Publications

4502

‘Candidatus Liberibacter asiaticus’ (Las) is the prominent species of Liberibacter associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts, whereas an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host.

‘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.

The devastating citrus disease huanglongbing (HLB) associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) has caused a more than 70% reduction in citrus production since its discovery in Florida in 2005. Most citrus scion cultivars are sensitive to HLB, whereas some cultivars used as rootstocks are tolerant. Using such tolerant rootstocks can help trees to cope better with the disease’s impact. Evaluating rootstock effects on a grafted scion in the field takes many years, but shorter-term evaluation is imperative to aid in rootstock selection for an HLB-endemic production environment. In this study, we investigated grafted healthy and CLas-infected citrus trees under controlled greenhouse conditions. The objectives were to identify traits suitable for assessing grafted tree tolerance in advance of longer-term field studies and aiding in the selection of superior rootstock cultivars. We assessed 10 commercially important rootstocks grafted with ‘Valencia’ sweet orange scion and with known field performance. At 6, 9, 15, and 21 months after graft inoculation (mai), leaf CLas titers were determined and canopy health was evaluated. Plants were destructively sampled at 21 mai to assess plant biomasses and other physiological and horticultural variables. There was little influence of the rootstock cultivar on CLas titers. Surprisingly, few HLB foliar disease symptoms and no differences in soluble and nonsoluble carbohydrate concentrations were measured in infected compared with healthy plants, despite high CLas titers and significant reductions in plant biomasses. Most trees on rootstocks with trifoliate orange parentage were less damaged by HLB than other rootstocks, although results did not always agree with reported field performance. Among the different variables measured, leaf size appeared to be most predictive for grafted tree assessment of HLB sensitivity. The results of this study provide a better understanding of the strengths and weaknesses of assessing rootstock influence on grafted tree performance in a controlled greenhouse environment. Although such studies provide valuable information for cultivar tolerance to HLB, other rootstock traits will ultimately contribute to field survival and productivity in an HLB endemic production environment.

Tomato (Solanum lycopersicum) is one of the most financially important vegetable crops. It is a species belonging to the Solanaceae family and is cultivated in many countries, including Turkey. The natural presence of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’) from the Stolbur group (16SrXII) in tomato plants is extensively characterized based on the 16S rRNA gene worldwide. Tomato plants displaying abnormality and sterility of flower, purpling, and bushy appearance were observed in the Van province, Turkey. DNA extraction from tomato leaves was performed, and the extracted DNA was used to amplify 16S rRNA sequences using universal primer pairs by double PCR assays. After confirming the presence of the probable agent (‘Ca. P. solani’), nested PCR testing was performed using an appropriate primer set amplifying the SecY and Vmp1 genes of the same DNAs. Amplified PCR yields were then cloned into a pGEM T-Easy vector and sequenced by New Generation System (NGS). Sequenced 1438 bp nucleotides for Vmp1 gene (MN104838) and 905 bp nucleotides for SecY gene (MN125054) revealed 97.38% and 100% maximum nucleotide similarity with formerly published Vmp1 and SecY gene sequences of ‘Ca. P. solani’ species belonged to the Stolbur group (16Sr-XII), respectively. According to the phylogenetic tree created using our gene sequences from ‘Ca. P. solani’, Vmp1, and SecY gene sequences showed a highly phylogenetic affinity with the same sequences of the same agent from Serbia and France, respectively. To the best of our knowledge, this study is the first report to reveal the phylogenetic relationships of ‘Ca. P. solani’ in stolbur group (16Sr XII-A) using SecY and Vmp1 genes in the tomato plant in Turkey based on the non-ribosomal genes.

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.