16S metabarcoding analysis reveals the influence of organic and conventional farming practices on bacterial communities from the rhizospheric of Coffea arabica L.

Coffea sp. is cultivated in many tropical countries. Brazil has always adopted intensive agricultural practices, but organic coffee farming is an alternative system based on the non-use of agrochemicals and the rational management of soils. Metabarcoding 16S analysis using next-generation sequencing has been developed to identify and compare the diversity of the Coffea arabica L. rhizospheric bacterial community in two farming areas in São Paulo, Brazil. Dourado uses conventional farming, while Ribeirão Corrente uses organic. We found broad taxonomic composition, with sequences from 24 phyla, 55 classes, 61 orders, 146 families, and 337genus. The three most abundant phyla were Proteobacteria (38.27%), Actinobacteria (15.56%), and Acidobacteria (16.10%). In organic farming, the top 3 were the family Sphingomonadaceae, order Rhizobiales, genus Nocardioides, and Gp6. The genus Gp2 and the phylum Candidatus Saccharibacteria were the most abundant OTUs exclusively present in conventional farming. In the organic farming practice, Proteobacteria, Actinobacteria, and Acidobacteria were also present among the exclusive OTUs; we also found OTUs belonging to Bacteroidetes, Firmicutes, and Verrucomicrobia. Our study indicates a positive effect of organic farming on microbial communities. Fertilization may directly affect soil microbiota, suggesting that a large and active microbial community low in functional diversity might not adapt to new climatic conditions. A diverse community could provide better resilience to environmental changes, improving the productivity of this important crop.

Endophytic fungi: an overview on biotechnological and agronomic potential.

Endophytic fungi colonize the inter- and/or intracellular regions of healthy plant tissues and have a close symbiotic relationship with their hosts. These microorganisms produce antibiotics, enzymes, and other bioactive compounds that enable them to survive in competitive habitats with other microorganisms. In addition, secondary metabolites confer protection to their host plant against other bacterial and fungal pathogens and/or can promote plant growth. Endophytic fungi are viewed as a promising source of bioactive natural products, which can be optimized through changes in growing conditions. The exploration of novel bioactive molecules produced by these microorganisms has been attracting attention from researchers. The chemical and functional diversity of natural products from endophytic fungi exhibits a broad spectrum of applications in medicine, agriculture, industry and the environment. Fungal endophytes can also enhance the photoprotective effects and photochemical efficiency in the host plants. Modern omic approaches have facilitated research investigating symbiotic plant-endophytic fungi interactions. Therefore, research on endophytic fungi can help discovery novel biomolecules for various biotechnological applications and develop a sustainable agriculture.

Potential of Mangrove-Associated Endophytic Fungi for Production of Carbohydrolases with High Saccharification Efficiency.

The endophytic fungi represent a potential source of microorganisms for enzyme production. However, there have been only few studies exploiting their potential for the production of enzymes of industrial interest, such as the (hemi)cellulolytic enzymatic cocktail required in the hydrolysis of lignocellulosic biomass. Here, a collection of endophytic fungi isolated from mangrove tropical forests was evaluated for the production of carbohydrolases and performance on the hydrolysis of cellulose. For that, 41 endophytic strains were initially screened using a plate assay containing crystalline cellulose as the sole carbon source and the selected strains were cultivated under solid-state fermentation for endoglucanase, ß-glucosidase, and xylanase enzyme quantification. The hydrolysis of a cellulosic material with the enzymes from endophytic strains of the Aspergillus genus resulted in glucose and conversion values more than twofold higher than the reference strains (Aspergillus niger F12 and Trichoderma reesei Rut-C30). Particularly, the enzymes from strains A. niger 56 (3) and A. awamori 82 (4) showed a distinguished saccharification performance, reaching cellulose conversion values of about 35% after 24 h. Linking hydrolysis performance to the screening steps played an important role towards finding potential fungal strains for producing enzymatic cocktails with high saccharification efficiency. These results indicate the potential of mangrove-associated endophytic fungi for production of carbohydrolases with efficient performance in the hydrolysis of biomass, thus contributing to the implementation of future biorefineries.

