The in Planta Effective Concentration of Oxytetracycline Against ‘Candidatus Liberibacter asiaticus’ for Suppression of Citrus Huanglongbing

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.

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Residue dynamics of streptomycin in citrus delivered by foliar spray and trunk injection and effect on ‘Candidatus Liberibacter asiaticus’ titer

Phytopathology ◽

10.1094/phyto-09-20-0427-r ◽

2020 ◽

Author(s):

Jinyun Li ◽

Vladimir Kolbasov ◽

Donghwan Lee ◽

zhiqian pang ◽

Huang Yixiao ◽

...

Keyword(s):

Foliar Spray ◽

Experimental Period ◽

Effective Concentration ◽

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Minimum Effective Concentration ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Trunk Injection ◽

Residue Levels

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. Following foliar spray, STR levels in leaves peaked at 2 to 7 days post-application (DPA) and gradually declined thereafter. The STR spray did not significantly affect CLas titers in leaves of treated plants. Following 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 g/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 required to suppress the CLas titer to an undetectable level (Ct ≥ 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.

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Probing the application of OmpA-derived peptides to disrupt the acquisition of ‘Candidatus Liberibacter asiaticus’ by Diaphorina citri

Phytopathology ◽

10.1094/phyto-06-21-0252-fi ◽

2021 ◽

Author(s):

Marcus Vinicius Merfa e Silva ◽

Eduarda Regina Fischer ◽

Mariana de Souza e Silva ◽

Carolina Sardinha Francisco ◽

Helvécio Coletta-Filho ◽

...

Keyword(s):

Control Strategies ◽

Insect Vector ◽

Diaphorina Citri ◽

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Host Interaction ◽

Vector Population ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Bacterial Outer Membrane

Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) and ‘Ca. Liberibacter americanus’ (CLam) are associated with HLB in Brazil, but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid – ACP) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison to CLas, suggesting a possible role on host-interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides aiming to evaluate the acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5 and Pep6 in artificial diet significantly reduced the acquisition of CLas, while increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas and sweet orange plants stably absorbed and maintained this peptide for as long as three months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.

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Sec-Delivered Effector 1 (SDE1) of ‘Candidatus Liberibacter asiaticus’ Promotes Citrus Huanglongbing

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-20-0123-r ◽

2020 ◽

Vol 33 (12) ◽

pp. 1394-1404

Author(s):

Kelley J. Clark ◽

Zhiqian Pang ◽

Jessica Trinh ◽

Nian Wang ◽

Wenbo Ma

Keyword(s):

Cysteine Proteases ◽

Citrus Paradisi ◽

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Altered Expression ◽

Model Plant Arabidopsis Thaliana ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Transgenic Citrus

Sec-delivered effector 1 (SDE1) from the huanglongbing (HLB)-associated bacterium ‘Candidatus Liberibacter asiaticus’ was previously characterized as an inhibitor of defense-related, papain-like cysteine proteases in vitro and in planta. Here, we investigated the contributions of SDE1 to HLB progression. We found that SDE1 expression in the model plant Arabidopsis thaliana caused severe yellowing in mature leaves, reminiscent of both ‘Ca. L. asiaticus’ infection symptoms and accelerated leaf senescence. Induction of senescence signatures was also observed in the SDE1-expressing A. thaliana lines. These signatures were apparent in older leaves but not in seedlings, suggesting an age-associated effect. Furthermore, independent lines of transgenic Citrus paradisi (L.) Macfadyen (Duncan grapefruit) that express SDE1 exhibited hypersusceptibility to ‘Ca. L. asiaticus’. Similar to A. thaliana, transgenic citrus expressing SDE1 showed altered expression of senescence-associated genes, but only after infection with ‘Ca. L. asiaticus’. These findings suggest that SDE1 is a virulence factor that contributes to HLB progression, likely by inducing premature or accelerated senescence in citrus. This work provides new insight into HLB pathogenesis. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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In Planta Distribution of ‘Candidatus Liberibacter asiaticus’ as Revealed by Polymerase Chain Reaction (PCR) and Real-Time PCR

Phytopathology ◽

10.1094/phyto-98-5-0592 ◽

2008 ◽

Vol 98 (5) ◽

pp. 592-599 ◽

Cited By ~ 151

Author(s):

Satyanarayana Tatineni ◽

Uma Shankar Sagaram ◽

Siddarame Gowda ◽

Cecile J. Robertson ◽

William O. Dawson ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Citrus Tree ◽

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Chain Reaction ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Polymerase Chain

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic α-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/μg 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.

