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Suppression of Botrytis rot in cut rose flowers by postharvest application of methyl jasmonate1
Postharvest Biology and Technology 13 (1998) 235 – 243
Suppression of Botrytis rot in cut rose flowers by postharvest application of methyl jasmonate1 Shimon Meir 2,*, Samir Droby2, Herman Davidson, Shoshana Alsevia, Lea Cohen, Batia Horev, Sonia Philosoph-Hadas Department of Posthar6est Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan, 50250, Israel Received 22 December 1997; accepted 30 March 1998
Abstract Grey mould, caused by Botrytis cinerea infection on rose (Rosa hybrida L.) petals, is a common disease of greenhouse roses, that significantly reduces the ornamental value of the cut flowers. Methyl jasmonate (MJ), a natural growth regulator postulated to induce plant defense responses, was tested for postharvest control of the grey mould disease in various cut rose cultivars (Mercedes, Europa, Lambada, Frisco, Sacha and Eskimo). Systemic protection against B. cinerea was evident for all cultivars examined, in flowers pulsed with 200 mM MJ following either natural or artificial infection. At this concentration, MJ also significantly reduced lesion size and appearance, as evaluated by a detached petal bioassay. However, local protection, following simultaneous application of B. cinerea spores and MJ directly to flower petals, was not provided by less than 300 mM MJ. These MJ concentrations neither caused any phytotoxicity on leaves and petals, nor impaired flower quality and longevity. A direct antifungal effect of 100 – 400 mM MJ on spore germination and germ-tube elongation of B. cinerea was obtained in vitro, with complete inhibition at 400 mM MJ. These results suggest that MJ pulsing provides systemic protection against Botrytis rot by inducing resistance mechanisms in the treated cut roses without impairing flower quality. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Botrytis cinerea; Grey mould; Cut flowers; Induced resistance; Methyl jasmonate; Rosa hybrida L.
* Corresponding author. Tel.: +972 3 9683667; fax: +972 3 9683622; e-mail: [email protected] 1 Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 2306-E, 1997 series. 2 These authors contributed equally to the experimental work presented in this study. 0925-5214/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0925-5214(98)00017-9
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1. Introduction Botrytis rot, caused by the ubiquitous pathogen Botrytis cinerea Pers.:Fr., is a widespread disease of greenhouse roses and many other flower crops (Coyier, 1985). Since flower petals infected with B. cinerea significantly reduce the ornamental value of cut roses, susceptibility to this pathogen is an important factor in determining vase life. Initially, the symptoms appear on infected petals as restricted lesions (Elad, 1988). Subsequently, these lesions become necrotic and spread to the whole petals and the receptacle, finally resulting in collapse of the flower head and petal drop (Elad, 1988). The problem is aggravated by latency of the petal infection, which may not present visible symptoms at the time of flower harvest, but would become apparent under humid (RH above 93%) and high-temperature (18–25°C) conditions prevailing during storage and transport (Elad, 1988). Currently, chemical fungicides are being used either as preharvest sprays in the greenhouse or as postharvest dips of rose flowers to prevent disease development. However, the efficacy of this control strategy is limited, since latent infections are not controlled efficiently by the various fungicide treatments (Elad, 1988; Elad et al., 1992). In the search for alternatives to fungicides, several plant growth regulators have been found to affect the development of Botrytis rot in rose flowers: while abscisic acid (ABA) promoted the disease (Shaul et al., 1996), gibberellic acid (GA3) suppressed it (Shaul et al., 1995a,b, 1996). However, GA3 had no direct effect on B. cinerea development in vitro (Shaul et al., 1995a, 1996). The suppressive effect of GA3 on development of Botrytis rot was shown to be mainly in detached rose petals, and in most of the rose cultivars examined the GA3 spray for whole cut roses was not very effective as a protective postharvest treatment against the disease (Shaul et al., 1996). Therefore, the efficacy of another promising growth regulator, methyl jasmonate (MJ), was examined as a means for postharvest control of Botrytis rot in cut rose flowers. Jasmonic acid (JA) and its esterified derivative, methyl jasmonate (MJ) are naturally occurring
compounds that have been identified in a wide variety of plant species (Sembdner and Parthier, 1993; Loake, 1996). They are widely regarded as endogenous plant growth substances, which play key roles in plant growth, development and responses to environmental stresses (Sembdner and Parthier, 1993; Creelman and Mullet, 1995; Meir et al., 1996). In addition, jasmonates have also been shown to be involved in direct protection against biotic stresses caused by diseases, and are considered to play a central role in the intracellular signaling cascades that activate inducible plant defenses (Gundlach et al., 1992; Mueller et al., 1993; Loake, 1996). Thus, JA and MJ were found to exhibit a direct antifungal activity (Neto et al., 1991; Schweizer et al., 1993), and jasmonates applied as a foliar spray protected potato or tomato plants against a challenge infection with Phytophthora infestans (Cohen et al., 1993), and barley plants against infection by Erysiphe graminis f. sp. hordei (Schweizer et al., 1993). Apart from their direct antifungal effects, evidence has accumulated that JA and MJ can activate many inducible genes, thereby leading to synthesis of secondary plant products which function as antimicrobial compounds of central importance to the plant defense response. Such jasmonate elicitor-inducible genes are: proteinase inhibitors (Farmer et al., 1992), phenylalanine ammonia-lyase (PAL) (Dittrich et al., 1992), chalcone synthase (CHS) (Dittrich et al., 1992), LOX (Bell and Mullet, 1991; Avdiushko et al., 1995) and hydroperoxide lyase (Avdiushko et al., 1995). Several antifungal compounds, such as hexanal, taxol, steroid-glycoalkaloids, glucosinolates and momilactone A, have been found to increase in various plant tissues after jasmonate application (Avdiushko et al., 1995; Doughty et al., 1995; Mirjalili and Linden, 1996; Nojiri et al., 1996). As MJ is mobile, pulsing cut roses with MJ was used for the control of B. cinerea. The present study describes the effects of MJ in various cut rose cultivars, following either artificial or natural infection. The effects of MJ on the development of B. cinerea, both in vitro and in detached rose petals were also examined.
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2. Materials and methods
2.1. Materials The following materials and products, developed by a local commercial company (Assia Reisel, Ramat Gan, Israel) for flower pulse treatment, were used in the experiments: two flower preservative solutions — TOG-4, which contains 200 ml l − 1 8-hydroxyquinoline citrate as the active ingredient formulated with surfactants, and TOG-6, which contains 5 mg ml − 1 active chlorine complexed as sodium dichloroisocyanureate. Silver thiosulphate (STS) was used in the form of the product STS-75 (Assia Reisel, Ramat Gan, Israel), which contains 37.5% (75 mM) STS. Methyl jasmonate (MJ) was used as the product TOGMJ-1 (Assia Reisel, Ramat Gan, Israel), which contains a 2% MJ (Aldrich Chemical Company Inc., USA) solution formulated with surfactants.
