Section Nigri Research Articles

FUNGAL CONTAMINATION AND MYCOTOXIN PRODUCTION BY ASPERGILLUS SPP. IN NUTS AND SESAME SEEDS

This work reports the occurrence of the fungal flora and evaluates the mycotoxigenic potential of Aspergillus genera in 63 samples of oil seeds and nuts (almonds, pistachio and sesame seeds). Fungal isolation and identification revealed the presence of 5 genera (Aspergillus, Penicillium, Cladosporium, Mucor and yeasts) with the predominance of Aspergillus section Nigri. A number of 138 strains of Aspergillus section Nigri and 91 of Aspergillus section Flavi were isolated and tested for their ability to produce ochratoxin A (OTA) and Aflatoxin, respectively. The detection of Aflatoxins and OTA production was carried out using thin-layer chromatography (TLC). Our results showed that 93.93% of Aspergillus carbonarius were able to produce OTA, but none of the Aspergillus niger aggregate was found to be an OTA-producer. Among the 91 Aspergillus section Flavi isolates, 88 were identified as Aspergillus flavus and 3 as Aspergillus parasiticus. All A. parasiticus were strong AFB and AFG producers. A percentage of 30.3% of A. flavus isolates produced AFB1, with levels ranging from 0.69 to 44.28 μg.g−1. The frequency of aflatoxigenic A. flavus strains was higher in pistachios (46.3%) than in almonds (30.3%) or sesame seeds (23.52%). Cyclopiazonic acid (CPA) and sclerotia production were carried out on CYA medium. All aflatoxigenic A. flavus strains produced CPA, whereas 29.2% produced sclerotia (L-type).

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).

Prospecting for the incidence of genes involved in ochratoxin and fumonisin biosynthesis in Brazilian strains of Aspergillus niger and Aspergillus welwitschiae

Aspergillus niger “aggregate” is an informal taxonomic rank that represents a group of species from the section Nigri. Among A. niger “aggregate” species Aspergillus niger sensu stricto and its cryptic species Aspergillus welwitschiae (=Aspergillus awamori sensu Perrone) are proven as ochratoxin A and fumonisin B2 producing species. A. niger has been frequently found in tropical and subtropical foods. A. welwitschiae is a new species, which was recently dismembered from the A. niger taxon. These species are morphologically very similar and molecular data are indispensable for their identification. A total of 175 Brazilian isolates previously identified as A. niger collected from dried fruits, Brazil nuts, coffee beans, grapes, cocoa and onions were investigated in this study. Based on partial calmodulin gene sequences about one-half of our isolates were identified as A. welwitschiae. This new species was the predominant species in onions analyzed in Brazil. A. niger and A. welwitschiae differ in their ability to produce ochratoxin A and fumonisin B2. Among A. niger isolates, approximately 32% were OTA producers, but in contrast only 1% of the A. welwitschiae isolates revealed the ability to produce ochratoxin A. Regarding fumonisin B2 production, there was a higher frequency of FB2 producing isolates in A. niger (74%) compared to A. welwitschiae (34%). Because not all A. niger and A. welwitschiae strains produce ochratoxin A and fumonisin B2, in this study a multiplex PCR was developed for detecting the presence of essential genes involved in ochratoxin (polyketide synthase and radHflavin-dependent halogenase) and fumonisin (α-oxoamine synthase) biosynthesis in the genome of A. niger and A. welwitschiae isolates. The frequency of strains harboring the mycotoxin genes was markedly different between A. niger and A. welwitschiae. All OTA producing isolates of A. niger and A. welwitschiae showed in their genome the pks and radH genes, and 95.2% of the nonproducing isolates did not contain these genes. The α-oxoamine synthase gene was detected in 100% and 36% of the A. niger and A. welwitschiae isolates, respectively. The loss of ochratoxin A production in A. niger and A. welwitschiae is highly associated with gene deletions within the ochratoxin biosynthetic gene cluster. The loss of fumonisin production in A. welwitschiae is associated with gene deletions within the fumonisin biosynthetic gene cluster, but this is not the case with A. niger.

