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Phylogenetic tree inferred from Bayesian Analysis of 28S rDNA Sordariomycetes sequences. Bayesian posterior probability of >70% are indicated above the nodes and bold lines indicate posterior probabilities of 1.00. The species in study are in bold. The tree is rooted with Pyxidiophora arvernensis.
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The first black mildew fungus associated with Apiaceae is collected from the state of Minas Gerais, Brazil. The new species is described, illustrated, and compared morphologically with species reported on hosts belonging to the related Araliaceae. Additionally, two efficient methods for extracting DNA from biotrophic fungi are described. Phylogenet...
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... The most widely used gene sequences for phylogenetic studies have been 28S rDNA genes because of their easy amplification due their high copy number and the availability of numerous universal primers (Miller & Huhndorf 2005, Tang et al. 2007). The present paper confirms that Meliolales comprises a monophyletic order within Sordariomycetes (Fig. 1). Although the presence of dark perithecia and inamyloid asci indicate that Meliolales belong to the subclass Sordariomycetidae ( Zhang et al. 2006), the phylogenetic position of the order is still uncertain because the phylogeny inferred from the 28S rDNA datasets are the least supported ( Tang et al. 2007). Miller & Huhndorf (2005) ...
Citations
... Total genomic DNA was extracted from mycelia grown on PDA plates for approximately 7 days at 25 °C in the dark using the Wizard ® Genomic Purification Kit (Promega), following the protocol described by Pinho et al. [34]. PCR amplifications were performed using the following primers: ITS5 [35] and LR6 [36] for the internal transcribed spacer 1 and 2 regions and intervening 5.8S rDNA region (ITS) and the nuclear 28S rDNA region (LSU), fRPB2-5F2 [37] and fRPB2-7cR [38] for the RNA polymerase second largest subunit (RPB2); Bt2a and Bt2b [39] for the partial betatubulin gene region (TUB), EF1-983F and EF1-2218R for the translation elongation factor 1-alpha gene (TEF) [40]. ...
Caves are unique environments characterized by spatial limitations, partial or total absence of direct light, and scarcity of organic carbon and nutrients. Caves are shelters for a variety of adapted animals and microorganisms such as fungi, many of which are still unknown. Amphichorda is a fungal genus belonging to the family Bionectriaceae, which includes cave-dwelling and entomopathogenic species with biotechnological applications. In this study, a new fungal species was identified using morphological and multi-locus phylogenetic analyses of the ITS, LSU, and TEF loci, in the Gruta Velha Nova limestone cave located in the Southern Espinhaço Range, Monjolos, Minas Gerais, Brazil. During the exposure of potato dextrose agar plates to the cave environment, an insect from the family Rhaphidophoridae passed by and fed on the culture medium, resulting in three fungal isolates. Phylogenetic analyses showed that these isolates formed a clade distinct from all known species, leading us to introduce a new species, Amphichorda monjolensis, which may be associated with this insect. Here, we also proposed two new combinations for species of acremonium-like fungi in the Bionectriaceae: Bulbithecium globosisporum (synonym: Acremonium globosisporum) and Hapsidospora curva (synonym: Acremonium curvum). The discovery of A. monjolensis highlights the potential of caves as shelters for new species with significant biotechnological importance.
... The mycelium was macerated in microtubes using L-Beader-6 (a Cell Disruptor of Loccus Biotecnologia). DNA extraction was conducted with the Wizard Genomic DNA Purification Kit (Promega's Corporation, WI, USA), according to Pinho et al. [43]. ...
Caves are underground and natural environments mainly found in rocky terrain. Caves have a very specific microclimate, which benefits the occurrence of specific fungi. In recent studies, researchers have observed that caves harbour a great diversity of fungi. However, studies on fungal diversity in Brazilian caves are still incipient. In September 2019, airborne spore and soil samples were collected from the Monte Cristo cave, in the Southern Espinhaço Range, Diamantina, Minas Gerais state, Brazil. Two Cladosporium single‐spore isolates, among other genera, were obtained from these samples. This study aimed to characterise these two fungal isolates based on their DNA sequence data and morphology. Phylogenetic analyses of the rDNA-ITS, ACT and TEF1-α loci revealed that the isolates belonged to the Cladosporium cladosporioides species complex. Both isolates did not cluster with any known species and were formally described and named herein as C. diamantinense and C. speluncae. This study presents taxonomic novelties and contributes to the knowledge about the fungal diversity in Brazilian caves.
