Biosynthesis Of Novel Chitosan-iron Nanocomposite And Its Mechanism To Control Rice Bacterial Leaf Blight

Bacterial leaf blight（BLB）disease caused by Xanthomonas oryzae pv.oryzae（Xoo）,is among the most devastating pathogen limiting rice production.Conventional management options for BLB include various antibiotics and chemical-based products;however,these options are harmful to human health,environmentally damaging,and can lead to pathogen resistance.Hence,there is an urgent need to develop novel approaches for BLB disease control to increase production and combat food insecurity.Nano-enabled agrochemicals have attracted increasing interest for potential applications in plant disease management as sustainable alternatives to conventional pest control techniques.The present work involves the synthesis of bioengineered chitosan-iron nanocomposites（BNCs）equipped with potential in vitro and in vivo antibacterial activity against the economically important rice pathogen Xoo.Moreover,in order to elucidate the mechanism of BNCs for the control of BLB disease,the impact of BNCs on Xoo in rice plants was determined by high-throughput sequencing technologies（transcriptomics,proteomics,and metagenomics）as well as the analysis of innate plant defense responses,biochemical-molecular mechanisms,nutritional status and endophytic microbial communities of rice.These findings demonstrate that BNCs have potential to serve as non-toxic,sustainable and highly efficient alternatives for plant disease management.Firstly,this study reported the biosynthesis and characterization of BNCs along with their in vitro bactericidal activity against Xoo.The biogenic iron oxide nanoparticles（Fe ONPs）synthesized using a native Bacillus aryabhattai RNT7,were used to bioengineered chitosan iron nanocomposite（BNCs）through one-pot synthesis method.The synthesis of BNCs were further confirmed by state-of-the-art material characterization techniques such as,ultraviolet-visible（UV-vis）spectroscopy,zeta potential,fourier transform infrared（FTIR）,X-ray diffraction（XRD）,scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,and energy-dispersive X-ray（EDS）analyses.The BNCs were spherically shaped with an average size of 86nm.In vitro antibacterial assays showed that BNCs significantly inhibited biological functions of the pathogen,including growth and biofilm formation at 250μg m L^-1concentration as compared with respective control.The fluorescent microscopic images demonstrated that BNCs treatments induced reactive oxygen species（ROS）formation in bacterial cells,which subsequently caused cellular oxidative stress that disrupted normal cellular functions and eventually lead to cell death.Moreover,electron microscopy（SEM and TEM）images showed that the application of BNCs severely perforated the cell wall and cell membrane that caused the leakage of cytoplasmic matrix.Overall,these results demonstrated that the destructive interaction of BNCs with cellular components and macromolecules（i.e.,DNA and protein）produced a cascade of biological activity that leads to Xoo cell death.Secondly,this study investigated the toxicity and molecular antibacterial mechanisms of BNCs against Xoo using transcriptomic and proteomic approaches.Transcriptomic data showed that BNCs exposure caused differential expression of genes related to cell membrane biosynthesis,antioxidant stress,DNA damage,flagellar biosynthesis and transcriptional regulator,which clearly showing the involvement of these various processes in the interaction of BNCs to Xoo pathogen.Moreover,the transcriptomic data was justified by q RT-PCR analysis,which demonstrated that BNCs amendments significantly upregulated the expression levels of Xoo Gls B,Xoo CAT,Xoo SOD,and Xoo DDR,whereby,the expression levels of（Xoo Pvs A,Xoo Flg B,Xoo Flg C,and Xoo Flg G）were significantly downregulated after BNCs treatment as compared to untreated control.Similarly,proteomic profiling showed that BNCs treatment significantly altered the levels of functional proteins involved in the integral component of the cell membrane,catalase activity,oxidation-reduction process and metabolic process in Xoo,which is consistent with the results of the transcriptomic analysis.Overall,this study suggested that BNCs has great potential to serve as an eco-friendly,sustainable,and non-toxic alternative to traditional agrichemicals to control the BLB disease in rice.Thirdly,this study reported the in vivo antibacterial activity and disease-suppressive potential as well as biochemical and ultrastructural change of BNCs against BLB disease caused by Xoo.A greenhouse experiment demonstrated that foliar exposure to 250 mg·L^-1BNCs significantly reduced the BLB disease incidence（67.1%）by improving the plant growth（32.1%）,antioxidant enzymes,such as superoxide dismutase（49.2%）,peroxidase（38.8%）and ascorbate peroxidase（53.4%）,and decreasing the reactive oxygen species（ROS）including,hydrogen peroxide（47.0%）,malondialdehyde（50.9%）,and superoxide radical（44.6%）as compared to untreated diseased control.BNCs amendment also improved photosynthesis efficiency by promoting production of total chlorophyll（43.2%）and carotenoids contents（60.0%）,and modulating the nutritional profile（iron,phosphorus,potassium,sodium and calcium）of rice plants as compared with untreated diseased control.The ultrastructure imaging showed that the BNCs enter the rice leaf through the stomata and disperse within the large spaces of spongy mesophyll cells without inducing any overt morphological damage and BNCs seemed to form a protective layer without penetrating the cell walls.Importantly,after BNCs treatment Xoo infected rice showed the normal cellular structure with intact cell membranes and recovery of cellular material integrity as compared with diseased controls.In addition,BNCs also inhibited Xoo proliferation and invasion within rice leaf cells,effectively decreasing the spread of BLB disease.These findings demonstrate that BNCs suppressed rice BLB disease by multiple ex-planta and in-planta mechanisms and highlight the value of this approach as a powerful tool to increase agricultural production and alleviate food insecurity.Lastly,this study performed the high-throughput sequencing（transcriptomic and metagenomic）analyses to determine the impact of BNCs on plant defense responses and diversity of the rice microbial community.Transcriptomic profiling identified a total of 2525 differentially expressed genes（DEGs）upregulated（1206）and downregulated（1319）in response to BNCs as compared with untreated control treatment.q RT-PCR analysis revealed that BNCs amendments significantly upregulated the expression of the selected growth-related genes（Os GRP2,Os PSII,Os ILR1,Os CML2）and defense related genes（Os PR1,Os POD,Os WRKY23,Os TLP1b,Os CAT,OSRGA2）in healthy and Xoo-infected rice plants as compared with control treatment,which is consistent with the results of the transcriptomic analysis.Taken together,RNA-seq-based transcriptomic profiling demonstrates that BNCs can trigger the expression of different subsets of growth and defense-related genes to promote plant growth and activate defense responses against Xoo infection in rice plants.Moreover,metagenomic analysis results revealed that BNCs amendment decreased the relative abundance of Xanthomonas（87.5%）by reshaping the phyllospheric and root-endophytic bacterial community of rice.In addition,BNCs increased the bacterial community diversity in healthy and diseased plants;significant increases in relative abundance of Ochrobactrum,Allorhizobium,Methylobacterium,Devosia,Pseudolabrys,Sphingomonas and Bradyrhizobium in BNCs-treated diseased and healthy plants was noted.Overall,we speculated that BNCs-mediated suppression of BLB disease results in the shift of bacterial abundance and diversity in diseased rice plants to maintain endophytic microbial populations that exist in healthy rice plants without Xoo stress.In summary,this study investigated the effect of BNCs on rice BLB disease control through nano-strategy for the first time worldwide,revealed its antibacterial mechanism,and used molecular-based framework to quantify the impact of BNCs on rice endophytic microbial community.