Citrus is a globally important, perennial fruit crop whose rhizosphere microbiome is thought to play an important role in promoting citrus growth and health. Here, we report a comprehensive analysis of the structural and functional composition of the citrus rhizosphere microbiome. We use both amplicon and deep shotgun metagenomic sequencing of bulk soil and rhizosphere samples collected across distinct biogeographical regions from six continents. Predominant taxa include Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes. The core citrus rhizosphere microbiome comprises Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia, some of which are potential plant beneficial microbes. We also identify over-represented microbial functional traits mediating plant-microbe and microbe-microbe interactions, nutrition acquisition and plant growth promotion in citrus rhizosphere. The results provide valuable information to guide microbial isolation and culturing and, potentially, to harness the power of the microbiome to improve plant production and health.

SUMMARY: Taxonomic status:  Bacteria, Proteobacteria, gamma subdivision, Xanthomodales, Xanthomonas group, axonopodis DNA homology group, X. axonopodis pv. citri (Hasse) Vauterin et al .  Microbiological properties:  Gram negative, slender, rod‐shaped, aerobic, motile by a single polar flagellum, produces slow growing, non‐mucoid colonies in culture, ecologically obligate plant parasite.  Host range:  Causal agent of Asiatic citrus canker on most Citrus spp. and close relatives of Citrus in the family Rutaceae.  Disease symptoms:  Distinctively raised, necrotic lesions on fruits, stems and leaves.  Epidemiology:  Bacteria exude from lesions during wet weather and are disseminated by splash dispersal at short range, windblown rain at medium to long range and human assisted movement at all ranges.  Crop loss:  Severe infections cause defoliation, blemished fruit, premature fruit drop, die‐back of twigs and general debilitation of the tree.  Distribution:  Citrus canker is not present in all subtropical to tropical regions of citriculture in the world, so considerable regulatory efforts are expended to prevent the introduction and spread of X. axonopodis pv. citri into areas in the Americas, Australia and elsewhere, with climates conducive to the disease.  Importance:  Limited strategies exist for suppression of citrus canker on more susceptible cultivars. Blemished fruit are unmarketable and exposed fruit are restricted in market access. The economic impact of loss of markets is much greater than that from yield and quality reductions of the crop.  Useful websites:  http://doacs.state.fl.us/canker , http://www.apsnet.org/education/lessonsplantpath/citruscanker/top.htm , http://www.apsnet.org/online/feature/citruscanker/ , http://www.plantmanagementnetwork.org/pub/php/review/citruscanker/ , http://www.abecitrus.com.br/fundecitrus.html , http://www.biotech.ufl.edu/PlantContainment/canker.htm , http://www.aphis.usda.gov/oa/ccanker/ .

Summary: Biological control of plant diseases has gained acceptance in recent years. B acillus subtilis UMAF 6639 is an antagonistic strain specifically selected for the efficient control of the cucurbit powdery mildew fungus P odosphaera fusca , which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that UMAF 6639 was able to induce systemic resistance ( ISR ) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by UMAF 6639. First, we examined the signalling pathways elicited by UMAF 6639 in melon plants, as well as the defence mechanisms activated in response to P . fusca . Second, we analysed the role of the lipopeptides produced by UMAF 6639 as potential determinants for ISR activation. Our results demonstrated that UMAF 6639 confers protection against cucurbit powdery mildew by activation of jasmonate‐ and salicylic acid‐dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of UMAF 6639 as a biological control agent.

Partial sequence analysis of the ribosomal operon in Xanthomonas axonopodis allowed discrimination among strains causing the A, B, and C types of citrus bacterial canker (CBC) and quantification of the relationship of these organisms with other species and pathovars in the same genus. Sets of primers based on sequence differences in the internally transcribed spacer and on a sequence from the plasmid gene pthA involved in virulence were designed for specific identification of xanthomonads causing CBC diseases. The two sets were validated with a collection of Xanthomonas strains associated with citrus species. The primer set based on ribosomal sequences had a high level of specificity for X. axonopodis pv. citri, whereas the set based on the pthA gene was universal for all types of CBC organisms. Moreover, the relationships among worldwide Xanthomonas strains causing CBC were analyzed by amplification of repetitive sequences (enterobacterial repetitive intergenic consensus and BOX elements). Under specific conditions, pathotypes of these Xanthomonas strains could be discerned, and subgroups of the pathotypes were identified. Subgroups of strains were associated with certain geographic areas of the world, and on this basis the origin of type A strains introduced into Florida could be inferred.

In the Wellington and Lake Worth areas of Palm Beach County, FL, citrus canker appeared on Key/Mexican lime (Citrus aurantiifolia) and alemow (C. macrophylla) trees over a period of about 6 to 7 years before detection, but nearby canker-susceptible citrus, such as grapefruit (C. × paradisi) and sweet orange (C. sinensis), were unaffected. Colonies of the causal bacterium, isolated from leaf, stem, and fruit lesions, appeared similar to the Asiatic group of strains of Xanthomonas axonopodis pv. citri (Xac-A) on the nutrient agar plate, but the growth on lima bean agar slants was less mucoid. The bacterium produced erumpent, pustule-like lesions of typical Asiatic citrus canker syndrome after inoculation into Key/Mexican lime, but brownish, flat, and necrotic lesions on the leaves of Duncan grapefruit, Madame Vinous sweet orange, sour orange (C. aurantium), citron (C. medica), Orlando tangelo (C. reticulata × C. × paradisi), and trifoliate orange (Poncirus trifoliata). The bacterium did not react with the Xac-A specific monoclonal antibody A1 using enzyme-linked immunosorbent assay (ELISA) and could not be detected by polymerase chain reaction (PCR)-based assays using primers selected for Xac-A. DNA reassociation analysis confirmed that the pathogen, designated as Xac-A^W, was more closely related to Xac-A and Xac-A* strains than X. axonopodis pv. aurantifolii or the citrus bacterial spot pathogen (X. axonopodis pv. citrumelo). The strain can be easily differentiated from Xac-A and Xac-A* using ELISA, PCR-based tests, fatty acid analysis, pulsed-field gel electrophoresis of genomic DNA, and host specificity.