Control of Pythium wilt and root rot of hydroponically grown lettuce by means of chemical treatment of the nutrient solution.

Transcription

1 Control of Pythium wilt nd root rot of hydroponiclly grown lettuce by mens of chemicl tretment of the nutrient solution. by Roger Bgnll Submitted in prtil fulfillment of the requirements for the degree Mgister Scientie (M.Sc.) in the Fculty of Nturl & Agriculturl Science University of Pretori Pretori April, 2007 University of Pretori

2 Control of Pythium wilt nd root rot of hydroponiclly grown lettuce by mens of chemicl tretment of the nutrient solution NAME: SUPERVISOR: R. Bgnll Prof. N. Lbuschgne CO-SUPERVISOR: Prof. T.A.S Aveling DEPARTMENT: DEGREE: SUMMARY: Microbiology nd Plnt Pthology M.Sc. (Plnt Pthology) Hydroponic production ws initilly explored s n lterntive to field production due to the ese of plnt growth control nd the hopes of preventing the mjority of disese cusing gents known to be present in generl soil environments. Of primry concern in terms of pthogens re the wter-borne nd wter-motile zoosporic fungi (especilly Pythium spp.) which re ble to spred esily throughout the system nd cuse root-rot nd wilting. Few pesticides re currently registered for use in hydroponic systems due to the high costs of registrtion, while registered pesticides crry high cost to the grower. Recent legisltive moves by numerous countries re lso resulting in trend towrds the re-use of hydroponic nutrient solution. As result such hydroponic solutions require greter level of disinfection to prevent disese outbreks but without resulting in chemicl buildup of phytotoxic nd environmentl concern. Snitiser formultion hs seen significnt chnges over the lst few yers resulting in snitisers being used in mny new res nd in more environmentlly friendly nture. Although snitisers re not designed to hve specific ction ginst micro-orgnisms (s is the cse with fungicides nd nti-microbil gents such s ntibiotics), most snitisers re ble to ct on cell membrnes due to the inherent surfctnt properties. This study ttempted to determine the suitbility of vrious snitisers nd chemicls s lternte mens of control of Pythium in recirculting grvel hydroponic systems by: 1). Exposing Pythium zoospores in wter suspension to the snitisers Actsol, Agrl 90, Fitosn, Prsin, Purogene, TecsClor, Sporekill nd copper (s copper (I) sulphte) which ll mnged to eliminte 80% or more of the vible inoculum within 10 minute exposure time t reltively low concentrtions. 2). Testing the bove snitisers for phytotoxicity effects on cucumber plnts in sttic hydroculture system under lbortory conditions nd lettuce plnts in grvel bed hydroponic system under greenhouse conditions. Purogene nd TecsClor exhibited slight growth promotion effect t low concentrtions, yet still cused negtive phytotoxic effects when dosed t high concentrtions. All other snitisers exhibited some mesure of phytotoxicity, observed s growth retrdtion nd lef discolourtion, with phytotoxic effects incresing with incresing concentrtions. Copper sulphte ws found to be the most phytotoxic chemicl tested

3 3). Addition of the snitisers to smll scle hydroponic system (greenhouse), s well s to semi-commercil scle (field) grvel bed hydroponic system rtificilly infested with Pythium nd cultivted with lettuce. The snitisers were lso compred to commercilly vilble fungicide, Phytex. Only Phytex nd Purogene mnged to effectively reduce disese incidence nd promote growth over n untreted, Pythium infested control. The results indicted tht Purogene ws the most effective for ppliction into grvel bed hydroponic system cultivted with lettuce, while no snitiser tretment ws ble to equl the improved growth nd disese control recorded with tretment of the commercil fungicide Phytex. Although ll the snitisers were ble to reduce levels of Pythium inoculum in the hydroponic nutrient solution, this beneficil effect did not trnslte into incresed yields, due to the growth retrdtion due to phytotoxic effects

4 CONTENTS Contents Acknowledgements Declrtion i v vi Chpter 1: Generl Introduction Introduction Motivtion for study Aim of the Study Objectives References 4 Chpter 2: Literture Review Hydroponics Overview Diseses in hydroponic systems Pthogens Pythium Rlstoni Fusrium Snitisers References 21 Chpter 3: In vitro efficcy of wter snitisers ginst Pythium zoospores in queous suspension Abstrct Introduction Mterils nd Methods Mintennce of cultures Inoculum preprtion Snitiser preprtion Experimentl procedure Dt nlysis 34 i

5 3.4 Results Actsol Prsin Purogene TecsClor Fitosn Agrl Copper sulphte Sporekill Discussion References 51 Chpter 4: In vivo ssessment of phytotoxicity of snitisers on cucumber nd lettuce plnts Abstrct Introduction Mterils nd Methods Cucumber model Cucumber vriety nd germintion Sttic hydroculture Growth conditions Phytotoxicity ssessment Anlysis Lettuce model Lettuce vriety nd germintion Smll scle grvel bed hydroponic system Growth conditions Phytotoxicity ssessment Anlysis Snitiser preprtion Results Cucumber model Actsol Prsin Purogene TecsClor Fitosn Copper sulphte Sporekill 66 ii

6 4.4.2 Smll scle grvel bed hydroponic system (lettuce model) Actsol Prsin Purogene TecsClor Discussion References Plte I 74 Chpter 5: Control of Pythium wilt nd root rot of lettuce by mens of chemicl tretment of the nutrient solution in re-circulting hydroponic systems in the greenhouse nd field Abstrct Introduction Method nd Mterils Smll scle grvel bed hydroponic system (greenhouse model) Lettuce vriety nd germintion Design of smll scle grvel bed hydroponic system Growth conditions Yield nd infesttion ssessments Anlysis Semi-commercil scle grvel bed hydroponic system in the field Growth conditions Yield nd infesttion ssessment Anlysis Snitiser preprtion Results Smll scle grvel bed hydroponic system (greenhouse model) evlution of snitisers individully t rnge of dosge rtes Actsol Prsin Purogene TecsClor Comprison of different snitisers t optimum dosge rtes in the greenhouse Preliminry experiment Comprison of primry snitisers in smll scle grvel bed hydroponic system (greenhouse model) Comprison of dditionl snitisers in smll scle grvel bed hydroponic system in the greenhouse 88 iii

7 5.4.3 Tretment comprisons in semi-commercil grvel bed hydroponic field system (multi-snitiser tril) Comprison of snitisers in semi-commercil scle grvel bed hydroponic system in the field Discussion References Plte II 100 Chpter 6: Generl conclusion Discussion References 107 Appendix I 109 Summry 112 iv

8 Acknowledgements I would like to thnk the following people: Prof Terry Aveling for ssisting beyond wht should be expected nd for constnt support nd encourgement. Wilm du Plooy, Andrew Gllgher nd Veloshinie Govender for their motivtion nd encourgement, s well s their help in terms of editing nd vluble input t criticl times. The compnies: SIDL BTC Products nd Services Rdicl Wters Helth & Hygiene for their prticiption in this project, for mking the products nd their expertise vilble, nd for dditionl funding of this study. Hydrotech nd its employees for the id in building the semi-commercil scle model nd during plnting nd hrvesting. THRIP nd NRF for iding in the funding of this reserch. v

9 DECLARATION I, the undersigned, declre tht the disserttion, which I hereby submit for the degree of Mster of Science t the University of Pretori, is my own work nd hs not previously been submitted by me for degree t this, or ny other, university Nme: Signture: Dte: vi

10 CHAPTER ONE GENERAL INTRODUCTION 1.1 Introduction Hydroponics nd soilless cultivtion systems of plnt production re used worldwide to grow flower, folige, bedding nd vegetble crops (Crruthers, 2002; Song et l., 2004). Certin crops cultivted in this mnner re of significnt economic importnce (Pulitz et l., 1992). Plnts re grown using nutrient solutions, with or without solid substrtes for root growth (Song et l., 2004). The nutrient solution cn either be re-circulted in closed system or drined fter use in n open system. Hydroponic systems hve become populr over the lst 20 yers ll over the world for the growth of high-vlue crops in glsshouses (Svvs et l., 2002). Use of hydroponic cultivtion systems in greenhouses offers unique sitution tht my mke conditions more fvourble for diseses. A hydroponic culture system is esily infected by soil-borne pthogens such s Fusrium nd Pythium spp. (Schwrz nd Grosch, 2003). Pthogens cnnot be completely excluded from the greenhouse environment. In hydroponic systems, Pythium zoospores re relesed from infected roots into the nutrient solution, where they re dispersed throughout the hydroponic system (Pulitz et l., 1992; vn West et l., 2003). Airborne spores enter through doors nd screens, soil-borne pthogens enter through dust or contminted soil on shoes, tools, or equipment, nd some pthogens re introduced on seeds or contminted propgting mterils (Pulitz, 1997; Schwrz nd Grosch, 2003). Fungl gnts hve lso been reported s probbly the most importnt vector of root pthogens (Stnghellini et l., 1996) - 1 -

11 Methods of control include the ppliction of systemic pesticides vi the nutrient solution in closed soilless culture systems (Wood nd Ling, 1992, s cited in Krrs et l., 2006). Other methods include the use of mono-potssium phosphtes (Reuveni et l., 2000). However, the pthogen resistnce to most pesticides mkes this solution temporry. Current methods of steriliztion such s ozontion nd the use of ultr-violet light re costly, difficult to mnge nd cn led to buildup of toxic compounds (Crrillo et l., 1996; Monrc et l., 2000). Stnghellini nd Miller (1997) hve shown tht surfctnts cn exhibit lytic ctivity ginst zoospores. Surfctnts cn be used to control root-infecting zoosporic plnt pthogens in hydroponic systems (Stnghellini et l., 1996). Synthetic surfctnts lso hve potentil to control lef-ttcking zoosporic plnt pthogens such s white rust (Irish, 2002, s cited by De Jonghe, 2005). In the current study rnge of snitisers were evluted for their efficcy in controlling Pythium infesttion in hydroponics by mens of tretment of the nutrient solution. Some dditionl compounds, such s copper sulphte, the surfctnt Agrl 90 nd the commercil fungicide potssium phosphonte (Phytex ), were included for comprison. 1.2 Motivtion for study Although hydroponic cultivtion is ble to exclude mny soil-borne pthogens, nd thus reduce disese vriety, number of wter-borne nd wter-disseminted pthogens (Schwrz nd Grosch, 2003) re still ble to infest these systems nd cuse severe crop devsttion, to n extent worse thn wht would be experienced in soil-cultivted crops. This requires tht ttention be pid to the disinfection of the re-circulting nutrient solution in order to eliminte or reduce disese pressure nd ssocited crop losses. There is now greter consumer wreness of grochemicl problems, such s the negtive impct on the environment from the use of hrmful nd liberlly pplied pesticides nd hrsh chemicl tretments (Sb nd Messin, 2003). After the initil work by Stnghellini et l. (1996) on disese control using surfctnts, vrious products with similr formultions or ctivity hve been identified s hving comprble effects

12 Previous studies hve generlly been limited in either trget pthogen or snitiser selection nd hve delt minimlly with possible phytotoxic effects. The current study includes wide vriety of commercilly vilble snitisers ginst three vstly different pthogens, while lso ssessing possible negtive effects due to phytotoxicity. 1.3 Aim of the Study Estblishing whether ny of the snitisers under investigtion would be suitble for ppliction in hydroponic systems to limit yield losses of lettuce crops due to infection cused by Pythium spp. 1.4 Objectives 1. To determine in vitro efficcy of rnge of snitisers nd the most pproprite dosge rtes of ech snitiser ginst three selected plnt pthogens, including Pythium spp. 2. Estblish whether snitisers re phytotoxic on both cucumber (rpid ssy) nd lettuce (hydroponic system). 3. Evlute the benefits of ppliction of snitisers t the pre-determined dosges (s estblished in Points 1 nd 2, bove) into Pythium infested hydroponic system, t both greenhouse level nd semi-commercil field system

13 1.5 References Crrillo, A., Puente, M.E. nd Bshn, Y Appliction of diluted chlorine dioxide to rdish nd lettuce nurseries insignificntly reduced plnt development. Ecotoxicology nd Environmentl Sfety 35: Crruthers, S Hydroponics s n griculturl production system. Prcticl Hydroponics & Greenhouses. Issue 63. (Mr/Apr) pp1-4. De Jonghe, K., De Dobbelere, I., Srrzynb, R. nd Höfte, M Control of brown root rot cused by Phytophthor cryptoge in the hydroponic forcing of witloof chicory (Cichorium intybus vr. foliosum) by mens of nonionic surfctnt. Crop Protection 24: Irish, B.M., Correll, J.C., Morelock, T.E The effect of synthetic surfctnts on disese severity of white rust on spinch. Plnt Disese 86: Krrs, G., Svvs, D., Ptkiouts, G. nd Pomonis, P Fte of cyromzine pplied in nutrient solution to gerber (Gerber jmesonii) crop grown in closed hydroponic system. Crop Protection 26: Monrc, S., Feretti, D., Collivignrelli, C., Guzzell, L., Zerbini, I., Bertnz, G., nd Pedrzzni, R The influence of different disinfectnts on mutgenicity nd toxicity of urbn wstewter. Wter Reserch 34: Pulitz, T.C., Zhou, T. nd Rnkin, L Selection of rhizosphere bcteri for biologicl control of Pythium phnidermtum on hydroponiclly grown cucumber. Biologicl Control 2: Pulitz, T.C Biologicl control of root pthogens in soilless nd hydroponic systems. HortScience 32:

14 Reuveni, R., Dor, G., Rviv, M., Reuveni, M. nd Tuzun, S Systemic resistnce ginst Spherothec fuligine in cucumber plnts exposed to phosphte in hydroponics system, nd its control by folir spry of mono-potssium phosphte. Crop Protection 19: Sb, A. nd Messin, F Attitudes towrds orgnic foods nd risk/benefit perception ssocited with pesticides. Food Qulity nd Preference 14: Svvs, D., Mnos, G., Kotsirs, A. nd Souvliotis, S., Effects of silicon nd nutrient-induced slinity on yield, flower qulity nd nutrient uptke of gerber grown in closed hydroponic system. Journl of Applied Botny 76: Schwrz, D. nd Grosch, R Influence of nutrient solution concentrtion nd root pthogen (Pythium phnidermtum) on tomto root growth nd morphology. Science Horticulture 97: Song, W., Zhou, L., Yng, C., Co, X., Zhng, L. nd Liu, X Tomto Fusrium wilt nd its chemicl control strtegies in hydroponic system. Crop Protection 23: Stnghellini, M.E., Kim, D.H., Rsmussen, S.L. nd Rorbugh, P.A Control of root rot of peppers cused by Phytophthor cpsici with nonionic surfctnt. Plnt Disese 80: Stnghellini, M.E. nd Miller, R.M Biosurfctnts. Their identity nd potentil efficcy in the biologicl control of zoosporic plnt pthogens. Plnt Disese 81: vn West, P., Appih, A.A. nd Gow, N.A.R Advnces in reserch on oomycete root pthogens. Physiologicl nd Moleculr Plnt Pthology 62: Wood, A.R. nd Ling, M.D., The control of fungl root pthogens of ornmentl folige plnts in hydroculture. In: Proccedings of the Eighth Interntionl Congress on Soiless Culture. ISOSC, Wgeningen, The Netherlnds, pp

15 CHAPTER 2 LITERATURE REVIEW The following text is intended s brief overview to elucidte the rtionle of the current study nd provide bckground on the concepts discussed during this study nd is not intended s n exhustive nd in-depth review. 2.1 Hydroponics Overview Hydroponics is the science of growing plnts in soilless (non-nutritive) substrte (Song et l., 2004), with nutrition being supplied rtificilly, most commonly in the wter supply, directly to the roots (Stnghellini nd Rsmussen, 1994), while folir feeding cn lso be used. The usul design of the hydroponic systems is such tht the plnt roots re exposed to the nutrient solution (liquid systems) (Stnghellini nd Rsmussen, 1994), or the nutrient solution is directly pplied to the root zone (ggregte systems) (Neiderweiser, 2001). More recent developments sometimes include ppliction of the fertignts to the lef surfces to supplement plnt nutrition by folir ppliction. The word hydroponics origintes from the Greek hydro, mening wter, nd ponos, mening work or lbour (Hrris, 1976), to indicte tht the min work for growth is provided by the wter in which the plnts re grown

16 Originlly hydroponics ws defined s the growth of plnts without soil, or lterntively in wter, nd ws used primrily in the more recent pst by scientists to chieve greter control of environmentl conditions in smll-scle trils (Fresh Produce Hydroponics, 2002). A more current definition which is more pplicble to commercil cultivtion is: Hydroponics or soil-less culture is the production of crops isolted from the soil, either with or without medium, with their totl wter nd nutrient requirements supplied by the system (Hnger, 1993; Jensen, 1999). The prctice of growing plnts in hydroponic system in vrious, bsic forms hs been utilised by frmers since severl hundred yers B.C. This is specificlly seen in hieroglyphs nd drwings from ncient Egyptin history (Fresh Produce Hydroponics, 2002). The Egyptins, Inc Indin tribes, the Aztecs, nd the Bbylonins re exmples of ncient civiliztions which prcticed hydroponic grdening without even relizing it, long before the word "hydroponics" ws ever thought of (Deutschmnn, 1998). It is quite possible tht the most primitive form of hydroponics ws the suspension of plnts in thin soil nd wter mixture which provided the bsic nutrients required. Hydroponics hs seen more widespred commercil use since the mid-1930 s, with Western Europe leding this trend (Zinnen, 1988). This commercil interest ws primrily due to the scientific development of specificlly designed fertiliser mixes for use in hydroponics, nd these mixes subsequently becoming more redily vilble to the commercil growers. Other spects iding the development of hydroponics included: The use of plstics (Fresh Produce Hydroponics, 2002) which llowed more cost effective nd less lbour intensive production of the physicl fcilities; New types of inert substrtes such s rockwool, perlite nd vermiculite being introduced nd used s growth substrtes - 7 -

