| The research of carbon-based nanomaterials in environmental management,energy and medical antibacterial treatment has developed rapidly in recent years,but there are few researches in agriculture,especially in the field of plant protection.In this study,urea was used to synthesize g-C3N4 nanosheets that are cheap and easy to obtain,non-toxic,chemically stable and thermally stable.In this study,g-C3N4 nanosheets were confirmed to have photocatalytic and antibacterial activity,and its mechanism of action was also studied.However,the effect of single non-metallic nanomaterials are often unsatisfactory.Therefore,after it is clear that Zn ONPs can better induce plant resistance,the composite material of g-C3N4@Zn ONPs was further synthesized,and its antibacterial and antibacterial effect was studied under lower dosage,and the synergistic mechanism of the composite material was also investigated.The main findings are as follows,1.The g-C3N4 nanosheets were prepared by high-temperature polymerization of urea and the antibacterial performance and mechanism of g-C3N4 nanosheets on tobacco wildfire bacteria(bacteria)were studied.The morphology and structure of g-C3N4 nanosheets were characterized using transmission electron microscopy(TEM),scanning electron microscopy(SEM),X-ray photoelectron spectroscopy(XPS),X-ray diffractometry(XRD),infrared spectroscopy(FT-IR)and potentiometric measurement(Zeta)for g-C3N4 nanosheets.The results show that g-C3N4 nanosheets are in the shape of Tremella,which is composed of abundant folded aggregates of nanosheets.And there are a large number of irregularly shaped in-plane void spaces in the carbon nitride layers.The antibacterial effect of g-C3N4 nanosheets on Tobacco wildfire bacteria had a significant dose-dependent and visible light irradiation time-dependent character.Compared with the control,g-C3N4 nanosheets at 0.5 mg/m L can significantly inhibit wildfire bacteria and control wildfire disease.The analysis of the antibacterial mechanism revealed that g-C3N4 nanosheets produced a large amount of ROS under visible light irradiation,including ROS within and outside the bacterial cell.After 2 hours of treatment,the stress of g-C3N4 nanosheets on Tobacco wildfire bacteria resulted in the inhibition of biofilm formation and motility,further damaging the cell membrane,causing cytoplasmic leakage and DNA damage,and ultimately leading to the death of the bacteria.On the other hand,the attachment of g-C3N4 nanosheets on the surface of bacteria is also an important synergistic physical effect.The transcriptome analysis results showed that after 1 h of treatment with g-C3N4 nanosheets,500 genes of the pathogen were differentially expressed.Among them,the expression of genes related to"antioxidant activity"and"membrane transport"was significantly up-regulated,and the expression of genes related to"bacterial chemotaxis","biofilm formation","energy metabolism"and"cell movement"was down-regulated.This result also explained the mechanism of g-C3N4 nanosheets inhibiting wildfire bacteria at the genetic level.2.The antibacterial effect and mechanism of g-C3N4 nanosheets on Phytophthora capsici(oomycetes)were studied.Transcriptome analysis were first carried out using three representative growth stages of Phytophthora capsici samples treated with 0.5 mg/m L g-C3N4 nanosheets under light conditions.The results showed that after treatment with g-C3N4 nanosheets,the antioxidant activity and genes related to structural components of Phytophthora capsici are significantly up-regulated,while genes related to metabolic pathways including complex adaptation processes such as ATP production,autophagy destruction,and membrane system disorders are down-regulated.Subsequent experiments further proved that under visible light irradiation,g-C3N4 nanosheets significantly inhibited all life cycles of Phytophthora capsici,including mycelial growth,sporangia formation,and zoospore amount.At the same time,the treatment of g-C3N4 nanosheets under light produces a large amount of ROS.With the help of the sharp structure of the nanosheets,the g-C3N4nanosheets also damage the morphology,ultrastructure,sporangia,and zoospores of Phytophthora capsici mycelium,which further confirms the transcriptome analysis results,in which contains the"membrane component"gene enrichment.In view of the fact that the antibacterial activity of g-C3N4nanosheets is derived from photocatalytic ROS generation and physical damage,and this complex mechanism make g-C3N4 4 nanosheets might be effective not limited to one single pathogen target but to multiple kinds of oomycetes,which indicates that g-C3N4 nanosheets can be used to control crop oomycete