|Year : 2021 | Volume
| Issue : 4 | Page : 346-351
Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhiziumys against Aedes aegypti larvae
Aline Teixeira Carolino1, Thais Berçot Pontes Teodoro1, Simone Azevedo Gomes1, Carlos Peres Silva2, Richard Ian Samuels1
1 Universidade Estadual do Norte Fluminense Darcy Ribeiro, CCTA-LEF Av. Alberto Lamego 2000 Campos dos Goytacazes RJ 28013-602, Brazil
2 UFSC Faculdade de Biociências, Florianópolis, SC, Brazil
|Date of Submission||29-Jun-2020|
|Date of Acceptance||21-Jan-2021|
|Date of Web Publication||25-Mar-2022|
Richard Ian Samuels
UENF-CCTA-LEF, Av. Alberto Lamego 2000, Campos dos Goytacazes, RJ 28013-602
Source of Support: None, Conflict of Interest: None
Background & objectives: Entomopathogenic fungi are being investigated for the biological control of a range of mosquitoes. Metarhizium conidiospores (conidia) effectively kill Aedes aegypti larvae and could be deployed as an alternative to chemical insecticides. Conidial yield and virulence of fungi when cultured on three different types of solid media, was investigated.
Methods: Three culture media were tested: a) Sabouraud dextrose agar (SDA); b) rice flour yeast agar (RYA) and c) rice grains. Conidia produced using these substrates were tested for virulence against Ae. aegypti larvae obtained from field collected eggs. Larvae (2nd – 3rd instar) were exposed to aqueous conidial suspensions and survival monitored over 7 days. Survival analysis was performed using Log-Rank and Kaplan Meier tests, while fungal growth and conidial yields were analyzed using a two-way ANOVA.
Results: There were only small differences between growth rates on RYA and SDA; however, ESALQ 818 showed the highest conidial yield on rice. Conidia produced on rice grains were more virulent, rapidly reducing survival rates of mosquito larvae. ESALQ 818 conidia produced on rice grains, RYA and SDA killed 100% of the larvae on the 2nd, 3rd and 4th day of exposure, respectively. IP 46 virulence of was consistently lower than ESALQ 818 for all the media tested.
Interpretation & conclusion: The choice of culture media can influence the virulence of fungal conidia to Ae. aegypti larvae, demonstrating the importance of not only selecting the most virulent isolate but also standardizing growth conditions when screening for virulence.
Keywords: Culture media; Entomopathogenic fungus; Virulence; Infection; Biological control
|How to cite this article:|
Carolino AT, Teodoro TB, Gomes SA, Silva CP, Samuels RI. Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhiziumys against Aedes aegypti larvae. J Vector Borne Dis 2021;58:346-51
|How to cite this URL:|
Carolino AT, Teodoro TB, Gomes SA, Silva CP, Samuels RI. Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhiziumys against Aedes aegypti larvae. J Vector Borne Dis [serial online] 2021 [cited 2022 May 21];58:346-51. Available from: https://www.jvbd.org/text.asp?2021/58/4/346/318315
| Introduction|| |
Aedes aegypti Linnaeus, 1762 (Diptera: Culicidae) is a vector of four highly important etiological agents: dengue, chikungunya, Zika and urban yellow fever. Application of synthetic insecticides is currently the most important method used to control these insects and reduce the incidence of diseases. However, the cumulative effects of using chemical insecticides, which select resistant insects, as well as damage the environment and cause human health concerns, have encouraged the search for alternative methods of mosquito control. Alternative control methods could help reduce the negative impact on the environment caused by the action of chemical agents,. The biological control of vector insects is promising, especially with new developments in the deployment and formulation of entomopathogenic fungi. Fungal encapsulation techniques, combining fungi with attractive baits and the use of blastospores rather than conidia, are some of the new approaches being investigated to improve the efficiency of fungal biological control agents. Furthermore, the bioactive metabolites produced by entomopathogenic fungi could also be a source of novel insecticidal compounds. A recent study showed that Metarhizium anisopliae metabolites had larvicidal activity against three species of vector mosquitoes, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus.
