Verticillium longisporum in oilseed rape

Verticillium longisporum (VL) is a host-adapted, amphihaploid, vascular fungal pathogen causing stem striping of oilseed rape (Brassica napus L. spp. oleifera) (Zeise & Tiedemann, 2001, 2002; Depotter et al. 2016). The increased  intensity of production have rendered this disease an increasing threat to oilseed rape (OSR) production particularly in Europe, but it recently started to spread also in Canadian canola fields. Unlike other Verticillium diseases VL does not induce wilting, but premature senescence and ripening which may severely reduce yields (Dunker et al., 2008; Figs. 1, 2). Artificially inoculated plants in the greenhouse show a distinct stunting of the shoots (Fig. 3).

Host pathogen interaction

A closely related species to VL is V. dahliae (VD). This ubiquitous soilborne fungus causes wilt diseases on many economically important crops, including cotton, tomato and potato. In earlier studies we investigated the host specificity of VL vs. VD and found a clear restriction of VD to non-Brassica hosts while VL was pathogenic only on Brassica species (Zeise u. Tiedemann, 2001, 2002a & b). We investigated the differential interactions of VL and VD on the root surface and in the root and shoot vascular system of B. napus by confocal laser scanning microscopy (CLSM), using GFP-tagged strains of VL and VD and conventional fluorescence dyes, acid fuchsin and acridin orange (Eynck et al. 2007). At 24 hours post inoculation (hpi), hyphae of VL and VD intensely interwove with the root hairs. VL hyphae covered the roots with a hyphal net following the grooves of the junctions of the epidermal cells (Fig. 4) and started to penetrate the root epidermal cells without any conspicuous infection structures (36 hpi). Hyphae grew through the root cortex and colonized the xylem while individual vessels were entirely filled with mycelium and conidia, adjacent vessels remained completely unaffected (Fig. 5). This may explain why no wilt symptoms occur in B. napus infected with VL. Root penetration and an intercellular invasion of the root tissue was also observed for VD, but no systemic spread into the shoots of B. napus. This study confirms that VD is non-pathogenic on B. napus and demonstrates that non-host resistance against this fungus materializes by restriction of systemic spread rather than inhibition of penetration.

Plant defense and resistance factors

Salicylic acid (SA) is a phytohormone involved in the regulation of plant defense reactions against biotrophic pathogens and induces systemic acquired resistance (SAR). Deposition of callose into plant cell walls, hypersensitive response, and expression of phytoalexins are typical resistance mechanisms triggered by SA. These patterns of resistance are not obviously activated in B. napus after VL infection. Plants infected with VL showed sigificant increases in the level of SA (Ratzinger et al., 2009; Siebold & Tiedemann, 2013) and up-regulation of related PR genes. Further phytohormones like jasmonic acid and its defense-related gene PDF1.2 did not respond to VL. The role of SA in Brassica plant defense against VL is currently studied with inoculation experiments using nahG transgenic B. napus plants (Fig. 6), which have been engineered to remove SA enzymatically. Recent studies indicate, that SA is essential for basal resistance of B. napus to VL. In cultivar resistance, SA may be also relevant in the early stages of infection, while later stages are governed by a diverse pattern of responses in the phenylpropanoid pathway leading to different intensities in lignin formation.

Recently, we identified some promising genotypes of cabbage (B. oleracea) with enhanced resistance to VL (Rygulla et al. 2007a). Using such resistant genotypes of B. oleracea as parental lines, resistant OSR lines were resynthesized (Rygulla et al. 2007b) and used for generation of double-haploid (DH) B. napus populations. Beside the phenotyping of the DH lines in the greenhouse and in the field we are identifying resistance mechanisms with histological and molecular methods. Resistant genotypes have significantly elevated levels of constitutive and faster induction of soluble and cell wall-bound phenols and of lignin in the hypocotyls tissue (Eynck et al., 2009). Genotypic resistance is quantitative and expressed in the hypocotyl. As a result, B. napus roots appear to be generally invaded by VL but resistant genotypes are able to hinder any further spread oft he pathogen into the shoot xylem. Recent studies did not confirm the occurrence of soluble antifungal compounds in the xylem of resistant B. napus cultivars (Lopisso et al., 2017b). Carbohydrate and protein content in the xylem sap was similar in susceptible and resistant cultivars of B. napus, but the content of these nutritional factors changed with plant age. Fungal growth in the xylem was significantly correlated with the levels of carbohydrates in the xylem suggesting a crucial role of the nutritional quality of xylem sap for the vascular fungal spread.


