Plant biostimulants: Definition, concept, main categories and regulation

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Scientia

Horticulturae

j o u r n a l h o m e 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

Plant

biostimulants:

Definition,

concept,

main

categories

and

regulation

Patrick

du

Jardin

PlantBiologyUnit,GemblouxAgro-BioTech,UniversityofLiège,Belgium,2,PassagedesDéportés,B-5030Gembloux,Belgium

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Articlehistory: Received21May2015

Receivedinrevisedform28August2015 Accepted17September2015 Availableonlinexxx Keywords: Biostimulant Biofertiliser Definition Regulation

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Aplantbiostimulantisanysubstanceormicroorganismappliedtoplantswiththeaimtoenhance nutri-tionefficiency,abioticstresstoleranceand/orcropqualitytraits,regardlessofitsnutrientscontent. Byextension,plantbiostimulantsalsodesignatecommercialproductscontainingmixturesofsuch sub-stancesand/ormicroorganisms.Thedefinitionproposedbythisarticleissupportedbyargumentsrelated tothescientificknowledgeaboutthenature,modesofactionandtypesofeffectsofbiostimulantsoncrop andhorticulturalplants.Furthermore,theproposeddefinitionaimsatcontributingtotheacceptance ofbiostimulantsbyfutureregulations,especiallyintheEU,drawingthelinesbetweenbiostimulants andfertilisers,pesticidesorbiocontrolagents.Manybiostimulantsimprovenutritionandtheydoso regardlessoftheirnutrientscontents.Biofertilisers,whichweproposeasasubcategoryofbiostimulants, increasenutrientuseefficiencyandopennewroutesofnutrientsacquisitionbyplants.Inthissense, microbialbiostimulantsincludemycorrhizalandnon-mycorrhizalfungi,bacterialendosymbionts(like Rhizobium)andPlantGrowth-PromotingRhizobacteria.Thus,microorganismsappliedtoplantscanhave adualfunctionofbiocontrolagentandofbiostimulant,andtheclaimedagriculturaleffectwillbe instru-mentalintheirregulatorycategorization.Thepresentreviewgivesanoverviewofthedefinitionand conceptofplantbiostimulants,aswellasthemaincategories.Thispaperwillalsobrieflydescribethe legalandregulatorystatusofbiostimulants,withafocusontheEUandtheUS,andoutlinesthedrivers, opportunitiesandchallengesoftheirmarketdevelopment.

©2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction...00

2. Maincategoriesofplantbiostimulants ... 00

2.1. Humicandfulvicacids...00

2.2. ProteinhydrolysatesandotherN-containingcompounds...00

2.3. Seaweedextractsandbotanicals ... 00

2.4. Chitosanandotherbiopolymers...00

2.5. Inorganiccompounds...00

2.6. Beneficialfungi...00

2.7. Beneficialbacteria...00

3. Commonfeaturesofbiostimulants...00

4. Definingplantbiostimulants:aimingataconsensus...00

5. Regulationofplantbiostimulants...00

6. Developingthemarket:opportunitiesandchallenges...00

7. Concludingremarks–lookingahead...00

Acknowledgements ... 00

References...00

E-mailaddress:patrick.dujardin@ulg.ac.be http://dx.doi.org/10.1016/j.scienta.2015.09.021

0304-4238/©2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4. 0/).

1. Introduction

Thewordbiostimulantwasapparentlycoinedbyhorticulture specialistsfordescribingsubstancespromotingplantgrowth with-outbeingnutrients,soilimprovers,orpesticides.Tracingbackthe firstdefinitionofthewordbiostimulantsidentifiesawebjournal dedicatedtoturfmaintenanceprofessionals,calledGround Mainte-nance(http://grounds-mag.com).Inthiswebjournalin1997,Zhang andSchmidtfromtheDepartmentofCropandSoilEnvironmental Sciences ofthe Virginia Polytechnic Instituteand State Univer-sitydefinedbiostimulantsas‘materialsthat,inminutequantities, promoteplantgrowth’.Byusingthewords‘minutequantities’for describingbiostimulants,theauthorsaimedatdistinguishing bios-timulantsfromnutrientsandsoilamendments,whichalsopromote plantgrowthbutareappliedinlargerquantities.The biostimu-lantsmentionedbythiswebarticlearehumicacidsandseaweed extracts.Laterpeer-reviewedpapersbythesameauthorsonthe sameorsimilarresearchdidnotnecessarilyusetheterm biostim-ulant.Forinstanceapaperdescribingtheuseofhumicacisand seaweedextractsforincreasingdroughttoleranceofturfgrassdid notusethetermbiostimulantatall(ZhangandSchmidt,2000).The paperfocusedonthehormone-likeactivitiesofthesecompounds andtheterm‘hormone-containingproducts’wasusedinsteadof biostimulants.Thischoicecouldalsobeexplained bythe regu-lationintheUnitedStates,wheretheEnvironmentalProtection Agency(‘EPA’)exempts‘vitamin-hormonehorticultureproducts’ fromregistration under certain conditions. Zhang and Schmidt explainedthebiostimulationactionbyhormonaleffectsand, sec-ondly,byprotectionagainstabioticstressbyantioxidants.Theterm ‘metabolicenhancers’wasalsousedinlaterpapers(Zhangetal., 2003).

In the scientific literature, the word biostimulant was first definedbyKauffmanetal.(2007)inapeer-reviewedpaper,with modifications:‘biostimulantsarematerials,otherthanfertilisers, thatpromoteplantgrowthwhenappliedinlowquantities.’Worth mentioningisthe additionof thewords‘otherthan fertilisers’, whichisinlinewiththedescriptionofZhangand Schmidt,but which was not explicitely included in their original definition. Kauffmanetal.(2007)attempttosummarizewhatbiostimulants are,by introducinga classification: ‘Biostimulantsare available inavarietyofformulationsandwithvaryingingredientsbutare generallyclassifiedintothreemajorgroupsonthebasisoftheir sourceandcontent.Thesegroupsincludehumicsubstances(HS), hormonecontainingproducts(HCP),andaminoacidcontaining products(AACP).HCPs,suchasseaweedextracts,contain identi-fiableamountsofactiveplantgrowthsubstancessuchasauxins, cytokinins,ortheirderivatives’.

Thewordbiostimulantwasincreasinglyusedbythescientific literatureover thefollowingyears, expandingtherange of sub-stances and of modes of actions (Calvo et al., 2014; du Jardin, 2012;Halpern et al., 2015). In fact,‘biostimulant’ appears as a versatiledescriptor of any substancebeneficial toplants with-outbeingnutrients,pesticides,orsoilimprovers.Tosomeextent, biostimulantsarefirstdefinedbywhattheyarenot,bydrawing aborderline betweenbiostimulantsandotherwidely used cat-egoriesofsubstancesappliedtoplantsandcrops:fertilisersand pesticides.Inasecondstage,itturnedoutthatthepositiveactions ascribedto thechemical biostimulants(of natural or synthetic origin)–growthpromotion,modulationofdevelopmentand of qualitytraits,increasedtolerancetoenvironmentalstress–canalso bedeliveredbybacteriaandfungi.Asanexample,PGPRsor‘plant growth-promotingrhizobacteria’aredefinedbybeneficialeffects ontheplants,withoutbeingnutrients,pesticidesorsoilimprovers. Likechemicalsubstances,theirnature(i.e.theirtaxonomicstatus inthiscase)canbeverydiverseandthePGPRcategoryisdefinedon thebasisofitsagricultural/horticulturaloutputs.‘Biofertilisers’and

‘biocontrolagents’arealsousedfordescribingPGPRs,referingto theexpectedagricultural/horticulturaloutputs.Therelationships betweentheseconceptsandtermswillbediscussedlaterinthis paper.

Industryisakeyplayerinthedefinitionandpromotionofthe conceptofbiostimulants,includingmicroorganisms.Companiesin thesectorhavecreatedassociations,likethe‘European Biostim-ulants Industry Council’(EBIC) inEurope andthe ‘Biostimulant Coalition’in theUSA,dialoguingwithotherstakeholders, regu-latorsandscientists.Thecorporatesectorhasalsosupportedthe organisationofinternationalsymposiums.The‘FirstWorldCongress ontheuseofBiostimulantsinagriculture’tookplaceinStrasbourg,in November2012andmayberegardedasamilestoneinthe accep-tanceofbiostimulantsintotheacademicarea.

Thepurposeofthisarticleistocontributetoabetter under-standingoftheconceptofplantbiostimulantonthebasisofthe theoreticalandpracticalknowledgeofthemaincategoriesof bios-timulantproductsusedinagricultureandhorticulture.Withthis aim,themaincategorieswillbebrieflydescribed.Theirmodesof actionwillbesummarized,providingthebasisofanydefinition.

