Prevention of Bacteriophage Adsorption on Bacillus megaterium Cells Via Two Mechanisms

Abstract

Background and Objective: Bacillus megaterium is commonly used as a phosphate dissolving bio-fertilizer. Presence of bacteriophages in the soil is likely to be the significant environmental agents effecting the protection and activities of such useful bacteria. Receptors play a definitive role in the development of bacterial resistance to bacteriophage infection, the adsorption resistance divided to three groups: Blocking of phage receptors, production of capsule layers and presence of competitive inhibitors. In this study efforts were made to protect B. megaterium against its phage infection. B. megaterium was immobilized system with different concentrations of alginate. Moreover, blocked phage adsorption receptors B. megaterium mutants resistant to phage infection were induced via exposure of B. megaterium to U.V. irradiation at wave length of 240 nm for 20 min. Methodology: Different concentration of sodium alginate (3, 5, 7 and 9 % w/v) and U.V. irradiation of 240 nm at distance of 60 cm from plates for 5, 10, 15….. up to 30 min were used for prepared two different formula of B. megaterium (wild type) bacteria as a resistant to phage infection. Results: Bacillus megaterium isolate efficient in dissolving phosphate and the isolate was non-lysogenic and susceptible to an isolate of lytic bacteriophage. Presence of phage had no effect on immobilized cells of B. megaterium with 7% alginate immobilized and blocked phage adsorption receptors B. megaterium mutant which obtained after 20 min. The blocked phage adsorption receptors B. megaterium was more efficient in decreasing the pH value in its liquid culture than the other mutant after 25 min, therefore, this mutant was selected to be as a bio-fertilizer inoculum. Under green house conditions fertilization of wheat plants inoculated with immobilized cells (7%) and blocked bacteriophage adsorption receptors Bacillus megaterium mutant resistant to phage infection, no significant effect for presence of phages was discover, as compared to those fertilized with the free cells in presence of bacteriophages. Conclusion: The results of this study showed that two mechanisms (Immobilized system and blocked phage adsorption receptors system) can be used to protect B. megaterium from phage attacking.

Full text

OPEN ACCESS Journal of Biological Sciences
ISSN 1727-3048
DOI: 10.3923/jbs.2018.338.345
Research Article
Prevention of Bacteriophage Adsorption on Bacillus megaterium
Cells Via Two Mechanisms
1Eman Mokhtar Marei, 2Tarek Hassan Elsharouny and 3Adel Mahmoud Hammad
1Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt
2Department of Agricultural Microbiology, Faculty of Agriculture, Sohag University, Sohag, Egypt
3Departmnt of Agricultural Microbiology, Faculty of Agriculture, Minia University, Minia, Egypt
Abstract
BackgroundandObjective:
Bacillusmegaterium
iscommonlyusedasaphosphatedissolvingbio-fertilizer.Presenceofbacteriophages
inthesoilislikelytobethesignificantenvironmentalagentseffectingtheprotectionandactivitiesofsuchusefulbacteria.Receptorsplay
adefinitiveroleinthedevelopmentofbacterialresistancetobacteriophageinfection,theadsorptionresistancedividedtothreegroups:
Blockingofphagereceptors,productionofcapsulelayersandpresenceofcompetitiveinhibitors.Inthisstudyeffortsweremadeto
protect
B.megaterium
againstitsphageinfection.
B.megaterium
wasimmobilizedsystemwithdifferentconcentrationsofalginate.
Moreover,blockedphageadsorptionreceptors
B.megaterium
mutantsresistanttophageinfectionwereinducedviaexposureof
B.megaterium
toU.V.irradiationatwavelengthof240nmfor20min.Methodology:Differentconcentrationofsodiumalginate
(3,5,7and9%w/v)andU.V.irradiationof240nmatdistanceof60cmfromplatesfor5,10,15.....upto30minwereusedforprepared
twodifferentformulaof
B.megaterium
(wildtype)bacteriaasaresistanttophageinfection
.
Results:
Bacillusmegaterium
isolate
efficientindissolvingphosphateandtheisolatewasnon-lysogenicandsusceptibletoanisolateoflyticbacteriophage.Presenceofphage
hadnoeffectonimmobilizedcellsof
B.megaterium
with7%alginateimmobilizedandblockedphageadsorptionreceptors
B.megaterium
mutantwhichobtainedafter20min.Theblockedphageadsorptionreceptors
B.megaterium
wasmoreefficientin
decreasingthepHvalueinitsliquidculturethantheothermutantafter25min,therefore,thismutantwasselectedtobeasabio-fertilizer
inoculum.Undergreenhouseconditionsfertilizationofwheatplantsinoculatedwithimmobilizedcells(7%)andblockedbacteriophage
adsorptionreceptors
Bacillusmegaterium
mutantresistanttophageinfection,nosignificanteffectforpresenceofphageswasdiscover,
ascomparedtothosefertilizedwiththefreecellsinpresenceofbacteriophages.Conclusion:Theresultsofthisstudyshowedthattwo
mechanisms(Immobilizedsystemandblockedphageadsorptionreceptorssystem)canbeusedtoprotect
B.megaterium
fromphage
attacking.
Keywords: 
Bacillusmegaterium
,bacteriophage,immobilization,mutant,blockedadsorptionreceptors
Received: April18,2018 Accepted: July24,2018 Published: September15,2018
Citation:  Eman Mokhtar Marei, Tarek Hassan Elsharouny and Adel Mahmoud Hammad, 2018. Prevention of bacteriophage adsorption on
Bacillus
megaterium
cellsviatwomechanisms.J.Biol.Sci.,18:338-345.
CorrespondingAuthor: EmanMokhtarMarei,DepartmentofAgriculturalMicrobiology,FacultyofAgriculture,AinShamsUniversity,Cairo,Egypt
Tel:00201003582480Fax:0020244444460
Copyright: ©2018EmanMokhtarMarei
etal
.ThisisanopenaccessarticledistributedunderthetermsofthecreativecommonsattributionLicense,
whichpermitsunrestricteduse,distributionandreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
CompetingInterest: Theauthorshavedeclaredthatnocompetinginterestexists.
DataAvailability: Allrelevantdataarewithinthepaperanditssupportinginformationfiles.

