Fusarium mirum sp. nov, intertwining Fusarium madaense and Fusarium andiyazi, pathogens of tropical grasses.

Many species in the Fusarium fujikuroi Species Complex (FFSC) have an affinity for grass species, with whom they live in an endophytic association or cause disease. We recovered isolates of Fusarium from agriculturally important grasses in Africa and Brazil, and characterized them with morphological markers, mating type, and Amplified Fragment Length Polymorphisms (AFLPs). We also conducted multi-locus phylogenetic analyses based on partial DNA sequences of translation elongation factor-1α (TEF1), ß-tubulin (TUB), and the second largest subunit of RNA polymerase (RPB2) gene regions. Sexual cross fertility was used to test the biological species concept and the sexual stage of F. madaense is described. A novel species within the FFSC, Fusarium mirum, that is different from the other known species in the complex, was formally described. Fusarium mirum, F. madaense, and Fusarium andiyazi are a tightly intertwined species trio that are morphologically identical, but phylogenetically distinguishable, and amongst whom interspecific genetic exchange may still occur. These three species are so close that they cannot be reliably distinguished if only sequences of the TEF1 gene are used. In pathogenicity tests, all tested isolates of F. madaense from sugarcane, sorghum, maize, millet and Brachiaria could induce stalk rot in sorghum, maize and millet, and pokkah boeng in sugarcane. This study increases our understanding of the diversity of species within the FFSC that cause disease in tropical grasses or act as endophytes, and their geographic distributions. The genetically close relationship between F. mirum, F. madaense, and F. andiyazi provides an opportunity to study and identify factors underlying their limited inter-specific cross-fertility and sympatric speciation.

Fusarium fujikuroi species complex in Brazilian rice: Unveiling increased phylogenetic diversity and toxigenic potential.

Fusarium fujikuroi species complex (FFSC) species are commonly encountered infecting rice, but knowledge of the diversity and toxigenic potential of the species is lacking in Brazil, the largest rice-producing country outside Asia. One hundred FFSC isolates obtained from national rice were identified using morphology and phylogeny of TEF, CAL and TUB genes. Eight previously known and one novel Fusarium species were identified. Three species accounted for around 60% of the strains: F. fujikuroi (n = 23), F. proliferatum (n = 22) and F. verticillioides (n = 16). The less frequent species were F. volatile (n = 8), F. anthophilum (n = 6), F. pseudocircinatum (n = 4), F. sterilihyphosum (n = 2) and F. begoniae (n = 1). The novel Fusarium species was represented by 18 isolates. All species produced at least one of the analyzed mycotoxins [beauvericin (BEA), fumonisins (FBs), moniliformin (MON) and enniatins (ENNs)]. BEA was produced by all species but F. verticillioides. The FBs (mainly FB1) were produced mostly by F. fujikuroi, F. proliferatum and F. verticillioides. F. begoniae and F. verticillioides did not produce ENNs and F. sterilihyphosum and F. begoniae did not produce MON, while the other species produced MON and ENNs. Our results add new knowledge of the diversity, geographical distribution and host range of FFSC species.

Fusarium incarnatum-equiseti species complex associated with Brazilian rice: Phylogeny, morphology and toxigenic potential.