Mycobiota of Slovak wine grapes with emphasis on Aspergillus and Penicillium species in the small carpathian area

The Slovak wine-growing region is divided into six viticulture areas. The largest in size and the most important over the centuries has been the Small Carpathian area (around 5800 ha of vineyards) spreads in the western of Slovakia. The objectives of this study were: to gain more knowledge about mycobiota on grapes originating from Slovakia, with a focus on genera Aspergillus and Penicillium and their ability to produce mycotoxins in in vitro conditions by thin layer chromatography method. From the twelve vineyards were collected 14 samples of wine grapes (white 6, blue 8) during harvesting 2011, 2012 and 2013. Fifty wine grapes per bunch (approximately 7-8 berries per plate) that showed no symptoms were randomly selected on Dichloran Rose Bengal Chloramphenicol agar medium. The plates were then incubated aerobically at 25 ±1 °C for 5 to 7 days in the dark. Of these samples were identified 22 genera. Ninety-three percent of samples were colonies by the genus Penicillium and 79% by the genus Aspergillus. During the survey, 251 isolates belonging to 14 Penicillium species (P. aurantiogriseum, P. citrinum, P. coprophylum, P. crustosum, P. expansum, P. funiculosum, P. glabrum, P. griseofulvum, P. chrysogenum, P. oxalicum, P. polonicum, P. purpurogenum, P. roqueforti and P. thomii) and 37 isolates belonging to 7 Aspergillus species (A. clavatus, A. flavus, A. section Nigri, A. ostianus, A. parasiticus, A. versicolor and A. westerdijkiae) were isolated and identified from exogenous contamination. The main occurring penicillium species of the samples were P. chrysogenum (36% Fr), followed P. crustosum (29% Fr), P. griseofulvum (21% Fr) and P. expansum (21% Fr). The main occurring aspergillus species of the samples were A. section Nigri (64%). Thirteen potentially toxigenic species were tested for their toxigenic ability. It was confirmed the production of various mycotoxins such as aflatoxin B1, G1, citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, roquefortin C and sterigmatocystin. Out of 124 strains, 84% produced at least one mycotoxin.

Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger.

BackgroundGalactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter.MethodsBased on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively.ResultsThe two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the Golgi.ConclusionA. niger contains two genes encoding UDP-Galf-transporters. Deletion and localization studies indicate that UgtA and UgtB have redundant functions in the biosynthesis of Galf-containing glycoconjugates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0541-2) contains supplementary material, which is available to authorized users.

Occurrence and biodiversity of Aspergillus section Nigri on ‘Tannat’ grapes in Uruguay

Ochratoxin A (OTA) is a nephrotoxic mycotoxin which has been found worldwide as a contaminant in wines. It is produced on grapes mainly by molds from Aspergillus section Nigri. This study has demonstrated for the first time the occurrence of black aspergilli on Tannat grapes from Uruguay, in a two year survey. Aspergillus uvarum (uniseriate) and Aspergillus welwitschiae (from Aspergillusniger aggregate) were the prevalent species whereas Aspergillus carbonarius which is considered the main OTA producing species was not detected. OTA production in culture medium was evaluated for native isolates from A. niger aggregate and compared to levels produced by a type strain of A. carbonarius. This work also includes the development of quick and easy molecular methods to identify black aspergilli to species level, avoiding sequencing.

Conidia survival of Aspergillus section Nigri, Flavi and Circumdati under UV-A and UV-B radiation with cycling temperature/light regime.

Bio-geographical differences in fungal infection distribution have been observed around the world, confirming that climatic conditions are decisive in colonization. This research is focused on the impact of ultraviolet radiation (UV) on Aspergillus species, based on the consideration that an increase in UV-B radiation may have large ecological effects. Conidia of six mycotoxigenic Aspergillus species isolated from vineyards located in the northeast and south of Spain were incubated for 15 days under light/dark cycles and temperatures between 20 and 30 °C per day. Additionally, 6 h of exposure to UV-A or UV-B radiation per day were included in the light exposure. UV irradiance used were 1.7 ± 0.2 mW cm(-2) of UV-A (peak 365 nm) and 0.10 ± 0.2 mW cm(-2) of UV-B (peak 312 nm). The intrinsic decrease in viability of conidia over time was accentuated when they were UV irradiated. UV-B radiation was more harmful. Conidial sensitivity to UV light was marked in Aspergillus section Circumdati. Conidia pigmentation could be related to UV sensitivity. Different resistance was observed within species belonging to sections Flavi and Nigri. An increase in UV radiation could lead to a reduction in the Aspergillus spp. inoculum present in the field (vineyards, nuts, cereal crops). In addition, it could unbalance the spore species present in the field, leading to a higher predominance of dark-pigmented conidia.