Conidiospores (conidia) are the most highly investigated and utilized agents for the microbial control of insects, although some studies have shown that blastospores can be more virulent than conidia,. The virulence of conidia against larvae and adult Ae. aegypti has been demonstrated under laboratory and field conditions,. Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) conidia differ from blastospores of the same fungus in that they possess a thick cell wall, which makes this structure more tolerant to the stress of natural environmental conditions.
The use of different culture media has been investigated with the aim of improving the efficiency of conidia and blastospores,. The influence of different substrates on virulence against Ae. aegypti larvae and adults was investigated for two species of entomopathogenic fungi, M. anisopliae and Beauveria bassiana. Maldonado-Blanco and co-workers demonstrated that the highest levels of virulence were recorded when M. anisopliae was cultured in casamino acids medium. Culture media can exert an effect on the production of key enzymes involved in the infection process, such as Pr1 and Pr2, and these enzymes are considered as virulence factors. For mass production of conidia, the use of rice grains as a substrate is economical and highly efficient, although alternative substrates have also been investigated such as sorghum, wheat bran, barley, sugar-cane bagasse and millet. When Metarhizium anisopliae was cultured on millet, the conidia were shown to have potential for the control of the Asian tiger mosquito Aedes albopictus larvae. However, rice continues to be an excellent substrate, producing high concentrations of infectious propagules.
The current study investigated the effect of three different culture media on conidial production and virulence. The virulence of two species of Metarhizium (Metarhizium anisopliae and Metarhizium humberi) was evaluated against Ae. aegypti larvae. The results highlight the importance of the correct choice of conidial production media when considering specific insect targets, not only to optimize yield, but also to maximize virulence.
| Material & Methods|| |
Mosquito collection and rearing
All of the experiments used larvae which were reared from eggs collected in the field (University Campus). One of the aims of this study was to test the virulence of fungal isolates against mosquito larvae from natural populations, rather than laboratory lines. It is probable that laboratory mosquito lines are not as fit as natural populations and therefore any new control strategies should be tested against genotypes that are present in the field.
Aedes aegypti eggs were collected in the field using “ovitraps”. Ovitraps were black plastic plant pots (12 cm in diameter x 15 cm in height) with 4 wooden strips (3 x 12 cm) placed vertically within the pots, which act as attractive surfaces for mosquito oviposition. About 300 mL of tap water was added to each ovitrap. The ovitraps were placed near the University Insectary (Latitude: -21°45’8.11”S; Longitude: -41°19’49.58”W) in positions that were protected from rain and direct sunlight. After 5 days, the wooden strips were dried at room temperature for 24 h. The strips were examined for the presence of eggs. One strip could normally collect on average up to 100 eggs. This technique specifically collects Aedes eggs and adult emergence studies by our group have shown that > 90% of the eggs were Aedes aegypti and <10% were Aedes albopictus (unpublished results).
Strips with eggs were then submerged in water to stimulate larval emergence. Larvae were maintained in plastic trays (approximately 100 larvae per 100 mL) and feed on freshly ground and autoclaved commercial fish food (Alcon®, Camboriú, SC, Brazil; 0.05 g per L). Second and third instar larvae were selected for bioassays as there was sufficient time before pupation occurred to evaluate differences in survival. Fourth and fifth instar larvae are not appropriate for this type of bioassay.
Metarhizium anisopliae (ESALQ 818) was obtained from the Universidade de São Paulo - Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ) collection in Piracicaba, São Paulo, Brazil. Metarhizium humberi (IP 46) was obtained from the Institute of Tropical Pathology and Public Health, at the Federal University of Goiás, Goiânia, Brazil. ESALQ 818 was chosen for this study as virulence of this isolate against Ae. aegypti larvae has been demonstrated by our research group. IP 46 was chosen for this comparative study as it is a promising candidate for mosquito biological control programs.
