Also available as PDF (figures and tables appear in the PDF).
Soil microbial communities are essential for maintaining ecosystem stability, nutrient cycling, agricultural productivity, and environmental sustainability. Metagenomic analysis has emerged as a powerful approach for investigating the diversity, structure, and functional potential of soil microorganisms under different agricultural systems. The present review evaluates the microbial dynamics associated with regenerative and conventional agriculture using advanced metagenomic tools including 16S rRNA sequencing and shotgun metagenomics. Findings from recent studies indicate that regenerative agricultural practices such as cover cropping, crop rotation, reduced tillage, and organic amendments significantly enhance microbial diversity, beneficial taxa abundance, nutrient cycling efficiency, and soil ecological resilience. In contrast, conventional agriculture characterized by intensive tillage and excessive chemical inputs is associated with reduced microbial diversity, increased pathogen prevalence, and enrichment of antimicrobial resistance genes. These microbial alterations have important implications for soil health, food safety, environmental quality, and public health within the One Health framework. The review highlights the importance of integrating metagenomic approaches into sustainable agricultural management and public health policy development.
Keywords: Metagenomics; Soil Microbial Communities; Regenerative Agriculture; Conventional Agriculture; Soil Health; Microbial Diversity; One Health; Sustainable Agriculture; SoilMicrobialCommunities; RegenerativeAgriculture; ConventionalAgriculture; SoilHealth; MicrobialDiversity; OneHealth; SustainableAgriculture
Soilmicrobialcommunitiesconstituteoneofthemost complex and functionally significant biological systemsonEarth,governingessentialecosystemservices suchasnutrientcycling,organicmatterdecomposition,soilstructureformation,andsuppressionofplant andhumanpathogens(1,2).Withadvancesinmolecularbiology,particularlymetagenomics,ithasbecome possibletocomprehensivelycharacterizethesemicrobialcommunitieswithoutthelimitationsofculturedependentmethods.Metagenomicanalysisenables thedirectextractionandsequencingofgeneticmaterialfromenvironmentalsamples,providinginsights intomicrobialdiversity,taxonomiccomposition,and functionalpotential(3, 4). Thishassignificantlyenhanced our understanding of how agricultural practicesinfluencesoilmicrobiomesandtheirdownstream impacts on environmental and public health. Agriculturalintensificationthroughconventionalfarming practices—characterizedbymonoculturesystems,excessive tillage, and heavy reliance on synthetic fertilizers and pesticides—has led to substantial alterations in soil microbial diversity and ecological balance (5, 6). Metagenomic studies have consistently reportedadeclineinmicrobialrichnessandevenness inconventionallymanagedsoils,alongwithareductioninbeneficialmicrobialtaxainvolvedinnutrient cycling and plant growth promotion (7, 8). Furthermore,thesesystemsoftenshowanincreasedprevalence of pathogenic microorganisms and antimicrobialresistancegenes,raisingseriousconcernsregarding food safety, environmental contamination, and public health risks (9, 10). In contrast, regenerative agriculturehasemergedasasustainablelandmanagementapproachaimedatrestoringsoilhealthand enhancingbiodiversitythroughpracticessuchasreducedtillage, croprotation, covercropping, andorganicamendments.Metagenomicanalysesofregenerdrivenbylandmanagementpractices(13,14). Additionally, functionalmetagenomicsprovidesinsights intometabolicpathways,resistancegenedissemination, and microbial interactions that are critical for understanding ecosystem health and sustainability. The implications of soil microbial dynamics extend beyondagricultureintothedomainofpublichealth. Soilservesasareservoirforbothbeneficialmicrobes andpotentialpathogens,aswellasantimicrobialresistancegenesthatcanbetransferredacrossenvironmental,animal,andhumaninterfaces(15,16).Disruptions insoilmicrobialcommunities,particularlyunderconventionalagriculturalsystems,canfacilitatetheemergenceandspreadofzoonoticdiseases,contamination offoodandwaterresources,andincreasedexposure toharmfulmicrobialagents.Conversely,regenerative practicesthatpromotemicrobialdiversityandecologicalbalancecanhelpmitigatetheserisksandsupport theprinciplesoftheOneHealthapproach,whichintegrates human, animal, and environmental health. Despitegrowingevidencesupportingthebenefitsof regenerativeagriculture,thereremainsaneedforcomprehensivesynthesisofmetagenomicfindingstobetterunderstandtheextentandmechanismsthrough whichdifferentagriculturalpracticesinfluencesoilmicrobiomes.Thisreviewfocusesontheapplicationof metagenomicanalysistoevaluatesoilmicrobialcommunitiesunderregenerativeandconventionalagriculture,highlightingkeydifferencesinmicrobialdiversity, functionalpotential,andpublichealthimplications. Byintegratingfindingsfromrecentstudies,thiswork aimstocontributetothedevelopmentofsustainable agriculturalstrategiesthatpromoteecosystemhealth andsafeguardpublichealth.
