Toxigenic Species Research Articles

Identifying the Source Organisms Producing Paralytic Shellfish Toxins in a Subtropical Bay in the South China Sea.

Identifying the exact phytoplanktonic sources of paralytic shellfish toxins (PSTs) is crucial for monitoring and preventing the buildup of toxin pollution, especially for causative species occurring at low levels. Phytoplankton and shellfish samples were simultaneously collected from representative mariculture zones in Daya Bay, China. Low concentration/low toxicity PSTs predominated with N-sulfocarbamoyl toxins 1, 2 (C1/2) being detected in phytoplankton (≤6.25 pmol L-1) and shellfish (≤0.21 μg STXeq g-1), which pose a potential risk of seafood poisoning. High-throughput sequencing of the phytoplankton samples based on 18S rDNA V4 regions identified 93 genera in 445 operational taxonomic units (OTUs). Five OTUs were assigned to Alexandrium hiranoi, Ambicodamus leei, Alexandrium pacificum, Alexandrium minutum, and an uncertain Alexandrium sp. A. pacificum and A. minutum are candidate PST producers and observed under the light microscope with densities of 66-972 cells L-1. Three strains of toxigenic species were successfully isolated and identified as A. pacificum and A. minutum based on their 18S rDNA V4 regions. The predominant toxins in A. pacificum were C1/2 (43.9-53.6 fmol cell-1) and resembled the toxins found in field samples predominated with C1/2. A. minutum produced only gonyautoxins 2/3 (8.03 fmol cell-1). Therefore, A. pacificum was identified as the predominant PST contributor in this area. This research makes a valuable contribution to the understanding of the traceability of phycotoxins in marine waters.

Bioburden in sleeping environments from Portuguese dwellings.

A wider characterization of indoor air quality during sleep is still lacking in the literature. This study intends to assess bioburden before and after sleeping periods in Portuguese dwellings through active methods (air sampling) coupled with passive methods, such as electrostatic dust cloths (EDC); and investigate associations between before and after sleeping and bioburden. In addition, and driven by the lack of information regarding fungi azole-resistance in Portuguese dwellings, a screening with supplemented media was also performed. The most prevalent genera of airborne bacteria identified in the indoor air of the bedrooms were Micrococcus (41%), Staphylococcus (15%) and Neisseria (9%). The major indoor bacterial species isolated in all ten studied bedrooms were Micrococcus luteus (30%), Staphylococcus aureus (13%) and Micrococcus varians (11%). Our results highlight that our bodies are the source of the majority of the bacteria found in the indoor air of our homes. Regarding air fungal contamination, Chrysosporium spp. presented the highest prevalence both in after the sleeping period (40.8%) and before the sleeping period (28.8%) followed by Penicillium spp. (23.47% morning; 23.6% night) and Chrysonilia spp. (12.4% morning; 20.3% night). Several Aspergillus sections were identified in air and EDC samples. However, none of the fungal species/strains (Aspergillus sections Fumigati, Flavi, Nidulantes and Circumdati) were amplified by qPCR in the analyzed EDC. The correlations observed suggest reduced susceptibility to antifungal drugs of some fungal species found in sleeping environments. Toxigenic fungal species and indicators of harmful fungal contamination were observed in sleeping environments.

ВЕТЕРИНАРНО-САНИТАРНАЯ ОЦЕНКА КОФЕЙНОГО СЫРЬЯ, ЗАГРЯЗНЕННОГО ПЛЕСНЕВЫМИ ГРИБАМИ И ИХ ТОКСИНАМИ

Coffee refers to the products of daily mass consumption around the world. At the same time, there is a risk of contamination by mold fungi at all stages of production of this product. The most dangerous species are those that are capable of producing mycotoxins, among which the most dangerous are ochratoxins and aflatoxins. In this regard, it is particularly important to conduct a veterinary and sanitary examination aimed at studying the content of mold fungi, assessing their species composition and, especially, toxigenic species in raw materials, thereby preventing risks to human health. When studying the internal mycoflora of green coffee beans, the contamination with mold fungi was found to be from 70 to 100%, depending on the region of origin. The main contaminants were fungi of the genus Aspergillus and Penicillum, among which potential producers were identified: ochratoxins A and B-A. carbonarius and A. sclerotioniger; ochratoxin A and fumonisins-A. niger.

