Apple replant disease (ARD) poses a significant threat to the health and sustainable development of the apple industry. Chemical fumigation, as the most effective method, is limited due to residual pollution mitigation and other problems. It is crucial to find green and pollution-free prevention and control measures to replace chemical fumigation. Fulvic acid (FA) demonstrates considerable potential in promoting plant growth, resisting soil pathogen infection, and improving soil quality. In this study, we conducted a pot experiment to compare the changes in photosynthetic capacity, reactive oxygen species (ROS) scavenging capacity, 15N uptake and utilization efficiency, and the abundance and community composition of soil bacteria and fungi in replanted apple rootstocks (Malus hupehensis Rehd.) after exogenous application of different concentrations of FA, and explored the biocontrol effect of FA on ARD. We found that application of FA at 2 g·L-1 effectively mitigated replanting-induced ROS accumulation by enhancing photosynthesis and increasing the activities of defense enzymes in M. hupehensis Rehd. seedling roots. Furthermore, FA supplementation improved 15N absorption and utilization by enhancing nitrogen-metabolizing enzyme activities in roots. Notably, significant changes were observed in the community structure of both soil bacteria and fungi, along with a substantial increase in bacterial and fungal abundance compared with treatments without FA addition. Additionally, FA supplementation significantly increased soil enzyme activities. Mantel test results indicated positive correlations between soil properties (except for pH), growth indices, 15N uptake rate, and utilization efficiency. Spearman correlation analysis further revealed relationships of these factors with bacterial/fungal community composition, suggesting that microbial community composition plays a pivotal role in mediating changes within the replant soil environment, thereby affecting plant growth and nutrient uptake and utilization. In conclusion, FA can effectively alleviate ARD by promoting plant nitrogen uptake and optimizing soil microbial community structure.

Stone cell formation, resulting from aberrant lignin deposition in parenchyma cells, is a key determinant of pear fruit quality. Although exogenous calcium application is known to inhibit lignin biosynthesis and stone cell development, the underlying molecular mechanism involving calcium sensor proteins remains poorly understood. This study aimed to elucidate the molecular pathway by which calcium signaling modulates lignin biosynthesis. Our findings demonstrate that the calmodulin-like protein PbCML46 significantly suppresses lignin accumulation. This function was validated through complementary approaches, including transient injection in pear fruit and stable overexpression in pear calli. A yeast two-hybrid screen revealed that PbCML46 specifically interacts with the bHLH transcription factor PbbHLH96, an interaction further confirmed by pull-down and luciferase complementation imaging (LCI) assays. Further yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and luciferase (LUC) reporter assays analysis showed that PbbHLH96 directly binds to the MYC cis-element in the PbCAD6 promoter and functions as a transcriptional repressor. Dual-luciferase reporter (DLR) and in vivo co-expression assays indicated that PbCML46 markedly enhances the repressive activity of PbbHLH96 on the PbCAD6 promoter, an effect strictly dependent on PbbHLH96. In conclusion, this study reveals a Ca2+-initiated regulatory cascade in which calcium signaling promotes the interaction between the sensor PbCML46 and the transcriptional repressor PbbHLH96, leading to cooperative suppression of the key lignin biosynthetic gene PbCAD6 and thereby negatively regulating lignin deposition in pear stone cells. These findings provide novel mechanistic insights into how calcium signaling improves fruit quality.

Apple replant disease (ARD) severely constrains the sustainability of apple production and is closely associated with unfavorable soil microbial communities in replanted orchards. With the global ban on methyl bromide, environmentally friendly alternatives for soil disinfection are urgently needed. In this study, four oxidants-peracetic acid, hydrogen peroxide, sodium hypochlorite, and potassium permanganate-were evaluated for their effects on soil properties, microbial communities, and growth of replanted Malus hupehensis Rehd. seedlings. Compared with untreated soil, most oxidant treatments significantly reduced phenolic acid accumulation, enhanced soil enzyme activities during recovery, and promoted root development and biomass accumulation. Among the tested oxidants, potassium permanganate exhibited the most consistent beneficial effects, increasing seedling fresh weight and dry weight by 186% and 300%, respectively. High-throughput sequencing revealed that potassium permanganate markedly reshaped the soil fungal community, reducing the relative abundance of Fusarium from 18.26% to below 1.94%, while bacterial community structure remained relatively stable. Furthermore, peat soil amendment accelerate microbial recolonization and increase microbial diversity, enhanced enzyme activities, and further improved seedling growth. These results indicate that potassium permanganate combined with peat soil amendment represents a promising strategy for alleviating ARD through targeted modulation of soil fungal communities.

