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Salicylic acid accumulates in the roots and hypocotyl after inoculation of cucumber leaves with Colletotrichum lagenarium
ARTICLE IN PRESS Journal of Plant Physiology 163 (2006) 1111—1117
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Salicylic acid accumulates in the roots and hypocotyl after inoculation of cucumber leaves with Colletotrichum lagenarium Masaharu Kubota, Kazufumi Nishi Laboratory of Plant Pathology, Department of Fruit Vegetables, National Institute of Vegetable and Tea Science (NIVTS), 360 Kusawa, Ano, Mie 514-2392 Japan Received 13 June 2005; accepted 26 September 2005
KEYWORDS Colletotrichum lagenarium; Cucumis sativus; Hypocotyl; Root; Salicylic acid
Summary Increased amounts of salicylic acid (SA) were detected in the roots and hypocotyl of cucumber plants (Cucumis sativus) using high-performance liquid chromatography following inoculation of the leaves with the anthracnose pathogen, Colletotrichum lagenarium. The concentrations of SA in the internodes immediately below the infected leaves increased to more than 1 mg/g fresh weight. In contrast, the concentrations of SA in stems distant from, or above the infected leaves increased to 100–300 ng/g. An increase in SA levels was observed in the upper stem 2 d after inoculation, followed by the hypocotyl with an increase detected 4 d after inoculation. An initial increase in the SA levels was detected in the stem, followed by an increase in SA levels in the root from a basal level of approximately 300 ng/g to more than 1 mg/g. The increased level of SA in the lower leaves was less than 100 ng/g. These results indicate that the levels of SA in the hypocotyl and root increased significantly following inoculation of the leaves with a microorganism capable of inducing SAR. & 2005 Elsevier GmbH. All rights reserved.
Introduction Abbreviation: HPLC, High-performance liquid chromatography; PHSA, Potato homogenate sucrose agar; SA, Salicylic acid; SAR, Systemic-acquired resistance Corresponding author. Tel.: +81 59 268 4641; fax: +81 59 268 1339. E-mail address: [email protected] (M. Kubota).
When plants are necrotized by biotic or abiotic agents they systemically develop resistance to diseases, which is known as systemic-acquired resistance (SAR) (Sticher et al., 1997). SAR is effective against a wide range of pathogens, including fungi, bacteria and viruses (Sticher
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.09.005
ARTICLE IN PRESS 1112 et al., 1997). Numerous reports have described SAR in leaves or stems located above the treated leaves (Sticher et al., 1997), and some cases of downward SAR have also been reported (Gessler and Kuc´, 1982; Guedes et al., 1980; Kubota and Abiko, 2000, 2001; Madi and Katan, 1998; Reglinski et al., 2001; Tjamos, 1979; Xue et al., 1998). In previous studies, salicylic acid (SA) was produced in leaves infected with SAR-inducing microbes and was exported systemically (Sticher et al., 1997). It was also produced in distant parts of the plant to those that were infected (Sticher et al., 1997). SA was required in the protected parts of tobacco plants as an endogenous signal, but it did not act as a systemic signal (Delaney et al., 1994; Gaffney et al., 1993; Pallas et al., 1996; Vernooij et al., 1994). However, the role of SA in the protected organs has been doubted in tomato and Arabidopsis plants (Cameron et al., 1999; Coquoz et al., 1995; Jeun et al., 2000). Induced SAR and systemic SA accumulation in the upper stem of cucumber plants have been observed following infection of the leaves with the anthracnose pathogen, Colletotrichum lagenarium (Me´traux et al., 1990; Sticher et al., 1997). However, this pathogen has not been used subsequently to induce SA accumulation. Infecting the leaves with this pathogen also induced resistance in the lower parts of cucumber plants (Gessler and Kuc´, 1982; Guedes et al., 1980; Kubota and Abiko, 2000, 2001). Until now, only Chen et al. (1999) reported increased amounts of SA in the roots of SAR-induced cucumber plants. They detected an increase in the levels of SA in a region of the plant distant from the root which was inoculated with a resistance-inducible bacterium. However, an increase in the levels of SA in the lower parts of the plant, including the hypocotyl and root, following inoculation of the leaves, has not been shown. In the present study, we describe SA accumulation in the root, hypocotyl, leaves and stem of cucumber plants after inoculation of cotyledons, first leaves or second leaves with C. lagenarium.
Materials and methods Plant and fungal inoculum Cucumber plants (Cucumis sativus L.) cultivar Shimoshirazuzihai were grown in pots (diameter: 9 cm) containing compost (Kureha Chem. Ind., Tokyo, Japan) in a chamber at 25 1C with 12 h of daylight. Colletotrichum lagenarium (Passerini)
M. Kubota, K. Nishi Ellis et Halsted strain 104-T, was incubated on potato homogenate sucrose agar (PHSA; 20% boiled potato homogenate, 2% sucrose and 3% cooking agar) at 24 1C for 5–8 d. A conidial suspension was created in water by rubbing the surface of the colony with a glass rod, followed by centrifugation at 1000g for 5 min. Conidia were washed once with water, recentrifuged and finally suspended in water at 107 conidia/mL for use as inoculum.
