Black Aspergilli Research Articles

Reliable and Simple Detection of Ochratoxin and Fumonisin Production in Black Aspergillus

To date, edible fungi such as black Aspergillus (Aspergillus niger aggregates) have been considered as safe. However, it has recently been reported that some strains have a mycotoxin biosynthetic capability, and this capability must be evaluated to determine the safety of edible fungi. In this study, we assessed the ability of mycotoxin production in A. niger aggregates isolated from various Korean foods using multiplex PCR and high-performance liquid chromatography (HPLC) analyses. Multiplex PCR and HPLC analyses of 32 A. niger aggregates showed that ochratoxin and fumonisin were produced only by strains exhibiting positive PCR patterns with ochratoxin and fumonisin biosynthesis genes. However, several strains did not produce mycotoxins, even though they contained mycotoxin biosynthesis genes. Using multiplex PCR pattern and HPLC analyses, we selected Aspergillus strains that do not produce mycotoxins, which will contribute to the development of safer fermented foods.

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 (0.83aw versus 0.87aw) 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 β-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.

Increase of Fumonisin B2 and Ochratoxin A Production by Black Aspergillus Species and Oxidative Stress in Grape Berries Damaged by Powdery Mildew

Powdery mildew (PM), caused by the fungus Erysiphe necator, is one of the most widespread fungal disease of grape and may cause extensive openings on the berry surface during the infection. We evaluated the effect of damage caused by PM in grape berries on the growth of and mycotoxin production by Aspergillus and on the oxidative stress in infected berries. Berries of Vitis vinifera L. cv. Negroamaro with sound skin (SS) and those naturally infected by PM were surface sterilized and inoculated with either fumonisin B2(FB2)-producing strains of Aspergillus niger or ochratoxin A (OTA)-producing strains of Aspergillus carbonarius and incubated at 20 and 30°C. The PM berries were significantly more susceptible to both Aspergillus colonization (5 to 15 times more susceptible) and OTA and FB2 contamination (2 to 9 times more susceptible) than were SS berries. The highest toxin concentration was detected in inoculated PM berries both for OTA (9 ng/g) at 20°C and for FB2 (687 ng/g) at 30°C. In inoculated SS and PM berries, although malondialdehyde and hydrogen peroxide concentrations did not increase, the two black Aspergillus species caused a significant decrease in ascorbate content, thus inducing a pro-oxidant effect. These results indicate that grape berries affected by PM are more susceptible to black Aspergillus growth and to production and/or accumulation of FB2 and OTA. Thus, preventive control of E. necator on grape berries could reduce the mycotoxicological risk from black Aspergillus infection.

Taxonomic re-evaluation of black koji molds

Black koji molds including its albino mutant, the white koji mold, have been widely used for making the distilled spirit shochu in Northeast Asia because they produce citric acid which prevents undesirable contamination from bacteria. Since Inui reported Aspergillus luchuensis from black koji in Okinawa in 1901, many fungal names associated with black koji molds were reported. However, some species are similar and differentiation between species is difficult. Fungal taxonomists tried to arrange a taxonomic system for black koji molds, but the results were not clear. Recently, multi-locus sequence typing has been successfully used to taxonomy of black Aspergillus. According to β-tubulin and calmodulin gene sequences, black koji molds can be subdivided in three species, A. luchuensis, Aspergillus niger, and Aspergillus tubingensis. Aspergillus awamori, Aspergillus kawachii, Aspergillus inuii, Aspergillus nakazawai, and Aspergillus coreanus are synonyms of A. luchuensis, Aspergillus batatae, Aspergillus aureus (or Aspergillus foetidus), Aspergillus miyakoensis, and Aspergillus usamii (including A. usamii mut. shirousamii) are synonyms of A. niger and Aspergillus saitoi and A. saitoi var. kagoshimaensis are synonyms of A. tubingensis. A. luchuensis mut. kawachii was suggested particular names for A. kawachii because of their industrial importance. The history and modern taxonomy of black koji molds is further discussed.

