Screening of Indanoyl-Type Compounds as Elicitors of Isoflavonoid Phytoalexins in Colombian Common Bean Cultivars

Eleven indanoyl derivatives were synthesized and, along with methyl jasmonate, evaluated as isoflavonoid-phytoalexin elicitors in two cultivars of common bean (Phaseolus vulgaris L. cvs. ICA-Cerinza and Uribe Rosado, tolerant and susceptible to anthracnose, respectively). Indanoyl derivatives (an ester, two amides, and eight indanoyl-amino acid conjugates) were obtained from 1-oxo-indane-4-carboxylic acid. In general, the accumulation of isoflavonoid-type phytoalexins, such as isoflavones (genistein, daidzein, and 2′-hydroxygenistein), isoflavanones (dalbergioidin and kievitone), isoflavan (phaseollinisoflavan), coumestrol, and pterocarpans (phaseollidin and phaseollin), was dependent on the common bean cultivar, the post-induction time, and the elicitor structure. Isoflavones, dalbergioidin, and coumestrol reached their highest amounts during the first 48 to 72 h, whereas kievitone, phaseollinisoflavano, and the pterocarpans reached maximum levels between 72 and 96 h. The 1-oxo-indanoyl-L-isoleucine methyl ester elicited the highest levels of phytoalexins (similar to those elicited by the methyl jasmonate) and showed no significant phytotoxic effects on common bean seedlings. The indanoyl-type synthetic elicitor, 1-oxo-indanoyl-L-isoleucine methyl ester, may represent a promising agronomic alternative for disease control in common bean by enhancing the accumulation of antimicrobial isoflavonoid phytoalexins.


Introduction
Plants often respond to microbial infection by producing antimicrobial, low-molecularweight secondary metabolites known as phytoalexins. These compounds also accumulate in plants in response to various agents called elicitors, including substances of biotic (from pathogens or the same plant) and abiotic origin, such as synthetic compounds. The exogenous application of elicitors allows for an increase the phytoalexin concentration and, consequently, enhances the resistance of plants to fungal infections [1]. Therefore, elicitors have been postulated as a friendly alternative to fungicides due to their nonbiocidal character. Unfortunately, very frequent applications or in applications in high concentrations of some synthetic elicitors may result in phytotoxicity symptoms [2,3].
Exogenous application of jasmonate-type elicitors, which are biologically active fatderived cyclopentanones present in the plant kingdom, such as jasmonic acid (JA) (Figure 1) and methyl jasmonate (MeJA), have been reported to activate the hyperproduction of various secondary metabolites [4][5][6]. In addition, coronatine, a bacterial toxin produced by Pseudomonas syringae that mimics the plant hormone jasmonoyl-L-isoleucine, together with some structurally-related compounds (such as indanoyl-amino acid conjugates) elicits secondary On the other hand, the common bean (Phaseolus vulgaris L.) is an important legume crop around the world, with an estimated annual production of around 12 million tons [14]. It represents the main source of protein for nearly five hundred million people in Africa, Latin America, and the Caribbean [15]. Additionally, common bean has a high content of dietary fiber, complex carbohydrates, vitamins, minerals, and phytochemicals [16]. Unfortunately, the high incidence of diseases and the use of susceptible cultivars limit the production of common beans. In South and Central America, the most limiting disease of the common bean is anthracnose caused by the fungus Colletotrichum lindemuthianum Sacc. and Magnus. Traditionally, the disease is controlled using fungicides, some of which have negative effects on the environment and humans due to their low selectivity and biocidal character [17]. In this sense, the elicitation of defensive responses in common bean emerges as a secure and ecofriendly alternative for replacing the current fungicides.
Elicitation of common bean increases the levels of isoflavonoid-type phytoalexins; two separate biosynthetic pathways have been proposed: 5-deoxy-and 5-hydroxyisoflavonoid ( Figure 2). The first pathway involves the conversion from daidzein to coumestrol or to pterocarpan-type phytoalexins (phaseollidin and phaseollin) and phaseollinisoflavan [13]. The 5-hydroxyisoflavonoid route passes from genistein to 2 -hydroxygenistein, then dalbergioidin and, finally, kievitone. The aim of the present work was to analyze the isoflavonoidtype phytoalexin elicitor effect of a series of 1-oxo-indane-4-carboxylic acid derivatives (indanoyl derivatives) in two Colombian common bean varieties (ICA-Cerinza and Uribe Rosado). Then, the effect of post-induction time and phytotoxicity for the most active derivative as elicitor was evaluated.

