CANOPY AND LEAF GAS EXCHANGE ACCOMPANYING PYTHIUM

ROOT ROT OF LETTUCE AND CHRYSANTHEMUM

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

In partial ful filment of requirements

for the degree of

Master of Science

January, 200 1

Q Melanie Beth Johnstone, 200 1

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ABSTRACT

CAKOPY AND LEAF GAS EXCHANGE ACCOMPANYING PYTHIUMROOT ROT OF LETTUCE AND CHRYSANTHEMUM

Melanie Beth Johnstone University of Guelph, 2000

Advisors: Professor B. Grodzinski Professor J.C. Sutton

The first charactenzation of host carbon assimilation in response to Pythium infection

is described. Hydroponic lettuce (Lactuca sativa L. cv. Bella Green) inoculated with

Pjtfhitrw dissofoctm experienced 3 M O % growth inhibition, amibuteci to decreased lea f and

whole plant photosynthesis. Leaf gas exchange and transpiration was significantly reduced

as early as 7 days afler inoculation, several days in advance of visible foliar symproms. P.

aphanidermarirm was mildly pathogenic in hydroponic chrysanthemum (Chrysanthemum

nzori/oliurn L. cv. Fina), exhibiting reduced whole plant photosynthesis only late in the

gowth cycle. Altentions in gas exchange may be useful in nondestructive, objective

diagnosis of plant disease, a critical step in pathogen control in recirculating nutrient

solutions. W irradiation of nutrient solutions was studied as a potential rnethod of pathogen

inactivation; however, copper and zinc toxicity even with inacfivated lamps confounded

evaluations. It is premature to recommend W as a remediation technology for recirculating

nutrient solutions in commercial greenhouses.

My sincere thanks are extended to my supervison, Drs. Bernard Grodzinski and John

Sutton, for their support and guidance throughout this study. 1 would also like to

acknowledge DE. Michael Dixon and J. Chris Hall, members of my advisory committee, and

Drs. John Proctor and M. Jim Tsujita, the Chair and extemal representative, respectively, of

the examination committee, for their involvement and critical suggestions for the

irnprovement of this manuscript.

I wish to express great appreciation to Dr. Hai Yu, Yaping Zheng, Dr. Weizhong Lu,

and Nathan Owen-Going for P ' i u m inoculum preparation in this collaborative project.

Bridging the gap between physiology and pathology would not have been possible without

their contributions. 1 would like to thank George Lin, Rodger Tschanz, Luke Lairson,

Carmrn Tse. and Emily Binnendyk for their invaluable assistance in maintenance of plant

materials and hydroponic systems.

1 am extremely grateful to Geoffiey Cloutier for statistical consultations and

programming support in S-Plus. This man knowç regression analysis like no other.

Financial assistance From the Centre for Research in Earth and Space Technologies

(CRESTech), Flowers Canada (Ont.) Ltd., CanAdapt, Natural Sciences and Engineering

Research Council (NSERC), Ontario Ministry of Agriculture, Food, and Rural Affairs

(OMAFRA), Ontario Greenhouse Vegetable Producers Marketing Board, and Trojan

Technologies, Inc., in support of this project is most gratefully achowledged. 1 would also

like to thank Rob Hansen of Erieview Acres Ltd. for providing chrysanthemum cumngs.

Lastly I wish to thank my family and fiends for their emotional support and

encouragement, especially Ryan Ramsey and Jeff Riggs for keepin' it real, my sistas in

crime in the Bw.r community, and my parents, Bill and Sue Johnstone, whose lifelong

encouragement is much appreciated.

TABLE OF CONTENTS

................................................................................................ ACKYOWLEDGEMENTS i

... ................................................................................................... TABLE OF CONTENTS 111

. . ............................................................................................................. LIST OF TABLES vil

... ........................................................................... ............................ LIST OF FIGURES , v111

.................................................. LIST OF DEFINED TERMS AiD ABBREVIATIONS x

CHAPTER 1: Introduction and Literature Review

......................................................................... ................... Chapter introduction .. 1

3 P ~ t h i m infection ........................ ................................................................................

Host Responses to Root infection

............................. 1 . Cellular responses within the mot zone of host plants 5

...................................................... 2. Metabolic responses of the whoie plant 7

........................................................................ i. infection by fithium 8

................................................. ii. Infection by other mot pathogens 9

.................................................. Root Rot Epidemics in Recirculating Hydroponics 1 1

Emerging Technologies for Pathogen Control in Commercial Hydroponic

................................................................................................. Systems 1 4

CHAPTER 2: Photosynthesis and Plant Productivity of Lettuce (Lacruca sativa L.

cv. Bella Green) Following Infection by Pythium dissotocum

Chapter Introduction ............................................................................................ 18

Materials and Methods ............................................................................................. 20

Experimental Results

influence of PpLium dissotoaim on growth of lettuce ................................ 27

................................... Whole plant gas exchange and carbon accumulation 27

Influence of irradiance on whole plant NCER ............................................. 30

................................ Influence of CO2 concentration on whole plant NCER 30

.............................................................................. Leaf chlorophyll content 33

.................................................. influence of irndiance on leaf gas exchange 33

Influence of stage of host maturity on leaf gas exchange and water

reIations ................ .. ........................................................................ 36

...................... Influence of inoculum density on seventy of disease effects 39

Specific leaf area ...................................................................................... .A2

Discussion .................................................................................. 4.c

Conclusion .............................................................................................................. 56

CHAPTER 3: Photosynthesis and Plant Productivity of Chrysanthemum

(Chrysa~ttlremum morifoliurn L . cv Fina) Following Infection by Pythium

aph art idermatum

Chapter Introduction ................................................................................................ 58

Materials and Methods .................... ,.,. ..................................................................... 60

Experimental Results

Influence ofPythium aphanidenncltum on growth ofchrysanthemum ......... 64

Whole plant gas exchange and carbon accumulation ................................. 64

Influence of Pyfhium inoculation on leaf physiology of plants

. . in smgle pot culture .......................................................................... 68

Effect of Pythi~trn inoculation on NFT-groown chrysanthemum ................. 68

Root hydraulic conductance.. . . . . . . ... ... ...... ... . . . . .. . .. . .... . . . . . . .. .. . . .. . . . . . . .. . . .. ... . . . . .68

Discussion.. .. . . . . . . . . . . . . . . . . .......... ............ ... ..... . ... . ... . . . . ... . . . .. . . . . . . .. . .. ... ... . . . . .. . .. . ..... . . . . .. . .. 72

Conclusion ....... ............................................ ............ ... ................... ................ .. ........ 76

CHAPTER 4: Use of Ultraviolet Irradiation of Recirculating Hydroponic Solution

in Trertment of Pythiurn Root Rot

Chapter Introduction ........................................................ ........ .............. ............. .. ... 78

Materials and Methods .......................... .................................................................. 80

Experirnental Results

Influence of concurrent W exposure and Pythiurn inoculation on

vegetative growth of chrysanthemum, Experirnent 1 .................... 85

Elernental analysis of non-inoculated plants grown in W-treated

nutrient solution .....,.......... .... .. ....... . ......... ........ ............................... ... 85

Influence of Pythium inoculation on physiological parameters in

plants exposed to UV-treated nutrient solution .............................. 85

Hydraulic conductance of chrysanthernums exposed to UV-treated

solution following Pyrhium inoculation .......................................... 90

Influence of exposure to inactivated UV system on yield of

chrysanthemum: Experiment 2. .................................................... -90

Elemental analysis of nutrient solution in contact with inactivated

W larnp system.. ........................................................................... .90

Influence of concurrent LN exposure and Pythiirm inoculation on

flowenng chrysanthemum, Experiment 3 ......................................... 95

Influence of exposure to b ra s or plastic fittings on chrysanthemum

growth: Experiment 4 ....................................................................... .95

Discussion ................................................................................................................... 99

Conclusion ............................................................................................................. LOS

.................................................................................................. GENERAL SUMRIARY 107

................................................................................................... LITEMTURE CITED 108

.APPELiDIX 1: Correlation of Nondestructive SPAD Measurements of Leaf

...................................... "Greenness" with Extractable Chlorophyll Content 1 24

APPENDIX 2: SAS Program for Calculation of Leaf Physiological

.......................................................................................................... Parameters 135

LIST OF TABLES

Table 2.1 Leaf physiological changes associated with Pythium root rot in lettuce

plants inoculated at 14, 2 1, or 28 DAP in Experirnent 1 ...................................... 38

Table 2.2 Leaf physiological changes associated with Pythilim root rot in lettuce

plants inoculated at 14 or 2 1 DAP in Expenment 2 ............................................. 40

Table 1.1 Elemental analysis of chrysanthemum leaf tissue, Experiment 1 ........................ 88

Table 4.2 Elemental analysis of chrysanthemurn root tissue, Expenment 1 .............. .......... 89

Table 4.3 influence of nutrient solution exposure to inactivated W lamp system

on growth of chrysanthemum, Experiment 2... .. .... .. . . .. . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . .... .... . . . .. -93

Table 4.4 Nutritional analysis of nutrient solution exposed to inactivated UV lamp

systern, Experiment 2 ................................................................................................ 94

Table 4.5 Influence of Pythium aphanidennatum inoculation and one hour UV

solution irradiation on growth of chrysanthemurn, Expenment 3 .......................... 3 6

Table 4.6 Influence of nutrient solution exposure to brass or plastic fittings on growth

of chrysanthemum, Expenment 4 ................................................. ........................... 97

Table 4.7 Nutrient content of fertilizer solutions exposed to brass or plastic fittings,

Experiment 4 ....... .... ..... .. ... .. ....... .... . ....... ....... ... . .. ..... ..... . .... ...... ..... ............ ............... 98

vii

LIST OF FIGURES

Figure 2.1 Growth of lettuce cv. Bella Green following inoculation with Pythiurn

dissotoczrm ........................................................................................................ 2 8

Figure 2.2 Diumal gas exchange and carbon accumulation in lettuce 28 d after

fythiirrn inoculation ................................................................................................. 29

Figure 2.3 Influence of irradiance on whole plant photosynthesis of

................................................................................... lettuce 28 DAI with Pythicrm 3 1

Figure 2.1 Influence of short rem variations in CO, concentration on

whole plant NCER in lemice 28 d after inoculation with Pythium ......................... 32

Figure 2.5 SPAD measurernents in lettuce following Pythium inoculation ...................... 34

Figure 2.6 Influence of irradiance on lettuce leaf physiology, 28 d post-inoculation

with Pythirrm ............................................................................................................ 35

Figure 2.7 Leaf NCER in lemice inoculated with Pythium at 14 DAP and statistical

interpretation of physiological parameten .............................................................. 37

Figure 2.8 hfluence of Pythium inoculurn density on growth and metabolism

of lettuce inocutated at 2 1 DAP ................................................................................ 4 1

Figure 2.9 Specific leaf area of lemice inoculated with Pyihiurn at different stages

..................................................................... of growth and two inoculum densities 43

Figure 3.1 Growth of chrysanthemum cv. Fina following inoculation with

........................................................................................ Pythiiini aphonidermatum -65

Figure 3.2 Whole plant gas exchange and carbon accumulation in chrysanthernum

25-28 days after inoculation with Pythium (Set 1 ) ................................................ 66

Figure 3.3 W hole plant gas exchange and carbon accumulation in chrysanthemum

29-32 days afier inoculation with Pythium (Set 2) ................................................ 67

Figure 3.4 Influence of Pythium inoculation on leaf physiology of chrysanthemurn

grown in individual pot culture, Experiment 1 .................................. ......................... 69

Figure 3.5 influence of Pyrhilm infection on Ieaf physiology of NFT

c hrysanthemum (Experiment 2) ....... ..... . ... .. . .. . ...... ......... ............. ..... . .. .. . . . ... .. . . . ..... . .. 70

Figure 3.6 Influence of Pythiim infection on hydraulic conductance of entire

c hrysanthemum mot systems ................. ....... .......................... ........... ....... ........ 7 1

Figure 1.1 Ultraviolet lamp system in line with NFT chrysanthemum ............................ 82

Figure 4.2 Biomass accumulation in chrysanthemurn cv. Fina following exposure to

UV-treated nutrient solution and Pythium inoculation, Experiment 1 ..................... 86

Figure 4.3 influence of üV solution irradiation on growth of chrysanthemum,

Experiment 1 ............................................................................................................. 87

Figure 4.4 Leaf gas exchange and water relations in chrysanthemurn following

inoculation with Pythium and exposure to W-treated solutions, Exp. 1 .... ............. 9 1

Figure 4.5 Hydraulic conductance of severed chrysanthemurn root systems,

Experiment 1 .............................................................................................................. 92

LIST OF DEFINED TERMS AM) ABBREVIATIONS

C,: intercellular CO,: concentration of CO, inside the mesophyll cells of a leaf; rneasured

in p ~ - ~ - '

DAI: days after inoculation

DAP: days after planting (transplanting to noughs)

E: transpiration, the loss of water vapour from the surface of leaves and other aboveground

pans of a plant; measured in mmol H,o*~"-s ' '

EC: electrical conductivity, reflecting the concentration of dissolved ions in a nutrient

solution; measured in pS-cm-'

Epidemiology: the study of the incidence and distribution of diseases, and of their control

and prevention

Etiology: the determination and snidy of the cause, or ongin, of a disease

GCO,: stomatal conductance for diffusion of CO,, a measure of stomatal opening; rneasured

in pmol CO,-mJ-s*'

Hydraulic Conductance: the rate of movement of water in the root system; rneasured in

,J4-'

Inoculate: to bring a pathogen into contact with a host plant or plant organ

IRGA: infra-red gas analyzer

NCER: net carbon exchange rate, an instantaneous measurement of carbon fixation in

photosynthesis; measured in pmol C~~.m".s"

NFT: nutrient film technique

PAR: photosynthetically active radiation, from 400-700 ,urnol-rn"-~-~

Pathogenesis: the rnanner of disease development within host tissues

Pathogenicity: the capability of a pathogen to cause disease

Virulence: the degree or measure of pathogenicity of a given pathogen

WUE: water use efficiency; as a ratio of NCER to E, this parameter is unitless

Zoospore: an asexual fungal spore, bearing flagella and capable of moving in water

Chapter 1: Introduction and Literature Review

Introduction

The established philosophy of hydroponics accepts that closed recirculating systems

are inherently more conducive to epidemics of root diseases than open drain systems

(Schuerger 1992). While the divenity of infectious root diseases seen in hydroponic crops

appears to be less than field-grown crops, disease severity is often intensified due to the

favourable environmental conditions and the dense monoculture of susceptible plants

(Zinncn 1988; Stanghellini 1988). Hydroponic systems genenlly lack the microbial diversity

and biological "buffering" found in soil, where many soilbome pathogens are limited by

antagonism and competition fiom other microorganisms (Paulitz 1997). A shifi in root-

infecting pathogens is observed in hydroponics, to species better adapted to survival and

dispersal in water. Species of Pjjthium and Phytophthora have been the most frequently

rrported root pathogens in recirculating hydroponics (Rey et al. 1997; Paulitz 1997;

Schuerger l992), well-suited to dispersa1 in aquatic environments with biflageellate, motiie

spores. Pyhiiim zoospores c m penetrate the root epidermis within 5 minutes of adhesion

to the root surface, followed by rapid development within the elongation zone of the roots

(Stanghellini 1988; Stanghellini and Rasmussen 1994). Virulent stmins can produce

secondary inoculum within 24 houe, which is reieased into the solution and potentially

spreads to other plants.

Classic symptoms of Pythirrm infection are brown, decayed roots coupled with

wil ting and stunting of the shoot (Rowe 1986; Agrios 1997). Generally considered to be

solely a cortical root pathogen, Pyrhium can advance into vascular tissues, like Fusaritirn and

Verticilli~rm (Chérif et al. 1991). P'h ium is commonly known to cause damping off of

seedlings (rotting at soi1 level), but young roots of plants at any stage of growth can be

aaacked by Pythium. Plants undergoing stress or flower production are also thought to be

more susceptible to infection, likely due to increased root exudation (Curl and Truelove

1986; Hamlen et al. 1972). Paulitz (1998) observed that Pyrhiunt aphaniderntartrm kills

younger hydroponically grown cucumber plants quicker than mature plants, and that wilt and

collapse of mature plants may not appear until fmit set.

The speed and severity of Pythium anack in hydroponic crops indicate the need for

remediation effons and greater understanding of the infection process From a whole plant

perspective. Ln order for an appropriate remediation method to be chosen and evaluated, a

thorough knowledge of the life and infection cycle of the pathogen is necessary. An

adequate understanding of the epidemiology is lacking for many fungal pathogens in

hydroponic systems, including Pythium. While many aspects of the mechanism or "mode

of action" of Pythium infection remain unclear, a picture of the infection process is

emerging.

Pythium Infection

There are approximately 120 species of Pythium, varying widely in host range,

virulence and pathogenicity (Martin and Loper 1999). Taxonomically, Pyrhium is classified

in the Phylum of Pseudofungi, Kùigdom Chromista (Corliss 1994), but is still commonly

refened to as a fungal species. Inoculum can be in the form of sporangia, zoospores,

mycelia, or oospores (Endo and Colt 1974). Some types of sporangia and oospores

rerminate directly by forming germ tubes but most function indirectly to produce zoospores. C

Numerous workers conclude that zoospores and mycelia function comrnonly as uni& of

inoculum (Endo & Colt 1974; Stanghellini 1988).

Infection of plants fiom zoospores follows a characteristic pre-penetration sequence

of zoospore taxis, settling, encystment, adhesion, cyst germination, and orientation of the

germ tube (Deacon 1988; Hardharn 1992; Martin and Loper 1999). Zoospore responses are

usually not host-specific and have been elucidated in vitro. Taxis occurs towards root and

seed exudates, mixtures of nutrients, and depending on the species, towards ethanol,

particular sugars and amino acids [glutamic and aspartic acid, among the most abundant

acids in root exudates (Jones et ai. 199 l)]. Zoospores also respond to light, electric fields or

water currents (Deacon 1988).

In analysis of zoospore behavior, videomicroscopy showed that Pythirrrn

aplianidennatirtn spores can swim for sevenl hours by consuming lipid reserves (Jones et

a(. 199 1). They crase moving when a chernoattractant is encountered, swell by altering

osmotic balance, and release an adhesive glycoprotein from pre-formed vesicles lying just

beneath the cell membrane (Estrada-Garcia et al. 1990). Flagellar contact directs the

orientation of the spore with respect to the source of the attractant. The naked ce11 produces

a cyst wall, made up largely of polysaccharide, then produces a germ tube which emerges

and grows towards the attracting source. The whole sequence from initial release of the

zoospore from sporangia to gem-tube emergence can take as little as 30-40 minutes. Deacon

(1985) points out it is "one of the fastest and most cornplex developmental sequences in

eukaryotic cells."

