effects of intermittent ozone exposures on powdery mildew of cucumber

Environmental and Experimental Botany 42 (1999) 163–171

Effects of intermittent ozone exposures on powdery mildewof cucumber

Mujeebur Rahman Khan a,*, M. Wajid Khan b

a Department of Plant Protection, Institute of Agriculture, Aligarh Muslim Uni6ersity, Aligarh 202 002, Indiab Department of Botany, Aligarh Muslim Uni6ersity, Aligarh 202 002, India

Received 20 October 1998; received in revised form 1 June 1999; accepted 5 June 1999

Abstract

Exposure of plants to O3 may influence foliar diseases caused by fungi. It is hypothesized that concentrations of O3

at or below economic threshold may enhance severity of the fungal disease, whereas higher levels may decrease theseverity. To test this hypothesis, effects of intermittent exposures at 50, 100 and 200 ppb O3 and powdery mildewinfection by Sphaerotheca fuliginea, using pre-, post- and concomitant-inoculation exposures were investigated oncucumber in closed-top exposure chambers. For impact assessment, plant growth, flowering, fruit-setting, foliar ozoneinjury, fungal colonization, conidia size and their germination were considered. Ozone (except 50 ppb) caused necroticlesions on leaves and reduced the plant growth and fruit-setting of cucumber. Plants inoculated with S. fuligineaconidia developed powdery colonies in the intercostal region of leaves. Ozone injury was relatively moderate onfungus-inoculated plants. Powdery mildew development was, however, severe on the plants exposed to 50 ppb O3, butat higher concentrations there was significant decline in fungus colonization. Conidia examined from such plants(exposed to 100 or 200 ppb) were smaller in size and contained fewer fibrosin bodies and showed poor germination.Ozone exposures at 50 ppb, however, stimulated the conidial germination. In an in–vitro experiment, the conidiadirectly exposed to ozone on glass slides showed more or less similar response to the gas. Ozone at 50 ppb and S.fuliginea interacted synergistically and caused significantly greater decrease in the number of fruits/plant. At 200 ppbO3, the mutual effects were antagonistic. Fungus infection provided partial protection to plants against 200 ppbozone, and mildew infection was also mild. © 1999 Elsevier Science B.V. All rights reserved.

Keywords: Cucumber; Cucumis sati6a ; Fungus disease; Ozone; Plant growth; Sphaerotheca fuliginea

www.elsevier.com/locate/envexpbot

1. Introduction

Ozone is considered the most economically im-portant air pollutant and it causes decline ofmany forest trees (Woodbury et al., 1994; Lau-

rence et al., 1996) and reduces growth and yield ofcrop plants in America (Pell and Pearson, 1984;Kress et al., 1986; Temple, 1990) and Europe(Ashmore, 1984; Grandjean and Fuhrer, 1989;Adaros et al., 1991). Ambient concentration of O3

in USA (NESCAUM, 1993) and Europe (Volzand Kley, 1988; Bytnerowicz et al., 1993) may* Corresponding author.

S0098-8472/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.

PII: S0098 -8472 (99 )00029 -5

M.R. Khan, M.W. Khan / En6ironmental and Experimental Botany 42 (1999) 163–171164

range between 50 and 100 ppb or above 120 ppbduring summer. In Asian countries, O3 has beenreported to occur at relatively lower concentra-tion, such as 24–46 ppb in Japan (Kobayashi,1988), 2–69 ppb in India (Khemani et al., 1995;Khan and Khan, 1997) and 36 ppb in Pakistan(Wahid et al., 1995a,b). Existing ambient concen-trations of O3 in India may cause foliar injury andreduce growth and yield of crop plants (Bam-bawale, 1986).

