Insect-toxic secreted proteins and virulence of the entomopathogenic fungus Beauveria bassiana

Fungal virulence has been mostly associated with cuticle-degrading enzymes that can be regulated depending on nutrient conditions. However, few studies have related fungal virulence to insect-toxic secreted proteins. Here, we describe how the presence of secreted toxic proteins may be linked to conidial virulence, which can be affected by nutrient factors. In this study we evaluated: (1) the virulence of the conidia of four Beauveria bassiana strains (EABb 01/103-Su, EABb 01/12-Su, EABb 01/88-Su and EABb 01/110-Su) grown on three different media (malt extract agar (MA), Rice (Rice), Sabouraud dextrose agar (SDA) and harvested from the cadavers of fungal-infected Galleria mellonella larvae (CAD) and (2) the toxicity of the crude soluble protein extracts (CSPEs) obtained from Adamek’s liquid medium inoculated with these conidia. Conidial suspensions were obtained from the four media, assessed on G. mellonella larvae and used to produce CSPEs that were injected into healthy G. mellonella larvae. The larvae were also injected with conidia obtained from MA and CAD cultures to expose them to in vivo-secreted proteins. For all isolates, the CAD conidia were by far the most virulent, followed by conidia grown on SDA, Rice and MA. The injected CSPEs showed the same toxicity trends as the conidial suspensions. In addition, the outcomes of injection of the in vivo-secreted proteins showed that the toxic proteins secreted in vitro by the EABb 01/110-Su strain are not produced in vivo. However, the other strains produced toxic proteins both in vivo and in vitro, suggesting that these toxic proteins may be virulence factors involved in invertebrate pathogenesis.     

Alkane-grown Beauveria bassiana produce mycelial pellets displaying peroxisome proliferation,oxidative stress,and cell surface alterations

The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons, inducing alkane assimilation pathways and concomitantly increasing virulence against insect hosts. In this study, we describe some physiological and molecular processes implicated in growth, nutritional stress response, and cellular alterations found in alkane-grown fungi. The fungal cytology was investigated using light and transmission electron microscopy while the surface topography was examined using atomic force microscopy. Additionally, the expression pattern of several genes associated with oxidative stress, peroxisome biogenesis, and hydrophobicity were analysed by qPCR. We found a novel type of growth in alkane-cultured B. bassiana similar to mycelial pellets described in other alkane-free fungi, which were able to produce viable conidia and to be pathogenic against larvae of the beetles Tenebrio molitor and Tribolium castaneum. Mycelial pellets were formed by hyphae cumulates with high peroxidase activity, exhibiting peroxisome proliferation and an apparent surface thickening. Alkane-grown conidia appeared to be more hydrophobic and cell surfaces displayed different topography than glucose-grown cells. We also found a significant induction in several genes encoding for peroxins, catalases, superoxide dismutases, and hydrophobins. These results show that both morphological and metabolic changes are triggered in mycelial pellets derived from alkane-grown B. bassiana.     

Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

Light conditions during fungal growth are well known to cause several physiological adaptations in the conidia produced. In this study, conidia of the entomopathogenic fungi Metarhizium robertsii were produced on: 1) potato dextrose agar (PDA) medium in the dark; 2) PDA medium under white light (4.98 W m^?2); 3) PDA medium under blue light (4.8 W m^?2); 4) PDA medium under red light (2.8 W m^?2); and 5) minimum medium (Czapek medium without sucrose) supplemented with 3 % lactose (MML) in the dark. The conidial production, the speed of conidial germination, and the virulence to the insect Tenebrio molitor (Coleoptera: Tenebrionidae) were evaluated. Conidia produced on MML or PDA medium under white or blue light germinated faster than conidia produced on PDA medium in the dark. Conidia produced under red light germinated slower than conidia produced on PDA medium in the dark. Conidia produced on MML were the most virulent, followed by conidia produced on PDA medium under white light. The fungus grown under blue light produced more conidia than the fungus grown in the dark. The quantity of conidia produced for the fungus grown in the dark, under white, and red light was similar. The MML afforded the least conidial production. In conclusion, white light produced conidia that germinated faster and killed the insects faster; in addition, blue light afforded the highest conidial production.     

