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

  • María MarinaEmail author
  • Fernando M. Romero
  • Natalia M. Villarreal
  • Andrés J. Medina
  • Andrés Gárriz
  • Franco R. Rossi
  • Gustavo A. Martinez
  • Fernando L. Pieckenstain


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.


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.


Arabidopsis thaliana Botrytis cinerea Callose Oxalotrophic bacteria Plant cell wall Plant defence Sclerotinia sclerotiorum Stenotrophomonas spp. 



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.

Author 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.

Supplementary material

11103_2019_888_MOESM1_ESM.pdf (47 kb)
Supplemental Table 1 Primer sequences used in PCR and qRT-PCR analysis (PDF 46 kb)
11103_2019_888_MOESM2_ESM.pdf (468 kb)
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 (38 kb)
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 (51 kb)
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 (50 kb)
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 (5 mb)
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 (4.5 mb)
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 (22 kb)
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)


  1. Asselbergh B, Höfte M (2007) Basal tomato defences to Botrytis cinerea include abscisic acid-dependent callose formation. Physiol Mol Plant Pathol 71:33–40CrossRefGoogle Scholar
  2. Bateman DF, Beer SV (1965) Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis by Sclerotiorum rolfsii. Phytopathol 55:204–211Google Scholar
  3. Bravo D, Braissant O, Cailleau G, Verrecchia E, Junier P (2015) Isolation and characterization of oxalotrophic bacteria from tropical soils. Arch Microbiol 197:65–77CrossRefGoogle Scholar
  4. Cantu D, Vicente AR, Labavitch JM, Bennett AB, Powell ALT (2008) Strangers in the matrix: plant cell walls and pathogen susceptibility. Trends Plant Sci 13:610–617CrossRefGoogle Scholar
  5. Carpita NC, McCann M (2000) The cell wall. In: Buchanan BB, Gruissem W, Jones RL (eds) Biochemistry and molecular biology of plants. American Society of Plant Biologists, Rockville, pp 45–110Google Scholar
  6. Cecchini NM, Monteoliva MI, Alvarez ME (2011) Proline dehydrogenase contributes to pathogen defense in Arabidopsis. Plant Physiol 155(4):1947–1959CrossRefGoogle Scholar
  7. Cessna SG, Sears VE, Dickman MB, Low PS (2000) Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12:2191–2199CrossRefGoogle Scholar
  8. Chipps TJ, Gilmore B, Myers JR, Stotz HU (2005) Relationship between oxalate, oxalate oxidase activity, oxalate sensitivity, and white mold susceptibility in Phaseolus coccineus. Phytopathol 95(31):292–299CrossRefGoogle Scholar
  9. da Silva LF, Dias CV, Cidade LC et al (2011) Expression of an oxalate decarboxylase impairs the necrotic effect induced by Nep1-like protein (NLP) of Moniliophthora perniciosa in transgenic tobacco. Mol Plant Microbe Interact 24(7):839–848CrossRefGoogle Scholar
  10. de Nogueira EM, Vinagre F, Masuda HP, Vargas C, de Pádua VLM, da Silva FR, dos Santos RV, Baldani JI, Ferreira PCG, Hemerley (2001) Expression of sugarcane genes induced by inoculation with Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans. Genet Mol Biol 24:99–206CrossRefGoogle Scholar
  11. Dias B, Cunha W, Morais L (2006) Expression of an oxalate decarboxylase gene from Flammulina sp. in transgenic lettuce (Lactuca sativa) plants and resistance to Sclerotinia sclerotiorum. Plant Pathol 55:187–193CrossRefGoogle Scholar