Inoculation with C. lagenarium and extraction of salicylic acid Ten drops of the inoculum were placed onto the cotyledons of cucumber plants 6–14 d after sowing. Samples (0.3 g) of the first leaf, hypocotyl and root were excised 14 d after sowing (0–8 d after the inoculation) and were heated for 10 min in 3 mL of boiled water containing 2% acetic acid (Nakayama et al., 1996). After inoculation with 20 drops of the conidial suspension on the first leaf of 11- to 19-dold plants, extracts from the second leaf, hypocotyls and roots were prepared in 2% acetic acid 19 d after sowing (0–8 d after the inoculation). In a further experiment, following inoculation of the second leaf of 14- to 22-d-old plants, extracts from the first leaf, third leaf, hypocotyl, and the internodes between the first and third leaves were prepared 22 d after sowing (0–8 d after the inoculation).
Conditions for high-performance liquid chromatography (HPLC) Extracts were filtered with 0.25-mm mesh and then analyzed using HPLC. The extract (50 mL) was injected into a C-18 reverse-phase column (diameter length: 4.6 250 mm) (Wakosil-II 5C18 HC, Wako Pure Chem. Ind., Osaka, Japan, or Shodex C18 M 4E, Shoko, Tokyo, Japan) at 40 1C and equilibrated in 50 mM sodium acetate containing 25% methanol flowing at 1 mL/min, and detected using a fluorescence spectrometer (821-FP; JASCO International, Tokyo, Japan) with an excitation wavelength of 290 nm and an emission of 402 nm. The peak areas on the chromatograph were analyzed using a recorder (Chromatocorder 21J; System Instruments, Tokyo, Japan). The amount of SA was calculated in comparison to 10–7 g/mL (v/w) SA. In the extraction, only free SA was detected (Nakayama et al., 1996) and the detection limit was approximately 10 ng/g of intact tissues. The peak of SA was detected about 6 min after injection of the sample into the HPLC.
ARTICLE IN PRESS Salicylic acid in roots and hypocotyl after inoculation of leaves
Results Accumulation of SA after inoculation of the cotyledons SA levels increased in the first leaf and hypocotyl 4 d after inoculation (Fig. 1). When compared to the levels of SA in the mock-inoculated plants, a significant increase was observed in the levels of SA in the hypocotyl after 5 d and in the first leaf after 6 d following inoculation with C. lagenarium. In the root, levels of SA increased 6 d after the inoculation with C. lagenarium. The SA concentration increased to approximately 600 ng/g of tissue in the first leaf, and to approximately 2 mg/g of tissue in the hypocotyl and root. In the root of the control plants, the basal amount of SA was greater than that found in the first leaf and hypocotyl, reaching approximately 300 ng/g.
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Accumulation of SA after inoculation of the first leaf Concentrations of SA in the second leaf, hypocotyl and root noticeably increased 4 d after inoculation. The difference in concentration of SA between the inoculated and mock-inoculated plants was statistically significant in the hypocotyl, second leaf and root after 3, 4 and 5 d, respectively (Fig. 2). In the second leaf, 7 d after the inoculation, the concentration of SA had increased to about 2.5 mg/g. Similar levels of SA were detected in the hypocotyl and root.
Accumulation of SA after inoculation of the second leaf The level of SA did not increase significantly in the first leaf until 8 d after inoculation of the second leaf
Figure 1. SA accumulation after inoculation of the cotyledons with C. lagenarium. SA levels in: A, first leaf; B, hypocotyl; C, root. E, inoculated; m, mock-inoculated; * indicates a significant difference (Po0:05) between the inoculated and mock-inoculated plants. The bars indicate standard errors.
Figure 2. SA accumulation after inoculation of the first leaf with C. lagenarium. SA levels in: A, second leaf; B, hypocotyl; C, root. E, inoculated; m, mock-inoculated; * indicates a significant difference (Po0:05) between the inoculated and mock-inoculated plants. The bars indicate standard errors.
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Figure 3. SA accumulation after inoculation of the second leaf with C. lagenarium. SA levels in: A, first leaf; B, third leaf. E, inoculated; m, mock-inoculated; * indicates a significant difference (Po0:05) between the inoculated and mock-inoculated plants. The bars indicate standard errors.
Figure 4. SA accumulation after inoculation of the second leaf with C. lagenarium. SA levels in: A, internode between the third and second leaf; B, internode between the second and first leaf; C, hypocotyl. E, inoculated; m, mockinoculated; * indicates a significant difference (Po0:05) between the inoculated and mock-inoculated plants. The bars indicate standard errors.
when compared to the control plants (Fig. 3). In the third leaf, the amount of SA increased after 3 d and this increase became significant 4 d after inoculation with levels of approximately 400 ng/g. In the internode between the third and second leaf, the level of SA increased to about 200 ng/g 2 d after inoculation (Fig. 4). In the internode between the second and first leaves, the concentration of SA increased to more than 1 mg/g 2 d after inoculation. Finally, in the hypocotyl, the increased SA level stabilized at approximately 100 ng/g 4 d after inoculation.
