Triterpenoid Glycosides from Medicago sativa as Antifungal Agents

Oct 31, 2014 - The evidenced antifungal properties of the tested metabolites allowed some considerations on their structure–activity relationship. R...
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Triterpenoid Glycosides from Medicago sativa as Antifungal Agents against Pyricularia oryzae Pamela Abbruscato,† Solveig Tosi,‡ Laura Crispino,† Elisa Biazzi,§ Barbara Menin,† Anna M. Picco,‡ Luciano Pecetti,§ Pinarosa Avato,∥ and Aldo Tava*,§ †

Genomica del Riso, Parco Tecnologico Padano, Via Einstein Località Cascina Codazza, 26900 Lodi, Italy Laboratorio di Micologia, Dipartimento di Scienza della Terra e dell’Ambiente, Università di Pavia, Via Sant’Epifanio 14, 27100 Pavia, Italy § Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero Casearie (CRA-FLC), Viale Piacenza 29, 26900 Lodi, Italy ∥ Dipartimento di Farmacia, Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, Via Orabona 4, 70125 Bari, Italy ‡

ABSTRACT: The antifungal properties of saponin mixtures from alfalfa (Medicago sativa L.) tops and roots, the corresponding mixtures of prosapogenins from tops, and purified saponins and sapogenins against the causal agent of rice blast Pyricularia oryzae isolates are presented. In vitro experiments highlighted a range of activities, depending upon the assayed metabolite. The antifungal effects of the most promising prosapogenin mixture from alfalfa tops were confirmed by means of in planta tests using three different Italian cultivars of rice (Oryza sativa L. ssp. japonica), known to possess high, medium, and low blast resistance. The evidenced antifungal properties of the tested metabolites allowed some considerations on their structure−activity relationship. Results indicate that prosapogenins are active compounds to prevent the fungal attack of P. oryzae on different rice cultivars. Therefore, if properly formulated, these substances could represent a promising and environmentally friendly treatment to control rice blast. KEYWORDS: alfalfa, saponins, prosapogenins, hederagenin, medicagenic acid, antifungal activity, Pyricularia oryzae



INTRODUCTION The fungus Pyricularia oryzae Cavara (anamorph of Magnaporthe oryzae B. Couch sp. nov.)1 is a severe hemibiotroph pathogen of rice (Oryza sativa L.) causing the well-known rice blast disease, having a worldwide distribution. Rice is an important staple crop, and yield loss because of this pathogen may reach 50−70%, when environmental factors favor disease development to epidemic proportions, destroying each year as much rice needed to feed more than 60 million people.2,3 P. oryzae damages any aerial part of rice plants, although the leaves and panicles (necks) are the most commonly affected. Leaf infection reduces the photosynthetic efficiency of the plant, and panicle infection reduces the crop yield.4 Strategies to deal with rice blast disease include the use of cultural techniques (e.g., crop rotation, proper fertilization, right flood level, etc.), availability of rice-resistant genotypes (e.g., improving genetic resistance in the different rice races/ strains/varieties), and use of chemical fungicides.5 The control of rice blast with genetic resistance has long been the main strategy of successful rice production. The major limitation of this strategy is the presence of many races/strains of the pathogen in the field, while rice cultivars contain single genes conferring resistance to a specific strain. Over time, rice resistance can be overcome by the development of new races of the pathogen able to infect the plants.6 The use of chemical fungicides has long been viewed as a last resort to control rice blast. However, attention on environmental safety and human and animal health, along with the obligation of gradual dismission of synthetic formulations [by European Union © 2014 American Chemical Society

