ABSTRACT Carnivorous plant species, such as Utricularia spp., capture and digest prey. This digestion can occur through the secretion of plant digestive enzymes and/or by bacterial digestive enzymes. To comprehend the physiological mechanisms of carnivorous plants, it is essential to understand the microbial diversity related to these plants. Therefore, in the present study, we isolated and classified bacteria from different organs of Utricularia breviscapa (stolons and utricles) and from different geographic locations (São Paulo and Mato Grosso). We were able to build the first bacterium collection for U. breviscapa and study the diversity of cultivable bacteria. The results show that U. breviscapa bacterial diversity varied according to the geographic isolation site (São Paulo and Mato Grosso) but not the analyzed organs (utricle and stolon). We reported that six genera were common to both sample sites (São Paulo and Mato Grosso). These genera have previously been reported to be beneficial to plants, as well as related to the bioremediation process, showing that these isolates present great biotechnological and agricultural potential. This is the first report of an Acidobacteria isolated from U. breviscapa. The role of these bacteria inside the plant must be further investigated in order to understand their population dynamics within the host.

Abstract The bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC) and has been associated with important losses in commercial orchards of all sweet orange [Citrus sinensis (L.)] cultivars. The development of this disease depends on the environmental conditions, including the endophytic microbial community associated with the host plant. Previous studies have shown that X. fastidiosa interacts with the endophytic community in xylem vessels as well as in the insect vector, resulting in a lower bacterial population and reduced CVC symptoms. The citrus endophytic bacterium Methylobacterium mesophilicum can trigger X. fastidiosa response in vitro, which results in reduced growth and induction of genes associated with energy production, stress, transport, and motility, indicating that X. fastidiosa has an adaptive response to M. mesophilicum. Although this response may result in reduced CVC symptoms, the colonization rate of the endophytic bacteria should be considered in studies that intend to use this endophyte to suppress CVC disease. Symbiotic control is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace pathogens. Candidate endophytes for symbiotic control of CVC must occupy the xylem of host plants and attach to the precibarium of sharpshooter insects to access the pathogen. In the present review, we focus on interactions between endophytic bacteria from sweet orange plants and X. fastidiosa, especially those that may be candidates for control of CVC.

ABSTRACT The microorganism-microorganism or microorganism-host interactions are the key strategy to colonize and establish in a variety of different environments. These interactions involve all ecological aspects, including physiochemical changes, metabolite exchange, metabolite conversion, signaling, chemotaxis and genetic exchange resulting in genotype selection. In addition, the establishment in the environment depends on the species diversity, since high functional redundancy in the microbial community increases the competitive ability of the community, decreasing the possibility of an invader to establish in this environment. Therefore, these associations are the result of a co-evolution process that leads to the adaptation and specialization, allowing the occupation of different niches, by reducing biotic and abiotic stress or exchanging growth factors and signaling. Microbial interactions occur by the transference of molecular and genetic information, and many mechanisms can be involved in this exchange, such as secondary metabolites, siderophores, quorum sensing system, biofilm formation, and cellular transduction signaling, among others. The ultimate unit of interaction is the gene expression of each organism in response to an environmental (biotic or abiotic) stimulus, which is responsible for the production of molecules involved in these interactions. Therefore, in the present review, we focused on some molecular mechanisms involved in the microbial interaction, not only in microbial-host interaction, which has been exploited by other reviews, but also in the molecular strategy used by different microorganisms in the environment that can modulate the establishment and structuration of the microbial community.

The entomopathogenic fungi of the genus Metarhizium have several subtilisin-like proteases that are involved in pathogenesis and these have been used to investigate genes that are differentially expressed in response to different growth conditions. The identification and characterization of these proteases can provide insight into how the fungus is capable of infecting a wide variety of insects and adapt to different substrates. In addition, the pr1A gene has been used for the genetic improvement of strains used in pest control. In this study we used quantitative RT-PCR to assess the relative expression levels of the pr1A gene in M. anisopliae and M. acridum during growth in different culture conditions and during infection of the sugar cane borer, Diatraea saccharalis Fabricius. We also carried out a pathogenicity test to assess the virulence of both species against D. saccharalis and correlated the results with the pattern of pr1A gene expression. This analysis revealed that, in both species, the pr1A gene was differentially expressed under the growth conditions studied and during the pathogenic process. M. anisopliae showed higher expression of pr1A in all conditions examined, when compared to M. acridum. Furthermore, M. anisopliae showed a greater potential to control D. saccharalis. Taken together, our results suggest that these species have developed different strategies to adapt to different growing conditions.

