Colletotrichum siamense, a Mycovirus-Carrying Endophyte, as a Biological Control Strategy for Anthracnose in Guarana Plants

Abstract Guarana, the fruit of Paullinia cupana, is known for its stimulating and medicinal properties by the Amazonian indigenous population and communities. However, it presents serious phytopathological problems, such as anthracnose disease caused by Colletotrichum spp. The objective of this study was to verify if C. siamense, a mycovirus-carrying endophytic fungus, could protect guarana seedlings, by reducing or by eliminating characteristic disease symptoms. Other physiological changes in the plant caused by the presence of this endophyte were also evaluated. The cuttings of the Cereçaporanga cultivar were dipped in a biological control suspension and planted in a specific substrate. After four months in the greenhouse, the seedlings were sprayed with a suspension of phytopathogen conidia, and a portion of these seedlings received the fungicide indicated for the crop to be compared with the control seedlings. After 28 days, the number of lesions, morphophysiological and macro characteristics, and leaf micronutrients were evaluated. The seedlings treated with C. siamense showed a lower percentage of lesions and an increased aerial part and root system compared to the other treatments. There were no significant differences between treatments regarding the percentage of macronutrients and micronutrients.

Additive and heterozygous (dis)advantage GWAS models reveal candidate genes involved in the genotypic variation of maize hybrids to Azospirillum brasilense.

Maize genotypes can show different responsiveness to inoculation with Azospirillum brasilense and an intriguing issue is which genes of the plant are involved in the recognition and growth promotion by these Plant Growth-Promoting Bacteria (PGPB). We conducted Genome-Wide Association Studies (GWAS) using additive and heterozygous (dis)advantage models to find candidate genes for root and shoot traits under nitrogen (N) stress and N stress plus A. brasilense. A total of 52,215 Single Nucleotide Polymorphism (SNP) markers were used for GWAS analyses. For the six root traits with significant inoculation effect, the GWAS analyses revealed 25 significant SNPs for the N stress plus A. brasilense treatment, in which only two were overlapped with the 22 found for N stress only. Most were found by the heterozygous (dis)advantage model and were more related to exclusive gene ontology terms. Interestingly, the candidate genes around the significant SNPs found for the maize-A. brasilense association were involved in different functions previously described for PGPB in plants (e.g. signaling pathways of the plant's defense system and phytohormone biosynthesis). Our findings are a benchmark in the understanding of the genetic variation among maize hybrids for the association with A. brasilense and reveal the potential for further enhancement of maize through this association.

Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth

ABSTRACT Mangroves are ecosystems located in the transition zone between land and sea that serve as a potential source of biotechnological resources. Brazil's extensive coast contains one of the largest mangrove forests in the world (encompassing an area of 25,000 km2 along all the coast). Endophytic bacteria were isolated from the following three plant species: Rhizophora mangle, Laguncularia racemosa and Avicennia nitida. A large number of these isolates, 115 in total, were evaluated for their ability to fix nitrogen and solubilize phosphorous. Bacteria that tested positive for both of these tests were examined further to determine their level of indole acetic acid production. Two strains with high indole acetic acid production were selected for use as inoculants for reforestation trees, and then the growth of the plants was evaluated under field conditions. The bacterium Pseudomonas fluorescens (strain MCR1.10) had a low phosphorus solubilization index, while this index was higher in the other strain used, Enterobacter sp. (strain MCR1.48). We used the reforestation tree Acacia polyphylla. The results indicate that inoculation with the MCR1.48 endophyte increases Acacia polyphylla shoot dry mass, demonstrating that this strain effectively promotes the plant's growth and fitness, which can be used in the seedling production of this tree. Therefore, we successfully screened the biotechnological potential of endophyte isolates from mangrove, with a focus on plant growth promotion, and selected a strain able to provide limited nutrients and hormones for in plant growth.

Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth.

Mangroves are ecosystems located in the transition zone between land and sea that serve as a potential source of biotechnological resources. Brazil's extensive coast contains one of the largest mangrove forests in the world (encompassing an area of 25,000km2 along all the coast). Endophytic bacteria were isolated from the following three plant species: Rhizophora mangle, Laguncularia racemosa and Avicennia nitida. A large number of these isolates, 115 in total, were evaluated for their ability to fix nitrogen and solubilize phosphorous. Bacteria that tested positive for both of these tests were examined further to determine their level of indole acetic acid production. Two strains with high indole acetic acid production were selected for use as inoculants for reforestation trees, and then the growth of the plants was evaluated under field conditions. The bacterium Pseudomonas fluorescens (strain MCR1.10) had a low phosphorus solubilization index, while this index was higher in the other strain used, Enterobacter sp. (strain MCR1.48). We used the reforestation tree Acacia polyphylla. The results indicate that inoculation with the MCR1.48 endophyte increases Acacia polyphylla shoot dry mass, demonstrating that this strain effectively promotes the plant's growth and fitness, which can be used in the seedling production of this tree. Therefore, we successfully screened the biotechnological potential of endophyte isolates from mangrove, with a focus on plant growth promotion, and selected a strain able to provide limited nutrients and hormones for in plant growth.

