Transcriptome and Secretome Analyses of Endophyte Methylobacterium mesophilicum and Pathogen Xylella fastidiosa Interacting Show Nutrient Competition.

Xylella fastidiosa is the causal agent of several plant diseases affecting fruit and nut crops. Methylobacterium mesophilicum strain SR1.6/6 was isolated from Citrus sinensis and shown to promote plant growth by producing phytohormones, providing nutrients, inhibiting X. fastidiosa, and preventing Citrus Variegated Chlorosis. However, the molecular mechanisms involved in the interaction among these microbes are still unclear. The present work aimed to analyze physiological and molecular aspects of M. mesophilicum SR1.6/6 and X. fastidiosa 9a5c in co-culture. The transcriptome and secretome analyses indicated that X. fastidiosa down-regulates cell division and transport genes and up-regulates stress via induction of chaperones and pathogenicity-related genes including, the lipase-esterase LesA, a protease, as well as an oligopeptidase in response to M. mesophilicum competition. On the other hand, M. mesophilicum also down-regulated transport genes, except for iron uptake, which was up-regulated. Secretome analysis identified four proteins in M. mesophilicum exclusively produced in co-culture with X. fastidiosa, among these, three are related to phosphorous uptake. These results suggest that M. mesophilicum inhibits X. fastidiosa growth mainly due to nutrient competition for iron and phosphorous, thus promoting X. fastidiosa starvation, besides producing enzymes that degrade X. fastidiosa cell wall, mainly hydrolases. The understanding of these interactions provides a direction for control and management of the phytopathogen X. fastidiosa, and consequently, helps to improve citrus growth and productivity.

Mechanisms of Mucor sp. CM3 isolated from the aquatic macrophyte Eichhornia crassipes (Mart.) Solms to increase cadmium bioremediation.

Bioremediation of toxic metals is a feasible and low-cost remediation tool to reduce metal contamination. Plant-fungus interactions can improve this technique. Eichhornia crassipes (Mart.) Solms is a macrophyte reported to bioremediate contaminated water. Thus, the present study aimed to isolate endophytic fungi from E. crassipes, select a highly cadmium (Cd) tolerant isolate and evaluate its bioremediation potential. This was evaluated by (1) the fungus tolerance and capacity to accumulate Cd; (2) Cd effects on cell morphology (using SEM and TEM) and on the fungal antioxidant defense system, as well as (3) the effect on model plant Solanum lycopersicum L. cultivar Calabash Rouge, inoculated with the endophyte fungus and exposed to Cd. Our results selected the endophyte Mucor sp. CM3, which was able to tolerate up to 1000 g/L of Cd and to accumulate 900 mg of Cd/g of biomass. Significant changes in Mucor sp. CM3 morphology were observed when exposed to high Cd concentrations, retaining this metal both in its cytoplasm and in its cell wall, which may be linked to detoxification and metal sequestration mechanisms related to the formation of Cd-GSH complexes. In addition, Cd stress induced the activation of all tested antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) - in this endophytic fungus. Moreover, when inoculated in tomato plants, this fungus promoted plant growth (in treatments without Cd) and induced an increased metal translocation to plant shoot, showing its potential to increase metal bioremediation. Therefore, this study indicates that the isolated endophyte Mucor sp. CM3 can be applied as a tool in different plant conditions, improving plant bioremediation and reducing the environmental damage caused by Cd, while also promoting plant growth in the absence of contaminants.

The auxin-producing Bacillus thuringiensis RZ2MS9 promotes the growth and modifies the root architecture of tomato (Solanum lycopersicum cv. Micro-Tom).