diseases and have great potential serving as a new non-metallic antibacterial agents for bacterial diseases control.More importantly,in addition to inhibiting the pathogenicity of Phytophthora capsici,g-C3N4 nanosheets can also promote the growth of host pepper plants,which further increases the application prospects of g-C3N4 nanosheets.3.The mechanism of g-C3N4 nanosheets inducing disease resistance of Nicotiana benthamiana were revealed.g-C3N4 nanosheets at 0.25 mg/m L dosage were sprayed on N.benthamiana.And TMV/Phytophthora capsici/Pseudomonas syringae tomato pathogenic species Pst DC3000 were inoculated after 24 hours.g-C3N4 nanosheets were found can enhance the resistance of N.benthamiana to these three pathogenic microorganisms,while urea has no similar inducing effect,indicating that the g-C3N4 nanosheet structure is the reason for resistance enhancing.Further study shows that spraying g-C3N4 nanosheets can activate a series of resistance physiological responses of N.benthamiana,including the accumulation of reactive oxygen species and callose,and up-regulated expression of multiple disease-related genes such as MAPK kinase phosphorylation and PR1.Further,through quantitative analysis of expression of hormone synthesis-related genes,determination of plant endogenous hormones,and silencing of related genes of N.benthamiana,the disease resistance of N.benthamiana stimulated by g-C3N4 nanosheets were found depending on coordinated regulation of multiple signal pathways,which include salicylic acid,brassinolide,ethylene and abscisic acid,etc.Transcriptome data analysis of N.benthamiana treated with g-C3N4nanosheets for 12 h,24 h and 36 h showed that the response of plants to g-C3N4 treatment is a dynamic process that changes over time.The enrichment of a large number of genes related to disease resistance up-regulated differential expression were found within 24 h after treatment,verifying that g-C3N4 nanosheets stimulated multiple disease resistance-dependent signaling pathways.And this stress response gradually returned to normal after 36 h after treatment.It was also found that 7d treatment of g-C3N4 nanosheets can promote the photosynthesis and plant growth of N.benthamiana,which can be seen that g-C3N4 nanosheets transformed from immune regulatior to nutrition regulator.Therefore,24 h of g-C3N4 nanosheet treatment on N.benthamiana can trigger plant immunity similar to the PTI response triggered by PAMP molecules.And the resistance to stress gradually decreases after 36 hours,which is transformed into plant nutrition regulation and promote plant growth.4.In order to screen materials that can be composited with g-C3N4 nanosheets,Zn ONPs were obtained by the green starch synthesis method.And the mechanism of Zn ONPs inducing TMV resistance in N.benthamiana plants was studied.Structure characterization were carried out using dynamic light scattering(DLS)and TEM.Zn ONPs are spherical particles with a diameter of 20 nm,partially aggregated,and have a stable particle size distribution in deionized water.The effect of Zn ONPs on plant disease resistance and growth was further studied.The results showed that after 2h pretreatment with Zn ONPs or Si O2NPs in vitro,TMV particles aggregated and fractured.These mixtures were inoculated on tobacco plants.Although the accumulation of inoculated leaf virus was lower than the control group after 2 days of inoculation,there was no difference in the systemic infection and accumulation of virus after 7 days of inoculation.The continuous foliar spraying of nanomaterials for 12 days before virus inoculation can significantly inhibit the accumulation of TMV.Analysis of the induction mechanism found that after Zn ONPs treatment,the accumulation of active oxygen,antioxidant enzyme activity,and disease-related resistance genes PR1 and PR1 and PR2were all up-regulated.And the content of salicylic acid and abscisic acid increased by 162%and517%,respectively.At the same time,Zn ONPs also promoted the dry weight and fresh weight of N.benthamiana as compared with the control.Electron microscopy analysis further revealed that Zn ONPs were absorbed by tobacco leaves and transported throughout the plant,which may be one of the reasons why it improves the disease resistance of tobacco.Therefore,Zn ONPs has resistance inducing and growth promoting effect,and is a potential hybrid material for g-C3N4 nanosheets.5.Using electrostatic adsorption method of Zn ONPs,g-C3N4@Zn ONPs heterostructure was constructed to enhance the photocatalytic activity of g-C3N4 nanosheets,and the antibacterial,resistance inducing as well as synergistic activity and mechanism of this composite was studied.Since the