Discussion In this study, we extracted free SA by boiling plant tissues in 2% acetic acid (Nakayama et al.,
1996). The SA detected in the leaves and stems of the SAR-induced cucumber plants was either produced there or was transported from the necrotized leaves (Meuwly and Me´traux, 1993; Meuwly et al., 1995; Mo ¨lders et al., 1996; SmithBecker et al., 1998). The production of SA in the roots following inoculation with Pseudomonas spp. into the roots has been previously suggested (Chen et al., 1999). Glucoside SA has also been detected in the leaves and phloem of cucumber plants (Meuwly and Me´traux, 1993; Meuwly et al., 1995; Mo ¨lders et al., 1996). However, the conjugated SA was inactive in SAR induction (Meuwly et al., 1995; Sticher et al., 1997), and we interpreted that the detection of free SA was sufficient to evaluate the SAR induction. The detection limit of this method was approximately 10 ng/g fresh weight of plant tissue, a sufficient level to recognize increases in
ARTICLE IN PRESS Salicylic acid in roots and hypocotyl after inoculation of leaves the SA concentration in the immunized cucumber plants. We previously reported SAR in the root and hypocotyl of cucumber plants after infection of the leaves with C. lagenarium (Kubota and Abiko, 2000, 2001). Our present results confirm the accumulation of SA in the root and hypocotyl below the inoculated leaves, although the increase in the lower leaf was not significant. SA accumulation in cucumber roots after inoculation with Pseudomonas lachrymans into the cotyledons was mentioned by Meuwly et al. (1994), but was not described in detail. The increase in concentration of SA in the root occurred subsequently to the increase in the upper leaves and stem. The increase in concentration of SA in the hypocotyl also occurred subsequent to the increases observed in the upper stem and in the internodes immediately below the inoculated leaves. There appeared to be a physiological barrier to signal transduction between the hypocotyl and the upper parts of the plant. The time sequence of increases in SA in all of the stem parts, except the hypocotyl, was similar to that induced in the upper stem using the same pathogen, as described by Me´traux et al. (1990). The increase in concentration of SA in the upper leaves also occurred later than in the stem parts (Me´traux et al., 1990), possibly suggesting a time lag in the translocation of the signal. The transport of signals to the lower, lateral and opposite sides of plants to induce SAR and SA accumulation has suggested the involvement of pathways other than the vascular bundles (Chen et al., 1999; Gessler and Kuc´, 1982; Guedes et al., 1980; Jenns and Kuc ´, 1977; Kiefer and Slusarenko, 2003; Kubota and Abiko, 2000, 2001; Madi and Katan, 1998; Meuwly et al., 1994; Reglinski et al., 2001; Tjamos, 1979; Xue et al., 1998). The signal is presumed to use both the phloem and alternative pathways. In the hypocotyl, SAR to C. lagenarium and to Pythium ultimum Trow var. ultimum was effective 3 and 4 d after their inoculation into the cotyledons, respectively, and was complete 1 d later (Kubota and Abiko, 2000). C. lagenarium started to penetrate into the host tissues 1 d after inoculation with the conidial suspension, and the P. ultimum mycelia interacted directly with the host tissues (Xuei et al., 1988) resulting in an apparent effect 4 d after inoculation of the cotyledons. The time sequence of SAR induction in the upper leaves was similar to that in the hypocotyl (data not shown) (Me´traux et al., 1990). Meanwhile, levels of SA increased dramatically in the immunized parts. Levels of SA reached approximately 100 and 1000 ng/g in the first leaf and hypocotyl, respectively, with SAR becoming complete 5 d after
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inoculation. However, as both SA increase and SAR induction were intermediated at 4 d after the inoculation, we were unable to determine minimal levels of SA necessary for SAR induction. The accumulation of SA in the internode immediately below an infected leaf was 1.5 to 3.5 mg/g, 10-fold greater than that detected in other internodes. Although the rate of SA production and import in the stem were not clear, we speculate that nodes obstruct the movement of SA to a degree, but not the movement of the SAR signal. The increase in SA to a similar level in both the hypocotyl and root suggests that there may not be such an obstacle between these organs. The increase in levels of SA in the upper stem to approximately 200 ng/g was similar to that detected by Me´traux et al. (1990) using the same pathogen, but was lower than that induced using Pseudomonas syringae (Smith-Becker et al., 1998). The accumulation of SA in the upper leaves was between 400 and 3000 ng/g, which was greater than that induced by the Tobacco necrotic virus, and similar or greater than that induced by P. lachrymans in cucumber plants (Meuwly and Me ´traux, 1993; Meuwly et al., 1994; Mo ¨lders et al., 1994, 1996). The magnitude of the increase in SA differed according to the species of pathogen and the inoculation method used. The basal concentration of SA in the cucumber root was about 300 ng/g, which was higher than in the leaves and stem, as reported by Chen et al. (1999). It was not clear whether Chen et al. (1999) conducted their experiments under sterilized conditions. However, in the present study, cucumber plants were not grown under sterilized conditions; therefore, biases, such as the effects of microbes around the root, might be impacting upon the data, although the induction of SAR and the accumulation of SA in the root were significant (Kubota and Abiko, 2001). The amount of SA in the root after induction was similar to that shown by Chen et al. (1999), who inoculated Pseudomonas spp. into the root. The small increase in SA (less than 100 ng/g) in the lower leaf in this study may have been enough to induce a weaker SAR in the lower leaf compared with that in the upper leaves (Guedes et al., 1980). We speculate three possible factors, directional preference of the SAR signal, reactivity of each organ to the signal, and capacity of SA synthesis in the organ. These factors may affect the level of SA and sequent SAR in cucumber plants.
Acknowledgments We thank Dr. Masaharu Nakayama (Takeda Chem. Ind. Ltd.) and Dr. Tsukasa Nunome (NIVTS) for
ARTICLE IN PRESS 1116 advice on the analytical conditions, Mr. Hidekazu Ito (NIVTS) for technical advice about the HPLC, and Dr. Takashi Shirakawa, Ms. Toshiko Umezawa (NIVTS), Dr. Kazuo Abiko (JA Shiga, formerly NIVTS) and Dr. Mamoru Satou (National Agriculture and Bio-orientated Research Organization, Headquarters, formerly NIVTS) for laboratory support.
