RESEARCH PAPER Thermal imaging of cucumber leaves affected ... · Wang et al., 2004), and for assessment of plant–pathogen interaction by monitoring patterns of surface leaf temper-ature

RESEARCH PAPER

Thermal imaging of cucumber leaves affected bydowny mildew and environmental conditions

E-C. Oerke*, U. Steiner, H-W. Dehne and M. Lindenthal

Institute for Plant Diseases, University of Bonn, Nussallee 9, D-53115 Bonn, Germany

Received 21 October 2005; Accepted 22 February 2006

Abstract

Pathogenesis of Pseudoperonospora cubensis caus-

ing downy mildew of cucumber resulted in changes in

the metabolic processes within cucumber leaves in-

cluding the transpiration rate. Due to the negative

correlation between transpiration rate and leaf temper-

ature, digital infrared thermography permitted a non-

invasive monitoring and an indirect visualization of

downy mildew development. Depending on the stage

of pathogenesis and the topology of chloroses and

necroses, infection resulted in a typical temperature

pattern. Spatial heterogeneity of the leaf temperature

could be quantified by the maximum temperature

difference (MTD) within a leaf. The MTD increased

during pathogenesis with the formation of necrotic

tissue and was related to disease severity as described

by linear and quadratic regression curves. Under

controlled conditions, changes in temperature of in-

fected leaves allowed the discrimination between healthy

and infected areas in thermograms, even before

visible symptoms of downy mildew appeared. Envi-

ronmental conditions during thermographic measure-

ment, in particular air temperature and humidity, as

well as water content and age of the leaf influenced

the temperature of its surface. Conditions enhancing

the transpiration rate facilitated thedetectionof changes

in leaf temperature of infected leaves at early stages

of infection. As modified by environmental conditions,

MTD alone is not suitable for the quantification of

downy mildew severity in the field.

Key words: Cucumis sativus, digital thermography, leaf

temperature, Pseudoperonospora cubensis, transpiration.

Introduction

Leaf temperature of plants is the result of external and

internal (physiological) factors. The environmental factors

solar radiation, air temperature, and relative humidity (RH),

and the water status of the shoot tissue determine the

temperature of plants via stomatal transpiration. There is

a correlation between leaf temperature and water status, as

water is the primary source of infrared absorption in plant

tissue (Kummerlen et al., 1999).In addition to water supply and overall metabolic activity

regulated by environmental conditions, pathogenic organ-

isms may affect both cuticular and stomatal conductance

of plant tissue, resulting in significant modifications in

leaf temperature. As leaf temperature may be measured

remotely and with high spatial resolution, digital infrared

thermography may have the potential for the identification

of management zones in disease control.The detection of modifications in plants or canopies asso-

ciated with low disease severity in the early stages of disease

epidemiology is crucial for the targeted, site-specific or on-

demand application of fungicides in integrated disease

control. The sensing of ethylene associated with tissue

damage from pathogens (Boller, 1983) and optical methods

assessing the reflection and fluorescence characteristics of

plants associated with photosynthetic activity (Scholes and

Rolfe, 1996; Coops et al., 2003; Laudien et al., 2004;

Franke et al., 2005) are some approaches. Thermography

allows the quantitative analysis of spatial and dynamic

physiological information on the plant status (Jones, 2004).In plant biology, infrared thermography is used to study

spatial variability of stomatal conductance (Jones, 1999a;

Omasa and Takayama, 2003; Prytz et al., 2003), to

schedule irrigation (Gebhardt, 1990; Jones 1999b), for

monitoring of ice-nucleation or temperature stress in plants

(Wisniewski et al., 1997; Yang et al., 2003), to screen for

* To whom correspondence should be addressed. E-mail: [email protected]: MTD, maximum temperature difference; RH, relative humidity.

Journal of Experimental Botany, Vol. 57, No. 9, pp. 2121–2132, 2006

doi:10.1093/jxb/erj170 Advance Access publication 19 May, 2006

ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

mutants with altered stomatal control (Merlot et al., 2002;Wang et al., 2004), and for assessment of plant–pathogeninteraction by monitoring patterns of surface leaf temper-ature (Chaerle et al., 1999).

For remote detection, identification, and quantificationof plant diseases and associated pathogens, sensors have tobe sensitive to physiological disorders associated withfungal attack and disease resulting from pathogen attackand tissue colonization. In contrast to weeds which can beremotely detected and identified in crops according to theirmacroscopic shape early after emergence (Gerhards andChristensen, 2003), micro-organisms causing plant diseasesmay be detected only by their effect on plant tissue; visiblesymptoms often appear only after latent colonization ofthe plant tissue. Digital infrared thermography has beenshown in previous studies to be a useful tool for the pre-symptomatic detection of cucumber downy mildew causedby Pseudoperonospora cubensis (Berk. et Curt.) Rostovzev(Lindenthal et al., 2005; Oerke et al., 2005). The maximumtemperature difference (MTD) within a leaf or a canopyturned out to be suitable for the differentiation of infectedand non-infected tissue under controlled conditions.

