Interpretation of intraspecific variability in mtDNAs of Aspergillus niger strains and rearrangement of their mtDNAs following mitochondrial transmissions

Physical and functional maps of mitochondrial DNAs of Aspergillus niger strains representing different mitochondrial DNA RFLP patterns were constructed and compared. In spite of the high similarity in the organisation of mitochondrial DNAs among examined strains, differences could be easily recognised by applying molecular markers, such as the different intron content of the cox1 genes, the sequence of the intergenic regions between the Met- and His-tRNA genes and downstream of the tRNA-Gly gene. Intraspecific mitochondrial transfers between the heterokaryon incompatible mitochondrial oligomycin-resistant A. niger strain, as the donor, and other A. niger-sensitive strains bearing different RFLP patterns resulted in oligomycin-resistant progeny possessing either rearranged or unchanged donor mitochondrial DNA and recipient nuclei. Since the intergenic marker sequences of mitochondrial DNAs turned out to be identical in the donor and the progeny, it can be assumed that the oligomycin-resistant progeny inherit the mitochondrial DNA of the donor strain; this may either remain unchanged or may be modified by a mobile intron of the cox1 gene of the recipient mitochondria.     

Effects of water activity and temperature on the survival of Aspergillus carbonarius spores in vitro

AIMS: To determine the effect of water activity (a(w)) and temperature on the survival of Aspergillus carbonarius spores. METHODS AND RESULTS: Spores of A. carbonarius were dried onto filter membranes. These filters were held at 1.0, 0.9, 0.8, 0.6 and 0.4 a(w) and at 1, 15, 25 and 37 degrees C for up to 618 d. At intervals, spores were recovered from filters and assessed for viability by enumeration on dichloran rose bengal chloramphenicol agar. Survival and subsequent growth of spores was prolonged at low temperatures and at a(w) below 0.6. Above 15 degrees C, 0.6-0.9 a(w) were often more deleterious than 1.0. However, at 1 degrees C and 1.0 a(w), spores lost viability more rapidly than at lower a(w). CONCLUSIONS: Increased incidence of black Aspergillus spp. in dry soils and from grapes in dry conditions may result partly from prolonged survival of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Delineating the direct effect of a(w) and temperature on survival of A. carbonarius spores may aid in understanding the incidence of this ochratoxin A-producing species in vineyard soils and on grapes.     

Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples

AIMS: The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. CONCLUSIONS: No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.     

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.     

Partitioning of ochratoxin A in mycelium and conidia of Aspergillus carbonarius and the impact on toxin contamination of grapes and wine

AIMS: Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus which is responsible for toxin contamination of grapes and wine. The objectives of this study were to examine the partitioning of OTA in mycelium and conidia of a range of A. carbonarius strains on artificial grape juice and defined media, to determine the excretion patterns of OTA from these spores, and the effect of organic acids used in wine production on OTA excretion from conidia. METHODS AND RESULTS: The results showed that 60-70% of the OTA was accumulated in the conidia of a number of different isolates of A. carbonarius. Calculations showed that on different defined media, an amount of 0.011- to 0.1-pg OTA was present per conidium. The OTA in spores was found to be rapidly excreted into the medium during the initial few hours after conidial germination leading to an increase of OTA in must during maceration for wine production. The presence of tartaric acid inhibited OTA production, but malic acid enhanced this production during mycelial growth. These acids were also shown to affect the time course of germination and the rate of OTA excretion from conidia during germination. CONCLUSIONS: This study is the first to examine and show the partitioning of OTA into spores of strains of A. carbonarius and that rapid excretion of OTA from spores could be a reason for OTA accumulation in musts during wine production. SIGNIFICANCE AND IMPACT OF THE STUDY: Conidia of A. carbonarius could be a major source of OTA contamination of grapes used in wine production. This information could help in the development of effective prevention strategies to minimize wine contamination with this important mycotoxin.     

Aspergillus species producing ochratoxin A: isolation from vineyard soils and infection of Semillon bunches in Australia

AIMS: The incidence of toxigenicity among Australian isolates of Aspergillus niger and Aspergillus carbonarius was assessed. Aspergillus rot and concomitant production of ochratoxin A (OA) in bunches inoculated with A. carbonarius were also investigated. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated from vineyard soils. Aspergillus niger was more widespread than A. carbonarius, and two restriction fragment length polymorphism types of A. niger, N and T, were present. Three of 113 A. niger isolates and all 33 A. carbonarius isolates produced OA. Aspergillus carbonarius was inoculated onto Semillon bunches with and without damage in the month before harvest. Damaged berries at greater than 12.3 (o) Bx were particularly susceptible to Aspergillus rot and production of OA, which was concentrated in severely mouldy berries. CONCLUSIONS: OA in Australian grapes results mainly from infection of berries by A. carbonarius. It is concentrated in discoloured, shrivelled berries. The potential for Aspergillus rot and OA production appears to commence after veraison and increase with berry damage and ripeness. SIGNIFICANCE AND IMPACT OF THE STUDY: Minimizing damage to grapes between veraison and harvest significantly reduces Aspergillus rot and OA formation. Monitoring the extent of Aspergillus rot in bunches infected with toxigenic Aspergillus spp. may give some indication of OA contamination.     

