Isolation of entomopathogenic fungi from Northern Thailand and their production in cereal grains

Spore productivity in six entomopathogenic fungal strains isolated from insect cadavers at four locations in Chiang Mai province was evaluated in five cereal grains: white-rice, wheat, rye, corn and sorghum. According to sequence analysis of the internal transcribed spacer regions of these isolates, they were closely related to Beauveria bassiana (2 isolates), Metarhizium flavoviride (1 isolate), Metarhizium anisopliae (1 isolate), Paecilomyces lilacinus (1 isolate) and Isaria tenuipes (1 isolate). Among all fungal isolates, the maximum amount of spores (530.0?×?10⁹ conidia/g) was yielded P. lilacinus CMUCDMT02 on sorghum grain followed by white-rice (399.3?×?10⁹ conidia/g). Moreover, the highest number of spore in M. flavoviride was 102.8?×?10⁹ conidia/g sorghum whereas white-rice yielded the greatest amount of spore for B. bassiana CMUCDMF03 (141.0?×?10⁹ conidia/g) after 60?days incubation. The fungal growth rate was found highest in corn for all strains and rye showed the lowest with the exception of P. lilacinus CMUCDMT02 among the tested grains. Spore viability was over 80?% for all isolates that had been inoculated for 60?days. Fungal conidia suspension of P. lilacinus obtained highest virulence against Bactrocera spp. at a concentration of 1?×?10⁶ spore/ml. The strains isolated, exhibited good production of conidia suggesting a promising strategy for the mass production of inoculum as biocontrol agents with low production cost.     

Selection of promising fungal biological control agent of the western flower thrips Frankliniella occidentalis (Pergande)

Aims: Larval stages of Frankliniella occidentalis are known to be refractory to fungal infection compared with the adult stage. The objective of this study was to identify promising fungal isolate(s) for the control of larval stages of F. occidentalis. Methods and Results: Ten isolates of Metarhizium anisopliae and eight of Beauveria bassiana were screened for virulence against second‐instar larvae of F. occidentalis. Conidial production and genetic polymorphism were also investigated. Metarhizium anisopliae isolates ICIPE 7, ICIPE 20, ICIPE 69 and ICIPE 665 had the shortest LT₅₀ values of 8·0–8·9 days. ICIPE 69, ICIPE 7 and ICIPE 20 had the lowest LC₅₀ values of 1·1 × 10⁷, 2·0 × 10⁷ and 3·0 × 10⁷ conidia ml^?1, respectively. Metarhizium anisopliae isolate ICIPE 69 produced significantly more conidia than M. anisopliae isolates ICIPE 7 and ICIPE 20. Internally transcribed spacers sequences alignment showed differences in nucleotides composition, which can partly explain differences in virulence. Conclusion: These results coupled with the previous ones on virulence and field efficacy against other species of thrips make M. anisopliae isolate ICIPE 69 a good candidate. Significance and Impact of the Study: Metarhizium anisopliae isolate ICIPE 69 can be suggested for development as fungus‐based biopesticide for thrips management.     

The plant beneficial effects of Metarhizium species correlate with their association with roots

Metarhizium species have recently been found to be plant rhizosphere associates as well as insect pathogens. Because of their abundance, rhizospheric Metarhizium could have enormous environmental impact, with co-evolutionary implications. Here, we tested the hypothesis that some Metarhizium spp. are multifactorial plant growth promoters. In two consecutive years, corn seeds were treated with entomopathogenic Metarhizium spp. and field tested at the Beltsville Facility in Maryland. Seed treatments included application of green fluorescent protein (GFP)-tagged strains of Metarhizium brunneum, Metarhizium anisopliae, Metarhizium robertsii, and M. robertsii gene disruption mutants that were either avirulent (Δmcl1), unable to adhere to plant roots (Δmad2), or poorly utilized root exudates (Δmrt). Relative to seeds treated with heat-killed conidia, M. brunneum, M. anisopliae, and M. robertsii significantly increased leaf collar formation (by 15, 14, and 13 %), stalk length (by 16, 10, and 10 %), average ear biomass (by 61, 56, and 36 %), and average stalk and foliage biomass (by 46, 36, and 33 %). Their major impact on corn yield was during early vegetative growth by allowing the plants to establish earlier and thereby potentially outpacing ambient biotic and abiotic stressors. Δmcl1 colonized roots and promoted plant growth to a similar extent as the parent wild type, showing that Metarhizium populations are plant growth promoters irrespective of their role as insect pathogens. In contrast, rhizospheric populations and growth promotion by Δmrt were significantly reduced, and Δmad2 failed to colonize roots or impact plant growth, suggesting that colonization of the root is a prerequisite for most, if not all, of the beneficial effects of Metarhizium.     

Pathogenicity of entomopathogenic fungus,Metarhizium anisopliae MET-GRA4 isolate on dengue vectors,Aedes albopictus and Aedes aegypti mosquito larvae (Diptera: Culicidae)

Considering the rapid transmission of the dengue virus, substantial efforts need to be conducted to ward-off the epidemics of dengue viruses. The control effort is depending on chemical insecticides and had aroused undesirable conflicts of insecticide resistance. Here, we study the entomopathogenic fungus, Metarhizium anisopliae as a promising new biological control agent for vector control. The pathogenicity effects of Metarhizium anisopliae against field and laboratory strains of Aedes albopictus and Aedes aegypti larvae were tested using the larvicidal bioassay technique. The results demonstrate that the treatments using M. anisopliae isolate MET-GRA4 were highly effective and able to kill 100% of both Ae. albopictus and Ae. aegypti mosquito larvae at a conidia concentration of 1 × 10?/ml within 7 days of the treatment period. The fungus displayed high larvicidal activity against laboratory and field strain of Ae. aegypti larvae with LC₅₀ values (9.6 × 10³/ml, 1.3 × 10³/ml) and LC₉₅ values (1.2 × 10?/ml, 5.5 × 10⁵/ml) respectively. For Ae. albopictus, LC₅₀ values for laboratory and field strains were (1.7 × 10⁴/ml, 2.7 × 10⁴/ml) and the LC₉₅ values were (2.1 × 10?/ml, 7.0 × 10⁵/ml) respectively. Interestingly, the susceptibility of field strain towards M. anisopliae was higher as compared to the laboratory strain Aedes larvae. In which, the causative agents of all the dead larvae were verified by the virulence of M. anisopliae and caused morphological deformities on larval body. The findings from this study identify this isolate could be an effective potential biocontrol agent for vector mosquitoes in Malaysia.     

LABORATORY SUSCEPTIBILITY OF PLUTELLA XYLOSTELLA TO METARHIZIUM ANISOPLIAE AND NOMURAEA RILEYI*

Abstract Five isolates of Metarhizium anisopliae and one isolate of Nomuraea rileyi were bioassayed against larvae of Plutella xylostella. Larvae were treated by exposing cabbage leaf discs previously immersed in conidial suspension, and were then incubated at 25^oC and observed daily. One of M. anisopliae isolates, F11248, originally from Mastotermes darwiniensis was found being most virulent against P. xylostella. The LC₅₀ was estimated as 2.03 times 10⁴conidia/ml with 9 d mortality data, and the LT₅₀ was 4.97 d at concentration of 10⁷conidia/ml. Isolate F163 (N. rileyi) showed virulence to P. xylostella in both tests, but no cadavers sporulated.     

Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation

We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l^?1 with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS–DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or ?20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1–7.3 × 10⁶ l^?1 after 3 days growth with maximum MS yields (0.7–1.1 × 10⁷ l^?1) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS–DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 10⁹ conidia g^?1). All MS granules showed similar stability after storage at either 26 or ?20 °C for 3.5 months.     

Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of growth substrate on conidial survival and virulence against mosquitoes

Metarhizium anisopliae was grown on six complex mycological media and on three types of rice at three moisture levels to determine the effect of growth substrate on conidial yield, viability, and virulence against mosquitoes immediately after spore maturation and after the storage of conidia at four different temperature-relative humidity (RH) combinations over a 1-year period. Conidial yields varied with the mycological media, but the viability and virulence of conidia against mosquitoes produced on all substrates were similar when spores were stored under the same conditions. The storage conditions were more critical to spore survival and virulence than the substrate upon which conidia were produced. The comparison of rice types for conidial production indicated that conidial yield, viability, and virulence to mosquitoes were more dependent upon the moisture level during growth and on the storage conditions that upon the rice used. The best storage conditions among those tested for the retention of both spore viability and virulence against mosquitoes were 19°C–97% RH and 4°C–0% RH.     

Entomopathogenic fungus as a biological control agent against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat

To evaluate the pathogenicity of Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes) a bioassay was designed under laboratory conditions against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat. The fungus was applied at the dose rates of 8 × 10³, 8 × 10⁵, 8 × 10⁷ and 8 × 10⁹ conidia/kg of wheat and the bioassay was conducted at 25°C with 60% relative humidity. The data regarding the mortality was recorded after 7 and 14 days exposure intervals. All the treatments gave the significant mortality of R. dominica and M. anisopliae of 8 × 10⁹ conidia/kg was found to be the most effective after a 14-day exposure interval. There was greater production of progeny when the low rate of M. anisopliae was applied to wheat. Overall, our study showed that M. anisopliae is vigorous when applied at a high dose rate which revealed an effective control of R. dominica and also played a pivotal role in the integrated pest management program (IPM) of stored wheat insect pests.     

Species of the Metarhizium anisopliae complex with diverse ecological niches display different susceptibilities to antifungal agents

Species of the Metarhizium anisopliae complex are globally ubiquitous soil-inhabiting and predominantly insect-pathogenic fungi. The Metarhizium genus contains species ranging from specialists, such as Metarhizium acridum that only infects acridids, to generalists, such as M. anisopliae, Metarhizium brunneum, and Metarhizium robertsii that infect a broad range of insects and can also colonize plant roots. There is little information available about the susceptibility of Metarhizium species to clinical and non-clinical antifungal agents. We determined the susceptibility of 16 isolates comprising four Metarhizium species with different ecological niches to seven clinical (amphotericin B, ciclopirox olamine, fluconazole, griseofulvin, itraconazole, tebinafine, and voriconazole) and one non-clinical (benomyl) antifungal agents. All isolates of the specialist M. acridum were clearly more susceptible to most antifungals than the isolates of the generalists M. anisopliae sensu lato, M. brunneum, and M. robertsii. All isolates of M. anisopliae, M. brunneum, and M. robertsii were resistant to fluconazole and some were also resistant to amphotericin B. The marked differences in susceptibility between the specialist M. acridum and the generalist Metarhizium species suggest that this characteristic is associated with their different ecological niches, and may assist in devising rational antifungal treatments for the rare cases of mycoses caused by Metarhizium species.     

金龟子绿僵菌在森林土壤中的分布及对松墨天牛致病性测定

松墨天牛是重大森林植物检疫性病害——松材线虫病的主要媒介昆虫。本研究于2005年8月～2006年8月从福建、江西两省共110个林分样区(其中松林88个样区)采集土壤样品330份,采用选择性培养基分离土壤中的金龟子绿僵菌。从21个样区的26份土样中分离出的金龟子绿僵菌占采集样区的19．1％和样品的7．9％,成菌落数(CFU)500～72500CFU/g,表明金龟子绿僵菌在森林土壤中有较为广泛的分布。对分离到的9个产孢量高的菌株,采用浸渍法(1×10~7孢子/mL)接种3～4龄健康松墨天牛幼虫,采用跗节接种法接种2～15日龄健康成虫,测定其致病力。结果表明,MaYTTR-03、MaYTTR-04菌株对松墨天牛幼虫和成虫均有较高致病力,表现出良好的生防潜力。     