J.Biol.Sci.,18(7):338-345,2018
INTRODUCTION
Applicationofgrowth-promotingrhizobacteria(PGPR)
asan alternative tochemical fertilizers tosoilis commonly
used in ecological and sustainable agriculture1.
Bacillus
megaterium
is one of the plant growth-promoting
rhizobacteria (PGPB) which can stimulate plant growth in
association with the root systems of plants and it is
widespreadinsoil,playsansignificantroleinprovisioningthe
increasing plants with a soluble forms of phosphorus by
producing organic acids and CO2 which decrease the soil
alkalinityandtransformtheinsolublephosphorusformsinto
solubleones2.
Zayed3 andHammad4reportedthat bacteriophages of
Bacillusmegaterium
hadabadeffectontheactivityofthis
bacteriumindissolvingphosphate.
Bacteriophageadsorptionusuallydependsonelementary
contact, compatibility and specialization between the
receptorsandacceptorofthebacteriophageandthebacterial
hostandthedifferentbindingtosucceedtheattachmentand
adsorbentsteps5-7.Chemicalstructureofthebacterialcellwall
generally and cell wall G+ bacteria are a significant role in
succeed infection. This explain the significant role these
structuresmayplayintheadsorptionofbacteriophagetoG+
bacteria8-10.
Aphage loses its infectivityto the bacterial hostifthe
acceptorsbecomeblockedornoncomplementaryandnon
specialization.So,theblockedofbacterialacceptorsorlossof
bacteriophage receptors this can use to adsorption
resistance11-14.
Hammad15andFathy16foundthatimmobilizedcellswere
providedhighdefenseto
Bacillusmegaterium
versus their
bacteriophages and increased their activity in soluble
phosphate in soil. Mutations affecting bacteriophage
receptors represent the most recurrent reason of phage
resistance11,17.
Thisstudy was investigated to conduct as aneffort to
protect such required bacterium against bacteriophage
infectionviaimmobilizationofthebacterialcellsinalginate
beads and blocked receptors of
B. megaterium
 via U.V.
irradiationresistanttophageinfection
.
MATERIALSANDMETHODS
Sourceof
Bacillusmegaterium
isolate:
Bacillusmegaterium