2.2. Plant material and experimental procedures Experiments were performed with cut rose (Rosa hybrida L.) flowers of various cultivars (Mercedes, Europa, Lambada, Frisco, Sacha and Eskimo), obtained from local commercial greenhouses. Freshly cut flowers with 30-cm long stems, harvested at the marketing stage of bud development, were brought to the laboratory in water. The cut stem ends were trimmed by 2 cm, and lower leaves were removed up to 10 cm from the stem base. Flowers were then pulsed in the standard pulsing solution recommended by the Israeli Flower Board for roses, composed of the preservative TOG-4 (0.2%)+0.15 mM STS. Where indicated, MJ was added as the product TOG-MJ-1 to this pulsing solution at concentrations ranging between 50 and 600 mM. All pulsing treatments and subsequent incubations were performed for 24 h in a conditioned room maintained at 20°C with 60 – 70% RH and 12-h photoperiod at a light intensity of 14 mmol m − 2 s − 1, provided by cool-white fluorescent tubes. After the pulsing treatments, flowers were transferred to 600-ml glass cylinders (ten flowers per cylinder), each containing 300 ml of TOG-6 solution as a bacteriocide, and divided into three
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groups, each of which underwent a different treatment. Group I was used to examine the effect of MJ treatments on flower opening and phytotoxicity during vase life: these flowers were kept in the controlled conditions for daily measurements of flower diameter and visual evaluation of possible toxicity symptoms. Group II was used to examine the effect of MJ treatments on disease incidence following natural infection: to enhance the development of Botrytis rot from natural infections the glass cylinders with the flowers were covered with perforated polyethylene (PE) bags, and incubated for 2 additional days at 20°C. The decay index of flower petals and receptacle was evaluated daily after removal of the PE bags. Group III was used to examine the effect of MJ treatments on disease incidence following artificial inoculation: these flowers were sprayed to run-off with a spore suspension of B. cinerea (104 spores ml − 1), covered with perforated PE bags and incubated for 2 additional days at 20°C. The PE bags were then removed, and the decay index evaluated 5 days later, as described for flowers of group II. In one experiment (Table 1), MJ was added to the Botrytis suspension and applied by spraying to the flower petals, instead of application by pulsing.
2.3. Preparation of spore suspension A culture of B. cinerea was obtained from infected rose petals and grown on potato dextrose agar (PDA, Difco). Spore suspensions were prepared from the sporulating edges of 2-week-old B. cinerea cultures. Spores were gently removed with a bacteriological loop, suspended in sterile distilled water (DW) and filtered through four layers of sterile cheese cloth to remove remaining mycelia. The spore concentration was determined with a haemocytometer and adjusted to 104 spores ml − 1.
2.4. Disease assessment The extent of the Botrytis rot development, expressed as a decay index of flower petals and receptacle, was evaluated according to Hazendonk et al. (1995), with several modifications. Visual rating, on a relative scale of 0–5, was
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defined as follows: 0, no symptoms; 1, appearance of one to four lesions on the petals; 2, appearance of more than four lesions; 3, appearance of fully necrotic petals; 4, necrosis of the petals and receptacle; 5, collapse of flower head and petal drop.
2.5. E6aluation of flower opening and phytotoxicity Flower opening was evaluated by measuring the flower diameter every second day in flowers that did not have any visible Botrytis rot symptoms. Additionally, all treated flowers were examined for injury symptoms such as necrotic lesions on leaves and petals, leaf drop and change in petal colour.
2.6. Detached petal bioassay A bioassay for testing the effect of MJ pulsing treatment on the susceptibility of rose flower petals to B. cinerea was performed with petals detached from control and MJ-treated flowers. The four outermost petals of each flower were discarded and the next four petals were used. A total of 20 such inner petals, collected from five different flowers, were placed on nylon net pads mounted on plastic trays, and the center of each petal was wounded with a sterile microbiological needle. A 30-ml drop of spore suspension of B. cinerea (104 spores ml − 1) was then placed into each wound, and the wounded petals incubated
under high RH conditions (\ 95%) at 20°C. Infection percentage and lesion size were determined 3 or 4 days after inoculation. Since the lesions were oval in shape, an average of width and length measurements is presented. In one experiment (Table 1), MJ (TOG-MJ-1) was applied directly to the detached petals with the spore suspension, instead of by the pulsing treatment.
2.7. Effect of MJ on de6elopment of Botrytis cinerea in 6itro The effects of MJ on spore germination and germ-tube elongation of B. cinerea were tested in a filter-sterilized solution of glucose (1%), supplemented with various concentrations of MJ. Spores of B. cinerea were suspended in the 1% glucose solution at a final concentration of 106 spores ml − 1. Aliquots (900 ml) containing fungal spores were transferred to each well of tissue culture plates (Costar 24-cell tissue culture clusters), and 100-ml aliquots of various MJ (TOG-MJ-1) concentrations were added to make final concentrations ranging between 0 and 400 mM. Similarly, 100-ml aliquots of the surfactants mixture in which the MJ was formulated, were added at the same dilutions to another series of 900-ml aliquots of spore suspensions that served as controls. Samples of the various test solutions (30-ml drops), containing fungal spores with either TOG-MJ-1 or the surfactants mixture of this product, were placed on ethanol-washed microscope slides (three
Table 1 Effect of MJ concentration, applied with the spore suspension of B. cinerea, on incidence of Botrytis rot in attached or detached petals of cut rose flowers cvs. Frisco and Sacha MJ concentration (mM)
0 50 300
Decay index in attached petals (0 – 5)a
Detached petals bioassay
Frisco
% Infection
3.5 a 3.7 a 2.3 b
Sacha
1.9 a 2.1 a 1.3 b
Lesion size (mm)
Frisco
Sacha
Frisco
Sacha
84 50 0
100 100 0
13.2 a 4.4 b n.d.
32.0 a 28.2 a n.d.
A total of 30 flowers or 20 petals per treatment were assayed. Means within each column followed by different letters are significantly different (P = 0.05). a On a scale of 0= no symptoms to 5= severe symptoms.
S. Meir et al. / Posthar6est Biology and Technology 13 (1998) 235–243 Table 2 Effect of MJ concentration, applied in the pulsing solution, on development of natural infection of Botrytis rot in attached petals of cut rose flowers cvs. Frisco and Sacha MJ concentration (mM)
0 50 100 200 400 600
the petal bioassay. Results were analyzed using SigmaStat statistical software (Jandel Scientific Software, San Rafael, CA). Paired t-test, one way ANOVA, and Student–Newman–Keuls multiple range test combined with Kruskall–Wallis ANOVA on ranks were performed where indicated.
Decay index (0–5)a Frisco
Sacha
1.95 1.35 1.45 0.61 1.74 0.85
1.21 1.67 1.74 0.80 0.95 1.10
a abc abc c ab bc
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a a a a a a
3. Results Pulsing Frisco and Sacha cut rose flowers with 50 or 100 mM MJ had no significant effect on the development of Botrytis rot symptoms following natural infection for 2 days at 20°C (Table 2). Whereas pulsing Sacha flowers with MJ concentrations up to 600 mM was still ineffective, the rot symptoms were significantly suppressed in Frisco flowers following pulsing with 600 mM MJ (Table 2). Similarly, pulsing Mercedes, Europa and Frisco cut flowers with low MJ concentrations (25, 50 and 100 mM) had no effect on disease incidence following either natural or artificial infection (data not shown). Pulsing Frisco and Lambada flowers with MJ concentrations of 200 mM and above, significantly suppressed the development of Botrytis rot resulting from artificial inoculation (Table 3). This inhibitory effect of MJ was saturated at a concentration of 200 mM for both rose cultivars; the same MJ concentration
Flowers (n =30) were evaluated 5 days after removal of the perforated PE bags. Means within each column followed by different letters are significantly different (P= 0.05). a On a scale of 0= no symptoms to 5= severe symptoms.
drops per slide) in Petri dishes padded with moistened filter paper, and incubated for 24 h at 25°C in darkness. Spore germination and germ-tube elongation were observed under a light microscope, in three microscope fields, each containing 20 – 40 spores.
2.8. Statistical analysis Experiments were repeated two to four times, with 30 flower replicates for each treatment in the cut flower assays, and with 20 detached petals in
Table 3 Effect of MJ concentration, applied in the pulsing solution, on development of Botrytis rot in artificially or naturally infected attached petals of cut rose flowers cvs. Frisco and Lambada MJ concentration (mM)
Decay index (0–5)a Artificial infectionb
0 200 400 600
Natural infectionc
Frisco
Lambada
Frisco
Lambada
2.6 1.7 1.6 1.4
3.8 2.3 2.7 2.8
0.6 0.9 0.9 0.9
4.3 1.9 3.3 3.3
a b b b
a b b b
a a a a
a b a a
A total of 30 flowers per treatment were assayed. Means within each column followed by different letters are significantly different (P =0.05). a On a scale of 0= no symptoms to 5= severe symptoms. b Evaluated 4 days after inoculation. c Evaluated 5 days after removal of the perforated PE bags.