Auxin is a central plant hormone, and organic acids are key determinants of fruit acidity and flavor; however, the molecular mechanism connecting auxin signaling to organic acids remains elusive. Here, we report a negative correlation between auxin levels and malate, the predominant organic acid in apple (Malus domestica), in progenies of 'Gala' × 'Mato 1' and across fruit developmental stages. This suggests that auxin reduces malate levels independently of Ma1, an aluminum-activated malate transporter (ALMT) gene that is the causal gene for the major QTL determining fruit acidity in apple. Integrated bulked segregant analysis and RNA-seq identified MdARF16 and MdARF17 as auxin-responsive transcription factors that repress malate accumulation. Auxin represses two tonoplast-localized malate transporter genes, the ALMT gene MdALMT9L and the MdMATEL2 gene, which encodes a functionally distinct multidrug and toxic compound extrusion (MATE) protein. However, auxin does not repress Ma1. This repression requires the cooperative action of MdARF16 and MdARF17, which directly bind the promoters of the malate transporter genes to inhibit transcription. Decreased malate transporter levels then restrict cytosolic-to-vacuolar malate transport. These findings reveal a Ma1-independent auxin-MdARF16/17 pathway controlling vacuolar malate transport in apple, offering a molecular framework for the auxin-responsive improvement of organic acid traits.

Breeding red-fleshed apples with enhanced health benefits is a primary objective of modern apple improvement, yet high acidity and small fruit size limit their commercial potential. Resolving trade-offs among flesh color, flavor, and appearance and dissecting their underlying genetic relationships remain major challenges. In this study, using the red-fleshed 'CSR6R6' (Malus sieversii) and the cultivated 'Royal Gala' (M. domestica) as parents, we developed an F1 population comprising 140 individuals and performed integrative large-scale multi-omics analyses, identifying 13,331,096 SNPs and 2,134 metabolites. Red flesh correlated positively with malic acid but negatively with fruit weight. A major QTL linked to red flesh was mapped, and six genes regulating anthocyanin and proanthocyanidin biosynthesis were functionally validated, including MdUGT89A2.1, MdUGT89A2.2, MdALMT4, MdALMT4-like, MdTT1, and MdLAR1. Notably, MdALMT4 and MdALMT4-like encode malate transporters that coordinate malate accumulation and anthocyanin biosynthesis, suggesting a genetic link between acidity and pigmentation. Integrative multi-omics analysis revealed a large-scale regulatory network, revealing gene-metabolite-phenotype interactions and accurately predicting pathways involving the anthocyanin regulator MdTT1 and the fruit shape gene MdMADS13. Leveraging this network, we identified opposing effects of red-flesh QTL haplotypes across traits, defining the genetic basis of phenotypic antagonism, and resolved 11 positive and 13 negative haplotypes affecting red flesh, malic acid, and fruit weight. Pseudo-backcrossing further demonstrated that aggregating positive haplotypes substantially mitigates trade-offs among flesh color, flavor, and appearance, enabling their concurrent improvement. These findings will advance omics-assisted apple breeding, offering potential strategies for genetic improvement of other perennial fruit trees.

Flavonoid compounds, including anthocyanins and proanthocyanidins, are significant secondary metabolites in plants and play crucial roles in various aspects of plant growth, development, and environmental stress responses. In the present study, we identified a key transcription factor from the NAC family, designated as MdNAC72-like, which had a strong correlation with the anthocyanin content during the apple fruit ripening process. Through techniques including yeast one-hybrid analysis, electrophoretic mobility shift assays, and luciferase reporter assays, we illustrated that MdNAC72-like directly interacts with the promoters of the MdMYB9, MdLAR, and MdUFGT genes. This interaction enhances their transcriptional activity, leading to a favorable impact on the biosynthesis of anthocyanins and proanthocyanidins in plants. Furthermore, utilizing yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays, we demonstrated that MdERF1B forms an interaction with MdNAC72-like, which in turn augments the transcriptional activation ability of MdNAC72-like on the downstream structural genes MdMYB9, MdLAR, and MdUFGT. In conclusion, MdNAC72-like presents significant research potential, and these results offer a theoretical framework for understanding the regulatory mechanisms governing anthocyanin and proanthocyanidin synthesis in apple.

