Primers for characterization of tandem repeats in four <i>loci</i> of the genomes of ‘<i>Candidatus</i> Liberibacter asiaticus’ isolates.

<p>Primers for characterization of tandem repeats in four <i>loci</i> of the genomes of ‘<i>Candidatus</i> Liberibacter asiaticus’ isolates.</p

Validation of ‘Variable Number of Tandem Repeat’-Based Approach for Examination of ‘<i>Candidatus</i> Liberibacter asiaticus’ Diversity and Its Applications for the Analysis of the Pathogen Populations in the Areas of Recent Introduction

<div><p>Citrus greening (Huanglongbing, HLB) is one of the most destructive diseases of citrus worldwide. In South Asia HLB has been known for more than a century, while in Americas the disease was found relatively recently. HLB is associated with three species of ‘<i>Candidatus</i> Liberibacter’ among which ‘<i>Ca.</i> Liberibacter asiaticus’ (<i>C</i>Las) has most wide distribution. Recently, a number of studies identified different regions in the <i>C</i>Las genome with variable number of tandem repeats (VNTRs) that could be used for examination of <i>C</i>Las diversity. One of the objectives of the work presented here was to further validate the VNTR analysis-based approach by assessing the stability of these repeats upon multiplication of the pathogen in a host over an extended period of time and upon its passaging from a host to a host using <i>C</i>Las populations from Florida. Our results showed that the numbers of tandem repeats in the four <i>loci</i> tested display very distinguishable “signature profiles” for the two Florida-type <i>C</i>Las haplotype groups. Remarkably, the profiles do not change upon passage of the pathogen in citrus and psyllid hosts as well as after its presence within a host over a period of five years, suggesting that VNTR analysis-based approach represents a valid methodology for examination of the pathogen populations in various geographical regions. Interestingly, an extended analysis of <i>C</i>Las populations in different locations throughout Florida and in several countries in the Caribbean and Central America regions and in Mexico where the pathogen has been introduced recently demonstrated the dispersion of the same haplotypes of <i>C</i>Las. On the other hand, these CLas populations appeared to differ significantly from those obtained from locations where the disease has been present for a much longer time.</p></div

Polymerase chain reaction analyses of ‘<i>Candidatus</i> Liberibacter asiaticus’ isolates from Florida using primer set that amplifies Motif A-containing locus.

<p>Letters and numbers on top designate the following lanes: M, DNA markers; C-, control DNA from a healthy plant; C_HA and C_HB, positive controls obtained by using DNA from plants known to be infected with the HA or HB haplotypes, respectively; 1–9, various samples from Florida. Note double bands in lines 2, 3, and 6. Numbers on the right indicate DNA fragments sizes.</p

Analysis of ‘<i>Candidatus</i> Liberibacter asiaticus’ populations in the Caribbean and Central America countries and Mexico.

a<p>Analysis was done based on examination of the number of repeats in Motifs A and B.</p

Examination of a few ‘<i>Candidatus</i> Liberibacter asiaticus’ isolates from China, Japan, and Brazil.

<p>Examination of a few ‘<i>Candidatus</i> Liberibacter asiaticus’ isolates from China, Japan, and Brazil.</p

Analysis of the stability of tandem repeats upon transmission of the pathogen from field-grown mandarin infected with HB haplotype of ‘<i>Candidatus</i> Liberibacter asiaticus’.

a<p><i>C</i>Las-infected field-grown mandarin (Man-Field) used as inoculum source for graft transmission of the pathogen.</p>b<p>MV, <i>Citrus macrophylla</i> (CM), and DG plants graft-inoculated using tissue from the source Man-Field.</p>c<p>Number of clones contained a particular number of repeats out of total clones sequenced.</p

Analysis of the stability of tandem repeats over a period of five years.

a<p>N/A, not assayed.</p

Distribution of ‘<i>Candidatus</i> Liberibacter asiaticus’ haplotypes in Florida counties.

a<p>Geographical location of counties in Florida: C, Central; N, North; S, South; W, West; E, East.</p

Analysis of the stability of tandem repeats upon transmission of the pathogen from Duncan grapefruit infected with HA haplotype of ‘<i>Candidatus</i> Liberibacter asiaticus’.

a<p><i>C</i>Las-infected greenhouse-grown Duncan grapefruit (DG-GH) used as inoculum source for graft or psyllid transmission of the pathogen.</p>b<p>Psyllids that acquired the pathogen after feeding on DG-GH plant.</p>c<p>DG and MV plants graft-inoculated using tissue from the source DG-GH.</p>d<p>Receptor plants (DG, MV, <i>Citrus macrophylla</i>, Sun Chu Sha and Clementine mandarins, sour orange) that became infected upon psyllid transmission of the bacterium from the source DG.</p>e<p>Number of clones contained a particular number of repeats out of total clones sequenced.</p

Typical APCI-HPLC-MS chromatograms of ‘Hamlin’ and ‘Valencia’ leaf extracts and mass-spectra and UV spectra of unknown 5, 6, 7, and 9.

<p>A) selective ion recording chromatograms of “<i>m/z</i>” specie of 177 in the positive ion mode; <b>B</b>) negative electrospray ionization mass spectra ; <b>C</b>) UV spectra of unknown <b>5</b>, <b>6</b>, <b>7</b>, and <b>9</b>. </p