(EU) recommendations], have led researchers to look for alternative strategies, including the biological control and use of bioactive metabolites from natural sources to be used as natural fungicides. Recent studies on biocontrol of the rice blast disease are focused on the identification of microorganisms and their extracts able to control P. oryzae growth and development.7−9 Additional investigations are assessing the fungicidal activity of plant extracts, which include essential oils, phenolic compounds, coumarin derivatives, and other metabolites, such as saponins.10−14 Saponins are secondary plant metabolites distributed in the plant kingdom in several species, and they encompass triterpenoids, steroids, and steroidal alkaloids glycosylated with one or more sugar chains.15 Different properties have been reported for this class of substances, and their biological activities have been extensively investigated,16−18 making these molecules attractive compounds also for industrial application. Saponins isolated from different species within the genus Medicago (Leguminosae or Fabaceae) have been reported to possess antimicrobial, fungicidal, cytotoxic, and insecticidal activities.16,17 More recently, their nematicidal potential18,19 has awakened interest in these substances as important bioactive compounds to be employed in the agrochemical industry. The chemical composition of saponins extracted from Medicago spp. Received: Revised: Accepted: Published: 11030

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Figure 1. Chemical structure of purified saponins (1−12) and sapogenins (13−15) used in this investigation (I, hederagenin; II medicagenic acid; III, zanhic acid; and IV, soyasapogenol B).

has been extensively investigated.17,20−22 In some cases, their structure−activity relationship has been assessed.16,23 In addition, the main genes involved in their biosynthesis have been identified.24−27 In the present study, data on the antifungal properties of saponin extracts from alfalfa (Medicago sativa L.) against the causal agent of rice blast P. oryzae were obtained. The P. oryzae strains used in these investigations were isolated from five different rice varieties cultivated in Lombardy, in the Po valley, a typical Italian area of rice production. Saponins from M. sativa tops and roots, the corresponding mixture of prosapogenins, and pure saponins and sapogenins were tested against P. oryzae isolates in in vitro experiments. A prosapogenin mixture from alfalfa leaves was used to evaluate their antifungal activity by means of the in planta tests. These assays were performed in controlled conditions, using three different rice cultivars known to possess high, medium, and low pathogen resistance, infected with a mixture of the isolated strains of P. oryzae. Results of the antifungal properties of saponins from M. sativa are also discussed on the basis of their structure−activity relationship. To our knowledge, no previous investigations have been

reported on the antifungal activity of saponins from M. sativa against the rice pathogen P. oryzae in both in vitro and in planta tests.



MATERIALS AND METHODS

Chemicals and Reagents. Ethanol (EtOH) and penicillin G were purchased from Sigma-Aldrich s.r.l. (Milan, Italy). Water was deionized by a filter through a Direct-Q system (Millipore, Bedford, MA). Potato dextrose agar (PDA) was purchased from BioLife (Milan, Italy). Extraction and Purification of M. sativa Saponins and Preparation of Prosapogenins and Sapogenins. Alfalfa (M. sativa cv. Equipe) plants were grown in an open field at CRA-FLC (Lodi, Italy). Leaf samples (collected at anthesis) and roots (collected in autumn) were oven-dried at 50 °C for 2 days, ground, and used for saponin extraction. Saponins used in this study were newly extracted and purified, following general procedures previously reported.16,21,22,28,29 Pure saponins, compounds 1−12 (Figure 1), were obtained by a combination of chromatographic separation steps [silica gel open-column chromatograpy and semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC)] from the crude saponin mixtures from leaves and roots as described.21,22,28 The saponin mixture from leaves was used to prepare prosapogenins and 11031