Apple replant disease (ARD) severely restricts the growth and productivity of apple plants in replanted orchards. Soil C/N ratio is a key regulator of microbial community, with direct implications for plant growth and development. This study aimed to develop an integrated horticultural practice to mitigate ARD by synchronously adjusting the soil C/N ratio and applying biocontrol agents. Field investigations were conducted in multiple replanted orchards to assess soil C/N status. Laboratory screening identified suitable carbon and nitrogen sources, and the optimal C/N ratio for the biocontrol strains Bacillus velezensis XC1 and Trichoderma harzianum was determined. Pot and field experiments were then carried out to verify the effects of soil C/N adjustment and biocontrol agent application on plant growth and soil microecology. The results indicated an optimal soil C/N ratio of 12:1 for alleviating ARD symptoms. This ratio was achieved using dextrin and glutamic acid as sources of carbon and nitrogen. At this C/N level, both biocontrol strains exhibited optimal growth situation. The combined treatment significantly improved plant growth and systemic resistance, promoted soil element cycling, reduced the abundance of Fusarium, and increased the metabolic activity of the soil microbial community. Notably, the application of T. harzianum following C/N adjustment proved more effective than B. velezensis XC1 in enhancing plant health and reshaping the rhizosphere microbiome. In conclusion, adjusting the soil C/N ratio to 12:1 together with biocontrol agents (particularly T. harzianum) introduction can effectively suppress soil-borne pathogens, optimize rhizosphere microecology, and enhance plant vigor, providing a sustainable horticultural strategy for managing ARD in apple production.

The apple anthocyanin content is an important trait in apple breeding. Auxin, as an important plant hormone, plays significant roles in regulating the biosynthesis of anthocyanins. However, the molecular mechanism of how plants regulate auxin content and activity to affect anthocyanin accumulation remains unclear. In this study, through fruit anthocyanin content analysis and transcriptome sequencing of the hybrids derived from 'Golden Delicious' and 'Fuji Nagafu No. 2' crosses, a key gene for regulating apple anthocyanin accumulation, indole-3-acetic acid (IAA) methyltransferase (MdIAMT), was identified. Functional analyses showed that the apple calli and peel overexpressing MdIAMT accumulated more anthocyanin than that in Vec by regulating IAA homeostasis. Yeast two-hybrid assays, luciferase complementation imaging assays and co-immunoprecipitation assays revealed that MdCSN5, an important protein in light signal transduction, interacts with MdIAMT. More importantly, further research showed that the MdCSN5-MdIAMT module affected auxin signal transduction pathway by regulating IAA homeostasis, thus promoting anthocyanin accumulation. In summary, our findings elucidate a novel mechanism by which auxin-regulated anthocyanin accumulation via MdCSN5-MdIAMT module, deepening our knowledge of plant hormone signaling in anthocyanin biosynthesis.

Postharvest loss constitutes a critical issue during the storage period of pears. Based on previous studies, both 1-methylcyclopropene (1-MCP) treatment and modified atmosphere packaging (MAP) can delay fruit senescence and softening. To investigate whether the combination of these treatments achieves a superior storage effect, 'Shannong Su' pears were treated with 1 μL/L 1-MCP for 24 h, then packaged in sealed or perforated film bags or left unpackaged, and stored at 4.00 ± 0.50 °C for 120 days. Firmness, ethylene release, quality indicators, gene expression, and metabolite profiles were analyzed. Results showed that 1-MCP inhibited ethylene production and the expression of PbACS1/2 and PbACO1. The combined treatment (1-MCP + sealed film bag) achieved the lowest expression levels of nine key cell wall-degrading enzyme genes (PbXTH1/28, PbPL8/18, PbCGR3, PbPG, Pbα/β-GAL, Pbβ-GLU) and maintained higher firmness, antioxidant capacity, total sugar, protopectin, and cellulose content. Metabolomic analysis revealed that 1-MCP treatment altered the biosynthesis of plant secondary metabolites and fatty acids, suggesting it may influence the anabolic metabolism of defensive compounds and lipids in plants. Film bag packaging affected antioxidant stress responses, nitrogen metabolism, specific amino acid derivative metabolism, and cofactor biosynthesis, indicating that sealed film packaging may induce oxidative stress responses and activate specific defensive or protective metabolic pathways. In conclusion, the combined treatment preserves pear quality through synergistic inhibition of ethylene synthesis, suppression of cell wall-degrading genes, and modulation of metabolic pathways. This cost-effective method can reduce postharvest losses in 'Shannong Su' pears and guide the storage of other similar climacteric fruits.

