Iron homeostasis in the absence of ferricrocin and its consequences in fungal development and insect virulence in Beauveria bassiana.

The putative ferricrocin synthetase gene ferS in the fungal entomopathogen Beauveria bassiana BCC 2660 was identified and characterized. The 14,445-bp ferS encodes a multimodular nonribosomal siderophore synthetase tightly clustered with Fusarium graminearum ferricrocin synthetase. Functional analysis of this gene was performed by disruption with the bar cassette. ΔferS mutants were verified by Southern and PCR analyses. HPLC and TLC analyses of crude extracts indicated that biosynthesis of ferricrocin was abolished in ΔferS. Insect bioassays surprisingly indicated that ΔferS killed the Spodoptera exigua larvae faster (LT50 59 h) than wild type (66 h). Growth and developmental assays of the mutant and wild type demonstrated that ΔferS had a significant increase in germination under iron depletion and radial growth and a decrease in conidiation. Mitotracker staining showed that the mitochondrial activity was enriched in ΔferS under both iron excess and iron depletion. Comparative transcriptomes between wild type and ΔferS indicated that the mutant was increased in the expression of eight cytochrome P450 genes and those in iron homeostasis, ferroptosis, oxidative stress response, ergosterol biosynthesis, and TCA cycle, compared to wild type. Our data suggested that ΔferS sensed the iron excess and the oxidative stress and, in turn, was up-regulated in the antioxidant-related genes and those in ergosterol biosynthesis and TCA cycle. These increased biological pathways help ΔferS grow and germinate faster than the wild type and caused higher insect mortality than the wild type in the early phase of infection.

Identification and phylogenetic analysis of a collection of Beauveria spp. Isolates from Central America and Puerto Rico.

The genus Beauveria comprises economically important entomopathogenic fungi, widely used for biological control in agriculture. Interest in these organisms in Costa Rica prompted surveys and establishment of collections in the past two decades. However, there was neither a formal identification nor a characterization of the isolates. With that purpose, the morphology and genetic variation by microsatellites and partial sequencing of Bloc, TEF-1α and RPB2 regions were studied for 32 isolates of Beauveria, which included 26 from Costa Rica, five from Puerto Rico and one from Honduras. The isolates were identified as B. bassiana (29) and B. caledonica (3). Ninety-three percent of B. bassiana isolates belonged to a monophyletic group of African and Neotropical isolates. A total of 105 alleles were recorded with 11 SSR markers, and the results suggested high diversity within the collection. Mantel tests showed low association between geographic origin and the variation among isolates.

Gene diversity explains variation in biological features of insect killing fungus, Beauveria bassiana.

Beauveria bassiana is a species complex whose isolates show considerable natural genetic variability. However, little is known about how this genetic diversity affects the fungus performance. Herein, we characterized the diversity of genes involved in various mechanisms of the infective cycle of 42 isolates that have different growth rates, thermotolerance and virulence. The analysed genes showed general genetic diversity measured as non-synonymous changes (NSC) and copy number variation (CNV), with most of them being subjected to positive episodic diversifying selection. Correlation analyses between NSC or CNV and the isolate virulence, thermotolerance and growth rate revealed that various genes shaped the biological features of the fungus. Lectin-like, mucin signalling, Biotrophy associated and chitinase genes NSCs correlated with the three biological features of B. bassiana. In addition, other genes (i.e. DNA photolyase and cyclophilin B) that had relatively conserved sequences, had variable CNs across the isolates which were correlated with the variability of either virulence or thermotolerance of B. bassiana isolates. The data obtained is important for a better understanding of population structure, ecological and potential impact when isolates are used as mycoinsecticides and can justify industrialization of new isolates.

Mitochondrial evolution in the entomopathogenic fungal genus Beauveria.