Host–parasite relationships may be affecteddue to ozone (Manning and Tiedemann, 1995),because the gas exposure may influence parasitismby bacteria, fungi, etc. (Khan and Khan, 1993).Exposure of plants to 0.15–0.25 ppm promotedthe invasion by Botrytis cinerea on potato leaves(Manning et al., 1970). Woodbury et al. (1994)reported that 42–79 ppb ozone (9 h daily for 98days) predisposed hybrid Populus seedlings toSeptoria musi6a, leading to a greater number ofcankers, and many of the plants died during thesubsequent winter. Similarly, ozone exposures at40–160 ppb enhanced the infection of Rhizoctoniasolani on white clover (Kochhar et al., 1987).Intermittent exposure of pea seedlings to 60 or120 ppb O3 (6 h/day for 5 days before or afterinoculation) decreased the leaf colonization ofErysiphe polygoni f.sp. pisi and infected plantsdeveloped less foliar injury caused by O3 (Ruschand Laurence, 1993).

The above studies examining the effect of O3 onthe fungus–plant complex have demonstratedthat O3 may stimulate and/or suppress plantpathogenic fungi. It is hypothesized that the con-centration of the gas would control the kind ofeffect on a pathogen. A concentration lower thaneconomic threshold level may promote the dis-ease; higher concentrations may prove suppressivefor fungal pathogenesis. This hypothesis wastested against a powdery mildew fungus,Sphaerotheca fuliginea on cucumber (Cucumis sa-ti6a). Cucumber is an important vegetable cropthroughout the world and is regularly attacked byS. fuliginea (Khan, 1989). The powdery mildewdisease is characterised by the development ofwhite powdery colonies on leaves, flowers andstem. The disease is responsible for a considerableyield loss to cucumber each year. The experiment

was conducted during two consecutive years inclosed-top exposure chambers, using pre-, post-and concomitant-inoculation exposures at 17 (am-bient air), 50, 100 and 200 ppb O3.

2. Materials and methods

2.1. Exposure system

Four exposure chambers, each of 90×90×120cm (length×width×height), were used. Threechambers were used for ozone exposures (50, 100and 200 ppb) and the fourth one for ambient air(control). The chambers were made of transparentfibre glass with an exhaust duct at the top and adouble-layered bottom. Ozone was generated in agenerator by ionizing oxygen (O2) in the presenceof ultraviolet light. The exposure chambers andgenerators have been described in detail elsewhere(Khan and Khan, 1994b). Ambient concentrationof nitrogen dioxide and ozone at the Departmentof Botany, AMU, was recorded as 29 and 17 ppb,respectively. An air sampler was kept inside thechamber during each exposure to sample ozone,which was analysed immediately after the expo-sure by a colorimetric method (Anonymous,1986). The average concentration of ozone insidethe chambers after completion of 27 intermittentexposures was 5094.7, 10097.9 and 200912.3ppb. The fourth exposure chamber used for thecontrol set (without ozone), was not connected tothe ozone generator and, hence, received ambientair that contained around 1792.6 ppb ozone.Uniform air flow rates (1.9 m/s) were maintainedin all the chambers. Chambers were kept in theglasshouse where plants were cultured, but in aseparate room with similar conditions of tempera-ture (35 and 30°C during day and night), light(06:00–19:00 h photosynthetic photon flux den-sity 600925 mmol/m2 per s) and humidity (65–70%) in both years.

2.2. Treatments and plant culture

Clay pots (15 cm diameter) containing auto-claved field soil and compost (3:1) were kept onglasshouse benches (32 rows, five pots/row). Five

M.R. Khan, M.W. Khan / En6ironmental and Experimental Botany 42 (1999) 163–171 165

water-soaked seeds of cucumber, Cucumis sati6aL. were sown in each pot. A week after sowing,the germinated seedlings were thinned to oneseedling/pot. Two weeks later, some plants wereinoculated with the conidia of Sphaerothecafuliginea (Schlecht.) Poll. The conidia were ob-tained from cucumber plants grown in a green-house and experimental plants were inoculatedaccording to the ‘rolling’ method (Nair andEllingboc, 1962). Rather than depositing conid-iospores from source leaves onto a glass slide,the spores were directly applied to the leaves ofcucumber plants.

