2.2. Dose–Response of Elicitor Treatments
The use of SA as a plant defense inducer is limited by its phytotoxic activity. The effect of the concentration of SA on the synthesis of phytoalexins on common bean cotyledons was evaluated in the 0.36–14.50 mM range and after 96 h of incubation (
Figure 3). All cultivars showed significant increases in the phytoalexin levels after treatment with SA in relation to the control. Even at 0.36 mM SA, amounts of coumestrol were between 2- and 6-fold above that in the corresponding controls. In general, levels of phytoalexins in cotyledons increased steadily at 3.62 mM SA and below, in a dose-dependent manner. As shown in
Figure 3, phytoalexin contents progressively increased to reach their maximum concentrations at 3.62 mM SA for Cargamanto Rojo, Cargamanto Mocho, and ICA Quimbaya. Then at 7.25 mM SA, phytoalexin concentrations declined slightly for Cargamanto Mocho, and rapidly for Cargamanto Rojo and ICA Quimbaya. Unlike these cultivars, CORPOICA 106 reached the maximum level of phytoalexins at 14.50 mM SA. However after 96 h and at concentrations of 3.62 mM SA and above, common bean cotyledons started to show symptoms of necrosis, whereas controls and treatments below 3.62 mM SA remained green for this period (data not shown). Most of cotyledons treated with 7.25 mM SA turned brownish and exhibited wilting. Accordingly, under these conditions a hypersensitive response occurred, and our results suggest that SA at 3.62 mM and below, is safe and could be used as elicitor in common bean. Nonetheless, further studies about the physiological effects of SA in common bean cotyledons (and other tissues) are needed.
On the other hand, cotyledons from resistant cultivars (ICA Quimbaya and CORPOICA 106) accumulated significantly higher amount of phytoalexins as compared to the susceptible cultivars (Cargamanto Mocho and Rojo). For example, coumestrol reached maximal levels of about 37.11 and 32.75 µg/g for Cargamanto Rojo and Cargamanto Mocho, and near 73.65 and 92.37 µg/g for CORPOICA 106 and ICA Quimbaya respectively; almost two- and three times higher phytoalexin production in the resistant cultivars in comparison with the susceptible ones. It is noteworthy that cotyledons of CORPOICA 106 accumulated high amounts of phaseollidin and phaseollinisoflavan, being respectively about 13 and 20-fold higher that in the other cultivars. However, no substantial difference was observed in the amount of daidzein and genistein between resistant and susceptible cultivars.
Figure 3.
Accumulation of isoflavonoid phytoalexins on cotyledons of
Phaseolus vulgaris by SA at different concentrations.
, genistein;
, dalbergioidin;
, phaseollinisoflavan;
, phaseollidin;
, daidzein;
, 2’-hydroxygenistein;
, kievitone;
, coumestrol;
, phaseollin. Bars represent the mean concentrations of the isoflavonoids ± standard deviation (n = 3). Cultivars: CM, Cargamanto Mocho; CR, Cargamanto Rojo; IQ, ICA Quimbaya; CI, CORPOICA 106. For each compound, bars with different letters are significantly different (
p = 0.05; Fisher’s LSD test).
Figure 3.
Accumulation of isoflavonoid phytoalexins on cotyledons of
Phaseolus vulgaris by SA at different concentrations.
, genistein;
, dalbergioidin;
, phaseollinisoflavan;
, phaseollidin;
, daidzein;
, 2’-hydroxygenistein;
, kievitone;
, coumestrol;
, phaseollin. Bars represent the mean concentrations of the isoflavonoids ± standard deviation (n = 3). Cultivars: CM, Cargamanto Mocho; CR, Cargamanto Rojo; IQ, ICA Quimbaya; CI, CORPOICA 106. For each compound, bars with different letters are significantly different (
p = 0.05; Fisher’s LSD test).
