Inhibition of Phytopathogenic Fungi on Selected Vegetable. Crops by Catechins, Caffeine, Theanine and Extracts of. Camellia sinensis (L.) O.

Transcription

1 Inhiition of Phytopthogenic Fungi on Selected Vegetle Crops y Ctechins, Cffeine, Thenine nd Extrcts of Cmelli sinensis (L.) O. Kuntze y Michelle Wilmot Sumitted in prtil fulfilment of the requirements for the degree of Mgister Scientie In the Fculty of Nturl nd Agriculturl Science University of Pretori Pretori Septemer 2006 Supervisor: Co-supervisor: Prof. N. Luschgne Dr. Z. Apostolides University of Pretori

2 DECLARATION I, the undersigned, declre tht the work contined in this disserttion is my own originl work nd tht it hs not previously, in its entirety or prt, sumitted for degree to ny other university Michelle Wilmot 2

3 Tle of Contents Acknowledgements 5 Chpter 1: Cmelli sinensis extrcts s lterntive nti-microil compounds in plnt disese mngement: A review Introduction Cmelli sinensis Description nd origin Chemistry Medicinl properties Anti-microil properties Mechnisms which contriutes to Cmelli sisnensis nti-microil ctivity Conclusions References Chpter 2: In vitro inhiition of phytopthogenic fungi y cffeine, te ctechins, thenine, Polyphenon G, lck, green nd rooios tes Astrct Introduction Mterils nd Methods Results Discussion References

4 Chpter 3: Control of phytopthogenic fungi on selected vegetle crops with cffeine nd green te (Cmelli sinensis) extrcts under greenhouse conditions Astrct Introduction Mterils nd Methods Sttisticl nlysis Results Discussion References Chpter 4: Induction of defence rections in tomto nd lettuce plnts y ppliction to te (Cmelli sinensis) extrcts nd cffeine Astrct Introduction Mterils nd Methods Sttisticl nlysis Results Discussion References Chpter 3: Generl discussion References Resumé 133 4

5 ACKNOWLEDGEMENTS I sincerely thnk the following individuls nd institutions tht mde this study possile: Prof. Nico Luschgne, for his dvice, guidnce, support, llowing me the freedom to try my own ides with regrds to the reserch nd understnding in my often unique wys. A good resercher nd gret mn. Dr. Z. Apostolides, for his lterntive thinking, honesty nd the ility to encourge those round him to strive eyond their own cpilities nd chieving their gols. And ll the iochemistry stuff. The Technology nd Humn Resources for Industry Progrmme (THRIP) (funded y the Deprtment of Trde nd Industry (DTI) nd mnged y the Ntionl Reserch Foundtion (NRF), RSA) for funding in prt. Dr Hr of Mitsui Norin Co. Ltd for generously providing Polyphenon G, thenine, ctechins nd for funding, in prt, the reserch undertken. Mr Roger Bgnll nd Dr. Thierry Regnier for vlule ssistnce in conducting the lortory experiments. My mother for her exmple, encourgement nd love. Zeld Pieterse for her friendship, lwys nd ll wys. 5

6 CHAPTER 1 Generl Introduction Cmelli sinensis s n Alterntive Anti-microil Compound in Plnt Disese Mngement 6

7 INTRODUCTION Plnt pthogens re estimted to cuse yield reductions of lmost 20% in the principl food nd csh crops worldwide (Oerke et l, 1994). Although these losses my e minimized y the use of disese-tolernt cultivrs, crop rottion, or snittion prctices, fungicides re often essentil to mximize crop yields. Fungicides lso present sustntil enefits on food qulity. First, they contriute to food sfety y controlling mny of the fungi tht produce mycotoxins. Nerly one qurter of food crops worldwide re ffected y mycotoxins such s fltoxins, ergot toxins, Fusrium toxins, ptulin, nd tenuzonic cid (Pohlnd, 1993; Schneider nd Dickert, 1994). Second, plnt pthogenic fungi must e controlled if consumer demnd in developed countries for premium qulity, diverse foods is to e met; while highqulity fruit nd vegetles re n indictor of economic growth in developing countries (Mitchell, 1993). Disese control using chemicls egn during the 1850s when Bordeux mixture ws introduced to control downy mildew ((Plsmopr viticol (Berk. nd M. A. Curtis) Berl. nd De Toni) in French vineyrds (Brent, 1985). Pris green (copper cetorsenite), for instnce, originlly used in house pint s fungicide, nd ws ccidentlly found to control lef-eting insects. In the first hlf of this century, protection of mny crops ecme possile with the introduction of orgnic fungicides. In the 1960s, the development of systemic products le to penetrte plnt tissue nd deliver curtive properties permitted more flexile ppliction regime (Brent, 1985; Lyr, 1995). Despite the choice of effective fungicides ville, new nti-fungl chemicls re still needed to deliver improved yield nd qulity enefits. 7

8 An importnt motivtion for mintining reserch progrms is the growing demnd y the pulic for crop protection gents with low use rtes, enign environmentl profile, nd low toxicity to humns nd wildlife. For these resons, vrious plnt extrcts hve een screened in vitro. According to A (1996) the osmotic proteins in tocco plnts re le to inhiit spore germintion of some phytopthogenic fungi. The nti-microil ctivity of te (Aror nd Bhrwj, 1997; Aror nd Ohln, 1997) nd te polyphenols hs lso een demonstrted. The inhiitory ctivity of essentil oils on fungi hs een investigted (Mnohr et l, 2001). Recently it hs een shown tht certin compounds in olives (Del Rio et l, 2003) nmely: tyrosol, ctechins nd oleuropein hve nti-microil ctivity. Jsso de Rodríguez (2005) tested Aloe ver (L.) Miller pulp on the mycelium development of Rhizoctoni solni Kühn, Fusrium oxysporum Schlechtend, nd Colletotrichum coccodes Berk nd Broome. We decided to expnd on the erlier in vitro work of Aror (1997), testing Cmelli sinensis (L.) O. Kuntze wter extrcts s well s some individul compounds in te for possile nti-fungl ctivity in vitro nd in vivo. The reserch ws lso encourged y positive results otined y frmer s climing tht Polyphenon G ( dried concentrted green te extrct) prevents cteril infections on vegetle crops (Hr, Personl communictions). We lso imed to identify the mode of ction y which our tretment compounds control or llevite the effect of plnt pthogens with specific focus on induced resistnce. 8

9 CAMELLIA SINENSIS DESCRIPTION AND ORIGIN Te is the common nme for certin Cmelli species elonging to fmily consisting of mostly woody flowering plnts. This fmily of plnts is distriuted throughout tropicl nd sutropicl res, ut most species occur in estern Asi nd South Americ. C. sinensis is considered to e the most importnt plnt in the Cmelli genus, prticulrly from commercil point of view, s it hs ecome the second most populr everge, fter wter, throughout the world (Wng et l, 2000). Te ws first discovered in Chin where it hs een consumed for medicinl properties since 3000 BC (Blentine et l, 1997; Ferrr et l, 2001). The first exclusive ook on te, Ch' Ching mening 'Te Clssic' y the Chinese te expert Lu Yu ws pulished in AD 780 in which he hs descried vrious kinds of te, their cultivtion nd mnufcturing in Chin. Te my vry in flvour nd chrcteristics ccording to the genotype, type of soil, ltitude nd climtic conditions of the re in which it is grown (Grmz nd Korczk, 2005). Processing methods lso ffect the flvour nd chrcteristics, s does the lending of different tes from different cultures nd geogrphic regions. Three min ctegories of te re produced from C. sinensis, nmely green-, oolong- nd lck te. These vry only in the processing, which results in differences in the chemicl mke up of the individul types of te. The processing of green te is sed on the inctivtion of enzymes in fresh leves, either y firing or steming, to prevent the enzymtic oxidtion of ctechins. In lck te the polyphenol oxidse ctlysed oxidtion of fresh lef ctechins is encourged (Wng et l, 9

10 2000). Oolong te is semi-fermented nd the chrcteristics of this te re etween tht of green nd lck te. CHEMISTRY The everge mde from the leves of the C. sinensis plnt tht is n romtic stimulnt, contining vrious polyphenols, essentil oils such s cffeine (Figure 1.1), theoromine, nd theophylline, the principl lkloids, phenolic cids such s gllic cid nd the chrcteristic mino cid, thenine (Figure 1.2) (Hr, 2001). Polyphenols in green te include flvnols, flvndiols, flvonoids, nd phenolic cids; these compounds my ccount for up to 30% of the dry weight in young te leves. Most of the green te polyphenols re flvonols, commonly known s ctechins. As previously mentioned the processing of te chnges the te chemistry, thus chnging the polyphenolic component of the te. The polyphenols tht occur in te re divided into two min groups nmely the crude ctechins nd the crude theflvins nd theruigins (Fuki et l, 1991; Hrowy et l, 1997), with the ctechins predominntly occurring in green te nd the theflvins nd theruigins predominntly occurring in lck te (Hrowy et l, 1997; Wng et el, 2000; Hr, 2001). The min ctechins (Figure 1.3) in green te re epiglloctechin gllte (EGCg), epictechin gllte (ECg), epiglloctechin (EGC) nd epictechin (EC) (Wng et l, 2000). Herl tes, which contin vrious known, or unknown components, re quite different from te (C. sinensis). Rooios (Aspltus lineris, (L) Kuntze) te is n herl te tht ws lso included in our study ecuse it is indigenous to South Afric nd it is 10

11 considered to hve medicinl properties. Rooios te contins no cffeine nd very smll quntities of flvonoids. Asplthin is the min polyphenol in Rooios (Gdow et l, 1997). It is importnt to hve n understnding of the chemicl composition of different tes nd the vrince in composition of chemicls in the different tes to eventully understnd the mechnisms y which microil growth is inhiited. MEDICINAL PROPERTIES C. sinensis extrcts s well s specific compounds in these extrcts hve well documented medicinl properties. Green te is known to e very rich in flouride. A study using nturl toothpste (contining green te ioflvonoid/zinc scorte) ws conducted to determine the effect on cteril plque ccumultion. The results showed significnt decrese in totl vile plque iomss when compred with non-ctive control toothpste (Wolinsky et l, 2000). Another study showed the epictechins to hve properties tht prevent cteril dherence to teeth, inhiit humn nd cteril mylses nd inhiit glucosyl trnsferse, therey limiting the iosynthesis of sticky glucn (Wng, 2000). Using different niml models, mny lortories hve shown tht green te extrct, tken orlly or pplied to the skin, inhiits skin tumour formtion induced y chemicl crcinogens or ultr-violet rdition (UVB). The extrcts lso possess nti-inflmmtory ctivity (Kkegw et l, 1985; Wgner, 1989) tht, similrly to the nti-cncer ctivity (Wng et l, 1994), is owed to the polyphenolic constituents present therein. The polyphenol 11

12 minly responsile for the prevention of cncer formtion is EGCg. When pplied to mouse skin, EGCg prevents UVB-induced oxidtive stress. Mouse skin models hve illustrted extensive eneficil effects of green te extrcts nd lthough only few humn skin studies hve een conducted, mny cosmetic nd phrmceuticl compnies re supplementing their skin cre products with green te extrcts (Ktiyr nd Elmets, 2001) Te hs lso shown to hve nti-crcinogenic ctivity (Skmoto, 2000; Lmert nd Yng, 2003). A study (Nkchi et l, 2000) using 8552 residents, representtive of Jpn s popultion, tested whether or not green te ws n effective nti-crcinogenic. Results showed decresed reltive risk of cncer incidence for those consuming over ten cups, compred with those consuming elow three cups of green te per dy. In ddition, incresed consumption ws ssocited with significnt dely in the onset of cncer. The tumour incidence nd verge tumour yield in rts with chemiclly induced colon cncer were significntly reduced when the rts received (-)-epiglloctechin gllte, mjor polyphenolic constituent of green te (Kim et l, 1994). In study conducted t the New Jersey Medicl School, extrcts of oth lck nd green te significntly inhiited leukemi nd liver tumour cells from synthesizing DNA (Lee et l, 1993). C. sinensis extrcts hve lso shown to hve ntioxidnt ctivity which is known to oost the immune system (Serfini et l, 1996; Cmpnell et l, 2003). Some ntioxidnts re known to reverse endothelil dysfunction which could led to the reduced risk of crdiovsculr disese (Duffy et l, 2000) Although te hs proven medicl enefits there re lso possile helth risks. Cffeine cn induce insomni nd nervousness in some individuls. The U.S. Food nd Drug 12

13 Administrtion does not include cffeine on its generlly recognized s sfe (GRAS) list, ut cknowledges no cler evidence of hzrd t norml levels of use. Phenolic-rich te extrcts hs een shown to reduce the ility of humns to utilise dietry iron. Thus excessive intke of te should e voided y individuls who re prone to nemi nd lso y vegetrins (Smmn et l, 2001). ANTI-MICROBIAL PROPERTIES Humn nd niml pthogens Green nd lck tes hve een reported to possess nti-fungl, nticteril, nd ntivirl ctivity (Dreosti, 1996; Ann et l., 2003). Green te extrct hve een shown to inhiit cteril species such s Escherichi coli (IFO 3301), Streptococcus mutns (IFO 13955), Helicocter pylori (ATCC 6919), Propionicter cnes (ATCC 6919), Bcillus sterothermophilus (ATCC 12980) nd Stphylococcus ureus (ATCC 25923) in vitro to nme ut few (Ymmoto et l, 1997; Ktsuhiro et l, 1999). Glucn synthesis y the cteril glucosyltrnsferse is inhiited y ECg nd EGCg (Kuo et l, 1992). These gllted ctechins were lso shown to interfere with the sucrose-dependent dherence of cteril cells t much smller concentrtions thn those which were needed to inhiit the growth of the cteri (Muroi et l, 1993). C. sinensis extrct hve een shown to hve some nti-fungl ctivity on humn pthogens such s Cndid licns nd other filmentous fungi (Ym et l, 1998). Ctechins, cffeine nd tnnic cid hve proven ctivity ginst viruses such s influenz virus (Nkym et l, 1990), rotvirus (Ymmoto et l, 1997) nd humn immunodeficiency virus (Nkne nd Ono, 1990). 13

14 Plnt pthogens Fuki, Ishigmi nd Hr (1991) tested the nti-cteril ctivity of ctechins (EC, EGC, ECg nd EGCg) ginst phytopthogenic cteri in vitro. The strins tht were tested included Erwini spp., Pseudomons spp., Clvicter spp., Xnthomons spp. nd Agrocterium spp. Erwini spp.ws the most inhiited while Clvicter spp.ws the lest inhiited of the cteri. EGCg hd the most nti-cteril ctivity of the ctechins tested. Blck nd green te extrct hve lso een shown to hve ctivity ginst Bcillus spp., Pseudomons spp. nd Xnthomons spp. in vitro (Aror nd Bhrwj, 1997). Cffeine hs lso een documented s eing insecticidl, lrvicidl nd inhiitory to moulds, yest nd cteri (Nthnson, 1984; Person nd Mrth, 1990 nd Anej nd Ginfgn, 2001). Antifungl ctivity of ctechins ginst Bipolris cronum Nelson hs lso een reported (Chkrorty nd Sh, 1994). El-Gmml et l. (1986) showed tht the flvonols quercitin, kempferol nd myricetin hd ctivity ginst grm-positive cteri nd phytopthogenic fungi in screening test. Theflvins, the oxidized products formed from te lef ctechins during lck te fermenttion, showed n nti-virl ctivity on tocco mosic virus (TMV) (Okd et l, 1977). Little work hs een done on the effectiveness of C. sinesis extrcts nd some of the individul components thereof on phytopthogens. This hs therefore een min focus of our study. 14

