Ecology and Development Series No. 11, 2003

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1 Ecology nd Development Series No. 11, 2003 Editor-in-Chief: Pul L.G.Vlek Editors: Mnfred Denich Christopher Mrtius Nick vn de Giesen Huong Thi Thu Le Role of Pythium phnidermtum (Edson) Fitzp. in tomto sudden deth in the tropics with emphsis on integrted disese mngement Cuvillier Verlg Göttingen

2 ABSTRACT Sudden deth cuses severe dmge to tomto grown under field conditions following flooding t high soil tempertures. P. phnidermtum plys key role in sudden deth. In the present study it ws shown tht tomto sudden deth only occurred fter flooding for 48 hrs or more t soil tempertures of 28 to 32 C when the soil ws inoculted with P. phnidermtum. The disese ws reduced significntly t soil tempertures of 18 to 25 C. The fungus cused severe dmge to the root cortex, cused wilting nd plnt deth. In field experiment conducted in the hot growing seson between June nd September 2002 in Tiwn, 51, 72 nd 93-dy-old tomto plnts were flooded nd tested for susceptibility to sudden deth. When the plnts were 51 dys old, they survived in significntly higher numbers thn older plnts. Flood durtion plys significnt role in the deth of tomto in infested soil. Flooding for 24 hrs did not hve ny effect on the disese wheres flooding for 48 nd 72 hrs cused 100% of the plnts to wilt. Three isoltes of Trichoderm hrzinum, two of Trichoderm virens nd one of Streptomyces srceticus were evluted for biologicl control of tomto sudden deth due to P. phnidermtum in the greenhouse nd in the field. T. hrzinum nd T. virens isoltes were used to tret the seed nd the potting medium used for seedling production, nd lso incorported into the soil before trnsplnting. Streptomyces ws dded to the potting medium nd pplied dily to the field soil or greenhouse plots until 40 dys fter trnsplnting. The results reveled tht neither Trichoderm spp. nor Streptomyces gve significnt biocontrol of tomto sudden deth. The percentge of disesed tomto plnts growing in soil treted with either Trichoderm isoltes or Streptomyces fter flooding ws not significntly different when compred to the soil treted with P. phnidermtum lone. The soil mendments, SFMC, FNB-5A nd S-H Mixture tested in this study lso showed little promise for controlling tomto sudden deth due to P. phnidermtum. In both greenhouse nd field experiments conducted in the summer of 2001 in Tiwn, the number of wilted nd ded tomto plnts ws not significntly reduced over the control. The Trichoderm ureoviride dded to the soil mendment tretments lso gve no significnt improvement in control of P. phnidermtum with respect to tomto sudden deth. Similr poor results were observed in tretments with the fungicide Mefenoxm. Grfting tomto onto eggplnt rootstocks successfully protected the plnts ginst tomto sudden deth. In greenhouse tests, the number of disesed plnts ws significntly reduced on grfted tomto. The result in the greenhouse nd field experiments collborted ech other. The eggplnt roots were colonized by P. phnidermtum, but no extensive dmge ws cused to the plnt. The results of the present study indicte tht, with exception of grfting with eggplnt rootstocks on biocontrol or the use of orgnic mendments would not give dequte control in the field. More reserch on biocontrol nd orgnic mtter for control of tomto sudden deth is thus required.

3 Die Rolle von Pythium phnidermtum (Edson) Fitzp bei "tomto Sudden Deth" in den Tropen mit Schwerpunktpunkt integriertes Pflnzenschutzmngement ZUSAMMENFASSUNG "Sudden Deth" ruft schwere Schäden n Tomten hervor, die unter Gewächshus- und Freilndbedingungen nch Überflutung und hohen Bodentemperturen hernwchsen. In diesem Zusmmenhng spielt Pythium phnidermtum eine Schlüsselrolle. Die Erkrnkung tritt nur uf, wenn hohe Bodentemperturen (28-32 C) mit der Überflutung einhergehen und Pythium phnidermtum im Boden vorliegt oder der Boden nicht sterilisiert wurde. Bei niedrigeren Temperturen (18-25 C) wurde ds Ausmß des Schdens reduziert. Der Pilz rief eindeutige Schäden n der Wurzelrinde hervor, die zu Welkeerscheinungen und Absterben der Pflnzen führte, sowie ds Wurzelfrischgewicht reduzierte. Im Freilndversuch von Juni bis September 2002 wurden Tomtenpflnzen zu unterschiedlichen Zeitpunkten, 51, 72 und 93 Tge nch dem Auspflnzen geflutet. Pflnzen die 51 Tge nch dem Auspflnzen geflutet wurden überstnden besser ls solche, die zu einem späteren Zeitpunkt geflutet wurden. Die Überflutungsduer spielt eine Rolle beim "Sudden Deth" n Tomten uf infizierten Böden. Nch Flutungszeiten von 24 Stunden trten keine Symptome uf, ber es km zum Totlusfll, wenn die Überflutung 48 oder 72 Stunden nduerte. Drei Isolte von Trichoderm hrzinum, zwei Isolte Trichoderm virens und ein Isolt Streptomyces srceticus wurden uf ihre biologischen Kontrolleigenschften gegen "tomto Sudden Deth", welches durch Pythium phnidermtum hervorgerufen wird, im Gewächshus und unter Freilndbedingungen untersucht. Trichoderm hrzinum und Trichoderm virens Isolte wurden sowohl ins Ausstsubstrt ls uch in ds Kultursubstrt eingemischt. Streptomyces wurde erst ins Kultursubstrt und erneut nch 40 Tgen ppliziert. Die Ergebnisse zeigen, dss weder Trichoderm noch Streptomyces wenig Aussichten hben ls biologische Bekämpfungsmßnhmen gegen "tomto Sudden Deth" eingesetzt zu werden. Der Prozentstz n gesunden Tomtenpflnzen die in, mit Trichoderm oder Streptomyces Isolten, behndelten Böden herngezogen wurden, wr nicht signifiknt unterschiedlich, wenn mn sie mit der unbehndelten Kontrolle verglich. Keine Änderung der Bodenzusmmensetzung, ob mit SFMC, FNB-5A oder S-H, die in dieser Studie getestet wurden lässt vermuten, dss mn hierdurch einen Bekämpfungserfolg von Pythium phnidermtum erreichen knn. In keinem der beiden Gewächshus- und Freilndversuchen, die im Sommer 2001 durchgeführt wurden, konnten durch die Veränderungen der Bodenzusmmensetzung signifiknte Reduzierungen des Beflls erzielt werden. Trichoderm ureoviride, welches den veränderten Böden zugefügt wurde, zeigte keine Effektivität gegen Pythium phnidermtum. Ähnliche Resultte wurden für die mit Mefenoxm behndelte Kontrolle beobchtet. Veredeln der Pflnzen uf Auberginenunterlgen schützte die Tomten erfolgreich gegen "Sudden Deth". In Gewächshusversuchen wurde die Zhl der infizierten Pflnzen signifiknt reduziert und dies konnte in Freilndversuchen bestätigt werden. Die Auberginenwurzeln wurden von Pythium phnidermtum besiedelt ohne umfngreiche Schäden hervorzurufen.

4 TABLE OF CONTENTS 1 GENERAL INTRODUCTION 1 2 ROLE OF PYTHIUM APHANIDERMATUM IN TOMATO (Lycopersicon esculentum Mill.) SUDDEN DEATH AND EFFECT OF SOIL TEMPERATURE ON DISEASE DEVELOPMENT Introduction Mterils nd methods Tomto plnts Preprtion of Pythium phnidermtum inocultion Micro plot design Isolte Pythium phnidermtum from soil, floodwter nd soil Root weight Flooding Experimentl design Dt nd sttisticl nlysis Results The role of Pythium phnidermtum in tomto sudden deth The role of Pythium phnidermtum in tomto sudden deth in 18 nturlly nd rtificilly inoculted soil The role of Pythium phnidermtum in tomto sudden deth following 22 flooding between two regimes of soil temperture regimes Discussion PLANT AGE AND FLOOD DURATION EFFECTS ON TOMATO SUDDEN DEATH CAUSED BY PYTHIUM APHANIDERMATUM Introduction Mterils nd methods Plnt mterils Pythium phnidermtum recovery from the soil Field experiment design Dt collection Results Effect of plnt ge nd flood durtion on tomto sudden deth Pythium phnidermtum recovery from the soil Discussions EVALUATION OF BIOLOGICAL CONTROL AGENTS FOR MANAGEMENT OF TOMATO SUDDEN DEATH CAUSED BY PYTHIUM APHANIDERMATUM FOLLOWING FLOODING UNDER HIGH TEMPERATURE CONDITIONS Introduction Mterils nd methods Preprtions 45

5 4.2.2 Inoculums preprtion nd inocultion techniques Field experiment to evlute Trichoderm nd Streptomyces to control 47 tomto sudden deth due to Pythium phnidermtum Greenhouse experiment to evlute Trichoderm nd Streptomyces to control tomto sudden deth due to Pythium phnidermtum under different durtion of flooding Dt collection nd sttisticl nlysis Results Field experiment to evlute Trichoderm nd Streptomyces to control tomto sudden deth due to Pythium phnidermtum Greenhouse experiment to evlute Trichoderm nd Streptomyces to control tomto sudden deth due to Pythium phnidermtum fter 48 hrs of flooding Greenhouse experiment to evlute Trichoderm nd Streptomyces to control tomto sudden deth due to Pythium phnidermtum fter 72 hrs of flooding Discussions EFFECT OF SOIL AMENDMENTS TO CONTROL TOMATO SUDDEN DEATH DUE TO PYTHIUM APHANIDERMATUM FOLLOWING FLOODING IN HOT SEASON Introduction Mterils nd methods Preprtions Field experiment Greenhouse experiment Dt collection nd sttisticl nlysis Results Field experiment Greenhouse experiment Discussions RESPONSE OF TOMATO (Lycopersicon esculentum Mill.), EGGPLANT (Solnum melongen L.) AND TOMATO GRAFTED ONTO EGGPLANT ROOTSTOCK TO TOMATO SUDDEN DEATH DUE TO PYTHIUM APHANIDERMATUM Introduction Mterils nd methods Plnt mterils Field experiment Greenhouse experiments Dt collection nd sttisticl nlysis Results Field experiment Greenhouse experiment I... 90

6 6.3.3 Greenhouse experiment II Discussions SUMARY FUTURE PROSPECTS REFERENCES APPENDIX 113 ACKNOWLEDGEMENTS

7 Generl introduction 1 GENERAL INTRODUCTION The importnce of Pythium phnidermtum in tomto sudden deth disese Tomto (Lycopersicon esculentum Mill.) production in lowlnd res of tropicl nd subtropicl countries during the hot, wet seson fces number of constrints. Yield is generlly quite low compred to tht in the cool seson or from highlnd production res (Abdelhfeez et l. 1969). High temperture reduces fruit set (Abdelhfeez et l. 1975), hevy rinfll directly dmges eril plnt prts, soil wter logging reduces plnt vigor, nd short periods of soil flooding often result in wilt nd sudden deth of the tomto plnts (Kuo et l. 1982). Also contributing to the difficulties of growing tomtoes in the hot, wet seson re diseses such s dmping-off, bcteril wilt, southern blight, root-knot, trget lef spot, gry lef spot, erly blight, nd bcteril spot. The sudden deth disese is mjor nd growing problem in tomto production during the hot, wet seson (Villrel et l. 1978). Rpid wilting nd deth of tomto plnts fter short period of flooding re usully observed under hot nd humid conditions, which is likely due to the combintion of high temperture with flooding (Kuo et l. 1982). Tomto hs been shown to be one of the most flood-sensitive vegetbles (Iden 1956). Flood resistnt genotypes of tomto hve been investigted nd reported by Reid et l. (1969); Kuo et l. (1980), nd Kuo et l. (1982). However, the flood-tolernt genotypes re not sustinble, since some ccessions, which ppered flood tolernt in one tril, were not tolernt in nother. In ddition, plnts did not die when flooded t low temperture or in sterilized soil in greenhouse experiments, but died in the field following flooding t high temperture (Kuo et l. 1982). It hs been suggested tht there might be biologicl component responsible for plnt wilt nd sudden deth in field soils, since the disese ws eliminted by soil steriliztion. Mny fungl root pthogens hve been recorded s cusing soilborne diseses of tomto including Phytophthor spp., Fusrium spp., Rhizoctoni solni, Sclerotium rolfsii, nd Pythium spp. (Frnk 1995; McCrter 1997). Species of Pythium, including P. phnidermtum, P. ultimum nd P. debryn, hve worldwide distribution (Vn Der Plts-Niterink 1981) nd hve been recorded s the cuse of dmping-off nd stem root of mny crops such s tomto, chickpe, cucumber, 1

8 Generl introduction pepper, ben, spinch in both soil nd soilless cultivtions (Jenkins et l. 1983; Trperocs et l. 1990; Wulff et l. 1998). Among these, Pythium phnidermtum (Edson) Fitzp. is one of the most serious pthogens, cpble of cusing ctstrophic yield losses (Fvrin et l. 1988). This fungus is known to hve high temperture optimum of C nd it is lso known tht temperture gretly influences infection nd the subsequent dmge it cuses to its plnt host (Thomson et l. 1971; Kmmedhl et l. 1979). At lower tempertures, dmge is much less or negligible (Bolton 1980). Plnt ge nd susceptibility to soilborne diseses Plnt susceptibility to disese vries with plnt ge nd lso chnges with time of yer (Populer 1978). Susceptibility of plnts to soilborne diseses depends on plnt ge. Four generl clsses of chnge in susceptibility with plnt ge re recognized: (1) decresed susceptibility with the ge of plnt; (2) incresed susceptibility with ge nd intermedite ge susceptibility tht is either greter or less thn tht of younger or older plnts (Yrwood 1959; Populer 1978). Decresing susceptibility to soilborne pthogens with plnt ge my be relted to the structure nd function of different types of roots nd with their position in the overll root system (Zobel 1991). If the pthogen infects primrily first-older roots, the proportion of tissue susceptible to soilborne pthogens t different times or stges of host development will depend on the rtio of susceptible first-older roots to non-susceptible second roots (English et l. 1994). In ddition, mny other components of host development my contribute indirectly to chnges in susceptibility to root disese such s pthogens, beneficil microorgnisms, soil temperture, nd soil ph (English et l. 1994). The susceptibility of plnt to chnging environmentl fctors vries depending on field nd glsshouse conditions. The most importnt biotic fctors tht influence chnges in the susceptibility of hosts to root disese re light, soil moisture, temperture, ertion, minerl nutrition, hydrogen ion concentrtion, nd bulk density (English et l. 1994). Excess wter or flooded soil, for exmple, my increse disese severity by directly ffecting the chemicl nd physiologicl structures of the plnt root (Kou et l. 1980). It my lso directly influence the pthogen by promoting the relese of zoospores nd 2

9 Generl introduction stimulting the processes of infection by species of Phytophthor nd Pythium (Duniwy 1983). Cultivtion prctices my ply importnt direct or indirect roles in chnging host susceptibility (Rovir et l. 1990). Predisposition of the root to disese in monoculture or crop rottions under different cultivtion prctices my be consequence of utotoxicity or llelopthy (Rovir et l. 1990). If root development is slowed or roots re dmged by compction, susceptibility to disese my be ltered (English et l. 1994). Timing of crop mngement prctices cn gretly influence host susceptibility (Plty et l. 1997). Some Pythium spp. ffect plnt roots t tempertures fvorble for root growth but only cuse disese t higher tempertures (Mitchell, 1975). Biologicl control of soilborne diseses Biologicl control hs been known since 1874, when Roberts showed the suppressive ctivity of Penicillium glucum ginst bcteri nd regrded this phenomenon s ntgonism. For more thn 60 yers, the term biologicl control, mening control of one orgnism by nother, hs been used by both entomologists nd plnt pthologists. In biologicl control of plnt disese, three ntgonistic mechnisms re recognized: ntibiosis, which mens production of metbolites toxic or inhibitory to the plnt pthogens; competition for nutrients nd spce; nd prsitism, where the ntgonist directly extrcts nutrients from the pthogen (Bker et l. 1996). The biologicl control of plnt pthogens hs been defined by Cook et l. (1983) s the reduction of the mount of inoculum density or disese-producing ctivity of pthogen ccomplished by or through one or more orgnisms other thn mn. According to this brod definition, biologicl control includes culturl prctices tht crete n environment fvorble to ntgonists, hostplnt resistnce, mss introduction of ntgonists, nonpthogenic strins, or other beneficil orgnisms or gents such s compost (Cook et l. 1983). Biologicl control of soilborne pthogens hs emphsized the use of ntgonistic microorgnisms nd medites through mechnisms including competition, ntibiosis nd mycoprsitism (Bker et l. 1974; Tronsmo 1996). These ctivities occur mong fungi tht re not injurious to the plnts (Bcker et l. 1974). Competition, for exmple, occurs mong 3

10 Generl introduction microorgnisms for the resources: wter, oxygen, nutrients nd spce (Bker et l. 1974). Biocontrol mnipultes the environment round the crop plnt to fvor orgnisms tht contribute to plnt helth nd vigor. It is less disruptive to the ecosystem thn pplying chemicl pesticides (Hrmn et l. 1989). The term biologicl system mngement defined s the outside-in nd inside-out perspective hs been coined to describe full integrtion of biocontrol in crop production (Sikor 1997). Biologicl control of soilborne disese hs incresed in the pst two decdes. Beneficil fungi, especilly species of Trichoderm spp., hve been used extensively s seed tretment in order to reduce diseses (Hrmn et l. 1980; Lifshitz et l. 1986; Hrmn et l. 1989). The species Trichoderm hrzinum nd Trichoderm virens re mong the most intensively studied biocontrol gents. Tretment of seed or plnts with Trichoderm hrzinum hs promoted plnt helth nd hs lso effectively controlled Pythium dmpingoff (Liu et l. 1965; Hrmn et l. 1980; Mrshl 1982; Lifshitz et l. 1986; Hrmn et l. 1989). In ddition, Trichoderm hrzinum hs shown brod-spectrum ction to other soilborne pthogens such s Pythium ultimum, P. phnidermtum, Rhizoctoni solni, nd species of Phytophthor in mny crops (Chet et l. 1981; Eld et l. 1982; Sinv et l. 1984; Hrmn et l. 1989; Roiger et l. 1991). Biologicl control of soilborne diseses by soil orgnic mendments Orgnic mendments such s crop debris, green nd niml mnures nd other residues hve been used in prcticl crop mngement in old griculture systems in order to improve soil fertility (Rodríguez-Kábn et l. 1987). Erly ttempts were lso mde to use soil microorgnisms in prcticl biologicl control under field conditions (Bcker et l. 1974). Orgnic mendments my lter soil physicl nd chemicl conditions such s ph (Blcker et l. 1983), nd produce toxic fungl inhibitors (Spencer et l. 1982; Hung et l. 1993) tht hve importnt consequences for plnt growth nd the occurrence of soilborne diseses (Hung et l. 1993). Soil mendments hve either simple or complex mechnisms tht ffect plnt pthogens depending on the mendments nd pthogens involved (Hung et l. 1993). It directly ffects the pthogens by killing the pthogen propgules; indirectly through the 4

