Bacterial community structure and co-occurrence networks in the rhizosphere and root endosphere of the grafted apple.

BACKGROUND: Compared with aerial plant tissues (such as leaf, stem, and flower), root-associated microbiomes play an indisputable role in promoting plant health and productivity. We thus explored the similarities and differences between rhizosphere and root endosphere bacterial community in the grafted apple system. RESULTS: Using pot experiments, three microhabitats (bulk soil, rhizosphere and root endosphere) samples were obtained from two-year-old apple trees grafted on the four different rootstocks. We then investigated the bacterial community composition, diversity, and co-occurrence network in three microhabitats using the Illumina sequencing methods. Only 63 amplicon sequence variants (ASVs) out of a total of 24,485 were shared in the rhizosphere and root endosphere of apple grafted on the four different rootstocks (M9T337, Malus hupehensis Rehd., Malus robusta Rehd., and Malus baccata Borkh.). The core microbiome contained 8 phyla and 25 families. From the bulk soil to the rhizosphere to the root endosphere, the members of the phylum and class levels demonstrated a significant enrichment and depletion pattern. Co-occurrence network analysis showed the network complexity of the rhizosphere was higher than the root endosphere. Most of the keystone nodes in both networks were classified as Proteobacteria, Actinobacteriota and Bacteroidetes and were low abundance species. CONCLUSION: The hierarchical filtration pattern existed not only in the assembly of root endosphere bacteria, but also in the core microbiome. Moreover, most of the core ASVs were high-abundance species, while the keystone ASVs of the network were low-abundance species.

Wood biochar in soil enhances the promotion of KNO3 on sulfur accumulation in apple trees by regulating root sulfate assimilation.

To determine how potassium nitrate (KNO3) effect apple roots and sulfate assimilation in the presence of wood biochar in soil, KNO3 was applied to the root-zone soil without or with 150-day naturally aged wood biochar (1% w/w) in soil. Soil properties, root architecture, root activity, the accumulation and distribution of sulfur (S), enzyme activity, and gene expression related to sulfate uptake and assimilation in apple trees were analyzed. Results showed that KNO3 and wood biochar application exhibited synergistic effects on improving S accumulation and root growth. Meanwhile, KNO3 application increased the activities of ATPS, APR, SAT, OASTL and upregulated the expression of ATPS, APR, Sultr3;1, Sultr2;1, Sultr3;4, and Sultr3;5 in both roots and leaves, and the positive effects of KNO3 addition on both genes and enzyme activity were enhanced by wood biochar. Wood biochar amendment alone promoted the activities of enzymes described above, upregulated the expression of ATPS, APR, Sultr3;1, Sultr2;1, Sultr3;4, and Sultr4;2 in leaves, and enhanced S distribution in roots. KNO3 addition alone decreased S distribution in roots and increased that in stems. In the presence of wood biochar in soil, KNO3 application decreased S distribution in roots but increased that in both stems and leaves. These results indicated that the wood biochar in soil enhances the effect of KNO3 on S accumulation by promoting root growth and sulfate assimilation in apple trees.

Multi-walled carbon nanotubes promote the accumulation, distribution, and assimilation of 15N-KNO3 in Malus hupehensis by entering the roots.

