Filtrates from cultures of endophytic fungi isolated from leaves of Copaifera oblongifolia (Fabaceae) affect germination and seedling development differently / Fungos endofíticos isolados de folhas de Copaifera oblongifolia (Fabaceae) afetam diferentemente a germinação e o desenvolvimento das plântulas

Abstract Interactions between endophytic fungi (EFs) and their host plants range from positive to neutral to negative. The results of such interactions can vary depending on the organ of the infected host plant. EFs isolated from the leaves of some species of plants have potential for use as agents to inhibit seed germination and control invasive plants. The objectives of this study were to identify EFs present in the leaves of Copaifera oblongifolia and to evaluate the role of these fungi in seed germination and seedling development. A total of 11 species of EFs were isolated, which were identified using the internal transcribed spacers (ITS) sequence of the nuclear ribosomal DNA. The isolated species of EFs are generalists and probably are transmitted horizontally. Laboratory tests revealed that filtrates of these fungal isolates differently affect seed germination and seedling development of C. oblongifolia. The species Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum and Phomopsis sp. negatively affected seed germination, with N. parvum standing out for its negative effects, inhibiting seedling germination and survival in 89 and 222%, respectively. In addition, Cochliobolus intermedius negatively affected seedling development. Thus, the combined use of N. parvum and C. intermedius, or products from the metabolism of these microorganisms, in the control of invasive plants deserves attention from future studies.

Resumo As interações entre fungos endofíticos (FEs) e suas plantas hospedeiras variam de positivas, neutras a negativas. Os resultados destas interações podem variar dependendo do órgão da planta hospedeira infectada. FEs isolados de folhas de algumas espécies de plantas têm potencial para serem usados como agentes inibidores da germinação de sementes e no controle de plantas invasoras. Os objetivos deste estudo foram identificar os FEs presentes nas folhas de Copaifera oblongifolia e avaliar o papel destes fungos na germinação das sementes e no desenvolvimento das plântulas. Um total de 11 espécies de FEs foi isolado das folhas de C. oblongifolia e identificado através da sequência dos espaçadores internos transcritos do DNA ribossomal nuclear. As espécies de FEs isoladas são generalistas e provavelmente devem ser transmitidas horizontalmente. Os resultados dos testes de germinação mostraram que filtrados destes isolados fúngicos podem afetar diferentemente a germinação das sementes e o desenvolvimento das plântulas de C. oblongifolia. As espécies Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum e Phomopsis sp. afetaram negativamente a germinação das sementes de C. oblongifolia. Dentre estas espécies devemos destacar que N. parvum reduziu a germinação e a sobrevivência das plântulas em 89 e 222%, respectivamente. Além disso, Cochiliobolus intermedius afetou negativamente o desenvolvimento das plântulas. Assim, o uso combinado de N. parvum e C. intermedius, ou de produtos do metabolismo destas espécies de fungos, têm potencial para serem usados no manejo de plantas invasoras.

Filtrates from cultures of endophytic fungi isolated from leaves of Copaifera oblongifolia (Fabaceae) affect germination and seedling development differently / Fungos endofíticos isolados de folhas de Copaifera oblongifolia (Fabaceae) afetam diferentemente a germinação e o desenvolvimento das plântulas

Interactions between endophytic fungi (EFs) and their host plants range from positive to neutral to negative. The results of such interactions can vary depending on the organ of the infected host plant. EFs isolated from the leaves of some species of plants have potential for use as agents to inhibit seed germination and control invasive plants. The objectives of this study were to identify EFs present in the leaves of Copaifera oblongifolia and to evaluate the role of these fungi in seed germination and seedling development. A total of 11 species of EFs were isolated, which were identified using the internal transcribed spacers (ITS) sequence of the nuclear ribosomal DNA. The isolated species of EFs are generalists and probably are transmitted horizontally. Laboratory tests revealed that filtrates of these fungal isolates differently affect seed germination and seedling development of C. oblongifolia. The species Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum and Phomopsis sp. negatively affected seed germination, with N. parvum standing out for its negative effects, inhibiting seedling germination and survival in 89 and 222%, respectively. In addition, Cochliobolus intermedius negatively affected seedling development. Thus, the combined use of N. parvum and C. intermedius, or products from the metabolism of these microorganisms, in the control of invasive plants deserves attention from future studies.

