Highly sensitive fluorescent detection of EDIL3 overexpressed exosomes for the diagnosis of triple-negative breast cancer.

EDIL3 is a strong and highly accurate diagnostic marker for breast cancer, meanwhile, EDIL3 overexpressed exosomes are novel biomarkers for the early diagnosis of triple-negative breast cancer (TNBC). Here, we proposed a fluorescent detection method for EDIL3 overexpressed exosomes, which is simple and sensitive. Basically, we utilized a magnetic nanospheres (MNS) based liquid sandwich immunoassay strategy. MNS were modified with CD63 aptamers, which can immunologically bound to the CD63 protein on the surface of exosomes. Alexa Fluor 647 labeled anti-EDIL3 antibodies (Anti-EDIL3/AF647) were used as the fluorescent probes to recognize the EDIL3 on exosomes derived from a TNBC cell line (MDA-MB-231). With the target TNBC exosomes present, sandwich structures containing MNS, exosomes and fluorescent probes were formed. After magnetic purification, optical super resolution imaging of the products was conducted to check the specificity of the assay. In addition, fluorescence signals of the products were detected to quantitatively analyze the EDIL3 overexpressed exosomes. The linear range was found to be 7.78 × 101to 7.78× 106particlesµl-1. The detection limit was approximately 10 particlesµl-1. The feasibility of the method for the detection of exosomes in complex biological samples was also demonstrated. Such a simple and sensitive detection method for EDIL3 overexpressed exosomes holds a great potential in clinical diagnosis of TNBC.

Application of ultrasound artificial intelligence in the differential diagnosis between benign and malignant breast lesions of BI-RADS 4A.

BACKGROUND: The classification of Breast Imaging Reporting and Data System 4A (BI-RADS 4A) lesions is mostly based on the personal experience of doctors and lacks specific and clear classification standards. The development of artificial intelligence (AI) provides a new method for BI-RADS categorisation. We analysed the ultrasonic morphological and texture characteristics of BI-RADS 4A benign and malignant lesions using AI, and these ultrasonic characteristics of BI-RADS 4A benign and malignant lesions were compared to examine the value of AI in the differential diagnosis of BI-RADS 4A benign and malignant lesions. METHODS: A total of 206 lesions of BI-RADS 4A examined using ultrasonography were analysed retrospectively, including 174 benign lesions and 32 malignant lesions. All of the lesions were contoured manually, and the ultrasonic morphological and texture features of the lesions, such as circularity, height-to-width ratio, margin spicules, margin coarseness, margin indistinctness, margin lobulation, energy, entropy, grey mean, internal calcification and angle between the long axis of the lesion and skin, were calculated using grey level gradient co-occurrence matrix analysis. Differences between benign and malignant lesions of BI-RADS 4A were analysed. RESULTS: Significant differences in margin lobulation, entropy, internal calcification and ALS were noted between the benign group and malignant group (P = 0.013, 0.045, 0.045, and 0.002, respectively). The malignant group had more margin lobulations and lower entropy compared with the benign group, and the benign group had more internal calcifications and a greater angle between the long axis of the lesion and skin compared with the malignant group. No significant differences in circularity, height-to-width ratio, margin spicules, margin coarseness, margin indistinctness, energy, and grey mean were noted between benign and malignant lesions. CONCLUSIONS: Compared with the naked eye, AI can reveal more subtle differences between benign and malignant BI-RADS 4A lesions. These results remind us carefully observation of the margin and the internal echo is of great significance. With the help of morphological and texture information provided by AI, doctors can make a more accurate judgment on such atypical benign and malignant lesions.

Nitrous oxide emission and the related denitrifier community: A short-term response to organic manure substituting chemical fertilizer.

