Investigating the Effect of a Mixed Mycorrhizal Inoculum on the Productivity of Biomass Plantation

16 Pages·2017·1.21 MB·English

Article
Investigating the Effect of a Mixed Mycorrhizal
Inoculum on the Productivity of Biomass Plantation
Willows Grown on Marginal Farm Land
ThomasJosephPray1,WertherGuidiNissim2,MarcSt-Arnaud1andMichelLabrecque1,*
1 BiodiversityCentre,InstitutdeRechercheenBiologieVégétale,UniversitédeMontréalandJardin
BotaniquedeMontréal,Montréal,QCHIX2B2,Canada;[email protected](T.J.P.);
[email protected](M.S.-A.)
2 DepartmentofAgrifoodProductionandEnvironmentalScience,UniversityofFlorence,
4,50121Firenze,Italy;werther.guidinissim@unifi.it
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:28February2018;Accepted:30March2018;Published:4April2018
Abstract: Inoculationwithmycorrhizalfungi,provenmediatorsofsoilfertility,hasgreatpotential
inagriculturalandsilviculturalsystems. Thisisparticularlytrueinshort-rotationcoppices(SRCs),
where questions of food displacement and fertilization are causes of concern for researchers
and policy makers. We set out to thoroughly test if current inoculation methods, coupled
with reduced fertilization, can demonstrate a growth benefit in SRC willows on marginal lands.
Roughly21,600SalixmiyabeanaSeeman(‘SX61’and‘SX64’)wereplantedinahierarchicaldesignwith
inoculationtreatmentsrandomizedfirst,cultivarsrandomizedsecond,andfertilizationtreatments
randomized third. This process was repeated across three fields of different marginal soil type
(which,inourexperiment,weregiventhedescriptivenamesSandy,Rocky,andDry). Theinoculum
species, Rhizoglomus irregulare Błaszk., Wubet, Renker & Buscot Sieverd., G.A. Silva & Oehl and
Hebeloma longicaudum (Pers.) P. Kumm., were chosen as they are most likely to be commercially
available,andbecausetheyrepresentbotharbuscularandectomycorrhizalinoculumtypes. Growth
wasmeasuredover2.5years,orthreegrowingseasons. Fertilizationtreatment(75kg/haNitrogen),
however,wasonlyappliedduringthesecondgrowingseason. Ourresultsconclusivelyshowedno
benefitfrommycorrhizalinoculationacrossfieldsthatexhibitedsignificantlydifferentgrowthrates,
aswellassignificantdifferentiationfromfertilization.
Keywords: mycorrhizalfungi;willow;inoculation;agriculturalfieldexperiment;rhizosphericsoil
1. Introduction
Growingshort-rotationcoppiced(SRC)willowsforenergypurposesisparticularlypromising
onagriculturallymarginalland. Suchlandwouldneithercomeasasacrificetopristine,old-growth
wilderness,norwouldithaveasignificantimpactuponfoodproduction,asmuchofthismarginal
landhasbecome,orisbecoming,unviablewithinthecurrentcompetitiveagriculturalmarketplace[1].
However,themaindriverofeffortstodevelopbiomassplantations,globalwarming,canalsofuel
concernsaboutfertilizeruse[2]. Ingeneral,SRCwillowscanbegrownonmanytypesofagricultural
land (though wetter land is much better than dry) [3,4]. Due to high biomass yields though, they
removenutrientsatahighrate[5]. ThismeansthatpoorsitesarenotsuitableforSRCcultivation
unlessfertilizersaresupplied. Arelevantstudyshowedthatfertilizerrepresentsupto10–20%of
thecostofproductionoverseveralrotationsofawillowSRCcrop[6]. Estimatesbasedonnutrient
off-takemeasurementsvarybetween50–130kgN,60–83kgK,and8–16kgPperhectareperyearfor
willowSRC,althoughfarmersgenerallyonlyneedtoaddnitrogen(N)overthefirstseveralyears,
Forests2018,9,185;doi:10.3390/f9040185 www.mdpi.com/journal/forests
Forests2018,9,185 2of16
aspotassium(K)andphosphorus(P)usuallybuildupexcessivelyinfarmsoil[7].Thispaperaddresses
whetherthecentralchallengeoffertilitythatariseswithSRCfarminginacarbon-negativewayon
marginallandcouldbeaddressedormitigatedbyinoculationwithmycorrhizalfungi.
