Detection and diversity assessment of Xylella fastidiosa in field-collected plant and insect samples by using 16S rRNA and gyrB sequences

Abstract

The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease.

FIG. 1.

Phylogenetic trees based on the nucleotide sequences of the 16S rRNA (A) and gyrB (B) genes of X. fastidiosa strains. Sequences of gyrB and 16S rRNA genes were submitted to GenBank under the following first and second accession numbers, respectively. When only one accession number is shown, it represents a gyrB sequence: Temecula (AF53460 and AF536760), PCE-RR (AF53461), PLM G83 (AF53462 and AF536761), RGW-R (AF53463 and AF536762), MUL-1 (AF53464), MUL-3 (AF53465 and AF536763), ELM-1 (AF53466 and AF536764), B14 (AF53468 and AF536765), SL1 (AF53469 and AF536766), CRS2 (AF534670 and AF536768), PWT-22 (AF53471), P3 (AF53472 and AF536769), ALS-BC (AF53473 and AF536770), and CM1 (AF53474 and AF536767). Unrooted trees were constructed by using the neighbor-joining method (27). The sequences for Xanthomonas campestris pv. campestris were treated as the outgroup. All sequences were generated during the present study, except for strains Ann1, PD, and Dixon. The scale is the expected number of substitutions per position. The numbers at the nodes represent percentage bootstrap values of 1,000 resamplings.

FIG. 2.

(A) Scheme of relative position of the 16S rRNA gene-specific primers for X. fastidiosa. (B) Ethidium bromide-stained agarose gel of PCR products after amplification of the 16S rRNA gene with eubacterial (Eub) or X. fastidiosa-specific (sets A to C) primers. The expected sizes for specific primer sets A, B, and C are 1,348, 745, and 603 bp, respectively. Lanes: 1, molecular marker λ restricted with HindIII and EcoRI; 2, 8, 14, and 20, X. fastidiosa strain 9a5c; 3, 9, 15, and 21, strain Temecula; 4, 10, 16, and 22, strain MUL1; 5, 11, 17, and 23, Xanthomonas campestris pv. campestris; 6, 12, 18, and 24, Xanthomonas axonopodis pv. citri; and 7, 13, 19, and 25, negative control (H₂O).

FIG. 3.

Sensitivity of X. fastidiosa detection by multiplex PCR. (A) Leaf disks of a known diseased citrus plant used for DNA extraction. Subpanels: 1, nonsymptomatic area; 2, chlorotic area; 3, necrotic area. (B) Ethidium bromide-stained gel of coamplified PCR products from citrus leaf disks. The expected sizes are 745 and 429 bp for 16S rRNA (set B)- and gyrB-specific primers, respectively. Lanes: 1, molecular marker λ restricted with HindIII and EcoRI (947, 831, and 564 bp); 2 and 5, DNA extracted from nonsymptomatic area; 3 and 6, chlorotic areas; 4 and 7, necrotic area; 8, known healthy plant; 9, negative control (H₂O); 10, molecular marker (100-bp ladder).