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

The authors would like to thank Hort Pome, Hort Stone, and the Technology and Human Resources for Industry Programme (THRIP: TP14062571871) for funding the project.
ORCID: 
Letodi L. Mathulwe http://orcid.org/0000-0002-5118-3578
Antoinette P. Malan http://orcid.org/0000-0002-9257-0312
Nomakholwa F. Stokwe http://orcid.org/0000-0003-2869-5652

1. Source of fungal strains
<ol>
	<li>Use South African isolated fungal strains of both M. pinghaense 5 HEID (GenBank Accession number: MT367414/MT895630) and M. robertsii 6EIKEN (MT378171/MT380849), collected from apple orchards in the Western Cape province, South Africa.</li>
	<li>Grow cultures of each EPF isolate on 60 g of Sabouraud dextrose agar medium, supplemented with 1 g of yeast extract (SDAY) and 10 &#181;L of Streptomycin. 
	NOTE: Incubate EPF cultures at a c...

<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. 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The successful integration of microbial agents for the biological control of important agricultural insect pests in an agroecosystem depends on both success and ease of mass production of the entomopathogens as the first step under laboratory conditions. The mass production of EPF is important for the large-scale application and availability of EPF products for IPM programs using biological control9,10,11,<sup class="...

Entomopathogenic fungi (EPF) have gained importance as crop protection agents in the biological control of important agricultural insect pests1,2. The entomopathogens, which occur naturally in soil, cause epizootics in the populations of various pest species3. The species of EPF are host-specific and pose relatively few risks in terms of attacking nontarget species, and they are nontoxic to the environment4. EPF have a unique mechanism for invading their host, as well as for propagating and persisting in their immediate environment1. They attack the host mainly through asexual spores that attach to and penetrate the host cuticle to invade and proliferate in the host hemocoel. The host eventually dies due to depletion of the hemolymph nutrients or as a result of the toxemia caused by the toxic metabolites released by the fungus. Following death, under ideal environmental conditions, the fungus emerges on the outer surface (overt mycosis) of the host cadaver5,6.
Growing concerns regarding the negative effects of chemical residues on human health, environmental pollution, and the development of pest resistance have led to the global drive to reduce inputs of chemical-based insecticides and to find alternative, novel, and sustainable strategies for crop protection and pest control6,7,8. This has provided opportunities to develop microbial-based insecticides for use in Integrated Pest Management (IPM) programs, which are more ecologically favorable strategies than conventional chemical control3,8.
To develop a successful microbial control agent for an agricultural pest, a suitable organism must first be isolated, characterized, identified and its pathogenicity for the&#160;target pest confirmed. However, an easy, cost-effective method for large-scale production of the microbial agent is required to produce a viable product for use in biological control programmes9,10,11,12,13. Mass production of substantial quantities of good-quality entomopathogens depends on the microbial strain, the environment, the target pest, the formulation, the market, the application strategy, and the desired end product14,15,16. EPF can be mass-produced using liquid substrate fermentation to produce blastospores or the solid substrate fermentation process to produce aerial conidia6,17,18. However, the mass production and formulation process of entomopathogens directly influences the virulence, the cost, the shelf life, and the field efficacy of the final product. For successful use in IPM, the production process of the entomopathogens must be easy to run, require minimal labor, produce a high-yield concentration of virulent, viable, and persistent propagules, and be low in cost4,13,14,16.
Understanding the nutritional requirements of entomopathogens is important for mass cultivation with all culturing methods4,12. The nutritional components of the production medium have a significant impact on the attributes of the resulting propagules, including biocontrol efficacy, yield, desiccation tolerance, and persistence8,19,20,21. The optimization of production procedures is designed to address such factors22. For EPF, the main requirements for good growth, sporulation, and mass production of fungal conidia are adequate moisture, optimum growth temperature, pH, gas exchange of CO2 and O2, and nutrition, including good phosphorous, carbohydrate, carbon, and nitrogen sources18.
Jaronski and Jackson18 describe the solid substrate fermentation method as the most efficient and the closest approximation method to the natural process for EPF production relative to the liquid substrate fermentation method because, under natural conditions, the fungal conidium is borne on solid erect structures, like the surface of insect cadavers. Agricultural products and by-products containing starch are mostly used for the mass production of hypocrealean fungi, as the fungi readily decompose starch through secretion of highly concentrated hydrolytic enzymes from their hyphal tips, to penetrate the solid substance, and to access the nutrients present in the substance11,17,18,23. The grain products also provide the requirements for healthy biomass production because, when they are hydrated and sterilized, the substrates can absorb further nutrients from any liquid medium16,18,24.
Previously, several studies attempted to mass culture EPF species like Beauveria bassiana (Bals.) Vuil., Cordyceps fumosorosea (Wize) Kelper B. Shrestha &#38; Spatafora, Verticillium lecanii (Zimm.) Viegas and some of the Metarhizium anisopliae (Metschn.) Sorokin species complex isolates on various substrates16,23,24. Such mass-produced and commercially developed isolates include Green Muscle&#174; (strain IMI 330189), developed from M. anisopliae var Metarhizium acridum (Driver &#38; Milner) J.F. Bisch, Rehner &#38; Humber, Metarhizium 69 (Meta 69 strain ICIPE69), and Real Metarhizium 69 (L9281), developed from M. anisopliae, and Broadband&#174; (strain PPRI 5339) and Eco-Bb&#174;, developed from B. bassiana25,26. However, limited attempts have been made to mass culture Metarhizium robertsii J.F. Bisch., S.A. Rehner &#38; Humber and Metarhizium pinghaense Chen &#38; Guo. These two isolates were selected in a previous study as the most effective for the control of the mealybug, Pseudococcus viburni Signoret (Hemiptera: Pseudococcidae)27. Therefore, the current study aimed to formulate and mass-produce a sufficient number of resilient infective propagules of the local isolates of M. robertsii and M. pinghaense for commercial application against insect pests. The solid substrate fermentation method was used to mass-produce the fungal conidia for both EPF isolates. Two EPF inoculation methods, using conidial suspensions and the liquid fungal culture of blastospores, were used to inoculate the solid substrates.

