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.

Toxicity of ß-(1→3, 1→6)-á´ -glucans produced by Diaporthe sp. endophytes on Metarhizium anisopliae (Metschnikoff) Sorokin assessed by conidia germination speed parameter / Toxicidade de ß- (1 → 3, 1 → 6) -á´ -glucanas produzidas por endófitos Diaporthe sp. sobre Metarhizium anisopliae (Metschnikoff) Sorokin avaliada pelo parâmetro de velocidade de germinação dos conídios

We have previously reported that ß-(1→3,1→6)-ᴅ-glucans produced by endophytes Diaporthe sp. G27-60 and G65-65 (GenBank accession codes JF766998 and JF767007, respectively) are promising anti-proliferation agents against human breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG2-C3A) cells. However, the literature fails to describe the effects of Diaporthe exopolysaccharides (EPS) on eukaryotic healthy cells. The fungus Metarhiziumanisopliae has been employed as model-system to evaluate the toxicity of pharmaceutical and agricultural-interest substances, taking into account, among other parameters, the speed of conidia germination. Current study verified the effect of different concentrations of Diaporthe ß-glucans on the germination speed of M. anisopliae. Conidia were incubated with ß-glucans treatments (50, 200 and 400 µg/mL) at 28ºC, sampled during 24 h and analyzed by light microscopy. At the end of a 24-h incubation, the amount of germinated conidia reached ≈99% for controls and ranged between 97.7 and 98.6% for treatments. Bayesian analysis indicated that Diaporthe glucans had no toxicity on M. anisopliaeand the curve of germination occurred as expected for this fungal strain. Considering the validity of filamentous fungi as model-systems, results are important data on the toxicity of endophytic EPS on healthy cells and may be associated with our previous results obtained for these polymers against tumor cells.

Anteriormente, um estudo mostrou que ß-(1→3,1→6)-ᴅ-glucanas produzidas pelos endófitos Diaporthe sp. G27-60 e G65-65 (códigos de acesso no GenBank JF766998 e JF767007, respectivamente) são agentes promissores com ação antiproliferativa contra células HepG2-C3A (hepatoma humano) e MCF-7 (adenocarcinoma mamário humano). No entanto, os efeitos de exopolissacarídeos (EPS) produzidos por fungos do gênero Diaporthe em células eucarióticas sadias não estão descritos na literatura atual. O fungo Metarhiziumanisopliae tem sido utilizado como sistema-modelo para avaliar a toxicidade de substâncias de interesse farmacêutico e agronômico, considerando, entre outros parâmetros, a velocidade de germinação de conídios. O presente estudo teve como objetivo verificar os efeitos de diferentes concentrações de ß-glucanas produzidas por Diaporthe sp. sobre a velocidade de germinação de M. anisopliae. Os conídios foram incubados com os tratamentos de ß-glucanas (50, 200 e 400 µg/mL) a 28 ºC, com amostras coletadas ao longo de 24 h, e analisados por microscopia de luz. Ao final das 24 h de incubação, o total de conídios germinados nos controles foi de ≈99%, e variou entre 97,7 e 98,6% para os tratamentos. A análise bayesiana indicou que as glucanas de Diaporthe sp. não apresentaram toxicidade sobre M. anisopliae, e a curva de germinação atendeu ao esperado para essa linhagem fúngica. Considerando a validade dos fungos filamentosos como sistemas-modelo, esses resultados representam dados importantes sobre a toxicidade dos EPS de endófitos sobre células sadias e podem ser associados aos resultados anteriormente obtidos para esses polímeros em testes contra células tumorais.

Bioprospection and molecular phylogeny of culturable endophytic fungi associated with yellow passion fruit

Endophytic microorganisms live inside the plants without causing any damage to their hosts. In the agricultural field, these endophytes might be a strategy of biological control for phytopathogens. We aimed to isolate endophytic fungifrom yellowpassion fruit (Passiflora edulis) leaves, evaluating its biocontrol capacity by in vitroantagonism against phytopathogen Colletotrichum sp. CNPU378. We also carried out greenhouse experiments in bean seedlings. A high colonization frequency was obtained (89%), and the molecular identification based on DNA sequencing attested Colletotrichumas the most frequent genus and minor occurrence of Curvulariaendophytes. The endophytes tested showed different types of competitive interactions in in vitro antagonism inhibition rate ranging from 28.8 to 48.8%. There were 10 promising antagonists tested for their antagonist activity of crude extracts of secondary metabolites, in which strain PE-36 (20.8%) stood out among the other strains evaluated. In the greenhouse assay, plants inoculated only with endophyte Colletotrichumsp. PE-36 was symptomless and suggest that the endophyte strengthened the growth promotion in common bean plants, especially in the root length and number of leaves when compared to control plantsand other treatments. Despite many fungiof Colletotrichumgenus being described as causative agents of anthracnose, in this study, the plant sampled was colonized predominantly by Colletotrichumendophytes living in asymptomatic relationship. By the way,we come across a Colletotrichumsp. endophyte able to antagonize a Colletotrichumsp. pathogen

