Insect Pathogenic Fungus Research Articles

Differential divergences of obligately insect-pathogenicEntomophthoraspecies from fly and aphid hosts

Three DNA regions (ITS 1, LSU rRNA and GPD) of isolates from the insect-pathogenic fungus genus Entomophthora originating from different fly (Diptera) and aphid (Hemiptera) host taxa were sequenced. The results documented a large genetic diversity among the fly-pathogenic Entomophthora and only minor differences among aphid-pathogenic Entomophthora. The evolutionary time of divergence of the fly and the aphid host taxa included cannot account for this difference. The host-driven divergence of Entomophthora, therefore, has been much greater in flies than in aphids. Host-range differences or a recent host shift to aphid are possible explanations.

A phosphoketolase Mpk1 of bacterial origin is adaptively required for full virulence in the insect‐pathogenic fungus Metarhizium anisopliae

Pentose metabolism through the phosphoketolase pathway has been well characterized in bacteria. In this paper, we report the identification of a phosphoketolase homologue Mpk1 in the insect-pathogenic fungus Metarhizium anisopliae. Phylogenetic analysis showed that fungal phosphoketolases are of bacterial origin and diverged into two superfamilies. Frequent gene loss or lack of acquisition is evident in specific fungal lineages or species. The mpk1 gene is highly expressed when grown in trehalose-rich insect haemolymph but poorly induced by insect cuticle or carbohydrate-rich plant root exudate. In addition, mpk1 gene expression and enzyme activity could be upregulated by different sugars including xylose, trehalose, glucose or sucrose. mpk1 null mutants generated by homologous recombination grew similar to the wild type of M. anisopliae on medium amended with xylose as a sole carbon source. However, insect (tobacco hornworm, Manduca sexta) bioassays showed significantly reduced virulence in Deltampk1. The results of this study suggest that the horizontally transferred Mpk1 in M. anisopliae plays an important niche adaptation role for fungal propagation in insect haemocoel. Following the carbohydrate flux from plants to plant-feeding insects and insect pathogenic fungi, a tritrophic relationship is discussed in association with the requirement of fungal phosphoketolase pathway.

Total Synthesis of Hirsutellone B

Hirsutellones,[1] pyrrospirones,[2] and pyrrocidines[3] belong to a growing class of fungal secondary metabolites whose biological properties include antifungal and antibiotic activities. Particularly impressive are the activities of hirsutellones A and B (1a and 1b, Figure 1)[1] against Mycobacterium tuberculosis (MIC = 0.78 μg/mL), the causative pathogen of tuberculosis.[4] Isolated from the insect pathogenic fungus Hirsutella nivea BCC 2594, the hirsutellones share a number of unique structural features, including a 6,5,6-fused tricyclic core, a γ-lactam- or succinimide-containing moiety, a 12- or 13-membered p-cyclophane structural motif that encompasses an aryl ether linkage, and ten stereogenic centers. In view of their promising biological properties as lead compounds for new antituberculosis drugs and because of their challenging molecular architectures, the hirsutellones are deemed important synthetic targets.[5] Herein we report the total synthesis of hirsutellone B (1b) in its enantiomerically pure form through a strategy that features a number of novel cascade sequences and chemoselective reactions.

Wundinfektionen bei Raupen von Malacosoma neustria (L.) durch Penicillium brevi-compactum Dierckx

Summary On three caterpillars (L3) of Malacosoma neustria (L.) reared in the laboratory we observed a mycosis caused by Penicillium brevi-compactum Dierckx. The moribund larvae showed a mycelium, 2–3 mm wide, approximately girdle-like sourrounding the body. Histopathological investigations and infection experiments confirmed the suggestion that these were wound-infections caused by a fungus normally not pathogenic for insects. The wounds are closed by a coagulation plug, with a mass of hyphae in his exterior strongly melanized part adjoined by the outer layer of the macroscopically visible mycelium. Hyphae grow through the coagulation plug into the body of the larvae. Apparently the infestation spreads relatively slow towards the ends of the body. The hyphae preferably grow in the fat body, muscles, tracheal matrix, epidermis (hypodermis), and endocuticle. It is striking that in moribund caterpillars the muscularis of the midgut is densely infected, whereas the epithelium cells are free or almost free of hyphae. Finally, hyphae grow through the integument in outside direction preferring the insertion-points of the hairs. Contrary to the fungus diseases caused by the typical insect pathogenic fungi which spread in the whole body, here we have the picture of a localized, but also fatal mycosis. For infection experiments 20 larvae (L4 and L5) of M. neustria obtained dorsolaterally an incision of 1 mm in the region of the first pair of the abdominal feet. After 15 min. and again after 2 hours, the wounds were contamined with spores of P. brevi-compactum. For 48 hours the larvae remained in Petri dishes with a rel. humidity of about 100 per cent. 30 per cent of the larvae died of wound-mycosis, the average time of mortality being 7,8 days. The controls, wounded and uninfected on the one side, not wounded but spore dusted on the other, did not die of mycosis. Thus the saprophytic P. brevi-compactum is able to act as a wound parasite on larvae of M. neustria. The synergism (“enchainement”) of two harmless factors (wound and fungus) causes death within a relatively short time.