isolateefficientindissolvingphosphatewasobtainedfrom
themicrobialcollectionofAgric.MicrobiologyDept.,Fac.of
Agric.MiniaUniversity.
Detectionofprophagein
Bacillusmegaterium
isolate:For
detectedtothepresenceofprophageinthebacterialisolate,
liquidculture of
B.megaterium
wasU.V.irradiatedatwave
length 240 nm in dark as described by Prinsloo18 and
temperatephagewasdetectedbyspottest.
Source of lytic
 Bacillus megaterium
 phage: Lytic phage
isolatewaskindlysuppliedbyDept.ofMicrobiology,Fac.of
Agric.,MiniaUniv.,Minia,Egypt
.
Propagationoflyticphage suspension: The broth culture
propagation  technique  was  used  as  described  by
Elmaghraby
etal
.19toprepareahightiterphagesuspension.
The titer of
B. megaterium
phage after propagation was
estimatedasdescribedbyEl-Balkhi
etal
.20.
Preparation
Bacillus  megaterium
  inocula resistant to
phage infection: Two techniques were used as a try to
conserve the used bacteria (
B. megaterium
) against
bacteriophagesinfection.Thesetechniqueswereasfollow:
CAlginate-immobilized
B.megaterium
cells:Different
concentrationof sodium alginate(3, 5,7 and 9%w/v)
wereusedforpreparedtheimmobilizedcellsof
B.megaterium
asawildtypebacteria.Theimmobilized
methodforobtainingthebeadsof
B.megaterium
was
describedbyEl-Balkhi
etal
.20
CBlocked phage adsorption receptors
B. megaterium
resistantphagetoinfection:
Bacillus megaterium
(wildtype) was grown inpetri plates for24h at 30EC.
Plates were kept opened and exposed to U.V. light at
wavelengthof240nmatdistanceof60cmfromplates
for5,10,15.....upto30min.Aftereach5min,aplatewas
takenandtheirradiatedbacteriawereharvestedin5mL
ofnutrientbroth.Theharvestedbacteriawasaddedto
50mLnutrientbrothinaflaskandincubatedat30EC
for24h.Theabilityofirradiatedbacteriatoproduce
acids and dissolve phosphate also, their ability to
bacteriophageinfectionwereestimated.Liquidcultures
oftheirradiated bacteria were preparedtobeusedas
inocula.Flaskscontaining200mLofnutrientbrothwere
inoculatedwiththeirradiatedbacteriaandincubatedat
30ECfor48h
CFreecellsinoculum:
Bacillusmegaterium
isolate(wild
type)wasinoculatedin200mLofnutrientbrothmedium
andincubatedat30ECfor48 htobeusedasfreecells
inoculum20
339

J.Biol.Sci.,18(7):338-345,2018
Efficiencyofblockedphageadsorptionreceptors
B.megaterium
in acids prod uctio n:Flaskseachcontaining
200 mL of nutrient broth were prepared. Flasks were
inoculatedseparatelywith5mLofeachirradiated
B.megaterium
liquidcultureandincubatedat30ECfor
9days.ThepHwasmeasuredeverytwodays.Un-irradiated
bacteriawereinvolvedasacontrol.
Susceptibilityofblockedadsorptionreceptors
B.megaterium
tophageinfection:Eachirradiated
B.megaterium
wasusedasindicatorbacteriainagar
doublelayerplatscontainingthelyticbacteriophage.Plates
were  incubated  at 30ECfor24handinspectedfor
formationofsingleplaques.Aplate

containingun-irradiated
B. megaterium
 (wild type) as indicator bacteria was also
involved(control).
Evaluation of the prepared inocula: This experiment was
designedtoevaluatethesusceptibilityofthedifferentforms
of
B.megaterium
  prepared as inocula to phage infection.
Also,studiedtheefficienciesoftheirdifferenttreatmentsand
forms of
B. megaterium
  in dissolving phosphate under
cultivatedsoilconditions(
invivo
).
Invivo
study
Propertiesofsoilused:Aclayloamsoilwasobtainedfrom
thesurface15cmlayeroftheexperimentalfarmofFacultyof
Agriculture,AinShamsUniversity,Shoubra,Cairo,Egypt.The
Physic-chemical properties of the soil used is presented in
Table1.Thesoilwasautoclavedat121ECfor60min.Thissoil
wasusedforcultivationofwheatplants.Themechanicaland
chemical analysis of the used soil were carried out at the
centrallab,Fac.ofagric.,MiniaUniversity,Minia,Egypt.
Greenhouseexperiment:Undergreenhouseconditiona
potexperimentwasstudiedtotheeffectofpresenceofphage
ontheefficiencyof
B.megaterium
(phosphatedissolving
bacteria)asabio-fertilizerforwheatplantsfromyear2016to
2018. Wheat grains (Gemaza 11) were kindly supplied by
Agriculture Research Center (ARC) Cairo, Egypt. Sixty five
sterilizedplasticpots(30cmindiameter)werefilledwith
1kgsterilizedsoil/pot.Sevenwheatgrainswerecultivated
ineachpotandirrigatedwithtapwater.Whenplantswere
15daysold,thepotsweredividedintothirteengroups,each
groupcomprised5potsasreplicates.Thepreparedgroupsof
potsweresubjectedtothefollowingtreatments:
CFertilizationwithfreecells

