The Entomopathogenic Fungal Endophytes Purpureocillium lilacinum (Formerly Paecilomyces lilacinus) and Beauveria bassiana Negatively Affect Cotton Aphid Reproduction under Both Greenhouse and Field Conditions

The effects of two entomopathogenic fungal endophytes, Beauveria bassiana and Purpureocillium lilacinum (formerly Paecilomyces lilacinus), were assessed on the reproduction of cotton aphid, Aphis gossypii Glover (Homoptera:Aphididae), through in planta feeding trials. In replicate greenhouse and field trials, cotton plants (Gossypium hirsutum) were inoculated as seed treatments with two concentrations of B. bassiana or P. lilacinum conidia. Positive colonization of cotton by the endophytes was confirmed through potato dextrose agar (PDA) media plating and PCR analysis. Inoculation and colonization of cotton by either B. bassiana or P. lilacinum negatively affected aphid reproduction over periods of seven and 14 days in a series of greenhouse trials. Field trials were conducted in the summers of 2012 and 2013 in which cotton plants inoculated as seed treatments with B. bassiana and P. lilacinum were exposed to cotton aphids for 14 days. There was a significant overall effect of endophyte treatment on the number of cotton aphids per plant. Plants inoculated with B. bassiana had significantly lower numbers of aphids across both years. The number of aphids on plants inoculated with P. lilacinum exhibited a similar, but non-significant, reduction in numbers relative to control plants. We also tested the pathogenicity of both P. lilacinum and B. bassiana strains used in the experiments against cotton aphids in a survival experiment where 60% and 57% of treated aphids, respectively, died from infection over seven days versus 10% mortality among control insects. Our results demonstrate (i) the successful establishment of P. lilacinum and B. bassiana as endophytes in cotton via seed inoculation, (ii) subsequent negative effects of the presence of both target endophytes on cotton aphid reproduction using whole plant assays, and (iii) that the P. lilacinum strain used is both endophytic and pathogenic to cotton aphids. Our results illustrate the potential of using these endophytes for the biological control of aphids and other herbivores under greenhouse and field conditions.     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Effect of temperature and humidity on infection by Beauveria bassiana,Metarhizium anisopliae, and Paecilomyces farinosus

Larvae of the elm bark beetle, Scolytus scolytus, were inoculated with conidia of the entomogenous fungi Beauveria bassiana (two strains), Metarhizium anisopliae (two strains), and Paecilomyces farinosus (two strains) and incubated over a range of temperatures (2°, 6°, 10°, 15°, and 20°C). One strain each of B. bassiana and P. farinosus caused infection even at 2°C, whereas the two strains of M. anisopliae caused no infection below 10°C. Infection of adult beetles by B. bassiana (one strain) and M. anisopliae (one strain) was tested at 15°, 20°, and 25°C (B. bassiana) and at 15° and 20°C (M. anisopliae). Fungal infection occurred at all three temperatures, but at 25°C beetles tended to succumb to bacterial infection. The effect of relative humidity on infection of larvae by B. bassiana (one strain), M. anisopliae (one strain), and P. farinosus (one strain) was tested at 51, 74, 86, 90, 95, 97.5, and 100% relative humidity. B. bassiana and M. anisopliae caused some infection at all humidities: with P. farinosus there was no infection at the two lowest humidities. Mortality due to infection by these fungi was most rapid at the highest humidities.     

Nematicidal activity of culture filtrate of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> against <Emphasis Type="Italic">Meloidogyne hapla</Emphasis>

Culture filtrates of Beauveria bassiana at different concentrations were evaluated for nematicidal activity against the northern root knot nematode (Meloidogyne hapla); bioassays included egg hatching, mortality and infectivity on tomato plants in pots under glasshouse conditions. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filtrates of B. bassiana. Soil drenching with culture filtrate of B. bassiana significantly reduced nematode population densities in soil and in the roots and subsequent gall formation and egg-mass production by M. hapla under glasshouse conditions.     

