Development of a droplet digital PCR assay for population analysis of aflatoxigenic and atoxigenic <Emphasis Type="Italic">Aspergillus flavus</Emphasis> mixtures in soil

Aflatoxin B₁ is a potent hepatotoxin and carcinogen that poses a serious safety hazard to both humans and animals. Aspergillus flavus is the most common aflatoxin-producing species on corn, cotton, peanuts, and tree nuts. Application of atoxigenic strains to compete against aflatoxigenic strains of A. flavus has emerged as one of the most practical strategies for ameliorating aflatoxin contamination in food. Genes directly involved in aflatoxin biosynthesis are clustered on an 82-kb region of the genome. Three atoxigenic strains (CA12, M34, and AF123) were each paired with each of four aflatoxigenic strains (CA28, CA42, CA90, and M52), inoculated into soil and incubated at 28 °C for 2 weeks and 1 month. TaqMan probes, omtA-FAM, and norA-HEX were designed for developing a droplet digital PCR (ddPCR) assay to analyze the soil population of mixtures of A. flavus strains. DNA was extracted from each soil sample and used for ddPCR assays. The data indicated that competition between atoxigenic and aflatoxigenic was strain dependent. Variation in competitive ability among different strains of A. flavus influenced the population reduction of the aflatoxigenic strain by the atoxigenic strain. Higher ratios of atoxigenic to aflatoxigenic strains increased soil population of atoxigenic strains. This is the first study to demonstrate the utility of ddPCR to quantify mixtures of both atoxigenic and aflatoxigenic A. flavus strains in soil and allows for rapid and accurate determination of population sizes of atoxigenic and aflatoxigenic strains. This method eliminates the need for isolation and identification of individual fungal isolates from experimental soil samples.     

First report of an atypical new <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> isolates with nucleotide insertion in <Emphasis Type="Italic">aflR</Emphasis> gene resembling to <Emphasis Type="Italic">A. sojae</Emphasis>

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B₁, B₂, G₁, and G₂ and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153–1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.     

Investigation of stored wheat mycoflora,reporting the<Emphasis Type="Italic">Fusarium</Emphasis> cf.<Emphasis Type="Italic">langsethiae</Emphasis> in three provinces of Iran during 2007

Wheat is the most important cereal produced in Iran. A mycological survey was carried out for the first time, on the stored wheat samples in Tehran, East Azarbayejan and Mazandaran provinces in 2007. Exogenous and endogenous fungi, were isolated by the method of flotation with Malachite green agar (MGA 0.25) and Freeze blotter techniques respectively. In this study, 46 species belonging to 23 different genera were isolated.Cladosporium spp. (57.1–89.2%) andAlternaria spp. (82.4–100%) species were the predominant fungal species identified as endogenous mycoflora. The predominant exogenous fungi werePenicillium spp. (78.4–92.8%) andAspergillus spp. (71.4–85.7%) species.Fusarium proliferatum was the most prevalent species ofFusarium isolates.Aspergillus niger (39.4%) andAspergillus flavus (36.7%) were the predominantAspergillus species identified as exogenous mycoflora.Aspergillus flavus (26.6%) was the predominantAspergillus species identified as endogenous mycoflora. Flotation method with MGA 0.25 recommended for isolating of hyaline fungi from wheat cereals. In this study one isolate fromFusarium species was isolated on the basis of morphology and ribosomal internal transcribed spacer classified asFusarium langsethiae but on the basis of partial translation elongation factor-1alpha gene grouped withFusarium sporotrichioides. To our knowledge, this is the first report aboutF. cf.langsethiae in Iran and Asia.     

Isolation and characterization of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> strains in China

