Antimicrobial Activity and Biodiversity of Endophytic Fungi in <Emphasis Type="Italic">Dendrobium</Emphasis><Emphasis Type="Italic">devonianum</Emphasis> and <Emphasis Type="Italic">Dendrobium thyrsiflorum</Emphasis> from Vietman

Endophytic fungi are rich in orchids and have great impacts on their host plants. 53 endophytes (30 isolates from Dendrobium devonianum and 23 endophytic fungi from D. thyrsiflorum) were isolated, respectively, from roots and stems of Dendrobium species. All the fungi were identified by way of morphological and/or molecular biological methods. 30 endophytic fungi in D. devonianum were categorized into 11 taxa and 23 fungal endophytes in D. thyrsiflorum were grouped into 11 genera, respectively. Fusarium was the dominant species of the two Dendrobium species in common. Antimicrobial activity of ethanol extract of fermentation broth of these fungi was explored using agar diffusion test. 10 endophytic fungi in D. devonianum and 11 in D. thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among 6 pathogenic microbes (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus). Out of the fungal endophytes isolated from D. devonianum and D. thyrsiflorum, Phoma displayed strong inhibitory activity (inhibition zones in diameter >20 mm) against pathogens. Epicoccum nigrum from D. thyrsiflorum exhibited antibacterial activity even stronger than ampicillin sodium. Fusarium isolated from the two Dendrobium species was effective against the pathogenic bacterial as well as fungal pathogens. The study reinforced the assumption that endophytic fungi isolated from different Dendrobium species could be of potential antibacterial or antifungal resource.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Stimulation of saponin production in <Emphasis Type="Italic">Panax ginseng</Emphasis> hairy roots by two oligosaccharides from <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5–30 mg l⁻¹. HS and OS at 30 mg l⁻¹, fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to ∼20 g l⁻¹ from 13 g l⁻¹ and the total saponin content of roots by more than 1-fold to ∼3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.     

Clavatol and patulin formation as the antagonistic principle of <Emphasis Type="Italic">Aspergillus clavatonanicus</Emphasis>, an endophytic fungus of <Emphasis Type="Italic">Taxus mairei</Emphasis>

Many endophytic fungi are known to protect plants from plant pathogens, but the antagonistic mechanism has rarely been revealed. In this study, we wished to learn whether an endophytic Aspergillus sp., isolated from Taxus mairei, would indeed produce bioactive components, and if so whether (a) they would antagonize plant pathogenic fungi; and (b) whether this Aspergillus sp. would produce the compound also under conditions of confrontation with these fungi. The endophytic fungal strain from T. mairei was identified as Aspergillus clavatonanicus by analysis of morphological characteristics and the sequence of the internal transcribed spacers (ITS rDNA) of rDNA. When grown in surface culture, the fungus produced clavatol (2′,4′-dihydroxy-3′,5′-dimethylacetophenone) and patulin (2-hydroxy-3,7-dioxabicyclo [4.3.0]nona-5,9-dien-8-one), as shown by shown by NMR, MS, X-ray, and EI-MS analysis. Both exhibited inhibitory activity in vitro against several plant pathogenic fungi, i.e., Botrytis cinerea, Didymella bryoniae, Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, and Pythium ultimum. During confrontation with P. ultimum, A. clavatonanicus antagonized its growth of P. ultimum, and both clavatol as well as patulin were formed as the only bioactive components, albeit with different kinetics. We conclude that A. clavatonanicus produces clavatol and patulin, and that these two polyketides may be involved in the protection of T. mairei against attack by plant pathogens by this Aspergillus sp.     