  12. Dutton MV, Evans CS (1996) Oxalate production by fungi: its role in pathogenicity and ecology in the soil environment. Can J Microbiol 42:881–895CrossRefGoogle Scholar
  13. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  14. Ferrari S, Plotnikova JM, De Lorenzo G, Ausubel FM (2003) Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but Not SID2, EDS5 or PAD4. Plant J 35:193–205CrossRefGoogle Scholar
  15. Feys BJ, Benedetti CE, Penfold CN, Turner JG (1994) Arabidopsis mutants selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. Plant Cell 6:751–759CrossRefGoogle Scholar
  16. Franceschi VR, Loewus FA (1995) Oxalate biosynthesis and function in plants and fungi. In: Khan SR (ed) Calcium oxalate in biological systems. CRC Press, Boca Raton, pp 113–130Google Scholar
  17. Godoy G, Steadman JR, Dickman MB, Dam R (1990) Use of mutants to demonstrate the role of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on Phaseolus vulgaris. Physiol Mol Plant Pathol 37:179–191CrossRefGoogle Scholar
  18. Guimarães RL, Stotz HU (2004) Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection. Plant Physiol 136:3703–3711CrossRefGoogle Scholar
  19. Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914CrossRefGoogle Scholar
  20. Heller A, Witt-Geiges T (2013) Oxalic acid has an additional, detoxifying function in Sclerotinia Sclerotiorum pathogenesis. PLoS ONE 8:e72292. CrossRefGoogle Scholar
  21. Hu X, Bidney DL, Yalpani N, Duvick JP, Crasta O, Folkerts O, Lu G (2003) Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower. Plant Physiol 133:170–181CrossRefGoogle Scholar
  22. Iniguez AL, Dong Y, Carter HD, Ahmer BMM, Stone JM, Triplett EW (2005) Regulation of enteric endophytic bacterial colonization by plant defenses. Mol Plant Microb Interact 18:169–178CrossRefGoogle Scholar
  23. Kabbage M, Williams B, Dickman MB (2013) Cell death control: the interplay of apoptosis and autophagy in the pathogenicity of Sclerotinia sclerotiorum. PLoS Pathog 9:e1003287. CrossRefGoogle Scholar
  24. Kesarwani M, Azam M, Natarajan K, Mehta A, Datta A (2000) Oxalate decarboxylase from Collybia velutipes. Molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato. J Biolog Chem 275:7230–7238CrossRefGoogle Scholar
  25. Kim KS, Min JY, Dickman MB (2008) Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development. Mol Plant Microbe Interact 21:605–612CrossRefGoogle Scholar
  26. Kloepper JW, Ryu CM, Zhang S (2004) Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathol 94:1259–1266CrossRefGoogle Scholar
  27. Kost TNS, Agnoli K, Eberl L, Weisskopf L (2014) Oxalotrophy, a widespread trait of plant-associated burkholderia species, is involved in successful root colonization of lupin and maize by Burkholderia phytofirmans. Front Microbiol 4:1–9CrossRefGoogle Scholar
  28. Kumar V, Chattopadhyay A, Ghosh S, Irfan M, Chakraborty N, Chakraborty S, Datta A (2015) Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase. Plant Biotechnol J. Google Scholar
  29. Kunz C, Vandelle E, Rolland S et al (2006) Characterization of a new, nonpathogenic mutant of Botrytis cinerea with impaired plant colonization capacity. New Phytol 170:537–550CrossRefGoogle Scholar
  30. Le Gall H, Philippe F, Domon JM, Gillet F, Pelloux J, Rayon C (2015) Cell wall metabolism in response to abiotic stress. Plants 4:112–166CrossRefGoogle Scholar
  31. Marciano P, Di Lenna P, Magro P (1983) Oxalic acid, cell wall degrading enzymes and pH in pathogenesis and their significance in the virulence of two Sclerotinia sclerotiorum isolates on sunflower. Physiol Plant Pathol 22:339–345CrossRefGoogle Scholar