ENZYMATIC AND ANTAGONIST ACTIVITY OF ENDOPHYTIC FUNGI FROM Sapindus saponaria L. (SAPINDACEAE) / Actividad enzimática y antagonista de los hongos endofíticos de Sapindus saponaria L. (Sapindaceae)

ABSTRACT Endophyte microorganisms have great biotechnological interest, with features applicable to different areas and are potentially useful in agriculture. The current study determines the biotechnological potential of endophytic fungi, isolated from leaves of Sapindus saponaria, to control phytopathogenic fungi and evaluate their enzyme production. Molecular taxonomy was performed by sequencing of the ITS1-5.8S-ITS2 ribosomal DNA region, identifying the genera Phomopsis, Sordariomycetes, Diaporthe, and Colletotrichum. In vitro antagonism against phytopathogens showed better results against Fusarium solani and provided inhibition indices between 41.8 % and 67.5 %. The endophytic strain SS81 (Diaporthe citri) presented the highest antagonism index against the pathogen. Against Glomerella sp. and Moniliophthora perniciosa, inhibition rates ranged between 18.7 % and 57.4 % and between 38.3 % and 64.8 %, respectively. Enzyme assays revealed that strain SS65 (Diaporthe sp.) produced 1.16 UI μmol/min of amylase; strain SS77 (Diaporthe sp.) produced 2.74 UI μmol/min of pectinase, and strain SS08 (Diaporthe sp.) produced 1.51 UI μmol/min of cellulase. Thus, the current study shows evidence the importance of isolated endophytes with phytoprotective properties of plants with medicinal properties as alternatives for biological control and natural sources of products with biotechnological interest.

RESUMEN Los microorganismos endofíticos tienen gran interés biotecnológico, con características aplicables a diferentes áreas y potencialmente útiles en la agricultura. El presente estudio determinó el potencial biotecnológico de los hongos endofíticos, aislados de las hojas de Sapindus saponaria, en el control de hongos fitopatógenos y evaluación de su producción de enzimática. La taxonomía molecular fue realizada por la secuencia de la región ITS1-5.8S-ITS2 del ADN ribosomal, identificando los géneros Phomopsis, Sordariomycetes, Diaporthe y Colletotrichum. El antagonismo in vitro contra fitopatógenos mostró mejores resultados contra Fusarium solani y proporcionó índices de inhibición de entre el 41,8 % y el 67,5 %. El linaje endofítico SS81 (Diaporthe citri) presentó el mayor índice de antagonismo contra los patógenos. Contra Glomerella sp. y Moniliophthora perniciosa, las tasas de inhibición variaron entre el 18,7 % y el 57,4 % y entre el 38,3 % y el 64,8 %, respectivamente. El ensayo enzimático reveló que el linaje SS65 (Diaporthe sp.) produjo 1,16 UI μmol / min de amilasa; el linaje SS77 (Diaporthe sp.) produjo 2,74 UI μmol / min de pectinasa; y el linaje SS08 (Diaporthe sp.) produjo 1,51 UI μmol / min de celulasa. Así, el presente estudio evidencia la importancia de los endófitos aislados con propiedades fitoprotectoras como alternativas para el control biológico y como fuentes naturales de productos con interés biotecnológico.

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.

