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Phosphatidylcholine (PtdCho) is an unusual membrane phospholipid present in some endosymbiotic and intracellular pathogenic prokaryotes. ‘ Candidatus Liberibacter asiaticus’ (CLas) is a phloem-limited, uncultured, fastidious α-Proteobacterium associated with the devastating citrus “greening” disease (huanglongbing). Phylogenetically related but nonpathogenic Liberibacter crescens (Lcr) was used as a culturable surrogate to examine PtdCho biosynthesis in pathogenic CLas. Genes encoding key enzymes for two alternative PtdCho biosynthetic routes are present in the Lcr genome: the one-step cytidine diphosphate (CDP)-choline ( pcs-encoding phosphatidylcholine synthase) and the three-step methyl-transferase pathway ( pmt-encoding phospholipid N-methyltransferase). However, only the CDP-choline pathway genes for incorporating exogenous Cho were identified in the CLas genome. Exogenous Cho enhanced growth and alleviated osmotic stress in wild-type Lcr and in the pmt insertion mutant strains when cultured in a sugar-rich medium. Quantitative RT-PCR analyses confirmed active uptake and condensation of nutritional Cho into PtdCho by CLas in both its plant host and psyllid vector. CLas-infected grapefruit leaves showed transcriptional activation of Cho biosynthesis genes and 2.8-fold higher levels of Cho. In plant cells, the compatible osmolyte glycine-betaine (GlyBet) is also derived from Cho. Expression of GlyBet biosynthesis genes and the GlyBet content were similar in both CLas-infected and healthy leaf tissue. The data presented here suggest that CLas likely exploits the Cho biosynthetic pathway in citrus hosts to expand the nutritional Cho pool. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

As hemp ( Cannabis sativa) emerges as a commercial crop in Nevada, mild to severe abnormal growth has been observed from many plants in commercial fields throughout the growing season. Affected plants exhibited a group of symptoms typically including stunting, leaf yellowing, excessive apical branching, clusters of witches’ broom, leaf rolling upwards, and leaf mottling and mosaic. These symptoms mostly showed up together in a plant or a crop and are defined here as abnormal growth syndrome (AGS). Between 2017 and 2019, the Nevada State Plant Pathology and Molecular Diagnostic Lab received 67 symptomatic hemp samples from Nevada for diagnosis, and ‘ Candidatus Phytoplasma trifolii’ was detected in 14 samples (21%). To investigate additional biotic agents associated with AGS, PCR products generated by primers P1/P7 were cloned into a pGEM-T vector and sequenced, and Spiroplasma citri DNA was found in two samples (3%). All 67 DNA samples were further tested for beet curly top virus (BCTV), and 57 samples (85%) were found to be infected by BCTV. Twelve samples (18%) were coinfected by both ‘ Ca. Phytoplasma trifolii’ and BCTV, and two (3%) were coinfected by S. citri, ‘ Ca. Phytoplasma trifolii’, and BCTV. The findings suggest that BCTV is the most prevalent pathogen causing the hemp abnormal growth in Nevada, but ‘ Ca. Phytoplasma trifolii’ and S. citri may also contribute to the severity and complexity of symptoms. Thus, hemp abnormal growth can be attributed to single, dual, or triple infections of these three leafhopper-vectored mollicutes and virus. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

