ISOLATION OF Rhizobium.pdf

This PowerPoint Presentation intends to explore the isolation of Rhizobium and the contribution of microbes to soil fertility on a large scale.

1. General account about the microbes used as biofertilizer –
Rhizobium–isolation, identification, mass multiplication,
carrier based inoculants, Actinorrhizal symbiosis.

3. A Presentation by
Dr. N. Sannigrahi, Associate Professor,
Department of Botany,
Nistarini College, Purulia,
D.B. Road, Purulia (W.B) India-723101

4. Microbesarethetiny,invisiblebiologicalworldobservedunder
magnifyingdevices-microscope,ultramicroscopelikeSEMetc.
Theyincludesingle-andmulti-celledorganisms,bothprokaryotesand
eukaryotescanbemicroorganisms.Theycanbebacteria,archaea,fungi,or
protists.
Wecanonlyseeupto100micrometers(µm)withourbareeyes,so
organismssmallerthanthissizeareconsideredmicroorganisms(Fig.1).
Microorganismsarefoundineveryecosystemandcanalsobeclosely
associatedwithmanymulticellularorganisms.
Bacteriaaresingle-celledprokaryoticmicroorganismsthatcanlivefreely
orinassociationwithahostandareomnipresentwithinourenvironment.
Whenbacteriaaresaidtobeomnipresent,itmeansthattheyarepresentin
virtuallyeveryhabitatonEarth,fromthesoiltotheoceantowithinour
bodies.Bacteriaeveninhabitseeminglyinhospitableenvironments,suchas
withinthehotsoilinandnearvolcanoesandinradioactivewaste.

5. Archaeaareanothertypeofsingle-celledprokaryoticorganismand,until
relativelyrecently,werebelievedtobeanotherkindofbacteriaknownas
archaebacteria.Thisisduetothemanysimilaritiesthatarchaeaand
bacteriashare.
Fungiareeukaryoticorganismsthatcanbemicroscopicorvisible.Perhaps
thefungiyouaremostfamiliarwitharemushroomssincetheyarevisible
tothenakedeye(and,sometimes,edible)!
Despitethephysicalappearanceofmushrooms,fungiarenotplantsand
are,instead,membersofaseparatebiologicalkingdomuniquefrom
animalsandplants.Forourpurposes,weareonlyinterestedinmicroscopic
fungi,knownasmicrofungi.
Protistsaremostlymicroscopic,single-celledorganisms,includingalgae
species,amoebas,ciliates,slimemolds,andmore.Protistsappeartobea
verydiversegroupoforganisms,andprotisttaxonomyismainlyinastate
offluxduetocontinuallychangingclassificationsanddiscoveries.
Allthemicrobe4splayanumberofbeneficial;&harmfulattributesandthe
functionscanbesummarizedasbelow:

6. Bacteriaplayavitalroleinourenvironmentandwithinourbodies.The
presenceofbacteriaisessentialforallotherlifeonEarth.
Bacteriaarebothdecomposersandproducers-theydecomposedead
organicmatterandwasteintoinorganicmatterrichinnutrients,thus
allowingplantstogrow.Theseplantsthenfeedherbivores,whichinturn
feedcarnivores.
Insideandonanimals,includinghumans,bacteriacanbefoundaspartof
themicrobiome,playingvitalrolessuchasaidingdigestion.Infact,
disruptionofthemicrobiomeisbelievedtoplayaroleindeveloping
specificsyndromesanddiseases.Whilemostbacteriaareharmlessor
beneficialtohumans,somecancauseillness,disease,andevendeath.
Theseareknownaspathogenicbacteria.
Theycanactasthedecomposersandtobeusedasthefertilizersof
biologicalorigin.Someofthebacteriahavethepotentialtoactasbio-
fertilizers.
Someofthefungiplayaverysignificantroleandactsasthefertilizers
withthecloseassociationofthehigherplants-thesymbioticassessmentis
consideredasmycorrhizae.