17 (Niederweiser, 2001; Gul et l., 2005); nd lstly, the reserch, nd subsequent development of, more refined hydroponic growth systems such s the Nutrient Film Technique nd ebb-nd-flow systems where the plnts re not continuously immersed in sttic solution (Hrris, 1976). These developments hve lso gretly expnded the vriety of crops which cn now be cultivted in modern hydroponic systems. Hydroponic cultivtion is split into two brod ctegories, nmely liquid systems where no inert substrte is present, nd ggregte systems where n inert (non-nutritive) substrte such s snd, grvel or rockwool is used (Stnghellini nd Rsmussen, 1994). The purpose of the substrte is to provide physiclly supportive structure to enble the plnt to remin upright. Hydroponic systems cn further be divided into closed nd open systems (Stnghellini nd Rsmussen, 1994; Niederweiser, 2001) where closed nd open refer to the wter supply. Closed systems refer to those where the nutrient solution is collected nd re-used fter tretment nd djustment for nutrient losses nd then re-supplied to the plnt roots. This type of system is becoming the method of choice (Crruthers, 2002) due to reduction of constnt input costs nd thus improving the economic efficiency of the fertignts, while lso preventing environmentl pollution such s contmintion of sub-surfce wter sources. The Dutch government hs lredy pssed lws which enforce the use of only recirculting systems (Runi, 1994; Runi, 1995; Fresh Produce Hydroponics, 2002) to prevent dmge to the environment nd it is presumed tht other countries will follow this trend in the future

18 In open systems the wter is llowed to drin freely s wste-wter or is collected nd used for n lternte purpose such s irrigtion. This type of system is usully seen in bg-type production systems where collection of wste-wter is fr more of logistic problem thn in nutrient-flow bsed production (Niederweiser, 2001). The dvntge of hydroponic production ws initilly explored s n lterntive to field production due to the ese of plnt growth control nd the hopes of preventing the mjority of disese cusing gents known to overwinter or be present in generl soil environments (Zinnen, 1988; Stnghellini nd Rsmussen, 1994). Further dvntges of hydroponic systems were soon relized in tht crops cn be grown in res where there re problems with soil suitbility, in non-rble or borderline res (Svvs, 2003), or where environmentl fctors such s temperture or winds prevent cceptble yields s well s res where slope of lnd prevents ploughing (Pulitz et l., 1992). The environmentl conditions cn be overcome since much of the hydroponic systems re under some form of covering such s shde net, plstic tunnels or greenhouse (multispn) complexes. In environmentlly controlled greenhouses crops cn lso be grown yer round with the sme yields obtined during summer nd winter (Cornell CEA Homepge, 2002), while miniml form of environmentl consistency cn be obtined under shde net nd in plstic tunnels by mens of fns, heters nd specilised mist systems. Hydroponics in the current form is extremely beneficil in commercil sense s plnt growth is more controlled nd uniform, nd up to eight crops (in the cse of lettuce) cn be cultivted in 12-month cycle (Zinnen, 1988; Cornell CEA Homepge, 2002), compred - 9 -

19 to mximum of six crops or less when grown under regulr open field conditions. Furthermore higher yield per re is obtined from hydroponic production due to lesser spcing requirements necessry between plnts, nd consistent growth is chieved between crops s the nutrient supply remins constnt throughout the yer, in contrst to fields becoming more nutrient deficient nd requiring expensive griculturl inputs between crops (Svvs, 2003). A more consistent growth is lso chievble due to minimised sesonl vritions of light nd temperture when cultivtion occurs under controlled or prtilly-controlled environment. A tril greenhouse t Cornell University ws ble to chieve lettuce yields equivlent to tons of lettuce per cre per nnum, wheres typicl yields under field conditions re only tons per cre per nnum (Cornell CEA Homepge, 2002). Due to the control of environmentl conditions nd the supply of ll essentil nd required nutrients in the wter supply, hydroponic plnt growth is more rpid nd very good uniformity is obtined cross the entire plnting. This is very beneficil for commercil frmers who re required to supply specificlly sized plnt t certin time. Thus plnning nd supply become known fctors nd mechnistion in lrge greenhouses is lso possible (Vnchter, 1995), mking hydroponic crop production very cost effective in lbour terms. Although the mjority of crops re grown by providing nutrients only to the roots, dditionl nutrition cn be supplied vi folir ppliction. This method of nutrient ppliction lso hs the dvntge of iding temperture nd humidity control. This misting does unfortuntely dd n increse in cost, logistics nd generl mngement, nd s it is not currently widely used in South Afric this method ws not included in the scope of this reserch project

20 Even though hydroponic plnt production hs numerous benefits (Pulitz et l., 1992), some of which hve been discussed bove, nd commercil hydroponic crop production worldwide hs incresed to pproximtely cres producing crops worth $6 billion per nnum (2002 estimte) (Crruthers, 2002; Fresh Produce Hydroponics, 2002) there re certin inherent difficulties: Since hydroponic crop production is n intensive monoculture in reltively humid environments (due to the bundnt presence of wter) these crops re thus extremely prone to devsttion by smll number of diseses (Zinnen, 1988; Stnghellini nd Rsmussen, 1994; vn West et l., 2003). Coupled to this is the fct tht due to the intensive crop production methodology, the plnts re cultivted t the mximl possible rte. The result is tht the crop becomes very prone to stress should the environmentl conditions chnge or the nutrient supply cese for even short period (personl observtion). During these stress conditions susceptible seedlings hving survived erly infection, cn rpidly develop full-blown disese leding to serious outbreks, plnt deths nd yield losses (Wkehm et l., 1997), while plnts cn lso become more susceptible to pthogens present in the nutrient supply or develop disese from sub-clinicl infections (Stnghellini nd Kronlnd, 1986; Schwrz nd Grosch, 2003; vn West et l., 2003) Diseses in hydroponic systems The move to re-circulting hydroponic systems, lthough positive for economicl nd environmentl resons, could result in serious yield losses due to disese (Zinnen, 1988; vn West et l., 2003)

21 It ws soon relized tht the move to hydroponics would not prevent soil-borne diseses s initilly hoped, s vriety of pthogens cn nd do infect hydroponic crops (Stnghellini nd Rsmussen, 1994), yet the growth of plnts in greenhouses cn hve the benefit of estblishing n integrted crop mngement strtegy (Vn Assche nd Vngheel, 1989; Svvs, 2003) which cn id in preventing pest nd disese dmge. Of primry concern in terms of pthogens re the wter-borne nd wter-motile zoosporic fungi (specificlly Pythium spp.) which re ble to spred esily throughout the system (Stnghellini nd Rsmussen, 1994; vn West, 2003) nd cuse root-rot nd wilting. Due to the intensive cropping nd monoculture prctices in hydroponic production, infection cn led to severe losses, in mny cses without the usul visible root-rot or wilt symptoms of infection yet with yields being reduced by up to 54% by this sub-clinicl infection. (Stnghellini nd Kronlnd, 1986; Stnghellini nd Rsmussen, 1994; Schwrz et l., 2003). In recirculting systems ech plnt becomes ner neighbour of every other plnt supplied by the sme btch of nutrient solution. One infected plnt cn thus result in every plnt becoming infected (Zinnen, 1988) nd leding to devstting losses if disese develops fully. Conversely s ech plnt is ffected in the sme wy, in the cse of subclinicl infections the yield loss is hrdly ever noticed s ll the plnts re eqully reduced while ppering helthy (Stnghellini nd Kronlnd, 1986; Schwrz nd Grosch, 2003). Once recirculting hydroponic systems become infested, the entire system hs to be stopped, drined nd thoroughly disinfected (Stnghellini et l., 1996) before being put into economiclly vible production gin

22 Rlstoni is nother soil-borne pthogen of concern in hydroponic systems, specificlly on long-term crops such s tomtoes, peppers nd cucurbits (Lemy et l., 2003; Guo et l., 2004) usully grown in open-bg systems. Rlstoni cuses vsculr wilt nd soft-rot infection of the stlk t ground level (soil or wter / ir interfce) nd primrily cuses blockge of wter trnsport up the xylem resulting in devstting wilt. The motile nture of this bcterium lso ids in the spred between plnts (Lemy et l., 2003; Guo et l., 2004; Agrios, 2005). 2.2 Pthogens The three pthogens selected for the current study were Pythium, Fusrium nd Rlstoni nd their selection criteri re discussed below Pythium Pythium belongs to the Pythiceous group of fungi which hs motile zoospore stge in its life cycle (Kuchrek nd Mitchell, 2000). This zoospore is especilly well dpted to queous environments mking it severe pthogen in wterlogged or over-wtered fields nd especilly devstting in hydroponic systems which rely hevily on wter. Pythium lso hs very brod host rnge nd hs been known to infect lrge proportion of hydroponiclly cultivted crops (Stnghellini nd Rsmussen, 1994; Kuchrek nd Mitchell, 2000). The motile zoospore is ttrcted to the root zone of plnts by electricl fields (vn West et l., 2003) nd infects the roots, cusing root decy which cn initilly mnifest s root rot the first noticeble symptom (Stnghellini nd Kronlnd, 1986)

23 During this time the infected root cn relese millions of new zoospores ech dy which infect surrounding roots nd spred by their motility to nerby plnts (Kuchrek nd Mitchell, 2000). As the level of infection of plnt increses, the plnt root function is severely impcted, preventing dequte uptke of nutrients nd wter nd leding to the second noticeble symptom of generl plnt wilt. Once the plnt hs reched this stge of infection, recovery generlly ppers to be impossible (Personl observtion). Pythium infesttion ws lso shown in lettuce plnts where no observble symptoms were noted, yet reduction in yield (Stnghellini nd Kronlnd, 1986; Schwrz nd Grosch, 2003; vn West et l., 2003) ws demonstrted when compred to non-infected control. This sub-clinicl infection is lso problem in hydroponic crop production, lthough it is not yet recognised s such. Pythium cn lso overwinter in plnt (root) debris left in the substrte of hydroponic systems, cusing rpid re-infection of new seedlings plnted in the following cultivtion cycle (Kuchrek nd Mitchell, 2000). The level of infective mteril increses with ech growth cycle, resulting in n incresed disese pressure t the initition of the next growth cycle nd possible higher level of sub-clinicl infection (Stnghellini nd Kronlnd, 1986; Schwrz nd Grosch, 2003). Although Pythium cuses serious hydroponic diseses in the form of root rot nd wilt, nd once plnts re infected there re few curtive methods vilble, it is hypotheticlly esily prevented s the thin-wlled zoospore stge should be very susceptible to control by mens of chemicls (Stnghellini nd Tomlinson, 1987)

24 Figure 1: Typicl disese cycle of Pythium spp. (vn West et l., 2003) Rlstoni Rlstoni solncerum (Smith) Ybuuchi et l. is motile Grm negtive bcterium, previously clssified s Pseudomons solncerum (Smith) Smith. The grm negtive chrcteristics of this bcterium hypotheticlly mke it more resistnt to the effects of snitisers due to the complex boundry of cell wll nd cell membrnes which hve to be overcome by the snitisers, while it is lso considered model orgnism for plnt pthogenicity (Agrios, 2005). This bcterium is generl, yet severe, soil-borne plnt pthogen which ffects lrge rnge of hosts (Guo et l., 2004). Infection occurs t the roots fter which the xylem vessels of the plnt become clogged with bcteril growth cusing rpid nd devstting wilt (Lemy et l., 2003)

25 This orgnism is lso of gret concern in hydroponiclly grown tomtoes nd cucurbits where it is ble to devstte entire crops in miniml time due to the motile nture of the orgnism llowing cross infection between plnts, s well s the environmentl conditions being idel for infection (Lemy et l., 2003; Guo et l., 2004) Fusrium Fusrium solni (Mrt.) Scc. is common soil-borne plnt pthogenic fungus (Frvel nd Lrkin, 2004) which forms thick-wlled micro- nd mcro-conidi. These conidi re highly resistnt structures resulting in the fungus being ble to overwinter successfully s well s iding in mking Fusrium one of the most fungicide resistnt fungi (Agrios, 2005). Fusrium conidi were specificlly selected for this study due to their environmentl nd chemicl resistnce chrcteristic, s well s the fct tht the cell membrne is enclosed by the thick cell wll, possibly mking Fusrium more resistnt to snitisers which re theorised to cuse disruption of cell membrnes (Buck et l., 2002) Fusrium diseses re common nd destructive in mny hydroponic systems where the fungus ttcks the roots nd cuses dmping off, especilly in young seedlings (Frvel nd Lrkin, 2002; Song et l., 2004). Most commonly ffected re tomto nd cucurbit plnts such s cucumbers (Song et l., 2004)

26 2.3 Snitisers As with hydroponic development, snitiser formultion hs seen significnt chnges over the lst few yers resulting in snitisers being used in mny new res nd in more environmentlly friendly nture (Nlecz-Jwecki et l., 2003; Monrc et l., 2004). These formultions hve thus seen snitisers introduced into the food industry specificlly in plnt-product nd fresh fruit pckging processes to prevent post-hrvest diseses nd lso on redy-to-et products to reduce or prevent contmintion by humn pthogens (Do Socorro et l., 2005; Allende et l., 2006). New forms of snitisers, termed wter snitisers, re efficient products which, when dded to contminted wter supplies t low concentrtions, effect high level of snittion of the wter to llow the wter to be used without contminting the downstrem products nd processes (Rdziminski et l., 2002; Lee et l., 2004). A further benefit of mny snitisers is their bility nd effectiveness in biofilm control (Simoes et l., 2005), which cn rpidly ccumulte in piping used in hydroponic systems due to the high nutrient-slt content nd orgnic plnt exudtes nd debris relesed into the re-circulted hydroponic nutrient supply. This microbil polymer lyer pcks onto the internl wlls of pipes creting n orgnic nd inorgnic biofilm lyer, which both blocks pipes nd spryers nd is prime re for pthogens nd other micro-orgnisms to lodge nd reproduce or overwinter (Chen nd Stewrt, 2005). Although snitisers re not designed to hve specific ction ginst micro-orgnisms, s with fungicides nd nti-microbil gents such s ntibiotics, most snitisers re ble to ct on cell membrnes due to the inherent surfctnt properties (Stnghellini et l., 1996)

27 This cuses disruption of the cell membrne nd the resulting lysis nd subsequent deth of the cell. The ction of snitisers on more resistnt structures such s Fusrium conidi is more complex nd not understood s yet. It hs lso been shown tht certin surfctnts nd snitisers re rpidly broken down fter being introduced into hydroponic systems, while initil ntimicrobil efficcy is still mintined (Grlnd et l., 2000; Grlnd et l., 2004). This indictes tht the ntimicrobil effect is ttined rpidly on ddition of the surfctnts. A further benefit of this is tht the environmentl hzrd risk of using these products is lso miniml. Thus dding wter snitisers to hydroponic nutrient supply could hve possible threefold benefit nmely biofilm formtion is minimised while the nutrient solution is continully snitised of the mjor plnt pthogenic propgules, resulting in re-circulted wter being less infectious. Lstly the relese of toxic chemicls into the environment would lso be minimised. The snitisers selected for use in this study (Tble 1) hve ctive ingredients with known ctivity ginst micro-orgnisms with mny products being recommended for griculturl use. Fitosn, Sporekill nd Prsin re bsed on quternry mmonium compounds which re widely used for disinfection in medicl nd food environments (Sundheim et l ) nd hve been shown to hve ctivity ginst Pythium (O Neill, 1995). Fitosn nd Prsin lso contin gunidines which hve been shown to hve ntifungl nd ntibcteril ctivity (Hudson et l., 1986). Purogene nd TecsClor hve chlorine dioxide s n ctive ingredient which hs lso been well described s hving ntimicrobil ctivity (Ltshw, 1994; Foschino et l., 1998) s well s hving n effect on Pythium

28 (O Niell, 1995). Purogene hs lso specificlly been shown to hve ctivity on bcteri (Hrkeh, 1988). Actsol is bsed on the electrochemicl ctivtion (ECA) of wter nd brine solution to obtin solution contining brod rnge of mixed oxidising rdicls which hs been demonstrted to hve both ntibcteril nd ntifungl ctivity including ctivity ginst micro-orgnisms of concern in griculture (Csteel et l., 2000; Buck et l., 2002). Agrl 90 is non-ionic surfctnt contining lkryl polyglycol ether. Agrl 90 ws demonstrted by Stnghellini nd Tomlinson (1987) to hve ctivity ginst Pythium zoospores, while vrious surfctnts, including Agrl 90 were shown to hve ctivity ginst zoospores of Olpidium brssice (Woronin) P. A. Dng. (Tomlinson nd Fithfull, 1980). Pesticides bsed on copper s n ctive ingredient hve seen widespred use over mny yers (de Oliveir-Filho et l., 2004). Copper (II) sulphte ws selected s the chemicl compound providing source of the bsic form of copper used in this study. During the greenhouse nd field evlutions the commercilly vilble systemic fungicide Phytex ws selected s stndrd tretment due to it being commercilly registered for use ginst pythiceous fungi. The ctive ingredient of Phytex is phosphorous cid which hs been demonstrted to hve n effect ginst Pythium (Fenn nd Coffey, 1984)

29 Tble 1: Detiled informtion on snitisers selected for the current study. Nme used Active ingredient Type of product Agrl 90 90% m.m -1 lkryl Agriculturl polyglycol ether surfctnt Actsol Mixed oxidnt & Electro metstble species e.g chemiclly hypochlorous cid, ctivted hypochlorite, chlorte, wter perchlorte (180mg.l -1 totl) Copper sulphte Fitosn (F10 Agriculturl) Phytex (mrketed s Phytex 200SL) Prsin (mrketed s Prsin Agri ) Purogene (with ctivtor) Sporekill Tecs Clor Copper (II) sulphte penthydrte supplying Cu 2+ Quternry mmonium & bigunide (5.8%) Potssium phosphonte (200g.l -1 ) Polymetric bigunide hydrochloride & quternry mmonium (7%) Chlorine dioxide (3g.l -1 mx) N,N-Didecyl N,Ndimethyl mmoniumchloride (12%) Chlorine dioxide (2-3g.l -1 ) Supplier Kynoch chemicls Rdicl Wters Notes & Referenced in Formultion Nonnionic, SL Anionic, SL 3.4.1; ; ; ; ; Chemicl Merck 3.4.7; Agriculturl snitiser Helth & Hygiene Ctionic, SL 3.4.5; ; ; Fungicide Horticur SL ; Agriculturl snitiser Generl & griculturl snitiser Agriculturl snitiser Generl & griculturl snitiser SIDL cc Ctionic, SL 3.4.2; ; ; ; ; BTC products & services Hygrotech Seed BTC products & services Nonionic, SL 3.4.3; ; ; ; ; Nonionic, SL 3.4.8; Nonionic, SL 3.4.4; ; ; ; ;

