Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

3-Hydroxypropionic Acid as an Antibacterial Agent from Endophytic Fungi Diaporthe phaseolorum

Endophytic fungi are considered a rich source of active compounds resulting from their secondary metabolism. Fungi from marine environment grow in a habitat with unique conditions that can contribute to the activation of metabolic pathways of synthesis of different unknown molecules. The production of these compounds may support the adaptation and survival of the fungi in the marine ecosystem. Mangroves are ecosystems situated between land and sea. They are frequently found in tropical and subtropical areas and enclose approximately 18.1 million hectares of the planet. The great biodiversity found in these ecosystems shows the importance of researching them, including studies regarding new compounds derived from the endophytic fungi that inhabit these ecosystems. 3-hydroxypropionic acid (3-HPA) has been isolated from the mangrove endophytic fungus Diaporthe phaseolorum, which was obtained from branches of Laguncularia racemosa. The structure of this compound was elucidated by spectroscopic methods, mainly 1D and 2D NMR. In bioassays, 3-HPA showed antimicrobial activities against both Staphylococcus aureus and Salmonella typhi. The structure of this antibiotic was modified by the chemical reaction of Fischer-Speier esterification to evaluate the biologic activity of its chemical analog. The esterified product, 3-hydroxypropanoic ethyl ester, did not exhibit antibiotic activity, suggesting that the free carboxylic acid group is important to the pharmacological activity. The antibiotic-producing strain was identified with internal transcribed spacer sequence data. To the best of our knowledge, this is the first report of antibacterial activity by 3-HPA against the growth of medically important pathogens.

Production of a xylose-stimulated beta-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of beta-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 +/- A 411.2 U g(-1), while beta-glucosidase production was increased about 2.6-fold, reaching 20.7 +/- A 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis beta-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for beta-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The beta-glucosidase maintained about 95 % of its activity after 26 h in water at 55 A degrees C, with half-lives of 15.7 h at 60 A degrees C and 5.1 h at 65 A degrees C. The presence of xylose during heat treatment at 65 A degrees C protected beta-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 A degrees C. Xylose stimulated beta-glucosidase activity up to 1.7-fold, at 200 mmol L-1. The notable features of both xylanase and beta-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

Effects of Natural Radiation, Photosynthetically Active Radiation and Artificial Ultraviolet Radiation-B on the Chloroplast Organization and Metabolism of Porphyra acanthophora var. brasiliensis (Rhodophyta, Bangiales)

We undertook a study of Porphyra acanthophora var. brasiliensis to determine its responses under ambient conditions, photosynthetically active radiation (PAR), and PAR+UVBR (ultraviolet radiation-B) treatment, focusing on changes in ultrastructure, and cytochemistry. Accordingly, control ambient samples were collected in the field, and two different treatments were performed in the laboratory. Plants were exposed to PAR at 60 mu mol photons m(-2) s(-1) and PAR+UVBR at 0.35 W m(-2) for 3 h per day during 21 days of in vitro cultivation. Confocal laser scanning microscopy analysis of the vegetative cells showed single stellate chloroplast in ambient and PAR samples, but in PAR+UVBR-exposed plants, the chloroplast showed alterations in the number and form of arms. Under PAR+UVBR treatment, the thylakoids of the chloroplasts were disrupted, and an increase in the number of plastoglobuli was observed, in addition to mitochondria, which appeared with irregular, disrupted morphology compared to ambient and PAR samples. After UVBR exposure, the formation of carpospores was also observed. Plants under ambient conditions, as well as those treated with PAR and PAR+UVBR, all showed different concentrations of enzymatic response, including glutathione peroxidase and reductase activity. In summary, the present study demonstrates that P. acanthophora var. brasiliensis shows the activation of distinct mechanisms against natural radiation, PAR and PAR+UVBR.

Evaluation of the genetic diversity of Histoplasma capsulatum var. capsulatum isolates from north-eastern Brazil

Since the beginning of the HIV epidemic, there has been a significant increase in the number of histoplasmosis cases in Ceara, a state in north-east Brazil. The lack of epidemiological data on the genotypes circulating in the north-east region shows the importance of more detailed studies on the molecular epidemiology of Histoplasma capsulatum var. capsulatum in this region. Different molecular techniques have been used to better characterize the genetic profile of H. capsulatum var. capsulatum strains. The aim of this study was to analyse the genetic diversity of H. capsulatum var. capsulatum isolates in Fortaleza, the capital of Ceara, through the sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, and establish the molecular profile of these isolates, along with strains from south-east Brazil, by RAPD analysis, featuring the different clusters in those regions. The isolates were grouped into two clusters. Cluster 1 included strains from the south-east and north-east regions with separation of isolates into three distinct subgroups (subgroups 1a, 1 b and 1 c). Cluster 2 included only samples from north-east Brazil. Sequencing of the ITS1 -5.8S-ITS2 region allowed the detection of two major clades, which showed geographical correlation between them and their subgroups. Therefore, it can be concluded that the H. capsulatum var. capsulatum isolates from Ceara have a high degree of genetic polymorphism. The molecular data also confirm that populations of this fungus are composed of different genotypes in Brazil and worldwide.