References Cameron RK, Paiva NL, Lamb CJ, Dixon RA. Accumulation of salicylic acid and PR-1 gene transcripts in relation to the systemic acquired resistance (SAR) response induced by Pseudomonas syringae pv. tomato in Arabidopsis. Physiol Mol Plant Pathol 1999;55: 121–30. Chen C, Be ´langer RR, Benhamou N, Paulitz TC. Role of salicylic acid in systemic resistance induced by Pseudomonas spp. against Pythium aphanidermatum in cucumber roots. Eur J Plant Pathol 1999;105: 477–86. Coquoz JL, Buchala AJ, Meuwly P, Me´traux JP. Arachidonic acid induces local but not systemic synthesis of salicylic acid and confers systemic resistance in potato plants to Phytophthora infestans and Alternaria solani. Phytopathology 1995;85:1219–24. Delaney TP, Uknes S, Vernooij B, Friedrich L, Weymann K, Negretto D, et al. A central role of salicylic acid in plant disease resistance. Science 1994;266: 1247–50. Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, et al. Requirement of salicylic acid for the induction of systemic acquired resistance. Science 1993;261:754–6. Gessler C, Kuc´ J. Induction of resistance to Fusarium wilt in cucumber by root and foliar pathogens. Phytopathology 1982;72:1439–41. Guedes MEM, Richmond S, Kuc´ J. Induced systemic resistance to anthracnose in cucumber as influenced by the location of the inducer inoculation with Colletotrichum lagenarium and the onset of flowering and fruiting. Physiol Plant Pathol 1980;17:229–33. Jenns AE, Kuc´ J. Localized infection with tobacco necrosis virus protects cucumber against Colletotrichum lagenarium. Physiol Plant Pathol 1977;11:207–12. Jeun YC, Siegrist J, Buchenauer H. Biochemical and cytological studies on mechanisms of systemically induced resistance to Phytophthora infestans in tomato plants. J Phytopathol 2000;148:129–40. Kiefer IW, Slusarenko AJ. The pattern of systemic acquired resistance induction within the Arabidopsis rosette in relation to the pattern of translocation. Plant Physiol 2003;132:840–7. Kubota M, Abiko K. Induced resistance in hypocotyl of cucumber by infection with Colletotrichum lagenarium in leaves. J Gen Plant Pathol 2000;66: 128–31.
M. Kubota, K. Nishi Kubota M, Abiko K. Induced resistance in cucumbers against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani AG2-2 by infection of the cotyledons. J Phytopathol 2001;149:297–300. Madi L, Katan J. Penicillium janczewskii and its metabolites, applied to leaves, elicit systemic acquired resistance to stem rot caused by Rhizoctonia solani. Physiol Mol Plant Pathol 1998;53:163–75. Me´traux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, Gaudin J, et al. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 1990;250:1004–6. Meuwly P, Me ´traux JP. Ortho-anisic acid as internal standard for the simultaneous quantitation of salicylic acid and its putative biosynthesis precursors in cucumber leaves. Anal Biochem 1993;214: 500–5. Meuwly P, Mo ¨lders W, Summermatter K, Sticher L, Me´traux JP. Salicylic acid and chitinase in infected cucumber plants. Acta Hortic 1994;381:371–4. Meuwly P, Mo ¨lders W, Buchala A, Me´traux JP. Local and systemic biosynthesis of salicylic acid in infected cucumber plants. Plant Physiol 1995;109: 1107–14. Mo ¨lders W, Meuwly P, Summermatter K, Me´traux JP. Salicylic acid content in cucumber plants infected with Pseudomonas lachrymans and tobacco necrosis virus. Acta Hortic 1994;381:375–8. Mo ¨lders W, Buchala A, Me ´traux JP. Transport of salicylic acid in tobacco necrosis virus-infected cucumber plants. Plant Physiol 1996;112:787–92. Nakayama M, Matsuura K, Okuno T. Production of salicylic acid in tobacco and cowpea plants by a systemic fungicide ferimzone and induction of resistance to virus infection. J Pestic Sci 1996;21:69–72. Pallas JA, Paiva NL, Lamb C, Dixon RA. Tobacco plants epigenetically suppressed in phenylalanine ammonialyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus. Plant J 1996;10:281–93. Reglinski T, Whitaker G, Cooney JM, Taylor JT, Poole PR, Roberts PB, et al. Systemic acquired resistance to Sclerotinia sclerotiorum in kiwifruit vines. Physiol Mol Plant Pathol 2001;58:111–8. Smith-Becker J, Marois E, Huguet EJ, Midland SL, Sims JJ, Keen NT. 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 1998;116: 231–8. Sticher L, Mauch-Mani B, Me´traux JP. Systemic acquired resistance. Annu Rev Phytopathol 1997;35: 235–70. Tjamos EC. Induction of resistance to Verticillium wilt in cucumber (Cucumis sativus). Physiol Plant Pathol 1979;15:223–7. Vernooij B, Friedrich L, Morse A, Reist R, Kolditz-Jawhar R, Ward E, et al. Salicylic acid is not the translocated signal responsible for inducing systemic acquired
ARTICLE IN PRESS Salicylic acid in roots and hypocotyl after inoculation of leaves resistance but is required in signal transduction. Plant Cell 1994;6:959–65. Xue L, Charest PM, Jabaji-Hara SH. Systemic induction of peroxidase, 1,3-b-glucanase, chitinases, and resistance in bean plants by binucleate Rhizoctonia species. Phytopathology 1998;88:359–65.
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Xuei XL, Ja ¨rlfors U, Kuc´ J. Ultrastructural changes associated with induced systemic resistance of cucumber to disease: host response and development of Colletotrichum lagenarium in systemically protected leaves. Can J Bot 1988;66: 1028–38.