15 MECHANISMS WHICH CONTRIBUTES TO CAMELLIA SINENSIS ANTI-MICROBIAL ACTIVITY The mjor nti-microil ctivity of C. sinensis extrcts hs een contriuted to cffeine nd the ctechins in these extrcts. Cffeine is known inhiitor of norml cell division in plnt nd niml cells tht often results in inuclete cells (Anej nd Ginfgn, 2001), thus inhiiting spore germintion nd growth of microes. Gllted ctechins (EGCg nd ECg) hve een reported to dectivte proteins nd disrupts the cteril lipid ilyer y chnging the memrnes fluidity nd morphology (Ikigi et l, 1993). During the current study we imed t identifying whether cffeine nd ctechin rich C. sinensis extrct (PolyphenonG) induces phytolexins in tomto nd lettuce plnts. Induced resistnce is the phenomenon tht plnt, when given n pproprite stimultion y hrmless, inducer, will ecome le to comt more efficiently the infection y virulent pthogens through enhncement of its nturlly inherent defence mechnisms. Efforts were concentrted on the identifiction of ferulic cid, cffeic cid in the lettuce nd tomto root nd shoot extrcts, s these cids re known to e in these plnts or reported to e induced s prt of the plnts nturl defence mechnisms (Cspersen et l, 2000 nd Romni et l, 2002). 15

16 CONCLUSIONS Previous studies demonstrting some nti-microil ctivity of C. sinensis extrcts nd some of the mjor compounds in these extrcts provided the motivtion for the current study, with the im of investigting wider griculturl ppliction of these nturl products. Furthermore, understnding the underlying mode of ction of these compounds will contriute towrds improved ppliction of these products. The ims of the current study ws: 1. To test whether Polyphenon G (PPG), concentrted extrct from green te (C. sinesis) nd the individul compounds in PPG hd ctivity ginst phytopthogenic fungi in vitro nd in vivo. 2. To determine the mode of ction of these compounds using specific iochemicl methods imed t identifying possile induced resistnce. 16

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22 Nkym M., Suzuki K., Tod M., Okuu S., Hr Y., Shimmur T., Inhiition of the effectivity of influenz virus y te polyphenols. Antivir. Res. 21, 289. Nthnson J.A Cffeine nd relted methyl-xnthines: possile nturlly occurring pesticides. Sci. 226: Oerke EC, Dehne HW, Schoneck F, Weer A Crop Protection nd Crop Production. Amsterdm: Elsevier. Okd F., Tkeo T., Okd S., Tmems O., Anti virl effect of theflvins on Tocco mosic virus. Agri. Biol. Chem. 41, Person L.J., Mrth E.H.;1990. Behviour of Listeri monocytogenes in the presence of methylxnthines- cffeine nd theoromine. J. Food Prot. 53, Pohlnd AE Mycotoxins in review. Food Addit. Contm. 10, Romni A, Pinelli P., Glrdi C., Sni G., Cimto A., Heimler D., Polyphenols in greenhouse nd open ir grown lettuce. Food Chem. 79, Smmn S., Sndstrom B., Toft M.B., Bukhve K., Jensen M., Sorensen S.S. Hnsen M., Green te extrct dded to foods reduces nonheme-iron sorption. Am. J. Clin. Nutr. 73,

23 Schneider EF, Dickert KJ Helth costs nd enefits of fungicide use in griculture: literture review. J. Agromed. 1, Serfini M., Ghiselli A., Ferro Luzzi A., In vivo ntioxidnt effect of green te nd lck te in mn. Eur. J. Clin. Nutr. 50, Wgner H Serch for new plnt constituents with potentil ntiphlogistic nd ntillergic ctivity. Plnt. Med. 55, Wng H., Provn G.J., Helliwell K., Te flvonoids: their functions, utilistion nd nlysis. Trends Food Sci. Tech. 11, Wng Z.Y., Cheng S.J., Zhou Z.C., Athr M., Khn W.A., Bickers D.R., Mukhtr H., Antimutgenic ctivity of green te polyphenols. Mutt. Res. 223, Wolinsky LE., Cuomo J., Quesd K., Bto T., Cmrgo P.M., A comprtive study of the effects of dentifice contining green te ioflvonoids, snguinrine or triclosn on orl cteril iofilm formtion. J. Clin. Dent. 11,53-9. Ym T.S., Shh S. Hmilton-Miller J.M.T Microiologicl ctivity of whole nd frctionl crude extrcts of te (Cmelli sinensis), nd of te components. FEMS Microiol. Lett. 152, Ymmoto, T., Junej, L. R., Chu, D., Kim, M., Chemistry nd pplictions of green te. CRC Press, USA. 23

24 C H 3 N O N CH 3 O N N CH 3 Figure 1.1 Chemicl structure of cffeine HOOC H 2 N H 2 C C HH 2 O N H CH C 3 H 2 Figure 1.2 Chemicl structure of thenine (γ-ethyl mine glutmic cid) 24

25 OH OH OH OH HO O HO O OH (A) OH OH OH OH (B) OH OH OH OH HO O HO O OH OH O O OH OH O O OH (C) OH OH OH OH (D) Figure 1.3 Chemicl structures of (A) Epictechin (EC) (B) Epiglloctechin (EGC) (C) Epictechin gllte (ECg) nd (D) Epiglloctechin gllte (EGCg) 25

26 CHAPTER 2* In vitro Inhiition of Phytopthogenic Fungi y Cffeine, Te Ctechins, Thenine, Polyphenon G, Blck-, Green - nd Rooios te extrcts * The use of Polyphenon G nd cffeine s fungicide hs een ptented in 2006, ref numer 05P328 MN 26

27 ABSTRACT Commercil griculture relies hevily upon high inputs of chemicl pesticides to protect crops ginst pthogens nd pests. These prctices re currently eing re-evluted due to concern out the possile helth nd environmentl consequences. One option for n environmentlly sfe, ntimicroil compound is the use of te extrcts for control of phytopthogenic fungi. The present study reports on the sensitivity of twenty different phytopthogenic fungl species to extrcts from lck-, green- nd rooios te extrcts, concentrted green te extrct (Polyphenon G), cffeine, thenine, epiglloctechin gllte (EGCg), epictechin gllte (ECg), epiglloctechin (EGC), nd epictechin (EC), nd Polyphenon G comined with cffeine. The fungi included in the study were the following: Alternri solni, Aspergillus flvus, Botrytis cinere, Chlr elegns, Colletotrichum coccoides, Curvulri lunt, Dreschler sp., Fusrium oxysporum, F. solni, Mucor hiemlis, M. pusillus, Penicillium expnsum, Phytophthor cpsici, P. nicotine, Pythium F- group, Rhizopus stolonifer, Sclerotini sclerotiorum, Sclerotium rolfsii, Stemphylium herrum nd Verticillium fungicol. In vitro nti-fungl ctivity ws determined y mens of the gr-mendment method. For ech fungus, the dose response ws recorded nd the IC 50 vlue to the compounds ws clculted. The inhiition of fungl growth y the compounds ws s follows (in decresing order): cffeine > EGCg ECg > EGC EC > Polyphenon G > green te extrcts lck te extrcts > rooios te extrcts thenine. In some cses the Polyphenon G nd cffeine comintion reduced the IC 50 vlues for oth the compounds, indicting synergistic effect. P. nicotine nd P. cpsici were most sensitive to ll the compounds, while R. stolonifer nd P. expnsum were lest sensitive. These results suggest 27

28 the possiility tht these compounds will e effective ginst fungl diseses of crops when pplied in vivo. INTRODUCTION Crop growers continully ttle with fungl diseses ffecting their crops. Chemicl insecticides nd fungicides re helpful in improving the qulity nd dependility of food supplies when used correctly nd with cre. The resistnce of pests nd plnt pthogens ginst pesticides nd fungicides is rpidly ecoming serious prolem, nd some chemicls hve detrimentl effects on the environment. Growing consumer wreness towrds the use of orgniclly grown produce is eginning to direct frmers into the investigtion of lterntive chemicl control mesures such s nturl plnt extrcts (Quirog et l, 2001). In n effort to serch for novel environmentlly friendly ntimicroil gents, vrious plnt extrcts re eing screened in vitro. According to Ad (1996), the osmotic proteins in tocco plnts re le to inhiit spore germintion of some phytopthogenic fungi. The ntimicroil ctivity of te (Aror nd Bhrdwj, 1997) nd te polyphenols hs lso een demonstrted (Fuki et l., 1991; Kuo et l., 1992). Recently it hs een shown tht certin compounds in olives (Del Rio et l., 2003), such s tyrosol, ctechins nd oleuropein, hve ntimicroil ctivity. The ntimicroil properties of cffeine nd polyphenols re known to e prt of the chemicl defence mechnism in certin plnts (Hrorne, 1993). Three compounds, nmely ctechins, cffeine nd thenine (respectively 15%, 5% nd 4% of dry weight) re found in reltively high concentrtions in te (Cmelli sinensis (L.) Kuntze) (Hr, 2001). Te cultivrs tht synthesize high mounts of these compounds hve een selected over centuries for commercil te production, due to their good tste nd 28

29 disese resistnce. Despite the considerle metolic energy cost of producing such lrge mount of secondry metolites (Hrorne, 1993), the scrcity of pthogens on te plnts is proly due to the undnce of these compounds. The low toxicity of te on humns is well estlished, since dverse effects hve rrely een reported. Investigtion into the nti-fungl properties of te extrcts on fungl pthogens of selected vegetle crops, ll with low phenolic- nd no cffeine content, ws therefore undertken. In Jpn, frmers hve oserved positive effects ginst cteril diseses on vegetle crops when using Polyphenon G (Hr, personl communiction). In the current study, the nti-fungl ctivity of te extrcts nd individul compounds ws ssessed ginst rod spectrum of fungi. The nti-fungl ctivity of Rooios te extrct (Asplthus lineris) ws lso investigted. Rooios leves my e rewed into n herl te tht contins no cffeine nd smll quntities of ctechins. Asplthin is the min flvonoid in the rooios plnt (Gdow et l, 1997). The in vitro nti-fungl ctivity of extrcts from lck-, green- nd rooios te extrcts, concentrted green te extrct (Polyphenon G) nd the principle components thereof, nmely cffeine, thenine, epiglloctechin gllte (EGCg), epictechin gllte (ECg), epiglloctechin (EGC) nd epictechin (EC) were ssessed individully, using the grmendment method (Aror nd Ohln, 1997). The comined effect of Polyphenon G nd cffeine ws then investigted fter it ws determined tht these two compounds were the most effective nd suitle for in vivo ppliction. All of the ove-mentioned compounds were tested ginst selected fungl phytopthogens of vegetle crops nd post-hrvest pthogens, including Penicillium expnsum Link nd Aspergillus flvus Link: Fr, oth mycotoxin producers. P. expnsum is known to produce the toxin ptulin in pples (Errmplli, 2004), whilst A. flvus produces fltoxins in cerel crops such s mize nd 29

30 whet (Ptkr et l, 1994) under wrm nd humid storge conditions. Inhiition of these fungi y te compounds could hve oth economic nd helth importnce. MATERIALS AND METHODS Fungl phytopthogens tested The following fungl isoltes were otined from the culture collection of the Deprtment of Microiology nd Plnt Pthology, University of Pretori nd mintined on potto dextrose gr (PDA) (Biol, Johnnesurg, RSA): Alternri solni (Soruer), A. flvus (Link), Botrytis cinere (de Bry), Chlr elegns (Nj Rj nd W.B. Kendr.), Colletotrichum coccoides (Berk nd Broome), Curvulri lunt (Wkker), Dreschler sp., Fusrium oxysporum (Schltdl. Em. W.C. Snyder nd H.N. Hnsen), F. solni (Mrt.), Mucor hiemlis (Link.), M. pusillus (Link.), P. expnsum (Link), Phytophthor cpsici (Leonin), P. nicotine (Brend de Hn), Pythium F-group, Rhizopus stolonifer (Ehren.: Fr), Sclerotini sclerotiorum ((Li.) de Bry), Sclerotium rolfsii ((Curzi) Tu nd Kimr), Stemphylium herrum (E. Simmons) nd Verticillium fungicol (Kle.). These fungi were selected on the sis of their host specificity nd txonomic diversity. The diseses cused y these pthogens re shown in Tle 2.1. Chemicl compounds tested The chemicls nd concentrtions thereof used in the fungl inhiition studies re given in Tle 2.2. The composition of Polyphenon G, s provided y Mitsui Norin Co. Ltd. (MN), Jpn, is given in Tle 2.3. Polyphenon G in comintion with cffeine ws tested t 30

31 concentrtions tht vried etween pthogens. The verge IC 50 vlues, otined in previous single compound inhiition studies, were used in the Polyphenon G nd cffeine comintion tests. The fungi were divided into three ctegories nmely: sensitive, intermedite nd tolernt. The verge IC 50 vlue for ech group of fungi ws then qurtered, hlved, douled nd qudrupled to give totl of five doses per compound per fungus. These phytopthogens nd the investigted concentrtions re listed in Tle 2.4. Polyphenon G, thenine, EGCg, ECg, EGC nd EC were donted y MN. Cffeine ws purchsed from Sigm. Lipton lck te, Lipton rooios te nd Eve s green te were purchsed from locl supermrket. Preprtion of the te extrcts The te extrcts for lck-, green- nd rooios te were prepred y rewing in hot wter. The required mount of ech te smple ws weighed off, plced in muslin cloth g nd suspended in oiling wter for 15 minutes. Excess te extrct ws then mnully squeezed out of the cloth g. The extrct ws llowed to stnd for 10 minutes fter which it ws decnted into n Erlenmeyer flsk. The volume ws djusted to 1L. PDA ws mended with the te extrcts t the required concentrtions nd utoclved for 12 minutes t 121 C. Assy of the nti-fungl ctivity The gr-mendment method ws used to ssess nti-fungl ctivity (Aror nd Ohln, 1997). Concentrtions of the compounds used re given in Tle 2.2 nd Tle 2.4. Ech gr plte (60 mm dimeter) ws inoculted in the centre with fungl disc (5 mm dimeter) cut from the edge of n ctively growing fungl colony (seven dys old) on PDA. The pltes were incuted t 25 C in the drk. The colony dimeter of M. hiemlis, M. 31

32 pusillus, Pythium F-group, R. stolonifer nd S. rolfsii ws mesured fter three dys, nd the colony dimeters of the remining fungi were mesured fter seven dys due to their slower growth rte. Fungi were grown on unmended PDA s controls. PDA mended with selected commercil fungicides ws used for comprtive purposes. Fungicides were selected on the sis of known efficcy ginst the representtive groups of fungi included in the current tests. All fungicides were tested t concentrtion of 0.1g.i.L -1. The trde nme, mnufcturer nd recommended dosge of the fungicides re given in prentheses: enomyl WP, 500g.i.kg -1 (Benlte, du Pont, Hlfwy House, RSA g.l -1 ) for A. flvus, B. cinere, C. elegns, C. coccoides, F. oxysporum, F. solni, P. expnsum, S. sclerotiorum, nd V. fungicol; difenoconzole EC, 250g.i.L -1 (Score, Novrtis, Isndo, RSA g.l -1 ) for A. solni, C. lunt, Dreschler sp. nd S. herrum; propmocr SL, 722g.i.L -1 (Previcur, Snchem, Durn, RSA g.l -1 ) for Pythium F-group; tolclofos-methyl WP,500g.i.kg -1 (Rizolex, Snchem, Durn, RSA g.l -1 ) for M. hiemlis, M. pusillus, R. stolonifer nd S. rolfsii; nd metlxyl GR, 50g.i.kg -1 (Ridomil, Novrtis, Isndo, RSA - 2 g.l -1 ) for P. cpsici nd P. nicotine. Ech experiment ws repeted twice nd three replicte pltes were used for ech. Clcultions The percentge inhiition ws clculted ccording to the following formul: Percentge inhiition = (C T) x 100 C Eqution 1 C T = the dimeter of growth of the unmended control. = the dimeter of growth on the test plte. 32