Low temperature (LT) stress negatively impacts the yield and quality of key horticultural crops such as apples. While previous research has predominantly focused on white-fleshed apple varieties with limited cold tolerance, the mechanisms underlying low temperature responses in red-fleshed apples remain largely unexplored. In this study, we used red-fleshed apple seedlings that we developed previously to investigate the biochemical responses to varying temperature conditions. Notably, LT stress significantly enhanced the accumulation of anthocyanins, soluble sugars, soluble proteins, and other metabolites in the red-fleshed apple seedlings. Leveraging RNA-seq data, we identified a potential LT stress-responsive transcription factor, which we designated as MdbHLH51. Functional characterization revealed that overexpression of MdbHLH51 in ‘Orin’ calli significantly promoted anthocyanin accumulation and up-regulated the expression of all MdCBFs genes, thereby bolstering tolerance to cold stress. These findings provide valuable insights into the cold stress response mechanisms in red-fleshed apples, offering a theoretical foundation for the genetic breeding of cold-resistant red apple varieties.

In apples, fruit firmness is a crucial quality trait influencing fruit storability, transportability, shelf life and consumer preference. However, the genetic network underlying this trait remains unclear. Therefore, the present study investigated the changes in apple fruit at different stages of postharvest storage using a combination of transcriptomic and metabolomic analyses. With prolonged storage, we detected a significant increase in two metabolites, D-galacturonic acid (D-GalUA) and D-glucuronic acid (D-GlcA), which are associated with a key class 1 non-symbiotic haemoglobin (MdHb1). We innovatively found that MdHb1 regulates fruit softening by catalysing the conversion from protopectin to water-soluble pectin. Biochemical analysis demonstrated that MdMYB2/MdNAC14/MdNTL9 transcription factors directly bind to the MdHb1 promoter to activate its transcriptional expression and promote fruit softening. Further injection experiments in apple fruit and histological as well as transmission electron microscopy analyses of the fruit samples revealed that D-GalUA and D-GlcA reduce the transcription of MdHb1, or through the MdMYB2/MdNAC14/MdNTL9-MdHb1 regulatory module, thereby delaying fruit softening. Our study provides novel insights into the role of two important metabolites, D-GalUA and D-GlcA, in the regulation of MdHb1-mediated fruit softening in apples.

Apple replant disease (ARD) is mainly caused by biological factors, and it severely restricts the development of the apple industry. The use of biological control measures to alleviate ARD is critically important for the sustainable development of the apple industry. The effects of raw amino acid powder and Trichoderma harzianum fertilizer on plant biomass, leaf and root indexes, soil physical and chemical properties, soil enzyme activities, and the soil fungal community were studied under pot and field conditions using Malus hupehensis Rehd. seedlings and grafted trees (Fuji New 2001/M9T337) as experimental materials. We found that the application of the materials significantly promoted plant growth, increased the leaf photosynthesis and chlorophyll content, root respiration rate, root antioxidant enzyme activities, and soil enzyme activities, significantly reduced the number of Fusarium sp. in soil, and significantly increased the abundance of beneficial fungi. In conclusion, the mixed application of raw amino acid powder and T. harzianum fertilizer is an effective method for the prevention and management of ARD.

Previous studies have shown that the bacterial fertilizer Lactobacillus reuteri (LBR) significantly alleviates apple replant disease (ARD), but the mechanism behind its effectiveness remains unclear. This study investigated the effects of key LBR metabolites on the rhizosphere microbial community. The biocontrol function of extracellular polysaccharides (EPS) was examined and shown to be further enhanced after optimizing the fermentation conditions. The optimized fermentation conditions were found to generate intermediates involved in various plant metabolic pathways, leading to plant growth promotion, increased abundance of beneficial bacteria like Bacillus and Pseudomonas in the rhizosphere soil, and decreased abundance of pathogenic fungi. Through the isolation and identification of rhizosphere microorganisms, a strain of Pseudomonas monteilii with chemotaxis to EPS was isolated, which had growth promotion ability and effectively improved plant resistance and relieves ARD. To further understand the mechanism underlying the inhibitory effect on soil pathogens of microbial aggregations and development in the rhizosphere driven by beneficial bacteria metabolites. These findings offer valuable technical insights for utilizing biocontrol bacteria metabolites in ARD management.