For the virulence assays, three different media were used: (1) Sabouraud Dextrose Agar (SDA); (2) rice flour + yeast Agar media (RYA) and (3) rice grains. SDA (dextrose 10 g; peptone 2.5 g; yeast extract 2.5 g; agar 20 g in 1 L H20) was prepared following manufacture instructions (LABSYNTH Ltd. São Paulo, Brazil) and autoclaved for 20 min at 121°C. For RYA, 20 g of rice flour (SADIA Ltd. São Paulo, Brazil) was mixed with 2.5 g of yeast extract (LABSYNTH Ltd.) and 20 g of agar (KASVI Ltd. São Paulo, Brazil) in 1L H20 and autoclaved as described for SDA. Five hundred μL of conidial suspensions (1x107 conidia mL-1) of both Metarhizium isolates were inoculated onto SDA and RYA plates.
For rice grain media, 50g of parboiled white grain rice (Tio João, São Paulo, Brazil) were presoaked in 30 mL of water for 30 min, placed in 250 mL conical flasks and then autoclaved as above. A volume of 500 μL of conidial suspensions of either ESALQ 818 or IP 46 adjusted to 1x107 conidia mL-1, following conidial concentration evaluation using a Neubauer hemocytometer, was inoculated into the previously sterilized conical flasks containing rice. All fungal cultures were incubated at 27°C and 12L:12D photoperiod for 15 days before harvesting the conidia by rinsing the Petri dishes with aqueous Tween 80 (0.05%). These suspensions were used in the subsequent experiments.
Growth and conidi alyield of fungal isolates on different culture media
In order to determine the effect of different media on fungi growth and conidial yield, 10 μL of conidial suspensions of ESALQ 818 or IP 46 (1x106 conidia mL-1) were inoculated in the center of plastic Petri dishes (9 cm diameter) containing SDA or RYA media. All plates were incubated at 27 °C and 12L: 12D photoperiod. Conidial production on rice grains was determined following inoculation of 10 μL of fungal suspensions (1x106 conidia mL-1) onto 5 g of sterilized parboiled rice in glass vials (25 mL). The total radial growth (cm) of the colonies on SDA and RYA media were measured on the 15th day after fungal inoculation using a steel ruler. All experiments were performed three times.
The conidial production of both isolates on the three different media was estimated on the 15th day after inoculation. Conidia were harvested from SDA and RYA using a spatula and added to 10 mL of Tween (0.05%). The conidia grown on rice were rinsed off the grains by adding 10 mL water + Tween 80 to each tube and the tubes were vigorously agitated using a vortex mixer for 1 min. The conidial concentration in each suspension was then estimated using a Neubauer hemocytometer.
Survival rates of larvae exposed to conidia produced on different substrates
The bioassays were conducted to investigate the virulence of conidia produced on three different substrates. The experiments were carried out using Ae. aegypti larvae (L2-3) obtained from eggs collected in the field using a standard procedure. Conidia of both isolates cultivated on three substrates were harvested using a spatula and suspended in sterile distilled water + Tween 80 (0.01%). Conidial concentration was determined using a Neubauer hemocytometer and adjusted to 1x106 conidia mL-1 by serial dilution.
The assays were conducted by placing ten larvae in each plastic cup (8.4 cm height x 6.3 cm diameter) with 50 mL of conidial suspensions. Therefore, larvae were constantly exposed to the conidial suspension. Larval mortality was recorded daily for 7 days. The assays were conducted at room temperature (25–27 °C). Control treatments were carried out using Tween 80 (0.01%) only. For each treatment group 40 larvae were used. Therefore, a total of 160 larvae were used per isolate per repetition (40 larvae for each of the three substrates + 40 control larvae). All experiments were carried out four times.
