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.

MhCLC-c1, a Cl channel c homolog from Malus hupehensis, alleviates NaCl-induced cell death by inhibiting intracellular Cl- accumulation.

BACKGROUND: Overaccumulation of chloride (Cl) when plants suffer NaCl causes cell damage and death, and is regulated by Cl- channel protein (CLC). Apple roots are very sensitive to Cl-, but information associated with CLC is limited in apple crop that widely cultivated in the world. RESULTS: We identified 9 CLCs from the apple genome and divided them into two subclasses. Among them, MdCLC-c1 promoter contained the largest number of cis-acting elements associated with NaCl stress, and only the MdCLC-c1, MdCLC-d, and MdCLC-g were predicted that may be Cl- antiporters or channels. Expression analysis of MdCLCs homologs in the roots of Malus hupehensis showed that most of the MhCLCs expression were response to NaCl stress, especially MhCLC-c1 expression was upregulated continuously and rapidly expressed during NaCl treatment. Therefore, we isolated MhCLC-c1 and observed it was a plasma membrane-localized protein. The MhCLC-c1 suppression significantly increased sensitivity, reactive oxygen species content, and cell death of apple calli; while MhCLC-c1 overexpression decreased sensitivity, reactive oxygen species content, and cell death of apple calli and Arabidopsis by inhibiting intracellular Cl- accumulation under NaCl stress. CONCLUSIONS: The study selected and isolated a CLC-c gene MhCLC-c1 from Malus hupehensis based on identification of CLCs gene family in apple, and their homologs MhCLCs expression patterns during NaCl treatments, revealing that MhCLC-c1 alleviates NaCl-induced cell death by inhibiting intracellular Cl- accumulation. Our findings confer the comprehensive and in-depth upstanding of the mechanism that plants resist salt stress, and might also confer genetic improvement of salt tolerance in horticultural crops and the development and utilization of saline-alkali land.

Comparative physiological and transcriptomic analysis reveal MdWRKY75 associated with sucrose accumulation in postharvest 'Honeycrisp' apples with bitter pit.

BACKGROUND: Calcium (Ca) deficiency can cause apple bitter pit, reduce the quality and shelf life. WRKY transcription factors play essential role in plant response to multiple disorders. However, the underlying mechanisms causing bitter pit in apple fruit due to Ca deficiency during storage is extremely limited. RESULTS: In the present study, the nutritional metabolites and reactive oxygen species (ROS) were compared in Ca-deficient and healthy apple fruit (CK) during storage. Results showed that Ca-deficient apples sustained significantly higher production of ROS, PPO activity, flavonoids, total phenol, total soluble solids (TSS), and sucrose contents, but the contents of Ca, H2O2, titratable acids (TA), glucose and fructose were significantly lower than those of CK during storage. Principal component analysis (PCA) showed that TSS, •O2-, PPO, malondialdehyde (MDA) and Ca were the main factors, and TSS had a positive correlation with sucrose. Furthermore, transcriptome analysis revealed that WRKYs were co-expressed with sucrose metabolism-related enzymes (SWEETs, SS, SPS). qRT-PCR and correlation analysis indicated that MdWRKY75 was correlated positively with MdSWEET1. Moreover, transient overexpression of MdWRKY75 could significantly increase the sucrose content and promote the expression of MdSWEET1 in apple fruit. CONCLUSIONS: Calcium deficiency could decrease antioxidant capacity, accelerate nutritional metabolism and up-regulate the expression of WRKYs in apple with bitter pit. Overexpression of MdWRKY75 significantly increased sucrose accumulation and the expression of MdSWEET1. These findings further strengthened knowledge of the basic molecular mechanisms in calcium deficiency apple flesh and contributed to improving the nutritional quality of apple fruit.

H2 S improves salt-stress recovery via organic acid turn-over in apple seedlings.

