Toxigenic Species Research Articles

Development of a HOG-based real-time PCR method to detect stress response changes in mycotoxigenic moulds

There is a need to understand the mechanism of adaptation of toxigenic fungal species which are able to colonise highly specialised foods such as cured meats where there is a high osmotic stress due to the presence up to 20–22% NaCl during the ripening process. A new tool able to detect changes in stress related genes would be useful to understand the ecological reasons for the ability of these species to grow in specialised niches. In this work a real-time PCR (qPCR) using SYBR Green was developed. Primers were designed from the Hog1 gene involved in osmo-adaptation in fungi. For this, conserved regions resulting from the alignment of 26 published partial sequences of such gene were used. Specificity of primers HogF2/R2 was demonstrated when amplified, producing a unique 131-bp PCR product with a Tm value of 84 °C. The qPCR method showed an efficiency of 98%, R2 value > 0.99 and a detection limit of 0.7 log Hog1 gene copies. The qPCR method to measure changes in the Hog1 gene expression in relation to growth in ionic and non-ionic stressed environments (using 10–40% NaCl and sorbitol concentrations) was found to be suitable for two mycotoxigenic species (Penicillium nordicum, P. expansum). This assay will be a valuable tool for generating relevant Hog1 expression data from different mould species in relation to different stresses in food habitats. It will also be a good tool for a better understanding of the ability of xerophilic and xerotolerant species to colonise extreme environments.

Light regulation of mycotoxin biosynthesis: new perspectives for food safety

Mycotoxins are secondary metabolites produced by toxigenic fungi contaminating foods and feeds in pre-, post-harvest and processing, and represent a great concern worldwide, both for the economic implications and for the health of the consumers. Many environmental conditions are involved in the regulation of mycotoxin biosynthesis. Among these, light represents one of the most important signals for fungi, influencing several physiological responses such as pigmentation, sexual development and asexual conidiation, primary and secondary metabolism, including mycotoxin biosynthesis. In this review we summarise some recent findings on the effect of specific light wavelength and intensity on mycotoxin biosynthesis in the main toxigenic fungal genera. We describe the molecular mechanism underlying light perception and its involvement in the regulation of secondary metabolism, focusing on VeA, global regulator inAspergillus nidulans, and the White-Collar proteins, key components of light response inNeurospora crassa. Light of specific wavelength and intensity exerts different effects both on growth and on toxin production depending on the fungal genus. InPenicilliumspp. red (627 nm) and blue wavelengths (455-470 nm) reduce ochratoxin A (OTA) biosynthesis by modulating the level of expression of the ochratoxin polyketide synthase. Furthermore a mutual regulation between citrinin and OTA production is reported inPenicilliumtoxigenic species. InAspergillusspp. the effect of light treatment is strongly dependent on the species and culture conditions. Royal blue wavelength (455 nm) of high intensity (1,700 Lux) is capable of completely inhibit fungal growth and OTA production inAspergillus stenyiiandPenicillum verrucosum. InFusariumspp. the effect of light exposure is less effective; mycotoxin-producing species, such asFusarium verticillioidesandFusarium proliferatum, grow better under light conditions, and fumonisin production increased. This review provides a comprehensive picture on light regulation of mycotoxin biosynthesis and discusses possible new applications of this resource in food safety.

Occurrence, prevention and remediation of toxigenic fungi and mycotoxins in silage: a review.

Ruminants are considered to be less sensitive towards mycotoxins than monogastric animals because rumen microbiota have mycotoxin-detoxifying capacities. Therefore the effect of mycotoxins towards ruminants has been studied to a lesser extent compared with monogastric animals. Worldwide, a high proportion of the ruminant diet consists of silages made of forage crops (i.e. all parts of the crop above the stubble are harvested). In practice, silages are often contaminated with multiple mycotoxins. Exposure to a cocktail of mycotoxins can hamper animal production and have severe health consequences. In this article the different aspects associated with mycotoxin contamination of silage are reviewed 'from seed to feed'. An overview is given on the occurrence of toxigenic fungal species and their concomitant mycotoxins in forage crops before and after ensiling. The mycotoxin load of visually non-mouldy samples and mouldy hot spots within the same silo is also compared. Subsequently, this review delves into different problem-solving strategies. A logical first step is prevention of mould growth and mycotoxin production in the field, during harvest and during ensiling. If prevention should fail, several remediation strategies are available. These are listed, mainly focusing on the possibilities of microbial degradation of mycotoxins in vivo in silage. © 2015 Society of Chemical Industry.

