New morphological criteria and molecular characterization of black aspergilli aggregate from corn, sorghum and wheat grains.

Corn, sorghum and wheat grains are used as livestock feed in the world. Identification of black aspergilli associated with these grains is necessary to make sure of the safety of the grains because its occurrence is an indicator of mycotoxin production. Forty-five isolates were isolated from the samples collected from Upper Egypt's markets and identified morphologically based on colony color, conidia, stipe and vesicle size and molecularly by using ß-tubulin and calmodulin genes. Isolates were divided into 30 strains of Aspergillus welwitschiae and 15 strains of A. niger. We have found new criteria in the morphological identification of A. welwitschiae as its colony color was black to brown with yellow edge, but in A. niger was black with white edge, also A. welwitschiae sometimes produced finely-to-distinctly roughened brownish conidia on malt extract agar (MEA) media. Thirteen isolates of A. welwitschiae and six of A. niger were recognized as potential producers for ochratoxin A.

Different repartition of the cryptic species of black aspergilli according to the anatomical sites in human infections, in a French University hospital.

Black aspergilli of the section Nigri are rarely differentiated at the species level when originating from human specimens. We wondered whether some cryptic species could be more frequently observed in some clinical entities. We analyzed the 198 black isolates consecutively collected from the external ear canal (EEC; n = 66), respiratory specimens (n = 99), and environment (n = 33). DNA was extracted and species identification was performed upon the partial calmodulin gene. We identified by decreasing frequency: Aspergillus welwitschiae (35.3%), Aspergillus tubingensis (34.3%), Aspergillus niger (17.2%), Aspergillus luchuensis (4%), Aspergillus aff. welwitschiae (3%), Aspergillus neoniger (2%), Aspergillus piperis (1.5%), Aspergillus japonicus (1.0%), Aspergillus vadensis (0.5%), and two Aspergillus tubingensis clade (1%). The distribution of the three main cryptic species was different between EEC and respiratory samples (P < 0.001) but not different between respiratory and environment samples (P = 0.264). Aspergillus welwitschiae was more often associated with EEC (54.5%), whereas A. tubingensis and A. niger were predominant in respiratory samples (39.4 and 26.3%, respectively). Among the 99 respiratory isolates, only 10 were deemed responsible for probable invasive aspergillosis, of which six were mixed with other pathogenic moulds. This study shows the interest to pursue the identification of clinical isolates in the Aspergillus section Nigri to unravel some specific associations with clinical entities. The association of A. welwitschiae with otomycosis suggests a better fitness to infect/colonize the ear canal. Also, members of the Aspergillus section Nigri alone are rarely responsible for invasive aspergillosis. LAY SUMMARY: We analyzed 198 black aspergilli isolates collected from different samples type to determine their species identification. We observe a different distribution of species between ear canal and respiratory samples (P < 0.001), suggesting a better fitness of A. welwitschiae to infect the ear canal.

Rapid and accurate identification of black aspergilli from grapes using high-resolution melting (HRM) analysis.

BACKGROUND: Aspergillus is a diverse genus of fungi with high economic and social impact. Various species that belong to section Nigri (black aspergilli) are common agents of grape spoilage and potent producers of ochratoxin A (OTA), a mycotoxin associated with various nephrotoxic and immunotoxic effects in humans. Black aspergilli are difficult to classify following only phenotypic criteria; thus chemotaxonomic and molecular methods are employed in parallel with phenotypic ones for species characterization. These approaches, though accurate and replicable, require more than one individual step and are to a certain extent laborious when a rapid identification of these species is required. RESULTS: The aim of this study was to develop a high-resolution melting polymerase chain reaction (HRM-PCR) assay as a rapid method for identification of Aspergillus spp. section Nigri isolates and their detection in grape samples. Melt curve analysis of amplicons originating from the internal transcribed spacer 2 (ITS2) ribosomal region generated species-specific HRM curve profiles, enabling the accurate differentiation of the analyzed genotypes. Furthermore, the assay was able to identify A. carbonarius, A. tubingensis, A. niger, A. ibericus and A. japonicus in grape samples artificially inoculated with conidia of these fungi. CONCLUSION: To our knowledge this is the first report on the development of an HRM-PCR assay for the identification of black Aspergillus species in grape samples. © 2018 Society of Chemical Industry.

Whole-genome sequencing of Aspergillus tubingensis G131 and overview of its secondary metabolism potential.