As interações entre fungos endofíticos (FEs) e suas plantas hospedeiras variam de positivas, neutras a negativas. Os resultados destas interações podem variar dependendo do órgão da planta hospedeira infectada. FEs isolados de folhas de algumas espécies de plantas têm potencial para serem usados como agentes inibidores da germinação de sementes e no controle de plantas invasoras. Os objetivos deste estudo foram identificar os FEs presentes nas folhas de Copaifera oblongifolia e avaliar o papel destes fungos na germinação das sementes e no desenvolvimento das plântulas. Um total de 11 espécies de FEs foi isolado das folhas de C. oblongifolia e identificado através da sequência dos espaçadores internos transcritos do DNA ribossomal nuclear. As espécies de FEs isoladas são generalistas e provavelmente devem ser transmitidas horizontalmente. Os resultados dos testes de germinação mostraram que filtrados destes isolados fúngicos podem afetar diferentemente a germinação das sementes e o desenvolvimento das plântulas de C. oblongifolia. As espécies Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum e Phomopsis sp. afetaram negativamente a germinação das sementes de C. oblongifolia. Dentre estas espécies devemos destacar que N. parvum reduziu a germinação e a sobrevivência das plântulas em 89 e 222%, respectivamente. Além disso, Cochiliobolus intermedius afetou negativamente o desenvolvimento das plântulas. Assim, o uso combinado de N. parvum e C. intermedius, ou de produtos do metabolismo destas espécies de fungos, têm potencial para serem usados no manejo de plantas invasoras.

Filtrates from cultures of endophytic fungi isolated from leaves of Copaifera oblongifolia (Fabaceae) affect germination and seedling development differently

Abstract Interactions between endophytic fungi (EFs) and their host plants range from positive to neutral to negative. The results of such interactions can vary depending on the organ of the infected host plant. EFs isolated from the leaves of some species of plants have potential for use as agents to inhibit seed germination and control invasive plants. The objectives of this study were to identify EFs present in the leaves of Copaifera oblongifolia and to evaluate the role of these fungi in seed germination and seedling development. A total of 11 species of EFs were isolated, which were identified using the internal transcribed spacers (ITS) sequence of the nuclear ribosomal DNA. The isolated species of EFs are generalists and probably are transmitted horizontally. Laboratory tests revealed that filtrates of these fungal isolates differently affect seed germination and seedling development of C. oblongifolia. The species Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum and Phomopsis sp. negatively affected seed germination, with N. parvum standing out for its negative effects, inhibiting seedling germination and survival in 89 and 222%, respectively. In addition, Cochliobolus intermedius negatively affected seedling development. Thus, the combined use of N. parvum and C. intermedius, or products from the metabolism of these microorganisms, in the control of invasive plants deserves attention from future studies.

