Comparative genomic analysis of Bacillus atrophaeus HAB-5 reveals genes associated with antimicrobial and plant growth-promoting activities.

Bacillus atrophaeus HAB-5 is a plant growth-promoting rhizobacterium (PGPR) that exhibits several biotechnological traits, such as enhancing plant growth, colonizing the rhizosphere, and engaging in biocontrol activities. In this study, we conducted whole-genome sequencing of B. atrophaeus HAB-5 using the single-molecule real-time (SMRT) sequencing platform by Pacific Biosciences (PacBio; United States), which has a circular chromosome with a total length of 4,083,597 bp and a G + C content of 44.21%. The comparative genomic analysis of B. atrophaeus HAB-5 with other strains, Bacillus amyloliquefaciens DSM7, B. atrophaeus SRCM101359, Bacillus velezensis FZB42, B. velezensis HAB-2, and Bacillus subtilis 168, revealed that these strains share 2,465 CDSs, while 599 CDSs are exclusive to the B. atrophaeus HAB-5 strain. Many gene clusters in the B. atrophaeus HAB-5 genome are associated with the production of antimicrobial lipopeptides and polypeptides. These gene clusters comprise distinct enzymes that encode three NRPs, two Transat-Pks, one terpene, one lanthipeptide, one T3PKS, one Ripp, and one thiopeptide. In addition to the likely IAA-producing genes (trpA, trpB, trpC, trpD, trpE, trpS, ywkB, miaA, and nadE), there are probable genes that produce volatile chemicals (acoA, acoB, acoR, acuB, and acuC). Moreover, HAB-5 contained genes linked to iron transportation (fbpA, fetB, feuC, feuB, feuA, and fecD), sulfur metabolism (cysC, sat, cysK, cysS, and sulP), phosphorus solubilization (ispH, pstA, pstC, pstS, pstB, gltP, and phoH), and nitrogen fixation (nif3-like, gltP, gltX, glnR, glnA, nadR, nirB, nirD, nasD, narl, narH, narJ, and nark). In conclusion, this study provides a comprehensive genomic analysis of B. atrophaeus HAB-5, pinpointing the genes and genomic regions linked to the antimicrobial properties of the strain. These findings advance our knowledge of the genetic basis of the antimicrobial properties of B. atrophaeus and imply that HAB-5 may employ a variety of commercial biopesticides and biofertilizers as a substitute strategy to increase agricultural output and manage a variety of plant diseases.

First report of Lasiodipilodia theobromae Causing leaf spot of Agave sisalana in GuangXi, China.

