Evaluation of the pathogenicity and infectivity of entomopathogenic hypocrealean fungi,isolated from wild mosquitoes in Japan and Burkina Faso,against female adult Anopheles stephensi mosquitoes

We investigated the potential of entomopathogenic hypocrealean fungi that naturally occur in or on adult mosquitoes for use as biocontrol agents of vector mosquitoes. The fungi were isolated from wild mosquitoes collected in Japan and Burkina Faso using two isolation methods (with and without surface sterilization). Detected fungal species included Beauveria bassiana sensu lato, Isaria spp., Paecilomyces spp., Lecanicillium spp., and Simplicillium spp. These isolates were used in bioassays against adult female Anopheles stephensi mosquitoes. The median survival time ranged from 5.8 to 14.9 d (control, 17.0 d). Reduced survival times were observed in the isolates from surface-sterilized mosquitoes from Japan, with the isolate B. bassiana s.l. 60-2 exhibiting the highest virulence. This study indicates that adult mosquitoes are naturally infected with various entomopathogenic hypocrealean fungi, and that some of these isolates have the potential for use as fungal pesticides to control vector mosquitoes.     

Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae

Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely “regenerated” Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.     

Entomopathogenic fungi as biological control agents for the vector of the laurel wilt disease,the redbay ambrosia beetle,Xyleborus glabratus (Coleoptera: Curculionidae)

The redbay ambrosia beetle (RAB), Xyleborus glabratus, is a wood-boring insect that vectors the fungal pathogen, Raffaelea lauricola, which causes laurel wilt, a lethal disease of avocado. The objective of this study was to determine the susceptibility of RAB to infection and subsequent death by exposure to three commercial strains of entomopathogenic fungi [two strains of Isaria fumosorosea (Ifr 3581 and PFR), and strain GHA of Beauveria bassiana]. RAB females were dipped in fungal spore solutions and their median survivorship times (MST) determined. Contact with any of the biopesticides resulted in death of all RAB females. MSTs of RAB females ranged from 3 days (B. bassiana) to 5 days (I. fumosorosea PFR). B. bassiana killed RAB females faster, followed by Ifr 3581 and PFR. RAB females dipped in B. bassiana suspensions had the highest number of viable spores attached to their bodies, followed by Ifr 3581. Beetles dipped in PFR suspension had significantly less viable spores attached to their bodies. No significant differences were observed in the mortality of beetles exposed to entomopathogenic fungi by dipping in a fungal suspension or walking on treated avocado bolts. Beetles bored into the logs and constructed galleries, but they were found dead inside the galleries a few days after exposure to the entomopathogens. Entomopathogenic fungal infection in dead beetles was confirmed through molecular techniques. This is the first study to demonstrate that entomopathogenic fungi are potential biological control agents against RAB.     

Expression of a Toll Signaling Regulator Serpin in a Mycoinsecticide for Increased Virulence

Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The Drosophila melanogaster serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus Beauveria bassiana offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD₅₀) for the wild-type parent, the LD₅₀ of B. bassiana expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ∼3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ∼24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin > laminarin > lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassiana-derived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.     

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.     

Pathogenicity of Beauveria bassiana ANU1 to the red imported fire ant,Solenopsis invicta workers in Korea

The red imported fire ant (RIFA), Solenopsis invicta, is one of the globalized invasive pests. This study focused on pathogenicity and virulence of entomopathogenic fungi as one of the biological control agents to RIFA workers under different temperatures. The fungal pathogen, Beauveria bassiana ANU1 was isolated from Korea in 2015 and showed the pathogenicity to RIFA workers. A conidial suspension (1 × 10⁷ conidia/ml) induced a low mortality from day 2 after treatment and reached to 100% mortality at day 7 and day 8 after treatment for major and minor workers, respectively. The median lethal concentrations of B. bassiana ANU1 were calculated as 3.9 × 10³ for major and 4.6 × 10³ for minor workers at day 7 after treatment. Low temperatures decreased a virulence of B. bassiana ANU1 (1 × 10⁷ conidia/ml) to RIFA and showed mortality of 26.6% for major and 20% for minor workers. Based on bioassay results, this study provides one of possibilities of effective and successful strategy for controlling RIFA by entomopathogenic fungi.     

