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.
Highlights
IntroductionOften plant growth-associated measurements like shoot length and plant weight are used to assess the severity of Apple replant disease (ARD) (Mahnkopp et al 2018; Reim et al 2019; Yim et al 2013)
The replant disease of apple negatively affects the plant growth and fruit yield in apple nurseries and orchards worldwide (Geldart 1994; Mazzola and Manici 2012; Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Institute of Horticultural Production Systems, Section Phytomedicine, Leibniz University Hannover, Herrenhäuser Str. 2, 30419 Hannover, GermanyInstitute of Horticultural Production Systems, Section Woody Plant and Propagation Physiology, Leibniz University Hannover, Herrenhäuser Str. 2, 30419 Hannover, GermanyWinkelmann et al 2019)
Small clusters of light brown or clearly necrotic cells with cauliflower-like appearance (CF) structures were selected for further analyses (Fig. 1b–d)
Summary
Often plant growth-associated measurements like shoot length and plant weight are used to assess the severity of ARD (Mahnkopp et al 2018; Reim et al 2019; Yim et al 2013). Biotic factors are known to be involved in the etiology since the plant growth can be restored by soil disinfection treatments (Mai and Abawi 1981; Mazzola 1998; Yim et al 2013). Apple plants can produce phytoalexins to deal with biotic stress. These secondary metabolites were nearly absent in healthy plants, but were produced in high amounts in roots affected by ARD (Weiß et al 2017a). It was reported that an effective defense response was missing albeit genes encoding enzymes of phytoalexin biosynthesis were upregulated and the phytoalexins were produced (Weiß et al 2017a, b)
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