Search

Anthocyanins play important roles in plant secondary metabolism. Although previous studies have identified many transcription factors (TFs) that participate in the synthetic pathway of anthocyanins, the regulation mechanism of the pathway remain poorly understood. In this study, we identified a WRKY Group IIc TF, MdWRKY75, which contained a typical WRKYGQK heptapeptide sequence and a C2H2-zinc finger structure. Subcellular localisation assays found that MdWRKY75 was located in the nucleus. Overexpression of MdWRKY75 promoted the accumulation of anthocyanins in apple (Malus domestica L.) ‘Orin’ calli. MdWRKY75 mainly stimulated the accumulation of anthocyanins by binding to the promoter of MYB transcription factor, MdMYB1. Our research could provide new insights into how WRKY TFs regulate the accumulation of anthocyanins in apples.

Enhancing fruit sugar contents, especially for high-flavonoid apples with a sour taste, is one of the main goals of horticultural crop breeders. This study analyzed sugar accumulation and the underlying mechanisms in the F2 progenies of a hybridization between the high-sugar apple (Malus × domestica) variety “Gala” and high-flavonoid apple germplasm “CSR6R6”. We revealed that MdSWEET9b (sugars will eventually be exported transporter) helps mediate sugar accumulation in fruits. Functional characterization of MdSWEET9b in yeast mutants lacking sugar transport as well as in overexpressing and CRISPR/Cas9 knockdown apple calli revealed MdSWEET9b could transport sucrose specifically, ultimately promoting normal yeast growth and accumulation of total sugar contents. Moreover, MdWRKY9 bound to the MdSWEET9b promoter and regulated its activity, which responded to abscisic acid (ABA) signaling. Furthermore, MdWRKY9 interacted with MdbZIP23 (basic leucine zipper) and MdbZIP46, key ABA signal transducers, at the protein and DNA levels to enhance its regulatory effect on MdSWEET9b expression, thereby influencing sugar accumulation. Based on the contents of ABA in lines with differing sugar contents and the effects of ABA treatments on fruits and calli, we revealed ABA as one of the main factors responsible for the diversity in apple fruit sugar content. The results of this study have clarified how MdSWEET9b influences fruit sugar accumulation, while also further elucidating the regulatory effects of the ABA-signaling network on fruit sugar accumulation. This work provides a basis for future explorations of the crosstalk between hormone and sugar metabolism pathways.

This study investigated the effects of six crop rotation combinations on the soil quality of old apple orchard and seedling growth of Malus hupehensis Rehd. (apple rootstock) under pot conditions. The inhibitory effects of crops such as Allium fistulosum, Brassica juncea, and Triticum aestivum on four species of Fusarium were observed and compared in six treatments. These were continuous cropping (CK), fumigation with the methyl bromide (FM), rotating A. fistulosum only (R1), rotating A. fistulosum and T. aestivum (R2), rotating A. fistulosum, B. juncea, and T. aestivum (R3), and fallow (FC) in a year. The results showed that the biomass of Malus hupehensis Rehd. seedlings increased significantly. The root length increased and the root architecture was optimized. The respiration rate of the root system was increased by about 1 time after rotation. The treatments of R1, R2, R3, and FC increased bacterial count by 232.17%, 96.04%, 316.21%, and 60.02%, respectively. However, the fungi were reduced in varying degrees and bacteria/fungi ratio was increased by 5–10 times. The enzyme activities, pH, and organic matter were increased, but soil bulk density was decreased. Phenolic acids such as phloridzin was decreased significantly. The copy number of four Fusarium species declined by 85.59%, 74.94%, 69.68%, and 54.41% after rotating three different crops (R3 treatment). The root volatiles of three plants inhibited mycelial growth and spore germination of four Fusarium species.

The Luochuan area is an important area for apple production in China. With the renewal and transformation of aged apple orchards, the occurrence of apple replant disease (ARD) was inevitable and has seriously affected the sustainable development of apples. Therefore, we randomly selected 14 soil samples from aged apple orchards in the Luochuan area to study the structural changes in the soil fungal community. The results showed that there were significant differences in the diversity of fungal communities between different aged apple orchards. The harmful fungi Gibberella, Fusarium, and Cryptococcus existed in 14 aged apple orchards in the Luochuan area, but their abundances were different in different aged apple orchards. A FUN Guild analysis showed that fungi were mainly present in the aged apple orchards in Luochuan in the saprotroph and pathotroph nutrition modes. Pathogenic fungi were widely present, which increased the risk of disease and seriously affected the growth and development of fruit trees. To sum up, there was a strong correlation between the ages of orchards and the unbalanced microbial community structure. Therefore, pathogenic fungi could be prevented and controlled during the renewal and transformation of aged orchards to reduce the impact of ARD on the apple industry.

Sugar and anthocyanin are important indicators of fruit quality, and understanding the mechanism underlying their accumulation is essential for breeding high-quality fruit. We identified an R2R3-MYB transcription factor (TF) MdMYB305 in the red-fleshed apple progeny, which was positively correlated with fruit sugar content but negatively correlated with anthocyanin content. Transient injection, stable expression (overexpressing and CRISPR/Cas9 knockdown) and heterologous transformation tomato confirmed that MdMYB305 promotes the accumulation of sugar and inhibits the synthesis of anthocyanin. Through a series of molecular experiments (such as EMSA, LUC) confirmed that MdMYB305 combines with sugar-related genes (MdCWI1/MdVGT3/MdTMT2) and anthocyanin-related genes (MdF3H/MdDFR/MdUFGT), promote and inhibit their activities, and finally regulate the sugar and anthocyanin content of fruits. In addition, this study also found that MdMYB305 competes with MdMYB10 for the MdbHLH33 binding site to balance sugar and anthocyanin accumulation in the fruits, which provides a reference value for exploring more functions of MYB-bHLH-MYB complex and the balance relationship between sugar and anthocyanin in the future.

