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Phloem-limited bacterial " Candidatus Liberibacter" species are associated with incurable plant diseases worldwide. Antimicrobial treatments for these pathogens are challenging due to the difficulty of reaching the vascular tissue they occupy at bactericidal concentrations. Here, in vitro antimicrobial mechanisms of Zinkicide TMN110 (ZnK), a nonphytotoxic zinc oxide (ZnO)-based nanoformulation, were compared to those of bulk ZnO (b-ZnO) using as a model the only culturable species of the genus, Liberibacter crescens Minimum bactericidal concentration (MBC) determination and time-kill assays showed that ZnK has a bactericidal effect against L. crescens , whereas b-ZnO is bacteriostatic. When ZnK was used at the MBC (150 ppm), its antimicrobial mechanisms included an increase in Zn solubility, generation of intracellular reactive oxygen species, lipid peroxidation, and cell membrane disruption; all of these were of greater intensity than those of b-ZnO. Inhibition of biofilms, which are important during insect vector colonization, was stronger by ZnK than by b-ZnO at concentrations between 2.5 and 10 ppm in batch cultures; however, neither ZnK nor b-ZnO removed L. crescens preformed biofilms when applied between 100 and 400 ppm. In microfluidic chambers simulating source-to-sink phloem movement, ZnK significantly outperformed b-ZnO in Zn mobilization and bactericidal activity against L. crescens planktonic cells in sink reservoirs. In microfluidic chamber assays assessing antibiofilm activity, ZnK displayed a significantly enhanced bactericidal activity against L. crescens individual attached cells as well as preformed biofilms compared to that of b-ZnO. The superior mobility and antimicrobial activity of ZnK in microenvironments make this formulation a promising product to control plant diseases caused by " Candidatus Liberibacter" species and other plant vascular pathogens. IMPORTANCE " Candidatus Liberibacter" species are associated with incurable plant diseases that have caused billions of dollars of losses for United States and world agriculture. Chemical control of these pathogens is complicated, because their life cycle combines intracellular vascular stages in plant hosts with transmission by highly mobile insect vectors. To date, " Candidatus Liberibacter" species are mostly unculturable, except for Liberibacter crescens , a member of the genus that has been used as a model for in vitro assays. Here, we evaluated the potential of Zinkicide (ZnK) as an antimicrobial against " Candidatus Liberibacter" species in batch cultures and under flow conditions, using L. crescens as a biological model. ZnK displayed bactericidal activity against L. crescens in batch cultures and showed increased mobility and bactericidal activity in microfluidic devices resembling " Candidatus Liberibacter" species natural habitats. ZnK performance observed here against L. crescens makes this compound a promising candidate to control plant diseases caused by vascular pathogens., (Copyright © 2020 American Society for Microbiology.)

Citrus greening, also called Huanglongbing (HLB), is one of the most destructive citrus diseases worldwide. It is caused by the fastidious gram-negative α-proteobacteria bacterium Candidatus Liberibacter asiaticus (CLas) and vectored by the Asian citrus psyllid (ACP), Diaphorina citri. Currently, there is no cure for HLB, no compounds have been successful in controlling HLB, and no sustainable management practices have been established for the disease. Thus, searching for alternative citrus greening disease mitigation strategies is considered an urgent priority for a sustainable citrus industry. The aim of this study was to use compounds extracted from oak, Quercus hemisphaerica, and to assess the antibacterial effects of these against CLas-infected citrus plants. The application of aqueous oak leaf extracts showed substantial inhibitory effects against CLas in citrus plants and the activity of genes related to starch. Significant differences were also observed in plant phenotypic and physiological traits after treatments. Citrus plants treated with oak extracts displayed an increase in stomatal conductance, chlorophyll content and nutrient uptake concurrently with a reduction of CLas titer, when compared to citrus plants treated with just water. The information provided from this study suggests a new management treatment program to effectively deal with the HLB disease., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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.

