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.

Citrus is a highly valued tree crop worldwide, while, at the same time, citrus production faces many biotic challenges, including bacterial canker and Huanglongbing (HLB). Breeding for disease‐resistant varieties is the most efficient and sustainable approach to control plant diseases. Traditional breeding of citrus varieties is challenging due to multiple limitations, including polyploidy, polyembryony, extended juvenility and long crossing cycles. Targeted genome editing technology has the potential to shorten varietal development for some traits, including disease resistance. Here, we used CRISPR/Cas9/sgRNA technology to modify the canker susceptibility gene CsLOB1 in Duncan grapefruit. Six independent lines, DLOB2, DLOB3, DLOB9, DLOB10, DLOB11 and DLOB12, were generated. Targeted next‐generation sequencing of the six lines showed the mutation rate was 31.58%, 23.80%, 89.36%, 88.79%, 46.91% and 51.12% for DLOB2, DLOB3, DLOB9, DLOB10, DLOB11 and DLOB12, respectively, of the cells in each line. DLOB2 and DLOB3 showed canker symptoms similar to wild‐type grapefruit, when inoculated with the pathogen Xanthomonas citri subsp. citri (Xcc). No canker symptoms were observed on DLOB9, DLOB10, DLOB11 and DLOB12 at 4 days postinoculation (DPI) with Xcc. Pustules caused by Xcc were observed on DLOB9, DLOB10, DLOB11 and DLOB12 in later stages, which were much reduced compared to that on wild‐type grapefruit. The pustules on DLOB9 and DLOB10 did not develop into typical canker symptoms. No side effects and off‐target mutations were detected in the mutated plants. This study indicates that genome editing using CRISPR technology will provide a promising pathway to generate disease‐resistant citrus varieties.

"Candidatus Liberibacter" species are associated with economically devastating diseases of citrus, potato, and many other crops. The importance of these diseases as well as the proliferation of new diseases on a wider host range is likely to increase as the insects vectoring the "Ca. Liberibacter" species expand their territories worldwide. Here, we review the progress on understanding pathogenesis mechanisms of "Ca. Liberibacter" species and the control approaches for diseases they cause. We discuss the Liberibacter virulence traits, including secretion systems, putative effectors, and lipopolysaccharides (LPSs), as well as other important traits likely to contribute to disease development, e.g., flagella, prophages, and salicylic acid hydroxylase. The pathogenesis mechanisms of Liberibacters are discussed. Liberibacters secrete Sec-dependent effectors (SDEs) or other virulence factors into the phloem elements or companion cells to interfere with host targets (e.g., proteins or genes), which cause cell death, necrosis, or other phenotypes of phloem elements or companion cells, leading to localized cell responses and systemic malfunction of phloem. Receptors on the remaining organelles in the phloem, such as plastid, vacuole, mitochondrion, or endoplasmic reticulum, interact with secreted SDEs and/or other virulence factors secreted or located on the Liberibacter outer membrane to trigger cell responses. Some of the host genes or proteins targeted by SDEs or other virulence factors of Liberibacters serve as susceptibility genes that facilitate compatibility (e.g., promoting pathogen growth or suppressing immune responses) or disease development. In addition, Liberibacters trigger plant immunity response via pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharides), which leads to premature cell death, callose deposition, or phloem protein accumulation, causing a localized response and/or systemic effect on phloem transportation. Physical presence of Liberibacters and their metabolic activities may disturb the function of phloem, via disrupting osmotic gradients, or the integrity of phloem conductivity. We also review disease management strategies, including promising new technologies. Citrus production in the presence of Huanglongbing is possible if the most promising management approaches are integrated. HLB management is discussed in the context of local, area-wide, and regional Huanglongbing/Asian Citrus Psyllid epidemiological zones. For zebra chip disease control, aggressive psyllid management enables potato production, although insecticide resistance is becoming an issue. Meanwhile, new technologies such as clustered regularly interspaced short palindromic repeat (CRISPR)-derived genome editing provide an unprecedented opportunity to provide long-term solutions.