Mycological and chemical screening of maize at open Nigeria

The aim of the present study was to screen maize at the open markets in Osun state, Nigeria to determine species of fungi total aflatoxin content and changes inproximate, energy and some vitamins content of maize grains. A total of forty five mouldy maize samples were collected from the six different open markets within the region which were later bulked into six representative samples (20-50Kg) for each market while Aflasafe maize, obtained from the Internationa lInstitute of Tropical Agriculture (IITA) was used as a control.There presentative samples from the bulk were taken for proximate,selected vitamins and total aflatoxin contents determination.The quantitative enumeration of fungias colony forming units per gram of the grains (CFU/g) was performed using the surface spread method in different culture media. The results of fungi profile were further expressed as fungal isolation frequency (IF) and relative density (RD). Twelve (12) species and seven fungi genera were isolated in this study. The total fungi count ranged from 1.50x10 to 2.1X10 CFU/g in the six different locations. Aspergillus flavus, Rhyzopus stolonifer and Aspergillus glaucus were the most prevalent species Aspergillus and Rhizopus species occurred at 100% and 87% (IF) respectively. The toxigenic species of Aspergillus, Fusarium and Penicellium had 85.7%,14.3% and 14.3% RD respectively. The proximate contents of the mouldy maize reduced by 11.54% for crude protein, 12.72% for ether extract and 1.12% for ash while about 5% caloric loss was observed. Total Aflatoxin in mouldy maize rose from near zero in aflasafe maize to 267.41µg/Kg with an average value of 142.0µg/Kg in mouldy maize while retinol and tocopherol contents were depleted by 48.7% and 37.6% respectively. This study indicated substantial loss of nutrients and reduced feed value in maize due to mould contamination and the need for continuous assessment offered ingredients particularly maize for mycological and nutritive status in order to feed animals for optimal performance

СТРУКТУРА МИКОКОМПЛЕКСОВ ФИЛЛОПЛАНЫ БОБОВЫХ КУЛЬТУР

The importance of legumes as a component of biological crop rotations dictates the need to study the species composition of microscopic fungi in the phylloplan and to determine the presence of phytopathogenic, toxigenic, allergenic and opportunistic species in mycocomplexes. The aim was to study the structure of mycocomplexes of the phylloplan of cultivated peanuts, cowpea, sowing vetch, varicoloured clover, creeping clover, alfalfa, and hornbeam. The isolation of fungi from the phylloplan was carried out by washing from the leaf surface in 4 replicates. The prepared samples were transferred to Petri dishes with Saburo media. The experiment was repeated 3-5 times. The cultures were incubated in a thermostat at 25 ± 2 ºС for 5-14 days. The grown colonies were seeded into a test tube on an oblique nutrient medium. From the leaf surface of the studied legumes, 17 genera were identified, represented by 31 species of fungi, which are grouped according to the frequency of occurrence of species in the complex and the proportion in the group. A high proportion of A. alternata fungi was characteristic of the mycocomplexes of leaves of the common vetch, varicoloured elm, creeping clover, and alfalfa. F. sporotrichioides was found on creeping clover and alfalfa, F. moniliforme was found on horned clover, and both species were found on radiant cowpea. The content of toxigenic, opportunistic and allergenic species of fungi is least of all revealed under annual species: cultivated peanuts and radiant cowpea. The species composition of the phylloplan of leguminous crops should be taken into account in the development of plant protection measures and taking into account their correct placement in crop rotations.

