Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.

Apple replant disease (ARD) is a major limitation to the establishment of economically viable orchards on replant sites due to the buildup and long-term survival of pathogen inoculum. Several soilborne necrotrophic fungi and oomycetes are primarily responsible for ARD, and symptoms range from serious inhibition of growth to the death of young trees. Chemical fumigation has been the primary method used for control of ARD, and manipulating soil microbial ecology to reduce pathogen density and aggressiveness is being investigated. To date, innate resistance of apple rootstocks as a means to control this disease has not been carefully explored, partly due to the complex etiology and the difficulty in phenotyping the disease resistance. Molecular defense responses of plant roots to soilborne necrotrophic pathogens are largely elusive, although considerable progress has been achieved using foliar disease systems. Plant defense responses to necrotrophic pathogens consist of several interacting modules and operate as a network. Upon pathogen detection by plants, cellular signals such as the oscillation of Ca²⁺ concentration, reactive oxygen species (ROS) burst and protein kinase activity, lead to plant hormone biosynthesis and signaling. Jasmonic acid (JA) and ethylene (ET) are known to be fundamental to the induction and regulation of defense mechanisms toward invading necrotrophic pathogens. Complicated hormone crosstalk modulates the fine-tuning of transcriptional reprogramming and metabolic redirection, resulting in production of antimicrobial metabolites, enzyme inhibitors and cell wall refortification to restrict further pathogenesis. Transcriptome profiling of apple roots in response to inoculation with Pythium ultimum demonstrated that there is a high degree of conservation regarding the molecular framework of defense responses compared with those observed with foliar tissues. It is conceivable that the timing and intensity of genotype-specific defense responses may lead to different outcomes between rootstocks in response to invasion by necrotrophic pathogens. Elucidation of host defense mechanisms is critical in developing molecular tools for genomics-assisted breeding of resistant apple rootstocks. Due to their perennial nature, use of resistant rootstocks as a component for disease management might offer a durable and cost-effective benefit to tree performance than the standard practice of soil fumigation for control of ARD.

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The mechanisms of plant root defences against soilborne necrotrophic pathogens are poorly understood, particularly in perennial tree fruit crops such as apple. Pythium ultimum is a component of the pathogen complex that incites apple replant disease (ARD). Previous field evaluation has demonstrated that apple rootstocks have different levels of ARD tolerance, but the underlying molecular mechanisms are unknown. Genotype responses in tissue cultured apple plants to P. ultimum infection were investigated at the biological, microscopic and molecular levels using ARD tolerant G.935 and susceptible B.9 apple rootstocks. Distinct differences in survival rate, root biomass, shoot biomass and maximum root length were observed between these two genotypes. Microscopic observations indicated contrasting dynamics in symptom development. The B.9 rootstock demonstrated an inability to limit pathogen progression, resulting in rapid progression of necrosis across the entire root system. In contrast, effective constraint of pathogen progression was shown in roots of G.935, as indicated by a limited or localized root necrosis. Apple genes, including MdCHS, MdCHIB, MdBIS and MdGLUC, which correspond to annotated functions in plant defence responses, showed differential expression profiles during infection between these two genotypes. These observations suggest that discrete molecular and biochemical processes exist in the two apple genotypes in response to P. ultimum infection.