Biosurfactants produced by Microbacterium sp, isolated from aquatic macrophytes in hydrocarbon-contaminated area in the Rio Negro, Manaus, Amazonas / Biossurfactantes produzidos por Microbacterium sp, isolada de macrófita aquática em área impactada por hidrocarbonetos no rio Negro, Manaus, Amazonas

Endophytic bacteria isolated from Eichhornia crassipes (Mart) Solms., collected in oil contaminated wastewater of effluent generated by Petrobras refinery in Manaus were investigated to determine their potential for producing biosurfactants. Assay with 2.6-dichlorophenol indophenol (DCPIP) indicator to verify hydrocarbon biodegradation activity; oil emulsification test; drop-collapse method; surface tension and growth curve of biosurfactant production. The M87 Microbacterium sp. strain chosen for this work was identified by the sequencing of the rDNA region and the chemical characterization was performed by FTIR, UFLC/MS and 1H RMN techniques. The selected bacterial isolate provided 3g L-1 of biosurfactant, using diesel oil as sole carbon source, being efficient in biodegrading oil as demonstrated by the DCPIP test. Fractions obtained by column chromatography were efficient in reducing water surface tension around 40 mN m-1, especially fraction 1, which reduced it to 34.17 mN m-1. The different techniques of chemical analysis used for the identification of the biosurfactant isolate indicated that this is probably a long - chain fatty acid lipid type, which may be used in the future as both biosurfactant in decontamination processes of hydrocarbon-polluted areas or as bioemulsifier in countless processes, since it exhibited no toxicity as determined by Alamar Blue assay.

Foram investigadas bactérias endofíticas isoladas de Eichhornia crassipes (Mart.) Solms., coletadas em águas contaminadas com resíduos de petróleo em um afluente da refinaria da Petrobrás/Manaus, para avaliação da produção de biossurfactantes. Para selecionar o micro-organismos à produção e caracterização de biossurfactantes, foram realizados os seguintes testes: a descoloração do indicador 2,6 indofenol (DCPIP), emulsificação do diesel, colapso da gota, tensão superficial e curva de produção. A caracterização química foi realizada por meio das técnicas de FT-IR, UFLC/MS e RMN1H. A bactéria M87 Microbacterium neste estudo, foi identificada pelo sequenciamento da região rDNA e produziu 3g L-1 de biossurfactantes utilizando o diesel como fonte de carbono, mostrando-se eficiente na ação biodegradadora do petróleo, por meio do teste de Indofenol (DCPIP). As frações obtidas, mostraram-se eficazes na redução da tensão superficial da água abaixo de 40 mN m-1, com destaque para a fração 1 que reduziu a tensão superficial para 34,17 mN m-1. Pelas análises química utilizadas, pode-se inferir que, provavelmente, se trata de um ácido graxo de cadeia longa, que pode ser utilizado futuramente tanto como biossurfactante em processos de descontaminação de ambientes impactados por hidrocarbonetos, assim como bioemulsificante em inúmeros processos uma vez que não apresentou toxicidade por meio do teste realizado.

Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

Metabólitos Secundários de Interesse Farmacêutico Produzidos por Fungos Endofíticos / Secondary Metabolites Produced by Endophytic Fungi of Pharmaceutical Interest

Fungos endofíticos são grandes produtores de metabólitos secundários. No entanto, essa produção ainda não foi estimada, mas sabe-se que 80% dos fungos endofíticos estudados produzem metabólitos ativos, influenciados por condições ambientais, parâmetros de fermentação, tempo, temperatura, pH e nutrientes. Esses compostos são agrupados em diversas classes, e se apresentam tóxicos e até mesmo letais frente a outros microrganismos. Essas classes de compostos possuem atividades hormonais, antibióticas, antitumorais, antifúngicas, citotóxicas, antivirais, imunossupressoras, antiparasitárias, entre outras. Os produtos naturais obtidos de endófitos incluem principalmente alcalóides, esteróides, terpenóides, isocumarinas, quinonas, fenilpropanóides, ligninas, fenóis e ácidos fenólicos, metabólitos alifáticos, lactonas, citocatalasinas, flavonóides, peptídeos e xantonas. Considerando que os endófitos ainda são pouco estudados, especialmente em espécies tropicais, eles surgem como fonte de novos produtos para a incorporação de medicamentos e como forma de preservação de plantas medicinais. Salienta-se que a produção de metabólitos fúngicos realizada in vitro apresenta um menor custo e melhor otimização, bem como os microrganismos serem fontes facilmente renováveis e reprodutíveis quando comparadas com outras fontes naturais como plantas e animais. Várias técnicas têm sido utilizadas para aprimorar a utilização de microrganismos para a produção de novos princípios ativos, reunindo o conhecimento em química, bioquímica, microbiologia e biologia molecular. Este trabalho tem como objetivo mostrar a diversidade de produtos metabolicamente ativos produzidos por fungos endofíticos, assim como seu potencial farmacológico.