Strains of Bacillus thuringiensis (Bt) are commonly commercialized as bioinoculants for insect pest control, but their benefits go beyond their insecticidal property: they can act as plant growth-promoters. Auxins play a major role in the plant growth promotion. However, the mechanism of auxin production by the Bacilli group, and more specifically by Bt strains, is unclear. In previous work, the plant growth-promoting rhizobacterium (PGPR) B. thuringiensis strain RZ2MS9 increased the corn roots. This drew our attention to the strain's auxin production trait, earlier detected in vitro. Here, we demonstrate that in its genome, RZ2MS9 harbours the complete set of genes required in two pathways that are used for Indole acetic acid (IAA) production. We also detected that the strain produces almost five times more IAA during the stationary phase. The bacterial application increased the shoot dry weight of the Micro-Tom (MT) tomato by 24%. The application also modified MT root architecture, with an increase of 26% in the average lateral root length and inhibition of the axial root. At the cellular level, RZ2MS9-treated MT plants presented elongated root cortical cells with intensified mitotic activity. Altogether, these are the best characterized auxin-associated phenotypes. Besides that, no growth alteration was detected in the auxin-insensitive diageotropic (dgt) plants either with or without the RZ2MS9 inoculation. Our results suggest that auxins play an important role in the ability of B. thuringiensis RZ2MS9 to promote MT growth and provide a better understanding of the auxin production mechanism by a Bt strain.

Bacterial communities associated with anthracnose symptomatic and asymptomatic leaves of guarana, an endogenous tropical crop, and their pathogen antagonistic effects.

Plants are colonized by diverse microorganisms that can substantially impact their health and growth. Understanding bacterial diversity and the relationships between bacteria and phytopathogens may be key to finding effective biocontrol agents. We evaluated the bacterial community associated with anthracnose symptomatic and asymptomatic leaves of guarana, a typical tropical crop. Bacterial communities were assessed through culture-independent techniques based on extensive 16S rRNA sequencing, and cultured bacterial strains were evaluated for their ability to inhibit the growth of Colletotrichum sp. as well as for enzyme and siderophore production. The culture-independent method revealed that Proteobacteria was the most abundant phylum, but many sequences were unclassified. The emergence of anthracnose disease did not significantly affect the bacterial community, but the abundance of the genera Acinetobacter, Pseudomonas and Klebsiella were significantly higher in the symptomatic leaves. In vitro growth of Colletotrichum sp. was inhibited by 11.38% of the cultured bacterial strains, and bacteria with the highest inhibition rates were isolated from symptomatic leaves, while asymptomatic leaves hosted significantly more bacteria that produced amylase and polygalacturonase. The bacterial isolate Bacillus sp. EpD2-5 demonstrated the highest inhibition rate against Colletotrichum sp., whereas the isolates EpD2-12 and FD5-12 from the same genus also had high inhibition rates. These isolates were also able to produce several hydrolytic enzymes and siderophores, indicating that they may be good candidates for the biocontrol of anthracnose. Our work demonstrated the importance of using a polyphasic approach to study microbial communities from plant diseases, and future work should focus on elucidating the roles of culture-independent bacterial communities in guarana anthracnose disease.

Cultivated bacterial diversity associated with the carnivorous plant Utricularia breviscapa (Lentibulariaceae) from floodplains in Brazil

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.

Cultivated bacterial diversity associated with the carnivorous plant Utricularia breviscapa (Lentibulariaceae) from floodplains in Brazil.

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.

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.

Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth.

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 25,000km2 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.

Microbial interactions: ecology in a molecular perspective

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.

Microbial interactions: ecology in a molecular perspective.

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.

Diversity of Cultivated Fungi Associated with Conventional and Transgenic Sugarcane and the Interaction between Endophytic Trichoderma virens and the Host Plant.