Signalling roles of hydrogen sulphide (H2 S) in stress biology are widely reported but not sufficiently established to urge its use in agronomic practice. Our lack of quantitative understanding of the metabolic rewiring in H2 S signalling makes it difficult to elucidate its functions in stress tolerance on the biochemical level. Here, Malus hupehensis Rehd. var. pingyiensis seedlings were first treated with salt stress for 2 weeks and then treated with four different concentrations of NaHS. Through vigorous investigations, including phenotypic analysis, 13 C transient labelling and targeted metabolic and transcriptomic analysis, for the first time in the seedlings of a woody fruit crop, we found out that H2 S recycles fixed carbons through glycolysis and tricarboxylic acid cycle to inhibit the futile accumulation of carbohydrates, to maintain an efficient CO2 assimilation, to keep a balanced starch metabolism, to produce sufficient H2 O2 , to maintain malate/γ-aminobutyric acid homeostasis via an H2 O2 -induced anion channel (aluminium-activated malate transporter) and eventually to improve salt-stress recovery. Our results systematically demonstrate the vital roles of central carbon metabolism in H2 S signalling and clarify the mode of action of H2 S in apple seedlings. We conclude that H2 S signalling interacts with central carbon metabolism in a bottom-up manner to recover plant growth after salt stress.

H2S Enhanced the Tolerance of Malus hupehensis to Alkaline Salt Stress through the Expression of Genes Related to Sulfur-Containing Compounds and the Cell Wall in Roots.

Malus is an economically important plant that is widely cultivated worldwide, but it often encounters saline-alkali stress. The composition of saline-alkali land is a variety of salt and alkali mixed with the formation of alkaline salt. Hydrogen sulfide (H2S) has been reported to have positive effects on plant responses to abiotic stresses. Our previous study showed that H2S pretreatment alleviated the damage caused by alkaline salt stress to Malus hupehensis Rehd. var. pingyiensis Jiang (Pingyi Tiancha, PYTC) roots by regulating Na+/K+ homeostasis and oxidative stress. In this study, transcriptome analysis was used to investigate the overall mechanism through which H2S alleviates alkaline salt stress in PYTC roots. Simultaneously, differentially expressed genes (DEGs) were explored. Transcriptional profiling of the Control-H2S, Control-AS, Control-H2S + AS, and AS-H2S + AS comparison groups identified 1618, 18,652, 16,575, and 4314 DEGs, respectively. Further analysis revealed that H2S could alleviate alkaline salt stress by increasing the energy maintenance capacity and cell wall integrity of M. hupehensis roots and by enhancing the capacity for reactive oxygen species (ROS) metabolism because more upregulated genes involved in ROS metabolism and sulfur-containing compounds were identified in M. hupehensis roots after H2S pretreatment. qRT-PCR analysis of H2S-induced and alkaline salt-response genes showed that these genes were consistent with the RNA-seq analysis results, which indicated that H2S alleviation of alkaline salt stress involves the genes of the cell wall and sulfur-containing compounds in PYTC roots.

Improving litterfall production prediction in China under variable environmental conditions using machine learning algorithms.

Litterfall production is a major process within forest ecosystems that plays a crucial role in the global carbon cycle. Accordingly, studies have explored the abiotic and biotic features that influence litterfall production. In addition to traditional statistical models, the rapid development of nonparametric and nonlinear machine learning models, such as random forest (RF), light gradient boosting machine (LightGBM), and categorical boosting (CatBoost), have provided new methods of predicting the production of forest litterfall. Here, we evaluated the ability of the abovementioned models and mixed effect random forest (MERF) models to predict total annual litterfall production-based on several abiotic and biotic features-using 968 records from 314 forest sites covering the full geographical range of Chinese forests. In general, machine learning models were found to outperform linear mixed models. In particular, the MERF models ranked the highest in terms of performance (R2 = 0.7), which may be attributed to their ability to characterize nonlinear relationships between features and litterfall production. The key drivers were climate-related features and forest age, with the mean annual temperature and age positively correlated with litterfall production. Furthermore, the correlation between forest type and litterfall production was more significant for needleleaf forests than for other forest types. For needleleaf and broadleaf forests in several regions in China, the future litterfall production was predicted to be the highest under IPCC representative concentration pathway (RCP) 8.5, followed by RCP 4.5, RCP 2.6, and the original scenarios (sample data). Improved models to better understand and estimate litterfall production in forests at present and in the future are required for forest management planning to minimize the negative impacts of climate change on forest ecosystems.

