Northward migration past the nearctic biogeographical divide; neotropical Gyrodactylus spp. infecting Astyanax have crossed the Trans-Mexican Volcanic Belt.

The neotropical fish genus Astyanax (Characidae) and its associated helminths migrated northward from South America following the Great American Biotic Interchange (GABI): ca. 150 Astyanax spp. are found throughout South and Central America, up to the Mexico-USA border. Most characids are distributed south of the Trans-Mexican Volcanic Belt (TMVB), which bisects the country and represents a major transition zone between the neotropical and nearctic realms. Here, we characterize parasites of the monogenean genus Gyrodactylus infecting Astyanax spp. in Mexico: Astyanax aeneus south of the TMBV, Astyanax mexicanus north of it. Based on morphological, phylogenetic (internal transcribed spacer (ITS) and cytochrome oxidase subunit II (cox 2)) and statistical analyses of morphometric data, we confirmed the validity of Gyrodactylus pakan and Gyrodactylus teken, and erected two new species, Gyrodactylus aphaa n. sp. and Gyrodactylus ricardoi n. sp. These four gyrodactylids are part of a complex of morphologically cryptic species, which are phylogenetically closely related to each other, and sister species to Gyrodactylus carolinae and Gyrodactylus heteracanthus, parasites of characins in Brazil. Four gyrodactylid lineages (G. pakan, G. ricardoi n. sp., G. teken, Gyrodactylus sp. A) are distributed north of the TMVB; G. pakan is also widely distributed south of the TMVB, together with G. aphaa n. sp. Based on the ITS phylogeny, Brazilian parasites form a sister clade to all Mexican gyrodactylids, whose derived clades are distributed in progressively more northerly latitudes in Mexico - the three most-derived species north of the TMVB. This would suggest that gyrodactylid species diverged gradually, presumably as their characid fish hosts colonized and adapted to new environments north of the TMVB.

Morphological and molecular characterization of three new species of Gyrodactylus (Monogenea: Gyrodactylidae) infecting Sicydium salvini (Teleostei: Gobiidae) in Mexican rivers draining into the Pacific Ocean.

The genus Gyrodactylus von Nordmann, 1832 is one of the most diverse within the class Monogenea; it contains mainly parasites of freshwater and marine teleost fishes. Around 40 species of Gyrodactylus have been described from gobiid fishes; mainly in Europe, as only two species are known from the Americas. In this study, we describe three new gyrodactylids from the body surface and fins of the goby Sicydium salvini (Gobiidae, Sycydiinae), which has a wide distribution on the Pacific coast, from Mexico to Panama. We describe Gyrodactylus oaxacae n. sp., G. atoyacensis n. sp. and G. salvini n. sp. collected from rivers draining to the eastern Pacific in the state of Oaxaca, Mexico. Morphologically, G. atoyacensis n. sp. and G. salvini n. sp. are very similar, and both are easily differentiated from G. oaxacae n. sp. Phylogenetic hypotheses based on sequences of the Internal Transcribed Spacers (ITS1-5.8S-ITS2 rDNA) and the D2 + D3 domains of the large ribosomal subunit (28S rDNA) support the erection of the three new taxa; and suggest that G. atoyacensis n. sp. and G. salvini n. sp. are sister species. These gyrodactylids are the first monogeneans described from gobies of the genus Sicydium in Mexico.

Two New Species of Backusella (Mucorales, Mucoromycota) from Soil in an Upland Forest in Northeastern Brazil with an Identification Key of Backusella from the Americas.

During a survey of Mucorales from a forest located in Pernambuco state, Brazil, two new Backusella species were discovered and described based on morphological and molecular data (internal transcribed spacer and large subunit ribosomal DNA sequences). Both species were characterized as unbranched sporangiophores and sporangia with columellae of varied shapes forming. Multispored sporangiola were frequent, whereas unispored sporangiola were rare. URM 8395 forms sporangiophores that may support hyaline, slightly curved or circinate pedicels with multispored sporangiola at their apical portion, and abundant giant cells and chlamydospores. Columellae of sporangia are hyaline, conical (majority), or ellipsoidal with a truncate base, globose to subglobose or subglobose to conical, and, rarely, with slight medial constriction. URM 8427 does not form sporangiola from pedicels, giant cells are not observed, and columellae of sporangia are globose to subglobose, cylindrical with a truncate base, some with a slight constriction, applanate, obovoid, ellipsoidal, or, rarely, conical. Some columellae may have one side more swollen than the other and some are arranged obliquely on the sporangiophores. Sterile sporangia may or may not be formed on short sporophores. The detailed description and illustration of both novel species as well as an identification key for Backusella from the Americas are provided.