11 Generl introduction brekdown of chemicls, pesticides or fertilizers tht hve hrmful side-effects on the pthogens, or by incresing dormncy of propgules (Bker et l. 1974; Hung et l. 1993). Soilborne pthogens re in some cses indirectly controlled through combintion of fctors, which include improved vigor of the host plnts combined with enhnced ctivity of ntgonistic microbes (Bker et l. 1974; Hung et l. 1993). Adding mendments, for exmple, in the form of green legume crops, which re rich in vilble nitrogen nd crbon compounds, promotes host plnt growth (Bker et l. 1974; Plti 1981; Mndelbum et l. 1990). Soil ph chnges nd minerl vilbility increses for both soil microorgnisms nd plnts (Bker et l. 1974; Lin et l. 1990). In ddition, orgnic mendments with chitinous mterils fvour the development of chitinolytic microflor in the soil or even under soil-less cultivtion (Godoy et l. 1983). Mny microorgnisms in the soil microflor re ntibiotic-producing bcteri, ctinomycetes nd fungi ntgonistic to phytonemtodes nd other soilborne pthogens (Plti 1981; Sikor 1992; Hung et l. 1993; Rodríguez-Kábn et l. 1997). Mny composts used s soil mendment hve been investigted s prt of integrted biologicl control due to their bility to suppress soilborne plnt pthogens. This phenomenon hs been studied on wide rnge of pthogens such s Pythium phnidermtum, P. ultimum, Rhizoctoni solni, Sclerotium rolfsii, nd Phytophthor cinnmomi, using compost originted from wste mterils such s hrdwood or pine brk, grpe mrc, cttle mnure or other mterils (Nelson et l. 1983; Hdr et l. 1986; Mndelbum et l. 1990; Shiu et l. 1999). These compost mendments provide n improved environment to the susceptible plnt in which disese incidence is reduced (Hdr et l. 1992). The mendment nmed S-H, for exmple, hs direct effect on P. phnidermtum by inhibiting mycelil growth nd zoospore germintion. It gretly decreses disese incidence on cucumber under greenhouse nd field conditions (Lin et l. 1988; Lin et l. 1990). Compost mendments provide potentilly effective biologicl control of plnt pthogens, but the biologicl nd nturl spects of this phenomenon lso re ssocited with prcticl problems of use in griculture (Hdr et l. 1992). In ddition, orgnic mendments re not pnce, nd in some cses the mendments do not work well for deep-rooted crops or for plnts with long growing period (Lin et l. 1990). 5

12 Generl introduction Grfting to reduce soilborne diseses Using grfted plnts is currently populr in horticulturl production round the world. In Jpn, for exmple, 93% of wtermelons, 72% of cucumbers, 50% of eggplnts, 32% of tomtoes nd 30% of ll types of other melons re produced using grfted plnts (Od 1993). The grfted plnt ws developed for multi purposes tht protect the plnt roots in dverse soil environments. Abdelhfeez et l. (1975) first tested tomto grfted onto tomto or eggplnt rootstocks with the ssumption tht the tomto/eggplnt combintion would be of benefit for tomto yield under hot, rid conditions. The eggplnt rootstock hs n effect on vegettive growth only, wheres the production of trusses nd fruits lgged behind resulting in lower yield thn tht of the tomto/tomto grft. However, the tomto grfted onto eggplnt ws more beneficil thn tht of non-grfted plnts or the tomto/tomto grft under hot nd rinfll conditions (AVRDC 1999). Another min use of grfted plnts ws to reduce the overll problem of soilborne pthogens. Grfting plnts in most cses hs been shown to protect plnts ginst diseses due to nemtodes, bcteri, nd fungi (Od et l. 1994; Od 1995). Grfting on resistnt rootstocks is new component of integrted control of soilborne plnt pthogens (Ktn 1996). Grfting tomto onto disese-resistnt vrieties or onto eggplnt rootstocks hs multiple benefits. Grfted plnts were shown to be resistnt to severl soilborne diseses such s bcteril wilt, dmping-off or nemtodes (Peregrine et l. 1982; Chdh 1988; Mtsuzoe et l. 1993). A disdvntge of tomto grfted onto resistnt tomto rootstocks is tht the plnts re not protected ginst excess wter or flooding conditions (AVRDC 1999). In this cse, tomto grfted onto eggplnt rootstock is superior over the combintion of tomto/tomto (AVRDC 1999). 6

13 Generl introduction Scope of the study The objectives of the study presented here were to: 1. Assess the role of Pythium phnidermtum in sudden deth of tomto following flooding t high soil tempertures s well s the effects of soil tempertures on disese development. 2. Study the level of resistnce in the tomto plnt t different plnt ges to tomto sudden deth due to Pythium phnidermtum. 3. Investigte biologicl control efficcy of selected fungl ntgonists to sudden deth due to Pythium phnidermtum following flooding t high tempertures. 4. Evlute the effect of soil orgnic mendments on biologicl control of tomto sudden deth due to Pythium phnidermtum. 5. Determine whether tomto (Lycopersicon esculentum Mill.) grfted onto eggplnt (Solnum melongen L.) rootstocks protects ginst tomto sudden deth due to Pythium phnidermtum following flooding in high temperture conditions. 7

14 Role of Pythium phnidermtum on tomto sudden deth 2 ROLE OF PYTHIUM APHANIDERMATUM IN TOMATO (LYCOPERSICON ESCULENTUM MILL.) SUDDEN DEATH AND EFFECT OF SOIL TEMPERATURE ON DISEASE DEVELOPMENT 2.1 Introduction Pythium phnidermtum is known to hve high virulence t tempertures between 35 nd 40 C (Vn Der Plts-Niterink 1981). It is lso known tht temperture gretly influences infection nd subsequent dmge tht P. phnidermtum cuses to its host plnt (Thomson et l. 1971; Kmmedhl et l. 1979). Optimum temperture for oospore germintion is 30 C (Adms 1971). Tempertures over 30 C re considered to be most fvorble for disese development, while t lower tempertures dmge is much less or negligible (Thomson et l. 1971; Bolton 1980). Similrly, high soil moisture is usully fvorble for root disese cused by Pythium spp. (Kmmedhl et l. 1979). P. phnidermtum hs very fst growth rte nd is prolific producer of zoospores. P. phnidermtum hs been reported s cusing severl diseses on young tomto plnts such s dmping-off, root rot, nd cottony blight (Wells et l. 1954; Bolton 1980; McCrter 1997). It is lso mjor pthogen cusing series of diseses on other crops including cucumber, sugrbeet, spinch, cbbge, hot chilli, nd turf grss (Hine et l. 1969; Gold et l. 1984; Stij et l. 1987; Lo et l. 1997; Wulff et l. 1998; Shiu et l. 1999). The dmping-off disese hs been well documented from mny regions in the world. The disese hs been the min cuse of crop losses in tomto, cucumber nd pepper in Englnd, Americ, Cnd, Chin, Indi, nd Thilnd. It is often cited s the cusl gent of root diseses nd wilting of number of vegetble crops grown not only in hydroponic systems, but lso in the field (Wlker 1952; Yu et l. 1989; Zhng et l. 1990; Pulitz et l. 1992). Pthogenic mechnisms of P. phnidermtum for ttcking the plnt hve been investigted. Environmentl fctors such s soil temperture, soil moisture nd soil ph hve effects on the popultion densities of the fungus. High soil moisture hs been shown to hve positive influence on popultion densities of P. phnidermtum. In ddition, soil ph between 5-8 nd tempertures of 30 C nd higher hve been shown to positively ffect the penetrtion of the host plnt by P. phnidermtum (Bolton 1980). However, the true 8

15 Role of Pythium phnidermtum on tomto sudden deth cuse of sudden deth of tomto under hot, wet conditions is not clerly understood. During summer trils in recent yers t AVRDC (Tiwn), the occurrence of stem rot, wilting nd plnt deth cused by P. phnidermtum hve been observed fter hevy rinfll. The ims of this study were to: 1. Assess the role of Pythium phnidermtum in tomto sudden deth following flooding under high tempertures. 2. Investigte the effect of soil temperture on tomto sudden deth development due to Pythium phnidermtum following flooding. 2.2 Mterils nd methods Three different greenhouse experiments were conducted from summer 2001 to summer 2002 t AVRDC, Shnhu, Tiwn, in soil infested with P. phnidermtum. The fungus ws incorported into the soil 10 dys fter trnsplnting. Plstic continers (tubs) were used s micro experimentl plots Tomto plnts The tomto line CL D, which ws determined by AVRDC to be het tolernt nd virus resistnt, ws used. The seedlings were plnted in pet moss substrte for 30 dys in the greenhouse nd then trnsplnted into the tubs for experimenttion Preprtion of Pythium phnidermtum inoculum P. phnidermtum, strin number 4, isolted by the mycology unit t AVRDC, ws cultured on V-8 gr for 3 dys t 28 C before being inoculted onto rice seed for solid stte fermenttion. Ech 400 ml beker contining 150 ml of rice grin nd 75 ml of distilled wter ws utoclved twice prior to being used s the finl growth substrte. Two blocks of gr of 3-dy-old P. phnidermtum cultures were put into the rice grin in ech beker nd then incubted in n illuminted chmber t 28 C for 10 dys. One beker of 9

16 Role of Pythium phnidermtum on tomto sudden deth rice grin ws incorported into the upper 10 cm of soil of ech tub, 10 dys fter trnsplnting Micro plot design Plstic tubs (31 cm x 51 cm x 40 cm: height x width x length) were used s experimentl units. The tubs were sterilized with solution of hydrochloride (1%) before being filled with soil. Five kg of sterilized pebbles (1-2 cm dimeter) were plced in the bottom of the tub. A plstic net ws then lid over the pebbles in the bottom of the tub. A U-shded siphon ws ttched to the tub so tht wter could be siphoned out from under the net (Figure 2.1). Fifty-five kg of hevy cly soil, which hd been tken from AVRDC fields, (Tiwn) ws stem psteurized, nd then filled into ech tub. Soil ph ws evluted nd found to be 6.5. About 82.5 g of orgnic commercilly vilble grnulr fertilizer (N:P:K=4:4:4) nd 20 g chemicl fertilizer (N:P:K:Mg=15:15:15:4) ws incorported into the soil of ech tub before trnsplnting. In ddition, 3.5 g of chemicl fertilizer (N:P:K=20:20:20) per tub ws dded weekly until the plnts were hrvested. A B Figure 2.1: Greenhouse experiment t AVRDC, Shnhu, Tiwn (A) nd plstic tub with siphon tube (rrowed) for dringe (B). 10

17 Role of Pythium phnidermtum on tomto sudden deth Pythium phnidermtum recovery from soil, floodwter nd plnt roots Isoltion of Pythium phnidermtum from the soil Soil smples were tken rndomly from the tubs t 12-hr intervls during flooding s well s 1 nd 2 dys fter floodwter hs been removed. The soil smples were plced on pper towels nd llowed to dry t room temperture for 2 dys. Four dilutions 1/5, 1/10, 1/20, nd 1/40 of soil (g soil per ml 0.3 % wter gr) were used to quntify the popultion of P. phnidermtum (Burr et l. 1973). Ech dilution ws mixed on Vortex stirrer for t lest 15 min nd 1 ml liquot ws dispensed evenly cross the surfce of the selective medium for P. phnidermtum developed by Burr et l. (1973). The pltes were incubted t 35 C for time intervls rnging from 24 to 72 hrs, fter which the soil ws crefully wshed from the gr surfce nd the Pythium colonies were recorded. Isoltion of Pythium phnidermtum from floodwter Floodwter smpling ws used to ssess the fluctution of the popultion density of P. phnidermtum during flooding. A smple of 50 ml of floodwter ws tken from ech tub every 12 hrs during the flooding period. The wter smples were diluted to 1/1, 1/10, 1/100, 1/1000 (1 ml floodwter smple per 9 ml sterilized wter) nd spred over ABPDA gr medium (AVRDC selective medium). The pltes were incubted t 28 C for time intervls rnging from 24 to 72 hrs nd the Pythium colonies were recorded. Isoltion of Pythium phnidermtum from the roots The roots were collected from the tubs t 12-hr intervls during flooding s well s 1 nd 2 dys fter the floodwter hs been removed. The roots were wshed with running tp wter nd cut into 1 cm length. Ten root pieces were plced on plte of Mircetich medium (Mircetich 1971) for fungl colony production. The pltes were incubted t 28 C nd the colony-forming units (CFU) of P. phnidermtum recovered from ech root section were observed nd recorded fter 24 hrs incubtion. 11

18 Role of Pythium phnidermtum on tomto sudden deth Root weight Tomto roots were removed from the soil by crefully wshing with tp wter 7 dys fter floodwter dringe. The wshed roots were plced on pper towels t room temperture for 30 min to remove excess wter nd then fresh root weight ws determined. The wshed root ws dried in n oven t temperture of 50 C for 48 hrs fter which the root dry weight ws determined Flooding The plstic tubs were flooded 60 dys fter trnsplnting. The wter level ws mintined t 2-4 cm bove the soil surfce for 48 or 72 hrs, nd then quickly removed through the siphon tube (Figure 2.1, B) Experimentl design Role of Pythium phnidermtum in tomto sudden deth following flooding t high temperture in rtificilly inoculted soil The experiment ws conducted in August-November, The soil temperture ws mintined t C during the experiment. The soil nd ir tempertures s well s the humidity inside the greenhouse were recorded dily. Two tretments with 3 replictions ech were tested (Tble 2.1). Tble 2.1: Tretments for evluting the role of Pythium phnidermtum in tomto sudden deth in rtificilly inoculted soil Tretment Pythium phnidermtum inocultion Flood durtion Inoculted 10 dys fter trnsplnting 48 hrs Non-inoculted control (ck) None 48 hrs 12

19 Role of Pythium phnidermtum on tomto sudden deth Role of Pythium phnidermtum in tomto sudden deth following flooding t high temperture in nturlly nd rtificilly infested soil The experiment ws conducted between Jnury nd April 2002 in soils both nturlly nd rtificilly infested with P. phnidermtum to ssess the role of the fungus in tomto sudden deth. The soil ws tken from AVRDC fields where tomto sudden deth hd previously occurred. Hlf of the field soil ws used s the tretment with P. phnidermtum infested soil, nd the other hlf ws stem-sterilized for the tretments sterilized soil-inoculted nd sterilized non-inoculted control. Both tretments with infesttion were compred to the non-inoculted control. The tubs were flooded 60 dys fter trnsplnting for 48 hrs. During crop development, the temperture rnged between 15 nd 20 C due to nturl cool winter conditions. Seven dys before flooding, the temperture ws incresed to C nd styed t this level until 7 dys fter floodwter removl. The following tretments with three replictions ech were tested (Tble 2.2). Tble 2.2: Tretments to evlute the role of Pythium phnidermtum in cusing tomto sudden deth in nturlly nd rtificilly infested soil Tretments Pythium phnidermtum Flood inocultion durtion Field soil + nturl infesttion None 48 hrs Sterilized soil + inoculted 10 dys fter trnsplnting 48 hrs Sterilized soil + non-inoculted (ck) None 48 hrs Role of Pythium phnidermtum in tomto sudden deth following flooding in two soil temperture regimes The experiment ws conducted between Jnury nd April 2002 to investigte two temperture regimes in soil rtificilly infested with P. phnidermtum. Tempertures were mintined t C (Set I) nd C (Set II), respectively. The experiment ws conducted in the cool seson to mintin the greenhouse t C. Higher soil tempertures of C were mintined through heting system 7 dys prior to flooding 13

20 Role of Pythium phnidermtum on tomto sudden deth nd 7 dys fter flooding. All tubs of Set I were flooded 45 dys fter trnsplnting nd of Set II fter 60 dys. The wter level ws mintined t 2-4 cm bove the soil surfce for 48 hrs nd then quickly removed by the siphon system. The following tretments with three replictions ech were tested (Tble 2.3). Tble 2.3: Tretments to evlute the role of Pythium phnidermtum in cusing tomto sudden deth following flooding depending on soil temperture Tretments Flood durtion Set I Non-inoculted control (ck) 48 hrs C P. phnidermtum inoculted (P) 48 hrs Set II Non-inoculted control (ck) 48 hrs C P. phnidermtum inoculted (P) 48 hrs Dt nd sttisticl nlysis The number of wilted plnts from ll tretments ws recorded 2, 4 nd 7 dys fter floodwter ws removed. Seven dys fter flooding, plnts were hrvested nd root dry weights determined. The dt on incidence of permnent wilt of tomto were nlyzed with the SAS (SAS Institute Inc. 1989) progrm, using the generl liner model procedure, including nlysis of vrince, Duncn s multiple rnge test, lest significnt difference, (LSD) nd/or orthogonl contrsts. 2.3 Results Role of Pythium phnidermtum in tomto sudden deth following flooding t high temperture in rtificilly inoculted soil Incidence of permnent wilt: No infected tomto plnts were observed fter trnsplnting to soil inoculted with P. phnidermtum or in the non-inoculted control. However, fter flooding for 48 hrs, few plnts wilted in the non-inoculted controls. In the infested soil, 65% of the tomto plnts showed wilt symptoms 4 dys fter flooding. Seven dys fter 14

21 Role of Pythium phnidermtum on tomto sudden deth dringe, over 70% of the plnts showed permnent wilt symptoms. In the non-inoculted control, 3% of the plnts showed wilt symptoms, which ws significntly (P 0.01) lower when compred to the inoculted tretment (Figure 2.2). 4 dys fter flooding 7 dys fter flooding Permnent wilt (%) b b Pyth-inoc non-inoc Pyth-inoc non-inoc Figure 2.2: Effect of Pythium phnidermtum on incidence of permnent wilt of tomto plnts 4 nd 7 dys fter floodwter ws removed in greenhouse experiment between August nd November 2001 t AVRDC, Shnhu, Tiwn. Different letters show sttisticlly significnt differences between two tretments ccording to Duncn s multiple rnge test (P 0.01) n=6. Root dmge nd root dry weight: Seven dys fter the floodwter ws removed, severe dmge to the root system by P. phnidermtum ws observed. The root cortexes were sloughed-off or missing in ll plnts grown in infested soil. No injured roots were observed on tomto plnts grown in non-inoculted soil (Figure 2.3). Root dmge by P. phnidermtum resulted in reduced root biomss. Both fresh nd dry weights of the roots of tomto plnts grown in soil infested with P. phnidermtum were significntly (P 0.05) lower when compred to the root weight of plnts grown in the non-inoculted soil (Figure 2.4). 15