Arbuscular mycorrhiza (AM), which penetrate the root cells of their host plant,
andectomycorrhiza(EM),whichinteractjustasintimatelywiththeirhostplantbutthroughaform
ofrootinterfacethatdoesnotpenetratecells,arethetwomaintypesofmycorrhizalfungi(e.g.,[8]).
Inexchangeforthesugarsthattheplantproduces, bothAMandEMprovidenutrientsharvested
from surrounding soil (particularly those trapped in mineral form or difficult-to-degrade organic
molecules)withtheirextensivefungalnetworksandspecializeddegradationenzymes[9,10]. Thereis
alsoextensiveevidenceofAMandEMprovidingtheirhostswithprotectionfromdiseaseandaiding
withwaterstressduringdroughts[11–13].
Biomass shrub willows—though small and with multiple stems instead of a single
trunk—areneverthelessatreespeciesinthegenusSalixL.,andaretypicallyfoundwithEMsymbionts
insamplesoftheirrootsandrhizosphere(thenarrowsoilzoneundertheinfluenceofroots)[14–16].
Salixspecies,however,havebeenfoundtobeassociatedwithbothEMandAM(separatelyoratthe
sametime)[17–20]. MinorassociationwithAMcannotbeignored,assomeevidencesuggeststhat
mycorrhizacanprovidesignificantbenefitsfortheirhost,evenatlowlevelsofrootcolonization[21,22].
Because agricultural land (or marginal, formerly agricultural land) does not typically have
adiversecollectionoftreesgrowingonit,researchershaveconductedexperimentsinoculatingtrees
withmycorrhiza,reasoningthatthespecificEMspeciesthatwouldcolonizebiomasswillowscould
bemissing[23]. Similarly,asagriculturallandisoftenexhaustedoforganiccarbonandrepeatedly
leftbarrenofplanthosts(whencropsareharvestedinlatesummerorfall),evenAM,thatcoulduse
cropplantsashosts,mightbelowinnumberand/ordiversity[24,25]. Someexperimentsshowed
abenefitfrominoculation,butmanyotherswereinconclusive[26–30].Thesameistrueforexperiments
specificallyusingSalix(orcloselyrelatedPopulusL.)andAMorEMinoculum[31–33].
In most of these studies, the controlled conditions used are significantly different from what
willowgrowerswillactuallyencounterintheirfields.Evenwhenyoungtreeswereplantedintonatural
settingsinsomeoftheseexperiments,theseedlingswerefirstraisedincontainers. Thismeansthatthe
controlconditions,withcompletelysterilepottingsoil,couldstuntthetrees’earlygrowth,andtherefore
donotreflectanaturalcontrolwitharandommixofnativemycorrhiza. Experimentswithsterile
controlsoils,andsterilesoilsplusoneormoreinoculumspeciesthroughoutthemeasurementtime
period,whilevaluableforbasicresearch,areinherentlylimited. Agriculturalactivitiesalmostnever
dealwithsterilesoils,soforpracticalpurposes,farmersandagriculturalresearchersneedtoknow
whattheirtreatmentsdoinrelationtounsterilecontrols.
Thisstudyaddressesthesterilecontrolproblembyplantingdirectlyintointactfarmsoil. Thisis
possibleinpracticaltermsbecausewillowcuttingsdonotneedtobestartedinpots,andintheoretical
termsbecauseofthesheernumberofwillowcuttingsplanted. Themyriadofcombinationsofnative
mycorrhiza,aswellasofplantpathogens,etc.,thatwouldthreatenasmallerexperimentwithtoomany
confoundingvariables,arebetterdealtwiththroughreplicationacrosslargefields. Ifafewwillowsare
negativelyaffectedbysmallareasofsoilwithdiseasepathogens,orconverselyifafewwillowshave
increasedgrowthduetoasmallareaofparticularlybeneficialmycorrhiza,theirgrowthmeasurements
willnotsignificantlyshiftthemeanresultsfromthemanymoregrowthmeasurementswewereable
totakethroughafield-scaleinvestigation. Furthermore,thehighvariabilityinthegrowthofplants
infieldsthatarenaturallypatchyinsoilstructureandnutrientconcentrationcanbeaccountedfor
statisticallywithenoughdatapointsfromseveraldifferentblocks.This“random”variationwaspartof
ourANOVAmodels,andourfieldsizesallowedustodothis. Biomassfarmers,andthosewhoadvise
farmers,shouldtakenoteofthisstudy’sresults,astheyrelatecloselytoreal-worldconditions.Eventhe
equipmentusedtosetupandimplementtheexperimentwasconsistentwithmodernfarmingrealities.