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sapogenins. Prosapogenins were obtained after basic hydrolysis,19,30 and acid hydrolysis afforded the related sapogenins, compounds 13− 15 (Figure 1), that were obtained in a pure form after silica gel opencolumn chromatograpy.16,31 Analysis of Saponin Extracts. Purified mixtures of saponins were obtained as whitish powder in a purity of 90−95% and analyzed by silica gel 60H ready-to-use thin-layer chromatography (TLC) plates (Merck, Darmstadt, Germany) as previously described20 and by highperformance liquid chromatography (HPLC) as reported.21,22,28 Purified saponin and prosapogenin mixtures were characterized for their quantitative aglycone composition by gas chromatography−flame ionization detection (GC−FID) and gas chromatography−mass spectrometry (GC−MS) analyses of derivatized sapogenins.20,32 Detailed structural elucidation of pure saponins, compounds 1−12, and sapogenins, compounds 13−15, was obtained by nuclear magnetic resonance (NMR) and electrospray ionization−tandem mass spectrometry (ESI−MS/MS) analysis.17,21,22,27−29 Evaluation of the saponin composition in the plant extracts was also performed by liquid chromatography−tandem mass spectrometry (LC−MS/MS) experiments. Saponin Preparation for in Vitro and in Planta Bioassays. For the in vitro tests, crude saponin mixtures from alfalfa leaves and roots, prosapogenins, and pure saponins, compounds 1−12, were dissolved in EtOH/H2O solutions (from 30 to 70% EtOH according to their solubility), while pure sapogenins, compounds 13−15, were dissolved in 100% EtOH. All samples were prepared in a final concentration of 0.5 mg/mL. Serial dilutions (2-fold) were prepared from the starting standardized stock solutions to obtain a concentration range from 500 to 15.625 μg/mL for each sample. Sonication of each sample was generally adopted to facilitate solubilization of the metabolites. A series of EtOH/H2O control solutions at different concentrations were also tested against P. oryzae. For the in planta bioassays, under controlled conditions, prosapogenins were dissolved at 1% (w/v) in water with added 0.5% (w/v) gelatin, which is used in the rice infection standard protocol, with mild heating to facilitate solubilization. Prosapogenins were also tested in the same water/gelatin mixture at the same concentration in the in vitro experiments. Microorganism Growth Conditions. Antifungal activity was tested against different isolates of P. oryzae [formerly called Pyricularia grisea (Cooke) Saccardo],33 obtained from leaf lesions of the following rice Italian varieties: Augusto, Nembo, Italico, Karnak, and Vialone Nano, with isolate number PatVeg 202 a, b, c, d, and e, respectively, included in a large collection of P. oryzae partially used for the study of the genetic structure of the Italian pathogen population. The isolates were maintained in the Laboratory of Mycology of the University of Pavia and grown on potato dextrose agar or rice seed flour medium, adding 500 000 units of penicillin G. Petri dishes with the cultures were maintained at 25 °C in a growth chamber with a 12 h photoperiod for 7−9 days before the antifungal assays.34 Each of the five P. oryzae isolates from the above-cited rice varieties were individually used for the in vitro tests and further used as a mixture (50 000 conidia/mL each) for the in planta experiments. In Vitro Antifungal Assay. The in vitro antifungal activities of all of the tested saponins were determined as the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) against the P. oryzae isolates described above, following reported procedures,35−37 with some modifications. MIC was defined as the lowest drug concentration that completely inhibited the growth of the test fungal pathogens after 7 days of incubation at 25 °C. To determine the lowest concentration required to kill completely the test fungus (MFC), the initial inoculum was sub-cultured from 7-day-old microwell plates containing the extract where no fungal growth was observed (100% of inhibition) in a fresh cultural medium free of the extract and examined at 24 h intervals for 10 days. The MFC was defined as the lowest drug dilution, allowing for a total reduction (99.9%) of the initial inoculum on the culture medium.38 MIC and MFC values were determined by a 2-fold serial broth microdilution method in multi-well plates. All of the tested samples were diluted in a Sabouraud culture medium to the following final concentrations (1 mL final volume): 0.500, 0.250, 0.125, 0.060, 0.030, 0.015, and 0.0075 mg/

