Insect Pathogenic Fungus Research Articles

Effect of the Entomopathogenic Fungus Beauveria bassiana on the Development of Faba Bean (Vicia faba) Diseases in the Field Conditions.

Several ascomycetous entomopathogenic fungi, including species in the genera Beauveria, are plant symbionts/endophytes and are termed as endophytic insect-pathogenic fungi. It was shown that the fungus Beauveria bassiana (BBK-1 strain) successfully colonized Vicia faba bean plants in laboratory and field conditions of Western Siberia. The B. bassiana reisolate passed through the plants had significantly higher antagonistic activity against phytopathogens in comparison with the primary stem of entomopahogenic fungi. Pre-sowing faba bean seeds treatment reduced the level of infection of the seed material with phytopathogens, significantly decrease the development and prevalence of root rot disease. A decrease in the disease development index (chocolate spot, powdery mildew, fusariosis and other spots diseases) was found as a result of the use of B. bassiana. The effectiveness and prolonged action of B. bassiana on plants opens up new opportunities both in the creation of biological products and in molecular-genetic research and selection of certain pairs of plants and fungi based on the principle of the greatest synergy.

MaAreB, a GATA Transcription Factor, Is Involved in Nitrogen Source Utilization, Stress Tolerances and Virulence in Metarhizium acridum.

The nitrogen catabolite repression (NCR) pathway is involved in nitrogen utilization, in which the global GATA transcription factor AreA plays an indispensable role and has been reported in many fungi. However, relatively few studies are focused on AreB, another GATA transcription factor in the NCR pathway and the functions of AreB are largely unknown in entomopathogenic fungi. Here, we characterized MaAreB in the model entomopathogenic fungus Metarhizium acridum. Sequence arrangement found that MaAreB had a conserved GATA zinc finger DNA binding domain and a leucine zipper domain. Disruption of MaAreB affected the nitrogen utilization and led to decelerated conidial germination and hyphal growth, decreased conidial yield, and lower tolerances to UV-B irradiation and heat-shock. Furthermore, the MaAreB mutant (ΔMaAreB) exhibited increased sensitivity to CFW (Calcofluor white), decreased cell wall contents (chitin and β-1,3-glucan) and reduced expression levels of some genes related to cell wall integrity, indicating that disruption of MaAreB affected the cell wall integrity. Bioassays showed that the virulence of the ΔMaAreB strain was decreased in topical inoculation but not in intra-hemocoel injection. Consistently, deletion of MaAreB severely impaired the appressorium formation and reduced the turgor pressure of appressorium. These results revealed that MaAreB regulated fungal nitrogen utilization, cell wall integrity and biological control potential, which would contribute to the functional characterization of AreB homologous proteins in other insect fungal pathogens, and even filamentous fungi.

Activation of microlipophagy during early infection of insect hosts by Metarhizium robertsii

ABSTRACT The requirement of macroautophagic/autophagic machinery for filamentous fungal development and pathogenicity has been recognized, but the underlying effects and mechanisms remain elusive. The insect pathogenic fungus Metarhizium robertsii infects hosts by cuticular penetration through the formation of the infection structure appressoria. Here, we show that autophagic fluxes were highly activated during the appressorial formation of M. robertsii. Genome-wide deletion of the autophagy-related genes and insect bioassays identified 10 of 23 encoded MrATG genes with requirements for topical fungal infection of insect hosts. Besides the defect in forming appressoria on insects (two null mutants), these virulence-reduced mutants were largely impaired in penetrating cellophane membrane and insect cuticles, suggesting their failures in generating proper appressorium turgor. We found that the conidial storage of lipid droplets (LDs) had no obvious difference between strains, but autophagic LD degradation was impaired in different mutants. After induction of cell autophagy by nitrogen starvation, we found that LD entry into vacuoles was unaffected in the selected mutant cells with potential failures in forming autophagosomes. The finding therefore reveals a microlipophagy machinery employed in this fungus and that the direct engulfment of LDs occurs without inhibition by the downstream defective lipolysis. Our data first unveil the activation and contribution of microlipophagy to fungal infection biology. The obtained technique may benefit future detection of microlipophagy in different organisms by examining vacuolar or lysosomal engulfment of LDs in core autophagic gene deletion mutants. Abbreviations: AIM: Atg8-family interacting motif; ATG: autophagy-related; CM: complete medium; CMAC: 7-amino-4-chloromethylcoumarin; DTT: dithiothreitol; ER: endoplasmic reticulum; GFP: green fluorescent protein; LD, lipid droplet; MM: minimum medium; MM-N: minimum medium without nitrogen source; PDA: potato dextrose agar; PMSF: phenylmethylsulfonyl fluoride; RFP: red fluorescent protein; SDB: Sabouraud dextrose broth; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TAG: triacylglycerol; TEM: transmission electron microscopy; WT, wild type.

