Isolation and identification of antagonistic Bacillus amyloliquefaciens HSE-12 and its effects on peanut growth and rhizosphere microbial community

The HSE-12 strain isolated from peanut rhizosphere soil was identified as Bacillus amyloliquefaciens by observation of phenotypic characteristics, physiological and biochemical tests, 16S rDNA and gyrB gene sequencing. In vitro experiments showed that the strain possessed biocontrol activity against a variety of pathogens including Sclerotium rolfsii. The strain has the ability to produce hydrolytic enzymes, as well as volatile organic compounds with antagonistic and probiotic effects such as ethyleneglycol and 2,3-butanediol. In addition, HSE-12 showed potassium solubilizing (10.54 ± 0.19 mg/L), phosphorus solubilization (168.34 ± 8.06 mg/L) and nitrogen fixation (17.35 ± 2.34 mg/g) abilities, and was able to secrete siderophores [(Ar-A)/Ar × 100%: 56%] which promoted plant growth. After inoculating peanut with HSE-12, the available phosphorus content in rhizosphere soil increased by 27%, urease activity increased by 43%, catalase activity increased by 70% and sucrase activity increased by 50% (p < 0.05). The dry weight, fresh weight and the height of the first pair of lateral branches of peanuts increased by 24.7, 41.9, and 36.4%, respectively, compared with uninoculated peanuts. In addition, compared with the blank control, it increased the diversity and richness of peanut rhizosphere bacteria and changed the community structure of bacteria and fungi. The relative abundance of beneficial microorganisms such as Sphingomonas, Arthrobacter, RB41, and Micromonospora in rhizosphere soil was increased, while the relative abundance of pathogenic microorganisms such as Aspergillus, Neocosmospora, and Rhizoctonia was decreased

Contrast agent-free sonoporation: the use of an ultrasonic standing wave microfluidic system for the delivery of pharmaceutical agents

Sonoporation is a useful biophysical mechanism for facilitating the transmembrane delivery of therapeutic agents from the extracellular to the intracellular milieu. Conventionally, sonoporation is carried out in the presence of ultrasound contrast agents, which are known to greatly enhance transient poration of biological cell membranes. However, in vivo contrast agents have been observed to induce capillary rupture and haemorrhage due to endothelial cell damage and to greatly increase the potential for cell lysis in vitro. Here, we demonstrate sonoporation of cardiac myoblasts in the absence of contrast agent (CA-free sonoporation) using a low-cost ultrasound-microfluidic device. Within this device an ultrasonic standing wave was generated, allowing control over the position of the cells and the strength of the acoustic radiation forces. Real-time single-cell analysis and retrospective postsonication analysis of insonated cardiac myoblasts showed that CA-free sonoporation induced transmembrane transfer of fluorescent probes (CMFDA and FITC-dextran) and that different mechanisms potentially contribute to membrane poration in the presence of an ultrasonic wave. Additionally, to the best of our knowledge, we have shown for the first time that sonoporation induces increased cell cytotoxicity as a consequence of CA-free ultrasound-facilitated uptake of pharmaceutical agents (doxorubicin, luteolin, and apigenin). The US-microfluidic device designed here provides an in vitro alternative to expensive and controversial in vivo models used for early stage drug discovery, and drug delivery programs and toxicity measurements

Effects of <i>Bacillus subtilis</i> T6-1 on the Rhizosphere Microbial Community Structure of Continuous Cropping Poplar

The continuous cropping obstacles in poplar cultivation cause declines in wood yield and serious soil-borne diseases, mainly because of structural alterations in the microbial community and the aggregation of pathogenic fungi. Bacillus subtilis T6-1, isolated from poplar rhizospheric soil, has strong antagonistic effects on poplar pathogens. We aimed to investigate the effects of B. subtilis T6-1 on the structure of the microbial community in the poplar rhizosphere. Poplar seedlings were replanted in three successive generations of soil. The diameter at breast height, plant height, and the number of culturable bacteria of the poplars inoculated with T6-1 exceeded those in the non-inoculated control group. qPCR analysis revealed that the total abundance of T6-1 bacteria in the treated poplars was remarkably higher in contrast to that in the control group. Illumina MiSeq sequencing was employed to track the alterations in diversity and structure of the total microbial community in the poplar rhizosphere inoculated with B. subtilis T6-1. Fungal diversity and abundance in the T6-1 rhizosphere were remarkably lower in contrast with those in the control rhizosphere. The proportion of Bacillus sp. in the total bacterial community in the T6-1 and control groups was 3.04% and 2.38%, respectively, while those of the Rhizoctonia sp. was 2.02% and 5.82%, respectively. In conclusion, B. subtilis T6-1 has the potential to serve as a microbial agent, enhancing the structure of the rhizosphere microbial community as well as promoting tree growth in poplar cultivation

