Physiological and Molecular Plant Pathology (1990) 36, 277-287

Production of ethylene by tissues of tomato, pepper,French-bean and cucumber in response to infection byBotrytis cinerea-

YIGAL ELAD

Department of Plant Pathology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50 250, Israel

(Accepted for publication January 1990)

Ethylene production by detached leaves of tomato, pepper, bean and cucumber infected byBotrytis cinerea was much higher than that of noninfected leaves of the same hosts . Maximumproduction of ethylene was observed in leaves showing mild symptoms of the disease as comparedwith noninfected or completely infected leaves . The fungus produced negligible amounts ofethylene when grown on autoclaved leaves, with the exception of pepper leaves supplementedwith methionine (0 .14 nl g t h-4 ) . Exogenously supplied ethylene induced 75-370°,, morenecrosis than B. cinerea infections in all the crops . Applications of methionine increased necrosis toa lesser extent. Silver-thiosulphate (STS) at levels of 10-4 and 10 -5 M, aminooxyacetic acid(AOA 10-s and 10-4 M) and aminoethoxyvinylglycine (AVG 10-s and 10-4 M) reduced thedevelopment of grey mould disease significantly . AOA and AVG inhibited the production ofethylene by all the crops . Disease incidence in cucumber fruits was increased by growth at 4 °Cor treatment with ethephon prior to inoculation whereas STS treatments completely inhibiteddisease development. Disease development was also decreased in intact plants treated with theinhibitors .

INTRODUCTION

During normal plant growth the amount of ethylene produced is usually relatively low,but slight peaks in production occur mainly around germination, when the plant ismature, and during leaf abscission [9] . Abscission of diseased leaves is common inplants infected with fungi [4, 11] . It is known that stress factors such as chemicaltreatments, irradiation, drought, infestation with pests, diseases and mechanicalinjuries cause increases in ethylene production [3, 8, 16] .

Boller et al . [1] proposed that ethylene-induced chitinase in bean leaves may functionas a defence enzyme against fungal and bacterial invaders . However, Mauch et al . [10]

suggested later that ethylene production is a symptom of, and not a signal for theinduction of the enzyme in pea pods infected by Fusarium solani . Maintaining ethyleneconcentration in the storage atmosphere at < 0 . 01 µl 1 -1 increased the susceptibility oftomato fruits to infection by Botrytis cinerea Pers ., whereas higher concentrationsresulted in less infection [7] . Ethylene in the storage atmosphere has been reported tostimulate postharvest decay by other fungi also [6] . Infection of wheat seedlings by

tContribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, IsraelNo. 2144-E, 1987 series .

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Septoria nodorum was found to induce the formation of ethylene and chemical control ofinfections was associated with a reduction in ethylene production [15] .

Botrytis cinerea is a major pathogen of vegetables grown in Israel and elsewhere,especially under plastic cover during the winter . Severe damage is inflicted by greymould epidemics on cucumber [5], tomato, bean and pepper . The purpose of this workwas to study the possible involvement of ethylene in the development of grey moulddisease caused by B. cinerea on vegetable crops .

MATERIALS AND METHODS

Organisms and growth conditionsBotrytis cinerea Pers . was isolated from naturally infected female flowers of cucumber [5] .The fungus was maintained on potato dextrose agar (PDA Difco) and then tested onthe following host plants : bean (Phaseolus vulgaris L . cv . Brittle Wax or cv . Hilda),cucumber (Cucumis sativus L . cv. Delila), pepper (Capsicum annuum L . cv. Maor) andtomato (Lycopersicon esculentum Mill. cv. VF 198) . Plants or detached leaves wereinoculated with B. cinerea using agar disks infested with mycelium or conidialsuspensions as described below . A disk from the growing margin of a PDA culture ofB. cinerea was placed in the middle of each leaf. The area of necrotic tissue developingin the leaves around the inoculum was measured after different periods of incubation .Controls with sterile PDA did not produce necrosis when incubated under the sameconditions. A conidial suspension (1-3 x 10' spores ml -') from 2-week-old culturesgrown on PDA, supplemented with 0 . 1 M glucose, were sprayed on either leaves or onwhole plants . The inoculated leaves were placed over water in a plastic box . The boxwas enclosed in a plastic polyethylene bag to maintain humid conditions duringincubation . Inoculated plants were either incubated in a plastic bag or placed in a dewchamber. Plants and detached leaves were incubated in a growth chamber with 12 hphotoperiod at 20±2 °C. Disease severity was recorded on a scale of 0-5, where0 = healthy leaf and 5 = leaf completely destroyed .