Toxigenic molds in Tunisian and Egyptian sorghum for human consumption

The objective of this study was to characterize the mycoflora of sorghum grains commercialized in the Tunisian retail market and to identify aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) producing species. Sixty four samples of sorghum (37 samples of Tunisian sorghum and 27 samples of Egyptian sorghum) were analyzed. Dilution plating (CFU, colony forming units) was used for fungal enumeration. The isolation of mycobiota was carried out by plating of grains on PDA and malachite green medium. Aspergillus section Flavi and section Nigri and Fusarium isolates were sub-cultured in CYA to test their ability to produce AFs, OTA and ZEA, respectively. The selected Aspergillus section Flavi and section Nigri, Penicillium and Fusarium isolates were subjected to specific PCR assays using published species-specific primers. The results revealed the dominance of Fusarium (95.3%), followed by Aspergillus (87.2%) and Alternaria (81.2%) species. The fungal counts ranged from 100 to 1.3·104 CFU/g for Tunisian sorghum and from 100 to 5.7·103 CFU/g for Egyptian sorghum. Among Aspergillus section Flavi isolates identified by molecular biology, Aspergillus flavus was the most dominant (90.1%) while Aspergillus parasiticus represent 9.9% only. About Aspergillus section Nigri, results showed the dominance of Aspergillus niger aggregate species, including Aspergillus niger, Aspergillus tubingensis and other species. Among Fusarium species, Fusarium incarnatum was the most dominant in both Tunisian and Egyptian sorghum. Penicillium citrinum was the dominant Penicillium species in the studied samples. More than 890 isolates belonging to the genus Aspergillus and Fusarium were tested in order to test their capacity to produce AFs, OTA and ZEA. The percentage of mycotoxin producing isolates in Aspergillus section Flavi, A. section Nigri, and Fusarium was 30.0%, 4.6% and 11.1%, respectively.

English

The mycotoxin contamination in foods has become a growing threat and aroused many researches in science area. According to the food control authorities, Ochratoxin A (OTA) is among mycotoxins priority of food contaminants. The current work focuses on coffee cherries quality environment in Ivory Coast. The neglecting of good agricultural practices would lead to recurrent contamination of ivorian coffee in mycotoxinogen fungi and mycotoxin. We investigated and sampled during the post-harvest drying process of Robusta coffee bean, brought from Ivory Coast in 2008 and 2009. Morphological identification of total fungal flora and the determination of OTA producers of Aspergillus section Nigri have been performed. The reliability of the overall evaluated fungal contamination is estimated at 97%, being 7.03 in one coffee sample package of 300 g. Strains were isolated on potato dextrose agar (PDA) by the direct plating technique, and were grown at 25°C. Morphological study was performed using macroscopic and microscopic morphological characters. From the two hundred and eighteen strains of fungi isolated, the following were identified: Aspergillus section Nigri, Aspergillus section Fumigati, Penicillium, Mucor, Fusarium amongst others. Aspergillus section Nigri was found to be the most important group representing 52% of the population. Within this section OTA production was evaluated on czapeck yeast agar (CYA) and quantify by High-performance liquid chromatography (HPLC). Twenty percent of produced detectable OTA with concentrations ranging from 0.3 to 56 µg/g of agar medium. The objectives of this study is to define the risk contamination in post-harvest fungi on coffee.   Key words: Agriculture, coffee, process, filamentous fungi, mycelium, Aspergillus, mycotoxin, Ochratoxin A.

Diversity of black Aspergilli isolated from raisins in Argentina: Polyphasic approach to species identification and development of SCAR markers for Aspergillus ibericus

Aspergillus section Nigri is a heterogeneous fungal group including some ochratoxin A producer species that usually contaminate raisins. The section contains the Series Carbonaria which includes the toxigenic species Aspergillus carbonarius and nontoxigenic Aspergillus ibericus that are phenotypically undistinguishable. The aim of this study was to examine the diversity of black aspergilli isolated from raisins and to develop a specific genetic marker to distinguish A. ibericus from A. carbonarius. The species most frequently found in raisins in this study were Aspergillus tubingensis (35.4%) and A. carbonarius (32.3%), followed by Aspergillus luchuensis (10.7%), Aspergillus japonicus (7.7%), Aspergillus niger (6.2%), Aspergillus welwitschiae (4.6%) and A. ibericus (3.1%). Based on inter-simple sequence repeat (ISSR) fingerprinting profiles of major Aspergillus section Nigri members, a sequence-characterized amplified region (SCAR) marker was identified. Primers were designed based on the conserved regions of the SCAR marker and were utilized in a PCR for simultaneous identification of A. carbonarius and A. ibericus. The detection level of the SCAR-PCR was found to be 0.01ng of purified DNA. The present SCAR-PCR is rapid and less cumbersome than conventional identification techniques and could be a supplementary strategy and a reliable tool for high-throughput sample analysis.

