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Candidatus Liberibacter spp. are fastidious α-proteobacteria that cause multiple diseases on plant hosts of economic importance, including the most devastating citrus disease: Huanglongbing (HLB). HLB was reported in Asia a century ago but has since spread worldwide. Understanding the pathogenesis of Candidatus Liberibacter spp. remains challenging as they are yet to be cultured in artificial media and infect the phloem, a sophisticated environment that is difficult to manipulate. Despite those challenges, tremendous progress has been made on Ca. Liberibacter pathosystems. Here, we first reviewed recent studies on genetic information of flagellar and type IV pili biosynthesis, their expression profiles, and movement of Ca. Liberibacter spp. inside the plant and insect hosts. Next, we reviewed the transcriptomic, proteomic, and metabolomic studies of susceptible and tolerant plant genotypes to Ca. Liberibacter spp. infection and how Ca. Liberibacter spp. adapt in plants. Analyses of the interactions between plants and Ca. Liberibacter spp. imply the involvement of immune response in the Ca. Liberibacter pathosystems. Lastly, we reviewed how Ca. Liberibacter spp. movement inside and interactions with plants lead to symptom development. [ABSTRACT FROM AUTHOR]

In this review, we highlight studies in which whole-genome sequencing, comparative genomics, and population genomics have provided unprecedented insights into past and ongoing pathogen evolution. These include new understandings of the adaptive evolution of secretion systems and their effectors. We focus on Xanthomonas pathosystems that have seen intensive study and improved our understanding of pathogen emergence and evolution, particularly in the context of host specialization: citrus canker, bacterial blight of rice, and bacterial spot of tomato and pepper. Across pathosystems, pathogens appear to follow a pattern of bursts of evolution and diversification that impact host adaptation. There remains a need for studies on the mechanisms of host range evolution and genetic exchange among closely related but differentially host-specialized species and to start moving beyond the study of specific strain and host cultivar pairwise interactions to thinking about these pathosystems in a community context., Competing Interests: The author(s) declare no conflict of interest.

Plant diseases significantly impact food security and food safety. It was estimated that food production needs to increase by 50% to feed the projected 9.3 billion people by 2050. Yet, plant pathogens and pests are documented to cause up to 40% yield losses in major crops, including maize, rice, and wheat, resulting in annual worldwide economic losses of approximately US$220 billion. Yield losses due to plant diseases and pests are estimated to be 21.5% (10.1 to 28.1%) in wheat, 30.3% (24.6 to 40.9%) in rice, and 22.6% (19.5 to 41.4%) in maize. In March 2023, The American Phytopathological Society (APS) conducted a survey to identify and rank key challenges in plant pathology in the next decade. Phytopathology subsequently invited papers that address those key challenges in plant pathology, and these were published as a special issue. The key challenges identified include climate change effect on the disease triangle and outbreaks, plant disease resistance mechanisms and its applications, and specific diseases including those caused by Candidatus Liberibacter spp. and Xylella fastidiosa . Additionally, disease detection, natural and man-made disasters, and plant disease control strategies were explored in issue articles. Finally, aspects of open access and how to publish articles to maximize the Findability, Accessibility, Interoperability, and Reuse of digital assets in plant pathology were described. Only by identifying the challenges and tracking progress in developing solutions for them will we be able to resolve the issues in plant pathology and ultimately ensure plant health, food security, and food safety., Competing Interests: The author(s) declare no conflict of interest.

Citrus Huanglongbing (HLB) caused by ' Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves-late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB., Competing Interests: The author(s) declare no conflict of interest.

