Application of municipal biowaste derived products in Hibiscus cultivation: Effect on leaf gaseous exchange activity, and plant biomass accumulation and quality

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ContentslistsavailableatScienceDirect

Scientia

Horticulturae

j o u r n a l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / s c i h o r t i

Application

of

municipal

biowaste

derived

products

in

Hibiscus

cultivation:

Effect

on

leaf

gaseous

exchange

activity,

and

plant

biomass

accumulation

and

quality

Daniele

Massa

a,∗

,

Domenico

Prisa

a

_,

_Enzo

_Montoneri

b

_,

_Daniele

_Battaglini

a

_,

Marco

Ginepro

c

_,

_Michele

_Negre

d

_,

_Gianluca

_Burchi

a

a_Council_for_Agricultural_Research_and_Economics_(CREA),_Landscaping_plants_and_nursery_research_unit,_Via_dei_Fiori_8,₅₁₀₁₂_Pescia,_PT,_Italy b_Biowaste_Processing,_Via_XXIV_Maggio_25,₃₇₁₂₆_Verona,_Italy

c_Dipartimento_di_Chimica,_Università_di_Torino,_Via_P._Giuria_7,₁₀₁₂₅_Torino,_Italy

d_Dipartimento_di_Scienze_Agrarie_Forestali_e_Alimentari,_Università_di_Torino,_Largo_P._Braccini_2,₁₀₀₉₅_Grugliasco,_TO,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received1October2015

Receivedinrevisedform11March2016 Accepted23March2016

Availableonline26April2016 Keywords: Biowaste Biostimulant Photosynthesis Substrateculture Hibiscuspalustris

a

b

s

t

r

a

c

t

Twourbanbiowastematerials,fermentedunderanaerobicandaerobicconditions,theirsolubleand insolublealkalinehydrolysates,andacommercialbiostimulantproduct,werecomparedfortheircapacity toboostHibiscuscropproductionandquality.Plantsweregrownin4-litrepots,containingpeatand pumiceassubstrate,underoptimalgrowingconditions.Arandomized-blockexperimentaldesignwas adopted.Twotypes(mainfactorsofvariability)oftreatmentswereapplied,i.e.byblendingtheabove productswiththesubstrateattransplant,andbyfertigationusingthesolublehydrolysatesonly.Plant biomasscharacteristicsandecophysiologicalparametersweremeasured.Meaneffectoffactorsand theirinteractionswereassessedbytwo-wayANOVA.Principalcomponentanalysiswasperformedby usingthedifferentdependentvariablestosummarizethemostrelevantdifferencesamongtreatments. Comparedwiththecontrol(untreatedplants),theappliedtreatmentsenhancedmostoftheinvestigated parameters.Themostvaluableeffectswereobservedfortotalbiomassaccumulation(+25%),plantheight (+10%),leafchlorophyllSPADindex(+15%)andnetphotosynthesis(+24%).Thehydrolysatesperformed betterthanthepristinematerials.Theformeroneswerecomparabletothecommercialbiostimulant. Theresultsconﬁrmedthehypothesisthatbiowastederivedproductsinducebiostimulantactivityon Hibiscus;theirapplicationcanimprovecultivationsustainability.

Abbreviations: D,digestateobtainedfromtheanaerobicfermentationofthe organichumidfractionofurbanwastes;DAT1,daysafterT1(day);DW,plantdry

weight(gm−2);E,evapotranspirationrate(mmolm−2s−1);−F,fertigation treat-ments;FERT,treatmentsappliedduringthecultivationthroughfertigation;GC, greencompostobtainedfromtheaerobicfermentationofurbangardeningwastes; Gs,stomatal conductance (mmolm−2_s−1_); _ID, _insoluble _digestate_hydrolysate

obtainedfromalkalinehydrolysisofD;IGC,insolublecomposthydrolysateobtained fromthealkalinehydrolysisofGC;LAI,leafareaindex(m2_m−2_);_NO,_untreated;

NOVA,commercialbiostimulant;OM,organicmatter(kgm−3);Pn,net photosyn-theticrate(␮molm−2s−1);SD,solubledigestatehydrolysateobtainedfromthe alkalinehydrolysisofD;SGC,solublecomposthydrolysateobtainedfromthe alka-linehydrolysisofGC;SUB,treatmentsappliedtothesubstrateasapowderblended withthesubstrate;T1,beginningoftheexperimentation(30daysaftertransplant)

(day).

∗ Correspondingauthor.

E-mailaddress:daniele.massa@crea.gov.it(D.Massa).

1. Introduction

Soluble biobased substances isolated from the alkaline hydrolysateoffermentedurbanbiowasteshavebeenproven to performasefficientecofriendlychemicalauxiliariesindiversified fieldsofthechemicalindustry(Montonerietal.,2011), agricul-ture(Sortinoetal.,2014),andanimalhusbandry(Montonerietal., 2013).The productsare mixtures of moleculeswith molecular weightfrom5toseveralhundredskDa,comprisingaliphaticand aromaticCatomsbondedtoavarietyofacidandbasicfunctional groups.Thisisthelikelyreasonoftheirmultipurposeperformance (Montonerietal.,2011).Particularlyrelevantforagriculturearethe resultspublishedbySortinoetal.(2014)andGomisetal.(2014). Sortino et al. (2013, 2014) have shown that theabove soluble biobasedsubstancesaddedtosoilincreasedtomatoandredpepper plantphotosyntheticactivity,growthandproductivity.Thesame substances enhanced thephotochemical degradationof organic pollutantsinindustrialeffluent(Gomisetal.,2014).Thesefindings

http://dx.doi.org/10.1016/j.scienta.2016.03.033

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suggestedthattheymightpromoteeitherCﬁxationor mineral-ization,according tothedifferent operationalenvironments. In bothcases,itwassuggestedthat,bytheircapacitytocomplexFe ionsandkeeptheminsolutionatslightlyacidicoralkaline con-ditions,theabovesolublebiobasedsusbtancesmaycontributeto enhancephoto-Fentonprocesses.Onthisbasis,thedevelopmentof aself-sustainableecosystembasedoncyclingrenewableorganic Cbetweenwastesandaddedvalueproductsappearsa fascinat-ingreachablegoal,certainlyworthwhiletopursuit.Generally,the environmentalbeneﬁtsofthesesubstanceslieinthefactthatthese productsarepotentiallyalternativetocommercialsynthetic chem-icals.Thus,bytheiruse,onemayexpectareductionoffossilsource depletionandofCO2emissions.

Therealizationoftheaboveperspectivesdoesnotdependonly ontheproductpropertiesandperformance,butalsoon sustainabil-ityoftheproductionprocess.Rossoetal.(2015)havereportedthe hydrolysisofseveralmaterialsrecoveredfromdifferentstreamsof atypicalmunicipalwastetreatmentplantlocatedinNorthWest Italy.Thesematerialswerethedigestateoftheplantbiogasreactor performingtheanaerobicdigestionoftheorganichumidfraction, andseveralcompostsobtainedfrommixturesofdigestate,private andpublicgardeningresidues,and/orsewagesludge.The hydrol-ysisofthesematerialswascarriedoutinwateratpH13and60◦C. Inallcases,thesolublehydrolysatewasobtainedtogetherwith equalorhigheramountsofinsolublehydrolysate.Thepublished datashowedthatboththesolubleandinsolublehydrolysates con-tainorganicandmineralmatter,buttheorganic/mineralratiois higherintheformer.Thesolublehydrolysatescontainthelowest relativeamountoftotalmineralmatter,comparedwiththe insolu-blehydrolysatesandthepristinesourcingmaterials.Althoughthis process,performedat low temperatureand withcomplete sol-ventandreagentsrecycling,appearsecofriendly,a criticalpoint remainstheproductionoftheinsolublehydrolysate.Intheabsence ofdemonstrationofapromisinguseofthisproduct,itsdisposalis aneconomicburdenforthesolublehydrolysateproduction pro-cess.Yet,by itscontentofmineralelements, alsotheinsoluble hydrolysateproductmightbeusefulforagriculture.

Thepossibilityofboostingyieldandqualityofcropsbyusing biostimulantshasbeenlargelyinvestigatedinthelastyears(e.g. Bulgariet al., 2015; Calvo et al., 2014; du Jardin,2015). Many relationshipsbetweentheapplicationofbiostimulantsandcrop performance havebeen assessed inseveral experimentaltrials. However,themechanismsactivatedatplantlevelbythese prod-uctsarelargelyunknownanddifﬁculttoidentify.Biostimulants havebeenfoundtoenhanceplantgrowthand/orproductionand quality(Calvoetal.,2014),nutrientuptake(Saaetal.,2015), photo-syntheticactivity(Castroetal.,2012),plantresponsetobioticand abioticstresses(duJardin,2015),andseveralmetabolicprocesses (Bulgarietal.,2015;Calvoetal.,2014).Mostliteratureon biostim-ulantsreportstheirusebyfoliarapplication(e.g.Saaetal.,2015). However,thepossibilityofsupplyingthesesubstancesdirectlyto therootzoneisvaluableduetoimprovedefﬁciencyofdistribution anddosage.Thisisespeciallytrueiffertigationisadoptedforcover fertilization;obviously,thispracticeisrestrictedonlytosoluble compounds.

Muchresearchactivityontheuseofbiostimulantsin agricul-turehasbeencarriedoutundersoilconditionsforextensivecrops (Calvoetal.,2014;duJardin,2015),whilescarceliteratureis avail-ableforsubstratecultures(e.g.Bulgarietal.,2015).Forthelatter, rootzoneconditionsaresigniﬁcantlydifferentfromsoilintermsof bio-organicprocess(e.g.microbialactivity),climate(e.g. temper-ature,humidity)andchemo-physicalcharacteristics.Inintensive agriculture,suchascontainer-grownplantsystems,therequired agrochemicals,water,andenergyinputsperunitareaare signif-icantlyhigher, compared withthose neededfor extensivecrop systems.Theformersystemsapplytoornamentalplants,forwhich

thehighaestheticqualityisacriticalmarketabilityparameter.In this scenario,using natural productstoboost plants highyield andqualitycouldsigniﬁcantlyimprovetheuseefﬁciencyof non-renewableresources(e.g.freshwaterandchemicalfertilizers),thus increasingenvironmentalandeconomicsustainability.Thisposes worthwhileresearchscopeonsuchproductsfor understanding bettertheiractiononplantthroughtherootzoneandoptimizing theirdirectadministrationtothesubstrate.