Important staple foods (peanuts, maize and rice) are susceptible to contamination by aflatoxin (AF)-producing fungi such as Aspergillus flavus. The objective of this study was to explore non-aflatoxin-producing (atoxigenic) A. flavus strains as biocontrol agents for the control of AFs. In the current study, a total of 724 A. flavus strains were isolated from different regions of China. Polyphasic approaches were utilized for species identification. Non-aflatoxin and non-cyclopiazonic acid (CPA)-producing strains were further screened for aflatoxin B₁ (AFB₁) biosynthesis pathway gene clusters using a PCR assay. Strains lacking an amplicon for the regulatory gene aflR were then analyzed for the presence of the other 28 biosynthetic genes. Only 229 (32%) of the A. flavus strains were found to be atoxigenic. Smaller (S) sclerotial phenotypes were dominant (51%) compared to large (L, 34%) and non-sclerotial (NS, 15%) phenotypes. Among the atoxigenic strains, 24 strains were PCR-negative for the fas-1 and aflJ genes. Sixteen (67%) atoxigenic A. flavus strains were PCRnegative for 10 or more of the biosynthetic genes. Altogether, 18 new PCR product patterns were observed, indicating great diversity in the AFB₁ biosynthesis pathway. The current study demonstrates that many atoxigenic A. flavus strains can be isolated from different regions of China. In the future laboratory as well as field based studies are recommended to test these atoxigenic strains as biocontrol agents for aflatoxin contamination.     

Secondary metabolites of <Emphasis Type="Italic">Penicillium</Emphasis> fungi isolated from permafrost deposits as chemotaxonomic markers

Species identification of slow-growing fungi of the genus Penicillium isolated from ancient permafrost deposits was performed using micro- and macromorphological characteristics as well as the composition of secondary metabolites. The strains producing clavine ergot alkaloids fumigaclavines A and B and festuclavine were assigned to the species P. palitans Westling 1911, whereas the strains forming ochratoxins A and B were identified as P. verrucosum Dierckx 1901.     

Two novel <Emphasis Type="Italic">Aspergillus</Emphasis> species from hypersaline soils of The National Park of Lake Urmia,Iran

Two novel Aspergillus species, one belonging to the section Terrei and the other to section Flavipedes, were isolated from hypersaline soils of The National Park of Lake Urmia (Iran) and are here described as Aspergillus iranicus and Aspergillus urmiensis. A polyphasic taxonomic approach comprising extrolite profiles, phenotypic characters and molecular data (beta-tubulin, calmodulin and ribosomal polymerase II second largest subunit gene sequences) was applied to determine their novel taxonomic status. Aspergillus iranicus (CBS 139561^T) is phylogenetically related to A. carneus, A. niveus, A. allahabadii and A. neoindicus, and it can be differentiated from those species by a unique extrolite pattern (citrinin, gregatins, and a terrequinone) and its conidial colour. Aspergillus urmiensis (CBS 139558^T) shares a most recent common ancestor with A. templicola. The former species produces globose vesicles, and those of A. templicola are predominantly elongate. The Aspergillus urmiensis isolates produce several uncharacterized extrolites. Two other strains obtained during this study reside in a clade, together with the type strain of A. movilensis (CCF 4410^T), and are identified accordingly. Based on the phylogenetic data presented in this study, A. frequens is reduced to synonymy with A. micronesiensis and A. mangaliensis is considered to be a synonym of A. templicola.     

Strain differentiation of airborne opportunistic microorganisms within a municipal landfill area as assessed by PCR MP method

A municipal landfill is the site where occurs differentiation of microorganisms inclusive of several hazardous to human health. The aim of this study was to evaluate by a PCR melting profile (PCR MP) technique the level of genetic intraspecies relatedness of strains, representing several opportunistic bacteria and fungi commonly found in bioaerosol in the landfill site. In total, 27 strains representing four bacterial species (i.e. Escherichia coli, Proteus mirabilis, Staphylococcus sciurii, S. xylosus) and 36 fungal strains belonging to Aspergillus fumigatus and A. flavus were isolated from air samples collected by an Anderson impactor within the landfill area. The PCR melting profile approach clearly indicated that except E. coli, represented by one genotype, other microbial species underwent significant genetic variability in the active sector and surrounding the landfill forest and field areas. Although the genetic relatedness of some strains could testify to distribution of microbes from the active sector to the surroundings in the past, the bacterial and fungal isolates indicated site-specific genetic fingerprints. This is the first report on the distribution of airborne opportunistic microbial species within the landfill area, performing a comparison of their genotypes and evaluation of genetic relatedness between the isolates using the PCR MP method.     