Red-fleshed apples are characterized by high nutritional value due to their abundant anthocyanin content. However, their commercial development is often constrained by a decline in fresh-eating quality caused by excessive acidity. The molecular mechanism underlying the coordinated accumulation of anthocyanins and malic acid in apple flesh remains largely unclear. In this study, transcriptome analysis of an F1 hybrid population derived from a cross of Malus sieversii ‘CSR6R6’ and Malus domestica cv. ‘Royal Gala’ identified an R2R3-MYB transcription factor, MdMYB21, which was further confirmed as a key negative regulator of both metabolic pathways. Functional assays revealed that overexpressing MdMYB21 significantly inhibited the accumulation of anthocyanins and malic acid in apple calli and fruit tissues. Mechanistic investigations using chromatin immunoprecipitation sequencing (ChIP-Seq), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assays demonstrated that MdMYB21 directly binds to promoters of core genes involved in anthocyanin synthesis and transport—including the glycosyltransferase gene MdAGT1 and glutathione S-transferase genes MdGSTU15, MdGSTU16, and MdGSTU22—and acts as a transcriptional repressor that inhibits their expression. These findings uncover a novel mechanism by which MdMYB21 coordinately regulates color and flavor metabolism in apple, providing an important genetic target for breeding new red-fleshed apple varieties with a desirable balance of visual and sensory quality.

WRKY transcription factors are essential for mediating many developmental processes, such as fruit ripening, a highly controlled and intricate physiological phenomenon. In the current research, a novel transcription factor, MdWRKY9, was identified and categorized, which significantly promotes apple fruit ripening. Its role was validated through a combination of transient injection and stable overexpression-transformed tomato experiments. Notably, MdWRKY9 interacts with the fruit ripening suppressor MdERF5L at protein and DNA levels. This interaction counteracts MdERF5L-mediated MdACS1 repression. MdACS1 is an important ethylene biosynthesis enzyme; the mentioned process eventually allows fruit ripening. Furthermore, phosphorylation, a post-translational modification, regulates maturation and ethylene synthesis. Through liquid chromatography-tandem mass spectrometry, we identified phosphorylation sites within the MdWRKY9-GFP protein. MdMAPK6-MdWRKY9 interaction and MdMAPK6-mediated phosphorylation of MdWRKY9 were confirmed through protein-protein interaction assays, such as bimolecular fluorescence complementation (BIFC), yeast two-hybrid (Y2H), protein phosphorylation, luciferase complementation imaging (LCI), and pull-down assays. Specifically, MdMAPK6 phosphorylates MdWRKY9 at Tyr394 site, enhancing the stability and activity of MdWRKY9 and thus modulating its regulatory role in fruit maturation. These findings provide new directions and insights into the intricate regulatory network governing the ripening of apples.

Salt stress is an important abiotic stress affecting the growth and fruit quality of apple fruits. Although jasmonic acid (JA) hormones and WRKY transcription factors (TFs) have both been reported to be involved in plant salt stress responses, the molecular mechanisms by which JA-mediated WRKY TFs regulate salt stress in apples remain unclear. Here, we report the identification of a WRKY family TF from apple, MdWRKY9, and its involvement in apple salt tolerance by regulating the expression of Na+/H+ antiporters, MdNHX1, and MdSOS2. Furthermore, we show that the protein repressors MdJAZ5 and MdJAZ10 in the JA signaling pathway can both interact with MdWRKY9 to form a complex and inhibit its DNA-binding and transcriptional activation activity. The JA signal triggers the degradation of MdJAZ5 and MdJAZ10 proteins by the 26S proteasome, disrupting the JAZ-WRKY protein complex and thereby releasing MdWRKY9 to activate downstream gene expression, promoting salt tolerance in apples. These findings provide important insights into the molecular mechanism of the WRKY TFs in JA-mediated salt tolerance in plants.

Apple replant disease (ARD) caused by the pathogen Fusarium solani is a destructive disease in apple planting areas worldwide, which leads to the decline of apple quality and yield. WRKY transcription factors are involved in the process of plants responding to various environmental stresses, but the function of WRKY TFs in ARD is unclear. In this study, the expression of MdWRKY20 was significantly increased after infection of apple rootstock 'M9T337' with F. solani. Transgenic analysis showed that the resistance of apple callus and Arabidopsis to F. solani increased after overexpression of MdWRKY20. Ectopic expression of MdWRKY20 also significantly enhanced antioxidant capacity in Arabidopsis under treatment with F. solani. Then, MdWRKY20 was found to bind directly to the W-box II of the MdPR1 promoter and significantly promoted its expression. In summary, MdWRKY20 plays a positive role in regulating the resistance of apples to F. solani.