Ethylene determinationFive leaves (of 8-10 week old plants) were placed in a 32 ml glass test tube(25 mm diam . x 95 mm height) and the tube was closed with a rubber stopper . Forwhole plant measurements the aerial parts of 4-6-week-old plants were enclosed inplastic bags. Samples were incubated in the sealed containers for 2-5 h at 20 ° +2 °C .The ethylene present in gas samples removed from the containers was measured witha gas chromatograph (Gow-Mac Instrument Co ., Series 750) fitted with a flameionization detector and a 3 ft glass column packed with allumina . The rate of ethyleneproduction was expressed as nI g- ' fresh weight h - ', using pure ethylene as a standard .There was no detectable ethylene in empty glass test tubes but very low levels weremeasured in the atmosphere of empty plastic bags .B. cinerea was grown on PDA or on PDA supplemented with blended leaves of the

various plants . Leaves of the various hosts were laced in 32 ml bottles plugged withcotton wool and autoclaved for 20 min at 121 °C before inoculating with B. cinerea . L-methionine (Sigma Chemical Co ., U.S .A.) was added to autoclaved leaves beforeinoculation. On the eighth day the cotton wool plug was replaced with a rubberstopper and the ethylene present was measured a day later .

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Treatments with chemical substancesEthylene mixed with air was injected into a closed vessel (final concentration ofethylene 12 nl 1 - ') in which infected leaves were placed . The system was aerated everyday, resealed, and supplied with a fresh ethylene mixture . Solutions of L-methionine,(Sigma) aminooxyacetic acid (AOA, Sigma), aminoethoxyvinylglycine (AVG, Sigma)and silver thiosulfate (STS-prepared from silver nitrate and sodium thiosulfate,1 : 8 mol/mol) were sprayed on leaves or plants immediately before inoculation withB. cinerea . Fungicidal control was achieved with the fungicide isopropyl N-(3,4-diethoxyphenyl) carbamate (diethofencarb) plus carbendazim 50 % wp (Supplied byAgan Ltd ., Israel) at a concentration of 0 . 2 °o .

Experiments with female flowers of cucumberFemale flowers of a parthenocarpic cucumber cv . Kasem 292 were harvested from acommercial greenhouse in April 1987, one month after the final application of thefungicides. Some of the flowers were held at 4 °C for 12 h in a refrigerator . STS (5 mm),ethephon (20 ppm) and Botrytis conidia (l0' ml - ') supplemented with 0 . 1 M glucosewere sprayed on the open flowers 24 h after picking . Flowers were kept in 32 ml glassbottles at 100 11,/ humidity. Ethylene production was tested 3, 5 and 7 days after theflowers were picked .

Experimental designAll experiments included five or six replicates of each treatment . The experiments wererepeated at least three times and the results presented are from representativeexperiments .

RESULTS

Ethylene production by infected tissuesMeasurements of ethylene production by leaves of cucumber and bean showingdifferent levels of infection showed that maximum levels were emitted by leavesshowing a low disease index (Fig . 1) . Ethylene production decreased with increase indisease severity and leaves which were completely necrotic (disease index-5) producedsmall amounts of ethylene only or none at all .