Mycotoxins in Bovine Milk and Dairy Products: A Review.

This paper presents a literature review of the occurrence of several mycotoxins in bovine milk and dairy products, because it is the main type of milk produced and marketed worldwide. Mycotoxins are produced by different genera of filamentous fungi and present serious health hazards such as carcinogenicity and mutagenicity. Under favorable growth conditions, toxigenic fungi produce mycotoxins which contaminate the lactating cow's feedstuff. During metabolism, these mycotoxins undergo biotransformation and are secreted in milk. Data show that there is a seasonal trend in the levels of mycotoxins in milk, with these being higher in the cold months probably due to the prolonged storage required for the cattle feeds providing favorable conditions for fungal growth. Good agricultural and storage practices are therefore of fundamental importance in the control of toxigenic species and mycotoxins. Although aflatoxins (especially aflatoxin M1 ) are the mycotoxins of greater incidence in milk and dairy products, this review shows that other mycotoxins, such as fumonisin, ochratoxin A, trichothecenes, zearalenone, T-2 toxin, and deoxynivalenol, can also be found in these products. Given that milk is widely consumed and is a source of nutrients, especially in childhood, a thorough investigation of the occurrence of mycotoxins as well the adoption of measures to minimize their contamination of milk is essential.

Prevalence of toxigenic Penicillium species associated with poultry house in Telangana, India

ABSTRACTThe prevalence of mycotoxigenic Penicillium species in poultry houses of Telangana, India, was studied during 4 seasons between June 2009 and May 2010. Fungi belonging to 13 genera, including Penicillium, comprising 43 species were collected using petri plates. Fourteen Penicillium species demonstrated varying degrees of mycotoxigenicity. Chemical and chromatographic analysis of the different poultry feed samples revealed 8 different mycotoxins with ochratoxin A (OTA) predominating. The mean contamination rate of OTA was 38%. OTA quantities ranged between 5.78 and 6.73 µg/kg−1, 10.13 and 14.23 µg/kg−1, and 12.33 and 15.20 µg/kg−1 in starter, broiler, and layer feeds, respectively. Statistically significant positive correlation between prevalence of Penicillium species and the monsoon, autumn, and spring seasons and negative correlation between prevalence and the autumn, spring, and summer seasons were observed. These findings may serve as risk exposure indicators and contribute toward the initiation of a sustainable control program.

Mycobiota of Slovak wine grapes with emphasis on Aspergillus and Penicillium species in the small carpathian area

The Slovak wine-growing region is divided into six viticulture areas. The largest in size and the most important over the centuries has been the Small Carpathian area (around 5800 ha of vineyards) spreads in the western of Slovakia. The objectives of this study were: to gain more knowledge about mycobiota on grapes originating from Slovakia, with a focus on genera Aspergillus and Penicillium and their ability to produce mycotoxins in in vitro conditions by thin layer chromatography method. From the twelve vineyards were collected 14 samples of wine grapes (white 6, blue 8) during harvesting 2011, 2012 and 2013. Fifty wine grapes per bunch (approximately 7-8 berries per plate) that showed no symptoms were randomly selected on Dichloran Rose Bengal Chloramphenicol agar medium. The plates were then incubated aerobically at 25 ±1 °C for 5 to 7 days in the dark. Of these samples were identified 22 genera. Ninety-three percent of samples were colonies by the genus Penicillium and 79% by the genus Aspergillus. During the survey, 251 isolates belonging to 14 Penicillium species (P. aurantiogriseum, P. citrinum, P. coprophylum, P. crustosum, P. expansum, P. funiculosum, P. glabrum, P. griseofulvum, P. chrysogenum, P. oxalicum, P. polonicum, P. purpurogenum, P. roqueforti and P. thomii) and 37 isolates belonging to 7 Aspergillus species (A. clavatus, A. flavus, A. section Nigri, A. ostianus, A. parasiticus, A. versicolor and A. westerdijkiae) were isolated and identified from exogenous contamination. The main occurring penicillium species of the samples were P. chrysogenum (36% Fr), followed P. crustosum (29% Fr), P. griseofulvum (21% Fr) and P. expansum (21% Fr). The main occurring aspergillus species of the samples were A. section Nigri (64%). Thirteen potentially toxigenic species were tested for their toxigenic ability. It was confirmed the production of various mycotoxins such as aflatoxin B1, G1, citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, roquefortin C and sterigmatocystin. Out of 124 strains, 84% produced at least one mycotoxin.