Predictive assessment of ochratoxin A accumulation in grape juice based-medium by Aspergillus carbonarius using neural networks

Aims:  To study the ability of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict ochratoxin A (OTA) concentration over time in grape-based cultures of Aspergillus carbonarius under different conditions of temperature, water activity ( a _w) and sub-inhibitory doses of the fungicide carbendazim.
Methods and Results:  A strain of A. carbonarius was cultured in a red grape juice-based medium. The input variables to the network were temperature (20–28°C), a _w (0·94–0·98), carbendazim level (0–450 ng ml⁻¹) and time (3–15 days after the lag phase). The output of the ANNs was OTA level determined by liquid chromatography. Three algorithms were comparatively tested for MLP. The lowest error was obtained by MLP without validation. Performance decreased when hold-out validation was accomplished but the risk of over-fitting is also lower. The best MLP architecture was determined. RBFNs provided similar performances but a substantially higher number of hidden nodes were needed.
Conclusions:  ANNs are useful to predict OTA level in grape juice cultures of A. carbonarius over a range of a _w, temperature and carbendazim doses.
Significance and Impact of the Study:  This is a pioneering study on the application of ANNs to forecast OTA accumulation in food based substrates. These models can be similarly applied to other mycotoxins and fungal species.     

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, Structure elucidation, and antimycobacterial properties of dimeric naphtho-gamma-pyrones from the marine-derived fungus Aspergillus carbonarius

Two new dimeric naphtho-gamma-pyrones, compounds 1 and 2, were isolated from the AcOEt extract of the fungal strain WZ-4-11 of Aspergillus carbonarius, together with eight known analogues, including 10,10'-bifonsecin B (3), 6'-O-demethylnigerone (4), nigerone (5), isonigerone (6), fonsecin (7), rubrofusarin B (8), TMC 256A1 (9), and flavasperone (10). Their structures were elucidated by means of UV, CD, IR, and 1D- and 2D-NMR spectroscopy, in combination with HR-MS analysis. The fully assigned (1)H- and (13)C-NMR data of 3, and the (13)C-NMR data of 6 are reported for the first time. Compounds 1 and 2 showed weak antimycobacterial activities against Mycobacterium tuberculosis H37Rv, with MIC values of 43.0 and 21.5 microM, resp.     

Water and temperature relations of growth and ochratoxin A production by Aspergillus carbonarius strains from grapes in Europe and Israel

AIMS: This study investigated the in vitro effects of water activity (a(w); 0.85-0.987) and temperature (10-40 degrees C) on growth and ochratoxin A (OTA) production by two strains of Aspergillus carbonarius isolated from wine grapes from three different European countries and Israel on a synthetic grape juice medium representative of mid-veraison (total of eight strains). METHODS AND RESULTS: The synthetic grape juice medium was modified with glycerol or glucose and experiments carried out for up to 56 days for growth and 25 days for OTA production. The lag phase prior to growth, growth rates and ochratoxin production were quantified. Statistical comparisons were made of all factors and multiple regression analysis used to obtain surface response curves of a(w) x temperature for the eight strains and optimum growth and OTA production by A. carbonarius. The lag phase increased from <1 day at 25-35 degrees C and 0.98 a(w) to >20 days at marginal temperatures and water availabilities. Generally, most A. carbonarius strains grew optimally at 30-35 degrees C, regardless of solute used to modify a(w), with no growth at <15 degrees C. The optimum a(w) for growth varied from 0.93 to 0.987 depending on the strain, with the widest a(w) tolerance at 25-30 degrees C. There was no direct relationship among growth, environmental factors and country of origin of individual strains. Optimum conditions for OTA production varied with strain. Some strains produced optimal OTA at 15-20 degrees C and 0.95-98 a(w). The maximum OTA produced after 10 days was about 0.6-0.7 microg g(-1), with a mean production over all eight strains of 0.2 microg g(-1) at optimum environmental conditions. CONCLUSIONS: This work demonstrates that optimum conditions for OTA production are very different from those for growth. While growth rates differed significantly between strains, integration of the OTA production data suggests possible benefits for use of the information on a regional basis. SIGNIFICANCE AND IMPACT OF THE STUDY: Very little detailed information has previously been available on the ecology of A. carbonarius. This knowledge is critical in the development and prediction of the risk models of contamination of grapes and grape products by this species under fluctuating and interacting environmental parameters.     