Endophytes are large producing secondary metabolites. Although the production has not yet been estimated, it is known that 80% of endophytic fungi studied produce active metabolites, influenced by environmental conditions, fermentation parameters, time, temperature, nutrients and pH. These compounds are grouped into several classes, which are toxic and even lethal against other microorganisms. These classes of metabolites have hormonal, antibiotic, antitumor, antifungal, cytotoxic, antiviral, immunosuppressive, and antiparasitic activities. The products obtained from natural endophyte mainly include alkaloids, steroids, terpenes, isocumarins, quinones, phenylpropanoids, lignins, phenols and phenolic acids, aliphatic metabolites, lactones cytocatalasins, flavonoids, peptides and xanthones. Whereas the endophytes are still poorly studied, particularly in tropical species, they appear as a source of new products for the incorporation in drugs and as a means of preservation of medicinal plants. It is noted that the production of fungal metabolites performed in vitro has a lower cost and a better optimization. In addition, microorganisms are easily renewable and reproducible sources when compared with other natural sources such as plants and animals. Several techniques have been used to enhance the use of microorganisms for production of new active metabolites, gathering knowledge in chemistry, biochemistry, microbiology and molecular biology. This study aims to show the diversity of products produced by metabolically active endophytes as well as their pharmacological potential.

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.

Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51 percent and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.

The effect of different growth regimes on the endophytic bacterial communities of the fern, Dicksonia sellowiana hook (Dicksoniaceae)

Endophytic bacteria associated with the fern Dicksonia sellowiana were investigated. The bacterial communities from the surface-sterilized pinnae and rachis segments of the plants from the Brazilian Atlantic Rainforest that grew in native field conditions were compared with the bacterial communities from plants grown in greenhouses and plants that were initially grown in greenhouses and then transferred to the forest. From 540 pinnae and 540 rachis segments, 163 (30.2 percent) and 346 (64.2 percent) were colonized by bacteria, respectively. The main bacterial genera and species that were isolated included Bacillus spp. (B. cereus, B. megaterium, B. pumilus and B. subtilis), Paenibacillus sp., Amphibacillus sp., Gracilibacillus sp., Micrococcus sp. and Stenotrophomonas spp. (S. maltophilia and S. nitroreducens). B. pumilus was the most frequently isolated bacterial species. Amphibacillus and Gracilibacillus were reported as endophytes for the first time. Other commonly found bacterial genera were not observed in D. sellowiana, which may reflect preferences of specific bacterial communities inside this fern or detection limitations due to the isolation procedures. Plants that were grown in greenhouses and plants that were reintroduced into the forest displayed more bacterial genera and species diversity than native field plants, suggesting that reintroduction shifts the bacterial diversity. Endophytic bacteria that displayed antagonistic properties against different microorganisms were detected, but no obvious correlation was found between their frequencies with plant tissues or with plants from different growth regimes. This paper reports the first isolation of endophytic bacteria from a fern.

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.

Assessing the diversity of bacterial communities associated with plants / Avaliação da diversidade de comunidades bacterianas associadas às plantas

Plant-bacteria interactions result from reciprocal recognition between both species. These interactions are responsible for essential biological processes in plant development and health status. Here, we present a review of the methodologies applied to investigate shifts in bacterial communities associated with plants. A description of techniques is made from initial isolations to culture-independent approaches focusing on quantitative Polymerase Chain Reaction in real time (qPCR), Denaturing Gradient Gel Electrophoresis (DGGE), clone library construction and analysis, the application of multivariate analyses to microbial ecology data and the upcoming high throughput methodologies such as microarrays and pyrosequencing. This review supplies information about the development of traditional methods and a general overview about the new insights into bacterial communities associated with plants.

As interações planta-bactéria resultam de um reconhecimento recíproco de ambas espécies. Estas interações são responsáveis por processos biológicos essenciais para o desenvolvimento e a proteção das plantas. Este trabalho revisa as metodologias aplicadas na investigação de alterações nas comunidades bacterianas associadas às plantas. Uma descrição das técnicas é feita, desde o isolamento até a aplicação de técnicas independentes de cultivo, destacando as técnicas de qPCR, Gel de Eletroforese em Gradiente Desnaturante (DGGE), construção e análise de bibliotecas de clones, a aplicação de análise multivariada em dados de ecologia microbiana, e as novas metodologias de alto processamento de amostras como microarranjos e pirosequenciamento. Em resumo, esta revisão fornece informações sobre o desenvolvimento das técnicas tradicionais e uma visão geral sobre as novas tendências dos estudos de comunidades bacterianas associadas às plantas.