... Total genomic DNA extractions were performed by using biomass harvested from PDA colonies incubated from 7 to 10 days at 25 °C in the dark, using the Wizard® Genomic Purification Kit (Promega) protocol, with modifications described by Pinho et al. (2013). PCR amplifications were carried out using the following primers: ITS5 (White et al. 1990) and LR6 (Vilgalys and Hester 1990) for the internal transcribed spacer 1 and 2 regions and intervening 5.8S rDNA region (ITS) and nuclear 28S rDNA region (LSU); fRPB2-5F2 (Sung et al. 2007) and fRPB2-7cR (Liu et al. 1999) or RPB2AM-1bf and RPB2AM-7R (Miller and Huhndorf 2005) for the RNA polymerase second largest subunit (RPB2); T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) or Tub4RD (Groenewald et al. 2013) for the partial beta-tubulin gene region (TUB). ...
Interest in cave fungal diversity is flourishing because it may represent a reservoir of new species and metabolites. However, the mycobiota remains poorly studied in the underground environment, especially in neotropical regions. During surveys that aimed to investigate the fungal diversity in quartzite and limestone caves in the Southern Espinhaço Mountain in Brazil, six Chaetomiaceae isolates were obtained from different cave substrates. Five taxonomical novelties of Chaetomiaceae in Brazilian caves were discovered based on phylogenetic analyses using DNA sequences from the ITS, LSU, TUB, and RPB2 genes. Chaetomium meridionalense, Pseudohumicola alba, and Pseudohumicola lutea are new species found in Gruta da Extração and Gruta Velha Nova caves. Parahumicola is introduced as a new genus representing a novel phylogenetic lineage with unique morphological characteristics in the family Chaetomiaceae. This new monotypic genus is typified by P. guana, which was found in a bat guano sample in the Gruta Monte Cristo cave. Furthermore, this is the first report of Collariella bostrychodes in a neotropical cave. Overall, these findings emphasise that Brazilian caves constitute an untapped source of fungal resources.
... Mycelium was then transferred to 1.5 mL microtubes containing 30 μL of Tris-EDTA buffer. DNA extraction was performed with the aid of the Wizard Genomic DNA Purification kit (Promega Inc.) adapted by Pinho et al. (2012). Isolate identification was done by PCR reactions with primers from regions CAA5, GA4 and β-tubulin (see Table 2) as proposed by O'Donnell and Cigelnik (1997) and Dixon et al. (2009). ...
Brazilian soybean is mainly grown in the Cerrado, the South American Savannah. We report epidemiological studies of soybean target spot (STS), caused by Corynespora cassiicola in two Cerrado regions in the Federal District (DF) and the state of
Bahia (BA), relating the environmental parameters with disease intensities. Here we: i) characterize the feld response of soybean cultivars to STS and compute their respective latent periods; ii) relate STS severity to yield; and iii) verify the cross-reaction of C. cassiicola isolates of soybean and cotton to each of these hosts. Ten commercial soybean cultivars were cultivated in DF and BA felds. Additionally,
the cv. reactions were studied in greenhouse assays in the spring/summer and autumn/winter seasons.
Precipitation and relative humidity were higher in DF than in BA, whereas the temperature was cooler with fewer extremely hot days. Greater STS intensity was observed in DF, reaching 48% incidence and 19% severity. Fungicide applications reduced disease only in the DF trial. Cultivars were separated into four groups: highly susceptible, susceptible, moderately resistant, and resistant. Higher disease severities were recorded in the spring/summer than in the autumn/winter. Length of latent periods correlated with the cv. response to STS and climate conditions. Environmental diferences were the probable causes for the lower disease levels recorded in the BA felds and in the autumn/winter trial. A negative relationship was established between severity and yield. In the cross-inoculation studies, isolates of soybean and cotton were able to infect both hosts, but aggressiveness corrrelated with the host of origin.
... Genomic DNA was extracted from colonies originated from fragments of hyphae tip, grown in PDA, at 25 °C, with 12 daily hours of light, for 7 days. Fungal mycelium was scraped in the colony margins, and DNA extraction was carried out according to the protocol established by Pinho et. al (2012) [15], whose genomic DNA is extracted by Wizard® Genomic DNA Purification Kit (Promega Corporation, WI, U.S.A.) with adaptations. ...
... Fungal mycelium was scraped in the colony margins, and DNA extraction was carried out according to the protocol established by Pinho et. al (2012) [15], whose genomic DNA is extracted by Wizard® Genomic DNA Purification Kit (Promega Corporation, WI, U.S.A.) with adaptations. ...
