Identification and validation of early genetic biomarkers for apple replant disease

Abstract

Apple replant disease (ARD) is a serious threat to producers of apple trees and fruits worldwide. The ARD etiology is not unraveled and managing options are either economically not applicable or environmentally harmful. Thus, interest is given in biomarkers that allow to indicate ARD situations at early time points in order to classify soils according to ARD severity but also to analyze the effectiveness to potential countermeasures. This study aimed at (i) identifying ARD biomarkers on the transcriptional level in root tissue by analyzing the expression of previously identified candidate genes in ARD soils of different origin and texture and (ii) testing the specificity of these marker genes to ARD. In vitro propagated M26 plantlets were submitted to a bio-test with three ARD soils, either untreated or disinfected by γ-irradiation. Expression of seven candidate genes identified in a previous transcriptomic study was investigated by RT-qPCR in a time course experiment. Already three days after planting, a prominent upregulation of the phytoalexin biosynthesis genes biphenyl synthase 3 (BIS3) and biphenyl 4-hydroxylase (B4Hb) was observed in the untreated ARD variants of all three soils. The phytoalexin composition in roots was comparable for all three soils and the total phytoalexin content correlated with the expression of BIS3 and B4Hb. The third promising candidate gene that was upregulated under ARD conditions was the ethyleneresponsive transcription factor 1B-like (ERF1B). In a second experiment M26 plantlets were exposed to different abiotic stressors, namely heat, salt and nutrient starvation, and candidate gene expression was determined in the roots. The expression levels of BIS3 and B4Hb were highly and specifically upregulated in ARD soil, but not upon the abiotic stress conditions, whereas ERF1B also showed higher expression under heat stress. In conclusion, BIS3 and B4Hb are recommended as early ARD biomarkers due to their high expression levels and their high specificity.

Identification of Candidate Genes Associated With Tolerance to Apple Replant Disease by Genome-Wide Transcriptome Analysis

“…However, a relationship between ARD severity and the production of terpenoid phytoalexins in the plant has not been observed to date. Several studies on ARD demonstrated remarkably higher contents of biphenyl and dibenzofuran phytoalexins in plants cultivated in ARD soils compared to disinfected ARD soils ( Winkelmann et al, 2019 ; Reim et al, 2020 ; Rohr et al, 2020b ; Balbin-Suarez et al, 2021 ; Busnena et al, 2021 ). However, Reim et al (2020) showed that the genes coding for biosynthesis of these phytoalexins were more strongly induced in the susceptible rootstock ‘M9’ than in the ARD tolerant genotype Mr5, indicating that these phytoalexin compounds do not help against ARD.…”

“…Plants of the apple rootstock ‘M9’ and the wild apple genotype Malus ×robusta 5 (Mr5) accession MAL0991 were propagated in vitro via axillary shoots on a modified MS medium ( Murashige and Skoog, 1962 ; Rohr et al, 2020b ). Mr5 is known to be tolerant, whereas ‘M9’ is susceptible to ARD ( Reim et al, 2019 ).…”

“…To validate the RNA-seq results, 30 DEGs were selected that showed remarkable differences in gene expression between ‘M9’ and Mr5 and were assumed to play a possible role in the defense response to ARD. In addition, the three genes Biphenyl synthase 3 ( BIS3 ), biphenyl 4-hydroxylase ( B4H ), and ethylene-responsive transcription factor 1B-like ( ERF1B ) that had been shown in previous studies to be suitable biomarkers for early ARD diagnosis were analyzed ( Reim et al, 2020 ; Rohr et al, 2020a ). For the selected 30 candidate genes, primers were generated using Primer3 tool with the following parameters: primer length 18–24 bp, amplification product 100–200 bp, T M = 59°C–61°C, CG content 40%–60% ( Untergasser et al, 2012 ).…”

“…Recent studies revealed the induction of genes associated with biotic stress response in roots of plants grown in ARD-affected soil ( Zhu et al, 2014 ; Weiß et al, 2017a ; Reim et al, 2020 ; Rohr et al, 2020b ). In particular, phytoalexins of the biphenyl biosynthetic pathway were demonstrably activated in response to ARD, which were shown to have an antifungal and antibacterial effect in plants ( Chizzali et al, 2012 ).…”

Evaluation of Apple Root-Associated Endophytic Streptomyces pulveraceus Strain ES16 by an OSMAC-Assisted Metabolomics Approach

“…Furthermore, our results provide a basis for employing a similar approach to other Streptomyces strains and species or other apple rootassociated organisms to comprehend the chemical basis of their ecological functions and interactions. It would also be useful to investigate the phenotypic and metabolic effect of ARD biomarkers such as phytoalexins (Reim et al, 2020;Rohr et al, 2020) and apple root exudates or compounds therein (e.g., phlorizin) on apple root-associated microorganisms and vice versa. Further investigations on the etiology of ARD as well as designing effective and sustainable biocontrol remedies must take into account both the molecular and the chemical networks between associated ecological partners in order to gain a holistic view.…”