Section Nigri Research Articles

Survey on the occurrence of Aspergillus section Nigri species in grapes cultivated in Umbria (central Italy) and influence of several factors on their distribution

AbstractSecondary bunch rots caused by Aspergillus species belonging to the section Nigri, commonly called black aspergilli, pose a serious threat to grapes yield and quality. Due to their ability to produce ochratoxin A (OTA), they are considered the primary source of contamination of this mycotoxin in grapes. In this study, a three-year survey was realized to assess the occurrence of black aspergilli in grapes harvested in the Umbria region, a very important grapevine cultivation area in central Italy, by fungal isolation and phylogenetic analysis. In addition, the influence of growing season, vineyard location, farming system and grapes growth stages on black aspergilli distribution was examined. Phylogenetic analysis of the 208 obtained isolates showed the presence of three black aspergilli species: Aspergillus tubingensis, Aspergillus welwitschiae and Aspergillus uvarum. Interestingly, A. carbonarius, one of the most important OTA producers, was not detected in the surveyed years. A. tubingensis, whose ability to produce OTA is controversial, was always the most isolated species followed by A. welwistichiae (OTA producer). The species A. uvarum (unable to produce OTA) was detected only in one surveyed year with a low incidence. A. tubingensis was always the species with the highest incidence, regardless of location, and farming system. Growth stage appeared to influence the incidence of the three species, that, in the case of A tubingensis, was lower during setting in comparison to berries pea-size and berries harvest-ripe. Finally, in the setting and berries pea-size stages, a stereomicroscope analysis, showed that flower debris was the substrate from which black aspergilli mainly developed prior to colonizing berries and bunches.

Draft genome sequence of two Aspergillus aculeatus isolated from cashew nuts from coastal Kenya.

Aspergillus aculeatus is a common saprophyte and ubiquitous fungus belonging to section Nigri. They produce diverse secondary metabolites which are important in biological processes and industrial applications. We present the draft genome sequences of two A. aculeatus isolated from cashew nuts from coastal Kenya.

Investigating the Ochratoxin Production Potential of Aspergillus spp in Indian Rice: A Comprehensive Study

Rice (Oryza sativa), world’s second most important cereal crop and most commonly consumed grains in the world. Though more than 100,000 species of fungi are known to exist, majority of mycotoxigenic fungi belong to the species of Aspergillus, Penicillium, and Fusarium. Among Aspergillus spp, Aspergillus niger and Aspergillus ochraceus are known to produce Ochratoxin A (OTA), a notable mycotoxin having adverse effects due to ubiquitous presence, renal toxicity and lengthy persistence. Totally eighty-one samples including unpolished (n = 36) and polished rice samples (n = 45) were collected in various districts of Tamil Nadu to identify ochratoxigenic fungi particularly Aspergillus spp. Out of 81 samples, 62 % (50/81) of samples including unpolished (n = 24) and polished rice (n = 26) documented the occurrence of ochratoxigenic fungi. Extraction of OTA was carried out by agar plug method and the ability of fungus to produce OTA was quantified by RP HPLC-FLD analysis. Among the fungal genera, Aspergillus spp (80 %) recorded the predominant fungus in rice. Molecular confirmation of ochratoxigenic fungi A. niger and A. ochraceus isolates was performed by 18s rDNA analysis. OTA producing ability by RP HPLC-FLD analysis revealed A. ochraceus of section Circumdati recorded the highest OTA production than A. niger isolates of section Nigri. The concentration range of OTA by A. ochraceus vary between 12.33 - 196.84 ng/g and 0.18 - 2.82 ng/g respectively in A. niger isolates. Among the A. ochraceus isolates, potent isolate AO 9 documented the highest OTA production (196.84 ng/g) followed by AO 6 (104.74 ng/g). Similarly, in A. niger AN 1 showed highest production of 2.82 ng/g followed by AN 5 isolate (1.25 ng/g) respectively. The objective of the study is to determine and quantify the ochratoxin production potential of Aspergillus spp in Indian rice by RP HPLC-FLD analysis. The occurrence of Aspergillus spp in rice and the subsequent ochratoxin production raise concern about possible health risk to animal and human environment upon consumption. Hence there is need for improved storage practices and regular monitoring to prevent the prevalence of Aspergillus fungus and OTA contamination in rice supply chain.