金龟子绿僵菌在森林土壤中的分布及对松墨天牛致病性测定

松墨天牛是重大森林植物检疫性病害——松材线虫病的主要媒介昆虫。本研究于2005年8月～2006年8月从福建、江西两省共110个林分样区（其中松林88个样区）采集土壤样品330份,采用选择性培养基分离土壤中的金龟子绿僵菌。从21个样区的26份土样中分离出的金龟子绿僵菌占采集样区的19.1%和样品的7.9%,成菌落数（CFU）500～72500CFU/g,表明金龟子绿僵菌在森林土壤中有较为广泛的分布。对分离到的9个产孢量高的菌株,采用浸渍法（1×10⁷孢子/mL）接种3～4龄健康松墨天牛幼虫,采用跗节接种法接种2～15日龄健康成虫,测定其致病力。结果表明,MaYTTR-03、MaYTTR-04菌株对松墨天牛幼虫和成虫均有较高致病力,表现出良好的生防潜力。     

A rapid knockdown effect of Penicillium citrinum for control of the mosquito Culex quinquefasciatus in Thailand

Twenty local isolates of entomopathogenic fungi were determined for control of the larvae and adults of Culex quinquefasciatus. In a laboratory experiment, a Penicillium sp. CM-010 caused 100 % mortality of third-instar larvae within 2 h using a conidial suspension of 1 × 10⁶ conidia ml^?1. Its LC₅₀ was 3 × 10⁵ conidia ml^?1, and the lethal time (LT₅₀) was 1.06 h. Cloning and sequencing of its internal transcribed spacer region indicated that this Penicillium species is Penicillium citrinum (100 % identity in 434 bp). Mortality of the adult was highest with Aspergillus flavus CM-011 followed with Metarhizium anisopliae CKM-048 from 1 × 10⁹ conidia ml^?1. P. citrinum CM-010 at 1 × 10⁶ conidia ml^?1 killed 100 % larvae within 2 h while Bacillus thuringiensis var. israelensis at 5 ITU ml^?1 required 24 h. This P. citrinum CM-010 also greatly reduced survival of C. quinquefasciatus larvae in an unreplicated field test. Light and transmission electron micrographs showed that the fungal conidia were ingested by the larvae and deposited in the gut. The metabolite patulin was produced by P. citrinum CM-010 instead of citrinin.     

A Mutant of Metarhizium anisopliae var. acridum with Enhanced Submerged Conidiation

Summary An insertional mutant of Metarhizium anisopliae is described with enhanced submerged conidiation. In a 500 ml submerged culture, this mutant produces a mean of 4.05 × 10⁸ propagules ml⁻¹ from an inoculum of 1 × 10⁶ conidia, where the parental strain accumulates only 3.75 × 10⁴ propagules ml⁻¹.     

Comparison of the relative efficacy of an insect baiting method and selective media for diversity studies of Metarhizium species in the soil

Metarhizium are a commonly occurring group of entomopathogenic fungi normally found in soil. The most common methods to assess the diversity of Metarhizium species in soil are (i) the use of selective media and (ii) insect baiting using Galleria mellonella larvae. We compared the recovery efficiency from soil of four common species of Metarhizium (Metarhizium anisopliae, Metarhizium pingshaense, Metarhizium brunneum and Metarhizium robertsii) using these two methods. Firstly, we compared the number of colony forming units (CFU) produced in vitro when grown on two selective media, one containing chloramphenicol, thiabendazole and cycloheximidethe (CTC) and one based on the fungicide dodine (n-dodecylguanidine acetate) (DOD). Secondly, we artificially inoculated natural/non-sterile soil with the four fungal species at a rate of 2×10² and 2×10³ conidia g^?1of soil, baited with G. mellonella, and processed for evaluation using the selective media. The in vitro results showed that the greatest number of CFUs were recorded for M. brunneum. In contrast, when inoculated into soil, more G. mellonella larvae became infected by M. anisopliae. Finally, when using selective media, most CFUs recovered were for M. robertsii. The importance of our results in selecting a method to study the natural occurrence of Metarhizium in soil are discussed.     

