Molecular assessment of oat head blight fungus, including a new genus and species in a family of Nectriaceae.

Head blight (HB) of oat (Avena sativa) has caused significant production losses in oats growing areas of western China. A total of 314 isolates, associated with HB were collected from the major oat cultivating areas of Gansu, Qinghai, and Yunnan Provinces in western China. Based on morphological characters, the isolates were initially classified into three genera, as differentiation to species was a bit difficult. Taxonomic analysis of these isolates based on muti-gene phylogenetic analyses (ITS, TEF1, TUB2, and RPB2) revealed four known Fusarium species, F. proliferatum, F. avenaceum, F. poae, and F. sibiricum, and one Acremonium specie (A. sclerotigenum). In addition, a new genus Neonalanthamala gen. nov., similar to genus Nalanthamala was introduced herein with a new combination, Neonalanthamala graminearum sp. nov., to accommodate the HB fungus. The molecular clock analyses estimated the divergence time of the Neonalanthamala and Nalanthamala based on a dataset (ITS, TUB2, RPB2), and we recognized the mean stem ages of the two genera are 98.95 Mya, which showed that they evolved from the same ancestor. N. graminearum was the most prevalent throughout the surveyed provinces. Pathogenicity test was carried out by using two different methods: seed inoculation and head inoculation. Results showed that F. sibiricum isolates were the most aggressive on the seed and head. A. sclerotigenum isolates were not pathogenic to seeds, and were developed less symptoms to the head compared to other species. Data analyses showed that the correlation of the germination potential, germination index, and dry weight of seed inoculation and disease index of plant inoculation had a highly significant negative correlation (P < 0.001). These results showed that the development of HB might be predicted by seed tests for this species. A. sclerotigenum and N. graminearum causing HB are being firstly reported on oat in the world. Similarly, F. proliferatum, F. avenaceum, F. poae and F. sibiricum causing oat HB are firstly reported in China.

Assessment of mycoviral diversity in Pakistani fungal isolates revealed infection by 11 novel viruses of a single strain of Fusarium mangiferae isolate SP1.

An extensive screening survey was conducted on Pakistani filamentous fungal isolates for the identification of viral infections. A total of 396 fungal samples were screened, of which 36 isolates were found double-stranded (ds) RNA positive with an overall frequency of 9% when analysed by a classical dsRNA isolation method. One of 36 dsRNA-positive strains, strain SP1 of a plant pathogenic fungus Fusarium mangiferae, was subjected to virome analysis. Next-generation sequencing and subsequent completion of the entire genome sequencing by a classical Sanger sequencing method showed the SP1 strain to be co-infected by 11 distinct viruses, at least seven of which should be described as new taxa at the species level according to the ICTV (International Committee on Taxonomy of Viruses) species demarcation criteria. The newly identified F. mangiferae viruses (FmVs) include two partitivirids, one betapartitivirus (FmPV1) and one gammapartitivirus (FmPV2); six mitovirids, three unuamitovirus (FmMV2, FmMV4, FmMV6), one duamitovirus (FmMV5), and two unclassified mitovirids (FmMV1, FmMV3); and three botourmiavirids, two magoulivirus (FmBOV1, FmBOV3) and one scleroulivirus (FmBOV2). The number of coinfecting viruses is among the largest ones of fungal coinfections. Their molecular features are thoroughly described here. This represents the first large virus survey in the Indian sub-continent.

Quantitative detection of economically important Fusarium oxysporum f. sp. cubense strains in Africa in plants, soil and water.

Banana is an important food crop and source of income in Africa. Sustainable production of banana, however, is at risk because of pests and diseases such as Fusarium wilt, caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). Foc can be disseminated from infested to disease-free fields in plant material, water and soil. Early detection of Foc using DNA technologies is thus required to accurately identify the fungus and prevent its further dissemination with plants, soil and water. In this study, quantitative (q)PCR assays were developed for the detection of Foc Lineage VI strains found in central and eastern Africa (Foc races 1 and 2), Foc TR4 (vegetative compatibility groups (VCG) 01213/16) that is present in Mozambique, and Foc STR4 (VCG 0120/15) that occurs in South Africa. A collection of 127 fungal isolates were selected for specificity testing, including endophytic Fusarium isolates from banana pseudostems, non-pathogenic F. oxysporum strains and Foc isolates representing the 24 VCGs in Foc. Primer sets that proved to be specific to Foc Lineage VI, Foc TR4 and Foc STR4 were used to produce standard curves for absolute quantification, and the qPCR assays were evaluated based on the quality of standard curves, repeatability and reproducibility, and limits of quantification (LOQ) and detection (LOD). The qPCR assays for Foc Lineage VI, TR4 and STR4 were repeatable and reproducible, with LOQ values of 10-3-10-4 ng/µL and a LOD of 10-4-10-5 ng/µL. The quantitative detection of Foc strains in Africa could reduce the time and improve the accuracy for identifying the Fusarium wilt pathogen from plants, water and soil on the continent.