Antifungal activity of carvacrol-based solids and their effects on Whatman and Kraft paper

The degradation of cellulose-based materials by fungi represents a menace to the cultural heritage conservation. Carvacrol-based β-cyclodextrins and cocrystals proved effective antifungal remedies in vitro but their effects on paper structure and properties were not studied. The aim of this study was to investigate possible structural modifications and alterations of the mechanical, optical and chemical properties of artificially aged and unaged Whatman and Kraft paper subjected to the treatment with carvacrol-based β-ciclodextrins and cocrystals. The pH of the samples did not significantly change after the treatment, as well as no colour-related alterations were detected (1.00<ΔE<2.00). The tensile strength of both Whatman and Kraft paper was not affected by the vapours of carvacrol and spectroscopic analysis (FTIR and XRD) revealed no carvacrol-related damages of paper structure. The antifungal efficacy of the carvacrol-cocrystal was also proved on a book prototype made of Whatman and Kraft paper, kept under 98% of humidity for 28 days, and purposely inoculated with a mix of fungal species (A. alternata, Aspergillus sp. section Nigri, C. cladosporioides, and T. orientale). These results show the applicability of a carvacrol-releasing system, effective as antifungal remedy, and at the same time not harmful to Whatman and Kraft paper, as these materials did not show treatment-induced degradation.

Reduction in Ochratoxin A Occurrence in Coffee: From Good Practices to Biocontrol Agents.

Ochratoxin A (OTA) is a mycotoxin mainly produced by Aspergillus section Circumdati and section Nigri across the coffee chain. OTA is nephrotoxic and is a threat to human health. This review summarizes current knowledge on how to reduce OTA concentration in coffee from farm to cup. After a brief introduction to the OTA occurrence in coffee, current good management practices are introduced. The core of this review focuses on biocontrol and microbial decontamination by lactic acid bacteria, yeasts and fungi, and their associated enzymes currently reported in the literature. Special attention is given to publications closest to in vivo applications of biocontrol agents and microbial OTA adsorption or degradation agents. Finally, this review provides an opinion on which future techniques to promote within the coffee supply chain.

Evaluation of Primers OPF-01, P54, and 1253 to Identify A. fumigatus, A. flavus, and A. niger from Polymorphic Patterns Obtained by RAPD-PCR.

We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns' polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively.

ANTIBACTERIAL POTENTIAL AND MICROBIOLOGICAL QUALITY OF HONEY FROM SLOVAKIA

In this study, the antibacterial activity of 10 honeys at three concentrations 50%, 25%, and 12.5% was tested against two G- and two G+ strains, also the microbiological quality of the honeys in terms of the representation of total count of bacteria, coliforms, yeasts, and filamentous fungi was evaluated, and micromycetes to the species level were identified. Antibacterial activity of the honeys was assayed using well diffusion method, determination of microbial groups by the pour plate method and diversity of mycobiota in honey according to macro- and micromorphological characteristics. Results showed the antibacterial effects of Slovak honey collected from the Spiš region, against bacterial strains Escherichia coli, Pseudomonas fluorescens, Enterococcus faecalis, and Staphylococcus aureus, which are among the most common bacteria responsible for nosocomial infections. We found that honeydew honey was very effective against E. coli and S. aureus, rapeseed honey against P. fluorescens, and mixed honey (no. 10) against E. faecalis. Staphylococcus aureus was the most susceptible bacteria tested for all honeys. The presence of both yeasts and molds was detected in 3 honey samples at concentrations ranging from &lt;4x101 to 3.6x101 CFU/g, while the total count of bacteria was detected in 9 samples at concentrations ranging from 2.3x101 to 3.6x102 CFU/g (in 1 sample, the occurrence of microorganisms was not recorded even at the lowest dilution of 10-1). Coliforms were not isolated. A total of 3 strains belonging to the Aspergillus section Nigri were identified. The microbiological analyses of the samples indicates that the honeys were produced, processed and stored in accordance with the rules of good hygiene practice.

Ochratoxin A Contamination of California Pistachios and Identification of Causal Agents.