Genetic engineering of fungal biocontrol agents to achieve greater efficacy against insect pests

Molecular biology methods have elucidated pathogenic processes in several fungal biocontrol agents including two of the most commonly applied entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana. In this review, we describe how a combination of molecular techniques has: (1) identified and characterized genes involved in infection; (2) manipulated the genes of the pathogen to improve biocontrol performance; and (3) allowed expression of a neurotoxin from the scorpion Androctonus australis. The complete sequencing of four exemplar species of entomopathogenic fungi including B. bassiana and M. anisopliae will be completed in 2010. Coverage of these genomes will help determine the identity, origin, and evolution of traits needed for diverse lifestyles and host switching. Such knowledge combined with the precision and malleability of molecular techniques will allow design of multiple pathogens with different strategies to be used for different ecosystems and avoid the possibility of the host developing resistance.     

Efficacy of entomopathogenic hypocrealean fungi against Periplaneta americana

The American cockroach Periplaneta americana, one of the worlds' most important urban insect pests was tested with entomopathogenic fungi. Most promising Metarhizium anisopliae, Metarhizium robertsii and Beauveria bassiana killed nymphs (≥ 81.7% mortality, 25 days after treatment), and these fungi developed on all dead insects. Other fungi tested were less virulent (Metarhizium frigidum and Purpureocillium lilacinum) or avirulent (Isaria cateniobliqua, Isaria farinosa, Simplicillium lanosoniveum, Sporothrix insectorum and Tolypocladium cylindrosporum). Intrageneric and intraspecific variability of fungal activity was detected. Adults were highly susceptible, and oothecae proved to be more resistant than nymphs and adults to infection with M. anisopliae IP 46. Findings of the study underscore the potential of fungi as biocontrol agents against this pest.     

Molecular phylogeny of asexual entomopathogenic fungi with special reference to Beauveria bassiana and Nomuraea rileyi

The phylogenetic lineage, taxonomic affiliation and interrelationships of important asexual entomopathogenic fungal genera were studied using the sequences of partial regions of β-tubulin and rRNA genes. The species structures of Beauveria bassiana and Nomuraea rileyi were also investigated. A total of 147 fungal entries covering 94 species were analysed. Phylogenetic analysis placed all the asexual entomopathogenic fungal species analysed, in the family Clavicipitaceae of the order Hypocreales of Ascomycota. Deep phylogenetic lineages were observed in B. bassiana iterating the complex nature of this species. Some of the isolates assigned to this species separated out more distinctly than morphologically distinguishable genera. Cryptic speciation was also evident in N. rileyi. It is concluded that the asexual fungi with entomopathogenic habit arose from a single lineage in sexual Clavicipitaceae.     

Entomopathogenic fungi recorded from the harlequin ladybird, Harmonia axyridis

Entomopathogenic fungi were recorded from field samples of the harlequin ladybird Harmonia axyridis, an invasive coccinellid that has recently arrived in Denmark. Larvae, pupae and adults were found to be infected by Isaria farinosa, Beauveria bassiana and species of Lecanicillium. This is the first record of entomopathogenic fungi infecting larvae and pupae. Winter mortality due to fungal infection reached 17.9% in adults collected at one location. The larval stage was most susceptible to fungal infection, as confirmed through bioassay with I. farinosa.     

Natural occurrence of soil-borne entomopathogenic fungi in the Moroccan Endemic forest of <Emphasis Type="Italic">Argania spinosa</Emphasis> and their pathogenicity to <Emphasis Type="Italic">Ceratitis capitata</Emphasis>

