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‘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.

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.

A survey of weeds was undertaken in a palm nursery affected by lethal bronzing (LB) to identify a reservoir host of the causal phytoplasma. Three common species were identified; Urochloa maxima (Guineagrass), Sporobolus indicus (smut grass), and Cyperus esculentus (yellow nutsedge) and sampled over a period of 2 years. Each species was sampled 36 times and all three species were negative for the LB phytoplasma. However, three specimens of C. esculentus tested positive for the phytoplasma species ‘Candidatus Phytoplasma brasiliense’. These findings represent the first documented case of ‘Ca. P. brasiliense’ in North America, specifically in Florida, U.S.A., as well as a new host record for the phytoplasma and the first monocot host documented. Because of the impact this phytoplasma has on papaya and hibiscus in South America, it presents a unique threat to ornamental and agricultural sectors in south Florida. An area-wide survey for the phytoplasma and potential vectors is recommended.

Grafting a scion onto a rootstock results in physical and physiological changes in plant growth and development, which can affect tree vigor, productivity, and tolerance to stress and disease. Huanglongbing (HLB) is one of the most destructive citrus diseases and has become endemic in Florida since its introduction in 2005. It is associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), which cause severe metabolic disruptions in affected plants. Although most scion cultivars are highly susceptible, some rootstock cultivars are tolerant and allow the grafted tree to cope better with the disease. The objectives of this study were to identify rootstock traits that can be used to assess cultivars under controlled greenhouse conditions in advance of longer-term field trials. We used 10 commercially important rootstocks with different genetic backgrounds and known field performance in graft combination with ‘Valencia’ sweet orange scion. Trees were graft-inoculated with CLas and compared against mock-inoculated trees. Tree health and CLas populations were assessed regularly, and root growth was monitored using a minirhizotron imaging system. Plants were excavated and destructively sampled 21 months after inoculation to assess biomass distributions and other CLas-induced effects. We found significant differences between healthy and infected trees for most variables measured, regardless of the rootstock. In contrast to leaf CLas titers, root titers were significantly influenced by the rootstock, and highest levels were measured for ‘Ridge’ sweet orange and sour orange. Root growth and root biomasses were reduced upon infection but differences among rootstocks did not always agree with reported field performances. Despite severe biomass reductions plants maintained their relative distribution of biomass among different components of the root system, and no dead roots were observed. Root respiration was reduced by CLas infection and was overall higher in tolerant cultivars suggesting its potential as a physiological marker. This study improves our knowledge about the strengths and weaknesses of assessing rootstock traits of grafted trees in a controlled greenhouse setting. Results from the study suggest that in addition to HLB tolerance, other rootstock traits will ultimately have major contributions to field survival and productivity of the grafted trees in an HLB endemic production environment.

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 decade ago, shoot proliferation symptoms (i.e., witches’ broom) in carrots were believed to be the cause of ‘Candidatus Phytoplasma’ and Spiroplasma infection, yet in recent years this association appeared to have weakened, and a closer association was found with the yet-unculturable, psyllid-transmitted Gram-negative bacterium ‘Candidatus Liberibacter solanacearum’. In Israel, carrots are grown throughout the year, yet shoot proliferation symptoms tend to appear only in mature plants and mostly in late spring to early summer. We hypothesized that factors such as plant age, temperature, and vector load, which vary during the year, have a critical effect on symptom development and examined these factors under controlled conditions. Here we show that young carrot seedlings are as prone as older plants to develop shoot proliferation symptoms after ‘Ca. L. solanacearum’ inoculation. Surprisingly, we found that the local ‘Ca. L. solanacearum’ haplotype was extremely sensitive to constant temperature of 30°C, which led to a significant reduction in bacterial growth and symptom development compared with 18°C, which was very conducive to symptom development. We have also found that inoculations with 10 or 20 psyllids per plant results in faster symptom development compared with inoculations with two psyllids per plant; however, the difference between vector loads in disease progress rate was not significant. These data provide important insights to the effects of plant age, growth temperature, and vector load on ‘Ca. L. solanacearum’ and its associated symptoms and further strengthen the notion that ‘Ca. L. solanacearum’ is the main responsible agent for carrot witches’ broom in Israel. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

‘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.

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.

‘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.