GraphPad Prism 8 software was used for survival curve comparisons (Log-rank Mantel-Cox) and median survival time (S50) estimated from Kaplan-Meier analysis. Insects which survived the 7-day experimental period were regarded as “censored”. The data for fungal growth and spore production were submitted to normality (Shapiro-Wilk) and equality of variance tests. Normally distributed data was then analyzed using a two-way ANOVA and Tukey’s post-hoc test using SigmaPlot 12.
| Results|| |
Radial growth and conidial production
The study was conducted evaluate the radial growth rates of both species of fungi when cultured on either SDA or RYA. The Shapiro-Wilk test confirmed normality of this data (P= 0.899) and equality of variance (P = 0.412). The two-way ANOVA showed that there was a significant effect of fungal species on the colony growth (diameter) (F (1) = 7.511, P= 0.025), but no significantly effect of the different media on growth (F (1) = 2.844, P = 0.130). The average radial growth of ESALQ 818 on day 15 was 5.76 cm (± 0.21 SEM) on SDA and 6.03 cm (± 0.16) on RYA. The average mycelial growth of IP 46 was higher than that of ESALQ 818 with a mean of 6.20 cm (± 0.15) on SDA and 6.46 cm (± 0.03) 15 days after inoculation on RYA.
The data for conidial production on all three substrates (SDA, RYA and rice grains) was normally distributed (Shapiro-Wilk; P = 0.225). The result showed that there was a significant difference between conidial yields when comparing the two species of Metarhizium tested here cultured on the three different substrates (F (1) =1318.33; P < 0.001). ESALQ cultured on rice produced significantly more conidia (approximately x5) than IP 46 cultured on rice [Table 1].
|Table 1: Conidial yield of Metarhizium anisopliae (ESALQ 818) and Metarhizium humberi (IP 46) when cultured on different substrates|
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There was also a significant difference between the conidial yields when considering each fungus grown on the three substrates (F (2) = 1457.78; P < 0.001). ESALQ 818 produced the highest conidial yields when grown on rice grains when compared to SDA or RYA [Table 1]. IP 46 also produced the highest yields when grown on rice [Table 1].
Effect of culture media on virulence
ESALQ 818 conidia produced all three types of culture media caused significant reductions in Ae. aegypti larval survival when tested at a concentration of 1x106 conidia mL-1 [Figure 1]. The survival curves were significantly different when comparing all treatments, including control survival, 48h after exposing the larvae to conidia (χ2 = 552.9, df = 3, P < 0.001).
|Figure 1: Survival curves of Aedes aegypti larvae exposed to ESALQ 818 conidia which had been produced on SDA, RYA and Rice grains. Larvae were exposed to a concentration of 1x106 conidia mL-1. The bars represent the standard error of the mean.|
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Conidia produced on rice grains were more virulent than conidia produced on the two other substrates, causing 100% of mortality within 2 days (S50 = 1 day). When larvae were exposed to conidia produced on RYA and SDA, 100% mortality was only observed on day 3 and day 4, respectively (S50 = 2 days for both treatments). Control survival was high throughout the experiment (96.2 % on day seven).
The virulence of IP 46 conidia produced on rice grains was significantly different to that of conidia produced using the other two substrates (χ2 = 570.4, df = 3, P < 0.0001). These comparisons were made using Log-rank survival curve analysis [Figure 2]. When larvae were exposed to conidia produced on rice, 100% died within 72 hours. Conidia produced on SDA and RYA were less virulent, with 80% and 82% of the larvae still alive after 24 hours, respectively (S50 = 3 days for both treatments). At the end of the bioassay (day 7), conidia produced on RYA had killed higher percentages of the larvae (3% survival) than conidia produced on SDA (11% survival). Control larval survival was 96.8 % on day 7 [Figure 2].
|Figure 2: Survival curves of Aedes aegypti larvae exposed to IP 46 conidia which had been produced on SDA, RYA and Rice grains. Larvae were exposed to a concentration of 1x106 conidia mL-1. The bars represent the standard error of the mean.|
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| Discussion|| |
Different entomopathogenic fungi, even when closely related, can display different levels of virulence towards the same target insect. In the current study, when comparing the virulence of Metarhizium anisopliae (isolate ESALQ 818) to that of Metarhizium humberi (isolate IP 46),M. anisopliae was significantly more virulent than M. humberi against Ae. aegypti larvae. Differences in virulence of entomopathogenic fungi have also been observed when modifying culture conditions. However, few studies have investigated the effect of different culture media on the virulence of fungi against disease vectors such as Ae. aegypti. When testing the virulence of Metarhizium and Beauveria spores produced using a range of liquid substrates, no significant differences were seen in spore yield but only spores produced in one of the substrates (casamino acids) were virulent to Ae. aegypti larvae. The virulence levels seen in the current study were much higher than those observed by Maldonado-Blanco and co-workers.
