Tools to make Stachybotrys chartarum genetically amendable: Key to unlocking cryptic biosynthetic gene clusters.

The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.

Detection of Stachybotrys chartarum isolates from faba beans dust during threshing.

Stachybotrys (S.) chartarum had been related to dangerous health problems in animals and humans that take place when exposure to S. chartarum toxins. S. chartarum had been isolated from various substrates, ranging from inappropriately stored feed and culinary herbs to damp buildings. To evaluate the pathogenic potential of isolates, it is essential to identify them with different methods. The occurrence and genetic diversity of S. chartarum isolates from faba beans dust during threshing in Upper Egypt were investigated. Low counts of Stachybotrys were found (six isolates) and identified morphologically by single-spore isolation and molecularly by the amplification of the specific internal transcribed spacer (ITS) region and glyceraldehydes-3-phosphate dehydrogenase (gpd). The genetic diversity of the collected isolates was studied by specific genes random primer polymerase chain reaction (SGRP-PCR). The phylogenetic analysis of S. chartarum showed that the specific primers IT51 and StacR3 used by commercial laboratories for detecting S. chartarum were not able to differentiate species of S. chartarum from S. chlorohalonata and unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of SGRP fragments confirmed this result. The six isolates of S. chartarum were analyzed for the presence of trichodiene synthase (Tri5) gene, which needed in the early stage of the trichothecene synthesis path. All the tested isolates were positive for the Tri5 gene. Further study on the taxonomic status of the epithet S. chartarum is necessary and presence of sub species to S. chartarum might be acceptable depending on the variations of morphological characteristics which were confirmed by molecular techniques.

Transcriptional analysis of the lichenase-like gene cel12A of the filamentous fungus Stachybotrys atra BP-A and its relevance for lignocellulose depolymerization.

To rationally optimize the production of industrial enzymes by molecular means requires previous knowledge of the regulatory circuits controlling the expression of the corresponding genes. The genus Stachybotrys is an outstanding producer of cellulose-degrading enzymes. Previous studies isolated and characterized the lichenase-like/non-typical cellulase Cel12A of S. atra (AKA S. chartarum) belonging to glycosyl hydrolase family 12 (GH12). In this study, we used RT-qPCR to determine the pattern of expression of cel12A under different carbon sources and initial ambient pH. Among the carbon sources examined, rice straw triggered a greater increase in the expression of cel12A than 1% lactose or 0.1% glucose, indicating specific induction by rice straw. In contrast, cel12A was repressed in the presence of glucose even when combined with this inducer. The proximity of 2 adjacent 5'-CTGGGGTCTGGGG-3' CreA consensus target sites to the translational start site of cel12A strongly suggests that the carbon catabolite repression observed is directly mediated by CreA. Ambient pH did not have a significant effect on cel12A expression. These findings present new knowledge on transcriptional regulatory networks in Stachybotrys associated with cellulose/hemicellulose depolymerization. Rational engineering of CreA to remove CCR could constitute a novel strategy for improving the production of Cel12A.

Rapid and selective detection of macrocyclic trichothecene producing Stachybotrys chartarum strains by loop-mediated isothermal amplification (LAMP).

Cytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay's detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing.

Comparative Mitogenomics of Fungal Species in Stachybotryaceae Provides Evolutionary Insights into Hypocreales.

Stachybotrys chartarum is one of the world's ten most feared fungi within the family Stachybotryaceae, although to date, not a single mitogenome has been documented for Stachybotryaceae. Herein, six mitogenomes of four different species in Stachybotryaceae are newly reported. The S. chartarum mitogenome was 30.7 kb in length and contained two introns (one each in rnl and cox1). A comparison of the mitogenomes of three different individuals of S. chartarum showed few nucleotide variations and conservation of gene content/order and intron insertion. A comparison of the mitogenomes of four different Stachybotryaceae species (Memnoniella echinata, Myrothecium inundatum, S. chartarum, and S. chlorohalonata), however, revealed variations in intron insertion, gene order/content, and nad2/nad3 joining pattern. Further investigations on all Hypocreales species with available mitogenomes showed greater variabilities in gene order (six patterns) and nad2/nad3 joining pattern (five patterns) although a dominant pattern always existed in each case. Ancestral state estimation showed that in each case the dominant pattern was always more ancestral than those rare patterns. Phylogenetic analyses based on mitochondrion-encoded genes supported the placement of Stachybotryaceae in Hypocreales. The crown age of Stachybotryaceae was estimated to be approximately the Early Cretaceous (141-142 Mya). This study greatly promotes our understanding of the evolution of fungal species in Hypocreales.