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

ABSTRACT Background: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. Methods: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. Results: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. Conclusions: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

Isolation, identification, and characterization of cadmium-tolerant endophytic fungi isolated from barley (Hordeum vulgare L.) roots and their role in enhancing phytoremediation.

Four hundred endophytic fungi isolates with different colony morphologies were isolated from roots of Hordeum vulgare L. collected from un-engineered landfills (the measured cadmium was 0.9 mg kg-1) of Kermanshah province in West Iran. Based on morphology and phylogeny of DNA sequence data for the internal transcribed spacer (ITS) rDNA and comparing the sequences with that available in NCBI database, 11 isolates are identified as dark septate endophytes (DSE) including Alternaria alternata, Microdochium bolleyi, Bipolaris zeicola, Alternaria sp., and Pleosporales sp., and the other nine are not dark septate endophytes (non-DSE) including Fusarium redolens, Fusarium tricinctum, Fusarium monliforme, Clonostachys rosea, and Epicoccum nigrum. Tolerance of DSE and non-DSE strains for Cd were investigated in potato dextrose agar medium. Minimum inhibitory concentrations (MIC) of Cd from nitrate salt source (Cd (NO3)2) and EC50 were determined. The means of MIC and EC50 values for DSE fungi species were 1254.5 and 209.74 mg/kg, compared to 800 and 150.3 mg/kg for non-DSEs. Among the endophytic fungi isolated, Alternaria sp. (TBR5) and Bipolaris zeicola (Tw26) showed the highest tolerance to Cd with a MIC value of 2000 mg/L and 1800 mg/L, respectively. Barley plants were inoculated with TBR5 and Tw26 in Cd-added sands (0, 10, 30, 60 mg Cd/kg sand). In terms of Cd accumulation, our results showed that TBR5 and Tw26 inoculation increased the amount of Cd in the barley roots. TBR5 and Tw26 significantly improved (p < 0.05) plant growth in the presence of Cd by enhancing plant growth attributes such as chlorophyll content, root weight, plant length, fresh weight, and dry weight of plants. This is the first study on the abundance and identification of endophytic root fungi of barley in a cadmium-contaminated soil in Iran. The results of this study showed that DSE and non-DSE have the potential to improve the efficiency of phytoremediation.

Fungal diversity present on rocks from a polar desert in continental Antarctica assessed using DNA metabarcoding.

We evaluated the fungal diversity associated with carbonate veins and two types of salt encrustation in rocks in a polar desert region of continental Antarctica using DNA a metabarcoding approach. We detected 262,268 reads grouped into 517 amplicon sequence variants (ASVs) assigned to the phyla Ascomycota, Basidiomycota, Mortierellomycota and Mucoromycota. Fourteen ASVs belonging to the genera Trichosporon, Mortierella, Penicillium, Aspergillus, Cladosporium, Coprinellus, Pleurotus and Pseudogymnoascus were assessed to be dominant taxa. The fungal communities of the three habitats sampled displayed high diversity indices when compared with other habitats of Antarctica, although differing in detail, with the highest diversity indices in the gypsum crust type 2. Only 48 of the 517 ASVs (9.28%) were detected in all three habitats, including dominant, intermediate and minor components. In contrast with previous studies of fungal communities living in the ultra-extreme conditions of continental Antarctica, application of metabarcoding revealed the DNA of a rich and complex resident fungal community. The ASVs detected included fungi with different ecological roles, with xerophilic, human- and animal-associated, phytopathogenic, saprotrophic, mutualistic, psychrotolerant and cosmopolitan taxa. This sequence diversity may be the result of deposition of fungal propagules over time driven by air currents, precipitation or human activities in the region.

Species delimitation of Gyrodactylus (Monogenea: Gyrodactylidae) infecting the southernmost cyprinids (Actinopterygii: Cyprinidae) in the New World.