Thepresentworkwasundertakentoaddressseveralquestions posedinpreviousexperimentsontheperformanceoftheabove soluble biowaste hydrolysates as promoter of plant photosyn-theticactivity,growthandproductivity,andonthesustainability oftheirproductionprocess.Tothispurpose,Hibiscuswaschosenas testplant.Thesolublehydrolysateswereobtained,togetherwith thecorrespondinginsolublehydrolysate,byalkalinehydrolysisof twodifferentfermentedmunicipalbiowastematerials.The solu-bleandinsolublehydrolysates,andtheirsourcingmaterials,were distributedattransplantaspowder blendedwiththesubstrate, and/orsolubilisedintotheirrigationwaterandsuppliedthrough fertigation.Acommercialproduct,claimedbythevendortohave biostimulantproperties,wasalsotestedbysubstrateapplication. Thisproduct,underthetradeofNOVA@®GR,isproducedand dis-tributedbyBiolchimSpa,Medicina(BO),Italy.Theappliedproducts wereevaluatedfortheireffectsonseveralplantperformance indi-catorsrelatedtoplantbiomassaccumulationandquality,andto leafgasexchange activity.Main goalsof theexperiment were: (i)evaluationofthedifferenturbanbiowastesourced materials and/ortheirmixtures;(ii)assessmentoftheproductapplication methodology;(iii)evaluationoftheaboveurbanbiowastesourced materialswithrespecttoacommercialbiostimulant.

2. Materialsandmethods

2.1. Location,plantmaterialandgrowingconditions

Theexperimentwascarriedoutinthespring-summerof2014, under typicalMediterranean climateconditions, in anopen-air experimentalnursery locatedin Pescia (Tuscany, Italy; latitude 43.54N,longitude10.42E)attheLandscapingPlantsandNursery ResearchUnitoftheItalianCouncilforAgriculturalResearchand Economics.

Hibiscusseedlings(HibiscusmoscheutosL.subsp.Hibiscus palus-tris)were transplanted on10th of April into four-litrepots (Ø 18cm)containingabasesubstrate(peat50%andpumice50%,V/V) adjustedtopH6.

Plantswereleft30daysundershadenet(40%ofshading)for acclimatisationtoexternal(outdoor)conditions.Thirtydaysafter transplant (T1), potsweremoved to theexperimental site and arrangedinarandomized-blockexperimentaldesign,withthree replicatesper treatment(eight plants per replicate).Pots were positionedat0.40×0.60meachother,obtainingacropdensity of 4.2plantsm−2. Twenty days after T1 (DAT1), allplants were trimmedabovethefourthtrueleaftostimulatetheemissionof lat-eralshootsasrecommendedbythestandardcultivationtechnique incommercialnurseryproductionofHibiscuspalustris.The exper-imentwasconcludedat110DAT1withthedestructiveanalysisof plantmaterialperformedroughlyonemonthbeforethebeginning ofplantsenescence(basingonlocalclimateconditions).

Irrigationwaterwassuppliedthroughdripirrigationsystem(2 emittersperpotwithatotalﬂowrateof7.5lh−1,onaverage),using atimerfortriggingirrigationfourtimesperday(onaverage,overall thecultivationperiod).Irrigationschedulingwasadjustedweekly accordingtoclimateconditionsand leaching fraction(theratio betweenwaterdrainageandwatersupply),whichwascalculated bymeasuringthevolumeofwaterdrainedoutfromalimited(three

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perblock)numberofpots.Irrigationwasregulatedtokeepleaching fractiononatargetvalueofroughly10–15%.IrrigationwaterpH andelectricalconductivityranged6.2–6.6and0.42–0.60dSm−1, respectively.

Priortoaddingthebiobasedproductsunderinvestigation, stan-dardbasicfertilization wasappliedtothecultivationsubstrate, basedonplantnutrientuptake,andtakingintoaccountirrigation watercharacteristicsandpossiblefertilizerlossesduetoleaching. However,thefertilizationplanwasdisposedwiththeaimof pre-ventinganynutritionalstress.Macroandmicro-nutrientswerein partsuppliedatthetransplantdate,throughcontrolledrelease fer-tilizersblendedwiththesubstrate(3kgm−3ofOsmocotePro®3–4 months+3kgm−3ofOsmocotePro®5–6month).Anadditional fer-tigation,betweentheexponentialgrowthandincipientﬂowering phases,(from40to50DAT1)wasperformedwithwatersoluble saltsforatotalsupplyof0.21N,0.28P2O5and0.13K2Okgm−3of substrate.

Climate conditions were monitored continuously (5-minute basis) during the experiment by recording radiation, relative humidityandtemperature oftheairthrough theon-site mete-orological station (Dacagon Device, Pullman, WA 99163 USA). Minimum,meanandmaximumdailyaveragephotosynthetic pho-tonﬂuxdensityvalueswere109.2,568.3and750.5␮molm−2s−1, respectively.Meandailyglobalradiationaveraged21.7MJm−2d−1. Minimum,meanandmaximumdailyaverageofairtemperature valueswere11.6,20.5and22.3◦C,respectively.Meandailyrelative humidityofairaveraged64.5%.

2.2. Biobasedmaterialsunderinvestigation

Thesolubleandinsolublehydrolysateproductswereproduced andsuppliedbyStudioChionoedAssociatiinRivaroloCanavese, Torino,Italy.TheywereobtainedaspreviouslyreportedbySortino etal.(2014).Thepristinematerialswerethedigestaterecovered fromthe anaerobicfermentation of theorganichumid fraction ofmunicipalsolidwastefromseparatesourcecollection,andthe greencompostobtainedby180daysaerobicfermentationof pri-vategardeningandpublicparktrimmings.Thedigestateandthe compostmaterialswereprocessed ina pilotproduction facility comprisinga500lcapacityreactor.Thesolidmaterialwastreated under stirring with alkaline water at 4mlg−1 liquid/solid and 0.02w/wKOH/solidratiosat60◦Cfor4h.Thereactionmixwas allowedsettlingtoseparatethesupernatantliquidcontainingthe solublehydrolysatefractionfromthesolidinsolublehydrolysate residue.Thelatterresiduewaswashedoncewithfreshwaterat 4mlg−1 addedwater/insoluble hydrolysateratio. Thetotal col-lectedliquidphasewascentrifugedtoseparatefinesolidparticles, andrunthrougha5kDacutoffultrafitrationpolysulphone mem-brane.Themembraneretentatecontainingthesolublehydrolysate, andthesolidinsolublehydrolysateweredriedinventilatedoven at60◦C.Thefinaldriedproductswereanalyzedaccordingto pre-viously reportedmethods (Montoneri et al., 2011) and yielded theanalytical valuesreportedinTable1for thesourcing diges-tate(D)andgreencompost(GC)materials,andfortherespective hydrolysates, i.e. the soluble (SD) and insoluble (ID) digestate hydrolysates,andthesoluble(SGC)andinsoluble(IGC)compost hydrolysates.

2.3. Treatments

Treatments were arranged based on the following criteria and/orobjectives:(i)tosupplyamountsoftreatments’products per unit area or plant, which were negligible, compared with theapplieddoses of mostbiowaste sourcedmaterials(such as compost),whicharereportedinliterature(Dorais,2007);(ii)to supplyamountsofmacronutrientscontributedbythetreatments’

products, which were negligible, compared with the normally appliedamountsofchemicalfertilizers;(iii)toaccountforeffectsof treatments’productsasafunctionoftheirformulationand distri-butionmode;(iv)tostudypossibleinteractionsbetweenproducts obtainedbydifferentsourcingmaterials.

Twodifferenttypes(factorsofvariability)oftreatmentswere applied(Table2):(i)thesubstratetreatments(SUB)comprising 11 treatmentsandthecontrol,and thefertigation (FERT) treat-mentscomprising2treatmentsandthecontrol.Intheﬁrstcase, thesubstrateatseedlingstransplantwasblendedwiththe differ-entmaterials,whicharelistedinTable2.Thesearethecommercial NOVAbiostimulant,thegreencompost(GC),theinsoluble com-posthydrolysate(IGC),thesolublecomposthydrolysate(SGC),the compostmixedwithitssolublehydrolysate(GC/S),theinsoluble composthydrolysatemixedwiththecompostsolublehydrolysate (IGC/S),thedigestate(D),theinsolubledigestatehydrolysate(ID), thesolubledigestastehydrolysate(SD),thedigestatemixedwith the soluble digestate hydrolysate (D/S), the insolubledigestate hydrolysatemixedwiththedigestatesolublehydrolysate(ID/S).In thefertigationtreatments,onlythesolublecompost(SGC-F)and digestate(SD-F)hydrolysatesversustheuntreated controlwere tested.Inthiscase,thetotaldoseofeachsolublehydrolysate,which isreportedinTable2,wasdeliveredthroughtheirrigationsystem infourweeklyirrigations,startingat50DAT1.TheNOVAproduct wasnotincludedinthefertigationexperiments,asnoclaimforthis applicationmodewasreportedinthevendor’sproductdescription (Biolchim,2015).Thedaybeforetreatmentsirrigationwas inter-rupted,whichallowedalossofwateruptothesamequantityof easilyavailablewater (roughly400mlpot−1)inthepotvolume. Thisquantitywasthenrecoveredbysupplyingthesameamount ofwaterwiththedispensedsolubleproductsolution.This expedi-entallowedpreventingreductionoftheapplieddoseperplantper week,duetopossibleproducts’leaching.

Table2reportsalsodrymatter,andorganicmatter(OM)and carbon(C)applieddoses.Thesewerecalculatedbasingon previ-ousstudiescarriedoutwithsimilarproducts(Sortinoetal.,2013, 2014).Basedonthechemicalcompositionoftheappliedproducts (Table1), drymatterdoseswerearrangedinordertoapplythe sameamountofOMwitheachproduct.Theonlyexceptionwas thecommercialNOVAproduct,whichwasinevitablyappliedatthe dosesuggestedbytheproducer.Theproducts’mixturesfor treat-ment6,7,11and12(Table2)weremadeat4/1OMw/wratioof therespectiveingredients.

Theaboveexperimentalplan,containingthetwoSUBandFERT factors of variability (Table 2), allowedanalysing36 SUBxFERT combinations.Inthisfashion,threetypesofeffectscouldbe inves-tigated:i)effectsduetothenatureoftheappliedproduct;ii)effects relatedtotheproductapplicationmode;iii)effectsfromthe pos-sibleinteractionoftheproductnatureandapplicationmode.In additiontowhatreportedinTable2,sixmoretreatmentswere alsocarried out. In this case, the controlsubstrate and soluble hydrolysatesinfertigationtreatmentswerereplicatedbutwitha totalsupplyofnutrientsequaltoroughly1/3thedosereportedin Table2.Thesetreatmentswereexcludedfromstatisticalanalyses, exceptforthelinearregressionanalysisofphotosyntheticrateand leaftissuecharacteristics(i.e.leafNandchlorophyllcontent).To thispurpose,theadditionaltreatmentswerenecessarytohavea widerrangeofvalues,especiallybelowoptimalgrowingconditions (i.e.limitednutrientavailability).

