Atranones and dolabellanes with cardiomyocyte protective activity against cold ischemic injury from a coral-associated fungus Stachybotrys chartarum.

Five undescribed atranones, namely atranones V-Z (1-5), three undescribed dolabellane-type diterpenoids, namely stachatranones D-F (7-9), together with four known congeners (6 and 10-12), were obtained from a coral-associated strain of the toxigenic fungus Stachybotrys chartarum. Their structures were elucidated via extensive spectroscopic analyses, mainly including the HRESIMS and NMR data, single-crystal X-ray diffraction analysis, electronic circular dichroism calculation, and [Mo2(OAc)4] induced circular dichroism spectrum. The cardiomyocyte protective activity assay revealed that compound 9 significantly ameliorated cold ischemic injury at 24 h post cold ischemia (CI) in a dose-dependent manner. Moreover, compound 9 prevented CI induced dephosphorylation of phosphatidylinositol-3-kinase and RAC-α serine/threonine-protein kinase at 12 h post CI in a dose-dependent manner. In this work, the undescribed compound 9 could significantly protect cardiomyocytes against cold ischemic injury, highlighting the promising potential to be designed and developed as a novel cardioprotectant in heart transplant medicine.

Discovery of novel natural cardiomyocyte protectants from a toxigenic fungus Stachybotrys chartarum.

Stachybatranones A-F (1a/1b and 2-6) and three known analogues, namely methylatranones A and B (7 and 8) and atranone B (9), were isolated and identified from a toxigenic fungus Stachybotrys chartarum. Their structures and absolute configurations were elucidated via the extensive spectroscopic data, comparison of the experimental electronic circular dichroism (ECD) data, and single-crystal X-ray diffraction analyses. Structurally, compounds 2-6 belonged to a rare class of C-alkylated dolabellanes, featuring a unique five-membered hemiketal ring and a γ-butyrolactone moiety both fused to an 11-membered carbocyclic system, while compound 1 (1a/1b) represented the first example of a 5-11-6-fused atranone possessing a 2,3-butanediol moiety. The cardiomyocyte protective activity assay revealed that compounds 1-9 ameliorated cold ischemic injury at 24 h post cold ischemia (CI), with compounds 1 and 4 acting in a dose-dependent manner. Moreover, compound 1 prevented cold ischemia induced dephosphorylation of PI3K and AKT acting in a dose-dependent manner. In this study, a new class of natural products were found to protect cardiomyocytes against cold ischemic injury, providing a potential option for the development of novel cardioprotectants in heart transplant medicine.

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.

Phenylspirodrimane with Moderate Reversal Effect of Multidrug Resistance Isolated from the Deep-Sea Fungus Stachybotrys sp. 3A00409.

Two new phenylspirodrimanes, stachybotrins K and L (1 and 2), together with eight known analogues (3-10), were isolated from deep-sea-derived Stachybotrys sp. MCCC 3A00409. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Absolute configurations of new compounds were determined through a comparison of their circular dichroism (CD) spectra with other reported compounds. The possible reversal effects of all compounds were assayed in the resistant cancer cell lines. Stachybotrysin B (8) can reverse multidrug resistance (MDR) in ABCB1-overexpression cells (KBv200, Hela/VCR) at the non-cytotoxic concentration. Doxorubicin accumulation assay and molecular-docking analysis reveal that the mechanism of its reversal MDR effect may be related to the increase in the intracellular concentration of substrate anticancer drugs.

Exploring the catalytic diversity of two short-chain dehydrogenases/reductases from Stachybotrys chartarum.

Short-chain dehydrogenase/reductases (SDRs) belong to the NAD(P)(H)-dependent oxidoreductase superfamily, which have various functions of catalyzing oxidation/reduction reactions and have been generally used as powerful biocatalysts in the production of pharmaceuticals. In this study, ScSDR1 and ScSDR2, two new SDRs have been identified and characterized from Stachybotrys chartarum 3.5365. Substrate scope investigation revealed that both of the enzymes possessed the ability to oxidize ß-OH to ketone specifically, and exhibited substrate promiscuity and high stereo-selectivity for efficiently catalyzing the structurally different prochiral ketones to chiral alcohols. These findings not only suggest that ScSDR1 and ScSDR2 might be potent synthetic tools in drug research and development, but also provide good examples for further engineered enzymes with higher efficiency and stereo-selectivity.

A Chemically Defined Medium That Supports Mycotoxin Production by Stachybotrys chartarum Enabled Analysis of the Impact of Nitrogen and Carbon Sources on the Biosynthesis of Macrocyclic Trichothecenes and Stachybotrylactam.