Thepresentreviewwasconductedusingacomprehensiveandsystematicevaluationofpublishedscientific literaturerelatedtometagenomicanalysisofsoilmicrobialcommunitiesunderregenerativeandconventional agriculturalsystems.Relevantpeer-reviewedarticles were retrieved from electronic databases including PubMed,Scopus,WebofScience,andGoogleScholar usingkeywordssuchas“soilmetagenomics,”“regenerativeagriculturemicrobiome,”“conventionalfarmingsoilmicrobes,”“16SrRNAsequencing,”“shotgun metagenomics,”and“soilmicrobialdiversity”(17,18). Studiespublishedbetween2005and2025werepreferentiallyincludedtocapturerecentadvancementsin next-generationsequencingtechnologiesandmicrobialecology.Inclusioncriteriacomprisedstudiesinvestigatingsoilmicrobialdiversity,functionalgeneprofiling,microbialabundance,antimicrobialresistance genes, and ecological functions under regenerative andconventionalagriculturalpractices.Studiesbased solelyonculture-dependenttechniqueswereexcluded becauseoftheirinabilitytorepresentthetotalsoilmicrobialdiversity(19,20).
Mostmetagenomicstudiesincludedinthisreviewemployedrandomizedfieldsamplingfromagricultural landsmanagedunderregenerativeandconventional systems. Soilsamplesweregenerallycollectedfrom therhizosphereandbulksoilatdepthsrangingfrom 0–15cmusingsterileaugersandstoredat20°Cor80°C priortoDNAextractiontopreservemicrobialintegrity (21,22).
Regenerativeagriculturalsystemsincludedpractices suchas: • Covercropping • Croprotation • Organicmanureapplication • Reducedorzerotillage • Compostamendments • Agroforestryintegration Conventionalsystemswerecharacterizedby: • Intensivetillage • Monoculturecropping • Syntheticfertilizerapplication • Chemicalpesticideusage • Herbicidedependency Replicatedsamplingdesignswithseasonalandspatial variationswereconsideredtoensurereproducibility andecologicalrepresentation(23,24).
MicrobialDNAwasextractedfromsoilsamplesusing commerciallyavailablesoilDNAisolationkitssuchas: • QiagenDNeasyPowerSoilKit • MoBioPowerSoilDNAIsolationKit • NucleoSpinSoilKit Thesekitseffectivelyremovehumicacidcontaminants thatinterferewithdownstreamsequencingreactions (25). Afterextraction, DNAquantityandpuritywere assessedusingspectrophotometryandfluorometric quantificationmethodsincludingNanoDropandQubit fluorometeranalyses(26).High-qualityDNAsamples weresubjectedtometagenomicsequencingusingnextgenerationsequencing(NGS)platformssuchas:
• IlluminaMiSeq • IlluminaHiSeq • NovaSeq • OxfordNanopore • PacBioSMRTsequencing Twomajorsequencingapproacheswerecommonly employed: 1. 16S rRNA Gene Sequencing Used primarily forbacterialandarchaealtaxonomicprofiling throughamplificationofhypervariableregions suchasV3–V4andV4–V5(27).
2. ShotgunMetagenomicSequencingUsedforcomprehensivefunctionalandtaxonomicanalysisby sequencingtotalmicrobialDNAfromsoilsamples(28).
RawsequencingreadsobtainedfromNGSplatforms underwentqualityassessmentandpreprocessingusingstandardbioinformaticspipelines.
Low-qualityreads,sequencingadapters,andchimeric sequenceswereremovedusingsoftwaresuchas: • FastQC • Trimmomatic • Cutadapt • DADA2(29,30)
For16SrRNAsequencingdata,operationaltaxonomic units (OTUs) or amplicon sequence variants (ASVs) weregeneratedusing: • QIIME2 • Mothur
• RibosomalDatabaseProject(RDP)(32)
Functional gene annotation and pathway analyses wereconductedusingtoolssuchas: • MG-RAST • HUMAnN • MetaPhlAn • MEGAN • Kraken2(33,34) Functionalgenesrelatedto: • Nitrogencycling • Carbonmetabolism • Phosphorussolubilization • Antibioticresistance • Pathogenvirulence
Alphadiversityindicesincluding:
• Shannondiversityindex • Simpsondiversityindex • Chao1richnessestimator were calculated to evaluate microbial richness and evennesswithinsoilsamples(35).BetadiversityanalysesbasedonBray–CurtisdissimilarityandPrincipal CoordinateAnalysis(PCoA)wereemployedtocompare microbialcommunitycompositionacrossagricultural systems(36).
