Accessing the specialized metabolome of actinobacteria from the bulk soil of Paullinia cupana Mart. on the Brazilian Amazon: a promising source of bioactive compounds against soybean phytopathogens.

The Amazon rainforest, an incredibly biodiverse ecosystem, has been increasingly vulnerable to deforestation. Despite its undeniable importance and potential, the Amazonian microbiome has historically received limited study, particularly in relation to its unique arsenal of specialized metabolites. Therefore, in this study our aim was to assess the metabolic diversity and the antifungal activity of actinobacterial strains isolated from the bulk soil of Paullinia cupana, a native crop, in the Brazilian Amazon Rainforest. Extracts from 24 strains were subjected to UPLC-MS/MS analysis using an integrative approach that relied on the Chemical Structural and Compositional Similarity (CSCS) metric, GNPS molecular networking, and in silico dereplication tools. This procedure allowed the comprehensive understanding of the chemical space encompassed by these actinobacteria, which consists of features belonging to known bioactive metabolite classes and several unannotated molecular families. Among the evaluated strains, five isolates exhibited bioactivity against a panel of soybean fungal phytopathogens (Rhizoctonia solani, Macrophomina phaseolina, and Sclerotinia sclerotiorum). A focused inspection led to the annotation of pepstatins, oligomycins, hydroxamate siderophores and dorrigocins as metabolites produced by these bioactive strains, with potentially unknown compounds also comprising their metabolomes. This study introduces a pragmatic protocol grounded in established and readily available tools for the annotation of metabolites and the prioritization of strains to optimize further isolation of specialized metabolites. Conclusively, we demonstrate the relevance of the Amazonian actinobacteria as sources for bioactive metabolites useful for agriculture. We also emphasize the importance of preserving this biome and conducting more in-depth studies on its microbiota.

In Planta Study Localizes an Effector Candidate from Austropuccinia psidii Strain MF-1 to the Nucleus and Demonstrates In Vitro Cuticular Wax-Dependent Differential Expression.

Austropuccinia psidii is a biotrophic fungus that causes myrtle rust. First described in Brazil, it has since spread to become a globally important pathogen that infects more than 480 myrtaceous species. One of the most important commercial crops affected by A. psidii is eucalypt, a widely grown forestry tree. The A. psidii-Eucalyptus spp. interaction is poorly understood, but pathogenesis is likely driven by pathogen-secreted effector molecules. Here, we identified and characterized a total of 255 virulence effector candidates using a genome assembly of A. psidii strain MF-1, which was recovered from Eucalyptus grandis in Brazil. We show that the expression of seven effector candidate genes is modulated by cell wax from leaves sourced from resistant and susceptible hosts. Two effector candidates with different subcellular localization predictions, and with specific gene expression profiles, were transiently expressed with GFP-fusions in Nicotiana benthamiana leaves. Interestingly, we observed the accumulation of an effector candidate, Ap28303, which was upregulated under cell wax from rust susceptible E. grandis and described as a peptidase inhibitor I9 domain-containing protein in the nucleus. This was in accordance with in silico analyses. Few studies have characterized nuclear effectors. Our findings open new perspectives on the study of A. psidii-Eucalyptus interactions by providing a potential entry point to understand how the pathogen manipulates its hosts in modulating physiology, structure, or function with effector proteins.

Meta-omics integration approach reveals the effect of soil native microbiome diversity in the performance of inoculant Azospirillum brasilense.

