Publications

4498

‘Candidatus Liberibacter’ species are associated with severe, economically important diseases. Nearly all known species are putatively insect transmitted, specifically by psyllids. Detection of ‘Ca. Liberibacter’ in plants is complicated by their uneven distribution in host plants and largely fastidius nature. The death of black (Fraxinus nigra) and mancana (Fraxinus mandshurica) ash trees in Saskatchewan, Canada has been associated with infestation by the cottony ash psyllid (Psyllopsis discrepans). A combination of conventional PCR amplification and Sanger sequencing of the 16S recombinant DNA was used to detect and identify ‘Ca. Liberibacter’ in psyllids collected from ash trees in Saskatchewan. BLAST analysis of two 16S sequences that were 1,058 and 1,085 bp long (NTHA 5, GenBank accession number MK942379 and NTHA 6, GenBank accession number MK937570, respectively) revealed they were 99 to 100% similar to a ‘Ca. Liberibacter solanacearum’ sequence (GenBank accession number KX197200) isolated from the Nearctic psyllid (Bactericera maculipennis) of U.S. provenance. Sequencing the psyllid genes CO1 and Cyt-b confirmed that the psyllids from which the bacterial DNA was isolated were P. discrepans, based on comparisons with sequences in GenBank and BOLD and a reference sample from the United Kingdom. These results provide the first evidence that ‘Ca. Liberibacter solanacearum’ species are associated with psyllids collected from ash trees and specifically P. discrepans. The recent episodes of dieback of ash in Saskatchewan associated with psyllid feeding are consistent with disease symptoms caused by ‘Ca. Liberibacter’ pathogens, and this possibility warrants further study.

‘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 an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames 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, whereas 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.

‘Candidatus Liberibacter asiaticus’ (CLas), the devastating pathogen related to Huanglongbing (HLB), is a phloem-limited, fastidious, insect-borne bacterium. Rapid spread of HLB disease relies on CLas-efficient propagation in the vector, the Asian citrus psyllid Diaphorina citri, in a circulative manner. Understanding the intracellular lifecycle of CLas in psyllid midgut, the major organ for CLas transmission, is fundamental to improving current management strategies. Using a microscopic approach within CLas-infected insect midgut, we observed the entry of CLas into gut cells inside vesicles, termed Liberibacter-containing vacuoles (LCVs), by endocytosis. Endocytosis is followed by the formation of endoplasmic reticulum-related and replication permissive vacuoles (rLCVs). Additionally, we observed the formation of double membrane autophagosome-like structure, termed autophagy-related vacuole (aLCV). Vesicles containing CLas egress from aLCV and fuse with the cell membrane. Immunolocalization studies showed that CLas uses endocytosis- and exocytosis-like mechanisms that mediates bacterial invasion and egress. Upregulation of autophagy-related genes indicated subversion of host autophagy by CLas in psyllid vector to promote infection. These results indicate that CLas interacts with host cellular machineries to undergo a multistage intracellular cycle through endocytic, secretory, autophagic, and exocytic pathways via complex machineries. Potential tactics for HLB control can be made depending on further investigations on the knowledge of the molecular mechanisms of CLas intracellular cycle.

It has been nearly 100 years since citrus growers in two distinct regions in the northern provinces of South Africa noticed unusual symptoms in their citrus trees, causing significant crop losses. They had no idea that these symptoms would later become part of an almost global pandemic of a disease called greening or huanglongbing (HLB). The rapid spread of the disease indicated that it might be caused by a transmissible pathogen, but it took >50 years to identify the causative agent as ‘Candidatus Liberibacter africanus’. Recently, the disease appeared in more African countries, spreading by both infected planting material and Trioza erytreae. To date, five ‘Ca. L. africanus’ subspecies have been identified in various rutaceous species, with ‘Ca. L. africanus subsp. clausenae’ the only subspecies for which a biovar was detected in citrus. Efforts to detect and differentiate HLB-causing Liberibacter species are ongoing, and recent developments are discussed here. This review focuses on aspects of the African form of HLB, including its specific bacterial species and subspecies, its main insect vector, its geographic distribution, and current management strategies.