8. RhizobiumisagenusofbacteriabelongstoRhizobiaceaefamilyacclaimedfor
itsabilitytoinstitutemutualismwithleguminousplantsandultimatelyfix
elementaldinitrogen.Withthefurtheranceofresearch,Rhizobiumhasgained
expeditioustractionbecauseofwhichlegume–Rhizobiumtechnologyhasalso
risentopopularity.Amidsuchadvancement,hydroponicsisbeingappliedto
screensymbioticinteractionsofRhizobiuminordertoobtainmoreinformation
ontheirgeneticandmolecularmechanismsincludingfunctionalityinthehost
plant.Thebacteriaentertherootsofleguminousplantsandcreatenodules,
withinwhichmolecularnitrogenisreducedtoammonia,whichisthenusedby
theplanttosynthesizevitamins,proteins,andothernitrogen-containing
substances.Asaresult,theserootnodulesserveasammonium-ionstoragesites
(Flores-Félixetal.,2013).Rhizobiumincreasesgrowthinnon-leguminous
cropsbychangingtheirrootshapeandgrowthphysiology.Furthermore,
Rhizobiumsprayingboostscropproductivitybyincreasingplantheight,seed
germination,nitrogencontent,andleafchlorophyll(Saraetal.,2013).Rice
seedinoculationwithvariousRhizobiumstrainsatincreasinglevelsofnitrogen
increasesthecontentofstrawby4%–19%andtheyieldofricegrainby8%–
22%(Sammauriaetal.,2020).RhizobiaincludebacteriasuchasRhizobium,
Bradyrhizobium,Azorhizobium,Mesorhizobium,andSinorhizobiumetc.

10. Rhizobiumaresymbioticdiazotrophs,
Agram(-)soilbacteriaendo-symbioticassociationwithlegumes,
Rodshapednon-sporeformingbacteria,
Invadelegumerootthroughroothairs,
FormeffectiveredcoloredLHbintherootnodulesandfixatmospheric
nitrogen,
5importantgenera-Rhizobium-slow-growingrhizobiaformsacid,
Bradyrhizobium-fast–growingalkali,Azorhizobiouminfectsbothstem&
root,Sinorhiozobium,Photorhizobium,
DifferentRhizobiumgrowsondifferenthostslikeR.leguminosorumon
pea,R.phaseoliofBean,R.trifolionClover,R.melilotionalfa-alfa,R.
japonicumonsoyabean,R.lupineonlupinegroups.
Mostofthecases,thebasicnatureoftheinfectionsandthedevelopmental
stagesarealmostidenticalandthemultiplicationinlargestepsarealmost
identical.

12. Name of the
group of host
plant
Name of
RhizobiumSp.
Name of the
host plant
Nitrogen
fixation.kg/ year
Pea Group R. leguminosoriumPea ( Pisum
sativum), Lentil (
Lensculinaris)
62-132
Soybean Group R. Japonica Soybean ( Glycine
max)
57-105
Lupine Group R. lupini Lupinus arcticus70-90
Alfalfa group R. meliloti Melilotus indicus100-150
Beans Group R. phaseoli Phaseolus munga80-110
Clover group R. trifolii Trifolium repens130
Cowpea Group R. species Cicer arietinum,
Phaseolus munga
57-105

14. HealthynoduleswereisolatedfromthesixmontholdD.sissooseedlings
grownundernethouseconditions.
Thenoduleswerewashedintapwatertoremovetheadheringsoilparticles
onitssurface.
Nodulesweredippedin0.1%mercuricchloride(HgCl2)solutionfor30
secondandthenwashedsuccessivelyeighttotentimeswithsterilized
distilledwatertoremovethetracesofHgCl2.
Surfacesterilizednoduleswerecrushedinsterilizeddistilledwaterbyglass
rodtoobtainamilkysuspensionofbacteriods.
ThesuspensionwasstreakedonYEMAmediumandincubatedat28±2°C
for2-5days.
ThegrowthonYEMAmediumwascountedandexpressedascfu/g.
IsolatesobtainedfromnodulesofDalbergiasissoowerepurifiedon
YEMAmediumbystreakplatemethod
AuthenticationCongoredtestAllthepurifiedrhizobialisolateswere
streakedonCRYEMAmediumandwereobservedforabsorptionofCongo
reddye(Vincent,1970).

15. BromothymolbluetestTheYEMAmediumcontainingbro-mothymolblue
wasstreakedwithisolatedstrainsandwasobservedeitherforyellowcolor
duetoproductionofacidsorbluecolorduetoproductionofalkali(Norris,
1965).
Hofer’salkalinetestThistestisbasedonthefactthatRhizobiumisunable
togrowathigherpH11.0onyeastextractmannitolbroth(Hofer,1935).
Ketolactoseagartest:Ketolactoseagarplateswerestreakedwithisolated
microbes.Afterincubationfor4-6daysat28±2°C,theplateswereflooded
withBenedict’ssolution.ThistestisbasedonthefactthatRhizobiumis
unabletoutilizethelactose(BernaertsandDeley,1963).
PlantinfectiontestThedifferentisolatesweretestedfortheirabilityto
nodulateDalbergiasissooplantsgrowninplasticpots.SeedsofDalbergia
sissoowereinoculatedwithRhizobiumisolatesbysoakingseeds.Plants
werecarefullyuprootedafter75daysandobservedfornodulation.