Transcription activator-like effectors are key virulence factors of Xanthomonas . They are secreted into host plant cells and mimic transcription factors inducing the expression of host susceptibility (S) genes. In citrus, CsLOB1 is a direct target of PthA4, the primary effector associated with citrus canker symptoms. CsLOB1 is a transcription factor, and its expression is required for canker symptoms induced by Xanthomonas citri subsp. citri . Several genes are up-regulated by PthA4; however, only CsLOB1 was described as an S gene induced by PthA4. Here, we investigated whether other up-regulated genes could be direct targets of PthA4 or CsLOB1. Seven up-regulated genes by PthA4 were investigated; however, an expansin-coding gene was more induced than CsLOB1 . In Nicotiana benthamiana transient expression experiments, we demonstrate that the expansin-coding gene, referred here to as CsLOB1-INDUCED EXPANSIN 1 ( CsLIEXP1 ), is not a direct target of PthA4, but CsLOB1. Interestingly, CsLIEXP1 was induced by CsLOB1 even without the predicted CsLOB1 binding site, which suggested that CsLOB1 has other unknown binding sites. We also investigated the minimum promoter regulated by CsLOB1, and this region and LOB1 domain were conserved among citrus species and relatives, which suggests that the interaction PthA4- CsLOB1 - CsLIEXP1 is conserved in citrus species and relatives. This is the first study that experimentally demonstrated a CsLOB1 downstream target and lays the foundation to identify other new targets. In addition, we demonstrated that the CsLIEXP1 is a putative S gene indirectly induced by PthA4, which may serve as the target for genome editing to generate citrus canker-resistant varieties., Competing Interests: The author(s) declare no conflict of interest.

' Candidatus Liberibacter asiaticus' (CLas) is associated with the devastating citrus disease Huanglongbing (HLB). Young flushes are the center of the HLB pathosystem due to their roles in the psyllid life cycle and in the acquisition and transmission of CLas. However, the early events of CLas infection and how CLas modulates young flush physiology remain poorly understood. Here, transmission electron microscopy analysis showed that the mean diameter of the sieve pores decreased in young leaves of HLB-positive trees after CLas infection, consistent with CLas-triggered callose deposition. RNA-seq-based global expression analysis of young leaves of HLB-positive sweet orange with (CLas-Pos) and without (CLas-Neg) detectable CLas demonstrated a significant impact on gene expression in young leaves, including on the expression of genes involved in host immunity, stress response, and plant hormone biosynthesis and signaling. CLas-Pos and CLas-Neg expression data displayed distinct patterns. The number of upregulated genes was higher than that of the downregulated genes in CLas-Pos for plant-pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling, which are commonly associated with pathogen infections, compared with the healthy control. On the contrary, the number of upregulated genes was lower than that of the downregulated genes in CLas-Neg for genes involved in plant-pathogen interactions and peroxisome biogenesis/metabolism. Additionally, a time-course quantitative reverse transcription-PCR-based expression analysis visualized the induced expression of companion cell-specific genes, phloem protein 2 genes, and sucrose transport genes in young flushes triggered by CLas. This study advances our understanding of early events during CLas infection of citrus young flushes., Competing Interests: The author(s) declare no conflict of interest.

Competing Interests: The author(s) declare no conflict of interest.

Plant pathology plays a critical role in safeguarding plant health, food security, and food safety through science-based solutions to protect plants against recurring and emerging diseases. In addition, plant pathology contributed significantly to basic discoveries that have had broad impacts on the life sciences beyond plant pathology. In December 2021, The American Phytopathological Society (APS) conducted a survey among its members and among the readership of its journals to identify and rank key discoveries in plant pathology that have had broad impacts on science and/or practical disease management during the past half century. Based on the responses received, key discoveries that have broadly impacted the life sciences during that period include the Agrobacterium Ti plasmid and its mechanism in T-DNA transfer, bacterial ice nucleation, cloning of resistance genes, discovery of viroids, effectors and their mechanisms, pattern-triggered immunity and effector-triggered immunity, RNA interference and gene silencing, structure and function of R genes, transcription activator-like effectors, and type-III secretion system and hrp / hrc . Major advances that significantly impacted practical disease management include the deployment and management of host resistance genes; the application of disease models and forecasting systems; the introduction of modern systemic fungicides and host resistance inducers, along with a better understanding of fungicide resistance mechanisms and management; and the utilization of biological controls and suppressive soils, including the implementation of methyl-bromide alternatives. In this special issue, experts from the pertinent fields review the discovery process, recent progress, and impacts of some of the highest ranked discoveries in each category while also pointing out future directions for new discoveries in fundamental and applied plant pathology., Competing Interests: The author(s) declare no conflict of interest.