Apple replant disease (ARD) is a common problem, which occurs in all major apple-growing regions worldwide. It hinders the growth of apple trees and reduces apple yield and quality. Besides, ARD has substantially impeded the development of the apple industry. Fusarium solani, a soil-borne pathogen, is the causative agent of ARD. WRKY transcription factors (TFs) play a crucial role in plant response to biological stress; however, the functional role of apple WRKY-TFs in ARD remains unknown. In this study, F. solani infection apple rootstock ‘M9T337’ induced MdWRKY74 expression was observed. As per the significance analysis, MdWRKY74 expression was positively correlated to endochitinase activity. The MdWRKY74 overexpression in apple callus can significantly improve the resistance to F. solani. In F. solani infection, the relative expression levels of the endochitinase gene (MdECHT) and the activity of endochitinase in the OE-MdWRKY74 calli were higher than those in the control calli. Yeast one-hybrid and electrophoretic mobility transfer analysis revealed the interaction between MdWRKY74 and MdECHT promoter. As per the luciferase reporter analysis, MdWRKY74 significantly increased the activity of the MdECHT promoter. In conclusion, MdWRKY74 was found to be a positive regulator of disease resistance, which improved the apple plant resistance to F. solani infection. This study provides a scientific basis for disease resistance breeding for apple rootstocks.

In this study, the potential regulatory effects of fulvic acid extracted from paper mill effluent (PFA) in apple replant disease (ARD) were investigated through a comprehensive experimental evaluation of the effects of PFA on soil properties, growth inhibition of apple replant pathogens, and growth of replanted Malus hupehensis Rehd. seedlings. PFA with a relatively lower molecular weight was mainly composed of carbohydrates, lignin derivatives, and polysaccharides and was rich in functional groups such as carboxyl and phenolic hydroxyl groups. Treatment with PFA dosages ranging from 2 to 3 g/pot significantly increased available phosphorus (P) in soil by 47.5 to 57.5% when compared with the control without PFA, indicating that PFA had a positive effect in activating P. In addition, PFA stimulated the growth of replanted seedlings by promoting root elongation, enhancing leaf photosynthesis, and increasing the activity of root antioxidant enzymes including superoxide dismutase, peroxidase, and catalase. However, no convincing evidence was found that application of different dosages of PFA had remarkable effects on soil pH, inorganic nitrogen, available potassium, organic matter, and the numbers of bacteria and fungi. Notably, PFA had no effect on the copy number of the main pathogenic fungi causing ARD, including Fusarium oxysporum, Fusarium solani, Fusarium proliferatum, and Fusarium moniliforme. Overall, PFA can alleviate ARD to a certain extent mainly through its effects on improving the resilience of replanted young seedlings rather than by affecting soil microorganisms or providing nutrients.

Apple replant disease (ARD) causes enormous economic loss and threatens the survival of apple industry worldwide. Fusarium solani is one of the pathogens that has been proven to cause ARD. Samples were collected at different time periods to investigate the mechanism of defense responses of apple to F. solani infection by monitoring the biomass, reactive oxygen species (ROS), and antioxidant enzyme activities of the apple rootstock ‘M.9T337’. In addition, the abundance of transcription of four pathogenesis-related (PR) proteins involved in antifungal defense was monitored. The results showed that the apple root system was normal and had small brown areas. However, there is a rapid burst of ROS during the early infection stage, and the activities of antioxidant enzymes and transcription of PRs increased during this period. With the extension in infection time, the infected root tissues displayed dark brown necrosis, and the activities of antioxidant enzymes and abundance of transcription of PRs decreased gradually after reaching their peak. Eventually, the plant biomass decreased, and the plant died. In conclusion, the levels of ROS and activities of antioxidant enzymes played an active role during the early stage of resistance of ‘M.9T337’ apples to infection by F. solani. Infection by F. solani can destroy the ROS scavenging system, causing oxidative damage and inhibiting the growth of apple rootstocks.

CKS (cyclin-dependent kinase subunit) is one of cell cycle genes, and cell cycle in apple (Malus × domestic Borkh.) fruit controlled cell number and fruit size. In order to explore CKS expression during the development of apple fruit size, the cultivar ‘Fuji’ and ‘Ralls’ whose fruit size had obvious difference were used for MdCKS1 gene cloning and expressing. In ‘Fuji’ and ‘Ralls’, the greatest growth rates of fruit size and weight occurred during 7–21 days after full bloom (DAFB), but in ‘Fuji’ during 21–35 DAFB the growth rates of fruit size and weight was still faster, and ‘Fuji’ remained much rapider growth rate of fruit size and weight than ‘Ralls’ within 7–35 DAFB, so the majority of cell production in ‘Ralls’ occurred between 7–21 DAFB and in ‘Fuji’ between 7–35 DAFB, and the biggest difference of fruit size and weight between ‘Fuji’ and ‘Ralls’ occurred at 35 DAFB. MdCKS1 sequence was identical with MdCKS1;1 (CO868566) and apple CKS1 (EB141951). MdCKS1 expression peak in ‘Fuji’ was two-fold higher than in ‘Ralls’; MdCKS1 expression peak in ‘Ralls’ occurred at 21 DAFB and in ‘Fuji’ at 35 DAFB which coincided respectively with rapid growth rate of early fruit. During 140–161 DAFB, ‘Fuji’ fruit had MdCKS1 expression peak again, but ‘Ralls’ not; ‘Fuji’ fruit size and weight had a larger extent of increase than ‘Ralls’, and the second significant difference of fruit size and weight between ‘Fuji’ and ‘Ralls’ occurred at 161 DAFB. In callus, MdCKS1 expression was much higher in ‘Fuji’ than in ‘Ralls’, up-regulated by auxin (NAA) or cytokinin (6-BA) or co-existence of them, and depressed by lower culture temperature. Altogether, ‘Fuji’ fruit had much rapider growth rate than ‘Ralls’ which resulted in significant difference of fruit size and weight between ‘Fuji’ and ‘Ralls’ respectively at 35 DAFB and 161 DAFB, and contributed to their fruit size difference; MdCKS1 expression in ‘Fuji’ fruit was much higher than in ‘Ralls’ both at early stage for fruit cell division and later stage for fruit cell expansion; MdCKS1 expression in callus was up-regulated by auxin or/and cytokinin. Hence, difference of fruit size between ‘Fuji’ and ‘Ralls’ was correlated with growth rate during fruit development, MdCKS1 expression correlated positively with growth rate and cell production of early fruit, and involved in fruit size which maybe was regulated by endogenous auxin or/and cytokine level of genotype.