Partially substituting chemical fertilizer with organic manure can aid in the disposal of agricultural wastes via recycling into agricultural land, reduce chemical fertilizer application, and influence nitrogen (N) transformation. However, relatively few studies have investigated the association between soil physicochemical properties, denitrifier communities and N2O emission after short-term substitution of organic manure in vegetable fields. We conducted a short-term vegetable field experiment which included one control treatment (CT, no fertilizer) and three fertilization treatments containing equal amount of total N, phosphorus and potassium (CF, chemical fertilizer only; CMR, chemical fertilizer plus mushroom residue; COM, chemical fertilizer plus cattle manure). The results showed that partial substitution of chemical fertilizer with organic manure greatly increased cumulative N2O emissions, N2O emission factors and yield-scaled N2O emissions by 122-203%, 238-600% and 128-181%, respectively. Compared with the CF treatment, short-term substitution with organic manure reduced the abundance of nirS- and nosZ-type denitrifiers, and increased that of nirK-type denitrifiers. Modeling indicated that nirS abundance, together with soil available N, NIR activity, nirK abundance, SOC, NH4+, and NO3- were the primary factors associated with cumulative N2O emissions. The denitrifier community composition of the CF- treated soil was separated from that of soils treated with CMR and COM, and was primarily influenced by soil NH4+ concentration. NIR activity showed a significant correlation with denitrifier community composition. Overall, short-term substitution of chemical fertilizer with cattle manure (lower C/N ratio) reduced the abundance of nirS- and nosZ-type denitrifiers, but stimulated N2O emission.

[Value of sTREM-1 in serum and bronchoalveolar lavage fluid, APACHE II score, and SOFA score in evaluating the conditions and prognosis of children with severe pneumonia].

OBJECTIVE: To study the significance of the level of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in serum and bronchoalveolar lavage fluid (BALF), Acute Physiology and Chronic Health Evaluation II (APACHE II) score, and Sequential Organ Failure Assessment (SOFA) score in evaluating the conditions and prognosis of children with severe pneumonia. METHODS: A total of 76 children with severe pneumonia who were admitted from August 2017 to October 2019 were enrolled as the severe pneumonia group. According to the treatment outcome, they were divided into a non-response group with 34 children and a response group with 42 children. Ninety-four children with common pneumonia who were admitted during the same period of time were enrolled as the common pneumonia group. One hundred healthy children who underwent physical examination in the outpatient service during the same period of time were enrolled as the control group. The serum level of sTREM-1, APACHE II score, and SOFA score were measured for each group, and the level of sTREM-1 in BALF was measured for children with severe pneumonia. The correlation of the above indices with the severity and prognosis of severe pneumonia in children was analyzed. RESULTS: The severe pneumonia group had significantly higher serum sTREM-1 level, APACHEII score, and SOFA score than the common pneumonia group and the control group (P<0.05). For the children with severe pneumonia, the non-response group had significant increases in the levels of sTREM-1 in serum and BALF and SOFA score on day 7 after admission, while the response group had significant reductions in these indices, and there were significant differences between the two groups (P<0.05). Positive correlation was found between any two of serum sTREM-1, BALF sTREM-1, and SOFA score (P<0.05). APACHE II score was not correlated with serum sTREM-1, BALF sTREM-1, and SOFA score (P>0.05). CONCLUSIONS: The level of sTREM-1 in serum and BALF and SOFA score can be used to evaluate the severity and prognosis of severe pneumonia in children.

Epithelial-mesenchymal transition markers screened in a cell-based model and validated in lung adenocarcinoma.

BACKGROUND: Re-capture of the differences between tumor and normal tissues observed at the patient level in cell cultures and animal models is critical for applications of these cancer-related differences. The epithelial-mesenchymal transition (EMT) process is essential for tumor migratory and invasive capabilities. Although plenty of EMT markers are revealed, molecular features during the early stages of EMT are poorly understood. METHODS: A cell-based model to induce lung cell (A549) EMT using conditioned medium of in vitro cancer activated fibroblast (WI38) was established. High-throughput sequencing methods, including RNA-seq and miRNA-seq, and advanced bioinformatics methods were used to explore the transcriptome profile transitions accompanying the progression of EMT. We validated our findings with experimental techniques including transwell and immunofluorescence assay, as well as the TCGA data. RESULTS: We have constructed an in vitro cell model to mimic the EMT in patients. We discovered that several new transcription factors were among the early genes (3 h) to respond to cancer micro-environmental cues which could play critical roles in triggering further EMT signals. The early EMT markers also included genes encoding membrane transporters and blood coagulation function. Three of the nine-examined early EMT hallmark genes, GALNT6, SPARC and HES7, were up-regulated specifically in the early stages of lung adenocarcinoma (LUAD) and confirmed by TCGA patient transcriptome data. In addition, we showed that miR-3613, a regulator of EGFR pathway genes, was constantly repressed during EMT progress and indicative of an epithelial miRNA marker. CONCLUSIONS: The CAF-stimulated EMT cell model may recapture some of the molecular changes during EMT progression in clinical patients. The identified early EMT hallmark genes GALNT6, SPARC and HES7and miR-3613 provide new markers and therapeutic targets for LUAD for the further clinical diagnosis and drug screening.

