Characterization of kefir yeasts with antifungal capacity against Aspergillus species.

Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them-belonging to S. cerevisiae and P. kudriavzevii species-generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.

Selection of Antarctic yeasts as gray mold biocontrol agents in strawberry.

The postharvest disease popularly known as gray mold is considered one of the most limiting factors strawberry fruit production. The most effective way to control this disease is still the use of chemical fungicides. However, other alternative sources of control are being explored. Among these, psychrophilic yeasts adapted to extreme conditions, such as those found in the Antarctic region, may have great potential for use as biocontrol agents. Thus, the present study aimed to select psychrotolerant yeasts obtained from Antarctic region and to evaluate their potential for biocontrol under gray mold, caused by Botrytis cinerea in strawberries stored at low temperature. For this, 20 potential antagonist yeasts were evaluated in vitro (thermotolerance and enzymatic) assays. Debaryomyces hansenii, Rhodotorula mucilaginosa and Dioszegia hungarica were selected for growing in strawberry juice. However, only D. hansenii was selected for in vivo studies and showed a reduction in the incidence of gray mold by 82% for the tests performed on injury and 86% for the tests on non-injured fruits treated by immersion bath. Thus, demonstrating that the selection of this cold-adapted Antarctic yeast can be a promising strategy as a biocontrol agent used to curb the development of gray mold in strawberry fruits.

Production of microbial oils by the oleaginous yeast Rhodotorula graminis S1/2R in a medium based on agro-industrial by-products.

Biodiesel generated by transesterification of triglycerides from renewable sources is a clean form of energy that is currently used in many countries in blends with petrodiesel. It is mainly produced from food-grade vegetable oils obtained from oleaginous crops. High prices of these oils have made the sustainability of biodiesel production questionable. The use of nonedible feedstocks, such as intracellular triglycerides accumulated by oleaginous yeasts, appears as a feasible alternative. However, it has been demonstrated that an economically sustainable production of yeast oil could only be possible if low-cost media based on industrial subproducts, or wastes are used. In this work, we propose intracellular lipids production by a previously selected oleaginous yeast strain in a medium composed only by sugar cane vinasse and crude glycerol. Different culture strategies were studied. The highest biomass and lipid yields were obtained when the yeast R. graminis S1/2R was cultivated in batch without control of dissolved oxygen. The fatty acid methyl esters obtained under these conditions met the specification of international biodiesel standards.

Fungal diversity in the coastal waters of King George Island (maritime Antarctica).

Fungi have been reported as common inhabitants of the maritime waters in Antarctica by studies based on culture-dependent methods. More recently, results obtained using DNA sequencing technologies, revealed that fungal diversity worldwide has been underestimated by culture methods. The present study provides the first characterization of fungal communities in the coastal waters of King George Island (maritime Antarctica) using both culture-dependent and high-throughput sequencing (HTS) methods. HTS demostrated a higher level of fungal diversity than the obtained by culture methods. A high prevalence of basidiomycetous yeasts and ascomycetous filamentous fungi was confirmed by both methods, however, Chythriomycota, Rozellomycota, lichenized fungi and Malassezia spp. were detected only by HTS. Correspondingly, members of some genera, such as Metschnikowia, were only found by culture-dependent methods. Our results confirm that culturing and HTS, should be seen as complementary approaches that enable one to obtain a more comprehensive picture of the composition of microbial communities.

Debaryomyces hansenii F39A as biosorbent for textile dye removal.

Many industries generate a considerable amount of wastewater containing toxic and recalcitrant dyes. The main objective of this research was to examine the biosorption capacity of Reactive Blue 19 and Reactive Red 141 by the Antarctic yeast Debaryomyces hansenii F39A biomass. Some variables, including pH, dye concentration, amount of adsorbent and contact time, were studied. The equilibrium sorption capacity of the biomass increased with increasing initial dye concentration up to 350mg/l. Experimental isotherms fit the Langmuir model and the maximum uptake capacity (qmax) for the selected dyes was in the range of 0.0676-0.169mmol/g biomass. At an initial dye concentration of 100mg/l, 2g/l biomass loading and 20±1°C, D. hansenii F39A adsorbed around 90% of Reactive Red 141 and 50% of Reactive Blue 19 at pH 6.0. When biomass loading was increased (6g/l), the uptake reached up to 90% for Reactive Blue 19. The dye uptake process followed a pseudo-second-order kinetics for each dye system. As seen throughout this research study, D. hansenii has the potential to efficiently and effectively remove dyes in a biosorption process and may be an alternative to other costly materials.

