Multiple toxins and a protease contribute to the aphid-killing ability of Pseudomonas fluorescens PpR24.

Aphids are globally important pests causing damage to a broad range of crops. Due to insecticide resistance, there is an urgent need to develop alternative control strategies. In our previous work, we found Pseudomonas fluorescens PpR24 can orally infect and kill the insecticide-resistant green-peach aphid (Myzus persicae). However, the genetic basis of the insecticidal capability of PpR24 remains unclear. Genome sequencing of PpR24 confirmed the presence of various insecticidal toxins such as Tc (toxin complexes), Rhs (rearrangement hotspot) elements, and other insect-killing proteases. Upon aphids infection with PpR24, RNA-Seq analysis revealed 193 aphid genes were differentially expressed with down-regulation of 16 detoxification genes. In addition, 1325 PpR24 genes (542 were upregulated and 783 downregulated) were subject to differential expression, including genes responsible for secondary metabolite biosynthesis, the iron-restriction response, oxidative stress resistance, and virulence factors. Single and double deletion of candidate virulence genes encoding a secreted protease (AprX) and four toxin components (two TcA-like; one TcB-like; one TcC-like insecticidal toxins) showed that all five genes contribute significantly to aphid killing, particularly AprX. This comprehensive host-pathogen transcriptomic analysis provides novel insight into the molecular basis of bacteria-mediated aphid mortality and the potential of PpR24 as an effective biocontrol agent.

Microbial Catabolic Activity: Methods, Pertinence, and Potential Interest for Improving Microbial Inoculant Efficiency.

Microbial catabolic activity (MCA) defined as the degrading activity of microorganisms toward various organic compounds for their growth and energy is commonly used to assess soil microbial function potential. For its measure, several methods are available including multi-substrate-induced respiration (MSIR) measurement which allow to estimate functional diversity using selected carbon substrates targeting specific biochemical pathways. In this review, the techniques used to measure soil MCA are described and compared with respect to their accuracy and practical use. Particularly the efficiency of MSIR-based approaches as soil microbial function indicators was discussed by (i) showing their sensitivity to different agricultural practices including tillage, amendments, and cropping systems and (ii) by investigating their relationship with soil enzyme activities and some soil chemical properties (pH, soil organic carbon, cation exchange capacity). We highlighted the potential of these MSIR-based MCA measurements to improve microbial inoculant composition and to determine their potential effects on soil microbial functions. Finally, we have proposed ideas for improving MCA measurement notably through the use of molecular tools and stable isotope probing which can be combined with classic MSIR methods. Graphical abstract describing the interrelation between the different parts and the concepts developed in the review.

Is there sufficient Ensifer and Rhizobium species diversity in UK farmland soils to support red clover (Trifolium pratense), white clover (T. repens), lucerne (Medicago sativa) and black medic (M. lupulina)?

Rhizobia play important roles in agriculture owing to their ability to fix nitrogen through a symbiosis with legumes. The specificity of rhizobia-legume associations means that underused legume species may depend on seed inoculation with their rhizobial partners. For black medic (Medicago lupulina) and lucerne (Medicago sativa) little is known about the natural prevalence of their rhizobial partner Ensifer meliloti in UK soils, so that the need for inoculating them is unclear. We analysed the site-dependence of rhizobial seed inoculation effects on the subsequent ability of rhizobial communities to form symbioses with four legume species (Medicago lupulina, M. sativa, Trifolium repens and T. pratense). At ten organic farms across the UK, a species-diverse legume based mixture (LBM) which included these four species was grown. The LBM seed was inoculated with a mix of commercial inocula specific for clover and lucerne. At each site, soil from the LBM treatment was compared to the soil sampled prior to the sowing of the LBM (the control). From each site and each of the two treatments, a suspension of soils was applied to seedlings of the four legume species and grown in axenic conditions for six weeks. Root nodules were counted and their rhizobia isolated. PCR and sequencing of a fragment of the gyrB gene from rhizobial isolates allowed identification of strains. The number of nodules on each of the four legume species was significantly increased when inoculated with soil from the LBM treatment compared to the control. Both the proportion of plants forming nodules and the number of nodules formed varied significantly by site, with sites significantly affecting the Medicago species but not the Trifolium species. These differences in nodulation were broadly reflected in plant biomass where site and treatment interacted; at some sites there was a significant advantage from inoculation with the commercial inoculum but not at others. In particular, this study has demonstrated the commercial merit of inoculation of lucerne with compatible rhizobia.

