Aspergillus Section Nigri Species Research Articles

First report of Aspergillus welwitschiae causing maize ear rot in Serbia.

In recent years, countries in Southeast Europe are facing climate changes characterized by extreme hot weather, which contribute to the increased frequency of Aspergillus species. Because of these changes, Aspergillus parasiticus was isolated, for the first time, from maize grain in Serbia (Nikolic et al, 2018). The presence of black powdery mycelia on maize ears indicated occurrence of species of the genus Aspergillus section Nigri, which led to the need for detailed identification of these fungi. Disease incidence ranged from 10 and 15% in August 2013. Maize ears with black powdery symptoms were collected from field in Zemun Polje, Serbia. Symptomatic kernels were surface sterilized with 1% sodium hypochlorite solution for 3 min, rinsed three times with sterilized water, then incubated at 25°C in the dark for 7 days on potato dextrose agar (PDA). Twenty isolates were identified as genus Aspergillus section Nigri. Monospore cultures formed black cottony colonies with a yellowish border on PDA. The average colony diameter was 50 mm. In order to reliably identify, isolates were transferred to Malt Extract agar (MEA) and Czapek Yeast Autolysate agar (CYA) (Samson et al, 2014). On CYA fungal colonies consisted of a white mycelium, covered by a layer of black conidiophores. On MEA fungal colonies were dense, black, with yellowish border. The reverse side was colorless to pale yellow, with a yellow ring in the middle. The average size of conidia was 4.3 µm. The conidia were globose to sub-globose, smooth to roughened, which coincides with previous research (Silva et al, 2020). Given that the fungi Aspergillus niger and Aspergillus welwitschiae are morphologically indistinguishable (Susca et al, 2016), species level identification was completed by analysis of a partial sequence of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers) and calmodulin gene (CMD5/CMD6 primers) (Samson et al., 2014). The sequences were compared with the sequences of A. welwitschiae strains registered in the GenBank database based on nucleotide similarity, and results showed 99,64 and 100% similarity with ITS (OL711714) and calmodulin (KX894585), respectively. The sequence was deposited in GenBank with accession numbers OQ456471 (ITS) and OQ426518 (calmodulin). We also confirmed the presence of this species with specific primers (AWEL1/AWEL2) designed by Susca et al. 2020. Pathogenicity test was performed in Zemun Polje on the same maize hybrid from which the fungal species was isolated. Using artificial inoculations by the injecting conidial suspension into the silk channel, three days after 50% of plants reached the silking stage. Twenty ears were inoculated with each isolate, in four replicates (Reid et al, 1996). Inoculum was prepared from 7-day-old colonies on PDA, and 2 ml of a conidial suspension (1×106 spores/ml) was used. Control plants were inoculated with sterile water. All inoculated ears showed symptoms, similar to those from field infections. Control ears were symptomless. The fungus was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. Based on molecular, morphological and pathogenic properties, the isolates were identified as A. welwitschiae. This is the first report of A. welwitschiae as the causal agent of black maize ear rot not only in Serbia, but also in the other countries of the Western Balkans. Given that the fungus A. welwitschiae synthesizes both ochratoxin A (OTA) (Battilani et al, 2006) and fumonisin (FB) (Frisvad et al, 2011), further studies should be focused on assessment its aggressiveness and toxicological profile.

A Brief Snapshot of Aspergillus Section Nigri Isolated from Brazilian Peanuts and Soil

The occurrence of mycotoxigenic species in peanuts is a major concern, and has been investigated in depth for many years. However, most studies focus on the occurrence of aflatoxigenic fungi, such as Aspergillus section Flavi. The present study aimed to clarify the occurrence of Aspergillus section Nigri, a group that harbors species capable of producing ochratoxin A (OTA), which has scarcely been investigated in peanuts. A total of 52 peanut samples, collected in the field and from storage, were analyzed. Aspergillus section Nigri was isolated from 64% and 100% of field and storage samples, respectively, and 218 strains were obtained. Based on the multiloci phylogeny of the CaM and BenA loci, six species of Aspergillus section Nigri were identified: A. brasiliensis, A. niger, A. neoniger, A. welwitschiae, A. costaricaensis, and A. japonicus. The incidence of ochratoxigenic strains was 5.0% (11/218), and only A. niger and A. welwitschiae were able to produce OTA. The presence of OTA in peanuts was found in 6 field and 8 storage samples, with levels ranging from 0.106 to 0.387 and 0.090 to 0.160 µg/kg, respectively.