of
B.megaterium
(wildtype)
CInoculationwith thefree cells

of
B. megaterium
 (wild
type)

pluslyticphage
CFertilizationwith3%

alginateimmobilizedcellsof
B.megaterium
CInoculationwith3%

alginateimmobilizedcellsof
B.megaterium
pluslyticphage
CFertilizationwith5%

alginateimmobilizedcellsof
B.megaterium
CInoculationwith5%

alginateimmobilizedcellsof
B.megaterium
pluslyticphage
CFertilizationwith7%

alginateimmobilizedcellsof
B.megaterium
CInoculationwith7%

alginateimmobilizedcellsof
B.megaterium
pluslyticphage
CFertilizationwith9%

alginateimmobilizedcellsof
B.megaterium
CInoculationwith9%

alginateimmobilizedcellsof
B.megaterium
pluslyticphage
CFertilizationwithblockedadsorptionreceptors
B.megaterium
CInoculationwithblockedadsorptionreceptors
B.megaterium
pluslyticphage
CUn-inoculatedplantswerealsoinvolvedasacontrol
Intreatmentsinoculated with freecells(wildtype)and
blockedadsorptionreceptors
B.megaterium
cells,fivemlof
theintendedbrothcultureswereaddedtoeachpot.Incase
offertilizationwiththeimmobilizedcells,acalculatedweight
valueofbeadscontainingthesamenumberofbacterialcells
(inthe5mLofliquidculture)wasaddedtoeachpot.
In treatments which received phages, 5 mL of
bacteriophagesuspensionwereaddedtoeachpot.Potsofall
groupswereirrigatedwithtapwatertwiceweekly.
Numberof
B.megaterium
wasestimatedinrhizosphere
soilofwheatplantsineachgroupatintervalsof15daysupto
75daysdescribedbyHammad21.
Plantheight(cm/plant),wasdeterminedatintervalsof
15daysupto75days.While,freshanddryweight(g/plant)as
wellasP%inplantsweredeterminedwhenplantswere
75 days old. The plant P% analysis was carried out at the
CentralLab,Fac.ofagric.,MiniaUniversity,Minia,Egypt.
Table1:Physic-chemicalpropertiesofsoilused
Coarsesand(%) Finesand(%) Silt(%) Clay(%) Texturegrade TotalN(%) CaCO3(%) Organicmatter(%) TotalP(ppm) Ec(dsmG1)pH
2.9 25.71 30.61 39.78 Clayloam 0.33 2.57 6.79 14.31 2.47 8.06
340

J.Biol.Sci.,18(7):338-345,2018
Statisticalanalysis:DatawerestatisticallyanalyzedbyH.S.D
methodswasusedforevaluationofthesignificanceofmean
differences.TheH.S.Dwere usedforcalculationoftheleast
sig n i ficantdifferencesbetwe e n t h emaccordin g t o Gomezand
Gomez22.
RESULTS
Presence/absence of prophage in
Bacillus megaterium
:
Bacillusmegaterium
wastreatedwithU.V.radiationatwave
length 240 nm to find out if the tested bacteria contain a
prophageornot.Theresultsofthespottestshowedthatthe
tested
B.megaterium
areprophagefree.i.e.,
B.megaterium
isnotlysogenicbacterium.
Titerofthepreparedphagesuspension:Fordeterminedthe
titerofthepreparedphagesuspensionwasassayedbyplaque
assay technique and found to be 8.5×1010 pfu mLG1. The
phageformedcircularsingleplaquesclearinappearanceand
of2mmindiameter(Fig.1).
Susceptibilityofblockedadsorptionreceptors
B.megaterium
tothelyticphage:Thesusceptibilityofthe
U.V.irradiated
B.megaterium
tothelyticphagewasstudied
usingplaqueassaytechnique.Theobtainedresultsshowed
thatthewildtypeof
B.megaterium
andthoseexposedtoU.V.
for5,10and15minwerefoundtobesusceptibletothelytic
phage, whereas,
B. megaterium
which