Influence of Indanoyl-Derivative Structure on Phytoalexin Accumulation
A series of eleven indanoyl derivatives structurally related to coronatine was synthesized ( Figure 3), and its isoflavonoid-phytoalexin elicitor effect in hypocotyl-roots of the common bean was evaluated. All indanoyl derivatives were evaluated at 1.0 mM in hydroalcoholic solution (0.05%). Hypocotyl-roots treated with hydroalcoholic solution were used as control. The plant tissues were incubated for 72 h post-induction.
2.1.1. Effect of C4-Substituted Indanoyl Derivatives (4, 9, 10, and 11) on Phytoalexin Accumulation In order to select the most promising structural core as an elicitor, a screening based on the stimulation of the isoflavonoid phytoalexin production was carried out. A special interest was placed on the accumulation of phaseollin, a recognized antifungal substance biosynthesized in the later stages of the 5-deoxy isoflavonoid pathway. The results of isoflavonoid phytoalexin production in hypocotyl-roots of common bean (cv. ICA Cerinza) treated with an indanoyl ester (9), two indanoyl-alkyl amides (10 and 11), and an indanoylamino acid conjugate (4) are shown in Figure 4. Derivatives (4, 9, 10, and 11) significantly increased the concentration of phytoalexins in comparison to the control (tissues treated with hydroalcoholic solution, 0.05%), particularly genistein, phaseollin, and coumestrol. In the cultivar ICA Cerinza, the highest accumulation of phytoalexins was elicited by the indanoyl-amino acid conjugate (4), with genistein (35.6 µg/g), phaseollin (17.7 µg/g), and coumestrol (10.0 µg/g) as the major phytoalexins. Remarkably, the amount of phaseollin in tissues treated with (4) was seventeen times greater than that found in the control. Similarly, in the cultivar Uribe Rosado, the highest accumulation of the phytoalexins genistein (39.2 µg/g) and phaseollin (21.9 µg/g) was observed when the tissues were treated with the indanoyl derivative (4), which is of interest because phaseollin and genistein have been reported as strong antifungal compounds [18,19], the production of which is directly related to resistance to phytopathogenic micro-organisms in common bean [20]. Even phaseollin has been proposed as a chemical marker of resistance to fungal diseases in common bean [13]. The concentration of phaseollin in the tissues treated with (9), (10), (11), and the control was below that found for daidzein and coumestrol. However, after treatment with (4), tissues exhibited amounts of phaseollin substantially higher than daidzein and coumestrol, which demonstrates that the branch of the biosynthetic pathway leading to the pterocarpans in common bean becomes more rapid and efficient as a result of the application of indanoyl derivative (4). As can be seen in Figure 4, the indanoyl-L-isoleucine methyl ester conjugate elicited the highest levels of phytoalexins, particularly phaseollin. Interestingly, the levels of phytoalexins found in the tissues of seedlings treated with (5), (6), and (7) were similar to those detected in the control (hydroalcoholic solution-treated seedlings). This shows that the indanoyl derivatives containing the amino acids L-leucine methyl ester, L-valine methyl ester, and L-glycine ester, respectively, were biologically inactive. The induction of phytoalexins in common bean with indanoyl derivatives appears to be highly dependent on structural features; even small differences in structure are sufficient to differentially elicit the phytoalexins. The indanoyl-amino acid conjugate containing the L-isoleucine methyl ester is of particularly interest because this structural element is easily derived from the amino acid building block coronamic acid of coronatine by opening the C(2)-C(3) bond of the cyclopropyl amino acid [8].
Studies with different indanoyl-amino acid conjugates have reported that only those containing isoleucine and leucine are biologically active in inducing defense volatiles in Lima bean (P. lunatus L.); the authors of these studies report that the loss of a -CH 2 -group, as in the case of valine, leads to a total loss of biological activity [9,21]. The above suggests that the interaction of the indanoyl elicitor with the receptor that triggers biological activity is very specific, discriminating small differences in the carbon chain of the amino acid or even between the L and D conformation [9]. Such an active site seems to be optimized for the isoleucine side chain, preferably by the L-conformation. Considering that previous studies showed compound (4) as a promising phytoalexin elicitor in common bean, it was used as a structural template to obtain new derivatives. Structural modifications included bromination at position 6 of (2) and condensation with the L-isoleucine methyl ester to obtain compound (8), reduction of the carbonyl group to obtain alcohol (12), aldol condensation at carbon 2 of (2) to yield the 2-benzylidene (13), and preparation of imine (14). As can be seen in Figure 5, treatment of tissues with the indanoyl-L-isoleucine methyl ester derivatives increased the concentration of phytoalexins in ICA Cerinza, particularly dalbergioidin, daidzein, 2 -hydroxygenistein, phaseollin, coumestrol, and kievitone. In the case of phaseollin, significant differences in concentration were detected between tissues treated with (4), (8), and (12) compared to the control. However, a significant loss of isoflavonoid phytoalexin elicitor activity was evident in both cultivars as a result of the carbonyl group reduction (at C1) (13) and the formation of hydrazone (14). Thus, for the cultivar Uribe Rosado, the concentration of phaseollin in hypocotyl-roots was decreased by twelve times and three times by treatment with (13) and (14), respectively, with respect to the tissues treated with (4). Therefore, the carbonyl group plays a very important role in the recognition of the derivative as a chemical signal for the activation of phytoalexin production and accumulation, which coincides with that reported in the specialized literature [8]. The derivative substituted with bromine at C-6 (8) exhibited an elicitor effect and a significant accumulation of 2 -hydroxygenistein, daidzein, genistein, coumestrol, and phaseollin compared to the control. However, its effect was lower than that observed for compound (4). Finally, the 2-benzylidene derivative (13) did not elicit phytoalexin accumulation in any of the common bean cultivars studied. This inactivity was accompanied by lower water solubility for this derivative. The low solubility limits the practical use of indanoyl derivatives, as mentioned in [21].
As a result of the systematic analysis of the effect of structure on phytoalexin elicitation in common bean hypocotyl-root, it was concluded that the most promising derivative corresponds to compound (4), which elicited an isoflavonoid accumulation similar to that exhibited by the known elicitor MeJA. Therefore, a more detailed analysis of the effect of concentration of (4) on phytoalexin-eliciting activity and over time was carried out.