Resumo As interações entre fungos endofíticos (FEs) e suas plantas hospedeiras variam de positivas, neutras a negativas. Os resultados destas interações podem variar dependendo do órgão da planta hospedeira infectada. FEs isolados de folhas de algumas espécies de plantas têm potencial para serem usados como agentes inibidores da germinação de sementes e no controle de plantas invasoras. Os objetivos deste estudo foram identificar os FEs presentes nas folhas de Copaifera oblongifolia e avaliar o papel destes fungos na germinação das sementes e no desenvolvimento das plântulas. Um total de 11 espécies de FEs foi isolado das folhas de C. oblongifolia e identificado através da sequência dos espaçadores internos transcritos do DNA ribossomal nuclear. As espécies de FEs isoladas são generalistas e provavelmente devem ser transmitidas horizontalmente. Os resultados dos testes de germinação mostraram que filtrados destes isolados fúngicos podem afetar diferentemente a germinação das sementes e o desenvolvimento das plântulas de C. oblongifolia. As espécies Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum e Phomopsis sp. afetaram negativamente a germinação das sementes de C. oblongifolia. Dentre estas espécies devemos destacar que N. parvum reduziu a germinação e a sobrevivência das plântulas em 89 e 222%, respectivamente. Além disso, Cochiliobolus intermedius afetou negativamente o desenvolvimento das plântulas. Assim, o uso combinado de N. parvum e C. intermedius, ou de produtos do metabolismo destas espécies de fungos, têm potencial para serem usados no manejo de plantas invasoras.

Filtrates from cultures of endophytic fungi isolated from leaves of Copaifera oblongifolia (Fabaceae) affect germination and seedling development differently.

Interactions between endophytic fungi (EFs) and their host plants range from positive to neutral to negative. The results of such interactions can vary depending on the organ of the infected host plant. EFs isolated from the leaves of some species of plants have potential for use as agents to inhibit seed germination and control invasive plants. The objectives of this study were to identify EFs present in the leaves of Copaifera oblongifolia and to evaluate the role of these fungi in seed germination and seedling development. A total of 11 species of EFs were isolated, which were identified using the internal transcribed spacers (ITS) sequence of the nuclear ribosomal DNA. The isolated species of EFs are generalists and probably are transmitted horizontally. Laboratory tests revealed that filtrates of these fungal isolates differently affect seed germination and seedling development of C. oblongifolia. The species Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum and Phomopsis sp. negatively affected seed germination, with N. parvum standing out for its negative effects, inhibiting seedling germination and survival in 89 and 222%, respectively. In addition, Cochliobolus intermedius negatively affected seedling development. Thus, the combined use of N. parvum and C. intermedius, or products from the metabolism of these microorganisms, in the control of invasive plants deserves attention from future studies.

Obtainment, selection and characterization of a mutant strain of Kluyveromyces marxianus that displays improved production of 2-phenylethanol and enhanced DAHP synthase activity.

AIMS: Yeasts produce 2-phenylethanol (2-PE) from sugars via de novo synthesis; however, its synthesis is limited due to feedback inhibition on the isofunctional 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthases (Aro3p and Aro4p). This work aimed to select Kluyveromyces marxianus mutant strains with improved capacity to produce 2-PE from sugars. METHODS AND RESULTS: Kluyveromyces marxianus CCT 7735 mutant strains were selected from UV irradiation coupled with screening of p-fluoro-dl-phenylalanine (PFP) tolerant strains on culture medium without l-Phe addition. Most of them produced 2-PE titres higher than the parental strain and the Km_PFP41 mutant strain stood out for displaying the highest 2-PE specific production rate. Moreover it showed higher activity of DAHP synthase than the parental strain. We sequenced both ARO3 and ARO4 genes of Km_PFP41 mutant and identified mutations in ARO4 which caused changes in both size and conformation of the Aro4p. These changes seem to be associated with the enhanced activity of DAHP synthase and improved production of 2-PE exhibited by that mutant strain. CONCLUSIONS: The Km_PFP41 mutant strain presented improved 2-PE production via de novo synthesis and enhanced DAHP synthase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant strain obtained in this work may be exploited as a yeast cell factory for high-level synthesis of 2-PE.

Genotypic and Pathogenic Diversity of Colletotrichum sublineola Isolates from Sorghum (Sorghum bicolor) and Johnsongrass (S. halepense) in the Southeastern United States.

