Characterization of Terpenoids from the Ambrosia Beetle Symbiont and Laurel Wilt Pathogen Harringtonia lauricola.

Little is known concerning terpenoids produced by members of the fungal order Ophiostomales, with the member Harringtonia lauricola having the unique lifestyle of being a beetle symbiont but potentially devastating tree pathogen. Nine known terpenoids, including six labdane diterpenoids (1-6) and three hopane triterpenes (7-9), were isolated from H. lauricola ethyl acetate (EtOAc) extracts for the first time. All compounds were tested for various in vitro bioactivities. Six compounds, 2, 4, 5, 6, 7, and 9, are described functionally. Compounds 2, 4, 5, and 9 expressed potent antiproliferative activity against the MCF-7, HepG2 and A549 cancer cell lines, with half-maximal inhibitory concentrations (IC50s) ~12.54-26.06 µM. Antimicrobial activity bioassays revealed that compounds 4, 5, and 9 exhibited substantial effects against Gram-negative bacteria (Escherichia coli and Ralstonia solanacearum) with minimum inhibitory concentration (MIC) values between 3.13 and 12.50 µg/mL. Little activity was seen towards Gram-positive bacteria for any of the compounds, whereas compounds 2, 4, 7, and 9 expressed antifungal activities (Fusarium oxysporum) with MIC values ranging from 6.25 to 25.00 µg/mL. Compounds 4, 5, and 9 also displayed free radical scavenging abilities towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide (O2-), with IC50 values of compounds 2, 4, and 6 ~3.45-14.04 µg/mL and 22.87-53.31 µg/mL towards DPPH and O2-, respectively. These data provide an insight into the biopharmaceutical potential of terpenoids from this group of fungal insect symbionts and plant pathogens.

Cell detoxification of secondary metabolites by P4-ATPase-mediated vesicle transport.

Mechanisms for cellular detoxification of drug compounds are of significant interest in human health. Cyclosporine A (CsA) and tacrolimus (FK506) are widely known antifungal and immunosuppressive microbial natural products. However, both compounds can result in significant side effects when used as immunosuppressants. The insect pathogenic fungus Beauveria bassiana shows resistance to CsA and FK506. However, the mechanisms underlying the resistance have remained unknown. Here, we identify a P4-ATPase gene, BbCRPA, from the fungus, which confers resistance via a unique vesicle mediated transport pathway that targets the compounds into detoxifying vacuoles. Interestingly, the expression of BbCRPA in plants promotes resistance to the phytopathogenic fungus Verticillium dahliae via detoxification of the mycotoxin cinnamyl acetate using a similar pathway. Our data reveal a new function for a subclass of P4-ATPases in cell detoxification. The P4-ATPases conferred cross-species resistance can be exploited for plant disease control and human health protection.

Isolation of a highly virulent Metarhizium strain targeting the tea pest, Ectropis obliqua.