A pot experiment was performed to investigate the effects of Trichoderma harzianum on the root morphology of Malus hupehensis Rehd. seedlings and their soil environment under replant conditions. The experiment consisted of four treatments: continuously cropped soil (CK1), methyl bromide fumigation (CK2), carrier substrate control (T1), and T. harzianum fertilizer (T2). Plant growth parameters, soil phenolic acid content, abundance of soil microorganisms, and root respiration rate were measured. Compared with CK1, plant height, basal diameter, and fresh weight were 34.58%, 27.55%, and 32.91% greater in T2; 11.35%, 12.10%, and 18.33% greater in T1; and 54.34%, 57.64%, and 45.74% greater in CK2. These metrics were significantly higher in the CK2 treatment than in the other treatments. The second highest values were recorded in the T2 treatment. Differences in root architecture were consistent with differences in biomass. Application of T. harzianum fertilizer was associated with increases of 45.45%, 120.06%, 86.44%, and 268.29% in the activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), respectively, and there was little difference between T2 and CK2. The contents of phlorizin and phloretin were 39.39% and 51.70% less in T2, respectively, and 17.85% and 18.14% less in T1, respectively, compared with CK1. Trichoderma harzianum fertilizer increased the abundance of bacteria and actinomycetes while decreasing that of fungi. The gene copy numbers of Fusarium oxysporum and Fusarium moniliforme were 64.30% and 49.35% less, respectively, in the T2 treatment. The fungus population and the gene copy number of Fusarium oxysporum and Fusarium moniliforme was the least in CK2 because of the good sterilization effect. The T. harzianum fertilizer showed satisfactory effects in promoting the root growth of M. hupehensis, increasing the root resistance, decreasing the soil phenolic acid content, and significantly reducing the gene copy number of F. oxysporum and F. moniliforme. In summary, T. harzianum fertilizer is an effective and green alternative for the prevention and control of apple replant disease (ARD).

Background Cultivation of resistant rootstocks can effectively prevent apple replant disease (ARD), and grafting tests are an important means of evaluating the compatibility of rootstocks with scions. Methods The apple rootstocks 12–2 (self-named) and Malus hupehensis Rehd. (PYTC) were planted in a replanted 20-year-old apple orchard. The two rootstocks were grafted with scions of 13 apple varieties. Multiple aboveground physiological parameters of the grafted combinations were measured and evaluated to verify the grafting affinity of 12–2 with the scions as compared to Malus hupehensis Rehd. (PYTC). Results The graft survival rate and graft interface healing of 12–2 did not differ significantly from those of PYTC. Mechanical strength tests of the grafted interfaces showed that some mechanical strength indices of Redchief, Jonagold, Starking, Goldspur and Yinv apple varieties were significantly higher when they were grafted onto 12–2 compared to the PYTC control. The height and diameter of shoots and the relative chlorophyll content, photosynthetic and fluorescence parameters, antioxidant enzyme activities and malondialdehyde content of leaves showed that Fuji 2001, Tengmu No.1, RedChief, Gala, USA8, and Shoufu1 grew similarly on the two rootstocks, but Tianhong 2, Lvguang, Jonagold, Starking, Goldspur, Yinv and Luli grew better when grafted onto 12–2 than onto the PYTC control. The rootstock 12-2, therefore, showed good grafting affinity. Conclusion These results provide experimental materials and theoretical guidance for the cultivation of a new grafting compatible rootstock to the 13 studied apple cultivars.

Organic acids secreted by plants, such as p-hydroxybenzoic acid, ferulic acid, cinnamic acid, and benzoic acid, can inhibit seed germination and root growth. The effects of root and soil leaching liquor from orchards on the growth of

(1) Background: Cultivating resistant rootstocks is an effective way to mitigate apple replant disease (ARD), and we developed superior apple rootstock line 12-2 (self-named), which shows improved ARD resistance. (2) Methods: We used ARD-associated pathogen Fusarium proliferatum MR5 (MR5) to test the fungal infection in the 12-2 line. Seedlings of the 12-2, T337, and M26 rootstock lines were planted in a substrate with potato dextrose broth and MR5 spore solution, and aboveground physiological indicators were measured. (3) Results: MR5 had the greatest effect on the leaf growth of T337 and M26. The incidence rates of infectious symptoms in the T337 and M26 lines were 68 and 100%, respectively. MR5 significantly affected the leaf chlorophyll content, ETR, and NPQ of T337 and M26, as well as Pn and Tr of M26. MR5 tended to reduce the leaf photosynthetic parameters of T337, but the decreases were not significant. The leaf reactive-oxygen-species levels of T337 and M26, the leaf antioxidant-enzyme activities of M26, and the superoxide-dismutase activity of T337 were significantly affected by MR5. MR5 also had a significant effect on the leaf malondialdehyde, proline, and soluble-sugar contents of T337 and M26. None of these aboveground physiological indicators were affected by MR5 in the 12-2 rootstock. (4) Conclusions: The 12-2 rootstock was more resistant to ARD-associated MR5 and could serve as an important test material for resistant-apple-rootstock breeding in China.

The effects of different soil chemical amendments (T1, 1‰ quicklime + 1‰ superphosphate; T2, 1‰ quicklime; T3, 1‰ superphosphate) on the soil environment and growth of Malus hupehensis Rehd. seedlings in aged apple orchard soil were studied to provide new insight into the prevention and control of apple replant disease. The amendments differed in their ability to ameliorate the soil environment; nevertheless, they all promoted the growth of M. hupehensis Rehd. seedlings, and the greatest enhancement of growth was observed in T1. On August 15, 2018, soil urease, sucrase, phosphatase, and catalase activities were 1.67 times, 1.32 times, 1.62 times, and 1.35 times higher in T1 compared with CK, respectively. The soil pH increased, which alleviated soil acidification. T1 also promoted the renewal of the community structure and the diversity of soil microorganisms. The copy numbers of Fusarium solani and Fusarium oxysporum were 71.96 and 70.30% lower in T1 compared with CK, respectively. The seedling height and root length of M. hupehensis Rehd. seedlings increased by 40.97 and 289.69% in T1 compared with CK, respectively. Therefore, soil replanting obstacles can be overcome with the application of quicklime and superphosphate; these soil chemical amendments also improve the soil microbial ecological environment and promote the growth of M. hupehensis Rehd. seedlings.

