Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Fungal biodiversity in the air of Turin

The qualitative fungal composition of Turin 's atmospheric environment was surveyed, carrying out a twelve-month study and collecting with a single stage volumetric sieve sampler on Dermasel agar supplemented with 0.4 g l^–1 cycloheximide and 0.05 g l^–1 chloramphenicol. We isolated 165 species and 2 varieties of mesophilic fungi from 58 genera and 26 thermotolerant species from 12 genera. Penicillium, Aspergillus, Acremonium, Chrysosporium, Scopulariopsis, Malbranchea, Paecilomyces, Phialophora and Cladosporium were in sequence the genera most rich in mesophilic species; Aspergillus, Penicillium, Chrysosporium and Scopulariopsis the most rich in thermotolerant species. Many of the species isolated are rarely or never recorded in the atmospheric environment. Cycloheximide can thus be said to select among airborne fungi, giving a characteristic picture.Abbreviation CH Cycloheximide - cfu colony forming unit     

Development and evaluation of a real-time quantitative PCR assay for Aspergillus flavus

Aspergillus flavus is a ubiquitous mold and the most common mold contaminating foodstuffs. Many strains of A. flavus produce aflatoxins. In addition it is an allergen and an opportunistic pathogen of animals and plants. A. flavus often is underestimated in traditional culture analyses due to the expertise required and the cost associated with speciating members of the genus Aspergillus. The goal of this study was to develop and validate a primer and probe set for the rapid detection and quantitation of A. flavus in pure culture using real-time quantitative polymerase chain reaction (QPCR) amplification. Unique DNA regions were located in the genome of the target organism by sequence comparison with the GenBank database, and several candidate oligonucleotides were identified from the scientific literature for potential use with the TaqMan QPCR technology. Three primer and probe sets were designed and validated for specificity and sensitivity in laboratory experiments. Initial screening to test for sensitivity was performed with seven A. flavus isolates and selected nontarget fungi. Specificity testing was conducted with the selected primer and probe set, which amplified all nine A. flavus isolates tested, including an aflatoxin producing strain. The primers did not amplify DNA extracted from 39 other fungal species (comprising 16 genera), including 18 other Aspergillus species and six Penicillium species. No amplification of human or bacterial DNA was observed; however cross-reactivity was observed with Aspergillus oryzae. PCR analysis of DNA dilutions and the use of an internal positive control demonstrated that 67% of the fungal DNA samples assayed contained PCR inhibitors. The assay validated for the target organism is capable of producing PCR results in less than 1 h after DNA extraction. The results of this research demonstrate the capabilities of QPCR for the enhanced detection and enumeration of fungi of significance to human health.     

Mycoflora and Occurrence of Aflatoxin B 1 in Dried Yam Chips from Markets in Ogun and Oyo States,Nigeria

Seventy-six samples of dried yam chips locally called elubo isu were purchased in 2000 from markets in Ogun and Oyo States of southwestern Nigeria. The samples were assessed for pH, moisture content, associated fungi and aflatoxin B1 contamination. The pH of samples ranged from 5.6 to 6.1,while the moisture contents varied from 6.8 to 14.5% in Ogun samples, and 7.1 to 13.6% in samples from Oyo. Aspergillus and Penicillium were the two prevalent genera of fungi, and the number of colony forming units per gram of these two genera in the yam chips studied exceeded the tolerance limit in foodstuffs. The other fungal genera isolated included Botryodiplodia, Cladosporium, Fusarium, Rhizopus, Mucor, Aureobasidium and Paecilomyces. The two most frequent fungal species were A. niger and A. flavus. Thin layer chromatographic analysis showed that 17 samples or 22% contained aflatoxin B1 beyond the detection limit (5 ppb), but only three samples or 4% had toxin level above 30 ppb, the tolerance level in food for human consumption. The mean concentration of aflatoxin B1 in positive samples was 27.1 ppb.     

Evaluation of different methods for the extraction of DNA from fungal conidia by quantitative competitive PCR analysis.

Five different DNA extraction methods were evaluated for their effectiveness in recovering PCR templates from the conidia of a series of fungal species often encountered in indoor air. The test organisms were Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartarum, Cladosporium herbarum and Alternaria alternata. The extraction methods differed in their use of different cell lysis procedures. These included grinding in liquid nitrogen, grinding at ambient temperature, sonication, glass bead milling and freeze-thawing. DNA purification and recovery from the lysates were performed using a commercially available system based on the selective binding of nucleic acids to glass milk. A simple quantitative competitive polymerase chain reaction (QC-PCR) assay was developed for use in determining copy numbers of the internal transcribed spacer (ITS) regions of the ribosomal RNA operon (rDNA) in the total DNA extracts. These quantitative analyses demonstrated that the method using glass bead milling was most effective in recovering PCR templates from each of the different types of conidia both in terms of absolute copy numbers recovered and also in terms of lowest extract to extract variability. Calculations of average template copy yield per conidium in this study indicate that the bead milling method is sufficient to support the detection of less than ten conidia of each of the different organisms in a PCR assay.     