Transcription activator-like effectors (TALEs) are bacterial proteins that are injected into the eukaryotic nucleus to act as transcriptional factors and function as key virulence factors of the phytopathogen Xanthomonas . TALEs are translocated into plant host cells via the type III secretion system and induce the expression of host susceptibility (S) genes to facilitate disease. The unique modular DNA binding domains of TALEs comprise an array of nearly identical direct repeats that enable binding to DNA targets based on the recognition of a single nucleotide target per repeat. The very nature of TALE structure and function permits the proliferation of TALE genes and evolutionary adaptations in the host to counter TALE function, making the TALE-host interaction the most dynamic story in effector biology. The TALE genes appear to be a relatively young effector gene family, with a presence in all virulent members of some species and absent in others. Genome sequencing has revealed many TALE genes throughout the xanthomonads, and relatively few have been associated with a cognate S gene. Several species, including Xanthomonas oryzae pv. oryzae and X. citri pv. citri , have near absolute requirement for TALE gene function, while the genes appear to be just now entering the disease interactions with new fitness contributions to the pathogens of tomato and pepper among others. Deciphering the simple and effective DNA binding mechanism also has led to the development of DNA manipulation tools in fields of gene editing and transgenic research. In the three decades since their discovery, TALE research remains at the forefront of the study of bacterial evolution, plant-pathogen interactions, and synthetic biology. We also discuss critical questions that remain to be addressed regarding TALEs., Competing Interests: The author(s) declare no conflict of interest.

Citrus huanglongbing (HLB) caused by ' Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. Most commercial citrus cultivars are susceptible to HLB, with a few more tolerant exceptions such as 'LB8-9' Sugar Belle mandarin. Transcriptomic analyses have been widely used to investigate the potential mechanisms for disease susceptibility, resistance, or tolerance. Previous transcriptomic studies related to HLB mostly focused on single time point data collection. We hypothesize that changes in day length and temperature throughout the seasons have profound effects on citrus-CLas interactions. Here, we conducted RNA-seq analyses on HLB-susceptible Valencia sweet orange and HLB-tolerant mandarin 'LB8-9' in winter, spring, summer, and fall. Significant variations in differentially expressed genes (DEGs) related to HLB were observed among the four seasons. For both cultivars, the highest number of DEGs were found in the spring. CLas infection stimulates the expression of immune-related genes such as NBS-LRR, RLK, RLCK, CDPK, MAPK pathway, reactive oxygen species (ROS), and PR genes in both cultivars, consistent with the model that HLB is a pathogen-triggered immune disease. HLB-positive mandarin 'LB8-9' trees contained higher concentrations of maltose and sucrose, which are known to scavenge ROS. In addition, mandarin 'LB8-9' showed higher expression of genes involved in phloem regeneration, which might contribute to its HLB tolerance. This study shed light on the pathogenicity mechanism of the HLB pathosystem and the tolerance mechanism against HLB, providing valuable insights into HLB management.

Xanthomonas citri subsp. citri ( Xcc ) is the causal agent of citrus bacterial canker (CBC), one of the most devastating citrus diseases. Most commercial citrus varieties are susceptible to CBC. However, some citrus varieties and wild citrus germplasms are CBC resistant and are promising in genetic increases in citrus resistance against CBC. We aimed to evaluate citrus germplasms for resistance against CBC. First, we developed a rapid evaluation method based on enhanced yellow fluorescent protein (eYFP)-labeled Xcc . The results demonstrated that eYFP does not affect the growth and virulence of Xcc . Xcc-eYFP allows measurement of bacterial titers but is more efficient and rapid than the plate colony counting method. Next, we evaluated citrus germplasms collected in China. Based on symptoms and bacterial titers, we identified that two citrus germplasms ('Ichang' papeda and 'Huapi' kumquat) are resistant, whereas eight citrus germplasms ('Chongyi' wild mandarin, 'Mangshan' wild mandarin, 'Ledong' kumquat, 'Dali' citron, 'Yiliang' citron, 'Longyan' kumquat, 'Bawang' kumquat, and 'Daoxian' wild mandarin) are tolerant. In summary, we have developed a rapid evaluation method to test the resistance of citrus plants against CBC. This method was successfully used to identify two highly canker-resistant citrus germplasms and eight citrus germplasms with canker tolerance. These results could be leveraged in traditional breeding contexts or be used to identify canker resistance genes to increase the disease resistance of commercial citrus varieties via biotechnological approaches.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. X. citri subsp. citri causes canker symptoms by inducing the expression of canker susceptibility gene LOB1 via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of LOB1 . However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit ( Citrus paradisi ), which usually contain two alleles of LOB1 and thus, have two types of LOB1 promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBE PthA4 -LOBP in grapefruit. Using 'Duncan' grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#Dun Yao 7). X. citri subsp. citri caused canker symptoms on wild-type and nonbiallelic mutant plants but not on #Dun Yao 7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #Dun Yao 7, caused canker symptoms on both wild-type and #Dun Yao 7. No off-target mutations were detected in #Dun Yao 7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for 'Duncan' grapefruit, paving the way for using disease-resistant varieties to control canker.