SA is a recognized natural elicitor involved in the disease resistance of plants. It may improve the plant defensive capacity against a broad array of pathogens after appropriate treatment. In this study, the application of SA on bean cotyledons resulted in strong increases of phytoalexin content and antifungal activity of the extracts in comparison to controls. The data indicate that SA activated phytoalexin biosynthesis in all cultivars. Coumestrol, phaseollin, and 2'-hydroxygenistein were the major phytoalexins induced in cotyledons from Colombian bean varieties. This contrasts with an earlier study, in which kievitone was the major phytoalexin in cotyledons, whereas phaseollin predominates in hypocotyls [
18]. In addition, it was found that anthracnose-resistant cultivars treated with SA accumulated significantly higher levels of phytoalexins as compared to the susceptible ones. These results are in accordance with other results reported for common bean elicited by CuCl
2 [
18] and some other crops [
23,
24,
25,
26]. Hence, the induction of bean cotyledons with SA may contribute to the development of rapid and low-cost techniques for selecting resistant cultivars to anthracnose based on the biosynthesis of phytoalexins. According to course-time experiments, as a result of induction with SA, there was a gradual increase in phytoalexin content mainly for coumestrol, phaseollin, kievitone, 2'-hydroxygenistein, phaseollidin and phaseollinisoflavan. Meanwhile, genistein, daidzein, and dalbergioidin contents, precursors of phytoalexins kievitone, coumestrol and pterocarpans, remained almost constant over the whole period of the evaluation. Otherwise, dose-response experiments showed that when cotyledons were immersed on SA at 3.62 mM and below, the phytoalexin production was increased in a dose-dependent manner; this was appreciably higher for anthracnose-resistant cultivars. However, the application of SA at high concentrations (7.25 and 14.50 mM) resulted in a fast decline of phytoalexin contents, except for the cultivar CORPOICA 106. We hypothesize that the decreases in the biosynthesis of defense secondary metabolites can result in some effects related with the phytotoxic character of SA, which has been well documented [
27,
28]. The above results are in agreement with the necrosis symptoms observed in cotyledons treated with SA at 7.25 and 14.50 mM. Nevertheless, the molecular mechanism by which SA at high concentration inhibited the phytoalexin formation is unclear. Previously, War
et al. [
29] found that chickpea plants treated with SA at 2 mM showed phytotoxicity symptoms and a lower peroxidase and polyphenol oxidase activity, and phenol content. Accordingly, they suggested that SA at 1.5 mM is safe to these plants and could be utilized as plant defense inducer.
Thus, it was observed that the highest levels of phytoalexins in common bean cotyledons were achieved after 96 h of post-induction and using SA at 3.62 mM (for cv. Cargamanto Rojo, Cargamanto Mocho, and ICA Quimbaya) and at 14.50 mM (for CORPOICA 106). Interestingly, the phytoalexin amounts detected in our study from Colombian common bean cultivars are substantially lower compared with those reported for North American and European cultivars. Hynes
et al. [
21] reported accumulations of kievitone reaching 850 ± 251 µg/g f.w in wounded cotyledons (white bean cv. OAC Seaforth) following inoculation with
Fusarium solani f. sp.
phaseoli.
2.3. Antifungal Activity
The antifungal activity of extracts obtained from cotyledons (course-time experiments at 48, 96, and 144 h; dose-response assays at 0.72, 1.45, y 3.62 mM) of common bean cultivars Cargamanto Rojo (susceptible to anthracnose) and ICA Quimbaya (resistant to anthracnose), non-treated and treated with SA, in terms of radial growth inhibition of
C. lindemuthianum are summarized in
Figure 4. The inhibition of
C. lindemuthianum growth was dependent to the concentration of SA used in treatments and the incubation (post-induction) time. In general, the radial growth was inhibited in higher proportion during the first 24 h. As shown in
Figure 4a (top), inhibitions of
C. lindemuthianum using extracts from both cultivars treated with SA at 1.45 and 3.62 mM were significantly higher compared to the untreated cotyledons. At 24 h, it can be noticed that the extract obtained from cotyledons of ICA Quimbaya treated with 1.45 mM SA was more active (inhibition about 50%) toward
C. lindemuthianum than the extract proceeding from Cargamanto Rojo (inhibition 33%) under the same conditions. Nonetheless, the antifungal activity of cotyledons treated with 3.62 mM SA was similar for both cultivars. On the other hand, cotyledons of both cultivars elicited by 0.72 mM SA showed no substantial differences with respect to untreated cotyledons and solvent control. Thus, the increase in the radial growth inhibition of
C. lindemuthianum seems to be related with the upper phytoalexin levels presents in the extracts. Even so, the comparable antifungal effect against
C. lindemuthianum shown by Cargamanto Rojo and ICA Quimbaya cotyledons induced with SA at 3.62 mM results strange given their so different chemical profiles.