Risk factors for patient selection in ambulatory laparoscopic cholecystectomy: A Single-centre experience.

Backgrounds: Ambulatory laparoscopic cholecystectomy (LC) reduces healthcare cost and increases hospital bed capacity. Currently, there is no consensus on patient selection for ambulatory LC. Evaluation of risk factors for ambulatory discharge is essential. Materials and Methods: Consecutive patients who underwent LC in our centre throughout 2019 were collected. We evaluated the discharge fitness using the Post-Anaesthetic Discharge Scoring System at 8 h after the operation. The relations between pre-operative variables and dischargeable possibilities were analysed for screening risk factors. Furthermore, we performed a literature review to summarise all published information. Results: Six hundred and forty-one cases were included in this study. American Society of Anaesthesiologist (ASA) grading (odds ratio OR = 0.415, P = 0.001) and leucocytes (OR = 0.80, P < 0.001) significantly predicted the fitness of discharge. ASA contributed to lower activity (P = 0.002) and intake/output (P = 0.006) scores. Leucocytes influence the vital sign (P < 0.001) and pain or post-operative nausea and vomiting (PONV) (P < 0.001) scores. The prolonged operation could predict the inabilities of discharge with a cut-off value of 55 min by dropping vital signs (P = 0.011), activity (P < 0.001) and pain or PONV (P = 0.012) scores. Male sex (OR: 1.702, P = 0.010), body mass index (BMI) (OR: 1.087, P = 0.008), leucocytes (OR: 1.075, P = 0.017) and C-reactive protein (CRP) (OR: 1.018, P = 0.003) were predictors for prolonged operation (>55 min). Conclusions: We suggest that pre-operative ASA grading III and leucocytes are risk factors for the fitness of ambulatory discharge after LC and intraoperative time. Male, BMI and CRP predict complicated surgery, and they should be considered preoperatively.

Transcriptomics Reveals the ERF2-bHLH2-CML5 Module Responses to H2S and ROS in Postharvest Calcium Deficiency Apples.

Calcium deficiency usually causes accelerated quality deterioration in postharvest fruit, whereas the underlining mechanism is still unclear. Here, we report that calcium deficiency induced the development of bitter pit on the surface of apple peels compared with the healthy appearance in control apples during postharvest storage. Physiological analysis indicates that calcium-deficient peels contained higher levels of superoxide anion (O2•-), malondialdehyde (MDA), total phenol, flavonoid contents and polyphenol oxidase (PPO) activity, and reduced calcium, H2S production, anthocyanin, soluble protein content, and peroxidase (POD) activity compared with those in calcium-sufficient peels. The principal component analysis (PCA) results show that calcium content, ROS, and H2S production were the main factors between calcium-deficient and calcium-sufficient apple peels. Transcriptome data indicated that four calmodulin-like proteins (CMLs), seven AP2/ERFs, and three bHLHs transcripts were significantly differentially expressed in calcium-deficient apple peels. RT-qPCR and correlation analyses further revealed that CML5 expression was significantly positively correlated with the expression of ERF2/17, bHLH2, and H2S production related genes. In addition, transcriptional co-activation of CML5 by ERF2 and bHLH2 was demonstrated by apple transient expression assays and dual-luciferase reporter system experiments. Therefore, these findings provide a basis for studying the molecular mechanism of postharvest quality decline in calcium-deficient apples and the potential interaction between Ca2+ and endogenous H2S.

Improving Carbon Stock Estimates for In-Use Harvested Wood Products by Linking Production and Consumption-A Global Case Study.