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Fig. 1. Effect of 200 mM MJ, applied in the pulsing solution, on the time course of Botrytis rot development in rose flowers cvs. Frisco (A) and Mercedes (B) following artificial inoculation. Each bar represents the mean 9 S.E. of 30 replicates, evaluated 4 (dark bars) or 7 (empty bars) days after artificial inoculation.
was also the most effective in reducing rot symptoms after natural infection in Lambada, but not in Frisco flowers (Table 3). The apparent discrepancy between the two experiments in the MJ effects obtained for Frisco flowers following natural infection (Tables 2 and 3), may be ascribed to variations in the infection potential of these cultivars. When control Frisco flowers were highly infected (decay index of 1.95), pulsing with 200 mM MJ significantly suppressed the disease symptoms (Table 2), but when control Frisco flowers were not infected (decay index of 0.6), MJ pulsing was ineffective (Table 3). The effect of MJ at 200 mM, the most effective concentration in suppressing Botrytis rot in various rose cultivars, on the time course of disease development in artificially inoculated flowers, was also studied. Results depicted in Fig. 1 clearly demonstrate that MJ at this concentration significantly reduced disease incidence 4 and 7 days after inoculation of both Frisco and Mercedes roses. The effect of 200-mM MJ pulsing in suppressing rot development was also observed in the petal bioassay experiments (Fig. 2). The bioassay
was performed with detached petals taken from control or MJ-treated Eskimo and Lambada flowers, and wound-inoculated with B. cinerea spore suspensions. The results presented in Fig. 2 show that the MJ pulsing treatment inhibited lesion expansion by about 50% in both cultivars. The possibility that MJ suppresses Botrytis rot through a direct effect on B. cinerea development was examined in vitro. The results presented in Fig. 3 show that MJ significantly inhibited spore germination and germ-tube elongation of the pathogen. Inhibition was dose-dependent, very effective above 100 mM; 50% inhibition of germtube elongation was obtained with 200 mM MJ, while 50% inhibition of spore germination was obtained with MJ concentrations higher than 300 mM. Complete inhibition of spore germination was obtained with 400 mM MJ (Fig. 3). The control media of the product TOG-MJ-1, consisting of the surfactants mixture at dilutions equivalent to MJ in the 0–400-mM range, had no inhibitory effect on either germination or elongation (data not shown).
Fig. 2. Effect of 200 mM MJ, applied in the pulsing solution, on lesion size of Botrytis rot developed in detached petals of rose flowers cvs. Eskimo and Lambada, following wound-inoculation with B. cinerea. A total of 20 petals, taken from five control or MJ-treated flowers of each cultivar, were wounded and inoculated with a suspension of B. cinerea (104 spores ml − 1). Lesion sizes represent means of measurements of the length and width, monitored 3 (in Eskimo) or 4 (in Lambada) days after inoculation. Each bar represents the mean 9 S.E. of 20 replicates.
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mained more turgid, with a shiny appearance during vase life (data not shown).
4. Discussion
Fig. 3. Effect of MJ concentration on inhibition of spore germination and germ-tube elongation of B. cinerea in vitro. Each point represents the mean 9 S.E. of 60–120 spores from four experiments.
The possible direct effect of MJ on the pathogen was further manifested when rose flowers and detached petals were inoculated with B. cinerea spore suspensions containing various concentration of MJ (Table 1). When Frisco and Sacha flowers were artificially inoculated only 300 mM MJ, applied with the spores, significantly reduced the development of Botrytis rot (Table 1) to levels similar to those obtained for the naturally infected flowers that were not treated with MJ (Table 2). In the petal bioassay, application of 50 mM MJ with the spore suspension significantly inhibited petal disease incidence in Frisco flowers, but had no effect on Sacha petals (Table 1). The higher MJ concentration (300 mM), however, completely inhibited the petal infection in both cultivars (Table 1). No negative effects of MJ on flower quality (damage, flower opening or longevity) were observed in any of the experiments. Moreover, positive effects of MJ were observed in several experiments. Pulsing flowers with 200 mM MJ resulted in a better maintenance of the original yellow colour of Frisco petals, and inhibited colour fading of other cultivars during vase life. In addition, leaves of MJ-treated flowers re-
Inducible defense responses in plants are known to be mediated by signalling molecules that are produced or released at the site of pathogen attack, and may be transported either locally by diffusion or systemically through the vascular system (Farmer et al., 1992). A large body of evidence suggests that JA or MJ is a key component of such intracellular signalling in response to pathogen attack, and that its application may, therefore, induce disease resistance in a wide variety of plants (Creelman and Mullet, 1995). In this respect, since MJ is regarded as a natural plant growth regulator, it has the advantage of eliciting defense (Farmer et al., 1992; Cohen et al., 1993; Schweizer et al., 1993) or physiological (Meir et al., 1996) responses to its exogenous application to plants at low concentrations in a non-destructive manner. Our present study supports this possible role of MJ as an inducer of a defense response in plants, and demonstrates for the first time its potential for postharvest application as a means to elicit systemic and local protection of cut roses against Botrytis rot. MJ pulsing seems to provide systemic protection against Botrytis rot by inducing resistance mechanisms in the treated cut roses, as suggested previously for GA3 (Shaul et al., 1995a,b). This conclusion is based on the following findings of the present study. (a) Pulsing cut roses of various cultivars with MJ significantly reduced the development of Botrytis rot in flower petals following either natural (Tables 2 and 3) or artificial infection (Table 3 and Fig. 1), without causing any flower damage. (b) The threshold, optimal pulsing dose of MJ obtained for rot inhibition in these rose cultivars was 200 mM (Tables 2 and 3 and Fig. 1). (c) This 200-mM MJ pulsing concentration was also markedly effective in reducing disease incidence in the detached petal bioassay performed with Eskimo and Lambada cultivars (Fig. 2). (d) When MJ was applied by spray simultaneously with B. cinerea spores, higher MJ
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concentrations (300 mM) were required for protection of Frisco roses (Table 1). These results are in full agreement with previous studies which found a significant local protective effect of sprayapplied MJ against pathogen infection that involved MJ concentrations higher than 200 mM (Cohen et al., 1993; Schweizer et al., 1993). In addition to the effect of MJ in inducing flower resistance, the possibility that MJ exerts its inhibitory effects on Botrytis rot through its direct antifungal activity cannot be excluded. This direct antifungal effect of MJ was manifested both in in vitro inhibition of growth and germination of B. cinerea (Fig. 3), and in inhibition of Botrytis rot incidence in petals following simultaneous spray of spores and MJ (Table 1). The inhibition of B. cinerea growth in vitro was dose-dependent, being effective above 100 mM MJ (Fig. 3). However, the spray application of 50 mM MJ to detached Frisco petals through wounding provides a better local protection (Table 1), since it suppresses the rot possibly through a combination of direct and induced resistance mechanisms. The results presented in Table 1 show clearly that in the detached petal bioassay, MJ significantly reduced the infection percentage and lesion size for Frisco petals but was ineffective with Sacha petals. This suggests that the Sacha cultivar may be more susceptible to B. cinerea and less responsive to MJ, as indicated also by the data of Table 2. These results further imply that, apart from the direct effect of MJ on the fungus, the type of petal tissue may also play a role in the defense mechanism (Hammer and Evensen, 1994). Jasmonates have been previously reported to have a direct antifungal activity against various pathogens, although with varying efficiency. Whereas, in the present study, complete inhibition of B. cinerea spore germination and germ-tube elongation in vitro were already obtained at 400 mM MJ (Fig. 3), much higher concentrations, of about 1.2 mM JA or 3.5 mM MJ, were required for complete inhibition of in vitro spore germination of Pyricularia oryzae (Neto et al., 1991), or of mycelial growth of Phytophthora infestans (Cohen et al., 1993). Similarly, a concentration of about 5 mM JA was required for direct inhibition of appressoria differentiation of Erysiphe graminis,
but no inhibition of germination was obtained (Schweizer et al., 1993). These results suggest that MJ is more effective in suppression of development of B. cinerea than of other fungi. Unlike their effects on various seedling systems, in which high MJ or JA concentrations in the mM range caused leaf chlorosis or necrosis of leaf tips (Cohen et al., 1993; Schweizer et al., 1993), MJ applied as a pulse to cut roses in micromolar concentrations neither impaired the quality nor accelerated senescence of flowers of any of the cultivars examined. This may be due to the lower MJ doses required for inhibition of B. cinerea compared with other fungi. Alternatively, the lack of visible damage in MJ-treated roses, with or without STS in the pulsing solution, may also stem from the fact that senescence of most rose cultivars is not ethylene-regulated (Mor and Zieslin, 1987). This conclusion is further reinforced by comparison between the effects of MJ application to ethylene-sensitive or -insensitive flowers. Thus, application of 50–5000 mM MJ to petunia or dendrobium flowers, in which ethylene plays a major role in regulation of senescence processes, resulted in significant acceleration of ethylene production rates and flower senescence (Porat et al., 1993). It was, therefore, suggested that the effects of MJ in accelerating senescence of these two ethylene-sensitive flowers are ethylenemediated (Porat et al., 1993). In petals of tulip, however, which is an ethylene-insensitive flower, MJ did not accelerate ethylene production (Porat et al., 1993). It seems, therefore, that MJ-induced senescence in flowers is correlated with increased ethylene production and sensitivity to ethylene. Hence, application of MJ as an antifungal agent should not cause any adverse effects in ethyleneinsensitive flowers and/or in low ethylene producers. In ethylene-sensitive flowers, MJ may be applied with an inhibitor of ethylene action such as STS. Taken together, our results suggest the possible use of MJ as a postharvest application in low doses, for suppression of Botrytis rot in various cut rose cultivars without impairing flower quality. As such, it may provide flower growers with a better and more environmentally-friendly alternative to conventional fungicides for preventing decay development.