If the use of such energy intensive products as fertilizers can be limited through mycorrhizal
inoculation,andlikewisemarginalagriculturallandcanbebettertakenadvantageof,thenbiofuel
Forests2018,9,185 3of16
grown with carbon taken from the atmosphere would inarguably represent a net climate benefit.
Knowingwhetherinoculationiseffectiveornotisakeystepforthisindustry,andmoregenerally,
forthoseworkinginagricultureandsilviculture. Ourexperimentaimedtotakethisstep: ittested
whether or not a mycorrhizal inoculant can have a positive effect on the growth of SRC willows.
Furthermore,ourexperimentwasdesignedextremelyrobustly,testingtheinoculantseffectacross
twodifferentcultivarsofwillowinthreedifferentmarginalfield-types,andbothwithandwithout
nitrogenfertilization.
2. MaterialsandMethods
2.1. ExperimentalDesign
Threesimilarlydesignedexperimentalfieldsof108m×43.2m,or~4670m2each,wereestablished
inearlyJuneof2010atthreesitesontheAllardfamilyfarm(AgroÉnergieInc.,Saint-Roch-de-l’Achigan,
QC,Canada(45◦51(cid:48)00”N,73◦36(cid:48)00”W).Thefarmis~60mabovesealevel,flatandopen,butsparsely
wooded at the edges. It lies within the St. Lawrence River watershed, ~25 km north-northeast of
Montreal,QC,Canada. Adifferentsoiltypedefineseachexperimentalfield(detailsaresummarized
inTable1). Onelocation,referredtointhispaperastheSandyfield,isalmostpuresand,withalow
pHof6. Another,namedDryfield,issandy-loamwithaclosetoneutralpHof7. Thelastconsistsof
silty-loam,withahighpHof8,butwith~30%ofthesurfacecoveredwithsmallandmediumsized
rocks(~1–5cm)(thereforenamedRockyfield). Fieldshadbeencultivatedinthepastwiththestandard
NorthAmericanrotationofcornandsoybeans(cornmostrecently),buttheSandyfieldhadalsobeen
periodicallyplantedwithcarrotsbeforethat.
Table1.Characterizationofthethreefieldsites,includingasoilanalysisattwodepths.
Depth pH Nitrate Phosphorus OrganicMatter Clay Silt Sand SoilType
DryField(45◦49(cid:48)31”N,73◦37(cid:48)29”W)1
(0−20cm) 7.1 5.77ppm 130kg/ha 4.0% 21.0% 40.9% 38.1% Mediumloam
(20–40cm) 7.3 7.35ppm 81kg/ha 3.9% 19.9% 32.7% 47.4% Edgingtowardssandy
RockyField(45◦49(cid:48)38”N,73◦37(cid:48)36”W)2
(0–20cm) 7.9 6.87ppm 63kg/ha 3.5% 29.5% 39.2% 31.3% Mediumloam
(20–40cm) 7.9 5.77ppm 39kg/ha 2.8% 24.1% 43.5% 32.4% Edgingtowardsclay
SandyField(45◦49(cid:48)32”N,73◦37(cid:48)04”W)3
(0–20cm) 6.0 5.35ppm 256kg/ha 2.1% 2.5% 10.4% 87.1% Loamysand
Veryclosetopure
(20–40cm) 6.1 5.91ppm 192kg/ha 2.0% 3.4% 6.8% 89.8%
sand
1Definingqualitativefeatures:heavilydrainedwitha0.5mdeepditchalongitswestsideanda2mdeepchannel
alongitssouthside. 2Definingqualitativefeatures: ~30%ofitssurfacecoveredwithsmallandmediumsized
rocks(~1–5cm).3Definingqualitativefeatures:farremovedfromanydrainage,itoftenhadstandingwaterfor
aweekatatimeafteranyrainfall,betweenthefieldandtheforestborderingitssouthsideandthesouthernhalfof
itseastside.