From
Figure 4b (bottom), it can be seen that extracts from ICA Quimbaya cotyledons induced by 1.45 mM SA and incubated during 48 and 96 h showed radial growth inhibitions of 33.3 and 50.0% respectively. However, extracts obtained after 144 h presented less antifungal activity and no significant differences were found between this extract, untreated cotyledons, and the solvent control. This behavior in the antifungal activity correlates with the increased amounts of isoflavonoid phytolalexins detected in ICA Quimbaya cotyledons 96 h post-induction. Meanwhile, it can be found that extracts from Cargamanto Rojo cotyledons treated with 1.45 mM SA and incubated for 96 and 144 h showed inhibitions of about 33.3%. The above is in agreement with the higher levels of phytoalexins detected for these extracts as compared to untreated cotyledons and that obtained 48 h post-induction. In addition,
Figure 4 showed that inhibitory effects of extracts rapidly decreased with time, a fact that suggests that the fungus had a rapid adaptation to the medium. The above is consistent with the known capacity of phytopathogenic fungi, including
C. lindemuthianum, to circumvent some of the plant chemical defenses through metabolism and detoxification [
30]. However, further studies are necessary to establish the relationship between phytoalexin level and the inhibitory effects against
C. lindemuthianum.
Figure 4.
Antifungal activity against
C. lindemuthianum of extracts from common bean cotyledons induced by SA at different concentration (up) and post-induction time (down). Cultivars: CR, Cargamanto Rojo; IQ, ICA Quimbaya.
Figure 4a:
, solvent control;
, CR-untreated cotyledons;
, CR-0.72 mM SA;
, CR-1.45 mM SA;
, CR-3.62 mM SA;
, IQ-untreated cotyledons;
, IQ-0.72 mM SA;
, IQ-1.45 mM SA;
, IQ-3.62 mM SA.
Figure 4b:
, solvent control;
, CR-untreated cotyledons;
, CR-48 h post-induction;
, CR-96 h post-induction;
, CR-144 h post-induction;
, IQ-untreated cotyledons;
, IQ-48 h post-induction;
, IQ-96 h post-induction;
, IQ-144 h post-induction. For each time point, the bars headed by the same letter do not differ at
p = 0.05 (Fisher’s LSD test).
Figure 4.
Antifungal activity against
C. lindemuthianum of extracts from common bean cotyledons induced by SA at different concentration (up) and post-induction time (down). Cultivars: CR, Cargamanto Rojo; IQ, ICA Quimbaya.
Figure 4a:
, solvent control;
, CR-untreated cotyledons;
, CR-0.72 mM SA;
, CR-1.45 mM SA;
, CR-3.62 mM SA;
, IQ-untreated cotyledons;
, IQ-0.72 mM SA;
, IQ-1.45 mM SA;
, IQ-3.62 mM SA.
Figure 4b:
, solvent control;
, CR-untreated cotyledons;
, CR-48 h post-induction;
, CR-96 h post-induction;
, CR-144 h post-induction;
, IQ-untreated cotyledons;
, IQ-48 h post-induction;
, IQ-96 h post-induction;
, IQ-144 h post-induction. For each time point, the bars headed by the same letter do not differ at
p = 0.05 (Fisher’s LSD test).
