Effects of combining <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Nosema pyrausta</Emphasis> on the mortality of <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis>

We tested the combined effect of the fungus Beauveria bassiana and the microsporidium Nosema pyrausta on the European corn borer larvae, Ostrinia nubilalis, in the laboratory. The first instar of O. nubilalis larvae was the most sensitive to the B. bassiana infection followed by the fifth, second, third, and fourth instar (LC₅₀s were 4.91, 6.67, 7.13, 9.15, and 6.51 × 10⁵ conidia/ml for the first to fifth instars, respectively). Mortality of each instar increases positively with concentration of conidia. When B. bassiana and N. pyrausta were used in combination, mortality increased significantly in all instars. Relative to the B. bassiana treatment alone, the B. bassiana + N. pyrausta treatment decreased the LC₅₀s by 42.16%, 37.63%, 21.60%, 27.11%, and 33.95% for the first to fifth instars, respectively. The combined effects of the two pathogens were mostly additive. However, at the two highest concentrations the pathogens interacted synergistically in the first and second instar. Individuals that survived the B. bassiana and B. bassiana + N. pyrausta treatments and developed into adults had significantly shorter lifespans and females oviposited fewer eggs than non-exposed insects. The effects on the longevity and the egg production were most pronounced at high concentration of B. bassiana conidia.     

Diversity,prevalence and virulence of fungal entomopathogens in colonies of the ant <Emphasis Type="Italic">Formica selysi</Emphasis>

The richness of the parasitic community associated with social insect colonies has rarely been investigated. Moreover, understanding how hosts and pathogens interact in nature is important to interpret results from laboratory experiments. Here, we assessed the diversity, prevalence and virulence of fungal entomopathogens present around and within colonies of the ant Formica selysi. We detected eight fungal species known to be entomopathogenic in soil sampled from the habitat of ants. Six of these entomopathogens were found in active nests, abandoned nests, and corpses from dump piles or live ants. A systematic search for the presence of three generalist fungal entomopathogens in ant colonies revealed a large variation in their prevalence. The most common of the three pathogens, Paecilomyces lilacinus, was detected in 44% of the colonies. Beauveria bassiana occurred in 17% of the colonies, often in association with P. lilacinus, whereas we did not detect Metarhizium brunneum (formerly M. anisopliae) in active colonies. The three fungal species caused significant mortality to experimentally challenged ants, but varied in their degree of virulence. There was a high level of genetic diversity within B. bassiana isolates, which delineated three genetic strains that also differed significantly in their virulence. Overall, our study indicates that the ants encounter a diversity of fungal entomopathogens in their natural habitat. Moreover, some generalist pathogens vary greatly in their virulence and prevalence in ant colonies, which calls for further studies on the specificity of the interactions between the ant hosts and their fungal pathogens.     

Interaction between entomopathogenic nematodes and entomopathogenic fungi applied to third instar southern masked chafer white grubs,Cyclocephala lurida (Coleoptera: Scarabaeidae), under laboratory and greenhouse conditions

Four entomopathogenic nematode (EPN) species (Heterorhabditis bacteriophora Poinar, Heterorhabditis megidis Poinar, Jackson & Klein, Steinernema feltiae Filipjev and Steinernema riobrave Cabanillas, Poinar & Raulston) were tested for virulence against 3^rd instar southern masked chafer white grubs, Cyclocephala lurida Bland. H. bacteriophora and H. megidis, being the most virulent, were selected to evaluate the interaction with an entomopathogenic fungus (EPF), Beauveria bassiana (Balsamo) Vuillemin strain GHA or Metarhizium anisopliae (Metsch.) Sorokin strain F-52, under laboratory and greenhouse conditions. Nematodes and fungi were either applied alone or in combination, with nematodes added to fungi at different times. When applied alone, B. bassiana and M. anisopliae did not reduce grub numbers. Under laboratory conditions, additive interactions were found between H. megidis and B. bassiana, and between H. bacteriophora and B. bassiana or M. anisopliae in most combinations against chafer grubs; a few treatments showed synergism or antagonism. The combined effect did not differ significantly for nematode and fungal applications made simultaneously or at different times. Nematode infection and infective juveniles (IJs) production in grub carcasses were not significantly affected by the presence of a fungus. Efficacies of H. bacteriophora and M. anisopliae were affected by temperature, with grub mortality increasing at higher temperatures. Under greenhouse conditions, additive or synergistic interaction was found between H. bacteriophora and B. bassiana or M. anisopliae in different formulations in simultaneous applications or when the nematode was applied 4 weeks after the fungi, except between B. bassiana ES and H. bacteriophora. The impact of H. bacteriophora alone or in combination with M. anisopliae or B. bassiana on 3^rd instar C. lurida was comparable to that of an imidacloprid insecticide used as curative applications. More virulent fungal strains or species may be required to achieve a stronger interaction with nematodes in the management of C. lurida.     

Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution

Dual biological control, of both insect pests and plant pathogens, has been reported for the fungal entomopathogens, Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) and Lecanicillium spp. (Ascomycota: Hypocreales). However, the primary mechanisms of plant disease suppression are different for these fungi. Beauveria spp. produce an array of bioactive metabolites, and have been reported to limit growth of fungal plant pathogens in vitro. In plant assays, B. bassiana has been reported to reduce diseases caused by soilborne plant pathogens, such as Pythium, Rhizoctonia, and Fusarium. Evidence has accumulated that B. bassiana can endophytically colonize a wide array of plant species, both monocots and dicots. B. bassiana also induced systemic resistance when endophytically colonized cotton seedlings were challenged with a bacterial plant pathogen on foliage. Species of Lecanicillium are known to reduce disease caused by powdery mildew as well as various rust fungi. Endophytic colonization has been reported for Lecanicillium spp., and it has been suggested that induced systemic resistance may be active against powdery mildew. However, mycoparasitism is the primary mechanism employed by Lecanicillium spp. against plant pathogens. Comparisons of Beauveria and Lecanicillium are made with Trichoderma, a fungus used for biological control of plant pathogens and insects. For T. harzianum Rifai (Ascomycota: Hypocreales), it has been shown that some fungal traits that are important for insect pathogenicity are also involved in biocontrol of phytopathogens.     

Age-specific dose-mortality effects of Beauveria bassiana (Deuteromycotina: Hyphomycetes) on the European corn borer,Ostrinia nubilalis (Lepidoptera: Pyralidae)

Three isolates of Beauveria bassiana were evaluated under laboratory conditions for pathogenicity against the European corn borer, Ostrinia nubilalis. Dose-mortality regressions and nonparametric statistical analyses revealed significant differences in pathogenicity between isolates and across O. nubilalis instars. First instars were found to be the most susceptible of the five larval stages. Little difference was noted for instars 2, 3, and 5 within any of the three B. bassiana isolates. Fourth instar larvae were found to be the most tolerant in all cases.     

Influence of juvenile hormone analogue on food consumption and digestive enzyme activities in Spodoptera mauritia Boisd

Final instar larvae of S. mauritia treated topically on day 0, 1, 2 and day 3 with a daily dose of 20 microg juvenile hormone analogue (JHA) showed an increase in most of the nutritional parameters such as approximate digestibility, efficiency of conversion of ingested food, consumption index and growth rate. Also, the activities of digestive enzymes amylase, invertase, trehalase and protease increased significantly in JHA treated larvae. The supernumerary larvae formed after JHA treatments showed an increase in the activities of digestive enzymes. Neck-ligated larvae treated with 10 microg JHA exhibited a significant increase in the activities of trehalase and protease. The results demonstrate that treatments of JHA increase the activities of digestive enzymes in the last instar larvae of S. mauritia.     

Responses of midgut amylases of Helicoverpa armigera to feeding on various host plants

Midgut digestive amylases and proteinases of Helicoverpa armigera, a polyphagous and devastating insect pest of economic importance have been studied. We also identified the potential of a sorghum amylase inhibitor against H. armigera midgut amylase. Amylase activities were detected in all the larval instars, pupae, moths and eggs; early instars had lower amylase levels which steadily increased up to the sixth larval instar. Qualitative and quantitative differences in midgut amylases of H. armigera upon feeding on natural and artificial diets were evident. Natural diets were categorized as one or more members of legumes, vegetables, flowers and cereals belonging to different plant families. Amylase activity and isoform patterns varied depending on host plant and/or artificial diet. Artificial diet-fed H. armigera larvae had comparatively high amylase activity and several unique amylase isoforms. Correlation of amylase and proteinase activities of H. armigera with the protein and carbohydrate content of various diets suggested that H. armigera regulates the levels of these digestive enzymes in response to macromolecular composition of the diet. These adjustments in the digestive enzymes of H. armigera may be to obtain better nourishment from the diet and avoid toxicity due to nutritional imbalance. H. armigera, a generalist feeder experiences a great degree of nutritional heterogeneity in its diet. An investigation of the differences in enzyme levels in response to macronutrient balance and imbalance highlight their importance in insect nutrition.     

