Publications

4463

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.

‘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.

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.

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.

‘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.

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 .

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.

Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) and ‘Candidatus 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 with CLas, suggesting a possible role in host interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides, aiming to evaluate 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, whereas 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 3 months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.