Development of a PCR-RFLP method based on the transcription elongation factor 1-α gene to differentiate Fusarium graminearum from other species within the Fusarium graminearum species complex.

Fusarium head blight (FHB) is a destructive disease of cereals crops worldwide and a major food safety concern due to grain contamination with trichothecenes and other mycotoxins. Fusarium graminearum, a member of the Fusarium graminearum species complex (FGSC) is the dominant FHB pathogen in many parts of the world. However, a number of other Fusarium species, including other members of the FGSC, may also be present for example in Argentina, New Zealand, Ethiopia, Nepal, Unites States in cereals such as wheat and barley. Proper species identification is critical to research aimed at improving disease and mycotoxin control programs. Identification of Fusarium species is are often unreliable by traditional, as many species are morphologically cryptic. DNA sequence-based methods offer a reliable means of species identification, but can be expensive when applied to the analyses of population samples. To facilitate identification of the major causative agent of FHB, this work describes an easy and inexpensive method to differentiate F. graminearum from the remaining species within the FGSC and from the other common Fusarium species causing FHB in cereals. The developed method is based on a PCR-RFLP of the transcription elongation factor (TEF 1-α) gene using the restriction enzyme BsaHI.

Species composition, toxigenic potential and aggressiveness of Fusarium isolates causing Head Blight of barley in Uruguay.

Fusarium Head Blight (FHB) is a major constraint to barley production that substantially reduces yield and grain quality. FHB is also a major food safety concern because FHB pathogens contaminate grain with trichothecenes and other mycotoxins. DNA sequence-based analyses and in-vitro toxin assessments were used to characterize the species and trichothecene chemotype composition of FHB pathogens on barley in Uruguay. F. graminearum was the dominant species (89.7%), and three other members of the F. graminearum species complex (FGSC) were identified as FHB pathogens of barley in Uruguay for the first time. Other minor contributors to FHB species diversity included F. poae, F. avenaceum, F. pseudograminearum and an unnamed species from the F. incarnatum-equiseti species complex (FIESC). Most isolates (89.7%) had the 15-acetyldeoxynivalenol (15-ADON) trichothecene type. However, the results expanded the known area of occurrence within Uruguay for the nivalenol (NIV) toxin type, which was observed among isolates from three species of the FGSC, F. pseudograminearum, and F. poae. Isolates with the 3-acetyldeoxynivalenol (3-ADON) or NX-2 toxin types were not observed, although a previously published multilocus genotyping assay was updated to identify NX-2 strains. Analyses of population structure and comparisons with FHB isolates from wheat in Uruguay indicated that F. graminearum constitutes a single genetic population with no evidence of population differentiation related to the sampled hosts. Inter and intraspecific differences were observed in aggressiveness toward four barley genotypes with different levels of resistance to FHB, and in general nivalenol producers were the least aggressive isolates. Sensitivity to metconazole was approximately 10 times higher than was detected for tebuconazole. This is the first report regarding tebuconazole and metconazole sensitivity for Fusarium species causing FHB in barley in Uruguay, and constitutes an important starting point for monitoring temporal or spatial changes in FGSC sensitivity, which is critical to define FHB management practices.

Evaluation of yeasts obtained from Antarctic soil samples as biocontrol agents for the management of postharvest diseases of apple (Malus × domestica).