A tridecaptin-based fluorescent probe for differential staining of Gram-negative bacteria.

The traditional Gram-staining method, which was invented more than a century ago for differentiating bacteria as Gram positive or Gram negative, is still widely practiced in microbiology. However, Gram staining suffers from several problems which can affect the accuracy of the diagnosis. Here, we report a new Gram-negative-specific fluorescent probe, which is based on a narrow-spectrum antibiotic, tridecaptin A1, and allows selective staining of Gram-negative bacteria in different fixed bacterial samples. Solid-phase peptide synthesis was used to prepare the tridecaptin A1-fluorophore conjugate with a single structure. Labeling selectivity of the probe toward Gram-negative bacteria was confirmed by testing against a panel of bacterial species. By combining the use of a previously reported Gram-positive-specific fluorescent probe, we then further showed the capability of the new probe in differential labeling of a number of complex bacterial samples, which included a mouse gut microbiota cultured in vitro, as well as microbiotas collected from the human oral cavity, soil, and crude oil. High labeling selectivity and coverage were observed in most samples. This method offers a new Gram-negative-specific probe with a defined structure, which allows facile fluorescence-based differentiation of Gram-positive and Gram-negative bacteria for further microbial studies.

Genetic diversity and characterization of arsenic-resistant endophytic bacteria isolated from Pteris vittata, an arsenic hyperaccumulator.

BACKGROUND: Alleviating arsenic (As) contamination is a high-priority environmental issue. Hyperaccumulator plants may harbor endophytic bacteria able to detoxify As. Therefore, we investigated the distribution, diversity, As (III) resistance levels, and resistance-related functional genes of arsenite-resistant bacterial endophytes in Pteris vittata L. growing in a lead-zinc mining area with different As contamination levels. RESULTS: A total of 116 arsenite-resistant bacteria were isolated from roots of P. vittata with different As concentrations. Based on the 16S rRNA gene sequence analysis of representative isolates, the isolates belonged to Proteobacteria, Actinobacteria, and Firmicutes. Major genera found were Agrobacterium, Stenotrophomonas, Pseudomonas, Rhodococcus, and Bacillus. The most highly arsenite-resistant bacteria (minimum inhibitory concentration > 45 mM) were isolated from P. vittata with high As concentrations and belonged to the genera Agrobacterium and Bacillus. The strains with high As tolerance also showed high levels of indole-3-acetic acid (IAA) production and carried arsB/ACR3(2) genes. The arsB and ACR3(2) were most likely horizontally transferred among the strains. CONCLUSION: The results of this study suggest that P. vittata plants with high As concentrations may select diverse arsenite-resistant bacteria; this diversity might, at least partly, be a result of horizontal gene transfer. These diverse endophytic bacteria are potential candidates to enhance phytoremediation techniques.

[Risk factors analysis and security application discussion of Polygonum multiflorum based on retrospective study].

In recent years，hepatotoxicity problem of Polygonum multiflorum has caused high attention. Domestic scholars also explored the causes of liver damage caused by it. For example, the establishment of guideline for diagnosis and treatment of herb-induced liver injury, and the theory about relationship between hepatocyte toxicity and chemical composition, solvents, processing, use and pathological basis of patients and so on. To try to combine theory with practice，author analyzed risk factors about the case reports of P. multiflorum causing liver damage, and made some suggestions on P. multiflorum about individualized application, drug selection and requirements for taking. This for providing reference for the safe use of P. multiflorum.