A virulence-related lectin traffics into eisosome and contributes to functionality of cytomembrane and cell-wall in the insect-pathogenic fungus Beauveria bassiana

Lectins are characterized of the carbohydrate-binding ability and play comprehensive roles in fungal physiology (e.g., defense response, development and host–pathogen interaction). Beauveria bassiana, a filamentous entomopathogenic fungus, has a lectin-like protein containing a Fruit Body_domain (BbLec1). BbLec1 could bind to chitobiose and chitin in fungal cell wall. BbLec1 proteins interacted with each other to form multimers, and translocated into eisosomes. Further, the interdependence between BbLec1 and the eisosome protein PliA was essential for stabilizing the eisosome architecture. To test the BbLec1 roles in B. bassiana, we constructed the gene disruption and complementation mutants. Notably, the BbLec1 loss resulted in the impaired cell wall in mycelia and conidia as well as conidial formation capacity. In addition, disruption of BbLec1 led to the reduced cytomembrane integrity and the enhanced sensitivity to osmotic stress. Finally, ΔBbLec1 mutant strain displayed the weakened virulence when compared with the wild-type strain. Taken together, BbLec1 traffics into eisosome and links the functionality of eisosome to development and virulence of B. bassiana.

Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts.

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus. It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A. fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species.

Conidiation under illumination enhances conidial tolerance of insect-pathogenic fungi to environmental stresses

Light is an important signal for fungi in the environment and induces many genes with roles in stress and virulence responses. Conidia of the entomopathogenic fungi Aschersonia aleyrodis, Beauveria bassiana, Cordyceps fumosorosea, Lecanicillium aphanocladii, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium robertsii, Simplicillium lanosoniveum, Tolypocladium cylindrosporum, and Tolypocladium inflatum were produced on potato dextrose agar (PDA) medium under continuous white light, on PDA medium in the dark, or under nutritional stress (= Czapek medium without sucrose = MM) in the dark. The conidial tolerance of these species produced under these different conditions were evaluated in relation to heat stress, oxidative stress (menadione), osmotic stress (KCl), UV radiation, and genotoxic stress caused by 4-nitroquinoline 1-oxide (4-NQO). Several fungal species demonstrated greater stress tolerance when conidia were produced under white light than in the dark; for instance white light induced higher tolerance of A. aleyrodis to KCl and 4-NQO; B. bassiana to KCl and 4-NQO; C. fumosorosea to UV radiation; M. anisopliae to heat and menadione; M. brunneum to menadione, KCl, UV radiation, and 4-NQO; M. robertsii to heat, menadione, KCl, and UV radiation; and T. cylindrosporum to menadione and KCl. However, conidia of L. aphanocladii, S. lanosoniveum, and T. inflatum produced under white light exhibited similar tolerance as conidia produced in the dark. When conidia were produced on MM, a much stronger stress tolerance was found for B. bassiana to menadione, KCl, UV radiation, and 4-NQO; C. fumosorosea to KCl and 4-NQO; Metarhizium species to heat, menadione, KCl, and UV radiation; T. cylindrosporum to menadione and UV radiation; and T. inflatum to heat and UV radiation. Again, conidia of L. aphanocladii and S. lanosoniveum produced on MM had similar tolerance to conidia produced on PDA medium in the dark. Therefore, white light is an important factor that induces higher stress tolerance in some insect-pathogenic fungi, but growth in nutritional stress always provides in conidia with stronger stress tolerance than conidia produced under white light.