Diversity and enzyme activity of <Emphasis Type="Italic">Penicillium</Emphasis> species associated with macroalgae in Jeju Island

A total of 28 strains of 19 Penicillium species were isolated in a survey of extracellular enzyme-producing fungi from macroalgae along the coast of Jeju Island of Korea. Penicillium species were identified based on morphological and β-tubulin sequence analyses. In addition, the halo-tolerance and enzyme activity of all strains were evaluated. The diversity of Penicillium strains isolated from brown algae was higher than the diversity of strains isolated from green and red algae. The commonly isolated species were Penicillium antarcticum, P. bialowiezense, P. brevicompactum, P. crustosum, P. oxalicum, P. rubens, P. sumatrense, and P. terrigenum. While many strains showed endoglucanase, β-glucosidase, and protease activity, no alginase activity was detected. There was a positive correlation between halo-tolerance and endoglucanase activity within Penicillium species. Among 19 Penicillium species, three species–P. kongii, P. olsonii, and P. viticola–have not been previously recorded in Korea.     

New sterigmatocystin-producing species of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Versicolores</Emphasis> from indoor air in Croatia

Multilocus DNA sequence-based identification methods raised the number of known species assigned to the Aspergillus section Versicolores. Currently, there are 16 species accepted in the section, including A. amoenus, A. austroafricanus, A. creber, A. cvjetkovicii, A. fructus, A. griseoaurantiacus, A. hongkongensis, A. jensenii, A. protuberus, A. puulaauensis, A. subversicolor, A. sydowii, A. tabacinus, A. tennesseensis, A. venenatus, and A. versicolor. Based on morphological identifications, most of these species were identified as either A. sydowii or A. versicolor, with the latter reported to have a world-wide distribution, growing in many habitats. Aspergillus versicolor has been implicated in health hazards including sick building syndrome, human and animal mycoses, and contamination of food and feed were assigned primarily to this species. A. versicolor is still commonly isolated from indoor surveys, even though species such as A. jensenii and A. creber seem more common. From indoor air samples collected at a grain mill in Croatia, we isolated an undescribed species assigned to the Aspergillus section Versicolores. A polyphasic approach, including sequence-based methods, morphological and physiological studies, was used for species characterization and in this paper is described as Aspergillus pepii. Additionally, sterigmatocystin producing abilities have been confirmed. Based on a combined phylogenetic tree, morphological features and sterigmatocystin producing abilities, A. pepii is closely related to A. versicolor. Further studies should explore the frequency of the species in indoor environments and its medical, industrial, and environmental significance.     

Monitoring of ochratoxin A and ochratoxin-producing fungi in traditional salami manufactured in Northern Italy

Fungi have a crucial role in the correct maturation of salami, but special attention should be addressed to the production of the nephrotoxic, immunotoxic, and carcinogenic mycotoxin ochratoxin A (OTA). In a monitoring study conducted in Northern Italy, OTA was detected by liquid chromatography coupled with mass spectrometry in 13 out 133 samples of traditional salami (9.8% of the total count). Mycological analysis of these samples yielded 247 fungal isolates which were identified to species level. The most frequent species were Penicillium nalgiovense, P. solitum, and P. chrysogenum. P. nordicum, an OTA-producing species commonly found in proteinaceous food, was not found in these samples. Three isolates were found to be Aspergillus westerdijkiae, an OTA-producing species. In order to check the results of the microbiological identification, 19 different strains of Aspergillus and 94 of Penicillium were tested for the presence of a sequence common to OTA-producing fungi by real-time PCR. None of the studied isolates, including the three A. westerdijkiae, possessed the otanpsPN target which is common to OTA-producing strains. Two out of three isolates of the A. westerdijkiae were also PCR-negative for the otanpsPN gene and did not produce OTA in culture. Conversely, this target sequence was amplified from the DNA purified from 14 salami casings including three casings harboring A. westerdijkiae. The amplification of sequences specific for OTA-producing strains performed on total genomic DNA extracted directly from salami casings provided a more suitable approach than PCR analysis of isolates from salami for the OTA-related otanpsPN gene to evaluate the risk of OTA contamination.     

Black-headed gulls (<Emphasis Type="Italic">Chroicocephalus ridibundus</Emphasis>) - a natural reservoir of potentially pathogenic microfungi?