FIP-sch2, a new fungal immunomodulatory protein from Stachybotrys chlorohalonata, suppresses proliferation and migration in lung cancer cells.

Fungal immunomodulatory protein (FIP)-sch2, an immunomodulatory protein identified in the ascomycete Stachybotrys chlorohalonata by a sequence similarity search, is a novel member of the FIP family. FIP-sch2 shares high sequence identity, structure, and evolutionary conservation with previously reported FIPs. It was satisfactorily expressed in Escherichia coli with a glutathione S-transferase (GST) tag and purified by GST-affinity magnetic beads. To characterize the direct antitumor effects, human lung adenocarcinoma A549 cells were treated with different concentrations of recombinant FIP (rFIP)-sch2 in vitro, and the results showed that rFIP-sch2 could reduce cell viability dose-dependently with a half-maximal inhibitory concentration (IC50) of 9.48 µg/mL. Furthermore, rFIP-sch2 at 8 µg/mL could significantly induce apoptosis and interrupt migration in A549 cells. Notably, the antitumor effect of rFIP-sch2 was equivalent to that of rLZ-8 but was obviously increased compared to rFIP-fve. In addition, the exploration of the antitumor mechanism suggested that rFIP-sch2 induced lung cancer cell death by activating apoptosis and inhibiting migration. Our results indicated that rFIP-sch2 was a promising candidate for use in future cancer therapy.

Fungal DNA in dust in Swedish day care centres: associations with respiratory symptoms, fractional exhaled nitrogen oxide (FeNO) and C-reactive protein (CRP) in serum among day care centre staff.

PURPOSE: To study associations between fungal DNA in day care centres, fractional exhaled nitric oxide (FeNO) and inflammatory markers in day care centre staff. METHODS: Totally, 62 staff (90 %) from five day care centres in Sweden participated. All were females. Settled dust was collected and analysed for five sequences of fungal DNA by quantitative PCR. Levels of FeNO (NIOX MINO 50 ml/min) and serum levels of eosinophilic cationic protein, myeloperoxidase (MPO) and high-sensitivity C-reactive protein in blood (HsCRP) were measured. Dynamic spirometry was performed, and dyspnoea was measured. Biomarkers and dyspnoea ratings were log-transformed, and associations were analysed by linear mixed models, adjusting for age, atopy, smoking, body mass index (BMI), ETS and dampness/mould at home. RESULTS: Geometric mean (GM) for FeNO was 15.3 ppb, 6% were smokers, 14% were obese, 31% were overweight and 18% had atopy. GM concentration was 2.16 × 10(5) cell equivalents (CE)/g for total fungal DNA, 2310 CE/g for Aspergillus/penicillium (Asp/Pen) DNA, 17 CE/g for Aspergillus versicolor DNA and 14 CE/g dust for Streptomyces DNA. FeNO was associated with total fungal DNA (p = 0.004), Asp/Pen DNA (p = 0.005) and Streptomyces DNA (p = 0.03). HsCRP was associated with total fungal DNA (p = 0.03) and BMI (p = 0.001). Dyspnoea was associated with Asp/Pen DNA (p = 0.04). Subjects with ETS at home had lower lung function (FEV1) (p = 0.03), and those with dampness/mould at home had lower MPO (p = 0.03). CONCLUSION: Fungal contamination in day care centres, measured as fungal DNA, can be a risk factor for airway inflammation, and CRP is associated with BMI.

Recombinant expression of a GH12 ß-glucanase carrying its own signal peptide from Stachybotrys atra in yeast and filamentous fungi.

The ß-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 ß-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete.

[Expression in Aspergillus niger and characterization of ß-mannanases from Stachybotrys chartarum].