mL. Flutriafol Pestanal, containing the antifungal compound (R,S)-2,4difluoro(1H-1,2,4-triazol-1-ylmethyl)benzhydryl alcohol, was used at the following concentrations: 0.250, 0.125, 0.060, 0.030, 0.015, and 0.0075 mg/mL. The MIC and MFC of the EtOH/H2O solutions were also recorded at the concentrations described above to exclude any antifungal effect because of the solvent. All bioassays were performed in triplicate. In Planta Antifungal Assays. Two in planta bioassays were performed. The first experiment aimed to compare the in vitro results and was performed using the Italian rice (O. sativa L. ssp. japonica) cultivar Maratelli, highly susceptible to rice blast. The protection against the disease exerted by prosapogenins was compared versus that of commercial fungicide Flutriafol Pestanal. In a second assay, the prosapogenin effect on rice genotypes was evaluated using the rice cultivars Baldo and Selenio, characterized by medium and low susceptibility to blast disease, respectively. The resistance/susceptibility to P. oryzae of the three rice cultivars has been reported39 and was confirmed by previous pathogenicity assays (data not shown). In both assays, rice plants were grown from seeds in trays filled with compost kept in a greenhouse under a daytime temperature of 27 °C and a nighttime temperature of 22 °C, with a 12 h photoperiod and 60% humidity. Nitrogen fertilization with 8.6 g of nitrogen equivalent (corresponding to standard levels used during rice crop cultivation) was applied 2 days before inoculation with the fungus. The fungal conidia suspension used to infect the rice plants was obtained by mixing the same amounts of the five isolates of P. oryzae (50 000 conidia/mL each) with 0.5% (w/v) gelatin to ensure conidial adhesion on rice leaves. Inoculation was performed as described,40 by spraying 30 mL of conidia suspension on a block of 150 rice seedlings 4 weeks after sowing. Prosapogenin treatments were performed by spraying a solution [50 mL of 10 g/L in 0.5% (w/v) gelatin]. The Flutriafol Pestanal formulation was applied at field dosage (125−150 g/ha). All treatments (prosapogenin and fungicide) were applied at two different times, viz., 24 h before and 3 days after inoculation of rice plantlets with P. oryzae. Positive control plants were sprayed with conidial suspensions (30 mL as above) without further treatment. All bioassays were performed in three replicates. After 7 days, the macroscopic disease symptoms on the second rice leaf were scored on all plants to assess the effect of the treatments. The disease severity (DS) was estimated on the basis of a visual 5-level scale from 1 (no disease) to 5 (serious disease), as described in Figure 2. Efficacy classes were then created by merging DS score levels as follows: DS1 and DS2 were associated with high efficacy of the treatment, and the corresponding efficacy class was “protection” (P), with no reduction of the grain yield; DS3 was considered of moderate efficacy, and the class was “medium protection” (MP), with reduction of the grain yield; and DS4 and DS5 were associated with low efficacy of the treatment, and the efficacy class was “no protection” (NP), with considerable grain loss. Efficacy classes were subsequently used in the statistical analysis. Statistical Analysis. For each cultivar, treatment, and replicate, the frequency of the three efficacy classes was computed as a percentage of evaluated plants. The angular transformation was applied to the percentage values, and the transformed data were subjected to analysis of variance (ANOVA).41 A first ANOVA compared the frequency of the three efficacy classes in each treatment of each individual cultivar (Table 2). In a second ANOVA, the effect of treatments on plant protection was assessed by comparing, within each cultivar, the frequency of plants from different treatments within each efficacy class (Figure 3). In both ANOVAs, mean values were compared by Duncan’s multiple range test (at p < 0.05) when the F test from the analysis was significant at p < 0.05.



RESULTS AND DISCUSSION The saponin composition from alfalfa extracts was evaluated by TLC and HPLC analyses as previously reported.21,22,28 LC− MS/MS analyses of the total extracts were also performed, and several mass peaks corresponding to the different saponins were identified (data not shown). GC−FID and GC−MS analyses of the aglycone moieties, obtained by acid hydrolyses from the 11032

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Figure 2. DS scale of rice leaves based on a visual 5-level scale: DS1, highly resistant (no symptoms); DS2, resistant (necrotic spots); DS3, partially resistant (mixture of necrotic spots and small gray lesions); DS4, susceptible (typical diamond-shaped blast lesions, lengthened, gray inside and brown outside, and yellow edges); and DS5, highly susceptible (blast lesions with wide yellow edges on the whole leaf surface). The treatment efficacy classes were then created by merging DS score levels: (P) DS1 and DS2, (MP) DS3, and (NP) DS4 and DS5.