Plant growth promoting bacteria (PGPB) have been used as integrative inputs to minimize the use of chemical fertilizers. However, a holistic comprehension about PGPB-plant-microbiome interactions is still incipient. Furthermore, the interaction among PGPB and the holobiont (host-microbiome association) represent a new frontier to plant breeding programs. We aimed to characterize maize bulk soil and rhizosphere microbiomes in irradiated soil (IS) and a native soil (NS) microbial community gradient (dilution-to-extinction) with Azospirillum brasilense Ab-V5, a PGPB commercial inoculant. Our hypothesis was that plant growth promotion efficiency is a result of PGPB niche occupation and persistence according to the holobiont conditions. The effects of Ab-V5 and NS microbial communities were evaluated in microcosms by a combined approach of microbiomics (species-specific qPCR, 16S rRNA metataxonomics and metagenomics) and plant phenomics (conventional and high-throughput methods). Our results revealed a weak maize growth promoting effect of Ab-V5 inoculation in undiluted NS, contrasting the positive effects of NS dilutions 10-3, 10-6, 10-9 and IS with Ab-V5. Alpha diversity in NS + Ab-V5 soil samples was higher than in all other treatments in a time course of 25 days after sowing (DAS). At 15 DAS, alpha diversity indexes were different between NS and IS, but similar in all NS dilutions in rhizospheric samples. These differences were not persistent at 25 DAS, demonstrating a stabilization process in the rhizobiomes. In NS 10-3 +Ab-V5 and NS 10-6 Ab-V5, Ab-V5 persisted in the maize rhizosphere until 15 DAS in higher abundances compared to NS. In NS + Ab-V5, abundance of six taxa were positively correlated with response to (a)biotic stresses in plant-soil interface. Genes involved in bacterial metabolism of riboses and amino acids, and cresol degradation were abundant on NS 10-3 + Ab-V5, indicating that these pathways can contribute to plant growth promotion and might be a result of Ab-V5 performance as a microbial recruiter of beneficial functions to the plant. Our results demonstrated the effects of holobiont on Ab-V5 performance. The meta-omics integration supported by plant phenomics opens new perspectives to better understanding of inoculants-holobiont interaction and for developing better strategies for optimization in the use of microbial products.

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.

Draft Genome Sequence of the Plant Growth Promoter and Biocontrol Agent Bacillus velezensis Strain BIB0110.

Here, we report the draft genome sequence of Bacillus velezensis strain BIB0110, a broad-range biocontrol agent isolated from cultivated eucalyptus in Brazil. The genome has a size of 4.19 Mbp, with a GC content of 45.87%, and it was assembled into 32 scaffolds.

The key role of indole-3-acetic acid biosynthesis by Bacillus thuringiensis RZ2MS9 in promoting maize growth revealed by the ipdC gene knockout mediated by the CRISPR-Cas9 system.

The bacterial biosynthesis of indole-3-acetic acid (IAA) is often related to the beneficial effects of plant growth-promoting rhizobacteria (PGPR) on plant development. In PGPR belonging to the Bacillus genus, the synthesis of IAA may occur through different metabolic pathways that are still poorly understood. B. thuringiensis (Bt) is well known for its insecticidal properties; however, its beneficial features are not limited to pest control. Our group has been studed the beneficial effects of Bt strain RZ2MS9 as growth promoter in a range of plant crops, including soybean, tomato, and maize. We recently demonstrated that bacterial IAA biosynthesis plays an important role in the ability of RZ2MS9 to benefit plant development. However, the molecular involved mechanisms in the IAA biosynthesis by this bacterium in the beneficial interaction with plants remain unclear. Here, we investigated the genetic basis of IAA biosynthesis by RZ2MS9. We knocked out the ipdC gene, involved in IAA biosynthesis via the tryptophan-dependent IPyA pathway, using the CRISPR-Cas9 system. Our results showed that, by disrupting the IPyA pathway, the amount of IAA synthesized by the mutant RZ2MS9 (ΔipdC) in the presence of tryptophan drops 57%. The gene knockout did not affect the bacterial growth, but it did affect its ability to colonize maize. Moreover, deactivating the ipdC gene in RZ2MS9 significantly reduces its ability to promote maize growth. ΔipdC performed worse than RZ2MS9 in almost all evaluated plant parameters, including total root length, projected root area, lateral roots, aerial part dry matter, and germination speed index. Therefore, we demonstrated that tryptophan-dependent IAA biosynthesis via the IPyA pathway by RZ2MS9 is strongly influenced by the ipdC gene. Furthermore, IAA biosynthesis by RZ2MS9 is a major mechanism used by this PGPR to promote maize growth.

Opposite Sides of Pantoea agglomerans and Its Associated Commercial Outlook.

Multifaceted microorganisms such as the bacterium Pantoea colonize a wide range of habitats and can exhibit both beneficial and harmful behaviors, which provide new insights into microbial ecology. In the agricultural context, several strains of Pantoea spp. can promote plant growth through direct or indirect mechanisms. Members of this genus contribute to plant growth mainly by increasing the supply of nitrogen, solubilizing ammonia and inorganic phosphate, and producing phytohormones (e.g., auxins). Several other studies have shown the potential of strains of Pantoea spp. to induce systemic resistance and protection against pests and pathogenic microorganisms in cultivated plants. Strains of the species Pantoea agglomerans deserve attention as a pest and phytopathogen control agent. Several of them also possess a biotechnological potential for therapeutic purposes (e.g., immunomodulators) and are implicated in human infections. Thus, the differentiation between the harmful and beneficial strains of P. agglomerans is mandatory to apply this bacterium safely as a biofertilizer or biocontroller. This review specifically evaluates the potential of the strain-associated features of P. agglomerans for bioprospecting and agricultural applications through its biological versatility as well as clarifying its potential animal and human health risks from a genomic point of view.