Pathogenicity against hemipteran vector insects of a novel insect pathogenic fungus from Entomophthorales (Pandora sp. nov. inedit.) with potential for biological control

A new but still unpublished entomopathogenic fungus (ARSEF13372) in the genus Pandora (Entomophthorales: Entomophthoraceae) was originally isolated from Cacopsylla sp. (Hemiptera: Psyllidae). Several species of the genus Cacopsylla vector phloem-borne bacteria of the genus ‘Candidatus Phytoplasma‘, which cause diseases in fruit crops such as apple proliferation, pear decline and European stone fruit yellows. To determine Pandora’s host range and biocontrol potential we conducted laboratory infection bioassays; Hemipteran phloem-feeding insects were exposed to conidia actively discharged from in vitro produced mycelial mats of standardized area. We documented the pathogenicity of Pandora sp. nov. to species of the insect families Psyllidae and Triozidae, namely Cacopsyllapyri L., C.pyricola (Foerster), C.picta (Foerster, 1848), C.pruni (Scopoli), C.peregrina (Foerster), and Trioza apicalis Foerster. The occurrence of postmortem signs of infection on cadavers within 10 days post inoculation proved that Pandora sp. nov. was infective to the tested insect species under laboratory conditions and significantly reduced mean survival time for C.pyri (summer form and nymph), C.pyricola, C.picta, C.pruni, C.peregrina and T.apicalis. Assessing a potential interaction between phytoplasma, fungus and insect host revealed that phytoplasma infection (‘Candidatus Phytoplasma mali’) of the vector C.picta and/or its host plant apple Malus domestica Borkh. did not significantly impact the survival of C.picta after Pandora sp. nov. infection. The results from infection bioassays were discussed in relation to Pandora sp. nov. host range and its suitability as biocontrol agent in integrated pest management strategies of psyllid pests, including vector species, in orchards.

Empirical Support for the Pattern of Competitive Exclusion between Insect Parasitic Fungi.

Fungal entomopathogens are largely facultative parasites and play an important role in controlling the density of insect populations in nature. A few species of these fungi have been used for biocontrol of insect pests. The pattern of the entomopathogen competition for insect individuals is still elusive. Here, we report the empirical competition for hosts or niches between the inter- and intra-species of the entomopathogens Metarhizium robertsii and Beauveria bassiana. It was found that the synergistic effect of coinfection on virulence increase was not evident, and the insects were largely killed and mycosed by M. robertsii independent of its initial co-inoculation dosage and infection order. For example, >90% dead insects were mycosed by M. robertsii even after immersion in a spore suspension with a mixture ratio of 9:1 for B. bassiana versus M. robertsii. The results thus support the pattern of competitive exclusion between insect pathogenic fungi that occurred from outside to inside the insect hosts. Even being inferior to compete for insects, B. bassiana could outcompete M. robertsii during co-culturing in liquid medium. It was also found that the one-sided mycosis of insects occurred during coinfection with different genotypic strains of either fungi. However, parasexual recombination was evident to take place between the compatible strains after coinfection. The data of this study can help explain the phenomena of the exclusive mycosis of insect individuals, but co-occurrence of entomopathogens in the fields, and suggest that the synergistic effect is questionable regarding the mixed use of fungal parasites for insect pest control.