Bacteria from the genus Methylobacterium interact symbiotically (endophytically and epiphytically) with different plant species. These interactions can promote plant growth or induce systemic resistance, increasing plant fitness. The plant colonization is guided by molecular communication between bacteria-bacteria and bacteria-plants, where the bacteria recognize specific exuded compounds by other bacteria (e.g. homoserine molecules) and/or by the plant roots (e.g. flavonoids, ethanol and methanol), respectively. In this context, the aim of this study was to evaluate the effect of quorum sensing molecules (N-acyl-homoserine lactones) and plant exudates (including ethanol) in the expression of a series of bacterial genes involved in Methylobacterium-plant interaction. The selected genes are related to bacterial metabolism (mxaF), adaptation to stressful environment (crtI, phoU and sss), to interactions with plant metabolism compounds (acdS) and pathogenicity (patatin and phoU). Under in vitro conditions, our results showed the differential expression of some important genes related to metabolism, stress and pathogenesis, thereby AHL molecules up-regulate all tested genes, except phoU, while plant exudates induce only mxaF gene expression. In the presence of plant exudates there is a lower bacterial density (due the endophytic and epiphytic colonization), which produce less AHL, leading to down regulation of genes when compared to the control. Therefore, bacterial density, more than plant exudate, influences the expression of genes related to plant-bacteria interaction.

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.

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% 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.

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%) and 346 (64.2%) 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.

A mancha marrom ou mancha de Alternaria é uma doença causada pelo fungo Alternaria alternata f. sp. citri, encontrada no Brasil desde 2001 em plantas de tangerina e seus híbridos. Por se tratar de uma doença recente no Brasil, a epidemiologia e variabilidade genética deste patógeno compõem importantes pontos a serem estudados. Este trabalho teve como objetivo avaliar a variabilidade genética deste patógeno por meio da caracterização de vinte e quatro isolados de A. alternata f. sp. citri de plantas de citros juntamente com quatro isolados endofíticos de manga, sendo um deles identificado como Alternaria tenuissima e outros três como Alternaria arborescens. A análise de componentes principais (PCA) do perfil de bandas obtidos pela aplicação de duas técnicas de marcadores moleculares, Amplificação Aleatória de Polimorfismos de DNA (RAPD) e Polimorfismo de Comprimento de Fragmentos Amplificados (AFLP), mostrou a formação de quatro grupos distintos. Apesar do mais amplo perfil de análise por meio da técnica de AFLP, não foi observado nenhuma modificação significativa dentro dos grandes grupos obtidos quando comparado ao RAPD, exceto no posicionamento dos isolados LRS 39/3 e 25M. Além disso, em ambas as análises, somente os isolados de plantas de limão agruparam entre si. Considerando outros hospedeiros ou tecidos de planta não foi possível encontrar grupos específicos. Concluindo, ambas as análises (RAPD e AFLP) são eficientes no estudo de variabilidade genética de Alternaria spp., fornecendo informações sobre a diversidade genética desta espécie, servindo como base para futuramente correlacionar este estudo com estudos adicionais objetivando o controle da doença.

The Alternaria brown spot (ABS) is a disease caused in tangerine plants and its hybrids by the fungus Alternaria alternata f. sp. citri which has been found in Brazil since 2001. Due to the recent occurrence in Brazilian orchards, the epidemiology and genetic variability of this pathogen is still an issue to be addressed. Here it is presented a survey about the genetic variability of this fungus by the characterization of twenty four pathogenic isolates of A. alternata f. sp. citri from citrus plants and four endophytic isolates from mango (one Alternaria tenuissima and three Alternaria arborescens). The application of two molecular markers Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) had revealed the isolates clustering in distinct groups when fingerprintings were analyzed by Principal Components Analysis (PCA). Despite the better assessment of the genetic variability through the AFLP, significant modifications in clusters components were not observed, and only slight shifts in the positioning of isolates LRS 39/3 and 25M were observed in PCA plots. Furthermore, in both analyses, only the isolates from lemon plants revealed to be clustered, differently from the absence of clustering for other hosts or plant tissues. Summarizing, both RAPD and AFLP analyses were both efficient to detect the genetic variability within the population of the pathogenic fungus Alternaria spp., supplying information on the genetic variability of this species as a basis for further studies aiming the disease control.