The occurrence and abundance of entomopathogenic fungi were analysed in 203 soil samples of the Moroccan endemic forests of Argania spinosa, the world main refuge of the Mediterranean fruit fly, Ceratitis capitata. Using the Galleria baiting method and selective media, entomopathogenic fungi were isolated from 186 of the 203 (91.62%) soil samples, with only three species found: Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae (Metschnikoff) Sorokin and Paecilomyces lilacinus (Thom.) Samson. B. bassiana was the most widespread entomopathogenic fungi (90.64%) in the Argan forest whereas M. anisopliae was less common (15.27%) and P. lilacinus was very rare (1.48%). This is the first report of natural occurrence of M. anisopliae and P. lilacinus in Morocco. Furthermore, 118 Moroccan B. bassiana isolates were studied for their pathogenicity to C. capitata and thermotolerance. Most of these autochtonous B. bassiana isolates were virulent (86.44%) to Medfly pupae and tolerant (55.08%) to temperature stress at 45°C for 2 h. Only 60.17% of Moroccan B. bassiana isolates might be considered as highly entomopathogenic and will serve as a source of potential biological control agents to C. capitata. The percentage of thermotolerant and pathogenic B. bassiana to C. capitata were shown to decrease significantly at winter time characterized by low temperatures and absence of any noticeable medfly in the Argan forest. The occurrence, thermotolerance and virulence of B. bassiana isolates to C. capitata seemed to be related to the sampling periods and location. Our data are discussed with respect to fungal ecology and biocontrol potential of B. bassiana isolates in relation to their habitat.     

Attenuation of the entomopathogenic fungus Beauveria bassiana following serial in vitro transfers

Stability of pathogenicity in continuous in vitro cultivation is desirable for the purpose of large-scale production of a mycoinsecticide. Fungal biocontrol agents may lose virulence when maintain on artificial media, resulting in products of commercially inferior quality. In this research, two isolates (DEBI007 and DEBI008) of entomopathogenic fungus Beauveria bassiana were investigated for their stability following fifteen serial in vitro transfers assaying virulence to mealworm larvae, conidiation, and hyphal development on artificial culture as some fungal virulence determinants. Moreover, role of insect cuticle on fungal virulence restoration and protease 1 (Pr1) activity was considered as the most important factor. Although radial hyphal development and colony colour on in vitro culture was not affected following serial transfers, conidiation and Pr1 activity of both fungal isolates were reduced remarkably after fifteen transfers compared with control. Similarly, mean lethal concentration (LC₅₀) values were increased as the number of serial transfers on artificial diet increased, although these increases were not statistically significant in both isolates as the confidential limits of LC₅₀ values were overlapping. Our results revealed that attenuation of entomopathogenic fungi following serial in vitro transfers is a combination of interconnected factors. Other probable components such as pathogenicity determinants in this interaction should be explored in next researches.     

Proteomic and Phosphoproteomic Insights into a Signaling Hub Role for Cdc14 in Asexual Development and Multiple Stress Responses in Beauveria bassiana

Cdc14 is a dual-specificity phosphatase that regulates nuclear behavior by dephosphorylating phosphotyrosine and phosphoserine/phosphothreonine in fungi. Previously, Cdc14 was shown to act as a positive regulator of cytokinesis, asexual development and multiple stress responses in Beauveria bassiana, a fungal insect pathogen. This study seeks to gain deep insight into a pivotal role of Cdc14 in the signaling network of B. bassiana by analyzing the Cdc14-specific proteome and phosphoproteome generated by the 8-plex iTRAQ labeling and MS/MS analysis of peptides and phosphopeptides. Under normal conditions, 154 proteins and 86 phosphorylation sites in 67 phosphoproteins were upregulated in Δcdc14 versus wild-type, whereas 117 proteins and 85 phosphorylation sites in 58 phosphoproteins were significantly downregulated. Co-cultivation of Δcdc14 with NaCl (1 M), H₂O₂ (3 mM) and Congo red (0.15 mg/ml) resulted in the upregulation / downregulation of 23/63, 41/39 and 79/79 proteins and of 127/112, 52/47 and 105/226 phosphorylation sites in 85/92, 45/36 and 79/146 phosphoproteins, respectively. Bioinformatic analyses revealed that Cdc14 could participate in many biological and cellular processes, such as carbohydrate metabolism, glycerophospholipid metabolism, the MAP Kinase signaling pathway, and DNA conformation, by regulating protein expression and key kinase phosphorylation in response to different environmental cues. These indicate that in B. bassiana, Cdc14 is a vital regulator of not only protein expression but also many phosphorylation events involved in developmental and stress-responsive pathways. Fourteen conserved and novel motifs were identified in the fungal phosphorylation events.     

Colonization of Corn, Zea mays, by the Entomopathogenic Fungus Beauveria bassiana

Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin into corn, Zea mays L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located, indicating that B. bassiana does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how B. bassiana travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that B. bassiana does not lose virulence toward the European corn borer, Ostrinia nubilalis (Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities for biological control, including the use of indigenous fungal inocula as insecticides.     