2. Maincategoriesofplantbiostimulants

Despiterecenteffortstoclarifytheregulatorystatusof bios-timulants, there is no legal or regulatory definition of plant biostimulantsanywhereintheworld,includingintheEuropean UnionandintheUnitedStates.Thissituationprecludesadetailed listingandcategorizationofthesubstancesandmicroorganisms coveredbytheconcept.Despitethis,somemajorcategoriesare widelyrecognizedbyscientists,regulatorsandstakeholders(Calvo etal.,2014;duJardin,2012;Halpernetal.,2015),coveringboth substances and microorganisms. Microorganisms include bene-ficialbacteria, mainlyPGPRs, and beneficial fungi.They canbe free-living,rhizospheric or endosymbiotic.These categories are brieflyintroducedinthenextsectionandwillbefurtherdescribed bytheaccompanyingpapersofthisspecialissueonplant biostim-ulantsinhorticulture.

2.1. Humicandfulvicacids

Humic substances (HS) are natural constituents of the soil organicmatter,resulting fromthedecomposition ofplant, ani-malandmicrobialresidues,butalsofromthemetabolicactivity ofsoilmicrobesusingthesesubstrates.HSarecollectionsof het-erogeneouscompounds,originallycategorizedaccordingtotheir molecularweights andsolubility intohumins, humicacids and fulvicacids. Thesecompounds alsoshow complexdynamics of association/dissociationintosupra-molecularcolloids,andthisis influencedbyplantrootsviathereleaseofprotonsandexudates. Humicsubstancesandtheircomplexesinthesoilthusresultfrom theinterplaybetweentheorganicmatter,microbesandplantroots. Anyattempttousehumicsubstancesforpromotingplantgrowth and cropyield needs tooptimize theseinteractions to achieve theexpectedoutputs.Thisexplainswhytheapplicationofhumic sustances–solublehumicandfulvicacidsfractions–shows incon-sistent, yet globally positive, resultson plantgrowth. A recent random-effectmeta-analysisofHSappliedtoplants(Roseetal., 2014)concludedonanoveralldryweightincreaseof22±4%for shootsandofby21±6%forroots.

ThevariabilityineffectsofHSareduetothesourceoftheHS, theenvironmentalconditions,thereceivingplantandthedoseand mannerofHSapplication(Roseetal.,2014).Regardingthesources ofHS(duJardin,2012),theyareextractedfromnaturally humi-fiedorganicmatter(e.g.frompeatorvolcanicsoils),fromcomposts andvermicomposts,orfrommineraldeposits(leonardite,an oxida-tionformoflignite).Furthermore,agriculturalby-products,instead

ofbeingdecomposedinasoilorbycomposting,areamenableto controlledbreakdownandoxidationbychemicalprocesses, lead-ingto‘humic-likesubstances’whichareproposedassubstitutefor naturalHS(Eyheraguibeletal.,2008).

Humicsubstances havebeenrecognizedforlongasessential contributorstosoilfertility,actingonphysical,physico-chemical, chemicalandbiologicalpropertiesofthesoil.Mostbiostimulant effectsofHSrefertotheameliorationofrootnutrition,via differ-entmechanisms.Oneofthemistheincreaseduptakeofmacro-and micronutrients,duetotheincreasedcationexchangecapacityof thesoilcontainingthepolyanionicHS,andtotheincreased avail-abilityofphosphorusbyHSinterferingwithcalciumphosphate precipitation.AnotherimportantcontributionofHStoroot nutri-tionisthestimulationof plasmamembraneH+_{-ATPases, which} convertthefreeenergyreleasedbyATPhydrolysisintoa trans-membraneelectrochemicalpotentialusedfortheimportofnitrate andothernutrients.Besidesnutrientsuptake,protonpumpingby plasmamembraneATPasesalsocontributestocellwallloosening, cellenlargementandorgangrowth(Jindoetal.,2012).HSseemto enhancerespirationandinvertaseactivitiesprovidingCsubstrates. Hormonaleffectsarealsodescribed,butwhetherHScontain func-tional groups recognizedby thereception/signalling complexes ofplanthormonalpathways,liberateentrappedhormonal com-pounds,orstimulatehormone-producingmicroorganismsisoften unclear(duJardin,2012).Theproposedbiostimulationactivityof HSalsoreferstostressprotection.Phenylpropanoidmetabolism iscentraltotheproductionofphenoliccompounds,involvedin secondarymetabolism andin a widerange ofstressresponses. High-molecularmassHShavebeenshowntoenhancetheactivity ofkeyenzymesofthismetabolisminhydroponically-grownmaize seedlings,suggestingstressresponsemodulationbyHS(Olivares etal.,2015;Schiavonetal.,2010)..

2.2. ProteinhydrolysatesandotherN-containingcompounds Amino-acidsandpeptidesmixturesareobtainedbychemical andenzymaticproteinhydrolysisfromagroindustrialby-products, frombothplantsources(cropresidues)andanimalwastes(e.g. col-lagen,epithelialtissues)(duJardin,2012;Calvoetal.,2014;Halpern et al.,2015).Chemical synthesis canalso beused for singleor mixedcompounds.Othernitrogenousmoleculesincludebetaines, polyaminesand‘non-proteinaminoacids’,whicharediversifiedin higherplantsbutpoorlycharacterizedwithregardtotheir physio-logicalandecologicalroles(Vranovaetal.,2011).Glycinebetaineis aspecialcaseofaminoacidderivativewithwell-knownanti-stress properties(ChenandMurata,2011).

Casebycase,thesecompoundshavebeenshowntoplay mul-tiplerolesasbiostimulantsofplantgrowth(Calvoetal.,2014;du Jardin,2012,Halpernetal.,2015).Directeffectsonplantsinclude modulationof N uptake and assimilation,by the regulation of enzymesinvolvedinNassimilationandoftheirstructuralgenes, andbyactingonthesignallingpathwayofNacquisitioninroots. ByregulatingenzymesoftheTCAcycle,theyalsocontributeto thecross talkbetweenC and N metabolisms. Hormonal activi-tiesarealsoreportedincomplexproteinandtissuehydrolysates (Collaetal.,2014).Chelatingeffectsarereportedforsomeamino acids(likeproline)whichmayprotectplantsagainstheavy met-alsbutalsocontributetomicronutrientsmobilityandacquisition. Antioxidantactivityisconferredbythescavengingoffreeradicals bysomeofthenitrogeouscompounds,includingglycinebetaine andproline,whichcontributestothemitigationofenvironmental stress.

Indirecteffectsonplantnutritionandgrowtharealsoimportant intheagriculturalpracticewhenproteinhydrolysatesareapplied toplants and soils.Proteinhydrolysates are knowntoincrease microbialbiomassandactivity,soilrespirationand,overall,soil

fertility.Chelatingandcomplexingactivitiesofspecificaminoacids andpeptidesaredeemedtocontributetonutrientsavailabilityand acquisitionbyroots.

Several commercial products obtained from protein hydrolysates of plant and animal origins have been placed on themarket.Variable,butinmanycasessignificantimprovements inyieldandqualitytraitshavebeenreportedinagriculturaland horticulturalcrops(Calvoetal.,2014).Thesafetyofhydrolyzed proteinsofanimaloriginwasrecentlyassessedandno genotox-icity, ecotoxicity or phytotoxicty was reported on the basis of bioassaysusingyeastsandplantsastestorganisms(Corteetal., 2014).Nevertheless,there is agrowingsafety concernofusing protein hydrolysates derived from animal by-products in the foodchain.TheEUbannedtheapplicationofsuchanimalprotein hydrolysates on the edible parts of organic crops, through the Commission Implementing Regulation (EU) no 354/2014 with regardtoorganicproduction,labellingandcontrol.

2.3. Seaweedextractsandbotanicals

Theuseoffreshseaweedsassourceoforganicmatterandas fertiliseris ancientin agriculture,but biostimulanteffects have beenrecordedonlyrecently.Thispromptsthecommercialuseof seaweedextractsandofpurifiedcompounds,whichincludethe polysaccharideslaminarin,alginatesand carrageenansand their breakdownproducts.Otherconstituentscontributingtotheplant growthpromotionincludemicro-andmacronutrients,sterols, N-containingcompoundslikebetaines,andhormones(Craigie,2011; Khanetal.,2009).Severalofthesecompoundsareindeeduniqueto theiralgalsource,explainingtheincreasinginterestofthescientific communityandoftheindustryforthesetaxonomicgroups.Most ofthealgalspeciesbelongtothephylumofbrownalgae–with Ascophyllum,Fucus,Laminariaasmaingenera-,butcarrageenans originatefromredseaweeds,whichcorrespondtoadistinct phy-logeneticline.Productnamesofmorethant20seaweedproducts usedasplantgrowthbiostimulanthavebeenlistedbyKhanetal. (2009).