2.4. Plantbiomassanalyses

Plantgrowthwasmonitoredmeasuringleafareaindex(LAI),by portableceptometer(AccuPARLP-80,DecagonDevicesInc, Pull-man WA 99163 USA),and plant height,twiceper month. Two destructive plant analyses, at 50 (just before the beginning of

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Table1

pH,organicmatter(OM),carbonconcentration(C),C/Nratioandmineralelementsconcentrationofthetestedproducts.Concentrationvaluesreferredtodrymatter:averages andstandarddeviationcalculatedovertriplicatemeasurements.

Parameter Digestate(D) Insoluble digestate hydrolysate(ID) Solubledigestate hydrolysate(SD) Compost(GC) Insoluble compost hydrolysate(IGC) Solublecompost hydrolysate (SGC) Commercial product(NOVA) OM(g100g−1₎ _61.5_±_0.6 _55.5_±_1.5 _66.4_±_0.9 _41.0_±_0.6 _50.7_±_0.2 _64.6_±_1.3 _73.8_±_1.0 C(g100g−1₎ _34.0_±_0.8 _30.0_±_0.5 _40.0_±_0.4 _22.7_±_0.4 _28.4_±_1.2 _39.0_±_0.5 _23.6_±_0.5 N(g100g−1₎ _3.99_±_0.07 _1.36_±_0.08 _6.63_±_0.08 _2.30_±_0.09 _1.69_±_0.09 _4.49_±_0.21 _5.28_±_0.10 C/N 8.5 22.1 6.0 9.9 16.8 8.7 4.5 OM/(C+N) 1.6 1.8 1.4 1.6 1.7 1.5 2.6 P(g100g−1) 1.4±0.06 1.2±0.01 0.50±0.04 0.17±0.01 0.16±0.01 0.23±0.02 0.20±0.01 K(g100g−1₎ _0.72_±_0.03 _2.43_±_0.05 _1.59_±_0.06 _1.43_±_0.08 _1.11_±_0.02 _2.12_±_0.21 _0.27_±_0.02 Ca(g100g−1₎ _6.04_±_0.08 _6.50_±_0.05 _2.08_±_0.05 _3.21_±_0.04 _3.17_±_0.09 _2.86_±_0.38 _1.56_±_0.10 Mg(g100g−1₎ _0.82_±_0.02 _0.99_±_0.02 _0.27_±_0.01 _0.56_±_0.01 _0.58_±_0.01 _0.38_±_0.06 _0.57_±_0.02 Fe(g100g−1₎ _1.21_±_0.03 _1.15_±_0.01 _0.52_±_0.00 _1.54_±_0.03 _2.13_±_0.07 _0.83_±_0.04 _0.22_±_0.01 Si(g100g−1) 3.74±0.05 4.20±0.03 0.25±0.03 6.24±0.04 7.50±0.10 0.43±0.01 0.10±0.01 Al(g100g−1) 0.27±0.02 1.0±0.06 0.10±0.04 0.25±0.01 0.23±0.01 0.13±0.04 0.01±0.00 Na(g100g−1₎ _0.36_±_0.02 _0.31_±_0.01 _0.19_±_0.01 _0.50_±_0.04 _0.66_±_0.01 _0.30_±_0.01 _0.19_±_0.01 Se(mgkg−1₎ _<1 _<1 _1.65_±_0.11 _<1 _<1 _<1 _1.45_±_0.09 Cu(mgkg−1₎ ₁₀₁_±₂ ₆₆_±₄ ₂₆₂_±₁ ₈₇_±₁ ₅₄_±₃ ₂₆₄_±₄ ₅_±_0.1 Ni(mgkg−1₎ ₄₇_±₀ ₅₀_±₁ ₂₄_±₁ ₈₁_±₃ ₁₀₂_±₃ ₉₁_±₁ ₁₃_±₁ Zn(mgkg−1₎ ₂₉₁_±₆ ₁₄₄_±₄ ₃₆₁_±₄ ₁₉₆_±₁₃ ₁₄₀_±₃ ₃₀₃_±₁ ₂₄_±₁ Cr(mgkg−1) 50±1 48±1 15±0 49±1 104±3 32±1 13±0.5 Pb(mgkg−1) 81±2 71±1 46±2 45±2 45±1 65±1 0.9±0.03 Table2

Applied,drymatter,organicmatter(OM)andcarbon(C)dosesinthedifferentsubstrateandfertigationtreatments.DrymatterdoseswerenormalizedbytheOMcontent ofeachmaterial,inordertoapplythesameOMamountineachtreatment,exceptforNOVAappliedattheratesuggestedbytheproducer.

Treatment Drymatter

(kgm−3₎

OM(kgm−3₎ _C_(kg_m−3₎ _Applied_product_description

Substrate(SUB) Productssuppliedaspowderblendedwiththesubstrateattransplant

1NO 0 0 0 Notreatment(control)

2NOVA 1.90 1.33 0.42 Commercialbiostimulant

3GC 1.68 0.65 0.36 Greencompost

4IGC 1.32 0.65 0.37 Insolublecomposthydrolysate

5SGC 1.04 0.65 0.39 Solublecomposthydrolysate

6GC/S 1.55 0.65 0.37 MixofGCandSGC,and

relativecomposition

GC 1.34 0.52 0.29

SGC 0.21 0.13 0.08

7IGC/S 1.27 0.65 0.38 MixofIGCandSGC,

andrelative composition

IGC 1.06 0.52 0.30

SGC 0.21 0.13 0.08

8D 1.14 0.65 0.36 Digestate

9ID 1.21 0.65 0.35 Insolubledigestatehydrolysate

10SD 0.99 0.65 0.39 Solubledigestatehydrolysate

11D/S 1.11 0.65 0.37 MixofDandSD,and

relativecomposition

D 0.91 0.52 0.29

SD 0.20 0.13 0.08

12ID/S 1.16 0.65 0.36 MixofIDandSD,and

relativecomposition

ID 0.96 0.52 0.28

SD 0.20 0.13 0.08

Fertigation(FERT) Productssuppliedinaqueoussolutionthroughfertigation

13NO-F 0 0 0 Notreatment(control)

14SGC-F 1.04 0.65 0.39 Solublecomposthydrolysate

15SD-F 0.99 0.65 0.39 Solubledigestatehydrolysate

fertigationtreatments)and 110DAT1 (attheend ofthe

exper-iment)wereperformedto quantifytheeffect of treatmentson

physiological and biometric parameters, biomass accumulation

andpartitioning,andtissuemineralcontentoftheplants.Before

destructiveanalysis,SPADindexwasmeasuredthroughaportable

SPAD-502(KonicaMinoltaOptics,2970Ishikawa-machi,Hachioji,

Tokyo,Japan)onsix healthyleavesper plant,fromthebottom

tothetop,and thenaveraging a totalof 18 measurementsper

replicate.Plantheight,meandiameter(ofthecanopyprojectedto

thesoil)andnumberofﬂowersandstemsweremeasured.Plan

height(cmpt−1)andmeandiameter(cmpt−1)wereusedto

cal-culateplantellipsoidvolume(cm3_pt−1₎_and_the_plant_shape_index

(astheplantheighttodiameterratio).Afterward,theplant

mate-rialwasseparatedintoﬂowers,stemsandleaves,andweightedto

assessbiomassfreshweight.Aportion(roughly150g)ofleaves

wasusedformeasuringunitleafareathroughaleafareameter

(WinDIASImageAnalysisSystem,Delta-TDevices,U.K.).Unitleaf

areawasusedtocalculateleafareaindex(LAI,m2_m−2_)._Plant_fresh

materialwasdriedinaforced-airoven(80◦Cfor72h)andweighed

forassessingplantdrymatteraccumulation.

Leaftissueswereanalysedfortheorganicnitrogencontent(N)

by Kjeldhal method.Leaf dry matter(0.5g) wasweighted, put

intopyrextubeswithaseleniumcatalyser(potassiumsulphate

4.63g,coppersulphate0.28gandselenium0.09g)anddigested

with12mlofphosphosulfuricacidat370◦Cfor40.Attheendof

thedigestion,thesamplesweredistilledusingtheVELP-UDK127

apparatus(VELPScientiﬁca,UsmateMB,Italy)afteradding40ml

ofNaOH(40%w/V).Thedistillatewascollectedinaconicalﬂask

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colourindicator.Finally,thecontentofNwasdeterminedby

titra-tionwith0.1NHCl.

2.5. Leafgasexchangemeasurements

Leafgasexchangemeasurementswereperformedintwo

differ-entperiods,duringtheexperiment,at45DAT1,justbeforetheﬁrst

destructiveplantanalysis,or105DAT1.Aportablegasanalyzer

(Portable PhotosynthesisSystemCiras-2,PPSystems, Amesbury,

MA01913USA)wasusedtoperformonsitemeasurementsduring

the morning (between 9.00 and 12.00 am). During

measure-ments,tomaintaincomparableanalyticalconditions,thechamber

was set at a constant value of light saturating photosynthesis

(PPFD=1000␮molm−2s−1,primarilydeterminedthrough

photo-synthesislight-responsecurves),CO2(400gm−3),vapourpressure

deﬁcit(VPD=1.0±0.2kPa)andtemperature(27.5±1◦_C)._The

tem-peraturevaluewastheaverageofclimatedatarecordedwitha

datalogger,inthesamedailyperiodofmeasurements,overthe

threedayspriortomeasurementstartup.Twomatureandhealthy

leaves(secondandfourthleaf,completelyunfoldedabovetheapex

of the main stem) were chosen for gas exchange analysis. For

thesemeasurements,twoplantsperreplicatewereused.Atotal

oftwelvemeasurementspertreatmentcombinationateachstage

wascarriedout.Thisprocedureprovidedacquisitionofvaluesfor

currentnetphotosynthetic(carbonassimilation) rate(Pn,␮mol

m−2s−1),transpirationrate(E,mmolm−2s−1)andstomatal

con-ductance(Gs,mmolm−2s−1).Theformer twoparameterswere

ﬁnallyusedforthecomputationofwateruseefﬁciency(PnE−1,

␮molCO2mmol−1H2O).

2.6. Statistics

Collected data were analysed by two-way ANOVA, in order

toassesssigniﬁcant(P≤0.05,0.01and0.001)differencesamong

treatments.MeanvalueswerethenseparatedbyDuncan’smultiple

rangetest(P=0.05).Dataanalysisincludedalsomultiple-variable

analysis(correlation analysis,n=36),variables’ relationshipsby

linearregressions(n=42forleafNandchlorophyllcontentorn=36

forall othervariables)and principalcomponentanalysis(PCA).

Statisticsandgraphicsweresupportedbytheprograms

Statgraph-icsCenturionXV(StatPoint,Inc.,Herndon,VA,USA)andPrism5

(GraphPadSoftware,Inc.,LaJolla,CaliforniaUSA).