The pattern of ethylene production by healthy and wounded leaves of pepper,tomato and bean was compared with that by diseased leaves showing mild to severenecrosis (Fig. 2) . Very low production was observed in both healthy and woundedleaves during 7 days of incubation . Slow spreading necroses were associated with agradual increase in ethylene production, up to 5 .2 nl h-' g- ' fresh wt . i n pepper,30.0 nl h-' 9-' in tomato and 11 . 7 nl h - ' g - ' in bean. Fast spreading necroses gavepeaks of production around the fourth to sixth day after inoculation in pepper, thethird day in tomato and the fourth day in bean . Healthy leaves of tomato, bean andcucumber produced 0 .06, 0 and 0.06 nl g -' h - ' ethylene, respectively . Production ofethylene by wounded leaves of tomato was 0 .70 nl g - ' h-', of bean was 0.49 nl g -1 h- 'and of cucumber was 0 . 09 nl g -' h- ' . Infected leaves produced 34 (tomato), 54 (bean)and 52 (cucumber), times the amount of ethylene produced by wounded leaves .Production by B. cinerea after 9 days growth on autoclaved leaves of cucumber was

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FIG . 1 . Ethylene production by Botrytis cinerea infected leaves of bean cv . Brittle Wax (a) primaryleaf, (b) secondary leaf; cucumber true leaves (c) 4 days after inoculation . (Bar = ±SE) .

0 . 04 nl g -' h -1 on bean 0 and on pepper 0.06 nl h-1 g-1 . The amount of ethyleneproduced on autoclaved leaves supplemented with 1 mm methionine was 0 . 05 nl g - ' h"'on cucumber, 0 bean and 0 . 14 nl h -' g-1 pepper. The level of production by leavessupplemented with methionine was not statistically significant from that ofunsupplemented leaves . The fungus grew well on both supplemented and un-supplemented media . B. cinerea did not produce detectable amounts of ethylene whengrown on PDA or PDA supplemented with blended leaves of pepper, bean, tomato orcucumber .

Influence of exogenous ethylene, methionine, STS, AOA and AVG on the development ofB. cinerea infections in leaf tissueLeaves of the different species were inoculated with mycelium of B. cinerea andincubated in closed vessels in an atmosphere containing ethylene at a concentration of12 nl 1 - ' (Fig . 3) . The exogenously supplied ethylene increased the size of the necroticareas produced 5 days after inoculation in pepper by 87 %, in tomato by 90 % and inbean by 77 % . True leaves and cotyledons of cucumber were also inoculated withconidia of the pathogen . The disease index in untreated cucumber leaves was 1 . 0 after9 days but in ethylene-treated leaves it was 3 . 6 after the same period . On the otherhand ethylene did not increase necrosis severity in cotyledons of cucumber . Theapplication ofmethionine at concentrations of 5 or 50 mm increased disease development

Production of ethylene by tissues in infection by Botrytis cinerea

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FIG . 2 . The relationship between the amount of necrotic tissue (right) and production ofethylene (left) by leaves of (a) pepper, (b) tomato and (c) bean cv . Brittle Wax . The leaves wereinoculated with an agar block infested with Botrytis cinerea . Ethylene production by leaves in whichnecrosis developed slowly (V) was measured separately from those in which necrosis developedrapidly (A) . (Q) uninfected control (0) leaves wounded by scratching with a needle .(Bar= f SE) .

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FIG . 3 . The effect of exogenously supplied ethylene at a concentration of 12 nll - ' on thedevelopment of disease in detached leaves of (a) pepper, (b) tomato, and (c) bean cv . Brittle Wax .Leaves were inoculated with blocks of agar infested with mycelium (a-c) (Q), control . (e),infected (Bar= ±SE) .

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FIC . 4 . Development of necrosis in leaves inoculated with a block of agar infested withmycelium. (a) tomato, (b) bean cv . Brittle Wax, (c) pepper, (d) bean cv . Hilda. Detached leaveswere treated with silver thiosulfate at concentrations of 10 -e M (A) and 10-4 M (p) at the time ofinoculation . Untreated control (Q) . (Bar= ±SE) .

