Plant Science

‘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.

‘Candidatus Liberibacter’ species are associated with severe, economically important diseases. Nearly all known species are putatively insect transmitted, specifically by psyllids. Detection of ‘Ca. Liberibacter’ in plants is complicated by their uneven distribution in host plants and largely fastidius nature. The death of black (Fraxinus nigra) and mancana (Fraxinus mandshurica) ash trees in Saskatchewan, Canada has been associated with infestation by the cottony ash psyllid (Psyllopsis discrepans). A combination of conventional PCR amplification and Sanger sequencing of the 16S recombinant DNA was used to detect and identify ‘Ca. Liberibacter’ in psyllids collected from ash trees in Saskatchewan. BLAST analysis of two 16S sequences that were 1,058 and 1,085 bp long (NTHA 5, GenBank accession number MK942379 and NTHA 6, GenBank accession number MK937570, respectively) revealed they were 99 to 100% similar to a ‘Ca. Liberibacter solanacearum’ sequence (GenBank accession number KX197200) isolated from the Nearctic psyllid (Bactericera maculipennis) of U.S. provenance. Sequencing the psyllid genes CO1 and Cyt-b confirmed that the psyllids from which the bacterial DNA was isolated were P. discrepans, based on comparisons with sequences in GenBank and BOLD and a reference sample from the United Kingdom. These results provide the first evidence that ‘Ca. Liberibacter solanacearum’ species are associated with psyllids collected from ash trees and specifically P. discrepans. The recent episodes of dieback of ash in Saskatchewan associated with psyllid feeding are consistent with disease symptoms caused by ‘Ca. Liberibacter’ pathogens, and this possibility warrants further study.

A protocol to successfully transmit the huanglongbing (HLB) pathogen, ‘Candidatus Liberibacter asiaticus’, between citrus plants by using the Asian citrus psyllid (ACP) and an alternative way to help growers control ACP are proposed. Best results were obtained when pathogen acquisition by adults reared on fully symptomatic ‘Ca. Liberibacter asiaticus’-positive plants, latency, and inoculation occurred at ambient air temperatures ranging from 24 to 28°C and when a single infective adult ACP was confined for 7 days on soft, newly developing vegetative shoots (stages v2 to v4). No infection resulted from confinement of infective ACP adults on mature leaves (stage v6). Under the described conditions, single ACP adults could successfully transmit ‘Ca. Liberibacter asiaticus’ to an average of 56.5% (35 to 83%) of plantlets with v2 to v4 shoots growing in 0.3-liter tubes and to 80.5% (76 to 86%) of plants with v2 to v4 shoots growing in 4.7-liter pots. The use of single insects and plantlets reduces labor, space, and other resources necessary to undertake transmission tests. It also reduces time needed for transmission studies and should help accelerate research on HLB. The results were used to develop an index for favorability to infection (IFI) to determine orchard vulnerabilities to ‘Ca. Liberibacter asiaticus’. The IFI is based on the heterogeneous population of new shoots that occurs on tree canopies and may offer alternative or complementary alternatives to the laborious and costly insect surveys currently used in most instances to determine threshold levels for insecticide applications.

AbstractIn recent years, empty or poor grain bearing corn plants with leaf reddening symptoms have been observed in some commercial maize production areas in the Adana province of Turkey. The disease is increasing and causing economic losses. To verify the possible presence of phytoplasmas, leaf samples were tested with P1/Tint and R16F2n/R2 phytoplasma‐specific primer pairs after DNA extraction. The 16S ribosomal gene sequence analysis followed by sequence in silico enzyme digestions and phylogenetic analyses allowed the identification of ‘Candidatus Phytoplasma solani’, subgroup of 16SrXII‐A related‐strain in all symptomatic samples. The positive samples were characterized using tuf and vmp1 genes. In silico restriction fragment length polymorphism and sequence analyses allowed the inclusion of all the positive samples into tuf‐type b and V15 molecular types. The sequence analysis indicated the presence of a single genotype in all the tested samples (n = 12). This is the first molecular characterization of a ‘Ca. P. solani’ strain infecting maize plants in Turkey. The identified pathogen is listed in the EPPO A2 list.