A new mycoparasite, Aspergillus sp. ASP-4, parasitizes the sclerotia of Sclerotinia sclerotiorum

A species of Aspergillus (strain ASP-4), belonging to the Aspergillus aculeatus clade of the black aspergilli, which can parasitize and destroy the sclerotia of the economically important plant pathogen Sclerotinia sclerotiorum was isolated from a field plot where oilseed rape had been planted for nearly 20 years. This is the first time that a member of Aspergillus section Nigri has been described as a mycoparasite. Inoculation experiments of S. sclerotiorum sclerotia were conducted in both the laboratory and field plots over a three year period. The results clearly show that the effect of this mycoparasite on reducing numbers of germinating sclerotia and subsequent spread of this pathogen is highly significant. Therefore, Aspergillus sp. ASP-4 has the potential to be an important biocontrol agent. In addition, since Aspergillus sp. ASP-4 infects sclerotia, the application time and method for the prevention and control of Sclerotinia stem rot of oilseed rape can be made relatively easy compared to the critical timing of fungicide application during the early to mid-flowering season, as fungal spores can be sprayed directly on to soils after oilseed rape harvest.

The importance of structural diversity of spores in the taxonomy of Aspergillus (section Nigri)

Species of Aspergillus from section Nigri, also known as black aspergilli, are distributed worldwide and have been widely used for purposes of various types such as: biotechnological, industrial, medical, among others. They have been extensively studied for being the causing agents of biodeterioration of commodities and food.Within this section, new species have been recently described and among them Aspergillus ibericus and Aspergillus uvarum were both isolated from grapes. The polyphasic approach used in the analysis of these species, either by morphological techniques as well as by molecular methods, allowed not only their characterisation but also their consequent separation from all others in the section. Microbial taxonomy has the identification of species as one of its fundamental goals. Data such as: morphological characteristics description, physiological and biochemical properties, ecological roles, and societal risks or benefits, are key elements in any fungal identification process. But, this process is subjected to periodic changes due to frequent revisions of the taxonomic schemes, therefore becoming time consuming and more demanding and difficult, even for skilled specialists. Furthermore, each taxonomic group has specialised literature, terminology and characters. This takes place since identifications have difficulties of consensual naming, depending on the criteria used and the amount of information available when producing all data. It is increasingly becoming clear that, to better achieve a more accurate concept of species, microbial identification and authentication require a polyphasic approach to produce consistent, useful and quality data.Characterisation of morphologic and structural aspects of spores from Aspergillus strains, section Nigri, has been carried out using scanning electron microscopy (SEM) and intends to contribute to the associated data of the strains from this section.Colonies from 13 Aspergillus from section Nigri, from the Micoteca da Universidade do Minho (MUM), were grown at 25 ºC for 3 or 4 days in malt extract agar directly mounted in a sterilised SEM stub. The samples were covered with a mixture of gold and platinum (80/20%) and then examined in the SEM [NanoSEM - FEI NovaTM 200 (FEG/SEM); EDAX - Pegasus X4M (EDS/EBSD)]. A stereomicroscope (Leica MZ12.5) was used to have a clear image comparison of the size of the conidial head.Although the analysed strains presented dimensional, morphological and structural diversity, common or typical features could be inferred and related to each taxon like those represented in Fig 1.From the SEM analysis we were able to conclude that in the section Nigri of genus Aspergillus the spore wall ornamentation, and its size and shape continue to present themselves as important primary diagnostic traits for species differentiation.

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 3days after inoculation, but intracellular hyphal growth was more evident after 7days 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.

메주에서 분리한 검은 Aspergillus 균주의 동정

검은 Aspergillus 속균은 동양의 발효산업에서 매우 중요한 곰팡이로서 식품산업에 관련된 검은 Aspergillus를 흑국균이라고 한다. 흑국균은 A. luchuensis, A. niger, A. tubingensis로 구성이 되어 있는데 이 중에서 A. luchuensis는 동양의 발효산업에서 매우 중요한 곰팡이로 알려져 있다. 우리나라 전통 된장과 간장의 주요 원료가 되는 메주에서도 검은 Aspergillus가 흔히 발견되었다. 33균주의 검은 Aspergillus가 2008년부터 2011년 사이에 전국적으로 수집한 98개의 완성메주로부터 분리되었고 추가로, 발효과정에 있는 메주로부터 검은 Aspergillus 21균주를 분리하였다. <TEX>${\beta}$</TEX>-tubulin과 calmodulin 유전자 염기서열 분석을 통하여 이들을 동정한 결과 이들은 A. niger 21균주, A. luchuensis 14균주, A. tubingensis 10균주, A. welwitschiae 9균주로 동정되었다. 이 중 A. luchuensis는 생장이 활발하고 단백질, 지방, 탄수화물 분해력이 높은 것으로 조사되어 이들의 산업적 응용에 대한 추가의 연구가 필요한 것으로 사료된다. Black Aspergillus is important fungus for oriental fermentation industry. Black Aspergillus was frequently isolated from Korean traditional Meju, a fermented soybean starting material for soy sauce and soybean paste. Thirty three strains were isolated from 98 finished Meju collected in various regions of Korea from 2008 to 2011, and 21 strains were isolated from in-process Meju at various farms from 2010 to 2011. The isolated black Aspergillus were identified using DNA sequences of partial <TEX>${\beta}$</TEX>-tubulin and calmodulin genes. Of 54 black Aspergillus strains, 14 strains were identified as A. luchuensis and the others were composed of A. niger (n = 21), A. tubingensis (n = 10), and A. welwitschiae (n = 9).

