Development and Application of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Diagnosis of the Bat White-Nose Disease Fungus Pseudogymnoascus destructans.

Pseudogymnoascus destructans (= Geomyces destructans) is a psychrophilic filamentous fungus that causes White-Nose Disease (WND; the disease associated with White-Nose Syndrome, WNS) in hibernating bats. The disease has caused considerable reductions in bat populations in the USA and Canada since 2006. Identification and detection of the pathogen in pure cultures and environmental samples is routinely based on qPCR or PCR after DNA isolation and purification. Rapid and specific direct detection of the fungus in the field would strongly improve prompt surveillance, and support control measures. Based on the genes coding for ATP citrate lyase1 (acl1) and the 28S-18S ribosomal RNA intergenic spacer (IGS) in P. destructans, two independent LAMP assays were developed for the rapid and sensitive diagnosis of the fungus. Both assays could discriminate P. destructans from 159 tested species of filamentous fungi and yeasts. Sensitivity of the assays was 2.1 picogram per reaction (pg/rxn) and 21 femtogram per reaction (fg/rxn) for the acl1 and IGS based assays, respectively. Moreover, both assays also work with spores and mycelia of P. destructans that are directly added to the master mix without prior DNA extraction. For field-diagnostics, we developed and tested a field-applicable version of the IGS-based LAMP assay. Lastly, we also developed a protocol for preparation of fungal spores and mycelia from swabs and tape liftings of contaminated surfaces or infected bats. This protocol in combination with the highly sensitive IGS-based LAMP-assay enabled sensitive detection of P. destructans from various sources.

The Evolution of the Satratoxin and Atranone Gene Clusters of Stachybotrys chartarum.

Stachybotrys chartarum is frequently isolated from damp building materials or improperly stored animal forage. Human and animal exposure to the secondary metabolites of this mold is linked to severe health effects. The mutually exclusive production of either satratoxins or atranones defines the chemotypes A and S. Based upon the genes (satratoxin cluster, SC1-3, sat or atranone cluster, AC1, atr) that are supposed to be essential for satratoxin and atranone production, S. chartarum can furthermore be divided into three genotypes: the S-type possessing all sat- but no atr-genes, the A-type lacking the sat- but harboring all atr-genes, and the H-type having only certain sat- and all atr-genes. We analyzed the above-mentioned gene clusters and their flanking regions to shed light on the evolutionary relationship. Furthermore, we performed a deep re-sequencing and LC-MS/MS (Liquid chromatography-mass spectrometry) analysis. We propose a first model for the evolution of the S. chartarum genotypes. We assume that genotype H represents the most ancient form. A loss of the AC1 and the concomitant acquisition of the SC2 led to the emergence of the genotype S. According to our model, the genotype H also developed towards genotype A, a process that was accompanied by a loss of SC1 and SC3.

Discrimination of Tilletia controversa from the T. caries/T. laevis complex by MALDI-TOF MS analysis of teliospores.

The fungal genus Tilletia includes a large number of plant pathogens of Poaceae. Only a few of those cause bunt of wheat, but these species can lead to significant yield losses in crop production worldwide. Due to quarantine regulations and specific disease control using appropriate seed treatments for the different disease agents, it is of high importance to distinguish Tilletia caries and Tilletia laevis as causal agents of common bunt accurately from Tilletia controversa, the causal agent of the dwarf bunt. Several studies have shown that matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful tool to differentiate closely related fungal species. The aim of this study was to assess whether MALDI-TOF MS analysis is able to distinguish specimens of the three closely related pathogens T. caries, T. laevis, and T. controversa and whether it may constitute an alternative method to the morphology-based identification or germination tests. Spectral data are available via ProteomeXchange with identifier PXD030401. Spectra-based hierarchical cluster analysis (HCA) and discriminant analysis of principal components (DAPC) of the obtained mass spectra showed two main clusters. One cluster included specimens of T. controversa, whereas the second cluster comprised T. laevis and T. caries specimens. Even though main spectral profiles (MSPs) for species identification are missing, MALDI-TOF MS has proven to be a useful method for distinguishing between T. controversa and the two causal agents of common bunt, using direct analysis of teliospores, but was unable to separate T. caries and T. laevis species. KEY POINTS: • MALDI-TOF MS was developed to classify Tilletia species causing bunt of wheat. • Best results were achieved when combining HCA and DAPC analysis. • The method resulted in an accuracy of 98.51% testing 67 Tilletia specimens.

Characterization of the influence of carbon sources on fum1 gene expression in the fumonisin producer Fusarium verticillioides using RT - LAMP assay.