BackgroundRoots are the primary site for plant-microbe interactions. Among the three root-associated layers (i.e., rhizosphere, rhizoplane, and endorhiza), the rhizoplane is a key component serving a critical gating role that controls microbial entry into plant roots. The microbial communities colonizing the three layers are believed to be gradually enriched from the bulk soil inoculum. However, it is unknown how this enrichment process, particularly the rhizosphere to rhizoplane step, is affected by biotic stresses, such as disease. In this study, we address this question using the citrus root-associated microbiome as a model.ResultsWe identified the rhizosphere-to-rhizoplane-enriched taxonomic and functional properties of the citrus root-associated microbiome and determined how they were affected by Huanglongbing (HLB), a severe systemic disease caused by Candidatus Liberibacter asiaticus, using metagenomic and metatranscriptomic approaches. Multiple rhizoplane-enriched genera were identified, with Bradyrhizobium and Burkholderia being the most dominant. Plant-derived carbon sources are an important driving force for the enrichment process. The enrichment of functional attributes, such as motility, chemotaxis, secretion systems, and lipopolysaccharide (LPS) synthesis, demonstrated more active microbe-plant interactions on the rhizoplane than the rhizosphere. We observed that HLB impaired the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome in three ways: (1) by decreasing the relative abundance of most rhizoplane-enriched genera; (2) by reducing the relative abundance and/or expression activity of the functional attributes involved in microbe-plant interactions; and (3) by recruiting more functional features involved in autotrophic life cycle adaptation, such as carbon fixation and nitrogen nitrification in the HLB rhizoplane microbiome. Finally, our data showed that inoculation of Burkholderia strains isolated from the healthy citrus root-associated microbiome could trigger the expression of genes involved in induced systemic resistance in inoculated plants.ConclusionsHLB causes decreased relative abundance and/or expression activity of rhizoplane-enriched taxonomic and functional properties, collectively resulting in impaired plant host-microbiome interactions. Manipulation of the citrus root-associated microbiome, for instance, by inoculating citrus roots with beneficial Burkholderia strains, has potential to promote plant health. Our results provide novel insights for understanding the contributions of the community enrichment process of the root-associated microbiome to the plant hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0304-4) contains supplementary material, which is available to authorized users.

The root microbes play pivotal roles in plant productivity, nutrient uptakes, and disease resistance. The root microbial community structure has been extensively investigated by 16S/18S/ITS amplicons and metagenomic sequencing in crops and model plants. However, the functional associations between root microbes and host plant growth are poorly understood. This work investigates the root bacterial community of foxtail millet (Setaria italica) and its potential effects on host plant productivity. We determined the bacterial composition of 2882 samples from foxtail millet rhizoplane, rhizosphere and corresponding bulk soils from 2 well-separated geographic locations by 16S rRNA gene amplicon sequencing. We identified 16 109 operational taxonomic units (OTUs), and defined 187 OTUs as shared rhizoplane core OTUs. The β-diversity analysis revealed that microhabitat was the major factor shaping foxtail millet root bacterial community, followed by geographic locations. Large-scale association analysis identified the potential beneficial bacteria correlated with plant high productivity. Besides, the functional prediction revealed specific pathways enriched in foxtail millet rhizoplane bacterial community. We systematically described the root bacterial community structure of foxtail millet and found its core rhizoplane bacterial members. Our results demonstrated that host plants enrich specific bacteria and functions in the rhizoplane. The potentially beneficial bacteria may serve as a valuable knowledge foundation for bio-fertilizer development in agriculture.