Huanglongbing (HLB), also known as citrus greening, is a bacterial disease that poses a devastating threat to the citrus industry worldwide. To manage this disease efficiently, we developed and characterized a ternary aqueous solution (TSOL) that contains zinc nitrate, urea, and hydrogen peroxide. We report that TSOL exhibits better antimicrobial activity than commercial bactericides for growers. X-ray fluorescence analysis demonstrates that zinc is delivered to citrus leaves, where the bacteria reside. FTIR and Raman spectroscopy, molecular dynamics simulations, and density functional theory calculations elucidate the solution structure of TSOL and reveal a water-mediated interaction between Zn 2+ and H 2 O 2 , which may facilitate the generation of highly reactive hydroxyl radicals contributing to superior antimicrobial activity of TSOL. Our results not only suggest TSOL as a potent antimicrobial agent to suppress bacterial growth in HLB-infected trees, but also provide a structure-property relationship that explains the superior performance of TSOL.

Ternary solution (TSOL) is a novel Zn chelate-based systemic antimicrobial formulation designed for treating citrus bacterial pathogens 'Candidatus Liberibacter asiaticus' and Xanthomonas citri subsp. citri. TSOL is a component of MS3T, a novel multifunctional surface/sub-surface/systemic therapeutic formulation. Antimicrobial activity of TSOL was compared with the antimicrobial compound ZnO against X. citri subsp. citri and 'Ca. L. asiaticus' surrogate Liberibacter crescens in batch cultures. X. citri subsp. citri and L. crescens were also introduced into microfluidic chambers, and the inhibitory action of TSOL against biofilm formation was evaluated. The minimum inhibitory concentration of TSOL for both X. citri subsp. citri and L. crescens was 40ppm. TSOL was bactericidal to X. citri subsp. citri and L. crescens above 150 ppm and 200 ppm, respectively. On the contrary, ZnO was more effective as a bactericidal agent against L. crescens than X. citri subsp. citri. TSOL was more effective in controlling growth and biofilm formation of X. citri subsp. citri in batch cultures compared to ZnO. Time-lapse video imaging microscopy showed that biofilm formation of X. citri subsp. citri was inhibited in microfluidic chambers treated with 60 ppm TSOL. TSOL also inhibited further growth of already formed X. citri subsp. citri and L. crescens biofilms in microfluidic chambers. Leaf spraying of TSOL showed higher plant uptake and systemic movement in citrus (Citrus reshni) plants compared to that of ZnO, suggesting that TSOL is a promising antimicrobial compound to control vascular plant pathogens such as 'Ca. L. asiaticus'., Competing Interests: The authors have declared that no competing interests exist.

Huanglongbing (HLB, also known as citrus greening) is considered to be the most devastating disease that has significantly damaged the citrus industry globally. HLB is caused by the Candidatus Liberibacter asiaticus (CLas), the fastidious phloem-restricted gram-negative bacterium, vectored by the asian citrus psyllid. To date, there is no effective control available against CLas. To alleviate the effects of HLB on the industry and protect citrus farmers, there is an urgent need to identify or develop inhibitor molecules to suppress or eradicate CLas from infected citrus plant. In this paper, we demonstrate for the first time an in planta efficacy of two antimicrobial compounds against CLas viz. 2S albumin (a plant based protein; ~12.5 kDa), Nano-Zinc Oxide (Nano-ZnO; ~ 4.0 nm diameter) and their combinations. Aqueous formulations of these compounds were trunk-injected to HLB affected Mosambi plants (Citrus sinensis) grafted on 3-year old rough lemon (C. jambhiri) rootstock with known CLas titer maintained inside an insect-free screen house. The effective concentration of 2S albumin (330 ppm) coupled with the Nano-ZnO (330 ppm) at 1:1 ratio was used. The dynamics of CLas pathogen load of treated Mosambi plants was assessed using TaqMan-qPCR assay every 30 days after treatment (DAT) and monitored till 120 days. We observed that 2S albumin-Nano-ZnO formulation performed the best among all the treatments decreasing CLas population by 96.2%, 97.6%, 95.6%, and 97% of the initial bacterial load (per 12.5 ng of genomic DNA) at 30, 60, 90, and 120 DAT, respectively. Our studies demonstrated the potency of 2S albumin-Nano-ZnO formulation as an antimicrobial treatment for suppressing CLas in planta and could potentially be developed as a novel anti CLas therapeutics to mitigate the HLB severity affecting the citrus industry worldwide., Competing Interests: The authors have declared that no competing interests exist.