Isolation and characterization of endophytic taxol-producing fungi from <Emphasis Type="Italic">Taxus chinensis</Emphasis>

This study investigated the endophytic fungi diversity of Taxus chinensis and screened the taxol-producing fungi in the host. A total of 115 endophytic fungi isolates obtained from bark segments of T. chinensis were grouped into 23 genera based on the morphological traits and sequence analysis of the internal transcribed spacers (ITS1-5.8S-ITS2), indicating endophytic fungi in T. chinensis are diverse and abundant. Diaporthe, Phomopsis (anamorph of Diaporthe), Acremonium, and Pezicula were the dominant genera, whereas the remaining genera were infrequent groups. The 13 representative species of the distinct genera were capable of producing taxol verified by reverse-phase high performance liquid chromatography (HPLC). Among the taxol-producing fungi, the yield of taxol produced by the Metarhizium anisopliae, H-27 was 846.1 μg l⁻¹ in reformative potato dextrose liquid medium, and the fungal taxol was further validated by mass spectrometry (MS). The taxol-producing fungi (92.3%) were infrequent communities, suggesting that infrequent fungi associated with T. chinensis might be a fascinating reservoir of taxol-generating fungi.     

Study of endophytic fungal community from different parts of <Emphasis Type="Italic">Aegle marmelos</Emphasis> Correae (Rutaceae) from Varanasi (India)

Endophytic fungi were isolated from healthy, living, and symptomless tissues of inner bark, leaf, and roots of Aegle marmelos, a well-known medicinal plant, growing in different parts of India including Varanasi. A total of 79 isolates of endophytic fungi were isolated, representing 21 genera, adopting a standard isolation protocol. Members of the deuteromycotina were more prevalent than ascomycotina and others. The result was quite encouraging in terms of maximum isolates recovery from hyphomycetes (78.5%) followed by ascomycetes (8.9%) and coelomycetes (7.6%) respectively, which corroborates previous studies in same area. However, 5.1% isolates remained unidentified and were classified under Mycelia Sterilia. No isolate was obtained from either basidiomycotina or from zygomycotina. Fusarium spp. had maximum colonization frequency (8.00%) in this plant. The other dominant endophytic genera were Aspergillus spp., Alternaria sp., Drechslera sp., Rhizoctonia sp., Curvularia sp., Nigrospora sp., and Stenella sp. Only two ascomycetous members Chaetomium globosum and Emericella sp. (perfect state of Aspergillus sp.) were obtained from the bark sample. These results indicated that distribution of endophytic fungi within the A. marmelos is not even. Bark harbors more endophytic fungi than leaf and root.     

Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic,medicinal orchid (<Emphasis Type="Italic">Dendrobium nobile</Emphasis>): estimation and characterization

Although the terrestrial and temperate orchids–fungal biology have been largely explored, knowledge of tropical epiphytic orchids–fungus relationships, especially on the ecological roles imparted by non-mycorrhizal fungal endophytes, is less known. Exploitation of the endophytic fungal mycobiota residing in epiphytic orchid plants may be of great importance to further elucidate the fungal ecology in this special habitat as well as developing new approaches for orchid conversations. The composition of fungal endophytes associated with leaves, stems and roots of an epiphytic orchid (Dendrobium nobile), a famous Chinese traditional medicinal plant, was investigated. Microscopic imaging, culture-dependant method and molecular phylogeny were used to estimate their entity and diversity. Totally, there were 172 isolates, at least 14 fungal genera and 33 different morphospecies recovered from 288 samples. Ascomycetes, coelomycetes and hyphomycetes were three major fungal groups. There were higher overall colonization and isolation rates of endophytic fungi from leaves than from other tissues. Guignardia mangiferae was the dominant fungal species within leaves; while the endophytic Xylariaceae were frequently observed in all plant tissues; Colletotrichum, Phomopsis and Fusarium were also frequently observed. Phylogenetic analysis based on ITS gene revealed the high diversity of Xylariacea fungi and relatively diverse of non-Xylariacea fungi. Some potentially promising beneficial fungi such as Clonostachys rosea and Trichoderma chlorosporum were found in roots. This is the first report concerning above-ground and below-ground endophytic fungi community of an epiphytic medicinal orchid, suggesting the ubiquitous distribution of non-mycorrhizal fungal endophytes in orchid plants together with heterogeneity and tissue specificity of the endophyte assemblage. Possible physiological functions played by these fungal endophytes and their potential applications are also discussed briefly. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antimicrobial potentials of endophytic fungi residing in <Emphasis Type="Italic">Quercus variabilis</Emphasis> and brefeldin A obtained from <Emphasis Type="Italic">Cladosporium</Emphasis> sp.

