Bassiana Growth Research Articles

Influences of compound age and identity in the effectiveness of insect quinone secretions against the fungus Beauveria bassiana.

Chemical defences against parasites and pathogens can be seen in a wide range of animal taxa, including insect pests such as the red flour beetle Tribolium castaneum. Antimicrobial quinone-based secretions can be used by these beetles to defend against various parasites, particularly the fungal entomopathogen Beauveria bassiana. While quinone secretions can inhibit B. bassiana growth, it is unknown how long they remain effective or how individual secretion compounds contribute to growth inhibition. Here, we tested each individual component of the quinone secretions (methyl-1,4-benzoquinone, ethyl-1,4-benzoquinone, and 1-pentadecene), as well as two mixed solutions that represent the composition range found in natural T. castaneum secretions, after aging for 0, 24, or 72h. The two quinone compounds equally contributed to B. bassiana inhibition, but their efficacy was significantly reduced after 24h, with no growth inhibition after 72h. This indicates that quinones protect insects against B. bassiana for only a limited time, perhaps requiring constant secretion into the environment to effectively defend against this fungal threat. Future investigations may consider the extent to which quinone secretions are effective against other parasites, as well as how their ability to cause parasite damage changes with compound age.

A Sensitive Method for Detecting Beauveria bassiana, an Insecticidal Biocontrol Agent, Population Dynamics, and Stability in Different Substrates.

Beauveria bassiana is a well-known insecticidal biocontrol agent. Despite its broad field applications, its survival, colonization, and stability under field conditions remained unclear, mainly due to the lack of a quick and reliable detection method. In this study, we developed a quantitative real-time PCR technology to monitor the stability and population dynamics of B. bassiana in different substrates (water, soil, and on the cotton leaves surface), different spores of B. bassiana applied on Chinese cabbage leaves surface, and the lethality of Pieris rapae spraying with different spores of B. bassiana. Our results showed a decreased concentration of B. bassiana DNA in all three substrates from the 1st day till 9th day of post inoculation (dpi) period, possibly due to the death of B. bassiana. After this decrease, a quick and significant rebound of B. bassiana DNA concentration was observed, starting from the 11th dpi in all three substrates. The B. bassiana DNA concentration reached the plateau at about 13th dpi in water and 17th dpi in the soil. On cotton leaves surface, the B. bassiana DNA concentration reached the highest level at the 17th dpi followed by a small decline and then stabilized. This increase of DNA concentration suggested recovery of B. bassiana growth in all three substrates. We found that the most suitable killing effectiveness of P. rapae was the 1.0 × 107 spores/mL of B. bassiana. In summary, we have established a detection technology that allows a fast and reliable monitoring for the concentration and stability of B. bassiana under different conditions. This technology can benefit and help us in the development of proper management strategies for the application of this biocontrol agent in the field.

Metabolism of plant-derived toxins from its insect host increases the success of the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a soil fungus that parasitizes a large number of arthropod species, including numerous crop pests, causing white muscardine disease and is therefore used as a biological insecticide. However, some insects, such as the cabbage aphid (Brevicoryne brassicae), defend themselves chemically by sequestering dietary pro-toxins (glucosinolates) from their Brassicales host plants. Glucosinolates are accumulated by cabbage aphids and activated to form toxic isothiocyanates when under attack. While isothiocyanate formation protects aphids against most attackers, B. bassiana is still able to infect the cabbage aphid under natural conditions. We therefore investigated how this fungus is able to circumvent the chemical defense system of the cabbage aphid. Here, we describe how B. bassiana infection activates the cabbage aphid defense system, but the resulting toxins are metabolized by B. bassiana via the mercapturic acid pathway, of which the first step is catalyzed by glutathione-S-transferases of low substrate specificity. This detoxification pathway enhances B. bassiana growth when isothiocyanates are present in natural concentrations, and so appears to be an important factor in fungal parasitization of these chemically defended aphids.