Crop rotation in fruit trees is an effective approach for addressing some of the problems of continuous cropping. To determine whether aged peach orchard soil is suitable for planting apple trees, we studied the effects of two substances abundant in aged peach orchard soil—amygdalin and benzoic acid—on the soil microbial community structure, soil enzyme activity, and the growth of Malus hupehensis Rehd. seedlings. Soils treated with amygdalin (T1), benzoic acid (T2), and a mixed solution of amygdalin and benzoic acid (T3) were used to plant M. hupehensis Rehd. seedlings. Compared with fallow (control) soil, the soil microbial community structure, soil enzyme activities, and root protective enzyme activities, leaf chlorophyll content, and net photosynthetic rate decreased in the three treatments. The biomass and root index of M. hupehensis Rehd. seedlings significantly decreased. Compared with T3, the plant height, ground diameter, fresh weight, dry weight, root length, root surface area, root volume, and root respiration rate of M. hupehensis Rehd. seedlings in T2 in 2015 (2016 in parentheses) decreased by 19.3% (12.6%), 8.7% (7.1%), 21.2% (13.3%), 9.1% (19.6%), 7.9% (25.3%), 40.7% (28.8%), 46.2% (21.1%), and 44.2% (27.5%), respectively. Compared with T3, the same variables in T1 in 2015 (2016 in parentheses) decreased by 34.9% (16.7%), 27.6% (9.8%), 53.6% (19.4%), and 50% (20.5%), 24.1% (31.4%), 55.1% (37.6%), 63.2% (28.2%), and 47.0% (28.7%), respectively. Thus, the inhibitory effect of T3 was the strongest, followed by T2 and T1. In sum, amygdalin and benzoic acid are harmful substances in aged peach orchard soil that inhibit the growth of M. hupehensis Rehd. seedlings.

Exogenous application of nitrogen (N) and phosphate (P) has been demonstrated to alleviate apple replant disease (ARD). Yet, the effect of controlled-release diammonium phosphate (C-DAP), which continuously supply N and P for ARD control, is still poorly understood. Applying C-DAP markedly alleviated the typical symptoms of ARD. C-DAP maintained soil N and P at relatively high and stable levels during the entire growth period of the replanted seedlings, thus, limiting the copy number of the four key pathogenic

Identifying the genetic variation characteristics of phenotypic traits is important for fruit tree breeding. During the long-term evolution of fruit trees, gene recombination and natural mutation have resulted in a high degree of heterozygosity. Apple (Malus × domestica Borkh.) shows strong ecological adaptability and is widely cultivated, and is among the most economically important fruit crops worldwide. However, the high level of heterozygosity and large genome of apple, in combination with its perennial life history and long juvenile phase, complicate investigation of the genetic basis of fruit quality traits. With continuing augmentation in the apple genomic resources available, in recent years important progress has been achieved in research on the genetic variation of fruit quality traits. This review focuses on summarizing recent genetic studies on apple fruit quality traits, including appearance, flavor, nutritional, ripening, and storage qualities. In addition, we discuss the mapping of quantitative trait loci, screening of molecular markers, and mining of major genes associated with fruit quality traits. The overall aim of this review is to provide valuable insights into the mechanisms of genetic variation and molecular breeding of important fruit quality traits in apple.

Background Elaeagnus angustifolia L. is a deciduous tree in the family Elaeagnaceae. It is widely used to study abiotic stress tolerance in plants and to improve desertification-affected land because of its ability to withstand diverse types of environmental stress, such as drought, salt, cold, and wind. However, no studies have examined the mechanisms underlying the resistance of E. angustifolia to environmental stress and its adaptive evolution. Methods Here, we used PacBio, Hi-C, resequencing, and RNA-seq to construct the genome and transcriptome of E. angustifolia and explore its adaptive evolution. Results The reconstructed genome of E. angustifolia was 526.80 Mb, with a contig N50 of 12.60 Mb and estimated divergence time of 84.24 Mya. Gene family expansion and resequencing analyses showed that the evolution of E. angustifolia was closely related to environmental conditions. After exposure to salt stress, GO pathway analysis showed that new genes identified from the transcriptome were related to ATP-binding, metal ion binding, and nucleic acid binding. Conclusion The genome sequence of E. angustifolia could be used for comparative genomic analyses of Elaeagnaceae family members and could help elucidate the mechanisms underlying the response of E. angustifolia to drought, salt, cold, and wind stress. Generally, these results provide new insights that could be used to improve desertification-affected land.

The relationship between soil texture and the degree of apple replant disease (ARD) was analyzed from the perspective of the microbial community structure and diversity within the rhizosphere soil of Malus hupehensis Rehd. seedlings. Three different textured soils were taken from different apple orchards in Laizhou, Yantai. The soils were divided into two parts, one was kept in replanted conditions, and the other was fumigated with methyl bromide to act as a high standard control. The strength of ARD occurrence was examined by measuring fresh and dry weight suppression (%) of the M. hupehensis seedlings. Differences in the fungal community structure (especially in Fusarium) among the three soil texture types were analyzed using high-throughput sequencing. The results showed that replanted loam clay soil had the highest fungal diversity, followed by sandy loam soil and finally loam soil. The richness of fungi between soil textures, however, was not significantly different. At the genus level, the relative abundance of Fusarium was 1.96%, 0.78%, and 10.89% in replanted sandy loam, replanted loam soil, and replanted loam clay soil, respectively. Moreover, the gene copy number of Fusarium oxysporum, Fusarium solani, and the inhibition rate of fresh weight of M. hupehensis seedlings were the same in the three soil textures. The plant height, photosynthesis (net) (Pn), and stomatal conductance (gS) of the M. hupehensis seedlings were significantly less in the replanted soil compared with the control treatments, with the overall difference being greatest in replanted loam clay soil, followed by replanted sandy loam and then replanted loam soil.

