Publications

4468

‘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.

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.

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.

Summary Halorhodospira halophila , one of the most‐xerophilic halophiles, inhabits biophysically stressful and energetically expensive, salt‐saturated alkaline brines. Here, we report an additional stress factor that is biotic: a diminutive Candidate‐Phyla‐Radiation bacterium, that we named ‘ Ca . Absconditicoccus praedator’ M39‐6, which predates H . halophila M39‐5, an obligately photosynthetic, anaerobic purple‐sulfur bacterium. We cultivated this association (isolated from the hypersaline alkaline Lake Hotontyn Nur, Mongolia) and characterized their biology. ‘Ca . Absconditicoccus praedator’ is the first stably cultivated species from the candidate class‐level lineage Gracilibacteria (order‐level lineage Absconditabacterales). Its closed‐and‐curated genome lacks genes for the glycolytic, pentose phosphate‐ and Entner–Doudoroff pathways which would generate energy/reducing equivalents and produce central carbon currencies. Therefore, ‘Ca . Absconditicoccus praedator’ is dependent on host‐derived building blocks for nucleic acid‐, protein‐, and peptidoglycan synthesis. It shares traits with (the uncultured) ‘ Ca . Vampirococcus lugosii’, which is also of the Gracilibacteria lineage. These are obligate parasitic lifestyle, feeding on photosynthetic anoxygenic Gammaproteobacteria, and absorption of host cytoplasm. Commonalities in their genomic composition and structure suggest that the entire Absconditabacterales lineage consists of predatory species which act to cull the populations of their respective host bacteria. Cultivation of vampire : host associations can shed light on unresolved aspects of their metabolism and ecosystem dynamics at life‐limiting extremes.

‘Candidatus Liberibacter asiaticus’, the putative causal agent of citrus greening, is not available in pure culture yet. In addition to trees of citrus and citrus relatives, ‘Ca. L. asiaticus’ can grow in Madagascar periwinkle (Catharanthus roseus). Using gas chromatography-mass spectrometry, we compared the phloem sap composition in sweet orange ‘Valencia’ (Citrus sinensis) and periwinkle plants after the infection with ‘Ca. L. asiaticus’. Interestingly, in contrast to our previous studies of total leaf metabolites, we found that, compared with uninfected phloem sap, the organic acids implicated in the tricarboxylic acid cycle (TCA) cycle including citrate, isocitrate, succinate, fumarate, and malate were reduced significantly in the infected phloem saps of both species. As a result of the reduction of organic acids content, the pH of infected phloem saps was increased. We hypothesize that the bacterial growth induces the mitochondrial TCA cycle in parenchyma cells to produce more of these compounds to be used as a bacterial carbon source. Once these compounds reach a low level in the phloem sap, the bacterium may send a signal, yet to be identified, to initiate a feedback loop to further induce the TCA cycle. Phloem blockage might be another reason behind the reduced translocation of TCA cycle intermediates within the phloem. The net result, localized availability of organic acids, likely benefits bacterial growth and may explain the unequal distribution of ‘Ca. L. asiaticus’ within infected trees. These findings may help in designing media for the pure culturing of ‘Ca. L. asiaticus’.

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.

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.