Name: mass production of entomopathogenic fungi, metarhizium robertsii and metarhizium pinghaense, for commercial application against insect pests
Uploaded: 2022-03-31T13:10:00
Description: Watch this Scientific Journal Video about Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests at JoVE.com

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

1. 真菌菌株的来源
<ol><li>使用南非分离的来自南非西开普省苹果园的 M. pinghaense 5 HEID（GenBank加入编号：MT367414 / MT895630）和 M. robertsii 6EIKEN（MT378171 / MT380849）的 南非分离真菌菌株。</li>
	<li>在60g Sabouraud葡萄糖琼脂培养基上生长每种EPF分离物的培养物，并补充1g酵母提取物（SDAY）和10μL链霉素。 注意：在黑暗中将EPF培养物在±25°C的受控温度下孵育。</li>
</ol><p cla...

<ol>
	<li>Shah, P. A., Pell, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomopathogenic+fungi+as+biological+control+agents.">Entomopathogenic fungi as biological control agents.</a> Applied Microbiology and Biotechnology. 61 (5), 413-423 (2003).</li><li>Mathulwe, L. L., Malan, A. P., Stokwe, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> African Entomology. 29 (1), 1-16 (2020).</li><li>Ibrahim, L., Laham, L., Touma, A., Ibrahim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production,+yield,+quality,+formulation+and+efficacy+of+entomopathogenic+Metarhizium+anisopliae+conidia.">Mass production, yield, quality, formulation and efficacy of entomopathogenic Metarhizium anisopliae conidia.</a> Current Journal of Applied Science and Technology. 9 (5), 427-440 (2015).</li><li>Banu, J. G., Rajalakshmi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardisation+of+media+for+mass+multiplication+of+entomopathogenic+fungi.">Standardisation of media for mass multiplication of entomopathogenic fungi.</a> Indian Journal of Plant Protection. 42 (1), 91-93 (2014).</li><li>Roberts, D. W., Humber, R. A., Cole, G. T., Kendrick, W. 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> Biology of Conidial Fungi. , 201-236 (1981).</li><li>Feng, M. G., Poprawski, T. J., 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=Production,+formulation+and+application+of+the+entomopathogenic+fungus+Beauveria+bassiana+for+insect+control.+Current+status.">Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control. Current status.</a> Biocontrol Science and Technology. 4 (1), 3-34 (1994).</li><li>Karanja, L. W., Phiri, N. A., Oduor, G. I. <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+different+solid+substrates+on+mass+production+of+Beauveria+bassiana+and+Metarhizium+anisopliae+entomopathogens.">Effect of different solid substrates on mass production of Beauveria bassiana and Metarhizium anisopliae entomopathogens.</a> The Proceedings of the12th KARI Biennial Scientific Conference. , 8-12 (2010).</li><li>Prasad, C. S., Pal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+and+economics+of+entomopathogenic+fungus,+Beauveria+bassiana,+Metarhizium+anisopliae+and+Verticillium+lecanii+on+agricultural+and+industrial+waste.">Mass production and economics of entomopathogenic fungus, Beauveria bassiana, Metarhizium anisopliae and Verticillium lecanii on agricultural and industrial waste.</a> Scholars Journal of Agriculture and Veterinary Sciences. 1 (1), 28-32 (2014).</li><li>Ehlers, R. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+of+entomopathogenic+nematodes+for+plant+protection.">Mass production of entomopathogenic nematodes for plant protection.</a> Applied Microbiology and Biotechnology. 56 (5), 623-633 (2001).</li><li>Pham, T. A., Kim, J. J., Kim, S. G., Kim, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+blastospore+of+entomopathogenic+Beauveria+bassiana+in+a+submerged+batch+culture.">Production of blastospore of entomopathogenic Beauveria bassiana in a submerged batch culture.</a> Mycobiology. 37 (3), 218-224 (2009).</li><li>Bhadauria, B. P., Puri, S., Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+of+entomopathogenic+fungi+using+agricultural+products.">Mass production of entomopathogenic fungi using agricultural products.</a> The Bioscan. 7 (2), 229-232 (2012).</li><li>Latifian, M., Rad, B., Amani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+of+entomopathogenic+fungi+Metarhizium+anisopliae+by+using+agricultural+products+based+on+liquid-solid+diphasic+method+for+date+palm+pest+control.">Mass production of entomopathogenic fungi Metarhizium anisopliae by using agricultural products based on liquid-solid diphasic method for date palm pest control.</a> International Journal of Farming and Allied Sciences. 3 (4), 368-372 (2014).</li><li>Agale, S. V., Gopalakrishnan, S., Ambhure, K. G., Chandravanshi, H., Gupta, R., Wani, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+of+entomopathogenic+fungi+(Metarhizium+anisopliae)+using+different+grains+as+a+substrate.">Mass production of entomopathogenic fungi (Metarhizium anisopliae) using different grains as a substrate.</a> International Journal of Current Microbiology and Applied Sciences. 7 (1), 2227-2232 (2018).</li><li>Jackson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+nutritional+conditions+for+the+liquid+culture+production+of+effective+fungal+biological+control+agents.">Optimizing nutritional conditions for the liquid culture production of effective fungal biological control agents.</a> Journal of Industrial Microbiology and Biotechnology. 19 (3), 180-187 (1997).</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=Mycopesticides+production+by+fermentation.