Huanglongbing (HLB) is a devastating citrus disease worldwide. A three-pronged approach to controlling HLB has been suggested, namely, removal of HLB-symptomatic trees, psyllid control, and replacement with HLB-free trees. However, such a strategy did not lead to successful HLB control in many citrus-producing regions, such as Florida. We hypothesize that this is because of the small-scale or incomprehensive implementation of the program; conversely, a comprehensive implementation of such a strategy at the regional level can successfully control HLB. To test our hypothesis, we investigated the effects of region-wide comprehensive implementation of this scheme to control HLB in Gannan region, China, with a total planted citrus acreage of over 110,000 ha from 2013 to 2019. With the region-wide implementation of comprehensive HLB management, the overall HLB incidence in Gannan decreased from 19.71% in 2014 to 3.86% in 2019. A partial implementation of such a program (without a comprehensive inoculum removal) at the regional level in Brazil resulted in HLB incidence increasing from 1.89% in 2010 to 19.02% in 2019. Using dynamic regression model analyses with data from both Brazil and China, we constructed a model to predict HLB incidence when all three components were applied at 100%. It was predicated that in a region-wide comprehensive implementation of such a program, HLB incidence would be controlled to a level of less than 1%. We conducted economic feasibility analyses and showed that average net profits were positive for groves that implemented the comprehensive strategy, but groves that did not implement it had negative net profits over a 10-year period. Overall, the key for the three-pronged program to successfully control HLB is the large scale (region-wide) and comprehensiveness in implementation. This study provides valuable information to control HLB and other economically important endemic diseases worldwide.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Streptomycin (STR) has been used to control citrus huanglongbing (HLB) caused by ' Candidatus Liberibacter asiaticus' (CLas) via foliar spray. Here, we studied the residue dynamics of STR and its effect on CLas titers in planta applied by foliar spray and trunk injection of 3-year-old citrus trees that were naturally infected by CLas in the field. After foliar spray, STR levels in leaves peaked at 2 to 7 days postapplication (dpa) and gradually declined thereafter. The STR spray did not significantly affect CLas titers in leaves of treated plants as determined by quantitative PCR. After trunk injection, peak levels of STR were observed 7 to 14 dpa in the leaf and root tissues, and near-peak levels were sustained for another 14 days before significantly declining. At 12 months after injection, moderate to low or undetectable levels of STR were observed in the leaf, root, and fruit, depending on the doses of STR injected, with a residue level of 0.28 µg/g in harvested fruit at the highest injection concentration of 2.0 µg/tree. CLas titers in leaves were significantly reduced by trunk injection of STR at 1.0 or 2.0 g/tree, starting from 7 dpa and throughout the experimental period. The reduction of CLas titers was positively correlated with STR residue levels in leaves. The in planta minimum effective concentration of STR needed to suppress the CLas titer to an undetectable level (cycle threshold ≥36.0) was 1.92 µg/g fresh weight. Determination of the in planta minimum effective concentration of STR against CLas and its spatiotemporal residue levels in planta provides the guidance to use STR for HLB management.

' Candidatus Liberibacter asiaticus' (CLas) is the predominant causal agent of citrus huanglongbing, the most devastating citrus disease worldwide. CLas colonizes phloem tissue and causes phloem dysfunction. The pathogen population size in local tissues and in the whole plant is critical for the development of disease symptoms by determining the load of pathogenicity factors and metabolic burden to the host. However, the total population size of CLas in a whole plant and the ratio of CLas to citrus cells in local tissues have not been addressed previously. The total CLas population size for 2.5-year-old 'Valencia' sweet orange on 'Kuharske' citrange rootstock trees was quantified using quantitative PCR to be approximately 1.74 × 10 9 cells/tree, whereas 7- and 20-year-old sweet orange trees were estimated to be 4.3 × 10 10 cells/tree, and 6.0 × 10 10 cells/tree, respectively. The majority of CLas cells were distributed in leaf tissues (55.58%), followed by those in branch (36.78%), feeder root (4.75%), trunk (2.39%), and structural root (0.51%) tissues. The ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were within approximately 39 to 79, 44 to 124, 153 to 1,355, 191 to 1,054, and 561 to 3,760, respectively, representing the metabolic burden of CLas in different organs. It was estimated that the ratios of phloem cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples are approximately 0.39 to 0.79, 0.44 to 1.24, 1.53 to 13.55, 1.91 to 10.54, and 5.61 to 37.60, respectively. Approximately 0.01% of the total citrus phloem volume was estimated to be occupied by CLas, explaining the difficulty to observe CLas in most tissues under transmission electron microscopy. The CLas titer inside the leaf was estimated to be approximately 1.64 × 10 6 cells/leaf or 9.2 × 10 4 cells cm -2 in leaves, approximately 10 4 times less than that of typical apoplastic bacterial pathogens. This study provides quantitative estimates of phloem colonization by bacterial pathogens and furthers the understanding of the biology and virulence mechanisms of CLas.

