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

Abstract

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 ‘Candidatus Liberibacter’ among which ‘Ca. Liberibacter asiaticus’ (CLas) has most wide distribution. Recently, a number of studies identified different regions in the CLas genome with variable number of tandem repeats (VNTRs) that could be used for examination of CLas 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 CLas populations from Florida. Our results showed that the numbers of tandem repeats in the four loci tested display very distinguishable “signature profiles” for the two Florida-type CLas 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 CLas 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 CLas. 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.

Deep Characterization of the Microbiomes of Calophya spp. (Hemiptera: Calophyidae) Gall-Inducing Psyllids Reveals the Absence of Plant Pathogenic Bacteria and Three Dominant Endosymbionts

“…Psyllid DNA was extracted from three sub-samples of each population, using the DNeasy Blood & Tissue Kit (Qiagen, Venlo Netherlands) as reported for the extraction of DNA from the Asian citrus psyllid, Diaphorina citri [ 31 ]. DNA from twenty psyllids (a group) was extracted using the manufacturer’s instructions.…”

Huanglongbing: An overview of a complex pathosystem ravaging the world's citrus

“…They hypothesized that the pathogen was likely introduced to Thailand from China and/or the Philippines based on their phylogenetic analyses. Matos et al (2013) used four microsatellite markers to investigate diversity from HLB isolates from Florida, USA, Mexico and various parts of the Caribbean. Similarly to Chen et al (2010), lower genotypic diversity was found in Florida compared to the other regions sampled where the disease has been prevalent for a longer period of time.…”

Origin of the Outbreak in France of Pseudomonas syringae pv. actinidiae Biovar 3, the Causal Agent of Bacterial Canker of Kiwifruit, Revealed by a Multilocus Variable-Number Tandem-Repeat Analysis

“…MLVA was used in an epidemiological survey to trace the routes of Haemophilus influenzae outbreaks (8) or Bacillus anthracis (2,9) outbreaks. MLVA was applied to monomorphic plant-pathogenic bacteria belonging to different genera and species such as Xylella fastidiosa (10), Xanthomonas citri (11), Ralstonia solanacearum (12), "Candidatus Liberibacter asiaticus" (13), Erwinia amylovora (14), and Xanthomonas arboricola pathovars (15). MLVA was first applied on Pseudomonas syringae by Gironde and Manceau (16) and provided new insights into host specificity of P. syringae pathogenic on brassicaceous and solanaceous plants.…”