Joint treatments of ozone and fungus inocula-tion were done according to sequential (pre- andpost-) and concomitant-inoculation exposure.Plants were exposed intermittently to ozone at50, 100 and 200 ppb every third day for 7 hfrom 09:00 h for 75 days. Each treatment re-ceived 27 intermittent ozone exposures (7 h ev-ery third day). In pre-inoculation andconcomitant treatments, the 27th exposure wasgiven on the 96th day of seedling emergence.Last exposure of post-inoculation treatment wasfinished on 103rd day of seedling emergence.Four independent control sets of plants for eachtreatment corresponding to the day of inocula-tion were maintained and exposed to ambientair (17 ppb O3) in exposure chambers for thesame duration. For brevity they are referred as17 ppb O3.

In total, 16 treatments (four unioculated+three inoculated× four ozone concentrations, i.e.17 (ambient), 50, 100 and 200 ppb), were main-tained each year, with ten pots per treatment.Pots were placed on glasshouse benches in acompletely randomized block design. Plants wererotated among the four chambers on each expo-sure to reduce the differences in their microcli-mates. Plants were supported by bamboo stickserected in the pots and were irrigated with tapwater (250 ml/pot) on alternate days. Five outof the ten plants of each treatment were ran-domly harvested on the 103rd day of seedlingemergence and the following parameters weredetermined.

2.3. Foliar symptoms, dry matter production etc.

The plants were examined regularly for thesymptoms attributable to ozone damage and/orthe mildew infection. At the time of harvest, thefive plants not used to determine plant growthetc., were used to measure percent ozone-inducedfoliar injury and mildew colonization on theleaves by use of a planimeter. Flower buds formedduring the course of the experiment were counted.At termination of the experiment, the number offruits/plant were counted. A few hours beforetermination, the soil in each pot was saturatedwith water to facilitate plant root recovery. Rootsand shoots, after exposure to sunlight for 3 days,were kept in a hot air oven at 60°C for 24 h, foruniform drying to determine dry weights.

2.4. Size, germination and fibrosin bodies ofconidia from exposed plants

The conidia from the infected leaves of cucumberplants exposed to 17, 50, 100 and 200 ppb O3 weredusted on clean glass slides to measure their lengthand width under a microscope. Some additionalslides with conidia were placed on glass triangleskept in Petri plates containing sterilized water at thebottom. The plates were covered with a lid linedwith moistened cotton wool and incubated at 25°Cfor 36 h. After incubation the slides were examinedunder a microscope and conidial germination wasdetermined. Fibrosin bodies were counted from thefresh conidia mounted in 3% aqueous potassiumhydroxide solution. One hundred conidia for eachtreatment were examined to determine their size,germination and number of fibrosin bodies.

2.5. Effects of ozone on conidial germination onglass slides

Direct effects of ozone (17, 50, 100 and 200ppb) for different durations (3, 6, 9 and 12 h) onconidial germination were studied in a microgasexposure cabinet. The cabinet (33×23×3 cm)made of transparent fibreglass was placed insidethe exposure chamber. The cabinet has beendescribed elsewhere (Khan and Khan, 1994a). The


conidia from unexposed plants were dusted onclean glass slides, which were placed on glasstriangles in Petri plates containing sterilized wa-ter. The plates (without cover) were kept insidethe cabinets to expose the conidia directly toozone at the desired dosages. After exposure theplates were covered with lids lined with moistenedcotton wool and incubated at 25°C for 36 h.

2.6. Statistical analysis

Data obtained from five plants each year (1990and 1991) were averaged and considered as onereplicate. So there were two replicates (one repli-cate/year); the experiment was replicated overtime. The data on plant growth, flowering andfruit setting and ozone induced injury and funguscolonization on foliage were analysed by two-fac-tor analysis of variance (ANOVA) and least sig-

nificance differences (L.S.D.) were calculated atP=0.05. The two factors were ozone 17, 50, 100and 200 ppb and powdery mildew fungus (uninoc-ulated and pre-, post- and concomitant-inocula-tions). Single-factor ANOVA was employed tosignify the effects of ozone on the size, germina-tion and number of fibrosin bodies of the conidiaof S. fuliginea (Dospekhov, 1984).