Among 67 endophytic fungi isolated from Quercus variabilis, 53.7% of endophytic fungal fermentation broths displayed growth inhibition on at least one test microorganism, such as pathogenic fungi (Trichophyton rubrum, Candida albicans, Aspergillus niger, Epidermophyton floccosum, Microsporum canis) and bacteria (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens). Moreover, 19.4% of strains showed a broader antimicrobial spectrum, such as Aspergillus sp., Penicillium sp., Alternaria sp., 20.9% of strains showed strong inhibition (+++) to pathogenic bacteria, while only 7.5% displayed that to test fungi. The most active antifungal strain I(R)9-2, Cladosporium sp. was selected and fermented. From the broth, a secondary metabolite, brefeldin A was obtained. This is the first report on the antimicrobial potentials of endophytic fungi residing in Q. variabilis and isolation of brefeldin A produced by Cladosporium sp.     

The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Extensive host range of an endophytic fungus, <Emphasis Type="Italic">Guignardia endophyllicola</Emphasis> (anamorph: <Emphasis Type="Italic">Phyllosticta capitalensis</Emphasis>)

Isolation of endophytic species of Guignardia (anamorph: Phyllosticta) from healthy leaves of 94 plants (91 species and 3 varieties) in 69 genera, 42 families, was carried out in a test site (Kyoto Herbal Garden) to investigate the host range of Guignardia endophyllicola (anamorph: Phyllosticta capitalensis). Species of Guignardia and Phyllosticta were isolated from the leaves of 67 plants (66 species and 1 variety) belonging to 54 genera, 38 families. Among them, 53 isolates from different plants belonging to 43 genera in 36 families were similar in morphology, and sequence analysis of internal transcribed spacer (ITS) regions of ribosome DNA revealed these isolates to be conspecific with G. endophyllicola. In addition, this fungus was isolated from leaves of various plants collected in different places in Japan and Thailand. Thus, this endophytic fungus has been revealed to live within various vascular plants, angiosperms, gymnosperms, and pteridophytes.     

Revision of <Emphasis Type="Italic">Heterosavia</Emphasis>, stat. nov., with notes on <Emphasis Type="Italic">Gonatogyne</Emphasis> and <Emphasis Type="Italic">Savia</Emphasis> (Phyllanthaceae)

Heterosavia (Phyllanthaceae) is segregated from Savia (tribe Bridelieae), recognized at generic rank, and placed in tribe Phyllantheae. Floral, fruit, leaf anatomical, leaf venation, and pollen characters of the neotropical taxa previously united as Savia including Gonatogyne are discussed and illustrated. Keys to the three genera and to the species of Heterosavia are presented. Four species (all new combinations), Heterosavia bahamensis, H. erythroxyloides, H. laurifolia, and H. maculata, are recognized. The new combinations Heterosavia laurifolia var. intermedia and H. maculata var. clementis are proposed. The names Heterosavia, H. erythroxyloides, H. laurifolia, Savia clementis, S. clusiifolia, S. clusiifolia var. fallax, and S. longipes are lectotypified. Distribution maps and conservation assessments (IUCN ratings) of Heterosavia species and varieties are provided.     

<Emphasis Type="Italic">Fusarium</Emphasis> species of the <Emphasis Type="Italic">Gibberella fujikuroi</Emphasis> complex and fumonisin contamination of pearl millet and corn in Georgia,USA

