| Rice(Oryza sativa L.)is the staple food of nearly half of the world’s population and the main source of dietary minerals.Over the past 50 years,the world’s total population has exploded,which has put tremendous pressure on food production.Rice is a typical nitrogen-fond crop.As a large country of food production and consumption,the amount of nitrogen fertilizer applied to rice fields in China is far higher than the international level during the same period.Indica rice and japonica rice are the main cultivated subspecies in China.Among them,japonica has greater demand due to its higher protein content and better taste.However,nitrogen-use efficiency of japonica rice is lower than that of indica varieties in the field and often requires larger nitrogen fertilizer input.How to improve the nitrogen-use efficiency of japonica rice to avoid excessive nitrogen application is an urgent problem in agricultural production.In the natural environment,plants can reciprocally coexist with beneficial microorganisms and jointly resist the unfavorable external environment.Our group previously isolated a broad-spectrum endophytic fungus Phomopsis liquidambaris B3 from the inner bark of Bischofia polycarpa.Ph.liquidambaris could establish symbiotic relationship with many plants,such as rice,peanuts and arabidopsis.It has multiple advantages in enhancing the host’s resistance to pathogens,drought,nutrition deficiency and other stress environments.Previous studies have shown that the Ph.liquidambaris can promote the nitrogen absorption efficiency of "Wuyun Japonica23",a common cultivar in Jiangsu Province,and plays a key role in improving the habitat adaptability of rice.Although we have proposed some possible mechanisms,including promoting the development of root aeration tissue,regulating related plant hormones and enzyme activities,and increasing the abundance and diversity of microorganisms related to nitrogen transformation in the rhizosphere,research on this topic is still incomplete.In this study,we take "Wuyun Japonica 23"-Ph.liquidambaris as the main research system and explore from plant physiology and microecology.In this study,pot experiments showed that rice yield increased by 22.3% after inoculation with Ph.liquidambaris under low nitrogen conditions,and nitrogen loss in pot was reduced by 24.6%.Meanwhile,Ph.liquidambaris promotes the formation of iron film and the enrichment of mineral ions on the root surface,and induces the up-regulation of the expression of rice-related nutrient transporters that promote the host’ absorption and redistribution efficiency of various mineral nutrients.The contents of N,P,Fe,Mn,Zn,Mo and Se in rice grains increased by 20.5%,18.9%,18.2%,9.5%,17.9%,13.3% and 17.1%,respectively,with Ph.liquidambaris colonization;brown rice rate,milled rice rate and protein content were increased by11.5%,15.5% and 17.3% respectively.We also detected the colonization of endophytic fungi in rice vegetative organs and reproductive organs through specific primers and found that the endophytic fungi did not appear in the grain.These results indicate that Ph.liquidambaris can optimize the distribution of essential mineral elements in rice vegetative and reproductive organs by affecting the physiological structure and function of the host root.thereby alleviating the inhibitory effect of nitrogen deficiency on crop yield and nutritional quality.Microorganisms are the key driving force to promote soil element cycling.We detected the effects of Ph.liquidambaris on the structure and interaction of rice rhizosphere microbial community.The results showed that Ph.liquidambaris not only promoted the abundance of genes related to nitrogen fixation and nitrification process,but also inhibited the abundance of genes related to denitrification process.Illumina Mi Seq sequencing showed that the colonization of Ph.liquidambaris increased the abundance of indigenous beneficial microorganisms such as Bacillus,Pseudomonas,Actinobacteria,Acidobacteria,Bradyrhizobium and Nitrospira in rice’ rhizosphere.Species correlation network analysis further revealed that the symbiotic relationship changed the species coexistence relationship of microorganisms in the rice rhizosphere,making the beneficial bacteria in the soil occupy the key node of the microbial community and accelerating the turnover rate of nutrient elements.These results revealed the potential relationship between the rhizosphere microbial community and crop nutrient absorption.Ph.liquidambaris improved the supply of available nitrogen in the rhizosphere by optimizing the structure and function of the community,then indirectly promotes the nitrogen absorption efficiency of plants.We further compared the symbiosis effect of “Wuyun Japonica 23” and“Huiliangyou Indica 996” with