Effects Of Nitrogen Levels On Nitrogen Metabolism In Ericoid Mycorrhizal Fungi-Blueberry Symbiosis

Blueberries are suitable for growing in acidic soil with sufficient water,and their fruits have rich nutritional components and high economic value.In recent years,mycorrhizal fungi have been widely used in agricultural production.Among them,ericoid mycorrhizal fungi（EMF）can establish a symbiotic relationship with blueberries,promote the absorption and transportation of nitrogen by blueberries,and reduce their dependence on exogenous nitrogen fertilizer.In this study,pot experiments were conducted to investigate the effects of EMF on physiological characteristics,nitrogen absorption and metabolism,soil physical and chemical properties,and soil enzyme activity of blueberries at different nitrogen levels by setting up three nitrogen levels（1.25,2.5,and 5 m M）,and establishing symbiotic（E+）and non symbiotic（E-）experimental systems for EMF and blueberries.It will lay a foundation for further revealing the mechanism of action of EMF at different nitrogen levels on the absorption and utilization of nitrogen by blueberries seedlings.The main results are as follows:（1）The growth characteristics of EMF Oidiodendron maius strain 143 were analyzed.The mycelium growth rate was the highest from the 12th to 14th day of culture.After the 14th day of culture,the nutrients in the culture medium were exhausted,and the mycelium growth entered a decay stage,with the growth rate slowing down and the growth curve gradually tending to flatten out.The phosphorus solubilization ability of strain 143 was measured and it was found that the growth rate of phosphorus solubilization of strain 143 was the largest between 6 and 10 days,with the maximum phosphorus solubilization reaching 27.18 mg/L.Through plate qualitative testing,it was found that strain 143 can produce cellulase,pectinase,protease,amylase,and xylanase.Based on the characteristics of enzyme production,it is shown that it is beneficial for mycorrhizal fungi to destroy plant cell walls and invade plants,as well as for mycorrhizal fungi to decompose nutrients in soil that are difficult for plants to directly absorb.（2）Using blueberries as experimental materials,the effects of EMF inoculation on the root morphology and vitality of blueberries seedlings under different nitrogen levels were studied.It was found that the EMF colonization rate gradually decreased with the increase of nitrogen levels.Under low nitrogen levels,EMF inoculation significantly increased blueberries’root vitality,root length,root surface area,root volume,root average diameter,root tip number,root branching number,and root dry weight（P<0.05）.It has no significant effect on blueberry root area.This indicates that a low nitrogen environment may be more conducive to the colonization of mycorrhizal fungi,allowing more mycorrhizal fungi to invade plant roots,changing root activity and morphology,enhancing the ability of roots to absorb water and nutrients,thereby improving the adaptability of plants to nitrogen deficiency.In addition,the effects of EMF on the physiological characteristics of blueberries were also studied.It was found that inoculation with EMF significantly increased the contents of chlorophyll,soluble sugar,reducing sugar,and soluble protein in blueberries under low nitrogen levels（P<0.05）.In summary,low nitrogen levels are more conducive to the colonization and growth promotion of EMF.The phosphatase activity of blueberry plants was measured and it was found that under low nitrogen and medium nitrogen levels,EMF inoculation significantly promoted the activities of alkaline phosphatase（P<0.05）,it had no significant effect on acid phosphatase activity.Inoculation with EMF at high nitrogen levels had no significant effect on acid phosphatase and alkaline phosphatase.Under relatively deficient nutrient conditions,it is speculated that inoculation with EMF can promote the secretion of phosphatase by blueberry plants to activate phosphorus in the soil,thereby promoting phosphorus absorption.（3）The effect of EMF on nitrogen absorption of blueberry under different nitrogen levels was studied.It was found that inoculation with EMF at low nitrogen levels significantly increased the nitrogen content of blueberry plants（P<0.05）,no significant effect on free NO₃^-content in blueberry roots.EMF could promote plant nitrogen acquisition in nitrogen deficient environments.The activities of enzymes related to nitrogen metabolism were measured under different nitrogen levels.It was found that inoculation with EMF at low nitrogen levels significantly increased the activities of nitrate reductase（NR）,nitrite reductase（Ni R）,glutamine synthetase（GS）,glutamate synthetase（GOGAT）,and glutamine dehydrogenase（GDH）in the roots and leaves of blueberries（P<0.05）.Using fluorescence quantitative PCR technology to analyze the effect of EMF on the expression level of nitrogen metabolism related genes,similar to the results of enzyme activity assay,it was found that inoculation with EMF significantly upregulated the expression level of GS,GOGAT,and NRT genes in blueberry roots and leaves at low nitrogen levels（P<0.05）,no significant effect on the expression level of NR gene in blueberry roots.This indicates that in nitrogen deficient environments,EMF can improve the adaptability of plants to low nitrogen environments by inducing the expression of genes such as nitrogen transporters.（4）A study was conducted on the effects of EMF on soil physicochemical properties under different nitrogen levels.It was found that inoculation with EMF at low nitrogen levels did not significantly affect soil p H,available phosphorus,organic matter,total phosphorus,and total nitrogen content.It was found that inoculation with EMF at low nitrogen levels did not significantly affect the activity of acetylglucosidase（NAG）,but significantly increased soil urease（NM）,leucine aminopeptidase（LAP）,sucrase（ZM）,β-1,4-cellobiosidase（CBH）,β-xylosidase（BX）,β-1,4-glucosidase（BG）,phenol oxidase（PPO）,peroxidase（Per）,and phosphatase（AKP）indirectly increase the availability of soil nutrients.Analyzing the soil enzyme stoichiometry and vector characteristics,it was found that EMF can regulate the soil enzyme stoichiometry.Inoculating EMF at low nitrogen levels can increase enzyme C:N and vector angle,while reducing N:P,it has no significant impact on vector length.As nitrogen levels increase,data points shift from nitrogen limitation to carbon and phosphorus limitation,and inoculation with EMF alleviates nitrogen limitation in blueberries.Overall,EMF can alleviate the nitrogen limitation of plants in low nitrogen environments.It is speculated that a large amount of EMF colonization at low nitrogen levels can produce more extracellular enzymes,promote the decomposition of plant residues and other nutrients in the soil,and thereby release more inorganic nitrogen for plant absorption and utilization.