DIM5/KMT1 controls fungal insect pathogenicity and genome stability by methylation of histone H3K4, H3K9 and H3K36

ABSTRACT Mono-, di- and tri-methylation of histone H3 Lys 9, Lys 4, and Lys 36 (H3K_me1/me2/me3) required for mediation of DNA-based cellular events in eukaryotes usually rely upon the activities of histone lysine methyltransferases (KMTs) classified to the KMT1, KMT2, and KMT3 families, respectively. Here, an H3K9-specific DIM5/KMT1 orthologue, which lacks a C-terminal post-SET domain and localizes mainly in nucleus, is reported to have both conserved and noncanonical roles in methylating the H3 core lysines in Beauveria bassiana, an insect-pathogenic fungus serving as a main source of wide-spectrum fungal insecticides. Disruption of dim5 led to abolishment of H3K9me3 and marked attenuation of H3K4me1/me2, H3K9me1/me2 and H3K36me2. Consequently, the Δdim5 mutant lost the whole insect pathogenicity through normal cuticle infection, and was compromised severely in virulence through cuticle-bypassing infection (hemocoel injection) and also in a series of cellular events critical for the fungal virulence and lifecycle in vivo and in vitro, including reduced hyphal growth, blocked conidiation, impeded proliferation in vivo, altered carbohydrate epitopes, disturbed cell cycle, reduced biosynthesis and secretion of cuticle-degrading enzymes, and increased sensitivities to various stresses. Among 1,201 dysregulated genes (up/down ratio: 712:489) associated with those phenotypic changes, 92 (up/down ratio: 59:33) encode transcription factors and proteins or enzymes involved in posttranslational modifications, implying that the DIM5-methylated H3 core lysines could act as preferential marks of those transcription-active genes crucial for global gene regulation. These findings uncover a novel scenario of DIM5 and its indispensability for insect-pathogenic lifestyle and genome stability of B. bassiana.

Virulence of Beauveria bassiana Balsamo to red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

BackgroundThe red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), is a cosmopolitan pest of palms. Current management strategies largely depend on insecticides, and use of insect pathogenic fungi is needed in integrated pest management (IPM) programs.ResultsThe effectiveness of Beauveria bassiana was determined against 3rd instar R. ferrugineus larvae by using the following concentrations, i.e., 3 × 108, 2 × 108, 1 × 108, 1 × 107, and 1 × 106 spores/ml. The lowest LC50 value (1.3×107 spores/ml) was recorded at the Khyber Pakhtunkhwa (KPK) population, followed by those populations from Punjab, Sindh, and Baluchistan, which had LC50 values of 1.5×107, 5.3 ×107, and 1.02 ×108 spores/ml, respectively, on the 7th day post-treatment; the highest mortality rates were 90.0, 85.0, 77.5, and 75.0% for the KPK, Punjab, Sindh, and Baluchistan populations, respectively, at the highest tested concentration of B. bassiana.ConclusionThe findings of this study indicate the potential use of B. bassiana in IPM programs for R. ferrugineus that are more ecologically sound than those programs that are heavily reliant on insecticides.

Vital roles of Pks11, a highly reducing polyketide synthase, in fungal conidiation, antioxidant activity, conidial cell wall integrity, and UV tolerance of Beauveria bassiana

Fungal polyketide synthases play important and differential roles in synthesizing secondary metabolites and regulating several cell events, including asexual development, environmental adaptation, and pathogenicity. This study shows the important functions of a highly reducing polyketide synthase, Pks11, in Beauveria bassiana, a filamentous fungal insect pathogen used worldwide for pest biocontrol. The deletion of pks11 led to severe defects in conidial yields on different media and a decrease of 36.27% in the mean thickness of conidial cell wall under normal conditions. Compared with the wild-type, Δpks11 showed higher tolerance to oxidation and increased sensitivity to high temperature during colony growth. Moreover, the lack of pks11 caused a decrease in conidial germination after exposure to UV radiation but did not affect the virulence of B. bassiana against Galleria mellonella larvae via typical cuticle infection. These findings concurred with the alteration in the transcript levels of some phenotype-related genes. These data suggested that pks11 played vital roles in the asexual development, cell wall integrity, and fungal responses to oxidation, high temperature, and UV irradiation of B. bassiana.

A p53-like transcription factor, BbTFO1, contributes to virulence and oxidative and thermal stress tolerances in the insect pathogenic fungus, Beauveria bassiana.

