Regulatory Mechanism In Crop Nitrate Metabolism And Controlled-Release Effect Of Paecilomyces Variotii SJ1 Extract

Endophyte extract improves the nitrogen utilization efficiency of crops,but its mechanism is not clear.Moreover,its field activity is unstable,and the application is laborious.In this study,the extract from plant endophytic fungus Paecilomyces variotii SJ1(PVSE)was used as the research object.The basic physicochemical properties and characterization technique of PVSE were explored,using biochemical analysis technology.The process parameters for high-yield,high-quality,and stable PVSE production were optimized to ensure the stability of PVSE between batches,using response surface optimization,fingerprinting and enzyme-linked immunoassay.The effective active component in PVSE named P4 was separated and purified by a chromatographic column,and bioassays tracked its activity.Its structure was identified as uridine by liquid mass and nuclear magnetic resonance technology.Arabidopsis nitrogen-related gene transcriptome,q-PCR,post-transcriptional enzymes and physicochemical indexes,biological phenotypes and agronomic evaluation of pakchoi in laboratory and field and analysis of pakchoi to were further comprehensively used to reveal the regulatory pathways and mechanisms of PVSE involved in crop nitrate metabolism.Then,the corn field experiment was used to explore the main factors of the synergy between PVSE and controlled release fertilizer.Furthermore,bio-polyurethane was used as a carrier to achieve dual controlled release of PVSE and nitrogen to stabilize the microenvironment under which PVSE acts.Finally,the regulation of PVSE and nitrogen release laws were synchronized with the growth period of the crops combined with the developed PVSE detection technology in fertilizers,and agronomically evaluated the field effect on sweet potato after one-time application of controlled-release PVSE-coated urea.The above content provided theoretical basis and technical support for improving crop nitrogen utilization efficiency and realizing high-yield and high-efficiency agricultural production.The main research is as follows.(1)The average molecular weight of PVSE was less than 376 Da,mainly distributed between 70 and 500 Da,rich in aromatic and heterocyclic structures,and the maximum UV absorption peak is 210 nm.In organic matter,the contents of carbohydrate,protein,amino acid,nucleoside and lipid were 33.3%,19.2%,29.0%,7.4%and 3.8%respectively.It had temperature,acid base,light,organic reagent and urea stability.The ultrasonic extraction conditions of PVSE were optimized by response surface method,and the maximum yield extraction conditions were determined as material concentration 40%,alcohol concentration40%,extraction time 58.2 min and power 6 k W,respectively.Chromatographic fingerprinting and enzyme-linked immunosorbent assay were applied to evaluate the similarity and specificity of unknown effective components in PVSE,ensuring a similarity of more than90%and a quantitative accuracy of greater than 99.9%for the products from different batches.Overall,we have established PVSE extract technology system with high yield and high quality.PVSE was separated into 16 components by amino column using phosphate as mobile phase,and the bioassay resulted show that P4 has a significant activity in regulating nitrate nitrogen metabolism.(2)P4 stimulated the NLP family and hormone pathways to regulate nitrate metabolism and signal transduction.Under nitrogen deficiency,P4 regulate the response of nitrate-sensing genes NPF6.3 and NRT2.1 by inducing high expression of NPL family nitrogen-regulating genes in the nucleus.Then,it increased the absorption of nitrate by up-regulating the expression of high-affinity nitrate transport genes in NRT2 family,and mediated down-regulation of NAXT1 Genes to reduce the efflux of nitrate nitrogen from roots,thereby increasing nitrogen accumulation.Their root-crown-root signal transduction was mediated by the significant down-regulation of nitrogen signal peptide secretion pathway genes in CLE family,and feed back the nitrogen deficiency signal to the shoots of plants.Then,PA4increased the NPF7.3 gene to increase root nitrate nitrogen transfer to the above-ground xylem and inhibited the expression of NPF7.2 gene to reduce the above-ground nitrate nitrogen return to the xylem,thereby increasing the above-ground nitrate nitrogen input.Furthermore,the nitrogen nutrition accumulated