Assessment of Fusarium Infection and Mycotoxin Contamination of Wheat Kernels and Flour Using Hyperspectral Imaging.

Fusarium head blight (FHB) epidemics in wheat and contamination with Fusarium mycotoxins has become an increasing problem over the last decades. This prompted the need for non-invasive and non-destructive techniques to screen cereal grains for Fusarium infection, which is usually accompanied by mycotoxin contamination. This study tested the potential of hyperspectral imaging to monitor the infection of wheat kernels and flour with three Fusarium species. Kernels of two wheat varieties inoculated at anthesis with F. graminearum, F. culmorum, and F. poae were investigated. Hyperspectral images of kernels and flour were taken in the visible-near infrared (VIS-NIR) (400-1000 nm) and short-wave infrared (SWIR) (1000-2500 nm) ranges. The fungal DNA and mycotoxin contents were quantified. Spectral reflectance of Fusarium-damaged kernels (FDK) was significantly higher than non-inoculated ones. In contrast, spectral reflectance of flour from non-inoculated kernels was higher than that of FDK in the VIS and lower in the NIR and SWIR ranges. Spectral reflectance of kernels was positively correlated with fungal DNA and deoxynivalenol (DON) contents. In the case of the flour, this correlation exceeded r = -0.80 in the VIS range. Remarkable peaks of correlation appeared at 1193, 1231, 1446 to 1465, and 1742 to 2500 nm in the SWIR range.

Assessment of changes in the occurrence of Fusarium toxin and ochratoxin A in Poland related to extreme weather phenomena

Background: Mycotoxins ­ substances harmful to humans, are ubiquitous in the environment. Mycotoxins are generated primarily by Penicilium, Aspergillus and Fusarium genus fungi. Their presence is associated with the unavoidable presence of mold fungi in the environment. The presently observed adverse climatic changes could negatively affect agriculture, causing erosion and loss of organic matter from soil, promulgation of pests and plant diseases, including those originating from pathogenic molds, and also migration of certain mold species into new regions, ultimately creating more favorable conditions for generation of mycotoxins. Objective: The purpose of this work was to investigate contamination of cereals in Poland with Fusarium and ochratoxin A. Elucidating a correlation between precipitation levels in the individual Provinces and reported levels of the investigated mycotoxins, referring to the generally available meteorological databases, would result in more efficient planning of sampling processes and focusing further preventive actions associated with establishing sampling plans for the following years. Material and methods: Investigations were performed on cereal and cereal product samples taken by the official foodstuffs inspection staff. Some 100 samples were taken annually in the 2009-2012 period (357 samples in total). Tests were performed using high performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Precipitation data were obtained from the Central Office of Statistics, based on data received from the Institute of Meteorology and Water Management. Results: Analysis of the influence of precipitation levels during vegetation period on mycotoxin levels in the investigated foodstuffs was performed by associating each recorded content of deoxynivalenol (n=52, corresponding to 14.6% tested samples), zearalenone (n=30, 8.4%), total T-2 and HT-2 toxins (n=21, 5.9%) and ochratoxin A (n=88, 24.6%) above quantification limit with precipitation levels within the Province from which the sample originated. Deoxynivalenol and zearalenone levels show distinct variability corresponding with variability of precipitation levels, well reflecting the reported higher deoxynivalenol and zearalenone levels observed during the rainy years of 2011-2012. Variability in average ochratoxin A levels was not statistically significant. The relatively higher mycotoxin levels in 2009 may result from the heavy rainfall and flooding of 2007-2008. Dependence between the precipitation levels and number of samples showing levels above quantification limit has been also observed for deoxynivalenol. However, a similar analysis made for zearalenone and ochratoxin A does not point to any significant relationship. No data analysis was possible in reference to total T-2 and HT-2 toxins content due to the insufficient number of results available. However, it should be noted that 21% analyzed samples in 2009 contained T-2 and HT-2 levels above the quantification limit, with average of 8.9 µg/kg, whereas in 2010-2012 only one sample of the 263 tested contained contaminants in quantities above the quantification limit. Conclusions: The model used for forecasting presence of mycotoxins in cereals does not allow its practical application during routine generation of official control and monitoring plans on national scale. Notably, tests performed show that exceeding of maximum contamination levels occurred just incidentally, notwithstanding the adverse weather conditions. Further systematic collection of data on mycotoxin contamination of agricultural crops is required for effective continued investigations.