3. Results

3.1. Chemicalcompositionofbiowasteandcommercialproducts.

Table 1 reports the chemical composition of the municipal biowasteandthecommercialproductstestedfortheireffectsin thecultivationofHibiscus.NOVAisclearlydistinguishedforthe highestcontentofOM,therelativelylowestorganicCandhighest Ncontents.Indeed,theNOVAOM,C/NandOM/(C+N)valuesare 73.8%,4.5and2.6,respectively,whilethevaluesforthebiowaste productsrangefrom50.7to66.4%OM,from6to22.1C/Nandfrom 1.4to1.8OM/(C+N).Thesefeatures reﬂectthedifferentnature oftheproducts’sources.Inessence,themunicipalbiowaste prod-uctsarederived frommixturesof unsortedkitchenwastes and gardeningresidues,pre-treatedbyanaerobicand/oraerobic fer-mentationfollowedbychemicalhydrolysis.ThecommercialNOVA product(Biolchim,2015)isdescribedbythevendorasaproprietary mixofplantextractrichinfulvicacids,humicacids,aminoacids andglycinebetaineasbiostimulants.Also,thebiowasteproducts presentdifferences,onefromtheother.Comparedwiththepristine digestate(D)andcompost(GC)materials,andwiththeinsoluble digestate(ID)andcompost(IGC)hydrolysates,thesoluble diges-tate(SD)andcompost(SGC)hydrolysateshavethehighestcontent

oforganicmatter,CandN.Comparedwiththesolubledigestate hydrolysate(SD),thesolublecomposthydrolysate(SGC)hashigher contentofallmineralelements,exceptforPandN,whicharehigher inthesolubledigestatehydrolysate(SD).Itisalsoworthwhileto observethat,comparedwithallotherproductsinTable1,the insol-ublecomposthydrolysate(IGC)hasthehigestcontentofSiandFe, followedbythepristinecompost(GC)intheorderofdecreasingSi andFe.

3.2. Biomassaccumulationandbiometricparameters

Datacollectedduringthefirstdestructiveanalysis,carriedoutin themiddleoftheexperimentalperiod,beforetheinitiationof fer-tigationtreatments(50DAT1),didnotshowanysignificanteffect onbiomassaccumulationandotherbiometricparametersbythe substratetreatments(datanotshown).Onthecontrary,thedata (Table3)collectedbydestructiveanalysis,performedattheendof theexperiment(110DAT1),demonstratedsignificanteffectsondry biomassaccumulationandleafareaindex(LAI).Table3showsthat allsubstratetreatments,excepttreatment7bythemixof insolu-bleandsolublecomposthydrolysates,increasedsignificantlythe leafdryweight(DW),comparedwiththecontrolnotreatment1. SignificantincreasesinleafDWwerealsoobservedbythe ferti-gationtreatments14(SGC-F)and15(SD-F),comparedwiththe controlplants.SimilarresultswerefoundforLAI(Table3).A sig-nificantpositive correlationbetweenLAI and leafDW(R=0.90, P<0.001,)wasfound.Inparticular,inthefertigationtreatments 14(SGC-F)and15(SD-F),thesolublecompostandsoluble diges-tatehydrolysates,respectively,caused30.2%higherLAI,compared withthecontrolplantsinthenotreatment13(NO-F).

Thestem and flowerDW,and thetotal shoot DW(Table 3) showedhigherselectivityinestablishingtherankingorderofthe treatments effects. Shoot DW correlated better with stem and flower DW(R=0.99, P<0.001) than withtheleaf DW(R=0.92, P<0.001). In the substrate treatments, the highest increase (25–26%)was caused bythesoluble digestate hydrolysate(SD) treatment10only,comparedwiththecontrolnotreatment1.For thestemandflowerDW,theothertreatmentsdidnotappearto causesignificantlydifferenteffects,comparedwiththecontrolno treatment1.ForthetotalshootDW,onlythecommercialNOVA, and the insoluble(ID) and soluble (SD) digestate hydrolysates causedsignificantincrease,comparedwiththecontrolno treat-ment 1.In thefertigation treatments14 (SGC-F) and 15(SD-F) bythesolublecompostandsolubledigestatehydrolysatescaused significant24.2%stemandflowerDW,and22.4%totalshootDW increase,comparedwiththecontrolnotreatment13(NO-F).

UnlikethesigniﬁcantdifferencesfoundinDWaccumulation, thedifferenttreatmentsdidnotaffectatallneitherbiomass parti-tioningnorthepercentageofDWinplanttissues(datanotshown). Thelatterwas17.2%forﬂowers,27.3%forleaves,and29.8%for stems,calculatedastheaverageofalltreatmentcombinations.

Neitherthesubstratenorthefertigationtreatmentsinfluenced significantlythemainbiometricparameters,whichweremeasured duringthefirst destructiveanalysis(50 DAT1).Inthesubstrate treatments,theonlystatisticallysignificantproveneffectwasthe plantvolumeincrease,whichwascausedbythecommercialNOVA productintreatment2attheendofthecultivationcycle(Table3). On the other hand, thesoluble compost and soluble digestate hydrolysates,whichweresuppliedthroughfertigationtreatments 14(SGC-F)and15(SD-F),increasedsignificantlyplantheightand volume(Table3),comparedwiththecontrolnotreatment13 (NO-F).

Table3showsalsothattheplantshapeindexwasreducedbythe fertigationtreatments14(SGC-F)and15(SD-F),performedwith thesolublecompostandsolubledigestatehydrolysates,compared withthecontrolnotreatment13(NO-F).Indeed,thecontrolplants

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Table3

Dryweight(DW)ofleaves,stemsandﬂowers,andtotalshoot,andleafareaindex(LAI),pantheight,plantvolume,plantshapeindex(heighttodiameterratio)measuredat theﬁnaldestructiveanalysis(110DAT1).

Treatmenta _Leaf_DW(g_m−2₎ _Stem_and_ﬂower

DW (gm−2₎

TotalshootDW

(gm−2₎ LAI(m

2_m−2₎ _Plant_height

(cmpt−1₎ Plant_(cm3_ptvolume−1₎ Plantshapeindex

Substrate(SUB)

1NO 99.57c 191.58bc 291.15cd 1.16c 44.44 66.58b 0.85 2NOVA 122.64a 225.46ab 348.10ab 1.48a 46.94 75.74a 0.88 3GC 109.58abc 215.09abc 324.67a-d 1.35abc 46.44 54.28bc 1.01 4IGC 107.71abc 198.85bc 306.56bcd 1.35abc 43.56 44.09c 1.01 5SGC 116.85ab 217.15abc 334.00abc 1.42ab 44.22 50.44bc 0.99 6GC/S 105.87bc 214.96abc 320.83a-d 1.35abc 48.00 58.33bc 1.02 7IGC/S 100.62c 178.10c 278.73d 1.19bc 44.89 52.91bc 0.97 8D 117.76ab 202.91abc 320.66a-d 1.41ab 45.11 60.90b 0.94 9ID 122.79a 221.91ab 344.70ab 1.50a 45.78 55.28bc 1.00 10SD 121.61a 241.49a 363.11a 1.54a 44.72 56.65bc 0.95 11D/S 116.80ab 225.35ab 342.15abc 1.48a 44.83 54.02bc 0.96 12ID/S 102.71bc 196.72bc 299.43bcd 1.33abc 45.22 54.00bc 0.99 Fertigation(FERT)

13NO-F 99.31b 181.54b 280.85b 1.16b 43.26c 38.48c 1.06a 14SGC-F 118.13a 226.44a 344.57a 1.47a 45.26b 59.56b 0.93b 15SD-F 118.69a 224.41a 343.10a 1.51a 47.51a 72.76a 0.91b Signiﬁcanceb

SUB 0.001 0.043 0.011 0.008 n.s. 0.009 n.s.

FERT <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

SUBxFERT n.s. n.s. n.s. n.s. n.s. n.s. n.s.

a_{Abbreviations}_and_materials_description_as_in_Table₂_.

b _Statistical_analysis_performed_through_two-way_ANOVA;_P-value_or_n.s._are_reported_for_signiﬁcant_or_non_signiﬁcant_differences,_{respectively.}_Different_letters_within

eachcolumnandeachfactorofvariability(SUBorFERT)indicatesigniﬁcantdifferencesaccordingtoDuncan’smultiple-rangetest(at95%ofprobability).

Table4

Leafchlorophyll(SPADindex)andnitrogen(N)concentrationmeasuredattheﬁnal destructiveanalysis.

Treatmenta _SPAD _N(g₁₀₀_g−1₎

Substrate(SUB)

1NO 40.72c 2.45cd

2NOVA 43.52abc 2.80ab

3GC 43.80abc 2.70abc

4IGC 46.73a 2.85a

5SGC 45.16ab 2.54a-d 6GC/S 42.28bc 2.50bcd 7IGC/S 41.11c 2.36d 8D 43.77abc 2.75abc 9ID 43.44abc 2.55a-d 10SD 43.97abc 2.66a-d 11D/S 44.20abc 2.69a-d

12ID/S 45.78ab 2.81ab

Fertigation(FERT) 13NO-F 41.35c 2.58 14SGC-F 45.85a 2.73 15SD-F 43.92b 2.60 Signiﬁcanceb SUB 0.013 0.018 FERT <0.001 n.s. SUBxFERT 0.002 n.s.

b _Statistical_analysis_performed_through_two-way_ANOVA;_P-value_or_n.s._are

reportedforsignificantornonsignificantdifferences,respectively.Differentletters withineachcolumnandeachfactorofvariability(SUBorFERT)indicatesignificant differencesaccordingtoDuncan’smultiple-rangetest(at95%ofprobability).

showedlargerandshortervegetativehabitus,comparedwiththe plantstreatedbyfertigation.

3.3. Leafanalysisandgasexchangeactivity

The experimental data collected for leaf chlorophyll and N content, and for leaf gas exchange activity are reported in Tables4and5,respectively.Chlorophyll(representedinthiswork bytheSPADindex)andNcontentinleafsamples,didnotshow signiﬁcantdifferencesamongalltreatmentsintheﬁrst

destruc-tiveplantanalysis at50 DAT1. However,significantdifferences wereobservedlaterduringthelastperiodofanalyses(Table4).In thesubstratetreatments,theinsolublecomposthydrolysate(IGC) treatment4,themixoftheinsolublesolublehydrolysate(ID/S),and thesolublecomposthydrolysate(SGC)treatments12and5, respec-tively,causedthehighestSPADvaluecomparedwiththecontrol notreatment1.ForleafN,onlytheinsolublecomposthydrolysate (IGC)treatment4,themixoftheinsolubleandsolubledigestate hydrolysates(ID/S),andthecommercialNOVAproducttreatments 12and2,respectively,weresignificantlyhigherthanthecontrol notreatment1.Inthefertigationtreatments,thesolublecompost hydrolysatetreatment14(SGC-F)gavethehighestSPADvalue.This was4.4%and10.8%higherthantherespectivevalues,whichwere recordedforthesolubledigestatehydrolysatetreatment15(SD-F) andthecontrolnotreatment13.ForleafN,nofertigationtreatment gavesignificantlydifferentvaluesfromthevaluethatwasrecorded forthecontrolnotreatment13(NO-F).ThedatainTable4alsoshow thataninteractionwasfoundbetweenthemainexperimental fac-torsforchlorophyllSPADindex; suchaninteractionwasdueto thesignificantlyhigherresponseofthesolublecompost(treatment SGC-F)andthesolubledigestate(treatment SD-F)hydrolysates, whencombinedwiththeinsolubledigestatehydrolysate(ID).A heterogeneousresponsewasobservedfortheothercombinations (datanotshown).