The Importance of the Microbial N Cycle in Soil for Crop Plant Nutrition.

Nitrogen is crucial for living cells, and prior to the introduction of mineral N fertilizer, fixation of atmospheric N2 by diverse prokaryotes was the primary source of N in all ecosystems. Microorganisms drive the N cycle starting with N2 fixation to ammonia, through nitrification in which ammonia is oxidized to nitrate and denitrification where nitrate is reduced to N2 to complete the cycle, or partially reduced to generate the greenhouse gas nitrous oxide. Traditionally, agriculture has relied on rotations that exploited N fixed by symbiotic rhizobia in leguminous plants, and recycled wastes and manures that microbial activity mineralized to release ammonia or nitrate. Mineral N fertilizer provided by the Haber-Bosch process has become essential for modern agriculture to increase crop yields and replace N removed from the system at harvest. However, with the increasing global population and problems caused by unintended N wastage and pollution, more sustainable ways of managing the N cycle in soil and utilizing biological N2 fixation have become imperative. This review describes the biological N cycle and details the steps and organisms involved. The effects of various agricultural practices that exploit fixation, retard nitrification, and reduce denitrification are presented, together with strategies that minimize inorganic fertilizer applications and curtail losses. The development and implementation of new technologies together with rediscovering traditional practices are discussed to speculate how the grand challenge of feeding the world sustainably can be met.

The potato rhizosphere microbiota correlated to the yield of three different regions in Korea.

We examined potato rhizosphere bacterial and fungal communities across three regions: Cheongju, Pyeongchang, and Gangneung. These regions have varying soil and climate conditions, resulting in different yields. We found that precipitation was the main limiting factor in our study while soil physiochemical factors affect bacterial and fungal microbiota in correlation with yield. Both bacterial and fungal microbiota showed distinct patterns according to the regions. ASVs positively correlated with yield were predominantly found in the Pyeongchang region which also produced the highest yields, while ASVs negatively correlated with yield were associated with Gangneung where the lowest yields were observed. The greatest bacterial and fungal diversity was detected in Pyeongchang consisting of Propionibacteriales, Burkholderiales, and Vicinamibacteriales. Gangneung, on the other hand primarily belong to Sordariales, Mortierellales, Cystofilobasidiales, and Tremellales. The putative yield-negative ASVs detected in Gangneung may have been influenced by drought stress. This work has highlighted key bacterial and fungal taxa as well as core taxa that may potentially be associated with high and low yields of potato in relation to metadata which includes soil chemical and physical parameters as well as weather data. Taken together we suggest that this information can be used to assess site suitability for potato production.

Repeated exposure of wheat to the fungal root pathogen Bipolaris sorokiniana modulates rhizosphere microbiome assembly and disease suppressiveness.

BACKGROUND: Disease suppressiveness of soils to fungal root pathogens is typically induced in the field by repeated infections of the host plant and concomitant changes in the taxonomic composition and functional traits of the rhizosphere microbiome. Here, we studied this remarkable phenomenon for Bipolaris sorokiniana in two wheat cultivars differing in resistance to this fungal root pathogen. RESULTS: The results showed that repeated exposure of the susceptible wheat cultivar to the pathogen led to a significant reduction in disease severity after five successive growth cycles. Surprisingly, the resistant wheat cultivar, initially included as a control, showed the opposite pattern with an increase in disease severity after repeated pathogen exposure. Amplicon analyses revealed that the bacterial families Chitinophagaceae, Anaerolineaceae and Nitrosomonadaceae were associated with disease suppressiveness in the susceptible wheat cultivar; disease suppressiveness in the resistant wheat cultivar was also associated with Chitinophagaceae and a higher abundance of Comamonadaceae. Metagenome analysis led to the selection of 604 Biosynthetic Gene Clusters (BGCs), out of a total of 2,571 identified by AntiSMASH analysis, that were overrepresented when the soil entered the disease suppressive state. These BGCs are involved in the biosynthesis of terpenes, non-ribosomal peptides, polyketides, aryl polyenes and post-translationally modified peptides. CONCLUSION: Combining taxonomic and functional profiling we identified key changes in the rhizosphere microbiome during disease suppression. This illustrates how the host plant relies on the rhizosphere microbiome as the first line of defense to fight soil-borne pathogens. Microbial taxa and functions identified here can be used in novel strategies to control soil-borne fungal pathogens.

