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Acibenzolar-S-methyl induces resistance to Colletotrichum lagenarium and cucumber mosaic virus in cantaloupe
Crop Protection 22 (2003) 769–774
Acibenzolar-S-methyl induces resistance to Colletotrichum lagenarium and cucumber mosaic virus in cantaloupe J. Smith-Beckera, N.T. Keena,{, J.O. Beckerb,* a
Department of Plant Pathology, University of California, Riverside, CA 92521, USA b Department of Nematology, University of California, Riverside, CA 92521, USA Received 22 May 2002; accepted 17 February 2003
Abstract Acibenzolar-S-methyl (ASM) is a synthetic analogue of salicylic acid developed for use in a novel strategy for crop protection through systemic acquired resistance (SAR). ASM protected cantaloupe against a fungal pathogen, Colletotrichum lagenarium, and a viral pathogen, cucumber mosaic virus (CMV). ASM induced the systemic accumulation of chitinase, a marker protein for SAR, in both greenhouse and field grown seedlings. ASM at 50 or 100 mg/ml provided almost complete protection against the fungal pathogen and effectively delayed the spread of CMV in greenhouse trials. In two field trials, ASM had no negative effect on fruit production in the absence of CMV disease pressure. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Acibenzolar-S-methyl; Cantaloupe; CMV; Chitinase; Salicylic acid; SAR
1. Introduction Systemic acquired resistance (SAR) involves the activation of natural defense mechanisms in plants prior to pathogen attack (Kuc, 1982). In biologically induced resistance, necrosis caused by pathogens results in the synthesis of salicylic acid (SA) both at the site of infection and systemically in uninfected tissues (Meuwly et al., 1995; Shulaev et al., 1995). The pathogen induced signal that elicits SA synthesis is unknown, although reactive oxygen compounds such as hydrogen peroxide and superoxide have been shown to induce SA synthesis in several systems (Leon et al., 1995; Neuenschwander et al., 1995; Enyedi, 1999). Considerable evidence suggests that SA is the signal molecule that activates the synthesis of pathogen defense related proteins (PR proteins) in plant foliage (Ward et al., 1991; Uknes et al., 1992; Gaffney et al., 1993). The PR proteins provide useful markers for the establishment of SAR, since the level of accumulation of these proteins is correlated to the level of SAR induced. PR proteins shown to *Corresponding author. Tel.: +1-909-787-2185; fax: +1-909-7873719. E-mail address: [email protected] (J.O. Becker). { N. Keen passed away on April 18, 2002.
accumulate during SAR in cucurbits include chitinase, peroxidase and X -glucanase (Metraux et al., 1988; Hammerschmidt et al., 1982; Ji and Kuc, 1995). Chitinase is a particularly useful marker for SAR, since levels of the enzyme in non-induced tissues are low. Acibenzolar-S-methyl (1,2,3-benzothiadiazole-7-carbothioic acid S-methyl ester, ASM) mimics the effects of SA and has been developed for use as a crop protectant (Gorlach et al., 1996; Kunz et al., 1997). This compound offers promise as an alternative method to conventional disease control with pesticides. ASM has the same spectrum of activity as biologically induced resistance, and has been shown effective in protecting several crop species and Arabidopsis from fungal, bacterial and viral pathogens (Friedrich et al., 1996; Gorlach et al., 1996; Lawton et al., 1996). Huang et al. (2000) demonstrated that one application of ASM prior to flowering protected rock and Hami melon fruit from several post-harvest fungal diseases. Although ASM protects tobacco against tobacco mosaic virus (TMV), and Arabidopsis against turnip crinkle virus, no studies have reported efficacy against melon viral diseases. Mosaic diseases caused by arthropod-borne viruses pose one of the greatest threats to melon production. In California, the four major aphid-transmitted viruses are watermelon mosaic potyvirus 2, papaya ringspot
0261-2194/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0261-2194(03)00044-9
770
J. Smith-Becker et al. / Crop Protection 22 (2003) 769–774
potyvirus (watermelon strain), zucchini yellow mosaic potyvirus and cucumber mosaic cucumovirus (Nameth et al., 1986; Grafton-Cardwell et al., 1996). Infected melons often develop typical mosaic disease symptoms such as clearing of veins, systemic chlorotic speckling, stunting of leaves and inhibited fruit development. Control of viral pathogens is currently limited to the use of resistant varieties and control of the insect vectors. In this work we tested the activity of ASM in cantaloupe against cucumber mosaic virus (CMV), and Colletotrichum lagenarium, causal agent of anthracnose. C. lagenarium has been used extensively in studies of SAR in cucumber and melon (Hammerschmidt et al., 1982; Metraux et al., 1990). It produces slowly spreading necrotic lesions that are easily quantified in SAR assays.
