Establishment and persistence of the entomopathogenic fungus Metarhizium anisopliae in maize fields

The entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) was applied in maize fields to control the Western Corn Rootworm Diabrotica virgifera virgifera Le Conte (Coleoptera: Chrysomelidae). Establishment and persistence of two strains of M. anisopliae were investigated after application as ‘fungal colonized barley kernels’ (FCBK) into the soil and as a spore suspension on maize leaves and on the soil surface in 2006 and 2007 at two locations in Hungary. The applied fungal strains were able to establish at both locations and a long‐term persistence of at least 15 months could be recorded in the soil. A positive correlation between density of colony forming units (CFU) in the soil and the soil inhabiting stages of the host insect D. v. virgifera could be found. M. anisopliae spores applied on maize leaves were able to survive for no longer than 3 days after application, whereas on the soil surface a noticeably increase of fungus densities were found after treatments. Molecular markers were used to identify the applied M. anisopliae strains before and after application of FCBK into the soil of the maize field.     

Resporulation of Metarhizium anisopliae granules on soil and mortality of Tenebrio molitor: Implications for wireworm management in sweetpotato

In Australia, sweetpotato (Ipomoea batatas L.) is vulnerable to root feeding insect pests such as wireworms (e.g., Agrypnus spp.). The number of registered insecticides to control these insect pests is limited and often pest pressure, for example by wireworms, is severe close to harvest, further limiting what insecticides can be applied. Incorporating biological control agents such as entomopathogenic fungi (e.g., Metarhizium anisopliae) into integrated pest management programmes may be feasible in sweetpotato. M. anisopliae has been shown to be effective in controlling more than 200 insects and it is able to reside and grow in the rhizosphere and rhizoplane, suggesting that M. anisopliae could be a promising candidate against soil insect pests. In the study presented here, M. anisopliae was formulated into calcium alginate granules fortified with nutrients. The resporulation of the fungal granules was tested on four different soil types in the laboratory. The biocontrol efficacy of the resulting fungal growth was also examined using larval mealworms, Tenebrio molitor as a model insect in the laboratory and the glasshouse. Our results indicated that sterilised soil favoured optimal fungal resporulation, although different soil types did not have a significant effect on fungal resporulation. The resulting fungal resporulation and growth on sterilised soil caused high mortality (up to 76%) of larval mealworms in the glasshouse, whereas the fungal granules applied to non-sterile soil demonstrated poor resporulation that led to low mortality (13%) of larval mealworms. The result of this study indicates that the manipulation of microbial populations in field soil is required to enhance the fungal growth and potential insect control against wireworms in the field.     

Susceptibility of adult Aedes aegypti (Diptera: Culicidae) to infection by Metarhizium anisopliae and Beauveria bassiana: prospects for Dengue vector control

Dengue fever vectored by the mosquito Aedes aegypti is one of the most rapidly spreading insect-borne diseases, stimulating the search for alternatives to current control methods. Screening assays using a range of Metarhizium anisopliae and Beauveria bassiana isolates were performed against adult female Ae. aegypti. Four virulent isolates were selected for detailed study. Adult female mosquitoes were exposed to supports previously inoculated with fungal suspensions. Fungal isolates were suspended in Tween 80+8% vegetable oil. The isolates caused between 70 and 89% mortality as a result of fungal infection over the 7-day test period. Mean survival times varied between 3 and 5 days for treated insects, whilst control survival exceeded 40 days. The most promising isolate, M. anisopliae LPP133, based not only on virulence but facility for mass production, was used for lethal exposure time determinations. An exposure time of only 3.5 h was necessary to cause 50% mortality. Large cage trails were also carried out and mean survival time of insects exposed to fungus impregnated black cloths was significantly reduced. These results show that entomopathogenic fungi could be promising biological control agents for use against adult Ae. aegypti, by inoculating fungi onto surfaces on which the mosquitoes tend to rest. The subsequent mortality caused by the fungi could potentially reduce the populations of this insect thus reducing the incidence of Dengue.     