Anthracnose caused by Colletotrichum sublineola is an important disease of cultivated sorghum (Sorghum bicolor) worldwide. Anthracnose is also common on the ubiquitous wild sorghum relative Johnsongrass (S. halepense). Analysis of repetitive molecular fingerprinting markers revealed that isolates of C. sublineola from both hosts in the southeastern United States were genotypically diverse, with relatively few haplotypes found in more than one location. With few exceptions, isolates recovered from S. bicolor belonged to a population that was genetically distinct from the population recovered from S. halepense. Twenty-three isolates from cultivated sorghum were all pathogenic to at least one of 13 heritage inbred lines of S. bicolor. In all, 4 of 10 isolates from S. halepense were also pathogenic to one or more of the lines, while the rest caused no disease in greenhouse assays. The four pathogenic isolates from S. halepense were less aggressive, on average, than isolates from S. bicolor, although the ranges overlapped. Pathogenicity tests involving 15 representative pathogenic isolates from S. bicolor and S. halepense on eight heritage inbred lines of S. bicolor identified 12 races. The combined results of this study demonstrated that C. sublineola comprises two separate host-associated subpopulations in the field, even though some isolates from S. halepense were able to cause disease on S. bicolor under ideal greenhouse conditions. Nonetheless, the apparent existence of infrequent cross-infection events in the field, indicated by molecular fingerprinting, suggests that Johnsongrass has the potential to serve as a refuge and an incubator for genetic diversity in C. sublineola, which can complicate efforts to develop and deploy resistant sweet sorghum varieties in the region.

High genetic variability in endophytic fungi from the genus Diaporthe isolated from common bean (Phaseolus vulgaris L.) in Brazil.

AIMS: The goals of the present study were to identify, to analyse the phylogenetic relations and to evaluate the genetic variability in Diaporthe endophytic isolates from common bean. METHODS AND RESULTS: Diaporthe sp., D. infecunda and D. phaseolorum strains were identified using multilocus phylogeny (rDNA ITS region; EF1-α, ß-tubulin, and calmodulin genes). IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) molecular markers reveal the existence of high genetic variability, especially among D. infecunda isolates. CONCLUSIONS: It was concluded that the multilocus phylogenetic approach was more effective than individual analysis of ITS sequences, in identifying the isolates to species level, and that IRAP and REMAP markers can be used for studying the genetic variability in the genus Diaporthe particularly at the intraspecific level. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined use of molecular tools such as multilocus phylogenetic approach and molecular markers, as performed in this study, is the best way to distinguish endophytic strains of Diaporthe isolated from common bean (Phaseolus vulgaris L.).

Involvement of the ornithine decarboxylase gene in acid stress response in probiotic Lactobacillus delbrueckii UFV H2b20.

Amino acid decarboxylation is important for the maintenance of intracellular pH under acid stress. This study aims to carry out phylogenetic and expression analysis by real-time PCR of two genes that encode proteins involved in ornithine decarboxylation in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress. Sequencing and phylogeny analysis of genes encoding ornithine decarboxylase and amino acid permease in L. delbrueckii UFV H2b20 showed their high sequence identity (99%) and grouping with those of L. delbrueckii subsp. bulgaricus ATCC 11842. Exposure of L. delbrueckii UFV H2b20 cells in MRS pH 3.5 for 30 and 60 min caused a significant increase in expression of the gene encoding ornithine decarboxylase (up to 8.1 times higher when compared to the control treatment). Increased expression of the ornithine decarboxylase gene demonstrates its involvement in acid stress response in L. delbrueckii UFV H2b20, evidencing that the protein encoded by that gene could be involved in intracellular pH regulation. The results obtained show ornithine decarboxylation as a possible mechanism of adaptation to an acidic environmental condition, a desirable and necessary characteristic for probiotic cultures and certainly important to the survival and persistence of the L. delbrueckii UFV H2b20 in the human gastrointestinal tract.

Endophytic fungi from the genus Colletotrichum are abundant in the Phaseolus vulgaris and have high genetic diversity.