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium spp., which contaminates many crops, including pistachios. Pistachios contaminated with OTA may be subjected to border rejections resulting in significant economic losses to the United States agricultural revenues. The current study examined prevalence of OTA in California-grown pistachios and identified its causal agents. OTA was detected in 20% of samples from 2018 to 2021 (n = 809), with 18% of samples exceeding the European Union regulatory limit of 5 μg/kg. Fungi potentially responsible for OTA contamination were isolated from leaves, nuts, and soil collected from 14 pistachio orchards across California. A total of 1,882 isolates of Aspergillus section Nigri and 85 isolates of section Circumdati were recovered. Within section Nigri, 216 (11.5%) isolates were identified as potential OTA producers using a boscalid-resistance assay. Phylogenetic analyses of partial gene sequences for β-tubulin and calmodulin genes resolved section Circumdati into four species: A. ochraceus (33%), A. melleus (28%), A. bridgeri (21%), and A. westerdijkiae (19%). A. westerdijkiae produced the highest levels of OTA in inoculated pistachios (47 μg/g), followed by A. ochraceus (9.6 μg/g) and A. melleus (3.3 μg/g). A. bridgeri did not produce OTA. OTA production by section Circumdati was optimal from 20 to 30°C. All 216 boscalid-resistant isolates from section Nigri were identified as A. tubingensis, and representative isolates (n = 130) produced 3.8 μg/kg OTA in inoculated pistachios. This is the first detailed report on OTA contamination and causal fungi in California pistachios and will be helpful in devising effective management strategies.

Pre- and post-storage applications of rhamnolipids and cyprodinil + fludioxonil against Aspergillus section Nigri population and ochratoxin A contamination of grapes

Grapevines constitute a very important crop in Greece and ochratoxin A (OTA) contamination of grapes is one of the most serious problems. The main aim of this study was to investigate the fungicidal activity of the fungicide Switch 62.5WG (37.5% v/v cyprodinil + 25% v/v fludioxonil) and Rhapynal (80% rhamnolipids) to reduce the black Aspergillus spp. (section Nigri) population and OTA contamination of grapes grown in Greece. Applications with the above products were conducted both at the harvesting stage and after cold storage. The results showed that pre-harvest applications with Switch 62.5WG or Switch 62.5WG + Rhapynal generally reduced the black Aspergillus spp. population, but not the OTA contamination of grapes. However, a repeated application after cold storage further reduced the black Aspergillus spp. population and significantly reduced the OTA contamination of grapes. Generally, this study showed that it is possible to reduce the black Aspergillus spp. population, but not the ochratoxin A contamination of grapes by spray applications with the fungicide Switch 62.5WG. However, fungicide application to grapes immediately after cold storage (2 months) could reduce ochratoxin A contamination of grapes and further reduce the black Aspergillus spp. population.

First report of Aspergillus species in green pistachio of Bronte

Aspergillus contamination of pistachios causes significant product losses and potential presence of mycotoxins, particularly aflatoxin B1 (AFB1), and ochratoxin A (OTA). These toxins, which threaten human health, are strictly monitored by most nations. Italian pistachios produced in Bronte, Sicily, have high nutritional value and unique organoleptic properties, but the extent to which they contain these contaminants is unknown. Aspergillus spp. isolated from Bronte pistachios (cultivar Napoletana) were assessed for their ability to synthesize OTA or AFB1. Aspergillus occurrence in pistachio samples was measured at 1137 cfu g-1 for in shell pistachios and 770 cfu g-1 for kernels. The predominant isolated Aspergillus species was A. niger representing 74% of section Nigri (black isolates) and 47% of all Aspergillus isolates. Within section Flavi, A. flavus comprised 83% of green isolates. Only one black isolate (identified as A. carbonarius) had high OTA production, but all the A. flavus isolates had potential to produce AFG1 and AFB1, with AFB1 produced amount ranging from 0.1 to 8498 ng mL-1 of culture filtrate.

Assessment of mycobiota in Thai pigmented rice: Insights into ochratoxin A and citrinin production by Aspergillus and Penicillium species

Pigmented rice has gained interest for its nutritional and health benefits attributed to its bioactive compounds. Despite these advantages, the vulnerability of pigmented rice to mycotoxin contamination poses a major concern. This study examined the mycobiota of seven Thai pigmented rice varieties, with a focus on fungi capable of producing ochratoxin A (OTA) and citrinin. The study analyzed 110 pigmented rice samples and found that the Aspergillus section Aspergillus was predominant (50–82.35%), followed by Penicillium species (12.50–70.59%). One isolate of A. carbonarius from Aspergillus section Nigri (1/112) and two isolates from Aspergillus section Circumdati (2/61)—A. steynii and A. flocculosus—produced OTA. However, none of the Penicillium species produced OTA, though citrinin production was notable in P. citrinum (128/280). The study found that the Black Jasmine rice and Red Jasmine rice varieties were more susceptible to mycotoxin production, while the Luem Pua glutinous rice and Hom Nil rice varieties displayed more resistance to the accumulation of both mycotoxins. These findings should guide targeted strategies to mitigate mycotoxin risks and ensure the safety of pigmented rice in the food chain.