2. Materials and methods 2.1. Culture of pathogens CMV was maintained and increased in cantaloupe (Cucumis melo L. cv. Durango, Petoseed, Saticoy, CA). Inocula were prepared from young symptomatic leaves by homogenizing leaf tissue in 20 mm potassium phosphate buffer, pH 7.0. Homogenates were diluted 1:50 (fresh weight/volume buffer) and Celite (0.5% w/v) was added prior to inoculation. C. lagenarium (Pass.) Ellis and Halsted race 1 was cultured on potato dextrose agar (PDA) at 18 C in the dark. Conidial suspensions for use as inoculum were prepared from 7 to 10 day-old cultures as described previously (Kuc and Richmond, 1977). 2.2. Plant culture and inoculations All experiments were conducted in the greenhouse and repeated at least twice. Each treatment contained 10–15 plants individually potted in 4 in2 peat pots. All the leaves of three-week-old cantaloupe seedlings were sprayed with ASM (Actigard, Syngenta, Greensboro, NC, USA) at a concentration of active compound of 25, 50 or 100 mg/ml in water until runoff. ASM stock solutions were prepared from a wettable powder containing 50% active compound. Four days after the inducing treatments, all seedlings were inoculated on the second leaf with either CMV or C. lagenarium. CMV was inoculated by gently rubbing 100 ml of infected leaf homogenate onto one-half of the leaf. Fungal spores were applied by placing ten 10 ml drops of a 105 spores/ ml suspension on leaves. C. lagenarium inoculated plants were placed in plastic bags in the dark for 18 h after inoculation to allow spore germination. Viral and fungal symptoms were evaluated 8 and 10 days, respectively, after inoculation. In addition, CMV titer in the youngest expanded leaves was determined using polyclonal
antibodies to the virus in an indirect ELISA assay (Fang and Grumet, 1993). The ability of ASM to protect seedlings at the cotyledon stage was also evaluated. We were unable to infect with CMV through mechanical inoculation of cotyledons, so SAR in the first true leaves was evaluated using the fungal pathogen. One-week-old seedlings were sprayed with 10, 20 or 40 mg/ml ASM. Seven days after treatment, the first true leaves were inoculated with five drops of the fungal spore suspension as described above. 2.3. Measurement of chitinase The optimal concentration of ASM needed to induce SAR in cantaloupe was first determined by measuring the levels of the SAR marker protein chitinase in leaves 5 days after treatment with 25, 50 or 100 mg/ml ASM. Chitinase levels were measured in the second true leaves of seedlings using antibodies prepared to cucumber chitinase in a sandwich ELISA assay as described previously (Smith-Becker et al., 1998). Two leaf discs, 1.5 cm diameter, were collected from each leaf and stored at 80 C prior to assay. In experiments using one-week-old seedlings, chitinase was measured in the first true leaf 7 days after treatment. 2.4. Field trials Two field trials were conducted at the UCR Coachella Valley Agricultural Research Station, Thermal, CA. Melon production in that area is often severely affected by one or more viruses. The soil type was a Carsetas loamy sand (84% sand, 5% clay, and 0.8% organic matter, pH 7.3). Plot design was a randomized complete block with 4 treatments and 5 replications. The beds were one m wide with 20 plants per plot, spaced 0.3 m apart. Cantaloupe (cv. Durango) was seeded into nontreated soil and grown with a low-volume irrigation setup which provided both water and fertilizer according to local standard practice. In the first trial, the treatments consisted of a non-treated control and a single ASM foliar application at three weeks after seeding. Seedlings were sprayed with ASM at a rate of 37.5 mg/ml until runoff. Chitinase accumulation was measured in each treatment two weeks after the inducing inoculations. Two 1.5 cm diameter leaf discs were collected from the second true leaf of each of 5 randomly selected plants per plot. Leaf discs were placed immediately into 1.5 ml microfuge tubes on dry ice and stored at 80 C prior to chitinase assay. In the second trial, the four treatments consisted of a non-treated control, a single ASM treatment at 100 mg/ml, three ASM applications at 100 mg/ml in two week intervals and three applications at 50 mg/ml in two week intervals, each starting three weeks after seeding. At harvest, the total number of fruits, the number of marketable fruits and the weight of
J. Smith-Becker et al. / Crop Protection 22 (2003) 769–774
marketable fruits was determined. All data were subject to analysis of variance and mean separation with Fisher’s protected least significant difference.
771
ASM concentrations as low as 10 mg/ml were as effective as higher concentrations at inducing chitinase accumulation in the first leaf (Fig. 3). ASM concentrations higher than 50 mg/ml caused phytotoxicy in one-weekold seedlings.
3. Results 3.2. Protection against C. lagenarium and CMV 3.1. Chitinase accumulation The accumulation of chitinase increased with increasing concentrations of ASM (Fig. 1). ASM at 100 mg/ml induced the highest level of chitinase without causing any phytotoxic effects under greenhouse conditions, and this concentration was used for subsequent greenhouse experiments with three-week-old seedlings. Chitinase levels in treated leaves increased for at least the first 4 days after treatment (Fig. 2). In one-week-old seedlings,
Chitinase (ng/disc)
150
ASM at 50 or 100 mg/ml provided almost complete protection against the fungal pathogen (Fig. 4). ASM also provided protection against the viral pathogen CMV. This resistance was observed as an absence of viral symptoms 8 days after inoculation in ASM treated plants and a reduced viral titer in young leaves (Fig. 5). ASM treated plants first began to show viral symptoms 14 days after inoculation, indicating that several applications of ASM may be necessary to provide extended protection. ASM treatment of cotyledons was highly effective against C. lagenarium, and no lesions developed on the first true leaves of seedlings treated on the cotyledons with 10, 20 or 40 mg/ml.
100
3.3. Field trials
50
0 0
25
50
100
ASM (µg/ml) Fig. 1. Effect of ASM concentration on chitinase accumulation. Three-week-old cantaloupe seedlings were sprayed with 25, 50 or 100 mg/ml ASM. Chitinase was measured in discs from the second true leaves 5 days after treatment. Bars indicate standard error.
ASM caused a significant increase in chitinase accumulation in the field-grown plants compared to the non-treated control (Fig. 6). Despite the more stressful growing conditions in the field, the levels of chitinase in non-treated field-grown plants were no higher than in control plants grown in the greenhouse. None of the treatments in the first trial had a measurable effect on the cantaloupe yield (data not shown). In the 80
Chitinase (ng/disc)
60
40
20
Fig. 2. Time course of chitinase accumulation in response to ASM. Three-week-old cantaloupe seedlings were sprayed with water (control) or 100 mg/ml ASM. Chitinase levels were measured in the second leaves of treated plants for 4 days. Bars indicate standard error.
ASM (40 µg/ml)
ASM (20 µg/ml)
ASM (10 µg/ml)
Control
0
Fig. 3. Chitinase accumulation in young seedlings in response to ASM. One-week-old seedlings were sprayed on the cotyledons with 10, 20 or 40 mg/ml ASM. Chitinase was measured in the first true leaves 7 days after treatment. Bars indicate standard error.
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10
100
7.5
75 Chitinase ng/disc
Number of Lesions
772
5
2.5
50
25
Fig. 4. ASM induced resistance to C. lagenarium. Three-week-old greenhouse grown seedlings were sprayed with ASM (100 mg/ml). Four days after treatment, seedlings were inoculated on leaf 2 with 10 drops of a C. lagenarium spore suspension. C. lagenarium lesions were counted 10 days after inoculation. Bars indicate standard error.
