European Journal of Plant Pathology

, Volume 153, Issue 3, pp 743–757 | Cite as

Evaluation of Malus gene bank resources with German strains of Marssonina coronaria using a greenhouse-based screening method

  • Thomas WöhnerEmail author
  • Vadim Girichev
  • Stine Radatz
  • Beatrize Lauria-Baca
  • Hans Scheinpflug
  • Magda-Viola Hanke


Marssonina coronaria is considered a threat to organic apple production in Central Europe. Since the application of fungicides is limited in organic production, breeding for resistance seems to be a promising strategy to manage the disease in the future. In this study, an artificial inoculation method similar to procedures used in apple scab greenhouse screenings was developed using German strains of the pathogen M. coronaria for evaluating 110 Malus domestica cultivars. The strains were morphologically and molecularly characterized and confirmed as M. coronaria. Symptom development was significantly influenced by incubation method, conidia concentration and point of inoculation, but not by leaf position and different water types used for inoculation. The success of inoculation and spread of the fungus on infected leaves were confirmed by conventional PCR. Moreover, there was a difference in development of disease symptoms between inoculations with conidia from in vitro grown strains (in vitro inoculum) and conidia from diseased leaves from the orchard (field inoculum) in a time dependent manner. These differences which were first found with the susceptible cultivar ‘Topaz’ after artificial inoculation with in vitro and field inoculum were confirmed on 20 more apple cultivars inoculated with different in vitro grown strains. In summary, all tested cultivars, including 21 which are scab-resistant, developed symptoms of this disease. Results from three years of investigation indicate a decrease in virulence of M. coronaria strains, when cultivated on artificial culture (growth)-media. Hence field inoculum is recommended for artificial greenhouse screenings for the evaluation of disease resistance in Malus genetic resources.


Marssonina coronaria Field inoculum In vitro inoculum Malus domestica Genetic resources 



The authors acknowledge Thomas Nothnagel fore pictures and assistance during the microscopic preparation of samples. We also acknowledge the KOB Bavendorf and Jan Hinrichs-Berger for the delivery of symptomatic leaves. Special thanks to Sabine Bartsch for excellent technical assistance and, to the staff of the experimental orchard at JKI. This work was funded by the Julius Kühn-Institut.

Compliance with ethical standards

The authors can assure that this article does not contain any studies with human or animal subject.

Conflict of interests

The authors declare that there are no conflicts of interests.

Supplementary material

10658_2018_1588_MOESM1_ESM.docx (15 kb)
Table S1 (DOCX 14 kb)
10658_2018_1588_MOESM2_ESM.docx (14 kb)
Table S2 (DOCX 14 kb)
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Figure S1

Inoculation trial with M .coronaria in the greenhouse. (JPG 3358 kb)

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Figure S2

Growth characterization ofM. coronariaon different cultural media. Strain FU0021 was characterized on three cultural media (MA, PDA, PCDA) and water agar as control. The growth of M. coronaria mycelial bodies was evaluated in two experiments. Five mycelial bodies each were transferred to five petri dishes containing MA, PCDA, PDA or water agar and cultivated with permanent light in a growth chamber (CLF Plant Climatics, Donau-Wörth, Germany) or kept in the dark. Each colony was photographed with a camera (ColorView III, Olympus Soft Imaging Solutions GmbH) integrated into a stereo microscope (Stemi SV11, Zeiss). The area on the media of each mycelial body was measured using the software Cell^D (Version 2.7, Olympus Soft Imaging Solutions GmbH) after 0, 7 and 14 days growth at a temperature of 20 °C. - MA-malt agar; PCDA-potato carrot dextrose agar; PDA - potato dextrose agar (PNG 163 kb)

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High resolution image (TIF 8375 kb)
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Figure S3

Cross section of an apple leave after inoculation withM. coronaria. Discs from symptomatic areas of infected leaves were incubated at 90 °C for 10 min in Ethanol (99%) to release chlorophyll. The procedure was performed in a ThermoMixer in 1.5 ml Eppendorf tubes (Eppendorf AG, Hamburg, Germany). After bleaching, the leaf discs were embedded in Technovit 7100 (Heraeus Kulzer GmbH, Wehrheim, Germany) following the standard procedure of the kit. The leaf discs were sliced with an automated microtome and stained with aniline blue. (PNG 2906 kb)

10658_2018_1588_MOESM4_ESM.tif (10.5 mb)
High resolution image (TIF 10739 kb)