We developed a method to better estimate the carbon stocks of in-use harvested wood products (HWP) by using the Eora multiregional input-output tables to link global HWP production and end uses, compared to existing global-scale studies that focused on semifinished HWP. Using the new method, we allocated global HWP to country-specific end uses, including solid HWP used in (1) construction, (2) furniture production, and (3) other end uses, and as (4) household and sanitary paper and (5) other paper and paper products, while the HWP carbon stocks in these end uses were estimated using the Stock Change Approach. We reported that HWP produced globally contained an annual average of 277.7 teragram carbon in 1992-2015, of which 63.0, 12.6, 76.7, 9.1, and 116.3 teragram carbon were consumed by the above five end uses, respectively. By 2015, the carbon stocks of global in-use HWP produced since 1992 accumulated to 2938 teragrams of carbon, of which the above five HWP end uses accounted for 1489, 268, 890, 0, and 291 teragrams of carbon, respectively. Country-specific HWP production and consumption varied significantly, with the eight leading consuming countries (United States, China, Japan, Canada, Germany, Russia, United Kingdom, and France) accounting for 69% of the global in-use HWP carbon stocks.

Assessing the Greenhouse Gas Mitigation Potential of Harvested Wood Products Substitution in China.

Substituting harvested wood products (HWP) for greenhouse gas (GHG) intensive nonwood materials in long-lived end uses has the potential to significantly reduce GHG emissions. To determine the mitigation effects of HWP substitution, we produced China-specific wood displacement factors (DFs) by HWP end use subcategory, defined as tonnes of carbon (tC) of reduced emissions per tC contained by the HWP substituted for typical alternative nonwood materials. The weighted average DFs for substituting HWP for nonwood materials in construction and furniture production in China were estimated to be 3.48 tC/tC and 1.36 tC/tC, respectively, or 2.90 tC/tC for HWP substitution when these two sectors were combined. If annual solid HWP consumption in China increased by 10% on the basis of 2014 consumption (an increase of 25.9 million m3 of HWP) and these HWP were used to substitute for GHG-intensive materials in construction and furniture production, 18.76 Mt C of emission reduction can be achieved annually. Substituting HWP for nonwood materials in construction appeared to be more effective than in furniture manufacture in mitigating GHG emissions. Our study suggested that increasing HWP use in China, especially in the construction industry to substitute for nonwood materials can significantly contribute to China's emission reduction targets.

MhMAPK4 from Malus hupehensis Rehd. decreases cell death in tobacco roots by controlling Cd2+ uptake.

Cadmium (Cd) induces cell death in plant roots. Mitogen-activated protein kinase (MAPK) plays a role in the regulation of cell death induced by Cd in plant roots. In this study, MhMAPK4 was isolated from the roots of Malus hupehensis. Subcellular localization showed that the MhMAPK4 protein was located in the cell membrane and cytoplasm and is a transmembrane protein that is characterized by hydrophily. The expression of MhMAPK4 in the roots of M. hupehensis was up-regulated by Cd sulfate and Cd chloride. Phenotypic comparison under Cd stress showed that the growth of wild-type (WT) tobacco was lower than the transgenic lines overexpressing MhMAPK4. The fresh weight and the root length of WT also was lower than that of the transgenic tobacco. The net Cd2+ influx in the tobacco roots was decreased by the overexpression of MhMAPK4, as was root Cd accumulation. The recovery time of the Cd2+ influx to stable state in the transgenic tobacco was also shorter than the WT. The expression of iron-regulated transporter 1 (NtIRT1) and natural resistance associated macrophage protein 5 (NtNRAMP5) was relatively low in the transgenic lines under Cd stress. Cell death and apoptosis in the tobacco roots was reduced following the overexpression of MhMAPK4. The activity of vacuolar processing enzyme (VPE) and the transcript level of VPE in the transgenic tobacco was lower than that of WT under Cd stress. In addition, the electrolyte leakage and malondialdehyde and hydrogen peroxide contents in the transgenic tobacco were lower than those of WT, whereas the antioxidant enzyme activity and expression were higher. These results suggest that MhMAPK4 regulates Cd accumulation by mediating Cd2+ uptake by the roots, and controls Cd-caused cell death by adjusting VPE activity.