Our study also indicates that the activity of extracts from bean cotyledons treated with SA against C. lindemuthianum was only slightly enhanced. Nonetheless, it is important to note that the extracts were evaluated without an additional purification and at a relatively low concentration (<300 µg/mL), and in a static experimental system that not allow generation of new amounts of phytoalexins. In general, there were no significant differences in the fungitoxicity against C. lindemuthianum between untreated cotyledons and those treated at 0.62 mM SA, at least during the first 24 h of evaluation. Likewise, extracts obtained after 48 h (for Cargamanto Rojo) and 144 h (for ICA Quimbaya) of induction showed a similar inhibitory effect than untreated cotyledons (about 16%). The results also demonstrate that the application of 1.45 and 3.62 mM SA on cotyledons or s post-induction time of 96 and 144 h for Cargamanto Rojo and 48 and 96 h for ICA Quimbaya led to more active extracts (inhibitions between 33 and 50%). These findings are in agreement with the greater phytoalexin contents established for cotyledons elicited by 1.45 and 3.62 mM SA and post-incubation times longer than 96 h, in relation to untreated controls. Under these conditions, cotyledons of Cargamanto Rojo and ICA Quimbaya were found to accumulate the higher levels of coumestrol, 2’-hydroxygenistein, phaseollin, and kievitone, among other. Nonetheless, no marked differences in the antifungal activity were found between extracts from anthracnose-resistant and anthracnose-susceptible cultivars. Moreover, experimental data also suggest a rapid adaptation of phytopathogenic microorganisms to the medium containing the different extracts; the inhibitory effect for all extracts against C. lindemuthianum was nearly the same after 72 h of evaluation (<10% inhibition). It seems that the use of extracts without further purification and at low concentrations (<300 µg/mL) could be responsible for the quick detoxification of the medium by C. lindemuthianum, besides the modest antifungal activity after 72 h. Extracts at these concentrations may contain fungitoxic compounds at very low levels, being rapidly transformed into innocuous metabolites by biotransformation after 72 h. Thus, the lack of antifungal activity of extracts after 72 h may be due to metabolism of the phytoalexins by the fungus.
2.4. Inducer and Antifungal Effects of Structurally Related Compounds to SA
Currently, there is an increasing interest in the search for new elicitors for controlling important plant diseases. Here, the inducer effect of phytoalexins in bean cotyledons of some dihydro-quinazolinones and imines (
Figure 5), along with ABZ, INA, BTH, and 2-NBA was evaluated. Bean phytoalexins were grouped in three classes: isoflavones and isoflavanones (genistein, daidzein, dalbergioidin, 2’-hydroxygenistein, and kievitone), coumestan (coumestrol), and pterocarpans and isoflavans (phaseollidin, phaseollin, and phaseollinisoflavan). As can be seen in
Table 1, dihydro-quinazolinones and imines possess a strong elicitation effect, being even higher than that shown by the plant hormone, SA, and the structurally related compound, 2-NBA. The upper isoflavones/isoflavanones accumulation was found to be induced by
1, followed by
10 and
9. The isoflavones/isoflavanones content detected in response to
1 was near twice that found in cotyledons treated with SA. In addition,
1 induced high levels of coumestrol (73.55 µg/g f.w., the highest amount) and pterocarpans/isoflavan.
In fact, coumestrol content using 1, 6 and 10 as elicitors was increased in that order by about six, five, and three-fold in comparison to the cotyledons induced by SA. It is noteworthy that coumestrol was not detected in appreciable amounts in untreated cotyledons. Overall, pterocarpans/isoflavan levels of common bean cotyledons in response to dihydroquinazolinones and imines were always higher than that detected when SA was used as elicitor; only 2, 3, 5, and 6 showed a slightly higher increases in pterocarpans/isoflavan content.
Meanwhile, 10 had a stronger inducer effect on the pterocarpans/isoflavan content because these increased almost ten and five-fold the levels detected in untreated (control) and SA-treated cotyledons respectively. It is noteworthy that extracts proceeding from common bean cotyledons cv. Cargamanto Mocho treated with 10 also showed the higher inhibitory effects against C. lindemuthianum during the first 48 h.