Downy mildew of cucurbits is a devastating disease,especially in temperate regions of the world (Palti andCohen, 1980; Lebeda and Schwinn, 1994) where humidconditions favour disease spread; infection by zoosporesrequires free water on the lower leaf surface for at least 2 h,and production of zoosporangia in the dark occurs at an RHof >90% for at least 6 h (Cohen et al., 1971; Cohen, 1977;Zitter et al., 1996). First symptoms on leaves are small,slightly chlorotic to bright yellow areas on the upper surfacewithout loss of vitality in plant cells (Spencer, 1981).Lesions expand with time and may remain chlorotic oryellow or, depending on environmental conditions, becomenecrotic and brown. Further development of lesions resultsin the necrotization of progressively larger leaf areas, andin a few days the entire leaf may be destroyed.

The chronological and spatial dynamics in producingdisease symptoms seem to be rather limited to downymildew pathogens which have to colonize the new healthyleaf areas rapidly as—despite being biotrophic—the patho-gens damage the colonized plant tissue (Spencer, 1981).Water loss from infected leaf areas can increase due todestruction of the leaf cuticle (Bassanezi et al., 2002),increased permeability of leaf cell membranes (Chaerleet al., 2001), or inhibition of stomatal closure (Smith et al.,1986; Felle et al., 2004). Reduction of transpiration mayresult from stomatal closure (Chaerle et al., 2001), ob-struction of xylem elements and stomata (Wright et al.,2000; Bassanezi et al., 2002), and defoliation.

A negative correlation between transpiration rate and leaftemperature has been shown by Inoue et al. (1990). Anincrease in leaf temperature due to restricted water supplyof the shoot has been described for diseases caused by rootrot pathogens or wilt pathogens (Pinter et al., 1979;

Nilsson, 1985a, 1995; Lili et al., 1991; Chaerle and vander Straeten, 2001). Necrotrophic leaf pathogens such asPyrenophora spp. and Pseudomonas syringae as well asPhytophthora sojae in soybean and tobacco mosaic virusin tobacco cause stomatal closure, and consequently re-duce transpiration of infected leaves and increase canopytemperature (Nilsson 1985b, c; Di Giorgio et al., 1996;Chaerle et al., 1999; McDonald and Cahill, 1999). Incontrast, foliar temperature of susceptible wheat wasreduced by 0.2–1.0 8C during early sporulation of Pucciniastriiformis due to rust pustules rupturing the epidermis andpreventing stomatal closure, followed by a significanttemperature increase only in later stages of stripe rustdevelopment (Smith et al., 1986).

Transpiration is a process of diffusion with the rate oftranspiration depending on the air to leaf vapour pressuredeficit (ALVPD). Under humid, low-light, and high-windspeed conditions, the temperature of a dry and a wet leafmay differ by only a few degrees, whereas under highirradiance and low humidity this temperature range mayexceed 15 8C (Jones, 1999b). The effect of environmentalfactors on the spatial heterogeneity of transpiration causedby pathogen colonization, however, has not been investi-gated so far.

The objectives of this study were (i) to evaluate therelationship between disease severity and MTD of cucum-ber leaves infected by P. cubensis; and (ii) to assess theimpact of environmental conditions during measurement ofMTD in order to describe the potential of MTD for theassessment and quantification of downy mildew in the field.

Materials and methods

Plant material

Seeds of cucumber (Cucumis sativus L.), cultivar ‘Vorgebirgstraube’susceptible to P. cubensis were germinated on moist paper at 25/20 8Cfor 4 d. Germinated seeds were transplanted into plastic pots (Ø 11cm) with a 3:1 mixture of organic soil (Klasmann-Deilmann GmbH,Germany) and sand, and were grown in a greenhouse at 25/20 8C(day/night) with an RH of 70610% and a photoperiod of 16 h d�1

(>300 lmol m�2 s�1, Philips SGR 140, Hamburg, Germany). Plantswere watered daily with tap water and fertilized as required, and wereused for the experiments when the first true leaf had fully developed.

Pathogen

The obligate biotrophic oomycte (kingdom chromista) P. cubensis(Berk. et Curt.) Rostovzev was maintained on the first true leaves ofsusceptible cucumber cv. Vorgebirgstraube kept in the greenhouse at25/20 8C, 70610% RH with 16 h of light per day (>300 lE m�2 s�1).Sporulation of the pathogen was induced by placing plants with thefirst symptoms of downy mildew in a darkened moist chamber at20 8C and 100% RH for 18 h. Leaves bearing zoosporangia wereeither frozen at �20 8C for storage or used directly for inoculation.