Stachybotrys chartarum (Hypocreales, Ascomycota) is a toxigenic fungus that is frequently isolated from water-damaged buildings or improperly stored feed. The secondary metabolites formed by this mold have been associated with health problems in humans and animals. Several authors have studied the influence of environmental conditions on the production of mycotoxins, but these studies focused on undefined or complex substrates, such as building materials and media that impeded investigations of the influence of specific nutrients. In this study, a chemically defined cultivation medium was used to investigate the impact of several nitrogen and carbon sources on growth of S. chartarum and its production of macrocyclic trichothecenes (MTs) and stachybotrylactam (STLAC). Increasing concentrations of sodium nitrate were found to positively affect mycelial growth, the level of sporulation, and MT production, while ammonium nitrate and ammonium chloride had an inhibitory effect. Potato starch was the superior and most reliable carbon source tested. Additionally, we observed that the level of sporulation was correlated with the production of MTs but not with that of STLAC. In this study, we provide a chemically well-defined cultivation medium suitable for standardized in vitro testing of the capacity of S. chartarum isolates to produce macrocyclic trichothecenes. IMPORTANCE Macrocyclic trichothecenes (MTs) are highly toxic secondary metabolites that are produced by certain Stachybotrys chartarum strains, which consequently pose a risk for animals and humans. To identify hazardous, toxin-producing strains by analytical means, it is important to grow them under conditions that support MT production. Nutrients determine growth and development and thus the synthesis of secondary metabolites. Complex rich media are commonly used for diagnostics, but batch differences of supplements pose a risk for inconsistent data. We have established a chemically defined medium for S. chartarum and used it to analyze the impact of nitrogen and carbon sources. A key finding is that nitrate stimulates MT production, whereas ammonium suppresses it. Defining nutrients that support MT production will enable a more reliable identification of hazardous S. chartarum isolates. The new medium will also be instrumental in analyzing the biosynthetic pathways and regulatory mechanisms that control mycotoxin production in S. chartarum.

New Phenylspirodrimanes from the Sponge-Associated Fungus Stachybotrys chartarum MUT 3308.

Two phenylspirodrimanes, never isolated before, stachybotrin J (1) and new stachybocin G (epi-stachybocin A) (2), along with the already reported stachybotrin I (3), stachybotrin H (4), stachybotrylactam (5), stachybotrylactam acetate (6), 2α-acetoxystachybotrylactam acetate (7), stachybotramide (8), chartarlactam B (9), and F1839-J (10) were isolated from the sponge-associated fungus Stachybotrys chartarum MUT 3308. Their structures were established based on extensive spectrometric (HRMS) and spectroscopic (1D and 2D NMR) analyses. Absolute configurations of the stereogenic centers of stachybotrin J (1), stachybocin G (2), and stachybotrin I (3), were determined by comparison of their experimental circular dichroism (CD) spectra with their time-dependent density functional theory (TD-DFT) circular dichroism (ECD) spectra. The putative structures of seventeen additional phenylspirodrimanes were proposed by analysis of their respective MS/MS spectra through a Feature-Based Molecular Networking approach. All the isolated compounds were evaluated for their cytotoxicity against five aggressive cancer cell lines (MP41, 786, 786R, CAL33, and CAL33RR), notably including two resistant human cancer cell lines (786R, CAL33RR), and compounds 5, 6, and 7 exhibited cytotoxicity with IC50 values in the range of 0.3-2.2 µM.

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.

Differential effects of exposure to toxic or nontoxic mold spores on brain inflammation and Morris water maze performance.

People who live or work in moldy buildings often complain of "brain fog" that interferes with cognitive performance. Until recently, there was no published research on the effects of controlled exposure to mold stimuli on cognitive function or an obvious mechanism of action, fueling controversy over these claims. The constellation of health problems reported by mold-exposed individuals (respiratory issues, fatigue, pain, anxiety, depression, and cognitive deficits) correspond to those caused by innate immune activation following exposure to bacterial or viral stimuli. To determine if mold-induced innate immune activation might cause cognitive issues, we quantified the effects of both toxic and nontoxic mold on brain immune activation and spatial memory in the Morris water maze. We intranasally administered either 1) intact, toxic Stachybotrys chartarum spores; 2) ethanol-extracted, nontoxic Stachybotrys chartarum spores; or 3) control saline vehicle to mice. Inhalation of nontoxic spores caused significant deficits in the test of long-term memory of platform location, while not affecting short-term memory. Inhalation of toxic spores increased motivation to reach the platform. Interestingly, in both groups of mold-exposed males, numbers of interleukin-1ß-immunoreactive cells in many areas of the hippocampus significantly correlated with latency to find the platform, path length, and swimming speed during training, but not during testing for long-term memory. These data add to our prior evidence that mold inhalation can interfere with cognitive processing in different ways depending on the task, and that brain inflammation is significantly correlated with changes in behavior.

