M. Senthilkumar N. Amaresan A. Sankaranarayanan Plant-Microbe Interactions Laboratory Techniques S P H PRINGER ROTOCOLS ANDBOOKS Forfurther volumes: http://www.springer.com/series/8623 SpringerProtocols Handbooks collects adiverse range ofstep-by-steplaboratorymethods andprotocolsfromacrossthelifeandbiomedicalsciences.Eachprotocolisprovidedinthe Springer Protocol format: readily-reproducible in a step-by-step fashion. Each protocol openswithanintroductoryoverview,alistofthematerialsandreagentsneededtocomplete theexperiment,andisfollowedbyadetailedproceduresupportedbyahelpfulnotessection offeringtipsandtricksofthetradeaswellastroubleshootingadvice.Withafocusonlarge comprehensive protocol collections and an international authorship, Springer Protocols Handbooksareavaluableadditiontothelaboratory. Plant-Microbe Interactions Laboratory Techniques M. Senthilkumar Division of Basic Sciences, Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India N. Amaresan C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Surat, Gujarat, India A. Sankaranarayanan C.G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Surat, Gujarat, India M.Senthilkumar N.Amaresan DivisionofBasicSciences C.G.BhaktaInstituteofBiotechnology IndianInstituteofPulsesResearch UkaTarsadiaUniversity Kanpur,UttarPradesh,India Surat,Gujarat,India A.Sankaranarayanan C.G.BhaktaInstituteofBiotechnology UkaTarsadiaUniversity Surat,Gujarat,India ISSN1949-2448 ISSN1949-2456 (electronic) SpringerProtocolsHandbooks ISBN978-1-0716-1079-4 ISBN978-1-0716-1080-0 (eBook) https://doi.org/10.1007/978-1-0716-1080-0 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2021 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. 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Preface The world is much concerned about meeting the food demand of emerging population in thecontextofvariousbioticandabioticstressesaffectingthegrowthoftheplants,yield,and sustainable agricultural development. Agricultural research to improve and sustain food production especially using beneficial plant–microbe interactions is gaining importance. ImportantresearchprotocolshavebeenpresentedinPlant-MicrobeInteractions:Laboratory Techniquesfor thebenefitofglobalresearchersandstudentcommunity. This manual comprises of 83 protocols given under five major sections: biological nitrogen fixation,mineralsolubilization and mobilization,abiotic stress management,bio- controlofplantpathogens,andbiologicalcontrolofinsectsandnematodes.Ingeneral,this manualcoversvariousprotocolsrelatingtoplant–microbeinteractionswiththepreparation of reagents and important instructions/special note relating to the practicals. We devoted much care to the preparation and compiling of each and every protocol provided in the manual. We strongly believe that the lab manual will meet the practical and research needs of postgraduate students, research scholars, postdoctoral fellows, and scientists as well as teachers from various fields, including pathology, microbiology, entomology, and agron- omy. We thank the Springer Publications for their support and cooperation for bringing outthislabmanualinanicemannerandontime.Wehighlyappreciateyourvaluableinputs andconcretesuggestionsfor theimprovementofthemanual. Kanpur,UttarPradesh,India M.Senthilkumar Surat,Gujarat,India N.Amaresan Surat,Gujarat,India A.Sankaranarayanan v Contents PART I TECHNIQUES FOR STUDYING BIOLOGICAL NITROGEN FIXATION 1 Isolation,Characterization,andPreservationofRhizobiafromRoot NodulesofLegumes .................................................... 3 1 IsolationofRhizobiafromRootNodulesofLegumes ..................... 3 1.1 Materials ............................................................. 3 1.2 Methods ............................................................. 4 1.3 Observations ......................................................... 5 1.4 Precautions........................................................... 5 2 CharacterizationofRhizobia ............................................ 6 2.1 PresumptiveTests..................................................... 6 2.2 ConfirmativePlantInfectionTest...................................... 7 3 PreservationofRhizobia................................................ 7 References ............................................................... 8 2 EnumerationofRhizobia:Most-Probable-Number(MPN)Technique........ 9 1 Method ............................................................... 10 2 Precautions............................................................ 11 3 ResultsandAnalysis .................................................... 16 References ............................................................... 27 3 IsolationandEnumerationofAzospirillumbyMost-Probable-Number (MPN)Method......................................................... 29 1 Principle............................................................... 29 2 Procedure ............................................................. 30 Reference................................................................ 31 4 QuantitativeEstimationofLeghemoglobinContentinLegumeRoot Nodules ............................................................... 33 1 Principle............................................................... 33 2 Materials............................................................... 34 3 Method ............................................................... 34 4 Notes ................................................................. 34 Reference................................................................ 35 5 QuantitativeEstimationofNitrogenaseActivity:AcetyleneReductionAssay... 37 1 Principle............................................................... 37 2 DeterminationofNitrogenaseActivityofFreeLivingBacteria.............. 38 3 AcetyleneReductionAssayofRootNoduleSystem........................ 38 3.1 Procedure........................................................... 38 References ............................................................... 39 vii viii Contents 6 QuantitativeEstimationofNitrogenFixation:15N NaturalAbundanceStudy... 41 2 1 Procedure ............................................................. 42 Reference................................................................ 43 7 EstimationofGlutamineSynthetase(GS),GlutamateSynthase(GOGAT), andGlucoseDehydrogenase(GDH)...................................... 45 1 Method ............................................................... 45 1.1 EnzymeExtraction................................................... 45 2 InVitroGlutamineSynthetase(GS)Assay................................ 46 3 InVitroGlutamateSynthase(GOGAT)Assay............................. 46 4 InVitroGlutamateDehydrogenase(GDH)Assay......................... 46 References ............................................................... 47 8 ColorimetricAnalysisofUreide-N,Amino-N,andNitrate-Nitrogen.......... 49 1 DeterminationofN-FixingActivityandAbsorptionRate .................. 50 2 DeterminationofUreide-N ............................................. 51 3 DeterminationofAmino-NbytheNinhydrinMethod..................... 51 4 DeterminationofNitrate-N............................................. 52 References ............................................................... 52 PART II TECHNIQUES FOR STUDYING MINERAL SOLUBILIZATION AND MOBILIZATION 9 IsolationofMineralPhosphateSolubilizingMicrobesfromRhizosphereSoil... 55 1 Protocol............................................................... 55 2 IsolationofHalophilicP-SolubilizingMicrobesfromMarine............... 56 2.1 Protocol............................................................. 56 Reference................................................................ 