+Potential+and+challenges.">Mycopesticides production by fermentation. Potential and challenges.</a> Critical Reviews in Microbiology. 25 (3), 229-243 (1999).</li><li>Sahayaraj, K., Namasivayam, S. K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+of+entomopathogenic+fungi+using+agricultural+products+and+by+products.">Mass production of entomopathogenic fungi using agricultural products and by products.</a> African Journal of Biotechnology. 7 (12), 1907-1910 (2008).</li><li>Feng, K. C., Liu, L. B., Tzeng, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verticillium+lecanii+spore+production+in+solid-state+and+liquid-state+fermentations.">Verticillium lecanii spore production in solid-state and liquid-state fermentations.</a> Bioprocess Engineering. 23 (1), 25-29 (2000).</li><li>Jaronski, S. T., Jackson, M. A., Lacey, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+production+of+entomopathogenic+Hypocreales.">Mass production of entomopathogenic Hypocreales.</a> Manual of Techniques in Invertebrate Pathology 2nd edition. , 255-284 (2012).</li><li>Vega, F. E., Jackson, M. A., Mercandier, G., Poprawski, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+nutrition+on+spore+yields+for+various+fungal+entomopathogens+in+liquid+culture.">The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture.</a> World Journal of Microbiology and Biotechnology. 19 (4), 363-368 (2003).</li><li>El Damir, M. <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+growing+media+and+water+volume+on+conidial+production+of+Beauveria+bassiana+and+Metarhizium+anisopliae.">Effect of growing media and water volume on conidial production of Beauveria bassiana and Metarhizium anisopliae.</a> Journal of Biological Sciences. 6 (2), 269-274 (2006).</li><li>Pandey, A. K., Kanaujia, K. R. <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+different+grains+as+solid+substrates+on+sporulation,+viability+and+pathogenicity+of+Metarhizium+anisopliae+(Metschnikoff)+Sorokin.">Effect of different grains as solid substrates on sporulation, viability and pathogenicity of Metarhizium anisopliae (Metschnikoff) Sorokin.</a> Journal of Biological Control. 22 (2), 369-374 (2008).</li><li>Kassa, 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=Whey+for+mass+production+of+Beauveria+bassiana+and+Metarhizium+anisopliae.">Whey for mass production of Beauveria bassiana and Metarhizium anisopliae.</a> Mycological Research. 112 (5), 583-591 (2008).</li><li>Sharma, S., Gupta, R. B. L., Yadavam, C. P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+a+suitable+medium+for+mass+multiplication+of+entomofungal+pathogens.">Selection of a suitable medium for mass multiplication of entomofungal pathogens.</a> Indian Journal of Entomology. 64 (3), 254-261 (2002).</li><li>Bich, G. A., Castrillo, M. L., Villalba, L. L., Zapata, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+rice+by-products,+incubation+time,+and+photoperiod+for+solid+state+mass+multiplication+of+the+biocontrol+agents+Beauveria+bassiana+and+Metarhizium+anisopliae.">Evaluation of rice by-products, incubation time, and photoperiod for solid state mass multiplication of the biocontrol agents Beauveria bassiana and Metarhizium anisopliae.</a> Agronomy Research. 16 (5), 1921-1930 (2018).</li><li>Price, R. E., M&#252;ller, E. J., Brown, H. D., D'Uamba, P., Jone, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+trial+of+a+Metarhizium+anisopliae+var.+acridum+mycoinsecticide+for+the+control+of+the+red+locust+in+a+recognised+outbreak+area.">The first trial of a Metarhizium anisopliae var. acridum mycoinsecticide for the control of the red locust in a recognised outbreak area.</a> International Journal of Tropical Insect Science. 19 (4), 323-331 (1999).</li><li>Hatting, J. L., Moore, S. D., Malan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+control+of+phytophagous+invertebrate+pests+in+South+Africa.+Current+status+and+future+prospects.">Microbial control of phytophagous invertebrate pests in South Africa. Current status and future prospects.</a> Journal of Invertebrate Pathology. 165, 54-66 (2019).</li><li>Mathulwe, L. L., Malan, A. P., Stokwe, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+screening+of+entomopathogenic+fungi+and+nematodes+for+pathogenicity+against+the+obscure+mealybug,+Pseudococcus+viburni+(Hemiptera:+Pseudococcidae).">Laboratory screening of entomopathogenic fungi and nematodes for pathogenicity against the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae).</a> Biocontrol Science and Technology. , (2021).</li><li>Inglis, G. D., Enkerli, J., Goettel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+techniques+used+for+entomopathogenic+fungi:+Hypocreales.">Laboratory techniques used for entomopathogenic fungi: Hypocreales.</a> Manual of Techniques in Invertebrate Pathology. , 189-253 (2012).</li><li>Mehta, J., 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=Impact+of+carbon+&amp;+nitrogen+sources+on+the+Trichoderma+viride+(Biofungicide)+and+Beauveria+bassiana+(entomopathogenic+fungi).">Impact of carbon &amp; nitrogen sources on the Trichoderma viride (Biofungicide) and Beauveria bassiana (entomopathogenic fungi).</a> European Journal of Experimental Biology. 2 (6), 2061-2067 (2012).</li><li>Burges, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+of+mycoinsecticides.">Formulation of mycoinsecticides.</a> Formulation of Microbial Biopesticides. , 131-185 (1998).</li></ol>