Transcriptome analysis of Bacillus thuringiensis spore life, germination and cell outgrowth in a vegetable-based food model

Toxigenic species belonging to Bacillus cereus sensu lato, including Bacillus thuringiensis, cause foodborne outbreaks thanks to their capacity to survive as spores and to grow in food matrixes. The goal of this work was to assess by means of a genome-wide transcriptional assay, in the food isolate B. thuringiensis UC10070, the gene expression behind the process of spore germination and consequent outgrowth in a vegetable-based food model. Scanning electron microscopy and Energy Dispersive X-ray microanalysis were applied to select the key steps of B. thuringiensis UC10070 cell cycle to be analyzed with DNA-microarrays. At only 40 min from heat activation, germination started rapidly and in less than two hours spores transformed in active growing cells. A total of 1646 genes were found to be differentially expressed and modulated during the entire B. cereus life cycle in the food model, with most of the significant genes belonging to transport, transcriptional regulation and protein synthesis, cell wall and motility and DNA repair groups. Gene expression studies revealed that toxin-coding genes nheC, cytK and hblC were found to be expressed in vegetative cells growing in the food model.

Lessons from Paradise: The Many Subtleties of Tropical Sprue

Tropical Sprue (TS) classically presents as chronic diarrhea and symptoms of malabsorption in countries located within 30 degrees latitude of the equator, mostly affecting the indigenous population. Although the etiology is unclear, bacterial overgrowth, toxigenic bacterial species, and viral sources have all been theorized to play roles in the disease pathogenesis. Duodenal biopsies are the gold standard. However, TS is often a diagnosis of exclusion and histopathologic findings can mimic celiac disease. Timely diagnosis may be assisted by a dedicated GI pathologist when clinical suspicion for TS is high. Long term treatment with folic acid and broad spectrum antibiotics are the mainstay of therapy. We present a case of TS in a man visiting Haiti whose diagnosis was initially delayed due to misdiagnosis. Case: A 61 year old man with a medical history of Chronic Lymphocytic Leukemia presented after a six week trip to Haiti with watery, non-bloody diarrhea associated with bloating and a 20lb weight loss. From another GI provider, stool studies and routine blood-work were unremarkable and infectious and autoimmune work-ups were negative. After failing an empiric trial of antibiotics, Esophagastroduodenoscopy (EGD) and Colonoscopy (CF) were performed with unremarkable endoscopic findings. Duodenum, colon and ileum biopsies were interpreted as normal per the assigned pathologist. Four months later, the patient presented to our clinic seeking a second opinion. The prior EGD and CF biopsies were reviewed with an expert GI pathologist. CF biopsies were interpreted as normal. However, the duodenal biopsies were significant for moderate villous blunting and increased intraepithelial lymphocytosis consistent with indeterminate sprue. Having no relief with a gluten free diet and given his risk of TS, Doxycycline and folic acid were started, resulting in immediate improvement. He has since returned to his regular body weight and feels well. Conclusion: Our case highlights two important details. TS must be kept in the differential diagnosis for persons traveling to countries at risk for this infection. A high level of clinical suspicion must be maintained when diagnosing TS, as serologic testing does not exist and histopathology can be misleading. In addition, reviewing biopsies with a dedicated GI pathologist for patients with any GI disorder is imperative not only to improve our own knowledge base but to assure that our patients receive the best care possible.

