Stefan T. Jaronski

Ecological factors in the inundative use of fungal entomopathogens

Fungal entomopathogens have been developed in numerous countries as biocontrol agents with more than 100 mycoinsecticide products commercially available in 2006. The chief, perhaps sole, use of these mycoinsecticides has been as inundative agents, within a chemical paradigm. Large numbers of propagules are applied in an attempt to overwhelm by brute force many of the factors that keep a pathogen in nonepizootic equilibrium with its host. This review attempts to summarize what we know about the abiotic and biotic factors that affect the efficacy of these mycoinsecticides in both foliar and soil applications. Sunlight, humidity, temperature, and phylloplane-associated factors can affect both immediate efficacy and persistence on plants. Likewise, soil texture-moisture interactions, temperature, and a host of biotic factors can affect mycoinsecticides in the soil. Despite much research, our understanding of these ecological aspects is imperfect, especially in a holistic, dynamic sense.

Ecological considerations in producing and formulating fungal entomopathogens for use in insect biocontrol

Insect pests persist in a wide-variety of agricultural, arboreal and urban environments. Effective control with fungal entomopathogens using inundation biocontrol requires an understanding of the ecology of the target insect, fungal pathogen, and the insect-pathogen interaction. Historically, the development of production and formulation processes for biocontrol fungi has primarily focused on reducing costs by maximizing the yield of infective propagules, increasing storage stability, and improving product form for ease of application. These goals are critical for commercialization but are often in conflict with environmental and ecological considerations. Critical parameters for selecting a fungal pathogen for use in inundation biocontrol include the cost-effective production of a stable, infective propagule that is suited for use in the environment where the insect must be controlled. Production processes can be manipulated nutritionally and environmentally to produce efficacious propagules or to direct fungal differentiation to propagule forms that may be better suited for use in specific environments. Formulation development must also consider ecological and environmental factors to maximize biocontrol efficacy. A basic understanding of the surface chemistries of the fungal propagule and insect, the interactions between a fungal propagule and the insect cuticle that lead to infection, and the impact of the environment on this interaction can aid in the development of effective formulations.

Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects.

Microsclerotia (MS), overwintering structures produced by many plant pathogenic fungi, have not been described for Metarhizium anisopliae. Three strains of M. anisopliae--F52, TM109, and MA1200--formed MS in shake flask cultures using media with varying carbon concentrations and carbon-to-nitrogen (C:N) ratios. Under the conditions of this study, all strains produced MS, compact hyphal aggregates that become pigmented with culture age, in addition to more typical blastospores and mycelia. While all strains formed desiccation tolerant MS, highest concentrations (2.7-2.9 x 10(8) L(-1) liquid medium) were produced in rich media with C:N ratios of 30:1 and 50:1 by strain F52. All three strains of M. anisopliae produced similar biomass concentrations when media and growth time were compared. Strain MA1200 produced higher concentrations of blastospores than the other two strains of M. anisopliae with highest blastospore concentrations (1.6 and 4.2 x 10(8) blastospores ml(-1) on days 4 and 8, respectively) in media with the highest carbon and nitrogen concentrations. Microsclerotial preparations of M. anisopliae containing diatomaceous earth survived air-drying (to <5 % moisture) with no significant loss in viability. Rehydration and incubation of air-dried MS granules on water agar plates resulted in hyphal germination and sporogenic germination to produce high concentrations of conidia. Bioassays using soil-incorporated, air-dried MS preparations resulted in significant infection and mortality in larvae of the sugar beet root maggot, Tetanops myopaeformis. This is the first report of the production of sclerotial bodies by M. anisopliae and provides a novel approach for the control of soil-dwelling insects with this entomopathogenic fungus.

