scholarly journals An in vitro protocol for rapidly assessing the effects of antimicrobial compounds on the unculturable bacterial plant pathogen, Candidatus Liberibacter asiaticus

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Joseph Krystel ◽  
Qingchun Shi ◽  
Jefferson Shaw ◽  
Goutam Gupta ◽  
David Hall ◽  
...  
Keyword(s):  
Plant Pathogen ◽  
Antimicrobial Compounds ◽  
Candidatus Liberibacter Asiaticus ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Bacterial Plant Pathogen

scholarly journals Viability of ‘Candidatus Liberibacter asiaticus’ Prolonged by Addition of Citrus Juice to Culture Medium

2014 ◽  
Vol 104 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Jennifer K. Parker ◽  
Sarah R. Wisotsky ◽  
Evan G. Johnson ◽  
Faraj M. Hijaz ◽  
Nabil Killiny ◽  
...  
Keyword(s):  
Grapefruit Juice ◽  
Culture Medium ◽  
Quantitative Polymerase Chain Reaction ◽  
Chemical Characterization ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Greening Disease ◽  
Candidatus Liberibacter ◽  
The Media ◽  
Liberibacter Asiaticus

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’. Infection with ‘Ca. L. asiaticus’ is incurable; therefore, knowledge regarding ‘Ca. L. asiaticus’ biology and pathogenesis is essential to develop a treatment. However, ‘Ca. L. asiaticus’ cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of ‘Ca. L. asiaticus’ in vitro, ‘Ca. L. asiaticus’ inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima ‘Mato Buntan’) was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air–liquid interface of juice cultures contained ‘Ca. L. asiaticus’ cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining ‘Ca. L. asiaticus’ viability in vitro, which will contribute to future development of a culture medium for ‘Ca. L. asiaticus’.

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 .

Growth of ‘Candidatus Liberibacter asiaticus’ in commercial grapefruit juice-based media formulations reveals common cell density-dependent transient behaviors

2021 ◽  
Author(s):  
Marcus Vinicius Merfa e Silva ◽  
Eber Naranjo ◽  
Deepak Shantharaj ◽  
Leonardo De La Fuente
Keyword(s):  
Cell Density ◽  
Grapefruit Juice ◽  
Ex Vivo ◽  
Post Inoculation ◽  
Candidatus Liberibacter Asiaticus ◽  
Density Dependent ◽  
Wide Range ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

The phloem-restricted, insect-transmitted bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with Huanglongbing (HLB), the most devastating disease of citrus worldwide. The inability to culture CLas impairs the understanding of its virulence mechanisms and the development of effective management strategies to control this incurable disease. Previously, our research group used commercial grapefruit juice (GJ) to prolong the viability of CLas in vitro. In the present study, GJ was amended with a wide range of compounds and incubated under different conditions to optimize CLas growth. Remarkably, results showed that CLas growth ratios were inversely proportional to the initial inoculum concentration. This correlation is likely regulated by a cell density-dependent mechanism, since diluting samples between subcultures allowed CLas to resume growth. Moreover, strategies to reduce the cell density of CLas, such as subculturing at short intervals and incubating samples under flow conditions, allowed this bacterium to multiply and reach maximum growth as fast as 3 days post inoculation, although no sustained exponential growth was observed under any tested condition. Unfortunately, cultures were only transient, since CLas lost viability over time; nevertheless, we obtained populations of 105 genome equivalents/ml repeatedly. Finally, we established an ex vivo system to grow CLas within periwinkle calli that could be used to propagate bacterial inoculum in the lab. In this study we determined the influence of a comprehensive set of conditions and compounds on CLas growth in culture. We hope our results will help guide future efforts towards the long-sought goal of culturing CLas axenically.

scholarly journals Molecular signatures between citrus and Candidatus Liberibacter asiaticus

2021 ◽  
Vol 17 (12) ◽  
pp. e1010071
Author(s):  
Bin Hu ◽  
Muhammad Junaid Rao ◽  
Xiuxin Deng ◽  
Sheo Shankar Pandey ◽  
Connor Hendrich ◽  
...  
Keyword(s):  
Genomic Research ◽  
Molecular Signatures ◽  
Mechanistic Studies ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Diseases ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Prevalent Strain ◽  
Liberibacter Asiaticus

Citrus Huanglongbing (HLB), also known as citrus greening, is one of the most devastating citrus diseases worldwide. Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain associated with HLB, which is yet to be cultured in vitro. None of the commercial citrus cultivars are resistant to HLB. The pathosystem of Ca. Liberibacter is complex and remains a mystery. In this review, we focus on the recent progress in genomic research on the pathogen, the interaction of host and CLas, and the influence of CLas infection on the transcripts, proteins, and metabolism of the host. We have also focused on the identification of candidate genes for CLas pathogenicity or the improvements of HLB tolerance in citrus. In the end, we propose potentially promising areas for mechanistic studies of CLas pathogenicity, defense regulators, and genetic improvement for HLB tolerance/resistance in the future.

scholarly journals Restructuring citrus endophytic diversity through potential indigenous endophytes could eliminate huanglongbing pathogen Candidatus Liberibacter asiaticus

2020 ◽  
Author(s):  
Shahzad Munir ◽  
Yongmei Li ◽  
Pengfei He ◽  
Pengbo He ◽  
Pengjie He ◽  
...  
Keyword(s):  
Large Scale ◽  
Pathogen Resistance ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Grove ◽  
Fold Reduction ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
The Impact ◽  
Leaf Midrib

Abstract Background Huanglongbing (HLB) is a major botanical pandemic of citrus crops caused by Candidatus Liberibacter asiaticus (Clas). It is important to understand the different mechanisms involved in interaction of pathogen with plants to develop novel management strategy against HLB. However, until now there has been no control strategy to manage this disease in vitro and on large scale in citrus grove. We found that, indigenous endophyte Bacillus subtilis L1-21, a patented strain isolated from healthy citrus tree, may have the potential to reduce the impact of pathogen through restructuring of core endophytes. Results A novel half-leaf method was developed to test the efficacy of B. subtilis L1-21 against Clas. Concentration of B. subtilis L1-21 at 104 cfu ml− 1 resulted in a 1000-fold reduction in Clas copy densities per gram of leaf midrib (107 to 104) by 4 d after treatment. With endophytes, where HLB incidence was reduced to < 3% and Clas copy density was reduced from 109 to 104 pathogen g− 1 of diseased leaf midrib. We found that 16 of 93 tree samples became Clas-free and functional pathways and pathogen resistance genes were regulated in diseased citrus trees after treatment. Conclusions This is the first large-scale study using an indigenous endophyte and shows its potential utility in sustainable disease management through strengthening the citrus microbiome.

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