Production of ethylene by tissues in infection by Botrytis cinerea

283in leaves and cotyledons of cucumber and in leaves of cucumber, pepper and the twocultivars of bean, but not in tomato leaves . Ethylene did not affect conidialgermination and hyphal growth of B. cinerea on PDA .

Silver thiosulfate was sprayed at concentrations of 10 -4 and 10-s M onto leaves oftomato, bean and pepper prior to inoculation with conidia. Disease severity inuntreated controls reached indices of 3 . 7 (tomato), 4 . 2 (bean) and 4.6 (pepper) 8 daysafter inoculation . Disease severity was reduced by the treatments by 100-23 % 4-8 daysafter inoculation of the leaves . Silver thiosulfate also significantly reduced diseasedevelopment in tomato, bean and pepper leaves inoculated with mycelium (Fig . 4) .

AOA, sprayed at concentrations of 10 -3 and 10 -4 M prior to inoculation of leaves,reduced the necrotic area produced in cucumber, tomato and pepper but not in beancv. Brittle Wax, relative to untreated leaves . Ethylene production by the inoculatedleaves was significantly (P = 0 .05) reduced except in those of bean up to 8 days afterinoculation (Fig . 5) .AVG at a concentration of 5 x 10 -4 M inhibited disease development in bean leaves

8 days after inoculation by 61-76 "ô while 5 x 10 -3 M AVG prevented it completely .Disease development in tomato was inhibited by 5 x 10 -4 M AVG only by 26-50 % andit was completely prevented by 5 x 10 -3 M AVG (Table 1) . Ethylene production wasdrastically reduced in both tomato and bean leaves by treatments with AVG at aconcentration of 5-10 -'m but treatment with 5-10 -'m was effective in reducingethylene production only up to 7 days of incubation .

Exogenous ethylene added to the atmosphere surrounding leaves of tomato andbean, previously treated with AVG and AOA, overcame the inhibitory effects of thesecompounds on disease development .

Intact bean plants were treated with methionine, inhibitors of ethylene productionor of ethylene action, or with the fungicidal mixture diethofencarb+carbendazim(Fig. 6) . Methionine treatment increased disease severity by 130%, 3 days afterinfèction, but this effect declined later . AVG reduced disease development by 28-67 °o,AOA and STS by 80-33().,, while treatments with diethofencarb+carbendazimprevented it completely (Fig. 6) .

Ethylene production (in nl h -i g-1 ) by the bean plants was, for nontreated (2 . 4) ortreated with AOA (0), AVG (0), STS (2 . 8), methionine (3 . 7) and glucose (2 . 1), 13 daysafter treatment. All treatments except glucose significantly increased or reducedproduction over the control (P = 0.05) . B. cinerea was grown on PDA supplementedwith 10 -s -10-4 M AOA, AVG, STS or methionine . Linear growth of the fungus wasnot affected by any of the compounds . Conidia ofB. cinerea were inoculated on the samemedia but no effects on conidial germination were observed .

Influence of predisposition by cold temperature treatment or treatments with ethephon and STS onthe response offemale flowers of cucumber to Botrytis cinereaFemale flowers of cucumber were incubated at 20 °C for 7 days (Table 2) . Ethyleneproduction was highest after 5 days of incubation . Predisposing the flowers at 4 °Ccaused 20-37 % increase in ethylene production . A less pronounced effect was observedwith silverthiosulfate (5 mm) . Inoculation of flowers caused a significant increase inethylene production (Table 2) . The incidence of grey mould 7 days after inoculation

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Ftc . 5 . Development of necrosis (---( and production of ethylene ( ) in leaf tissues aftertreatment with aminooxyacetic acid (a) Cucumber, true leaves (b) cucumber cotyledons (c)tomato leaves (d) bean leaves cv . Brittle Wax (e) pepper leaves . AOA was sprayed immediatelyafter inoculation with mycelium, at rates of (V), 10-9 M and (Q) 10 -s M. (O), not treated(Bar= ±SD) .

was 57% . Ethephon (25 ppm) and the cold treatment increased disease incidence by

up to 100 %, whereas all of the STS-treated flowers remained healthy (Table 1) .