Fusarium incarnatum-equiseti species complex (FIESC) is commonly detected in Brazilian rice, but knowledge of the species limits and their toxigenic potential is lacking. Seventy strains morphologically identified as FIESC-like, isolated from the major rice-growing regions of Brazil, were subjected to sequencing of EF-1α gene. Among them, 18 strains were selected and analyzed for their RPB2 gene sequences. Nine phylogenetic species were identified, among which eight matched the previously reported FIESC 4 (F. lacertarum), 6, 16, 17 (F. pernambucanum), 20 (F. caatingaense), 24, 26 and 29. One new phylogenetic species was identified, and named FIESC 38. Five strains formed new singleton lineages. The most dominant species were FIESC 26 (22/70 strains) and FIESC 38 (21/70), the newly identified species. The incarnatum morphotype was dominant (10 phylogenetic species) over the equiseti (4 species). Among 46 strains selected to represent all species, only 16 strains produced detectable levels of mycotoxins in vitro. FIESC 26 produced ZEA and FIESC 38 produced both ZEA and DON. ZEA was produced by nine isolates of three other species, among which few isolates produced trichothecenes: DON (5/46), NIV (3/46), 4-ANIV (2/46), 15-ADON (1/46) and 3-ADON (1/46). The T-2 and HT-2 mycotoxins were not detected. Our results contribute novel information on species limits and mycotoxin production within cereal-infecting FIESC in the southern hemisphere and provide baseline data for further exploring morphological differences among the species.

Differential metabolism of diastereoisomeric diterpenes by Preussia minima, found as endophytic fungus in Cupressus lusitanica.

The plant diastereoisomeric diterpenes ent-pimara-8(14)-15-dien-19-oic acid, obtained from Viguiera arenaria, and isopimara-8(14)-15-dien-18-oic acid, isolated from Cupressus lusitanica, were distinctly functionalized by the enzymes produced in whole cell cultures of the fungus Preussia minima, isolated from surface sterilized stems of C. lusitanica. The ent-pimaradienoic acid was transformed into the known 7ß-hydroxy-ent-pimara-8(14)-15-dien-19-oic acid, and into the novel diterpenes 7-oxo-8 ß-hydroxy-ent-pimara-8(14)-15-dien-19-oic and 7-oxo-9ß-hydroxy-ent-pimara-8(14)-15-dien-19-oic acids. Isopimara-8(14)-15-dien-18-oic acid was converted into novel diterpenes 11α-hydroxyisopimara-8(14)-15-dien-18-oic acid, 7ß,11α-dihydroxyisopimara-8(14)-15-dien-18-oic acid, and 1ß,11α-dihydroxyisopimara-8(14)-15-dien-18-oic acid, along with the known 7ß-hydroxyisopimara-8(14)-15-dien-18-oic acid. All compounds were isolated and fully characterized by 1D and 2D NMR, especially 13C NMR. The diterpene bioproduct 7-oxo-9ß-hydroxy-ent-pimara-8(14)-15-dien-19-oic acid is an isomer of sphaeropsidin C, a phytotoxin that affects cypress trees produced by Shaeropsis sapinea, one of the main phytopathogen of Cupressus. The differential metabolism of the diterpene isomers used as substrates for biotransformation was interpreted with the help of computational molecular docking calculations, considering as target enzymes those of cytochrome P450 group.

Lactone Derivatives Produced by a Phaeoacremonium sp., an Endophytic Fungus from Senna spectabilis.

Three new isoaigialones, A, B, and C (1-3), along with aigialone (4), were isolated from the crude EtOAc extract of a Phaeoacremonium sp., an endophytic fungus obtained from the leaves of Senna spectabilis. The structures of these compounds were elucidated based on the analysis of spectroscopic data. Compounds 2 and 4 were active against the phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum. This is the first report of metabolites produced by an Phaeoacremonium sp., associated with S. spectabilis.

New cytotoxic furan from the marine sediment-derived fungi Aspergillus niger.

A fungal strain of Aspergillus niger was recovered from sediments collected in the Northeast coast of Brazil (Pecém's offshore port terminal). Cultivation in different growth media yielded a new ester furan derivative, 1, along with malformin A1, malformin C, cyclo (trans-4-hydroxy-L-Pro-L-Leu), cyclo (trans-4-hydroxy-L-Pro-L-Phe), cyclo (L-Pro-L-Leu), cyclo (L-Pro-L-Phe), pseurotin D, pseurotin A, chlovalicin, cyclo (L-Pro-L-Tyr) and cyclo (L-Pro-L-Val). Compound 1 was cytotoxic against HCT-116 cell line, showing IC50 = 2.9 µg/mL (CI 95% from 1.8 to 4.7 µg/mL).