Fungal Contamination in the Working Environ-Ment of Waste Sorting Facilities: A Review

Abstract In recent years, many studies have pointed to a relationship between the working activity in waste sorting facilities and the occurrence of health problems (respiratory, gastrointestinal, musculoskeletal, skin and eye irritations). Employees in these facilities are simultaneously exposed to many biological, chemical and physical factors. Due to the repeated detection of high concentrations of airborne fungi (reaching up to 1.8 x 106 CFU m–3) in the working environment involving potentially allergenic, infectious and toxigenic fungal species, it can be assumed that fungi may play an important role in the development of health problems. In terms of minimizing health risks, it is necessary to take several preventive and protective measures to reduce contamination of the working environment by biological agents. The basic recommendation is the consistent use of protective equipment as well as the observance of personal hygiene by the employees. Other necessary measures are the frequent air exchange inside the facility, educating the employees about health risks associated with waste sorting and establishment of regular medical check-ups.

Black aspergilli in Brazilian onions: From field to market

The occurrence of black aspergilli in onions has been reported as frequent, and this group of fungi harbors potentially toxigenic species. In addition, Aspergillus niger has been reported as the causative agent of black mold rot, an important postharvest disease that causes damage throughout the world. Brazil stands out as one of the world's largest onion producers. However, few studies have been conducted to investigate the mycobiota in Brazilian onions. For this reason, we investigated the mycobiota of 48 market (n = 25) and field (n = 23) onion bulb samples. Nineteen soil samples were collected from the same fields and evaluated. In field onions and soil samples, Penicillium spp. was the prevalent fungal group, whereas in market samples A. section Nigri was the most frequent group. Due to the taxonomic complexity of this group, species identification was supported by phylogenetic data (CaM gene). A. welwitschiae was the most prevalent species in market samples. Black aspergillus strains were evaluated for fumonisin B2 (FB2) and ochratoxin A (OTA) production. Overall, 53% and 2.2% of the strains produced FB2 and OTA, respectively. The occurrence of FB2 and OTA was also investigated in onion bulb samples but none showed contamination with these mycotoxins.

A Review of Potential Impacts of Climate Change on Coffee Cultivation and Mycotoxigenic Fungi.

Coffee is one of the most traded commodities in the world. It plays a significant role in the global economy, employing over 125 million people. However, it is possible that this vital crop is threatened by changing climate conditions and fungal infections. This paper reviews how suitable areas for coffee cultivation and the toxigenic fungi species of Aspergillus, Penicillium, and Fusarium will be affected due to climate change. By combining climate models with species distribution models, a number of studies have investigated the future distribution of coffee cultivation. Studies predict that suitable coffee cultivation area could drop by ~50% under representation concentration pathway (RCP) 6.0 by 2050 for both Arabica and Robusta. These findings agree with other studies which also see an altitudinal migration of suitable cultivation areas to cooler regions, but limited scope for latitudinal migration owing to coffee’s inability to tolerate seasonal temperature changes. Increased temperatures will see an overall increase in mycotoxin production such as aflatoxins, particularly in mycotoxigenic fungi (e.g., Aspergillus flavus) more suited to higher temperatures. Arabica and Robusta’s limited ability to relocate means both species will be grown in less suitable climates, increasing plant stress and making coffee more susceptible to fungal infection and mycotoxins. Information regarding climate change parameters with respect to mycotoxin concentrations in real coffee samples is provided and how the changed climate affects mycotoxins in non-coffee systems is discussed. In a few areas where relocating farms is possible, mycotoxin contamination may decrease due to the “parasites lost” phenomenon. More research is needed to include the effect of mycotoxins on coffee under various climate change scenarios, as currently there is a significant knowledge gap, and only generalisations can be made. Future modelling of coffee cultivation, which includes the influence of atmospheric carbon dioxide fertilisation and forest management, is also required; however, all indications show that climate change will have an extremely negative effect on future coffee production worldwide in terms of both a loss of suitable cultivation areas and an increase in mycotoxin contamination.

Intraseasonal variation of phycocyanin concentrations and environmental covariates in two agricultural irrigation ponds in Maryland, USA.