LeafNconcentrationwashighlycorrelatedwithSPAD(R=0.85, P<0.001)whileapoor,althoughsignificant,correlationwasfound withleaf DW (R=0.46, P=0.002) and total shootDW (R=0.59, P<0.001)ascanbealsodeducedbyFig.1.Therelationshipbetween SPADandNwassignificantly(P<0.001)representedbyalinear equation(SPAD=13.09N+0.72)withadeterminationcoefficient explaining73%oftheexperimentalvariability(Fig.1).

Gas exchange activity measured at 45 DAT1 did not show any significant difference among treatments for the measured photosyntheticrate(Pn),stomatal conductance (Gs), and evap-otranspiration (E). However, the measurements at 105 DAT1 (Table 5) showedsignificant treatmenteffects. Substrate treat-mentscausedsignificantincreasesofthephotosyntheticactivity,

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Table5

Photosyntheticrate(Pn),stomatalconductance(Gs),evapotranspirationrate(E)andwateruseefﬁciencyasphotosynthetic/evapotranspirationratesratio(PnE−1_),_measured

atsaturatinglightbeforeexperimentending(105DAT1).

Treatmenta _Pn_(␮mol_m−2_s−1₎ _Gs_(mmol_m−2_s−1₎ _E_(mmol_m−2_s−1₎ _Pn_E−1_(␮mol_mmol−1₎

Substrate(SUB)

1NO 17.47d 283.64bcd 3.42cd 5.54bc

2NOVA 20.25ab 307.59bcd 3.90abc 5.55bc

3GC 21.70a 410.29a 3.93ab 6.14ab

4IGC 21.09ab 412.39a 4.48a 4.79c

5SGC 20.25ab 366.65ab 3.88abc 5.63bc

6GC/S 17.65cd 245.50d 3.49bcd 5.77bc

7IGC/S 18.34cd 260.65cd 3.98a 6.16ab

8D 20.70ab 320.64a-d 3.76a-d 5.68bc

9ID 21.21ab 349.28abc 3.88abc 5.71bc

10SD 19.43bc 304.56bcd 3.29d 6.21ab

11D/S 20.96ab 366.94ab 3.63a-d 6.63ab

12ID/S 21.39a 266.70cd 3.40d 7.04a

Fertigation(FERT)

13NO-F 17.40b 286.70b 3.15b 5.97b

14SGC-F 21.62a 382.62a 4.55a 4.77c

15SD-F 21.09a 304.38b 3.55b 6.98a

Signiﬁcanceb

SUB <0.001 <0.001 <0.001 0.005

FERT <0.001 <0.001 <0.001 <0.001

SUBxFERT 0.046 n.s. n.s. n.s.

b_Statistical_analysis_performed_through_two-way_ANOVA;_P-value_or_n.s._are_reported_for_signiﬁcant_or_non_signiﬁcant_differences,_{respectively.}_Different_letters_within

eachcolumnandeachfactorofvariability(SUBorFERT)indicatesigniﬁcantdifferencesaccordingtoDuncan’smultiple-rangetest(at95%ofprobability).

Fig.1. Variableloadings(Fig.1a)anddatascores(Fig.1b)obtainedbyprincipal componentanalysis.Acronyms(Fig1a)representleafstomatalconductance(Gs), transpiration(E)andphotosynthesis(Pn),wateruseefﬁciency(Pn/E),leaf chloro-phyll(SPADindex)andnitrogen(N)content,plantvolume(V),height(H)andshape index(SI),andleaf(LeD),stem(StD)andtotalshoot(stems,leavesandﬂowers)dry weight(TD).Pointsandnumbers(Fig.1b)representtreatmentcombinations(see

Table2):(i)greennumbersshowcombinationswiththecontroltreatment1;(ii)red numbersshowcombinationswiththeNOVAtreatment2;(iii)bluenumbersshow combinationswiththenon-mixedhydrolysateproducts(treatment4,59,and10). Dashedlinesgroupallcombinationswiththedifferentfertigationtreatments:(i) dashedcircleforthecontroltreatment13;(ii)topdashedrectangleforthe solu-blegreencomposthydrolysatetreatment14;(iii)bottomdashedrectangleforthe solubledigestatehydrolysatetreatment15.(Forinterpretationofthereferencesto colorinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle).

compared with the control no treatment 1. Photosynthetic rate of treated plant averaged 20.4␮molm−2s−1 compared to 17.4␮molm−2s−1foruntreatedplants.Thecompost(GC) treat-ment 3gave thehighest24.3% Pnincrease,compared withthe controlnotreatment1.Inthefertigationtreatments,thesoluble compostandsolubledigestatetreatments14(SGC-F)and15 (SD-F)gavesignificantlyhigheraverage22.7%Pnincrease,compared withthecontrolnotreatment13(NO-F).Aheterogeneousplant responsetothedifferenttreatmentcombinationswashighlighted byasignificantinteractionofthemainexperimentalfactors.Inthis case,thegeneraltendencyofsolublecompostandsoluble diges-tatefertigationtreatments14(SGC-F)and15(SD-F)toincreasePn wassignificantlymorepronouncedforthefollowingcombinations: treatments1×14,5×14,8×14,11×14and9×15(seeTable2for combinations)withrespecttountreatedplants(datanotshown). Theseresultsweregenerallydenotingthat:(i)solublecomposthad amajoreffectonPnwithrespecttosolubledigestate;(ii) combi-nationsofproducts(betweensubstrateandfertigationtreatments) derivedfromthesameorganicmatrixeshadmajoreffectsrespect totheirmixtures.Thecollecteddatashowedastrongcorrelation betweenPnandSPAD(R=0.87,P<0.001)asconfirmedbydata anal-ysisreportedinFig.1,andapooreralthoughsignificantcorrelation withtotalshootDWaccumulatedattheendofthecultivationcycle (R=0.65,P<0.001).

Stomatalconductance(Table5)wassigniﬁcantlyincreased,by 45%onaverage,onlybytheinsolublecomposthydrolysate(IGC) andtheGCtreatments4and3,respectively,comparedwiththe control notreatment1. In thefertigation treatments,the solu-ble compost hydrolysatetreatment14 (SGC-F) increased Gsby 25.7%and33.5%,comparedwiththesolubledigestatehydrolysate treatment15 (SD-F)andtothecontrolnotreatment13(NO-F), respectively.Aroughlysimilarplantresponsetothedifferent treat-mentswasobservedfortheevapotranspirationrate(E)reported in Table 5. In the fertigation treatments, the soluble compost hydrolysatetreatment14(SGC-F) increasedthe evapotranspira-tionrateby28.2and44.4%,comparedwiththesolubledigestate hydrolysate treatment15 (SD-F) and the control no treatment 13(NO-F),respectively.Theevapotranspirationrateandthe

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sto-matalconductancewerefoundsigniﬁcantlycorrelated (R=0.61, P<0.001).

Finally,inthesubstratetreatments,thecropwateruseefficiency values(Table5)showednosignificantdifferenceamongmostof treatments.Theonlyexceptionwastreatment12bythemixofthe insolubleandsolubledigestatehydrolysates(ID/S),whichcaused a27.1%significantlyhigherwateruseefficiencyvalue,compared withthe controlno treatment1. In the fertigation treatments, thesolubledigestatehydrolysate(SD)treatment15(SD-F)caused 16.9%significantlyhigherwateruseefficiencyvaluecomparedwith thecontrolnotreatment13(NO-F).Onthecontrary,thesoluble composthydrolysatetreatment14(SGC-F)caused20.1% signifi-cantdecreaseofwateruseefficiency,comparedwiththecontrol notreatment13(NO-F).

3.4. Treatments’rankingorderandprincipalcomponentanalysis SummarizingthedatareportedinTables3–5,itmaybeobserved that,forsubstratetreatments,thebiowastesourcedproductsrank significantlyfirstinmostcases.Exceptionsaretheplantvolume, forwhichthecommercialNOVAbiostimulantranksfirst,andthe plantheightandshapeindex,whichshowednotreatmenteffects. Thesoluble(SD)and/orinsoluble(ID)digestatehydrolysatesrank significantlyfirstfortheireffectsonfourindicatorsconnectedto plantbiomassaccumulationandbiometricparameters(Table3). Thesearetheleaf,stemandflowers,andtotalshootDW,andLAI. Theinsolublecomposthydrolysate(IGC)and/orthecompost(GC) ranksignificantlyfirstfortheeffectsconnectedtothePn,leafN andSPADindex(Table4and5).Therankingorderofthefertigation treatmentssummarizestheresultsdescribedintheabove subsec-tions.Inessence,thesolubledigestateandcomposthydrolysates treatments14(SGC-F)and15(SD-F),comparedwiththecontrolno treatment13(NO-F),showedsignificanteffectsforallindicators. TheonlyexceptionwastheleafNcontent,forwhichno signifi-cantdifferenceoccurredamongtreatments14and15,andtheno treatment13(Table4).Treatment15wasbetterthantreatment14, fortheeffectonplantheightandvolume.Onthecontrary,forthe effectsonGs,EandSPADindex,thesolublecomposthydrolysatein treatment14(SGC-F)performedbetterthanthesolubledigestate hydrolysateintreatment15(SD-F).