Hospital environment fungal contamination and aspergillosis risk in acute leukaemia patients in Sousse (Tunisia).

Hospital environment is considered the main source of invasive aspergillosis (IA) in leukemic patients. This study aimed to describe Aspergillus colonisation in leukemic patients and their hospital environment and to test whether Aspergillus environmental contamination was associated with IA. For a 2-year period including 14-month renovation work, 91 acute leukaemia inpatients at the hematology department of University hospital in Sousse (Tunisia) were prospectively included. The incidence of probable IA (EORTC/MSG criteria) was 9.9%. Fifty-six Aspergillus were isolated from 53 (6.5%) of 811 sputa collected from 35 (38.5%) patients. Aspergillus spp. were isolated in 59.7% of 494 air samples and in 52.8% of 1579 surface samples taken in the patients' room. Aspergillus section Nigri (72.7%) was the most frequent. Aspergillus contamination peaked in autumn and winter on surface and in summer and autumn in air samples and was higher (P=0.03) during the renovation work period. Multivariate analysis showed that for each Aspergillus section Nigri CFU airborne contamination IA risk increased by 1.05 (P=0.04). In Tunisia, Aspergillus section Nigri and Flavi, but not Fumigati, are chiefly involved in IA. Our findings support swift implementation of airborne fungal contamination control measures in areas where immunocompromised patient are hospitalised.

Sexual recombination in Aspergillus tubingensis

Aspergillus tubingensis from section Nigri (black Aspergilli) is closely related to A. niger and is used extensively in the industrial production of enzymes and organic acids. We recently discovered sexual reproduction in A. tubingensis, and in this study we demonstrate that the progeny are products of meiosis. Progeny were obtained from six crosses involving five MAT1-1 strains and two MAT1-2 strains. We examined three loci, including mating type (MAT), RNA polymerase II (RPB2) and β-tubulin (BT2), and found that 84% (58/69) of progeny were recombinants. Recombination associated with sexual reproduction in A. tubingensis provides a new option for the genetic improvement of industrial strains for enzyme and organic acid production.

Molecular Characterization of BlackAspergillusSpecies from Onion and Their Potential for Ochratoxin A and Fumonisin B2 Production

Onion bulbs can become contaminated with various molds during the storage period, the most important causal agents being black aspergilli (Aspergillus section Nigri). Taxonomic studies have revealed that this group of Aspergillus contains many species that cannot be reliably identified using standard morphological methods. Therefore, it is necessary to define the fungus causing this problem in the onion exactly, especially since some species assigned to section Nigri are well known as ochratoxin and/or fumonisin producers. Sixty fungal isolates belonging to 10 fungal genera were isolated from 40 onion samples originated from the Taif region in Saudi Arabia. Black aspergilli were detected in 37 onion samples. Using primer pairs (awaspec and Cmd6) designed based on partial calmodulin gene sequence data, 37 isolates were identified as A. welwitschiae. The ochratoxin A and fumonisin B2 contents of the onion samples were examined. No ochratoxins were detected in the collected samples, while fumonisin B2 was detected in 37.5% of the onion samples. Eighteen of 37 isolates of Aspergillus welwitschiae were recognized as potential producers for fumonisin B2. Multiplex polymerase chain reactions designed to detect biosynthetic genes of fumonisins confirmed these results.

English

Section Nigri is among the most important from the genus Aspergillus, since it possesses economical and biotechnological properties. Its identification is considered quite hard and complex. Due to this fact, Aspergillus taxonomy has been carried out by morphological, biochemical and molecular data. Morphological characterization besides molecular analyses was performed to properly identify Aspergillus section Nigri. A set of 42 Aspergillus section Nigri isolates were obtained from Brazilian Atlantic Rainforest. From these isolates, 16.7% originated from leaf litter layer and 83.3%, from soil. The isolates were divided into three different groups according to analyzed characteristics. 26 were identified as Aspergillus aculeatus, 12 as Aspergillus aculeatinus and 4 belonged to a group called “Aspergillus niger Aggregate”, considered as biseriate. Key words: Fungi, taxonomy, morphological identification, polymerase chain reaction (PCR), ITS.