The UK Crop Microbiome Cryobank: a utility and model for supporting Phytobiomes research.

Plant microbiomes are the microbial communities essential to the functioning of the phytobiome-the system that consist of plants, their environment, and their associated communities of organisms. A healthy, functional phytobiome is critical to crop health, improved yields and quality food. However, crop microbiomes are relatively under-researched, and this is associated with a fundamental need to underpin phytobiome research through the provision of a supporting infrastructure. The UK Crop Microbiome Cryobank (UKCMC) project is developing a unique, integrated and open-access resource to enable the development of solutions to improve soil and crop health. Six economically important crops (Barley, Fava Bean, Oats, Oil Seed Rape, Sugar Beet and Wheat) are targeted, and the methods as well as data outputs will underpin research activity both in the UK and internationally. This manuscript describes the approaches being taken, from characterisation, cryopreservation and analysis of the crop microbiome through to potential applications. We believe that the model research framework proposed is transferable to different crop and soil systems, acting not only as a mechanism to conserve biodiversity, but as a potential facilitator of sustainable agriculture systems.

Identification of novel aphid-killing bacteria to protect plants.

Aphids, including the peach-potato aphid, Myzus persicae, are major insect pests of agriculture and horticulture, and aphid control measures are limited. There is therefore an urgent need to develop alternative and more sustainable means of control. Recent studies have shown that environmental microbes have varying abilities to kill insects. We screened a range of environmental bacteria isolates for their abilities to kill target aphid species. Tests demonstrated the killing aptitude of these bacteria against six aphid genera (including Myzus persicae). No single bacterial strain was identified that was consistently toxic to insecticide-resistant aphid clones than susceptible clones, suggesting resistance to chemicals is not strongly correlated with bacterial challenge. Pseudomonas fluorescens PpR24 proved the most toxic to almost all aphid clones whilst exhibiting the ability to survive for over three weeks on three plant species at populations of 5-6 log CFU cm-2 leaf. Application of PpR24 to plants immediately prior to introducing aphids onto the plants led to a 68%, 57% and 69% reduction in aphid populations, after 21 days, on Capsicum annuum, Arabidopsis thaliana and Beta vulgaris respectively. Together, these findings provide new insights into aphid susceptibility to bacterial infection with the aim of utilizing bacteria as effective biocontrol agents.

Identification of new single nucleotide polymorphism-based markers for inter- and intraspecies discrimination of obligate bacterial parasites (Pasteuria spp.) of invertebrates.

Protein-encoding and 16S rRNA genes of Pasteuria penetrans populations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of "cryptic" SNPs which were not present in the consensus sequences of any P. penetrans population. Additionally, hierarchical cluster analysis separated P. penetrans 16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among three Pasteuria species, namely, P. penetrans, P. hartismeri, and P. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination of Pasteuria at both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms.

Defining the wheat microbiome: Towards microbiome-facilitated crop production.

Wheat is one of the world's most important crops, but its production relies heavily on agrochemical inputs which can be harmful to the environment when used excessively. It is well known that a multitude of microbes interact with eukaryotic organisms, including plants, and the sum of microbes and their functions associated with a given host is termed the microbiome. Plant-microbe interactions can be beneficial, neutral or harmful to the host plant. Over the last decade, with the development of next generation DNA sequencing technology, our understanding of the plant microbiome structure has dramatically increased. Considering that defining the wheat microbiome is key to leverage crop production in a sustainable way, here we describe how different factors drive microbiome assembly in wheat, including crop management, edaphic-environmental conditions and host selection. In addition, we highlight the benefits to take a multidisciplinary approach to define and explore the wheat core microbiome to generate solutions based on microbial (synthetic) communities or single inoculants. Advances in plant microbiome research will facilitate the development of microbial strategies to guarantee a sustainable intensification of crop production.

Culture-based Methods for Studying the Bacterial Root Microbiome of Wheat.

Beneficial plant-microbe interactions are important and desirable for sustainable intensification of agriculture. Here, we describe methods to isolate microbes from the roots of field-grown wheat plants. This includes the rhizosphere and rhizoplane soil, as well as the root endosphere. We also describe a method to test for endosphere competence of putative endophytes.

Land Management Legacy Affects Abundance and Function of the acdS Gene in Wheat Root Associated Pseudomonads.

