Publications

3561

AbstractCitrus Greening disease (CG) in South Africa (SA) is associated with the fastidious bacterium ‘Candidatus Liberibacter africanus’ (Laf). It has been observed that Laf isolates obtained from different geographic localities in SA differed in the rate of transmission during grafting experiments leading to the hypothesis that genetic variation of Laf may exist in this country. To determine this, 167 Laf isolates obtained from Limpopo, North West, Mpumalanga and the Western Cape were subjected to microsatellite analyses, using four polymorphic markers. From UPGMA and STRUCTURE analysis, it was shown that most sources belong to one of two major genetic groups of Laf and these comprise 25 distinct haplotypes. Four samples included within this study did not group with these two major groups, suggesting a potential third and fourth genetic group of Laf being present, which can be validated by further sampling. Results further indicate that Laf populations in SA are formed by geographic locality. The high genetic diversity observed for Laf within this study is consistent with the hypothesis that Laf originated on the African continent, warranting further genetic analysis of Laf populations from Africa. This is the first study to unveil the genetic diversity of Laf.

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 .

The potato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) is a pest of solanaceous crops (order Solanales), including potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.). Feeding by high populations of nymphs causes psyllid yellows while adults and nymphs are vectors of the plant pathogen ‘Candidatus Liberibacter solanacearum’. Foliar symptoms that were consistent with either ‘Ca. L. solanacearum’ infection or psyllid yellows were observed in 2019 on tomatillo (Physalis ixocarpa Brot.; family Solanaceae) grown within an experimental plot located near Saltillo, Mexico. This study had three primary objectives: 9i) determine whether the foliar symptoms observed on tomatillo were associated with ‘Ca. L. solanacearum’ infection, (ii) identify the haplotypes of ‘Ca. L. solanacearum’ and potato psyllids present in the symptomatic plot, and (iii) use gut content analysis to infer the plant sources of ‘Ca. L. solanacearum’-infected psyllids. Results confirmed that 71% of symptomatic plants and 71% of psyllids collected from the plants were infected with ‘Ca. L. solanacearum’. The detection of ‘Ca. L. solanacearum’ in plants and psyllids and the lack of nymphal populations associated with psyllid yellows strongly suggests that the observed foliar symptoms were caused by ‘Ca. L. solanacearum’ infection. All infected plants and insects harbored the more virulent ‘Ca. L. solanacearum’ haplotype B but one psyllid was also coinfected with haplotype A. The potato psyllids were predominantly of the central haplotype but one psyllid was identified as the western haplotype. Molecular gut content analysis of psyllids confirmed the movement of psyllids between noncrop habitats and tomatillo and indicated that ‘Ca. L. solanacearum’ infection of psyllids was associated with increased plant diversity in their diet.

Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri) virescence in Michigan, USA, was selected as the reference strain for the novel taxon ‘Candidatus Phytoplasma asteris’. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

The beet leafhopper Circulifer tenellus is an important pest of agricultural crops in the United States, where it transmits beet curly top virus, beet leafhopper-transmitted virescence agent phytoplasma, and Spiroplasma citri to numerous crops, affecting yield and quality. Each of these pathogens have been linked to serious disease outbreaks within Washington State in the past century. To mitigate the risk of disease, growers target the beet leafhopper in their insect pest management programs. Knowledge of pathogen prevalence in beet leafhopper populations could help growers make better management decisions, but timely diagnostics is required. Four new assays were developed for the rapid detection of the beet leafhopper-associated pathogens. These include two assays that detect Beet leafhopper transmitted virescence agent (a PCR and a real-time PCR SYBR green assay), a duplex PCR assay that simultaneously detects beet curly top virus and Spiroplasma citri, and a multiplex real-time PCR assay for the simultaneous detection of all three pathogens. The screening of dilution series generated from plant total nucleic acid extracts with these new assays typically led to detection at levels 10- to 100-fold more sensitive than the conventional PCR assays currently used. These new tools will allow the rapid detection of beet leafhopper-associated pathogens in both plant and insect specimens and will have the potential to be used in diagnostic laboratories seeking to disseminate fast and accurate results to growers for implementation in their insect pest monitoring programs.

AbstractCandidatusLiberibacter asiaticus (CLas) is the causal agent of the devastating citrus Huanglongbing (HLB) disease. Our understanding of the pathogenicity mechanism and biology of CLas remain limited because CLas has not been cultured in artificial media. CLas encodes 1136 proteins of which 415 have unknown functions. Since genetic studies of CLas genes with unknown functions are impossible, we utilized genome-wide protein-protein interactions (PPIs) yeast-two-hybrid (Y2H) assays to help solve the mystery. PPIs are fundamental to all cellular processes and machinery and instrumental in investigating uncharacterized proteins and inferring biological pathways. In total, 916 bait and 936 prey proteins were included in the three-phase screening, which identified 4245 interactions. The false positive rate of the Y2H assay was estimated to be 3.1%. Pull-down assays confirmed the robustness of our Y2H. The average interactions per node for CLas Y2H interactome were approximately 15.6, significantly higher than free-living bacteria, indicating genome reduction has led to a multi-function of proteins. PPIs provide clues for functions of 371 uncharacterized proteins of CLas. Forty HUB node proteins were identified which might play critical roles in CLas, including a quinone oxidoreductase and LysR that are known to protect bacteria against oxidative stress. This explains why CLas survives well in the phloem even though it triggers immune-mediated disease, systemic and chronic production of reactive oxygen species, and phloem cell death. This PPI database facilitates the investigation of CLas cellular biochemistry and physiology, functions of uncharacterized proteins, and pathogenicity mechanisms of the pathogen.

Abstract Zebra chip, is a potato disease associated with the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) and vectored by the potato psyllid, Bactericera cockerelli Šulc. Potato psyllids are native to North America, where four haplotypes have been described. They are able to colonize a wide range of solanaceous species, crops, and weeds. The epidemiology of zebra chip disease is still poorly understood and might involve the different haplotypes of psyllids as well as two haplotypes of Lso. As several perennial weeds have been recognized as potential host for potato psyllids and Lso, a yearly monitoring of several patches of bittersweet nightshade (Solanum dulcamara) and field bindweed (Convolvulus arvensis) located in the potato-growing region of southern Idaho was conducted from 2013 to 2017, to gain insight into psyllid dynamics in non-potato hosts and Lso presence in the fields. Potato psyllids caught on each host were individually tested for Lso, and a subset were haplotyped based on the CO1 gene, along with the haplotyping of Lso in positive samples. On bittersweet nightshade, the Northwestern haplotype was numerically dominant, with around 2.7% of psyllids found to be carrying either Lso haplotype A or B, suggesting a limited role in zebra chip persistence, which has infected Idaho fields at a low occurrence since the 2012 outbreak. Field bindweed was found to be a transient, non-overwintering host for potato psyllid of Northwestern, Western and Central haplotypes late in the season, suggesting minor, if any, role in persistence of Lso and field infestation by potato psyllids.