Resistance of Black Aspergilli Species from Grape Vineyards to SDHI, QoI, DMI, and Phenylpyrrole Fungicides.

Fungicide applications constitute a management practice that reduces the size of fungal populations and by acting as a genetic drift factor, may affect pathogen evolution. In a previous study, we showed that the farming system influenced the population structure of the Aspergillus section Nigri species in Greek vineyards. The current study aimed to test the hypothesis that the differences in the population structure may be associated with the selection of fungicide-resistant strains within the black aspergilli populations. To achieve this, we determined the sensitivity of 102, 151, 19, and 22 for the A. uvarum, A. tubingensis, A. niger, and A. carbonarious isolates, respectively, originating either from conventionally-treated or organic vineyards to the fungicides fluxapyroxad-SDHIs, pyraclostrobin-QoIs, tebuconazole-DMIs, and fludioxonil-phenylpyrroles. The results showed widespread resistance to all four fungicides tested in the A. uvarum isolates originating mostly from conventional vineyards. In contrast, all the A. tubingensis isolates tested were sensitive to pyraclostrobin, while moderate frequencies of only lowly resistant isolates were identified for tebuconazole, fludioxonil, and fluxapyroxad. Sequencing analysis of the corresponding fungicide target encoding genes revealed the presence of H270Y, H65Q/S66P, and G143A mutations in the sdhB, sdhD, and cytb genes of A. uvarum resistant isolates, respectively. No mutations in the Cyp51A and Cyp51B genes were detected in either the A. uvarum or A. tubingensis isolates exhibiting high or low resistance levels to DMIs, suggesting that other resistance mechanisms are responsible for the observed phenotype. Our results support the initial hypothesis for the contribution of fungicide resistance in the black aspergilli population structure in conventional and organic vineyards, while this is the first report of A. uvarum resistance to SDHIs and the first documentation of H270Y or H65Q/S66P mutations in sdhB, sdhD, and of the G143A mutation in the cytb gene of this fungal species.

Aspergillus welwitschiae: A Potential amylases Producer.

Amylases, glycoside hydrolases widely used in several industrial processes, can be produced by many animals, plants, bacteria, and fungi. Fungal amylases from Aspergillus sp. hold remarkable importance in biotechnological applications for presenting a great catalysis efficiency in a wide range of pH and temperature. The production of amylases is mainly dependent on the genetic background of the species, i.e., Aspergillus strains, and abiotic factors. Among the major producers of amylases are the species of Aspergillus section Nigri, including Aspergillus welwitschiae. In this study, Aspergillus welwitschiae strains were evaluated for their ability to produce extracellular amylases. Among the 24 strains, wild Aspergillus welwitschiae UELAs 15.262 and mutant A. welwitschiae UELAs 15.262/35 strains showed greater potential for amylases production. The A. welwitschiae UELAs 15.262 produced more amylases (8645 U/mg) when compared to A. welwitschiae UELAs 15.262/35 (6666 U/mg). The amylases activity from partially purified crude enzymatic extract of A. welwitschiae UELAs 15.262 strain obtained at pH 5.5, 60°C, resulted in 1.98-fold (3837 U/mg) increase in enzymatic activity. Likewise, the amylases activity from partially purified crude extract of A. welwitschiae UELAs 15.262/35 obtained at pH 5.0, 60°C resulted in 2.2-fold (9077 U/mg) increase in amylases activity. The presence of metallic ions (Cu2+ and Fe3+) also provided an increase of amylases activity for both strains. To our knowledge, this is the first study reporting the ability of Aspergillus welwitschiae strains in order to produce amylases.