Host–pathogen interaction after infection of Galleria mellonella with the filamentous fungus Beauveria bassiana

The filamentous fungus Beauveria bassiana is a natural pathogen of the greater wax moth Galleria mellonella. Infection with this fungus triggered systemic immune response in G. mellonella; nevertheless, the infection was lethal if spores entered the insect hemocel. We observed melanin deposition in the insect cuticle and walls of air bags, while the invading fungus interrupted tissue continuity. We have shown colonization of muscles, air bags, and finally colonization and complete destruction of the fat body—the main organ responsible for the synthesis of defense molecules in response to infection. This destruction was probably not caused by simple fungal growth, because the fat body was not destroyed during colonization with a human opportunistic pathogen Candida albicans. This may mean that the infecting fungus is able to destroy actively the insect's fat body as part of its virulence mechanism. Finally, we were unable to reduce the extremely high virulence of B. bassiana against G. mellonella by priming of larvae with thermally inactivated fungal spores.     

Effects of three endophytic entomopathogens on sweet sorghum and on the larvae of the stalk borer Sesamia nonagrioides

Various endophytic fungi of native plants and crops are important entomopathogens. The objective of this study was to investigate the entomopathogenic action of Beauveria bassiana (Balsamo) Vuillemin, Metarhizium robertsii (Metchnikoff) Sorokin, and Isaria fumosorosea (Wize) Brown & Smith (all Ascomycota: Hypocreales) against larvae of Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae) artificially introduced into Sorghum bicolor L. (Moench) (Poaceae) plants under natural environmental conditions. Sorghum bicolor is an economically important crop cultivated for grain, fiber, forage, and lately for biofuel, and S. nonagrioides is its main pest in Mediterranean areas. Young sorghum plants were inoculated with the entomopathogens by spraying in the field. Plant water status, chlorophyll concentration, photosynthesis, and transpiration were not affected. Thirty days after endophyte establishment, plants were infested with fourth instars of S. nonagrioides. The endophytes prevented 50–70% of larvae from entering stalks. Larval mortality was 70–100% and tunnel lengths were reduced by 60–87%. Larval infestation resulted in reduced electron transport capacity and net photosynthetic rate, which was ameliorated in the presence of I. fumosorosea and reversed by B. bassiana and M. robertsii. The growth of sorghum was unaffected in all treatments during the experimental period. Beauveria bassiana and M. robertsii can protect sweet sorghum from damage induced by S. nonagrioides under natural environmental conditions without affecting plant physiology and growth.     

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.     

Pine processionary moth (Thaumetopoea pityocampa,Lepidoptera: Thaumetopoeidae) larvae are highly susceptible to the entomopathogenic fungi Metarhizium brunneum and Beauveria bassiana

The larvae of the pine processionary moth (PPM), Thaumetopoea pityocampa, feed on the needles of pine and cedar. The urticating hairs of older instars pose a threat to human and animal health. Strains of the entomopathogenic fungi, Metarhizium brunneum (V275, ARSEF 4556) and Beauveria bassiana (KTU-24), were assayed against first to fourth instar T. pityocampa using doses ranging from 1?×?10⁵ to 1?×?10⁸ conidia mL^?1. The three strains differed slightly in their virulence but caused 100% mortality of all instars at the highest dose. The newly emerged or first instar larvae were extremely susceptible with 100% mortality being achieved 2–4 days post inoculation with V275 at all but the lowest dose. The fourth instar larvae appeared to be less susceptible than earlier instars. There was good horizontal transmission of conidia from treated to un-inoculated larvae. However, mortality was higher in third and fourth instars and where the ratio of inoculated versus untreated larvae was high. This we presume is due to spores being more readily trapped by the urticating hairs found on third and older instar larvae. Injection of the nests offers a simple and environmentally friendly way of controlling the pest with reduced risk to operators.     

Occurrence and diversity of fungal entomopathogens in soils of low and high Arctic Greenland

Knowledge of the occurrence, distribution and diversity of pathogens of insects and arachnids (entomopathogens) in the Arctic is very limited. Climate change is expected to affect Arctic terrestrial arthropod communities and therefore also host–pathogen interactions, given that entomopathogens are present. We conducted a survey of fungal entomopathogens in soil samples collected at four localities in Greenland; two at low Arctic sites (Ritenbenk and Disko Island) and two at sites in the high Arctic (Zackenberg and Danmarkshavn). Fungi were isolated from soil samples using larvae of the insect species Galleria mellonella (Lepidoptera) and Tenebrio molitor (Coleoptera) as baits providing evidence that the fungal isolates were indeed entomopathogenic. Five fungal species (Ascomycota; Hypocreales) were found: Isaria fumosorosea Wize, Isaria farinosa (Holmsk.) Fr., Beauveria bassiana (Bals.) Vuill., Beauveria pseudobassiana Rehner and Humber and Tolypocladium inflatum W. Gams (syn.?=?T. niveum). I. farinosa was found at all four localities, while I. fumosorosea was detected in single samples at each of three localities including both high Arctic sites. Only the locality on Disko Island revealed B. bassiana, whereas B. pseudobassiana was isolated at the three other sites. T. inflatum was only found on Disko Island and only isolated with T. molitor as a bait insect. The results document that fungal entomopathogens are widely distributed in the soil environment in Greenland. Entomopathogens should therefore be included in future studies of arthropod ecology in the Arctic.     