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Acknowledgements The authors would like to express their gratitude to Yigal Slonim from the Assia Reisel Company for his helpful cooperation in conducting this research.
References Avdiushko, S., Croft, K.P.C., Brown, G.C., Jackson, D.M., Hamilton-Kemp, T.R., Hildebrand, D., 1995. Effect of volatile methyl jasmonate on the oxylipin pathway in tobacco, cucumber, and Arabidopsis. Plant Physiol. 109, 1227 – 1230. Bell, E., Mullet, J.E., 1991. Lipoxygenase gene expression is modulated in plants by water deficit, wounding, and methyl jasmonate. Mol. Gen. Genet. 230, 456–462. Cohen, Y., Grisi, U., Mosinger, E., 1993. Local and systemic protection against Phytophthora infestans induced in potato and tomato by jasmonic acid and methyl jasmonate. Phytopathology 83, 1054–1062. Coyier, D.L., 1985. Roses. In: Strider, D.L. (Ed.), Diseases of Floral Crops. Praeger, New York, pp. 405–488. Creelman, R., Mullet, J.E., 1995. Jasmonic acid action and distribution in plants: regulation during development and responses to biotic and abiotic stresses. Proc. Natl. Acad. Sci. USA 92, 4114 – 4119. Dittrich, H., Kutchan, T., Zenk, M.H., 1992. The jasmonate precursor, 12-oxo-phytodienoic acid, induces phytoalexin synthesis in Petroselinum hortense cell cultures. FEBS Lett. 309, 33 – 36. Doughty, K.L., Kiddle, G.A., Pye, B.J., Wallsgrove, R.M., Pickett, J.A., 1995. Selective induction of glucosinolates in oilseed rape leaves by methyl jasmonate. Phytochemistry 38, 347 – 350. Elad, Y., 1988. Latent infection of Botrytis cinerea in rose flowers and combined chemical and physiological control of the disease. Crop Prot. 7, 631–633. Elad, Y., Yunis, H., Katan, T., 1992. Multiple fungicide resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of Botrytis cinerea in Israel. Plant Pathol. 41, 41 –46. Farmer, E.E., Johnson, R.R., Ryan, C.A., 1992. Regulation of expression of proteinase inhibitor genes by methyl jasmonate and jasmonic acid. Plant Physiol. 98, 995–1002. Gundlach, H., Muller, M., Kutchan, T.M., Zenk, M.H., 1992. Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc. Natl. Acad. Sci. USA 89, 2389– 2393. Hammer, P.E., Evensen, K.B., 1994. Differences between rose cultivars in susceptibility to infection by Botrytis cinerea. Phytopathology 84, 1305–1312. .
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Hazendonk, H., ten Hoope, M., van der Wurff, T., 1995. Methods to test rose cultivars on their susceptibility to Botrytis cinerea during the postharvest stage. Acta Hortic. 405, 39 – 45. Loake, G.J., 1996. Jasmonates: global regulators of plant gene expression. In: Smallwood, M., Knox, J.P., Bowles, D.J. (Eds.), Specialized Function in Plants. Bios Scientific, Oxford, pp. 215 – 230. Meir, S., Philosoph-Hadas, S., Lurie, S., Droby, S., Akerman, M., Zauberman, G., Shapiro, B., Cohen, E., Fuchs, Y., 1996. Reduction of chilling injury in stored avocado, grapefruit and bell pepper by methyl jasmonate. Can. J. Bot. 74, 870 – 874. Mirjalili, N., Linden, J.C., 1996. Methyl jasmonate induced production of taxol in suspension cultures of Taxus cuspidata: ethylene interaction and induction models. Biotechnol. Prog. 12, 110 – 118. Mor, Y., Zieslin, N., 1987. Plant growth regulators in rose plants. Hort. Rev. 9, 53 – 73. Mueller, M.J., Brodschelm, W., Spannagl, E., Zenk, M.H., 1993. Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid. Proc. Natl. Acad. Sci. USA 90, 7490 – 7494. Neto, G.C., Kono, Y., Hyakuttake, H., Watanabe, M., Suzuki, Y., Sakurai, A., 1991. Isolation and identification of (−)-jasmonic acid from wild rice, Oryza officinalis, as an antifungal substance. Agric. Biol. Chem. 55, 3097 – 3098. Nojiri, H., Sugimori, M., Yamane, H., Nishimura, Y., Yamada, A., Shibuya, N., Kodama, O., Murofushi, N., Omori, T., 1996. Involvement of jasmonic acid in elicitorinduced phytoalexin production in suspension-cultured rice cells. Plant Physiol. 110, 387 – 392. Porat, R., Borochov, A., Halevy, A.H., 1993. Enhancement of petunia and dendrobium flower senescence by jasmonic acid methyl ester is via the promotion of ethylene production. Plant Growth Reg. 13, 297 – 301. Schweizer, P., Gees, R., Mosinger, E., 1993. Effect of jasmonic acid on the interaction of barley (Hordeum 6ulgare L.) with the powdery mildew Erysiphe graminis f. sp. hordei. Plant Physiol. 102, 503 – 511. Sembdner, G., Parthier, B., 1993. The biochemistry and the physiological and molecular actions of jasmonates. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 569 – 589. Shaul, O., Elad, Y., Zieslin, N., 1995a. Suppression of Botrytis blight in cut flowers with gibberellic acid: effects of concentration and mode of application. Postharvest Biol. Technol. 6, 321 – 330. Shaul, O., Elad, Y., Zieslin, N., 1995b. Suppression of Botrytis blight in cut rose flowers with gibberellic acid: effects of postharvest timing of the gibberellin treatment, conidial inoculation and cold storage. Postharvest Biol. Technol. 6, 331 – 339. Shaul, O., Elad, Y., Zieslin, N., 1996. Suppression of Botrytis blight in cut rose flowers with gibberellic acid: effects of exogenous application of abscisic acid and paclobutrazol. Postharvest Biol. Technol. 7, 145 – 150.