22 Role of Pythium phnidermtum on tomto sudden deth 1 2 A 1 2 B Figure 2.3: Helthy roots nd roots dmged by Pythium phnidermtum following 48 hrs flooding t C soil temperture in greenhouse experiment between August nd November 2001 t AVRDC, Shnhu, Tiwn. A(1): Root of plnt grown in soil infested with Pythium phnidermtum A (2): Root of plnt grown in non-inoculted soil B(1): Root cortexes missing on the plnt grown in soil infested with Pythium phnidermtum B (2): Root of plnt grown in non-inoculted control 16

23 Role of Pythium phnidermtum on tomto sudden deth 2,5 Fresh weight Dry weight 2 Averge weight (g) 1,5 1 b A B 0,5 0 Pyth-inoc non-inoc Pyth-inoc non-inoc Figure 2.4: Effect of Pythium phnidermtum on fresh nd dry weight of tomto roots, in greenhouse experiment between August nd November 2001 t AVRDC, Shnhu, Tiwn. Different letters show sttisticlly significnt differences between two tretments ccording to Duncn s multiple rnge test (P 0.05) n=6. P. phnidermtum recovery from soil, wter nd root: The popultion density of P. phnidermtum in the soil, floodwter nd roots, ws determined every 12 hrs during the flooding period. The soil nd roots were lso smpled 1 nd 2 dys fter floodwter ws removed. Due to high vrition in the colony-forming units (CFU) of the pthogen in the smples, sttisticl nlysis ws not possible. The results, however, re presented s the mens of the smples t the different smpling periods. P. phnidermtum popultion densities in the soil dropped slightly between 12 nd 48 hrs during flooding. However, fter floodwter removl, P. phnidermtum ws recovered in higher densities in the soil 1 nd 2 dys fter floodwter hs been drined (Figure 2.5). P. phnidermtum zoospores isolted from the floodwter were highest 12 hrs fter flooding nd then dropped 24, 36 nd 48 hrs therefter (Figure 2.5). The percentge of P. phnidermtum-infected roots remined more or less stble from 12 to 48 hrs fter flooding. However, one dy fter floodwter removl, the number of CFU incresed nd resulted in 3-fold higher number fter 2 dys compred to the initil CFU number t 12 hrs (Figure 2.5). 17

24 Role of Pythium phnidermtum on tomto sudden deth 120 CFU of P. phnidermtum Pyth inoc non inoc 0 12hr 24hr 36hr 48hr 1dy 2dy 12hr 24hr 36hr 48hr 12hr 24hr 36hr 48hr 1dy 2dy Soil CFU/g soil Floodwter CFU/ml of wter Roots (% ) infected root sections Figure 2.5: Pythium phnidermtum recovery from soil (CFU/g soil), floodwter (CFU/ml of flood wter), nd roots (% infected root sections) in greenhouse experiment to test the effect of rtificil inocultion on tomto sudden deth Role of Pythium phnidermtum in tomto sudden deth following flooding t high tempertures in nturlly nd rtificilly inoculted soil Permnent wilt: No significnt difference ws observed 4 dys fter flooding in the wilting rte of tomtoes grown in the tretments of field soil nturlly infested (20%) nd sterilized soil inoculted with P. phnidermtum (27%). Four dys fter flooding the control showed 10% wilted plnts. However, 7 dys fter flooding, 3.3 % of the plnts in the control hd wilted. The number of wilted plnts observed in the control ws significntly lower when compred to both nturlly infested nd rtificilly inoculted soil. There ws lso no obvious difference in wilt between the 4- nd 7-dy periods (Figure 2.6). 18

25 Role of Pythium phnidermtum on tomto sudden deth 4 dys fter flooding 7 dys fter flooding Permnent wilt (%) b b 0 Field soil ntur infes Sterile soil rtif inoc ck Field soil ntur infes Sterile soil rtif inoc ck Figure 2.6: Occurrence of sudden deth of tomtoes grown in field soil with infested nd sterilized soil inoculted with Pythium phnidermtum, 4 nd 7 dys fter floodwter removl in greenhouse experiment between Jnury nd April 2002 t AVRDC, Shnhu, Tiwn. Different letters show sttisticlly significnt differences between two tretments ccording to Duncn s multiple rnge test (P 0.05) n=9. Root dmge nd root dry weight: Seven dys fter flooding, the dmged to the root cortex by P. phnidermtum t high tempertures ws evluted. The root cortex sloughed off or ws missing in ll plnts grown in nturlly infested s well s in rtificilly inoculted soil. No dmged roots were observed on plnts grown in non-inoculted, sterilized soil (Figure 2.7). 19

26 Role of Pythium phnidermtum on tomto sudden deth Figure 2.7: Helthy roots (2) nd roots dmged (1 nd 3) by Pythium phnidermtum following 48 hrs flooding t C soil temperture in greenhouse experiment to test the effect of nturl nd rtificil inoculum on tomto sudden deth. 1: Roots of plnt grown in field soil nturlly infested 2: Roots of plnt grown in sterilized soil non-inoculted control 3: Roots of plnt grown in sterilized soil rtificilly inoculted with Pythium phnidermtum The root dmged by P. phnidermtum led to the reduction of root biomss. Both the root fresh nd dry weight of plnts grown in either field soil or inoculted soil with P. phnidermtum were significntly lower compred to tht of plnts grown in sterilized, non-inoculted soil (Figure 2.8). 20

27 Role of Pythium phnidermtum on tomto sudden deth Fresh weight Dry weight 9 8 b b Averge root weight (g) B B A 0 Field soil ntur infes Sterile soil rtif inoc ck Field soil ntur infes Sterile soil rtif inoc ck Figure 2.8: Effect of Pythium phnidermtum on root fresh nd dry weight of tomto plnts in greenhouse experiment between Jnury nd April 2002 t AVRDC, Shnhu, Tiwn. Different letters show sttisticl significnt differences mong the tretments ccording to Duncn s multiple rnge test (P 0.05) n=9. P. phnidermtum recovery from soil, floodwter nd roots: The popultion density of P. phnidermtum in soil, floodwter nd roots ws determined every 12 hrs during the flooding period. In ddition, the density of the fungus in the soil nd roots ws quntified 1 nd 2 dys fter floodwter removl. Becuse of the lrge vrition in colony-forming units (CFU) of the pthogen in the smples, sttisticl nlysis ws not possible. The results re presented s the mens of the smples t the different smpling times. The popultion density of P. phnidermtum isolted from soil smples, floodwter nd roots showed fluctution over time in both nturlly infested nd rtificilly inoculted soil. The number of P. phnidermtum in the soil remined more or less the sme in the time period 12 to 48 hrs during the flooding period. There ws lso no chnge 1 nd 2 dys fter floodwter hs been drined (Figure 2.9). In floodwter smples, the density of P. phnidermtum lso remined stble during the smpling period in nturlly infested nd rtificilly inoculted soil (Figure 2.9). The percentge of roots colonized by P. phnidermtum incresed during flooding both in nturlly infested nd rtificilly inoculted soil. A mximum of 80% infected roots ws observed 48 hrs fter 21

28 Role of Pythium phnidermtum on tomto sudden deth flooding when the soil hs been rtificilly inoculted. However, 1 nd 2 dys fter dringe, the percentge of colonized roots decresed to bout 40% (Figure 2.9). When the soil ws nturlly infested with P. phnidermtum, mximum of 20% infected roots ws observed 1 dy fter dringe, nd dropped slightly 2 dys fter floodwter removl. In the rtificilly inoculted soil, 30% of the roots were infested t 1 dy fter dringe, nd fter 2 dys the level of infection rose to 40% (Figure 2.9). 90 CFU of P. phnidermtum Field soil Sterile soil Pyt inoc Sterile soil non inoc 12hr 24hr 36hr 48hr 1dy 2dys 12hr 24hr 36hr 48hr 12hr 24hr 36hr 48hr 1dy 2dys Soil CFU/g soil Floodwter CFU/ml wter Root (%) infected root section Figure 2.9: Pythium phnidermtum recovery from soil, floodwter nd roots during 48 hrs flooding, 1 nd 2 dys fter dringe in greenhouse experiment compring nturlly infested nd rtificilly inoculted soil Role of Pythium phnidermtum in tomto sudden deth following flooding in two soil temperture regimes Permnent wilt: Temperture showed cler effect on pthogenicity of P. phnidermtum on tomto plnts. No wilted plnts were observed t low soil tempertures of C (Figure 2.10). At higher soil tempertures (28-32 C), 28% wilted plnts were observed in soil is tht ctully infested with P. phnidermtum, 4 dys fter flooding. Seven dys fter flooding, bout 25% of plnts grown in infested soil showed wilt symptoms. As shown in Figure 2.10, the percentge of wilted plnts in the control ws significntly lower 4 nd 7 dys fter flooding. 22

29 Role of Pythium phnidermtum on tomto sudden deth 4 dys fter flooding 7 dys fter flooding Permnent wilt (%) b 5 0 b b b b b P ck P ck P ck P ck C C C C Figure 2.10: Effect of temperture t the time of flooding on development of sudden deth of tomto plnts due to Pythium phnidermtum in greenhouse between Jnury nd April 2002 t AVRDC, Shnhu, Tiwn. P = Pythium phnidermtum inoculted; ck = non-inoculted control. Different letters show sttisticlly significnt differences mong the tretments ccording to Duncn s multiple rnge test (P 0.05) n=12. P. phnidermtum recovery from soil, floodwter nd roots: P. phnidermtum ws isolted from soil, floodwter nd plnt roots (Figure 2.11). The soil temperture showed no mjor effect on the development of the fungus in the soil. Independent of the soil tempertures, the popultion density of P. phnidermtum decresed during flooding nd then recovered nd remined constnt fter floodwter ws removed. In floodwter smples, the P. phnidermtum number incresed t soil tempertures of C. The highest CFU of the fungus ws isolted t C, 36 hrs fter flooding. Similr results were observed with root smples in the two soil temperture regimes. The percentge of roots infested with P. phnidermtum t C nd C were 66% nd 80%, 36 nd 48 hrs during flooding, respectively. Although the percentge of infected root sections hd incresed during flooding t 36 nd 48 hrs t low nd high tempertures, respectively, dringe resulted in reduction of infection. However, levels of infection were still elevted compred to the initil percentge found t 12 to 24 hrs. 23

30 Role of Pythium phnidermtum on tomto sudden deth CFU of P. phn Soil (CFU/g soil) Pyth. inoc non inoc 12hr 24hr 36hr 48hr 1dys 2dys 12hr 24hr 36hr 48hr 1dys 2dys C C Floodwter (CFU/ml wter) CFU of P.phn hr 24hr 36hr 48hr 12hr 24hr 36hr 48hr C C Percentge (%) of infected root sections 12hr 24hr 36hr 48hr 1dys 2dys 12hr 24hr 36hr 48hr 1dys 2dys C C Figure 2.11: Pythium phnidermtum recovery from soil (CFU/g soil), floodwter (CFU/ml of flood wter) nd roots (% infected root sections) in greenhouse experiment between Jnury nd April Plnts in both soil temperture regimes of C nd C, were flooded for 48 hrs. n=12. 24

31 Role of Pythium phnidermtum on tomto sudden deth 2.4 Discussion Assessment of the role of Pythium phnidermtum in tomto sudden deth following flooding t high soil tempertures Incidence of the disese: The im of this prt of the study ws to ssess the role of P. phnidermtum in tomto sudden deth following flooding t high soil tempertures. P. phnidermtum nd other fungi, including Rhizoctoni solni Kühn, Fusrium solni nd Phytophthor spp., re well known pthogens, which cuse dmping-off on vegetbles (Stnghellini et l. 1982; Menzies et l. 1996; McCrter 1997). In this study, the plnts tht were grown in the soil treted with P. phnidermtum showed wilting symptoms only when the experimentl plots were flooded for 48 hrs. The percentge of wilted plnts observed in inoculted tretments ws significntly higher thn tht of the untreted control. The results re in greement with other previous studies on the role of P. phnidermtum in wilting nd deth of vegetbles. Zhng et l. (1990) nd Gbor (1997) found tht P. phnidermtum is common pthogen of pre-emergence dmping-off of vegetbles tht tends to be most severe under conditions of high soil moisture. Hine et l. (1969) reported tht the highest incidence of disese in sugrbeets due to P. phnidermtum occurred during July-August when soil tempertures were highest, together with mximum rinfll. In ddition, P. phnidermtum hs been known s the most ggressive pthogen in the hydroponic system (Stnghelline et l. 1996; Wulff et l. 1998). Pythium species re well known s poorly competitive pthogens in the soil reltive to other root-colonizing orgnisms, nd often ct only s primry colonizers (Kmmedhl et l. 1979). Among these, P. phnidermtum hs been reported s less effective in cusing dmping-off thn the other species, including P. debrynum nd P. ultimum (Thomson et l. 1971). It seems to be the mjor pthogen, however, in the high tempertures during the summer seson. Under these conditions, the fungus ws the predominnt species isolted from infested plnts (Gold et l. 1984). In the present study, no significnt differences in the percentge of wilted plnts grown in field soil nturlly infested or soil sterilized nd rtificilly inoculted with P. phnidermtum ws observed. This my be due to the fct tht the environmentl fctors, including high soil tempertures 25

32 Role of Pythium phnidermtum on tomto sudden deth nd soil moisture, enhnce the ctivities P. phnidermtum (Stnghellini et l. 1975). Dmge my lso occur whenever minimum mount of inoculum is present in the soil. Fvrin et l. (1988) lso found tht P. phnidermtum showed higher pthogenicity to cucumber thn other Pythium species, such s P. irregulre, t high soil tempertures nd moisture levels. Root dmge: In the present study, the roots of tomto plnts grown in the soil infested with P. phnidermtum showed severe dmge fter the experimentl plot hs been flooded. The corticl root tissues were injured nd sloughed-off. This ws probbly due to the fct tht high number of zoospores of P. phnidermtum hd penetrted into the cell nd then creted n ppressorium to cesing the cyclosis of the cell (Krft et l. 1967). Zoospores ggregte primrily in the region of mturtion on uninjured roots nd only rrely in the differentition nd elongtion regions, except when the roots re injured in these regions (Krft et l. 1967). The growth nd reproduction process of the fungus fter penetrtion is bsed on the mucilge of the root (Zheng et l. 2000). However, the root mucilge in the nutrient solution did not correlte significntly with the colony density of Pythium in young tgged roots, while in the regression nlysis, root growth ws relted negtively to colony density (Zheng et l. 2000). Shorter root elongtion ws observed in cucumber plnts treted with P. phnidermtum when compred to the untreted control (Wulff et l. 1998). In the present study, the root dry weight of tomto plnts in soil infested with P. phnidermtum ws significntly lower thn in non-inoculted controls. The results re in greement with other reserch ssessing the role of P. phnidermtum in the wilting of vegetbles. Zhou et l. (1994) found tht P. phnidermtum cused reduced root volume nd root dry weight of cucumber. It showed stronger effects on the host thn other Pythium species, which ws indicted by lower fresh weight of the host plnt 6 dys fter inocultion. In ddition, plnt growth ws severely suppressed (Wulff et l. 1998). 26

33 Role of Pythium phnidermtum on tomto sudden deth The role of soil temperture in tomto sudden deth due to Pythium phnidermtum following flooding conditions In the present study, the soil temperture plyed n importnt role with respect to the pthogenicity of P. phnidermtum. Tomto plnts did not show wilting symptoms t soil tempertures of C. The occurrence of wilted plnts ws observed t higher tempertures (28-32 C) fter the experimentl plots were flooded. The findings re in greement with previous reserch on the reltionship between temperture nd pthogenicity done by Hine et l. (1969); Gold et l. (1984) nd Yu et l. (1989). P. phnidermtum is known to be virulent in temperture rnge of C nd shows little effect below 20 C (Thomson et l. 1971). The temperture rnge for fungus growth is between 10 nd 40 C nd the optimum is 35 to 40 C (Vn Der Plts-Niterink 1981). Another environmentl fctor, which ws not directly investigted in this experiment, ws soil moisture. It hs been reported s mjor fctor for pthogenicity of soilborne pthogens. For diseses cused by Pythium spp., high soil temperture nd bundnt moisture hve been identified s the two most importnt fctors (Hendrix et l. 1973). P. phnidermtum ws responsible for killing seedlings s well s mture tomto nd cucumber plnts tht were irrigted 3-4 times dily t soil tempertures bove 30 C (Stnghellini et l. 1975). In hydroponic systems, P. phnidermtum cuses severe stunting nd deth of spinch plnts within 3-4 dys fter inocultion t 21 nd 27 C but not t low temperture (17 C) (Gold et l. 1984). In the present study, the incidence of the disese due to P. phnidermtum occurred in the plnts only fter the experimentl plots were flooded for 48 hrs. Isoltion from soil, wter nd root smples showed tht P. phnidermtum ws recovered independent from soil tempertures rnging from 18 to 32 C. The temperture, however, hs direct effect on the fungus virulence. Although the fungus ws widely distributed in the soil, floodwter nd roots t low tempertures of 18 to 25 C, it ws less ggressive to the host plnt. The results re in greement with other previous studies done by Hine et l. (1969) nd Gold et l. (1984). They reported tht t 17 C soil temperture, the development of P. phnidermtum ws limited. 27

34 Role of Pythium phnidermtum on tomto sudden deth The role of P. phnidermtum on tomto sudden deth following flooding in the hot seson ws demonstrted. However, the roles of secondry pthogens including bcteri nd other soilborne fungi involved in the deth of host plnts ws considered. Studies relting to the effect of bcteri on disese symptoms re rre in the soil. More informtion is needed to understnd the complex mechnisms ffecting disese severity cused by rhizobcteri in the presence nd bsence of P. phnidermtum. 28