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Tween 20</td>
			<td>Lasec</td>
			<td></td>
			<td>Added to conidial suspensions to allow fungal spores to mix with water</td>
		</tr>
		<tr>
			<td>20 mL McCartney bottles</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to make conidial suspensions</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td></td>
			<td></td>
			<td>Used as a cover of the cotton wool plugs on 250-mL flask</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td></td>
			<td></td>
			<td>Used to sterilize materials and ingredients used for the conidia production process</td>
		</tr>
		<tr>
			<td>Autoclave bags</td>
			<td>Lasec</td>
			<td></td>
			<td>Fermentation bags or solid substrate containers</td>
		</tr>
		<tr>
			<td>Autoclave tape</td>
			<td>Lasec</td>
			<td></td>
			<td>To secure PVC pipes on the fermentation bags</td>
		</tr>
		<tr>
			<td>Brown Kraft paper bags</td>
			<td></td>
			<td></td>
			<td>Used to dry conidia cultures on agricultural grains</td>
		</tr>
		<tr>
			<td>Bunsen burnner</td>
			<td>Labnet (Labnet International, Inc.)</td>
			<td></td>
			<td>Used to flame equipment (surgical blades,inoculating loops and rims of flasks)</td>
		</tr>
		<tr>
			<td>Clear edge test sieve</td>
			<td></td>
			<td></td>
			<td>Used to separate fungal conidia from agricultural grain substrates</td>
		</tr>
		<tr>
			<td>Corn steep liquor</td>
			<td>SIGMA</td>
			<td>66071-94-1</td>
			<td>Ingredient of the blastospore liquid medium</td>
		</tr>
		<tr>
			<td>Cotton Wool</td>
			<td>Lasec</td>
			<td></td>
			<td>Used as plug of the neck for fermentation bags</td>
		</tr>
		<tr>
			<td>Duran laboratory bottles</td>
			<td>Neolab</td>
			<td></td>
			<td>Used to autoclave SDA medium and distilled water</td>
		</tr>
		<tr>
			<td>Electrical tape</td>
			<td></td>
			<td></td>
			<td>Used to tape and seal the sieve joints to prevent the escape of conidial dust</td>
		</tr>
		<tr>
			<td>ENDECOTTS test sieve</td>
			<td></td>
			<td></td>
			<td>Used to separate fungal conidia from agricultural grain substrates</td>
		</tr>
		<tr>
			<td>Erlenmeyer Flasks, Narrow neck,250-mL flask</td>
			<td>Lasec</td>
			<td></td>
			<td>Carrier of the blastospore liquid medium</td>
		</tr>
		<tr>
			<td>Ethanol (99%)</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to sterilize surgical blades and inoculating loops</td>
		</tr>
		<tr>
			<td>Flaked barley</td>
			<td>Health Connection Wholefoods</td>
			<td></td>
			<td>Agricultural grain used as a solid substrate growth medium for conidia of both M. pinghaense and M. robertsii</td>
		</tr>
		<tr>
			<td>Flaked oats</td>
			<td>Tiger brands</td>
			<td></td>
			<td>Agricultural grain used as a solid substrate growth medium for conidia of both M. pinghaense and M. robertsii</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td></td>
			<td>Ingredient of the blastospore liquid medium</td>
		</tr>
		<tr>
			<td>Growth Chamber/ incubators</td>
			<td></td>
			<td></td>
			<td>For growing fungal conidia culture</td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td></td>
			<td></td>
			<td>Used to determine conidial concentrations</td>
		</tr>
		<tr>
			<td>Inoculating loops</td>
			<td>Lasec</td>
			<td></td>
			<td>For harvesting spores to innoculate liquid medium for blastospores growth</td>
		</tr>
		<tr>
			<td>Kitchen rolling pin</td>