Agave sisalana, as an excellent fiber producing plant, is mainly planted in Guangxi Province, China. In November 2023, a foliar disease occured on A. sisalana at Liangjiang Town (108.3593 W, 23.4723 N), Wuming District, Nanning in GuangXi, China. Approximately 50 to 60% of the plants (n=200) had obvious leaf spots on more than 70% of the leaves. On the leaves of sisal, circular or irregularly shaped yellow brown spots can be seen, sunken, with no halo on the edges. As time goes on, the lesion gradually expands to the entire blade of the sword (Figure 1A, 1B). To identify the disease etiology, ten agave leaves were collected from GuangXi. Symptomatic midribs were cut into 3×3 mm pieces, surface sterilized with 75 % ethanol for 20 s, rinsed with sterilized distilled water three times, air dried on sterile filter paper, plated on photo dextrose agar (PDA) medium, and incubated at 28 ℃ in the dark. Five isolates (JM01, JM02, JM03, JM05, JM06) with similar morphology were obtained. Colonies on PDA medium were white to grayish-white with atrial mycelia growing initially upward and then forming clusters (Figure 1E). After five days, mycelia turned grayish black. Immature conidia were initially hyaline, aseptate, and ellipsoid. Mature conidia were dark brown, one septate, longitudinal striate, and 22.1 to 26.3×10.2 to 14.9 µm (Figure 1F). Morphologically , the isolates were identified as Lasiodiplodia theobromae (Alves et al. 2008). For molecular identification, genome DNA of five representative isolate was extracted using the Fungi Genomic DNA Purification kit. The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (TEF-1α) and ß-tublin (TUB) gene were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively, and sequenced. The ITS (PP209594), TEF-1α (PP234629), and TUB (PP234628) sequences of representative isolate JM01 were deposited in GeneBank. BLAST searches showed >99% nucleotide identity to sequences of L. theobromae (ITS, 99.26% to NR111174; TEF-1α, 99.69% to MM840490; TUB, 98.92% to MN172230). Phylogenetic analysis using maximum likelihood based on the combined ITS, TEF-1α, and TUB sequences of the isolates and reference sequences of Lasiodiplodias spp. from GenBank indicated the isolates obtained in this study formed a clade strongly supported based on bootstrap values to the ex-type isolate CBS164.96 sequences of L.theobromae (Figure 2). To test pathogenicity, JM01 was tested by inoculation leaves of one year old agave plants, the epidermis at the inoculation site, 10, 15 and 20 cm below to the crown, was wiped with a 75% alcohol cotton ball, washed three times with sterile water, and punctured (5 mm diameter) with a sterile inoculation needle. A 5 mm block of each isolate cultured on PDA for 3 days was attached to the inoculation site. Controls were inoculated with sterile PDA. The inoculation area was covered with plastic wrap. All plants were kept in a controlled greenhouse at 27℃, 80% relative humidity, and natural daylight, and watered weekly. Each treatment was repeated three times. Remove the block one day later. Three days after inoculation, all inoculated had typical symptoms,but control were healthy (Figure 1C, 1D). Fungal isolates were only recovered from symptomatic stems and were morphologically identical to L. theobromae, completing Koch's postulates. L. Theobromae has been reported as the cause of leaf rot on A. angustifolia in Mexico (Reyes-García et al. 2023). To our knowledge, this is the first report of L. theobromae causing leaf spot on A. sisalana in GuangXi, China. L. theobromae is primarily a plant pathogen that causes rotting and dieback in fruits and plants in tropical and subtropical regions (Puttanna 1967). This study is useful to focus on management strategies for leaf rot disease by L. theobromae of A. sisalana.

Biochar addition promotes soil organic carbon sequestration dominantly contributed by macro-aggregates in agricultural ecosystems of China.

Soil aggregates play pivotal roles in soil organic carbon (SOC) preservation and climate change. Biochar has been widely applied in agricultural ecosystems to improve soil physicochemical properties. However, the underlying mechanisms of SOC sequestration by soil aggregation with biochar addition are not well understood at a large scale. Here, we conducted a meta-analysis of 2335 pairwise data from 45 studies to explore how soil aggregation sequestrated SOC after biochar addition in agricultural ecosystems of China. Biochar addition markedly enhanced the proportions of macro-aggregates and aggregate stability, and the production of organic binding agents positively facilitated the formation of macro-aggregates and aggregate stability. Soil aggregate-associated organic carbon (OC) indicated a significantly increasement by biochar addition, which was attributed to direct and indirect inputs of OC from biochar and organic residues, respectively. Biochar stimulated SOC sequestration dominantly contributed by macro-aggregates, and it could be interpreted by a greater improvement in proportions and OC protection of macro-aggregates. Furthermore, the SOC sequestration of soil aggregation with biochar addition was regulated by climate conditions (mean annual temperature and precipitation), biochar attributes (biochar C/N ratio and pH), experimental practices (biochar addition level and duration), and agronomic managements (land type, cropping intensity, fertilization condition, and crop type). Collectively, our synthetic analysis emphasized that biochar promoted the SOC sequestration by improving soil aggregation in agricultural ecosystems of China.

Synthesis of Indolyl Phenyl Diketones through Visible-Light-Promoted Ni-Catalyzed Intramolecular Cyclization/Oxidation Sequence of Ynones.

The combination of visible light catalysis and Ni catalysis has enabled the synthesis of indolyl phenyl diketones through the cyclization/oxidation process of ynones. This reaction proceeded under mild and base-free conditions and showed a broad scope and feasibility for gram-scale synthesis. Several natural products and biologically interesting molecules could be readily postfunctionalized by this method.

Intermittent Theta-Burst Stimulation for Stroke: Primary Motor Cortex Versus Cerebellar Stimulation: A Randomized Sham-Controlled Trial.

