J, Bigirimana and M. H6fte (2002) Phytoparasitica 30(2):159-168

Induction of Systemic Resistance to Colletotrichum lindemuthianum in Bean by a Benzothiadiazole Derivative

and Rhizobacteria

J. Bigirimana and M. H6fte *,1

Plants have developed mechanisms to resist secondary infection upon inoculation with a necrotizing pathogen, chemical treatment as well as treatment with some non-pathogenic microorganisms such as rhizosphere bacteria. This phenomenon has been variously described as induced systemic resistance (ISR) or systemic acquired resistance. In the present study, the chemical benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH, acibenzolar- S-methyl), and the rhizobacteria Pseudomonas aeruginosa KMPCH and P. fluorescens WCS417 were tested for their ability to induce resistance to Colletotrichum lindemuthianum in susceptible and moderately resistant bean plants (Phaseolus vulgaris L.). BTH induced local and systemic resistance when bean leaves were immersed in 10 -3 to 10 -~ M BTH 3 days before the challenge inoculation. At a high concentration (10 -3 M), BTH induced resistance of the same order as resistance induced by the pathogen C. lindemuthianum, although at this high concentration BTH appeared to be phytotoxic. Soil and seed treatment with 1 mg kg-1 BTH protected beans against anthracnose. BTH-mediated induced resistance was effective in susceptible and moderately resistant plants. P. aeruginosa KMPCH induced resistance in bean against C. lindemuthianum only in a moderately resistant interaction. KMPCH-567, a salicylic acid mutant of KMPCH, failed to induce resistance, indicating that salicylic acid is important for KMPCH to induce resistance in the bean-C, lindemuthianum system. P. fluorescens WCS417 could induce resistance to C. lindemuthianum in a susceptible and in moderately resistant interactions. KEY WORDS: Bean anthracnose; BTH; acibenzolar-S-methyl; fluorescent pseudomonads; Pseudomonas aeruginosa; Pseudomonas fluorescens; induced resistance; Phaseolus vul- garis.

INTRODUCTION

After pathogen infection, plants can develop resistance against later attacks, locally on previously infected parts and even systemically on parts distant from the primary infection. It has been demonstrated that this phenomenon, named induced (acquired) resistance, is effective not only against the inducing pathogens, but also against a wide range of unrelated pathogens (10,18). In some cases, induced or acquired resistance is characterized in plants by an accumulation of salicylic acid (SA) (19), and the expression of a specific subset of pathogenesis-related (PR) genes, the so-called systemic acquired resistance (SAR) genes (31). SA is an essential molecule for induced resistance, since plants transformed with

Received Aug. 24, 2001; received in final form Nov. 30, 2001: hnp://www.phytoparasitica.org posting Jan. 16, 2002. 1 Laboratory of Phytopathology, Ghent University, Coupure Links 653. B-9000 Gent, Belgium. [*Fax: +32-9- 2646238: e-mall: monica.hofte@rug.ac.be].

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the NahG gene, which are unable to accumulate SA, are incapable of developing systemic resistance in response to necrotizing pathogens (8).

Systemic resistance can be induced also by chemicals (22) and non-pathogenic microorganisms (23,29). Resistance can, for instance, be induced by exogenous appli- cation of SA and functional analogs of SA such as 2,6-dichloro-isonicotinic acid and benzo(l,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH) (7,13). Induction of systemic resistance by plant growth-promoting rhizobacteria has been studied extensively (11,16,17,30,32). Although induced resistance mediated by Pseudomonas fluorescens WCS417 in Arabidopsis was shown to be independent of SA accumulation and PR gene expression (24), but was characterized by a pathway where jasmonic acid and ethylene play a crucial role (25,29), other rhizobacteria such as Pseudomonas aeruginosa 7NSK2 and a genetically modified SA-overproducing P. fluorescens P3 strain, induce systemic resistance via a SA-dependent pathway (21). It was shown that SA production by P. aeruginosa 7NSK2 is essential for induced resistance to Botrytis cinerea in bean and tomato and to tobacco mosaic virus in tobacco (1,4,6). In addition, P. aeruginosa 7NSK2 was unable to induce resistance in NahG tomato and tobacco plants (1,4). In the present study, induced resistance in Phaseolus vulgaris against Colletotrichum lindemuthianum, the causal agent of bean anthracnose, was investigated. We employed different concentrations of BTH and Pseudomonas strains that induce resistance via SA-dependent or SA-independent pathways and used susceptible and moderately resistant cultivars.

