Differentiation between Aspergillus flavus and Aspergillus parasiticus from Pure Culture and Aflatoxin-Contaminated Grapes Using PCR-RFLP Analysis of aflR-aflJ Intergenic Spacer

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification

Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature

A microarray analysis was performed to study the effect of varying combinations of water activity and temperature on the activation of aflatoxin biosynthesis genes in Aspergillus flavus grown on YES medium. Generally A. flavus showed expression of the aflatoxin biosynthetic genes at all parameter combinations tested. Certain combinations of aw and temperature, especially combinations which imposed stress on the fungus resulted in a significant reduction of the growth rate. At these conditions induction of the whole aflatoxin biosynthesis gene cluster occurred, however the produced aflatoxin B1 was low. At all other combinations (25 °C/0.95 and 0.99; 30 °C/0.95 and 0.99; 35 °C/0.95 and 0.99) a reduced basal level of cluster gene expression occurred. At these combinations a high growth rate was obtained as well as high aflatoxin production. When single genes were compared, two groups with different expression profiles in relation to water activity/temperature combinations occurred. These two groups were co-ordinately localized within the aflatoxin gene cluster. The ratio of aflR/aflJ expression was correlated with increased aflatoxin biosynthesis

Return of Aflatoxin Production in Aspergillus flavus and Aspergillus parasiticus after Serial Passaging

Undergraduate Theoretical Proposa

Aspergillus section Flavi and aflatoxins in Algerian wheat and derived products

Wheat and its derivatives are a very important staple food for North African populations. The aim of this study was to analyze populations of Aspergillus section Flavi from local wheat based on aflatoxins (AFs),cyclopiazonic acid (CPA) and sclerotia production, and also to evaluate AFs-contaminated wheat collected from two different climatic regions in Algeria. A total of 108 samples of wheat were collected during the following phases: pre-harvest, storage in silos and after processing. The results revealed that among the Aspergillus species isolated, those belonging to section Flavi were predominant. Of the 150 strains of Aspergillus section Flavi isolated, 144 were identified as Aspergillus flavus and 6 as Aspergillus tamarii. We showed that 72% and 10% of the A. flavus strains produced AFs and CPA, respectively. Among the 150 strains tested, 60 produced amounts of AFB1 ranging from 12.1 to 234.6 lg/g of CYA medium. Also, we showed that most strains produced large sclerotia. AFB1was detected by HPLC in 56.6% of the wheat samples and derived products (flour, semolina and bran) with contamination levels ranging from 0.13 to 37.42 lg/kg

Mycoflora and Aflatoxin Contamination of Some Foodstuffs

Analysis was made of the mycoflora and aflatoxin contamination of Rice (Oryza sativa), Beans (Phaseolus vulgaris), Corn (Zea mays), and Groundnut (Arachis hypogaea) sold in four different markets in Sango-Ota, Ogun state, Nigeria. Sixty four samples comprising of four samples of each foodstuff from four food vendors in four different markets was assayed. The samples were contaminated with different species of fungi to include Aspergillus flavus, Aspergillus tamarii, Aspergillus niger, Rhizopus nigricans, Rhizopus oryzae, Saccharomyces cerevisiae, Aspergillus parasiticus, Fusarium moniliforme, Fusarium verticilliodes, Aspergillus ochraceus, Cladosporium cladosporioide, Mucor spp, Trichodema spp, Rhizopus arrhizus and Aspergillus fumigates. Aspergillus flavus and Fusarium spp had the highest rate of occurrence among the isolated fungi. Aflatoxins B1, B2, G1 and G2 were found associated with the samples at concentration ranging from 9 - 25 ppb, 8 - 12 ppb, 6 - 21 ppb, 4 - 8 ppb respectively. The fungal counts were between 6.3 x 102 to 7.0 x 103 cfu/g. The moisture content and the pH of samples were between 10.9 to 28.0% and 6.20 to 6.66 respectively. Effective storage and adherence to HACCP principles will help prevent contamination of foodstuffs with aflatoxigenic fungi

Inhibition of Aflatoxin Formation in Aspergillus Species by Peanut (Arachis hypogaea) Seed Stilbenoids in the Course of Peanut− Fungus Interaction