Seaweedsactonsoilsandonplants(Craigieetal.,2008;Craigie, 2011;Khanetal.,2009).Theycanbeappliedonsoils,inhydroponic solutionsorasfoliartreatments.Insoils,theirpolysaccharides con-tribute togel formation,water retention and soilaeration. The polyanioniccompoundscontributetothefixationandexchangeof cations,whichisalsoofinterestforthefixationofheavymetalsand forsoilremediation.Positiveeffectsviathesoilmicrofloraarealso described,withthepromotionofplantgrowth-promotingbacteria andpathogenantagonistsinsuppressivesoils.Inplants,nutritional effectsviatheprovisionandmicro-andmacronutrientsindicate thattheyactasfertilisers,besidetheirotherroles.Impactsonseed germination,plantestablishmentandonfurthergrowthand devel-opmentisassociatedwithhormonaleffects,whichisviewedas majorcausesofbiostimulationactivityoncropplants.Although cytokinins,auxins,abscisicacid,gibberellinsandotherclassesof hormone-likecompounds,likesterolsandpolyamines,havebeen identifiedinseaweedextractsbybioassaysandbyimmunological tools(Craigie,2011),thereisevidencethatthehormonaleffectsof extractsofthebrownseaweedAscophyllumnodosumareexplained toalargeextentbythedown-andupregulationofhormone biosyn-theticgenesinplanttissues,andtoalesserextenttothehormonal contentsoftheseaweedextractsthemselves(Wallyetal.,2013a,b). Moleculargenetics,i.e.hormonemutantsinArabidopsisand tran-scriptanalysisbyRT-qPCR,wereusedtoreachthisconclusion.

Anti-stress effects are also reported and both protective compoundswithintheseaweedextracts,likeantioxidants,and reg-ulatorsofendogenousstress-responsivegenescouldbeinvolved (Calvoetal.,2014).

‘Botanicals’describesubstancesextractedfromplantswhichare usedinpharmaceuticalandcosmeticproducts,asfoodingredients, andalsoinplantprotectionproducts(Seiberetal.,2014).Compared withseaweeds,muchlessisknownregardingtheirbiostimulant activities,theattentionbeingfocusedontheirpesticidal proper-tiessofar.However,thereseemstobeopportunitiestousethemas biostimulantsaswell(Ertanietal.,2013;Ziosietal.,2012). Further-more,plantinteractionsinecosystemsareknowntobemediated byplantactivecompounds,refered toasallelochemicals,which arereceivingincreasingattentioninthecontextofsustainablecrop management.Althoughcroprotations,intercropping,covercrops andmulchingareusedinthefirstinstancetoexploitallelochemical interactionsbetweenplants(namedallelopathy),futherattention shouldbepaidtothesechemicalinteractionsforthedevelopment ofnewbiostimulants.

2.4. Chitosanandotherbiopolymers

Chitosanisadeacetylatedformofthebiopolymerchitin, pro-ducednaturallyandindustrially.Poly-andoligomersofvariable, controlledsizesareusedinthefood,cosmetic,medicaland agri-culturalsectors. Thephysiological effects of chitosan oligomers inplantsaretheresultsofthecapacityofthispolycationic com-poundtobindawiderangeofcellularcomponents,includingDNA, plasmamembraneandcellwallconstituents,butalsotobind spe-cificreceptorsinvolvedindefensegeneactivation,inasimilarway asplantdefenseelicitors(ElHadramietal.,2010;Hadwiger,2013; Katiyaretal.,2015;Yinetal.,2010).Chitinandchitosan appar-entlyusedistinctreceptorsandsignallingpathways.Amongthe cellularconsequencesofthebindingofchitosantomoreorless specificcellreceptors,hydrogenperoxideaccumulationandCa2+ leakageintothecellhavebeendemonstrated,whichareexpected tocauselargephysiologicalchanges,asthesearekeyplayersin thesignallingofstressresponsesandinthedevelopment regula-tion.Analysisoftheproteome(Ferrietal.,2014)ortranscriptome (Povero etal.,2011)ofplanttissuestreatedwithchitosan con-firmthisassumption.Inconsequence,agriculturalapplicationsof chitosanhavebeendevelopedover theyears, focusingonplant protectionagainstfungalpathogens,butbroaderagriculturaluses bearontolerancetoabioticstress(drought,salinity,coldstress) andonqualitytraitsrelatedtoprimaryandsecondarymetabolisms. StomatalclosureinducedbychitosanviaanABA-dependent mech-anism(Iritietal.,2009)participatestotheenvironmentalstress protectionconferedbythisbiostimulant.

Severalpoly-andoligomersofbiologicaloriginor(hemi-) syn-theticvariantsareincreasinglyusedinagricultureaselicitorsof plantdefense,includingseaweedpolysaccharideswhichwehave alreadymentioned.Agoodexampleislaminarin,astorageglucanof brownalgae,ofwhichpurifiedpreparationsareusedinagricultural applications.Althoughadistinctionhastobemadebetween bio-controlandbiostimulation(e.g.enhancingabioticstress),signalling pathwaysmaybeinterconnectedandbotheffectsmaypractically resultfromtheapplicationofthesameinducers(GozzoandFaoro, 2013).

2.5. Inorganiccompounds

Chemical elements that promote plant growth and may be essentialtoparticulartaxabutarenotrequiredbyallplantsare calledbeneficialelements(Pilon-Smitsetal.,2009).Thefivemain beneficialelementsareAl,Co,Na,SeandSi,presentinsoilsandin plantsasdifferentinorganicsaltsandasinsolubleformslik amor-phoussilica(SiO2.nH2O)ingraminaceaousspecies.Thesebeneficial functionscanbeconstitutive,likethestrengtheningofcellwallsby silicadeposits,orexpressedindefinedenvironmentalconditions, likepathogenattackforseleniumandosmoticstressforsodium.

Definitionofbeneficialelementsisthusnotlimitedtotheir chem-icalnatures, but mustalso refer to thespecial contexts where thepositiveeffectsonplantgrowthandstressresponsemaybe observed.Itmaybeassumedthatthebioactivityofsomecomplex biostimulants,likeextractsofseaweeds,ofcropresiduesor ani-malwastes,involvesthephysiologicalfunctionsofthecontained beneficialelements.

Many effects ofbeneficial elementsare reportedby the sci-entific litterature, which promote plant growth, the quality of plantproductsandtolerancetoabioticstress.Thisincludescell wallrigidification,osmoregulation,reducedtranspirationby crys-taldeposits,thermalregulationviaradiationreflection,enzyme activitybyco-factors,plantnutritionviainteractionswithother elements during uptake and mobility, antioxidant protection, interactions withsymbionts, pathogenand herbivoreresponse, protectionagainstheavymetalstoxicity,planthormonesynthesis andsignalling(Pilon-Smitsetal.,2009).

Inorganic salts of beneficial and essential elements – chlo-rides, phosphates, phosphites, silicates and carbonates – have beenusedasfungicides(Deliopoulosetal.,2010).Althoughthe modesofactionarenotyetfullyestablished,theseinorganic com-poundsinfluence osmotic,pHand redoxhomeostasis,hormone signallingand enzymesinvolvedinstressresponse (e.g. peroxi-dases).Theirfunctionasbiostimulantofplantgrowth,actingon nutritionefficiencyandabioticstresstolerance,hencedistinctfrom theirfungicidalactionandfromtheirfertiliserfunctionassources ofnutrients,deservesmoreattention.