35 The effect of plnt ge on tomto sudden deth 3 PLANT AGE AMD FLOOD DURATION EFFECTS ON TOMATO SUDDEN DEATH CAUSED BY PYTHIUM APHANIDERMATUM 3.1 Introduction Studies concerning the effect of plnt ge on susceptibility to soilborne diseses hve been conducted for mny yers. These studies reveled tht the dte of plnting or trnsplnting cn be used to control diseses. Populer (1978) demonstrted tht plnts vry in their susceptibility to disese with ge. Severl reports hve shown tht older plnts re more resistnt to Pythium spp. thn younger ones (Mellno et l. 1970; Populer 1978; Nelson 1984). Tomto seedlings of different ges differ in susceptibility to ttck by P. phnidermtum nd 5-week-old tomto plnts were shown to be resistnt to P. phnidermtum (Blck 2000, unpublished dt). In hydroponic systems, older tomto plnts were lso not s susceptible to dmge by Pythium spp. s younger plnts. Dmge cn be quite severe on older plnts (Jenkins et l. 1983). The most importnt biotic fctors tht influence chnges in the susceptibility of the host plnt to soilborne pthogens include soil moisture nd temperture (English et l. 1994). It hs been reported tht flooding of tomto plnts for 24 hrs does not hve ny significnt effects on the plnts, wheres flooding for over 72 hrs seriously dmges tomto crops (Kuo et l. 1980; Kent et l. 1981). P. phnidermtum is well known pthogen of tomto tht hs been shown to cuse dmping-off, stem rot, root disese, nd fruit rot. In summer tomto production in tropicl growing res, the stem rot cused by P. phnidermtum occurred in scttered plnts following hevy rins tht did not result in soil flooding. The pthogen hs very fst growth rte nd is prolific producer of zoospores, which re relesed under high moisture conditions. P. phnidermtum is lso the cusl gent of root diseses nd wilting of number of vegetble crops grown in hydroponics systems (McCrter 1997; Postm et l. 2000; Stnghellini et l. 2000). 29

36 The effect of plnt ge on tomto sudden deth The objectives of the present field study were to: 1. Study the pttern of popultion development of Pythium phnidermtum during the cropping seson. 2. Determine the effect of tomto plnt ge on susceptibility to tomto sudden deth. 3. Exmine the effect of flood durtion on severity of tomto sudden deth. 3.2 Mterils nd methods Plnt mterils The tomto vriety PT4723 F1, which ws developed by AVRDC s het tolernt, bcteri nd virus resistnt, ws used. The seeds were sown t intervls of pproximtely 2 weeks in the greenhouse for the different trnsplnting schedules in the field. The first sowing ws on My 1 st, followed by My 16 th nd June 2 nd Pythium phnidermtum recovery from the soil Chnges in pthogen popultion density in the field soil were mesured during the crop production period from June to August P. phnidermtum ws detected nd quntified bsed on the method of Burr et l. (1973). Five soil smples were tken rndomly from the upper 20 cm long the digonl of the field by cylinder (5 cm dimeter). The first smple ws tken shortly before trnsplnting nd smples were then tken on monthly bsis. In the flooded fields, the quntittive chnge of P. phnidermtum popultions due to flooding ws lso checked t 24, 48 nd 72 hrs fter flooding. In order to quntify the number of spores of P. phnidermtum in the soil, 3 soil smples were tken rndomly from the upper 20 cm between 2 rows of plnts of ech tretment plot. The soil smples were left to dry t room temperture for 2 dys nd then used to quntify popultion densities of P. phnidermtum. Soil dilutions of 1/5, 1/10, 1/20, nd 1/40 g soil per ml 0.3 % wter gr were used to quntify the popultion of P. phnidermtum (Burr et l. 1973). Ech dilution ws mixed on Vortex stirrer for t lest 15 min nd 1 ml liquot ws dispensed evenly cross 30

37 The effect of plnt ge on tomto sudden deth the surfce of the selective medium for P. phnidermtum developed by Burr et l. (1973). The pltes were incubted t 35 C for time intervls rnging from 24 hrs to 72 hrs fter which the soil ws crefully wshed from the gr surfce nd the Pythium colonies were recorded Field experiment design This experiment ws set up in the summer seson from June to August 2002 in the experimentl field of AVRDC in Shnhu, Tiwn. The field ws divided into 12 plots with 3 replictions of 4 flood-durtion tretments. The tretments re listed in Tble 3.1. Tble 3.1: Tretments to ssess the effects of plnt ge nd flood durtion on tomto sudden deth cused by Pythium phnidermtum Flood durtion Time between trnsplnting nd flooding 51 dys 72 dys 93 dys Non-flooded hrs hrs hrs A field covering 1400 m 2 ws weeded, ploughed nd fertilized 15 dys prior to trnsplnting. Twelve rectngulr blocks (15 m x 3 m = length x width) were prepred. A distnce of 5 m between the blocks ws estblished with 1-m-deep trenches, which were used to regulte the wter level during nd fter the flood period. In ech block, 3 subblocks (1 m x 5 m = width x length) were lid out for the 51, 72 nd 93 dy-old plnts. Ech flood durtion (0, 24, 48 nd 72 hrs) tretment ws replicted 3 times (Figure 3.1). 31

38 The effect of plnt ge on tomto sudden deth Non-flood 24hrs 48 hrs 72 hrs Rep. I Rep. II Rep. III Figure 3.1: Experimentl lyout in field experiment between June nd August 2002 t AVRDC, Shnhu, Tiwn. Twenty-four tomto seedlings (30 dys old) were plnted into the field for the 93- dy-old plnts trils. The plnts were tied to bmboo sticks to void dmge through hevy rin nd strong wind. Three weeks lter, nother 24 tomto seedlings were trnsplnted into the bed of the 72-dy-old plnts trils. Six weeks fter the first trnsplnting, the tril of 51 dy-old plnts ws trnsplnted. The field experiment ws flooded 3 weeks fter the lst trnsplnting (9 weeks fter the first trnsplnting) using controlled irrigtion system. The wter level ws mintined t 10 cm bove the upper soil surfce for 0, 24, 48 or 72 hrs nd then removed by nturl dringe through cnl system. 32

39 The effect of plnt ge on tomto sudden deth Dt collection nd sttisticl nlysis The number of wilted plnts ws recorded t 2, 4 nd 7 dys fter the floodwter hs been removed. The dt on the incidence of permnent wilt of tomto plnts were nlyzed by SAS (SAS Institute Inc. 1989), using the generl liner model procedure, including nlysis of vrince, Duncn s multiple rnge test, lest significnt difference (LSD) test, nd orthogonl contrst. 3.3 Results Effect of plnt ge nd flood durtion on tomto sudden deth The wilting symptoms were first observed on the upper leves two dys fter the floodwter hs been drined. Wilting then stedily progressed to the lower leves of the plnt. The percentge of wilted plnts incresed shrply from 1 nd 2 dys to 4 dys fter flooding. The incidence of tomto permnent wilt observed for ll tretments nd plnt ges ws significntly lower 7 dys fter floodwter dringe thn fter 4 dys (Figures 3.2 nd 3.3). Plnt ge clerly ffected susceptibility to tomto sudden deth cused by P. phnidermtum. The percentge of plnts with permnent wilt ws significntly (P 0.05) lower in younger 51-dy-old plnts thn in the older 72- nd 93-dy-old plnts in the sme flood regime (Figure 3.2). Hundred percent wilted plnts were observed in the 72-dy-old tretment t 4 dys nd 7 dys fter flooding (Figures 3.2 nd 3.3). In the 93-dy-old plnt tretment, the rte of wilting ws slightly lower 7 dys fter flooding thn fter 4 dys (Figure 3.2). However, the differences were not significnt when 72-dy-old plnts were compred with 93-dy-old plnts in the sme flooding regime (Figures 3.2 nd 3.4). 33

40 The effect of plnt ge on tomto sudden deth 4 dys fter flooding dys Permnent wilt (%) dys 93 dys b b 20 b 0 0 hr 24 hrs 48 hrs 72 hrs Flood durtion 7 dys fter flooding dys 72 dys Permnent wilt (%) dys b b 20 0 b 0 hr 24 hrs 48 hrs 72 hrs Flood durtion Figure 3.2: Effect of plnt ge (51, 72 nd 93 dys) nd durtion of flooding (0, 24, 48, 72 hrs) on incidence of permnent wilt cused by Pythium phnidermtum on tomto 4 nd 7 dys fter flooding in field experiment between June nd September, 2002 t AVRDC, Shnhu, Tiwn. Different letters in group of columns indicte sttisticlly significnt differences between two tretments ccording to Duncn s multiple rnge test (P 0.05). n=48. 34

41 The effect of plnt ge on tomto sudden deth The results clerly show tht tomto sudden deth cused by P. phnidermtum incresed in response to flooding under hot conditions. Flooding for 24 hrs cused less dmge thn flood regimes of 48 nd 72 hrs, regrdless of plnt ge. The level of wilting recorded fter 24 hrs flooding on dy 4 ws significntly (P 0.05) lower compred with tretments of 48 nd 72 hrs flooding (Figure 3.3). There were no significnt differences between the flood regimes of 48 nd 72 hrs for ny of the plnt ge tretments. Wilting exceeded 90% in both flood regimes 4 dys fter floodwter ws removed. Seven dys fter flooding, 80% of the older plnts wilted fter 72 hrs flooding. The level of wilting here ws not significntly different to the 48 hrs tretment. 35

42 The effect of plnt ge on tomto sudden deth 4 dys fter flooding hr 24 hrs 48 hrs 72 hrs 100 Permnent wilt (%) b b b 20 b 0 51 dys 72 dys 93 dys Plnt ge t flood 7 dys fter flooding hr 24 hrs 48 hrs 72 hrs 100 Permnent wilt (%) b b b 20 b 0 51 dys 72 dys 93 dys Plnt ge t flood Figure 3.3: Effect flood durtion (0, 24, 48, 72 hrs) nd plnt ge (51, 72 nd 93 dys) on incidence of permnent wilt cused by Pythium phnidermtum in tomto t 4 nd 7 dys fter floodwter removl in field experiment between June nd September, 2002 t AVRDC, Shnhu, Tiwn. Different letters in group of columns indicte sttisticlly significnt differences between two tretments ccording to Duncn s multiple rnge test (P 0.05). n=48. 36

43 The effect of plnt ge on tomto sudden deth A B Figure 3.4: Wilted tomtoes 3 dys fter flooding (A) nd ded plnts t 42 dys fter flooding in tretment of 72 nd 93 dy-old plnts (B) in field experiment from June to August 2002 t AVRDC, Shnhu, Tiwn. 37

44 The effect of plnt ge on tomto sudden deth Pythium phnidermtum recovery from the soil Fluctutions in the popultions of P. phnidermtum in the soil were found to be correlted to soil moisture nd soil temperture. About 0.51 CFU of P. phnidermtum were isolted per grm of soil before trnsplnting compred to up to 1 CFU/g of soil nd 2 CFU/g 30 nd 60 dys fter trnsplnting, respectively. There were no differences in the number of P. phnidermtum CFUs recovered from the soil between tretments of different flood regimes in August 2002 (Figure 3.5). The number of P. phnidermtum CFU isolted 7 dys fter flooding ws similr in the non-flooded control compred to the 72 hrs flood tretment. During the pre-experiment phse from June to August, 1-2 CFU/ g soil ws detected (Figure 3.5). The highest ir nd soil temperture ws reched during My-July (Figure 3.6). 3 Non flood 24hr 48hr 72hr 2.5 Averge CFU/g soil Trnsplnt Flood Drin dys 60 dys 63 dys 65 dys 70 dys June July August Figure 3.5: Number of CFU/g soil of Pythium phnidermtum in the soil before flooding nd 7 dys fter flooding under different flood conditions in field experiment between June nd August 2002 t AVRDC, Shnhu, Tiwn (Results not sttisticlly nlyzed). 38

45 The effect of plnt ge on tomto sudden deth Tm Ts 30 Temperture ( C) Dec-01 Jn-02 Feb-02 Mr-02 Apr-02 My-02 Jun-02 Jul-02 Aug-02 Sep-02 Oct-02 Figure 3.6: Dily men ir temperture (Tm), men soil temperture (Ts) in the experimentl field between June nd September 2002 t AVRDC, Shnhu, Tiwn. 3.4 Discussion The min gol of this study ws to determine how plnt ge ffects susceptibly to P. phnidermtum following flooding t high soil tempertures. The susceptibility of plnts to soilborne diseses is known to decrese with the ge of the plnt (Yrwood 1959; Populer 1978; Seem 1988). Mximum susceptibility of host plnts to pthogens hs been shown to coincide with the period of mximum growth of the plnt (Populer 1978). However, in this study, the highest percentge of surviving plnts ws observed in the tretment with young plnts. Here, the level of survivl ws significntly higher thn in the tretment with older plnts. This conflicts with previous results. The susceptibility of plnts to Pythium spp. in the nursery or erly seedling stge differs gretly from tht of older plnts grown under field conditions. Mellno et l. (1970) tested the susceptibility of Antirrhinum mjus seedlings to P. ultimum nd found tht the fungus penetrted the elongtion nd mturtion regions of the root tips of Antirrhinum mjus, but rrely the mture root portions nd never the meristem or the differentition region just behind it. Seedlings inoculted fter 15 dys or erlier were killed, nd growth of the pthogen ws 39

46 The effect of plnt ge on tomto sudden deth rpid in the root, stem nd leves. On seedlings tht were inoculted fter 20 dys, the fungus esily colonized tertiry nd quternry roots, but ws unble to colonized the mture portions of secondry nd primry roots, nd the plnts survived. The reson for decresing severity of wilt with plnt ge t the time of infection is due to restricted fungl growth in the plnt vessels rther thn to decresed infectibility of roots (Nyvll et l. 1976). In the present study, ll plnts in the field experiment t the time of flooding were over 50 dys old nd therefore beyond the susceptible nursery or seedling stge. The results obtined re due to the fct tht often-older tomto plnts re more susceptible to Pythium spp. thn younger ones (Jenkins et l. 1983). Another explntion could be tht biotic nd biotic environmentl fctors ffect susceptibility through chnges in the host prior to infection or in the host-pthogen interction fter infection hs occurred (Levitt 1972; Schoeneweiss 1975; Burdon 1987). Furthermore, under field conditions where complex interctions between soil microflor nd micro mcrofun occur, both within nd round the plnts, sudden chnges in plnt-microbe densities nd interctions due to flooding my result in incresed ttck by specific soilborne pthogens fvoured by high moisture (Duniwy 1983). The root system lso plys n importnt role in the susceptibility of host plnts to soil pthogens. Dmge to the root system due to excess wter s result of physiologicl nd chemicl processes re often ssocited with the infection process (Mtheron et l. 1985; Duniwy 1983; Schffer et l. 1992). For exmple, oxygen deficiency is induced by flooding nd my inhibit wter nd nutrient uptke, this in turn my result in chlorosis, wilting nd subsequent predisposition to ttck (Plti 1981; Kwse 1981). In the present study, the entire root system of the tomto plnts ws dmged by P. phnidermtum 7 dys fter flooding. This ws indicted by encysted zoospores occurring inside ll of the injured roots. Dmge lso my be due to direct influences on P. phnidermtum by flooding, such s promotion of zoospore relese nd medition of the infection process (Duniwy 1983). The ppernce of new dventitious roots reduced the level of permnent wilt in the 51-dy-old plnts, which tended to recover (Figure 3.6). However, plnts 72 nd 93 dys old t flooding did not recover fter dringe. This my be due to the fct tht younger plnts were better ble to recover from flooding by producing new roots to supply 40

47 The effect of plnt ge on tomto sudden deth their comprtively smller shoots with wter nd nutrients. Older plnts hd lredy invested energy in shoots now too lrge to be supplied with wter nd nutrients by the dmged root system. The result ws permnent wilting. Flooding s n environmentl fctor is of mjor importnce concerning chnges in susceptibility to root disese (Burdon 1987). It my increse disese severity by predisposing plnts to infection through dverse effects on host physiology (Fredrich et l. 1989; Schffer et l. 1992). Depletion of soil oxygen which restricts tissue growth cn lso enhnce disese regenertion from necrotic roots (Stolzy et l. 1984; Schffer et l. 1992). In the present study, tomto plnts flooded for 24 hrs were not ffected, even though tomto is considered one of the vegetbles most sensitive to flooding (Kuo et l. 1982). This my be due to the fct tht the tomto plnt is cpcity to trnsport sufficient O 2 from the shoot to sustin the root respirtory system during shorter periods of flooding (McNmr et l. 1989). The growth of the roots nd stem of tomto plnts is inhibited when the plnt is flooded for 48 or 72 hrs (Kent et l. 1981; Kuo et l. 1982; McNmr et l. 1989). Flooding for this durtion cuses collpse nd deth of the plnt. Another fctor gretly contributing to sudden deth is the fct tht the flooding increses disese severity by direct influence on the pthogens. Flooding promotes the relese of zoospores nd medites the infection processes by species of Phytophthor nd Pythium (Duniwy 1979; 1983). Under the multiple side effects cused by flooding listed bove, the survivl bility of tomto plnts, even when dventitious roots develop, is gretly limited. Therefore, even though 51-dy-old plnts survived, they were very smll nd probbly poor yielding. Fluctutions in the popultion density of P. phnidermtum in the soil were relted to soil temperture nd soil moisture. The number of P. phnidermtum recovered from the soil ws lower in the smple recovered 30 dys fter trnsplnting thn in the one recovered fter 60 dys. Even higher densities were recovered in the soil t the end of the cropping seson (August-October) when the field ws fllowed. This my be due to P. phnidermtum moving into the roots of the host plnts, reducing the density of the fungus in the soil (Zhng et l. 1990). Chnges in the popultion density of this fungus were drmtic. At times, lrge numbers of propgules were detected in the soil but t other times could not be detected t ll. The popultion of P. phnidermtum ws highest in 41

48 The effect of plnt ge on tomto sudden deth October, when soil tempertures were low, nd disese infested plnts were not present. In contrst, lower popultion densities of the fungus were found in the soil during June-July, when high numbers of wilted nd ded plnts were observed. This vrition could be due to the fct tht P. phnidermtum is typicl plnt pthogen of wrm regions (Vn der Plts-Niterink 1981) nd reproduces to higher levels in hot plnt tissue thn in soil. The response of tomto plnts of different ges to tomto sudden deth due to P. phnidermtum under flooding t high temperture my be useful for culturl mngement prctices. Timing the plnting of the crop cn gretly influence host susceptibility (English et l. 1994). Plnting dtes should thus be selected to void exposing susceptible tomto plnts to the disese during times when sesonl environmentl influences such s high soil tempertures nd flooding increse the disese ttck potentil. Predictive models using long-term wether dt could help in selecting plnting dtes less prone to erly flooding. Future reserch should be conducted in psteurized soil to ssess the response of differently ged tomto plnts to P. phnidermtum. More informtion is needed to understnd the complex microbil mechnisms involved in incresing the susceptibility of older plnts to P. phnidermtum following flooding t high tempertures under field conditions. 42