30 2.4 References Agrios, G.N Plnt Pthology. 5 th Ed. Elsevier Acdemic Press, UK. Allende, A., Toms-Brbern, F.A. nd Gil, M.I Miniml processing for helthy trditionl foods. Trends in Food Science nd Technology 17: Buck, J.W., vn Iersel, M.W., Oetting, R.D. nd Hung, Y.C In vitro fungicidl ctivity of cidic electrolyzed oxidizing wter. Plnt Disese 86: Crruthers, S Hydroponics s n griculturl production system. Prcticl Hydroponics & Greenhouses 63: 4-7. Csteel, M.J., Sobsey, M.D. nd Arrowood, M.J Inctivtion of Cryptosporidium prvum oocysts nd other microbes in wter nd wstewter by electrochemiclly generted mixed oxidnts. Wter Science nd Technology 41: Chen, X. nd Stewrt, P.S Disinfectnt efficcy of chlorite nd chlorine dioxide in drinking wter biofilms. Wter Reserch 39: Cornell CEA Homepge Accessed: 20/06/2002. de Oliveir-Filho, E.C., Lopes, R.M. nd Pumgrtten, F.J.R Comprtive study on the susceptibility of freshwter species to copper-bsed pesticides. Chemosphere 56: Deutschmnn, G.V The history of hydroponics. Accessed: 05/12/2006. Do Socorro, M., Bstos, R., de Ftim Ferreir Sores, N., de Andrde, N.J., Arrud, A.C. nd Alves, R.E The effect of the ssocition of snitizers nd surfctnt in the microbiot of the cntloupe (Cucumis melo L.) melon surfce. Food Control 16:

31 Fenn, M.A. nd Coffey, M.D Studies on the in vitro nd in vivo ntifungl ctivity of fosetyl-l nd phosphorous cid. Phytopthology 74: Foschino, R., Nervegn, I., Mott, A. nd Glli, A Bctericidl ctivity of chlorine dioxide ginst Escherichi coli in wter nd on hrd surfces. Journl of Food Protection 61: Frvel, D.R. nd Lrkin, R.P Reduction of Fusrium wilt of hydroponiclly grown bsil by Fusrium oxysporum strin CS-20. Crop Protection 21: Fresh Produce Hydroponics Produce Mrketing Assocition. Accessed: 20/06/2002. Grlnd, J.L., Levine, L.H., Yorio, N.C., Adms, J.L. nd Cook, K.L Grywter processing in recirculting hydroponic systems: Phytotoxicity, surfctnt degrdtion, nd bcteril dynmics. Wter Reserch 34: Grlnd, J.L., Levine, L.H., Yorio, N.C. nd Hummerick, M.E Response of grywter recycling systems bsed on hydroponic plnt growth to three clsses of surfctnts. Wter Reserch 38: Gul, A., Erogul, D. nd Ongun, A. R Comprison of the use of zeolite nd perlite s substrte for crisp-hed lettuce. Scienti Horticulture 106: Guo, J., Qi, H., Guo, Y., Ge, H., Gong, L., Zhng, L. nd Sun, P Biocontrol of tomto wilt by plnt growth-promoting rhizobcteri. Biologicl Control 29: Hnger, B Hydroponics: The world, Austrlin, nd South Pcific islnds scene. In: Commercil Hydroponics in Austrlsi, A Guide for Growers. Austrlin Hydroponics Assocition Inc. Pro-Set Pty Ltd, Hobrt, NZ. Hrkeh, S., Illescs, A. nd Mtin, A Inctivtion of bcteri by Purogene. Journl of Applied Bcteriology 64:

32 Hrris, D.A Hydroponics: The Grdening Without Soil. Purnell, Cpe Town, South Afric. Hudson, H.R., Ojo, I.A.O. nd Pink, M Gunidines with ntifungl (nd ntibcteril) ctivity review. Interntionl Pest Control 28: Jensen, M.H Hydroponics worldwide. In: Proceedings interntionl symposium on growing medi nd hydroponics, Ontrio, Cnd My 1997, Ed. Ppdopoulos, A.P. Act Horticulture 481: Kuchrek, T. nd Mitchell, D Diseses of gronomic nd vegetble crops cused by Pythium. Plnt Pthology Fct Sheet pp-53. Florid Coopertive Extension Service, Institute of Food nd Agriculturl Sciences, University of Florid, Christine Wddill, Den. Ltshw, C.L Chlorine dioxide: effective, brod-spectrum biocide for white wter systems. Tppi Journl 78: Lee, S., Gry, P.M., Dougherty, R.H. nd Kng, D The use of chlorine dioxide to control Alicyclobcillus cidoterrestris spores in queous suspension nd on pples. Interntionl Journl of Food Microbiology 92: Lemy, A., Redlin, S., Fowler, G. nd Dirni, M Pest Dt Sheet: Rlstoni solncerum rce 3 biovr 2. USDA/APHIS/PPQ, Center for Plnt Helth Science nd Technology, Plnt Epidemiology nd Risk Anlysis Lbortory, Rleigh, NC. Monrc, M., Zni, C., Richrdson, S.D., Thruston Jr, A.D., Moretti, M., Feretti, D. nd Villrini, M A new pproch to evluting the toxicity nd genotoxicity of disinfected drinking wter. Wter Reserch 38: Nlecz-Jwecki, G., Grbinsk-Sot, E. nd Nrkiewicz, P The toxicity of ctionic surfctnts in four biossys. Ecotoxicology nd Environmentl Sfety 54:

33 Niederweiser, J.G Guide to Hydroponic Vegetble Production. Agriculturl Reserch Council, Roodeplt, South Afric. O Niell, T.M Evlution of disinfectnts ginst Pythium spp. nd Thielviopsis bsicol. Test of Agrochemicls nd Cultivrs, Annls of Applied Biology 126 (Supplement): Pulitz, T.C., Zhou, T. nd Rnkin, L Selection of rhizosphere bcteri for biologicl control of Pythium phnidermtum on hydroponiclly grown cucumber. Biologicl Control 2: Rdziminski, C., Bllntyne, L., Hodson, J., Creson, R., Andrews, R.C. nd Churet, C Disinfection of Bcillus subtilis spores with chlorine dioxide: A bench-scle nd pilot-scle study. Wter Reserch 36: Runi, W Disinfection of recircultion wter from closed cultivtion systems with ozone. Act Horticulture 361: Runi, W A review of possibilities for disinfection of recircultion wter from soilless cultures. Act Horticulture 382: Svvs, D Hydroponics: A modern technology supporting the ppliction of integrted crop mngement in greenhouse. Food, Agriculture nd Environment 1: Schwrz, D. nd Grosch, R Influence of nutrient solution concentrtion nd root pthogen (Pythium phnidermtum) on tomto root growth nd morphology. Scienti Horticulture 97: Simoes, M., Pereir, M.O. nd Vieir, M.J Action of ctionic surfctnt on the ctivity nd removl of bcteril biofilms formed under different flow regimes. Wter Reserch 39: Song, W., Zhou, L., Yng, C., Co, X., Zhng, L. nd Liu, X Tomto Fusrium wilt nd its chemicl control strtegies in hydroponic system. Crop Protection 23:

34 Stnghellini, M.E. nd Kronlnd, W.C Yield loss in hydroponiclly grown lettuce ttributed to subclinicl infection of feeder rootlets by Pythium dissotocum. Plnt Disese 70: Stnghellini, M.E. nd Rsmussen, S.L Hydroponics : A solution for zoosporic pthogens. Plnt Disese 78: Stnghellini, M.E., Rsmussen, S.L., Kim, D.H. nd Rorbugh, P.A Efficcy of nonionic surfctnts in the control of zoospore spred of Pythium phnidermtum in recirculting hydroponic system. Plnt Disese 80: Stnghellini, M.E. nd Tomlinson, J.A Inhibitory nd lytic effects of nonionic surfctnt on vrious sexul stges in the life cycle of Pythium nd Phytophthor species. Phytopthology 77: Sundheim, G., Lngsrud, S., Heir, E. nd Holck, A.L Bcteril resistnce to disinfectnts contining quternry mmonium compounds. Interntionl Biodeteriortion nd Biodegrdtion 41: Tomlinson, J.A. nd Fithfull, E.M Studies on the control of lettuce big-vein disese in recirculted nutrient solutions. Act Horticulture 98: Vn Assche, C. nd Vngheel, M Plnt protection in hydroponics. Act Horticulture 260: vn West, P., Appih, A.A. nd Gow, N.A.R Advnces in reserch on oomycete root pthogens. Physiologicl nd Moleculr Plnt Pthology 62: Vnchter, A Development of Olpidium nd Pythium in the nutrient solutions of NFT grown lettuce, nd possible control methods. Act Horticulture 382: Wkehm, A.J., Pettitt, T.R. nd White, J.G A novel method for detection of vible zoospores of Pythium in irrigtion wter. Annls of Applied Biology 131:

35 Zinnen, T.M Assessment of plnt diseses in hydroponic culture. Plnt Disese 72:

36 CHAPTER 3 IN VITRO EFFICACY OF WATER SANITISERS AGAINST PYTHIUM ZOOSPORES IN AQUEOUS SUSPENSION 3.1 Abstrct Although the use of re-circulting hydroponic systems hs its dvntges, it is lso prone to infesttion by pthogens such s Pythium. Current methods of steriliztion of hydroponic nutrient solution, such s chlorintion nd ozontion, re costly nd difficult to mnge. Severl snitisers re now vilble tht re consumer friendly nd environmentlly sfe nd these were tested for their efficcy in controlling Pythium zoospores in wter suspension. Testing ws performed by ddition of vrious concentrtions of these snitisers into volume of wter contining Pythium zoospores, llowing specific exposure time nd then determining the vible zoospores remining. Two other plnt pthogens (Fusrium nd Rlstoni) were lso tested for comprison. All the snitisers were mde up t the recommended rtes. Actsol demonstrted very good efficcy ginst ll the test orgnisms nd erdicted Pythium from the test suspension t ll the concentrtions tested, nd with the shortest exposure time of 10 min. Prsin (5mg.l - 1 ) nd TecsClor (25mg.l -1 ) chieved the desired 80% kill of Pythium zoospores t 10 nd exposure times respectively. Pythium zoospores were effectively eliminted within when exposed to Fitosn concentrtion of 7.5mg.l -1. Agrl 90 ws ble to chieve the desired kill rte of 80% t 1mg.l -1 nd exposure time. Exposure to Sporekill gve percentge kill of bove 80% of Pythium zoospores t concentrtion of 5mg.l -1 with exposure time. For most of the snitisers, Fusrium nd Rlstoni exhibited typicl dose-response where the kill rtes incresed with incresed exposure time. However, these two pthogens proved to be more resistnt to the snitisers thn Pythium. This dt shows tht the ddition of the bove products to Pythium-infested wter supply would effectively eliminte Pythium nd lso gretly reduce Fusrium nd Rlstoni inoculum levels, indicting possible use for disinfection of recirculting hydroponic nutrient solutions

37 3.2 Introduction The use of surfce- nd wter-snitisers hve seen incresed usge s new chemicl formultions re developed which re less hrmful in terms of humn helth nd environmentl concerns thn the toxic snitisers such s chlorine, which hve lso been shown to produce mutgens nd crcinogens (Andrews et l., 2002). Thus the new snitisers re more consumer nd environmentlly friendly lterntive s well s being more effective t lower concentrtions, resulting in both reduced risk nd reduced cost, while meeting stricter stndrds for effluents (Adler et l., 2003). The use of snitisers lso does not crry the sme stigm s specificlly formulted chemicls (nti-microbil nd ntibiotics) for use in control of pthogens nd pests. This meets consumer demnds for products tht re grown under conditions where pesticides re not liberlly pplied (Sb nd Messin, 2003). Additionlly there is lso reduced risk of the pthogens developing resistnce to these products. Although primrily used for snittion nd disinfection of fixed surfces (Peng et l., 2002), numerous snitisers nd detergents, when pplied in wter, re ble to effect snittion, or even totl sterilistion, of the wter volume (Lee et l., 2004). Due to their resonbly sfe nture nd use t low concentrtions, snitisers hve lso seen widespred use in posthrvest clening of fruits nd lso fresh-cut vegetbles to both remove pthogens nd spoilge orgnisms (Singh et l., 2002; De Socorro et l., 2005) Current methods of sterilistion of recirculted hydroponic nutrient solution, such s chlorintion, ozontion, iodintion nd ultr-violet (UV) sterilistion re costly, difficult to mnge nd often ineffective due to high orgnic lod nd concentrtion of slts (Runi, 1994; Runi, 1995). Chlorintion, lthough reltively chep nd esy to pply, is not widely used due to the phytotoxic nture of chlorine. Additionlly ll the previously mentioned controls hve, in certin instnces, been shown to produce toxic by-products or degrdtion products (Monrc et l., 2004). Due to these setbcks, it is thought tht the snitisers, such s those under investigtion in this study, will be more effective in snitising wter, with fewer hrmful side-effects in recirculting hydroponic systems

38 The life-cycles of mny Pythium species include motile stge where flgellted zoospores re produced (Roux nd Both, 1997). These flgellted zoospores hve been implicted s the mjor gents in disese spred (Stnghellini et l., 1996; Stnghellini nd Miller, 1997). Since zoospores only hve thin cell membrnes, which re esily disrupted by surfctnts (Stnghellini nd Tomlinson, 1987; Stnghellini et l., 1996; De Jonghe et l., 2005), zoospores were thus identified s the most vulnerble trget of wter snitiser ctivity. In hydroponic systems, zoospores re relesed from infected roots into the nutrient solution, where they cn then be dispersed throughout the hydroponic system, resulting in rpid disese spred nd increse in disese pressure (Kuchrek nd Mitchell, 2000). Thus elimintion or inctivtion of plnt pthogens (in this cse specificlly Pythium zoospores), or even totl sterilistion of the nutrient solution, is of importnce, especilly with regrds to recirculting hydroponic systems, where rpid increse in inoculum cn occur, s inoculum is continully being dded nd recirculted. The primry im of this study ws to determine whether selected snitisers re effective in killing Pythium zoospores in hydroponic nutrient solutions. To chieve this, the snitisers were tested for efficcy ginst three pthogens (Pythium spp. zoospores, Fusrium solni (Mrt.) Scc. conidi nd Rlstoni solncerum (Smith) Ybuuchi et l. plnktonic cells) in queous suspension using sterile wter. The results of this exposure would determine whether vible control, or elimintion, of the pthogens could be chieved, s well s the lowest concentrtion t which this could be chieved

39 3.3 Mterils nd Methods Mintennce of cultures Initil experiments were performed using nturlly infested runoff wter tken from commercil hydroponic system known to hve Pythium infesttion. Although positive results were obtined from these experiments, the vrition in the results indicted tht consistency could not be mintined between experiments. A novel method of obtining fresh Pythium zoospores ws developed, which llowed for consistent results, s well s the elimintion of other infectious propgules nd orgnic mtter which my hve ffected the experimentl outcome. Pythium cultures were mintined on V8 juice gr (De Jonghe et l., 2005) s well s the Pythium selective medi BNPRA (Roux nd Both, 1997). Fusrium cultures were mintined on Potto Dextrose Agr (PDA) (Biolb C100, Merck, South Afric), nd Rlstoni on Nutrient Agr (NA) (Biolb C150, Merck, South Afric) s well s the selective medi TZC (2,3,5-Triphenyltetrzolium chloride) (Merck, South Afric)(vn Broekhuizen, 2002). For ech experiment fresh cultures of ech orgnism were grown from stock culture with the verge ge of cultures during the experiments being six dys

40 3.3.2 Inoculum preprtion Pythium zoospores were obtined from n rtificilly infested sttic hydroponic system where 5l continers were plnted with Butter lettuce (Lctuc stiv L. vr cpitt L. cv Ndine) seedlings in sterile tp wter. The continers were then inoculted with mcerted 7d old Pythium Group F cultures on V8 gr medium (De Jonghe et l., 2005). This culture ws previously isolted from commercil hydroponic system nd stored in n internl culture collection s UP 92/00, lter deposited t the Ntionl Mycologicl Herbrium (Agriculturl Reserch Council, Vredehuis, Pretori, South Afric) culture collection with reference number PPRI Mcertion ws done by plcing three V8 gr pltes contining the Pythium growth into 800ml sterile wter in n lcohol-sterilised kitchen blender nd pulsing for 0.5s followed by 3s stnding period until visully homogenous suspension ws obtined. This suspension ws then dded to the sttic hydroponic system t rte of 200ml per 5l continer. Regulr Pythium biting (Grimm nd Alexnder, 1973) ws crried out on this wter to ensure the consistent presence (n incidence rting of 70% or greter) of zoospore inoculum. Fusrium solni (isolted from citrus roots in previous study) conidi were hrvested by pouring 5ml sterile deionised wter over fresh culture on PDA medi nd brushing lightly with sterile etleur. The resulting conidil suspension ws removed nd spore count ws done using hemocytometer. Rlstoni solncerum Biovr 3 (isolted from tomto plnts by vn Broekhuizen, 2002) cells were hrvested by pouring 5ml sterile wter over fresh culture on TZC medi nd brushing lightly with sterile etleur. The resulting cell suspension ws removed from the Petri dish nd cells counted using Petroff-Huser counting chmber Snitiser preprtion Prsin (SIDL, South Afric), Fitosn (Helth & Hygiene, South Afric), TecsClor (BTC Products, South Afric), Agrl 90 (Kynoch Chemicls, South Afric) nd Sporekill (Hygrotech Seeds, South Afric) were provided by the vrious mnufctures nd used undiluted