Adhesion and encystment are reponed to be regulated by fucosyl residues of root

surface mucilage (Longman and CalIow 1 98'?), pectin and other uronates (Grant et al. 1 98 5) ,

lecrins, some monoclonal antibodies, arnino acids and ions, principally Ca" (Jones et al.

199 1 ; Donaldson and Deacon 1992; lser et al. 1989; Grant et al. 1985). Proteinaceous

components on the surface of the encysted zoospore interact with terminal glycosyl residues

on mucilage in the zone of root elongation just behind the root cap (Wulff et al. 1998;

Longrnan and Callow 1987; Hinch and Clarke 1980). The elongation zone of the root and

sites of mot hair emergence and wounding are preferred sires of adhesion (Hardham 1992;

Jones er al. 199 1 ).

Cell wall drgrading enzymes are produced abundantly and diffused some distance

from the point of fungal contact (Rey et al. 1995; Benharnou and Coté 1992). Pythirrm

an-hrnomanes, P. irregulare. and P. trltinirim produced in vitro metabolites that caused

browning of root tissue. root stunting, inhibition of root hair formation, reduced fresh and

dry root weights, and sninted vegetative growth in wheat and geranium (Mojhedi ei al. 1990;

Désilets and Bélanger 199 1). P. ultimum secretes polygaIacmronases, P- l,4-glucanases and

a large diversity of cellulase isoenzyrnes (Campion et al. 1997). Highly virulent isolates

produced more cellulolytic and pectolytic enzymes than moderately and weakly pathogenic

isolates (Sadik et al. 1983). Cornparing ultrastructural degradation events caused by P.

ulthtum infection with those associated with exposure to partiaily purified culture filtrates,

similar alterations were found, indicating that observed ce11 damage is largely induced by

similar biochemical processes by toxic, difisible metabolites produced by P. ultimum

(Désilets et al. 1994), as opposed to mechanical force.

Using imrnunoenzymatic staining, Rey et al. (1997) found that formation of

sporangia on the root surface was the fint indication of Pythium establishment in tomato

roots. Exoglucanase-gold cytochemical labelling and TEM were used to study the

ultrastructural host reactions in cucumber seedlings infected with P. ultimum (Chérif et ai.

199 1 ) and in asymptomatic infection of tomato by Pythium F (Rey et al. 1998). Penemtion

of the epidermal cells by constricted hyphae or "penetration pegs" occurred just 2-7 houn

after inoculation, and hyphae reached the f i s t outer cortical layers between 24 and 48 houe,

developing through most of the inner root tissues including the cortex, endodermis,

paratracheal parenchyma cells and vascular stele within 48 to 72 hours afier inoculation.

Hyphae grew both within and between ce11 wails.

Host Responses to Pythium Infection

1. Cellular responses within the root zone of host plants

Invasion of tomato and cucumber seedlings by Pythiuni conelated with severe host

ceIl damage. Cells in the outer cortex expenenced organelle disintegration, cytoplasmic

aggregation, swelling, and shredding of ce11 walls, which showed a marked decrease in

electron density. Ce11 wall hydrolytic enzymes appeared to be produced, weakening host

cell walls and middle lameila matrices, demonstrated by an altered pattern of cellulose

distribution over walls of invaded and adjacent epidermal and cortical cells (Chérif et al.

1991: Reyer al. 1998).

Intercellular spaces of outer cortical cells of tomato were coated with a band of

electron-opaque material (Rey et ai. 1998). in the inner cortex, fiom 48 to 72 hours afier

inoculation, osrniophilic material accumulated to form large polymorphic aggregates at the

fungus/host wall interface. This material partially surrounded the invading hyphae, which

displayed disordered ce11 structure fiom increased vacuolation to mitochondrial disruphon.

The massive deposition and accumulation of osmiophilic material at sites of attempted

fungal entry suggests that inner root cells are signalled to mobilize a number of defence

strategies to halt the movement of the pathogen towards the vascular stele.

Tomato host ce11 responses to Pythium F invasion were enhanced in the pantracheal

parenchyma cells, forming hemisphenc protuberances or papillae at sites of fungal

penetration (Rey et al. 1998). Several colonized host cells filled with fibrillar aggregates,

foming a dense netsvork around the fungal wall. Both infected and uninvaded host cells

occluded with opaque flecks of electron-dense aggregated matenals. It appeared that

released aggregates rnignted inside the host cells and surrounded the fungal cells, causing

lysis. Parenchyma cells near the vascular bundles showed ce11 wall impregnation with an

osmiophilic material. Based on its texture and increased elecuon density, this matenal

contained phenolic compounds, likely lignin (Benhamou 1995). Although fythium F was

able to penetrate to the xylern vessels, the fungal ultrastructure was significantly changed.

Most invading hyphae were similar to ghost cells, with imperceptible cytoplasm and

organelles (Rey et al. 1998). Tornato xylem vessels reacted to hyphal invasion by coating

the secondary host walls with osmiophilic droplets and callose (Benhamou 1995).

Cucumber host defence reactions to P. ultimum were not as successful. By 72-96

hours afier inoculation, P. ultirnirm colonization of the vascular stele in cucurnber seedlings

proceeded by invasion of the endodermis, the pencycle and the paratracheal parenchyrna

ce1 ls (Chérif et al. 199 1 ) . Paratracheal parenchyma cells showed protoplasm disintegration,

organelle alteration, and disruption of the middle lamella matrices (Chérif et al. 1991).

Invasion of the xylem tissue occurred directly through the pit membrane between

paratracheal parenchyrna cells and xylem vessels or benveen adjacent vessels. Vessels were

frequently found to be coated by a thick layer of an amorphous osmiophilic material which

also accumulated along pit membranes and secondary thickenings of some vessels and

estended a short distance into the vesse1 lumen. In some cases, the vesse1 Iumen was

completely occluded by the emergence of tyloses fiom adjacent parûtracheal parenchyrnâ

cells.

Early in the infection process, some degradation of cucumber xylem vesse1 ce11 walls

and pit membranes was noticeable (Chérif et al. 199 1). Later, with extensive colonization

of xylem parenchyma cells and abundant hyphae present in xylem vessels, degradation of

the secondary walls and the dismption of cellulosic constinients were more pronounced. At

this point, pronounced wilting and root rot were visible. The researchers concluded that the

extensive ce11 wail degradation and colonization of xylem vessels were similar to the

vascular wilts Fusarium and Verticillium. Other Pythiltm species known to reach the

vascular elements are P. sylvaticitrn and P. dissotocurn (Nemec 1972).

2. Metabolic responses of the whole plant

Vascular occlusion or degndation has implicit consequences for host plant

metabolism, potentially interrupting evapotranspiration. Altered stomatal functioning can

influence fluxes of water and CO2 in mesophyll ceils (Ayres 198 1 b). Transpiration has been

observed to increase, decrease or not change at a11 as a result of pathogen infection.

Epidermal breakage and higher tissue permeability (Daly 1976; Agrios 1997) have been

implicated in increased water loss in host plants following infection. In contrast, restricted

wae r loss has been associated with induced stomatal closure and obstruction of vascuIar

tissue by hyphae, defence molecuies, or ce11 wall fragments (Ayres 198 1 a; MacHardy et al.

1976; Bowden et al. 1990; Abd El-Rahim et al. 1998). Stomatal apemire is also known to

be affected by hormones, such as ABA, produced during penods of water stress (Nobel

199 1).

Plant productivity and growth rely primarily on photosynthesis (Jiao et al. 1997).

Biomass production correlates strongly with accumulation of carbon fixed in gas exchange.

Daily carbon gain, less nightly C respiratory losses, provides assimilates available for plant

growth and development. in a broad range of pathosystems, a reduction in leaf and whole

plant carbon assimilation has been reponed following infection (Ploetz and Schaffer 1989;

Shtienberg 1992; Saeed et al. 1997; Jiao et al. 1999), despite an initial stimulation of leaf

photosynthesis following infection with sorne foliar pathogens (Roberts and Walters 1986;

Scholes er al. 1994). Reductions in photosynthetic activity have been amibuted to the

srnaller leaf area observed with stunted growth in infected plants, or to decreased

photosyntheric efficiency, itself related to chlorophyll content and chloroplast functioning

(Roberts and Walters 1988; Madeira and Clark 1995). Understanding the physiology

underlying disease expression in host plants is vital to devise appropriate remedial efforts.

i. hjècrion by P-vthiirm

Very few reports have dealt with the physiological basis of disease effects caused by

Pyrhiurn. ui homogenized stem tissue of highly resistant pumpkin infected with Pythium

~rltimunl, oxygen consurnption was 2.5-3.5 times greater than in healthy tissue (Takahashi

1958). Activity of oxidases, peroxidases, and polyphenoloxidase in diseased pumpkin stem

tissue was increased tsvo-to-three foid over healthy tissue. Daelmans eî al. (1972, as cited

in Endo and Colt 1974) found that peroxidase activity was not increased in Pyrhitim-infected

seedlings of tomatoes, pea and cauliflower but polyphenoloxidase activity was significantly

increased. P. ultirnrirn and P. sylvaticum elicited accumulation of the phytoalexins kievitone

and phaseollin in root tissue of hydroponic beans (Liu et al. 1995).

i i In fecrion by oiher root pathogens

An early plant response to disease, among other stresses, is stomatal closure (Ayres

198 1 a; Ayres 198 1 b). Consequently, researchers have used stomatal function and associated

gas exchange and transpiration to monitor host response in disease snidies. This has not yet

been descnbed for Fyihizirn infection, but Phyroplirhora, a cortical root pathogen related to

P i reduced net CO, assimilation 7 days after avocado plants were transplanted to

infected soi1 (Ploetz and Schaffer 1989). By 14 days, CO, assimilation was 75% lower for

infected plants. Reductions in assimilation, conductance and transpiration preceded

developrnent of shoot syrnptorns (wilting and defoliation) by several days. Phytophlhora

infection did not significantiy reduce the xylem pressure potential in avocado, measured 3

rnonths after inoculation (Nevin et al. 1990). in maize infected with Cephalosporizrni

ma~*clis, a vascular wilt pathogen, leaves of infected plants increased proline content by

about nvo-fold compared to healthy plants (Abd El-Rahim et al. 1998), a characteristic

response of plants to water deficit. The authon concluded that vascular occlusion may be

the principal cause of symptom development in late wilt disease.

in potato, significant changes in stornatal conductance and gas exchange occurred

2 weeks after inoculation with V. dahliae (Haverkort et al. 1990). Infection led to decreased

stomatal conductance, transpiration and photosynthetic rates even in nonsymptomatic leaves.

Infection reduced carbon assimilation rate in high light but not always in low light, and did

not affect dark respiration (Bowden and Rouse 1991b). InterceIlular CO, concentration

decreased and leaf water use eficiency increased (Haverkort et al. 1990).

Tomato infected by Furarium oqsporum f. sp. Iycopersici, another vascular wilt

pathogen, experienced marked reductions in photosynthesis and transpiration 15 days after

inoculation, measured in the youngest hilly expanded leaf (Duniway and Slatyer 197 1). Leaf

water content and water potential measurernents indicated that increased stomatal and

intracellular resistances preceded the onset of water stress in a given leaf and were associated

with a reduction in dark respiration, but not with a decrease in Rubisco activity, indicating

that decreased carbon metabolism was not directly related to reduced carboxylation

potential. Comparable levels of water stress caused liale increase in inrracellular resistance

of healrhy leaves.

Localized and whole plant metabolic responses to Pythiitm infection are critical in

understanding syrnptom development in host plants. The physiological bais of disease

caused by other root pathogens indicates several possible mechanisrns of Pythium

pathogenesis. Degradation of mot cells and the resulting cortical occlusions may reduce

hydraulic conductance, leading to stomatal closure, a subsequent decline in mesophyll

intercellular CO, concentration, and reduced CO, assimilation, with the end result of sninted

growth. Altemativeiy, roxic metabolites of fungal ongin, stress hormones produced by the

plant in response to infection, or reduced ion uptake by infected roots (Cook 1992) may

decrease CO, assimilation more directly, the backlog leading to increased intercellular CO,

concentration, encouraging reduced stomatal conductance. The reality may be a combination

of these factors.

Chapters hvo and three of this thesis will investigate whole plant and leaf

physiological responses to Pythiwn infection, in two hosts, Iettuce and chrysanthernurn.

Hypothesizing that alterations in canopy and leaf gas exchange in host plants accompany

Pythiunt root rot, the elucidation of the cascade of host physiological responses to Fythium

infection could contribute to funire efforts to combat the pathogen, such as by identibng

key traits of host resistance, finding new means to minimize rates of epidemic increase, or

improving efficacy of existing treatment rnethods.

Root Rot Epidemics in Recirculating Hydroponics

Hydroponic systems have becorne an industry standard for many greenhouse crops,

including letnice, tomato, cucumber and several ornamentals. Careh1 control of light,

temperature, nutrition and water can provide continuously optimal growing conditions,

minimizing plant stress as well as rnaximizing use of available space (MacFadyen 1984;

Srrayer 1994), resulting in better quality production and control of crop scheduling (Paulitz

1997). Maximum yields are possible (Paulitz 1997) although Hanan ( 1998) cautions that,

"quite often, the high yields amibutable to hydroponics are heralded to the point of being

misleading." Yields are higher because greenhouse hydroponic culture is intensive: al1

elements in the nutrient solution are available to the plant, so nutrient cornpetition can be

reduced, and greater plant densities c m be used (Paulitz 1997). in addition to higher yields

and speed of crop growth, advocates of hydroponics a&rn several advantages over soil

culture. Instead of soil, chemically inert growing media like sand and rockwool supply

physical support for the plants. These tend to provide more consistent rooting conditions for

the crop by having a more uniform stmcture. Hydroponic systems without substrates inchde

the nutrient film technique (NFT), with plant roots mspended in a charnel or uough over

which the nutrient solution flows in a thin film, deep flow systems, with the roots

submerged in solutions of greater depth. and ebb-and-flow, where the plants are supponed

in an inert moting medium and penodically flooded and drained by nutrient solution. An

additional advantage to hydroponics is that plant nutrition is supplied exclusively in solution

by the imgation system and not by the rooting medium, so pH and nutrient concentration are

entirely under the grower's control.

To avoid buildup of pathogen populations, ion imbalance, and potential problems

with allelopathy, commercial growers oflen discard used nutrient solutions into the

environment. As most greenhouse operations employ large solution volumes and are located

near urbanized areas, concems about environmental protection demand that pollution of

surface and groundwaten be minimized. These concerns have spurred initiation of zero

runoff recirculatlng systems in many areas, encouraged by govemmental guidelines (British

Columbia Ministry of Agriculture and Food 1998) and the possibility of future legislation

requinng extended recirculation.

Root-infecting fungi such as Pythirm and other pathogens are ubiquitous in almost

al1 soils (Hendrix 1974) and in and around greenhouse facilities. These fungi produce spores

or other reproductive structures, often in great quantities, which, borne by the wind, in dust,

raindrops, irrigation water, on insects, workers' shoes or clothing, wili eventually find their

way into the greenhouse (Hockenhull and Funck-Jensen 1983; Paulitz 1997; White 1998).

Once a root-infecting pathogen has been introduced to a hydroponic system, its dispersal

may occur by solution recirculation or by root-to-root contact (Stanghellini 1988). Many

fungal root pathogens can grow, by hyphae, from an infected to a healthy root. Rapid and

uniform dispersal of spores is enhanced by recirculation of infected nutrient solution.

Stanghellini ( 1988) asserts that, "any infective unit, upon entry into the nutrient solution, will

eventually make contact with a root. The probability of a chance encounter is very high

when one takes into consideration the density and confinement of roots in a recirculating

system, particularly those employing the nutrient film technique."

Substantial infection and disease loss can result from a minute quantity of initial

inoculum of the pathogen. Uniform infection of one individual mature lemice plant, with

approximately 20 m of roots, could result in the production and release of about 8 million

zoospores (Stanghellini 1988). As few as 20 zoospores of fythium aphanidermatum,

introduced into the 100 L reservoir in an NFT system, caused significant losses in cucumber

production (Menzies er al. 1996). One infected transplant can spread Pyrhium to other plants

in less than a week (Rowe 1986; Jenkins and Averre 1983). Commercial production of

spinach in Arizona was abandoned due to P. aphanidermartim and P. dissotocum (Bates and

Stanghellini 1984); infected plants showed extensive root rot, wilt, severe stunting, and

dearh. Of perhaps greater concern is the ability of some fungi to cause subclinical disease:

yield reductions of up to 54% were caused by P. dissotocum in lettuce, in the absence of

visible root or foliar symptoms (Stanghellini and Kronland 1986). Yield losses by

subclinical disease are ofien not detected because al1 plants in the system are affected and

appear healthy and normal (Stanghellini 1988).

Irrespective of whether the pathogen causes clear or asyrnptomatic infection,

pathogen control in recirculating systems is abundantly necessary. in order to reduce

environmental damage by dischargmg used nutient solutions, and comply with future

govemmental regulations, recirculation systems must be implemented and recirculation time

extended. in a closed system, conditions become for favourable for disease over time, as

root exudates and other pathogen substrates accumulate. Consequently, for recirculation to

be viable, remediation of the nutrient solution is a necessary cornponent to lower, if not

eliminate, the presence of pathogens in hydroponic systems.

Emerging Technologies for Pathogen Control in Commercial Hydroponic Systems

There are several reviews available on methods of controlling pathogen populations

in recirculating nutrient solutions (Vestergard 1988; Schuerger 1992; Evans 1994; Menzies

and Bélanger 1996). It seems clear that complete sterilization is impractical, and likely

impossible. Control of root zone pathogens has frequently been directed at reduction of

rnicrobial populations or inactivation of pathogens (Evans 1994) to prevent reproduction to

problematic levels. Effective pathogen control involves sanitation practices at every point

in plant production, including the incoming water source, plant materials, growing system

surfaces, and most imponmtly in a recirculating system, the nutrient solution.

The use of füngicides in commercial hydroponics requires legal registration. and only

one is currently registered. The systemic fungicide Previcur (AgrEvo 2000), registered

spccifically for cucumber, is limited to systems involving substrate. More innovative

rechniques are required for disinfection of hydroponic solutions with wider crop ranges.

Commercially available sterilization methods include ozone, filtration, and ultraviolet

radiation (UV). Studies have shown the effectiveness, in vitro. of ozone against a wide

variety of microorganisms (Vanachter et al. 1988; Yamamoto et al. 1 990). However, several

of these researchers reponed problems with the stability of iron chelates with ozone

treatrnent. Ulnafiltration requires frequent cleaning or filter replacement due to biofilms

(Evans 1994), reducing the long term effectiveness of this method. Slow sand filtration can

effectively remove propagules of fungal and bacterial pathogens from nutrient solutions

(Wohanka 1992; Barth 1995; van Os et al. 1999). There is a trend in the greenhouse industry

to combine physical and biological remediation technologies; for example, establishment

of a biological agent such as Trichodema in a slow sand filter can increase the eficacy fiom

80% to 98.5% (Brand et al. 1998).

