Pre-soil fumigation with ammonium bicarbonate and lime modulates the rhizosphere microbiome to mitigate clubroot disease in Chinese cabbage.

Background: Plasmodiophora brassicae is an ever-increasing threat to cruciferous crop production worldwide. Aims and methods: This study investigated the impact of pre-soil fumigation with ammonium bicarbonate (N) and lime (NB) to manage clubroot disease in Chinese cabbage through 16S rRNA gene amplification sequencing. Results: We found that soil fumigation with N and NB suppressed disease incidence by reducing the soil acidity and population of P. brassicae in the rhizosphere. Minimum disease incidence and maximum relative control effect of about 74.68 and 66.28% were achieved in greenhouse and field experiments, respectively, under the combined application of ammonium bicarbonate and lime (LNB) as compared with N, NB, and control (GZ). Microbial diversity analysis through Miseq sequencing proved that pre-soil fumigation with N, NB, and LNB clearly manipulated rhizosphere microbial community composition and changed the diversity and structure of rhizosphere microbes compared with GZ. Bacterial phyla such as Proteobacteria, Bacteriodetes, and Acidobacteria and fungal phyla including Olpidiomycota and Ascomycota were most dominant in the rhizosphere of Chinese cabbage plants. Soil fumigation with N and NB significantly reduced the abundance of clubroot pathogen at genus (Plasmodiophora) level compared with GZ, while decreased further under combined application LNB. Microbial co-occurrence network analysis showed a highly connected and complex network and less competition for resources among microbes under combined application LNB. Conclusion: We conclude that for environmentally friendly and sustainable agriculture, soil fumigation with combined ammonium bicarbonate and lime plays a crucial role in mitigating Chinese cabbage clubroot disease by alleviating soil pH, reducing pathogen population, and manipulating the rhizosphere microbiome.

Whole-genome sequencing and comparative genome analysis of Xanthomonas fragariae YM2 causing angular leaf spot disease in strawberry.

Background: Angular leaf spot disease caused by plant pathogenic bacterium Xanthomonas fragariae seriously threatens strawberry crop production globally. Methods: In this study, we sequenced the whole genome of X. fragariae YM2, isolated from Yunnan Province, China. In addition, we performed a comparative genome analysis of X. fragariae YM2 with two existing strains of X. fragariae YL19 and SHQP01 isolated from Liaoning and Shanghai, respectively. Results: The results of Nanopore sequencing showed that X. fragariae YM2 comprises one single chromosome with a contig size of 4,263,697 bp, one plasmid contig size of 0.39 Mb, a GC content ratio of 62.27%, and 3,958 predicted coding genes. The genome of YM2 comprises gum, hrp, rpf, and xps gene clusters and lipopolysaccharide (LPS), which are typical virulence factors in Xanthomonas species. By performing a comparative genomic analysis between X. fragariae strains YM2, YL19, and SHQP01, we found that strain YM2 is similar to YL19 and SHQP01 regarding genome size and GC contents. However, there are minor differences in the composition of major virulence factors and homologous gene clusters. Furthermore, the results of collinearity analysis demonstrated that YM2 has lower similarity and longer evolutionary distance with YL19 and SHQP01, but YL19 is more closely related to SHQP01. Conclusions: The availability of this high-quality genetic resource will serve as a basic tool for investigating the biology, molecular pathogenesis, and virulence of X. fragariae YM2. In addition, unraveling the potential vulnerabilities in its genetic makeup will aid in developing more effective disease suppression control measures.

Crop Rotation with Marigold Promotes Soil Bacterial Structure to Assist in Mitigating Clubroot Incidence in Chinese Cabbage.

Clubroot caused by Plasmodiophora brassicae is an economically important soilborne disease of Chinese cabbage worldwide. Integrated biological control through crop rotation is considered a good disease management approach to suppress the incidence of soilborne diseases. In this study, we evaluated the effect of a marigold plant (root exudates, crude extract, and powder) on the germination and death of resting spores of P. brassicae in vitro assays. Additionally, we also performed 16S high throughput sequencing, to investigate the impact of marigold-Chinese cabbage crop rotation on soil bacterial community composition, to manage this devastating pathogen. This study revealed that the marigold root exudates, crude extract, and powder significantly promoted the germination and death of P. brassicae resting spores. Under field conditions, marigold-Chinese cabbage crop rotation with an empty period of at least 15 days enhanced the germination of P. brassicae resting spores, shifted the rhizosphere bacterial community composition, and suppressed the incidence of clubroot by up to 63.35%. Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, and Verrucomicrobia were the most dominant phyla and were present at high relative levels in the rhizosphere soil of Chinese cabbage. We concluded that crop rotation of Chinese cabbage with marigold can significantly reduce the incidence of clubroot disease in the next crop. To our knowledge, this is the first comprehensive study on the prevention and control of clubroot disease in Chinese cabbage through crop rotation with marigold.