Symbiotic Bacteria System of Locusta migratoria Showed Antifungal Capabilities against Beauveria bassiana

The stability of symbiotic flora is an important indicator of the health of an organism. Symbiotic bacteria have been proven to be closely involved in the immune process of organisms. The pathogenicity of Beauveria bassiana was studied in relation to symbiotic bacteria on the surface and inside of the migratory locust (Locusta migratoria). The results showed that the surface disinfection of test locusts contributed to the pathogenicity of B. bassiana to locusts. Most of the surface bacteria of L. migratoria caused some inhibition of B. bassiana growth, and LM5-4 (Raoultella ornithinolytica), LM5-2 (Enterobacter aerogenes), and LM5-13 (Citrobacter freundii) showed the highest inhibitory effect on the growth of B. bassiana. The inoculation of locusts with additional surface symbiotic bacteria reduced the virulence of B. bassiana to L. migratoria. Infection by different strains of B. bassiana caused similar changes in the symbiotic flora of migratory locusts. The inoculation of locusts with additional intestinal symbiotic bacteria (Enterobacter sp.) reduced the virulence of B. bassiana to L. migratoria. These findings illustrate the effect of bacterial communities on fungal infections in L. migratoria when seen from the perspective of ecology in a microenvironment. The active antifungal substances of such bacteria and their mechanisms of action need further study.

Beauveria bassiana Growth and Development in Various Liquid Media

Beauveria bassiana is entomopathogenic fungi that can be cultivated on solid or liquid media. The cultivation of B. bassiana on cracked corn medium leaves unwanted waste. Therefore, cultivating B. bassiana on liquid media is expected to shorten the cultivation time and leave no waste. The purpose of the research was to analyze the physical characteristics of the B. bassiana colony, nutritional changes of the liquid media, and the pH level changes of the liquid media. This research used an experimental complete randomized design with six treatments and 4 times repetitions. The main phases of this research were the subculture of B. bassiana isolate; the preparation of liquid media consisting of distilled water, Potato Dextrose Broth (PDB), Potato Sugar extract (PSE), PSE+NPK 1%, PSE+coconut water, PSE+NPK 1%+coconut water each for 150 ml; B. bassisana inoculation into the liquid media; incubation process for 30 days; and measurement of fungal blastospore density, medium pH, and carbohydrate, protein, and lipid content. The results obtained from the morphological observation of B.bassiana on liquid media showed that the colonies are white, growing on the surface of the media with a powdery texture. There are some differences in the nutrient content of the media after 30 days of B. Bassiana incubation. After the incubation, there is an increase in carbohydrates and a decrease in protein and lipid content, as well as an increase in the pH level of the media.

Contributions of a Histone Deacetylase (SirT2/Hst2) to Beauveria bassiana Growth, Development, and Virulence.

Sirtuins are a class of histone deacetylases that promote heterochromatin formation to repress transcription. The entomopathogenic fungus Beauveria bassiana contains six sirtuin homologs. The class III histone deacetylase, BbSir2, has been previously shown to affect the regulation of carbon/nitrogen metabolism and asexual development, with only moderate effects on virulence. Here, we examine another class III histone deacetylase (BbSirT2) and show that it contributes to deacetylation of lysine residues on histone H4-K16ac. Directed gene-knockout of BbSirT2 dramatically reduced conidiation, the ability of the fungus to metabolize a range of carbon and nitrogen sources, and tolerances to oxidative, heat, and UV stress and significantly attenuated virulence in both intrahemocoel injection and topical bioassays using the Greater wax moth (Galleria mellonella) as the insect host. ΔBbSirT2 cells showed alterations in cell cycle development and hyphal septation and produced morphologically aberrant conidia. Comparative transcriptomic analyses of wild type versus ΔBbSirT2 cells indicated differential expression of 1148 genes. Differentially expressed genes were enriched in pathways involved in cell cycle and rescue, carbon/nitrogen metabolism, and pathogenesis. These included changes in the expression of polyketide synthases (PKSs) and LysM effector proteins that contribute to degradation of host toxins and target host pathways, respectively. These data indicate contributions of BbSirT2 in helping to mediate fungal stress and development, with the identification of affected gene targets that can help account for the observed reduced virulence phenotype.