The above-summarizedresults were used as a base for the interpretationofrelationshipsamongtreatments,between treat-mentsandnatureofthepristinematerials,andbetweentreatments andinvestigatedvariables.Tothisend,principalcomponent anal-ysis(PCA)wasperformedbyusingtheinvestigated parameters (Fig.1a) tosummarizemain differencesamong treatments and sourcingmaterials underinvestigation(Fig.1b).Asreported in Fig.1,theﬁrstprincipalcomponent1(PC1)separatestreatedand untreatedplants.Such a separationis moremarkedfor fertiga-tiontreatmentsthataregroupedinthreemainareasrelatedto: (i)notreatment13(100%ofitscombinations);(ii)solublegreen composthydrolysatetreatment14(92%ofitscombinations);(iii) solubledigestatehydrolysatetreatment15(83%ofits combina-tions).Thelattertwotreatmentswerethenseparatedbythesecond principalcomponent2(PC2).Accordingtotheloadings(Fig.1), treatmentcombinationswithsolublegreencomposthydrolysate (treatment14)weremorecorrelatedwithecophysiological(Pn,E andGs)parametersandtissuecharacteristics(leafNcontentand SPAD)thancombinationstreatmentsobtainedbysolubledigestate treatment15.However,byPC1itwasclearthatbothtreatments were,ingeneral,morecorrelatedwithbiomassaccumulation,plant heightandvolume,SPAD,leafNcontent,andphotosynthesisthan untreatedplants(notreatment13).Withinthethreemain clus-ters,treatmentcombinationswithNOVAandhydrolysateproducts weremorecorrelatedwiththeaboveparametersthanuntreated plants(controltreatment1),whichperformedatthelowestlevel.

Moreindetails,hydrolysatesproducts(treatment4,5,9and10) were, on average, more correlated with the above parameters thanpristinematerials(treatment3and8)and/ormixed treat-ments(treatment6,7,11and12)thatinsteadgaveheterogeneous responses.

4. Discussion

Toavoidanynutritionalplantstress,thecropreceivedan opti-malamountof nutrientsby standardchemical fertilization(see Section2.1.Theappliedproductsunderinvestigationcontaineda quantityofpotentialplantnutrients(forinstanceroughly2–6%N ondrymatterbasis)thatvaried,asafunctionofproduct chem-ical nature (Table 1).As well known, any increase in nutrient supply,aboveanoptimalthreshold,doesnotproduceany signif-icanteffectonplantyieldanddrybiomassaccumulation(Massa et al., 2009;Reid 2002;Silberbush et al.,2005).Therefore, the amountsofpotentialadditionalnutrients,whichwerecontributed bytheaddedexperimentalbiowasteandcommercialNOVA prod-ucts(Tables1and2),weredeemednegligible(2–5%)compared withtheamountsofnutrients,whichwereavailabletothecrop bythestandardchemicalfertilization.Thisconsiderationappears highlyrelevantforthefollowingdiscussion.

ThedatareportedinTables3–5showthattheplantresponse totheappliedtreatmentswasgenerallypositive,showinghigher accumulation of fresh (data not shown) and dry biomass and photosynthesis,comparedwithuntreatedplants.Consideringthe relativelylowamount(Table2)ofproductsblendedwiththe sub-strate(inthesubstratetreatments),and/ordeliveredthroughthe fertigationsystem(inthefertigationtreatments),theincreasein mostofthemeasuredplantparameters(Tables3–5)was remark-able.Infact,thetotalquantityofproductsusedwasintheorderof 5–15%oftheminimumdoseforcommonorganicfertilizersand/or genericamendments,whichare normallyappliedinagriculture (Dorais,2007).

In recent on-field red pepper and tomato cultivation trials, Sortinoetal.(2013,2014)foundthattheapplicationof700kgha−1 ofurbanbiowastes’hydrolysatesdidnotaltersignificantlythesoil chemicalcomposition.Yet,plantphotosyntethicactivity,growth, andproductivitywereenhancedsignificantlybytheadded solu-blehydrolysates.Theauthorsconcludedthatthetestedproducts enhancedtheplantphotosyntethicactivityandthat,inturn,the increaseofphotosyntheticactivitywasthemainfactor increas-ingplantgrowthandproductivity.Morerecently(Fascellaetal., 2015),thishypothesiswasconfirmedbyapplyingthesamesoluble hydrolysatesforthecultivationofEuphorbiainpots.Thedata col-lectedonHibiscusseemtosupporttheroleofthetestedproductsas promotersoftheplantphotosyntheticactivity(Table5).Inrelation tothesepreviousworks,andtothepresentwork,therearetwokey issuesdeservingfurtherdiscussion.Theseare(i)therelationship betweenleafchemicalfeatures,gasexchangeactivity,andbiomass accumulation,and(ii)therelationshipoftheobservedeffectswith theappliedproductschemicalnatureand/orcomposition.

Several authors (Bulgari et al., 2015; Calvo et al., 2014; du Jardin,2015;Ertanietal.,2013a)haveassociatedtheincreasein drybiomassofagriculturalcrops,treatedwithproductsderived fromorganicmatrixes,tothepresenceoforganicmoleculesthat stimulate plants. More generally, biostimulant substances aug-mentbiomassaccumulationandyield,improve nutrientuptake andenhancemetabolicfunctions.Also,manybiostimulantorganic substances,suchasalgaeand/orseaweedextract,soilandwater humicandfulvicacid,havebeenfoundtoimprovephotosynthetic capacityinseveraldifferentplantspecies,whicharegrowneither underoptimalconditions(Castroetal.,2012;Janninetal.,2012)or inpresenceofabioticstress(Anjumetal.,2011;Ertanietal.,2013b).

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Humic-likesubstances,obtainedfromdifferentorganicwastes,fall inthiscategory,eitherfortheirchemicalnatureorfortheir bios-timulantactivity(Eyheraguibeletal.,2008;Morardetal.,2011). Thebiowastederivedproductstestedinthisworkbelongtothe categoryof“complexorganicmaterial”(duJardin,2015)andbear structuralsimilaritieswithnaturalhumicsubstances(Montoneri etal.,2011).Theyarerichinmolecules,whicharetypicallyfound inproductsclaimedtohavebiostimulantactivity(e.g.Biolchim, 2015).

FortheindicatorsconnectedtoHibiscusbiomassaccumulation inthesubstratetreatments,theproducts’rankingdependsonthe plantparameterandorgan(Table3).Bycomparison,inthe fertiga-tiontreatments,thesolubleproducts(SGC-FandSD-F)improved most of theabove indicators, compared with thecontrol (NO-F).However,itshouldbehighlightedthatfertigationtreatments practicallyreceivedaquantityofproductshigherthansubstrate treatments(Table2),whichcouldraisethehypothesisofan addi-tiveand/ordoseresponseeffectofthetestedproductsasobserved forexamplebySortinoetal.(2013)withsolublehydrolysates.As matteroffact,plantresponsetofertigationtreatmentswasmore pronouncedanddeﬁnedthanforsubstratetreatments(Tables3–5 andFig.1).

Attemptstocorrelatetheobservedeffectrankingorderwiththe products’chemicalcharacteristics(Table1)didnotallowassessing definiteclearproduct-propertiesrelationships.Itistruethatthe investigatedproductsdifferfortheconcentrationofmacro-and micro-nutrients.However,whentheproductsaresuppliedtothe plantsatthedosesreportedinTable2,themineralcomposition dif-ferencesarelikelytobelevelledoutbythehigherrelativelyamount ofnutrientssuppliedbytheconventionalchemicalfertilizers.Itwas worthtodeterminetheamountofbeneficialelementssuchasSe andSiamongtheoligoelementsinthetextedproducts.Selenium canhaveagrowth-promotingeffectformanyplantspecies (Pilon-Smitsetal.,2009),forexamplebyincreasingNreductaseactivity (Nowaketal.,2004).Itnaturallyoccursastraceelementinmost soils,attypicallevelsbelow1mgkg−1(Pilon-Smitsetal.,2009). Seleniumin theinvestigated products(Table 1)hasbeenfound slightlyabovethislevel,onlyinthesolubledigestatehydrolysate (SD)andthecommercialproduct(NOVA).However,neitherany significantvariationofSenorcorrelationwithNcontentwasfound inleaftissues (datanot shown).Thesefindings didnothelpto explaintheproducts’performancerankingorderandwerein agree-mentwithpreviousworks(Hawrylak-Nowaketal.,2015;Riosetal., 2013).

LeafNcorrelated(Fig.1)wellwithSPAD,butlesswellwithPn, andbiomassaccumulationparameters.Weakcorrelationsbetween PnandleafNwereobservedbyotherauthors(e.g.Kenzoetal., 2015).Inthepresentexperiment,itislikelythattheoptimal nutri-entavailability intherootallowedplantstakinguptheneeded quantityofnutrientstosupportthehighergrowthrateinduced intreatedplants,withoutaffectingleafNcontent(Table4).The highcorrelationbetweenPnandSPAD,asobservedinthepresent work(Fig.1),isusual.Veryoften,theincreaseinPn,in biostimu-latedplants,iscoupledtohigherchlorophyllandnutrientcontent inplanttissues(Bulgarietal.,2015;Calvoetal.,2014).Tothis pur-pose,mostofliteratureisbasedonthestudyofextensivecrops, cultivatedonsoilandtreatedthroughfoliarapplication(e.g.Anjum etal.,2011;Janninetal.,2012).Bycomparison,verylittleisknown onpottedornamentalplantscultivated insubstrateandtreated directlyintherootzone.

Thepositiveinﬂuenceoftestedproductsonchlorophyllcontent andPn(Tables4and5)isinagreementwithpreviousworks con-ductedonplantbiostimulationobtainedwithvarioussubstances andproducts(e.g.Amandaetal.,2009;Anjumetal.,2011;Ertani etal.,2013b; Fascellaetal.,2015; Janninet al.,2012). Further-more,therelationshipbetweenSPADindexandleafcolour(e.g.

Papasavvasetal.,2008;Shibayamaetal.,2012)helpstoassessa relevantcommercialparameterforornamentalplantssuchasleaf greenness.Tothispurpose,Lohetal.(2002)proposedSPADmeter aseffectivetoolfortheevaluationofplantqualityinornamental crops.

The data collected on tissue characteristics and leaf gas exchanges (Tables 4 and 5) support the intriguing role of the biowastederivedproductsasphotosensitizers(BiancoPrevotetal., 2011;andGomisetal.,2014),promotersofphotosynthetic activ-ity,inagreementwithSortinoetal.(2013,2014)andFascellaetal. (2015).Thishypothesis is consistentlywithsimilar ﬁndingsfor humicacids(Bulgarietal.,2015;Calvoetal.,2014),bearing struc-turalsimilaritieswiththeabovebiowasteproduct(Montonerietal., 2011).