Mycological studies of selected populations of black-headed gulls were carried out in response to the increasing interest in wild birds as reservoirs of potentially pathogenic fungi and links in the epidemiological chain of mycoses hazardous to human and animals. The biological material comprised swabs from the beaks and cloacae of adult and young birds subjected to standard mycological diagnostics. 79.5% of samples were positive, comprising 22 fungal species belonging to 10 genera, mainly Candida, Rhodotorula, Aspergillus, Fusarium, Cryptococcus, and Trichosporon. The most frequently isolated species were Candida albicans and Rhodotorula rubra, found in the beaks of females and young birds and in the cloacae of young birds with comparable frequency. Cryptococcus laurentii, Cr. neoformans, Aspergillus niger, A. flavus, and Rh. muscilaginosa were isolated relatively frequently from all birds. The results highlight the ecological importance of wild birds in the circulation of potentially pathogenic fungi in the biosphere.     

Polysaccharide-Degrading Activity in Marine and Terrestrial Strains of Mycelial Fungi

The activity of extracellular polysaccharide-degrading enzymes and glycosidases from mycelial fungi towards various carbohydrates and carbohydrate derivatives from plant and algal cell walls has been screened. Twenty-three strains of mycelial fungi isolated from the marine sediment and dung were grown by submerged cultivation on a plant-based substrate (a by-product of the grain processing industry) for previous screening for their biomass and protein productivity. Molecular identification allowed for the assignment of marine fungal strains to the following species: Sirastachys phyllophila, Ochroconis mirabilis, Pseudallescheria boydii, Pseudallescheria ellipsoidea, Beauveria felina, Scopulariopsis brevicaulis, Cladosporium sp., and Trichoderma sp. The terrestrial strains belonged to the species Thermomyces thermophilus, Thermomyces dupontii, Thermomyces lanuginosus, Fusarium avenaceum, Mycothermus thermophilum, and Thermothelomyces thermophila. Seven strains of thermophilic terrestrial fungal species T. thermophila, T. thermophilus, T. dupontii and M. thermophilus and two marine fungal strains of S. brevicaulis and Beauveria felina exhibited the highest protein yields and a wide range of polysaccharide-degrading activity when the cultures were cultivated at 22–25°C. The cellulolytic thermophilic strain M. thermophilus 55 isolated from dung demonstrated unusual specificity, most intensive increase of mycelial biomass, and high activity towards algal polysaccharides after seven days of cultivation. The specific activity of laminarinase was one order of magnitude higher than in the marine strains and amounted to 1180 U/mg, and the alginate lyase, carrageenase, polymannuronate lyase, agarase, and fucoidanase activity levels (from 208 to 500 U/mg) were also higher than in all marine strains. All active polysaccharide-degrading strains of thermophilic terrestrial and marine fungi identified in the present study are of considerable interest, as the potential of these fungi for polysaccharide degradation can be applied in the transformation of various agricultural and maricultural waste of plant origin and in the modification of carbohydrate-containing substances in structural research and biotechnology.     

Culturable microorganisms from the earthworm digestive tract

The cultured aerobic copiotrophic bacteria and fungi from food-free digestive tracts of Aporrectodea caliginosa, Lumbricus terrestris, and Eisenia fetida earthworms, soil (compost), and fresh earthworm excrements were investigated. The microorganisms were isolated on nutrient media and identified by sequencing the fragments of bacterial 16S rRNA and fungal 28S rRNA (D1/D2 domain) gene sequences with subsequent phylogenetic analysis. Bacteria isolated from the digestive tracts of earthworms belonged to the families Aeromonadaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Moraxellaceae, Pseudomonadaceae, and Sphingobacteriaceae (Bacteroidetes), as well as Actinobacteria. For five strains, namely Ochrobactrum sp. 341-2 (α-Proteobacteria), Massilia sp. 557-1 (β-Proteobacteria), Sphingobacterium sp. 611-2 (Bacteroidetes), Leifsonia sp. 555-1, and a bacterium from the family Microbacteriaceae, isolate 521-1 (Actinobacteria), the similarity to known 16S rRNA sequences was 93–97%; they therefore, probably belong to new species and genera. Bacterial groups isolated from the digestive tracts of earthworms were significantly different from those isolated from soil and excrements. Some bacterial taxa occurred in different sections of A. caliginosa intestine and in intestines of different earthworm species; however, the overall composition of bacterial communities in these objects is different. Existence of bacterial groupings symbiotically associated with intestines is proposed. Among the fungi, Bjerkandera adusta and Syspastospora parasitica were isolated from the cleaned digestive tracts as light-colored, sterile mycelium, as well as Geotrichum candidum, Acremonium murorum (A. murorum var. felina), Alternaria alternata, Aspergillus candidus, A. versicolor, Cladosporium cladosporioides, Rhizomucor racemosus, Mucor hiemalis, Fusarium (F. oxysporum, Fusarium sp.), and Penicillium spp. These fungi survive for a long time in the earthworm’s digestive environment. Investigation of the functional characteristics and role in the host organism is required to confirm the symbiotic status of the microorganisms associated with the earthworm digestive tract.     

