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Bacillus subtilis IAB/BS03 as a potential biological control agent

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Abstract

We describe the efficacy of Bacillus subtilis strain IAB/BS03 in reducing disease incidence of B. subtilis IAB/BS03 as a foliar treatment against Botrytis cinerea and Pseudomonas syringae on greenhouse-grown tomato (Solanum lycopersicon) plants. We also tested the effect of foliar treatments on lettuce (Lactuca sativa) against lettuce downy mildew caused by Bremia lactucae in multiple trials under different field conditions. All the assays indicated that B. subtilis IAB/BS03 reduced disease. To ascertain the mechanism of action, the induction of pathogenesis-related (PR) proteins, the accumulation of salicylic acid and the activation of peroxidase caused by foliar or root treatments with B. subtilis IAB/BS03 were studied in tomato. A salicylic acid-independent induction of the antifungal protein PR1 was observed after treatment with B. subtilis IAB/BS03, with the strongest induction due to root treatment compared with foliar application. A metabolic analysis of B. subtilis IAB/BS03 culture broth using Ultra Performance Liquid Chromatography coupled with ultraviolet and mass spectrometric detection determined surfactin and iturin A isomers. These compounds have been described as antifungal and antibiotic lipopeptides. The results indicated that B. subtilis IAB/BS03 could be effectively used as a biocontrol agent.

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Acknowledgments

This work was funded by IAB S. L. (Investigaciones y Aplicaciones Biotecnológicas, S. L.), and by grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness. Mayte Castellano was the recipient of a research grant also funded by IAB S. L. The authors would like to thank Cristina Torres (IBMCP, UPV-CSIC) for her excellent technical assistance.

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Authors and Affiliations

  1. IAB S.L. (Investigaciones y Aplicaciones Biotecnológicas, S.L.), Polígono Industrial Moncada III, Avenida Paret del Patriarca 11-B, 46113, Moncada, Valencia, Spain

    Estefanía Hinarejos

  2. Instituto de Biología Molecular y Celular de Plantas. Universitat Politècnica de València (UPV) - Consejo Superior de Investigaciones Científicas (CSIC), Ciudad Politécnica de la Innovación (CPI), Ingeniero Fausto Elio s/n, 46022, Valencia, Spain

    Mayte Castellano, Ismael Rodrigo, José María Bellés, Vicente Conejero, María Pilar López-Gresa & Purificación Lisón

Authors
  1. Estefanía Hinarejos
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  2. Mayte Castellano
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  3. Ismael Rodrigo
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  4. José María Bellés
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  5. Vicente Conejero
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Corresponding author

Correspondence to Ismael Rodrigo.

Additional information

María Pilar López-Gresa and Purificación Lisón contributed equally to this work.

Electronic Supplementary Material

Fig. S1

(A) Symptoms of disease caused by Botrytis cinerea on greenhouse-grown Solanum lycopersicum. (B) Symptoms of disease caused by Pseudomonas syringae on greenhouse Solanum lycopersicum. (C) Symptoms of lettuce downy mildew caused by Bremia lactucae on field-grown Lactuca sativa. (PDF 4115 kb)

Fig. S2

Evaluation of efficacy (based on Abbot’s formula) of applications of B. subtilis IAB/BS03 against B. cinerea in greenhouse-grown tomato plants: foliar disease. Tomato plants were either untreated or treated with B. subtilis IAB/BS03 at three different doses (1, 10 and 100 g hL−1) compared with a standard fungicide (Scala 0.15 %). Five different foliar applications (A, B, C, D and E) were applied, and disease incidence (A) or disease severity (B) on leaves was assessed at the different time periods after application (dpt-X indicates days after the X application; 0 dpt-X indicates just prior to the X application). Comparisons among treatments were performed by analysis of variance (ANOVA) for each time point and a Newman-Keuls means comparison (where A ≠ B ≠ C) was performed (α = 5 %). Means with the same letter indicate that there are no significant differences. * Barlett’s test (on transformed data) did not permit us to verify homogeneity of variances and Kruskal-Wallis test for non-parametric data were applied. (PDF 35 kb)