33 The IC 50 vlues for green nd lck te were determined visully from dose response grphs. The IC 50 vlues for cffeine, EGCg, Polyphenon G nd comintions of Polyphenon G nd cffeine were clculted using the Clcusyn 1.1 (Biosoft) softwre pckge (Chou nd Hyll, 1996). The generl eqution for dose-effect s derived y Chou (1996) correltes dose nd the effect in the simplest possile form: f /f u = (D/D m ) m Eqution 2 D D m f = the dose of the compound = the medin-effect dose signifying the potency = the frction effected y the dose f u m = the frction unffected, fu = 1- f = n exponent signifying the sigmoidicity of the dose curve. The comintion results were processed ccording to the Chou-Tlly comintion index (CI) eqution using the Clcusyn progrm version 2 (Chou nd Hyll, 1996). The eqution determines only the dditive effect rther thn synergistic or ntgonistic effect. However, synergism is defined s more thn dditive effect, whilst ntgonism is defined s less thn dditive effect. Thus, CI < 1 indictes synergism, CI = 1 indictes dditive effect nd CI > 1 indictes ntgonism. Using this CI eqution hs the dded enefit of the utomted clcultion of the dose reduction index (DRI). DRI is n importnt prmeter s fvourle DRI (i.e. > 1) reduces toxicity while retining the therpeutic effect (Hn et l, 2004). 33

34 RESULTS Thenine hd no inhiitory effect on ny of the fungi tested. The rooios te extrct inhiited only P. nicotine t n IC 50 vlue of less thn 100 g.l -1 whilst the IC 50 vlues for ll the other pthogens ws higher thn 200g.L -1. The IC 50 vlues for cffeine, EGCg, Polyphenon G, comintions of Polyphenon G nd cffeine, lck nd green te extrcts re indicted in Tle 2.5. The in vitro fungl inhiition otined with four ctechins t 2.5g.L -1 is shown in Tle 2.6. The clculted CI nd DRI vlues for vrious concentrtions of the Polyphenon G nd cffeine comintions re given in Tle 2.7. DISCUSSION The present study investigted the in vitro sensitivity of twenty different fungi to te extrcts, nd to vrious individul compounds from te. The mount of inhiition of fungl growth y the compounds ws s follows (in decresing order): cffeine > EGCg ECg > EGC EC > Polyphenon G > green te extrcts lck te extrcts > rooios te extrcts thenine. The comined effect of Polyphenon G nd cffeine ws lso investigted nd in some instnces the comintion of Polyphenon G nd cffeine reduced the IC 50 vlues for oth the compounds individully therey indicting synergistic effect. Cffeine is the most undnt lkloid in te (Hr, 2001). The nti-fungl ctivity of cffeine ginst wood-rotting pthogens ws previously reported y Aror nd Ohln (1997). It hs lso een reported tht cffeine ccumultes round wound res in coffee plnts s nturl, ntimicroil defence mechnism (Anej nd Ginfgn, 2001). With the exception 34

35 of P. expnsum nd F. solni, ll the fungi were completely inhiited t concentrtions of less thn 5 g.l -1 of cffeine. The effect of cffeine on S. rolfsii is shown in Figure 2.2. During the studies y Aror nd Bhrdwj (1997) on the nti-fungl ctivity of lck nd green tes on phytopthogens, only one concentrtion of te extrct ws used, nmely 150 g.l -1. Their results showed 49% inhiition of F. solni with green te extrct nd 45% inhiition y lck te extrct, while the current study found 51% inhiition of the sme fungus with green te extrct nd 40% inhiition with lck te extrct, when tested t the sme concentrtion, indicting similr findings. Of the three tes tested green te extrcts showed the highest nti-fungl ctivity followed y lck te extrcts, while rooios te extrcts exhiited very little or no nti-fungl ctivity (effect on S. sclerotiorum illustrted in Figure 2.3). Cffeine, EGCg nd Polyphenon G showed much higher nti-fungl ctivity thn the lck-, green- nd rooios te extrcts. Thenine hd no nti-fungl effect t the highest dose tested (20 g.l -1 ). Rooios te extrcts hd negligile nti-fungl effect. The lck te extrcts exhiited less nti-fungl ctivity thn green te extrcts. Blck te is enzymticlly oxidised nd contins fewer monomeric polyphenols (Chun et l., 1999) thus, even though oth lck- nd green te extrcts contin cffeine, green te extrcts still exhiited greter nti-fungl ctivity, possily due to the presence of the monomeric ctechins. PPG ws five times more effective thn green te extrcts. EGCg ws ten times more effective thn green te extrcts. The effect of EGCg on F. oxysporum is shown in Figure 2.4. Cffeine ws 100 times more effective thn green te extrcts. Thus cffeine cts synergisticlly with PPG to mke this crude extrct of green te s effective s pure EGCg. The monomeric ctechins tested included EC, EGC, ECg nd EGCg. All of these compounds were tested t concentrtion of 2.5 g.l -1 to determine which ctechin in Polyphenon G exhiits the gretest nti-fungl properties. Overll, the 35

36 gllted ctechins exhiited greter nti-fungl ctivity thn the non-gllted ctechins. The nti-fungl efficcy seems to increse with moleculr mss nd numer of hydroxyl groups on the ctechin i.e. EGCg ECg > EGC EC. This reltionship cn e seen for C. coccodes (Figure 2.5), Dreschler sp., P. cpsici nd P. nicotine. The increse in percent growth inhiition y ctechins with increse in moleculr weight is shown in Figure 2.1. This grees with the nticteril properties reported y Fuki et l. (1991) nd Ymmoto et l, (1997) nd the ntimutgenic properties reported y Apostolides et l. (1997). This ner liner reltionship my e useful in predicting the ntimicroil properties of other ioflvonoids. Polyphenon G hd n IC 50 of < 16 g.l -1 for 11 of the 20 pthogens. EGCg hd n IC 50 of < 5g.L -1 for 11 of the 20 pthogens. Cffeine hd n IC 50 of < 2 g.l -1 for 18 of the 20 pthogens gin indicting rod-spectrum of ctivity. Overll, the in vitro efficcies of commercil fungicides (t lower concentrtion thn the experimentl compounds) were etter thn the experimentl compounds. In some cses the experimentl compounds used t higher concentrtions thn the fungicides preformed s well s the fungicides. The comintion index (CI) eqution is sed on the multiple drug-effects derived from enzyme kinetic models. The DRI defines the degree of dose reduction tht is possile in comintion for given degree of effect s compred with the concentrtion of ech drug lone. Thus CI vlue less thn one nd DRI vlue for oth chemicls greter thn one indictes synergism. The CI nd DRI vlues for ll concentrtions of the PPG cffeine comintions re noted in Tle 2.7. The comintions tht showed synergistic effect re indicted in old (ornge), whilst those tht showed n ntgonistic effect re indicted in itlics (lue). Those tht re neither synergistic nor ntgonistic re indicted in lck. 36

37 The comintions of Polyphenon G nd cffeine reduced the mount of one or oth compounds required to otin the IC 50 doses for 17 of the 20 pthogens. The PPG + cffeine mixture hlved the concentrtions needed for ech chemicl individully to otin IC 50 vlues in the cse of A. solni, B. cinere, M. pusillus nd V. fungicol. Thus the PPG + cffeine mixture showed significnt synergism for four of the 20 fungi tested nd showed mild synergism for 13 of the 20 fungi tested. It cn e concluded from these studies tht te extrcts, nd some of the mjor compounds in te, hve the potentil to e effective nti-fungl gents with specific use ginst fungl diseses on vegetle crops. These compounds my e of economic importnce nd lso hve potentil use s rod-spectrum nti-microil gents. Greenhouse trils will hve to e conducted to determine whether these compounds will e effective ginst the diseses cused y these pthogens on their respective hosts. 37

38 REFERENCES Ad, L.R., D Urzo, M.P., Liu, D., Nrsimhn, M.L., Reuveni, M., Zhu, J.K., Niu, X., Sigh, N.K., osmotin hs Hsegw, P.M., Bressn, R.A., Anti-fungl ctivity of tocco specificity nd involves plsm memrne permeiliztion. Plnt Sci. 118, Anej, M., Ginfgn, T., Induction nd ccumultion of cffeine in young, ctively growing leves of coco (Theorom cco L.) y wounding or infection with Crinipellis pernicios. Physiol. Mol. Plnt Pthol. 59, Apostolides, Z., Blentine, D.A., Hrowy, M.E., Hr, Y., Weisurger, J.H., Inhiition of PhIP mutgenicity y ctechins, nd y theflvins nd gllte esters. Mutt. Res. 389, Aror, D.S., Bhrdwj, S.K., Antimicroil ctivity of te (Cmelli sinensis) ginst some plnt pthogens. GEOBIOS. 24, Aror, D.S., Ohln D., In vitro studies on nti-fungl ctivity of te (Cmelli sinensis) nd coffee (Coffe ric) ginst wood rotting fungi. J. Bsic Microiol. 37,

39 Chou, T.C., Hyll, M.P., Clcusyn: Windows softwre for dose effect nlysis. Biosoft, Cmridge. Chun, C.C., Leu, L.L., Thom C.K, Antimicroil ctivity of te s ffected y the degree of fermenttion nd mnufcturing seson. J. Food Microiol. 48, Del Rio J.A., Bidez, A.G., Boti, J.M., Ortuno, A., Enhncement of phenolic compounds in olive plnts (Ole europe L.) nd their influence on resistnce ginst Phytophthor spp. Food Chem. 83, Errmplli D., Effect of fludioxonil on germintion nd growth of Penicillium expnsum nd decy in pple cvs. Empire nd Gl. Crop Prot. 24, Fuki, K, Ishigm, T., Hr, Y., Anticteril ctivity of te polyphenols ginst phytopthogenic cteri. Agric. Biol. Chem. 55, Gdow, V., Jouert, E., Hnsmn, C.F., Comprison of the ntioxidnt ctivity of rooios te (Asplthus lineris) with green-, oolong nd lck te. Food Chem. 60, Hn, T., Fernndez, M., Chou, T., Rm, P., Agrwl 2-Chloro-2 -deoxydenosine synergisticlly enhnces zidothymidine cytotoxicity in zidothymidine resistnt T- lymphoid cells. Biochem. Biophys. Res. Com. 301, Hr, Y., Green te. Helth Benefits nd Applictions. Mrcel Dekker Inc., New York. 39

40 Hrorne, J.B., Introduction to Ecologicl Biochemistry. 4th ed. Acdemic Press, London. Kuo, I., Muroi, H., Himejim, M., Antimicroil ctivity of green te flvour components nd their comintion effects. J. Agric. Food Chem. 40, Ptkr, K.L., Ush, C.M., Shetty, H.S., Pster, N., Lcey, J., Effects of spice oil tretment of rice on moulding nd mycotoxin contmintion. Crop Prot. 13, Quirog, E.M., Smpietro, A.M., Vttuone, M.A., Screening nti-fungl ctivities of selected medicinl plnts. J. Ethnophrmcol. 74: 89. Ymmoto, T., Junej, L. R., Chu, D., Kim, M., Chemistry nd pplictions of green te. CRC Press, USA. 40

41 Tle 2.1 Fungi tht were tested in vitro, the hosts they infect nd the diseses they cuse Host Disese Pthogen Cucumer diseses Wilt Fusrium oxysporum (Cucumis stivus L.) Wilt Fusrium solni Lettuce diseses (Lctuc stiv L.) Tomto diseses (Lycopersicon esculentum Mill.) Vrious hosts Rot Root rot Lef spot Lef spot Erly light Grey mould Blck root rot Anthrcnose Wilt Wilt Root rot Buckeye rot Lef spot Post-hrvest rot Mycotoxin producers Sclerotini sclerotiorum Pythium F-group Curvulri lunt Stemphylium herrum Alternri solni Botrytis cinere Chlr elegns Colletotrichum coccoides Sclerotium rolfsii Verticillium fungicol Phytophthor cpsici Phytophthor nicotine Dreschler sp. Mucor hiemlis Mucor pusillus Rhizopus stolonifer Penicillium expnsum Aspergillus flvus 41

42 Tle 2.2 Compounds nd concentrtion rnges tht were tested for in vitro nti-fungl ctivity Compound Concentrtion (g.l -1 ) Polyphenon G 2.5, 5, 10, 15 nd 20 Cffeine 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 5 nd 10 Thenine 2.5, 5, 10, 15 nd 20 Blck te extrct (Lipton) 100, 150 nd 200 Green te extrct (Eve s) 100, 150 nd 200 Rooios te extrct (Lipton) 100, 150 nd 200 Epiglloctechin gllte (EGCg) 0.5, 1, 2, 2.5 nd 5 Epictechin gllte (ECg) 2.5 Epiglloctechin (EGC) 2.5 Epictechin (EC)

43 Tle 2.3 Composition of Polyphenon G* Compound % Dry weight Epiglloctechin (EGC) 8.4 Epictechin (EC) 2.1 Epiglloctechin gllte (EGCg) 13.8 Epictechin gllte (ECg) 2.6 Glloctechin gllte (ECg) 0.3 Ctechin gllte (CG) 0.2 Cffeine 6.1 Totl 33.2 * Provided y Mitsui Norin Co. Ltd. (MN), Jpn 43

44 Tle 2.4 Polyphenon G, cffeine nd comintions of the two compounds nd concentrtion rnges tht were tested ginst the selected pthogens Concentrtion (g.l -1 ) Fungl pthogens Polyphenon G Cffeine PPG+CAF* (PPG) (CAF) Alternri solni, Botrytis cinere, :0.5 Colletotrichum coccoides, :1 Tolernt fungi Chlr elegns, Fusrium solni, Penicillium expnsum, Rhizopus stolonifer, Sclerotium rolfsii nd :2 40:4 80:8 Sclerotini sclerotiorum Aspergillus flvus, :0.375 Intermeditly sensitive Dreschler sp., Fusrium oxysporum, Mucor pusillus nd Verticillium fungicol : :1.5 30:3 60:6 Curvulri lunt, Mucor hiemlis, :0.25 Sensitive fungi Phytophthor cpsici, Pythium F-group, Phytophthor nicotine nd :0.5 5:1 10:2 Stemphylium herrum :4 * PPG:CAF signifies the concentrtion of Polyphenon G nd cffeine used in ech cse, e.g. 5:0.5 is comintion of 5g of Polyphenon G nd 0.5 g of cffeine in 1L of potto dextrose gr (PDA). 44

45 Tle 2.5 The IC 50 vlues for in vitro inhiition of fungl growth y cffeine, epiglloctechin gllte (EGCg), Polyphenon G, comintions of cffeine nd Polyphenon G, green-, lck- nd rooios te extrcts Fungl Pthogens IC 50 (g.l -1 ) PPG Cffeine PPG:CAF*** EGCg Green te Blck te Rooios te extrcts extrcts extrcts Alternri solni : >200 >200 >200 Aspergillus flvus : >200 >200 Botrytis cinere : >200 >200 >200 Chlr elegns : >200 Colletotrichum coccoides : < >200 Curvulri lunt : >200 Dreschler sp : < >200 Fusrium oxysporum : >200 Fusrium solni : >200 >200 Mucor hiemlis : < >200 Mucor pusillus : < >200 Penicillium expnsum : >200 >200 >200 Phytophthor cpsici < 1.25:0.25* 4.02 < 100 < 100 >200 Phytophthor nicotine < 1.25:0.25* 0.10 < 100 < 100 < 100 Pythium F-group : < 100 < 100 >200 45

46 Rhizopus stolonifer :2 >5.0* >200 >200 >200 Sclerotini sclerotiorum : >200 >200 Sclerotium rolfsii :1 >5.0* 100 < 100 >200 Stemphylium herrum : < 100 < 100 >200 Verticillium fungicol : < 100 < 100 >200 Averge IC 50 (g.l-1) ** ** ** * Indicted vlues tht were excluded from verge IC 50 clcultions s they could not e clculted from the dt. ** The verge IC 50 vlues could not e clculted for these compounds s most of the vlues re < or > vlues. *** PPG:CAF signifies the concentrtion of Polyphenon G nd cffeine used in ech cse. Eg. 5:0.5 is comintion of 5g of Polyphenon G nd 0.5 g of cffeine in 1L of potto dextrose gr (PDA). 46

47 Tle 2.6 In vitro inhiition of fungl growth y epictechin (EC), epiglloctechin (EGC), epictechin gllte (ECg) nd epiglloctechin gllte (EGCg) t concentrtion of 2.5g.L -1 Percentge inhiition y ctechin type Fungl Pthogens EC EGC ECg EGCg Alternri solni Aspergillus flvus Botrytis cinere Chlr elegns Colletotrichum coccoides Curvulri lunt Dreschler sp Fusrium oxysporum Fusrium solni Mucor hiemlis Mucor pusillus Penicillium expnsum Phytophthor cpsici Phytophthor nicotine Pythium F-group Rhizopus stolonifer Sclerotini sclerotiorum Sclerotium rolfsii Stemphylium herrum Verticillium fungicol Averge percentge inhiition