MICROFUNGI AND MYCOTOXINS OF GRAPES FROM EASTERN SLOVAK WINE REGION

The current study investigated an endogenous mycobiota of grapes in Eastern wine region, Slovakia and detection a potentially pathogenic isolates to produce selected mycotoxins. Intact berries from four wine grape cultivars were tested. Seven/eight berries superficially sterilized from each samples were placed on a Dichloran Rose Bengal Chloramphenicol agar in a total of 50 and incubated at 25 °C, 5 - 7 days. A total of 582 isolates were obtained that belonged to ten genera: Alternaria, Aspergillus, Botrytis, Cladosporium, Epicoccum, Fusarium, Mucor, Penicillium, Rhizopus, Trichoderma and one unidentified genus Mycelium sterillium without creation fruiting bodies. The most frequent were genera Alternaria, Aspergillus, Botrytis and Penicillium with 100 % frequention. The most frequent Aspergillus species was Aspergillus section Nigri (100 %) and Penicillium species was Penicillium chrysogenum (50 %). The largest number of isolates belonged to Alternaria (275 isolates), Cladosporium (114 isolates) and Penicillium (92 isolates). For that reason the relative density of both genera were the highest 42 %, 19.6 % and 15.8 %, respectively. The selected isolates – Aspergillus flavus, Aspergillus section Nigri, Penicillium citrinum, Penicillium expansum and Penicillium chrysogenum were tested for patulin, citrinin, penitrem A, roquefortin C, ochratoxin A, aflatoxin B1, G1 and cyclopiazonic acid producing ability. Out of 11 strains 54 % produced at least one mycotoxin. In our research ochratoxigenic microfungi (some species of Aspergillus section Nigri) were found in grape samples but without production of OTA.

MICROFUNGI AND MYCOTOXINS OF GRAPES FROM TOKAJ WINE REGION

The aim of this study was to investigate a surface mycobiota of grapes and detection a potentially pathogenic isolates to produce selected mycotoxins. Three samples of wine grapes Furmint, Lipovina and Yellow muscat were collected in the Tokaj wine region in Viničky during the November harvest in 2012. Seven/eight berries from each sample were placed on a Dichloran Rose Bengal Chloramphenicol agar in a total of 50 and incubated at 25 °C for 5 - 7 days. After incubation from the samples were isolated following filamentous fungi: Alternaria, Aspergillus, Botrytis, Cladosporium, Mucor, Penicillium, Phoma, Rhizopus, Trichoderma and Mycelium sterillium. The most frequent were genera Aspergillus, Botrytis and Penicillium with 100 % frequention. The largest number of isolates belonged to Penicillium (368 isolates) and Botrytis (334 isolates). For that reason the relative density of both genera were the highest 47 % and 43 %, respectively. The selected isolates - Aspergillus section Nigri, Penicillium crustosum, Penicillium expansum and Penicillium chrysogenum were tested for patulin, citrinin, penitrem A, roquefortin C and ochratoxin A producing ability. A higher percentage of Penicillium crustosum isolates (79 %) were positive for penitrem A and all were positive for roquefortin C. A higher percentage of Penicillium expansum (83 %) were positive for patulin, 72 % for citrinin and they were all positive for roquefortin C. All isolates of Penicillium chrysogenum produced roquefortin C. As would be expected, ochratoxin A was not detected from Aspergillus section Nigri.

Effect of Water Activity, Temperature, and Mixed Fungal Spore Interactions on Ochratoxin A Production by Aspergillus carbonarius

The purpose of this work was to investigate the potential of two nontoxigenic Aspergillus section Nigri species (Aspergillus tubingensis and Aspergillus japonicus) to influence the in vitro ochratoxin A (OTA) production of three toxigenic Aspergillus carbonarius isolates (Ac-28, Ac-29, and Ac-33) from Greek vineyards of different geographical areas. OTA accumulation was evaluated by inoculation of 0:100, 25:75, 50:50, 75:25, and 100:0 ratios of mixed spore suspensions on a synthetic grape juice medium for up to 28 days at different temperatures (15, 20, and 25°C), water activity (aw) levels (0.95 and 0.98 aw) and incubation time (7, 14, 21, and 28 days). Results confirmed that environmental factors and fungal species had a significant effect on OTA production. Specifically, maximum OTA concentration for Ac-28 (3.21 μg g−1) and Ac-29 (7.69 μg g−1) was observed at 20°C/0.98 aw and for Ac-33 (9.13 μg g−1) at 15°C/0.95 aw, regardless of incubation time. Moreover, A. tubingensis had no significant influence on OTA concentration of all toxigenic isolates assayed, regardless of temperature, aw, and incubation time. On the other hand, the presence of A. japonicus slightly inhibited OTA production of Ac-29 and Ac-33, while for Ac-28, stimulation of OTA was observed in some cases. Overall, lower aw levels reduced OTA accumulation for Ac-28 and Ac-29, regardless of temperature, inoculum ratio, and time. On the contrary, for Ac-33, low aw increased OTA production, regardless of the investigated parameters. The importance of this study concerns the understanding of interspecific interactions on OTA diffusion by A. carbonarius in an attempt to find ways to prevent the presence of toxins in grapes and their derivatives.

