Soil Bacteria In Organic Farming Systems And Their Effects On Antioxidant Accumulation In Pakchoi

Organic farming improves soil and plant quality,which is one of sustainable production systems.Soil microbes is the main driver of ecological functions and highly associated to soil conditions and plant health.However,most studies were focused on the soil physical and chemical properties,while little is known about the interaction among microbial community composition,soil environment and crop quality.In the light of microbial ecology,studies on the microbial communities in the bulk soil and rhizosphere of agricultural crops could provide a better understanding of the microbial roles in soil environment and crop quality.In this study,high-throughput sequencing was used to investigate the responses of microbial community composition to horticultural soils under different management and cultivation type.Subsequently,we conducted a pakchoi?Brassica campestris ssp.chinensis L.?pot trial amended with chemical and organic fertilization,respectively.We investigated the effects of cultivation time and rhizosphere effect on microbial community compositions in bulk soil and rhizosphere of pakchoi,as well as the relationship between rhizobacteria and plant quality.We then applied a sterile agar plate system to verify the effects of Bacillus spp.inoculation on phenolic compounds accumulation in pakchoi.The main results were as follows:1.Characteristics of soil properties and microbial taxa in organic farming systemsComparative analysis of the soil properties and microbial communities in horticultural soils under different managements and cultivation types,including organic management in open fields?OF?,organic management in plastic tunnel?OP?,conventional management in open fields?CF?and conventional management in plastic tunnel?CP?.Organic farming increased the nutrient-related soil variables compared to conventional farming regardless of cultivation type,especially for the available N and available P,which were increased by 137%and 711%,respectively,in OP compared to CP.Additionally,OP had the highest microbial abundance and diversity among treatments.Linear discriminate analysis showed that OP possessed diverse differential bacteria,which mainly related to the organic material turnover?e.g.,Roseiflexus spp.,Planctomyces spp.and Butyrivibrio spp.?and plant growth promotion?e.g.,Nostoc spp.,Glycomyces spp.and Bacillus spp.?.These bacteria is positively correlated with nutrient variables?e.g.,EC,available N and available P?,which implied their preference of copiotrophic condition.Redundancy analysis showed that soil pH,EC,nutrient levels?e.g.,available N and P?and total Zn content were significant correlated to the microbial community structure.These results indicated that agricultural management affected the soil microbial community composition and structure by changing soil conditions.2.Dynamic changes of soil microbial community composition across pakchoi growth periodWe conducted a pakchoi pot trial with organic open-field soil to investigated the effects of cultivation time?D1,D7 and D45 represent the day after transplantation?and fertilizer treatment?control,chemical and organic fertilization,respectively?on soil chemical properties and microbial community composition.Fertilization had no effect on the microbial abundance and diversity in the bulk soil across plant growth period.However,the microbial community composition was significantly affected by fertilization.Most of the differential genera among the cultivation period were enriched on D1?e.g.,Methylophaga spp.?,which might be associated to drought condition.Organic fertilizer application was clearly introduced a large amount of microbes related to compost fermentation?e.g.,the phylum of Firmicutes and the genera of Alkaliphilus?into soil on D1.However,the relative abundance of these bacteria were decreased significantly in the soil on D7,indicating that the soil possessed resistance to the exogenous microbes.Redundancy analysis showed that total carbon,NH₄⁺-N,pH and available phosphorus were significantly correlated to microbial community structure.In summary,fertilization affected soil microbial community composition was mainly attributed to the changed soil condition rather than introduced exogenous microbes.3.The differential taxa and network analysis between bulk soil and rhizosphere of pakchoiThe difference and correlation of microbial community composition between bulk soil and rhizosphere under different fertilizations were analyzed by pyrosequencing.The rhizosphere effect enriched copiotrophs in the rhizosphere of pakchoi.For example,bacteria related to copiotrophic condition?e.g.,Protebobacteria?was found in higher abundance in rhizosphere when compared to the bulk soil.In contrast,the abundance of oligotrophs?e.g.,Actinobacteria?was lower in the rhizosphere than bulk soil of pakchoi in all