2013 Annual Report
1a.Objectives (from AD-416):
1. Identify the effectors and other pathogenesis-related genes of ‘Ca. Liberibacter solanacearum’.
2. Use the genome information to generate genetic markers, such as Simple Sequence Repeat (SSR), for detection and differentiations of the PY and ZC bacterial complex.
3. Use the primers LJ-ZC07f/07r with LJ-NZC07p primers designed from this research to further investigate the biological significance of Type 1 (asymptomatic) and Type 2 (symptomatic) Liberibacter solanacearum (Lso) populations.
1b.Approach (from AD-416):
1. Comparative genomics of Liberibacter psyllaurous (Lps)/Liberibacter solanacearum (Lso) and Liberibacter asiaticus (Las) will be carried out by using reciprocal BLAST searches to identify the set of genes common to all isolates and those genes unique to each isolate. Results will be compared using Glimmer. Orthologs will be defined as reciprocal best BLAST hits between two isolates where the alignments with > 70% sequence identity span a minimum query length of 70% (Blom et. Al. 2009). Syntenic regions between these orthologs will be detected using MUMmer and SyMAP software. Since prophage/phages that have been identified in these genomes play important role in the evolution and horizontal gene transfer, the analyses will focus on them and how they mediate the genome evolution.
2. Generate genetic markers for detection and differentiation of Psyllid Yellow (PY) and Zebra Chip (ZC) strains of Lps/Lso and to further elucidate the biological importance of Type 1 and Type 2 Lso. Archived DNA from PY and ZC affected potato plants will be used in this analysis as well as DNA from PY and ZC grafted plants and DNA obtained from PY and ZC affected potato plants in the field. DNA extracted from existing psyllid colonies and those obtained from various geographical locations in the USA will also be used in these assays. Using clonal populations of Type 1 Lso-infected psyllids and type 2 Lso-infected psyllids we will also study the biological significance of these two types of Lso. Pathogen-free, tissue culture derived tubers will be used to generate disease-free potato plants in the growth chamber. Individual potato plants will be challenged with Type 1 Lso-infected psyllids or Type 2 Lso-infected psyllids separately and monitored for disease development. Additional potato plants will be challenged with a mixed colony containing Type 1 and Type 2 psyllids and also monitored for disease development. Lso- psyllids (without Lso) will be used in acquisition experiments to determine the frequency with which they can acquire each type of Lso and to determine whether or not both types of Lso can inhabit the same psyllid or whether or not Type 1 and Type 2 Lso are mutually exclusive in psyllids.
This research relates to inhouse project objective: 1. Characterize ecology, biology, epidemiology, molecular genetics, and vector and host (crop and weed) interactions of domestic, exotic, newly emerging, and re-emerging pathogens, and 2. Develop/refine rapid, sensitive reliable detection/sampling methods for pathogens.
In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained a complete genome sequence of Candidatus Liberibacter solanacearum. The complete genome of haplotype A ‘Candidatus Liberibacter solanacearum’ (LsoA) has been sequenced using Life Technology’s Ion-Torrent, and sequence assembly is in process. Comparison of previously published LsoB and the draft LsoA genome sequence was conducted. Nucleotide variation between haplotypes A and B is low, generally 1-2%, with the exception of the putatively designated phage regions. Two regions in the Lso haplotype B genome were previously identified as phage-like based on homology with the related bacteria ‘Candidatus Liberibacter asiaticus’ (Las) and its characterized phages. Nucleotide variation in the phage regions varies from 5-20% and appears to account for most of the genetic variation between the two haplotypes. Similarities with known phages indicate that the Las and Lso phages are active temperate phages. The increased nucleotide variation in the phage regions is likely the result of an increased error rate in the phage polymerases. Experiments to characterize the phages and their significance in Lso biology and pathology are underway.
The two genome sequences have also been used to design PCR primer sets to specifically distinguish the two haplotypes. The primer targets were distributed across the genomes to test for recombination between haplotypes. No recombination was detected as all samples were consistently A or B. Quantitative PCR assays of mixed infections were also able to quantify distinct levels for both haplotypes.