Psychrotrophic yeasts were isolated from Antarctic soils, selected based on their ability to grow in apple juice at low temperatures, and were evaluated as potential biocontrol agents for the management of postharvest diseases of apple during cold storage. Among the species recovered, an isolate of Leucosporidium scottii, designated At17, was identified as a good biocontrol agent for blue and gray mold of two apple cultivars. The selected isolate produced soluble and volatile antifungal substances that were inhibitory to apple pathogens. Siderophore production was also demonstrated, but it did not appear to play a role in pathogen inhibition. The selected yeast had the capacity to form a biofilm when grown in apple juice, which is considered an important attribute of postharvest antagonists to successfully colonize wounds and intact fruit surfaces. At17 was resistant to commonly used postharvest fungicides, so application of a combination of low-dose fungicide along with the biocontrol agent could be used as an integrated management practice.

Effect of moisture on wheat grains lipid patterns and infection with Fusarium graminearum.

Suitable conditions of temperature and humidity are required to maintain wheat grains quality, but during processing and storage, the grains can be exposed to adverse environmental conditions and presence of infectious fungi. Fusarium graminearum, the main causal agent of Fusarium head blight on wheat, affects crop yields and grain quality by alteration of their biochemical components and mycotoxin contamination, which reduces the possibilities of wheat end use and compromises food safety. Lipid degradation by hydrolytic, oxidative and microbial deterioration is the predominant cause of the loss of sensory acceptability, nutritional value and baking quality. The aim of this research was to determine the influence of adverse environmental conditions -as the increasing moisture - on lipid patterns of whole wheat flours contaminated with F. graminearum in relation to the infection degree. In vitro cultures of F. graminearum were carried out on wheat grains under different degrees of relative humidity (11, 50, 75 and 100%) throughout 45 days of incubation at 28 °C. The fungal biomass measured by q-PCR increased proportionally with the humidity. A decrease in the signals of saturated (palmitic and estearic) and unsaturated (oleic, linoleic and linolenic) fatty acids, analyzed as fatty acid methyl esters (FAMEs) by GC-MS, was observed in relation with the humidity and infection degree. The degradation rate of the lipids was high during the first 15 days of incubation, reaching the fatty acids content, values around 20-40% of those found in the control. From that moment on, the rate of degradation was slower or even null. It was observed that in all treatments, the linolenic acid reached the highest degradation ratio in comparison with the other fatty acids, which may be caused by the action of lipoxygenases. The lipase activity and the content of deoxynivalenol were also determinate on the flours. The lipase activity increased until day 25 of incubation reaching twice the initial value. The deoxynivalenol content also increased along incubation while fatty acids decreased. Our results demonstrated that the magnitude in the signal of fatty acids in whole wheat flours varied in relation to the degree of humidity and fungal infection of the grains from which they were obtained. Otherwise, lipids and their oxidation products are related with the pathogenesis and production of mycotoxins. These observations highlight the importance of an adequate manipulation of wheat grains on the processing chain to prevent quality changes and mycotoxins contamination.

Biocontrol of Aspergillus flavus in Ensiled Sorghum by Water Kefir Microorganisms.

The capacity of microorganisms from water kefir (WK) to control Aspergillus flavus growth during the aerobic phase of ensiled sorghum grains was determined. Sorghum inoculated with A. flavus was treated with filter-sterilized and non-sterilized water kefir, ensiled, and incubated 7 days at 25 °C. A. flavus growth was quantified by qPCR after incubation. Mold growth was inhibited in the presence of water kefir while no inhibition was observed when filter-sterilized water kefir was applied, demonstrating the relevant role of the microorganisms in the kefir water in the biocontrol process. Fungal and bacterial diversity in treated sorghum mini-silos was analyzed by high-throughput sequencing. Firmicutes was the predominant bacterial phyla and Lactobacillus represented the most abundant genus, while Ascomycota was the predominant fungal phyla with Saccharomyces and Pichia as the major genera. Bacterial and yeast counts before and after incubation indicated that the microbial community obtained from WK was able to grow in the sorghum mini-silos in the presence of A. flavus. Results of the present work indicate that the use of a mixed inoculum of microorganisms present in WK may represent an alternative management practice to avoid the growth of A. flavus in ensiled sorghum grains and the concomitant contamination with aflatoxins.

