Study of Spanish grape mycobiota and ochratoxin A production by Isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.     

Identification of Ochratoxigenic Aspergillus Section Nigri Isolated from Grapes by ITS‐5.8S rDNA Sequencing Analysis and In Silico RFLP

To characterize Aspergillus section Nigri strains involved in the ochratoxin A (OTA) contamination of Tunisian wine and table grapes, a total of 33 strains were analysed. A molecular characterization of the isolates was performed by the amplification of internal transcribed spacer (ITS1‐5.8S rDNA‐ITS2) region combined with amplicon sequencing. Analysis of similarity between the obtained sequences and those deposited in the GenBank database was performed. Twelve strains were confirmed to belong to the Aspergillus carbonarius species. Strains belonging to the Aspergillus niger aggregate group were classified by in silico RFLP assay into two patterns N and T, corresponding to A. niger and Aspergillus tubingensis. Among the 21 OTA producing isolates analysed, 13 showed the T‐type pattern and 8 showed the N‐type pattern. The presented method showed to be a reliable alternative to the classic RFLP method. Our findings unambiguously revealed that multiple aspergilli species isolated from wine and table grape in Tunisia are able to produce OTA.     

Biodiversity and ITS-RFLP Characterisation of Aspergillus Section Nigri Isolates in Grapes from Four Traditional Grape-Producing Areas in Greece

A study on the occurrence of Aspergillus section Nigri species on grapes from four traditional grape-producing areas in Greece during the 2011/2012 vintage, and their capability to produce OTA was conducted. One hundred and twenty-eight black aspergilli isolates were characterised at the species level initially by the use of morphological criteria in accordance with appropriate keys, followed by molecular characterisation performed with Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) of the 5.8 ribosomal RNA gene Internal Transcribed Spacer region (5.8 rRNA ITS). Restriction enzyme digestion of the ITS amplicons using the HhaI, HinfI and RsaI, endonucleases distinguished eleven different patterns of restriction fragment length polymorphism (RFLP), four for each of the HhaI and RsaI digests and three for HinfI. From a total number of 128 individual isolates, 124 were classified into four Aspergillus species corresponding to A. carbonarius, A. tubingensis, A. japonicus and A. ibericus, and the remaining 4 were classified as members of the A. niger aggregate. A. carbonarius and A. tubingensis being the main representative species were equally counted, with higher geographical representation of the former in southern and the latter in northern regions, respectively. All isolates were tested for their ochratoxigenic potential by use of High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immuno Sorbent Assay (ELISA), resulting in significant interspecies differences in OTA production.     

Aspergillus niger and Aspergillus carbonarius in Corinth Raisin and Wine-Producing Vineyards in Greece: Population Composition, Ochratoxin A Production and Chemical Control

Vineyard surveys of Corinth raisin cultivar carried out in the Peloponnese region of Greece during 2002 and of wine‐producing grape cultivars (Cabernet Sauvignon and Grenache Rouge) on the island of Rhodes, Greece, during 2003, demonstrated the occurrence of various Aspergillus spp. in berries of bunches at harvest. Aspergillus niger and A. carbonarius were predominantly isolated from sampled berries. Although the prevailing Aspergillus spp. isolates belonged mainly to A. niger aggregate, isolates of A. carbonarius were by far the most efficient Ochratoxin A (OTA) producers as revealed by the enzyme‐linked immunosorbent assay test. This study provides the first evidence concerning the composition of Aspergillus populations in raisin and wine‐producing vineyards and offers convincing data for their ability to produce various levels of OTA in Corinth raisins and wine‐producing grapes in Greece. Furthermore, it demonstrates that chemical applications with the fungicide Switch, especially under low to intermediate Aspergillus infection of vineyards, could both significantly reduce the occurrence of OTA‐producing Aspergillus spp. and restrict sour rot severity. In contrast, vineyard applications with the fungicides Carbendazim or Chorus were ineffective in controlling the fungus in Corinth raisin cultivar.     