The p53-like transcription factor (TF) NDT80 plays a vital role in the regulation of pathogenic mechanisms and meiosis in certain fungi. However, the effects of NDT80 on entomopathogenic fungi are still unknown. In this paper, the NDT80 orthologue BbTFO1 was examined in Beauveria bassiana, a filamentous entomopathogenic fungus, to explore the role of an NDT80-like protein for fungal pest control potential. Disruption of BbTFO1 resulted in impaired resistance to oxidative stress (OS) in a growth assay under OS and a 50% minimum inhibitory concentration experiment. Intriguingly, the oxidation resistance changes were accompanied by transcriptional repression of the two key antioxidant enzyme genes cat2 and cat5. ΔBbTFO1 also displayed defective conidial germination, virulence and heat resistance. The specific supplementation of BbTFO1 reversed these phenotypic changes. As revealed by this work, BbTFO1 can affect the transcription of catalase genes and play vital roles in the maintenance of phenotypes associated with the biological control ability of B. bassiana.

Manipulation of host ecdysteroid hormone levels facilitates infection by the fungal insect pathogen, Metarhizium rileyi.

Entomopathogenic fungi such as Metarhizium rileyi and Beauveria bassiana are widely used insect biological control agents. Little, however, is known concerning genetic or enzymatic factors that differentiate the mechanisms employed by these two fungal pathogens to infect target hosts. Infection by either of these organisms is known to increase levels of the growth and molting hormone, ecdysone, which also regulates the expression of a number of innate immune pathways. M. rileyi, but not B. bassiana, has apparently evolved an ecdysteroid-22-oxidase (MrE22O) that inactivate ecdysone. We show that deletion of MrE22O impaired virulence compared with the wild-type strain, with an increase in ecdysone titer seen in hosts that was coupled to an increase in the expression of antimicrobial genes. An M. rileyi strain engineered to overexpress MrE22O (MrE22OOE ), as well as trans-expression in B. bassiana (Bb::MrE220OE ) resulted, in strains displaying enhanced virulence and dampening of host immune responses compared with their respective wild-type parental strains. These results indicate that ecdysone plays an important role in mediating responses to fungal infection and that some insect pathogenic fungi have evolved mechanisms for targeting this hormone as a means for facilitating infection.

MripacC regulates blastosphere budding and influences virulence of the pathogenic fungus Metarhizium rileyi

Fungal dimorphism is the ability of certain fungi to switch between two different cellular forms, yeast and mycelial forms, in response to external environmental factors. The pacC/Pal signal transduction pathway responds to neutral and alkaline environments and is also involved in the fungal dimorphic transition. In this study, we investigated the function of the pacC homolog, MripacC, which regulates the dimorphic transition and modulates virulence of the insect pathogenic fungus Metarhizium rileyi. MripacC expression was upregulated under alkaline condition, with increased number of yeast-like cells compared to the number of hyphae cells. A MripacC deletion mutant (ΔMripacC) was obtained by homologous replacement and exhibited decreased blastospore budding, with direct development of conidia into hyphae without entering the yeast-like stage when cultured on alkaline medium. Observation of host hemolymph morphology and analysis of samples to detect the main immune factors revealed a decreased ability of ΔMripacC to evade the host immune system. The results of insect bioassays showed that ΔMripacC had decreased virulence with extended median lethality time. Together, the results suggested that MripacC not only regulated adaptation to acidic and alkaline environments, but also influenced virulence by budding blastospores. This elucidation of the function of MripacC adds to our understanding of blastospore budding and virulence of this fungal pathogen.

Characterization and Toxicity of Crude Toxins Produced by Cordyceps fumosorosea against Bemisia tabaci (Gennadius) and Aphis craccivora (Koch).