Sugarcane growth promotion by the endophytic bacterium Pantoea agglomerans 33.1.

The promotion of sugarcane growth by the endophytic Pantoea agglomerans strain 33.1 was studied under gnotobiotic and greenhouse conditions. The green fluorescent protein (GFP)-tagged strain P. agglomerans 33.1::pNKGFP was monitored in vitro in sugarcane plants by microscopy, reisolation, and quantitative PCR (qPCR). Using qPCR and reisolation 4 and 15 days after inoculation, we observed that GFP-tagged strains reached similar density levels both in the rhizosphere and inside the roots and aerial plant tissues. Microscopic analysis was performed at 5, 10, and 18 days after inoculation. Under greenhouse conditions, P. agglomerans 33.1-inoculated sugarcane plants presented more dry mass 30 days after inoculation. Cross-colonization was confirmed by reisolation of the GFP-tagged strain. These data demonstrate that 33.1::pNKGFP is a superior colonizer of sugarcane due to its ability to colonize a number of different plant parts. The growth promotion observed in colonized plants may be related to the ability of P. agglomerans 33.1 to synthesize indoleacetic acid and solubilize phosphate. Additionally, this strain may trigger chitinase and cellulase production by plant roots, suggesting the induction of a plant defense system. However, levels of indigenous bacterial colonization did not vary between inoculated and noninoculated sugarcane plants under greenhouse conditions, suggesting that the presence of P. agglomerans 33.1 has no effect on these communities. In this study, different techniques were used to monitor 33.1::pNKGFP during sugarcane cross-colonization, and our results suggested that this plant growth promoter could be used with other crops. The interaction between sugarcane and P. agglomerans 33.1 has important benefits that promote the plant's growth and fitness.

Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants.

Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003. Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for M. mesophilicum. The assays are specific for M. mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., X. fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria.

Interaction between endophytic bacteria from citrus plants and the phytopathogenic bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis.

AIMS: To isolate endophytic bacteria and Xylella fastidiosa and also to evaluate whether the bacterial endophyte community contributes to citrus-variegated chlorosis (CVC) status in sweet orange (Citrus sinensis [L.] Osbeck cv. Pera). METHODS AND RESULTS: The presence of Xylella fastidiosa and the population diversity of culturable endophytic bacteria in the leaves and branches of healthy, CVC-asymptomatic and CVC-symptomatic sweet orange plants and in tangerine (Citrus reticulata cv. Blanco) plants were assessed, and the in vitro interaction between endophytic bacteria and X. fastidiosa was investigated. There were significant differences in endophyte incidence between leaves and branches, and among healthy, CVC-asymptomatic and CVC-symptomatic plants. Bacteria identified as belonging to the genus Methylobacterium were isolated only from branches, mainly from those sampled from healthy and diseased plants, from which were also isolated X. fastidiosa. CONCLUSIONS: The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the first evidence of an interaction between citrus endophytic bacteria and X. fastidiosa and suggests a promising approach that can be used to better understand CVC disease.

RAPD profile and antibiotic susceptibility of Xylella fastidiosa, causal agent of citrus variegated chlorosis.

AIMS: The aim of this study was to evaluate the diversity of Xylella fastidiosa isolated from citrus trees affected by Citrus Variegated Chlorosis (CVC). METHODS AND RESULTS: The antibiotic susceptibility by agar disc diffusion and minimum inhibitory concentration (MIC) methods was observed for all drug evaluated, except for penicillin-G. Genetic diversity by RAPD analysis revealed three major groups (citrus, coffee and grapevine), being the citrus group more similar with the coffee group than with the grapevine group. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the possibility to use these antibiotics susceptibility as markers in the development of a cloning vector and penicillin-G could be used as a selective marker for the isolation of X. fastidiosa from citrus plants.