Fusarium paranaense sp. nov., a member of the Fusarium solani species complex causes root rot on soybean in Brazil.

Isolates of Fusarium obtained from soybean plants showing symptoms of root rot collected in subtropical southern and tropical central Brazil were characterized based on phylogenetic analyses, sexual crossing, morphology, and pathogenicity tests. A novel species within the Fusarium solani species complex (FSSC) causing soybean root rot is formally described herein as Fusarium paranaense. This species can be distinguished from the other soybean root rot pathogens in the FSSC, which are commonly associated with soybean sudden death syndrome (SDS) based on analyses of the combined DNA sequences of translation elongation factor 1-α and the second largest subunit of RNA polymerase II and on interspecies mating compatibility. Bayesian and maximum parsimony phylogenetic analyses showed that isolates of F. paranaense formed a distinct group in clade 3 of the FSSC in contrast to the pathogens currently known to cause SDS, which are in clade 2. Female fertile tester strains were developed that can be used for the identification of this new species in the FSSC based on sexual crosses. All isolates were heterothallic and belonged to a distinct mating population. Fusarium tucumaniae, a known SDS pathogen, was found in the subtropical southern region of the country.

Bioprospection of cytotoxic compounds in fungal strains recovered from sediments of the Brazilian coast.

The cytotoxic activities of extracts (50 µg/ml) from 48 fungal strains, recovered from sediments of Pecém's offshore port terminal (Northeast coast of Brazil), against HCT-116 colon cancer cell lines were investigated. The most promising extract was obtained from strain BRF082, identified as Dichotomomyces cejpii by phylogenetic analyses of partial RPB2 gene sequence. Thus, it was selected for bioassay-guided isolation of the cytotoxic compounds. Large-scale fermentation of BRF082 in potato dextrose broth, followed by chromatographic purification of the bioactive fractions from the liquid medium, yielded gliotoxin (4) and its derivatives acetylgliotoxin G (3), bis(dethio)bis(methylsulfanyl)gliotoxin (1), acetylgliotoxin (5), 6-acetylbis(dethio)bis(methylsulfanyl)gliotoxin (2), besides the quinazolinone alkaloid fiscalin B. All isolated compounds were tested for their cytotoxicities against the tumor cell lines HCT-116, revealing 4 and 3 as the most cytotoxic ones (IC50 0.41 and 1.06 µg/ml, resp.).

Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast.

A fungal strain of Aspergillus sp. (BRF 030) was isolated from the sediments collected in the northeast coast of Brazil, and the cytotoxic activity of its secondary metabolites was investigated against HCT-116 tumour cell line. The cytotoxicity-guided fractionation of the extracts from this fungus cultured in potato-dextrose-sea water for 14 days at room temperature yielded the hetero-spirocyclic γ-lactams pseurotin A (1), pseurotin D (2) and pseurotin FD-838 (7), the alkaloids fumitremorgin C (5), 12,13-dihydroxy fumitremorgin C (6), methylsulochrin (4) and bis(dethio)bis(methylthio)gliotoxin (3). Among them, fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6) were the most active. The cytotoxic activities of the extracts from Aspergillus sp. grown from 7 to 28 days were investigated, and they were associated with the kinetic production of the compounds. The most active extracts (14 and 21 days) were those with the highest relative concentrations of the compounds fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6).

Diversity of Clonostachys species assessed by molecular phylogenetics and MALDI-TOF mass spectrometry.