Land management practices can vastly influence belowground plant traits due to chemical, physical, and biological alteration of soil properties. Beneficial Pseudomonas spp. are agriculturally relevant bacteria with a plethora of plant growth promoting (PGP) qualities, including the potential to alter plant physiology by modulating plant produced ethylene via the action of the bacterial enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS). This study evaluated the impact of land management legacy on the selection and function of wheat root associated culturable pseudomonad isolates. Three distinct previous land uses prior to wheat culture (grassland, arable, and bare fallow) were tested and culturable pseudomonad abundance, phylogeny (gyrB and acdS genes), function (ACC deaminase activity), and the co-selection of acdS with other PGP genes examined. The pseudomonad community could to some extent be discriminated based on previous land use. The isolates from rhizosphere and root compartments of wheat had a higher acdS gene frequency than the bulk soil, particularly in plants grown in soil from the bare fallow treatment which is known to have degraded soil properties such as low nutrient availability. Additionally, other genes of interest to agriculture encoding anti-fungal metabolites, siderophores, and genes involved in nitrogen metabolism were highly positively associated with the presence of the acdS gene in the long-term arable treatment in the genomes of these isolates. In contrast, genes involved in antibiotic resistance and type VI secretion systems along with nitrogen cycling genes were highly positively correlated with the acdS gene in bare fallow isolated pseudomonad. This highlights that the three land managements prior to wheat culture present different selection pressures that can shape culturable pseudomonad community structure and function either directly or indirectly via the influence of wheat roots.

Inorganic Chemical Fertilizer Application to Wheat Reduces the Abundance of Putative Plant Growth-Promoting Rhizobacteria.

The profound negative effect of inorganic chemical fertilizer application on rhizobacterial diversity has been well documented using 16S rRNA gene amplicon sequencing and predictive metagenomics. We aimed to measure the function and relative abundance of readily culturable putative plant growth-promoting rhizobacterial (PGPR) isolates from wheat root soil samples under contrasting inorganic fertilization regimes. We hypothesized that putative PGPR abundance will be reduced in fertilized relative to unfertilized samples. Triticum aestivum cv. Cadenza seeds were sown in a nutrient depleted agricultural soil in pots treated with and without Osmocote® fertilizer containing nitrogen-phosphorous-potassium (NPK). Rhizosphere and rhizoplane samples were collected at flowering stage (10 weeks) and analyzed by culture-independent (CI) amplicon sequence variant (ASV) analysis of rhizobacterial DNA as well as culture-dependent (CD) techniques. Rhizosphere and rhizoplane derived microbiota culture collections were tested for plant growth-promoting traits using functional bioassays. In general, fertilizer addition decreased the proportion of nutrient-solubilizing bacteria (nitrate, phosphate, potassium, iron, and zinc) isolated from rhizocompartments in wheat whereas salt tolerant bacteria were not affected. A "PGPR" database was created from isolate 16S rRNA gene sequences against which total amplified 16S rRNA soil DNA was searched, identifying 1.52% of total community ASVs as culturable PGPR isolates. Bioassays identified a higher proportion of PGPR in non-fertilized samples [rhizosphere (49%) and rhizoplane (91%)] compared to fertilized samples [rhizosphere (21%) and rhizoplane (19%)] which constituted approximately 1.95 and 1.25% in non-fertilized and fertilized total community DNA, respectively. The analyses of 16S rRNA genes and deduced functional profiles provide an in-depth understanding of the responses of bacterial communities to fertilizer; our study suggests that rhizobacteria that potentially benefit plants by mobilizing insoluble nutrients in soil are reduced by chemical fertilizer addition. This knowledge will benefit the development of more targeted biofertilization strategies.

Identification of microbial signatures linked to oilseed rape yield decline at the landscape scale.