			<td></td>
			<td></td>
			<td>Used to manipulate the solid grain substrate bed</td>
		</tr>
		<tr>
			<td>Laminar flow Cabinet</td>
			<td>ESCO Laminar Flow Cabinet</td>
			<td></td>
			<td>Provide as sterile environment during substrate inoculation</td>
		</tr>
		<tr>
			<td>Metarhizium pinghaense conidia</td>
			<td>Stellenbosch University</td>
			<td>5HEID</td>
			<td>Cultures used to mass culture conidia of Metarhizium pinghaense</td>
		</tr>
		<tr>
			<td>Metarhizium robertsii conidia</td>
			<td>Stellenbosch University</td>
			<td>6EIKEN</td>
			<td>Cultures used to mass culture conidia of Metarhizium robertsii</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEIZZ (Scope. A1)</td>
			<td></td>
			<td>Used to determine conidial concentrations and conidial viability</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>IncoShake- LABOTEC</td>
			<td></td>
			<td>Used for the blastospore production process</td>
		</tr>
		<tr>
			<td>Parboiled rice</td>
			<td>Spekko</td>
			<td></td>
			<td>Agricultural grain used as a solid substrate growth medium for conidia of both M. pinghaense and M. robertsii</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>SIGMA</td>
			<td></td>
			<td>Added to the SDA medium to prevent bacterial contamination</td>
		</tr>
		<tr>
			<td>Petri-dishes</td>
			<td>Lasec</td>
			<td></td>
			<td>Containers for the SDA medium</td>
		</tr>
		<tr>
			<td>Pipettes and pipette tips</td>
			<td>Labnet (BioPette PLUS)</td>
			<td></td>
			<td>Used to measure liquids ingredients</td>
		</tr>
		<tr>
			<td>Polyvinylchloride Marley waste pipe</td>
			<td></td>
			<td></td>
			<td>Used to create a neck for the fermentation bag</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic (K2HPO4)</td>
			<td>SIGMA-ALDRICH</td>
			<td></td>
			<td>Ingredient of the blastospore liquid medium</td>
		</tr>
		<tr>
			<td>Rubber band</td>
			<td></td>
			<td></td>
			<td>Used to secure the secure the surgical paper over the fermentation bag PVC pipe necks</td>
		</tr>
		<tr>
			<td>Sabaroud dextrose agar (SDA)</td>
			<td>NEOGEN Culture Media</td>
			<td></td>
			<td>Medium used to culture spores of both Metarhizium pinghaense and Metarhizium robertsii</td>
		</tr>
		<tr>
			<td>Sterile distilled water</td>
			<td></td>
			<td></td>
			<td>To hydrate agricultural grains, to make conidial suspensions</td>
		</tr>
		<tr>
			<td>Sticky pad</td>
			<td></td>
			<td></td>
			<td>Used to secure the seives on the vibratory shaker</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to scrape off spores from fungal cultures</td>
		</tr>
		<tr>
			<td>Surgical paper</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to cover the PVC necks and cotton wool plugs of the fermentation bag</td>
		</tr>
		<tr>
			<td>Vibratory shaker</td>
			<td></td>
			<td></td>
			<td>Used to shake conidia off the agricultural grain substrates</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Labnet (Labnet International, Inc.)</td>
			<td></td>
			<td>Used to mix conidial suspensions in Mc Cartney bottles</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Biolab</td>
			<td></td>
			<td>Added to the SDA medium to improve spore germination and growth</td>
		</tr>
		<tr>
			<td>Zipper-lock bags</td>
			<td>GLAD</td>
			<td></td>
			<td>Used to to store harvested fungal conidia</td>
		</tr>
	</tbody>
</table>