Exposure assessment of infants and young children on selected Fusarium toxins

Background: Mycotoxins belong to substances harmful to human health. They are found mainly in cereal products and their preparations. In particular, infants and young children who consume cereal products, including porridge and gruel, are exposed to these substances. Objective: The aim of the study is to assess the exposure of infants and young children in Poland to micotoxins (ochratoxin A. deoxynivalenol, nivalenol, fumonisins B1 and B1, T-2 and HT-2 toxins) derived from cereal products intended for infants and children. Material and methods: Samples of products (302) were taken from all over the country in the following three years (2011, 2012 and 2013). HPLC-MS / MS method was used to determine the test compounds. Results: Using the HPLC-MS / MS method, the assessment of population exposure in Poland to mikototoxins (ochratoxin A, deoxynivalenol, nivalenol, fumonisins B1 and B1, T-2 and HT-2 toxins) derived from cereal products (porridge, gruel) intended for infants and small children. Samples (302) were taken from across the country over the next three years. The exposure values obtained in the average exposure scenario range from 0.2 to 3% compared to the reference toxicological parameters. Considering that in the case of infants and young children, the tested products constitute a quantitatively significant part of the balanced diet of these consumers, and the remaining groups of foodstuffs, including vegetable products. fruit and meat and dairy products do not contribute significant amounts of mycotoxins to the diet can be accepted. that the level of contamination of cereal products does not pose a significant risk to the health of consumers. In the case of high exposure, it did not exceed 10% of the reference values for deoxynivalenol and the sum of fumonisins B1 and B2. These values were assessed as not relevant for the exposure of infants and young children. In contrast, in the case of zearalenone, the high level of exposure corresponded to 36% of the value of tolerable daily intake (TDI), and for the sum of T-2 and HT-2 toxins, the value of 48% of tolerable daily intake. In both cases, the contribution of pollutants to the diet was significant, but still remained 2-3 times less than the tolerable daily intake. Given, that cereal products are the main source of these contaminants, it can be estimated that exceeding the TDI value in relation to the total diet of infants and young children is unlikely. Conclusions: The exposure values obtained in the average exposure scenario range from 0.2 to 3% compared to the reference toxicological parameters. In the case of zearalenone, the high level of exposure corresponded to 36% of the TDI value. and for the sum of T-2 and HT-2 toxins, 48% TDI. The contribution of pollutants to the diet in both cases was significant. however, it still remained 2-3 times less than the tolerable daily intake. Considering, that cereal products are the main source of these pollutants can be assessed. that exceeding the TDI value for the total diet of infants and young children is unlikely.

Assessment of Toxigenic Fusarium Species and Their Mycotoxins in Brewing Barley Grains.

Fusarium species threaten yield and quality of cereals worldwide due to their ability to produce mycotoxins and cause plant diseases. Trichothecenes and zearalenone are the most economically significant mycotoxins and are of particular concern in barley, maize and wheat. For this reason, the aim of this study was to characterize the Fusarium isolates from brewing barley and to assess deoxynivalenol and zearalenone contamination in grains. Characterization of the Fusarium strains was carried out by the phylogeny based on two loci (EF-1α and RPB2). Mycotoxin detection and quantification were performed by LC-MS. The results show that Fusarium was the predominant genus. Phylogenetic study demonstrated that the majority of the strains clustered within the Fusarium sambucinum species complex followed by the Fusarium tricinctum species complex. The results revealed high incidence of deoxynivalenol (DON) and zearalenone (ZEA) contamination (90.6% and 87.5%, respectively). It was observed that 86% of the samples contaminated with ZEA were above the limits set by the EU and Brazilian regulations. These results may highlight the importance of controlling Fusarium toxins in barley, mainly because of its use in the brewing industry and the resistance of various mycotoxins to food processing treatments.