Suppression of Botrytis rot in cut rose flowers by postharvest application of methyl jasmonate1 Shimon Meir 2,*, Samir Droby2, Herman Davidson, Shoshana Alsevia, Lea Cohen, Batia Horev, Sonia Philosoph-Hadas Department of Posthar6est Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan, 50250, Israel Received 22 December 1997; accepted 30 March 1998
Abstract Grey mould, caused by Botrytis cinerea infection on rose (Rosa hybrida L.) petals, is a common disease of greenhouse roses, that significantly reduces the ornamental value of the cut flowers. Methyl jasmonate (MJ), a natural growth regulator postulated to induce plant defense responses, was tested for postharvest control of the grey mould disease in various cut rose cultivars (Mercedes, Europa, Lambada, Frisco, Sacha and Eskimo). Systemic protection against B. cinerea was evident for all cultivars examined, in flowers pulsed with 200 mM MJ following either natural or artificial infection. At this concentration, MJ also significantly reduced lesion size and appearance, as evaluated by a detached petal bioassay. However, local protection, following simultaneous application of B. cinerea spores and MJ directly to flower petals, was not provided by less than 300 mM MJ. These MJ concentrations neither caused any phytotoxicity on leaves and petals, nor impaired flower quality and longevity. A direct antifungal effect of 100 – 400 mM MJ on spore germination and germ-tube elongation of B. cinerea was obtained in vitro, with complete inhibition at 400 mM MJ. These results suggest that MJ pulsing provides systemic protection against Botrytis rot by inducing resistance mechanisms in the treated cut roses without impairing flower quality. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Botrytis cinerea; Grey mould; Cut flowers; Induced resistance; Methyl jasmonate; Rosa hybrida L.
* Corresponding author. Tel.: +972 3 9683667; fax: +972 3 9683622; e-mail: [email protected] 1 Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 2306-E, 1997 series. 2 These authors contributed equally to the experimental work presented in this study. 0925-5214/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0925-5214(98)00017-9
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1. Introduction Botrytis rot, caused by the ubiquitous pathogen Botrytis cinerea Pers.:Fr., is a widespread disease of greenhouse roses and many other flower crops (Coyier, 1985). Since flower petals infected with B. cinerea significantly reduce the ornamental value of cut roses, susceptibility to this pathogen is an important factor in determining vase life. Initially, the symptoms appear on infected petals as restricted lesions (Elad, 1988). Subsequently, these lesions become necrotic and spread to the whole petals and the receptacle, finally resulting in collapse of the flower head and petal drop (Elad, 1988). The problem is aggravated by latency of the petal infection, which may not present visible symptoms at the time of flower harvest, but would become apparent under humid (RH above 93%) and high-temperature (18–25°C) conditions prevailing during storage and transport (Elad, 1988). Currently, chemical fungicides are being used either as preharvest sprays in the greenhouse or as postharvest dips of rose flowers to prevent disease development. However, the efficacy of this control strategy is limited, since latent infections are not controlled efficiently by the various fungicide treatments (Elad, 1988; Elad et al., 1992). In the search for alternatives to fungicides, several plant growth regulators have been found to affect the development of Botrytis rot in rose flowers: while abscisic acid (ABA) promoted the disease (Shaul et al., 1996), gibberellic acid (GA3) suppressed it (Shaul et al., 1995a,b, 1996). However, GA3 had no direct effect on B. cinerea development in vitro (Shaul et al., 1995a, 1996). The suppressive effect of GA3 on development of Botrytis rot was shown to be mainly in detached rose petals, and in most of the rose cultivars examined the GA3 spray for whole cut roses was not very effective as a protective postharvest treatment against the disease (Shaul et al., 1996). Therefore, the efficacy of another promising growth regulator, methyl jasmonate (MJ), was examined as a means for postharvest control of Botrytis rot in cut rose flowers. Jasmonic acid (JA) and its esterified derivative, methyl jasmonate (MJ) are naturally occurring
compounds that have been identified in a wide variety of plant species (Sembdner and Parthier, 1993; Loake, 1996). They are widely regarded as endogenous plant growth substances, which play key roles in plant growth, development and responses to environmental stresses (Sembdner and Parthier, 1993; Creelman and Mullet, 1995; Meir et al., 1996). In addition, jasmonates have also been shown to be involved in direct protection against biotic stresses caused by diseases, and are considered to play a central role in the intracellular signaling cascades that activate inducible plant defenses (Gundlach et al., 1992; Mueller et al., 1993; Loake, 1996). Thus, JA and MJ were found to exhibit a direct antifungal activity (Neto et al., 1991; Schweizer et al., 1993), and jasmonates applied as a foliar spray protected potato or tomato plants against a challenge infection with Phytophthora infestans (Cohen et al., 1993), and barley plants against infection by Erysiphe graminis f. sp. hordei (Schweizer et al., 1993). Apart from their direct antifungal effects, evidence has accumulated that JA and MJ can activate many inducible genes, thereby leading to synthesis of secondary plant products which function as antimicrobial compounds of central importance to the plant defense response. Such jasmonate elicitor-inducible genes are: proteinase inhibitors (Farmer et al., 1992), phenylalanine ammonia-lyase (PAL) (Dittrich et al., 1992), chalcone synthase (CHS) (Dittrich et al., 1992), LOX (Bell and Mullet, 1991; Avdiushko et al., 1995) and hydroperoxide lyase (Avdiushko et al., 1995). Several antifungal compounds, such as hexanal, taxol, steroid-glycoalkaloids, glucosinolates and momilactone A, have been found to increase in various plant tissues after jasmonate application (Avdiushko et al., 1995; Doughty et al., 1995; Mirjalili and Linden, 1996; Nojiri et al., 1996). As MJ is mobile, pulsing cut roses with MJ was used for the control of B. cinerea. The present study describes the effects of MJ in various cut rose cultivars, following either artificial or natural infection. The effects of MJ on the development of B. cinerea, both in vitro and in detached rose petals were also examined.
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2. Materials and methods
2.1. Materials The following materials and products, developed by a local commercial company (Assia Reisel, Ramat Gan, Israel) for flower pulse treatment, were used in the experiments: two flower preservative solutions — TOG-4, which contains 200 ml l − 1 8-hydroxyquinoline citrate as the active ingredient formulated with surfactants, and TOG-6, which contains 5 mg ml − 1 active chlorine complexed as sodium dichloroisocyanureate. Silver thiosulphate (STS) was used in the form of the product STS-75 (Assia Reisel, Ramat Gan, Israel), which contains 37.5% (75 mM) STS. Methyl jasmonate (MJ) was used as the product TOGMJ-1 (Assia Reisel, Ramat Gan, Israel), which contains a 2% MJ (Aldrich Chemical Company Inc., USA) solution formulated with surfactants.
2.2. Plant material and experimental procedures Experiments were performed with cut rose (Rosa hybrida L.) flowers of various cultivars (Mercedes, Europa, Lambada, Frisco, Sacha and Eskimo), obtained from local commercial greenhouses. Freshly cut flowers with 30-cm long stems, harvested at the marketing stage of bud development, were brought to the laboratory in water. The cut stem ends were trimmed by 2 cm, and lower leaves were removed up to 10 cm from the stem base. Flowers were then pulsed in the standard pulsing solution recommended by the Israeli Flower Board for roses, composed of the preservative TOG-4 (0.2%)+0.15 mM STS. Where indicated, MJ was added as the product TOG-MJ-1 to this pulsing solution at concentrations ranging between 50 and 600 mM. All pulsing treatments and subsequent incubations were performed for 24 h in a conditioned room maintained at 20°C with 60 – 70% RH and 12-h photoperiod at a light intensity of 14 mmol m − 2 s − 1, provided by cool-white fluorescent tubes. After the pulsing treatments, flowers were transferred to 600-ml glass cylinders (ten flowers per cylinder), each containing 300 ml of TOG-6 solution as a bacteriocide, and divided into three
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groups, each of which underwent a different treatment. Group I was used to examine the effect of MJ treatments on flower opening and phytotoxicity during vase life: these flowers were kept in the controlled conditions for daily measurements of flower diameter and visual evaluation of possible toxicity symptoms. Group II was used to examine the effect of MJ treatments on disease incidence following natural infection: to enhance the development of Botrytis rot from natural infections the glass cylinders with the flowers were covered with perforated polyethylene (PE) bags, and incubated for 2 additional days at 20°C. The decay index of flower petals and receptacle was evaluated daily after removal of the PE bags. Group III was used to examine the effect of MJ treatments on disease incidence following artificial inoculation: these flowers were sprayed to run-off with a spore suspension of B. cinerea (104 spores ml − 1), covered with perforated PE bags and incubated for 2 additional days at 20°C. The PE bags were then removed, and the decay index evaluated 5 days later, as described for flowers of group II. In one experiment (Table 1), MJ was added to the Botrytis suspension and applied by spraying to the flower petals, instead of application by pulsing.