49 The effect of plnt ge on tomto sudden deth Figure 3.6: Root system of plnts 42 dys fter 48 hrs flooding in field experiment between June nd September : Root of the 51-dy-old plnt, non-flood control 2: Root of the 51-dy-old plnt, flooded for 48 hrs 3: Root of the 72-dy-old plnt, flooded for 48 hrs 4: Root of the 93-dy-old plnt, flooded for 48 hrs 43

50 Evlution of biocontrol gents to control tomto sudden deth 4 EVALUATION OF BIOLOGICAL CONTROL AGENTS FOR MANAGEMENT OF TOMATO SUDDEN DEATH CAUSED BY PYTHIUM APHANIDERMATUM FOLLOWING FLOODING UNDER HIGH TEMPERATURE CONDITIONS 4.1 Introduction Biologicl control is promising strtegy for mnging soilborne nd other disese in wide rnge of crops (Bker 1987; Cook 1993). Biocontrol through beneficil microbes introduced into the environment round crop plnt to enhnce the presence nd/or beneficil effects of these orgnisms cn contribute significntly to plnt helth. This method is less disruptive to ecosystems thn the use of chemicl pesticides (Cook et l. 1983). Severl beneficil orgnisms, which prsitize or re ntgonistic to number of soilborne fungi, hve been detected. Well known beneficil fungi tht hve been used for the control of Pythium phnidermtum include Trichoderm hrzinum, Trichoderm virens, nd Trichoderm hmtum (Hdr et l. 1979; Hrmn et l. 1980; Sivn et l. 1984; Lo et l. 1997). Previous studies hve shown tht T. hrzinum reduced the incidence of dmping-off disese on ben due to P. phnidermtum, Rhizoctoni solni nd other pthogens such s Sclerotium rolfsi (Eld et l. 1980; Sivn et l. 1984; Lo et l. 1997). Despite the success of Trichoderm spp. in the control of soilborne pthogens cusing pre-emergence dmping-off, these beneficil fungi hve not been used to control sudden deth disese due to Pythium phnidermtum, which occurs t lter stge in plnt development following flooding t high temperture. The objective of the experiment described in this chpter ws to evlute isoltes of Trichoderm hrzinum, Trichoderm viren, nd Streptomyces srceticus with respect to biologicl control of tomto sudden deth due to Pythium phnidermtum following flooding t high tempertures under greenhouse micro-plot nd field conditions. 44

51 Evlution of biocontrol gents to control tomto sudden deth 4.2 Mterils nd methods Preprtions Preprtion of Pythium phnidermtum inocultion P. phnidermtum strin number 4 ws cultured on V8 gr in Petri dishes for 3 dys t 28 C before being inoculted onto rice seed for solid stte fermenttion. Ech 400 ml beker contining 150 ml of rice grin nd 75 ml distilled wter ws utoclved twince before being used s the finl growth substrte. Two blocks of gr of 3-dy-old culture of P. phnidermtum were plced into the rice grin in ech beker nd then incubted in n illuminted chmber t 28 C for 10 dys. One beker of rice grin ws incorported into the upper 10 cm of soil of ech micro-plot continer 10 dys fter trnsplnting. Tomto plnt The tomto line CL D, which ws determined by AVRDC to be het tolernt nd virus resistnt, ws used in both greenhouse nd field tests. The seedlings were plnted in pet moss substrte for 30 dys in the greenhouse nd then trnsplnted into the micro-plot continer or into the field plots for experimenttion Inoculum preprtion nd inocultion techniques Two isoltes ech of Trichoderm hrzinum nd Trichoderm virens from the Tiwn Agriculturl Reserch Institute (TARI) nd one strin of Trichoderm hrzinum isolted from n AVRDC field were tested. An isolte of Streptomyces srceticus, which hd been introduced into the mrket s commercil product by Ntionl Chunghsing University (Tiwn), ws lso tested. The isoltes tested were: Trichoderm hrzinum: Th-Gl-6 nd Th-R1-6 Trichoderm hrzinum: AVRDC (Th-3) Trichoderm viren: Tv-Y3-7 nd Tv-R4-2 Streptomyces srceticus 45

52 Evlution of biocontrol gents to control tomto sudden deth Seed inocultion Trichoderm isoltes were grown on PDA medium for conidi development. They were incubted for 5 dys t 28 C, except Th-G1-6, which ws incubted for 8 dys. The conidi, fter being removed from the medium by dding 20 ml distilled sterilized wter with dditionl scrping by glss rod, were used for seed nd substrte inocultion. The conidil suspension, djusted to 5 x 10 9 conidi/ml tp wter, ws supplemented with 1 ml of CMC 1% (Crboxymethylcellulose) s sticker nd spreder. Tomto seeds were surfce sterilized with 0.1% hydrochloride before being treted with the biocontrol gents. Three milliliter of suspension of ech test orgnism ws used to cot 10 g of tomto seeds. The inoculted seeds were then plced inside sterile trnsfer hood to dry prior to sowing (Lo et l. 1997). All experiments were crried out with untreted surfce-sterilized seeds s controls. Potting soil inocultion Five hundred ml of substrte mixture of pet moss nd whet brn (1:1=v/v) t 40 % (w/w) moisture ws put into beker (1000 ml) before being utoclved for 1 hr t C (Sivn et l. 1984). The substrte mixture ws inoculted seprtely with 10 ml conidil suspensions contining 2 x 10 4 of ech of the five isoltes of Trichoderm nd incubted in n illuminted chmber for 14 dys t 28 C. The culture medi of the five Trichoderm isoltes were then mixed with pet moss (5g/kg) nd filled into 5 cm dimeter pots (Sivn et l. 1984). Seeds treted with the Trichoderm isoltes were then sown into these pots, which contined pet moss pre-inoculted with the sme isolte. These seedlings were used for the field nd greenhouse tub experiments. Field soil nd greenhouse micro-plot inocultion The culture medi (5g/plnt) from ech of the five Trichoderm isoltes s described bove were plced in the plnting hole 2 dys before the seedlings were trnsplnted into the field. The plnts seed-treted with the isoltes of Trichoderm nd were growing in inoculted potting soil were plced into similr holes t trnsplnting. 46

53 Evlution of biocontrol gents to control tomto sudden deth The commercil Streptomyces product ws diluted in wter (1/1000) nd wtered dily onto the potted plnts in pure pet moss fter the seedlings hd emerged, nd tretment continued with 1/500 dilution until 40 dys fter trnsplnting Field experiment to evlute the effect of Trichoderm spp. nd Streptomyces srceticus for controlling tomto sudden deth due to Pythium phnidermtum Generl methods The field experiment to test the efficcy of Trichoderm species for controlling tomto sudden deth ws estblished in field in AVRDC, Tiwn. The experiment ws conducted from July 15 th to October 15 th 2001, in pddy rice field (cly soil), where dmping-off disese of tomto due to P. phnidermtum hd been prevlent in previous yers. The field ws left fllow for one month for drying nd the rice strw ws burned prior to ploughing. Twenty-eight plots of 5 m 2 (1m x 5 m) were estblished to host seven tretments of ech isolte of Trichoderm nd the control. The tretments were set up in rndom mnner long the length of the field nd repeted in four blocks long the width. The tretments were rndomly distributed to the plots nd plots lbeled one week before trnsplnting. Empty bnds were left between plots to seprte these from ech other nd to void cross contmintion between tretments. The plots were covered by plstic to control weeds nd limit dmge by insect pests. A totl of 210 kg (N:P:K=4:4:4) orgnic fertilizer ws brodcst over the field prior to the estblishment of the plots. In ddition, totl of 65 kg (N:P:K:Mg=15:15:15:4) of chemicl fertilizer ws used s top dressing. Five soil smples were tken rndomly from the upper 20 cm long the digonl of the field with 5-cm-dimeter cylinder before trnsplnting. In order to mesure ph nd quntify the popultion of P. phnidermtum (see 2.3.2), the soil smples were mixed nd plced on pper towel t room temperture for two dys until dry. Twenty grms of this dried soil ws dissolved in 0.01M clcium chloride (CCl 2 ) on rotry shker for one hour to check the ph. The soil ph ws mesured s n verge of three sub-smples nd found to be

54 Evlution of biocontrol gents to control tomto sudden deth Isoltion of Pythium phnidermtum from the soil The soil smples were tken rndomly from the field prior to trnsplnting, s mentioned bove. They were tken 30 nd 60 dys fter trnsplnting nd gin 7 dys fter floodwter removl. The soil smples were plced on pper towels nd llowed to dry t room temperture for 2 dys. Four dilutions of the soil of 1/5, 1/10, 1/20, nd 1/40 g soil/ml 0.3 % wter gr were used to quntify the popultion of P. phnidermtum (Burr et l. 1973). Ech dilution ws mixed on Vortex stirrer for t lest 15 min nd 1 ml liquot ws dispensed evenly cross the surfce of the selective medium developed for P. phnidermtum developed by Burr et l. (1973) (see 2.1.3). The pltes were incubted t 35 C for time intervls rnging from 24 hrs to 72 hrs, fter which the soil ws crefully wshed from the gr surfce nd the Pythium colonies were recorded. Experimentl design The im of this experiment ws to test the efficcy of 3 isoltes of T. hrzinum, 2 isoltes of T. virens nd 1 isolte of Streptomyces to control tomto sudden deth in the field. The following tretments, ech with four replictes re listed in Tble 1. Twenty 30-dy-old seedlings were trnsplnted into ech plot in 2 rows of 10 plnts ech nd ttched to bmboo sticks to void dmge from strong wind nd hevy rinfll. The experiment ws flooded 2 months fter trnsplnting, when the soil temperture reched C. The wter level ws mintined 10 cm bove the upper soil surfce for 48 hrs nd then drined by cnl system round the blocks. 48

55 Evlution of biocontrol gents to control tomto sudden deth Tble 4.1: Tretments to ssess the efficcy of Trichoderm spp. nd Streptomyces srceticus with respect to biologicl control of tomto sudden deth due to Pythium phnidermtum Isolte Tretment Th-G1-6 - treted seed - treted seedling potting medium in the nursery - incorported into field soil t time of trnsplnting Th-R1-6 - treted seed - treted seedling potting medium in the nursery - incorported into field soil t time of trnsplnting Th-3 - treted seed - treted seedling potting medium in the nursery - incorported into field soil t time of trnsplnting Tv-Y3-7 - treted seed - treted seedling potting medium in the nursery - incorported into field soil t time of trnsplnting Tv-R4-2 - treted seed - treted seedling potting medium in the nursery - incorported into field soil t time of trnsplnting Streptomyces srceticus Control - treted seedling potting medium in the nursery - drench tretment of plnts until 40 dys fter trnsplnting in the field Non-treted 49

56 Evlution of biocontrol gents to control tomto sudden deth Greenhouse experiments to evlute the effect of Trichoderm spp. nd Streptomyces srceticus on control of tomto sudden deth due to Pythium phnidermtum under flooding different durtions Generl methods Plstic continers (tub) of size 31 cm x 51 cm x 40 cm (height x width x length) were used s experimentl units. The tubs were sterilized with solution of 1% hydrochloride before being filled with soil. Five kg of sterilized pebbles (1-2 cm dimeter) were plced in the bottom of the tub nd covered by plstic netting. A U-shded plstic siphon ws ttched to the tub so tht wter could be siphoned out from under the net (see Figure 1, Chpter 2). Fifty-five kg of hevy cly soil, which hd been tken from AVRDC fields, ws stempsteurized, nd then filled into ech tub. Soil ph ws evluted prior to filling the soil into the tubs nd found to be 6.5. About 82.5 g of orgnic commercilly vilble grnulr fertilizer (N:P:K=4:4:4) nd 20 g chemicl fertilizer (N:P:K:Mg=15:15:15:4) ws incorported into the soil of ech tub before trnsplnting. In ddition, 3.5 g of chemicl fertilizer (N:P:K=20:20:20) were dded to ech tub weekly until the plnts were hrvested. Flooding nd temperture control The plstic tubs were flooded 60 dys fter trnsplnting. The wter level ws mintined t 2-4 cm bove the soil surfce for 48 or 72 hrs, respectively, nd then removed rpidly through the drinpipe system. The temperture ws mintined t C for the entire period of ech experiment. Root weight Tomto roots were removed from the soil by creful wshing with tp wter 7 dys fter floodwter ws removed. The wshed roots were plced on pper towels t room temperture for 30 min to remove excess wter nd then dried in n oven t temperture of 50 C for 48 hrs followed by determintion of root dry weight. 50

57 Evlution of biocontrol gents to control tomto sudden deth Experiments design Two different greenhouse experiments were conducted in the summer sesons of 2001 nd In the first experiment, ll 6 isoltes of the biocontrol gents were used. Flooding lsted done for 48 hrs. The second experiment ws conducted with 3 biocontrol gents nd extended flooding time of 72 hrs. The soil ws inoculted with isoltes of T. hrzinum (Th-R1-6) nd T. virens (Tv-Y3-7) t the time of trnsplnting (see 2.2.3) nd inoculted with P. phnidermtum 10 dys fter trnsplnting (see 2.1.1). Ten treted tomto plnts per tretment were trnsplnted in two rows in ech micro-plot. Greenhouse experiment to evlute the effect of Trichoderm spp. nd Streptomyces srceticus on control of tomto sudden deth due to Pythium phnidermtum fter 48 hrs of flooding The experiment ws conducted between August nd November Twenty-four plstic continers were used for 3 replictes of the 5 tretments of ech isolte of Trichoderm spp., Streptomyces srceticus nd two controls. Tomtoes were trnsplnted on August 5 th, P. phnidermtum ws grown on rice grin nd incorported into the soil 10 dys fter trnsplnting. The continers were flooded for 48 hrs, 60 dys fter trnsplnting. Tble 4.2: Tretments to ssess the effect of Trichoderm spp. nd Streptomyces srceticus on biologicl control tomto sudden deth due to Pythium phnidermtum. Tretments Pythium phnidermtum inocultion Flood period T. hrzinum Th-G dys fter trnsplnting 48 hrs T. hrzinum Th-R dys fter trnsplnting 48 hrs T. hrzinum Th-3 10 dys fter trnsplnting 48 hrs T. virens Tv-Y dys fter trnsplnting 48 hrs T. virens Tv-R dys fter trnsplnting 48 hrs Streptomyces srceticus 10 dys fter trnsplnting 48 hrs Pythium phnidermtum lone (P) 10 dys fter trnsplnting 48 hrs Non-inoculte control (ck) None 48 hrs 51

58 Evlution of biocontrol gents to control tomto sudden deth Greenhouse experiment to evlute the effect of Trichoderm spp. nd Streptomyces srceticus to control tomto sudden deth due to Pythium phnidermtum fter 72 hrs of flooding The experiment ws conducted between My nd August Twenty-four tubs for 8 tretments ech with 3 replictes were used. Tomto plnts were plnted into the tubs on My 1 st Biocontrol gents were inoculted both in the soil infested with P. phnidermtum nd in the non-infested soil. The tubs were flooded for 72 hrs 60 dys fter trnsplnting. Tble 4.3: Tretments to ssess the effect of Trichoderm spp. nd Streptomyces srceticus on biologicl control of tomto sudden deth due to Pythium phnidermtum Tretment Pythium phnidermtum inocultion Flood period T. hrzinum Th-R1-6 None 72 hrs T. virens Tv-Y3-7 None 72 hrs Streptomyces srceticus None 72 hrs T. hrzinum Th-R dys fter trnsplnting 72 hrs T. virens Tv-Y dys fter trnsplnting 72 hrs Streptomyces srceticus 10 dys fter trnsplnting 72 hrs Pythium phnidermtum lone (P) 10 dys fter trnsplnting 72 hrs Control (ck) None 72 hrs Dt collection nd sttisticl nlysis The number of wilted plnts in ll tretments were recorded t 2, 4 nd 7 dys fter the floodwter ws removed. Seven dys fter flooding, the plnts were hrvested nd root dry weights mesured nd recorded. The dt on incidence of permnent wilt of tomto were nlyzed with the SAS (SAS Institute Inc. 1989) progrm, using the generl liner model procedure, including nlysis of vrince, Duncn s multiple rnge test, lest significnt difference (LSD) nd /or test orthogonl contrst. 52

59 Evlution of biocontrol gents to control tomto sudden deth 4.3 Results Field experiment to evlute Trichoderm spp. nd Streptomyces srceticus control of tomto sudden deth due to Pythium phnidermtum fter 48 hrs of flooding Pythium phnidermtum recovery from the soil: The detection of 1.6 CFU/g soil of Pythium phnidermtum prior to trnsplnting confirmed tht the field soil ws nturlly infested by the fungus. The popultion density of P. phnidermtum chnged during the cropping seson nd chieved the highest density month fter tomto trnsplnting. There ws no difference in density directly before or fter flooding (Figure 1) CFU/g soil Trnsplnt Flood July August Sep. Oct. Figure 4.1: Popultion density of Pythium phnidermtum in soil in field experiment between July nd October 2001 t AVRDC, Shnhu, Tiwn. The soil ws smpled before, 30 nd 60 dys fter trnsplnting nd 7 dys fter flooding. Permnent wilt: Four dys fter flooding, the percentge of wilted tomto plnts in the soil treted with T. virens Tv-Y3-7 ws significntly (P 0.05) lower thn the control tretment. There were no significnt differences between percentge of wilted plnts observed in tretments with the ntgonists tested (Figure 2). Seven dys fter flooding, Tv-Y3-7 did not hve significnt effect on tomto sudden deth. Similr results were observed in the tretments with Streptomyces nd the other isoltes of T. hrzinum: Th-G1-6, Th-R1-6, Th-3; nd T. virens: Tv-R4-2 (Figure 2). 53

60 Evlution of biocontrol gents to control tomto sudden deth Permnent wilt (%) dys fter flooding b b b b b b Th-G1-6 Th-R1-6 Th-3 Tv-Y3-7 Tv-R4-2 Strep. ck Permnent wilt (%) dys fter flooding n.s * Th-G1-6 Th-R1-6 Th-3 Tv-Y3-7 Tv-R4-2 Strep. ck Figure 4.2: Effect of biocontrol gents on permnent wilt cused by Pythium phnidermtum on tomto plnts 4 nd 7 dys fter 48 hrs flooding in field experiment between July nd October, 2001 t AVRDC, Shnhu, Tiwn. Th- G1-6, Th-R1-6 nd Th-3 = isoltes of Trichoderm hrzinum; Tv-Y3-7 nd Tv-R4-2 = isoltes of Trichoderm virens; Strep. = Streptomyces srceticus; ck = control. Tretment indicted by the sme letters re not significntly different ccording to Duncn s multiple rnge test (P 0.05), n.s* = not significnt. n=28. 54