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.

Analysis of mold and mycotoxins in naturally infested indoor building materials.

Health issues of residents of mold-infested housing are reported on a regular basis, and reasons for the arising impairments can be manifold. One possible cause are the toxic secondary metabolite produced by indoor microfungi (mycotoxins). To enable a more thorough characterization of the exposure to mycotoxins in indoor environments, data on occurrence and quantities of mycotoxins is essential. In the presented study, 51 naturally mold-infested building material samples were analyzed applying a previously developed method based on ultra-high performance liquid chromatography (UHPLC) separation in combination with triple-quadrupole mass spectrometry (TQMS) detection. A total of 38 secondary metabolites derived from different indoor mold genera like Aspergillus, Fusarium, Penicillium, and Stachybotrys were analyzed, of which 16 were detectable in 28 samples. As both the spectrum of target analytes and the investigated sample matrices showed high chemical varieties, an alternative calibration approach was applied complementary to identify potentially emerging matrix effects during ionization and mass spectrometric detection. Overall, strong alterations of analyte signals were rare, and compensation of considerable matrix suppression/enhancement only had to be performed for certain samples. Besides mycotoxin determination and quantification, the presence of 18 different mold species was confirmed applying microbiological approaches in combination with macro- and microscopic identification according to DIN ISO 16000-17:2010-06. These results additionally highlight the diversity of mycotoxins potentially arising in indoor environments and leads to the assumption that indoor mycotoxin exposure stays an emerging topic of research, which has only just commenced.

In Vitro Metabolism of Phenylspirodrimanes Derived from the Indoor Fungus Stachybotrys.

Fungi belonging to the genus Stachybotrys are frequently detected in water-damaged indoor environments, and a potential correlation between emerging health problems of inhabitants of affected housing and the fungi is controversially discussed. Secondary metabolites (i.e., mycotoxins) produced by Stachybotrys, such as the highly toxic macrocyclic trichothecenes (MCTs), are of potential concern to human health. The present study, however, focused on the potential effects of the more broadly and abundantly formed group of phenylspirodrimanes (PSDs). The phase I and II metabolism of four structurally different PSDs were investigated in vitro using hepatic models in combination with high-performance liquid chromatography high-resolution mass spectrometry (HPLC-HRMS) analysis. In addition to metabolite detection by HRMS, isolation and structure elucidation by nuclear magnetic resonance spectroscopy (NMR) was part of the conducted study as well.

SMTP-44D Exerts Antioxidant and Anti-Inflammatory Effects through Its Soluble Epoxide Hydrolase Inhibitory Action in Immortalized Mouse Schwann Cells upon High Glucose Treatment.

Diabetic neuropathy (DN) is a major complication of diabetes mellitus. We have previously reported the efficacy of Stachybotrys microspora triprenyl phenol-44D (SMTP-44D) for DN through its potential antioxidant and anti-inflammatory activities. However, the mechanisms underlying the antioxidant and anti-inflammatory activities of SMTP-44D remain unclear. The present study aimed to explore the mechanism of these effects of SMTP-44D in regard to its inhibition of soluble epoxide hydrolase (sEH) in immortalized mouse Schwann cells (IMS32) following high glucose treatment. IMS32 cells were incubated in a high glucose medium for 48 h and then treated with SMTP-44D for 48 h. After incubation, the ratio of epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), oxidative stress markers, such as NADPH oxidase-1 and malondialdehyde, inflammatory factors, such as the ratio of nuclear to cytosolic levels of NF-κB and the levels of IL-6, MCP-1, MMP-9, the receptor for the advanced glycation end product (RAGE), and apoptosis, were evaluated. SMTP-44D treatment considerably increased the ratio of EETs to DHETs and mitigated oxidative stress, inflammation, RAGE induction, and apoptosis after high glucose treatment. In conclusion, SMTP-44D can suppress the induction of apoptosis by exerting antioxidant and anti-inflammatory effects, possibly through sEH inhibition. SMTP-44D can be a potential therapeutic agent against DN.

FGFC1 Exhibits Anti-Cancer Activity via Inhibiting NF-κB Signaling Pathway in EGFR-Mutant NSCLC Cells.