Interactions among vegetable-infesting aphids,the fungal pathogen Metarhizium anisopliae (Ascomycota: Hypocreales) and the predatory coccinellid Cheilomenes lunata (Coleoptera: Coccinellidae)

Entomopathogenic fungi are among biocontrol agents being considered for the control of aphids on a variety of crops. Predatory coccinellids, although generalist, are also among important natural enemies that must be conserved for aphid management. Laboratory studies were carried out to investigate the interaction between three vegetable-infesting aphids, Metarhizium anisopliae isolate ICIPE 62 and the coccinellid predator Cheilomenes lunata. At a concentration of 1?×?10⁸?conidial?ml^–1, the fungus was found to cause mortality of 7.5% to C. lunata, compared to 2.5% mortality in the control at 10?days post-treatment. Female adult C. lunata to which fungus-infected aphids were offered as prey never accepted them as food source in non-choice bioassays. However, live and dead non-infected aphids were fed upon. In choice bioassay, a total of 1–3 out of 24 infected non-sporulating aphids per species (average of 0.1–0.4 aphids per arena) were consumed by 48?h-starved C. lunata within a period of 60?min, but avoided sporulating cadavers. Foraging adult C. lunata enhanced the spread of conidia of M. anisopliae from infected cadavers to fourth instars Aphis gossypii feeding on okra (0.8–15.0% mortality), Brevicoryne brassicae (3.3–15.0% mortality) and Lipaphis pseudobrassicae (0.8–14.2% mortality) on kale plants. Results of this study demonstrate compatibility between M. anisopliae and C. lunata, and could provide a sustainable strategy for effective management of aphids on crucifers and okra cropping systems.     

The potential of Metarhizium anisopliae and Beauveria bassiana isolates for the control of Aedes aegypti (Diptera: Culicidae) larvae

Fungal isolates were screened against Aedes aegypti larvae. Exposure of larvae to conidial suspensions resulted in 6–90% mortality. An inoculum persistence assay using one of the most virulent isolates showed an approximate half-life of 10 days in water containers. Not all larvae surviving to form pupae resulted in adult emergence.     

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC₅₀ values differed by up to ~10-fold (5.3×10⁶?4.5×10⁵ conidia/ml) and LT₅₀ values by ~40% (9.8?7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.     

Impact of Metarhizium brunneum (Hypocreales: Clavicipitaceae) on pre-imaginal Rhagoletis indifferens (Diptera: Tephritidae) within and on the surface of orchard soil

When last instar laboratory-reared Rhagoletis indifferens were allowed to pupate within non-sterile orchard soil containing incorporated Metarhizium brunneum isolate F52 conidia, a dose-related proportion died from developmental abnormalities and mycosis. When larvae entered soil superficially treated with M. brunneum, over 80% of the pupae died of developmental abnormalities.     

Polymorphism in Metarhizium anisopliae var. anisopliae (Hypocreales: Clavicipitaceae) based on internal transcribed spacer-RFLP, ISSR and intron markers

Isolates of entomopathogenic fungus Metarhizium anisopliae var. anisopliae were characterized using internal transcribed spacer-RFLP, ISSR and intron splice site primers. Thirty-seven isolates were studied, most of which were obtained from the sugar cane pest, Mahanarva fimbriolata (Hemiptera: Cercopidae) from Tangará da Serra, Southwest Mato Grosso State, Brazil. Internal transcribed spacer-RFLP did not differentiate the isolates of M. anisopliae var. anisopliae, while ISSR and intron primers identified three distinct groups. Variability among these groups was 96% for (GTG)(5) and 100% for the other primers. We found considerable genetic variability, even among isolates from the same geographical origin and host.     

An improved cage system for the bioassay of Metarhizium anisopliae on Thrips tabaci (Thysanoptera: Thripidae)

The ability of thrips and other minute insects to escape from conventional assay cups led to development of a successful assay chamber. In the present study, we evaluated three bioassay systems for thrips; Tashiro cage, microtube assay and leaf sandwich assay and introduced an improved thrips entomopathogenic bioassay system (TEBS). Our objective was to investigate the effect of Metarhizium anisopliae on Thrips tabaci using an easy-to-use and inexpensive holding chamber that would produce bioassay results with acceptable levels of accuracy and precision. In our assays, we found that escape of second-instar larvae from TEBS was significantly less than those of other methods which we tested. Our results indicated that our assay system was more suitable than the other conventional assays for the bioassay of entomopathogenic fungi on T. tabaci.     

Method for determining the best hydrophilic-lipophilic balance (HLB) number for a compatible non-ionic surfactant in formulation development for aerial conidia of Metarhizium anisopliae (Hypocreales: Clavicipitaceae)

A direct dropping method was developed to determine the best hydrophilic-lipophilic balance number of Metarhizium anisopliae conidia at different water content levels during dehydration. Results showed the best HLB number of the tested M. anisopliae conidia was 8; dehydration did not change the best HLB number but significantly increased wetting time.     

