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

Studies on bacterial community composition are affected by the time and storage method of the rumen content

The objective of this study was to investigate three storage methods and four storage times for rumen sampling in terms of quality and yield of extracted metagenomic DNA as well as the composition of the rumen bacterial community. One Nellore steer fitted with a ruminal silicone- type cannula was used as a donor of ruminal contents. The experiment comprised 11 experimental groups: pellet control (PC), lyophilized control (LC), P-20: pellet stored frozen at -20ÊC for a period of 3, 6, and 12 months, P-80: pellet stored frozen at -80ÊC for a period of 3, 6, and 12 months, and L-20: lyophilized sample stored frozen at -20ÊC for a period of 3, 6, and 12 months. Metagenomic DNA concentrations were measured spectrophotometrically and fluorometrically and ion torrent sequencing was used to assess the bacterial community composition. The L-20 method could not maintain the yield of DNA during storage. In addition, the P-80 group showed a greater yield of metagenomic DNA than the other groups after 6 months of storage. Rumen samples stored as pellets (P-20 and P-80) resulted in lower richness Chao 1, ACE, and Shannon Wiener indices when compared to PC, while LC and PC were only different in richness ACE. The storage method and storage time influenced the proportions of 14 of 17 phyla identified by sequencing. In the P-20 group, the proportion of Cyanobacteria, Elusimicrobia, Fibrobacteres, Lentisphaerae, Proteobacteria, and Spirochaetes phyla identified was lower than 1%. In the P-80 group, there was an increase in the proportion of the Bacteroidetes phylum (p = 0.010); however, the proportion of Actinobacteria, Chloroflexi, SR1, Synergistetes, TM7, and WPS.2 phyla were unchanged compared to the PC group (p > 0.05). The class Clostridium was the most abundant in all stored groups and increased in its proportion, especially in the L-20 group. The rumen sample storage time significantly reduced the yield of metagenomic DNA extracted. Therefore, the storage method can influence the abundance of phyla, classes, and bacterial families studied in rumen samples and affect the richness and diversity index

Análise do microbioma procarionte de fêmeas e cultura celular embrionária de Rhipicephalus sanguineus linhagens tropical e temperada de duas localidades no Brasil

Two lineages of Rhipicephalus sanguineus are known in Brazil: the temperate or southern and the tropical or northern populations. The distribution patterns of both lineages of R. sanguineus have epidemiological implications that can affect vectorial competence concerning Ehrlichia canis, the agent of canine monocytic ehrlichiosis. Intending to identify the microbiomes of both lineages and compare microorganisms in R. sanguineus, we used the 16S rRNA (V4-V5 region) gene-based metataxonomic approach, through NGS sequencing on the MiSeq Illumina platform. We selected specimens of females from the environment and samples of primary embryonic cell cultures, from both lineages, and this was the first study to investigate the prokaryotic microbiome in tick cell cultures. The results showed that many bacterial taxa detected in the samples were typical members of the host environment. A significant diversity of microorganisms in R. sanguineus females and in embryonic cell cultures from both lineages was found, with emphasis on the presence of Coxiella in all samples, albeit in different proportions. The Coxiella species present in the two lineages of ticks may be different and may have co-evolved with them, thus driving different patterns of interactions between ticks and the pathogens that they can harbor or transmit to vertebrate hosts.Duas linhagens de Rhipicephalus sanguineus são conhecidas no Brasil: populações da linhagem temperada ou do sul, e tropical ou do norte. Os padrões de distribuição de ambas as linhagens de R. sanguineus têm implicações epidemiológicas, podendo afetar a competência vetorial de Ehrlichia canis, o agente etiológico da erliquiose monocítica canina. Com a intenção de identificar os microbiomas de ambas as linhagens e comparar microrganismos de R. sanguineus, foi utilizada a metataxonomia, baseada no gene 16S rRNA (região V4-V5), por meio do sequenciamento de nova geração na plataforma MiSeq Illumina. Foram selecionadas amostras de fêmeas do ambiente e cultivo primário de células embrionárias, considerando-se as duas linhagens conhecidas do Brasil. Este é o primeiro estudo que investiga o microbioma procariótico de células de cultura de carrapato. Os resultados mostram que muitos grupos de bactérias detectadas nas amostras são membros típicos do ambiente do hospedeiro. Uma diversidade significativa de microrganismos em fêmeas e cultura de células embrionárias nas duas linhagens de R. sanguineus foi encontrada, com ênfase na presença de Coxiella em todas as amostras, ainda que em diferentes proporções. Possivelmente, as espécies de Coxiella presentes nas duas linhagens de carrapatos são diferentes e co-evoluíram com essas linhagens, conduzindo a diferentes padrões de interação entre carrapatos e patógenos que podem abrigar ou transmitir aos hospedeiros vertebrados

Long-Term Encapsulated Nitrate Supplementation Modulates Rumen Microbial Diversity and Rumen Fermentation to Reduce Methane Emission in Grazing Steers