Theexperimentaldesignwasamodifiedsplit-plotdesignandhadtwelvefullblocksrepeated
in each field. In this hierarchical design, inoculation treatments were randomized before cultivar,
andfertilizationtreatmentswererandomizedlast. Localsoilconditionsandweatherdeterminedour
partnerfarmer’sbestpracticesforpreparingaweed-free,flat,andloosesoilbed(amixofplowing
anddisking).Smallcuttings(~30cmlong,and~1–2cmthick)oftwocultivarsofwillow,Salixmiyabeana
Seeman‘SX64’and‘SX61’wereplanted. Plantingwasdoneusingamodified3-rowcabbageplanter.
Rowswere1.8mapart,andwillowswereplantedevery36cm,foradensityof16,103treesperhectare.
Flaggedstakesmarkedevery18m,showingtheedgesofeachexperimentblock(6×12-row-wide
blocksrunningdowneachhalfofthefield,for12intotalineachfield—seeSupplementaryMaterial,
FigureS1fordiagram). Themodifiedaspectofthesplit-plotdesigncamewiththerandomizationof
thecultivar. Insteadofbeingrandomizedwitheachblock,therandomselectionfromthefirstblock
was continued for the entire length of the field, in order to facilitate timely and accurate planting
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at this large scale (again, see Supplementary Material, Figure S1 for diagram). The three farmers
sittingontheplanterwereinformedaboutwhichofthetwowillowtypestofeedintotherotating
plantercylindersatthebeginningofeachnewgroupofthreerows. However,duringplanting,water
deliveryoftheinoculantwasturnedoffandonbyhandastheplanterstoppedandstartedeachblock
section,thusallowingatruerandomizationbetweeneachexperimentalblock. Smallerflagsonwire
stakes subdivided the twelve blocks at 9 m (or six-row) intervals, but these subdivisions directed
nitrogenfertilization,whichwasnotapplieduntilthesecondgrowingseason(whenitwasappliedby
hand,duetotherelativelysmallsizeofeachtreatmentplot—seenextparagraphforapplicationrates).
Therefore, duringthefirstyeareachexperimentalblockhadfourtreatmentsubplotswithinthem,
whichthenbecameeighttreatmentsubplotswithineachblockfromthesecondyearonSimilarblocks,
albeitwithadifferentrandomlyassignedtreatmentpattern,weresetupinthethreedifferentfields.
InoculationwasdoneusingamixedinoculumofAMandEMfungi,providedbyBioSyneterra
SolutionsInc. (L’Assomption,QC,Canada). Itwasdeliveredviaawater-suspensiontoeachcutting
at planting as ~350 propagules of the AM and ~250 of the EM in 50 mL of water. The AM strain
used was a Rhizoglomus irregulare (Błaszk., Wubet, Renker & Buscot) Sieverd., G.A. Silva & Oehl
DAOM197198[34,35])fromPontRouge,QC,Canada. TheEMwasaproprietaryHebelomalongicaudum
(Pers.)P.Kumm.Viabilityoftheinoculumwascheckedbytakingasampleoftheinoculumsuspension
onthedayofplanting,andinoculatingseveralpottedwillowsinautoclavedfarmsoil. Viabilitywas
qualitativelyconfirmedthefollowingdaywithrootstainingandmicroscopicvisualizationofAMand
EMstructures,aswellaswithmacroscopicidentificationofEMfructifications. Some7200cuttings
wereplantedoneachsite,foratotalof21,600plants. Theexperimentranfortwoandahalfyears
(throughthreegrowingseasons). Onlyduringthesecondgrowingseasondidhalfofthetreesreceive
nitrogenfertilization(75kg/haN,aspelletedchickenfeathercompost,scatteredbyhandinMay).
2.2. SamplingandMeasurements
Before planting had taken place, a baseline soil analysis was conducted. Soil was collected
using a 1000 cm3, screw-boring hand sampler, combining seven samples taken from a diagonal
line across each field. Two depths were sampled, 0–20 cm and 20–40 cm. All of the soil from
eachsampletypewasmixedthoroughly. Asubsamplewasthentakenbacktothelaboratoryand
air dried for chemical analysis using recommended methods [36]. Total nitrogen was measured
using Kjeldal’s method. Total phosphorus was extracted with nitric-perchloric acid or stannous
chloride(forthesludge), andmeasuredusingplasmaemissionspectroscopy. Potassium, calcium,
and magnesium were extracted with NH Ac, and analyzed by atomic absorption. Metals were
4
extractedwithnitricorhydrochloricacid,andtheirdosagewasdoneusingICP(InductivelyCoupled
Plasma)[37].