Recently, cyanobacteria blooms have become a concern for agricultural irrigation water quality. Numerous studies have shown that cyanotoxins from these harmful algal blooms (HABs) can be transported to and assimilated into crops when present in irrigation waters. Phycocyanin is a pigment known only to occur in cyanobacteria and is often used to indicate cyanobacteria presence in waters. The objective of this work was to identify the most influential environmental covariates affecting the phycocyanin concentrations in agricultural irrigation ponds that experience cyanobacteria blooms of the potentially toxigenic species Microcystis and Aphanizomenon using machine learning methodology. The study was performed at two agricultural irrigation ponds over a 5-month period in the summer of 2018. Phycocyanin concentrations, along with sensor-based and fluorometer-based water quality parameters including turbidity (NTU), pH, dissolved oxygen (DO), fluorescent dissolved organic matter (fDOM), conductivity, chlorophyll, color dissolved organic matter (CDOM), and extracted chlorophyll were measured. Regression tree analyses were used to determine the most influential water quality parameters on phycocyanin concentrations. Nearshore sampling locations had higher phycocyanin concentrations than interior sampling locations and "zones" of consistently higher concentrations of phycocyanin were found in both ponds. The regression tree analyses indicated extracted chlorophyll, CDOM, and NTU were the three most influential parameters on phycocyanin concentrations. This study indicates that sensor-based and fluorometer-based water quality parameters could be useful to identify spatial patterns of phycocyanin concentrations and therefore, cyanobacteria blooms, in agricultural irrigation ponds and potentially other water bodies.

Antifungal activity of Euphorbia species against moulds responsible of cereal ear rots.

This work aimed to identify secondary metabolites from aerial parts of Euphorbia species functional for control of toxigenic Fusarium species responsible of cereal grain rots. Aerial parts of Euphorbia serpens, Euphorbia schickendantzii and Euphorbia collina were sequentially extracted with hexane, ethyl acetate and methanol. The extracts were tested against strains of Fusarium verticillioides and Fusarium graminearum by microdilution tests. The hexane extract of E. collina provided the lowest IC50 s on both fungal species. Further fractionation showed that cycloartenol (CA) and 24-methylenecycloartanol are associated to the moderate inhibitory effect of the hexane extract on fungal growth.Sublethal concentrations of CA and 24MCA blocked deoxynivalenol (DON) and fumonisins production.CA and 24MCA co-applied with potassium sorbate, a food preservative used for Fusarium control, synergized the growth inhibition of fungi. The mixtures reduced mycotoxins accumulation when applied at sublethal concentrations. CA and 24MCA inhibited both fungal growth and mycotoxins production. This fact is an advantage respect to potassium sorbate which increased the mycotoxins accumulation at sublethal concentrations. CA and 24MCA synergized potassium sorbate and their mixtures offer a lower mycotoxigenic risk than potassium sorbate for control of the Fusarium species.

Potential Control of Mycotoxigenic Fungi and Ochratoxin A in Stored Coffee Using Gaseous Ozone Treatment

