Development of Primers for Improved PCR Detection of the Potato Zebra Chip Pathogen, 'Candidatus Liberibacter solanacearum'.

Zebra chip disease poses a major economic threat to potato production. The causative agent is a phloem-limited bacterium identified as 'Candidatus Liberibacter solanacearum' that is transmitted by the potato/ tomato psyllid. Currently, there are no effective controls and existing control strategies depend largely on the early detection of the pathogen via polymerase chain reaction (PCR) assays. Most primer sets used for PCR detection target a region of the bacterial 16S rDNA gene, and detection of the pathogen in symptomatic potato tissue with existing primers has been variable depending on the specific primer sets used. This study describes the development of two new primer sets that target a conserved intergenic region between the 16S and 23S rDNA genes and a conserved bacterial housekeeping gene, adenylate kinase (adk). Results demonstrate that the new primer sets are more reliable in detecting 'Ca. L. solanacearum' in field and glasshouse samples than the currently used LsoF/OI2 primers. The newly developed primers differentiated between 'Ca. L. solanacearum' and a closely related 'Ca. Liberibacter' spp. and were more sensitive than the LsoF/OI2 primers. The low detection limit for the new primers was four times lower (0.65 ng) than the limit (2.5 ng) for the LsoF/OI2 primers.

[1]  C. Tamborindeguy,et al.  Translocation of 'Candidatus Liberibacter solanacearum', the Zebra Chip pathogen, in potato and tomato. , 2011, Phytopathology.

[2]  J. Elphinstone,et al.  Rapid phylogenetic identification of members of the Pseudomonas syringae species complex using the rpoD locus , 2011 .

[3]  D. Daffonchio,et al.  'Candidatus Liberibacter europaeus' sp. nov. that is associated with and transmitted by the psyllid Cacopsylla pyri apparently behaves as an endophyte rather than a pathogen. , 2011, Environmental microbiology.

[4]  Wenbin Li,et al.  Detection, Distribution, and Genetic Variability of 'Candidatus Liberibacter' Species Associated with Zebra Complex Disease of Potato in North America. , 2009, Plant disease.

[5]  S. Pennycook,et al.  'Candidatus Liberibacter solanacearum', associated with plants in the family Solanaceae. , 2009, International journal of systematic and evolutionary microbiology.

[6]  K. Kubota,et al.  Evaluation of genetic diversity among 'Candidatus Liberibacter asiaticus' isolates collected in Southeast Asia. , 2009, Phytopathology.

[7]  Yongping Duan,et al.  Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip. , 2009, Journal of microbiological methods.

[8]  C. Nansen,et al.  Knockdown Mortality, Repellency, and Residual Effects of Insecticides for Control of Adult Bactericera cockerelli (Hemiptera: Psyllidae) , 2009, Journal of economic entomology.

[9]  S. D. De Boer,et al.  Association of 'Candidatus Liberibacter solanacearum' with Zebra Chip Disease of Potato Established by Graft and Psyllid Transmission, Electron Microscopy, and PCR. , 2009, Plant disease.

[10]  J. Crosslin,et al.  Evidence that the Zebra Chip Disease and the Putative Causal Agent Can be Maintained in Potatoes by Grafting and In Vitro , 2009, American Journal of Potato Research.

[11]  L. Ward,et al.  A New 'Candidatus Liberibacter' Species Associated with Diseases of Solanaceous Crops. , 2009, Plant disease.

[12]  M. López,et al.  Molecular Tools for Detection of Plant Pathogens 13 1 Are Molecular Tools Solving the Challenges Posed by Detection of Plant Pathogenic Bacteria and Viruses ? , 2016 .

[13]  J. Anderson,et al.  A New 'Candidatus Liberibacter' Species in Solanum tuberosum in New Zealand. , 2008, Plant disease.

[14]  T. Paine,et al.  A New Huanglongbing Species, “Candidatus Liberibacter psyllaurous,” Found To Infect Tomato and Potato, Is Vectored by the Psyllid Bactericera cockerelli (Sulc) , 2008, Applied and Environmental Microbiology.

[15]  U. Sagaram,et al.  In planta distribution of 'Candidatus Liberibacter asiaticus' as revealed by polymerase chain reaction (PCR) and real-time PCR. , 2008, Phytopathology.

[16]  J. Crosslin,et al.  Association ofBactericera cockerelli (Homoptera: Psyllidae) with “Zebra Chip,” a New Potato Disease in Southwestern United States and Mexico , 2007, Journal of economic entomology.

[17]  Wenbin Li,et al.  Evaluation of DNA Amplification Methods for Improved Detection of "Candidatus Liberibacter Species" Associated with Citrus Huanglongbing. , 2007, Plant disease.

[18]  M. Maiden.  Multilocus sequence typing of bacteria. , 2006, Annual review of microbiology.

[19]  J. Côté,et al.  Phylogenetic analysis of gamma-proteobacteria inferred from nucleotide sequence comparisons of the house-keeping genes adk, aroE and gdh: comparisons with phylogeny inferred from 16S rRNA gene sequences. , 2006, The Journal of general and applied microbiology.

[20]  J. Bové,et al.  Huanglongbing: a destructive, newly-emerging, century-old disease of citrus [Asia; South Africa; Brazil; Florida] , 2006 .

[21]  Jun Liu,et al.  Monitoring the expression patterns of potato genes associated with quantitative resistance to late blight during Phytophthora infestans infection using cDNA microarrays , 2005 .

[22]  Howard Ochman,et al.  Identification and phylogenetic sorting of bacterial lineages with universally conserved genes and proteins. , 2004, Environmental microbiology.

[23]  M. Maiden,et al.  Multi-locus sequence typing: a tool for global epidemiology. , 2003, Trends in microbiology.

[24]  J. Burdon,et al.  Conflicting phylogeographic patterns in rRNA and nifD indicate regionally restricted gene transfer in Bradyrhizobium. , 2002, Microbiology.

[25]  J. Young,et al.  The glutamine synthetases of rhizobia: phylogenetics and evolutionary implications. , 2000, Molecular biology and evolution.

[26]  T. Iwanami,et al.  Comparison of 16S rDNA and 16S/23S Intergenic Region Sequences Among Citrus Greening Organisms in Asia. , 2000, Plant disease.

[27]  M. Achtman,et al.  Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[28]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[29]  J. Bové,et al.  Comparison of the 16S/23S ribosomal intergenic regions of "Candidatus Liberobacter asiaticum" and "Candidatus Liberobacter africanum," the two species associated with citrus huanglongbing (greening) disease. , 1997, International journal of systematic bacteriology.

[30]  H. Philippe,et al.  16S rRNA and 16S to 23S internal transcribed spacer sequence analyses reveal inter- and intraspecific Bifidobacterium phylogeny. , 1996, International journal of systematic bacteriology.

[31]  J. Bové,et al.  The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria. , 1994, International journal of systematic bacteriology.

[32]  F. Gannon,et al.  The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria. , 1991, PCR methods and applications.

[33]  J. Doyle,et al.  Isolation of plant DNA from fresh tissue , 1990 .