' Candidatus Liberibacter asiaticus' (CLas), the causal agent of citrus huanglongbing (HLB), colonizes inside the phloem and is naturally transmitted by the Asian citrus psyllid (ACP). Here, we investigated spatiotemporal CLas colonization in different tissues after ACP transmission. Of the nine plants successfully infected via ACP transmission, CLas was detected in the roots of all trees at 75 days postremoval of ACPs (DPR) but in the mature leaf of only one tree; this finding is consistent with the model that CLas moves passively from source to sink tissues. At 75 and 365 DPR, CLas was detected in 11.1 and 43.1% of mature leaves not fed on by ACPs during transmission, respectively, unveiling active movement to the source tissue. The difference in colonization timing of sink and source tissues indicates that CLas is capable of both passive and active movement, with passive movement being dominant. At 225 DPR, leaves fed on by ACPs during the young stage showed the highest ratio of HLB symptomatic leaves and the highest CLas titer, followed by leaves that emerged after ACP removal and mature leaves not fed on by ACPs. Importantly, our data showed that ACPs were unable to transmit CLas via feeding on mature leaves. It is estimated that it takes 3 years at most for CLas to infect the whole tree. Overall, spatiotemporal detection of CLas in different tissues after ACP transmission helps visualize the infection process of CLas in planta and subsequent HLB symptom development and provides evidence showing that young leaves should be the focus of HLB management.

Poplar trees ( Populus spp.) are important and are widely grown worldwide. However, the extensive occurrence of leaf rust disease caused by Melampsora spp. seriously inhibits their growth and reduces their biomass. In our previous study, a high-quality genetic map was constructed for the poplar F1 population I-69 × XYY by using next-generation sequencing-based genotyping-by-sequencing. Here, we collected phenotypic data on leaf rust disease resistance on three different dates for all 300 progenies of the F1 population. Combining a high-quality genetic map and phenotypic data, we were able to detect 11 major quantitative trait loci (QTLs) for leaf rust disease resistance. Among these 11 QTLs, two pairs were detected on at least two dates. In the corresponding genomic sequence, we found that resistance ( R ) gene clusters were located in these two QTL regions. By using genome resequencing, PCR confirmation and statistical analysis, a 611-bp deletion within an R gene in one QTL region was found to be associated with variation in leaf rust disease resistance. A PCR-based examination of this 611-bp deletion was performed. This 611-bp deletion was also found to affect mRNA splicing and form a new protein with the loss of some key protein domains. Based on this study, we were able to determine the genetic architecture of variation in poplar leaf rust disease resistance, and the 611-bp deletion in the R gene could be used as a diagnostic marker for future poplar molecular breeding.

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.

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by ' Candidatus Liberibacter asiaticus' and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of ' Ca. L. asiaticus' inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of ' Ca. L. asiaticus'. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after ' Ca. L. asiaticus' transmission by ACP. In this study, we report a new method for targeted early detection of ' Ca. L. asiaticus' in cultivar Valencia sweet orange ( Citrus sinensis ) before HLB symptom expression. We take advantage of the fact that ' Ca. L. asiaticus' remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect ' Ca. L. asiaticus' in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting ' Ca. L. asiaticus' inoculum removal and preventing HLB from spreading.