Ochratoxin A and ochratoxin-producing fungi on cereal grain in China: a review

The mycotoxin ochratoxin A (OTA) is known to be the main contaminant of cereal grain and has become increasingly important in recent years. Therefore, a survey of ochratoxigenic fungi and OTA contamination in China is a special challenge. This paper summarises data on cereals and moulds (Aspergillus niger, Penicillium verrucosum, Aspergillus ochraceus, for example) and on grain and OTA from 1973 by searching Chinese information databases (NCKI, VIP, DuXiu etc.), calculating the OTA-producing mould detection rate, referring to sampling locations, latitude and temperature, and also combining six grain-producing areas of ochratoxigenic fungi and OTA positive rate through a comprehensive analysis. It is concluded that in China rice (excluding shell rice) has less OTA contamination than wheat or maize. The contamination of cereal grains with Aspergillus section Nigri (formerly of the A. niger group) is a serious problem in China, and these fungi may be the main ochratoxigenic fungi on cereals.

Identification Key for <i>Aspergillus</i> Species Isolated from Maize and Soil of Nandi County, Kenya

The aim of this study was to identify Aspergillus species isolated from maize kernels and soils of maize fields of Nandi County using macro and micro morphological characteristics. A cross sectional research design was used in the study and purposive sampling was employed to determine districts of Nandi County and sub locations where sampling was done. This study was part of a larger project whose aim was to survey aflatoxin exposure in the maize value chain. Aspergillus species were isolated from maize and soil samples using quarter strength potato dextrose agar and modified Rose Bengal agar respectively. Pure cultures of the isolates were sub cultured and transferred onto differential media; malt extract agar, czapek yeast extract agar and czapek dox agar for species identification using macro morphological characteristics. Fungal slides were prepared from pure cultures on potato dextrose agar media after three days to identify micro morphological characteristics. Based on morphological characteristics, seven sections of Aspergillus namely: Flavi, Fumigati, Nigri, Circumdati, Clavati, Nidulantes and Candidi were identified. Aspergillus section Flavi was the most predominant with 57% followed by section Nigri with 27% from maize and 58% of section Flavi followed by 26% of section Nigri from the soil across the three locations. Aspergillus sections Nidulantes and Candidi were rare and only recovered from the soil samples of Kaptumo location. All the Aspergillius flavus that formed sclerotia both from the soils or maize kernels were of the L strains. In conclusion Aspergillus section Flavi was most frequent during the isolation process and dominated with Aspergillus flavus from both the maize and soil. Morphological characteristics remain the primary tool for detection and identification of Aspergillus species. The significance for high incidence of Aspergillus section Flavi is in regard to their aflatoxin production profiles that poses a health threat to the community and it is of public health concern. Morphological characteristics as a primary tool for Aspergillus identification should be embraced and more personnel with the knowledge are required since modern and faster techniques are scarce and expensive.

Detection and discrimination of four Aspergillus section Nigri species by PCR.

Species of Aspergillus section Nigri are not easily distinguished by traditional morphological techniques, and typically are identified by DNA sequencing methods. We developed four PCR primers to distinguish between Aspergillus niger, Aspergillus welwitschiae, Aspergillus carbonarius and Aspergillus tubingensis, based on species-conserved differences in the calmodulin gene sequence. PCR amplification from total DNA using these primers was species specific; no amplification occurred from nontarget species DNA for each primer pair. Species-specific PCR could distinguish between species in mixed DNA templates, indicating a utility in determining culture uniformity of isolated Aspergillus strains. In addition, with these primer sets, each species could be detected in soil following mixed-species inoculation with Aspergillus spores. This indicates that PCR with these species-specific primers may be useful in determining the distribution of Aspergillus species in environmental samples without the need for species identification from isolated strains, as well as detecting species that may be infrequently isolated by culture-based methods.