Fig. S3

Evaluation of efficacy (based on Abbot’s formula) of applications of B. subtilis IAB/BS03 against P. syringae in greenhouse-grown tomato plants: disease on tomato footstalks and calyxes. Tomato plants were either untreated or treated with B. subtilis IAB/BS03 at three different doses (1, 10 and 100 g hL−1) compared with a standard bactericide (ZZ Cuprocol 0.3 %). Five different foliar applications (A, B, C, D and E) were applied, and disease incidence (A) or disease severity (B) on tomato footstalks and calyxes was assessed at the different time periods after application (dpt-X indicates days after the X application; 0 dpt-X indicates just prior to the X application). Comparisons among treatments were performed by analysis of variance (ANOVA) for each time point and a Newman-Keuls means comparison (where A ≠ B ≠ C) was performed (α = 5 %). Means with the same letter indicates that there are no significant differences. * Barlett’s test (on transformed data) did not permit us to verify homogeneity of variances, and Kruskal-Wallis and Mann-Whitney tests for non-parametric data were applied. (PDF 28 kb)

Fig. S4

Evaluation of efficacy (based on Abbot’s formula) of applications of B. subtilis IAB/BS03 against Bremia lactucae on field-grown, ‘Carrascoy’ lettuce in Vinaròs (Spain). Lettuce plants were either untreated or treated with B. subtilis IAB/BS03 at three doses (50, 100 and 150 g hL−1). The reference product was a fungicide (Curzate M 0.44 %). Five different foliar applications (A, B, C, D and E) were performed. Disease incidence (A) or disease severity (B) on leaves was assessed at the different time periods after application (dpt-X indicates days after the X application). Comparisons among treatments were performed by analysis of variance (ANOVA), for each time point and a Newman-Keuls means comparison (where A ≠ B ≠ C) was performed (α = 5 %). Means with the same letter indicates that there are no significant differences (p value <0.05). (PDF 26 kb)

Fig. S5

HPLC-MS (ESI-) chromatograms of methanol extracts obtained from B. subtilis IAB/BS03 culture broths incubated at 28 °C or 37 °C showing the (A) surfactin, (B) iturin isomers. (PDF 93 kb)

Table S1

Weather conditions for efficacy trials of B. subtilis IAB/BS03 against Bremia lactucae in lettuce in different years and locations in Europe. (PDF 19 kb)

Table S2

Statistical parameters of the efficacy of B. subtilis IAB/BS03 against B. cinerea in greenhouse tomato plants: foliar disease. A) Results of ANOVA test for disease incidence. B) Results of ANOVA test for disease severity. C) Kruskal-Wallis test for severity at 0 dpt-D. D) p-values of Mann-Whitney test for severity at 0dpt-D. (PDF 19 kb)

Table S3

Results of ANOVA of the efficacy of B. subtilis IAB/BS03 against P. syringae in greenhouse tomato plants: disease on main stems. A) Disease incidence. B) Disease severity. (PDF 18 kb)

Table S4

Statistical parameters of the evaluation of the efficacy of B. subtilis IAB/BS03 against P. syringae in greenhouse tomato plants: disease on footstalks and calyxes. A) Results of ANOVA test for disease incidence. B) Kruskal-Wallis test for incidence at 0 dpt-E. C) Results of ANOVA test for disease severity. D) Kruskal-Wallis test for severity at 0, 8 and 14 dpt-E. E) p-values of Mann-Whitney test for severity at 14 dpt E. (PDF 19 kb)

Table S5

Results of ANOVA of the evaluation of the efficacy of B. subtilis IAB/BS03 against Bremia lactucae on field-grown ‘Carrascoy’ lettuce in Vinaròs (Spain): disease on leaves. A) Disease incidence. B) Disease severity. (PDF 17 kb)

Table S6

Results of ANOVA of the evaluation of the efficacy of B. subtilis IAB/BS03 against Bremia lactucae in additional experiments on lettuce. A total of 8 additional experiments were performed. Several varieties were used at the different locations in Europe in 2011 and 2012. (PDF 19 kb)

Table S7

Summary of the 9 efficacy trials showing the performance of B. subtilis IAB/BS03 and reference products against Bremia lactucae on field-grown lettuce. A) Incidence of lettuce downy mildew on leaves. B) Severity of lettuce downy mildew on leaves. (PDF 37 kb)

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Hinarejos, E., Castellano, M., Rodrigo, I. et al. Bacillus subtilis IAB/BS03 as a potential biological control agent. Eur J Plant Pathol 146, 597–608 (2016). https://doi.org/10.1007/s10658-016-0945-3

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