Reassessing the Etiology of Aspergillus Vine Canker and Summer Bunch Rot of Table Grapes in California.

Fungal taxonomy is in constant flux, and the advent of reliable DNA barcodes has enabled the enhancement of plant pathogen identification accuracy. In California, Aspergillus vine canker (AVC) and summer bunch rot (SBR) are economically important diseases that affect the wood and fruit of grapevines, respectively, and their causal agents are primarily species of black aspergilli (Aspergillus section Nigri). During the last decade, the taxonomy of this fungal group has been rearranged several times using morphological, physiological, and genetic analyses, which resulted in the incorporation of multiple cryptic species that are difficult to distinguish. Therefore, in this study, we aimed to reassess the etiology of AVC and SBR using a combination of morphological observations with phylogenetic reconstructions based on nucleotide sequences of the calmodulin (CaM) gene. Results revealed that the isolates causing AVC from recent isolations corresponded to A. tubingensis, whereas the isolates obtained from initial surveys when the disease was discovered were confirmed as A. niger and A. carbonarius. Similarly, the isolates obtained from table grapes with SBR symptoms and from spore traps placed in those vineyards were identified primarily as A. tubingensis, followed by A. niger and A. carbonarius. Notably, the A. niger isolates formed a subclade with strains previously known as A. welwitschiae, which is a species that was recently synonymized with A. niger. Overall, the most prevalent species was A. tubingensis, which was associated with both AVC and SBR, and representative isolates recovered from AVC-symptomatic wood, berries SBR symptoms, and spore traps were equally pathogenic in healthy wood and berries of 'Red Globe' grapevines. This study also constitutes the first report of A. tubingensis causing AVC and SBR of grapes in California and in the United States.

Cyp51A mutations, protein modeling, and efflux pump gene expression reveals multifactorial complexity towards understanding Aspergillus section Nigri azole resistance mechanism

Black Aspergillus species are the most common etiological agents of otomycosis, and pulmonary aspergillosis. However, limited data is available on their antifungal susceptibility profiles and associated resistance mechanisms. Here, we determined the azole susceptibility profiles of black Aspergillus species isolated from the Indian environment and explored the potential resistance mechanisms through cyp51A gene sequencing, protein homology modeling, and expression analysis of selected genes cyp51A, cyp51B, mdr1, and mfs based on their role in imparting resistance against antifungal drugs. In this study, we have isolated a total of 161 black aspergilli isolates from 174 agricultural soil samples. Isolates had variable resistance towards medical azoles; approximately 11.80%, 3.10%, and 1.24% of isolates were resistant to itraconazole (ITC), posaconazole (POS), and voriconazole (VRC), respectively. Further, cyp51A sequence analysis showed that non-synonymous mutations were present in 20 azole-resistant Aspergillus section Nigri and 10 susceptible isolates. However, Cyp51A homology modeling indicated insignificant protein structural variations because of these mutations. Most of the isolates showed the overexpression of mdr1, and mfs genes. Hence, the study concluded that azole-resistance in section Nigri cannot be attributed exclusively to the cyp51A gene mutation or its overexpression. However, overexpression of mdr1 and mfs genes may have a potential role in drug resistance.

Unveiling ochratoxin A and ochratoxigenic fungi in Brazilian artisanal Cheeses: Insights from production to consumption

Ochratoxin A (OTA) is a toxin produced by several Aspergillus species, mainly those belonging to section Circumdati and section Nigri. The presence of OTA in cheese has been reported recently in cave cheese in Italy. As artisanal cheese production in Brazil has increased, the aim of this study was to investigate the presence of ochratoxin A and related fungi in artisanal cheese consumed in Brazil.A total of 130 samples of artisanal cheeses with natural moldy rind at different periods of maturation were collected. Of this total, 79 samples were collected from 6 producers from Canastra region in the state of Minas Gerais, since this is the largest artisanal cheese producer region; 13 samples from one producer in the Amparo region in the state of São Paulo and 36 samples from markets located in these 2 states. Aspergillus section Circumdati occurred in samples of three producers and some samples from the markets. A. section Circumdati colony counts varied from 102 to 106 CFU/g. Molecular analysis revealed Aspergillus westerdijkiae (67 %) as the most frequent species, followed by Aspergillus ostianus (22 %), and Aspergillus steynii (11 %). All of these isolates of A. section Circumdati were able to produce OTA in Yeast Extract Sucrose Agar (YESA) at 25 °C/7 days. OTA was found in 22 % of the artisanal cheese samples, ranging from 1.0 to above 1000 µg/kg, but only five samples had OTA higher than 1000 µg/kg. These findings emphasize the significance of ongoing monitoring and quality control in the artisanal cheese production process to minimize potential health risks linked to OTA contamination.