This study investigated the long-term effects (13 months) of encapsulated nitrate supplementation (ENS) on enteric methane emissions, rumen fermentation parameters, ruminal bacteria, and diversity of archaea in grazing beef cattle. We used a total of thirty-two Nellore steers (initial BW of 197 ± 15.3 kg), 12 of which were fitted with rumen cannulas. For 13 months, the animals were maintained in 12 paddocks and fed a concentrate of ground corn, soybean meals, mineral supplements, and urea (URS) or encapsulated nitrate (EN) containing 70 g of EN/100 kg of BW (corresponding to 47 g NO3-/100 kg BW). Encapsulated nitrate supplementation resulted in similar forage, supplement and total DMI values as URS (P > 0.05), but ENS tended to increase (+48 g/d; P = 0.055) average daily weight gain. Daily reductions in methane emissions (-9.54 g or 18.5%) were observed with ENS when expressed as g of CH4/kg of forage dry matter intake (fDMI) (P = 0.037). Lower concentrations of NH3-N and a higher ruminal pH were observed in ENS groups 6 h after supplementation (P < 0.05). Total VFA rumen concentration 6 h (P = 0.009) and 12 h after supplementation with EN resulted in lower acetate concentrations in the rumen (P = 0.041). Steers supplemented with EN had a greater ruminal abundance of Bacteroides, Barnesiella, Lactobacillus, Selenomonas, Veillonella, Succinimonas, Succinivibrio, and Duganella sp. (P < 0.05), but a lower abundance of Methanobrevibacter sp. (P = 0.007). Strong negative correlations were found between daily methane emissions and Proteobacteria, Erysipelotrichaceae, Prevotellaceae, and Roseburia, Kandleria, Selenomonas, Veillonella, and Succinivibrio sp. (P < 0.05) in the rumen of ENS steers. Encapsulated nitrate is a feed additive that persistently affects enteric methane emission in grazing steers, thereby decreasing Methanobrevibacter abundance in the rumen. In addition, ENS can promote fumarate-reducer and lactate-producer bacteria, thereby reducing acetate production during rumen fermentation

Análise comparativa das seqüências de genes da ilha simbiótica entre Bradyrhizobium elkanii e Bradyrhizobium japonicum

Bactérias simbiontes conhecidos como rizóbios interagem com raízes de leguminosas e induzem a formação de nódulos fixadores de nitrogênio. Em rizóbios, genes essenciais para simbiose são compartimentalizados em ilhas simbióticas ou em ilhas no cromossoma. Para o entendimento da estrutura e evolução dessas ilhas simbióticas, é necessário analisar seu contexto genético e sua organização. O genoma parcial de B. elkanii SEMIA 587 apresenta hoje um total de 5.821.111 pb seqüenciados contendo 62,6 % de GC em 3941 Contigs, onde foram identificados 5381 ORFs. Uma suposta ilha simbiótica foi localizada através da identificação de genes relacionados à fixação e nodulação em comparação à ilha simbiótica de B. japonicum, encontrando 267 ORFs em B. elkanii, onde 17 ORFs foram identificadas como transposases. Assim, através destes resultados parciais pode ser possível averiguar que B. elkanii provavelmente sofreu um rearranjo genético no cromossoma, já que ele apresenta um tamanho menor que B. japonicum em relação ao tamanho do genoma.Symbiont bacteria, commonly known as rhizobia interact with root legume and induce the formation of Nitrogen-fixing nodules. Among rhizobia, essential genes for symbioses are compartmentalized either in symbiotic islands or in chromosomal islands. For better understanding of the structure and evolution of these symbiotic islands, it is necessary to analyze their genetic context and organization. The work had as objective to analyze genes related to the symbiotic island in the partial genoma of the B. elkanii strain 587 has 5.821.111 bp sequenced, containing 62,6 % of GC in 3941 Contigs, been identified 5381 ORFs. A purported symbiotic island was localized through of the identification of related genes of fixing nitrogen and nodulation in comparison with the symbiotic island of B. japonicum, founding 267 ORFs in B. elkanii, where 17 ORFs were identified as transposases. So through these partial results could be possible to infer that B. elkanii probably shows a symbiotic island with genetic rearrange into chromosome, because it showed lower genome size than B. japonicum.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Evaluation of Xylella fastidiosa genetic diversity by fAFPL markers