Thecompost(GC)andtheinsolublecomposthydrolysate(IGC) rankfirstfortheeffectsonPninthesubstratetreatments(Table5). ThesedatawereconsistentwiththePCAshowninFig.1.Table1 showsthattheseproductscontainthehighestamountsofFeand Si,comparedwithallothertestedproducts.Thecontributionof thecompost(GC)andinsolublecomposthydrolysate(IGC)tothe totalamountofFeandSiwasroughly64%and177%,respectively, higherthan theaveragevaluecontributed bytheotherapplied productslistedin Table1.BothSi(Houbenetal., 2014)and Fe (Milleretal.,1995)areknowntohaveimportantrolein photo-synthesis.Gomisetal.(2014)haveproposedthatFeinthesoluble composthydrolysateisresponsibleofthephotosensitizing proper-tiesofthismaterial,whichweredemonstratedfortheremediation ofchemicalindustrialwastewaterscontainingorganicpollutants. Otherworks(duJardin,2015;Liet al.,2015;Pilon-Smitsetal., 2009)reportthatSiisabeneficialelementforplants,inasmuch asitplaysakeyroleagainstseveralbioticandabioticstress,and promotesplantgrowthanddevelopment,chlorophyll concentra-tionandphotosyntheticactivity.Thedatacollectedinthepresent workseemconsistentwiththeaboveliteraturefindings.However, consideringalltreatments,adefinitecorrelationofphotosynthetic activityand/orSPADindexvs.theamountsofappliedSiand Fe amountscouldnotbeestablished.Verylikely,theeffects ofthe abovemineralelementsdonotrelytotheappliedamounts,but dependalsoonthenatureandsolubilityoftheorganicmatterto whichtheyarebonded.

Theconfirmation that thebiowaste-derived products, which weretested inthepresentwork,promote plantphotosynthetic activity is certainly relevant. Nevertheless, under the adopted experimentalconditions,manyotherfactorscouldcontributeto theobservedplantgrowthanddevelopment.Theweak,although significant,correlationfoundbetweenPnandthetotalshootDW (R=0.65,P<0.001;seealsoFig.1)leadstosupposethatnot all appliedproductsinthepresentworkwereabletoinduceareal improvedefficiencyintheconversionofcarbonassimilatesinplant biomass.Asmatteroffact,onlythesoluble(SD)andinsoluble(ID) digestatehydrolysates, andthecommercialNOVAbiostimulant, inthesubstratetreatments,andthesolublecompost(SGC)and solubledigestate(SD),infertigationtreatments,causeda signifi-canthigheraccumulationintotalshootDW(Table3).Thiscould alsoimplylikelyfurtherpositiveeffectsbytheabovementioned treatmentsondifferentaspectsofplantprimary andsecondary metabolism,otherthanphotosynthesis.Forexample,other com-plexorganicmaterialshavebeenfoundimprovingnutrientuptake (e.g.Ertanietal.,2013b;Morardetal.,2011)andothermetabolic processes(duJardin,2015;Ertanietal.,2013a).Ontheotherhand, a highcarbon intakedoesnot necessarily resultsin high long-termcarbonstorage.Indeed,notallassimilatedCO2isconverted instructuralbiomassduetodifferentrespirationefficiency dur-ingdarkhoursandcarbonallocationtothesecondarymetabolism, as for example related to volatile organic compounds (Herms and Mattson 1992). However, the use of products stimulating

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photosyntheticactivityappearsofgreatinteresttoornamental cul-tivationforboostinghighproductionandquality,andforimproving theefﬁciencyofthecultivationprocess.Photosynthesisisa pro-cessverysensitivetobioticandabioticstresses(AshrafandHarris, 2013).Theapplicationofthetestedproductscouldbeapractice forovercomingand/orcontainingmoderatestressfulconditions, which affect photosynthesis (e.g. Flexas et al., 2004; Lieth and Pasian1991;Yamorietal.,2005),withpositiveeffectson produc-tionsustainabilityandmarketcompetition(e.g.shortercultivation cycle).

Inthepresentwork,togetherwiththehighestenhancementof Pn,thecompost-derivedproductscausedthehighestEandGs val-uescomparedtountreatedplants,thusdecreasingcropwateruse efﬁciency(Table5).Generallyspeaking,thesupplyofwatercan notbea limitingfactorincontainer-grownplants.Thisis espe-ciallythecase of ornamentalswhere theaestheticvalueofthe plantisthefundamentalmarketparameter.However,wateruse efﬁciencymustbehighlyconsideredinagricultureforits environ-mentalimplications.

Thecollecteddataallowdrawingsomeconclusionsonsome importantissues,whichhavebotheconomicandenvironmental relevance.Thesearerelated notonly toagriculture,butalsoto urbanbiowasteprocesses and product development.One main issueisthevalue ofthebiowastehydrolysates, comparedwith their sourcing materials. The data reported in Tables 3–5 and Fig.1indicatethatthesolubleand/orinsolublehydrolysatesappear more efﬁcient than the sourcing digestate or compost materi-als.Undoubtedly,themostremarkableanddeﬁnedeffectswere obtainedwithsolublehydrolysatesappliedbyfertigation(Fig.1); sucheffectswerealsoduetoalikelyhigherbioavailabilityof bios-timulantsubstancesfortheaboveproducts.Asmatteroffact,the PCA(Fig.1)highlightedthepresenceofthreemainclusters,related tofertigationtreatments;treatedplantsweremorecorrelatedwith biomassaccumulation,plantheightandvolume,SPADindex,leaf Ncontent,andphotosynthesisthancontroltreatment13.Within thesemacro-clusters,hydrolysateproductsweremorecorrelated withtheabove parametersthanuntreated plants and/orplants treatedonlywiththepristine materials.Theevidencethat both thesolubleandinsolublehydrolysatesarevaluableproducts,more thantheirsourcingbiowastematerials,offerspositiveperspectives fortheirproductionprocess.

A furtherissueaddressed by thepresent work is how gen-eraltheeffectsofthebiowaste-derivedproductsarefordifferent plant species (ornamentals), considering previous experiments reportedonotherplantspecieswiththesameorsimilarfermented biowastes and hydrolysates. Fascella et al. (2015) compared the same soluble digestate hydrolysates (SD) with the soluble hydrolysateobtainedfromacompostedmixoftheabove diges-tate,andurbangardeningwastesandsewagesludge.Theyshowed that,inthecaseofEuphorbiacultivation,thesolublehydrolysate obtainedfromthecompostwasmoreefficientthanthesoluble digestatehydrolysate(SD).Sortinoetal.(2013,2014)compared thesameabovesolubledigestate(SD)andsolublecompost(SGC) hydrolysatesforgreenhousetomatocultivation.Theyfoundthat thesolublecomposthydrolysate(SGC)aswellorbetterthanthe solubledigestatehydrolysate(SD).Roveroetal.(2015)compared thecomposted mix of the above digestate, and urban garden-ing wastes and sewage sludge, and its soluble and insoluble hydrolysates,inmaizecultivationtrialsperformedinopenfield. Noaddedbenefitswereshown fromtheuseofthesoluble and insolublecomposthydrolysates,comparedwiththesource com-post.Theresultsoftheabovereportedstudies,andtheresultsof thepresentwork,demonstratethatproductperformancedepends muchonthecultivatedplantandontheboundary (environmen-tal)conditions.However,differentproductsmaybeobtainedfrom avarietyofdifferentmunicipalbiowastessourcedfromdifferent

locationsandpre-treatedbyanaerobicandaerobicfermentation (Rossoetal.,2015).Thisofferstheprospecttoobtainawide vari-etyofproducts,whichcanbeusedadhocfordifferentcultivations. Itseemsthereforethattheresultsandconclusionsofthepresent workofferworthwhilescopeforfurtherresearchaimingtodevelop biowaste-sourcedproductstailoredforspeciﬁcplants’studiesand cultivation.Tothis purpose, it shouldbeunderlinedthat many commercialbiostimulantproductsofferscarceandoften insufﬁ-cientandorvagueinformationonproductdosageanddistribution. OneofthereasonsforselectingtheNOVAproduct,asreference commercialproductinthepresentwork,wasthedetailed infor-mationreportedbytheproduceraboutthedosageforsubstrate cultivations.Thecomparativedatareportedinthiswork,forthe biowaste-sourcedproductsand for thecommercialNOVA bios-timulant,prove that thebiowaste-derived productshave great potentialinintensiveagriculture.

5. Conclusions

Theresultsobtainedinthepresent workassesstheeffectof biowaste-sourcedproductsoncontainer-grownHibiscus.All treat-mentshavebeenfoundtoenhancemostoftheinvestigatedplant parameters, atdifferent degreeand dependingontheir nature, compared to untreated plants. The most valuable effects were observedonbiomassaccumulation,relativegrowthrate,net assim-ilationrate,SPADindexandgasexchangeactivity.Thecomparison of the above biowaste-sourced products with the commercial NOVAbiostimulantdemonstratesthatthebiowaste-sourced prod-uctshave acommerciallyexploitablepotential.Thecomparison oftheresultsobtainedinthepresentwork,withthosepreviously reportedontheperformanceofsimilarproductsinthecultivation ofotherornamentalandfoodplants,showsthat awidevariety ofbiowaste-sourcedproductsispotentiallyobtainable,whichmay betargetedtothecultivationofdifferentplantspecies.Thiswork offersscopeforfurtherworthwhileinvestigationonotherplant species,usingotherdifferentbiowastes,inordertoassessthefull potentialofmunicipalbiowasteassourceofaddedvalueproducts foruseinagriculture.Thiswouldalsoinvolveabetter understand-ingof thebiomolecular mechanisms rulingthe effects ofthese products.

Acknowledgements

ThisworkwascarriedoutpartlywithfundsfromtheItalian Min-istryofAgricultureaspartofthe“Agrienergia”project.Theauthors aregratefultoDr.A.DiNardo,Dr.L.IncrocciandDr.B.Nesifortheir manuscriptrevisionandcomments.

References

Amanda,A.,Ferrante,A.,Valagussa,M.,Piaggesi,A.,2009.Effectofbiostimulants onqualityofbabyleaflettucegrownunderplastictunnel.ActaHortic.807, 407–412.

Anjum,S.A.,Wang,L.,Farooq,M.,Xue,L.,Ali,S.,2011.Fulvicacidapplication improvesthemaizeperformanceunderwell-wateredanddroughtconditions. J.Agron.CropSci.197,409–417,http://dx.doi.org/10.1111/j.1439-037X.2011. 00483.x.

Ashraf,M.,Harris,P.J.C.,2013.Photosynthesisunderstressfulenvironments:an overview.Photosynthetica51,163–190, http://dx.doi.org/10.1007/s11099-013-0021-6.

BiancoPrevot,A.,Avetta,P.,Fabbri,D.,Laurenti,E.,Marchis,T.,Perrone,D.G., Montoneri,E.,Boffa,V.,2011.Waste-derivedbioorganicsubstancesfor light-inducedgenerationofreactiveoxygenatedspecies.ChemSusChem4, 85–90,http://dx.doi.org/10.1002/cssc.201000237.

Biolchim,2015.NOVA@®_GR_Granular_biostimulant_based_on_plant_extracts,_http://

www.biolchim.it/index.php?lang=en&page=products/biostimulants/novagr granularbiostimulantbasedonplantextracts.html(accessed01.09.15.). Bulgari,R.,Cocetta,G.,Trivellini,A.,Vernieri,P.,Ferrante,A.,2015.Biostimulants

andcropresponses:areview.Biol.Agric.Hortic.31,1–17,http://dx.doi.org/10. 1080/01448765.2014.964649.