Antibiotics produced by Streptomyces

Streptomyces is a genus of Gram-positive bacteria that grows in various environments, and its shape resembles filamentous fungi. The morphological differentiation of Streptomyces involves the formation of a layer of hyphae that can differentiate into a chain of spores. The most interesting property of Streptomyces is the ability to produce bioactive secondary metabolites, such as antifungals, antivirals, antitumorals, anti-hypertensives, immunosuppressants, and especially antibiotics. The production of most antibiotics is species specific, and these secondary metabolites are important for Streptomyces species in order to compete with other microorganisms that come in contact, even within the same genre. Despite the success of the discovery of antibiotics, and advances in the techniques of their production, infectious diseases still remain the second leading cause of death worldwide, and bacterial infections cause approximately 17 million deaths annually, affecting mainly children and the elderly. Self-medication and overuse of antibiotics is another important factor that contributes to resistance, reducing the lifetime of the antibiotic, thus causing the constant need for research and development of new antibiotics.

Antibiotics produced by Streptomyces.

Streptomyces is a genus of Gram-positive bacteria that grows in various environments, and its shape resembles filamentous fungi. The morphological differentiation of Streptomyces involves the formation of a layer of hyphae that can differentiate into a chain of spores. The most interesting property of Streptomyces is the ability to produce bioactive secondary metabolites, such as antifungals, antivirals, antitumorals, anti-hypertensives, immunosuppressants, and especially antibiotics. The production of most antibiotics is species specific, and these secondary metabolites are important for Streptomyces species in order to compete with other microorganisms that come in contact, even within the same genre. Despite the success of the discovery of antibiotics, and advances in the techniques of their production, infectious diseases still remain the second leading cause of death worldwide, and bacterial infections cause approximately 17 million deaths annually, affecting mainly children and the elderly. Self-medication and overuse of antibiotics is another important factor that contributes to resistance, reducing the lifetime of the antibiotic, thus causing the constant need for research and development of new antibiotics.

Cytological characterization of an Aspergillus nidulans mutant from a strain with chromosomic duplication

A development mutant, named V103, was obtained spontaneously from the A strain of A. nidulans. The A strain contains a duplicated segment of chromosome I that has undergone translocation to chromosome II (I ¨ II). It is mitotically unstable and generates phenotypically deteriorated types, some with enhanced stability. The deteriorated variants of A. nidulans show abnormal development, exhibiting slower colony growth, variations in colony pigmentation and changes in conidiophore structure. The alterations observed in the conidiophore include fewer metulae and phialides, further elongation and ramification of these structures, delayed nuclear migration and the presence of secondary conidiophores.

High molecular diversity of the fungus Guignardia citricarpa and Guignardia mangiferae and new primers for the diagnosis of the citrus black spot

RAPD markers were used to investigate the distribution of genetic variability among a group of Guignardia citricarpa, G. mangiferae, and Phyllosticta spinarum isolates obtained from several hosts in Brazil, Argentina, Mexico, Costa Rica, Thailand, Japan, United States and South Africa. Pathogenic isolates G. citricarpa Kiely (anamorph form P. citricarpa McAlp Van Der Aa) are the etiological agent of the Citrus Black Spot (CBS), a disease that affects several citric plants and causes substantial injuries to the appearance of their fruits, thus preventing their export. Several previous studies have demonstrated the existence of an endophytic species with high morphological similarity to the causal agent of CBS that could remain latent in the same hosts. Consequently, the identification of the plants and fruits free from the causal agent of the disease is severely hampered. The RAPD analysis showed a clear discrimination among the pathogenic isolates of G. citricarpa and endophytic isolates (G. mangiferae and P. spinarum). In addition, a Principal Coordinate Analysis (PCO) based on a matrix of genetic similarity estimated by the RAPD markers showed four clusters, irrespective of their host or geographical origin. An Analysis of Molecular Variance (AMOVA) indicated that 62.8 percent of the genetic variation was found between the populations (G. citricarpa, G. mangiferae, P. spinarum and Phyllosticta sp.). Substantial variation was found in the populations (37.2 percent). Exclusive RAPD markers of isolates of G. citricarpa were cloned, sequenced and used to obtain SCARS (Sequence Characterized Amplified Regions), which allowed the development of new specific primers for the identification of G. citricarpa PCR (Polymerase Chain Reaction) analysis using a pair of primers specific to pathogenic isolates corroborating the groupings obtained by the RAPD markers, underscoring its efficiency in the identification of the causal agent of CBS.