in the above-ground part during the vegetative period mediates the transfer of old leaves to new leaves through the NPF2.13 gene,and accelerates the recycling of nitrogen.At the same time,increased the expression of vacuolar storage nitrate nitrogen related genes in NPF5 family to increase the efflux of nitrate nitrogen.It inhibited the high expression of BT1 and BT2 genes to improve the efficiency of nitrate utilization under nitrogen-deficient conditions.To be exact,it highly expressed GLN1.3,GLN1.4 and GLN1.3 to increase the synthesis of amino acids,and up-regulate the NPF8.2gene to increase the enrichment of dipeptide compounds and their transport to the moss.Furthermore,nitrogen enriched in the moss was transferred to the seeds through the up-regulation of the NPF2.12 transfer gene,and the up-regulation of NPF2.7 gene mediates the storage of nitrate nitrogen in plant seed vacuoles.It is worth mentioning that the regulation of the nitrogen response,assimilation,metabolism and circulation pathways of PVSE and P4 was accompanied by hormone synthesis and signal transduction pathways.They mediated NPF4.1,NPF4.5 and NPF5.3 to accelerate the accumulation of ABA,and regulated the transfer of hormones such as abscisic acid(ABA),GA1/3/4,and JA-Ile through the NPF5 family.In turn,they promoted the ripening of fruits and improves the nitrogen utilization rate of Arabidopsis thaliana.(3)Mechanism verification experiments showed that PVSE and nitrogen concentration synergistically affect the biological phenotype,endogenous hormone content,nutrient absorption,yield and quality of crops,and nitrogen concentration is the main influencing factor.It improved the activities of NR,NIR,GS,GDH and other nitrogen assimilation related enzymes and increased nitrogen metabolism and photosynthesis.It further increased accumulation of nitrogen and soluble protein and promoted crop growth.Under low temperature,it increased the activity of SOD,POD,CAT and other oxidases,and alleviated cell membrane damage,which stabilized cell membrane structure.Under the nitrogen reduction by 1/3 and the normal nitrogen application level,the combined application of PVSE could improve nitrogen agronomic efficiency(NAE)and nitrogen partial factor productivity(PFPN)of pakchoi.It improved the yield by 10.5%?19.6%,and increased the net income by0.43?9100 RMB/hm~2,so that achieve synergy effect,which verified the mechanism of PVSE regulating the nutrient transfer between crop roots and shoots.At the same time,nitrogen reduction by 1/3 combined with PVSE(2/3 UP)did not form a significant difference in yield and net income compared with conventional nitrogen treatment,and NAE and PFPN were significantly increased by 37.8%and 45.6%,thus,The goal of reducing nitrogen by 1/3without reducing production has been achieved.(4)Under the normal nitrogen application conditions,the fertilization method was the main factor affecting the NUE,NAE and PFPN of maize.The yield,NUE and net income of epoxy resin CRU combined with PVSE increased by 5.7%,1.85 times and 1311.61 RMB/hm~2respectively compared with urea combined with PVSE.The synergistic effect of PVSE and slow-controlled release fertilizers on corn agricultural production was realized and the mechanism of PVSE regulating the transfer of crop nutrients to grains was verified.Using environmentally friendly bio-based polyurethane materials as the carrier to achieve controlled release of PVSE and urea.It made the external nutrient supply meet the fertilizer requirement of sweet potato.At the same time,the sweet potato was induced by PVSE,which increased the activity of activity of enzymes related to nitrogen metabolism,promoted photosynthesis,increased the accumulation of nutrients,and promoted nutrient distribution during the tuber expansion period.So that the mechanism of PVSE regulating nutrient transfer between crown and tuber was verified.Compared with treatment groups including farmers'conventional fertilization,controlled-release fertilizer(CRF1P0)and controlled-release fertilizer that outer membrane coated with PVSE,the yield of sweet potato in controlled-release fertilizer whose inner membrane coated with PVSE(CRF1P1)increased by 29.3%,23.2%,and 7.0%,respectively,and its income increased by 24.7%,15.9%and 7.6%respectively.Compared with CRF1P0,the content of reducing sugar and VC in sweet potato of CRF1P1 increased by10.7%and 19.3%,respectively,and improve yield,economic efficiency and quality of crop.