ABSTRACT It is know that many plants live in symbiosis with microorganisms that can be found on their interior, the endophytes. Environment and tissue type are modulating factors of this community, in which most of these microorganisms produce important antimicrobial molecules and they may be powerful biocontrol agents in agriculture. Thus, the aim of this study was to evaluate the community of Endophytic fungi from Eremanthus erythropappus in anthropogenic and natural areas (with human interaction, natural habitat and planned planting) of Minas Gerais State, Brazil, through cultivation-based approach and verify their antimicrobial activity against phytopathogenic fungal and pathogenic bacteria. The endophytic fungi isolated were identified by sequencing of the ITS region and subjected an in vitro antagonism test. The antagonisms that show antibiosis were submitted to tests on split plates to verify the volatile compound production. In the pairing testes, the endophytic fungi of the genera Cryptosporiopsis, Diaporthe, Xylaria, Paraconiothyrium and Camarosporium presented antibiosis against phytopathogenic fungi by releasing compounds in the medium. To our knowledge, this paper is the first report on the isolation of twelve genera fungi in E. erythropappus besides verifying their antagonist capacity, which opens the way for discovery of bioactive substances produced by endophytic fungi that inhibit pathogens.

Abstract Bacterial endophytes are considered to have a beneficial effect on host plants, improving their growth by different mechanisms. The objective of this study was to investigate the capacity of four endophytic Bacillus strains to solubilize iron phosphate (Fe-P), produce siderophores and indole-acetic acid (IAA) in vitro, and to evaluate their plant growth promotion ability in greenhouse conditions by inoculation into pearl millet cultivated in a P-deficient soils without P fertilization, with Araxá rock phosphate or soluble triple superphosphate. All strains solubilized Fe-P and three of them produced carboxylate-type siderophores and high levels of IAA in the presence of tryptophan. Positive effect of inoculation of some of these strains on shoot and root dry weight and the N P K content of plants cultivated in soil with no P fertilization might result from the synergistic combination of multiple plant growth promoting (PGP) traits. Specifically, while B1923 enhanced shoot and root dry weight and root N P content of plants cultivated with no P added, B2084 and B2088 strains showed positive performance on biomass production and accumulation of N P K in the shoot, indicating that they have higher potential to be microbial biofertilizer candidates for commercial applications in the absence of fertilization.

A produção de maçã vem sendo comprometida pela ocorrência de muitas doenças, entre as quais se destaca a Mancha Foliar de Glomerella (MFG), causada por Colletotrichum spp. O primeiro relato dessa doença em maçã foi registrado em plantas próximas a pomares de citrus no Estado de São Paulo, Brasil. A origem da MFG ainda não está bem clara, e estudos baseados na filogenia permitirão relacionar o organismo evolutivamente, possibilitando caracterizar possíveis mecanismos divergentes de evolução. A amplificação da região 5.8S-ITS (espaçador interno transcrito) do rDNA de 51 isolados de Colletotrichum patogênicos em de maçã, goiabeira serrana e citrus produziu um fragmento de aproximadamente 600 pares de bases (pb) para todos os isolados analisados. Os fragmentos amplificados foram digeridos por enzimas de restrição, sendo obtidos fragmentos de 90 a 500pb. O sequenciamento dessa região permitiu a construção de uma árvore filogenética com a distribuição dos isolados em cinco grupos, independentemente de seus hospedeiros. A partir da comparação in silico, foi possível verificar uma variação de 93 a 100% de similaridade entre as sequências estudadas e o banco de dados do GenBank. O agente causal da MFG está relacionado (agrupado) a isolados de goiabeira serrana e aos isolados padrões de citrus.

Several diseases have affected apple production, among them there is Glomerella leaf spot (GLS) caused by Colletotrichum spp. The first report of this disease in apple was in plants nearby citrus orchards in São Paulo State, Brazil. The origin of this disease is still not clear, and studies based on the molecular phylogeny could relate the organisms evolutionarily and characterize possible mechanisms of divergent evolution. The amplification of 5.8S-ITS (Internal Transcribed Spacer) of rDNA of 51 pathogenic Colletotrichum spp. isolates from apples, pineapple guava and citrus produced one fragment of approximately 600 bases pairs (bp) for all the isolates analyzed. The amplified fragments were cleaved with restriction enzymes, and fragments from 90 to 500bp were obtained. The sequencing of this region allowed the generation of a phylogenetic tree, regardless of their hosts, and 5 isolated groups were obtained. From the "in silico" comparison, it was possible to verify a variation from 93 to 100% of similarity between the sequences studied and the Genbank data base. The causal agent of GLS is nearly related (clustered) to isolates of pineapple guava and to the citrus isolates used as control.