Zoosporangia formed on the lower leaf side were dislodged withan artist’s soft brush in tap water containing 0.01% Tween-20. Thesuspension was filtered through cheesecloth and the concentrationwas adjusted to 2.53104�53105 sporangia ml�1, depending on the

2122 Oerke et al.

disease severity desired, using a Fuchs–Rosenthal haemacytometer.About 5 ml of zoosporangia suspension of P. cubensis was spray-inoculated onto the lower surface of the first true leaves of cucumberplants using a hand sprayer. Immediately after inoculation, plantswere placed into a moist chamber at 25 8C under natural lightconditions for 6 h in order to provide optimum infection conditions.For control, non-inoculated plants of the same age sprayed with watercontaining 0.01% Tween-20 were kept under the same conditions.Subsequently, inoculated plants and control plants were kept in thegreenhouse at 25/20 8C, 70610% RH, and a photoperiod of 16 h(>300 lE m�2 s�1).

Disease assessment

Plants inoculated with P. cubensis were assessed daily for downymildew development. At the first visible symptom, disease severitywas assessed by visual rating of the percentage of leaf area showingcharacteristic symptoms of downy mildew. The necrotic area as wellas the yellowish halo and the faded area surrounding the lesions wereall included in the assessment of disease severity. Plants were scoredvisually for pathogen presence on a scale of 0, 1, 3, 5, 10, 20, . . . 90and 100% of leaf area covered with symptoms of downy mildewusing a standard area diagram according to Gaunt (1987).

Thermographic measurements, data acquisition, and

analysis

Plants were equilibrated in the laboratory for 1 h before thermalimages were recorded between 9 and 12 a.m. In general, airtemperature in the laboratory was 2361 8C, RH varied between45% and 65%, and photosynthetic active radiation was 250650 lEm�2 s�1. Variation was compensated by measuring the differenttreatments in an alternating sequence. For measurements undervarious environmental conditions, air temperature and RH in theroom were set to 16, 21, or 26 8C, and at 60, 80, or 90% RH.

Digital thermal images were obtained using a VARIOSCAN 3201ST (Jenoptic Laser, Jena, Germany) sterling-cooled infrared scanningcamera with a spectral sensitivity from 8 to 12 lm and a geometricresolution of 1.5 mrad (2403360 pixels focal plane array anda 3083208 field of view lens with a minimum focus distance of;0.2 m). Thermal resolution is 0.03 K, and accuracy of absolutetemperature measurement less than 62 K. Digital thermograms wereanalysed with the software package IRBIS Plus V 2.2 (Infratec,Dresden, Germany) which allowed for correction of object emissivityafter images had been recorded. However, leaf emissivity was set to 1since relative differences in leaf temperature resulting from pathogendevelopment were the main factors of interest of these experiments.

Colour reflectance images were taken with a digital camera (JD4100 Z3, Jenoptic, Jena, Germany).

The transpiration rate (E, mmol H2O m�2 s�1) and assimilationrate (A, lmol CO2 m�2 s�1) of inoculated and non-inoculated tissuewere measured for three areas (2.5 cm2) with a portable porometertype CIRAS-1 with automated gas mixing and a Parkinson leafchamber type PLC-B (PP Systems, Hitchin, UK). Flow rates werekept at 290 ml min�1 and CO2 concentration was adjusted to480 ppm. The average leaf temperature was calculated for the threeareas representing the measuring areas for the transpiration rate onhealthy and infected leaf tissue. Thermographic as well as gasexchange measurements were done on one leaf per plant using six re-plicates per treatment.

The maximum temperature difference within healthy and infectedleaves was studied by taking thermal and colour reflectance imagesfrom control leaves and inoculated leaves and recording diseaseseverity day by day for up to 8 d after inoculation. Using IRBIS PlusV 2.2, a polygon was placed on the area representing a leaf byredrawing the cucumber leaf’s outline omitting mixed pixels on theleaf edge. For every polygon, the maximum temperature difference

[K] was automatically recorded as the difference between the highestand lowest temperature within the polygon. Subsequently the soft-ware produced histograms of leaf temperature from all pixels of themarked area.

Stomatal aperture

The abaxial side of cucumber leaves was coated with transparent nailpolish and peeled off with transparent scotch tape. Epidermalimprints were placed on microscope slides and analysed with a LeitzDMRB photomicroscope (Leica, Wetzlar, Germany). The width ofstomatal aperture was measured for 30 stomata each from non-inoculated and inoculated leaves using the software Diskus 4.2(Hilgers, Konigswinter, Germany) and averaged over three replicates.