Feasibility of growing Moringa oleifera as a mix-crop along with tomato for control of Meloidogyne incognita and Rotylenchulus reniformis in Egypt

Impact of nematicidal potential of drumstick, Moringa oleifera Lam. (Moringaceae), as a medicinal plant (1–4 plants per pot) is obtained when it is used as a mix-crop along with tomato cv. Super Strain B against Meloidogyne incognita and Rotylenchulus reniformis was evaluated under a greenhouse conditions (30?±?5?°C) at the National Research Centre, Egypt. The nematodes final population and their rate of build-up as well as the root gall index were significantly affected by the number of moringa plants when grown with tomato together. There was a negative correlation between the number of moringa seedlings and the final population of both nematodes. The lowest nematode final population and rate of build-up were determined at the highest number of moringa plants (four plants per pot). The highest number of root gall index (4.1) was found on roots of tomato grown alone, while, the lowest one (0.6) was found on roots of tomato grown with four plants of moringa. This type of control is considered easy, inexpensive and pollution-free.     

Marigold,Castor Bean,and Chrysanthemum as Controls of Meloidogyne incognita and Pratylenchus alleni

Root and soil populations of Meloidogyne incognita were significantly fewer from marigold, castor bean, and chrysanthemum than from tomato roots and soil, but not from fallow soil. Root populations of Pratvlenchus alleni were significantly fewer from marigold, castor bean, and chrysanthemum than from tomato: marigold had the fewest. Root populations of M. incognita and P. alleni from tomato simultaneously cultivated with marigold, castor bean, and chrysanthemum were significantly fewer than from tomato cultivated alone. Aborted giant cells and dead M. incognita (larvae and females) were observed in roots of marigold and castor bean, but not in chrysanthemum or tomato. Significantly more males than females occurred in castor bean roots. lnfcction sites of P. alleni appeared normal in all hosts. Thin-layer and column chromatography of alcoholic extracts from castor bean revealed no nematicidal thiophenc derivatives.     

The effects of exotoxin (Thuringiensin) from Bacillus thuringiensis on Meloidogyne incognita and Caenorhabditis elegans

A partially purified preparation as well as two formulations of exotoxin from Bacillus thuringiensis (thuringiensin) were evaluated for nematicidal activity. The methods used in our evaluations included direct contact nematicidal assays, hatching tests, infection tests in seed pouches using the cucumber/root-knot nematode (Meloidogyne incognita) system, and greenhouse test using the root-knot nematode. While contact nematicidal activity was not observed against juveniles of M. incognita, 100% mortality occurred when the free-living nematode, Caenorhabditis elegans, was used as the test organism. Nematode infection evaluations in the seed pouch assay showed reduced root galling at relatively high concentrations (>10 mg kg^-1). Greenhouse assays indicated significant reduction in the soil population. However, the degree of control in relation to the amount of material applied, as measured by the gall numbers, larvae from soil/roots, and plant growth parameters, was not considered adequate. Data on the plant response in relation to treatment with different formulations of the toxin are presented.     

Pasteuria penetrans for Control of Meloidogyne incognita on Tomato and Cucumber,and M. arenaria on Snapdragon

Meloidogyne incognita and Meloidogyne arenaria are important parasitic nematodes of vegetable and ornamental crops. Microplot and greenhouse experiments were conducted to test commercial formulations of the biocontrol agent Pasteuria penetrans for control of M. incognita on tomato and cucumber and M. arenaria on snapdragon. Three methods of application for P. penetrans were assessed including seed, transplant, and post-plant treatments. Efficacy in controlling galling and reproduction of the two root-knot nematode species was evaluated. Seed treatment application was assessed only for M. incognita on cucumber. Pasteuria treatment rates of a granular transplant formulation ranged from 1.5 × 10⁵ endospores/cm³ to 3 × 10⁵ endospores/cm³ of transplant mix applied at seeding. Additional applications of 1.5 × 10⁵ endospores/cm³ of soil were applied as a liquid formulation to soil post-transplant for both greenhouse and microplot trials. In greenhouse cucumber trials, all Pasteuria treatments were equivalent to steamed soil for reducing M. incognita populations in roots and soil, and reducing nematode reproduction and galling. In cucumber microplot trials there were no differences among treatments for M. incognita populations in roots or soil, eggs/g root, or root condition ratings. Nematode reproduction on cucumber was low with Telone II and with the seed treatment plus post-plant application of Pasteuria, which had the lowest nematode reproduction. However, galling for all Pasteuria treatments was higher than galling with Telone II. Root-knot nematode control with Pasteuria in greenhouse and microplot trials varied on tomato and snapdragon. Positive results were achieved for control of M. incognita with the seed treatment application on cucumber.     