0.2
Absorbance 405
0.15
ASM
0 Control
ASM (100 µg/ml)
ASM (50 µg/ml)
Control
0
Fig. 6. Effect of ASM on chitinase accumulation in field-grown plants. Three-week-old field grown plants were sprayed with 37.5 mg/ml ASM. Leaf discs for chitinase assay were collected from the second leaves two weeks after the inducing treatment. Bars indicate standard error.
second trial, repeated treatments with ASM had no deleterious effect on the quantity or quality of fruit production and a single treatment of 100 mg/ml resulted in a significant increase in the number of marketable fruit (Table 1). No obvious CMV symptoms were observed in both field trials. Thus, although the marker protein chitinase indicated that plants in the field were induced, we were unable to evaluate the level of viral resistance.
4. Discussion 0.1
0.05
ASM (100 µg/ml)
ASM (50 µg/ml)
Control
0
Fig. 5. ASM induced resistance to CMV. Three-week-old greenhouse grown seedlings were sprayed with water (control) or ASM at 50 or 100 mg/ml. Four days after treatment, seedlings were inoculated on leaf 2 with CMV. Eight days after CMV inoculation, the youngest expanded leaves (leaves 4 or 5) were collected and assayed for CMV using an indirect ELISA assay. Bars indicate standard error.
ASM induced accumulation of the SAR marker protein chitinase and provided almost complete protection against C. lagenarium in cantaloupe. One application of ASM also effectively delayed the spread of the viral pathogen CMV in greenhouse trials. Although several PR proteins have been identified as enzymes with potential antimicrobial activity against bacteria and fungi, none have been associated with a function related to viral inhibition (Linthorst et al., 1989). Prior to the discovery of SA as an endogenous signal molecule, a derivative of SA, acetylsalicylic acid, was shown to greatly reduce the accumulation of TMV in a susceptible tobacco cultivar (Antoniw and White, 1980; White et al., 1983). Consistent with the function of ASM as an analogue of SA, Friedrich et al. (1996) showed that ASM treatment of susceptible tobacco resulted in reduced levels of TMV RNAs in inoculated plants.
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Table 1 Effect of foliar applications of ASM on cantaloupe harvested from a field trial in Californiaa Treatment (number of applications)
Total number of fruit/plot
Number of marketable fruitb
Weight (kg) of marketable fruit
Non-treated check 100 mg/ml ASM (1) 100 mg/ml ASM (3) 50 mg/ml ASM (3)
31.671.6a 31.271.4a 33.872.2a 32.872.1a
9.070.5a 11.971.2b 10.370.4ab 10.170.7ab
0.5570.04a 0.5770.03a 0.5670.02a 0.6070.02a
a
Data are means7std error of 5 replicate plots. Means of a given columns followed by the same letter are not significantly different (Pp0:05) according to Fisher’s protected least significant difference. b Fruit diameter>12 cm.
One of the first biological roles described for SA in plant metabolism was its ability to induce the alternate respiratory pathway in the thermogenic Arum lily (Raskin et al., 1987). SA also induced cyanide resistant respiration and thermogenicity in tobacco, indicative of the activation of the alternative respiration pathway (Kapulnik et al., 1992; Van Der Straeten et al., 1995). Rhoads and McIntosh, (1993) showed that the promoter of the SA inducible alternative oxidase (AOX) in tobacco shared sequence similarity to promoters of SA inducible PR proteins, suggesting that the AOX is regulated coordinately with other defense related proteins during SAR. Chivasa et al. (1997) investigated the possibility that SA induced resistance to TMV in tobacco is dependent on the known effects of SA on the AOX. They demonstrated that salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial AOX, compromised SA induced resistance to TMV without affecting induced resistance to a bacterial or fungal pathogen. In addition, SHAM treatment did not reduce the level of PR-1 protein accumulation in response to SA. The authors suggest that SA induced resistance to viruses is distinct from resistance to fungal and bacterial pathogens in the requirement for AOX activity or other as yet unidentified SHAM sensitive activity. In order to study the mechanism of SA induced resistance to viruses, Naylor et al. (1998) investigated the effects of SA treatment on the replication and movement of potato virus X (PVX) and CMV in tobacco. SA treatment reduced the replication of PVX in inoculated leaves, but had no effect on CMV replication. However, plants treated with SA showed delayed symptoms of CMV, and the authors were able to attribute this delay to an inhibition of systemic movement of the virus. Surprisingly, SHAM treatment effectively eliminated the resistance induced by SA to both PVX and CMV. Thus the ability of SA to inhibit the replication of certain viruses and the movement of others is somehow linked through a SHAM sensitive mechanism. It is likely that the mode of action of ASM in reducing CMV spread in melon is related to the ability of SA to reduce CMV movement in tobacco. While we were not able to confirm the efficacy of ASM in the field due to the lack of disease pressure, the
levels of chitinase measured in field-grown plants demonstrated that ASM application induced SAR to similar levels as in greenhouse trials. ASM treatment also had no negative impact on fruit quality or quantity, even when applied several times throughout the growing season. In tomato, ASM residues were detected in tissues only during the first 72 h after treatment (Scarponi et al., 2001). Similarly, a detailed study of ASM residues in pepper showed that the compound was undetectable by 5 days after treatment (Buonaurio et al. 2002). Although repeated booster applications of ASM may prove advantageous for an extended resistance period, early protection has the most important impact economically. Plants infected early, before fruit set, produce fewer flowers and may abort fruits, resulting in extensive production losses (Nameth et al., 1986). ASM may therefore become an important component of an integrated pest management approach to reduce viral and fungal disease impact on melons.
Acknowledgements This study was supported in part by a grant from the California Melon Research Board.