Time-Course Experiments with 1-Oxo-Indanoyl-L-Isoleucyl Methyl Ester
It is observed that the concentration of phytoalexins increases significantly with post-induction time compared to the control ( Figure 6). During the first 48 h, in ICA Cerinza, maximum concentrations were detected for the isoflavones 2 -hydroxygenistein (9.0 µg/g) and the isoflavanone dalbergioidin (5.1 µg/g), after which the accumulation of the isoflavone and the isoflavanone decreased. Consequently, the concentration of coumestrol and pterocarpan-type phytoalexins increased, reaching maximum levels after 72 and 96 h post-induction, respectively. In ICA Cerinza, the maximum concentration of phaseollin (17.2 µg/g; 39 times higher than in the control) and coumestrol (9.8 µg/g) was reached at 72 h post-induction. Similar results were found in the cultivar Uribe Rosado; in particular, the maximum concentration of phaseollin (21.0 µg/g; 6.5 times higher than in the control) and coumestrol (15.9 µg/g; two times greater than in the control) was reached after 72 and 96 h post-induction, respectively. The amount of kievitone remained almost constant (below 5.0 µg/g) during the period of analysis for both cultivars. The high concentrations of genistein in the two bean varieties but low levels of 2hydroxygenistein and elevated levels of dalbergioidin and kievitone clearly indicate that there is some metabolic blockage in the biosynthetic pathway of the 5-hydroxyisoflavonoids (genistein to 2 -hydroxygenistein to dalbergioidin to kievitone). Additionally, the low amount of kievitone may be the result of the tissue studied. Goossens et al. [22] found that the highest levels of this phytoalexin occur in the cotyledons and decrease in the hypocotyls and roots. In contrast, the 5-deoxyisoflavonoid biosynthetic pathway appears to be quite efficient in the tissues (hypocotyl-root) of both cultivars in response to treatment with 1-oxo-indanoyl-L-isoleucine methyl ester (4), as despite the low levels of the precursor daidzein detected, a relatively high concentration of phaseollin and coumestrol is elicited between 72 and 96 h post-induction. The low levels of phaseollidin clearly indicate that conversion to phaseollin is rapid (daidzein to phaseollidin to phaseollin). The metabolism of phaseollin to phaseollinisoflavan has been hypothesized to result from the need to reduce the phytotoxic effect of the phytoalexin phaseollin [23]. The low levels of phaseollinisoflavan detected as a result of elicitation with (4) suggest that the phaseollin concentrations achieved in both cultivars do not represent toxic levels to the tissues [23].