17. ThemultiplicationoftheRhizobiumisveryimportantforthelargescale
inoculationofthemicrobesinthehosttissuetoimprovetheplanthealthby
makingtheavenuesaccessibletothestandingcrops.Onlytheinoculums
ofthisparticularspeciesisnotenough,italsoneedstheaddressingofthe
processingalongwiththepackagingfortransportationfromthelaboratory
tothefields.
ThemassproductionofRhizobiumcomprisesthefollowingstepsforthe
successfulmultiplicationofthemicrobesinthelargescale:
Inoculumspreparation,
Processingofcarriermaterial,
Selectionofidealcarriermaterial,
Preparationofcarriermaterial,
Mixingthecarrier&brothcultureandpacking,
Preparationoftheinoculantspacking.

18. Theinoculumspreparationneedsthepreparationbeforetheprocessandit
needstheindepthknowledgeaboutthemicrobialpureculturetechniques
garnishedwiththefollowingapparatuses-
Autoclaveforsterilization-formakingthemedia&glassgoodsalongwith
desiredmaterialscontaminationfree
Hotairoven-Growthofthemicrobesatdesiredtemperature
LaminarAirFlowchamber-Transferofinoculumsinasepticenvironment
Incubator-Incubationofthemicrobes
RotaryandShaker-Aerationofthegrowthofthemicrobes
pHmeter-TomaintainthedesiredpHofthemicrobes
Refrigerator-Topreservetheinoculantsatlowtemperature
Fermentor-Alargevesselformassproductionofmicrobesincontrolled
condition
Inadditiontothese,themediapreparationalongwiththedesiredgrowth
materialsandcontinuouselectricsupplyisessentialinordertomaintainthe
asepticenvironmentforthesuccessfulexecutionoftheentiremicrobiological
process.

19. INOCULUMS PREPARATION:
Theinoculumspreparationneedstheunderstandingofthefollowing
featuresoftheisolationoftheRhizobiumfromtherootnodules.
MaterialsRequired:RootsoftheLegumeplants,steriledistilledwater,
pipettes,testtubes,YEMAplates,70%ethanol,0.1%Mercuricchloride
solution,asepticconditionsbyusingthedisinfectantsandsterilizing
materialsalongwiththebasicingredientsofthemicrobiologicalattributes.
1.Therootnodulesarecollected,washedwiththerunningtapwaterand
treatedwith0.1%mercuricchlorideor3-5%Hydrogenperoxide,
2.Afterrepeatedrunningwiththetapwater,thenodulesarepassedthrough
70%ethylalcohol,
YEMAplatesarepreparedwiththesterilizedenvironment,
1gmofnodularextractispreparedwith10mlofdistilledwaterandmixed
properly,
Serialdilutionismadeupto10
-8

20. Suspension of 0.1 ml from 10
-8
is taken and poured over the plates
prepared previously and made it, spreads throughout the plate,
In the suitable environmental conditions at 32℃ is incubated for further
analysis.
PREPARATION OF STOCK CULTURE
After incubation, after 4-5 days, the Rhizobium colony will appeared on
the plate and it is identified with congored dye . Most of the Rhizobium
colony arte white in color and took the stain of congored.
The colony took the strain are Rhizobium,
The pH of the stock culture to be kept at 6.8.
For large scale production, production of starter culture is required; the
starter culture is prepared in suitable broth,
In the starter culture, the conical flasks are kept at the suitable temperature
for the same in the large scale production.

21. PRODUCTION OFCARRIERBASEDINOCULUM
Aftertheproductionofsemi-liquidinoculumsfromthefermentor,the
carriermaterialsaretaken,
Thecarriermaterialsmaybepit,lignite,vermiculite,charcoalofthe
farmyardmanure,
Thecarriermaterialsmustbecheap,easilyavailable,lowtoxiccontents
alongwithhighorganiccontentstoaddresstheeconomicfeasibility,
Waterholdingcapacityofthecarriermaterialsmustbemorethan50%,
ThecarriermaterialsmustbetransformedintodustandthepHmustbe
maintainedwithcalciumcarbonatepowder,
Theallsortsofmaterialtobeautoclavedproperlyformakingthe
substancesfreefromthecontamination.
Thecarriermaterialsmusthavesomeadhesivepropertysoastoholdwith
therootsurfacewheretheinoculationtobeexercised