Feasibility research of treating advanced gastric carcinoma with laparoscopy.

PURPOSE: To determine the feasibility and safety of treating patients with advanced gastric cancer with laparoscopy. METHODS: We retrospectively analyzed 180 patients with advanced gastric carcinoma and divided them into either the laparoscopy group (96 cases) or the laparotomy group (84 cases). RESULTS: The number of lymph nodes dissected during surgery and the surgical time were similar in both groups. The incision length, total amount of bleeding during the operation, postoperative exhaust recovery time, and the length of hospital stay were significantly improved in the laparoscopy group when compared to the laparotomy group. The rate of postoperative complications was also significantly lower in the laparoscopy group. The levels of C-reactive protein (CRP) at 1, 7, and 10 days after surgery were significantly lower in the laparoscopy group. CONCLUSION: Overall, the laparoscopic radical operation for advanced gastric carcinoma demonstrated higher safety, shorter incision less bleeding, faster postoperative recovery, and lower rate of postoperative complications compared to the laparotomy group. Thus, this study has shown clear advantages for shifting to laparoscopy for the treatment of advanced gastric carcinoma.

Activin A induces growth arrest through a SMAD- dependent pathway in hepatic progenitor cells.

BACKGROUND: Activin A, an important member of transforming growth factor-ß superfamily, is reported to inhibit proliferation of mature hepatocyte. However, the effect of activin A on growth of hepatic progenitor cells is not fully understood. To that end, we attempted to evaluate the potential role of activin A in the regulation of hepatic progenitor cell proliferation. RESULTS: Using the 2-acetaminofluorene/partial hepatectomy model, activin A expression decreased immediately after partial hepatectomy and then increased from the 9th to 15th day post surgery, which is associated with the attenuation of oval cell proliferation. Activin A inhibited oval cell line LE6 growth via activating the SMAD signaling pathway, which manifested as the phosphorylation of SMAD2/3, the inhibition of Rb phosphorylation, the suppression of cyclinD1 and cyclinE, and the promotion of p21WAF1/Cip1 and p15INK4B expression. Treatment with activin A antagonist follistatin or blocking SMAD signaling could diminish the anti-proliferative effect of activin A. By contrast, inhibition of the MAPK pathway did not contribute to this effect. Antagonizing activin A activity by follistatin administration enhanced oval cell proliferation in the 2-acetylaminofluorene/partial hepatectomy model. CONCLUSION: Activin A, acting through the SMAD pathway, negatively regulates the proliferation of hepatic progenitor cells.

Metagenomic and metabolomic analysis of the effect of bleaching on unsaturated fatty acid synthesis pathways in coral symbionts.

Unsaturated fatty acids (UFAs) are known to play a vital role in regulating stress resistance and metabolism in corals. Nevertheless, a comprehensive understanding of the microbial and functional composition of the UFA synthesis pathway (UFASP) remains lacking. This study employed metagenome and metabolome to investigate the microbial community, function, and metabolic response of UFASP in reef-building corals inhabiting the Nansha Islands. Our findings revealed significantly higher diversity for the UFASP microbe in bleached corals compared to unbleached corals. Furthermore, principal coordinates analysis (PCoA) and taxonomy assessments exhibited notable distinctions in the microbe between the two coral states. Notably, the dominant microorganisms involved in UFASP were Dinophyceae, Sordariomycetes, Ulvophyceae, and Chlorophyceae. Bleaching resulted in a considerable increase in fungal abundance within coral symbionts. A total of 12 KEGG Orthology (KO) were identified in UFASP, with PCoA analysis indicating significant differences in their abundance between bleached and unbleached corals. UFASP's beta-Oxidation module exhibited reduced abundance in bleached corals. Contribution analysis highlighted the participation of Symbiodiniaceae, Ascomycota, Chlorophyta, Proteobacteria, and Actinobacteria in UFASP. Notably, Symbiodiniaceae and Ascomycota were the major contributors to two UFASP modules, with the latter displaying greater involvement in bleached corals. Furthermore, significant differences in n3 and n6-family metabolites were observed between bleached and unbleached corals. Notably, bleaching induced a reduction in metabolites of Symbiodiniaceae, while an increase in the multiple UFAs abundance was detected in bleached corals. These findings suggest that bleaching-induced alterations coral symbionts composition directly impact the functionality of UFASP, ultimately affecting the corals' capacity to adapt to stress.