57 10 EstimationofPhosphateSolubilizingCapacityofMicroorganisms............ 59 1 Principle............................................................... 59 2 Procedure ............................................................. 59 3 Procedure ............................................................. 59 Reference................................................................ 60 11 Isolation,EnumerationofSpores,andInoculumProductionofArbuscular Mycorrhizal (AM)Fungi................................................. 61 1 Procedure ............................................................. 61 2 MassProductionofAMFungi........................................... 62 3 QuantificationofAMFungiInfectioninPlantRoots...................... 62 Reference................................................................ 63 12 IsolationofZnSolubilizingBacteria ...................................... 65 1 Materials............................................................... 65 2 Methods............................................................... 65 3 Observation............................................................ 66 4 Precautions/Note...................................................... 66 References ............................................................... 66 Contents ix 13 QuantitativeEstimationofZnSolubilizationEfficiencyofMicroorganisms .... 67 1 InVitroAssessmentofZnSolubilizationEfficiency........................ 67 1.1 Method............................................................. 67 2 InVitroLiquidAssayforDeterminationofZnSolubilization .............. 68 2.1 Method............................................................. 68 References ............................................................... 68 14 IsolationofSulfur-OxidizingBacteria ..................................... 69 1 Materials............................................................... 69 2 Methods............................................................... 70 Reference................................................................ 70 15 EnrichmentIsolationofSulfur-OxidizingBacteria.......................... 71 1 Materials............................................................... 71 2 Methods............................................................... 72 Reference................................................................ 72 16 QuantitativeEstimationofSulfateProducedbySulfur-OxidizingBacteria..... 73 1 SulfateDetectionbySpectrophotometry ................................. 73 2 SulfateDetectionbyAtomicAbsorptionSpectrometry(AAS) .............. 74 3 DetectionofSulfateIonProduction ..................................... 74 4 ModifiedMethodofDetectionofSulfateIonProduction.................. 74 5 SulfideOxidaseAssay................................................... 75 References ............................................................... 75 PART III TECHNIQUES FOR STUDYING ABIOTIC STRESS MANAGEMENT 17 EnrichmentIsolationof1-Aminocyclopropane-1-CarboxylicAcid(ACC) DeaminaseProducingBacteria ........................................... 79 1 Principle............................................................... 79 2 Materials............................................................... 79 3 Methodology .......................................................... 80 4 Note.................................................................. 80 Reference................................................................ 80 18 EstimationofACCDeaminaseActivityinBacterialCells .................... 81 1 Principle............................................................... 81 2 Materials............................................................... 81 3 Methodology .......................................................... 82 4 Notes ................................................................. 82 References ............................................................... 83 19 PCR-BasedDetectionofACCDeaminaseProducingBacteria................ 85 1 Principle............................................................... 85 2 Materials............................................................... 85 3 Methodology .......................................................... 86 4 Notes ................................................................. 87 References ............................................................... 87 x Contents 20 ClassicalTripleResponseBioassayforDeterminingStressMitigatingPotential ofACCDeaminaseProducingBacteria.................................... 89 1 Principle............................................................... 89 2 Materials............................................................... 90 3 Methodology .......................................................... 90 4 Notes ................................................................. 90 References ............................................................... 90 21 EstimationofEthyleneinPlant-BioassaySystem:GasChromatography ....... 91 1 Principle............................................................... 91 2 Materials............................................................... 91 3 Methodology .......................................................... 92 3.1 SurfaceSterilizationofSeeds ......................................... 92 3.2 PreparationofHeadspaceGCVialswithSeeds ........................ 92 3.3 MeasurementofEthylenewithaGasChromatograph.................. 93 3.4 CalculationofEthyleneConcentrationfromtheSamples............... 93 4 Notes ................................................................. 93 References ............................................................... 93 22 EstimationofProlineContentinPlantTissues............................. 95 1 EstimationofProlinebyIsatinPaperAssay ............................... 96 1.1 Principle ............................................................ 96 1.2 Materials............................................................ 96 1.3 Methodology........................................................ 96 1.4 Notes............................................................... 96 2 EstimationofProlinebyColorimetricAssay .............................. 97 2.1 Principle ............................................................ 97 2.2 Materials............................................................ 97 2.3 Methodology........................................................ 97 2.4 Note................................................................ 98 References ............................................................... 98 23 DeterminationofGlycineBetainebyPeriodideMethod..................... 99 1 Principle............................................................... 99 2 Materials............................................................... 99 3 Methodology .......................................................... 100 4 Notes ................................................................. 100 References ............................................................... 100 24 EstimationofBetaineAldehydeDehydrogenase(BADH)Activity............ 101 1 Principle............................................................... 101 2 Materials............................................................... 101 3 Methodology .......................................................... 102 4 Notes ................................................................. 102 Reference................................................................ 102 25 EstimationofMalondialdehyde(MDA)byThiobarbituricAcid(TBA)Assay... 103 1 Principle............................................................... 103 2 Materials............................................................... 103