Conventional vs. organic vineyards: Black Aspergilli population structure, mycotoxigenic capacity and mycotoxin contamination assessment in wines, using a new Q-TOF MS-MS detection method

Aspergillus bunch rot, caused by several mycotoxigenic species of Black Aspergilli section Nigri is one of the most severe diseases of grapevine, while mycotoxin contaminated products may pose a risk to the consumers health. During a 2-year (2018–2019) survey, the impact of conventional and organic farming systems on Black Aspergilli population structure in Greek vineyards was thoroughly investigated. In total, 300 isolates of Aspergillus spp. were selected and identified by amplicon sequencing of the ITS-region, β-tubulin and calmodulin genes. Four different Aspergillus section Nigri species (A. tubingensis, A. uvarum, A. carbonarius and A. niger) were identified as the casual agents of the disease. During both sampling years, higher frequencies of A. tubingensis and A. uvarum were found in the organic and conventional vineyards, respectively. In vitro production of ochratoxin A (OTA) and fumonisin B2, B3 and B4 (FB2, FB3, and FB4) was evaluated in two selective media. The analysis revealed a low frequency of mycotoxigenic isolates, while, most of these isolates originated from conventional vineyards. As expected, A. carbonarious was identified as the main OTA producer (0.01–9.2 μg/g), whereas A. niger was the leading producer of FBs. In total, 74 wine samples originating from either organic or conventional vineyards of the same region, were analyzed using a new Quadrupole Time-of-Flight Mass Spectrometry (QTOF MS-MS) analytical method developed to detect and quantify Alternariol (AOH), Alternariol monomethyl ether (AME), and Citrinin (CIT) in addition to the aforementioned Aspergillus mycotoxins (OTA, FB1, FB2 FB3), using a modified QuEChERS extraction protocol. Interestingly, a low frequency of mycotoxin-contaminated wines was detected. At the same time, fumonisins (FBs) were identified as the main mycotoxins in Greek wines. This is the first study deciphering the impact of conventional and organic farming systems on Aspergillus section Nigri species in Greek vineyards, suggesting that farming system may affect the species composition within the vineyard. Furthermore, evidence was provided for a higher risk of selection of Aspergillus strains with higher mycotoxigenic capacity in conventional vineyards, associated with higher risk of mycotoxin contamination in wines originating from these vineyards.

Invasive Factors Recognition in Aspergillus Section Nigri Isolates from Patient and Environmental Samples in the Centre Region, Cameroon

Background: Aspergillus section Nigri species are invasive opportunistic pathogens, seen in individuals with various immune disorders. The invasive capacity involves the production of varieties of enzymes such as lipases, phospholipase, proteases and hyaluronidase. The determination of proteinase, phospholipase, esterase and biofilm production in patient and environmental isolates approve the pathogenic strength of the species.
 Aims: To evaluate the invasive capacity of Aspergillus section Nigri isolates from patients and environmental samples.
 Methods: Our study is cross sectional and experimental, performed at the outpatient clinic of the otorhinolaryngology department of Central and University teaching hospital during a period of 12 months from March 2018 to February 2019. To determine the invasive capacity of Aspergillus section Nigri species, 400 samples were evaluated in the study (that is; 250 from patients and 150 samples from environment). Patient samples and hospital environment samples were evaluated by standard phenotypic methods for detecting of Proteinase, phospolipase, esterase and biofilm. The variables were statistically analyzed using Chi-square test of independent and SPSS (Version 16.0).
 Results: The isolates recovered from the patient sample shows maximum invasive capacity as compared to the environmental isolates, that is for 44 isolates; 42 isolates showed proteinase activity and biofilm production, followed by phospholipase activity 36, and then esterase 32.The isolates recovered from the hospital environment also showed the production of the various invasive factors, that is for 16 isolates; 15 isolates showed biofilm production, followed by proteinase activity 6, phospholipase 5 and esterase 4.The disparities of the invasive capacity in patient and environment isolates virulence were statistically significant for proteinase, phospholipase and esterase ( that is; p-value <0.05). Majority the isolates recovered from patients and the environment were potential producers of biofilm.
 Conclusion: The isolates recovered from patients sample showed high invasive capacity as compare to the isolates recovered from the environment. This highlights the implications of phospholipase enzyme, proteinase enzyme, esterase enzyme and biofilm used by Aspergillus section Nigri isolates as means of survival in the host system.