Virulence of the two species of fungi investigated here was influenced by cultivation on different substrates. Cultivation of both species of fungi on rice grains resulted in the highest virulence (lowest S50 values) against natural populations of Ae. aegypti larvae, and in the case of ESALQ 818, the use of rice media also resulted in significantly greater conidial yields [Table 1].
One of the most commonly used media for fungal culture is SDA. However, as shown here, the virulence of conidia produced on this media was lower than that of conidia produced on either RYA or rice grains. It is therefore important to evaluate virulence of conidia produced on rice grains otherwise possible candidates for biological control programs could be underestimated. An interesting alternative for small scale conidial production in the laboratory is the use of RYA. In all of the experiments carried out here, the virulence of conidia produced on this substrate was higher than that of conidia produced on SDA and larger amounts of conidia were also obtained using RYA when compared to SDA for both species of fungi [Table 1].
It is known that nutritional factors can directly affect virulence. The amount of carbon and nitrogen stored in conidia can alter the ability of the fungus to germinate and infect organisms. In addition, the level of stress under which entomopathogens are cultured may also influence germination and production of cuticle degrading enzymes with consequences for virulence levels. When tested against Tenebrio molitor (Coleoptera), M. anisopliae cultivated under nutritive stress on minimal medium, supplemented with lactose, produced highly virulent conidia with enhanced rates of adhesion to the host cuticle when compared to conidia produced using nutrient rich media.
Metarhizium humberi (IP 46) is considered to be a highly promising candidate for vector control as it has been shown to be pathogenic to adult malaria mosquitoes Anopheles gambiae s.s. and Anopheles arabiensis. It is also pathogenic to Ae. aegypti eggs, larvae and adults. However, those studies were carried out using conidia produced using SDA and PDA (potato dextrose agar) media, which could have negatively influenced the results. Here, we have shown that IP 46 conidia produced on SDA media were significantly less virulent than conidia produced on RYA or rice grains when tested against Ae. aegypti larvae. These results highlight the need to standardize the substrates used when comparing fungal virulence.
It is not known which factors provided by rice flour increased virulence when compared to nutrient rich SDA, although studies have shown that limiting nutrients can have a positive effect on virulence. Conidia produced on rice grains tolerate stress and produce higher levels of destruxins and proteases such as Pr1 when compared to conidia cultivated on other substrates. Rice is the most commonly used substrate for commercial conidial production. It is economical and has positive effects on fungal germination, providing a surface area that allows rapid fungal colonization and has the capacity to absorb moisture, contributing to faster germination and high conidial viability. Another factor to be considered is post-harvest storage. A study of the effect of different substrates on conidial production showed that storage conditions were more critical to virulence against Culex pipiens pipiens larvae than the substrate used to produce the conidia.
| Conclusion|| |
The present study showed that the choice of substrate used for conidial production is an important consideration for optimizing virulence against Ae. aegypti larvae. Further studies should be carried out to determine whether other substrates could further increase the virulence of the fungi, with concomitant improvement in their potential for use in mosquito biological control strategies.
Conflict of interest: None
| Acknowledgements|| |
We wish to thank UENF for the post-doctoral research grant awarded to ATC. We also thank FAPERJ (E26/201.336/2016 & E26/202.923/2019) and CNPq (431766/2016-9) for financial support. RIS is a CNPq Research Fellow.
| References|| |
Powell JR. Mosquito-Borne Human Viral Diseases: Why Aedes aegypti
? Am J Trop Med Hyg
Smith LB, Kasai S, Scott JG. Pyrethroid resistance in Aedes aegypti
and Aedes albopictus:
Important mosquito vectors of human disease. Pestic Biochem Physiol
Benelli G, Jeffries CL, Walker T. Biological control of mosquito vectors: past, present, and future. Insects
2016; 7(4): 52.