corresponding saponin mixtures, showed that saponins from alfalfa leaves are characterized by a high concentration of medicagenic acid (51% of the total sapogenins), followed by zanhic acid (25%), soyasapogenol B (16%), bayogenin (3%), hederagenin (1%), and soyasapogenol A (1%), while saponins from alfalfa roots are characterized by the presence of medicagenic acid (68%), hederagenin (18%), soyasapogenol B (6%), bayogenin (3%), and zanhic acid (2%) (Figure 1). Composition of purified saponin mixtures used in these assays was consistent with previously obtained results.19,20,29 From the TLC and HPLC analyses, a different saponin composition was confirmed for the two plant organs, e.g., the presence of compound 4, the 3-O-β-D-glucopyranosyl derivative of medicagenic acid (Figure 1), only in roots.17 In addition, monodesmosidic saponins, compounds 2, 4, and 12, were found to be more abundant in alfalfa root extracts, representing about 45% of the total saponins, while saponins from tops were predominantly composed of bidesmosidic compounds 1, 3, and 5−11, accounting for about 80% of the total saponins (data not shown). Prosapogenins that were obtained from the leaf saponin mixture after basic hydrolyses showed the same chemical aglycone composition of the saponins from which they were prepared, but they were entirely composed of monodesmosidic compounds. Individual pure saponins, compounds 1−12, and sapogenins, compounds 13−15, were obtained by means of chromatographic purification steps, and

Figure 3. Frequency of plants (percentage of evaluated plants) classified in three efficacy classes (protection, medium protection, and no protection) after treatments against P. oryzae infection (untreated control and application of prosapogenins or Flutriafol Pestanal fungicide before and after plant inoculation with the pathogen) in three rice cultivars. For a symptom description of efficacy classes, see Figure 2. ns, ∗, and ∗∗, variation among treatments within each cultivar and efficacy classes not significant and significant at p < 0.05 and p < 0.01, respectively. In each cultivar and efficacy class, treatment mean values followed by different letters are different at p < 0.05 according to Duncan’s multiple range test.

their chemical structure (Figure 1) was determined by LC− MS/MS and NMR experiments. The in vitro biological effects of saponin extracts from alfalfa leaves and roots, single purified saponins, prosapogenins, and sapogenins were investigated for activity against five strains of the rice fungal pathogen P. oryzae isolated from five different Italian rice varieties. All of these isolates showed a similar response (in terms of MIC/MFC) to the different tested compounds. Results from these bioassays are reported as MIC and MFC values in Table 1. All of the tested compounds showed a broad spectrum of antifungal activity. The lowest 11033