BACKGROUND: Stroke survivors with impaired balance and motor function tend to have relatively poor functional outcomes. The cerebellum and primary motor cortex (M1) have been suggested as targets for neuromodulation of balance and motor recovery after stroke. This study aimed to compare the efficacy and safety of intermittent theta-burst stimulation (iTBS) to the cerebellum or M1 on balance and motor recovery in patients with stroke. METHODS: In this randomized, double-blind, sham-controlled clinical trial, patients with subacute stroke were randomly divided into 3 groups: M1-, cerebellar-, and sham-iTBS (n=12 per group; 15 sessions, 3 weeks). All outcomes were evaluated before intervention (T0), after 1 week of intervention (T1), after 3 weeks of intervention (T2), and at follow-up (T3). The primary outcome was the Berg balance scale score at T2. Secondary outcomes include the Fugl-Meyer assessment scale for lower extremities, the trunk impairment scale, the Barthel index, the modified Rankin Scale, the functional ambulation categories, and cortical excitability. RESULTS: A total of 167 inpatients were screened, 36 patients (age, 57.50±2.41 years; 10 women, 12 ischemic) were enrolled between December 2020 and January 2023. At T2, M1- or cerebellar-iTBS significantly improved Berg balance scale scores by 10.7 points ([95% CI, 2.7-18.6], P=0.009) and 14.2 points ([95% CI, 1.2-27.2], P=0.032) compared with the sham-iTBS group. Moreover, the cerebellar-iTBS group showed a significantly greater improvement in Fugl-Meyer assessment scale for lower extremities scores by 5.6 points than the M1-iTBS ([95% CI, 0.3-10.9], P=0.037) and by 7.8 points than the sham-iTBS ([95% CI, 1.1-14.5], P=0.021) groups at T2. The motor-evoked potential amplitudes of the M1- and cerebellar-iTBS groups were higher than those of the sham-iTBS group (P<0.001). CONCLUSIONS: Both M1- and cerebellar-iTBS could improve balance function. Moreover, cerebellar-iTBS, but not M1-iTBS, induced significant effects on motor recovery. Thus, cerebellar-iTBS may be a valuable new therapeutic option in stroke rehabilitation programs. REGISTRATION: URL: https://www.chictr.org.cn/; Unique identifier: ChiCTR2100047002.

Prevalence and clinical predictors of spasticity after intracerebral hemorrhage.

BACKGROUND: Spasticity is a common complication of intracerebral hemorrhage (ICH). However, no consensus exists on the relation between spasticity and initial clinical findings after ICH. METHODS: This retrospective study enrolled adult patients with a history of ICH between January 2012 and October 2020. The modified Ashworth scale was used to assess spasticity. A trained image analyst traced all ICH lesions. Multivariable logistic regression was used to examine the association between ICH lesion sites and spasticity. RESULTS: We finally analyzed 304 patients (mean age 54.86 ± 12.93 years; 72.04% men). The incidence of spasticity in patients with ICH was 30.92%. Higher National Institutes of Health stroke scale (NIHSS) scores were associated with an increased predicted probability for spasticity (odds ratio, OR = 1.153 [95% confidence interval, CI 1.093-1.216], p < .001). Logistic regression analysis revealed that lower age, higher NIHSS scores, and drinking were associated with an increased risk of moderate-to-severe spasticity (OR = 0.965 [95% CI 0.939-0.992], p = .013; OR = 1.068 [95% CI 1.008-1.130], p = .025; OR = 4.809 [95% CI 1.671-13.840], p = .004, respectively). However, smoking and ICH in the thalamus were associated with a reduced risk of moderate-to-severe spasticity (OR = 0.200 [95% CI 0.071-0.563], p = .002; OR = 0.405 [95% CI 0.140-1.174], p = .046, respectively) compared with ICH in the basal ganglia. CONCLUSIONS: Our results suggest that ICH lesion locations are at least partly associated with post-stroke spasticity rather than the latter simply being a physiological reaction to ICH itself. The predictors for spasticity after ICH were age, NIHSS scores, past medical history, and ICH lesion sites.

Combined application of chemical fertilizer with green manure increased the stabilization of organic carbon in the organo-mineral complexes of paddy soil.