  1. Badiu, D., Arion, F. H., Muresan, I. C., Lile, R., & Mitre, V. (2015). Evaluation of economic efficiency of apple orchard investments. Sustainability, 7(8), 10521–10533. Scholar
  2. Bertani, G. (2004). Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. Journal of Bacteriology, 186(3), 595–600.CrossRefGoogle Scholar
  3. Bus, V. G. M., Laurens, F. N. D., van de Weg, W. E., Rusholme, R. L., Rikkerink, E. H. A., Gardiner, S. E., Bassett, H. C. M., Kodde, L. P., & Plummer, K. M. (2005). The Vh8 locus of a new gene-for-gene interaction between Venturia inaequalis and the wild apple Malus sieversii is closely linked to the Vh2 locus in Malus pumila R12740-7A. New Phytologist, 166, 1035–1049.CrossRefGoogle Scholar
  4. Dang, J. L., Gleason, M. L., Niu, C. K., Liu, X., Guo, Y. Z., Zhang, R., & Sun, G. Y. (2017). Effects of fungicides and spray application interval on controlling Marssonina blotch of apple in the loess plateau region of China. Plant Disease, 101(4), 568–575.CrossRefGoogle Scholar
  5. Davis, J. J. (1903). Third supplementary list of parasitic fungi of Wisconsin. Transaction of the Wisconsin Academy of Science, Art and Letters, 14(1), 83–106.Google Scholar
  6. Didelot, F., Caffier, V., Orain, G., Lemarquand, A., & Parisi, L. (2016). Sustainable management of scab control through the integration of apple resistant cultivars in a low-fungicide input system. Agriculture, Ecosystems Environment, 217, 41–48.CrossRefGoogle Scholar
  7. Flachowsky, H., Szankowski, I., Fischer, T. C., Richter, K., Peil, A., Höfer, M., Dörschel, C., Schmoock, S., Gau, A. E., Halbwirth, H., & Hanke, M.-V. (2010). Transgenic apple plants overexpressing the Lc gene of maize show an altered growth habit and increased resistance to apple scab and fire blight. Planta, 231(3), 623–635.CrossRefGoogle Scholar
  8. Funes, I., Aranda, X., Biel, C., Carbó, J., Camps, F., Molina, A., Herralde, F., Grau, B., & Savé, R. (2016). Future climate change impacts on apple flowering date in a Mediterranean subbasin. Agricultural Water Management, 164(1), 19–27.CrossRefGoogle Scholar
  9. Harada, Y., Sawamura, K., & Konno, K. (1974). Diplocarpon mali sp. Nov., the perfect stage of apple blotch fungus Marssonina coronaria. Annals of the Phytopathological Society of Japan, 40, 412–418.CrossRefGoogle Scholar
  10. Hartmann, J. R., Parisi, L., & Bautrais, P. (1999). Effect of leaf wetness duration, temperature, and conidial inoculum dose on apple scab infections. Plant Disease, 83, 531–534.CrossRefGoogle Scholar
  11. Hinrichs-Berger, J., & Müller, G. (2013). Zum Auftreten von Marssonina coronaria an Apfel in Baden-Württemberg. Journal für Kulturpflanzen, 65, 347–350.Google Scholar
  12. Hong, K. H., & Hwang, K. H. (1998). Influence of inoculums densitiy, wetness duration, plant age, inoculums method, and cultivar resistance on infection of pepper plants by Colletotrichum coccodes. Plant Disease, 82, 1079–1083.CrossRefGoogle Scholar
  13. Lee, H., & Shin, H. (2000). Taxonomic studies on the genus Marssonina coronaria in Korea. Mycobiology, 281, 39–46.CrossRefGoogle Scholar
  14. Lee, D. H., Back, C., Win, N. K. K., Choi, K., Kim, K., Kang, I., et al. (2011). Biological characterization of Marssonina coronaria associated with apple blotch disease. Mycobiology, 39(3), 200–205.Google Scholar
  15. Leschenne, V. (2016). Understanding of the Population structure of Marssonina coronaria (Mc) causing apple leaf blotch in Swiss and European orchards. Master thesis Plant Pathology, ETH-Zürich, Zürich.Google Scholar
  16. Li, Y., Hirst, P. M., Wan, Y., & Liu, Y. (2012). Resistance to Marssonina coronaria and Alternaria alternata apple pathotype in the major apple cultivars and rootstocks used in China. Horticultural Science, 47(9), 1241–1244.Google Scholar
  17. Lu, S.-W., Kroken, S., Lee, B.-N., Robbertse, B., Churchill, A. C. L., Yoder, O. C., & Turgeon, B. G. (2003). A novel class of gene controlling virulence in plant pathogenic ascomycete fungi. PNAS, 100(10), 5980–5985.CrossRefGoogle Scholar
  18. McKinney, H. H. (1923). Influence of soil temperature and moisture on infection of wheat seedlings by Helminthosporium sativum. Journal of Agricultural Research, 26, 195–217.Google Scholar
  19. Oberhänsli, T., Vorley, T., Tamm, L., & Schärer, H. J. (2014). Development of a quantitative PCR for improved detection of Marssonina coronaria in field samples. Ecofruit(2013) Short Contribution, 187–190.Google Scholar
