production, yield, quality, formulation and efficacy of entomopathogenic Metarhizium anisopliae conidia. Current Journal of Applied Science and Technology. 9 (5), 427-440 (2015).
- Banu, J. G., Rajalakshmi, S. Standardisation of media for mass multiplication of entomopathogenic fungi. Indian Journal of Plant Protection. 42 (1), 91-93 (2014).
- Roberts, D. W., Humber, R. A., Cole, G. T., Kendrick, W. B. Entomogenous fungi. Biology of Conidial Fungi. , 201-236 (1981).
- Feng, M. G., Poprawski, T. J., Khachatourians, G. G. Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control. Current status. Biocontrol Science and Technology. 4 (1), 3-34 (1994).
- Karanja, L. W., Phiri, N. A., Oduor, G. I. Effect of different solid substrates on mass production of Beauveria bassiana and Metarhizium anisopliae entomopathogens. The Proceedings of the12th KARI Biennial Scientific Conference. , 8-12 (2010).
- Prasad, C. S., Pal, R. Mass production and economics of entomopathogenic fungus, Beauveria bassiana, Metarhizium anisopliae and Verticillium lecanii on agricultural and industrial waste. Scholars Journal of Agriculture and Veterinary Sciences. 1 (1), 28-32 (2014).
- Ehlers, R. U. Mass production of entomopathogenic nematodes for plant protection. Applied Microbiology and Biotechnology. 56 (5), 623-633 (2001).
- Pham, T. A., Kim, J. J., Kim, S. G., Kim, K. Production of blastospore of entomopathogenic Beauveria bassiana in a submerged batch culture. Mycobiology. 37 (3), 218-224 (2009).
- Bhadauria, B. P., Puri, S., Singh, P. K. Mass production of entomopathogenic fungi using agricultural products. The Bioscan. 7 (2), 229-232 (2012).
- Latifian, M., Rad, B., Amani, M. Mass production of entomopathogenic fungi Metarhizium anisopliae by using agricultural products based on liquid-solid diphasic method for date palm pest control. International Journal of Farming and Allied Sciences. 3 (4), 368-372 (2014).
- Agale, S. V., Gopalakrishnan, S., Ambhure, K. G., Chandravanshi, H., Gupta, R., Wani, S. P. Mass production of entomopathogenic fungi (Metarhizium anisopliae) using different grains as a substrate. International Journal of Current Microbiology and Applied Sciences. 7 (1), 2227-2232 (2018).
- Jackson, M. A. Optimizing nutritional conditions for the liquid culture production of effective fungal biological control agents. Journal of Industrial Microbiology and Biotechnology. 19 (3), 180-187 (1997).
- Deshpande, M. V. Mycopesticides production by fermentation. Potential and challenges. Critical Reviews in Microbiology. 25 (3), 229-243 (1999).
- Sahayaraj, K., Namasivayam, S. K. R. Mass production of entomopathogenic fungi using agricultural products and by products. African Journal of Biotechnology. 7 (12), 1907-1910 (2008).
- Feng, K. C., Liu, L. B., Tzeng, Y. M. Verticillium lecanii spore production in solid-state and liquid-state fermentations. Bioprocess Engineering. 23 (1), 25-29 (2000).
- Jaronski, S. T., Jackson, M. A., Lacey, L. A. Mass production of entomopathogenic Hypocreales. Manual of Techniques in Invertebrate Pathology 2nd edition. , 255-284 (2012).
- Vega, F. E., Jackson, M. A., Mercandier, G., Poprawski, T. J. The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture. World Journal of Microbiology and Biotechnology. 19 (4), 363-368 (2003).
- El Damir, M. Effect of growing media and water volume on conidial production of Beauveria bassiana and Metarhizium anisopliae. Journal of Biological Sciences. 6 (2), 269-274 (2006).
- Pandey, A. K., Kanaujia, K. R. Effect of different grains as solid substrates on sporulation, viability and pathogenicity of Metarhizium anisopliae (Metschnikoff) Sorokin. Journal of Biological Control. 22 (2), 369-374 (2008).
- Kassa, A., et al. Whey for mass production of Beauveria bassiana and Metarhizium anisopliae. Mycological Research. 112 (5), 583-591 (2008).
- Sharma, S., Gupta, R. B. L., Yadavam, C. P. S. Selection of a suitable medium for mass multiplication of entomofungal pathogens. Indian Journal of Entomology. 64 (3), 254-261 (2002).
- Bich, G. A., Castrillo, M. L., Villalba, L. L., Zapata, P. D. Evaluation of rice by-products, incubation time, and photoperiod for solid state mass multiplication of the biocontrol agents Beauveria bassiana and Metarhizium anisopliae. Agronomy Research. 16 (5), 1921-1930 (2018).
- Price, R. E., Müller, E. J., Brown, H. D., D’Uamba, P., Jone, A. A. The first trial of a Metarhizium anisopliae var. acridum mycoinsecticide for the control of the red locust in a recognised outbreak area. International Journal of Tropical Insect Science. 19 (4), 323-331 (1999).
- Hatting, J. L., Moore, S. D., Malan, A. P. Microbial control of phytophagous invertebrate pests in South Africa. Current status and future prospects. Journal of Invertebrate Pathology. 165, 54-66 (2019).
- Mathulwe, L. L., Malan, A. P., Stokwe, N. F. Laboratory screening of entomopathogenic fungi and nematodes for pathogenicity against the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae). Biocontrol Science and Technology. , (2021).
- Inglis, G. D., Enkerli, J., Goettel, M. S. Laboratory techniques used for entomopathogenic fungi: Hypocreales. Manual of Techniques in Invertebrate Pathology. , 189-253 (2012).
- Mehta, J., et al. Impact of carbon & nitrogen sources on the Trichoderma viride (Biofungicide) and Beauveria bassiana (entomopathogenic fungi). European Journal of Experimental Biology. 2 (6), 2061-2067 (2012).
- Burges, H. D. Formulation of mycoinsecticides. Formulation of Microbial Biopesticides. , 131-185 (1998).