41 Fresh Purogene (BTC Products, South Afric) ws generted for ech experiment ccording to the lbel instructions (ddition of one prt supplied ctivtor to ten prts Purogene ). This ws llowed to rect for 5min before use. Fresh Actsol ws generted for ech experiment using n ECA (ElectroChemicl Activtion) device provided by Rdicl Wters (Midrnd, South Afric) nd freshly prepred brine solution [2.5g NCl (Merck, South Afric) per litre wter] to chieve n Actsol solution of verge ph 7.2 nd ORP 800mV. This freshly prepred solution ws used in ll the experiments. Copper (II) sulphte crystls (Merck, South Afric) were used to provide copper ions when dissolved in wter nd diluted to the finl volume of wter. Detils of ech snitiser re provided in Appendix I: B. Contct detils of ech supplier cn be found in Appendix I: C. The bove snitisers were tested t rnge of concentrtions nd exposure times, s described in Tble 1 below. Tble 1: Concentrtion nd exposure time of chemicls tested in the current study. Product Product concentrtions Exposure times Actsol 1:10, 1:20 nd 100% 10, 30, 60 nd 120min Prsin 5, 7.5, 10, 20, 100, 150, 200, 250 nd 500mg.l -1 10, 30 nd 60min Purogene 5, 10, 25, 50 nd 100mg.l -1 10, 30 nd 60min TecsClor 10, 25, 50 nd 100mg.l -1 10, 30 nd 60min Fitosn 1, 5, 7.5 nd 10mg.l -1 Agrl 90 1, 2.5, 5 nd 10mg.l nd Copper (II) sulphte 0.5, 1, 2, 5, 10 nd 20mg.l nd Sporekill 1, 2.5, 5 nd 10mg.l nd Concentrtions referred to re product concentrtions, i.e. concentrtions mde directly from the stock solutions. Active ingredient concentrtions for ech product re stipulted in Appendix I: B

42 3.3.4 Experimentl procedure For most of the tests, sterilized 500ml Erlenmeyer flsks were filled with 500ml of sterile deionised wter. The exceptions were the Pythium tests where rtificilly infested wter ws used, nd the Actsol tests where the Actsol solution ws diluted with sterile wter to give finl volume of 500ml t the test dilution. Ech product ws then diluted into the Erlenmeyer flsks to give the test dilutions described Tble 1. For ech orgnism n untreted control (no snitiser) ws included. Fusrium inoculum ws dded to give finl concentrtion of pproximtely 10 5 cfu.ml -1, while the Rlstoni ws diluted to n pproximte concentrtion of 10 7 cells.ml -1. Pythium infested wter from the sttic hydroculture described in Section ws used s source of Pythium zoospores, hving concentrtion pproching 10 4 zoospores.ml -1. An dequte smple volume ws tken, stirred to ensure homogenous distribution of zoospores nd then divided eqully into sterile Erlenmeyer flsks. A smple ws lso observed microscopiclly to confirm the presence of zoospores. Directly fter ddition of the inoculum, t time 0, smple ws tken from ech untreted control, with further smples tken t 5min; ; nd 60min, nd processed s described below. A further control smple ws lso tken t the mximum time. For enumertion 50ml smple of the zoospore suspension nd 20ml smple for Fusrium nd Rlstoni ws drwn out of the flsks using sterile 25ml syringe nd filtered through 25cm syringe filter (Osmonics Acette Plus, Seprtions, South Afric) of pore sizes 0.22µm for Rlstoni nd 1.2µm for Pythium nd Fusrium. The filters of the Fusrium nd Rlstoni smples were then plced in 10ml sterile wter in test-tube nd vortexed for 10s, fter which 10x seril dilution of the resulting suspension ws prepred. For Fusrium nd Rlstoni 100µl of ech dilution ws plted out on PDA nd NA respectively, using the spred-plte technique. Pltes were then incubted in drkness t 25ºC for 3d, fter which colony forming units (cfu) were counted nd the cfu/ml clculted. Pythium ws enumerted by biting the suspension from the vortexed test tube contining the filter ccording to modifiction of the biting method described by Grimm nd Alexnder (1976) where citrus lef discs re floted on the surfce of the suspension for

43 24h s opposed to 48h. After 24h the discs were trnsferred to the Pythium selective medium (BNPRA) nd incubted for 3d, fter which the lef discs showing fungl growth were microscopiclly exmined to verify Pythium growth. The number of discs rendering Pythium were counted nd the percentge incidence of the fungus clculted s n indiction of the proportion of live zoospores remining in the suspension. This procedure constitutes semi-quntittive ssessment. Ech experiment ws done in duplicte, with two replicte Petri-dishes being used t ech step Dt nlysis For the respective pthogens, percentge kill ws clculted ccording to the following eqution: T 0 -T x T 0 x 100 Where T 0 = smple tken t time = 0 minutes (control) T x = smple tken fter x minutes. The dt ws sttisticlly nlysed using the SAS for Windows progrm (version 8e) pplying Duncn s Multiple Rnge test t P = A percentge kill of 80%, or higher, ws considered to be positive result

44 3.4 Results Actsol Actsol demonstrted good efficcy ginst ll the test orgnisms nd totlly erdicted Pythium from the test suspension t ll the concentrtions tested, including the shortest exposure time of (Fig. 1). Actsol ws shown to hve dequte efficcy for Pythium kill t the highest dilution of 1:20. Fusrium exhibited typicl dose-response where the kill rte incresed with incresed exposure time, nd this trend ws more noticeble t the lower snitiser concentrtion (Fig. 1b). Rlstoni demonstrted similr trend to Fusrium, lbeit to lesser extent. No significnt (P=0.05) differences were observed between tretments (Fig. 1c). A 100% kill ws recorded for ll pthogens min % Kill 60min 100% 1:10 1:20 Tretment Figure 1: Efficcy of Actsol t vrious concentrtions nd exposure times on Pythium zoospores in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

45 100 b b c b min 120min % Kill 60min 120min 100% 1:10 1:20 Tretment Figure 1b: Efficcy of Actsol t vrious concentrtions nd exposure times on Fusrium conidi in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) % Kill min 60min 100% 1:10 1:20 Tretment Figure 1c: Efficcy of Actsol t vrious concentrtions nd exposure times on Rlstoni plnktonic cells in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Prsin When exposed to Prsin t 5mg.l -1 Pythium exhibited the expected dose response over time. With 5mg.l -1 concentrtion, Prsin chieved greter thn 80% kill of Pythium zoospores t exposure time. Higher snitiser concentrtions resulted in 100% kill of Pythium zoospores within exposure time (Fig. 2)

46 The sme dose response trend ws demonstrted for both Fusrium (Fig. 2b) nd Rlstoni (Fig. 2c), lthough totl kill ws only chieved fter 60min exposure time t 100mg.l -1 concentrtion. Fusrium conidi were less ffected thn Rlstoni cells t the sme concentrtion nd exposure time. 100 b b b b b b b b 80 % Kill min 60min 5mg/l 7.5mg/l 10mg/l 20mg/l Tretment Figure 2: Efficcy of Prsin t vrious concentrtions nd exposure times ginst Pythium zoospores in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). 100 e e d c b bc min 60min 60min 60min % Kill 60min 100mg/l 150mg/l 200mg/l 250mg/l 500mg/l Tretment Figure 2b: Efficcy of Prsin t vrious concentrtions nd exposure times ginst Fusrium conidi in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

47 100 e e e e e e d b c b min 60min 60min 60min % Kill 60min 60min 10mg/l 20mg/l 100mg/l 150mg/l 250mg/l 500mg/l Tretment Figure 2c: Efficcy of Prsin t vrious concentrtions nd exposure times ginst Rlstoni plnktonic cells in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Purogene Exposure of Pythium zoospores to Purogene rendered typicl dose response t 5mg.l -1 snitiser concentrtion where n increse in kill ws chieved with incresing exposure time (Fig. 3). A exposure time t this concentrtion chieved the desired 80% kill. Snitiser concentrtions of 10mg.l -1 or bove chieved 100% kill within exposure time (Fig. 3). Fusrium (Fig. 3b) nd Rlstoni (Fig. 3c) showed similr dose response trends t 20mg.l -1 snitiser concentrtion. Similr results were chieved for both orgnisms t this concentrtion. A snitiser concentrtion of 50mg.l -1 or higher chieved 100% kill within exposure time

48 100 d c b min 60min % Kill 60min 60min 5mg/l 10mg/l 25mg/l 50mg/l Tretment Figure 3: Efficcy of Purogene t vrious concentrtions nd exposure times ginst Pythium zoospores in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). 100 f f e d c b min 60min 60min % Kill 60min 10mg/l 20mg/l 50mg/l 100mg/l Tretment Figure 3b: Efficcy of Purogene t vrious concentrtions nd exposure times ginst Fusrium conidi in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

49 100 f f e d c b min 60min 60min % Kill 60min 10mg/l 20mg/l 50mg/l 100mg/l Tretment Figure 3c: Efficcy of Purogene t vrious concentrtions nd exposure times ginst Rlstoni plnktonic cells in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) TecsClor Exposure of Pythium zoospores to TecsClor t 10mg.l -1 did not result in typicl dose response with incresing time, lthough totl kill of zoospores ws chieved within exposure to 25mg.l -1 TecsClor concentrtion (Fig. 4). The typicl dose response ws observed for Fusrium (Fig. 4b) nd Rlstoni (Fig. 4c) t 50mg.l -1 snitiser concentrtion where incresed percentge kill ws observed with incresing exposure time. Rlstoni cells lso showed slightly higher sensitivity thn Fusrium conidi t this concentrtion, with higher level of kill chieved with Rlstoni t the sme concentrtion nd time exposure

50 100 d c b min 60min % Kill 60min 60min 10mg/l 25mg/l 50mg/l 100mg/l Tretment Figure 4: Efficcy of TecsClor t vrious concentrtions nd exposure times ginst Pythium zoospores in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). 100 g f f f f e d c b min 60min 60min % Kill 60min 10mg/l 20mg/l 50mg/l 100mg/l Tretment Figure 4b: Efficcy of TecsClor t vrious concentrtions nd exposure times ginst Fusrium conidi in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

51 100 f f f f f e d c b min 60min 60min % Kill 60min 10mg/l 20mg/l 50mg/l 100mg/l Tretment Figure 4c: Efficcy of TecsClor t vrious concentrtions nd exposure times ginst Rlstoni plnktonic cells in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test with P= Fitosn Fitosn ws only tested ginst Pythium zoospores for exposure time, where clssic dose response ws observed with stedy increse in zoospore kill being obtined with incresing snitiser concentrtion (Fig. 5). Totl kill of zoospores ws chieved t 7.5mg.l -1 nd 10mg.l -1 concentrtions. 100 c b 80 % Kill mg/l 5mg/l 7,5mg/l 10mg/l Tretment Figure 5: Efficcy of Fitosn t vrious concentrtions nd exposure time on Pythium zoospores in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

52 3.4.6 Agrl 90 Pythium zoospore survivl showed n typicl trend when exposed to incresing Agrl 90 concentrtions t exposure time (Fig. 6). This observtion showed trend which is effectively n inverse of the expected dose response, where decresed efficcy ws noted with n incresed snitiser concentrtion. This trend ws lso demonstrted by Fusrium where 10mg.l -1 concentrtion for both nd exposure time showed lower level of efficcy thn 1mg.l -1 or 5mg.l -1 concentrtion t the sme exposure times (Fig. 6b). Rlstoni cells showed high levels of tolernce to Agrl 90 with the desired 80% kill level not being chieved in the tested concentrtion rnge nd exposure time (Fig. 6c). These results showed similr trend to those observed with the Pythium nd Fusrium tests. 100 b c 80 % Kill mg/l 5mg/l 10mg/l Tretment Figure 6: Efficcy of Agrl 90 t vrious concentrtions t exposure time on Pythium zoospores in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

53 100 b b b 80 % Kill mg/l 5mg/l 10mg/l Tretment Figure 6b: Efficcy of Agrl 90 t vrious concentrtions nd exposure times on Fusrium conidi in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). 100 c c d d d c b % Kill 1mg/l 2.5mg/l 5mg/l 10mg/l Tretment Figure 6c: Efficcy of Agrl 90 t vrious concentrtions nd exposure times on Rlstoni plnktonic cells in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Copper sulphte Pythium zoospores, when exposed to incresing levels of copper ions for, demonstrted regulr dose response with higher levels of kill being chieved with n increse in copper ion concentrtion (Fig. 7)

54 Copper ion concentrtions of 5mg.l -1 nd higher chieved 100% kill of zoospores, while 1mg.l -1 concentrtion chieved percentge kill of over 80%. Both Fusrium (Fig. 7b) nd Rlstoni (Fig. 7c) demonstrted similr dose responses with higher level of efficcy being noted t incresed copper ion concentrtions nd longer exposure times. Rlstoni ws shown to be less sensitive thn Fusrium, with lower levels of efficcy observed with Rlstoni cells t the sme concentrtion nd exposure time. 100 b 80 % Kill mg/l 5mg/l 10mg/l 20mg/l Tretment Figure 7: Efficcy of copper ions t vrious concentrtions t exposure time on Pythium zoospores in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). 100 c b % Kill 1mg/l 5mg/l 10mg/l Tretment Figure 7b: Efficcy of copper ions t vrious concentrtions nd exposure times on Fusrium conidi in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

55 100 d c e c b % Kill 0.5mg/l 1mg/l 2mg/l Tretment Figure 7c: Efficcy of copper ions t vrious concentrtions nd exposure times on Rlstoni plnktonic cells in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Sporekill When exposed to incresing concentrtions of Sporekill for, Pythium zoospore survivl ws decresed t n esclting level (Fig. 8). At 5mg.l -1 totl elimintion ws chieved. This is the expected dose response. Fusrium conidi lso demonstrted this clssic dose response with ner liner increse in efficcy with incresing exposure time or concentrtion, with the exception of exposure t 5mg.l -1 which showed n unexpected decrese in efficcy, below tht expected from the other results (Fig. 8b). Rlstoni cells initilly lso demonstrted n expected dose response with the exception of exposure t 2.5mg.l -1 which yielded result higher thn would be expected (Fig. 8c). Excluding this nomlous singulrity the other results demonstrted the expected trend

56 100 b 80 % Kill mg/l 5mg/l 10mg/l Tretment Figure 8: Efficcy of Sporekill t vrious concentrtions t exposure time ginst Pythium zoospores in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). 100 f d c e b % Kill 1mg/l 5mg/l 10mg/l Tretment Figure 8b: Efficcy of Sporekill t vrious concentrtions nd exposure times ginst Fusrium conidi in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

57 100 d c b 80 % Kill mg/l Tretment 2.5mg/l Figure 8c: Efficcy of Sporekill t vrious concentrtions nd exposure times ginst Rlstoni plnktonic cells in queous suspension. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

58 3.5 Discussion All the products were effective ginst Pythium zoospores in n queous suspension t reltively low concentrtions. Presumbly due to the presence of cell wll nd more complex brrier, Fusrium nd Rlstoni required exposure to higher concentrtions of the snitisers before effective reduction in orgnism survivl ws noted. This ws to be expected since the zoospores with only simple cell membrne re likely to be more vulnerble to the snitisers. This ws in greement with results obtined by Stnghellini nd Tomlinson (1987) who showed tht the non-ionic surfctnt Agrl 90 ws ble to cuse lysis of Pythium zoospores nd inhibit root infection nd growth. In prllel trils (described in Chpter 4) the phytotoxic nture of Sporekill nd copper ws determined, these chemicls were subsequently tested t lower concentrtions ginst Rlstoni to scertin whether testing t lower concentrtions would be indicted. As the lowered concentrtions ginst Rlstoni did not exhibit dequte efficcy, these low concentrtions were not tested dditionlly ginst Pythium nd Fusrium. From these experiments the most effective dosges for control of Pythium in wter for the respective compounds t exposure time were: Actsol t 1:10 dilution (one prt Actsol to ten prts wter); Prsin t concentrtion of 5mg.l -1 ; Purogene t concentrtion of 10mg.l -1 ; TecsClor t concentrtion of 25mg.l -1 ; Fitosn t concentrtion of 7.5mg.l -1 ; Agrl 90 t concentrtion of 1mg.l -1 ; copper sulphte t concentrtion of 1mg.l -1 nd Sporekill t concentrtion of 5mg.l -1. The Agrl 90 results ws consistent throughout ll the experiments, yet did not follow the expected trend, nor did these results concur with those of Stnghellini nd Tomlinson (1987) who demonstrted incresing ctivity with incresing concentrtion. A concentrtion of 1mg.l -1 did, however, show similr effect on the zoospores. The nomly in the current experiment cn possibly be explined by the fct tht the higher concentrtions cuse rpid encystment of the Pythium zoospores (vn West et l., 2003) with n ssocited increse in resistnce to the snitiser, while lower concentrtions ffect the zoospores directly nd cuse lysis before encystment cn occur. This inverse trend is not demonstrted to the sme degree in the results of tests conducted on Rlstoni nd

59 Fusrium, indicting tht the more complex nture of the cell wlls nd cell membrnes of these orgnisms my id in the resistnce to Agrl 90. The trends shown by the results of wter tretment with the other snitisers were s expected, where decrese in inoculum survivl ws seen with incresing snitiser concentrtions. An incresed exposure time generlly resulted in miniml increse in kill rte, indicting tht the effects of the snitisers re more of n immedite nture s opposed to cumultive effect over time. The results further confirm the hypothesis tht the more complex cell wll nd cell membrne structures found in Rlstoni nd Fusrium the less effective the snitiser. This sme trend hs lso previously been reported by Hudson et l. (1986), Koponen et l. (1992) nd Meblds et l. (1997) where orgnisms with incresing brrier complexity showed decresing sensitivity to wter snitisers. It is possible tht the complex Grm negtive structure of the Rlstoni cell wlls nd cell membrnes resulted in the gretest resistnce to the effects of the snitisers s well s the destructive effects of the copper ion tretment. This dt lso indictes tht the simple membrne of Pythium zoospore results in this structure being highly sensitive to the effects of wter snitisers. From this dt it cn be presumed tht the ddition of the bove products to Pythium infested wter supply of hydroponic system would effectively inctivte or kill Pythium inoculum nd lso gretly reduce Fusrium nd Rlstoni inoculum levels. However, it must be borne in mind tht these experiments were conducted in the bsence of orgnic mtter nd other contminnts which would be present in commercil system. Therefore, in recirculting hydroponic system the exposure time is not considered criticl since, if effective mixing occurs, the product will remin in the solution until the solution is replced or the product dissiptes s would be the cse with the Actsol, Purogene nd TecsClor where the ctive ingredients will tend to voltilise, or be degrded s would lso be expected with Prsin nd Fitosn