The devastating citrus disease huanglongbing (HLB) associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) has caused a more than 70% reduction in citrus production since its discovery in Florida in 2005. Most citrus scion cultivars are sensitive to HLB, whereas some cultivars used as rootstocks are tolerant. Using such tolerant rootstocks can help trees to cope better with the disease’s impact. Evaluating rootstock effects on a grafted scion in the field takes many years, but shorter-term evaluation is imperative to aid in rootstock selection for an HLB-endemic production environment. In this study, we investigated grafted healthy and CLas-infected citrus trees under controlled greenhouse conditions. The objectives were to identify traits suitable for assessing grafted tree tolerance in advance of longer-term field studies and aiding in the selection of superior rootstock cultivars. We assessed 10 commercially important rootstocks grafted with ‘Valencia’ sweet orange scion and with known field performance. At 6, 9, 15, and 21 months after graft inoculation (mai), leaf CLas titers were determined and canopy health was evaluated. Plants were destructively sampled at 21 mai to assess plant biomasses and other physiological and horticultural variables. There was little influence of the rootstock cultivar on CLas titers. Surprisingly, few HLB foliar disease symptoms and no differences in soluble and nonsoluble carbohydrate concentrations were measured in infected compared with healthy plants, despite high CLas titers and significant reductions in plant biomasses. Most trees on rootstocks with trifoliate orange parentage were less damaged by HLB than other rootstocks, although results did not always agree with reported field performance. Among the different variables measured, leaf size appeared to be most predictive for grafted tree assessment of HLB sensitivity. The results of this study provide a better understanding of the strengths and weaknesses of assessing rootstock influence on grafted tree performance in a controlled greenhouse environment. Although such studies provide valuable information for cultivar tolerance to HLB, other rootstock traits will ultimately contribute to field survival and productivity in an HLB endemic production environment.

The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, ‘Candidatus Liberibacter asiaticus’ (CLas), which is associated with citrus greening disease. The citrus relative Murraya paniculata (orange jasmine) is a host plant of D. citri but is more resistant to CLas compared with all tested Citrus genotypes. The effect of host switching of D. citri between Citrus medica (citron) and M. paniculata plants on the acquisition and transmission of CLas was investigated. The psyllid CLas titer and the proportion of CLas-infected psyllids decreased in the generations after transfer from CLas-infected citron to healthy M. paniculata plants. Furthermore, after several generations of feeding on M. paniculata, pathogen acquisition (20 to 40% reduction) and transmission rates (15 to 20% reduction) in psyllids transferred to CLas-infected citron were reduced compared with psyllids continually maintained on infected citron. Top-down (difference gel electrophoresis) and bottom-up (shotgun MS/MS) proteomics methods were used to identify changes in D. citri protein expression resulting from host plant switching between Citrus macrophylla and M. paniculata. Changes in expression of insect metabolism, immunity, and cytoskeleton proteins were associated with host plant switching. Both transient and sustained feeding on M. paniculata induced distinct patterns of protein expression in D. citri compared with psyllids reared on C. macrophylla. The results point to complex interactions that affect vector competence and may lead to strategies to control the spread of citrus greening disease.

‘Candidatus Liberibacter asiaticus’ (Las) is an emergent bacterial pathogen that is associated with the devastating citrus huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful, and dual-transcriptome analyses aiming to profile gene expression in both Las and its hosts have a low coverage of the Las genome because of the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, a lack of understanding of the Las transcriptome remains a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited high expression in citrus include transporters, ferritin, outer membrane porins, specific pilins, and genes involved in phage-related functions, cell wall modification, and stress responses. We also found 106 genes to be differentially expressed in citrus versus Asian citrus psyllids. Genes related to transcription or translation and resilience to host defense response were upregulated in citrus, whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. Finally, we determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

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 probably regulated by a cell density-dependent mechanism, because 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 early as 3 days after inoculation, although no sustained exponential growth was observed under any tested condition. Unfortunately, cultures were only transient, because CLas lost viability over time; nevertheless, we obtained populations of about 105genome 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 toward the long-sought goal of culturing CLas axenically.