2.6. Beneficialfungi

Fungiinteractwithplantrootsindifferentways,from mutualis-ticsymbioses(i.e.whenbothorganismsliveindirectcontactwith eachotherandestablishmutuallybeneficialrelationships)to para-sitim(BehieandBidochka,2014).Plantsandfungihaveco-evolved sincetheoriginofterrestrialplantsandtheconceptofmutualism –parasitismcontinuumisusefultodescribetheextendedrangeof relationshipsthatdevelopedovertheevolutionarytimes(Bonfante andGenre,2010;JohnsonandGraham,2013).Mycorrhizalfungi are a heterogeneous group of taxa which establish symbioses withover 90 %of allplant species. Amongthe differentforms ofphysicalinteractionsandtaxainvolved,theArbuscule-Forming Mycorrhiza(AMF)areawidespreadtypeofendomycorrhiza asso-ciatedwithcropand horticulturalplants, wherefungal hyphae ofGlomeromycotaspeciespenetraterootcorticalcellsandform branchedstructurescalledarbuscules(BonfanteandGenre,2010; BehieandBidochka,2014).Thereisanincreasinginterestforthe useofmycorrhizatopromotesustainableagriculture,considering thewidelyacceptedbenefitsofthesymbiosestonutrition effi-ciency(forbothmacronutrients,especiallyP,andmicronutrients), waterbalance,bioticandabioticstressprotectionofplants(Augé, 2001;Gianinazzietal.,2010;HamelandPlenchette,2007;Harrier andWatson, 2004;Siddiquiet al.,2008;vanderHeijdenetal., 2004).Recentknowledgealsopointstotheexistenceofhyphal net-workswhichinterconnectnotonlyfungalandplantpartnersbut alsoindividualplantswithinaplantcommunity.Thiscouldhave significantecologicalandagriculturalimplicationssincethereis evidencethatthefungalconduitsallowforinterplantsignalling (JohnsonandGilbert,2015;Simardetal.,2012).Asafurtherarea ofresearch,AMFformtripartiteassociationswithplantsand rhi-zobacteriawhicharerelevantinpracticalfieldsituations(Siddiqui etal.,2008).Inordertoreapthebenefitsofthemycorrhizal asso-ciations,cropmanagementpracticesandplantcultivarsshouldbe adaptedtotheinteractionwithmicroorganisms(Gianinazzietal., 2010;HamelandPlenchette,2007;Plenchetteetal.,2005;Sheng etal.,2011).Metagenomicsareaninterestingtooltomonitorand studymicrobialassociationsintherhizosphere.Inoculationofplant

propagulesandsoilscomplementstheseapproches(Candidoetal., 2013,2015;Collaetal.,2015;Sarkaretal.,2015;Sensoyetal.,2007; Sorensenetal.,2008).

Fungal-based products applied to plants to promote nutri-tionefficiency,tolerancetostress,cropyieldandproductquality shouldfallundertheconceptofbiostimulants.Majorlimitations ontheiruseare thetechnicaldifficultytopropagateAMF ona largescale,duetotheirbiotrophiccharacter(DalpéandMonreal, 2004), and, more fundamentally, the lack of understanding of thedeterminantsofthehostspecificitiesandpopulation dynam-icsofmycorrhizalcommunitiesinagroecosystems.Nevertheless, otherfungalendophytes,likeTrichodermaspp.(Ascomycota)and Sebacinales(Basidiomycota,withPiriformosporaindicaasmodel organism),distinctfromthemycorrhizalspecies,areabletoliveat leastpartoftheirlifecycleawayfromtheplant,tocolonizeroots and,asshownrecently,totransfernutrientstotheirhosts,using poorlyunderstoodmechanisms(BehieandBidochka,2014).They arereceivingincreasingattention,both asplantinoculants eas-iertomultiplyinvitroandasmodelorganismsfordissectingthe mechanismsofnutrienttransferbetweenfungalendosymbionts andtheirhosts.Someofthesefungi,mainlyTrichodermaspp.,have beenextensivelystudiedandusedfortheirbiopesticidal (myco-parasitic)andbiocontrol(inducerofdiseaseresistance)capacities, andhavebeenexploitedassourcesofenzymesby biotechnologi-calindustries(Mukherjeeetal.,2012;Nicolásetal.,2014).Thereis convincingevidencethatmanyplantresponsesarealsoinduced, includingincreasedtolerancetoabioticstress,nutrientuse effi-ciencyandorgangrowthandmorphogenesis(Collaetal.,2015; Shoreshetal.,2010).Onthebasisoftheseeffects,thesefungal endo-phytesmayberegardedasbiostimulants,thoughtheiragricultural usesarecurrentlysupportedbyclaimsasbiopesticides.

2.7. Beneficialbacteria

Bacteriainteractwithplantsinallpossibleways(Ahmadetal., 2008):(i)asforfungithere isacontinuumbetweenmutualism andparasitism;(ii)bacterialnichesextendfromthesoil tothe interiorofcells,withintermediatelocationscalledtherhizosphere andtherhizoplane;(iii)associations mybetransientor perma-nent,somebacteriabeingevenverticallytransmittedviatheseed; (iv)functionsinfluencingplantlifecoverparticipationtothe bio-geochemicalcycles,supply ofnutrients,increaseinnutrientuse efficiency,inductionofdiseaseresistance,enhancementofabiotic stresstolerance, modulationofmorphogenesisbyplantgrowth regulators.

With regard to the agricultural uses of biostimulants, two main types should be considered within this taxonomic, func-tionalandecologicaldiversity:(i)mutualisticendosymbiontsof thetypeRhizobiumand(ii)mutualistic,rhizosphericPGPRs(‘plant growth-promotingrhizobacteria’).Rhizobiumandrelatedtaxaare commercialisedasbiofertilisers,i.e.microbialinoculants facilitat-ingnutrients acquisitionby plants (see glossaryin Box 1).The biologyand agriculturalusesoftheRhizobium-basedsymbioses havebeenextensivelyreviewedbythescientificliteratureandin textbooks.PGPRsaremultifunctionalandinfluenceallaspectsof plantlife:nutritionandgrowth,morphogenesisanddevelopment, responsetobioticandabioticstress,interactionswithother organ-ismsintheagroecosystems(Ahmadetal.,2008;Babalola,2010; Berendsenetal.,2012;Bergetal.,2014;BhattacharyyaandJha, 2012;Gaieroetal.,2013;Philippotetal.,2013;Vacheronetal., 2013).Severalofthesefunctionsaregenerallyfulfilledbythesame organisms,somearestrain-specific,othersaredependenton syn-ergismswithinbacterialconsortia.AgriculturalusesofPGPRsare constrainedbythiscomplexity,bythevariableresponsesofthe plantcultivars andthereceivingenvironments.Alsothe techni-caldifficultiesassociatedwiththeformulationoftheinoculants

Box 1: Glossary of ‘biosolutions’ contributing to sus-tainable plant productions

Biostimulant: A plant biostimulant is any substance or microor-ganism applied to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance and/or crop quality traits, regardless of its nutrients content. By extension, plant bios-timulants also designate commercial products containing mixtures of such substances and/or microorganisms. Biofertiliser: A biofertiliser is any bacterial or fungal inoculant applied to plants with the aim to increase the availability of nutrients and their utilization by plants, regardless of the nutri-ent contnutri-ent of the inoculant itself. Biofertilisers may also be defined as microbial biostimulants improving plant nutrition efficiency.

Biocontrol: The control of one organism by another. Biocontrol agents used in plant productions are living organisms protect-ing plants against their enemies, i.e. reducprotect-ing the population of pests or diseases to acceptable levels. Modes of action may include competition, antibiosis, parasitism and also Induced Systemic Resistance which is mediated by the plant.

give riseto inconsistent resultsin practice (Aroraet al., 2011; BrahmaprakashandSahu,2012).Despitethis,theworldmarket ofbacterialbiostimulantsisgrowingandPGPRinoculantsarenow regardedassomekindofplant‘probiotics’,i.e.efficientcontributors toplantnutritionandimmunity(Berendsenetal.,2012).

3. Commonfeaturesofbiostimulants

Acommondesignationofbiostimulantsisonlyjustifiedifthe describedsubstancesandmicroorganismssharesomeimportant characteristicsregardingtheirnatures,functionsand/oruses.Such characteristicswouldthenbethegroundforanydefinition.

Fromthebibliographicreview,thefollowingconclusionsmay bedrawn:

1.Thenatureofbiostimulantsisdiverse.Substancesand microor-ganisms are involved. Substances can be single compounds (e.g.glycinebetaine)orgroups ofcompoundsofsingle natu-raloriginofwhichthecompositionandbioactivecomponents are not fully characterized (e.g. seaweed extracts); the sub-stances commented by this review are naturally produced organiccompounds,orinorganicmolecules,butsynthetic com-poundsshouldnotbeexcluded,especiallyifcertainplantgrowth regulators are included within biostimulants (for example, nitro-phenolates are described and commercialized as ‘bios-timulants’butaresyntheticphenoliccompoundsregisteredas plantproductionproductsaccordingtotheEULaw,seePrzybysz et al.,2014).Microbialinoculants maycontainsingle strains (e.g.ofBacillussubtilis)ormixtures ofmicroorganisms show-ingadditiveorsynergisticeffects(e.g.severalproductsonthe market).Dependingonthepeer-reviewedand‘grey’scientific literatureand onthedocumentationprovided bycompanies, biostimulantsmayrefertothebioactiveingredientsortothe commercializedproductscombiningthemandoftenaddedto fertilisers orcropprotectionproducts.Anyregulatory defini-tionwillhavetoclarifywhetheringredientsorfinalproducts (orboth)areactuallycovered.

