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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.

Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to agricultural cropping systems.

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.

Severe Verticillium dahliae attacks have occurred in artichoke crops in the Comunidad Valenciana region of eastern-central Spain since the late 1990s. Knowledge of genetic and virulence diversity in the pathogen population is a key factor for the management of the disease through disease risk assessment as well as development and use of resistant cultivars. V. dahliae isolates from artichoke (109 isolates) and cotton (three isolates) in that region were characterized by vegetative compatibility grouping (VCG), and specific polymerase chain reaction assays using three sets of primer pairs that differentiate the cotton-defoliating (D) and -nondefoliating (ND) V. dahliae pathotypes. In all, 35 and 39 V. dahliae isolates representative of the identified VCGs and geographic origins were tested for virulence to artichoke cvs. Nun 6374 and Nun 9444, and cotton cv. Acala SJ-2, respectively. Four VCGs were identified among 107 artichoke isolates, and 2 isolates were heterokaryon self-incompatible: VCG1A (one isolate), VCG2A (31 isolates), VCG2B (72 isolates), and VCG4B (three isolates). The three cotton isolates were VCG1A. Isolates in VCG2B were distributed across the region and were the most prevalent isolates in the northern part. Conversely, 83.9% of isolates in VCG2A were recovered from the southern part of the region. Two subgroups of isolates were identified in VCG2B based on heterokaryon compatibility with either international or local tester isolates, which further showed diversity in the amplification of 334- and 824-bp DNA fragments which are markers of the D and ND pathotypes, respectively. Virulence of isolates to artichoke and cotton correlated with VCG but the pattern of correlation varied with the host. VCG1A isolates from artichoke and cotton induced defoliation in cotton but not in artichoke. Collectively, isolates of VCG2B and VCG4B were the most virulent and isolates of VCG1A or HSI were the least virulent to artichoke; but isolates of VCG1A were more virulent to cotton than those of any other VCG. Also, molecular subgrouping in VCG2B determined by amplification of the 334- and 824-bp markers correlated with virulence of isolates to the two hosts tested.

In Spain, Verticillium wilt, caused by Verticillium dahliae, is the most important disease of cotton and olive. Isolates of V. dahliae infecting these crops can be classified into highly virulent, defoliating (D), and mildly virulent, nondefoliating (ND), pathotypes. Infested soil is the primary source of inoculum for Verticillium wilt epidemics in cotton and olive, and severity of disease relates to the prevailing V. dahliae pathotype. In this work we have adapted the use of previously developed primer pairs specific for D and ND V. dahliae for the detection of these pathotypes by nested PCR in artificial and natural soils. Success in the detection procedure depends upon efficiency in extracting PCR-quality DNA from soil samples. We developed an efficient DNA extraction method from microsclerotia infesting the soil that includes the use of acid washed sand during the grinding process and skimmed milk to avoid co-purification of Taq-polymerase inhibitors with DNA. The specific nested-PCR procedure effectively detected 10 or more microsclerotia per gram of soil. The detection procedure has proven efficient when used with a naturally infested soil, thus demonstrating usefullness of the diagnostic method for rapid and accurate assessment of soil contamination by V. dahliae pathotypes.

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.