A soil-free method for assessing pathogenicity of fungal isolates from apple roots
- 58 Downloads
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.
KeywordsBiotest 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.
- 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
- 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
- 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 61:1323–1330Google Scholar
- 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
- Hoestra H (1968) Replant diseases of apple in the Netherlands. Ph.D. thesis. Meded. Landbouwhogesch., Wageningen, The NetherlandsGoogle Scholar
- Köhn S (1972) Ein Biotest zum schnellen und routineartigen Nachweis von Corynebacterium fascians. Nachrichtenblatt Deutscher Pflanzenschutzdienst 24:51–53Google Scholar
- 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. https://doi.org/10.1016/j.apsoil.2013.07.011 CrossRefGoogle Scholar
- Mazzola M, Manici LM (2012) Apple replant disease: role of microbial ecology in cause and control. Annu Rev Phytopathol 50:45–65. https://doi.org/10.1146/annurev-phyto-081211-173005 CrossRefGoogle Scholar
- 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
- 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. https://doi.org/10.1016/j.soilbio.2011.05.014 CrossRefGoogle Scholar
- 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 76:583–586Google Scholar
- 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
- 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. https://doi.org/10.21775/cimb.030.089 CrossRefGoogle Scholar