Identification of a Penicillium urticae metabolite which inhibits Beauveria bassiana

A metabolite of a common soil fungus, Penicillium urticae, which inhibits conidia germination and growth of Beauveria bassiana, was identified. The production, extraction from the culture, and purification of the metabolite is described. Two-dimension thin-layer chromatography, reverse-phase chromatography, mass spectrophotometer and bioassay data indicate that the metabolite is patulin. The implication of patulin inhibition of B. bassiana and its subsequent effect on the potential role of B. bassiana as a control agent of soil-inhabiting insects is discussed.     

The entomopathogenic fungus Beauveria bassiana and its compatibility with triflumuron: effects on the twospotted spider mite Tetranychus urticae

Laboratory studies were conducted to determine the effects of the mycoinsecticide Naturalis-L (Beauveria bassiana conidial formulation) on the twospotted spider mite Tetranychus urticae. Compatibility of B. bassiana with triflumuron (benzoylphenyl urea typically used as an insecticide, but also with acaricide effects), was also investigated in order to incorporate both in the control of this pest. For each juvenile stage, 180–22,800 viable conidia/mL on deutonymphs, 380–12,160 viable conidia/mL on protonymphs, and 712–7480 viable conidia/mL on larvae were evaluated. For the adult stage, the concentrations ranged from 213 to 54,720 viable conidia/mL. The mortality data used in the analysis were those accumulated after 5 days of treatment for deutonymphs and protonymphs, 7 days for larvae and 9 days for adults. The lethal concentration to kill 50% (LC₅₀) for the juvenile stages was 3184 viable conidia/mL (their probit-log concentration regression lines were the same), and 1949 viable conidia/mL for adults. No significant differences in mortality were observed among egg age classes (24-, 48-, 72-, and 96-h-old eggs) at the tested concentrations (1400–22,800 viable conidia/mL). When B. bassiana was combined with 0.25 g Alsystin (25% triflumuron as a wettable powder)/L, mite egg mortality decreased significantly. Triflumuron reduced mycelial growth but not conidial germination of B. bassiana. This fungus is a possible candidate to be included in integrated pest management programs with triflumuron of T. urticae. In such programs, the possible antagonist effects of triflumuron should be considered.     

Differential susceptibility of Amblyomma maculatum and Amblyomma americanum (Acari:Ixodidea) to the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae

Nymph and adult ticks from Ambylomma americanum and Ambylomma maculatum were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (90517) and Metarhizium anisopliae (20500). Fungal suspensions of conidia harvested from potato dextrose plates containing 10⁸ conidia/ml caused greater than 90% mortality in adult A. maculatum but less than 10% mortality in adult A. americanum over a 28 day time course. Similarly, infection with M. anisopliae (10⁸ conidia/ml) resulted in 60 and 15% mortality in A. maculatum and A. americanum, respectively. Nymphs of both tick species were more susceptible to fungal infection reaching mortality rates of almost 100% for A. maculatum and over 35% for A. americanum. Scanning electron microscopy of infected ticks showed rapid attachment, germination, and proliferation of fungal spores on A. maculatum cuticles, and to a much lesser extent on A. americanum cuticles. Pentane extracts of A. americanum cuticle hydrocarbons inhibited germination and hyphal growth of B. bassiana conidia, whereas no inhibition was observed using A. maculatum extracts. Significant mortality towards A. americanum was observed (>60%, 28 days) only when the ticks were treated with B. bassiana directly from the growth medium (10⁷ blastospores/ml, grown for 3–4 days in Sabouraud dextrose + 0.5% yeast extract liquid media). These results indicate tick species display differential susceptibility to the entomopathogenic fungi B. bassiana and M. anisopliae, and that the ability to overcome fungistatic compounds present in the tick epicuticle may determine the likelihood of successful infection and virulence.     