First Report of Bunch Rot Caused by Aspergillus tubingensis of Shine Muscat Grape in Korea

Shine Muscat (Vitis vinifera L.) is an increasingly popular grape cultivar in South Korea because of the sweet and unique flavors and large berries. Fungal diseases, including bunch rot, are a continual challenge for grape growers, causing pre- and postharvest fruit decay. In September 2018, Shine Muscat plants showing bunch rot were collected from an orchard in Gimcheon, South Korea. Initial symptoms of the disease consisted of brown spots on the fruits. Whole fruit became rotten as the disease progressed and were covered by black powdery spores. The pathogens were isolated from diseased fruit tissue to potato dextrose agar (PDA). To avoid contamination, small sections of diseased tissue were cut from affected fruits, immersed in 0.5% NaOCl for 1 min, rinsed in sterile distilled water, and blotted with sterile tissue. Then, sterilized sections of infected tissue were placed on PDA plates and incubated at 25°C in the dark. Pure cultures were obtained by transferring hyphal tips to a fresh PDA plate. Two pure cultures (AS181 and AS182) were used to examine morphological characteristics. Seven-day-old colonies were initially white and then covered with dark gray conidial heads. Conidial heads were large and radiate, and vesicles were globose. Conidia were globose or subglobose (3.6 to 5.5 μm in diameter) and dark brown to black in color. The morphological characteristics of isolates were typical of Aspergillus spp. of Aspergillus section Nigri (black Aspergilli), including Aspergillus tubingensis (Mirhendi et al. 2016; Zulkifli and Zakaria 2017). The species of Aspergillus section Nigri cannot be distinguished using only morphological characteristics (Zulkifli and Zakaria 2017). Total genomic DNA was isolated from mycelium of AS181. The internal transcribed spacer (ITS) region and β-tubulin (TUB) gene were amplified using primers ITS-1F/ITS4 and Bt2a/Bt2b, respectively. PCR products were purified and sequenced by Macrogen (Seoul, South Korea) and deposited in GenBank. BLAST search analysis of the ITS sequence (LC467944) showed 602/604 (99.7%) similarity with the sequence from A. tubingensis strain CMXY25850 (GenBank accession no. MG991653). TUB (LC467945) showed 559/562 (99.5%) similarity with the sequence from A. tubingensis strain IHEM 23902 (MH614560). Phylogenetic analysis using maximum likelihood and Bayesian inference methods based on concatenated sequences of ITS and TUB showed that isolate AS181 clustered together with previously identified A. tubingensis strains (USML03, USML13, and USMJ02) (Zulkifli and Zakaria 2017). Molecular analysis using TUB sequences data is an important tool for species delineation of black Aspergillus isolates (Zulkifli and Zakaria 2017). To confirm pathogenicity, 1 × 10⁶ conidia/ml (conidial suspension of isolate AS181) was sprayed on five healthy bunches of fruit. The same number of bunches were sprayed with sterile distilled water as noninoculated controls. The treated fruit bunches were placed in a separate sterile plastic box containing moist tissue paper and incubated at 25°C in the dark. Ten days after inoculation, symptoms similar to naturally infected fruits developed on 20% of inoculated fruits, whereas control fruits remained asymptotic. One pure culture was reisolated from artificially infected fruits and identified as A. tubingensis following procedures described above. In a previous study, A. tubingensis was reported as the causal agent of vine canker disease of grapevine in Italy (Vitale et al. 2012). A. tubingensis has also been reported as the causal agent of postharvest fruit rot of Prunus salicina and leaf spot of Jatropha curcas. (Guo et al. 2017; Wang et al. 2012). To our knowledge, this is the first report of A. tubingensis casing bunch rot on V. vinifera in South Korea.

