Details
| Original language | English |
|---|---|
| Pages (from-to) | 571-582 |
| Number of pages | 12 |
| Journal | Journal of Plant Diseases and Protection |
| Volume | 127 |
| Issue number | 4 |
| Early online date | 16 May 2020 |
| Publication status | Published - Aug 2020 |
Abstract
Apple replant disease (ARD) negatively affects growth and yield of apple plants worldwide. Fungi belonging to the Nectriaceae have often been isolated from roots grown in replant soils and thus are proposed among others as one biotic cause of the disease complex. Microscopic analyses of ARD-affected roots revealed characteristic symptoms associated with fungal infection sites. Here, two extraction methods of such tissue sites were applied to directly identify an unknown fungus that forms typical cauliflower-like structures in diseased root cortex cells. Punching small tissue samples of about 0.5 mm3 volume with the Harris Uni-Core is a quick and easy method to harvest symptomatic material. Secondly, a laser microdissection (LMD) protocol for apple roots was established. This technique allows the extraction of defined cell or tissue fractions from thin cryo-sections. Tissue harvesting was followed by the identification of fungi via PCR amplification of two gene fragments and Sanger sequencing. For Harris samples, Chelex was used for DNA stabilization, while LMD samples were directly submitted to PCR. In Harris samples, mainly the Nectriaceae species Dactylonectria torresensis, Ilyonectria robusta and Rugonectria rugulosa were identified. In addition to these, in LMD samples Cylindrocladiella sp. and Ilyonectria europaea were detected. Thus, the intracellular CF structures contained different species of Nectriaceae in the ARD-affected cortex cells. These results contribute considerably to the etiology of the ARD. Both protocols offer the possibility to identify fungi from selected symptomatic small root sections by molecular tools avoiding isolation and subsequent axenic pure cultures of single fungal isolates.
Keywords
- Cryo-sections, Microscopy, Necrosis, Root pathogens, Root symptoms
ASJC Scopus subject areas
- Agricultural and Biological Sciences(all)
- Agronomy and Crop Science
- Agricultural and Biological Sciences(all)
- Plant Science
- Agricultural and Biological Sciences(all)
- Horticulture
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In: Journal of Plant Diseases and Protection, Vol. 127, No. 4, 08.2020, p. 571-582.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Molecular identification of Nectriaceae in infections of apple replant disease affected roots collected by Harris Uni-Core punching or laser microdissection
AU - Popp, C.
AU - Wamhoff, D.
AU - Winkelmann, Traud
AU - Maiss, Edgar
AU - Grunewaldt-Stöcker, Gisela
N1 - Funding Information: Our thanks go to Mrs. Ewa Schneider for providing in vitro propagated and acclimatized M26 plants and Mrs. Birgit Milde for technical support in the cloning of PCR amplificates for sequencing, both members of IGPS Leibniz University Hannover. We also thank Mrs. Marion Langeheinrich for her support in cryo-sectioning and Mrs. Ines Blume for her invaluable help in working with the LMD system, both members of the Anatomical Institute of the University of Veterinary Medicine Hannover (TiHo). The cooperation and hospitality of Prof. Dr. med. vet. Christiane Pfarrer offered to GGS and DW during their work at TiHo are highly appreciated. The German Federal Ministry of Research and Education funded this work in the project ORDIAmur (FKZ 031B0512A) within the framework of the BonaRes program.
PY - 2020/8
Y1 - 2020/8
N2 - Apple replant disease (ARD) negatively affects growth and yield of apple plants worldwide. Fungi belonging to the Nectriaceae have often been isolated from roots grown in replant soils and thus are proposed among others as one biotic cause of the disease complex. Microscopic analyses of ARD-affected roots revealed characteristic symptoms associated with fungal infection sites. Here, two extraction methods of such tissue sites were applied to directly identify an unknown fungus that forms typical cauliflower-like structures in diseased root cortex cells. Punching small tissue samples of about 0.5 mm3 volume with the Harris Uni-Core is a quick and easy method to harvest symptomatic material. Secondly, a laser microdissection (LMD) protocol for apple roots was established. This technique allows the extraction of defined cell or tissue fractions from thin cryo-sections. Tissue harvesting was followed by the identification of fungi via PCR amplification of two gene fragments and Sanger sequencing. For Harris samples, Chelex was used for DNA stabilization, while LMD samples were directly submitted to PCR. In Harris samples, mainly the Nectriaceae species Dactylonectria torresensis, Ilyonectria robusta and Rugonectria rugulosa were identified. In addition to these, in LMD samples Cylindrocladiella sp. and Ilyonectria europaea were detected. Thus, the intracellular CF structures contained different species of Nectriaceae in the ARD-affected cortex cells. These results contribute considerably to the etiology of the ARD. Both protocols offer the possibility to identify fungi from selected symptomatic small root sections by molecular tools avoiding isolation and subsequent axenic pure cultures of single fungal isolates.
AB - Apple replant disease (ARD) negatively affects growth and yield of apple plants worldwide. Fungi belonging to the Nectriaceae have often been isolated from roots grown in replant soils and thus are proposed among others as one biotic cause of the disease complex. Microscopic analyses of ARD-affected roots revealed characteristic symptoms associated with fungal infection sites. Here, two extraction methods of such tissue sites were applied to directly identify an unknown fungus that forms typical cauliflower-like structures in diseased root cortex cells. Punching small tissue samples of about 0.5 mm3 volume with the Harris Uni-Core is a quick and easy method to harvest symptomatic material. Secondly, a laser microdissection (LMD) protocol for apple roots was established. This technique allows the extraction of defined cell or tissue fractions from thin cryo-sections. Tissue harvesting was followed by the identification of fungi via PCR amplification of two gene fragments and Sanger sequencing. For Harris samples, Chelex was used for DNA stabilization, while LMD samples were directly submitted to PCR. In Harris samples, mainly the Nectriaceae species Dactylonectria torresensis, Ilyonectria robusta and Rugonectria rugulosa were identified. In addition to these, in LMD samples Cylindrocladiella sp. and Ilyonectria europaea were detected. Thus, the intracellular CF structures contained different species of Nectriaceae in the ARD-affected cortex cells. These results contribute considerably to the etiology of the ARD. Both protocols offer the possibility to identify fungi from selected symptomatic small root sections by molecular tools avoiding isolation and subsequent axenic pure cultures of single fungal isolates.
KW - Cryo-sections
KW - Microscopy
KW - Necrosis
KW - Root pathogens
KW - Root symptoms
UR - http://www.scopus.com/inward/record.url?scp=85084852011&partnerID=8YFLogxK
U2 - 10.1007/s41348-020-00333-x
DO - 10.1007/s41348-020-00333-x
M3 - Article
AN - SCOPUS:85084852011
VL - 127
SP - 571
EP - 582
JO - Journal of Plant Diseases and Protection
JF - Journal of Plant Diseases and Protection
SN - 1861-3829
IS - 4
ER -