Citrus Huanglongbing (HLB), also known as greening, is a destructive disease caused by the fastidious, phloem-colonizing bacteria Candidatus Liberibacter spp.; 'Ca. Liberibacter asiaticus' (Las) is the most prevalent of the species causing HLB. The Asian citrus psyllid (ACP, Diaphorina citri) transmits Las. HLB is threatening citrus production worldwide, and there is no cure for infected trees. Management strategies targeting diseased trees at different stages of colonization by Las are needed for sustainable citrus production in HLB-endemic regions. We evaluated the effect of the combinations of plant defense elicitors, nitrogen (N) fertilizer, and compost on mildly diseased trees. We tested thermotherapy on severely diseased trees and assessed tree protectors to prevent feeding by ACP, thus preventing Las from being transmitted to new plantings that replaced HLB-moribund trees. After four applications over two consecutive growing seasons we found that the combination of compost, urea, and plant defense elicitors β-aminobutyric acid, plus ascorbic acid and potassium phosphite with or without salicylic acid, slowed down the progression of HLB and reduced disease severity by approximately 18%, compared with the untreated control. Our data showed no decline in fruit yield, indeed treatment resulted in a higher yield compared with the untreated control. Thermotherapy treatment (55°C for 2 min) exhibited a suppressive effect on growth of Las and progress of HLB in severely diseased trees for 2 to 3 months after treatment. The tree protectors prevented feeding by ACP, and therefore young replant trees remained healthy and free from infection by Las over the 2-year duration of the experiment. Taken together, these results may contribute to a basis for developing a targeted approach to control HLB based on stage of host colonization, application of plant defense elicitors, N fertilizer, compost, thermotherapy, and tree protectors. There is potential to implement these strategies in conjunction with other disease control measures to contribute to sustainable citrus production in HLB-endemic regions.

Citrus canker, caused by Xanthomonas citri subsp. citri, is an important citrus disease that causes significant economic losses worldwide. All commercial citrus varieties are susceptible to citrus canker. Currently, chemical control with copper based products is the main approach to control X. citri subsp. citri dispersal and plant colonization. However, extensive use of copper compounds can result in copper-resistant strains and cause adverse effects on the environment. Alternatives to chemical control involve the activation of citrus immunity to control the disease. Here, we investigated the ability of multiple rhizobacteria to induce a systemic defense response in cultivar Duncan grapefruit. Burkholderia territorii strain A63, Burkholderia metallica strain A53, and Pseudomonas geniculata strain 95 were found to effectively activate plant defense and significantly reduce symptom development in leaves challenged with X. citri subsp. citri. In the priming phase, root application of P. geniculata induced the expression of salicylic acid (SA)-signaling pathway marker genes (PR1, PR2, PR5, and salicylic acid carboxyl methyltransferase [SAM-SACM]). Gene expression analyses after X. citri subsp. citri challenge showed that root inoculation with P. geniculata strain 95 increased the relative levels of phenylalanine ammonia lyase 1 and SAM-SACM, two genes involved in the phenylpropanoid pathway as well as the biosynthesis of SA and methyl salicylate (MeSA), respectively. However, hormone analyses by UPLC-MS/MS showed no significant difference between SA in P. geniculata-treated plants and control plants at 8 days post-beneficial bacteria root inoculation. Moreover, P. geniculata root-treated plants contained higher reactive oxygen species levels in aerial tissues than control plants 8 days post-treatment application. This study demonstrates that rhizobacteria can modulate citrus immunity resulting in a systemic defense response against X. citri subsp. citri under greenhouse conditions.

The Huanglongbing (HLB) disease pyramid is composed of Liberibacters, psyllid vectors, citrus hosts, and the environment. The epidemiological outcomes for Liberibacter-associated plant diseases are collectively determined by the inherent relationships among plant-Liberibacters-psyllids, and how various environmental factors affect plant-Liberibacter-psyllid interactions. Citrus-Liberibacter-psyllid interactions occur in a complex microbiome system. In this review, we focus on the progress in understanding the HLB disease pyramid, and how the microbiome affects the HLB disease pyramid including the interaction between HLB and the citrus microbiome; the interaction between Liberibacters and psyllids; the interaction between Liberibacters and gut microbiota in psyllids; and the effect of HLB on selected above- and belowground citrus pathogens. Their implications for HLB management are also discussed.

Citrus huanglongbing (HLB) or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing 'Candidatus Liberibacter asiaticus' (Las) in Florida. In this study, we evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected 'Hamlin' sweet orange trees on 'Swingle' citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by high-performance liquid chromatography method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root system was achieved 2 days postinjection. High levels of OTC (>850 µg/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 µg/kg) persisted for more than 9 months. Reduction of Las populations in root system and leaves of OTC-treated trees were over 95% and 99% (i.e., 1.76 and 2.19 log reduction) between 2 and 28 days postinjection. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB.