Draft genome sequence of the cyanobacterium Sphaerospermopsis aphanizomenoides BCCUSP55 from the Brazilian semiarid region reveals potential for anti-cancer applications.

Sphaerospermopsis aphanizomenoides is a filamentous nitrogen-fixing and bloom-forming cyanobacterium, which biomass can fertilize natural water with nutrients, especially through nitrogen fixation. The Sphaerospermopsis aphanizomenoides strain BCCUSP55 was previously isolated from a water supply reservoir in the Brazilian semiarid region, and its draft genome assembly coupled with the gene contents are reported here. The obtained BCCUSP55 draft genome comprised 254 scaffolds with a genome size estimated of 6,096,273 bp. In addition, it has 5250 predicted coding sequences (CDS) and the G + C content is 38.5%. Further, the BCCUSP55 draft genome presented the putative nocuolin A gene complete cluster, a natural oxadiazine that triggers apoptosis in human cancer cells. Thus, our results contribute to extend the knowledge on the genus Sphaerospermopsis and reveal its biotechnological potential.

Bacillus thuringiensis RZ2MS9, a tropical plant growth-promoting rhizobacterium, colonizes maize endophytically and alters the plant's production of volatile organic compounds during co-inoculation with Azospirillum brasilense Ab-V5.

The beneficial features of Bacillus thuringiensis (Bt) are not limited to its role as an insecticide; it is also able to promote plant growth interacting with plants and other plant growth-promoting rhizobacterium (PGPR). The PGPR Bt strain RZ2MS9 is a multi-trait maize growth promoter. We obtained a stable mutant of RZ2MS9 labelled with green fluorescent protein (RZ2MS9-GFP). We demonstrated that the Bt RZ2MS9-GFP successfully colonizes maize's roots and leaves endophytically. We evaluated whether RZ2MS9 has an additive effect on plant growth promotion when co-inoculated with Azospirillum brasilense Ab-V5. The two strains combined enhanced maize's roots and shoots dry weight around 50% and 80%, respectively, when compared to the non-inoculated control. However, non-differences were observed comparing RZ2MS9 alone and when co-inoculated with Ab-V5, In addition, we used co-inoculation experiments in glass chambers to analyse the plant's volatile organic compounds (VOCs) production during the maize-RZ2MS9 and maize-RZ2MS9-Ab-V5 interaction. We found that the single and co-inoculation altered maize's VOCs emission profile, with an increase in the production of indoles in the co-inoculation. Collectively, these results increase our knowledge about the interaction between the Bt and maize, and provide a new possibility of combined application with the commercial inoculant A. brasilense Ab-V5.

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.

Revealing the high variability on nonconserved core and mobile elements of Austropuccinia psidii and other rust mitochondrial genomes.

Mitochondrial genomes are highly conserved in many fungal groups, and they can help characterize the phylogenetic relationships and evolutionary biology of plant pathogenic fungi. Rust fungi are among the most devastating diseases for economically important crops around the world. Here, we report the complete sequence and annotation of the mitochondrial genome of Austropuccinia psidii (syn. Puccinia psidii), the causal agent of myrtle rust. We performed a phylogenomic analysis including the complete mitochondrial sequences from other rust fungi. The genome composed of 93.299 bp has 73 predicted genes, 33 of which encoded nonconserved proteins (ncORFs), representing almost 45% of all predicted genes. A. psidii mtDNA is one of the largest rust mtDNA sequenced to date, most likely due to the abundance of ncORFs. Among them, 33% were within intronic regions of diverse intron groups. Mobile genetic elements invading intron sequences may have played significant roles in size but not shaping of the rust mitochondrial genome structure. The mtDNAs from rust fungi are highly syntenic. Phylogenetic inferences with 14 concatenated mitochondrial proteins encoded by the core genes placed A. psidii according to phylogenetic analysis based on 18S rDNA. Interestingly, cox1, the gene with the greatest number of introns, provided phylogenies not congruent with the core set. For the first time, we identified the proteins encoded by three A. psidii ncORFs using proteomics analyses. Also, the orf208 encoded a transmembrane protein repressed during in vitro morphogenesis. To the best of our knowledge, we presented the first report of a complete mtDNA sequence of a member of the family Sphaerophragmiacea.