[Summary and analysis of safety warning on clinical application of anti-cold Chinese patent medicine preparations].

In China, many surveys have shown that most people do not have a correct understanding about cold and administration of anti-cold Chinese patent medicine preparations. The author conducted a systematic summary and analysis on the actual application of anti-cold Chinese patent medicine preparations as well as the warning on safe application of anti-cold Chinese patent medicine preparations in Clinical Medication Information of China Pharmacopoeia, in the expectation of reducing the blind application of anti-cold Chinese patent medicine preparations and providing traditional Chinese medicine pharmacists new ideas in monitoring the safe application of exterior syndrome-relieving Chinese patent medicine preparations.

LPS preconditioning ameliorates intestinal injury in a rat model of hemorrhagic shock.

OBJECTIVE AND DESIGN: Previous studies indicate that endotoxin preconditioning may decrease the inflammatory response and alleviate intestinal mucosal damage caused by sepsis. However, it is not known whether preconditioning with endotoxin might protect the intestinal mucosa after hemorrhagic shock. In this study, we investigated the effect of lipopolysaccharide (LPS) preconditioning on the intestinal mucosa following hemorrhagic shock in a rat model. Given that intestinal toll-like receptor 4 (TLR4) signaling is exaggerated in response to LPS, we further investigated the role of TLR4 signaling in endotoxin tolerance. METHODS: Animals were pre-treated with intra-peritoneal Escherichia coli LPS for 5 days prior to hemorrhagic shock. Animals were bled to achieve a mean arterial pressure (MAP) of 35-40 mmHg, then resuscitated with Ringer solution and the heparinized shed blood to maintain MAP between 90 and 100 mmHg. The distal ileum was harvested after resuscitation and graded for mucosal damage. TNF-α, TLR4, cleaved caspase-3, and intestinal trefoil factor 3 (TFF3) levels were measured at different time points. RESULTS: Pretreatment with LPS significantly reduced intestinal mucosal damage and protein levels of cleaved caspase-3. Furthermore, animals pre-treated with LPS experienced reduction of TNF-α and increased mucosal expression of TFF3. LPS tolerance was associated with reduced TLR4 expression. CONCLUSIONS: Endotoxin preconditioning can lessen the effects of ischemia and reperfusion injury in intestinal mucosa of a rat model with hemorrhagic shock. It is hypothesized that this effect is mediated via inhibition of TLR4 over-expression.

A rare case report: Myopathy related to the interaction between azathioprine and infliximab in the treatment of ulcerative colitis and ankylosing spondylitis.

Key Clinical Message: Administering azathioprine or infliximab for UC and AS treatment carries a significant risk of adverse reactions. Here, we present the case diagnosed with UC and AS, who received treatment with azathioprine and infliximab for 10 months, and subsequently developed drug-induced myopathy affecting the right vastus medialis muscle. Abstract: Drug-induced myopathy is an uncommon form of muscle injury that can arise in patients without preexisting muscle conditions when exposed to therapeutic doses of certain medications. Administering azathioprine or infliximab for ulcerative colitis (UC) and ankylosing spondylitis (AS) treatment carries a significant risk of adverse reactions, including drug-induced myopathy and increased susceptibility to opportunistic infections. However, occurrences of myopathy induced by the combination of azathioprine and infliximab are rarely reported in clinical practice. Here, we present the case of a 37-year-old male patient diagnosed with UC and AS, who received treatment with azathioprine and infliximab for 10 months. Despite the resolution of symptoms and improvement in intestinal mucosal inflammation observed via endoscopy, the patient subsequently developed drug-induced myopathy affecting the right vastus medialis muscle.

Comparative analysis of two-hour creatinine clearance and the C-G formula for renal function assessment in critically ill patients.