A rapid increase in endogenous ethylene production during ripening is related to fruit softening in the apple (Malus domestica Borkh.) cultivar ‘Taishanzaoxia’. Two genes involved in the ethylene response, MdERF1.1 and MdERF2.1, were isolated and characterized. MdERF1.1 and MdERF2.1 were predicted to encode proteins of 252 and 334 amino acids, respectively. MdERF1.1 and MdERF2.1 were expressed in Escherichia coli, and their molecular weights were determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and western blotting analyses (28 and 37 kD, respectively). Sequence comparisons and phylogenetic and secondary structure indicated that MdERF1.1 and MdERF2.1 are members of the ERF family. Both of them include a typical GCC‑box binding domain consisting of an α-helix and a three-stranded anti-parallel β-sheet. Sequence comparisons showed that MdERF1.1 is highly identity to MdERF1 from ‘Golden Delicious’, but Gly replaces Ala at position 110 in β-strand 2. This amino acid variation may be involved in fruit softening. Similarly, MdERF2.1 is highly identity to MdERF2 from ‘Golden Delicious’ but differs at three amino acids (Ala, Thr and Glu replace Pro, Ala and Asp at positions 97, 139, and 269, respectively). These three amino acid differences in MdERF2.1 might also be related to fruit softening. In ripening fruit, MdERF1.1 transcript levels rapidly increased during fruit softening in ‘Taishanzaoxia’, and this increase was associated with ethylene production. MdERF2.1 was predominantly expressed in softening ‘Taishanzaoxia’ fruit. The results indicate that ethylene signaling and MdERF1.1 and MdERF2.1 are involved in fruit softening in ‘Taishanzaoxia’.

The replant disease refers to abnormal crop growth caused by continuous cultivation of the same or related species on the same soil, and often occurring in crops and orchards. Recently, replant dis...

Apple replant disease (ARD) has been reported in all major fruit-growing regions of the world and is often caused by biotic factors (pathogen fungi) and abiotic factors (phenolic compounds). Soil chemical fumigation can kill soil pathogenic fungi; however, the traditionally used fumigant methyl bromide has been banned because of its ozone-depleting effects. There is thus a need to identify greener fumigant candidates. We characterized the effects of different fumigants on the replanted soil environment and the growth characteristics of Malus hupehensis Rehd. seedlings. All five experimental treatments [treatment 1 (T1), metham-sodium; treatment 2 (T2), dazomet; treatment 3 (T3), calcium cyanamide; treatment 4 (T4), 1,3-dichloropropene; and treatment 5 (T5), methyl bromide] promoted significantly the biomass, root growth, and root respiration rate of M. hupehensis seedlings and the ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3–-N) contents of replanted soil. Metham sodium (T1) and dazomet (T2) had stronger effects compared with 1,3-dichloropropene (T4) and calcium cyanamide (T3). At 172 days after T1, the height, root length, and root respiration rate of Malus hupehensis Rehd. seedlings, and the NH4+-N and NO3–-N contents of replanted soil increased by 91.64%, 97.67%, 69.78%, 81.98%, and 27.44%, respectively, compared with the control. Thus, dazomet and metham sodium were determined to be the optimal fumigants for use in practical applications.

Soil salinization is one of the most important abiotic stresses adversely affecting plant growth and yield. In this study, we cloned and characterized a putative salt tolerance gene encoding a bHLH (basic helix-loop-helix) transcription factor, MdbHLH106L (MdbHLH106-like), in the genome of apple (Malus domestica Borkh.). The expression level and promoter activity of MdbHLH106L are increased under salt stress. Overexpressing MdbHLH106L in ‘Orin’ calli can promote the expression level of MdNHX1 (Na+/H+ exchanger 1). Further analysis showed that MdbHLH106L is able to bind the MdNHX1 promoter. Moreover, we also confirmed the interaction between MdbHLH106L and MdZAT10. In conclusion, MdbHLH106L participates in the salt-stress signaling pathway and may mediate salt tolerance by activating the MdNHX1 promoter and interacting with MdZAT10. Our findings may serve as a theoretical basis for further investigating molecular mechanisms of stress responses and for enriching genetic resources of stress-tolerant apple cultivars.

‘Shannongsu’ and ‘Xincixiang’ were two new late-ripening pear cultivars with high quality, that were bred by our team. In order to clear the postharvest storage characteristics, mature pears were collected and stored at room temperature. Several fruit characteristics were evaluated over time, such as firmness, ethylene release rate, content of aroma components, softening-related enzyme activity, and gene expression. Both ‘Shannongsu’ and ‘Xincixiang’ were crisp and juicy stored after 60 d at room temperature, which exhibited excellent storage characteristics. The storability may be comprehensive result of low levels of ethylene production, aroma synthesis, softening-related activities, and gene expression. The research cleared the storage characteristics of ‘Shannongsu’ and ‘Xincixiang’ at room temperature for the first time, which will provide scientific theoretical guidance for fruit production and marketing.

Melatonin, as a plant growth regulator, is involved in plant stress resistance. We studied the effects of different concentrations (0, 10, 50, 100, 150, and 200 µmol · L−1) of melatonin on the growth and physiological characteristics of strawberry under cadmium (Cd) stress. The results represented that the growth of strawberry seedlings was inhibited under Cd stress, and the seedling biomass, chlorophyll content and the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) decreased. These toxic effects were, however, effectively remediated by exogenous melatonin pretreatment. Melatonin treatment slowed the inhibitory effect of seedling growth and significantly increased seedling biomass, chlorophyll content, antioxidant enzyme activities, and soluble protein levels in leaves and roots compared with the control. Melatonin treatment also reduced the malondialdehyde (MDA) content and the oxidative stress reactions, increased the anthocyanin content, and slowed the senescence rate, thereby reducing the toxic effects of Cd on strawberries. This indicates that foliar application of melatonin can effectively remediate the adverse effects of Cd on strawberry seedlings; the greatest effect was obtained with melatonin concentration of 100 µmol · L−1.