Comparative study between Larval Packet Test and Larval Immersion Test to assess the effect of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> on <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> tick larvae

Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT₅₀ values obtained were 134.6, 27.2 and 24.8 days for concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT₅₀ values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 10⁶, 10⁷ and 10⁸ conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.     

Effect of entomopathogenic Aspergillus strains against the pea aphid,Acyrthosiphon pisum (Hemiptera: Aphididae)

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Entomopathogenic fungi are valuable tools for potential aphid control. As part of a selection process, laboratory bioassays were carried with five different concentrations of Aspergillus clavatus (Desmazières), Aspergillus flavus (Link) and Metarhizium anisopliae ((Metschnikoff) Sorokin) spores against the pea aphid, Acyrthosiphon pisum (Harris). Aspergillus isolates induced higher mortalities than M. anisopliae, which is a well-known entomopathogen in the literature. Lethal concentrations (LC₅₀ and LC₉₀) were 1.23 × 10³ and 1.34 × 10⁷ spores/ml for A. flavus, 4.95 × 10² and 5.65 × 10⁷ spores/ml for A. clavatus, and 3.67 × 10³ and 9.71 × 10⁷ spores/ml for M. anisopliae 5 days after treatment. Mycelia development and sporulation on adult cadavers were observed 48 h after incubation. The intrinsic growth rate of A. pisum decreased with increased spore concentration for all fungal strains, suggesting an increase in pathogen fitness related to a consumption of host resources. In conclusion, Aspergillus species could be useful in aphid control as pest control agents despite their saprophytic lifestyle. This is also to our knowledge the first report of A. clavatus and A. flavus strains pathogenic to aphids.     

Production and quality of conidia by <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">lepidiotum</Emphasis>: critical oxygen level and period of mycelium competence

Mycoinsecticides application within Integral Pest Management requires high quantities of conidia, with the proper quality and resistance against environmental conditions. Metarhizium anisopliae var. lepidiotum conidia were produced in normal atmospheric conditions (21 % O₂) and different concentrations of oxygen pulses (16, 26, 30, and 40 %); conidia obtained under hypoxic conditions showed significantly lower viability, hydrophobicity, and virulence against Tenebrio molitor larvae or mealworm, compared with those obtained under normal atmospheric conditions. Higher concentrations of oxygen (26 and 30 %) improved conidial production. However, when a 30 % oxygen concentration was applied, maximal conidial yields were obtained at earlier times (132 h) relative to 26 % oxygen pulses (156 h); additionally, with 30 % oxygen pulses, conidia thermotolerance was improved, maintaining viability, hydrophobicity, and virulence. Although conidial production was not affected when 40 % oxygen pulses were applied, viability and virulence were diminished in those conidia. In order to find a critical time for mycelia competence to respond to these oxidant conditions, oxygen pulses were first applied either at 36, 48, 60, and 72 h. A critical time of 60 h was determined to be the best time for the M. anisopliae var. lepidiotum mycelia to respond to oxygen pulses in order to increase conidial production and also to maintain the quality features. Therefore, oxygen-enriched (30 %) pulses starting at 60 h are recommended for a high production without the impairment of quality of M. anisopliae var. lepidiotum conidia.     

Cellular and humoral immune defenses of Oxya japonica (Orthoptera: Acrididae) to entomopathogenic fungi Metarhizium anisopliae

One Indonesian isolate of the fungus Metarhizium anisopliae, named Majalengka strain, was evaluated not only for its virulence but also for the immune response of rice grasshopper Oxya japonica (Orthoptera: Acrididae) as a target organism. Five aqueous suspensions with different conidia concentrations in logarithmic series were prepared. The fungus showed high virulence as it caused 100% mortality at low conidia concentration (1.5 × 10² conidia/mL). Remarkable changes in the cellular and humoral responses were also observed when adult grasshoppers were infected with the fungus. The number of hemocytes decreased significantly within 12 h after infection. In addition, the total number of granulocytes increased rapidly in the first 12 h then gradually decreased 24 and 48 h after infections, while the number of coagulocytes fluctuated over time. The infection influenced the humoral response by increasing the phenoloxidase activity.     