Samuels RI, Paula AR, Carolino AT, Gomes SA, Morais COP, Cypriano MBC, et al
. Entomopathogenic organisms: conceptual advances and real-world applications for mosquito biological control. Open Access Insect Physiol
2016; 6: 25-31.
Vivekanandhan P, Bedini S, Shivakumar, MS. Isolation and identification of entomopathogenic fungus from Eastern Ghats of South Indian forest soil and their efficacy as biopesticide for mosquito control. Parasitol Inter
Silva LEI, Paula AR, Ribeiro A, Butt TM, Silva CP, Samuel RI. A new method of deploying entomopathogenic fungi to control adult Aedes aegypti
mosquitoes. J Appl Entomol
Liu CP, Liu SD. Formulation and characterization of the microencapsulated entomopathogenic fungus Metarhizium anisopliae
MA126. J Microencapsul
2009; 26(5): 377-384.
Paula AR, Silva LEI, Ribeiro A, Butt TM, Silva CP, Samuels RI. Improving the delivery and efficiency of fungus-impregnated cloths for control of adult Aedes aegypti
using a synthetic attractive lure. Parasit Vectors
2018; 11(1): 285.
Alkhaibari AM, Carolino AT, Yavasoglu SI, Maffeis T, Mattoso TC, Bull JC, et al. Metarhizium brunneum
blastospore pathogenesis in Aedes aegypti
larvae: attack on several fronts accelerates mortality. PLoS Pathog
2016; 12(7): e1005715.
Vivekanandhan P, Swathy K, Kalaimurugan D, Ramachandran M, Yuvaraj A, Kumar AN, et al
. Larvicidal toxicity of Metarhizium anisopliae
metabolites against three mosquito species and non-targeting organisms. PLoS ONE
Carolino AT, Gomes SA, Teodoro TBP, Mattoso TC, Samuels RI. Aedes aegypti
pupae are highly susceptible to infection by Metarhizium anisopliae
blastospores. J Pure Appl Microbiol
2019; 13(3): 1629-1634.
Carolino AT, Paula AR, Silva CP, Butt TM, Samuels RI. Monitoring persistence of the entomopathogenic fungus Metarhizium anisopliae
under simulated field conditions with the aim of controlling adult Aedes aegypti
(Diptera: Culicidae). Parasit Vectors
2014; 7(1): 198.
Gomes SA, Paula AR, Ribeiro A, Moraes COP, Santos JWAB, Silva CP, et al
. Neem oil increases the efficiency of the entomopathogenic fungus Metarhizium anisopliae
for the control of Aedes aegypti
(Diptera: Culicidae) larvae. Parasit Vectors
Rangel DEN, Anderson AJ, Roberts DW. Growth of Metarhizium anisopliae
on non-preferred carbon sources yields co-nidia with increased UV-B tolerance. J Invertebr Pathol
Safavi SA, Shah FA, Pakdel AK, Rasoulian GR, Bandani AR, Butt TM. Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana. FEMS Microbiol Lett
Mascarin GM, Kobori NN, Jackson MA, Dunlap CA, Delalibera Jr I. Nitrogen sources affect productivity, desiccation tolerance and storage stability of Beauveria bassiana
blastospores. J Appl Microbiol
Maldonado-Blanco MG, Gallegos-Sandova JL, Fernández-Peña G, Sandoval-Coronado CF, Elías-Santos M. Effect of culture medium on the production and virulence of submerged spores of Metarhizium anisopliae
and Beauveria bassiana
against larvae and adults of Aedes aegypti
(Diptera: Culicidae). Biocontrol Sci Technol
2014; 24: 180-189.