Huanglongbing (HLB) is one of the most destructive citrus plant diseases worldwide. It is associated with the fastidious phloem-limited α-proteobacteria 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' and 'Ca. Liberibacter americanus'. In recent years, HLB-associated Liberibacters have extended to North and South America. The causal agents of HLB have been putatively identified, and their transmission pathways and worldwide population structure have been extensively studied. However, very little is known about the epidemiologic relationships of Ca. L. asiaticus, which has limited the scope of HLB research and especially the development of control strategies. HLB-affected plants produce damaged fruits and die within several years. To control the disease, scientists have developed new compounds and screened existing compounds for their antibiotic and antimicrobial activities against the disease. These compounds, however, have very little or even no effect on the disease. The aim of the present review was to compile and compare different methods of HLB disease control with newly developed integrative strategies. In light of recent studies, we also describe how to control the vectors of this disease and the biological control of other citrus plant pathogens. This work could steer the attention of scientists towards integrative control strategies.

LdtR is a master regulator of gene expression in Liberibacter asiaticus, one of the causative agents of citrus greening disease. LdtR belongs to the MarR-family of transcriptional regulators and it has been linked to the regulation of more than 180 genes in Liberibacter species, most of them gathered in the following Clusters of Orthologous Groups: cell motility, cell wall envelope, energy production, and transcription. Our previous transcriptomic evidence suggested that LdtR is directly involved in the modulation of the zinc uptake system genes (znu) in the closely related L. crescens. In this report, we show that LdtR is involved in the regulation of one of the two encoded zinc uptake mechanisms in L. asiaticus, named znu2. We also show that LdtR binds zinc with higher affinity than benzbromarone, a synthetic effector inhibitory molecule, resulting in the disruption of the LdtR:promoter interactions. Using site-directed mutagenesis, electrophoretic mobility shift assays (EMSAs), and isothermal titration calorimetry, we identified that residues C28 and T43 in LdtR, located in close proximity to the Benz1 pocket, are involved in the interaction with zinc. These results provided new evidence of a high-affinity effector molecule targeting a key player in L. asiaticus' physiology and complemented our previous findings about the mechanisms of signal transduction in members of the MarR-family.

Huanglongbing (HLB; "citrus greening" disease) has caused significant damages to the global citrus industry as it has become well established in leading citrus-producing regions and continues to spread worldwide. Insecticidal control has been a critical component of HLB disease management, as there is a direct relationship between vector control and Candidatus Liberibacter spp. (i.e., the HLB pathogen) titer in HLB-infected citrus trees. In recent years, there have been substantial efforts to develop practical strategies for specifically managing Ca. Liberibacter spp.; however, a literature review on the outcomes of such attempts is still lacking. This work summarizes the greenhouse and field studies that have documented the effects and implications of chemical-based treatments (i.e., applications of broad-spectrum antibiotics, small molecule compounds) and nonchemical measures (i.e., applications of plant-beneficial compounds, applications of inorganic fertilizers, biological control, thermotherapy) for phytopathogen control. The ongoing challenges associated with mitigating Ca. Liberibacter spp. populations at the field-scale, such as the seasonality of the phytopathogen and associated HLB disease symptoms, limitations for therapeutics to contact the phytopathogen in planta, adverse impacts of broad-spectrum treatments on plant-beneficial microbiota, and potential implications on public and ecosystem health, are also discussed.

Citrus huanglongbing (HLB) or greening is a devastating disease of citrus worldwide and no effective control measure is currently available. Plant defense activators environmentally friendly compounds capable of inducing resistance against many plant pathogens. Earlier studies showed that foliar spray of plant defense inducers could slow down HLB disease progress. In this study, eight plant defense activators and three antibiotics were evaluated in three field trials for their effect to control HLB by trunk injection of young and mature sweet orange trees. Results showed that four trunk injections of several activators, including salicylic acid, oxalic acid, acibenzolar-S-methyl, and potassium phosphate, provided significant control of HLB by suppressing 'Candidatus Liberibacter asiaticus' titer and disease progress. Trunk injection of penicillin, streptomycin, and oxytetracycline hydrochloride resulted in excellent control of HLB. In general, antibiotics were more effective in reduction of 'Ca. L. asiaticus' titer and HLB symptom expressions than plant defense activators. These treatments also resulted in increased yield and better fruit quality. Injection of both salicylic acid and acibenzolar-S-methyl led to significant induction of pathogenesis-related (PR) genes PR-1 and PR-2 genes. Meanwhile, injection of either potassium phosphate or oxalic acid resulted in significant induction of PR-2 or PR-15 gene expression, respectively. These results suggested that HLB diseased trees remained inducible for systemic acquired resistance under field conditions. In summary, this study presents information regarding controlling HLB via trunk injection of plant defense activators and antibiotics, which helps citrus growers in decision making regarding developing an effective HLB management program.