<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

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.

A decline in the content mass of the cultures on rice for both the M. pinghaense and the M. robertsii was observed over time during the drying stage of the fungal cultures, with no, or little, change being observed in the mass once the cultures were dry (Figure 5). The harvested dry fungal conidia powder of both the M. pinghaense and the M. robertsii is shown in Figure 6.
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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

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.

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. ...

This method provides new information on how to mass produce infective conidia of entomopathogenic fungi to control important insect pests in commercial agro-ecosystems, given the current situation where most insecticides are phased out due to concerns of their toxic effect on the environment and human health. The technique describes the method used to ensure the optimum yield of EPF conidia during mass production. The method is helpful for integrated pest management and biological control by providing insight into the effectiveness of mass produced conidia for large-scale management of damaging insecticide resistance insect pests in the agro-ecosystems.This procedure was developed and demonstrated by Dr.Letodi Luki Mathulwe, Dr.Nomakholwa Faith Stokwe, and Professor Antoinette Paula Malan. Heat one liter distilled water and switch off the heat before reaching the boiling point. Now, add 30 grams of glucose, four grams of potassium phosphate dibasic, 20 grams of yeast extract, 15 milliliters of corn steep liquor, and bring the contents to a gentle boil for two to three minutes.Pour a total of 100 milliliters of the medium into nine different 250-milliliter flasks. Place a cotton wool plug on each flask and cover the cotton wool with aluminum foil as a stopper. Autoclave the medium in the flasks for 55 minutes at 121 degrees Celsius.Later, allow the medium in the flasks to cool and add 10 milligrams per milliliter of streptomycin to the medium in each flask. Collect two to three bacterial loops of fungal conidia from two-to three-week-old fungal culture plates for both the EPF isolates. Transfer the fungal conidia to each 100 milliliters of liquid media in the 250 milliliter flasks under sterile conditions and seal the flasks.Incubate the liquid culture flasks at approximately 25 degrees Celsius on an orbital shaker at 140 RPM for three days and cease the incubation once the cultures show signs of high turbidity with fungal blastospore growth. To detect any possible bacterial contamination from the cultures, draw a 100-microliters sample from each liquid culture flask after 24 hours of incubation and plate on three SDA plates per isolate. Incubate the plates for 48 hours at a controlled temperature of plus or minus 25 degrees Celsius.Use a small polyvinyl chloride waste pipe to create a neck for the fermentation bag at the open end of the autoclave bag and use autoclave tape to secure the pipe. Close the pipe with a sterile cotton wool plug to allow sufficient gas exchange during fermentation. Use parboiled long grain white rice as a solid substrate medium for the blastospores of both Metarhizium pinghaense and Metarhizium robertsii.For each fermentation bag, add one kilogram of rice and 300 milliliters of sterile distilled water. Gently mix the contents of the fermentation bags. Place them in outer autoclave bags in an upright position and autoclave at 121 degrees Celsius for 55 minutes.Following the autoclaving, allow the substrates to cool down for about 45 minutes under sterile conditions. Remove the closure of each of the liquid culture flasks of both Metarhizium pinghaense and Metarhizium robertsii under a laminar flow and flame the rim of each flask for 10 seconds. Pour 100 milliliters of liquid cultures into the fermentation bags by removing the cotton wool plugs from their necks.Place the cotton plugs back on and cover the top of the bag's neck with surgical paper secured with a rubber band. Twist the top part of the bag and mix the bag's contents by shaking and manipulating the substrate by massage. Incubate the bags at about 25 degrees Celsius by flattening the substrate in the bags to prevent the formation of thick beds.Two to three days after inoculation and incubation, when visible mycelial growth and the binding of the substrate by the fungus had occurred, break the substrate in the fermentation bags by massaging the bags'contents. Following fermentation, transfer the sporulated cultures into 26-to 30-kilogram brown paper bags and dry the fungal cultures for 10 to 12 days before their use in trials. To improve the tensile strength of the paper bags, horizontally cut off 1/3 of the top part of the bag and line the bottom of the bag.Gently crumble the substrate in each fermentation bag. Cut off the corner of each bag and slowly transfer the whole culture to the paper bags through the space left by the cut off corner. Label each paper bag, fold over the top end of each bag twice, and close with staples to create a triangular tent structure.Place the bags on wire drying racks to allow proper, even drying at a controlled temperature of about 25 degrees Celsius and low humidity of 30 to 40%Harvest fungal conidia from the cultures using three nested sieves mounted on a collection pan. Slowly load the dry culture sample on the ETS mesh number 35 and sieve. Place a lid on the sieve to prevent the release of fungal conidia into the air.Add 10 to 12 glass marbles to the sieves to assist the passage of the fungal conidia through the mesh screens and avoid the retention of the conidia in the sieve, which can result in reduced spore recovery. Tape and seal the sieve joints using electrical tape. Place the sieves on a vibratory shaker fitted with a sticky pad to secure the collection pan and sieves for 20 to 25 minutes at a motion of 560 to 640 vibrations per minute.Remove the test sieves from the collection pan, collect conidia, and store the collected conidia in airtight and water-impermeable zipper-lock bags for long-term storage. An average of approximately 1.83 grams of dry Metarhizium robertsii conidia was harvested from the flaked barley substrate, whereas zero Metarhizium pinghaense was harvested from the substrate. No dry fungal conidia were harvested from the flaked oat substrate for either Metarhizium species.A significant difference was observed between the two species of Metarhizium in the estimated average conidia per gram harvested from the rice grains. Metarhizium robertsii had slightly higher average conidia count per gram than Metarhizium pinghaense. No significant difference was observed between the two species of Metarhizium in the estimated number of conidia present on the spent rice substrate.However, Metarhizium pinghaense had slightly higher conidia on the rice substrate than Metarhizium robertsii No significant difference is estimated in the two species of Metarhizium in their overall conidia yield obtained from the rice substrate cultures. However, Metarhizium pinghaense produced a slightly higher conidia yield than did Metarhizium robertsii, which produced a lower conidial yield. The challenging part of this technique is the substrate contamination through inoculation with a contaminated blastospores inoculum.Thus, always ensure sterility while working. This demonstration helps the students efficiently reproduce the technique without clumsy mistakes that can result in the termination of the entire process.