On the genetic architecture in a public tropical maize panel of the symbiosis between corn and plant growth-promoting bacteria aiming to improve plant resilience.

Exploring the symbiosis between plants and plant growth-promoting bacteria (PGPB) is a new challenge for sustainable agriculture. Even though many works have reported the beneficial effects of PGPB in increasing plant resilience for several stresses, its potential is not yet widely explored. One of the many reasons is the differential symbiosis performance depending on the host genotype. This opens doors to plant breeding programs to explore the genetic variability and develop new cultivars with higher responses to PGPB interaction and, therefore, have higher resilience to stress. Hence, we aimed to study the genetic architecture of the symbiosis between PGPB and tropical maize germplasm, using a public association panel and its impact on plant resilience. Our findings reveal that the synthetic PGPB population can modulate and impact root architecture traits and improve resilience to nitrogen stress, and 37 regions were significant for controlling the symbiosis between PGPB and tropical maize. In addition, we found two overlapping SNPs in the GWAS analysis indicating strong candidates for further investigations. Furthermore, genomic prediction analysis with genomic relationship matrix computed using only significant SNPs obtained from GWAS analysis substantially increased the predictive ability for several traits endorsing the importance of these genomic regions for the response of PGPB. Finally, the public tropical panel reveals a significant genetic variability to the symbiosis with the PGPB and can be a source of alleles to improve plant resilience. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01257-6.

Fast Screening of Bacteria for Plant Growth Promoting Traits.

Plant Growth Promoting Bacteria (PGPB) are a group of beneficial microorganisms that can positively influence plant fitness and development by improving nutrient acquisition, influencing global plant hormone levels (direct effect), or by reducing the detrimental effects of various pathogens on plant development (indirect effect). The use of PGPB in agriculture as formulated bioinoculants is a potential approach to reduce the negative environmental impacts caused by the continuous application of chemical fertilizers and pesticides. The evaluation of a great number of bacteria in the laboratory for key traits involved in the improvement of plant fitness is a suitable strategy to find prospective candidates for bioinoculants. This chapter presents the main methods described in the literature to quickly screen potential candidates from a bacterial collection to directly and indirectly promote the plant growth.

Gene Knockout of Beneficial Plant-associated Bacillus spp. Using the CRISPR-Cas9 Double Plasmid System.

Bacillus spp. have great agricultural potential as a plant growth promoter and biocontrol agent. However, little is known concerning the bacterial molecular basis for the improvement of plant fitness. Thus, it is highly desirable to develop techniques that can contribute to the elucidation of the genetic basis for the mechanisms involved in beneficial bacterium-plant interactions. In this context, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 is a powerful tool based on programmable molecular scissors that perform precise incisions in any DNA sequence. CRISPR-Cas9 can alter gene sequences and constitutes a cutting-edge tool to elucidate the role and function of bacterial genes associated with the benefits of plant interactions. The method described here uses a feasible CRISPR-Cas9 system in a double plasmid, one plasmid harboring the Cas9 endonuclease and the other the sgRNA, to promote gene knockout/editing in the Bacillus genus. This approach favors high efficiency in generating mutants for one or more genes in continuous or multiplex editing. Additionally, due to its universality, it can be applied to genera other than Bacillus.

Rehydration of dry corn preserves the desirable bacterial community during ensiling.

This study evaluated the rehydration approach of mature corn grains as an alternative for high-moisture corn grain silage production in distinct corn hybrids, storage period, cultivation locations and kernel maturity at plant harvest. High-moisture corn was used as a control. The dry matter content and pH of the silage were measured, and the bacterial community associated with corn grains pre- and post-ensiling was also assessed through 16S rRNA high-throughput sequencing. The decrease in pH value was directly linked to an ecological microbial succession of Enterobacteriales and Actinomycetales to Lactobacillales in the silage at 120 days after storage, either in rehydrated or high-moisture corn. These results were similar for both maize production locations and hybrids tested. Finally, the similarity between the ensiling processes including rehydrated corn and the high-moisture corn grain silages proves the reliability of the rehydration approach as an alternative for the maintenance of a successful bacterial community structure and composition capable of producing high-quality silages from dent and flint corn hybrids in tropical conditions.