Species in the fungal genus Beauveria are pathogens of invertebrates and have been commonly used as the active agent in biopesticides. After many decades with few species described, recent molecular approaches to classification have led to over 25 species now delimited. Little attention has been given to the mitochondrial genomes of Beauveria but better understanding may led to insights into the nature of species and evolution in this important genus. In this study, we sequenced the mitochondrial genomes of four new strains belonging to Beauveria bassiana, Beauveria caledonica and Beauveria malawiensis, and compared them to existing mitochondrial sequences of related fungi. The mitochondrial genomes of Beauveria ranged widely from 28,806 to 44,135 base pairs, with intron insertions accounting for most size variation and up to 39% (B. malawiensis) of the mitochondrial length due to introns in genes. Gene order of the common mitochondrial genes did not vary among the Beauveria sequences, but variation was observed in the number of transfer ribonucleic acid genes. Although phylogenetic analysis using whole mitochondrial genomes showed, unsurprisingly, that B. bassiana isolates were the most closely related to each other, mitochondrial codon usage suggested that some B. bassiana isolates were more similar to B. malawiensis and B. caledonica than the other B. bassiana isolates analyzed.

Production of Diverse Beauveriolide Analogs in Closely Related Fungi: a Rare Case of Fungal Chemodiversity.

Fungal chemodiversity is well known in part due to the production of diverse analogous compounds by a single biosynthetic gene cluster (BGC). Usually, similar or the same metabolites are produced by closely related fungal species under a given condition, the foundation of fungal chemotaxonomy. Here, we report a rare case of the production of the cyclodepsipeptide beauveriolides (BVDs) in three insect-pathogenic fungi. We found that the more closely related fungi Beauveria bassiana and Beauveria brongniartii produced structurally distinct analogs of BVDs, whereas the less-close relatives B. brongniartii and Cordyceps militaris biosynthesized structurally similar congeners under the same growth condition. It was verified that a conserved BGC containing four genes is responsible for BVD biosynthesis in three fungi, including a polyketide synthase (PKS) for the production of 3-hydroxy fatty acids (FAs) with chain length variations. In contrast to BVD production patterns, phylogenetic analysis of the BGC enzymes or enzyme domains largely resulted in the congruence relationship with fungal speciation. Feeding assays demonstrated that an FA with a chain length of eight carbon atoms was preferentially utilized, whereas an FA with a chain longer than 10 carbon atoms could not be used as a substrate for BVD biosynthesis. Insect survival assays suggested that the contribution of BVDs to fungal virulence might be associated with the susceptibility of insect species. The results of this study enrich the knowledge of fungal secondary metabolic diversity that can question the reliability of fungal chemotaxonomy.IMPORTANCE Fungal chemotaxonomy is an approach to classify fungi based on the fungal production profile of metabolites, especially the secondary metabolites. We found an atypical example that could question the reliability of fungal chemical classifications in this study, i.e., the more closely related entomopathogenic species Beauveria bassiana and Beauveria brongniartii produced structurally different congeners of the cyclodepsipeptide beauveriolides, whereas the rather divergent species B. brongniartii and Cordyceps militaris biosynthesized similar analogs under the same growth condition. The conserved biosynthetic gene cluster (BGC) containing four genes present in each species is responsible for beauveriolide production. In contrast to the compound formation profiles, the phylogenies of biosynthetic enzymes or enzymatic domains show associations with fungal speciation. Dependent on the insect species, production of beauveriolides may contribute to fungal virulence against the susceptible insect hosts. The findings in this study augment the diversity of fungal secondary metabolisms.

Morphology, molecular characterization, and virulence of Beauveria pseudobassiana isolated from different hosts.

Beauveria pseudobassiana has great potential for use in the management of various insect pests. In the present study, we aimed to explore the the virulence of B. pseudobassiana isolated from a diversity of hosts to Bombyx mori and Tenebrio molitor larvae. To this end, 15B. pseudobassiana isolates from 10 different geographical locations were identified based on morphological characteristics and molecular data. The phylogenetic positions of the isolates were evaluated according to morphological features and phylogenetic inferences based on six loci (nrSSU, nrLSU, TEF, RPB1, RPB2 and Bloc). In addition to growth in soil, the B. pseudobassiana isolates in our study were isolated from a wide host range that extended to 5 orders, 11 families, and 14 species. Moreover, anamorphically typified B. pseudobassiana was grown for the first time from teleomorph stromata. Pathogenicity of the B. pseudobassiana isolates from the different hosts was determined with two bioassays using B. mori and T. molitor larvae. The results indicated that mortality of B. mori caused by the lepidopteran isolates was significantly higher than that of isolates from other hosts, and virulence of the coleopteran isolates to T. molitor was significantly higher than that of isolates from other hosts. The host specificity of B. pseudobassiana should be studied in more detail before future consideration of isolates for use in biological control of pests.