Isolation and Sequencing of a New Glucoamylase Gene from an Aspergillus niger Aggregate Strain (DSM 823) Molecularly Classified as Aspergillus tubingensis

Based on morphological characteristics the taxa included in the Aspergillus aggregate can hardly be differentiated. For that reason the phylogeny of this genus was revised several times as different criteria, from morphological to later molecular, were used. We found, comparing nucleotide sequences of the ITS-region, that the strain Aspergillus niger (DSM 823) which is claimed to be identical to the strains ATCC 10577, IMI 027809, NCTC 7193 and NRRL 2322 can be molecularly classified as Aspergillus tubingensis, exhibiting 100% identity with the A. tubingensis CBS strains 643.92 and 127.49. We amplified, cloned and sequenced a new glucoamylase gene (glaA) from this strain of A. tubingensis (A. niger DSM 823) using primers derived from A. niger glucoamylase G1. The amplified cDNA fragment of 2013 bp contained an open reading frame encoding 648 amino acid residues. The calculated molecular mass of the glucoamylase, deduced from the amino acid sequence, was 68 kDa. The nucleotide sequence of glaA showed 99% similarity with glucoamylases from Aspergillus kawachii and Aspergillus shirousami, whereas the similarity with the glucoamylase G1 from A. niger was 92% An erratum to this article is available at .     

Ochratoxin A Production and Amplified Fragment Length Polymorphism Analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger Strains Isolated from Grapes in Italy

Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than Aspergillus carbonarius within Aspergillus section Nigri to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and β-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of A. carbonarius produced ochratoxin A (6 to 7,500 μg/liter), 5 of 20 strains of A. tubingensis produced ochratoxin A (4 to 130 μg/liter), 3 of 15 strains of A. niger produced ochratoxin A (250 to 360 μg/liter), and none of the 19 strains of Aspergillus “uniseriate” produced ochratoxin A above the level of detection (4 μg/liter). These findings indicate that A. tubingensis is able to produce ochratoxin and that, together with A. carbonarius and A. niger, it may be responsible for the ochratoxin contamination of wine in Italy.     

The Molecular Identification and Antifungal Susceptibilities of Aspergillus Species Causing Otomycosis in Tochigi,Japan

Aspergillus species are the most common pathogenic fungi involved in otomycosis, an infection of the outer ear canal. In this study, we examined the incidence of Aspergillus infections and the antifungal susceptibilities of 30 Aspergillus species isolates from patients with otomycosis who visited Saiseikai Utsunomiya Hospital between August 2013 and July 2016. Based on the morphological test results, the strains were identified as Aspergillus niger sensu lato (20 strains), A. terreus sensu lato (7 strains), and A. fumigatus sensu lato (3 strains). In contrast, the molecular identifications based on analyzing the isolates’ partial β-tubulin gene sequences revealed them to be A. niger sensu stricto (12 strains), A. tubingensis (8 strains), A. terreus sensu stricto (7 strains), and A. fumigatus sensu stricto (3 strains). The antifungal susceptibility test results indicated that strains of A. tubingensis and A. niger sensu stricto displayed lower susceptibilities to ravuconazole, compared with the other isolates. The Aspergillus strains from this study showed low minimum inhibitory concentrations toward the azole-based drugs efinaconazole, lanoconazole, and luliconazole. Therefore, these topical therapeutic agents may be effective for the treatment of otomycosis.

     

Mycoremediation of crude oil contaminated soil by specific fungi isolated from Dhahran in Saudi Arabia

Crude oil biodegrading microorganism considers the key role for environmental preserving. In this investigation, crude oil biodegrading fungal strains have been isolated in polluted soil of crude-oil at khurais oil ground in Kingdom of Saudi Arabia. Among of 22 fungal isolates, only three isolates reflected potential capability for oil degradation. These isolates were identified and submitted to GenBank as (A1) Aspergillus polyporicola (MT448790), (A2) Aspergillus spelaeus (MT448791) and (A3) Aspergillus niger (MT459302) through internal-transcribed spacer-regions (ITS1&ITS2) for sequencing in molecular marker. Comparing with controls, strain (A1) Aspergillus niger was superior for biodegradation ability (58%) comparing with Aspergillus polyporicola and Aspergillus spelaeus degrading were showed 47 and 51% respectively. Employed CO₂ evolution as indicator for petroleum oil biodegradation by the fungal isolates reflected that, Aspergillus niger emission highest CO2 (28.6%) comparing with Aspergillus spelaeus and Aspergillus polyporicola which showed 13% and 12.4% respectively. capability of Aspergillus sp. to tolerate and adapted oil pollutants with successful growth rate on them, indicated that it can be employed as mycoremediation agent for recovering restoring ecosystem when contaminated by crude oil.     