The genus Gyrodactylus von Nordmann, 1832 represents one of the most diverse and widespread taxa within Monogenea, with approximately 500 species described worldwide. Thirty-three species of Gyrodactylus have been recorded in Mexico, and in the last two decades, at least 26 new species have been described mainly from freshwater fish families such as poeciliids, goodeids, profundulids, characids, and cichlids. In this study, we describe two new species of Gyrodactylus infecting freshwater cyprinids based on morphological and molecular characteristics. Gyrodactylus ticuchi n. sp. and Gyrodactylus tobala n. sp. were recovered from Notropis moralesi de Buen and N. imeldae Cortés, respectively, captured in five localities from the State of Oaxaca, Mexico. The new species differ slightly from their congeners in the morphology of the haptoral hard parts and the male copulatory organ. Sequences of the Internal Transcribed Spacers rDNA (ITS1-5.8S-ITS2), cytochrome oxidase subunit I (cox1), and the D2 + D3 domains of the large subunit (28S rDNA) were obtained from multiple specimens and analyzed using Maximum Likelihood (ML) and Bayesian Inference (BI). Phylogenetic hypotheses using ITS rDNA, cox1, and 28S rDNA genes recovered two new species of Gyrodactylus from N. moralesi and N. imeldae; we briefly discuss their phylogenetic relationship with other congeners. These gyrodactylids represent the first species described in species of Notropis from southern Mexico, the cyprinids exhibiting the southernmost distribution in the New World.

Multiplex TaqMan Real-Time PCR Assay for Sensitive Detection of Two Weevil Species (Coleoptera: Curculionidae).

Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus. This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/µl to 1 × 10-6 pg/µl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.

A new species of Creptotrematina (Trematoda: Allocreadiidae) from characid fishes of Brazil: morphological and molecular data.

A new species of Creptotrematina Yamaguti, 1954 was collected from characid fishes, Astyanax fasciatus (Cuvier, 1819) and Astyanax lacustris Lucerna & Soares, 2016 from the Batalha River in the State of Sao Paulo, Brazil. The new species most closely resembles Creptotrematina aguirrepequenoi, but differs by the elongated shape of vitelline follicles, the extension of these follicles in the posterior end of body and the fact that they are not confluent. The morphological differences were confirmed through molecular data. Three specimens were sequenced, and molecular analyses were based on the internal transcribed spacers 2 and D1-D3 domains of the 28S ribosomal RNA gene. The obtained topologies showed the new species as a sister taxon of C. aguirrepequenoi, a species originally described from Astyanax mexicanus in Mexico, and later found in Astyanax aeneus in Costa Rica. Isolates of the new species are reciprocally monophyletic, and genetic distance values are similar to those observed in other species pairs within Allocreadiidae. These findings corroborate that the genus Creptotrematina is mostly a parasite of characids, and widely extended across the Americas, with representative species occurring between Argentina and northern Mexico.

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological, molecular and ecophysiological data.

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20-25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.

ITS rDNA Gene Analysis Versus MALDI-TOF MS For Identification of Neoscytalidium dimidiatum Isolated from Onychomycosis and Dermatomycosis Cases in Medellin (Colombia).

Within the Neoscytalidium genus, N. dimidiatum, N. oculus, N. orchidacearum, and N. novaehollandiae have been recognized. Although these species are frequently found in soil, N. dimidiatum has been identified as an etiologic agent of onychomycosis or dermatomycosis, and N. oculus has been identified as an etiologic agent of an ocular lesion. All these species can be cultured in vitro, but their morphological identification by macroscopic and microscopic traits is difficult and imprecise due to their similarity. In this study, 34 isolates of Neoscytalidium spp. from 32 onychomycosis and two dermatomycosis cases in Medellin (Colombia) were identified at the species level using sequencing of the ITS1+5.8S+ITS2 nuclear rDNA region and MALDI-TOF mass spectrometry (MS). Neoscytalidium dimidiatum strain MUM 17.21 was used to construct the reference spectrum in the in-house library to identify the clinical isolates by MALDI-TOF MS. Additionally, N. dimidiatum PPC-216 and PLAB-055 strains were used to validate the in-house constructed reference spectra. Although four groups were observed in the dendrogram obtained from the proteins of each isolate profile, MALDI-TOF MS and sequencing results are in accordance, since all isolates were identified as N. dimidiatum.

Ceratocorys mariaovidiorum sp. nov. (Gonyaulacales), a new dinoflagellate species previously reported as Protoceratium reticulatum.