Objective and rationale: To investigate if the 2-h creatinine clearance (Ccr2) provides a more precise and timely assessment of renal function in critically ill patients compared to the Cockcroft-Gault formula (CrC-G). Materials and methods: This cohort study incorporated 74 patients who were hospitalized for more than 48 h in the Intensive Care Unit over 6 months. A 24-h urine collection protocol was observed, and concurrently, 316 2-h urine specimens were obtained. Then calculated and analyzed the correlation and consistency between Ccr2, CrC-G, and 24-h creatinine clearance (Ccr24) values. The rates of change in Ccr2(ΔCcr2) and CrC-G(ΔCrC-G) were compared over two consecutive samples. Results: The R-values of Ccr2 and Ccr24 in the early, middle and late 24 h were 0.640, 0.886 and 0.854 (P < 0.001), with biases of -2.1, 1.7, and 6.3 ml/min/1.73 m2, respectively. Meanwhile, the R-values for CrC-G and Ccr24 at these time points were 0.618, 0.822, and 0.828(P < 0.001), with biases of -14.0, -5.2, and -1.8 ml/min/1.73 m2, respectively. For patients with Ccr24≥60 ml/min/1.73 m2, the R-value of Ccr2 and Ccr24 during the middle 2 h was 0.852(P < 0.001), while the R-values for CrC-G and Ccr24 were 0.763(P < 0.001), with biases of -2.3 ml/min/1.73 m2 and -14.2 ml/min/1.73 m2 respectively. For the group with Ccr24 ≥ 120 ml/min/1.73 m2 (n = 72), both Ccr2 and Ccr24 displayed a statistically significant elevation compared to CrC-G (P < 0.001), yet no significant difference was observed between Ccr2 and Ccr24 (P = 0.289). Out of 50 patients, 46(92 %) experienced a ΔCcr2≥20 % at least once, compared to 20(40 %) with a ΔCrC-G≥20 %(P < 0.001). 25(50 %) with a ΔCcr2≥50 %, compared to 3(6 %) with a ΔCrC-G≥50 %(P < 0.001). Conclusion: Ccr2 demonstrates a more accurate and more timely indicator of renal function in critically ill patients than CrC-G.

Effects of adjunctive Chinese patent medicine on outcome of viral encephalitis in children: A multicenter retrospective study in China.

BACKGROUND: Some patients with viral encephalitis in China seek treatment with Chinese patent medicine (CPM) to improve their symptoms, but few studies have focused on the impact of CPM on the prognosis of viral encephalitis (VE). The aim of this multicenter retrospective study was to assess the benefit of adjunctive CPM therapy on the outcome of children with VE in China. METHODS: This study retrospectively included 834 children with viral encephalitis who were hospitalized at five medical institutions from 2018 to 2021. Univariate and multivariate logistic regression was used to assess the effect of CPM on sequelae in patients with VE. 1:1 propensity score matching was used to exclude the effect of confounding factors. Forest plots were used to observe the effect of CPM on the prognosis of VE in different subgroups. RESULTS: There were fewer patients with sequelae in the group of patients using CPM regardless of whether they were matched or not. The results of multivariate logistic regression analysis showed that the use of CPM was an independent protective factor for the development of sequelae in VE patients (OR = 0.063, 95 % CI: 0.011-0.350, p = 0.002). Subgroup analyses showed that CPM was a protective factor for the development of sequelae regardless of the presence or absence of coma and comorbidities. In addition, we evaluated other outcome indicators and found shorter duration of illness, fever and headache in children with EV in the CPM group. CONCLUSION: Adjunctive CPM therapy may significantly reduce sequelae in children with VE, as well as effectively alleviate patients' clinical symptoms. However, more prospective studies and clinical trials are needed to further evaluate its efficacy and safety.

Improvement of hypoxia-ischemia-induced white matter injury in immature rat brain by ethyl pyruvate.

Ethyl pyruvate (EP) has been reported to be neuroprotective in several models of brain injury, yet its influence on periventricular leukomalacia still remains elusive. Here we investigated whether repeated administration of EP could protect against white matter injury after hypoxia-ischemia (HI) (right common carotid artery ligation and 6 % O2 for 60 min) in post-natal 3 day rat pups. EP was injected (50 mg/kg, intraperitoneally) 10 min, 1 and 24 h after HI insult. Treatment with EP significantly reduced HI-induced ventricular enlargement, loss of developing oligodendrocytes, and hypomyelination. We further demonstrated a marked inhibitory effect of EP on inflammatory responses, as indicated by the decreased number of activated microglia and astrocytes and the reduced release of proinflammatory cytokines. Moreover, EP down-regulated the expression of cleaved caspase-3 and Bax, and up-regulated Bcl-2 expression after HI exposure. In conclusion, our results demonstrated that EP was able to provide potent protection on white matter injury through blocking the cerebral inflammatory responses and modulating the apoptotic death program of oligodendrocytes, indicating a potential neuroprotective agent in neonatal brain injury.