60 3.6 References. Adler, P.R., Summerfelt, S.T., Glenn, D.M. nd Tked, F Mechnistic pproch to phytoremedition of wter. Ecologicl Engineering 20: Andrews, L.S., Key, A.M., Mrtin, R.L., Grodner, R. nd Prk, D.L Chlorine dioxide wsh of shrimp nd crwfish: An lterntive to queous chlorine. Food Microbiology 19: De Jonghe, K., De Dobbelere, I., Srrzyn, R. nd Hofte, M Control of brown root rot cused by Phytophthor cryptoge in the hydroponic forcing of witloof chicory (Cichorium intybus vr. foliosum) by mens of nonionic surfctnt. Crop Protection 24: Do Socorro, M., Bstos, R., de Ftim Ferreir Sores, N., de Andrde, N.J., Arrud, A.C. nd Alves, R.E The effect of the ssocition of snitisers nd surfctnt in the microbiot of the cntloupe (Cucumis melo L.) melon surfce. Food Control 16: Grimm, G.R. nd Alexnder, A.F Citrus lef pieces s trps for Phytophthor prsitic from soil slurries. Phytopthology 63: Hudson, H.R., Ojo, I.A.O. nd Pink, M Gunidines with ntifungl (nd ntibcteril) ctivity review. Interntionl Pest Control: Koponen, H., Avikinen, H. nd Thvonen, R The effect of disinfectnts on fungi in pure culture nd on different surfce mterils. Agriculturl Science Finlnd 1: Kuchrek, T. nd Mitchell, D Diseses of gronomic nd vegetble crops cused by Pythium. Plnt Pthology Fct Sheet PP-53. Florid Coopertive Extension Service, Institute of Food nd Agriculturl Sciences, University of Florid, Christine Wddill, Den

61 Lee, S., Gry, P.M., Dougherty, R.H. nd Kng, D The use of chlorine dioxide to control Alicyclobcillus cidoterrestris spores in queous suspension nd on pples. Interntionl Journl of Food Microbiology 92: Meblds, M., Bnkier, M. nd Berdsell, D Wter disinfections control pthogens. Austrlin Horticulture, April 1997: Monrc, M., Zni, C., Richrdson, S.D., Thruston Jr, A.D., Moretti, M., Feretti, D. nd Villrini, M A new pproch to evluting the toxicity nd genotoxicity of disinfected drinking wter. Wter Reserch 38: Peng, J., Tsi, W. nd Chou, C Inctivtion nd removl of Bcillus cereus by snitiser nd detergent. Interntionl Journl of Food Microbiology 77: Roux, C. nd Both, W.J An introduction to the Pythicee in South Afric. Agriculturl Reserch Council, Plnt Protection Reserch Institute, Roodeplt. Runi, W Disinfection of recircultion wter from closed cultivtion systems with ozone. Act Horticulture 361: Runi, W A review of possibilities for disinfection of recircultion wter from soilless cultures. Act Horticulture 382: Sb, A. nd Messin, F Attitudes towrds orgnic foods nd risk/benefit perception ssocited with pesticides. Food Qulity nd Preference 14: Singh, N, Singh, R.K., Bhuni, A.K. nd Stroshine, R.L Efficcy of chlorine dioxide, ozone, nd thyme essentil oil or sequentil wshing in killing Escherichi coli O157:H7 on lettuce nd bby crrots. Food Science nd Technology 35: Stnghellini, M.E. nd Tomlinson, J.A Inhibitory nd lytic effects of nonionic surfctnt on vrious sexul stges in the life cycle of Pythium nd Phytophthor species. Phytopthology 77:

62 Stnghellini, M.E., Kim, D.H., Rsmussen, S.L. nd Rorbugh, P.A Control of root rot of peppers cused by Phytophthor cpsici with nonionic surfctnt. Plnt Disese 80: Stnghellini, M.E. nd Miller, R.M Biosurfctnts: Their identity nd potentil efficcy in the biologicl control of zoosporic plnt pthogens. Plnt Disese 81: vn Broekhuizen, W Detection, chrcteristion nd suppression of Rlstoni solncerum. Appendix 2, M.Sc Disserttion, University of Pretori, South Afric. vn West, P., Appih, A.A. nd Gow, N.A.R Advnces in reserch on oomycete root pthogens. Physiologicl nd Moleculr Plnt Pthology 62:

63 CHAPTER 4 IN VIVO ASSESSMENT OF PHYTOTOXICITY OF SANITISERS ON CUCUMBER AND LETTUCE PLANTS 4.1 Abstrct In Chpter Three it ws demonstrted tht the snitisers being tested were ble to eliminte Pythium infesttion from volume of wter while lso reducing levels of Fusrium nd Rlstoni. The im of this Chpter ws to evlute the phytotoxic effect of the snitisers on cucumber nd lettuce plnts in vivo. Two plnt models were used to ssess phytotoxic effects nd estblish threshold dosges (in terms of phytotoxicity) of the vrious snitisers. These models were rpid model using cucumber seedlings (Cucumis stiv L.) exposed to the snitisers in sttic hydroponic system under controlled conditions, nd model using Butter lettuce (Lctuc stiv L.) cultivted in greenhouse-scle grvel bed recirculting hydroponic system under controlled greenhouse conditions. The snitisers tht were observed to be highly phytotoxic on cucumber plnts were: Actsol, Copper, Prsin nd Sporekill, with copper being most phytotoxic t concentrtions bove 2mg.l -1. Phytotoxicity mnifested minly s stunting of growth nd lef development nd reduction in fresh biomss of both folir plnt prts nd roots, when compred to the untreted control. Actsol nd copper tretments resulted in yellowing of the leves. An interesting spect observed in the cucumber model ws tht the chlorine-dioxide bsed snitisers (Purogene nd TecsClor ) cused slight growth stimulting effect on the cucumber seedlings nd no observble phytotoxic effects, t concentrtions lower thn 50mg.l -1. In the lettuce model, t lower Actsol concentrtions of 1:50 nd 1:100 phytotoxic effects were reduced, while concentrtion of 100mg.l -1 Prsin cused n unexpected result in tht lesser reduction in fresh mss ws observed when compred with the 7.5mg.l -1 tretment. Tretment of the nutrient solution with TecsClor did not result in ny visible or mesurble phytotoxic effects on lettuce plnts fter four week exposure time t concentrtions up to 100mg.l -1. The finl conclusion from the current study is tht the snitisers could further be tested t low concentrtions for disese control or yield enhncement in pthogen infested hydroponic systems

64 4.2 Introduction Although it hs been shown tht mny snitisers cn effectively reduce the levels of Pythium zoospores in wter suspension (Koponen et l., 1992; Meblds et l., 1997), this tretment cnnot necessrily be pplied directly into hydroponic nutrient solution tht feeds hydroponic plnt roots since exposure my result in phytotoxic effects on the plnts (Nlecz-Jwecki et l., 2003). An intermedite step is necessry to scertin whether the snitisers under investigtion hve ny phytotoxic effects, in terms of growth reduction, discolourtion or ny other effects, which would disdvntge the mrketbility of the crop in question, nmely Butter lettuce (Lctuc stiv L. vr cpitt L. cv Ndine). This study imed to ddress this by subjecting two models (discussed below) to the snitisers t concentrtion rnge centred on the most effective concentrtions s estblished in previous chpter (Chpter 3 of this study). The two plnt models used were: 1) A rpid lbortory model, using fst growing crop which is known for sensitivity to phytotoxic effects (cucumber) nd cultivted under ccurtely controlled climtic conditions, nd 2) A scle model using slower growing crop (lettuce) grown under greenhouse conditions which pproximte field conditions. The im of this study ws to estblish the phytotoxicity thresholds of the two crops to ech snitiser, with the im of estblishing whether the effective concentrtions could be included in nutrient solution in recirculting hydroponic system. For the rpid model, cucumber (Cucumis stiv L.) ws selected s this plnt is both n importnt hydroponic crop (Pulitz et l., 1992) nd it cn be rpidly cultivted to n ge where ny phytotoxicity effects would be evident. Cucumbers re lso susceptible to infection by ll the pthogens ssessed in the previous chpter (Chpter 3) (Pulitz et l., 1992; Frvel nd Lrkin, 2002; Lemy et l., 2003). Cucumber plnts hve been reported s hving sensitivity to numerous chemicls nd re therefore suitble s monitors of environmentl contmintion (Migliore et l., 2003). Phytotoxic effects hve lso been well documented for this crop (Vinit-Dunnd et l., 2002; Wng et l., 2002). This dt indicted tht cucumbers would llow for rpid ssessment of even miniml phytotoxic effects

65 Hund-Rinke nd Kordel (2003) lso demonstrted the benefits nd incresed rte t which phytotoxic effects cn be observed with lbortory scle experiments under controlled conditions s precursor to more lengthy nd complicted field-scle experiments. Butter lettuce ws selected s the crop used in the greenhouse hydroponic system s it is commercilly importnt hydroponic crop nd will be the min focus of this study. Butter lettuce is lso bundntly vilble nd is less sensitive to phytotoxic effects by t lest one of the snitisers under investigtion in this study (Crrillo et l., 1996). Lettuce still remins sensitive enough to phytotoxic effects to be considered n cceptble crop to be used s monitor of phytotoxic effects (Migliore et l., 2003). Furthermore, both lettuce nd cucumbers re listed s cceptble crops in the Ecologicl Effects Test Guidelines (1996), which describes procedures for phytotoxicity evlutions on non-trget crops. Thus the results obtined from this study would give n indiction of the likely effects these snitisers would hve on the mjority of crops

66 4.3 Mterils nd Methods Cucumber model Cucumber vriety nd germintion Disese-free seeds of commercil vriety (Dlt 22) of prthenocrpic English cucumber (Cucumis stiv L.) were obtined from Hygrotec Seeds (South Afric). The seeds were plnted in sterilised vermiculite (utoclved t 121 C for 15min with n inclusion of 100ml tp wter.kg -1 vermiculite), which ws liberlly moistened with sterilised tp wter (utoclved t 121 C for 15min). The seeds were then germinted for 7d in n environmentlly controlled growth cbinet (Conviron ) with conditions set t 25 C with 65% reltive humidity (RH) nd no light Sttic hydroculture Distilled wter ws used to prepre 1l btches of stndrd hydroponic nutrient solution (Appendix I: A, Solution 1). Approprite volumes of ech snitiser were then dded to the nutrient solution to chieve the required test concentrtions. The solution ws mixed thoroughly by mnul gittion for 10s. The resulting solution ws then dispensed into clen 250ml plstic continers. Eight continers were prepred for ech snitiser concentrtion. For ech snitiser concentrtion rnge control ws lso prepred s bove, with no snitiser dded to the nutrient solution. The entire volume of nutrient solution ws replced with freshly prepred nd treted nutrient solution fter one week of growth to mintin idel nutrient growth conditions nd constnt snitiser concentrtions. Cucumber seedlings of equivlent size nd ppernce were selected nd plnted singly into the prepred continers. Seedlings were kept upright by mking n incision into the lid of ech continer nd plcing the seedling into this incision in such wy tht the roots were completely immersed in nutrient solution (Plte 1: A). The stems were supported by thin strip of fom rubber to prevent dmge

67 Growth conditions The continers contining the cucumber seedling were plced in Conviron controlled environment growth cbinet set in cycle of 25 C, 66% RH, with simulted dylight for 12h followed by conditions of 20 C, 60% RH nd totl drkness for 12h. The plnts were visully observed dily for signs of phytotoxicity Phytotoxicity ssessment After 14d of growth the seedlings were gin observed for ny visible signs of phytotoxicity such s colour chnges in leves, generl lef size nd development nd root development. The plnts were then hrvested, the roots excised nd fresh weight of shoots nd roots determined by weighing (Vinit-Dunnd et l., 2002) Anlysis Root nd shoot mss dt ws sttisticlly nlysed using Duncn s Multiple Rnge test t P = 0.05, utilizing the SAS for Windows version 8.0e softwre pckge Lettuce model Lettuce vriety nd germintion Disese-free Butter lettuce seeds were germinted t commercil hydroponics grower (Hydrotec, South Afric) under conditions preventing generl disese infesttion. Clen seedling trys were filled with stem psteurised vermiculite nd pet mixture (80:20) tht ws used s the germintion medium. The seeds were wtered every 20min, during dylight hours, by overhed emitters, supplied with pthogen-free wter. Seedlings were germinted t environmentl conditions under shde net structure

68 Smll scle grvel bed hydroponic system A smll scle grvel bed hydroponic system (bsed on the grvel film technique) ws ssembled in n environmentlly controlled greenhouse (Plte 1: B). This system consisted of ten 100l reservoirs, ech contining 100l het psteurized tp wter nd hydroponic nutrient mixture (Appendix I: A, Solution 1). Ech reservoir supplied nutrient solution to three plstic (PVC) troughs of equl lengths (2.5m) by mens of submersible pump within ech reservoir. Ech trough ws filled to level of 8cm with wshed grvel (crushed dolerite / grnite chips of pproximtely 15mm). Nutrient solution flow ws limited to 400mg.min -1.trough -1. The outflow solution ws collected t the lower end of ech trough due to grdient nd recirculted bck into the 100l reservoir by mens of grvity flow. The entire volume of nutrient solution in ech reservoir ws replced on weekly bsis. Snitisers were dded to ech reservoir during this preprtion of the nutrient solution t the necessry dosges required to chieve the required test concentrtion rnges. Ech of the three troughs supplied by single reservoir were plnted with 15 28d Butter lettuce seedlings plced equidistnt from ech other, resulting in totl of 45 plnts per tretment (15 plnts per trough) (Plte I: B). Plnts were llowed to grow nturlly for totl of 28d nd were inspected every 2d for ny visible symptoms of phytotoxicity or growth problems Growth conditions Environmentl conditions were mintined within the greenhouse t n verge RH of 65%, n verge mximum dytime temperture of 28 C nd verge minimum nightly temperture of 18 C. Light conditions were s nturl nd no supplementtion ws dded, resulting in verge length of dylight being pproximtely 13h

69 Phytotoxicity ssessment After 28d the lettuce plnts were observed for ny visible signs of phytotoxic effects fter which they were hrvested. The shoots nd roots were seprted from ech other nd their fresh mss determined seprtely (Migliore et l., 2003). Hrvesting nd weighing ws completed before 10m for ech experiment to minimize possible growth-cycle differences. Selected root smples were nlysed for Pythium infection by plting 3mm root tip pieces t rte of five per Petri-dish on Pythium-selective medium (Roux nd Both, 1997) to determine the bsence or presence of infection Anlysis Root nd shoot mss dt ws sttisticlly nlysed by mens of Duncn s Multiple Rnge test t P = 0.05, utilizing the SAS for Windows version 8.0e softwre pckge Snitiser preprtion For both models snitisers were prepred s follows, with the concentrtions tested in ech model detiled in Tble 1. Prsin (SIDL, South Afric), Fitosn (Helth & Hygiene, South Afric), TecsClor (BTC Products, South Afric), Agrl 90 (Kynoch chemicls, South Afric) nd Sporekill (Hygrotech, South Afric) were used directly from the solution provided by the mnufcturer. Fresh Purogene (BTC Products, South Afric) ws generted for ech experiment ccording to lbel instructions (ddition of one prt supplied ctivtor to ten prts Purogene ). This ws llowed to rect for 5min before use. Fresh Actsol ws generted for ech experiment using n ECA (ElectroChemicl Activtion) device provided by Rdicl Wters (Midrnd, South Afric) nd freshly prepred brine solution [2.5g NCl (Merck, South Afric) per litre wter] to chieve n Actsol solution of verge ph 7.2 nd ORP 800mV. This solution ws used directly in the experiments

70 Copper (II) sulphte crystls (Merck, South Afric) were dissolved in de-ionised wter nd diluted to the finl volume of wter. Detils of ech snitiser re provided in Appendix 1: B). Contct detils of ll suppliers re provided in Appendix I: C. Tble 1: Concentrtions of snitisers tested in the cucumber nd lettuce models Product Cucumber Model Lettuce Model Actsol 1:10; 1:20; 1:50; 1:100 nd 1:200 1:10; 1:20 nd 1:50 Prsin 5mg.l -1 ; 7.5mg.l -1 nd 100mg.l mg.l -1 ; 5mg.l -1 ; 7.5mg.l -1 nd 100mg.l -1 Purogene 2.5mg.l -1 ; 5mg.l -1 ; 10mg.l -1 ; 25mg.l -1 nd 50mg.l mg.l -1 ; 10mg.l -1 nd 50mg.l -1 TecsClor 10mg.l -1 ; 50mg.l -1 nd 100mg.l -1 25mg.l -1 ; 50mg.l -1 nd 100mg.l -1 Fitosn 1mg.l -1 ; 2.5mg.l -1 ; 5mg.l -1 ; 7.5mg.l -1 ; 10mg.l -1 nd Not tested 15mg.l -1 Copper 1mg.l -1 ; 2mg.l -1 ; 5mg.l -1 ; 10mg.l -1 nd 20mg.l -1 Not tested Sporekill 1mg.l -1 ; 5mg.l -1 ; 7.5mg.l -1 nd 10mg.l -1 Not tested Concentrtions referred to re product concentrtions, i.e. concentrtions mde directly from the stock solutions. Active ingredient concentrtions for ech product re specified in Appendix I: B