48 Tle 2.7 Comintion index (CI) nd dose reduction index (DRI) vlues for concentrtions indicting synergism during nti-fungl ssys with Polyphenon G (PPG) nd cffeine (CAF) in vrious comintions. These vlues were clculted using the Clcusyn 1.1 (Biosoft) pckge Pthogens Concentrtions (g.l -1 ) PPG 5 CAF 0.5 PPG 10 CAF 1 PPG 20 CAF 2 PPG 40 CAF 4 PPG 80 CAF 8 CI DRI DRI CI DRI DRI CI DRI DRI CI DRI DRI CI DRI DRI PPG CAF PPG CAF PPG CAF PPG CAF PPG CAF Alternri solni Botrytis cinere Colletotrichum coccodes High dose Tolernt fungi Chlr elegns Fusrium solni Penicillium expnsum Rhizopus stolonifer Sclerotium rolfsii Sclerotini sclerotiorum

49 Medium dose Intermeditely sensitive fungi PPG 3.75 CAF PPG 7.5 CAF 0.75 PPG 15 CAF 1.5 PPG 30 CAF 3 PPG 60 CAF 6 Aspergillus flvus Dreschler spp Fusrium oxysporium Mucor pusillus Verticillium fungicol PPG 1.25 CAF 0.25 PPG 2.5 CAF 0.5 PPG 5 CAF 1 PPG 10 CAF 2 PPG 20 CAF 4 Curvulri lunt Mucor hiemlis Low dose Sensitive fungi Phytophthor cpsici Pythium F-group Photosphere nicotine Stemphylium herrum * CI vlues < 1 nd DRI vlues > thn 1 indictes synergism ** Vlues in old (ornge) indicte synergism, while vlues in itlics (lue) indicte ntgonism 49

50 Figure 2.1 The nti-fungl efficcy of epictechin (EC), epiglloctechin (EGC), epictechin gllte (ECg) nd epiglloctechin gllte (EGCg) t concentrtion of 2.5g.L -1 with regrds to moleculr mss 50

51 Figure 2.2 The in vitro effect of cffeine on the colony dimeter of Sclerotium rolfsii. The tretments (g.l -1 ) re indicted on the lels Figure 2.3 The in vitro effect of lck-, green- nd rooios- te extrcts on the colony dimeter of Sclerotini sclerotiorum. The tretments (g.l -1 ) re indicted on the lels 51

52 Figure 2.4 The in vitro effect of epiglloctechin gllte (EGCg) on the colony dimeter of Fusrium oxysporum. The tretments (g.l -1 ) re indicted on the lels Figure 2.5 The in vitro effect of thenine, Polyphenon G, cffeine, epiglloctechin gllte (EGCg), epiglloctechin (EGC), epictechin gllte (ECg) nd epictechin (EC) t concentrtion of 2.5g.L -1, on the colony dimeter of Colletotrichum coccodes 52

53 CHAPTER 3* Control of Phytopthogenic Fungi on Selected Vegetle Crops with Cffeine nd Green Te (Cmelli sinensis) Extrcts Under Greenhouse Conditions * The use of Polyphenon G nd cffeine s fungicide hs een ptented in 2006, ref numer 05P328 MN 53

54 ABSTRACT In the current study te (Cmelli sinensis) extrcts hve een evluted for their in vivo ctivity ginst phytopthogenic fungi. These compounds hve potentil s lterntive, environmentlly friendly plnt disese control gents. The fungicidl ctivity of Polyphenon G, cffeine, nd comintions of Polyphenon G nd cffeine were tested for in vivo ctivity ginst seven phytopthogenic fungi including Fusrium oxysporum nd Fusrium solni on cucumer, Phytophthor cpsici nd Sclerotium rolfsii on tomto, Sclerotini sclerotiorum nd Pythium F-group on lettuce nd Spherothec fuligine on zucchini sqush plnts. For ech pthogen, tretment with commercil fungicide effective ginst tht prticulr pthogen ws included s stndrds. The comintions of Polyphenon G nd cffeine gve the est overll results nd effectively controlled Fusrium solni on cucumer, Phytophthor cpsici nd Sclerotium rolfsii on tomto, Sclerotini sclerotiorum nd Pythium F-group on lettuce. This is the first report of in vivo fungicidl ctivity of Polyphenon G, cffeine nd comintions of these compounds. In some instnces the test compounds performed etter thn the commercil fungicides t the recommended doses. The results indicte tht Polyphenon G, cffeine or comintions of these compounds hve potentil s lterntive, environmentlly friendly plnt disese control gents on selected vegetle crops. 54

55 INTRODUCTION Alterntive strtegies employing the products of plnt extrcts for plnt disese control hve gined populrity in recent yers (Ad et l 1996; Singh et l, 1998; Lee et l, 2001; Quirog et l, 2001; Bnos-Butist et l, 2003; Del Rio et l, 2003; Jsso de Rodríguez et l, 2004). Plnt extrcts re rich in ioctive chemicls nd my e potentil lterntive to synthetic fungicides (Hedin, 1982). These nturl compounds re iodegrdle, resulting in the production of non-toxic intermedites (Lee et l, 2001). Certin plnt extrcts nd phytochemicls ct on different types of plnt disese complexes through vrious mechnisms nd my e pplied to crop in the sme wy s other synthetic chemicls. Vrious other plnt derived compounds, such s phenolics, terpenoids, lkloids nd lignns, my lso hve nti-microil ctivity (Hrorne, 1993). Anti-microil ctivity of commercil tes nd some of the individul compounds in te hve previously een reported (Fuki et l, 1991; Aror nd Bhrdwj, 1997). During previous in vitro studies (Chpter 2), we ssessed the ntifungl ctivity of extrcts from lck (Cmelli sinensis (L) Kuntze), green, nd rooios (Aspltus lineris (L) Kuntze) tes, concentrted green te extrct (Polyphenon G), nd the principle components thereof, including: cffeine, thenine, epiglloctechin gllte (EGCg), epictechin gllte (ECg), epiglloctechin (EGC) nd epictechin (EC). In the previous tests, cffeine (CAF) showed the gretest ntifungl ctivity followed y EGCg, Polyphenon G (PPG), green-, lck- nd rooios te extrcts in decresing order. Thenine hd no nti-fungl effect. Polyphenon G nd cffeine eing the most effective compounds in vitro, were tested in these trils 55

56 for their efficcy in vivo, on rnge of fungl pthogens on selected vegetle crops under greenhouse conditions. MATERIALS AND METHODS Fungl phytopthogens Pure cultures of the following six fungl species were otined from the University of Pretori culture collection nd mintined on potto dextrose gr (PDA) (Biol, Merck) pltes: Fusrium oxysporum (Schltdl. Em. W.C. Snyder nd H.N. Hnsen), Fusrium solni (Mrt.), Phytophthor cpsici (Leonin), Pythium F- group, Sclerotini sclerotiorum ((Li.) de Bry) nd Sclerotium rolfsii ((Curzi) Tu nd Kimr). Cultures were stored on PDA slnts t 25 C. Spherothec fuligine ((Schlecht.) Pollcci) ws mintined on live host plnts (Zucchini sqush (Cucurit pepo L.)) under greenhouse conditions, t tempertures vrying etween 25 C nd 31 C, for the durtion of the study. The pthogens nd diseses cused on the respective hosts re shown in Tle 3.1. Inoculum preprtion nd Inocultion The soilorne phytopthogens (F. solni, F. oxysporum, P. cpsici, Pythium F-group, S. sclerotiorum nd S. rolfsii) were grown on 90 mm dimeter PDA pltes t 25ºC until the pltes were completely covered. Millet (Penniselum glucem L.) seeds (200g) were steeped in wter (200ml) for 24h, trnsferred to utoclvle polyethylene gs, nd utoclved twice for 45min t 120 C on two consecutive dys. 56

57 Ten 5mm dimeter gr discs were cut with cork orer from the ctively growing edge of ech fungl colony nd mycelil discs were septiclly trnsferred to ech of the sterilized millet seed gs. The gs were incuted for 28 dys t 25 C. The millet seed inoculum of ech individul pthogen ws dded to the soil (Weidemn nd Wehner, 1993) t the following concentrtions: 100 g.l -1 for F. oxysporum, F. solni nd P. F-group; 70, 50 nd 20 g.l -1 for P. cpsici, S. sclerotiorum nd S. rolfsii, respectively. Zucchini sqush plnts were nturlly infected with S. fuligine y trnsferring helthy plnts to greenhouse with infected plnts nd plcing the helthy plnts mongst the infected plnts (Bettiol, 1999). Soil preprtion Sndy lom soil (Conrdie Orgnics, Pretori, Guteng, South Afric) nd potting soil mix consisting of composted krl mnure nd rk (Just Nture Orgnics, Pretori, Guteng, South Afric) ws stem psteurized seprtely nd mixed in 1:1 rtio (v:v) sed on results otined during preliminry trils. Host plnts Seeds of susceptile commercil vrieties of cucumer (Cucumis stivus L., cv Volcno), lettuce (Lctuc stiv L., cv Ndine), tomto (Lycopersicon esculentum Mill., cv Money Mker) nd zucchini sqush (Cucurit pepo, cv. Csert) were otined from Hygrotec (Pretori-North, Guteng, South Afric). The cucumer nd 57

58 zucchini sqush seeds were pre-germinted in sterile vermiculite in Conviron (Fisons, Fi-totron 600H) growth cinet t 25 ºC nd susequently trnsplnted into psteurized potting soil. The seedlings were grown until four true leves hd developed efore inocultion. The tomto nd lettuce seeds were germinted nd grown t commercil nursery (Multiplnt, Brits, North West South Afric) until five weeks old. Green house trils Tretments: The tretments included in this study were untreted uninfected nd infected controls respectively, fungicide stndrd nd five concentrtions of Polyphenon G, cffeine nd comintions of the two compounds (totl of 18 tretments per experiment). The tretment concentrtions re give in Tle 3.2. All the synthetic fungicide stndrds were tested t concentrtion of 0.1g.i.L -1. The trde nme, mnufcturer nd recommended dosge of the fungicides re given in prentheses: enomyl WP, 500g.i.kg -1 (Benlte, du Pont, Hlfwy House, RSA g.l -1 ) for F. oxysporum, F. solni nd S. sclerotiorum, propmocr SL, 722g.i.L -1 (Previcur, Snchem, Durn, RSA g.l -1 ) for P. F-group; tolclofosmethyl WP, 500g.i.kg -1 (Rizolex, Snchem, Durn, RSA g.l -1 ) for S. rolfsii; metlxyl GR, 50g.i.kg -1 (Ridomyl, Novrtis, Isndo, RSA - 2 g.l -1 ) for P. cpsici; triforine SL, 190 g.i.l -1 (Funginex, Efekto, Pretori, RSA 0.53 g.l -1 ) for S. fuligine. 58

59 Soilorne pthogens: Soil contining millet seed inoculum of the vrious soilorne pthogens ws trnsferred to 700 ml plstic pots. Seedlings were trnsplnted into the inoculted soil nd treted with 100 ml of the vrious compounds s soil drench. Soil inocultion, trnsplnting of the plnts into the inoculted soil nd first tretment of plnts were ll done on the sme dy. Tretments were pplied second time fter 14 dys. The experimentl design consisted of rndomised lock design with 18 tretments nd 8 replictes per tretment. Ech replicte comprised of single plnt per pot. Pots were rotted once week to ensure even exposure to greenhouse conditions. Ech experiment ws repeted twice. Greenhouse tempertures were mintined t 25 to 35ºC during the experiments. Twenty-seven dys fter inocultion the plnts were removed from the soil nd ny soil dhering to the roots ws rinsed off under running tp wter. Roots nd shoots of plnts were excised nd weighed seprtely. Root rot ws visully ssessed ccording to 0-4 scle (0 = helthy, 1 = 25%, 2 = 50%, 3 = 75% nd 4 = 100% root rot). Rndomly selected root segments (pproximtely 5 mm long) were excised from ech plnt, surfce sterilized in 70% ethnol for 1 min nd plted on the pproprite medium s descried elow. Three pltes were used per plnt with ten root segments plted on ech. From this, the percentge infection ws clculted nd pthogens were re-isolted (Stnghellini nd Kronlnd, 1986). Root segments from seedlings inoculted with F. solni, F. oxysporum nd S. sclerotiorum were plted onto semiselective RBGU medium (Vn Wyk et l 1986), contining: glycerol (UniL, Merck) 10 ml, ure (Biol, Merck) 1g, L-Alnine (Sigm) 0.5g, quintozene (750g 59

60 (ctive ingredient (.i.))kg -1, Plschem) 1g, Rose Bengl (Sigm) 0.5g nd Chlormphenicol (RoL, Norvtis) 0.25g. These chemicl compounds were dissolved in 20ml 70% Ethnol nd dded to 12g Agr cteriologicl (Biol, Merck) fter it hs een utoclved. Root segments from seedlings inoculted with P. F-group (Figure 3.1) were plted onto BNPRA medium (Msgo et l, 1977): tolclofos-methyl (500g.i.kg -1, Snchem) 0.2g, quintozene (750g.i.kg -1, Plschem) 0.05g, enomyl (500g.i.kg -1, Du Pont) 0.01g, nysttin (Sigm) 0.025g, mpicillin (BeTs phrmceuticls) 0.25g, rifmpicin (Rol, Norvtis) 0.025g These chemicl compounds were dissolved in 20ml 70% Ethnol nd dded to 12g Agr cteriologicl fter it hs een utoclved. Roots inoculted with P. cpsici were plted on BNPRAH medium which contins the sme ingredients s the BNPRA medi with n ddition of 1ml hymexzol (360 g (.i)l -1, Agrishel) (Msgo et l, 1977). Roots inoculted with S. rolfsii were plted on PDA mended with 0.05g.L -1 rifmpicin. Folir pthogen: The zucchini plnts infected with S. fuligine were treted only fter the onset of disese (seven dys fter eing moved to the greenhouse comprtment with high inoculum density). Zucchini sqush plnts were spryed with the vrious compounds until runoff, on weekly sis. No uninfected control ws included during this tril (McGrth nd Shiskoff, 1996). Experiments were lid out in rndomized design with five replictes per tretment. Ech repliction consisted of one pot contining one plnt. The pots were rotted on the greenhouse ench on weekly sis to ensure even exposure to conditions in greenhouse. The durtion of the experiment extended 60

61 over period of three weeks. Ech independent experiment ws repeted twice. The tempertures in the greenhouse during the experiments were mintined t 26 to 32 C. The severity of the S. fuligine infesttion ws visully evluted on ech individul lef, y rting ccording to 0-4 scle (0 = helthy, 1 = 25%, 2 = 50%, 3 = 75% nd 4 = 100% disese development). The disese rting scle for S. fuligine on zucchini sqush used during the experiments is shown in Figure 3.2 (Tzeng et l., 1996; Reuveni et l, 1996). STATISTICAL ANALYSIS Significnt differences were determined using Fisher s protected lest significnt difference test (F-test), nd re indicted on the grphs using letter nottion. The comprison wise error rte ws P<0.05. Stndrd error of the mens re indicted s error rs on the grphs. RESULTS Dt on the verge fresh shoot nd root mss is given in Figures 3.3, 3.5, 3.7, 3.9, 3.11 nd The dt on the verge percentge infection nd root rot rtings re shown in Figure 3.4, 3.6, 3.8, 3.10, 3.12 nd The verge percentge disese ssessment rtings for S. fuligine on zucchini sqush plnts re shown in Figure