Occurrence of ochratoxin A, fumonisin B2 and black aspergilli in raisins from Western Greece regions in relation to environmental and geographical factors

The presence of ochratoxin A (OTA), fumonisin B2 (FB2) and black aspergilli in raisins from Western Greece regions (Messinia, Corinthia, Achaia, Ilia and Zante Island) was investigated in relation to the different geographic and climatic conditions in the 2011 growing season. The biseriate species Aspergillus niger “aggregate” and A. carbonarius were mainly identified. The population of A. niger “aggregate” species occurred in all raisin samples at colony-forming units (CFU) concentrations significantly higher (mean 2.2 × 105 CFU g−1 homogenate) than those of A. carbonarius population (mean 4.9 × 103 CFU g−1 homogenate), which occurred in 80% of the raisin samples. OTA was found in 73% of the samples at levels ranging from 0.1 µg kg−1 to 98.2 µg kg−1, with the highest level occurring in a raisin sample from Ilia that also contained the highest level of A. carbonarius. The European Union legal limit for OTA was exceeded in 15% of the raisin samples. FB2 was found in 29% of the raisin samples at levels ranging from 7.1 µg kg−1 to 25.5 µg kg−1, with 20% of the samples co-occurring with OTA. Principal-component analysis was applied to levels of mycotoxins, fungal contamination, geographical data and environmental conditions recorded in the harvesting (August) or drying (September) period. Principal-component analysis clearly indicated a good direct correlation of rainfall and relative humidity with OTA and A. carbonarius contamination. A lack of clustering was observed when A. niger and FB2 contamination were considered. This is the first report on the co-occurrence of the mycotoxins OTA and FB2 in dried vine fruits from Greece.

Multilocus sequence analysis of Aspergillus Sect. Nigri in dried vine fruits of worldwide origin

Dried vine fruits may be heavily colonized by Aspergillus species. The molecular biodiversity of an Aspergillus population (234 strains) isolated from dried vine fruit samples of worldwide origin were analyzed by investigating four housekeeping gene loci (calmodulin, β-tubulin, elongation factor 1-α, RPB2). Aspergillus Sect. Nigri was dominant and the strains were identified as A. tubingensis (138), A. awamori (38), A. carbonarius (27), A. uvarum (16) and A. niger (11). Four Aspergillus flavus strains were also identified from Chilean raisins. Two clusters closely related to the A. tubingensis species with a significant bootstrap (60% and 99%) were identified as distinct populations. Among the four loci, RPB2 showed the highest genetic variability. This is the first complete study on the worldwide distribution of black Aspergilli occurring on dried vine fruits identified by a molecular approach.

Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop-mediated isothermal amplification (LAMP) reaction

To develop two assays based on the loop-mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes. Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction. The two LAMP assays permitted to quickly and specifically identify DNA from OTA-producing black aspergilli, as well as isolates grown in pure culture. Monitoring vineyards for the presence of OTA-producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA-producing black aspergilli.