'Shannongsu' pear is a new high-quality cultivar. To ascertain the storage characteristics of 'Shannongsu' pears at low temperatures (0 ± 0.5 °C), the following parameters were determined: fruit firmness, ethylene, aromatic compounds, sugar content, acidity, ascorbic acid, and the expression levels of ethylene-related genes and texture-softening genes. The firmness of 'Shannongsu' pears changed less than that of the control, decreasing by only 18.8% after 170 days of storage. Low temperatures suppressed the expression of key genes associated with PbACS1a and PbACO1. Moreover, the expression of key genes related to fruit softening (PbPG1, PbXET, PbPME, and Pbα-L-Af) was suppressed during storage at low temperatures and remained at low levels. Therefore, the low levels of ethylene biosynthesis and the expression of key genes involved in fruit softening might play a major role in the excellent storage characteristics of the 'Shannongsu' cultivar. After 170 days of storage, 'Shannongsu' pears did not show significant changes in key quality dimensions such as firmness, sugar, acid, sugar-acid ratio, and ascorbic acid content. Therefore, low temperatures could help maintain the freshness, flavor, and nutritional quality of the 'Shannongsu' pear. Our findings reveal for the first time the low-temperature storage characteristics of 'Shannongsu' pears, providing a new scientific theoretical basis for pear production and marketing.

The rhizosphere microbiome, as the second genome of plant immunity, forms a critical ecological barrier in plant-pathogen interactions. However, its functional mechanism in resisting the replanting disease pathogenic Fusarium proliferatum MR5 in apples has not been systematically elucidated. This study employed an integrated multi-omics approach to investigate the rhizosphere mechanisms of resistant (CG935) and sensitive (M9T337) apple rootstocks, aiming to uncover the metabolic and microbial interactions underlying apple replant disease resistance. Multiple omics joint analysis found that the infection of Fusarium proliferatum MR5 triggered the activation of a specific lysine biosynthesis pathway in resistant rootstocks, and the expression levels of key rate limiting enzymes aspartate kinase and dihydrodipicolinate synthase were significantly upregulated by 2.79 6.81 times compared to M9T337. Along with the metabolic reprogramming process, the efflux of lysine from the rhizosphere increased, and Bacillus with broad-spectrum antibacterial activity were specifically recruited, increasing its relative abundance by 40.73

The cultivation of apples in replanted orchards is essential given limitations in land resources. However, the presence of Fusarium and phenolic acids in the replanted soil harms the soil environment, which impedes the sustainable development of the apple industry. In this study, earthworm was used as the fermentation precursor protein to optimize the fermentation conditions, and the inhibition mechanism of the fermentation product on Fusarium and its potential to repair the apple replant soil environment were explored. Laboratory experiments showed that the optimum initial pH, temperature and time of earthworm fermentation were 7, 37 °C and 10 d, respectively. The inhibition rates of earthworm fermentation products against F. oxysporum, F. solani, F. proliferatum, and F. moniliforme were 79.8%, 75.1%, 78.7% and 79.2%, respectively. The inhibition rates of spore germination on F. oxysporum, F. solani, F. proliferatum, and F. moniliforme were 83.8%, 87.3%, 83.2% and 84.8%, respectively. In the field, use 300 mL of earthworm fermentation products for each planting pits before planting. The experimental results showed that, compared with the control, the content of soil pathogenic Fusarium and phenolic acid in Wantou (W3) were decreased by 75.1% and 59.8%, respectively, after treatment with earthworm fermentation products in 2019. Soil urease, phosphatase, sucrase and catalase activities increased by 383.2%, 78.2%, 130.3% and 43.5%, respectively. The fruit weight, anthocyanin content, soluble sugar, sugar-acid ratio, total ester ratio, total ester concentration and yield increased by 80.7%, 60.6%, 25.6%, 50.3%, 19.7%, 262.4% and 193.5%, respectively, while titratable acid content decreased by 16.9%. In conclusion, earthworm fermentation products can be used as a sustainable amendment to control apple replant disease.