71 4.4 Results Cucumber model Actsol Young cucumber plnts demonstrted severe phytotoxic effects when exposed to high concentrtions of Actsol, while plnt growth (s mesured by fresh biomss differences) ws significntly reduced (P=0.05) to less thn hlf tht observed in the untreted control, with the exception of the 1:200 concentrtion (Fig. 1). Stunting nd reduced growth ws visully observed within 7d fter exposure with miniml growth being observed fter the initil exposure when subjected to concentrtions of 1:10 nd 1:20. The trend displyed does follow the expected dose response with incresed effects being noted with n increse in Actsol concentrtion (Fig. 1), lthough the effects were more severe thn expected. Actsol ws observed to be highly phytotoxic cross the entire rnge of tested concentrtions, exhibiting symptoms such s stunting of growth nd lef development nd n ssocited reduction in fresh biomss of both eril plnt prts (shoot mss) nd roots (root mss). Generl root development ws reduced when compred to the untreted control (Fig. 1). No observble discolourtion ws however noted on leves. Averge fresh biomss (g) c c c b b d d d c b Control 1:10 1:20 1:50 1:100 1:200 Concentrtion Root Mss Shoot Mss Figure 1: Phytotoxic effects of Actsol on cucumber seedlings grown for 14d in sttic hydroponic nutrient solution. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

72 Prsin Prsin demonstrted n expected dose response trend. The cucumber seedlings demonstrted incresed phytotoxic effects with n increse in snitiser concentrtion (Fig. 2). A concentrtion of 100mg.l -1 prevented ll further growth nd development over the initil size of the seedlings. The phytotoxic effects mnifested s stunting of growth nd development of both eril plnt prts (lef size nd formtion) nd roots (reduced development) nd n ssocited decrese in fresh biomss fter two weeks exposure (Fig. 2). No distinguishble discolourtion or yellowing of the leves ws noted. Averge fresh biomss (g) b c d b c Control 5mg/l 7.5mg/l 100mg/l Concentrtion Root Mss Shoot Mss Figure 2: Phytotoxic effects of Prsin on cucumber seedlings grown in sttic hydroculture for 14d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Purogene After 14d exposure to vrious concentrtions of Purogene cucumber seedlings demonstrted no significnt (P=0.05) difference to the untreted control, even t high concentrtion of 50mg.l -1 (Fig. 3). Both lef nd root growth nd development ws comprble to tht of the untreted control, nd no visible signs of phytotoxicity were observed

73 Averge fresh biomss (g) b b b b Control 2.5mg/l 5mg/l 10mg/l 25mg/l 50mg/l Concentrtion Root Mss Shoot Mss Figure 3: Effects of Purogene on cucumber seedlings grown in sttic hydroculture for 14d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) TecsClor Cucumber seedlings exposed to TecsClor concentrtions of up to 100mg.l -1 demonstrted no significnt (P=0.05) differences between ny of the concentrtions in fresh root or shoot mss (Fig. 4). Root nd lef growth nd development ws comprble cross ll tretments nd showed no visible symptoms of phytotoxicity. Averge fresh biomss (g) Control 10mg/l 50mg/l 100mg/l Concentrtion Root Mss Shoot Mss Figure 4: Effects of TecsClor on cucumber seedlings grown in sttic hydroculture for 14d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

74 Fitosn After 14d exposure to Fitosn concentrtions cucumber seedlings exhibited n expected dose response rection where decrese in fresh root nd shoot biomss ws observed with incresing concentrtion up to concentrtion of 7.5mg.l -1, fter which no further effects on development, s evluted by fresh mss, were observed (Fig. 5). A high concentrtion of 15mg.l -1 resulted in visibly lighter green lef, nd reduced development of the 3 rd true lef. Averge fresh biomss (g) b b c d d d b b b b b Control 1mg/l 2.5mg/l 5mg/l 7.5mg/l 10mg/l 15mg/l Concentrtion Root Mss Shoot Mss Figure 5: Phytotoxic effects of Fitosn on cucumber seedlings grown in sterile hydroponic nutrient solution, expressed s chnge in fresh biomss fter 14d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Copper sulphte Copper sulphte t concentrtions of bove 2mg.l -1 resulted in visible severe phytotoxic effects on cucumber seedlings fter the first week of growth. During the second week of growth no further development ws noted t the 5mg.l -1 nd 10mg.l -1 concentrtions nd plnt deth ws seen t the 20mg.l -1 concentrtion (Fig. 6). Plnt development rte ws reduced when compred to the untreted control, nd visible lightening in colour of the leves ws observed (Plte 1: C)

75 Averge fresh biomss (g) b b b b b b b b Control 1mg/l 2mg/l 5mg/l 10mg/l 20mg/l Concentrtion Root Mss Shoot Mss Figure 6: Phytotoxic effects of copper t vrious concentrtions on cucumber seedlings grown in hydroponic nutrient solution fter 14d growth. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Sporekill An incresing concentrtion of Sporekill did not result in either the expected liner or exponentil increse in phytotoxic effects on 14d old cucumber seedlings but rther n inconsistent increse in phytotoxic effects (Fig. 7). Concentrtions of 1mg.l -1 nd 5mg.l -1 resulted in significnt (P=0.05) stunting of plnt development, s mesured by reduction in fresh root nd shoot mss, both these concentrtions yielded similr nd insignificntly different results from ech other. The higher concentrtions of 7.5mg.l -1 nd 10mg.l -1 resulted in further, significnt, decrese in plnt root nd shoot mss over the untreted control but were insignificntly different between them (Fig. 7). Averge fresh biomss b b c c b b c c Control 1mg/l 5mg/l 7.5mg/l 10mg/l Concentrtion Root Mss Shoot Mss Figure 7: Phytotoxic effects of Sporekill t vrious concentrtions on cucumber seedlings grown in hydroponic nutrient solution fter 14d growth. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

76 4.4.2 Smll scle grvel bed hydroponic system (lettuce model) Actsol Lettuce seedlings exposed to 1:10 concentrtion of Actsol showed extreme phytotoxic effects in terms of wilting nd lef discolourtion within three dys of exposure nd totl plnt deth occurred during the second week of exposure. Actsol concentrtions of 1:20 nd 1:50 were significntly (P=0.05) less thn the untreted control (in terms of fresh lef plnt mss) (Fig. 8). These two tretments gve equivlent results which did not differ significntly. Averge fresh biomss (g) c b b b Control 1:10 1:20 1:50 Concentrtion Root Mss Shoot Mss Figure 8: Phytotoxic effects of Actsol on lettuce plnts grown in grvel bed hydroponic system for 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Prsin Prsin tretment of the nutrient solution t incresing concentrtions resulted in the lettuce plnts exhibiting n expected dose response, with decresing fresh plnt root nd shoot mss up to concentrtion of 7.5mg.l -1 (Fig. 9). A dosge concentrtion of 100mg.l -1 gve n unexpected nd nomlous result in tht lower reduction in fresh mss ws observed when compred to the 7.5mg.l -1 tretment

77 Averge fresh biomss (g) b bc d c b b b b Control 2.5mg/l 5mg/l 7.5mg/l 100mg/l Concentrtion Root Mss Shoot Mss Figure 9: Phytotoxic effects of Prsin on lettuce plnts grown in grvel bed hydroponic system for 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Purogene Exposure of lettuce plnts to incresing concentrtions of Purogene resulted in n inverted dose response where n increse in fresh plnt root nd shoot mss ws observed up to 10mg.l -1 (Fig. 10). A dosge of 50mg.l -1 resulted in stunting of plnt development nd reduction in fresh plnt root nd shoot mss in comprison to the untreted control. Averge fresh biomss (g) c b d b Control 2.5mg/l 10mg/l 50mg/l Concentrtion Root Mss Shoot Mss Figure 10: Phytotoxic effects of Purogene on lettuce plnts grown in grvel bed hydroponic system for 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

78 TecsClor Tretment of the nutrient solution with TecsClor did not result in ny visible or mesurble phytotoxic effects on lettuce plnts fter 28d exposure time t concentrtions up to 100mg.l -1 (Fig. 11). A minor increse in fresh plnt root nd shoot mss compred to the untreted control ws observed for ech tretment, lthough not sttisticlly significntly in terms of fresh plnt mss (P=0.05). Averge fresh biomss (g) b b Control 25mg/l 50mg/l 100mg/l Concentrtion Root Mss Shoot Mss Figure 11: Effects of TecsClor on lettuce plnts grown in grvel bed hydroponic system for 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

79 4.5 Discussion The results obtined from the cucumber model indicted tht the selection nd design of the model ws pproprite for the initil testing since, where pplicble, visible phytotoxic effects were seen within the first week of exposure to the snitisers. This model is therefore suitble for the purpose of the study in ccordnce with the report of Hund-Rinke nd Kordel (2003). The cucumber model lso showed tht copper nd quternry mmonium compoundcontining snitisers (Sporekill, Fitosn nd Prsin ) hd definite phytotoxic effects s mesured in terms of fresh biomss reduction (Migliore et l., 2003), confirming previous results where similr phytotoxic effects were demonstrted (Wng et l., 2001; Vinit- Dunnd et l., 2002; Nlecz-Jwecki et l., 2003). At these low concentrtions, phytotoxicity of the bove products mnifested only s stunting of growth, s opposed to other visible symptoms, which implies tht these tretments should not result in ny negtive consumer impct. Therefore, t the lowest concentrtions these snitisers could still be considered s vible wter tretment options if the increse in yield due to disese control outweighs the cost of tretment nd crop yield losses due to phytotoxicity. The most interesting spect observed in the cucumber model ws tht the chlorine-dioxide bsed snitisers (Purogene nd TecsClor ) hd slight growth stimulting effect on the cucumber seedlings nd no observble phytotoxic effects t concentrtions lower thn 50mg.l -1. These findings re confirmed by Crrillo et l. (1996) where single dose of chlorine-dioxide ws lso shown to hve growth enhncing effects, while high dosge levels resulted in phytotoxic effects. The trend seen in the cucumber model ws gin observed in the smll scle grvel bed hydroponic system (lettuce model), where ll the snitiser tretments resulted in similr effects to those seen in the cucumber model. Phytotoxic effects in the lettuce model were limited to growth (lef nd root development) stunting or totl deth of the plnts within two weeks. No visible signs of wilting, yellowing or other folir symptoms were observed. As with the cucumber model, the chlorine-dioxide bsed snitisers resulted in growth enhncement, while the quternry mmonium compound (QAC) bsed snitisers resulted in reduction in fresh biomss, indicting growth stunting effect

80 The growth enhncement seen in the chlorine-dioxide bsed snitisers could be ttributed to the fct tht the ctive ingredient is voltile, hving lower vpour pressure thn wter, (detiled in the mteril sfety nd dt sheet (MSDS)). Thus there would be rpid initil effect nd interction with the plnt roots, fter which the ctive ingredient would voltilise, resulting in the snitiser returning to benign stte without further ction on the roots. This is in direct contrst to the QAC-bsed snitisers, which do not voltilise nd remin in solution for the durtion of the tril (vpour pressure equl to wter s described in the MSDS). This constnt interction could be either direct result on the plnt roots due to the minimlly toxic nture of the ctive ingredient, or n dditive effect over time s the plnt roots tke up the QAC. Actsol, unique product, which cts s n oxidising biocide, showed severe phytotoxic effects in both the cucumber nd lettuce models, resulting in rpid plnt deth t the highest concentrtions. These observtions re in contrst to results obtined by Pernezy et l. (2005) where folir ppliction resulted in miniml phytotoxic effects. However, one of the ctive ingredients of Actsol hs been shown to hve phytotoxic effects (Monrc et l., 2004). This is possibly due to the Actsol ffecting the regulr functioning of the roots due to the combintion of chemicl nd electro-chemicl effect of the Actsol, likely preventing norml moisture nd nutrient uptke by the roots, which then results in plnt deth observed. At lower concentrtions of 1:50 nd 1:100 the phytotoxic effects were gretly reduced. The finl result nd conclusion from the current study is tht the snitisers could further be tested t the following concentrtions for disese control nd possible crop yield enhncements in pthogen infested hydroponic systems: Actsol t dilution of 1:50 (one prt Actsol in 50 prts wter); Prsin t concentrtion of 5mg.l -1 ; Purogene nd TecsClor t concentrtion of 10mg.l -1 nd Fitosn t concentrtion of 5mg.l -1. It could further lso be concluded tht copper sulphte nd Sporekill t concentrtion of 1mg.l -1 could be vible options for snittion of hydroponic nutrient solutions, yet the severe phytotoxicity of these products mke it unlikely tht ny benefits would be observed s possible yield reduction due to phytotoxic stunting my outweigh the benefit gined from lowered levels of Pythium infesttion

81 4.6 References Crrillo, A., Puente, M.E. nd Bshn, Y Appliction of diluted chlorine dioxide to rdish nd lettuce nurseries insignificntly reduced plnt development. Ecotoxicology nd Environmentl Sfety 35: Ecologicl Effects Test Guidelines: OPPTS , Terrestril Plnt Toxicity, Tier I (Vegettive Vigour). United Sttes Environmentl Protection Agency. Prevention, Pesticides nd Toxic Substnces (7101) EPA 712 C April Frvel, D.R. nd Lrkin, R.P Reduction of Fusrium wilt of hydroponiclly grown bsil by Fusrium oxysporum strin CS-20. Crop Protection 21: Hund-Rinke, K. nd Kordel, W Underlying issues in bioccessibility nd biovilbility: Experimentl methods. Ecotoxicology nd Environmentl Sfety 56: Koponen, H., Avikinen, H. nd Thvonen, R The effect of disinfectnts on fungi in pure culture nd on different surfce mterils. Agriculturl Science Finlnd 1: Lemy, A., Redlin, S., Fowler, G. nd Dirni, M Pest Dt Sheet: Rlstoni solncerum rce 3 biovr 2. USDA/APHIS/PPQ, Center for Plnt Helth Science nd Technology, Plnt Epidemiology nd Risk Anlysis Lbortory, Rleigh, NC. Meblds, M., Bnkier, M. nd Berdsell, D Wter disinfections control pthogens. Austrlin Horticulture. April 1997: Migliore, L., Cozzolino, S. nd Fiori, M Phytotoxicity to nd uptke of enrofloxcin in crop plnts. Chemosphere 52: Monrc, M., Zni, C., Richrdson, S.D., Thruston Jr, A.D., Moretti, M., Feretti, D. nd Villrini, M A new pproch to evluting the toxicity nd genotoxicity of disinfected drinking wter. Wter Reserch 38:

82 Nlecz-Jwecki, G., Grbinsk-Sot, E. nd Nrkiewicz, P The toxicity of ctionic surfctnts in four biossys. Ecotoxicology nd Environmentl Sfety 54: Pulitz, T.C., Zhou, T. nd Rnkin, L Selection of rhizosphere bcteri for biologicl control of Pythium phnidermtum on hydroponiclly grown cucumber. Biologicl Control 2: Pernezny, K., Rid, R.N., Hvrnek, N. nd Snchez, J Toxicity of mixed-oxidnt electrolyzed oxidizing wter to in vitro nd lef surfce popultions of vegetble bcteril pthogens nd control of bcteril diseses in the greenhouse. Crop Protection 24: Roux, C. nd Both, W.J An introduction to the Pythicee in South Afric. Agriculturl Reserch Council. Plnt Protection Reserch Institute, Roodeplt. Vinit-Dunnd, F., Epron, D., Aloui-Sosse, B. nd Bdot, P Effects of copper on growth nd on photosynthesis of mture nd expnding leves in cucumber plnts. Plnt Science 163: Wng, X., Sun, C., Go, S., Wng, L. nd Shuokui, H Vlidtion of germintion rte nd root elongtion s indictor to ssess phytotoxicity with Cucumis stivus. Chemosphere 44: Wng, X., Sun, C., Wng, Y., nd Wng, L Quntittive structure-ctivity reltionships for the inhibition toxicity to root elongtion of Cucumis stivus of selected phenols nd interspecies correltion with Tetrhymen pyriformis. Chemosphere 46:

83 4.7 Plte I A: Cucumber in sttic hydroculture, top nd side views. B: Grvel Flow Technique hydroponic system in the greenhouse plnted with Butter lettuce t 42d. C: Visible lightening of cucumber true lef fter 14d exposure to 2mg.l -1 copper sulphte solution

84 CHAPTER 5 CONTROL OF PYTHIUM WILT AND ROOT ROT OF LETTUCE BY MEANS OF CHEMICAL TREATMENT OF THE NUTRIENT SOLUTION IN RE-CIRCULATING HYDROPONIC SYSTEMS IN THE GREENHOUSE AND FIELD 5.1 Abstrct Results from previous chpters showed tht the tested snitisers were ble to control Pythium infesttion in wter volume while lso reducing the levels of Fusrium nd Rlstoni. Secondly, the phytotoxic effects of the snitisers were determined using two plnt models (cucumber nd Butter lettuce) grown in hydroponic systems. The im of the current study ws to further test these snitisers for the control of Pythium in vivo using greenhouse nd semi-commercil scle hydroponic systems. The hydroponic systems were rtificilly infested by introducing Pythium infected seedlings. The hydroponic nutrient solution ws subsequently treted with the snitisers. Phytex, Prsin nd Fitosn significntly reduced the Pythium zoospore levels in the nutrient solution ssessed t the end of the finl week of growth. Purogene chieved totl erdiction (no significnt difference from the untreted, uninfected control) of the zoospores. In the semicommercil field system, Phytex nd Purogene tretments were ble to improve lettuce yield compred to the untreted, Pythium-infested control. Agrl 90, Sporekill nd Actsol resulted in yield decreses when compred to the untreted, Pythium infested control. In generl, Phytex nd Purogene rendered the most consistent nd positive yield improvements in both the greenhouse nd field models. Purogene lso ppered to hve two-fold benefit in tht growth ws enhnced, while pthogen levels were simultneously decresed. Prsin nd Fitosn resulted in some degree of phytotoxicity, while lso chieving some mesure of Pythium control. Although no mjor yield improvement ws obtined, there were no dditionl negtive effects to pplying these snitisers to the nutrient solution. Comprisons between the snitisers under greenhouse field conditions re discussed