BACKGROUND: Black Aspergilli represent one of the most important fungal resources of primary and secondary metabolites for biotechnological industry. Having several black Aspergilli sequenced genomes should allow targeting the production of certain metabolites with bioactive properties. RESULTS: In this study, we report the draft genome of a black Aspergilli, A. tubingensis G131, isolated from a French Mediterranean vineyard. This 35 Mb genome includes 10,994 predicted genes. A genomic-based discovery identifies 80 secondary metabolites biosynthetic gene clusters. Genomic sequences of these clusters were blasted on 3 chosen black Aspergilli genomes: A. tubingensis CBS 134.48, A. niger CBS 513.88 and A. kawachii IFO 4308. This comparison highlights different levels of clusters conservation between the four strains. It also allows identifying seven unique clusters in A. tubingensis G131. Moreover, the putative secondary metabolites clusters for asperazine and naphtho-gamma-pyrones production were proposed based on this genomic analysis. Key biosynthetic genes required for the production of 2 mycotoxins, ochratoxin A and fumonisin, are absent from this draft genome. Even if intergenic sequences of these mycotoxins biosynthetic pathways are present, this could not lead to the production of those mycotoxins by A. tubingensis G131. CONCLUSIONS: Functional and bioinformatics analyses of A. tubingensis G131 genome highlight its potential for metabolites production in particular for TAN-1612, asperazine and naphtho-gamma-pyrones presenting antioxidant, anticancer or antibiotic properties.

Grape and environmental mycoflora monitoring in old, traditionally cultivated vineyards on Mount Etna, southern Italy.

BACKGROUND: Grape contamination by several fungal species occurs during a vineyard's preharvest and harvest. Agronomic management and microclimatic conditions can affect fungi occurrence and epidemiology, thus explaining qualitative differences in mycoflora composition, including the presence of phytopathogenic or mycotoxigenic fungi. In this study a two-year grape, air and soil mycoflora monitoring programme was undertaken in vineyards on Mount Etna (eastern Sicily, Italy). The mycoflora composition was investigated at pea berry and veraison phenological phases from air and soil and at ripening from sample grapes. RESULTS: Mycoflora in air and soil varied according to the phenological stage. In the air samples, penicillia were dominant over aspergilli at the pea berry phase, but their ratio was inverted at early veraison. Black aspergilli (BA) were isolated from the vine environment and grape samples, where BA were represented mainly by Aspergillus niger aggregate, which showed no or low ochratoxin A (OTA) production. Aspergillus carbonarius was either not identified or identified at low frequency, although most of the isolates produced OTA. CONCLUSION: Monitoring focused on the environmental mycoflora composition and highlighted the good health profile of various Sicilian autochthonous grape cultivars. In addition, data suggest that the lower relative humidity occurring at the highest altitudes reduces BA incidence. © 2016 Society of Chemical Industry.

Variation in the fumonisin biosynthetic gene cluster in fumonisin-producing and nonproducing black aspergilli.

The ability to produce fumonisin mycotoxins varies among members of the black aspergilli. Previously, analyses of selected genes in the fumonisin biosynthetic gene (fum) cluster in black aspergilli from California grapes indicated that fumonisin-nonproducing isolates of Aspergillus welwitschiae lack six fum genes, but nonproducing isolates of Aspergillus niger do not. In the current study, analyses of black aspergilli from grapes from the Mediterranean Basin indicate that the genomic context of the fum cluster is the same in isolates of A. niger and A. welwitschiae regardless of fumonisin-production ability and that full-length clusters occur in producing isolates of both species and nonproducing isolates of A. niger. In contrast, the cluster has undergone an eight-gene deletion in fumonisin-nonproducing isolates of A. welwitschiae. Phylogenetic analyses suggest each species consists of a mixed population of fumonisin-producing and nonproducing individuals, and that existence of both production phenotypes may provide a selective advantage to these species. Differences in gene content of fum cluster homologues and phylogenetic relationships of fum genes suggest that the mutation(s) responsible for the nonproduction phenotype differs, and therefore arose independently, in the two species. Partial fum cluster homologues were also identified in genome sequences of four other black Aspergillus species. Gene content of these partial clusters and phylogenetic relationships of fum sequences indicate that non-random partial deletion of the cluster has occurred multiple times among the species. This in turn suggests that an intact cluster and fumonisin production were once more widespread among black aspergilli.

Development of a machine learning model for systematics of Aspergillus section Nigri using synchrotron radiation-based fourier transform infrared spectroscopy.