Effects of Selected Fungicides and the Timing of Fungicide Application on Beauveria bassiana-Induced Mortality of the Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Four fungicides used for controlling foliar diseases of potato (Solanum tuberosum) were evaluated under field and laboratory conditions for their effects on the infectivity and sporulation of Beauveria bassiana when used as a control for the Colorado potato beetle, Leptinotarsa decemlineata (CPB). We investigated the direct effects of fungicides on B. bassiana-induced CPB mortality and the effect of time between fungicide and B. bassiana application. Effects of fungicide on conidial survival in the soil and on foliage were examined in the field. Significantly more larval mortality was observed when larvae were sprayed with B. bassiana than with the water control. Fungicide had no significant effect on larval mortality in the field. In the laboratory, survival of larvae was significantly lower among larvae fed fungicide-treated foliage. B. bassiana-induced mortality in the laboratory was observed only when larvae were fed foliage treated with copper hydroxide or water. Larvae fed mancozeb- or chlorothalonil-treated foliage experienced high mortality regardless of B. bassiana treatment. While there was no significant effect of fungicide on B. bassiana sporulation on cadavers in the field, a pattern emerged that indicated higher proportions of cadavers producing conidia in plots sprayed with water or copper hydroxide than in plots sprayed with chlorothalonil or mancozeb. Survival of B. bassiana conidia in the soil and on foliage was significantly greater in plots treated with copper hydroxide or water than in plots treated with mancozeb or chlorothalonil. Fungicides such as copper hydroxide may be less deleterious to the fungus than mancozeb and chlorothalonil.     

Nutrient-mediated germination of Beauveria bassiana conidia on the integument of the bark beetle Dendroctonus ponderosae (Coleoptera:Scolytidae)

Field-collected adult mountain pine beetles, Dendroctonus ponderosae, were inoculated with the white muscardine fungus, Beauveria bassiana, and thoroughly examined externally with a scanning electron microscope. Germinating conidia were found at a very low incidence, and only on antennal clubs. However, mortality of inoculated controls was high, and B. bassiana was confirmed as the causative agent. Germination on the cuticle was greatly increased by sonication of adult beetles. The hypothesis that hemolymph released through sonication-damaged membranes provided a nutrient stimulus that enhanced conidial germination was tested by placing hemolymph or yeast-malt extract broth on the cuticle of otherwise nontreated beetles, which were then inoculated with conidia. Significant germination occurred on the cuticle of these beetles. Therefore, a limiting factor for conidial germination on the sclerotized cuticle of D. ponderosae is probably a lack of sufficient nutrients.     

Method To Enhance Growth and Sporulation of Pelletized Biocontrol Fungi

The biocontrol fungi Trichoderma harzianum, used to control soilborne plant pathogens, and Beauveria bassiana, used to control insect pests, were formulated as mycelial biomass in alginate pellets with wheat bran added. After drying for 0, 4, or 16 h, pellets were placed in water or in aqueous solutions of polyethylene glycol (PEG) 8000 for 4 to 24 h and then allowed to continue drying. PEG-treated pellets containing T. harzianum showed significantly greater proliferation of hyphae in soil than untreated pellets or pellets treated with water. Production of conidia of T. harzianum from PEG-treated pellets was lower than production from untreated pellets after 4 days, although rates were equivalent after 7 days. In contrast, production of conidia of B. bassiana was significantly more rapid from PEG-treated pellets than from untreated pellets. Biocontrol of soilborne plant pathogens or insect pests may be enhanced by rapid hyphal growth of T. harzianum in soil or rapid sporulation of B. bassiana on foliage, respectively. Therefore, PEG treatment may improve the efficacy of these biocontrol agents.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi Beauveria bassiana and Metarhizium anisopliae

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO₄, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20–35°C in both fungal species.     