Furthermore, although bean cotyledons treated with 6 resulted in a marked increase of coumestrol, the antifungal activity was relatively weak (55.9 ± 8.8 and 44.2 ± 20.9 after 24 and 48 h, respectively). This result suggests a low fungitoxic effect of coumestrol against C. lindemuthianum. In general, inhibitory effect of extracts was rapidly decreased; at 24 h, fungal inhibitions were almost twice that found after 48 h except for 6 and 7.
Additionally, we also evaluated the direct antifungal properties of these potential elicitors. In general, dihydroquinazolinones and imines displayed a moderate to weak fungistatic activity against
C. lindemuthianum. As can be seen in
Table 1, the highest radial growth inhibition was exhibited by
10 (76.9% after 48 h at 200 μg/mL), followed by
8 and
6. Remarkably,
10,
6, and
1, which showed higher phytoalexin-inducing effect, also displayed inhibitions of 76.9 ± 0.0, 57.7 ± 13.3, and 38.5 ± 6.7, so dihydroquinazolinones and imines may have dual mode of action for controlling of fungal diseases; elevating host resistance and reducing pathogen inoculum.
Figure 5.
Chemical structures of compounds evaluated as elicitors.
Figure 5.
Chemical structures of compounds evaluated as elicitors.
Table 1.
Elicitor effect of some dihydro-quinazolinones and imines related structurally to SA (at 1.45 mM and after 96 h post-induction) in cotyledons of common bean cv. Cargamanto Mocho, and antifungal activity of the extracts from induced cotyledons.
Table 1.
Elicitor effect of some dihydro-quinazolinones and imines related structurally to SA (at 1.45 mM and after 96 h post-induction) in cotyledons of common bean cv. Cargamanto Mocho, and antifungal activity of the extracts from induced cotyledons.
Compound | Isoflavones/Isoflavanones * (µg/g f.w.) | Coumestan (µg/g f.w.) | Pterocarpans/Isoflavan † (µg/g f.w.) | Radial growth inhibition (%) of C. lindemuthianum |
---|
| | | | Elicited-cotyledon extracts | Elicitor |
| | | | 24 h | 48 h | 48 h |
SA | 20.20 ± 1.47 d | 13.07 ± 1.49 a | 11.53 ± 1.42 | n.d. | 9.5 ± 4.1 |
2-NBA | 22.97 ± 2.33 a,c,d | 13.72 ± 1.12 a | 17.72 ± 5.45 | n.d. | n.d. |
1 § | 43.04 ± 9.36 a,b,c,d | 79.99 ± 22.02 a,b,c,d | 46.67 ± 10.52 a,b,c,d | 82.4 ± 0.0 | 33.7 ± 19.8 | 38.5 ± 6.7 |
2 § | 15.57 ± 3.23 | 12.93 ± 6.68 a | 13.73 ± 2.85 | 38.2 ± 8.8 | 19.8 ± 3.5 | 11.5 ± 6.7 |
3 § | 20.16 ± 4.06 b,c | 14.37 ± 0.20 a | 19.92 ± 9.89 a,c | 38.2 ± 8.8 | 19.8 ± 3.5 | 42.3 ± 0.0 |
4 § | 24.13 ± 6.84 a,c,d | 14.98 ± 1.75 a | 34.90 ± 16.37 a,c,d | 47.1 ± 0.0 | 23.3 ± 0.0 | 53.8 ± 11.5 |
5 § | 20.81 ± 6.81 c | 21.25 ± 9.26 a | 11.94 ± 5.21 | 52.9 ± 5.1 | 26.7 ± 10.5 | 42.3 ± 0.0 |
6 § | 29.26 ± 14.19 a,b,c,d | 68.58 ± 18.89 a,b,c,d | 12.05 ± 8.51 | 55.9 ± 8.8 | 44.2 ± 20.9 | 57.7 ± 13.3 |
7 § | 17.64 ± 6.46 c | 24.81 ± 9.79 a | 36.03 ± 9.33 a,c,d | 64.7 ± 0.0 | 58.1 ± 7.0 | 38.5 ± 6.7 |
8 ‡ | 17.96 ± 2.86 a,b,c | 23.08 ± 3.11 a | 28.29 ± 5.00 a,c,d | 64.7 ± 0.0 | 33.7 ± 3.5 | 65.4 ± 0.0 |
9 ǂ | 32.60 ± 16.75 a,b,c,d | 18.02 ± 2.69 a | 26.96 ± 4.08 a,c,d | 47.1 ± 0.0 | 23.3 ± 0.0 | 50.0 ± 0.0 |
10 ǂ | 37.88 ± 11.98 a,b,c,d | 38.47 ± 4.40 a,b,c,d | 60.85 ± 17.05 a,b,c,d | 70.6 ± 10.2 | 45.3 ± 17.4 | 76.9 ± 0.0 |
11 ‡ | 13.85 ± 3.88 | 10.99 ± 3.01 a | 34.71 ± 6.11 a,c,d | 47.1 ± 17.6 | 33.7 ± 3.5 | 53.8 ± 0.0 |
ABZ | 10.58 ± 1.58 | 17.74 ± 3.27 a,b | 9.32 ± 0.41 | n.d. | n.d. |
BHT | 7.90 ± 0.64 | 7.58 ± 1.83 a | 6.19 ± 1.57 b | n.d. | n.d. |
INA | 14.64 ± 2.53 | 18.58 ± 5.14 a | 22.61 ± 6.51 a | n.d. | n.d. |