2.Thephysiologicalfunctionsarediverse.Byphysiologicalfunction, we mean any action onplant processes (Table 1).Examples ofphysiologicalfunctionsaretheprotectionofphotosynthetic machineryagainstphotodamage,ortheinitiationoflateralroots. Functionsaresupportedbycellularmechanisms,likereactive oxgenscavengingbyantioxidantsorincreasedsynthesisofauxin transporters,tocarryonwiththetwopreviousexamples.

Phys-Table1

Effectsofbiostimulantsoncropproductions,fromtheircellulartargetsinplantstowhole-plantphysiologicalfunctions,toagricultural/horticulturalfunctions,andultimately toexpectedeconomicandenvironmentalbenefits(Dobbelaereetal.,1999;Huangetal.,2010;Shabalaetal.,2012).

Humic acids Seaweed extracts

Protein

hydrolysate Glycine betaine

Plant Growth-promoting Rhizobacteria Cellular mechanism

(i.e. interaction with cellular components and processes)

â

Activate plasma

membrane pr

oton-pumping ATPases, promote cell wall loosening and cell elongation in

maize roots (Zea

mays) (Jindo et al., 2012) Ascophyllum nodosumextracts stimulate expression of genes encoding transporters of

micronutrients (e.g.

Cu, Fe, Zn) in

oilseed rape

(Brassicanapus)

(Billard et al., 2014)

Enzymatic hydrolysate from alfalfa (Medicaco sativa) stimulates phenylalanine ammonia-lyase

(PAL) enzyme and

gene expression,

and production of

lavonoids under salt stress (Ertaniet al., 2013) Protects photosystem II against salt-induced photodamage in quinoa (Shabala et

al., 2012), likely via

activation of

scavengers of

reactive oxygen

(Chen & Murata ,

2011)

Azospirillum brasilensereleases

auxins and activates auxin-signalling pathways

involved in root

morphogenesis in

winter wheat

(Triticumaestivum)

(Dobbelaere et al.,

1999)

Physiological function

(i.e.action on whole-plant processes) â Increasedlinear growth of roots, root biomass Increasedtissue

concentrations and

root to shoot transport of micronutrients Protection by lavonoids against UV and oxidative damage (Huang et al., 2010) Maintenanceofleaf photosynthetic activity under salt

stress

Increasedlateral

root density and

surface of root

hairs

Agricultural/horticultural function

(i.e. output traits relevant for crop performance)

â

Increasedroot

foraging capacity,

enhancednutrient

use eficiency

Improvedmineral

composition of

plant tissues

Increasedcrop

tolerance to abiotic

(e.g.salt) stress

Increasedcrop

tolerance to abiotic (e.g. high salinity)

stress

Increased root

foraging capacity,

enhancednutrient

use eficiency

Economic and environmental beneits

(i.e. changes in yield,

products quality, ecosystem services)

Higher crop yield, savings of fertilisers and

reduced losses to

the environment

Enhanced

nutritional value,

‘biofortiication’ of

plant tissues

(increased contents

in S, Fe, Zn, Mg, Cu)

Higher crop yield

understress

conditions (e.g. high salinity)

Higher crop yield

understress

conditions(e.g.

high salinity)

Higher crop yield, savings of fertilisers and

reduced losses to

the environment

iologicalfunctionsandtheunderlyingcellularmechanismsmay

bereferedtoas‘modesofactions’ofthebiostimulants,

collec-tively.Finally,thesemodesofactionsexplaintheagricultural

functionsof biostimulants,e.g.increased tolerancetoabiotic

stress(causingoxidativestress),orincreasedNuseefficiency

(whichdependsoftheforagingcapacityofroots,henceon

lat-eralrootdensity).Agricultural functionsmayfinallytranslate

intoeconomic andenvironmentalbenefits:highercropyield,

savingsoffertilisers,increasedqualityandprofitabilityofcrop

products,enhancedecosystemservices,etc.

3.Thescientificallydemonstratedeffectsofallbiostimulants

con-verge to at least one or several of the following agricultural

functions:theyenhancenutritionefficiency,abioticstress

tol-erance and/orcrop qualitytraits. Quality traits mayrefer to

nutritionalvalue, grainprotein content, shelf life, etc. These

convergingactionsshouldbethebasisofanydefinitionof

bios-timulants.Stimulation of pathogenresponse by elicitors and

plant gene regulators is achieved by many of the described

biostimulantsaswell(chitosan,laminarin,somePGPRs,etc.).

However,thereisagrowingconsensusamongregulatorsand

stakeholders to keep biostimulation and biocontrol separate

fromaregulatorypointofview.Bioticstressistakenoutofthe

scopeofthedefinition,accordingly.

4.Definitionofeconomicandenvironmentalbenefitsdependson

agri-culturalandenvironmentalpolicies,bothintermsofobjectivesand

assessmentendpoints.Althoughincentivesfordeveloping

bios-timulantsarelinkedtotheseaspects,theyshouldnotbethe

groundforascience-baseddefinitionofbiostimulants.

Inconclusionanydefinitionofbiostimulantsshouldfocuson

theagriculturalfunctionsof biostimulants,notonthenatureof

theirconstituentsnorontheirmodesofactions,astheyhavebeen

definedabove.

4. Definingplantbiostimulants:aimingataconsensus

Inlinewiththeaboveconsiderations,thefollowingdefinition

isproposed(Box1):

«Aplantbiostimulantisanysubstanceormicroorganism appliedtoplantswiththeaimtoenhancenutritionefficiency, abioticstresstoleranceand/orcropqualitytraits,regardlessof itsnutrientscontent.»Thisdefinitioncouldbecompletedby:«By extension,plantbiostimulantsalsodesignatecommercialproducts containingmixturesofsuchsubstancesand/ormicroorganisms.»

Acoupleofremarks:

1.Thenatureofthebiostimulantisnot restrictive:it canbea substance or a microorganism. A substance maybe either a singlechemicalcompoundoragroupofcompoundshavinga wellestablished biological origin,e.g. plant extracts,but not necessarilya fullycharacterizedcomposition.Inthissense,it fitswiththemeaningoftheword«substance»inexisting

reg-ulations.ThisincludestheeuropeanREACHregulation(ECNo 1907/2006)concerningtheRegistration,Evaluation, Authorisa-tionandRestrictionofChemicals,whichrecognizesacategory ofsubstancesofvariablecomposition:‘UVCBsubstances (sub-stances of unknown or variable composition, complex reaction products orbiological materials) maybe registered as a single substanceunderthisRegulation,despitetheirvariablecomposition, providedthatthehazardouspropertiesdonotdiffersignificantly andwarrantthesameclassification’.Anotherexampleofcomplex substancespotentiallycomprisingmanychemicalconstituents areplant extractsreferedto as‘botanicalactivesubstances ‘ andas‘basicsubstances‘andapprovedunderregulation(EC) No 1107/2009 on plant protection in the EU. The European Commission‘guidancedocumentonthebotanicalactive sub-stances used in plant protection product ‘ (http://ec.europa. eu/food/plant/pesticides/guidancedocuments/docs/guidance documentbotanicalsrev8en.pdf)defines:‘A‘botanical active substance’consistsofoneormorecomponentsfoundinplantsand obtainedbysubjectingplantsorpartsofplantsofthesamespecies toaprocesssuchaspressing,milling,crushing,distillationand/or extractions’.Clearly,themulticomponentnatureofsubstances ofplantoriginisacknowledgedhere,asit isin international forumsoftheOECDonbiopesticides(http://www.oecd.org/env/ ehs/pesticides-biocides/env-jm-mono-2012-36-core%20report. pdf). The word substance in the definition of biostimulants shouldbeunderstoodinasimilarway.Microorganismsshould beidentifiedatthelevelofthestrain,consideringthatmany biologicalactivitiesareindeedstrain-specific.Whenmixtures (i.e.intentionalblends)ofmicroorganismsareused,the result-ingproductswouldbereferedtoasbiostimulants,followingour proposaltoextendthedefinitiontocommercialpreparations. 2.The agricultural functions form the core of the definition.

