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.
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 ± 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 ± 4.38 g and 6 g ± 2 g harvested from the substrate, respectively.
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 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 & 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® (strain IMI 330189), developed from M. anisopliae var Metarhizium acridum (Driver & Milner) J.F. Bisch, Rehner & Humber, Metarhizium 69 (Meta 69 strain ICIPE69), and Real Metarhizium 69 (L9281), developed from M. anisopliae, and Broadband® (strain PPRI 5339) and Eco-Bb®, developed from B. bassiana25,26. However, limited attempts have been made to mass culture Metarhizium robertsii J.F. Bisch., S.A. Rehner & Humber and Metarhizium pinghaense Chen & 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.
1. Source of fungal strains
2. Metarhizium pinghaense and M. robertsii conidial suspension inoculation
3. Blastospore inoculation
Figure 1: Liquid culture medium in 250-mL flasks. (A) Before autoclaving. (B) After autoclaving and inoculation with EPF spores. (C) Turbid medium with fungal blastospores. Please click here to view a larger version of this figure.
Figure 2: Prepared blastospore liquid culture medium. (A) Metarhizium robertsii and (B) Metarhizium pinghaense prior to the inoculation of rice as a solid substrate. Please click here to view a larger version of this figure.
4. Drying of fungal cultures
Figure 3: Preparation of paper bags, drying procedure of cultures, and packaging. (A,B) The preparation of brown paper bags. The drying procedure of Metarhizium species cultures grown on (C,E) parboiled rice and (D,F) flaked barley. (G) Paper bags closed with staples to create a triangular tent structure. Please click here to view a larger version of this figure.
5. Harvest of fungal conidia
Figure 4: Harvesting of fungal spores from dried Metarhizium robertsii cultures on rice and flaked barley. (A) 10-12 glass marbles added to the sieves to assist the passage of the fungal conidia through the mesh screens. M. robertsii conidia harvested from cultures on (B) rice, and (C) flaked barely. (D) Sieves on a vibratory shaker. Please click here to view a larger version of this figure.
6. Quantification of fungal conidia produced
7. Data analysis
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.
Figure 5: The change in the bag content mass of the Metarhizium robertsii and the M. pinghaense cultures on rice (95% confidence interval) over 10 days (ANOVA; F5,35 = 2.21; p = 0.08). Different letters above the bars indicate the significant difference (p < 0.05) obtained in bag content mass over the days. Please click here to view a larger version of this figure.
Figure 6: Harvested conidia. (A) Harvested conidia in the collection pan. (B) Metarhizium robertsii. (C) M. pinghaense. Please click here to view a larger version of this figure.
No significant difference was observed in the average conidial powder that was harvested from each grain type for both M. pinghaense and M. robertsii. Harvested dry conidia for both had an average of >90% conidial viability for both M. pinghaense and M. robertsii. On the rice substrate, M. pinghaense produced an average of 8.2 g ± 4.38 g of conidial powder, which slightly exceeded the amount that was obtained for M. robertsii (6 g ± 2 g). An average of 1.83 g ± 1.47 g of dry M. robertsii conidia was harvested from the flaked barley substrate, whereas zero M. pinghaense was harvested from the substrate. No dry fungal conidia were harvested from the flaked oats substrate for either M. pinghaense or M. robertsii (Figure 7).
Figure 7: Average amount of conidia powder. The average amount of conidia powder of Metarhizium pinghaense and M. robertsii, measured in grams (95% confidence interval) harvested from the three solid substrates: parboiled rice, flaked barley, and flaked oats (ANOVA: F2,27 = 2.82; p = 0.08). Please click here to view a larger version of this figure.
A significant difference in the estimated average conidia per gram, harvested from the rice grains, was observed between M. pinghaense (3.51 x 107/g ± 0.43 x 107/g) and M. robertsii (4.31 x 107/g ± 0.38 x 107/g). M. robertsii had a slightly higher average conidia count per gram than M. pinghaense (Figure 8).
Figure 8: Average conidia per gram. The estimated average conidia per gram for Metarhizium pinghaense and M. robertsii (95% confidence interval) from the rice cultures (ANOVA: F 1,7 = 8.47; p = 0.02). Please click here to view a larger version of this figure.
No significant difference in the estimated number of conidia present on the spent rice substrate was observed between M. pinghaense (5.02 x 107/mL ± 1.47 x 107/mL) and M. robertsii (3.70 x 107/mL ± 0.91 x 107/mL) (Figure 9). However, M. pinghaense had a slightly higher number of conidia present on the rice substrate than M. robertsii.
Figure 9: Average fungal conidia. The estimated average fungal conidia of Metarhizium pinghaense and M robertsii (95% confidence interval) from the cultures (F1,7 = 2.45; p = 0.16) present on the spent rice culture substrates. Please click here to view a larger version of this figure.
No significant difference in the estimated overall conidia yield was observed between the M. pinghaense (5.09 x 107/g ± 1.35 x 107/g) and the M. robertsii (3.55 x 107/g ± 0.85 x 107/g) obtained from the rice substrate cultures. However, M. pinghaense produced a slightly higher conidial yield than did M. robertsii, which produced a lower conidial yield (Figure 10).
Figure 10: Total conidia yield. The estimated total conidia yield of Metarhizium pinghaense and M. robertsii (95% confidence interval) from the cultures on rice (F1,7 = 3.91; p = 0.09). Please click here to view a larger version of this figure.
