Occurrence and genetic diversity of Verticillium longisporum lineages in Europe and their interaction with oilseed rape, Brassica napus L.

Abstract

The current threat that V. longisporum exerts on oilseed rape production, as well as the knowledge gaps in the biology of this pathogen, have motivated the performance of this study, which aimed to further characterize the genetic and pathogenicity diversity of V. longisporum. Additionally, due to limited resistance against V. longisporum in the current oilseed rape varieties (Friedt and Snowdon 2010), oilseed rape double haploid lines (DH) were evaluated for future quantitative trait locus (QTL) mapping and breeding purposes. The phylogenetic analysis by Inderbitzin et al. (2011), based on five protein-coding genes and the nuclear ribosomal internal transcribed spacer region (ITS), revealed that V. longisporum consists of three lineages (A1/D1, A1/D2, and A1/D3) that originated from three independent hybridization events of four haploid Verticillium parents. The first objective of this study was the performance of an extensive lineage monitoring in the main oilseed rape growing regions in Europe and Canada. The lineage monitoring, which was carried out with a multiplex-PCR according to Inderbitzin et al. (2013), revealed that A1/D1 was the dominant lineage in oilseed rape. A phylogenetic analysis of A1/D1 isolates based on genotyping by sequencing (GBS) was conducted to determine if genetic subgroups exist within the A1/D1 lineage. The A1/D1 genetic clusters were, in general, independent of geographic region or field. The pathogenicity characterization in the greenhouse of A1/D1 isolates revealed pathogenicity diversity within the lineage, also within one genetic cluster and among isolates from the same field. The limited availability of control measures calls for the investigation of new alternatives, such as the use of microorganisms as biocontrol agents (Deketelaere et al. 2017; Yadeta and Thomma 2013). Thus, the biocontrol ability of the non-aggressive A1/D2 lineage was investigated in oilseed rape. The study revealed a biocontrol effect of A1/D2 against a V. longisporum aggressive A1/D1 isolate with root-dip inoculation. The induction of salicylic acid (SA), a signal in basal and cultivar-related resistance against V. longisporum (Zheng et al. 2019a), was not involved in the biocontrol mechanism. Applying a biocontrol agent as a seed coat is the most practical and economically feasible application method in fields of arable crops (Alabouvette et al. 2009; Rocha et al. 2019). However, the biocontrol effect of A1/D2 was lost with this application technique. Confocal microscopy analyses revealed that seed coating resulted in less A1/D2 hyphal establishment in the roots than the root-dip inoculation, which might explain the lack of A1/D2 biocontrol effect with a seed coat. The non-aggressive interaction of V. longisporum lineage A1/D2 with oilseed rape was compared to the aggressive interaction of an A1/D1 isolate. Phenotypic assessments in the greenhouse confirmed the non-aggressiveness of A1/D2 on oilseed rape. The external root colonization assessment by confocal microscopy revealed sporulation, hyphal coiling, and unorganized outward hyphal growth by A1/D2 at the rhizosphere, in contrast to the organized net-like hyphal growth of A1/D1 close to the root surface. Electron microscopy and qPCR assays revealed minimal vascular colonization by A1/D2 in the roots and stems, respectively. Additionally, electron microscopy observations showed a more intense xylem vessel coating in A1/D2-treated samples than in A1/D1-treated samples, as well as stronger secondary vessel wall degradation by A1/D1 than by A1/D2. The results indicate that low penetration at the root surface, a low vascular colonization by A1/D2, and a higher intensity of plant defense reactions upon A1/D2 infection in comparison to A1/D1 might explain the low pathogenicity of the lineage A1/D2. Intensive breeding for quality traits in the past decades and the subsequent reduction of the genetic basis of the species has led to limited resistance against V. longisporum in the current oilseed rape varieties (Friedt and Snowdon 2010). Thus, the final goal of this study consisted of the evaluation of oilseed rape double haploid lines (DH) for future QTL mapping and breeding purposes. The greenhouse and field assessments revealed relatively high variability within the DH populations in their response to V. longisporum. There was a low correlation between greenhouse and field symptoms, which evidences the difficulty of performing disease screenings in field conditions.