References Antoniw, J., White, R., 1980. The effects of aspirin and polyacrylic acid on soluble leaf proteins and resistance to virus infection in five cultivars of tobacco. Phytopathol. Z. 99, 331–341. Buonaurio, R., Scarponi, L., Ferrara, M., Sidoti, P., Bertona, A., 2002. Induction of systemic acquired resistance in pepper plants by acibenzolar-S-methyl against bacterial spot disease. Eur. J. Plant Pathol. 108, 41–49. Chivasa, S., Murphy, A., Naylor, M., Carr, J., 1997. Salicylic acid interferes with tobacco mosaic virus replication via a novel salicylhydroxamic acid-sensitive mechanism. Plant Cell 9, 547–557. Enyedi, A.J., 1999. Induction of salicylic acid biosynthesis and systemic acquired resistance using the active oxygen species generator rose bengal. J. Plant Physiol. 154, 106–112. Fang, G., Grumet, R., 1993. Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene. Mol. Plant-Microbe Interact. 6, 358–367. Friedrich, L., Lawton, K., Ruess, W., Masner, P., Specker, N., Gut-Rella, M., Mejer, B., Dincher, S., Staub, T., Uknes, S., Metraux, J-P., Kessmann, H., Ryals, J., 1996. A benzothiadiazole
774
J. Smith-Becker et al. / Crop Protection 22 (2003) 769–774
derivative induces systemic acquired resistance in tobacco. Plant J. 10, 61–70. Gaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, G., Uknes, S., Ward, E., Kessmann, H., Ryals, J., 1993. Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261, 754–756. Gorlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.-H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H., Ryals, J., 1996. Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8, 629–643. Grafton-Cardwell, E., Perring, T., Smith, R., Valencia, J., Farrar, C., 1996. Occurrence of mosaic viruses in melons in the Central Valley of California. Plant Dis. 80, 1092–1097. Hammerschmidt, R., Nuckles, E.M., Kuc, J., 1982. Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol. Plant Pathol. 20, 73–82. Huang, Y., Deverall, B.J., Tang, W.H., Wang, W., Wu, F.W., 2000. Foliar application of acibenzolar-S-methyl and protection of postharvest rock melons and Hami melons from disease. Eur. J. Plant Pathol. 106, 651–656. Ji, C., Kuc, J., 1995. Purification and characterization of an acidic B-1,3-glucanase from cucumber and its relationship to systemic disease resistance induced by Colletotrichum lagenarium and tobacco necrosis virus. Mol. Plant Microbe Interact. 8, 899–905. Kapulnik, Y., Yalpani, N., Raskin, I., 1992. Salicylic acid induces cyanide-resistant respiration in tobacco cell-suspension cultures. Plant Physiol. 100, 1921–1926. Kunz, W., Schurter, R., Maetzke, T., 1997. The chemistry of benzothiadiazole plant activators. Pestic. Sci. 50, 275–282. Kuc, J., 1982. Induced immunity to plant disease. Bioscience 32, 854–860. Kuc, J., Richmond, S., 1977. Aspects of the protection of cucumber against Colletotrichum lagenarium by Colletotrichum lagenarium. Phytopathology 67, 533–536. Lawton, K., Friedrich, L., Hunt, M., Weymann, K., Delaney, T., Kessmann, H., Staub, T., Ryals, J., 1996. Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway. Plant J. 10, 71–82. Leon, J., Lawton, M., Raskin, I., 1995. Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Plant Physiol. 108, 1673–1678. Linthorst, H., Meutwissen, R., Kaufman, S., Bol, J., 1989. Constitutive expression of pathogenesis-related proteins PR-1, GRP and PR-S in tobacco has no effect on virus infection. Plant Cell 1, 285–291. Metraux, J.-P., Streit, L., Staub, T., 1988. A pathogenesis-related protein in cucumber is a chitinase. Physiol. Mol. Plant Pathol. 33, 1–9. Metraux, J.-P., Signer, H., Ryals, J., Ward, E., Wyss-Benz, E.M, Gaudin, J., Raschdorf, K., Schmid, E., Blum, W., Inverardi, B.,
1990. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250, 1004–1006. Meuwly, P., Molders, W., Buchala, A., Metraux, J.-P., 1995. Local and systemic biosynthesis of salicylic acid in infected cucumber plants. Plant Physiol. 109, 1107–1114. Nameth, S., Dodds, J., Paulus, A., Laemmlen, F., 1986. Cucurbit viruses of California: an ever-changing problem. Plant Dis. 70, 8–12. Naylor, M., Murphy, A.M., Berry, J.O., Carr, J.P., 1998. Salicylic acid can induce resistance to plant virus movement. Mol. Plant Microbe Interac. 11, 860–868. Neuenschwander, U., Vernooij, B., Friedrich, L., Uknes, S., Kessmann, H., Ryals, J., 1995. Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance? Plant J. 8, 227–233. Raskin, I., Ehmann, A., Melander, W., Meeuse, B., 1987. Salicylic acid: a natural inducer of heat production in Arum lilies. Science 257, 1601–1602. Rhoads, D., McIntosh, L., 1993. Salicylic acid-inducible alternative oxidase gene Aox1 and genes encoding pathogenisis-related proteins share regions of sequence similarity in their promoters. Plant Mol. Biol. 21, 615–624. Scarponi, L., Buonaurio, R., Martinetti, L., 2001. Persistence and translocation of a benzothiadiazole derivative in tomato plants in relation to systemic acquired resistance against Pseudomonas syringae pv tomato. Pest Manag. Sci. 57, 262–268. Shulaev, V., Leon, J., Raskin, I., 1995. Is salicylic acid a translocated signal of systemic acquired resistance in tobacco? Plant Cell 7, 1691–1701. Smith-Becker, J., Marois, E., Huguet, E.J., Midland, S., Sims, J.J., Keen, N., 1998. Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems. Plant Physiol. 116, 231–238. Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S., Dincher, S., Chandler, D., Slusarenko, A., Ward, E., Ryals, J., 1992. Acquired resistance in Arabidopsis. Plant Cell 4, 645–656. Van Der Straeten, D., Chaerle, L., Sharkov, G., Lambers, H., Van Montagu, M., 1995. Salicylic acid enhances the activity of the alternative pathway of respiration in tobacco leaves and induces thermogenicity. Planta 196, 412–419. Ward, E., Uknes, S., Williams, S., Dincher, S., Wiederhold, D., Alexander, D., Ahl Goy, P., Metraux, J.-P., Ryals, J., 1991. Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3, 1085–1094. White, R., Antoniw, J., Carr, J., Woods, R., 1983. The effects of aspirin and polyacrylic acid on the multiplication and spread of TMV in different cultivars of tobacco with and without the N-gene. Phytopathol. Z. 107, 224–232.