The objective of this study was to examine the effect of treatment of Arabica green coffee beans with gaseous ozone (O3) for the control of ochratoxigenic fungi and ochratoxin A (OTA) contamination by Aspergillus westerdijkiae, A. ochraceus, and A. carbonarius during storage. Studies included (i) relative control of the populations of each of these three species when inoculated on irradiated green coffee beans of different initial water availabilities using 400 and 600 ppm gaseous O3 treatment for 60 min at a flow rate of 6 L−1 and on OTA contamination after 12 days storage at 30 °C and (ii) effect of 600 ppm O3 treatment on natural populations of green stored coffee beans at 0.75, 0.90, and 0.95 water activity (aw) or with additional inoculum of a mixture of these three ochratoxigenic fungi after treatment and storage for 12 days at 30 °C on fungal populations and OTA contamination. Exposure to 400 and 600 ppm O3 of coffee beans inoculated with the toxigenic species showed that there was less effect on fungal populations at the lowered aw (0.75). However, toxigenic fungal populations significantly increased 48 h after exposure and when stored at 0.90 and 0.95 aw for 12 days. All three species produced high amounts of OTA in both O3 treatments of the wetter coffee beans at 0.90 and 0.95 aw. Gaseous O3 (600 ppm) treatment of naturally contaminated green coffee beans had little effect on fungal populations after treatment, regardless of the initial aw level. However, after storage, there was some reduction (26%) observed in coffee at 0.95 aw. In addition, no fungal populations or OTA contamination occurred in the 0.75 and 0.90 aw treatments after exposure to 600 ppm gaseous O3 and storage for 12 days. It appears that under wetter conditions (≥0.90–95 aw) it is unlikely that fungal populations and OTA contamination of stored coffee beans, even with such high O3 concentrations would be controlled. The results are discussed in the context of potential application of O3 as an intervention system for stored coffee post-fermentation and during medium term storage and transport.

Insight into Unprecedented Diversity of Cyanopeptides in Eutrophic Ponds Using an MS/MS Networking Approach.

Man-made shallow fishponds in the Czech Republic have been facing high eutrophication since the 1950s. Anthropogenic eutrophication and feeding of fish have strongly affected the physicochemical properties of water and its aquatic community composition, leading to harmful algal bloom formation. In our current study, we characterized the phytoplankton community across three eutrophic ponds to assess the phytoplankton dynamics during the vegetation season. We microscopically identified and quantified 29 cyanobacterial taxa comprising non-toxigenic and toxigenic species. Further, a detailed cyanopeptides (CNPs) profiling was performed using molecular networking analysis of liquid chromatography-tandem mass spectrometry (LC-MS/MS) data coupled with a dereplication strategy. This MS networking approach, coupled with dereplication, on the online global natural product social networking (GNPS) web platform led us to putatively identify forty CNPs: fourteen anabaenopeptins, ten microcystins, five cyanopeptolins, six microginins, two cyanobactins, a dipeptide radiosumin, a cyclooctapeptide planktocyclin, and epidolastatin 12. We applied the binary logistic regression to estimate the CNPs producers by correlating the GNPS data with the species abundance. The usage of the GNPS web platform proved a valuable approach for the rapid and simultaneous detection of a large number of peptides and rapid risk assessments for harmful blooms.

Mycotoxins Content of Smoked Fishes Sampled on Market Stalls of Ngaoundere and Antifungal Activity of Essential Oils of Cymbopogon citratus and Ocimum basilicum against Mycotoxin Producing Strains

Aim: The aim of this study was to evaluate the sanitary quality of smoked fish by determining their mycotoxin content, and also the chemical composition, antifungal activity of Cymbopogon citratus and Ocimum basilicum essential oils from Cameroon against some mycotoxigenic fungal strains responsible of the smoked fish biodegradation.
 Place and Duration of Study: Laboratory of Food Microbiology and Biotechnology, National School of Agro-Industrial Sciences, University of Ngaoundere, Cameroon, from August 2019 to April 2020.
 Methodology: Fifteen samples of smoked fishes have been collected in August 2019 from “petit marché” market (Ngaoundere, Cameroon). Physico-chemical parameters of smoked fishes were evaluated. Mycotoxin (AFB1, CIT, and OTA) contents have been determined with HPLC. Isolation and identification of molds were done using their macroscopic and microscopic characteristics, and the identity of the strains was done by PCR sequencing methods. For plants, 15 kg of Cymbopogon citratus leaves and 5 kg of Ocimum basilicum leaves have been harvested on 10 September 2019 in Ngaoundere and used for the essential oil’s extraction. Essential oil extraction has been done through hydrodistillation and the determination of its chemical composition done with GC/MS.
 Results: Major part of samples contains at least one mycotoxin, in quantities which are beyond the safe dose. Eleven species of molds have been identified: Aspergillus spp., Penicillium citrinum, and Mucor hiemalis. Amongst the isolated species, those which can produce mycotoxins are: Penicillium citrinum, Aspergillus versicolor, Aspergillus fumigatus, Aspergillus carbonarius, Aspergillus niger, and Fusarium moniliforme. Geranial (42.4%), Neral (33.5%) and Myrcene (10.8%) are major compounds found in the essential oil of C. citratus while monoterpens (60.8%), Eugenol (30.7%), Linalol (29.4%) and 1,8-Cineol (14.3%) are the major compounds found in the essential oil of O. basilicum. 
 Conclusion: Essential oils of C. citratus and O. basilicum are efficient against isolated toxigenic species. C. citratus being more efficient than O. basilicum.

