Molecular Variability Within and Among Verticillium dahliae Vegetative Compatibility Groups Determined by Fluorescent Amplified Fragment Length Polymorphism and Polymerase Chain Reaction Markers

Abstract

A degree of genetic diversity may exist among Verticillium dahliae isolates within vegetative compatibility groups (VCGs) that bears phytopathological significance and is worth investigating using molecular tools of a higher resolution than VCG characterization. The molecular variability within and among V. dahliae VCGs was studied using 53 artichoke isolates from eastern-central Spain, 96 isolates from cotton, 7 from cotton soil, and 45 from olive trees in countries of the Mediterranean Basin. Isolates were selected to represent the widest available diversity in cotton- and olive-defoliating (D) and -nondefoliating (ND) pathotypes, as well as for VCG. The VCG of 96 cotton and olive isolates was determined in this present study. Molecular variability among V. dahliae isolates was assessed by fluorescent amplified fragment length polymorphism (AFLP) analysis and by polymerase chain reaction (PCR) assays for DNA fragments associated with the D (462 bp) and ND (824 bp) pathotypes, as well as a 334-bp amplicon associated with D pathotype isolates but also present in some VCG2B isolates. Isolates from cotton were in VCG1A, VCG1B, VCG2A, VCG2B, and VCG4B and those from olive trees were in VCG1A, VCG2A, and VCG4B. Artichoke isolates included representatives of VCG1A, VCG2A, VCG2B (including a newly identified VCG2Ba), and VCG4B. AFLP data were used to generate matrixes of genetic distance among isolates for cluster analysis using the neighbor-joining method and for analysis of molecular variance. Results demonstrated that V. dahliae isolates within a VCG subgroup are molecularly similar, to the extent that clustering of isolates correlated with VCG subgroups regardless of the host source and geographic origin. VCGs differed in molecular variability, with the variability being highest in VCG2B and VCG2A. For some AFLP/VCG subgroup clusterings, V. dahliae isolates from artichoke grouped in subclusters clearly distinct from those comprising isolates from cotton and olive trees. In addition, VCG2B isolates from artichoke formed two distinct clusters that correlated with PCR markers of 334 bp (VCG2B(334)) or 824 bp (VCG2B(824)). Artichoke isolates in the VCG2B(334)/2beta(334) cluster were molecularly similar to isolates of VCG1A. The molecular difference found among artichoke isolates in VCG2B correlates with virulence of isolates to artichoke and cotton cultivars demonstrated in a previous study.

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

“…Other VCGs, either less prevalent or localized in well-defined geographical areas, have been reported as well (namely VCG3, VCG5, VCG6, VCGBr or VCGBa) (Bhat and Subbarao 1999;Bhat et al 2003;Joaquim and Rowe 1990;Strausbaugh et al 1992;Jiménez-Díaz et al 2006). Regarding to V. dahliae isolates infecting olive, only representatives of VCG1A, VCG2A, VCG2B, and VCG4B have so far been identified (Pérez-Artés et al 2000;Cherrab et al 2002;Tantaoui et al 2002;Tsror and Levin 2003;Bellahcene et al 2005b;Sanei et al 2005;Collado-Romero et al 2006;Dervis et al 2007Dervis et al , 2010 (Fig. 4).…”

“…Interesting examples pointing to an association between genetic groups and host specificity are: i) the predominance of isolates assigned to VCG4 in potato (Solanum tuberosum L.) infections in the United States and Israel (Joaquim and Rowe 1991;Strausbaugh et al 1992;Korolev et al 2000); ii) the aforementioned association of the highly-virulent, D-pathotype isolates of cotton (Gossypium hirsutum L.) with their assignment to VCG1A described in various areas such as Central Asia, China, Israel, Peru, Spain, Turkey and USA (Daayf et al 1995;Korolev et al 2001;Zhengjun et al 1998;Korolev et al 2008;Dervis et al 2008), in what may constitute an example of dispersal of isolates from an original focus of infected cotton genotypes; or iii) the prevalence of VCG2A and 2B isolates infecting artichoke (Cynara scolymus L.) in a defined artichokecultivating region of eastern-central Spain, with VCG2B being the most virulent in this host . Studies regarding to olive-infecting V. dahliae isolates, are indicating the following scenario: 1) highly-virulent isolates, inducing severe syndromes in olive, generally belong to the D pathotype; 2) all olive D-pathotype isolates have so far been assigned to VCG1A in all countries where this group/pathotype has been reported (Pérez-Artés et al 2000;Collado-Romero et al 2006;Dervis et al 2010); and 3) olive isolates assigned to other VCGs (namely, VCG2 and VCG4) have been characterized as ND pathotype either by pathogenicity testing or by PCR-based molecular pathotyping (Fig. 4).…”

“…Indeed, from the variety of molecular markers identified, proper selection can be made from those ones clearly associated with specific groups or linked to a particular phenotypic trait (i.e., pathotype). Thus, specific DNA sequences can be identified to design VCG-specific or pathotype-specific primers to be used in PCR-based detection and diagnosis procedures of V. dahliae isolates infecting olive or any other host (Pérez-Artés et al 2000;Mercado-Blanco et al 2003b;Collado-Romero et al 2006, 2009 (Fig. 4).…”

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

“…In Spain, the D pathotype was first reported infecting cotton in a restricted area of intensive cultivation at the southern part of Andalusia in 1981 (2). This pathotype has now appeared to distant cotton-and olive-growing areas all over Andalusia (8), but has not yet been reported in other olive growing areas in northern Spain or other countries in the Mediterranean Basin, except Greece (11) and Israel (25).…”

Scientific opinion on the pest categorisation of Verticillium dahliae Kleb

“…The D pathotype was first recognised on cotton and olive in California, but it now occurs worldwide, e.g. in Peru, China, Iran, Tajikistan, Turkey and Israel (Dervis et al, 2007;Jiménez-Díaz et al, 2012;Milgroom et al, 2014) as well as in Greece, Italy and Spain (Bejarano-Alcázar et al, 1996;Collado-Romero et al, 2006;Colella et al, 2008;Jiménez-Diaz et al, 2011). The increased virulence of D isolates on olive compared with that of ND isolates is of significance for management of the disease because (i) the inoculum density of D pathotype needed for the development of severe Verticillium wilt is much lower than that of the ND pathotype and (ii) cultivars tolerant to the ND pathotype are highly susceptible to the D pathotype (Jiménez-Díaz et al, 2012).…”