Dissertation zur Erlangung des Doktorgrades der Agrarwissenschaften (Dr. agr.) der

Transcription

1 Underground networks of rusculr mycorrhizl fungi development nd functioning of the externl mycelium of Glomus mossee nd G. intrrdices in soil sustrte nd plnt residues Disserttion zur Erlngung des Doktorgrdes der Agrrwissenschften (Dr. gr.) der Nturwissenschftlichen Fkultät III Agrr und Ernährungswissenschften, Geowissenschften und Informtik der Mrtin Luther Universität Hlle Wittenerg vorgelegt von Anj-Christine Müller ge. m in Berlin Gutchter: 1. Prof. Dr. Edgr Peiter 2. Prof. Dr. Eckhrd George Tg der mündlichen Prüfung:

2 Contents 1 GENERAL INTRODUCTION Mycorrhiz Arusculr mycorrhizl symiosis generl chrcteristics AM fungl morphology nd development AM fungl presymiotic growth nd plnt root colonistion AM fungl extr-rdicl growth Photosynthte costs in the AM symiosis Host plnt enefits y AM fungl colonistion Indirect enefits AM fungl contriution to plnt P nutrition AM fungl contriution to plnt N nutrition The plnt nutritionl sttus nd the outcome of the AM symiosis AM fungl inoculum production nd the request for dequte inoculum formultions Agriculturl prctices tht ffect AM fungl symiosis Ojectives of the study GENERAL MATERIALS AND METHODS Description nd preprtion of experimentl plnt growth sustrte Preprtion of fungl comprtments Preprtion of fungl comprtment sustrte Extrction of the extr-rdicl mycelium from fungl comprtments nd estimtion of hyphe length nd spore numer AM fungl isoltes Estlishment of non-inoculted control plnts Estimtion of the AM fungl colonised root length Nutrient nlysis in plnt tissue Experimentl loction...23 i

3 CONTENTS 3 THE SYMBIOTIC RECAPTURE OF NITROGEN FROM DEAD MYCORRHIZAL AND NON-MYCORRHIZAL ROOTS OF TOMATO PLANTS Astrct Introduction Mterils nd methods Pre-cultivtion of plnt mteril Preprtion of growth sustrte nd plnting units Arusculr mycorrhizl inocultion nd instlltion of fungl comprtments Plnt cultivtion, 15 N ppliction nd set-up of the donor plnt tretments Hrvest nd nlysis of plnt nd AM fungl mteril Nutrient nlysis nd sttistics Results Dry weight nd nutrient sttus of the donor plnts Intr- nd extr-rdicl AM fungl development Nitrogen concentrtion nd content in the extr-rdicl mycelium Dry weight nd nutrient sttus of the receiver plnts Receiver plnt dry weight nd P sttus Receiver plnt sttus of totl nitrogen nd 15 N Discussion Estlishment of experimentl conditions to quntify AM fungl derived interplnt N trnsfer Symiotic N trnsfer from mycorrhizl nd non-mycorrhizl ded roots AM fungl medited N trnsfer s ffected y the presence of mycelium within the donor root Effect of soil disruption on N trnsfer to receiver plnts Conclusions DETACHED EXTRA-RADICAL MYCELIUM NETWORKS OF DIFFERENT AM FUNGI COLONISATION POTENTIAL AND PLANT GROWTH PROMOTION AFTER MYCELIUM DISRUPTION Astrct Introduction Mterils nd methods Production of experimentl plnts Preprtion of rhizooxes, sustrte filling nd AM fungl inocultion Preprtion nd insertion of fungl tues Plnting, experimentl set-up nd growth conditions Hrvest nd nlysis of plnt nd AM fungl mteril Nutrient nlysis nd sttistics Results Mize plnts in experimentl phse Sweet potto plnt iomss, AM fungl root colonistion rte nd P sttus AM fungl ERM development in fungl tues...62 ii

4 CONTENTS 4.5 Discussion Mize plnt colonistion nd growth in experimentl phse Detched excised extr-rdicl mycelium s source of AM fungl colonistion The growth response nd P uptke of sweet potto plnts in reltion to AM fungl colonistion The growth pttern of the AM fungl extr-rdicl mycelium The effect of soil disturnce on the infectivity of the excised ERM Root distriution with depth Conclusions AM FUNGAL SPORULATION WITHIN DEAD TRAP ROOTS SPORE QUANTITIES AND DISTRIBUTION PATTERN Astrct Introduction Mterils nd Methods Nurse plnt pre-cultivtion nd AM fungl inocultion Production nd preprtion of trp roots Preprtion nd filling of trp root comprtments Experimentl set-up nd growth conditions Hrvest nd quntifiction of AM fungl propgules in roots Sttisticl nlysis Results Nurse plnt root AM fungl colonistion nd spore density in pot sustrte AM fungl colonistion nd sporultion in trp roots Nurse plnt root AM fungl colonistion nd spore density in pot sustrte AM fungl colonistion nd sporultion in trp roots Discussion Experimentl conditions nd AM fungl sporultion pttern in trp roots Trp roots s possile source of nutrients Sporultion intensity per trp roots of different origin Sporultion quntity ccording to trp root dimeter Sporultion in trp root lyers of different thickness Conclusions GENERAL DISCUSSION Inter-plnt N trnsfer through common rusculr mycorrhizl mycelium network Relevnce of AM fungl N trnsfer for the receiver plnt growth Relevnce of AM fungl N trnsfer for N-cycling The common mycorrhizl mycelium network s n underground trnsport mens ffecting inter-plnt communiction nd competition The impct of soil disruption on AM fungl development nd functioning The effects of tillge systems on the infectivity of AM fungi The colonistion of root y detched ERM is followed y erly plnt growth promotion iii

5 CONTENTS 6.3 AM fungl sporultion in ded roots is strtegic The preferred prolifertion of ERM towrds orgnic mtter nd the ttrction of AM fungl sporultion y root frgments Root frgments s low-weight crrier mteril for future of AM fungl inoculum production? SUMMARY / ZUSAMMENFASSUNG Summry Zusmmenfssung REFERENCES ACKNOWLEDGMENTS iv

6 CONTENTS Arevitions AM DAP DAS DC DS DW ERM FC/ FT IRM LC RC v/v w/w Arusculr mycorrhiz Dys fter plnting Dys fter sowing Donor root comprtment Dry (soil) sustrte Dry weight Extr-rdicl mycelium Fungl comprtment/ Fungl tue Intr-rdicl mycelium Lelled root comprtment Receiver root comprtment Volume per volume Weight per weight v

7 Chpter 1 1 Generl introduction The development of symiotic ssocitions etween different orgnisms is strtegy tht hs evolved over millenni to llow species to cope with the lrge vriety of environmentl conditions existing on erth. Plnts hve developed different strtegies for dption nd diversifiction, one of which is the co-evolution with microorgnisms to cquire nutritionl elements from the soil. Specil importnce cn e ttched to the symiotic interction of plnts with soil-orne fungi, mycorrhizl fungi. Fossil finds of plnts, dted to the Ordovicin / Devonin period (Redecker et l. 2002), hve reveled mycorrhizl colonistion of plnt roots s erly s 400 million yers go, indicting tht this inter-specific connection hs existed since plnts first inhited terrestril ecosystems. It is therefore not stonishing tht mycorrhizl symioses cn e found in the vst mjority of lnd plnt species occupying ll different terrestril ecosystems, thus mking them one of the most widespred plnt-microil ssocitions. From the point of view of the plnt, one min enefit within this reltionship is the fungl derived nutrient supply to the plnt. This chpter will provide some informtion on the mycorrhizl symiosis nd its significnce for plnt nutrition. 1.1 Mycorrhiz The erliest experimentl studies on mycorrhiz, descriing the symiotic connection etween fungi nd plnt roots, hve een pulished in the lte 19 th century. Frnk (1885) ws the first to coin the term mycorrhiz which comes from the Greek words: mykes (fungus) nd rhiz (root). It hs een shown tht the fungus is supplied with cron y the host plnt nd in return provides minerl nutrients to the plnt. The type of symiosis minly reported for mycorrhizl ssocitions is mutulistic form of symiosis, where in most cses the plnt enefits from the 1

8 1 GENERAL INTRODUCTION fungl colonistion (Smith nd Red 2008). From the nutritionl point of view, when chrcterising mycorrhiz, it should e considered tht this symiosis (similr to other symiotic forms) is the result of cost-enefit rtio etween plnt nd fungl nutrient contriution nd consumption. In n optiml, lnced cse, oth prtners would enefit from ech other. Previous studies, quntifying costs nd enefits of mycorrhizl symioses, hve indicted tht not ll mycorrhizl ssocitions re mutulistic ut rther shift into one-sided enefit within the reltionship. This underlines the complexity of this symiosis nd mkes it importnt to understnd processes tht influence the outcomes of the mycorrhizl symiosis. Mycorrhiz is formed y fungi elonging to the phylum Glomeromycot (Redecker nd R 2006) nd includes oth septte fungi, elonging to Glomeromycetes, nd septte fungi, elonging to Ascomycetes nd Bsidiomycetes. Referring to their specific morphology nd chrcteristics of the ssocition etween the fungl mycelium nd plnt roots, the mycorrhiz fungi re sudivided into severl different types: ectomycorrhiz, ectendomycorrhiz, endomycorrhiz, ericoid mycorrhiz, rutoid mycorrhiz, monotropoid mycorrhiz nd orchid mycorrhiz (Smith nd Red 2008). The most undnt groups of these mycorrhiz re the ectomycorrhiz nd the endomycorrhiz. Ectomycorrhizl fungi form thickened mycelium cpsules round host plnt roots, termed the Hrtig net, nd mycelium growth is limited to the intercellulr spce etween root corticl cells (Mssicotte et l. 1989; Finly 2008). The common plnt types hosting ectomycorrhiz re woody perennil species (Smith nd Red 2008). In contrst to ectomycorrhiz, the growth of endomycorrhizl fungi occurs in oth, inter- nd intrcellulr spces of corticl cells. Endomycorrhiz forms complex intr-rdicl mycelium (IRM) within the root cortex which is differentited into hyphe, ruscules nd vesicles. One importnt memer of this group is the rusculr mycorrhiz, the min suject of this thesis. 1.2 Arusculr mycorrhizl symiosis generl chrcteristics To complete their life-cycle, rusculr mycorrhizl fungi depend on the crohydrte supplied y their host plnt nd re therefore clssified s oligte iotrophs (Prniske 2008). They colonise the outstnding mjority of known lnd plnt species, elonging to ll lnd plnt phyl, nd re estlished in very diverse terrestril ecosystems. To dte, out 200 morphospecies hve trditionlly een descried, distinguished y fetures of the spore wll (Smith nd Red 2008). The wy the spore is formed on the hyphe is used to circumscrie gener nd fmilies, nd the lyered structure of spore wlls is used to distinguish species 2

9 1 GENERAL INTRODUCTION (Morton nd Benny 1990; Blszkowski et l. 2010). The following fmilies were distinguished within the clss Glomeromycetes: Aculosporcee, Amisporcee, Archeosporcee, Diversisporcee, Entrophosporcee, Geosiphoncee, Gigsporcee, Glomercee, Pcisporcee nd Prglomercee (Redecker nd R 2006). AM fungi re multinuclete with severl hundreds to thousnds of nuclei within single spore (Becrd nd Pfeffer 1993; Mrleu et l. 2011). Nuclei migrte through hyphe nd ggregte in developing spores nd re formed y mitosis (Mrleu et l. 2011). Since the nucler popultion within spore or hyphe frgment is hetero-kryotic, genetic vrition within individul AM fungi is high. It hs een shown tht glomlen spores originted from singlespore cultures hold different genetic fingerprints (Zeze et l. 1997; Koch et l. 2004). It cn e ssumed tht the multi-genomic chrcter of these fungi is necessry, since they hve to fce huge vriility of micro-environmentl conditions, differentiting inside plnt roots nd proliferting extr-rdiclly into the soil. At the sme time they re chllenged y mcroenvironmentl iotic nd iotic fctors. AM fungl species cn colonise wide spectrum of plnt species nd re known to e minly host unspecific. One fctor tht influences the outcome of the symiosis is thought to e the host plnt dependence on the AM symiosis for nutrient uptke nd growth, vrying from lmost independent to highly dependent. The AM symiosis seems to e prticulrly eneficil when plnts possess reltively low cpcity for nutrient uptke vi their own root system or when nutrient vilility in soils is limited y iotic fctors (Mosse 1977; Sif 1987; Smith nd Red 2008). When quntifying net enefit derived from AM colonised compred with uncolonised host plnts, reserch hs rought vrile results in terms of nutrient uptke nd plnt growth. A etter understnding of the processes tht influence the outcome of AM symiosis in terms of plnt nutrition my contriute to improve mngement strtegies for plnt production in sustinle griculture. 1.3 AM fungl morphology nd development This section descries the structures nd growth processes of AM fungi involved in the AM life cycle, strting with the resting propgule, followed y the colonistion of plnt root, the formtion of extr-rdicl structures up to the ending of the life cycle. 3

10 1 GENERAL INTRODUCTION AM fungl presymiotic growth nd plnt root colonistion Root colonistion y AM fungi cn e initited in different wys: i) Asymiotic infection originted from spores, mycelium frgments, or from AM fungl colonised plnt roots; ii) Symiotic infection originted from neighouring roots of the sme or different plnts nd plnt species. In terms of symiotic infection nd estlishment of new colonies, spores re importnt inoculum sources nd therefore re studied in the present work. Depending on the AM fungl species, spore dimeters rnge etween 15 nd 800 µm (Sieverding 1991). Spores contin cytoplsm nd storge lipids, their energy source, nd cn mintin their germinility for severl yers in the soil despite eing exposed to hrsh nd chnging environmentl conditions. By these mens spores re the min genertive orgns for AM fungi which is in contrst to excised mycelium frgments tht cn only mintin their viility for reltively short period. Spores nd mycelium frgments differ in life-spn depending on the fungl species nd their relevnce s propgules to estlish new colonistion vries etween fungl fmilies. For exmple, memers of the fmily Glomercee re le to infect effectively from spores nd mycelium frgments while representtives of Gigsporcee infect only from spores (Klironomos nd Hrt 2002). The life cycle of AM fungi usully strts with the germintion of propgule, either resting spore or mycelium frgment locted within the ulk soil or within former AM colonised root frgment. The germintion process hppens in sence of the host plnt during presymiotic growth nd is chrcterised y the germ tue development nd elongtion which is usully interrupted fter few millimetres when no potentil plnt root is present, so tht stored resources re used economiclly (Koske 1981). In this stte the propgules exist in n symiotic wy nd re not influenced y the presence of host plnt ut merely y iotic fctors, predominntly soil moisture, soil ph nd temperture (Dniels nd Trppe 1980; Siqueir et l. 1982; Clrk 1997). In the cse tht propgules germinte in the presence of plnt, germintion is triggered y signl molecules such s strigolctones, flvonoids nd phytoestrogenes contined in plnt root exudtes (Akiym et l. 2005; Steinkellner et l. 2007). These compounds of root exudtes my e detected y the fungus s chemotropic guidnce to ccelerte host root loction nd therefore reduce energy loss during presymiotic growth (Srn nd Giovnnetti 2005). Prior to the contct with the root, AM fungi produce the so clled MYC-fctor stimulting formtion of AM symiosis s well s root rnching in host plnts (Smith nd Red 2008). 4

11 1 GENERAL INTRODUCTION As soon s the hyph comes in contct with root it differentites to form hyphl swellings on the root surfce, termed ppressori (Huse nd Fester 2005). Nturl plnt defence responses re incresed t this erly stge of ssocition, ut re suppressed to low levels very soon therefter (Kpulnik et l. 1996). The fungl entry into the host plnt root cortex is ccompnied y vrious chnges in the root cell, including cell wll loosening (Blestrini et l. 2005), reorgnistion of corticl cell orgnelles nd finlly the formtion of pre-penetrtion pprtus tht finlly forms hollow tue in the plnt cell fcilitting the fungl hyphe growth through the root epidermis (Genre et l. 2005). At this stge the hyphe prolifertes intensively longitudinlly etween prenchym cortex cells nd develops mnifold side rnches tht form chrcteristic intr-rdicl structures including ruscules nd, depending on the fungl species, lso vesicles. It is recognised tht two different types of AM fungi cn e distinguished in terms of the structures they form in corticl cells: Arum type which is chrcterised y ruscules nd Pris type, tht forms hyphl coils (Smith nd Red 2008). There is evidence tht given AM fungus cn develop either ruscules or hyphl coils depending on the host plnt (Dickson 2004). The present description refers to the Arum type which ws oserved in the host-am interctions exmined in the experiments of this study. Until ruscules re formed, the fungus relies on its propgule resources for development. The ruscule formtion strts with hyphl penetrtion into corticl cell which susequently rnches dichotomously into tree-shped structure, the ruscule (Huse nd Fester 2005). The pronounced rnching llows for incresed surfce contct etween the interfces of oth symiotic prtners nd this is ssumed to e the loction where crohydrtes re exchnged for nutrients (Hrrison 1999). Aruscule formtion thus mrks the eginning of the symiotic phse. Aruscules, like other intr-rdicl structures, remin in the poplst nd re lwys seprted from plnt cell cytoplsm. The seprtion consists of thin mtrix including the fungl cell wll, the plnt-derived poplst nd the perirusculr memrne which origintes from the plnt cell plsm memrne (Dexheimer nd Prgney 1991; Hrrison 1999; Prniske 2008). Susequent to their formtion, ruscules remin ctive for out seven dys (Alexnder et l. 1988; Huse nd Fester 2005) efore they senesce nd degrde. After ruscule development, mny AM fungl species ggregte their resources within hyphl swellings, the vesicles contining high levels of cytoplsm s well s storge lipids nd functioning s propgules within root frgments (Smith nd Red 2008). 5

12 1 GENERAL INTRODUCTION AM fungl extr-rdicl growth During intr-rdicl colonistion the fungus is supplied with crohydrte y the host. The extr-rdicl mycelium (ERM) development occurs y spreding intensively out of the root nd into the sustrte eyond the rhizosphere. Hyphe tht develop extr-rdicl nd spred into ulk soil differ in dimeter etween 1 nd 20 µm (Sieverding 1991). Fine hyphe with dimeters etween 1 nd 5 µm re ssumed to e responsile for nutrient uptke, since they form rnched soring structures (BAS) with incresed surfces, similr to ruscules (Bgo et l. 1998). Corse hyphe (5-20 µm) cn e oserved to run longitudinlly long the root surfce (runner hyphe) nd pper to serve minly for extension nd fst spred of the fungl colony (Friese nd Allen 1991). By re-colonistion of roots the fungus connects not only neighouring roots of the sme plnt ut lso connects root systems elonging to different host plnts. Depending on the host crohydrte supply, the extr-rdicl mycelium (ERM) prolifertes into the surrounding sustrte out 15 cm distnt from the host root surfce (Jns et l. 2003) nd therefore cn cquire nutrients fr eyond the rhizosphere. Prolifertion strtegies, in terms of spred intensity into the root surrounding sustrte, differ etween AM fungl species (Mikkelsen et l. 2008). Once colonistion is well dvnced (etween 3 weeks nd 6 months post initition of root colonistion), depending on the fungl species, sexul spores cn form on the ERM (Sieverding 1991). The importnce of spores s infective units vries etween fungl species, the locl undnce of the fungus, nd the environmentl conditions. Spores re the most stle nd effective propgules to estlish infection compred with other inoculum sources such s colonised roots or excised hyphe (Bellgrd 1993). 1.4 Photosynthte costs in the AM symiosis For their prolifertion nd mintennce, AM fungi depend on the crohydrte supplied y their host. Sustntil mounts of mycelium iomss cn e present within roots nd mycorrhizl roots cn receive 4-20% more photosynthtes thn non-inoculted roots (Douds et l. 1988; Jkosen nd Rosendhl 1990). By pulse lelling of extensively colonised plnts with stle isotopes, Jkosen nd Rosendhl (1990) clculted tht out 20% of the totl plnt fixed cron (C) cn e ttriuted to AM fungl use. It is possile tht AM fungl colonistion cn cuse plnt growth depressions, due to the C drin to the fungus especilly under conditions were C reserves of young plnts fil to meet AM fungl crohydrte demnd (Mortimer et l. 2005). Nevertheless, due to the nutritionl enefits provided y the fungus, the 6

13 1 GENERAL INTRODUCTION plnt is usully le to compenste the C costs of the fungus y the increse of photosynthesis per unit lef re (Mortimer et l. 2008). The photosyntheticlly fixed C is trnslocted to the plnt sink orgns, predominntly in the form of sucrose which is lost from the plnt cell long concentrtion grdient nd then relesed into the poplstic interfce. Sucrose first hs to e cleved y cytosolic sucrose synthse or y invertses efore eing sored y the fungus s hexose, minly in form of glucose nd lso fructose. The hexoses re rpidly incorported into trehlose nd glycogen which re supposed to uffer excess glucose ccumultion in the cell (Smith nd Red 2008). It is ssumed, tht hexose sorption is conducted vi the plsm memrne of intr-rdicl orgns including hyphe, ruscules nd hyphl coils (Smith nd Red 2008). 1.5 Host plnt enefits y AM fungl colonistion Indirect enefits The most importnt enefit of AM symiosis for the host plnt is the AM fungl function with respect to nutrient trnsfer to the plnt prtner nd therewith the involvement in nutrient cycling processes. Indirect enefits for host plnts medited y AM fungl colonistion include the following: i) Allevition of the dverse effects of drought (reviewed y Augé 2001), slt stress (reviewed y Evelin et l. 2009), nd high concentrtions of hevy metls in AM fungl colonised host plnts. AM fungi my function s n effective sink for hevy metl surpluses nd pssively dsor hevy metl ions y inding them to the fungl cell wll (Joner et l. 2000) nd to glycoproteins secreted y the fungi (Gonzlez-Chvez et l. 2004). ii) The formtion of eneficil reltionships etween AM fungi nd other rhizosphere microorgnisms, such s nitrogen fixing nd plnt growth-promoting cteri which cn physiclly ttch to the fungl surfce (Gerdemnn nd Trppe 1974; Ho 1988; Binciotto et l. 2001; reviewed y Artursson et l. 2006). iii) Improvement of soil structure due to the formtion of wter stle ggregtes s result of the secretion of glycoproteins y AM fungi (Rillig et l. 2002). iv) Incresed host plnt resistnce to soil-orne pthogens nd nemtodes, thought to e induced y stimultion of defence responses (Volpin et l. 1994; Morndi 1996; Li et l. 2006) or y competition with pthogens for root infection sites (Muchovej et l. 1991). 7

14 1 GENERAL INTRODUCTION It is firly widely cknowledged tht soil-orne cteri, present in the myco-rhizosphere re closely ssocited with AM fungi. Some such cteri re cple of producing plnt ville minerl nutrients y decomposing soil orgnic mtter nd consequently ply crucil role in nutrient cycling. Therefore, when exmining AM fungl contriution to plnt nutrient supply, the impct of soil-orne cteri should not e neglected. Bcteril species known to e eneficil for plnt growth, due to their nitrogen fixtion, P-soluilising or io-degrdtive properties, cn e ttched to hyphe nd spore surfces (Toljnder et l. 2006). Severl mechnisms hve een proposed to e involved in this inter-specific interction: the ccommodtion of cteri y fungl secretion of soil ggregte stilising polyscchrides (Binciotto et l. 2001), the improved fungl growth nd estlishment in presence of certin cteri (Xvier nd Germid 2003), or lterntively the orgnisms could lso e in competition for nutrients (Rvnskov et l. 1999). By spreding into soil, the lrge surfce of the AM fungl extr-rdicl mycelium my not only directly tke up nutrients ville in the ulk soil distnt from the host rhizosphere, ut could lso function s mens of trnsport for cteri. Soil cteri occurring together with AM fungi increse the nutrient vilility from orgnic sources (Hodge et l. 2001) nd therey enhnce AM fungl ility to promote plnt growth. A possile function of externl hyphe s pthwy for soil solutes other thn minerl nutrients ws recently demonstrted y Brto et l. (2011), who oserved trnsfer of hydrophilic nd lipophilic sustnces etween two colonised root systems of two plnts interconnected y common AM fungl hyphe network. Allowing for solute movement (either on hyphl surfces or in the interior of hyphe), AM fungl myceli my lso serve s highwy for sustnces such s signlling molecules tht enle chemicl communiction etween plnts. In this thesis the emphsis will e plced on the function of AM fungi in nutrient trnsfer, the fungi s most direct contriution to plnt growth. Even though the production of externl mycelium vries considerly etween AM fungl species (Aott nd Roson 1985; Jkosen et l. 1992; Smith et l. 2004), ll develop extensively rnched, sorptive structures (Bgo et l. 1998) tht enle them to ccess the soil solution cptured within fine soil pores, otherwise unville to plnt roots. Furthermore, hyphe spred cn explore out 12 cm³ of soil volume per centimetre of colonised root length, compred with soil volume of out 1-2 cm³ for similr length of n uncolonised root (Sieverding 1991). Therefore during the symiotic ssocition, AM fungi my forge for nutrients fr eyond the soil volume of the rhizosphere y ridging nrrow depletion zones, especilly those of reltively immoile nutrients such s 8