Microbial Consortia: An Engineering Tool to Suppress Clubroot of Chinese Cabbage by Changing the Rhizosphere Bacterial Community Composition.

Clubroot disease, caused by Plasmodiophora brassicae, is a serious threat to Chinese cabbage (Brassica rapa subsp. pekinensis) production, which results in extensive yield losses. At present, clubroot control mainly depends upon pesticides, which provoke food-safety concerns, and the application of sole biocontrol agents cannot successfully control the disease. In this study, we investigated the effect of Bacillus cereus BT-23, Lysobacter antibioticus 13-6, and Lysobacter capsici ZST1-2 as sole strains, intra-/inter-genus co-culture, and microbial consortia on clubroot disease, plant growth, and rhizosphere bacterial diversity in a field experiment. The microbial consortia efficiently controlled the incidence of clubroot disease, with a biocontrol effect of about 65.78%, by decreasing the soil acidity and enhancing the yield (17,662.49 kg/acre). The high-throughput sequencing results demonstrated that the phyla Proteobacteria and Bacteroidetes were present in high relative abundance in the rhizosphere soil of the Chinese cabbage. Furthermore, Firmicutes was found as a unique phylum in the rhizosphere soil of CK-H and T1-T7, except for CK-D. The application of microbial consortia recovers the imbalance in indigenous microbial communities. Therefore, we conclude that microbial consortia can reduce the clubroot incidence in Chinese cabbage by decreasing the soil acidity and altering the diversity and structure of rhizosphere bacterial communities. This study highlights the potential of microbial consortia as an engineering tool to control devastating soilborne diseases in commercial crops.

Exploiting the antibacterial mechanism of phenazine substances from Lysobacter antibioticus 13-6 against Xanthomonas oryzae pv. oryzicola.

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most destructive diseases affecting rice production worldwide. In this study, we extracted and purified phenazine substances from the secondary metabolites of Lysobacter antibioticus 13-6. The bacteriostatic mechanism of phenazine substances against Xoc was investigated through physiological response and transcriptomic analysis. Results showed that phenazine substances affects the cell membrane permeability of Xoc, which causes cell swelling and deformation, blockage of flagellum synthesis, and imbalance of intracellular environment. The changes in intracellular environment affect the physiological and metabolic functions of Xoc, which reduces the formation of pathogenic factors and pathogenicity. Through transcriptomic analysis, we found that among differentially expressed genes, the expression of 595 genes was induced significantly (275 up-regulated and 320 down-regulated). In addition, we observed that phenazine substances affects three main functions of Xoc, i.e., transmembrane transporter activity, DNA-mediated transposition, and structural molecular activity. Phenazine substances also inhibits the potassium ion transport system that reduces Xoc resistance and induces the phosphate ion transport system to maintain the stability of the internal environment. Finally, we conclude that phenazine substances could retard cell growth and reduce the pathogenicity of Xoc by affecting cell structure and physiological metabolism. Altogether, our study highlights latest insights into the antibacterial mechanism of phenazine substances against Xoc and provides basic guidance to manage the incidence of bacterial leaf streak of rice.

Diversity of transcription activator-like effectors and pathogenicity in strains of Xanthomonas oryzae pv. oryzicola from Yunnan.

The present study aimed to evaluate transcriptional activator-like effector (TALE) genes in 86 Xanthomonas oryzae pv. oryzicola strains collected from 8 rice-growing regions in Yunnan, and to examine the relationship between TALE genotypes and virulence in 6 differential rice lines. Besides, the geographical areas, distribution of these genotypes were studied in detail. Genetic diversity was analyzed through the number and size of putative TALE genes based on TALE gene avrXa3 as a probe. We found that X. oryzae pv. oryzicola strains consist of variable number (13-27) of avrXa3-hybridizing fragments (putative TALE genes). Test strains were classified into 8 genotypes (G1-G8) with major genotypes G3 and G7 widely distributed in Yunnan. Pathogenicity of X. oryzae pv. oryzicola was evaluated by inoculating 6 differential rice lines with a single resistance gene into 9 pathotypes clusters (I-IX), the dominant Genotypes G3 and G7 consist of pathotypes I, II, and IV. Furthermore, we also detected the known TALE target genes expression in susceptible rice cultivar (cv. nipponbare) after inoculating 8 genotypes-representative X. oryzae pv. oryzicola strain. Correlation between the numbers of putative TALE genes of X. oryzae pv. oryzicola and relevant target genes in nipponbare confirmed up-regulation. Altogether, this study has given insights into the population structure of X. oryzae pv. oryzicola that may inform strategies to control BLS in rice.