Assessment of the diversity of Brazilian entomopathogenic fungi in the genus Beauveria.

We combined matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) along with sequencing of the B locus intergenic region (Bloc) to assess the diversity of Brazilian species within the anamorphic genus Beauveria. A total of 121 strains maintained in a government-owned culture collection and isolated from a range of hosts/substrates over a long time span (1981-2015) were assessed. Strains were collected in five of six Brazilian biomes, mostly in the Atlantic Forest (42.2%) and Cerrado (29.8%), primarily from insect pests of crops. All strains were subjected to MS, and those not accurately identified by this technique were genomically analyzed. Among the outcomes of this study, four taxa from the genus Beauveria were recognized, with the great majority of strains belonging to B. bassiana s.str. (93.4%), followed by B. caledonica (2.5%), B. pseudobassiana (2.5%) and B. amorpha (1.6%). B. bassiana s.str. was found in all biomes and isolated from a wide range of hosts/substrates. Due to low numbers, associations of the remaining Beauveria species with specific hosts or habitats/biomes were not clear, except that all three B. caledonica strains were found only in the Cerrado biome and were associated with adults of the banana weevil, Cosmopolites sordidus (Col.:Curculionidae). B. pseudobassiana is reported for the first time on the South American continent, in a subtropical region and from two insect orders not yet associated with this taxon. We also showed that some strains previously ascribed to B. brongniartii were misidentifications. The biodiversity of Beauveria analyzed in our study was comparatively low. The geographic origins of strains used in our study were biased towards biomes with intense human interventions. Future surveys on more conserved, less environmentally disturbed biomes, such as Caatinga, Pampa, Pantanal, and Amazon are needed for a more comprehensive picture of the diversity of Beauveria and related genera in Brazil.

Genetic diversity of Beauveria bassiana in semi natural and agricultural habitats and its biocontrol potential against cowpea aphid, Aphis craccivora Koch.

Information on the biology and ecology of Beauveria bassiana in different habitats could provide essential knowledge in their development as biocontrol agents of insect pests. In this study, phylogenetic and genotypic information was used to evaluate the genetic diversity of B. bassiana within semi natural and agricultural habitats in Karnataka State of South India and assessed their extracellular chitinase activity and pathogenicity against cowpea aphid, Aphis craccivora. Multilocus phylogeny and microsatellite genotyping of B. bassiana conjointly resolved three phylogenetic species, Bb_1, Bb_2, and Bb_3, in semi natural and agricultural habitats. None of the three phylogenetic species of B. bassiana were associated with crop plants in agroecosystem or insect hosts in semi natural habitat. All the three phylogenetic species were detected with four genotypes each. All isolates of B. bassiana were pathogenic to A. craccivora in greenhouse bioassays. Isolate GKVK 01_13 caused a significantly high mortality of aphids and detected with an increased level of chitinase activity. The study results suggest that application of indigenous virulent strain of B. bassiana could provide effective control of native insect pest A. craccivora.

Occurrence and diversity of entomopathogenic fungi (Beauveria spp. and Metarhizium spp.) in Australian vineyard soils.

Entomopathogenic Ascomycetes: Hypocreales fungi occur worldwide in the soil; however, the abundance and distribution of these fungi in a vineyard environment is unknown. A survey of Australian vineyards was carried out in order to isolate and identify entomopathogenic fungi. A total of 240 soil samples were taken from eight vineyards in two states (New South Wales and Victoria). Insect baiting (using Tenebrio molitor) and soil dilution methods were used to isolate Beauveria spp. and Metarhizium spp. from all soil samples. Of the 240 soil samples, 60% contained either Beauveria spp. (26%) or Metarhizium spp. (33%). Species of Beauveria and Metarhizium were identified by sequencing the B locus nuclear intergenic region (Bloc) and elongation factor-1 alpha (EFT1) regions, respectively. Three Beauveria species (B. bassiana, B. australis and B. pseudobassiana) and six Metarhizium species (M. guizhouense, M. robertsii, M. brunneum, M. flavoviride var. pemphigi, M. pingshaense and M. majus) were identified. A new sister clade made up of six isolates was identified within B. australis. Two potentially new phylogenetic species (six isolates each) were found within the B. bassiana clade. This study revealed a diverse community of entomopathogenic fungi in sampled Australian vineyard soils.