15 1 GENERAL INTRODUCTION phosphorus (P), zinc (Zn), copper (Cu) nd mmonium (NH + 4 ). AM fungl colonistion hs een shown to increse plnt uptke of the elements mentioned ove nd lso tht of sulphur (S) nd potssium (K) (Smith nd Red 2008). The following susections give rief overview of plnt P nd N nutrition s ffected y the rusculr mycorrhizl symiosis AM fungl contriution to plnt P nutrition For plnt nutrition phosphorus (P) is one of the ll-importnt mcro-elements, required y the plnt in reltively lrge quntities. Being structurl component of mcromolecules, P is most prominent in nucleic cids, the phospholipids of io-memrnes nd in the energy-rich intermedites nd coenzymes involved in iosynthesis nd degrdtion processes (Mrschner 1995). In the plnt tissue, P is very moile nd is trnsported within the phloem during plnt development, depending on the demnd of the respective orgn (Biddulph et l. 1958; Rusch nd Bucher 2002). Soil P is contined in orgnic s well s minerl P pools (Shrpley nd Smith 1985). Inorgnic - P is considered to e the most importnt form of P tken up y plnt roots either s H 2 PO 4 or HPO 2-4. P-ions in soils re esily ound to C, resulting into the formtion of hrdly solule C-phosphtes minly in high ph soils. P cn lso e ound to Fe or Al, forming hrdly solule complexes minly in low ph soils (Scheffer nd Schchtschel 2009). The strong ffinity of soils for P-ions my result in P immoilistion nd in low concentrtions of plnt ville P in the soil solution, especilly under lkline or cid soil conditions nd in soils with high C content (Koide 1991). As result of these rections, P depletion zones my develop rpidly round plnt roots (Mrschner 1995). To some extent, microil minerlistion of P from soil orgnic mtter cn increse P concentrtions nd moility in the soil solution (Seeling nd Zsoski 1993). Thus, lthough the totl content of P in the soil my e high, it is often present in unville forms. More thn 80% of the soil P sources cn ecome immoile ecuse of dsorption, precipittion, or microil conversion into immoile orgnic forms (Scheffer nd Schchtschel 2009), nd this insufficient P vilility hs often een oserved to limit plnt growth in nturl soils (Bucher 2007). Under such conditions, the vlue of the AM symiosis for sustinle griculture nd re-vegettion prctices my e gret, since AM fungi re usully eneficil for plnts in terms of improved P cquisition. Accordingly, AM fungl contriution to plnt P uptke is most significnt under conditions of low P vilility in the soil solution (Mrschner nd Dell 1994). 9

16 1 GENERAL INTRODUCTION To forge for P locted in the immedite vicinity of the root rhizosphere, terrestril plnts hve developed efficient phenotypic nd physiologicl dpttions. The most importnt physicl dpttions re mximistion of the sorptive surfce re y incresing root length densities in ulk soil nd root hir development per unit root length (Lynch 1995; Brer 1995). Furthermore, plnts ctively induce cidifiction of the soil solution y proton relese from root tips (Krus et l. 1987; Tng et l. 2004). Depending on the plnt species, complex of root exudtes re produced, which cn include orgnic cids to soluilise inorgnic P frctions or cid phosphtse to ctlyse the minerlistion of orgnic P frctions (Li et l. 1997). In ddition to these P cquisition methods, plnts cn lso e supplied with P y the mycorrhizl pthwy. The extr-rdicl mycelium (ERM) of AM fungi spreds into the ulk soil eyond the depletion zone of plnt roots, thus creting lrger P soring surfce. In terms of plnt P cquisition, the increse of vilility of P is the min dvntge of the ssocition with AM fungi. The fungl hyphe cn enter soil pores with very smll dimeters tht re inccessile to roots (Drew et l. 2003). Moreover, it hs een reported tht the fungi hve gret ptitude for mining P from the soil solution. They hve the ility to excrete enzymes, nmely phosphtses, which enle the moilistion of P from orgnic mtter (Joner nd Johnsen 2000). When intercting synergisticlly with P-soluilising microorgnisms, AM fungi re thought to contriute lso to the soluilistion of P from rock phosphte sources (Antunes et l. 2007). Moreover, AM fungl colonistion cn reduce the severity of wter stress to plnts (Nelsen nd Sfir 1982; Neumnn et l. 2009), n effect tht hs een ttriuted to n incresed P nutrition through the mycorrhizl pthwy under dry soil conditions (Neumnn et l. 2009). When in symiosis with AM fungi, plnts usully respond to improved P nutrition y the development of lower root, ut higher shoot growth, compred with non-mycorrhizl plnts. This is noticele in the higher shoot-root dry weight rtio typiclly oserved in mycorrhizl plnts (Mrschner 1995). Under conditions of pronounced P deficiency, root P uptke my not stisfy the plnt s P requirement. In such cses, the enefit of mycorrhizl P delivery ecomes incresingly importnt for plnt growth, so tht the resultnt plnt iomss ccumultion is enhnced compred to tht of non-mycorrhizl plnts (Sieverding 1991). The trnsport of P from the AM fungi to plnts hs een studied using comprtmented pot systems where lelled phosphorus isotopes were supplied to the fungus (Jkosen et l. 1992; Person nd Jkosen 1993; Smith et l. 2003; Smith et l. 2004). These studies reveled tht fungl-derived P rnges 10

17 1 GENERAL INTRODUCTION from smll percentge to lmost ll of the P cquired y the plnt, nd huge vritions exist depending on the plnt/fungus comintion (Person nd Jkosen 1993; Smith et l. 2003; Smith et l. 2004). Although P is delivered through the mycorrhizl pthwy, plnts my not necessrily respond to mycorrhizl colonistion with incresed iomss production or incresed net P uptke when compred with non-mycorrhizl plnts (Smith et l. 2003; Smith et l. 2011). This effect hs een explined y down-regultion of the plnt high-ffinity Pi trnsporters (PiTs; usully expressed in ctively P soring root tissue) in the root epidermis of AM colonised plnts (Smith et l. 2011). Therefore the reduced direct pthwy might e compensted y the independent AM fungl pthwy resulting into similr quntities of totl P uptke in mycorrhizl compred to non-mycorrhizl plnts (Smith et l. 2011). The concentrtion of P in soil solution is usully lower thn in plnt roots nd fungl cytoplsm, nd to counterct the concentrtion grdient, P uptke y the extr-rdicl mycelium requires energy. Therefore, inorgnic P (P i ) is ctively sored y the ERM nd enters the fungl cytoplsm driven y H + /P i symporters, whilst the required proton grdient is produced y plsm memrne H + -ATPses. After eing tken up, P i is incorported into polyphosphtes, which re trnslocted within the mycelium (Bücking nd Shchr-Hill 2005). When fungl P uptke is higher thn demnd, surplus of P ccumultes in vcuoles where it is stored for lter use. When required, P trnsport through the interfcil poplst is ssumed to e regulted y the intrcellulr P i concentrtion within the hyphe (Bücking nd Shchr-Hill 2005). The vcuolr P pool contins minly polyphosphtes which proly ply importnt roles in fungl derived P supply to the plnt (Ezw et l. 2002). The intr-rdicl mycelium (IRM) of AM fungi is likely supplied with P derived from vcuolr components (Ezw et l. 2002), nd the trnsport my occur long motile tuulr vcuole system (Olsson et l. 2002; Uetke et l. 2002). The exct mechnism of P rekdown in the IRM is still not well understood, ut it is ssumed tht the polyphosphte molecules re reduced in size y hydrolystion in the intr-rdicl hyphe (Ohtomo nd Sito 2005), nd then relesed to the host s P i. The min site of nutrient exchnge etween the two symionts is proposed to e the interfce etween the fungl rusculr memrne nd the plnt perirusculr memrne (Cox nd Tinker 1976). P i supposedly exits through the fungl plsm memrne into the interfcil poplst where it is ctively trnsported into plnt cells (Ezw et l. 2002). 11

18 1 GENERAL INTRODUCTION AM fungl contriution to plnt N nutrition Nitrogen (N) is mcro-nutrient required in the highest quntities y the plnt. N plys centrl role in the synthesis of plnt mcro-molecules nd is component of structurl proteins, enzymes nd mino- nd nucleic cids (Mrschner 1995). Therefore, plnt growth is first of ll determined y the vilility of N in the soil. Nitrogen ecomes ville s result of the continuous cycling of inorgnic nd orgnic compounds crucilly ffected y the ctivity of soil-orne micro-orgnisms. The soil N pool consists predominntly (out 90%) of orgnic forms, such s mino cids, mino sugrs nd N-contining heterocyclic compounds. Orgnic N molecules cn e rpidly decomposed y heterotrophic microes tht moilise N from orgnic sources y the conversion into mmonium (NH + 4 ) which then underlies the nitrifiction process y microil trnsformtion into nitrte (NO - 3 ). A considerle contriution to the soil N input is medited y Rhizoi cteril N fixtion of gseous nitrogen, nd lso soil orgnic mtter is n importnt N pool relesing plnt ville N susequent to microil degrdtion. Following the minerlistion process, reltively smll proportion of the soil N pool (out 5%) is plnt ville in the form of inorgnic N. Stedily produced mmonium is unlikely ccumulted in most soils, since the conversion to nitrte occurs fster thn mmonifiction (Scheffer nd Schchtschel 2009). In well erted soils, minerl N is predominntly present s nitrte, reltively moile component susceptile to e lost to deeper soil lyers y leching. The concentrtion of minerl N (N min ) in griculturl field soils vries gretly; e.g kg/h N min in dry soil from the top lyer fter crop hrvest (Herle et l. 2004; Sdej nd Przekws 2008). Low sttus of ville N in field soils re usully compensted y fertiliser ppliction, since N demnd of crop plnts is reltively high. The tissue N concentrtion of well-nourished crop plnts rnges etween 2 nd 5% depending on the plnt species, the developmentl sttus nd the considered orgn (Mrschner 1995). + The mjor forms of inorgnic N tken up y plnt roots re NH 4 nd NO - 3. Depending on the plnt N demnd nd species specific preferences, NO - 3 is ssimilted in the root plstids nd in the shoot chloroplsts. NO - 3 is redily moile in the xylem tissue nd in cse of surplus it is + stored in the cell vcuoles of different plnt orgns. In contrst, NH 4 hs to e ssimilted immeditely y the plnt into mino cids t the site of uptke to prevent toxic effects of this - compound. Plnts incorporte the mjor prt of the sored NO 3 into essentil orgnic compounds. Therefore, NO - 3 hs to e trnsformed y enzymtic reduction to NH + 4. Finlly, 12

19 1 GENERAL INTRODUCTION NH 4 + derived from either NO 3 - reduction or root uptke, serves s sis to uild-up essentil mino cids nd other orgnic compounds relevnt for plnt development (Tiz nd Zeiger 1999). It hs een shown tht N is tken up y AM fungi nd trnsported to host plnts, thus the ctul significnce of AM fungl N cquisition for overll plnt nutrition remins uncler. The cpcity of AM fungi to improve N vilility to colonised host plnts cn e explined y its intense hyphl prolifertion in soil enling etter sptil explortion of N. Utilising 15 N lelled N, it hs een reported tht considerle mounts of N re tken up y AM fungi, trnsported through the ERM network nd supplied to the host plnts (Johnsen et l. 1992; Frey nd Schüepp 1993; Surmnin nd Chrest 1999; Tnk nd Yno 2005). When high mounts of N were supplied only to fungl comprtments, thus seprted from plnt roots, the percentge of plnt totl N ttriuted to hyphl uptke were up to 20-30% (Ames et l. 1983; Frey nd Schüepp 1993). In contrst, AM fungl plnt-to-plnt N trnsfer hs een shown to not increse plnt N uptke when compred with uncolonised plnts (Johnsen nd Jensen 1996). The uptke y the hyphe occurs in the form of NH + 4 nd NO - 3 (Johnsen et l. 1992; Surmnin nd Chrest 1999) nd lso mino cids (Hwkins et l. 2000). When tken up either s NH + 4 or NO - 3, oth forms re likely ssimilted into rginine s the min trnsport form within hyphe (Govindrjulu et l. 2005), nd therefter, N is proly trnsferred in the form of NH + 4 to the plnt (Govindrjulu et l. 2005; Tnk nd Yno 2005). Results of previous studies hve successfully highlighted the potentil for AM fungl medited N trnsfer to the host ut filed to provide cler evidence for considerle contriution to plnt N nutrition. The experimentl conditions used in former reports hve een lrgely sed on rtificil sustrtes nd hve used high quntities of inorgnic N (offered only to the fungus). Not only did the results of these experiments differ drmticlly etween the individul trils, they proly lso did not dequtely simulte nturl field site conditions. In nture, however, it seems likely tht AM fungl contriution to plnt N uptke could ecome importnt under circumstnces where plnt N demnd exceeds N vilility, for exmple under conditions of immoilised N sources or during drought (Tor et l. 1994; Surmnin nd Chrest 1999). Soil orgnic mtter is possile nutrient source for AM fungi, nd only little informtion is ville on the quntities of N tken up nd trnsferred from decomposing roots or litter (e.g. Johnsen nd Jensen 1996; Hodge et l. 2001; Hodge nd Fitter 2010). 13

20 1 GENERAL INTRODUCTION Therefore, more studies in soil re needed in order to understnd the contriution of AM fungl N supply under field conditions, especilly when N is tken from plnt residues The plnt nutritionl sttus nd the outcome of the AM symiosis Plnt species differ in their requirements for AM symiosis, minly due to root morphologicl or physiologicl fetures nd their demnd for P. Plnt species with corse, poorly rnched root systems nd smll surfce res (Hetrick 1991), nd/or low ility to excrete P- moilising root exudtes, enefit the most from n AM symiosis (Mrschner 1995). More thn tht, progress nd eventul outcomes of the plnt /AM fungl ssocition depend gretly on the plnt nutritionl sttus, in prticulr the plnt P sttus. A high plnt vilility of soil P reduces AM fungl root colonistion (Son nd Smith 1988; Amijee et l. 1993; Vierheilig 2004), ruscule development nd lso decreses the spred of the externl mycelium in soil (Smith nd Red 2008). In generl, reltively pronounced eneficil effects of the AM symiosis re oserved when plnt ville soil P is low (Mrschner nd Dell 1994), or when plnts hve high P demnd, ut root P uptke cpcity is restricted y some mens. Therefore, seedlings re highly responsive to AM fungl colonistion (Fisher nd Jychndrn 2002; Guissou 2009). When highly receptive to AM symiosis nd grown in low P soils, mycorrhizl plnts my tke up three to four times more P thn non-mycorrhizl plnts (Smith nd Red 2008). In contrst, under conditions where plnt ville P enles optiml P uptke y the roots, the extent of fungl colonistion declines. The mechnisms ehind re still not fully understood (Smith nd Red 2008). It hs een suggested tht this suppression of mycorrhiz development my result from reduced crohydrte lloction from roots to the fungus y the plnt in response to high P sttus (Grhm et l. 1997; Olsson 2002). For their development, AM fungi rely on the C contined in sugrs synthesized nd delivered y their host. Thus, ny fctors (such s irrdition, ville nutrients or drought) tht restrict photosynthte production or C distriution in the plnt my lso ffect AM fungl colonistion. 1.6 AM fungl inoculum production nd the request for dequte inoculum formultions The volume of AM fungl inoculum trded worldwide incresed considerly within the pst twenty yers (more thn 5-fold gin etween 1999 nd 2003; Grotkss et l. 2005), nd regions with the most predominnt demnd hve een Germny nd North-Americ (Feldmnn 2008). A relistion of the enefits of AM fungi for supplying nutrients under unsuitle iotic soil conditions nd their ility to ct synergisticlly with other soil-orne micro- 14

21 1 GENERAL INTRODUCTION orgnisms (see Section 1.5) hve contriuted to the success of AM inoculum products. They re eing considered more nd more in griculture, horticulture s well s for re-cultivtion ctivities. The most promising pplictions of AM fungl inoculum for plnt production re: i) To sustin or estlish functionl AM fungl popultions in low-input (gro-) ecosystems (Sieverding 1991; Douds et l. 2005; Plenchette et l. 2005). ii) To improve plnt estlishment for re-cultivtion processes of degrded or polluted sites which hve een distured y nthropogenic mens (Menge 1983; Cuenc et l. 1998; Joner nd Leyvl 2003). iii) To improve the development of cuttings (Douds et l. 1995; Druege et l. 2006) nd micropropgted plnts fter trnsplnting into non-sterile sustrtes, inoculted with AM fungi (Brnznti et l. 1992; Vesterg et l. 2004; Crretero et l. 2009). The oligte iotrophic nture of AM fungi mens tht fungl propgtion must tke plce in the presence of host plnt. This fct complictes nd hinders cost-efficient mss propgtion of AM fungl inoculum, nd s consequence, commercil production is still in its infncy. However, in response to the growing demnd for AM fungl inoculum in the lst decdes, producers nd scientists re working specificlly towrds the development of lrge-scle production (Ijdo et l. 2011). At present, inoculum is produced for commercil purposes using severl simple, nd some more complicted techniques. The most importnt of these include (in scending order of technicl stndrd nd cost expenses): i) Production on inoculted plnts within open field or nursery eds using soil (Sieverding 1991). ii) Production in continers or rised eds, where plnts re inoculted nd grown under greenhouse conditions within different sustrtes. As strting inoculum individul AM fungl strins cn e used, e.g. otined from sterile cultures provided y gene nks (Ijdo et l. 2011; Feldmnn nd Schneider 2008). iii) Production on pre-inoculted plnts in hydroponic or eroponic systems (Hung nd Sylvi 1988; Hwkins nd George 1997; Mohmmd et l. 2000). iv) Axenic production of pure AM fungl strins in vitro on trnsformed roots or utotrophic plnts (Becrd nd Fortin 1988; Declerck et l. 1996; Voets et l. 2009). This technique produces crrier-free inoculum, suitle for mny pplictions. Disdvntges my include the reltively complicted nd cost-intensive technologicl setup. Furthermore, not ll AM fungl species cn e propgted successfully on sterile medi (Gininzzi nd Vostk 2004). 15

22 1 GENERAL INTRODUCTION In generl, the procedure for formulting AM fungl inoculum involves plcing fungl propgules (colonised root frgments, spores nd hyphe frgments) into given crrier mteril (e.g. snd, clcined cly, vermiculite, pet, etc.). Inoculum from sustrte-sed production therefore contins not only AM fungi ut lso ssocited microorgnisms, nd the producer hs to ensure tht those re not hrmful to plnts (Feldmnn 2008). The finl configurtion of the formultion is determined y the trget inoculum ppliction method (mixing or surfce incorportion y hnd or mchine, inocultion of re roots, continer sustrte, seeds, culture sustrtes, field soils, etc.), nd it is possile to dpt the crrier mteril to the demnd of the user (Feldmnn 1998). For certin ppliction methods, solid crrier mteril my function s protective unit, for exmple for the mendment on roof tops expnded cly cn prevent spore dmge during high-pressure ppliction processes (Feldmnn 2008). However, in mny cses solid crrier mteril is undesirle, since the dditionl weight nd volume increses the effort required for trnsport nd ppliction, nd ultimtely leds to higher costs for the user. Developing chep nd crrier-free inocul, esy to pply, would certinly increse the cceptnce of AM inoculum mong potentil costumers nd my llow the expnsion into new fields of ppliction. Accordingly, n incresing effort into reserch hs een mde over the pst few yers (Gininzzi nd Vostk 2004; Ijdo et l. 2011). Still more studies re requested y inoculum producers (Feldmnn 2008; C. Schneider, 2011, personl communiction) therefore the present work should contriute to tht. 1.7 Agriculturl prctices tht ffect AM fungl symiosis Since the middle of the lst century the use of fossil fuels for input production hs llowed griculture to ecome intensified in temperte regions, nd more recently, lso in tropicl res (Crswell nd Krjlinen 1990). As result of this, wide rnge of different cropping systems hve een estlished, of which the most intensive forms hve chieved gret increses in yields. The pronounced rise in the use of grochemicls for crop or energy plnt production inevitly increses not only field opertions nd input costs, ut lso the costs for the environment. Associted with inpproprite griculturl mngement methods, consequences my include surfce wter pollution from leching of fertilisers nd pesticides (Flury 1996; Olrewju et l. 2009), loss of soil C stocks due to insufficient orgnic fertilistion (Guo nd Gifford 2002) nd erosion due to fllow periods nd due to soil compction y pssing over with mchinery (reviewed y Hmz nd Anderson 2005). 16

23 1 GENERAL INTRODUCTION AM fungi hve coexisted nd coevolved with plnts for millions of yers (Remy et l. 1994). These fungi re hence commonly found in nturl nd griculturl soils nd worldwide where they re symioticlly ssocited with oth wild- nd cultivted plnt species (Sieverding 1991). It is thought, however, tht intensive forms of cropping cn e detrimentl to soil-orne microil symionts, such s AM fungi. By ltering the iotic nd iotic soil conditions, inproprite griculturl prctices cn impct the development of AM fungi in the following wys: i) High levels of P fertilistion cn reduce AM fungl colonistion of host plnts (Hymn et l. 1975; Brunerger et l. 1991; Vierheilig 2004), nd lso decrese the susequent plnt growth response to mycorrhizl colonistion (Schuert nd Hymn 1986; Smith nd Red 2008). In intensive conventionl plnt production systems where P is pplied regulrly, the contriution to plnt nutrition y AM fungi is negligile. Sufficient P supply y fertilistion inevitly leds to decresed dependency on the symiotic fungl prtner which my ccount for reduced AM fungl undnce in such sites. When nturl ecosystems re trnsformed into griculturl fields, over time this effect my led to reduced genetic vriility in AM fungl species popultions (Schenck et l. 1989; Oehl et l. 2003). When compred with permnent grsslnd high-input field site might select for fst developing AM fungl species, so clled generlists (Oehl et l. 2003). ii) The infective potentil of AM fungl propgules in soil my e ltered y severl griculturl prctices. Crop rottions tht include considerle proportion of non-mycorrhizl plnt species (e.g. sugr eet, rpeseed) nd/or fllow periods cn reduce drmticlly AM fungl infectivity for the following growth seson (Hrinikumr nd Bgyrj 1988; Douds et l. 1997; Kir et l. 1999). The sence of mycorrhizl plnts during the vegettive period of the fungi my cuse the most extensive hrm to the survivl of AM fungl species (Kir et l. 1999; Plenchette et l. 2005). iii) Soil disturnce (ploughing) hs een shown to decrese AM fungl development nd contriution to plnt P uptke (Evns nd Miller 1988; Firchild nd Miller 1988), nd cused reduction in AM fungl species richness (Brito et l. 2012). However, AM fungl species oviously differ in their susceptiility to disturnce (Hrt nd Reder 2004; Brito et l. 2012). Within the soil depth profile of nturl grsslnd, AM fungl spores re minly present in the top 20 cm of soil, nd only smll portion of AM fungl spores re lso locted in deeper lyers of cm depth (Aott nd Roson 1991; Oehl et l. 2005). Mouldord ploughing my led to spore reloction to deeper soil lyers, or to reduced density of propgules y diluting top soil (hrouring higher spore densities) with deeper soil lyers. Accordingly, it hs 17

24 1 GENERAL INTRODUCTION een shown tht AM fungl colonistion nd erly P uptke were higher in mize plnts grown in no-till or ridge-tillge mngement compred to mouldord ploughed plots (McGonigle nd Miller 1993). Moreover, s consequence of mechnicl soil disruption the infection potentil of n AM fungl network might e reduced. Olsen et l suggested tht the estlishment nd colonistion y frgments of disrupted extr-rdicl mycelium might need more C expenditure from the susequent plnt, compred with n intct mycelium. Especilly moderte forms of mechnicl soil tretment (such s pplied in reduced tillge systems) re not precisely studied with respect to their effects on the AM symiosis nd the present study gives more informtion out tht. In order to tke dvntge of the AM symiosis in griculture, conditions must e met tht support AM fungl development. Most importntly this includes the use of sustinle mngement systems with reduced tillge, voidnce of non-mycotrophic plnts in the rottion nd the prevention of P ccumultion in soils y fertilistion. In cses where the former AM fungl popultions could not e mintined due to mngement prctices, trgeted ppliction of selected AM fungl inoculum might e recommendle. Inocultion in the field with efficient AM fungl isoltes cn e n effective mens of re-estlishing AM symioses nd improving plnt yield nd qulity fter trnsition from conventionl to orgnic frming. It might lso e n opportunity for frmers in regions were minerl P fertilisers re too costly. Any AM fungl strins directly selected from the frm itself re likely etter dpted to the present soil conditions. To ttin lrge quntities of the desired strins, inoculum could esily e propgted on-frm with low operting costs (Sieverding 1991). 1.8 Ojectives of the study This study ims t improving our understnding on some morphologicl nd physiologicl spects of the AM symiosis. It focuses on investigting the N uptke from decomposing plnt roots nd delivery to the host plnt, especilly considering growth nd development of the AM fungl extr-rdicl mycelium (ERM). It lso ddresses the question of inoculum potentil of the ERM with respect to its developmentl stge nd sptil distriution in soil. Furthermore, the ERM s n infective unit for host plnt colonistion ws studied with respect to its susceptiility to mechnicl disruption typiclly for mny griculturl soil mngement prctices. The study lso gives more informtion out AM fungl spore production, s spores re the most importnt propgules in soil. This study lso nlysed quntities nd ptterns of 18

25 1 GENERAL INTRODUCTION fungl spore production occurring within ded plnt roots which re uiquitous in vegetted soils. In view of these ojectives the following hypotheses were formulted: 1. Nitrogen is sored y the extr-rdicl mycelium of the AM fungi from dying donor plnt root nd delivered to living receiver plnt. Therey fungl trnsfer of N to the receiver plnt will e higher from AM colonised donor roots compred to uncolonised roots. 2. AM fungl N trnsfer to colonised host plnt will e reduced when soil, contining estlished mycorrhizl networks, is mechniclly distured. 3. When fungl colonistion of plnts is estlished exclusively y the ERM, AM fungl isoltes with higher extent of ERM prolifertion in the soil volume prior to mycelium excision will hve higher inoculum potentil nd growth promoting effect on the susequent plnt. 4. The mechnicl frgmenttion of detched ERM, induced y soil disturnce, reduces AM fungl inoculum potentil nd consequently reduces fungl contriution to P uptke nd growth of the next plnt. 5. Spore development within ded plnt roots will not depend on whether the root originted from host or non-host plnt species, ut rther will increse with root dimeter. The outcomes of the present study im to contriute to our knowledge on the ecology of AM fungi nd their potentil to improve plnt nutrition. Findings my lso ssist the development of suitle mngement prctices to improve the use of AM fungi in griculturl systems for more sustinle plnt production. 19