mass production of entomopathogenic fungi, metarhizium robertsii and metarhizium pinghaense, for commercial application against insect pests

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。

在真菌培养物的干燥阶段，随着时间的推移，观察到 M. pinghaense 和 M. robertsii 的水稻培养物含量质量下降，一旦培养物干燥，在质量上没有或几乎没有变化（图5）。收获的 M. pinghaense 和 M. robertsii 的干燥真菌分生孢子粉如图 6所示。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63246...

Watch this Scientific Journal Video about Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests at JoVE.com

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

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。

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. ...

鉴于目前大多数杀虫剂由于担心其对环境和人类健康的毒性作用而被逐步淘汰的情况，这种方法为如何大规模生产昆虫病原真菌的感染分生孢子以控制商业农业生态系统中的重要害虫提供了新的信息。该技术描述了用于在批量生产过程中确保EPF分生孢子最佳产量的方法。该方法有助于虫害综合治理和生物防治，为大规模生产的分生孢子对农业生态系统中破坏性杀虫剂的大规模管理的有效性提供见解。该程序由Letodi Luki Mathulwe博士，Nomakholwa Faith Stokwe博士和Antoinette Paula Malan教授开发和演示。加热一升蒸馏水，并在达到沸点之前关闭热量。现在，加入30克葡萄糖，四克磷酸钾二元，20克酵母提取物，15毫升玉米浸泡液，并将内容物轻轻煮沸两到三分钟。将总共100毫升培养基倒入九个不同的250毫升烧瓶中。在每个烧瓶上放一个棉絮塞，并用铝箔作为塞子盖住棉絮。将培养基在烧瓶中以121摄氏度高压灭菌55分钟。之后，让烧瓶中的培养基冷却，并向每个烧瓶中的培养基中加入每毫升链霉素10毫克。从两到三周龄的真菌培养板中收集两到三个真菌分生孢子的细菌环，用于两种EPF分离物。在无菌条件下将真菌分生孢子转移到250毫升烧瓶中的每100毫升液体培养基中，并密封烧瓶。将液体培养瓶在约25摄氏度的轨道振荡器上以140 RPM孵育三天，一旦培养物显示出高浊度和真菌囊孢子生长的迹象，就停止孵育。为了检测培养物中任何可能的细菌污染，在孵育24小时后从每个液体培养瓶中抽取100微升样品，并在每个分离物的三个SDA板上板。将板在正负25摄氏度的受控温度下孵育48小时。使用一个小的聚氯乙烯废液管在高压灭菌袋的开口端为发酵袋创建一个颈部，并使用高压灭菌胶带固定管道。用无菌棉絮塞关闭管道，以便在发酵过程中进行足够的气体交换。使用煮熟的长粒白米作为Metarhizium pinghaense和Metarhisrhizium robertsii的囊孢子的固体基质介质。对于每个发酵袋，加入一公斤大米和300毫升无菌蒸馏水。轻轻混合发酵袋的内容物。将它们放在外部高压灭菌袋中，以直立姿势放置，并在121摄氏度下高压灭菌55分钟。高压灭菌后，让基质在无菌条件下冷却约45分钟。在层流下取下Metarhizium pinghaense和Metarhisiu robertsii的每个液体培养瓶的封闭，并在每个烧瓶的边缘燃烧10秒钟。将100毫升液体培养物倒入发酵袋中，方法是从脖子上取下棉絮。将棉塞放回原处，并用橡皮筋固定的手术纸盖住袋子的颈部顶部。扭转袋子的顶部，通过摇晃和按摩操纵基质来混合袋子的内容物。通过在袋子中压平基材，在约25摄氏度下孵育袋子，以防止形成厚床。接种和孵育后两至三天，当发生可见的菌丝生长和真菌对底物的结合时，通过按摩袋子的内容物来破坏发酵袋中的底物。发酵后，将孢子培养物转移到26至30公斤的牛皮纸袋中，并在试验中使用之前将真菌培养物干燥10至12天。为了提高纸袋的抗拉强度，水平切掉袋子顶部1/3，并衬在袋子的底部。轻轻粉碎每个发酵袋中的底物。切掉每个袋子的角落，并通过切角留下的空间将整个培养物慢慢转移到纸袋上。为每个纸袋贴上标签，在每个袋子的顶端折叠两次，并用订书钉关闭以创建三角形帐篷结构。