  32. Marina M, Maiale SJ, Rossi FR, Romero MF, Rivas EI, Gárriz A, Ruiz OA, Pieckenstain FL (2008) Apoplastic polyamine oxidation plays different roles in local responses of tobacco to infection by the necrotrophic fungus Sclerotinia sclerotiorum and the biotrophic bacterium Pseudomonas viridiflava. Plant Physiol 147:2164–2178CrossRefGoogle Scholar
  33. Marina M, Vera Sirera F, Rambla JL, Gonzalez ME, Blázquez MA, Carbonell J, Pieckenstain FL, Ruiz OA (2013) Thermospermine catabolism increases the resistance to Pseudomonas viridiflava in Arabidopsis thaliana. J Exp Bot 64(5):1393–1402CrossRefGoogle Scholar
  34. Maxwell DP, Lumsden RD (1970) Oxalic acid production by Sclerotinia sclerotiorum in infected bean and in culture. Phytopathol 60:1395–1398CrossRefGoogle Scholar
  35. Miché L, Battistoni F, Gemmer S, Belghazi M, Reinhold-Hurek B (2006) Upregulation of jasmonate-inducible defense proteins and differential colonization of roots of Oryza sativa cultivars with the endophyte Azoarcus sp. Mol Plant Microbe Interact 19:502–511CrossRefGoogle Scholar
  36. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  37. Nardi CF, Villarreal NM, Rossi FR, Martínez S, Martínez GA, Civello PM (2015) Overexpression of the carbohydrate binding module of strawberry expansin2 in Arabidopsis thaliana modifies plant growth and cell wall metabolism. Plant Mol Biol 88:101–117CrossRefGoogle Scholar
  38. Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New YorkGoogle Scholar
  39. Noyes RD, Hancock JG (1981) Role of oxalic acid in the Sclerotinia sclerotiorum wilt of sunflower Helianthus annuus. Physiol Plant Pathol 18:123–132CrossRefGoogle Scholar
  40. Partridge-Telenko DE, Hu J, Livingstone DM, Shew BB, Phipps PM, Grabau E (2011) Sclerotinia blight resistance in Virginia-type peanut transformed with a barley oxalate oxidase gene. Phytopathol 101:786–793CrossRefGoogle Scholar
  41. Perini MA, Sin IN, Villarreal NM, Marina M, Powell ALT, Martínez GA, Civello PM (2017) Overexpression of the carbohydrate 1 binding module from Solanum lycopersicum Expansin 1 (Sl-EXP1) modifies tomato fruit firmness and Botrytis cinerea susceptibility. Plant Physiol Biochem 113:122–132CrossRefGoogle Scholar
  42. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefGoogle Scholar
  43. Pfaffl M, Horgan G, Dempfle L (2002) Relative expression software tool 24 (REST(C)) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36CrossRefGoogle Scholar
  44. Ramirez V, Agorio A, Coego A, Garcia-Andrade J, Hernandez MJ, Balaguer B, Ouwerkerk PBF, Zarra I, Vera P (2011) MYB46 modulates disease susceptibility to Botrytis cinerea in Arabidopsis. Plant Physiol 155(4):1920–1935CrossRefGoogle Scholar
  45. Romero FM, Marina M, Pieckenstain FL (2016) Novel components of leaf bacterial communities of field-grown tomato plants and their potential for plant growth promotion and biocontrol of tomato diseases. Res Microbiol 167:222–233CrossRefGoogle Scholar
  46. Romero FM, Rossi FR, Gárriz A, Carrasco P, Ruíz OA (2019) A bacterial endophyte from apoplast fluids protects canola plants from different phytopathogens via antibiosis and induction of host resistance. Phytopathol 109(3):375–383CrossRefGoogle Scholar
  47. Rosenblueth M, Martinez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant Microbe Interact 19:827–837CrossRefGoogle Scholar
  48. Rossi FR, Gárriz A, Marina M, Romero FM, Gonzalez ME, Collado I, Pieckenstain FL (2011) The sesquiterpene botrydial produced by Botrytis cinerea induces the hypersensitive response and its toxicity is modulated by host signaling pathways mediated by salicylic acid and jasmonic acid. Mol Plant Microbe Interact 24:888–896CrossRefGoogle Scholar
