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Xanthomonas citri pv. citri (Xcc) causes the devastating citrus canker disease. Xcc is known to have been introduced into Florida, USA in at least three different events in 1915, 1986 and 1995 with the first two claimed to be eradicated. It was questioned whether the Xcc introduction in 1986 has been successfully eradicated. Furthermore, it is unknown how Xcc has spread throughout the citrus groves in Florida. In this study, we investigated the population structure of Xcc to address these questions. We sequenced the whole genome of 343 Xcc strains collected from Florida groves between 1997 and 2016. Our analysis revealed two distinct clusters of Xcc. Our data strongly indicate that the claimed eradication of the 1986 Xcc introduction was not successful and Xcc strains from 1986 introduction were present in samples from at least 8 counties collected after 1994. Importantly, our data revealed that the Cluster 2 strains, which are present in all 20 citrus-producing counties sampled in Florida, originated from the Xcc introduction event in the Miami area in 1995. Our data suggest that Polk County is the epicenter of the dispersal of Cluster 2 Xcc strains, which is consistent with the fact that three major hurricanes passed through Polk County in 2004. As copper-based products have been extensively used to control citrus canker, we also investigated whether Xcc strains have developed resistance to copper. Notably, none of the 343 strains contained known copper resistance genes. Twenty randomly selected Xcc strains displayed sensitivity to copper. Overall, this study provides valuable insights into the introduction, eradication, spread, and copper resistance of Xcc in Florida. Author summary: Sequencing and analyzing the genomes of 343 Xcc strains collected from 1997 to 2016 have enabled us to gain a deeper understanding of its introduction, spread, and eradication. This information is critical for optimizing strategies for responding to the invasion of quarantine pathogens. We have conclusively demonstrated that the Xcc introduction in 1986 was not successfully eradicated, three major hurricanes in 2004 play a critical role in spreading Xcc throughout Florida, and Polk County is the epicenter of Xcc dispersal. Additionally, none of the tested Xcc strains displayed resistance to copper, consistent with the absence of copper resistance genes in their genome. [ABSTRACT FROM AUTHOR]

'Candidatus Liberibacter' species are insect‐transmitted, phloem‐limited α‐Proteobacteria in the order of Rhizobiales. The citrus industry is facing significant challenges due to huanglongbing, associated with infection from 'Candidatus Liberibacter asiaticus' (Las). In order to gain greater insight into 'Ca. Liberibacter' biology and genetic diversity, we have performed genome sequencing and comparative analyses of diverse 'Ca. Liberibacter' species, including those that can infect citrus. Our phylogenetic analysis differentiates 'Ca. Liberibacter' species and Rhizobiales in separate clades and suggests stepwise evolution from a common ancestor splitting first into nonpathogenic Liberibacter crescens followed by diversification of pathogenic 'Ca. Liberibacter' species. Further analysis of Las genomes from different geographical locations revealed diversity among isolates from the United States. Our phylogenetic study also indicates multiple Las introduction events in California and spread of the pathogen from Florida to Texas. Texan Las isolates were closely related, while Florida and Asian isolates exhibited the most genetic variation. We have identified conserved Sec translocon (SEC)‐dependent effectors likely involved in bacterial survival and virulence of Las and analysed their expression in their plant host (citrus) and insect vector (Diaphorina citri). Individual SEC‐dependent effectors exhibited differential expression patterns between host and vector, indicating that Las uses its effector repertoire to differentially modulate diverse organisms. Collectively, this work provides insights into the evolution of 'Ca. Liberibacter' species, the introduction of Las in the United States and identifies promising Las targets for disease management. [ABSTRACT FROM AUTHOR]

Cultivable diversity of bacteria associated with citrus was investigated as part of a larger study to understand the roles of beneficial bacteria and utilize them to increase the productive capacity and sustainability of agro-ecosystems. Citrus roots from Huanglongbing (HLB) diseased symptomatic and asymptomatic citrus were used in this study. A total of 227 and 125 morphologically distinct colonies were isolated and characterized from HLB asymptomatic and symptomatic trees, respectively. We observed that the frequency of bacterial isolates possessing various plant beneficial properties was significantly higher in the asymptomatic samples. A total of 39 bacterial isolates showing a minimum of five beneficial traits related to mineral nutrition [phosphate (P) solubilization, siderophore production, nitrogen (N) fixation], development [indole acetic acid (IAA) synthesis], health [production of antibiotic and lytic enzymes (chitinase)], induction of systemic resistance [salicylic acid (SA) production], stress relief [production of 1-amino-cyclopropane-1-carboxylate deaminase] and production of quorum sensing [ N-acyl homoserine lactones] signals were characterized. A bioassay using ethidium monoazide (EMA)-qPCR was developed to select bacteria antagonistic to Candidatus Liberibacter asiaticus. Using the modified EMA-qPCR assay, we found six bacterial isolates showing maximum similarity to Paenibacillus validus, Lysinibacillus fusiformis, Bacillus licheniformis, Pseudomonas putida, Microbacterium oleivorans, and Serratia plymutica could significantly reduce the population of viable Ca. L. asiaticus in HLB symptomatic leaf samples. In conclusion, we have isolated and characterized multiple beneficial bacterial strains from citrus roots which have the potential to enhance plant growth and suppress diseases. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) or greening currently is the most devastating citrus disease worldwide. The fastidious phloem-colonizing bacterium ' Candidatus Liberibacter asiaticus' (CLas) is the causal agent of citrus HLB in Florida. Bactericides containing the active ingredient oxytetracycline (OTC) have been used in foliar spray to control citrus HLB in Florida since 2016. However, the minimum concentration of OTC required to suppress CLas in planta remains unknown. We developed a new method for evaluating the effects of OTC treatment on CLas titers in infected plants and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In both greenhouse and field, OTC spray did not reduce the titers of CLas, and it produced undetectable or mild levels of OTC residue in leaves within 7 days post-application (DPA). In greenhouse, OTC injection at 0.05 g per tree decreased CLas titers to an undetectable level (cycle threshold value ≥ 36.0) from 7 to 30 DPA and produced a residue level of OTC at 0.68 to 0.73 µg/g of fresh tissue over this period. In the field, OTC injection at 0.50 g per tree resulted in the decline of CLas titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27 to 0.33 µg/g of fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for CLas achieved. The results suggested that the minimum concentrations of OTC required to suppress CLas populations in planta to below the detection limit are 0.68 and 0.86 µg/g and that the minimum concentrations of OTC required for initial inhibition of CLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target CLas in infected plants for effective control of citrus HLB.