Variances in trichothecene chemotype distribution in Lithuanian wheat grain and within pure culture Fusarium graminearum isolated from the same grain samples

The Fusarium head blight emerged as the main disease of wheat in Lithuania in 2012. The chemotype diversity of FHB pathogens was not investigated prior that time. In this study chemotype determination of pure F. graminearum cultures, the main cause of FHB in wheat, isolated from grain and chemotype detection rates in homogenized grain were compared. A total of 105 pure culture F. graminearum isolates from 2013 and 144 homogenized wheat grain samples from 2013 and 2014 were analysed for presence and quantification of 15ADON, 3ADON and NIV chemotype DNA. Results show that 15ADON chemotype was clearly dominant in pure culture isolates, but not in homogenized grain, where 3ADON incidence was almost as high. Also the results allow dismissal of other toxigenic Fusarium species, F. culmorum, as either a source of NIV or the main source of 3ADON chemotype DNA in grain.

Identification of toxigenic Aspergillus species from diet dairy goat using a polyphasic approach

Some species of filamentous fungi that infest agricultural commodities are able to produce mycotoxins, contaminating feed and animal products. The aim of this research was to identify the mycoflora present in the feed and forage for dairy goat and to isolate and characterize the Aspergillus flavus and A. parasiticus strains based on a morphological and molecular characterization and mycotoxigenic ability. The goat dairy diets were collected monthly from 11 goat milk farms, totaling 129 and 106 samples of concentrate and forage, respectively. For the isolation of the mycobiota the surface plating method was used. Aspergillus, Penicillium, and Fusarium were the main fungi producing mycotoxins isolated. The morphological and molecular characterization and mycotoxigenic ability were used for A. flavus and A. parasiticus identification. The Aspergillus spp. from feed 39% produced aflatoxins B1 and B2, 17% produced cyclopiazonic acid (CPA), 18% produced both toxins, and 42% had no toxigenic ability. Only 2.0% of the strains produced aflatoxins B1, B2, G1, and G2, but no CPA. The strains from forage were producers of aflatoxins B1 and B2 (37%), CPA (14%), 14% of both mycotoxins, whereas 49% have shown no toxigenic ability. The aflD and aflR genes were used by PCR and PCR-RFLP, respectively. The presence of toxigenic species in samples of feed for lactating goats indicates a potential risk of contamination of dairy products, if they are exposed to environmental conditions favorable to fungal growth and mycotoxin production.

Diversity of black Aspergilli isolated from raisins in Argentina: Polyphasic approach to species identification and development of SCAR markers for Aspergillus ibericus

Aspergillus section Nigri is a heterogeneous fungal group including some ochratoxin A producer species that usually contaminate raisins. The section contains the Series Carbonaria which includes the toxigenic species Aspergillus carbonarius and nontoxigenic Aspergillus ibericus that are phenotypically undistinguishable. The aim of this study was to examine the diversity of black aspergilli isolated from raisins and to develop a specific genetic marker to distinguish A. ibericus from A. carbonarius. The species most frequently found in raisins in this study were Aspergillus tubingensis (35.4%) and A. carbonarius (32.3%), followed by Aspergillus luchuensis (10.7%), Aspergillus japonicus (7.7%), Aspergillus niger (6.2%), Aspergillus welwitschiae (4.6%) and A. ibericus (3.1%). Based on inter-simple sequence repeat (ISSR) fingerprinting profiles of major Aspergillus section Nigri members, a sequence-characterized amplified region (SCAR) marker was identified. Primers were designed based on the conserved regions of the SCAR marker and were utilized in a PCR for simultaneous identification of A. carbonarius and A. ibericus. The detection level of the SCAR-PCR was found to be 0.01ng of purified DNA. The present SCAR-PCR is rapid and less cumbersome than conventional identification techniques and could be a supplementary strategy and a reliable tool for high-throughput sample analysis.

English

The presence of fungi of the genus Aspergillus must be investigated because it is related to the diminishment of quality and consequently to the risk to health of consumers due to the possible production of mycotoxins. In this context, this study was performed with the aim to evaluate the presence of Aspergillus species in cocoa beans produced in the state of Rondônia, Brazil, as well as verify the toxigenic potential of these species and the effect of the geographical origin on the incidence of toxigenic and non-toxigenic species of fungi. The technique of logistic regression was used to estimate the probability of occurrence of the genus Aspergillus. A total of 185 Aspergillus were identified. Among the tested Aspergillus carbonarius, 79.41% produced ochratoxin on levels from 0.08 to 44.09 µg/g in the culture medium. For the other Aspergillus species, the production potential was only assessed, and 3.13% of the A. niger presented potential to produce OTA and 83.33% of A. flavus which was able to produce aflatoxins. The presence of these toxigenic species indicates a potential risk of mycotoxin in cocoa beans and in their derived products in the studied region. Besides, the occurrence of Aspergillus species differed in function of geographical coordinates and temperature, presenting higher probability of occurrence on cocoa beans of municipalities located in Southern Rondônia, Brazil. Key words: Cocoa beans, Aspergillus, mycotoxins, ochratoxin A, aflatoxins, logistic regression, thin layer chromatography, high performance liquid chromatography.