Endophytic fungi from Vitis labrusca L. ('Niagara Rosada') and its potential for the biological control of Fusarium oxysporum

We investigated the diversity of endophytic fungi found on grape (Vitis labrusca cv. Niagara Rosada) leaves collected from Salesopolis, SP, Brazil. The fungi were isolated and characterized by amplified ribosomal DNA restriction analysis, followed by sequencing of the ITS1-5.8S-ITS2 rDNA. In addition, the ability of these endophytic fungi to inhibit the grapevine pathogen Fusarium oxysporum f. sp herbemontis was determined in vitro. We also observed that the climatic factors, such as temperature and rainfall, have no effect on the frequency of infection by endophytic fungi. The endophytic fungal community that was identified included Aporospora terricola, Aureobasidium pullulans, Bjerkandera adusta, Colletotrichum boninense, C. gloeosporioides, Diaporthe helianthi, D. phaseolorum, Epicoccum nigrum, Flavodon flavus, Fusarium subglutinans, F. sacchari, Guignardia mangiferae, Lenzites elegans, Paraphaeosphaeria pilleata, Phanerochaete sordida, Phyllosticta sp, Pleurotus nebrodensis, Preussia africana, Tinctoporellus epiniltinus, and Xylaria berteri. Among these isolates, two, C. gloeosporioides and F. flavus, showed potential antagonistic activity against F. oxysporum f. sp herbemontis. We suggest the involvement of the fungal endophyte community of V. labrusca in protecting the host plant against pathogenic Fusarium species. Possibly, some endophytic isolates could be selected for the development of biological control agents for grape fungal disease; alternatively, management strategies could be tailored to increase these beneficial fungi.

Diversity and genetic structure in natural populations of Hancornia speciosa var. speciosa Gomes in northeastern Brazil

Hancornia speciosa Gomes is a fruit tree native from Brazil that belongs to Apocinaceae family, and is popularly known as Mangabeira. Its fruits are widely consumed raw or processed as fruit jam, juices and ice creams, which have made it a target of intense exploitation. The extractive activities and intense human activity on the environment of natural occurrence of H. speciosa has caused genetic erosion in the species and little is known about the ecology or genetic structure of natural populations. The objective of this research was the evaluation of the genetic diversity and genetic structure of H. speciosa var. speciosa. The genetic variability was assessed using 11 allozyme loci with a sample of 164 individuals distributed in six natural populations located in the States of Pernambuco and Alagoas, Northeastern Brazil. The results showed a high level of genetic diversity within the species (e= 0.36) seeing that the most of the genetic variability of H. speciosa var. speciosa is within its natural populations with low difference among populations ( or = 0.081). The inbreeding values within ( = -0.555) and among populations ( =-0.428) were low showing lacking of endogamy and a surplus of heterozygotes. The estimated gene flow ( m ) was high, ranging from 2.20 to 13.18, indicating to be enough to prevent the effects of genetic drift and genetic differentiation among populations. The multivariate analyses indicated that there is a relationship between genetic and geographical distances, which was confirmed by a spatial pattern analysis using Mantel test (r = 0.3598; p = 0.0920) with 1000 random permutations. The high genetic diversity index in these populations indicates potential for in situ genetic conservation.

Efeito de restritores hídricos sobre a germinação, comprimento da radícula e níveis de detecção de Colletotrichum gossypii var. cephalosporioides em sementes de algodão

O fungo Colletotrichum gossypii var. cephalosporioides, agente causal da ramulose do algodoeiro, é transmitido pela semente que se constitui em uma das mais importantes fontes de inóculo inicial e de introdução da doença em áreas indenes. Para que se possa identificar sua presença em lotes de sementes, é importante que se empreguem métodos de detecção rápidos e seguros. O mais empregado é o do papel de filtro, que se baseia na avaliação de sinais do patógeno desenvolvidos sobre as sementes, seguida da sua identificação morfológica. O método apresenta a desvantagem do crescimento das plântulas no período de incubação das sementes que pode favorecer o desenvolvimento de outros fungos e prejudicar a caracterização do patógeno. Para minimizar este problema vem sendo empregada a técnica da restrição hídrica. O presente trabalho teve como objetivo avaliar o efeito de três solutos em dois potenciais osmóticos, comparados ao tratamento padrão de água destilada, ao congelamento e ao 2,4 D, sobre a germinação, comprimento da radícula e detecção do agente causal da ramulose, durante o teste de sanidade. Os solutos Manitol e NaCl foram mais eficientes em inibir a germinação e favorecer a incidência do patógeno no potencial osmótico de -0,8 MPa. O KCl mostrou-se eficiente em inibir a germinação nos dois potenciais osmóticos testados, -0,6 e -0,8 MPa, porém reduziu a incidência do patógeno no potencial de -0,8 MPa. Os solutos Manitol, nos potenciais osmóticos de -0,8 e -0,6 MPa e o NaCl no potencial osmótico de -0,8 foram eficientes em reduzir o comprimento da radícula, sem interferir negativamente nos níveis de detecção de C. gossypii var. cephalosporioides, podendo ser recomendados para uso em análises sanitárias de rotina.