Species identification, antifungal susceptibility, and clinical feature association of Aspergillus section Nigri isolates from the lower respiratory tract.

Species of Aspergillus section Nigri are generally identified by molecular genetics approaches, whereas in clinical practice, they are classified as A. niger by their morphological characteristics. This study aimed to investigate whether the species of Aspergillus section Nigri isolated from the respiratory tract vary depending on clinical diagnosis. Forty-four Aspergillus section Nigri isolates isolated from the lower respiratory tracts of 43 patients were collected from February 2012 to January 2017 at the National Hospital Organization (NHO) Tokyo National Hospital. Species identification was carried out based on β-tubulin gene analysis. Drug susceptibility tests were performed according to the Clinical and Laboratory Standards Institute (CLSI) M38 3rd edition, and the clinical characteristics were retrospectively reviewed. A. welwitschiae was isolated most frequently, followed by A. tubingensis. More than half of the A. tubingensis isolates exhibited low susceptibility to azoles in contrast to only one A. welwitschiae isolate. Approximately three quarters of the patients from whom A. welwitschiae was isolated were diagnosed with colonization, whereas more than half the patients from whom A. tubingensis was isolated were diagnosed with chronic pulmonary aspergillosis (CPA). More attention needs to be given to the drug choice for patients with CPA with Aspergillus section Nigri infection because A. tubingensis, which was found to be frequently azole-resistant, was the most prevalent in these patients.

Species identification, antifungal susceptibility, and clinical feature association of Aspergillus section Nigri isolates from the lower respiratory tract.

Species of Aspergillus section Nigri are generally identified by molecular genetics approaches, whereas in clinical practice, they are classified as A. niger by their morphological characteristics. This study aimed to investigate whether the species of Aspergillus section Nigri isolated from the respiratory tract vary depending on clinical diagnosis. Forty-four Aspergillus section Nigri isolates isolated from the lower respiratory tracts of 43 patients were collected from February 2012 to January 2017 at the National Hospital Organization (NHO) Tokyo National Hospital. Species identification was carried out based on β-tubulin gene analysis. Drug susceptibility tests were performed according to the Clinical and Laboratory Standards Institute (CLSI) M38 3rd edition, and the clinical characteristics were retrospectively reviewed. A. welwitschiae was isolated most frequently, followed by A. tubingensis. More than half of the A. tubingensis isolates exhibited low susceptibility to azoles in contrast to only one A. welwitschiae isolate. Approximately three quarters of the patients from whom A. welwitschiae was isolated were diagnosed with colonization, whereas more than half the patients from whom A. tubingensis was isolated were diagnosed with chronic pulmonary aspergillosis (CPA). More attention needs to be given to the drug choice for patients with CPA with Aspergillus section Nigri infection because A. tubingensis, which was found to be frequently azole-resistant, was the most prevalent in these patients.

Pathogenicity of Fumonisin-producing and Nonproducing Strains of Aspergillus Species in Section Nigri to Maize Ears and Seedlings

Species of Aspergillus section Nigri are commonly associated with maize kernels, and some strains can produce fumonisin mycotoxins. However, there is little information about the extent to which these fungi contribute to fumonisin contamination in grain, the damage they cause to maize ears, or their effects on maize seed germination and seedling health. We compared fumonisin-producing and nonproducing strains of A. niger, A. welwitschiae, A. phoenicis, A. tubingensis, and A. carbonarius from the United States and Italy in laboratory and field studies to assess their ability to contribute to fumonisin contamination, to cause maize ear rot, and to affect seed germination and seedling growth. In laboratory experiments, some strains of each Aspergillus species reduced germination or seedling growth, but there was high variability among strains within species. There were no consistent differences between fumonisin-producing and nonproducing strains. In field studies in Iowa and Illinois, strains were variable in their ability to cause ear rot symptoms, but this was independent of the ability of the Aspergillus strains to produce fumonisins. Contamination of grain with fumonisins was not consistently increased by inoculation with Aspergillus strains compared with the control, and was much greater in F. verticillioides-inoculated treatments than in Aspergillus-inoculated treatments. However, the ratio of the FB analogs FB2 and FB1 was altered by inoculation with some Aspergillus strains, indicating that FB2 production by Aspergillus strains occurred in the field. These results demonstrate the pathogenic capabilities of strains of Aspergillus in section Nigri, but suggest that their effects on maize ears and seedlings are not related to their ability to produce fumonisins, and that fumonisin contamination of grain caused by Aspergillus spp. is not as significant as that caused by Fusarium spp.