The advent of genomics has advanced science into a new era, providing a plethora of "toys" for researchers in many related and disparate fields. Genomics has also spawned many new fields, including proteomics and metabolomics, furthering our ability to gain a more comprehensive view of individual organisms and of interacting organisms. Genomic information of both bacterial pathogens and their hosts has provided the critical starting point in understanding the molecular bases of how pathogens disrupt host cells to cause disease. In addition, knowledge of the complete genome sequence of the pathogen provides a potentially broad slate of targets for the development of novel virulence inhibitors that are desperately needed for disease management. Regarding plant bacterial pathogens and disease management, the potential for utilizing genomics resources in the development of durable resistance is enhanced because of developing technologies that enable targeted modification of the host. Here, we summarize the role of genomics studies in furthering efforts to manage bacterial plant diseases and highlight novel genomics-enabled strategies heading down this path.

Citrus canker, caused by Xanthomonas citri subsp. citri, is a devastating disease of most commercial citrus varieties. In our previous study, we analyzed the transcriptional response of 'Valencia' sweet orange to X. citri subsp. citri wild-type and pthA4 mutant infection at 48 h postinoculation (hpi). Using microarray analysis, two PthA4 targets, CsLOB1 and CsSWEET1, were identified. We have shown that PthA4 binds to the effector binding element (EBE) of CsLOB1 and activates gene expression of this susceptibility gene. However, how PthA4 modulates host genes at different stages of infection remains to be determined. In this study, we compared the transcriptional profiles between citrus leaf tissue inoculated with Xcc306 and those inoculated with a pthA4-deletion mutant strain (Xcc306∆pthA4) at 6, 48, and 120 hpi. At both 48 and 120 hpi, the PthA4-mediated infection significantly upregulated expression of a variety of genes involved in cell-wall degradation and modification, DNA packaging, G-protein, protein synthesis, sucrose metabolism, and cell division functions, while the downregulated genes were mainly enriched in photosynthesis, transport, secondary metabolism, cytochrome P450, and various plant defense-associated mechanisms. To validate microarray results, gene expression of 26 genes representing genes associated with cell-wall-associated, immunity system, and carbohydrate metabolism was confirmed using quantitative reverse-transcription polymerase chain reaction. Expression patterns of these genes at 48 and 120 hpi were consistent with the microarray results. We also identified putative EBE for PthA4 (EBEPthA4) in the promoter regions of multiple genes upregulated by PthA4, to which PthA4 might bind directly to control their gene expression. Our study provided a dynamic picture of citrus genes regulated by PthA4 during the X. citri subsp. citri infection of citrus leaves at different stages. This study will be useful in further understanding the virulence mechanism of X. citri subsp. citri and identifying potential targets of PthA4.

Huanglongbing (HLB) is currently the most economically devastating disease of citrus worldwide and no established cure is available. Defense inducing compounds are able to induce plant resistance effective against various pathogens. In this study the effects of various chemical inducers on HLB diseased citrus were evaluated in four groves (three with sweet orange and one with mandarin) in Florida (United States) for two to four consecutive growing seasons. Results have demonstrated that plant defense inducers including β-aminobutyric acid (BABA), 2,1,3-benzothiadiazole (BTH), and 2,6-dichloroisonicotinic acid (INA), individually or in combination, were effective in suppressing progress of HLB disease. Ascorbic acid (AA) and the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DDG) also exhibited positive control effects on HLB. After three or four applications for each season, the treatments AA (60 to 600 µM), BABA (0.2 to 1.0 mM), BTH (1.0 mM), INA (0.1 mM), 2-DDG (100 µM), BABA (1.0 mM) plus BTH (1.0 mM), BTH (1.0 mM) plus AA (600 µM), and BTH (1.0 mM) plus 2-DDG (100 µM) slowed down the population growth in planta of 'Candidatus Liberibacter asiaticus', the putative pathogen of HLB and reduced HLB disease severity by approximately 15 to 30% compared with the nontreated control, depending on the age and initial HLB severity of infected trees. These treatments also conferred positive effect on fruit yield and quality. Altogether, these findings indicate that plant defense inducers may be a useful strategy for the management of citrus HLB.

