Identification of an emergent and atypical Pseudomonas viridiflava lineage causing bacteriosis in plants of agronomic importance in a Spanish region

Abstract

Pseudomonas strains with an atypical LOPAT profile (where LOPAT is a series of determinative tests: L, levan production; O, oxidase production; P, pectinolitic activity; A, arginine dihydrolase production; and T, tobacco hypersensibility) can be regarded as emergent pathogens in the Principality of Asturias (Spain), where they have been causing, since 1999, severe damage in at least three taxonomically unrelated orchard plants of agronomic importance: common bean (Phaseolus vulgaris), kiwifruit (Actinidia deliciosa), and lettuce (Lactuca sativa). These strains are mainly differentiated by production of yellowish mucoid material in hypersucrose medium, used for the levan test, and by a variable pectinolytic activity on different potato varieties. The atypical organisms were identified as Pseudomonas viridiflava based on their 16S rRNA sequences. Among them a certain intraspecies genetic heterogeneity was detected by randomly amplified polymorphic DNA (RAPD) typing. To differentiate between isolates of P. viridiflava and Pseudomonas syringae pathovars, a 16S ribosomal DNA restriction fragment length polymorphism method employing the restriction endonucleases SacI and HinfI was developed. This could be used as a means of reliable species determination after the usual phenotypical characterization, which includes the LOPAT tests.

FIG. 1.

Differential LOPAT tests of atypical Pseudomonas isolates. Growth on hypersucrose medium (L test) (A to C) and pectinolytic activity on potato slices (D to F) are shown. (A) P. viridiflava CECT 458 (negative control); (B) P. syringae pv. syringae CECT 4429 (positive control); (C) atypical P. viridiflava LPPA 144; (D) P. viridiflava CECT 458 (positive control); (E) P. syringae pv. syringae CECT 4429 (negative control); (F) atypical P. viridiflava LPPA 144.

FIG. 2.

Inferred phylogenetic relationships among the atypical isolates and validly described members of the genus Pseudomonas (sensu stricto). See Materials and Methods for accession numbers and details on the construction of the tree.

FIG. 3.

Restriction profiles of PCR-amplified 16S rDNA fragments from different phytopathogenic Pseudomonas. (A) Digestion with SacI; (B) digestion with HinfI. Lane L, molecular size standards; lanes 1 to 3, RFLP profiles corresponding to atypical isolates of P. viridiflava; lane 4, P. viridiflava CECT 458; lane 5, P. syringae pv. syringae LPPA 93/01; lane 6, P. syringae pv. syringae LPPA 136/00; lane 7, P. syringae pv. syringae CECT 4429; lane 8, P. syringae pv. phaseolicola CECT 321.

FIG. 4.

RAPD typing of atypical Pseudomonas isolates performed with primer S. Lanes 1 to 6, profiles R-1 to R-6, respectively, generated by atypical P. viridiflava isolates; lanes 10 to 13, R profiles corresponding to P. syringae pv. syringae strains; lane 20, R profile of P. syringae pv. phaseolicola; lane L, molecular size standards. The distribution of the isolates within different profiles is shown in Table 1.