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Assessment of latent infection with Verticillium longisporum in field-grown oilseed rape by qPCR

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Improvement of cultivar resistance is the key strategy to control the host-specialized pathogen Verticillium longisporum in oilseed rape (OSR). A special feature of this pathogen is its systemic, non-homogenous and delayed colonization of the plant xylem resulting in an extended symptomless period of latency. As a result, severity of infection in the field is difficult to score as it becomes apparent only at crop maturity stages when it may be confused with natural senescence. Assessment of Verticillium disease severity in OSR by visual scoring of microsclerotia on harvested stubbles unsatisfactorily reflects genotypic resistance as it is strongly affected by the ripening stage of the plant. To overcome these limitations, we developed a qPCR method, which unambiguously differentiates levels of quantitative resistance to V. longisporum in OSR genotypes under field conditions. The specificity and sensitivity of two primer pairs targeting ITS or tubulin loci in the V. longisporum genome were tested. While tubulin primers showed a high specificity to V. longisporum isolates, ITS primers exhibited a significantly higher sensitivity in detecting fungal DNA in stem tissue (limit of quantification =0.56 fg DNA) of field-grown pre-symptomatic plants. The best discrimination of resistant and susceptible OSR cultivars based on fungal DNA analysis in stem tissue was achieved at growth stage 80, at the transition of fungal vascular growth in viable plants to saprotrophic colonization of senescent stem tissues. Field screening data obtained with qPCR at growth stage 80 confirmed results from greenhouse testing thus corroborating the relevance and reliability of seedling assays for determining cultivar responses to V. longisporum in the field, as a useful tool for breeders in first selection of elite OSR genotypes with improved resistance to Verticillium.

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This work was financially supported by GFP (an association for the promotion of private plant breeding in Germany), BMEL (German Federal Ministry for Food and Agriculture) and FNR (Federal Agency for Renewable Resources, Gülzow, Germany). We thank the breeding companies within GFP for cooperation and providing plant material. Jutta Schaper, Dagmar Tacke and Heike Rollwage are gratefully acknowledged for their excellent technical assistance in the field, greenhouse and laboratory experiments. We thank Sabine Nutz for her support in ROC curve data analysis.

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Correspondence to Jessica Knüfer.

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Supplementary Fig. 1

Receiver operating characteristic (ROC) curve from quantitative real-time PCR with ITS primers amplification of V. longisporum DNA extracted from stems of field grown oilseed rape. (PDF 24 kb)

Supplementary Fig. 2

Correlation between visual disease assessment (stubble disease index) and qPCR analyses (fungal DNA in stem tissue) in four field grown susceptible and resistant winter oilseed rape cultivars. VL, Verticillium longisporum. DW, dry weight. (PDF 4 kb)

Supplementary Fig. 3

Net AUDPC values (A) and relative stunting (B) showing V. longisporum disease severity in susceptible and resistant winter oilseed rape cultivars under greenhouse conditions at 28 dpi. Data are means of three independent experiments. Bars indicate standard deviations. Express and Oase are resistant reference cultivars. Laser and Falcon are susceptible reference cultivars. Means with the same letter are not significantly different at P = 0.05. (PDF 351 kb)


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Knüfer, J., Lopisso, D.T., Koopmann, B. et al. Assessment of latent infection with Verticillium longisporum in field-grown oilseed rape by qPCR. Eur J Plant Pathol 147, 819–831 (2017).

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