Characterization of rue extract and its potential for controlling rice blast

The objective of this work was to purify and standardize the rue (Ruta graveolens) extract and evaluate its effect on Magnaporthe oryzae as an alternative to the integrated management of rice blast. The drug was characterized, the liquid extract was obtained, and the methodology for quantifying the standard markers psoralen and bergapten was validated. Rue extract and the markers, solely or in combination, were assayed in vitro, as well as in greenhouse conditions, for their ability to suppress leaf blast, by the evaluation of mycelial growth, conidial germination, and appressorium formation. Rue extract inhibited M. oryzae mycelial growth (100%), conidial germination (LD₅₀=0.237 mg), and the appressorium formation (LD₅₀=0.121 mg); besides, the extract reduced leaf blast severity by 80.84%. Fluorescence microscopy showed that rue extract did not damage M. oryzae cell wall and plasma membrane, indicating another mode of action. Rue extract has a great potential for controlling rice leaf blast.

Index terms:
Magnaporthe oryzae; Ruta graveolens; bergapten; psoralen

Authorship

Karinna Bannach Reis

Universidade Federal de Goiás, Faculdade de Farmácia, Laboratório de Pesquisas de Produtos Naturais, Caixa Postal 131, CEP 74001-970 Goiânia, GO, Brazil. E-mail: kaka.bch@gmail.com, pjrpaula@gmail.com, farmacotecnicaufg@yahoo.com.brUniversidade Federal de GoiásBrazilGoiânia, GO, BrazilUniversidade Federal de Goiás, Faculdade de Farmácia, Laboratório de Pesquisas de Produtos Naturais, Caixa Postal 131, CEP 74001-970 Goiânia, GO, Brazil. E-mail: kaka.bch@gmail.com, pjrpaula@gmail.com, farmacotecnicaufg@yahoo.com.br

Marcio Vinicius de Carvalho Barros Côrtes

Embrapa Arroz e Feijão, Rodovia GO-462, Km 12, Zona Rural, Caixa Postal 179, CEP 75375-000 Santo Antônio de Goiás, GO, Brazil. E-mail: marcio.cortes@embrapa.br, cristina.filippi@embrapa.brEmbrapa Arroz e FeijãoBrazilSanto Antônio de Goiás, GO, BrazilEmbrapa Arroz e Feijão, Rodovia GO-462, Km 12, Zona Rural, Caixa Postal 179, CEP 75375-000 Santo Antônio de Goiás, GO, Brazil. E-mail: marcio.cortes@embrapa.br, cristina.filippi@embrapa.br

Frederico Severino Martins

Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, CEP 14040-903 Ribeirão Preto, SP, Brazil. E-mail: fsmfarmacia@hotmail.comUniversidade de São PauloBrazilRibeirão Preto, SP, BrazilUniversidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, CEP 14040-903 Ribeirão Preto, SP, Brazil. E-mail: fsmfarmacia@hotmail.com

Marta Cristina Corsi de Filippi

José Realino de Paula

Edemilson Cardoso da Conceição

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (7)
Tables (1)

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Figure 1:
Inhibition of Magnaporthe oryzae (mm) mycelial growth, after standardized Ruta graveolens extract treatment at different concentrations (0, 3.38, 6.76, 13.52, 27.04, 40.56, 54.08, 67.61 mg mL^-1). Means followed by equal letters do not differ significantly, according to Duncan's test, at 5% probability

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Figure 2:
Petri dishes showing inhibition of Magnaporthe oryzae mycelial growth, after standardized Ruta graveolens extract treatment at different concentrations (mg mL^-1): A, 0; B, 3.38; C, 6.76; D, 13.52; E, 27.04; F, 40.56; G, 54.08; H, 67.61.

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Figure 3:
Germination of Magnaporthe oryzae conidia. A, concentration of standardized Ruta graveolens extract (mg): T1, 0.0; T2, 0.10; T3: 0.20; T4: 0.30; T5 0.40; T6: 0.51; T7: 0.61; T8: 0.71; T9: 0.81; T10: 0.91; T11: 1.01. Columns followed by equal letters did not differ, according to Duncan's test, at 5% probability. B, effect of Ruta graveolens extract concentrations on the inhibition of conidial germination. Probit - log dosage curve.

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Figure 4:
Magnaporthe oryzae appressorium formation. A, concentration of standardized Ruta graveolens extract (mg): T1, 0.0; T2, 0.10; T3, 0.20; T4, 0.30; T5, 0.40; T6, 0.51; T7, 0.61; T8, 0.71; T 9, 0.81; T10, 0.91; T11, 1.01. Columns followed by equal letters did not differ significantly, according to Duncan's test, at 5% probability. B, effects of Ruta graveolens extract concentrations on appressorium inhibition. Probit - log dosage curve.

