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

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.

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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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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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Susceptibility of Sixteen Citrus Genotypes to ‘Candidatus Liberibacter asiaticus’

Plant Disease ◽

10.1094/pdis-08-15-0940-re ◽

2016 ◽

Vol 100 (6) ◽

pp. 1080-1086 ◽

Cited By ~ 10

Author(s):

Greg McCollum ◽

Mark Hilf ◽

Mike Irey ◽

Weiqi Luo ◽

Tim Gottwald

Keyword(s):

The United States ◽

Insect Vector ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Germplasm ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Florida Citrus ◽

Citrus Macrophylla ◽

Free Ranging

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and ‘Ca. L. asiaticus’ inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of ‘Ca. L. asiaticus’ infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to ‘Ca. L. asiaticus’, with 100% infection by the end of the test period in three trials, while the complex genetic hybrids ‘US 1-4-59’ and ‘Fallglo’ consistently were least susceptible, with approximately 30% infection. Results obtained in this greenhouse experiment showed good agreement with trends observed in the orchard, supporting the validity of our approach for screening citrus germplasm for susceptibility to HLB.

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

Phytopathology ◽

10.1094/phyto-06-19-0198-r ◽

2019 ◽

Vol 109 (12) ◽

pp. 2046-2054 ◽

Cited By ~ 10

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.

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Metabolome and Microbiome Signatures in the Roots of Citrus Affected by Huanglongbing

Phytopathology ◽

10.1094/phyto-03-19-0103-r ◽

2019 ◽

Vol 109 (12) ◽

pp. 2022-2032 ◽

Cited By ~ 3

Author(s):

Emily M. T. Padhi ◽

Nilesh Maharaj ◽

Shin-Yi Lin ◽

Darya O. Mishchuk ◽

Elizabeth Chin ◽

...

Keyword(s):

High Throughput Sequencing ◽

Water Soluble ◽

Resonance Spectroscopy ◽

Its Gene ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Citrus Leaves ◽

Citrus Varieties ◽

And Control ◽

The Impact

Huanglongbing (HLB) is a severe, incurable citrus disease caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas). Although citrus leaves serve as the site of initial infection, CLas is known to migrate to and colonize the root system; however, little is known about the impact of CLas infection on root metabolism and resident microbial communities. Scions of ‘Lisbon’ lemon and ‘Washington Navel’ orange grafted onto ‘Carrizo’ rootstock were grafted with either CLas-infected citrus budwood or uninfected budwood. Roots were obtained from trees 46 weeks after grafting and analyzed via 1H nuclear magnetic resonance spectroscopy to identify water-soluble root metabolites and high-throughput sequencing of 16S rRNA and ITS gene amplicons to determine the relative abundance of bacterial and fungal taxa in the root rhizosphere and endosphere. In both citrus varieties, 27 metabolites were identified, of which several were significantly different between CLas(+) and control plants. CLas infection also appeared to alter the microbial community structure near and inside the roots of citrus plants. Nonmetric multidimensional scaling (NMDS) and a principal coordinate analysis (PCoA) revealed distinct metabolite and microbial profiles, demonstrating that CLas impacts the root metabolome and microbiome in a manner that is variety-specific.

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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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Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing

Metabolites ◽

10.3390/metabo10120477 ◽

2020 ◽

Vol 10 (12) ◽

pp. 477

Author(s):

Nabil Killiny ◽

Shelley E. Jones ◽

Faraj Hijaz ◽

Abdelaziz Kishk ◽

Yulica Santos-Ortega ◽

...

Keyword(s):

Leaf Morphology ◽

Bacterial Pathogen ◽

Gas Chromatography Mass Spectrometry ◽

Insect Vector ◽

Diaphorina Citri ◽

Metabolomic Analysis ◽

Citrus Industry ◽

Candidatus Liberibacter ◽

Citrus Maxima ◽

Liberibacter Asiaticus

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen “Candidatus Liberibacter asiaticus” and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

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