Temporal and spatial distribution of Azadinium species in the inland and coastal waters of the Pacific northwest in 2014–2018

Azaspiracids, produced by some species of the dinoflagellate genera Azadinium and Amphidoma, can cause a syndrome in humans called azaspiracid shellfish poisoning (AZP). In 1995, mussels from the Irish west coast contaminated with azaspiracids were, for the first time, linked to this human illness that has symptoms of nausea, vomiting, severe diarrhea, and stomach cramps. The only confirmed cases of AZP to date in the United States occurred in Washington State in 2008 from mussels imported from Ireland. Shortly after this case, several others involving similar gastrointestinal symptoms were reported by shellfish consumers from Washington State. However, no detectable diarrhetic shellfish toxins or Vibrio contamination were found. Cursory analysis of Solid Phase Adsorption Toxin Tracking (SPATT) samplers suggested the presence of azaspiracids in Washington State waters and motivated a study to evaluate the presence and distribution of Azadinium species in the region. During the spring and summer months of 2014–2015, quantitative polymerase chain reaction (qPCR) analyses detected the presence of the toxigenic species Azadinium poporum and A. spinosum on the outer coast and throughout the inland waters of Washington State. In 2016–2018, standard curves developed using A. poporum isolated from Puget Sound and A. spinosum isolated from the North Sea were used to quantify abundances of up to 10,525 cells L−1 of A. poporum and 156 cells L−1 of A. spinosum at shore-based sites. Abundances up to 1,206 cells L−1 of A. poporum and 30 cells L−1 of A. spinosum were measured in the coastal waters of the Pacific Northwest in 2017. Other harmful genera, including Alexandrium, Dinophysis, and Pseudo-nitzschia, were observed using light microscopy at coastal sites where A. poporum was also observed. In some samples where both A. poporum and A. spinosum were absent, an Amphidomataceae-specific qPCR assay indicated that other species of Azadinium or Amphidoma were present. The identification of Azadinium species in the PNW demonstrates the need to assess their toxicity and to incorporate their routine detection in monitoring programs to aid resource managers in mitigating risks to azaspiracid shellfish poisoning in this region.

Toxigenic fungal species and natural occurrence of mycotoxins in crops harvested in Argentina

Mycotoxins are secondary metabolites produced by fungal species that mainly belong to Aspergillus, Fusarium, Penicillium and Alternaria, which can grow in a variety of crops including cereals, oilseeds and fruits. Consequently, their prevalence in foods and by-products not only affects human and animal health but also causes important losses in both domestic and international markets. This review provides data about toxigenic fungal species and mycotoxin occurrence in different crops commonly grown in Argentina. This information will be relevant to establish adequate management strategies to reduce the impact of mycotoxins on human food and animal feed chains and to implement future legislation on the maximum permitted levels of these fungal metabolites.