85 5.2 Introduction Pythium hs been shown to cuse severe disese outbreks nd crop losses over brod rnge of hydroponiclly cultivted vegetble crops, with lettuce nd tomto crops being most ffected (Stnghellini nd Kronlnd 1986; Pulitz et l., 1992; Schwrz nd Grosch, 2003; Song et l., 2004). Thus Pythium is considered one of the most serious pthogens of hydroponic systems (Song et l., 2004), with infection nd yield losses often going unnoticed due to the bility of this pthogen to cuse subclinicl infections (Stnghellini nd Kronlnd, 1986). In the recent pst, control of this pthogen hs been successful with systemic fungicides (Vnchter, 1995; Song et l., 2004). Chnges in worldwide regultions hve resulted in mny hydroponic growth systems being of recirculting nture to reduce both environmentl contmintion nd wter utilistion (Runi 1994). Recirculting hydroponic nutrient solution is n idel trnsport medium for pthogen inoculum to rpidly spred throughout nd entire hydroponic system (Zinnen, 1988; Stnghellini nd Rsmussen 1994; Vnchter 1995). Current methods of sterilistion of the recirculted nutrient solution re costly or lbour intensive while not constntly effective (Schwrtzkopf et l., 1987; Frvel nd Lrkin, 2002). Other methods rely on the use of toxic chemicls or substnces which hve been shown to produce toxic by-products (Dte et l., 2005). While good pthogen control hs lso been chieved with fungicides nd pesticides (Zinnen, 1988; Song et l., 2004), current consumer demnd hs tended towrds preference for products on which pesticide use hs been reduced or eliminted (Sb nd Messin, 2003). To stisfy this consumer demnd for minimised use of pesticides, while lso obtining consistent sterilistion of the hydroponic nutrient solution, sfer lterntive chemicls such s surfctnts nd snitisers (Crillo et l., 1996; Allende et l., 2006) hve been investigted with positive results (Crillo et l., 1996; Stnghellini et l., 1996) In Chpter 3 it ws determined tht certin wter snitisers pplied to n queous suspension of plnt pthogens would result in lowered contmintion level. It ws then demonstrted in Chpter 4 tht certin of these snitisers, when pplied t low concentrtions to hydroponic nutrient solution, should not result in severe phytotoxic effects or impct negtively on consumer demnds

86 The im of this current study ws to determine whether the snitisers re ble to reduce crop losses due to Pythium infesttion, when they re pplied in semi-commercil hydroponic system

87 5.3 Method nd Mterils Two hydroponic systems were designed nmely both n experimentl scle greenhouse system s well s semi-commercil scle field system. To chieve infection of plnts by Pythium, inoculum ws rtificilly introduced into the hydroponic systems to ensure high level of infesttion Smll scle grvel bed hydroponic system (greenhouse model) Lettuce vriety nd germintion Disese-free Butter lettuce (Lctuc stiv L. vr cpitt L. cv Ndine) seeds were germinted t commercil hydroponics grower (Hydrotec, South Afric)) under conditions preventing pthogen infesttion. Seedling trys were clened with chlorinted wter nd filled with stem psteurised vermiculite nd pet mixture (80:20) tht ws used s the germintion medium. The seeds were wtered every 20min, during dylight hours, by overhed emitters, supplied with pthogen-free borehole wter. Seedlings were germinted t optimum environmentl conditions under shde net structure Design of smll scle grvel bed hydroponic system A smll scle grvel bed hydroponic system (bsed on the grvel flow technique) ws ssembled in n environmentlly controlled greenhouse. This system consisted of ten 100l reservoirs, ech contining 100l het psteurized tp wter nd hydroponic nutrient mixture (Appendix I: Solution 2). Ech reservoir supplied nutrient solution to three plstic troughs of equl lengths (2.5m) nd widths (0.15m) by mens of submersible pump within ech reservoir. Ech trough ws filled to level of 8cm with wshed grvel (crushed dolerite / grnite chips of pproximtely 15mm). Nutrient solution flow ws limited to 400mg.min -1.trough -1. Outflow solution ws collected t the lower end of ech trough due to grdient nd recirculted bck into the 100l reservoir by mens of grvity flow (Plte II: A)

88 The entire volume of nutrient solution in ech reservoir ws replced on weekly bsis. Snitisers were dded to ech reservoir during this preprtion of the nutrient solution t the necessry dosges required to chieve the test concentrtion. Ech trough ws plnted with 15 28d old Butter lettuce seedlings plced equidistnt from ech other, resulting in totl of 45 plnts per tretment (Plte I: B) Plnts were llowed to grow nturlly for totl of 28d nd were inspected every 2d for ny visible symptoms of phytotoxicity or growth problems. To chieve nd ensure Pythium infesttion in the hydroponic system, 12 seedlings per tretment were exposed to Pythium Group F (PPRI #7079) zoospores for two dys prior to plnting. This exposure ws done by immersing the seedling roots into wter volume contining Pythium zoospores t n pproximte concentrtion of 10 3 zoospores.ml -1, obtined by mcerting two 7d old cultures of Pythium on V8-juice gr in 400ml of sterile deionised wter. Four of these infested seedlings were then plnted t the hed of ech trough, to serve s continuous source of zoospore inoculum into the nutrient solution, which would ensure infection long the entire length of the trough Growth conditions Environmentl conditions were mintined within the greenhouse t n verge RH of 65%, n verge mximum dytime temperture of 28 C nd verge minimum nightly temperture of 18 C. Initil experiments exposed lettuce plnts to rnge of concentrtions of ech snitiser, while two finl experiments compred the optiml concentrtions of ll the snitisers ginst ech other. For ech experiment the following controls were included: n untreted, uninfested control; Pythium infested, untreted control nd Pythium infested control treted with the fungicide Phytex t the mnufcturers recommended dosge rte of 1ml.l -1 wter

89 Yield nd infesttion ssessments After 28d the lettuce plnts were observed for ny visible signs of phytotoxic effects fter which they were hrvested. The shoots nd roots were seprted from ech other nd their fresh mss determined seprtely (Migliore et l., 2003). Hrvesting nd weight determintion were completed before 10m for ech experiment to minimize possible growth-cycle differences. Recirculted nutrient solution ws tested for Pythium incidence using the citrus lef disc biting procedure described by Grimm nd Alexnder (1973) nd plting on Pythiumselective medium (BNPRA) (Roux nd Both, 1997) Anlysis Root nd shoot mss dt ws sttisticlly nlysed using Duncn s Multiple Rnge test t P = 0.05, utilising the SAS for Windows version 8.0e softwre pckge Semi-commercil scle grvel bed hydroponic system in the field A semi-commercil scle grvel bed hydroponic system (bsed on the grvel film technique) ws constructed under 20% grey shde net structure on the University of Pretori experimentl frm (Plte II: B), modelled on commercil frming system (Plte II: C). Eighteen troughs of 20m lengths were constructed nd filled to depth of 6cm with clen grvel (crushed dolerite / grnite chips of pproximtely 15mm). Ech trough ws fed by 500l reservoir contining submersible pump supplying constnt flow of 2l.hr -1 t the hed of ech trough. Runoff ws collected t the lower end of ech bed nd chnnelled bck into the reservoir by mens of grvity. A commercilly vilble hydroponic nutrient solution pre-mix ws used (Appendix I: Solution 2) s the fertignt solution, nd the ph ws mintined t 6.4 by the ddition of

90 nitric cid. The fertignt solution ws replced weekly with fresh mixture to which the snitisers were dded t the estblished dosges (detiled in Tble 1). Ech bed ws plnted with n verge of 350 lettuce seedlings eqully spced long the length of the bed in sets of three spced in tringulr shpe with single seedling t ech point of the tringle. Seedlings were llowed to grow nturlly for 42d. To ensure even infesttion of Pythium cross the entire length of ech bed, s well s cross seprte beds, 96cm Petri dish contining 7d old Pythium culture on V8 medium ws cut into four equl sections (3.6cm 2 pieces). Four of these culture pieces were then plced underneth the grvel in contct with plnt roots t distnces of 0m, 5m, 10m nd 15m long ech bed Growth conditions Environmentl conditions fluctuted due to nturl climtic conditions. Averge dytime tempertures rnged from C nd verge night time tempertures from 9-14 C For ech experiment n untreted, uninfested control nd Pythium infested, untreted control were included Yield nd infesttion ssessments After 28d the lettuce plnts were observed for ny visible signs of phytotoxic effects fter which they were hrvested. The shoots nd roots were seprted from ech other nd their fresh mss determined seprtely (Migliore et l., 2003). Recirculted nutrient solution ws tested for Pythium incidence using citrus lef disc biting procedure s described previously (Grimm nd Alexnder, 1973; Roux nd Both, 1997) Anlysis Shoot mss dt ws sttisticlly compred using Duncn s Multiple Rnge test t P=0.05, utilising the SAS for Windows version 8.0e softwre pckge. Root mss ws not sttisticlly nlysed s root mss does not contribute to the mrketble yield

91 5.3.3 Snitiser preprtion For both hydroponic systems snitisers were prepred s follows, with the concentrtions tested in ech instnce detiled in Tble 1. Prsin (SIDL, South Afric), Fitosn (Helth & Hygiene, South Afric), TecsClor (BTC Products, South Afric), Agrl 90 (Kynoch Chemicls, South Afric), Sporekill (Hygrotech Seeds, South Afric) nd Phytex (Horticur, South Afric) were used directly from the solution provided by the mnufcturer. Fresh Purogene (BTC Products, South Afric) ws generted for ech experiment by following lbel instructions (ddition of one prt supplied ctivtor to ten prts Purogene ). This ws llowed to rect for 5min before use. Actsol ws freshly prepred nd delivered weekly by Rdicl Wters. Actsol solution hd n verge ph of 7.2 nd ORP of 800mV. This solution ws used directly in the experiments. Copper (II) sulphte crystls (Merck, South Afric) were dissolved in de-ionised wter nd diluted to give the finl concentrtion required. Contct detils of ll suppliers re provided in Appendix I: C. Detils of ech snitiser re provided in Appendix 1: B) Tble 1: Concentrtions of snitisers tested in the greenhouse system (both individully nd in comprison) nd in the field-scle system. Product Greenhouse system Greenhouse comprison experiment Field system Actsol 1:10; 1:20 nd 1:50 1:20 nd 1:50 1:20 Prsin 2,5; 5 nd 7.5mg.l mg.l mg.l -1 Purogene 10; 25 nd 50mg.l -1 10mg.l -1 10mg.l -1 TecsClor 25; 50 nd 75mg.l -1 25mg.l -1 Not tested Fitosn Not tested 7.5mg.l mg.l -1 Phytex Not tested 1ml.l -1 1ml.l -1 Sporekill Not tested 5mg.l -1 5mg.l -1 Agrl 90 Not tested 5mg.l -1 5mg.l -1 Copper Not tested 5mg.l -1 Not tested Concentrtions referred to re product concentrtions, i.e. concentrtions mde directly from the stock solutions. Active ingredient concentrtions for ech product cn be found in Appendix I: B

92 5.4 Results Smll scle grvel bed hydroponic system (greenhouse model) evlution of snitisers individully t rnge of dosge rtes Actsol Actsol t dilution of 1:10 nd 1:20 into Pythium infested hydroponic lettuce system resulted in severe phytotoxic effects, with plnt deth occurring t the 1:10 dilution fter period of 14d nd severely reduced growth nd development of plnts exposed to 1:20 dilution fter 28d (Fig. 1). A 1:50 dilution resulted in lettuce plnts hving significnt (P=0.05) higher fresh shoot mss thn n infested nd untreted control, while lso being significntly reduced in fresh shoot mss when compred to n uninfested nd untreted control fter 28d (Fig. 1). Averge fresh biomss (g) Figure 1: b c e d b b d Clen Control Infested Control 1:10 1:20 1:50 Tretment Root Mss Shoot Mss Effect of tretment of the nutrient solution with Actsol on lettuce yield in the presence of Pythium infesttion in smll scle grvel bed hydroponic system in the greenhouse. Plnts were grown for 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). c

93 Prsin Prsin dosed t concentrtion of 7.5mg.l -1 into the nutrient supply resulted in the fresh shoot mss being significntly (P=0.05) lower thn the untreted, uninfested control yet higher thn the untreted, infested control (Fig. 2). Prsin tretments of 2.5mg.l -1 nd 5mg.l -1 did not result in ny significnt fresh shoot mss differences from n untreted, Pythium infested control, but were significntly lower thn the untreted, uninfested control. Averge fresh biomss (g) Figure 2: c c c b b b b Clen Control Infested Control 2.5mg/l 5mg/l 7.5mg/l Tretment Root Mss Shoot Mss Effect of tretment of the nutrient solution with Prsin on lettuce yield in the presence of Pythium infesttion in smll scle grvel bed hydroponic system in the greenhouse. Plnts were grown for 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). b Purogene Tretment of the nutrient solution with Purogene t concentrtions of 10mg.l -1 nd 25mg.l -1 resulted in no significnt (P=0.05) differences when compred to n untreted, uninfested control (Fig. 3). None of the Purogene tretments demonstrted significnt difference to the untreted, Pythium infested control. Only the 50mg.l -1 tretment resulted in significnt difference in root mss when compred to the uninfested, untreted control lthough significnt increse in root mss ws lso observed t 25mg.l -1 concentrtion

94 Averge fresh biomss (g) Figure 3: b b bc c b b b b Clen Control Infested Control 10mg/l 25mg/l 50mg/l Tretment Root Mss Shoot Mss Effect of tretment of the nutrient solution with Purogene on lettuce yield in the presence of Pythium infesttion in smll scle grvel bed hydroponic system in the greenhouse. Plnts were grown for 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) TecsClor TecsClor t ll tretment concentrtions did not result in ny significnt (P=0.05) differences in fresh shoot mss when compred to n untreted, Pythium infested control, nor between tretment concentrtions (Fig. 4). All tretments did result in significnt reduction in fresh shoot mss when compred to n untreted, uninfested control. Root mss ws lso significntly decresed by ll tretments when compred to the uninfested, untreted control, while 25mg.l -1 concentrtion resulted in significnt increse in root mss when compred to n untreted, Pythium infested control

95 Averge fresh biomss (g) Figure 4: b b b b c b bc bc Clen Control Infested Control 25mg/l 50mg/l 75mg/l Tretment Root Mss Shoot Mss Effect of tretment of the nutrient solution with TecsClor on lettuce yield in the presence of Pythium infesttion in smll scle grvel bed hydroponic system in the greenhouse. Plnts were grown for 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Comprison of different snitisers t optimum dosge rtes in the greenhouse Preliminry experiment The preliminry comprison experiment showed tht the untreted, uninfested control exhibited significntly (P=0.05) reduced growth s mesured by fresh shoot mss (Fig. 5). Root mss ws lso reduced. This biomss ws less thn n untreted, Pythium infested control, indicting tht Pythium contmintion hd likely occurred in the uninfested control, which ws lter confirmed by root pltings on Pythium selective medi. This dt ws therefore considered unrelible. The dt presented here shows tht Actsol t 1:20 dilution resulted in totl plnt deth, while other tretments indicted tht Prsin t 7.5mg.l -1, TecsClor t 25mg.l -1, Phytex t 1ml.l -1, Fitosn t 7.5mg.l -1 nd Purogene t 10mg.l -1 could be most effective in decresing order

96 Averge fresh biomss (g) Figure 5: d b cd f e bc d Clen control Infested control Phytex 1ml/L Actsol 1:20 Prsin 5mg/l Tretment Purogene 10mg/l TecsClor 25mg/l Fitosn 7.5mg/l Root Mss Shoot Mss Effect of snitisers t optimum dosges on yield of Pythium infested lettuce in smll scle grvel bed hydroponic system in greenhouse, over period of 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Comprison of primry snitisers in smll scle grvel bed hydroponic system (greenhouse model) Due to the results obtined in the preliminry experiment, the procedure ws repeted, with Actsol reduced to 1:50 dilution. The untreted, uninfested nd untreted, Pythium infested controls showed significnt (P=0.05) difference in shoot mss indicting tht Pythium infection resulted in 29% reduction in yield (Fig. 6). Phytex t 1ml.l -1 resulted in significnt increse in fresh shoot mss when compred to the untreted, uninfested control whilst Purogene t 10mg.l -1 showed no significnt difference in fresh shoot mss. Both these tretments resulted in significnt increse in fresh shoot mss when compred to the untreted, Pythium infested control chieving 69% nd 39% increse respectively. Prsin t 7.5mg.l -1 nd Fitosn t 7.5mg.l -1 did not differ significntly from ech other, nor from the untreted, Pythium infested control. Actsol t 1:50 dilution nd TecsClor t 25mg.l -1 showed significnt decrese in fresh shoot mss in comprison to the untreted, Pythium infested control (Fig. 6). Phytex, Prsin nd Fitosn significntly reduced the Pythium zoospore levels in the nutrient solution t the end of the finl week of growth, while only Purogene chieved totl elimintion (sme s the untreted, uninfested control) (Fig. 7). Plnts treted with Actsol nd TecsClor showed no significnt difference in Pythium levels when compred to the untreted, Pythium infested control

97 Averge fresh biomss (g) b c d c b d c Clen control Infested control Phytex 1ml/L Actsol 1:20 Prsin 5mg/l Tretment Purogene 10mg/l TecsClor 25mg/l Fitosn 7.5mg/l Root Mss Shoot Mss Figure 6: Effect of snitisers t optimum dosge on yield of Pythium infested lettuce in smll scle grvel bed hydroponic system in greenhouse, over period of 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). 100 b b c b % Incidence Clen Control Infested Control Phytex 1ml/L Actsol 1:20 Prsin 5mg/l Purogene 10mg/l TecsClor 25mg/l Fitosn 7.5mg/l Tretment Figure 7: Effect of chemicl tretments on Pythium infesttion in recirculted nutrient solution t the end of the 28d of lettuce growth in smll scle grvel bed hydroponic system in the greenhouse. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Comprison of dditionl snitisers in smll scle grvel bed hydroponic system in the greenhouse All snitiser tretments, nd the untreted, Pythium infested control showed significnt (P=0.05) reduction in fresh shoot mss in comprison to the untreted, uninfested control (Fig. 8). Phytex dosed t 1ml.l -1 nd Agrl 90 dosed t 5mg.l -1 resulted in significnt