BACKGROUND: The plant microbiome plays a vital role in determining host health and productivity. However, we lack real-world comparative understanding of the factors which shape assembly of its diverse biota, and crucially relationships between microbiota composition and plant health. Here we investigated landscape scale rhizosphere microbial assembly processes in oilseed rape (OSR), the UK's third most cultivated crop by area and the world's third largest source of vegetable oil, which suffers from yield decline associated with the frequency it is grown in rotations. By including 37 conventional farmers' fields with varying OSR rotation frequencies, we present an innovative approach to identify microbial signatures characteristic of microbiomes which are beneficial and harmful to the host. RESULTS: We show that OSR yield decline is linked to rotation frequency in real-world agricultural systems. We demonstrate fundamental differences in the environmental and agronomic drivers of protist, bacterial and fungal communities between root, rhizosphere soil and bulk soil compartments. We further discovered that the assembly of fungi, but neither bacteria nor protists, was influenced by OSR rotation frequency. However, there were individual abundant bacterial OTUs that correlated with either yield or rotation frequency. A variety of fungal and protist pathogens were detected in roots and rhizosphere soil of OSR, and several increased relative abundance in root or rhizosphere compartments as OSR rotation frequency increased. Importantly, the relative abundance of the fungal pathogen Olpidium brassicae both increased with short rotations and was significantly associated with low yield. In contrast, the root endophyte Tetracladium spp. showed the reverse associations with both rotation frequency and yield to O. brassicae, suggesting that they are signatures of a microbiome which benefits the host. We also identified a variety of novel protist and fungal clades which are highly connected within the microbiome and could play a role in determining microbiome composition. CONCLUSIONS: We show that at the landscape scale, OSR crop yield is governed by interplay between complex communities of both pathogens and beneficial biota which is modulated by rotation frequency. Our comprehensive study has identified signatures of dysbiosis within the OSR microbiome, grown in real-world agricultural systems, which could be used in strategies to promote crop yield. Video abstract.

Wheat dwarfing influences selection of the rhizosphere microbiome.

The development of dwarf wheat cultivars combined with high levels of agrochemical inputs during the green revolution resulted in high yielding cropping systems. However, changes in wheat cultivars were made without considering impacts on plant and soil microbe interactions. We studied the effect of these changes on root traits and on the assembly of rhizosphere bacterial communities by comparing eight wheat cultivars ranging from tall to semi-dwarf plants grown under field conditions. Wheat breeding influenced root diameter and specific root length (SRL). Rhizosphere bacterial communities from tall cultivars were distinct from those associated with semi-dwarf cultivars, with higher differential abundance of Actinobacteria, Bacteroidetes and Proteobacteria in tall cultivars, compared with a higher differential abundance of Verrucomicrobia, Planctomycetes and Acidobacteria in semi-dwarf cultivars. Predicted microbial functions were also impacted and network analysis revealed a greater level of connectedness between microbial communities in the tall cultivars relative to semi-dwarf cultivars. Taken together, results suggest that the development of semi-dwarf plants might have affected the ability of plants to recruit and sustain a complex bacterial community network in the rhizosphere.

Multitrophic interactions in the rhizosphere microbiome of wheat: from bacteria and fungi to protists.

Plants modulate the soil microbiota by root exudation assembling a complex rhizosphere microbiome with organisms spanning different trophic levels. Here, we assessed the diversity of bacterial, fungal and cercozoan communities in landraces and modern varieties of wheat. The dominant taxa within each group were the bacterial phyla Proteobacteria, Actinobacteria and Acidobacteria; the fungi phyla Ascomycota, Chytridiomycota and Basidiomycota; and the Cercozoa classes Sarcomonadea, Thecofilosea and Imbricatea. We showed that microbial networks of the wheat landraces formed a more intricate network topology than that of modern wheat cultivars, suggesting that breeding selection resulted in a reduced ability to recruit specific microbes in the rhizosphere. The high connectedness of certain cercozoan taxa to bacteria and fungi indicated trophic network hierarchies where certain predators gain predominance over others. Positive correlations between protists and bacteria in landraces were preserved as a subset in cultivars as was the case for the Sarcomonadea class with Actinobacteria. The correlations between the microbiome structure and plant genotype observed in our results suggest the importance of top-down control by organisms of higher trophic levels as a key factor for understanding the drivers of microbiome community assembly in the rhizosphere.

Evidence for diversifying selection of genetic regions of encoding putative collagen-like host-adhesive fibers in Pasteuria penetrans.