The family Ceratocoryaceae includes the genera Ceratocorys, Protoceratium, and Schuettiella, whose phylogenetic relationships are poorly known. Here, the new non-yessotoxin-producing species of the genus Ceratocorys, Ceratocorys mariaovidiorum sp. nov., previously reported as the toxic Protoceratium reticulatum, is described from examinations by light and scanning electron microscopy, molecular phylogeny, and toxin analyses. The species description is made from culture samples of strains CCMP1740 and CCMP404 from USA waters. Ceratocorys mariaovidiorum is globular and has thick and strongly reticulated plates with one pore within each reticule, just like P. reticulatum, but the key difference between the two species is the presence of five precingular plates in C. mariaovidiorum instead of six as in P. reticulatum. The thecal plate formula is Po, 4', 0a, 5″, 6c, ~7s, 5‴, 0p, 2''''. The apical pore plate is oval with a λ-shaped pore. The first apical plate is narrow with a ventral pore on the right anterior side; it contacts the apical pore plate and its contact with the anterior sulcal plate is slight or absent. The fourth precingular plate of other Gonyaulacales is absent. Ceratocorys mariaovidiorum may have small spines on the second antapical plate. A phylogenetic study based on internal transcribed spacer/5.8SrDNA supports the morphological classification of C. mariaovidiorum as a new species of Ceratocorys and in a different clade from P. reticulatum.

Growth, mortality and susceptibility of oyster Crassostrea spp. to Perkinsus spp. infection during on growing in northeast Brazil / Crescimento, mortalidade e susceptibilidade de ostras Crassostrea spp. à infecção por Perkinsus spp. em cultivo no Nordeste do Brasil

Abstract Crassostrea rhizophorae and C. gasar oysters are cultivated in the northeast region. Perkinsus parasites infect bivalves, and their effects on oysters from tropical regions are poorly understood. This study evaluated the impact of Perkinsus infection on the productive traits of native oysters. Oysters were sampled bimonthly during 7 months, from July 2010 to February 2011, to evaluate growth rate, mortality and shell color patterns (white and dark-gray) (n = 500), and to determine the prevalence and intensity of Perkinsus (n = 152). Perkinsus and Crassostrea species were determined using molecular tools. Results showed that most dark-gray (90%, n = 20) and white (67%, n = 18) oysters were C. gasar and C. rhizophorae, respectively. Oysters showed a high growth rate and moderate cumulative mortality (44%). C. gasar oysters grew better and showed lower mortality and lower incidence of Perkinsus compared to C. rhizophorae. The mean prevalence of Perkinsus was moderate (48%), but the infection intensity was light (2.2). Perkinsosis affected very small oysters (19.4 mm). In conclusion, native oysters, especially C. gasar, have a great potential for culture, mortality is not associated with perkinsosis, and the shell color of oysters can be used to improve selection for spats with better performance.

Resumo Crassostrea rhizophorae e C. gasar são cultivadas na região Nordeste. Parasitas Perkinsus infectam bivalves e seus efeitos em ostras de regiões tropicais são pouco compreendidos. Este estudo avaliou o impacto da infecção por Perkinsus em parâmetros de produção de ostras nativas. Ostras foram coletadas bimestralmente durante 7 meses, de julho de 2010 a fevereiro de 2011, para avaliar crescimento, mortalidade e padrão de coloração da concha (branca e cinza-escura) (n = 500); além da presença e intensidade de Perkinsus (n = 152). Perkinsus e Crassostrea foram identificados por abordagem molecular. Os resultados mostraram que as ostras cinza-escuras (90%, n = 20) e brancas (67%, n = 18) eram C. gasar e C. rhizophorae, respectivamente. As ostras mostraram uma boa taxa de crescimento e mortalidade acumulada moderada (44%). C. gasar cresceu melhor com menor mortalidade e menor incidência de Perkinsus que C. rhizophorae. A prevalência média de Perkinsus foi moderada (48%), mas a intensidade de infecção foi leve (2,2). A perkinsiose afetou ostras pequenas (19,4 mm). Em conclusão, ostras nativas, especialmente C. gasar, têm grande potencial de produção; sem mortalidade associada à perkinsiose; e, a cor da concha pode ser usada para melhorar a seleção de sementes com melhor desempenho.