Objective: Using Aspergillus niger as host to express ß-mannanases from Stachybotrys chartarum. Methods: Through sequence analysis of Stachybotrys chartarum genome, two ß-mannanase genes (s16942 and s331) were identified. The primers were designed based on the DNA sequence and the ß-mannanase genes (s16942 and s331) were obtained, and then inserted to the vector pGm. The expression plasmids were transferred into Aspergillus niger. ß-mannanase producing strains (G1-pGm-s16942 and G1-pGm-s331) were isolated after screening several transformants using amdS selection plates and confirmed by PCR fragment sequencing. Results: The molecular weight of the enzymes from G1-pGm-s16942 and G1-pGm-s331 were about 48 kDa and 60 kDa respectively by SDS-PAGE gel analysis, and the recombinant proteins did not present in the negative control. Assays of enzymatic property using the crude enzyme preparations indicated that the enzyme from G1-pGm-s16942 exhibited maximum activity (521 U/mL) under the optimum. Conclusion: This was the first study of the heterologous expression of the ß-mannanase genes from Stachybotrys chartarum in Aspergillus niger host and the ß-mannanase genes could be expressed successfully with high activities and protein titers.

Comparative genome sequencing reveals chemotype-specific gene clusters in the toxigenic black mold Stachybotrys.

BACKGROUND: The fungal genus Stachybotrys produces several diverse toxins that affect human health. Its strains comprise two mutually-exclusive toxin chemotypes, one producing satratoxins, which are a subclass of trichothecenes, and the other producing the less-toxic atranones. To determine the genetic basis for chemotype-specific differences in toxin production, the genomes of four Stachybotrys strains were sequenced and assembled de novo. Two of these strains produce atranones and two produce satratoxins. RESULTS: Comparative analysis of these four 35-Mbp genomes revealed several chemotype-specific gene clusters that are predicted to make secondary metabolites. The largest, which was named the core atranone cluster, encodes 14 proteins that may suffice to produce all observed atranone compounds via reactions that include an unusual Baeyer-Villiger oxidation. Satratoxins are suggested to be made by products of multiple gene clusters that encode 21 proteins in all, including polyketide synthases, acetyltransferases, and other enzymes expected to modify the trichothecene skeleton. One such satratoxin chemotype-specific cluster is adjacent to the core trichothecene cluster, which has diverged from those of other trichothecene producers to contain a unique polyketide synthase. CONCLUSIONS: The results suggest that chemotype-specific gene clusters are likely the genetic basis for the mutually-exclusive toxin chemotypes of Stachybotrys. A unified biochemical model for Stachybotrys toxin production is presented. Overall, the four genomes described here will be useful for ongoing studies of this mold's diverse toxicity mechanisms.

Characterization and transcriptional regulation of Stachybotrys elegans mitogen-activated-protein kinase gene smkA following mycoparasitism and starvation conditions.

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in the development and conidiation of fungal pathogens on their hosts and the sensing of host-derived cues. Mycoparasitism is a fungus-fungus interaction comprising host-pathogen cross talk. Until now, only little information is available on the role of the MAPK signaling pathway during this interaction. Here, we report on the differential expression of a MAPK/ERK gene in the mycoparasite Stachybotrys elegans in response to direct parasitism of different vegetative structures of the plant pathogen Rhizoctonia solani (i.e., carbon-rich condition) and to nutrient starvation (i.e., carbon-poor condition). Western blot analysis against ERK1/2 highlighted an increase in their phosphorylated forms when S. elegans was grown under starvation condition compared to that detected in response to mycoparasitism. A higher abundance of phosphorylated ERK1/2 at the third day of interaction compared to that estimated under starvation condition was detected applying LC-MS/MS. At the transcriptional level, smkA, a YERK1 class member, was significantly induced in response to hyphal parasitism compared to parasitized sclerotia at 3, 4, and 5 days of interaction. However, under starvation condition, smkA levels were significantly induced after 7 days of growth. Southern blot analysis revealed that smkA is member of a small gene family. Collectively, these results suggest that smkA could be implicated in the mycoparasitic process in S. elegans as well as in stress-activated pathways. These results may be of wider significance in other fungus-fungus interactions.