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Spatiotemporal dynamics of Candidatus Liberibacter asiaticus colonization inside citrus plant and Huanglongbing disease development

Phytopathology ◽

10.1094/phyto-09-20-0407-r ◽

2020 ◽

Author(s):

Sheo Shankar Pandey ◽

Fernanda N.C. Vasconcelos ◽

Nian Wang

Keyword(s):

Passive Movement ◽

Infection Process ◽

Active Movement ◽

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Mature Leaves ◽

Young Leaves ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Different Tissues

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

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The flagella of ‘ Candidatus Liberibacter asiaticus’ and its movement in planta

Molecular Plant Pathology ◽

10.1111/mpp.12884 ◽

2019 ◽

Vol 21 (1) ◽

pp. 109-123 ◽

Cited By ~ 2

Author(s):

Maxuel O. Andrade ◽

Zhiqian Pang ◽

Diann S. Achor ◽

Han Wang ◽

Tingshan Yao ◽

...

Keyword(s):

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus

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Identification of a chromosomal deletion mutation and the dynamics of two major populations of Candidatus Liberibacter asiaticus in its hosts

Phytopathology ◽

10.1094/phyto-08-21-0325-fi ◽

2021 ◽

Author(s):

Cheryl Armstrong ◽

Lijuang Zhou ◽

Weiqi Luo ◽

Ozgur Batuman ◽

Olfemi Alabi ◽

...

Keyword(s):

Mixed Population ◽

Open Reading Frames ◽

Genetic Element ◽

Candidatus Liberibacter Asiaticus ◽

Wild Type ◽

In Planta ◽

Depth Study ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Genome Comparisons

Candidatus Liberibacter asiaticus (Las) is the prominent species of Liberibacter associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of a ~8.3 kb DNA region of the Las genome containing eight putative open reading frames (ORFs) flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild-type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild-type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild-type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild-type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts while an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host.

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A Secreted ‘Candidatus Liberibacter asiaticus’ Peroxiredoxin Simultaneously Suppresses Both Localized and Systemic Innate Immune Responses In Planta

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-03-18-0068-r ◽

2018 ◽

Vol 31 (12) ◽

pp. 1312-1322 ◽

Cited By ~ 12

Author(s):

Mukesh Jain ◽

Alejandra Munoz-Bodnar ◽

Shujian Zhang ◽

Dean W. Gabriel

Keyword(s):

Plant Defense ◽

Transcriptional Activation ◽

Lipid Membranes ◽

Transcript Abundance ◽

Housekeeping Gene ◽

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Hypersensitive Cell Death ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus

The oxidative (H2O2) burst is a seminal feature of the basal plant defense response to attempted pathogen invasions. In ‘Candidatus Liberibacter asiaticus’ UF506, expression of the SC2 prophage-encoded secreted peroxidase (F489_gp15) increases bacterial fitness and delays symptom progression in citrus. Two chromosomal 1-Cys peroxiredoxin genes, CLIBASIA_RS00940 (Lasprx5) and CLIBASIA_RS00445 (Lasbcp), are conserved among all sequenced ‘Ca. L. asiaticus’ strains, including those lacking prophages. Both LasBCP and LasdPrx5 have only a single conserved peroxidatic Cys (CP/SH) and lack the resolving Cys (CR/SH). Lasprx5 appeared to be a housekeeping gene with similar moderate transcript abundance in both ‘Ca. L. asiaticus’–infected psyllids and citrus. By contrast, Lasbcp was expressed only in planta, similar to the expression of the SC2 peroxidase. Since ‘Ca. L. asiaticus’ is uncultured, Lasbcp and Lasprx5 were functionally validated in a cultured surrogate species, Liberibacter crescens, and both genes significantly increased oxidative stress tolerance and cell viability in culture. LasBCP was nonclassically secreted and, in L. crescens, conferred 214-fold more resistance to tert-butyl hydroperoxide (tBOOH) than wild type. Transient overexpression of Lasbcp in tobacco suppressed H2O2-mediated transcriptional activation of RbohB, the key gatekeeper of the systemic plant defense signaling cascade. Lasbcp expression did not interfere with the perception of ‘Ca. L. asiaticus’ flagellin (flg22Las) but interrupted the downstream activation of RbohB and stereotypical deposition of callose in tobacco. Critically, LasBCP also protected against tBOOH-induced peroxidative degradation of lipid membranes in planta, preventing subsequent accumulation of antimicrobial oxylipins that can also trigger the localized hypersensitive cell death response.

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UAV color images for determination of citrus plant parameters

The Journal of Agriculture of the University of Puerto Rico ◽

10.46429/jaupr.v103i2.18226 ◽

2019 ◽

pp. 141-153

Author(s):

Sunil K. Mathanker ◽

Consuelo Estévez de Jensen ◽

Andrés M. Pagán-López ◽

Luis R. Pérez-Alegría

Keyword(s):

Puerto Rico ◽

Color Images ◽

Citrus Greening ◽

Effective Control ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Plant ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Supplemental Nutrients ◽

Citrus Spp

Unmanned aerial vehicles (UAVs) or drones are being studied for many agricultural applications. One application is plant phenotyping to reduce the time and effort required in collecting field data. This study aims to explore the use of a UAV, 4K-color camera and a commercial image analysis service to measure citrus plant parameters that are important to a crop scientist or grower with limited technical background and resources. Citrus spp. are important crops in Puerto Rico and the United States. Currently, the citrus industry is struggling to contain the devastating effects of citrus greening or Huanglongbing disease. The disease is associated with a phloem-limited bacteria, Candidatus Liberibacter asiaticus (CLAs), vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. The use of insecticides for vector control is the primary strategy used in nurseries and orchards. However, once the citrus plant is infected, there is no effective control available for the disease. In Puerto Rico this disease has reduced Citrus spp. yields by more than 50%; studies are underway to find effective control measures such as supplemental nutrients, vector management practices, planting disease-free vegetative material and protective screen structures. An experiment at the Fortuna Agricultural Experiment Substation, in Juana Díaz, Puerto Rico, was conducted to address the challenges posed by citrus greening. The experiment was established in a four-year-old grove of Tahiti lime (Citrus latifolia Tan.) on Cleopatra mandarin (Citrus reshni hort. ex Tanaka), naturally infected with Candidatus Liberibacter asiaticus. The experiment was arranged in a randomized complete block design with four replicates and three treatments: supplemental nutrients, supplemental nutrients + salicylic acid, and granular fertilization. Tree growth parameters were measured, and laboratory analyses were carried out to determine nutrient levels and disease severity levels from the leaf samples. The color camera, on board the UAV, was employed to acquire images of the experimental plot. Drone Deploy application was used for planning the UAV flights and image analysis. Field-measured plant height and canopy diameter compared well with the parameters determined from the color images. The average errors in measuring canopy diameter (14.5%) and plant height (22.4%) could be considered within an acceptable range, especially for comparing different treatments or crop varieties. However, the average errors in measuring canopy volume (47.5%) were high and can be considered unacceptable. It appears that the assumed conical shape of the trees could be one of the main reasons, besides the algorithms used in calculating plant volume, and built-in inaccuracies of the single frequency GPS (global positioning system) used in estimating altitude. Further studies could help in reducing errors and exploring other applications. The method used can be of importance in evaluating fruit trees.

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