This study was designed to identify and compare the Fusarium species of the Gibberella fujikuroi complex on pearl millet (Pennisetum glaucum (L.) R. Br) and corn (Zea mays L.) crops grown in southern Georgia, and to determine their influence on potential fumonisin production. Pearl millet and corn samples were collected in Georgia in 1996, 1997 and 1998. Three percent of the pearl millet seeds had fungi similar to the Fusarium species of the G. fujikuroi species complex. One hundred and nineteen representative isolates visually similar to the G. fujikuroi species complex from pearl millet were paired with mating population A (Fusarium verticillioides (Sacc.) Nirenberg), mating population D (F. proliferatum (Matsushima) Nirenberg) and mating population F (F. thapsinum (Klittich, Leslie, Nelson and Marasas) tester strains. Successful crosses were obtained with 50.4%, 10.1% and 0.0% of these isolates with the A, D and F tester strains, while 39.5 of the isolates did not form perithecia with any tester strains. Two of the typical infertile isolates were characterized by DNA sequence comparisons and were identified as Fusarium pseudonygamai (Nirenberg and ODonnell), which is the first known isolation of this species in the United States. Based on the pattern of cross-compatibility, conidiogenesis, colony characteristics and media pigmentation, a majority of the infertile isolates belong to this species. Fumonisins FB₁ and FB₂ were not detected in any of the 81 pearl millet samples analyzed. The species of the G. fujikuroi species complex were dominant in corn and were isolated from 84%, 74% and 65% of the seed in 1996, 1997 and 1998, respectively. Representative species of the G. fujikuroi species complex were isolated from 1996 to 1998 Georgia corn survey (162, 104 and 111 isolates, respectively) and tested for mating compatibility. The incidence of isolates belonging to mating population A (F. verticillioides) ranged from 70.2% to 89.5%. Corn survey samples were assayed for fumonisins, and 63% to 91% of the 1996, 1997 and 1998 samples were contaminated. The total amount of fumonisins in the corn samples ranged from 0.6 to 33.3 g/g.     

Isolation and characterization of <Emphasis Type="Italic">Azotobacter</Emphasis> and <Emphasis Type="Italic">Azospirillum</Emphasis> strains from the sugarcane rhizosphere

Bacteria with the ability to grow on nitrogen-free media and with nitrogenase activity under aerobic or microaerobic conditions were isolated from sugarcane roots collected from four different agricultural locations in Granada (Spain). Isolates were Gram negative rods and were identified as Azotobacter chroococcum and Azospirillum brasilense. Our results suggest that Azotobacter isolates do not have a particular affinity for sugarcane rhizospheres and that, on the contrary, Azospirillum isolates show specific association and perhaps endophytic colonization of sugarcane. However, obligate endophytes (Gluconacetobacter diazotrophicus) were not found in the apoplastic fluid of the stems and macerates extracts of sugarcane tissues with the procedure applied. Population of this microorganism might be in low number in the Spanish sugarcane varieties studied which is also discussed.     

Mycorrhizal synthesis between <Emphasis Type="Italic">Boletus edulis</Emphasis> species complex and rockroses (<Emphasis Type="Italic">Cistus</Emphasis> sp.)

Ectomycorrhizas of Boletus aereus, Boletus edulis, and Boletus reticulatus were synthesized with Cistus sp. under laboratory conditions using synthesis tubes filled with a mixture of sterilized peat-vermiculite and nutrient solution. The fungal strains isolated from sporocarps were identified by molecular techniques. The inoculated seedlings were grown for 4–5 months. The ectomycorrhizas formed were described based on standard morphological and anatomical characters. The three ectomycorrhizas described were very similar, with white monopodial-pinnate morphology, a three-layered plectenchymatous mantle on plan view and boletoid rhizomorphs.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

<Emphasis Type="Italic">Erysiphe monascogera</Emphasis> sp. nov., an unusual powdery mildew fungus found on fruits of <Emphasis Type="Italic">Styrax japonica</Emphasis>

Morphological observations using light and scanning electron microscopes and molecular phylogenetic analysis revealed that the fungus growing on the surface of fruits or sepals of Styrax japonica collected at Nagano, Japan, is a new powdery mildew with an unusual morphology, described here as Erysiphe monascogera. This fungus has mainly a single ascus in a chasmothecium, but molecular phylogenetic analysis and the shape of the hyphal appressoria suggest that it is an Erysiphe species. Erysiphe monascogera is a sister-species to E. nomurae on Symplocos chinensis var. leucocarpa f. pilosa, although there are obvious morphological differences between the two species. This inconsistency between molecular phylogeny and morphology may be explained by the unique habitat of E. monascogera. Erysiphe monascogera and E. nomurae are included in a clade composed of the E. alphitoides complex, which suggests that these two species diverged by host jumping of the E. alphitoides complex, having oaks as major host plants.     