Aspergillus section Nigri (black aspergilli) fungi are economically important food spoilage agents. Some species in this section also produce harmful mycotoxins in food. However, it is remarkably difficult to identify this fungal group at the species level using morphological and chemical characteristics. The molecular approach for classification is preferable; however, it is time-consuming, making it inappropriate for rapid testing of large numbers of samples. To address this, we explored synchrotron radiation-based Fourier transform infrared microspectroscopy (SR-FTIR) as a rapid method for obtaining data suitable for species classification. SR-FTIR data were obtained from the mycelia/conidia of 22 black aspergilli species. The Convolutional Neural Network (CNN) approach, a supervised deep learning algorithm, was used with SR-FTIR data to classify black aspergilli at the species level. A subset of the data was used to train the CNN model, and the model classification performance was evaluated using the validation data subsets. The model demonstrated a 95.97% accuracy in species classification on the testing (blind) data subset. The technique presented herein could be an alternative method for identifying problematic black aspergilli in the food industry.

ß-tubulin paralogue tubC is frequently misidentified as the benA gene in Aspergillus section Nigri taxonomy: primer specificity testing and taxonomic consequences.

ß-tubulin (benA, tub-2) and calmodulin (caM) are crucial genes in the taxonomy of Aspergillus section Nigri. Widely used ß-tubulin primers are not specific for the benA gene for some taxa and preferentially amplify the tubC paralogue. Sequences of the tubC paralogue are widely combined with benA sequences in recent taxonomical works as well as other works, resulting in incongruent trees. In this study we newly provide benA sequences for several ex-type strains, which were characterised using the tubC gene only. We designed a highly specific forward primer to benA designated Ben2f for use in Aspergillus section Nigri, and tested specificity of numerous primer combinations to ß-tubulin paralogs. The primer pairs with the highest specificity to the benA gene and functional across species in section Nigri includes Ben2f/Bt2b, Ben2f/T22 and T10/T22. We also provide tools based on codon usage bias analysis that reliably distinguish both paralogues. Exon/intron arrangement is the next distinctive characteristic, although this tool is not valid outside section Nigri. The species identity of taxa from the A. aculeatus clade used in previous molecular studies was revised using combined molecular data (ITS, benA, caM). These data together with two different PCR-fingerprinting methods indicated that A. japonicus should be treated as a synonym of A. violaceofuscus. Similarly, A. fijiensis is reduced to synonymy with A. brunneoviolaceus.

Ochratoxin A and related fungi in Brazilian black pepper (Piper nigrum L.).

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, genotoxic, teratogenic and carcinogenic properties, produced by several species of Aspergillus, mainly those belonging to the A. section Circumdati and A. section Nigri. Although this toxin has been detected in spices and condiments, in black pepper (Piper nigrum L.) few studies have investigated the mycobiota (based on a molecular approach) and the presence of OTA in this food. The aim of this study was to investigate the presence of potentially ochratoxigenic species and ochratoxin A in black pepper marketed in Brazil, one of the largest producers in the world. A total of 60 samples of black pepper (29 in powder and 31 in grain) were collected in markets. The presence of OTA was investigated in black pepper samples using High-Performance Liquid Chromatography (HPLC), OTA was detected in 55% of the samples, with levels ranging from 0.05 to 13.15 µg/kg, all of which were below the Brazilian legal tolerances. A. section Nigri and A. section Circumdati were found in 80% of the samples, but the species of A. section Nigri were significantly more frequent than those of A. section Circumdati. The potential for OTA production by fungal isolates was tested using the agar plug technique and confirmed by HPLC. Among the isolates belonging to A. section Nigri (n = 1,083) and A. section Circumdati (n = 129), 3.7% and 3.8%, respectively, were able to produce OTA in Yeast Extract Sucrose Agar (YESA). A total of 25 strains from A. section Circumdati and 64 from A. section Nigri were identified using molecular data. The following potentially ochratoxigenic species were found in black pepper: A. niger, A. welwitschiae, A. carbonarius, A. westerdijkiae and A. ochraceus. The occurrence of these species denotes the need for continuous monitoring of black pepper by regulatory bodies in order to safeguard consumers' health.

Growth Enhancement of Arabidopsis (Arabidopsis thaliana) and Onion (Allium cepa) With Inoculation of Three Newly Identified Mineral-Solubilizing Fungi in the Genus Aspergillus Section Nigri.