Cordyceps fumosorosea, an insect pathogenic fungus, produces different toxins/secondary metabolites which can act as pest control agents. This study reports the extraction and characterization of crude mycelial extracts of C. fumosorosea isolate SP502 along with their bio-efficacy against Bemisia tabaci and Aphis craccivora. Fourier transform infrared spectroscopy, liquid chromatography, mass spectrometery and nuclear magnetic resonance analysis of C. fumosorosea isolate SP502 extracts showed the presence of five major compounds—Trichodermin, 5-Methylmellein, Brevianamide F, Enniatin and Beauvericin—which all may potentially be involved in insecticidal activity. The HPLC analysis of C. fumosorosea mycelial extracts and Beauvericin standard showed similar chromatographic peaks, with the content of Beauvericin in the crude toxin being calculated as 0.66 mg/ml. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of A. craccivora were 46.35, 54.55, 68.94, and 81.92 µg/mL, respectively. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of B. tabaci were 62.67, 72.84, 77.40, and 94.40 µg/mL, respectively. Our results demonstrate that bioactive compounds produced by C. fumosorosea isolate SP502 have insecticidal properties and could, therefore, be developed into biopesticides for the management of B. tabaci and A. craccivora.

Ubr1-mediated ubiquitylation orchestrates asexual development, polar growth, and virulence-related cellular events in Beauveria bassiana.

The E3 ubiquitin ligase Ubr1 is a core player in yeast ubiquitylation and protein quality control required for cellular events including proteasomal degradation and gene activity but has been rarely explored in filamentous fungi. We show here an essentiality of orthologous Ubr1-mediated ubiquitylation for the activation of central developmental pathway (CPD) and the CPD-controlled cellular events in Beauveria bassiana, a filamentous fungal insect pathogen that undergoes an asexual cycle in vitro or in vivo. As a result of ubr1 disruption, intracellular free ubiquitin accumulation increased by 1.4-fold, indicating an impaired ability for the disruptant to transfer ubiquitin to target proteins. Consequently, the disruptant was compromised in polar growth featured with curved or hook-like germ tubes and abnormally branched hyphae, leading to impeded propagation of aberrant hyphal bodies in infected insect hemocoel and attenuated virulence. In the mutant, sharply repressed expression of three CDP activator genes (brlA, abaA, and wetA) correlated well with severe defects in aerial conidiation and submerged blastospore (hyphal body) production in insect hemolymph or a mimicking medium. Moreover, the disruptant was sensitive to cell wall perturbation or lysing and showed increased catalase activity and resistance to hydrogen peroxide despite null response to high osmolarity or heat shock. Most of the examined genes involved in polar growth and cell wall integrity were down-regulated in the disruptant. These findings uncover that the Ubr1-mediated ubiquitylation orchestrates polar growth and the CDP-regulated asexual cycle in vitro and in vivo in B. bassiana. KEY POINTS: • Ubr1 is an E3 ubiquitin ligase essential for ubiquitylation in Beauveria bassiana. • Ubr1-mediated ubiquitylation is required for activation of central development pathway. • Ubr1 orchestrates polar growth and asexual cycle in vitro and in vivo.

Antimicrobial, antimalarial, and cytotoxic substances from the insect pathogenic fungus Beauveria asiatica BCC 16812

Abstract Six new compounds 1–6 and two new naturally occurring compounds 7 and 8, together with nine known compounds 9–14, beauverioride I, dipicolinic acid, and 6-(methoxycarbonyl) picolinic acid were isolated from a wasp-pathogenic fungus Beauveria asiatica, strain BCC 16812. Their structures were determined by extensive spectroscopic analyses. The absolute configurations of compound 6 were established by the application of modified Mosher’s method and ECD calculation. Compounds 9–11 exhibited antimalarial (IC50 0.47–1.79 u g/mL), antimicrobial (MIC 3.13–50.0 u g/mL), and cytotoxic (IC50 0.26–1.23 u g/mL) activities, while compounds 1, 2, 8, and 14 showed only cytotoxicity against Vero cell lines, with IC50 values range 12.96–47.03 u g/mL.

Altered Expression of Chemosensory and Odorant Binding Proteins in Response to Fungal Infection in the Red Imported Fire Ant, Solenopsis invicta.