Complete Genome Sequence of Bacillus sp. Strain RZ2MS9, a Multitrait Plant Growth Promoter.

Here, we report the complete genome sequence of Bacillus sp. strain RZ2MS9, a plant growth-promoting bacterium isolated from the rhizosphere of guarana, a native crop from Amazonas, Brazil. The assembled genome comprises 5.35 Mbp, no plasmids, and a GC content of 35.22%.

Beneficial Plant-Associated Microorganisms From Semiarid Regions and Seasonally Dry Environments: A Review.

Semiarid regions are apparently low biodiversity environments; however, these environments may host a phylogenetically diverse microbial community associated with plants. Their microbial inhabitants are often recruited to withstand stressful settings and improve plant growth under harsh conditions. Thus, plant-associated microorganisms isolated from semiarid and seasonally dry environments will be detailed in the present review, focusing on plant growth promotion potential and the microbial ability to alleviate plant abiotic stress. Initially, we explored the role of microbes from dry environments around the world, and then, we focused on seasonally dry Brazilian biomes, the Caatinga and the Cerrado. Cultivable bacteria from semiarid and seasonally dry environments have demonstrated great plant growth promotion traits such as plant hormone production, mobilization of insoluble nutrients, and mechanisms related to plant abiotic stress alleviation. Several of these isolates were able to improve plant growth under stressful conditions commonly present in typical semiarid regions, such as high salinity and drought. Additionally, we highlight the potential of plants highly adapted to seasonal climates from the Caatinga and Cerrado biomes as a suitable pool of microbial inoculants to maintain plant growth under abiotic stress conditions. In general, we point out the potential for the exploitation of new microbial inoculants from plants growing in dry environments to ensure a sustainable increase in agricultural productivity in a future climate change scenario.

Additive and heterozygous (dis)advantage GWAS models reveal candidate genes involved in the genotypic variation of maize hybrids to Azospirillum brasilense.

Maize genotypes can show different responsiveness to inoculation with Azospirillum brasilense and an intriguing issue is which genes of the plant are involved in the recognition and growth promotion by these Plant Growth-Promoting Bacteria (PGPB). We conducted Genome-Wide Association Studies (GWAS) using additive and heterozygous (dis)advantage models to find candidate genes for root and shoot traits under nitrogen (N) stress and N stress plus A. brasilense. A total of 52,215 Single Nucleotide Polymorphism (SNP) markers were used for GWAS analyses. For the six root traits with significant inoculation effect, the GWAS analyses revealed 25 significant SNPs for the N stress plus A. brasilense treatment, in which only two were overlapped with the 22 found for N stress only. Most were found by the heterozygous (dis)advantage model and were more related to exclusive gene ontology terms. Interestingly, the candidate genes around the significant SNPs found for the maize-A. brasilense association were involved in different functions previously described for PGPB in plants (e.g. signaling pathways of the plant's defense system and phytohormone biosynthesis). Our findings are a benchmark in the understanding of the genetic variation among maize hybrids for the association with A. brasilense and reveal the potential for further enhancement of maize through this association.

Gloeosporiocide, a new antifungal cyclic peptide from Streptomyces morookaense AM25 isolated from the Amazon bulk soil.

Actinobacteria are known by their ability to produce several antimicrobial compounds of biotechnological interest. Thus, in this study, we isolated and identified by partial 16S RNA sequencing ∼100 actinobacteria isolates from guarana (Paullinia cupana) bulk soil. Besides, we isolated from the actinobacteria Streptomyces morookaense AM25 a novel cyclic peptide, named gloeosporiocide, molecular formula C44H48N11O7S3 (calculated 938.2901), and characterized by the presence of cyclized cysteins to form three thiazols. The novel compound had activity against the plant pathogen Colletotrichum gloeosporioides, assayed by the paper disk diffusion method (42.7% inhibition, 0.1 mg disk-1) and by the microdilution assay (1.25 g L-1). Our results reveal the potential of the actinobacteria from the Amazon rhizospheric soils as biocontrol agents as well as producers of new compounds with antifungal activity. Thus, this work constitutes a step forward in the development of the biotechnology of actinobacteria in the production of compounds of agronomic interest.

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.