Membrane injury

Leakage of electrolytes of P. cubensis inoculated leaf tissue wasinvestigated 5 d post-inoculation. Three types of leaf tissue wereconsidered using chlorotic tissue, tissue surrounding chloroses, andnon-inoculated leaf tissue. Leakage was assessed using a modifiedmethod described by Prohens et al. (2004). For the cucumber leaves,eight discs (Ø 22 mm) were punched out of one leaf and washed threetimes with distilled water in order to eliminate electrolytes fromtruncated cells. Leaf discs were incubated in 100 ml of 0.4 Mmannitol for 3 h. Subsequently, electric conductivity (lS cm�1) wasmeasured with a microprocessor conductivity meter (LF 539, WTW,Weilheim, Germany) equipped with a standard cell (Tetra Con 96,WTW, Weilheim, Germany); 0.4 M mannitol was used for reference.After the first measurement, leaf samples were autoclaved at 121 8Cfor 20 min to destroy the tissue completely and measured again aftercooling down to 25 8C. The ratio between the first and secondmeasurement characterizes the percentage electrolyte leakage.

According to Premachandra and Shimada (1987), the degree ofmembrane injury was calculated using the formula

MI = ½1 �ð1�D1=D2Þ=ð1�H1=H2Þ�3100

where MI is membrane injury (%); D1, electrolyte leakage of diseasedtissue prior to autoclaving; D2, electrolyte leakage of diseased tissueafter autoclaving; H1, electrolyte leakage of non-diseased tissue priorto autoclaving; and H2, electrolyte leakage of non-diseased tissueafter autoclaving.

Leaf water content was determined by comparing fresh weight anddry weight of leaf discs (3.2 cm2) punched out of non-diseased anddiseased leaves.

Statistical analysis

All analyses were conducted using the Superior Performing SoftwareSystem SPSS 11.0 (SPSS Inc., Chicago, IL, USA). Data wereanalysed by standard analysis of variance (ANOVA). For significantF-values, mean comparisons were performed using a significancelevel of P=0.05. Data series were related to each other by the Pearsoncoefficient (r). All experiments were conducted at least twice.

Results

Relationship between leaf temperature andtranspiration rate

Under controlled conditions, the transpiration rate ofcucumber tissue proved to be linearly related to the leaftemperature as measured by digital infrared thermogra-phy. Measurements of non-inoculated and P. cubensis-inoculated leaves were made on six consecutive daysfollowing inoculation. Analysis of data for healthy and

Thermal imaging of cucumber leaves affected by downy mildew 2123

P. cubensis-infected tissue developing the typical sequenceof downy mildew symptoms showed similar regressionlines, with the slope for healthy leaves being a little bitsteeper (Fig. 1). Since there was no statistically significantdifference between the two regression lines, an overall cor-relation between leaf temperature and transpiration ratey=28.21–0.90x was calculated which proved to be highlysignificant (r=�0.84, P <0.01).

Effect of downy mildew on leaf temperature

As early as 2 d after inoculation, infection with P. cubensisincreased the overall temperature of cucumber leavesaffected by almost 2 8C (Fig. 2). One day before theappearance of typical early disease symptoms (water-soaked flecks) at day 3, the MTD within the leaves wassignificantly larger than the MTD of non-inoculated plants.The MTD of these leaves remained largely constant duringthe experiment, while the MTD of infected leaves sub-stantially increased with the appearance of chlorosesassociated with a locally decreased tissue temperature.Two and three days after inoculation, the calculated averagelargely resulted from the temperature of unaffected tissue;infection sites resulted in a few outliers. With the appear-ance of the first necroses 5 d after inoculation, the averageleaf temperature was closer to the lowest temperature ofchlorotic leaf tissue, while the substantial increase intemperature of dead tissue was restricted to some leafspots. The concurrent presence of tissue with increased anddecreased transpiration resulted in an MTD >3.5 K.

In Fig. 3, histograms demonstrate the distribution of leaftemperatures within a cucumber leaf as measured in ther-mograms taken 0, 3, 4, and 6 d after P. cubensis incubation.The variation in skewness with the time of incubationillustrates the dynamics in leaf temperature distributionduring pathogenesis of P. cubensis: with the appearance ofthe first chloroses showing increased transpiration, theGaussian distribution of healthy leaves changed to a left-skewed distribution, which changed again to a right-skeweddistribution when the first necroses were produced. Withthe progression of pathogenesis, the frequency of leaf areasof the same temperature markedly decreased.

The effects of P. cubensis infection on leaf temperaturerepresenting leaf transpiration during pathogenesis—aninitial decrease in temperature due to the formation ofchloroses followed by an increase due to the necrotizationof tissue—could also be demonstrated in the spatial distribu-tion of leaf transpiration around infection sites. Seven daysafter inoculation, the necrotic host tissue in the centre ofinfection sites showing a tissue temperature 0.6 8C higherthan non-infected leaf areas was surrounded by a distinctsmall ring of cool chlorotic tissue—1.2 8C cooler than thenon-infected tissue at 21.66 8C—with increased transpira-tion (Fig. 4). Between these zones and the non-infectedtissue, zones of intermediate temperatures were identified.