Susceptibility of Culicoides biting midge larvae to the insect-pathogenic fungus, Metarhizium anisopliae: Prospects for bluetongue vector control

Culicoides biting midges (Diptera: Ceratopogonidae) are responsible for the spread of several arboviruses of livestock and humans that are of international importance. This study assesses the virulence of 18 insect-pathogenic fungal strains from the genera Metarhizium, Beauveria, Isaria and Lecanicillium to larval stages of Culicoides nubeculous Meigen as a means of examining their potential as biocontrol agents. In initial screening, six strains of M. anisopliae (ERL700, CA1, V275, LRC181A, ARSEF 3291 and ARSEF 4556) outperformed the other tested genera and were found to cause between 90% and 100% larval mortality in all larval instars of this species at 72 h post inoculation. The virulence of the most effective strain, M. anisopliae V275, was then further tested by exposing larvae to doses which ranged from 10 4–10 8 conidia/ml and recording mortality at 24, 48 and 72 h in a 24-multi-well plate with each well containing 600 μl of water and at 24 and 48 h in 250 ml plastic cups containing 50 ml of water. Sensitivity of larvae was extremely high in the multi-well plates, with LC 50 values of 4.3–4.5 × 10 3 conidia/ml and no significant differences between larval instars. In the 250 ml cups, M. anisopliae V275 caused mortalities of between 70% and 100% to larvae and later instars exhibited higher mortality rates. The results are discussed in relation to incorporation of M. anisopliae into biocontrol programmes to control arboviruses vectored by Culicoides.

Imbibitional damage in conidia of the entomopathogenic fungi Beauveria bassiana, Metarhizium acridum, and Metarhizium anisopliae

When dried organisms are immersed in water, rapid imbibition may cause severe damage to plasma membranes; in unicellular organisms, such damage is usually lethal. This study investigated effects of water activity (dryness) of organisms and immersion temperature on imbibitional damage in three insect pathogenic fungi. Conidial powders of Beauveria bassiana (Bb), Metarhizium anisopliae (Ma) and Metarhizium acridum (Mac) were dried/hydrated to a broad range of water activities (aw) (0.023–0.961) prior to immersion in water at 0.5–33°C. Imbibitional damage in conidia of each fungus occurred rapidly, with no differences in viabilities observed following immersion for 2 vs. 60min. Damage increased with decreasing water activity of the conidia and decreasing temperature of the immersion water. Dry (aw⩽0.333) Metarhizium spp. conidia were highly susceptible to imbibitional damage, with viability declining to ⩽5% after immersion at 0.5°C and ⩽63% following immersion at 15°C. Germination of the driest Ma conidia was reduced to 66% after treatment at 25°C. In contrast, Bb was highly tolerant to damage, with significant reductions in viability (to levels as low as 43–65%) occurring only when dry conidia were immersed at 0.5°C. Damage was prevented when conidia were slowly rehydrated by humidification prior to immersion and immersion temperature was increased to 33–34°C; germination of all fungi was ⩾94% under these optimal conditions. However, immersion of the driest Bb, Ma, and Mac powders in warm water (33°C) also resulted in high viabilities (95%, 89%, and 94%, respectively), and slow-rehydrated conidia also retained high viability (87%, 92%, and 83%, respectively) after immersion in ice-cold water (0.5°C). Formulation of conidia in pure (non-emulsifiable) paraffinic oil provided considerable protection from imbibitional damage. This study underscores a need for establishing standard protocols for preparing aqueous suspensions of sensitive fungi for both research and commercial applications.