A promising method for physical controi of large volumes of nutrient solution is

ultraviolet radiation. Several authors have demonstrated the efEcacy of using UV to reduce

pathogen populations in hydroponic applications (Ewart and Chrimes 1980; Buyanovsky et

ai. 198 1 ; Daughtrey and Schippen 1980; Schwartzkopf et al. 1987; Wohanka 1992);

however, UV did not consistently reduce disease symptoms in tomato (Zhang and Tu 2000)

The cffectiveness of W radiation in allowing plant recovery after initial infection, as

opposed to preventing disease in the first place, remains to be seen. Most experirnentors to

date have not measured the applied dosages required for reducing viability of various

pathogenic and nonpathogenic organisms. In in vitro collimated beam assays, a dosage of

40 rn~-cm-' reduced survival of P. aphanidernratun~ zoospores (Zhang and Tu 1999). Sutton

er ai. (2000) recommended a dosage of 30-40 rn~-s*crn- ' to inactivate Pytkium and

Fiisuriu»r. using flowthrough systems comparable to commercial UV systems.

UV ndiation is germicidal in the 200-300 nm range, with peak effectiveness at

around 260 nm (Schenck 1981). Absorption of W by microorganisms resulis in

photochernical reactions in nucleic acids and proteins that are vital for reproductive and

rnetabolic hinctions (Camgan and Sakamoto 1990; Camgan and Cairns 199 1). W radiation

is therefore not technically a "sterilizing" process but rather one of inactivation. The

effectiveness relies on the dosage applied, the product of intensity and exposure time

(Morowitz 1 950; Qualls and Johnson 1983). Pathogens differ greatly in their sensitivity to

W, due to differences in ceil walls detennining U V penetration, the presence of surface

proteins capable of LN absorption, and structural differences in the DNA (Carrigan and

Sakamoto 1990). There is evidence indicating that some microorganisms can recover by

pho to-reactivation or dark incubation (Evans 1994; Zhang aiid Tu 1999). Furthermore, UV,

like ozonation, can reduce availability of some essential elements in hydroponic media.

Most of the authors conducting the above UV studies reported significant

precipitation of Fr, and Mn to a lesser extent, out of the numeot solution due to absorption

at 254 nm. The stability order of cornmon iron chelates was identified as EDDHA> DTPA>

EDTA (Acher et al. 1997) although another account lists iron citrate> DTPA > EDDHA>

EDTA (Evans 1994). Commercial mals used continuous dosing of 0.5 ppm Fe-EDTA afier

UV treatment (Evans 1994) with occasional adjustments. Mina (1 998) related that growers

in Alberta found precipitation of iron and manganese to be a problem in recirculating

systems and they preferred to use the lamps only on incoming water. W disinfection is not

recommended for plants in peat substrates, due to the humic acid released which absorbs UV

and reduces the efficacy of W treatment (Menzies and Bélanger 1996).

Vestergard (1988) concluded regarding possible methods of solution treatment, "If

the quantity [of solution] is small, pasteunzation is the most economic agent," especially

with efficient heat exchangen. "If the heat can not be reused and the recirculation rate is

high. UV sterilization must be considered the best alternative, and if the quantity of solution

is very large, ozone and different combinations using ozone [Le. ozone and UV] will be the

cheapest way to assure an adequate sterilization." While many options for remediation of

nutrient solutions are currently available commercially, and more are under development (for

example, biological control agents), many of these techniques require M e r research and

testing to handle the large volumes of solution and increasing organic load over time (Sutton

et al. 2000). There are no clear recornmendations for commercial growers as to which

treatment options are best suited to different cultural conditions. There are also no guidelines

regarding which rernediation techniques are better suited to continuous, preventative use and

which are preferable for short-term, intermittent use to control existing pathogen

contamination. Chapter 4 of this thesis deals with a senes of experiments in which UV

lamps were incorporated into recirculating hydroponic systerns, using spot treatments or

çhon term UV doses to allow plants a recovery period afier initial infection and possibly

prevent plant-to-plant secondary transmission.

Chapter 2: Photosynthesis and Plant Productivity of Lettuce (Luctuca sntiva L. cv Bella

Green) Following Infection by Pythium dissotocum

II1TRODUCTION

Regarded as one of the most important vegetables grown in Nonh Amenca, lettuce

(L<zcntca saliva L.) has a very large consumption per capita despite its relatively low

nutri tional value (Peirce 1987). Greenhouse lettuce production is a S 1 O.3M industry annually

in Canada (Agriculture and Agri-Food Canada 1999a); currently, there are 20.7 ha of

meenhouses dedicated to lettuce production (Agriculture and Agri-Food Canada 2000). in Y

1999, 100% of Ontario greenhouse lettuce was grown with the NFT method. Greenhouse

cultivars generally grown are leaf and the higher value butterhead, also referred to as Bibb

or Boston types (Peirce 1987). Bella Green, a butterhead cultivar, has cmmpled leaves with

a soft buttery texture, developing loose heads (Decoteau 2000). Lettuce is commercially

hamested while in the vegetative growth stage, pnor to the physiological demands of

fl owennç and seed formation. The relatively rapid groowth cycle, compared to greenhouse

crops such as tomato, cucumber and pepper, renders lemice an optimal mode1 system for

repeatable hydroponic pathology experiments.

The ability of pathogenic Pyihium species, including P. aphanidermafurn. P.

iwegulare. and P. ulrimum. to induce root rot, shoot wilting, and stunting is well docurnented

(Stanghellini and Rasmussen 1994; Favrin et al. 1988; Bates and S tanghellini 1984; Jenkins

and Averre 1983). A major pathogen of hydroponically grown lettuce, P. dissot~cum caused

significant yeld reductions (35-54%) with no visible root or foliar symptoms (Stanghellini

and Kronland 1986). The objectives of the experiments in this chapter were several: 1) to

quanti@ effects of root infection of lettuce by a P. dissotocurn isolate on yield as an indirect

means to estimate aggressiveness of the pathogen; 2) quanti& effects of root infection by

Pytlzilrni on physiological parameters of individual leaves and 3) whole lemice plants; and

1) Examine relationships of stage of crop development and inoculum density on severity of

disease caused by this isolate.

MATERIALS AlND METHODS

Plant Material

Seeds of Lactuca sativa L. cv. Bella Green M.I. were obtained from Stokes Seeds

Ltd. (St. Catharines, ON) and germinated in small rockwool cubes (SBSA 36-77, Grodan).

At 14 days after seeding, the plants were placed in hydroponic NFT troughs (Rehau

Industries, inc., Baie-D'Urfe, PQ) spaced 30 cm (1 2 in) apart, with 10 plants per aough.

Plants were grown to commercially harvestable maturity in a research greenhouse at 2 1 OC

day, 1 6 T night ternperatures, with ambient CO, (360 rnL*Le1) and approximately 30-60%

relative humidity. To rninimize mutual leaf shading, a planting density of 10.76 plants-m*'

( 1 plant-ft") was used. Plants were grown throughout September 1998-December 1999, with

supplementai HPS (high pressure sodium) Iighting (SonAgro 430W, Philips, Somerset, NJ)

when required to maintain a 16-hour photoperiod. Each trough had sepante nutrient

solution, continuously recirculated by a submersible pump (Little Salty mode1 1 -EUAA-MD,

Little Giant Pump Co., Oklahoma City, OK) at a flow rate of approximately 1 Lmin". Ail

solutions ( 1.15 g*L" Plant Prod 7-1 1-27 N:P:K supplemented with 0.775 g-L'l calcium

nitrate. Plant Products inc., Brampton, ON), completely changed at 14 day intervals, were

maintained at pH 6.0, adjusted by addition of NaOH when necessary, and an electncal

conductivity (EC) of approximately 2.0 mS*cm". EC and pH were continuously monitored

in each 20 L reservoir by electrodes suspended at the solution surface (Cole P m e r

Instrument Co., Vernon Hills, IL) using an Argus control system (SM 12 modules, Argus for

Windows v 1.1, White Rock, B.C.). Solution tempenture averaged 19'C.

Pythitlnt Inocirlation Protocol

Zoospore suspensions of Pythium dLrsotontm Drechsler, isolate HYüLETl,

previously isolated from commercial hydroponic lettuce, were prepared following standard

p rocedure (Dhingra and Sinclair 1995; Rahimian and Banihashemi L 979). Lemice plants

were inoculated 14,2 1, or 28 days afier transplanting (DAP) by a direct root-dipping method

for 30 minutes in spore suspensions in autoclaved 100 mL beaken at an inoculum density

of 5x10' z o ~ s ~ o r e s m L - ~ unless othenvise specified. Following inoculation, plants were

returned to the troughs. At the end of the experiment, al1 troughs, lids, reservoirs, tubing, and

pumps were scrubbed with a 0.5% Virkon (Dispar, Vétoquinol Canada hc., Joliette, PQ)

disinfecting solution. The solution recirculated through the system a minimum of 16 h,

followed by rinsing with deionized water for a minimum of 24 h.

Cliuracterizution of Growth Parameters

At intervals of 3 to 5 days throughout the growth cycle, one plant was randomly

chosen from each trough for destructive harvest for ffesh weight, leaf area, and dry weight

analysis. Statistical significance of differences between treatments was assessed by two-way

ANOVA (S-Plus 2000 Professional Release 1, MathSoft, Inc., Seattle, WA). Identification

of when differences became significant (time elapsed post-inoculation) was detetmined by

plotting linear rnodels and corresponding 95% confidence intervals. Areas where the

confidence intervals did not overlap were considered statistically different.

To determine qualitative differences in shoot appearance, SPAD measurements were

taken. The portable, handheld, dual-wavelength SPAD reader (mode1 502, Minolta Co., Ltd.,

Osaka, Japan) measures "greenness" of a leaf and was used to indicate the appearance of

foliar symptoms visible to the human eye. Six separate measurements were taken of a single

leaf (several leaves, when the plants were very small) of each of 3 subsampled plants per

trough. A standard curve reiating SPAD measurements to total extractable chlorophyll in

iV.iV-Dimethylformamide (DMF) was performed following the protocol of Inskeep and

Bloom ( l985), presented in Appendix L. Statistical significance of SPAD measurements was

determined using a hvo-way ANOVA (S-Plus 2000, MathSoft Inc.).

Iflzole Plant Net CO, Erchange

Whole plant net gas exchange was measured as described previously by Dutton et

al. ( 1988) and Leonardos et al. (1994). in late afternoon the day before measurements of net

carbon exchange rate (NCER) were made, mature plants, approxirnately 28 days afier

inoculation (DAI), were nansferred fiom the greenhouse trough system to 2 L pots

containing half-strength nutrient solution. Two plants were placed in each of 4 sealed plant

chambers with temperature, humidity and CO, concentration maintained by cornputer

controls. Unless specified othenvise, temperature was set to 2 1 OC dayll6"C night, relative

humidity was set at 50%, and the COz concentration set to 350 c<L.L*' (350 ppm). High

pressure sodium lamps (1000W, Lumalu- LU 1000; GTE Sylvania Canada Ltd.) provided

photosynthetically active radiation (PAR, 400-700 nm) £Yom 400 to 1600 c<rnoI.m"*s-' as

measured by quantum sensors (Q399L-4; LI-COR, Lincoln, NE) at the top of the plant

canopy. NCER was calculated fiom initial and final CO, concentrations in the chamber

atmosphere and pure CO, injection measurements. The leafarea of the plant canopy in each

chamber was measured destructively at the end of the daily CO2 measurements using a leaf

area meter (LI-3000, LI-COR). This tissue was then dned for 48 h at 75 "C to estimate dry

weight.

Diumal gas exchange and carbon accumulation was rneasured for 36 hours

commencing with the daylight period (800 ~mol.m-'-s-l PAR) following an ovemight

equilibntion of 12 h. Carbon accumulation was calculated by integrating NCER over the 36-

hour penod. Statistical significance of total carbon accumulation was assessed by a one-way

ANOVA (S-Plus 2000).

Light response curves were generated for healthy and diseased plants by adjusting

the irradiance levels randomly throughout the day, using neutral density shading screens to

achieve irradiances of under 400 prnol-m''~s*'. Regression lines were fitted using a second-

order polynomial function and 95% confidence intervals generated (Statistical Analysis

Systern Release 6.12, SAS Institute inc., Cary, NC). Light responses were interpreted as

statistically different due to P y h h infection based on the results of a hvo-way ANOVA.

Specific irradiance levels with significant difference in NCER response were judged to be

where the confidence intervals did not overlap.

Carbon dioxide response c w e s were generated by varying the CO, levels throughout

the day. ranging From 4 0 0 PL-L" (100 ppm) to over 1500 PL-L" (1500 ppm). The

irradiance was set at 1000 irmol-m''d PAR. A two-way ANOVA on the entire data set

determined srastistical significance. Linear models were fitted using a second-order

po l ynomial function and 95% confidence intervals generated. Plant p hotosynthetic response

to CO, availability was interpreted as significantly different where the confidence intervals

did not overlap.

Leu f Gus Ekchange and Transpiration

The afiemoon pnor to an experiment, plants were transferred from the trough to a 2

L pot containing dilute nutrient solution and moved to an illuminated charnber. Steady-state

ças exchange of attached leaves was measured using an open-flow gas analysis system

described previously (Leonardos et al. 1996). Briefly, a portion of the youngest fully

rxpanded leaf was enclosed in a leaf cuvette and exposed to overhead irradiance, provided

by three JOOW HPS lamps (Lumalux. Sylvania GTE), measured at the surface Ievel of the

leaf with a quantum sensor (model LI-189, LI-COR, Lincoln, NE). The CO2 concentration

in the gas stream and its changes resulting from leaf metabolisrn were monitored with an

infrared gas analyzer (IRGA; model LI-6262, LI-COR). The dew point in the gas stream and

its changes resulting From leaf transpiration were measured with a digital humidity analyser

(model 99 1 Dew-All, EG&G). The flow rate remained constant at 0.5 L-min", monitored by

Buw meten (Cole-Parmer Instrument Cornp.). Unless otherwise noted, plants were exposed

ro an inadiance of 1050 ,urnol.rn''* s" PAR and CO, concentration in the cuvette of 350

,LL L.L" (3 50ppm). Leaf area measurements were made by imaging scanned, naced, outlines

of the exposed portion of the leaf (Scion Image, release Beta 3b, Scion Corporation).

The data obtained were used to calculate IeafNCER and transpiration at steady-state,

using the equations of von Caemmerer and Farquhar (198 1). Water use efficiency (WUE)

was determined as the ratio of leaf photosynthesis to transpiration. Stomatal conductance

for CO, was calculated fiom transpiration rates and vapour pressure differences. COz

concentration within intercellular air spaces between mesophyll cells (Ci) was calculated

from stornatal conductance, transpiration, and photosynthesis measurements (Farquhar and

Sharkey 1982). Calculations for al1 physiological parametes were based on those described

by Leonardos (1999). The SAS (SAS Institute Inc.) program containing these formulae is

provided in Appendix 2.

Light response curves of NCER, transpiration, and stomatal conductance at the leaf

level were generated by varying the irradiance frorn 50 pmol-m"d to 1300 pmol~rn~2~s"

PAR. General linear rnodels were fitted using a second-order polynomial function and 95%

confidence intervals calculated (S-Plus 2000). Responses were evaluated with a two-way

ANOVA and were considered to be significantly different at light levels where the

confidence intervals do not overlap.

Changes in photosynthetic ability and other physiological panmeters through time

were measured in lemice inocuiated with Pythium at three different stages of maturity: 14,

21, and 28 DAP. This experiment (Expenment 1) was performed From 20 January to 8

March. 1999. Experiment 2, analyzed separately, involved 14 and 2 1 DAP inoculations only

and utilized plants grown from 6 May to 18 June, 1999. Daia sets for each inoculation time

were analyzed with a two-way ANOVA. Linear or quaàratic models were fitted and 95%

confidence intervals generated. The time, measured as DAI, at which metabolic parameten

were considered significantly different was detemined by comparing confidence interval

overlap.

A dose-response study was performed fiom 21 September to 2 November, 2000

(Experirnent 3), measuring leaf photosynthesis and transpiration in lettuce inoculated at 2 1

DAP with Pythirrin at the standard inoculum density (5x 10' zoospores.&') and double that

concentration ( 1 x 1 O" zoosporesmL~'). Leaf physiological parameters were measured every

4 to 5 DAI; immediately thereafter, leaf area (LI-3000, LI-COR) and fresh weight data was

collected for each plant. Shoot tissue and roots extending f?om the rockwool cube were

placed in an oven at 75 "C for 48 hours to measure biomass gain. Data was analyzed using

hw-way ANOVA.

Specific Leaf Area

As a measure of plant "leafiness" on a dry weight basis (Beadle 1985), specific leaf

area was calculated as the ratio of leaf area to shoot dry weight. Specific leaf area was

examined in plants destnictively harvested in two experiments, comparing the effecr of

inoculation at different plant ages (14 and 28 DAP inoculations; growth characterization

study 30 March to 24 A p d , 1999) and hvo inoculum densities (5~10'zoospores~rnL~' and

1 x 1 o4 zoospores*mL~'; Experiment 3 descnbed previously) on allocation of fixed carbon to

leaf tissue.

RESULTS

Influence of Pytitium dissoiocurn on growth of lettuce

The leaf area and shoot dry weight of lettuce plants following inoculation with

P~~iliirrm dissoroctrm at 14 or 28 DAP is s h o w in Figure 2.1. Commencing at 22 DAI, and

continuing until final harvest, plants inoculated at 14 DAP showed a significant difference

in shoot fresh weight (P=O.O252) and leaf area (P=O.O l36), but not shoot (P=O.O577) or root

dry weight (P=0.3907; data not shown) cornpared to non-inoculated control plants. The

signi ficant difference in shoot:root ratio (P=0.0 169) reflects the stunted shoot growth. At

maturity, these Pythim-infected plants displayed 36.1% smaller leaf area, 35.4% less shoot

fresh weight, and 39.1% reduced dry weight compared to controls. The 28 DAP inoculation

showed no significant differences in any growth parameters compared to both controls and

14 DAP-inoculated plants. Leaves of plants inoculated at 14 DAP were flatter and less

crowded than leaves of control plants.

Whole plant gns exchange and carbon nccumulation

Whole plant photosynthesis and dark respiration rates were measured over a 36-h

period with plants at commercial maturity, approximately 28 DAI (Figure 2.2A). While there

was no appreciable difference in dark respiration rates between treatments, it was observed

that SCER of Pyhitrm-infected plants was initially lower than that of the control plants at

the start of each dayiight period, not approaching control levels of photosynthesis until mid-

aftemoon. Whole plant NCER was integrated over the 36-h period to calculate total carbon

accumulation, as shown in Figure 2.2B. Afier 36 houn enclosure, infected plants

accumulated significantly less carbon (P=0.0 144), a 16.1 % reduction compared to non-

inoculated controls. Data shown is expressed by plant dry weight.

