Genome-based Comparative Analysis And Molecular Specific Detection Of Xylella Fastidiosa

Xylella fastidiosa is a Gram-negative, nutrition-fastidious, xylem-inhabiting and insecttransmitted bacterium. This bacterium has a very wide host range, affecting more than 30 plant families including both mono- and dicotyledonous plants. Agriculturally important plant diseases caused by strains of X. fastidiosa include Pierce?s disease of grape in the United States, and citrus variegated chlorosis in Brazil. X. fastidiosa also causes bacterial leaf scorch in many important woody ornamentals including mulberry, sycamore, oleander, etc. The genomic sequences of the strains infecting landscape trees are remaining unknown and the molecular detection methods for differentiating and diagnosing these strains and the diseases that they cause are still limited. Focusing on the stains that cause bacterial leaf scorch of landscape tress and ornamentals, this study determined the genomic sequences of the strain Mul-MD and Sy-VA. Based on the genomic comparison, we designed and validated two molecular detection methods using real-time quantitative PCR and conventional PCR assay. This study would be advantageous for epidemiological and etiological studies on the diseases caused by X. fastidiosa.1. Determined the draft genomes of the strains Mul-MD and Sy-VA that cause the bacterial leaf scorch of mulberry and sycamore, respectively. Sequenced the genomic sequences of the two strains using Roche 454 GS, and then submitted to NCBI with assembly and annotation. The Mul-MD genome contains 2,543,372 bp and has a GC content of 51.65%. A total of 2,286 protein-encoding genes were predicted. The Sy-VA genome contains 2,477,829 bp and has a GC content of 51.64%. A total of 2,231 protein-encoding genes were predicted. In addition, an ~25-kb plasmid sequence was found that is similar to the four plasmids that are present individually in four California mulberry strains of X. fastidiosa.2. Created a TaqMan-based real time PCR assay for specific detection and quantification of X. fastidiosa strains causing oleander leaf scorch. A Bi-directional BLAST including five genomes of X. fastidiosa was carried out to find out unique sequences that are present in the genome of oleander strain Ann-1. We designed a specific primer pair and fluorescent probe based on the unique sequence and tested pure cultures and genomic DNAs of 30 bacteria. This specific primer pair and probe only recognized the oleander strains, not other strains of X. fastidiosa strains nor other plant-associated bacteria tested. The assay is also sensitive, with a detection limit of 10.4 fg DNA of X. fastidiosa per reaction in vitro and in planta. This assay can also be developed into a multiplex q PCR assay if needed in the future. Such a multiplexed assay would be advantageous for epidemiological and etiological studies, since it would identify and quantify the strain(s) of X. fastidiosa that are harbored by a particular plant or insect vector to improve management of the diseases caused by X. fastidiosa.3. Developed a conventional PCR assay for specifically detecting the strains causing mulberry leaf scorch. We designed and validated two sets of PCR primers specific for mulberry-infecting strains of X. fastidiosa based on the nucleotide sequence of a unique open reading frame identified only in mulberry-infecting strains among all the North and South American strains of X. fastidiosa sequenced so far. PCR using either set of primers allowed specific detection and identification of mulberry-infecting X. fastidiosa strains in cultures and in samples collected from naturally infected mulberry trees. In addition, no mixed infections with or unspecific detections of the mulberry-infecting strains of X. fastidiosa were found in naturally-infected oak, elm and sycamore trees that tested positive for X. fastidiosa from the same region where naturally infected mulberry trees were detected. This genotype-specific PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. Unexpectedly, the unique open reading frame conserved only in the mulberry-infecting strains in the U. S. was also identified in the recently sequenced olive strain Co Di RO isolated from Italy, so the two primer sets might also be useful for specific detection and differentiation of the olive strains in Italy to aid a better understanding of the evolution and biology of this new group of X. fastidiosa strains.