Involvement of the opportunistic pathogen Aspergillus tubingensis in osteomyelitis of the maxillary bone: a case report

BackgroundAspergillus tubingensis is a black Aspergillus belonging to the Aspergillus section Nigri, which includes species that morphologically resemble Aspergillus niger. Recent developments in species determination have resulted in clinical isolates presumed to be Aspergillus niger being reclassified as Aspergillus tubingensis by sequencing. We present a report of a patient with an osteomyelitis of the maxillary bone with a probable invasive Aspergillus tubingensis infection.Case presentationWe describe an immune compromised patient suffering from osteomyelitis of the maxillary bone after tooth extraction. The osteomyelitis probably resulted in dentogenic pansinusitis presenting as an acute ethmoiditis. Histologic examination of biopsy samples showed osteomyelitis, and inflammation of the surrounding connective tissue. Cultures of the alveolar wound grew Aspergillus tubingensis. The patient was treated with liposomal amphoterocin B, which was changed to oral treatment with voriconazole based on susceptibility testing (MIC for voriconazole was 1 μg/ml).ConclusionThis case shows that Aspergillus tubingensis may have the potential to cause severe invasive infections in immunocompromised hosts. A larger proportion of Aspergillus tubingensis isolates are less susceptible to azoles compared to Aspergillus niger. Therefore, correct species identification and susceptibility testing is crucial for the choice of anti-fungal treatment, screening of azole resistance, and characterization of the pathogenic potential of the various species within Aspergillus section Nigri.

Ochratoxin A Management in Vineyards by Lobesia botrana Biocontrol

Grape berries attacked by Lobesia botrana larvae are more easily infected by Aspergillus section Nigri (black aspergilli) ochratoxigenic species. Two-year field trials were carried out in Apulia (Italy) to evaluate a bioinsecticide control strategy against L. botrana and the indirect effect on reducing ochratoxin A (OTA) contamination in vineyards. A commercial Bacillus thuringiensis formulate and an experimental Beauveria bassiana (ITEM-1559) formulate were tested in two vineyards cultivated with the same grape variety, Negroamaro, but with two different training systems (espalier and little-arbor techniques). In both years and training systems the treatments by B. bassiana ITEM-1559 significantly controlled L. botrana larvae attacks with effectiveness similar to B. thuringensis (more than 20%). A significant reduction of OTA concentrations (up to 80% compared to untreated controls) was observed only in the first year in both training systems, when the metereological parameters prior to harvest were more favorable to the insect attack. Results of field trials showed that B. bassiana ITEM-1559 is a valid bioinsecticide against L. botrana and that grape moth biocontrol is a strategy to reduce OTA contamination in vineyard in seasons with heavy natural infestation.

Aspergillus species as mycotoxin producers in agricultural products in central Europe

Aspergillus species are able to produce a range of mycotoxins, includ?ing e.g. aflatoxins, ochratoxins, fumonisins and patulin. Aflatoxins are mainly produced by members of Aspergillus section Flavi, and they contaminate various agricultural products in several parts of the world. Several recent reports have indicated that aflatoxin-producing fungi and consequently aflatoxin contamination occur in agricultural commodities in a number of European countries which have not been faced with this problem before. Indeed, recent surveys have clarified that concentrations of aflatoxins in maize products and milk has been exceeding the EU limit in several regions of Central Europe including Serbia, Slovenia, Croatia, Northern Italy and Romania. However, aflatoxin contamination and aflatoxin-producing Aspergillus species have not been identified yet in maize in Hungary. We examined the presence of potential aflatoxin-producing Aspergilli in maize samples collected in southern parts of Hungary. Several A. flavus isolates were identified, and pre?liminary results indicated that some of the isolates were able to produce aflatoxins. Con?tamination of other agricultural products with aflatoxins can also pose problems in Central Europe due to global warming. Ochratoxin contamination of grapes and grape-derived products is usually caused by black Aspergilli, especially by A. carbonarius and A. niger, although these species have been rare in Central European vineyards due to climatic fac?tors. Ochratoxin contamination of other agricultural products including spices and cereals was also observed in the region. Besides, ochratoxin producing Aspergilli are frequently isolated from imported products including coffee beans, dried fruits and spices, and ochra?toxin contamination of these samples was also observed. Fumonisins are produced mainly by Fusarium species, and by the recently identified producers Aspergillus niger and A. awamori. We examined fumonisin producing abilities of A. niger / A. awamori isolates col?lected from the variety of substrates including raisins, figs, dates, maize and onions. The isolates, which came from dried vine fruits, produced several fumonisin isomers also pre?sent in the raisin samples, indicating that fumonisin contamination of these products was probably caused by black Aspergilli. Besides, strains collected from figs, dates and onions were also able to produce fumonisins, and preliminary data indicated that figs and onions were also contaminated with low but significant amount of fumonisins. Potential fumonisin producing A. awamori isolates were also identified on maize samples. Further studies on the examination of the occurrence of fumonisins and their potential producers in other agricultural products are in progress. Regarding patulin, contamination of apple based products is a serious problem in the region, mainly caused by Penicillium species. Although patulin producing Aspergilli have also been identified in cereals, patulin contamination of cereals and cereal based products is usually low in Central Europe.