Combining vegetable oil and sub-lethal concentrations of Imidacloprid with Beauveria bassiana and Metarhizium anisopliae against adult guava weevil Conotrachelus psidii (Coleoptera: Curculionidae)

Of the insect pests that attack guava fruits, the guava weevil, Conotrachelus psidii Marshall, 1922 (Coleoptera: Curculionidae), is one of the most important in Brazil. In search of alternatives to chemical pesticides, this study was performed to select fungal isolates of Beauveria bassiana and Metarhizium anisopliae as potential candidates for the control of adult C. psidii. Tests were carried out using three products applied with the entomopathogenic fungi: Tween 80, sunflower oil and Imidacloprid (IMI). A sub-lethal concentration of IMI was determined (100 ppm). The results demonstrated that LPP 19 and LPP 114 were the most effective isolates when used in combination with all of the products. The least virulent isolate, ESALQ 818, when applied in Tween 80 caused only 26.6% mortality, however, this isolate showed significantly improved efficiency when applied together with either sunflower oil or IMI, causing 57.3 and 88.6% mortality, respectively. The efficiency of all the isolates tested here improved when applied together with IMI, with LT₅₀ values of 5.3–10.3 days when compared to LT₅₀ values in Tween alone of 9.5–17 days. The isolate that produced the highest number of conidia on the cadavers of adult C. psidii was LPP138, independent of the product used; however, conidial production was slightly reduced when fungi were applied together with IMI. These results are promising for developing new formulations of the isolates to be tested in the field.     

Efficacy of Metarhizium anisopliae in controlling the two‐spotted spider mite Tetranychus urticae on common bean in screenhouse and field experiments

The efficacy of aqueous and emulsifiable formulations of the fungus Metarhizium anisopliae isolate ICIPE78 was evaluated on the population density of Tetranychus urticae infesting common bean plants under screenhouse and field conditions. Synthetic acaricide abamectin was included as a check. Bean plants were artificially infested with T. urticae and allowed to multiply. Three treatments were applied in the screenhouse and 1 treatment in field trials. Mite density was recorded 2 d before spraying and weekly postspraying. The number of pods per plant, number of seeds per pod, and the dry weight of seeds per plant were recorded only in the screenhouse trials. In both screenhouse and field trials, fungal formulations applied at the concentration of 10⁸ conidia/mL and the acaricide reduced the population density of mites as compared to the controls. There were significant differences in T. urticae population densities between the treatments at the various post‐spraying sampling dates. In the screenhouse, the mite densities were near zero from 3‐week postspraying in the treated leaves. At 4‐week postspraying, there were no more leaves in the untreated control (T1) and in the control water + Silwet‐L77 (T2). Fungal formulations were as effective as abamectin in reducing mite densities in both screenhouse and field experiments. There were significant differences in the production parameters during the 2 screenhouse trials, with fungal and abamectin treatments generally having the highest yield. Results of this study underline the potential of the M. anisopliae isolate ICIPE78 as an alternative to acaricides for T. urticae management.     

Persistence of Beauveria bassiana sensu lato and Metarhizium anisopliae sensu lato in Vermont (USA) forest soil

Persistence of granular formulations of Beauveria bassiana sensu lato, strains GHA726 and ERL1170, and Metarhizium anisopliae sensu lato, strains ERL824 and ERL701, applied to forest soil was documented. One formulation was prepared by producing fungi on millet and at application adding cooked, dried millet to provide additional nutrients for fungal growth. The other was made by producing fungi on corn starch ‘packing peanuts’, a biodegradable packaging material, adding additional packing peanuts as a nutrient base. The trial was conducted in three sites in a forest predominating in sugar maple (Acer saccharum) in Underhill, VT, USA. In each site, 33 dominant sugar maples were randomly selected. A 1-m² plot located 1 m from the bole of the tree was established. Soil samples were collected before application, and 1 h, 2, 4, 8, 12 and 16 wk post-application. Fungal levels were estimated based on number of colony-forming units (CFUs). A significant increase in CFUs was observed 2 wk post-application in plots treated with isolates GHA726, ERL1170 and ERL824 as a result of fungal reproduction on nutrients in the formulations. Evidence of greater persistence for the B. bassiana than the M. anisopliae isolates was observed, based on CFU levels over time. Isolates formulated with millet proliferated more than those on packing peanuts. Use of entomopathogenic fungi in forest soil has potential for managing soil-dwelling insect pests such as pear thrips, Taeniothrips inconsequens, and the black vine weevil, Otiorhynchus sulcatus, though the type of formulation and the specific isolate used must be selected carefully.     