Monitoring of ochratoxin A and ochratoxin-producing fungi in traditional salami manufactured in Northern Italy

Fungi have a crucial role in the correct maturation of salami, but special attention should be addressed to the production of the nephrotoxic, immunotoxic, and carcinogenic mycotoxin ochratoxin A (OTA). In a monitoring study conducted in Northern Italy, OTA was detected by liquid chromatography coupled with mass spectrometry in 13 out 133 samples of traditional salami (9.8% of the total count). Mycological analysis of these samples yielded 247 fungal isolates which were identified to species level. The most frequent species were Penicillium nalgiovense, P. solitum, and P. chrysogenum. P. nordicum, an OTA-producing species commonly found in proteinaceous food, was not found in these samples. Three isolates were found to be Aspergillus westerdijkiae, an OTA-producing species. In order to check the results of the microbiological identification, 19 different strains of Aspergillus and 94 of Penicillium were tested for the presence of a sequence common to OTA-producing fungi by real-time PCR. None of the studied isolates, including the three A. westerdijkiae, possessed the otanpsPN target which is common to OTA-producing strains. Two out of three isolates of the A. westerdijkiae were also PCR-negative for the otanpsPN gene and did not produce OTA in culture. Conversely, this target sequence was amplified from the DNA purified from 14 salami casings including three casings harboring A. westerdijkiae. The amplification of sequences specific for OTA-producing strains performed on total genomic DNA extracted directly from salami casings provided a more suitable approach than PCR analysis of isolates from salami for the OTA-related otanpsPN gene to evaluate the risk of OTA contamination.     

Survey of Philippine coffee beans for the presence of ochratoxigenic fungi

In 2012 to 2014, Philippine green coffee beans from Coffea arabica in Benguet and Ifugao; Coffea canephora var. Robusta in Abra, Cavite, and Ifugao; and Coffea liberica and Coffea excelsea from Cavite were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The presence of fungal species was evaluated both before and after surface sterilization. There were remarkable ecological and varietal differences in the population of OTA-producing species from the five provinces. Aspergillus ochraceus, A. westerdijkiae, and Penicillium verruculosum were detected from Arabica in Benguet and Ifugao while Aspergillus carbonarius, Aspergillus niger, and Aspergillus japonicus were isolated in Excelsa, Liberica, and Robusta varieties from Abra, Cavite, and Davao. Contamination by Aspergillus and Penicillium species was found on 59 and 19 %, respectively, of the 57 samples from five provinces. After disinfection with 1 % sodium hypochlorite, the levels of infection by Aspergillus and Penicillium fell to 40 and 17 %, respectively. A total of 1184 fungal isolates were identified to species level comprising Aspergillus sections Circumdati (four species), Clavati (one), Flavi (one), Fumigati (one), Nigri (three), and Terrie (one). Within section Circumdati, 70 % of A. ochraceus produced OTA as high as 16238 ng g^?1 while 40 % of A. westerdijkiae produced maximum OTA of 36561 ng g^?1 in solid agar. Within section Nigri, 16.76 % of A. niger produced OTA at the highest 18439 ng g^?1, 10 % of A. japonicus at maximum level of 174 ng g^?1, and 21.21 % of A. carbonarius yielded maximum OTA of 1900 ng g^?1. Of the 12 species of Penicillium isolated, P. verruculosum was ochratoxigenic, with a maximum OTA production of 12 ng g^?1.     