Introduction: Tea is one of the most widely consumed beverages around the world. Larvae of the moth, Ectropis obliqua Prout (Geometridae, Lepidoptera), are one of the most destructive insect pests of tea in China. E. obliqua is a polyphagus insect that is of increasing concern due to the development of populations resistant to certain chemical insecticides. Microbial biological control agents offer an environmentally friendly and effective means for insect control that can be compatible with "green" and organic farming practices. Methods: To identify novel E. obliqua biological control agents, soil and inset cadaver samples were collected from tea growing regions in the Fujian province, China. Isolates were analyzed morphologically and via molecular characterization to identity them at the species level. Laboratory and greenhouse insect bioassays were used to determine the effectiveness of the isolates for E. obliqua control. Results: Eleven isolates corresponding to ten different species of Metarhizium were identified according to morphological and molecular analyses from soil and/or insect cadavers found on tea plants and/or in the surrounding soil sampled from eight different regions within the Fujian province, China. Four species of Metarhizium including M. clavatum, M. indigoticum, M. pemphigi, and M. phasmatodeae were documented for the first time in China, and the other species were identified as M. anisopliae, M. brunneum, M. lepidiotae, M. majus, M. pinghaense, and M. robertsii. Insect bioassays of the eleven isolates of Metarhizium revealed significant variation in the efficacy of each isolate to infect and kill E. obliqua. Metarhizium pingshaense (MaFZ-13) showed the highest virulence reaching a host target mortality rate of 93% in laboratory bioassays. The median lethal concentration (LC50) and median lethal time (LT50) values of M. pingshaense MaFZ-13 were 9.6 × 104 conidia/mL and 4.8 days, respectively. Greenhouse experiments and a time-dose-mortality (TDM) models were used to further evaluate and confirm the fungal pathogenic potential of M. pingshaense MaFZ-13 against E. obliqua larvae. Discussion: Isolation of indigenous microbial biological control agents targeting specific pests is an effective approach for collecting resources that can be exploited for pest control with lowered obstacles to approval and commercialization. Our data show the presence of four different previously unreported Metarhizium species in China. Bioassays of the eleven different Metarhizium strains isolated revealed that each could infect and kill E. obliqua to different degrees with the newly isolated M. pingshaense MaFZ-13 strain representing a particularly highly virulent isolate potentially applicable for the control of E. obliqua larvae.

Characterization of a fungal competition factor: Production of a conidial cell-wall associated antifungal peptide.

Competition is one of the fundamental driving forces of natural selection. Beauveria bassiana is a soil and plant phylloplane/root fungus capable of parasitizing insect hosts. Soil and plant environments are often enriched with other fungi against which B. bassiana competes for survival. Here, we report an antifungal peptide (BbAFP1), specifically expressed and localized to the conidial cell wall and is released into the surrounding microenvironment inhibiting growth of competing fungi. B. bassiana strains expressing BbAFP1, including overexpression strains, inhibited growth of Alternaria brassicae in co-cultured experiments, whereas targeted gene deletion of BbAFP1 significantly decreased (25%) this inhibitory effect. Recombinant BbAFP1 showed chitin and glucan binding abilities, and growth inhibition of a wide range of phytopathogenic fungi by disrupting membrane integrity and eliciting reactive oxygen species (ROS) production. A phenylalanine residue (F50) contributes to chitin binding and antifungal activity, but was not required for the latter. Expression of BbAFP1 in tomato resulted in transgenic plants with enhanced resistance to plant fungal pathogens. These results highlight the importance of fungal competition in shaping primitive competition strategies, with antimicrobial compounds that can be embedded in the spore cell wall to be released into the environment during the critical initial phases of germination for successful growth in its environmental niche. Furthermore, these peptides can be exploited to increase plant resistance to fungal pathogens.

Disruption of an adenylate-forming reductase required for conidiation, increases virulence of the insect pathogenic fungus Metarhizium acridum by enhancing cuticle invasion.

BACKGROUND: Metarhizium acridum, is a specific acridid pathogen developed for use against the migratory locust (Locusta migratoria manilensis). Adenylate-forming reductases (AFRs) include enzymes that are involved in natural product biosynthesis. Here, we genetically characterize the functions of a class IV AFR in M. acridum (MaAfrIV ) on fungal development and virulence. RESULTS: Gene expression analyses indicated MaAfrIV was induced on locust wings early during the infection process. Surprisingly, loss of MaAfrIV increased virulence (25.20% decrease in the median lethal time) against the locust in topical bioassays but was no different than the wild type when the cuticle was bypassed by direct infection of conidia into the insect hemocoel. Virulence markers including protease (Pr1) expression and appressorial turgor pressure were higher in the mutant than the parent strain. No difference was seen in the expression of host immune genes (Toll pathway) or in polyphenol oxidase (PPO) activity in locusts infected by the ΔMaAfrIV or wild type strains. However, the ΔMaAfrIV strain was unable to successfully sporulate on dead cadavers. CONCLUSION: Disruption of MaAfrIV increased fungal virulence by promoting insect cuticle invasion without altering host immune response or fungal immune evasion. Although loss of MaAfrIV conferred an apparent benefit to the fungus in terms of enhanced virulence, a significant trade-off was seen in the inability of the fungus to sporulate on the cadaver. As conidiation on the cadaver is essential for subsequent propagation in the environment, loss of MaAfrIV can reduce the engineering strains survivability in the field and improve the safety. © 2019 Society of Chemical Industry.

