Publications

4620

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.

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.

‘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.

Candidatus Liberibacter spp. are fastidious α-proteobacteria that cause multiple diseases on plant hosts of economic importance, including the most devastating citrus disease: Huanglongbing (HLB). HLB was reported in Asia a century ago but has since spread worldwide. Understanding the pathogenesis of Candidatus Liberibacter spp. remains challenging as they are yet to be cultured in artificial media and infect the phloem, a sophisticated environment that is difficult to manipulate. Despite those challenges, tremendous progress has been made on Ca. Liberibacter pathosystems. Here, we first reviewed recent studies on genetic information of flagellar and type IV pili biosynthesis, their expression profiles, and movement of Ca. Liberibacter spp. inside the plant and insect hosts. Next, we reviewed the transcriptomic, proteomic, and metabolomic studies of susceptible and tolerant plant genotypes to Ca. Liberibacter spp. infection and how Ca. Liberibacter spp. adapt in plants. Analyses of the interactions between plants and Ca. Liberibacter spp. imply the involvement of immune response in the Ca. Liberibacter pathosystems. Lastly, we reviewed how Ca. Liberibacter spp. movement inside and interactions with plants lead to symptom development.

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.

‘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 considerable economic losses in citrus associated with ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of ‘Ca. Liberibacter’ associated with “huanglongbing” disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phytoplasmas in citrus worldwide.