Several endophytic fungi have been reported to have produced bioactive metabolites. Some of them, including the Induratia species, have the capacity to emit volatile compounds with antimicrobial properties with broad spectrum against human and plant pathogens. The present study aimed to prospect the Induratia species producing volatile organic compounds (VOCs), in carqueja plants used in alternative medicine and coffee plants in Brazil. A total of 11 fungal isolates producing volatile metabolites were obtained by a parallel growth technique, using I. alba 620 as a reference strain. Phylogenetic relationships revealed the presence of at least three distinct species, I. coffeana, I. yucatanensis, and Induratia sp. SPME/GC/MS analyses of the VOCs in the headspace above the mycelium from Induratia species cultured for 10 days on PDA revealed the volatile profile emitted by I. coffeana CCF 572, I. coffeana COAD 2055, I. yucatanensis COAD 2062, and Induratia sp. COAD 2059. Volatile organic compounds produced by I. coffeana isolates presented antimicrobial activity against Aspergillus ochraceus, A. sclerotiorum, A. elegans, A. foetidus, A. flavus, A. tamari, A. tubingensis, A. sydowii, A. niger, A. caespitosus, A. versicolor, and A. expansum, sometimes by decreasing the growth rate or, mainly, by fully inhibiting colony growth. Fifty-eight percent of the target species died after 6 days of exposure to VOCs emitted by I. coffeana CCF 572. In addition, VOCs emitted by the same fungus inhibited the growth in A. ochraceus inoculated into coffee beans, which indicates that plants which have I. coffeana as an endophyte may be protected from attacks by this plant pathogen.
... These fungi are mandatory biotrophics and must to interact with living plant cells for growth and reproduction, without causing the kill of its host. They are usually host specific or have a very narrow host range (Pinho et al. 2012). It cannot be cultured in artificial media and their identification is based on morphological characteristics and, when possible, amplification of genes regions (Pinho et al. 2012, Zeng et al. 2020. ...
... They are usually host specific or have a very narrow host range (Pinho et al. 2012). It cannot be cultured in artificial media and their identification is based on morphological characteristics and, when possible, amplification of genes regions (Pinho et al. 2012, Zeng et al. 2020. ...
... The leaves were dried and reserved frozen for seven days, and the specimens were deposited at the Herbarium Padre Camille Torrend (URM). Some black mildew fungi were also identified based according to the extraction protocol DNA of Pinho et al. (2012). The electrospherograms were analyzed using the software Sequencer 4.7 (Gene Codes, Ann Arbor, Michigan, USA), from which the consensus nucleotide sequences were obtained and exported as a FASTA file. ...
Five species of Meliola and one of Prillieuxina (black mildews) were recorded in agroecological transitional areas in polyculture crop (consortium of plants-COPs-and agroforestry systems-AFSs) located in Pernambuco State, Brazilian northeast. Meliola anacardii on Anacardium occidentale, M. annonacearum on Annona montana, M. mangiferae on Mangifera indica, M. rhoina on Schinus terebinthifolius, M. trichostroma on Psidium guajava and Prillieuxina winteriana on A. muricata and A. montana. These species differed for host relationship and taxonomic structures. Illustrations of P. winteriana, Meliola spp. and its epiparasites are provided. Resumo: Cinco espécies de Meliola e uma de Prillieuxina (Míldios negros) foram registradas em policultivos de áreas de transição agroecológica (consórcio de plantas-COPse sistemas agroflorestais-AFSs) localizados no estado de Pernambuco, nordeste do Brasil. Meliola anacardii em Anacardium occidentale, M. annonacearum em Annona montana, M. mangiferae em Mangifera indica, M. rhoina em Schinus terebinthifolius, M. trichostroma em Psidium guajava e Prillieuxina winteriana em A. muricata e A. montana. Estas espécies diferiram pelo hospedeiro a quem estão relacionadas e estruturas taxonômicas. Ilustrações de P. winteriana, Meliola spp. e seus epiparasitas são apresentadas. Termos para indexação: Asterina winteriana, Fungos micoparasitas, Meliolaceae, Spiropes sp., Trichothyrium reptans.
... Single-spore isolates were grown on PDA for eight days at 22 °C in the dark, after which the mycelium was harvested to a sterilized microcentrifuge tube (1.5 ml) and grinding in liquid nitrogen. The genomic DNA extraction was performed using the Wizard Genomic DNA Purification (Promega Corporation, WI, USA) according to the protocol of Pinho et al. (2012). Partial sequences of the ITS, TUB, and TEF gene regions were obtained using combinations of primers ITS1/ITS4 (White et al. 1990), T1/Bt-2b (Glass and Donaldson 1995;O'Donnell and Cigelnik 1997), and EF1-728F/EF-2 (Carbone and Kohn 1999;O'Donnell et al. 1998), respectively, in the stages of amplification and sequencing. ...