Isolation and evaluation of South African isolates of Beauveria bassiana (Hypocreales: Cordycipitaceae) on Rhipicephalus microplus (Acari: Ixodidae)

Beauveria bassiana (Hypocreales: Cordycipitaceae) is an entomopathogenic fungus that has shown promising results as a biocontrol agent of ticks. Locally isolated B. bassiana are better acclimatised to the natural conditions of their geographical origin; therefore, they are essential in developing effective biocontrol agents for ticks. The current study aimed to isolate native strains of B. bassiana that are pathogenic to Rhipicephalus microplus ticks. The virulence of the isolates was tested against R. microplus larvae using a formulation containing 15% avocado oil, 0.05% adjuvant and 10⁸ conidia mL^?1. The two best strains were further evaluated for various biological parameters on adult engorged female ticks. Breakthru® or Ballista® (adjuvant) was mixed with the formulation to compare their effect on the isolates' virulence. In total 61 entomopathogenic fungi were isolated from the 360 greater wax moth larvae, Galleria mellonella (Lepidoptera: Pyralidae) used. The virulence test identified Bb-40 and Bb-41 to be the most virulent isolates against R. microplus larvae with mortalities of 91 and 93% and LT₅₀ values of 5.8 and 6.2 days, respectively. Compared to the control, both strains significantly affected all the measured biological parameters. The type of adjuvant also considerably affected the susceptibility of ticks to the fungi. In conclusion, the two isolates combined with adjuvants can be used as a biocontrol agent to control R. microplus.

     

Enhanced xylanase and endoglucanase production from Beauveria bassiana SAN01, an entomopathogenic fungal endophyte

This study was undertaken to explore alternative applications of the widely known entomopathogenic/endophytic fungus, Beauveria bassiana, besides its sole use as a biocontrol agent. B. bassiana SAN01, was investigated for the production of two glycoside hydrolases, xylanase and endoglucanase under submerged conditions. Among the different biomass tested, wheat bran provided the best results for both xylanase and endoglucanase, and their production levels were further enhanced using response surface methodology. Under optimised conditions, heightened yields of 1061 U/ml and 23.03 U/ml were observed for xylanase and endoglucanase, respectively, which were 3.44 and 1.35 folds higher than their initial yields. These are the highest ever production levels reported for xylanase and endoglucanase from any B. bassiana strain or any known entomopathogenic fungi. Furthermore, the efficacy of xylanase/endoglucanase cocktail in the saccharification of sugarcane bagasse was evaluated. The highest amount of reducing sugar released from the pretreated biomass by the action of the crude Beauveria enzyme cocktail was recorded at 30°C after 8 h incubation. The significant activities of the hydrolytic enzymes recorded with B. bassiana in this study thus present promising avenues for the use of the entomopathogen as a new source of industrial enzymes and by extension, other biotechnological applications.     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.     

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.     

Management of Frankliniella occidentalis (Thysanoptera: Thripidae) with granular formulations of entomopathogenic fungi

Western flower thrips (WFT), Franklinella occidentalis, is a major pest of ornamentals. Mycotized millet grains with entomopathogenic fungi applied to soil of potted marigold plants were tested to target the soil-dwelling stages of thrips. Two experimental fungal isolates, (Beauveria bassiana [ARS7060] and Metarhizium anisopliae [ERL1171]), were compared with the registered B. bassiana strain GHA [commercialized as BotaniGard®] and untreated controls in greenhouse caged trials. Mycotized millet grains were mixed into the upper surface of the potting soil in pots of flowering ‘Hero Yellow’ marigolds (4 g/pot). One week after application five mated WFT females were released onto each plant (four plants per cage). At 8 weeks post-infestation, the mean total number of thrips per plant was 81% and 90% less in the ERL1171 and ARS 7060 treatments, respectively, than in the control. The mean numbers of thrips per plant for the control and GHA treatments were not significantly different. Plant damage was 60% less on plants treated with the experimental fungi than the control and GHA treatments. At 10 weeks post-application, 75–90% of WFT collected from the treatments of the experimental fungi were infected with the fungal species applied. These results demonstrate that soil applications of entomopathogenic fungi can reduce WFT populations significantly and prevent major damage.