Biostimulants are defined by intended agricultural outputs. ‘Nutrition efficiency’ may cover nutrient mobilization and uptakefromthesoil,rootdevelopment,transport,storageand assimilation(i.e.conversionofinorganictoorganicforms)of nutrientsintheplant.‘Abioticstress’referstoanyphysicalor chemicalstressorofnonbiologicalorigin(drought,salinity,cold, etc.).‘Qualitytraits’maybediverseandrangefromnutritional valuetoshelflifeorflowerpigmentation.Anyoftheseeffects shouldbedistinctfromthoseresultingfromthenutrient con-tentofthebiostimulant.Biostimulantsarenotfertilisersinthe sensetheydonotcontainnutrientsintendedtobedeliveredto theplant.However,theymayfacilitatenutrientacquisition,e.g. bymobilizingelementsintherhizosphereorbydevelopingnew routesofnutrientacquisition,likefixationofatmosphericNby therecruitmentofbacterialendosymbionts.

3.Theproposeddefinitionisinlinewiththefew existing defi-nitionsunderdiscussionbetweenregulatorsandtheindustry. TheassociationEBICproposesthefollowing:‘Plantbiostimulants containsubstance(s)and/ormicro-organismswhosefunctionwhen appliedtoplantsortherhizosphereistostimulatenaturalprocesses toenhance/benefitnutrientuptake, nutrientefficiency,tolerance toabioticstress,andcropquality’.Thisisconsistentwiththe proposalofthisarticle.However,biostimulantsappliedtothe rhizospherecanbecoveredbythewording‘appliedtoplants’ (fertilisersandpesticidescanalsobedescribedasbeingapplied toplantseveniftheyaresprayedontheparcel,includingplants andsoils,includingtherhizosphere).Bysaying‘appliedtoplants’ inourdefinition,theintentionisnottoberestrictive,butonthe contrarytocoverallmodesofapplicationtargetingtheplant attheend.Furthermore,abiostimulantisdefinedasbeing pri-marilythesubstanceand/orthemicroorganismexertingsome effect,notwhatcontainssubstancesandmicroorganisms.EBIC seemstoreferinthefirstinstancetothecommercializedproduct containingactiveingredients.abiostimulantisdefinedfor

des-ignatingtheactivesubstanceormicroorganisminthefirstplace, andsecondlyanycommercialpreparationcontainingthem.

IntheUS,theBiostimulantCoalition,agroupofinterested par-ties equivalenttotheEBIC,hasattemptedtocoalescearounda definitionandtoreachanagreementwiththeAmerican Associa-tionofPlantFoodControlOfficials(AAPFCO),whichisinstrumental inharmonizingfertilizerandsoilamendmentlawsbetweenStates. Unfortunately,noagreementonadefinitionofbiostimulantscould bereached.However,abreakthroughtookplaceinFebruary2014 whenAAPFCOagreedtoexpandthedefinitionoftheexisting cat-egoryof‘beneficialsubstances’inordertoincludebiostimulants. Beneficialsubstancesaredefinedas“anysubstanceorcompound otherthanprimary,secondary,andmicroplantnutrientsthatcanbe demonstratedbyscientificresearchtobebeneficialtooneormore species of plants, when applied exogenously.” (AAPFCO, 2012) As such, many important biostimulants(e.g.all microbial biostim-ulants) areexcluded fromthe definition.The current approach aimsatincludingbiostimulantsassubcategoriesofbeneficial sub-stances, each having its own specification and definition. The initialsubcategoriesproposedbytheBiostimulantCoalitionare: antioxidants, amino acids materials, biomolecule/biomolecular, enzymatic extracts, fulvicacid materials,humic acid materials, microbial inoculants, microbial soil amendments, mycorrhizal fungi,PGPRs,phytohormones,seaweedextractmaterials.

5. Regulationofplantbiostimulants

Theregulatorysituationofbiostimulantsisverycomplextoday, intheabsenceofanyspecificandharmonizedframeworkineither theEUortheUSA.Oneofthemainreasonsforthis situationis thelackofformaldefinitionandacceptanceoftheconceptby reg-ulatorybodies.InEuropetoday,biostimulantsareplacedonthe marketbyfollowingeitheroftworoutes:oneisthenational regu-lationsonfertilisers,theotheroneistheeuropeanpesticideslaw, which combinesboth supranationaland nationalprovisions for introducingplantprotectionproductsonthemarket.InEurope,the currentsituationisthattheECregulationNo1107/2009onplant protectionproducts(‘PPPs’)isapplicabletoallcategoriesof bios-timulants,consideringtheverybroaddefinitionofPPPs.Indeed, Article2ofthis regulationreads:‘ThisRegulationshallapplyto products,intheforminwhichtheyaresuppliedtotheuser,consisting oforcontainingactivesubstances,safenersorsynergists,andintended foroneofthefollowinguses:

(a)(...)

(b)influencingthelifeprocessesofplants,suchassubstances influ-encingtheirgrowth,otherthanasanutrient.’

Asanybiostimulantisintendedtoinfluencethelifeprocesses ofplantsbyotherwaysthanasanutrient,itmayberegardedas a«plantprotectionproduct»fromastrictregulatoryviewpoint. Syntheticandnaturalsubstances(includingbotanicalsandbasic substancesasmentionedbefore),andmicroorganisms,areall cov-eredbythisregulation.Allplantgrowthregulatorsandherbicide safenershavebeenregisteredunderthisPPPregulationuntilnow andthesearesubstancesthatinteractwiththephysiologyofthe plant,eventhoughtheydonotprotecttheplantagainstpestsor diseases.

DuetothelengthyandcostlyprocedurestoplaceaPPPonthe europeanmarket,takingintoconsiderationthatmanycompanies developingbiostimulantsareSMEsandthatimprovedplant nutri-tionandgrowtharethemainscopeofbiostimulants,analternative routehasbeenchosen,namelythe‘fertilisersroute’inwhichcase nationallegislationisapplied.WhynottheeuropeanlawonEC fer-tilisers(regulation(EC)No2003/2003)?Becausethedefinitionof

fertiliserslaiddownbythisregulationisveryrestrictiveandcannot includebiostimulants.Indeed,Article2reads:

‘ForthepurposesofthisRegulationthefollowingdefinitionsshall apply:

(a)‘Fertiliser’meansmaterial,themainfunctionofwhichistoprovide nutrientsforplants.

(b)‘Primarynutrient’meanstheelementsnitrogen,phosphorusand potassiumonly.

(c)‘Secondary nutrient’ meansthe elementscalcium, magnesium, sodiumandsulphur.

(d)‘Micro-nutrients’meanstheelementsboron,cobalt,copper,iron, manganese,molybdenumandzincessentialforplantgrowthin quantitiesthat aresmallcompared withthose of primaryand secondarynutrients.’

Anyfertilisermustprovidenutrientasitsmainfunction.Thisis clearlynotthecaseofbiostimulants,whichbydefinitionpromote plantgrowthbyothermeansthanbyprovidingnutrients.AnnexI ofthe(EC)No2003/2003regulationonECfertilisersliststypesof fertilisers,whichareallinorganicmaterialsprovidingmacro-and micronutrients,butalsochelatingandcomplexingagentsintended to optimize the delivery of micronutrients to plants, allowing chelatedandcomplexedmicronutrientstobeplacedonthe mar-ket bythe way of this regulation. It was later considered that othercompoundsusedasfertilisersadditives,i.e.nitrificationand ureaseinhibitors,shouldalsobegrantedmarketaccessvia this regulation.Thisledtoabreakthroughintheeuropeanfertiliser reg-ulation,whichwasamendedbythe(EC)No1107/2008regulation inordertointroducematerialswhicharenotprovidersof nutri-ents(fertiliserssensustricto)butadditivesoffertilisersenhancing fertilisersperformance.Manybiostimulantsmaybeconsideredas enhancersoffertilisersperformanceandthisregulatoryadvance seemedtopavethewaytotheinclusionofbiostimulantsintothe EUfertiliserslaw.However,thisoptionisnotrealisticasamending regulationsisalaboriousprocedurewhichcannotbefollowedfor allbiostimulants.Whenthenationalfertiliserslawsareusedfor introducingbiostimulantsontheeuropeanmarket(mainlythose enhancingnutritionandgrowth,e.g.humicacids,seaweedextracts andproteinhydrolysates),markeddifferencesexistbetween mem-berstatesintermsofdatarequirementsforefficacy,toxicityand ecotoxicityassessment(Traonetal.,2014;LaTorreetal.,2015). Tocompletethisoverview,itisworthtomentionthatlegal provi-sionsexistwithintheEUtopromote«mutualrecognition»between memberstates(Regulation(EC)No764/2008),i.e.‘fasttracks’exist fortheplacingonthemarketofmembersstateswhenan authorisa-tionhasbeengrantedinoneofthem.However,basedoninterviews withrepresentativesofstakeholdersandcompetentauthorities,It isrealistictothinkthatthissystemisnotefficientenoughandis notexpectedtodevelopinthefuture.