Large-scale analysis of the genome of the rare alkaline-halophilic Stachybotrys microspora reveals 46 cellulase genes.

Fungi are of great importance in biotechnology, for example in the production of enzymes and metabolites. The main goal of this study was to obtain a high-coverage draft of the Stachybotrys microspora genome and to annotate and analyze the genome sequence data. The rare fungus S. microspora N1 strain is distinguished by its ability to grow in an alkaline halophilic environment and to efficiently secrete cellulolytic enzymes. Here we report the draft genome sequence composed of 3715 contigs, a genome size of 35 343 854 bp, with a GC content of 53.31% and a coverage around 20.5×. The identification of cellulolytic genes and of their corresponding functions was carried out through analysis and annotation of the whole genome sequence. Forty-six cellulases were identified using the fungicompanion bioinformatic tool. Interestingly, an S. microspora endoglucanase selected from those with a low isoelectric point was predicted to have a halophilic profile and share significant homology with a well-known bacterial halophilic cellulase. These results confirm previous biochemical studies revealing a halophilic character, which is a very rare feature among fungal cellulases. All these properties suggest that cellulases of S. microspora may have potential for use in the biofuel, textile, and detergent industries.

Production of Satratoxin G and H Is Tightly Linked to Sporulation in Stachybotrys chartarum.

Stachybotrys chartarum is a toxigenic fungus that is frequently isolated from damp building materials or improperly stored forage. Macrocyclic trichothecenes and in particular satratoxins are the most potent mycotoxins known to be produced by this fungus. Exposure of humans or animals to these secondary metabolites can be associated with severe health problems. To assess the pathogenic potential of S. chartarum isolates, it is essential to cultivate them under conditions that reliably promote toxin production. Potato dextrose agar (PDA) was reported to be the optimal nutrition medium for satratoxin production. In this study, the growth of S. chartarum genotype S strains on PDA from two manufacturers led to divergent results, namely, well-grown and sporulating cultures with high satratoxin concentrations (20.8 ± 0.4 µg/cm2) versus cultures with sparse sporulation and low satratoxin production (0.3 ± 0.1 µg/cm2). This finding is important for any attempt to identify toxigenic S. chartarum isolates. Further experiments performed with the two media provided strong evidence for a link between satratoxin production and sporulation. A comparison of three-point and one-point cultures grown on the two types of PDA, furthermore, demonstrated an inter-colony communication that influences both sporulation and mycotoxin production of S. chartarum genotype S strains.

Characterization of human antigenic proteins SchS21 and SchS34 from Stachybotrys chartarum.

BACKGROUND: SchS21 and SchS34 are proteins from Stachybotrys chartarum sensu latto that are antigenic in goats, mice and humans. Monoclonal antibodies to these proteins react with spores of S. chartarum and S. chlorohalonata but do not cross-react with a diverse taxonomic and ecological array of other fungi. METHODS: Based on partial sequences of the 21- and 34-kDa proteins, obtained from tandem mass spectra and Edman degradation, degenerate primers were designed for touchdown PCR and the resulting amplicons were sequenced. Subsequently, inverse-PCR was used to obtain genomic DNA sequences encoding SchS21 and SchS34. RT-PCR products were sequenced to predict the mature protein sequences of SchS21 and SchS34. Based on the speculation that SchS21 protein was a DNase, the enzymatic properties were investigated. RESULTS: Sequences of 435 and 666 bp in length were obtained from SchS21 and SchS34 cDNAs. The SchS21 open reading frame encodes a mature protein of 144 amino acids, while that of SchS34 is 221 amino acids in length. SchS21 is a secretory, alkaline, Mg-dependent exodeoxyribonuclease, while SchS34 is a secretory protein of unknown function. His-tagged forms of the mature SchS21 and SchS34 proteins were separately overexpressed in Escherichia coli and purified using Ni-NTA columns (0.5 mg/l yield). CONCLUSIONS: Based on Western blots, the expressed proteins were similar in molecular weight and bound to the respective monoclonal antibodies to SchS21 and SchS34 proteins from S. chartarum. Interactions with human sera IgE confirmed the expressed forms of SchS21 and SchS34 as naturally occurring allergens.

