Study On Ecotoxicological Effect Mechanism Of Florfenicol On Wheat-Soil-Microbial System

Antibiotics are widely used in the treatment of bacterial infections in humans and animals because of their high biological activity and wide bactericidal effect.However,because of incomplete metabolism,antibiotics enter the environment via excretion,which poses a threat to the environment.Despite the global attention to the ecological impact of antibiotics,studies of florfenicol toxicity are still limited.The objective of this study was to determine the toxic effects of florfenicol on wheat（Triticum aestivum L.）by combining proteomics,metabolomics,microbiomics,and macrogenomics with respect to basic physiological properties,photosystem,oxidative stress system,root secretion,microbial community structure between roots,and resistance genes in wheat.The aim of this study is to provide theoretical support for the rational application of florfenicol antibiotics and its ecological risk assessment by analysing the effects of florfenicol antibiotics on the physiological and ecological traits of wheat seedlings and the mechanism of ecotoxicological effect of root-to-root microorganisms.The principal components are:（1）Florfenicol stress significantly inhibited the growth of wheat seedlings,and led to the bleaching of wheat leaves.As days of cultivation increased,the stem length of wheat seedlings in the low florfenicol treatment regained growth and the whitened leaves gradually regained green.Florfenicol interfered with the homeostasis of reactive oxygen species（ROS）in wheat seedlings.ROS marker hydrogen peroxide（H₂O₂）and the oxidative stress marker malondialdehyde（MDA）responded to florfenicol stress.There was an overall trend towards increased cell membrane permeability,with different levels of damage in new and old leaves.Florfenicol increased the activity of superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）in wheat seedlings,building a defence against ROS and maintaining normal cellular physiological activity.IBR values of six biomarkers（H₂O₂,MDA,cell membrane permeability,SOD,POD and CAT）of oxidative stress system showed that the level of oxidative stress in wheat exposed to florfenicol increased with increasing antibiotic concentration.（2）The content of photosynthetic pigments（chlorophyll a（Chla）,chlorophyll b（Chlb）and carotenoids（Car））and the precursors for chlorophyll synthesis in wheat leaves was reduced.After 21 d of florfenicol treatment,photosynthetic pigment content was significantly lower in older leaves than that of 14 d.The chlorophyll precursors protoporphyrin IX（Proto IX）,magnesium protoporphyrin（Mg-proto IX）and protochlorophyllide（Pchlide）in wheat leaves all decreased with increasing concentration.The respiratory oxygen consumption rate of wheat leaves decreased gradually and the photosynthetic oxygen release rate increased and then decreased,with an imbalance in intracellular homeostasis caused by florfenicol.After 21 d of florfenicol treatment,the photosynthetic rate of older leaves showed an overall decrease,while the respiratory rate of newer leaves was less inhibited due to reduced toxicity or their own protective mechanisms.Cellular chloroplasts were swollen and deformed,with blurred membrane surfaces,irregularly arranged basal grains and damaged vesicle-like lamellar structures.The number of stromal spheres was significantly increased and tightly packed,and starch granules were increased and in a degraded state.The mitochondrial surface was blurred,the cristae were lost and cavities appeared.Florfenicol significantly inhibited chlorophyll fluorescence parameters（F0,Fm,Fv/Fm）and fast light response curves（Y（II）,ETR,Y（NPQ）and Y（NO））of wheat leaves.After 21 d of florfenicol treatment,damage to older leaves was consistent with changes from the 14 d treatment.While new leaves showed significant changes,the area of severely damaged leaf tips decreased in all treatment except for the highest concentration,and the treatment with low concentration showed no significant difference from the control,showing a recovery trend.