2.3. Preparation of spore suspension A culture of B. cinerea was obtained from infected rose petals and grown on potato dextrose agar (PDA, Difco). Spore suspensions were prepared from the sporulating edges of 2-week-old B. cinerea cultures. Spores were gently removed with a bacteriological loop, suspended in sterile distilled water (DW) and filtered through four layers of sterile cheese cloth to remove remaining mycelia. The spore concentration was determined with a haemocytometer and adjusted to 104 spores ml − 1.
2.4. Disease assessment The extent of the Botrytis rot development, expressed as a decay index of flower petals and receptacle, was evaluated according to Hazendonk et al. (1995), with several modifications. Visual rating, on a relative scale of 0–5, was
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defined as follows: 0, no symptoms; 1, appearance of one to four lesions on the petals; 2, appearance of more than four lesions; 3, appearance of fully necrotic petals; 4, necrosis of the petals and receptacle; 5, collapse of flower head and petal drop.
2.5. E6aluation of flower opening and phytotoxicity Flower opening was evaluated by measuring the flower diameter every second day in flowers that did not have any visible Botrytis rot symptoms. Additionally, all treated flowers were examined for injury symptoms such as necrotic lesions on leaves and petals, leaf drop and change in petal colour.
2.6. Detached petal bioassay A bioassay for testing the effect of MJ pulsing treatment on the susceptibility of rose flower petals to B. cinerea was performed with petals detached from control and MJ-treated flowers. The four outermost petals of each flower were discarded and the next four petals were used. A total of 20 such inner petals, collected from five different flowers, were placed on nylon net pads mounted on plastic trays, and the center of each petal was wounded with a sterile microbiological needle. A 30-ml drop of spore suspension of B. cinerea (104 spores ml − 1) was then placed into each wound, and the wounded petals incubated
under high RH conditions (\ 95%) at 20°C. Infection percentage and lesion size were determined 3 or 4 days after inoculation. Since the lesions were oval in shape, an average of width and length measurements is presented. In one experiment (Table 1), MJ (TOG-MJ-1) was applied directly to the detached petals with the spore suspension, instead of by the pulsing treatment.
2.7. Effect of MJ on de6elopment of Botrytis cinerea in 6itro The effects of MJ on spore germination and germ-tube elongation of B. cinerea were tested in a filter-sterilized solution of glucose (1%), supplemented with various concentrations of MJ. Spores of B. cinerea were suspended in the 1% glucose solution at a final concentration of 106 spores ml − 1. Aliquots (900 ml) containing fungal spores were transferred to each well of tissue culture plates (Costar 24-cell tissue culture clusters), and 100-ml aliquots of various MJ (TOG-MJ-1) concentrations were added to make final concentrations ranging between 0 and 400 mM. Similarly, 100-ml aliquots of the surfactants mixture in which the MJ was formulated, were added at the same dilutions to another series of 900-ml aliquots of spore suspensions that served as controls. Samples of the various test solutions (30-ml drops), containing fungal spores with either TOG-MJ-1 or the surfactants mixture of this product, were placed on ethanol-washed microscope slides (three
Table 1 Effect of MJ concentration, applied with the spore suspension of B. cinerea, on incidence of Botrytis rot in attached or detached petals of cut rose flowers cvs. Frisco and Sacha MJ concentration (mM)
0 50 300
Decay index in attached petals (0 – 5)a
Detached petals bioassay
Frisco
% Infection
3.5 a 3.7 a 2.3 b
Sacha
1.9 a 2.1 a 1.3 b
Lesion size (mm)
Frisco
Sacha
Frisco
Sacha
84 50 0
100 100 0
13.2 a 4.4 b n.d.
32.0 a 28.2 a n.d.
A total of 30 flowers or 20 petals per treatment were assayed. Means within each column followed by different letters are significantly different (P = 0.05). a On a scale of 0= no symptoms to 5= severe symptoms.
S. Meir et al. / Posthar6est Biology and Technology 13 (1998) 235–243 Table 2 Effect of MJ concentration, applied in the pulsing solution, on development of natural infection of Botrytis rot in attached petals of cut rose flowers cvs. Frisco and Sacha MJ concentration (mM)
0 50 100 200 400 600
the petal bioassay. Results were analyzed using SigmaStat statistical software (Jandel Scientific Software, San Rafael, CA). Paired t-test, one way ANOVA, and Student–Newman–Keuls multiple range test combined with Kruskall–Wallis ANOVA on ranks were performed where indicated.
Decay index (0–5)a Frisco
Sacha
1.95 1.35 1.45 0.61 1.74 0.85
1.21 1.67 1.74 0.80 0.95 1.10
a abc abc c ab bc
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a a a a a a
3. Results Pulsing Frisco and Sacha cut rose flowers with 50 or 100 mM MJ had no significant effect on the development of Botrytis rot symptoms following natural infection for 2 days at 20°C (Table 2). Whereas pulsing Sacha flowers with MJ concentrations up to 600 mM was still ineffective, the rot symptoms were significantly suppressed in Frisco flowers following pulsing with 600 mM MJ (Table 2). Similarly, pulsing Mercedes, Europa and Frisco cut flowers with low MJ concentrations (25, 50 and 100 mM) had no effect on disease incidence following either natural or artificial infection (data not shown). Pulsing Frisco and Lambada flowers with MJ concentrations of 200 mM and above, significantly suppressed the development of Botrytis rot resulting from artificial inoculation (Table 3). This inhibitory effect of MJ was saturated at a concentration of 200 mM for both rose cultivars; the same MJ concentration
Flowers (n =30) were evaluated 5 days after removal of the perforated PE bags. Means within each column followed by different letters are significantly different (P= 0.05). a On a scale of 0= no symptoms to 5= severe symptoms.
drops per slide) in Petri dishes padded with moistened filter paper, and incubated for 24 h at 25°C in darkness. Spore germination and germ-tube elongation were observed under a light microscope, in three microscope fields, each containing 20 – 40 spores.
2.8. Statistical analysis Experiments were repeated two to four times, with 30 flower replicates for each treatment in the cut flower assays, and with 20 detached petals in
Table 3 Effect of MJ concentration, applied in the pulsing solution, on development of Botrytis rot in artificially or naturally infected attached petals of cut rose flowers cvs. Frisco and Lambada MJ concentration (mM)
Decay index (0–5)a Artificial infectionb
0 200 400 600
Natural infectionc
Frisco
Lambada
Frisco
Lambada
2.6 1.7 1.6 1.4
3.8 2.3 2.7 2.8
0.6 0.9 0.9 0.9
4.3 1.9 3.3 3.3
a b b b
a b b b
a a a a
a b a a
A total of 30 flowers per treatment were assayed. Means within each column followed by different letters are significantly different (P =0.05). a On a scale of 0= no symptoms to 5= severe symptoms. b Evaluated 4 days after inoculation. c Evaluated 5 days after removal of the perforated PE bags.