Phytotoxicity Effects of 1-Oxo-Indanoyl-L-Isoleucyl Methyl Ester on Bean Seedlings
It is important to evaluate the phytotoxic effects of elicitors on common bean, from seed germination to seedling growth, because a large number of negative effects has been reported [24]. Systematic analysis of the effects produced by the application of the synthesized elicitors at various stages of the seedling life cycle on common bean can yield information concerning the optimal conditions for the application and use of elicitors in the field. For this purpose, 1.0 mM 1-oxo-indanoyl-L-isoleucyl methyl ester (4), along with 1.0 mM MeJA and 0.05% ethanol, were separately applied over common bean seeds and seedlings, and their phytotoxic effects were evaluated. Seeds of the cultivar Uribe Rosado were used for the assays. In a Petri dish on wet filter paper, forty common bean seeds were generously sprayed with the treatments daily for five days. The seed germination (%) and cotyledon hardness are presented in Table 1. After three days, the emerged radicle was evident. Interestingly, on the third day, the application of MeJA and (4) resulted in a significant increase in the percentage of germinated seeds: 56.9 and 71.7%, respectively. For the remaining days, no significant differences in seed germination percentage were observed. It has been shown that treatment with some elicitors, such as salicylic or acetylsalicylic acid, significantly improved the germination percentages in carrot seeds [25]. Table 1. Effect of spraying with 1-oxo-indanoyl-L-isoleucyl methyl ester (4) and MeJA on seed germination (%), cotyledon hardness (puncture and compression tests), and radicle number and length of common bean (P. vulgaris L. cv. Uribe Rosado). All values are presented as mean ± standard deviation (n = 3); for each trial, eight seeds were used. On day 5, the length and number of secondary roots were measured. The results showed no significant differences in the number of roots and their length between the water-treated seedlings and those with 1.0 mM MeJA. However, significant differences were evident with the application of (4), resulting in a reduction in the length of roots, a total inhibition in the growth of secondary roots, and a reduction in the hardness of cotyledons. These effects on root length and growth have been documented in studies of coronalon (an indanoyl derivative) analogs [8]. Similarly, a significant reduction in the hardness of cotyledons was observed with the application of (4), possibly due to histological and structural changes and differences in the activation of the enzymatic machinery of the common bean seeds [26]. Rao et al. [27] studied the effect of the application of coronatine and MeJA on tomatoes, observing changes in chloroplast structure, cell wall thickening, accumulation of proteinase inhibitors, induction of anthocyanins, and root growth inhibition.

Effect of 1-Oxo-Indanoyl-L-Isoleucyl Methyl Ester on Common Bean Seedlings and Chlorophyll Content
Important variables related to the physiology and functioning of the seedlings and roots were monitored, such as seedling size, stem size, leaf size, length, number of secondary roots (Table 2), and chlorophyll content. The results show that there was no significant difference in the variables studied for days 7, 9, 11, and 13. Considering the above results, it is observed that the spraying of 1.0 mM of derivative (4) on common bean seedlings had no significant effect on hypocotyls and roots during this stage of development. The above results and the inhibition of secondary root growth in the germination stage may suggest that it is more convenient to apply derivative (4) during a later stage after seed germination. Other authors have found negative effects on seedling, hypocotyl, and leaf size after exogenous application of MeJA in common bean and lupin, respectively [28,29]. Table 2. Effect of spraying with 1-oxo-indanoyl-L-isoleucyl methyl ester (4) and MeJA on growth of common bean seedlings (P. vulgaris L. cv. Uribe Rosado). Ten seedlings were used each day; all values are presented as mean ± standard deviation. The exogenous application of cinnamic acid derivatives in common bean [28] and salicylic acid in barley [24] have shown that as the concentration of the derivative increases, there is a greater inhibitory effect on seedling root growth. In the case of salicylic acid, which is known to be a plant growth regulator, it was observed that a low concentration increased root size, but at high concentrations, the inhibition of growth was significant.