  20. Peil, A., Garcia-Libreros, T., Richter, K., Trognitz, F. C., Trognitz, B., Hanke, M.-V., & Flachowsky, H. (2007). Strong evidence for a fire blight resistance gene of Malus robusta located on linkage group 3. Plant Breeding, 126(5), 470–475.CrossRefGoogle Scholar
  21. Peil, A., Kellerhals, M., Höfer, M., & Flachowsky, H. (2011). Apple breeding - from origin to genetic engineering. Fruit Vegetables and Cereal Science and Biotechnology, 5(Special Issue 1), 118–138.Google Scholar
  22. Perez-Nadales, E., Nogueira, M. F. A., Baldin, C., Castanheira, S., Ghalid, M. E., Grund, E., et al. (2014). Fungal model systems and the elucidation of pathogenicity determinants. Fungal Genetics and Biology, 70, 42–67.CrossRefGoogle Scholar
  23. Persen, U., Steffek, R., Freiding, C., & Bedlan, G. (2012). Erstnachweis von Diplocarpon mali an Malus domestica in Österreich. Journal für Kulturpflanzen, 64(5), 168–170.Google Scholar
  24. Reddy, S., Spencer, J. A., & Newman, S. E. (1992). Leaflet surfaces of blackspot-resistant and susceptible roses and their reactions to fungal invasion. Hortscience, 27(2), 133–135.CrossRefGoogle Scholar
  25. Sharma, N., Thakur, V. S., Mohan, S. M., Khurana, S. M. P., & Sharma, S. (2011). Epidemiology of Marssonina bloth (Marssonina coronaria) of apple India. Indian Phytopathology, 62(3), 348–359.Google Scholar
  26. Shou, Y. Y., Li, C. M., Zhao, Y. B., Chen, D. M., & Zhang, X. Z. (2009). In vitro evaluation of resistance to Marssonia mali. in Apple. Journal of Fruit Science, 26(6), 912–914 (in Chinese).Google Scholar
  27. Spotts, R. A., & Cervantes, L. A. (2001). Disease incidence-inoculum dose relationships for botrytis cinerea and Penicillium expansum and decay of pear fruit using dry, airborne conidia. Plant Disease, 85, 755–759.CrossRefGoogle Scholar
  28. Stielow, J. B., Lévesque, C. A., Seifert, K. A., Meyer, W., Irinyi, L., Smiths, D., et al. (2015). One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes. Persoonia, 35, 242–263. Scholar
  29. Sutton, T.B., Aldwinckle, H.S., Agnello, A.M., Walgenbach, J.F. (2014). Compendium of apple and pear diseases and pests, second edition. APS Press, 1, 48–49.Google Scholar
  30. Vorley, T., Oberhänsli, T., Tamm, L., & Schärer, H. J. (2014). Testing susceptibility of apple cultivars against Marssonina coronaria. Ecofruit(2013) Short contributions, 191–194.Google Scholar
  31. Weibel, F. P., Daniel, C., Tamm, L., Willer, H., & Schwartau, H. (2012). Development of organic fruit in Europe. Acta Horticulturae, 1001, 19–34.Google Scholar
  32. White, T. J., Bruns, T., Lee, S. B., & Taylor, J. W. (1990). Amplification and direct sequencing of fungal ribosomal DNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols: A guide to methods and applications (pp. 315–322). New York: Academic Press, Inc..Google Scholar
  33. Würdig, J., Flachowsky, H., Saß, A., Peil, A., & Hanke, M. V. (2015). Improving resistance of different apple cultivars using the Rvi6 scab resistance gene in a cisgenic approach based on the Flp/FRT recombinase system. Molecular Breeding, 35, 1–18.CrossRefGoogle Scholar
  34. Yin, L., Wang, P., Li, M., Ke, X., Li, C., Liang, D., Wu, S., Ma, X., Li, C., Zou, Y., & Ma, F. (2013a). Exogenous melatonin improves Malus resistance to Marssonina apple blotch. Journal of Pineal Research, 54, 426–434. Scholar
  35. Yin, L., Li, M., Ke, X., Li, C., Zou, Y., Liang, D., & Ma, F. (2013b). Evaluation of Malus germplasm resistance to Marssonina apple blotch. European Journal of Plant Pathology, 136, 597–602.CrossRefGoogle Scholar
  36. Zhao, H., Huang, L., Xiao, C. L., Liu, J., Wei, J., & Gao, X. (2010). Influence of culture media and environmental factors on mycelial growth and conidial production of Diplocarpon mali. Letters in Applied Microbiology, 50(6), 639–644.CrossRefGoogle Scholar
  37. Zhao, H., Han, Q., Wang, J., Gao, X., Xiao, C., Liu, J., & Huang, L. (2013). Cytology of infection of apple leaves by Diplocarpon mali. European Journal of Plant Pathology, 136, 41–49.CrossRefGoogle Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2018

Authors and Affiliations

  1. 1.Julius Kühn-Institut, Federal Research Center for Cultivated PlantsInstitute for Breeding Research on Fruit CropsDresdenGermany
  2. 2.DresdenGermany
  3. 3.DubrauGermany
  4. 4.SingaporeRepublic of Singapore
  5. 5.LeverkusenGermany

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