The influence of the combined application of chemical fertilizer with green manure on the stabilization of organic carbon (C) was explored in the organo-mineral complexes of paddy soil. The organo-mineral complexes were isolated from paddy soil treated with no fertilizer, chemical fertilizer alone, and chemical fertilizer combined with increasing amounts of Chinese milk vetch (CMV). The stability (reflected by mineralizable carbon proportion), the content and chemical composition of organic C, the Fe/Al oxides and their associated organic C in the organo-mineral complexes were investigated. The application of chemical fertilizer in combination with CMV significantly improved the stability of organic C in the organo-mineral complexes. The combined application of chemical fertilizer with CMV slightly decreased the proportion of O-alkyl C (easily decomposed) yet somewhat increased the proportions of carbonyl C and aromatic C (difficultly decomposed) and aromaticity index in the organo-mineral complexes. The treatments of chemical fertilizer combined with CMV showed more Fe oxides and Fe/Al-associated organic C and higher proportion of Fe/Al-associated organic C in the total organic C of the organo-mineral complexes. The mineralizable carbon proportion displayed significantly negative correlations with carbonyl C and Fe/Al oxide-associated organic C in the organo-mineral complexes. The Fe/Al oxides were likely to be preferentially bound with the aromatic C and carbonyl C in the organo-mineral complexes. Overall, the combined application of chemical fertilizer with CMV facilitated the association of difficultly decomposed carbon and Fe/Al oxides, which significantly improved the stabilization of organic C in the organo-mineral complexes of paddy soil.

Polymer Topology Reinforced Synergistic Interactions among Nanoscale Molecular Clusters for Impact Resistance with Facile Processability and Recoverability.

The intrinsic conflicts between mechanical performances and processability are main challenges to develop cost-effective impact-resistant materials from polymers and their composites. Herein, polyhedral oligomeric silsesquioxanes (POSSs) are integrated as side chains to the polymer backbones. The one-dimension (1D) rigid topology imposes strong space confinements to realize synergistic interactions among POSS units, reinforcing the correlations among polymer chains. The afforded composites demonstrate unprecedented mechanical properties with ultra-stretchability, high rate-dependent strength, superior impact-resistant capacity as well as feasible processability/recoverability. The hierarchical structures of the hybrid polymers enable the co-existence of multiple dynamic relaxations that are responsible for fast energy dissipation and high mechanical strengths. The effective synergistic correlation strategy paves a new pathway for the design of advanced cluster-based materials.

Analysis of the Taxonomy and Pathogenic Factors of Pectobacterium aroidearum L6 Using Whole-Genome Sequencing and Comparative Genomics.

Soft rot pectobacteria are devastating plant pathogens with a global distribution and a broad host range. Pectobacterium aroidearum L6, previously isolated from leaves of Syngonium podophyllum, is a pectolytic bacterial pathogen that causes typical soft rot on S. podophyllum. There is a shortage for genome data of P. aroidearum, which seriously hinders research on classification and pathogenesis of Pectobacterium. We present here the complete genome sequence of P. aroidearum L6. The L6 strain carries a single 4,995,896-bp chromosome with 53.10% G + C content and harbors 4,306 predicted protein-coding genes. We estimated in silico DNA-DNA hybridization and average nucleotide identity values in combination with the whole-genome-based phylogeny from 19 Pectobacterium strains including P. aroidearum L6. The results showed that L6 and PC1 formed a population distinct from other populations of the Pectobacterium genus. Phylogenetic analysis based on 16S rRNA and genome sequences showed a close evolutionary relationship among Pectobacterium species. Overall, evolutionary analysis showed that L6 was in the same branch with PC1. In comparison with 18 Pectobacterium spp. reference pathogens, strain L6 had 2,712 gene families, among which 1,632 gene families were identified as orthologous to those strains, as well as 1 putative unique gene family. We discovered 478 genes, 10.4% of the total of predicted genes, that were potentially related to pathogenesis using the Virulence Factors of Pathogenic Bacteria database. A total of 25 genes were related to toxins, 35 encoded plant cell-wall degrading enzymes, and 122 were involved in secretion systems. This study provides a foundation for a better understanding of the genomic structure of P. aroidearum and particularly offers information for the discovery of potential pathogenic factors and the development of more effective strategies against this pathogen.