Introduction: Multi-walled nanotubes (MWCNTs) consist of multiple rolled layers of graphene. Nitrogen plays an important role in apple growth. The effect of MWCNTs on nitrogen utilization in apple needs to be further investigated. Methods: In this study, the woody plant Malus hupehensis seedlings were used as plant materials, the distribution of MWCNTs in the roots was observed, and the effects of MWCNTs on the accumulation, distribution, and assimilation of nitrate by the seedlings were explored. Results: The results showed that MWCNTs could penetrate the roots of Malus hupehensis seedlings, and the 50, 100, and 200 µg·mL-1 MWCNTs significantly promoted the root growth of seedlings, increased root number, root activity, fresh weight, and nitrate content of seedlings, and also increased nitrate reductase activity, free amino acid, and soluble protein content of roots and leaves. 15N tracer experiments indicated that MWCNTs decreased the distribution ratio of 15N-KNO3 in Malus hupehensis roots but increased its distribution ratio in stems and leaves. MWCNTs improved the utilization ratio of 15N-KNO3 in Malus hupehensis seedlings, with the values being increased by 16.19%, 53.04%, and 86.44% following the 50, 100, and 200 µg·mL-1 MWCNTs, respectively. The RT-qPCR analysis showed that MWCNTs significantly affected the expression of genes (MhNRTs) related to nitrate uptake and transport in roots and leaves, and MhNRT1.4, MhNRT1.7, MhNRT1.8, MhNRT2.1, MhNRT2.5, and MhNRT2.7 were notably up-regulated in response to 200 µg·mL-1 MWCNTs. Raman analysis and transmission electron microscopy images indicated that MWCNTs could enter the root tissue of Malus hupehensis and were distributed between the cell wall and cytoplasmic membrane. Pearson correlation analysis showed that root tip number, root fractal dimension, and root activity were the main factors affecting root uptake and assimilation of nitrate. Conclusions: These findings suggest that MWCNTs promoted root growth by entering the root, stimulated the expression of MhNRTs, and increased NR activity, thereby enhancing the uptake, distribution, and assimilation of nitrate by root, and ultimately improved the utilization of 15N-KNO3 by Malus hupehensis seedlings.

Occurrence and prevalence of antibiotic resistance genes in apple orchard after continual application of anaerobic fermentation residues of pig manure.

Fermented organic fertilizers made from pig manure contaminated with antibiotics are widely used in fruit tree production. However, their effects on the residual antibiotics and the spread of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in apple orchards are still largely unknown. In the present study, we detected 100 ARGs and 10 MGEs that were transferred from pig manure to an apple orchard. Compared with the original pig manure, significantly greater concentrations of tetracycline, chlortetracycline, oxytetracycline, sulfadiazine, and salfamethyldiazine were observed in anaerobic fermentation residues of the pig manure. The total relative abundance levels of ARGs on the apple pericarp surface, in the orchard soil treated with biogas slurry, and in the orchard soil treated with biogas residue were 122.5, 5.2, 1.4 times higher than those in pristine soil, respectively, which were primarily attributed to the increase in the relative abundance of some ARG subtypes, including blaCTX-M, blaTEM, ermC, sul2, tetO, vgaB, and vgb. Long-term biogas slurry and biogas residue applications to orchard soil enriched bioaccumulation of 10 ARGs and 1 MGEs on the apple pericarp surface with 67.98 the highest factor. This research indicates that the application of anaerobic fermentation residues of pig manure promoted the spread of ARGs in the soil and fruits and increased the level of ARG pollution in the orchard. Results of this study highlight the importance of assessing the ecological safety of organic fertilizers from the perspective of ARGs and indicate that efforts should be devoted to further reducing ARG levels in pig manure before its application to farmland.

Regulation effects of carbonized apple branches on absorption, distribution, and utilization of 15N single-labeled ammonium nitrate (15NH4NO3 or NH415NO3) in Malus hupehensis.