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disease and, hence, assayed as a standard for P. oryzae positive control. Previous investigations on the structure−activity relationship of saponins have led to contrasting results:16,42,43 some authors suggested that sugar moieties are important for their antifungal effects, while according to others, sapogenins are more active. The data reported here clearly demonstrate that medicagenic acid, the main aglycone among M. sativa saponins, and its derivatives are responsible for the highest antifungal inhibition against P. oryzae (in comparison to hederagenin and zanhic acid saponins). In addition, as previously stated,16,17,42,43 the monodesmosidic compounds (prosapogenins from M. sativa tops and compound 4) are, in general, more active against rice blast pathogen than the corresponding bidesmosides. Concerning the mechanism of action of these antifungal compounds, some considerations on their biocidal activities can be deduced. It is known that Flutriafol Pestanal is a triazole derivative, and it is able to inhibit the ergosterol biosynthesis, causing the rupture of fungi cell membranes.44 In addition, there is evidence that saponins are able to alter membrane permeability, to complex cholesterol or other membrane components, leading to the rupture of the cell.15,45 The results obtained in our experiments show that monodesmosides of medicagenic acid and compound 4 are as bioactive as Flutriafol and support a mechanism of action affecting the cell membrane integrity. On the basis of the highest antifungal activity shown in the in vitro tests by the monodesmosidic prosapogenins, these compounds were employed for the in planta assays on three Italian rice varieties, Maratelli, Baldo, and Selenio, chosen for their different levels of susceptibility to the pathogen. The in planta assays confirmed the different degrees of susceptibility to the disease shown by the three cultivars evaluated. The frequency of highly symptomatic plants (represented by the efficacy class “no protection”) was indeed generally very high in cv. Maratelli, regardless of the applied treatment, moderate or low in cv. Selenio, and somewhat intermediate between the previous two in cv. Baldo (Table 2). In the tolerant cv. Selenio, the frequency of the three efficacy classes (protection, medium protection, and no protection) did not differ in the inoculated untreated condition, whereas with either application of prosapogenins, the frequency of “protection” was significantly greater (p < 0.05) than those of the two other classes (Table 2). Interestingly, in both the susceptible cv. Maratelli and the moderately susceptible cv. Baldo, the three efficacy classes did not differ under the pre-inoculation prosapogenin application, as a consequence of an increase of frequency of protected plants and a decrease of frequency of non-protected plants, in comparison to the other treatments in each cultivar (Table 2). The latter finding is shown better in Figure 3, where the results of the second ANOVA are graphically reported. In particular, in Figure 3A, it is evident that, for cv. Maratelli, the pre-inoculation application of prosapogenins enhanced remarkably the frequency of plants with “protection” and “medium protection” and reduced the frequency of those with “no protection” compared to the control and all other treatments. Similarly, the application of saponins before the inoculation also increased the frequency of protected plants and decreased that of non-protected plants in cv. Baldo compared to the control and post-inoculation application (Figure 3B). Only in the tolerant cv. Selenio was the post-inoculation application of prosapogenins able to exert some protective efficacy against the

Table 1. Antifungal Activity (MIC and MFC) of Alfalfa Saponins, Prosapogenins, and Sapogenins Tested in Vitro against the Fungus P. oryzae Isolated from O. sativaa samples total saponins prosapogenins pure saponins

pure sapogenins

Flutriafol Pestanal

M. sativa tops M. sativa roots M. sativa tops 1 2 3 4 5 6 7 8 9 10 11 12 13, hederagenin 14, medicagenic acid 15, zanhic acid

MIC (mg/mL)

MFC (mg/mL)

2.0 0.25 0.03 >0.5 0.25 >0.5 0.03 0.125 0.125 0.25 0.5 0.125 0.25 >0.5 >0.5 >0.5 0.01 0.25 0.01

nd 0.5 0.25 >0.5 nd >0.5 0.125 0.25 nd 0.5 >0.5 >0.5 0.5 >0.5 >0.5 >0.5 0.03 0.25 0.01

a

Flutriafol Pestanal was tested as a positive control. For chemical structures, see Figure 1.

activity (MIC = 2.0 mg/mL) was observed for the saponin mixture from alfalfa leaves, while the corresponding prosapogenins showed the highest inhibition activity, with MIC and MFC values of 0.03 and 0.25 mg/mL, respectively. Intermediate values (MIC = 0.25 mg/mL, and MFC = 0.5 mg/mL) were instead recorded for pure saponins, compounds 1−12 (Table 1). Among pure saponins, the bidesmosidic hederagenin derivatives, compounds 1 and 3 (Figure 1), did not show any antifungal activity (MIC and MFC values of >0.5 mg/mL) against P. oryzae, while a moderate inhibition (MIC and MFC values from 0.125 to 0.5 and from 0.125 to >0.5 mg/ mL, respectively) was in general observed for the bidesmosidic saponins of medicagenic and zanhic acid (compounds 5−10). The only exception was saponin 11, a bidesmoside of zanhic acid, which revealed no activity (MIC and MFC values of >0.5 mg/mL). A moderate inhibition (MIC = 0.25 mg/mL) was observed for saponin 2, a trisaccharide monodesmoside of hederagenin, while a strong inhibition was observed for compound 4, the 3-O-β-D-glucopyranosyl derivative of medicagenic acid, for which a MIC of 0.03 mg/mL and a MFC of 0.125 mg/mL were recorded. As expected, no growth inhibition was instead observed for compound 12, a saponin of soyasapogenol B, that is known for its very low or null antifungal activity.17 A similar trend (Table 1) could also be observed for the three pure sapogenins 13−15: no activity was recorded for hederagenin 13 (MIC and MFC values of >0.5 mg/mL); a moderate activity was observed for zanhic acid 15 (MIC and MFC values of 0.25 mg/mL); and a high antifungal activity was observed for medicagenic acid 14 with a MIC = 0.01 mg/mL and MFC = 0.03 mg/mL. Among all of the tested compounds, this last sapogenin and its 3-O-β-D-glucopyranosyl derivative, compound 4, showed the highest antifungal activity similar to that of the Flutriafol Pestanal, which is a systemic fungicide currently applied in rice fields to control the blast 11034