Carbohydrate Specificity of Antibodies against Phytopathogenic Fungi of the <Emphasis Type="Italic">Aspergillus</Emphasis> Genus

Brush rabbits were immunized with injections prepared from the fungi Aspergillus fumigatus, Aspergillus niger, and Aspergillus repens. A library of synthetic biotinylated oligosaccharides containing the key fragments of antigenic polysaccharides of the fungal cell wall—galctomannan, α- and β-glucans, mannan, and chitin—was used to analyze carbohydrate specificity. The anticarbohydrate antibodies obtained from animals immunized with preparations from A. fumigatus and A. repens predominantly recognized epitopes containing galactofuranoside residues, while the majority of the antibodies against A. niger bound the chitooligosaccharide ligand. These results are the basis for the identification of specific markers required for the development of immunoenzyme test systems.     

Characterization of the velvet regulators in <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

Fungal development and secondary metabolism are closely associated via the activities of the fungal NK-kB-type velvet regulators that are highly conserved in filamentous fungi. Here, we investigated the roles of the velvet genes in the aflatoxigenic fungus Aspergillus flavus. Distinct from other Aspergillus species, the A. flavus genome contains five velvet genes, veA, velB, velC, velD, and vosA. The deletion of velD blocks the production of aflatoxin B1, but does not affect the formation of sclerotia. Expression analyses revealed that vosA and velB mRNAs accumulated at high levels during the late phase of asexual development and in conidia. The absence of vosA or velB decreased the content of conidial trehalose and the tolerance of conidia to the thermal and UV stresses. In addition, double mutant analyses demonstrated that VosA and VelB play an inter-dependent role in trehalose biosynthesis and conidial stress tolerance. Together with the findings of previous studies, the results of the present study suggest that the velvet regulators play the conserved and vital role in sporogenesis, conidial trehalose biogenesis, stress tolerance, and aflatoxin biosynthesis in A. flavus.     

Phylogenetic analysis of <Emphasis Type="Italic">Plectosphaerella</Emphasis> species based on multi-locus DNA sequences and description of <Emphasis Type="Italic">P. sinensis</Emphasis> sp. nov.

Species in Plectosphaerella are well known as pathogens of several plant species causing fruit, root and collar rot and collapse. In an investigation of endophytic fungi associated with cucurbit plants in China, we isolated 77 strains belonging to the genus Plectosphaerella. To identify the isolated strains, we collected the type or reference strains of all currently accepted species in Plectosphaerella except P. oratosquillae and conducted a phylogenetic analysis. Phylogenetic analysis of the partial 28S rDNA sequences showed that all species in Plectosphaerella were located in one clade of Plectosphaerellaceae. Based on multi-locus phylogenetic analysis of the ITS, CaM, EF1, TUB and morphological characteristics, all species in Plectosphaerella were well separated. Three endophytic strains from stems of Cucurbita moschata, Citrullus lanatus and Cucumis melo from North China were assigned to a new species described as P. sinensis in this paper. The new species differs morphologically from other Plectosphaerella species by irregular chlamydospores, and the dimensions of phialides and conidia. The other endophytic strains from several cucurbit plants were identified as P. cucumerina.     