Impact of local pre-harvest management practices in maize on the occurrence of Fusarium species and associated mycotoxins in two agro-ecosystems in Tanzania

Knowledge on the presence of mycotoxins in Africa is fragmentary, although it can be assumed that both concentrations and prevalence in food commodities is high. The present study focuses on the presence of Fusarium species and their associated mycotoxins in maize from two geographically distant agro ecological systems in Tanzania. In a two-year survey, both Fusarium species and concomitant mycotoxins were surveyed in the Northern highlands (Hanang district) and the Eastern lowlands (Kilosa district). Parallel with this, a questionnaire on agricultural practices in both agro-ecosystems was included in this study. This allowed us to put the presence of the toxigenic Fusarium species and their mycotoxins within a relevant agricultural framework.Fusarium verticillioides, Fusarium graminearum and Fusarium poae were the predominant species in both locations although the population in the Eastern lowlands was slightly more complex comprising also Fusarium culmorum, Fusarium avenaceum and Fusarium sporotrichioides. The predominant presence of F. verticillioides resulted in a high prevalence of fumonisins in both regions. The importance of F. graminearum in the population was reflected by the presence of deoxynivalenol in the mycotoxin analysis. Although the agricultural practices differed significantly amongst both locations, only few significant correlations were detected between mycotoxin presence and crop rotation, storage conditions, and insect control measures.

Distribution of disease symptoms and mycotoxins in maize ears infected by Fusarium culmorum and Fusarium graminearum.

Red ear rot an important disease of maize cultivated in Europe is caused by toxigenic Fusarium species like Fusarium graminearum and Fusarium culmorum. To get detailed information on the time course of the infection process leading to the accumulation of Fusarium mycotoxins in maize ears, a field study was conducted over 2 years with two maize varieties, which were inoculated with F. culmorum or F. graminearum isolates at the stage of female flowering. Every fortnight after inoculation, infection and contamination progress in the ears was followed by visually evaluating disease signs and analysing Fusarium toxin concentrations in the infected ear tissues. In principle, infection and mycotoxin distribution were similar in respect of pathogens, varieties, and years. External infection symptoms showing some small pale or brown-marbled kernels with dark brown pedicels were mainly seen at the ear tip, whereas internal infection symptoms on the rachis were much more pronounced and spread in the upper half showing greyish brownish or pink discoloration of the pith. Well correlated with disease symptoms, a top-down gradient from high to low toxin levels within the ear with considerably higher concentrations in the rachis compared with the kernels was observed. It is suggested that both Fusarium pathogens primarily infect the rachis from the tip toward the bottom, whereas the kernels are subsequently infected via the rachillae connected to the rachis. A special focus on the pronounced disease symptoms visible in the rachis may be an approach to improve the evaluation of maize-genotype susceptibility against red ear rot pathogens. It has to be underlined that the accumulation of Fusarium mycotoxins in the rachis greatly accelerated 6 weeks after inoculation; therefore, highest contamination risk is indicated for feedstuffs containing large amounts of rachis (e.g., corn cob mix), especially when cut late in growing season.

SEASONAL AND SPATIAL PATTERNS OF TOXIGENIC SPECIES OF Dinophysis AND Pseudo-nitzschia IN A SUBTROPICAL BRAZILIAN ESTUARY