DISCUSSION

Many workers have reported increased ethylene production by diseased plants[6-8, 10-12, 14, 15] . The production of ethylene by diseased plants was recentlyreviewed by Goodman et al . [8] . Studies with a number of plant species suggest thatincreased ethylene synthesis and tissue senescence are responses to stress [2] .

Ethylene production by the crop species studied in this work was significantly higher

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Production of ethylene by tissues in infection by Botrytis cinerea

TABLE 1

The effect of aminoethoxyvinylglycine on disease development and ethylene production in bean and tomatoleaves infected with Botrytis cinerea

Leaves of bean or tomato were inoculated by applying an agar block infested with myceliumand then sprayed with aminoethoxyvinylglycine (AVG) . Values in each column for each cropfollowed by a common letter are significantly different according to Duncan's Multiple RangeTest (P < 0. 05) .

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from tissues infected by B. cinerea than from wounded tissues . Ethylene was producedby the healthy cells surrounding the degenerating tissue . B. cinerea grown on variousnutrient sources did not produce ethylene . The application of ethylene or of itsprecursor methionine to inoculated tissues accelerated disease development . Conidialgermination and mycelial growth of B. cinerea was not affected by methionine at theconcentrations used and it is concluded that the ethylene is produced mostly by theplant tissue with its production being triggered by the infecting pathogen. However,the mechanism which triggers production is unknown . The increases in ethyleneproduction paralleled initial disease development and was at its maximum whenmoderate symptoms were observed .

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DAYS AFTER INOCULATION

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(M)

Necrotic area (cm 5 l

Ethylene production(til h -i g``

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8Days after inoculation

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TABLE 2Effect of inoculation with B . cinerea, predisposing to 4 °C and treatments with stlcerthrosnlfale (Si S) and

ethephon on ethylene production and incidence of grey mould offèmale flowers of racism her

Female flowers of cucumber were picked in a greenhouse and incubated in the laboratorywith their basal side dipped in water in 32 ml glass bottles at 100% RH . The flowers wereincubated at 4 °C or 20 °C for 12 h prior to incubation at 20 °C .

° Flowers were sprayed with either 5 mm silverthioselfate (STS) or ethephon (20 ppm) .'Flowers were inoculated by spraying 103 ml - ' conidia of B. cinerea supplemented with

0 . 1 m glucose . Flowers were then incubated in 100 °~ RH .Treatments in each column followed by a common letter are not significantly different from

each other according to Duncan's Multiple Range Test (P < 0 .05) . Production of ethylene byinoculated flowers was significantly (P = 0. 05) higher than production of ethylene byuntreated flowers at all dates and treatments .

The application of AOA and AVG, known inhibitors of ethylene production[1, 4, 13], decreased both disease development and ethylene production . Theapplication of silver thiosulfate also reduced disease development . Although AVG is aninhibitor of ethylene production it may also inhibit protein synthesis . FurthermoreAOA inhibits ACC synthase, an enzyme involved in ethylene production but it alsoinhibits transaminases such as phenylalanine ammonia lyase (PAL) and so couldinterfere with the development of the necrotic reaction . The pathogen itself was notinfluenced by STS, AOA and AVG in concentrations at which the substances wereapplied to and shown to be effective in disease reduction in the hosts .

Disease in stored vegetable crops was correlated with the presence of ethylene in theair around the crop [7, 12] . Therefore, Reyes & Smith [12] recommended the removalof ethylene from the atmosphere of stored products . Ethylene regulates leaf senescenceand is detected in parasitized tissue . It was suggested that several hydrolytic enzymesare present at higher levels along with ethylene [8] . Our work indicates that ethyleneincreases the severity of the disease developed in response to infection by Botrytis in allthe crops tested and that inhibition of ethylene biosynthesis or action in the tissues hasthe opposite affect .