Management of entomopathogenic fungi in cultures of Tenebrio molitor (Coleoptera: Tenebrionidae)

Larvae of mealworms Tenebrio molitor L. (Coleoptera: Tenebrionidae) have been used as animal feed, but fungal pathogens rapidly downsize the populations, resulting in economic losses. In this work, we established an effective management strategy for fungal pathogens. An entomopathogenic fungus, Beauveria bassiana, was isolated from mealworm cadavers. The bioassay of some isolates of this species at >90% relative humidity revealed that the ERL1575 isolate had the highest virulence. At 20–30% RH, ERL1575 conidia when ingested produced 80% mortality but when sprayed topically produced only <10% mortality. Mealworms that had ingested conidia were exposed to 20, 25, 30 and 35°C and high humidity (>95%) for 5 days. This experiment produced about 90% mortality except at 35°C where mortality was <20%. When 40 fungicides were assayed against ERL1575, fluazinam (1000‐fold) and mancozeb (667‐fold) significantly inhibited conidial germination and/or hyphal growth. When fluazinam and mancozeb were added to the mealworm diet of conidia‐inoculated wheat bran, most were alive 3 days post application. However, 100% mortality resulted 3 days post application in the conidia‐inoculated wheat bran without any fungicides. In conclusion, B. bassiana isolates are pathogenic at <30°C when they are ingested by mealworms but fluazinam and mancozeb can be used for management to control the pathogen in their cultures.     

Biotic and abiotic factors affecting stability of Beauveria bassiana conidia in soil

Beauveria bassiana conidia were stored in sterile and nonsterile soil under various temperature, relative humidity, soil water content, and pH regimes. Survival of the conidia was primarily dependent on temperature and soil water content. Conidia half-lives ranged from 14 days at 25°C and 75% water saturation to 276 days at 10°C and 25% water saturation. Conidia held at ?15°C exhibited little or no loss in viability regardless of water content, relative humidity, or pH. Conidia were not recoverable after 10 days from soils held at 55°C. Conidia survival in nonsterile soil that was amended with carbon sources, nitrogen sources, or combinations of carbon and nitrogen was greatly decreased and loss was often complete in less than 22 days whereas sterile soil treated in the same manner showed dramatic increases in number, demonstrating that B. bassiana is capable of growth in sterile soil. The obvious fungistatic effect in amended nonsterile soils was possibly related to Penicillium urticae which was routinely isolated from the soils and is shown to produce a water-soluble inhibitor of B. bassiana. The fungistatic effect was shown to be an active inhibition rather than due to competition.     

Selection of surfactant compounds to enhance the dispersion of Beauveria bassiana

This study aimed to select surfactant compounds to improve the dispersion of emulsifiable conidial suspensions of Beauveria bassiana for field application while maintaining high conidia viability. Surfactant compatibility was initially evaluated by colony growth in culture media amended with increasing doses of surfactants. In a second experiment, the conidia germination rate was evaluated after temporary exposure to the compounds under laboratory conditions. The effect of the hydrophilic–lipophilic balance (HLB) of the surfactants on conidia dispersion and viability was assessed using mixtures of the most compatible products and concentration initially selected. Unitol L/20 and Ultranex NP/100 did not show deleterious effect on colony growth on culture media, while Surfom 3409 strongly inhibited conidial germination. Unitol L/20, Ultranex NP/100 and Ultratan D did not interfere in the germination of conidia during the first 10 minutes of exposure. After 120 minutes, germination was greatly reduced at a 0.5% concentration, with the exception of Ultranex NP/100. The higher conidial dispersion and germination were obtained with HLB values between 8 and 12 and lower remaining clusters at HLB 10. Moreover, there was no significant effect of HLB values on insect mortality. Unitol L/20 and Ultranex NP/100 were the most compatible surfactants in concentrations lower than 0.5%. The HLB value selected for dispersion of B. bassiana on water based formulations was 10.     

Distribution of the Entomopathogenic Fungus Beauveria bassiana in Rice Ecosystems and Its Effect on Soil Enzymes