Effect on water content and electrolyte leakage ofcucumber leaves

The water content of healthy cucumber leaf tissue largelyremained constant at 18.161 mg cm�2 equivalent to 91%of fresh mass throughout the experiment. Inoculation withP. cubensis slightly decreased the water content 2 d afterinoculation, which substantially increased when the first

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]

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Non-inoculated

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Imax

Imin

Iave

Nmax

Nmin

Nave

Fig. 2. Effect of Pseudoperonospora cubensis infection on averagetemperature, minimum, and maximum temperature of cucumber leaves7 d after inoculation. The light grey area depicts the maximum temper-ature difference (MTD) of non-inoculated leaves; the dark grey areaillustrates the MTD of inoculated leaves (n=6; the asterisk marks asignificant difference from non-inoculated leaves, t test, P <0.05).

inoculated

y = 28.01 – 0.87 x

r = -0.91 **

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543210

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Fig. 1. Correlation of transpiration rate with leaf temperature of non-inoculated and Pseudoperonospora cubensis-inoculated cucumber leaftissue under controlled conditions (P <0.01; measurements were madeon six consecutive days following inoculation with P. cubensis).

2124 Oerke et al.

symptoms of water-soaked tissue appeared on ;80% ofleaf area 3 d after inoculation (Fig. 5). Due to increasedtranspiration of chlorotic tissue, subsequently the watercontent rapidly decreased and continued to decline to reach;50% of that of healthy tissue 7 d after inoculation when70% of leaf area had become necrotic. The damaging effectof P. cubensis infection on the integrity of cucumberplasmalemma was shown to be associated with the forma-tion of chloroses. Five days after pathogen inoculation,the electrolyte leakage of chlorotic tissue reached almostthree times the value of healthy leaf tissue (17% comparedwith 6%). Even the green symptomless tissue neighbour-ing chloroses showed an increased leakage of electrolytes

intermediate to the chlorotic and the unaffected tissue,respectively. Using the formula given by Premachandra andShimada (1987), membrane damage around chloroses was7% and reached 12.5% for chloroses themselves. The deadtissue of necroses was not investigated.

Measurements of stomatal aperture during the earlystages of pathogenesis indicated that the decreased watercontent of infected tissue 2 d after inoculation coincidedwith a slight increase in stomatal opening. In darkness, theaperture of stomata which had an average area of 25.5 lm2

in non-inoculated leaves dramatically increased due to thedevelopment of downy mildew and reached 160% and280%, respectively, 3 d and 6 d after inoculation (Fig. 6).

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-5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 -1.5

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0 d p.i. 3 d p.i.

4 d p.i. 6 d p.i.

Average -3.00MTD 0.91




Fig. 3. Histograms of leaf temperature (relative to air temperature) taken from thermograms of cucumber leaves inoculated with Pseudoperonosporacubensis 0, 3, 4, and 6 d, respectively, after inoculation (measurements at 25 8C, 50% RH; vertical line represents leaf average).

Fig. 4. Zones of different temperature of cucumber leaves 7 d after inoculation with Pseudoperonospora cubensis; reflectance image (left) andthermogram (right) of a leaf inoculated with two 100 ll drops of zoosporangia on the lower leaf surface (measurement at 22 8C, 50% RH).


With illumination, stomatal aperture of infected leaves wassignificantly affected only 5 d after inoculation, and theeffect was less pronounced.

Relationship between disease severity and MTD

Time series experiments showed an increase in MTD withtime of incubation and hence the increase in downy mildewsymptoms. Using cucumber leaves inoculated with fourlevels of zoospore inoculum, the relationship between theseverity of downy mildew and the MTD within a leaf wasinvestigated at three stages of symptom development (Fig.7). After 6, 7, and 8 d of incubation, corresponding toleaves showing almost exclusively chloroses (6 d) and anincreasing portion of necrotic lesions (days 7 and 8), there

was a strong linear correlation between the percentagediseased leaf area and MTD. The slope of the regressioncurve increased with the portion of necrotic symptoms(compare days 6 and 8). For all times assessed, however,quadratic regression analysis resulted in a higher regressioncoefficient and turned out to be more suitable to describethe relationship.

Influence of environmental conditions duringmeasurements

Environmental conditions during thermographic measure-ments substantially influenced the transpiration of cucum-ber tissue and thereby leaf temperature. At 21 8C, anincrease of RH from 60% to 80% significantly reducedtranspiration of healthy leaves and resulted in an increaseof leaf temperature from 18.3 8C to 20.5 8C. Similarly, theincrease of RH to 90% at 26 8C led to a reduced coolingeffect of transpiration, resulting in a leaf temperature of25.5 8C compared with 24.4 8C at 60% RH.