Citrus Huanglongbing (HLB) is the most destructive citrus disease worldwide. HLB is associated with three species of the phloem-limited, gram-negative, fastidious α-proteobacteria: Candidatus Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf) with Las being the most widespread species. Las has not been cultured in artificial media, which has greatly hampered our efforts to understand its virulence mechanisms. Las contains a complete Sec-translocon, which has been suggested to transport Las proteins including virulence factors into the extracytoplasmic milieu. In this study, we characterized the Sec-translocon dependent, signal peptide containing extracytoplasmic proteins of Las. A total of 166 proteins of Las-psy62 strain were predicted to contain signal peptides targeting them out of the cell cytoplasm via the Sec-translocon using LipoP, SigalP 3.0, SignalP 4.1, and Phobius. We also predicated SP containing extracytoplasmic proteins for Las-gxpsy and Las-Ishi-1, Lam, Laf, Ca. L. solanacearum (Lso), and L. crescens (Lcr). For experimental validation of the predicted extracytoplasmic proteins, Escherichia coli based alkaline phosphatase (PhoA) gene fusion assays were conducted. A total of 86 out of the 166 predicted Las proteins were experimentally validated to contain signal peptides. Additionally, Las-psy62 lepB (CLIBASIA_04190), the gene encodes signal peptidase I, was able to partially complement the amber mutant of lepB of E. coli. This work will contribute to the identification of Sec-translocon dependent effector proteins of Las, which might be involved in virulence of Las.

SaCas9/sgRNA, derived from Staphylococcus aureus, is an alternative system for genome editing to Streptococcus pyogenes SpCas9/sgRNA. The smaller SaCas9 recognizes a different protospacer adjacent motif (PAM) sequence from SpCas9. SaCas9/sgRNA has been employed to edit the genomes of Arabidopsis, tobacco and rice. In this study, we aimed to test its potential in genome editing of citrus. Transient expression of SaCas9/sgRNA in Duncan grapefruit via Xcc-facilitated agroinfiltration showed it can successfully modify CsPDS and Cs2g12470. Subsequently, binary vector GFP-p1380N-SaCas9/35S-sgRNA1:AtU6-sgRNA2 was developed to edit two target sites of Cs7g03360 in transgenic Carrizo citrange. Twelve GFP-positive Carrizo transformants were successfully established, designated as #Cz1 to #Cz12. Based on targeted next generation sequencing results, the mutation rates for the two targets ranged from 15.55 to 39.13% for sgRNA1 and 49.01 to 79.67% for sgRNA2. Therefore, SaCas9/sgRNA can be used as an alternative tool to SpCas9/sgRNA for citrus genome editing.

Background Xanthomonas citri subsp. citri pathotypes cause bacterial citrus canker, being responsible for severe agricultural losses worldwide. The A pathotype has a broad host spectrum, while A* and Aw are more restricted both in hosts and in geography. Two previous phylogenomic studies led to contrasting well-supported clades for sequenced genomes of these pathotypes. No extensive biogeographical or divergence dating analytic approaches have been so far applied to available genomes. Results Based on a larger sampling of genomes than in previous studies (including six new genomes sequenced by our group, adding to a total of 95 genomes), phylogenomic analyses resulted in different resolutions, though overall indicating that A + AW is the most likely true clade. Our results suggest the high degree of recombination at some branches and the fast diversification of lineages are probable causes for this phylogenetic blurring effect. One of the genomes analyzed, X. campestris pv. durantae, was shown to be an A* strain; this strain has been reported to infect a plant of the family Verbenaceae, though there are no reports of any X. citri subsp. citri pathotypes infecting any plant outside the Citrus genus. Host reconstruction indicated the pathotype ancestor likely had plant hosts in the family Fabaceae, implying an ancient jump to the current Rutaceae hosts. Extensive dating analyses indicated that the origin of X. citri subsp. citri occurred more recently than the main phylogenetic splits of Citrus plants, suggesting dispersion rather than host-directed vicariance as the main driver of geographic expansion. An analysis of 120 pathogenic-related genes revealed pathotype-associated patterns of presence/absence. Conclusions Our results provide novel insights into the evolutionary history of X. citri subsp. citri as well as a sound phylogenetic foundation for future evolutionary and genomic studies of its pathotypes.