Presence of Zearalenone in Cereal Grains and Its Exposure Risk Assessment in Indian Population.

Zearalenone (ZEA) is a toxic metabolite of Fusarium genera that frequently contaminates cereal grains. India being a tropical country provides suitable conditions for fungal invasion to the cereals. In the absence of any regulatory limits for ZEA in India, the present study was carried out to analyze the contamination levels of ZEA in different cereal samples consumed by Indian population and its exposure assessment through intake. Out of 117 cereal samples comprising of wheat, rice, corn, and oats, 70 (84%) were found to be positive for ZEA contamination, among which 24 (33%) samples exceeded the permissible limits proposed by European Union when analyzed by high-performance liquid chromatography. The positive samples were further validated by Liquid Chromatography-Mass Spectroscopy (LC-MS) analysis. Based on the quantitative estimation of ZEA contamination in cereals and their daily consumption values, the probable daily intake of ZEA was found to be 16.9- and 7.9-fold higher in rice and wheat samples, respectively, than the tolerable daily intake prescribed by European Food Safety Authority. The presence of ZEA at high levels indicates a higher exposure risk for Indian population as wheat and rice are staple foods in India. Thus, there is an immediate need to set the permissible levels of ZEA in India to safeguard the health of 1.34 billion people. PRACTICAL APPLICATION: High levels of ZEA contaminated wheat and rice samples suggest that the consumers are at a greater exposure risk. The study will help the Indian regulatory bodies to set the permissible level of ZEA in different cereal grains so as to safeguard the health of common masses. This can happen by simply adopting to European Food Safety Authority standards or depending on the consumption pattern of food and its occurrence, the new safe limit can be prescribed in India like in other Asian countries.

Effectiveness of a full-scale horizontal slow sand filter for controlling phytopathogens in recirculating hydroponics: From microbial isolation to full microbiome assessment.

The microbial disinfestation efficiency of an innovative horizontal-flow slow sand filter (HSSF) for treating nutrient solution spent from an experimental closed-loop nursery was evaluated by means of a combination of culture-dependent and independent molecular techniques. A dense inoculum of the fungal plant pathogen Fusarium oxysporum f.sp. lycopersici was applied in the fertigation system (106 cells per mL). Indigenous and introduced populations of eubacteria and fungi were assessed in the nutrient solution, the HSSF influent/effluent, and a sand bed transect by isolation on selective media, as well as by quantitative qPCR and next-generation sequencing (NGS) on target ribosomal genes. The HSSF effectively reduced viable Fusarium propagules and fungal gene content with an efficiency consistently above 99.9% (5 orders of magnitude down). On the other hand, Fusarium cells accumulated in the sand bed, indicating that physical entrapment was the main removal mechanism. The viability of retained Fusarium cells tended to decrease in time, so that treatment efficiency might be enhanced by antagonistic species from the genera Bacillus, Pseudomonas, and Trichoderma, also identified in the sand bed. Indigenous bacterial populations from the HSSF effluent were reduced by 87.2% and 99.9% in terms of colony forming units and gene counts, respectively, when compared to the influent. Furthermore, microbial populations from the HSSF effluent were different from those observed in the sand bed and the influent. In summary, the HSSF microbial disinfestation efficiency is comparable to that reported for other more intensive and costly methodologies, while allowing a significant recovery of water and nutrients.

Mycotoxin risk assessment for consumers of groundnut in domestic markets in Nigeria.

The fungal and multi-mycotoxin profiles of groundnuts sold in domestic markets in Nigeria as well as the associated risk to consumers were assessed in the present study. Four hundred fungal isolates representing mainly Aspergillus [58.6%: Aspergillus section Flavi (37.1%) and A. niger-clade (21.5%)], Penicillium (40.9%) and Fusarium (0.5%) were isolated from 82 (97.6%, n=84) groundnut samples collected from four agro-ecological zones (AEZs) of Nigeria. The incidence of aflatoxin-producing A. flavus isolates (71%) was significantly (p<0.05) higher in the groundnuts than that of the non-aflatoxigenic isolates (29%). Fifty-four fungal metabolites [including aflatoxins (AFB1, AFB2, AFG1, AFG2 and AFM1), beauvericin (BEAU), cyclopiazonic acid (CPA), moniliformin, nivalenol and ochratoxin A] and four bacterial metabolites were detected in the groundnuts by liquid chromatography tandem mass spectrometry. Aflatoxins (39%; max: 2076µg/kg; mean: 216µg/kg) were detected in more samples than any other mycotoxin. About 25, 23 and 14% of the samples respectively were above the 2µg/kg AFB1, 4 and 20µg/kg total aflatoxin limits of the European Union and US FDA respectively. The mean margins of exposure of AFB1 and total aflatoxins for adult consumers were 1665 and 908, respectively, while mean estimated daily intake values for infants, children and adults were <0.1% for BEAU and 4% for CPA. Consumers of mycotoxin contaminated groundnuts in Nigeria may therefore be at a risk of liver cancer in addition to other combinatory effects of mycotoxin/metabolite cocktails. There is need for increased targeted interventions in the groundnut value chain in Nigeria for public health benefits.

