Journal of Plant Diseases and Protection

, Volume 126, Issue 4, pp 329–341 | Cite as

A soil-free method for assessing pathogenicity of fungal isolates from apple roots

  • C. PoppEmail author
  • G. Grunewaldt-Stöcker
  • E. Maiss
Original Article


Apple replant disease is a problem in tree nurseries and apple orchards worldwide. Its cause is still unknown, but fungi are discussed to contribute to a complex of causal biotic factors. Several fungi are claimed to be replant disease pathogens but have not been significantly confirmed in experiments. Therefore, it seems indispensable to study fungal root endophytes in pathogenicity tests. Bioassays conducted in green house pot cultures using peat substrate or disinfected natural soil have been time and labor intensive and often resulted in only low infection rates. A quick biotest using the inert material perlite under controlled conditions is presented as an improved method to assess the effects of fungal isolates from replant-diseased root tissue. In vitro cultivated M26 rootstock plantlets were grown for 3 weeks in a Petri dish growth box with perlite substrate inoculated with selected fungal isolates. Symptom ratings for shoot wilting started after only 2 days; root symptoms appeared later and were assessed microscopically. Necroses in root tissue as well as hyphae, chlamydospores, and macroconidia could be detected. The tested endophytic isolates led to the following plant reactions: (1) negative (Cadophora, Calonectria, Dactylonectria, Ilyonectria, and Leptosphaeria) or (2) neutral (Plectosphaerella, Pleotrichocladium, and Zalerion). After re-isolation, most of the Nectriaceae isolates were confirmed as pathogens for M26 plants by fulfilling Koch’s postulates in a subsequent test. We recommend this perlite biotest to facilitate studies on root endophyte interactions with their hosts.


Biotest Perlite Malus domestica Nectriaceae Cylindrocarpon-like species Apple replant disease 



The authors are grateful to Mrs. Ewa Schneider and to Ms. Jenny Rebentisch for technical assistance. Dr. Christine Dieckhoff provided helpful support in improving the English version of this manuscript. The German Federal Ministry of Research and Education funded this work in the project ORDIAmur (FKZ 031B0025A) within the framework of the BonaRes program.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.