Table 1. ComparativeMicrobialCharacteristicsunder AgriculturalSystems
Microbial High Reduced Diversity Beneficial Increased Decreased Bacteria SoilOrganic Enhanced Declined Carbon Antibiotic Resistance Lower Higher Genes Pathogen Reduced Increased Abundance Nutrient Cycling Improved Impaired Efficiency
Caporasoetal. 2012 DevelopedQIIMEplatformforhigh-throughputmicrobialcommunityanalysis Knightetal. 2018 Advancedmicrobiomeanalyticalstandardsformetagenomics Venteretal. 2016 Regenerativesystemsshowedhighermicrobialrichness Banerjeeetal. 2019 Soilmicrobiomesinfluenceecosystemmultifunctionality Sunetal. 2015 Conventionalfarmingincreasedantibioticresistancegenes Schmidtetal. 2019 Covercroppingimprovedbeneficialmicrobialabundance
Figure 2. GeneralWorkflowofSoilMetagenomic Analysis
↓
↓
↓
↓
↓
↓
↓
↓
↓
Metagenomicanalysishassignificantlytransformed the understanding of soil microbial ecology by enabling comprehensive characterization of microbial communitiesandtheirfunctionalcapacitiesunderdifferent agricultural systems. The findings reviewed in the present study demonstrate substantial differencesinmicrobialdiversity,abundance,andecologicalfunctionsbetweenregenerativeandconventional agriculturalpractices. Regenerativeagriculturalsys-
↓
↓
↓
↓
↓
↓
temsconsistentlyexhibitedgreatermicrobialrichness, highertaxonomicdiversity,andimprovedfunctional geneabundancecomparedtoconventionalsystems (38,39).Theseobservationsindicatethatregenerative managementpracticessupporthealthierandmoreresilientsoilecosystems.Oneofthemajorfindingsfrom metagenomicstudiesistheincreasedabundanceof beneficialmicrobialtaxaunderregenerativeagriculture.SoilmicroorganismssuchasActinobacteria,Proteobacteria,Firmicutes,andarbuscularmycorrhizal fungiplayessentialrolesinnutrientcycling,nitrogen fixation,phosphorussolubilization,andsuppression ofsoil-bornepathogens(40, 41). Regenerativepracticesincludingcovercropping,croprotation,compost application,andreducedtillagecreatefavorableenvironmentalconditionsformicrobialproliferationbyincreasingsoilorganicmatterandreducingphysicaldisturbance(42).Enhancedmicrobialdiversityimproves soilaggregation,waterretention,andnutrientavailability, thereby promoting sustainable crop productionandecologicalstability.Conversely,conventional agriculturalsystemswereassociatedwithsignificant reductions in microbial diversity and functional stability.Intensivetillagedisruptssoilstructureandmicrobialhabitats,whileexcessiveuseofsyntheticfertilizersandpesticidesnegativelyaffectsbeneficialmicrobialpopulations(43,44). Metagenomicanalyses haveshownthatprolongedchemicalexposureleads tothedominanceofstress-tolerantandopportunistic microbialtaxawhilesuppressingecologicallybeneficialorganisms(45). Suchalterationscanimpairnutrient cycling efficiency, reduce soil fertility, and increase susceptibility to plant diseases. Another importantobservationistheincreasedprevalenceofantimicrobialresistancegenes(ARGs)andpathogenicmicroorganismsinconventionallymanagedsoils.Studieshavereportedthatexcessiveapplicationofagrochemicalsandanimalmanurecontainingantibiotic residuescontributestotheenrichmentanddisseminationofARGswithinsoilmicrobialcommunities(46, 47). Soilactsasamajorenvironmentalreservoirfor resistance genes, which may subsequently transfer tohumanandanimalpathogensthroughhorizontal gene transfer mechanisms. This poses serious publichealthconcernsduetotheincreasingglobalburdenofantimicrobialresistance. Metagenomicinvestigationsalsorevealedthatregenerativeagriculture contributestoenhancedecosystemmultifunctionalityandenvironmentalsustainability.Functionalgene profilingdemonstratedhigherabundanceofgenesassociatedwithcarbonsequestration,methaneoxidation,nitrogenmetabolism,andbiodegradationpathways in regenerative systems (48, 49). These microbial functions are critically important for mitigating