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.

Aims: Huanglongbing (HLB)-affected citrus often display zinc deficiency symptoms. In this study, supplemental zinc was applied to citrus to determine its effect on Candidatus Liberibacter asiaticus (Las) titre, HLB symptoms, and leaf microbiome., Methods and Results: HLB-affected citrus were treated with various amounts of zinc. The treatments promoted Las growth and affected microbiomes in citrus leaves. Phylochip(™) -based results indicated that 5475 of over 50 000 known Operational Taxonomic Units (OTUs) in 52 phyla were detected in the midribs of HLB-affected citrus, of which Proteobacteria was the most abundant, followed by Firmicutes and Actinobacteria. In comparison, the microbiomes of zinc-treated diseased plants had overall more OTUs with higher amounts of Proteobacteria, but decreased percentages of Firmicutes and Actinobacteria. In addition, more OTUs of siderophore-producing bacteria were present. Only zinc-sensitive Staphylococcaceae had higher OTU's in the diseased plants without zinc treatments., Conclusions: Although HLB-affected citrus appear zinc deficient, zinc amendments increased the pathogen levels and shifted the microbiome., Significance and Impact of the Study: HLB is currently the most devastating disease of citrus worldwide. Zinc is often applied to HLB-affected citrus due to zinc deficiency symptoms. This study provided new insights into the potential effects of zinc on HLB and the microbial ecology of citrus., (© 2016 The Society for Applied Microbiology.)

Plant pathogens can manipulate the odor of their host; the odor of an infected plant is often attractive to the plant pathogen vector. It has been suggested that this odor-mediated manipulation attracts vectors and may contribute to spread of disease; however, this requires further broad demonstration among vector-pathogen systems. In addition, disruption of this indirect chemical communication between the pathogen and the vector has not been attempted. We present a model that demonstrates how a phytophathogen (Candidatus Liberibacter asiaticus) can increase its spread by indirectly manipulating the behavior of its vector (Asian citrus psyllid, Diaphorina citri Kuwayama). The model indicates that when vectors are attracted to pathogen-infected hosts, the proportion of infected vectors increases, as well as, the proportion of infected hosts. Additionally, the peak of infected host populations occurs earlier as compared with controls. These changes in disease dynamics were more important during scenarios with higher vector mortality. Subsequently, we conducted a series of experiments to disrupt the behavior of the Asian citrus psyllid. To do so, we exposed the vector to methyl salicylate, the major compound released following host infection with the pathogen. We observed that during exposure or after pre-exposure to methyl salicylate, the host preference can be altered; indeed, the Asian citrus psyllids were unable to select infected hosts over uninfected counterparts. We suggest mechanisms to explain these interactions and potential applications of disrupting herbivore host preference with plant volatiles for sustainable management of insect vectors.

Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, 'Candidatus Liberibacter asiaticus'. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced in leaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.

In this study, we developed and validated a method for the extraction, identification, and quantitation of penicillin G and its metabolites (penilloic acid and penillic acid) in a variety of citrus fruits by employing sequential liquid/liquid and solid-phase extraction techniques in conjunction with UHPLC-MS/MS. Two product ion transitions per analyte were required for identification, which contributes to a high degree of selectivity. Corrected recoveries of penicillin G using an isotopically labeled internal standard were 90-100% at fortification levels of 0.1, 0.25, 1, and 10 ng/g. Absolute recoveries for penillic acid and penilloic acid were 50-75% depending on the matrix used. The limit of detection (LOD) of penicillin G and its metabolites was found to be 0.1 ng/g when 2 g of citrus was extracted. This method is useful in determining residue levels of penicillin G and its metabolites in citrus trees infected with huanglongbing bacteria after antibiotic treatment.