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Salicylic acid accumulates in the roots and hypocotyl after inoculation of cucumber leaves with Colletotrichum lagenarium Masaharu Kubota, Kazufumi Nishi Laboratory of Plant Pathology, Department of Fruit Vegetables, National Institute of Vegetable and Tea Science (NIVTS), 360 Kusawa, Ano, Mie 514-2392 Japan Received 13 June 2005; accepted 26 September 2005
KEYWORDS Colletotrichum lagenarium; Cucumis sativus; Hypocotyl; Root; Salicylic acid
Summary Increased amounts of salicylic acid (SA) were detected in the roots and hypocotyl of cucumber plants (Cucumis sativus) using high-performance liquid chromatography following inoculation of the leaves with the anthracnose pathogen, Colletotrichum lagenarium. The concentrations of SA in the internodes immediately below the infected leaves increased to more than 1 mg/g fresh weight. In contrast, the concentrations of SA in stems distant from, or above the infected leaves increased to 100–300 ng/g. An increase in SA levels was observed in the upper stem 2 d after inoculation, followed by the hypocotyl with an increase detected 4 d after inoculation. An initial increase in the SA levels was detected in the stem, followed by an increase in SA levels in the root from a basal level of approximately 300 ng/g to more than 1 mg/g. The increased level of SA in the lower leaves was less than 100 ng/g. These results indicate that the levels of SA in the hypocotyl and root increased significantly following inoculation of the leaves with a microorganism capable of inducing SAR. & 2005 Elsevier GmbH. All rights reserved.
Introduction Abbreviation: HPLC, High-performance liquid chromatography; PHSA, Potato homogenate sucrose agar; SA, Salicylic acid; SAR, Systemic-acquired resistance Corresponding author. Tel.: +81 59 268 4641; fax: +81 59 268 1339. E-mail address: [email protected] (M. Kubota).
When plants are necrotized by biotic or abiotic agents they systemically develop resistance to diseases, which is known as systemic-acquired resistance (SAR) (Sticher et al., 1997). SAR is effective against a wide range of pathogens, including fungi, bacteria and viruses (Sticher
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.09.005
ARTICLE IN PRESS 1112 et al., 1997). Numerous reports have described SAR in leaves or stems located above the treated leaves (Sticher et al., 1997), and some cases of downward SAR have also been reported (Gessler and Kuc´, 1982; Guedes et al., 1980; Kubota and Abiko, 2000, 2001; Madi and Katan, 1998; Reglinski et al., 2001; Tjamos, 1979; Xue et al., 1998). In previous studies, salicylic acid (SA) was produced in leaves infected with SAR-inducing microbes and was exported systemically (Sticher et al., 1997). It was also produced in distant parts of the plant to those that were infected (Sticher et al., 1997). SA was required in the protected parts of tobacco plants as an endogenous signal, but it did not act as a systemic signal (Delaney et al., 1994; Gaffney et al., 1993; Pallas et al., 1996; Vernooij et al., 1994). However, the role of SA in the protected organs has been doubted in tomato and Arabidopsis plants (Cameron et al., 1999; Coquoz et al., 1995; Jeun et al., 2000). Induced SAR and systemic SA accumulation in the upper stem of cucumber plants have been observed following infection of the leaves with the anthracnose pathogen, Colletotrichum lagenarium (Me´traux et al., 1990; Sticher et al., 1997). However, this pathogen has not been used subsequently to induce SA accumulation. Infecting the leaves with this pathogen also induced resistance in the lower parts of cucumber plants (Gessler and Kuc´, 1982; Guedes et al., 1980; Kubota and Abiko, 2000, 2001). Until now, only Chen et al. (1999) reported increased amounts of SA in the roots of SAR-induced cucumber plants. They detected an increase in the levels of SA in a region of the plant distant from the root which was inoculated with a resistance-inducible bacterium. However, an increase in the levels of SA in the lower parts of the plant, including the hypocotyl and root, following inoculation of the leaves, has not been shown. In the present study, we describe SA accumulation in the root, hypocotyl, leaves and stem of cucumber plants after inoculation of cotyledons, first leaves or second leaves with C. lagenarium.
Materials and methods Plant and fungal inoculum Cucumber plants (Cucumis sativus L.) cultivar Shimoshirazuzihai were grown in pots (diameter: 9 cm) containing compost (Kureha Chem. Ind., Tokyo, Japan) in a chamber at 25 1C with 12 h of daylight. Colletotrichum lagenarium (Passerini)
M. Kubota, K. Nishi Ellis et Halsted strain 104-T, was incubated on potato homogenate sucrose agar (PHSA; 20% boiled potato homogenate, 2% sucrose and 3% cooking agar) at 24 1C for 5–8 d. A conidial suspension was created in water by rubbing the surface of the colony with a glass rod, followed by centrifugation at 1000g for 5 min. Conidia were washed once with water, recentrifuged and finally suspended in water at 107 conidia/mL for use as inoculum.
Inoculation with C. lagenarium and extraction of salicylic acid Ten drops of the inoculum were placed onto the cotyledons of cucumber plants 6–14 d after sowing. Samples (0.3 g) of the first leaf, hypocotyl and root were excised 14 d after sowing (0–8 d after the inoculation) and were heated for 10 min in 3 mL of boiled water containing 2% acetic acid (Nakayama et al., 1996). After inoculation with 20 drops of the conidial suspension on the first leaf of 11- to 19-dold plants, extracts from the second leaf, hypocotyls and roots were prepared in 2% acetic acid 19 d after sowing (0–8 d after the inoculation). In a further experiment, following inoculation of the second leaf of 14- to 22-d-old plants, extracts from the first leaf, third leaf, hypocotyl, and the internodes between the first and third leaves were prepared 22 d after sowing (0–8 d after the inoculation).