Plant-associated fungi are considered a vast source for biotechnological processes whose potential has been poorly explored. The interactions and diversity of sugarcane, one of the most important crops in Brazil, have been rarely studied, mainly concerning fungal communities and their interactions with transgenic plants. Taking this into consideration, the purpose of this study was, based on culture dependent strategy, to determine the structure and diversity of the fungal community (root endophytes and rhizosphere) associated with two varieties of sugarcane, a non-genetically modified (SP80-1842) variety and its genetically modified counterpart (IMI-1, expressing imazapyr herbicide resistance). For this, the sugarcane varieties were evaluated in three sampling times (3, 10 and 17 months after planting) under two crop management (weeding and herbicide treatments). In addition, a strain of Trichoderma virens, an endophyte isolated from sugarcane with great potential as a biological control, growth promotion and enzyme production agent, was selected for the fungal-plant interaction assays. The results of the isolation, characterization and evaluation of fungal community changes showed that the sugarcane fungal community is composed of at least 35 different genera, mostly in the phylum Ascomycota. Many genera are observed at very low frequencies among a few most abundant genera, some of which were isolated from specific plant sites (e.g., the roots or the rhizosphere). An assessment of the possible effects upon the fungal community showed that the plant growth stage was the only factor that significantly affected the community's structure. Moreover, if transgenic effects are present, they may be minor compared to other natural sources of variation. The results of interaction studies using the Green fluorescent protein (GFP)-expressing T. virens strain T.v.223 revealed that this fungus did not promote any phenotypic changes in the host plant and was found mostly in the roots where it formed a dense mycelial cover and was able to penetrate the intercellular spaces of the root epidermis upper layers. The ability of T. virens to colonize plant roots suggests a potential for protecting plant health, inhibiting pathogens or inducing systemic resistance.

Microbial interactions: ecology in a molecular perspective

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.

Genes related to antioxidant metabolism are involved in Methylobacterium mesophilicum-soybean interaction.

The genus Methylobacterium is composed of pink-pigmented methylotrophic bacterial species that are widespread in natural environments, such as soils, stream water and plants. When in association with plants, this genus colonizes the host plant epiphytically and/or endophytically. This association is known to promote plant growth, induce plant systemic resistance and inhibit plant infection by phytopathogens. In the present study, we focused on evaluating the colonization of soybean seedling-roots by Methylobacterium mesophilicum strain SR1.6/6. We focused on the identification of the key genes involved in the initial step of soybean colonization by methylotrophic bacteria, which includes the plant exudate recognition and adaptation by planktonic bacteria. Visualization by scanning electron microscopy revealed that M. mesophilicum SR1.6/6 colonizes soybean roots surface effectively at 48 h after inoculation, suggesting a mechanism for root recognition and adaptation before this period. The colonization proceeds by the development of a mature biofilm on roots at 96 h after inoculation. Transcriptomic analysis of the planktonic bacteria (with plant) revealed the expression of several genes involved in membrane transport, thus confirming an initial metabolic activation of bacterial responses when in the presence of plant root exudates. Moreover, antioxidant genes were mostly expressed during the interaction with the plant exudates. Further evaluation of stress- and methylotrophic-related genes expression by qPCR showed that glutathione peroxidase and glutathione synthetase genes were up-regulated during the Methylobacterium-soybean interaction. These findings support that glutathione (GSH) is potentially a key molecule involved in cellular detoxification during plant root colonization. In addition to methylotrophic metabolism, antioxidant genes, mainly glutathione-related genes, play a key role during soybean exudate recognition and adaptation, the first step in bacterial colonization.

Differential gene expression in Xylella fastidiosa 9a5c during co-cultivation with the endophytic bacterium Methylobacterium mesophilicum SR1.6/6.

Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC), colonizes plant xylem, reducing sap flow, and inducing internerval chlorosis, leaf size reduction, necrosis, and harder and smaller fruits. This bacterium may be transmitted from plant to plant by sharpshooter insects, including Bucephalogonia xanthopis. The citrus endophytic bacterium Methylobacterium mesophilicum SR1.6/6 colonizes citrus xylem and previous studies showed that this strain is also transferred from plant to plant by B. xanthopis (Insecta), suggesting that this endophytic bacterium may interact with X. fastidiosa in planta and inside the insect vector during co-transmission by the same insect vector. To better understand the X. fastidiosa behavior in the presence of M. mesophilicum, we evaluated the X. fastidiosa transcriptional profile during in vitro interaction with M. mesophilicum SR1.6/6. The results showed that during co-cultivation, X. fastidiosa down-regulated genes related to growth and up-regulated genes related to energy production, stress, transport, and motility, suggesting the existence of a specific adaptive response to the presence of M. mesophilicum in the culture medium.