Insights into the relevance between bacterial endophytic communities and resistance of rice cultivars infected by Xanthomonas oryzae pv. oryzicola.

Bacterial leaf streak (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc), impacts the production of rice. However, several rice cultivars displayed resistance to Xoc in the field, but scarce information is available about the role of endophytic microbiota in disease resistance. In the present study, the endophytic bacterial communities of resistant and susceptible rice cultivars "CG2" and "IR24", respectively, were analyzed using high throughput 16S rRNA gene amplified sequencing and culture dependent method was further used for bacterial isolation. A total of 452,716 high-quality sequences representing 132 distinct OTUs (Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes) and 46 isolates of 16 genera were explored from rice leaves infected with Xoc. Community diversity of endophytic bacteria were higher in the leaves of the resistant cultivars compared to susceptible cultivars upon Xoc infection. Strikingly, this diversity might contribute to natural defense of the resistant cultivar against pathogen. Pantoea, which is pathogen antagonist, was frequently detected in two cultivars and higher abundance were recorded in resistant cultivars. Different abundance genus includes endophytic isolates with marked antagonistic activity to Xoc. The increased proportions of antagonistic bacteria, may contribute to resistance of rice cultivar against Xoc and the Pantoea genus was recruited by Xoc infection play a key role in suppressing the development of BLS disease in rice. Taken together, this work reveals the association between endophytic bacteria and BLS resistance in rice and identification of antagonism-Xoc bacterial communities in rice. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02979-2.

Blockade of NaV1.8 Increases the Susceptibility to Ventricular Arrhythmias During Acute Myocardial Infarction.

SCN10A/NaV1.8 may be associated with a lower risk of ventricular fibrillation in the setting of acute myocardial infarction (AMI), but if and by which mechanism NaV1.8 impacts on ventricular electrophysiology is still a matter of debate. The purpose of this study was to elucidate the contribution of NaV1.8 in ganglionated plexi (GP) to ventricular arrhythmias in the AMI model. Twenty beagles were randomized to either the A-803467 group (n = 10) or the control group (n = 10). NaV1.8 blocker (A-803467, 1 µmol/0.5 mL per GP) or DMSO (0.5 mL per GP) was injected into four major GPs. Ventricular effective refractory period, APD90, ventricular fibrillation threshold, and the incidence of ventricular arrhythmias were measured 1 h after left anterior descending coronary artery occlusion. A-803467 significantly shortened ventricular effective refractory period, APD90, and ventricular fibrillation threshold compared to control. In the A-803467 group, the incidence of ventricular arrhythmias was significantly higher compared to control. A-803467 suppressed the slowing of heart rate response to high-frequency electrical stimulation of the anterior right GP, suggesting that A-803467 could inhibit GP activity. SCN10A/NaV1.8 was readily detected in GPs, but was not validated in ventricles by quantitative RT-PCR, western blot and immunohistochemistry. While SCN10A/NaV1.8 is detectible in canine GPs but not in ventricles, blockade of NaV1.8 in GP increases the incidence of ventricular arrhythmias in AMI hearts. Our study shows for the first time an influence of SCN10A/NaV1.8 on the regulation of ventricular arrhythmogenesis via modulating GP activity in the AMI model.

Functional analysis of pde gene and its role in the pathogenesis of Xanthomonas oryzae pv. oryzicola.

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a devastating disease of rice worldwide, including China. The second messenger c-di-GMP plays an important role in the transduction of intercellular signals. However, little is known about the function of EAL domain protein in c-di-GMP that regulates the virulence in Xoc. In this study, the function of EAL domain protein encoded by pde (FE36_09715) gene in the regulation of c-di-GMP was investigated. Results of this study, showed that the deletion of pde gene led to a significant reduction in the virulence of Xoc and was positively related to the reduction of exopolysaccharides production, biofilm formation, and flagellar motility. However, these significantly impaired properties from the ∆pde mutant strain were partially recovered in the complementary strain. In addition, the deletion of pde gene in Xoc strain YM15 had no visible effect on the colony morphology, amylase, and protease activities of Xoc. It is concluded that, as a regulator for the c-di-GMP level, the pde gene plays an important role in partial biological processes in Xoc and is essential for its virulence.