Assessment of the microbial contamination of mechanical protection gloves used on waste sorting industry: A contribution for the risk characterization

In Portugal, mechanical protection gloves (MPG) are of mandatory use and during their use sweat is released and, consequently, the humidity of the material increases leading to conditions favorable to the growth of microorganisms. However, no studies have been conducted in MPG to assess the bioburden. This study intended to determine the bioburden present in MPG and their biological effects, and to discuss the possibility to use MPG as a passive method to assess occupational exposure to microbial contamination. Fungal burden was characterized through molecular tools for fungal toxigenic species, and antifungal resistance and mycotoxins profiles were determined. Cell viability was determined in swine kidney (SK) monolayer and hepatocellular carcinoma (Hep G2) cell lines. All MPG samples presented Gram-negative bacteria. The fungal contamination ranged from 0 CFU.m−2 in both MEA and DG18, to 5.09 × 106 and 2.75 × 106 and the most commonly fungi found was Aspergillus spp. (50.46%). Azole resistant Aspergillus sections were found in azole supplemented media. Aspergillus sections (Circumdati, Flavi, Fumigati and Versicolores) were detected by molecular tools in 66 out of 67 samples. The most reported mycotoxin was mycophenolic acid (89.6%). HepG2 cells appear to be more sensitive to MPG contamination, with high cytotoxicity (IC50 < 0.05 mm2/ml) observed for 18 out of 57 gloves.MPG can be used in passive sampling to assess occupational exposure to bioburden in waste sorting industries and contribute for risk characterization. Some contaminants of MPG had cytotoxic potential and affected the biology of hepatic cells more than renal cells.

Distribution and abundance of azaspiracid-producing dinophyte species and their toxins in North Atlantic and North Sea waters in summer 2018.

Representatives of the marine dinophyte family Amphidomataceae produce lipophilic phycotoxins called azaspiracids (AZA) which may cause azaspiracid shellfish poisoning (AZP) in humans after consumption of contaminated seafood. Three of the four known toxigenic species are observed frequently in the eastern North Atlantic. In 2018, a research survey was performed to strengthen knowledge on the distribution and abundance of toxigenic Amphidomataceae and their respective toxins in Irish coastal waters and in the North Sea. Species-specific quantification of the three toxigenic species (Azadinium spinosum, Azadinium poporum and Amphidoma languida) was based on recently developed qPCR assays, whose performance was successfully validated and tested with specificity tests and spike experiments. The multi-method approach of on-board live microscopy, qPCR assays and chemical AZA-analysis revealed the presence of Amphidomataceae in the North Atlantic including the three targeted toxigenic species and their respective AZA analogues (AZA-1, -2, -33, -38, -39). Azadinium spinosum was detected at the majority of Irish stations with a peak density of 8.3 x 104 cells L-1 and AZA (AZA-1, -2, -33) abundances up to 1,274 pg L-1. Amphidoma languida was also present at most Irish stations but appeared in highest abundance in a bloom at a central North Sea station with a density of 1.2 x 105 cells L-1 and an AZA (AZA-38, -39) abundances of 618 pg L-1. Azadinium poporum was detected sporadically at the Irish south coast and North Sea and was rather low in abundance during this study. The results confirmed the wide distribution and frequent occurrence of the target species in the North Atlantic area and revealed, for the first time, bloom abundances of toxigenic Amphidomataceae in this area. This emphasizes the importance of future studies and monitoring of amphidomatacean species and their respective AZA analogues in the North Atlantic.

Fate of free and modified Alternaria mycotoxins during the production of apple concentrates