Neonatal immune challenge exacerbates seizure-induced hippocampus-dependent memory impairment in adult rats.

Our aim was to examine whether neonatal lipopolysaccharide (LPS) exposure is associated with changes in microglia and whether these alternations could influence later seizure-induced neurobehavioral outcomes. Male pups were first injected intraperitoneally with either LPS or saline on postnatal day 3 (P3) and postnatal day 5 (P5). Immunohistochemical analysis showed that LPS-treated animals exhibited increased microglia activation that persisted into adolescence. At P45, seizures were induced in rats by intraperitoneal injection of kainic acid (KA). Rats treated with LPS neonatally showed significantly greater proinflammatory responses and performed significantly worse in the Y-maze, Morris water maze, and inhibitory avoidance tasks after KA insult. Treatment with minocycline at the time of neonatal LPS exposure to block LPS-induced microglia alternation attenuated the exaggerated neuroinflammatory responses and alleviated memory impairment associated with the KA insult. Our findings suggest that neonatal immune activation can predispose the brain to exacerbated behavioral deficits following seizures in adulthood, possibly by priming microglia.

Discrepant diversity patterns and function of bacterial and fungal communities on an earthquake-prone mountain gradient in Northwest Sichuan, China.

Patterns of microbial diversity on elevational gradients have been extensively studied, but little is known about those patterns during the restoration of earthquake-fractured alpine ecosystems. In this study, soil properties, soil enzyme activities, abundance and diversity of soil bacterial and fungal communities at four positions along a 2.6-km elevational gradient in the Snow Treasure Summit National Nature Reserve, located in Pingwu County, Southwest China. Although there were no significant changes in the soil chemical environment, bacterial and fungal communities were significantly different at different elevations. The overall fungal community presented an N-shaped diversity pattern with increasing elevation, while bacterial diversity decreased significantly with elevation. Changes in microbial diversity were associated with soil phosphorus, plant litter, and variations in dominant microbial taxa. Differences in enzyme activities among elevations were regulated by microbial communities, with changes in catalase and acid phosphatase activities mainly controlled by Acidobacteria and Planctomycetaceae bacteria, respectively (catalase: p < 0.001; acid phosphatase: p < 0.01), and those in ß-glucosidase, sucrase, and urease activities mainly controlled by fungi. The ß-glucosidase and sucrase were both positively correlated with Herpotrichiellaceae, and urease was positively correlated with Sebacinaceae (p < 0.05). These findings contribute to the conservation and management of mountain ecosystems in the face of changing environmental conditions. Further research can delve into the specific interactions between microbial communities, soil properties, and vegetation to gain deeper insights into the intricate ecological dynamics within earthquake-prone mountain ecosystems.

[Effects of Chemical Fertilizer Reduction Substitute with Organic Fertilizer on Soil Functional Microbes and Lemon Yield and Quality].