Ph.liquidambaris.The results showed that Ph.liquidambaris and rice varieties have a symbiosis preference.The plant height and root length of japonica rice were increased by 23.7% and 19.9%;the content of available nitrogen in rhizosphere and nitrogen use efficiency were increased by 33.7%and 38.5% respectively after inoculation of Ph.liquidambaris,but no significant influence on the growth of indica rice.Biolog-ECO and metagenomic sequencing further analyzed the metabolic capacity and functional gene set of the rhizosphere microbial community.The results showed that the utilization of organic acids,amino acids,and polysaccharides by the microbial community in the rhizosphere of the japonica rice was significantly increased after inoculation of Ph.liquidambaris.The abundance of genes related to the cycle of nutrient elements(such as carbon,nitrogen,phosphorus,sulfur,and iron),biomass synthesis,resistance levels,oxidative stress levels,signal transduction and chemotactic movement have also increased significantly.The prediction results of KEGG metabolic pathway showed that the regulation of rhizosphere nitrogen by endophytic fungi was mainly reflected in the inhibition of nir K and nor B,with the promotion of nif H and amo A.The prediction of Network X showed that the key microbial species related to nitrogen metabolism in the japonica rhizosphere increased from 9 to 14 after inoculation with Ph.liquidambaris,which has more pathways related to nitrogen metabolism and cycling with more complicated function,thereby promoting the bioavailability of soil nitrogen and the absorption of plants.Interestingly,the rhizosphere microbial community of japonica rice optimized by Ph.liquidambaris was almost at the same level as that of indica.These results provide metabolic and genetic evidence to confirm the directional optimization effect of endophytic fungi on the microbial community in the rhizosphere of japonica,which also lays a foundation for synthetic community(Syn Com).Syn Com is an effective means to study the interaction between plant phenotype and microbial community.Based on the plant’s ability to shape the root microbiome,we designed a chemotaxis device to eliminate the unnecessary microorganisms and more than 80% of key functional species were efficiently isolated,and the chemotaxis of microbes toward to the root exudates were showed on the plate.These recruited microbial are used to construct Syn Com,which generally have good abilities of fixing nitrogen,producting siderophore,producing biofilm and using secretions as a carbon source for growth.Ph.liquidambaris-Japonica-enriched Syn Com includes Azotobacter,Brevibacillu,Streptomyces,Xanthobacter,Novosphingobium,Rhizobium,Nitrosospira,Burkholderia and Serratia.Additionally,Syn Com can promote the mineralization of organic nitrogen to inorganic nitrogen and restore the growth of rice under nitrogen-deficient environment(clay and natural soil).It was also found that the growth-promoting function and and the genes abundance of nitrogen transformation of Syn Coms were at the same level as the original flora.These results emphasize the important role of root exudates in the process of microbial assembly.Ph.liquidambaris regulates the microbial community by mediating the root exudates of japonica rice.We obtained the representative synthesis community of rice rhizosphere microbial community at the seedling stage,and verified the key role of functional microorganisms in plant nutrient absorption and growth.This study clarified that the endophytic fungus Ph.liquidambaris relieved the restriction of poor soil on plant growth and nutrient utilization efficiency through promoting the level of nutrient supply in the rhizosphere and improving the ability of plants to absorb nutrients.We confirmed the regulation of endophytic fungi on the structure and function of the rhizosphere microbial community,which further deepened our understanding of the interaction mechanism of endophytic fungi and rice.Ph.liquidambaris strengthens the connection between plants and soil beneficial microorganisms as a medium and helps japonica integrate soil microbial resources at the seedling stage,then recruits beneficial species in rhizosphere,corrects the niche of rhizosphere microorganisms and establishes the nutrient-efficient abovegroundunderground interaction model.To summary,the endophytic fungus Ph.liquidambaris has shown great application potential in the construction of the nitrogen-efficient "plant-root-rhizosphere-indigenous microorganism" rhizobiont of rice.This study not only provides new microbial resources and ideas for managing the field microbiome to promote crop nutrient absorption,but also has important guiding significance for the intensive ecological agriculture system. |
PDF Full Text Request