Preparation And Mechanism Of Galla Chinensis Film For Preventing And Controlling Rhizoctonia Solani In Rice

Rice sheath blight is a fungal disease caused by Rhizoctonia solani,which is one of the"three major diseases"seriously affecting the safety of rice production in China.In view of the fact that chemical control is still the main control measure of rice sheath blight and the related problems,the research and development of efficient,green and safe botanical pesticides to control rice sheath blight has become a research hotspot.Therefore,the screening of plant extracts with good inhibitory effect on Rhizoctonia solani showed that galla chinensis extract and its main compounds had good antifungal activity;A galla chinensis film agent for controlling rice sheath blight was developed with lauric acid and natural film-forming materials as raw materials;The antifungal mechanism of lauric acid,the main component of galla chinensis film agent,against Rhizoctonia solani was analyzed from physiological,biochemical and molecular levels.The results can provide new ideas for the research and development of botanical fungicides and green control of rice sheath blight.The main results are as follows:1.It was found that the crude extract of galla chinensis and its active component lauric acid had good antifungal activity against Rhizoctonia solani.Theantifungal activities of 25 plant extracts against Rhizoctonia solani were determined.The results showed that the extract had good antifungal activity against Rhizoctonia solani,and its EC50 value was 41.84μg/m L.The results of LC-MS/MS showed that the extract contained tannic acid,gallic acid,pyrogallic acid,lauric acidβ-The results showed that lauric acid,tannic acid,gallic acid and pyrogallic acid had antifungal activity against Rhizoctonia solani,and lauric acid had strong inhibitory effect on mycelial growth of Rhizoctonia solani,with EC₅₀value of 52.21μg/m L.The inhibitory rates of lauric acid on the germination of Rhizoctonia solani at 100,200,and 400μg/m L were23.33%,60.00%,and 80.00%,respectively.Jinggangmycin at 100,200,and 400μg/m L had the inhibition rate of germination of sclerotia was 0.2.The results showed that the combination of tannic acid and lauric acid had obvious synergistic effect on rice sheath blight.Seven active compounds in galla chinensis were identified by LC-MS/MS and their biological activities were tested against Rhizoctonia Solani,tannin acid,gallic acid acid,pyrogenic gallic acid acid and lauric acid were the only substances with inhibitory effect on rice sheath blight.When the concentration of lauric acid and Jinggangmycin was 400μg/m L,the content of lauric acid in galla chinensis was 13.9%,the EC₅₀of rice sheath blight against Rhizoctonia Solani was 52.21μg/m L,the inhibition rates to sclerotia germination of Rhizoctonia Solani were 80%and 100%respectively,Lauric acid could inhibit the germination of sclerotia to some extent.The results showed that the combination of lauric acid and tannic acid（20:1）with EC₅₀of 51.44μg/m L and CTC of 408.65 had significant synergism.3.The galla chinensis film was prepared with the synergistic combination of lauric acid and tannic acid as the main effective component.By optimizing the film-forming material of 22.1%galla chinensis film,the formulation of the film was determined as follows:2.1%galla chinensis active ingredient（2%lauric acid+0.1%tannic acid）+12%guar gum+8%chitosan+1%sodium benzoate+2%glycerol+5%op-15+69.9%water.The film has good extensibility,drawability,contact angle and dilubility The elongation at break and oxygen transmittance were 52.54%and52.54%,respectively,52.54±0.0929 N/mm²,2.49±0.0677%and（8.37±0.0677）×10-⁹cm³/m²·d·Pa.Field test results showed that the control effect of 22.1%of the galla chinensis film dosage of 400 g/667 m²was 88.76%at 7 days after the drug,and the control effect was still 80.37%at the 14-day after the medicine;the dosage of22.1%galla chinensis film was 50 g/667 m²The control effect was 61.72%after 7days after the drug,and the control effect still reached 61.61%after 14 days after the drug.However,the 15%Jinggangmycin WP and lauric acid·tannic acid combination with a dosage of 50 g/667 m²was used 14 days after the drug.The control effect is only 24.71%and 14.23%.It can be seen that the 22.1%galla chinensis film agent delays the duration of the active combination of lauric and tannin acid.4.The inhibition activity and physiological and biochemical mechanism of22.1%galla chinensis film agent with lauric acid as the main effective component were clarified.The activities of PPO,POD and PAL and MDA content of rice defense enzymes were stable after 24 hours of application of galla chinensis film and water spraying alone;The defense enzymes of other treatments changed to different degrees after inoculation with rice blight.It was suggested that the application of galla chinensis film could prevent the infection of rice blight,which may affect the identification of rice.The antifungal mechanism of lauric acid to rice blight is to destroy the organelles such as mitochondria and endoplasmic reticulum,reduce the content of ergosterol,enhance the permeability of cell membrane and destroy the structural integrity of cell membrane;Lauric acid can also increase the SOD activity,cat enzyme activity and superoxide anion clearance rate of rice blight mycelium,and decrease the activity of SDH and MDH,which are the key enzymes of tricarboxylic acid cycle,and inhibit the mycelium growth after destroying the cell environment.5.The molecular mechanism of action of lauric acid on Rhizoctonia solani was elucidated.The gene changes of Rhizoctonia Solani after different time treatment with lauric acid were analyzed by means of transcriptomics.For gene GO enrichment analysis,40 genes were involved in the classification of significant functional GO,and the significant ones were metabolic process,cellular process,single-organism regulation,localization.The analysis of KEGG showed that DEGs could be divided into biosynthetic of antibiotics（KO01130）,Protein processing in in endoplasmic reticulum（KO04141）,Carbon metabolism（KO01200）,Starch and sucrose metabolism（KO00500）.The primary mechanism is that lauric acid induces up-regulation of glucanase and Fatty acid metabolism related genes,reduces the amount of ergosterol in cell membrane,causes cell membrane damage,down-regulates Cytochrome C oxidase and up-regulates iron reductase,the accumulation of ROS（H₂O₂）and the overproduction of ROS resulted in the breakdown of cell membrane potential,the oxidation of cell membrane,the enhancement of cell membrane permeability and electrolyte leakage,which led to the destruction of cell plasma membrane.In addition,the accumulation of ROS caused DNA fragmentation and ER stress,activated phosphite enzyme,and caused the apoptosis of Rhizoctonia Solani.