Day
On the other hand, chlorophyll content is a very important variable in the normal functioning of seedlings due to its participation in the photosynthesis process; therefore, on day 13, chlorophyll content was measured, taking into account the reports of [30]. The chlorophyll contents in leaves of common bean seedlings after treatments with 0.05% ethanol, 1.0 mM MeJA, and 1.0 mM (4) were 142.6 ± 37.2, 181.0 ± 35.7, and 126.3 ± 20.9 mg/g fresh weight, respectively. The obtained results do not show significant differences in chlorophyll content after applying the elicitor solutions for 8 days. In addition, no color changes or signs of chlorosis were observed in the leaves of the common bean seedlings during treatment with the elicitors. In contrast with these results, Pancheva et al. [24] observed a decrease in the amount of chlorophylls produced in wheat seedlings with the application of salicylic acid, where the amount of chlorophylls was concentration-and contact-time-dependent. Likewise, Xie et al. [31] reported a reduction in chlorophyll production in cotton seedlings as a result of treatment with coronatine. Therefore, the low phytotoxicity and the strong isoflavonoid phytoalexin elicitor effect convert to indanoyl-type synthetic compound (4) in a promising option in the long or medium term as an agronomic alternative for disease control in common bean.

Discussion
Stimulating the natural defense mechanisms of plants, such as phytoalexins, through the application of elicitors offers new alternatives for disease control in crops of importance [13]. Therefore, it is important to continue with the design of new elicitors that allow for greater diversity and the generation of knowledge about plant-elicitor interactions, as well as the main factors that influence this response [3]. Indanoyl elicitors can modify the chemical response of common bean seedlings in Colombian cultivars, such as ICA Cerinza and Uribe Rosado, generating a mixture of phytoalexins with possible potential for non-biocidal control of diseases, such as anthracnose. However, the search for new elicitors should not focus only on phytoalexin accumulation studies; it is also necessary to evaluate the effects on the growth and development of the seedlings treated with the elicitor, which will allow for progress towards the application of elicitors with greater potential in crops. In this work, the elicitor process, as well as phytotoxicity, of 1-oxo-indanoyl-L-isoleucine methyl ester was evaluated in bean seedlings of two Colombian bean varieties.
(i) Structure of the elicitor. Systematic modifications of the 1-oxo-indane-4-carboxylic acid system showed the importance of different functional groups and substituents in inducing activity of isoflavonoid phytoalexins. First, a change at position 4 by another substituent different from the amino acid L-isoleucine methyl ester resulted in a significant loss of the abovementioned biological activity; second, the carbonyl group of the oxo-cyclopentyl system plays an important role in the elicitor activity; reduction of this group or nucleophilic addition for hydrazone formation had negative effects on the activity. Finally, incorporation of a bulky substituent, such as bromine, at position 6 significantly reduced the elicitor activity. It is clear that it is possible to modulate phytoalexin production in beans with the use of indanoyl-type elicitors. (ii) Elicitation process. Phytoalexin accumulation depends on the common bean cultivar, the post-induction time, and the elicitor structure. The maximum concentrations of isoflavonoid phytoalexins were elicited after treatment with the compound 1-oxoindanoyl-L-isoleucine methyl ester (4) and a post-induction time of 72 h. Additionally, the major phytoalexins in the elicitation process were genistein and phaseollin, which are recognized for their antimicrobial effect. Likewise, the application of indanoyl derivatives showed that the 5-deoxyisoflavonoid production pathway, which uses daidzein as a precursor, was preferentially channeled to the pterocarpans biosynthetic pathway, specifically phaseollin. In the case of the 5-hydroxyisoflavonoid biosynthetic pathway, a high concentration of genistein was observed but a low concentration of 2 -hydroxygenistein, with considerably reduced levels of dalbergioidin and kievitone; this may suggest that the 5-hydroxyisoflavonoid phytoalexin pathway is repressed or blocked in the tissues of the cultivars ICA Cerinza and Uribe Rosado. (iii) Phytotoxicity test. The compound 1-oxo-indanoyl-L-isoleucine methyl ester exhibited a low phytotoxic effect on different bean tissues. The elicitor should be applied during a later stage after germination because inhibition of root growth was evidenced. The insignificant or null effects on seedling size, stem and leaves, chlorophyll content, number of secondary roots, and root length, along with the strong phytoalexin-elicitor capacity of 1-oxo-indanoyl-L-isoleucine methyl ester, make it a potential elicitor for disease control that could be applied in the field.