Fungal entomopathogens, especially Beauveria bassiana, are often studied within the context of their use in biological pest control; however, there is limited knowledge of their distributions in host plants and soil ecosystem. We examined the distribution of B. bassiana and its influence on rice plants and paddy soils. B. bassiana could only be detected on the foliar surfaces of rice plants within 15 days under Bb-4 (7.5 × 10⁴ conidia/mL) and Bb-7 (7.5 × 10⁷ conidia/mL) treatments. The endophytic colonization of B. bassiana could not be found in stems, roots, or seeds of rice plants under Bb-4 and Bb-7 treatments. The fungus was found only in the leaves of rice plants under Bb-4 and Bb-7 treatments at 15 days after inoculation. Moreover, B. bassiana was absent from paddy soils under Bb-4 and Bb-7 treatments at all times. Enzyme activity (urease and phosphatase) in the paddy soils of Bb-4 and Bb-7 treatments showed no significant difference from the control. It is possible that B. bassiana was not able to colonize paddy soil. Detailed understanding of distribution and ecological interactions of B. bassiana is helpful for understanding and predicting the effects of fungal entomopathogens on host populations, and the interactions among fungal entomopathogens and other organisms in the community.     

Effects of soil-treatment with fungal biopesticides on pupation behaviors,emergence success and fitness of tea geometrid,Ectropis grisescens (Lepidoptera: Geometridae)

Ectropisgrisescens Warren (Lepidoptera: Geometridae) is one of the most severe defoliating pests of tea plants. Synthetic pesticides have been widely applied to control this pest in tea plantations, but pesticide residues may decrease the quality and safety of tea products. In the present study, we hypothesized that soil treatment with the two commercial biopesticides (Shuiguxin^®) based on Metarhizium anisopliae (Metchnikoff) Sorokin and Beauveria bassiana (Balsamo) could reduce the survivorship and fitness of E. grisescens. Wandering larvae of E. grisescens were allowed to pupate in soil treated with each biopesticide, and the concentrations of M. anisopliae (Shuiguxin^®) and B. bassiana (Shuiguxin^®) that produced the 50% mortality values (LC₅₀) were 2.9 × 10⁶ and 1.6 × 10⁷ conidia/g soil, respectively. Artificial burying the pupae using soil treated with M. anisopliae (Shuiguxin^®) and B. bassiana (Shuiguxin^®) (1 × 10⁸ or 1 × 10⁹ conidia/g soil for both biopesticides) also significantly reduced emergence success of E. grisescens. In addition, choice tests showed that soil treated with the high concentration of M. anisopliae (Shuiguxin^®) or B. bassiana (Shuiguxin^®) had repellent effects on pupating E. grisescens. However, sublethal concentrations (LC₂₅ and LC₅₀) of both biopesticides did not significantly affect fecundity, fertility and longevity of post-emerged adults. Our study showed that soil treatment with the two commercial biopesticides caused direct mortality of pupating E. grisescens, but may not effectively suppress E. grisescens populations at sublethal concentrations. The realistic application of the fungal dosage in fields should be determined in future studies     

Germination polarity of Beauveria bassiana conidia and its possible correlation with virulence

Three different germination types of conidia; unidirectional, bidirectional and multidirectional, were revealed through microscopic observations for eight Beauveria bassiana isolates germinated on Sabouraud dextrose agar. Canonical correlation analysis indicated that there is a positive correlation between unidirectional germination and virulence against diamondback moth, Plutella xylostella and the Colorado potato beetle, Leptinotarsa decemlineata. Scanning electron microscopy revealed different in vivo behaviors for unipolar- and bipolar-germinated conidia. Unipolar-germinated conidia produced a strong germ tube with mostly appressorium-like structures while bipolar-germinated conidia continued to invasive hyphal growth without any penetration, indicating that germination polarity in one way or another may be an indicator of pathogenic ability.     

Nutritional requirements for conidial germination and hyphal growth of Beauveria bassiana

Nutritional requirements for germination and growth of the entomopathogenic fungus Beauveria bassiana are not complex. For germination to occur, a utilizable source of carbon must be present; however, a nitrogen source is needed for continued hyphal growth, otherwise lysis ensues. Compounds that can serve as utilizable carbon-energy sources for germination include glucose, N-acetylglucosamine, glucosamine, chitin, starch, lanolin, hydrocarbons in crude oil, and some longer-chain fatty acids. Both organic and inorganic sources of nitrogen are readily utilized for growth. Conidia undergo active metabolism soon after being placed in a suitable growth medium, indicating that conidia are released from their state of dormancy several hours before emergence of the germ tube can be observed. Because of the nutritional versatility of B. bassiana, this fungus should be able to survive and be infective in several types of natural environments.     