Regulation of soil aggregate size under different fertilizations on dissolved organic matter, cellobiose hydrolyzing microbial community and their roles in organic matter mineralization.

The mineralization of soil organic matter is closely related to climate change. Labile organic matter and microbial community are vital intrinsic factors in controlling the mineralization of soil organic matter. Regulation of soil aggregate size on dissolved organic matter (DOM), the cellobiose hydrolyzing microbial community, and their roles in organic matter mineralization remains unclear. The mineralization of organic matter in large macroaggregates (LMA, >2 mm), small macroaggregates (SMA, 0.25-2 mm), and microaggregates (MI, <0.25 mm) from an Ultisol treated with long-term non-fertilizers (Ck), chemical fertilizers (NPK) and animal manure (AM) was observed in this study. The concentration and structure of DOM, activity of ß-glucosidase, and the abundance, diversity, and community composition of GH1 (glycoside hydrolase family 1) microbial ß-glucosidase encoding genes were investigated. The cumulative CO2-C emissions occurred in the order LMA < SMA < MI in each fertilization treatment and followed the sequence Ck < NPK < AM in each size of aggregate. The concentration of DOM in the soil aggregates increased as the aggregate size decreased, while the structural complexity of DOM followed the opposite trend. The activity of ß-glucosidase in the smaller aggregates was higher than that in the larger aggregates, and the abundance and diversity of the GH1 microbial ß-glucosidase genes generally echoed the same trend. The dominant microbial classes harboring GH1 ß-glucosidase genes in the soil aggregates were Actinobacteria, Alphaproteobacteria, Gammaproteobacteria, Flavobacteria, Eurotiomycetes, and Sordariomycetes. The relative abundance of Actinobacteria, Sordariomycetes, and Eurotiomycetes revealed significant differences among the aggregates. Redundancy analysis confirmed that microbial GH1 ß-glucosidase community in the soil aggregates was primarily regulated by DOM concentration and pH. Structural equation modelling revealed that soil aggregates mainly regulated the ß-glucosidase activity and DOM concentration and then the abundance and diversity of the GH1 microbial ß-glucosidase genes in controlling organic matter mineralization.

Comparative Genomics Analysis Provides New Strategies for Bacteriostatic Ability of Bacillus velezensis HAB-2.

Biocontrol formulations prepared from biocontrol bacteria are increasingly applied in sustainable agriculture. Notably, inoculants prepared from Bacillus strains have been proven efficient and environmentally friendly alternatives to chemical bactericides. The bacterium Bacillus velezensis HAB-2 (formerly classified as B. amyloliquefaciens HAB-2) is used as a biological control agent in agricultural fields. In this study, we reported a high-quality genome sequence of HAB-2 using third-generation sequencing technology (PacBio RS II). The 3.89 Mb genome encoded 3,820 predicted genes. Comparative analysis among the genome sequences of reference strains B. velezensis FZB42, B. amyloliquefaciens DSM7 and B. subtilis 168 with the HAB-2 genome revealed obvious differences in the variable part of the genomes, while the core genome shared by FZB42 and HAB-2 was similar (96.14%). However, there were differences in the prophage region among the four strains. The numbers of prophage regions and coding genes in HAB-2 and FZB42 were smaller than the other two strains. The HAB-2 genome showed superior ability to produce secondary metabolites and harbored 13 gene clusters involved in synthesis of antifungal and antibacterial acting secondary metabolites. Furthermore, there were two unique clusters: one cluster which encoded lanthipeptide was involved in mersacidin synthesis and another cluster which encoded ladderane was shown to direct an unknown compound. Multidomain enzymes, such as non-ribosomal peptide synthetase and polyketide synthase, control the biosynthesis of secondary metabolites and rely on 4'-phosphopantetheinyl transferases (PPTases). Key genes lpaH2 and a encoded PPTases in HAB-2 encoded 224 and 120 amino acids, respectively. The genomic features revealed that HAB-2 possesses immense potential to synthesize antimicrobial acting secondary metabolites by regulating and controlling gene clusters. The prophage regions and genes encoding PPTases may provide novel insight for the bacteriostatic mechanism of Bacillus in the biological control of plant diseases.