Dry Shoot Weight [g]

I . -- . -

O ..A N

Shoot:Root Ratio

Leaf Area [cm'] A - r N N W W

U l O U i O f n O U l 0 0 0 0 0 0 0

a 0 0 0 0 0 0 0

P

I --

O 10 20 30

Time [ h ]

Figure 2.2 Diurnal gas exchange, A, and carbon accumulation, B, in lettuce plants measured 28 d after Pythium inoculation. Each point represents the hourly mean and standard error of 4 chambers, expressed by dry weight of enclosed plants. Noninoculated control plants ( ), inoculated plants (@). Plants were measured at ambient CO2 (350 &=L*') and 800 umol-rn4*s" PAR irradiance. L/D indicates Iight (0) and dark (I ) periods. The horizontal mis represents tirne of NCER rneasurements comrnencing with the start of the first phoropenod.

Influence of irradiance on whole plant NCER

The influence of varying irradiance levels on photosynthesis of manire plants is

s h o w in Figure 2.3. Absolute measurements of NCER, on a per chamber basis (two plants

were enclosed in each chamber), is displayed in Figure 2.3A. Control plants exhibited higher

photosynthetic rates at al1 light levels above 300 ~rno1m"s-'. Correcting for the larger size

of the control plants, Figures 2.3B and 2.3 C express NCER by leaf area and dry weight

respectively, indicating the reverse trend: at most levels of irradiance, fphium-infected

plants showed higher NCER. The differences are significant (Pc0.000 1 ) for al1 light levels

except the extrernes, c 180 pmol~m"~s*l and > 1400 ,umol-m"d NCER of infected plants was

saturatcd at 1 100 pmol.m-'.s-', while control plants do not appear to reach saturation even

at 1.1100 i r m ~ l m % - ~ PAR. infected plants had a slightly higher light compensation point, but

overlapping confidence intervals in this area indicate the difference is not statistically

significant.

Influence of CO, Concentration

In studies of short terni variations in CO, concentration in relation to net carbon

exchange of mature plants (28 DAI), whole plant photosynthesis increased with increasing

CO1 concentration. Data in Figure 2.4A demonstrate that, measured per chamber,

photosynthetic rates of non-inoculated control plants were higher than in inoculated plants

at al1 CO, concentrations measured. Adjusting for the increased biomass of control plants,

Figures 2.4B and 2.4C express NCER in tems of leaf area and dry weight. NCER in

PJ-rhictnz-infected plants was significantly higher across the range of CO2 concentrations

measured than in control plants (P<0.000 1). CO, saturation was estimated at 1000 &L-'

for both inocufated and noninoculated plants, with a maximum NCER of 6 pmol.m'2d in

-200 O 2Q@ 4C0 600 800 1000 1200 1400 1600

lrradiance [ .m~lm-~-s ' ' PAR]

Figure 2.3 Influence of irradiance on whole plant photosyuthesis of lettuce 28 DAI with Pyhium. Noninoculated controls ( a), inoculated plants (O). A, NCER exaressed per chamber; i of fitted regression lines and 95 % confiden& intervals = 0.96 for both control and inoc- ulated plants. B, NCER expressed by leaf area of enclosed plants; ?= 0.96 for both control and inoculated plants. C, NCER expressed by dry weight of enclosed plants; i= 0.97 and 0.95 for control and inoculated plants, respectively. Second order polynomial functions were used to fit the data. Plants were rneasured at ambient CO2 (350 p ~ * L - ' ) and 2 1 " C.

CO, Concentration [ PL-L-'1

Figure 2.4 Influence of short term variations in CO, concen- tration on whole plant NCER in lettuce 28 d afier inoculation with Pythium. Noninoculated controls ( 0 ), inoculated plants ( O ). A, NCER expressed per chamber, with ?= 0.89 and 0.90 for the fitted regression lines and 95 % confidence intervals for noninoculated controls and inoculated plants, respectively. B, NCER expressed by leaf area of enclosed plants, ?=0.85 and 0.88 for control and inoculated plants. C, NCER expressed by dry weight, 0.76 and 0.78 for control and inoculated plants.

infected plants and 4 pmolm-L-~' in control plants, measured on a leaf area basis.

Leaf chlorophyll content

SPAD values were measured in the greenhouse at several time points post-

inoculation in both inoculated and control plants. The expenment was perfomed hvice, but

due to seasonal variations in recorded SPAD values, observations of the expenmental

repetitions could not be pooled. Figure 2.5 represents SPAD values in lemice grown during

fipinter. Chlorop hyll content was significantly higher in Pythium-inoculated plants when

compared to controls (P<0.000 1) and visible to the human eye as a slight darkening of the

leaves at approxirnately 20 DAI. There was no difference between plants inoculated at 14

and Z 1 DAP (P=0.9847). SPAD measurements taken in early surnmer followed the same

trend (inoculated plants statistically different from controls, P=0.0002).

Influence of irradiance on leaf gas exchange

The influence of vaiying levels of irradiance on NCER of individual leaves of

healthy and infected plants is seen in Figure 2.6A. At light levels at and above 800

umol-m"*s", control plants had significantly higher NCER (P=0.0386). Leaves of non-

inoculated plants saturated at 1000 pmol-m"d PAR, while those of Pythium-infected plants

saturated at approximately 700 ,umolm"d and even appeared to decrease in NCER at

higher light intensities. Wilting was evident in infected plants at the highest light level so

those data were excluded, as leaves of those plants were not at steady-state. Measurements

of transpiration (Figure 2.6B) and stornatal conductance (Figure 2.6C) demonstrated similar

patterns, with significantly different responses above 400-500 pmol~m~2*s-' irradiance

(P<O.OOO 1 ) .

5 10 15 20 25 30

Days After Inoculation

Figure 2.5 SPAD measurements in lettuce following PytItium inocuiatioa. Shown are noninoculated controls ( ), plants inoculated at 14 DAP ( ), and 21 DAP-inoculated ( ) plants. Each bar represents the mean and standard error of 5 troughs, with 3 sampled plants per nough and 6 subsampled Ieaf measurements From each plant. The expenment was performed twice. A representative graph fiom plants grown under winter conditions as described in Materials and Methods is shown. 14 and 2 1 DAP treatments resulted in significantly higher SPAD measurements cornmencing at 20 DAI.

1 rradiance ~ l - r n - ~ d PAR]

Figure 2.6 Influence of irradiance on lettuce leaf physiology, 28 d post-inoculation with Pythium. Control plants ( ); inoculated plants (O). Panel A shows leaf NCER with fitted regession lines and 95 confidence intervals (?=0.96 for control plants, 0.81 for inoculated plants). B, leaf transpiration, and C, stomatal conductance, display similar trends. ~ L 0 . 9 3 and 0.8 1 for leaf transpiration of control and inoculated plants, respectively, and 0.90 and 0.70 for stomatai conductance for COZ.

Influence of stage of host maturity on leaf gas exchange and water relations

Altered gas exchange following initial P. dissotocum infection was investigated

fùrther at the level of individual leaves. Steady-state IeafNCER of 14 DAP-inoculated plants

in Experiment 1 is shown in Figure 2.7A. Individual plots with fitted regression lines and

95% confidence intervals are displayed in Figure 2.7B, to illustrate the manner in which

statistical ly significant response variables were assessed. Table 2.1 presents al1 physiological

parameters measured in plants inoculated at 14,11, and 28 DAP in Experiment I , including

leaf NCER, transpiration, water use efficiency, stomatal conductance for CO,, and mesophyll

intercellular CO2 concentration. Metabolic changes are expressed as a percentage of control

values corresponding to the three treanent groups. Also listed is the time elapsed following

Pphitm inoculation before physiological alterations were significant, and the P-value for

the significance of each physiological factor over the entire period of disease development.

Leaf photosynthesis, transpiration, and stomatal conductance were each significantly

reduced by 16 DAI in plants inoculated ai 14 DAP. Intercellular CO, decreased significantly

at 25 DAI, and water use efficiency increased at 25 DA[. Leaf NCER was reduced by 13.6%

at maturity, transpiration by 28.6%, stomatal conductance by 44.5%, and intercellular CO,

concentration by 1 1.1%. Water use eficiency increased by 5.5%.

Ln plants inoculated at 2 1 DAP, none of the metabolic parameters was significantly

altered over the course of the growth cycle. Transpiration was reduced by 5.8%, and stomatal

conductance for CO2 by 13.6%, compared to non-inoculated controls, but due to high

variation, did not differ statistically. Water use efficiency increased by 12.7%. Physiological

changes associated with inoculation at 28 DAP in Experiment 1 were similarly insignificant

even at plant maturity.

Days After inoculation

Figure 2.7A. Reduction in leaf NCER in lettuce following Pythium inoculation 14 DAP, under wintedspring conditions in Experiment 1. Each bar represents the mean and standard error of a minimum of 5 replicate plants; Noninoculated controls (m), Pythium-inoculated plants (0 ). Measurernents were taken at an irradiance of 1050 ,umol=m"*s~' PAR and ambient COt (360 uL-L"). B, Statistical interpretation of physiological parameten. ANOVA was performed on the entire data set of each treatment. In cases where the ANOVA indicated a significant difference in response, regression lines were fitted and 95 % confidence intervals generated. DAI values where the confidence intervals did not overlap were considered to be significantly different.

Table 2.1 Leaf physiological changes associated with Pythiurn root rot in lettuce plants grown under wintedspring conditions (Expenment 1) as descnbed in Marerials and Methods. KS= no significant differences were observed.

Mstabolic Inoculation Time of Change From P- Parameter Time (DAP) Significant Effects Controls at Final Value

( D M Harvest (%)

NCER

[prnol Co,-rn'.~-']

Transpiration

[rnmol ~ ,@m"-s - ' ]

Water use efficiency

S tomata t conductance

[ prno 1 C O ~ ~ - ' . S - ' ]

Intercellular CO,

[ p ~ - ~ ' l ]

The same metabolic parameters were assessed in Experiment 2, under summer

conditions. To increase repetition and reduce variation, plants were inoculated at 14 and 2 1

DAP only. Transpiration, stomatal conductance, and intercellular CO, showed significant

alterations 13 DAI in plants inoculated at 14 DAP (see Table 2.2), with significant changes

in NCER occumng at 10 DAI. WUE was not significantly affected. At rnaturity,

transpiration was reduced by 36.1% in infected plants, stomatal conductance by 44.3%, and

intercellular CO, by 15.7%. Water use efficiency was increased by 2 1.1% in infected plants

compared to noninoculated controls.

in plants inoculated at 21 DAP in Experiment 2, leaf NCER and most other

parameters showed significant changes by 7 DAI, with WUE by I O DAI. NCER was reduced

3 .3%. and transpiration by 38.6%. Again, stomatal conductance was the most greatly

affected measured variable, with a 40.9% reduction by final hamesr.

Visible shoot symptoms in inoculated plants in both experiments were not observed

until at least 2 1 DAI, and appeared as a slight darkening of leaf tissue, stunted shoot growth.

and occasionally wilting on particularly hot, high irradiance days.

Influence of inoculum density on severity of disease effects

Lemice was inoculated with Pyrhiurn at two different inoculum densities: the

standard 5x 1 o3 zoospores~ml" and 1 x 1 O" sporesmL-'. Figures 2.8.4-D show that the higher

inoculum density did not intensiQ disease effects. While the higher density resulted in

significantly reduced leaf area (P=0.0276), leaf photosynthesis (P=0.0073), and

transpiration (P=0.006 1) compared to the healthy control commencing at 18 DAI, shoot dry

weight wûs not significantly different (P=0.0944) even at the end of the experiment. Plants

inoculated with the higher inoculurn density exhibited higher leaf area (P=0.0009) and shoot

Table 2.2 Leaf physiological changes associated with Pythiurn root rot in inoculated lemice plants grown under sumrner conditions (Experiment 2) as described in Materials and Methods. NS= no significant differences were observed.

Metabolic Inoculation Tirne of Change From P- Parameter Time (DAP) Significant Effects Controls at Final Value

(DAI) H m e s t (%)

KCER 14 I O - 18.7 <O.OOO 1

Transpiration 14 13

[rnmol H ~ O . ~ * ' . S - ' ] 2 1 7

Water use efficiency 14 NS t21.1 0.099 1

2 1 I O +15.7 0.0067

S tomatal conductance 14 13

[pmol ~ O , ~ m - ' ~ s " ] 2 1 7

Days After Inoculation [DAI] Days After Inoculation [DAI]

Figure 2.8 Influence of Pythium Inoculum Density on Growth and Metabolism of Lettuce inoculated at 21 DAP. A, Leaf area, B, Shoot dry weight, C, Leaf NCER, and D, leaf transpiration. Noninoculated controls ( O ), plants inoculated with 5x 1 o3 zoospores. m ~ " ( a ), and plants inoculated with 1x1 O' zoosporesmL-'( N ). Each bar represents the mean and standard error of a minimum of 4 replicate plants. Plants were grown under fall conditions as described in Materials and Methods.

dry weight (P=0.0027), but not NCER (P=0.6775) or transpiration (P=O.6 1 15) compared

to plants inoculated with the standard inoculum density.

Specific Leaf Area

The allocation of fixed carbon to leaf tissue, based on dry weight, is presented in

Figure 2.9. In the growth charactenzation study, there were no differences in specific leaf

area between controls and plants inoculated at either 14 or 28 DAP (P=0.4872; Figure

2.9.4). Plants in Experiment 3, inoculated at nvo different inoculum densities, also showed

no significant differences in specific leaf area compared to noninoculated controls

(P=0.7007; Figure 2.98).

Days After inoculation [DAI]

Figure 2.9 Specific leaf area of lettuce inoculated with Pytirium at different stages of growth and two inoculum densities. A, Noninoculated controls ( ), inoculated at 13 ( ) and 28 ( Q ) DAP. B, Noninoculated controls ( D, inoculated with 5x 1 o3 ~ o o s ~ o r e s m L ' ~ ( ), and inoculated with l x 1 O' zoosporesml-' ( ). Each bar rep- resents the mean of a minimum of 3 replicate plants. Plants in panels A and B were gram under spnng and aunimn conditions, respectively, as described in Materials and Methods.

DISCUSSION

These data are the first showing the influence of a Pythium root rot pathogen on

carbon assimilation of host plants. In addition, the mode1 systern used of P. dissotocum in

lemice is the first such description of plant-pathogen interactions in a hydmponic systern.

Measurement of gas exchange characteristics as an indirect means to produce disease

progress curves bas been used only rarely in plant disease epidemiology. The gas exchange

methodoiogy is nondestructive, objective, and physiologically based, which may prove

important in many pathosystems where visible symptoms are delayed or insufficient, as in

the case of subclinical disease. An additional advantage in root disease studies is the ready

accessibility of the shoot compared to roots for physiological or disease measurements.

Understanding host physiological responses associated with Pythium infection of roots may

provide insishts that could lead to improved technologies to control the disease.

CVhole pianr yield and gas exchange

initial experiments to determine the aggressiveness of the P. dissotocum isolate

established that final velds were reduced by approximately 35-40% when plants were

inoculated 14 days after transplanting (Figure 2. l), which agreed with observations in

previous studies of other isolates of this species on lemice (cv. Salina; Stanghellini and

Kronland 1986) and spinach (Gold and Stanghellini 1985) at similar nutrient solution

temperatures. Final shoot yield of control plants was comparable to that of lemice in earlier

studies (Wheeler et al. 1994), and leaf area was similar to that in cornmercially-produced

hydroponic butterhead lemice purchased from a local vendor (data not shown),

demonstraiing that the cultural systern was sufficient to provide normal growth conditions.

The measurements of whole plant photosynthesis and estimation of carbon

accumulation presented in Figure 2.2 substantiated the observed growth effects associated

with Pythiirm root rot in lemice. Most of the reduction in carbon accumulation was

accounted for by the inhibition of photosynthesis in infected plants in the fint six houn of

the photoperiod, possibly due to delayed stomatal opening. A similar response was observed

in potato plants infected with Verticillium dahliae (Bowden and Rouse 1991b). in the

Irttuce studies, integration of NCER measurements during only 36 hours of enclosure

indicated that 16% less carbon accumulated in inoculated compared to noninoculated plants.

This difference, considered over the entire growth period, provides clear suppon that

reduced photoassimilation is involved in the stunted shoot growth of Pythium-infected

plants.

The lack of measured difference in dark respiration rates between healthy controls

and inoculated plants contrasted with numerous reports in the literanire in which respiration

rates increased in plants infected by pathogens. Respiration typically increases dunng the

initial hours or days after infection, but declines to normal levels at later stages of disease

development (Agrios 1997; Daly 1976). increased respiration is also associated with greater

activity of the pentose phosphate pathway in infected plants, the chief source of phenolic

cornpounds important in defense rnechanisms. It is possible that respiration initially

increased in diseased plants, but any alterations in respiration were undetectable when this

expenment was performed on the mature plants 28 DAI.

The light response curves presented in Figure 2.3 showed some unexpected findings.

Photosynthesis on a whole plant bais (Le. per charnber; Figure 2.3A) over a broad range of

light levels (100 to N400 pmol*m'f-f' PAR) was reduced in infected plants, which was

expected given the approximately 35-40% reduction in photosynthetically active leaf area

(Figure 2.1 A). However, when NCER was normalized for leaf area or dry weight (Figures

2.3B and C) of the enclosed plants, infected plants consistently displayed higher rates of gas

exchange, at al1 irradiances except the extremes. There are several possible explanations for

these observations. Differences in canopy architecture between healthy and infected plants

undoubtedly affected light interception, the efficiency of which is determined by leaf

orientation, distribution of leaf area, and canopy height (Boote and Loomis 1991). The

stunted shoot growth of Pythium-inoculated plants resulted not only in reduced leaf area and

plant height. but reduced leaf angles so each leaf of diseased plants was oriented more

perpendicularly to the light source than in control leaves, increasing light interception.

Leavrs of control plants also experienced much greater mutual shading from higher leaves

on the same plant and From the canopy provided by other enclosed plants. Whatever the

contributing factors, the end result was a much higher efficiency of light interception in

inoculated plants, as indicated by the greater rates of NCER when expressed on the basis of

biomâss.

Besides plant architecture, biochemical factors possibly contributed to increases in

photosynthetic rates in infected lemice plants. The rate of electron transport limits

photosynthesis at low irradiances, whereas NCER is fiequently limited by nbulose-1,s-

bisphosphate carboxylase-oxygenase (Rubisco) at higher irradiances (Ogren and Evans

1993). Host plant responses to disease affecting either of these factors, confounded by the

high evapontive demand in high intensity light, may be involved. Another possibility is that

plants compensate for overail reduced metabolism associated with disease by increasing

photosynthetic efficiency in some parts of the plant (Madeira and Clark 1995).