In contrast to the large variation in absolute values, thevariation of transpiration within non-infected cucumberleaves hardly varied with environmental conditions (Table1). The MTD of healthy leaves showed no significant dif-ferences throughout the experiment, irrespective of the me-asuring conditions. Three days after inoculation, infectedleaves had a higher MTD, at least at 21 8C and 26 8C. Withthe appearance of downy mildew symptoms 4 d afterinoculation—the average disease severity increased from20% chloroses 4 days after inoculation to 40% at 5 d afterinoculation and to 70% (40% necroses, 30% chloroses) at6 d after inoculation, respectively—the MTD of infectedleaves further increased during pathogenesis, especially athigher air temperature and reduced RH. Figure 8 demon-strates the effect of temperature and RH during measure-ment on the temperature distribution of a non-infected and


Wat

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Fig. 5. Effect of Pseudoperonospora cubensis on water content (left) and electrolyte leakage (right) of cucumber leaves; electrolyte leakage wasassessed 5 d post-inoculation for non-infected leaf areas, chloroses due to Pseudoperonospora cubensis, and the tissue adjacent to these chloroses(n=6; measurements at 2261 8C). *P <0.05, t test; values with the same letter are not significantly different, Tukey test, P <0.05.

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Stom

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rtur

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illuminated

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Fig. 6. Influence of light conditions on stomatal behaviour of cucumberleaves after 2–6 d of incubation with Pseudoperonospora cubensis;stomatal aperture of non-inoculated and inoculated abaxial leaf sur-faces after 2 h in darkness and after 2 h in the light, respectively(2261 8C, n=30). *Significantly different from non-inoculated leaves(t test, P <0.05).

2126 Oerke et al.

a P. cubensis-infected cucumber leaf for four differentmeasuring conditions 5 d after inoculation; the leaves wereadapted to the environmental conditions for 1 h beforemeasurement. In the last column of Fig. 8, image param-eters were set to maximize the temperature contrast withinthe displayed leaves. In contrast to the healthy leaf showingan MTD of 0.5860.04 8C, the effect of environmentalconditions on diseased leaves was very pronounced; at 268C, 60% RH, MTD was more than three times higher than at

16 8C, 80% RH. Since restrictions in the transpiration ofdiseased leaf areas (some necroses had developed) limitedthe cooling effect, the average leaf temperature of infectedleaves was only 1.5 8C lower than the air temperature,compared with 2.8 8C for non-infected leaves.

The relationship between MTD and disease severity wasalso affected by the environmental conditions duringmeasurement. The scattered plot in Fig. 9 demonstratesthe positive correlation (P <0.01) between both param-eters, irrespective of measuring conditions. Nevertheless, at26 8C and 60% RH, the steep regression curve explainedalmost 85% of the variation in the percentage of diseasedleaf area. Under measuring conditions reducing leaftranspiration (16 8C, 80% RH), the slope of the regressioncurve was not significantly different from zero and acc-ounted for only 14% of the variation in disease severity.

Discussion

Digital infrared thermography proved to be a highly suit-able tool for the spatial analysis of the effects of P. cubensison transpiration of cucumber leaves under controlled condi-tions. After penetrating the leaf through stomata, thispathogen rapidly colonizes the mesophyll of its host cellproducing intercellular hyphae and intracellular haustoriafor the uptake of nutrients (Michelmore et al., 1988; Zitteret al., 1996). As experimental conditions were not condu-cive to the formation of sporangiophores through stomatawhich requires almost 100% RH for at least 6 h (Cohen,1981), the oomycete was confined to the leaf mesophyll andhad no direct effects on the leaf boundary layers. Conse-quently, the relationship between transpiration rate and leaftemperature was similar for healthy and infected leaves.For infected tissue, however, transpiration rates showedhigher variation depending on disease symptoms: trans-piration varied from 1 to 4 mmol m�2 s�1 for non-infectedleaves, but varied from almost 0, for necrotic tissue at laterstages of pathogenesis, to 5.5 mmol m�2 s�1, for chloroticcucumber tissue, the first visible disease symptom occur-ring 2–3 d after inoculation.

Growth of P. cubensis in the mesophyll affected stomatalconductance of cucumber leaves prior to the formation ofvisible disease symptoms (Lindenthal et al., 2005). Diseasesymptoms result from the physiological changes in plantmetabolism brought about by the pathogen. The change inmembrane semi-permeability is one of the earliest host reac-tions to pathogen attack (Wheeler, 1978; Novacky, 1983;Lucas, 1998). Modifications in the primary and secondarymetabolism of plants are the results of this change. In earlystages of pathogenesis (;2 d after inoculation), pathogen-induced membrane injury is likely to result in a localdecrease of cell turgor and a slightly reduced water contentof tissue associated with a transient increase in overall leaftemperature. Only recently, Scharte et al. (2005) demonstrated

Fig. 7. Regression between downy mildew severity and maximumtemperature difference of cucumber leaves 6, 7, and 8 d, respectively,after inoculation with Pseudoperonospora cubensis (n=8; measurementsat 2662 8C, 60610% RH; P <0.05).