We assessed the species diversity among 45 strains of Clonostachys from different substrates and localities in Brazil using molecular phylogenetics, and compared the results with the phenotypic classification of strains obtained from matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Phylogenetic analyses were based on beta tubulin (Tub), ITS-LSU rDNA, and a combined Tub-ITS DNA dataset. MALDI-TOF MS analyses were performed using intact conidia and conidiophores of strains cultivated on oatmeal agar and 4% malt extract agar. Six known species were identified: Clonostachys byssicola, Clonostachys candelabrum, Clonostachys pseudochroleuca, Clonostachys rhizophaga, Clonostachys rogersoniana, and Clonostachys rosea. Two clades and two singleton lineages did not correspond to known species represented in the reference DNA dataset and were identified as Clonostachys sp. 1-4. Multivariate cluster analyses of MALDI-TOF MS data classified the strains into eight clusters and three singletons, corresponding to the ten identified species plus one additional cluster containing two strains of C. rogersoniana that split from the other co-specific strains. The consistent results of MALDI-TOF MS supported the identification of strains assigned to C. byssicola and C. pseudochroleuca, which did not form well supported clades in all phylogenetic analyses, but formed distinct clusters in the MALDI-TOF dendrograms.

An HPLC evaluation of cytochalasin D biosynthesis by Xylaria arbuscula cultivated in different media.

A quantitative profile of cytochalasin D production by Xylaria arbuscula was followed by growing the fungus in rice, Czapek, Czapek enriched with yeast extract, wheat, and corn. This cytochalasin producer, X. arbuscula, was collected as an endophytic fungus from healthy tissues of Cupressus lusitanica (Cupressaceae). A new HPLC method was developed using a synthetic N-acetyl-L-phenylalanine ethyl ester as internal standard, which showed a good correlation coefficient (r2 = 0.9995). The results varied from 6.40 to 39.55 mg per 100 g of culture medium, with wheat being the best medium for cytochalasin D production. The level of any free amino acids in the medium, not necessarily phenylalanine, appeared to be an important factor to enhance cytochalasin D biosynthesis.

Fusarium tupiense sp. nov., a member of the Gibberella fujikuroi complex that causes mango malformation in Brazil.

Fusarium tupiense, the main causal agent of mango malformation in Brazil, is described through a combination of morphological, biological and molecular markers. This new species belongs to the Gibberella fujikuroi species complex (GFSC) and has an anamorph morphologically similar to Fusarium mangiferae and F. sterilihyphosum. F. tupiense can be differentiated from other species in the G. fujikuroi species complex on the basis of sexual crosses, amplified fragment length polymorphism (AFLP) markers and partial sequences of the tef1 and tub2 genes. Female fertility for field isolates of F. tupiense appears to be low. PCR with primers specific for the mating type (MAT) alleles and sexual crosses identified this species as heterothallic with two idiomorphs. Female-fertile tester strains were developed for the identification of field strains of this species through sexual crosses.

Chemical and molecular characterization of Phomopsis and Cytospora-like endophytes from different host plants in Brazil.

Phomopsis and related taxa comprise important endophytic and plant pathogenic species, and are known for the production of a diverse array of secondary metabolites. Species concepts within this group based on morphological characters and assumed host specificity do not reflect phylogenetic affinities. Additional phenotypic characters, such as profiles of secondary metabolites, are needed for practical species recognition. We investigated 36 strains of Phomopsis spp. and Cytospora-like fungi, obtained as endophytes of different host plants in Brazil, using metabolite profiling based on HPLC-UV/liquid chromatography -mass spectrometry (LC-MS) combined with cluster analysis of the results. Strains were also subjected to phylogenetic analyses based on internal transcribed spacer (ITS) rDNA. Six chemotypes were identified. Chemotypes 1-5 contained Phomopsis strains, while Cytospora-like strains formed the chemotype 6. Strains of chemotype 1 typically produced alternariols, altenusin, altenuene, cytosporones, and dothiorelones. Alternariol and seven unknown compounds were consistently produced by strains of chemotype 2. Members of chemotypes 3-5 produced poor metabolite profiles containing few chemical markers. Cytospora-like endophytes (chemotype 6) produced a characteristic set of metabolites including cytosporones and dothiorelones. Bayesian and Maximum Parsimony (MP) trees classified strains of each chemotype into single phylogenetic lineages or closely related groups. Strains of chemotypes 1 and 2 formed a monophyletic group along with Diaporthe neotheicola. The remaining Phomopsis strains formed monophyletic (chemotype 4) or polyphyletic (chemotypes 3 and 5) lineages inside a large and well supported clade. Cytospora-like strains formed a monophyletic lineage located at an intermediary position between Diaporthe/Phomopsis and Valsa/Cytospora clades. The combined results show that the production of secondary metabolites by Phomopsis and related Diaporthales may be species-specific, giving support to the use of metabolite profiling and chemical classification for phenotypic recognition and delimitation of species.