Agroindustrial composts to reduce the use of peat and fungicides in the cultivation of muskmelon seedlings.

BACKGROUND: Environmental concerns about peat extraction in wetland ecosystems have increased. Therefore, there is an international effort to evaluate alternative organic substrates for the partial substitution of peat. The aim of this work was to use different composts (C1-C10) obtained from the fruit and vegetable processing industry (pepper, carrot, broccoli, orange, artichoke residues, sewage sludge (citric and pepper) and vineyard pruning wastes) to produce added-value composts as growing media with suppressive effect against Fusarium oxysporum f.sp. melonis (FOM) in muskmelon. RESULTS: Composts showed values of water-soluble carbon fractions and dehydrogenase activity that allowed them to be considered mature and stabilized. All compost treatments produced significantly (F = 7.382; P < 0.05) higher fresh shoot weight than peat, treatment T-C2 showing the highest values. Treatments T-C5, T-C7 and T-C8 showed percentages of disease incidence that were significantly (F = 16.052; P < 0.05) the lowest, relative to peat, followed by T-C6, T-C10, T-C1 and T-C9 with values below 50%. CONCLUSION: Composts produced are suitable components of mixed compost-peat growing media, providing a 50% substitution of peat. Furthermore, some of these composts also showed an added value as a suppressive organic medium against Fusarium wilt in muskmelon seedling, a fact probably related to high pH and pepper wastes and high content of pruning waste as initial raw materials. © 2016 Society of Chemical Industry.

Patogenicidad de especies de Fusarium asociadas a la pudrición basal del ajo en el centro norte de México / Fusarium species associated with basal rot of garlic in North Central Mexico and its pathogenicity

El ajo en México es uno de los cultivos de hortalizas más rentables, más del 83% de esta superficie es aportada por los estados de Zacatecas, Guanajuato, Sonora, Puebla, Baja California y Aguascalientes. La pudrición basal ocasionada por Fusarium spp. se encuentra ampliamente distribuida a nivel mundial; esta enfermedad se ha convertido en una limitante en zonas productoras de cebolla y ajo, no solo en México, sino también en otros países, En México, se ha informado la presencia de Fusarium oxysporum en plantas en Guanajuato y en semillas de ajo en Aguascalientes. En el estado de Morelos se ha reportado la presencia de Fusarium culmorum en cultivares de cebolla. Asimismo, en Aguascalientes se tienen antecedentes de otras especies como Fusarium proliferatum, Fusarium verticillioides, Fusarium solani y Fusarium acuminatum. Para este trabajo se planteó como objetivo identificar las especies de Fusarium encontradas en los estados de Zacatecas, Guanajuato y Aguascalientes, y evaluar su patogenicidad. Se realizaron recolectas de plantas con síntomas de la enfermedad en los estados antes mencionados. De los muestreos realizados se identificaron las especies F. oxysporum, F. proliferatum, F. verticillioides, F. solani y F. acuminatum; las cepas de Aguascalientes identificadas como AGS1A (F. oxysporum), AGS1B (F. oxysporum) y AGSY-10 (F. acuminatum) fueron las que presentaron bajo condiciones de invernadero un mayor índice de severidad.