Pathogenicity of the Fungus, Aspergillus clavatus, isolated from the locust, Oedaleus senegalensis, against larvae of the mosquitoes Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus.

The use of insect pathogenic fungi is a promising alternative to chemical control against mosquitoes. Among the Hyphomycetes isolated from insects for mosquito control, the genus Aspergillus remains the least studied. In September 2005, four fungi were isolated from the Senegalese locust, Oedaleus senegalensis Kraus (Orthoptera: Acrididae), collected in Dakar, Senegal. One of these fungi, identified as Aspergillus clavatus, Desmazières (Eurotiales: Trichocomaceae) was highly pathogenic against larvae of the mosquitoes Aedes aegypti L., Anopheles gambiae s.l. Giles and Culex quinquefasciatus Say (Diptera: Culicidae). An application of 1.2 mg/ml dry conidia yielded 100% mortality after 24 hours against both Ae. aegypti and Cx. quinquefasciatus while with An. gambiae it was 95%. With unidentified species in the genus Aspergillus, mortality after 24 h was <5% against all the larval species. Application of A. clavatus produced in a wheat powder medium using doses ranging between 4.3 to 21×107 spores/ml, caused 11 to 68% mortality against Cx. quinquefasciatus at 24h, and 37 to 100% against Ae. aegypti. Microscopic observations showed fungal germination on both Ae. aegypti and Cx. quinquefasciatus larvae. Histological studies revealed that A. clavatus penetrated the cuticle, invaded the gut and disintegrated its cells. Some Cx. quinquefasciatus larvae, treated with A. clavatus reached the pupal stage and produced infected adults. However, the infection was mainly located on the extremity of their abdomen. These results suggest that A. clavatus could be an effective tool to manage mosquito proliferation.

Identification of genes differentially expressed in vivo by Metarhizium anisopliae in the hemolymph of Locusta migratoria using suppression-subtractive hybridization

Metarhizium anisopliae is an important insect pathogenic fungus widely used in biological pest control. The aim of this study was to identify genes differentially expressed in vivo by M. anisopliae CQMa102 in the hemolymph of infected Locusta migratoria. Suppression-subtractive hybridization was performed using cDNA generated from hyphal bodies purified from hemolymph and the fungus germinating and differentiating on locust wings. A total of 350/1,600 random clones screened by cDNA array dot blotting were sequenced, resulting in 120 uniquely expressed sequence tags (ESTs) that were up-regulated during colonization of hemolymph. Among these 120 ESTs, 42 (35.0%) had matches in the NR protein database, and 29 (24.2%) were significantly similar to known proteins involved in various cellular processes, including general metabolism, cell wall remodeling, protein synthesis, signal transduction and stress responses. In contrast, the remaining 78 ESTs (65.0%) either had low similarity in the NR database or represented novel genes. Semi-quantitative RT-PCR analysis of five randomly selected genes revealed that all were highly expressed in the host hemolymph. These results provide new insight into the underlying molecular mechanisms of adaptation to host hemolymph and may increase understanding of host-pathogen interactions.

Torrubiellutins A–C, from insect pathogenic fungus Torrubiella luteorostrata BCC 12904

Investigation of the insect pathogenic fungus Torrubiella luteorostrata led to the isolation of three new macrocyclic torrubiellutins A–C ( 1– 3) and the known pyrone diterpene 4. Structures were elucidated by spectroscopic data including 1D, 2D NMR, and MS spectral data. The absolute stereochemistry was determined by chemical means using Mosher reactions and Marfey's reagent, together with NOESY spectral data. Torrubiellutin C showed biological activities against KB, MCF-7, NCI-H187, and Vero cell lines with IC 50 varying from 0.78 to 4.36 μg/mL, while compound 4 exhibited antimalaria and anti-inflammatory activity with IC 50 values of 3.49 and 1.21 μg/mL, respectively.

Isocoumarin Glucosides from the Scale Insect Fungus Torrubiella tenuis BCC 12732

Three new isocoumarin glucosides (1, 3, and 4), 6,8-dihydroxy-3-methylisocoumarin (2), and 6,8-dihydroxy-3-hydroxymethylisocoumarin (5) were isolated from the scale insect pathogenic fungus Torrubiella tenuis BCC 12732. Structures of these compounds were elucidated using NMR spectroscopic and MS spectrometric analyses. Compound 5 exhibited moderate anti-HSV-1 and antimycobacterial activities with IC(50) and MIC values of 50 and 25 microg/mL, respectively.