greenhousegasemissionsandimprovingclimateresilience. Increased microbial-mediated carbon storageunderregenerativeagriculturefurthersupports globaleffortstowardclimatechangemitigationand sustainablelandmanagement.Fromtheperspective ofpublichealthandOneHealth,soilmicrobialdiversityiscloselylinkedwithfoodsafety,environmental quality, anddiseaseprevention. Healthysoilmicrobiomessuppresspathogenicorganismsthroughmicrobialcompetitionandproductionofantimicrobial compounds(50). Reducedmicrobialdiversityunder conventionalagriculturemayfacilitatepathogenemergence,contaminationofagriculturalproduce,andincreasedtransmissionofzoonoticdiseases.Regenerativesystems,bymaintainingecologicalbalanceand microbialresilience,maythereforecontributetoimprovedfoodqualityandreducedhealthrisks.Theapplicationofadvancedmetagenomictoolssuchasshotgunsequencing,metatranscriptomics,andfunctional genomicshasfurtherenhancedunderstandingofmicrobialinteractionsandecosystemprocesses. BioinformaticsplatformsincludingQIIME2,MG-RAST,HU- MAnN,andKraken2haveenabledlarge-scalecomparativeanalysesofmicrobialcommunitiesacrossdiverse agroecosystems(51,52).Thesetechnologiesallowpreciseidentificationofmicrobialbiomarkersassociated withsoilhealthandagriculturalsustainability.Despite significantadvancements,severalchallengesremain insoilmetagenomicresearch.Soilmicrobialcommunitiesarehighlyheterogeneousandinfluencedbyclimaticconditions,soiltype,cropspecies,andmanagementintensity(53). Variationsinsamplingmethods, sequencingdepth,andbioinformaticspipelinesmay alsoaffectreproducibilityandinterpretationofresults. Furthermore, many soil microorganisms remain unculturableandfunctionallyuncharacterized,limiting theunderstandingoftheirecologicalroles.Futureresearchintegratingmulti-omicsapproaches,long-term fieldexperiments,andmachinelearning-basedanalysesmayprovidedeeperinsightsintosoilmicrobiome dynamicsandtheirimplicationsforsustainableagricultureandpublichealth.Overall,thereviewedevidence stronglysupportsthehypothesisthatregenerativeagriculturalpracticesenhancesoilmicrobialdiversity,improveecologicalfunctioning,andreduceenvironmentalandpublichealthriskscomparedtoconventional farmingsystems. Metagenomicanalysisservesasa powerfulscientifictoolformonitoringsoilhealthand guidingevidence-basedagriculturalpoliciesaimedat achievingsustainabilityandOneHealthgoals.
Metagenomicanalysishasemergedasanadvanced andhighlyeffectiveapproachforunderstandingthe structure, diversity, and functional potential of soil microbial communities under different agricultural systems. Thefindingsreviewedinthisstudyclearly demonstratethatregenerativeagriculturepromotes greatermicrobialdiversity,improvedfunctionalgene abundance,enhancednutrientcycling,andincreased ecologicalresiliencecomparedtoconventionalagriculturalpractices.Incontrast,conventionalfarmingsystemscharacterizedbyintensivetillageandexcessive chemicalinputscontributetomicrobialimbalance,reducedsoilfertility,enrichmentofpathogenicmicroorganisms,andincreaseddisseminationofantimicrobial resistancegenes.Theapplicationofnext-generation sequencingtechnologiesandbioinformaticstoolshas providedvaluableinsightsintothecomplexinteractionsbetweensoilmicrobes,agriculturalmanagement, environmentalsustainability,andpublichealth. Regenerativeagriculturalpracticesnotonlyimprovesoil qualityandcropproductivitybutalsosupportclimate changemitigation,ecosystemstability,andfoodsafety withinthebroaderOneHealthframework.Therefore, integratingmetagenomicmonitoringintoagricultural managementandpublichealthpoliciescanplayacriticalroleinpromotingsustainablefoodsystemsand environmentalprotection. Futureresearchfocusing on long-term multi-omics studies and standardized metagenomicmethodologieswillfurtherstrengthen theunderstandingofsoilmicrobiomesandfacilitate thedevelopmentofresilientagriculturalecosystems forglobalhealthsustainability.