Genetic Diversity and Population Differentiation of the Causal Agent of Citrus Black Spot in Brazil

One of the most important diseases that affect sweet orange orchards in Brazil is the Citrus Black Spot that is caused by the fungus Guignardia citricarpa. This disease causes irreparable losses due to the premature falling of fruit, as well as its severe effects on the epidermis of ripe fruit that renders them unacceptable at the fresh fruit markets. Despite the fact that the fungus and the disease are well studied, little is known about the genetic diversity and the structure of the fungi populations in Brazilian orchards. The objective of this work was study the genetic diversity and population differentiation of G. citricarpa associated with four sweet orange varieties in two geographic locations using DNA sequence of ITS1-5.8S-ITS2 region from fungi isolates. We observed that different populations are closely related and present little genetic structure according to varieties and geographic places with the highest genetic diversity distributed among isolates of the same populations. The same haplotypes were sampled in different populations from the same and different orange varieties and from similar and different origins. If new and pathogenic fungi would become resistant to fungicides, the observed genetic structure could rapidly spread this new form from one population to others

Characterization of rhizobia indicated for inoculant production using 16S rRNA partial sequencing

O objetivo deste trabalho foi confrontar as sequências parciais do gene 16S rRNA de estirpes padrão de rizóbios com as de estirpes recomendadas para a produção de inoculantes no Brasil, com vistas à verificação da confiabilidade do sequenciamento parcial desse gene para a identificação rápida de estirpes. Foram realizados sequenciamentos através de reação em cadeia da polimerase (PCR) com iniciadores relativos à região codificadora do gene 16S rRNA entre as bactérias estudadas. Os resultados foram analisados pela consulta de similaridade de nucleotídeos aos do "Basic Local Alignment Search Tool" (Blastn) e por meio da interpretação de árvores filogenéticas geradas usando ferramentas de bioinformática. A classificação taxonômica das estirpes Semia recomendadas para inoculação de leguminosas com base em propriedades morfológicas e especificidade hospedeira não foi confirmada em todas as estirpes. A maioria das estirpes estudadas, consultadas no Blastn, é consistente com a classificação proposta pela construção de árvores filogenéticas das sequências destas estirpes, com base na similaridade pelo sequenciamento parcial do gene considerado.The aim of this work was to compare the partial sequences of 16S rRNA gene of rhizobia strain patterns already classified with strains recommended for the production of inoculants in Brazil, in order to verify the reliability of partial sequencing of the gene for the purpose of rapid identification of strains. (PCR) sequencing using primers on the coding region of the 16S rRNA gene among the bacteria studied was conducted. The results were analyzed by consulting the nucleotides' similarity based on Basic Local Alignment Search Tool (Blastn) and by interpreting the phylogenetic trees generated by bioinformatic tools. The taxonomic classification of Semia strains recommended for legume inoculation based on morphological properties and host specificity was not confirmed in all strains. The similarity of the Blastn consultation by partial sequencing of the gene found in strains studied is consistent with the classification proposed by the construction of a phylogenetic tree of sequences of strains in most cases

Caracterização genética e capacidade de fixação de nitrogênio de estirpes de Rhizobium em feijoeiro

This study aimed to genetically characterize four new Rhizobium strains, and to evaluate their nodulation and fi xation capacity compared to commercial strains and to native rhizobia population of aBrazilian Rhodic Hapludox. Two experiments were carried out in randomized blocks design, under greenhouse conditions, in 2007. In the fi rst experiment, the nodulation and nitrogen fi xation capacity of new strains were evaluated, in comparison to the commercial strains CIAT-899 and PRF-81 and to native soil population. It was carried out in plastic tubes fi lled with vermiculite. DNA extractions and PCR sequencing of the intergenic space were made from the isolated pure colonies, in order to genetically characterize the strains and the nativerhizobia population. In the second experiment, the nodulation and productivity of common beans Perola cultivar were determined, with the use of evaluated strains, alone or in mixture with PRF-81 strain. It was carried out in pots fi lled with soil. The native soil population was identifi ed as Rhizobium sp. and was ineffi cient in nitrogen fi xation. Three different Rhizobium species were found among the four new strains. The LBMP-4BR and LBMP-12BR new strains are among the ones with greatest nodulation and fi xation capacity and exhibit differential responses when mixed to PRF-81.O objetivo deste trabalho foi a caracterização genética de quatro novas estirpes de Rhizobium e a avaliação de sua capacidade de fi xação de N2 e nodulação, comparadas a estirpes comerciais e à populaçãonativa de rizóbios de um Latossolo Vermelho. Dois experimentos foram conduzidos em blocos ao acaso, em casa de vegetação. No primeiro experimento, conduzido em tubetes com vermiculita, avaliaram-se a nodulação e a capacidade de fi xação das novas estirpes, em comparação com as estirpes comerciais CIAT-899 e PRF-81 ecom a população nativa do solo. Das colônias puras isoladas, extraiu-se o DNA genômico e realizou-se o seqüenciamento do espaço intergênico, para a caracterização genética das estirpes e da população nativa derizóbios. O segundo experimento foi realizado em vasos com solo, para determinação da produtividade e da nodulação do feijoeiro, cultivar Pérola, com o uso das estirpes isoladamente ou em mistura com a PRF-81. A população nativa do solo foi identifi cada como Rhizobium sp. e se mostrou inefi ciente na fi xação de nitrogênio.Foram encontradas três espécies de Rhizobium entre as quatro novas estirpes. As estirpes LBMP-4BR e LBMP-12BR estão entre as que têm maior capacidade de nodulação e fi xação de N2, e apresentam respostas diferenciadas quando misturadas à PRF-81