A primeira bactéria fitopatogênica a ter seu genoma totalmente seqüenciado foi detectada e isolada em diferentes hospedeiros em diferentes regiões geográficas. Embora seja causadora de doenças em culturas economicamente importantes, como citros, cafeeiro e videira, pouco se conhece acerca das relações genéticas estabelecidas entre isolados da bactéria. Atualmente, todos os isolados são agrupados como uma única espécie, Xylella fastidiosa, apesar de colonizarem diferentes hospedeiros que desenvolvem sintomas diferenciados e possuir diferentes condições fisiológicas e microbiológicas. A existência de diversidade genética entre isolados da bactéria foi detectada da por métodos culturais e moleculares, embora pouco se conheça acerca das relações genéticas de isolados brasileiros obtidos de citros e cafeeiro. Este trabalho teve por objetivo verificar, através de marcadores moleculares fAFLP a existência de diversidade genética e as relações filogenéticas estabelecidas pelos isolados das bactérias brasileiras e estrangeiras. Estes marcadores foram selecionados por sua alta reproducibilidade e por ser de uso comum para tipagem e classificação bacteriana. Os resultados obtidos mostraram que os isolados brasileiros apresentam diversidade genética e que os isolados deste estudo agruparam-se de acordo com o hospedeiro e origem geográfica, a saber, citros-cafeeiro, temécula-videira-amoreira e ameixeira-elmo.The first phytopathogenic bacterium with its DNA entirely sequenced is being detected and isolated from different host plants in several geographic regions. Although it causes diseases in cultures of economic importance, such as citrus, coffee, and grapevine little is known about the genetic relationships among different strains. Actually, all strains are grouped as a single species, Xylella fastidiosa, despite colonizing different hosts, developing symptoms, and different physiological and microbiological observed conditions. The existence of genetic diversity among X. fastidiosa strains was detected by different methodological techniques, since cultural to molecular methods. However, little is know about the phylogenetic relationships developed by Brazilian strains obtained from coffee and citrus plants. In order to evaluate it, fAFLP markers were used to verify genetic diversity and phylogenetic relationships developed by Brazilian and strange strains. fAFLP is an efficient technique, with high reproducibility that is currently used for bacterial typing and classification. The obtained results showed that Brazilian strains present genetic diversity and that the strains from this study were grouped distinctly according host and geographical origin like citrus-coffee, temecula-grapevine-mulberry and plum-elm.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Phylogenetic diversity of methanogenic archae in diets with different hay proportions Diversidade filogenética de archaea metanogênica em dietas com diferentes proporções de feno

The objective of this research was to detect the presence of archaea in the bovine rumen using genetic sequences from the conserved region of the 16S rDNA. Samples were collected from bovines that were fed two different experimental dietetic ratios of roughage to concentrate. The 16S rDNA region was amplified using PCR and analyzed using the Phred/Phrap/Consed programs. For the treatment with 70% hay diet 96 sequences related to the Methanobacteriaceae family, 47 sequences from non-cultured archaea, and 60 sequences from unknown archaea were identified by the BLAST analysis. For the treatment with 30% hay diet the BLAST analysis identified 125 sequences belonging to the Methanobacteriaceae fa-mily 42 sequences from non-cultured archaea, and 32 sequences from unknown archaea. The analysis of the 16S rDNA sequences of archaea collected from the bovine rumen, allows more sequences matching the unknown archaea were identified in the treatment with 70% hay.O objetivo deste trabalho foi detectar a presença de arqueias no rúmen bovino por meio das sequências gênicas da região conservada 16S rDNA. As amostras foram coletadas de bovinos alimentados com duas dietas experimentais contendo diferentes relações volumoso e concentrado. Para amplificação da região ribossomal 16S rDNA foi feita PCR e a análise das sequências foi realizada pelos programas Phred/Phrap/Consed. As análises do BLAST permitiram identificar no tratamento com 70% de feno, 96 sequências relacionadas à família Methanobacteriaceae, 47 sequências a arqueias não cultiváveis e 60 sequências foram de arqueias desconhecidas e no tratamento com 30% de feno foram 125 sequências relacionadas à família Methanobacteriaceae, 42 sequências a arqueias não cultiváveis e 32 sequências foram de arqueias desconhecidas. A análise das sequências da região 16S rDNA de arqueias do rúmen bovino permitiu detectar maior número de sequências relacionadas com arqueias desconhecidas no tratamento com 70% de feno

A transcriptomic survey of Migdolus fryanus (sugarcane rhizome borer) larvae.

Sugarcane, a major crop grown in the tropical and subtropical areas of the world, is produced mainly for sucrose, which is used as a sweetener or for the production of bioethanol. Among the numerous pests that significantly affect the yield of sugarcane, the sugarcane rhizome borer (Migdolus fryanus, a cerambycidae beetle) is known to cause severe damage to the crops in Brazil. The absence of molecular information about this insect reinforces the need for studies and an effective method to control this pest. In this study, RNA-Seq technology was employed to study different parts of M. fryanus larvae. The generated data will help in further investigations about the taxonomy, development, and adaptation of this insect. RNA was extracted from six different parts (head, fat body, integument, hindgut, midgut, and foregut) using Trizol methodology. Using Illumina paired-end sequencing technology and the Trinity platform, trimming and de novo assembly was performed, resulting in 44,567 contigs longer than 200 nt for a reunion of data from all transcriptomes, with a mean length of 1,095.27 nt. Transcripts were annotated using BLAST against different protein databanks (Uniprot/Swissprot, PFAM, KEEG, SignalP 4.1, Gene Ontology, and CAZY) and were compared for similarity using a Venn diagram. Differential expression patterns were studied for select genes through qPCR and FPKM comprising important protein families (digestive peptidases, glucosyl hydrolases, serine protease inhibitors and otopetrin), which allowed a better understanding of the insect's digestion, immunity and gravity sensorial mechanisms