Identification and Diferentiation of Verticillium Species with PCR Markers and Sequencing of ITS Region

The genus Verticillium is a group of ascomycete fungi, including plant-pathogenic species capable of affecting the vasculature of many agricultural crops, and therefore causes major economic losses worldwide. In 2011, a new taxonomic classification of the genus was proposed, which is now referred to as Verticillium sensu stricto, comprising ten species: V. dahliae V. albo-atru, V. alfalfae, V. longisporum, V. nonalfalfae, V. tricorpus, V. zaregamsianum, V. nubilum, V. isaacii and V. klebahnii.

We screened our Verticillium spp. collection with the new taxonomic PCR-markers using simplex and multiplex PCR assays. In addition, we also evaluated an alternative ITS-sequence based approach for the identification purposes.

The new PCR-marker analysis enabled us to confirm the identity of 88 isolates from various geographic locations in Europe, North America and Japan and from various host plants, including hop, potato, tomato, cotton, olive and alfalfa. According to the new taxonomic classification, 41 isolates were identified as V. nonalfalfae, 28 as V. dahliae, 6 as V. albo-atrum, 5 as V. alfalfa, 3 as V. isaacii, 2 as V. tricorpus, 1 as V. longisporum lineage A1/D1 and 1 as V. nubilum. Identification and differentiation of V. longisporum linegaes was performed by multiplex PCR assay.  

We were also able to determine all isolates by means of sequence ITS analysis, reflecting two main groups of isolates, Flavexudans and Flavnonexudans. Moreover, based on ITS sequencing analysis, we were able to obtain additional information on the subgroups within the species V. albo-atrum and V. dahliae that were not revealed by PCR-marker analysis.