  49. Rossi FR, Marina M, Pieckenstain FL (2015) Role of arginine decarboxylase (ADC) in Arabidopsis thaliana defence against the pathogenic bacterium Pseudomonas viridiflava. Plant Biol 17:831–839CrossRefGoogle Scholar
  50. Ryan RP, Monchy S, Cardinale M, Taghavi S, Crossman L, Avison MB, Berg G, van der Lelie D, Dow JM (2009) The versatility and adaptation of bacteria from the genus Stenotrophomonas. Nat Rev Microbiol 7:514–525CrossRefGoogle Scholar
  51. Sahin N (2003) Oxalotrophic bacteria. Res Microbiol 154:399–407CrossRefGoogle Scholar
  52. Schoonbeek H, Jacquat-Bovet AC, Mascher F, Métraux JP (2007) Oxalate-degrading bacteria can protect Arabidopsis thaliana and crop plants against Botrytis cinerea. Mol Plant Microbe Interact 20:1535–1544CrossRefGoogle Scholar
  53. Sneath PH, Sokal RR (1973) Numerical taxonomy. W. H. Freeman and Co, San FranciscoGoogle Scholar
  54. Stone JM, Heard JE, Asai T, Ausubel FM (2000) Simulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants. Plant Cell 12:1811–1822CrossRefGoogle Scholar
  55. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefGoogle Scholar
  56. Thomma BP, Eggermont K, Penninckx IAMA, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF (1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. PNAS 95:15107–15111CrossRefGoogle Scholar
  57. Thomma BP, Eggermont K, Tierens KFMJ, Broekaert WF (1999) Requirement of functional Ethylene-Insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea. Plant Physiol 121:1093–1101CrossRefGoogle Scholar
  58. Versalovic J, Schneider M, De Bruijn F, Lupski J (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5:25e40Google Scholar
  59. Villarreal NM, Marina M, Nardi CF, Civello PM, Martínez GA (2016) Novel insights of ethylene role in strawberry cell wall metabolism. Plant Sci 252:1–11CrossRefGoogle Scholar
  60. Vogel J, Somerville S (2000) Isolation and characterization of powdery mildew-resistant Arabidopsis mutants. PNAS 97:1897–1902CrossRefGoogle Scholar
  61. Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414:562–565CrossRefGoogle Scholar
  62. Williams B, Kabbage M, Kim HJ, Britt R, Dickman MB (2011) Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment. PLoS Pathog 7:e1002107. CrossRefGoogle Scholar
  63. Zhao-Xia J, Wang C, Chen W, Chen X, Li X (2007) Induction of oxalate decarboxylase by oxalate in a newly isolated Pandoraea Sp. OXJ-11 and its ability to protect against Sclerotinia sclerotiorum infection. Can J Microbiol 53:1316–1322CrossRefGoogle Scholar
  64. Zhou T, Boland GJ (1999) Mycelial growth and production of oxalic acid by virulent and hypovirulent isolates of Sclerotinia sclerotiorum. Can J Plant Pathol 21:93–99CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • María Marina
    • 1
    Email author
  • Fernando M. Romero
    • 1
  • Natalia M. Villarreal
    • 1
  • Andrés J. Medina
    • 2
  • Andrés Gárriz
    • 1
  • Franco R. Rossi
    • 1
  • Gustavo A. Martinez
    • 1
    • 3
  • Fernando L. Pieckenstain
    • 1
  1. 1.Instituto Tecnológico ChascomúsUniversidad Nacional de General San Martín-Consejo Nacional de Investigaciones Científicas y Técnicas (INTECH/UNSAM-CONICET)ChascomúsArgentina
  2. 2.Centro de Investigaciones Cardiovasculares “Horacio Cingolani” Facultad de Ciencias Médicas (UNLP)La PlataArgentina
  3. 3.Instituto de Fisiología Vegetal (INFIVE), Facultad de Ciencias Agrarias y Forestales - Facultad de Ciencias Naturales y Museo (UNLP-CONICET)La PlataArgentina

Personalised recommendations