Wemeasuredtheheightofthelongeststem,itsdiameteratadistanceof10cmfromtheground,
aswellasthetotalnumberofstems,inordertoassessshrubgrowth.Growthmeasurementsweretaken
oneveryothertreealongthemiddlerowofthethree-rowtreatmentgroupsinearlyNovember,2010.
Thismeantthat16treesweremeasuredineachtreatmentgroup. Withfourtreatmentgroupsduring
thefirstyearand12blocksrepeatedineachfield,thattotaled2304treesmeasured(theactualtotalwas
slightlylowerasoneblockwithintheSandyfieldwasnotmeasuredinthefirstyear,duetoexcessive
mortalityinthatblock,presumablyasrootshadnotdevelopedenoughtocompensateforthehigher
drainageinanalmost100%sandpatch).
At the end of the second growing season (early December 2011), a total of 1152 trees were
measured for growth, with 576 also selected randomly to be cut and weighed. A representative
2–3kgofstempieces(withoutleaves)werefurthersubsampledfromeachfieldtomeasuremoisture
percentage. AlthoughablockintheSandyfieldhadbeendroppedfromtheanalysisinthefirstyear
due to the aforementioned high mortality, it was kept during the second year. This was because
manyofthetreesthathaddiedwereactuallyconcentratedinthemiddleoftheblock,andtherewere
Forests2018,9,185 5of16
enoughlefttogiveanaccuraterepresentationofallthetreatmentswiththeadditionofanincreased
fertilizationsubdivision.
Attheendofthethirdgrowingseason(lateNovember2012),twotreespertreatmentsubgroup
perblockweremeasuredandweighed(atotalof288trees).
Thirty-sixwholerootsampleswerealsodugupandcollectedinlateOctober,2010,afterthefirst
growingseason;theseconsistedofthreereplicatesofthetwelvetreatmentcombinationstheninplace
(randomlychosenfromthetentreesinthemiddleofeachtreatmentgroup,withinthreerandomly
chosenblocksineachfield). Becausenitrogenfertilizationwasnotapplieduntilthesecondseason,
the twelve treatment combinations consisted of the two willow cultivar types in each of the three
fields,inoculatedandnot. Aftershakingoffunattachedorexcesssoil,eachplant’srootsystemwas
separatelybaggedandstoredat−30◦Cinthelaboratory’sfreezersthesameday.
Rootsystemswerelaterthawedandvigorouslyrinsedandagitatedbyhandindistilledwater.
The dirty rinse water was then allowed to settle, and the sediment was set aside and refrozen as
36 rhizospheric soil samples. After cutting up the roots into 1 cm pieces, they were well mixed
and further homogenized (with a washed and sterilized commercial food-beverage blender in
milli-Q/0.1TEbuffersolution(Black&Decker,Montréal,QC,Canada)andfrozenas36rootsamples
forthenextmolecularanalysissteps.
2.3. MolecularFungalCommunityAnalysis
DNA from the first-year’s 36 root and 36 rhizosphere samples was extracted using a MoBio
LaboratoriesPowerSoilExtractionkit(MoBioLaboratoriesInc.,Carlsbad,CA,USA)accordingtothe
manufacturer’sinstructions,exceptthatinsteadofthestandardhomogenizationandlysisstep,anMP
BiomedicalsFastPrepmachine(MPBiomedicalsInc.,Solon,OH,USA)atsetting4wasusedfor25s
andsixrepetitions.
Forcloning-sequencing,allreplicatesofbothwillowcultivarswerepooledtogetherbyfieldand,
inoculationtreatment. TheDNAextractedfromrhizosphericsoilwasusedtolookforAMwhileDNA
fromrootsamplestolookforEMsincepreliminaryeffortsrevealedatendencyoftheAMspecific
primers(AML1/AML2[38])toamplifywillowDNA,attemptsweremadetodetectbothAMinthe
DNAextractedfromrhizosphericsoil,andEMintheDNAoftherootsamples. Conversely,inorderto
lookforEM,universalfungalprimers(ITS1F/ITS2[39])wereused,sincenoEMfungi-specificprimer
wasavailable. AsearchforAMwasundertakeninbothrootextractsfromtheSandyfieldandthe
rhizosphericsoilextract,assoilextractshadnotproducedanyAMsequences. Allofthisleftsixpooled
samplesforAMPCR-cloningandsixforEMPCR-cloning.