Isolation and Characterization of Antagonistic Bacteria Paenibacillus jamilae HS-26 and Their Effects on Plant Growth

Soilborne pathogens affect plant growth and food production worldwide. The application of chemical fertilizers and pesticides to control plant diseases has harmful effects; fortunately, plant growth-promoting rhizobacteria can be used as a potential alternative strategy. Here, Paenibacillus jamilae HS-26 was selected for its highly antagonistic activity against several soilborne pathogens. The bacterium synthesized hydrolytic enzymes and released extracellular antifungal metabolites and volatile organic compounds—primarily, N, N-diethyl-1, 4-phenylenediamine, which was detected by gas chromatography-mass spectrometry and shown to inhibit fungal mycelial growth. Furthermore, HS-26 was useful for nitrogen fixation, phosphate and potassium solubilization, and siderophore and indoleacetic acid production. In vitro tests and pot experiments revealed that HS-26 considerably increased plant biometric parameters. Illumina MiSeq sequencing data showed a significant reduction in soilborne pathogens and increase in beneficial bacteria in the wheat rhizosphere after treatment with strain HS-26

Effects of channel wall wettability on gas–liquid dynamics mass transfer under Taylor flow in a serpentine microchannel

The wall wettability of microchannels plays an important role in the gas–liquid mass transfer dynamics under Taylor flow. In this study, we regulated the contact angle of the wall surface through surface chemical grafting polymerization under controlled experimental conditions. The dynamic changes of CO2 bubbles flowing along the microchannel were captured by a high-speed video camera mounted on a stereo microscope, whilst a unit cell model was employed to theoretically investigate the gas–liquid mass transfer dynamics. We quantitatively characterized the effects of wall wettability, specifically the contact angle, on the formation mechanism of gas bubbles and mass transfer process experimentally. The results revealed that the gas bubble velocity, the overall volumetric liquid phase mass transfer coefficients (kLa), and the specific interfacial area (a) all increased with the increase of the contact angle. Conversely, gas bubble length and leakage flow decreased. Furthermore, we proposed a new modified model to predict the gas–liquid two-phase mass transfer performance, based on van Baten's and Yao's models. Our proposed model was observed to agree reasonably well with experimental observations

Combined application of microbial inoculant and kelp-soaking wastewater promotes wheat seedlings growth and improves structural diversity of rhizosphere microbial community

Abstract Industrial processing of kelp generates large amounts of kelp-soaking wastewater (KSW), which contains a large amount of nutrient-containing substances. The plant growth-promoting effect might be further improved by combined application of growth-promoting bacteria and the nutrient-containing KSW. Here, a greenhouse experiment was conducted to determine the effect of the mixture of KSW and Bacillus methylotrophicus M4-1 (MS) vs. KSW alone (SE) on wheat seedlings, soil properties and the microbial community structure in wheat rhizosphere soil. The available potassium, available nitrogen, organic matter content and urease activity of MS soil as well as the available potassium of the SE soil were significantly different (p < 0.05) from those of the CK with water only added, increased by 39.51%, 36.25%, 41.61%, 80.56% and 32.99%, respectively. The dry and fresh weight of wheat seedlings from MS plants increased by 166.17% and 50.62%, respectively, while plant height increased by 16.99%, compared with CK. Moreover, the abundance and diversity of fungi in the wheat rhizosphere soil were significantly increased (p < 0.05), the relative abundance of Ascomycetes and Fusarium spp. decreased, while the relative abundance of Bacillus and Mortierella increased. Collectively, the combination of KSW and the plant growth-promoting strain M4-1 can promote wheat seedlings growth and improve the microecology of rhizosphere microorganisms, thereby solving the problems of resource waste and environmental pollution, ultimately turning waste into economic gain