Whole plant CO, response c w e s , generated to investigate the effect of short-term

increases in COz concentration on photosynthesis of lettuce inoculated with Pythizrm (Figure

2.41, were similar to those of increasing irradiance, with infected plants showing less

productivity at ail CO2 levels when expressed per plant. Corrected for leaf area or dry weight

of the enclosed plants, infected plants had higher NCER at al1 CO, concentrations except the

lowest. Connol plants had the same photosynthetic rates at LOO0 ~ L * L " (1000 ppm) as

Pytki~inz-infected plants did at ambient CO?. This suggests that CO, enrichment in

commercial greenhouses might be able to offset crop productivity and rninirnize yeld

reducrions in infected plants pnor to harvest. Edwards (1970) found similar results in barley

leaves affected by powdery mildew and concluded that dysfunction of the photosynthetic

apparatus was unlikely to account for the low photosynthetic rates in diseased plants at

normal CO,.

Chlorophyii content in in fecied plants

Chlorophyll content of leaves, a frequently-used indicator of plant stress, usually

correlates strongly with photosynthetic capacity. Visual estimation of leaf greenness is

highly subjective, so a portable chlorophyll meter may provide more reliable evaluation. The

unitless nurnbers caiculated (SPAD measurements) correspond to actual leaf chlorophyll

concentration, estimated on a leaf area basis by traditional destructive colonmetric methods

(Monj e and Bugbee 1992; Dwyer et al. 199 1). Using the extraction procedure and equations

of Inskeep and Bloom (1985) with N,Ndimethylformamide (DMF), the equation of

y= -5 . ~ O ~ + O . ~ ~ ~ X - O . O O ~ X '

was generated, where x=SPAD value, to quanti@ the relationship of SPAD measurements

and extractable total chlorophyll in Bella Green lettuce in our greenhouse environment

(Appendix 1). Transformation of SPAD values obtained in lettuce following inoculation with

Pyrhicim (Figure 2.6) indicated that chlorophyll content increased approximately 2% in

infected plants by the end of the experiment. This observation may contribute to an

rxplanation of higher whole plant photosynthesis in inoculated plants when expressed on a

leaf area or dry weight basis.

A darkening of infected leaves was the first aboveground symptom identified in

infected plants, visible to the casual observer at approximately 2 1 DAI. A similar darkening

in leaves of potato plants infected with Venicillium duhliae was observed, associated with

the systemic phase of disease progression, in which al1 upper leaves were significantly

affected (Bowden and Rouse 199 1 a). However, the darker green leaf colour in potato was

temporary, whereas in Pythiiim-infected lettuce the colour change penisted until the

temination of the experiment. The range of SPAD values collected for control plants of

Bella Green were consistent with those recorded in a previous study using an unspecified

Icttwe cultivar (Fukada et al. 2000). It rnay be that increased chlorophyll concentration in

infectcd leaves is a snategy to compensate for reduced leaf area, or to facilitate stress

responses or defense mechanisms.

Leaf 'gas exchange and water relations

Leaf responses to varying irradiances ofien differ fiom whole plant responses, such

as saturation of NCER at lower light intensity and attainment of much higher photosynthetic

rates (Jiao et al. 1997). Lemice was no exception, as the light response curve of leaf NCER

(Figure 2.6A) was the opposite of those at the whole plant level after correction for leaf area

or dry weight: there was a higher NCER in leaves of noninoculated compared to inoculated

plants. A similar light response for leaf NCER was reported for potato infected with

Verticillizirn (Bowden and Rouse 199 1 b). Leaves of both control and inoculated plants

reached light saturation at approximately 300 ~ r n ~ l * r n * ~ . s ' ' lower light intensity than whole

p!ants.

Leaf gas exchange, mediated through stomata, must meet the cornpeting demands of

maximizing CO1 uptake while minimizing water loss. There exist several important

limitations to carbon assimilation and transpiration in the leaf, including diffûsional

resistance from leaf air spaces through stomatal pores, and boundary layer resistance, which

consists of the layer of relatively unstirred air next to the leaf surface (Taiz and Zeiger 1998).

Transpiration has the additional resistances involved in water and solute aquisition at the

root surface as well as transport through the xylem. Funher resistance considerations for CO,

in the leaf include diffusion into mesophyll cells and into chloroplasts (Nobel 1991). The

diffusion of both water vapour and CO, is controlled by concentration gradients. Therefore,

the extent of stomatal opening is somewhat dependent on guard cell CO2 concentration,

which reflects C, within the leaf. in this manner, COz enrichment of the greenhouse

atmosphere could overcome partial stomatal closure in Pytliium-infected plants.

The existence of several feedback loops complicates the detemination of stomatal

and biochemical constmints in the control of gas exchange in transpiration and NCER

(Nobel 199 1 ; Jones 1998). Biochernical limitations relate to the photosynthetic apparatus and

to enzymes and substrates involved in photosynthesis. Alterations in stomatal conductance,

the inverse of resistance, effect changes in photosynthetic or transpiration rates that c m

subsequentl y modiQ intercellular CO,, influencing conductance. Determining the nature of

the cause-and-effect relationships between stomatal conductance, intercellular CO2,

photosynthesis and transpiration is cornplex, especially considering that calculations for Ci

and stomatal conductance rely on values derived from transpiration and NCER. It must be

acknowledged that the interdependency of calculated values dernands rhat any inferred

conclusions of causality be speculative rather than absolute.

Currently, it is theoretically impossible to calculate a value to specify the stomatal

contribution relative to the mesophyll contribution to a reduction in NCER (Sharkey 1984).

To determine the limitation of biochemical photosynthetic capacity, the relationship between

NCER and C, should be developed (Sharkey 1984). For example, a situation where NCER

decreases while C, remains constant c m be interpreted as a saong biochemical as opposed

to stomatal limitation of photosynthesis (Farquhar and Sharkey 1982; PospiSilova and

Santnitek 1997). in the potato- Verticilliiirn pathosystem, the relationship between carbon

assimilation rate and intercellular CO, was unaltered compared to corirrol plants (Bowden

et al. 1990), implicating stomatal closure as responsible for initial reductions in

photosynthesis. The situation is further complicated by the realization that standard

calculations of Ci may be inaccurate if significant stomatal heterogeneity or patchy stomatal

closure exists (Jones 19%). Stomatai patchiness can be defined as the nonuniform behaviour

of stornatal apertures and the resuiting variable distribution of stomatal conductance

(PospiSilova and Santrii~ek 1997), and can be induced by npid (but not gradual) water

stress. Minimal patchiness was observed in leaves of potato plants infected with Verticilliurn,

tested by autoradiography (Bowden et al. 1990).

The reductions in carbon assimilation and transpiration observed in Ieaves of

Pyrhiurn-infected plants were generally concomitant with alterations to stomatal conductance

to CO2 and Ci (Tables 2.1 and 2.2). Stomatal conductance was the parameter rnost affected

in al1 cases except in plants inoculated at 28 DAI? in Experiment 1 (Table 2.1). The time

course of physiological alterations associated with Pythiltm infection indicated that plant age

at inoculation was a significant determinant of susceptibility, with younger plants more

affected than older plants. There was also a strong seasonal component to the variation in

disease effects observed, possibly resulting fiom differential incidences of infection by

zoospores influenced by solution temperature, and by higher evaporative demand under the

higher irndiances and temperatures experienced by summer-grown plants in Experiment 2.

Reduced water potential could affect NCER as a reduced capacity for RuBP

regeneration (Farquhar and Sharkey 1982). In a situation in which C, is higher after water

stress than in controls, despite decreased stomatal conductance, it can be concluded that

stomatal closure is not the main determinant of reduced NCER (Farquhar and Sharkey 1982).

The same conclusion rnay be drawn when Ci is unchanged despite reductions in conductance

and assimilation rate. It is evident that loss of root integrity associated with browning and

rotting, as in Pyrhitrm infection, may have a significant effect on the hydraulic efficiency of

the plant as a whole, inducing water stress in spite of the plentiful water availability in

hydroponic solutions. Defence responses in the xylem such as phenolic deposition and tylose

formation may impede the bulk flow of water to an even greater extent. Future work to

detennine the relationship of assimilation to Ci across a broad range of intercellular CO2

concentrations may clai@ the physiological responses involved in reduced NCER in

Pythirtm-infected plants. That water use efficiency, the ratio of fixed CO2 to H20 lost as

transpiration, was the only calculated parameter to increase consistently at each inoculation

time (Tables 2.1 and 2.2) indicated that stomatal closure in infected plants was a beneficial

response in maintaining intemal water statu. WUE is maximized by reductions in stomatal

opening and C, mTobel 199 1 ; Bowden and Rouse 199 1 b), as transpiration is decreased more

than NCER by partial stomatal closure.

Because lettuce roots were considered too delicate for direct measurement of

hydraulic conductance, a future experiment that may elucidate stomatal closure related to

vascular dysfunction would involve increasing relative humidity in the armosphere

surrounding infected plants, allowing opening of partially closed stomata. Results From a

humidity experirnent with potato inoculated with Verticiliim indicated that when the vapour

pressure gradient between air and leaf was reduced, stomata of diseased leaves were capable

of opening (Bowden 1989, as cited in Bowden et al. 1990). If this was found to occur with

Pj*thiton- in fected hydroponic lemice, the reduced water supp ly would be 1 inked to reduced

hydraulic conductance.

Export of photoassimiIates out of source leaves was not directly measured in these

experiments so it is difficult to definitively state that reductions in carbon assimilation were

solely responsible for the stunted growth of infected plants. However, in other esperirnents

on export of radiolabelled carbon in Bella Green lettuce accidentally infected with fythium,

export of newly fixed carbon was reduced by approximately 20% (data not shown).

Geranium inoculated with the foliar bactenal pathogen Xanthonlonas campestris pv.

peiargonii showed reduced photosynthesis and export at both ambient and enriched CO2

(Jiao et al. 1999). Export in the dark was completely inhibited pnor to the appearance of any

visible symptoms. Daytirne export was more affected than NCER under both CO, regimes

and at al1 tirne points rneasured following inoculation, indicating that reduced biomass

accumulation could be attributable to differential translocation of fixed carbon in addition

to reduced capacity for fixation. Further study of expon in Pyrhizirn-infected lettuce could

determine more accurately the cause underlying decreased biomass accumulation leading

to stunting of the shoot.

Determination of specific leaf area, a more traditional method of estimating

allocation of fixed carbon in photoçynthesis, indicated no differences between noninoculated

sontrols and Pythim-infected plants in two experiments (Figure 2.9). These observations

provided some evidence that carbon partitioning was not a major factor causing reduced

shoot expansion in infected plants.

Cotnparisons ivich previous w r k in other pathosystems

The findings that Pythium infection significantly affected photosynthesis, and hence

productivity and growth, in lettuce augmented similar findings in potato infected with

Verricillirrrn (Saeed et al. 1997; Bowden and Rouse 199 1 b; Bowden et al. 1990; Haverkort

et al. 1990). tomato with Fzrsarium (Duniway and Slatyer 197 1), and avocado affected by

P/zv~ophthora (Ploetz and Schaffer 1989). Stomatal conductance was ofien found to be the

most sensitive indicator of disease, in ternis of both timing and magnitude of effect (Bowden

and Rouse 199 1 b). The relative timing of gas exchange effects with the differing pathogens

was variable. possibly reflecting differences in types of disease, pathogen multiplication

within host plants, and rapidity and extent of host defence responses. Previously rnost

authon have concluded that CO, limitation resulted from stomatal closure rather than direct

effects on the photosynthetic process in the mesophyll (Duniway and Slatyer 197 1; Bowden

el al. 1990), indicating opportunity to fix CO2 was the limiting factor in growth of infected

plants, rather than photosynthetic capacity. Although it seems a valid explmation, further

experiments and deeper analyses of existing data are necessary to extend that conclusion

definitively to lemice infected with Pyrhium.

In most pathosystems including lettuce-Pythium, reductions in gas exchange were

observed before the onset of visible syrnptoms. This introduces the possibility that gas

exchange rnay be used in early indication of disease. As drought stress produced similar

physiological patterns as Verticillium infection, namely decreased assimilation rate, stornatal

conductance, and Ci, in potato, cautious interpretation of gas exchange alterations will be

necessary (Bowden and Rouse 199 1 b). Fluorescence analysis in conjunction with gas

exchange rneasurement may prove useful in determining direct effects on photosynthetic

biochernistry during disease development especially when visible symptoms are not present

(Daley 1995). This may allow development of remote sensing equipment suitable for early

disease or nutrient stress detection in commercial greenhouses. The earliest possible

recognition of disease problems is crucial in order to apply appropriate treatments to prevent

epidemic development.

Eficr oj'irtoarlirrn densiy on disease severiry

Dose-response relationships between Pythium inoculum density and severity of

infection were investigated in Experiment 3 (Figure 2.8). The standard inoculum density

(5x 1 O' zoosporesml") actually produced more severe disease than did the higher

concentration, an unusual finding. In cucumber infected with Pythium aphonidermatum,

increasing pathogen inoculum concentration decreased plant dry weight, number and weight

of marketable fruit per plant, as well as h i t shelf life in a Iinear fashion (Menzies et al.

1996). Potato inoculated with Verticillium dahliae showed a delay in disease progress by 10-

15 days as a result of increasing the inoculum density by a factor of four (Bowden and Rouse

199 1 a).

Heightened cornpetition for binding sites on the lettuce root surface, possibly

accompanied by increased concentrations of cell-wall-degrading enzymes secreted by

zoospores, may have elicited a hypersensitive response or other defence responses in plants

exposed to the higher inoculum density. The hypersensitive response is a localized induced

ce11 death at the site of pathogen infection in host plants (Agios 1997) which is thought to

provide resistance to the host by limiting pathogen growth within rhe plant. Klisiewicz

(1968) reported a hypersensitive response to Pythiurn in resistant safflower. Mutual

inhibition of zoospore germination in high spore densities might be another possible

explanation for the observations in Experiment 3.

CONCLUSION

Bella Green lemice inoculated with this isolate of Pythium dissotocurn experienced

35-40% reduced growth, as measured by leaf area and shoot fresh and dry weights,

indicating that this isolate possessed moderate pathogenicity, not high enough to cause plant

death, but substantial enough to exceed subclinical classification. A major factor in the

decreased yield of inoculated plants was the reduced supply of photoassimilates provided

by gas exchange. Whole plant photosynthesis of infected plants was significantly inhibited

for the first six hours of the daylight penod, measured at commercial rnaturity, 28 DAI.

lntegrated over the 36 hour enclosure period, carbon accumulation, the main contributor to

biomass gain, was decreased by an estimated 16%, indicating that reduced photosynthetic

activi ty was at least partially responsible for stunting of Pythium-infected plants.

Whole plant photosynthetic measurernents in response to varymg levels of irradiance

and atmospheric COI in infected plants were decreased per plant compared to healthy

controls. When these measurements were nonnalized for the reduced biornass and leaf area

available for gas exchange, infected plants displayed higher NCER than controls at al1

intemediate light and CO, levels. This apparent increase in productivity was partially due

to greater light interception associated with reduction of leaf shading and the more

perpendicular leaf orientation of infected plants, and to increased chlorophyll concentration

observed in Pyrhium-infected leaves. Additional expenments such as Rubisco activity or

oxidative enzyme assays could indicate whether other biochemical factors are involved.

Exposure to higher inoculum densities did not significantly increase severity of

disease. It appears that there is a threshold for inoculum concentration surrounding root tips,

above which defence responses are induced. Investigation of NCER and transpiration at the

leaf level allowed calculations of other physiological factors that may help to hilly determine

the mechanism(s) underiying reduced productivity in inoculated plants. Several workers

shidying other pathosystems have suggested that reduced hydraulic efficiency stemming

from vascular dysfunction was the primary cause of stomatal alterations and subsequent

decreases in carbon assimilation. The physiological evidence appears to support this

conclusion with Pyrhiurn; however, further expenments are necessary to determine in finer

detail the cascade of events involved in Pythicrrn infection.

The rime course of physiological events accompanying Pythilrm infection in lettuce

drscnbed in [hie chapter is the first such study. This research confimed previous reports that

younscr seedlings are more susceptible to Pythium infection than older plants, that seasonal

variations exist with regards to disease progression, and that gas exchange alterations

precede the appearance of visible syrnptoms. The latter point substantiates recommendations

that gas exchange charactenstics rnay be usehl in early diagnosis of disease and facilitates

the exploration of fluorescence and other remote sensing equipment, already used in

quantification of saline or metal stress, as possible tools in diagnosing plant disease. Early

diagnosis of disease is cntical in optimizing remediation technologies, especially in

recirculating hydroponic systems where the nsk of rapid dispersal and epidemic

development is high. Chapter 3 of this thesis extends these gas exchange determinations to

an ornamental crop at the vegetative growth stage, chtysanthemum, infected with another

P y t h i m species.

Chrpter 3: Photosyuthesis and Plant Productivity of Chrysanthemum (Chrysanthemum

wurifolium L. cv. Fina) Following Infection by Pythium aphaiiidermatum

INTRODUCTION

Floriculture sales have continually nsen in Canada over the past decade, reaching

S903 million in 1998 (Agriculture and Agi-Food Canada 1999b). Chrysanthemum

(Cht?~anrhemrrni morifolizirn L.) is second only to rose in production terms, with 20.9

million stems hanrested as cut flowen and 12.8 million cuttings taken annually (Agriculture

and Ap-Food Canada 1999b). Chrysanthemum is also one of the major pot plants grown.

The inflorescence exhibits the typical composite floral morphology, radially sytnmetrical

with an outer ring consisting of conspicuous, petallike, n y fiowers surrounding a central

clustrr of minute tubular disk flowen, usually with both stamens and pistils. A series of

modified leaves (bracts) arising from the base of the flower stalk supports the head. The

flowers are edible, although generally not marketed as such. A popular cultivar in

commercial greenhouses in Ontario, Fina produces a spray of white flowers.

Single stem chrysanthemum production in soi1 is one ofthe most intensive greenhouse

cultivation systems (Hansen 1999). Improvements in production eficiencies, crop yields and

quality are in demand, as in the floriculture industry as a whole, to counter the rising influx

of cut flowers from South Amenca and other regions (Agriculture and Agri-Food Canada

1999b). As root rots caused by Pyrhium and other pathogens are a serious probiem in

c hrysanthemum (de Kreij and Patemotte 1999), commercial growen have viewed

recirculating hydroponics as both roo expensive and too high a risk of disease. Recently,

however, a modified NFT system has been developed for cut chrysanthemum production.