Efficacy of Metarhizium anisopliae microsclerotial granules

Abstract The entomopathogenic fungus Metarhizium anisopliae has very recently been shown to produce microsclerotia (MS) – compact, heavily melanised, hyphal aggregates – in liquid media. Soil incorporation bioassays of dried MS preparations of three isolates of M. anisopliae were conducted using third instar Tetanops myopaeformis (sugarbeet root maggot) in clay and/or clay loam field soils as a model system to demonstrate efficacy. At rates as low as 23 mg MS granules/100 g dry soil, the biocontrol efficacy of MS granules of M. anisopliae Strain F52 produced in liquid media with a high carbon concentration (36 g/L) and high C:N ratios (30:1, 50:1) were superior to MS preparations produced in low carbon (8 g carbon/L) media and a high carbon medium with a 10:1 C:N ratio. Bioassays using MS formulations of M. anisopliae strains MA1200 and TM109 produced in high carbon and high C:N ratio media were superior in efficacy to the other MS production media tested. MS preparations of M. anisopliae F52 showed superior efficacy against the sugarbeet root maggot in comparison with more conventional, conidia-covered nutritive (corn grit) granules in a clay and clay soil. The MS granules were also highly efficacious against the sugarbeet root maggot at soil moisture levels as low as 0.983 A w (−2.33 MPa). Granular preparations incorporating Metarhizium MS can serve as a viable formulation for the use of this fungus against soil insects.

Mass production of entomopathogenic Hypocreales

The Hypocreales, Beauveria bassiana , Metarhizium anisopliae sensu lato, Isaria fumosorosea and I. farinosus , Lecanicillium spp., and Nomuraea rileyi , have become important insect control agents in recent times and consequently subjects of much scientific study and development. Mass production of infective stages is important for obtaining sufficient quantities of infective stages for field studies of these fungi. Conidial production on agar media in Petri dishes is generally insufficient for such purposes. There are several methods for mass production, involving solid substrate fermentation for aerial conidia, and liquid fermentation for blastospores or microcycle conidia, and for a novel structure, evidently specific to Metarhizium spp., the microsclerotium. This chapter presents basic techniques for all these procedures, techniques that should allow any laboratory to produce hypocrealean fungi for their use, as well as provide a theoretical basis to adapt mass production to local requirements and devise technical improvements.

Susceptibility of preimaginal western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae) to Beauveria bassiana (Balsamo) Vuillemin Clavicipitaceae (Hypocreales).

Last-instar larvae of the western cherry fruit fly, Rhagoletis indifferens, were subjected to Beauveria bassiana GHA incorporated into sterile sand and non-sterile orchard soil. Mycosis in the pupal stage was observed in >20% of buried R. indifferens pupae and >80% of larvae entering sand treated with either of two B. bassiana isolates. When pre-pupal larvae burrowed into conidium-treated non-sterile cherry orchard soil, the incidence of mycosis, on both the puparia and internally developing pupae, increased with dose. Internal pupal tissues were found to contain B. bassiana. Increasing the soil moisture level from 20% to 35% water holding capacity did not have an effect on the percentage of mycosed pupae. This is the first evidence that the preimaginal stages of R. indifferens are susceptible to infection by B. bassiana.

A distributed delay routine-based simulation model of Beauveria bassiana conidial stability in response to environmental stressors

Using published data and equations on therelationship between spore longevity of theentomopathogenic hyphomycetes, Metarhiziumanisopliae var. acridum and Beauveria bassiana (Balsamo) Vuillemin(Deuteromycota: Hyphomycetes) and temperatureand moisture content, a model of sporeviability was constructed based on adistributed-delay routine. The model ismodified via average spore survival time or byincluding an additional attrition (mortality)rate. The model was parameterized usingpublished values from studies on M. a.var. acridum spores, and output comparedfavorably with germination data and with apreviously-developed model. After initializingthe model using parameter estimates of B.bassiana spores from the laboratory andpublished data on changes in (1) spore viabilitywith respect to temperature and moisturecontent, and (2) spore moisture content withrespect to temperature and relative humidity,the model was run using daily min/maxtemperature and relative humidity data andcompared with data from four field experimentsof Mycotech B. bassiana isolate GHAsprayed on canteloupe plants. For two of theexperiments, observed viability trends werecompared to model outputs using weather datafrom both a weather station and fromwithin-canopy temperature and humidity probes. Output using weather station data fitobservations much better than output usingwithin-canopy probe data. For the tworemaining sets of field data, both earlier inthe season, only weather station data wereavailable and the resulting output fitobservations poorly. An attrition rate of 98%was needed to fit output to field data early inthe growing season, and a rate of 74% wasneeded for data collected four weeks later. These attrition rates can be consideredestimates for the proportion of spores dyingfor reasons other than temperature and relativehumidity, and they were attributed largely toUVB radiation due to the more open canopyearlier in the season.