Alkalitolerant micromycetes in acidic and neutral soils of the temperate zone

A wide diversity of micromycetes from various taxonomic groups in acidic and neutral soils is known from the literature data. In the present work, the fungi isolated from these soils and capable of growth at high pH are analyzed. The fungi were isolated from acidic sod-podzol and neutral cultivated soils by plating on alkaline agar (pH 10.0–10.5). Their identification was carried out using morphological, cultural, and molecular genetic criteria. Phylogenetic analysis was performed and the rates of linear growth within a broad pH range (4.0–10.4) were determined. The isolates represented a polyphyletic group of ascomycetes (Sordariomycetes), which included members of Plectosphaerellaceae (5 species) and various families of Hypocreales (4 species). The most common species were Gibellulopsis nigrescens, Acrostalagmus luteoalbus, Chordomyces antarcticum, and Plectosphaerella spp. Investigation of fungal growth at different pH values revealed all isolates to be alkalitolerant, with no alkaliphilic fungi isolated from acidic sod-podzol and neutral cultivated soils. Although the group of isolates was polyphyletic and its members originated from different ecological and trophic niches, most alkalitolerant isolates exhibited common morphological traits with acremonium- and verticillium-like conidial spore formation, abundant slime formation, and a tendency for aggregation of their mycelium in bundles. Our research confirmed the presence of fungi with alkalitolerant adaptation to external pH in the sod-podzolic and cultivated soils of the Moscow region.     

Survey of <Emphasis Type="Italic">Aspergillus</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> species and their mycotoxins in raw materials and poultry feeds from Córdoba,Argentina

The aims of the present work were: (1) to determine both mycobiota in raw materials and finisher poultry feed, as well as the ability to produce aflatoxin B₁ by A. flavus strains, and (2) to evaluate the natural co-occurrence of aflatoxins (AFs), fumonisins (FBs), gliotoxin, diacetoxyscirpenol (DAS), HT-2 toxin, and T-2 toxin in poultry feed by LC-MS/MS. Nineteen percent of raw materials and 79% of finisher poultry feed samples exceeded the maximum allowed total fungal count (1?×?10⁴ CFU g^?1) to ensure hygienic quality. Aspergillus flavus was the only species belonging to section Flavi which was isolated while Fusarium verticilliodes was the prevalent species. Forty-seven percent of A. flavus strains were aflatoxin B₁ producers and the highest frequency of aflatoxigenic strains was isolated from finisher poultry feeds. Principal component analysis showed that corn grains are closely related with total fungal and Fusarium counts. This positive relationship suggests that total fungal and Fusarium spp. counts in poultry feed might come mainly from corn grains. Regarding poultry feeds, in ground finisher type, Aspergillus spp. counts increased as water activity (a_w) diminished. A positive relationship among a_w, total fungal and Fusarium spp. counts was observed in both ground finisher and ground starter feed. Several mycotoxins were monitored in feeds by applying the LC MS/MS technique. One hundred percent of poultry samples were contaminated with FB₁, and the highest levels were detected in pelleted finisher poultry. AFB₁, gliotoxin, DAS, HT-2 toxin, and T-2 toxin were not detected in any poultry feed. The scarcity of available mycotoxicological studies from Argentinean poultry feed using a multitoxin analysis technique enhances the contribution of the findings of this report.     

Endophytism and bioactivity of endophytic fungi isolated from <Emphasis Type="Italic">Combretum lanceolatum</Emphasis> Pohl ex Eichler

We report the isolation and identification of endophytic fungi from Combretum lanceolatum Pohl ex Eichler. Further, we evaluated the relationships of fungi with the host plant and tested bioactivities of isolates. The fungi were isolated from disinfected root fragments and plated onto potato dextrose agar. Root pieces were also used to quantify fungal structures associated with the roots. Identification of fungi was carried out by characterization of morphological features and sequencing of the ITS region. Endophytism was confirmed by inoculation of endophyte-free seedlings followed by microscopic examination. The extract was obtained by maceration of the mycelium in ethyl acetate for antioxidant and antimicrobial evaluations. A total of 112 strains belonging to nine different species were isolated, the major classes were Dothideomycetes and Sordariomycetes. C. lanceolatum is colonized by dark septate endophytes (DSE), evidenced by the presence of microsclerotia and melanized hyphae. There is also co-colonization with mycorrhizal fungi in the same root fragments. Seedling inoculation experiments revealed that C. perangustum-95C and M. phaseolina-46C showed association with the seedlings of C. lanceolatum and differentiated microsclerotia and dark septate hyphae, indicating that these species are DSE. In the antimicrobial test, the D. phaseolorum-92C extract had the highest zones of inhibition against S. aureus and E. coli, respectively. The results showed that 100 % of the extracts have antioxidant activity ranging from low to moderate. All endophyte species had antioxidant and antimicrobial activities that were directly proportional to the dose-responses. Future research will involve chemical characterization and structural elucidation of bioactive compounds.