MATERIALS AND METHODS

Microorganisms: Characteristics and culture conditions Physiological races 385 and 485 of C. lindemuthianum originating from Burundi (2) were used in this study. Isolates were grown to sporulate at 25~ on Mathur medium (20) composed of glucose (2.8 g l-1), MgSO4.7H20 (1.23 g i-1), agar (20 g l-1), Neopeptone (2 g 1-1), KH2PO4(2.72 g 1-1) and H20 (1 1). After sporulation (10-day-old cultures), plates were flooded with 10 ml of demineralized water and spores were scraped from plates using a brush. The spore �9 suspension was filtered through four layers of 0.2 x 0.2 nun nylon mesh, the number of spores was estimated with a haemacytometer and the inoculum concentration adjusted to 105 spores m1-1 before inoculation. Different bacterial species and strains were tested for their ability to induce resistance in bean: P aeruginosa KMPCH, a pyoverdin- and pyochelin-negative, SA-producing mutant of the PGPR P aeruginosa 7NSK2 (12); P. aeruginosa KMPCH-567, a SA-negative mutant of KMPCH obtained by gene replacement (5); and P. fluorescens WCS417 (15). Bacteria were cultured on iron limited King's B medium (14) for 12-15 h at 37~ for KMPCH and KMPCH-567, and at 28~ for WCS417. Plates with bacterial colonies were flooded with 10 ml of physiological solution containing 8.5 g NaC1 per liter of demineralized water. Colonies were scraped from plates and the concentration was estimated optically.

Bean cultivars, growing conditions and induction treatments Two bean cultivars of P vulgaris were used, cvs. 'Prelude' and 'Boterkoning'. Previous studies (3) have shown that Boterkoning is sensitive to races 385 and 485, whereas the reaction of Prelude depends on the bean developmental stage: 8-day-old plants were susceptible to race 385, whereas a resistant reaction was observed on 12- or 14-day-old plants. An intermediate reaction was observed on 10- and 1 l-day-old plants. Before sowing bean seeds, the soil (Klassmann- Deilman, Geeste, Germany) was mixed with different chemicals or microorganisms to

160 J. Bigirimana and M. H0fle

be tested for induced resistance: Pseudomonas strains were suspended in H20 before use. BTH (Bion 50 WG), formulated as a water-dispersible granule containing 50% active ingredients (code number CGA 245704, Ciba-Geigy), was dissolved in H20 for use, The same quantity of water was added to the control soil to maintain an identical relative humidity in all the treatments. Concentrations were adjusted to 108 cfu g-1 soil for Pseudomonas spp.; BTH was tested at a concentration of 1 mg kg -1 in the soil. Prior to sowing, seeds were soaked for 5 min in the bacterial suspension (108 cfu m1-1) for the Pseudomonas spp. treatments or in 1 mg 1-1 BTH for BTH treatments, and in demineralized water for the control. Bean plants were grown in the greenhouse at 28-1-3 ~ under a 16:8 h, L:D photoperiod.

Leaf treatment to induce resistance was performed 3 days before challenge inoculation. Primary bean leaves were immersed in one of the following solutions: C. lindemuthianum race 485, 5.104 spores m1-1, or BTH at different concentrations varying from 10 -3 to 10 -7 M. Control leaves were immersed in demineralized water. Plants treated with C. lindemuthianum were placed in a humid chamber (>92% r.h.) at 19-21 ~

Challenge inoculation, experimental design, disease evaluation and data analysis Eleven-day-old bean plants were challenge-inoculated with races 385 and 485 of C. lindemuthianum, 105 spores m1-1. Cv. Prelude was also inoculated at its most susceptible stage, 8-day-old plants (3). The inoculation was performed using the detached leaf method (3). Three or four bean leaves per treatment were placed in each tray (according to the number of treatments) and four trays were used per experiment. Each experiment was repeated at least once. On C. lindemuthianum-inoculated leaves, anthracnose severity was evaluated 7 days after inoculation using a scale of 1 to 9, where 1 = no visible symptoms and 9 = severe disease. Score ratings were distributed into three classes for visual presentation, as follows: class 1, ratings 1 to 3; class 2, ratings 4 to 6; class 3, ratings 7 to 9. Score ratings were analyzed statistically, using the Kruskal-Wallis multiple comparison tests completed by the Mann-Whitney comparison test. For all techniques a = 0.05 was used with Bonferroni correction for non-parametric tests.