Carbonyl Group Reduction
1-oxo-indanoyl-L-isoleucyl methyl ester (4) was reduced using NaBH 4 dissolved in a mixture of methanol and dry DCM, 2:3, v/v according to [9]. analyze the samples according to [13]. Methanol and water (0.05% acetic acid) were used as solvents. The mobile phase was from 10 to 70% methanol in 40 min, then 70 to 90% methanol in 20 min, followed by 10% methanol in 3 min and re-equilibration of the column in 8 min. A Luna C18 column (5 µm, 150 mm × 4.6 mm i.d.) at 30 • C, with a guard column Phenomenex ODS (4 mm × 3.0 mm i.d.) and a flow rate of 0.7 mL/min were used for the separation of isoflavonoids. A sample volume of 20 µL was injected. Detection of isoflavonoid compounds was performed at wavelengths of 248, 259, 278, 286, and 343 nm. Results are expressed as µg isoflavonoid/g fresh material (g f.w.).

Phytotoxicity Tests
The phytotoxic effect on common bean of the elicitor that generated the highest phytoalexin accumulation was evaluated at a concentration of 1.0 mM. In addition, MeJA (1.0 mM) and ethanol (0.05%, v/v) were used as positive and negative control, respectively, for comparison purposes. Tests were carried out on the cultivar Uribe Rosado, analyzing seed germination, cotyledon hardness, and seedling and root growth after regular and periodic spraying with the elicitor or ethanol (0.05%, v/v). Eight seeds were used in each plate, and tests were performed in quintuplicate.

Assays on Common Bean Seeds and Cotyledons Hardness
For the evaluation of seed germination, the methodology reported in [28] was used, with some modifications. In a Petri dish on wet filter paper, 1.0 mM indanoyl derivative, 1.0 mM MeJA, or 0.05% ethanol was applied daily to forty seeds. On the fifth day of growth, the tegument was carefully removed; the presence or absence of secondary roots was examined, and the length of radicles was measured. In addition, cotyledons were used to measure their hardness. Puncture and compression tests were performed on cotyledons according to [35].

Assays on Common Bean Seedlings and Chlorophyll Content
Common bean seedlings (cv. Uribe Rosado) with 5-day growth were generously sprayed with the treatments (1.0 mM indanoyl derivative, 1.0 mM MeJA, or 0.05% ethanol). This process was repeated every two days for one week. On the non-treatment days, seedlings were sprayed with water to avoid wilting. On the seventh day, seedlings were removed from the vermiculite; root, seedling, stem, and leaf size, along with the number of secondary roots and leaf coloration, were measured. Seedlings were exposed to diffused light at room temperature and 80% relative humidity. Chlorophyll content: fresh leaves (200 mg) were extracted with an aqueous solution of ethanol (80%) for 20 min in a water bath at 80 • C. The mixture was then filtered, and the filtrate was adjusted to a volume of 5 mL. The absorbance of the solution was measured at 654 nm in a spectrophotometer, and the chlorophyll content was calculated by taking into account the extinction coefficient of chlorophyll. The results were expressed as mg/g fresh weight [36].

Statistical Analysis
Values are expressed as mean ± standard deviation (s.d.). Data within the groups were analyzed using one-way analysis of variance (ANOVA), followed by Fischer's least significant differences (LSD) test at p ≤ 0.05.

Conclusions
The accumulation of isoflavonoid phytoalexins in common bean tissues was found to depend on the time after induction and the structure of the elicitor. During the first 48 h, the biosynthetic precursors, isoflavones and isoflavanones, were the main phytoalexins; then, coumestrol and pterocarpan-type phytoalexins increased, reaching maximum levels. A metabolic blockage in the biosynthetic pathway in the 5-hydroxyisoflavonoid biosynthetic pathway could be suggested. Analysis of a series of eleven indanoyl derivatives revealed that some structural requirements are necessary for the elicitor effect. Varia-tions in the C-4 or C-6 position of the indanoyl system and the carbonyl group of the oxo-cyclopentyl system resulted in significant changes in activity. The compound 1-oxoindanoyl-L-isoleucine methyl ester is a promising elicitor, stimulating the synthesis and accumulation of phytoalexins in common bean tissues without significant phytotoxic effects on seeds and seedlings.