Transcriptomic analysis of Sur7-mediated response ofBeauveria bassianato different nutritional conditions

ABSTRACTIntegrity of the cell wall is requisite for fungal growth and function. Sur7 governs cell wall composition, and affects conidial sporulation and germination in Beauveria bassiana, a filamentous entomopathogenic fungus. The role of Sur7 in fungal growth on various nutrients remains unclear. We have previously reported that Sur7 deletion results in the attenuation of B. bassiana growth on supplemented Sabouraud dextrose agar (SDAY) and minimal Czapek–Dox agar (CDA) compared to wild type (WT). Here, we used transcriptomic analysis to compare WT and Sur7 mutant (ΔSur7) responses to CDA and SDAY. Growth on CDA, compared with that on SDAY, affected the expression of more genes in the WT than in the mutant. Differentially expressed genes were enriched for transportation process terms in the ΔSur7 mutant and metabolic process terms in the WT. Different processes were repressed in the ΔSur7 (metabolic process) and WT (ribosome synthesis) cells. Despite the shared enrichment of nitrogen metabolism genes, differentially expressed genes were enriched in distinct saccharide-energy metabolism terms in each strain. We conclude that Sur7 ensures the growth of B. bassiana in a minimal medium by influencing the expression of genes involved in the consumption of sucrose via specific energy metabolism pathways.

Plant secondary metabolites and low temperature are the major limiting factors for Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) growth and virulence in a bark beetle system

Beauveria bassiana is a ubiquitous entomopathogen and widely used as a biological control agent for a variety of arthropod pests, including bark beetles. The North American spruce beetle (Dendroctonus rufipennis) is a major pest in forest landscapes and recent studies show that B. bassiana is pathogenic to beetles in the lab but successful field applications have been limited by abiotic and biotic factors within the study system. To understand how habitat conditions impact fungal ecology, we evaluated variation in B. bassiana radial growth across conditions representative of the spruce beetle habitat, incorporating a range of temperatures, competition with spruce beetle symbiotic fungi, as well as exposure to Engelmann spruce tree secondary metabolites, nutrient limitations, osmotic potentials, and sunlight. Pathogenicity to spruce beetle was subsequently quantified in tests varying beetle origin, temperature, and bioassay arena substrate. Three major findings emerged: (1) growth of genetically similar B. bassiana isolates varied considerably in response to abiotic conditions, suggesting significant within-haplotype phenotypic diversity; (2) low ambient temperatures and exposure to Engelmann spruce tree secondary metabolites, two conditions which are prevalent in spruce beetle habitat, strongly inhibit B. bassiana growth; and (3) pathogenicity varied across environments: all isolates appeared pathogenic under room conditions, but when beetles inhabited in planta bioassays under in natura conditions most isolates were not pathogenic. This is the first study to evaluate the inhibitory effects that a series of tree secondary metabolites have on B. bassiana growth, virulence and pathogenicity. These collective findings have implications for field applications of B. bassiana but also the interpretation of entomopathogenicity, and could help to explain discrepancies between laboratory and field tests.

The autophagy gene BbATG5, involved in the formation of the autophagosome, contributes to cell differentiation and growth but is dispensable for pathogenesis in the entomopathogenic fungus Beauveria bassiana

Autophagy is a highly conserved process, representing the major eukaryotic degradative pathway of cellular components. Autophagy-mediated recycling of cellular materials contributes to cell differentiation, tissue remodelling and proper development. In fungi, autophagy is required for normal growth and cell differentiation. The entomopathogenic fungus Beauveria bassiana and its invertebrate targets represent a unique model system with which to examine host-pathogen interactions. The ATG5 gene is one of 17 involved in autophagosome formation, and the B. bassiana homologue (BbATG5) was identified. The role of autophagy in B. bassiana growth and virulence was investigated via construction of a targeted gene knockout of BbATG5. The mutant strain displayed increased sensitivity to nutrient limitation, with decreased germination and growth as compared with the wild-type parent. Conidiation was severely compromised and conidia derived from the ΔBbATG5 strain were altered in morphology. Cell differentiation into blastospores was also greatly reduced. Despite the significant growth and developmental defects, insect bioassays using the oriental leafworm moth, Spodoptera litura, indicated a modest (~40 %) decrease in virulence in the ΔBbATG5 strain. The phenotypic defects of the ΔBbATG5 strain could be restored by introduction of an intact copy of BbATG5. These data suggest that unlike several plant and animal pathogenic fungi, where ATG5 is required for infection, in B. bassiana it is dispensable for pathogenesis.