61 Evlution of biocontrol gents to control tomto sudden deth Greenhouse experiment to evlute the effect of Trichoderm spp. nd Streptomyces srceticus on the control of tomto sudden deth due to Pythium phnidermtum fter 48 hrs of flooding Permnent wilt: Most tomto plnts hd wilted fter 48 hrs flooding t 30 C soil temperture in the greenhouse test. The percentge of wilted tomtoes grown in the soil inoculted with the biocontrol gents ws significntly higher compred to tht of plnts grown in untreted soil (Figure 3). Seven dys fter flooding, the percentge of wilted tomtoes observed in the tretment with P. phnidermtum lone ws not different from the tretments of the T. hrzinum isoltes, T. virens isoltes or Streptomyces srceticus. T. hrzinum (Th-R1-6) reduced sudden deth symptoms by only 10% compred to the P. phnidermtum control nd, therefore, ws not effective in reducing P. phnidermtum on tomto to economic levels. The percentge of wilted plnts observed in ll the tretments with biocontrol gents ws significntly higher thn the untreted control (Figure 4.3). 55

62 Evlution of biocontrol gents to control tomto sudden deth 4 dys fter flooding Permnent wilt(%) b b b b 20 c 0 Th-G1-6 Th-R1-6 Th-3 Tv-Y3-7 Tv-R4-2 Strep. P ck Permnent wilt(%) dys fter flooding b Th-G1-6 Th-R1-6 Th-3 Tv-Y3-7 Tv-R4-2 Strep. P ck Figure 4.3: Effect of different biocontrol gents on permnent wilt of tomto plnt cused by Pythium phnidermtum 4 nd 7 dys fter 48 hrs flooding in greenhouse experiment conducted between August nd November 2001 t AVRDC, Shnhu, Tiwn. Th-G1-6, Th-R1-6 nd Th-3 = isoltes of Trichoderm hrzinum; Tv-Y3-7 nd Tv-R4-2 = isoltes of Trichoderm virens; Strep. = Streptomyces srceticus; P = Pythium phnidermtum inoculted; ck = noninoculted control. Tretments indicted by the sme letters re not significntly different ccording to Duncn s multiple rnge test (P 0.05). n=24. Root dry weight: Root dry weight of tomtoes grown in soil infested with P. phnidermtum lone ws significntly lower compred to the non-inoculted (Figure 4). The root weight of plnts treted with Th-G1-6 nd Th-R1-6 in the soil infested by P. phnidermtum were significntly higher. The isoltes Tv-Y3-7, Tv-R4-2, nd Th-3 cused non-significnt increse in root weight compred to the P. phnidermtum 56

63 Evlution of biocontrol gents to control tomto sudden deth inoculted control (Figure 4). Generlly, none of the Trichoderm isoltes ffected root weight compred to the untreted control. There ws lso no significnt difference in root dry weight between the Streptomyces srceticus tretment nd the P. phnidermtum inoculted control (Figure 4). However, root weight ws significntly lower in this tretment compred to tht with Trichoderm isoltes. Root dry weights (g) b b b c bc Th-G1-6 Th-R1-6 Th-3 Tv-Y3-7 Tv-R4-2 Strept P ck Figure 4.4: Effect of biocontrol gents nd Pythium phnidermtum on root dry weight of tomto in greenhouse experiment on August-November, 2001 in AVRDC, Shnhu, Tiwn. Th-G1-6, Th-R1-6 nd Th-3 = isoltes of Trichoderm hrzinum; Tv-Y3-7 nd Tv-R4-2 = isoltes of Trichoderm virens; Strep. = Streptomyces srceticus; P = Pythium phnidermtum; ck = non-inoculted control. Different letters show sttisticlly significnt differences mong tretments ccording to Duncn s multiple rnge test (P 0.05). n= Greenhouse experiment to evlute the effect of Trichoderm spp. nd Streptomyces srceticus on the control of tomto sudden deth due to Pythium phnidermtum following 72 hrs of flooding Permnent wilt: There were no significnt differences in the percentge of wilted plnts mong tretments with biocontrol gents in P. phnidermtum infested soil compred to the non-infested control 4 or 7 dys fter flooding (Figure 5). Permnent wilt of tomtoes grown in the soil treted with Th-R1-6, Tv-Y3-7 or Streptomyces ws slightly lower thn in the untreted control but not significntly different. High levels of wilting were lso observed in the Pythium-free soil (Figure 5). 57

64 Evlution of biocontrol gents to control tomto sudden deth 60 4 dys fter flooding 50 n.s.* Permnent wilt (%) Th-R1-6 Tv-Y3-7 Strep P Th-R1-6 Tv-Y3-7 Strep ck Inoculte Non- inoculte 60 7 dys fter flooding 50 n.s.* Permnent wilt (%) Th-R1-6 Tv-Y3-7 Strept P Th-R1-6 Tv-Y3-7 Strept ck Inoculted Non-inoculte Figure 5: Effect of biocontrol gents on permnent wilt of tomto plnts cused by Pythium phnidermtum 4 nd 7 dys fter 72 hrs flooding in greenhouse experiment conducted between My nd August 2002 t AVRDC, Shnhu, Tiwn. Th- R1-6 = Trichoderm hrzinum; Tv-Y3-7 = Trichoderm virens; Strep. = Streptomyces srceticus; P = Pythium phnidermtum inoculted control; ck = non-inoculted control. n.s.* = not significnt, n=24. 58

65 Evlution of biocontrol gents to control tomto sudden deth Root dry weight: The biocontrol gents slightly incresed root dry weight in P. phnidermtum infested soils compred to the control. Th-R1-6 incresed root dry weight in soil both infested nd not infested with P. phnidermtum, but not significntly. Inocultion with P. phnidermtum did not significntly ffect root dry weight compred to the untreted control (Figure 6). 0,9 Roots dry weight (g) 0,8 0,7 0,6 0,5 0,4 0,3 0,2 n.s.* 0,1 0 Th-R1-6 Tv-Y3-7 Strep P Th-R1-6 TV-Y3.7 Strept ck Inoculte Non-inoculted Figure 6: Effect of biocontrol gents nd inocultion with Pythium phnidermtum on root dry weight of tomto in greenhouse experiment conducted between My nd August 2002 t AVRDC, Shnhu, Tiwn. Th-R1-6 = Trichoderm hrzinum; Tv-Y3-7 = Trichoderm virens; Strep. = Streptomyces srceticus; P = Pythium phnidermtum inoculted control; ck = non-inoculted control. n.s.* = not significnt, n=24. 59

66 Evlution of biocontrol gents to control tomto sudden deth 4.4 Discussion Field experiment to evlute Trichoderm spp. nd Streptomyces srceticus for the control of tomto sudden deth due to Pythium phnidermtum Field experiments give true picture of the biocontrol potentil of fungl ntgonists s influenced by the biotic nd biotic conditions existing in the ecosystem (Merrin et l. 1990; Pusey 1990). It is lso known tht results of biocontrol experiments re often reproducible in in-vitro or in greenhouse experiments, but not under field conditions (Mhffee et l. 1993). In the present study, field experiment ws conducted in soil nturlly infested with P. phnidermtum. The soil nlysis demonstrted tht P. phnidermtum ws ubiquitous nd the popultion density fluctuted during the cropping seson. The highest popultion density ws detected one month fter trnsplnting of tomtoes nd ws ffected by soil temperture (30-32 C) nd high soil moisture. Of the biocontrol gents tested here, only T. virens isolte Tv-Y3-7 showed some effect on tomto sudden deth due to P. phnidermtum under high temperture following flooding conditions fter 4 dys. None of the tested isoltes protected tomto effectively ginst P. phnidermtum infection fter flooding. This my be becuse Trichoderm spp. mycoprsitism does not occur until 24 hrs fter contct with the pthogen nd then t low frequency (Chet et l. 1981). Thus, mycoprsitism ws not initited soon enough to protect ginst P. phnidermtum under flooding t high tempertures, conditions tht fvor the relese of P. phnidermtum zoospores nd host ttck (Vn der Plts-Niterink 1981). So fr, effectiveness of Trichoderm spp. for control of P. phnidermtum ws shown in non-flooding or cool conditions. Lo et l. (1997) found tht T. hrzinum tretment significntly reduced disese incidence up to 83 percent cused by P. phnidermtum in turfgrss in non-flooding conditions. Sivn et l. (1984) reported tht n isolte of T. hrzinum reduced the incidence of dmping-off disese due to P. phnidermtum up to 87% on tomto nd 90% on pepper seedlings in non-flooded conditions. In this study, there ws no evidence to support reports of Streptomyces srceticus biocontrol of tomto sudden deth due to P. phnidermtum. The reson for the poor results with Streptomyces on tomto sudden deth is unknown. It my be due to the direct effect of flooding on the popultion density of Streptomyces in the soil, which leds to 60

67 Evlution of biocontrol gents to control tomto sudden deth reduced ntibiotic ctivity. Bolton (1980) found tht Streptomyces spp. hd little ffected on P. phnidermtum in hydroponic culture when the concentrtion dropped below 10 7 propgules/ml. The poor results obtined in the present study my be due to the influence of biotic fctors, such s soil moisture nd soil temperture, on the biocontrol gents. Soil microorgnisms my lso ply direct role in limiting the potentil level of biocontrol over long periods of time during the growing seson. In the present study, tretment with the ntgonists ws followed by flooding the soil 2 months fter trnsplnting for 48 or 72 hrs. It is well known tht P. phnidermtum survives s oospores in soil nd on plnt debris nd s mycelium in infected roots where the biocontrol gents my not be ctive. Wet or flooded soil fvors the production nd relese of zoospores nd infection of roots or stems of the host. In ddition, high soil temperture meets the virulence requirements of the pthogen for development of the disese (Hine et l. 1969; Vn der Plts-Niterink 1981). The high density of infective zoospores of the pthogen in the soil relesed during flooding my hve led to unsucessful biocontrol becuse of the mssive relese of infective zoospores. It is lso possible tht the gents used re not suitble for effective control of P. phnidermtum ttcking tomto s the plnts grow out of the seedling stge. New isoltes tht come from fields where flooding does not led to sudden deth of the tomto need to be tested. Greenhouse experiments to evlute the effect of Trichoderm spp. nd Streptomyces srceticus on the control of tomto sudden deth due to Pythium phnidermtum fter 48 nd 72 hrs of flooding Wilting: In the greenhouse experiment conducted in the hot seson between August nd November 2001, three isoltes of T. hrzinum, two isoltes of T. virens, nd Streptomyces srceticus were tested. There ws no evidence of biocontrol on tomto sudden deth due to P. phnidermtum. The greenhouse test ws repeted between My nd August 2002 with 72 hrs of flooding to evluted the ntgonistic potentil of Trichoderm nd Streptomyces towrd P. phnidermtum nd their effects on tomto growth. The biocontrol gents hd no 61

68 Evlution of biocontrol gents to control tomto sudden deth significnt effect on disese incidence compred to the infested control. There were lso no significnt differences in dry root weight mong tretments or in the tretments compred to the untreted control. However, T. hrzinum nd T. virens hd slightly positive effect on plnt growth compred to the tretment Streptomyces srceticus lone. The results seem to be in conflict with those of previous studies (Sivn et l. 1984). They found tht Trichoderm spp. reduced the disese incidence of dmping-off due to severl pthogens including P. phnidermtum nd Rhizoctoni solni. Plnts treted with T. hrzinum reduced the level of dmping-off by 87% in tomto seedlings in soils rtificilly infested with P. phnidermtum. Bolton (1980) found tht Streptomyces delyed infection of P. phnidermtum, which cused root rot nd sudden wilt of poinsetti, in soilless culture. The poor results observed in the present study could be due to the filure of the Trichoderm spp. mycoprsitism of P. phnidermtum. Under nonflooded conditions, Trichoderm spp. cn prsitize the hyphe of Pythium spp. (Lifshitz et l. 1986). Eld et l. (1982) lso reported tht the bility of isoltes of T. hrzinum to control dmping-off cused by P. phnidermtum ws correlted to the level of hydrolytic enzyme production in the soil. Hydrolytic enzymes were the sole crbon source when the biologicl gent ws grown on fungl cell wll components (Eld et l. 1982). This enzyme my be negtively ffected by flooding or even dispper during flooding periods, leding to ineffective biocontrol of P. phnidermtum. The poor results recorded from both greenhouse experiments regrdless of durtion of flooding my be due to the effect of flooding on host plnt susceptibility to P. phnidermtum. It is well known tht the tomto plnt is the most flood-sensitive vegetble (Kuo et l. 1982). Flooding of the tomto plnts for 48 hrs or longer dmges the root system leding to brekdown of the plnt s disese resistnce (Plti 1981). In ddition, flooding soil fvors the production nd relese of zoospores nd increses infection of roots or stems of the host. Wilting my result from injury to the root system cused by flooding nd the ggressive pthogen. The filure of Trichoderm nd Streptomyces to control P. phnidermtum fter flooding my be relted to their popultion density in the environment round the host 62

69 Evlution of biocontrol gents to control tomto sudden deth plnt. Bolton (1980) found tht in soil-less culture Streptomyces spp. nd T. viride hd little impct on P. phnidermtum when the concentrtion ws below 10 7 propgules/ml. Biomss: In the experiment in 2001, the plnt treted with the T. hrzinum isoltes Th-G1-6 nd Th-R1-6 hd significntly lrger dry root weight thn infested controls. The dry root weights of plnts treted with T. virens in infested soil were slightly higher but were not significntly different from the infested control. In the My-August 2002 experiment, the biocontrol gents cused limited growth promotion to tomto plnt. However, there were not significnt differences between the root dry weights of tomto plnt grown in the soil treted with Trichoderm spp. or Streptomyces compred to the control. The results re in greement with reserch on the use of T. hrzinum nd T. virens for control of other plnt pthogenic diseses. Windhm et l. (1986) found tht tomto root nd shoot dry weights grown in soil treted with Trichoderm spp. incresed % nd % respectively, over control. There could be severl resons for this. Conversely, they found tht tretments with Streptomyces resulted in root weights lower thn the infested control. The dry shoot weights of tomto plnts grown in the P. phnidermtum infested soil treted with Streptomyces spp. ws lso significntly lower thn in the untreted control nd tht of the tretments with Trichoderm spp. The increse in root dry weight my be due to two mechnisms tht explin incresed growth response induced by soil microflor (Windhm et l. 1986). The enhnced growth of plnts induced by Pseudomonds is predominntly due to biologicl control of minor pthogens (Slt 1978; Suslow et l. 1979; Kloepper et l. 1981). The other reson could be tht microbil gent produces growth-regulting metbolites tht ffect plnt growth (Lindsey et l. 1967; Windhm et l. 1986). Despite intensive reserch on biologicl control of soil born diseses, the effect of flooding on biologicl control of soilborne pthogens is not well understood. Future reserch to increse the efficcy of fungl ntgonists should concentrte on the effects of flooding, soil temperture nd root coloniztion on the biocontrol gents before, during nd fter flooding. Becuse of the nture of this interction, biocontrol with soil-bsed 63

70 Evlution of biocontrol gents to control tomto sudden deth ntgonists my not be effective. The isoltes used here, for exmple, were developed for dmping-off control nd not for long seson effects. There is lso need to look t fungl nd bcteril endophytes for control. They cn be pplied to the seedlings nd live inside the root tissue; they would thus not be ffected by flooding (Sikor 1992, Hllmn 2001). 64

71 Effect of soil mendments to control sudden deth 5 EFFECT OF SOIL AMENDMENTS TO COTNROL TOAMTO SUDDEN DEATH DUE TO PYTHIUM APHANIDERMATUM FOLLOWING FLOODING IN HOT SEASON 5.l Introduction Pythium phnidermtum (Edson) Fitzp. is serious soilborne pthogen in high temperture regions ffecting mny high vlue crops worldwide. It cuses dmping-off, root rot nd fruit rot in tomto, cucumber, pepper, lettuce, spinch, nd ben in both fieldplnted nd hydroponic cultures (Sumner et l. 1976; Jenkins et l. 1983; Btes et l. 1984; Pulitz et l. 1992; McCrter 1997). P. phnidermtum hs been detected during tomto production in hot summer in the tropics fter hevy rinfll, nd it is considered the min cusl gent of the occurrence of stem rot, wilting, nd ded plnts (Bolton 1980; McCrter 1997). Mny control methods hve been suggested nd tested to solve the disese problem. The use of soil mendments, resistnt vrieties, ppliction of fungicides, s well s other methods hve been tested (Plti 1981; Btes et l. 1984; Plti et l. 1997). New products bsed on orgnic mendments hve been developed in Tiwn in ttempts to control P. phnidermtum. Previous reports indicte tht dding orgnic mtter to the soil is effective in reducing the incidence of certin soilborne pthogens nd in promoting helthy growth of the host plnts (Hung et l. 1993). Lin et l. (1988) found tht the soil mendments clled S-H mixture reduced the incidence of cucumber dmping-off due to P. phnidermtum. Other soil mendments composed of mushroom compost (SFMC) hve been shown to reduce dmping-off disese cused by P. phnidermtum on cucumber (Wng et l. 2000). Complex mixtures (FNB-5A) hve shown control of dmping-off on cbbge seedlings due to Rhizoctoni solni (Shiu et l. 1999). Orgnic mtter hs been used in generl to improve soil fertility, increse plnt growth nd stimulte soil microbil densities, thereby improving plnt helth (Plti 1981). The gol of the investigtion reported here ws to test the efficcy of soil orgnic mendments nd Trichoderm spp. enhnced mendments ginst tomto sudden deth cused by P. phnidermtum under flooded nd high soil temperture conditions in the field nd in greenhouse micro-plot experiments. 65

72 Effect of soil mendments to control sudden deth 5.2 Mterils nd methods Preprtions Preprtion of Pythium phnidermtum inocultion Pythium phnidermtum strin number 4 ws cultured on V8 gr in Petri dishes for 3 dys t 28 C before incorportion into rice seed for solid stte fermenttion. Ech 400 ml beker contining 150 ml of rice grin nd 75 ml distilled wter ws utoclved twice prior to being used s the finl growth substrte. Two blocks of gr of 3-dy-old culture of P. phnidermtum were inserted into the rice grin in ech beker nd then incubted under illuminted light t 28 C for 10 dys. One beker of rice grin ws incorported into the upper 10 cm of ech tub 10 dys fter trnsplnting. Culture medi The medi used in these studies were utoclved for 20 minutes t temperture of 121 C. All the ntibiotics were dded to the respective medium fter utoclving nd fter cooling to 50 C. The composition of the medi is given below: Burr nd Stnghellini medium (Burr et l. 1973) 17g Corn Mel Agr (Difco) 100mg Pimricin 200mg Streptomycin sulfte 150mg Rose Bengl 5mg Benomyl 1000ml distilled H 2 O Potto Dextrose Broth Agr (PDA) 37g PDA (Difco) 1000ml distilled H 2 O 66