Fusarium verticillioides is one of the major fumonisin producers. The ingestion of this mycotoxin represents a risk for both human and animal health. The development of F. verticillioides is associated with environmental conditions, especially carbon sources. We developed a reliable and fast reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and determined fum1 gene expression upon growth of two F. verticillioides strains isolated from maize and wheat in Czapek's medium containing four different sugars as sole carbon sources. Fumonisin B1 (FB1) production was determined by LC-MS/MS analysis. High growth and production of FB1 were observed in fructose-containing medium for the strain that originated from maize. Less production of FB1 occurred using maltose as sole carbon source for both strains. The fum1 gene expression started between 2 and 4 days of incubation, and positive signals were detected prior to the initial production of FB1. The RT-LAMP assay was effective in the detection of fum1 gene expression at very early stages of F. verticillioides growth and allowed the prediction of FB1 formation.

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.

Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison.

Tilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

Influence of fermentation conditions on the secretion of seripauperin 5 (PAU5) by industrial sparkling wine strains of Saccharomyces cerevisiae.

The yeast mannoprotein seripauperin 5 (PAU5) from Saccharomyces cerevisiae is a negative gushing biomarker in sparkling wine with a direct gushing-reducing effect. The knowledge about the influence of the yeast strain and the fermentation conditions on the PAU5 content in the final product could reduce the gushing potential of sparkling wines and avoid economic losses for sparkling wine producers. The potential of 30 different commercially used (sparkling) wine S. cerevisiae strains to produce PAU5 was analyzed by RP-HPLC. The experiments revealed great differences between strains and identified three high-PAU5-producing yeast strains. The influence of different fermentation conditions was analyzed in two selected strains. Cultivation conditions of elevated temperature and lower inoculation density, as well as cultivation under diffuse daylight significantly enhanced PAU5 production by the tested S. cerevisiae strains as compared to standard conditions. However, cultivation as agitated cultures and co-cultivation with Metchnikowia pulcherrima or Torulaspora delbrueckii led to a reduction of PAU5 production as compared to standard conditions.

Studies on the gushing potential of Penicillium expansum.

Gushing describes the spontaneous excessive over-foaming of carbonated beverages leading to considerable economic losses and reputational damages to the beverage industry. Surface-active proteins produced by filamentous fungi are involved in the induction of gushing. In the current study, the role of Penicillium expansum in sparkling wine gushing was investigated. Almost 40 P. expansum strains were analyzed regarding their ability to secrete surface-active proteins and to induce gushing in carbonated water as a model system and in sparkling wine. The majority of the strains produced surface-active compounds and induced gushing. The severity of gushing depended on the volume of culture supernatant added to carbonated liquids. Moreover, sparkling wine showed more severe gushing than carbonated water. A protein with a molecular mass of 20 kDa was prominent in gushing-inducing P. expansum culture supernatants. It was identified as PEX2_044840 from P. expansum. This protein was heterologously expressed in Pichia pastoris (Komagataella phaffi). The purified recombinant protein induced gushing in sparkling wine after addition of at least 30 µg/mL of protein sample.

Development and optimization of a loop-mediated isothermal amplification (LAMP) assay for the species-specific detection of Penicillium expansum.

Penicillium expansum is the main cause of Blue Mold Decay, which is the economically most significant postharvest disease on fruits. It occurs especially on pomaceous fruits such as apples and pears but also on a wide range of other fruits such as grapes or strawberries. Besides its negative economic effects on the industry, the fungus is also of health concern as it produces patulin, a mycotoxin known to provoke harmful effects in humans. A specific and rapid detection of this fungus therefore is required. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized for the species-specific detection of P. expansum. The assay showed high specificity during tests with genomic DNA of 187 fungal strains. The detection limit of the developed assay was 25 pg genomic DNA of P. expansum per reaction. The assay was successfully applied for the detection of the fungus on artificially contaminated apples, grapes, apple juice, apple puree, and grape juice. The developed assay is a promising tool for rapid, sensitive, specific, and cost-efficient detection of P. expansum in quality control applications in the food and beverage industry.

Universal law for diffusive mass transport through mycelial networks.

Filamentous fungal cell factories play a pivotal role in biotechnology and circular economy. Hyphal growth and macroscopic morphology are critical for product titers; however, these are difficult to control and predict. Usually pellets, which are dense networks of branched hyphae, are formed during industrial cultivations. They are nutrient- and oxygen-depleted in their core due to limited diffusive mass transport, which compromises productivity of bioprocesses. Here, we demonstrate that a generalized law for diffusive mass transport exists for filamentous fungal pellets. Diffusion computations were conducted based on three-dimensional X-ray microtomography measurements of 66 pellets originating from four industrially exploited filamentous fungi and based on 3125 Monte Carlo simulated pellets. Our data show that the diffusion hindrance factor follows a scaling law with respect to the solid hyphal fraction. This law can be harnessed to predict diffusion of nutrients, oxygen, and secreted metabolites in any filamentous pellets and will thus advance the rational design of pellet morphologies on genetic and process levels.