Garlic in Mexico is one of the most profitable vegetable crops, grown in almost 5,451 ha; out of which more than 83% are located in Zacatecas, Guanajuato, Sonora, Puebla, Baja California and Aguascalientes. Blossom-end rot caused by Fusarium spp is widely distributed worldwide and has been a limiting factor in onion and garlic production regions, not only in Mexico but also in other countries. The presence of Fusarium oxysporum has been reported in Guanajuato and Aguascalientes. Fusarium culmorum has been reported in onion cultivars of Morelos; and Fusarium proliferatum, Fusarium verticillioides, Fusarium solani and Fusarium acuminatum have been previously reported in Aguascalientes. The goal of this work was identifying the Fusarium species found in Zacatecas, Guanajuato and Aguascalientes, to assess their pathogenicity. Plants with disease symptoms were collected from hereinabove mentioned States. The samples resulted in the identification of: F. oxysporum, F. proliferatum, F. verticillioides, F. solani and F. acuminatum species; out of which Aguascalientes AGS1A (F. oxysporum), AGS1B (F. oxysporum) and AGSY-10 (F. acuminatum) strains showed higher severity under greenhouse conditions.

Re-purposing bridging flocculation for on-site, rapid, qualitative DNA detection in resource-poor settings.

Developing molecular diagnostics in resource-poor settings is challenging. As such, we purpose-built a novel bridging flocculation assay for qualitative evaluation of isothermally amplified DNA by naked eye. The flocculation assay was dependent on pH, DNA polymer amounts and lengths. The method was first applied to the rapid and sensitive detection of important plant pathogens and subsequently extended to other pathogens across the animal kingdom to demonstrate the wide applications of our approach.

Development of DArT markers and assessment of diversity in Fusarium oxysporum f. sp. ciceris, wilt pathogen of chickpea (Cicer arietinum L.).

BACKGROUND: Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt of chickpea is highly variable and frequent recurrence of virulent forms have affected chickpea production and exhausted valuable genetic resources. The severity and yield losses of Fusarium wilt differ from place to place owing to existence of physiological races among isolates. Diversity study of fungal population associated with a disease plays a major role in understanding and devising better disease control strategies. The advantages of using molecular markers to understand the distribution of genetic diversity in Foc populations is well understood. The recent development of Diversity Arrays Technology (DArT) offers new possibilities to study the diversity in pathogen population. In this study, we developed DArT markers for Foc population, analysed the genetic diversity existing within and among Foc isolates, compared the genotypic and phenotypic diversity and infer the race scenario of Foc in India. RESULTS: We report the successful development of DArT markers for Foc and their utility in genotyping of Foc collections representing five chickpea growing agro-ecological zones of India. The DArT arrays revealed a total 1,813 polymorphic markers with an average genotyping call rate of 91.16% and a scoring reproducibility of 100%. Cluster analysis, principal coordinate analysis and population structure indicated that the different isolates of Foc were partially classified based on geographical source. Diversity in Foc population was compared with the phenotypic variability and it was found that DArT markers were able to group the isolates consistent with its virulence group. A number of race-specific unique and rare alleles were also detected. CONCLUSION: The present study generated significant information in terms of pathogenic and genetic diversity of Foc which could be used further for development and deployment of region-specific resistant cultivars of chickpea. The DArT markers were proved to be a powerful diagnostic tool to study the genotypic diversity in Foc. The high number of DArT markers allowed a greater resolution of genetic differences among isolates and enabled us to examine the extent of diversity in the Foc population present in India, as well as provided support to know the changing race scenario in Foc population.

Assessment of rose bengal versus riboflavin photodynamic therapy for inhibition of fungal keratitis isolates.

PURPOSE: To compare the in vitro effect of rose bengal and riboflavin as photosensitizing agents for photodynamic therapy (PDT) on fungal isolates that are common causes of fungal keratitis. DESIGN: Experimental study. METHODS: Three isolates (Fusarium solani, Aspergillus fumigatus, Candida albicans) recovered from patients with confirmed fungal keratitis were used in the experiments. Isolates were grown on Sabouraud-Dextrose agar, swabbed, and prepared in suspension, and 1 mL aliquots were inoculated onto test plates in triplicate. Test plates were separated into 5 groups: Group 1, no treatment; Group 2, 0.1% rose bengal alone; Group 3, 518 nm irradiation alone; Group 4, riboflavin PDT (riboflavin + 375 nm irradiation); and Group 5, rose bengal PDT (rose bengal + 518 nm irradiation). Irradiation was performed over a circular area using either a green light-emitting diode (LED) array (peak wavelength: 518 nm) or an ultraviolet-A LED array (peak wavelength: 375 nm). Test plates were irradiated with an energy density of 5.4 J/cm(2). Later, plates were placed in a 30 C incubator and observed for growth. RESULTS: Rose bengal-mediated PDT successfully inhibited the growth of all 3 fungal isolates in the irradiated area. All other groups exhibited unrestricted growth throughout the plate. CONCLUSIONS: Rose bengal-mediated PDT successfully inhibited the growth of 3 types of fungi. No other experimental groups, including riboflavin-mediated PDT, had any inhibitory effect on the isolates. The results might be useful for the treatment of patients suffering from corneal infection.