Comparative virulence of Beauveria bassiana isolates against lepidopteran pests of vegetable crops

Forty-three isolates of the entomopathogenic fungus Beauveria bassiana were screened for virulence against second-instar larvae of diamondback moth (Plutella xylostella) (DBM), European corn borer (Ostrinia nubilalis) (ECB), corn earworm (Helicoverpa zea) (CEW), and fall armyworm (Spodoptera frugiperda) (FAW); 30 of these isolates were tested against beet armyworm (Spodoptera exigua) (BAW). Highly virulent isolates were also tested against black cutworm (Agrotis ipsilon) (BCW), and the most virulent isolate was also assayed against imported cabbage worm (Pieris rapae) (ICW) and cabbage looper (Trichoplusia ni) (CL). All lepidopteran species tested were susceptible to B. bassiana. Corn earworm and beet armyworm were most susceptible to fungal infection, and fall armyworm was least susceptible. Limited testing suggested low susceptibility of black cutworm and cabbage looper. B. bassiana isolate 1200 exhibited virulence against all pest species greater than or equal to commercial strain GHA of B. bassiana currently registered in the USA as BotaniGard®. In assays in which larvae were topically sprayed and maintained on the treated substrate for 24 h at 100% relative humidity, 6-day (25 °C) median lethal concentrations (LC₅₀s) of this isolate against CEW, BAW, DBM, FAW, ICW, ECB, CL, and BCW were 4, 5, 7, 11, 12, 98, 125, and 273 conidia/mm², respectively. The respective LC₅₀s of commercial strain GHA against these pest species were 9, 67, 97, 1213, 29, 1668, 541, and 3504 conidia/mm². Use of LC₅₀ versus median lethal concentration ratios (comparing LC₅₀s of each isolate to a “standard” strain) generated similar rankings of isolate virulence. Results from parametric ANOVAs of log LC₅₀ values followed by Tukey HSD multiple comparisons tests and those from Kruskal-Wallis nonparametric analyses followed by sequential Bonferroni tests for means comparisons were nearly identical.     

Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae are being developed as alternatives to chemical insecticides. They infect insects by direct penetration of the cuticle using a combination of physical pressure and extracellular hydrolytic enzymes such as proteases and chitinases. Previously we found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana, respectively. In this study, we found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 degraded insect cuticle in vitro more efficiently than either CDEP1 or Bbchit1 alone. Based on this we produced three plasmid constructs; (1) Bbchit1, (2) CDEP1, and (3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1:Bbchit1) penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT₅₀, without a reduction in LC₅₀. The LT₅₀ of the transformant expressing CDEP1:Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1:Bbchit1 resulted in a 60.5% reduction in LC₅₀, more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.     

The oxygen concentration in cultures modulates protein expression and enzymatic antioxidant responses in Metarhizium lepidiotae conidia

Conidia from Metarhizium spp. are used for integrated pest control; however, environmental factors diminish the effectivity of these programs. Several approaches tried to improve conidia resistance to overcome this limitation, although little is known about the mechanisms involved in this effect. Here we measured the activity of antioxidant enzymes and conidia virulence, comparing the proteomic profiles of Metarhiziumlepidiotae CP-OAX conidia produced under normal (21% O₂) and high oxygen atmospheres (pulses with 30% O₂). We detected a higher virulence against Tenebrio molitor larvae, in addition to an increase in ultraviolet light tolerance in conidia produced under 30% O₂, which correlates with increased glutathione reductase activity. Two-dimensional gel electrophoresis (2D SDS–PAGE) of proteins extracted in conidia harvested from both experimental conditions revealed a group of proteins that was observed only in conidia from oxidant atmospheres. Some of those proteins were directly involved in oxidative stress responses, whereas others were involved in conidial virulence, thermo-tolerance, and the central metabolism. Thus, a high atmospheric oxygen concentration (30%) activates antioxidant defence and general stress response mechanisms involved in conidia resistance to adverse environmental factors, which can ultimately translate into higher effectivity for the use of entomopathogenic fungi conidia in pest control.     