AIMS: To evaluate the diversity of endophytic fungi from the leaves of the common bean and the genetic diversity of endophytic fungi from the genus Colletotrichum using IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism) analyses. METHODS AND RESULTS: The fungi were isolated by tissue fragmentation and identified by analysing the morphological features and sequencing the internal transcribed spacer (ITS) regions and the rDNA large subunit (LSU). Twenty-seven different taxa were identified. Colletotrichum was the most commonly isolated genera from the common bean (32.69% and 24.29% of the total isolates from the Ouro Negro and Talismã varieties, respectively). The IRAP and REMAP analyses revealed a high genetic diversity in the Colletotrichum endophytic isolates and were able to discriminate these isolates from the phytopathogen Colletotrichum lindemuthianum. CONCLUSIONS: Fungi from the genus Colletotrichum are abundant in the Phaseolus vulgaris endophytic community, and the IRAP and REMAP markers can be used to rapidly distinguish between C. lindemuthianum and other Colletotrichum members that are frequently found as endophytes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the diversity of endophytic fungi present in the common bean and the use of IRAP and REMAP markers to assess the genetic diversity of endophytic fungi from the genus Colletotrichum.

Increased expression of clp genes in Lactobacillus delbrueckii UFV H2b20 exposed to acid stress and bile salts.

The ability to survive in harsh environments is an important criterion to select potential probiotics strains. The objective of this study was to identify and carry out phylogenetic and expression analysis by quantitative real-time PCR of the clpP, clpE, clpL and clpX genes in the probiotic strain Lactobacillus delbrueckii UFV H2b20 exposed to the conditions prevailing in the gastrointestinal tract (GIT). Phylogenetic trees reconstructed by Bayesian inference showed that the L. delbrueckii UFV H2b20 clpP, clpL and clpE genes and the ones from L. delbrueckii ATCC 11842 were grouped. The exposure of cells to MRS broth of pH 3.5 for 30 and 60 min resulted in an increased expression of the four genes. Exposure of the L. delbrueckii UFV H2b20 cells for 30 and 60 min to MRS broth containing 0.1% bile salts increased the expression of the clpP and clpE genes, while the expression level of the clpL and clpX genes increased only after 30 min of exposure. The involvement of the studied genes in the responses to acid stress and bile salts suggests a possible central role of these genes in the survival of L. delbrueckii UFV H2b20 during the passage through the GIT, a characteristic necessary for probiotic strains.

Carboxyl ester hydrolase from Penicillium expansum: cloning, characterization and overproduction by Penicillium griseoroseum.

AIMS: In this study, a gene that encodes a carboxylesterase (carb) in Penicillium expansum GF was cloned, sequenced and overexpressed by Penicillium griseoroseum PG63, and the enzyme was characterized. METHODS AND RESULTS: The recombinant strain, P. griseoroseum T55, obtained upon transformation using the plasmid pAN-52-1-carb, showed integration of the carb gene into at least two heterologous sites of the genome by Southern blotting. Furthermore, the recombinant strain T55 exhibited almost a fourfold increase in carboxylesterase activity compared with PG63 strain when both were cultured without inducers. Based on the secondary structure and multiple sequence alignments with carboxylesterases, cholinesterase and lipase, a three-dimensional model was obtained. The α/ß barrel topology, that is typical of esterases and lipases, was indicated for the CARB protein with Ser(213)-Glu(341)-His(456) as the putative catalytic triad. CARB preferentially hydrolysed acyl chains with eight carbon atoms, and its activity was optimal at a pH of 7·0 and a temperature of 25°C. CARB exhibited stability in alkaline pH, high activity under mesophilic conditions and stability in organic solvents. CONCLUSION: The CARB protein is potentially useful in bioremediation, food and chemical/pharmaceutical industries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to report the development of a recombinant strain superproducing a Penicillium sp. carboxylesterase.