Procedure-Related Complications of Left Bundle Branch Pacing: A Single-Center Experience.

Background: Although left bundle branch pacing (LBBP) has emerged as a novel physiological pacing strategy with a low and stable threshold, its safety has not been well-documented. In the present study, we included all the patients with procedure-related complications at our centre to estimate these LBBP cases with unique complications. Methods: We enrolled 612 consecutive patients who received the procedure in Zhongshan Hospital, Fudan University, between January 2018 and July 2020. Regular follow-ups were conducted (at 1, 3, and 6 months in the first year and every 6-12 months from the second year), and the clinical data of the patients with complications were collected and analyzed. Results: With a mean follow-up period of 12.32 ± 5.21 months, procedure-related complications were observed in 10 patients (1.63%) that included two postoperative septum perforations (2/612, 0.33%), two postoperative lead dislodgements (2/612, 0.33%), four intraoperative septum injuries (4/612, 0.65%), and two intraoperative lead fractures (2/612, 0.33%). Pacing parameters were stable during follow-up, and no major complications were observed after lead repositioning in the cases of septum perforation and lead dislodgement. Conclusion: The incidence of procedure-related complications for LBBP, namely postoperative septum perforation, postoperative lead dislodgement, intraoperative septum injury, and intraoperative lead fracture, were low. No adverse clinical outcomes were demonstrated after successful repositioning of the lead and appropriate treatment.

Effect of culture medium optimization on the secondary metabolites activity of Lysobacter antibioticus 13-6.

Fermentation products of Lysobacter antibioticus 13-6 have antagonistic activity against devastating phytopathogenic bacerium Xanthomonas oryzae pv. oryzicola. The production of Lysobacter antibioticus 13-6 secondary metabolites was increased by optimizing the fermentation medium; using a single-factor screening test, Plackett-Burman Design, and Box-Behnken Design. The medium's final formulation for active secondary metabolites high-yield included peptone 5 g/L, glucose 4.73 g/L, MgSO4·7H2O 2.33 g/L, and K2HPO4 2.21 g/L. We compared phenazine-1-carboxylic acid (PCA) contents of L. antibioticus 13-6 in the initial and optimized mediums through HPLC. It was found PCA contents of the optimized medium are two folds more than in the initial medium. We also detected the relative expression of five phenazine genes of L. antibioticus 13-6 via RT-qPCR, and it was found that genes: phzB, C, S, and NO1 have more significant expression compared with the initial medium, while gene phzD has found just significant. Further, we revealed that the optimal fermentation conditions for secondary metabolites were: fermentation time 60 hours, shaking speed 160 rpm, inoculum size 3%, and the initial pH = 7.0. In the end, it was determined that the antimicrobial activity and quality of L. antibioticus 13-6 secondary metabolites were increased by about 41.75% and 2-times, respectively, after the optimization of the fermentation medium.

Probiotic Consortia: Reshaping the Rhizospheric Microbiome and Its Role in Suppressing Root-Rot Disease of Panax notoginseng.

Root-rot disease caused by Fusarium oxysporum is a growing problem in agriculture for commercial cultivation of Panax notoginseng. Diverse microbes colonize plant roots, and numerous earlier studies have characterized the rhizospheric microbiome of P. notoginseng; nevertheless, the function of probiotic consortia on the rhizospheric microbiome against the root-rot disease remain elusive. We have compared and described the rhizospheric microbiome of lightly and severely diseased P. notoginseng as well as the interactions of the probiotic consortia and rhizospheric microbiome, and their function to alleviate the plant diseases were explored by inoculating probiotic consortia in bulk soil. From the perspective of microbial diversity, the rhizospheric dominant bacterial and fungal genera were utterly different between lightly and severely diseased plants. Through inoculating assembled probiotic consortia to diseased plant roots, we found that the application of probiotic consortia reshaped the rhizosphere microbiome, increasing the relative abundance of bacteria and fungi, while the relative abundance of potential pathogens was decreased significantly. We developed a microcosm system that provides a preliminary ecological framework for constructing an active probiotic community to reshape soil microbiota and restrain the disease. Microbial community structure differs between lightly and seriously diseased plants. The application of probiotic consortia changes the imbalance of micro-ecology to a state of relative health, reducing plant mortality. Plant disease suppression may be achieved by seeking and applying antagonistic microbes based on their direct inhibitory capability or by restructuring the soil microbiome structure and function.

