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

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.

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

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.

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

2019 ◽  
Vol 109 (12) ◽  
pp. 2046-2054 ◽  
Author(s):  
Jinyun Li ◽  
Zhiqian Pang ◽  
Shuo Duan ◽  
Donghwan Lee ◽  
Vladimir G. Kolbasov ◽  
...  
Keyword(s):  
Field Experiments ◽  
Foliar Spray ◽  
Effective Control ◽  
Candidatus Liberibacter Asiaticus ◽  
In Planta ◽  
Fresh Tissue ◽  
Minimum Concentration ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Residue Levels

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.

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

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 .

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

2008 ◽  
Vol 98 (5) ◽  
pp. 592-599 ◽  
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.

Temporal Dynamics of Candidatus Liberibacter asiaticus Titer in Mature Leaves from Citrus sinensis cv Valencia Are Associated with Vegetative Growth

2019 ◽  
Vol 113 (2) ◽  
pp. 589-595 ◽  
Author(s):  
Freddy Ibanez ◽  
Lukasz L Stelinski
Keyword(s):  
Vegetative Growth ◽  
Citrus Sinensis ◽  
Temporal Dynamics ◽  
Critical Role ◽  
Insect Vector ◽  
In Planta ◽  
Mature Leaves ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
The Impact

Abstract Huanglongbing, a highly destructive disease of citrus species, is associated with a fastidious, gram-negative, phloem-limited bacteria (Candidatus Liberibacter spp.). In Florida, the causative agent of Huanglongbing (HLB) is C. Liberibacter asiaticus (CLas) and it is transmitted by the insect vector, Asian citrus psyllid (Diaphorina citri Kuwayama). Previous investigations have revealed systemic infection of CLas with an erratic and uneven distribution of pathogen in tree phloem. However, previous investigations did not consider the potential impact of plant vegetative growth on presence/absence of CLas in planta. Our objectives were to determine: 1) the effect of vegetative growth of Citrus sinensis (L.) Osbeck cv Valencia on detection of CLas in mature leaves, and 2) the impact of CLas inoculation frequency on progression of CLas titer in citrus leaves through the first year of infection. Temporal dynamics of CLas detection were associated with vegetative flush growth. Surprisingly, there was no difference in CLas titer detected between plants exposed to infected vectors for a one-time 7 d inoculation access period, as compared with plants exposed to continuously breeding CLas-infected insects over the course of an entire year of plant infection. Our results suggest that the CLas bacterium is transported through phloem during annual movement of carbon compounds needed for vegetative plant growth, including transportation from roots to mature leaves. These results highlight the importance of vegetative growth on temporal dynamics of CLas in citrus, and suggest a critical role of the sink-source interaction on presence/absence of CLas in leaves.

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

2019 ◽  
Vol 21 (1) ◽  
pp. 109-123 ◽  
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

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

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.

A protocol for successful transmission of ‘Candidatus Liberibacter asiaticus’ from citrus to citrus using Diaphorina citri

2021 ◽  
Author(s):  
Silvio Aparecido Lopes ◽  
Juan Camilo Cifuentes-Arenas
Keyword(s):  
Ambient Air ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Mature Leaves ◽  
Air Temperatures ◽  
Tree Canopies ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Time Required ◽  
Transmission Studies

A protocol to successfully transmit the huanglongbing (HLB) pathogen, ‘Candidatus Liberibacter asiaticus’ (Las), from citrus-to-citrus plants using the Asian citrus psyllid (ACP), and an alternative way to help growers control ACP are proposed. Best results were obtained when pathogen acquisition by adults reared on fully symptomatic Las-positive plants, latency, and inoculation, occurred at ambient air temperatures ranging from 24 to 28°C, and by confining single infective adult ACP for seven days on soft newly developing vegetative shoots (stages v2 to v4). No infection resulted from confinement of infective ACP adults on mature leaves (stage v6). Under the described conditions, single ACP adults could successfully transmit Las to an average of 56.5% (35 to 83%) of plantlets with v2 to v4 shoots growing in 0.3 L tubes, and to 80.5% (76 to 86%) of plants with v2 to v4 shoots growing in 4.7 L pots. The use of single insects and plantlets reduces labor, space and other resources required to undertake transmission tests. It also reduces time required for transmission studies and should help accelerate research on HLB. The results were used to develop an index for favorability to infection (IFI) to determine orchard vulnerabilities to Las. The IFI is based on the heterogeneous population of new shoots that occurs on tree canopies and may offer alternative or complementary alternatives to the laborious and costly insect surveys currently used in most instances to determine threshold levels for insecticide applications.

scholarly journals Improvement in the Excised Citrus Leaf Assay to Investigate Inoculation of ‘Candidatus Liberibacter asiaticus’ by the Asian Citrus Psyllid Diaphorina citri

Plant Disease ◽  
2017 ◽  
Vol 101 (3) ◽  
pp. 409-413 ◽  
Author(s):  
Laudecir L. Raiol-Junior ◽  
Ana D. B. Baia ◽  
Fernanda Q. B. F. Luiz ◽  
Camila G. Fassini ◽  
Viviani V. Marques ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Leaf Age ◽  
Infection Process ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Leaf Petiole ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Leaves ◽  
Time Required

Huanglongbing (HLB) is a difficult-to-control and highly destructive citrus disease that, in Brazil, is associated mainly with the bacterium ‘Candidatus Liberibacter asiaticus’ transmitted by the psyllid Diaphorina citri. The aim of this study was to improve our understanding of the ‘Ca. L. asiaticus’ infection process by exposing excised, fully expanded, immature citrus leaves in 50-ml Falcon tubes to one, four, or eight adults from a ‘Ca. L. asiaticus’-exposed colony for 1-, 3-, 7-, or 15-day periods for access to inoculation (IAP). The leaves were incubated at 26°C for 1, 3, 7, 15, and 21 days (incubation period [IP]). Infection frequencies and ‘Ca. L. asiaticus’ titers were assessed by quantitative polymerase chain reaction (qPCR). ‘Ca. L. asiaticus’ infection was a function of leaf age, number of insects, IAP, and IP. In general, higher infection rates were observed on younger leaves inoculated with higher numbers of insects and after longer IAP and IP. The immature excised leaf method allowed determination of 3 to 7 days as the range of time required by ‘Ca. L. asiaticus’ to reach qPCR detectable levels. Even though leaf survival could be prolonged by the maintenance of a branch segment at the base of the leaf petiole, leaf degradation, visible after about 15 days IP, did not allow observation of the entire infection process which, in the intact plant, culminates with the appearance of the blotch mottling symptom on leaf blades.

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

2018 ◽  
Vol 31 (12) ◽  
pp. 1312-1322 ◽  
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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