antibacterial effect of g-C3N4 nanosheets is still restricted by bottlenecks such as slow charge transfer,giant exciton effect and low conductivity,and we have confirmed that Zn ONPs have good plant resistance and can promote plant growth.In this chapter,a simple electrostatic self-assembly method was used to modify Zn ONPs with g-C3N4 nanosheets as a photocatalytic carrier to obtain g-C3N4@Zn ONPs composite material,which greatly improves the photocatalytic activity of g-C3N4 nanosheets.The composite was characterized by XRD,TEM and FT-IR,and the results show that there is a strong interaction between g-C3N4 nanosheets and Zn ONPs rather than simple physical adsorption,which is due to the formation of a heterostructure.The antibacterial and antiviral effects as well as synergistic mechanism of g-C3N4@Zn ONPs were further investigated.The results show that g-C3N4@Zn ONPs produces more ROS than g-C3N4 nanosheets under visible light irradiation.And g-C3N4@Zn ONPs is adsorbed on the surface of the bacteria cell.The bacteria cell is then inhibited by both the physical membrane damage and the chemical damage of metals and ROS.And the presence of Zn ONPs caused g-C3N4@Zn ONPs to have a certain degree of antibacterial activity even without visible light irradiation.The results of transcriptome analysis of Tobacco wildfire bacteria showed that the expression of 463 same genes were down-regulated after treatment with g-C3N4@Zn ONPs and g-C3N4 nanosheets,indicating that the resistance mechanisms of g-C3N4@Zn ONPs and g-C3N4 nanosheets to tobacco wildfire are partially overlapped.However,g-C3N4@Zn ONPs has a significantly stronger antibacterial effect than g-C3N4 nanosheets,which is due to the stronger inhibition of the expression of genes related to the bacterial membrane structure,motility and energy metabolism.g-C3N4@Zn ONPs not only hindered the vegetative growth of Phytophthora capsici hyphae,but also significantly damaged and inhibited its reproductive growth(the number of spores,sporangia formation and spore germination)than g-C3N4 nanosheets.Combined with the results of transcriptome analysis,it can be seen that destroying the membrane structure and inhibiting energy metabolism is the main inhibiting mechanism of g-C3N4@Zn ONPs to Phytophthora capsici.Transcriptome and proteome analysis of N.benthamiana treated with the three materials showed that after continuous spraying for 3 days,g-C3N4@Zn ONPs and g-C3N4nanosheets both activated plant disease resistance and up-regulated the synthesis of plant endogenous hormones,while Zn ONPs mainly affects photosynthesis-related and various growth and metabolism pathways.And hormone signal transduction pathways related to plant growth,including activating the auxin and cytokinin pathways while inhibiting the response to external stress hormones,were found significantly enriched in all three treatments,which indicate that the three nanomaterials are beneficial to plant growth after continuous 3 d treatments.On the other hand,the differentially expressed genes in g-C3N4@Zn ONPs are also more enriched in the interaction pathway between plants and pathogens,indicating that g-C3N4@Zn ONPs has better and sustained induction plant resistance than Zn ONPs and g-C3N4 nanosheets.Therefore,compared with g-C3N4nanosheets,g-C3N4@Zn ONPs has more efficient antibacterial activity at lower dosage,and stronger induction of disease resistance and photosynthesis.In summary,g-C3N4 nanosheets were obtained by high-temperature polymerization of urea and was confirmed to have photocatalytic inhibition of Tobacco pyrotechnica and Phytophthora capsici with the mechanism of synergistic light-dependent chemical damage and physical damage.Mechanism investigation revealed that g-C3N4 nanosheets can stimulate N.benthamiana to produce PTI-like disease resistance that depends on the activation of ROS,MAPK kinase phosphorylation and plant hormone(SA,ABA,ET,and BR)pathways.And 7d treatment of g-C3N4 nanosheets to N.benthamiana change the resistance inducing state to a nutrient regulation stage,which mainly promoted photosynthesis and plant growth.Zn ONPs was further confirmed to have resistance inducing and growth promoting effect,and is used as the composite material for g-C3N4 nanosheets.Electrostatic adsorption method was employed to obtain g-C3N4@Zn ONPs composite materials with higher photocatalytic activity,which has more efficient antibacterial activity,resistance inducing and photosynthesis promoting activity at low-dosage.The research results can provide scientific basis for the application of photocatalytic nano-monomers as well as their composite materials in plant disease control. |
PDF Full Text Request