Common soil fungi, Aspergillus flavus and Aspergillus parasiticus, are opportunistic pathogens that invade preharvest peanut seeds. These fungi often produce carcinogenic aflatoxins that pose a threat to human and animal health through food chains and cause significant economic losses worldwide. Detection of aflatoxins and further processing of crops are mandated to ensure that contaminated agricultural products do not enter food channels. Under favorable conditions, the fungus-challenged peanut seeds produce phytoalexins, structurally related stilbenoids, capable of retarding fungal development. The purpose of the present study was to evaluate the potential influence of peanut phytoalexins on fungal development and aflatoxin formation in the course of peanut−fungus interaction. The present research revealed that during such interaction, aflatoxin formation was completely suppressed in A. flavus and A. parasiticus strains tested, when low concentrations of spores were introduced to wounded preincubated peanuts. In most of the experiments, when fungal spore concentrations were 2 orders of magnitude higher, the spores germinated and produced aflatoxins. Of all experimental seeds that showed fungal growth, 57.7% were aflatoxin-free after 72 h of incubation. The research provided new knowledge on the aflatoxin/phytoalexin formation in the course of peanut−fungus interaction

Survey and aflatoxigenic characterization for Aspergillus section Flavi from three maize production regions of Argentina

Argentina is one of the main exporters of maize. Soil is the main source of inoculum for the species Aspergillus section Flavi determining grain colonization and the subsequent aflatoxin production. The objective of this study was to evaluate the Aspergillus section Flavi incidence in soil and corn kernels from different regions of Argentina and evaluate the sclerotial type and the aflatoxin B1 (AFB1) producing capacity. Maize kernels and maize soil samples were collected at harvest from the north of Argentina and from the center and south of the province of Córdoba. Analysis was performed by comparing the distribution of culturable fungal and Aspergillus section Flavi strains. The type of sclerotia and the production of aflatoxin B1 (AFB1) were evaluated. The Aspergillus section Flavi counts observed in soil samples from the southern region of Córdoba were similar than those observed in samples from the central region. Severity by Aspergillus section Flavi did not exceed 9% and 2.5% in southern and northern Córdoba. 75% of the strains from the northern region of Argentina produced L sclerotia while a 25% were not sclerotia producers and showed high levels of AFB1. The highest percentages of strains producingL sclerotia (95%) and the lowest number of S strains (5%) were isolated from the southern part of Córdoba. 61% of the strains from the central region produced L sclerotia while approximately 20% produced S sclerotia and the highest AFB1 levels. This study contributes to the knowledge of aflatoxigenic strains from three regions of Argentina and to the development of further aflatoxin control and prevention strategies.Argentina es uno de los principales países exportadores de maíz. El suelo constituye el principal inóculo de Aspergillus sección Flavi determinando la colonización de los granos y la posterior producción de aflatoxinas. El objetivo de este trabajo fue comparar la incidencia de hongos totales y de Aspergillus sección Flavi en suelos y granos de maíz de diferentes regiones de Argentina y evaluar el tipo de esclerocios y la capacidad de producir aflatoxina B1 (AFB1). Se recolectaron muestras de suelo y granos de maíz de tres regiones maiceras de Argentina, comparando la distribución fúngica y de Aspergillus sección Flavi. Se evaluó el tipo de esclerocios y la producción de AFB1. Los recuentos de Aspergillus sección Flavi fueron similares en suelos de la región sur y centro de Córdoba. La severidad de infección en granos de maíz fue similar en el norte de Argentina y en el sur de Córdoba. La severidad por Aspergillus sección Flavi no superó el 9% y el 2,5% en el sur de Córdoba y el norte de Argentina, respectivamente. Los mayores porcentajes de cepas con esclerocios L (95%), presentaron la mayor capacidad toxicogénica, y el menor número de cepas S (5%) fueron aislados del sur de Córdoba. El 61% de las cepas de la región central de Córdoba produjeron escleróticos L, y el 20% escleróticos S con los más altos niveles de AFB1. Este estudio contribuye al conocimiento de las cepas aflatoxigénicas presentes en Argentina, así como al desarrollo de nuevas estrategias prevención de aflatoxinas.Fil: Benito, Nicolas. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; ArgentinaFil: Carranza, Cecilia Soledad. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; ArgentinaFil: Magnoli, Carina Elizabeth. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; ArgentinaFil: Barberis, Carla Lorena. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología. Cátedra de Micología; Argentin

Identification of dothistromin biosynthetic pathway genes : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand

Dothistromin is a polyketide-derived toxic secondary metabolite produced by the filamentous fungus Dothistroma pini which causes the disease Dothistroma needle blight in Pinus radiata. Dothistromin is considered to be an important component in the disease process, although its exact function is yet to be identified. By isolating and identifying genes involved in dothistromin biosynthesis, and subsequently obtaining mutants blocked or altered in the synthesis of dothistromin, the role of this toxin in pathogenicity will be able to be assessed. Dothistromin is structurally related to the mycotoxins, aflatoxin (AF) from Aspergillus parasiticus and A. flavus, and sterigmatocystin (ST) from A. nidulans. Three intermediates in the ST and AF biosynthetic pathways (averantin, averufin, and versicolorin B) are thought to also be intermediates dothistromin biosynthesis. Due to these similarities, cloned AF pathway genes were used as heterologous probes in Southern hybridisation analysis to provide a direct method for identifying dothistromin biosynthetic genes. A fragment of the A. parasiticus nor-1 gene, encoding a reductase involved in the conversion of norsolorinic acid (NA) to averantin (AVN) in the AF biosynthetic pathway, was used as a probe to detect a region of sequence similarity to D. pini genomic DNA. A D. pini genomic library was then constructed and screened, resulting in clone λCGN2. However, Southern hybridisation analysis suggested that this clone did not contain a homologue of the nor-1 gene from A. parasiticus. A fragment of the Aspergillus parasiticus ver-1 gene, encoding a reductase involved in the conversion of versicolorin A (VA) to ST in the AF biosynthetic pathway, was also used as a probe to detect a region of sequence similarity to D. pini genomic DNA. The D. pini genomic library was then screened. Two clones, λCGV1 and λCGV2, were isolated and Southern hybridisation analysis confirmed that these clones contained sequences hybridising to the A. parasiticus ver-1 gene fragment. Fragments of these clones which hybridised were then sequenced and compared to the GenBank database. The amino acid coding sequence of a 0.8 kb SalI region from clone λCGV1 exhibited a high degree of similarity with the A. nidulans verA and A. parasiticus ver-1 genes, involved in the ST and AF biosynthetic pathways, and the Magnaporthe grisea ThnR, and Colletotrichum lagenarium Thr1 genes, involved in melanin biosynthesis. This data suggested a ver-1 homologue is present in the D. pini genome. Limited sequence analysis of a 2.1 kb region from clone λCGV2 suggested that a second independent copy of a ver-1-like gene may also be present in the genome

Antifungal activity of lactic acid bacteria

Enrichment culture techniques produced more than 1200 isolates of lactic acid bacteria (LAB) that were screened for antifungal activity against the indicator mould Aspergillus fumigatus. Approximately 10% of the LAB were active, but only 4% had medium or strong activity in an agar plate assay. The majority of isolates with strong antifungal activity were Lactobacillus coryniformis strains, but Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified. Some of the isolates lost activity during storage but most maintained their fungal inhibitory effect. Large variations in sensitivity were observed between different moulds and yeasts. Antifungal cyclic dipeptides and phenyllactic acid were detected in culture filtrates from several of the LAB isolates. Lactobacillus coryniformis subsp. coryniformis strain Si3 produced an antifungal compound that lost activity when treated with proteinases. The antifungal peptide(s) was heat stable, with a size of approx. 3kDa and had maximum activity at pH 3.0 to 4.5. Addition of ethanol to the growth medium of strain Si3 prevented a decline in observed antifungal activity during the stationary phase. Glycerol addition to agar plates with L. coryniformis strains, overlaid with soft agar suspensions of yeast cells or fungal spores, strongly enhanced the antifungal effect. This was particularly true with spoilage moulds and yeasts, e.g. Penicillium roqueforti and Pichia anomala, not normally affected by the antifungal metabolites of L. coryniformis. Chemical and genetic data suggested that reuterin (3-hydroxypropionaldehyde) was the cause of this effect. The glycerol/diol dehydratase operon of L. coryniformis was partially elucidated and found to be similar to that Lactobacillus collinoides. Bioassay-guided isolation of new metabolites from LAB revealed that Lactobacillus plantarum MiLAB 14 produces hydroxylated fatty acids with strong antifungal effects. 3-Hydroxydecanoic acid, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid and 3-hydroxy-5-cis-dodecenoic acid were characterized from the supernatant of MiLAB 14. The hydroxy fatty acids had total inhibitory effects in the range 10 to >100 µg ml-1 against several moulds and yeasts