Ocorrência do fungo entomopatogênico Isaria javanica (Frieder. & Bally) Samson & Hywell-Jones (Fungi, Sordariomycetes) em lagartas de Lonomia obliqua Walker (Lepidoptera, Saturniidae, Hemileucinae) / Occurrence of the entomopathogenic fungus Isaria javanica (Frieder. & Bally) Samson & Hywell-Jones (Fungi, Sordariomycetes) infecting Lonomia obliqua Walker (Lepidoptera, Saturniidae, Hemileucinae) caterpillars

Este fungo foi isolado pela primeira vez de lagartas de L. obliqua de uma agregação em plátano (Platanus acerifolia (Aiton) Wild - Platanaceae), em Bento Gonçalves, RS, Brasil. Após isolamento, purificação e caracterização, realizou-se um teste de patogenicidade com lagartas sadias de L. obliqua para corroborar, sua infectividade pelo postulado de Koch. Constatou-se correspondência morfológica e molecular entre o inóculo e o reisolado, comprovando sua patogenicidade a L. obliqua.

It is recorded for the first time the occurrence of the entomopathogenic fungus Isaria javanica (Frieder. & Bally) Samson & Hywell-Jones (Fungi: Sordariomycetes) infecting Lonomia obliqua Walker (Lepidoptera: Saturniidae: Hemileucinae) caterpillars. This fungus was isolated from L. obliqua individuals collected from Platanus acerifolia (Aiton) Wild- Platanaceae in Rio Grande do Sul state, Brazil. After isolation, purification and characterization, fungal conidia were inoculated on healthy L. obliqua caterpillars and from dead caterpillars the fungal isolates were again obtained. New isolates and the original isolate did not differ when compared by morphological and molecular tests.

Bioassay assessment of metarhizium anisopliae (metchnikoff) sorokin (deuteromycota: hyphomycetes) against Oncometopia facialis (signoret) (hemiptera: cicadellidae)

Citrus Variegated Chlorosis (CVC) is an economically important, destructive disease in Brazil and is caused by Xylella fastidiosa and transmitted by sharpshooter insects. In this study, the efficacy of the fungus Metarhizium anisopliae in controlling the sharpshooter Oncometopia facialis was studied by bioassay conditions. In the bioassay, insects were sprayed with a suspension containing 5 X 10(7) conidia mL-1. Adults captured in the field were treated in groups of 10 in a total of 11 replications per treatment. Significant differences between the natural mortality and the mortality of insects treated with the fungus were observed 6 days after inoculations (P<0.05). These significant differences increased until 10 days after treatment. The fungus caused 87.1 percent mortality, with the LT50 varying from 5 to 6 days. The LC50 was 1.2 X 10(6) conidia mL-1, varying from 7.7 X 10(5) to 2 X 10(6) conidia mL-1. The results showed that the sharpshooter O. facialis was susceptible to the entomopathogenic action of M. anisopliae in controlled condition during bioassay.

A Clorose Variegada dos Citros (CVC) é uma doença economicamente importante e destrutiva no Brasil e é causada pela bactéria Xylella fastidiosa e transmitida por insetos vetores tal como Oncometopia facialis. Nesse estudo, a eficácia do fungo Metarhizium anisopliae em controlar o inseto vetor O. facialis foi estudada em condições de bioensaio. Nesse bioensaio, insetos foram pulverizados com uma suspensão de 5 X 10(7) conídio mL-1. Insetos-adultos capturados no campo foram tratados em grupos de 10, em um total de 11 replicatas por tratamento. Diferenças significativas entre a mortalidade natural e a mortalidade dos insetos tratados com o fungo foram observadas em 6 dias após a inoculação (P<0.05). Estas diferenças significativas aumentaram antes do décimo dia após o tratamento. O fungo causou uma mortalidade de 87,1 por cento, com uma LT50 variando entre 5 e 6 dias. A LC50 foi de 1,2 X 10(6) conídio mL-1, variando de 7,7 X 10(5) a 2 X 10(6) conídio mL-1. Estes resultados mostraram que o vetor O. facialis foi susceptível a ação entomopatogênica de M. anisopliae em condições controladas durante o bioensaio.