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Figure 5:
Germination and formation of appressorium. Magnaporthe oryzae conidia mixed with 7.09 mg (A), 0.71 mg (B) and 0.10 mg (C) of Ruta graveolens extract after 48 hours of deposition on a hydrophobic surface under optical microscope. Magnaporthe oryzae conidia mixed with water (D, control) and 0.304 mg (E) of Ruta graveolens extract after 72 hours of deposition on a hydrophobic surface, added of calcofluor white reagent. The image was obtained using a fluorescent microscope at 400X magnification.

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Figure 6:
Disease severity (A) and the blast lesion type (B) on rice leaves. Treatments: 1, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia; 2, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + plant extract; 3, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia +18.26 μg mL^-1 of psoralen; 4, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 29.14 μg mL^-1 of bergapten; 5, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 18.26 μg mL^-1 of psoralen + 29.14 μg mL^-1 of bergapten. Means followed by equal letters did not differ, according to Duncan's test, at 5% probability

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Figure 7:
Sample of rice leaves representing how disease severity (A) and blast lesion type (B) developed. Treatments: 1, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia; 2, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + plant extract; 3, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 18.26 μg mL^-1 of psoralen; 4, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 29.14 μg mL^-1 of bergapten; 5, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 18.26 μg mL^-1 of psoralen + 29.14 μg mL^-1 of bergapten.

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Table 1:
Treatments of blast disease assessment under greenhouse conditions.

Figure 1: Inhibition of Magnaporthe oryzae (mm) mycelial growth, after standardized Ruta graveolens extract treatment at different concentrations (0, 3.38, 6.76, 13.52, 27.04, 40.56, 54.08, 67.61 mg mL^-1). Means followed by equal letters do not differ significantly, according to Duncan's test, at 5% probability

Figure 2: Petri dishes showing inhibition of Magnaporthe oryzae mycelial growth, after standardized Ruta graveolens extract treatment at different concentrations (mg mL^-1): A, 0; B, 3.38; C, 6.76; D, 13.52; E, 27.04; F, 40.56; G, 54.08; H, 67.61.

Figure 3: Germination of Magnaporthe oryzae conidia. A, concentration of standardized Ruta graveolens extract (mg): T1, 0.0; T2, 0.10; T3: 0.20; T4: 0.30; T5 0.40; T6: 0.51; T7: 0.61; T8: 0.71; T9: 0.81; T10: 0.91; T11: 1.01. Columns followed by equal letters did not differ, according to Duncan's test, at 5% probability. B, effect of Ruta graveolens extract concentrations on the inhibition of conidial germination. Probit - log dosage curve.

Figure 4: Magnaporthe oryzae appressorium formation. A, concentration of standardized Ruta graveolens extract (mg): T1, 0.0; T2, 0.10; T3, 0.20; T4, 0.30; T5, 0.40; T6, 0.51; T7, 0.61; T8, 0.71; T 9, 0.81; T10, 0.91; T11, 1.01. Columns followed by equal letters did not differ significantly, according to Duncan's test, at 5% probability. B, effects of Ruta graveolens extract concentrations on appressorium inhibition. Probit - log dosage curve.

Figure 5: Germination and formation of appressorium. Magnaporthe oryzae conidia mixed with 7.09 mg (A), 0.71 mg (B) and 0.10 mg (C) of Ruta graveolens extract after 48 hours of deposition on a hydrophobic surface under optical microscope. Magnaporthe oryzae conidia mixed with water (D, control) and 0.304 mg (E) of Ruta graveolens extract after 72 hours of deposition on a hydrophobic surface, added of calcofluor white reagent. The image was obtained using a fluorescent microscope at 400X magnification.

Figure 6: Disease severity (A) and the blast lesion type (B) on rice leaves. Treatments: 1, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia; 2, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + plant extract; 3, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia +18.26 μg mL^-1 of psoralen; 4, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 29.14 μg mL^-1 of bergapten; 5, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 18.26 μg mL^-1 of psoralen + 29.14 μg mL^-1 of bergapten. Means followed by equal letters did not differ, according to Duncan's test, at 5% probability

Figure 7: Sample of rice leaves representing how disease severity (A) and blast lesion type (B) developed. Treatments: 1, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia; 2, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + plant extract; 3, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 18.26 μg mL^-1 of psoralen; 4, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 29.14 μg mL^-1 of bergapten; 5, 3x10⁵ mL^-1 of Magnaporthe oryzae conidia + 18.26 μg mL^-1 of psoralen + 29.14 μg mL^-1 of bergapten.

Table 1: Treatments of blast disease assessment under greenhouse conditions.

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Characterization of rue extract and its potential for controlling rice blast

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