Genetic diversity of Pierce's disease strains and other pathotypes of Xylella fastidiosa

Abstract

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.

FIG. 1

REP-PCR analysis of X. fastidiosa strains. ERIC-PCR profiles of X. fastidiosa strains from diverse plant hosts or geographical locations are shown. Lane 1, grape isolate from Georgia (strain R116V3); lane 2, grape from Florida (PD95-2); lane 3, grape from California (Stags Leap); lane 4, maple from California; lane 5, grape from California (Conn Creek); lane 6, almond (ALS1); lane 7, almond (ALS2); lane 8, almond (ALS3); lane 9, 1-kb size standard; lane 10, oak from Florida (88-9); lane 11, oak from Georgia (OLS#2); lane 12, oleander (Ann1); lane 13, oleander (TR1); lane 14, water control.

FIG. 2

Agarose gel electrophoresis of RAPD-PCR products (primer OP-AA-01) synthesized using the template DNA of various strains. Lane 1, 1-kb ladder; lane 2, strain ALS2; lane 3, ALS3; lane 4, ALS4; lane 5, ALS5; lane 6, ALS7; lane 7, ALS9; lane 8, Dixon; lane 9, ALS6; lane 10, Contra Costa; lane 11, ALS1; lane 12, Manteca; lane 13, Tulare; lane 14, Conn Creek; lane 15, Douglas; lane 16, Hopland; lane 17, Medeiros; lane 18, Meyley; lane 19, Moore Park; lane 20, Oxford; lane 21, 1-kb ladder; lane 22, Santa Cruz; lane 23, Traver; lane 24, UCLA; lane 25, 95-2; lane 26, 95-4; lane 27, 95–9; lane 28, R116V3; lane 29, R118V3-4; lane 30, Maple; lane 31, T1c; lane 32, TR1; lane 33, Plum 2#4; lane 34, 4S3; lane 35, 5R1; lane 36, 5S2; lane 37, Oak 88-9; lane 38, Oak 92–3; lane 39, Oak 92–10; lane 40, Stucky.

FIG. 3

Phylogram based on distance data obtained by RAPD-PCR analysis. A distance matrix was constructed by pairwise comparison of the RAPD PCR products among strains. The Jaccard similarity coefficient (32) was used (S = 2n_xy/n_x + n_y), where n_xy is the number of PCR products common to strains x and y, n_x is the number of bands present in strain x, and n_y is the number of bands present in strain y. The scale bar indicates relative dissimilarity between strains. A, almond; G, grape; M, maple; O, oak; Ol, oleander; P, peach; Pl, plum.

FIG. 4

CHEF-agarose gel electrophoresis of the genomic DNA of various strains digested with enzyme NotI digests. Lane 1, one concatemer; lane 2, UCLA; lane 3, Douglas; lane 4, 95-4; lane 5, R118V3-4; lane 6, ALS1; lane 7, Manteca; lane 8, Maple; lane 9, ALS2; lane 10, ALS3; lane 11, ALS5; lane 12, ALS6; lane 13, 5S2; lane 14, 5R1; lane 15, Plum 2#4; lane 16, Oak 92-3; lane 17, Stucky; lane 18, Ann1; lane 19, TR1; lane 20, one concatemer.

FIG. 5

Phylogram based on distance data obtained by CHEF-gel electrophoresis of NotI- and SpeI-restricted genomic DNA. A distance matrix was constructed by pairwise comparison of the RAPD-PCR products among strains. The Jaccard similarity coefficient (32) was used. The scale bar indicates relative dissimilarity between strains. A, almond; G, grape; M, maple; O, oak; Ol, oleander; P, peach; and Pl, plum.

FIG. 6

Agarose gel electrophoresis of HindIII-digested plasmid DNA from various X. fastidiosa strains. Lane 1, 1-kb ladder; lane 2, strain UCLA; lane 3, Tulare; lane 4, ALS4; lane 5, ALS6; lane 6, 95-2; lane 7, 95-4; lane 8, peach; lane 9, Plum 2#4; lane 10, Oak 88-9; lane 11, Ann1; lane 12, 1-kb ladder.