Acibenzolar-S-methyl induces resistance to Colletotrichum lagenarium and cucumber mosaic virus in cantaloupe J. Smith-Beckera, N.T. Keena,{, J.O. Beckerb,* a
Department of Plant Pathology, University of California, Riverside, CA 92521, USA b Department of Nematology, University of California, Riverside, CA 92521, USA Received 22 May 2002; accepted 17 February 2003
Abstract Acibenzolar-S-methyl (ASM) is a synthetic analogue of salicylic acid developed for use in a novel strategy for crop protection through systemic acquired resistance (SAR). ASM protected cantaloupe against a fungal pathogen, Colletotrichum lagenarium, and a viral pathogen, cucumber mosaic virus (CMV). ASM induced the systemic accumulation of chitinase, a marker protein for SAR, in both greenhouse and field grown seedlings. ASM at 50 or 100 mg/ml provided almost complete protection against the fungal pathogen and effectively delayed the spread of CMV in greenhouse trials. In two field trials, ASM had no negative effect on fruit production in the absence of CMV disease pressure. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Acibenzolar-S-methyl; Cantaloupe; CMV; Chitinase; Salicylic acid; SAR
1. Introduction Systemic acquired resistance (SAR) involves the activation of natural defense mechanisms in plants prior to pathogen attack (Kuc, 1982). In biologically induced resistance, necrosis caused by pathogens results in the synthesis of salicylic acid (SA) both at the site of infection and systemically in uninfected tissues (Meuwly et al., 1995; Shulaev et al., 1995). The pathogen induced signal that elicits SA synthesis is unknown, although reactive oxygen compounds such as hydrogen peroxide and superoxide have been shown to induce SA synthesis in several systems (Leon et al., 1995; Neuenschwander et al., 1995; Enyedi, 1999). Considerable evidence suggests that SA is the signal molecule that activates the synthesis of pathogen defense related proteins (PR proteins) in plant foliage (Ward et al., 1991; Uknes et al., 1992; Gaffney et al., 1993). The PR proteins provide useful markers for the establishment of SAR, since the level of accumulation of these proteins is correlated to the level of SAR induced. PR proteins shown to *Corresponding author. Tel.: +1-909-787-2185; fax: +1-909-7873719. E-mail address: [email protected] (J.O. Becker). { N. Keen passed away on April 18, 2002.
accumulate during SAR in cucurbits include chitinase, peroxidase and X -glucanase (Metraux et al., 1988; Hammerschmidt et al., 1982; Ji and Kuc, 1995). Chitinase is a particularly useful marker for SAR, since levels of the enzyme in non-induced tissues are low. Acibenzolar-S-methyl (1,2,3-benzothiadiazole-7-carbothioic acid S-methyl ester, ASM) mimics the effects of SA and has been developed for use as a crop protectant (Gorlach et al., 1996; Kunz et al., 1997). This compound offers promise as an alternative method to conventional disease control with pesticides. ASM has the same spectrum of activity as biologically induced resistance, and has been shown effective in protecting several crop species and Arabidopsis from fungal, bacterial and viral pathogens (Friedrich et al., 1996; Gorlach et al., 1996; Lawton et al., 1996). Huang et al. (2000) demonstrated that one application of ASM prior to flowering protected rock and Hami melon fruit from several post-harvest fungal diseases. Although ASM protects tobacco against tobacco mosaic virus (TMV), and Arabidopsis against turnip crinkle virus, no studies have reported efficacy against melon viral diseases. Mosaic diseases caused by arthropod-borne viruses pose one of the greatest threats to melon production. In California, the four major aphid-transmitted viruses are watermelon mosaic potyvirus 2, papaya ringspot
0261-2194/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0261-2194(03)00044-9
770
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potyvirus (watermelon strain), zucchini yellow mosaic potyvirus and cucumber mosaic cucumovirus (Nameth et al., 1986; Grafton-Cardwell et al., 1996). Infected melons often develop typical mosaic disease symptoms such as clearing of veins, systemic chlorotic speckling, stunting of leaves and inhibited fruit development. Control of viral pathogens is currently limited to the use of resistant varieties and control of the insect vectors. In this work we tested the activity of ASM in cantaloupe against cucumber mosaic virus (CMV), and Colletotrichum lagenarium, causal agent of anthracnose. C. lagenarium has been used extensively in studies of SAR in cucumber and melon (Hammerschmidt et al., 1982; Metraux et al., 1990). It produces slowly spreading necrotic lesions that are easily quantified in SAR assays.