Taking into consideration the need for harmonization of legislationonbiostimulants,but alsoofothercategories of fer-tilising materials and additives, – i.e. nutrients performance enhancers,organicandorgano-mineralfertilisers,soilimprovers, growing media, liming materials – the European Commis-sionand itsFertilisers Working Grouprepresenting competent authoritites of member states and stakeholders has initiated an ambitious reform of its fertilisers regulation (see pub-lic reports at http://ec.europa.eu/transparency/regexpert/index. cfm?do=groupDetail.groupDetail&groupID=1320).

The situation in the USA is to some extent similar to the europeansituation:noapproveddefinitionofbiostimulants,no harmonizationbetweenthe50states,useoffertiliserslawsforthe placingonthemarketofcertainbiostimulantsatthestatelevel, andworkinprogressbetweenstakeholders,representativesof reg-ulatorybodiesandfederalagenciestoimprovethelegalcertainty

surroundingbiostimulants.TheroleoftheAmericanAssociation ofPlantFoodControlOfficialshasalreadybeenunderlined, espe-ciallyregardingthedefinitionsandformalrecognitionofcategories offertilisers(AAPFCO,2012).Thefuturewillindicatehowthe fed-eralagenciesEPAandUSDAwillregulatebiostimulantproducts. Aplausiblescenariotodayseemsthatsomeofthebiostimulants couldfallunderEPAjurisdiction,whiletheotherswouldbe reg-isteredasfertilisersorsoilamendmentsatthestatelevel(David Beaudreau,BiostimulantCoalition,personalcommunication). Fur-thermore,USDA, viaitsNatural ResourcesConservation Service (NRCS)Agency,couldacknowledgethecapacityofcertain biostim-ulantstoreducenutrientrunoffbyincludingbiostimulantproducts inalistofsoilhealth-promotingpractices.

6. Developingthemarket:opportunitiesandchallenges

Duetothelackoflegalacceptanceoftheconceptof biostimu-lants,maketdataarescarceandoflimitedreliability.Theregulatory statusofbiostimulantsisindeeddiverse,dependingonwhether ornottheyareregisteredundertheREACHregulation,as fertil-isingmaterialsundernationallaws,aspesticidesundereuropean legislations,authorizedornotinorganicproductions,etc. Biostim-ulantsarespreadovermanyregulations,withoutbeingnamedas such,andthissituationconstraintstheestablishmentofregistersof productsandofreliablestatisticsoftheiruses.Still,theassociation EBIChasissuedeconomicoverviewsofthebiostimulantssectorin Europe,basedonsurveysofitsmembers(EBIC,2013).Although thedataarequalitativeratherthanstatistical,asacknowledgedby EBIC,theyindicateasteadilygrowingmarket(ofabout10%ormore peryear),whateverindicatorisused(sales,treatedhectares, num-berofusers).Themaincropsonwhichbiostimulantsareapplied todayinEuropeareindicatedinTable2.Anothermarket analy-sisreportwasissuedrecently,whichseemstoconfirmtheoutlines byEBIC(Marketsandmarkets.com,2014,seealsoatwww.agra-net. com).

Itisproblablymorerelevanttoidentifythedriversofthis grow-inginterest.Themaindriversarerelatedtogeneralagriculturaland environmentalpolicies,butotherdrivingfactorsaremorespecific tothebiostimulantssector.Regardingthefirstaspect,thereisan increasingawarenessoftheneedtopromotesustainable agricul-tureworldwide,combininghighproductivityandhighresourceuse efficiency(Garnettetal.,2013;SCAR,2011;TheGovernmentOffice forScience,2011;Tilmanetal.,2002).

Productive and resource efficient agrosystems should face future needsfor food and non foodmaterials,but theyshould alsodeliverecosystemserviceswichcontributethepreservation ofsoils,waterandair.Sustainableintensificationofcrop produc-tionscallsfortherecruitmentofecologicalfunctionalitiesincase of protectionand nutrition ofplants (Box 1).Biocontrol agents (e.g.predatororparasitoidinsects,antagonisticbacteriaproducing toxins and antibiotics, etc.)and biofertilisers (e.g. root growth-promoting,N-fixing PGPRs) benefit plants plants via ecological interactions in the cultivated ecosystem. Organic farming and agroecologypromotetheuseofsuchbiologicalsolutionsandof materialsofbiologicalorigin.InEurope,severalbiocontrolagents andbiostimulantsareincludedinregistersofmaterialsauthorized in organicproduction (Regulation (EC)No 889/2008). Inclusion intheseregistersisnotequivalenttoamarketingauthorization, whichdependsonseparateregulationsonfertilisersandplant pro-tectionproducts.

Regardingthefactorsrelatedtothebiostimulantssector,the EBICsurveysandmarketanalysespointtothespreadof biostimu-lantstonewgeographicalareasandnewcropproductions,fromthe pioneercountriesandapplicationsectors(e.g.fromhorticultural toagriculturalcrops,fromorganictoconventionalproductions).

Table2

IndicativelistofsomecropstowhichbiostimulantsarecurrentlyappliedinEurope(adaptedfromEBIC,2013,withpermission).

Treesandvinecrops Agriculturalcrops Vegetableandlegumecrops Otherhorticulturalcrops Citrus Barley Broccoli Onions Ornamentals

Pomefruits Maize Cabbage Peppers Nursery Stonefruits Rice Carrots Potato Turf Grapes(table) Wheat Cauliflower Lettuce

Grapes(wine) Oilseedrape Cucumber Squash Sugarbeet Eggplant Strawberry

Garlic Tomato Melons Watermelon

Anotherdriverofmarketdevelopmentistherelativelyhigh

invest-mentofcompaniesinresearchanddevelopment(between3%and

10%oftheannualturnoverinthesurveyedmembersofEBIC),which

contributestoanexpandinglistofbiostimulantsandofindustrial

processesfortheirproductionandformulation.

Challengesforthedevelopmentofbiostimulantsareof

scien-tific,technicalandregulatorynature.

Themainscientificchallengeisthecomplexityofthe

physiolog-icaleffectsofbiostimulants.Ingeneralterms,theprimaryeffects

ofbiostimulantsaretoinducephysiologicalresponsesintheplant.

Manyoftheseresponsesbearonprimarymetabolism,growthand

development.These processes are subject totight homeostatic

regulationswhichoriginatefrommillionsofyearsofbiological

evo-lutionandexplainwhyplantsoccupyspecificecologicalnichesand

display characteristicphenotypic responsesto fluctuating

envi-ronments.Actingonsuchbiologicalprocessesischallengingand

attentionshouldbepaidto themanycross talksbetween

pro-cessesandpathwaysinplantorganismsintheirresponsetotheir

environment.Furthermore,theuseofbiostimulantscanonlybe

successfulifthetripartiteinteractionsbetweenthebiostimulant,

theplantandtheenvironmentcanbeproperlyaddressed.Asan

example,phosphorus mobilizationbythehelp of

phosphatase-releasingPGPRsmaycontributetoplantgrowthandcropyieldif

soilinorganicphosphateisindeedlimitingandiftheplant

con-tributestothemaintenanceandactivityofthePGPRinoculantin

therhizosphere,e.g.viaitsexudates.

Technicalchallengesincludetheformulationandblendingof

biostimulantswithotherfertilisingmaterialsand/orplant

protec-tionproducts.Manybiostimulantsaimatimprovingnutrientuse

efficiencyandcombinationsbetweenfertilisersandbiostimulants

willneedtobeoptimized.Formulationofbiofertilisersisspecially

complex,andpositiveinteractionsbetweenmicrobiological

com-ponentsofthebiostimulantsmixturesononehand,andbetween

thebiostimulant inoculant and the resident rhizo-/endospheric

microbiotaontheotherhand,havetobesearchedfor.Technical

difficultiesalsoariseforthemonitoringofcropsandfordecidingon

whether,whenandhowbiostimulantsshouldbeapplied.The

situ-ationismorecomplexthanwithanyplantprotectionproducts,for

whichtheincidenceofpestsanddiseasesisrelativeyeasytodetect

andquantify,andforwhichepidemiologicalmodelsareavailable

tooptimizepesticidesapplications.Wearefarfromsuchasituation

withabioticstressors,whichofteninteractinthefield,aredifficult

toassessandareusuallyquantifiedexpostbyyieldpenalties.

