Detection of the nondefoliating pathotype of Verticillium dahliae in infected olive plants by nested PCR

Abstract

An increasing incidence and distribution of verticillium wilt has occurred in the last few years in newly established olive orchards in southern Spain. This spread of the disease may result from use of Verticillium dahliae-infected planting material. The early in planta detection of the pathogen would aid the implementation of certification schemes for pathogen-free planting material. In this work, a nested polymerase chain reaction (PCR) method was developed for the in planta detection of the nondefoliating (ND) V. dahliae pathotype, aimed especially at nurseryproduced olive plants. For this purpose, specific primers were designed from the sequence of a 1958-bp random amplified polymorphic DNA (RAPD) marker of ND V. dahliae, and a procedure for the extraction of PCR-quality total genomic DNA from infected root and stem tissues of young olive plants was tested and further optimized. Nested PCR assays detected ND V. dahliae in 4-to 14-month-old artificially infected plants of three olive cultivars. The ND-specific PCR product was not amplified from total genomic DNA extracted from olive plants infected with the defoliating V. dahliae pathotype. Detection of the ND pathotype was effective from the very earliest moments following artificial inoculation of olive plants with a V. dahliae conidial suspension. Also, detection was achieved in inoculated, though symptomless, olive plants as well as in plants that were symptomatic but became symptomless by 217 days after inoculation.

Verticillium wilt of olive: a case study to implement an integrated strategy to control a soil-borne pathogen

“…As discussed above, heavy colonization of olive tissues by the pathogen clearly precedes disease symptoms development, as observed by both microscopy (Rodríguez-Jurado 1993;Prieto et al 2009) and molecular studies (Mercado-Blanco et al 2001, 2003a. However, little is known on mechanisms responsible of the wilting syndrome or on host defense responses in olive upon V. dahliae infection with the exception of those reported by Baídez et al (2007) and by Markakis et al (2010).…”

“…An EYFP (enhanced yellow fluorescent protein)-tagged derivative (VDAT-36I) of an olive-infecting D pathotype isolate was produced by ATMT. Interestingly, these processes were examined under non-gnotobiotic conditions, using nursery-produced olive plants with fully developed root systems and therefore harboring abundant and diverse naturally-resident microorganisms (Mercado-Blanco et al 2001). This provided an experimental design resembling conditions much closer to natural environments rather than those present in gnotobiotic systems frequently utilized in this type of study.…”

“…Detection of each V. dahliae pathotype DNA was effective from the very earliest moments after inoculation; that is, positive pathogen DNA detection was achieved in symptomless plants, much earlier than first VWO symptoms developed. Interestingly, olive plants inoculated with the ND pathotype which developed VWO but became symptomless 7 months after inoculation (i.e., they showed the recovery phenomenon, see below), were positive to the presence of pathogen DNA when examined by the nested-PCR assay (Mercado-Blanco et al 2001). These results confirmed that asymptomatic plants may constitute a reservoir of V. dahliae propagules, as previously discussed in this review.…”

“…Thus, an improved duplex, nested-PCR procedure was developed eliminating occasional ambiguities observed when detecting D isolates, and reducing time and cost for an effective detection of D and ND V. dahliae in olive tissues (Mercado-Blanco et al 2003b). The duplex, nested-PCR procedure is based on the simultaneous amplification of both the previously described ND PCR markers (Mercado-Blanco et al 2001) and a D-associated PCR marker designed from a V. dahliae species-specific genomic sequence , which has been revealed as highly polymorphic in subsequent studies (Collins et al 2005;Collado-Romero et al 2006;Collado-Romero et al 2010). The procedure was effective in the rapid, unequivocal and simultaneous detection of the two infecting V. dahliae pathotypes in both artificiallyinoculated, self-rooted olive plants and naturallyinfected adult trees of different cultivar, age and growing conditions (Mercado-Blanco et al 2003b).…”

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance

“…However, some improvements in the relative concentrations of the external and internal primers have permitted to perform the two reactions in a single closed tube supporting high throughput. This method has been widely used for detection and/or further characterisation of numerous fungi (Aroca & Raposo, 2007;Grote et al, 2002;Hong et al, 2010;Ippolito et al, 2002;Langrell et al, 2008;Meng and Wang 2010;Mercado-Blanco et al, 2001;Qin et al, 2011;Wu et al, 2011). …”