 exposed  to  U.V.
radiation for 20 and 25 min were found to be resistant to
phageinfection.Moreover,
B.megaterium
whichU.V.
irradiatedfor30minfailedtogrowonnutrientbroth
medium.AsshowninFig.2resistanceoftheU.V.irradiated
B.megaterium
for20mintophageinfection(Fig.2a)
andsusceptibilityofthewildtype(Fig.2b)canbeclearly
seen.
Abilitiesoftheblockedphageadsorptionreceptors
B.megaterium
andthewildtypetoreducethepHvaluesin
theirliquidculturesweretested.Datapresented in Table 2
indicatethat,thelowestpHvaluesinbrothculturesofthewild
typeof
B.megaterium
an dthos eexposedt oU.V. radia tionf or
20and25minwereachievedatthe7thdayafter
inoculation.Moreover,
B.megaterium
whichexposedtoU.V.
radiationfor 20 minwas showed themost efficient onein
reducingthepHvalue.
Fig.1: Apetridishcontainingcircularclearsingleplaquesof
thelyticphagespecificto
B.megaterium
Fig.2(a-b): Aplate containingblockedadsorptionreceptors
B.megaterium
andinoculatedwiththelyticphage(a)Aplate
containingthewildtypeof
B.megaterium
andinoculatedwiththelyticphageand(b)Lysisofthewildtypeandno
lysisoftheresistanceirradiatedbacteriacanbeclearlyseen
341
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J.Biol.Sci.,18(7):338-345,2018
Table2:pHvaluesofmediainoculatedwithwildtypeandU.V.irradiated
B.megaterium
for20and25minandincubatedfor9daysat30EC
pH
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------
Time(days)
B.megaterium
(wildtype)
B.megaterium
exposedtoU.V.for20min
B.megaterium
exposedtoU.V.for25min
1 4.85 4.77 4.81
3 4.00 3.51 4.54
5 3.75 3.69 3.87
7 3.50 3.39 3.65
9 6.90 6.62 6.70
Table3: Densitiesof
B.megaterium
inrhizospheresoil(CFUgG1drysoil)ofwheatinoculatedwithfree,immobilizedcellsandblockedphageadsorptionreceptors
B.megaterium
,inpresence/absenceofspecificphage
Numberof
B.megaterium
×107
-----------------------------------------------------------------------------------------------
Daysafterinoculation
-----------------------------------------------------------------------------------------------
Treatments Zerotime 15 30 45 60 75
Control Zero 13.5 20.2 27 32 25
Freecells 58 69 75 85 90 60
Freecellsplusphage 58 31 35 43 69 38
Immobilizedcells3% 58 72 78 82 89 79
Immobilizedcells3%plusphage 58 60 56 64 61 57
Immobilizedcells5% 58 66 74 79 85 77
Immobilizedcells5%plusphage 58 62 71 76 83 74
Immobilizedcells7% 58 84 91 94 98 89
Immobilizedcells7%plusphage 58 79 88 91 96 85
Immobilizedcells9% 58 70 77 84 87 75
Immobilizedcells9%plusphage 58 68 74 79 82 72
Blockedphageadsorptionreceptors
B.megaterium
exposedtoU.V.for20min 58 65 83 87 91 82
Blockedphageadsorptionreceptors
B.megaterium
exposedtoU.V.for20minplusPhage 58 61 79 83 89 70
Green  house  experiment:Effectoflyticphageon
efficienciesofdifferentformsof
B.megaterium
inocula
(i.e., free cells, immobilized cells and blocked adsorption
receptors
B.megaterium
)inpresenceandabsenceofphage
undercultivatedsoilconditionsinpotexperiments.
Totalcount of
B. megaterium
: Data presented in Table 3
illustratedbyFig.3indicatedthat,numberof
B.megaterium
in rhizosphere soil of wheat plants in different treatments
gradually increased after 60 days after inoculation then
decreased.
Inpresenceofphage,numberof
B. megaterium
markedlydecreasedinrhizospheresoilofwheatinoculated
with the wild type of
B. megaterium
 and 3% alginate
immobilizedcells.Whereas,presenceofbacteriophage
hadnoeffectonnumberof
B.megaterium
inrhizosphere
soilofwheatinoculatedwith5,7,9%alginateimmobilized
cells of
B. megaterium
andblockedphageadsorption
receptors
B. megaterium
exposedtoU.V.for20min.The
highest number  of
B. megaterium
 was recorded  in
rhizosphere soil of wheat inoculated with 7% alginate
immobilized
B.megaterium
an d b locked p hageads o rption 
receptors
B.megaterium
exposedtoU.V.for20minafter
60daysfrominoculation
.
Different parameters of fertilized wheat plants: Data
presented in Table4 indicated that values ofplant height,
freshanddryweight/plantand%Pinplantsinoculated
with the free cells of
B. megaterium
  plus phages were
significantlylowerthanthoseof plantsinothertreatments.
Moreoverfertilization ofwheatplantswith7% immobilized
cells of
B. megaterium
 and blocked phage adsorption
receptors
B. megaterium
 exposed to U.V. for 20 min.
significantlyincreasedthestudiedparametersascomparedto
thoseofplantsintheothertreatments,eveninthepresence
ofphages.
Presence of phage had no effect on the studied
parametersinplantsinoculatedwith7%alginateimmobilized
B. megaterium
and those inoculated with blocked phage
adsorptionreceptors
B.megaterium
exposedtoU.V.for
20min.Thehighestvaluesofthestudiedparameterswere
recordedinplantsinoculatedwith7%alginateimmobilized
B.megaterium
andblockedphageadsorptionreceptors
B.megaterium
exposedtoU.V.for20min.
Onthebasisoftheobtainedresults,itisobviousthatthe
immobilization system and blocking phage adsorption
receptors of
 B. megaterium
protectthebacterialinocula
againstphageattackandinfection.
342