（3）The protein expression of wheat seedlings treated with 5 mg kg^-1 florfenicol was significantly changed.The toxic effects of florfenicol activated the response（GO:0009636）and detoxification（GO:0098754）processes to toxic substances and oxidative stress in wheat,and reduced florfenicol-induced oxidative damage by building antioxidant defence.In addition,florfenicol significantly down-regulated photosynthesis（GO:0015979）and precursor metabolite and energy production（GO:0006091）pathways,cystoid membrane（GO:0042651）,photosynthetic membrane（GO:0034357）,cystoid（GO:0009579）,chloroplast（GO:0009507）,chlorophyll binding（GO.0016168）,and ATPase activity（GO:00016887）were significantly enriched in down-regulated differentially expressed proteins（down-DEPs）,indicating that florfenicol adversely affected wheat photosynthesis.PPI network analysis revealed significant increases in the expression of ribosomal proteins,heat shock protein 90（HSP90）,and TCA cycle-associated proteins.In the photosynthetic module enriched by down-DEPs,significant reductions in Psb A and Psb D expression revealed an imbalance in the electron transport chain of the photosynthetic system.At the same time,florfenicol significantly reduced the expression of light-trapping chlorophyll a-b binding protein（LHCB）,which is highly correlated with the chlorophyll reduction.High concentrations of florfenicol caused significant changes in the type and content of metabolites in wheat leaves,resulting in changes in metabolic pathways.Combining the results of the proteomic and metabolomic analyses,the TCA cycle was the common pathway significantly up-regulated and the photosynthetic pathway significantly down-regulated in the proteomics.Substrate and enzyme activities of TCA cycle processes were significantly elevated in wheat seedlings after florfenicol exposure,suggesting that wheat adjusted components of the TCA cycle in response to florfenicol stress.In the photosynthetic system,PSI,PSII,the cytochrome b6/f complex,f-type ATPase and photosynthetic electron transport-related proteins were significantly down-regulated.（4）Wheat rhizosphere soil fluorescein diacetate（FDA）hydrolase showed an overall upward and then downward trend with increasing florfenicol concentration.An up-regulation of FDA hydrolase activity at low concentrations(0.05-1 mg kg^-1)was observed in both time periods.Soil dehydrogenase（DHA）activity increased with increasing florfenicol concentrations.Urease（Urease）activity was slightly promoted by florfenicol at low concentrations,but was negatively affected by florfenicol at high concentrations.Increasing concentrations of florfenicol significantly reduced soil sucrase（Sucrase）activity.Florfenicol stress of 14 d and 21 d significantly altered wheat root secretion content and affected changes in root secretion pathways.A total of 11 metabolic pathways were significantly affected by the treatment of 14 d cultivation in wheat root secretion,including:1)phosphatidylinositol metabolism;2)arginine biosynthesis;3)glyoxylate and dicarboxylate metabolism;and 4)propionate metabolism.4)metabolism of propionate,etc.,whereas wheat root secretions had only 2 significantly affected metabolic pathways after 21d cultivation,namely biosynthesis of secondary metabolites and metabolism of glycerolipids.（5）PCoA analysis of rhizosphere microorganisms showed similar species structure for Control1 and Control2,and that florfenicol application significantly affected the species composition structure of wheat rhizosphere soil microorganisms,resulting in a significant reduction in wheat rhizosphere microbial community abundance and community diversity.Florfenicol exposure significantly altered the phylum-level and genus-level structure of the inter-rhizosphere microbial community,and the microbial community may experience antibiotic shock,death of less competitive strains or segregation of strains to different spatial ecological niches,leading to changes in microbial community abundance and shifts in dominant strains.Florfenicol induces inter-rhizosphere microbes to modulate their genetic and metabolic processes to functionally adapt to different habitats.Analysis of changes in the abundance of inter-root microbial resistance genes（ARGs）resulted in the detection of 36 antibiotic ARGs including peptides,rifamycins,fluoroquinolones,tetracyclines,macrolides and chloramphenicol.identification of florfenicol ARGs revealed that cat B2,Paer＿cat B7 and cat B9 ARGs derived from chloramphenicol acetyltransferase The abundance values of cat B2,Paer＿cat B7 and cat B9 ARGs from chloramphenicol acetyltransferase as well as chloramphenicol phosphotransferase cmlv ARGs were not generally upregulated as expected,but instead decreased significantly,which may be related to bacterial resistance mechanisms.The significant up-regulation of the relative abundance of chloramphenicol-like ARGs cml A4,cmx,flo R and pp-flo in the form of effluxed antibiotics was hypothesized to induce bacterial resistance in the form of effluxed antibiotics by florfenicol.