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Fig. 1. Effect of 200 mM MJ, applied in the pulsing solution, on the time course of Botrytis rot development in rose flowers cvs. Frisco (A) and Mercedes (B) following artificial inoculation. Each bar represents the mean 9 S.E. of 30 replicates, evaluated 4 (dark bars) or 7 (empty bars) days after artificial inoculation.
was also the most effective in reducing rot symptoms after natural infection in Lambada, but not in Frisco flowers (Table 3). The apparent discrepancy between the two experiments in the MJ effects obtained for Frisco flowers following natural infection (Tables 2 and 3), may be ascribed to variations in the infection potential of these cultivars. When control Frisco flowers were highly infected (decay index of 1.95), pulsing with 200 mM MJ significantly suppressed the disease symptoms (Table 2), but when control Frisco flowers were not infected (decay index of 0.6), MJ pulsing was ineffective (Table 3). The effect of MJ at 200 mM, the most effective concentration in suppressing Botrytis rot in various rose cultivars, on the time course of disease development in artificially inoculated flowers, was also studied. Results depicted in Fig. 1 clearly demonstrate that MJ at this concentration significantly reduced disease incidence 4 and 7 days after inoculation of both Frisco and Mercedes roses. The effect of 200-mM MJ pulsing in suppressing rot development was also observed in the petal bioassay experiments (Fig. 2). The bioassay
was performed with detached petals taken from control or MJ-treated Eskimo and Lambada flowers, and wound-inoculated with B. cinerea spore suspensions. The results presented in Fig. 2 show that the MJ pulsing treatment inhibited lesion expansion by about 50% in both cultivars. The possibility that MJ suppresses Botrytis rot through a direct effect on B. cinerea development was examined in vitro. The results presented in Fig. 3 show that MJ significantly inhibited spore germination and germ-tube elongation of the pathogen. Inhibition was dose-dependent, very effective above 100 mM; 50% inhibition of germtube elongation was obtained with 200 mM MJ, while 50% inhibition of spore germination was obtained with MJ concentrations higher than 300 mM. Complete inhibition of spore germination was obtained with 400 mM MJ (Fig. 3). The control media of the product TOG-MJ-1, consisting of the surfactants mixture at dilutions equivalent to MJ in the 0–400-mM range, had no inhibitory effect on either germination or elongation (data not shown).
Fig. 2. Effect of 200 mM MJ, applied in the pulsing solution, on lesion size of Botrytis rot developed in detached petals of rose flowers cvs. Eskimo and Lambada, following wound-inoculation with B. cinerea. A total of 20 petals, taken from five control or MJ-treated flowers of each cultivar, were wounded and inoculated with a suspension of B. cinerea (104 spores ml − 1). Lesion sizes represent means of measurements of the length and width, monitored 3 (in Eskimo) or 4 (in Lambada) days after inoculation. Each bar represents the mean 9 S.E. of 20 replicates.
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mained more turgid, with a shiny appearance during vase life (data not shown).
4. Discussion
Fig. 3. Effect of MJ concentration on inhibition of spore germination and germ-tube elongation of B. cinerea in vitro. Each point represents the mean 9 S.E. of 60–120 spores from four experiments.
The possible direct effect of MJ on the pathogen was further manifested when rose flowers and detached petals were inoculated with B. cinerea spore suspensions containing various concentration of MJ (Table 1). When Frisco and Sacha flowers were artificially inoculated only 300 mM MJ, applied with the spores, significantly reduced the development of Botrytis rot (Table 1) to levels similar to those obtained for the naturally infected flowers that were not treated with MJ (Table 2). In the petal bioassay, application of 50 mM MJ with the spore suspension significantly inhibited petal disease incidence in Frisco flowers, but had no effect on Sacha petals (Table 1). The higher MJ concentration (300 mM), however, completely inhibited the petal infection in both cultivars (Table 1). No negative effects of MJ on flower quality (damage, flower opening or longevity) were observed in any of the experiments. Moreover, positive effects of MJ were observed in several experiments. Pulsing flowers with 200 mM MJ resulted in a better maintenance of the original yellow colour of Frisco petals, and inhibited colour fading of other cultivars during vase life. In addition, leaves of MJ-treated flowers re-
Inducible defense responses in plants are known to be mediated by signalling molecules that are produced or released at the site of pathogen attack, and may be transported either locally by diffusion or systemically through the vascular system (Farmer et al., 1992). A large body of evidence suggests that JA or MJ is a key component of such intracellular signalling in response to pathogen attack, and that its application may, therefore, induce disease resistance in a wide variety of plants (Creelman and Mullet, 1995). In this respect, since MJ is regarded as a natural plant growth regulator, it has the advantage of eliciting defense (Farmer et al., 1992; Cohen et al., 1993; Schweizer et al., 1993) or physiological (Meir et al., 1996) responses to its exogenous application to plants at low concentrations in a non-destructive manner. Our present study supports this possible role of MJ as an inducer of a defense response in plants, and demonstrates for the first time its potential for postharvest application as a means to elicit systemic and local protection of cut roses against Botrytis rot. MJ pulsing seems to provide systemic protection against Botrytis rot by inducing resistance mechanisms in the treated cut roses, as suggested previously for GA3 (Shaul et al., 1995a,b). This conclusion is based on the following findings of the present study. (a) Pulsing cut roses of various cultivars with MJ significantly reduced the development of Botrytis rot in flower petals following either natural (Tables 2 and 3) or artificial infection (Table 3 and Fig. 1), without causing any flower damage. (b) The threshold, optimal pulsing dose of MJ obtained for rot inhibition in these rose cultivars was 200 mM (Tables 2 and 3 and Fig. 1). (c) This 200-mM MJ pulsing concentration was also markedly effective in reducing disease incidence in the detached petal bioassay performed with Eskimo and Lambada cultivars (Fig. 2). (d) When MJ was applied by spray simultaneously with B. cinerea spores, higher MJ
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concentrations (300 mM) were required for protection of Frisco roses (Table 1). These results are in full agreement with previous studies which found a significant local protective effect of sprayapplied MJ against pathogen infection that involved MJ concentrations higher than 200 mM (Cohen et al., 1993; Schweizer et al., 1993). In addition to the effect of MJ in inducing flower resistance, the possibility that MJ exerts its inhibitory effects on Botrytis rot through its direct antifungal activity cannot be excluded. This direct antifungal effect of MJ was manifested both in in vitro inhibition of growth and germination of B. cinerea (Fig. 3), and in inhibition of Botrytis rot incidence in petals following simultaneous spray of spores and MJ (Table 1). The inhibition of B. cinerea growth in vitro was dose-dependent, being effective above 100 mM MJ (Fig. 3). However, the spray application of 50 mM MJ to detached Frisco petals through wounding provides a better local protection (Table 1), since it suppresses the rot possibly through a combination of direct and induced resistance mechanisms. The results presented in Table 1 show clearly that in the detached petal bioassay, MJ significantly reduced the infection percentage and lesion size for Frisco petals but was ineffective with Sacha petals. This suggests that the Sacha cultivar may be more susceptible to B. cinerea and less responsive to MJ, as indicated also by the data of Table 2. These results further imply that, apart from the direct effect of MJ on the fungus, the type of petal tissue may also play a role in the defense mechanism (Hammer and Evensen, 1994). Jasmonates have been previously reported to have a direct antifungal activity against various pathogens, although with varying efficiency. Whereas, in the present study, complete inhibition of B. cinerea spore germination and germ-tube elongation in vitro were already obtained at 400 mM MJ (Fig. 3), much higher concentrations, of about 1.2 mM JA or 3.5 mM MJ, were required for complete inhibition of in vitro spore germination of Pyricularia oryzae (Neto et al., 1991), or of mycelial growth of Phytophthora infestans (Cohen et al., 1993). Similarly, a concentration of about 5 mM JA was required for direct inhibition of appressoria differentiation of Erysiphe graminis,
but no inhibition of germination was obtained (Schweizer et al., 1993). These results suggest that MJ is more effective in suppression of development of B. cinerea than of other fungi. Unlike their effects on various seedling systems, in which high MJ or JA concentrations in the mM range caused leaf chlorosis or necrosis of leaf tips (Cohen et al., 1993; Schweizer et al., 1993), MJ applied as a pulse to cut roses in micromolar concentrations neither impaired the quality nor accelerated senescence of flowers of any of the cultivars examined. This may be due to the lower MJ doses required for inhibition of B. cinerea compared with other fungi. Alternatively, the lack of visible damage in MJ-treated roses, with or without STS in the pulsing solution, may also stem from the fact that senescence of most rose cultivars is not ethylene-regulated (Mor and Zieslin, 1987). This conclusion is further reinforced by comparison between the effects of MJ application to ethylene-sensitive or -insensitive flowers. Thus, application of 50–5000 mM MJ to petunia or dendrobium flowers, in which ethylene plays a major role in regulation of senescence processes, resulted in significant acceleration of ethylene production rates and flower senescence (Porat et al., 1993). It was, therefore, suggested that the effects of MJ in accelerating senescence of these two ethylene-sensitive flowers are ethylenemediated (Porat et al., 1993). In petals of tulip, however, which is an ethylene-insensitive flower, MJ did not accelerate ethylene production (Porat et al., 1993). It seems, therefore, that MJ-induced senescence in flowers is correlated with increased ethylene production and sensitivity to ethylene. Hence, application of MJ as an antifungal agent should not cause any adverse effects in ethyleneinsensitive flowers and/or in low ethylene producers. In ethylene-sensitive flowers, MJ may be applied with an inhibitor of ethylene action such as STS. Taken together, our results suggest the possible use of MJ as a postharvest application in low doses, for suppression of Botrytis rot in various cut rose cultivars without impairing flower quality. As such, it may provide flower growers with a better and more environmentally-friendly alternative to conventional fungicides for preventing decay development.