Conditions for high-performance liquid chromatography (HPLC) Extracts were filtered with 0.25-mm mesh and then analyzed using HPLC. The extract (50 mL) was injected into a C-18 reverse-phase column (diameter length: 4.6 250 mm) (Wakosil-II 5C18 HC, Wako Pure Chem. Ind., Osaka, Japan, or Shodex C18 M 4E, Shoko, Tokyo, Japan) at 40 1C and equilibrated in 50 mM sodium acetate containing 25% methanol flowing at 1 mL/min, and detected using a fluorescence spectrometer (821-FP; JASCO International, Tokyo, Japan) with an excitation wavelength of 290 nm and an emission of 402 nm. The peak areas on the chromatograph were analyzed using a recorder (Chromatocorder 21J; System Instruments, Tokyo, Japan). The amount of SA was calculated in comparison to 10–7 g/mL (v/w) SA. In the extraction, only free SA was detected (Nakayama et al., 1996) and the detection limit was approximately 10 ng/g of intact tissues. The peak of SA was detected about 6 min after injection of the sample into the HPLC.
ARTICLE IN PRESS Salicylic acid in roots and hypocotyl after inoculation of leaves
Results Accumulation of SA after inoculation of the cotyledons SA levels increased in the first leaf and hypocotyl 4 d after inoculation (Fig. 1). When compared to the levels of SA in the mock-inoculated plants, a significant increase was observed in the levels of SA in the hypocotyl after 5 d and in the first leaf after 6 d following inoculation with C. lagenarium. In the root, levels of SA increased 6 d after the inoculation with C. lagenarium. The SA concentration increased to approximately 600 ng/g of tissue in the first leaf, and to approximately 2 mg/g of tissue in the hypocotyl and root. In the root of the control plants, the basal amount of SA was greater than that found in the first leaf and hypocotyl, reaching approximately 300 ng/g.
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Accumulation of SA after inoculation of the first leaf Concentrations of SA in the second leaf, hypocotyl and root noticeably increased 4 d after inoculation. The difference in concentration of SA between the inoculated and mock-inoculated plants was statistically significant in the hypocotyl, second leaf and root after 3, 4 and 5 d, respectively (Fig. 2). In the second leaf, 7 d after the inoculation, the concentration of SA had increased to about 2.5 mg/g. Similar levels of SA were detected in the hypocotyl and root.
Accumulation of SA after inoculation of the second leaf The level of SA did not increase significantly in the first leaf until 8 d after inoculation of the second leaf
Figure 1. SA accumulation after inoculation of the cotyledons with C. lagenarium. SA levels in: A, first leaf; B, hypocotyl; C, root. E, inoculated; m, mock-inoculated; * indicates a significant difference (Po0:05) between the inoculated and mock-inoculated plants. The bars indicate standard errors.
Figure 2. SA accumulation after inoculation of the first leaf with C. lagenarium. SA levels in: A, second leaf; B, hypocotyl; C, root. E, inoculated; m, mock-inoculated; * indicates a significant difference (Po0:05) between the inoculated and mock-inoculated plants. The bars indicate standard errors.
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Figure 3. SA accumulation after inoculation of the second leaf with C. lagenarium. SA levels in: A, first leaf; B, third leaf. E, inoculated; m, mock-inoculated; * indicates a significant difference (Po0:05) between the inoculated and mock-inoculated plants. The bars indicate standard errors.
Figure 4. SA accumulation after inoculation of the second leaf with C. lagenarium. SA levels in: A, internode between the third and second leaf; B, internode between the second and first leaf; C, hypocotyl. E, inoculated; m, mockinoculated; * indicates a significant difference (Po0:05) between the inoculated and mock-inoculated plants. The bars indicate standard errors.
when compared to the control plants (Fig. 3). In the third leaf, the amount of SA increased after 3 d and this increase became significant 4 d after inoculation with levels of approximately 400 ng/g. In the internode between the third and second leaf, the level of SA increased to about 200 ng/g 2 d after inoculation (Fig. 4). In the internode between the second and first leaves, the concentration of SA increased to more than 1 mg/g 2 d after inoculation. Finally, in the hypocotyl, the increased SA level stabilized at approximately 100 ng/g 4 d after inoculation.
Discussion In this study, we extracted free SA by boiling plant tissues in 2% acetic acid (Nakayama et al.,
1996). The SA detected in the leaves and stems of the SAR-induced cucumber plants was either produced there or was transported from the necrotized leaves (Meuwly and Me´traux, 1993; Meuwly et al., 1995; Mo ¨lders et al., 1996; SmithBecker et al., 1998). The production of SA in the roots following inoculation with Pseudomonas spp. into the roots has been previously suggested (Chen et al., 1999). Glucoside SA has also been detected in the leaves and phloem of cucumber plants (Meuwly and Me´traux, 1993; Meuwly et al., 1995; Mo ¨lders et al., 1996). However, the conjugated SA was inactive in SAR induction (Meuwly et al., 1995; Sticher et al., 1997), and we interpreted that the detection of free SA was sufficient to evaluate the SAR induction. The detection limit of this method was approximately 10 ng/g fresh weight of plant tissue, a sufficient level to recognize increases in
ARTICLE IN PRESS Salicylic acid in roots and hypocotyl after inoculation of leaves the SA concentration in the immunized cucumber plants. We previously reported SAR in the root and hypocotyl of cucumber plants after infection of the leaves with C. lagenarium (Kubota and Abiko, 2000, 2001). Our present results confirm the accumulation of SA in the root and hypocotyl below the inoculated leaves, although the increase in the lower leaf was not significant. SA accumulation in cucumber roots after inoculation with Pseudomonas lachrymans into the cotyledons was mentioned by Meuwly et al. (1994), but was not described in detail. The increase in concentration of SA in the root occurred subsequently to the increase in the upper leaves and stem. The increase in concentration of SA in the hypocotyl also occurred subsequent to the increases observed in the upper stem and in the internodes immediately below the inoculated leaves. There appeared to be a physiological barrier to signal transduction between the hypocotyl and the upper parts of the plant. The time sequence of increases in SA in all of the stem parts, except the hypocotyl, was similar to that induced in the upper stem using the same pathogen, as described by Me´traux et al. (1990). The increase in concentration of SA in the upper leaves also occurred later than in the stem parts (Me´traux et al., 1990), possibly suggesting a time lag in the translocation of the signal. The transport of signals to the lower, lateral and opposite sides of plants to induce SAR and SA accumulation has suggested the involvement of pathways other than the vascular bundles (Chen et al., 1999; Gessler and Kuc´, 1982; Guedes et al., 1980; Jenns and Kuc ´, 1977; Kiefer and Slusarenko, 2003; Kubota and Abiko, 2000, 2001; Madi and Katan, 1998; Meuwly et al., 1994; Reglinski et al., 2001; Tjamos, 1979; Xue et al., 1998). The signal is presumed to use both the phloem and alternative pathways. In the hypocotyl, SAR to C. lagenarium and to Pythium ultimum Trow var. ultimum was effective 3 and 4 d after their inoculation into the cotyledons, respectively, and was complete 1 d later (Kubota and Abiko, 2000). C. lagenarium started to penetrate into the host tissues 1 d after inoculation with the conidial suspension, and the P. ultimum mycelia interacted directly with the host tissues (Xuei et al., 1988) resulting in an apparent effect 4 d after inoculation of the cotyledons. The time sequence of SAR induction in the upper leaves was similar to that in the hypocotyl (data not shown) (Me´traux et al., 1990). Meanwhile, levels of SA increased dramatically in the immunized parts. Levels of SA reached approximately 100 and 1000 ng/g in the first leaf and hypocotyl, respectively, with SAR becoming complete 5 d after
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inoculation. However, as both SA increase and SAR induction were intermediated at 4 d after the inoculation, we were unable to determine minimal levels of SA necessary for SAR induction. The accumulation of SA in the internode immediately below an infected leaf was 1.5 to 3.5 mg/g, 10-fold greater than that detected in other internodes. Although the rate of SA production and import in the stem were not clear, we speculate that nodes obstruct the movement of SA to a degree, but not the movement of the SAR signal. The increase in SA to a similar level in both the hypocotyl and root suggests that there may not be such an obstacle between these organs. The increase in levels of SA in the upper stem to approximately 200 ng/g was similar to that detected by Me´traux et al. (1990) using the same pathogen, but was lower than that induced using Pseudomonas syringae (Smith-Becker et al., 1998). The accumulation of SA in the upper leaves was between 400 and 3000 ng/g, which was greater than that induced by the Tobacco necrotic virus, and similar or greater than that induced by P. lachrymans in cucumber plants (Meuwly and Me ´traux, 1993; Meuwly et al., 1994; Mo ¨lders et al., 1994, 1996). The magnitude of the increase in SA differed according to the species of pathogen and the inoculation method used. The basal concentration of SA in the cucumber root was about 300 ng/g, which was higher than in the leaves and stem, as reported by Chen et al. (1999). It was not clear whether Chen et al. (1999) conducted their experiments under sterilized conditions. However, in the present study, cucumber plants were not grown under sterilized conditions; therefore, biases, such as the effects of microbes around the root, might be impacting upon the data, although the induction of SAR and the accumulation of SA in the root were significant (Kubota and Abiko, 2001). The amount of SA in the root after induction was similar to that shown by Chen et al. (1999), who inoculated Pseudomonas spp. into the root. The small increase in SA (less than 100 ng/g) in the lower leaf in this study may have been enough to induce a weaker SAR in the lower leaf compared with that in the upper leaves (Guedes et al., 1980). We speculate three possible factors, directional preference of the SAR signal, reactivity of each organ to the signal, and capacity of SA synthesis in the organ. These factors may affect the level of SA and sequent SAR in cucumber plants.
Acknowledgments We thank Dr. Masaharu Nakayama (Takeda Chem. Ind. Ltd.) and Dr. Tsukasa Nunome (NIVTS) for
ARTICLE IN PRESS 1116 advice on the analytical conditions, Mr. Hidekazu Ito (NIVTS) for technical advice about the HPLC, and Dr. Takashi Shirakawa, Ms. Toshiko Umezawa (NIVTS), Dr. Kazuo Abiko (JA Shiga, formerly NIVTS) and Dr. Mamoru Satou (National Agriculture and Bio-orientated Research Organization, Headquarters, formerly NIVTS) for laboratory support.