Molecular characterization and toxigenic profile of Aspergillus section Nigri populations isolated from the main grape-growing regions in Argentina

Aims: The objective of this study was to evaluate the biodiversity of Aspergillus section Nigri populations from Argentinean vineyards by morphological, toxigenic and AFLP analysis. Materials and methods: Five hundred and thirty‐eight strains were isolated from grapes during 2006/07 and 2007/08 vintages. The morphological identification and toxigenic profile for all strains isolated were performed. Eighty‐eight strains were selected for characterization at species level by AFLP markers. Cluster analysis showed a clear separation into four main groups: A. carbonarius, A. tubingensis, A. niger‘aggregate’ and Aspergillus‘uniseriate’. A. carbonarius strains constituted a homogeneous group, while a high degree of genetic diversity was found within the A. niger‘aggregate’ and ‘A. uniseriate’ clusters. The A. tubingensis cluster was the most prevalent group and was clearly separated from A. niger‘aggregate’. Ten strains showed 45% homology with A. tubingensis FRR 5720 ex‐type strain and were considered as ‘atypical’ or a closely related species. AFLP results indicate that no genotypical differences can be established between ochratoxigenic and nonochratoxigenic strains. Conclusions: Aspergillus section Nigri populations on grapes were represented mainly by four groups. A. tubingensis species were separated from A. niger‘aggregate’ group and some of their strains produced OTA. Significance and Impact of the Study: This study provides new data on molecular characterization of Aspergillus section Nigri populations in Argentina.     

Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material

The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.     

Aspergillosis by cryptic Aspergillus species: A case series and review of the literature

BackgroundThe cryptic Aspegillus species are rare, these microorganisms are usually more resistant to common antifungal therapies. Therefore, a correct identification is important when evaluating the impact of such species in aspergillosis.AimsWe aimed to describe the frequency, clinical and microbiological characteristics, and the outcomes of those cases of aspergillosis caused by cryptic species in a tertiary hospital.MethodsWe retrospectively identified all microbiologically documented cases of aspergillosis between January 2013 and December 2018. Definitive species identification of clinically significant isolates was achieved via sequencing methods. The polymerase chain reaction (PCR) products were sequenced, and the results obtained were compared to sequences deposited in GenBank. Antifungal susceptibility testing was performed using the Sensititre® YeastOne® panel.ResultsA total of 679 Aspergillus isolates were recovered from 489 patients, of which 109 were clinically relevant. Ten (9.2%) isolates were identified as cryptic species: Aspergillus arcoverdensis (2), Aspergillus lentulus (2), Aspergillus ellipticus (2), Aspergillus alliaceus (1), Aspergillus nomius (1), Aspergillus tubingensis (1) and Aspergillus montevidensis (1). Most patients already suffered some type of immunosuppression. Half of these patients had required intensive care before the infection showed up, and most of them had a pulmonary infection. Mortality at the 100-day follow-up was 40%. Antifungal susceptibility testing was performed on three of the isolates (A. arcoverdensis, A. tubingensis and A. nomius), which showed high minimum inhibitory concentrations (MIC) for azoles and amphotericin B.ConclusionsThe frequency of cryptic species in our centre was 9.2%. Most patients had some degree of immunosuppression, and the mortality rate was 40%.     

Impact of some environmental factors on growth and production of ochratoxin A of/by <Emphasis Type="Italic">Aspergillus tubingensis,A. niger</Emphasis>, and <Emphasis Type="Italic">A. carbonarius</Emphasis> isolated from moroccan grapes

The effects of temperature, water activity (a_w), incubation time, and their combinations on radial growth and ochratoxin A (OTA) production of/by eight Aspergillus niger aggregate strains (six A. tubingensis and two A. niger) and four A. carbonarius isolated from Moroccan grapes were studied. Optimal conditions for the growth of most studied strains were shown to be at 25°C and 0.95 a_w. No growth was observed at 10°C regardless of the water activity and isolates. The optimal temperature for OTA production was in the range of 25°C∼30°C for A. carbonarius and 30°C∼37°C for A. niger aggregate. The optimal a_w for toxin production was 0.95∼0.99 for A. carbonarius and 0.90∼0.95 for A. niger aggregate. Mean OTA concentration produced by all the isolates of A. niger aggregate tested at all sampling times shows that maximum amount of OTA (0.24 μg/g) was produced at 37°C and 0.90 a_w. However, for A. carbonarius, mean maximum amounts of OTA (0.22 μg/g) were observed at 25°C and 0.99 a_w. Analysis of variance showed that the effects of all single factors (a_w, isolate, temperature and incubation time) and their interactions on growth and OTA production were highly significant.     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA‐producing A. carbonarius and B. cinerea were grown on grape‐like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape‐like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA‐spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day^?1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA‐producing Aspergillus in some wine‐growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation.     