Dynamic characterization of nanoparticles production in a droplet-based continuous flow microreactor

The present work was focused on the dynamic characterization of nanoparticles production under continuous flow in a droplet-based spiral-shaped microreactor. A novel drainage design was incorporated into the microreactor channel network for eliminating the commonly encountered problems of clogging and coalescence of microdroplets containing nanoparticles in the microchannel. Through precise manipulation of droplets formation for investigating the precipitation process of iron oxide, one-to-one pairing and fusion of reactant droplets were obtained and monitored in a real-time manner through digital imaging, where optimised flow rate ratios (in a range of 0.3–0.5) of aqueous phase to oil phase were applied. The nanoprecipitation process was observed and further analysed, indicating that the formation of precipitates was determined by the diffusion of OH- after coalescence of reactants droplets. By characterising the Fe3O4 nanoparticles produced, it demonstrated that smaller droplets were beneficial to obtaining high quality nanoparticles in terms of size control and size distribution. Moreover, the kinetics of the nucleation process was investigated through statistic calculation. Initial results suggested that heterogeneous nucleation occurring inside droplets was likely due to the presence of impurities-mediated (e.g. surfactants) or collision-induced nucleation events

Biocontrol of Two Bacterial Inoculant Strains and Their Effects on the Rhizosphere Microbial Community of Field-Grown Wheat

Biocontrol by inoculation with beneficial microbes is a proven strategy for reducing the negative effect of soil-borne pathogens. We evaluated the effects of microbial inoculants BIO-1 and BIO-2 in reducing soil-borne wheat diseases and in influencing wheat rhizosphere microbial community composition in a plot test. The experimental design consisted of three treatments: (1) Fusarium graminearum F0609 (CK), (2) F. graminearum + BIO-1 (T1), and (3) F. graminearum F0609 + BIO-2 (T2). The results of the wheat disease investigation showed that the relative efficacies of BIO-1 and BIO-2 were up to 82.5% and 83.9%, respectively. Illumina MiSeq sequencing revealed that bacterial abundance and diversity were significantly higher (P<0.05) in the treatment groups (T1 and T2) than in the control, with significantly decreased fungal diversity in the T2 group. Principal coordinates and hierarchical clustering analyses revealed that the bacterial and fungal communities were distinctly separated between the treatment and control groups. Bacterial community composition analysis demonstrated that beneficial microbes, such as Sphingomonas, Bacillus, Nocardioides, Rhizobium, Streptomyces, Pseudomonas, and Microbacterium, were more abundant in the treatment groups than in the control group. Fungal community composition analysis revealed that the relative abundance of the phytopathogenic fungi Fusarium and Gibberella decreased and that the well-known beneficial fungi Chaetomium, Penicillium, and Humicola were more abundant in the treatment groups than in the control group. Overall, these results confirm that beneficial microbes accumulate more easily in the wheat rhizosphere following application of BIO-1 and BIO-2 and that the relative abundance of phytopathogenic fungi decreased compared with that in the control group

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides

Mogrosides are a group of health-promoting natural products that extracted from Siraitia grosvenorii fruit (Luo-han-guo or monk fruit), which exhibited a promising practical application in natural sweeteners and pharmaceutical development. However, the production of mogrosides is inadequate to meet the need worldwide, and uneconomical synthetic chemistry methods are not generally recommended for structural complexity. To address this issue, an in-fusion based gene stacking strategy (IGS) for multigene stacking has been developed to assemble 6 mogrosides synthase genes in pCAMBIA1300. Metabolic engineering of Nicotiana benthamiana and Arabidopsis thaliana to produce mogrosides from 2,3-oxidosqualene was carried out. Moreover, a validated HPLC-MS/MS method was used for the quantitative analysis of mogrosides in transgenic plants. Herein, engineered Arabidopsis thaliana produced siamenoside I ranging from 29.65 to 1036.96 ng/g FW, and the content of mogroside III at 202.75 ng/g FW, respectively. The production of mogroside III was from 148.30 to 252.73 ng/g FW, and mogroside II-E with concentration between 339.27 and 5663.55 ng/g FW in the engineered tobacco, respectively. This study provides information potentially applicable to develop a powerful and green toolkit for the production of mogrosides