将袋子放在丝状干燥架上，以便在约25摄氏度的受控温度和30%至40%的低湿度下适当，均匀地干燥，使用安装在收集盘上的三个嵌套筛子从培养物中收获真菌分生孢子。将干培养样品缓慢加载到ETS网35号和筛子上。在筛子上盖上盖子，以防止真菌分生孢子释放到空气中。在筛子中加入10至12个玻璃弹珠，以协助真菌分生孢子通过网筛，避免分生孢子保留在筛子中，从而减少孢子回收。用电工胶带用胶带固定筛子接头并密封。将筛子放在装有粘性垫的振动筛上，以每分钟560至640次的振动将收集盘和筛子固定20至25分钟。从收集盘中取出测试筛，收集分生孢子，并将收集的分生孢子存放在密闭和不透水的拉链锁袋中，以便长期储存。平均从片状大麦底物中收获约1.83克干Metarhizium robertsii分生孢子，而从底物中收获的Metarhizium pinghaense为零。没有从片状燕麦底物中收获任何一种Metarhizium物种的干燥真菌分生孢子。在两种Metarhizium之间观察到从米粒中收获的每克平均分生孢子的估计差异显着。Metarhizium robertsii每克的平均分生孢子计数略高于Metarhizium pinghaense。两种Metarhizium在用过的水稻基质上存在的估计分生孢子数量上未观察到显着差异。然而，水稻基质上的分生孢子略高于Metarhizium robertsii，两种Metarhizium在水稻基质培养物获得的总分生孢子产量方面没有显着差异。然而，Metarhizium pinghaense产生的分生孢子产量略高于Metarhizium robertsii，后者产生的分生孢子产量较低。该技术具有挑战性的部分是通过接种受污染的囊孢子接种物来污染底物。因此，在工作时始终确保无菌。该演示可帮助学生有效地重现该技术，而不会出现可能导致整个过程终止的笨拙错误。

Research

JoVE Journal

Biology

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

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

重组昆虫细胞中的mCD1d的蛋白质的生产方法。

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

科研

生物学

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

膜和细胞壁选择性荧光染料在丝状真菌活细胞成像中的应用

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

使用杆状病毒-昆虫细胞培养系统生产和纯化腺相关病毒（AAV）2载体的工艺开发

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera ...

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

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 ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

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 ...

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

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

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

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

<meta charset="utf8"></head><body>探索森林蛀木虫的昆虫病原真菌资源

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

<meta charset="utf8"></head><body>作者聚焦：野生 Monochamus alternatus 种群中昆虫病原真菌的评估，用于森林蛀木虫的生物防治应用

森林蛀木虫昆虫病原真菌的分离、行为鉴定和致病性评估

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

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

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