The suitability of clay pots for indoor sampling of mosquitoes in an arid area in northern Tanzania

Water storage clay pots have been recently explored as method for outdoor mosquito sampling and as novel device for administrating insect-pathogenic fungi to mosquitoes. Their suitability for indoor mosquito sampling in natural conditions is unknown. We tested clay pots as indoor resting sites alongside catches by CDC light trap in an area of low malaria endemicity in northern Tanzania. Mosquitoes were caught by clay pots although the rate of female Anopheles mosquito catches was 22.64 (95% CI 11.26–45.52) times greater for CDC light traps. The proportion of fed female Anophelines was significantly higher for clay pots compared to CDC light trap ( p < 0.001), indicating these methods sample different populations of mosquitoes. Although we were able to identify households with a consistently higher exposure to mosquitoes by CDC light trap, there was no apparent heterogeneity in mosquito catches by clay pots. We conclude that clay pots are not a reliable tool to sample mosquitoes in the dry season in an area of low transmission intensity with Anopheles arabiensis as principle vector.

Mitogen-activated protein kinase hog1 in the entomopathogenic fungus Beauveria bassiana regulates environmental stress responses and virulence to insects.

Beauveria bassiana is an economically important insect-pathogenic fungus which is widely used as a biocontrol agent to control a variety of insect pests. However, its insecticide efficacy in the field is often influenced by adverse environmental factors. Thus, understanding the genetic regulatory processes involved in the response to environmental stress would facilitate engineering and production of a more efficient biocontrol agent. Here, a mitogen-activated protein kinase (MAPK)-encoding gene, Bbhog1, was isolated from B. bassiana and shown to encode a functional homolog of yeast HIGH-OSMOLARITY GLYCEROL 1 (HOG1). A Bbhog1 null mutation was generated in B. bassiana by targeted gene replacement, and the resulting mutants were more sensitive to hyperosmotic stress, high temperature, and oxidative stress than the wild-type controls. These results demonstrate the conserved function of HOG1 MAPKs in the regulation of abiotic stress responses. Interestingly, DeltaBbhog1 mutants exhibited greatly reduced pathogenicity, most likely due to a decrease in spore viability, a reduced ability to attach to insect cuticle, and a reduction in appressorium formation. The transcript levels of two hydrophobin-encoding genes, hyd1 and hyd2, were dramatically decreased in a DeltaBbhog1 mutant, suggesting that Bbhog1 may regulate the expression of the gene associated with hydrophobicity or adherence.

Identification of genes that are preferentially expressed in conidiogenous cell development of Metarhizium anisopliae by suppression subtractive hybridization

The insect pathogenic fungus Metarhizium anisopliae is widely used as an insect biocontrol agent. The M. anisopliae conidium plays an important role in pathogenesis and disease transmission. The aim of this study was to identify genes whose expression is up-regulated during conidiogenous cell development. This is a powerful strategy for obtaining insight into the molecular events that regulate conidiation. We isolated genes that are preferentially expressed in the developing conidiophores of the common fungal locust pathogen M. anisopliae CQMa102 using suppression subtractive hybridization. Based on the results of cDNA array dot blotting, we identified 109 unique expressed sequence tags (ESTs) that were up-regulated more than fivefold during conidiophore formation. Among these 109 ESTs were 45 (41.3%) with significant similarity to NCBI annotated hypothetical proteins, 35 (32.1%) with low similarity to known or predicted genes that might represent novel genes, and 29 (26.6%) with significant similarity to known proteins involved in various cell and molecular processes, such as ell structure and function, cell metabolism, protein metabolism, stress response, nucleic acid metabolism, and cell cycle and growth. We confirmed the up-regulation of 11 randomly selected genes with real-time reverse transcriptase-PCR analysis. The results of this study provide a preliminary description of genes that may be involved in the molecular regulation of fungal conidiogenesis.