73 Effect of soil mendments to control sudden deth Vegetble broth (V-8) 200ml V-8 vegetble juice (lbi) 3g CCO 3 15g Agr 1000ml distilled H 2 O Tomto plnt The tomto vriety CL D, which ws determined by AVRDC to be het tolernt nd virus resistnt, ws used in both greenhouse nd field tests. The seedlings were grown in pet moss substrte for 30 dys in the greenhouse nd then trnsplnted into tubs or into field plots. Soil mendment preprtion nd inocultion with Trichoderm Trichoderm ureoviride, which ws isolted by scientist from the Plnt Pthology Deprtment of Chung Hsing University, Tiwn, ws dded to the composts to enhnce control. The fungus ws grown on PDA medium nd incubted for 5 dys t 28 C for conidi development. The conidi, fter being removed from the medium by dding 20 ml distilled sterilized wter with dditionl scrping by glss rod, were used to inoculte the SFMC nd FBN-5A composts. Soil mendments developed by scientists in the Plnt Pthology Deprtment of Chung Hsing University, Tiwn, were lso tested. The orgnic mendments used were: 1. Spent forest mushroom compost (SFMC) 2. Fish mel, blood mel, NH 4 N0 3, lime, SFMC, llyl lcohol (FBN-5A) 3. Bgsse, rice husks, oyster shell, ure, KN0 3, CSO 4, minerl sh (S-H Mixture) Seedling substrte tretment: The soil mendment SFMC ws mixed with pet moss No.4 (Bs Vn Burren) nd wter t the rte of 2:1:1 (v/v). FBN-5A ws mixed with pet moss t the rte of 2:1000 (v/v) (Shiu et l. 1999). Both mixtures were inoculted with conidi of Trichoderm ureoviride (2 x 10 5 conidi/g medium) 7 dys prior to being filled 67

74 Effect of soil mendments to control sudden deth into trys (Recommendtion from Plnt Pthology Deprtment of Chung Hsing University, Tiwn). Tomto seeds were surfce-sterilized with sodium hydrochloride (1%) for 3 minutes nd then sown into the trys. One month lter, they were trnsplnted into the greenhouse tubs or into field plots for experimenttion. The S-H mixture without Trichoderm ureoviride ws pplied t trnsplnting. The dose, 1kg/m 2 (Lin et l. 1988), ws incorported into the upper 10 cm of the soil in the greenhouse tubs or field experimentl plots by hnd, 7 dys prior to trnsplnting. Tomto plnts tht hd grown in stndrd pet moss potting medium were trnsplnted to these plots Field experiment on effect of soil mendments on the control of tomto sudden deth due to Pythium phnidermtum Generl method The experiment ws conducted from July 15 th to October 15 th 2001 in pddy rice field t AVRDC, Shnhu, Tiwn, with cly soil, where dmping-off cused by P. phnidermtum hd been prevlent in previous yers. Rice strw ws burned nd the field left to fllow for one month for drying prior to ploughing. Sixteen plots of 5 m 2 (1m x 5m) were estblished nd lbeled to host the tretments. Four plots for ech of the tretments were set up in rndom mnner long the length of the field nd repeted in four blocks long the width. Empty bnds were left between plots to seprte them from ech other nd to void cross contmintion between tretments. The plots were covered with plstic to control weeds nd limit insect dmge. A totl of 210 kg (N:P:K=4:4:4) orgnic fertilizer ws brodcsted over the field prior to the estblishment of the plots. In ddition, totl of 65 kg (N:P:K:Mg=15:15:15:4) of chemicl fertilizer ws used s top dressing. The tretments were rndomly distributed within the plots. Five soil smples were tken rndomly from the upper 20 cm long the digonl of the field with 5-cm-dimeter cylinder before trnsplnting, in order to mesure soil ph nd to quntify the popultion of P. phnidermtum (see 2.2.2). The soil smples were mixed nd plced on pper towel t the room temperture for two dys until they becme dry. Twenty grm of this dried soil ws dissolved in 0.01M clcium chloride (CCl 2 ) on 68

75 Effect of soil mendments to control sudden deth rotry shker for one hour to check the ph. The soil ph ws mesured s n verge of three sub-smples nd found to be Twenty 30-dy-old seedlings were trnsplnted into ech plot in 2 rows of 10 plnts ech nd ttched to bmboo sticks to void dmge from strong wind nd hevy rin. The experiment ws flooded 2 months fter trnsplnting when the soil temperture reched C. The wter level ws mintined 10 cm bove the upper soil surfce for 48 hrs nd then drined by cnl system between nd round the 4 blocks. Isoltion of Pythium phnidermtum from the soil The soil smples were tken rndomly from the field prior to trnsplnting, s mentioned bove. In ddition, the soil ws smpled 30 nd 60 dys fter trnsplnting s well s 7 dys fter floodwter removl. The soil smples were plced on pper towel nd llowed to dry t room temperture for 2 dys. Four dilutions of the soil of 1/5, 1/10, 1/20, nd 1/40 g soil per ml 0.3 % wter gr were used to quntify the popultion of P. phnidermtum (Burr et l. 1973). Ech dilution ws mixed on Vortex stirrer for t lest 15 min nd 1 ml liquot ws dispensed evenly cross the surfce of the selective medium for P. phnidermtum developed by Burr et l. (1973). The pltes were incubted t 35 C for time intervls rnging from 24 to 72 hrs fter which the soil ws crefully wshed from the gr surfce nd the Pythium colonies recorded. 69

76 Effect of soil mendments to control sudden deth Experimentl design The following tretments with four replictes were used: Tble 5.1: Tretments to study the effect of soil orgnic mendments with nd without Trichoderm on tomto sudden deth due to Pythium phnidermtum under flooding nd hot conditions Amendment Tretment description SFMC + Trichoderm - Treted seedling potting medium in the nursery - Incorported into the field soil t the time of trnsplnting FBN-5A + Trichoderm S-H mixture treted seedling potting medium in the nursery - Treted seedling potting medium in the nursery - Incorported into the field soil t the time of trnsplnting Control - Stndrd seedling substrte without Trichoderm - No field incorported Greenhouse experiment on effect of soil mendments enriched with nd without Trichoderm spp. on tomto sudden deth due to Pythium phnidermtum Generl methods The experiment ws conducted from August to November 2001 in greenhouse micro-plot test t AVRDC, Shnhu, Tiwn. Plstic tubs 31 cm x 51 cm x 40 cm (height x width x length) were used s experimentl units. The tubs were sterilized with 1% solution of hydrochloride before being filled with soil. Five kg of sterilized pebbles (1-2 cm dimeter) were plced in the bottom of the tub nd covered by plstic netting. An inverted U-shded siphon ws ttched to the tub so tht wter could be siphoned out from under the net (see Figure 2.1, Chpter 2). Fifty-five kg of hevy cly soil, which hd been tken from AVRDC fields t Shnhu, Tiwn, ws stem-psteurized, nd then filled into ech tub. Soil ph ws evluted nd found to be

77 Effect of soil mendments to control sudden deth About 82.5 g of orgnic commercilly vilble grnulr fertilizer (N:P:K=4:4:4) nd 20 g chemicl fertilizer (N:P:K:Mg=15:15:15:4) ws incorported into the soil of ech tub before trnsplnting. In ddition, 3.5 g of chemicl fertilizer (N:P:K=20:20:20) ws dded weekly to ech tub until the plnt hrvest. Root weight Tomto roots were removed from the soil by crefully wshing with tp wter 7 dys fter floodwter dringe. The wshed roots were plced on pper towels t room temperture for 30 min to remove excess wter nd then dried in n oven t temperture of 50 C for 48 hrs fter which root dry weight ws determined. Experimentl design The experiment included 3 soil mendments nd 3 control tretments with Mefenoxm + P. phnidermtum, P. phnidermtum lone nd untreted control. Mefenoxm ws incorported t 2 g/m 2 t the time of trnsplnting nd gin 57 dys fter plnting. The tretments with three replictions re described in Tble 5.2. Tble 5.2: The tretments to test the effect of soil mendments enhnce with nd without Trichoderm spp. on tomto sudden deth due to P. phnidermtum under flooding nd hot conditions. Tretments Flood period Pythium phnidermtum inocultion SFMC + Trichoderm 48 hrs 10 dys fter trnsplnting FBN-5A + Trichoderm 48 hrs 10 dys fter trnsplnting S-H mixture 48 hrs 10 dys fter trnsplnting Mefenoxm 48 hrs 10 dys fter trnsplnting Pythium phnidermtum lone (P) 48 hrs 10 dys fter trnsplnting Non-inoculte control (ck) 48 hrs None 71

78 Effect of soil mendments to control sudden deth Ten tomto seedlings, pre-treted with soil mendment, were trnsplnted into the tub in the greenhouse. Tubs were inoculted with P. phnidermtum 10 dys fter trnsplnting. Sixty dys fter trnsplnting, the tubs were flooded. The wter level ws mintined t 2-4 cm bove the soil surfce for 48 hrs nd then removed rpidly through the siphon tube. The temperture in the greenhouse ws mintined t C for the durtion of the experiment Dt collection nd sttisticl nlysis The number of wilted plnts from ll tretments ws recorded t 2, 4 nd 7 dys fter floodwter removl. In ddition, 7 dys fter flooding, plnts were hrvested nd root dry weight determined. The dt on incidence of permnent wilt of tomto were nlyzed with the SAS (SAS Institute Inc. 1989) progrm, using the generl liner model procedure, including nlysis of vrince, Duncn s multiple rnge test, lest significnt difference (LSD) nd /or orthogonl contrsts. 5.3 Results Field experiment on effect of soil mendment nd Trichoderm enhncement on the biologicl control of tomto sudden deth due to Pythium phnidermtum Pythium phnidermtum recovery from the soil: The detection of 1.6 CFU/g soil of P. phnidermtum prior to trnsplnting confirmed tht the field soil ws nturlly infested by the fungus. The popultion density of P. phnidermtum chnged during the cropping seson nd reched the highest density one month fter tomto trnsplnting (Figure 5.1). 72

79 Effect of soil mendments to control sudden deth CFU/g soil Trnsplnt Flood July August Sep. Oct. Figure 5.1: Density of Pythium phnidermtum (CFU/g soil) in soil in field experiment between July nd October 2001 t AVRDC, Shnhu, Tiwn. The soil ws smpled before trnsplnting, 30 nd 60 dys fter trnsplnting s well s 7 dys fter flooding. Permnent wilt: The soil mendments SFMC, FBN-5A or S-H Mixture hd no effect on tomto sudden deth due to P. phnidermtum. Permnent wilt of tomto plnts grown in the treted soil ws not significntly different compred to the control (Figure 5.2). Furthermore, orgnic mtter enhnced with Trichoderm ureoviride did not increse the level of control. 73

80 Effect of soil mendments to control sudden deth 4 dys fter flooding 7 dys fter flooding 100 n.s.* 90 n.s.* Permnent wilt (%) SFMC FBN-5A S-H ck Tretments Permnent wilt (%) SFMC FBN-5A S-H ck Tretments Figure 5.2: Effect of the orgnic soil mendments SFMC, FBN-5A, nd S-H Mixture on permnent wilt of tomto cused by Pythium phnidermtum 4 nd 7 dys fter 48 hrs flooding in field experiment between July nd October ck = non-treted control. n.s.* = not significnt, n= Greenhouse experiment on effect of soil mendments nd Mefenoxm on tomto sudden deth due to Pythium phnidermtum Permnent wilt: None of the soil mendments reduced the rte of wilting determined 4 nd 7 dys fter flooding. Enhncement of the soil with Trichoderm ureoviride did not hve ny dditionl effect, lthough slight increse ws seen in FBN-5A over S-H nd the control. The percentge of wilted tomtoes grown in treted, infested soil ws not significntly different in comprison to the untreted, infested control (Figure 3). In ddition, Mefenoxm, which ws used s stndrd chemicl control tretment, did not reduce sudden deth in infested soil (Figure 5.3). 74

81 Effect of soil mendments to control sudden deth dys fter flooding Permnent wilt (%) b b b 20 0 SFMC FBN S-H Mefen P ck Tretments b Permnent wilt (%) dys fter flooding SFMC FBN-5A S-H Mefen P ck Tretments b Figure 5.3: Effect of the orgnic mendments, SFMC, FNB-5A nd S-H Mixture, on permnent wilt of tomto cused by Pythium phnidermtum 4 nd 7 dys fter 48 hrs flooding in greenhouse experiment between August nd November 2001 t AVRDC, Shnhu, Tiwn. Mefen = Mefenoxm; P = Pythium phnidermtum inoculted; ck = non-inoculted control. Different letters show sttisticlly significnt differences mong tretments ccording to Duncn s multiple rnge test (P 0.05). n=18. Root dry weight: Root dry weight of tomto plnts grown in the soil treted with soil mendments or Mefenoxm ws slightly but not significntly incresed compred to the 75

82 Effect of soil mendments to control sudden deth untreted control. However, significnt increses in root dry weight due to SFMC, FBN- 5A, nd Mefenoxm tretments were observed when compred to P. phnidermtum inoculted control (Figure 5.4). In the tretment with the S-H Mixture, the root dry weight of tomto plnts ws not higher thn tht in the infested control Root dry weight (g) b b SFMC FBN-5A S-H Mefen P ck Figure 5.4: Effects of the soil mendments, SFMC, FNB-5A, S-H Mixture, nd Pythium phnidermtum on root dry weight of tomto in greenhouse experiment between August nd November 2001 t AVRDC, Shnhu, Tiwn. Mefen = Mefenoxm; P = Pythium phnidermtum inoculted; ck = non-inoculted control. Different letters show sttisticlly significnt differences mong tretments ccording to Duncn s multiple rnge test (P 0.05). n= Discussion Field experiment on effect of soil mendments for biologicl control of tomto sudden deth due to Pythium phnidermtum The gol of the present study ws to ssess whether different soil mendments re cpble of controlling or limiting tomto sudden deth under high temperture conditions following flooding. Soil mendments re known to modify the soil environment, shifting the ecologicl blnce to the disdvntge of crop pthogens (Hung et l. 1993). The mechnisms by which soil mendments ffect plnt pthogens cn be simple or complex depending on the mendments nd the pthogens. In some cses, control is chieved by 76

83 Effect of soil mendments to control sudden deth direct killing of the pthogen propgules, in other cses, disese suppression is the result of combintion of multiple fctors, including direct toxicity to the pthogen or indirect effects due to enhnced microbil ctivity or improved vigor of plnts (Hung et l. 1993). Sun et l. (1985) reported tht the S-H Mixture soil mendment hs indirect effects on P. phnidermtum by incresing the popultion of ntgonistic fungi from 2 to 25 times in wtermelon field soil. In ddition, it gretly reduced the incidence of dmping-off disese on cucumber due to P. phnidermtum in the field (Lin et l. 1988). However, in the present study, S-H Mixture did not significntly reduce disese incidence of tomto sudden deth cused by P. phnidermtum. Similrly, the soil mendments SMFC nd FBN-5A plus Trichoderm ureoviride showed no significnt impct on P. phnidermtum. The lck of effectiveness of these soil mendments to control tomto sudden deth could be relted to effects of flooding combined with the soil mendments on the microbil environment in the soil. Under non-flooded conditions, the S-H mixture hs been shown to improve plnt vigor ginst pthogens (Hung et l. 1993) by producing mmoni in the soil, which inhibits myceli growth nd zoospore germintion of P. phnidermtum (Lin et l. 1988). SFMC nd FBN-5A lso enrich nd enhnce the ctivity of ntgonistic microbes in the soil ginst soilborne pthogens (Shiu et l. 1999; Wng et l. 2000). However, their effectiveness my hve been reduced or lessened due to the nerobic conditions resulting from the 48 hrs flooding. The interction between flooding nd soil mendments nd their influence on soilborne pthogens cusing sudden deth by quick relese of infective zoospores needs to be studied in future reserch. The exponentil increse in number of infective propgules nd the wekening of plnts defence mechnisms seems to limit the mendments to control this disese. Greenhouse experiment on effects of soil mendments nd Mefenoxm on tomto sudden deth due to Pythium phnidermtum The soil mendments SFMC, FBN-5A, nd S-H mixture did not significntly inhibit P. phnidermtum on tomto. In fct, higher levels of disese incidence following tretment of SFMC, FBN-5A, nd S-H Mixture compred to the P. phnidermtum infested control were observed. The results re in contrst to other studies relted to the use of soil 77

84 Effect of soil mendments to control sudden deth mendments nd control of dmping-off diseses due to soilborne pthogens including P. phnidermtum nd Fusrium spp. Shiu et l. (1999) nd Liu et l. (2000) studied the effect of FBN-5A lone on Fusrium oxysporum nd Rhizoctoni solni. They concluded tht treting potting unit with FBN-5A t rte of 1000:1 (Pet moss: FBN-5A = v:v) significntly reduced disese incidence on rdish nd cbbge seedlings. Wng et l. (2000) reported tht SFMC lone ws more effective in suppressing dmping-off of cucumber seedlings cused by P. phnidermtum compred to other potting substrtes like BVB pet moss. SFMC, however, ws ineffective in the control of dmping-off of cbbge cused by Rhizoctoni solni (Hung et l. 2000). Lin et l. (1988) found tht under greenhouse conditions, S-H t 2% or 5% (S-H Mixture: soil=w:w) completely inhibited dmping-off disese on cucumber by P. phnidermtum. It is importnt to hve in mind tht control in the bove-mentioned studies occurred t the seedling stge nd not on older plnts growing under field conditions tht re ffected in the present experiments by sudden deth. The poor results chieved fter flooding could be relted to the negtive effect of flooding on the predeposition of the plnts to disese (Gold et l. 1984). In the present study, the rte of wilting of tomto plnts ws not significntly reduced by the mendments when compred to the infested control when plnts were flooded 2 months fter trnsplnting. The lck of control is probbly due to severl fctors relted to interctions between soil moisture, environment nd the popultion dynmics of the pthogen in the soil. Soil mendments probbly only provide short- to medium-term protection ginst P. phnidermtum under erobic or norml growing conditions. Lin et l. (1990) reported tht S-H mixture is not pnce. It does not work well for deep-rooted crops or for plnts with long growing period, becuse the period of disese suppression lsts bout 40 dys. Under the long-term growing conditions in the present tests, the positive short-term effects of orgnic mendments were not ble to cope with disese pressure. Lrge numbers of wilted nd ded tomtoes were observed in ll tretments fter flooding, wheres only few or no ded plnts were found in the period before flooding. Lck of effective control by soil mendments could lso be due to the level of Pythium inoculum used, which my hve been too high for effective control. Similr tests 78