This study investigated the spatial and temporal distributions of toxigenic phytoplankton species between October 2010 and April 2012 in Guaratuba Bay, Paraná state, Brazil, where aquaculture has been intensified in recent years. The highest abundances of Pseudo-nitzschia calliantha and P. pungens (up to 7.3 x 104 cells L-1), diatoms which cause amnesic shellfish poisoning (ASP), were correlated with higher values of temperature (from December to April, salinity (>20) and silicate concentrations (ranging from 6.0 to 90.0 µm). The occurrence of these harmful diatoms in the bay also appeared to depend upon the development of seeding mechanisms in the adjacent coastal water and its subsequent advection by tidal currents to the interior of the estuary. During the study period, Pseudo-nitzschia cell abundance remained low to moderate probably as a result of growth limitation by phosphate shortage, especially in the outer area of the estuary. In addition, harmful species of Dinophysis, dinoflagellates responsible for diarrhetic shellfish poisoning (DSP) events, were recorded in every sampling campaign. The highest abundances of D. acuminata, D. caudata and D. tripos were associated with the upper halocline layer in regions of the bay where water column stratification was more frequent. On some occasions, cell abundances of D. acuminata (up to 3.2 x 103 cells L-1) exceeded levels considered harmful in areas used to cultivate bivalve mollusks for human consumption. These novel results strongly indicate the necessity of implementing a monitoring program for harmful microalgae in Guaratuba Bay.

English

Aflatoxin contamination of food and feed commodities, caused by Aspergillus section Flavi group of fungi, is a serious problem worldwide. Exposure through consumption of contaminated food and feed has deleterious effects on human and animal health. Therefore, aflatoxin contaminated products are a barrier to international trade of agricultural commodities. Not all fungi from Aspergillus section Flavi produce aflatoxins. Hence it is important to differentiate Aspergillus spp. into toxigenic and atoxigenic species to better understand their population structure in a specific environment. A range of methods are available today, including cultural, analytical and molecular methods, to identify the toxin producing ability of isolates from section Flavi. A comprehensive review of these methods would be of great use for researchers in developing nations where fully equipped aflatoxin detection laboratories are lacking. In this paper we critically reviewed the cultural and molecular methods of detecting aflatoxigenic Aspergillus species and their precision. Key words: Toxigenic Aspergillus, atoxigenic Aspergillus, cultural methods, PCR based methods.

ENDOGENAL COLONIZATION OF GRAPES BERRIES

The aim of study was to detect the microscopic filamentous fungi from wine surface of sterilized grapes berries of Slovak origin. We analyzed 21 samples of grapes, harvested in the year 2012 of various wine-growing regions. For the isolation of species we used the method of direct plating surface-sterilized berries (using 0.4% freshly pre-pared chlorine) on DRBC (Dichloran Rose Bengal Chloramphenicol agar). The cultivation was carried at 25±1°C, for 5 to 7 days. A total number of 2541 fungal isolates pertaining to 18 genera including Mycelia sterilia were recovered. Isolates of genus Alternaria were found in all of tested samples with the highest relative density 56.4%. The second highest isolation frequency we detected for genus Fusarium (90.48% positive samples), but with low relative density (31 isolates and 2.99% RD). Another genera with higher isolation frequency were Cladosporium (Fr 85.71%, RD 14.6%), Mycelia sterilia (Fr 85.71%, RD 4.25%), Penicillium (Fr 80.95%, RD 13.42%), Botrytis (Fr 71.43%, RD 2.95%) Rhizopus (Fr 66.66%, RD 1.34%), Aspergillus (Fr 57.14%, RD 0.87%), Epicoccum (Fr 47.62%, RD 1.22%), Trichoderma (Fr 42.86%, RD 1.26%). Isolation frequency of another eight genera (Arthrinium, Dichotomophtora, Geotrichum, Harzia, Chaetomium, Mucor, Nigrospora and Phoma) was less than 10% and relative density less than 0.5%. Chosen isolates of potential producers of mycotoxin (species of Alternaria, Aspergillus, Fusarium and Penicillium) were tested for the ability to produce relevant mycotoxins in in vitro conditions using TLC method. None isolate of Aspergillus niger aggregate (13 tested) did not produce ochratoxin A – mycotoxin monitored in wine and another products from grapes berries. Isolates of potentially toxigenic species recovered from the samples were found to produce another mycotoxins: aflatoxin B1, altenuene, alternariol, alternariol monomethylether, citrinin, diacetoxyscirpenol, deoxynivalenol, HT-2 patulin, penitrem A and T-2 toxin in in vitro conditions. In conclusion, another research should be performed to detect the occurrence of these mycotoxins in grapes, must, wine and another products from grape.