To clarify the effects of non-rhizosphere/rhizosphere soil functional microbes (nitrifiers, denitrifiers, and phosphorus-solubilizing microorganisms) on lemon yield and quality, the lemon fruit and non-rhizosphere/rhizosphere soil were selected as subjects. To explore the correlation between non-rhizosphere/rhizosphere soil functional microbes and lemon yield and quality under a chemical fertilizer reduction substitute with organic fertilizer, traditional fruit quality determination and multiple molecular techniques were used. The results showed that:① 30% chemical fertilizer reduction substitute with organic fertilizer increased the nitrification intensity and phosphatase activity but effectively controlled the denitrifying enzyme activity. ② The chemical fertilizer reduction substitute with organic fertilizer significantly decreased the abundances of nitrifiers and nirS/nirK-harboring denitrifiers and significantly increased the abundances of nosZ-harboring denitrifier and phoD-harboring microorganisms. However, the diversities of functional microbial community structure did not have clear regularity under chemical fertilizer reduction substitute with organic fertilizer. ③ Compared with that under the application of chemical fertilizer and organic fertilizer alone, lemon yield and quality were the highest under the 30% reduction of chemical fertilizer substitute with organic fertilizer. ④ Nitrogen and its related microbes significantly affected lemon yield through internal and external quality. Phosphorus and its related microbes affected lemon yield mainly through internal quality. In addition, the influence factors of non-rhizosphere soil and rhizosphere soil on lemon intrinsic quality were obviously different. Altogether, these results showed that the 30% reduction of chemical fertilizer substitute with organic fertilizer significantly affected soil nitrogen and phosphorus functional microorganisms and further improved lemon yield and quality.

[Effect of Chemical Fertilizer and Manure Combined with Biochar on Denitrification Potential and Denitrifying Bacterial Community in Rhizosphere Soil].

To clarify the effect of chemical fertilizer and manure combined with biochar on denitrifying microorganisms and denitrification potential of rhizosphere soil, a pot experiment growing lemon was conducted involving five treatments, namely no fertilization (CK), chemical fertilizer (CF), manure (M), chemical fertilizer combined with biochar (CFBC), and manure combined with biochar (MBC). We determined the characteristics of the rhizosphere soil nirS-, nirK-, and nosZ-type denitrifying bacteria populations; denitrification potential; and soil environmental factors to clarify the effects of chemical and manure combined with biochar on denitrification. Our results showed that compared with that in CK, the CF treatment reduced the rhizosphere soil denitrification potential by 47.7%, whereas the M and MBC treatments increased the denitrification potential by 2192.7% and 1989.9%, respectively. The M and MBC treatments increased the gene copy number of nirS and nosZ, the CF and CFBC treatments decreased the gene copy number of nirS and nosZ, and all four fertilization treatments increased the gene copy number of nirK. Stepwise regression analysis showed that pH was the main factor for the abundance of nirS-type denitrifying bacteria and SOM and NH+4-N were the main factors for the abundance of nirK-type denitrifying bacteria, whereas pH, NO-3-N, and N/P were main factors for the abundance of nosZ-type denitrifying bacteria. The results of partial least squares analysis indicated that the abundance of nirS-and nosZ-type denitrifying bacteria, pH, TN, and N/P were the main factors affecting rhizosphere denitrification potential. Therefore, in acidic purple soil, nirS- and nosZ-type denitrifying bacteria were the main drivers of the soil denitrification process in lemon rhizospheres under chemical fertilizer and pig manure combined with biochar, whereas fertilizer affected the rhizosphere soil denitrification process by regulating soil pH, TN, and N/P.

[Effects of Organic Fertilizer Combined with Biochar on Denitrifying Microorganisms and Enzyme Activities in Orchard Soil].

To study the effects of organic fertilizer combined with biochar on soil denitrification and denitrifying microbial community structure, this study took lemon orchard soil as the research object and adopted a pot experiment, setting up five fertilization treatments:no fertilization(CK), conventional fertilization(F), organic fertilizer(P), fertilizer+biochar(FP), and organic fertilizer+biochar(PP). The abundance and community structure of denitrifying microorganisms were studied using real-time quantitative PCR and T-RFLP. Redundancy analysis(RDA) was used to explore the environmental factors affecting the denitrifying microbial community structure, and PLS-PM analysis was used to explore the environmental factors affecting the denitrification potential of lemon orchard soil. The results showed as follows:① compared with that under the single fertilizer treatment(F), the organic fertilizer and biochar(P, FP, and PP) treatments significantly increased the denitrification potential of the soil, ranging from 147.8% to 1445.3%. The denitrification rate of soil treated with organic fertilizer combined with biochar was 23.8% lower than that treated with organic fertilizer alone. ② Compared with that in the CK treatment, fertilization treatment significantly increased the abundance of nirS and nirK denitrification microorganisms. Fertilizer treatments(F and FP) significantly reduced the abundance of nosZ denitrifying microorganisms. Biochar treatment significantly changed the diversity and uniformity of denitrifying microorganisms, but the specific law and mechanism quality remained unclear. ③ The results of RDA analysis showed that fertilization could affect the community structure of nirS, nirK, and nosZ denitrifying microorganisms by changing C/N, WC, NO3--N, SOC, AK, and AP. ④ PLS-PM analysis showed that soil denitrification was positively correlated with pH and the abundance of nirK denitrification microorganisms, and NO3--N indirectly affected soil denitrification by affecting the abundance of nirK denitrification microorganisms. In addition, the nirK microbial community was the dominant microbial community in soil denitrification in lemon orchards. In conclusion, organic fertilizer directly affected soil denitrification by regulating soil pH, whereas regulating NO3--N content affected nirK denitrification microbial abundance, indirectly affecting soil denitrification. The application of organic fertilizer combined with biochar could slow down the improvement of soil denitrification caused by single application of organic fertilizer, which is more suitable for promotion in orchards in this region.