Alum/CpG adjuvant promotes immunogenicity of inactivated SARS-CoV-2 Omicron vaccine through enhanced humoral and cellular immunity.

The SARS-CoV-2 Omicron variant, which was classified as a variant of concern (VOC) by the World Health Organization on 26 November 2021, has attracted worldwide attention for its high transmissibility and immune evasion ability. The existing COVID-19 vaccine has been shown to be less effective in preventing Omicron variant infection and symptomatic infection, which brings new challenges to vaccine development and application. Here, we evaluated the immunogenicity and safety of an Omicron variant COVID-19 inactivated vaccine containing aluminum and CpG adjuvants in a variety of animal models. The results showed that the vaccine candidate could induce high levels of neutralizing antibodies against the Omicron variant virus and binding antibodies, and significantly promoted cellular immune response. Meanwhile, the vaccine candidate was safe. Therefore, it provided more foundation for the development of aluminum and CpG as a combination adjuvant in human vaccines.

Using protein granularity to extract the protein sequence features.

The feature extraction of protein sequences is a challenging problem. It might need a lot of theoretical and practical knowledge from many fields. The difficulty would increase when investigators extract the features solely from protein sequences. In this paper, we present a method of protein granularity. The concepts of protein granularity, granularity order, granularity bound, granularity limit, and granularity increment are given respectively. The protein granularity can dig out the useful information solely from protein sequences. We provide an approach to construct the feature vectors. The feature vectors include the amino acid composition information, the sequence-order information, the same amino acid 'neighbor' information, and the sequence length information. Hence, the feature vectors can better represent protein sequences. Our feature extraction method does obviously consider the protein sequence length effects. An experiment of the protein structure class prediction was carried out. The prediction achieved 96.6% overall accuracy, and the success rate for each subset is all-α 92.3%, all-ß 100%, α/ß 100%, α+ß 93.5%, respectively. The last three success rates for subsets are equal to the best success rates in the published literatures. The overall accuracy of PG-SVM prediction is the second best result only having one protein prediction error difference with the first best result. The theoretical and experimental results demonstrate the application of protein granularity succeeds in the feature extraction of protein sequences.

Metabolic and metatranscriptional characteristics of corals bleaching induced by the most severe marine heatwaves in the South China Sea.

Coral bleaching significantly affects the function and health of coral reef ecosystems; however, the mechanisms underlying metabolism and transcription in corals remain unclear. In this study, untargeted metabolomics and metatranscriptomic analyses were performed to analyze the differences between unbleached and bleached Pocillopora corals during the most severe marine heatwaves. Difference analysis showed that bleached corals had significant metabolomic characteristics compared with those in unbleached corals. These differences were significant (p < 0.05) according to partial least squares discriminant analysis (PLS-DA). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the metabolites were significantly enriched in numerous pathways in bleached or unbleached corals, such as steroid hormone biosynthesis, biosynthesis of unsaturated fatty acids, and pyrimidine metabolism. Bleaching greatly affects coral reproduction as well as the tolerance of coral symbionts to heat stress. In metatranscriptomic analysis, we observed large gene expression differences between unbleached and bleached corals. Three Gene Ontology directed acyclic graphs (DAGs) were constructed to show the significantly differentially expressed genes (DEGs). Many biological and molecular processes were significantly enriched between bleached corals to unbleached corals, such as metabolic processes, lipid metabolic processes, oxidation-reduction processes, single-organism metabolic processes, and protein metabolic processes. Metabolome and metatranscriptome analyses showed that bleaching caused substantial physiological damage to corals. This study provides insight into the metabolic and transcriptional changes that occur in corals during bleaching.

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.

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.

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.