Identifying the genetic variation characteristics of phenotypic traits is important for fruit tree breeding. During the long-term evolution of fruit trees, gene recombination and natural mutation have resulted in a high degree of heterozygosity. Apple (

Background Apple replant disease is a soilborne disease caused by Fusarium proliferatum f. sp. malus domestica strain MR5 (abbreviated hereafter as Fpmd MR5) in China. This pathogen causes root tissue rot and wilting leaves in apple seedlings, leading to plant death. A comparative transcriptome analysis was conducted using the Illumina Novaseq platform to identify the molecular defense mechanisms of the susceptible M.26 and the resistant M9T337 apple rootstocks to Fpmd MR5 infection. Results Approximately 518.1 million high-quality reads were generated using RNA sequencing (RNA-seq). Comparative analysis between the mock-inoculated and Fpmd MR5 infected apple rootstocks revealed 28,196 significantly differentially expressed genes (DEGs), including 14,572 up-regulated and 13,624 down-regulated genes. Among them, the transcriptomes in the roots of the susceptible genotype M.26 were reflected by overrepresented DEGs. MapMan analysis indicated that a large number of DEGs were involved in the response of apple plants to Fpmd MR5 stress. The important functional groups identified via gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were responsible for fundamental biological regulation, secondary metabolism, plant-pathogen recognition, and plant hormone signal transduction (ethylene and jasmonate). Furthermore, the expression of 33 up-regulated candidate genes (12 related to WRKY DNA-binding proteins, one encoding endochitinase, two encoding beta-glucosidases, ten related to pathogenesis-related proteins, and eight encoding ethylene-responsive transcription factors) were validated by quantitative real-time PCR. Conclusion RNA-seq profiling was performed for the first time to analyze response of apple root to Fpmd MR5 infection. We found that the production of antimicrobial compounds and antioxidants enhanced plant resistance to pathogens, and pathogenesis-related protein (PR10 homologs, chitinase, and beta-glucosidase) may play unique roles in the defense response. These results provide new insights into the mechanisms of the apple root response to Fpmd MR5 infection.

In this study, an endophytic phlorizin-degrading Bacillus licheniformis XNRB-3 was isolated from the root tissue of healthy apple trees, and its control effect on apple replant disease (ARD) and how it alleviates the pathogen pressure via changes in soil microbiomes were studied. The addition of strain XNRB-3 in Fusarium infested soils significantly reduced the number of pathogens in the soil, thus resulting in a lower disease incidence, and the relative control effect on Fusarium oxysporum reached the highest of 66.11%. The fermentation broth can also protect the roots of the plants from Fusarium oxysporum, Fusarium moniliforme, Fusarium proliferatum, and Fusarium solani infection. These antagonistic effects were further validated using an in vitro assay in which the pathogen control was related to growth and spore germination inhibition via directly secreted antimicrobial substances and indirectly affecting the growth of pathogens. The secreted antimicrobial substances were identified using gas chromatography-mass spectrometry (GC-MS) technology. Among them, alpha-bisabolol and 2,4-di-tert-butylphenol had significant inhibitory effects on many planted pathogenic fungi. Butanedioic acid, monomethyl ester, and dibutyl phthalate promoted root development of Arabidopsis plants. Strain XNRB-3 has multifarious plant growth promoting traits and antagonistic potential. In pot and field experiments, the addition of strain XNRB-3 significantly promoted the growth of plants, and the activity of enzymes related to disease resistance [superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)] was also significantly enhanced. It also reduced the abundance of four species of Fusarium and the content of phenolic acids in the rhizosphere soil, improved soil microbial community structure and nutritional conditions, and increased soil microbial diversity and activity, as well as the soil enzyme activity. The above results indicated that B. licheniformis XNRB-3 could be developed into a promising biocontrol and plant-growth-promoting agent.

Fruit aroma is an important organoleptic quality, which influences consumer preference and market competitiveness. Aroma compound synthesis pathways in plants have been widely identified of which the lipoxygenase pathway is crucial for fatty acid catabolism to form esters in apple. However, the regulatory mechanism of this pathway remains elusive. In this study, linear regression analysis and transgene verification revealed that the lipoxygenase MdLOX1a participates in ester biosynthesis. Yeast one-hybrid library screening indicated that a novel abiotic stress gene, MdASG1 (ABIOTIC STRESS GENE 1), was a positive regulator of the MdLOX1a promoter and ester production based on yeast one-hybrid and dual-luciferase assays, and correlation analysis among eight apple cultivars. Overexpression of MdASG1 in apple and tomato stimulated the lipoxygenase pathway and increased the fatty acid-derived volatile content, whereas the latter was decreased by MdASG1 silencing. Furthermore, MdASG1 overexpression enhanced the salt-stress tolerance of tomato and apple ‘Orin’ calli accompanied by a higher content of fatty acid-derived volatiles compared with that of non-stressed transgenic tomato fruit. Collectively, these findings indicate that MdASG1 activates MdLOX1a expression and participates in the lipoxygenase pathway, subsequently increasing the accumulation of aroma compounds especially under moderate salt stress treatment. The results also provide insight into the regulation of aroma production, and the potential strategy of prudent development and utilization of saline-alkali land to produce high-quality fruit, thereby reducing pressure on arable land and ensuring national food security.One-sentence SummaryMdASG1 directly activates MdLOX1a expression to promote aroma volatiles accumulation especially under moderate salt stress.

Salt stress is an important environmental factor affecting the growth and production of agricultural crops and fruits worldwide, including apple (Malus × domestica). In this study, we demonstrate that a salt-responsive MYB transcription factor (TF), designated as MdMYB63, promotes survival under salt stress. Overexpression of MdMYB63 in apple calli significantly enhanced salt tolerance. Screening of the AP2/ERF family of TFs identified MdERF106 as an interaction partner of MdMYB63. Further analyses showed that the MdMYB63–MdERF106 complex significantly promotes the expression of downstream MdSOS1, thereby improving the Na+ expulsion and salt tolerance of apple. These functional analyses of MdMYB63 have provided valuable insights into the regulatory network of salt tolerance, and lay a theoretical foundation for the cultivation of new salt-tolerant apple varieties.