Identification and colonization of endophytic fungi from soybean (Glycine max (L) Merril) under different environmental conditions

A total of 297 endophytic fungi were isolated from 1728 leaf and stem fragments collected about twenty and forty days after germination from soybean (Glycine max (L.) Merril) plants grown in the field and a greenhouse. The fungi belonged to eight groups, six dematiaceous genera (Alternaria, Cladosporium, Chaetomium, Curvularia, Drechslera and Scopulariopsis) and the non-dematiaceous genera Acremonium, Aspergillus, Colletotrichum, Fusarium, Paecilomyces and Penicillium along with some Mycelia sterilia.. There were qualitative and quantitative differences in the type and number of isolates obtained from greenhouse and field-grown plants, with more isolates being obtained from the latter. No difference was found in the number of fungi isolated from leaves and stems irrespective of where the plants was grown. For was field-grown plants, the number of isolates decreased as the plants aged and more fungi were found in tissues near the soil, while for greenhouse-grown plants the number of isolates increased as the plants aged but in this case no more fungi were isolated from those tissues nearer the soil. These results could have biotechnological relevance for the biological control of pests or plant growth promotion.

A partir de 1728 fragmentos de hastes e folhas de soja (Glycine max (L.) Merril). provenientes de plantas do campo e de casa de vegetação, coletadas cerca de 20 e 40 dias após a germinação das sementes, 297 fungos endofíticos foram isolados. Os gêneros encontrados foram: Alternaria, Cladosporium, Curvularia, Chaetomium, Scopulariopsis, Drechslera (todos dematiáceos) além de Colletotrichum, Fusarium, Acremonium, Aspergillus, Penicillium, Paecilomyces e Mycelia sterilia. Foram detectadas diferenças qualitativas e quantitativas entre os isolados, em relação a micobiota de hospedeiros provenientes do campo e de casa de vegetação com maior frequência de fungos isolados de plantas no campo em comparação com as de casa de vegetação. Houve diminuição da frequência de fungos com a idade das plantas crescidas no campo, ocorrendo o inverso com as da casa de vegetação, mas em ambos os casos, não houve diferença no número de fungos isolados de hastes e de folhas. Houve em plantas crescidas no campo uma maior incidência nas regiões próximas ao solo o que não ocorreu nas plantas provenientes da casa de vegetação. Os resultados podem ser de utilidade do ponto de vista biotecnológico pelo uso de endófitos introduzidos deliberadamente em plantas visando o controle biológico de pestes ou promoção de crescimento vegetal.

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

Pulsed field gel electrophoresis reveals chromosome length and number differences in Brazilian strains of Metarhizium Anisopliae

Cariótipos de oito linhagens selvagens do fungo entomopatogênico Metarhizium anisopliae var. anisopliae foram obtidos em gel, por eletroforese em campo pulsado. As linhagens foram isoladas de insetos provenientes de seis estados brasileiros. As moléculas de DNA cromossômico de três linhagens foram separadas em sete bandas e, de cinco linhagens, em oito bandas. Polimorfismo de tamanho cromossômico também foi observado. O tamanho do DNA cromossômico de todas as linhagens variou de 7,7 a 0,9 Mb, utilizando-se DNA cromossômico de Aspergillus nidulans como padrão. O tamanho do genoma total foi estimado em pelo menos 29,7 Mb. Algumas correlações entre semelhanças e diferenças no cariótipo eletroforético e a ocorrência do ciclo parassexual como também a especificidade com insetos hospedeiros foram discutidas.

Direct RAPD evaluation of bacteria without conventional DNA extraction

O presente trabalho mostra a amplificação de DNA das bactérias Pantoea agglomerans e Bacillus pumilus por meio da técnica de RAPD (Amplificação ao acaso de DNA polimórfico). Para esta análise, o DNA molde foi obtido sem a utilização de técnicas de extração convencional, ou seja, sem a purificação do DNA. Bactérias foram cultivadas por 20 horas em 5 mL de meio LB, centrifugado e ressuspendido em tampão TE. A suspensão resultante foi fervida por 5 min., diluída e 2,0 µL foram usados em reações de 15 µL. Os resultados mostraram que os padrões observados com o DNA obtido pela fervura das células não apresentou diferenças significativas daquele obtido com DNA extraído e purificado com fenol, sugerindo a possibilidade da utilização deste método para o estudo da variabilidade genética de populações microbianas.