Citrus canker caused by the bacterium Xanthomonas citri subsp. citri is an economically important disease of citrus worldwide. Biofilm formation plays an important role in early infection of X. citri subsp. citri on host leaves. In this study, we assessed the hypothesis that small molecules inhibiting biofilm formation reduce X. citri subsp. citri infection and enhance the control of citrus canker disease. D-leucine and 3-indolylacetonitrile (IAN) were found to prevent biofilm formation by X. citri subsp. citri on different abiotic surfaces and host leaves at a concentration lower than the minimum inhibitory concentration (MIC). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis indicated that IAN repressed expression of chemotaxis/motility-related genes in X. citri subsp. citri. In laboratory experiments, planktonic and biofilm cells of X. citri subsp. citri treated with D-leucine and IAN, either alone or in combination, were more susceptible to copper (CuSO4) than those untreated. In greenhouse assays, D-leucine and IAN applied alone or combined with copper reduced both the number of canker lesions and bacterial populations of X. citri subsp. citri on citrus host leaves. This study provides the basis for the use of foliar-applied biofilm inhibitors for the control of citrus canker alone or combined with copper-based bactericides.

Citrus huanglongbing (HLB) is one of the oldest citrus diseases and has been known for over a century. HLB is caused by 'Candidatus Liberibacter' spp. that are phloem-limited, fastidious α-proteobacteria and infect hosts in different Kingdoms (i.e., Animalia and Plantae). When compared with well-characterized, cultivatable plant-pathogenic Gram-negative bacteria, the interactions of uncultured insect-vectored plant-pathogenic bacteria, including 'Ca. Liberibacter' spp., with their hosts remain poorly understood. 'Ca. Liberibacter' spp. have been known to cause HLB, which has been rapidly spreading worldwide, resulting in dramatic economic losses. HLB presents an unprecedented challenge to citrus production. In this review, we focus on the most recent research on citrus, 'Candidatus Liberibacter asiaticus', and psyllid interactions, specifically considering the following topics: evolutionary relationships among 'Ca. Liberibacter' spp., genetic diversity, host range, genome analysis, transmission, virulence mechanisms, and the ecological importance of HLB. Currently, no efficient management strategy is available to control HLB, although some promising progress has been made. Further studies are needed to understand citrus, 'Ca. L. asiaticus', and psyllid interactions to design innovative management strategies. Although HLB has been problematic for over a century, we can only win the battle against HLB with a coordinated and deliberate effort by the citrus industry, citrus growers, researchers, legislatures, and governments.

ABSTRACT Xylella fastidiosa regulates traits important to both virulence of grape as well as colonization of sharpshooter vectors via its production of a fatty acid signal molecule known as DSF whose production is dependent on rpfF. Although X. fastidiosa rpfF mutants exhibit increased virulence to plants, they are unable to be spread from plant to plant by insect vectors. To gain more insight into the traits that contribute to these processes, a whole-genome Agilent DNA microarray for this species was developed and used to determine the RpfF-dependent regulon by transcriptional profiling. In total, 446 protein coding genes whose expression was significantly different between the wild type and an rpfF mutant (false discovery rate < 0.05) were identified when cells were grown in PW liquid medium. Among them, 165 genes were downregulated in the rpfF mutant compared with the wild-type strain whereas 281 genes were over-expressed. RpfF function was required for regulation of 11 regulatory and σ factors, including rpfE, yybA, PD1177, glnB, rpfG, PD0954, PD0199, PD2050, colR, rpoH, and rpoD. In general, RpfF is required for regulation of genes involved in attachment and biofilm formation, enhancing expression of hemagglutinin genes hxfA and hxfB, and suppressing most type IV pili and gum genes. A large number of other RpfF-dependent genes that might contribute to virulence or insect colonization were also identified such as those encoding hemolysin and colicin V, as well as genes with unknown functions.

Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium 'Candidatus Liberibacter spp.' In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 33,879 expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB-affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since 'Ca. Liberibacter asiaticus' does not form aggregate in citrus. The up-regulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic alpha-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of 'Candidatus Liberibacter asiaticus,' respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that 'Ca. Liberibacter asiaticus' was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/mug of total DNA in different tissues. A relatively high concentration of 'Ca. Liberibacter asiaticus' was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that 'Ca. Liberibacter asiaticus' was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.