(11)

Calvo,P.,Nelson,L.,Kloepper,J.W.,2014.Agriculturalusesofplantbiostimulants. PlantSoil383,3–41.

Castro,J.,Vera,J.,González,A.,Moenne,A.,2012.Oligo-carrageenansstimulate growthbyenhancingphotosynthesis,basalmetabolism,andcellcyclein tobaccoplants(varBurley).JPlantGrowth.Regul.31,173–185,http://dx.doi. org/10.1007/s00344-011-9229-5.

Dorais,M.,2007.Organicproductionofvegetables:stateoftheartandchallenges. Can.J.PlantSci.87,1055–1066.

Ertani,A.,Nardi,S.,Altissimo,A.,2013a.Review:long-termresearchactivityonthe biostimulantpropertiesofnaturalorigincompounds.ActaHortic.1009, 181–188.

Ertani,A.,Schiavon,M.,Muscolo,A.,Nardi,S.,2013b.Alfalfaplant-derived biostimulantstimulateshort-termgrowthofsaltstressedZeamaysL.plants. PlantSoil364,145–158,http://dx.doi.org/10.1007/s11104-012-1335-z. Eyheraguibel,B.,Silvestre,J.,Morard,P.,2008.Effectsofhumicsubstancesderived

fromorganicwasteenhancementonthegrowthandmineralnutritionof maize.Bioresour.Technol.99,4206–4212,http://dx.doi.org/10.1016/j. biortech.2007.08.082.

Fascella,G.,Montoneri,E.,Ginepro,M.,Francavilla,M.,2015.Effectofurban biowastederivedsolublesubstancesongrowth,photosynthesisand ornamentalvalueofEuphorbia×lomi.Sci.Hortic.197,90–98,http://dx.doi.org/ 10.1016/j.scienta.2015.10.042.

Flexas,J.,Bota,J.,Loreto,F.,Cornic,G.,Sharkey,T.D.,2004.Diffusiveandmetabolic limitationstophotosynthesisunderdroughtandsalinityinC3plants.Plant Biol.6,269–279,http://dx.doi.org/10.1055/s-2004-820867.

Gomis,J.,BiancoPrevot,A.,Montoneri,E.,GonzÃ¡lez,M.C.,Amat,A.M.,MÃ¡rtire, D.O.,Arques,A.,Carlos,L.,2014.Wastesourcedbio-basedsubstancesfor solar-drivenwastewaterremediation:photodegradationofemerging pollutants.Chem.Eng.J.235,236–243,http://dx.doi.org/10.1016/j.cej.2013.09. 009.

Hawrylak-Nowak,B.,Matraszek,R.,Pogorzelec,M.,2015.Thedualeffectsoftwo inorganicseleniumformsonthegrowth,selectedphysiologicalparameters andmacronutrientsaccumulationincucumberplants.ActaPhysiol.Plant.37,

http://dx.doi.org/10.1007/s11738-015-1788-9.

Herms,D.A.,Mattson,W.J.,1992.Thedilemmaofplants:togrowordefend.Q.Rev. Biol.67,283–335,http://dx.doi.org/10.2307/2830650.

Houben,D.,Sonnet,P.,Cornelis,J.T.,2014.BiocharfromMiscanthus:apotential siliconfertilizer.PlantSoil374,871–882, http://dx.doi.org/10.1007/s11104-013-1885-8.

Jannin,L.,Arkoun,M.,Ourry,A.,Laîné,P.,Goux,D.,Garnica,M.,Fuentes,M., Francisco,S.S.,Baigorri,R.,Cruz,F.,Houdusse,F.,Garcia-Mina,J.M.,Yvin,J.C., Etienne,P.,2012.MicroarrayanalysisofhumicacideffectsonBrassicanapus growth:involvementofN,CandSmetabolisms.PlantSoil359,297–319.

Kenzo,T.,Inoue,Y.,Yoshimura,M.,Yamashita,M.,Tanaka-Oda,A.,Ichie,T.,2015. Height-relatedchangesinleafphotosynthetictraitsindiverseBornean tropicalrainforesttrees.Oecologia177,191–202,http://dx.doi.org/10.1007/ s00442-014-3126-0.

Li,P.,Song,A.,Li,Z.,Fan,F.,Liang,Y.,2015.Siliconamelioratesmanganesetoxicity byregulatingbothphysiologicalprocessesandexpressionofgenesassociated withphotosynthesisinrice(OryzasativaL.).PlantSoil,http://dx.doi.org/10. 1007/s11104-015-2626-y.

Lieth,J.H.,Pasian,C.C.,1991.Asimulationmodelforthegrowthanddevelopment ofﬂoweringroseshoots.Sci.Hortic.46,109–128.

Loh,F.C.W.,Grabosky,J.C.,Bassuk,N.L.,2002.UsingtheSPAD502metertoassess chlorophyllandnitrogencontentofbenjaminﬁgandcottonwoodleaves. Horttechnology12,682–686.

Massa,D.,Mattson,N.S.,Lieth,H.J.,2009.Effectsofsalinerootenvironment(NaCl) onnitrateandpotassiumuptakekineticsforroseplants:aMichaelis–Menten modellingapproach.PlantSoil318,101–115,http://dx.doi.org/10.1007/ s11104-008-9821-z.

Miller,G.W.,Huang,I.J.,Welkie,G.W.,Pusnik,J.C.,1995.Functionofironinplants withspecialemphasisonchloroplastandphotosyntheticactivity.In:Abadia,J. (Ed.),IronNutritioninSoilsandPlants.KluwerAcademicPublishers,Dordecht, pp.19–28.

Montoneri,E.,Mainero,D.,Boffa,V.,Perrone,D.G.,Montoneri,C.,2011. Biochemenergy:aprojecttoturnanurbanwastestreatmentplantinto bioreﬁneryfortheproductionofenergy,chemicalsandconsumer’sproducts withfriendlyenvironmentalimpact.Int.J.GlobalEnviron.(11),170–196,

http://dx.doi.org/10.1504/ijgenvi.2011.043528.

Montoneri,C.,Montoneri,E.,Tomasso,L.,Piva,A.,2013.Compostderived substancesdecreasefeedproteinNmineralizationinswinececal fermentation.J.Agric.Sci.13,31–44,http://dx.doi.org/10.5539/jas.v5n3p. Morard,P.,Eyheraguibel,B.,Morard,M.,Silvestre,J.,2011.Directeffectsof

Humic-Likesubstanceongrowth,water,andmineralnutritionofvarious species.J.PlantNutr.34,46–59,http://dx.doi.org/10.1080/01904167.2011. 531358.

Nowak,J.,Kaklewski,K.,Ligocki,M.,2004.Inﬂuenceofseleniumonoxidoreductive enzymesactivityinsoilandinplants.SoilBiol.Biochem.36,1553–1558,

http://dx.doi.org/10.1016/j.soilbio.2004.07.002.

Papasavvas,A.,Triantafyllidis,V.,Zervoudakis,G.,Kapotis,G.,Samaras,Y.,Salahas, G.,2008.CorrelationofSPAD-502meterreadingswithphysiological parametersandleafnitratecontentinBetavulgaris.J.Environ.Prot.Ecol.9, 351–356.

Pilon-Smits,E.A.,Quinn,C.F.,Tapken,W.,Malagoli,M.,Schiavon,M.,2009. Physiologicalfunctionsofbeneﬁcialelements.Curr.Opin.PlantBiol.12, 267–274,http://dx.doi.org/10.1016/j.pbi.2009.04.009.

Reid,J.B.,2002.Yieldresponsetonutrientsupplyacrossawiderangeof conditions.1.Modelderivation.FieldCrop.Res.77,161–171.

Rios,J.J.,Blasco,B.,Leyva,R.,Sanchez-Rodriguez,E.,Rubio-Wilhelmi,M.M., Romero,L.,Ruiz,J.M.,2013.Nutritionalbalancechangesinlettuceplantgrown underdifferentdosesandformsofselenium.J.PlantNutr.36,1344–1354,

http://dx.doi.org/10.1080/01904167.2013.790427.

Rosso,D.,Fan,J.,Montoneri,E.,Negre,M.,Clark,J.,Mainero,D.,2015.Conventional andmicrowaveassistedhydrolysisofurbanbiowastestoaddedvalue lignin-likeproducts.GreenChem.17,3424–3435,http://dx.doi.org/10.1039/ c5gc00357a.

Rovero,A.,Vitali,M.,Rosso,D.,Montoneri,E.,Chitarra,W.,Tabasso,S.,Ginepro,M., Lovisolo,C.,2015.Sustainablemaizeproductionbyurbanbiowasteproducts. Int.J.Agron.Agric.Res.6,75–91.

Saa,S.,Olivos-DelRio,A.,Castro,S.,Brown,P.H.,2015.Foliarapplicationof microbialandplantbasedbiostimulantsincreasesgrowthandpotassium uptakeinalmond(Prunusdulcis[Mill.]D.AWebb).Front.PlantSci.6,87,

http://dx.doi.org/10.3389/fpls.2015.00087.

Shibayama,M.,Sakamoto,T.,Takada,E.,Inoue,A.,Morita,K.,Yamaguchi,T., Takahashi,W.,Kimura,A.,2012.Estimatingriceleafgreenness(SPAD)using ﬁxed-pointcontinuousobservationsofvisibleredandnearinfrared narrow-banddigitalimages.PlantProd.Sci.15,293–309,http://dx.doi.org/10. 1626/pps.15.293.

Silberbush,M.,Ben-Asher,J.,Ephrath,J.E.,2005.Amodelfornutrientandwater ﬂowandtheiruptakebyplantsgrowninasoillessculture.PlantSoil271, 309–319.

Sortino,O.,Dipasquale,M.,Montoneri,E.,Tomasso,L.,Avetta,P.,BiancoPrevot,A., 2013.90%yieldincreaseofredpepperwithunexpectedlylowdosesof compostsolublesubstances.Agron.SustainableDev.33,433–441.

Sortino,O.,Montoneri,E.,Patanè,C.,Rosato,R.,Tabasso,S.,Ginepro,M.,2014. Beneﬁtsforagricultureandtheenvironmentfromurbanwaste.Sci.Total Environ.487,443–451,http://dx.doi.org/10.1016/j.scitotenv.2014.04.027. Yamori,W.,Noguchi,K.,Terashima,I.,2005.Temperatureacclimationof

photosynthesisinspinachleaves:analysesofphotosyntheticcomponentsand temperaturedependenciesofphotosyntheticpartialreactions.PlantCell Environ.28,536–547.

duJardin,P.,2015.Plantbiostimulants:deﬁnition,concept,maincategoriesand regulation.Sci.Hortic.196,3–14,http://dx.doi.org/10.1016/j.scienta.2015.09. 021.