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Acknowledgements The authors would like to express their gratitude to Yigal Slonim from the Assia Reisel Company for his helpful cooperation in conducting this research.
References Avdiushko, S., Croft, K.P.C., Brown, G.C., Jackson, D.M., Hamilton-Kemp, T.R., Hildebrand, D., 1995. Effect of volatile methyl jasmonate on the oxylipin pathway in tobacco, cucumber, and Arabidopsis. Plant Physiol. 109, 1227 – 1230. Bell, E., Mullet, J.E., 1991. Lipoxygenase gene expression is modulated in plants by water deficit, wounding, and methyl jasmonate. Mol. Gen. Genet. 230, 456–462. Cohen, Y., Grisi, U., Mosinger, E., 1993. Local and systemic protection against Phytophthora infestans induced in potato and tomato by jasmonic acid and methyl jasmonate. Phytopathology 83, 1054–1062. Coyier, D.L., 1985. Roses. In: Strider, D.L. (Ed.), Diseases of Floral Crops. Praeger, New York, pp. 405–488. Creelman, R., Mullet, J.E., 1995. Jasmonic acid action and distribution in plants: regulation during development and responses to biotic and abiotic stresses. Proc. Natl. Acad. Sci. USA 92, 4114 – 4119. Dittrich, H., Kutchan, T., Zenk, M.H., 1992. The jasmonate precursor, 12-oxo-phytodienoic acid, induces phytoalexin synthesis in Petroselinum hortense cell cultures. FEBS Lett. 309, 33 – 36. Doughty, K.L., Kiddle, G.A., Pye, B.J., Wallsgrove, R.M., Pickett, J.A., 1995. Selective induction of glucosinolates in oilseed rape leaves by methyl jasmonate. Phytochemistry 38, 347 – 350. Elad, Y., 1988. Latent infection of Botrytis cinerea in rose flowers and combined chemical and physiological control of the disease. Crop Prot. 7, 631–633. Elad, Y., Yunis, H., Katan, T., 1992. Multiple fungicide resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of Botrytis cinerea in Israel. Plant Pathol. 41, 41 –46. Farmer, E.E., Johnson, R.R., Ryan, C.A., 1992. Regulation of expression of proteinase inhibitor genes by methyl jasmonate and jasmonic acid. Plant Physiol. 98, 995–1002. Gundlach, H., Muller, M., Kutchan, T.M., Zenk, M.H., 1992. Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc. Natl. Acad. Sci. USA 89, 2389– 2393. Hammer, P.E., Evensen, K.B., 1994. Differences between rose cultivars in susceptibility to infection by Botrytis cinerea. Phytopathology 84, 1305–1312. .
243
Hazendonk, H., ten Hoope, M., van der Wurff, T., 1995. Methods to test rose cultivars on their susceptibility to Botrytis cinerea during the postharvest stage. Acta Hortic. 405, 39 – 45. Loake, G.J., 1996. Jasmonates: global regulators of plant gene expression. In: Smallwood, M., Knox, J.P., Bowles, D.J. (Eds.), Specialized Function in Plants. Bios Scientific, Oxford, pp. 215 – 230. Meir, S., Philosoph-Hadas, S., Lurie, S., Droby, S., Akerman, M., Zauberman, G., Shapiro, B., Cohen, E., Fuchs, Y., 1996. Reduction of chilling injury in stored avocado, grapefruit and bell pepper by methyl jasmonate. Can. J. Bot. 74, 870 – 874. Mirjalili, N., Linden, J.C., 1996. Methyl jasmonate induced production of taxol in suspension cultures of Taxus cuspidata: ethylene interaction and induction models. Biotechnol. Prog. 12, 110 – 118. Mor, Y., Zieslin, N., 1987. Plant growth regulators in rose plants. Hort. Rev. 9, 53 – 73. Mueller, M.J., Brodschelm, W., Spannagl, E., Zenk, M.H., 1993. Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid. Proc. Natl. Acad. Sci. USA 90, 7490 – 7494. Neto, G.C., Kono, Y., Hyakuttake, H., Watanabe, M., Suzuki, Y., Sakurai, A., 1991. Isolation and identification of (−)-jasmonic acid from wild rice, Oryza officinalis, as an antifungal substance. Agric. Biol. Chem. 55, 3097 – 3098. Nojiri, H., Sugimori, M., Yamane, H., Nishimura, Y., Yamada, A., Shibuya, N., Kodama, O., Murofushi, N., Omori, T., 1996. Involvement of jasmonic acid in elicitorinduced phytoalexin production in suspension-cultured rice cells. Plant Physiol. 110, 387 – 392. Porat, R., Borochov, A., Halevy, A.H., 1993. Enhancement of petunia and dendrobium flower senescence by jasmonic acid methyl ester is via the promotion of ethylene production. Plant Growth Reg. 13, 297 – 301. Schweizer, P., Gees, R., Mosinger, E., 1993. Effect of jasmonic acid on the interaction of barley (Hordeum 6ulgare L.) with the powdery mildew Erysiphe graminis f. sp. hordei. Plant Physiol. 102, 503 – 511. Sembdner, G., Parthier, B., 1993. The biochemistry and the physiological and molecular actions of jasmonates. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 569 – 589. Shaul, O., Elad, Y., Zieslin, N., 1995a. Suppression of Botrytis blight in cut flowers with gibberellic acid: effects of concentration and mode of application. Postharvest Biol. Technol. 6, 321 – 330. Shaul, O., Elad, Y., Zieslin, N., 1995b. Suppression of Botrytis blight in cut rose flowers with gibberellic acid: effects of postharvest timing of the gibberellin treatment, conidial inoculation and cold storage. Postharvest Biol. Technol. 6, 331 – 339. Shaul, O., Elad, Y., Zieslin, N., 1996. Suppression of Botrytis blight in cut rose flowers with gibberellic acid: effects of exogenous application of abscisic acid and paclobutrazol. Postharvest Biol. Technol. 7, 145 – 150.