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 control9,10,11,12,13. The success of mass production of different EPF isolates is influenced by the nutritional components of the production substrate or medium, which significantly impact the attributes of the resulting conidia, including their efficacy, desiccation tolerance, field persistence, and conidial yield8,19,20,21. Therefore, developing knowledge of the nutritional requirements of mass-produced entomopathogens during the cultivation and fermentation process is important29.
In the current study, the mass production of both M. robertsii and M. pinghaense was conducted using three different agricultural products as fermentation substrates: parboiled rice, flaked barley, and flaked oats. The three different grain substrates were observed to influence the conidial yield of the fungal isolates significantly. The parboiled rice was highly favorable for mass production of both isolates, while the flaked barley grains favored the production of M. robertsii only. The observations were made based on the yield of dry fungal conidia powder following fermentation. Little or no, conidia were harvested from the flaked oats after fermentation. The flaked oats were found to retain higher levels of moisture in the fermentation bags during the fermentation process, which might have resulted in the observed poor performance of the grain as a substrate for the production of M. pinghaense and M. robertsii. The flaked oats substrate was prone to a white vegetative mycelial overgrowth of the fungal isolates in some instances, which greatly influenced the conidial yield, as sporulation did not occur in the fermentation bags containing the overgrowth. The fermentation bags with the flaked oats substrate were also observed to have high levels of bacterial contamination relative to the fermentation bags containing flaked barley substrates.
High levels of saprophytic fungi or yeast contamination were observed concerning the mass production of M. pinghaense using flaked barley as a substrate. The saprophytic fungal contamination was observed at a later stage of the fermentation process through the growth and conidiogenesis of a fungus other than the one used to inoculate the substrate. Diagnosing substrate contamination at an initial stage of fermentation, prior to conidial sporulation, was difficult. Bacterial and yeast contamination during the fermentation process is thought to be manifested by wet patches in the substrate, and both flaked oats and barley grains tend to absorb and retain moisture for longer than rice grains. This was a limitation to using these two substrates as fermentation substrates for the mass production of the two Metarhizium isolates.
The observations made during the current study showed that the type of inoculation method used to inoculate each grain substrate affected conidial production. The conidial suspension inoculation method was found to be relatively less effective, as the inoculated flaked oats and flaked barley showed an increased level of contamination relative to when the liquid-cultured blastospore inoculation method was used on rice grains. Unlike the liquid-cultured blastospore inoculation method, the conidial suspension method does not allow for the detection of possible contaminants in the suspensions prior to inoculation of the substrates, resulting in the high levels of contamination that were observed in the fermentation bags. Jaronski and Jackson18 described several factors that could lead to the possible contamination of the substrates used, such as the lack of complete sterilization of the substrate, nonsterile inoculation, and improper handling of the fermentation substrate in the bags. The observed contamination in the fermentation bags containing flaked barley and flaked oats might also have resulted from a lack of complete sterilization of the substrate, which could have occurred during the autoclaving process. Jaronski and Jackson18 also described the use of conidial suspensions as inoculants as being disadvantageous, as it results in increased chances of contamination during the conidial harvest from an agar medium.
In the field, during the infection of suitable hosts, the fungus first builds up biomass in the insects' hemocoel through proliferation, prior to its emergence and sporulation on the outer solid surface of the insects30. Such a process was mimicked when using the liquid-blastospore fermentation method. Accordingly, the broth represented the insect's hemocoel, the inoculation of the broth with fungal conidia represented the initial contact and infection of the insect, and the formation of blastospores in the broth represented the proliferation of the blastospore process that occurs inside the infected insect's hemocoel. The final inoculation of the rice grains and sporulation of the fungus on the grains over time represented what occurs on the insect's cuticle once the fungus begins to emerge from inside the insect's cadaver. This can possibly explain why the liquid blastospore fermentation method worked better than the conidial suspension inoculation method.
Jaronski and Jackson18 described flaked oats and flaked barley grains as the preferred agricultural grains for mass culture of Metarhizium species, compared to rice grains, as the two grain types were found to maintain their moisture content during the fermentation process. However, the current study contradicts the aforementioned researchers' observations and conclusions, as both the flaked oats and the flaked barley were not found to be good solid fermentation substrates for either M. pinghaense or M. robertsii. The rice was found to be a good grain substrate for both isolates, as it did not decompose into small particles that could mix with the final product, as was suggested in previous studies.
The current study has shown that isolates of the Metarhizium species can be successfully cultured and mass-produced using rice grains and that the different grains tend to influence the conidial sporulation and yield differently. This could possibly be due to the fact that different grains differ in terms of such nutrition composition, e.g., carbon: nitrogen ratios and carbon concentration. The carbon concentration and carbon: nitrogen ratio of a growth medium are known to affect the yield of conidia, as well as it's quality in terms of viability and virulence22. The inoculation techniques used to inoculate the substrates have also been shown to influence the degree of success attained in the mass production of EPF isolates on specific agricultural grain substrates. Therefore, the liquid blastospore fermentation technique described in the current study is recommended as being the best inoculation method for agricultural grain substrates for the mass production of both M. pinghaense and M. robertsii isolates. Using such a method allows for the possible detection of contamination prior to the use of the inoculants, which can hinder the mass production of the desired EPF isolate relative to the use of the conidial suspension inoculation technique. It is also recommended that rice grains be used as the solid substrates for mass production of conidia of both M. pinghaense and M. robertsii, rather than flaked barley and flaked oats. The described technique is important for future mass production and commercial application of the conidia under field conditions against important insect pests in the agroecosystems.
The authors have nothing to disclose.
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
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 |