26 Chpter 2 2 Generl mterils nd methods Mterils nd methods routinely used in the experiments re descried in this chpter. Applictions nd modifictions relted to specific experiments re descried in the relevnt sections. 2.1 Description nd preprtion of experimentl plnt growth sustrte Susoil otined from the C-horizon of Luvisol from Weihenstephn, Southern Germny (48 25 N, E) ws used s growth (soil-) sustrte. The sustrte ws clssified s lomy snd (45.2% snd, 42.0% silt, 13% cly) nd it contined (mg kg -1 ): 5.2 nd 3.4 CCl 2 ( M)-extrctle NH 4 + nd NO 3 -, respectively. The orgnic mtter content ws 0.3% in DS, with sustrte ph (CCl 2 ) of 7.7 nd CCO 3 -equivlent of 23%. After het sterilistion, the sustrte contined (mg kg -1 DS) 6.5 cette lctte-extrctle P (CAL, Schüller, 1969); 65.7 CAL-extrctle K; nd 1.9 (Fe), 15.0 (Mn), 0.3 (Zn), 0.9 (Cu), 0.09 (B) nd 0.04 (Mo) CATextrctle micronutrients (Alt nd Peters 1993). Sustrte chrcteristics nd plnt ville nutrients were nlysed y LUFA Rostock ccording to VDLUFA, Prior to experimentl use the sustrte ws sieved through 5 mm sieve to homogenise nd to exclude lrger stones nd other prticles. It ws then het sterilised in drying oven t 85 C for 48 h to eliminte ll fungl propgules. Before use the sustrte ws fertilised with 200 mg K (K 2 SO 4 ), 200 mg N (NH 4 NO 3 ), 100 mg Mg (MgSO 4 ), 50 mg P (KH 2 PO 4 ), 10 mg Fe (Fe-EDTA), 10 mg Cu (CuSO 4 ), 10 mg Zn (ZnSO 4 ) kg -1 dry sustrte. All nutrients were dissolved in deionised wter nd then mixed homogeneously into the dry sustrte. The plnting pots were filled with the fertilised sustrte t ulk density of 1.3 g cm

27 2 GENERAL MATERIALS AND METHODS 2.2 Preprtion of fungl comprtments Fungl comprtments for the insertion into the growth sustrte were constructed from 60 ml plstic tues (height 6 cm, Ø 3 cm) with ltticed wll. The wlls of the tues nd the two open ends were covered with 30 µm mesh memrne (Sefr Nitex, Sefr AG, Switzerlnd) tht llowed hyphe, ut not roots, to grow into the comprtments. The memrne ws fixed to the wlls of the tues using fungicide-free silicone selnt (Prou, Buhus AG, Germny). 2.3 Preprtion of fungl comprtment sustrte The sustrte preprtion nd the extrction of the extr-rdicl mycelium (ERM) were done y modified method of Neumnn nd George (2005). The sustrte consisted of 1:1 mixture of wet sieved susoil (prticle size < 40 µm) nd glss eds (Ø mm; Crl Roth GmH Krlsruhe, Germny), nd with 20% w/w wter. This mixture llows for the extrction of lmost intct fungl ERM fter hrvest. The sustrte used for the sieving ws similr with tht used for the plnting pot sustrte. To prepre the comprtment sustrte the susoil ws thoroughly mixed with wter in ucket y stirring. The soil suspension ws llowed to stnd for few seconds to llow lrger prticles to settle to the ottom. It ws then poured over 40 µm sieve. The superntnt ws decnted repetedly nd the remining sludge ws dried t 65 C in drying oven for 48 h. The temperture ws then incresed to 85 C for 48 h to eliminte fungl propgules. This mteril ws then mixed with glss eds nd deionised wter contining dissolved nutrients. The rte of fertilistion ws similr to tht of the plnting pot sustrte. 2.4 Extrction of the extr-rdicl mycelium from fungl comprtments nd estimtion of hyphe length nd spore numer To extrct the ERM, the content of the fungl comprtments ws mixed with deionised wter in owl. After descent of the glss eds to the ottom the wter including the fungl ERM nd sustrte prticles were poured through 40 µm sieve. Tp wter ws used to wsh remining sustrte prticles through the sieve, leving only the ERM. The ERM ws susequently freeze-dried t -30 C for four dys. After the dry weight (DW) of the ERM hd een determined, susmples of pproximtely 0.5 mg were trnsferred to 2.5 ml Eppendorf tues nd stined overnight t room temperture with 0.05% trypn lue in lctic cid. Stined smples were trnsferred to lortory lender (Wring Blender 7009G, Wring, USA) with 21

28 2 GENERAL MATERIALS AND METHODS 300 ml tp wter, nd lended t low speed for 40 s. Aliquots of 90 ml of the suspension were filtered onto gridded (3 x 3 mm) 0.5 µm nitrocellulose memrne (Micronsep; GE Wter & Process Technologies, USA) following the modified memrne filter method of (Hnssen et l. 1974). The memrne filter ws mounted onto microscopic glss slide. Hyphe length ws estimted y modified gridline intersection method (Newmn 1966) under the microscope t 200 x mgnifiction. The numer of spores ws ssessed y counting AM spores visile on defined re with 50 x mgnifiction. 2.5 AM fungl isoltes The following tle presents list of ll AM fungl isoltes used in this study: Tle 2.1: Identity nd sources of the AM fungl isoltes used in this study. AM fungl isolte Glomus mossee (Gm IFP S/08) Glomus intrrdices (Glintr IFP S/08) Source Commercilly ville single-strin inoculum; Crrier mteril: qurtz snd (INOQ GmH Schneg, Germny) Glomus mossee BEG 12 Glomus intrrdices BEG 110 Field soil with indigenous AM fungi Self propgted on mize in C-Loess (sustrte tretment nd fertilistion similr s in the experiments) Soil smple from the top 10 cm of lomy snd soil, collected from field site ner Bnd Aceh, Indonesi, The phylum Glomeromycot hs recently een re-nmed, ccordingly Glomus mossee Gerd. & Trppe (1974) is now Funneliformis mossee nd Glomus intrrdices N.C. Schenck & G.S. Sm. (1982) is now Rhizophgus intrrdices ( Thus, similr to other recent pulictions lso in the present work the former nmes were kept on using to fcilitte comprison with other works studying the sme fungi. 2.6 Estlishment of non-inoculted control plnts To compre mycorrhizl [+AM] with non-inoculted [-AM] tretments, it is necessry to ensure similr distriution of nutrients nd microorgnisms other thn AM fungi. Therefore, [-AM] tretments received the sme mount of sterilised (heted t 85 C for 48 h) AM fungl inoculum s in [+AM] tretments, plus filtrte from living inoculum. The filtrte ws otined y mixing fresh inoculum with deionised wter (100 ml wter per 50 g dry inoculum) nd then filtering through Blue Rion filter pper (Schleicher nd Schüll, Germny). 2.7 Estimtion of the AM fungl colonised root length Plnt roots were wshed free from sustrte nd representtive smple of the fresh roots (pproximtely one g) tken nd stined with 0.05% trypn lue in lctic cid ccording to 22

29 2 GENERAL MATERIALS AND METHODS Koske nd Gemm (1989). The extent of AM fungl root colonistion ws determined ccording to modified gridline intersection method using stereo microscope with trnsmitted illumintion nd 50 x mgnifiction (Kormnik nd McGrw 1982). Between 250 nd 300 intersections were counted per smple. 2.8 Nutrient nlysis in plnt tissue Phosphorus The plnt mteril ws dried for 48 h in drying oven t 65 C nd the DW ws estimted. Susmples (200 mg) of ground plnt mteril (prticle size 0.25 mm) were dry-shed t 550 C for 4 h, oxidised with 5 ml 21% HNO 3, nd tken up into 25 ml 2% HCl. After stining with mmonium-molydte-vndte solution, the P concentrtion in the smples ws estimted colorimetriclly with spectrophotometer (EPOS nlyser, Eppendorf, Germny) t wvelength of 436 nm (Gericke nd Kurmies 1952). Totl nitrogen nd tom% 15 N excess For quntifiction of nitrogen nd 15 N concentrtions in plnt mteril, 10 mg of dried, ground shoot nd root smples were nlysed in n elementl nlyser (Elementr Vrio EL, Elementr, Germny) following the DUMAS method. After totl N mesurement, the N frction of the comustion gs ws utomticlly trnsferred to coupled emission spectrometer (NOI 7; Fischer Anlysen Instrumente, Leipzig, Germny) where the tom% 15 N excess ws determined, mening the percentge 15 N toms of ll N toms ove the nturl undnce. 2.9 Experimentl loction The experiments were conducted in controlled climte glsshouse locted t the Institute of Vegetle nd Ornmentl Crops (IGZ) in Grosseeren, Germny (52 22 N, E). Plnts were grown in single-glzed Venlo glsshouse cin (effective re 60 m 2 ; Width 6.4 m; Ridge height 4 m; Ridge ertion doule sided; Light trnsmission fctor 0.7). 23

30 Chpter 3 3 The symiotic recpture of nitrogen from ded mycorrhizl nd non-mycorrhizl roots of tomto plnts Astrct The im ws to quntify the nitrogen (N) trnsferred vi the extr-rdicl mycelium of the rusculr mycorrhizl fungus Glomus intrrdices from oth ded host nd ded non-host donor root to receiver tomto plnt. The effect of physicl disruption of the soil contining donor plnt roots nd fungl mycelium on the effectiveness of N trnsfer ws lso exmined. The root systems of the donor (wild-type tomto plnts or the mycorrhiz-defective rmc mutnt tomto) nd the receiver plnts were seprted y 30 µm mesh, penetrle y hyphe ut not y the roots. Both donor genotypes produced similr quntity of iomss nd hd similr nutrient sttus. Two weeks fter the supply of 15 N to split-root prt of donor plnts, the shoots were removed to kill the plnts. The quntity of N trnsferred from the ded roots into the receiver plnts ws mesured fter further two weeks. Up to 10.6% of donor-root 15 N ws recovered in the receiver plnts when inoculted with the rusculr mycorrhizl (AM) fungus. The quntity of 15 N derived from the mycorrhizl wildtype roots clerly exceeded tht from the only wekly surfce-colonised rmc roots. Hyphl length in the donor rmc root comprtments ws only out hlf tht in the wild-type comprtments. The disruption of the soil led to significntly incresed fungl-medited trnsfer of N to the receiver plnts. The trnsfer of N from ded roots cn e enhnced y AM fungi especilly when the donor roots hve een formerly colonised y AM fungi. The trnsfer cn e further incresed with higher hyphe length densities, nd the present dt lso suggest tht direct link etween receiver mycelium nd internl fungl structures in ded roots my in ddition fcilitte N trnsfer. A mechnicl disruption of soil contining ded roots my increse the susequent vilility of nutrients, thus promoting mycorrhizl N uptke. When ssocited with living plnt, the externl mycelium of G. intrrdices is redily le to re-estlish itself in the soil following disruption nd functions s trnsfer vessel. 1 Pulished in modified version s: Müller A, George E, Griel-Neumnn E (2012) The symiotic recpture of nitrogen from ded mycorrhizl nd non-mycorrhizl roots of tomto plnts. Plnt nd Soil; DOI /s

31 CHAPTER Introduction In terrestril ecosystems, root turnover is key component of elow-ground nutrient cycling, nd so provides n importnt source of nutrients for plnt growth. The quntity of nutrient relesed from ded roots cn e sustntil, lthough it differs from plnt species to plnt species. Aerts et l. (1992) estimted the volume of orgnic nitrogen (N) turnover in soil ssocited with root decy to e 1.7 g N m - ² yr -1 in Deschmpsi nd 19.7 g N m -2 yr -1 in Molini grsslnds. Detched Holcus grss roots lose up to 87% of their initil N within 42 dys nd pproximtely 40% of it is tken up y other plnts (vn der Krift et l. 2001). The ctivity of rusculr mycorrhizl (AM) fungi enhnces the ility of plnts to recycle nutrients from decying roots (Grime et l. 1987). AM fungl networks my lso e ssocited with different mycorrhizl plnt species nd so provide ccess to N derived from the roots of distnt plnts. Interconnected mycorrhizl plnts my e more competitive thn non-mycorrhizl species or those which re less responsive to mycorrhiz (Hrtnett et l. 1993). The use of isotope-lelled phosphorus hs shown tht AM fungl myceli cn trnsfer nutrients over distnce of s much s 50 cm (Wlter et l. 1996). The ppliction of 15 N-enrichment technology in AM fungl comprtments (ccessile to AM fungi ut not to roots) hs enled the quntifiction of soil-to-plnt N trnsfer vi the AM fungl extr-rdicl mycelium (ERM) from inorgnic s well s orgnic N sources (Ames et l. 1983; Frey nd Schüepp 1993; Johnsen et l. 1992; Johnsen et l. 1994; Hwkins et l. 2000; Mäder et l. 2000; Hwkins nd George 2001; Hodge et l. 2001; Cheng et l. 2008), For exmple, out 30% of receiver plnt N content derived from AM fungl N trnsfer (Ames et l. 1983; Frey nd Schüepp 1993; Mäder et l. 2000) suggesting tht AM fungi my hve lrge potentil to improve N nutrition of host plnts. Only few studies hve investigted N trnsfer etween live mycorrhizl plnts where roots hve een seprted y n AM fungl ccessile rrier (Hysted et l. 1988; Bethlenflvy et l. 1991; Hmel et l. 1991; Ikrm et l. 1994; Johnsen nd Jensen 1996; Jlonen et l. 2009; Li et l. 2009). A possile undesirle side-effect of AM fungl colonistion is incresed root iomss (which produces lrger nutrient pool) occurring especilly in legume species (Hysted et l. 1988; Li et l. 2009) nd the resulting sustntil level of N trnsfer ecomes difficult to interpret. The extent of AM fungl medited N trnsfer is only minor from the live root, while killing the root y removl of the shoot clerly rises the level (Johnsen nd Jensen 1996). The impliction is tht decying roots re much more effective source of trnsferrle 25

32 CHAPTER 3 N thn re the root exudtes from living plnts. However, the reltive contriutions of live roots, ded roots nd rhizodeposition remin s yet to e clrified. The direct uptke of N from the inner cortex of live roots y hyphe is unlikely, s it would contrdict the ccepted ide out two-sided mycelium functioning, i.e. the site of N uptke nd nolic ssimiltion into the fungl tissue is thought to e the ERM, while N is ctolised within the intr-rdicl mycelium (IRM) nd efore eing relesed to the host plnt vi the ruscules (Govindrjulu et l. 2005; Tin et l. 2010). Wht occurs susequent to the dieck of colonised donor plnt roots is uncler. It ppers possile, however, tht the AM symiosis cn fcilitte the efficient (re-) sorption of root N, so tht this root N is trnsferred directly to the receiver host plnt, rther thn to the rhizosphere soil, soil-orne microorgnisms or non-host plnts. The initil ojective of the present study ws to quntify the extent of mycorrhizl N trnsfer from the ded roots of donor plnt to receiver plnt. The working hypothesis ws tht greter quntity of N is trnsferred from ded mycorrhizl roots thn from ded nonmycorrhizl ones. To test this, comprison ws mde etween wild-type [WT] tomto (Solnum lycopersicum L. cv. RioGrnde 76R) nd mycorrhiz-defective [rmc] mutnt tomto. The ltter cnnot support intr-rdicl colonistion y Glomus intrrdices (Brker et l. 1998) ut its ove nd elow ground iomss production is similr to tht of the WT (Bgo et l. 2006; Cvgnro et l. 2006). The second im ws to sses the ility of the ERM to sor nd susequently trnsfer N following physicl dmge to the AM fungl network cused y tillge which hs een repetedly een shown to reduce the infectivity of mycelium (McGonigle et l. 1990; Jsper et l. 1991). Furthermore, the re-estlishment of the network nd fungl medited N trnsport cn e clerly reduced following the severe disruption of the ERM (Frey nd Schüepp 1993). Nevertheless, vrious AM fungl isoltes cn differ considerly from one nother in terms of their sensitivity to mechnicl disruption (Dun et l. 2011). 3.3 Mterils nd methods Pre-cultivtion of plnt mteril Seeds of the mycorrhiz-defective [rmc] mutnt tomto (Brker et l. 1998) nd the wild-type [WT] progenitor Solnum lycopersicum (L.) cv. RioGrnde 76R were germinted in the drk etween two lyers of pper soked with sturted CSO 4 solution. To otin seedlings with root system suitle to split etween two pots, plnts were pre-cultivted in nutrient solution. 26

33 CHAPTER 3 Therefore, t height of 5-6 cm, germinted seedlings were trnsferred to n erted nutrient solution (ph 6.8) composed of the following: 5 mm N (hlf C(NO 3 ) 2, hlf NH 4 NO 3 ); 0.7 mm P (KH 2 PO 4 ); 4 mm K (KH 2 PO 4 nd K 2 SO 4 ); 2.5 mm C (C(NO 3 ) 2 nd CSO 4 ); 1 mm Mg (MgCl 2 ); 4 mm S (CSO 4 nd K 2 SO 4 ); 10 µm Fe (Fe-EDTA); 10 µm B (H 3 BO 4 ), 5 µm Mn (MnSO 4 ); 1 µm Zn (ZnSO 4 ); 0.7 µm Cu (CuSO 4 ); 0.5 µm Mo ((NH 4 ) 6 Mo 7 O 24 ). Fourteen dys fter trnsfer to nutrient solution, the min root of ech tomto plnt ws cut off one cm ove the tip to rek picl dominnce. The plnts were grown nother two weeks efore trnsplnttion to the experimentl plnting units Preprtion of growth sustrte nd plnting units Triprtite plnting units were constructed consisting of three squre plstic pots (Teku-Tiner, Pöppelmnn, Germny), plced in row nd fstened together with dhesive tpe. One of the outer pots (comprtments) with volume of 0.5 L, served s the 15 N lelling comprtment (LC). The other two comprtments, with volume of 1.2 L, served s donor (DC) nd receiver (RC) root comprtment, respectively (see Fig 3.1). To llow for the growth of AM fungl myceli ut not of roots etween the two lrger comprtments, fungl window (height = 7 cm; width = 6 cm) comprising of 30 µm mesh memrne (Sefr Nitex; Sefr AG, Switzerlnd) ws cut into the two djoining wlls. The window ws covered y 30 µm mesh memrne (Sefr Nitex; Sefr AG, Switzerlnd), tht llowed fungl hyphe, ut not plnt roots to grow through. Ech 1.2 L nd 0.5 L comprtment ws filled with 1.4 kg nd 0.6 kg dry sustrte, respectively. The preprtion nd fertilistion of the sustrte is descried in Chpter

34 CHAPTER 3 Donor [WT] or [rmc] Receiver FC LC DC RC Fungl ERM Fungl window c d DC RC DC RC Tretment [U] Tretment [X] Fig. 3.1:. Photogrph of the tomto plnts used in this experiment, four weeks fter plnting.. The roots of donor plnt (either wild-type (WT) or mycorrhiz-defective (rmc) mutnt) were split etween the donor root comprtment (DC) nd the 15 N-lelling comprtment (LC). The receiver root comprtment (RC) contined WT tomto plnt in ech cse. The RC nd DC root comprtments were seprted from nother y 30 µm mesh memrne penetrle y AM fungl hyphe ut not y roots. Both root comprtments contined one fungl comprtment (FC) ech. Susequent to two week lelling period, the LC nd the donor shoots were removed nd the sustrte in DC ws either (c.) left undistured (tretment [U]) or (d.) ws mechniclly disrupted (tretment [X]) Arusculr mycorrhizl inocultion nd instlltion of fungl comprtments Inoculum of the AM fungus Glomus intrrdices ws used (Glintr IFP S/08; provided y INOQ GmH; Schneg; Germny). It consisted of mixture of AM fungl colonised roots with dhering growth sustrte (qurtz snd) nd extr-rdicl mycelium with spores. To prepre [+AM] tretments, inoculum ws mixed with the experimentl growth sustrte t rte of 7% (w/w). [-AM] tretments were prepred s descried in Chpter

35 CHAPTER 3 Fungl comprtments (FC) were constructed from 60 ml plstic tues (see Chpter 2.2) nd filled with FC sustrte prepred s descried in Chpter 2.3. One fungl comprtment ws verticlly inserted into the DC nd RC of ech plnting unit. They were locted in opposite corners, ner the fungl window (see Fig 3.1-d) Plnt cultivtion, 15 N ppliction nd set-up of the donor plnt tretments At the ge of 28 dys, one wild-type tomto [WT] receiver plnt ws plnted into the centre of the receiver comprtment, RC. At tht time lso one donor plnt, either [WT] or [rmc], ws trnsferred into the lelling comprtment (LC) nd donor comprtment (DC) with its root system split (see Fig 3.1 ). The min root of ech split-root donor plnt ws directed into the DC nd four to five upper lterl roots with length of 5-8 cm were directed into the LC. In totl, 32 pots were estlished. Thirty dys fter plnting, the sustrte in the LC ws supplied once with dditionlly 240 mg N kg -1 DS s C(NO 3 ) 2 tht contined 10 tom% 15 N isotope (Chemotrde GmH, Leipzig, Germny). Fourteen dys fter 15 N ppliction, ll LCs together with the split-root prts contined therein, were completely removed from the donor plnts nd the plnting units. At tht time ll donor plnt shoots were hrvested one cm ove the soil surfce The growth sustrte in the DC of hrvested plnts ws either left undistured [U] or ws disrupted [X; Fig. 3.1 c, d; Tle 3.1]. To crete disruption, the sustrte inside the DC ws cut verticlly into columns of pproximtely one centimetre size nd verticlly mixed y hnd using sptul. Fungl comprtments were removed from the DC during this process nd were re-instlled fterwrds. The experimentl plnts were grown for 72 dys in glsshouse etween Septemer nd Novemer. The verge dy nd night tempertures in the glsshouse were 22 C nd 17 C, respectively, nd the reltive ir humidity verged 71%. For the lst 42 dys the plnts received dditionl light for 8 h t rte of 380 µmol m -2 s -1 t plnt height provided y 400 W lmps (SON-T Agro; Philips, Germny). Dily wter loss from the plnting units ws estimted grvimetriclly nd replced with deionised wter. The irrigtion wter ws distriuted mong the three comprtments of ech plnting unit, in order to mintin verge wter content in the sustrte of ech comprtment t pproximtely 18% (w/w). 29

36 CHAPTER 3 Tle 3.1: Overview of the experimentl tretments. The donor sustrte tretment ws set up fter the 15 N-lelling period. Ech tretment ws replicted four times. Donor sustrte tretment [U] [X] Donor genotype Receiver genotype [WT] [WT] [+AM] [-AM] [rmc] [WT] [+AM] [-AM] [WT] [WT] [+AM] [-AM] [rmc] [WT] [+AM] [-AM] Mycorrhiz inocultion of donor nd receiver plnt [U] sustrte in donor comprtment undistured [X] sustrte in donor comprtments distured [WT] wild-type tomto plnt [rmc] mycorrhiz-defective tomto plnt [+ AM] inoculted with G. intrrdices [ AM] non-inoculted tretment Hrvest nd nlysis of plnt nd AM fungl mteril Receiver plnts nd the roots in the donor comprtments (DC) were hrvested nother 14 dys fter termintion of the 14-dys- 15 N lelling period nd the cutting off of the donor shoots (see Section 3.3.4). All roots were wshed from sustrte nd stined to estimte the extent of AM fungl root colonistion s descried in Chpter 2.7. As intr-rdicl AM fungl structures were sent from rmc roots, vlues for these plnts represent root surfce colonistion y ppressori nd ttched hyphe only. The ERM in the fungl comprtments ws extrcted nd freeze-dried nd the spore numer nd hyphe length were ssessed s descried in Chpter 2.4. The hrvested plnt mteril (shoot or root) ws dried for 48 h t 65 C efore DW ws estimted. Biomss nlyses for the donor split-root-prts LC nd DC were conducted seprtely Nutrient nlysis nd sttistics Dried plnt mteril (shoot or root) ws finely ground nd P concentrtion, N concentrtion nd tom% 15 N excess were nlysed s descried in Chpter 2.8. P nd N nlyses for the donor splitroot-prts LC nd DC were conducted seprtely. 30

37 CHAPTER 3 The results on 15 N concentrtion mesured with the method s descried in Chpter 2.8 were used to clculte totl 15 N uptke into the donor nd receiver plnt tissue. Assuming tht 14 N nd 15 N re oth tken up nd trnsferred in equl quntities, the reltive mount of N trnsferred from the donor to receiver plnt (%N trnsfer ) ws estimted from the rtio etween 15 N content in the receiver plnt nd the sum of 15 N contents in oth the receiver nd donor plnt. The %N trnsfer ws clculted using the donor plnt totl 15 N content comprising the lelled N contents in shoot nd oth split-root prts from LC nd DC. %N trnsfer = 15 N content Receiver x 100 / ( 15 N content Donor + 15 N content Receiver ) (1) where 15 N content plnt = tom% 15 N excess plnt x totl N content plnt / 100 (2) Since donor shoots nd the LC were removed 14 dys fter lelling nd 14 dys efore the hrvest of the receiver plnts, it my lso e meningful to estimte the N trnsfer percentge y tking into ccount only the N content in donor roots from the DC. Accordingly, the percentge N trnsferred to receiver plnts from donor roots (%Root N trnsfer ) ws clculted s (ccording to Johnsen nd Jensen (1996)): %Root N trnsfer = 15 N content Receiver x 100 / ( 15 N content Donor root DC + 15 N content Receiver ) (3) The mount of N (mg per plnt) trnsferred from the donor root (Root N trnsfer ) ws estimted with the following eqution: Root N trnsfer = %Root N trnsfer x N content Donor root DC / (100 - %Root N trnsfer ) (4) The % of totl N recovered in the receiver, derived from trnsfer (%N dft ), ws clculted s: % N dft = Root N trnsfer x 100 / N content Receiver (5) Four replictes per tretment were used. Provided tht results pssed the test for norml distriution (Kolmogorov-Smirnov test; p > 0.05) nd homogeneity of vrince (Levene test; p >0.05), dt were sujected to three-wy ANOVA. Dt for 15 N contents in receiver plnt tissue were normlised y squre root trnsformtion prior to sttisticl nlysis. In cses where the ANOVA indicted significnt effect of ny fctor, the multiple comprison Tukey-test ws used to estimte differences etween mens of ll tretments. P vlues elow 0.05 otined in oth tests were interpreted s indicting significnt effects. Sttistic clcultions were conducted using SPSS softwre, version 15.0 (SPSS Inc., USA). Results in tles nd figures re presented s tretment mens ± stndrd devition. 31