62 Lettuce infected with Pythium F-group Tretment of Pythium infected lettuce plnts with the comintions of PPG nd cffeine t concentrtions of 2.5 g.l g.l -1 nd 5 g.l g.l -1 respectively resulted in shoot mss comprle to tht of the untreted uninfected control s well s zero percent root rot (Figure 3.3; 3.16). Tretment with PPG t concentrtion of 12.5 g.l -1 resulted in significntly higher shoot mss in comprison with the propmocr tretment. Tretments with PPG t concentrtions of 7.5 g.l -1 nd 10 g.l -1, cffeine t 0.25 g.l -1, 0.5 g.l -1, 1 g.l -1 nd 1.25 g.l -1 s well s comintions of PPG nd cffeine t concentrtions of 7.5 g.l g.l -1, 10 g.l g.l -1 nd 12.5 g.l g.l- -1 respectively, gve shoot mss comprle to tht of the propmocr tretment. The ltter comintion tretments lso resulted in percentge infection comprle to tht of the propmocr tretment, while ll other tretments gve significntly higher infection vlues (Figure 3.4). Significnt differences were not s prevlent with regrds to root mss nd were, for most tretments, comprle to tht of the uninfected untreted control. Exceptions to this were the untreted infected control nd tretments with PPG t 12.5 g.l -1, cffeine t 1.25 g.l -1 nd comintions of PPG nd cffeine t concentrtions of 7.5 g.l g.l -1, 10 g.l g.l-1 nd 12.5 g.l g.l -1 respectively. Excluding the tretments with PPG t 12.5 g.l -1 nd cffeine t 1.25 g.l -1 ll other tretments resulted in percentge root rot comprle to or significntly lower thn tht of the propmocr tretment (Figure 3.4). Lettuce plnts infected with Pythium resulted in 50% reduction in shoot nd root mss respectively when compring the untreted uninfected nd untreted infected controls. 62

63 Lettuce plnts infected with Sclerotini sclerotiorum Tretment of S. sclerotiorum infected lettuce plnts with PPG t concentrtion of 10 g.l -1 nd the comintion of PPG nd cffeine t the concentrtion of 25 g.l g.l -1 respectively resulted in shoot mss comprle to tht of the untreted uninfected control (Figure 3.5). Tretments with PPG t concentrtions of 15 g.l -1, 20 g.l -1 nd 25 g.l -1 nd the comintions of tretments with PPG nd cffeine t concentrtions of 5 g.l g.l -1, 10 g.l g.l -1, 15 g.l g.l -1 nd 25 g.l g.l -1 respectively, gve shoot mss comprle to tht of the enomyl tretment. The root mss of the uninfected control s well s tretments with enomyl, PPG t 10 g.l -1 nd ll the comintion tretments did not significntly differ. The percentge infection (Figure 3.6) decresed s the concentrtions of the tretments incresed for ll tretments included. The lowest percentge root rot (Figure 3.6) ws oserved with tretments of enomyl, PPG t 10 g.l -1 nd the comintion of PPG nd cffeine t concentrtion of 5 g.l g.l -1 respectively. Lettuce plnts infected with S. sclerotiorum resulted in 50% reduction in shoot nd root mss respectively when compring the untreted uninfected nd untreted infected controls. Tomtoes infected with Phytophthor cpsici Tretment of P. cpsici infected tomto plnts with the comintions of PPG nd cffeine t the concentrtions of 5 g.l g.l -1, 7.5 g.l g.l -1, 10 g.l g.l -1 nd 12.5 g.l g.l -1 respectively resulted in shoot mss comprle to tht of the untreted uninfected control (Figure 3.7, 3.17). The tretment with the 63

64 comintion of PPG nd cffeine t concentrtion of 7.5 g.l g.l -1 resulted in root mss comprle to tht of the uninfected untreted control. The shoot nd root mss of the 2.5 g.l g.l -1 PPG nd cffeine comintion were not significntly different from tht of the commercil fungicide control (metlxyl). The percentge root rot (Figure 3.8) for ll comintion tretments ws zero lthough the percentge infection still remined etween 57 nd 85%. The tretments with PPG nd cffeine individully did not effectively control the disese. Tomto plnts infected with Phytophthor resulted in 90% reduction in shoot nd root mss respectively when compring the untreted uninfected nd untreted infected controls. Tomtoes infected with Sclerotium rolfsii Tretment of S. rolfsii infected tomto plnts with ll ut the tretments with 5 g.l -1 nd 10 g.l -1 PPG, respectively, nd the comintion of PPG nd cffeine t the concentrtion of 25 g.l g.l -1 respectively resulted in shoot mss comprle to tht of the untreted uninfected control (Figure 3.9). Tretments with 1 g.l -1, 1.5 g.l -1 2 g.l -1 nd 3 g.l -1 cffeine nd the comintion with PPG nd cffeine t concentrtion of 25 g.l g.l -1 respectively resulted in root mss comprle to the infected untreted control while ll other tretments resulted in root mss comprle to the uninfected untreted control. The percentge infection (Figure 3.10) decresed s the concentrtions of the tretments incresed for ll tretments included. The percentge root rot of the tolchlofos-methyl tretment, the PPG tretments nd the comintion tretments did not differ significntly from ech other. Tomto plnts infected with S. rolfsii resulted in 45% reduction in shoot mss nd 50% reduction in root mss when compring the untreted uninfected nd untreted infected controls. 64

65 Cucumer plnts infected with Fusrium solni Tretment of F. solni infected cucumer plnts with the comintions of PPG nd cffeine t the concentrtions of 20 g.l g.l -1 nd 25 g.l g.l - 1, respectively, resulted in shoot nd root mss not significntly higher thn tht of the untreted uninfected control (Figure 3.11; 3.18). Tretments with cffeine t concentrtion of 2 nd 2 g.l -1 s well s the PPG nd cffeine comintions of 5 g.l g.l -1, 10 g.l g.l -1 nd 15 g.l g.l -1 resulted in shoot mss tht did not differ significntly from the commercil fungicide, enomyl. PPG nd cffeine comintion tretments t the concentrtions of 20 g.l g.l -1 nd 25 g.l g.l -1, oth resulted in zero percent root rot (Figure 3.12), while the ltter tretment lso gve the lowest percentge infection of ll infected plnts. Cucumer plnts infected with F. solni resulted in 77% reduction in shoot mss nd 60% reduction in root mss when compring the untreted uninfected nd untreted infected controls. Cucumer plnts infected with Fusrium oxysporum Tretment of F. oxysporum infected cucumer plnts with comintion tretments of PPG nd cffeine s well s cffeine t concentrtions of 1 nd 1.5 g.l -1 did showed disese control, giving shoot mss significntly higher thn the infected control, which still only resulted in 50% of the potentil mss when compred to the uninfected untreted control (Figure 3.13). Percentge infection decresed s the concentrtions of the tretment compounds incresed (Figure 3.14). Percentge root rot decrese for PPG nd the PPG nd cffeine comintion treted plnts s the concentrtions of the tretments incresed. Percentge root rot initilly decresed fter 65

66 tretments with cffeine etween 1g.L -1 nd 2g.L -1 nd then incresed for etween tretments of 2g.L -1 nd 3 g.l -1 Cucumer plnts infected with F. oxysporum resulted in 73% reduction in shoot mss nd 60% reduction in root mss when compring the untreted uninfected nd untreted infected controls. Zucchini sqush plnts infected with Spherothec fuligine Tretment of S. fuligine infected zucchini plnts with PPG significntly reduced the percentge infection (Figure 3.15). The lowest disese incidence ws chieved with PPG t 12.5 g.l -1. Tretments with 7.5 nd 10 g.l -1 PPG nd the PPG nd cffeine comintions t concentrtions of 2.5 g.l g.l -1, 5 g.l g.l -1 nd 7.5 g.l -1 nd 0.75 g.l -1 resulted in percentge infection not significntly different from tht of the commercil fungicide tretment, triforine. DISCUSSION The ntimicroil ctivity of te nd te polyphenols hs een demonstrted efore (Fuki et l., 1991; Aror nd Bhrdwj, Hrorne (1993) showed the ntimicroil properties of cffeine nd polyphenols s prt of the chemicl defence mechnism in certin plnts. However, no scientific pulictions courewqld e foiurewnd reporting on in vivo efficcy of te extrct or its individul components ginst plnt diseses. The present study on the in vivo sensitivity of seven different fungi to PPG, cffeine nd comintions thereof indicte tht the comintion of PPG nd cffeine 66

67 is the most effective tretment followed y individul tretments with cffeine nd then PPG. These results identified the most effective concentrtions ginst the vrious pthogens ssessed during the study. The comintion of Polyphenon G nd cffeine effectively controlled F. solni on cucumer, P. cpsici nd S. rolfsii on tomto, S. sclerotiorum nd Pythium F-group on lettuce. PPG individully significntly inhiited the development of S. fuligine on zucchini sqush plnts. These results confirmed Aror nd Ohln (1997) in vitro work on the nti-fungl ctivity of C. sinenis nd cffeine. Phytotoxicity symptoms were oserved in infected lettuce nd cucumer plnts treted with cffeine (pplied s soil drench) t concentrtions of 2.5g.L -1 nd higher. The results lso show tht in these instnces, the comintion of PPG nd cffeine does pper to reduce the phytotoxicity of cffeine, while cffeine ppers to ugment the disese suppressing efficcy of PPG. On zucchini sqush plnts phytotoxicity ws visile t concentrtion of 1g.L -1 cffeine (pplied s folir spry) nd t higher concentrtions, whether it ws mended with PPG or not. These results indicte tht phytotoxic concentrtions re dependnt on oth crop type nd method of ppliction. A possile solution to the prolem of phytotoxicity nd still mintining effective control could e to reduce the concentrtions of the compounds whilst incresing the numer of pplictions. Cucumer plnts inoculted with F. solni showed no disese symptoms when treted with comintion of PPG nd cffeine. Cucumer plnts infected with F. oxysporum showed n verge loss in shoot mss of 50% when compred to the 67

68 uninfected control. These results indicte tht tretments re spescie spesific nd tht individul host/pthogen trils will hve to e preformed to ccurtely determine efficcy nd tretment concentrtion. All the pthogens tested during the greenhouse pot trils were inhiited y one or more of the pplied tretments, with the exception of F. oxysporum which ws less ffected. In some instnces the tretments only delyed the onset of disese. This is still of importnce s dely in the onset of disese could still ffect the tril yield. Compring tretment compounds to fungicide controls it ws oserved tht the comintion of PPG nd cffeine performed similr to or etter thn the commercil fungicide in six of the seven host/pthogen studies. PPG individully performed similr to or etter thn the commercil fungicide in four of the seven host/pthogen studies. Cffeine individully preformed similr to or etter thn the commercil fungicide in three of the seven host/pthogen studies. The incresing incidence of resistnce to fungicides y plnt pthogens nd the loss of existing chemicl compounds for disese control re two importnt fctors driving the need to serch for new griculturl fungicides. In ddition, the desire for sfer grochemicls with less environmentl nd humn toxicity is mjor concern. Prticulrly desirle is the discovery of novel nti-microil gents representing new chemicl clsses tht lck cross-resistnce to chemicls currently used. Bsed on the results nd erlier findings (Aror nd Ohln, 1997) in the in vitro ctivity of these compounds, we conclude tht the PPG nd cffeine comintions hve potentil to ct s environmentlly friendly plnt disese control gents. 68

69 REFERENCES Ad, L.R., D Urzo, M.P., Liu, D., Nrsimhn, M.L., Reuveni, M., Zhu, J.K., Niu, X., Sigh, N.K., Hsegw, P.M., Bressn, R.A., Anti-fungl ctivity of tocco osmotin hs specificity nd involves plsm memrne permeiliztion. Plnt Sci. 118, Aror, D.S., Bhrdwj, S.K., Antimicroil ctivity of te (Cmelli sinensis) ginst some plnt pthogens. GEOBIOS. 24, Aror, D.S., Ohln D., In vitro studies on nti-fungl ctivity of te (Cmelli sinensis) nd coffee (Coffe ric) ginst wood rotting fungi. J. Bsic Microiol. 37, Bnos-Butist S., Hernndez-Lopez M., Bosquez-Molin E. nd Wilson C.L., Effects of chitosn nd plnt extrcts on growth of Colletotrichum gloeosporioides, nthrcnose levels nd qulity of ppy fruit. Crop Prot. 22, Bettiol W., Effectiveness of cow's milk ginst zucchini sqush powdery mildew (Spherothec fuligine) in greenhouse conditions. Crop protection 18,

70 Del, Rio J.A., Bidez, A.G., Boti, J.M., Ortuno, A., Enhncement of phenolic compounds in olive plnts (Ole europe L.) nd their influence on resistnce ginst Phytophthor spp. Food Chem. 83, Fuki, K, Ishigmi, T., Hr, Y., Anticteril ctivity of te polyphenols ginst phytopthogenic cteri. Agric. Biol. Chem. 55, Hrorne J.B., Introduction to Ecologicl Biochemistry. 4th ed. Acdemic Press, London. Hedin, P.A., New concepts nd trends in pesticide chemistry. J. Agric. Food Chem. 30, pp Jsso de Rodríguez D., Hernández-Cstillo D., Rodríguez-Grcí R., Angulo- Sánchez J. L., Anti-fungl ctivity in vitro of Aloe ver pulp nd liquid frction ginst plnt pthogenic fungi. Ind. Crops Prod. 21, Lee, S.-E., Prk, B.-S.., Kim, M.-K.., Choi, W.-S., Kim, H.-T., Cho Kwng-Yun, Lee, S.-G., nd Lee, H.-S., Fungicidl ctivity of pipernnline, pipperidine lkloid derived from long pepper, Piper longum L. ginst phytopthogenic fungi. Crop Prot. 20, Msgo, H., Yoshikw, M., Fukud, M. nd Nknishi, N., Selective inhiition of Pythium spp on medium for direct isoltion of Phytophthor spp. from soil nd plnts. Phytopthology 67:

71 McGrth, M.T. nd Shiskoff, N., Evlution of AQ10 (Ampelomyces quisqulis) for cucurit powdery mildew under field conditions. Phytopthology. 86; 553. Quirog E. M., Smpietro A.M., Vttuone M.A., Screening nti-fungl ctivities of selected medicinl plnts. J. of Ethnophrm Reuveni M., Agpov V., Reuveni F.L Controlling powdery mildew cused y Spherothec fuligine in cucumer y folir sprys of phosphte nd potssium slts. Crop Prot.15, Singh, S.P., Ro, G.P., Updhyy, P.P., Fungitoxicity of essentil oils of some romtic plnts ginst sugrcne pthogens. Sugr Cne 2; Stnghellini, M.E., Kronlnd, W.C., Yield loss in hydroponiclly grown lettuce ttriuted to suclinicl infection of feeder rootlets y Pythium dissotocum. Plnt Dis. 70, Tzeng D. D., Tzeng H. C., Chen R., Chengs A., TsiiI C.C, Chenc C., Hwng T., Yeh Y., DeVy J. E The use of MR formultion s novel nd environmentlly sfe photodynmic fungicide for the control of powdery mildews. Crop Prot.15, Vn Wyk, P. S., Scholtz, D. J., Los, O A selective medium for the isoltion of Fusrium spp. from soil deris. Phyto phlctic. 19,

72 Weidemn, H., Wehner, F.C Greenhouse evlution of Trichoderm hrzinum nd Fusrium oxysporum for iologicl control of citrus root rot in soils nturlly nd rtificilly infected with Phytophthor nicotine. Phytophylctic. 25,

73 Tle 3.1 List of the fungl pthogens, their host plnts nd the diseses cused Host Pthogen Disese Cucumer (Cucumis stivus L.) Lettuce (Lctuc stiv L.) Tomto (Lycopersicon esculentum L.) Zucchini Fusrium oxysporum Fusrium solni Sclerotini sclerotiorum Pythium F-group Sclerotium rolfsii Phytophthor cpsici Root rot Root rot Root rot Root rot Root nd stem rot Root rot (Cucurit pepo L.) Spherothec fuligine Powdery mildew on leves 73