Horizontal Transmission of Entomopathogenic Fungi by the Diamondback Moth

The relative potential of the pathogenic fungi Beauveria bassiana and Zoophthora radicans for use as autodisseminated biological control agents of the diamondback moth (Plutella xylostella) was compared. The LC₅₀ of B. bassiana conidia to third instar larvae was 499 conidia/mm² of leaf surface and individual cadavers of mycosed fourth instar larvae yielded a mean of 67.5 × 10⁶ (±7.5 × 10⁶) conidia. All concentrations of B. bassiana tested in inoculation chambers (0.24, 2.4, and 6.2 μg/mm²) induced 100% mortality in adult male moths within 7 days. The times to death and sporulation were concentration and exposure duration dependent. A standard procedure for inoculating male moths resulted in >85% mortality from Z. radicans and >93% mortality from B. bassiana. Pairing of inoculated males with clean moths of both sexes yielded higher rates of passive transmission of B. bassiana than Z. radicans, but there was no evidence for sexual transmission of either pathogen. Similarly, B. bassiana was more effectively transmitted from inoculated male moths to larvae foraging on whole plants. Single sporulating cadavers producing B. bassiana or Z. radicans conidia placed on plants infested with larvae resulted in a similar rate of transmission for both pathogens. However, an increase of the density of sporulating cadavers from one to three/plant increased Z. radicans transmission (greater than fourfold) but had no effect on B. bassiana transmission. Simultaneous inoculations of larvae with conidia of both fungi reduced the mortality induced by each pathogen, the reduction being most acute for B. bassiana-induced mortality. Inoculation of adults with both fungi showed that, at concentrations required for effective passive transmission to larvae, B. bassiana severely inhibited Z. radicans mycosis in adults.     

Use of Beauveria bassiana in combination with commercial insecticides to manage Phauda flammans (Walker) (Lepidoptera: Phaudidae): Testing for compatibility and synergy

An entomopathogenic fungal strain, Beauveria bassiana PfBb, was identified from Phauda flammans (Lepidoptera: Phaudidae) larvae. The compatibility and synergy of B. bassiana PfBb employed in combination with three concentrations (i.e., recommended concentration, 20% and 10% of the recommended concentration) of five commercial insecticides were determined. Beta cypermethrin at 10% of recommended concentration had the lowest inhibitory effect on the mycelial growth of B. bassiana PfBb compared with other insecticides. Insecticides utilized at recommended concentration had no significant effect on the sporulation of B. bassiana PfBb, while the extent of their effect at 20% and 10% of recommended concentration differed among insecticides. Insecticides at 10% of recommended concentration had the lowest inhibition of sporulation and conidial germination compared with other concentrations. The conidial germination of B. bassiana PfBb was the highest after treatment with beta cypermethrin at 10% of recommended concentration. The cumulative mortality for 1 × 10⁷ spores/mL B. bassiana PfBb combined with each insecticide at 10% of recommended concentration was higher than that observed with the application of insecticides alone. The percent cadavers of Phauda flammans larvae observed after treatment with B. bassiana PfBb combined with beta cypermethrin at 10% of recommended concentration were not significantly different from those observed after infection with B. bassiana PfBb alone. Our findings demonstrate that B. bassiana PfBb combined with beta cypermethrin at 10% of recommended concentration could increase the efficiency of this insecticide.     

Growth and insect assays of Beauveria bassiana with neem to test their compatibility and synergism

Beauveria bassiana is being used as a biopesticide for many insect pests. Neem oil (azadirachtin) is an eco-safe popular botanical pesticide. A biopesticde with a neem compatible isolate of B. bassiana will enable their simultaneous use in pest management. A sample of 30 isolates of B. bassiana from culture collections was screened for compatibility with a commercial formulation of neem oil (Margoside®) at the field recommended dose (0.3%, v/v). Compatibility was tested in vitro through germination and growth assays. In all isolates, conidial germination was delayed but not significantly decreased by neem. In the growth assays, 23 isolates were found compatible with neem. In the neem sensitive isolates, growth was decreased but not totally inhibited. The effect of combined treatment with B. bassiana and neem in comparison to single treatments with either of them on Spodoptera litura Fabricius was tested in laboratory bioassays. The combined treatment was found to have synergistic effect on insect mortality when a B. bassiana isolate compatible with neem was used, while, with an isolate sensitive to neem, an antagonistic effect was observed.     