Description and management of Aspergillus section Nigri causing post-harvest bulbs rot of onion

When onions are improperly stored, a post-harvest disease known as black mold of onion bulbs can result in considerable economic losses. Aspergillus section Nigri, one of many species, has been implicated in the development of black mold. In the present study, rot onion bulbs were collected from markets in Qena, Egypt. Thirteen Aspergillus section Nigri isolates were obtained and identified by morphological and molecular characterization. The ochratoxins potential of isolated A. section Nigri was tested, and three isolates were producers at the range of 1.5–15 ppm. For the presence of pks gene, no amplification product was detected. Using the fungal growth inhibition test, the isolates of A. niger were inhibited by eco-friendly materials Cement and Zeolite. Cement exhibited maximum percentage growth inhibition against the tested isolates at 74.7–86.7%. The pathogenicity activity of the A. niger isolates was tested by inoculation of healthy onion bulbs, other onion bulbs covered with Cement and Zeolite before inoculation by A. niger was used. The two treatments significantly reduced bulbs rot disease of onion than untreated bulbs. Seven and nine isolates showed 0% rot on covered bulbs by Cement and Zeolite, respectively as compared with inoculated onions, which exhibited rot ranging from 55 to 80%. Using eco-friendly materials with efficiency against post-harvest bulbs rot of onion was evaluated in this study.

Development of a machine learning model for systematics of Aspergillus section Nigri using synchrotron radiation-based fourier transform infrared spectroscopy

Aspergillus section Nigri (black aspergilli) fungi are economically important food spoilage agents. Some species in this section also produce harmful mycotoxins in food. However, it is remarkably difficult to identify this fungal group at the species level using morphological and chemical characteristics. The molecular approach for classification is preferable; however, it is time-consuming, making it inappropriate for rapid testing of large numbers of samples. To address this, we explored synchrotron radiation-based Fourier transform infrared microspectroscopy (SR-FTIR) as a rapid method for obtaining data suitable for species classification. SR-FTIR data were obtained from the mycelia/conidia of 22 black aspergilli species. The Convolutional Neural Network (CNN) approach, a supervised deep learning algorithm, was used with SR-FTIR data to classify black aspergilli at the species level. A subset of the data was used to train the CNN model, and the model classification performance was evaluated using the validation data subsets. The model demonstrated a 95.97% accuracy in species classification on the testing (blind) data subset. The technique presented herein could be an alternative method for identifying problematic black aspergilli in the food industry.

Aspergillus population diversity and its role in aflatoxin contamination of cashew nuts from coastal Kenya.

Cashew nuts are among the main cash crops in coastal Kenya, due in large part to their high nutritional value. Unfortunately, they also make them highly susceptible to mold contamination, resulting in biodeterioration of the nutritional value and potential contamination with toxic secondary metabolites, such as aflatoxins, that cause them to be rejected for sale at the market. We determined the population diversity of the Aspergillus species and their role in aflatoxin contamination in cashew nuts in selected coastal regions of Kenya. Fifty raw cashew nut samples were collected from post-harvest storage facilities across three counties in Kenya's coastal region and examined for moisture content and the presence of Aspergillus fungi. About 63 presumptive isolates were recovered from the cashew nuts. ITS and 28S rDNA regions were sequenced. The aflD, aflM and aflR genes were amplified to identify the potentially aflatoxigenic from the Aspergillus isolates. The Aflatoxins' presence on the isolates was screened using UV and the ammonia vapour test on coconut milk agar and validated using ELISA assay. A comparison of cashew moisture content between the three counties sampled revealed a significant difference. Sixty-three isolates were recovered and identified to section based on morphological characters and their respective ITS regions were used to obtain species identifications. Three sections from the genus were represented, Flavi and Nigri, and Terrei with isolates from the section Nigri having slightly greater abundance (n = 35). The aflD, aflM and aflR genes were amplified for all isolates to assess the presence of the aflatoxin biosynthesis pathway, indicating the potential for aflatoxin production. Less than half of the Aspergillus isolates (39.68%) contained the aflatoxin pathway genes, while 22.22% isolates were aflatoxigenic, which included only the section Flavi isolates. Section Flavi isolates identification was confirmed by calmodulin gene. The presence of species from Aspergillus section Flavi and section Nigri indicate the potential for aflatoxin or ochratoxin in the cashew nuts. The study established a foundation for future investigations of the fungi and mycotoxins contaminating cashew nuts in Kenya, which necessitates developing strategies to prevent infection by mycotoxigenic fungi, especially during the storage and processing phases.