High genetic diversity of the entomopathogenic fungus Beauveria bassiana in Colima, Mexico.

The entomopathogenic fungus Beauveria bassiana is used widely as a biological control agent against a wide range of insect pests globally. In this study, 44 Beauveria isolates from the state of Colima, Mexico harbored in the "Colección de Hongos Entomopatógenos" of the "Centro Nacional de Referencia de Control Biológico" and from different substrates, insect-hosts, and localities were characterized with molecular markers. All isolates were identified using a Bayesian phylogenetic analysis of translation elongation factor 1-α (TEF) and nuclear intergenic Bloc region. Forty-three isolates were identified as B. bassiana and grouped into two sub-clades, i.e., AFNEO_1 (n = 22; previously defined as a clade with African and Neotropical origin) and Bb clade (n = 21; closely associated with ex-type strain ARSEF 1564), and one isolate was identified as B. pseudobassiana. The fixation index (FST = 0.493) established the genetic differentiation between AFNEO_1 and Bb clades. High genotype richness and genetic diversity of AFNEO_1 and Bb clades were revealed in sequence analysis of Bloc region and SSR genotyping. Moreover, the AFNEO_1 and Bb clades were confirmed as two independent clonally structured assemblages. Finally, the AMOVA detected no significant association between any combination of substrate, insect-host or geographical origin. High genetic variation of B. bassiana in Colima, Mexico could suggest a functional diversity among isolates that may include those effective against a specific insect pest.

Thermotolerant isolates of Beauveria bassiana as potential control agent of insect pest in subtropical climates.

The use of Beauveria bassiana in biological control of agricultural pests is mainly hampered by environmental factors, such as elevated temperatures and low humidity. These limitations, further amplified in a global warming scenario, could nullify biological control strategies based on this fungus. The identification of thermotolerant B. bassiana isolates represents a possible strategy to overcome this problem. In this study, in order to maximize the probability in the isolation of thermotolerant B. bassiana, soil samples and infected insects were collected in warm areas of Syria. The obtained fungal isolates were tested for different biological parameters (i.e., growth rate, sporulation and spore germination) at growing temperatures ranging from 20°C to 35°C. Among these isolates (eight from insects and 11 from soil samples), the five with the highest growth rate, spore production and germination at 30°C were tested for their entomopathogenicity through in vivo assays on Ephestia kuehniella larvae. Insect mortality induced by the five isolates ranged from 31% to 100%. Two isolates, one from Phyllognathus excavatus and one from soil, caused 50% of the larval mortality in less than four days, reaching values exceeding 92% in ten days. These two isolates were molecularly identified as B. bassiana sensu stricto by using three markers (i.e., ITS, Bloc and EF1-α). Considering these promising results, further studies are ongoing, testing their efficiency in field conditions as control agents for agricultural insect pests in Mediterranean and Subtropical regions.

Identification of hypocrealean reptile pathogenic isolates with MALDI-TOF MS.