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.

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.

O objetivo deste trabalho foi estudar a produção de sideróforos pelas bactérias endofíticas Methylobacterium spp., que ocupam o mesmo nicho ecológico que Xylella fastidiosa subsp. pauca (Xfp), em plantas cítricas. A produção de sideróforos, pelas linhagens de Methylobacterium, foi testada por meio do ensaio de cromoazarol-ágar (chromeazurol agar assay-CAS), teste de Csáky (tipo hidroxamato) e do teste de Arnow (tipo catecol). Além disso, a habilidade de Xfp em utilizar sideróforos produzidos por bactérias endofíticas, como fonte de ferro, in vitro, foi avaliada. Todas as 37 linhagens de Methylobacterium spp. testadas foram positivas para a produção de sideróforos, pelo teste CAS-ágar. Methylobacterium spp. foram capazes de produzir sideróforos do tipo hidroxamato, mas não do tipo catecol. O crescimento in vitro de Xfp foi estimulado pela presença de sideróforos no sobrenadante de Methylobacterium mesophilicum endofítica.

The objective of this work was to study the production of siderophores by endophytic bacteria Methylobacterium spp., which occupy the same ecological niche as Xylella fastidiosa subsp. pauca (Xfp) in citrus plants. The siderophore production of Methylobacterium strains was tested according to chromeazurol agar assay test (CAS), Csáky test (hydroxamate-type) and Arnow test (catechol-type). In addition, the ability of Xfp to use siderophores, in vitro, produced by endophytic bacteria as source of iron, was evaluated. All 37 strains of Methylobacterium spp. tested were CAS-positive for siderophore production. Methylobacterium spp. produced hydroxamate-type, but not catechol-type siderophores. In vitro growth of Xfp was stimulated by the presence of supernatant siderophores of endophytic Methylobacterium mesophilicum.

O objetivo deste trabalho foi avaliar a resistência de Guignardia citricarpa aos fungicidas carbendazim e piraclostrobina, por meio de avaliação do crescimento em meio de cultura, decomposição de tecido foliar e produção de corpos de frutificação. Para isso, o fungo G. citricarpa foi isolado de lesões de frutos de laranja (Citrus sinensis), produzidos em área com intensa aplicação de fungicida. Os isolados obtidos foram avaliados quanto à sensibilidade aos fungicidas piraclostrobina e carbendazim, nas dosagens de 0,5, 1 e 2 µg mL-1 de i.a., para se verificar o efeito da pressão de seleção causada pelo uso destes compostos em áreas citrícolas. Embora tenha sido observada redução efetiva no número de estruturas reprodutivas e na decomposição de folhas e frutos infectados com G. citricarpa, após a aplicação dos fungicidas, 7,5% dos isolados avaliados sobre meio de cultura apresentaram resistência a esse fungicida, o que indica que pode ocorrer seleção de isolados resistentes no campo. Para a piraclostrobina não foi observada resistência, o que indica que pode ser um composto alternativo para ser utilizado de forma alternada com carbendazim, para diminuir as chances de ocorrência de resistência do patógeno.

The objective of this work was to evaluate the resistance of Guignardia citricarpa to pyraclostrobine and carbendazim fungicides, through: growth analysis in culture media amended with the fungicides; leaf decomposition; and production of reproductive structures on leaves naturally infected with these fungi. G. citricarpa was isolated from symptomatic fruits of sweet orange (Citrus sinensis) cultivated in area with intense fungicide application. The sensibility to fungicides of G. citricarpa isolates was evaluated with pyraclostrobine and carbendazim, in the doses 0.5, 1 e 2 µg mL-1 a.i., to verify the effect of selection pressure caused by continuous use of these compounds. Although, reduction on leaf decomposition and on the number of reproductive structures were observed after application of pyraclostrobine and carbendazim, 7.5% of the isolates, evaluated in culture media, exhibited resistance to carbendazim, suggesting that the use of this agrochemical must be combined to other active principles in a consortium to reduce the chances of resistance occurrence. No resistance to pyraclostrobine was observed among tested isolates, what indicates that this could be an alternative compound to be used in combination to carbendazim, to minimize the chance of resistance occurrence.