Novel antimicrobial secondary metabolites from a Penicillium sp. isolated from Brazilian cerrado soil / Nuevos metabolitos antimicrobianos secundarios de Penicillium sp. aislada en suelo del cerrado de Brasil

The morphological features of a Penicillium, isolated from Brazilian cerrado soil, were characterized and showed to be distinctly different from all well-defined Penicillium species. Chemical and biological investigation on the ethyl acetate extract of this Penicillium isolate resulted in the isolation of three new naphthalenoids: a major metabolite, methyl 6-acetyl-4-methoxy-5,7,8-trihydroxynaphthalene-2-carboxylate and two minor ones, methyl 6-acetyl-4-methoxy-7,8-dihydroxynaphthalene-2-carboxylate and methyl 6-acetyl-4-methoxy-5,8-dihydroxynaphthalene-2-carboxylate. Their structures were determined based on their mono and bidimensional nuclear magnetic resonance data. Acetyl, allyl and methoxyl derivatives of the major metabolite were prepared in order to establish structure-activity relation. Antimicrobial activity of the major natural product and its semi-synthetic derivatives was screened by macro dilution methodology and the corresponding minimum inhibitory concentrations were determined. Natural secondary metabolite methyl 6-acetyl-4-methoxy-5,7,8-trihydroxynaphthalene-2-carboxylate, isolated in a very high yield (0.3175 mg.L-1) showed to be the most active compound, possessing expressive activity against Candida albicans (minimum inhibitory concentration (MIC) 32 ug/mL), Listeria monocitogenes and Bacillus cereus (MIC 64 µg/mL for both).

Phthalates production from Curvularia senegalensis (Speg.) Subram, a fungal species associated to crops of commercial value.

The fungal species Curvularia senegalensis was isolated from a soil sample collected at a Brazilian region of cerrado transition. This microorganism was grown in vitro and the extract of the culture medium was fractionated by chromatographic methods yielding an oil rich in phthalates, from which seven derivatives were identified by infrared, 1H and 13C NMR and mass spectrometry as 1-hexyl-2-propylphthalate, 1-ethyl-2-heptylphthalate, 1-hexyl-2-butylphthalate, 1-heptyl-2-proylphthalate, 1-propyl-2-nonylphthalate and two positional isomers of 1-decyl-2-butane phthalate. This is the first report on the phthalates production by Curvularia senegalensis revealing a scientific basis for the use of this species on biodegradation experiments. Since C. senegalensis is a very common pathogen in some commercial crops, presence of highly toxic phthalates on the final feed products should be investigated.

Cadinane sesquiterpenoids of Phomopsis cassiae, an endophytic fungus associated with Cassia spectabilis (Leguminosae).

Five cadinane sesquiterpenes derivatives were isolated by bioassay-guided fractionation from Phomopis cassiae, an endophytic fungus isolated from Cassia spectabilis. The structures of the two diastereoisomeric 3,9,12-trihydroxycalamenenes (1, 2); 3,12-dihydroxycalamenene (3); 3,12-dihydroxycadalene (4) and 3,11,12-trihydroxycadalene (5) were established on the basis of analyses of 1D and 2D NMR and HRTOFMS experiments. Antifungal activity of the isolates was evaluated against Cladosporium sphaerospermum and Cladosporium cladosporioides, revealing 5 as the most active compound.