Multilocus Sequence Analysis of Aspergillus Species from Clinical Samples and the Surge of Cryptic Aspergillosis in Southern India: A Descriptive Cross-sectional Study

Introduction: Aspergillus species cause a broad range of infections, from Allergic Bronchopulmonary Aspergillosis (ABPA) to invasive aspergillosis. Accurate species-level identification is crucial for diagnosis and treatment, especially since some rare Aspergillus species exhibit increased resistance to common antifungal agents. Aim: To identify Aspergillus species at the species complex level and determine the emergence of cryptic Aspergillus species using Multilocus Sequence Analysis (MLSA). Materials and Methods: This descriptive cross-sectional study was conducted over eight months, from January 2023 to August 2023, in three tertiary care centres in Chennai, India. Sixty Aspergillus strains were isolated from various clinical samples. The primary inclusion criteria were patients with suspected aspergillosis, categorised based on the European Organszation for Research and Treatment of Cancer Mycoses Study Group (EORTC-MSG) criteria. Isolates were grown on Oatmeal Agar or Sabouraud Dextrose Agar (SDA) and phenotypically identified using tease mount and slide culture techniques. Genomic fungal Deoxyribonucleic acid (DNA) was extracted and amplified using specific primers for Internal transcribed spacer (ITS), beta-tubulin, hydrophobin, and calmodulin genes, followed by nucleic acid sequencing. Results: Among the 60 Aspergillus isolates, the Aspergillus flavus complex comprised 26 (43.3%), the Aspergillus fumigatus complex 16 (26.7%), the Aspergillus niger complex 12 (20%), and the Aspergillus terreus complex 6 (10%). Initial speciation relied on microscopic and cultural characteristics. Isolates were recovered from various clinical specimens: bronchial wash 6 (10%), Bronchoalveolar lavage (BAL) fluid 10 (16.7%), sputum 3 (5%), ear swab 20 (33.3%), endotracheal aspirate 8 (13.3%), pus and tissue from the nasal cavity and paranasal sinus 11 (18.3%), and tissue from other sites 2 (3.3%). MLSA identified common species such as A. flavus, A. fumigatus, A. terreus, and A. niger, as well as rare species including A. awamori, A. carneus, A. lentulus, A. oryzae, A. tamarii, A. tubingensis, and A. welwitschiae. Sequence identity ranged from 99% to 100%, confirming species diversity. Phylogenetic analysis using ITS, beta-tubulin, hydrophobin, and calmodulin genes supported species identification with robust bootstrap support (1000 replications). Conclusion: Cryptic Aspergillus species are prevalent in clinical samples from the South Indian population, with a notable presence from the section Nigri and the first-time isolation of Aspergillus carneus in clinical samples from India. Molecular identification is essential for determining the invasive potential and guiding appropriate antifungal therapy.

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.

Simultaneous detection of mycotoxigenic Aspergillus species of sections Circumdati and Flavi using multiplex digital PCR.

Populations of ochratoxin-producing Aspergillus section Circumdati species and aflatoxin-producing Aspergillus section Flavi species frequently coexist in soil and are the main sources of mycotoxin contamination of tree nuts. Identification of mycotoxigenic Aspergillus species in these sections is difficult using traditional isolation and culture methods. We developed a multiplex digital PCR (dPCR) assay to detect and quantify Aspergillus ochraceus, Aspergillus westerdijkiae, and Aspergillus steynii (section Circumdati), as well as Aspergillus flavus and Aspergillus parasiticus (section Flavi), in environmental samples based on species-specific calmodulin gene sequences. Relative quantification of each species by dPCR of mixed-species templates correlated with corresponding DNA input ratios. Target species could be detected in soil inoculated with conidia from each species. Non-target species of sections Circumdati, Flavi, and Nigri were generally not detectable using this dPCR method. Detected non-target species (Aspergillus fresenii, Aspergillus melleus, Aspergillus sclerotiorum, and Aspergillus subramanianii) were discernible from A. ochraceus in dual-template dPCR reactions based on differential fluorescence intensity.