Plant-Microbe Interactions: Laboratory Techniques

276 Pages·2021·5.739 MB·English

by  M. Senthilkumar, N. Amaresan, A Sankaranarayanan

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.
Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply,
evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations
andthereforefreeforgeneraluse.
Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto
betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty,
expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen
made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer
Nature.
Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A.
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