Optimizing modes of inoculation of <Emphasis Type="Italic">Rhipicephalus</Emphasis> ticks (Acari: Ixodidae) with a mitosporic entomopathogenic fungus in the laboratory

The process of strain selection is an important step in the development of insect pathogens for biological control. Bioassays were conducted in the laboratory to evaluate the efficacy of different methods of inoculation using Rhipicephalus pulchellus Gerstäcker (Acari: Ixodidae) as a model. Initially, an oil-based formulation of Metarhizium anisopliae (Metsch.) Sorok. (Ascomycota: Hypocreales) titred at 10⁹ conidia ml^?1 was applied to R. pulchellus adults using a Burgerjon spray tower or a microapplicator. Inoculation by microapplicator yielded poor results (25.0% tick mortality) compared to Burgerjon’s spray tower (52.3% tick mortality), although the mean number of fungal conidia on R. pulchellus adults was lower (1.5 × 10⁴ ± 1.1 × 10³ conidia ml^?1) after spraying by Burgerjon’s spray tower compared to 1 × 10⁶ conidia ml^?1 obtained with the microapplicator. Thus, inoculation by Burgerjon’s spray tower was selected for further investigations. Different modes of inoculation were tested and included direct spray of inoculum on the tick and substrate (SS), direct spray on the substrate and tick followed by transfer of the tick to clean uncontaminated Petri dish (SP) or indirect inoculation of ticks through substrate (SW). The LC₅₀ values following contamination of nymphs (LC₅₀ = 1.4 × 10⁷ conidia ml^?1) and adults (LC₅₀ = 6.7 × 10⁷ conidia ml^?1) in SS were significantly lower compared to SP; nymphs (LC₅₀ = 5.7 × 10⁸ conidia ml^?1) and adults (LC₅₀ = 5.3 × 10⁹ conidia ml^?1) and SW; nymphs (LC₅₀ = 5 × 10⁸ conidia ml^?1). Although the LC₅₀ value in SS was the lowest, it recorded the highest tick mortality among control ticks (24.2% at 2 weeks post-treatment) and (23.3% at 3 weeks post-treatment) in nymphs and adults respectively compared to SP (2.5 and 5.8%, respectively) and SW (0.0 and 0.0). Results show that among the modes of inoculation tested, SP was the most appropriate for inoculating R. pulchellus adults. SW and SP were identified as appropriate techniques for infecting the R. pulchellus nymphs with conidia formulated in oil.     

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)

Three Metarhizium anisopliae and three Beauveria bassiana isolates were cultivated in media containing casamino acids, soybean flour or sunflower seed flour and were shaken for three days. M. anisopliae presented similar yields of around 10⁶ submerged spores/ml without significant differences among them, whereas B. bassiana produced yields of around 10⁸ spores/ml, of which GHA strain produced more submerged spores in the casamino acids medium. The other two strains showed no significant difference in the production of submerged spores in the three media used. Differences in mortality on Aedes aegypti larvae were observed with the submerged spores of Metarhizium depending on isolate and medium used. M. anisopliae 2157 caused significantly higher mortality (40%) when cultivated in casamino acids medium. It presented an LC₅₀ of 8.93 × 10⁵ submerged spores/ml water against mosquito larvae five days after application, whereas it caused 27% mortality in Ae. aegypti adults 10 days after application. In conclusion, fungal nutrition affected virulence of some isolates of M. anisopliae against Ae. aegypti larvae while such an effect was not noted for B. bassiana isolates.