Summary Co‐infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a‐based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namely Apple necrotic mosaic virus (ApNMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide‐conjugated gold nanoparticles. The CRISPR/Cas12a‐RT‐RPA platform exhibited comparable sensitivity to RT‐qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT‐PCR detection for 52 samples. This novel Cas12a‐based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.

Apple (Malus domestica Borkh.), an economically important tree fruit worldwide, frequently suffers from temperature stress during growth and development, which strongly affects the yield and quality. Heat shock protein 20 (HSP20) genes play crucial roles in protecting plants against abiotic stresses. However, they have not been systematically investigated in apple. In this study, we identified 41 HSP20 genes in the apple 'Golden Delicious' genome. These genes were unequally distributed on 15 different chromosomes and were classified into 10 subfamilies based on phylogenetic analysis and predicted subcellular localization. Chromosome mapping and synteny analysis indicated that three pairs of apple HSP20 genes were tandemly duplicated. Sequence analysis revealed that all apple HSP20 proteins reflected high structure conservation and most apple HSP20 genes (92.6%) possessed no introns, or only one intron. Numerous apple HSP20 gene promoter sequences contained stress and hormone response cis-elements. Transcriptome analysis revealed that 35 of 41 apple HSP20 genes were nearly unchanged or downregulated under normal temperature and cold stress, whereas these genes exhibited high-expression levels under heat stress. Subsequent qRT-PCR results showed that 12 of 29 selected apple HSP20 genes were extremely up-regulated (more than 1,000-fold) after 4 h of heat stress. However, the heat-upregulated genes were barely expressed or downregulated in response to cold stress, which indicated their potential function in mediating the response of apple to heat stress. Taken together, these findings lay the foundation to functionally characterize HSP20 genes to unravel their exact role in heat defense response in apple.

Responses of growth and antioxidant system to root-zone hypoxia stress were comparatively studied in two Malus species (M. hupenensis and M. toringoides) differing in hypoxia tolerance. 50-day-old seedlings were hydroponically grown for 20 days in normoxic and hypoxic nutrient solutions. Hypoxia stress inhibited the growth of both species. Compared with M. hupenensis, M. toringoides was more responsive to hypoxia stress, resulting in larger decreases in leaf number, root length, plant height, and biomass production. The contents of superoxide radicals O 2 Á ð Þ and hydrogen peroxide (H 2 O 2 ) significantly increased in roots of both species exposed to hypoxia stress, and resulted in lipid peroxidation, which was indicated by accumulated concentration of malonaldehyde (MDA). In addition, a significant increase in O 2 Á, H 2 O 2 and MDA contents was found in M. toringoides under hypoxia stress. In responses to hypoxia stress, peroxidse (POD), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities increased during the early part of the hypoxia stress, but decreased in the late period; the activities of SOD, POD and APX were more increased in M. hupenensis than in M. toringoides. Ascorbic acid (AsA) and glutathione (GSH) accumulation was also higher in M. hupenensis than in M. toringoides in the early period under hypoxia stress. These results suggest that the hypoxia-tolerant M. hupenensis has a larger protective capacity against oxidative damage by maintaining higher induced activities of antioxidant system than the hypoxia-sensitive M. toringoides.

We examined the potential differences in tolerance to hypoxia by two species of apple rootstocks. Stomatal behavior and photosynthesis were compared between Malus sieversii and Malus hupehensis. Plants were hydroponically grown for 15 days in normoxic or hypoxic nutrient solutions. Those of M. sieversii showed much greater sensitivity, with exposure to hypoxia resulting in higher leaf concentrations of abscisic acid (ABA) that prompted stomatal closure. Compared with the control plants of that species, stomatal density was greater in both new and mature leaves under stress conditions. In contrast, stomatal density was significantly decreased in leaves from M. hupehensis, while stomatal length was unaffected. Under stress, the net photosynthetic rate, stomatal conductance and chlorophyll contents were markedly reduced in M. sieversii. The relatively hypoxia-tolerant genotype M. hupehensis, however, showed only minor changes in net photosynthesis or chlorophyll content, and only a slight decrease in stomatal conductance due to such treatment. Therefore, we conclude that the more tolerant M. hupehensis utilizes a better protective mechanism for retaining higher photosynthetic capacity than does the hypoxia-sensitive M. sieversii. Moreover, this contrast in tolerance and adaptation to stress is linked to differences in their stomatal behavior, photosynthetic capacity and possibly their patterns of native distribution.

Tree architecture is an important, complex and dynamic trait affected by diverse genetic, ontogenetic and environmental factors. 'Wijcik McIntosh', a columnar (reduced branching) sport of 'McIntosh' and a valuable genetic resource, has been used intensively in apple-breeding programs for genetic improvement of tree architecture. The columnar growth habit is primarily controlled by the dominant allele of gene Co (columnar) on linkage group-10. But the Co locus is not well mapped and the Co gene remains unknown. To precisely map the Co locus and to identify candidate genes of Co, a sequence-based approach using both peach and apple genomes was used to develop new markers linked more tightly to Co. Five new simple sequence repeats markers were developed (C1753-3520, C18470-25831, C6536-31519, C7223-38004 and C7629-22009). The first four markers were obtained from apple genomic sequences on chromosome-10, whereas the last (C7629-22009) was from an unanchored apple contig that contains an apple expressed sequence tag CV082943, which was identified through synteny analysis between the peach and apple genomes. Genetic mapping of these five markers in four F(1) populations of 528 genotypes and 290 diverse columnar selections/cultivars (818 genotypes in total) delimited the Co locus in a genetic interval with 0.37 % recombination between markers C1753-3520 and C7629-22009. Marker C18470-25831 co-segregates with Co in the 818 genotypes studied. The Co region is estimated to be 193 kb and contains 26 predicted gene in the 'Golden Delicious' genome. Among the 26 genes, three are putative LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) containing transcription factor genes known of essential roles in plant lateral organ development, and are therefore considered as strong candidates of Co, designated MdLBD1, MdLBD2, and MdLBD3. Although more comprehensive studies are required to confirm the function of MdLBD1-3, the present work represents an important step forward to better understand the genetic and molecular control of tree architecture in apple.