Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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) Lso populations. Approach (from AD-416): 1. Comparative genomics of Lps/Lso and 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 PY and 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 related 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. Previous research has identified two primary genotypes of Candidatus Liberibacter solanacearum( Lso). The two types are distinguished by nucleotide level variation in the 16S and 50S genes. Screening of the genome with a selection of primers produced two primer sets that differentially amplified across samples. The differential primer sets have tentatively been linked to the nucleotide variation in the 16S and 50S genes. Also, two phage-like regions in the Lso genome have been identified based on homology to the related bacteria Candidatus Liberibacter asiaticus (Las). Polymerase chain reaction (PCR) screening of these areas has revealed significant variation between Lso isolates. Screening and typing of collected samples has been complicated by what we believe are infections with both Lso genotypes. In order to understand the significance of Lso genotype, we have established Lso colonies from different submitted samples of infected plant tissues and insects. We have several colonies containing either one or both genotypes that we can now screen for differential disease symptomatology. We will also be able to observe the interactions between haplotypes during dual infections. Testing of dual infected plants will help us modify our sample screening methods to account for dual infections. The B haplotype of Lso has previously been sequenced. We have isolated and will now have the A haplotype of the bacteria sequenced for comparison. Lso is vectored by the potato psyllid insect Bactericerca cockerelli. Coincidentally, two bio-types of B. cockerelli have been identified. We have both psyllid bio-types in colonies and are planning experiments to test for psyllid bio-type versus Lso haplotype interactions.
Impacts
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Publications
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