Comparison of peptidase activities in some fungi pathogenic to arthropods

Peptidases, highly specific toward several synthetic chromogenic peptides, were found in the mycelia of four arthropod pathogenic fungi: Aphanomyces astaci, Beauveria bassiana, Metarrhizium anisopliae, and Paecilomyces farinosus. A. astaci peptidases had high hydrolyzing activities toward most of the peptides, especially those with arginine in the P1 position, while those of B. bassiana and P. farinosus readily hydrolyzed peptides with valine and arginine, as well as proline and tyrosine in the P2 and P1 positions, respectively. The hydrolyzing capacities of M. anisopliae peptidases were similar to A. astaci, but showed lower specific activities. Casein or azocoll was only hydrolyzed by A. astaci peptidases. B. bassiana and M. anisopliae had a very low hydrolyzing capacity toward casein and could not degrade azocoll. P. farinosus had no hydrolyzing activity toward casein or azocoll. Only peptidases from the crayfish pathogen A. astaci could degrade the crayfish cuticle. The peptidase preparations of A. astaci and B. bassiana hydrolyzing MeO-Suc-Arg-Pro-Tyr-pNA or Bz-Phe-Val-Arg-pNA were of the serine type. The possible importance of peptidase activity of arthropod pathogenic fungi in the infection process is discussed.     

Effects of varying environmental factors on the biological control of Meloidogyne incognita in tomato by Bacillus cereus strain BCM2

The root-knot nematode (Meloidogyne spp.), which represents a global threat to agricultural production, can cause serious losses in both the yield and quality of many crops. Endophytic bacteria are known to have great potential against Meloidogyne incognita. The colonisation ability of endophytic Bacillus cereus BCM2 in tomato roots and its biological control efficacy of M. incognita were investigated. By the end of the growth period of tomato plants, the population of BCM2 in the rhizosphere soils and roots of the tomato were 5.86 and 3.38 log CFU g^?1, respectively, indicating that BCM2 can colonise tomato roots for long periods of time. Pre-inoculation with BCM2 resulted in a significant reduction in the population of M. incognita and the gall index of tomato compared to the untreated control, and there was an increase in the tomato yield of 47.4%. Colony counts showed that the population of BCM2 in tomato roots was affected by soil type and pH, and the colonisation of BCM2 in tomato rhizosphere soils was influenced by soil water and organic matter contents. We observed that the biocontrol effects of BCM2 were best when soil pH was 7. Pre-inoculation with BCM2 can inhibit the formation of tomato galls more effectively when soil water content is 25%, and rich organic matter content was conducive to a reduction in the number of M. incognita second stage juveniles (J2s) in soil. These results demonstrated that B. cereus BCM2 has great potential for controlling M. incognita in tomato plants.     