2. Materials and methods 2.1. Culture of pathogens CMV was maintained and increased in cantaloupe (Cucumis melo L. cv. Durango, Petoseed, Saticoy, CA). Inocula were prepared from young symptomatic leaves by homogenizing leaf tissue in 20 mm potassium phosphate buffer, pH 7.0. Homogenates were diluted 1:50 (fresh weight/volume buffer) and Celite (0.5% w/v) was added prior to inoculation. C. lagenarium (Pass.) Ellis and Halsted race 1 was cultured on potato dextrose agar (PDA) at 18 C in the dark. Conidial suspensions for use as inoculum were prepared from 7 to 10 day-old cultures as described previously (Kuc and Richmond, 1977). 2.2. Plant culture and inoculations All experiments were conducted in the greenhouse and repeated at least twice. Each treatment contained 10–15 plants individually potted in 4 in2 peat pots. All the leaves of three-week-old cantaloupe seedlings were sprayed with ASM (Actigard, Syngenta, Greensboro, NC, USA) at a concentration of active compound of 25, 50 or 100 mg/ml in water until runoff. ASM stock solutions were prepared from a wettable powder containing 50% active compound. Four days after the inducing treatments, all seedlings were inoculated on the second leaf with either CMV or C. lagenarium. CMV was inoculated by gently rubbing 100 ml of infected leaf homogenate onto one-half of the leaf. Fungal spores were applied by placing ten 10 ml drops of a 105 spores/ ml suspension on leaves. C. lagenarium inoculated plants were placed in plastic bags in the dark for 18 h after inoculation to allow spore germination. Viral and fungal symptoms were evaluated 8 and 10 days, respectively, after inoculation. In addition, CMV titer in the youngest expanded leaves was determined using polyclonal
antibodies to the virus in an indirect ELISA assay (Fang and Grumet, 1993). The ability of ASM to protect seedlings at the cotyledon stage was also evaluated. We were unable to infect with CMV through mechanical inoculation of cotyledons, so SAR in the first true leaves was evaluated using the fungal pathogen. One-week-old seedlings were sprayed with 10, 20 or 40 mg/ml ASM. Seven days after treatment, the first true leaves were inoculated with five drops of the fungal spore suspension as described above. 2.3. Measurement of chitinase The optimal concentration of ASM needed to induce SAR in cantaloupe was first determined by measuring the levels of the SAR marker protein chitinase in leaves 5 days after treatment with 25, 50 or 100 mg/ml ASM. Chitinase levels were measured in the second true leaves of seedlings using antibodies prepared to cucumber chitinase in a sandwich ELISA assay as described previously (Smith-Becker et al., 1998). Two leaf discs, 1.5 cm diameter, were collected from each leaf and stored at 80 C prior to assay. In experiments using one-week-old seedlings, chitinase was measured in the first true leaf 7 days after treatment. 2.4. Field trials Two field trials were conducted at the UCR Coachella Valley Agricultural Research Station, Thermal, CA. Melon production in that area is often severely affected by one or more viruses. The soil type was a Carsetas loamy sand (84% sand, 5% clay, and 0.8% organic matter, pH 7.3). Plot design was a randomized complete block with 4 treatments and 5 replications. The beds were one m wide with 20 plants per plot, spaced 0.3 m apart. Cantaloupe (cv. Durango) was seeded into nontreated soil and grown with a low-volume irrigation setup which provided both water and fertilizer according to local standard practice. In the first trial, the treatments consisted of a non-treated control and a single ASM foliar application at three weeks after seeding. Seedlings were sprayed with ASM at a rate of 37.5 mg/ml until runoff. Chitinase accumulation was measured in each treatment two weeks after the inducing inoculations. Two 1.5 cm diameter leaf discs were collected from the second true leaf of each of 5 randomly selected plants per plot. Leaf discs were placed immediately into 1.5 ml microfuge tubes on dry ice and stored at 80 C prior to chitinase assay. In the second trial, the four treatments consisted of a non-treated control, a single ASM treatment at 100 mg/ml, three ASM applications at 100 mg/ml in two week intervals and three applications at 50 mg/ml in two week intervals, each starting three weeks after seeding. At harvest, the total number of fruits, the number of marketable fruits and the weight of
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marketable fruits was determined. All data were subject to analysis of variance and mean separation with Fisher’s protected least significant difference.
771
ASM concentrations as low as 10 mg/ml were as effective as higher concentrations at inducing chitinase accumulation in the first leaf (Fig. 3). ASM concentrations higher than 50 mg/ml caused phytotoxicy in one-weekold seedlings.
3. Results 3.2. Protection against C. lagenarium and CMV 3.1. Chitinase accumulation The accumulation of chitinase increased with increasing concentrations of ASM (Fig. 1). ASM at 100 mg/ml induced the highest level of chitinase without causing any phytotoxic effects under greenhouse conditions, and this concentration was used for subsequent greenhouse experiments with three-week-old seedlings. Chitinase levels in treated leaves increased for at least the first 4 days after treatment (Fig. 2). In one-week-old seedlings,
Chitinase (ng/disc)
150
ASM at 50 or 100 mg/ml provided almost complete protection against the fungal pathogen (Fig. 4). ASM also provided protection against the viral pathogen CMV. This resistance was observed as an absence of viral symptoms 8 days after inoculation in ASM treated plants and a reduced viral titer in young leaves (Fig. 5). ASM treated plants first began to show viral symptoms 14 days after inoculation, indicating that several applications of ASM may be necessary to provide extended protection. ASM treatment of cotyledons was highly effective against C. lagenarium, and no lesions developed on the first true leaves of seedlings treated on the cotyledons with 10, 20 or 40 mg/ml.
100
3.3. Field trials
50
0 0
25
50
100
ASM (µg/ml) Fig. 1. Effect of ASM concentration on chitinase accumulation. Three-week-old cantaloupe seedlings were sprayed with 25, 50 or 100 mg/ml ASM. Chitinase was measured in discs from the second true leaves 5 days after treatment. Bars indicate standard error.
ASM caused a significant increase in chitinase accumulation in the field-grown plants compared to the non-treated control (Fig. 6). Despite the more stressful growing conditions in the field, the levels of chitinase in non-treated field-grown plants were no higher than in control plants grown in the greenhouse. None of the treatments in the first trial had a measurable effect on the cantaloupe yield (data not shown). In the 80
Chitinase (ng/disc)
60
40
20
Fig. 2. Time course of chitinase accumulation in response to ASM. Three-week-old cantaloupe seedlings were sprayed with water (control) or 100 mg/ml ASM. Chitinase levels were measured in the second leaves of treated plants for 4 days. Bars indicate standard error.
ASM (40 µg/ml)
ASM (20 µg/ml)
ASM (10 µg/ml)
Control
0
Fig. 3. Chitinase accumulation in young seedlings in response to ASM. One-week-old seedlings were sprayed on the cotyledons with 10, 20 or 40 mg/ml ASM. Chitinase was measured in the first true leaves 7 days after treatment. Bars indicate standard error.
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10
100
7.5
75 Chitinase ng/disc
Number of Lesions
772
5
2.5
50
25
Fig. 4. ASM induced resistance to C. lagenarium. Three-week-old greenhouse grown seedlings were sprayed with ASM (100 mg/ml). Four days after treatment, seedlings were inoculated on leaf 2 with 10 drops of a C. lagenarium spore suspension. C. lagenarium lesions were counted 10 days after inoculation. Bars indicate standard error.