Biotechnological and agronomic potential of endophytic pink-pigmented methylotrophic Methylobacterium spp.

The genus Methylobacterium is composed of pink-pigmented facultative methylotrophic (PPFM) bacteria, which are able to synthesize carotenoids and grow on reduced organic compounds containing one carbon (C1), such as methanol and methylamine. Due to their high phenotypic plasticity, these bacteria are able to colonize different habitats, such as soil, water, and sediment, and different host plants as both endophytes and epiphytes. In plant colonization, the frequency and distribution may be influenced by plant genotype or by interactions with other associated microorganisms, which may result in increasing plant fitness. In this review, different aspects of interactions with the host plant are discussed, including their capacity to fix nitrogen, nodule the host plant, produce cytokinins, auxin and enzymes involved in the induction of systemic resistance, such as pectinase and cellulase, and therefore plant growth promotion. In addition, bacteria belonging to this group can be used to reduce environmental contamination because they are able to degrade toxic compounds, tolerate high heavy metal concentrations, and increase plant tolerance to these compounds. Moreover, genome sequencing and omics approaches have revealed genes related to plant-bacteria interactions that may be important for developing strains able to promote plant growth and protection against phytopathogens.

Differential responses of the antioxidant system of ametryn and clomazone tolerant bacteria.

The herbicides ametryn and clomazone are widely used in sugarcane cultivation, and following microbial degradation are considered as soil and water contaminants. The exposure of microorganisms to pesticides can result in oxidative damage due to an increase in the production of reactive oxygen species (ROS). This study investigated the response of the antioxidant systems of two bacterial strains tolerant to the herbicides ametryn and clomazone. Bacteria were isolated from soil with a long history of ametryn and clomazone application. Comparative analyses based on 16S rRNA gene sequences revealed that strain CC07 is phylogenetically related to Pseudomonas aeruginosa and strain 4C07 to P. fulva. The two bacterial strains were grown for 14 h in the presence of separate and combined herbicides. Lipid peroxidation, reduced glutathione content (GSH) and antioxidant enzymes activities were evaluated. The overall results indicated that strain 4C07 formed an efficient mechanism to maintain the cellular redox balance by producing reactive oxygen species (ROS) and subsequently scavenging ROS in the presence of the herbicides. The growth of bacterium strain 4C07 was inhibited in the presence of clomazone alone, or in combination with ametryn, but increased glutathione reductase (GR) and glutathione S-transferase (GST) activities, and a higher GSH concentration were detected. Meanwhile, reduced superoxide dismutase (SOD), catalase (CAT) and GST activities and a lower concentration of GSH were detected in the bacterium strain CC07, which was able to achieve better growth in the presence of the herbicides. The results suggest that the two bacterial strains tolerate the ametryn and clomazone herbicides with distinctly different responses of the antioxidant systems.

Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules

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.

Methylobacterium-plant interaction genes regulated by plant exudate and quorum sensing molecules.

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.

Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association.

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.

The diversity of endophytic methylotrophic bacteria in an oil-contaminated and an oil-free mangrove ecosystem and their tolerance to heavy metals.

Methylobacterium strains were isolated from mangrove samples collected in Bertioga, SP, Brazil, from locations either contaminated or uncontaminated by oil spills. The tolerances of the strains to different heavy metals were assessed by exposing them to different concentrations of cadmium, lead, and arsenic (0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM). Additionally, the genetic diversity of Methylobacterium spp. was determined by sequence analysis of the 16S rRNA genes. The isolates from the contaminated locations were grouped, suggesting that oil can select for microorganisms that tolerate oil components and can change the methylotrophic bacterial community. Cadmium is the most toxic heavy metal assessed in this work, followed by arsenic and lead, and two isolates of Methylobacterium were found to be tolerant to all three metals. These isolates have the potential to bioremediate mangrove environments contaminated by oil spills by immobilizing the heavy metals present in the oil.