Mouldy core is a frequent apple fungal disease, mainly caused by toxigenic Alternaria species. Mouldy core is hardly detected in pre-selection procedures when the apples are destined for industrialization, and to date no information is available on the fate of Alternaria toxins during apple concentrate production. Therefore, we evaluated the effect of this process on the natural contamination levels of 10 Alternaria metabolites: alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tenuazonic acid (TeA), tentoxin (TEN), altertoxin-I (ATX-I), alternariol 3-sulfate (AOH-3-S), alternariol 3-glucoside (AOH-3-G), alternariol monomethyl ether 3-sulfate (AME-3-S), and alternariol monomethyl ether 3-glucoside (AME-3-G). Six stages (grinding, turbos, decanter muds, pre-concentration, concentrate and rejection) of five independent Red Delicious and one of Granny Smith apple concentrate processes were sampled. Four out of the six processes included clarification, while two did not. The Granny Smith raw material was the least contaminated one, both in quality and quantity of Alternaria mycotoxins. Quantifiable levels of AOH, AME, TeA and TEN, were observed in the ground apples of the Red Delicious processes. Regarding the modified mycotoxins, only AME-3-S was present in the raw material; nevertheless, AOH-3-S and AOH-3-G were detected along the process. ALT, ATX-I, and AME-3-G were not detected at any stage. Clear and cloudy processes showed similar variations on mycotoxin quantities until the clarification step, in which all the mycotoxins analysed underwent a significant reduction to non-quantifiable levels. Only TeA remained at detectable levels in one of the clarified final products. The concentration in the final cloudy product increased with respect to the raw material for AOH (301%), AME (221%), TEN (872%) and TeA (1024%). This is the first report of AOH-3-S and AME-3-S in apple-by-products. The clarification stage in apple concentrate production has a relevant role in reducing Alternaria toxins to safe levels in the final products. A major risk might be associated with cloudy apple-by-products.

Molecular Characterization of Mycobiota and Aspergillus Species from Eupolyphaga sinensis Walker Based on High-Throughput Sequencing of ITS1 and CaM

Eupolyphaga sinensis Walker is a valuable traditional Chinese animal medicine first recorded in Shennong Bencao. Previous research has shown that E. sinensis is easily contaminated by aflatoxins (AFs), which are highly toxic mycotoxins, during harvest, storage, and transport, thereby posing a considerable threat to consumer health. Most often, these AFs are produced by Aspergillus species. In this study, we contrast the traditional culture-based dilution plating method to the high-throughput sequencing (HTS) technology for fungal identification in TCM E. sinensis. Both of the methods used internal transcribed spacer 1 (ITS1) and calmodulin (CaM) sequencing for fungal molecular identification. The new CaM primer we designed in the study is suitable for MiSeq PE300 sequencing used for identification of Aspergillus species in community DNA samples. More fungal species were found in the E. sinensis samples based on HTS than those found using the culture-based dilution plating method. Overall, combining the sequencing power of ITS1 and CaM is an effective method for the detection and monitoring of potential toxigenic Aspergillus species in E. sinensis. In conclusion, HTS can be used to obtain a large amount of sequencing data about fungi contaminating animal medicine, allowing earlier detection of potential toxigenic fungi and ensuring the efficient production and safety of E. sinensis.

Asparagine-based production of hydrogen peroxide triggers cell death in the diatom Phaeodactylum tricornutum

ABSTRACT As a major component of coastal phytoplankton blooms, diatoms are an important source of dissolved organic matter (DOM) in the ocean. While this DOM pool fuels the growth of diverse bacterial populations, diatom population dynamics itself is considered unaffected by it. Here, we identified strong negative effects of the amino acid asparagine on the growth of the marine model diatom Phaeodactylum tricornutum. Using microscopic analyses and physiological assays, we showed that while asparagine fuelled photosynthetic growth, its supplementation to NO3 − medium significantly decreased the culture’s cell density and photosynthetic capacity (Fv/Fm) during the stationary phase of the growth cycle. We further showed that these effects resulted from asparagine-based production of hydrogen peroxide (H2O2) that triggered a massive cell death event. Effective lethal asparagine concentrations were as low as 12.5 µM, while 1 μM affected Fv/Fm, but did not trigger culture collapse. Based on sequence similarity with the characterized H2O2-producing L-amino acid oxidase (LAAO) of Chlamydomonas reinhardtii, LAO1, we identified putative LAO1 orthologs in the genomes of P. tricornutum and the toxigenic pennate species Pseudo-nitzschia multiseries. The molecular phylogeny of this gene indicates a shared ancestry with other algal LAAOs and suggests a conserved oxidative deaminase function that could mediate DOM release from diatom populations through the triggering of autocatalytic cell death events.