The Mosquito Melanization Response Is Implicated in Defense against the Entomopathogenic Fungus Beauveria bassiana

Mosquito immunity studies have focused mainly on characterizing immune effector mechanisms elicited against parasites, bacteria and more recently, viruses. However, those elicited against entomopathogenic fungi remain poorly understood, despite the ubiquitous nature of these microorganisms and their unique invasion route that bypasses the midgut epithelium, an important immune tissue and physical barrier. Here, we used the malaria vector Anopheles gambiae as a model to investigate the role of melanization, a potent immune effector mechanism of arthropods, in mosquito defense against the entomopathogenic fungus Beauveria bassiana, using in vivo functional genetic analysis and confocal microscopy. The temporal monitoring of fungal growth in mosquitoes injected with B. bassiana conidia showed that melanin eventually formed on all stages, including conidia, germ tubes and hyphae, except the single cell hyphal bodies. Nevertheless, melanin rarely aborted the growth of any of these stages and the mycelium continued growing despite being melanized. Silencing TEP1 and CLIPA8, key positive regulators of Plasmodium and bacterial melanization in A. gambiae, abolished completely melanin formation on hyphae but not on germinating conidia or germ tubes. The detection of a layer of hemocytes surrounding germinating conidia but not hyphae suggested that melanization of early fungal stages is cell-mediated while that of late stages is a humoral response dependent on TEP1 and CLIPA8. Microscopic analysis revealed specific association of TEP1 with surfaces of hyphae and the requirement of both, TEP1 and CLIPA8, for recruiting phenoloxidase to these surfaces. Finally, fungal proliferation was more rapid in TEP1 and CLIPA8 knockdown mosquitoes which exhibited increased sensitivity to natural B. bassiana infections than controls. In sum, the mosquito melanization response retards significantly B. bassiana growth and dissemination, a finding that may be exploited to design transgenic fungi with more potent bio-control activities against mosquitoes.

Pseudomonas contamination of a fungus-based biopesticide: Implications for honey bee (Hymenoptera: Apidae) health and Varroa mite (Acari: Varroidae) control

The ectoparasitic mite Varroa destructor is a major honey bee pest, and its control using pathogen-based biopesticides would resolve many of the problems, such as contamination and pesticide resistance, experienced with chemical control. A biopesticide, formulated with commercially-prepared conidia of a strain of Beauveria bassiana isolated from V. destructor was tested against the mites in bee colonies in southern France. The impact of treatment on hive survivorship, weight and mite infestation levels were very different from those of previous experiments using laboratory-prepared conidia: bee hives treated with the biopesticide died at a higher rate, lost more weight, and had higher mite densities at the end of the study than control hives. The biopesticide was subsequently found to be contaminated with bacteria. Two strains of bacteria were identified, by biotyping and sequencing data of the 16S rRNA and rpoB regions, and while the strains were distinct both were Pseudomonas sp. belonging to the P. fluorescens group. In dual cultures B. bassiana growth was slowed or suppressed when bacterial cfu density was about equal or greater than that of B. bassiana. Experiments using caged adult bees showed that bees ingesting diet and sugar solution treated with B. bassiana and kept at 30°C had significantly lower survival times than those treated with one of the bacterial strains, but the opposite was true at 33°C. Because one arthropod (honey bees) was treated for infestation by another (V. destructor), the impact of bacterial contamination was likely more noticeable than in most uses of biopesticides, such as treating plants against phytophagous insects. To reduce such risk in biopesticide development, a systematic screening for bacterial contamination prior to field application is recommended.