We characterized indigenous common bean rhizobia from five districts of the state of Minas Gerais, Brazil. The isolates were trapped by two common bean varieties, the Mineiro Precoce (Andean origin) and Ouro Negro (Mesoamerican origin). Analysis by BOX-PCR of selected isolates detected a high level of genetic diversity.

O objetivo deste trabalho foi avaliar a diversidade funcional e genética de bactérias associadas à rizosfera de genótipos de milho contrastantes quanto à eficiência de uso de fósforo, por meio do teste de fontes de carbono no sistema EcoPlate e da eletroforese em gel de gradiente desnaturante (DGGE) dos fragmentos amplificados dos genes 16S ribossomais (rDNA) das bactérias. Foram coletadas amostras de solo da rizosfera de linhagens e híbridos contrastantes quanto à eficiência de uso de fósforo, cultivados em Latossolo Vermelho-Escuro fase cerrado, com baixo e alto teor de P. Bactérias da rizosfera de híbridos e linhagens eficientes, sob estresse de P, analisadas pelo sistema EcoPlate, tenderam a se agrupar conforme a análise de componentes principais, o que indica que utilizaram fontes de carbono semelhantes. Não houve diferença na diversidade bacteriana, analisada pela DGGE, entre bactérias associadas a genótipos eficientes e ineficientes no uso de P. Com base no sequenciamento do 16S rDNA, foi verificado que a rizosfera de genótipos de milho sob estresse de P parece selecionar grupos específicos de bactérias. A estrutura populacional genética e metabólica de bactérias da rizosfera foi mais influenciada pelo teor de fósforo no solo do que pela eficiência das plantas em usar o fósforo.

The objective of this work was to evaluate the functional and genetic diversity of bacteria associated to the rhizosphere of maize genotypes contrasting for phosphorus use efficiency by means of the EcoPlate carbon source test and denaturing gradient gel electrophoresis (DGGE) of amplified 16S ribosomal DNA (rDNA) fragments of bacteria. Rhizosphere soil samples of maize genotypes (hybrids and lineages) contrasting for phosphorus use efficiency cultivated in an Oxisol with high and low P content were collected. Bacteria from the rhizosphere of P-efficient maize genotypes under P stress conditions analyzed by the EcoPlate system tended to group together according to main components analysis, which indicates that these bacteria used similar carbon sources. Microbial diversity analyzed by DGGE did not differ between P-efficient and inefficient maize genotypes. Based on the 16S rDNA fragment sequencing, the rhizosphere of maize genotypes growing on low-P soils seemed to select specific bacteria species. The genetic and metabolic structure of the rhizosphere bacterial community was more strongly influenced by the level of P in the soil than by the genotypes with contrasting P-use efficiency.

Bactérias endofíticas desempenham papel importante na agricultura, melhorando a performance e adaptação de plantas contra estresses bióticos e abióticos. No presente estudo, métodos moleculares foram empregados para identificar bactérias endofíticas do gênero Bacillus isoladas de cultivares de milho doce brasileiro. SDS-PAGE de extratos protéicos totais de quarenta e dois isolados revelaram elevado número de bandas escrutináveis. Vinte e quatro isolados formaram nove grupos diferentes de réplicas bactérianas e dezoito foram considerados como únicos. Entre os isolados, alguns polipeptídios, de tamanhos variados, foram altamente acumulados. Seqüenciamento parcial do gene ribosomal 16S revelou que todos os isolados pertencem ao gênero Bacillus e que, entre treze isolados com padrão protéico similar, dois eram linhagens diferentes. Entre os quarenta e dois isolados identificados por seqüenciamento de rDNA, Bacillus subtilis e B. pumilus foram mais frequentes (15 e 12 isolados, respectivamente), seguido por, B. licheniformes (7 isolados), B. cereus (5 isolados) e B. amiloliquefascens (3 isolados). Baseado nos resultados, conclui-se que a técnica de SDS-PAGE poderá ser usada como primeiro procedimento, rápido e barato, para identificar variação inter-específica em coleções de bactérias endofíticas isoladas do milho, enquanto o método de seqüenciamento de rDNA poderá ser aplicado para analisar variações intra-específica entre isolados com padões similares de proteínas e estudos de taxonomia.

Endophytic bacteria play an important role in agriculture by improving plant performance and adaptation against biotic and abiotic stresses. In the present study molecular methods were used for identifying Bacillus endophytic bacteria isolated from Brazilian sweet corn. SDS-PAGE of wholecell protein extract of fortytwo isolates revealed a high number of scrutinable bands. Twenty-four isolates were identified in nine different groups of duplicated bacteria and eighteen were identified as unique. Some high-accumulated polipeptides with variable length were observed in almost isolates. Partial sequencing of 16S ribosomal gene revealed that all isolates are Bacillus sp. and among thirteen isolates with similar protein profiles, two were different strains. Among the forty-two isolates identified by rDNA sequencing, Bacillus subitilis and B. pumilus were the most frequenty species (15 and 12 isolates, respectively) followed by B. licheniformes (7 isolates), B. cereus (5 isolates) and B. amiloliquefascens (3 isolates). According to present results, SDS-PAGE technique could be used as a fast and cheap first tool for identifying interspecific variation in maize endophytic bacterial collections while rDNA sequencing could be applied for analyzing intraspecific variation among isolates with similar protein profile as well as for taxonomic studies.