Some soil fungi play an important role in supplying elements to plants by the solubilizing of insoluble minerals in the soil. The present study was conducted to isolate the mineral-solubilizing fungi from rhizosphere soil in some agricultural areas in northern Thailand. Seven fungal strains were obtained and identified using a polyphasic taxonomic approach with multilocus phylogenetic and phenotypic (morphology and extrolite profile) analyses. All obtained fungal strains were newly identified in the genus Aspergillus section Nigri, Aspergillus chiangmaiensis (SDBR-CMUI4 and SDBR-CMU15), Aspergillus pseudopiperis (SDBR-CMUI1 and SDBR-CMUI7), and Aspergillus pseudotubingensis (SDBR-CMUO2, SDBR-CMUO8, and SDBR-CMU20). All fungal strains were able to solubilize the insoluble mineral form of calcium, copper, cobalt, iron, manganese, magnesium, zinc, phosphorus, feldspar, and kaolin in the agar plate assay. Consequently, the highest phosphate solubilization strains (SDBR-CMUI1, SDBR-CMUI4, and SDBR-CMUO2) of each fungal species were selected for evaluation of their plant growth enhancement ability on Arabidopsis and onion in laboratory and greenhouse experiments, respectively. Plant disease symptoms were not found in any treatment of fungal inoculation and control. All selected fungal strains significantly increased the leaf number, leaf length, dried biomass of shoot and root, chlorophyll content, and cellular inorganic phosphate content in both Arabidopsis and onion plants under supplementation with insoluble mineral phosphate. Additionally, the inoculation of selected fungal strains also improved the yield and quercetin content of onion bulb. Thus, the selected strains reveal the potential in plant growth promotion agents that can be applied as a biofertilizer in the future.

Interacting climate change factors (CO2 and temperature cycles) effects on growth, secondary metabolite gene expression and phenotypic ochratoxin A production by Aspergillus carbonarius strains on a grape-based matrix.

Little is known on the impact that climate change (CC) may have on Aspergillus carbonarius and Ochratoxin A (OTA) contamination of grapes, especially in the Mediterranean region where in CC scenarios temperature are expected to increase by +2-5 °C and CO2 from 400 to 800/1200 ppm. This study examined the effect of (i) current and increased temperature in the alternating 11.5 h dark/12.5 h light cycle (15-28 °C vs 18-34 °C), representative of the North Apulia area, South Italy and (ii) existing and predicted CO2 concentrations (400 vs 1000 ppm), on growth, expression of biosynthetic genes (AcOTApks, AcOTAnrps, AcOTAhal, AcOTAp450, AcOTAbZIP) and regulatory genes of Velvet complex (laeA/veA/velB, "velvet complex") involved in OTA biosynthesis and OTA phenotypic production by three strains of A. carbonarius. The experiments made on a grape-based matrix showed that elevated CO2 resulted in a general stimulation of growth and OTA production. These results were also supported by the up-regulation of both structural and regulatory genes involved in the OTA biosynthesis. Our work has shown for the first time that elevated CO2 concentration in the Mediterranean region may result in an increased risk of OTA contamination in the wine production chain.

Studies on the efficacy of electrolysed oxidising water to control Aspergillus carbonarius and ochratoxin A contamination on grape.

Ochratoxin A (OTA) occurrence in grapes is caused by black Aspergilli (Aspergillus carbonarius followed by A. niger) vineyards contamination. It depends on climatic conditions, geographical regions, damage by insects, and grape varieties. Good agricultural practices, pesticides, and fungicides seem adequate to manage the problem during low OTA risk vintages, but the development of new strategies is always encouraged, especially when an extremely favourable condition occurs in the vineyard. Electrolysed oxidising water (EOW) has become an interesting alternative to chemicals in agriculture, mainly during the post-harvest phase. This study tested the fungicidal efficacy of EOW generated by potassium chloride, in vitro, on black Aspergilli conidia, and detached grape berries infected by A. carbonarius. Then, during field trials on Primitivo cv vineyard treated with EOW, A. carbonarius contamination, and OTA levels were compared with Switch® fungicide treatment (0.8 g/l). Black Aspergilli conidia were killed on plate assay after 2 min of treatment by EOW containing >0.4 g/l of active chlorine. EOW (0.6 g/l active chlorine) treatment reduced the rate of A. carbonarius infections in vitro of about 87-92% on detached berries and, more than half in the field trials, although Switch® showed better performance. A significant reduction in the OTA concentration was observed for the EOW and Switch® treatments in vitro (92% and 96%, respectively), while in the field trials, although the average decrease in OTA was recorded in the treated grapes, it was not statistically significant. These results highlighted that EOW could be considered effective, as a substitute for fungicides, to reduce the contamination of A. carbonarius and OTA on grapes.