Susceptibility of apple clearwing moth larvae,Synanthedon myopaeformis (Lepidoptera: Sesiidae) to Beauveria bassiana and Metarhizium brunneum

Apple clearwing moth larvae, Synanthedon myopaeformis (Lepidoptera: Sesiidae) were found to be susceptible to infection by two entomopathogenic fungi: an indigenous fungus isolated from S. myopaeformis cadavers and identified as Metarhizium brunneum (Petch); and Beauveria bassiana isolate GHA. In laboratory bioassays, larvae exhibited dose related mortality after exposure to both the M. brunneum and Beauveria bassiana with 7 day LC₅₀'s of 2.9×10⁵ and 3.4×10⁵ spores/mL, respectively. Larval mortalities caused by the two isolates at 1×10⁶ spores/mL were not significantly different and 73% of the M. brunneum-treated, and 76% of the B. bassiana-treated larvae were dead 7 days post treatment, with LT₅₀'s of 5.5 and 5.1 days, respectively.     

Seasonal patterns of persistence and infectivity of Metarhizium anisopliae var. acridum in grasshopper cadavers in the Sahel

Field-based experiments were conducted to evaluate the fate and infectivity of the entomopathogenic fungus Metarhizium anisopliae var. acridum (Deuteromycotina: Hyphomycetes) in grasshopper cadavers in the Sahel. Unlike uninfected cadavers, which were rapidly scavenged, those infected with the fungus persisted in the environment for a number of weeks. The environmental factor most associated with cadaver disappearance was rainfall. The high environmental humidity associated with rainfall was also required for sporulation of the fungus on host cadavers, although the likelihood of sporulation differed between microsites. Characteristics of the infection profile from infective cadavers were investigated by the sequential exposure of uninfected hosts to sporulating cadavers in field cages. This experiment revealed that cadavers remained infective for > 30 days, with the net infectivity changing through time. The most likely explanation for these changes is climatic influences on both the fungus and host. High humidity was not required for infection. A measurement of the transmission coefficient between healthy hosts and sporulating cadavers in the field was obtained at a realistic density of infectious cadavers. This revealed a figure of 0.45 m² day^–1. Overall, these experiments show that following host death, M. anisopliae var. acridum can be persistent in the environment, sporulate on host cadavers and reinfect new hosts at a realistically low field density, although at least in arid or semi-arid areas, rainfall may be critical to the horizontal transmission of this pathogen.     

Infection and mortality of Microtheca ochroloma (Coleoptera: Chrysomelidae) by Isaria fumosorosea (Hypocreales: Cordycipitaceae) under laboratory conditions

Microtheca ochroloma Stål, the yellowmargined leaf beetle, is a pest in crucifer crops during the late fall and winter months in Florida. On organic farms, it is difficult to control due to the restricted use of insecticides, in addition to the lack of specific natural enemies. The objective of this study was to evaluate a blastospore-formulated product of Isaria fumosorosea (PFR-97^TM 20% WDG) against this beetle. In the first experiment, four of the beetle's life stages were treated with a suspension of 3 × 10⁷ blastospores/ml. Mean corrected mortality of treated insects was significantly higher in 1st and 3rd instars than in the egg, pupal, and adult stages. Larvae infected by I. fumosorosea exhibited reduced growth and unsuccessful molting. The second experiment quantified mortality of first instars of M. ochroloma by four concentrations of PFR-97^TM. Mean corrected larval infection/treatment was significantly (2.6 times) higher with a concentration of 4?g of product per 100?ml of water compared to concentrations of 1–3?g per 100?ml of water. Different factors that might have affected the pathogenicity of I. fumosorosea against M. ochroloma are discussed.     