Plant eolonization by Pseudomonas spp. and effect of BTH and SA on C. linde- muthianum growth in vitro At the inoculation stage of bean plants, the colonization by Pseudomonas spp. was investigated. One gram of plant material was macerated in sterile demineralized water and serial dilutions were plated on selective media. For Pseudomonas spp., King's B medium was used, adding kanamycin 200 mg 1-1 and rifampicin 50 mg 1-1 for KMPCH strains and WCS417, respectively. KMPCH strains were incubated at 37~ WCS417 was incubated at 28~ Colonies were counted after 1 day for KMPCH and KMPCH-567, and after 2 days for WCS417.

BTH and SA were added to sterilized Mathur medium to investigate an eventual direct antagonistic effect on the development of C. lindemuthianum in vitro. Different BTH or SA concentrations in the medium were tested, from 10 -7 to 10 -4 M. C. lindemuthianum race 385 was grown and the diameter of colonies was recorded for 10 days.

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RESULTS

Local induced resistance to C. lindemuthianum on bean Primary leaves of the bean cultivar Boterkoning were immersed in different BTH concentrations, from 10 -3 to 10-TM. Treated bean leaves were inoculated 3 days later with C. lindemuthianum race 485, to which Boterkoning normally shows a susceptible reaction. Results are presented in Figure 1. Leaves treated with 10 -z M BTH were resistant (90% of leaves were scored 1 to 3), whereas the control displayed a susceptible reaction (85% of Boterkoning leaves were in class 3, scores 7 to 9). Even a concentration of 10 - r M gave significant protection (78% of leaves in class 2) compared with the control (85% of leaves in class 3). The best results were obtained with the highest BTH concentrations tested (10 -3 to 10 -5 M). Growth abnormalities were observed on plants tested with high BTH concentrations. Bean leaves immersed in 10 -3 and 10 -4 M BTH became dark green and one week later were much smaller than control leaves. For 10 -3 M BTH, similar abnormalities were observed on trifoliate leaves of treated plants. Such plants had shorter internodes than plants treated with BTH concentrations < 10 -5 M.

Fig. 1. Influence of leaf treatment with BTH on the virulence of Colletotrichum lindemuthianum race 485. Primary leaves of bean cv. Boterkoning were immersed in various BTH-concentrations (10 -3 to 10 -7 M) and inoculated 3 days later with 105 spores m1-1. Seven days after inoculation, disease symptoms were evaluated and scores were grouped into three classes: class 1, ratings 1 to 3; class 2, ratings 4 to 6; and class 3, ratings 7 t~ 9. Bars with a common letter do not differ significantly using the Mann-Whitney comparison test at a = 0.05, n = 32.

Systemic induced resistance to C. lindemuthianum on bean Induction by bean leaf treatment: One of the two primary bean leaves was immersed

in 10 -3 M BTH or in a C. linder~uthianum suspension of 5.10 4 spores mi-1. Three days later, the opposite primary leaves were challenge-inoculated with 10 5 C. lindemuthianum

162 J. Bigirimana and M. HOfle

Fig. 2. Influence of leaf treatment with BTH or Colletotrichum lindemuthianum race 485 on bean anthracnose severity. One primary leaf of bean cv. Boterkoning was treated and the opposite leaf was inoculated 3 days later with 105 spores m1-1. Seven days after inoculation, disease symptoms were evaluated and scores were grouped into three classes: class 1, ratings 1 to 3; class 2, ratings 4 to 6; and class 3, ratings 7 to 9. Bars with a common letter do not differ significantly using the Mann- Whitney comparison test at c~ = 0.05. n = 24. 2A: Bean leaves treated with H20 (control), BTH 10 -z M or C. lindemuthianum race 485, 5.104 spores m1-1. 2B: Bean leaves treated with various BTH-concentrations, from 10 -4 to L0 -~ M.

spores m1-1. The induction and challenge-inoculations were performed using the same pathogenic race 485 of C. lindemuthianum. Symptoms were scored 7 days later and results are presented {n Figure 2A. Resistance was observed on BTH-treated bean plants, compared with the susceptible control plants (cv. Boterkoning). The resistance observed on plants treated with 10 -3 M BTH was of the same order as the s3,stemic resistance

induced by inoculation with a moderate concentration of C. lindemuthianum. Different concentrations of BTH were tested further and results are presented in Figure 2B. The best

protection was observed with the highest BTH concentration tested (10 -4 M). The lowest concentration used in this experiment (10 - 7 M) also induced resistance: a 50% reduction

of the number of susceptible leaves, compared with the control.