O objetivo deste trabalho foi avaliar a patogenicidade de 24 isolados de Beauveria contra larvas de Spodoptera frugiperda e caracterizá-los molecularmente, por meio do seqüenciamento da região ITS do rDNA e de marcadores RAPD. O seqüenciamento de fragmentos de 570 pares de bases, da região ITS do rDNA, possibilitou a identificação dos isolados como B. bassiana e B. brongniarti, pela comparação com seqüências depositadas no GenBank. Sessenta e sete fragmentos polimórficos de RAPD foram capazes de diferenciar 20 entre 24 isolados de Beauveria, e agrupá-los de acordo com o inseto hospedeiro e com a patogenicidade contra a lagarta-do-cartucho do milho. Os marcadores RAPD foram altamente associados à patogenicidade contra S. frugiperda e explicaram até 67% da variação fenotípica. Além da identificação e caracterização molecular de isolados de Beauveria, o seqüenciamento da região ITS, aliado aos marcadores RAPD, é útil na seleção de isolados potencialmente eficazes contra larvas de S. frugiperda e no monitoramento de liberações desses microrganismos em programas de biocontrole.

The objective of this work was to evaluate the pathogenicity of 24 Beauveria isolates to Spodoptera frugiperda larvae, and characterize them molecularly through rDNA-ITS sequencing and RAPD markers. Sequencing of rDNA-ITS fragments of 570 bp allowed the identification of isolates as B. bassiana or B. brongniarti by sequence comparison to GenBank. Sixty seven polymorphic RAPD fragments were capable to differentiate 20 among 24 Beauveria isolates, grouping them according to the derived host insect and to pathogenicity against maize fall armyworm larvae. Three RAPD markers were highly associated to the pathogenicity against S. frugiperda, explaining up to 67% of the phenotypic variation. Besides identification and molecular characterization of Beauveria isolates, ITS sequence and RAPD markers proved to be very useful in selecting the isolates potentially effective against S. frugiperda larvae and in monitoring field release of these microorganisms in biocontrol programs.

O objetivo deste trabalho foi caracterizar a variabilidade genética de isolados de fitoplasma e de Spiroplasma kunkelii obtidos de plantas de milho, apresentando sintomas de enfezamento, coletados em diferentes regiões do Brasil. Um fragmento de 500 pares de bases (pb) do gene que codifica a espiralina de S. kunkelii foi amplificado e um produto de amplificação de 1.200 pb foi gerado a partir do gene 16S rDNA de fitoplasma. As seqüências parciais do gene da espiralina mostraram similaridade de 98% entre os isolados de S. kunkelii analisados. Essas seqüências foram comparadas com a seqüência do gene da espiralina de outras espécies de Spiroplasma depositadas no GenBank, resultando em similaridade variável entre 76,9% e 88,1%. As seqüências do gene 16S rDNA dos isolados de fitoplasma foram completamente similares entre todos os isolados brasileiros e apresentaram até 98% de similaridade com seqüências do mesmo gene de outros fitoplasmas já publicadas. Uma variabilidade genética muito estreita foi detectada para esses genes entre os isolados de fitoplasma e Spiroplasma analisados. No entanto, outras regiões genômicas, que apresentem um maior polimorfismo precisam ser identificadas para melhor avaliar a diversidade genética dentro da população desses microrganismos.

The objective of this work was to characterize the genetic variability of phytoplasma and Spiroplasma kunkelii isolated from maize plants showing symptoms of stunt collected from different Brazilian geographic regions. A DNA fragment of 500 base pairs (bp) was amplified from the spiralin gene in S. kunkelii and one fragment of 1,200 bp was generated from 16S rDNA gene in phytoplasma. The partial sequences of the spiralin gene showed similarity of 98% among the isolates of S. kunkelii analyzed. These sequences were compared with the sequence of the spiralin gene from other Spiroplasma species deposited in the GenBank, resulting in a similarity varying from 76.9% to 88.1%. The 16S rDNA sequence from the phytoplasma were completely similar within the Brazilian isolates and showed up to 98% of the similarity with sequences already found from other phytoplasmas. A very narrow genetic variability was detected by these gene fragments within phytoplasma and Spiroplasma analyzed. However, other genomic regions with higher polymorphic levels shall be identified in order to better evaluate the genetic diversity within these microorganisms population.