J.Biol.Sci.,18(7):338-345,2018
Control
Blocked receptors free cells
bacterial after 20 min+Phage
Blocked receptors free cells
bacterial after 20 min
Immobilized cells 9% (+) phage
Immobilized cells 9%
Immobilized cells 7% (+) phage
Immobilized cells 7%
Immobilized cells 5% (+) phage
Immobilized cells 5%
Immobilized cells 3% (+) phage
Immobilized cells 3%
Free cells
Free ce lls (+) phage
Treatments
100
90
80
70
60
50
40
30
20
10
0
Number of P-dissolving bacteria 10 g soil
H
7
0 day
60 days
75 days
Fig.3: Densitiesof
B.megaterium
inrhizospheresoilofwheatafter60and75daysinoculatedwithfree,immobilizedcellsand
blockedphageadsorptionreceptors
B.megaterium
,inpresence/absenceofspecificphage
Table4: Growthandphosphoruspercentinwheatplantsinoculatedwith
B.megaterium
infreecells,immobilizedcellsformandblockedphageadsorptionreceptors
B.megaterium
exposedtoU.V.for20mininpresence/absenceofphages
Plantheight Freshweight Dryweight
Treatments (cm) (g/plant) (g/plant) P(%)
Control 58.40opq 8.20g4.58f0.19h
Freecells 73.40ijk 12.50f7.70de 0.33g
Freecellsplusphage 59.60nop 7.08h3.90f0.18h
Immobilizedcells(3%alginate) 83.80ef 15.06cd 8.94ab 0.47de
Immobilizedcells(3%alginate)plusphage 80.40fgh 13.92e7.96bcde 0.41f
Immobilizedcells(5%alginate) 89.00bcde 15.84bc 8.20abcde 0.54c
Immobilizedcells(5%alginate)plusphage 83.20ef 14.98cde 7.38e0.49cd
Immobilizedcells(7%alginate) 96.20a17.26a9.08a0.65a
Immobilizedcells(7%alginate)plusphage 91.00abc 16.70ab 8.46abcd 0.61ab
Immobilizedcells(9%alginate) 93.40ab 16.22ab 8.74abc 0.60b
Immobilizedcells(9%alginate)plusphage 90.60abcd 15.78bc 8.16abcde 0.55c
Blockedphageadsorptionreceptors
B.megaterium
exposedtoU.V.for20min 89.20bcde 16.28ab 8.28abcde 0.44ef
Blockedphageadsorptionreceptors
B.megaterium
exposedtoU.V.for20minplusPhage 84.00ef 14.14de 7.76cde 0.41f
H.S.D.5% 5.65 1.07 1.03 0.047
*Valuesfollowedbythesameletterarenotsignificantlydifferent
DISCUSSION
Inthisstudy
B.megaterium
wasusedasabio-fertilizerin
soil.Theusedbacteriumwasfoundtobenon-lysogenic
and susceptible to an isolate of lytic bacteriophage. The
bacteriophageisolateformedcircularsingleplaquesof2mm
in diameter and clear in appearance. Similar results were
obtainedbyElmaghraby
etal
.19.
Thedepressiveeffectofbacteriophagesonbacterial
bio-fertilizers was detected by many investigators4,16.
Therefore,inthisstudyeffortsweremadetoprotect
B.megaterium
asabio-fertilizeragainstbacteriophageattack.
Two  mechanisms  were used to protect
B. megaterium
against

bacteriophageattack.Thefirstonewasproduction
of
 B. megaterium
 in immobilized form using different
concentrations of sodium alginate. Zayed3, Hammad15 and
Fathy16reportedthatpresenceofthebacterialimmobilized
beads,mayblockthedirectadsorptionofbacteriophage
particleson the surfaceofbacterial cell walland hence no
infectioncanbehappened.
Thesecondmechanismwasblockingphageadsorption
receptorsof
B.megaterium
viaexposureofthebacteriumto
343