Induction by soil and seed treatment: BTH, P aeruginosa KMPCH (SA+), P.

aeruginosa KMPCH-567 ( S A - ) and P fluorescens WCS417 were tested for their ability

to induce resistance to C. lindemuthianum race 385. Tested on a susceptible cuhivar

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Fig. 3. Influence of soil and seed treatment with BTH 1 mg kg I , Pseudomonas aeruginosa KMPCH, P. aeruginosa KMPCH-567, and P.fluorescens WCS417 on bean anthracnose severity. Symptoms on primary bean leaves were evaluated 7 days after inoculation with 105 spores ml-1 of Colletotrichum lindemuthianum race 385; scores were grouped into three classes: class 1, ratings 1 to 3; class 2, ratings 4 to 6; and class 3, ratings 7 to 9. Bars with a common letter do not differ significantly using the Mann-Whitney comparison test at o~ = 0.05, n = 24. 3A: Cv. Boterkoning, inoculation on 11- day-old plants: susceptible interaction. 3B: Cv. Prelude, inoculation on 8-day-old plants: susceptible interaction. 3C: Cv. Prelude, inoculation on 1 l-day-old plants: moderately resistant interaction.

(Boterkoning), KMPCH and its SA-negative mutant KMPCH-567 did not show any significant difference from the control (Fig. 3A). On the contrary, WCS417 and BTH significantly reduced the number o f susceptible Boterkoning leaves. Reduction of class 3 leaves was of the order of 30% and 47% for WCS417 and BTH, respectively. There was no significant difference between WCS417 and BTH. Similar results were obtained when cv, Prelude was tested at its susceptible developmental stage, 8-day-old plants: induced resistance was observed in WCS417- or BTH-treated plants, whereas plants treated with KMPCH and KMPCH-567 were as sensitive as the control (Fig. 3B). Different results were observed when cv. Prelude was inoculated on 11-day-old plants. At this developmental

164 J. Bigirimana and M. HOfle

stage, Prelude displays an intermediate reaction to race 385 of C. lindemuthianum 105 spores m1-1 (3). Inoculated at this stage, KMPCH-treated Prelude plants were significantly more resistant than control plants (Fig. 3C). KMPCH treatment increased the number of resistant bean leaves by 20% and reduced the number of susceptible ones by 10%. No increase of resistance was observed on KMPCH-567-treated plants (Fig. 3C). This SA-negative mutant of KMPCH did not improve resistance in susceptible or intermediate interactions between C. lindemuthianum and P. vulgaris.

Bean colonization by Pseudomonas spp., effect o f BTH and SA on C. lindemuthianum growth in vitro All the bacterial strains tested colonized bean roots. Their superior colonization limit was around 2 x 106 cfu g - t roots. Their concentration on bean roots was 2.27 x 106, 1.72 x 10 e and 2.22 x 106 cfu g-1 roots for KMPCH, KMPCH-567 and WCS417, respectively. BTH and SA did not have any influence on C. lindemuthianum growth. For the highest concentration tested (10 -4 M), the diameters recorded (mean of five petri plates) were 28.5, 26.0 and 28.1 mm for the control, BTH and SA plates, respectively. BTH and SA did not affect C. lindemuthianum sporulation.