98 increse in fresh shoot mss when compred to the untreted, uninfested control, while copper sulphte dosed t 5mg.l -1 nd Sporekill dosed t 5mg.l -1 resulted in significnt decrese in shoot mss in comprison to the untreted, Pythium infested control. Averge fresh biomss (g) Figure 8: b c d f e Clen control Infested control Phytex 1ml/L Agrl 90 5mg/l Tretment Copper 5mg/l Sporekill 5mg/l Root Mss Shoot Mss Effect of dditionl snitisers t optiml dosges on yield of Pythium infested lettuce in smll scle grven bed hydroponic system in the greenhouse, over period of 28d. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). Tretment with Sporekill resulted in the highest level of Pythium in the nutrient solution t the end of the finl week of the 28d growth period, which ws significntly greter thn the untreted, Pythium infested control (Fig. 9). Phytex resulted in significnt reduction in Pythium incidence, while Agrl 90 nd copper sulphte tretments resulted in complete erdiction of Pythium in the nutrient solution, which ws the sme s the untreted, un-infested control

99 100 d b c d d % Incidence Clen Control Infested Control Phytex 1ml/L Agrl 90 5mg/l Copper 5mg/l Sporekill 5mg/l Tretment Figure 9: Effect of chemicl tretments on Pythium incidence in recirculted nutrient solution t the end of the 28d of lettuce growth in smll scle grvel bed hydroponic system in the greenhouse. Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05) Tretment comprisons in semi-commercil grvel bed hydroponic field system (multi-snitiser tril) Comprison of snitisers in semi-commercil scle grvel bed hydroponic system in the field In two trils in the semi-commercil grvel bed hydroponic field system, only Phytex dosed t 1ml.l -1 ws ble to chieve significnt (P=0.05) increse (37%) in fresh shoot mss over the untreted, Pythium infested control. Purogene dosed t 10mg.l -1 ws ble to chieve 7% improvement in mss compred to the untreted, Pythium infested control, yet this ws not sttisticlly significnt (Figs. 10 nd 11). With the exception of Phytex, which chieved the mximum lettuce yield, no snitiser tretment ws ble to chieve growth levels equivlent to or significntly greter thn the untreted, uninfested control. Tretments with Prsin nd Fitosn, both t 7.5mg.l -1, yielded fresh shoot biomss significntly equivlent to the untreted, Pythium infested control (Fig. 10), while Agrl 90 nd Sporekill, ech pplied t 5mg.l -1, showed severe reductions in shoot mss of

100 15% nd 20% respectively, which were not significntly different from the untreted, Pythium infested control (Fig. 11). Actsol dosed t 1:20 dilution showed the gretest yield reduction of 61% which ws significntly different from both the untreted, Pythium infested control, s well s the untreted, uninfested control. Averge fresh biomss (g) Figure 10: +24% +37% +1% +7% -3% b c c bc c Clen control Infested control Phytex 1ml/L Prsin 7.5mg/l Tretment Purogene 10mg/l Fitosn 7.5mg/l Root Mss Shoot Mss Effect of chemicl tretments on yield of Pythium infested lettuce in semi-commercil scle grvel bed hydroponic system in the field, fter 42d growth (vlues t top indicte yield increse over infested control). Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05). Averg fresh biomss (g) Figure 11: +10% -15% -20% -61% b b b c Clen control Infested control Agrl 90 5mg/l Sporekill 5mg/l Actsol 1:20 Tretment Root Mss Shoot mss Effect of chemicl tretments on yield of Pythium infested lettuce in semi-commercil scle grvel bed hydroponic system in the field, fter 42d growth (vlues t top indicte yield increse over infested control). Brs with the sme letter do not differ significntly ccording to Duncn s Multiple Rnge test (P=0.05)

101 5.5 Discussion In the greenhouse trils, Actsol exhibited trend of incresing yield with incresing dilution which is ttributed to incresed phytotoxicity t the higher concentrtions cusing phytotoxic stress nd root dmge with relted increse in susceptibility to disese. This ws gin confirmed in the comprison tril where 1:20 dilution resulted in plnt deth. Only 1:50 dilution resulted in significnt yield improvement over the Pythium-infested, untreted control, yet this ws significntly lower thn the untreted, uninfested control. This could be ttributed to the fct tht disese control ws not complete nd low levels of phytotoxicity being present, both fctors preventing optiml growth. The high levels (not significntly different to the untreted, Pythium infested control) of Pythium inoculum recorded in the comprison tril further indicte tht disese control ws not mximl nd ws not ffected by reduction of Pythium in the nutrient solution but rther t n infection stge t root level. Prsin exhibited n inverse trend to ll other snitisers tested in the greenhouse tril where significnt yield increse over n untreted, Pythium infested control ws only noted t the highest concentrtion tested (7.5mg.l -1 ) with lower concentrtions not ppering to hve significnt beneficil effects. This ws s expected, since previous reserch showed tht Prsin ws most effective ginst Pythium zoospores in suspension t 7.5mg.l -1 concentrtion with exposure time, while not being s effective t lower concentrtions. In the snitiser comprison experiment, it ws shown tht Pythium infesttion ws significntly lowered in comprison to the untreted, Pythium-infested control, further vlidting the hypothesis tht the yield improvement noted ws due to disese control effect. None of the tretments resulted in optimum growth which my hve been due to combintion of indequte disese control nd vrious phytotoxic effects. This indicted tht, s with Actsol, there ws both disese control benefit s well s phytotoxic effect. Unlike Actsol the disese control ppers to be s result of inoculum reduction within the nutrient solution, which lso explins the poor performnce of the low concentrtion tretments where filure to improve plnt yields my be due to the Pythium inoculum not being sufficiently reduced

102 With the exception of Purogene t 50mg.l -1 concentrtion, both Purogene nd TecsClor (which hve chlorine dioxide s n ctive ingredient) exhibited equivlent trends, which were similr to Actsol tretments. An increse in concentrtion of these snitisers resulted in lowered yields, which did not differ significntly. A 50mg.l -1 Purogene tretment resulted in significntly reduced yield. This is ttributed to the higher phytotoxic effects of chlorine-dioxide t high concentrtions nd is similr to findings by Crillo et l. (1996) where chlorine dioxide ppliction did not significntly reduce lettuce plnt development under nursery conditions. In the snitiser comprison tril under greenhouse conditions it ws shown tht the 10mg.l -1 tretment of Purogene ws ble to totlly erdicte Pythium inoculum from the nutrient solution indicting tht the growth improvement my be due to inoculum reduction in the nutrient solution. The incresed concentrtions would expectedly hve the sme effect on disese severity, yet the phytotoxic effects would be incresed, preventing optiml growth. These results do not follow the expected trend directly since dul benefit ws expected t tretment with lower concentrtions where disese incidence would be lowered or erdicted with simultneous growth enhncement s described in Chpter 4, with the growth enhncing spect of low concentrtions of chlorine dioxide lso being described by Lee et l. (2004) nd Pernezy et l. (2005). The reduced yield t the highest concentrtion, ttributed to phytotoxicity, ws expected nd the phytotoxic nture of high concentrtions of chlorine dioxide hs lso been previously described (Lee et l., 2004; Pernezy et l., 2005). TecsClor ws unble to chieve significnt improvement in yield over n untreted, Pythium infested control. As TecsClor previously exhibited miniml phytotoxicity (Chpter 4) nd low levels of chlorine dioxide hve been shown to be minimlly phytotoxic (Crillo et l., 1996) with possible growth enhncing fctor (Lee et l., 2004 nd Pernezy et l., 2005) the filure of TecsClor to effect n improved yield my be due to poor disese control. In the snitiser comprison experiment it ws shown tht TecsClor t 25mg.l -1 ws unble to significntly reduce Pythium inoculum levels in the nutrient solution in comprison to the untreted, Pythium infested control. The lck of improvement in yield my hve been due to miniml disese control, combined with miniml phytotoxicity nd growth enhncing fctors which resulted in the tretments being very similr to n untreted, Pythium infested control

103 In one of the preliminry experiments compring ll snitisers under greenhouse conditions the untreted, uninfested control showed poorer growth thn n untreted, Pythium infested control. This ws lter determined by rndom root plting (dt not shown) to be due to contmintion of the uninfested control by Pythium. Actsol t 1:20 dilution ws gin shown to be phytotoxic to level resulting in plnt deth. Purogene nd TecsClor demonstrted n expected trend, possibly due to phytotoxic effects where the Purogene tretment resulted in reduced growth in comprison to the TecsClor tretment. Prsin nd Fitosn did not conform to ny expected trend since results were expected to be similr between the two tretments. In the first successful comprison experiment the expected trends were observed for ll the tretments with the exception of the Phytex tretment which resulted in significntly incresed yield when compred to n untreted, uninfested control. Phytex ws lso unble to eliminte Pythium inoculum from the hydroponic nutrient solution, lthough this ws significntly reduced in comprison to levels noted in the untreted, Pythium-infested control. This ws understndble becuse Phytex is systemic fungicide bsed on phosphorous cid, exerting disese control within the plnt nd plnt roots (Fenn nd Coffey, 1984) s opposed to directly ffecting pthogen inoculum in the nutrient solution. Phosphorous cid hs lso been observed to hve growth stimulting effect on plnts when pplied t low concentrtions (Chluvrju et l., 2004) nd this my hve resulted in the mximum growth observed with Phytex tretment. Purogene, Fitosn, Prsin, TecsClor nd Actsol resulted in progressively decresing lettuce yields in scending order, long with incresing Pythium inoculum presence in the nutrient solution. These results followed n expected trend noted in the previous trils where only Purogene ws ble to effect significntly incresed yield over n untreted, Pythium infested control long with totl erdiction of Pythium inoculum from the nutrient solution. Fitosn nd Prsin (both hving similr ctive ingredients) resulted in similr, insignificntly different, yields, while Fitosn ws unexpectedly shown to hve the greter effect on Pythium inoculum. This my be ttributed to the higher level of phytotoxicity exerted by Fitosn, resulting in lowered yields even though disese control ws more effective thn Prsin tretment. TecsClor nd Actsol were not significntly different from ech other with neither ble to significntly reduce Pythium inoculum levels in the nutrient solution, which might hve resulted in the poor growth nd yield in these

104 tretments. Furthermore, phytotoxic stresses cused by these tretments my hve resulted in the significntly lowered yield when compred to the untreted, Pythium infested control s plnts my hve been more susceptible to disese. In the second greenhouse comprison experiment Phytex ws gin demonstrted to significntly increse lettuce yield (plnt mss) over n untreted, Pythium infested control while not completely eliminting Pythium inoculum in the nutrient solution, lthough significnt reduction ws chieved. Unlike the previous experiment no dditionl increse over n untreted, uninfested control ws noted. Neither Agrl 90, Sporekill or copper sulphte t 5mg.l -1 were ble to improve lettuce yield over n untreted, Pythium infested control lthough both Agrl 90 nd copper sulphte were ble to eliminte Pythium inoculum from the nutrient solution. This effect of Agrl 90 on zoospores hs previously been reported by Stnghellini nd Tomlinson (1987) nd Stnghellini et l. (1996). The low lettuce yield observed even though Pythium control ws high, is probbly due to the high levels of phytotoxicity of copper sulphte, s seen in Chpter 4 of this study, while growth reduction by non-ionic surfctnts hs been reported by Grlnd et l. (2004). When tested over two experiments in semi-commercil scle grvel bed hydroponic system in the field, the trends seen in previous experiments were gin observed. Only Phytex ws ble to chieve significntly incresed lettuce yield in comprison to the untreted, Pythium infested control, with further insignificnt improvement over n untreted, uninfested control. In the semi-commercil field system, Phytex nd Purogene tretments were ble to improve lettuce yield over tht of n untreted, Pythium-infested control, while Prsin nd Fitosn chieved yields similr to this control. Agrl 90, Sporekill nd Actsol showed yield decreses when compred to the untreted, Pythium-infested control. As previously shown, Purogene ws the only other tretment to result in n improved lettuce yield over n untreted, Pythium-infested control, lthough this improvement ws not sttisticlly significnt. Following previous trends Prsin, Fitosn, Agrl nd Sporekill did not chieve significntly different yield in comprison to the untreted, Pythium-infested control, while Actsol gin showed significntly reduced yield

105 Phytex t 1ml.l -1 nd Purogene t 10mg.l -1 demonstrted the most consistent nd positive yield improvements under both greenhouse nd field conditions. This yield improvement my be due to two spects where Pythium inoculum in the nutrient solution is reduced (or eliminted in the cse of Purogene tretment) long with growth stimultion effect on the lettuce plnts. Phytex my lso hve third spect where disese control is effected by the systemic nture of this product. Both Phytex nd Purogene re thus indicted s hving beneficil effects when dosed into hydroponic nutrient solution. Prsin, Fitosn, Agrl nd Sporekill tretments did not result in improved lettuce yields even though some mesure of disese control ws exerted. This my be due to combintion effect of benefits due to disese control coupled to yield reduction cused by the phytotoxic nture of these products. Although no direct benefit ws seen in the current tril setup using only Pythium, ddition of these snitisers in commercil hydroponic systems my result in yield improvement due to generl pthogen inoculum reduction in the nutrient solution nd the Pythium control my be beneficil under stress conditions when plnts re more susceptible to infection. Actsol consistently showed poor inoculum control from the nutrient solution long with decresed yield mss when compred to n untreted, Pythium infested control, indicting tht the tested concentrtions re not suited for ppliction into hydroponic nutrient solutions nd negtive impct is observed

106 5.6 References Allende, A., Toms-Brbern, F.A. nd Gil, M.I Miniml processing for helthy trditionl foods. Trends in Food Science nd Technology 17: Crrillo, A., Puente, M.E. nd Bshn, Y Appliction of diluted chlorine dioxide to rdish nd lettuce nurseries insignificntly reduced plnt development. Ecotoxicology nd Environmentl Sfety 35: Chluvrju, G., Bsvrju, P., Shetty, N.P., Deepk, S.A., Amruthesh, K.N. nd Shetty, H.S Effect of some phosphorous-bsed compounds on control of perl millet downy mildew disese. Crop Protection 23: Dte, S., Terbyshi, S., Kobyshi, Y. nd Fujime, Y Effects of chlormines concentrtion in nutrient solution nd exposure time on plnt growth in hydroponiclly cultured lettuce. Scienti Horticulture 103: Fenn, M. nd Coffey, M.D Studies on the in vitro nd in vivo ntifungl ctivity of fosetyl-a1 nd phosphorous cid. Phytopthology 74: Frvel, D.R. nd Lrkin, R.P Reduction of Fusrium wilt of hydroponiclly grown bsil by Fusrium oxysporum strin CS-20. Crop Protection 21: Grlnd, J.L., Levine, L.H., Yorio, N.C. nd Hummerick, M.E Response of grywter recycling systems bsed on hydroponic plnt growth to three clsses of surfctnts. Wter Reserch 38: Grimm, G.R. nd Alexnder, A.F Citrus lef pieces s trps for Phytophthor prsitic from soil slurries. Phytopthology 63: Lee, S., Gry, P.M., Dougherty, R.H. nd Kng, D The use of chlorine dioxide to control Alicyclobcillus cidoterrestris spores in queous suspension nd on pples. Interntionl Journl of Food Microbiology 92:

107 Migliore, L., Cozzolino, S. nd Fiori, M Phytotoxicity to nd uptke of enrofloxcin in crop plnts. Chemosphere 52: Pulitz, T.C., Zhou, T. nd Rnkin, L Selection of rhizosphere bcteri for biologicl control of Pythium phnidermtum on hydroponiclly grown cucumber. Biologicl Control 2: Pernezny, K., Rid, R.N., Hvrnek, N. nd Snchez, J Toxicity of mixed-oxidnt electrolyzed oxidizing wter to in vitro nd lef surfce popultions of vegetble bcteril pthogens nd control of bcteril diseses in the greenhouse. Crop Protection 24: Roux, C. nd Both, W.J An introduction to the Pythicee in South Afric. Agriculturl Reserch Council: Plnt Protection Reserch Institute, Roodeplt, Pretori. Runi, W Disinfection of recircultion wter from closed cultivtion systems with ozone. Act Horticulture 361: Sb, A. nd Messin, F Attitudes towrds orgnic foods nd risk/benefit perception ssocited with pesticides. Food Qulity nd Preference 14: Schwrtzkopf, S.H., Dudzinski, D. nd Minners, R.S The effects of nutrient solution sterilistion on the growth nd yield of hydroponiclly grown lettuce. HortScience 22: Schwrz, D. nd Grosch, R Influence of nutrient solution concentrtion nd root pthogen (Pythium phnidermtum) on tomto root growth nd morphology. Scienti Horticulture 97: Song, W., Zhou, L., Yng, C., Co, X., Zhng, L. nd Liu, X Tomto Fusrium wilt nd its chemicl control strtegies in hydroponic system. Crop Protection 23:

108 Stnghellini, M.E. nd Kronlnd, W.C Yield loss in hydroponiclly grown lettuce ttributed to subclinicl infection of feeder rootlets by Pythium dissotocum. Plnt Disese 70: Stnghellini, M.E. nd Rsmussen, S.L Hydroponics: A solution for zoosporic pthogens. Plnt Disese 78: Stnghellini, M.E. nd Tomlinson, J.A Inhibitory nd lytic effects of nonionic surfctnt on vrious sexul stges in the life cycle of Pythium nd Phytophthor species. Phytopthology 77: Stnghellini, M.E., Kim, D.H., Rsmussen, S.L. nd Rorbugh, P.A Control of root rot of peppers cused by Phytophthor cpsici with nonionic surfctnt. Plnt Disese 80: Vnchter, A Development of Olpidium nd Pythium in the nutrient solutions of NFT grown lettuce, nd possible control methods. Act Horticulture 382: Zinnen, T.M Assessment of plnt diseses in hydroponic culture. Plnt Disese 72:

109 5.7 Plte II A: Semi-commercil grvel bed hydroponic system in the field B: Semi-commercil grvel bed hydroponic system in the field, plnted with butter lettuce seedlings C: Commercil grvel bed hydroponic system plnted with butter lettuce