Cloning and characterization of the ribosomal protein L3 (<Emphasis Type="Italic">RPL3</Emphasis>) gene family from <Emphasis Type="Italic">Triticum aestivum</Emphasis>

Plant pathogenic fungi of the genus Fusarium can cause severe diseases on small grain cereals and maize. The contamination of harvested grain with Fusarium mycotoxins is a threat to human and animal health. In wheat production of the toxin deoxynivalenol (DON), which inhibits eukaryotic protein biosynthesis, is a virulence factor of Fusarium, and resistance against DON is considered to be part of Fusarium resistance. Previously, single amino acid changes in RPL3 (ribosomal protein L3) conferring DON resistance have been described in yeast. The goal of this work was to characterize the RPL3 gene family from wheat and to investigate the potential role of naturally existing RPL3 alleles in DON resistance by comparing Fusarium-resistant and susceptible cultivars. The gene family consists of three homoeologous alleles of both RPL3A and RPL3B, which are located on chromosomes 4A (RPL3-B2), 4B (RPL3-B1), 4D (RPL3-B3), 5A (RPL3-A3), 5B (RPL3-A2) and 5D (RPL3-A1). Alternative splicing was detected in the TaRPL3-A2 gene. Sequence comparison revealed no amino acid differences between cultivars differing in Fusarium resistance. While using developed SNP markers we nevertheless found that one of the genes, namely, TaRPL3-A3 mapped close to a Fusarium resistance QTL (Qfhs.ifa-5A). The potential role of the RPL3 gene family in DON resistance of wheat is discussed.     

Isolation and characterization of melanin pigment from <Emphasis Type="Italic">Pleurotus cystidiosus</Emphasis> (telomorph of <Emphasis Type="Italic">Antromycopsis</Emphasis><Emphasis Type="Italic">macrocarpa</Emphasis>)

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of bacteria and fungi. For more than 40 years, fungi have been known to produce pigments called melanins. Melanin pigment production by mushrooms was not intensively studied. The present study was carried out on isolation and characterization of melanin from an edible mushroom Pleurotus cystidiosus var. formosensis. The mushroom produced dark mucous mass of hyaline arthrospores on mycelium. The coremia exclusively produced dikaryotic arthrospores with the remnant of a clamp connection. Continuous cell extension and division in the coremium stipe supplied cells for arthroconidiation at the coremium apex, which is surrounded by a liquid droplet (coremioliquid). The black coloured coremea (conidia) were produced by Antromycopsis macrocarpa (anamorph of P. cystidiosus) when cultured on potato dextrose agar medium. The agar plate was incubated at continuous light illumination for high amount of pigment (coremea) production. The slimy layer of the coremea was extracted and partially purified by alkaline and acid treatment. The black pigment was confirmed as melanin based on UV, IR and EPR spectra apart from chemical analysis. This is the first report on characterization of melanin obtained from Pleurotus cystidiosus var. formosensis.     

Molecular analysis of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> isolated from Brazil nuts

Brazil nuts are an important export market in its main producing countries, including Brazil, Bolivia, and Peru. Approximately 30,000 tons of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs with subsequent production of aflatoxins. In our study, Aspergillus section Flavi were isolated from Brazil nuts (Bertholletia excelsa), and identified by morphological and molecular means. We obtained 241 isolates from nut samples, 41% positive for aflatoxin production. Eighty-one isolates were selected for molecular investigation. Pairwise genetic distances among isolates and phylogenetic relationships were assessed. The following Aspergillus species were identified: A. flavus, A. caelatus, A. nomius, A. tamarii, A. bombycis, and A. arachidicola. Additionally, molecular profiles indicated a high level of nucleotide variation within β-tubulin and calmodulin gene sequences associated with high genetic divergence from RAPD data. Among the 81 isolates analyzed by molecular means, three of them were phylogenetically distinct from all other isolates representing the six species of section Flavi. A putative novel species was identified based on molecular profiles.