A rubelose é uma doença causada pelo fungo Erythricium salmonicolor que atinge muitos hospedeiros, como citros, café, seringueira, eucalipto, Acacia sp., infectando principalmente os galhos. Rubelose é um sério problema para o Brasil, reduzindo a produção de citros em valores próximos de 10%. A diversidade do fungo E. salmonicolor em cultivares brasileiras ainda não foi avaliada. Este trabalho teve como objetivos: i) avaliar a variabilidade genética, por meio de RAPD, de 19 isolados de E. salmonicolor provenientes de diferentes regiões citrícolas de São Paulo e Minas Gerais, ii) avaliar a compatibilidade vegetativa e fusão de hifas do fungo E. salmonicolor. Após a análise por RAPD, foram observados 6 grupos distintos, os quais não apresentaram correlação com o local de origem e espécie hospedeira. No teste de compatibilidade vegetativa, houve encontro de hifas em todos os cruzamentos e 84% destes apresentaram fusão entre elas. Foi verificada compatibilidade entre linhagens, embora não tenha sido observada correlação com os haplótipos. Os resultados observados neste trabalho indicam a importância de futuros estudos sobre a fase sexual do fungo E. salmonicolor, uma vez que a anastomose de hifas precede a formação de heterocário, onde ocorrem os processos de recombinação sexual e parassexual responsáveis pela variabilidade genética em fungos filamentosos.

The Pink Disease is caused by Erythricium salmonicolor, which attacks broad hosts, such as citrus, coffee, rubber, Eucalyptus spp. and Acacia spp., infecting mainly branches. This disease became a serious problem in Brazil, reducing the citrus production up to 10%. However the genetic diversity and compatibility of the fungus E. salmonicolor from Brazilian citrus plants is not yet evaluated. Therefore, the aims of this study were to evaluate: i) the genetic variability of E. salmonicolor in the São Paulo and Minas Gerais States by the RAPD technique, and ii) the vegetative compatibility between these isolates. After RAPD analysis, six distinct groups were observed without correlation between the isolation site or host species. In the vegetative compatibility test, the contact of fungal hyphae between all evaluated crosses was observed, of which 84% presented hyphal fusion. Although the compatibility between strains was observed, no correlation between RAPD haplotypes and hyphal anastomosis was verified. These results show the importance of future studies on the sexual cycle of E. salmonicolor, since hyphal fusion, which precedes the formation of heterokaryons (sexual and parasexual reproduction) that could be responsible for the genetic variability in this species.

O objetivo deste trabalho foi caracterizar a comunidade bacteriana endofítica de plantas assintomáticas (escapes) e afetadas pela clorose variegada dos citros (CVC) por meio de isolamento em meio de cultura, técnica de gradiente desnaturante em gel de eletroforese (DGGE) e detecção de Methylobacterium mesophilicum e Xyllela fastidiosa por meio de PCR específico, para estudar esta comunidade e sua relação com a ocorrência da CVC. A análise da comunidade bacteriana via DGGE permitiu a detecção de X. fastidiosa, bem como Klebsiella sp. e Acinetobacter sp. como endófitos de citros. Foram observados também Curtobacterium sp., Pseudomonas sp., Enterobacter sp. e Bacillus spp. Utilizando primers específicos, Methylobacterium mesophilicum e X. fastidiosa também foram observadas, reforçando hipóteses de que estas bactérias podem estar interagindo no interior da planta hospedeira.

The aim of this work was to characterize endophytic bacterial community of assintomatic (escape) and Citrus Variegated Chlorosis (CVC)-affected citrus plants using isolation in culture medium, denaturing gradient gel electrophoresis (DGGE) technique and Methylobacterium mesophilicum as well as Xylella fastidiosa specific PCR, allowing to assess this community and its interactions with CVC. The study of bacterial community by DGGE analysis allowed the detection of X. fastidiosa, as well as Klebsiella sp. e Acinetobacter sp., which were not detected previously. Curtobacterium sp., Pseudomonas sp., Enterobacter sp. and Bacillus spp. were also observed as endophyte in citrus plants. Using specific primers Methylobacterium mesophilicum and X. fastidiosa were observed, reinforcing that these bacteria could interact inside the host plant.

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.