3. Results

3.1. Foliar symptoms

Powdery mildew fungus, S. fuliginea, causedcharacteristic circular white powdery colonies onupper and lower leaf surfaces. These symptomswere discerned 1 week after inoculation and be-came prominent in 5–6 weeks. The fungus colo-

Table 1Effects of ozone and/or Sphaerotheca fuliginea on O3-induced leaf injury and fungal colonization of the leaves of cucumbera

Treatment Ozone (ppb) Ozone injury (%) Fungal colonization

0.0 (0.0)17 0.0 (0.0)Uninoculated50 7.2 (15.6)* 0.0 (0.0)

100 18.5 (25.5)* 0.0 (0.0)200 26.1 (30.7)* 0.0 (0.0)

52.7 (46.6)Pre-inoculation exposure 17 0.0 (0.0)60.2 (50.9)*50 6.9 (15.2)*49.5 (44.7)17.4 (24.6)*100

200 20.5 (26.9)* 26.8 (31.2)*

17Post-inoculation exposure 0.0 (0.0) 50.9 (45.5)66.2 (54.4)*6.8 (15.1)*50

18.0 (25.1)* 47.5 (43.6)100200 21.3 (27.5)* 23.2 (28.8)*

17 0.0 (0.0) 52.0 (46.2)Concomitant-inoculation exposure50 7.1 (15.4)* 64.7 (53.6)*

100 16.9 (24.3)* 48.3 (44.1)200 19.2 (26.0)* 25.7 (30.5)*

L.S.D. (P=0.05) 5.32.15

F value:Ozone (df=3) 63.6** 29.5**Fungus (df=3) 12.7** 81.3**

8.7**9.2**Interaction (df=9)

a Values in parentheses are transformed angular values.* Significantly different from the control (17 ppb) at P=0.05.** Significant at P=0.05.


Table 2Effects of ozone and Sphaerotheca fuliginea on dry matter production, flowering and fruit setting of cucumbera

Dry weight (g)Ozone (ppb) Number/plantTreatment

Shoot Root Flowers Fruits

8.3 2.19Uninoculated 27.417 27.38.2 2.12 26.750 26.47.9 2.03*100 26.1 24.9*7.3* 1.86* 25.8200 24.1*

7.5* 2.10Pre-inoculation exposure 26.217 25.0*7.2 1.98 26.050 21.9*7.2 1.95100 25.9 21.4*7.0* 1.94* 25.7200 21.0*

7.4* 2.12Post-inoculation exposure 26.417 24.8*6.9* 1.97 25.950 22.3*6.8* 1.94100 25.9 22.3*6.7* 1.92* 25.6200 21.8*

7.4* 2.09Concomitant-inoculation exposure 26.217 24.9*7.0 1.98 26.050 23.06.9* 1.93100 25.8 22.6*6.9* 1.90* 25.7 22.5*200

0.49 0.12L.S.D. (P=0.05) 2.5 2.2

F value:12.7** 8.2** NSOzone (df=3) 6.9**16.4**Fungus (df=3) NS NS 11.4**8.5** 4.9** NS NSInteraction (df=9)

a Each value is mean of two replicates. NS, not significant at P=0.05.* Significantly different from the control (17 ppb) at P=0.05.** Significant at P=0.05.

nized more than 50% of the leaf area of controlplants (exposed to 17 ppb O3) (Table 1). Funguscolonization was 22.8% greater (P=0.05) on theleaves of plants exposed to 50 ppb, being greatestwith post-inoculation treatment compared with 17ppb. Exposures with 200 ppb O3, however, re-sulted in a drastic decrease in the colonization; onaverage it was half of that developed on 17 ppb-O3 exposed plants. The mildew symptoms werelowest with post-inoculation treatment (Table 1).Ozone at 100 ppb, however, did not induce anysignificant effect on the mildew development.

Intermittent exposure of plants to ozone causednumerous small chlorotic and/or necrotic lesionsin the intercostal leaf area. Ozone at 200 ppbcaused 26.1% lesions on the leaf surface of uni-noculated plants; the intensity of the leaf injurywas, however, significantly decreased on fungus-

inoculated plants compared with uninoculatedplants (Table 1). Fungus colonization did notaffect the gas injury at 100 ppb, and 16.9–18%chlorosis/necrosis developed on both groups ofplants. A very mild chlorosis developed on inocu-lated or uninoculated plants exposed to 50 ppbO3.