Occurrence and biodiversity of Aspergillus section Nigri on 'Tannat' grapes in Uruguay.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin which has been found worldwide as a contaminant in wines. It is produced on grapes mainly by molds from Aspergillus section Nigri. This study has demonstrated for the first time the occurrence of black aspergilli on Tannat grapes from Uruguay, in a two year survey. Aspergillus uvarum (uniseriate) and Aspergillus welwitschiae (from Aspergillusniger aggregate) were the prevalent species whereas Aspergillus carbonarius which is considered the main OTA producing species was not detected. OTA production in culture medium was evaluated for native isolates from A. niger aggregate and compared to levels produced by a type strain of A. carbonarius. This work also includes the development of quick and easy molecular methods to identify black aspergilli to species level, avoiding sequencing.

Process conditions for a rapid in situ transesterification for biodiesel production from oleaginous yeasts

BACKGROUND: Microbial oils produced by diverse microorganisms are being considered as alternative sources of triglycerides for biodiesel production. However, the standalone production of biodiesel from microorganisms is not currently economically feasible. In case of yeasts, the use of low-value nutrient sources in microbial production and the implementation of cost-efficient downstream processes could reduce costs and make microbial lipids competitive with other commodity-type oils in biodiesel production. Industrial biodiesel synthesis from oleaginous seeds is currently based on a multistep process. However, a simple process called in situ transesterification (ISTE), which takes place within the biomass without a previous lipid extraction step, is receiving increasing interest. In this work, the optimal conditions for an ISTE process to obtain biodiesel from previously selected oleaginous yeast (Rhodotorula graminis S1/S2) were defined using the response surface methodology (RSM). RESULTS: Using the RSM approach, the optimal conditions for the maximum yield with minimum reaction time included a methanol-to-biomass ratio of 60:1, 0.4 M H2SO4, and incubation at 70°C for 3 h. The optimized in situ process yield was significantly higher (123%) than that obtained with a two-step method in which fatty acids from saponifiable lipids were first extracted and then esterified with methanol. The composition of the fatty acid methyl ester mixture obtained from R. graminis S1/S2 by ISTE met Uruguayan standards for biodiesel. CONCLUSION: The characteristics achieved by the optimized method make microbial oil a potential alternative for biodiesel production from yeast at an industrial scale.

Characterization of kefir yeasts with antifungal capacity against Aspergillus species / Caracterización de levaduras de kéfir con capacidad antifúngica frente a especies de Aspergillus

Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them—belonging to S. cerevisiae and P. kudriavzevii species—generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.(AU)

Candidiasis urinaria y cerebral en un neonato con malformación urinaria bilateral congénita: hallazgos ultrasonográficos / Neonatal brain and urinary candidal infection: US findings

El pronóstico de este trabajo es describir los hallazgos ecográficos y demostrar la utilidad del ultrasonido en el diagnóstico precoz de la candidiasis urinaria y cerebral, por lo que realizamos ecografía prenatal de una paciente en la que se diagnosticó la presencia de un feto con uronefrosis bilateral congénita y ecografías tempranas al neonato pretérmino. La detección por US de la presencia de bolas fúngicas dentro de la vía excretora previamente dilatada fue determinante para realizar el cateterismo de ambos uréteres por medio del cual se obtuvo material para cultivo y sirvió para derivación urinaria. Concluimos que el US es el mejor método de diagnóstico por imágenes para ser utilizado en los neonatos prematuros de bajo peso y de alto riesgo (AU)

Candidiasis urinaria y cerebral en un neonato con malformación urinaria bilateral congénita: hallazgos ultrasonográficos / Neonatal brain and urinary candidal infection: US findings

El pronóstico de este trabajo es describir los hallazgos ecográficos y demostrar la utilidad del ultrasonido en el diagnóstico precoz de la candidiasis urinaria y cerebral, por lo que realizamos ecografía prenatal de una paciente en la que se diagnosticó la presencia de un feto con uronefrosis bilateral congénita y ecografías tempranas al neonato pretérmino. La detección por US de la presencia de bolas fúngicas dentro de la vía excretora previamente dilatada fue determinante para realizar el cateterismo de ambos uréteres por medio del cual se obtuvo material para cultivo y sirvió para derivación urinaria. Concluimos que el US es el mejor método de diagnóstico por imágenes para ser utilizado en los neonatos prematuros de bajo peso y de alto riesgo