Hussain A, Tian MY, He YR, Ruan L, Ahmed S. In vitro and in vivo culturing impacts on the virulence characteristics of serially passed entomopathogenic fungi. J Food Agric Environ
2010; 8: 481-487.
St. Leger RJ, Joshi L, Bidochka MJ, Rizzo NW, Roberts DW. Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae
in infected insect cuticles. J Appl Environ Microbiol
1996; 64(4): 1257-1264.
Taylor B, Edgington S, Luke B, Moore D. Yield and germination of the entomopathogenic fungus Beauveria bassiana
when grown on different rice preparations. J Stored Prod Res
Dhar P, Kaur G. Response surface methodology for optimizing process parameters for the mass production of Beauveria bassiana
conidiospores. Afr J Microbiol Res
2011; 4(17): 2399-2406.
Kim JJ, Xie L, Han JH, Lee SY. Influence of additives on the yield and pathogenicity of conidia produced by solid state cultivation of an Isaria javanica
Dalla Santana HS, Sousa NJ, Brand D, Dalla Santana OR, Pansdey A, Sobotka M, et al
. Conidia production of Beauveria
sp. by solid-state fermentation for biocontrol of Ilex paraguariensis
caterpillars. Folia Microbiol
(Praha). 2004; 49(4): 418-422.
Lee SJ, Kim S, Yu JS, Kim JC, Nai Y-S, Kim, JS. Biological control of Asian tiger mosquito, Aedes albopictus
(Diptera: Culicidae) using Metarhizium anisopliae
JEF-003 millet grain. J Asia-Pacific Entomol
Lopez-Perez M, Rodriguez-Gomez D, Loera O. Production of conidia of Beauveria bassiana
in solid-state culture: current status and future perspectives. Crit Rev Biotechnol
2015; 35(3): 334-341.
Pereira CR, Paula AR, Gomes SA, Pedra Jr PCO, Samuels RI. The potential of Metarhizium anisopliae
and Beauveria bassiana
isolates for the control of Aedes aegypti
(Díptera: Culicidae) laval. Biocontrol Sci Technol
Humber RA. Fungi: Preservation of cultures. In: Lacey L, editor. Manual of Techniques in Insect Pathology. Academic Press 1997; 269-279.
Shah FA, Wang CS, Butt TM. Nutrition influences growth and virulence of the insect-pathogenic fungus Metarhizium anisopliae. FEMS Microbiol Lett
2005; 251(2): 259-266.
Ansari MA, Butt TM. Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi. J Appl Microbiol
Rangel DEN, Alston DG, Roberts DW. Effects of physical and nutritional stress conditions during mycelial growth on conidial germination speed, adhesion to host cuticle, and virulence of Metarhizium anisopliae
, an entomopathogenic fungus. Mycol Res
Mnyone LL, Russell TL, Lyimo IN, Lwetoijera DW, Kirby MJM, Luz C. First report of Metarhizium anisopliae
IP 46 pathogenicity in adult Anopheles gambiae
s.s. and An. arabiensis
(Diptera; Culicidae). Parasit Vectors
Sousa NA, Lobo LS, Rodrigues J, Luz C. New insights on the effectiveness of Metarhizium anisopliae
formulation and application against Aedes aegypti
eggs. Lett Appl Microbiol
2013; 57(3): 193-199.
Falvo ML, Medina PA, Rodrigues J, Lastra CCL, García JJ, Fernandes EKK, et al
. Effect of UV-B irradiation on water-suspended Metarhizium anisopliae
s.l. (Hypocreales: Clavi-cipitaceae) conidia and their larvicidal activity in Aedes aegypti
(Diptera: Culicidae). J Med Entomol
Lobo LS, Rodriguez J, Luz C. Effectiveness of Metarhizium an-isopliae
formulations against dengue vectors under laboratory and field conditions. Biocontrol Sci Technol
Daoust RA, Roberts DW. Studies on the prolonged storage of Metarhizium anisopliae
conidia: Effect of growth substrate on conidial survival and virulence against mosquitoes. J Invertebr Pathol
[Figure 1], [Figure 2]