The author wishes to thank Ms Hanne Volpin, Ms Gilly Shimshoni, Mr H . Yunis, MrN. Ayash and Mr O . Kleifeld for their help and advice and Drs Susan Luria and RuthBen-Arie for their help with the gas chromatograph . The research was partly supported

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Production of ethylene by tissues in infection by Botrytis cinerea

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by grants from the Funds for Priority Projects and the Council of Vegetable Growersand by the National Council for Research and Development .

REFERENCES

1 . BEN DAVID, ANAT, BASI AN, Y . & OKON, Y . (1986) . Ethylene production in pepper (Capiscum annuuinleaves infected with Xanthomonas campestris pv . vesicatoria . Physiological and Alolecular Plant Pathology 29,

305--316 .2 . BOLLER, T., GEHRi, A ., MAUCH, F. & VOGEL!, U. (1983) . Chitinase in bean leaves : induction by

ethylene, purification, properties, and possible function . Planta 157, 27-31 .3 . BRADFORD, K . J. & YANG, S . F . (1980) . Stress induced ethylene production in the ethylene-requiring

tomato diagotropica . Plant Physiology 65, 327-330 .4 . COHEN, R., Riov, J ., LISKER, N. & KATAN, J. (1986) . Involvement of ethylene in herbicide-induced

resistance to Fusarium oxysporum f. sp. melonis . Phytopathology 78, 1281-1285 .5 . ELAD, Y. (1987) . Ultrastructural scanning electron microscopical study of parasitism of Botrytis cinerea on

flowers and fruit of cucumber . Transactions of the British Mycological Society 91, 185-190 .6 . EL-KAZZAZ, M . K ., SAMMER, N . F . & KADER, A. A. (1983) . Ethylene effect on in vitro and in vivo growth

of certain postharvest fruit infecting fungi . Phytopathology 73, 998--1001 .7 . GEESON, J . D ., BROWNE, K . M . & GUARALDI, F . (1986) . The effect of ethylene concentration in

controlled atmosphere storage of tomatoes . Annals of Applied Biology 108, 605-610 .8 . GOODMAN, R . N., KIRALY, Z . & WOOD, K . R . (1986) . The Biochemistry and Physiology of Plant Disease .

University of Missouri Press, Columbia .9 . LIEBERMAN, M . (1979) . Biosynthesis and action of ethylene. Annual Review of Plant Physiology 30, 533-591 .

10 . MAUGH, F., HADWIGER, L. A . & BOLLER, T. (1984) . Ethylene : symptom, not signal for the induction ofchitinase and ß 1,3-glucanase in pea pods by pathogens and elicitors . Plant Physiology 76, 607-611 .

11 . REUVENI, R ., PERL, M . & ROTEM, J . (1976) . Inhibition of shedding of pepper leaves infected withpowdery mildew (Leveillula taurica) by application of auxins . Phytoparasitica 4, 197-199 .

12 . REYES, R . A. & SMITH, R . B . (1986) . Controlled atmosphere effects on the pathogenicity of fungi oncelery and on the growth of Botrytis cinerea . Horticultural Science 21, 1162-1167 .

13 . Riov, J . & YANG, S . F . (1982) . Effects of exogenous ethylene on ethylene production in citrus leaf tissue .Plant Physiology 70, 136-141 .

14. WIESE, M . V . & DEVAY, J . E . 1970) . Growth regulator changes in cotton associated with defoliationcaused by Verticillium albo-atrum . Plant Physiology 45, 304-309 .

15 . WURZER-FASSNACHT, U . & HOFFMANN, G . M. (1986) . Ethylene formation of wheat germlings infectedby Seploria nodorum and the effect of .seed dressing . Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz 93,347--355 .

16 . Yu, Y . B . & YANG, S . F. (19801 . Biosynthesis of wound ethylene . Plant Physiology 66, 281-285 .