Characterization and comprehensive analysis of the ecological interaction networks of bacterial communities in Paullinia cupana var. sorbilis by 16S rRNA gene metabarcoding.

Endophytes improve the host performance in areas of high plant endemicity. Paullinia cupana is an Amazonia plant species of economic and social importance due to the high caffeine concentration in its seeds. An interesting strategy to identify endophytic microorganisms with potential biotechnological application is to understand the factors that influence the endophytic community to rationalize the host management programs. We used the next-generation sequencing for bacterial 16S rRNA gene to examine how the P. cupana organ, genotype, and geographic location influenced its endophytic bacterial community. We obtained 1520 operational taxonomic units (OTUs) distributed in 19 phyla, 32 classes, 79 orders, 114 families and 174 genera. The P. cupana roots and leaves were specifically colonized by the bacterial genera Acidothermus and Porphyromonas, respectively, with high relative frequency. The plant organ type influenced the endophytic community's richness, diversity, OTUs composition, relative abundance of phyla and genera, and genera interaction network. However, the host plant genotype and geographic location influenced the composition and interaction among genera in the network analysis. Prevotella is a super-generalist genus in the interaction network of endophytic bacteria of P. cupana. This study revealed endophytic bacterial groups of importance to P. cupana and stressed that the host plant organ modulates the structure and interactions within this community. Our results indicated that the microbial community adapted to colonize P. cupana by adjusting to its composition and interaction network. The isolation of abundant and super-generalist bacterial genera shall help to examine their functionality to the composition and fitness of the endophytic community of P. cupana.

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.

The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose

ABSTRACT Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46 µmol mL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02 µmol mL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose.

Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46µmolmL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02µmolmL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

Diversity of cultivable fungal endophytes in Paullinia cupana (Mart.) Ducke and bioactivity of their secondary metabolites.

Paullinia cupana is associated with a diverse community of pathogenic and endophytic microorganisms. We isolated and identified endophytic fungal communities from the roots and seeds of P. cupana genotypes susceptible and tolerant to anthracnose that grow in two sites of the Brazilian Amazonia forest. We assessed the antibacterial, antitumor and genotoxic activity in vitro of compounds isolated from the strains Trichoderma asperellum (1BDA) and Diaporthe phaseolorum (8S). In concert, we identified eight fungal species not previously reported as endophytes; some fungal species capable of inhibiting pathogen growth; and the production of antibiotics and compounds with bacteriostatic activity against Pseudomonas aeruginosa in both susceptible and multiresistant host strains. The plant genotype, geographic location and specially the organ influenced the composition of P. cupana endophytic fungal community. Together, our findings identify important functional roles of endophytic species found within the microbiome of P. cupana. This hypothesis requires experimental validation to propose management of this microbiome with the objective of promoting plant growth and protection.

Diversity of cultivable bacterial endophytes in Paullinia cupana and their potential for plant growth promotion and phytopathogen control.

Endophytic bacteria occupy the same niche of phytopathogens and may produce metabolites that induce the host plant systemic resistance and growth. Host and environmental variables often determine the endophytic community's structure and composition. In this study, we addressed whether the plant genotype, organ, and geographic location influence the structure, composition, and functionality of endophytic bacterial communities in Paullinia cupana. To characterize the communities and identify strains with potential application in agriculture, we analyzed two P. cupana genotypes cultivated in two cities of the State of Amazonas, Brazil. Endophytic bacteria were isolated from surface-disinfested root, leaf, and seed tissues through the fragmentation and maceration techniques. The colonization rate, number of bacteria, richness, diversity, and functional traits were determined. The plant growth-promoting ability of selected bacterial strains was assessed in Sorghum bicolor. We identified 95 bacterial species distributed in 29 genera and 3 phyla (Proteobacteria, Actinobacteria, and Firmicutes). The colonization rate, richness, diversity, and species composition varied across the plant organs; the last parameter also varied across the plant genotype and location. Some strains exhibited relevant plant growth-promoting traits and antagonistic traits against the main phytopathogens of P. cupana, but they were not separated by functional traits. The main bacterial strains with plant growth-promoting traits induced S. bicolor growth. Altogether, our findings open opportunities to study the application of isolated endophytic bacterial strains in the bioprospection of processes and products.