Pestalotioid fungi associated with leaf spot disease of several species of palms (Arecaceae) were investigated in nurseries located in the state of Minas Gerais, Brazil. Symptomatic seedlings of Caryota mitis, Cocos nucifera, Dypsis lutescens, Dypsis madagascariensis, Euterpe edulis, Licuala grandis, Phoenix roebelenii, Ptychosperma elegans, and Wodyetia bifurcata were examined and fungal isolates were identified molecularly using the internal transcribed spacer (ITS), partial β-tubulin (TUB) and partial translation elongation factor 1-α (TEF). Based on concordant phylogenetic and morphological results, as well as ecological specificities, one previously described species, as well as one new species, were recognized from the collection. These findings contribute to the knowledge of the diversity of Neopestalotiopsis in Brazil and may be helpful for further studies on quarantine programs and management measures.
... Probably because the fungus is biotrophic and cannot be cultivated in culture, there is no sequence of Meliola species on in the Araliaceae family in the GenBank. There is a need to propose improved protocols for DNA extraction from Meliola species (Pinho et al., 2012b) so that doubts about host specificity of Meliola species can be resolved. ...
... Next, the samples were mixed and crushed in the L-Beader 6 (Loccus Biotecnologia) for 30 s at 4,000 rpms. After maceration, the extraction proceeded as described by Pinho et al. (2013). The samples were deposited in the DNA collection (CDA) at the "Laboratório de Micologia e Etiologia de Doenças Fúngicas de Plantas," Viçosa, Minas Gerais, Brazil. ...
Pereskia aculeata is a non-conventional vegetable belonging to the family Cactaceae, native to the Americas. The leaves are very appreciated in cooking in some Brazilian regions, specially Minas Gerais. Plants of P. aculeata showing severe leaf spot diseases and stem rot were observed in Viçosa, state of Minas Gerais, Brazil. Colletotrichum sp. was observed on the diseased plant tissue and since no records of this fungus (and anthracnose disease) are reported for this host, the present study has aimed to identify the Colletotrichum sp. associated to P. aculeata and elucidate the etiology of the anthracnose disease. Based on the analyses of morphological characters combined with phylogenetic data from ITS, GAPDH, CHS-1, HIS3, ACT, and TUB2, a new species, namely Colletotrichum pereskiae sp. nov., is proposed, which belongs to the C. orchidearum species complex. A representative isolate was inoculated on healthy P. aculeata plants and reproduced the severe symptoms observed in the field. Colletotrichum pereskiae is the etiological agent of anthracnose on Pereskia aculeata in Brazil.
... Then, the samples were mixed and crushed in an L-Beader 6 (Loccus Biotecnologia). After maceration, the extraction proceeded as described by Pinho et al. (2012). Primers ITS5 (White et al., 1990) and LR6 (Vilgalys & Hester, 1990) were used to amplify the rDNA-ITS region (containing internal transcribed spacer 1, 5.8S ribosomal RNA, and internal transcribed spacer 2) and the D1/D2 domain of large subunit rRNA gene, respectively. ...
In Brazil, sour rot is an important postharvest disease on fruits and vegetables. Geotrichum candidum (synonym Galactomyces candidus) has been reported as the main species causing this disease. However, the identity of the causal agent is still uncertain. This research aimed to determine the identity of 165 fungal isolates associated with sour rot, obtained from fruits and vegetables in Brazil and to evaluate the effect of different temperatures on the incidence of sour rot on artificially inoculated tomato fruits. Based on phylogenetic analyses of DNA sequences from the D1/D2 domain of the large subunit (LSU) rRNA gene and morphological analyses, 129 samples belong to the Galactomyces candidus, 15 to G. candidum var. citri‐aurantii, 6 to G. phurueaense, 2 to Gal. pseudocandidus, 1 to Hyphopichia burtonii, 1 to H. khmerensis, 3 to Saccharomycopsis crataegensis, 1 to S. vini, 1 to Magnusiomyces tetrasperma, 1 to Trichosporon coremiiforme and 1 to Zygoascus meyerae. Two new species were found, namely, Geotrichum solani (on potato) and Geotrichum spondiadis (on red mombin). All isolates were pathogenic when inoculated on healthy tomato fruits, including the new species of Geotrichum, which were also inoculated into their respective hosts, i.e., potatoes and red mombins. To the best of our knowledge, this study is the first to report the presence of five other genera besides Geotrichum associated with sour rot on fruits and vegetables in Brazil, which demonstrates the diversity of fungi and yeasts associated with this disease.