Aspergillus labruscus sp. nov., a new species of Aspergillus section Nigri discovered in Brazil

A novel fungal species, Aspergillus labruscus sp. nov., has been found in Brazil during an investigation of the fungal species present on the surface of grape berries (Vitis labrusca L.) for use in the production of concentrated grape juice. It seems to be associated to V. labrusca, and has never been recovered from Vitis vinifera. This new species belonging to Aspergillus subgenus Circumdati section Nigri is described here using morphological characters, extrolite profiling, partial sequence data from the BenA and CaM genes, and internal transcribed spacer sequences of ribosomal DNA. Phenotypic and molecular data enabled this novel species to be clearly distinguished from other black aspergilli. A. labruscus sp. nov. is uniseriate, has yellow mycelium, poor sporulation on CYA at 25 °C, abundant salmon to pink sclerotia and rough conidia. Neoxaline and secalonic acid D were consistently produced by isolates in this taxon. The type strain of A. labruscus sp. nov. is CCT 7800 (T) = ITAL 22.223 (T) = IBT 33586 (T).

Comments on "Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes. Toxins 2016, 8, 333"-In Reporting Ochratoxin A Production from Strains of Aspergillus, Penicillium and Talaromyces.

Recently a species in the genus Talaromyces, a uniseriate species of Aspergillus section Nigri and an isolate each of two widespread species, Penicillium rubens and P. commune, were reported to produce ochratoxin A. This claim was based on insufficient biological and chemical data. We propose a list of criteria that need to be met before an unexpected mycotoxin producer is reported. There have only been convincing data on ochratoxin A production for Penicillium verrucosum, P. nordicum, P. thymicola, all from Penicillium series Verrucosa, and from species in three sections of Aspergillus: section Circumdati, section Nigri and section Flavi.

Characterization of Aspergillus section Nigri species populations in vineyard soil using droplet digital PCR.

This is the first study to demonstrate the utility of ddPCR as a means to quantify species of Aspergillus section Nigri in soil. This method eliminates the need for isolation and sequence identification of individual fungal isolates, and allows for greater throughput in measuring relative population sizes of important (i.e. mycotoxigenic) Aspergillus species within a population of morphologically indistinguishable species.

Diversity in Secondary Metabolites Including Mycotoxins from Strains of Aspergillus Section Nigri Isolated from Raw Cashew Nuts from Benin, West Africa.

In a previous study, raw cashew kernels were assayed for the fungal contamination focusing on strains belonging to the genus Aspergillus and on aflatoxins producers. These samples showed high contamination with Aspergillus section Nigri species and absence of aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, the species of A. section Nigri may produce and thus threaten to contaminate the raw cashew kernels, 150 strains were isolated from cashew samples and assayed for their production of secondary metabolites using liquid chromatography high resolution mass spectrometry (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological and chemical identification: A. tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonarius (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). From these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A, produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalent and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxins groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), and secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts. Thirty strains of black Aspergilli were randomly sampled for verification of species identity based on sequences of β-tubulin and calmodulin genes. Among them, 27 isolates were positive to the primers used and 11 were identified as A. niger, 7 as A. tubingensis, 6 as A. carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names as based on morphology and chemical features. These strains clustered in 5 clades in A. section Nigri. Chemical profile clustering also showed also 5 groups confirming the species specific metabolites production.