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Table 2. Comparison among Efficacy Classes within Individual Treatments against P. oryzae Infection (Untreated Control and Application of Prosapogenins or Flutriafol Pestanal before and after Plant Inoculation with the Pathogen) in Three Rice Cultivarsa

treatment untreated prosapogenins pre-inoculation prosapogenins post-inoculation Flutriafol pre-inoculation Flutriafol post-inoculation

medium protection

no protection

cv. Maratelli 0.0001 0.2 b ns 36.3 a

0.7 b 26.5 a

99.1 a 41.5 a

0.05

5.8 b

9.2 b

85.0 a

0.01

8.9 b

9.1 b

82.0 a

0.01

6.3 b

9.2 b

84.5 a

untreated prosapogenins pre-inoculation prosapogenins post-inoculation

0.05 ns

cv. Baldo 14.1 b 39.8 a

25.6 b 32.0 a

60.3 a 28.2 a

0.05

19.2 c

33.7 c

47.1 a

untreated prosapogenins pre-inoculation prosapogenins post-inoculation

ns 0.05

cv. Selenio 33.5 a 85.9 a

35.2 a 11.9 a

31.2 a 2.2 b

0.05

64.8 a

22.0 a

13.2 b

AUTHOR INFORMATION

Corresponding Author

*Telephone: +39-0371-40471. Fax: +39-0371-31853. E-mail: [email protected]. Funding

treatment efficacy classb F test probabilityc protection

Article

This work was supported by the Regione Lombardia within the framework of the project Biogesteca (Decreto 4779 14052009). Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors are grateful to Prof. M. Mella (Università di Pavia) and and Mr. G. Dipinto (Università degli Studi di Bari Aldo Moro) for their valuable help in the chemical analysis of saponins.



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a

Values are percentages of plants recorded in each class for each treatment and cultivar. For a symptom description of efficacy classes, see Figure 2. bIn each cultivar and treatment, class values followed by different letters are different at p < 0.05 according to Duncan’s multiple range test. Analysis was carried out after angular transformation of original percentage values reported in the table for the sake of clarity. cAmong efficacy classes within each treatment; ns, not significant.

fungus, while the pre-inoculation treatment had the same positive effect exhibited for the two other cultivars (Figure 3C). The results reported herein indicate that prosapogenins are active compounds against P. oryzae, especially in prevention of the fungal attack, and they were active against all five P. oryzae isolates used in both the in vitro and in planta experiments. The toxicity of the commercial formulation Flutriafol Pestanal has been poorly described.46,47 Nevertheless, this compound is extremely persistent in soil, possesses high mobility potential, and is considered a groundwater contaminant.48 Therefore, saponins from M. sativa could represent, if properly formulated, a promising and environmentally friendly treatment for control of rice blast. In conclusion, data presented here make a point about the potential use of saponins, related prosapogenins, or aglycones from M. sativa for new fungicidal formulations. The highest antifungal activity shown by prosapogenins makes these saponin-derived compounds good candidates for practical use in agriculture. The large biomass produced by alfalfa, together with the selection of new varieties with high content of selected saponins with a defined chemical structure,49 should make the industrial extraction of saponins economically viable. 11035

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