Biotyper analysis of Nannizziopsis guarroi, a fatal fungal pathogen in lizards, was described recently. Hypocrealean fungal infections in captive reptiles appear with an increasing frequency during the last decade. Therefore, the aim of this study was to proof Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) as diagnostic tool for the identification of reptile pathogenic hypocrealean fungi. Ten fungal isolates obtained from nine reptiles with fungal glossitis, disseminated visceral mycosis, pneumomycosis, and fungal keratitis were analyzed. Phylogeny consisted of fragments of the large subunit of nuclear encoded ribosomal DNA (D1/D2, LSU) and the internal transcribed spacer region 1 of nuclear encoded ribosomal DNA (ITS1) as well as the protein coding gene translation elongation factor 1 alpha (TEF). Results revealed unanimously two Metarhizium granulomatis genotypes in a total of three isolates, various M. viride genotypes (n = 3), two different Purpureocillium lilacinum isolates as well as one isolate of each P. lavendulum and Beauveria bassiana. Purpureocillium lilacinum and B. bassiana are likewise frequently employed as a mycoinsecticide and mycoacaricide in agriculture on a worldwide scale and have occasionally been reported in man, causing fungal keratitis, sclerokeratitis, nosocomial infections in immunosuppressed patients, as well as cavitary pulmonary disease and cutaneous hyalohyphomycosis in immunocompetent patients. According to the results establishment of Biotyper analysis for faster differentiation of reptile-associated fungal pathogens is entirely justified.

Phylogenetic relationships and effectiveness of four Beauveria bassiana sensu lato strains for control of Haemaphysalis longicornis (Acari: Ixodidae).

Haemaphysalis longicornis (Acari: Ixodidae) is an important vector tick that is widely distributed around the world. In many regions, this tick acts as vector of a wide range of pathogens to humans and animals, and its control is mainly based on the use of chemical pesticides. However, the occurrence of some adverse effects, such as tick resistance to pesticides and food and environmental contamination, are driving the need to develop more effective and environmentally sound approaches to control and prevent ticks. As an alternative control strategy, entomopathogenic fungi have been extensively used for the control of pests and cause high mortality in various ticks. In this study, we identified four isolates of Beauveria bassiana sensu lato from insects and investigated their pathogenicity against different developmental stages of H. longicornis (eggs, unfed larvae, unfed nymphs and engorged females). Phylogenetic analysis demonstrated that the four isolates of B. bassiana clustered into two clades. Four isolates showed different acaricidal qualities: the isolate from Cerambycidae (EF3) exhibited the highest pathogenicity to all developmental tick stages tested. High doses (1 × 107 conidia/ml) of the clade I fungi collected from Cryptotympana atrata fabricus (Cicadidae) (EF1), Cimicidae (EF2), and Boettcherisca peregrine (Sarcophagidae) (EF4) also showed virulence against H. longicornis, with high doses of the fungi application causing higher mortality than control group. Altogether, this study demonstrated that all four isolates of B. bassiana showed high virulence toward different developmental stages of H. longicornis, and therefore, they can be of potential use as biological control agents of ticks.

Identification and evaluation of a new entomopathogenic fungal strain against Riptortus pedestris (Hemiptera: Alydidae) and its two egg parasitoids.

A strain (ARP14) of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin was isolated from field-collected Riptortus pedestris (Fabricius) (Hemiptera: Alydidae). The lethal median concentration of the ARP14 strain was compared with that of a commercialized strain (GHA) of the same fungus against R. pedestris and its two egg parasitoids, Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) and Gryon japonicum (Ashmead) (Hymenoptera: Platygastridae). Mortality and mycosis rates were evaluated after exposure to five concentrations of the fungus, i.e., 1×109, 1×108, 1×107, 1×106, and 1×105 conidia/mL, using a glass scintillation vial as an exposure arena in 25.0 ± 0.5°C and 93.7 ± 2.9% RH. The lethal median concentrations (LC50) for 2nd and 4th instar nymphs, and adults of R. pedestris were not significantly different between the two strains of B. bassiana. However, the mycosis rate of ARP14 was 1.3 and 1.8 times higher than that of the GHA strain in 4th instar nymphs and adult females of R. pedestris, respectively, at the 1×108 conidia/mL concentration. More interestingly, the mycosis rates at 1×108 conidia/mL concentration in the parasitoids G. japonicum and O. nezarae were much lower in the ARP14 strain (15.0 and 0%) than in the GHA strain (73.3 and 66.0%), respectively, suggesting that the B. bassiana strain ARP14 is less virulent to these parasitoids than the commercially available strain. Our results suggest that B. bassiana ARP14 may be a potential new biopesticide against R. pedestris with fewer negative effects on beneficial parasitoids than currently available options.