Evaluation of the effectiveness of potential biocontrol yeasts against black sur rot and ochratoxin A occurring under greenhouse and field grape production conditions

The efficacy of two strains of Lanchancea thermotolerans in preventing the growth and ochratoxin A (OTA) accumulation of ochratoxigenic fungi under greenhouse and field conditions were evaluated during three consecutive year trials. The data from this study showed that both yeast strains were able to control Aspergillus section Nigri species ochratoxin A accumulation in wine grapes at harvest stage. The inhibitory effects were dependent on the ochratoxigenic species, yeast strains, and year analyzed. Over all conditions assayed, ochratoxin A accumulation was reduced from 27% to 100%, depending on the conditions evaluated. These results are promising for future development of a bio-pesticide.

Population Dynamics of Aspergillus Section Nigri Species on Vineyard Samples of Grapes and Raisins

Several species of Aspergillus section Nigri, including potential mycotoxin producers, are common residents of grape vineyards, but the relative population size of individual species throughout the growing season is difficult to determine using traditional isolation and identification methods. Using a quantitative droplet digital PCR (ddPCR) method in combination with dilution plating, total Aspergillus section Nigri populations and relative proportions of A. niger, A. welwitschiae, A. carbonarius, and A. tubingensis were measured from vineyard samples without the need for identifying individual fungal isolates. Grapes were sampled from two raisin vineyards (vineyards A and B) at berry set, veraison, harvest, and raisin stages in two consecutive years. Plate counts showed that the total population of Aspergillus section Nigri present on the fruit increased from berry set to raisin and became a larger component of the total recovered fungal population in both vineyards in both years. Results from ddPCR analysis showed that the relative proportion of A. carbonarius among the four species assayed increased later in the season (harvest and raisin) in comparison to earlier in the season (berry set and veraison). Total fungal and Aspergillus section Nigri plate counts were not significantly different between vineyards in either year. However, vineyard A generally showed higher proportions of A. carbonarius in harvest and raisin samples than vineyard B. This coincided with higher incidence and levels of ochratoxin A in vineyard A harvest and raisin fruit than in vineyard B fruit. This work demonstrates that this ddPCR method is a useful tool for culture-independent monitoring of populations of mycotoxigenic Aspergillus species during grape and raisin production.

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.

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.

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.

In vitro effect of pyrimethanil on the fungal growth of ochratoxigenic and non-ochratoxigenic species of Aspergillus tubingensis and Aspergil-lus foetidus isolated from Moroccan grapes

The contamination of Moroccan grapes by ochratoxin A is mainly due to the contamination of grapes by some species of Aspergillus section Nigri with prevalence of A. niger aggregate. Pyrimethanil, systemic fungicide of the anilinopyrimidine family, is commonly used in vineyard to control grey mould caused by the fungus Botrytis cinerea. In the current study, the effica-cy of this chemical compound for controlling growth of eight strains of A. tubingensis and A. foetidus (4 ochratoxigenic and 4 non-ochratoxigenic strains) isolated from Moroccan grapes was examined at 25°C on CYA medi-um. The results of molecular identification showed that similarity of all iso-lates studied compared with various sequences of references strains of A. tubingensis and A. foetidus from GenBank ranged from 100 to 99%, differing then by one to 4 positions. OTA produced by ochratoxigenic strains varied from 0.02 to 0.13 μg/g of CYA. Pyrimethanil effectively reduced the growth rate of A. tubingensis and A. foetidus. In general, there was a decrease of the RGR and an increase of lag phase as the fungicide dose increased regardless of strain. The EC50 concentrations ranged from 0.00013 to 2.89 mg/L (average = 1.15 mg/L) and EC90 varied from 26.75 to 502.81 mg/L. Statistical analysis revealed that fungicide concentrations, strains and their two inter-actions had significant effects on radial growth rate (p-values = 0.000). At concentration above 0.5xRD, growth of all strains was inhibited. Low doses of pyrimethanil (0.01xRD and 0.02xRD) reduced significantly the mycelia growth but the ochratoxigenic isolates were favoured.