Growth, mortality and susceptibility of oyster Crassostrea spp. to Perkinsus spp. infection during on growing in northeast Brazil / Crescimento, mortalidade e susceptibilidade de ostras Crassostrea spp. à infecção por Perkinsus spp. em cultivo no Nordeste do Brasil

Crassostrea rhizophorae and C. gasar oysters are cultivated in the northeast region. Perkinsus parasites infect bivalves, and their effects on oysters from tropical regions are poorly understood. This study evaluated the impact of Perkinsus infection on the productive traits of native oysters. Oysters were sampled bimonthly during 7 months, from July 2010 to February 2011, to evaluate growth rate, mortality and shell color patterns (white and dark-gray) (n = 500), and to determine the prevalence and intensity of Perkinsus (n = 152). Perkinsus and Crassostrea species were determined using molecular tools. Results showed that most dark-gray (90%, n = 20) and white (67%, n = 18) oysters were C. gasar and C. rhizophorae, respectively. Oysters showed a high growth rate and moderate cumulative mortality (44%). C. gasar oysters grew better and showed lower mortality and lower incidence of Perkinsus compared to C. rhizophorae. The mean prevalence of Perkinsus was moderate (48%), but the infection intensity was light (2.2). Perkinsosis affected very small oysters (19.4 mm). In conclusion, native oysters, especially C. gasar, have a great potential for culture, mortality is not associated with perkinsosis, and the shell color of oysters can be used to improve selection for spats with better performance.(AU)

Crassostrea rhizophorae e C. gasar são cultivadas na região Nordeste. Parasitas Perkinsus infectam bivalves e seus efeitos em ostras de regiões tropicais são pouco compreendidos. Este estudo avaliou o impacto da infecção por Perkinsus em parâmetros de produção de ostras nativas. Ostras foram coletadas bimestralmente durante 7 meses, de julho de 2010 a fevereiro de 2011, para avaliar crescimento, mortalidade e padrão de coloração da concha (branca e cinza-escura) (n = 500); além da presença e intensidade de Perkinsus (n = 152). Perkinsus e Crassostrea foram identificados por abordagem molecular. Os resultados mostraram que as ostras cinza-escuras (90%, n = 20) e brancas (67%, n = 18) eram C. gasar e C. rhizophorae, respectivamente. As ostras mostraram uma boa taxa de crescimento e mortalidade acumulada moderada (44%). C. gasar cresceu melhor com menor mortalidade e menor incidência de Perkinsus que C. rhizophorae. A prevalência média de Perkinsus foi moderada (48%), mas a intensidade de infecção foi leve (2,2). A perkinsiose afetou ostras pequenas (19,4 mm). Em conclusão, ostras nativas, especialmente C. gasar, têm grande potencial de produção; sem mortalidade associada à perkinsiose; e, a cor da concha pode ser usada para melhorar a seleção de sementes com melhor desempenho.(AU)

Mycorrhizal fungi isolated from native terrestrial orchids of pristine regions in Córdoba (Argentina) / Hongos micorrízicos aislados de orquídeas terrestres nativas de regiones pristinas en Córdoba (Argentina)

Orchidaceae is a highly dependent group on the Rhizoctonia complex that includes Ceratorhiza, Moniliopsis, Epulorhiza and Rhizoctonia, for seed germination and the development of new orchid plants. Thus, the isolation and identification of orchid mycorrhizal fungi are important to understand the orchid-fungus relationship, which can lead to the development of efficient conservation strategies by in vivo germination of seeds from endangered orchid plants. The aim of our work was to isolate and characterize the different mycorrhizal fungi found in roots of terrestrial orchids from Córdoba (Argentina), and, to learn about the natural habit and fungal associations in the Chaco Serrano woodland pristine region. In this study, bloomed orchid root and rhizosphere soil samples were obtained in two times from Valle de Punilla during spring of 2007; samples were kept in plastic bags until processed within 48 hours, and mycorrhizal condition confirmed assessing peloton presence. A total of 23 isolates of the orchideous mycorrhizal Rhizoctonia complex were obtained. The isolates were studied based on morphological characters and ITS-rDNA sequences. Morphological characteristics as color of colonies, texture, growth rate, hyphal diameter and length and presence of sclerotia were observed on culture media. To define the number of nuclei per cell, the isolates were grown in Petri dishes containing water-agar (WA) for three days at 25°C and stained with Safranine-O solution. The mycorrhizal fungi were grouped into binucleate (MSGib, 10 isolates) and multinucleate (MSGim, 13 isolates) based on morphological characteristics of the colonies. We obtained the ITS1-5.8s-ITS4 region that was amplified using primers ITS1 and ITS4. Based on DNA sequencing, isolates Q23 and Q29 were found to be related to species of Ceratobasidium. Isolates Q24 and Q4 were related to the binucleated anastomosis group AG-C of Rhizoctonia sp. The rest of the isolates grouped in the Ceratobasidium clade without grouping. From our knowledge this is the first report of the association of the AG-C testers with terrestrial orchids. A high specificity was observed in the symbiotic relationship. As the mycorrhizal fungal isolates were obtained from native orchids, they could be incorporated in conservation programes of endangered orchids in Argentina.