85 Effect of soil mendments to control sudden deth t lower inoculum densities my hve been more effective, since orgnic mendments re often not effective t high disese pressure. Plnts flooded t soil temperture C re more susceptible to P. phnidermtum (Hine et l. 1969; Vn Der Plts-Niterink 1981; Yu et l. 1989). Flooding probbly triggers mssive relese of zoospores in the field soil. This mssive relese coupled with root tissue injury my cncel ny positive effects orgnic mtter my hve on P. phnidermtum infection erlier in the seson. Therefore, despite the fct tht the soil mendments SFMC, FBN-5A, nd S-H Mixture hve been reported to be highly successful s mens of protecting vegetble seedlings, they were ineffective towrd tomto sudden deth due to P. phnidermtum following flooding t high tempertures condition in the present tests. The Trichoderm ureoviride dded with the soil mendment SFMC nd FBN-5A did not enhnce control of tomto sudden deth due to P. phnidermtum. This could be due to the fct tht in previous studies, T. ureoviride ws only effective on Pythium seedling disese. The fungus my lso be unble to grow in cly soil over long periods of time to levels effective in control. Hoitink et l. (1996) reported tht excessively stbilized orgnic mendments such s highly decomposed pets in the soil do not support the ctivity of biologicl control gents. Biologicl control gents inoculted into these types of orgnic mtter decline in popultion density nd re not ble to induce sustined biologicl control of Pythium root rot (Hoitink et l. 1996). This my hve occurred in the present test over the long growing seson till sudden deth took plce. There is further need to study the control of P. phnidermtum t high soil tempertures nd under short-term nerobic conditions. Future reserch should concentrte on other types of soil mendments in order to enhnce control of tomto sudden deth. The ppliction rte nd ppliction time of orgnic mendments to soil cn lso be modified in the future to chieve better control of P. phnidermtum sudden deth which occurs in mture plnts nd not in seedlings. 79

86 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth 6 RESPONSE OF TOMATO (LYCOPERSICON ESCULENTUM MILL.); EGGPLANT (SOLANUM MELONGENA L.) AND TOMATO GRAFTED ONTO EGGPLANT ROOTSTOCK TO TOMATO SUDDEN DEATH DUE TO PYTHIUM APHANIDERMATUM (EDSON) FITZP. 6.1 Introduction In tomto production in lowlnd res of tropicl nd subtropicl countries during the hot, wet seson, yield is generlly quite low compred to tht obtined in the cool seson or from highlnd production res. High tempertures reduce fruit set, hevy rinfll directly dmges eril plnt prts, soil wter logging reduces plnt vigor, nd short periods of soil flooding often result in wilt nd sudden deth of tomto plnts. Pythium phnidermtum is responsible for the deth of seedlings s well s of mtured tomto nd cucumber plnts tht were irrigted 3-4 times dily t soil tempertures bove 30 C (Stnghellini et l. 1975). The pthogen hs very fst growth rte nd is prolific producer of zoospores. It is often cited s the cusl gent of root diseses nd wilting for number of vegetble crops grown in hydroponic systems (Pulitz et l. 1992). P. phnidermtum is well known pthogen of tomto tht hs been shown to cuse dmping-off, stem rot, root disese, nd fruit rot (Bolton 1980; McCrter 1997). Grfting techniques hve been widely pplied in mny crops such s tomto, eggplnt, cucumber, nd wtermelon (Crlsson 1963; Tubbs 1974; Zijlstr et l. 1987; Chdh 1988; Cohen et l. 2002). Grfting hs been used effectively s strtegy to control number of soilborne diseses nd nemtodes ffecting vegetbles including those ttcking cucumber, tomto, eggplnt nd melons (Peregrine et l. 1982; Chdh 1988; Mtsuzoe et l. 1993; Edelstein et l. 1999). In Jpn, 93% of the wtermelons, 72% of the cucumbers, 50% of the eggplnts, 30% of the tomtoes, nd 30% of ll types of melon hve been improved using grfted plnts (Od 1993). Grfting of the tomto (Lycopersicon esculentum Mill.) onto tomto rootstocks, which re resistnt to soilborne diseses such s bcteril wilt or root-knot nemtodes, provides protection ginst the disese, but it does not give ny protection ginst sudden deth cused by soil flooding (AVRDC 1999). When the tomto scion is grfted onto eggplnt rootstocks (Solnum melongen L.), positive effects on tomto production ginst 80

87 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth bcteril wilt under high temperture summer conditions in tropicl res were demonstrted (Peregrine et l. 1982; Mtsuzoe et l. 1993). The strong eggplnt root system seemed to stimulte plnt growth t high ir nd soil tempertures (Abdelhfeez et l. 1975). In ddition, eggplnt roots hve shown high level of survivl under wter logged or flooding conditions (AVRDC 1999). The ims of the present study were to: 1. Test the bility of tomto plnts grfted onto eggplnt rootstocks to limit tomto sudden deth due to Pythium phnidermtum following flooding under high tempertures under field conditions. 2. Study the susceptibility of tomto, eggplnt nd tomto grfted onto eggplnt rootstocks to sudden deth due to Pythium phnidermtum under controlled greenhouse conditions. 6.2 Mterils nd methods Plnt mteril Tomto plnts of the line CL D, which ws determined by AVRDC to be het tolernt nd virus resistnt, were used in both non-grfted nd grfted tests in the greenhouse nd field. Eggplnts of the vriety EG203, which were reported by AVRDC to be flood resistnt, were lso used. The tomto nd eggplnt seeds were sown in pet-moss substrte nd kept in the greenhouse for dys, fter which they were trnsplnted into the experimentl plstic tub (see 6.2.3) or field (see 6.2.2). To produce grfted tomto, the scion of the tomto line CL D ws grfted onto rootstock of the eggplnt vriety line EG dys fter sowing (Figure 6.1). The grfted tomtoes were plced in growth chmber with tempertures rnging from C nd over 85% humidity. Five dys lter, the grfted plnts were moved to net house for 7 dys prior to being trnsplnted into the field or greenhouse tubs. 81

88 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth A B 82

89 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth C Figure 6.1: Tomto scion (A) grfted onto eggplnt rootstock (B nd C) in AVRDC, Shnhu, Tiwn (Source: Dr. Blck LL; AVRDC, Shnhu, Tiwn, 2002) Field experiment on response of tomto grfted onto eggplnt rootstock to tomto sudden deth due to Pythium phnidermtum Generl methods The field experiment ws conducted between July nd October 2001 in n AVRDC field t Shnhu, Tiwn, nturlly infested with P. phnidermtum. The experiment ws set up in the sme field s the experiment on soil mendments nd biocontrol gent (see Chpter 5). The field ws left fllow for one month to dry prior to ploughing. Rice strw ws burned in the field. Eight plots of 5 m 2 (1m x 5m) were estblished nd lbeled to host the tretments with grfted nd non-grfted tomtoes. The tretments were set up in rndom mnner long the length of ech block nd repeted in four blocks long the width of the field. The tretments were rndomly distributed to the blocks. Empty border rows were left between plots to seprte them from ech other nd void cross contmintion between tretments. The plots were covered by plstic to control weeds nd limit dmge by insect 83

90 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth pests. A totl of 210 kg (N:P:K=4:4:4) orgnic fertilizer nd 65 kg (N:P:K:Mg=15:15:15:4) chemicl fertilizer were brodcsted over the entire field prior to the estblishment of the plots. Five soil smples were tken rndomly from the upper 20 cm long the digonl of the field with 5 cm dimeter cylinder before trnsplnting. In order to mesure soil ph nd quntify the popultion of P. phnidermtum (see 2.2.2), the soil smples were mixed nd stored t room temperture for two dys until they becme dry. Twenty grms of this dried soil ws dissolved in 0.01M of Clcium chloride (CCl 2 ) on rotry shker for one hour to check the ph. The ph ws mesured s n verge of three sub-smples nd ws found to be Twenty plnts (30 dys old) of either tomto or tomto grfted onto eggplnt rootstocks were trnsplnted into ech plot nd tixed to bmboo sticks to void dmge from strong wind nd hevy rin. The experiment ws flooded for 48 hrs 2 months fter trnsplnting, when soil tempertures were between 30 nd 32 C. Isoltion of Pythium phnidermtum from the soil Soil smples were tken rndomly from the field prior to trnsplnting, s mentioned in section 2.2.1, 30 nd 60 dys fter trnsplnting s well s 7 dys fter floodwter removl. Four dilutions 1/5, 1/10, 1/20, nd 1/40 of soil (g soil per ml 0.3 % wter gr) were used to quntify the popultion of P. phnidermtum (Burr et l. 1973). Ech dilution ws mixed on Vortex stirrer for pproximtely 15 min nd 1 ml liquot of mixed soil nd 0.3% wter gr ws dispensed evenly cross the surfce of the P. phnidermtum selective medium developed by Burr et l. (1973). The pltes were incubted t 35 C from 24 to 72 hrs fter which the soil ws crefully wshed from the gr surfce nd the Pythium colonies were counted. 84

91 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth Greenhouse experiments on the response of tomto, eggplnt nd tomto grfted onto eggplnt rootstock to tomto sudden deth cused by Pythium phnidermtum Two experiments were conducted in the greenhouse to study the susceptibility of tomtoes grfted onto eggplnt rootstocks to P. phnidermtum. The experiments were conducted between August nd November 2001 nd gin between August nd November 2002 t the AVRDC experimentl site in Shnhu, Tiwn. Preprtion of fungus substrte nd ppliction P. phnidermtum strin number 4, isolted by the mycology unit t AVRDC, ws cultured on V8 gr for 3 dys before incorportion into rice seed for solid stte fermenttion. Ech 400 ml beker contining 150 ml of rice grin nd 75 ml distilled wter ws utoclved twice prior to being used s the finl growth substrte. Two blocks of gr of 3-dy-old culture of P. phnidermtum were plced into the rice grin in ech beker nd then incubted in n illuminted chmber t 28 C for 10 dys. One beker of rice grin ws incorported into the upper 10 cm of soil of ech tub 10 dys fter trnsplnting. Experimentl design Plstic continers (tub) of size 31 cm x 51 cm x 40 cm (height x width x length) were used s experimentl micro-plot units. The tubs were sterilized with solution of 1% hydrochloride before being filled. Five kg of sterilized pebbles (1-2 cm dimeter) were plced in the bottom of the tub nd covered by perforted plstic net. A U-shped plstic siphon tube ws then ttched for drining (Figure 6.2). Fifty-five kg of hevy cly soil tken from the AVRDC field t Shnhu, Tiwn, ws stem psteurized nd then filled into the tub. Soil ph ws evluted nd found to be 6.5. About 83 g of orgnic commercilly vilble grnulr fertilizer (N:P:K=4:4:4) nd 20 g chemicl fertilizer (N:P:K:Mg=15:15:15:4) were incorported into the soil of ech tub before trnsplnting. In ddition, 3.5 g inorgnic fertilizer (N:P:K=20:20:20) ws dded weekly to ech tub until the plnts were hrvested. 85

92 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth A B Figure 6.2: The experiment in the greenhouse in AVRDC, Shnhu, Tiwn: (A) Greenhouse experiment (B) Plstic tub with siphon tube (rrowed) for dringe Flooding nd temperture control The plnts were flooded 60 dys fter trnsplnting. The wter level ws mintined t 2-4 cm bove the soil surfce for 48 or 72 hrs nd then removed rpidly through the siphon tube. The temperture ws mintined t C for the entire durtion of ech experiment. Greenhouse experiment I: Response of non-grfted tomto nd tomto grfted onto eggplnt rootstock to Pythium phnidermtum infection fter 48 hrs of flooding The experiment ws conducted between August nd November 2001 to test the susceptibility of non-grfted tomto nd tomto grfted onto eggplnt rootstock to Pythium phnidermtum following flooding t high soil temperture. Ech tretment ws tested with three replictions (Tble 6.1). Tble 6.1: Tretments to test response of non-grfted tomto nd tomto grfted onto eggplnt rootstock to tomto sudden deth due to Pythium phnidermtum under flooding nd hot conditions Tretment P. phnidermtum inocultion Flood period Non-grfted tomto 10 dys fter trnsplnting 48 hrs Non-grfted tomto None 48 hrs Grfted tomto 10 dys fter trnsplnting 48 hrs 86

93 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth Greenhouse experiment II: Response of non-grfted tomto, tomto grfted onto eggplnt rootstock, nd eggplnt to Pythium phnidermtum fter 72 hrs of flooding Experimentl design The im of this experiment conducted between August nd November 2002 ws to study the response of tomto, tomto grfted onto eggplnt rootstock, nd eggplnt to P. phnidermtum fter 72 hrs flooding t high temperture. The tretments, ech with three replictions were tested (Tble 6.2). Tble 6.2: Tretments to test response of tomto, eggplnt, nd tomto grfted onto eggplnt rootstock to Pythium phnidermtum under flooding nd hot conditions Tretments P. phnidermtum inocultion Flood durtion Eggplnt, inoculted 10 dys fter trnsplnting 72 hrs Eggplnt, non-inoculted None 72 hrs Eggplnt, non-inoculted None None Tomto, inoculted 10 dys fter trnsplnting 72 hrs Tomto, non-inoculted None 72 hrs Tomto, non-inoculted None None Grfted, tomto inoculted 10 dys fter trnsplnting 72 hrs Grfted, tomto non-inoculted None 72 hrs Grfted, tomto non-inoculted None None Isoltion of Pythium phnidermtum from the roots The roots of tomto nd eggplnt collected from the tubs treted with P. phnidermtum 2 dys before nd 1 dy fter flooding were wshed in running tp wter nd cut into 1 cm lengths. Ten root pieces were plced on plte of Mircetich medium for fungl detection (Mircetich, 1971) nd then incubted t 28 C for 24 hrs. The colony-forming units (CFU) 87

94 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth of P. phnidermtum growing out of ech root section were recorded fter 24 hrs incubtion. Root weight Roots of tomto, eggplnt, nd grfted tomto plnts from tubs inoculted or not inoculted with P. phnidermtum were wshed with tp wter. The wshed root were plced on pper towels t room temperture for 30 min to remove excess wter nd then dried in n oven t 50 C for 48 hrs fter which the root dry weight ws determined Dt collection The number of wilted plnts in both field nd greenhouse micro-plot trils were recorded t 2, 4 nd 7 dys fter floodwter removl. Plnts in greenhouse experiment II were lso hrvested to mesure the root dry weight. The dt on incidence of permnent wilt of tomto, eggplnt, nd grfted tomto were nlyzed with the SAS (SAS Institute Inc., 1989) progrm, using the generl liner model procedure, including nlysis of vrince, Duncn s multiple rnge test, lest significnt difference (LSD) nd /or orthogonl contrsts. 6.3 Results Field experiment to ssess the response of tomto nd tomto grfted onto eggplnt rootstock to sudden deth Pythium phnidermtum Permnent wilting: No wilted plnts due to P. phnidermtum of neither tomto grfted onto eggplnt rootstock nor non-grfted were observed prior to flooding. Four dys fter 48 hrs flooding, over 90% of the non-grfted tomto s showed wilt symptoms. After 7 dys, 70% of the non-grfted tomtoes showed severe wilting. The wilting of non-grfted tomto plnts ws significntly higher compred to grfted tomto plnts during both observtion periods (Figure 6.3). Only 2% of the tomto plnts grfted onto eggplnt exhibited wilt symptoms in both periods. 88

95 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth 4 dys fter flooding 7 dys fter flooding Permnent wilt (%) b b Grfted Non-grfted Grfted Non-grfted Figure 6.3: Permnent wilt of tomto CL5915 nd tomto grfted onto eggplnt (E203) rootstock 4 nd 7 dys fter 48 hrs flooding in field experiment during July- October 2001 in AVRDC, Shnhu, Tiwn. Different letters show sttisticl differences between the tretments ccording Duncn s multiple rnge test (P 0.05), n=8. Pythium phnidermtum recovery from the soil: The popultion density of 1.6 CFU/g soil of P. phnidermtum mesured prior to trnsplnting confirmed tht the field soil ws nturlly infested by the fungus. The popultion density of P. phnidermtum fluctuted during the cropping seson nd reched the highest density one month fter trnsplnting (Figure 6.4). 89

96 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth CFU/g soil Trnsplnt Flood July August Sep. Oct. Figure 6.4: Popultion density (CFU/g soil) of Pythium phnidermtum in soil in field experiment between July nd October 2001 t AVRDC, Shnhu, Tiwn. Soil ws smpled before, 30 nd 60 dys fter trnsplnting nd 7 dys fter flooding Greenhouse experiment I: response of non-grfted tomto nd tomto grfted onto eggplnt rootstock to sudden deth tomto due to Pythium phnidermtum fter 48 hrs flooding A high number of non-grfted tomto plnts exhibited wilt symptoms in soil infested with P. phnidermtum compred to the non-infested control. The number of plnts infected with P. phnidermtum in the tretment with non-grfted tomtoes observed 4 dys fter flooding ws 65% nd incresed to 80% fter 7 dys (Figure 6.5). The grfted plnts grown in the soil infested with P. phnidermtum were s helthy s the non-grfted nd noninoculted controls, 4 nd 7 dys fter flooding. 90

97 Response of tomto, eggplnt & tomto grfted onto eggplnt rootstock to tomto sudden deth 4 dys fter flooding 7 dys fter flooding Permnent wilt (%) non-grft inoc b Grfted inoc b non-grft non-inoc non-grft inoc b Grfted inoc b non-grft non-inoc Figure 6.5: Permnent wilt cused by Pythium phnidermtum of non-grfted tomto nd tomto grfted onto eggplnt rootstocks 4 nd 7 dys fter 48 hrs flooding in greenhouse experiment between August nd November 2001 t AVRDC, Shnhu, Tiwn. Different letters show sttisticl difference mong tretments ccording to Duncn s multiple rnge test (P 0.05), n=9. Figure 6.6: Plnt wilt due to Pythium phnidermtum 7 dys fter 48 hrs flooding in greenhouse experiment conducted between August nd November 2001 t AVRDC, Shnhu, Tiwn. From left to right: tomto + Pythium phnidermtum; tomto + non-inoculted control; tomto grfted onto eggplnt rootstock + Pythium phnidermtum. 91