Effect of infection by Metarhizium anisopliae (Hypocreales: Clavicipitaceae) on the feeding and oviposition of the pea leafminer Liriomyza huidobrensis (Diptera: Agromyzidae) on different host plants

The effect of fungal infection by Metarhizium anisopliae on feeding and oviposition of adult Liriomyza huidobrensis was examined on three host plants, faba bean (Vicia faba), French bean (Phaseolus vuklgaris) and snow pea (Pisum sativum) in the laboratory. Flies were contaminated with dry conidia and allowed to feed and oviposit on the different host plants. Mortality in L. huidobrensis varied between 14% and 20% in the controls and between 77% and 100% in fungal treatments 120 h post-infection for the three host plants. L. huidobrensis made more punctures (47.3–52.6 cm^?2) in the control than in the fungal treatments (23.1–26.9 cm^?2) for the three host plants. The cumulative average number of punctures cm^?2/female by L. huidobrensis was higher in the controls than in fungal treatments from 72 h post-treatment in faba bean (12.2 vs. 8.2) and French bean (14.8 vs. 8.9), and from 48 h post-inoculation in snow pea (8.5 vs. 5.7). Female L. huidobrensis laid more eggs in the control (0.6–6.1) than in fungal treatments (0.2–1.5) across the host plants tested. The cumulative mean number of eggs cm^?2/female was significantly higher in the controls than in fungal treatments from 48 h post-treatment in faba bean (0.4 vs. 0.2) and French bean (0.1 vs. 0), and 96 h post-inoculation in snow pea (0.2 vs. 0.1). The host plant did not affect the average total number of punctures but had a significant effect on egg laying, with faba bean harboring greater number of eggs in both control and fungal treatments. A proper timeline application of the fungus before onset of feeding and oviposition peaks will be crucial in field suppression of the pest using M. anisopliae. In addition, a great consideration must be given to the target host plants prior to application of the fungus.     

Application of Metarhizium anisopliae (Metsch.) Sor. conidia to control Otiorhynchus sulcatus (F.) (Coleoptera: Curculionidae) larvae on glasshouse pot plants

The efficacy of the entomogenous fungus Metarhizium anisopliae was assessed against vine weevil (Otiorhynchus sulcatus) larvae in the glasshouse. Prophylactic application of M. anisopliae conidia to begonia resulted in total larval control, but curative applications were less effective with only 65% control when conidial application was delayed until 8 weeks after egg infestation. Prophylactic applications also provided effective larval control on begonia plants which received multiple egg applications over a six week period. Larval mortality was monitored on cyclamen plants which had received a prophylactic drench of M. anisopliae conidia. The population was reduced by 78% within 5 weeks of egg application and control rose to 90% after 17 weeks, although the increase was not significant. Prophylactic conidial drenches were compared with a similar number of compost incorporated conidia on cyclamen, but there was no significant difference between the two spore application strategies. Application of M. anisopliae conidia to impatiens modules before potting-on resulted in over 89% larval control compared to over 97% control when a similar number of conidia were applied to the plants after potting. Larval control was further reduced to 79% when the module drenches were reduced to one quarter of the highest dose (5 × 10⁷ compared to 2 × 10⁸ conidia per module). The persistence of three M. anisopliae strains was examined over a 20 week period on impatiens. There was no overall decline in efficacy over this period, although there was variability in the performance of the different strains and it was suggested that this was linked to temperature. The results of these experiments suggest that M. anisopliae has considerable potential as a microbial control agent for O. sulcatus on glasshouse ornamentals.     

Lethal effects of Gliocladium virens,Beauveria bassiana and Metarhizium anisopliae on the malaria vector Anopheles albimanus (Diptera: Culicidae)

Control of Anopheles albimanus, the main vector of malaria on the coast of the State of Chiapas, is based mainly on application of chemical insecticides, which has resulted in resistance to most registered insecticides. Strategies for biological control may provide sustainable alternatives. We report on the lethal effects of a native isolate of Gliocladium virens on An. albimanus larvae and adults, compared to that of strains of Beauveria bassiana and Metarhizium anisopliae. Conidial suspensions of G. virens, B. bassiana and M. anisopliae cultured on Sabouraud agar were tested in bioassays with An. albimanus larvae and adults. Mosquito larvae were more susceptible to all fungi, compared to adults. On early and late instar larvae, M. anisopliae showed the most pathogenic effect (LC₅₀ of 1.4×10⁵ conidia/mL in early instars; 1.1×10⁵ conidia/mL in late instars), followed by G. virens (LC₅₀ of 3.3×10⁵ conidia/mL in early instars and 3.5×10⁶ conidia/mL in late instars). Metarhizium anisopliae sensu lato and the native G. virens could be considered good choices for An. albimanus control in southern Mexico.