Structural and functional characterization of the Colletotrichum lindemuthianum nit1 gene, which encodes a nitrate eductase enzyme.

Colletotrichum lindemuthianum is the causal agent of plant bean anthracnose, one of the most important diseases affecting the common bean. We investigated the structure and expression of the nit1 gene (nitrate reductase) of C. lindemuthianum. The nit1 gene open reading frame contains 2787 bp, interrupted by a single 69-bp intron. The predicted protein has 905 amino acids; it shows high identity with the nitrate reductase of C. higginsianum (79%) and C. graminicola (73%). Expression of nit1 in C. lindemuthianum was evaluated in mycelia grown on different nitrogen sources under conditions of activation and repression. The gene was expressed after 15 min of induction with nitrate, reaching maximum expression at 360 min. The transcription was repressed in mycelia grown in media enriched with ammonia, urea or glutamine. Twenty nit1⁻ mutants were obtained in a medium treated with chlorate. Ten of these mutants were characterized by DNA hybridization, which identified point mutations, a deletion and an insertion. These rearrangements in the nit1 gene in the different mutants may have occurred through activity of transposable elements.

Damage of Whitefly (Bemisia Tabaci) (Gennadius, 1889 ) (Hemiptera: Aleyrodidae) and vertical distribution of nymphs in soybean Glycine Max (L.) merril cultivars in the greenhouse / Danos da Mosca- Branca Bemisia Tabaci (Genn. ) e distribuição vertical das ninfas em cultivares de soja em casa de vegetação

The whitefly (Bemisia tabaci) causes damage in the soybean crop by sucking the plant sap, transmission of viruses and promoting sooty mold that affect the growing and the soybean production. The goal of this work was to evaluate the direct damage of the whitefly, and the vertical nymph distribution, on soybean cultivars under greenhouse conditions. The experimental design was randomized blocks with 10 cultivars in a 10 x 2 factorial scheme (infestation and without infestation) and 4 replications. Counts were made of the number of eggs and nymphs in the upper, middle and lower third of the plants. The productive indices were: number of pods per plant and grains per pod, weight of 100 grains, and weight of 2 plants. The data were submitted to variance analysis and F test (; 0.05), and the means were compared by Tukey test at 5% probability, obtaining the least significant difference between treatments. The cultivars were not affected by the B. tabaci attack at the infestations levels registered. CD 219 RR was the cultivar most infested by the whitefly nymphs. For better efficiency and use of time in the evaluations, in greenhouse studies it is suggested that they be carried out in the upper and middle thirds of the soybean plants.

A mosca-branca Bemisia tabaci causa danos em plantas de soja em decorrência da sucção de seiva, transmissão de vírus e favorecimento da fumagina, que afetam o desenvolvimento e a produção da cultura. O objetivo deste trabalho foi avaliar os danos diretos da mosca-branca e a distribuição vertical de ninfas em cultivares de soja em condições de casa de vegetação. O delineamento experimental foi de blocos ao acaso, com 10 cultivares no esquema fatorial 10 x 2 (com e sem infestação) e 4 repetições. Contabilizou-se o número de ovos e ninfas em todos os folíolos e, separadamente, em um folíolo do terços superior, médio e inferior das plantas Os índices de produtividade analisados foram: número de vagens por planta e de grãos por vagem, massa de 100 grãos e massa total de duas plantas. Os dados foram submetidos à análise de variância e ao teste F (; 0,05) e as médias comparadas pelo teste de Tukey ao nível de 5% de probabilidade, obtendo-se as diferenças mínimas significativas entre os tratamentos. As cultivares estudadas não foram afetadas pelo ataque de B. tabaci nos níveis de infestação registrados. CD 219 RR foi o cultivar mais infestada por ninfas de mosca-branca. Para melhorar a eficiência e o uso de tempo nas avaliações, em estudos em casa de vegetação, indica-se que estas sejam realizadas nos terços superior e médio das plantas.