The Beauveria bassiana Gas3 ß-Glucanosyltransferase Contributes to Fungal Adaptation to Extreme Alkaline Conditions.

Fungal ß-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungus Beauveria bassiana displays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a ß-1,3-glucanosyltransferase gene (Bbgas3). Bbgas3 expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout of Bbgas3 resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3 aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and ß-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3 mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3 mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).IMPORTANCE Little is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling ß-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungus Beauveria bassiana to grow at extreme pH. The loss of Bbgas3 resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.

Phylogenomic analysis supports multiple instances of polyphyly in the oomycete peronosporalean lineage.

The study of biological diversification of oomycetes has been a difficult task for more than a century. Pioneer researchers used morphological characters to describe this heterogeneous group, and physiological and genetic tools expanded knowledge of these microorganisms. However, research on oomycete diversification is limited by conflicting phylogenies. Using whole genomic data from 17 oomycete taxa, we obtained a dataset of 277 core orthologous genes shared among these genomes. Analyses of this dataset resulted in highly congruent and strongly supported estimates of oomycete phylogeny when we used concatenated maximum likelihood and coalescent-based methods; the one important exception was the position of Albugo. Our results supported the position of Phytopythium vexans (formerly in Pythium clade K) as a sister clade to the Phytophthora-Hyaloperonospora clade. The remaining clades comprising Pythium sensu lato formed two monophyletic groups. One group was composed of three taxa that correspond to Pythium clades A, B and C, and the other group contained taxa representing clades F, G and I, in agreement with previous Pythium phylogenies. However, the group containing Pythium clades F, G and I was placed as sister to the Phytophthora-Hyaloperonospora-Phytopythium clade, thus confirming the lack of monophyly of Pythium sensu lato. Multispecies coalescent methods revealed that the white blister rust, Albugo laibachii, could not be placed with a high degree of confidence. Our analyses show that genomic data can resolve the oomycete phylogeny and provide a phylogenetic framework to study the evolution of oomycete lifestyles.

Screening of Metarhizium anisopliae UV-induced mutants for faster growth yields a hyper-virulent isolate with greater UV and thermal tolerances.

The insect pathogenic fungus Metarhizium anisopliae is an important insect biological control agent commercialized for use worldwide. Fungal infection is percutaneous, and rapid germination and growth has been linked to virulence. Using a simple in vitro growth screen to isolate mutants with increased virulence, M. anisopliae SM04 conidia were exposed to UV radiation for 20, 40, and 60 min, and mutants were subsequently screened for more rapid growth on standard potato dextrose agar. From a screen of >6,000 colonies, mutants were selected based on larger colony diameters as compared to the wild-type parent. Insect bioassays using the diamondback moth, Plutella xylostella, revealed one mutant, designated as MaUV-40.1 as displaying both more rapid growth and increased virulence. The mean lethal time to kill (LT50 using 106 conidia/ml) was 57.6 and 115.4 h for the MaUV-40.1 mutant and wild-type strains, respectively. Total conidial production, UV and thermal tolerances of the MaUV-40.1 strain were increased, but a reduced secretome was seen for the mutant compared to wild type. Analyses of culture supernatants indicated significant shifts in secondary metabolite production in the mutant. The insecticidal activity of EthOAc extracts derived from MaUV-40.1 mutant cell-free culture supernatants were ~20 times more potent that wild-type extracts. These data indicate that mutagenesis coupled to a growth screen can be a simple approach to isolate strains with greater stress resistance and virulence and that cell-free extracts may hold promise as an alternative to the living organism for insect control.

The Ifchit1 chitinase gene acts as a critical virulence factor in the insect pathogenic fungus Isaria fumosorosea.