A nested PCR reaction was run with samples used for AM detection. The initial PCR
used the primer pair NS1/NS41 in a 25 µL reaction with reagents from the MoBio PCR CoreKit
(MoBioLaboratoriesInc.,Carlsbad,CA,USA). Initialdenaturationwasat94◦Cfor3min,followedby
30cyclesof94◦Cfor45s,55◦Cfor1min,and72◦Cfor1min(modifiedfrom[40]).Thereactionended
with10minat72◦COnemicroliterofproductfromthisfirstPCRwasthenamplifiedinasecond25µL
reactionusingtheAML1/AML2primerpair. Initialdenaturationwasat94◦Cfor3min,followed
by30cyclesof94◦Cfor30s,58◦Cfor45s,and72◦Cfor45s,andendingwith10minat72◦C[40].
EppendorfMasterCyclerProthermocyclers(EppendorfCanadaLtd.,Mississauga,ON,Canada)were
used, and PCR products visualized on 1% agarose gels, according to the directions for Biotium’s
GelReddye(Biotium,Inc.,Fremont,CA,USA)andBioRad’sMolecularImagerGelDocXR(Bio-Rad
Laboratories,Hercules,CA,USA).
ThesamplesusedforEMdetectionwereamplifiedinasinglestepusingthesameMoBioreagents
(MoBioLaboratoriesInc.,Carlsbad,CA,USA)andtheprimerpairITS1F/ITS2.Initialdenaturationwas
at94◦Cfor3min,followedby30cyclesof94◦Cfor45s,60◦Cfor1min,and72◦Cfor1min(modified
from[41]). Thereactionendedwith10minat72◦C.Thesamethermocyclersandvisualizationmethod
wereusedasdescribedabove.
Forests2018,9,185 6of16
PCRproductswereclonedusingaTOPOTACloningKit(Invitrogen,Carlsbad,CA,USA)for
sequencing,fromLifeTechnology,accordingtothemanufacturer’sdirections. Forty-eightclonesfor
eachsamplewerereamplified,andtheproductssequenced,atMcGillUniversityandGénomeQuébec
InnovationCentreinMontreal,QC,Canada,usingthecommonSanger-sequencingmethod.
Sequences were grouped by CD-HIT (http://weizhongli-lab.org/cd-hit/) into operational
taxonomicunit(OTUs)of98%similarity. OnerepresentativesequencefromeachOTUgroupwasthen
randomlyselectedforanalysisusingtheBLASTsearchtoolintheNationalCenterforBiotechnology
Information(NCBI)database. OnlyOTUsthatwerematchedwithAMorganismsweretabulated(the
18SgeneisahighlyconservedregionandtheAML1/AML2primerpairisknowntoamplifyother
eukaryoticorganismsaswell).
2.4. DataAnalysis
Afull-factorialANOVAwasperformedonthemeasuredgrowthdata. Growthispresentedin
thispaperasstembasalareaperhectare(SBAm2/ha)(modifiedfrom[42]). WedeterminedSBAby
calculatingtransectionalstemareasfromdiametermeasurements,multipliedbymeanstemnumber,
andthendividedbytheaverageareaoflandoccupiedbyonewillow(inhectares). SBAwascalculated
foreachmeasuredtreebeforerunningtheANOVA,therebynormalizingdiametermeasurementsfor
differentmortalityratesineachfield.Theactualaveragedensityofwillowswasquitedifferentbetween
fields,aftermortalitysufferedduringdryconditionsintheweeksfollowingplanting(almost~25%in
Dryfield,~15%inSandy,and~5%inRocky;butscatteredenoughineachfieldtoberoughlyequivalent
acrosstreatmentcombinationsandblocks). Finally,becauseoffertilizerspilloverfromanearbyfield
duringthethirdyear,anentirerowoftheSandyfieldhadtobedroppedfromtheanalysesandthe
blocksinthatfieldredrawntomaintainthecorrectnumberofeachtreatmentcombination(reducing
thenumberofblocksinthatfieldbyfour).