Laboratory studies on the competition for insect haemocoel between Beauveria bassiana and Steinernema ichnusae recovered in the same ecological niche

Laboratory assays evaluated the combined action of the entomopathogenic nematode Steinernema ichnusae and the entomopathogenic fungus Beauveria bassiana isolated from the same ecological niche, an oak forest in Sardinia (Italy). Galleria mellonella larvae were used as the test insect with the aim of understanding what happens in host haemocoel during a simultaneous infection with two different entomopathogens. Larval mortality assays were performed using nematodes and fungi both alone and together, at the same and different times, and in different concentrations. No additive or synergistic effects were observed, but there was a clear antagonism and competition for survival space in the haemocoel. Moreover, mutual effects between the symbiotic bacteria of the entomopathogenic nematode Xenorhabdus bovienii and entomopathogenic fungi were investigated. In laboratory experiments, X. bovienii crude extracts were tested for their activity against fungal growth. Compounds produced by B. bassiana were tested for their activity against the growth of bacteria, revealing that X. bovienii and B. bassiana are antagonistic to each other.     

Laboratory and field evaluations of the susceptibility of immature Choristoneura rosaceana (Lepidoptera: Tortricidae) to Beauveria bassiana (Hypocreales: Cordycipitaceae)

Abstract

The susceptibility of immature Choristoneura rosaceana (Harris) to Beauveria bassiana-GHA (BotaniGard® 22WP) was evaluated under laboratory and field conditions. Although egg masses ≤24 h old were susceptible to infection by topically sprayed B. bassiana spores in the laboratory and resulting mycosis significantly reduced the percent of neonates able to emerge, no significant egg mass infections resulted from orchard treatment of ≤24–48 h eggs. Exposure to high levels of the B. bassiana product on apple leaf surfaces in the laboratory caused significant dose-related mortality in first to fourth instar C. rosaceana. First instar C. rosaceana were the most susceptible of the larval stages assayed in the laboratory although only 36% of neonates introduced into the orchard prior to Beauveria treatment and 27% of the neonates emerging from orchard treated egg masses became infected. Fourth instar mortalities were similar but moderate when treated with 1×10⁸ spores mL^?1 in the orchard and on leaf surfaces in the laboratory. Beauveria bassiana-induced larval mortalities were significantly higher when the spores were applied directly to the larval integument as opposed to the leaf surface where the insect would encounter the pathogen in a treated orchard. Feeding of C. rosaceana larvae on antibiotic containing meridic diet prior to their use in trials did not impact the susceptibility of the larvae to B. bassiana. Antibiotic containing meridic diet significantly reduced larval C. rosaceana mortality when treated larvae or the spores were placed directly on the diet as opposed to leaf tissue.     

Dissemination of Microbial Agents Using an Autoinoculating Device and Several Insect Species as Vectors

A device for autoinoculating insects with marker dye and active agents, such as biocompetitors of plant pathogens (e.g., Trichoderma harzianum, Trichoderma polysporum, Bacillus subtilis) and entomopathogens (e.g., Bacillus thuringiensis, Beauveria bassiana) is described. Laboratory tests using a powdered dye indicated that the device worked for sap beetles, house flies, pomace dies, and moths. Quantitative studies with blue dye (as an indicator of dispersal) and some bioactive agents demonstrated that a high percentage of sap beetles became contaminated with the material placed inside the autoinoculator in a short period of time (minutes). In the laboratory, sap beetles carried B. bassiana from the autoinoculator to unexposed sap beetles, causing high mortality. In the field, sap beetles entered the baited traps attached to the autoinoculator, became contaminated with the dye, and carried it to damaged corn or apples.     

