Phylogeny and ultrastructure of Myxobolus rangeli n. sp. (Myxozoa, Bivalvulida), a histozoic parasite in Siluriformes fish from the Amazon region.

A new species of Myxobolus parasitizing the arterial bulb and cardiac musculature of the freshwater fish Pimelodus ornatus Kner, 1858, from the Arari river in the municipality of Cachoeira do Arari, island of Marajó, Pará, Brazil, was described. In the present study, the observed prevalence of myxozoan parasites in the heart tissue of the hosts was 20% (6/30). The myxozoans observed had mature biconvex spores, slightly rounded, an anterior end with two pyriform polar capsules and a posterior end with very evident sporoplasm, measuring 8 ± 0.2 µmin length. The spore width was 5.8 ± 0.4 µm, with a thickness of 3.4 ± 0.2µm. The length of the polar capsules was 3.6 ± 0.3 µm and the width was 1.2 ± 0.2µm, with 6 to 7 turns of the polar filament. The divergences observed, regarding the morphometric and genetic structure of SSU rDNA, in relation to other Myxobolidae already described in the literature, confirm the description of the new species Myxobolus rangeli n. sp.

Utrastructure and molecular phylogeny of the myxozoan Kudoa ocellatus n. sp. (Myxozoa: Kudoidae), a parasite of the Oscar, Astronotus ocellatus (Agassiz, 1831; Teleostei: Cichlidae), a fish from northern Brazil.

Kudoa ocellatus n. sp. was found in the musculature of Astronotus ocelattus (Agassiz, 1831) from the Arari River on Marajó Island in Pará, Brazil. The new species forms pseudocysts in the epaxial and hypaxial musculature composed of various spores that are pseudoquadrate in the apical view. In the lateral view, the spores were triangular or pyramidal. In the lateral view, the spores were 46 ± 0.11 µm (4.5-4.8) in length and 6.6 ± 0.3 µm (6.2-7.2) in width, with four pyriform polar capsules of equal size that measured 2.0 ± 0.16 µm (1.8-2.2) in length and 1.5 ± 0.18 µm (1.3-1.8) in width. Based on the partial (1418 bps) sequence of the SSU rDNA gene, Kudoa ocellatus n. sp. was distinct from all the other Kudoa species deposited in GenBank. The phylogenetic Bayesian Inference and P distance placed the new species together with the other Kudoa species that parasitize freshwater Amazonian fish. The morphological evidence, together with the SSU rDNA gene sequence, supported the description of Kudoa ocellatus n. sp., a distinct new species of the genus, which parasitizes a freshwater Amazonian cichlid.

A new Piper nigrum cysteine proteinase inhibitor, PnCPI, with antifungal activity: molecular cloning, recombinant expression, functional analyses and molecular modeling.

MAIN CONCLUSION: A new Piper nigrum cysteine proteinase inhibitor, PnCPI, belonging to group I of phytocystatins, with inhibitory activity against papain and growth of Fusarium solani f. sp. piperis, was isolated and characterized. Previous studies (de Souza et al. 2011) have identified a partial cDNA sequence of putative cysteine proteinase inhibitor differentially expressed in roots of black pepper (P. nigrum L.) infected by F. solani f. sp. piperis. Here, we aimed to isolate the full-length cDNA and genomic sequences of the P. nigrum cysteine proteinase inhibitor gene, named PnCPI. Sequence analyses showed that the PnCPI gene encodes a deduced protein of 108 amino acid residues with a predicted molecular mass of 12.3 kDa and isoelectric point of 6.51. Besides the LARFAV-like sequence, common to all phytocystatins, PnCPI contains three conserved motifs of the superfamily cystatin: a glycine residue at the N-terminal region, the QxVxG reactive site more centrally positioned, and one tryptophan in the C-terminal region. PnCPI, belonging to group I of phytocystatins, showed high identity with cystatins isolated from several plant species. Sequence analyses also revealed no putative signal peptide at the N-terminal of PnCPI, as well as no introns within the genomic sequence corresponding to the PnCPI coding region. Molecular modeling showed the ability of PnCPI to interact with papain, while its inhibitory activity against this protease was confirmed after heterologous expression in Escherichia coli. The effects of heat treatments on the inhibitory activity of recombinant PnCPI, rPnCPI, were evaluated. In addition, rPnCPI exhibited in vitro activity against F. solani f. sp. piperis, revealing a new cystatin with the potential antifungal application. The identification of PnCPI as a functional cystatin able to inhibit the in vitro growth of F. solani f. sp. piperis indicates other factors contributing to in vivo susceptibility of black pepper to root rot disease.

Culture medium and inoculation methodology for the study of soft root rot caused by Phytopythium sp / Meio de cultura e metodologia de inoculação para o estudo da podridão mole de raízes causada por Phytopythium sp

ABSTRACT: Cassava (Manihot esculenta Crantz) is an important crop in Brazil and Pará is the major producer of roots. High temperature and humidity of tropical regions favor the development of various diseases, among them the cassava root rot. The objective of this study was to evaluate the effect of luminosity and culture medium on the mycelial growth and sporulation of Phytopythium sp. associated with different methods of inoculation on cassava roots. In vitro tests for pathogen growth were established in a 2 x 6 factorial design (luminosity x culture medium) with five replicates and the means were compared by t test (P≤0.05). The culture medium containing sweet cassava root produced greater mycelial development and higher pathogen sporulation and it was the most suitable medium for pathogen culture. The culture under absence of light generated better mycelial growth than culture under 12 hour of light. Regarding the type of inoculation, the response was better when deeper injuries were induced.