Horizontal transmission of mycotic infection in adult tsetse,Glossina morsitans morsitans

AdultGlossina morsitans morsitans exposed to wet conidia ofBeauveria bassiana andMetarhizium anisopliae suffered high mortalities ranging from 90 to 100% by 2 weeks post-exposure. Infected ♂ ♂ maintained in the same cages with non-infected ♀♀ throughout the experimental period transmitted the fungal infection to the ♀♀ resulting in mortalities of 65% withB. bassiana and 55% withM. anisopliae. Likewise, infected ♀♀ maintained together with non-infected ♂♂ transmitted the infection to the ♂♂ resulting in mortalities of 75% withB. bassiana and 45% withM. anisopliae. Female tsetse flies infected withB. bassiana andM. anisopliae and maintained in the same cages with non-infected ♀♀ also transmitted infection to the non-infected tsetse resulting in mortalities of 62% and 48% withB. bassiana andM. anisopliae respectively. Infected tsetse exposed to non-infected tsetse of the opposite sex for only 30 min were also able to transmit the fungal infection. Pupae produced by female tsetse infected withB. bassiana andM. anisopliae exhibited higher pupal mortality than those produced by non-infected ♀♀. However, pupae exposed directly to dry spores ofB. bassiana andM. anisopliae had no increase in pupal mortality but adults emerging from theB. bassiana-exposed pupae had markedly reduced longevity.      

Potential of Metarhizium anisopliae and Beauveria bassiana to control Dinoderus porcellus (Coleoptera: Bostrychidae) infesting yam chips

The beetle Dinoderus porcellus Lesne is a serious storage insect pest that causes important losses by destroying stocks of yam chips. In the aim to found an alternative control method to the use of synthetic insecticides for its management, the virulence of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin (isolate Bb115) and Metarhizium anisopliae (Metschnikoff) Sorokin (isolate Met 31) against adults of D. porcellus was evaluated under laboratory conditions (25 ± 2 °C and 70 ± 5% RH). Then, the effectiveness of the most virulent entomopathogenic fungus as biological agent against D. porcellus was assessed under farmer storage conditions. For each entomopathogenic fungus isolate, four conidial concentration (0, 10⁵, 10⁷, and 10⁹ conidia/mL) at the dose of 1 µL were inoculated topically on D. porcellus adults (3–5 days old). Observations focused on insect mortality, cadaver sporulation and weight loss of yam chips. Lethal dose and lethal time values were estimated using probit analysis. Both fungal isolates at all conidial dose caused more than 50% mortality on day 7, with the highest mortality (94.44%) achieved using B. bassiana at the 10⁹ conidia/mL. LT₅₀ values for B. bassiana and M. anisopliae isolates were 2.63 and 3.35 days, respectively, while their LT₉₀ values were 6.15 and 9.87 days, respectively. Yielding the lower LD₉₀ values and the highest rates of cadaver sporulation, B. bassiana isolate appeared as the most virulent against D. porcellus. After 3 months of storage, comparatively to the control, the B. bassiana isolate at the highest conidial dose (10⁹ conidia/mL) significantly reduced D. porcellus populations, and weight loss of yam chips. This study revealed the potential of B. bassiana and M. anisoplae isolates as biological control agent against D. porcellus for yam chips protection.     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Pathogenicity of Beauveria bassiana,metarhizium anisopliae, and Paecilomyces farinosus to larvae and adults of S. scolytus

The entomogenous fungi Beauveria bassiana (nine isolates), Metarhizium anisopliae (seven isolates), and Paecilomyces farinosus (four isolates) were tested as pathogens of larvae of the elm bark beetle, Scolytus scolytus. Single isolates of B. bassiana and M. anisopliae were also tested against adult beetles. Of the 21 isolates tested as conidial suspensions against larvae, all proved pathogenic. The three most and least virulent isolates were, respectively, isolates of B. bassiana and M. anisopliae. The other isolates fell between these two extremes, with the four P. farinosus isolates all moderately virulent. Spore retention on larvae following inoculation was estimated by washing conidia off the larvae. From the results it was possible to relate larval mortality to the approximate spore dose causing infection at different spore concentrations. Thus, application of spores of the three pathogens at a concentration of 10³ spores/ml resulted in limited mortality. At this concentration, an average of only a single spore was recovered from the inoculated larva. Adult bark beetles also proved susceptible to infection by isolates of B. bassiana and M. anisopliae. They were exposed to discs of elm bark dipped in a conidial suspension. It was estimated that a dose of less than 100 spores could cause infection of beetles following feeding on the elm bark discs.     

Growth and sporulation of Beauveria bassiana and Metarhizium anisopliae in a basal medium containing various carbohydrate sources

Naturally occurring isolates of two entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, pathogenic to the pecan weevil, Curculio caryae, were cultured in flasks of a liquid synthetic culture medium containing one of 22 individual carbohydrate sources to determine growth and sporulation responses. B. bassiana grew best on d-melizitose but sporulated best on d-sucrose, d-trehalose, and d-glucose. M. anisopliae grew best on d-mannose but sporulated best on i-inositol and glycerol. B. bassiana grew least on l-rhamnose but M. anisopliae grew least on d-arabinose. Both fungi sporulated least on d-sorbose. Both fungi utilized d-trehalose, a major component of insect hemolymph.     

Growth and sporulation of Beauveria bassiana and Metarrhizium anisopliae on media containing various amino acids

Natural isolates of two entomogenous fungi, Beauveria bassiana and Metarrhizium anisopliae, were cultured in liquid culture media containing 24 amino acids and KNO₃ to determine their effect on growth and sporulation. In addition, the growth and medium pH changes for each isolate grown on an asparagine-containing medium were compared. Tryptophan and alanine were most effective for growth and sporulation of B. bassiana, although glutamine and KNO₃ also produced large numbers of regularly shaped spores. Tryptophan, glutamic acid, and histidine were all well utilized for both growth and sporulation of M. anisopliae. Nitrogen sources containing sulfur were poorly utilized for sporulation by M. anisopliae. In general, B. bassiana produces greater mycelial mass and much larger numbers of spores than M. anisopliae. Both fungi attained nearly the same growth maximum on asparagine medium though B. bassiana exhibited an initially more rapid growth rate. In both fungi this rapid growth phase was accompanied by a decline in medium pH followed by a rise in pH during the decline phase of growth.     

Survival of Paecilomyces lilacinus in Selected Carriers and Related Effects on Meloidogyne incognita on Tomato

Laboratory and microplot experiments were conducted to determine the influence of carrier and storage of Paecilomyces lilacinus on its survival and related protection of tomato against Meloidogyne incognita. Spores of P. lilacinus were prepared in five formulations: alginate pellets (pellets), diatomaceous earth granules (granules), wheat grain, soil, and soil plus chitin. Fungal viability was high in wheat and granules, intermediate in pellets, and low in soil and chitin-amended soil stored at 25 ± 2 C. In 1985 P. lilacinus in field microplots resulted in about a 25% increase in tomato yield and 25% gall suppression, compared with nematodes alone. Greatest suppression of egg development occurred in plots treated with P. lilacinus in pellets, wheat grain, and granules. In 1986 carryover protection of tomato against M. incognita resulted in about a threefold increase in tomato fruit yield and 25% suppression of gall development, compared with plants treated with nematodes alone. Higher numbers of fungus-infected egg masses occurred in plots treated with pellets (32%) than in those treated with chitin-amended soil (24%), wheat (16%), granules (12%), or soil (7%). Numbers of fungal colony-forming units per gram of soil in plots treated with pellets were 10-fold greater than initial levels estimated at planting time in 1986.     

Behavioral and electrophysiological responses of Coptotermes formosanus Shiraki towards entomopathogenic fungal volatiles