Interactions Among Bt Maize, Entomopathogens, and Rootworm Species (Coleoptera: Chrysomelidae) in the Field: Effects on Survival, Yield, and Root Injury

ABSTRACT A 2 yr field study was conducted to determine how a blend of entomopathogens interacted with Bt maize to affect mortality of Diabrotica spp. (Coleoptera: Chrysomelidae), root injury to maize (Zea maize L.) and yield. The blend of entomopathogens included two entomopathogenic nematodes, Steinernema carpocapsae Weiser and Heterorhabditis bacteriophora Poinar, and one entomopathogenic fungus, Metarhizium brunneum (Metschnikoff) Sorokin. Bt maize (event DAS59122–7, which produces Bt toxin Cry34/35Ab1) decreased root injury and survival of western corn rootworm (Diabrotica virgifera virgifera LeConte) and northern corn rootworm (Diabrotica barberi Smith & Lawrence) but did not affect yield. During year 1 of the study, when rootworm abundance was high, entomopathogens in combination with Bt maize led to a significant reduction in root injury. In year 2 of the study, when rootworm abundance was lower, entomopathogens significantly decreased injury to non-Bt maize roots, but had no effect on Bt maize roots. Yield was significantly increased by the addition of entomopathogens to the soil. Entomopathogens did not decrease survival of corn rootworm species. The results suggest that soil-borne entomopathogens can complement Bt maize by protecting roots from feeding injury from corn rootworm when pest abundance is high, and can decrease root injury to non-Bt maize when rootworm abundance is low. In addition, this study also showed that the addition of entomopathogens to soil contributed to an overall increase in yield.

Effects of Entomopathogens on Mortality of Western Corn Rootworm (Coleoptera: Chrysomelidae) and Fitness Costs of Resistance to Cry3Bb1 Maize

ABSTRACT Fitness costs can delay pest resistance to crops that produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt), and past research has found that entomopathogens impose fitness costs of Bt resistance. In addition, entomopathogens can be used for integrated pest management by providing biological control of pests. The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of maize and is currently managed by planting of Bt maize. We tested whether entomopathogenic nematodes and fungi increased mortality of western corn rootworm and whether these entomopathogens increased fitness costs of resistance to Cry3Bb1 maize. We exposed western corn rootworm larvae to two species of nematodes, Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and Steinernema feltiae Filipjev (Rhabditida: Steinernematidae), and to two species of fungi, Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) (strain GHA) and Metarhizium brunneum (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) (strain F52) in two assay types, namely, seedling mat and small cup. Larval mortality increased with the concentration of H. bacteriophora and S. feltiae in the small cup assay, and with the exception of S. feltiae and B. bassiana in the seedling mat assay, mortality from entomopathogens was significantly greater than zero for the remaining entomopathogens in both assays. However, no fitness costs were observed in either assay type for any entomopathogen. Increased mortality of western corn rootworm larvae caused by these entomopathogens supports their potential use in biological control; however, the lack of fitness costs suggests that entomopathogens will not delay the evolution of Bt resistance in western corn rootworm.

A Novel Bioassay to Evaluate the Potential of Beauveria bassiana Strain NI8 and the Insect Growth Regulator Novaluron Against Lygus lineolaris on a Non-Autoclaved Solid Artificial Diet

Abstract A non-autoclaved solid diet was used to evaluate the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) strain NI8 and the insect growth regulator novaluron (Diamond® 0.83EC insecticide) for control of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae). The diet was composed of toasted wheat germ, ground lima bean meal, soy flour, yolk of chicken eggs, inhibitor, and agar. It was prepared in one step by blending the ingredients in boiling water. The diet was used to bioassay L. lineolaris from the second instar to the adult stage. Fourth and fifth instars and adults of L. lineolaris were more susceptible than second and third instars to infection by B. bassiana , whereas second, third, and fourth instars had higher mortality than fifth instars 10 days after exposure to novaluron. No effects on longevity were observed in adults treated with novaluron when compared with the control, but longevity was significantly different from that of adults exposed to B. bassiana . Adults of L. lineolaris were maintained for over a month without changing the diet. The non-autoclaved diet is semi-liquid before it cools, which facilitates the mechanics of diet packaging similar to food packaging or lepidopteran diet preparation. This solid artificial diet for Lygus bugs provides improved research capacity for studying the ecology and susceptibility of Lygus spp. to a number of different control agents, including beneficial organisms, insect pathogens, and insecticidal toxins being developed for transgenic technologies.