Plant Disease

‘Candidatus Liberibacter asiaticus’ is the unculturable causative agent of citrus huanglongbing disease. Here, we report the first citrus root metagenome sequence containing the draft genome of ‘Ca. L. asiaticus’ strain AHCA17, obtained from a pummelo tree in California. The assembled genome was 1.2 Mbp and resulted in 37 contigs (N50 = 158.7 kbp) containing 1,057 predicted open reading frames and 45 RNA-coding genes. This draft genome will provide a valuable resource in further study of ‘Ca. L. asiaticus’ genome diversity and pathogen epidemiology.

‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). PCR procedures that accurately confirm or exclude CLas infection in citrus tissue/Asian citrus psyllid (ACP) samples are critical for HLB management. When CLas was described in 1994, a 23-bp signature oligonucleotide sequence (OI1) in the 16S rRNA gene (rrs, three genomic copies) was identified based on Sanger sequencing. OI1 contains single nucleotide polymorphisms (SNPs) distinguishing CLas from non-CLas species. The SNPs were used to design the primer HLBas, a key primer for a commonly used TaqMan PCR system (HLBas-PCR) for CLas detection. Recent developments in next-generation sequencing technology have led to the identification of 15 CLas whole genome sequence strains (WGSs). Analyses of CLas WGSs have generated a significant amount of biological information that could help to improve CLas detection. Utilizing the WGS information, this study re-evaluated the sequence integrity of OI1/HLBas and identified and/or confirmed a missing nucleotide G in the two primers. Replacement primers for OI1 and HLBas are proposed. At low cycle threshold (Ct) values (e.g., <30), HLBas-PCR remained reliable in CLas determination. At high Ct values (e.g., >30), HLBas-PCR alone was unreliable in differentiating whether samples contain low CLas titers or whether CLas is not present. The availability of ribonucleotide reductase (RNR)-PCR derived from the five-copy nrdB gene helped to resolve this problem. To further enhance low CLas titer detection, a 4CP-PCR system, based on a four-copy genomic locus, was developed. Evaluation of 107 HLB samples (94 citrus and 13 ACP) showed that 4CP-PCR was more sensitive than HLBas-PCR and shared similar sensitivity with RNR-PCR.

‘Candidatus Liberibacter solanacearum’ (Lso), transmitted by the potato psyllid (Bactericera cockerelli), is the putative causal agent of potato zebra chip disease. The bacterial pathogen infects a wide range of solanaceous plants (both wild and cultivated species), among which are peppers, potatoes, and tomatoes. Currently there are two commonly detected, genetically distinct haplotypes of Lso (A and B) identified from potatoes in the United States. To determine whether there are interactions between Lso haplotypes and different solanaceous hosts, experiments were conducted in the greenhouse in which pepper, potato, and tomato plants were infested with psyllids carrying Lso A, B, or an A and B mix (AB) or with psyllids free of Lso. Host plants were grown in pots in cages on the greenhouse benches and infested with six psyllids per plant. In addition, eight pepper cultivars were similarly infested for deeper understanding of host–haplotype interactions. Approximately 7 weeks after infestation, adult psyllids in each cage were counted to determine the impact of Lso haplotype–host interactions on psyllid survival and plants were sampled and tested molecularly for Lso. Individual psyllids carrying haplotypes B or AB and those free of Lso copiously reproduced on all three hosts, and leaf tissue from each plant tested positive for the respective Lso except those infested with Lso-negative psyllids. However, psyllids carrying Lso A did not survive on peppers but survived and abundantly reproduced on potatoes and tomatoes. In addition, samples from peppers infested with psyllids carrying Lso A tested negative for Lso. However, peppers infested with individual psyllids carrying Lso AB tested positive for Lso A, indicating that the presence of B may be required for infection by Lso A and psyllid survival on peppers. The different pepper cultivars infested with psyllids carrying Lso A showed similar results to the haplotype–host interaction tests, suggesting that cultivar may not be a factor in Lso A–pepper host interactions. Results from these studies suggest that Lso A may affect host selection by psyllids either for nutrition or laying of eggs. Mechanisms involved in preventing psyllid reproduction on peppers, once identified, will have significant implications for potential psyllid management.