RESUMO: A mandioca (Manihot esculenta Crantz) é uma importante cultura para o Brasil, onde o Pará é o principal produtor de raízes. Regiões tropicais com alta umidade e temperatura favorecem o desenvolvimento de diversas doenças, como as podridões de raiz. O presente estudo tem como objetivo avaliar o efeito da luminosidade e de meios de cultura no crescimento micelial e na esporulação de Phytopythium sp. e analisar métodos de inoculação do patógeno em raízes de mandioca destacadas. Os ensaios in vitro foram instalados em esquema fatorial 2x6 (luminosidade x meio de cultura), com cinco repetições e as médias comparadas pelo teste t (p≤0,05). O meio de cultura contendo raiz de mandioca mansa proporcionou maior desenvolvimento micelial e maior esporulação do patógeno e é o mais adequado para o cultivo do patógeno. O cultivo sob ausência de luz gerou melhor crescimento micelial do que o cultivo sob 12 horas de luz. Quanto ao tipo de inoculação, a resposta foi melhor nas raízes que obtiveram ferimentos mais profundos.

Ellipsomyxa arariensis n. sp. (Myxozoa: Ceratomyxidae), a new myxozoan parasite of Pygocentrus nattereri Kner, 1858 (Teleostei: Characidae) and Pimelodus ornatus Kner, 1858 (Teleostei: Pimelodidae) from Marajó Island, in the Brazilian Amazon region.

Ellipsomyxa arariensis n. sp. was found in the gallbladder of Pygocentrus nattereri Kner, 1858 and Pimelodus ornatus Kner, 1858 from the Arari River on Marajó Island in Pará, Brazil. The new species has disporous plasmodium that varies in size and shape, with ellipsoidal mature spores in the sutural view that have a curved suture line. The spores are 12.6 (12.0-13.4) µm in length and 7.3 (6.7-8.0) µm in width. The two polar capsules present in the spore are pyriform and of equal size, with subterminal openings that project in opposite directions. The polar capsules are 3.5 (3.4-4.0) µm long and 2.6 (2.5-3.2) µm wide. Based on the partial sequences of the SSU rRNA gene of the Ellipsomyxa arariensis n. sp. spores found in Pygocentrus nattereri Kner, 1858 (1325 bps) and Pimelodus ornatus Kner, 1858 (1240 bps), the new species is clearly distinct from all the other myxozoan sequences deposited in GenBank. Based on Bayesian inference and p distances, the new species belongs to the "Ellipsomyxa clade", together with all the other Ellipsomxa species, reinforcing the monophyletic status of this genus. Overall, the morphological data and the partial sequences of the SSU rRNA gene provide a conclusive diagnosis of Ellipsomyxa arariensis n. sp. as a species distinct from all the other Ellipsomyxa species described previously.

Molecular cloning and characterization of a cassava translationally controlled tumor protein gene potentially related to salt stress response.

Cassava (Manihot esculenta Crantz) is one of the most important tropical crops showing tolerance to abiotic stress and adaptations to a wide range of environmental conditions. Here, we aimed to isolate and characterize the full-length cDNA and genomic sequences of a cassava translationally controlled tumor protein gene (MeTCTP), and evaluate its potential role in response to salt stress. The MeTCTP full-length cDNA sequence encodes for a deduced protein with 168 amino acid residues, with theoretical isoelectric point and molecular weight of 4.53 and 19 kDa, respectively, containing two putative signatures of TCTP family and one site for myristoylation. The MeTCTP genomic sequence includes four introns and five exons within a 1,643 bp coding region, and a 264 bp partial promoter sequence containing several putative cis-acting regulatory elements, among them, two putative GT-1 motifs, which may be related to response to sodium chloride (NaCl) and pathogen infection. Semi-quantitative RT-PCR assays showed that MeTCTP transcripts were higher in roots than leaves, and were significantly increased in detached leaves treated with NaCl. Furthermore, the recombinant MeTCTP conferred a protective function against salt stress in bacterial cells. We report for the first time the molecular cloning and characterization of a cassava TCTP with potential role in salt-stress response. Since salinity is one the most important abiotic factors affecting the production of crops worldwide, the MeTCTP gene could be a candidate gene for generation of salt tolerant crops.

Recent molecular advances on downstream plant responses to abiotic stress.

Abiotic stresses such as extremes of temperature and pH, high salinity and drought, comprise some of the major factors causing extensive losses to crop production worldwide. Understanding how plants respond and adapt at cellular and molecular levels to continuous environmental changes is a pre-requisite for the generation of resistant or tolerant plants to abiotic stresses. In this review we aimed to present the recent advances on mechanisms of downstream plant responses to abiotic stresses and the use of stress-related genes in the development of genetically engineered crops.