Publications

4527

‘Candidatus Liberibacter asiaticus’ is associated with the devastating citrus disease Huanglongbing (HLB). It is transmitted by grafting infected material to healthy plants and by the feeding of the Asian citrus psyllid (Diaphorina citri). Previously, we demonstrated that a metabolomics approach using proton-nuclear magnetic resonance spectroscopy discriminates healthy from diseased plants via grafting. This work assessed the capability of this technology in discriminating healthy and diseased plants when the bacterium is vectored by psyllids. One-year-old greenhouse-grown ‘Lisbon’ lemon trees were exposed to either carrier psyllids (exposed, n = 10), or psyllids that were free of ‘Candidatus Liberibacter asiaticus’ (control, n = 6). Leaf metabolites were tracked for 1 year and disease diagnosis was made using quantitative PCR. Overall, 31 water-soluble metabolites were quantified in leaves, including four sugars and 12 amino acids. Analysis via nonmetric multidimensional scaling and principal component analysis revealed significant differences between the leaf metabolome of control versus infected trees beginning at 8 weeks postexposure, including alterations in glucose and quinic acid concentrations. These findings provide a longitudinal overview of the metabolic effects of HLB during the early phases of disease, and confirm previous experimental work demonstrating that infection elicits changes in the leaf metabolome that enables discrimination between healthy and infected plants. Here we demonstrate that the mode of inoculation (i.e., graft versus psyllid) results in a similar pathology.

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.

Objective: Coxiella burnetii and Coxiella-like endosymbionts (CLEs) have been widely discovered in various ticks, animals, and even human beings. To estimate the possible origin of C. burnetii and its relatives CLEs, the prevalence of C. burnetii and CLEs has been intensively surveyed all over the world. Method: In the present study, the possible infection of C. burnetii and CLEs in host-seeking Haemaphysalis concinna was performed with meta-transcript analysis with tick specimens harvested from Mudanjiang City, Heilongjiang province, China. The meta-transcript results were subsequently confirmed by the specific sequence of partial 16S rRNA. Results: A total of three arrays of gene transcripts were harvested, including pyrophosphate-fructose 6-phosphate 1-phosphotransferase-eda-thiol-disulfide isomerase and thioredoxin-greA, carB-carA-DnaJ-DnaK-grpE-ppnk, ropC-ropB, and ubiA-non-canonical purine NTP pyrophosphatase-hemK-prfA, which suggest the infection of Candidatus Coxiella mudorwiae in H. concinna. The high identity of the 16S rRNA gene of Candidatus C. mudorwiae achieved in our study strongly supports our meta-transcripts analysis. Conclusion: The prevalence of Candidatus C. mudorwiae in hard ticks has been discovered in China. More detailed surveys are imperative to clarify the emergence of CLEs and their implication in the epidemiologic characteristics of Q fever.

A large (47.75 ± 3.56 µm in diameter) Thiovulum bacterial strain forming white veils is described from a marine mangrove ecosystem. High sulfide concentrations (up to 8 mM of H2S) were measured on sunken organic matter (wood/bone debris) under laboratory conditions. This sulfur-oxidizing bacterium colonized the organic matter, forming a white veil. According to conventional scanning electron microscope (SEM) observations, bacterial cells are ovoid and slightly motile by numerous small flagella present on the cell surface. Large intracytoplasmic internal sulfur granules were observed, suggesting a sulfidic-based metabolism. Observations were confirmed by elemental sulfur distribution detected by energy-dispersive X-ray spectroscopy (EDXS) analysis using an environmental scanning electron microscope (ESEM) on non-dehydrated samples. Phylogenetic analysis of the partial sequence of 16S rDNA obtained from purified fractions of this Epsilonproteobacteraeota strain indicates that this bacterium belongs to the Thiovulaceae cluster and could be one of the largest Thiovulum ever described. We propose to name this species Candidatus Thiovulum sp. strain imperiosus.

‘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.

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.

Huanglongbing, or citrus greening disease, is the most serious disease of citrus worldwide and is associated with plant infection by ‘Candidatus Liberibacter asiaticus’ (CLas) and other Liberibacter species. CLas is transmitted by Diaphorina citri, the Asian citrus psyllid, in a circulative propagative manner. Circulative propagative transmission is a complex process comprising at least three steps: movement of the pathogen into vector tissues, translocation and replication of the pathogen within the vector host, and pathogen inoculation of a new host by the vector. In this work, we describe an excised leaf CLas acquisition assay, which enables precise measurements of CLas acquisition by D. citri in a streamlined laboratory assay. Briefly, healthy fourth and fifth instar D. citri nymphs acquire CLas from excised CLas-positive leaves, where the insects also complete their developmental cycle. CLas titer in the resulting adults is measured using quantitative PCR and CLas-specific 16S rRNA gene primers. We observed positive correlations between CLas titer in each leaf replicate and the CLas titer that developed in the insects after acquisition (rs = 0.78; P = 0.0002). This simple assay could be used to detect CLas acquisition phenotypes and their underlying genotypes, facilitate assessment of plant factors that impact acquisition, and screen for compounds that interfere with CLas acquisition by delivering these compounds through the excised leaf.

In Brazil, citrus huanglongbing (HLB) is associated with ‘Candidatus Liberibacter americanus’ (CLam) and ‘Ca. Liberibacter asiaticus’ (CLas). However, there are few studies about HLB epidemiology when both Liberibacter spp. and its insect vector, the Asian citrus psyllid (ACP, Diaphorina citri), are present. The objective of this work was to compare the transmission of HLB by ACP when both CLam and CLas are present as primary inoculum. Two experiments were performed under screenhouse conditions from April 2008 to January 2012 (experiment 1) and from February 2011 to December 2015 (experiment 2). The experiments were carried out with sweet orange plants infected with CLam or CLas as inoculum source surrounded by sweet orange healthy plants. One hundred Liberibacter-free adult psyllids were monthly confined to the source of inoculum plants for 7 days with subsequent free movement inside the screenhouse. Fortnightly, nymphs and adults of psyllids were monitored. Psyllid and leaf samples were collected periodically for Liberibacter detection by PCR or quantitative PCR. CLas was detected more frequently than CLam in both psyllid and leaf samples. No mixed infections were detected in the psyllids. A clear prevalence of CLas over CLam was observed in both experiments. The final HLB incidences were 16.7 and 14.5% of Liberibacter-positive test plants, and CLas was detected in 92.3 and 93.1% of these infected plants. Mixed infection was observed only in 3.8% of infected test plants in experiment 1. These results endorse the shift in the prevalence of CLam to CLas observed in citrus orchards of São Paulo, Brazil.