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Research

JoVE Journal

Biology

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

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

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

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

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

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

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

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

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 highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

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

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

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

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.

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

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.

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

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).

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.)

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.

Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

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.

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

Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

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

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Author Spotlight: Evaluation of Entomopathogenic Fungi in Wild Monochamus alternatus Populations for Biocontrol Applications in Forest Wood Borers

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

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

Entomopathogenic Fungi (EPF) Preparation and Virulence Screening Against Mustard Aphids

For the EPF recovery from the fungal library, start by smearing the fungal isolates on a quarter strength SDA plate. Incubate it in darkness at 25 degrees Celsius for 10-14 days before conidia harvest. Following this, pipette 2-3 mL of 0.03%Tween 80 onto a cultured plate.Then use an inoculation loop to scrape the conidia by. Next, transfer the fungal suspension to a centrifuge tube. Vigorously vortex the suspension at the highest speed, then pipette out a sample into a hemocytometer to count the number of conidia.Dilute the conidia within the suspension using 0.03%Tween 80 til desired concentration is obtained. To prepare the inoculation chamber, first cut a 9 centimeter diameter leaf disk using the bottom Petri dish as a mold or with scissors. Next, prepare 1.5%water agar for each dish using a microwave.Pour 30 mL of the cooled water agar into the Petri dish and let it solidify in a laminar flow hood. When the agar surface is semisolid, place the leaf disk with its abaxial surface facing up, then press it slightly to imbed it in the agar. Now, place 20 apterous adults on the leaf disk.Open the cover of the inoculation chamber to spray 0.3 mL of conidia suspension directly onto the aphids and the leaf disk. Once the suspension has dried, close the cover to prevent the drowning of the mustard aphids. Subsequently, seal the inoculation chamber using paraffin film to maintain a high mount of humidity.Place the chamber in an incubator set at a temperature of 25 degrees Celsius with a light and dark photoperiod of 12 hours each. Use a stereomicroscope to count the mortality every 12 hours for 5 days. The virulence screening revealed a consistent survival rate for mustard aphids with the control group exhibiting an 85%survival rate.Cordyceps cateninnulata demonstrated the fastest aphid-killing ability, resulting in 50%and 90%moralities at 3 days and 4.5 days post-inoculation, or DPI. Beauveria strains 141, 143, and 153 showed slow aphid-killing abilities with only 5%mortality at 3 DPI. Apart from Purpureocillium, most EPF isolates exhibited corrected mortality rates higher than 70%within 5 DPI.Beauveria bassiana demonstrated the highest mortality rate of 100%among EPF isolates. EPF mycosis was observed on cadavers of mustard aphids infected with Metarhizium species, Beauveria species, Purpureocillium lilacinum, and Cordyceps cateniannulata during the virulence screening.