Systemic Protection ofPapaver somniferumL. AgainstIraella luteipes(Hymenoptera: Cynipidae) by an Endophytic Strain ofBeauveria bassiana(Ascomycota: Hypocreales)

The poppy stem gall wasp, Iraella luteipes (Thompson) (Hymenoptera: Cynipidae), is one of the main pests of the opium poppy, Papaver somniferum L., an economically important pharmaceutical crop cultivated worldwide. In a previous study, we obtained from I. luteipes larvae a strain of the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales) that can become established endophytically in opium poppy plants. A field experiment was conducted to study the ability of this B. bassiana strain to provide systemic protection against damage by I. luteipes in opium poppy in southern Spain for three seasons. Conidial suspensions were applied as seed dressings, leaf sprays, or soil sprays. The effect of the treatment was studied by harvesting fully ripened plants and dissecting I. luteipes larvae from the stem. The effect of treatment on growth and yield was also evaluated. Emergence of I. luteipes adults was not uniform over the 3 yr, with important differences exhibited in the duration of the emergence period, although the flight peaks tended to occur in mid-late April. B. bassiana seed dressings, leaf sprays at the fourth true-leaf stage, and soil sprays were not significantly different in their ability to reduce the number of larvae per plant compared with the controls, with percentage reductions of 36.5-58.5, 64.4-73.4, and 51.9-57.2% in 2005, 2006, and 2007, respectively. Even though the population level of I. luteipes increased over the 3 yr, the efficacy of the fungal inoculation in reducing the larval population was maintained throughout the study period. No significant differences between inoculation methods were detected in the percentage of leaf pieces showing fungal growth when placed on B. bassiana selective medium, with mean values in the range of 10-15% for the three seasons. Leaf pieces from controls did not exhibit any sign of B. bassiana growth when placed on B. bassiana-selective medium. Neither adverse effects on growth and yield nor symptomatic tissues were observed in B. bassiana-treated plants compared with controls in any of the three seasons.

Real-time quantitative PCR for analysis of candidate fungal biopesticides against malaria: Technique validation and first applications

Recent research has indicated that fungal biopesticides could augment existing malaria vector control tools. Here we present a set of methodologies to monitor the in vivo kinetics of entomopathogenic fungi in Anopheles in the presence or absence of malaria parasites using quantitative real-time PCR. Three qPCR assays were successfully developed for counting fungal genomes: “specific” assays capable of distinguishing two well characterized fungal entomopathogens Beauveria bassiana isolate IMI391510 and Metarhizium anisopliae var. acridum isolate IMI330189, both of which have previously been shown to be virulent to Anopheles mosquitoes, and a “generic” fungal assay for determining any fungal burden. A fourth assay to Plasmodium chabaudi enabled quantification of co-infecting malarial parasites. All qPCR assays provide sensitive, target-specific, and robust quantification over a linear range of greater than five orders of magnitude (seven orders of magnitude for the fungal assays). B. bassiana growth within mosquitoes exposed to three different conidial challenge doses was monitored using the B. bassiana-specific assay and represents the first description of entomopathogenic fungal replication within an insect host. This revealed that, irrespective of challenge dose, after several days of relatively little replication, a sudden on-set of substantial nuclear division occurs, accompanied by physical fungal growth (hyphae) within the mosquito haemocoel shortly before death. Exposure to higher densities of conidia resulted in significantly greater pick-up by mosquitoes and to elevated fungal burdens at each time point sampled. High fungal burdens, comparable to those identified in cadavers, were attained more rapidly and mortalities occurred earlier post-exposure with increasing challenge dose. The lines of research made possible by the qPCR assays described here will contribute to optimization of fungal biopesticides against malaria and other vector-borne diseases.