Control | 9.01 ± 0.52 | Traces | 5.02 ± 0.33 b | n.d. | n.d. |
The results concerning the ability to stimulate the phytoalexin biosynthesis of compounds structurally related to SA showed that dihydroquinazolinones and imines exhibit a strong elicitor effect. Lately, new synthetic compounds have been assayed as elicitors in different plant tissues [
31,
32]. Thus, some chemicals such as 2,6-dichloroisonicotinic acid (INA) and S-methyl benzo-1,2,3-thiadiazole-7-carbothioic acid (acibenzolar-S-methyl or BTH) have been found to be effective inducers of plant defenses [
13,
14,
15,
16,
17]. Both compounds were discovered as a result of screening assays of elicitors of broad-spectrum resistance in cucumber (
Cucumis sativus L.) [
15,
33]. However, only BTH is commercially available (from Syngenta) under the names Actigard and Bion. The present study clearly indicated that 2-NBA,
2,
3,
5 induced phytoalexin levels almost similar to that detected when bean cotyledons were treated with SA, a well-known elicitor; particularly, pterocarpans/isoflavan contents were nearly twice that found in untreated controls. Remarkably, phytoalexin accumulation in response to some dihydroquinazolinones and imines (for example,
1,
4,
6, and
10) was even higher than that found for SA and some recognized synthetic elicitors, such as INA, ABZ, and BHT. In fact, pterocarpans/isoflavan levels on cotyledons were increased by as much as 106 and 169% by
1 and
10 respectively, compared with INA. Additionally, the amounts of coumestrol and pterocarpans/isoflavan for cotyledons treated with dihydroquinazolinones and imines were higher than for SA. The presence of nitrogen-containing functional groups (such as an aryl amide and a nitrogenated substituent in position 2 resembling the structure of SA) in 2-NBA and dihydro-quinazolinones may be an important structural feature for their phytoalexin-inducing activity. Some of the potential synthetic elicitors that have recently been reported are nitrogen-rich compounds. For instance, synthetic pyrazine-2-carboxamide derivatives were found to act as potential elicitors in tissue cultures of
Ononis arvensis,
Silybum marianum, and
Genista tinctoria [
34,
35]. Thus, the application of 5-
tert-butyl-6-chloro-
N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide in callus culture of
Genista tinctoria enhanced the genistin production about 57 times compared to untreated control [
35]. Furthermore, the application of 1 µM 2-(2-fluoro-6-nitrobenzylsulfanyl) pyridine-4-carbothioamide significantly increased the production of the isoflavonoids genistin (11.60 mg/g dry weight), daidzein (8.31 mg/g dry weight), and genistein (1.50 mg/g dry weight) on
Trifolium pretense L. suspension culture after 48 h of application as compared to the control by 152, 151 and 400% respectively, which was recently reported by Kašparová
et al. [
36]. Similarly, 3,5-dichloroanthranilic acid (DCA), efficiently induced defense reactions in Arabidopsis (
Arabidopsis thaliana) plants to the phytopathogens
Hyaloperonospora parasitica and
Pseudomonas syringae [
37]. Authors also indicate that the removal of the amino group from DCA significantly reduces its biological activity. Moreover, the synthetic substances 2-pyrazinecarboxylic acid, picolinic acid, 2,6-pyridinedicarboxylic acid, 2,3-pyridinedicarboxylic acid, pyrrole-2-carboxylic acid, oxonic acid, among others, increase the phytoalexin(phytocassanes and momilactone A) contents in rice plants [
38].