Expression of genes of Rhizoctonia solani and the biocontrol Stachybotrys elegans during mycoparasitism of hyphae and sclerotia.

Knowledge of mycoparasitism has been focused on how antagonists affect pathogens in relation to mechanisms, metabolites and gene expression. Just as microbial antagonists use a diverse arsenal of mechanisms to dominate interactions with hosts, hosts also have diverse responses to counteract antagonism. In this study differential gene expression of eight mycoparasitism-induced genes and eight host-response genes was monitored during in vivo interactions between the mycoparasite Stachybotrys elegans and hyphae and sclerotia of the host, Rhizoctonia solani over 5 d of interaction. Using real time reverse transcription polymerase chain reaction, comparative analyses demonstrated that hyphal and sclerotial structures triggered different expression patterns. These results indicated that multiple regulatory mechanisms might be involved. The high elevated expression of some genes belonging to the mycoparasite and the host suggest that these genes play an important role during the mycoparasitic process and host defense respectively.

Truncated satratoxin gene clusters in selected isolates of the atranone chemotype of Stachybotrys chartarum (Ehrenb.) S. Hughes.

The fungus Stachybotrys (S.) chartarum was isolated from culinary herbs, damp building materials, and improperly stored animal forage. Two distinct chemotypes of the fungus were described that produced either high-cytotoxic macrocyclic trichothecenes (S type) or low-cytotoxic atranones (A type). Recently, two distinct gene clusters were described that were found to be necessary for the biosynthesis of either macrocyclic trichothecenes (21 SAT (Satratoxin) genes) or atranones (14 ATR (Atranone) genes). In the current study, PCR primers were designed to detect SAT and ATR genes in 19 S. chartarum chemotype S and eight S. chartarum chemotype A strains. Our analysis revealed the existence of three different genotypes: satratoxin-producing strains that harbored all SAT genes but lacked the ATR gene cluster (genotype S), non-satratoxin-producing strains that possessed the ATR genes but lacked SAT genes (genotype A), and a hitherto undescribed hybrid genotype among non-satratoxin-producing strains that harbored all ATR genes and an incomplete set of SAT genes (genotype H). In order to improve the discrimination of genotypes, a triplex PCR assay was developed and applied for the analysis of S. chartarum and S. chlorohalonata cultures. The results show that genes for macrocyclic trichothecenes and atranones are not mutually exclusive in S. chartarum. Correlation of the new genotype-based concept with mycotoxin production data shows also that macrocyclic trichothecenes are exclusively produced by S. chartarum genotype S strains.

Humidity governs the wall-inhabiting fungal community composition in a 1600-year tomb of Emperor Yang.

Biodeterioration caused by filamentous fungi is often a threat to the architectural heritage (i.e. tombs and historic sites). To specifically understand the deterioration phenomena caused by microorganisms in tombs and how these are shaped due to various environmental factors, the fungal communities in the coffin chamber of the Chinese emperor Yang (BC 569-618) were investigated at different heights using denaturant gradient gel electrophoresis (DGGE) fingerprinting. The associated environmental conditions, such as humidity, temperature, height and illumination, were also assessed. The results showed that a great diversity of fungal species (Cordyceps, Fusarium, Harpochytrium, Emericellopsis, Volutella, Cladosporium, Stachybotrys, Trichoderma, Cochlonema and two unknown fungal species) was present in emperor Yang's coffin chamber. The predominant species were Stachybotrys, Fusarium, Trichoderma and Cochlonema. Redundancy analysis (RDA) indicated that humidity, temperature, height and illumination were the most significantly related factors shaping the fungal communities. Humidity showed the highest degree of variance description (19.2%) than all other environmental factors, followed by illumination (18.3%) and height (12.8%). Furthermore, fungal richness and diversity indices showed a positive correlation with humidity (p < 0.05). These results help in understanding the fungal community in tombs, promoting the mitigation of deterioration phenomena of such building heritage for the present and future.