Genetic Diversity and Population Differentiation of Guignardia mangiferae from “Tahiti” Acid Lime

Among the citrus plants, “Tahiti” acid lime is known as a host of G. mangiferae fungi. This species is considered endophytic for citrus plants and is easily isolated from asymptomatic fruits and leaves. G. mangiferae is genetically related and sometimes confused with G. citricarpa which causes Citrus Black Spot (CBS). “Tahiti” acid lime is one of the few species that means to be resistant to this disease because it does not present symptoms. Despite the fact that it is commonly found in citric plants, little is known about the populations of G. mangiferae associated with these plants. Hence, the objective of this work was to gain insights about the genetic diversity of the G. mangiferae populations that colonize “Tahiti” acid limes by sequencing cistron ITS1-5.8S-ITS2. It was verified that “Tahiti” acid lime plants are hosts of G. mangiferae and also of G. citricarpa, without presenting symptoms of CBS. Populations of G. mangiferae present low-to-moderate genetic diversity and show little-to-moderate levels of population differentiation. As gene flow was detected among the studied populations and they share haplotypes, it is possible that all populations, from citrus plants and also from the other known hosts of this fungus, belong to one great panmictic population

Influence of Vinasse Application in the Structure and Composition of the Bacterial Community of the Soil under Sugarcane Cultivation

Although the use of vinasse as a waste helps replenish soil nutrients and improves the quality of the sugarcane crop, it is known that vinasse residues alter the diversity of bacteria naturally present in the soil. The actual impacts of vinasse application on the selection of bacterial taxa are not understood because no studies have addressed this phenomenon directly. Analysis of 16S rRNA gene clone sequences from four soil types showed that the soil planted with sugarcane and fertilized with vinasse has a high diversity of bacteria compared to other biomes, where Acidobacteria were the second most abundant phylum. Although the composition and structure of bacterial communities differ significantly in the four environments (Libshuff 's test), forest soils and soil planted with sugarcane without vinasse fertilizer were similar to each other because they share at least 28 OTUs related to Rhizobiales, which are important agents involved in nitrogen fixation. OTUs belonging to Actinomycetales were detected more often in the soil that had vinasse applied, indicating that these groups are more favored by this type of land management

Biodegradation of hepatotoxin (D-Leu1) - microcystin-LR by bacteria in carbon biological filters

A persistência das microcistinas (MCs) em ambientes aquáticos e sua difícil remoção no tratamento convencional de água representam um desafio às companhias de saneamento. Contudo, as MCs são susceptíveis à degradação por bactérias presentes na água, sedimentos e efluentes de esgotos. Neste estudo, avaliou-se a biodegradação de MCs por microrganismos presentes em filtros de carvão com atividade biológica (CAB) e sua identificação filogenética pelo sequenciamento do gene 16S RNA. Foi utilizada uma água de estudo contendo MCs com diferentes composições, acrescida de efluente de filtros CAB. Os resultados demonstraram que as MCs foram biodegradadas por microrganismos presentes no biofilme. Este estudo infere sobre a capacidade de biodegradação de MCs por bactérias presentes em filtros CAB e o possível uso destes microorganismos como alternativa de remoção de MCs no tratamento de água potável.The persistence of MCs in aquatic environments and their difficult removal in the conventional water treatment is a challenge to companies of sanitation. However, the MCs are susceptible to degradation by bacteria present in water, sediment and sewage effluents. In this study, we investigated the biodegradation of MCs by microorganism present in carbon filters with biological activity (BAC) and their phylogenetic identification by sequencing gene 16S RNA. A study of water containing MCs was used, with different compositions, plus a filters BAC effluent. The results showed that of MCs were biodegraded by microorganism present in the biofilm. This study provides the ability to complete biodegradation of MCs by bacteria present in BAC filters and the possible use of these microorganisms as alternative of the removal of MCs in the treatment of drinking wate

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Estudos sobre impacto do Eucalyptus spp. em solos brasileiros têm focalizado propriedades químicas do solo e isolamento de microrganismos de interesse. No Brasil há pouco enfoque em ecologia e diversidade microbiana, devido às limitações dos métodos tradicionais de cultivo e isolamento. A utilização de métodos moleculares no estudo da ecologia microbiana baseados na amplificação por PCR do 16S rDNA têm enriquecido o conhecimento da biodiversidade microbiana dos solos. O objetivo deste trabalho foi comparar e estimar a diversidade bacteriana de comunidades simpátricas em solos de duas áreas: uma floresta nativa (NFA) e outra adjacente com arboreto de eucaliptos (EAA). Oligonucleotídeos iniciadores foram utilizados para amplificar o 16S rDNA metagenômico do solo, o qual foi clonado usando pGEM-T e seqüenciado para determinar a diversidade bacteriana. Foram analisados 134 clones do solo NFA e 116 clones do solo EAA. As seqüências foram comparadas às depositadas no GenBank. Análises filogenéticas revelaram diferenças entre os tipos de solos e alta diversidade em ambas comunidades. No solo de arboreto de Eucalyptus spp. foi encontrada maior diversidade bacteriana em comparação com o solo da área de floresta nativa.Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA) and an eucalyptus arboretum (EAA). PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area