Pest Management and Ochratoxin A Contamination in Grapes: A Review.

Ochratoxin A (OTA) is the most toxic member of ochratoxins, a group of toxic secondary metabolites produced by fungi. The most relevant species involved in OTA production in grapes is Aspergillus carbonarius. Berry infection by A. carbonarius is enhanced by damage to the skin caused by abiotic and biotic factors. Insect pests play a major role in European vineyards, and Lepidopteran species such as the European grapevine moth Lobesia botrana are undoubtedly crucial. New scenarios are also emerging due to the introduction and spread of allochthonous pests as well as climate change. Such pests may be involved in the dissemination of OTA producing fungi even if confirmation is still lacking and further studies are needed. An OTA predicting model is available, but it should be integrated with models aimed at forecasting L. botrana phenology and demography in order to improve model reliability.

Putting the Mess in Order: Aspergillus welwitschiae (and Not A. niger) Is the Etiological Agent of Sisal Bole Rot Disease in Brazil.

Approximately 75% of the worldwide production of hard natural fibers originates from sisal, an industrial crop from arid and semiarid tropical regions. Brazil is the world's largest producer of sisal fiber, accounting for more than 40% of the worldwide production, and sisal bole rot disease has been the main phytosanitary problem of this crop. All previous studies reporting Aspergillus niger as the causal agent of the disease were based on the morphological features of fungal isolates from infected plant tissues in pure cultures. Black aspergilli are one of the most complex and difficult groups to classify and identify. Therefore, we performed an integrative analysis of this disease based on the isolation of black aspergilli from the endospheres and soils in the root zones of symptomatic adult plants, in vivo pathogenicity tests, histopathology of symptomatic plants, and molecular phylogeny and worldwide genetic variability of the causal agent. All sisal isolates were pathogenic and unequivocally produced symptoms of bole rot disease in healthy plants. In all tree-based phylogenetic methods used, a monophyletic group formed by A. welwitschiae along with all sisal isolates was retrieved. Ten A. welwitschiae haplotypes have been identified in the world, and three occur in the largest sisal-producing area. Most of the isolates are from a unique haplotype, present in only the sisal-producing region. A. welwitschiae destroyed parenchymatic and vascular cylinder cells and induced the necrosis of internal stem tissues. Therefore, sisal bole disease is probably the consequence of a saprotrophic fungus that opportunistically invades sisal plants and behaves as a typical necrotrophic pathogen.

Occurrence and fumonisin B2 producing potential of Aspergillus section Nigri in Brazil nuts.

Bertholletia excelsa is the tree that produces Brazil nuts which have vast economic importance in the Amazon region and as an export commodity. The aim of this study was to assess the presence of Aspergillus section Nigri in Brazil nut samples at different stages of its production chain and to verify the toxigenic potential for fumonisin B2 (FB2) production of these isolates along with the presence of this mycotoxin in Brazil nut samples. The fungal infection ranged from 0 to 80% at the different stages of the harvest and processing chain and the water activity of the nuts from 0.273 to 0.994. A total of 1052 A. section Nigri strains were isolated from Brazil nuts and 200 strains were tested for their ability to produce FB2: 41 strains (20.5%) were FB2 producers with concentrations ranging from 0.09 to 37.25 mg/kg; 2 strains (1%) showed traces of FB2, less than the detection limit (0.08 mg/kg); and 157 (78.5%) were not FB2 producers. Although several samples showed high contamination by A. section Nigri, no sample was contaminated by FB2.

Differentiation and identification of grape-associated black aspergilli using Fourier transform infrared (FT-IR) spectroscopic analysis of mycelia.