Marcadores de RAPD foram utilizados para investigar a distribuição da variabilidade genética de linhagens de Guignardia citricarpa, G. mangiferae, e Phyllosticta spinarum isolados em diversos hospedeiros no Brasil, Argentina, México, Costa Rica, Tailândia, Japão, EUA e África do Sul. O fungo Guignardia citricarpa Kiely (Phyllosticta citricarpa McAlp Van Der Aa) é o agente causal da Mancha Preta dos Citros (CBS), uma doença que afeta diversas plantas cítricas, causando dano a aparência dos frutos, prejudicando a exportação. Diversos estudos têm demonstrado a existência de uma espécie endofítica muito semelhante morfologicamente a G. citricarpa, e que permanece de forma endofítica no mesmo hospedeiro. Dificultando assim, a identificação de plantas e frutos livres do agente causa da CBS. A análise do perfil de RAPD revelou uma clara discriminação entre isolados patogênicos de G. citricarpa e isolados endofíticos (G. mangiferae e P. spinarum). A Análise de Coordenadas Principais (PCO) baseada na matriz de similaridade genética dos marcadores RAPD, demonstrou a formação de quatro grupos, sem relação com origem geográfica ou com hospedeiros utilizados. A análise de Variância de Marcadores Moleculares (AMOVA) indicou que 62,8 por cento da variação genética é encontrada entre as populações (G. citricarpa, G. mangiferae, P. spinarum and Phyllosticta sp.). Entretanto, variação substancial foi encontrada dentro destas populações (37,2 por cento). Bandas de RAPD exclusivas de isolados de G. citricarpa foram clonadas, sequenciadas e utilizadas na obtenção de SCARS (Sequence Characterized Amplified Regions), que permitiram o desenvolvimento de novos primers específicos para a identificação de G. citricarpa. Reações de PCR (Polymerase Chain Reaction) utilizando este par de primers corroboraram os agrupamentos obtidos pelos marcadores de RAPD, revelando sua eficiência na identificação do agente causal da CBS.

The biology and potential for genetic research of transposable elements in filamentous fungi

Recently many transposable elements have been identified and characterized in filamentous fungi, especially in species of agricultural, biotechnological and medical interest. Similar to the elements found in other eukaryotes, fungal transposons can be classified as class I elements (retrotransposons) that use RNA and reverse transcriptase and class II elements (DNA transposons) that use DNA. The changes (transposition and recombination) caused by transposons can supply wide-ranging genetic variation, especially for species that do not have a sexual phase. The application of transposable elements to gene isolation and population analysis is an important tool for molecular biology and studies of fungal evolution

Recurrent mutation-selection to improve rennet production in Candida tsukubaensis

The yeast Candida tsukubaensis is of industrial importance for the production of microbial milk-clotting enzyme. Milk-clotting enzyme is an enzymatic complex capable of coagulating milk for cheese manufacturing. High clotting activity (CA) and low proteolytic activity (PA) are desirable qualities. To study the genetic nature of the CA and PA traits, we analyzed 179 colonies obtained after mutagenic treatment. Analysis of the data obtained for this populations showed that CA and PA are traits controlled by polygenes and that they are correlated (r = 0.3). The existence of a positive correlation indicates that selection for one trait without considering the other may alter one of the traits in an undesirable direction, since the objective of selection would be an increase in clotting activity and a decrease in proteolytic activity. Three cycles of recurrent mutation-selection were carried out to obtain improved strains. The ultraviolet light dose permitting a 5 per cent rate of cell survival was sufficient to generate genetic variability in the three selection cycles. At the end of the third cycle there was an increase of about 98 per cent in clotting activity and a decrease of about 20 per cent in proteolytic activity. Analysis of variance of the selective cycles showed that the linear effects were significant (P < 0.01) for both traits. Estimates of genetic variances and heritabilities of the three selection cycles are presented.