To improve the utilization of nitrogen fertilizer and orchard waste, the apple branches were separated and pyrolyzed into carbonized wood and carbonized bark, and then applied to root-zone soil of potted Malus hupehensis. The physiological characteristics of leaves and roots were detected, and the absorption, utilization, and distribution of 15NH4NO3 and NH415NO3 in plants were analyzed using the 15N isotope tracer technique. The results indicated that the net photosynthetic rate and water use efficiency of leaves, the root growth, and the activity of nitrate reductase and glutamine synthetase were greatest increased by 1.0% (w:w) carbonized bark and carbonized wood, and the effect of carbonized bark was more effective. The carbonized bark more effectively increased nitrogen derived from fertilizer (Ndff) value in all organs, the distribution of 15N in roots, and utilization of the 15NH4NO3 and NH415NO3 of Malus hupehensis compared with carbonized wood at the same application ratio, and 1.0% ratio performed better than other ratios in these terms. The Malus hupehensis treated with carbonized bark had the highest utilization ratio of 15NH4NO3 (10.54%) when the application ratio was 1.0%, and the corresponding parameter of NH415NO3 was 12.98%. The soil immobilization capacity of 15N was improved, and carbonized bark resulted in the greatest decrease in the loss ratios of 15NH4NO3 and NH415NO3 under 1.0% ratio, which decreased by 27.33% and 30.08%, respectively. For reducing nitrogen loss and improving nitrogen utilization, carbonized bark was more effective than carbonized wood, mainly because bark contained more cellulose and less lignin than wood.

Genome-Wide Identification of Sultr Genes in Malus domestica and Low Sulfur-Induced MhSultr3;1a to Increase Cysteine-Improving Growth.

Sulfur is an essential nutrient for plant growth and development. Sulfate transporters (Sultrs) are critical for sulfate ( SO 4 2 - ) uptake from the soil by the roots in higher plants. However, knowledge about Sultrs in apples (Malus domestica) is scarce. Here, nine putative MdSultrs were identified and classified into two groups according to the their phylogenetic relationships, gene structures, and conserved motifs. Various cis-regulatory elements related to abiotic stress and plant hormone responsiveness were found in the promoter regions of MdSultrs. These MdSultrs exhibited tissue-specific expression patterns and responded to low sulfur (S), abscisic acid (ABA), indole-3-acetic acid (IAA), and methyl jasmonate (MeJA), wherein MdSultr3;1a was especially expressed in the roots and induced by low S. The uptake of SO 4 2 - in cultivated apples depends on the roots of its rootstock, and MhSultr3;1a was isolated from Malus hupehensis roots used as a rootstock. MhSultr3;1a shared 99.85% homology with MdSultr3;1a and localized on the plasma membrane and nucleus membrane. Further function characterization revealed that MhSultr3;1a complemented an SO 4 2 - transport-deficient yeast mutant and improved the growth of yeast and apple calli under low S conditions. The MhSultr3;1a-overexpressing apple calli had a higher fresh weight compared with the wild type (WT) under a low-S treatment because of the increased SO 4 2 - and cysteine (Cys) content. These results demonstrate that MhSultr3;1a may increase the content of SO 4 2 - and Cys to meet the demands of S-containing compounds and improve their growth under S-limiting conditions.

[Carbonized Apple Branches Decrease the Accumulation and Damage of Cadmium on Apple Rootstock by Reducing DTPA-Cd in Soil].

To explore the effects of carbonized apple branches on cadmium(Cd) accumulation and its damage to apple rootstock, the rootstocks of apple(Malus hupehensis Rehd.) in pots containing soil together with 0.5% and 1%(ω) carbonized apple branches were irrigated by a nutrient solution containing CdSO4. The content of DTPA-Cd(cadmium extracted by diethylenetriamine pentaacetic acid) in the potting soil, and the accumulation of Cd in the roots, stems, and leaves of apple rootstocks, were subsequently monitored. The activities of antioxidant enzymes in roots and leaves, root cell death, and the net photosynthesis rate were further analyzed. The results showed that the concentration of DTPA-Cd in the potting soil with carbonized apple branches was significantly lower than that without carbonized apple branches(Cd-only). Compared with the Cd-only treatment, the concentration of DTPA-Cd in the potting soil decreased by 17.50% and 25.55% in the treatment with 0.5% and 1%(ω) carbonized apple branches for 12 days. The Cd accumulation in roots, stems, and leaves; the accumulations of superoxide anions(·O2-), hydrogen peroxide(H2O2), and malondialdehyde(MDA) in roots and leaves; and the amount of cell death in the roots of apple rootstock treated by carbonized apple branches were significantly lower compared to the Cd-only treatment. However, the activities of superoxide dismutase(SOD), peroxidase(POD), and catalase(CAT) in the roots and leaves, and the net photosynthesis rate of apple rootstock treated by carbonized apple branches, were significantly higher than under the Cd-only treatment. Compared with the Cd-only treatment, Cd accumulation in roots decreased by 29.49% and 37.18% in the treatment with 0.5% and 1%(ω) carbonized apple branches for 12 days, and the amount of cell death decreased by 22.73% and 29.09%, respectively. Our results show that carbonized apple branches reduce the uptake and accumulation of Cd in apple rootstock by reducing the content of DTPA-Cd in the soil, thereby alleviating the damaging effect of Cd on cells and photosynthesis. Moreover, the use of 1%(ω) carbonized apple branches was more effective than 0.5%(ω).