38 CHAPTER Results Dry weight nd nutrient sttus of the donor plnts Donor plnt dry weight nd phosphorus uptke Across ll tretments the dry weight of the respective donor plnt prts verged 10.5 ± 0.7 g (shoot), 1.5 ± 0.3 g (root in donor root comprtment DC) nd 0.8 ± 0.2 g (root in 15 N-lelling comprtment LC) per plnt. The plnt prts lone or the totl plnt dry weight were not ffected y genotype nd AM fungl inocultion. Donor shoot phosphorus (P) concentrtion ws not ffected y ny of the tretments nd verged 1.4 ± 0.2 mg g -1 DW. The lelling comprtment (LC) ws removed from the growth unit fter the lelling period, nd the vlues mesured for the nutritionl sttus of roots from the LC in ll cses reflected the results shown for the split-root prt from the DC. Therefore no further results for root prts from the LC re shown. AM fungl inocultion led to significntly higher root P concentrtions in WT donor roots compred to non-inoculted controls. In contrst, rmc mutnt plnts showed no significnt response to the presence of mycorrhiz (Tle 3.2). The totl plnt P content ws not ffected y AM fungl inocultion or genotype (Tles 3.3). As result of disruption of roots nd mycelium in [X] tretments, P concentrtion nd P content in donor roots were reduced y out one third compred to the undistured [U] tretment (Tle 3.2). Tle 3.2: P concentrtion nd P content in roots from the donor root comprtment (DC). Shown re the men vlues ± SD for wild-type [WT] or mycorrhiz-defective [rmc] mutnt tomto plnts inoculted [+AM] or noninoculted [-AM] with Glomus intrrdices. The donor shoots were cut off y the end of the lelling period nd the sustrte in the donor root comprtment ws either undistured [U], or ws mnully disrupted [X]. Mens followed y different letters re significntly different from ech other ccording to multiple comprison Tukey-test (p < 0.05). Root P concentrtion (mg g -1 DW) Root P content (mg per plnt) Donor sustrte tretment Donor genotype +AM -AM +AM -AM [U] [WT] 3.12 d ± c ± ± ± 1.35 [rmc] 2.45 c ± c ± ± ± 0.46 [X] [WT] 2.12 c ± ± ± ± 0.18 [rmc] 1.84 ± ± ± ±

39 CHAPTER 3 Tle 3.3: Three-wy-ANOVA results of donor root P concentrtion nd content nd totl plnt P content. A significnt effect of the donor genotype (G), AM fungl inocultion (M) or donor sustrte tretment (T) is indicted with lck dot (n.s. = not significnt). Interction G M T G x M G x T M x T G x M x T Root P concentrtion in DC n. s. n. s. n. s. Root P content in DC n.s. n. s. n. s. n. s. n. s. n. s. Plnt P content n. s. n. s. n. s. n. s. n. s. n. s Donor plnt totl nitrogen nd 15 N Across ll tretments the verge shoot N concentrtion of donor plnts verged 18.2 ± 1.8 mg g -1 DW nd ws not ffected y the genotype or AM fungl inocultion tretments. The root N concentrtion in inoculted tretments ws y trend higher compred to non-inoculted plnts, significnt in the cse of the [rmc / X] tretment. Root N content (Tle 3.4) nd totl plnt N content (Tle 3.6) were not significntly different due to ny of the tretments. Sustrte disturnce did not significntly ffect root N concentrtion or content. Tle 3.4: Totl N concentrtion nd N content in roots from the donor root comprtment (DC). For revitions nd sttistics see Tle 3.2. Donor root N concentrtion (mg g -1 DW) Donor root N content (mg per plnt) Donor sustrte tretment Donor genotype +AM -AM +AM -AM [U] [WT] 15.9 ± ± ± ± 6.3 [rmc] 14.5 ± ± ± ± 2.8 [X] [WT] 14.6 ± ± ± ± 2.9 [rmc] 15.4 ± ± ± ± 3.0 At hrvest, the verge tom% 15 N excess in shoots ws 5.3 ± 0.2% nd it ws not ffected y the genotype or AM fungl inocultion. Atom% 15 N excess, s well s 15 N content in roots from the DC were similr irrespective of ny of the experimentl fctors (Tle 3.5), lthough the ANOVA detected significnt effect on the 15 N sttus due to the AM fungl inocultion ut not due to the donor genotype or sustrte tretment (Tle 3.6). Independent of the tretments, 33

40 CHAPTER 3 the verge quntity of 15 N recovered in the whole donor plnt ws 65 ± 11% of the mount pplied to the lelling comprtment of donor plnts (out 16 mg 15 N ws pplied per plnt; dt not shown). Tle 3.5: Atom% 15 N excess nd totl 15 N content in roots from the donor root comprtment (DC) t hrvest. For revitions nd sttistics see Tle 3.2. Atom% 15 N excess in donor root 15 N content in donor root (mg per plnt) Donor sustrte tretment Donor genotype +AM -AM +AM -AM [U] [WT] 3.3 ± ± ± ± 0.2 [rmc] 3.8 ± ± ± ± 0.2 [X] [WT] 3.4 ± ± ± ± 0.1 [rmc] 3.6 ± ± ± ± 0.1 Tle 3.6: Three-wy ANOVA results for uptke of totl nitrogen nd of 15 N into donor plnt tissue. For revitions nd sttistics see Tle 3.3. Interction G M T G x M G x T M x T G x M x T Root N concentrtion in DC n. s. n. s. n. s. n. s. n.s. n. s. Root N content in DC n. s. n. s. n. s. n. s. n. s. n. s. n. s. Plnt totl N content n. s. n. s. n. s. n. s. n. s. n. s. n. s. Root tom% 15 N excess in DC n. s. n. s. n. s. n. s. n. s. n. s. Root 15 N content in DC n. s. n. s. n. s. n. s. n. s. n. s. Plnt totl 15 N content n. s. n. s. n. s. n. s. n. s. n. s. n. s Intr- nd extr-rdicl AM fungl development The AM fungl colonised root length of ll AM fungl inoculted WT donor roots ws 50-60% (Tle 3.7), including ppressori on the root surfce with ttched extr-rdicl hyphe, spores, s well s intr-rdicl fungl structures. Donor roots of rmc mutnt plnts showed colonistion rte etween 12% nd 16% (Tle 3.7). These plnts showed surfce colonistion consisting only of ppressori nd ttched extr-rdicl hyphe nd spores. No intr-rdicl fungl structures were found inside of decomposing rmc mutnt roots, with the exception of 34

41 CHAPTER 3 few instnces where intr-rdicl AM fungl spores were present. These spore clusters colonised root length of not more thn 1.2 ± 0.9%. Receiver root colonistion rtes (WT only) rnged etween 60% nd 70% nd were unffected y the sustrte tretments in the DC (Tles 3.7 nd 3.8). No AM fungl colonistion ws oserved in non-inoculted tretments. Tle 3.7: Percentge of AM fungl colonised root length of roots from either the donor (DC) or the receiver (RC) comprtment. For revitions nd sttistics see Tle 3.2. AM fungl colonised root length (%) Donor sustrte tretment Donor genotype DC RC [U] [WT] 61.8 ± ± 20.3 [rmc]* 12.3 ± ± 6.2 [X] [WT] 48.0 ± 8.7 [rmc]* 16.2 ± ± ± 7.8 *= surfce colonistion At the end of the experiment, in ll AM fungl inoculted tretments the verge dry weights of the ERM from the donor fungl comprtments ws 0.3 ± 0.1 mg cm -3 cross ll tretments (dt not shown). No fungl mteril ws found in [ AM] comprtments. When the donor root ws left untreted [U], the externl mycelium in WT donor comprtments developed pproximtely four times higher hyphe length nd spore mounts per volume sustrte compred to the ERM of the rmc donor fungl comprtments (Fig. 3.2 nd ). Also, differences of externl mycelium rchitecture were oserved etween the genotypes: Specific hyphe length nd spore numer per unit dry weight of mycelium in WT comprtments were significntly higher thn those found in rmc comprtments (Fig. 3.2 c nd d). The disruption tretment [X] did not significntly ffect the hyphe length nd spore density (Fig. 3.2,). In contrst, the development nd rchitecture of the ERM otined from fungl comprtments of the receiver root comprtments (RC) were not significntly ffected y genotype or disruption of the neighouring donor plnt root (see Fig. 3.2 nd Tle 3.8). 35

42 CHAPTER 3 Hyphe length (m cm -3 sustrte) Spore density (nr. cm -3 sustrte) c Specific hyphe length (m mg -1 DW of ERM) d Numer of spores (nr. mg -1 DW of ERM [WT] [rmc] [WT] [rmc] [WT] [rmc] [WT] [rmc] [U] [X] [U] [X] ERM in DC ERM in RC Fig. 3.2: Development of the extr-rdicl mycelium (ERM) otined from fungl comprtments, hrvested t the end of the experiment. Shown re the results for. Hyphe length density in sustrte nd. Spore density in sustrte; c. Specific hyphe length per mg DW nd d. Numer of spores per mg DW of ERM otined from either donor (DC; figures left) or receiver (RC; figures right) root comprtments. Different letters indicte significntly different men vlues (multiple comprison Tukey-test; p < 0.05) due to the donor genotype [WT vs. rmc] or donor sustrte tretment [U vs. X]. 36

43 CHAPTER 3 Tle 3.8: Two-wy-ANOVA results for the percentge of the AM fungl colonised root length of donor nd receiver plnts nd ERM development in fungl comprtments (for dt see Tle 3.7 nd Fig. 3.2). The plnt roots nd the ERM were otined from either the donor comprtment (DC) or the receiver comprtment (RC). A significnt (p < 0.05) effect of the donor genotype (G), or donor sustrte tretment (T) is indicted y lck dot, n.s. = not significnt. Interction G T G x T Roots from DC AM fungl colonised root length n.s. n.s. Roots from RC AM fungl colonised root length n.s. n.s. n.s. ERM from DC Hyphe length density (m cm -3 sustrte) n.s. n.s. Spore density (numer cm -3 sustrte) n.s. n.s. Specific hyphe length (m mg -1 DW of ERM) n.s. n.s. Numer of spores (numer mg -1 DW of ERM) n.s. n.s. ERM from RC Hyphe length density (m cm -3 sustrte) n.s. n.s. n.s. Spore density (numer cm -3 sustrte) n.s. n.s. n.s. Specific hyphe length (m mg -1 DW of ERM) n.s. n.s. n.s. Numer of spores (numer mg -1 DW of ERM) n.s. n.s. n.s Nitrogen concentrtion nd content in the extr-rdicl mycelium At the end of the experiment AM fungl tissue N concentrtion nd contents were estimted. The AM fungl extr-rdicl mycelium (ERM) showed significntly decresed nitrogen concentrtion nd totl N content when growing in comprtments with rmc compred to comprtments with WT donor roots (Fig. 3.3), n effect tht ws significntly relted to the donor genotype ut not to the disturnce tretment (Tle 3.9). N sttus of ERM hrvested from the receiver root comprtments did not significntly differ due to ny of the experimentl fctors (Fig. 3.3 nd Tle 3.9). 37

44 CHAPTER 3 N concentrtion in ERM (mg g -1 DW) 15,0 10,0 5,0 5 c c 15,0 10,0 5,0 5 0,0 0 0,0 0 N content of the ERM (mg per comprtment) 0, , , , , , ,00 [WT] [rmc] [WT] [rmc] 0,00 [WT] [rmc] [WT] [rmc] [U] [X] [U] [X] ERM in DC ERM in RC Fig. 3.3: Nitrogen sttus of the ERM from the fungl comprtments hrvested t the end of the experiment. Shown re. N concentrtion nd. N content in the ERM from either donor (DC; figures left) or receiver (RC; figures right) root comprtments. For revitions nd sttistics see Figure 3.2. Tle 3.9: Two-wy-ANOVA for the N sttus of the ERM from fungl comprtments locted in either the donor comprtment (DC) or the receiver comprtment (RC). For revitions nd sttistics see Tle 3.8. Interction G T G x T Mycelium from DC N concentrtion n.s. n.s. N content n.s. n.s. Mycelium from RC N concentrtion n.s. n.s. n.s. N content n.s. n.s. n.s Dry weight nd nutrient sttus of the receiver plnts Receiver plnt dry weight nd P sttus Across ll tretments, the receiver plnt dry weight verged 15.9 ± 0.5 g per plnt. Totl plnt iomss nd the rtio of shoot-to-root DW (dt not shown) were not ffected y donor plnt genotype, donor tretment or AM fungl root colonistion. The totl P content of receiver plnt 38

45 CHAPTER 3 tissue did not differ due to the neighour plnt s genotype or sustrte tretment (Tles 3.10 nd 3.11). When inoculted with AM fungi the shoot nd root P concentrtion s well s the totl plnt P content in receiver plnts were significntly incresed compred to non-inoculted plnts (Tle 3.10 nd 3.11). Tle 3.10: Phosphorus concentrtion nd totl P content in the receiver plnt tissue. Receiver plnts were cultivted with their root system neighoured to 15 N lelled donor plnt wild-type [WT] or [rmc] mutnt root system. Both plnts were either inoculted with Glomus intrrdices [+AM] or non-inoculted [-AM]. After the 15 N lelling period the donor shoots were removed nd the sustrte in the donor root comprtment ws disrupted (tretment [X]) or ws left undistured (tretment [U]). Different letters indicte significntly different men vlues (Tukey-test; p < 0.05) due to the tretments. Shoot P concentrtion (mg g -1 DW) Root P concentrtion (mg g -1 DW) Plnt P content (mg per plnt) Donor tretment Donor genotype +AM -AM +AM -AM +AM -AM [U] [WT] 1.55 ± ± ± ± ± ± 2.36 [rmc] 1.52 ± ± ± ± ± ± 1.71 [X] [WT] 1.53 ± ± ± ± ± ± 2.11 [rmc] 1.49 ± ± ± ± ± ± 1.71 Tle 3.11: Three-wy ANOVA results for the receiver plnt phosphorus sttus. A significnt effect of AM fungl inocultion (M), donor genotype (G), or donor sustrte tretment (T) is indicted with lck dot (n.s. = not significnt). Interction G M T G x M G x T M x T G x M x T Shoot P concentrtion n.s. n.s. n.s. n.s. n.s. n.s. Root P concentrtion n.s. n.s. n.s. n.s. n.s. n.s. Plnt P content n.s. n.s. n.s. n.s. n.s. n.s. 39

46 CHAPTER Receiver plnt sttus of totl nitrogen nd 15 N Shoot N concentrtion (Tle 3.12) nd lso totl shoot N content (dt not shown) were not significntly ffected y ny of the tretments. When the neighouring donor plnt ws n undistured rmc plnt, significntly higher N concentrtion (Tle 3.12) nd content (dt not shown) were recorded in [+AM] receiver roots compred to the [ AM] tretment. However, the totl N content of the receiver plnt ws similr mong ll the tretments (Tles 3.12 nd 3.13). Tle 3.12: Nitrogen concentrtion in shoot nd root nd totl plnt N content of the receiver plnts. For revitions nd sttistics see Tle Shoot N concentrtion (mg g -1 DW) Root N concentrtion (mg g -1 DW) Plnt N content (mg per plnt) Donor sustrte tretment Donor genotype +AM -AM +AM -AM +AM -AM [U] [WT] 13.1 ± ± ± ± ± ± 23.6 [rmc] 13.3 ± ± ± ± ± ± 11.3 [X] [WT] 13.6 ± ± ± ± ± ± 12.9 [rmc] 13.1 ± ± ± ± ± ± 5.6 Tle 3.13: Three-wy ANOVA results for nitrogen concentrtion in shoot nd root nd totl plnt N content of the receiver plnts. For sttistics nd revitions see Tle Interction G M T G x M G x T M x T G x M x T Shoot N concentrtion n.s. n.s. n.s. n.s. n.s. n.s. n.s. Root N concentrtion n.s. n.s. n.s. n.s. Plnt N content n.s. n.s. n.s. n.s. n.s. n.s. n.s. 15 N trnsfer from the donor to the receiver plnt ws clerly ffected y the tretments: Significntly higher contents of 15 N were oserved in AM fungl inoculted thn in nonmycorrhizl receiver plnts (Tle 3.14). Only when AM fungl-inoculted, the quntity of 15 N 40

47 CHAPTER 3 derived from WT plnts clerly exceeded tht from rmc donor plnts. In undistured nd AM fungl-inoculted tretments the quntity of 15 N in receiver plnts originting from rmc mutnt roots of donor plnts ws low nd in similr rnge to tht of non-inoculted plnts. After the disruption of the donor plnt sustrte [X] tretment, AM fungl-inoculted receiver plnts otined t lest twice the mount of lelled N compred to the undistured [U] tretment, irrespective of the donor plnt genotype (Fig. 3.4). Tle 3.14: 15 N content in shoot nd root tissue of receiver plnts. For revitions see Tle Different letters indicte significntly different men vlues. Prior to multiple comprison Tukey-test (p < 0.05), dt were squre root trnsformed. 15 N content in receiver tissue (µg per plnt) Shoot Root Donor sustrte tretment Donor genotype +AM -AM +AM -AM [U] [WT] 8.7 ± ± ± ± 1.5 [rmc] 0.2 ± ± ± ± 1.6 [X] [WT] 21.0 ± ± c ± ± 2.1 [rmc] 3.3 ± ± ± ± 5.8 The mount of totl N trnsferred during the experiment (%N trnsfer ; see Section 3.3.6; eqution 1 nd 2) ws up to 1.5 ± 0.5% in WT plnts nd up to 0.5 ± 0.2% in rmc plnts. The highest percentge of receiver totl N content tht derived from fungl trnsfer (%N dft ; eqution 4 nd 5) ws found in WT tretments nd mounted up to 0.4 ± 0.1% in the undisrupted [U] tretment nd 1.1 ± 0.5% in the disrupted [X] tretment. The %Root N trnsfer to receiver plnts (eqution 3) ws significntly higher when donor roots were AM fungl inoculted [+AM] compred to the very low levels of non-inoculted [-AM] plnts (Fig. 3.4). When AM fungl symiosis ws present, the verge %Root N trnsfer from WT donor roots (3.4 ± 1.6%) clerly exceeded tht from [rmc] roots (0.3 ± 0.4%). This effect ws further enhnced y the disruption of donor roots: sustrte disruption incresed the mount of N trnsfer from AM fungl-inoculted roots of WT to 10.6 ± 4.8% nd tht of rmc plnts to

48 CHAPTER 3 ± 1.5% (Fig. 3.4). The interction etween donor genotype nd AM fungl inocultion ws sttisticlly significnt (Tle 3.15) c %Root N trnsfer Donor genotype [WT] [rmc] [WT] [rmc] [WT] [rmc] [WT] [rmc] AM inoculum [+AM] [-AM] [+AM] [-AM] Donor tretment [U] [X] Fig. 3.4: %Root N trnsfer to receiver plnts. For revitions see Tle Different letters indicte rs with significntly different mens, estimted using the multiple comprison Tukey-test (p < 0.05). Prior to sttistics, dt were normlised y squre root trnsformtion. Tle 3.15: Three-wy ANOVA results for receiver plnt 15 N uptke (for dt see Tle 3.14 nd Fig. 3.4). For revitions see Tle Significnces of men differences were clculted using the multiple comprison Tukey-test (p < 0.05) fter dt were normlised y squre root trnsformtion. Interction G M T G x M G x T M x T G x M x T 15 N content in receiver shoot n.s. n.s. n.s. n.s. 15 N content in receiver root n.s. n.s. n.s. %N trnsfer to receiver n.s. n.s. n.s. %Root N trnsfer to receiver n.s. n.s. n.s. 42

49 CHAPTER Discussion Estlishment of experimentl conditions to quntify AM fungl derived interplnt N trnsfer Mny tomto cultivrs re unresponsive to AM fungi in terms of growth (Bryl nd Koide 1990), including RioGrnde 76R used in the present experiment (Neumnn nd George 2005). Furthermore, the use of the tomto rmc mutnt llows quntifying the cpcity of AM fungl mycelium to trnsfer N etween roots which differed with respect to their ility to support mycorrhizl colonistion ut without confounding effects of differences in plnt iomss. In fct, neither the dry mtter production nor the totl N nd P content of donor nd receiver plnts ws significntly ffected y the genotype of the donor. Therewith, ll receiver plnts hd similr nutrient demnd when grown either djcent to wild-type or to n rmc mutnt plnt nd on the other hnd the donor plnts ll represented n N source of equivlent mgnitude Symiotic N trnsfer from mycorrhizl nd non-mycorrhizl ded roots As lso reveled y Johnsen nd Jensen (1996), the volume of N trnsferred to receiver plnt from ded roots of donor ws significntly incresed when the roots were mycorrhizl. The two root systems were physiclly isolted from one nother y nylon mesh which, nevertheless, llowed limited extent of direct trnsfer etween djcent non-inoculted roots. For exmple, in undisrupted tretments direct trnsfer in the non-inoculted WT tretment ws pproximtely 7% of tht mesured in the inoculted WT tretment. This form of direct N trnsfer is most likely to reflect the re-sorption of donor root N-losses y the receiver root, s lso demonstrted y Li et l. (2009). After two week-period fter shoot removl from donor plnts, the mount of 15 N present in ech receiver plnts incresed from 2-8 µg (not inoculted) to µg per plnt (inoculted with AM fungi). The proportion of the donor root N trnsferred (%RootN trnsfer ) reched 13%. Tht ws out one sixth of the donor root N content still ville t the end of the experiment hd een recovered y the receiver plnts. Relted to the totl N content of receiver plnts the proportion of N derived from fungl trnsfer (%N dft ) ws <1%, irrespective of soil disturnce. Similr levels of N trnsfer etween root systems connected y common AM fungl mycelium hve een reported y Johnsen nd Jensen (1996). This indictes tht under the present experimentl conditions the quntity of AM fungl N trnsfer from plnt residues cnnot e sufficient to hve positive impct on plnt N nutrition compred to totl plnt N 43

50 CHAPTER 3 uptke, presumly mostly y roots. Fresh plnt residues in soil in mny circumstnces re rpidly minerlised (Nett et l. 2010), nd hence re direct source for N for susequent nd neighouring plnts. Also under the present experimentl conditions N losses from donor roots would hve incresed with longer time of 15 N exposure, s lso shown y Ames et l. (1983) nd Jlonen et l. (2009). The contriution of AM fungi to plnt N nutrition my e more importnt in field sitution, where mycorrhizl plnts grow rther slowly nd/or plnt N demnd exceeds its vilility. This sitution rises when, for exmple, N sources re present in n immoile form, or when drought stress limits the ility of roots to sor nutrients from soil (Tor et l. 1994; Surmnin nd Chrest 1999) AM fungl medited N trnsfer s ffected y the presence of mycelium within the donor root Possile sources of fungl-medited 15 N uptke nd trnsfer included (1) N in the sustrte round donor roots, derived from rhizodeposition y live donor roots during the lelling period nd from losses y root decy fter shoot removl, nd (2) N from inside the colonised donor root. The ltter ws ccessile to AM mycelium connected to the receiver plnt either directly from the cortex vi the former intr-rdicl mycelium (IRM), or moilised from fungl storge structures inside the root (vesicles). The use of the rmc mutnt (lcking intr-rdicl colonistion) in the present experiment llowed for the seprte quntifiction of N trnsfer sed on the uptke vi the pthwy (1) (WT nd rmc plnts) nd pthwy (2) (WT plnts only). Here it ws shown tht the extent of symiotic N recpture ws clerly determined y the donor plnt s genotype - i.e., mycorrhizl (WT) s opposed to non-mycorrhizl (rmc mutnt). Nerly three times more N ws trnsferred from inoculted WT thn from the corresponding rmc mutnt donor root. Since the mjor source of trnsferred N ws in the sustrte relesed y ded donor roots, hyphl length close to the donor root my e relevnt fctor. Note tht the externl mycelium in the rmc donor comprtments ws llowed to enter y mens of the fungl window inserted etween oth neighouring plnts nd therefore the fungus ws likely in symiosis with the receiver root. We oserved tht the fungl iomss nd hyphe length in the WT comprtments douled tht found in the rmc comprtments. Bsed on isotope-lelled fertilistion of fungl comprtments, it hs een shown tht hyphl length density in the soil is positively correlted with the cpcity of the AM fungi to sor nd trnsfer oth N (Ames et l. 1983) nd P (Smith et l. 2004; Jns et l. 2005). Therefore, the oserved difference in N trnsfer etween the WT nd rmc roots my t lest prtly e 44