J.Biol.Sci.,18(7):338-345,2018
U.V. radiation for 20, 25 and 30 min. Two blocked phage
adsorptionreceptorsmutantsof
B.megaterium
resistantto
phageinfectionwereinducedviaexposureof
B.megaterium
toU.V.radiationatwavelengthof248nmfor20and25min.
Whereas,
B.megaterium
failedtogrowafterexposuretoU.V.
for30min.Thismaybebecauselethaleffectofthehighdose
of U.V. radiation. Moreover, the blocked phage adsorption
receptorsmutantof
B.megaterium
whichobtainedafter
20minexposuretoU.V.wasfoundtobeofhighefficiencyin
reducing the pH value in its broth culture than the other
mutant (obtained by exposure to U.V. for 25 min). The
resistantoftheinducedmutantstophageinfectionmaybe
duetotheactionofU.V.radiationonthebacterialcellswhich
mayresultinblockingthereceptorsofphageadsorptionon
the bacterial cell wall. Similar results were obtained by
Chatterjee and Rothenberg7 and Hyman and Abedon14.
Mutations affecting phage receptors represent the most
frequentcauseofphageresistance11,17.
Inapot experiment, the differentfertilization of wheat
plants with immobilized cells and blocked bacteriophage
adsorption receptors mutants of
  Bacillus  megaterium
absence and presence of phages the obtained resulted
showed that the increase in numbers of
B. megaterium
reachedtheirmaximaafter60daysold,thendecreasedinthe
rhizospheresoilandsignificantincreaseplantP%,freshand
dryg/plant, ascomparedtoplantsinoculatedwith thefree
cellsandfreecellsinoculatedplantsbacteriophage.Similar
resultswereobtainedbyLabrie
etal
.11,Fathy16and
El-Balkhi
etal
.20.
Dependingontheobtainedresultsduringtheexperiment
periodfortwoyearsrespectively,wecanrecommendtouse
B.megaterium
inoculainformofimmobilizedcellson7%
alginate beads

or blocked phage adsorption receptors
mutants resistant to phage infection (after 20 min by U.V.
irradiation)asabio-fertilizerapplicationtoavoidtheharmful
effectofpresenceofbacteriophage.
CONCLUSION
Generally,bacteriophagewasfoundtohaveabadeffect
on
B. megaterium
 when applied as P-dissolving bacterial
inoculum for wheat plants. Whereas, no marked effect for
presenceofphagewasobservedwhen
B.megaterium
was
usedasinoculumforwheatplantsinformofimmobilizedcells
on7%alginatebeadsorblockedphageadsorptionreceptors
mutant.Therefore,productionof
B.megaterium
inoculain
formofimmobilized cells on 7% alginate beads

orblocked
phage adsorption receptors mutants resistant to phage
infectioncanbeusedasagoodabio-fertilizerinthesoil.
SIGNIFICANTSTATEMENT
Thisstudyconfirmedthattheproductionof
B.megaterium
inoculainformofimmobilizedcellson 7%
alginate beads