Mass spectrometry as a tool for the selective profiling of destruxins; their first identification in Lecanicillium longisporum

Mass spectrometry was applied to the identification of the destruxins (dtxs), cyclic peptides that are commonly produced by the fungal insect-pathogen, Metarhizium anisopliae. The aim of the study was to optimise a methodology in order to firstly determine whether these compounds were present in other species and to determine the effect of differing growth conditions upon the dtx content detected. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) was initially used to analyse the dtxs, but limitations were indicated. Nano-scale high-performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) and automated 'data-dependent' tandem mass spectrometric (MS/MS) analysis were also applied, utilising characteristic neutral losses during fragmentation to confirm the presence of the dtxs. This latter approach distinguished the dtx E and B isoforms by retention time and diagnostic neutral losses during fragmentation allowing extraction of the destruxin data from a complex dataset. This process revealed the presence of a number of dtxs in the fungal species Lecanicillium longisporum, a species previously not known to produce dtxs, and dtx production in this species was shown to be significantly higher in aerated cultures compared with still cultures.

Insect-Specific Polyketide Synthases (PKSs), Potential PKS-Nonribosomal Peptide Synthetase Hybrids, and Novel PKS Clades in Tropical Fungi

Polyketides draw much attention because of their potential use in pharmaceutical and biotechnological applications. This study identifies an abundant pool of polyketide synthase (PKS) genes from local isolates of tropical fungi found in Thailand in three different ecological niches: insect pathogens, marine inhabitants, and lichen mutualists. We detected 149 PKS genes from 48 fungi using PCR with PKS-specific degenerate primers. We identified and classified 283 additional PKS genes from 13 fungal genomes. Phylogenetic analysis of all these PKS sequences the comprising ketosynthase (KS) conserved region and the KS-acyltransferase interdomain region yielded results very similar to those for phylogenies of the KS domain and suggested a number of remarkable points. (i) Twelve PKS genes amplified from 12 different insect-pathogenic fungi form a tight cluster, although along with two PKS genes extracted from genomes of Aspergillus niger and Aspergillus terreus, in reducing clade III. Some of these insect-specific fungal PKSs are nearly identical. (ii) We identified 38 new PKS-nonribosomal peptide synthetase hybrid genes in reducing clade II. (iii) Four distinct clades were discovered with more than 75% bootstrap support. We propose to designate the novel clade D1 with 100% bootstrap support "reducing clade V." The newly cloned PKS genes from these tropical fungi should provide useful and diverse genetic resources for future research on the characterization of polyketide compounds synthesized by these enzymes.

Steps towards sustainable harvest of Ophiocordyceps sinensis in Bhutan

The insect-pathogenic fungus Ophiocordyceps sinensis (better known as Cordyceps sinensis) is harvested over much of the Himalayan plateau as a highly prized remedy in traditional Oriental medicine. Over the past 10 years its financial value has increased dramatically, with collectors paid as much as US $12,500 kg−1 for top-quality material. This is causing significant distortion to local economies, and there is widespread concern that the current rate of collection is unsustainable. This paper introduces the fungus and its insect hosts, documents some of the biological and social constraints to achieving sustainability, describes the socioeconomic climate within which harvest and sale occurs in Bhutan, and details the measures put in place by the Royal Government of Bhutan to promote wise management of this valuable natural resource.

Conidiation Color Mutants of Aspergillus fumigatus Are Highly Pathogenic to the Heterologous Insect Host Galleria mellonella

The greater wax moth Galleria mellonella has been widely used as a heterologous host for a number of fungal pathogens including Candida albicans and Cryptococcus neoformans. A positive correlation in pathogenicity of these yeasts in this insect model and animal models has been observed. However, very few studies have evaluated the possibility of applying this heterologous insect model to investigate virulence traits of the filamentous fungal pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. Here, we have examined the impact of mutations in genes involved in melanin biosynthesis on the pathogenicity of A. fumigatus in the G. mellonella model. Melanization in A. fumigatus confers bluish-grey color to conidia and is a known virulence factor in mammal models. Surprisingly, conidial color mutants in B5233 background that have deletions in the defined six-gene cluster required for DHN-melanin biosynthesis caused enhanced insect mortality compared to the parent strain. To further examine and confirm the relationship between melanization defects and enhanced virulence in the wax moth model, we performed random insertional mutagenesis in the Af293 genetic background to isolate mutants producing altered conidia colors. Strains producing conidia of previously identified colors and of novel colors were isolated. Interestingly, these color mutants displayed a higher level of pathogenicity in the insect model compared to the wild type. Although some of the more virulent color mutants showed increased resistance to hydrogen peroxide, overall phenotypic characterizations including secondary metabolite production, metalloproteinase activity, and germination rate did not reveal a general mechanism accountable for the enhanced virulence of these color mutants observed in the insect model. Our observations indicate instead, that exacerbated immune response of the wax moth induced by increased exposure of PAMPs (pathogen-associated molecular patterns) may cause self-damage that results in increased mortality of larvae infected with the color mutants. The current study underscores the limitations of using this insect model for inferring the pathogenic potential of A. fumigatus strains in mammals, but also points to the importance of understanding the innate immunity of the insect host in providing insights into the pathogenicity level of different fungal strains in this model. Additionally, our observations that melanization defective color mutants demonstrate increased virulence in the insect wax moth, suggest the potential of using melanization defective mutants of native insect fungal pathogens in the biological control of insect populations.