Antifungal and Biocontrol Evaluation of Four Lysobacter Strains Against Clubroot Disease.

The effect of crude extract (Ce), seed coating agent (SCA) and whole bacterial broth culture (WBC) of Lysobacter strains was evaluated against the causal agent of clubroot formation in Cruciferous vegetables. The ability of four Lysobacter strains (L. antibioticus 6-B-1, L. antibioticus 6-T-4, L. antibioticus 13-B-1 and L. capsici ZST1-2) inhibited Plasmodiophora brassicae of resting spores and disease. Application of WBC of four Lysobacter strains inhibited clubroot disease, indicating that the disease suppression was due to antifungal compounds produced by the biocontrol bacterium in the culture. Development of clubroot on Chinese cabbage was inhibited when the WBC and SCA were applied before P. brassicae inoculation. Crude extract (Ce) of culture filtrate was effective in arresting the germination of resting spores of P. brassicae on slides. However, Lysobacter strains differed in their biocontrol effects, the strain L. capsci ZST1-2 recorded a high level of disease limiting effect.

Effect of Linker Flexibility on the Catalytic Features of a Glycoside Hydrolase Family 45 Endoglucanase from Rhizopus stolonifer.

This study investigated an endoglucanase (EGII) from Rhizopus stolonifer var. reflexus TP-02 that consists of a C-terminal catalytic domain and an N-terminal carbohydrate-binding module joined by a linker rich in glycine, serine, threonine, and alanine. Site-directed mutagenesis was applied to characterize the conformation and dynamics of the linker. Mutants were expressed in Escherichia coli BL21 and purified by Ni-chelating column. Structural analysis indicated that glycine provided flexibility in the enzymatic process. G67P, G91Y, G101Y, G108Y, G109Y, G112P, H61G, H75G, and Y103G were selected on the basis of the results of the bioinformatics and Ramachandran plot analysis for the linker. The catalytic activities of EGII and its mutants on CMC-Na, microcrystalline cellulose (Avicel), and phosphoric acid-swollen celluloses (PASC) showed that flexible amino acids strengthened the activity of the enzyme. It indicated that flexible amino acids could improve the flexibility of the linker. Overall, the linker affected the catalytic efficiency of the endoglucanase in hydrolyzing cellulose chains.

Draft Genome Sequence of Antagonistic Agent Lysobacter antibioticus 13-6.

Lysobacter antibioticus 13-6, isolated from the roots of Chinese cabbage, effectively controls the pathogens Plasmodiophora brassicae, Xanthomonas oryzae pv. oryzicola, X. oryzae pv. oryzae, Xanthomonas axonopodis pv. dieffenbachiae, and Pseudomonas syringae pv. tabaci. We report the first draft genome sequence of the L. antibioticus species in China.

[Expression of aquaporin-8 and bcl-2 protein in human cervical carcinoma and their correlations].

OBJECTIVE: To investigate the expression of aquaporin-8 (AQP8) and apoptosis associated bcl-2 protein in human cervical carcinoma and their relationship. METHODS: The expression of AQP8 and bcl-2 protein in 74 cases of cervical carcinoma (46 cases of squamous-cell carcinoma of the uterine cervix, 28 cases of adenocarcinoma of the uterine cervix), 34 cases of cervical intraepithelial neoplasia (CIN) and 15 cases of normal cervices were detected by immunohistochemical technique, and their clinical significance were analyzed. RESULTS: The expression of AQP8 and bcl-2 protein were detected in intracytoplasm of atypia cells in CIN, squamous-cell carcinoma and adenocarcinoma of the uterine cervix. The positive rates of AQP8 and bcl-2 in squamous-cell carcinoma, adenocarcinoma, CIN and normal cervical epithelium were 98%, 74%; 61%, 71%; 71%, 53% ; 53%, 20% respectively. There were significant differences between squamous-cell carcinoma of the uterine cervix and other groups in AQP8 (P < 0.01), but no significant differences were found in any other groups. There were significant differences between squamous-cell carcinoma of the uterine cervix and CIN or normal cervical epithelium in bcl-2, so were between adenocarcinoma of the uterine cervix. The expression of AQP8 was positively correlated with bcl-2 in human cervical carcinoma( r(s) = 0.463, P = 0.000). CONCLUSIONS: There is a close relationship between high expression of AQP8 and development of human cervical carcinoma. The expression of AQP8 protein is positively correlated with bcl-2 protein in human cervical carcinoma. AQP8 protein may have anti-apoptosis function, although the detailed mechanism in human cervical carcinoma remains to be clarified.