Oxaloacetate hydrolase gene links the cytoplasmic route of oxalate formation to differentiation and virulence of entomopathogenic fungus Beauveria bassiana

Oxalic acid is used as a functional molecule by most filamentous fungi, and produced via cytoplasmic pathway and mitochondrial pathway. The cytoplasmic pathway of oxalate production from oxaloacetate is a one-step reaction catalyzed by oxaloacetate hydrolase. The entomopathogenic fungus Beauveria bassiana contains a unique oxaloacetate hydrolase gene (BbOAH). The role of cytoplasmic pathway of oxalate production in B. bassiana development and virulence was studied via construction of a targeted gene disruption mutant of BbOAH. Disruption of BbOAH resulted in a slight decrease (~ 30%) in oxalate production, but has no significant influence on fungal growth. The mutant strain displayed a significant delay at early stage of conidial development, and a significant defect in dimorphic transition. Additionally, bioassay using the greater waxmoth as host indicated a slight (~ 20%) decrease in mortality caused by the gene disruption strain. The phenotypic defects of the ΔBbOAH strain could be restored by ectopic integration of BbOAH. Our findings indicate that BbOAH gene connects the cytoplasmic route of oxalate production to fungal development and virulence, although the cytoplasmic route is not indispensable for oxalate production in B. bassiana.     

Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni

Termites encounter a diverse array of potentially useful and harmful fungi in their subterranean habitats. These vary from symbiotic to harmful species with varying levels of virulence. How these hemiedaphic insects survive in habitats with infective fungi is not well understood. Possible mediation of olfactory signals in avoiding contact with entomopathogenic fungi has been explored by a number of workers. In the present study, we initially found that Macrotermes michaelseni detected a virulent isolate of Metarhizium anisopliae from some distance and avoided direct physical contact. We hypothesized that there may be a relationship between virulence and repellency of different isolates of M. anisopliae and Beauveria bassiana to the termite. We compared these for selected isolates of the two fungi. Positive correlations between the two parameters for both sets of isolates of the fungi were obtained. The results show an interesting co-evolutionary phenomenon in which the termite's response to either M. anisopliae or B. bassiana is directly related to potential harm these fungi can inflict on the insect and that the virulent strains are more likely to be recognized from some distance and avoided.     

Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the Red Palm Weevil, Rhynchophorus ferrugineus

The potential of a strain of Beauveria bassiana (Ascomycota: Clavicipitaceae) obtained from a naturally infected Rhynchophorus ferrugineus (Coleoptera: Curculionidae) pupa as a biological control agent against this weevil was evaluated both in the laboratory and in semi-field assays. Laboratory results indicate that this strain of B. bassiana can infect eggs, larvae and adults of R. ferrugineus (LC₅₀ from 6.3 × 10⁷ to 3.0 × 10⁹ conidia per ml). However, mortality was not the only indicator of treatment efficacy because adults of either sex inoculated with the fungus efficiently transmitted the disease to untreated adults of the opposite sex, with male-to-female and female-to-male rates of transmission of 55% and 60%, respectively. In addition, treatment with B. bassiana significantly reduced fecundity (up to 62.6%) and egg hatching (32.8%) in pairing combinations with fungus-challenged males, females or both sexes. Likewise, 30-35% increase in larval mortality was observed in larvae obtained from eggs from fungus-challenged females or from untreated females coupled with inoculated males, resulting in an overall 78% progeny reduction. Semi-field preventive assays on potted 5-year old Phoenix canariensis palms, with efficacies up to 85.7%, confirmed the potential of this strain as a biological control agent against R. ferrugineus.     