0.2
Absorbance 405
0.15
ASM
0 Control
ASM (100 µg/ml)
ASM (50 µg/ml)
Control
0
Fig. 6. Effect of ASM on chitinase accumulation in field-grown plants. Three-week-old field grown plants were sprayed with 37.5 mg/ml ASM. Leaf discs for chitinase assay were collected from the second leaves two weeks after the inducing treatment. Bars indicate standard error.
second trial, repeated treatments with ASM had no deleterious effect on the quantity or quality of fruit production and a single treatment of 100 mg/ml resulted in a significant increase in the number of marketable fruit (Table 1). No obvious CMV symptoms were observed in both field trials. Thus, although the marker protein chitinase indicated that plants in the field were induced, we were unable to evaluate the level of viral resistance.
4. Discussion 0.1
0.05
ASM (100 µg/ml)
ASM (50 µg/ml)
Control
0
Fig. 5. ASM induced resistance to CMV. Three-week-old greenhouse grown seedlings were sprayed with water (control) or ASM at 50 or 100 mg/ml. Four days after treatment, seedlings were inoculated on leaf 2 with CMV. Eight days after CMV inoculation, the youngest expanded leaves (leaves 4 or 5) were collected and assayed for CMV using an indirect ELISA assay. Bars indicate standard error.
ASM induced accumulation of the SAR marker protein chitinase and provided almost complete protection against C. lagenarium in cantaloupe. One application of ASM also effectively delayed the spread of the viral pathogen CMV in greenhouse trials. Although several PR proteins have been identified as enzymes with potential antimicrobial activity against bacteria and fungi, none have been associated with a function related to viral inhibition (Linthorst et al., 1989). Prior to the discovery of SA as an endogenous signal molecule, a derivative of SA, acetylsalicylic acid, was shown to greatly reduce the accumulation of TMV in a susceptible tobacco cultivar (Antoniw and White, 1980; White et al., 1983). Consistent with the function of ASM as an analogue of SA, Friedrich et al. (1996) showed that ASM treatment of susceptible tobacco resulted in reduced levels of TMV RNAs in inoculated plants.
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773
Table 1 Effect of foliar applications of ASM on cantaloupe harvested from a field trial in Californiaa Treatment (number of applications)
Total number of fruit/plot
Number of marketable fruitb
Weight (kg) of marketable fruit
Non-treated check 100 mg/ml ASM (1) 100 mg/ml ASM (3) 50 mg/ml ASM (3)
31.671.6a 31.271.4a 33.872.2a 32.872.1a
9.070.5a 11.971.2b 10.370.4ab 10.170.7ab
0.5570.04a 0.5770.03a 0.5670.02a 0.6070.02a
a
Data are means7std error of 5 replicate plots. Means of a given columns followed by the same letter are not significantly different (Pp0:05) according to Fisher’s protected least significant difference. b Fruit diameter>12 cm.
One of the first biological roles described for SA in plant metabolism was its ability to induce the alternate respiratory pathway in the thermogenic Arum lily (Raskin et al., 1987). SA also induced cyanide resistant respiration and thermogenicity in tobacco, indicative of the activation of the alternative respiration pathway (Kapulnik et al., 1992; Van Der Straeten et al., 1995). Rhoads and McIntosh, (1993) showed that the promoter of the SA inducible alternative oxidase (AOX) in tobacco shared sequence similarity to promoters of SA inducible PR proteins, suggesting that the AOX is regulated coordinately with other defense related proteins during SAR. Chivasa et al. (1997) investigated the possibility that SA induced resistance to TMV in tobacco is dependent on the known effects of SA on the AOX. They demonstrated that salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial AOX, compromised SA induced resistance to TMV without affecting induced resistance to a bacterial or fungal pathogen. In addition, SHAM treatment did not reduce the level of PR-1 protein accumulation in response to SA. The authors suggest that SA induced resistance to viruses is distinct from resistance to fungal and bacterial pathogens in the requirement for AOX activity or other as yet unidentified SHAM sensitive activity. In order to study the mechanism of SA induced resistance to viruses, Naylor et al. (1998) investigated the effects of SA treatment on the replication and movement of potato virus X (PVX) and CMV in tobacco. SA treatment reduced the replication of PVX in inoculated leaves, but had no effect on CMV replication. However, plants treated with SA showed delayed symptoms of CMV, and the authors were able to attribute this delay to an inhibition of systemic movement of the virus. Surprisingly, SHAM treatment effectively eliminated the resistance induced by SA to both PVX and CMV. Thus the ability of SA to inhibit the replication of certain viruses and the movement of others is somehow linked through a SHAM sensitive mechanism. It is likely that the mode of action of ASM in reducing CMV spread in melon is related to the ability of SA to reduce CMV movement in tobacco. While we were not able to confirm the efficacy of ASM in the field due to the lack of disease pressure, the
levels of chitinase measured in field-grown plants demonstrated that ASM application induced SAR to similar levels as in greenhouse trials. ASM treatment also had no negative impact on fruit quality or quantity, even when applied several times throughout the growing season. In tomato, ASM residues were detected in tissues only during the first 72 h after treatment (Scarponi et al., 2001). Similarly, a detailed study of ASM residues in pepper showed that the compound was undetectable by 5 days after treatment (Buonaurio et al. 2002). Although repeated booster applications of ASM may prove advantageous for an extended resistance period, early protection has the most important impact economically. Plants infected early, before fruit set, produce fewer flowers and may abort fruits, resulting in extensive production losses (Nameth et al., 1986). ASM may therefore become an important component of an integrated pest management approach to reduce viral and fungal disease impact on melons.
Acknowledgements This study was supported in part by a grant from the California Melon Research Board.