Preliminary analysis indicates that the system may result in a 50% increase in stem per unit

area production while lowenng labour and material costs (Hansen 1999). To assess the

potential for adapting recirculating hydroponics to chrysanthemum culture, experiments were

performed using physiological parameters to quanti@ susceptibility, prior to flowering, of

chrysanthemum cv. Fina to infection by Pyfhium aphanidennatrrrn in an NFT systern. The

more robust root system of chrysanthemum, compared to the fine roots of lettuce, allowed

measurement of root hydraulic conductance to determine what role changes in root hydraulic

cfficiency might play in the infection process.

bL4TElUALS AND METHODS

Plant lbl~rerial

Chrysanthemum cv. Fina cutrings were obtained fiom Erieview Acres, a commercial

chrysanthemurn greenhouse in Kingsville, Ontario, and rooted in perlite (Holiday, VIL

Vermicuiite inc., Toronto, ON) or rockwool cubes (SBSA 36-77, Grodan) for three weeks

under regular misting. Plants grown in single pot culture (Experiment 1) were rinsed in tap

\irater to remove perlite from the roots and suspended in a 1.9 L pot with a plastic foarn insert

(Identi-plugs, 14- 1 X-4OE, Fisher Scientific Ltd.) in the lid. The pots were filled with nutrient

solution ( 1.15 g . ~ " Plant Prod 7-1 1-27 N:P:K supplemented with 0.775 g*L-' calcium nitrate,

Plant Products Inc., Brampton, ON), of pH 4.5-5.5 and EC of 2.0 mS.cm", continuously

aerated with aquarium air Stones (Aqua-Fizw, A-962, Rolf C. Hagen hc., Montreal, PQ).

Chrysanthemums in Expenment 1 were grown fiom 15 February to 27 March 2000 without

supplernental lighting.

Plants rooted in rockwool cubes were transplanted into NFT troughs (Rehau industries

Inc.. Baie-D'Urfe, PQ) and fertilized with a modifieci Hoagland's solution with an EC of

approxirnately 2.1 mS.cm*' and pH of 4.5-5.5, maintained with the addition of H,PO, and

KOH as necessary. Each trough had separate nutrient solution, continuousiy recirculating by

a submersible pump (Little Salty mode1 1-EUAA-MD, Little Giant Purnp Co.? Oklahoma

City, OK) at a flow rate of 1Umin. EC and pH were continuously monitored in each 20 L

reservoir by suspended electrodes (Cole Parmer Instrument Co., Vernon Hills, iL) relaying

rneasurements (Argus SM 12 modules, Argus for Windows v 1.1, White Rock, B.C.) to a

cornputer. NFT chrysanthemums were g r o w from 16 Apnl to 8 June 2000 without

supplemental lighting (Expenment 2).

Pythium Inoculation Protocol

Zoospore suspensions of Pythium aphanidermafum (Edson) Fitzp. isolate 167,

previously isolated fkom commercial hydroponic cucumber, were prepared by standard

procedure (Dhingra & Sinclair 1995; Rahimian & Banihashemi 1979). Chrysanthemum plants

were inoculated 2 1 days afier transplanting (DAP) by a direct root-dipping method for 30

minutes in spore suspensions in autoclaved 100 mL beakers at an inoculum density of 104

zoospores.m~-'. Following inoculation, plants were retumed to their respective pots or

troughs. At the end of each study, al1 pots, troughs, lids, reservoirs, tubing, and pumps were

scrubbed with a 0.5% Virkon (Dispar, Vétoquinol Canada Inc., Joliette, PQ) disinfecting

solution. The solution recirculated through the system a minimum of 16 h, followed by

rinsing with deionized water for a minimum of 24 h. Air Stones used in single pot culture

were soaked in 2% hydrochloric acid solution for 30 minutes, rinsed several tirnes, soaked

in Aqua Plus water conditioner (Fincare #A-7655, Rolf C. Hagen Inc., Montreal, PQ) for 30

minutes to remove residual chlorine, nnsed again, and dned prior to re-use.

Wtole Plant Photosynthesis

From 25-32 DAI in Experiment 1, plants were removed fiom the troughs, placed in

1.9 L pots containing nutrient solution, and transported to the whole plant chamben descnbed

in Chapter 2. The daytime lighting period was fiom 0600h to 2000h at 1000 , u r n ~ l ~ r n ' ~ ~ s ~ '

PAR. Plants were allowed to equilibrate ovemight for a minimum of 12 h. Whole plant

photosynthesis and night respiration were measured for two sets of 36 h. The plant canopy

in each chamber was quantified after removal £kom the chambes with a leaf area meter (LI-

3000, LI-COR), followed by oven drymg at 75°C for 48 h. The total leaf area and dry weight

of plants enclosed in each chamber were used to nomalize NCER values for differences

among plant sizes. Carbon accumulation was calculated by integrating NCER over each 36

h period. Statistical significance of total carbon accumulation was assessed by a one-way

ANOVA (S-Plus 2000 Professional Release 1, MathSoft Inc., Seattle, WA).

Leaf C O Exchange and Transpiration

A procedure similar to that described in Chapter 2 was followed. Physioiogical

parameters were measured at several time points during the infection cycle in plants grown

both in single pots (Experimenr 1) and in NFT noughs (Expenment 2). Statistically

significant responses to Pythiurn inoculation were assessed by a two-way ANOVA. A linear

mode1 was fitted to the data, and 95 % confidence intervals generated. Time points where

confidence intervals did not overlap were considered io be significantly different.

Hydratrlic Conductance of Severed Root Systems

Following leaf photosynthesis measurements, NFT plants were refngerated overnight

in the dark at 4°C. Maximal hydntion was achieved by placing a plastic bag over the plants,

considered to be in a "pre-dam" state upon emergence from the refngerated chamber. Plant

stems were quickly severed approximately 5 cm (2 inches) from the base. The root system

was placed in a 10 mM NaCI solution within a pressure charnber (Soilmoisture) and

pressurized to 0.5 bar (50 kPa), foilowing the procedure of Radin & Eidenbock ( 1984). After

several minutes of equilibration, the exudate, referred to as xylem "sap", was collected for

30 min by attaching mbber tubes filled with absorbent paper to the excised stem. The amount

of collected Sap was detennined by weighing the tubes. The rate of hydraulic conductance

(expressed as pl*g"s*') was calculated using the total volume of fluid collected divided by

collection time and the dry weight of the root systern. Differences in hydraulic conductance

were determined with a two-way ANOVA.

Destrwtive Harvest

Following severance of the root systern, the shoot was separated into Ieaf and stem

portions and dried for 48 hours at 75 O C . Afier conductance rneasurements, roots extending

from the rockwool cube were removed for drymg as well. The data was used to construct

growth curves relating accumulation of biomass through time. Data was analyzed wirh a two-

way AXOVA.

RESULTS

Influence of Pytltium aphanidermatum on growth of chrysanthemum

Chrysanthemum plants in Experiment 2 were destnictively harvested periodically

following inoculation by Pythium aphanidermaturn, as shown in Figure 3.1. Infected plants

showed a trend towards inhibited shoot growth, as seen in Figure 3.1 A, but this trend was not

significant (P=O. 1 107). Root dry weight, represented in Figure 3. L B. was not different

benveen treared plants and noninoculated controls (P=0.3811). The shoot:root ratio,

displayed in Figure 3. lC, reflects the increased shoot dry weight of control plants but was

also statistically insignificant (P=O.lOM).

Whole plant gas exchange and carbon accumulation

Two groups of whole plant NCER measurements were taken from plants in

Experiment 2, Set 1 from 25-28 days afier inoculation with Pythirrm. and Set 2 fiom 29-32

DAI. As the two sets were significantly different (P=0.0053), they were analyzed separately.

Similar patterns were seen whether expressed by leaf area or dry weight of the plants enclosed

in the chambers (data not shown). Data collected in Set 1 are presented in Figure 3.2A. M o l e

plant NCER was not appreciably affected by Pythium inoculation. [ntepted NCER

measurements are shown in Figure 3.2B and indicate a reduction in carbon accumulation of

4.5% in infected plants, which was not statistically significant (P=0.55 15).

Slightly older plants, measured at 29-32 DAI, did show a decrease in whole plant

NCER associated with Pythium infection (Figure 3.3A). Reduction in NCER for the first

several hours of the fust daylight penod was sirnilar to that observed in lettuce. However, the

pattern was not repeated the following day; the photosynthetic rate was steadily inhibited

throughout the second illumination period. Carbon accumulation over 36 hours was

Days After Inoculation [DAI]

Figure 3.1 Growth of chrysanthemurn cv. Fina followiog inoculation with Pyrhium aphanidermatum. A, shoot dry weight, B, root dry weight, C, shoot:root ratio; there are no statistically significant differences due to inoculation. Bars represent the mean and standard error of a minimum of 5 replicate plants. Noninoculated controls ( ) and inoculated plants ( [7 ), were grown in NFT troughs under summer conditions as described in Materials and Methods.

.

O 1 O 20 30 4 0

Tim e [hl

Figure 3.2 Whole plant gas exchange, A, and carbon rccumulntion, B, in chrysanthemum 25-28 days after inoculation with Pythium (Set 1). Each point represents the mean and standard error of 4 replicate chamben, expressed as dry weight of enclosed plants. Measurements were taken of control plants (e) and inoculated plants (@) at 1000 pmol*m'2~s-' PAR and ambient COz (350 PL-L"). L/D indicates light ( O ) and dark ( ) periods. The horizontal axis represents time of NCER measurements commencing with the start of the first photopenod.

Time [hl

Figure 3.3 Whole plant grs exchange, A, and carbon accumulation, B, in chrysanthernum 29-32 days after inoculation with fythiurn (Set 2). Values are expressed by dry weight of enclosed plants. Each point represents the mean and standard error of 4 replicate chambers. Measurements were taken of non- inoculated controls (.) and inoculated plants (O) at 1000 p n ~ l * r n ~ ~ s ~ ' PAR and ambient CO, (350 PL-L") . UD indicates light ( 0 ) and dark (l ) periods. The horizontal axis represents tirne of NCER measurements commencing with the start of the first photopenod.

significantly reduced in infected plants (P=0.0227), by 20.5% at the termination of enclosure.

Influence of Pytltium inoculation on leaf physiology of plants in single pot culture

Figures 3.4A and 3.48 demonstrate alterations in leaf photosynthesis and transpiration

associated with inoculation by Pythium aphanidermorum of chrysanthemum plants grown in

single pot culture (Experiment 1). Despite evidence of browning of root tips within 24 hours

of inoculation, there were no significant differences in leafNCER (P=O.39 16) or transpiration

(P=0.5948) at any point in the 30 days following inoculation. Water use efficiency, stomatal

conductance and intercellular COZ showed similar response patterns (data not shown).

Effect of Pythium inoculation on NFT-grown chrysanthemum

Leaf physiology measurements collected in chrysanthemums grown in troughs in

Experiment 2 are displayed in Figures3.5A-D. Browning of roots, especially root tips, was

observed within 24 hours of inoculation. Analysis of the data indicates that there were no

significant differences between infected and control plants for any of the measured

parameters: photosynthesis (Figure 3SA, P=0.0823), transpiration (Figure 3SB, P=0.5920),

stomatal conductance (Figure 3SC, P=O.j7 17), or intercellular CO, concentration (Figure

MD. P=0.4564).

Root hydraulic conductance

Hydraulic conductance, interpreted as a measure of root hydraulic efficiency, of

severed chrysanthemurn stems is presented in Figure 3.6. While control plants appear to have

a higher conductance rate immediately following inoculation (3 DAI); overall, there is no

signi ficant effect on root hydraulic conductance between infected and healthy plants

(P=0.3082).

Days After Inoculation [DAI]

Figure 3.4 Influence of Pyihium inoculation on leaf physiology of chrysanthernum grown in individual pot culture, Experiment 1 . A, leaf NCER, and B, transpiration. Each bar represents the mean and standard error of a minimum of 5 replicate plants. Noninoculated controls (1 ) and inoculated plants (O ) were grown during spring summer conditions as descnbed in Materials and Methods.

Oays After Inoculation

5 10 15 M 25 30

Oays After Inoculation

Figure 3.5 Influence of Pyrhium infection on leaf physiology of NFT chrysanthemum: A, leaf NCER, B, transpiration, C, stomatal conductance for CO2, and D, intercellular CO, concentration. Each bar represents the mean and standard error of 5 replicate plants. There were no significant differences between noninoculated controls (u ) and inoculated plants (0 ) as determined by ANOVA. Plants were grown under summer conditions (Experiment 2) as described in Materials and Methods.

10 20 30

Days After Inoculation [DAI]

Figure 3.6 Influence of Pyrhium infection on hydraulic conductance of entire chrysanthemurn root systems. Each bar represents the mean and standard error of 4 replicate plants. Noninoculated controls (u ) and inoc- ulated plants (O) were grown in NFT troughs under sumrner conditions (Expenment 2) as described in Matenals and Methods.

DISCUSSION

Gas exchange and yield in chysanthemum post-inoculation

Vegetative chrysanthemum inoculated with Pythizrrn uphanidermatum manifested few

of the disease effects observed in lemice infected by P. dissotocum, as only minimal growth

reduction occurred in infected chrysanthemum plants (Figure 3.1). No changes in whole plant

gas exchange measurements were visible in inoculated plants from 25-28 DAI (Figure 3.2),

but some reduction in NCER was observed fiom 29-32 DAI (Figure 3.3). Infected

chrysanthemum plants at 29-32 DAI showed the same pattern as lemice of reduced

photosynthesis for several hours afier illumination initiation, before approaching control

levels. Dark respiration levels were not altered in infected plants. The second d y of enclosure

in the whole plant chambers, infected plants did not repeat the gradua1 nse in NCER seen in

lemice, remaining at a relatively constant value of approximately 65% of controls. Carbon

accumulation over the 36 h enclosure period was decreased in infected plants. Whether these

reductions in whole plant gas exchange would affect flowenng or final yield remains

uncertain.

These whole plant and yield observations indicated that this particular erbium

apita~~idermotwn isolate was only weakly pathogenic at the vegetative growth stage.

Physiological parameters measured in plants cultured both in individual pots (Expen~nent 1)

and NF?' (Experiment 2) indicate no differences between noninoculated controls and infected

plants. -4s no visible shoot syrnptoms were expressed, one possible explmation is that this

isolate was responsible for subclinical disease in chrysanthemum.

Asymptomatic infection caused by Pythium aphanidematum, and P. dissotocum, has

been observed in lettuce (Stanghellini 1988; Stanghellini and Kronland 1986) and other

species. It is known that the virulence of these species is temperature-dependent. P.

dissotocitm generally predominates at solution temperatures below 2 1-23"C, whereas P.

aplianiderrnatum produces stronger disease effects at higher temperatures (Gold and

Stanghellini 1985; Bates and Stanghellini 1984). As the temperature of the nunient solution

in these experiments averaged 2 1 T, it is possible that the solution did not provide an optima!

environment for P. aphanidennafum to sufficiently infect chrysanthemum roots.

Temperature was found to influence anachment of Fioarium solani f.sp. phaseoli to

roots of mung bean (Vigna radiata), as was pH (Schuerger and Mitchell 1992). Possible

mechanisms appeared to be influences on adhesion to root surfaces and post-attachent

processes including the production of secondary inoculum on or in the host plant. pH ranging

from 4.2-7.8 did not affect susceptibility to Pythicim in lettuce (Funck-Jensen and Hockenhull

1983). The pH of chrysanthemum solutions was kept low on grower recommendations (4.5-

5); however, the pH of spore suspensions dunng inoculation, where adhesion of 64-68% of

accumulated Pythittrn spores occurs (Gold and Stanghellini 19851, was approximately 6-7.

It is a reasonable conclusion that solution temperature, pH, or interactions between both

factors negatively affected P. aphanidermatum infectivity in these studies with

chrysanthemum.

fi*drarrlic Conductance

Roots comprise one of the main resistances to water movement within plants. Most

root resistance affecting metabolism and water flow is attributed to the cortex and epidermis

(Duniway l976), where initial infection and defence responses occur. Estimates of hydraulic

efficiency in roots of diseased plants are few. ModiQmg the traditional pressure bomb

technique involving severed leaves, we applied pressure to a solution surrounding the entire

root system and measured the resulting flow rate. While it appeared that initial measurements

of hydraulic conductance were decreased in exised chrysanthemum roots (Figure 3.6; 3 DA[),

possibly reflecting activated cortical defence responses simiiar to those obsemed in

subclinical Pyrhium F infection of tomato (Rey et ai. 1998), the ovenll conductance did not

significantly differ over the 30 days following inoculation. Other vascular pathogens such as

L'euticil/iwn and Fzisarium have increased the resistance of stem segments by 4 to 60 times

the control value (Duniway 1973; Duniway 1976). In exised roots ofbarley infected with mst,

a foliar pathogen, hydnulic conductivity decreased compared to control plants starting 5 DAI

(Berryman, et al. 199 l ) , increasing to control levels at 8 DAI.

It is important to consider that changes to hydraulic conductance may be direct, by

affecting the permeability of ce11 membranes or by defensive vascular or cortical occlusions,

or indirect. via a coupling of water and solute 80w (Steudle 1989). For that matter, metabolic

alterations resulting from putative decreases in hydraulic conductance may be influenced by

ion deficiencies due to reduced uptake of nutrients as well as water. For example, low P

concentration in nutrient solutions induced decreased root hydraulic conductance in Cotton,

preceding effects on leaf area expansion (Radin and Eidenbock 1984). Peterson et al. (1 993)

found that the endodermis of corn roots was the main barrier to radial ion movement, but not

water, which was limited by ceIl membranes and the apoplast. These authon also observed

that epidermal and cortical injury resulted in minimal effect on root pressure, whereas injury

to endodermal ceils produced an immediate decrease in root pressure. Root cortex wounding

was repaired within 4 days in onion (Moon ei al. 1984). If chrysanthemum mots are found

to behave similarly, a reasonable hypothesis to investigate further may be that this isolate of

erthiurn aphanidennaturn was weakly pathogenic, causing some cortical damage and

provoking defensive responses, but not extending to the endodermis. This could explain the

initial reduction in hydraulic conductance and the subsequent negligible effects on growth,

physiological parameters, and hydraulic conductance.

CONCLUSION

Growth of chrysanthemum inoculated with Pythium aphanidermarum isolare 167 was

minimal1 y reduced. Alterations in whole plant NCER and accumulated carbon were visible

only later in the infection cycle. Measurements of leaf physiological characteristics indicated

no significant reductions in productivity associated with Pythi~im infection in plants grown

in nvo culture systems. Hydraulic conductance, intended to eludicate the interrelationships

of SCER, transpiration, and stomatal conductance observed in lettuce inoculated with P.

~lissofocirm, was observed to have an initial reduction at 3 DAI in entire root systems of

infected chrysanthemum plants, but by 8 DAI was unaltered compared to control plants. The

use of hydnulic conductance as an analytical tool was limited by the weak pathogeniciry of

the isolate used in these experirnents. This marginal virulence may have been an artefact of

the pH and temperature of the hydroponic solutions. Future experiments with a more

aggressive isolate under conditions more conducive to infection may be necessary to clariQ

the relationship between hydraulic efficiency, vascular occlusion, stomatal closure and

reductions in NCER associated with pathogen infection in other plant-pathogen relationships.