74 Tle 3.2 Applied tretments nd included controls for ech phytopthogenic fungi used during in vivo studies Pthogen Tretments (g.l -1 ) Uninfected control Infected control Fungicide control (.i.) PPG Fusrium oxysporum Wter* Wter Benomyl 5, 10, , 20, 25 Fusrium solni Wter Wter Benomyl 5, 10, , 20, 25 Phytophthor Wter Wter Metlxyl 2.5, 5, cpsici , 10, 12.5 Pythium F-group Wter Wter Propmocr 2.5, 5, , 10, 12.5 Sclerotini Wter Wter Benomyl 5, 10, sclerotiorum , 20, 25 Sclerotium rolfsii Wter Wter Tolclofosmethyl 5, 10, 15, 20, PPG = Polyphenon G.i. = ctive ingredient * Tp wter Cffeine 1, 1.5, 2, 2.5, 3 1, 1.5, 2, 2.5, , 0.5, 0.75, 1, , 0.5, 0.75, 1, , 1.5, 2, 2.5, 3 1, 1.5, 2, 2.5, 3 Comintions of PPG nd Cffeine 5:1, 10:1, 15:1.5, 20:2, 25:2.5 5:1, 10:1, 15:1.5, 20:2, 25: :0.25, 5:0.5, 7.5:0.75, 10:1, 12.5: :0.25, 5:0.5, 7.5:0.75, 10:1, 12.5:1.25 5:1, 10:1, 15:1.5, 20:2, 25:2.5 5:1, 10:1, 15:1.5, 20:2, 25:2.5 74

75 Figure 3.1 Colonies of Pythium growing from root segments of inoculted lettuce plnts fter plting on BNPRA medium Figure 3.2 Disese rting scle used for zucchini sqush plnts infected with S. fuligine. 0 = helthy, 1 = 25%, 2 = 50%, 3 = 75% nd 4 = 100% disese severity 75

76 Mss (g) e c d d c c c c d c c c c c Men shoot mss Men root mss Uninfected control Infected control Propmocr 0.1 PPG 2.5 PPG 5 PPG 7.5 PPG 10 PPG 12.5 CAF 0.25 CAF 0.5 CAF 0.75 CAF 1 Tretments (g/l) (g.l -1 ) c CAF 1.25 PPG 2.5 CAF 0.25 PPG 5 CAF 0.5 PPG 7.5 CAF 0.75 PPG 10 CAF 1 PPG 12.5 CAF 1.25 Figure 3.3 Effect of soil drench tretments with Polyphenon G (PPG), cffeine (CAF) nd comintions of the two compounds on fresh shoot nd root mss of Pythium F-group inoculted lettuce plnts under greenhouse conditions. Uninfected nd infected controls were included. Propmocr tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test) 76

77 Percentge (%) A C f C C C C B c C C C C d B d e e D f Uninfected control Infected control Propmocr 0.1 PPG 2.5 PPG 5 PPG 7.5 PPG 10 PPG 12.5 CAF 0.25 CAF 0.5 CAF 0.75 CAF 1 CAF 1.5 PPG 2.5 CAF 0.25 PPG 5 CAF 0.5 PPG 7.5 CAF 0.75 PPG 10 CAF 1 PPG 12.5 CAF 1.25 D f D f Men percentge root rot Men percentge incidence of Pythium F- group Tretments (g/l) (g.l -1 ) Figure 3.4 Percentge root rot nd percentge infection on lettuce plnts inoculted with Pythium F-group. Tretments included Polyphenon G (PPG), cffeine (CAF) nd mixtures of the two compounds. Uninfected nd infected controls were included. Propmocr tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test). Lower cse percentge incidence. Upper cse percentge root rot 77

78 Men shoot mss Men root mss Mss (g) d d d c c e f Uninfected control d Infected control Benomyl 0.1 PPG 5.0 PPG 10 PPG 15 e d c c PPG 20 PPG 25 CAF 1.0 CAF 1.5 CAF 2.0 CAF 2.5 Tretments (g/l) (g.l -1 ) e CAF 3.0 PPG 5.0 CAF 1.0 PPG 10 CAF 1.5 PPG 15 CAF 2.0 PPG 20 CAF 2.5 PPG 25 CAF 3.0 Figure 3.5 Effect of soil drench tretments with Polyphenon G (PPG), cffeine (CAF) nd comintions of the two compounds on fresh shoot nd root mss of Sclerotini sclerotiorum inoculted lettuce plnts under greenhouse conditions. Uninfected nd infected controls were included. Benomyl tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test) 78

79 Percentge (%) B G f C G c E e E f F f D C d B e A g A h G c F d F d F h F h Men percentge root rot Men percentge incidence of Sclerotini sclerotiorum 0 Uninfected control Infected control Benomyl 0.1 PPG 5.0 PPG 10 PPG 15 PPG 20 PPG 25 CAF 1.0 CAF 1.5 CAF 2.0 Tretment (g/l) (g.l -1 ) CAF 2.5 CAF 3.0 PPG 5.0 CAF 1.0 PPG 10 CAF 1.5 PPG 15 CAF 2.0 PPG 20 CAF 2.5 PPG 25 CAF 3.0 Figure 3.6 Percentge root rot nd percentge infection on lettuce plnts inoculted with Sclerotini sclerotiorum. Tretments included Polyphenon G (PPG), cffeine (CAF) nd mixtures of the two compounds. Uninfected nd infected controls were included. Benomyl tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test). Lower cse percentge incidence. Upper cse percentge root rot 79

80 Men shoot mss Mss (g) e d e e e e e e e d d d d d d c d c c c Men root mss -8 Uninfected control Infected control Metlxyl 0.1 PPG 2.5 PPG 5 PPG 7.5 PPG 10 PPG 12.5 CAF 0.25 CAF 0.5 CAF 0.75 CAF 1 Tretments (g/l) (g.l -1 ) CAF 1.25 PPG 2.5 CAF 0.25 PPG 5 CAF 0.5 PPG 7.5 CAF 0.75 PPG 10 CAF 1 PPG 12.5 CAF 1.25 Figure 3.7 Effect of soil drench tretments with Polyphenon G (PPG), cffeine (CAF) nd comintions of the two compounds on fresh shoot nd root mss of Phytophthor cpsici inoculted tomto plnts under greenhouse conditions. Uninfected nd infected controls were included. Propmocr tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test) 80

81 Percentge (%) Uninfected control A Infected control f Metlxyl 0.1 A A A A A A d B e B C f f PPG 2.5 PPG 5 PPG 7.5 PPG 10 PPG 12.5 CAF 0.25 CAF 0.5 CAF 0.75 CAF 1 Tretments (g/l) (g.l -1 ) C CAF 1.5 g c PPG 2.5 CAF 0.25 c PPG 5 CAF 0.5 PPG 7.5 CAF 0.75 e e PPG 10 CAF 1 e PPG 12.5 CAF 1.25 Men percentge root rot Men percentge incidence of Phytophthor cpsici Figure 3.8 Percentge root rot nd percentge infection on tomto plnts inoculted with Phytophthor cpsici. Tretments included Polyphenon G (PPG), cffeine (CAF) nd mixtures of the two compounds. Uninfected nd infected controls were included. Metlxyl tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test). Lower cse percentge incidence. Upper cse percentge root rot 81

82 Mss (g) Men shoot mss Men root mss Uninfected control Infected control Tolclofos-methyl 0.1 PPG 5.0 PPG 10 PPG 15 PPG 20 PPG 25 CAF 1.0 CAF 1.5 CAF 2.0 CAF 2.5 Tretments (g/l) (g.l -1 ) CAF 3.0 PPG 5.0 CAF 1.0 PPG 10 CAF 1.5 PPG 15 CAF 2.0 PPG 20 CAF 2.5 PPG 25 CAF 3.0 Figure 3.9 Effect of soil drench tretments with Polyphenon G (PPG), cffeine (CAF) nd comintions of the two compounds on fresh shoot nd root mss of Sclerotium rolfsii inoculted tomto plnts under greenhouse conditions. Uninfected nd infected controls were included. Propmocr tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test) 82

83 Percentge (%) B D e D c D c D c e D A B c c C c d C d d d D f D D D D D f D f Men percentge root rot Men percentge incidence of Sclerotium rolfsii 0.00 Uninfected control Infected control Tolclofos-methyl 0.1 PPG 5.0 PPG 10 PPG 15 PPG 20 PPG 25 CAF 1.0 CAF 1.5 CAF 2.0 CAF 2.5 Tretments (g/l) (g.l -1 ) CAF 3.0 PPG 5.0 CAF 1.0 PPG 10 CAF 1.5 PPG 15 CAF 2.0 PPG 20 CAF 2.5 PPG 25 CAF 3.0 Figure 3.10 Percentge root rot nd percentge infection on tomto plnts inoculted with Sclerotium rolfsii. Tretments included Polyphenon G (PPG), cffeine (CAF) nd mixtures of the two compounds. Uninfected nd infected controls were included. Tolclofos-methyl tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test). Lower cse percentge incidence. Upper cse percentge root rot 83

84 20 Men shoot mss Mss (g) e c e c c d d d d e e e c c c Men root mss Uninfected control c Infected control Benonmyl 0.1 PPG 5.0 PPG 10 PPG 15 c c c PPG 20 PPG 25 CAF 1.0 CAF 1.5 CAF 2.0 CAF 2.5 CAF 3.0 PPG 5.0 CAF 1.0 PPG 10 CAF 1.5 PPG 15 CAF 2.0 Tretments (g/l) (g.l -1 ) PPG 20 CAF 2.5 PPG 25 CAF 3.0 Figure 3.11 Effect of soil drench tretments with Polyphenon G (PPG), cffeine (CAF) nd comintions of the two compounds on fresh shoot nd root mss of Fusrium solni inoculted cucumer plnts under greenhouse conditions. Uninfected nd infected controls were included. Benomyl tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test) 84

85 Percentge (%) A C c C D C C A C C c c E B d A d D d D d de de e Men percentge root rot Men percentge incidence of Fusrium solni 10 0 Uninfected control Infected control Benonmyl 0.1 PPG 5.0 PPG 10 PPG 15 PPG 20 PPG 25 CAF 1.0 CAF 1.5 CAF 2.0 (g.l -1 ) Tretments (g/l) CAF 2.5 CAF 3.0 PPG 5.0 CAF 1.0 PPG 10 CAF 1.5 PPG 15 CAF 2.0 PPG 20 CAF 2.5 PPG 25 CAF 3.0 Figure 3.12 Percentge root rot nd percentge infection on cucumer plnts inoculted with Fusrium solni. Tretments included Polyphenon G (PPG), cffeine (CAF) nd mixtures of the two compounds. Uninfected nd infected controls were included. Benomyl tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test). Lower cse percentge incidence. Upper cse percentge root rot 85

86 25 Men shoot mss Mss (g) d d d d d d c c c c c c c d d d Men root mss Uninfected control d Infected control c Benonmyl 0.1 c PPG 5.0 c PPG 10 c PPG 15 c PPG 20 c c PPG 25 CAF 1.0 CAF 1.5 CAF 2.0 CAF 2.5 Tretments (g/l) (g.l -1 ) d CAF 3.0 c PPG 5.0 CAF 1.0 c PPG 10 CAF 1.5 c PPG 15 CAF 2.0 c PPG 20 CAF 2.5 c PPG 25 CAF 3.0 Figure 3.13 Effect of soil drench tretments with Polyphenon G (PPG), cffeine (CAF) nd comintions of the two compounds on fresh shoot nd root mss of Fusrium oxysporum inoculted cucumer plnts under greenhouse conditions. Uninfected nd infected controls were included. Benomyl tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test) 86

87 Percentge (%) A E e B A c D C d C d A B B C d d d D E E e e e e e E e F D e Men percentge root rot Men percentge incidence of Fusrium oxysporum 10 0 Uninfected control Infected control Benonmyl 0.1 PPG 5.0 PPG 10 PPG 15 PPG 20 PPG 25 CAF 1.0 CAF 1.5 CAF 2.0 CAF 2.5 Tretments (g/l) (g.l -1 ) CAF 3.0 PPG 5.0 CAF 1.0 PPG 10 CAF 1.5 PPG 15 CAF 2.0 PPG 20 CAF 2.5 PPG 25 CAF 3.0 Figure 3.14 Percentge root rot nd percentge infection on lettuce plnts inoculted with Fusrium oxysporum. Tretments included Polyphenon G (PPG), cffeine (CAF) nd mixtures of the two compounds. Uninfected nd infected controls were included. Benomyl tretment ws included s commercil fungicide stndrd. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test). Lower cse percentge incidence. Upper cse percentge root rot 87

88 Percentge (%) Infected control cd Triforine PPG 2.5 c PPG 5 cd PPG 7.5 cd PPG 10 d PPG 12.5 c CAF 0.25 c CAF 0.5 c CAF 0.75 CAF 1 CAF 1.25 cd PPG 2.5 CAF 0.25 cd PPG 5 CAF 0.5 cd PPG 7.5 CAF 0.75 PPG 10 CAF 1 PPG 12.5 CAF 1.25 Tretment (g/l) (g.l -1 ) Figure 3.15 Men percentge disese incidence on zucchini sqush plnts infected with Spherothec fuligine. Tretments included Polyphenon G (PPG), cffeine (CAF) nd mixtures of the two compounds. Infected untreted plnts nd commercil fungicide (triforine) tretment were included s controls. Columns represent the mens of three independent experiments with eight plnts per tretment per experiment. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05 using the F-test) 88

89 Figure 3.16 Effect of soil drench tretments with Polyphenon G (P), cffeine (C) nd comintions of the two compounds on lettuce plnts inoculted with Sclerotini sclerotiorum. Concentrtions of compounds re indicted on lels 90

90 Figure 3.17 Effect of soil drench tretments with Polyphenon G (P), cffeine (C) nd comintions of the two compounds on tomto plnts inoculted with Phytophthor cpsici. Concentrtions of compounds re indicted on lels 91

91 Figure 3.18 Effect of soil drench tretments with Polyphenon G (P), cffeine (C) nd comintions of the two compounds on cucumer plnts inoculted with Fusrium solni. Concentrtions of compounds re indicted on lels 92

92 CHAPTER 4 Induction of Defence Rections in Tomto nd Lettuce Plnts y Appliction of Te (Cmelli sinensis) Extrcts nd Cffeine 93

93 ABSTRACT One of the mjor prolems concerning the production of food crops is the difficulty of controlling plnt diseses. One pproch to the control of plnt diseses is through the induction nd enhncement of the plnt's own defence mechnisms which would not involve the ppliction of toxic compounds to plnts. Recently, the ctivity of Polyphenon G (PPG) nd cffeine ginst phytopthogenic fungi hs een demonstrted (Chpter 2 nd 3). During the current study the im ws to investigte induced resistnce s possile mode of ction in tomto nd lettuce plnts. Totl phenolic content (TPC) ws determined in root nd shoot extrcts of tomto nd lettuce plnts fter tretment with PPG, cffeine nd comintions of PPG nd cffeine s well s commercil plnt defence inducing compound (fosetyl-l for tomtoes nd potssium phosphonte for lettuce) s positive control. Smples were tken t 0, 24, 48 nd 96 hours fter tretments were pplied. In the cse of lettuce Pythium inoculted plnts were lso included. The TPC ssy showed n increse in phenolic levels in root extrcts from tomto nd lettuce plnts for ll tretments when compred to the untreted plnts. TLC nlyses coupled with iossy reveled the presence of fungitoxic compound in ll infected lettuce smples s well s the uninfected comintion tretment nd potssium phosphonte tretment. The fungitoxic compound ws identified s ferulic cid y compring Rf-vlues to those of stndrds nd y retention time on reversed phse HPLC. The presence of slicylic cid nd cffeic cid in selected root nd shoot smples ws lso indicted y HPLC. These results confirm tht tomto nd lettuce plnts treted with PPG, cffeine nd comintion of PPG nd cffeine cn induce compounds in plnts similr to those produced y commercil defence system inducers. 94