Fungal flora of the digestive tract of 5 species of triatomines vectors of Trypanosoma cruzi,Chagas 1909

A study of the mycobiota in the digestive tract of 5 important species of triatomines, Triatoma brasiliensis, T. infestans, T. sordida, T. pseudomaculata and T. vitticeps, was made. The digestive tracts of 164 adults and 535 nymphs of those triatomines were studied and 393 fungal strains were isolated.The genera with the greatest number of species were Penicillium (19 species), Aspergillus (17 species) and Acremonium (5 species) and the most frequent species, in decreasing order, were Penicillium corylophilum, Aspergillus niger, Penicillium fellutanum, Cladosporium herbarum, Penicillium waksmanii, Aspergillus awamori and Paecilomyces variotii. Among the isolated fungi, we found species that are recognized as entomopathogenic and pathogenic for humans and animals.This revised version was published online in October 2005 with corrections to the Cover Date.     

Identification of fungi associated with rotylenchulus reniformis

The objective of this work was to isolate and identify fungi associated with R. reniformis in cotton roots. Soil samples were collected in cotton fields naturally infested with R. reniformis and from cotton stock plants cultured in the greenhouse. Nematodes extracted from the soil were observed under the stereoscope, and discolored eggs and vermiform stages colonized with mycelia were cultured on 1.5% water agar supplemented with antibiotics, and incubated at 27°C. Identification of the nematophagous fungi was based on the morphological characters, and the ITS regions and 5.8S rDNA amplified by PCR using the primers ITS1 and ITS4. The parasitism percentage on vermiform nematodes from greenhouse samples was 21.2%, and the percentages from cotton fields in Limestone, Henry, and Baldwin counties in Alabama were 3%, 23.2%, and 5.6%, respectively. A total of 12 fungi were identified from R. reniformis vermiform stages and eggs. The most frequently isolated fungi were Arthrobotrys dactyloides (46%) and Paecilomyces lilacinus (14%), followed by Phoma exigua (4.8%), Penicillium waksmanii and Dactylaria brochophaga (3.6%), Aspergillus glaucus group (2.4%). Cladosporium herbarum, Cladosporium cladiosporioides, Fusarium oxysporum, Torula herbarum, Aspergillus fumigatus, and an unidentified basidiomycete were less frequent (1.2%). A high percentage (16.8%) of fungi from colonized nematodes was not cultivable on our media. Out of those 12 fungi, only four have been previously reported as nematophagous fungi: three isolates of Arthrobotrys dactyloides, and one isolate of Dactylaria brochopaga, Paecilomyces lilacinus, and Fusarium oxysporum. Molecular identification of Arthrobotrys dactyloides and Dactylaria brochopaga was consistent with the morphological identification, placing these two fungi in the new genus Drechslerella as proposed in the new Orbilaceae classification.     

Mycoflora of Iranian Maize Harvested in the Main Production Areas in 2000

Maize is one of the most important cereals produced in, and imported into, Iran. The incidences of seed-borne fungi were determined in Iranian maize harvested in 2000 from four major production areas with different climatic conditions, namely Fars, Khuzestan, Kermanshah and Mazandaran provinces. This is the first study to compare the mycoflora of maize in the aforementioned areas. Mycological analyses showed a predominance of Fusarium species (38.5%), followed by Aspergillus species (8.7%), Rhizopus species (4.8%), Penicillium species (4.5%), Mucor species (1.1%), and four other fungal genera. Fusarium verticillioides was the most prevalent species (83% of Fusarium isolates and 52% of the total isolations), with the highest incidence in Mazandaran (59%), a region of Iran with the highest rainfall and relative humidity, high rate of esophageal cancer (EC) and high levels of fumonisins in maize. Aspergillus flavus was the most widely recovered Aspergillus species and 38% of samples were contaminated with this potentially aflatoxigenic fungus. The incidence of A. flavus was highest in Kermanshah, the province with lowest mean minimum temperature. Penicillium species were seen in all the samples and Fars had the highest incidence, with highly significant differences when compared to the other three provinces. Diplodia species were not isolated from any of the samples examined.     

Fibrinolytic activity of some fungi isolated from self-heated composted fertilizer

Mesophilic fungi isolated from organic fertilizer compost samples accounted for 70.94% of the total fungal count, while thermophilic and thermotolerant fungi constituted 29.05% of that count. Eight mesophilic fungal species, namelyAspergillus niger, Monilia sitophila, Paecilomyces divaricata, Penicillium chrysogenum, P. fellutanum, Scopulariopsis brevicaulis, S. brumptii andZygorhynchus japonicus; two thermophilic fungiHumicola grisea andOidiodendron flavum and three thermotolerant speciesAspergillus fumigatus, Thermomyces lanuginosus andZygorhynchus vuilleminii were isolated during the study. Most of the tested fungi showed a proteolytic activity and liquified gelatin in the test tube method and in cup plates. The thermophilic fungusO. flavum was the most potent proteolytic fungus. The comparative fibrinolytic assay revealed the following sequence in the ability of the tested fungi to hydrolyse fibrin:O. fiavum>S. brevicaulis>H. grisea>A. fumigatus>T. lanuginosus.     

Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi

Termites, Coptotermes formosanus Shiraki, reared individually, were highly susceptible to entomopathogenic fungi, Paecilomyces fumosoroseus and Beauveria brongniartii and Metarhizium anisopliae, while termites reared in groups were highly resistant. Quantitative assays with an epifluoresent microscope revealed a significant difference in the number of conidia attachments among three entomopathogenic fungi. The conidia of B. brongniartii and P. fumosoroseus bound to termite cuticles more effectively than M. anisopliae conidia. Our results also suggested that self-grooming behavior is less effective, but mutual grooming is very effective in the removal of conidia from cuticles of their nestmates. Statistical analysis of removal rates indicated that conidia of P. fumosoroseus and B. brongniartii were removed more rapidly than M. anisopliae conidia from termite cuticles.