Genetic diversity analysis of isolates of the fungal bean pathogen Pseudocercospora griseola from central and southern Brazil.

Planting resistant varieties is the most effective control measure against the angular leaf spot of dry beans, a fungal disease caused by Pseudocercospora griseola. However, dry bean varieties with durable resistance are not easily obtained. Knowledge about the genetic variability of the pathogen population is key for the success of dry bean breeding programs aimed at developing resistant materials, but finding suitable operationally simple and genetically accurate markers is not an easy task. The aim of this study was to assess the suitability of the ISSR-PCR technique to quantify the genetic variability of P. griseola isolates. Total DNA of 27 P. griseola isolates from Goiás, Minas Gerais, Espírito Santo, and Paraná States was extracted and amplified using specific primers for ISSR. Using cluster analysis, 27 genotypes were identified. The ISSR-PCR technique was suitable for assessing intraspecific variability of P. griseola. The ISSR-PCR marker was found to be highly sensitive to genetic variation and can aid in elucidating the genetic structure of the population of this plant pathogen as a support tool for the dry bean breeding programs.

Improved pectinase production in Penicillium griseoroseum recombinant strains.

AIMS: To obtain recombinant strains of Penicillium griseoroseum that produce high levels of pectin lyase (PL) and polygalacturonase (PG) simultaneously. METHODS AND RESULTS: A strain with high production of PL was transformed with the plasmid pAN52pgg2, containing the gene encoding PG of P. griseoroseum, under control of the gpd promoter gene from Aspergillus nidulans. Southern blot analysis demonstrated that all strain had at least one copy of pAN52pgg2 integrated into the genome. The recombinant strain P. griseoroseum T20 produced levels of PL and PG that were 266- and 27-fold greater, respectively, than the wild-type strain. Furthermore, the extracellular protein profile of recombinant T20 showed two protein bands of c. 36 and 38 kDa, associated with PL and PG, respectively. CONCLUSIONS: This recombinant strain T20 produces PL and PG using carbon sources of low costs, and an enzyme preparation that is free of cellulolytic and proteolytic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: PL and PG play an important role in the degradation of pectin. Owing to their use in the juice and wines industries, there is a growing interest in the inexpensive production of these enzymes. This work describes an efficient system of protein expression and secretion using the fungus P. griseoroseum.

Chronic arthritis directly induces quantitative and qualitative bone disturbances leading to compromised biomechanical properties.

OBJECTIVES: Rheumatoid arthritis (RA) is associated with an increased risk of fragility fractures. In RA patients, the direct effect of inflammation on bone is difficult to study because their skeleton is also affected by medication with corticosteroids and other drugs as well as aging and menopause, which contribute to bone fragility. This study used an animal model of chronic arthritis to evaluate the direct impact of chronic inflammation on biomechanical properties and structure of bone. METHODS: In the SKG mouse chronic arthritis model three point bending tests were performed on femoral bones and compression tests on vertebral bodies. Collagen structure was analysed using second-harmonic generation (SHG) imaging with a two-photon microscope, ultramorphology by scanning electron microscopy (SEM) coupled with energy dispersive x-ray spectroscopy (EDS) and bone density using water pycnometer. RESULTS: Arthritic bones had poor biomechanical quality compared to control bones. SHG, SEM and pycnometry disclosed variable signs of impaired collagen organization, poor trabecular architecture and low bone density. CONCLUSION: Present data demonstrate for the first time that chronic inflammation per se, without confounding influence of drugs and aging, leads to impairment of bone biomechanics in terms of stiffness, ductility and ultimate strength (fracture).

Differential expression of plg genes from Penicillium griseoroseum: plg1 a pectinolytic gene is expressed in sucrose and yeast extract.