Recombination of lac- mal+ cells with lac- mal- variants arisen spontaneously from different Candida albicans strains

Cepas de Candida albicans, mantidas em meios de cultura usuais, originaram colônias que em testes de assimilaçäo de fontes de carbono mostraram-se lac+ mal-. Esferosplastos obtidos a partir de células normais, lac- mal+, foram submetidos à fusäo e produziram apenas células lac+ mal+. Após reduçäo na ploidia,essas células deram origem a colônias lac- mal+ (82 por cento), lac- mal (13,3 por cento) e lac+ mal+ (4,4 por cento). Discute-se neste trabalho, ploidia dos produtos de fusäo, ocorrência de heterocariose nesses produtos e a perda de cromossomos durante a segregaçäo

Genetic recombination after protoplast fusion in Candida sp

O objetivo deste trabalho foi descrever o ciclo parassexual em Candida sp uma levedura que näo apresenta ciclo sexual. As condiçöes para isolamento e regeneraçäo de protoplastos foram estabelecidas para a linhagem selvagem de Candida sp. O polietileno glicol foi utilizado para induzir a fusäo dos protoplastos isolados a partir das linhagens auxotroficas (pab bio e ura arg). As colonias prototróficas originais após fusäo foram selecionadas por complementaçäo em meio mínimo. A partir de um produto de fusäo nuclear selecionado, foram obtidos vários recombinantes monoauxotróficos, os quais tiveram diametro nuclear similar às linhagens parenterais. este fato sugere que os recombinantes auxotróficos se originaram de perdas cromossomicas do produto de fusäo nuclear

Cloning of A Bacillus thuringiensis crystal protein gene

Um fragmento de DNA contendo o gene da -endotoxina de um novo isolado de Bacillus thuringiensis, o SPL407 (serotipo H1), foi clonado usando o vetor pUC18 e um novo vetor, o pHT3101, que contém a regiäo de origem de replicaçäo de um plasmídeo nativo de B. thuringiensis. As clonagens foram feitas a partir do DNA total: na primeira um fragmento de DNA de 12 kb Bam HI/Pst, foi clonado em E. coli usando o pUC18 como vetor, na segunda um fragmento de DNA de 12kb Smal/Pst foi clonado no pHT3101 digerido por Smal/Pst; e na terceira um fragmento de DNA de 7 kb Hpa/Smal foi inserido no pHT3101 digerido no Smal. Os plasmídios construídos usando o pHT3101 foram introduzidos em E. coli e em B. thuringiensis. A análise dos plasmídios dos clones recombinantes de B. thuringiensis indicou que eles näo sofreram rearranjos moleculares e parecem ser estruturalmente estáveis

Efeitos da acriflavina em diferentes espécies de streptomyces produtores de actinomicina / Effects of acriflavine on three actionomycin producer species of Streptomyces

O tratamento de três diferentes espécies de Streptomyces com acriflavina, resultou na perda da produçäo de actinomicina em 5-12% das colônias isoladas nestas linhagens. S. felleus e S. regensis mostraram mais instabilidade do que S. parvulus. A produçäo de pigmento amarelo característica destas linhagens produtoras de actinomicina foi totalmente eliminada. A instabilidade genética destas linhagens pode ser atribuída a elementos extracromossômicos

Mutant derivation and parasexuality in a cellulolytic strain of Aspergillus niger

Um estudo genético foi realizado em uma linhagem industrial de Aspergillus niger. O crescimento em diferentes meios de cultura foi testado e mutantes morfológicos e auxotróficos foram isolados. Foram também produzidos hererocários entre pares de mutantes auxotróficos, sendo que colônias prototróficas foram recuperads. Provavelmente, estas colônias eram recombinantes haplóides prototróficos derivados de um processo parameiótico

Resíduos de mandioca induzem uma lata produçäo de enzimas celuloliticas / Cassava residues induce high production of cellulolytic enzymes

O objetivo deste trabalho foi verificar a produçäo de celulase a partir do uso de dois resíduos de procesamento da mandioca (a casca e o farelo). Esta produçäo foi comparada com a produçäo em farelo de trigo, um resíduo tradicionalmente usado pelas indústrias na produçäo enzimas. Os resultados mostraram que a casca de mandioca induziu cerca de 40 (por cento) maior produçäo de celulase (FPA) do que o farelo de trigo (au)

Melhoramento genético de microrganismo produtores de celulase / Genetical improvement of cellulolytic microorganisms

A celulose é o recurso orgânico mais abundante existente na natureza. As aplicaçöes econômicas potenciais da degradaçäo enzimática da celulose säo inúmeras. Contudo, nenhum processo significativo utilizando celulases foi até agora elaborado por causa do alto custo da enzima no custo total do alto custo da enzima no custo total do processo. Para superar estes problemas, programas de melhoramento genético foram desenvolvidos visando a seleçäo de microorganismos hipercelulolíticos, fundamentalmente fungos pertencentes ao gênero Trichoderma. Nesse sentido, o objetivo dessa pequena revisäo foi a busca dos principais trabalhos publicados nos últimos anos relativos ao melhoramento genético de microrganismos produtores de celulase