Phylogenetic Relationships and Acaricidal Effects of Beauveria bassiana Obtained from Cattle Farm Soils Against Rhipicephalus microplus.

The objectives of the present study were to isolate Beauveria bassiana strains from cattle farm soils, analyze the phylogenetic relationships among the fungal strains isolated from these soils, and determine the acaricidal effect of B. bassiana isolates on engorged Rhipicephalus microplus tick strains resistant or susceptible to chemical acaricides. Six strains of B. bassiana were obtained and isolated from cattle farm soils in the Mexican tropics using the Galleria bait method, and their acaricidal effect was assessed against 2 populations of R. microplus ("Media Joya" chemical acaricide-resistant strain or "CLAR" chemical acaricide-susceptible strain) using the adult immersion test. The BbV03 strain produced 86.7% and 60% mortality in resistant and susceptible ticks on day 20, respectively, whereas the BbV04 strain produced 66.7% and 53.5% mortality in resistant and susceptible ticks on day 20, respectively. The BbV03 and BbV04 strains reduced egg laying on both R. microplus populations. There was no statistical difference in the acaricidal effect of B. bassiana strains among chemical acaricide-susceptible or -resistant R. microplus populations ( P > 0.05). The BbV03 strain was the most virulent against R. microplus with an LC50 of 2 × 107 and LC99 of 7 × 108 conidia/ml. We found that the 6 B. bassiana isolated clustered in the same clade with other previously reported B. bassiana strains (from GenBank) but were separated into 3 different sub-clades. This study shows that some B. bassiana strains are a promising coadjuvant alternative for biological tick control, including tick populations that are resistant to chemical acaricides. Beauveria bassiana is present in the pastures of tropic cattle farms, and there are genetic variations between B. bassiana strains living in this ecosystem that might play an important role in the natural control of R. microplus in cattle farm paddocks.

Elevational distribution and morphological attributes of the entomopathogenic fungi from forests of the Qinling Mountains in China.

Entomopathogenic fungi are considered to be a safe microbiological pesticide alternative to chemical control. Efforts are underway to understand precisely their taxonomy and natural distribution through mycological and biodiversity studies based on molecular markers. Here, we present descriptions of the diversity of the entomopathogenic fungi in the genera Metarhizium and Beauveria found along the elevational gradients of the Qinling subtropical and temperate forests of Shaanxi province in China, using morphological aspects and molecular markers. Molecular characterization using the Mz_IGS3 intergenic region revealed that Metarhizium isolates phylogenetically clustered in the PARB clade with four different distinguishable species, but the 5'-TEF gene allowed only ambiguous delimitation of Metarhizium species. Beauveria isolates were characterized by sequence analyses of the translation elongation factor 1-α and the Bloc region. The richness of Metarhizium species decreased with increasing elevation, with Metarhizium robertsii s.l. being the most abundant species along the elevational gradient. Our bioassay suggests that certain species of Metarhizium are significantly pathogenic to the insect model Tenebrio molitor at both the adult and larvae stages and could potentially serve as a control of insect pests of forests.

The Stress-Responsive and Host-Oriented Role of Nonribosomal Peptide Synthetases in an Entomopathogenic Fungus, Beauveria bassiana.

Beauveria bassiana infects a number of pest species and is known to produce insecticidal substances, such as the nonribosomal peptides (NRPs) beauvericin and bassianolide. However, most NRPs and their biological roles in B. bassiana remain undiscovered. To identify NRPs that potentially contribute to pathogenesis, the 21 predicted NRP synthetases (NRPSs) or NRPS-like proteins of B. bassiana ARSEF 2860 were primarily ranked into three functional groups: basic metabolism (7 NRPSs), pathogenicity (12 NRPSs), and unknown function (2 NRPSs). Based on the transcript levels during in vivo growth on diamondback moth (Plutella xylostella (Linnaeus)), half of the Group II NRPSs were likely to be involved in infection. Given that the metabolites biosynthesized by these NRPSs remain to be determined, our result underlines the importance of the NRPSome in fungal pathogenesis, and will serve as a guide for future genomic mining projects to discover functionally essential and structurally diverse NRPs in fungal genomes.