‘ Candidatus Phytoplasma pruni’ is the causative agent of X-disease on peach ( Prunus persica) trees. Infected trees exhibit premature yellowing, leaf necrosis causing a shot-hole appearance, limb dieback, and eventual death. How pathogen infection leads to these symptoms is unknown. This study undertook a modern characterization of the disease by assessing the physiological and transcriptomic consequences of phytoplasma infection. Phytoplasma titer was high in the symptomatic tissues and undetected or low in asymptomatic tissues. Symptomatic leaves had a significant decrease in chlorophyll a, chlorophyll b, and carotenoids. Transcriptomic analysis showed alterations in genes related to phytohormone synthesis and signaling, circadian rhythms, lignification, and sugar synthesis and transport. Several transcripts that might be related to symptom development were identified. Collectively, these data give a much clearer picture of symptom development in ‘ Ca. P. pruni’-infected P. persica and provide several avenues for further research in determining how ‘ Ca. P. pruni’ interacts with its host to elicit the observed symptoms. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Huanglongbing (HLB), caused by phloem-limited ‘ Candidatus Liberibacter asiaticus’ (CLas), is the primary limiting factor of production in most citrus regions of the world. After infection, CLas is transported systemically throughout the phloem tissues following the source-sink movement. Split-root rhizoboxes and one-sided graft inoculation above the split trunk was used to understand if the vertical distance of the inoculum source and different anatomical structures (grafted or seedling trees) can affect the speed of the CLas movement, as well as the effects of the seasonality on these movements. The time for CLas to reach the roots was not affected by either distance of the inoculum source or tree type. The seasonal infection period appears to have an important effect on CLas movement. Trees inoculated in the summer had fast and uniform movement (first detection at 4 weeks after inoculation). Plants inoculated in the winter had a slow and uneven movement (first CLas detection at 14 weeks after inoculation). Our results indicate that summer and spring are the seasons of CLas down and lateral movement, but this is independent of the vertical distance of the inoculum source or anatomical structures of the plants. The findings from this study aid in the management of HLB in the field, as well as improve the methods for CLas detection. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

An updated real-time multiplex quantitative polymerase chain reaction (qPCR) assay was designed and validated for the simultaneous detection of three ‘ Candidatus Liberibacter species’ (CLsp), ‘ Ca. Liberibacter asiaticus’ (CLas), ‘africanus’ (CLaf), and ‘americanus’ (CLam), associated with the huanglongbing disease of citrus. The multiplex assay was designed based on the qPCR assay published in 2006 by Li et al., considering all available CLsp 16S rRNA gene sequences in GenBank and the MIQE guidelines and workflow for qPCR optimization, which became available after 2006. When using the updated multiplex CLsp qPCR assay compared with singleplex qPCR, no significant increase in quantitative cycle (Cq) values was detected. The specificity and sensitivity of the updated qPCR assay was optimal, and measuring the intra- and interassay variations confirmed the reproducibility and repeatability of the assay. The assay was also successfully used with a large number of diverse samples at independent laboratories in four countries, thus demonstrating its transferability, applicability, practicability, and robustness as different qPCR reaction conditions or instruments had a minor effect on Cq values. This updated multiplex CLsp qPCR assay can be used in a variety of citrus surveys, germplasm, or nursery stock programs that require different pathogen detection tools for their successful operation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Comparison of the metabolic changes prior to symptom development upon infection with Candidatus Liberibacter asiaticus (CLas), the bacterium associated with citrus greening disease, between citrus hosts with different tolerances is lacking. The objective of this study was to compare the early response of Lisbon lemon (Citrus limon) and Washington navel orange (Citrus sinensis [L.] Osbeck), two citrus species commercially important to California, to CLas through graft inoculation. Here, we compare the transcriptome, proteome, and metabolome response, using RNA sequencing, liquid chromatography tandem mass spectrometry, and 1H nuclear magnetic resonance, respectively, from our two recently published studies examining the response of the lemon and navel oranges separately, and introduce new micronutrient data from inductively coupled plasma mass spectrometry analysis, focusing on lemons at 10 and 14 weeks postgrafting (wpg), and navels at 8 and 18 wpg, prior to symptom development. Several micronutrients accumulated in presymptomatic infected lemons compared with controls, whereas little change was observed in the navels. Photosynthesis proteins were substantially altered by CLas infection in navels, with fewer changes observed in lemons. The metabolome differed between control and infected navels throughout infection, although differences between control and infected lemons were identified only after symptom expression. Taken together, these findings highlight differences in response to CLas between two varieties with differing tolerances. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .