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Mechanisms of plant protection against two oxalate-producing fungal pathogens by oxalotrophic strains of Stenotrophomonas spp.

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Abstract

Key message

Oxalotrophic Stenotrophomonas isolated from tomato rhizosphere are able to protect plants against oxalate-producing pathogens by a combination of actions including induction of plant defence signalling callose deposition and the strengthening of plant cell walls and probably the degradation of oxalic acid.

Abstract

Oxalic acid plays a pivotal role in the virulence of the necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum. In this work, we isolated two oxalotrophic strains (OxA and OxB) belonging to the bacterial genus Stenotrophomonas from the rhizosphere of tomato plants. Both strains were capable to colonise endophytically Arabidopsis plants and protect them from the damage caused by high doses of oxalic acid. Furthermore, OxA and OxB protected Arabidopsis from S. sclerotiorum and B. cinerea infections. Bacterial inoculation induced the production of phenolic compounds and the expression of PR-1. Besides, both isolates exerted a protective effect against fungal pathogens in Arabidopsis mutants affected in the synthesis pathway of salicylic acid (sid2-2) and jasmonate perception (coi1). Callose deposition induced by OxA and OxB was required for protection against phytopathogens. Moreover, B. cinerea and S. sclerotiorum mycelial growth was reduced in culture media containing cell wall polysaccharides from leaves inoculated with each bacterial strain. These findings suggest that cell walls from Arabidopsis leaves colonised by these bacteria would be less susceptible to pathogen attack. Our results indicate that these oxalotrophic bacteria can protect plants against oxalate-producing pathogens by a combination of actions and show their potential for use as biological control agents against fungal diseases.

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Acknowledgements

This work was supported by grants of Consejo Nacional de Ciencia y Técnica (PIP 0256 and PIP 0440, Argentina), Agencia Nacional de Promoción Científica y Tecnológica (PICT 2147, Argentina). F.M.R has a post doctoral fellowship of Agencia Nacional de Promoción Científica y Tecnológica. M.M, N.M.V, AG, F.R.R, G.A.M and F.L.P are members of the Research Career of CONICET. The authors are grateful to José Luis Burgos (CIC) and Patricia Alejandra Uchiya (CIC) for their valuable technical assistance.

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Authors and Affiliations

  1. Instituto Tecnológico Chascomús, Universidad Nacional de General San Martín-Consejo Nacional de Investigaciones Científicas y Técnicas (INTECH/UNSAM-CONICET), Av. Intendente Marino Km 8,200 CC164, Chascomús, Argentina

    María Marina, Fernando M. Romero, Natalia M. Villarreal, Andrés Gárriz, Franco R. Rossi, Gustavo A. Martinez & Fernando L. Pieckenstain

  2. Centro de Investigaciones Cardiovasculares “Horacio Cingolani” Facultad de Ciencias Médicas (UNLP), La Plata, Argentina

    Andrés J. Medina

  3. Instituto de Fisiología Vegetal (INFIVE), Facultad de Ciencias Agrarias y Forestales - Facultad de Ciencias Naturales y Museo (UNLP-CONICET), La Plata, Argentina

    Gustavo A. Martinez

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  1. María Marina
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Contributions

MM designed the study. MM, FMR, NMV, AJM, FRR and AG performed the experimental studies. All results and data were analyzed and interpreted by MM, FMR, NMV, AG, GAM and FLP. MM wrote the manuscript with the contribution of GAM and FLP.

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Correspondence to María Marina.