Synergies between biological and neonicotinoid insecticides for the curative control of the white grubs Amphimallon majale and Popillia japonica

Synergistic combinations of biological and chemical insecticides might yield promising alternatives for soil insect pest management. In turfgrass of the Northeast U.S., control of root-feeding scarab larvae is highly dependent on conventional insecticides. Studies on interactions between entomopathogenic nematodes and neonicotinoid insecticides, however, demonstrate the feasibility of synergies as an approach for reduced-risk curative control. To understand the breadth of potential synergies, we screened numerous combinations of biological control agents with sublethal doses of neonicotinoids against third instars. Interactions were characterized as synergistic, additive or antagonistic. The most promising combinations identified in laboratory bioassays were advanced to greenhouse pot studies and then to field trials featuring microplots with artificially infested populations. To reveal variation across scarab species, trials were conducted on Amphimallon majale and Popillia japonica. Synergies were consistent across trials and specific to white grub species. For A. majale, synergistic combinations of Heterorhabditis bacteriophora with imidacloprid and clothianidin were discernible in laboratory, greenhouse and field trials. For P. japonica, synergistic combinations of Beauveria bassiana and Metarhizium anisopliae with both neonicotinoids were discernible in the laboratory and greenhouse, but not in the field. For both species, antagonistic interactions were discernible between Bt-products and both neonicotinoids. While nematode-neonicotinoid synergies among scarab larvae have been examined before, fungi-neonicotinoid synergies are unreported. In the context of previous studies, however, no patterns emerge to explain variation across target species or control agent. Further study of non-additive interactions will guide how biological and chemical products could be combined to enhance soil insect pest management.     

Effectiveness of Heterohabditis bacteriophora strain GPS11 applications targeted against different instars of the Japanese beetle Popillia japonica

Field and laboratory tests were conducted from 2001 through 2007 to assess the effectiveness of entomopathogenic nematode Heterorhabditis bacteriophora strain GPS11 applications targeted against different instars of the Japanese beetle, Popillia japonica. During summer flight, P. japonica adults were trapped and caged on turfgrass plots for oviposition. Larval development was monitored for the occurrence of each instar. Nematodes were applied in the field against each developing instar at 2.5 × 10⁹ infective juveniles/ha. In 2001, field data obtained in October resulted in 75%, 53%, and 33% control with the applications targeted against the first, second, and third instars, 69, 28, and 9 days after treatment (DAT), respectively. In 2002 field trial, data obtained in October indicated 97%, 88%, and 0% control when the applications were targeted against the first, second, and third instars at 66, 43, and 14 DAT, respectively. Additional plots established in 2002 to determine efficacy against each instar at 14 DAT showed control of the first, second, and third instars to be 55%, 53%, and 0%, respectively. In laboratory tests conducted in 2002, 2004, and 2007, P. japonica collected from the field at the occurrence of each instar were exposed to H. bacteriophora at concentrations of 0, 10, 33, 100, 330, or 1000 infective juveniles/grub. Probit analysis of the mortality from three of the four sets of tests conducted showed the first instar to be significantly more susceptible to H. bacteriophora than the third instar at the LC₅₀ level and all tests showed the first instar to be significantly more susceptible than the third instar at the LC₉₀ level. In addition to the observed decrease in the third instar susceptibility to H. bacteriophora, soil temperatures in the mid-western United States during late September and October rapidly decline often reaching below 15 °C by the beginning of October when grubs are in the third instar stage of development. Therefore, we conclude that the applications of the nematodes made in August or September will provide higher control than those made in October, due to the more appropriate temperature for nematode activity and the presence of more susceptible larval stages. Early nematode applications may also provide an opportunity for nematodes to recycle and cause secondary infections.     

Isoforms of trehalase and invertase of Fusarium oxysporum

Enzymatic assays and native PAGE were used to study trehalase and invertase activities, depending on culture age and different sugar conditions, in cell-free extracts, culture filtrates and ribosomal wash of Fusarium oxysporum. The activity of invertase preceded that of trehalase; in the exponential phase of growth, mainly invertase activity was produced, whereas trehalase activity was high in the stationary phase. In this last phase of growth, the activity of intracellular trehalase was repressed by monosaccharides, whereas disaccharides, especially lactose and starch, enhanced the activity of intracellular and extracellular trehalase. However, invertase activity was not repressed under these conditions and had the maximal activity in the presence of saccharose. Intracellular trehalase appeared in a single, high-molecular weight (120 kDa) form, whereas the extracellular enzyme appeared in a single, low-molecular weight (60 kDa) form. The activity pattern of invertase isoforms indicated the occurrence of three forms of intracellular enzyme with the main activity band at 120 kDa and two isoforms of extracellular enzyme. In the ribosomal wash, high-molecular weight isoforms of both trehalase and invertase were identified. A possible role of trehalase and invertase in carbohydrate metabolism of fungal pathogens is also discussed.