The filamentous fungus, Isaria fumosorosea, is a promising insect biological control agent. Chitinases have been implicated in targeting insect cuticle structures, with biotechnological potential in insect and fungal control. The I. fumosorosea chitinase gene, Ifchit1, was isolated and determined to encode a polypeptide of 423 amino acids (46 kDa, pI = 6.53), present as a single copy in the I. fumosorosea genome. A split marker transformation system was developed and used to construct an Ifchit1 gene knockout. The ΔIfchit1 strain displayed minor alterations in mycelial growth on diverse media at 26 °C compared to the wild type and complemented (ΔIfchit1::Ifchit1) strains; however, colony morphology was affected, and the mutant strain had a temperature sensitive phenotype (32 °C). Although sporulation was delayed for the mutant, overall conidial production was almost twice than that of wild type. Biochemical assays indicated decreased chitinase activity during growth in Czapek-Dox liquid media for the ΔIfchit1 strain. Insect bioassays using diamondback moth, Plutella xylostella, larvae revealed decreased infectivity, i.e., increased LC 50 (threefold to fourfold) and a significantly delayed time to death, LT 50 from 3 to 6 days, for the ΔIfchit1 strain compared to the wild type and complemented strains. These data indicate an important role for the Ifchit1 chitinase as a virulence factor in I. fumosorosea.

Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards Anopheles gambiae and reduces fecundity in the target mosquito.

BACKGROUND: Adult and larval mosquitoes regulate food digestion in their gut with trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by the ovaries and the neuroendocrine system. TMOF is currently being developed as a mosquitocide, however, delivery of the peptide to the mosquito remains a significant challenge. Entomopathogenic fungi offer a means for targeting mosquitoes with TMOF. FINDINGS: The efficacy of wild type and transgenic Beauveria bassiana strains expressing Aedes aegypti TMOF (Bb-Aa1) were evaluated against larvae and sugar- and blood-fed adult Anopheles gambiae mosquitoes using insect bioassays. Bb-Aa1 displayed increased virulence against larvae, and sugar and blood fed adult A. gambiae when compared to the wild type parent strain. Median lethal dose (LD50) values decreased by ~20% for larvae, and ~40% for both sugar and blood-fed mosquitoes using Bb-Aa1 relative to the wild type parent. Median lethal time (LT50) values were lower for blood-fed compared to sugar-fed mosquitoes in infections with both wild type and Bb-Aa1. However, infection using Bb-Aa1 resulted in 15% to 25% reduction in LT50 values for sugar- and blood fed mosquitoes, and ~27% for larvae, respectively, relative to the wild type parent. In addition, infection with Bb-Aa1 resulted in a dramatic reduction in fecundity of the target mosquitoes. CONCLUSIONS: B. bassiana expressing Ae. aegypti TMOF exhibited increased virulence against A. gambiae compared to the wild type strain. These data expand the range and utility of entomopathogenic fungi expressing mosquito-specific molecules to improve their biological control activities against mosquito vectors of disease.

Contribution of the gas1 gene of the entomopathogenic fungus Beauveria bassiana, encoding a putative glycosylphosphatidylinositol-anchored beta-1,3-glucanosyltransferase, to conidial thermotolerance and virulence.