[Effects of Chemical Fertilizer Reduction Combined with Organic Fertilizer Application on Bacterial Community Structure in Rhizosphere/Non-Rhizosphere Soil of Lemon].

The aim of this study was to examine the effects of different fertilization methods on the physicochemical properties and bacterial community structure of lemon rhizosphere/non-rhizosphere soil in order to provide theoretical basis for scientific and rational fertilization of orchards. A pot experiment was carried out, and six fertilization treatments were set up:control (CK), conventional fertilization (FM), organic fertilizer (P), fresh organic fertilizer (NP), 70% chemical fertilizer+30% organic fertilizer (70FP), and 50% chemical fertilizer+50% organic fertilizer (50FP). Chemical analysis, real-time fluorescence quantitative PCR, and terminal restriction fragment length polymorphism (T-RFLP) were used to study the effects of different fertilization treatments on the physicochemical properties of rhizosphere and non-rhizosphere soils, the abundance of the bacterial 16S rRNA gene, and bacterial community structure. Redundancy analysis (RDA) was used to explore the environmental factors affecting the bacterial community structure of lemon rhizosphere/non-rhizosphere soil. The results showed the following:① the pH and contents of organic matter, alkali-hydrolyzed nitrogen, available phosphorus, available potassium, and nitrate nitrogen in rhizosphere/non-rhizosphere soil were significantly increased by reducing the amount of chemical fertilizer and applying organic fertilizer (50FP and 70FP) (P<0.05). Compared with conventional fertilization (FM) and single application of organic fertilizer (P and NP), the soil available P content, available K content, and nitrate nitrogen content increased by 24.76%-97.98%, 6.87%-45.11%, and 18.42%-55.82%, respectively. ② Fertilizer reduction combined with organic fertilizer significantly increased the abundance of soil bacteria and soil respiration intensity (P<0.05), and the abundance of soil rhizosphere bacteria and soil respiration intensity under the 50FP treatment increased by 15.83%-232.98% and 8.0%-162.5% compared with that under conventional fertilization and organic fertilizer alone, respectively. The bacterial abundance of rhizosphere soil was positively correlated with the pH and contents of organic matter, total nitrogen, and total phosphorus. ③ The PCoA and RDA analysis results showed that the single organic fertilizer and organic fertilizer and chemical fertilizer de-weighting of rhizosphere bacterial community structure and not adding fertilizer had a bigger difference between processing, and the main environmental factors influencing the rhizosphere/non rhizosphere bacterial community structure were organic matter, total nitrogen, total phosphorus, total potassium, alkali solution nitrogen, nitrate nitrogen, and available potassium. Fertilizer reduction combined with organic fertilizer could significantly increase soil nutrient content, increase soil bacterial abundance, and change the bacterial community structure of rhizosphere soil, and the 50FP treatment yielded better results. Therefore, 50% Chemical fertilizer+50% organic fertilizer (50FP) was a better fertilization method to improve the physical and chemical properties of orchard soil, increase the abundance of soil bacteria, and improve the soil respiration intensity.