  1. Blok WJ, Bollen GJ (1995) Fungi on roots and stem bases of asparagus in the Netherlands: species and pathogenicity. Eur J Plant Pathol 101:15–24. CrossRefGoogle Scholar
  2. Braun PG (1995) Effects of Cylindrocarpon and Pythium species on apple seedlings and potential role in apple replant disease. Can J Plant Pathol 17:336–341. CrossRefGoogle Scholar
  3. Cabral A, Groenewald JZ, Rego C, Oliveira H, Crous PW (2012a) Cylindrocarpon root rot: multi-gene analysis reveals novel species within the Ilyonectria radicicola species complex. Mycol Prog 11:655–688. CrossRefGoogle Scholar
  4. Cabral A, Rego C, Crous PW, Oliveira H (2012b) Virulence and cross-infection potential of Ilyonectria spp. to grapevine. Phytopathol Mediterr 51:340–354Google Scholar
  5. Chu-Chou M (1979) Mycorrhizal fungi of Pinus radiata in New Zealand. Soil Biol Biochem 11:557–562. CrossRefGoogle Scholar
  6. Crous PW, Groenewald JZ, Risède J-M, Simoneau P, Hywel-Jones NL (2004) Calonectria species and their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Stud Mycol 50:415–430Google Scholar
  7. Dullahide SR, Stirling GR, Nikulin A, Stirling AM (1994) The role of nematodes, fungi, bacteria, and abiotic factors in the etiology of apple replant problems in the Granite Belt of Queensland. Aust J Exp Agric 34:1177–1182. CrossRefGoogle Scholar
  8. Franke-Whittle IH, Manici LM, Insam H, Stres B (2015) Rhizosphere bacteria and fungi associated with plant growth in soils of three replanted apple orchards. Plant Soil 395:317–333. CrossRefGoogle Scholar
  9. García-Jiménez J, Velázquez MT, Jordá C, Alfaro-García A (1994) Acremonium species as the causal agent of muskmelon collapse in Spain. Plant Dis 78:416–419. CrossRefGoogle Scholar
  10. Geldart GH (1994) The impact of replant problem on the economics of high density apple plantings. Acta Hortic 363:11–18. CrossRefGoogle Scholar
  11. Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–1330PubMedPubMedCentralGoogle Scholar
  12. Grunewaldt-Stöcker G, von Alten H (2003) Plant health effects of Acremonium root endophytes compared to those of Arbuscular mycorrhiza. In: Abe J (ed) The dynamic interface between plants and the earth: the 6th symposium of the international society of root research, 11–15 November 2001, Nagoya, Japan. Springer, Dordrecht, pp 445–454Google Scholar
  13. Grunewaldt-Stöcker G, von Alten H (2016) Is the root-colonizing endophyte Acremonium strictum an ericoid mycorrhizal fungus? Mycorrhiza 26:429–440. CrossRefPubMedGoogle Scholar
  14. Grunewaldt-Stöcker G, Mahnkopp F, Popp C, Maiss E, Winkelmann T (2019) Diagnosis of apple replant disease (ARD): microscopic evidence of early symptoms in fine roots of different apple rootstock genotypes. Sci Hortic 243:583–594. CrossRefGoogle Scholar
  15. Hoestra H (1968) Replant diseases of apple in the Netherlands. Ph.D. thesis. Meded. Landbouwhogesch., Wageningen, The NetherlandsGoogle Scholar
  16. Köhn S (1972) Ein Biotest zum schnellen und routineartigen Nachweis von Corynebacterium fascians. Nachrichtenblatt Deutscher Pflanzenschutzdienst 24:51–53Google Scholar
  17. Mahnkopp F, Simon M, Lehndorff E, Pätzold S, Wrede A, Winkelmann T (2018) Induction and diagnosis of apple replant disease (ARD): a matter of heterogeneous soil properties? Sci Hortic 241:167–177. CrossRefGoogle Scholar
  18. Mai WF, Abawi GS (1981) Controlling replant disease of pome and stone fruit in northeastern United States by preplant fumigation. Plant Dis 65:859–864CrossRefGoogle Scholar
  19. Manici LM, Ciavatta C, Kelderer M, Erschbaumer G (2003) Replant problems in South Tyrol: role of fungal pathogens and microbial population in conventional and organic apple orchards. Plant Soil 256:315–324CrossRefGoogle Scholar
  20. Manici LM, Kelderer M, Franke-Whittle IH, Rühmer T, Baab G, Nicoletti F, Caputo F, Topp A, Insam H, Naef A (2013) Relationship between root-endophytic microbial communities and replant disease in specialized apple growing areas in Europe. Appl Soil Ecol 72:207–214. CrossRefGoogle Scholar
  21. Manici LM, Caputo F, Saccà ML (2017) Secondary metabolites released into the rhizosphere by Fusarium oxysporum and Fusarium spp. as underestimated component of nonspecific replant disease. Plant Soil 415:85–98. CrossRefGoogle Scholar
  22. Manici LM, Kelderer M, Caputo F, Saccà ML, Nicoletti F, Topp AR, Mazzola M (2018) Involvement of Dactylonectria and Ilyonectria spp. in tree decline affecting multi-generation apple orchards. Plant Soil 425:217–230. CrossRefGoogle Scholar
  23. Mazzola M (1997) Identification and pathogenicity of Rhizoctonia spp. isolated from apple roots and orchard soils. Phytopathology 87:582–587. CrossRefPubMedGoogle Scholar
  24. Mazzola M (1998) Elucidation of the microbial complex having a causal role in the development of apple replant disease in Washington. Phytopathology 88:930–938. CrossRefPubMedGoogle Scholar
  25. Mazzola M, Manici LM (2012) Apple replant disease: role of microbial ecology in cause and control. Annu Rev Phytopathol 50:45–65. CrossRefPubMedGoogle Scholar
  26. Menzel W, Jelkmann W, Maiss E (2002) Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. J Virol Methods 99:81–92. CrossRefPubMedGoogle Scholar
  27. O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7:103–116. CrossRefPubMedGoogle Scholar
  28. Reis P, Cabral A, Nascimento T, Oliveira H, Rego C (2013) Diversity of Ilyonectria species in a young vineyard affected by black foot disease. Phytopathol Mediterr 52:335–346Google Scholar
  29. Sackston WE, Vimard B (1988) Leaf disk immersion (LDI) inoculation of sunflower with Plasmopara halstedii for in vitro determination of host pathogen relationships. Plant Dis 72:227–229CrossRefGoogle Scholar
  30. Tewoldemedhin YT, Mazzola M, Labuschagne I, McLeod A (2011a) A multi-phasic approach reveals that apple replant disease is caused by multiple biological agents, with some agents acting synergistically. Soil Biol Biochem 43:1917–1927. CrossRefGoogle Scholar
  31. Tewoldemedhin YT, Mazzola M, Mostert L, McLeod A (2011b) Cylindrocarpon species associated with apple tree roots in South Africa and their quantification using real-time PCR. Eur J Plant Pathol 129:637–651. CrossRefGoogle Scholar
  32. Utkhede RS, Vrain TC, Yorston JM (1992) Effects of nematodes, fungi and bacteria on the growth of young apple trees grown in apple replant disease soil. Plant Soil 139:1–6. CrossRefGoogle Scholar
  33. Vleugels T, Baert J, van Bockstaele E (2011) Construction of a bio-test for infection red clover plants with Sclerotinia trifoliorum. Commun Agric Appl Biol Sci 76:583–586PubMedGoogle Scholar
  34. Weber RWS, Entrop A-P (2017) Dactylonectria torresensis as the main component of the black root rot complex of strawberries and raspberries in Northern Germany. Erwerbs-Obstbau 59:157–169. CrossRefGoogle Scholar
  35. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, New York, pp 315–322Google Scholar
  36. Winkelmann T, Smalla K, Amelung W, Baab G, Grunewaldt-Stöcker G, Kanfra X, Meyhöfer R, Reim S, Schmitz M, Vetterlein D, Wrede A, Zühlke S, Grunewaldt J, Weiß S, Schloter M (2019) Apple replant disease: causes and mitigation strategies. Curr Issues Mol Biol 30:89–106. CrossRefPubMedGoogle Scholar
  37. Yim B, Smalla K, Winkelmann T (2013) Evaluation of apple replant problems based on different soil disinfection treatments—links to soil microbial community structure? Plant Soil 366:617–631. CrossRefGoogle Scholar

Copyright information

© Deutsche Phytomedizinische Gesellschaft 2019

Authors and Affiliations

  1. 1.Institute of Horticultural Production Systems, Section PhytomedicineLeibniz Universität HannoverHannoverGermany

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