A loop-mediated isothermal amplification (LAMP) based assay for the rapid and sensitive group-specific detection of fumonisin producing Fusarium spp.

Fumonisins are mycotoxins that contaminate maize and maize-based food products, and feed. They have been associated with nerve system disorders in horses, pulmonary edema in swine as well as neural tube defects and esophageal cancer in humans. The fum1 gene codes for a polyketide synthase involved in the biosynthesis of fumonisins. It is present in the genomes of all fumonisin producing Fusarium spp. Reliable detection of fum1 can provide an estimate of the toxicological potential of cultures and food sources. Therefore, a fum1 specific LAMP assay was developed and tested with purified DNA of 48 different species from the Fusarium fujikuroi species complex (FFSC). The fum1 gene was detected in 22 species among which F. fujikuroi, F. globosum, F. nygamai, F. proliferatum, F. subglutinans and F. verticillioides were the most prominent fumonisin producers. None out of 92 tested non-Fusarium species showed cross reactions with the new assay. The lowest limit of detection (LOD) was 5 pg of genomic DNA per reaction for F. fujikuroi, F. nygamai and F. verticillioides. Higher LODs were found for other LAMP positive species. Apart from pure genomic DNA, the LAMP assay detected fumonisin-producers when 103 conidia/reaction were used as template after mechanical lysis. LAMP-results were well correlated with FB1 production. This is the first report on fumonisin production in strains of F. annanatum, F. coicis, F. mundagurra, F. newnesense, F. pininemorale, F. sororula, F. tjataeba, F. udum and F. werrikimbe. Usefulness of the LAMP assay was demonstrated by analyzing fumonisin contaminated maize grains. The new LAMP assay is rapid, sensitive and reliable for the diagnosis of typical fumonisin producers and can be a versatile tool in HACCP concepts that target the reduction of fumonisins in the food and feed chain.

Correction to: truncated satratoxin gene clusters in selected isolates of the atranone chemotype of Stachybotrys chartarum (Ehrenb.) S. Hughes.

The original version of this paper was published with error. The supplementary materials originally provided were not captured during article production.

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.

MALDI-TOF MS fingerprinting for identification and differentiation of species within the Fusarium fujikuroi species complex.

Members of the Fusarium fujikuroi species complex (FFSC) are commonly involved in devastating diseases of many economically important plants. They invade developing seeds and other plant tissues in the field causing significant annual losses. In addition, fungal spoilage can also affect human and animal health because some species in this group, especially F. proliferatum and F. verticillioides, are mycotoxin producers occurring in food/feed worldwide. Since morphology-based species identification is of limited value in the FFSC, the development of new methods is fundamental for accurate identification of the molds to species level. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis of subproteomes has been applied as a promising tool for the discrimination of closely related species in many microorganisms. In the present study, MALDI-TOF MS was applied to distinguish closely related species in the FFSC and to validate the effectiveness of a standardized protocol by identifying field isolates that fulfilled the morphological characteristics of FFSC species. Forty-nine of the currently described 61 species were identified by DNA sequencing analysis and their mass spectra were included as reference in a supplementary MALDI-TOF MS database. The discriminative potential of the database was evaluated with more than 80 non-reference FFSC isolates and resulted in 94.61% of correct identifications at the species level. We demonstrate that MALDI-TOF MS is a suitable and accurate technology for the identification and differentiation of species within the FFSC as well as an innovative, time-efficient alternative to multilocus sequencing technology (MLST).

Development and optimization of a group-specific loop-mediated isothermal amplification (LAMP) assay for the detection of patulin-producing Penicillium species.

The mycotoxin patulin is a toxic fungal secondary metabolite occurring in food worldwide. Methods for rapid, simple and specific detection of patulin-producing fungi in food and feed are therefore urgently needed. In the current study, a loop-mediated isothermal amplification (LAMP) assay based on the isoepoxydon dehydrogenase (idh) gene of the patulin biosynthetic pathway was developed and optimized for the group-specific detection of patulin-producing Penicillium species. By testing purified DNA of 174 fungal strains representing 31 genera, the assay was demonstrated to be highly specific for the detection of patulin-producing species in Penicillium, Byssochlamys and Paecilomyces. The assay had a detection limit of 2.5 pg of purified genomic DNA of P. expansum per reaction. Moreover, the assay was demonstrated to detect patulin-producers when conidia were directly added to the master mix as template without any sample preparation. The applicability of the assay in food analyses was successfully tested on artificially contaminated grapes and apples requiring minimal sample preparation. A screening of grapes from the 2018 harvest from different locations in Germany revealed no presence of patulin-producers. The developed LAMP assay is a promising tool for rapid diagnosis in quality control applications in the food and beverage industry.