Verticillium resistance and drought stress in oilseed rape

We investigated whether plant damage through ‘Verticillium stem striping’ is due to impaired plant water relations, whether VL affects responses of a susceptible B.napus variety to drought stress and whether drought stress in turn affects plant responses to VL (Lopisso et al. 2017a). Two-factorial experiments on a susceptible cultivar of B. napus infected or non-infected with VL and exposed to three watering levels (30, 60 and 100% field capacity) revealed that drought stress and VL impaired plant growth by entirely different mechanisms. While both stresses similarly affected plant growth parameters (plant height, hypocotyl diameter, shoot and root dry matter), infection of <>B. napus with VL did not affect any drought related, physiological or molecular genetic plant parameters including transpiration rate, stomatal conductance, photosynthesis rate, water use efficiency, relative leaf water content, leaf proline content or the expression of drought-responsive genes. Thus, this study provides comprehensive physiological and molecular genetic evidence explaining the lack of wilt symptoms in <>B. napus infected with VL. Likewise, drought tolerance of B. napus was unaffected by VL as was the level of disease by drought conditions, thus excluding a concerted action of both stresses in the field. While it is evident that drought and vascular infection with VL impair plant growth by different mechanisms, it remains to be determined by which other factors VL causes crop loss.


Role of root exudates in host plant sensing of V. longisporum and Plasmodiophora brassicae (clubroot) during early stages of disease initiation in B. napus

Currently, the majority of research into clubroot and Verticillium longisporum in B. napus  is focusing on the disease development in the plant, however, the factors triggering germination of the dormant resting structures (resting spores, microsclerotia)  in the soil and guiding infective propagules (zoospores, hyphae) towards the host roots are not clearly understood.  Therefore we have initiated research on the metabolomics and transcriptomics level to unravel the signaling and early gene expression events underlying the initial contact of these pathogens with their hosts.
Germination of soil-borne resting spores (RS) or microsclerotia (MS) is an essential factor for root infection in cruciferous hosts. Germination of dormant P. brassicae RS occurs spontaneously at low rates but can be stimulated by various biotic and abiotic factors in the soil. Root exudates of several host and non-host plants can stimulate RS and MS germination under experimental conditions. We will study the different root exudate components derived from a metabolite profiling through comparing the activity of root exudates from host plants and non-host plants on the germination of RS and MS. The goal is to identify germination stimulating compounds and to assess their specificity and role in the soil. Based on the interaction between soil microbes and plant roots, we will explore which soil microbes may be involved in mediating root signals to dormant fungal propagules. From these studies, we expect to reveal the mechanisms by which dormancy of RS and MS is broken and germination is induced in the soil related to plant root exudation and to modulation by the root and soil microbiome. The goal is to understand the early processes in soil directing these pathogens to the roots of their hosts and initiating infection as a potential approach to develop novel tools and strategies for the integrated sustainable control of these notorious soilborne pathogens in oilseed rape.

Parts of this research are supported by the German Ministery of Agriculture (BMELV) or conducted in the framework of a DFG-funded research group (DFG-Forschergruppe 546).