Termites adjust their response to entomopathogenic fungi according to the profile of fungal volatile organic compounds (VOCs). This study demonstrates the pathogenicity of Metarhizium anisopliae, Beauveria bassiana and Isaria fumosorosea (=Paecilomyces fumosoroseus) towards the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Using no-choice assays, M. anisopliae was found to be highly virulent (LT₅₀ 3.10 d) when compared to B. bassiana (LT₅₀ 6.62 d) and I. fumosorosea (LT₅₀ 12.39 d). Also using choice assays, the foraging behavior of C. formosanus was determined in the presence of pathogenic fungi. The highly pathogenic fungi (M. anisopliae) elicited a repellent response, causing most of the termites to forage in a safe zone farthest from the fungal source. This repellency resulted in relatively low mortality similar to the controls. The repellency of M. anisopliae conidia can be used to protect human belongings and timber from termites. While I. fumosorosea cultures were not repellent to C. formosanus workers, the termites were highly susceptible to infection. Electroantennographic responses of workers showed approximately 47% and 78% lower level of response to conidia of B. bassiana and I. fumosorosea, respectively, as compared to M. anisopliae. The VOC profile of repellent cultures of M. anisopliae mainly consisted of paraffins (60.97%), while the major proportion of the I. fumosorosea profile consisted of branched and cyclic alkanes (84.41%). From the above findings, we conclude that the incorporation of I. fumosorosea may increase the control potential of bait.     

Glucosinolate content and nematicidal activity of Brazilian wild mustard tissues against Meloidogyne incognita in tomato

The wild mustard (Brassica juncea L.), an invasive weed of winter crops in Brazil, was evaluated for glucosinolate content of its plant tissues and nematicidal activity of its dry leaf meal (LM), whole seed meal (WSM) and hexane defatted seed meal (DSM) against Meloidogyne incognita on tomato plants. Sinigrin was the major glucosinolate in LM, WSM and DSM, occurring at concentration of 0.11, 12.2 and 21.9 mg/gdw, respectively. Allyl isothiocyanate (AITC) was the major degradation product and its concentration was highest in DSM followed by WSM and LM. The number of galls, egg masses and eggs on tomato plants was reduced by over 90% by amending soil with 1.6% LM, 0.2% WSM, or 0.05% DSM. Exposure to the volatiles from the amended soils reduced egg eclosion. The soil amendment with LM, WSM and DSM killed the second stage juveniles of M. javanica, M. enterolobii (=M. mayaguensis) and Heterodera glycines. The efficacy of the LM, WSM and DSM for nematode suppression was related to the amount of AITC released in soil.     

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.     

Effect of Soils from Six Management Systems on Root-knot Nematodes and Plant Growth in Greenhouse Assays

The effects of soil management systems on root-knot nematode (Meloidogyne incognita) eggs and gall incidence on tomato (Lycopersicon esculentum) and cucumber (Cucumis sativus) following tomato were evaluated. Soil was collected from a replicated field experiment in which six management systems were being assessed for vegetable production. Soil management systems were conventional production, organic production, bahiagrass (Paspalum notatum) pasture, bahiagrass: Stylosanthes (Stylosanthes guianensis) pasture, bare ground fallow, and weed fallow. Soil was collected from field plots and used in greenhouse experiments. Identification of egg-parasitic fungi and the incidence of root-knot nematode galling were assessed both on tomato and cucumber planted in the same pots following the removal of tomato plants. Organic, bare ground fallow and conventional production treatments reduced galling both on tomato and on cucumber following tomato. Although no treatment consistently enhanced egg-parasitic fungi, management system did affect egg viability and the types of fungi isolated from parasitized eggs.     

Age of three dipteran hosts as a factor governing the pathogenicity of Beauveria bassiana and Metarrhizium anisopliae

The age of three adult dipteran hosts was tested as a factor governing the virulence of two fungal pathogens. The subjects tested were 1-, 5-, 10-, 15-, 20-, 25-, and 30-day-old animals. The age of the house fly, Musca domestica, the age of the black blowfly, Phormia regina, and the age of the onion fly, Hylemya antiqua, do not significantly affect the virulence of either Beauveria bassiana or Metarrhizium anisopliae. B. bassiana kills M. domestica and P. regina more quickly than does M. anisopliae, but B. bassiana and M. anisopliae kill H. antiqua at the same rate. The ages of all three flies had no significant effect on average death time after inoculation with either pathogen.