Saving costs through the decontamination of the packaging of unused medical supplies using hydrogen peroxide vapor.

OBJECTIVE. Individually packaged sterile supply items may become contaminated and act as vectors for nosocomial transmission of multidrug-resistant organisms (MDROs). Thus, many hospitals have a policy to dispose of these unused, packaged supply items at patient discharge from the hospital, which has considerable cost implications. We evaluated the frequency of contamination of these items, the efficacy of hydrogen peroxide vapor (HPV) in disinfecting them, and costs associated with discarded supplies. DESIGN. Before-after study. METHODS. A pilot study was performed in the rooms of 20 patients known to be colonized or infected with vancomycin-resistant enterococci (VRE), and a follow-up study was performed in an additional 20 rooms of patients under precautions for various MDROs in 6 high-risk units. Five pairs of supply items were selected. One item of each pair was sampled without exposure to HPV, and the other was sampled after HPV exposure. The cost of discarded supplies was calculated by examining stock lists of supplies stored on the study units. RESULTS. Seven (7%) of 100 items were contaminated with VRE in the pilot study, and 9 (9%) of 100 items were contaminated with MDROs in the follow-up study. None of the items were contaminated after exposure to HPV (P < .02 in both the pilot and the follow-up study). The annual cost of supplies discarded at patient hospital discharge was $387,055. This figure does not include the cost of waste disposal and is therefore likely to be an underestimation of the financial burden. CONCLUSIONS. HPV effectively disinfected the packaging of supply items, which could generate considerable financial and environmental benefits.

Fungal and mycotoxin assessment of dried edible mushroom in Nigeria.

In order to determine whether dried mushrooms are a foodstuff that may be less susceptible to infection by toxigenic molds and consequently to mycotoxin contamination, 34 dried market samples were analyzed. Fungal population was determined in the samples by conventional mycological techniques and molecular studies, while the spectrum of microbial metabolites including mycotoxins was analyzed by a liquid chromatography tandem mass spectrometric method covering 320 metabolites. Molds such as Fusarium, Penicillium, Trichoderma and aflatoxigenic species of Aspergillus (Aspergillus flavus and Aspergillus parvisclerotigenus) were recovered from all samples at varying levels. None of the mycotoxins addressed by regulatory limits in the EU was positively identified in the samples. However, 26 other fungal metabolites occurred at sub- to medium µg/kg levels in the samples, including aflatoxin/sterigmatocystin bio-precursors, bis-anthraquinone derivatives from Talaromyces islandicus, emerging toxins (e.g. enniatins) and other Fusarium metabolites, and clavine alkaloids. Although little is known on the toxicology of these substances, the absence of aflatoxins and other primary mycotoxins suggests that dried mushrooms may represent a relatively safe type of food in view of mycotoxin contamination.

Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals.

A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin-BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73-109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 µg kg(-1), respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 µg kg(-1), respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC-MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

Deoxynivalenol and zearalenone in Fusarium-contaminated wheat in Mexico City.

Fusarium spp. invasion causes head blight, a destructive disease in the world's main wheat-growing areas, and deoxynivalenol (DON) and zearalenone (ZEA) contamination in cereal-based products. No data are available on the relationship between Fusarium spp. on commercial wheat samples in Mexico City and the presence of mycotoxins. A total of 30 wheat samples were subject to a PCR method involving genes of the trichothecene and zearalenone biosynthesis pathways to detect the presence of Fusarium. Detection and quantification of DON and ZEA was performed using liquid chromatography coupled to UV detection. PCR indicated the presence of the Tri5 and PKS4 genes in 16.7 and 23.3% of samples, respectively. DON and ZEA contamination was found in 51.2 and 71.4% of samples, respectively, where a positive amplification was obtained. This work presents up-to-date information on mycotoxin contamination in Mexico, where improved contamination/exposure data and firm control/monitoring measures are needed.