La Familia Orchidaceae se encuentra estrechamente relacionada con hongos micorrízicos que pertenecen al complejo Rhizoctonia, e incluyen los géneros Ceratorhiza, Moniliopsis, Epulorhiza y Rhizoctonia. Esta asociación es esencial para el desarrollo de nuevas plantas ya que favorecen el proceso de germinación de las semillas. Por lo tanto, el conocimiento de la naturaleza de esta interacción es importante para que los resultados de los programas de conservación de orquídeas sean efectivos. La fragmentación del bosque Chaqueño Serrano en el centro de Argentina, ha alcanzado un punto crítico en los últimos años, afectando el funcionamiento del ecosistema. El objetivo de este trabajo fue: a) aislar y caracterizar hongos micorrízicos presentes en orquídeas terrestres de la provincia de Córdoba (Argentina) y b) conocer el hábitat natural y las asociaciones fúngicas que se establecen en esta región prístina. A partir de las raíces de orquídeas terrestres, se obtuvieron 23 aislamientos de hongos micorrízicos que pertenecen al complejo Rhizoctonia. Estos aislamientos fueron caracterizados con base en caracteres morfológicos y moleculares. Las características morfológicas (color y textura de las colonias, cinética de crecimiento, diámetro y largo de la hifa y presencia de esclerocios) fueron observados en PDA y MEA a 25ºC. El número de núcleos por célula se observó en cultivos crecidos en AA (agar-agua) y teñidos con una solución de Safranine-O. La región ITS se amplificó usando los primers ITS1 e ITS4. Con base en las características morfológicas de la colonia, los aislamientos fueron agrupados en binucleados (MSGib) y multinucleados (MSGim). De acuerdo al cladograma obtenido con las secuencias de ADN, los aislamientos Q23 y Q29 están relacionados a especies de Ceratobasidium, aisladas de raíces de orquídeas. Los aislamientos Q24 y Q4 se asocian con el grupo de anastomosis de Rhizoctonia AG-C. Finalmente, se observó una alta variabilidad en el grado de especificidad existente en la simbiosis que se establece entre las raíces de estas orquídeas terrestres y los aislamientos obtenidos a partir de ellas. Este es el primer reporte de la asociación entre el grupo de anastomosis AG-C y orquídeas terrestres. Dado que estos aislamientos se obtuvieron de orquídeas terrestres nativas, podrían ser incorporados como nuevos patrones para micorrizas de orquídeas terrestres en Argentina. Este trabajo contribuye al conocimiento de la relación simbiótica que se establece entre orquídeas y hongos micorrízicos, así como también al desarrollo de estrategias de conservación de orquídeas terrestres nativas del bosque Chaco Serrano.

A case of Exophiala spinifera infection in Southern Brazil: Molecular identification and antifungal susceptibility.

We report a case of an 80-year-old Brazilian man, farmer, with lesions on the dorsum of the hand. A direct mycological examination, cultivation and microculture slide observation was performed. The sequencing of ITS1-5.8S rDNA-ITS2 region was carried out and the etiological agent confirmed as Exophiala spinifera. The in vitro susceptibility of this isolate to antifungal agents alone and in combination was evaluated. This is the third case of phaeohyphomycosis caused by Exophiala spinifera in Brazil.

Molecular Analysis in the differentiation of Colletotrichum gloeosporioides isolates from the cashew and mango trees

The aim of the present work was to analyze the molecular methods in the differentiation of Colletotrichum gloeosporioides isolates obtained from the cashew and mango trees. The different molecular taxonomic methods used proved to be efficient regarding intraspecific characterization. Similarly, molecular methods also proved to be efficient in differentiation of the C. gloeosporioides isolates in relation to host specificity. In the analysis of the ITS sequence of the ribosomal DNA, all the isolates amplified with the CgInt and ITS4 primers, confirming that they pertained to C. gloeosporioides. The results from this study suggested that methods based on the pathogenicity, isozyme analysis and RAPD were effective in differentiating C. gloeosporioides isolates from the cashew and mango trees.