treatments.Under genus level,four genera?Opitutus,Agrobacterium,Microbacterium and Oscillochloris?were enriched in the rhizosphere treated with chemical fertilizer while three genera?Flavisolibacter,Dyadobacter and Sinorhizobium?were enriched by organic fertilization.Network analysis showed that Pseudomonas in rhizosphere and Phormidium in the bulk soil with the highest betweenness centrality were 0.15 and 0.13,respectively.These two genera were the keystone genera to maintain the microbial network structure between bulk soil and rhizosphere.Compared to the control and chemical fertilizer treatments,organic fertilization enriched several plant growth-promoting rhizobacteria?e.g.,Devosia spp.,Bacillus spp.?in the rhizosphere,which may further improve the growth and quality of pakchoi.4.Shifts of pakchoi antioxidants and metabolic functions of rhizobacterial community under different fertilizationsThe pakchoi was harvested after 45 days of cultivation in the pot trial.The plant growth parameters and antioxidant levels were recorded and then correlated to the compositions and metabolic functions of rhizobacterial community.The phenolic content,phenolic acid and antioxidant activities of pakchoi treated with organic fertilizer were significantly higher than other treatments.Chemical fertilizer significantly reduced phenolic content,flavonoids and antioxidant activities by 23%,36%and 24%,respectively,when compared to the control.Correlation analysis between rhizobacteria and plant performance showed that a total of 13 genera were positively correlated to crop yield or quality,which may be potential beneficial rhizobacteria.Among them,Bacillus spp.and Planctomyces spp.were positively correlated to pakchoi biomass and phenolic acid.Predictive functional profiles of the rhizobacterial community involved in amino acid metabolism and lipid metabolism were significantly increased under organic fertilization,which may reflect the enrichment of organic nitrogen source in the rhizosphere.Both of these functions were positively correlated with plant antioxidant activity.In addition,the dry weight of pakchoi was positively correlated with the functions of rhizobacterial community related to nutrient turnover and metabolism?e.g.,energy metabolism,nucleic acid metabolism,enzyme family?,which indicated that the improvement of rhizosphere nutrient cycling may contribute to the accumulation of dry matter of pakchoi.Fertilization may affect crop yield and quality by alerting the composition and functions of rhizobacterial community of pakchoi.5.Antioxidant accumulation of pakchoi regulated by Bacillus spp.inoculationThe effects of Bacillus spp.?Bacillus subtilis,Bacillus amyloliquefaciens and Bacillus mucilaginosus Krassilnikov?on the antioxidant level of pakchoi were evaluated under sterile condition.Our results showed that pakchoi root inoculation with Bacillus subtilis reduced the fresh weight of shoot and root by 55%and 54%,respectively,when compared to control.However,Bacillus subtilis inoculation increased the phenolic content,flavonoids and antioxidant activity by+112%,+165%and+85%,respectively,when compared to control.The results generated from metabolomics and qPCR showed that phenolic metabolites?e.g.,kaempferol3-O-sophoroside-7-O-glucoside?and their gene expression levels?PAL,CHS and CHI?significantly increased in the shoot of pakchoi inoculated by Bacillus subtilis.Similarly,Bacillus amyloliquefaciens promoted phenolic content and flavonoids while Bacillus mucilaginosus Krassilnikov improved antioxidant activity in pakchoi when compared to control.These findings showed that the selected Bacillus spp.had positive effects on the accumulation of antioxidants in pakchoi.Overall,this study explored the differential taxa in the respective cultivation system in Shanghai through pyrosequencing.This could provide useful information to the preference of soil microbes for specific soil condition.Meanwhile,we proposed that organic fertilization might improve antioxidants accumulation of pakchoi by alerting the compositions and functions of rhizobacterial community.The rhizobacteria,Bacillus spp.,may play important roles in improving the quality of pakchoi.We then confirmed that pakchoi inoculated with Bacillus subtilis could upreculate the genes related to phenolic compounds biosynthesis and thus improved the accumulation of antioxidants in pakchoi.Our study identified the characteristics of horticultural soils under different cultivation systems and explored the relationship between rhizobacteria community and plant performance,and then confirmed the regulation of Bacillus spp.on pakchoi antioxidant levels.We provided a better understanding of the relationship between pakchoi nutrient physiology and rhizobacterial ecology in the respect of microbe,and provide fundamental information for the improved crop quality by changed rhizobacterial community composition in organic farming system.