TaqMan Quantitative PCR Detection of Xylella taiwanensis in Taiwan.

Xylella taiwanensis (Xt) is a nutritionally fastidious bacterial pathogen causing pear leaf scorch disease (PLSD) in Taiwan. The disease causes early defoliation, loss of tree vigor, and reduction in fruit yield and quality. No cure for PLSD is available. The only option for growers to control the disease is to use pathogen-free propagation material, which requires early and accurate detection of Xt. Currently, only one simplex PCR method is available for the diagnosis of PLSD. We developed five Xt-specific TaqMan quantitative PCR (TaqMan qPCR) systems (primers-probe sets) for the detection of Xt. The PCR systems target three conserved genomic loci commonly used in bacterial pathogen detection: the 16S rRNA gene (rrs), the 16S-23S rRNA intergenic transcribed sequence (16S-23S rRNA ITS), and the DNA gyrase gene (gyrB). BLAST analysis using the GenBank nr sequence database, including whole genome sequences of 88 Xanthomonas campestris pv. campestris (Xcc) strains, 147 X. fastidiosa (Xf) strains, and 32 Xt strains, showed that all primer and probe sequences were specific only to Xt. Single nucleotide polymorphisms (SNPs) provided the primer/probe specificity to Xt. The PCR systems were evaluated by using DNA samples from pure cultures of two Xt strains, one Xf strain, one Xcc strain, and 140 plant samples collected from 23 pear orchards in four counties in Taiwan. The two-copy rrs and 16S-23S rRNA ITS-based PCR systems (Xt803-F/R, Xt731-F/R, and Xt16S-F/R) showed higher detection sensitivity than the two single-copy gyrB-based systems (XtgB1-F/R and XtgB2-F/R). A metagenomic analysis of a representative PLSD leaf sample detected the presence of non-Xt proteobacteria and fungal pathogens that should be taken into consideration in PLSD, as they might interfere with diagnosis.

Genetic differentiation of Xylella fastidiosa following the introduction into Taiwan.

The economically important plant pathogen Xylella fastidiosa has been reported in multiple regions of the globe during the last two decades, threatening a growing list of plants. Particularly, X. fastidiosa subspecies fastidiosa causes Pierce's disease (PD) of grapevines, which is a problem in the USA, Spain, and Taiwan. In this work, we studied PD-causing subsp. fastidiosa populations and compared the genome sequences of 33 isolates found in Central Taiwan with 171 isolates from the USA and two from Spain. Phylogenetic relationships, haplotype networks, and genetic diversity analyses confirmed that subsp. fastidiosa was recently introduced into Taiwan from the Southeast USA (i.e. the PD-I lineage). Recent core-genome recombination events were detected among introduced subsp. fastidiosa isolates in Taiwan and contributed to the development of genetic diversity. The genetic diversity observed includes contributions through recombination from unknown donors, suggesting that higher genetic diversity exists in the region. Nevertheless, no recombination event was detected between X. fastidiosa subsp. fastidiosa and the endemic sister species Xylella taiwanensis, which is the causative agent of pear leaf scorch disease. In summary, this study improved our understanding of the genetic diversity of an important plant pathogenic bacterium after its invasion to a new region.

Complete Genome Sequence of Xylella taiwanensis and Comparative Analysis of Virulence Gene Content With Xylella fastidiosa.

The bacterial genus Xylella contains plant pathogens that are major threats to agriculture in America and Europe. Although extensive research was conducted to characterize different subspecies of Xylella fastidiosa (Xf), comparative analysis at above-species levels was lacking due to the unavailability of appropriate data sets. Recently, a bacterium that causes pear leaf scorch (PLS) in Taiwan was described as the second Xylella species (i.e., Xylella taiwanensis; Xt). In this work, we report the complete genome sequence of Xt type strain PLS229T. The genome-scale phylogeny provided strong support that Xf subspecies pauca (Xfp) is the basal lineage of this species and Xylella was derived from the paraphyletic genus Xanthomonas. Quantification of genomic divergence indicated that different Xf subspecies share ∼87-95% of their chromosomal segments, while the two Xylella species share only ∼66-70%. Analysis of overall gene content suggested that Xt is most similar to Xf subspecies sandyi (Xfs). Based on the existing knowledge of Xf virulence genes, the homolog distribution among 28 Xylella representatives was examined. Among the 11 functional categories, those involved in secretion and metabolism are the most conserved ones with no copy number variation. In contrast, several genes related to adhesins, hydrolytic enzymes, and toxin-antitoxin systems are highly variable in their copy numbers. Those virulence genes with high levels of conservation or variation may be promising candidates for future studies. In summary, the new genome sequence and analysis reported in this work contributed to the study of several important pathogens in the family Xanthomonadaceae.

Draft Genome Sequence of "Candidatus Sulcia muelleri" Strain KPTW1 from Kolla paulula, a Vector of Xylella fastidiosa Causing Pierce's Disease of Grapevine in Taiwan.

The genome of "Candidatus Sulcia muelleri" strain KPTW1 from Kolla paulula, a vector of Xylella fastidiosa that causes Pierce's disease (PD) of grapevine in Taiwan, was sequenced. The strain has a genome size of 253,942 bp, GC content of 22.7%, 237 predicted protein-coding genes, and 34 RNA genes.