New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.     

Isolation and identification of ochratoxin A-producing Aspergillus section Nigri strains from California raisins

Aims: To determine incidence and levels of ochratoxin A (OTA) in California raisins and to isolate and characterize OTA‐producing fungi from California raisin vineyard populations. Methods and Results: Forty raisin clusters sampled from four California vineyards in the San Joaquin Valley were analysed for OTA content using immunoaffinity and HPLC methods. OTA was detected in 93% of the samples, at levels from 0·06 to 11·4 ng g^?1. From these raisin samples, a total of 400 strains of Aspergillus were isolated and analysed for OTA production. Twelve isolates (3%), from five raisin samples, produced OTA. These isolates were identified as Aspergillus carbonarius, based on morphological characteristics and multilocus sequence analysis. Levels of OTA produced by these isolates on raisin agar ranged from 0·9 to 15 μg g^?1. Conclusions: OTA is a common contaminant of raisin vineyards, but average levels are much lower than EU regulatory limits for dried fruit. The primary species responsible for OTA contamination in California raisins is A. carbonarius. Significance and Impact of the Study: This study illustrates that low‐level OTA contamination of raisins occurs in California and that ecological studies of A. carbonarius within the Aspergillus section Nigri population on raisins are warranted to monitor ochratoxigenic potential of the crop.     

A New Black Aspergillus Species, A. vadensis, Is a Promising Host for Homologous and Heterologous Protein Production

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.     

Survey of mycobiota,black <Emphasis Type="Italic">Aspergillus</Emphasis> and ochratoxin A occurrence on Brazilian wine grapes

The southern state of Brazil is responsible for 90 % of the wine produced in the country; however, the mycobiota and the occurrence of ochratoxin A (OTA) on wine grapes produced in this region has never been fully characterized. To evaluate the fungal and OTA contamination in wine grapes of two varieties cultivated in southern Brazil, a survey was conducted in three regions with slightly different climates. From each region, samples were obtained at early ripeness and at harvest stage. Eight genera were isolated; Alternaria and Trichoderma were the most prevalent. Within the Aspergillus genus, the section Nigri was predominant. One uniseriate black Aspergillus was able to produce OTA under the conditions tested but no detectable levels of OTA were found in the grape samples. The low overall incidence of black Aspergillus and the absence of OTA on the grapes may be explained by the meteorological conditions at the analyzed regions, and characterizes a high quality of this product regarding mycotoxigenic contamination. This is the first study to fully describe the mycological biota of two wine grape varieties produced at three different regions of Rio Grande do Sul state.     

Growth and ochratoxin A production by Aspergillus carbonarius at different pHs and grape maturation stages

Aims: The objective of this work was to study the effect of some factors, linked to grape composition during ripeness process, on the growth and ochratoxin A (OTA) production of Aspergillus carbonarius isolated from grapes. Methods and Results: Aspergillus carbonarius isolates were tested (i) in vitro, in Czapek yeast autolysate agar (CYA) at different pHs (2·5–4·5) and incubation times (2–6 days), and (ii) in situ, in fresh grapes collected at different maturation stages. Aspergillus carbonarius was able to grow with the same intensity at the different maturation stages and pH levels tested. In general, a similar trend of OTA production by A. carbonarius in response to acidity in media and in grape was observed. Low pH level seemed optimal for maximum OTA production. Conclusions: Aspergillus carbonarius strains can strongly grow and produce OTA on grape from the early stages of maturation. Extrinsic environmental conditions at the harvest period and skin thickness are, probably, the mains factors contributing to OTA contamination of grapes at the end of maturation. Significance and Impact of the Study: The results lead to a better understanding of the critical point during grape maturation for the growth of ochratoxigenic fungi and the toxin production.