Related publications

  • Depotter, J.R.L., S. Deketelaere, P. Inderbitzin, A. von Tiedemann, M. Höfte, K. Subarao, T.A. Wood, B.P.H.J. Thomma (2016). Verticillium longisporum, the invisible threat of oilseed rape and other Brassicaceous plant hosts. Molecular Plant Pathology, 17(7), 1004-16. DOI: 10.1111/mpp.12350.
  • Dunker S, Keunecke H, Steinbach P, von Tiedemann A. (2008). Impact of Verticillium longisporum on yield and morphology of winter oilseed rape (Brassica napus) in relation to systemic spread in the plant. Journal of Phytopathology 156, 698–707. DOI: 10.1111/j.1439-0434.2008.01429.x.
  • Eynck, C., B. Koopmann, G. Grunewaldt-Stoecker, P. Karlovsky, A. v. Tiedemann (2007). Differential interactions of Verticillium longisporum and V. dahliae with Brassica napus detected with molecular and histological techniques. European Journal of Plant Pathology, 118:259-272. DOI: 10.1007/s10658-007-9144-6.
  • Eynck, C., B. Koopmann, P. Karlovsky, A. v. Tiedemann (2009). Internal resistance in winter oilseed rape inhibits systemic spread of the vascular pathogen Verticillium longisporum. Phytopathology, 99 (7), 802-811. DOI: 10.1094/PHYTO-99-7-0802.
  • Eynck, C., B. Koopmann, A. v. Tiedemann (2009). Identification of Brassica accessions with enhanced resistance to Verticillium longisporum under controlled and field conditions. Journal of Plant Diseases and Protection, 116 (2), 63-72.
  • Kamble, A., B. Koopmann, A. v. Tiedemann (2012). Induced resistance to Verticillium longisporum in Brassica napus by ß-aminobutyric acid. Plant Pathology, 62 (3), 552–561. DOI: 10.1111/j.1365-3059.2012.02669.x .
  • Knüfer, J., D. T. Lopisso, B. Koopmann, P. Karlovsky, A. von Tiedemann (2016). Assessment of latent infection with Verticillium longisporum in field-grown oilseed rape by qPCR. European Journal of Plant Pathology 147 (4), 819–831. DOI: 10.1007/s10658-016-1045-0.
  • Lopisso D. T., Knüfer J., Koopmann B., von Tiedemann A. (2017a). The vascular pathogen Verticillium longisporum does not affect water relations and plant responses to drought stress of its host, Brassica napus. Phytopathology 107, 444-454. DOI: 10.1094/PHYTO-07-16-0280-R.
  • Lopisso D. T., Knüfer J., Koopmann B., von Tiedemann A. (2017b). Growth of Verticillium longisporum in Brassica napus xylem sap is independent from cultivar resistance but promoted by plant ageing. Phytopathology 107, 1047-1054. DOI: 10.1094/PHYTO-02-17-0043-R .
  • Novakazi, F., P. Inderbitzin, G. Sandoya, R.J. Hayes, A. von Tiedemann, K.V. Subbarao (2015). The three lineages of the diploid hybrid Verticillium longisporum differ in virulence and pathogenicity. Phytopathology 105, 662-673. DOI: 10.1094/PHYTO-10-14-0265-R.
  • Obermeier, C., M. A. Hossain, R. Snowdon, J. Knüfer, A. v. Tiedemann, W. Friedt (2013). Genetic analysis of phenylpropanoid metabolites associated with resistance against Verticillium longisporum in Brassica napus. Molecular Breeding 31, 347-361. DOI: 10.1007/s11032-012-9794-8.
  • Ratzinger, A., N. Riediger, A. v. Tiedemann, P. Karlovsky (2009). Salicylic acid and salicylic acid glucoside in xylem sap of Brassica napus infected with Verticillium longisporum. J Plant Research, 122, 571-579. DOI: 10.1007/s10265-009-0237-5.
  • Rygulla W, F. Seyis, C. Eynck, A. v. Tiedemann, W. Friedt, W. Lühs, RJ Snowdon (2007). Combination of resistance to Verticillium longisporum from zero erucic acid Brassica oleracea and oilseed Brassica rapa genotypes in resynthesized rapeseed (Brassica napus) lines. Plant Breeding 126 (6), 596-602. DOI: 10.1111/j.1439-0523.2007.01414.x.
  • Rygulla W, RJ. Snowdon, C. Eynck, B. Koopmann, A. v. Tiedemann, W. Lühs, W. Friedt (2007). Broadening the genetic basis of Verticillium longisporum resistance in Brassica napus by interspecific hybridisation. Phytopathology 97: 1391-1396. DOI: 10.1094/PHYTO-97-11-1391.
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  • Singh S., S. A. Braus-Stromeyer, C. Timpner, G. Lohaus, Van Tuan Tran, M. Reusche, J. Knüfer, T. Teichmann, A. von Tiedemann & G.H. Braus (2009). Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem. Appl Microbiol Biotechnol 85 (6), 1961-1976, DOI: 10.1007/s00253-009-2269-0c.
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  • Avinash Kamble, Department of Botany, University of Pune, India
  • Petr Karlovsky, Molecular Phytopathology and Mycotoxin Research, University Göttingen
  • Wolfgang Friedt, Rod Snowdon, Christian Obermeier, Institute of Agronomy nad Plant Breeding, University Giessen
  • German rapeseed breeding companies
  • Members of the DFG research group 546.
  • Hossein Borhan, AAFC, Saskatoon, Canada
  • Krishna V. Subbarao, UC Davis, USA

Scientific staff
Currently involved: Marta Vega Marin, Antonia Wilch, Xiarong Zheng, Daniel Lopisso; Birger Koopmann; Andreas von Tiedemann
Formerly involved:  Sarah Bartsch, Nayuf Valdez; Eiko Tjaden; Jessica Knüfer; Nadine Riediger; Arne Weiberg; Avinash Kamble; Christina Eynck; Harald Keunecke; Karin Zeise; Ruben Gödecke; Niklas Heseker;