Simultaneous Detection of the Defoliating and Nondefoliating Verticillium dahliae Pathotypes in Infected Olive Plants by Duplex, Nested Polymerase Chain Reaction

Abstract

Pathogen-free certified planting material and accurate detection of Verticillium dahliae pathotypes infecting the plant are key components of successful management of Verticillium wilt of olive. Use of a nested-polymerase chain reaction (PCR) procedure developed in earlier studies for in planta detection of the defoliating (D) and nondefoliating (ND) V. dahliae pathotypes resulted in ambiguous detection of the pathogen in some cases, due to heterologous amplification of the D-associated marker in ND-infected olive plants. In the present study, an improved procedure was developed that eliminates ambiguity and reduces time and cost for detection of D and ND V. dahliae in olive. The improved procedure is based on the simultaneous amplification of both an ND- and a new D-specific marker by means of duplex, nested PCR. The procedure was effective in the rapid and unequivocal detection of the D and ND V. dahliae in both artificially inoculated, own-rooted olive plants and naturally infected adult olive trees of different cultivar, age, and growing conditions. Furthermore, the duplex, nested-PCR procedure detected simultaneously the D and ND pathotypes in adult olive trees naturally infected by both pathotypes and in young olive plants that were double-inoculated with D and ND isolates under controlled conditions.

Spatiotemporal Analysis of Spread of Infections by Verticillium dahliae Pathotypes Within a High Tree Density Olive Orchard in Southern Spain

“…dahliae isolates were further characterized to species and pathotype by multiplex PCR assay using three primers, DB19/ DB22/espdef01 (33). The use of these three primers in a single PCR assay yields a polymorphic amplicon of 539 or 523 bp which are specific to V. dahliae and differentially associated with D and ND V. dahliae, respectively.…”

“…The use of these three primers in a single PCR assay yields a polymorphic amplicon of 539 or 523 bp which are specific to V. dahliae and differentially associated with D and ND V. dahliae, respectively. PCR amplifications and gel electrophoresis were conducted as described previously (33).…”

“…This knowledge may be particularly relevant for Verticillium wilt of olive (Olea europaea L.) in southern Spain, caused by the soilborne fungus Verticillium dahliae Kleb., because of the increasing importance and distribution of the disease and occurrence of a highly virulent defoliating (D) V. dahliae pathotype in irrigated olive orchards during the last decade (23,33,42).…”

“…V. dahliae isolates infecting olive can be classified into defoliating (D) or nondefoliating (ND) pathotypes, based on their ability to cause defoliation or no defoliation of green leaves from shoots and twigs (28,40). These two pathotypes can be differentiated in infected plants by means of specific polymerase chain reaction (PCR) markers (33). Infections by the D pathotype can be lethal to the plant, whereas olive trees infected by the ND pathotype eventually can show complete remission from symptoms (23,33,40).…”

“…These two pathotypes can be differentiated in infected plants by means of specific polymerase chain reaction (PCR) markers (33). Infections by the D pathotype can be lethal to the plant, whereas olive trees infected by the ND pathotype eventually can show complete remission from symptoms (23,33,40). Differential virulence of V. dahliae isolates also occurs in upland cotton (Gossypium hirsutum L.), with isolates from cotton and olive showing cross-virulence (40,43).…”