Fig. 8. Effect of environmental conditions on temperature distribution, average leaf temperature (Ø), and maximum temperature difference (MTD) ofa non-inoculated and a Pseudoperonospora cubensis-inoculated cucumber leaf, respectively, 5 d after inoculation (the thermogram in the right row isshown with the settings to demonstrate maximum heterogeneity within the leaf).

Table 1. Influence of environmental conditions on maximum temperature difference (MTD) of non-inoculated and Pseudoperonosporacubensis-inoculated cucumber leaves during pathogenesis of downy mildew (n=6)

Values within the same column with the same letter are statistically not significantly different, Tukey test, P <0.05.

Measuring condition Inoculation Days post-inoculation

0 1 2 3 4 5 6

16 8C, 80% RH � 0.70 a 0.52 a 0.60 ab 0.50 c 0.60 d 0.66 d 0.49 d+ 0.62 a 0.62 a 0.64 ab 0.69 c 0.94 bc 1.21 c 1.11 c

21 8C, 80% RH � 0.60 a 0.58 a 0.61 ab 0.56 c 0.53 d 0.58 d 0.58 d+ 0.57 a 0.63 a 0.69 ab 1.00 a 1.08 ab 1.16 c 2.65 b

26 8C, 60% RH � 0.71 a 0.61 a 0.76 ab 0.77 bc 0.74 cd 0.82 cd 0.68 d+ 0.67 a 0.62 a 0.80 a 1.00 a 1.14 a 3.18 a 3.37 a

26 8C, 90% RH � 0.74 a 0.59 a 0.57 b 0.53 c 0.52 d 0.73 cd 0.60 d+ 0.66 a 0.63 a 0.68 ab 0.79 b 0.81 c 1.94 b 2.34 b

2128 Oerke et al.

initial stomatal closure of tobacco leaves in the incom-patible interaction with the oomycetous pathogen Phy-tophthora nicotianae. A temporary decrease in wateravailability due to a transient restriction of the xylem hasbeen reported for the hypersensitive reaction of Nicotianaedwardsonii with tobacco mosaic virus (Wright et al.,2000). It is not known whether these effects also occur inthe described compatible host–pathogen interactions.

Membrane injury which could already be detected inlatent infected, symptomless tissue progressed in the highlydynamic process of downy mildew development and wasassociated with disintegration of mesophyll cells; catabolicactivity in chloroplasts resulted in the appearance ofchloroses. The loss of electrolytes from infected tissue islikely to increase the osmotic value of diseased leaf areas,which, in combination with the sink activity of thebiotrophic pathogen, induced a transient increase in thewater content of tissue 3 days after inoculation. Stomatalopening in the non-colonized epidermal layer induceda significant decrease in leaf temperature at this time ofpathogenesis. The aperture of stomata is a function of theturgor difference between guard cells and neighbouring(epidermal) cells (Eschrich, 1995). The turgor is regulatedby the selective influx of potassium into the guard cells andthe accumulation of KCl and K2 malate (Outlaw, 2003;Tallman, 2004). Barley infection by Rhynchosporiumsecalis caused stomatal opening due to damage of epider-mal cells associated with a change in the turgor balancebetween guard cells and the other epidermal cells (Ayresand Jones, 1975). Three days after inoculation, stomatalregulation of cucumber leaves was still impaired. Theexcessive transpiration of colonized tissue rapidly led toa decrease in water content. This malfunction of stomata

finally results in the desiccation of leaf tissue and total lossof transpiration—cell death associated with the formationof visible necroses. The first appearance of necroses wasassociated with a supra-optimal opening of stomata in thelight. However, being a biotroph, P. cubensis may causesignificant damage to its host plants already in early stagesof pathogenesis (Lindenthal et al., 2005).

A sequence in thermal modifications resulting frompathogen attack—pre-symptomatic cooling followed bya temperature increase due to tissue desiccation—has alsobeen described for sugar beet leaves infected with Cercos-pora beticola (Chaerle et al., 2004). The authors explainedthe initial cooling effect of infection by the production ofa membrane-active toxin of the fungus. The biotrophicpathogen P. cubensis, in contrast, is not reported to producetoxins. The simultaneous occurrence of both symptoms inone leaf and the effect of infection on membrane functionin symptomless tissue also indicate the activity of amobile compound for the interaction of the oomycete andcucumber leaves.