AIMS: To study the regulation of the plg1 and plg2 genes of Penicillium griseoroseum, in order to identify the industrial potential of their products in alternative carbon sources that are cheaper and widely available in Brazil. METHODS AND RESULTS: RT-PCR and Northern blot were used to investigate if plg1 and plg2 expression is under influence of catabolic repression, ambient pH and cAMP. Results demonstrated that the genes were differentially regulated depending on the carbon sources in the culture medium and pH. Sucrose, a noninducing carbon source of the pectinolytic system, was able to promote plg1 transcription but only when yeast extract was added into the culture medium. CONCLUSIONS: The plg genes are differentially expressed. The plg1 gene is more attractive for industrial use due to its expression in alternative carbon sources like sucrose and yeast extract. SIGNIFICANCE AND IMPACT OF THE STUDY: In recent years, industries have been trying to replace the toxic conventional treatments employed in these processes by more eco-friendly enzyme treatment. Alternative carbon sources will be tested with the aim to reduce the costs associated to pectin lyase production in Brazil.

Delayed neutrophil apoptosis in very early rheumatoid arthritis patients is abrogated by methotrexate therapy.

OBJECTIVES: To analyse the activation state and apoptosis of circulating neutrophils in untreated very early rheumatoid arthritis (VERA) and after exposure to low dose corticosteroids and methotrexate (MTX). METHODS: Neutrophils were isolated from the peripheral blood of VERA patients at 3 different times: before any treatment was started, 2 weeks after starting a low dose of prednisone (5-10 mg) and 4 months after reaching more than 20mg/week of MTX. The expression of different activation markers (CD11b, CD64, CD86 and CD69) in freshly isolated neutrophils was analysed by flow cytometry. Apoptosis was measured by the loss of DNA content, which was analysed by flow cytometry using propidium iodide. RESULTS: Compared to neutrophils from healthy controls, we have found a delayed neutrophil apoptosis within 6 h and 22 h of cultured polymorphonuclear leukocytes (PMN) derived from VERA patients without any treatment or treated with corticosteroids. The delay of PMN apoptosis was restored to control levels after treatment with MTX. CONCLUSION: The treatment of VERA patients with corticosteroids did not affect the delay of neutrophil apoptosis. However, delayed apoptosis was restored to control levels after treatment with low dose MTX, which highlights the importance of early RA treatment with MTX.

BCG lymphadenopathy detected in a BCG-vaccinated infant

Large-scale vaccination with BCG, the live attenuated strain of Mycobacterium bovis, is being adopted around the world, although sporadic complications have occurred after the procedure. Lymphadenopathy is not uncommon especially in babies under one year (0.73 percent of vaccinated infants), but the swelling subsides within 2 months in most cases, with no medical or surgical treatment. Brazil adopted BCG vaccination program earlier in the seventies and by 1995 more than 96 percent of the infant population received this immunization. We report here the occurrence of lymphadenopathy in a two-year-old child vaccinated with the Brazilian BCG strain. The diagnosis was made using a lymph node biopsy and intestinal aspirates that yielded a positive mycobacterial culture. The isolate was resistant to isoniazid, rifampicin, pyrazinamide and thiophen-2-carbonic acid hydrazide, sensitive to streptomycin, ethambutol, and p-nitrobenzoic acid, and reacted positively to cyclo-serine and negatively to niacin. The pncA gene involved in bacterial activation of pyrazinamide contains in M. bovis a point mutation that renders pyrazinamidase unable to catalyze drug activation. Therefore, this polymorphism is a good option for developing methods to differentiate M. bovis and M. tuberculosis. Taking advantage of this difference we further analyzed the isolates by single-stranded conformation polymorphism electrophoresis of DNA following PCR of the pncA gene. The isolate identity was confirmed by RFLP electrophoretic analysis of the amplified fragment following Eco065I digestion, which selectively cleaves M. tuberculosis DNA. From this result it is proposed that RFLP of pncA gene represents an alternative for differential diagnosis of M. bovis.