94 INTRODUCTION Appliction of chemicls to plnts in order to prevent or inhiit disese development is fundmentl mens of mnging diseses cused y fungi. However, these chemicl methods of disese mngement re expensive nd cn detrimentlly ffect the ecosystem. The ovious pollution prolems due to indiscriminte use of synthetic pesticides nd their toxic effect on non-trget orgnisms hve prompted investigtions on exploiting pesticides of plnt origin. Resistnce to fungicides in plnt pthogen popultions is one of the most significnt prolems in the re of chemicl disese mngement. The use of fungicides will continue to ply mjor role in disese mngement for the foreseele future, nd development of strtegies for resistnce mngement will e necessry to mintin useful rsenl of the most effective fungicides. Such strtegies re required if we re to prolong the useful life of these disese control gents. Nturl plnt products re importnt sources of new grochemicls for the control of plnt diseses (Crdellin, 1988; Gulter, 1988). Furthermore, iocides of plnt origin re non-phytotoxic, systemic, esily iodegrdle nd possily ugment existing fungicides (Mson nd Mthew, 1996; Qsem nd Au-Bln, 1966) Knowledge of the effectiveness of prticulr compounds is importnt for chieving effective disese control. Eqully importnt is n understnding of the underlying physiologicl mode of ction of plnt disese mngement mterils (Schmourlo et l, 2005). Investigtions into mechnisms of disese suppression y plnt products hve suggested tht these plnt products my either ct on the pthogen directly (Amdioh, 2000), or induce systemic resistnce in host plnts resulting in 95

95 reduction of disese development (Olivieri et l, 1996; Nrwl et l, 2000; Pul nd Shrm, 2002; Schneider nd Ullrich, 1994). The min nti-microil ctivity of C. sinensis extrcts hs een contriuted to cffeine nd the ctechins present in these extrcts. Cffeine is known inhiitor of norml cell division in plnt nd niml cells tht often results in inuclete cells (Anej nd Ginfgn, 2001), thus inhiiting spore germintion nd growth of microes. Gllted ctechins (EGCg nd ECg) hve een reported to dectivte proteins nd disrupt the cteril lipid ilyer y chnging the memrnes fluidity nd morphology (Ikigi et l, 1993). In n effort to find lterntive or dditive modes of ction, we investigted induced resistnce s possile mechnism of ction of Polyphenon G (PPG) nd cffeine s well s comintions of the two compouds. Induced mechnisms hve received considerle ttention, hving een studied y mens of moleculr, iochemicl nd phytochemicl methods (Ng et l., 1987, Wu et l., 1995; Zng nd Lewis, 1997, Romni et l, 2002). Induced resistnce is the phenomenon wherey plnt, when given n pproprite stimultion y hrmless inducers, will ecome le to comt more efficiently the infection y virulent pthogens through enhncement of its nturlly inherent defence mechnisms. It is our hypothesis tht the profile of phytochemicls tht re present in te (Cmelli sinensis (L.), Kuntze) present rod spectrum of nti-microil ility nd potentilly cn induce disese resistnce in plnts. The present study ws thus undertken to determine if plnts treted with PPG (C. sinensis extrct), cffeine nd mixtures of the two compounds increses the totl 96

96 solule phenolic content in plnts using the Folin-Cioclteu method. Thin lyer chromtogrphy iossys were used for the detection of ctive nti-fungl compounds in plnt extrct while chnges in phenolic profiles were nlyzed using high performnce liquid chromtogrphy (HPLC). MATERIALS AND METHODS Inoculum preprtion Pythium F-group nd Cldosporium cldosporioides (Fres.) de Vries were otined from the University of Pretori culture collection nd mintined on potto dextrose gr (PDA) (Biol, Merck) pltes. Pythium F-group ws grown on 90 mm dimeter PDA pltes t 25ºC until the pltes were completely covered. Millet (Penniselum glucem L.) seeds (200g) were steeped in wter (200 ml) for 24h, trnsferred to utoclvle polyethylene gs, nd utoclved twice for 45min t 120 C on two consecutive dys. Ten 5 mm dimeter gr discs were cut with cork orer from the ctively growing edge of ech fungl colony. Mycelil discs were septiclly trnsferred to ech of the sterilized millet seed gs nd the gs were incuted for 28 dys t 25 C. The millet seed inoculum (100g.L -1 soil) ws dded to the soil (Weidemn nd Weher, 1993). 97

97 Soil preprtion Sndy lom soil (Conrdie Orgnics, Pretori, Guteng, South Afric) nd potting soil consisting of composted krl mnure nd rk (Just Nture Orgnics, Pretori, Guteng, South Afric) ws stem psteurized seprtely nd mixed in 1:1 rtio (v:v) sed on results otined during preliminry trils. Plnt preprtion Lettuce (Lctuc stiv L., cv Ndine) nd tomto (Lycopersicon esculentum Mill., cv Money Mker) seeds were otined from Hygrotec (Pretori-North, Guteng, South Afric). The tomto nd lettuce seeds were germinted nd grown t commercil nursery (Multiplnt, Brits, North-West, South Afric) until five weeks old. The five week old tomto plnts were trnsplnted into seedling trys in the soil mixture nd drenched with 7 ml/cell of (i) 12.5 g.l -1 Polyphenon G, (ii) 1.25 g.l -1 cffeine (Sigm), mixture of (iii) 12.5 g.l -1 Polyphenon G nd 1.25 g.l -1 cffeine, nd (iv) 100 ml.l -1 fosetyl-l WP 800 g.kg -1 ctive ingredient (.i.) (Aliette, Rhone- Poulenc, RSA) respectively. Untreted (v) (treted with wter only) plnts were included s controls. Untreted tomto plnts were hrvested t time 0h. Therefter plnts from ll tretments were hrvested t 24h, 48h nd 96h respectively. Fourty plnts with n pproximte comined fresh weight of 35g were hrvested per tretment per dy. A totl of 640 plnts were used per experiment. Plnt roots nd 98

98 shoots were seprted nd frozen t -4 ºC efore eing freeze dried for further nlysis. Ech experiment ws repeted once. Lettuce plnts were trnsplnted into seedling trys in the ove mentioned soil mixture. Greenhouse tempertures were mintined etween 23 nd 28 ºC, with mient humidity nd nturl dylight for the durtion of the experiment. Inocultion of specific plnts with P. F-group ws done 24h efore tretment of the plnts with the vrious compounds. Time 0h strted fter the 24h infection period nd untreted (treted with wter only) lettuce plnts were hrvested t this time. Uninfected nd infected plnts were drenched with 7 ml/cell of (i) 12.5 g.l -1 Polyphenon G, (ii)1.25 g.l -1 cffeine, the mixture of (iii) 12.5 g.l -1 Polyphenon G nd 1.25 g.l -1 cffeine, (iv) nd 100 ml.l -1 potssium phosphonte 200g.L -1.i. SL (Phytex, Horticur, RSA) respectively. Uninfected nd infected controls (treted with wter only) were lso included. Therefter plnts from ll tretments were hrvested t 24h, 48h nd 96h respectively. Thirty plnts with n pproximte comined weight of 50g were hrvested per tretment per dy. A totl of 930 plnts were used per experiment. Plnt roots nd shoots were seprted nd frozen t -4 ºC efore eing freeze dried for further nlysis. Ech experiment ws repeted once. Extrctions For extrction of root tissue, freeze dried roots were ground using mortr nd pestle. The ground root tissue (0.25g) ws then dded to 5ml of methnol (MeOH) contining 0.1% utylted hydroxyl-tolune (BHT, Sigm) nd then sonicted in sonifer th for 30 minutes. The suspensions were susequently centrifuged t

99 rpm for 10 min. The superntnts were used s the test solution (Simonovsk et l, 2003). For the extrction of lef tissue, freeze dried leves were ground using mortr nd pestle nd 8ml methnol ws then dded to 1.0g of the ground lef tissue. The suspension ws then sonicted (50% duty cycle; power setting 3 microtip on Brnson 30 sonifier) for 5min. The solutions were vcuum filtered once through Whtmn No 1 filter pper nd the MeOH ws removed y roto-evportion t 70±2 C (Rémus-Borel et l, 2005). After evportion, extrcts were dissolved in 12ml wter. Chlorophyll ws removed during extrctions (3 x 6 ml) with petroleum ether (Anlr, Merck), retining the ottom lyer. The phenolic compounds were then extrcted (5 x 6ml) with ethyl cette (Anlr, Merck), retining the top lyer (Simonovsk et l, 2003). Totl phenolic content Totl phenolic content (TPC) of the extrcts were determined using the Folin Cioclteu regent (Sigm) (Yu et l, 2003). The volumes were modified for the wells in microtiter pltes. Ech rection mixture contined 5µl smple solution, 175µl distilled-deionized wter, 25µl Folin Cioclteu regent nd 50µl of queous sodium cronte (N 2 CO 3 ) (20g.100ml -1 ). The regent lnk contined 5µl distilled deionised wter insted of smple. The solution of ech well ws mixed with micropipette until the yellow color fded. Pltes were then incuted in microplte incutor t 40 C for 30min. The sorency of the vrious extrcts ws mesured with Multiscn Ascent VI (version 1.3.1) t 690 nm. The phenolic 100

100 content ws expressed in mg gllic cid equivlents (GEA)/ ml extrct from the stndrd curve using the eqution: GAE/ml extrct = x (R 2 = ) Eqution 1 Asorency t 690 nm TPC ws expressed s (GAE) nd clculted s percentge of the dry weight using the following eqution: mg GAE/g dry weight = GAE mg/ml x V x 100 M Eqution 2 V M = Volume of originl smple in ml = Mss of originl smple in g The nlysis ws repeted for ech smple, i.e., four independent experiments, with three replictes per smple. Thin-lyer chromtogrphy (TLC) io-utogrphy The TLC io-utogrphy ws preformed on ll the smples. Thin-lyer chromtogrphy ws preformed on Silic gel 60 (Merck) pltes. Pltes were developed in chloroform-methnol (1:1, v/v) to the top nd dried for 30min t 110 ºC 101

101 to ctivtes the pltes. Ech smple (0.1ml) ws spotted on line 10mm from the ottom of the TLC pltes. Pltes were developed in developing chmer using n- hexne : ethyl cette : formic cid (20:19:1) (Simonovsk et l, 2003). Slicylic cid (Sigm), Cffeic cid (Sigm) nd Ferulic cid (Fluk) t concentrtions of 20 g.l -1, 5g.L -1 nd 2.5 g.l -1 respectively, were included in the study fter these compounds were identified through HPLC to e present in the extrcts. After the TLC pltes were developed, they were ssessed for the presence of nti-fungl compounds ccording to the io-ssy procedure decried y Homns nd Fuchs, (1970). A suspension comprising of 7g KH 2 PO 4 (Merck), 3g N 2 HPO 4 2H 2 0 (Merck), 4g KNO 3 (Merck), 1g MgSO 4 7H 2 0 (Merck), 1g NCl (Merck) per litre of tp wter ws prepred nd utoclved t 121 ºC for 20min. Ten ml of 30 % queous solution of glucose ws dded per 60ml of solution. The suspension ws poured onto n ctively growing colony of C. cldiosporiodes on PDA pltes nd the spores were loosened using n etuleur. Conidi in the resulting suspension were counted with hemcytometer, djusted to spores/ml nd trnsferd to 200ml spry ottle (Zinuri et l, 2001). Developed chromtogrms were spryed with the spore suspensions of C. cldosporioides nd susequently incuted t 100% RH nd 25 C for 3 6 dys. The presence of nti-fungl ctivity ws recorded s zones of fungl inhiition where mycelil growth nd sporultion ws inhiited (Klrmn nd Stnford, 1968, Terry et l, 2004). The Rf-vlues (Eqution 3) of inhiition zone were determined y tking mesurements from the line on which the compound ws spotted (origin) to the distnce the compound moved (middle of inhiition zone). 102

102 R F = Distnce moved y smple spot from origin Distnce moved y solvent front from origin Eqution 3 For ech smple the experiment ws repeted twice i.e. for the two independent plnt experiments two smples were otined per tretment nd ech smple ws then used three times in this ssy. High performnce liquid chromtogrphy (HPLC) The extrct smples descried ove were diluted in 1:1 rtio with distilled wter nd methnol (1:1). HPLC nlyses were preformed on ll the 48h tretment extrcts y mnully injecting 20 µl of the filtrte onto the HPLC column. The chromtogrphic system used ws n Agilent 1100 Series consisting of QutPump high pressure pumps nd Phenomenex Lun 3u C18 (2) column. The diode rry detector ws ttched to n nlyticl computer with dt storge system (ChemSttion LC). The moile phse consisted of wter uffer (ph 2.6) nd cetonitrile (Srchem). STATISTICAL ANALYSIS Significnt differences were determined using Fisher s protected lest significnt difference test, nd re indicted on the grphs using letter nottion. The comprison wise error rte ws P<0.05. Stndrd error of the mens re indicted s error rs on the grphs. 103

103 RESULTS Totl phenolic content Tomto nd lettuce shoot smples showed no increse in TPC for ny of the tretments. However, significnt increses in TPC were oserved for oth tomto (Figure 4.1) nd lettuce (Figure 4.2) root smples for ll tretments pplied. The tomto root smples (Figure 4.1) showed no significnt increse or decrese in TPC for untreted plnts over 96h period. Tretment with fosetyl-l nd PPG respectively resulted in the most significnt increse in TPC, followed y cffeine nd the comintion of PPG nd cffeine. The TPC of root smples treted with fosetyl-al, PPG nd cffeine respectively did not increse significntly, within these tretment groups, over 96 h period. Root smples treted with comintion of PPG nd cffeine showed significnt increse etween 24h nd 48h ut no significnt increse etween 48h nd 96h fter tretment. Lettuce root smples tht were untreted nd uninfected showed significnt increse in TPC over 24h period, lthough not sttisticlly significnt etween 24 nd 96h (Figure 4.2). Untreted lettuce plnts inoculted with Pythium showed no initil increse in TPC over the first 48h ut showed decresed etween 48 nd 96h. Potssium phosphonte tretment gve the highest TPC followed y the comintion of PPG nd cffeine, PPG nd cffeine in decresing order. PPG treted root smples showed higher overll TPC for infected thn uninfected plnts. Uninfected root smples of PPG treted plnts showed significnt increse in TPC over the first 48h 104

104 period nd susequent significnt decrese etween 48 nd 96h. No significnt chnge in TPC ws oserved in the roots of infected lettuce plnts treted with PPG over the 96h period. Root smples of cffeine treted plnts showed higher TPC for infected thn uninfected plnts. Root smples of plnts treted with comintion of PPG nd cffeine did not differ significntly in TPC etween uninfected nd infected plnts. Uninfected nd infected plnts treted with comintion of PPG nd cffeine resulted in no significnt increse etween 24h nd 48h period ut susequently incresed etween 48h nd 96h. Root smples of plnts treted with Potssium phosphonte did not significntly differ for uninfected nd infected smples. Thin-lyer chromtogrphy io-utogrphy The dt is presented in Tle 4.1. The stndrds, introduced susequently to HPLC identifiction, hd the following R F vlues: 0.64 for slicylic cid, 0.44 for ferulic cid nd 0.22 for cffeic cid. Cler inhiition zones were present t R F -vlues etween 0.42 nd 0.45 for ll lettuce plnts infected with P. F-group, independent of the tretment. Cler zones were lso visile for uninfected plnts treted with the comintion of PPG nd cffeine s well s plnts treted with potssium phosphonte t similr R F -vlues. None of the other smples included in this experiment formed cler inhiition zones. High performnce liquid chromtogrphy (HPLC) In the HPLC chromtogrm it ws oserved tht more smller peks t lower concentrtions were present for untreted uninfected root nd shoot smples thn for 105

105 ny of the infected nd/or treted plnts (which hd fewer nd lger peks). All treted nd/or infected smples were significntly different from the untreted uninfected control smples. Stndrds included in the HPLC dt se llowed for the identifiction nd clcultion of the concentrtion of three of the mjor peks in treted nd/or infected plnt extrcts. The peks tht were identified included cffeic cid, slicylic cid nd ferulic cid. The percentge dry weight of these compounds in root nd shoot smples were clculted nd is given in Figures 4.3 nd 4.4. HPLC nlysis of tomto root nd shoot smples (Figure 4.3) resulted in peks tht were identified s cffeic cid nd slicylic cid for shoot smples nd slicylic cid for root smples. Slicylic cid ws present in tomto shoot smples treted with cffeine nd fosetyl-l lthough the percentge of dry weight did not differ significntly etween the two smples. Cffeic cid ws present in ll the treted shoot smples nd the percentge of dry weight did not differ significntly etween tretments. Slicylic cid ws present in ll tomto root smples ut the fosetyl-l smple resulted in the lrgest mount in terms of percentge dry weight followed y PPG nd the comintion of PPG nd cffeine which did not differ significntly from ech other. HPLC nlysis of lettuce root nd shoot smples resulted in peks tht were identified s cffeic cid, slicylic cid nd ferulic cid for shoot nd root smples (Figure 4.4). Root smples contined higher concentrtions of these compounds when compred to shoot smples. In shoot smples slicylic cid ws present in untreted infected, PPG uninfected, cffeine uninfected nd infected potssium phosphonte smples, where the first three did not differ significntly from ech other nd the 106