Genetic study of Mediterranean and South American populations of tomato leafminer Tuta absoluta (Povolny, 1994) (Lepidoptera: Gelechiidae) using ribosomal and mitochondrial markers.

BACKGROUND: Before its introduction into Europe at the end of 2006, Tuta absoluta (Povolny, 1994) was confined solely to South America. Currently, this invasive pest is well established in various European and Mediterranean countries, causing important economic losses to tomato (Lycopersicon esculentum Mill.) crops. In order to study the genetic variability of this pest, 23 Mediterranean and ten native South American populations were analysed with nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) markers. RESULTS: The internal transcribed spacers 1 (ITS1) and 2 (ITS2) of rDNA and a fragment in the mtDNA gene encoding cytochrome oxidase I (COI) were PCR amplified and sequenced in T. absoluta. Sequence analyses consistently revealed neither intrapopulation nor interpopulation variation in either genomic region. CONCLUSIONS: High genetic homogeneity was detected in T. absoluta populations from the Mediterranean Basin and South America, based on mtCOI and ITS rDNA sequence analysis. A single genetic type was identified in this pest.

Diversity analysis of Bemisia tabaci biotypes: RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region

The Bemisia tabaci complex is formed by approximately 41 biotypes, two of which (B and BR) occur in Brazil. In this work we aimed at obtaining genetic markers to assess the genetic diversity of the different biotypes. In order to do that we analyzed Bemisia tabaci biotypes B, BR, Q and Cassava using molecular techniques including RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region. The analyses revealed a high similarity between the individuals of the B and Q biotypes, which could be distinguished from the BR individuals. A phylogenetic tree based on ITS1 rDNA sequence was constructed. This is the first report of the ITS1 rDNA sequence of Bemisia tuberculata and of the BR biotype of B. tabaci.

Morphological, anatomical, macro and micromolecular markers for Solanum cernuum identification

The plants consumed as remedy by the population may have imprecise taxonomical identification. If these plants are used for the production of phytomedicines such misidentification may affect the quality of the product. Hereby, we describe markers for identification of the entire plant or grounded plant material or the crude extract of Solanum cernuum Vell. (Solanaceae). Specimens from four localities were collected, analyzed and compared. Morphological characters were used to identify the plant when it is not grounded or extracted. However, when the plant material is grounded, the set of trichomes may be used as anatomical marker. The region ITS1, 5.8S and ITS2 of the nuclear ribosomal DNA was cloned and sequenced. The sequence, with length of about 600 base pairs, being 48.1 percent AT , was deposited in GenBank under the accession number DQ837371. Once this sequence is specific to S. cernuum, it was used as marker for this species. For the crude extract, chromatographic profiles of the leaves extracts were obtained by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Two flavonoids were isolated and identified as quercitrin and afzelin. So, this study presents morphological, anatomical, macro and micromolecular markers to identify S. cernuum.

Plantas consumidas como remédio nem sempre são identificadas taxonomicamente de maneira correta. Se estas plantas forem utilizadas para obtenção de uma droga vegetal ou um fitoterápico, tal erro pode afetar a qualidade do produto final. Neste trabalho são descritos marcadores para a identificação de Solanum cernuum Vell. (Solanaceae), esteja a planta íntegra, triturada ou como extrato bruto. Indivíduos de quatro localidades de Minas Gerais foram coletados, analisados e comparados. Os caracteres morfológicos foram utilizados para a planta íntegra. Para a planta triturada, o conjunto dos tricomas foi utilizado como marcador anatômico. Um marcador macromolecular também foi determinado. Para tal a região ITS1, 5.8S e ITS2 do DNAr foi clonada e seqüenciada. A seqüência, com cerca de 600 pares de bases dos quais 48,1 por cento são AT, foi depositada no GenBank sob o número de acesso DQ837371. Por ser uma seqüência específica para S. cernuum, ela pode ser usada como marcador desta espécie. Para o extrato bruto foram determinados perfis cromatográficos de extratos das folhas por cromatografia em camada delgada e por cromatografia líquida de alta eficiência. Dois flavonóides foram isolados e identificados como quercitrina e afzelina. Assim, neste trabalho foram determinados marcadores morfológicos, anatômicos, macro e micromoleculares para identificar S. cernuum.