Endophytic cultivable bacterial community obtained from the Paullinia cupana seed in Amazonas and Bahia regions and its antagonistic effects against Colletotrichum gloeosporioides.

Guarana (Paullinia cupana var. sorbilis) is a plant from the Amazonas region with socio-economic importance. However, guarana production has been increasingly affected by unfavorable conditions resulting from anthracnose, caused by the Colletotrichum fungal genus, which primarily affects mainly the Amazonas region. The aim of the present study was to isolate bacterial endophytes from the seeds of guarana plants obtained from Amazonas region and the Northeast state of Bahia, a region where this disease is not a problem for guarana plantations. The number of bacterial Colony Forming Units (CFU/g seeds) was 2.4 × 10(4) from the Bahia and 2.9 × 10(4) from the Amazonas region. One hundred and two isolated bacteria were evaluated in vitro against the phytopathogenic strain Colletotrichum gloeosporioides L1. These isolates were also analyzed for the enzymatic production of amylase, cellulase, protease, pectinase, lipase and esterase. Approximately 15% of isolates, showing high antagonistic activity, and the production of at least one enzyme were identified through the partial sequencing of 16S rDNA. The genus Bacillus was the most frequently observed, followed by Paenibacillus, Ochrobactrum, Microbacterium and Stenotrophomonas. Proteolytic activity was observed in 24 isolates followed by amylolytic, pectinolytic and cellulolytic activities. No esterase and lipase production was detected. Most of the isolates, showing antagonistic effects against C. gloeosporioides and high enzymatic activities, were isolated from the anthracnose-affected region. A biocontrol method using the endophytes from guarana seeds could be applied in the future, as these bacteria are vertically transferred to guarana seedlings.

Seasonal dynamics of arbuscular mycorrhizal fungi in plants of Theobroma grandiflorum Schum and Paullinia cupana Mart. of aN agroforestry system in Central Amazonia, Amazonas State, Brazil

A dinâmica sazonal de fungos micorrízicos arbusculares (FMA) foi investigada na rizosfera de duas espécies frutíferas em um ecossistema de terra firme na Amazônia Central. Adotou-se o delineamento inteiramente casualizado, em arranjo fatorial 2 x 9, onde os fatores representaram duas espécies frutíferas (Theobroma grandiflorum e Paullinia cupana) e nove meses de coleta (agosto, setembro e dezembro/1998, fevereiro, abril, maio e dezembro/1999, fevereiro e maio/2000), com cinco repeticões. O percentual de colonizacão micorrízica para as duas espécies atingiu valores máximos nos meses de fevereiro e maio de 2000 (estacão chuvosa). Similarmente, nos meses de abril e maio de 1999, fevereiro e maio de 2000 (estacão chuvosa) foram registrados os maiores números de esporos de FMA para ambas espécies. A precipitacão pluvial foi significativamente e positivamente correlacionada com o número de esporos para as duas espécies, e significativamente correlacionada apenas com a colonizacão micorrízica de P. cupana. O teor de umidade do solo foi positivamente correlacionado com o número de esporos e colonizacão por FMA para ambas espécies. A colonizacão micorrízica e o número de esporos de FMA foram positivamente correlacionados com os teores de Mg e K no solo. O número de esporos foi ainda negativamente correlacionado com a CTC efetiva do solo. A colonizacão micorrízica e o número de esporos de FMA na rizosfera de P. cupana foram positivamente correlacionadas com o pH e a concentracão de Mn no solo. A colonizacão micorrízica foi também positivamente correlacionada com o número de esporos de FMA para as duas espécies avaliadas. Em conclusão, esse estudo mostrou que tanto a colonizacão micorrízica como a esporulacão são sazonais e dependentes da espécie de planta hospedeira, precipitacão pluvial, teor de umidade e da química do solo, nas condicões da Amazônia Central.