The purpose of this study was to evaluate the potential of FT-IR spectroscopy as a high-throughput method for rapid differentiation among the ochratoxigenic species of Aspergillus carbonarius and the non-ochratoxigenic or low toxigenic species of Aspergillus niger aggregate, namely A. tubingensis and A. niger isolated previously from grapes of Greek vineyards. A total of 182 isolates of A. carbonarius, A. tubingensis, and A. niger were analyzed using FT-IR spectroscopy. The first derivative of specific spectral regions (3002-2801cm-1, 1773-1550cm-1, and 1286-952cm-1) were chosen and evaluated with respect to absorbance values. The average spectra of 130 fungal isolates were used for model calibration based on Discriminant analysis and the remaining 52 spectra were used for external model validation. This methodology was able to differentiate correctly 98.8% in total accuracy in both model calibration and validation. The per class accuracy for A. carbonarius was 95.3% and 100% for model calibration and validation, respectively, whereas for A. niger aggregate the per class accuracy amounted to 100% in both cases. The obtained results indicated that FT-IR could become a promising, fast, reliable and low-cost tool for the discrimination and differentiation of closely related fungal species.

Effects of different drying treatments on fungal population and ochratoxin A occurrence in sultana type grapes.

This study aimed to determine the changes in mould and ochratoxin A (OTA) occurrence in sultanas under three different conventional drying conditions. Five different vineyards were chosen, and the three different treatments were applied to these grapes while drying. At the end of the drying process, total mould and black aspergilli (BA) populations in the samples varied from 2.45 to 5.61 log colony-forming units (CFU) g(-)(1) and from 0 to 4.92 log CFU g(-)(1), respectively. Significant increases (p < 0.05) occurred in mould loads depending on the extending drying period. However, independent of vineyard location, all the samples treated with cold dipping solution showed the lowest fungal loads. These results indicate that dipping solution treatment was the most effective drying method to minimise fungal infection of grapes. The expected results could not be achieved by drying grapes artificially contaminated with ochratoxigenic Aspergillus carbonarius spores. Seventy-one of 96 isolates (73.95%) obtained during drying were Aspergillus spp., and the remaining (n = 25, 26.05%) belonged to other genera, such as Penicillium, Trichoderma and Cladosporium. Grape juice-based agar medium was used to determine the realistic OTA production capacities of the isolated mould strains. The highest OTA production capacities were 809.70 ± 9.19, 87.58 ± 16.89 and 45.44 ± 18.78 ng g(-1) in 50% grape juice agar (GJ50), all five of which were from A. niger isolates. OTA was not present in any sample during the drying period; however, OTA was detected in two samples at 0.32 ± 0.15 and 0.52 ± 0.36 µg kg(-)(1) after the end of the drying process. The limit of detection (LOD) and limit of quantitation (LOQ) of the method used for detecting OTA in samples were 0.1 and 0.3 µg kg(-)(1), respectively.

Ecophysiological characterization of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger isolated from grapes in Spanish vineyards.

The aim of this study was to evaluate the diversity of black aspergilli isolated from berries from different agroclimatic regions of Spain. Growth characterization (in terms of temperature and water activity requirements) of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger was carried out on synthetic grape medium. A. tubingensis and A. niger showed higher maximum temperatures for growth (>45 °C versus 40-42 °C), and lower minimum aw requirements (0.83 aw versus 0.87 aw) than A. carbonarius. No differences in growth boundaries due to their geographical origin were found within A. niger aggregate isolates. Conversely, A. carbonarius isolates from the hotter and drier region grew and produced OTA at lower aw than other isolates. However, little genetic diversity in A. carbonarius was observed for the microsatellites tested and the same sequence of ß-tubulin gene was observed; therefore intraspecific variability did not correlate with the geographical origin of the isolates or with their ability to produce OTA. Climatic change prediction points to drier and hotter climatic scenarios where A. tubingensis and A. niger could be even more prevalent over A. carbonarius, since they are better adapted to extreme high temperature and drier conditions.

Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants.

Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli-maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP1) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri.

Two novel species of Aspergillus section Nigri from indoor air.

Aspergillus floridensis and A. trinidadensis spp. nov. are described as novel uniseriate species of Aspergillus section Nigri isolated from air samples. To describe the species we used phenotypes from 7-d Czapek yeast extract agar culture (CYA), creatine agar culture (CREA) and malt extract agar culture (MEA), with support by molecular analysis of the ß-tubulin, calmodulin, RNA polymerase II (RPB2), and translation elongation factor-alpha (TEF) gene amplified and sequenced from 56 air isolates and one isolate from almonds belonging to Aspergillus sectionNigri.Aspergillus floridensis is closely related to A. aculeatus, and A. trinidadensis is closely related to A. aculeatinus. Aspergillus brunneoviolaceus (syn. A. fijiensis) and A. uvarum are reported for the first time from the USA and from the indoor air environment. The newly described species do not produce ochratoxin A.