Two to three days after inoculation, the heterogeneity ofthe transpiration rate within infected leaves was signifi-cantly higher than for healthy leaves. The MTD proved tobe a simple but reliable parameter for this heterogeneity andmay be used for the discrimination of healthy leaves orcanopies and those with downy mildew (Lindenthal et al.,2005; Oerke et al., 2005). MTD of leaves increased duringpathogenesis as initial symptoms show an increase intranspiration rate, while at later stages leaf tissue becamenecrotic associated with a transpiration approaching zeroand a drastic increase of local temperature; the average leaftemperature may be largely unaffected. The simultaneouspresence of chloroses and necroses in leaves results in thehighest values of MTD, which slowly decreases when theleaf becomes completely necrotic.

Repeated measurements of various leaves differing indowny mildew severity showed a strong linear correlationbetween disease severity and MTD irrespective of the typeof visible symptoms. The percentage leaf area affected, i.e.the number of both types of lesions, chloroses and necroses,was more important than the type of symptom; neverthe-less, the appearance of necroses increased the slope of theregression curve. For all stages of pathogenesis, however,statistical analysis produced higher correlation coefficientsfor quadratic regressions, indicating that the positivecorrelation may be used only until 60% disease severity.Higher percentages of leaf area affected by P. cubensisdid not increase the MTD but tended to decrease this in-dicator of heterogeneity in the spatial distribution of leaftemperature. Heterogeneity decreased as the leaves becamealmost completely diseased.

The effect of environmental conditions on the averageleaf temperature and its relationship to air temperature havebeen described many times (Jones, 2004). Conditions fa-vouring transpiration result in a strong cooling effect; under

rlin = 0.37**

rsq = 0.38**

rlin = 0.92**

rsq = 0.94**

Max

imum

tem

pera

ture

dif

fere

nce

[K]

Diseased leaf area [%]

0 20 40 60 80 100

26 °C, 60 % RH

16 °C, 80 % RH4.0

3.5

3.0

2.5

2.0

1.5

1.0

4.5

0.5

0

Fig. 9. Influence of environmental conditions on the correlation ofdowny mildew severity and maximum temperature difference ofcucumber leaves (measurements 4, 5, and 6, d after inoculation,P <0.01).


conditions suppressing stomatal conductance, the differ-ence between air temperature and overall leaf temperatureis often minimal. In contrast to the absolute average leaftemperature, MTD of healthy cucumber leaves was largelyunaffected by various environmental conditions duringmeasurements. Diseased leaves, however, showed markeddifferences in the spatial heterogeneity of transpiration—indicated by MTD values—depending on the measurementconditions. MTD of diseased leaves was high underenvironmental conditions favouring leaf transpiration. Ac-cording to Jones (1999b), the sensitivity of thermographyfor the detection of changes in leaf conductance increaseswith air to leaf vapour pressure deficit, net radiationabsorbed, and wind speed. Downy mildew caused markedchanges in tissue transpiration only when stomatalconductance of cucumber leaves was not restricted bya high RH or low temperature. Since the transpiration rateis related to a vapour pressure deficit, the effect of RH ontranspiration was more pronounced than that of air tem-perature. The correlation between disease severity andMTD was also affected by environmental conditions. Bothparameters were significantly correlated under the differ-ent measurement conditions, but the coefficient of corre-lation was markedly lower for conditions restricting leaftranspiration.

As the variability of leaf temperature between healthytissue and disease symptoms is modified by environmentalconditions, the MTD cannot be used either for the quan-tification of disease severity or as a reliable parameter forthe discrimination between healthy and downy mildew-infected cucumber plants. Its dependence on environmentalconditions makes it impossible to derive a threshold valuefor MTD. In this study, however, digital infrared thermo-graphy proved to be a powerful tool for the characterizationof the different stages in the host–pathogen interaction asthis remote sensing technique interferes as little as possiblewith the plant. MTD is a very sensitive parameter for spatialmodifications in leaf temperature due to pathogen attack,which is generally restricted to a low number of cells inearly pathogenesis. The mean leaf temperature (cf. Fig. 2),variance, as well as upper and lower percentiles of leaftemperature values (data not shown) proved to be lesssensitive.

Digital infrared thermography alone seems not to besuitable for disease detection in the field, a prerequisite fora more demand-based use of fungicides or site-specificdisease control (Paveley et al., 1996; West et al., 2003).This sensor has to be combined with other remote sensingmethods offering additional spectral information andsystems for the recognition of optical patterns in plantcanopies (Chaerle et al., 2003; Omasa and Takayama,2003; Jones, 2004). Also the use of reference areas or plantsmay be suitable (Jones, 2004), especially for the identifi-cation of wet and dry canopies.

Acknowledgements

This study was performed within the research training group ‘Useof information systems for precision crop protection’ at the Facultyof Agricultural Sciences, Rheinische Friedrich-Wilhelms-University ofBonn, and was funded by the German Research Foundation (DFG).

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