Beauveria bassiana is a mycoinsecticide alternative to chemicals for use in biological pest control. The fungus-insect interaction is also an emerging model system to examine unique aspects of the development, pathogenesis, and diversity of fungal lifestyles. The glycoside hydrolase 72 (GH72) family includes ß-1,3-glucanosyltransferases that are glycosylphosphatidylinositol (GPI)-anchored cell wall-modeling enzymes affecting fungal physiology. A putative B. bassiana GPI-anchored ß-1,3-glucanosyltransferase (Bbgas1) was isolated and characterized. B. bassiana targeted gene knockouts lacking Bbgas1 were affected in Congo red and salt sensitivity but displayed minor growth defects in the presence of sorbitol, SDS, or calcofluor white. Lectin and antibody mapping of surface carbohydrates revealed increased exposure of carbohydrate epitopes, including ß-1,3-glucans, in the ΔBbgas1 strain. Transmission electron micrographs revealed localized destabilization of the cell wall in ΔBbgas1 conidia, in which fraying of the outer cell wall was apparent. Heat shock temperature sensitivity profiling showed that in contrast to the wild-type parent, ΔBbgas1 conidial spores displayed decreased germination after 1 to 4 h of heat shock at temperatures >40°C, and propidium iodide exclusion assays revealed decreased membrane stability in the knockout strain at temperatures >50°C. The ΔBbgas1 knockout showed reduced virulence in Galleria mellonella insect bioassays in both topical and intrahemocoel-injection assays. B. bassiana ΔBbgas1 strains complemented with the complete Bbgas1 open reading frame were indistinguishable from the wild-type parent in all phenotypes examined. The Bbgas1 gene did not complement the phenotype of a Saccharomyces cerevisiae ß-1,3-glucanosyltransferase Δgas1 mutant, indicating that this family of enzymes likely possess discrete cellular functions.

Expression and purification of a functionally active class I fungal hydrophobin from the entomopathogenic fungus Beauveria bassiana in E. coli.

Hydrophobins represent a class of unique fungal proteins that have low molecular mass, are cysteine rich, and can self-assemble into two-dimensional arrays at water/air interfaces. These highly surface-active proteins are able to decrease the surface tension of water, thus allowing fungal structures to penetrate hydrophobic-hydrophilic barriers. Due to their unusual biophysical properties, hydrophobins have been suggested for use in a wide range of biotechnological applications. Here we describe a simple method for producing a functionally active class I hydrophobin derived from the entomopathogenic fungus, Beauveria bassiana, in an E. coli host. N-terminal modifications were required for proper expression and purification, and the hydrophobin was expressed as a fusion partner to a cleavable N-terminus chitin-binding domain-intein construct. The protein was purified and reconstituted from E. coli inclusion bodies. Self-assembly of the recombinant hydrophobin was followed kinetically using a thioflavin T fluorescence binding assay, and contact angle measurements of purified recombinant hydrophobin protein (mHyd2) films on a variety of substrata demonstrated its surface modification ability, which remained stable for at least 4 months. Filament or fibril-like structures were imaged using atomic force and transmission electron microscopy. These data confirmed the functional properties of the purified protein and indicate amino acid flexibility at the N-terminus, which can be exploited for various applications of these proteins.

Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host-pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simplicifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.

Surface characteristics of the entomopathogenic fungus Beauveria (Cordyceps) bassiana.

Marked differences in surface characteristics were observed among three types of single-cell propagules produced by the entomopathogenic fungus Beauveria bassiana. Atomic force microscopy (AFM) revealed the presence of bundles or fascicles in aerial conidia absent from in vitro blastospores and submerged conidia. Contact angle measurements using polar and apolar test liquids placed on cell layers were used to calculate surface tension values and the free energies of interaction of the cell types with surfaces. These analyses indicated that the cell surfaces of aerial conidia were hydrophobic, whereas those of blastospores and submerged conidia were hydrophilic. Zeta potential determinations of the electrostatic charge distribution across the surface of the cells varied from +22 to -30 mV for 16-day aerial conidia at pH values ranging from 3 to 9, while the net surface charge ranged from +10 to -13 mV for submerged conidia, with much less variation observed for blastospores, +4 to -4 mV, over the same pH range. Measurements of hydrophobicity using microbial adhesion to hydrocarbons (MATH) indicated that the surfaces of aerial conidia were hydrophobic, and those of blastospores hydrophilic, whereas submerged conidia displayed cell surface characteristics on the borderline between hydrophobic and hydrophilic. Insect pathology assays using tobacco budworm (Heliothis virescens) larvae revealed some variation in virulence among aerial conidia, in vitro blastospores and submerged conidia, using both topical application and haemocoel injection of the fungal cells.