51 CHAPTER 3 ttriutle to differences in hyphl density in donor root comprtments, s prts of these hyphe were ssocited with receiver plnts. The pttern of root colonistion is importnt in the context of n N source derived from the internl structure of the root. The proportion of the WT root length successfully colonised y AM fungi following inocultion ws 50-70%, while in the rmc root, AM fungi were restricted to the root surfce (12-16%) nd formed only ppressori. The extent of the rmc mutnt root surfce colonised y mixture of Glomus mossee nd Glomus intrrdices ws of the sme order (Neumnn nd George 2005). Even fter the demise of the rmc donor roots, the only intr-rdicl colonistion oserved ws the presence of smll numer of intr-rdicl spores occupying not more thn 2% of the root length. Thus, N trnsfer vi the IRM from the inner root cortex could hve een ffected in the WT ut not in the rmc mutnt tretment. Root internl vesicles hve relevnt potentil to estlish new root infection (Biermnn nd Lindermn 1983), nd represent significnt loction for the storge of nutrient reserves (vn Arle nd Olsson 2003), to e exported to the ERM s the fungus grows (Bgo et l. 2002). In view of the differences in ERM density etween the WT nd the rmc donor root comprtments, it remins uncler to wht extent intr-rdicl fungl structures in colonised WT donor roots contriuted to the quntity of N trnsferred. However, following the demise of the root, the former IRM my hve een le to grow nd lter fuse with the symiotic ERM originting from the receiver root comprtment, fcilitting the trnsfer of N lso from root-internl fungl structures to the receiver Effect of soil disruption on N trnsfer to receiver plnts The effect of ERM disruption during the non-symiotic growth of AM fungi is rther inconsistent. In some cses, reduction in the cpcity to colonise the host plnt hs een recorded fter tillge in the field (Evns nd Miller 1988; Jsper et l. 1989; Jsper et l. 1991), in some cses resulting into decresed growth of the host plnt (McGonigle et l. 1990). In contrst, effects due to the disruption tretment hve not een oserved (McGonigle nd Miller 2000). Tillge lso ffects the AM fungl propgule density in the soil profile (Smith 1978; Kir et l. 1998), nd high propgule density cn compenste for the negtive effect of tillge (Jsper et l. 1991; McGonigle nd Miller 2000). The effect of disruption of the hyphe during the plnt growth period nd the resulting consequences for AM fungl nutrient trnsfer is less well explored. Periodic mechnicl disruption of the ERM locted in root-free nd isotope-lelled fungl comprtments hs een shown to reduce the soil-to-plnt trnsfer of 45

52 CHAPTER 3 oth N (Frey nd Schüepp 1993) nd P (Tuffen et l. 2002; Dun et l. 2011). Such repeted nd severe disruption of the mycelium network must reduce the cpcity of the AM fungi to sor nutrients, s lso suggested erlier (Evns nd Miller 1990). Here, mycelium ws disrupted only once (s in single mixing procedure) nd root residues were used s N source (s they re usully present in vegetted soils). Under these conditions, the disruption in donor root comprtments led to higher 15 N contents in the receiver plnts compred with undisrupted tretments. This effect ws unexpected in light of erlier studies where disruption hd decresed fungl nutrient trnsfer. Two resons my e responsile for the higher N trnsfer y hyphe fter soil disruption in the present experiment. Firstly, root deth cn e followed y sustntil loss of nutrients from the root tissue due to utolysis (Wichern et l. 2007). For exmple, excised roots of rye grss incuted in soil for three weeks lose up to, respectively, 60% nd 70% of their initil N nd P (Eson nd Newmn 1990), nd y such mens lost nutrients rpidly ecome ville to plnt roots (Ritz nd Newmn 1985; Eissenstt 1990). Within few dys fter mechnicl disturnce, soil smples tken from tilled field site showed higher level of net N minerlistion ccompnied y the continuous ccumultion of nitrte susceptile to leching thn did soil smpled from n undistured site (Jckson et l. 2003). A similr contrst hs een shown to pply in the comprison etween sieved nd non-sieved field soil smples (Clderon et l. 2000). The mjor effect of soil disruption in the present study included the frgmenttion of the 15 N-lelled donor roots which very likely resulted in n incresed root surfce re exposed to microil degrdtion therey incresing N d P losses from roots. Indeed, when the soil ws disrupted P concentrtions were reduced compred to undistured donor roots, suggesting tht more nutrients were ville to hyphe in disrupted soil perhps ecuse of leching from dmged tissue. A etter ertion in disrupted tretments my hve dditionlly fcilitted nutrient minerlistion processes in these pots. Secondly, single disturnce my e quickly overcome y hyphe of some AM fungi. Representtives of the Glomus fmily typiclly develop rpidly in the soil, nd the hyphl network of Glomus intrrdices ppers to e quite insensitive to soil disruption with respect to following root colonistion (Dun et l. 2011). Mikkelsen et l. (2008) recorded rte of dvnce of the hyphl front in soil of up to 3.8 mm per dy, nd Giovnnetti et l. (1993) mesured the elongtion of germinted hyphe of up to pproximtely 5 mm per dy. Injured hyphe of Glomus isoltes re le to nstomose within minutes (de l Providenci et l. 2005), reflecting the species well-developed cpcity to repir its ERM network following disturnce. Here, provided tht the fungl mycelium ws in continuous symiotic ssocition 46

53 CHAPTER 3 with the (undistured) receiver plnt, the two-week intervl etween soil disruption nd hrvest ws pprently sufficient for the fungus to enter the donor root comprtment. Spreding from the receiver comprtment, the mycelium my hve entered the donor root comprtment, uilding linkges cross the frgmented mycelium. This process would hve enled the ERM network to function once more with respect to N uptke nd trnsfer, whether the donor ws mycorrhizl or non-mycorrhizl plnt. Note tht the N concentrtion in ERM from fungl comprtments in the rmc donor root comprtments ws significntly reduced y disruption. Attempts to grow gin fter the disruption nd possile N losses from the fungl tissue might hve led to dilution of nutrients within the fungl tissue. Since the ERM from WT tretments developed significntly higher quntity of spores per unit hyphe length, it proly possessed lrger N reserves for distriution within the tissue compred to the mycelium from rmc tretments. This my explin the higher extent of N dilution in rmc mycelium fter disruption. Together the new estlishment of the fungus in the donor comprtment nd supposle incresed vilility of N from roots frgmented y soil disturnce could explin the higher AM fungl N trnsfer from oth the inoculted WT nd the rmc mutnt donor roots compred with the non-inoculted tretments Conclusions It hs een possile to confirm tht the quntity of N trnsferred etween two root systems cn e enhnced y the presence of mycorrhizl extr-rdicl myceli. The quntity of N trnsferred during the short experimentl durtion ws sustntil compred to the totl mount of N in the ded roots, ut reltively smll compred to the totl N demnd of fst growing plnt. Mycorrhizl N trnsfer from dying roots ws further incresed when these roots were AM fungl colonised efore deth. This difference cn e resoned y higher mycelium densities in the soil round the roots nd in ddition y the export of N reserves from root internl fungl structures through linkges to the receiver mycelium. The mechnicl disruption of soil contining ded roots cn increse the vilility of nutrients nd therefore ssist the process of mycorrhizl nutrient uptke nd trnsfer. When ssocited with living plnt, G. intrrdices ppers to hve high potentil to re-estlish its network in the soil fter disruption, nd to function s vehicle of N trnsfer. Agriculturl prctices, including reduced tillge my increse nutrient vilility from plnt residues nd rther hve positive effect on AM symiosis when involving fungi unsusceptile to single mechnicl disruption. 47

54 Chpter 4 4 Detched extr-rdicl mycelium networks of different AM fungi Colonistion potentil nd plnt growth promotion fter mycelium disruption 4.1 Astrct The im ws to study the potentil of detched extr-rdicl mycelium (ERM) network of different AM fungi to colonise susequent host plnt. Therefore the horizontl nd verticl distriution of the mycelium in soil ws determined s well s the effect of mechnicl disruption of the ERM in the context of the resulting re-estlishment nd contriution to the growth und P uptke of sweet potto plnts. A pot experiment ws conducted where receiver sweet potto plnt cuttings were plnted into comprtments contining previously estlished ERM of either Glomus intrrdices BEG 110 [GI]; Glomus mossee BEG 12 [GM]; or of AM fungi from n griculturl soil [AS]. At time of sweet potto plnting the ERM network ws seprted from its nurse plnt where it hs een estlished nd then the ERM ws either mechniclly disrupted y soil mixing or left untreted. All tested AM fungl inoculnts effectively colonised the sweet potto plnts within four weeks, leding to nerly doule the quntity of iomss nd P uptke compred to non-inoculted tretments, irrespective of the initil mechnicl disruption of the ERM. Both the Glomus isoltes produced the highest hyphe length nd spore density in soil, nd they colonised roots more intense in deeper soil sections. Accordingly, these fungi contriuted more to oth the nurse plnt nd the receiver plnt growth s did AM fungi from the field soil in [AS] tretments. Although the ltter developed much lower ERM densities in the sustrte compred with [GI] nd [GM] tretments, [AS] tretments incresed plnt growth nd P uptke drsticlly in sweet potto plnts, indicting high nutrient uptke efficiency of these fungi. The results showed tht n estlished, detched AM fungl ERM network cn efficiently colonise host plnts in sence of ny mycorrhizl root frgments. A high sptil distriution nd density of ERM in soil nd high fungl specific nutrient uptke efficiency my scertin fst fungl root colonistion nd erly contriution to plnt nutrient uptke. Moderte soil disturnce such s pplied in reduced tillge systems my not reduce the infection potentil of AM fungi. 48

55 CHAPTER Introduction Different AM fungl species possess diverse chrcteristics, such s specific life-cycles (Gvito nd Olsson 2008), explortion ptterns in soil (Boddington nd Dodd 1998) nd P uptke efficiencies of their mycelium (Drew et l. 2003). Accordingly, AM fungl species cn contriute differently to plnt P uptke during the period of the symiosis, nd the outcome of the plnt-fungl reltionship is depending on comintion of oth prtners. An importnt ttriute chrcterising fungl species is their forging pttern in ulk soil. The ERM network cn e locted predominntly ner the colonised root, or cn hve n extensive sptil distriution wy from the root (Smith et l. 2000) ridging horizontl distnces of out 15 cm (Mikkelsen et l. 2008). The fungl ERM not only spreds horizontlly, ut to some extent lso follows root growth into deeper soil lyers. The velocity t which the ERM spred into the soil fter the estlishment of the symiosis is diverse (Mikkelsen et l. 2008). AM fungl species tht hve een shown to spred fster nd further into soil (Glomus intrrdices) re le to contriute erly to plnt P uptke y hyphl explortion of P resources distnt from plnt roots (Avio et l. 2006). Within the first weeks of colonising host plnt, AM fungi with slow ERM spred development (e.g. Gigspor spp.) seem to contriute less to plnt P nutrition nd growth compred to fster developing species, such s Glomus spp. (Smith et l. 2004), t lest in the short term. After the termintion of life-cycle, the ERM network cn serve s n inoculum for following plnt. Depending on the species, infective AM fungl propgules my lso e predominntly those structures present in mycorrhizl roots, such s intr-rdicl vesicles nd intr-rdicl spores. The extr-rdicl mycelium functions s n importnt infective unit in tht it produces stle nd long lsting spores eing importnt propgules (Biermnn nd Lindermn 1983). Representtives of the Glomercee fmily re known to infect new roots lso y mens of hyphe, while memers of Gigsporcee likely depend on spores only nd lck the ility to infect y mens of externl hyphe (Klironomos nd Hrt 2002). The existing sptil spred nd density of the ERM in soil must therefore e crucil for the infection potentil. So fr, the infectivity of n excised ERM in reltion to its sptil distriution with respect to horizontl nd verticl mycelium prolifertion in the soil hs not een quntified in the sence of infective mycorrhizl roots. The first im in this study ws to determine the infective potentil of n excised ERM network, previously estlished on nurse plnt nd therefter re-estlishing on receiver plnt. It ws hypothesized tht the AM fungi with the 49

56 CHAPTER 4 highest extent of verticl nd horizontl prolifertion in the soil prior to mycelium detchment will lso hve the highest infective potentil nd growth promoting effect on the following plnt. The horizontl mycelium spred ws studied in terms of hyphe length-density nd spore density in soil distnt from the nurse plnt root. Moreover, verticl AM fungl spred ws mesured, chrcterised y the soil depth up to where root colonistion occurred, nd the resulting outcome of the symioses in terms of plnt P uptke nd growth ws quntified. To exclude tht the outcome of the symiosis with the tested AM fungl inocul my e plnt species specific, two different plnt species were used to follow one nother s nurse (Mize) nd receiver (sweet potto) plnt. Sweet potto ws chosen for receiver plnts s it is n importnt tuer crop produced in the tropics even on mrginl lnd (Woolfe 1992) nd cn chieve significnt growth enefits in symiosis with AM fungi (Sieverding 1991). As the AM symiosis is common nd widely distriuted ssocition it is importnt to study effects of griculturl prctices on its development. Soil disturnce, such s tillge hs een reported to reduce AM fungl root colonistion, leding to reduced fungl contriution to plnt P uptke (Kir et l. 1997; McGonigle et l. 1999). Tilled field soils hve een oserved to hve reduced sporultion of some species nd AM fungl community structures dominted y Glomus species (Jns et l. 2002). Fst spreding species my etter compenste for hyphe disruption nd therefore hve een oserved to dominntly occur in distured griculturl field sites (Oehl et l. 2003). Thus, the destruction of hyphe networks not lwys hve negtive effect on fungl colonistion nd the resulting growth enefit of the colonised plnt (see McGonigle nd Miller 2000; Dun et l. 2011). Previous studies ttriuted negtive effects of mechnicl disturnce to decrese of the fungl P uptke effectiveness due to hyphe frgmenttion nd the resultnt necessity to re-estlish mycelium network y mens of mycorrhizl root frgments or spores (McGonigle nd Miller 2000). However, erlier reports on the effects of soil disturnce on the functioning of AM symiosis remined inconsistent nd the underlying mechnisms hve not een fully understood. Possily, AM fungi tht typiclly spred fst nd intensive will e less ffected in terms of re-estlishment on following host plnt fter eing disrupted thn would species colonising less intensive. The scrce knowledge out this issue led us to the second im of this experiment which ws to otin certinty out the effect of soil disturnce on the infection potentil of mycelium network contining spores nd hyphe only. It ws hypothesized tht soil disturnce reduces the totl infectivity of n excised AM fungl ERM nd therefore reduces fungl contriution to plnt P uptke nd growth of following crop. 50

57 CHAPTER Mterils nd methods In the present experiment, using specil rhizooxes with two root comprtments divided y hyphe permele mesh memrne, it ws possile to seprte the root system of mize nurse plnt from tht of sweet potto receiver plnt. This llowed for the study of AM fungl colonistion nd plnt growth unffected y interferences of root soil occuption or nutrient sorption y the nurse plnt. Using rhizooxes of 40 cm depth, root growth nd root colonistion rte in reltion to soil depth could e mesured for three different AM fungl isoltes Production of experimentl plnts Shoot cuttings of sweet potto (Ipomoe tts L.) plnts were rooted nd grown for 30 dys in nutrient solution (ph 6.8) composed of the following: 5 mm N (hlf C(NO 3 ) 2, hlf NH 4 NO 3 ); 0.7 mm P (KH 2 PO 4 ); 4 mm K (KH 2 PO 4 nd K 2 SO 4 ); 2.5 mm C (C(NO 3 ) 2 nd CSO 4 ); 1 mm Mg (MgCl 2 ); 4 mm S (CSO 4 nd K 2 SO 4 ); 10 µm Fe (Fe-EDTA); 10 µm B (H 3 BO 4 ), 5 µm Mn (MnSO 4 ); 1 µm Zn (ZnSO 4 ); 0.7 µm Cu (CuSO 4 ); 0.5 µm Mo ((NH 4 ) 6 Mo 7 O 24 ). The cuttings were trnsplnted into experimentl plnting units t the 4 th lef stge with n dventitious root length of out 20 cm Preprtion of rhizooxes, sustrte filling nd AM fungl inocultion To oserve root growth nd development of the fungl ERM in the sustrte, specific twocomprtmented rhizooxes were used (Fig 4.1 -e). This llowed young plnt to grow into n lredy existing AM fungl ERM in sence of the mize plnt roots. One such plnting unit ws constructed from 8 mm thick PVC pltes (ottom, sides) nd removle, trnsprent cryl glss pltes (front nd ck) nd mesured inside 18 x 9 x 39 cm (length, width, height) resulting in totl volume of 3.3 L. The plnting unit ws hlved verticlly y porous 0.4 cm thick PVC plte resulting to two djcent nd ttched equl sized soil comprtments of 4.3 cm width. To llow hyphe ut not roots to grow through the rrier, one side of the PVC plte ws covered with nylon memrne of 30 µm mesh size (Sefr AG; Switzerlnd) seled using silicone (Prou, Buhus AG, Germny). Ech soil comprtment ws filled to 3 cm elow the top with 3600 g dry soil-sustrte (for properties, preprtion nd fertilistion, see Chpter 2.1) t ulk density of 1.3 g cm 3, to soil depth of 36 cm. AM fungl inoculum ws dded ccording to the tretment only to the soil comprtment (Cpt A) of the nurse plnt y mixing it homogenously into the sustrte, while the neighouring soil comprtment (Cpt B) of the susequent receiver plnt ws not inoculted (Fig 4.1 e). 51

58 CHAPTER 4 c d Acryl glss pltes AM fungl ERM e 30 µm memrne Fungl tues Cpt A Cpt B AM fungl inocultion sustrte tretment (U vs. X) Fig. 4.1: Photogrph of the soil oxes in experimentl phse 1 with mize plnts in Cpt A, 25 dys fter sowing (DAS) nd : experimentl phse 2 with sweet potto plnts in Cpt B, 6 DAP. c. & d: Verticl cross-sectionl view of rhizoox, seprted into Cpt A nd Cpt B y 30 µm hyphe permele memrne. The front sides were covered y n cryl glss plte. The rhizooxes were kept in n ngle of 45 to encourge root growth towrds nd long the cryl glss plte nd visile roots were recorded weekly. To estlish n ERM network, mize plnt inoculted or not with AM fungi ws grown in Cpt A nd AM fungl hyphe hd ccess to oth comprtments. d & e: Susequent to the removl of the mize plnt from Cpt A, the sustrte in Cpt B ws either disrupted (X) or ws left untreted (U) nd following the sweet potto cuttings were plnted therein. e: Root growth ws studied in four different soil depths s indicted centred etween oth comprtments. Two fungl tues were inserted into Cpt B t two different soil depths ech. Three different inoculum types were used in Cpt A: i) Glomus intrrdices BEG 110 [GI]; ii) Glomus mossee BEG 12 [GM], oth self propgted on similr sustrte nd iii) smple 52

59 CHAPTER 4 from the top lyer of n griculturl soil [AS] including different AM fungl species, see Chpter 2.5. To ensure similr sustrte conditions in ll tretments, Cpt A received lwys ll three inoculum types, ut for ech AM fungl tretment only one inoculum type ws used live while the other two inoculum types consisted of sterilised inoculum. In non-inoculted [-AM] tretments, the root comprtments otined sterilised AM fungl inoculum s mixture of ll used inoculum types nd filtrte s descried in Chpter Preprtion nd insertion of fungl tues Fungl tues (FT) were constructed from 25 ml (6.3 cm length nd 2.2 cm dimeter) plstic cylinders with ltticed wll. The outer surfce of the plstic cylinder ws covered with nylon memrne hving mesh width of 30 µm (Sefr AG; Switzerlnd) tht llowed hyphe ut not roots to grow into the tues. The nylon memrne ws fixed to the plstic cylinders using silicone (Prou, Buhus AG, Germny). The FT sustrte ws prepred s descried in Chpter 2.3 nd fertilised t the sme rte nd with the sme compounds s the rhizoox sustrte. Four fungl tues were inserted verticlly into the sustrte of Cpt B (Figure 4.1 d nd e). Two FT were plced t 4-10 cm depth, while the remining two were plced t cm depth. All FT were horizontlly centred nd were locted close to the cryl glss plte of the rhizooxes. This permitted the study of the ERM development in two different soil depths t two different hrvest times Plnting, experimentl set-up nd growth conditions The experiment ws divided into two phses. i) The experimentl phse 1 served for the estlishment of the AM fungl extr-rdicl mycelium in the [+AM] tretments (Fig. 4.1 ). Two seeds of Ze mys (L.) Gold were germinted within the sustrte in the centrl position of Cpt A. The wter content of the sustrte ws mintined t 18%. After the emergence of the second lef, seedlings were reduced to one per rhizoox. The sides of the rhizooxes were covered with n opque plstic wrpping to prevent light exposure to roots. The surfce of the sustrte in the comprtments ws covered with foil to reduce wter loss y evportion. The rhizooxes were rrnged rndomised on shelf nd inclined t n ngle of 45 with the plnted comprtment downwrds to encourge root growth towrds nd long the cryl glss plte, where root growth could e trced weekly. After seven weeks mize plnts were hrvested, sustrte ws removed from Cpt A nd soil disturnce tretment in Cpt B ws conducted: The cryl glss pltes were opened nd the sustrte ws cut into squres of pproximtely one cm edge length. The sustrte ws then mixed with sptul respecting the 53

60 CHAPTER 4 verticl sections of the soil profile such tht no disloction etween the depth sections occurred. During the disturnce process fungl tues were removed from the oxes nd were reinstlled fterwrds. ii) Experimentl phse 2: Directly fter the conduction of soil disturnce the oxes were closed gin nd rooted sweet potto (Ipomoe tts L.) cutting ws plnted into the neighour comprtment Cpt B (Fig. 4.1 ). Sweet potto cuttings were prepred s descried in Section The experiment consisted of 32 two-prted rhizooxes including four different inoculum tretments [GI], [GM], [AS] nd [-AM], comined with two different sustrte tretments, where the sustrte ws untreted [U] or ws distured y soil mixing [X], see Tle 4.1. Tle 4.1: Overview of the tretments in the experimentl phse 2 with sweet potto plnts. Tretments were replicted four times. Sustrte tretment [U] [X] AM fungl inoculum [GI] [GI] [GM] [GM] [AS] [AS] [ AM] [ AM] [GI]: G. intrrdices [GM]: G. mossee [AS]: Agriculturl soil [ AM]: Non-inoculted tretment [U]: Sustrte non-distured [X]: Sustrte disrupted The experimentl plnts were grown in glsshouse. Mize plnts were grown for 50 dys from August to Septemer nd sweet potto plnts for 28 dys during Octoer. Throughout the growth period, verge dy nd night tempertures in the glsshouse were 27 C nd 21 C, respectively. The reltive ir humidity verged 65%. During the lst 21 dys of the experimentl period, the plnts received dditionl light during 6 h with 350 µmol photons m -2 s -1 t plnt height, provided y 400 W lmps (SON-T Agro; Philips, Germny). The plnt wter uptke ws estimted grvimetriclly twice week nd replced with deionised wter, pplied to the top of the rhizoox. In etween the grvimetric estimtions, clculted mount of the expected wter uptke ws given to ll plnts s deionised wter. Root growth ws monitored once week y trcing roots growing long the cryl glss plte with permnent mrker using different colours for ech week. The length of roots ws lter estimted using digitl mp reder (Wyfinder MR H; Huger Electronics, Germny) Hrvest nd nlysis of plnt nd AM fungl mteril Mize shoots nd roots were hrvested 50 DAS nd sweet potto plnts 28 dys fter plnting nd shoot fresh weight (FW) ws recorded. For mize, the iomss of the reproductive orgns 54

61 CHAPTER 4 (flowers) ws estimted seprted from the residul shoot prts. Sweet potto plnts hd not developed ny tuers t time of hrvest. The plnt root system ws divided into four sections: 0-12, 12-21, nd cm depth within the growth sustrte. Root mteril from ech section ws wshed free from the sustrte, FW determined, nd representtive smples (0.5 g) of fresh roots were collected nd stored in 15% ethnol. These were therefter used to determine the AM fungl root length colonistion rte, s descried in Chpter 2.7. All plnt prts were dried t 65 C for three dys nd the dry weight (DW) ws recorded. Root DW from the different soil depth ws dded to result in totl root DW. The fungl tues (FT) were hrvested sequentilly. One FT from ech plcement depth ws hrvested t time of sweet potto plnting (t 0 ), nd the other t hrvest of sweet potto plnts (t 1 ). The ERM in the FT ws extrcted nd freeze-dried nd spore numer nd hyphe length were ssessed s descried in Chpter Nutrient nlysis nd sttistics Susmples of 200 mg ground plnt mteril (shoot or root) were digested nd P concentrtion ws nlysed s descried in Chpter 2.8. For mize shoot nutrient nlysis, ll shoot prts including stem, leves nd flowers were pulverised. Four replictes per tretment were used. Provided tht results pssed the test for norml distriution (Kolmogorov-Smirnov test; p > 0.05) nd homogeneity of vrince (Levene test; p > 0.05), dt were sujected to two-wy ANOVA. In cses where the ANOVA indicted significnt effect of ny fctor, the multiple comprison Tukey-test ws used to estimte differences etween mens of ll tretments. P vlues elow 0.05 otined in oth tests were interpreted s indicting significnt effects. Sttistic clcultions were conducted using SigmStt softwre, version 3.5 (Systt Softwre, Inc., USA). Results in tles nd figures re presented s tretment mens ± stndrd devition. 4.4 Results Mize plnts in experimentl phse 1 At hrvest, the dry weight (DW) of mize flower, totl shoot nd totl plnt differed ccording to the mycorrhiz tretment, i.e. [GI] nd [GM] inoculted plnts showed pproximtely doule the mount of tht oserved in [AS] nd [-AM] tretments (Tle 4.2). On the other hnd, the root iomss ws unffected y the AM fungl tretment. This led to significntly higher shoot-to-root rtio of DW in [GI] nd [GM] plnt compred to [AS] nd [ AM] plnts. Almost doule the mount of shoot P concentrtion ws found in [GI] nd [GM] plnts compred with 55