MhNRAMP1 From Malus hupehensis Exacerbates Cell Death by Accelerating Cd Uptake in Tobacco and Apple Calli.

Excessive cadmium (Cd) damages plants by causing cell death. The present study discusses the function of natural resistance-associated macrophage protein (NRAMP) on cell death caused by Cd in Malus hupehensis. MhNRAMP1 was isolated from M. hupehensis roots, and its protein was located in the cell membrane as a transmembrane protein characterized by hydrophobicity. MhNRAMP1 expression in the roots was induced by Cd stress and calcium (Ca) deficiency. MhNRAMP1 overexpression increased Cd concentration in yeasts and enhanced their sensitivity to Cd. Phenotypic comparisons of plants under Cd stress revealed that the growth of transgenic tobacco and apple calli overexpressing MhNRAMP1 was worse than that of the wild type (WT). The Cd2+ influx of transgenic tobacco roots and apple calli was higher, and the recovery time of the Cd2+ influx to a stable state in transgenic apple calli was longer than that of the WT. Cd accumulation and the percentage of apoptotic cells in transgenic lines were higher. Correspondingly, the caspase-1-like and vacuolar processing enzyme (VPE) activities and MdVPEγ expression were higher in transgenic apple calli, but the expression levels of genes that inhibit cell death were lower than those in the WT under Cd stress. Moreover, the Cd translocation from the roots to leaves was increased after MhNRAMP1 overexpression, but the Cd translocation from the leaves to seeds was not affected. These results suggest that MhNRMAP1 exacerbated Cd-induced cell death, which was accomplished by mediating Cd2+ uptake and accumulation, as well as stimulating VPE.

Genome-Wide Identification and Characterization of Vacuolar Processing Enzyme Gene Family and Diverse Expression Under Stress in Apple (Malus × Domestic).

Vacuolar processing enzymes (VPEs) play an important role in stress resistance and development of plants. Despite their diverse roles, little information is available in apple (Malus × domestic). This study firstly presents the genome-wide identification of VPE family genes in apple, resulting in 20 family members those are unevenly distributed across six out of the 17 chromosomes. Phylogenetic analysis assigned these genes into four groups. Analysis of exon-intron junctions and motifs of each candidate gene revealed high levels of conservation within and between phylogenetic groups. Cis-element including w box, ABRE, LTR, and TC-rich repeats were found in promoters of MdVPEs. NCBI-GEO database shown that the expression of MdVPEs exhibited diverse patterns in different tissues as well as the infection of Pythium ultimum and Apple Stem Grooving Virus. Furthermore, qRT-PCR showed that MdVPE genes were responsive to salt, cadmium, low-temperature, and drought. Overexpression of MDP0000172014, which was strongly induced by salt and drought stress, significantly decreased Arabidopsis tolerance to salt stress. The genome-wide identification and characterization of MdVPEs in apple provided basic information for the potential utilization of MdVPEs in stress resistance.