3.2. Dry matter production and yield

Inoculation of plants with S. fuliginea or expo-sure to 200 ppb O3 caused significant decrease inthe dry weight of shoots compared with the con-trol (Table 2). Significant decrease in the dryweight of roots occurred due to 100 or 200 ppbO3, but not due to fungus inoculation. Exposureswith 50 ppb O3 slightly enhanced the negativeeffect of the mildew fungus leading to a greater


decrease in the dry weight, being significant foronly shoot dry weight due to post-inoculationexposure compared with the sum of individualeffects of the fungus and 50 ppb O3 (Table 2).The fungus and 200 ppb O3 jointly causedgreater decline in the dry weights, but the effectswere significantly less than the sum of decreasescaused by them individually (Table 2). Dryweight of shoots of inoculated–exposed (100ppb) plants were more or less equal to the treat-ment of fungus+50 ppb. The combined effectof 100 ppb O3 and S. fuliginea were largely nearto additive.

Flower production was not influenced butfruit setting was considerably affected (Table 2).Ozone at 100 or 200 ppb or S. fuliginea causedsignificant decrease in the number of fruits/plantcompared with the control (17 ppb O3). Inocu-lated plants exposed at 50 ppb exhibited greaterdecrease in fruit number compared to the sumof individual effects. The decrease in fruit num-ber caused by the fungus and 100 or 200 ppbO3 jointly was equal or less than the sum ofindividual effects.

3.3. Conidial characters of Sphaerotheca fuliginea

The conidia examined from the leaves of O3-exposed plants were smaller in size comparedwith the control (17 ppb O3) (Table 3). Therewas a decrease in the length and width ofconidia with an increase in the concentration ofO3, being significant at 200 ppb O3. In post-in-oculation at 100 ppb O3, the number of fibrosinbodies per conidium decreased, being significantat 100 and 200 ppb (Table 3). Germination ofconidia was also significantly inhibited at 100and 200 ppb O3. At 50 ppb O3, a slight increase(nonsignificant) in the number of germinatedconidia was recorded (Table 3). Mode of inocu-lation exposures did not influence the conidialcharacters.

Conidia on glass slides exposed to a singledose of ozone also showed variation in the ger-mination (Table 4). Ozone at 50 ppb (except 3 hduration) significantly enhanced the conidial ger-mination, being highest for 6 h exposure com-pared with 50 ppb for 0 h or 17 ppb treatments.Whereas 100 (except 3 h) and 200 ppb expo-

Table 3Effects of ozone on the size, fibrosin bodies and germination of conidiospores of Sphaerotheca fuligineaa

Conidia size (mm) ConidialOzoneTreatment Fibrosin bodies/germination (%)conidium

WidthLength

17 24.9Pre-inoculation exposure 13.2 61.46.864.513.124.650 6.6

100 23.8 12.6 6.3* 51.0*23.1* 12.3*200 5.5* 39.2*

60.96.713.2Post-inoculation exposure 25.11750 24.6 13.0 6.4 62.8

100 24.6 12.8 6.1* 47.5*34.7*5.6*200 12.0*23.3*

17 25.1Concomitant-inoculation exposure 13.3 6.7 61.150 24.4 13.1 6.5 63.7

100 24.0 13.9 6.0* 46.2*200 23.4* 12.4* 5.6* 35.9*

1.38L.S.D. (P=0.05) 0.85 0.42 4.7

10.4**F value (df=3) 7.2** 18.5** 21.2**

a Each value is based on 200 observations (100/year).* Significantly different from the control (17 ppb) at P=0.05.** Significant at P=0.05.