Growth was also measured in the second and third years by cutting down and weighing
above-groundbiomass,andreportedasovendrytonsperhectare(ODT/ha). Thiswascalculatedfrom
wetmassmeasurementstakeninthefield,usingaconversionof0.53in2011and0.61in2012,found
fromdryingsamplesinthelab. Thesamedensityvaluesforeachfieldwerethenusedtocalculate
perha,aswithSBA/ha.
InthemodelforourANOVA,experimentalblocksneededtobenestedinthefield,sinceblock1
oftheDryfieldwasnotthesameasblock1oftheSandyfield,andsoon. Themodelwasmodified
accordingly,keepingafull-factorialcombinationofallvariablesbesidesthoseinstanceswherefield
and(nownested)blockswerecombined. Furthermore,allcombinationsofblockshadtobedesignated
asrandomcomponents,sincetheydidnotrepresentavariablewewereinterestedintesting,butwere
necessaryfortheANOVAtoincorporaterandomheterogeneitywithinthefields. ANOVAresiduals
didindicateheteroscedasticity,whichwascorrectedbylogarithmictransformingourmeasureddata.
3. Results
The experiment found no productivity difference between uninoculated and inoculated
plantationwillows. AsFigure1shows,duringthefirst2years,whenthemosttreesweremeasured,
nodifferencewasshownbetweentheobservedresultsandinoculationtreatmentpredictedmean
SBA/havalues.Similarly,whilethethirdyearhadfewermeasuredtrees,anddidseeminglyseeasmall
differencebetweenSBA/hameanpredictedvalues,thisdifferencewasnotstatisticallysignificant.
Thisresultheldtruewhethertreeheightwasmeasured,orwholetreeswerecutdownandweighed
(seeSupplementaryMaterial,TablesS1–S10).
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Figure1.CumulativegrowthofSalixmiyabeana(‘SX61’and‘SX64’)duringestablishmentonmarginal
land,treatedwithmycorrhizalinoculum.Errorbarsarestandarderror(SE).Withineachyear,similar
letters(a,b)abovethebarsindicatethattheANOVAdidnotfindsignificantdifferences(p<0.05)
betweenpredictedmeansforinoculationtreatment.
Table 2 shows the results of the ANOVA on 2011 SBA/ha data in more depth. This ANOVA
confirmsthatthefieldsitesusedweredifferentenoughtoaffectthewillows’productivity,andthat
thenitrogenfertilizationtreatmentwassignificantaswell. Table3providestheeffectsize,showing
that fertilization gave a predicted mean increase of 27% SBA/ha in 2011. Even more strikingly,
the Sandy field showed a predicted mean increase of 55% SBA/ha over the Rocky field in 2011.
Similarly, significant biological and statistical effect sizes were seen in other years, and with the
heightandweightdata, thoughfertilizationwasonlyappliedin2011, andtherefore, showedless
effect in 2012 (see Supplementary Material, Tables S1–S14). The overall patterns in Figure 1 and
Tables2and3arerepresentativeofthoseseenoverthethreeyears,withheightandweightasseenin
theSupplementaryMaterial(TablesS1–S10). AsseeninFigure2,thedifferentfieldsdidyielddifferent
resultsrelativetooneanother. WhiletheSandyfieldshowedmarkedlyhighercumulativegrowththe
firsttwoyears,cumulativegrowthintheDryfieldcaughtupandequaledthatoftheSandyfieldby
thethirdyear(Figure2).
Forests2018,9,185 8of16
Table2.ANOVAoftheeffectsofsoiltype,mycorrhizalinoculationandnitrogenfertilizationonstem
basalarea/ha(LOGtransformed)ofwillowsafteroneseason’sgrowthinmarginalland.
Source Nparm DF DFDen FRatio Prob>F
Field 2 2 33 21.0017 <0001*
Inoc 1 1 33 0.0175 0.8955
Field×inoc 2 2 33 1.1145 0.3401
Fert 1 1 33 105.1391 <0001*
Field×fert 2 2 33 1.8694 0.1702
Inoc×fert 1 1 33 0.0187 0.8920
Field×inoc×fert 2 2 33 0.2658 0.7682
Cultivar 1 1 33 0.0465 0.8305
Field×cultivar 2 2 33 0.4076 0.6686
Inoc×cultivar 1 1 33 0.8624 0.3598
Field×inoc×cultivar 2 2 33 0.2301 0.7957
Fert×cultivar 1 1 33 0.1629 0.6891
Field×fert×cultivar 2 2 33 0.4619 0.6341
Inoc×fert×cultivar 1 1 33 2.0731 0.1593
Field×inoc×fert×cultivar 2 2 33 0.602 0.5536
Allcombinationsoftheblocktreatment,byitselfandwiththeothertreatmentvariables,werepartofthemodel.