Apple (Malus domestica Borkh.) is an important fruit tree species worldwide. Apple fruits are favored by consumers because of their antioxidative, anti-inflammatory, and antitumor effects as well as their protective effects against cardiovascular diseases and other health benefits. There is considerable interest in red-fleshed apple fruits among breeders because of their high flavonoid and anthocyanin contents. However, the flavonoids extracted from red-fleshed apple fruits must still be functionally characterized, especially regarding their protective effects against certain pathologies. In this study, the flavonoid components and contents in the extracts prepared from red-fleshed apple cultivar "Meihong" were determined. Additionally, the in vitro antioxidant activities and protective effects of the extracts against CCl4 -induced acute liver injury were investigated. The red-fleshed apple flesh flavonoid extract (RAFF) exhibited strong in vitro antioxidant activities. Compared with the model control mice treated with CCl4 , the mice pretreated with high (800 mg/kg·bw), middle (400 mg/kg·bw), and low (200 mg/kg·bw) RAFF doses had significantly lower CCl4 -induced serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities. Moreover, the RAFF pretreatment also significantly decreased the liver malondialdehyde activity and prevented the CCl4 -induced decrease in liver superoxide dismutase, glutathione peroxidase, catalase, and reduced glutathione levels. Furthermore, a histopathological examination revealed that RAFF inhibited the inflammatory cell infiltration and cell boundary loss caused by CCl4 in the liver. Thus, RAFF is a natural antioxidant with significant antioxidative activities and liver protective effects. The results of this study may be relevant for enhancing the application of the red-fleshed apple fruit extract as a food additive. PRACTICAL APPLICATION: We took the self-selected red-fleshed apple cultivar "Meihong" as the unique research material, and the active ingredients of its flavonoid extract, in vitro antioxidant activity and hepatoprotective effect were analyzed. It is of great significance to promote the development of the red-fleshed apple industry, and also provides an important reference for the development of natural antioxidants.

Phloridzin is a phenolic acid associated with the genus Malus (Mill.) that causes apple replant disease. The removal of phloridzin is of great significance to alleviate this condition, and hence bi...

A two-year field experiment was carried out in order to study the occurrence degree and mechanism of apple replant disease (ARD) in the apple orchards with different soil textures. So we can adopt appropriate control measures according to the severity of ARD. Healthy two-year-old seedlings with consistent growth were selected, of which the root stock was T337 and the scion was Yanfu 3. There were significant differences in biomass between methyl bromide fumigation and replanted treatments, and the difference was the largest in clay loam, followed by sandy loam, and loam, which verified ARD in clay loam was most serious, followed by sandy loam and loam. Based on high-throughput sequencing of fungi in soil samples, fungal richness and diversity were the highest in clay loam, followed by sandy loam, and loam. The relative abundance of Fusarium in SX, SL, FX, FL, WX and WL was 7.33%, 19.32%, 2.70%, 4.24%, 10.71% and 23.87%, respectively. Based on Real-time quantitative analysis, there were significant differences in the number of Fusarium oxysporum and Fusarium solani between methyl bromide fumigation and replanted treatments, i.e., clay loam > sandy loam > loam. Fusarium was the main pathogen causing ARD. This shows that ARD is the most serious under replanted clay loam condition. High-throughput sequencing technology was used to prove the difference in Fusarium was one of the important reasons for ARD under different soil textures. This technology provides a new idea for the prevention and control of ARD.

Summary Methylation at the MdMYB1 promoter in apple sports has been reported as a regulator of the anthocyanin pathway, but little is known about how the locus is recognized by the methylation machinery to regulate anthocyanin accumulation. In this study, we analysed three differently coloured ‘Fuji’ apples and found that differences in the transcript levels of MdMYB1, which encodes a key regulator of anthocyanin biosynthesis, control the anthocyanin content (and therefore colour) in fruit skin. The CHH methylation levels in the MR3 region (−1246 to −780) of the MdMYB1 promoter were found to be negatively correlated with MdMYB1 expression. Thus, they were ideal materials to study DNA methylation in apple sports. The protein of RNA‐directed DNA methylation (RdDM) pathway responsible for CHH methylation, MdAGO4, was found to interact with the MdMYB1 promoter. MdAGO4s can interact with MdRDM1 and MdDRM2s to form an effector complex, fulfilling CHH methylation. When MdAGO4s and MdDRM2s were overexpressed in apple calli and Arabidopsis mutants, those proteins increase the CHH methylation of AGO4‐binding sites. In electrophoretic mobility shift assays, MdAGO4s were found to specifically bind to sequence containing ATATCAGA. Knockdown of MdNRPE1 did not affect the binding of MdAGO4s to the c3 region of the MdMYB1 promoter in 35S::AGO4 calli. Taken together, our data show that the MdMYB1 locus is methylated through binding of MdAGO4s to the MdMYB1 promoter to regulate anthocyanin biosynthesis by the RdDM pathway.

Apple replant disease (ARD) is a complex syndrome caused by various biotic and abiotic stresses contained in replanted soil, leading to reduced plant growth and fruit yields and causing serious economic loss. Breeding disease-resistant varieties is an effective and practical method to control ARD. Effective plant defense depends in part on the plant immune responses induced by the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). BAK1 participates in the regulation of plant immunity as an important PRR-binding protein. In this study, MdBAK1 overexpression activated indeterminate immune responses in tissue-cultured apple plants. MdBAK1-overexpressing rooted apple plants exhibited enhanced resistance to ARD, as the inhibition of plant growth was significantly alleviated during the replanted soil treatment. In addition, MdBAK1-overexpressing apple plants showed abolished growth inhibition, wilting and root rot induced by Fusarium oxysporum, which is the main pathogen that causes ARD in China. MdBAK1 overexpression changed the microbial community structure in the rhizosphere soil, as reflected by the increase in bacterial content and the decrease in fungal content, and the root exudates of MdBAK1-overexpressing plants inhibited F. oxysporum spore germination compared with that of wild-type plants. Furthermore, the constitutive immunity and cell necrosis induced by the upregulation of MdBAK1 expression were involved in the inhibition of colonization and expansion of F. oxysporum in host plants. In short, MdBAK1 plays an important role in the regulation of apple resistance to ARD, suggesting that MdBAK1 may be a valuable gene for molecular breeding of ARD resistance.