or blocked phage adsorption receptors
mutantsresistanttophageinfectioncanbeuseasa
bio-fertilizer to phosphate dissolving in the soil. Also, it is
product recommended to avoid the harmful effect of
presenceofbacteriophage.
REFERENCES
1. Chan,Y.K.,M.E.Savard,L.M.Reid,T.Cyr,W.A.McCormickand
C.Seguin,2009.Identificationoflipopeptideantibioticsofa
Bacillus subtilis
 isolate and their control of
Fusarium
graminearum
diseasesinmaizeandwheat.BioControl,
54:567-574.
2. Ali,B.,A.N.Sabri,K.LjungandS.Hasnain,2009.Quantification
ofindole-3-aceticacid from plant associated
Bacillus
spp.
andtheirphytostimulatoryeffecton
Vignaradiata
(L.).World
J.Microbiol.Biotechnol.,25:519-526.
3. Zayed,G.,1998.Canimmobilizationof
Bacillusmegaterium
cellsinalginatebeadsprotectthemagainstbacteriophages?
PlantSoil,197:1-7.
4. Hammad,A.M.M.,1999.Inductionofbacteriophage-resistant
mutantsofnitrogenfixingandphosphatedissolvingbacteria.
Ann.Agric.Sci.Cairo,44:479-493.
5. Duckworth, D.H., 1987. History and Basic Properties of
BacterialViruses.In: Phage Ecology, Goyal,S.M.,C.P.Gerba
andG.Bitton(Eds.).JohnWileyandSons,NewYork,pp:1-43.
6. Garen, A. and T.T. Puck, 1951. The first two steps of the
invasionofhostcellsbybacterialviruses.II.J.Exp.Med.,
94:177-189.
7. Chatterjee, S. and E. Rothenberg, 2012. Interaction of
bacteriophage λwithits
E.coli
receptor,LamB.Viruses,
4:3162-3178.
8. Willey,J.M.,L.M.Sherwood,C.J.Woolvertonand
L.M.Prescott,2008.Prescott,HarleyandKlein'sMicrobiology.
7thEdn.,McGraw-HillHigherEducation,NewYork,
ISBN-13:9780071102315,Pages:1088.
9. Pommerville, J.C., 2010. Alcamo's Fundamentals of
Microbiology. 9th Edn., Jones and Bartlett Publishers,
Sudbury,Canada,ISBN:9781449655822,Pages:860.
10. Madigan,M.T.,J.M.Martinko,D.A.SthalandD.P.Clark,2012.
Brock Biology of Microorganisms. 13th Edn., Benjamin
Cummings, San Francisco, USA., ISBN-13: 9780321649638,
Pages:1043.
11. Labrie,S.J.,J.E.SamsonandS.Moineau,2010.Bacteriophage
resistancemechanisms.Nat.Rev.Microbiol.,8:317-327.
12. Lindberg, A.A., 1973. Bacteriophage receptors. Annu. Rev.
Microbiol.,27:205-241.
344

J.Biol.Sci.,18(7):338-345,2018
13. Wayne, R. and J.B. Neilands, 1975. Evidence for common
bindingsitesforferrichromecompoundsandbacteriophage
phi80inthecellenvelopeof
Escherichiacoli
.J.Bacteriol.,
121:497-503.
14. Hyman,P.andS.T.Abedon,2010.BacteriophageHostRange
and Bacterial Resistance. In: Advances in Applied
Microbiology,Laskin,A.I.,G.M.GaddandS.Sariaslani(Eds.).
Vol.70,ElsevierInc.,SanDiego,ISBN:9780123809926,
pp:217-248.
15. Hammad,A.M.M.,1998.Evaluationofalginate-encapsulated
Azotobacter chroococcum
 as a phage-resistant and an
effectiveinoculum.J.BasicMicrobiol.,38:9-16.
16. Fathy,S.H.,2004. Protection of certain nitrogenfixing and
phosphatedissolvingbacteriaagainstbacteriophageattack.
M.Sc.Thesis,MiniaUniversity,Egypt.
17. Heller, K.J., 1992. Molecular interaction between
bacteriophageandthegram-negativecellenvelope.Arch.
Microbiol.,158:235-248.
18. Prinsloo,H.E.,1966.Bacteriocinsandphages
producedby
Serratiamarcescens
.J.Gen.Microbiol.,
45:205-212.
19. Elmaghraby,I.,F.Carimi,A.Sharaf,E.M.Mareiand
A.M.M. Hammad, 2015. Isolation and identification  of
Bacillus megaterium
 bacteriophages via AFLP technique.
Curr.Res.Bacteriol.,8:77-89.
20. El-Balkhi, M.A., O.A.O. Saad and A.M.M. Hammad, 2012.
Protection of phosphate dissolving bacteria against
bacteriophageattack.J.BasicSci.,22:43-57.
21. Hammad, A.M.M., 1993. Occurrence of bacteriophages of
Bradyrhizobiumjaponicum
in rhizosphere soil of soybean.
MiniaJ.Agric.Res.Dev.,15:609-624.
22. Gomez,K.A.andA.A.Gomez,1976.StatisticalProceduresfor
AgriculturalResearchwith Emphasis on Rice. International
RiceResearchInstitute,Los-Banos,Manila,Philippines,
pp:75-88.
345