Light stimulates conidiation of the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a commercially important entomopathogenic fungus. Like other insect fungal pathogens, B. bassiana usually produces asexual reproductive bodies, conidia, for dispersal, transmission and infection of insects. Adequate mass-production of high quality conidia is crucial to development of an efficient B. bassiana insecticide. However, little is known about details of conidiation in this fungus in response to environmental signals, which limits understanding of the mechanism of conidiation and improvement in conidia production. Here, morphologenetic changes of B. bassiana under different light conditions are reported. When cultured in total darkness, B. bassiana hyphae can grow continuously with few reproductive structures differentiated, while illumination with white light resulted in prolific formation of conidiophores bearing abundant conidia, indicating that light could stimulate conidiation of B. bassiana. Among the single colour lights tested, blue light was the most effective to stimulating sporulation. Colonies became adapted for blue light stimulus only after hyphae had grown in total darkness for at least 96 h, whereas the photoadaptation obviously declined after 144 h. For the exposure time, 3 min of blue light pulse was enough to stimulate conidiation in the photoadapted mycelia, while prolonged light exposure over 3 min resulted in a decrease in conidia yield. Our results provided useful clues for understanding the mechanism of conidiation mediated by light in B. bassiana.

Late Stage Oxidations during the Biosynthesis of the 2-Pyridone Tenellin in the Entomopathogenic Fungus Beauveria bassiana

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana were investigated by a combination of gene knockout, antisense RNA, and gene coexpression studies. Open reading frames (ORF) 3 and 4 of the tenellin biosynthetic gene cluster were previously shown to encode a trans-acting enoyl reductase and a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), respectively, which together synthesize the acyltetramic acid pretenellin-A. In this work, we have shown that ORF1 encodes a cytochrome P450 oxidase, which catalyzes an unprecedented oxidative ring expansion of pretenellin-A to form the 2-pyridone core of tenellin and related metabolites, and that this enzyme does not catalyze the formation of a hydroxylated precursor. Similar genes appear to be associated with PKS-NRPS genes in other fungi. ORF2 encodes an unusual cytochrome P450 monooxygenase required for the selective N-hydroxylation of the 2-pyridone which is incapable of N-hydroxylation of acyltetramic acids.

Insect-associated fungi in soils of field crops and orchards

Insect-pathogenic fungi are important natural enemies of insect pests but their dynamics in the soils of different agroecosystems are incompletely understood. In the present study, the seasonal occurrence and species diversity of insect-associated fungi in soil were investigated by baiting soil samples with larvae of Galleria mellonella. The survey included insect-pathogenic fungi, opportunistic insect pathogens, and secondary colonizers of insect cadavers. Soil samples from two habitat types (field crops and orchards) were collected from 2004 to 2005 at 2-month intervals except during wintertime. A total of 29 species were isolated and identified, with 25 species in field crop soils and 20 species in orchard soils. Although the common insect-pathogenic species, such as Beauveria bassiana, Metarhizium anisopliae var. anisopliae, and Paecilomyces fumosoroseus (Ascomycota: Hypocreales), were detected in both field crop and orchard soils, their frequency in the two agroecosystems differed significantly: B. bassiana and P. fumosoroseus were more frequently detected in orchard soils, while M. anisopliae var. anisopliae was more frequently detected in field crop soils. The diversity of insect-pathogenic fungi was greater in field crop soil than in orchard soil. The number of Galleria larvae that died from fungal infection was larger in orchard soil than in field crop soil. B. bassiana was the most abundant species in orchard soil, and the number of larvae killed by B. bassiana was larger in June and December than in April and August. However, Mucor spp. (opportunistic pathogens) were the most abundant species in field crop soils, and the number of larvae killed by them was larger in August than at any other time of the year.