Nutri-entuseefficiencyisalsodifficulttoevaluateanddecisiontoapplya

biostimulantthatwouldtargetthistraitishardlyjustifiedby

mea-surableplantcharacteristicsinthefield.Biostimulantslooklikea«

nice-to-have»technologyinsteadofa«need-to-have»technology

(ascomparedwithpesticides)andconsiderationofboth

immedi-ateanddelayedbenefitstothefarmers,includingresourcesavings

andecosystemservices,shouldbepromotedinthelongterm.

Regulatorychallengesarerelatedtothecategorizationand

pre-marketassessmentofbiostimulants,andtointellectualproperty.It

issofarunclearwhetherandwherebiostimulantswillberegulated

assuch,i.e.recognizedasaspecificregulatorycategorybynational

orsuprationallaws.Currentlawsonfertilisersandpesticidesallow

forthesafeplacingonthemarketofbiostimulantsinmostplaces

oftheworld,andreasonsformakingthemadistinctregulatory

categoryshouldbemadeexplicit.Onesuchargumentisthe

pecu-liarityofbiostimulantsregardingtheirmodeofaction–viathe

plantresponse-andoftheintendedeffects–onnutrientuse

effi-ciency,abioticstresstolerance,productquality–whichmayjustify

specificprovisionsforefficacyassessment.Providingguidanceand

ensuringregulatorycertaintyintheefficacyassessment,besides

riskassessment,ofbiostimulantsisimportantfordevelopingthe

technologyandthemarket,andthiscouldbestreamlinedby

supra-nationalregulations.Anotherargumentismarketharmonization,

aslinkingbiostimulantstofertilisershasledsofartoagreat

hetero-geneityinthewaytheyareplacedonthemarketfromonecountry

totheother.Furthermore,developinga suigenerisapproachfor

biostimulantscouldhelpregulatorsoptimizedatabridgingwith

existingregulationsandregisters,likethoseoftheREACH

regu-lation(ECNo1907/2006concerningtheregistration,evaluation,

authorisationandrestrictionofchemicals).

Biostimulants pose specific difficulties for the protection of

intellectual property. Patentability and prevention of copies/

reverse engineering ofthe biostimulant productsare often

dif-ficult. Originality of the product and its status of invention,as

demandedbypatentability,issometimesdifficulttoestablish,and

companiestendtopatenttheindustrialprocessesusedforthe

pro-ductionofbiostimulants.Dataprotectionmechanismscoupledto

themandatoryregistrationofbiostimulantswouldstrengthen

pro-tectionofintellectualproperty.Datasharingmechanismslinked

totheregistrationofbiostimulantsubstancesormicroorganisms

wouldsupportthedevelopmentofthebiostimulantsmarketby

promotingindustrialexchangesandpartnerships.TheREACH

reg-ulationinEuropehaspavedthewayforsuchdevelopment,butit

coverschemicalsandexcludesmicroorganisms,whichare

impor-tantcategoriesofbiostimulants.

7. Concludingremarks–lookingahead

Thefutureofplantbiostimulantsshouldbedrivenbythe

fol-lowinglinesofforce.

Fromthelaboratorytothefield:weunderstandthephysiology

ofplantstodaybetterthanever,thankstoscientificandtechnical

breakthroughsinmanydisciplinesoverthelastdecades.Mostof

theseachievementshaveusedalimitednumberofmodel

organ-ismsincontrolledenvironments.Achallengeisnowtousethis

knowledgeandthesetoolsforthecharacterisationof

biostimu-lantsand theireffects ona widerange ofcultivated plants.For

example,high-throughputplantphenotypingplatformshavebeen

developedforthecharacterisationofmutantsproducedin

func-tionalgenomicsstudies,buttheyshould(anddo)inspirestudies

forunderstandingthemodesofactionofbiostimulantsandtheir

interactions withenvironmentalstressorsand withplant

geno-types.Bridgingthegapbetweenmanylaboratorydataonsingle

biostimulantsand field dataonmixtures (oftencombinedwith

Fig.1.InductionoflateralrootformationonseedlingsofthemodelgrassBrachypodiumdistachyon(lineBd21)byvolatilecompoundsemittedbythePGPRBacilluspumilus C26,co-cultivatedonaverticalplatewithinasharedatmosphere(Delaplaceetal.,2015).

rootgrowthpromotionbysoilbacteriacanbeconsistently demon-stratedinlaboratoryconditions(Fig.1),butthissayslittleabout possiblebeneficialeffectsinpracticalfieldsituations.

Fromthe fieldtothe laboratory:thedevelopmentof biostim-ulantsmayfollow aclassical‘pharmacological’approach, where candidateactive substances or microorganismsare screened in controlledconditions and a stepwiseprocedure is followed for selecting promising candidates, moving from the laboratoryto morerealisticconditions.Thiscanbeefficientbutthestringentand stepwiseselectionofactivesubstancesormicroorganismsresults inhighdevelopmentcostswhich arehardlyjustifiedin market sectorscreatinglimitedaddedvalue,likeinplantnutrition and agriculture.Analternativewaywouldstartfromfieldobservations andleadbacktothelaboratoryforthesystematisationofthe sci-entificquestionsraised.Togiveanexample,soilmicrobiologists andecologistsarepointingoutthevariabilityintheway individ-ualplantcultivarsinteractwithrhizosphericbacteriaandmodulate thecompositionofthebacterialpopulations,evenoverthegrowing seasonofanannualcroplikemaize(Airaetal.,2010;Philippotetal., 2013).Whetherthesegenotype-dependentchangesinthe rhizo-sphericmicrobiome impactplantgrowthand healthisanopen question.Suchobservationscanbeastartingpointfor understand-ingthekeystosuccessfulinteractionsbetweenPGPRsandplants. Fromamorepracticalviewpoint,novelcommercialapproachesare beingdevelopedwhichaimatamplifyinglocalbeneficial micro-biotainsteadofinoculatingstandardizedmicrobialproducts.This approachismotivatedbytheempiricalfactthatalimitingfactor

whenusingmicrobialbiostimulantsisthecapacityofthe inocu-lanttoestablishandmaintainsufficientactivityintherhizosphere. Aparallelcanbemadewiththeintestinalmicrobiotainhuman medecine:addinginoculants(i.e.‘probiotics’)isonething,but feed-ingbeneficialbacteriawithprebioticsseemsevenmoreimportant. Healthbenefitscanbeobtainedbyusingprebioticsalone,which modulatetheintestinalmicrobiome (Rastalland Gibson,2015). Thisinspiresnewavenues tosustainablecropmanagement,by developingnewfertilisingmaterialsandbybreedingplantswith enhancedcapacityto‘manage’theirrhizosphericandendospheric microbiota.

Agriculturalandhorticulturaluseofbiostimulantswillrequire locallyandtemporallyadaptedsolutions.Monitoringtoolsforthe efficacyof biostimulantswillbeneeded andstewardship plans optimisingtheirusedefined.Longer termeffects,viaecological servicesandbiogeochemicalcycles,shouldalsobeassessedand integratedin thedecision-making processonthefarm. Compa-niesdevelopingbiostimulantswillhavetocontributetointegrated solutionsattheagrosystem,farmandlandscapelevels,ofwhich biostimulantsareonlyoneelement.Involvementofstakeholders, farmers,publicresearchandregulatorybodieswillbeneededto reapthebenefitsthat biostimulantscanbringtoprofitableand sustainableplantproductions.Onthislongway,publicactionis awaitedtoharmonisepoliciesandregulations,andtobuildupa robustriskassessmentframeworkwhichrespectstheprincipleof proportionalityandavoidsduplicationofdatarequirementsacross regulations.

Acknowledgements

Iwish tothankEricLiégeoisfromtheEuropeanCommission for providing impetusand supportto the scientific analysis of theconceptofbiostimulants,myuniversitycolleaguesand stu-dents for the fruitful discussions and theresearch staff of my laboratoryfortheirstimulatingworkonthephysiologyofplants, includingresponsestobiostimulants.Ithankrepresentativesof industry associationsand consultants—specially Kristen Sukalac (EBIC),DavidBeaudreau(BiostimulantCoalition)andDanielTraon (ArcadiaInternational)—forsharingtheirexpertknowledgeduring thewritingofthispaperandinotheroccasions.Ithankmy collab-oratorsPierreDelaplaceforhiscarefulreadingofthemanuscript, CarolineBaudson,AnthonyDigradoandBenjaminDeloryfortheir supportduringthepreparationofthegraphicalmaterialsofthis article.Iapologizetothemanyscientistshavingpublishedon bios-timulantswhocouldnotbecitedduetothelimitedspaceofthis reviewarticle.

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