62 CHAPTER 4 [AS] nd [ AM] plnts (Tle 4.2). Root P concentrtion ws significntly higher in ll AM inoculted plnts compred with [ AM] plnts. All mycorrhizl tretments showed significntly higher P contents compred with the [ AM] tretment, nd the highest mount of totl plnt P content ws oserved in [GI] nd [GM]. In inoculted tretments, totl AM fungl colonised root length s the verge from ll soil depths, significntly differed etween mycorrhiz tretments nd ws incresed in the order of [AS] < [GM] < [GI] (Tle 4.2). Tle 4.2: Mize plnt iomss, totl AM fungl root colonistion rte nd P sttus, 50 DAS. Shown re mens ± SD. For the pre-experimentl phse, mize plnts were grown during 50 dys in Cpt A, inoculted with Glomus intrrdices [GI], G. mossee [GM], griculturl field soil [AS] or were non-inoculted [-AM]. AM fungl colonised root length (%) mesured in the different soil depths ws verged to the totl AM fungl root colonistion rte. Mens within row followed y different letters re significntly different (Tukey-test; p < 0.05; n = 8). Plnt DW (g per plnt) Flower DW (g per plnt) Shoot DW (g per plnt) Root DW (g per plnt) Shoot-to-root rtio [GI] [GM] [AS] [-AM] ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.59 Totl AM fungl colonised root length (%) ± ± ± Plnt P content (mg per plnt) Shoot P concentrtion (mg g -1 DW) c ± ± c ± ± ± ± ± ± 0.11 Root P concentrtion (mg g -1 DW) 1.14 c ± c ± ± ± 0.06 In the soil depths down to 29 cm, AM fungl root length colonistion in [GI] nd [GM] tretments rnged from pproximtely 60 to 90%, nd exceeded tht from [AS] tretments more thn twofold (Fig. 4.2). In the soil depth of cm, the root colonistion rte ws significntly incresed in the order of [AS] < [GM] < [GI]. In the lowest soil depth (29-36 cm), less thn 20% colonised root length ws oserved, nd the vlues were highly vrile nd not significntly different mong the mycorrhiz tretments. Roots from ll mycorrhiz tretments, 56

63 CHAPTER 4 [AS], [GM] nd [GI] were oserved to e colonised y AM fungl ruscules (dt not shown). Plnt roots from the non-inoculted tretment [-AM] were free from AM fungl colonistion cm cm cm cm [GI] [GM] [AS] soil depth A soil depth B soil depth C soil depth D [GI] [GM] [AS] [GI] [GM] [AS] [GI] [GM] [AS] c Mize plnt % colonised root length in reltion to soil depth AM fungl colonised root length (%) Fig. 4.2: AM fungl colonised root length (%) of mize plnts, in the soil depths of 0-12; cm; cm nd cm. Shown re mens ± SD. Mize plnts were inoculted with Glomus intrrdices [GI], G. mossee [GM] or griculturl field soil [AS] or were non-inoculted [-AM]. Non-inoculted plnts were not colonised y AM fungi. Within ech depth ctegory, mens followed y different letters re significntly different from ech other (Tukey-test; p < 0.05; n = 8). Totl root lengths of mize plnts trced on the cryl glss plte were not significntly different mong ll mycorrhizl tretments up to the time of 45 DAS (Fig. 4.3). During the growth period higher root lengths were mesured in [GI] compred to non-inoculted [-AM] tretments nd this effect ws incresed to significnt extent until 50 DAS. Root lengths from plnts of [GM] nd [AS] tretments were y trend higher compred to [-AM] ut the vlues did not gin significnt difference during the growth period. During the cultivtion period, in ll tretments the totl root length trced on the glss pltes ws out cm per plnt. 57

64 CHAPTER 4 Root length on cryl glss plte (cm per plnt) M ize plnt root length increment (cm), mesured y trcing roots on cryl glss plte [GI] G. intrrdices [GM] G.mossee [AS] ceh soil [-AM] Dys fter sowing ds18 ds31 ds38 ds45 ds50 Fig. 4.3: Totl root length (cm) of mize plnts trced on the cryl glss plte fter 18, 31, 38, 45 nd 50 DAS. Shown re mens ± SD. Mize plnts were inoculted with Glomus intrrdices [GI], G. mossee [GM], griculturl field soil [AS] or were non-inoculted [-AM]. For ech dte, mens followed y different letters re significntly different from ech other (Tukey-test; p < 0.05; n = 8) Sweet potto plnt iomss, AM fungl root colonistion rte nd P sttus At hrvest, twenty-eight dys fter plnting (DAP), sweet potto plnt DW ws higher in [GI] nd [GM] compred with [AS] nd [-AM] tretments (Tles 4.3 nd 4.4). This ws due to incresed shoot ut not root DW. According to the incresed shoot growth, the shoot-to-root rtio ws significntly higher in mycorrhizl compred with [-AM] tretments. The shoot DW in [AS] tretments rnged etween tht of [-AM] nd [GI] s well s [GM] tretments (Tle 4.3). Soil disturnce hd no significnt effect on plnt iomss (Tle 4.4). Sweet potto plnt roots from the non-inoculted [-AM] tretment were free from AM fungl colonistion. In mycorrhizl nd undisrupted tretments, AM fungl root colonistion rte ws similr in [GM] nd [AS] plnts, nd compred to these, it ws significntly higher when inoculted with [GI] (Tle 4.3). When the sustrte ws disrupted, AM fungl colonistion rte ws significntly decresed in [GI/X] plnts compred with [GI/U] tretments (Tle 4.3 nd 4.4). Roots from ll mycorrhiz tretments, [AS], [GM] nd [GI] were frequently colonised with AM fungl ruscules (dt not shown). 58

65 CHAPTER 4 Tle 4.3: Sweet potto plnt iomss nd AM fungl root colonistion rte fter hrvest. Shown re mens ± SD. AM fungl mycelium ws estlished on mize plnts in the neighour comprtment, inoculted with G. intrrdices [GI], G. mossee [GM] or griculturl field soil [AS]. At time of sweet potto plnting, the sustrte in Cpt B ws untreted [U] or distured [X]. AM fungl colonised root length (%) mesured in the different soil depths ws verged to the totl AM fungl root colonistion rte. Non-inoculted plnts [-AM] were not colonised y AM fungi. Mens followed y different letters re significntly different from ech other (Tukey-test; p < 0.05; n = 4). [GI] [GM] [AS] [-AM] [U] [X] [U] [X] [U] [X] [U] [X] Plnt DW (g per plnt) 1.45 ± ± ± ± ± ± ± ± 0.20 Shoot DW (g per plnt) 1.11 ± ± ± ± ± ± ± ± 0.17 Root DW (g per plnt) 0.34 ± ± ± ± ± ± ± ± 0.03 Shoot-to-root rtio 3.30 ± ± ± ± ± ± ± ± 0.45 Totl AM fungl colonised root length (%) ± ± ± ± ± ± 8.46 / / Tle 4.4: Two-wy-ANOVA results for iomss nd AM fungl colonistion rte of sweet potto plnts fter hrvest (see Tle 4.3). A significnt effect of AM fungl inoculum (M) or disturnce tretment (D) is indicted with lck dot (n.s. = not significnt). Interction M D M x D Plnt DW (g per plnt) n.s. n.s. Shoot DW (g per plnt) n.s. n.s. Root DW (g per plnt) n.s. n.s. n.s. Shoot-to-root rtio n.s. n.s. AM fungl colonised root length (%) n.s. Sweet potto root DW ws lrgest in the soil depth of cm (Fig. 4.4). This resulted in out 35-45% root DW of totl root iomss in this depth lyer. In this depth, the root DW percentge ws significntly higher in [GI] nd [GM] compred to [AS] nd [ AM] tretments. In the upper nd the two deepest lyers, not more thn 25% of totl root DW ws developed in ech lyer, irrespective of the AM fungl tretment. In the deepest root zone (29-36 cm) significntly lower percentge of root DW ws present in [GI] nd [GM] compred to [AS] nd [ AM] tretments (Fig. 4.4). Over ll soil depths, the disturnce tretment [X] hd no 59

66 CHAPTER 4 significnt effect on the percentge root DW distriution compred with undistured [U] tretments, irrespective of the mycorrhiz tretment (dt not shown) cm [GI] [GM] depth A [AS] [-AM] G. i ntr r di ces G. mossee f i el d soi l cm no i nocul um [GI] [GM] depth B [AS] [-AM cm [GI] [GM] depth C [AS] [-AM] cm [GI] [GM] depth D [AS] [-AM] Root DW distriution (% of totl root iomss) Fig. 4.4: Root DW distriution of sweet potto plnt in % of totl root iomss in the soil depths of 0-12 cm; cm; cm nd cm. Dt re verged over disturnce tretments. Prior to the cultivtion of sweet potto plnts, AM fungl mycelium ws estlished on mize plnts in the neighour comprtment, inoculted with G. intrrdices [GI], G. mossee [GM], griculturl field soil [AS] or non-inoculted [-AM]. Dt ws squre root trnsformed efore eing nlysed sttisticlly. Within ech depth ctegory, mens followed y different letters re significntly different from ech other (Tukey-test; p < 0.05; n = 8). The AM fungl root colonistion rte in different soil depths ws highest in cm depth (Fig. 4.5). In depth of cm the mycorrhizl tretments differed significntly. In this depth root colonistion rtes incresed in the order of [AS] < [GM] < [GI], nd the fungl colonistion in the [AS] tretment ws very low. Roots from ll mycorrhiz tretments were oserved to e colonised y AM fungl ruscules (dt not shown). No colonistion ws found in roots locted deeper thn 29 cm. A significntly decresed root colonistion rte with 60

67 CHAPTER 4 G. intrrdices in depths of cm ws oserved in distured [GI/X] compred to untreted [GI/U] tretments (Fig. 4.5) cm cm cm cm [GI] [GM] [AS] [GI] [GM] [AS] [GI] [GM] [AS] soil depth A soil depth B soil depth C soil depth D c 60 [GI] [GM] AM fungl colonised root length (%) Two-wy-ANOVA results M D M x D n.s. n.s. n.s. n.s. n.s. n.s. / / / [AS] Fig. 4.5: AM fungl root colonistion rte (%) of sweet potto plnts, oserved in the sustrte depths of 0-12; cm; cm nd cm. Shown re mens ± SD. AM fungl mycelium ws estlished in the plnted nurse plnt comprtment, inoculted with G. intrrdices [GI], G. mossee [GM] or griculturl field soil [AS]. At time of sweet potto plnting, the sustrte ws untreted [U] (plin rs) or distured [X] (dotted rs). In the soil deeper thn 29 cm, no AM fungl root colonistion occurred. Within ech depth ctegory, mens followed y different letters re significntly different from ech other (Tukey-test; p < 0.05; n = 4). Columns on the right show the two-wy-anova results for the AM fungl root colonistion rte. For revitions nd sttistics see Tle 4.4. Shoot nd root P concentrtion in sweet potto plnts ws lowest in [-AM] tretments; [GI] nd [GM] plnts showed out doule the shoot P concentrtion compred to [-AM] tretments (Figs. 4.6, ; Tle 4.7). [AS] plnt shoot P concentrtion remined etween the [-AM] tretment nd the tretments inoculted with either [GI] or [GM]. The P concentrtion in roots ws similr compred to tht of the shoots. Plnt totl P content in the different mycorrhizl tretments ws not significntly different ut ws much higher thn tht of non-inoculted tretments. Soil disturnce hd no significnt effect on P concentrtion or content in ny plnt prt, irrespective of the inocultion tretment (Fig. 4.6,, c; Tle 4.7). 61

68 CHAPTER 4 Shoot P concentrtion (mg g -1 DW) c c c c sw eet potto shoot P concentrtion (mg per g DW) Root P concentrtion (mg g -1 DW) c c c c sw eet potto root P concentrtion (mg per g DW) 0 G. intrrdices G.mossee field soil no inoculum [U] [X] [U] [X] [U] [X] [U] [X] 0 G. intrrdices G.mossee field soil no inoculum [U] [X] [U] [X] [U] [X] [U] [X] [GI] [GM] [AS] [-AM] [GI] [GM] [AS] [-AM] c 7 6 sw eet potto plnt P content (mg per plnt) Plnt P content (mg per plnt) G. intrrdices G.mossee field soil no inoculum [U] [X] [U] [X] [U] [X] [U] [X] [GI] [GM] [AS] [-AM] Fig. 4.6: Sweet potto plnt P sttus fter hrvest.. Shoot P concentrtion,. Root P concentrtion nd c. Totl P content in the plnt. Prior to the cultivtion of sweet potto plnts, AM fungl mycelium ws estlished in the plnted neighour comprtment, inoculted with G. intrrdices [GI], G. mossee [GM], griculturl field soil [AS], or non-inoculted [-AM]. At time of sweet potto plnting, the sustrte ws untreted [U] or distured [X]. Mens followed y different letters re significntly different from ech other (Tukey-test; p < 0.05; n = 4). Tle 4.5: Two-wy-ANOVA results for P sttus of sweet potto plnts fter hrvest (see Fig. 4.6). A significnt effect of AM fungl inoculum (M), or disturnce tretment (D) is indicted with lck dot (n.s. = not significnt). Interction M D M x D Plnt P content (mg per plnt) n.s. n.s. Shoot P concentrtion (mg g -1 DW) n.s. n.s. Root P concentrtion (mg g -1 DW) n.s. n.s AM fungl ERM development in fungl tues When plnts were non-inoculted [-AM], no AM fungl hyphe or spores were oserved in fungl tues (FT) from oth studied profile depths (4-10 nd cm). At the time of sweet potto plnting (t 0 ), the ERM DW from [GI] nd [GM] from the upper depth verged from

69 CHAPTER 4 to 5.8 mg per FT, this ws significntly higher compred to tht of [AS] tretment (Tle 4.6). The ERM DW in FT from the [AS] tretment ws less thn hlf milligrm. At the finl hrvest dte (t 1 ), ERM DW in FT of [GI] nd [GM] tretments ws t similr mgnitude compred with tht of the erlier hrvest dte (t 0 ), nd still significntly exceeded tht of the [AS] tretment. In the [AS] tretment, in the upper soil depth mycelium DW ws incresed t t 1 compred with t 0. As shown y the two-wy-anova, the disturnce tretment s min fctor did not ffect the ERM DW significntly t hrvest time (t 1 ) (Tle 4.6; right side), though fungl DW ws significntly lower in the deeper FT of [GM/X] compred with [GM/U] tretment (Tle 4.6; left side). Tle 4.6: AM fungl extr-rdicl mycelium DW (mg per 25 ml FT). Fungl tues from the receiver comprtment from 4-10 cm nd cm soil depth nd were exerted t time of sweet potto plnting (t 0 ) or hrvest (t 1 ), respectively. Prior to the cultivtion of sweet potto plnts, AM fungl mycelium ws estlished in the plnted neighour comprtment, inoculted with G. intrrdices [GI], G. mossee [GM] or griculturl field soil [AS]. At time of sweet potto plnting, the sustrte ws untreted [U] or distured [X]. Mens within row followed y different letters re significntly different (Tukey-test; p < 0.05; n = 4). The right-sided columns of the tle show the results of the sttisticl nlysis using two-wy-anova, for revitions nd sttistics see Tle 4.5. Dt ws squre root trnsformed efore eing nlysed sttisticlly. In cse mens elong to the smpling dte t 0, the fctor disturnce (D) ws not included in the ANOVA-nlysis, since dt were otined efore the set-up of the disturnce tretment. [GI] [GM] [AS] Interction Dte Soil depth [U] [X] [U] [X] [U] [X] M D M x D t cm 4.73 ± ± ± ± ± ± /- -/ cm 3.74 ± ± ± ± ± ± /- -/- t cm 5.72 c ± c ± c ± ± ± ± 0.65 n.s. n.s cm 3.08 c ± c ± c ± ± ± ± 0.07 n.s. n.s. At the time of sweet potto plnting (t 0 ), the hyphe length, spore density nd numer of spores per unit hyphe length in the sustrte of the FT otined from the soil depth of 4-10 cm ws similr in oth Glomus inoculted tretments nd ws significntly higher when compred with the [AS] tretment (Fig. 4.7,, c; Tle 4.7). 63

70 CHAPTER 4 10 d 10 Hyphe length (m cm -3 sustrte) Hyphe length (m cm -3 sustrte) c c c Spore density (nr. cm -3 sustrte) G. int rrdices G. mossee indon. Soil G. int rrdices G. mossee indon. Soil e Spore density (no. cm -3 sustrte) G. int rrdices G. mossee indon. Soil c c c G. int rrdices G. mossee indon. Soil Spores per unit hyphe length (nr. m -1 hyphe) c f Spores per unit hyphe length (no. m -1 hyphe) G. int rrdices [U] [X] G. mossee [U] [X] indon. Soil [U] [X] 0 G. int rrdices [U] [X] G. mossee [U] [X] indon. Soil [U] [X] [GI] [GM] [AS] [GI] [GM] [AS] t 0 t 1 Fig. 4.7: AM fungl ERM development in fungl tues otined from the upper 4-10 cm of soil in the receiver comprtment. Fungl tues were smpled efore set-up of disturnce tretments t time of sweet potto plnting (t 0 ; figures left) or four weeks fter the conduction of disturnce tretments t sweet potto hrvest (t 1 ; figures right). Shown re hyphe length density (. nd d.), spore density in the sustrte (. nd e.) nd numer of spores per unit hyphe length (c. nd f.). For revitions nd sttistics see Fig Dt ws squre root trnsformed efore sttisticl nlysis. At time of sweet potto hrvest (t 1 ), the hyphe length density in FT ws out 3.4 m cm -3 in [GI] nd out 2.8 m cm -3 in [GM] inoculted tretments (Fig. 4.7 d) nd therey comprised significntly higher hyphe length densities compred to [AS] tretments with up to 0.4 m cm -3. A similr difference etween fungl tretments ws shown for spore densities in the FT (Fig. 4.7 e). At (t 1 ) the numer of spores in FT of [AS] inoculted rhizooxes verged 33 ± 41 [U] nd 27 ± 23 [X] spores cm -3. The numer of spores per meter hyphe length ws similr etween [GI] nd [GM] ut much higher compred with tht of the [AS] tretment (Fig. 4.7 f). Soil disturnce did not hve significnt effect on the totl hyphe length, ut significntly decresed spore density in FT of the [GM] tretment (Fig. 4.7 d, e; Tle 4.7). A significnt 64

71 CHAPTER 4 interction etween AM fungl inoculum nd soil disturnce occurred in the cse of spore density in fungl tues (Tle 4.7) which ws relted to significntly decresed spore density in FT fter disturnce of the [GM] tretments only (Fig. 4.7 e). Tle 4.7: Two-wy-ANOVA results for the ERM development in fungl tues from the upper 4-10 cm t plnting (t 0 ) or t hrvest (t 1 ) of sweet potto plnts (see Fig. 4.7). For revitions nd sttistics see Tle 4.5. Dt ws squre root trnsformed efore eing nlysed sttisticlly. In cse mens elong to the smpling dte t 0, the fctor disturnce (D) ws not included in the ANOVA-nlysis, since dt were otined efore the set-up of the disturnce tretment. Interction M D M x D Hyphe length (m cm -3 sustrte) t 0 -/- -/- t 1 n.s. n.s. Spore density (nr. cm -3 sustrte) t 0 -/- -/- t 1 n.s. Numer of spores per unit hyphe t 0 -/- -/- length t 1 n.s. n.s. 4.5 Discussion Mize plnt colonistion nd growth in experimentl phse 1 During experimentl phse 1 (for ERM estlishment on mize plnts), lrge functionl differences were oserved etween the inocul utilised. The extent of root length colonistion ws clerly higher in [GI] nd [GM] tretments (60%) compred with [AS] plnts which hd low AM fungl root colonistion rte of out 25%. Similr results were shown y Douds et l. (1993), who reported AM fungl colonistion rtes of out 30% of field-soil inoculted, fourweek old mize plnts grown in the greenhouse. The oserved higher colonistion rtes of [GI] nd [GM] compred with [AS] inocul might indicte tht the Glomus inocul hd development dvntge due to etter dption to the experimentl soil conditions wherein the fungi hve een propgted efore. It might lso reflect the species specific growth pttern of the respective fungi in soil nd in roots, determining the inoculum potentil of fungus (McGee et l. 1999; vn der Heijden et l. 2006). AM fungl inocultion with [GI] nd [GM] incresed mize plnt growth nd totl P content out two- nd three-fold, respectively, compred with non-inoculted mize plnts. [AS] plnts showed similr iomss production ut contined significntly more P compred to the 65

72 CHAPTER 4 non-inoculted plnts. In the lst week of cultivtion, mize root growth on the cryl glss pltes of [GI] ws significntly higher nd tht of [GM] nd [AS] plnts ws y trend higher compred with non-inoculted plnts (see Fig 4.3). Also, P concentrtion in roots nd plnt totl P uptke ws incresed in the mycorrhizl plnts (see Tle 4.2). These findings indicte tht the P uptke of the mize plnts ws incresed y ll used sources of AM fungl inocultion compred with non-inoculted plnts fter the time period of seven weeks, nd tht the Glomus inocul incresed plnt growth more effectively compred to field soil inocultion [AS]. After the pre-cultivtion phse (t 0 ), the extent of verticl distriution of colonistion rte nd ERM undnce in fungl tues in the receiver comprtment depended on the utilised AM fungl inocul nd decresed in the order [GI] < [GM] < [AS]. Differences in fungl development led to distinct distriution ptterns nd spred intensities of the mycelium within the receiver root comprtments efore the mize root comprtment ws emptied. The following prgrphs will discuss the consequences for sweet potto AM fungl colonistion pttern nd mycorrhizl contriution to plnt growth fter the mycelium hd een detched from the former host y mize plnt removl Detched excised extr-rdicl mycelium s source of AM fungl colonistion The present experiment llowed for the uild-up of AM fungl colonistion from detched extr-rdicl myceli (ERM), nd this successfully enled AM root colonistion of sweet potto plnts. As eing removed from the neighour comprtment efore plnting of sweet potto plnts, AM colonised roots of mize plnts were not present during the experimentl phse 2. Therefore, intr-rdicl vesicles were sent, structures tht re considered s importnt propgules for Glomus species (Tommerup nd Aott 1981; Biermnn nd Lindermn 1983) nd usully re present in soil-sed AM fungl inoculum (Ijdo et l. 2011). Thus, the excised ERM in this study consisted only of spores nd hyphe. Consequently, root colonistion ws limited y the use of such structures serving s propgules. Not ll fungl species re le to estlish new root colonistion using hyphe frgments only: Representtives of the order Glomine were shown to estlish from hyphe frgments s well s spores, while memers of Gigsporcee depend solely on spores (Klironomos nd Hrt 2002). However, the present study does not llow distinction, whether hyphe frgments or spores were more importnt for susequent colonistion of sweet potto roots. Knowledge out species relted estlishment from extr-rdicl propgules in sence of mycorrhizl 66

73 CHAPTER 4 roots is scrce, nd the numer of studies on infectivity rtes of individul AM fungl structures still is limited (Tommerup nd Aott 1981; Biermnn nd Lindermn 1983; Klironomos nd Hrt 2002). Nevertheless, the excised mycelium llowed considerle colonistion rte of out 20-35% fter four weeks in sweet potto roots. This rte ws similr for oth the Glomus s well s the [AS] tretment. Considering the short time period, this colonistion rte is high compred to tht oserved in the study of O'Keefe nd Sylvi (1993), who otined similr vlues more thn eight weeks fter plnting of sweet potto in the field. In the present study, colonistion y mens of the excised ERM in ll used AM fungl inocul ws followed y cler improvement of sweet potto plnt growth. Therefore, it cn e stted tht the infection potentil of the excised ERM studied here ws high The growth response nd P uptke of sweet potto plnts in reltion to AM fungl colonistion Aout one third of the sweet potto plnt root length ws colonised irrespective of eing inoculted with the Glomus strins [GI], [GM] nd with field soil [AS]. The results reveled high response of sweet potto plnts to mycorrhizl root colonistion, since ll AM fungl inocul incresed drmticlly the growth of the host plnt. Totl P content of the plnt s well s P concentrtion in shoot nd root ws incresed y AM fungl inocultion (pproximtely two-fold in [GI] nd [GM] nd pproximtely 1.8-fold in the [AS] tretment). Accordingly, shoot P concentrtion in the dry mtter rnged etween 0.25 nd 0.30% in mycorrhizl sweet potto plnts compred to out 0.12% in non-inoculted plnts. This indictes tht noninoculted sweet potto plnts were clerly P-deficient (crop plnts re chrcterised s P- deficient with shoot P concentrtions elow 0.2% (Mrschner 1995)). The significnt enhncement of tissue P concentrtion from n insufficient to sufficient nutritionl P sttus due to AM fungl colonistion reflects the high mycorrhizl dependency of sweet potto plnts. A cler increse of net P uptke in inoculted plnts ws chieved within only four weeks in n erly plnt growth stge. Under conditions of restricted P vilility in soil, AM fungl colonistion my e very eneficil for sweet potto growth. Additionl P stored in plnt tissue my serve s resource for lter plnt growth, especilly t phses where plnt nutrient demnd is high, e.g. during rpid plnt growth or t storge-root formtion (O'Keefe nd Sylvi 1993). In the present study, AM fungl ruscules were oserved in roots of [GI], [GM] nd of field soil [AS] inoculted tretments, underlining the presence of functionl AM colonistion in ll mycorrhizl tretments. Both Glomus inocul led to clerly higher root colonistion rtes nd showed more thorough extension of root colonistion into the deeper soil sections compred 67