Toxic components on the larval surface of the corn earworm (Heliothis zea) and their effects on germination and growth of Beauveria bassiana

Caprylic acid is present on the surface of corn earworm, Heliothis zea, and fall armyworm, Spodoptera fragiterda, larvae. Because caprylic acid inhibits germination of Beauveria bassiana, presence of this compound will be determined to the establishment of an infection of larvae by this fungus. Other free fatty acids present on the surface of the H. zea and S. frugiterda are tentatively identified as valeric and nonanoic acids; these also possess mycostatic activity toward B. bassiana. Depending on concentration, caprylic acid inhibits germination of conidia for different amounts of time (R. J. Smith and E. A. Grula, 1981, J. Invertebr. Pathol., 37, 222–230). We now further report that inhibition and/or growth is also related to the source of carbon, nitrogen, and energy present in the growth medium. This observation of selective toxicity in the presence of different nutrients was also observed using nonanoic acid. Our data therefore make it necessary to interpret the effects of certain fatty acids on germination and growth of B. bassiana (and probably other fungi as well) in terms of nutrients for the germination process.     

Beauveria bassiana: Synergistic effect with acaricides against the tick Hyalomma anatolicum anatolicum (Acari: Ixodidae)

Owing to the need to combat the spread of chemical acaricide resistance in ticks, we evaluated the efficacy of a mixture of the entomopathogenic fungus Beauveria bassiana AT17 and acaricides for the control of Hyalomma anatolicum anatolicum in China. A mixture of B. bassiana AT17 at the concentration of 10⁸ conidia/mL and the synthetic pyrethroid deltamethrin at concentrations of 2500, 250, 25, 5, 2.5, 0.5 and 0.25 ppm was tested in vitro. The germination capability, vegetative growth, conidia production, and viability of B. bassiana AT17 were assessed and the efficacy of the mixture in killing engorged H. anatolicum anatolicum females was measured. High mortality rates were achieved when the entomopathogen was combined with different concentrations of deltamethrin. Neither B. bassiana AT17 nor deltamethrin alone at the same concentrations could cause the higher mortality rates seen with the combination. In addition the combination killed the ticks more rapidly than did either agent alone (3–5 days more rapidly). Our results indicate that the mixture of B. bassiana AT17 and deltamethrin has potential as a new type of reagent for integrated control of H. anatolicum anatolicum.     

Effects of crop type on persistence and control efficacy of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> against the two spotted spider mite

The influence of five different crops (beans, cucumber, eggplant, maize, and tomato) on persistence and the control efficacy of Beauveria bassiana (Balsamo) Vuillemin to Tetranychus urticae Koch was investigated. Plants were artificially infested with T. urticae. B. bassiana conidia were suspended in Break-thru^® solution (0.01%) at three different concentrations (0.5, 1, 2 g l^?1) and sprayed once onto plants. Plants in the control treatment were sprayed with Break-thru^® solution alone. The number of conidia and levels of mite mortality were evaluated. Colony forming unit counts of conidia recovered from plant leaves declined over time regardless of the concentrations of B. bassiana applied. The rate of decline was different among crops and significantly higher on maize. However, more than 50% of the initial population of conidia were still viable on crops three weeks after application. The longer persistence of B. bassiana on crops did not result in better control of mites. Rather, mortality was positively correlated to the amount of inoculum deposited on leaves immediately after spraying. Mortality of mites was concentration-dependent and influenced by the host crop, with less control observed on maize and tomato than eggplant, beans and cucumber. Results were consistent between trials conducted and confirmed the hypothesis that the type of crop can influence the efficacy of B. bassiana against T. urticae.