22. PACKING:
Duringthepackaging,thebacterialculturemustbekeptinametallictray
withthesuitablecarrierandwiththehelpofmixtureorusinghandgloves,
themixturemustbemixedproperlybeforestorage,
Thesuitablemeasuretobeadoptedforthepropersterilizationtodothe
procedurefortheoptimumbenefitthattobeexpectedinthisconsequences.
Thepolythenebagsshouldbelowdensitygradewithathicknessof50-75
micron,
Thepackageshouldcontainthefollowinginformation-thenameofthe
manufacturer,nameofthestrain,thecropwhichtorecommendedalong
withthemethodofinoculation,dateofmanufacturer,date,batchnumber,
priceetc,
Fulladdressofthemanufactureralongwithstorage,
Instructionstothefarmersforitsusesandapplication

23. STORGAE OF BIO-FERTILIZER PACKET
Thepacketsshouldbestoredinacoolplacefarfromtheheat&sunlight,
Thepacketsshouldbestoredinroomtemperatureorincoldstorage
conditionsinlotsinplasticcratesorthegunnybags,
Thepopulationoftheinoculantsmustbecheckedatthe15daysinterval
foritsoptimumutilization,
Thereshouldbemorethan10tothepower9inoculantsatthetimeof
packagingand10tothepower7inoculantsatthetimeofdisposal,

24. Theterm"actinorhiza"refersbothtothefilamentousbacteriaFrankia,an
actinomycete,andtotherootlocationofnitrogen-fixingnodules.
Actinorhizalplantsareclassifiedintofoursubclasses,eightfamilies,and
25generacomprisingmorethan220species.Althoughontogenically
relatedtolateralroots,actinorhizalnodulesarecharacterizedby
differentiallyexpressedgenes,supportingtheideaoftheuniquenessofthis
neworgan.Twopathwaysforrootinfectionhavebeendescribedfor
compatibleFrankiainteractions:
roothairinfectionorintercellularpenetration.Molecularphylogeny
groupingsofhostplantscorrelatewithmorphologicandanatomicfeatures
ofactinorhizalnodules.Fourcladesofactinorhizalplantshavebeen
defined,whereasFrankiabacteriaareclassifiedintothreemajor
phylogeneticgroups.Althoughthephylogeniesofthesymbiontsarenot
fullycongruent,acloserelationshipexistsbetweenplantandbacterial
groups.

25. Amodelforactinorhizalspecificityisproposedthatincludesdifferent
levelsordegreesofspecificityofhost-symbiontinteractions,fromfully
compatibletoincompatible.Intermediate,compatible,butdelayedor
limitedinteractionsarealsodiscussed.Actinorhizalplantsundergo
feedbackregulationofsymbiosisinvolvingatleasttwodifferentand
consecutivesignalsthatleadtoamechanismcontrollingrootnodulation.
Thesesignalsmediatetheopeningorclosingofthewindowof
susceptibilityforinfectionandinhibitinfectionandnoduledevelopmentin
thegrowingroot,independentlyofinfectionmechanism.

27. Soilfertility&soilhealthareimportantforcropproductivity.
Thenaturalrestorationofsoilfertilityisthecallofthetimedueto
eutrophicationandsoilqualitydeterioration,
Thedifferentmicrobesingeneralandsomebacteria,BGAandfungiin
particularplayaverysignificantroleinthisregard,
FreelivingbacteriaAzotobacter&clostridiumetcplayimportantroletofix
atmosphericnitrogen,
Differentsymbioticproducerootnodulesandtheactivelyfoxnitrogenby
Rhizobiunleguminosarumproducenoduleinthelegumeplants,
Bymeansofbiochemicalprocess,theatmosphericfreenitrogenarefixedby
thesymbioticmechanisms,
BacterialikeFrankiafixnitrogeninassociationwithangiospermslikeAlnus,
Casuarinaetc
Thehugedemandofbiofertilizersinthecontextoforganicfarmingis
increasingdaytodayandtheproductionofbio-fertlizerscanbedonebythe
processofindustrialprocess,
Theisolationfollowedbylargescaleproduction,packagingandtotransportto
thefieldsareveryimportantinthiscontext.

28. References:
1. Fundamental Botany-Sen & Giri
2. A text of Fungi-Vasistha,
3. A Textbook of Microbiology-R.P. Singh,
4.Textbook of Microbiology-Dubey & Maheswari
5. Soil Microbiology-N.S. Subba Rao
6. Agricultural Microbiology-G. Rangaswami
7. Google for images
8. Different WebPages for information.
Disclaimer:ThisPPThasbeenmadetoenrichfreeonlinestudy
resourceswithoutanypleasureoffinancialinterest.