74 CHAPTER 4 to plnts inoculted with field soil [AS]. Accordingly, AM fungl contriution to sweet potto plnt growth nd net P uptke ws higher for oth Glomus compred with [AS] tretments. A similr pttern of AM fungl colonistion nd resulting plnt growth promotion y the respective AM fungl inocul ws lso oserved with the preceding mize plnts. Besides other indictors, the totl degree of hyphl extension in the soil volume is likely reflected y the root colonistion pttern ccording to depth. Therefore, n improved contriution to host plnt growth nd nutrient uptke might e ttriuted to higher sorptive surfce re of the fungl mycelium in the soil. Underlining this, AM fungl medited improvement of host plnt P nutrition cn not e predicted directly y the percentge of totl colonised root length (Jones et l. 1998; Burleigh et l. 2002) The growth pttern of the AM fungl extr-rdicl mycelium In order to estimte the extent of mycelium spred in the receiver sustrte, fungl tues (FT) were inserted into the Cpt B in two different soil depths. A considerle mount of ERM (2.4 to 6.3 mg per 25-ml-FT) ws hrvested from the Glomus inoculted plnts. Hyphe length ws etween 2.8 nd 3.4 m cm -3 sustrte which ws within the rnge reported y Hwkins nd George (2001) nd Hrt nd Reder (2002). The FT from the [AS] tretment contined reltively low mounts of spores (up to 30 per cm 3 ), similr to the mounts reported for griculturl field soil (Hymn 1970; Gosling et l. 2010) nd for nturl grsslnds (Oehl et l. 2003). Here, AM fungi of oth Glomus inocul produced significntly higher mounts of spores per unit hyphe length compred with fungi of the [AS] tretment. Typiclly, Glomus species (lso termed s r-strtegists ) hve higher specific spore density in the mycelium compred to the representtives of Gigsporcee (de l Providenci et l. 2005). It is likely tht AM fungi of the ltter fmily were lso prt of the field soil inoculum [AS] used in the present study. Representing C sources, spores my crucilly support lter fungl prolifertion into soil during the long term continution of the symiosis, s reported for Scutellospor isoltes (fmily Gigsporcee) (Gvito nd Olsson 2008). However, long-term effects or strtegies of ERM formtion in different AM fungi were not studied in the present experiment. During the experimentl phse 1, efore eing detched from their host plnt, AM fungi proliferted into the sustrte nd into FT of the plnt-free root comprtment Cpt B. The ERM in FT hrvested t the first smpling dte (t 0 ) represented the sptil spred into the sustrte s it ws chieved in sence of sweet potto plnts. At time t 0, ERM DW, hyphe length nd spore numer recovered from FT of Glomus inoculted comprtments ws pproximtely two- 68

75 CHAPTER 4 fold compred with those in the [AS] tretment. These differences etween the AM fungl inocul were consistent lso t the second hrvest (t 1 ) when AM fungi hve een ssocited with sweet potto plnts since four weeks. ERM mounts from FT estimted t time t 0 nd t 1 were not clerly different. However, mycelium usully gets lost y mens of turnover (Stddon et l. 2003), nd the proportion of vile mycelium is unknown ecuse it ws not estimted here. It is lso possile tht fungl growth fter the plnting of the sweet potto plnt ws predominntly concentrted on the colonistion of roots t the expense of the prolifertion into the sustrte. A simultneous support of oth ctivities would cost n inpproprite energetic effort for the fungus. After sweet potto roots ecme present in the receiver root comprtment, the mycelium spred in FT (locted next to the roots) my hve declined to void the close vicinity of roots. A preferred mycelium prolifertion into ulk soil tht is severl centimetres wy from the rhizosphere hs een demonstrted erlier (Mikkelsen et l. 2008). It hs een reported tht the extr-rdicl growth pttern nd resulting P uptke strtegies re different mong AM fungl species. Using lelled P the externl mycelium of Glomus isolte (fmily Glomercee) ws shown to hve tken up much P from root-distnt fungl comprtments nd contriuted most to plnt P supply, while n isolte of Scutellospor (fmily Gigsporcee) otined P predominntly from soil close to the host plnt root (Smith et l. 2000). AM fungl P delivery to host plnts is often highest for such AM fungi tht hve the highest mount of ERM in root-free soil (Jkosen et l. 1992; George et l. 1995; Smith et l. 2000). In ddition, AM fungl contriution to plnt P uptke ws lso positively correlted to hyphe length in specific fungl comprtments when compring different AM fungl species (Avio et l. 2006) or individul strins of one AM fungl species (Munkvold et l. 2004; Smith et l. 2004). Although the ERM prolifertion into the fungl tues ws low s oserved in the present study, [AS] inoculted sweet potto plnts reched two-third of the iomss nd totl P uptke compred with tht for the Glomus inoculted plnts. It is possile tht AM fungi contined in the [AS] inoculum hd higher P uptke efficiency compred to oth Glomus species, so tht ll mycorrhizl tretments resulted in clerly improved plnt growth compred with the non-inoculted tretment The effect of soil disturnce on the infectivity of the excised ERM Directly efore sweet potto plnting, in tretment [X] the sustrte of the receiver comprtment ws disrupted once y mens of cutting nd horizontl mixing of the sustrte. During this process, ll pre-defined verticl soil sections were mintined in the respective depth. 69

76 CHAPTER 4 The AM fungus showing the highest ERM DW in fungl tues ws [GI]. Despite prtly decresed root colonistion rte, this fungus ws pprently lmost unffected y the disturnce tretment, s ws shown t hrvest (t 1 ) where ERM DW nd hyphe length in fungl tues (FT) were similr irrespective of the disturnce tretment. In contrst, the inocultion with [GM] yielded lower mount of ERM DW in fungl tues fter soil disruption: ERM DW in deeper soil lyers nd spore density in [GM] tretments were significntly decresed. Concerning the formtion potentil of the externl mycelium susequent to soil disturnce, in pot experiments Dun et l. (2011) showed tht Glomus intrrdices is reltively insensitive, nd the uthors hve ttriuted the tolernce to very rpid estlishment of ERM spred from propgules in the soil. In the present study, the [GI] isolte showed higher extent of sptil spred in soil thn the [GM] isolte, s expressed y deeper root colonistion of oth experimentl plnts. Possily, more pronounced fungl prolifertion ehviour leds to lower susceptiility to soil disturnce. This my explin the reduction of mycelium DW in FT in [GM] tretments susequent to disruption, while the effect ws lcking for [GI] tretments. Nutrient uptke y the ERM from soil is the most direct contriution of AM fungi to plnt growth (Smith nd Red 2008). According to Olsen et l. (1999), mechnicl soil disruption induces reduced infection potentil of n AM fungl network, ecuse the estlishment y mens of disrupted extr-rdicl mycelium might need more C expenditures from the following plnt compred with n intct mycelium. In the present experiment it ws ssumed tht without soil disturnce ([U] tretments) the intct ERM would contriute more to plnt P uptke thn would distured fungi in the tretment [X]. This ws not oserved, i.e. sweet potto plnts colonised y the different AM fungl inocul showed similr iomss production nd P uptke irrespective of eing disrupted [X] or not [U]. The results therefore show no indiction of distinct differences in ERM estlishment from n intct compred with disrupted ERM network when inoculted with either Glomus species. The mount of the externl mycelium in fungl tues in [AS] ws very low, so tht n ERM disruption effect could perhps not e expected in this tretment. Former studies reported reduced AM fungl root colonistion rtes nd ERM growth fter the externl mycelium hd een disrupted y sieving the experimentl sustrte through meshes of 4 mm size or smller (Firchild nd Miller 1990; Jsper et l. 1991; Hrt nd Reder 2004), or y soil ploughing in field studies (Kir et l. 1997; Jns et l. 2002). However, negtive effects on the AM fungl colonistion rte tht hve een reported fter ploughing my lso e explined y the resulting effect of turning the soil 70

77 CHAPTER 4 verticlly. It is possile tht deeper soil sections, comprising lower microil ctivity nd fungl undnce, might overly the top soil s the min loction of plnt root ssocited AM fungl structures. The min undnce of AM fungl structures hs een oserved in the top first centimetres of the field soil profile (Oehl et l. 2005). This corresponds with Kir et l. (1998) who found AM fungl popultions to e gretest in up to 15 cm depth, nd the uthors stted tht ploughing to more thn 15 cm depth reduces propgule density in the rooting zone y dilution, nd therewith lso reduces mycorrhiz formtion. Less intensive opertions, where soil is loosened ut not turned re mngement methods such s disking, which is common method used in reduced tillge systems in griculture of the temperte zones (Cnnell 1985). In the present study, disturnce ws conducted modertely y mixing the sustrte y hnd, such tht lower or upper sections were not dislocted or diluted. This opertion in some respect simulted cultivtion method typiclly used in reduced tillge systems. The results of the present study indicte tht ll used inocul comprised high potentil to overcome moderte mechnicl soil disturnce, nd the outcome of the AM symiosis ws not ffected y this intervention. In cses where the density of vile spores in soil is low, differences in spore numers my ecome more importnt for those AM fungl species tht re fully dependnt on spores s propgules. An insufficient presence of infective spores might occur fter certin crop mngement ctivities such s ploughing or fter crop plnt hrvest t n erly stge of AM symiosis, where AM fungl hyphe prolifertion could still e higher thn spore formtion. In such situtions, mycorrhizl infectivity my depend more on the presence of infective mycorrhizl root frgments or on n intct ERM s hs een suggested erlier (Jns et l. 2002; Hrt nd Reder 2004). Furthermore, fter repeted soil disturnce nd cultivtion of crops tht mture within short time periods, AM fungl species with chrcteristic lte nd less intense spore development might lose competitiveness compred with species tht complete their life cycle within shorter time period. This mtches with the field study of Oehl et l. (2003), who demonstrted in intensively mnged gro-ecosystems selection for species forming spores rpidly, nd lso the intensity of lnd use hs een negtively correlted with AM fungl species richness. Accordingly, Glomus species were predominntly present in intensely tilled fields, while representtives of the Gigsporcee fmily were more prevlent in non-tilled soils (Jns et l. 2002). 71

78 CHAPTER Root distriution with depth Besides the totl plnt root iomss of sweet potto, the root DW distriution in four different verticl soil sections ws mesured. A chnged shoot-to-root DW-rtio towrds higher shoot iomss ws oserved, s it usully occurs due to mycorrhizl infection (Bert et l. 1995), ut lso root morphologicl fetures were chnged. In non-inoculted plnts, root DW ws out 50% oth in the upper 0-21 cm nd ottom cm soil depths, respectively. In these tretments, roots were more evenly distriuted cross ll depths compred to those of [GI] nd [GM] inoculted plnts with their root DW predominntly locted in the upper hlf of the sustrte (out 70%). Concomitntly, in the ottom soil (29-36 cm) fungl root colonistion ws clerly low in [GI] nd [GM] tretments, nd root iomss ws significntly lower thn tht of non-inoculted plnts. Apprently, the inocultion with oth Glomus isoltes shifted the verticl root distriution towrds the upper soil sections, possily ecuse the upper 21 cm contined the min prt of the AM fungl colonised root length. A continuous P delivery through the fungl network to roots my hve occurred minly in this soil section nd therefore induced root rnching therein. This pttern of response is consistent with the suggestion mde y Helgson nd Fitter (2009): When AM fungi trnsfer P to the plnt cross the rusculr memrne, there will e locl increse in P concentrtion in the root. Thus, the plnt will unlikely distinguish fungl medited trnsfer from tht tken up y the plnt itself vi the root epidermis. Therefore, the plnt will respond y differentil trnsport of hexoses to the site of incresed P uptke, nd consequently the root rnching nd growth in the section will e incresed (Helgson nd Fitter 2009). However, n exct experimentl proof for this interprettion is still lcking. At lest it is known tht n individul plnt root system increses rnching nd consequently lso the numer of lterl roots within soil ptches contining higher P concentrtion levels (Drew 1975; Lyons et l. 2008) Conclusions Directly fter detchment from former host plnt the ERM of the studied AM fungi showed high inoculum potentil on new plnt roots, reflected y n erly root colonistion of sweet potto plnts. After reltively short cultivtion period, sweet potto cuttings enefited enormously from AM fungl colonistion, s ll AM fungi led to drmtic increse of plnt growth nd P uptke. According to the outlined hypothesis, the fungl species tht showed the highest sptil ERM spred in soil contriuted most to the growth of susequent plnt fter the estlishment of the symiosis. The inocultion with G. mossee nd G. intrrdices showed the highest extents 72

79 CHAPTER 4 of externl mycelium undnce in root distnt sustrte, nd the outcome of the symiosis with oth Glomus isoltes ws more eneficil to the host plnt in terms of plnt P uptke nd growth promotion thn with the AM fungi from the field soil inoculum. Despite their lower extension of ERM in soil, the AM fungi from the field soil cused similr extent of totl root colonistion rte in sweet potto compred with the Glomus species, nd significntly incresed plnt growth, showing high specific P uptke nd trnsfer efficiency. The results support former studies, indicting tht eneficil outcome of the ssocition with certin AM fungus cn e predicted rther y the AM fungl specific P trnsfer efficiency thn y the extent of the totl root colonistion rte. It hs een hypothesized tht mechnicl disturnce of n excised AM fungl mycelium reduces the susequent fungl contriution to plnt growth or P uptke compred with n intct mycelium. In contrst to the outlined hypothesis, soil disturnce in most instnces did not ffect root colonistion nd in no cse ffected nutrient uptke or growth of newly colonised plnt. This model study indictes tht s long s the density of fungl propgules in soil is sufficient, non-turning, moderte soil mngement prctices used in reduced tillge systems my not ffect the inoculum potentil of AM fungi nd their following estlishment. 73

80 Chpter 5 5 AM fungl sporultion within ded trp roots Spore quntities nd distriution pttern 5.1 Astrct The pttern nd density of AM fungl sporultion within ded roots ( trp roots ) excised from different plnt genotypes (host or non-host) ws estimted in regrd to root dimeter nd to thickness of trp root lyers. Experiments were conducted y inserting trp root comprtments into the sustrte of precultivted mize nurse plnts inoculted with Glomus mossee to otin n infective AM fungl extr-rdicl mycelium [vileam]. Comprtments contined either trp roots, or n empty spce s control, nd were covered with 30 µm mesh memrne to llow fungl mycelium ut not plnt roots to enter. Non-infective [dedam] tretments were otined y killing the mycorrhizl, pre-cultivted nurse plnts y shoot removl efore trp root comprtments were inserted into the pots. Trp roots from the non-infective [dedam] tretments were free from AM fungl structures. In the infective [vileam] tretments, fter two-week incution period, AM fungl spores were oserved on the surfce nd inside the cortex of trp roots, irrespective of the genotypic origin. The clculted spore density per unit trp root volume ws up to spores per cm 3. It surmounted tht of similr volume of the colonised sustrte y more thn hundred-fold. No sporultion occurred in the spce etween empty nylon meshes of control comprtments. When similr totl length of corse nd fine trp roots were provided, higher percentge of corse trp root length (dimeter > 150 µm) contined spores compred with finer roots. AM fungl hyphe nd spores were oserved in trp root lyers of up to 5 mm in thickness. The results indicte tht ded roots cn ttrct AM fungl growth nd sporultion, possily ecuse they function s nutrient source or supply protected spce. This demonstrted ility to yield spores within ded roots my represent potentil technique to otin AM fungl spores in low-weight, orgnic crrier mteril. In ddition to this discovery, method for the fst nd simple quntifiction of spores nd vesicles contined in trp roots ws developed. 74

81 CHAPTER Introduction Arusculr mycorrhizl fungi form symiotic ssocition y colonising the corticl cells of plnt prtner, however the symiosis is not restricted to the intr-rdicl spce, with hyphe extending out through the rhizosphere nd eyond into the distnt soil hitt. Once into this extr-rdicl spce, the fungl mycelium spreds nd rnches within the ulk soil producing spores continuously during the whole growth period. Formed from cytoplsm nd storge lipids nd protected y thick-wlled cell memrne, spores hve longer life-spn thn hyphe nd re le to lst severl yers in the soil nd overcome dverse iotic nd iotic conditions (Brundrett 1991). By these mens, spores ply n importnt role s supportive structures for the estlishment of new colonies. To gret extent, AM fungl development is influenced y the nutritionl sttus of the host plnt. An elevted plnt phosphorus demnd (while other nutrients re not limited) cn led to incresed fungl root colonistion, higher development of extrrdicl mycelium (ERM) nd consequently elevted spore production (Verkde nd Hmilton 1983; Douds nd Schenck 1990; Douds 1994; Sito et l. 2011). Moreover, AM fungl spore production during the symiosis could e modulted directly ccording to cron derived from the host plnt (Ijdo et l. 2010). Thus, irrespective of the host plnt cron supply, different AM fungl species hve specific extr-rdicl colonistion strtegies. For exmple, memers of the suorder Glominee estlish colonistion from spores, vesicles nd hyphe frgments, while most memers of Gigsporinee were oserved to use only spores s propgules (Biermnn nd Lindermn 1983; Klironomos nd Hrt 2002). Besides sporultion in ulk soil, different Glomus species were lso oserved to sporulte within empty seed cvities nd glss cpillries (Ter 1982; Rydlov et l. 2004), in ded spores of AM fungi (Koske 1984), in nemtode cysts (Frncl nd Dropkin 1985), in nodules of legumes (Vidl-Dominguez et l. 1994) nd in root frgments (Dniels-Hetrick 1984). Concentrted AM fungl spores cn usully e found within the decomposing root frgments contined in commercil inoculum (own oservtion). However, it could not e determined whether these spores in the root residues were emplced there directly or whether they were former vesicles, lter trnsformed into spores fter the deth of the host plnt. Presently, it is not well understood wht fctors might stimulte or induce sporultion into hollow odies. Rydlov et l. (2004), who oserved spore gglomertion within glss cpillries or empty seed cvities, suggested tht AM fungi my seek shelter to elude predtors such s soil insects. Ded roots re uiquitous in soils, for exmple s residues from root turnover in nturl ecosystems or s post-hrvest remins of min nd cover crops in griculturl fields. When deposited 75

82 CHAPTER 5 within root frgments, AM fungl spores my e etter protected ginst some unfvourle iotic conditions or ginst insect feeding. To dte, neither AM fungl spore colonistion pttern nor spore colonistion quntities within root frgments ( trp roots ) hve een descried, the present study thus ims to fill this knowledge gp. The first ojective of this study ws to ssess the effect of the genotypic origin of trp roots (host vs. non-host species) on sporultion intensity within the trp roots, nd to compre this with sporultion within the ulk soil. It ws hypothesised tht trp roots from either host or non-host plnts will e colonised to similr extent y AM fungl spores nd externl hyphe. Therefore, the sporultion quntity within roots excised from non-host species (rmc tomto plnts nd of Pk Choi) ws compred with tht in host species (wild-type tomto nd Tropeolum mjus). Under the ssumption tht root frgments serve s hollow spces ttrctive to AM fungl prolifertion, the trp root geometry ws lso tken into ccount. The different trp root genotypes differed to significnt extent in their volume per unit length nd therefore this criterion ws estimted nd relted to the sporultion intensity. It ws hypothesised tht higher frequency of sporultion will occur in corse compred to thin trp roots. A second ojective in this study ws to estimte AM fungl prolifertion nd sporultion intensity within different lyers of trp root mteril. Mycelium growth hs een reported to e more dense in sustrtes distnt from the rhizosphere compred to sustrtes within the rhizosphere (St- Arnud et l. 1996; Mikkelsen et l. 2008; Neumnn et l. 2009), indicting mycelium growth into soil volumes likely occurs in the sence of living plnt roots. Some uthors oserved AM fungl hyphe colonistion of different types of orgnic mtter (Wrner nd Mosse 1980, Hepper nd Wrner 1983). Lrge gglomertions of ded root mteril occur, for exmple in grsslnds where roots re dying-off for sesonl resons (winter or drought period) or ecuse of shoot removl y hrvesting or niml grzing. In temperte mountin grsslnds, within the first ten centimetres of soil depth, the iomss of ded roots cn ccount for hlf of tht of living roots (Puchet et l. 2004). Accumulted ded roots my represent considerle prt of nturlly undnt elowground orgnic mtter nd could possily e colonised y the ERM. It ws hypothesized tht AM fungl hyphl growth nd sporultion is not restricted to single root frgments scttered in ulk soil ut lso prolifertion of ERM occurs into dense lyers of trp roots. AM fungl colonistion within ptches (comprtments) filled with trp roots could e used to yield mycorrhizl hyphe nd spores in n orgnic crrier mteril free from solid sustrte. To test the hypotheses, comprtments contining trp roots were constructed nd inserted into the sustrtes of mycorrhizl plnts. AM fungl ERM growth nd sporultion intensity within trp 76

83 CHAPTER 5 roots were compred with nurse plnt root colonistion nd spore density within the growth sustrte. A method for fst nd convenient quntifiction of spores nd vesicles contined in trp roots ws developed. 5.3 Mterils nd Methods Nurse plnt pre-cultivtion nd AM fungl inocultion Experiment 1: Seeds of Ze mys (L.) Gold were germinted in the drk in sturted CSO 4 solution. Seedlings with fully estlished primordil lef were trnsplnted into one litre plstic plnting pots (TEKU-Tiner; Pöppelmnn, Germny) contining 1.3 kg of het sterilised (85 C for 48 h) dry sustrte (for soil properties, preprtion nd fertilistion see Chpter 2.1). One plnt ws grown per pot. All plnts were inoculted y mixing 10% (w/w) AM fungl inoculum of Glomus mossee (Glm IFP S/08; INOQ GmH, Schneg, Germny) with the sustrte. After plnting, wter content in the sustrte ws mintined t 18% (w/w) y irrigtion with deionised wter. Once week wter loss ws clculted grvimetriclly nd from this mesurement dily wter loss ws estimted. The plnts were grown under greenhouse conditions for 49 dys etween April nd My. Throughout the growth period the dy/night temperture verged 24/19 C nd the men reltive ir humidity ws 64%. Experiment 2: Seeds of Ze mys (L.) Gold were germinted in the drk in sturted CSO 4 solution. Three weeks fter germintion seedlings were trnsplnted into 2 L plnting pots (TEKU-Continer BC 17; Pöppelmnn, Germny) contining 3.4 kg of wshed nd het sterilised (85 C for 48 h) dry qurtz snd (prticle size 1-2 mm). Plnts were fertilised once week with nutrient solution (ph 6.8) contining the following elementl concentrtion: 9 mm N (C(NO 3 ) 2 nd NH 4 NO 3 ); 0.7 mm P (KH 2 PO 4 ); 6 mm K (K 2 SO 4 ); 3 mm C (C(NO 3 ) 2 nd CSO 4 ); 1.2 mm Mg (MgCl 2 ); 4 mm S (CSO 4 nd K 2 SO 4 ); 80 µm Fe (Fe-EDTA); 40 µm B (H 3 BO 4 ), 7 µm Mn (MnSO 4 ); 6 µm Zn (ZnSO 4 ); 0.7 µm Cu (CuSO 4 ) nd 0.05 µm Mo ((NH 4 ) 6 Mo 7 O 24 ). Wter content in the sustrte ws mintined t 20% (w/w) y irrigtion with deionised wter. Two mize plnts were grown per pot. All plnts were inoculted y mixing 5% (w/w) AM fungl inoculum of Glomus mossee (Glm IFP S/08; INOQ GmH, Schneg, Germny) with the growth sustrte. The plnts were grown under greenhouse conditions for 95 dys etween June nd August. Throughout the growth period the dy/night temperture verged 25/19 C nd the men reltive ir humidity ws 70%. Experiment 3: Seeds of Ze mys (L.) Gold were germinted nd pre-cultivted s descried in Experiment 1. All plnts were inoculted y mixing 10% (w/w) AM fungl inoculum of self- 77

84 CHAPTER 5 propgted Glomus mossee BEG 12 (Schenck & Smith) with the growth sustrte. The single strin inoculum used for plnt inocultion ws self-propgted on mize using the sme experimentl sustrte (see Chpter 2.5) nd consisted of AM fungl colonised roots with surrounding growth medium contining spores nd hyphe. After plnting, wter content in the sustrte ws mintined t 18% (w/w) y irrigtion with deionised wter. Once week wter loss ws clculted grvimetriclly nd from this mesurement, dily wter loss estimted. The plnts were grown under greenhouse conditions for 45 dys etween Mrch nd April. Throughout the growth period the dy/night temperture verged 22/17 C nd men reltive ir humidity ws 68% Production nd preprtion of trp roots Experiment 1 nd 2: To otin trp root mteril, seeds of Solnum lycopersicum (L.) cv. RioGrnde 76R (WT); mycorrhiz-defective (rmc) mutnt tomto (Brker et l. 1998); Tropeolum mjus (L.) ( Monks Cress ); Brssic rp (L.) ssp. chinensis ( Pk Choi ) nd Chloris gyn ( Rhodes grss ) were germinted in the drk in sturted CSO 4 solution. Seedlings were trnsferred to n erted nutrient solution (ph 6.8) contining the following elementl concentrtion: 5 mm N (hlf C(NO 3 ) 2, hlf NH 4 NO 3 ); 0.7 mm P (KH 2 PO 4 ); 4 mm K (KH 2 PO 4 nd K 2 SO 4 ); 2.5 mm C (C(NO 3 ) 2 nd CSO 4 ); 1 mm Mg (MgCl 2 ); 4 mm S (CSO 4 nd K 2 SO 4 ); 10 µm Fe (Fe-EDTA); 10 µm B (H 3 BO 4 ), 5 µm Mn (MnSO 4 ); 1 µm Zn (ZnSO 4 ); 0.7 µm Cu (CuSO 4 ) nd 0.5 µm Mo ((NH 4 ) 6 Mo 7 O 24 ). The nutrient solution ws exchnged twice week. Prior to the experimentl use, the verge specific root length of four susmples (1 g fresh weight ech) ws determined y modified line intercept method (Newmn 1966). The verge root dimeter ws mesured y mens of ten rndomly chosen frgments within these susmples. Results re shown in Tle 5.1. Susmples were dried t 85 C for 48 h to estimte the dry weights necessry to determine the specific root length. Tle 5.1: Averge trp root dimeter nd specific trp root length of the trp root mteril prior to experimentl use. Root mteril ws otined from different plnt species, grown for 50 dys. Shown re mens ± SD, estimted on susmples of four replictes. Plnt species Averge root dimeter (µm) Specific root length (m g -1 DW) Experiment 1 S. lycopersicum 76R 332 ± ± 11 S. lycopersicum rmc 313 ± ± 8 T. mjus 338 ± ± 6 B. rp ssp. chinensis 171 ± ± 21 Experiment 2 C. gyn 221 ± ± 41 78