Carbon utilization pattern as a potential quality control criterion for virulence of Beauveria brongniartii

The registered entomopathogenic fungus Beauveria brongniartii (BIPESCO 2) was tested for its virulence after one, five and 10 times sub-culturing on four types of selective synthetic nutrient media. Bioassays with third instar Melolontha melolontha larvae showed that sub-culturing negatively affects the virulence of the fungus after 10 transfers. With the Biolog™ SF-P2 and Biolog™ SF-N2 microtiter plate systems the sub-cultivated B. brongniartii conidia were monitored for any change in the carbon utilization pattern of 128 carbon sources. With the help of Spearman’s rank correlation, principal components analysis and canonical correspondence analysis, respectively, six carbon sources were identified as potential virulence indicators for BIPESCO 2 (pyruvic acid, maltose, glycyl-l-glutamic acid, malonic acid, glucuronamide and phenylethylamine). The Biolog™ microtiter plate system is suggested as a simple and inexpensive test-system for virulence determination of B. brongniartii strain BIPESCO 2 in routine quality control.     

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.     

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

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

Debilitation in conidia of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae and implication with respect to viability determinations and mycopesticide quality assessments

Germination of Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) conidia determined from a fast-rehydration (FR) protocol were compared to those obtained when dry conidia were subjected to slow rehydration (SR) by holding under high humidity conditions prior to aqueous suspension. Differences in viability estimates obtained using the FR vs. SR protocols increased markedly after conidia were exposed to various stress factors in storage (high a_w, temperature, and O₂ concentrations), with the SR protocol producing higher estimates of viability in all cases. After Bb conidia were stored under moist conditions for 21 days at 25 °C, the SR estimate of viability was >21% greater than the FR estimate. In jars flushed with different O₂ concentrations and stored at 50 °C for 34 days, proportional differences between protocols varied, depending on water activity, from 18-44% in jars flushed with 0% O₂ (100% N₂) to as high as 63-93% when treated with 21-22% O₂. For conidia stored over a broad range of moderate to high temperatures in the absence of O₂, SR-FR differences were ?9% at 25-40 °C but 30% at 50 °C. Germination of stressed Bb and Ma conidia increased substantially when incubation time on the germination substrate was increased from 24 to 72 h, whereas germination of non-stressed conidia showed little change. Conidia debilitated by stress were characterized by hypersensitivity to lethal imbibitional damage (damage that is mitigated by slow rehydration) and slow germination. Viability protocols that may provide more reliable assessments of overall mycopesticide quality are discussed.     

The influence of a single-spore isolate and repeated subculturing on the pathogenicity of conidia of the entomophagous fungus Beauveria bassiana

The potential of monospore isolations for detecting strains of the entomophagous fungus Beauveria bassiana with increased virulence was investigated as well as the effect of repeated subculturing of both multispore parent strains and 28 newly obtained single-spore isolates on virulence. The use of the singlespore method enabled the disclosure of six spontaneous mutants surpassing the mother strain in pathogenicity, i.e., in a quicker effect and a higher virulence. The use of an optimal medium enabled an enhancement of virulence of all types of isolates. Differences among spontaneous mutants were indicated by the slope of the probit line. Our newly formed positive mutants retained their high pathogenicity both in repeated subculturing and in materials subcultured after a 12-month storage.     

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and ¼SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45 °C for 45 min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14 d were more hydrophobic and exhibited greater thermotolerance than those produced on ¼SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

Selection of Beauveria bassiana isolates for control of the whiteflies Bemisia tabaci and Trialeurodes vaporariorum on the basis of their virulence, thermal requirements, and toxicogenic activity