An X-ray microtomography-based method for detailed analysis of the three-dimensional morphology of fungal pellets.

Filamentous fungi are widely used in the production of biotechnological compounds. Since their morphology is strongly linked to productivity, it is a key parameter in industrial biotechnology. However, identifying the morphological properties of filamentous fungi is challenging. Owing to a lack of appropriate methods, the detailed three-dimensional morphology of filamentous pellets remains unexplored. In the present study, we used state-of-the-art X-ray microtomography (µCT) to develop a new method for detailed characterization of fungal pellets. µCT measurements were performed using freeze-dried pellets obtained from submerged cultivations. Three-dimensional images were generated and analyzed to locate and quantify hyphal material, tips, and branches. As a result, morphological properties including hyphal length, tip number, branch number, hyphal growth unit, porosity, and hyphal average diameter were ascertained. To validate the potential of the new method, two fungal pellets were studied-one from Aspergillus niger and the other from Penicillium chrysogenum. We show here that µCT analysis is a promising tool to study the three-dimensional structure of pellet-forming filamentous microorganisms in utmost detail. The knowledge gained can be used to understand and thus optimize pellet structures by means of appropriate process or genetic control in biotechnological applications.

LAMP-based group specific detection of aflatoxin producers within Aspergillus section Flavi in food raw materials, spices, and dried fruit using neutral red for visible-light signal detection.

Aflatoxins can be produced by 21 species within sections Flavi (16 species), Ochraceorosei (2), and Nidulantes (3) of the fungal genus Aspergillus. They pose risks to human and animal health due to high toxicity and carcinogenicity. Detecting aflatoxin producers can help to assess toxicological risks associated with contaminated commodities. Species specific molecular assays (PCR and LAMP) are available for detection of major producers, but fail to detect species of minor importance. To enable rapid and sensitive detection of several aflatoxin producing species in a single analysis, a nor1 gene-specific LAMP assay was developed. Specificity testing showed that among 128 fungal species from 28 genera, 15 aflatoxigenic species in section Flavi were detected, including synonyms of A. flavus and A. parasiticus. No cross reactions were found with other tested species. The detection limit of the assay was 9.03pg of A. parasiticus genomic DNA per reaction. Visual detection of positive LAMP reactions under daylight conditions was facilitated using neutral red to allow unambiguous distinction between positive and negative assay results. Application of the assay to the detection of A. parasiticus conidia revealed a detection limit of 211 conidia per reaction after minimal sample preparation. The usefulness of the assay was demonstrated in the analysis of aflatoxinogenic species in samples of rice, nuts, raisins, dried figs, as well as powdered spices. Comparison of LAMP results with presence/absence of aflatoxins and aflatoxin producing fungi in 50 rice samples showed good correlation between these parameters. Our study suggests that the developed LAMP assay is a rapid, sensitive and user-friendly tool for surveillance and quality control in our food industry.

Foam-stabilizing properties of the yeast protein PAU5 and evaluation of factors that can influence its concentration in must and wine.

The absence of the yeast protein seripauperin 5 (PAU5) from Saccharomyces cerevisiae has been suggested as a biomarker for the occurrence of gushing in sparkling wine as samples lacking PAU5 were found to be more susceptible to gushing. In this study, further characterization of PAU5 regarding its foam-stabilizing properties was performed to elucidate whether PAU5 has foam-stabilizing properties and therefore, to elucidate a direct influence on the gushing potential of sparkling wines. PAU5 was successfully purified from non-gushing sparkling wine using reversed-phase high-performance liquid chromatography (RP-HPLC). Pure protein was added to grape juice as a model system for grape must prior to foam stability testing. The results revealed that the protein PAU5 has foam-stabilizing properties. Furthermore, the influence of heat and sulfur treatment in the presence of Botrytis cinerea was analyzed with regard to the amount of PAU5 produced by S. cerevisiae fermented in grape juice. Fermentation experiments using two different S. cerevisiae strains were performed, and the concentration of PAU5 in the samples was compared by RP-HPLC analysis. Unlike sulfur treatment, heat treatment prevented the protein degradation induced by B. cinerea and resulted in even higher amounts of PAU5 compared to the juice fermented with yeast without a previous botrytization. The two different yeast strains applied secreted PAU5 into the surrounding medium in different amounts. In further experiments, the fining process of the wine with bentonite was examined for its potential to remove PAU5 from the wine. RP-HPLC of wines processed with different fining agents revealed that bentonite treatment affected PAU5 concentrations in the final product. The extent of PAU5 removal depended on the type of bentonite applied and on the time of addition during the production process.