Social insects have evolved acute mechanisms for sensing and mitigating the spread of microbial pathogens within their communities that include complex behaviors such as grooming and sanitation. Chemical sensing involves detection and transport of olfactory and other chemicals that are mediated by at least two distinct classes of small molecular weight soluble proteins known as chemosensory- and odorant binding proteins (CSPs and OBPs, respectively) that exist as protein families in all insects. However, to date, a systematic examination of the expression of these genes involved in olfactory and other pathways to microbial infection has yet to be reported. The red imported fire ant, Solenopsis invicta, is one of the most successful invasive organisms on our planet. Here, we examined the temporal gene expression profiles of a suite of S. invicta CSPs (SiCSPs1-22) and OBPs (SiOBPs1-16) in response to infection by the broad host range fungal insect pathogen, Beauveria bassiana. Our data show that within 24 h post-infection, i.e., before the fungus has penetrated the host cuticle, the expression of SiCSPs and SiOBPs is altered (mainly increased compared to uninfected controls), followed by suppression of SiCSP and select SiOBP expression 48 h post-infection and mixed responses at 72 h post-infection. A smaller group of SiBOPs, however, appeared to respond to fungal infection, with expression of SiOBP15 consistently higher during fungal infection over the time course examined. These data indicate dynamic gene expression responses of CSPs and OBPs to fungal infection that provide clues to mechanisms that might mediate detection of microbial pathogens, triggering grooming, and nest sanitation.

Diversity of the insect pathogenic fungi in the genus Metarhizium in New Zealand

ABSTRACT Invertebrate-pathogenic fungi in the genus Metarhizium are commonly found throughout the world on a wide range of different arthropod hosts. Some strains have been developed as biopesticides and many others have shown potential for controlling important insect pests. Given the current interest in using these fungi, precise identification at species level is crucial, both for understanding their diversity and ecology and for regulatory approval and monitoring of new biopesticides. Metarhizium has undergone extensive systematic revision over the last decade, resulting in a large number of name changes and new species. This has created regulatory issues in New Zealand and importation of biocontrol strains has become difficult due to a lack of knowledge of which Metarhizium species are present in the country. This study identified Metarhizium isolates held in culture collections in New Zealand following analysis of sequence data from the ef-1α, β-tubulin, rpb1, and ITS regions. Our results show that M. anisopliae, M. brunneum, M. frigidum, M. novozealandicum, M. pemphigi, M. rileyi and M. robertsii occur naturally in New Zealand on native and introduced insect hosts. Exotic Metarhizium species from several countries, stored in New Zealand collections, include M. acridum, M. lepidiotae, M. majus and M. pingshaense.

A novel transcription factor negatively regulates antioxidant response, cell wall integrity and virulence in the fungal insect pathogen, Beauveria bassiana.

Genomic data have identified a class of fungal specific transcription factors (FsTFs) that are thought to regulate unique aspects of fungal gene expression, although the functions of many of these proteins remain unknown. Here, a novel FsTF (BbStf1), which features a leucine zipper dimerization domain and a fungal transcription factor regulatory middle homology region, was characterized in Beauveria bassiana, a filamentous insect fungal pathogen. Transcriptional activation and nuclear localization were experimentally confirmed for BbStf1. Disruption of Bbstf1 resulted in increased tolerance to oxidative stress and cell wall perturbation, accompanied by increased peroxidase (POD) and superoxide dismutase (SOD) activities and ratio of reduced/oxidized glutathione (GSH/GSSG), and by thickened cell wall and altered composition. Gene expression profile analysis revealed that transcription patterns of antioxidant enzyme and cell wall integrity-involved genes were altered in the ∆Bbstf1, including some BbStf1-targeted genes clarified with evidence. The ∆Bbstf1 strain displayed greater virulence to Galleria mellonella in the bioassays through both topical infection and intrahaemocoel injection due to more rapid proliferation in the haemocoel as compared to the wild-type strain. Altogether, BbStf1 acts as a negative regulator of antioxidant response, cell wall integrity and virulence in B. bassiana.