FGFC1, an active compound isolated from the culture of marine fungi Stachybotrys longispora FG216, elicits fibrinolytic, anti-oxidative, and anti-inflammatory activity. We have previously reported that FGFC1 inhibited the proliferation, migration, and invasion of the non-small cell lung cancer (NSCLC) cells in vitro. However, the precise mechanisms of FGFC1 on NSCLC and its anti-cancer activity in vivo remains unclear. Hence, this study was focused to investigate the effects and regulatory mechanisms of FGFC1 on two NSCLC cell lines, EGFR-mutant PC9 (ex19del) and EGFR wild-type H1299. Results suggested that FGFC1 significantly inhibited proliferation, colony formation, as well as triggered G0/G1 arrest and apoptosis of PC9 cells in a dose- and time-dependent manner, but no obvious inhibitory effects were observed in H1299 cells. Subsequently, transcriptome analysis revealed that FGFC1 significantly down-regulated 28 genes related to the NF-κB pathway, including IL-6, TNF-α, and ICAM-1 in the PC9 cells. We further confirmed that FGFC1 decreased the expression of protein p-IKKα/ß, p-p65, p-IκB, IL-6, and TNF-α. Moreover, NF-κB inhibitor PDTC could strengthen the effects of FGFC1 on the expression of CDK4, Cyclin D1, cleaved-PARP-1, and cleaved-caspase-3 proteins, suggesting that the NF-κB pathway plays a major role in FGFC1-induced cell cycle arrest and apoptosis. Correspondingly, the nuclear translocation of p-p65 was also suppressed by FGFC1 in PC9 cells. Finally, the intraperitoneal injection of FGFC1 remarkably inhibited PC9 xenograft growth and decreased the expression of Ki-67, p-p65, IL-6, and TNF-α in tumors. Our results indicated that FGFC1 exerted anti-cancer activity in PC9 cells via inhibiting the NF-κB signaling pathway, providing a possibility for FGFC1 to be used as a lead compound for the treatment of NSCLC in the future.

Stachybotranes A-D, phenylspirodrimanes from the wetland fungus Stachybotrys chartarum with cytotoxic activities.

Four new phenylspirodrimanes, stachybotranes A-D (1-4), along with seven known analogues (5-11), were isolated from the extract of a wetland fungal strain Stachybotrys chartarum DTH12-9. The structures of new compounds were determined by spectroscopic analyses, X-ray crystallographic analysis, and the CD analysis of the in situ formed [Rh2(OCOCF3)4] complex. Compounds 1-3, 5-8, and 10 were evaluated for in vitro cytotoxicity against five human cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7, and SW-480. Compounds 1, 2, and 7 exhibited moderate cytotoxic potency against various human cancer cell lines.

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.

Potent efficacy of Stachybotrys microspora triprenyl phenol-7, a small molecule having anti-inflammatory and antioxidant activities, in a mouse model of acute kidney injury.

Acute kidney injury (AKI) increases the risk of chronic kidney disease (CKD), complicates existing CKD, and can lead to the end-stage renal disease. However, there are no approved effective therapeutics for AKI. Recent studies have suggested that inflammation and oxidative stress are the primary causes of AKI. We previously reported the potential anti-inflammatory and antioxidant activities of Stachybotrys microspora triprenyl phenol-7 (SMTP-7). The aim of the present study was to evaluate the efficacy of SMTP-7 in AKI model mice. AKI was induced in mice by ischemia of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after the removal of right kidney. The efficacy of SMTP-7 was determined by measuring the renal function using urine and serum samples and morphological assessment. For deciphering the mechanism of action of SMTP-7, inflammatory cytokines and oxidative stress in kidney were detected. SMTP-7 (0.01, 0.1, 1, 10 mg/kg) dose-dependently improved the renal function. In addition, it improved the damage to renal tubules and exhibited anti-inflammatory and antioxidant activities in the kidney of AKI mice. These results indicate the potential of SMTP-7 as a medicinal compound for the treatment of AKI.

Natural Compounds Isolated from Stachybotrys chartarum Are Potent Inhibitors of Human Protein Kinase CK2.

A large number of secondary metabolites have been isolated from the filamentous fungus Stachybotrys chartarum and have been described before. Fourteen of these natural compounds were evaluated in vitro in the present study for their inhibitory activity towards the cancer target CK2. Among these compounds, stachybotrychromene C, stachybotrydial acetate and acetoxystachybotrydial acetate turned out to be potent inhibitors with IC50 values of 0.32 µM, 0.69 µM and 1.86 µM, respectively. The effects of these three compounds on cell proliferation, growth and viability of MCF7 cells, representing human breast adenocarcinoma as well as A427 (human lung carcinoma) and A431 (human epidermoid carcinoma) cells, were tested using EdU assay, IncuCyte® live-cell imaging and MTT assay. The most active compound in inhibiting MCF7 cell proliferation was acetoxystachybotrydial acetate with an EC50 value of 0.39 µM. In addition, acetoxystachybotrydial acetate turned out to inhibit the growth of all three cell lines completely at a concentration of 1 µM. In contrast, cell viability was impaired only moderately, to 37%, 14% and 23% in MCF7, A427 and A431 cells, respectively.

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.

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.