However,theblockwastreateddifferentlysinceitwasnestedinthefieldvariable,andlabeledasarandomattribute.
Therefore,itdoesnotappearinthistable.Anasterisk(*)nexttothep-valuedenotesa5%statisticalsignificance.
Table3.Stembasalareaperhectare(m2/ha)ofwillowsafteroneseason’sgrowthinmarginalland.
ANOVApredictedvaluesandaposterioritestresultsaccordingtosoiltype,mycorrhizalinoculation,
nitrogenfertilizationorwillowcultivar.
Experimental
LeastSquaresMean StandardError Test
Treatments
PanelA:Field Tukey’stest
Dry 11.029889 0.66402733 B1
Rocky 10.307269 0.66402733 B
Sandy 15.964387 0.66402733 A
PanelB:Inoculation1 Student’sT-test
Notinoculated 12.478451 0.43045506 A
Inoculated 12.389246 0.43045506 A
PanelC:Nitrogenfertilization Student’sT-test
Fertilized 13.921206 0.4122257 A
Unfertilized 10.946491 0.4122257 B
PanelD:Cultivar Student’sT-test
‘SX64’ 12.486569 0.44855227 A
‘SX61’ 12.381127 0.44855227 A
1Differentletters(AandB)withineachpaneldenoteasignificant(≥0.05)differencebetweenmeans.
Forests2018,9,185 9of16
Figure2.CumulativegrowthofSalixmiyabeana(‘SX61’and‘SX64’)duringestablishmentonmarginal
land, in three different fields. Stem basal area predicted values (untransformed) and test results
(LOGtransformed)byfieldforallthreeyears.Note:2012representsonlyonecultivartype(‘SX61’),
becauseoftreatmentinteraction;however,2010and2011arethefullmodel.Errorbarsarestandard
errors.Letters(a,b)abovethebarsindicatethattheTukey’stestfoundasignificantdifference(p<0.05)
betweenpredictedmeansforeachfield(aseparateanalysiseachyear).n=768eachforNormaland
Rockyfieldsin2010;n=704forSandyfieldin2010;n=384in2011foreachfield;n=96in2012for
eachfield.
Figure3showstheeffectofnitrogenfertilizationongrowth,usingSBA/hafrom2011and2012to
illustratethedifferenceineffectineachyear. Figure4showsthefertilizationeffectonmass,using
ODT/ha(Ovendrytonsperhectare),asopposedtoSBA/ha. Figure4alsofocuseson2011,theyear
ofthemainfertilizationeffect,andtakingintoaccountaninteractionbetweenfieldandfertilization
asindicatedbytheANOVA,breaksdowntheeffectbyfield. Theeffectoffertilizationisevenmore
markedinthiscase, asfertilizationgaveapredictedmeanincreaseof51%ODT/hain2011inthe
Sandyfield.
Forests2018,9,185 10of16
Figure3.CumulativegrowthofSalixmiyabeana(‘SX61’and‘SX64’)duringestablishmentonmarginal
land.Stembasalareapredictedvalues(untransformed)andANOVAresults(LOGtransformed)for
theyearinwhichfertilizationtreatmentwasapplied(2011),aswellasforthefollowingyear(past
growth,plusanyresidualnitrogeninthesoil).Note:Errorbarsarestandarderrors.Differentletters
(a,b)abovethebarsindicateasignificantdifference(p<0.05). n=576in2011foreachfertilization
treatment;n=144in2012foreachfertilizationtreatment.
Figure4.CumulativegrowthofSalixmiyabeana(‘SX61’and‘SX64’).Ovendrytonsperhapredicted
values(untransformed)byfieldfor2011.Note:Errorbarsarestandarderrors.Differentletters(a,b)
abovethebarsindicateasignificantdifference(p<0.05).TheANOVA(performedonLOGtransformed
values),includingallvariablesinthemodel,foundalikelyinteractionbetweenfieldandfertilization,
promptingthebreakdownbyfield.n=192foreachfieldandfertilizationtreatment.