Pasteuria spp. belong to a group of genetically diverse endospore-forming bacteria (phylum: Firmicutes) that are known to parasitize plant-parasitic nematodes and water fleas (Daphnia spp.). Collagen-like fibres form the nap on the surface of endospores and the genes encoding these sequences have been hypothesised to be involved in the adhesion of the endospores of Pasteuria spp. to their hosts. We report a group of 17 unique collagen-like genes putatively encoded by Pasteuria penetrans (strain: Res148) that formed five different phylogenetic clusters and suggest that collagen-like proteins are an important source of genetic diversity in animal pathogenic Firmicutes including Pasteuria. Additionally, and unexpectedly, we identified a putative collagen-like sequence which had a very different sequence structure to the other collagen-like proteins but was similar to the protein sequences in Megaviruses that are involved in host-parasite interactions. We, therefore, suggest that these diverse endospore surface proteins in Pasteuria are involved in biological functions, such as cellular adhesion; however, they are not of monophyletic origin and were possibly obtained de novo by mutation or possibly through selection acting upon several historic horizontal gene transfer events.

Land Management and Microbial Seed Load Effect on Rhizosphere and Endosphere Bacterial Community Assembly in Wheat.

Microbial community ecology studies have traditionally utilized culture-based methodologies, though the advent of next-generation amplicon sequencing has facilitated superior resolution analyses of complex microbial communities. Here, we used culture-dependent and -independent approaches to explore the influence of land use as well as microbial seed load on bacterial community structure of the wheat rhizosphere and root endosphere. It was found that niche was an important factor in shaping the microbiome when using both methodological approaches, and that land use was also a discriminatory factor for the culture-independent-based method. Although culture-independent methods provide a higher resolution of analysis, it was found that in the rhizosphere, particular operational taxonomic units (OTUs) in the culture-dependent fraction were absent from the culture-independent fraction, indicating that deeper sequence analysis is required for this approach to be exhaustive. We also found that the microbial seed load defined the endosphere, but not rhizosphere, community structure for plants grown in soil which was not wheat adapted. Together, these findings increase our understanding of the importance of land management and microbial seed load in shaping the root microbiome of wheat and this knowledge will facilitate the exploitation of plant-microbe interactions for the development of novel microbial inoculants.

In vivo expression technology (IVET) selection of genes of Rhizobium leguminosarum biovar viciae A34 expressed in the rhizosphere.

IVET was used to identify genes that are specifically expressed in the rhizosphere of the pea-nodulating bacterium Rhizobium leguminosarum A34. A library of R. leguminosarum A34 cloned in the integration vector pIE1, with inserts upstream of a promoter-less purN:gfp:gusA, was conjugated into purN host RU2249 and recombined into the genome. After removal of colonies that expressed the reporter genes of the vector under laboratory conditions, the library was inoculated into a nonsterile pea rhizosphere. The key result is that 29 rhizosphere-induced loci were identified. Sequence analysis of these clones showed that a wide variety of R. leguminosarum A34 genes are expressed specifically in the rhizosphere including those encoding proteins involved in environmental sensing, control of gene expression, metabolic reactions and membrane transport. These genes are likely to be important for survival and colonization of the pea rhizosphere.

Co-occurrence patterns of litter decomposing communities in mangroves indicate a robust community resistant to disturbances.

BACKGROUND: Mangroves are important coastal ecosystems known for high photosynthetic productivity and the ability to support marine food chains through supply of dissolved carbon or particular organic matter. Most of the carbon found in mangroves is produced by its vegetation and is decomposed in root associated sediment. This process involves a tight interaction between microbial populations, litter chemical composition, and environmental parameters. Here, we study the complex interactions found during litter decomposition in mangroves by applying network analysis to metagenomic data. METHODS: Leaves of three species of mangrove trees typically found in the southeast of Brazil (Rhizophora mangle, Laguncularia racemosa, and Avicennia schaueriana) were collected in separate litter bags and left on three different mangroves for 60 days. These leaves were subsequently used for metagenome sequencing using Ion Torrent technology. Sequences were annotated in MG-RAST and used for network construction using MENAp. RESULTS: The most common phyla were Proteobacteria (classes Gamma and Alphaproteobacteria) followed by Firmicutes (Clostridia and Bacilli). The most abundant protein clusters were associated with the metabolism of carbohydrates, amino acids, and proteins. Non-metric multidimensional scaling of the metagenomic data indicated that substrate (i.e., tree species) did not significantly select for a specific community. Both networks exhibited scale-free characteristics and small world structure due to the low mean shortest path length and high average clustering coefficient. These networks also had a low number of hub nodes most of which were module hubs. DISCUSSION: This study demonstrates that under different environmental pressures (i.e., plant species or mangrove location) the microbial community associated with the decaying material forms a robust and stable network.