85 CHAPTER 5 To check for fungl contmintion, roots of ech plnt species were nlysed microscopiclly prior to experimentl use. Four susmples of the freshly hrvested root mteril were stined with trypn lue ccording to the procedure explined in Chpter 2.7 nd exmined microscopiclly (150 x mgnifiction). All root smples were free from fungl colonistion. The roots were dried (60 C; 48h) nd stored t room temperture until ppliction. Before ppliction, the plnt roots were cut into pproximtely 2 cm long pieces, sterilised y trnsfer into 70% ethnol for 30 seconds, nd remining ethnol ws removed y lotting the root surfce with pper towels. Prepred roots were inserted into the trp root comprtments s descried in the following section (Section 5.3.3). Experiment 3: To otin homogenously grown plnt roots free from fungl colonistion, roots of hydroponiclly grown cucumer plnts (Cucumis stivus L.) were used (De Kreij et l. 1997). The cucumer plnts were otined from the Institute for Ornmentl Crops in Großeeren where they were produced from Ferury to April 2010 in nutrient film chnnels supplied with stndrd nutrient solution. After three months growth in nutrient film, the root systems of the cucumer plnts hd formed long, flt nd intensely interwoven lyers within the chnnel. Sections (0.18 x 1 m) were cut from res of the roots which ppered to hve the most homogenous growth pttern (Fig. 5.2 c). Hrvested roots were wshed crefully in tp wter, ir dried t 40 C for 12 h nd stored t room temperture for further use. Five susmples of the hrvested fresh root mteril were stined with 0.05% trypn lue (ccording to the procedure explined in Chpter 2.7) nd microscopiclly exmined t 150 x mgnifiction. All root smples were free from fungl colonistion. Using four replicte smples of cucumer root mteril, the verge root dimeter nd specific root length (ccording to the line intercept method of (Newmn 1966) were estimted to e 248 ± 9 µm nd 267 ± 41 m g -1 DW, respectively Preprtion nd filling of trp root comprtments Experiment 1: Trp root gs were constructed from nylon memrne (30 µm mesh size), seled with silicone (Prou, Buhus AG, Germny). This construction llowed AM fungl mycelium ut not plnt roots to grow into the gs. Using strips of silicon, ech g ws closed t the edges nd sudivided into twelve comprtments of similr sizes (3 x 4 cm ech; see Fig. 5.1 c). The comprtmented trp root gs were 52 cm in length so tht they could fit round the circumference of the trnsplnted mize plnt root. When prepring the trp root comprtments, 100 mg (DW) of roots were rndomly selected nd put into ech comprtment. To test whether sporultion nd hyphe growth occur inside ir gps etween synthetic 79

86 CHAPTER 5 surfces, control comprtment ws creted y comining three lyers of nylon mesh (2 mm mesh size; 3 x 4 cm) nd seling it similr s the trp root comprtments. After filling, ll comprtments (trp root nd controls) hd thickness of pproximtely 3 mm. Ech trp root g contined eight trp roots comprtments of ll four genotypes nd four control comprtments (Fig. 5.1 c). Becuse of the higher specific root length of Brssic rp (L.) ssp. chinensis (see Tle 5.1), only 50 mg (DW) of root frgments were used to ensure consistency cross tretments. The prepred trp root gs were inserted immeditely into the plnting pots s descried in the following section (Section 5.3.4). Mycorrhizl nurse plnt Fungl ERM c L. esculentum WT L. esculentum rmc T. mjus B. rp ssp. chin. G G G G G = stcked nylon mesh lyers. Fig. 5.1: Illustrtion of the experimentl plnting units including trp root g (experiment 1).. AM fungl inoculted mize plnts were pre-cultivted to estlish n ERM network on their root system.. Colonised mize plnts were trnsplnted into igger pots where the upper prt of the trnsplnted root system ws surrounded y the comprtmented trp root g. c. Trp root gs contining trp roots of ll different genotypes (s indicted) or control (stcked nylon mesh lyers; G). Experiment 2: Single trp root comprtments, mesuring 3 x 4 x 0.3 cm ech, were constructed from 30 µm mesh size nylon memrne seled with silicone, s descried in experiment 1. A mss of 50 mg of dry C. gyn root frgments (prepred s descried in Section 5.3.2) ws filled into ech comprtment. Ech comprtment hd thickness of out 3 mm. 80

87 CHAPTER 5 Experiment 3: Trp root comprtments were constructed from plstic frmes mesuring 11 cm in length nd 3 cm in width. Three different comprtment volumes (S c 1, S c 2 nd S c 3) were constructed y vrying the frme depth which ws 0.3 cm, 1 cm or 1.6 cm (Fig. 5.2 ). Both the front nd ck sides of the open frmes were covered with nylon mesh (1 mm) nd nylon memrne (30 µm) (Sefr AG; Switzerlnd) llowing hyphe ut not roots to grow into the trp root comprtments. The memrne ws fixed with fungicide-free silicone selnt (Prou, Buhus AG, Germny). The comprtments were filled with lyers of cucumer roots (prepred s descried in Section 5.3.2). Prior to ppliction, roots were sterilised y trnsferring into 70% ethnol for 30 seconds nd retining ethnol ws soked with pper towels from the root surfce. The root mts were cut to fit the frme size of 11 x 3 cm. To prevent the two root lyers from sticking together, they were seprted y single lyer of glss eds (Ø 1-2 mm). The totl dry weights of the trp root mteril inserted into the [S c 1], [S c 2] nd [S c 3] comprtments, were 0.32, 2.0 nd 3.2 g, respectively, nd this trnslted into trp root densities of 8, 25 nd 60 mg (DW) cm -3, respectively Experimentl set-up nd growth conditions Experiment 1: Forty-nine dys fter germintion, when roots were tested positive for AM fungl colonistion, the root system together with the ttched sustrte of the pre-cultivted mize nurse plnts (Fig. 5.1 ) were removed from their former plnting pots nd trnsplnted into 2 L plnting pots (TEKU continer MXA 17; Pöppelmnn, Germny) contining 2 kg of het sterilised (85 C for 48 h) dry sustrte. For sustrte properties nd preprtion see Chpter 2.1. One plnt ws grown per pot. Trnsplnting the nurse plnts required 3 distinct steps. First, one third of the new sustrte ws filled into the ottom of the plnting pots to serve s underlyment for the trnsplnted root system of nurse plnt. Next, one filled trp root g ws wrpped horizontlly round the upper four centimetres of the plnt root system nd fixed with stinless steel needle. Finlly, the gp etween the root system nd the pot wll ws filled with the remining sustrte (Fig. 5.1 ). Fourteen pots were produced. Experiment 2: Two comprtments filled with trp roots were inserted into the upper 4 cm of ech pot sustrte when the nurse plnts were 95 dys old. Ten replictes were produced per tretment. Experiments 1 nd 2: To otin tretment contining ded AM fungi [dedam] ut with composition of microorgnisms similr to the mycorrhizl tretment [vileam], the shoots of the nurse plnts of four prepred pots were removed one dy prior to the trp root insertion. 81

88 CHAPTER 5 Experiment 3: After AM fungl root colonistion ws detected, the pre-cultivted mize plnts (nurse plnts) were trnsplnted into lck, round 2 L plstic plnting pots (TEKU continer MXA 17; Pöppelmnn, Germny) contining 2.4 kg of het sterilised (85 C for 48 h) dry sustrte (for sustrte properties nd preprtion, see Chpter 2.1). Two plnts were grown per pot. During pot filling, plstic rectngulr-shped dummy comprtment ws plced verticlly into the upper 6 cm of the sustrte, etween the two mize plnt root systems, in order to crete spce tht would lter e filled with the trp root comprtments. Trp root contining comprtments were fitted twenty eight dys fter trnsplnttion nd ech comprtment size ws replicted five times. To otin tretment contining ded AM fungi [dedam] ut with composition of microorgnisms similr to the vile mycorrhizl tretment, four extr nurse plnt pots were prepred in similr wy to the [vileam] tretments with the smllest trp comprtment size [S c 1], ut nurse plnts were killed y shoot removl one dy prior to the trp root insertion. Experiment 1, 2 nd 3: After their insertion into the experimentl pots, the trp root comprtments were incuted for 14 dys during continued cultivtion of the nurse plnts. Wter content in the pot sustrte ws mintined t 18% (w/w), nd dily wter loss to e replced ws estimted grvimetriclly twice wek. 82

89 CHAPTER 5 Mycorrhizl nurse plnts S c 1 trp root lyer S c 2 fungl ERM S c 3 fungl window (30 µm memrne) c glss ed lyer 5 cm Fig. 5. 2: View of the plnting units, trp root comprtments nd the rw root mteril used in experiment 3.. Side view of 2 L plnting unit contining two mize plnts nd one trp root comprtment uried in the sustrte, tretment [vileam].. Individul components of the comprtments fillings. In ech cse, one glss ed lyer seprted two trp root lyers. Single or stcked trp root lyers were filled into the comprtments to totl thickness of 0.3 cm [S c 1], 1 cm [S c 2] nd 1.6 cm [S c 3], respectively. Both sides of the trp root comprtments were covered y 30 µm, hyphe permele memrne. c. Photogrph (top view) of the flt mt formed y hydroponiclly grown, interwoven cucumer roots used s trp root mteril. These mts were cut into segments so s to fit into the inner frme re of the comprtment Hrvest nd quntifiction of AM fungl propgules in roots Experiment 1 nd 2: Trp root comprtments were removed 14 dys fter insertion nd t the sme time, nurse plnt roots were lso hrvested for nlysis. Nurse plnt roots nd trp roots were prepred nd stined s descried in Chpter 2.7. The stcked nylon mesh lyers from ech control comprtment were lso stined y the sme method efore eing exmined under the microscope for ny signs of AM fungi. For oth the nurse plnt roots nd trp roots, the percentge of AM fungl colonised root length ws quntified using modified gridline intersection method, s descried in Chpter 2.7. Spore density per unit root trp root length ws determined y counting the spore numer within ten rndomly chosen trp root segments of 3 mm length (using 50 x mgnifiction). The percentge of trp root length un-colonised nd colonised with intr-rdicl AM spores ws estimted in reltion to three root dimeter clsses: <150 µm, µm nd >300 µm. Four 83

90 CHAPTER 5 replictes of ech trp root genotype, consisting of three pooled trp root comprtments, tken rndomly, were exmined. The smples were distriuted homogenously onto glss plte contining n underlying grid (0.5 cm squres). When plnt root ws found to intersect the underlying grid lines, it ws t this point exmined. For ech smple, out 150 intersections were exmined under stereo microscope with trnsmitted illumintion (100 x mgnifiction). At ech intersection the root ws positioned underneth hirline micrometer, locted in the oculr, nd the size mesured. Concurrently, the percentge of trp root length colonised with intr-rdicl spores ws estimted seprtely for ech dimeter clss. In ddition, ll intersections were clssified seprtely s roots without (when free from AM fungl spores) or with spores (when contining AM fungl intr-rdicl spores). At lest 20 intersections per dimeter clss were exmined. Experiment 3: Nurse plnts were hrvested 14 dys fter the insertion of trp root comprtments into the growth sustrte. Nurse plnt roots s well s trp root mteril from the comprtments were nlysed for occurrence of spores. Trp root top (T) nd centrl (C) positioned lyers of the trp root comprtments [S c 2] nd [S c 3] were extrcted nd nlysed seprtely for ech replicte. AM fungl undnce in trp roots ws quntified using the following methods: i) Gridline intersection counting: Prior to the estimtion of colonistion rte nd spore density, roots were ir-dried t 60 C, weighed, nd susequent to stining, exmined under microscope s descried for experiments 1 nd 2 in Section In ddition, the proportion of intr- to extr-rdicl spores ws quntified. To chieve this, during estimtion of spore density, spores found within the cortex were counted seprtely from those ttched to the cortex surfce y hyphl connections. Nurse plnt (mize) root smples (pproximtely 1 g, n = 4) were tken rndomly from ech root system nd fter stining with trypn lue, AM fungl colonistion ws quntified (procedure s descried in Chpter 2.7). ii) Filtrtion method: The propgule quntity of trp roots nd nurse plnt roots were estimted fter reking up nd filtrting smples. Stined roots otined from the gridline intersection counting (see ove) were shred into pieces of less thn 0.5 mm length, in 300 ml of wter, using lender (Wring Blender 7009G, Wring, USA). By shredding, root cells were frctured nd AM fungl propgules contined therein were relesed. The resultnt suspension could esily e mounted on memrne filter which ws used for susequent microscopy, s descried in Chpter 2.4. For experiments 1 nd 3, representtive susmples from the sustrte were tken (pproximtely 200 g, n = 4). From these susmples, spores were extrcted y wet sieving (40 84

91 CHAPTER 5 µm mesh) nd then seprted from foreign prticles y centrifugtion (2000 rpm for 2 minutes) in 70% sucrose solution, following the methods of Gerdemnn nd Nicolson (1963). After stining with trypn lue for 24 h, the spores were mounted on memrne filter with 3 mm squred grid nd counted s descried in Chpter Sttisticl nlysis Provided tht results pssed the test for norml distriution (Kolmogorov-Smirnov test; p > 0.05) nd homogeneity of vrince (Levène test; p > 0.05), dt were sujected to one-wy ANOVA. The multiple comprison Tukey-test ws used to estimte differences etween mens. In oth tests, p vlues elow 0.05 were interpreted s indicting significnt effects. Dt which did not show homogeneity of vrince ws sujected to the Kruskl-Wllis-test (p < 0.05). Sttistic clcultions were conducted using SPSS softwre, version 15.0 (SPSS Inc., USA). Results in tles nd figures re presented s tretment mens ± stndrd devition. 5.4 Results Nurse plnt root AM fungl colonistion nd spore density in pot sustrte Experiment 1: Mize plnt root iomss fter hrvest verged 4.2 ± 0.8 g DW, with root density in the sustrte of 14 ± 5 cm per cm 3, nd specific root length of 72 ± 3 m g -1. The percentge of the totl root length colonised with Glomus mossee verged 83 ± 15%, with 24 ± 6% contining ruscules, nd 20 ± 5% contining intr-rdicl vesicles. The estimted spore density within the plnting pots verged 51 ± 11 spores per cm 3 of sustrte. Experiment 2: Mize plnt root iomss fter hrvest verged 18.4 ± 2.1 g DW per pot. The percentge of root length colonised y Glomus mossee verged 78 ± 26%, with 31 ± 9% contining ruscules nd 25 ± 3% intr-rdicl vesicles AM fungl colonistion nd sporultion in trp roots Experiment 1 nd 2: The comprtmented trp root gs were hrvested 14 dys fter insertion. Nurse plnt roots did not cross the 30 µm memrne so no root growth ws oserved in the trp root comprtments. The trp roots which were inserted into the [dedam] pots were free from AM fungl structures. In mycorrhizl [vileam] tretments, the control comprtments contining only nylon mesh lyers were free from AM fungl spores, ut some AM fungl hyphe growth cross the nylon mesh lyers ws oserved (Fig. 5.3 h, see rrow). 85

92 CHAPTER 5 c d e f g h Fig. 5.3: Microphotogrphs of trp roots fter 14-dys incution within the sustrte of pre-cultivted mize plnts inoculted with Glomus mossee in [vileam] tretments (experiment 1 nd 2). Lterl hyphe growth nd differently sized spores found etween corticl cells of:. Rmc tomto, nd. Tropeolum trp roots. c. Intercellulr hyphe growth longitudinl to the trp root cortex of T. mjus, s generlly found for ll studied trp root genotypes. d. Trp roots originted from Pk Choi with dimeters smller thn 150 µm, colonised extrrdiclly with spores nd hyphe. e. A corse trp root of C. gyn colonised intr-rdiclly y AM fungl spores. Stelr cylinders were frequently colonised y AM fungl spores, shown in dissected Tropeolum smple (f) nd in n intct C. gyn smple (g). h. Top view of nylon mesh lyer, excised from stined mesh control. Corse nd finely rnched hyphe were oserved to cross the mesh surfce ut no spores were found. All trp roots were free from ny fungl colonistion efore eing used for incution. Brs indicte 100 µm. 86

93 CHAPTER 5 In [vileam] tretments, hyphe growth of G. mossee (dimeter of hyphe 3 to 15 µm) ws oserved on the surfce nd long the trp root min xis (Fig. 5.3, see rrows), s well s etween corticl cells (Fig. 5.3 c). Hyphe were spred ll through the trp root tissue nd showed rnching, nd occsionlly, inter-connection y h-ridges. G. mossee developed thick-wlled, ovl or gloose shped spores (dimeter up to 150 µm) within the cortex (Fig. 5.3, nd e). To lesser extent gloose shped spores were lso oserved outside of the trp root tissue (Fig 5.3 d) nd within the stelr cylinder (Fig 5.3 f nd g). No ruscules were found in trp roots. AMF hyphl colonistion (% of trp root length) R tomto rmc tomto Tropeolum Pk Choi AMF intr-rdicl spore colonistion (% of trp root length) c Rtio of percentge hyphe-to-spore colonised trp root length R tomto rmc tomto Tropeolum Pk Choi 76R tomto WT tomto rmc Tropeolum T. mjus Pk Pk Choi Tomto Fig. 5.4: Percentge of AM fungl colonised trp root length nd rtio of percentge hyphl-to-spore colonistion (Experiment 1). Trp roots from different plnt genotypes (nmed on x-xis) were inserted for 14-dy period into sustrte contining pre-cultivted mize plnt inoculted with the AM fungus Glomus mossee.. Percentge of root length with AM fungl hyphe colonistion.. Percentge of root length colonised with intrrdicl spores. Here, spores outside of the root cortex were not counted. c. Rtio of percentge hyphl-to-spore colonised root length. Brs represent mens ± SD. Different letters indicte significnt different mens (multiple comprison Tukey-test, p < 0.05; n = 4). 87

94 CHAPTER 5 Trp roots originted from wild-type tomto (host), Tropeolum (host) nd rmc mutnt tomto (non host), did not differ in colonistion rtes nd verged etween 55-80% for hyphl colonistion nd etween 20-30% for intr-rdicl spore colonistion (Fig. 5.4 nd ). For Pk Choi (non host), the trp root length colonised with spores ws up to 12% which ws significntly lower (Fig. 5.4 ) thn for the other trp root genotypes. In ddition to this, the pttern of colonistion in Pk Choi ws different thn in the other genotypes. The cortex of Pk Choi trp roots were colonised with mny spores nd hyphe (see Fig 5.3 d), while intrrdiclly, hyphe dominted nd reltively few spores were present. This pttern ws lso reflected in the high rtio of hyphe-to-spores in Pk Choi roots (Fig. 5.4 c). The spore density per unit length of trp roots originted from Pk Choi ws lower thn for the other trp root genotypes (Fig. 5.5 ). A similr result ws lso oserved for spore density per unit trp root DW (Fig. 5.5 ). AMF intr-rdicl spore density (no. cm -1 trp root length) R tomto rmc tomto Tropeolum Pk Choi AMF intr-rdicl spore density (no. mg -1 trp root DW) R tomto rmc tomto Tropeolum Pk Choi WT rmc T. mjus Pk Choi tomto Fig. 5.5: Intr-rdicl spore densities per unit trp root (Experiment 1), estimted s. per cm trp root length nd. per mg trp root DW. Brs represent mens ± SD. Different letters indicte significnt different mens (multiple comprison Tukey-test, p < 0.05; n = 4). In [vileam], the trp roots of C. gyn hd specific root length of 221 ± 155 m g -1 nd n verge totl length of 11 ± 0.3 m per comprtment. The totl AM fungl colonised length of trp roots verged 39 ± 4%, with 13 ± 2% intr-rdicl spores. 88

95 CHAPTER 5 Within distinct root dimeter size clsses, the totl trp root length in the comprtment, the trp root length colonised with AM intr-rdicl spores, nd the trp root length not colonised with AM intr-rdicl spores, were estimted for ech genotype used in experiments 1 nd 2. Since oth wild-type nd rmc tomto trp roots were similr in their fetures nd were colonised in similr wy with AM fungl structures, dt for rmc roots re not shown ny further. Roots were seprted into three dimeter size clsses, defined s <150 µm (fine), µm (intermedite) nd >300 µm (corse) (Fig 5.6 -d). Tomto roots predominntly consisted of dimeters lrger thn 150 µm (Fig 5.6 ) with very low proportion of roots eing thinner thn 150 µm. This trend could lso e oserved in T. mjus trp roots (Fig 5.6 ) which were completely lcking the finest root clss. In contrst to this, trp roots originted from Pk Choi nd C. gyn showed reltively high proportion of thin dimeter clsses (37% nd 30% of totl length in comprtment; Fig. 5.6 c nd d). C. gyn trp roots exhiited reltively homogenous distriution pttern of ll size clsses. The percentge of trp root length colonised with intr-rdicl spores (Fig 5.6 e-h) ws low in fine trp roots nd mrkedly incresed with incresing dimeter size. This trend ws oservle in ll studied genotypes, except for T. mjus trp roots which lcked of the thinnest dimeter size clss (Fig. 5.6 f), ut ws most pronounced in Pk Choi nd C. Gyn (Fig. 5.6 g nd h). Both these genotypes, who exhiited root size clsses in pproximtely the sme dimensions, showed tht up to four times more corse trp root length ws colonised with intr-rdicl spores thn for the fine clss. 89

96 CHAPTER WT tomto men clss prtition of totl root length (m/ trp ptch) e WT tomto men % prtition spore colonized within dim. clss Totl trp root length (m per comprtment) c ø (µm) <= >=310 T. mjus men clss prtition of totl root length (m/ trp ptch) ø (µm) <= >=310 Pk Choi men clss prtition of totl root length (m/ trp ptch) Trp root length colonised with AM fungl intr-rdicl spores (%) ø (µm) <= >=310 f g men % prtition spore colonized within dim. clss ø (µm) <= >=310 men % prtition spore colonized within dim. clss T. mjus Pk Choi c Exp. 1 Exp. 2 Totl trp root length (m per comprtment) d ø (µm) <= >=310 C. gyn clss prtition of totl root length (m/ trp ptch) ø (µm) <= >=310 < 150 µm µm > 300 µm Trp root length colonised with AM fungl intr-rdicl spores (%) h ø (µm) <= >=310 men % prtition spore colonized within dim. clss C. gyn ø (µm) <= >=310 < 150 µm µm > 300 µm Fig. 5.6: Totl trp root length (m per comprtment) (figures on the left) nd percentge of trp root length colonised with AM fungl intr-rdicl spores (figures on the right). Vlues were estimted for ech root dimeter size (see x-xis). Trp roots were excised from the plnt species indicted on top of the respective digrm. Brs represent mens ± SD. Different letters (figures on the right) indicte significntly different mens (multiple comprison Tukey-test, p < 0.05; n = 4). Dt were squre root trnsformed prior to sttisticl nlysis. 90

97 CHAPTER 5 Experiment Nurse plnt root AM fungl colonistion nd spore density in pot sustrte At hrvest 14 dys post trp root insertion into sustrte, nurse plnt root dry mtter verged 10.4 ± 0.7 g per pot. The percentge of root colonistion with Glomus mossee verged 71 ± 4% of which 21 ± 2% included ruscules nd 19 ± 3% intr-rdicl spores nd vesicles. The estimted spore density within the pot sustrte verged 34 ± 14 spores per cm³ AM fungl colonistion nd sporultion in trp roots Susequent to the incution within the pot sustrte, s intended, no nurse plnt roots penetrted the fungl windows covered y the 30 µm memrne. The trp root mteril inserted into the control pots contining the killed nurse plnts [dedam] ws free from fungl structures such s spores or hyphe. In mycorrhizl tretments [vileam], hyphe of G. mossee were found on surfces nd within the trp root cortex, nd spores were found within nd outside of the cortex (Fig d). More thn doule the mount of spores were locted inside the inner root tissues compred with outside. Irrespective of the trp comprtment size [S c 1-3], the rtio of extr- to intr-rdicl spores verged 0.34 ± 0.09 in the top lyer. Spores were up to 120 µm in dimeter nd were shped differently ccording to their loction, e.g. round spores were developed outside of roots, round or ovl spores were oserved etween corticl cells nd only ovl spores were oserved within stelr cylinder tissue (Fig. 5.7 d). No ruscules were found in the trp roots. 91

98 CHAPTER 5 d es eh is c eh e S c ih R eh H Fig. 5.7: Microphotogrphs of the cucumer trp roots fter 14-dys incution within the sustrte of mycorrhizl mize plnts inoculted with Glomus mossee, [vileam] tretments (experiment 3). Trp roots efore (.) nd fter (.) stining showed intense colonistion with AM fungl spores nd hyphe. Frequently, extr-rdicl spores (es) nd hyphe (eh) were oserved on the surfce of trp roots. c. Stined trp roots showed intr-rdicl hyphe (ih) growth longitudinlly long the root xis, nd externl hyphe (eh) connections etween trp roots. d. Spores were lso locted within the stelr cylinder (is c ) of cucumer roots. Spore dimeter vried nd ws up to pproximtely 120 µm. e. Using the filtrtion method for spore quntifiction, stined trp roots were frgmented in lender nd therefter, extrcted spores (S), hyphe (H) nd root cell residues (R) were mounted on gridded memrne filter. All trp roots were free from ny fungl colonistion efore eing used for incution. Brs indicte 200 µm. The percentge root length colonised with AM ws estimted vi the gridline intersection counting method. Top (ll comprtments) nd centrl lyers (only [S c 2] nd [S c 3]) were counted seprtely. (Fig. 5.8 nd ). Therefore, the top lyers (T) were tken from the first 3 mm of the inserted root mteril in every cse, while the centrl lyers (C) were otined from 5 mm [S c 2] nd 8 mm [S c 3] depth. Approximtely hlf of the root length ws colonised with AM fungl hyphe in (T) nd this did not differ significntly etween comprtment sizes (Fig. 92