Aspergillus fijiensis n. sp. isolated from bronchial washings in a human case of bronchiectasis with invasive aspergillosis: the first report

Introduction The black aspergilli (Aspergillus section Nigri) are an important group of species in food mycology, medical mycology and biotechnology. Many species cause food spoilage, but are also used in the fermentation industry. More recently they were shown to pose hazards to human and animal health as producers of ochratoxin A,

カビ汚染乾燥果実から分離された黒色コウジカビの同定とフモニシンB2産生

カビ汚染乾燥果実から分離された黒色コウジカビの同定とフモニシンB₂産生

Black Aspergilli and fumonisin contamination in onions purchased in Hungary

Black mould rot caused by black Aspergilli is an important post-harvest disease of onion worldwide. Usually Aspergillus niger is cited as the causative agent based on morphological criteria. In this study, the mycobiota and fumonisin contamination of mouldy onion bulbs purchased in Hungary were examined. All except one of the examined mouldy samples were found to be contaminated with black Aspergilli, which could be isolated both from the outer dry and the inner fleshy scales of onion bulbs. Species assignment of the isolates was carried out using sequence analysis of part of the calmodulin gene. Sequence data revealed that all 35 black Aspergilli isolated from onions belong to the Aspergillus awamori species. The range of fumonisin isomers present in the onion samples was also examined using reversed-phase high-performance liquid chromatography/electrospray ionization-ion trap mass spectrometry. Two of the examined onion samples were found to be contaminated with fumonisins at a rate of about 0.3 mg kg−1...

Evaluation of &lt;i&gt;Streptomyces &lt;/i&gt;&lt;i&gt;Au&lt;/i&gt;&lt;i&gt;reofaciens&lt;/i&gt;&lt;i&gt; &lt;/i&gt;and &lt;i&gt;Rhodotorulaglutinis&lt;/i&gt;&lt;i&gt; &lt;/i&gt;Against Ochratoxin A Producing &lt;i&gt;Aspergillusnigr&lt;/i&gt;in Grapevines

OchratoxinA (OTA) is a mycotoxin, produced by filamentous fungi, toxic to humans and animals and naturally found in a wide range of different agricultural products, includingfruits (grapevines).Members of <i>Aspergillus</i>section Nigri(black aspergilli) are mainly responsible for OTA accumulation in seeds. This research investigate the biological control of ochratoxin A (OTA) production by <i>A. niger</i>, using, that proved variable antagonistic activity against <i>A. niger</i>. A general inhibition effect was observed with tested <i>Rhodotorulaglutinis</i> strain followed by <i>Streptomyces aureofaciens</i>. A field experiment conducted during 2011 and 2012 vintages in a vineyard indicated that yeast spraying was found to be very efficient for reducing <i>A. niger</i> development. Grape berries sprayed with either with <i>Streptomyces aureofaciens</i> or<i>Rhodotorulaglutinis</i>. <i>Rhodotorulaglutinis</i> completely inhibited OchratoxinAproducing<i>A. niger</i> for 40 days of storage.

Recent Literature

Recent Literature

Efficacy of Biological Control Agents for Management of Postharvest Black Rot Pathogen Aspergillus carbonarius in Grapes

Grape berries were treated with different biological agents to test their efficacy in reducing post harvest incidence of black mould rot and minimize the loss. The agents Pseudomonas, Bacillus, Trichoderma and yeast isolates were individually screened against the black rot pathogen Aspergillus carbonarius. B. subtilis strains EPC-8 and EPCO-16 and T. harzianum (Th Co) showed high mycelial growth suppression of A. carbonarius in vitro . The effective biological control agents were tested in pre, post and combined inoculation studies against A. carbonarius in grape berries. In the pre inoculation, B. subtilis (EPC-8) showed 57.80 per cent reduction in the incidence of A. carbonarius followed by T. harzianum (Th Co) (48.43 percent). The same trend of effectiveness was also found in the post-inoculation and combined inoculation tests under storage conditions at room temperature in grape berries.