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4381

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.

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.

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.

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.

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.

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 .

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 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.