Efficacy of natamycin for control of growth and ochratoxin A production by Aspergillus carbonarius strains under different environmental conditions

AIMS: To examine the efficacy of natamycin produced by Streptomyces natalensis against strains of Aspergillus carbonarius growth and ochratoxin A (OTA) production under different environmental factors on a grape juice-based medium. METHODS AND RESULTS: Detailed studies in the range 0-20 ng ml(-1) for control of growth and ochratoxin production by strains of A. carbonarius at 0.98, 0.96 and 0.94 water availabilities (a(w)) and 15-25 degrees C on a fresh red grape extract medium were examined. Inhibition of growth was depending on temperature and a(w) level. At 15 degrees C, 5-10 ng ml(-1) natamycin was effective in reducing growth almost completely. However, at 20-25 degrees C and all the three a(w) levels, growth was only slightly inhibited by 5-10 ng ml(-1) natamycin. There were strain differences with regard to inhibition of OTA production. At 15 degrees C and 0.98 a(w), 10 ng ml(-1) was required to inhibit production by >90%. However, at 0.96 and 0.94 a(w), almost complete inhibition occurred. At 20 degrees C, OTA production was only significantly inhibited by 10 ng ml(-1) natamycin at 0.94 a(w). At 0.96 and 0.98 a(w), some inhibition occurred with 5-10 ng ml(-1), but greater concentrations would be required for effective inhibition. At 25 degrees C, 5 ng ml(-1) was effective at all a(w) levels. However, at 15 degrees C and 25 degrees C and a wide range of a(w) levels, natamycin effectively controlled OTA production. CONCLUSIONS: Natamycin appears to be a very effective for controlling growth and OTA production by strains of A. carbonarius over a range of a(w) and temperature conditions on grape-based media. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study to demonstrate the impact of natamycin against A. carbonarius. This study suggests that use of natamycin at 50-100 ng ml(-1) can give complete inhibition of growth of A. carbonarius and OTA production over a range of environmental conditions. Natamycin could be an important component of a system to prevent OTA contamination of wine as well during the drying and production of vine fruits.     

A low rate of replacement substitutions in two major Ovis aries mitochondrial genomes

Two mitochondrial DNA molecules which represent major Ovis aries mtDNA haplogroups were cloned and comparatively sequenced to assess the degree of intraspecific variation. A total of 9623 bp that correspond to 58% of both mitochondrial genomes were determined. The control region, the Cyt b , ND2, ND3, ND4L, COIII and 12 tRNA genes, including the origin of L-strand replication, were completely characterized. Partial sequence information was obtained from the 12S and 16S rRNA and an additional six protein coding and six tRNA genes. The control regions of the two mtDNAs showed a nucleotide divergence of 4·34% while coding regions differed by 0·44%. The number of sheep coding region substitutions was similar to values observed in intraspecific comparisons of mitochondrial DNAs that represent remote points in genealogical trees of mice and humans. However, replacement substitutions were only observed at ∼30% of the rate in mice and ∼20% of the rate in humans. Nucleotide substitutions with a potential for phenotypic effects were found in the 12S and 16S rRNA and in the ND1 and COIII genes.     

Physical mapping of the mitochondrial DNA from Aspergillus niger

Abstract The mitochondrial DNA was isolated from Aspergillus niger WU-2223L, a citric acid-production strain, and characterized by restriction-endonuclease mapping. Cloned fragments which covered the total range of the mitochondrial DNA were assembled and utilized to construct the restriction-endonuclease map for nine restriction enzymes. This map showed that the mitochondrial DNA was a circular molecule of 32.6 kb.     

Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium

Aims: To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a_w). Methods and Results: A full factorial design was followed between the factors considered. The a_w levels assayed were 0·850, 0·880, 0·900, 0·920, 0·940, 0·960, 0·980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40°C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3·4–4·1% false‐positives and 0·7–1·4% false‐negatives. No growth was observed at 10°C and 40°C for all a_w levels assayed, with the exception of 0·980 a_w/40°C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0·7–1·4% accounting for false‐positives and 2·0–2·7% false‐negatives. No OTA production was detected at 10°C or 40°C regardless of a_w, and at 0·850 a_w at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15–20°C) as opposed to the growth boundary that shifted to higher temperature levels (25–30°C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false‐positive and three false‐negative predictions out of 68 growth cases and 13 false‐positive predictions out of 45 OTA production cases. Conclusions: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a_w. Significance and Impact of the Study: The proposed modelling approach helps the understanding of fungal‐food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.     

Characterization of fall armyworm mitochondrial DNA (Lepidoptera: Noctuidae)

Mitochondrial DNA from the fall armyworm, Spodoptera frugiperda (J.E. Smith), was cloned and characterized using restriction enzyme mapping. Genome size is approximately 16.3 kilobase (Kb), consistent with that of most animals. Three fragments, 8.1 Kb, 6.2 Kb, and 2.0 Kb, were produced by digestion with restriction enzyme Xbal and cloned into a pUC19 vector. Twenty-nine restriction enzymes were used to generate a detailed physical restriction enzyme map of the three cloned fragments. These data represent the first detailed characterization of a lepidopteran mitochondrial genome. © 1992 Wiley-Liss, Inc.