Although SA, 2-NBA, AIN, ABZ and BTH are functionally different compounds, they share some common structural features. Thus, all compounds have the benzoyl fragment, and an adjacent electronegative group (substituted at the 2-position, except for INA). Also, all compounds present an electron withdrawing group bonded to the carbonyl group (forming a carboxylic acid for SA, ABZ and INA, an amide for 2-NBA, and a thioester for BTH). Nevertheless, while SA is an acid compound and 2-NBA is basic, both induce similar phytoalexin contents. Hence, the above results suggest that the acidity/basicity of compounds has no effect on the phytoalexin-inducing character.
The cotyledons exposed to dihydroquinazolinones 2 and 3 produced similar phytoalexin levels than those found when 2-NBA (the synthetic precursor) was used. It indicates that the presence of the N-nitrophenyl group in these compounds had no effect on the phytoalexin accumulation. In contrast, dihydroquinazolinones having a N-methoxyphenyl (1) and N-benzodioxoyl (6) group improved the synthesis of coumestrol as compared to 2-NBA. Also, 1 elicited in bean cotyledons high contents of pterocarpans/isoflavan. These results suggest that the eliciting effects can be related with the functional features presents in the dihydroquinazolinones. The presence of electron-donating groups (like methoxy-, and methylidendioxy- group) in the C-phenyl moiety of the tested dihydroquinazolinones seems to be an important requirement for the coumestrol-eliciting effect. Furthermore, it was observed that the pterocarpans/isoflavan accumulated in a greater concentration when cotyledons were treated with 4 and 7. Both compounds have a nitrogen-containing heterocyclic ring, pyridine and pyrrole respectively. Interestingly, when the dihydroquinazolinones presented a furan ring instead a nitrogen-containing heterocyclic system (5 vs. 7) there was a substantial loss of eliciting activity of pterocarpans/isoflavan. On the other hand, acyl hydrazones 9 and 10 had a strong effect on isoflavones/isoflavanones content. Also, 10 enhanced the production of coumestrol and pterocarpans/isoflavan.
In addition, dihydroquinazolinones and imines displayed a moderate to weak direct antifungal activity. Besides their promissing phytoalexin-inducing activity, dihydroquinazolinones and imines
1,
6, and
10 also inhibit the radial growth of
C. lindemuthianum between 38.5 and 76.9%. These results are in agreement with previous reports that establish that Schiff bases and dihydroquinazolinones have antifungal properties [
39,
40]. Thereby, the direct fungistatic properties of dihydroquinazolinones and imines offer an additional advantage over SA, and other elicitors. Therefore, these compounds have the potential to offer a dual mode of action with both direct inhibitory effects again
C. lindemuthianum and the capacity of enhance the phytoalexin content, and consequently the plant resistance. These results indicate that the dihydroquinazolinones and imines are promissing elicitors of phytoalexins in common bean cotyledons. To the best of our knowledge, this is the first report about the phytoalexin-inducer effect of dihydroquinazolinones and some imines.