By 2014, huanglongbing (HLB), the most destructive disease of citrus, and its insect vector, the Asian citrus psyllid (ACP), Diaphorina citri (Kuwayama), became established in all major citrus-growing regions of the world, including the United States, with the exception of California. At present, application of insecticides is the most widely followed option for reducing ACP populations, while application of antibiotics for suppressing HLB disease/symptoms is being practiced in some citrus-growing regions. Application of insecticides during the dormant winter season, along with cultivation of HLB-free seedlings and early detection and removal of symptomatic and asymptomatic trees, has been very effective in managing ACP. Area-wide management of ACP by application of insecticides at low volume in large areas of citrus cultivation has been shown to be effective in managing HLB and reducing management costs. As insecticide resistance is a major problem in sustainable management of ACP, rotation/alternation of insecticides with different chemistries and modes of action needs to be followed. Besides control of the insect vector, use of antibiotics has temporarily suppressed the symptoms of HLB in diseased trees. Recent efforts to discover and screen existing as well as new compounds for their antibiotic and antimicrobial activities have identified some promising molecules for HLB control. There is an urgent need to find a sustainable solution to the HLB menace through chemical control of ACP populations and within HLB-infected trees through the judicious use of labeled insecticides (existing and novel chemistries) and antibiotics in area-wide management programs with due consideration to the insecticide resistance problem., (© 2014 Society of Chemical Industry.)

Huanglongbing (HLB) is the most devastating disease of citrus. The global citrus industry is in urgent need of effective chemical treatments for HLB control because of its rapid spreading worldwide. Due to the fastidious nature of the pathogens, and the poor permissibility of citrus leaf surfaces, effective screening of chemicals for the HLB control can be challenging. In this study, we developed a graft-based chemotherapy method to rapidly screen potential HLB-controlling chemical compounds. In addition, we improved transmission efficiency by using the best HLB-affected scion-rootstock combination, and demonstrated the HLB bacterial titer was the critical factor in transmission. The HLB-affected lemon scions had a high titer of HLB bacterium, survival rate (83.3%), and pathogen transmission rate (59.9%). Trifoliate, a widely used commercial rootstock, had the highest survival rate (>70.0%) compared with grapefruit (52.6%) and sour orange (50.4%). Using this method, we confirmed a mixture of penicillin and streptomycin was the most effective compounds in eliminating the HLB bacterium from the HLB-affected scions, and in successfully rescuing severely HLB-affected citrus germplasms. These findings are useful not only for chemical treatments but also for graft-based transmission studies in HLB and other Liberibacter diseases.

Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide and is threatening the survival of the Floridian citrus industry. Currently, there is no established cure for this century-old and emerging disease. As a possible control strategy for citrus HLB, therapeutic compounds were screened using a propagation test system with 'Candidatus Liberibacter asiaticus'-infected periwinkle and citrus plants. The results demonstrated that the combination of penicillin and streptomycin (PS) was effective in eliminating or suppressing the 'Ca. L. asiaticus' bacterium and provided a therapeutically effective level of control for a much longer period of time than when administering either antibiotic separately. When treated with the PS, 'Ca. L. asiaticus'-infected periwinkle cuttings achieved 70% of regeneration rates versus <50% by other treatments. The 'Ca. L. asiaticus' bacterial titers in the infected periwinkle plants, as measured by quantitative real-time polymerase chain reaction, decreased significantly following root soaking or foliar spraying with PS. Application of the PS via trunk injection or root soaking also eliminated or suppressed the 'Ca. L. asiaticus' bacterium in the HLB-affected citrus plants. This may provide a useful tool for the management of citrus HLB and other Liberibacter-associated diseases.

A new medium designated Liber A has been designed and used to successfully cultivate all three 'Candidatus Liberibacter spp.,' the suspect causative agents of huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of 'Ca. Liberibacter spp.' for four or five single-colony transfers before viability declined. Colonies, positive for 'Ca. L. asiaticus' by a 16s-based rDNA real-time polymerase chain reaction (RT-PCR) assay and sequencing, were irregular-shaped, convex, and 0.1 to 0.3 mm after 3 to 4 days. Suspect 'Ca. L. asiaticus' and 'Ca. L. americanus' cells were observed in infected tissue and on agar culture by scanning electron microscopy. The cells were ovoid to rod shaped, 0.3 to 0.4 by 0.5 to 2.0 microm, often with fimbriae-like appendages. Two strains of 'Ca. L. asiaticus' and one of 'Ca. L. americanus' grown on Liber A medium were pathogenic on citrus and could be isolated from noninoculated tissues of inoculated trees and seedlings 9 and 2 months later, respectively. The identity was confirmed by RT-PCR and 16s rDNA sequencing. This is the first report of the cultivation and pathogenicity of 'Ca. L. asiaticus' and 'Ca. L. americanus' associated with symptoms of HLB.