Table 4Effect of ozone dosages on conidial germination of Sphaerotheca fuliginea on glass slidesa

Exposure duration (h) L.S.D.Ozone F value(P=0.05) (df=4)

3 6 9 120

57.8 57.5 57.5 56.9 2.5 NS17 58.360.3 65.8ab 65.0ab58.8 63.9ab50 4.8 21.3**

58.3100 58.4 53.2ab 50.1ab 40.5ab 4.3 20.7**200 49.0ab58.7 41.4ab 35.9ab 26.2ab 4.7 32.5**

4.1 3.9 4.4L.S.D.(P=0.05) 4.21.9

F value (df=4) NS 7.7** 15.2** 29.0** 41.3**

a Percent conidial germination: values in rows marked ‘a’ are significantly different from the control (0 h) at P=0.05. Values incolumns marked ‘b’ are significantly different from the control (17 ppb) at P=0.05; ** significant at P=0.05; NS, not significantat P=0.05.

sures caused significant decrease in the conidialgermination compared with respective controls, thelowest number of conidia germinated after 200 ppbO3 exposure for 12 h. This treatment also causeddistortion and shrinking of conidia.

4. Discussion

Ozone at 100 or 200 ppb caused considerablefoliar chlorosis/necrosis on cucumber plants, indi-cating that the plant species was susceptible toozone injury. Fungus infection, however, reducedthe gas injury. Infection with powdery mildew fungidecreased transpiration due to partial closure/clog-ging of stomata (Martin et al., 1975). Probably, forthis reason, less O3 injury developed on the leavescolonized by S. fuliginea. Because partial stomatalclosure or decrease in transpiration rate may leadto a reduced diffusion of ozone in the leaf tissue(Khan and Khan, 1997). In addition, mildew colo-nization may have reduced the leaf tissue availablefor ozone damage because the cells already killedby the fungus cannot be injured by O3. Rusch andLaurence (1993) observed less O3 injury on peaseedlings infected with E. polygoni f.sp. pisi.

Germination of conidia is a vital step in thepathogenesis of S. fuliginea and any effect on theirgermination may have corresponding effect on thedisease severity. Accordingly, stimulation inconidial germination due to 50 ppb O3 led togreater colonization by the fungus. In addition to

the direct stimulatory effect of O3 on the germina-tion as occurred on glass slides, host-mediatedeffects may also have contributed in increasing theseverity of the disease. Low levels of O3 are knownto promote fungal diseases (Khan and Khan, 1993).Heagle and Strickland (1972) have reported en-hancement in the severity of mildew of barleycaused by E. graminis f.sp. hordei due to exposureto low levels of O3. Greater colonization led tosynergistic interaction between 50 ppb O3 and S.fuliginea, and subsequently the yield declines weregreater than the sum of their individual effects.

Higher concentration of ozone apparentlycaused plasmolysis of conidia (Khan and Khan,1994a), as a result the conidia appeared distortedor deshaped after exposure to 200 ppb O3. Fewerfibrosin bodies in the conidia indicates that ozoneexposures either caused their depletion or theirnormal development could not occur due to thestress. Fibrosin bodies are considered to nourishconidia during germination. Hence, their fewernumbers may also have been partially responsiblefor lower germination of conidia (especially sec-ondary), and subsequently a reduced disease due to200 ppb O3. Rusch and Laurence (1993) havereported similar antagonistic effects of 100 ppb O3

on the powdery mildew of pea caused by E.polygoni f.sp. pisi.

The study revealed that the response of plants topathogens may vary with ozone concentration. Atlower concentrations (50–100 ppb), powderymildew may remain uninfluenced, whereas the


higher level may suppress the disease. Ozone fre-quently occurs at concentrations above 100 or 120ppb in USA (NESCAUM, 1993) and Europe(Volz and Kley, 1988; Bytnerowicz et al., 1993).Hence, cucurbits grown in such areas may exhibitless damage from powdery mildew fungus andozone. The present investigation conducted underartificial treatment conditions shall be considereda basis for further research under ambient condi-tions. Because air circulation and some other un-controllable factors influence the microclimate ofa plant inside the chamber, as a result plants mayrespond differently to the same concentrations ofthe gas when exposed in ambient conditions.

Acknowledgements

Financial support from the Council of Scientificand Industrial Research, New Delhi, India,through a research project no. 37(31)/85 EMR-IIis gratefully acknowledged.

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effects of intermittent ozone exposures on powdery mildew of cucumber

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