Analysis of the hydrographic conditions and cyst beds in the San Jorge Gulf, Argentina, that favor dinoflagellate population development including toxigenic species and their toxins

The overlay of cooler nutrient enriched Beagle–Magellan water with warmer nutrient depleted shelf water and a strong stratification of the water column in the San Jorge Gulf region, Argentina, coincided with relatively high dinoflagellate abundances in April 2012, up to 34,000cellsL−1. This dinoflagellate proliferation was dominated by Ceratium spp., but environmental conditions also favored to a lesser amount the occurrence of toxigenic dinoflagellates, such as Alexandrium tamarense and Protoceratium reticulatum, whose toxins were hardly detected in any other areas along the expedition transect of the R/V Puerto Deseado between 38 and 56°S (Ushuaia–Mar del Plata) in March/April 2012. Generally vegetative cells of A. tamarense and P. reticulatum co-occurred with their respective phycotoxins in the water column and their cysts in the upper sediment layers. Two strains of A. tamarense were isolated from the bloom sample and morphologically characterized. Their PSP toxin profiles consisted of C1/2, gonyautoxins 1/4 and to a lesser amount of neosaxitoxin and confirmed earlier data from this region. The ratios between autotrophic picoplankton and heterotrophic bacteria were higher in shelf waters in the north than in Beagle–Magellan waters in the south of San Jorge Gulf.

Diversity and temporal pattern of Pseudo-nitzschia species (Bacillariophyceae) through the molecular lens

In diatoms, as in other organisms, many genetically distinct and reproductively isolated species may show identical or highly similar morphological features. Such groups of species are defined as cryptic and pseudo-cryptic species, respectively. The difficulty of discriminating them with optical means impairs the study of their temporal patterns and geographic ranges. This is also the case for Pseudo-nitzschia, a worldwide distributed planktonic diatom genus which includes several toxigenic species. Using a Pseudo-nitzschia-specific pair of large sub-units ribosomal DNA (LSU rDNA) primers, we generated clone libraries from 19 samples collected at the Long Term Ecological Station MareChiara (LTER-MC) in the Gulf of Naples (GoN) from 2009 to 2010 and compared sequence records with light microscopy (LM) counts from the same samples. Our aim was to elucidate the diversity and the seasonal patterns of taxa within Pseudo-nitzschia. Most of the Pseudo-nitzschia species already known from the GoN were identified within the 1643 obtained sequences. In addition, two species known from elsewhere and three un-described ribotypes were detected. Several cryptic species showed distinct temporal patterns of occurrence, with most species confined to restricted periods and only a few present year-round. Microscopic and molecular results generally concurred for species recognizable using LM, while clone libraries tended to overestimate the relative abundance of some of the species. Due to its high resolution and detection power, the DNA-barcoding approach used in our study is an optimal tool to trace the distribution of cryptic and toxigenic Pseudo-nitzschia species and the diversity of this key diatom genus in the natural environment.

Intraspecific facilitation by allelochemical mediated grazing protection within a toxigenic dinoflagellate population.

Dinoflagellates are a major cause of harmful algal blooms (HABs), with consequences for coastal marine ecosystem functioning and services. Alexandrium fundyense (previously Alexandrium tamarense) is one of the most abundant and widespread toxigenic species in the temperate Northern and Southern Hemisphere and produces paralytic shellfish poisoning toxins as well as lytic allelochemical substances. These bioactive compounds may support the success of A. fundyense and its ability to form blooms. Here we investigate the impact of grazing on monoclonal and mixed set-ups of highly (Alex2) and moderately (Alex4) allelochemically active A. fundyense strains and a non-allelochemically active conspecific (Alex5) by the heterotrophic dinoflagellate Polykrikos kofoidii. While Alex4 and particularly Alex5 were strongly grazed by P. kofoidii when offered alone, both strains grew well in the mixed assemblages (Alex4 + Alex5 and Alex2 + Alex5). Hence, the allelochemical active strains facilitated growth of the non-active strain by protecting the population as a whole against grazing. Based on our results, we argue that facilitation among clonal lineages within a species may partly explain the high genotypic and phenotypic diversity of Alexandrium populations. Populations of Alexandrium may comprise multiple cooperative traits that act in concert with intraspecific facilitation, and hence promote the success of this notorious HAB species.