The reduction of whole plant photosynthesis and accumulated carbon seen 29-32 DAI

in chrysanthemum affected by Pythium root rot confims that gas exchange analysis may be

useful in disease diagnosis even with subclinical disease, when visible symptoms such as

stunted growth or root browning are absent or misleading. in the chrysanthemum-fyhium

pathosystem, reduced whole plant gas exchange several weeks afier inoculation raises the

possibili ty of negatively affected flower production later in the growth cycle. To address this

issue, studies in C hap ter 4 were designed, incorporating W sterilization equipment into the

troughing systems in order to observe growth of Howering chrysanthemum integrated with

solution remediation technology and interactions with Pythium inoculation.

Chapter 4: Use of Ultraviolet Irradiation of Recirculating Hydroponic Solution in

Treatment of Pythium Root Rot

INTRODUCTION

Ultraviolet radiation is a commercially available technology with documented success

in treating municipai and industrial waste water. Several reports published over the last 15

years indicate that ü V is effective, if used continuously, in reducing population numbea of

target pathogens in hydroponic solutions (Stanghellini et al. 1984; Schwartzkopf et al. 1987;

Wohanka 1992; Evans 1994); however, reduction of disease symptoms has been inconsistent

(Zhang and Tu 2000) and authors of most published studies did not accurately measure

applied dosage. Non-target bacteria in nutient solutions are also significantly affected by W

irradiation (Zhang and Tu 2000; Sutton et al. 2000).

Ln order to limit precipitation of iron chelates as well as negative effects on beneficial

microorganisms resulting fiom continuous usage, W irradiation could be used more

precisely as a short-terni, high-dosage treatrnent immediately upon detection of disease

syrnptoms or once pathogens are at predetermined critical levels. There are no published data

on the efficacy of UV in providing protection against disease increase from existing infection.

It has been proposed that UV could provide an opportunity for root regrowth and host plant

recovery while reducing or stopping the spread of hyphae or secondary spores from plant to

plant via the recirculating solution.

Minimizing problems associated with iron chelation and inadvertent elimination of

beneficial microorganisms, the efficacy of short terrn UV applications was explored in this

chapter. A one-hou, high-dosage treatment suficient to reduce pathogen populations was

appiied to reduce disease incidence. Physiological parameters were measured to provide both

an indication of plant disease expression and stress associated with exposure to W-treated

solution. However, due to technical problems encountered with modifications to the W

system, and weak pathogenicity of the Pythium inoculum as descnbed in Chapter 3, a limited

number of tests best described as preliminary are descnbed in this chapter.

MATERIALS AND METHODS

Plant lkfaterial

Cuttings of Chrysanthemum morzYofii L. cv. Fina were rooted in rockwool cubes

For 3 weeks and transplanted into NFT troughs as described in Cbapter 3. Plants were grown

frorn 16 April to 8 June (Experirnent 1) and 13 July to 17 August 2000 (Experiment 2) with

an average day temperature of 23 O C and night temperature of 18 O C . Experiment 3 was

perfotmed without supplemental lighting fiom 19 September to 3 November 2000, under the

samr temperature regirne. Plants had initiated flower production at time of inoculation in

Experiment 3 in response to the reduced photoperiod.

in Expenment 1, plants were not exposed to the UV lamp system until inoculation,

allowing roots to develop for several weeks pnor to UV treatment. Ln Expenments 2 and 3

nutrient solutions were in contact with the UV lamp system commencing when the plants

were transplanted, allowing assessrnent of root development in nutrient solutions exposed to

the lanp housing irrespective of lamp activation.

Pythiim inocztiation

In Experiment 1, plants were inoculated for 30 minutes with a zoospore suspension

of Pytlti~trn aphanidermalum isolate 167 as described in Chapter 3. Plants in Experiment 2

were not inoculated P. aphanidennatum isolate p6 was used for inoculation in Experiment

3, following the inoculation protocol described previously.

UV Treatrnent

Imrnediately following inoculation, W iamps (Aqua W Advantage 2, Trojan

Technologies inc., London, Ont.) were activated for 60 min. The lamps were placed in line

with the recirculating solution so that solutions were exposed to U V pnor to entering the

noughs. Lamp housings were attached to the troughs with brass (Expenments 1 and 2) or

plastic (Experiment 3) fittings. Each trough receiving W irradiation had one W lamp

insralled. which was outfirted with asmall quartz window Aush with the stainless steel casing,

prrmanently attached with silver solder, to allow accurate measurement of the minimum

radiation intensity (Figure 4.1). Measurement of radiation intensity was performed dunng

the 1 -h exposure with a ndiometer (mode1 iL 1400A, International Light, Inc., Newburyport,

MA) equipped with a photodiode detector (SEL240, International Light, Lnc.). Dosage was

calculated as the product of radiation intensity and exposure time, which was estimated fiom

the solution flow rate and volume of the stainless steel casing enclosing the Iamp. The mean

dosages in Experiments 1 and 3 were 27.86 mW.s+cm" and 3 1.59 m ~ . s * c m " , respectively.

As preliminary trials indicated rapid dissociation of iron chelate, 0.2 14 g Fe-DTPA

(7% Fe, Plant Products inc, Brampton, Ont.) was added to each solution after the larnps were

turned off to replenish the available iron in solution to a level of 0.1%. Experiments 1 and

3 were organized as a randomized complete block design with four treatments: control,

control + üV, Pyrhium-inoculated, and Pythîum-inoculated + U V .

Experiment 2 employed a randomized complete block design with w o treatments:

control, and W-exposed. This experiment was performed with the W lamps in place but not

activated, and did not involve Pythium inoculation. After five weeks of recirculation, solution

sarnples were taken at the completion of the experirnent and analyzed for macro and micro

nutrients, in addition to Zn, Cu, Al, and Ni.

Figure 4.1. Cntraviolet lamp system in line with NFT chrysrnthemum. The stainless steel casing (C) has a quartz observation window (QW) flush with the interior surface to allow accurate estimation of radiation intensity. Nutrient solution is pumped frorn the reservoir, through a brass (Experiments 1 and 2) or plastic (Expenment 3) fitting (F), and flows through the lamp casing at a depth of 1 cm surrounding the W lamp. irradiated solution reaches the NFT troughs (T) through another brass or plastic fitting and flows over plant (P) roots before retuming to the reservoir.

Bionioss acctrrnrrlation and tissue analysis

Plants in Experiment 1 were hawested periodically throughout the growth period.

Data were analyzed using a two-way ANOVA (S-Plus 2000, MathSoft Inc., Seattle, WA).

After the final harvest, dried samples were analyzed for elemental content, including macro

and micro nutrients, Fe, Al, Cr, Ni, and Ti. Nutrient content was evaluated statistically using

a one-way ANOVA. Plants in Expenments 2 and 3 were harvested at the end of the

rxperirnent and dried at 75°C for 48 h. Growth parameten were evaluated using two-way

ANOVA in Experiment 2 and by Scheffé's multiple mean cornparison in Expenment 3.

Leaf'Gas Exchange and Warer Relations

Physiological parameters measured in Experiment 1 were the same as in Chapter 2.

Statistically significant responses to Pythium inoculation under UV treatment were assessed

using hvo-way ANOVA.

/-@tir-aulic Conductance ofsevered Root Systerns

Root hydnulic conductance was measured in UV-exposed control and infected plants

in Experiment 1 as described in Chapter 3. Statistical analysis was perfomed using two-way

AKOVA.

Eiposirre to Brass or PIastic Fiftings

Experiment 4 was designed with 10 rooted cuttings grown in aerated single pots as

described in Chapter 3. One brass or plastic fitting, identical to those used to afix W lamp

systems to troughs, was piaced in each pot to simulate exposure of recirculating nutrient

solutions to the different fittings. Plants were grown from 11 October to 2 November 2000

and ini tiated flower production during the study. At the end of the experiment, the plants were

hanrested destructively and dried for 48 h at 75 O C . Harvest parameters were analyzed using

one-way ANOVA. Solution samples were taken at the termination of the study and analyzed

for macro ion concentrations using one-way ANOVA.

RESULTS

Influence of concurrent UV exposure and fythium inoculation on vegetative growth of

chrysanthemum: Experiment 1

Desmictive harvest measurements over 30 days following Pytlliurn inoculation and

UV solution treatment in chrysanthemum during Experiment 1 are presented in Figure 4.2A-

C. Although inoculated plants were generally smaller than controls, there were no significant

differences in shoot dry weight (Figure 4.2A; P=0.9642), root dry weight (Figure 4.2B;

P=0.529 i ), or shoot:root ratio (Figure 4.2C; P=0.9 146).

By the time of final harvest, W-exposed plants showed inhibited growth, regardless

of Pyrhilun inoculation, as seen in Figure 4.3. W exposure resulted in reduced shoot (Figure

43A; P=0.000 1) and root (Figure 4.38; P=O.O 145) dry weight in both control and inoculated

plants. Many plants exposed to W-treated nutrient solution were chlorotic throughout the

rxperiment.

Elementnl inalysis of non-inoculated plants grown in W-treated nutrient solution

Nutritional analysis of shoot and root tissue collected from plants grown in W-treated

solution is shown in Tables 4.1 and 4.2, respectively. Increased Cu and Zn concentrations

were observed in both leaf and root tissue of W-exposed plants. Additionally, UV-exposed

leaves accumulated higher P and Fe concentrations. Roots of control plants not exposed to

UV-aeated nutrient solution contained higher concentrations of Mg, Al, and Cr.

Influence of fythium inoculation on physiological parameten in plants exposed to W-

treated nutrient solution

No significant differences were observed in metabolic parameters between inoculated

plants and noninoculated controls exposed to W-treated solutions in Experiment 1 (Figure

Days After Inoculation [DAI] Figure 4.2 Biomass accumulation in chrysanthemum cv. Fina following exposure to UV-treated nutrient solution and Pyihiunr inoculation, Experiment 1. A, shoot dry weight, B, root dry weight, C, shoot:root ratio. Noninoc- ulated controls ( ); Inoculated plants ( ). Each bar rep- resents the mean and standard error of 3 plants, grown under sumrner conditions as described in Materials and Methods.

O 5 10 15 20 25 30

Days After Inoculation

Figure 4.3 Influence of UV solution irradiation on growth of chrysanthernum, Experiment 1. A, Shoot dry weight B, Root dry weight. Noninoculated controls not exposed to UV-treated solution (O), Noninoculated controls exposed to irradiated solution (a ), Pythium-inoculated plants not exposed to UV-beated solution (m), Pythium-inocuiated plants exposed to irradiated solution (H). Each bar represents the mean and standard error of 3 replicate plants. Plants were grown under summer conditions as descnbed in Materials and Methods. UV irradiation was of 1 h duration.

Table 4.1 Elemental Analysis of Chrysanthemum Leaf Tissue, Experiment 1. Data represent the mean i standard of 8 replicate plants, grown under sumrner conditions as described in Materials and Methods. UV-treated plants were grown in nutrient solution that had been irradiated for 1 h. P-Values were genented using two-way ANOVA (S-Plus 2000). BDL= Below detection limits; 5.0 mg-kg-' for Cr, Ni, and Ti. NS= No statistical analysis possible.

Element Control LN-Treated P-Value

N [%] 5.3 1 * 0.08 5.15 * 0.29 0.6 166

P [%] 0.75 k 0.03 1.20 * 0.1 1 0.0047

K [%] 7.75 * 0.3 1 7.76 * 0.47 0.9898

Ca [%] 2.02 k 0.04 2.1 1 10 .1 1 0.5 154

Mg [%] 0.23 I 0.0 1 0.25 i 0.02 0.4689

Cu [mgkg] 16.08 k 2.59 35.04 * 3.67 0.00 L 4

Zn [m&d 34.78 st 2.86 118.38 * 8.71 <O.OOO 1

Mn [mg/kgl 7 1.55 * 2.83 73.85 k 7.3 1 0.6590

B [mdk/kgl 48.43 k 1.4 1 46.0 1 3.52 0.55 17

Fe [mfiikgl 69.13 * 4.04 1 17.38 k 7.86 0.000 1

Al [mdkgl 22.50 i 2.22 18.25 3.38 0.3332

Cr b%hl BDL BDL NS

Ni [mg/kg] BDL BDL NS

Ti bg/kgl BDL BDL NS

Table 4.2 Eiemental Analysis of Chrysanthemum Root Tissue, Experiment 1. Each value represents the rnean standard error of a minimum of 7 replicate plants, grown under summer conditions as described in Materials and Methods. UV-treated plants were grown in nutrient solutions that had been subjected to 1 h irradiation. P-Values were generated using two-way ANOVA (S-Plus 2000). BDL= Below detection limits; 5.0 mg-kg-' for Ni.

Element Control UV-Treated P-Value

4.41 st 0.07

1.561 0.11

8.80 I 0.07

2.77 i 0.40

0.48 k 0.02

488.71 * 46.92

456.57 * 50.08

243.86 k 9 .O6

66.43 h 5.79

3494.29 k 23 1 .O9

857.25 * 1 18.9

17.75 k 1-60

BDL

126.0 k 10.1 1

4.4; P-values for NCER E, WUE, GCO,, and Ci, respectively = 0.0823, 0.5920, 0.6738,

0.57 17, and 0.4564).

Hydraulic conductance of chrysanthemums exposed to UV-treated solution following

fythium inoculation

No significant differences were observed in hydraulic conductance beween

inoculated plants and healthy controls exposed to W-treated solutions in Experiment I

(Figure 45A; P-value=0.734 1). Compared to plants not exposed to W-treated solutions,

there was no difference in the hydraulic conductance of exposed plants (P= 0.3 17 1).

Influence of exposure to ioactivated W system on yield of chrysanthemum:

Experiment 2

Plants in Experiment 2 experienced dramatic growth reductions when exposed to

nutrient solutions continuously recirculating through the UV lamp system, even when the

lamps were not tumed on (Table 4.3). Exposed plants had 9 1.5% and 94.3% decreased shoot

and root biomass (Pc0.000 1 ), and 48.5% increased shoot:root ratio (P=U.Z 5) compared to

control plants grown in nutrient solutions not exposed to the W lamp system.

Elemental analysis of nutrient solution in contact with inactivated UV lamp system

Samples of nutrient solutions exposed to the W lamp system in Experiment 2

contained higher NO,', Mi,-, and K* concentrations but lower PO,)-, sO,", Mg'&, and CaL-

concentrations than solutions not exposed to the UV system (Table 4.4). Of the metals

analyzed, solutions exposed to the inactivated W lamp system contained higher

concentrations of Al, Cu, and Zn than control solutions, while controls had higher Fe

content.

Water Use Efficiency A

O O O O rd O W

O /+, F rn

3 r - I q

O $ j g g g ô ; ; p : o ; a ; g q P P S ' E g s g 0 0 0 0 0 N W

Stomatal Conduclance [jimol.m~2~s~1] Transpiration [ r n m ~ l ~ m ~ ~ ~ s - ' ]

Days After Inoculation [DAI]

Figure 4.5 Hydraulic conductance of severed chrysanthemum root systems, Experiment 1. A, following W irradiation of the nutrient solution (m) and con- current Pythium inoculation and ü V solution treatment (O). B, Cornparison to non- W exposed plants. Noninoculated controls without exposure to W-treat- ed solution ( a), Noninoculated control plants grown in UV-treated solution (a, fythium- inoculated plants not exposed to UV-treated solution ( H), and plants inoculated with Pythiurn and grown in irradiated solution ( a). Each bar rep- resents the mean and standard error of a minimum of 3 repiicate plants. Pythium inoculation and plant growth under summer conditions was as described in Mat- erials and Methods. Recirculating nutrient solutions were irradiated with UV for one hour immediately following inoculation.

Table 1.3 Influence of nutrient solution exposure to innctivated UV lamp system on growth of chrysanthemum, Experiment 2. Data present the mean * standard error of 8 replicate noughs with 4 subsampled plants per trough. P-Values were generated with nvo- way XNOVA (S-Plus 2000). Plants were gram under summer conditions as described in Materials and Methods and harvested after 5 weeks of continuous solution recirculation.

Yield Parameter Control Exposed to P-Value Inactivated W Lamp System

- - -

Shoot Dry Weight [g] 1 1.372 * 0.474 0.971 * 0.062 <O.OOO 1

Root DN Weight [gl 1.1 19 5t 0.094 0.063 * 0.0 14 <O.OOO 1

Shoot:Root Ratio 10.453 20.288 0.0255

Table 4.4 Nutritional analysis of nutrient solution exposed to ioactivated UV lamp system, Erperiment 2. Solutions were sampled after 5 weeks of recirculation. Data represent the mean + standard error of 8 replicate troughs. P-Values were generated with one-way ANOVA (S-Plus 2000). BDL= Below detection limits; NS= No statistical analysis possible. Detection limits for AI=0.02 mg.^"; Cr-0.04 mg.^“.

Ion or Element Control Exposed to Inactivated P-Value [mg- L" ] W Lamp System

O

O

333.38 I 13.73

339.47 * 23.82

131.01 i 7.01

O

O

6.14 k 3.03

21.58 1.04

205.8 1 i: 9.22

BDL

BDL

0.2 1 k 0.02

1-40 k 0.13

0.24 * 0.0 1

1.68 i 0.82

O

5 17.98 i 35.27

135.82 r 9.02

84.70 * 5.37

O

10.92 i 1.32

140.62 ft 9.94

10.76 i 1.01

1 10.64 k 6.88

O. 15 k 0.05

BDL

5.99 k 0.62

0.06 * 0.02

3.95 * 0.25

Influence of concurrent W exposure and Pythium inoculation on flowering

chrysanthemum: Experiment 3

Control plants in Experiment 3 did not express significantly inhibited growth

associated with solution UV treatment or exposure to inactivated W lamp systems, presented

in Table 4.5. in plants not exposed to UV treatment, Pythium-inoculated plants showed no

significant differences compared to controls in any growth parameters including root

browning. Among UV-exposed plants, noninoculated controls exhibited a Iower incidence

of brown roots and increased leaf and total shoot dry weight than infected plants. Flower

production was not affected by either Pyrhium infection or exposure to solution irradiation.

UV irradiation of the nutnent so!ution did not appear to offer any protection fiom existing

Pytlii~rm infection as the total shoot dry weight of inoculated plants was smaller in plants

exposed to UV-treated solution. This isolate of Pyihium appears slightly more aggressive than

that used in Experiment 1 and in Chapter 3.