Trichoderma asperellum strain 6S-2 with biocontrol effects and potential growth-promoting properties was made into a fungal fertilizer for the prevention of apple replant disease (ARD). 6S-2 fertilizer not only promoted the growth of Malus hupehensis Rehd seedlings in greenhouse and pot experiments, but also increased the branch elongation growth of young apple trees. The soil microbial community structure changed significantly after the application of 6S-2 fertilizer: the relative abundance of Trichoderma increased significantly, the relative abundance of Fusarium (especially the gene copy numbers of four Fusarium species) and Cryptococcus decreased, and the relative abundance of Bacillus and Streptomyces increased. The bacteria/fungi and soil enzyme activities increased significantly after the application of 6S-2 fertilizer. The relative contents of alkenes, ethyl ethers, and citrullines increased in root exudates of M. hupehensis Rehd treated with 6S-2 fertilizer and were positively correlated with the abundance of Trichoderma. The relative contents of aldehydes, nitriles, and naphthalenes decreased, and they were positively correlated with the relative abundance of Fusarium. In addition, levels of ammonium nitrogen (NH4-N), nitrate nitrogen (NO3-N), available phosphorus (AP), available potassium (AK), organic matter (SOM), and pH in rhizosphere soil were also significantly related to changes in the microbial community structure. In summary, the application of 6S-2 fertilizer was effective in alleviating some aspects of ARD by promoting plant growth and optimizing the soil microbial community structure.

Evidence indicates that Allium and Brassica species which release bioactive compounds are widely used in bio-fumigation to suppress soil-borne diseases. However, the active molecules of such plant residues are easily volatilized. In this study, we conducted mixed cropping of the apple tree with Allium fistulosum or Brassica juncea; the results demonstrated that such mixed cropping significantly improved the growth of the grafted apple seedlings and alleviated apple replant disease (ARD) for two years. The terminal-restriction fragment length polymorphism profile results showed that the soil fungal community demonstrated distinct variation and diversity in terms of composition. A. fistulosum and B. juncea significantly improved the Margalef, Pielou, and Shannon indices. In addition, the analyses of clone libraries showed that A. fistulosum and B. juncea promoted the proliferation of antagonistic fungi such as Mortierella, Trichoderma, and Penicillium, and inhibited the proliferation of pathogens such as Fusarium. Fusarium. Proliferatum(F. proliferatum) was abundant in replanted soil and proved to be an aggressive pathogen of apple seedlings. Our findings thus indicate that apple tree mixed cropping with A. fistulosum and B. juncea was an effective long-term method for modifying the resident fungal community and alleviating ARD.

Apple replant disease (ARD), caused largely by soil-borne fungal pathogens, has seriously hindered the development of the apple industry. The use of antagonistic microorganisms has been confirmed as a low-cost and environmentally friendly means of controlling ARD. In the present study, we assessed the effects of Bacillus amyloliquefaciens QSB-6 on the growth of replanted apple saplings and the soil microbial environment under field conditions, thus providing a theoretical basis for the successful use of microbial biocontrol agents. Four treatments were implemented in three apple orchards: untreated replant soil (CK1), methyl bromide fumigation (CK2), blank carrier treatment (T1), and QSB-6 bacterial fertilizer treatment (T2). The plant height, ground diameter, and branch length of apple saplings treated with T2 in three replanted apple orchards were significantly higher than that of the CK1 treatment. Compared with the other treatments, T2 significantly increased the number of soil bacteria, the proportion of actinomycetes, and the activities of soil enzymes. By contrast, compared with the CK1 treatments, the phenolic acid content, the number of fungi, and the abundance of Fusarium oxysporum, Fusarium moniliforme, Fusarium proliferatum, and Fusarium solani in the soil were significantly reduced. PCoA and cluster analysis showed that soil inoculation with strain QSB-6 significantly decreased the Mcintosh and Brillouin index of soil fungi and increased the diversity of soil bacteria in T2 relative to CK1. The soil bacterial community structure in T2 was different from the other treatments, and the soil fungal communities of T2 and CK2 were similar. In summary, QSB-6 bacterial fertilizer shows promise as a potential bio-inoculum for the control of ARD.

Sugar content and cold tolerance are important apple (Malus × domestica) characteristics targeted by breeding programs. Here, a vacuolar membrane sucrose transporter, MdSWEET16, was cloned and characterized. A phylogenetic tree analysis found that MdSWEET16 was on the same polygenetic branch as Arabidopsis thaliana SWEET16 and SWEET17. MdSWEET16 was located on chromosome 2 and consists of six exons and five introns. The recombinant protein was obtained by prokaryotic induction, and the amino acid sequence and transmembrane domain were analyzed by bioinformatics. The encoded ~ 30-kDa protein has seven transmembrane domains and is localized on the tonoplasts of ‘Orin’ callus protoplasts. The expression of MdSWEET16 changed in response to sucrose and low temperature in ‘Orin’ calli. In addition, we also analyzed the expression level of MdSWEET16 during different fruit developmental stages using qRT-PCR. MdSWEET16 was highly expressed in young fruit, and its expression level during fruit development was significantly negatively correlated with sucrose content as assessed by a quantitative fluorescence analysis. Overexpression of MdSWEET16 in ‘Orin’ calli could reduce their sucrose content but increased their cold tolerance compared with MdSWEET16 RNA interference calli, which indicated that MdSWEET16 is involved in the sucrose transport and cold tolerance of apple. MdSWEET16’s expression level was significantly correlated with the sucrose content and low temperature, and was induced by sucrose and low temperatures. Overexpression of MdSWEET16 increased ‘Orin’ calli cold tolerance. The prokaryotic expression of its recombinant protein can be used to further study the function of the MdSWEET16 protein in apple.