Comparative genomics of Beauveria bassiana: uncovering signatures of virulence against mosquitoes.

BACKGROUND: Entomopathogenic fungi such as Beauveria bassiana are promising biological agents for control of malaria mosquitoes. Indeed, infection with B. bassiana reduces the lifespan of mosquitoes in the laboratory and in the field. Natural isolates of B. bassiana show up to 10-fold differences in virulence between the most and the least virulent isolate. In this study, we sequenced the genomes of five isolates representing the extremes of low/high virulence and three RNA libraries, and applied a genome comparison approach to uncover genetic mechanisms underpinning virulence. RESULTS: A high-quality, near-complete genome assembly was achieved for the highly virulent isolate Bb8028, which was compared to the assemblies of the four other isolates. Whole genome analysis showed a high level of genetic diversity between the five isolates (2.85-16.8 SNPs/kb), which grouped into two distinct phylogenetic clusters. Mating type gene analysis revealed the presence of either the MAT1-1-1 or the MAT1-2-1 gene. Moreover, a putative new MAT gene (MAT1-2-8) was detected in the MAT1-2 locus. Comparative genome analysis revealed that Bb8028 contains 163 genes exclusive for this isolate. These unique genes have a tendency to cluster in the genome and to be often located near the telomeres. Among the genes unique to Bb8028 are a Non-Ribosomal Peptide Synthetase (NRPS) secondary metabolite gene cluster, a polyketide synthase (PKS) gene, and five genes with homology to bacterial toxins. A survey of candidate virulence genes for B. bassiana is presented. CONCLUSIONS: Our results indicate several genes and molecular processes that may underpin virulence towards mosquitoes. Thus, the genome sequences of five isolates of B. bassiana provide a better understanding of the natural variation in virulence and will offer a major resource for future research on this important biological control agent.

Fungal Keratitis Due to Beauveria bassiana in a Contact Lenses Wearer and Review of Published Reports.

Fungal keratitis is a severe ocular infection that primarily affects subjects engaged in outdoor activities. Risk factors include allergic conjunctivitis, previous eye surgery, previous treatment with wide-spectrum antimicrobial agents and corticosteroids and using contact lenses. Corneal infection is usually secondary to trauma involving organic material, which is often the only predisposing factor. Early diagnosis based on clinical examination and microbiological investigation (microscopy, cultures and molecular techniques) is crucial to selecting the appropriate antifungal therapy and prevent progression. We report the case of a patient with keratitis due to Beauveria bassiana, an opportunistic and entomopathogenic filamentous fungus that is used as a biological insecticide and which is a rare cause of corneal infection. We review previous cases reports of B. bassiana keratitis published and its main features to compare with our case, a female occasional agriculture worker who had not suffered any trauma involving organic material. The patient received topical and oral antifungal therapy and debridement surgery, with a satisfactory outcome.

Development of a method for detection and quantification of B. brongniartii and B. bassiana in soil.

A culture independent method based on qPCR was developed for the detection and quantification of two fungal inoculants in soil. The aim was to adapt a genotyping approach based on SSR (Simple Sequence Repeat) marker to a discriminating tracing of two different species of bioinoculants in soil, after their in-field release. Two entomopathogenic fungi, Beauveria bassiana and B. brongniartii, were traced and quantified in soil samples obtained from field trials. These two fungal species were used as biological agents in Poland to control Melolontha melolontha (European cockchafer), whose larvae live in soil menacing horticultural crops. Specificity of SSR markers was verified using controls consisting of: i) soil samples containing fungal spores of B. bassiana and B. brongniartii in known dilutions; ii) the DNA of the fungal microorganisms; iii) soil samples singly inoculated with each fungus species. An initial evaluation of the protocol was performed with analyses of soil DNA and mycelial DNA. Further, the simultaneous detection and quantification of B. bassiana and B. brongniartii in soil was achieved in field samples after application of the bio-inoculants. The protocol can be considered as a relatively low cost solution for the detection, identification and traceability of fungal bio-inoculants in soil.