DISCUSSION

Induced resistance to C. lindemuthianum on bean by BTH C. lindemuthianum virulence was reduced when bean leaves were inoculated 3 days after immersion in BTH solutions. Strong local protection was observed when Boterkoning, a susceptible cultivar, was infected with race 485 of C. lindemuthianum (Fig. 1). The bean protection mediated by BTH is systemically effective (Fig. 2) and was detected also when BTH was applied in the planting substrate before sowing bean seeds (Fig. 3). It is known that the BTH compound moves systemically in the plant and the ability of BTH to improve plant resistance to diseases has been demonstrated in other plant pathogen interactions (27). In the bean- C. lindemuthianum interaction, it has been shown before that BTH can induce resistance (26). In the present work, however, we show that increased resistance is observed even at very low concentrations (10 -7 M) (Figs. 1, 2B). The level of resistance appeared to be dose-dependent, but concentrations of 10 -3 and 10 -4 M led to phytotoxicity: treated bean leaves became smaller and darker green. Such abnormalities were observed for plants treated with 10 -3 M BTH. Undesirable effects of BTH have been reported on some other plants: in burley type tobacco, for example, yellowing and stunting can sometimes result from repeated BTH treatments; in green bell peppers, yields can be severely reduced by BTH applications (27). BTH did not have any antifungal activity. The mycelial growth and the fructification of C. lindemuthianum were not affected by the presence of BTH in the culture medium. Against bean anthracnose, induced resistance by BTH was effective in a susceptible bean-C, lindemuthianum interaction as well as in an intermediate case.

Induced resistance to C. lindemuthianum on bean by Pseudomonas spp. In the present work, some rhizobacteria strains were tested for their ability to stimulate resistance mechanisms in bean plants against C. lindemuthianum, the bean anthracnose agent. P. aeruginosa KMPCH, P. aeruginosa KMPCH-567 and P. fluorescens WCS417 were tested on different bean-C, lindemuthianum interactions including: (i) race 385 on susceptible cv. Boterkoning; (ii) race 385 on cv. Prelude at a susceptible developmental stage; and (iii) race 385 on Prelude at a developmental stage transition between a susceptible and a resistant stage. On. the susceptible Boterkoning cultivar and on Prelude at a susceptible

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stage, KMPCH and KMPCH-567 could not induce resistance to C. lindemuthianum. Only induced resistance by WCS417 and BTH was observed. On cv. Prelude at the transition phase, however, only KMPCH-567 failed to induce resistance. KMPCH, WCS417 and BTH showed a significant increase in the bean resistance to race 385. These results show that the SA-mutant KMPCH-567 does not induce resistance in either susceptible or moderately resistant interactions, whereas induced resistance is observed for KMPCH in the latter case (Fig. 3C). Since the only difference between the two strains is SA production, we concluded that SA is important. This could suggest that KMPCH induces resistance to bean anthracnose through a SA-dependent pathway, as is the case for induced resistance to Botrytis cinerea in bean and tomato (1,5,6) and to tobacco mosaic virus in tobacco (4). However, for WCS417 it was shown that, at least in Arabidopsis, resistance is induced via a SA-independent, but jasmonate- and ethylene-dependent, signal transduction pathway (29).

The results of this study indicate clearly that different PGPR strains can induce systemic resistance, via different pathways. For that reason the use of the term SAR for pathogen-induced systemic resistance, and the term ISR for rhizobacteria-mediated systemic resistance as was suggested in the earlier literature (19,29), may be questioned, since these types of resistance do not necessarily differ mechanistically from each other. Hammerschmidt et al. (9) proposed to use the terms ISR and SAR synonymously. Our results also suggest that KMPCH induces resistance in a moderately resistant interaction, whereas BTH and WCS417 increase the resistance level of susceptible and moderately resistant cultivars. These results indicate that at least in the bean-C, lindemuthianum interaction, a minimum level of plant resistance may be needed for induced resistance by KMPCH. However, BTH and KMPCH apparently induce the same defense response, so it seems unlikely that one requires a basal level of plant resistance and the other does not. KMPCH is probably less effective than BTH and may be able to induce resistance only in the event of low disease pressure. An initial resistance level was reported to be necessary for induced resistance on carnation: WCS417 could reduce the incidence of Fusarium wilt in a moderately resistant carnation cultivar, but the same strain was inefficient in a susceptible cultivar (29). Moreover, on Arabidopsis thaliana, WCS417 was unable to induce resistance on highly susceptible cultivars without any basal resistance to Pseudomonas syringae pv. tomato DC3000 (28). Against C. lindemuthianum, however, WCS417 was able to induce resistance on susceptible and on moderately resistant bean cultivars. It appears thus that this criterion differs from one plant-pathogen system to another.

ACKNOWLEDGMENTS

J. Bigirimana was supported by a grant from the Belgian Administration for Development Cooperation. We thank Peter Bakker (Utrecht University, the Netherlands) for supplying Pseudomonasfluorescens WCS417 and Novartis for supplying BTH. We thank R. Fontaine for his help in bean experiments.

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