106 potssium phosphonte smple gve significntly higher concentrtion. Cffeic cid ws present in ll ut the untreted uninfected smple, where none of the concentrtions differed significntly. Ferulic cid ws present in ll infected smples s well s uninfected smples treted with comintion of PPG nd cffeine nd the uninfected smples treted with potssium phosphonte, ut none of these concentrtions were significntly different. DISCUSSION Phenolic compounds in plnts constitute mjor clss of io-ctive secondry plnt metolites with potentilly nti-microil ctivities. During this study our im ws to identify whether tretments with PPG, cffeine nd the comintion of the two compounds induces such compounds in tomto nd lettuce plnts. The totl phenolic content (TPC) ws determined in tomto shoot nd roots treted with fosetyl-l, PPG, cffeine nd comintions of PPG nd cffeine over 96h period. TPC ws lso determined in shoots nd roots of similrly treted lettuce plnts, including rnge of Pythium infected plnts over 96h period. The shoot smples of oth tomto nd lettuce plnts showed no increse in TPC with ny of the tretments which could possily e ttriuted to the tretments eing pplied s root drench. Tretments with the fungicide stndrds (fosetyl-l nd potssium phosphonte) resulted in the highest TPC levels in oth tomto nd lettuce roots, confirming tht these compounds re indeed inducers of resistnce mechnisms in plnts. Tomto plnts treted with PPG nd lettuce plnts treted with 107

107 comintion of PPG nd cffeine respectively, resulted in the highest TPC of the experimentl tretment compounds. These results indicte tht the phenolic compounds in the PPG tretments (i) could hve een ccumulted in the roots, (ii) induced iochemicl pthwys in the plnts to increse the production of phenolic compounds or (iii) contined precursors for the production of these phenolic compounds. Bio-utogrphy ws used to determine whether ny of the ccumulted compounds in the tomto nd lettuce roots where nti-fungl compounds. Bioutogrphy methods were developed for TLC where compounds re seprted ccording to hydrophilicity nd C. cldosporioides pplied to the TLC plte, cler zones indicte inhiition (Nrsimhchri nd Rmchndrn, 1967; Homm et l.,1989; Hmurger nd Cordell, 1987). Direct sprying of conidil suspensions is n esy technique to use, giving the most relile results for detection of fungitoxic compounds (Homns nd Fuchs, 1970). The dt from the TLC-utogrphy nlysis (Tle 4.1) showed tht the R f vlue of the nti-fungl compounds corresponded to the R f vlue of the ferulic cid stndrd. The presence of ferulic cid in these smples ws confirmed y mens of HPLC nlysis. HPLC nlysis reveled the presence of slicylic cid nd cffeic cid in specific root nd shoot smples. Once gin tretment with the synthetic fungicide stndrd resulted in the highest levels of phenolic cids per grm dry weight. The most significnt oservtion ws the high concentrtion of ferulic cid nd cffeic cid present in uninfected lettuce roots treted with comintion of PPG nd 108

108 cffeine (Figure 4.5), s most of the other increses in the levels of these compounds were ssocited with infection. HPLC nlysis detected these cids in the roots s well s the shoots wheres the Folin-Cioclteu method did not pick up significnt increses in the TPC of the shoot smples. A possile explntion for this could e tht when the originl extrction method for the shoot smples were decided, the Folin-Cioclteu method ws not s yet included in the protocol. The Folin-Cioclteu method relies on the principle tht the rection will occur in n lkline environment while extrction with ethyl cette creted n cidic environment. Efforts mde to evporte the ethyl cette nd re-dissolve the smples in methnol were unsuccessful. Severl studies of the phenol nd polyphenol composition in different cultivrs of lettuce hve een performed nd two min clsses of products hve een identified nmely cffeic cid derivtives (Ke nd Sltveit, 1988) nd flvonols (Hermnn, 1976). Cspersen (2000) lso showed tht under stress conditions ferulic cid, precursor for lignin formtion (Locher et l., 1994), ccumultes in hydroponiclly grown lettuce. Thus the presence of these phenolic cids in our extrcts is not suprising ut wht is of importnce is the effect of the tretments on the levels of these compounds. Yo (2004) identified six phenolic cids in C. sinensis using HPLC nmely: gllic cid, iso-chlorogenic cid, chlorogenic cid, p-coumric cid, courmrylquinic cid nd hydroxyphelpropionic cid. Cffeic- nd ferulic cids re produced in plnts from phenyllnine nd tyrosine vi the shikimte pthwy, forming p-coumric cid, 109

109 which undergoes further hydroxyltion to cffeic cid nd susequent O-methyltion to ferulic cid s shown in Figure 4.6 (Pnnl et l., 1998). These compounds usully occur nturlly s vrious conjugted forms resulting from enzymic hydroxyltion, O-methyltion, O-glycosyltion or esterifiction of p-coumric cid, principlly s the quinic or crohydrte esters, for exmple chlorogenic cid eing n ester of cffeic- nd quinic cid (Pruidze et l, 2003). From this we cn deduce tht y treting plnts with PPG s plnt tretment, one would e dding vlule precursors for phenolic cids such s ferulic- nd cffeic cid s well s esters of these cids tht my e hydrolysed y process in the plnts or surrounding environment. Slicylic cid is plnt hormone involved in the sic metolic pthwy involved in resistnce ginst plnt pthogens nd lso in induced resistnce. The HPLC nlysis lso showed n increse in the slicylic cid concentrtions for tretments on tomto plnts ut no significnt increse occurred in lettuce plnts. Further experimentl work will hve to e done to confirm the up-regultion of slicylic cid in plnts treted with PPG, cffeine or comintions thereof. Full chrcteristion of the nti-fungl compounds in lettuce nd tomto plnts s well s the elucidtion of pthwys involved in their iosynthesis is still required. More detiled informtion will llow for precise definition of the role these ntifungl compounds ply in plnt defence. In turn, such knowledge my enle strtegies to enhnce the levels of these compounds in plnts (Joyce nd Johnson, 1999). In ddition, n increse in the concentrtion of these nti-fungl compounds, mny of which re phenolics, my led to incresed helth enefits for consumers (Törrönen nd Määttä, 2002). 110

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115 Weidemn, H., Wehner, F.C Greenhouse evlution of Trichoderm hrzinum nd Fusrium oxysporum for iologicl control of citrus root rot in soils nturlly nd rtificilly infected with Phytophthor nicotine. Phytophylctic. 25: Wu A.M., Jing Y.J., Hwng P.Y., Shen F.S., Chrcteriztion of the okr mucilge y interction with Gl, GlNAc nd GlcNAc specific lectins, Biochimic et Biophysic Act 1243, Yo L., Jing Y., Dtt N., Singnusongc R., Liud X., Dun J., Rymonte K., Lislee A., Xu Y., HPLC nlyses of flvnols nd phenolic cids in the fresh young shoots of te (Cmelli sinensis) grown in Austrli. Food Chem. 84, Yu L., Perret J., Hrris M., Wilson J., Hley S., Antioxidnt properties of rn extrcts from Akron whet grown t different loctions, J. of Agri. nd Food Chem. 67, Zng Y., Lewis K., Ftins: new ntimicroil plnt peptides, FEMS Microiology Lett. 149, Tle 4.1 R f -vlues of Slicylic cid, Ferulic cid nd Cffeic cid stndrds s well s lettuce root extrcts tht gve cler inhiition zones with thin-lyer chromtogrphy ioutogrphy 116

116 Compound tested Men R F -vlue Stndrd devition of the men Slicylic cid Ferulic cid Cffeic cid IR 24 h IR 48 h IR 96 h PPG t IR 24 h PPG t IR 48 h PPG t IR 96 h CAF t IR 24 h CAF t IR 48 h CAF t IR 96 h COM t UN 24 h COM t UN 48 h COM t UN 96 h COM t IR 24 h COM t IR 48 h COM t IR 96 h Potssium Phosphonte t UN 24 h Potssium Phosphonte t UN 48 h

117 Potssium Phosphonte t UN 96 h Potssium Phosphonte t IR 24 h Potssium Phosphonte t IR 48 h Potssium Phosphonte t IR 96 h IR UR = Infected roots = Uninfected roots PPG t = Polyphenon G tretment t 12.5 g.l-1 CAF t = Cffeine tretment t 1.25 g.l-1 COM t = Comintion of Polyhenon G (12.5 g.l-1) nd cfiiene (1.25 g.l-1) 118

118 d d d d c Untreted Uninfected 0h Untreted Uninfected 24h Untreted Uninfected 48h Untreted Uninfected 96h PPG (12.5 g/l) Uninfected 24h PPG (12.5 g/l) Uninfected 48h PPG (12.5 g/l) Uninfected 96h CAF (1.25 g/l) Uninfected 24h CAF (1.25 g/l) Uninfected 48h CAF (1.25 g/l) Uninfected 96h PPG:CAF (12.5:1.25 g/l) Uninfected 24h PPG:CAF (12.5:1.25 g/l) Uninfected 48h PPG:CAF (12.5:1.25 g/l) Uninfected 96h Fosetyl-Al (100ml/L) Uninfected 24h Fosetyl-Al (100ml/L) Uninfected 48h Fosetyl-Al (100ml/L) Uninfected 96h Percentge of Dry Weight Tretments Figure 4.1 Percentge of dry weight of totl phenolics in extrcts of tomto roots treted with Polyphenon G (PPG), cffeine (CAF), the comintion PPG:CAF nd fosetyl-l over 96h period. Columns represent the mens. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05) 119

119 c c c c 0 Untreted Uninfected 0h Untreted Uninfected 24h Untreted Uninfected 48h Untreted Uninfected 96h Untreted Infected 24h Untreted Infected 48h Untreted Infected 96h PPG (12.5 g/l) Uninfected 24h PPG (12.5 g/l) Uninfected 48h PPG (12.5 g/l) Uninfected 96h PPG (12.5 g/l) Infected 24h PPG (12.5 g/l) Infected 48h PPG (12.5 g/l) Infected 96h CAF (1.25 g/l) Uninfected 24h CAF (1.25 g/l) Uninfected 48h CAF (1.25 g/l) Uninfected 96h CAF (1.25 g/l) Infected 24h CAF (1.25 g/l) Infected 48h CAF (1.25 g/l) Infected 96h PPG:CAF (12.5:1.25 g/l) Uninfected 24h PPG:CAF (12.5:1.25 g/l) Uninfected 48h PPG:CAF (12.5:1.25 g/l) Uninfected 96h PPG:CAF (12.5:1.25 g/l) Infected 24h PPG:CAF (12.5:1.25 g/l) Infected 48h PPG:CAF (12.5:1.25 g/l) Infected 96h Potssiumphosphonte (100ml/L) Uninfected Potssiumphosphonte (100ml/L) Uninfected Potssiumphosphonte (100ml/L) Uninfected Potssiumphosphonte (100ml/L) Infected 24h Potssiumphosphonte (100ml/L) Infected 48h Potssiumphosphonte (100ml/L) Infected 96h Percentge of Dry Weight Tretments Figure 4.2 Percentge of dry weight of totl phenolics in extrcts of lettuce roots treted with Polyphenon G (PPG), cffeine (CAF), the comintion PPG:CAF nd potssium phosphonte over 96h period. Columns represent the men. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05) 120

120 0.12 Percentge of Dry weight c SAs SAr CAs 0.00 Untreted Uninfected 48h PPG (12.5 g/l) Uninfected 48h CAF (1.25 g/l) Uninfected 48h PPG:CAF (12.5:1.25 g/l) Uninfected 48h Fosetyl-Al (100ml/L) Uninfected 48h Tretments Figure 4.3 Percentge of dry weight of slicylic nd cffeic cids in extrcts of tomto roots nd shoots treted with Polyphenon G (PPG), cffeine (CAF), the comintion PPG:CAF nd fosetyl-l over 48h period. Columns represent the men. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05) SAs = Slicylic cid in shoots. SAr = Slicylic cid in roots. CAs = Cffeic cid in shoots. 121

121 Percentge of Dry Weight Untreted Uninfected 48h Untreted Infected 48h PPG (12.5 g/l) Uninfected 48h PPG (12.5 g/l) Infected 48h CAF (1.25 g/l) Uninfected 48h CAF (1.25 g/l) Infected 48h Tretments PPG:CAF (12.5:1.25 g/l) Uninfected 48h PPG:CAF (12.5:1.25 g/l) Infected 48h Potssium phosphonte (100ml/L) Uninfected 48h Potssium phosphonte (100ml/L) Infected 48h SAs SAr FAs FAr CAs CAr Figure 4.4 Percentge of dry weight of slicylic, ferulic nd cffeic cids in extrcts of lettuce roots nd shoots treted with Polyphenon G (PPG), cffeine (CAF), the comintion PPG:CAF nd potssium phosphonte over 48h period. Columns represent the men. Error rs indicte the stndrd error of the men. Tretments with different letter nottion re significntly different (clculted t P < 0.05) SAs = Slicylic cid in shoots SAr = Slicylic cid in roots FAs = Ferulic cid in shoots FAr = Ferulic cid in roots CAs = Cffeic cid in shoots CAr = Cffeic cid in root 122

122 c Figure 4.5 Reverse phse high preformnce liquid chromtogrphy chromtogrm of () lettuce root smple 48h fter eing treted with comintion of 12.5 g.l-1 Polyphenon G nd 1.25 g.l-1 cffeine () cffeic cid stndrd (c) ferulic cid stndrd 123

123 Figure 4.7 Thin lyer chromtogrphy io-ssy pltes preformed on lettuce root extrcts. Lnes 1 nd 12: Slicylic cid Lnes 2,3,4, nd 5: Uninfected untreted extrct smples hrvested t 0, 24, 48 nd 96 hours respectively Lnes 6,7 nd 8: Infected untreted extrct smples hrvested t 24, 48 nd 96 hours respectively Lnes 9, 10 nd 11: PPG treted (12.5 g.l -1 ) uninfected extrct smples hrvested t 24, 48 nd 96 hours respectively 125

124 Figure 4.7 Thin lyer chromtogrphy io-ssy pltes preformed on lettuce root extrcts. Lnes 13 nd 23: Slicylic cid Lnes 14,15 nd 16: PPG treted (12.5 g.l -1 ) infected extrct smples hrvested t 0, 24, 48 nd 96 hours respectively Lnes 17,18 nd 19: Cffeine treted (1.25 g.l -1 ) uninfected extrct smples hrvested t 24, 48 nd 96 hours respectively Lnes 20,21,nd 22: Cffeine treted (1.25 g.l -1 ) infected extrct smples hrvested t 24, 48 nd 96 hours respectively 126

125 Figure 4.7c Thin lyer chromtogrphy io-ssy pltes preformed on lettuce root extrcts. Lnes 24 nd 37: Slicylic cid Lnes 25,26 nd 27: Comintion of PPG (12.5 g.l -1 ) nd cffeine (1.25 g.l -1 ) treted uninfected extrct smples hrvested t 0, 24, 48 nd 96 hours respectively Lnes 28,29 nd 30: Comintion of PPG (12.5 g.l -1 ) nd cffeine (1.25 g.l -1 ) treted infected extrct smples hrvested t 24, 48 nd 96 hours respectively Lnes 31,32 nd 33: Potssium phosphonte treted (100ml.L -1 ) uninfected extrct smples hrvested t 24, 48 nd 96 hours respectively Lnes 34,35 nd 36: Potssium phosphonte treted (100ml.L -1 ) infected extrct smples hrvested t 24, 48 nd 96 hours respectively 127