References Antoniw, J., White, R., 1980. The effects of aspirin and polyacrylic acid on soluble leaf proteins and resistance to virus infection in five cultivars of tobacco. Phytopathol. Z. 99, 331–341. Buonaurio, R., Scarponi, L., Ferrara, M., Sidoti, P., Bertona, A., 2002. Induction of systemic acquired resistance in pepper plants by acibenzolar-S-methyl against bacterial spot disease. Eur. J. Plant Pathol. 108, 41–49. Chivasa, S., Murphy, A., Naylor, M., Carr, J., 1997. Salicylic acid interferes with tobacco mosaic virus replication via a novel salicylhydroxamic acid-sensitive mechanism. Plant Cell 9, 547–557. Enyedi, A.J., 1999. Induction of salicylic acid biosynthesis and systemic acquired resistance using the active oxygen species generator rose bengal. J. Plant Physiol. 154, 106–112. Fang, G., Grumet, R., 1993. Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene. Mol. Plant-Microbe Interact. 6, 358–367. Friedrich, L., Lawton, K., Ruess, W., Masner, P., Specker, N., Gut-Rella, M., Mejer, B., Dincher, S., Staub, T., Uknes, S., Metraux, J-P., Kessmann, H., Ryals, J., 1996. A benzothiadiazole
774
J. Smith-Becker et al. / Crop Protection 22 (2003) 769–774
derivative induces systemic acquired resistance in tobacco. Plant J. 10, 61–70. Gaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, G., Uknes, S., Ward, E., Kessmann, H., Ryals, J., 1993. Requirement of salicylic acid for the induction of systemic acquired resistance. Science 261, 754–756. Gorlach, J., Volrath, S., Knauf-Beiter, G., Hengy, G., Beckhove, U., Kogel, K.-H., Oostendorp, M., Staub, T., Ward, E., Kessmann, H., Ryals, J., 1996. Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. Plant Cell 8, 629–643. Grafton-Cardwell, E., Perring, T., Smith, R., Valencia, J., Farrar, C., 1996. Occurrence of mosaic viruses in melons in the Central Valley of California. Plant Dis. 80, 1092–1097. Hammerschmidt, R., Nuckles, E.M., Kuc, J., 1982. Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol. Plant Pathol. 20, 73–82. Huang, Y., Deverall, B.J., Tang, W.H., Wang, W., Wu, F.W., 2000. Foliar application of acibenzolar-S-methyl and protection of postharvest rock melons and Hami melons from disease. Eur. J. Plant Pathol. 106, 651–656. Ji, C., Kuc, J., 1995. Purification and characterization of an acidic B-1,3-glucanase from cucumber and its relationship to systemic disease resistance induced by Colletotrichum lagenarium and tobacco necrosis virus. Mol. Plant Microbe Interact. 8, 899–905. Kapulnik, Y., Yalpani, N., Raskin, I., 1992. Salicylic acid induces cyanide-resistant respiration in tobacco cell-suspension cultures. Plant Physiol. 100, 1921–1926. Kunz, W., Schurter, R., Maetzke, T., 1997. The chemistry of benzothiadiazole plant activators. Pestic. Sci. 50, 275–282. Kuc, J., 1982. Induced immunity to plant disease. Bioscience 32, 854–860. Kuc, J., Richmond, S., 1977. Aspects of the protection of cucumber against Colletotrichum lagenarium by Colletotrichum lagenarium. Phytopathology 67, 533–536. Lawton, K., Friedrich, L., Hunt, M., Weymann, K., Delaney, T., Kessmann, H., Staub, T., Ryals, J., 1996. Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway. Plant J. 10, 71–82. Leon, J., Lawton, M., Raskin, I., 1995. Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Plant Physiol. 108, 1673–1678. Linthorst, H., Meutwissen, R., Kaufman, S., Bol, J., 1989. Constitutive expression of pathogenesis-related proteins PR-1, GRP and PR-S in tobacco has no effect on virus infection. Plant Cell 1, 285–291. Metraux, J.-P., Streit, L., Staub, T., 1988. A pathogenesis-related protein in cucumber is a chitinase. Physiol. Mol. Plant Pathol. 33, 1–9. Metraux, J.-P., Signer, H., Ryals, J., Ward, E., Wyss-Benz, E.M, Gaudin, J., Raschdorf, K., Schmid, E., Blum, W., Inverardi, B.,
1990. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250, 1004–1006. Meuwly, P., Molders, W., Buchala, A., Metraux, J.-P., 1995. Local and systemic biosynthesis of salicylic acid in infected cucumber plants. Plant Physiol. 109, 1107–1114. Nameth, S., Dodds, J., Paulus, A., Laemmlen, F., 1986. Cucurbit viruses of California: an ever-changing problem. Plant Dis. 70, 8–12. Naylor, M., Murphy, A.M., Berry, J.O., Carr, J.P., 1998. Salicylic acid can induce resistance to plant virus movement. Mol. Plant Microbe Interac. 11, 860–868. Neuenschwander, U., Vernooij, B., Friedrich, L., Uknes, S., Kessmann, H., Ryals, J., 1995. Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance? Plant J. 8, 227–233. Raskin, I., Ehmann, A., Melander, W., Meeuse, B., 1987. Salicylic acid: a natural inducer of heat production in Arum lilies. Science 257, 1601–1602. Rhoads, D., McIntosh, L., 1993. Salicylic acid-inducible alternative oxidase gene Aox1 and genes encoding pathogenisis-related proteins share regions of sequence similarity in their promoters. Plant Mol. Biol. 21, 615–624. Scarponi, L., Buonaurio, R., Martinetti, L., 2001. Persistence and translocation of a benzothiadiazole derivative in tomato plants in relation to systemic acquired resistance against Pseudomonas syringae pv tomato. Pest Manag. Sci. 57, 262–268. Shulaev, V., Leon, J., Raskin, I., 1995. Is salicylic acid a translocated signal of systemic acquired resistance in tobacco? Plant Cell 7, 1691–1701. Smith-Becker, J., Marois, E., Huguet, E.J., Midland, S., Sims, J.J., Keen, N., 1998. Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems. Plant Physiol. 116, 231–238. Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S., Dincher, S., Chandler, D., Slusarenko, A., Ward, E., Ryals, J., 1992. Acquired resistance in Arabidopsis. Plant Cell 4, 645–656. Van Der Straeten, D., Chaerle, L., Sharkov, G., Lambers, H., Van Montagu, M., 1995. Salicylic acid enhances the activity of the alternative pathway of respiration in tobacco leaves and induces thermogenicity. Planta 196, 412–419. Ward, E., Uknes, S., Williams, S., Dincher, S., Wiederhold, D., Alexander, D., Ahl Goy, P., Metraux, J.-P., Ryals, J., 1991. Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3, 1085–1094. White, R., Antoniw, J., Carr, J., Woods, R., 1983. The effects of aspirin and polyacrylic acid on the multiplication and spread of TMV in different cultivars of tobacco with and without the N-gene. Phytopathol. Z. 107, 224–232.