As part of a 3-fold approach to select potential mycoinsecticides for whitefly control, we evaluated infectivity, thermal requirements, and toxicogenic activity of the entomopathogenic fungus Beauveria bassiana (Ascomycota: Clavicipitaceae) under laboratory conditions. Twenty-five native B. bassiana isolates and a commercially available mycoinsecticide (based on B. bassiana) were evaluated for virulence to fourth instar nymphs of sweetpotato whitefly, Bemisia tabaci, and greenhouse whitefly, Trialeurodes vaporariorum, at a concentration of 1 × 10⁷ conidia/ml. All isolates were pathogenic for both whitefly species, whereas mortality rates varied from 3 to 85%. A second series of bioassays was conducted on 10 selected isolates using four 10-fold concentrations ranging from 1 × 10⁵ to 1 × 10⁸ conidia/ml. Median lethal concentrations (LC₅₀) of the four most virulent isolates varied from 1.1 × 10⁵ to 6.2 × 10⁶ conidia/ml and average survival time (AST) of treated nymphs from 5.9 to 7.4 days. T. vaporariorum were significantly more susceptible to all B. bassiana isolates than B. tabaci. The thermal biology of the eight most virulent isolates to both whitefly species was investigated at six temperatures (10–35 °C). The colony radial growth rate was estimated from the slope of the linear regression of colony radius on time and data were then fitted to a modified generalized β function that accounted for 90.5–99.3% of the data variance. Optimum temperatures for extension rate ranged from 23.1 to 27.1 °C, whereas maximum temperatures for fungal growth varied from 31.8 to 36.6 °C. On the basis of their virulence and thermal requirements, three isolates showed promise as candidates for whitefly management in Mediterranean greenhouses. Whilst in vitro production of macromolecular compounds toxic to Galleria mellonella larvae was not a requisite for virulence, ASTs of larvae injected with Sephadex G-25 fractions from candidate isolates ranged from 1.4 to 3.7 days compared with 5–6 days for non-toxic G-25 fractions. In addition, proteinase K treatment significantly reduced their toxic activity suggesting that they were proteins and revealing the potential of these isolates to be further improved through biotechnology to kill the pest more quickly.     

Characterization of Beauveria bassiana neutral trehalase (BbNTH1) and recognition of crucial stress-responsive elements to control its expression in response to multiple stresses

A neutral trehalase (NTH1) of fungal entomopathogen Beauveria bassiana was characterized for the first time as a 743-aa enzyme (84.4 kDa). To identify crucial stress-responsive elements (STREs) to control the expression of the NTH-coding gene (BbNTH1) in response to different stresses, the full-length promoter (−2713 bp) upstream of its open reading frame and three upstream-truncated fragments (−1912, −1060 and −560 bp) were fused to the reporter gene eGFP and then transformed into B. bassiana, respectively. Consequently, eGFP was well expressed as intensive fluorescence in mycelia, conidiogenic cells and forming conidia controlled by the full-length promoter with five STREs. Surprisingly, transformants controlled by the shortest fragment with last two STREs at −315 and −274 bp exhibited consistently brightest fluorescence in mycelia under 3-h oxidative adaption of 0.3-1.2 mM menadione, and in colonies under 6-day osmotic stress of 0.5-1 M NaCl and thermal stress of 15-540 min at 40 °C after 3-day growth at 25 °C. Single or dual site-directed mutations of the two STREs from CCCCT to CATCT significantly altered the gene response to the multiple stresses. Thus, the two STREs in the downstream 560-bp region of the promoter are crucial to regulating not only constitutive but stress-inducible expression of the target gene.     

The rdgB gene in drosophila: Retinal degeneration in different mutant alleles and inhibition of degeneration by norpA

We used histology, optical measurements of photoreceptor integrity and electrophysiology to characterize rdgB mutants of Drosophila which have hereditary retinal degeneration. We studied different mutant alleles of rdgB which had been reported to have differing receptor involvement in degeneration. However, we found similar profiles of degeneration: compound eye receptors R1-6 degenerate structurally over one week in a diurnal lighting cycle while R7/8 are preserved. We confirmed earlier findings that norpA transduction mutants inhibit degeneration in rdgB. Our micrographs show degeneration in the lamina (first synaptic neuropil) but none in the medulla (second synaptic neuropil).     

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.