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Supplemental Table 1 Primer sequences used in PCR and qRT-PCR analysis (PDF 46 kb)

11103_2019_888_MOESM2_ESM.pdf

Supplementary Supplemental Fig. 1: Potassium oxalate toxicity on A. thaliana leaves. Five-μl aliquots of potassium oxalate solutions (3, 6 and 20 μM) in water (pH 4.0) were dispensed on both sides of the central vein of the adaxial surface of detached A. thaliana Col-0 leaves, which were placed in Petri dishes containing 0.8% (p/v) water-agar and incubated in a plant growth chamber. Leaves treated with water (pH 4.0) were used as controls. Figure shows the development of the lesions 72 h after potassium oxalate application observed with the naked eye (PDF 467 kb)

11103_2019_888_MOESM3_ESM.pdf

Supplemental Fig. 2: Lesions caused by B. cinerea on tomato leaves inoculated by OxA and OxB. Detached tomato leaves previously inoculated with OxA or OxB cell suspensions were inoculated with B. cinerea conidea (5 x 104 conidia ml−1). The size of the lesion area around the inoculation site was determined at 48 and 72 hpi using the Image-ProPlus V 4.1 software (Media Cybernetics). Results are means of 30 replicates ± SD and different letters indicate statistically significant differences between plant treatment at each time, according to one-way ANOVA and Tukey’s test (PDF 38 kb)

11103_2019_888_MOESM4_ESM.pdf

Supplemental Fig. 3: Direct in vitro confrontation assay between B. cinerea and OxA or OxB. Direct confrontation cultures on PDA medium between fungal pathogen B. cinerea and bacterial isolates OxA or OxB. The cultures were incubated at 25 ºC until 3 and 7 days post-inoculation (PDF 50 kb)

11103_2019_888_MOESM5_ESM.pdf

Supplemental Fig. 4: Lesions caused by B. cinerea and S. sclerotiorum on Arabidopsis Col-0, sid2-2 and coi1 leaves. Detached Arabidopsis leaves were inoculated with (A)B. cinerea conidea (5 x 104 conidia ml−1) or (B)S. sclerotiorum mycelium. Col-0 leaves were used as controls. The size of the lesion area around the inoculation site was determined at 24, 48 and 72 hpi using the Image-ProPlus V 4.1 software (Media Cybernetics). Results are means of 30 replicates ± SD and different letters indicate statistically significant differences between plant lines, according to one-way ANOVA and Tukey’s test (PDF 50 kb)

11103_2019_888_MOESM6_ESM.pdf

Supplemental Fig. 5: Lesions caused by 20 mM potassium oxalate on Arabidopsis Col-0, sid2-2 and coi1 leaves. Five-μl aliquots of potassium oxalate solution (20 μM) in water (pH 4.0) were dispensed on both sides of the central vein of the adaxial surface of detached A. thaliana Col-0, sid2-2 and coi1 leaves, which were placed in Petri dishes containing 0.8% (p/v) water-agar and incubated in a plant growth chamber. The size of the lesion area around the inoculation site was determined at 48 hpt using the Image-ProPlus V 4.1 software (Media Cybernetics). Results are means of 24 replicates ± SD and statistically analyzed by to one-way ANOVA and Tukey’s test (PDF 5138 kb)

11103_2019_888_MOESM7_ESM.pdf

Supplemental Fig. 6: A. thaliana leaves previously inoculated by OxA or OxB. (A) Fluorescence microscopy was used to analyse the accumulation of phenolic compounds (autofluorescence) in Arabidopsis leaves 24 h post-inoculation with OxA or OxB. (B) Fluorescence microscopy was used to analyse callose deposition after aniline-blue staining 6 h post inoculation (hpi). Control leaves were mock-inoculated with MgCl2 10 mM pH 7.0 (PDF 4652 kb)

11103_2019_888_MOESM8_ESM.pdf

Supplemental Fig. 7: AIRs extracted from Arabidopsis leaves inoculated with OxA or OxB. Cell wall polysaccharides were obtained as alcohol insoluble residues (AIR) from Arabidopsis leaves previously (48 h before) inoculated with OxA or OxB. Leaves treated with MgCl2 10 mM pH 7.0 were used as controls. Results are means of 3 replicates ± SEM no statistically significant differences between treatments and controls were found, according to one-way ANOVA and Tukey’s test (PDF 21 kb)

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Marina, M., Romero, F.M., Villarreal, N.M. et al. Mechanisms of plant protection against two oxalate-producing fungal pathogens by oxalotrophic strains of Stenotrophomonas spp.. Plant Mol Biol 100, 659–674 (2019). https://doi.org/10.1007/s11103-019-00888-w

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