A multi-gene phylogeny for Stachybotrys evidences lack of trichodiene synthase (tri5) gene for isolates of one of three intrageneric lineages.

Members of the mitosporic fungal form-genus Stachybotrys, including common indoor contaminants Stachybotrys chartarum, Stachybotrys echinata and Stachybotrys chlorohalonata, are capable of producing potent, protein synthesis-inhibiting, trichothecene mycotoxins. A combined multi-gene approach was used to investigate relationships among species of Stachybotrys against which the presence/absence of the trichothecene biosynthetic pathway gene, trichodiene synthase (tri5), was evaluated. Phylogenetic analyses partitioned species of Stachybotrys into three strongly supported lineages, two of which contained common indoor taxa. No tri5 PCR product was amplified from members of the third clade, which included the only member of the group with a known sexual state, Stachybotrys albipes. Isolates grouped with S. albipes also tested negative for tri5 in Southern analyses. The phylogenetic distribution of tri5 was consistent with known toxin production for the group. For isolates with tri5 product, Bayesian analysis suggested that signal from amino acid determining sites conflicted with the combined phylogeny. Incongruence however, was not supported by either SH-test results or maximum likelihood analyses. Moreover, sites rates analysis showed that tri5 was highly conserved at the amino acid level suggesting that identity at variable sites, among otherwise divergent taxa, might be the result of chance events.

Molecular and bioinformatics analyses reveal two differentially expressed intracellular GH1 ß-glucosidases from the rare alkalophilic fungus Stachybotrys microspora.

The present study reports the isolation and analysis of two novel GH1 ß-glucosidases from the alkalophilic fungus Stachybotrys microspora, using PCR and Nested-PCR. Three major gene fragments were obtained by PCR: the first two are very similar and constitute a novel gene, which was named Smbgl1A, and the third PCR fragment is part of a different gene, named Smbgl1B. The truncated gene sequences were completely filled using the recent partial whole genome sequencing data of S. microspora (data not yet published). Moreover, we investigated the relative effects of glucose in comparison to cellulose rather than evaluate their absolute effects. In fact, RT-PCR analysis showed that while Smbgl1A was expressed when the fungus was grown in the presence of cellulose but not when grown with glucose, Smbgl1B was equally expressed under both conditions. The putative catalytic residues and the conserved glycone binding sites were identified. Zymogram analysis showed the intracellular production of ß-glucosidases in S. microspora. The predicted secondary structure exhibited a classical (ß/α)8 barrel fold, showing that both SmBGL1A and SmBGL1B belong to the GH1 family. Phylogenetic studies showed that SmBGL1A and SmBGL1B belong to the same branch as ß-glucosidases from Stachybotrys chlorohalonata and Stachybotrys chartarum. However, SmBGL1A and SmBGL1B form two distinct clades.

Biological and Chemical Control of Sclerotinia sclerotiorum using Stachybotrys levispora and Its Secondary Metabolite Griseofulvin.

Sclerotinia sclerotiorum is responsible for the white mold of soybeans, and the difficulty to control the disease in Brazil is causing million-dollar damages. Stachybotrys levispora has shown activity against S. sclerotiorum. In our present investigation, we analyzed the chemical basis of this inhibition. Eight compounds were isolated, and using spectroscopic methods, we identified their structures as the known substances 7-dechlorogriseofulvin, 7-dechlorodehydrogriseofulvin, griseofulvin, dehydrogriseofulvin, 3,13-dihydroxy-5,9,11-trimethoxy-1-methylbenzophenone, griseophenone A, 13-hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone, and 12-chloro-13-hydroxy-3,5,9,11-tetramethoxy-1-methylbenzophenone. Griseofulvin inhibited the mycelial growth of S. sclerotiorum at 2 µg mL-1. Thus, the antagonistic effect of S. levispora to S. sclerotiorum may well be due to the presence of griseofulvins. Our results stimulate new work on the biosynthesis of griseofulvins, to locate genes that encode key enzymes in these routes and use them to increase the production of these compounds and thus potentiate the fungicide effect of this fungus. S. levispora represents an agent for biocontrol, and griseofulvin represents a fungicide to S. sclerotiorum.