Influence of exposure to brass or plastic fittings on chrysanthemum growth:

Experiment 4

Exposure to nutient solutions in contact with brass fittings resulted in a much higher

incidence of root browning and formation of fewer flower buds, as shown in Table 4.6. Plants

exposed to the bnss fitrings developed a significantly higher shoot:root ratio, reflecting the

inhibited root growh of brass-exposed plants. Plants grown in brass-exposed nutrient

solutions displayed reduced nutrient uptake, as evidenced by the higher concentrations of

most ions in solution, presented in Table 4.7. Nutrient solutions in contact with brass fittings

accumulated significantly higher NO,, SO,'; NH,', K*, Mg+, Ca2-, Cu, and Zn.

Table 1.5 Influence of Pphium inoculation and one hour W solution irradiation on growth of chrysanthernum (Experiment 3). Plants had initiated flower production at time of inoculation. The rnean k standard error of 3 replicate troughs with 5 subsarnpled plants per trough are show. Values within a row with the same superscript are not significantly different as determined by Scheffé's multiple means cornpanson (Pc0.05; S-Plus 2000). Plants were grown under autumn conditions as described in Materials and Methods.

- -

Control, Control, Pythium- Pyihitim- Harvest Non-exposed to Exposed to W inoculated, inoculated, Parameter W Non-exposed to Exposed to W

UV

Roo t Browning [%]

Root Dry Weight [g]

Leaf Area [cm']

Leaf Dry Weight [g]

Xumber of Open FIowers

Xumber of Immature Flower Buds

Total Flowers

Stem and Flower Dry Weight [g]

Total Shoot Dry Weight k 1

Table 1.6. Effect of nutrient solution exposure to brass or plastic fittings on growth of chrysanthemum. Each value represents the mean * standard error of 5 replicate plants. Rooted cuttings were grown in nutrient solution exposed to either a brass or plastic fitting for 3 weeks before hmest , as described in Matenals and Methods.

Parameter Bmss Plastic P-Value

Root Browning [%]

Leaf Xrea [cm']

Leaf Dry Wright [g]

Stem Dry Weight [g]

Total Shoot Dry Weight [g]

Root Dry Weight [g]

Shoot: Root Ratio

Number of Open Flowers

Number of Flower Buds

Table 4.7. Nutrient Content of Fertiluer Solutions Exposed to Brass or Plastic Fittings, Experiment 4. Plants were grown for 3 weeks in individual pots with nutrient solutions in contact with one brass or plastic fimng (used in afixing UV lamp housing to NFT troughs) to simulate exposure. Each value represents the mean i standard error of 5 replicate solutions. P-Values were calcluated with one-way ANOVA (S-Plus 2000). NS= no statistical analysis applicable. BDL=Below detection limit of 0.05 mg. L" for Fe.

Ion or Element B rass-Exposed Plastic-Exposed P-Value [mg-L-' 1 Solution Solu lion

74.82 k 2.97

O

1047.44 * 22.87

522.02 * 1 1.93

L89.78 * 7.40

36.56 * 1.26

90.70 * 1.85

277.70 * 3.03

48.28 5 1.67

L 54.64 * 5.82

o. 12 * 0.02

6.92 I 0.32

BDL

4.80 k 0.33

DISCUSSION

Ultraviolet irradiation of recirculating nunient solutions had a negative effect on plant

growth, irrespective of Pyihium inoculation. Exposure to the UV lamp housing system, and

not solution irradiation per se, was responsible for observed negative correlations between

solution UV treatment and growth of both shoots and roots. Tissue analyses indicated copper

and zinc toxicity as the main cause of growth inhibition in plants grown in solutions exposed

to the UV system. The release of toxic Ievels of these and other elements to the nutrient

solution from lamp components appeared to be temporary, however, as plants grown in

Experiment 3 did not exhibit inhibited growth. The use of plastic instead of bras fittings in

Experiment 3 partially explains the amelioration of growth reduction associated with

exposure to the UV lamp system. Any evaluation of W as a remediation technique in

reducing incidence of disease in recirculating hydroponic solutions çhould be strictly

speculative at this point.

Copper and Zinc Toxiciîy

Growth of chrysanthemum cunings in nutrient solution exposed to the W lamp

system was affected by the extent of root development prior to exposure. Plants in

Experiment 1 with a well-developed root system were able to continue development

following exposure to nutrient solutions in contact with the W lamp housing system,

although rnuch reduced compared to non-exposed plants (Figure 4.2). In contrast, plants with

lirnited root systems in Experiment 2 were cornpletely unable to grow when exposed to

nutrient solutions in contact witb the W system (Table 4.3). The larger shoot:root ratio in

Experirnent 2 confirms that root growth was more affected than shoot growth.

Previous researchers documenting reduced plant growth as a result of exposure to W-

treated nutrient solutions suggested that growth inhibition was a result of ozone andor free

radical generation at 185 nm (Schwartzkopf et al. 1987) or excess chelate precipitation

(Acher et al. 1997) related to higher U V intensity. As our UV lamps were coated with TiOt

to filter out ozone-generating wavelengths, it is assumed that other factors were responsible

for the growth inhibition observed in Experiments 1 and 2. Acher et al. (1997) used nutrient

solution continuously irndiated with W. It would be expected that a one hour treatment? as

used in the present experiments, would avoid such massive chelate precipitation.

Tissue analyses in Experiment 1 indicated much higher concentrations of copper and

zinc in plants grown in nutrient solutions exposed to UV irradiation. Marschner (1995) states

that critical toxicity levels for these elements in leaves of crop plants are above 20-30 pg

Cwg" dry weight and 100 to >300 pg-g-' for zinc, but cautions that roots are sites of

preferential accumulation. In Experiment 1, concentration of copper in the leaves was above

rhese limits, and zinc concentration was within the toxic range (Table 4.1). W-exposed roots

had four to five times the concentration seen in non-exposed plants (Table 4.2), and displayed

extreme root inhibition in addition to discolouration. These classic signs of both copper and

zinc toxicity (Marschner 1995; Woolhouse 1983; Robson and Reuter 198 1) were

accompanied by leaf chlorosis, even though UV-exposed leaf tissue contained sipificantly

higher iron concentrations, and magnesium concentrations were comparable to non-W-

exposed controls.

It has been suggested that chlorosis associated with copper toxicity is related to lipid

peroxidation, membrane destruction (Sandmann & Boger 1983, as cited in Marschner 1995),

and acceleration of chlorophytl breakdown (Woolhouse 1983), although chlorosis is also

associated with zinc toxicity (Rauser 1973), so it is difficult to definihvely isolate the cause

of this response. CU?' and Cu3' ions blocked photosynthetic electron transport in isolated

spinach chloroplasts (Sandmann & Boger 1983, as cited in Marschner(l995). Woolhouse

( 1 983) noted that effects of Zn are less severe than Cu in tems of both root growth inhibition

and leaf chlorosis. Corn grown in hydroponic solution with high zinc concentrations showed

intemeinal chlorosis without reduced leaf Fe content (Rosen er al. 1977). Cornpetition

benveen Zn and Fe for binding sites in chloroplasts is one possible explanation of chlorosis

in zinc toxic plants with sufficient Fe content, as Zn" and Fe'havc identical ionic radii, 0.083

nm (0.74 A; Handbook of Chemisûy and Physics 1964).

in field plants, copper and zinc toxicity are often associated with acid (pH <5) soils

(Robson and Reuter 198 1; Woolhouse 1983; Marschner 1995). The low pH used in

recircuiating nutrient solutions may have been responsible for mobilizing copper and zinc

ions from cornponents of the UV lamp housing. Plumbing components of brass, an alloy of

both copper and zinc, occasionally supplemented with lead, tin, or aluminum (Encyclopaedia

Britannica 2000), are an obvious possible source of toxicity. A significant increase in root

browning associated with root exposure to the brass fittings is shown in Table 4.6

(Experirnent 4). However, the Iack of significant growth inhibition of root and shoot tissue

in that experiment, and the decrease in total shoot dry weight in Pythium-inoculated plants

exposed to W compared to inocuiated plants not exposed in Experiment 3 where plastic

fittings were used, indicates that other factors must bave connibuted to the toxicity observed

in Experiments 1 and 2.

The reduced concentrations of aluminum and chromium found in roots of W-exposed

plants (Table 4.2) suggests that these elernents were not dissolved from the stainless steel

casing and made available for plant uptake. Levels of nickel in roots of W-exposed and

control plants were similar, indicating that nickel was not rernoved from the stainless steel.

As there were no significant differences observed in titanium concentrations in exposed and

control roots, one can conclude the TiOZ lamp coating did not effect growth inhibition.

The remaining component of the W lamp system that could be affected by contact

with low pH nutrient solution is the silver solder used to anach the quartz observation

window on each lamp casing (Figure 4.1). The solder composition was listed as 56% Ag,

220h Cu, 19% Zn and 3% Sn (Weiler 2000). While manufacturing specifications listed this

solder as a braising alloy suitable for stainless steel in food applications, copper and zinc on

the surface exposed to the acidic solution could have been mobilized in contact with low pH

solution, contributing to the observed toxicity.

After several months of continuous recircuIation, al1 available ions on the inside

surface of the stainless steel casing may have been Ieached, leading to the disappearance of

toxic symptoms in Experiment 3. An alternative explanation is that iron or other elements

precipitated from the solution could have formed a protective coating over the soldered area.

Additionally, plants in Experiment 3 were undergoing flower production. At this stage root

activi ty and subsequent nutrient uptake declines as flowers become a si gni ficant carbohydrate

sink (Marschner 1995). Mobilized copper and zinc ions may not have accumulated in root

tissue at toxic levels due simply to reduced uptake.

Analysis of ions present after 5 weeks of recirculation in Experirnent 2 showed that

several ions (NO,', NH,', Kd) and metals (Al, Cu, Zn) accumulated in W system-exposed

nutrient solutions (Table 4.4). The extremely limited uptake resulting fkom minimal root

development in plants affected by copper and zinc toxicity is the probable cause of

accumulation of these elements in solution. This conclusion is supported by the observation

of significant ion accumulation in aerated nutrient solutions exposed to brass finings (Table

4.7). The reduction in pOd3-, sO,', Mg', Ca2'and Fe concentrations in W-exposed solutions

in Expenment 2 may reflect complexation with leached copper or zinc, or precipitation due

to the low pH.

U Y Dosage Consideraiions

In the hour that the UV lamps were activated, each unit volume of solution passed

through the lamp 7 times owing to the small reservoir size. Applied dosage is a function not

only of intensity and exposure time, but flow dynamics within the lamp casing, lamp age, and

reduced transmittance from precipitation or biofiims (Evans 1994; Sutton et al. 2000). The

lack of reliable literature relating UV radiation dose, pathogen inactivation, and reduction of

symptom expression (Sutton et a[. 2000; Evans 1994) confounds direct cornparisons of

results from Experiment 3 with previous work. [n a long-term, continuous irradiation study,

Zhang and Tu (2000) found no significant differences in root rot severity at several W doses

in tomatoes inoculated with P. aphanidermatztm. although a decline in Pythium populations

was observed. irradiation for 3 hours daily reduced the solution microorganism population

but correlation with disease symptorns was not investigated in this study (Buyanovsky er al.

198 1 ) .

The applied dosage in Expenments 1 and 3 was sufficient to inactivate spores,

according to previous collimated beam and flowthrough studies (Zhang and Tu 1999; Sutton

et ai. 7000). However, üV irradiation can only affect zoospores suspended in the nutrient

solution, not those attached to root surfaces. UV treatment could inactivate secondary spores

produced and released to the nutrient solution once initial Pyhium inoculum is established

as hyphae, but will not affect spore dynarnics and infectivity in the rhizosphere. There was

little evidence in Experiment 3 to support the contention that short term W solution

irradiation affords any oppominity for root regrowth in plants with existing Pythium

infection, or protection against secondary zoospore production, although longer term

experiments would be necessary to fully elucidate the risks associated with secondary spore

production in recirculating systems. Exposure to UV-treated nutrient solurions did not affect

flower production in chrysanthemum. More experiments investigating the interrelationships

between dosage, pathogen inactivation, and disease incidence are required to determine the

rfficacy of üV in pathogen control in recirculating hydroponic systems.

CONCLUSION

When root disease is detected in a hydroponic crop grown in recirculating nutrient

solution, either through early diagnosis with remote sensing equipment or by observation of

visible symptoms, removal of affected plants and treatment of the nutrient solution are

required to limit spread of disease and subsequent crop losses. in initial studies, inhibited

grotvth was obsewed in plants grown in W-treated recirculating nunient solution. The toxic

effects were expressed even when the W lamps were inactivated. Exposure to the UV lamp

system resulted in significant accumulation of copper and zinc in leaf and root tissue. It was

detemined that brass fittings used to affix the UV lamp housing to the trough could be

responsible for the increase in root browning, and could contribute to reduced nutrient uptake

and growth in brass-exposed plants. Another potential source of copper and zinc ions,

sonsidering the increased mobility of ions in low pH solution, was the silver solder on the

stainless steel casing.

Toxic effects associated with exposure to the LN lamp system were not observed in

Expenment 3, however, when plastic fittings were used in place of brass and most available

ions from the silver solder had already dissolved into solution. There was linle evidence in

Experiment 3 to suggest that short term W solution irradiation provided any protection or

afforded the oppominity for root regrowth in Pythium-infected plants, although future

experiments with a more aggressive Pythiirm isolate are necessary before any firm

conclusions may be drawn. There was no observed effect on flower production in plants

exposed to UV-treated solutions. It is possible that UV may be bener directed as a

continuous, low-dosage treatment to control pathogen populations. Considenng the paucity

of available reliable data regarding control of pathogens in recirculating hydroponic systems

with UV, and the difficulties associated with development of UV as a consistently effective

remediation tool, it is premature to evaluate the efficacy of UV as a potential treatment

tec hno l o g for commercial greenhouses.

The studies reported in this thesis confirm the hypothesis that alterations in canopy

and leaf gas exchange accompany Pythium root rot infection. Whole plant responses,

integnted as daily carbon accumulation, were obsemed in two host plants, lettuce and

chrysanthemum, inoculated with different species of Pythiurn. indicating that general

reductions in carbon fixation are not pathosystem-specific. In lettuce, leaf gas exchange and

transpiration were significantly reduced as early as 7 days after inoculation, well in advance

of visible foliar symptoms, Iending support to the possibility that gas exchange characteristics

may be used in early diagnosis of disease, a vital issue in pathogen control in recirculating

hydroponic solutions. A proposed merhod of pathogen inactivation, W irradiation of nutrient

solutions was tested in greenhouse trials; however, copper and zinc toxicity fiom the lamp

housing was observed even with lamp inactivation. Modifications to the W lamp system,

including b r a s fittings to attach lamps to NFT t~oughs, and silver solder used in allowing

accurate estimation of minimum radiation intensity, are likely sources of toxicity. Future

experiments are necessary to elucidate the relationships of alterations in gas exchange

parameten and hydraulic efficiency of roots affected by PythN~rn rot, possible negative effects

of root infection on fiower formation, and eficacy of UV in control of Pythium infection in

recirculating hydroponic systems.

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Appendix 1. Correlation of Nondestructive SPAD Measuremeats of Leaf

HGreenness9' with Extractable Chlorophyll Content

14 16 18 20 22 24 26 28 30 32

SPAD Values

Figure 1. Relationship of SPAD measurements to total chlorophyll content of Bella Green lettuce, extracted with DMF and expressed on a leaf area basis. Each point represents the mean of 6 SPAD values per leaf; 4 leaves per plant were measured. ~'0.85 1 for the fitted second order polynornial function.

AppendLu 2. SAS Program for Calculation of Leaf Physiological Parameters

I* This program calculates photosynthesis and transpiration rates, stomatal conductance, intercellular CO2 concentration,

for Dr. Grodzinski's leaf gas exchange system.

By Demos Leonardos */

Options 1s = 90;

Data A; In fi le1C:WyFiles\May99.prn'; Input Date S plant S DTreat Ptreat Block Chamber CO2Conc ChamTemp LeafTemp

FlowRate LeafArea CO2Diff DewSam DewRefer;

!* input variables must be in the following units: COZConc and CO2Diff (ppm), ChamTemp, LeafTemp and AirTemp (OC), FlowRate (mumin), LeafArea (cm2), and DewSam and DewRefer (OC) */

i'* Conversion of leaf area Crom cm2 to m2 */ LeafArea = LeafArea * 1 O**-4;

/* Conversion of flow rate from ml/min to m3/s */ FlowRate = FlowRate * 16.67 * 10**-9;

i* Density of pure CO2 corrected for temperature (pmoVrn3) */ COZDens = ,044643 * (273 / (ChamTemp + 273)) * 10**9;

/* Conversion of CO2Diff fiom ppm to m3/m3 */ C02Diff = C02Diff * IO**-6;

!* Calculation of photosynthesis rate (pmoVmUs) */ Pn = (FlowRate * CO2Dens * COZDifY) / LeafArea;

i* Vapour pressures (kPa) - Tetens equation's */ VPSam = 6.10800 1 * EXP((17.27 * DewSam) / (DewSam + 237.3)) * . l ; VPRefer = 6.10800 1 * E n ( ( 17.27 * DewRefer) / @ewRefer + 237.3)) * . l ; VPLeaf = 6.108001 * EXP((17.27 * Leaffemp) / (Leaffemp + 237.3)) * .1; VPCham = 6.108001 * EXP((17.27 * CharnTemp) / (ChamTemp + 237.3)) * . l ;

/* Vapour pressure for air over leaf wa) */ VPAir = (VPSam + VPRefer) / 2;

/* Saturation vapour pressure difference between leaf and air (kPa) */ SVPD = VPLeaf - VPAir;

/* Water densities (mmoYm3) */ WatRefer = ((VPRefer / ((273 + ChamTemp) * 4.62)) * 10**7) 1 18; WatSam = ((VPSam / ((273 + ChamTemp) * 4.62)) * 10**7) 1 18; WatLeaf = ((VPLeaf / ((273 + Leafi'emp) * 4.62)) * 1 O**7) 1 18;

!* Calculation of transpiration rate (mmoVmZ/s) */ E = (WatSam - WatRefer) * (FlowRate / LeafArea);

!* Water Use Efficiency (pmol CO2 / mm01 H20) *I WUE = Pn/ E;

:* Atrnospheric pressure (kPa) */ AtmPres = 10 1.325;

.'* Calculation of stomatal conductance for HZ0 (mmoVm2/s) */ GHz0 = (E * (AtmPres - (VPLeaf + VPAir) / 2)) 1 (VPLeaf - VPAir);

i* Calculation of stomatal conductance for CO2 (mmoVmZ/s) */ GC02 = GHz0 / 1.6;

i* Concentration of CO2 over leaf (m3/m3) */ C02Air = ((COZConc + (CO2Conc - (COZDiff * 10**6))) / 2) * IO**-6;

i* Calculation of intercellular CO2 concentration @pm) */ Ci = ((((GCOZ - (E 1 2 ) ) * CO2Air) - (Pn * IO**-3)) / (GCOZ + (E 12))) * 10**6;