Temperature Effects on the Susceptibility of the Colorado Potato Beetle (Coleoptera: Chrysomelidae) to Beauveria bassiana (Balsamo) Vuillemin in Poland, the Czech Republic and the United States2

Beauveria bassiana (Balsamo) Vuillemin is a fungus with broad spectrum insecticidal activity. As a biological control agent used against Colorado potato beetles (Leptinotarsa decemlineata (Say)), this fungus has performed erratically in various field studies. This inconsistent performance has been attributed to formulation problems, UV sensitivity, and humidity. In a multi-site test, B. bassiana controlled Colorado potato beetle larvae in both Poland and the Czech Republic, but not in Maryland. Control was measured by reduction in populations of beetle larvae. One of the major differences among these sites was temperature. In Poland, the mean temperature ranged from 5°C to 23°C; in the Czech Republic the average temperature ranged from 6.7°C to 18.7°C; and in Maryland, temperatures at time of application exceeded 45°C at canopy level. This led us to examine B. bassiana growth in vitro.While B. bassiana grew in the laboratory from 16 to 30°C, the B. bassiana from a formulated product (Mycotrol™, Mycotech, Butte, MT) did not germinate at temperatures above 37°C. Germination and subsequent development of this entomopathogenic fungi are critical factors in the infection and control of the Colorado potato beetle. As a consequence of the inability to germinate at high temperatures, B. bassiana would not be expected to effectively control pest insects in climates with hot summers. This fungus, however, may be suitable for insect control in early spring or in cool temperature climates during the growing season.

Effects of <I>Beauveria bassiana</I> on <I>Lygus hesperus</I> (Hemiptera: Miridae) Feeding and Oviposition

Effects of Beauveria bassiana (Balsamo) Vuillemin infection on feeding and ovipositional behavior of Lygus hesperus Knight were investigated in the laboratory. In the feeding experiment, bugs were inoculated with different concentrations (LC10, LC50, LC90) of B. bassiana conidia and feeding damage was measured on flower buds of Wisconsin fast plants, Brassica rapa L., after 5 d. We detected greater feeding damage by inoculated bugs compared with noninoculated bugs, particularly bugs that died during the experiment. In the oviposition experiment, bugs were inoculated with a high concentration of conidia (LC90 at 25°C) and incubated for 6 d at 25 or 35°C, temperatures that are optimal and suboptimal, respectively, for B. bassiana growth. Most inoculated bugs died at 25°C but survived at 35°C during the 6-d experiment. Incubation temperature did not significantly affect L. hesperus oviposition. B. bassiana inoculation reduced the oviposition rate of lygus bug, particularly in bugs that did not show sporulation at the end of the experiment. If B. bassiana is used for lygus bug control in seed crop fields, the behavioral changes of infected bugs are unlikely to contribute to a reduction in seed damage.

Compatibility of Phloxine B, an Insecticidal Photoactive Dye, with Selected Biocontrol Fungi

The incorporation of the photoactive red food dye, phloxine B, into integrated pest management strategies has been suggested for the control of various insect pests. Inclusion of such a chemical pest control agent may interfere with a microbial pest control agent. Thus, representative fungi with potential for biocontrol were tested for their responses to phloxine B (0.01%) at different temperatures. Growth rates at selected temperatures were inhibited by dye in the presence of light for Coniothyrium minitans and Verticillium lecanii. Trichoderma virens growth was inhibited in light and dark. At optimal growth temperatures, Beauveria bassiana growth was inhibited only by the photoactivated dye species, while growth of Stilbella erythrocephala was not affected by the dye even in the presence of light.

Production, formulation and application of the entomopathogenic fungusBeauveria bassianafor insect control: current status

This review summarizes the progress and achievements made in the last decade in mass production formulation and application technology of the entomopathogenic fungus Beauveria bassiana. Reports published on relevant research from Belgium, Canada, China, Cuba, Czechoslovakia (former), France, Germany, Great Britain, Philippines, Poland, Switzerland, USA and USSR (former) regarding this topic have been covered. Much of the non‐English language literature, particularly that from Eastern European and Chinese sources, has not been translated and is inaccessible to most English or other western language readers. We have done this translation and through this review provide technological details about mass production of B. bassiana in China. Various aspects of B. bassiana growth, substrate use, production of mycelia, conidiospore and blastospores, process technologies associated with separation, drying and milling, formulation, storage and ‘shelf‐life’, and field efficacy are reviewed. Data are presented on: a m...