References Cameron RK, Paiva NL, Lamb CJ, Dixon RA. Accumulation of salicylic acid and PR-1 gene transcripts in relation to the systemic acquired resistance (SAR) response induced by Pseudomonas syringae pv. tomato in Arabidopsis. Physiol Mol Plant Pathol 1999;55: 121–30. Chen C, Be ´langer RR, Benhamou N, Paulitz TC. Role of salicylic acid in systemic resistance induced by Pseudomonas spp. against Pythium aphanidermatum in cucumber roots. Eur J Plant Pathol 1999;105: 477–86. Coquoz JL, Buchala AJ, Meuwly P, Me´traux JP. Arachidonic acid induces local but not systemic synthesis of salicylic acid and confers systemic resistance in potato plants to Phytophthora infestans and Alternaria solani. Phytopathology 1995;85:1219–24. Delaney TP, Uknes S, Vernooij B, Friedrich L, Weymann K, Negretto D, et al. A central role of salicylic acid in plant disease resistance. Science 1994;266: 1247–50. Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, et al. Requirement of salicylic acid for the induction of systemic acquired resistance. Science 1993;261:754–6. Gessler C, Kuc´ J. Induction of resistance to Fusarium wilt in cucumber by root and foliar pathogens. Phytopathology 1982;72:1439–41. Guedes MEM, Richmond S, Kuc´ J. Induced systemic resistance to anthracnose in cucumber as influenced by the location of the inducer inoculation with Colletotrichum lagenarium and the onset of flowering and fruiting. Physiol Plant Pathol 1980;17:229–33. Jenns AE, Kuc´ J. Localized infection with tobacco necrosis virus protects cucumber against Colletotrichum lagenarium. Physiol Plant Pathol 1977;11:207–12. Jeun YC, Siegrist J, Buchenauer H. Biochemical and cytological studies on mechanisms of systemically induced resistance to Phytophthora infestans in tomato plants. J Phytopathol 2000;148:129–40. Kiefer IW, Slusarenko AJ. The pattern of systemic acquired resistance induction within the Arabidopsis rosette in relation to the pattern of translocation. Plant Physiol 2003;132:840–7. Kubota M, Abiko K. Induced resistance in hypocotyl of cucumber by infection with Colletotrichum lagenarium in leaves. J Gen Plant Pathol 2000;66: 128–31.
M. Kubota, K. Nishi Kubota M, Abiko K. Induced resistance in cucumbers against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani AG2-2 by infection of the cotyledons. J Phytopathol 2001;149:297–300. Madi L, Katan J. Penicillium janczewskii and its metabolites, applied to leaves, elicit systemic acquired resistance to stem rot caused by Rhizoctonia solani. Physiol Mol Plant Pathol 1998;53:163–75. Me´traux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, Gaudin J, et al. Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 1990;250:1004–6. Meuwly P, Me ´traux JP. Ortho-anisic acid as internal standard for the simultaneous quantitation of salicylic acid and its putative biosynthesis precursors in cucumber leaves. Anal Biochem 1993;214: 500–5. Meuwly P, Mo ¨lders W, Summermatter K, Sticher L, Me´traux JP. Salicylic acid and chitinase in infected cucumber plants. Acta Hortic 1994;381:371–4. Meuwly P, Mo ¨lders W, Buchala A, Me´traux JP. Local and systemic biosynthesis of salicylic acid in infected cucumber plants. Plant Physiol 1995;109: 1107–14. Mo ¨lders W, Meuwly P, Summermatter K, Me´traux JP. Salicylic acid content in cucumber plants infected with Pseudomonas lachrymans and tobacco necrosis virus. Acta Hortic 1994;381:375–8. Mo ¨lders W, Buchala A, Me ´traux JP. Transport of salicylic acid in tobacco necrosis virus-infected cucumber plants. Plant Physiol 1996;112:787–92. Nakayama M, Matsuura K, Okuno T. Production of salicylic acid in tobacco and cowpea plants by a systemic fungicide ferimzone and induction of resistance to virus infection. J Pestic Sci 1996;21:69–72. Pallas JA, Paiva NL, Lamb C, Dixon RA. Tobacco plants epigenetically suppressed in phenylalanine ammonialyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus. Plant J 1996;10:281–93. Reglinski T, Whitaker G, Cooney JM, Taylor JT, Poole PR, Roberts PB, et al. Systemic acquired resistance to Sclerotinia sclerotiorum in kiwifruit vines. Physiol Mol Plant Pathol 2001;58:111–8. Smith-Becker J, Marois E, Huguet EJ, Midland SL, Sims JJ, Keen NT. 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 1998;116: 231–8. Sticher L, Mauch-Mani B, Me´traux JP. Systemic acquired resistance. Annu Rev Phytopathol 1997;35: 235–70. Tjamos EC. Induction of resistance to Verticillium wilt in cucumber (Cucumis sativus). Physiol Plant Pathol 1979;15:223–7. Vernooij B, Friedrich L, Morse A, Reist R, Kolditz-Jawhar R, Ward E, et al. Salicylic acid is not the translocated signal responsible for inducing systemic acquired
ARTICLE IN PRESS Salicylic acid in roots and hypocotyl after inoculation of leaves resistance but is required in signal transduction. Plant Cell 1994;6:959–65. Xue L, Charest PM, Jabaji-Hara SH. Systemic induction of peroxidase, 1,3-b-glucanase, chitinases, and resistance in bean plants by binucleate Rhizoctonia species. Phytopathology 1998;88:359–65.
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Xuei XL, Ja ¨rlfors U, Kuc´ J. Ultrastructural changes associated with induced systemic resistance of cucumber to disease: host response and development of Colletotrichum lagenarium in systemically protected leaves. Can J Bot 1988;66: 1028–38.