Pseudomonas simiae effects on the mycotoxin formation by fusaria and alternaria in vitro and in a wheat field

  • Thomas MüllerEmail author
  • Peter Lentzsch
  • Undine Behrendt
  • Dietmar Barkusky
  • Marina E. H. Müller
Original Article


Fluorescent pseudomonads colonizing wheat ears have a high antagonistic potential against phytopathogenic fungi. To check this hypothesis, the bacterial antagonist Pseudomonas simiae 9 rif+/kan+ was spray-inoculated onto the ears of winter wheat in a locally demarcated experimental field plot. Fusarium and Alternaria fungi naturally occurring on the ears and the formation of their mycotoxins in the ripe grains were investigated. Inoculated bacteria were recovered from the plants in the inoculation cell, but not in the untreated neighboring plots or in the air above the plants. Growth of fusaria and alternaria on the ears was not influenced by the bacterial antagonist. Wheat kernels were co-inoculated in vitro with the antagonist and one mycotoxin-producing strain of Fusarium and Alternaria, respectively. Mycotoxin production was almost completely suppressed in these approaches. Concentrations of zearalenone, deoxynivalenol, alternariol, and tenuazonic acid were also significantly reduced in ripe grains in the field, but to a lesser extent than in vitro. The results of this and previous studies suggest that widespread biological control of the growth of fusaria and alternaria and their mycotoxin formation by naturally occurring pseudomonads with antagonistic activity is rather unlikely.


Biological control Wheat Pseudomonas Fusarium Alternaria Mycotoxins 





Alternariol monomethyl ether




Colony-forming units




Dry mass


Fresh mass


High-performance liquid chromatography


Microbial biocontrol agents




Tenuazonic acid





We thank Petra Lange, Martina Peters, and Grit von der Waydbrink, for excellent technical assistance.We are also grateful to E. Garcia-Valdes and J. Lalucat (Balearic Islands University) for rpoD gene sequencing of the antagonist used in this study.

Source of funding

This work was financially supported by Deutsche Forschungsgemeinschaft in the framework of the BioMove Research Training Group (DFG-GRK 2118/1).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12550_2019_379_MOESM1_ESM.pptx (74 kb)
ESM_1. pptx: Field plot design. (PPTX 74 kb)
12550_2019_379_MOESM2_ESM.pptx (89 kb)
ESM_2. pptx: Temperature and rainfall during field investigations in 2017 and 2018 (PPTX 88 kb)
12550_2019_379_MOESM3_ESM.xlsx (20 kb)
ESM_3. xlsx:In vitro-antagonism test on a natural substrate with Fusarium graminearum 23 and Pseudomonas simiae 9rif+/kan+. Microbiological and mycotoxin data (XLSX 20 kb)
12550_2019_379_MOESM4_ESM.xlsx (19 kb)
ESM_4. xlsx:In vitro-antagonism test on a natural substrate with Alternaria tenuissima 220 and Pseudomonas simiae 9rif+/kan+. Microbiological and mycotoxin data (XLSX 19 kb)
12550_2019_379_MOESM5_ESM.xlsx (31 kb)
ESM_5. xlsx: Field experiments in 2017 and 2018. Microbiological data (XLSX 31 kb)
12550_2019_379_MOESM6_ESM.pptx (238 kb)
ESM_6. pptx: Field experiments. Densities of culturable filamentous fungi on wheat ears. (PPTX 238 kb)


  1. Altalhi AD, El-Deeb B (2008) Localization of zearalenone detoxification gene(s) in pZEA-1 plasmid of Pseudomonas putida ZEA-1 and expressed in Escherichia coli. J Hazard Mater 161:1166–1172. CrossRefPubMedGoogle Scholar
  2. Bale JS, van Lenteren JC, Bigler F (2008) Biological control and sustainable food production. Philos Trans R Soc B 363:761–776. CrossRefGoogle Scholar
  3. Berg G, Zachow C, Müller H, Philipps J, Tilcher R (2013) Next-generation bio-products sowing the seeds of success for sustainable agriculture. Agronomy 3:648–656. CrossRefGoogle Scholar
  4. Berg G, Grube M, Schloter M, Smalla K (2014) Unraveling the plant microbiome: looking back and future perspectives. Front Microbiol 5:48. CrossRefGoogle Scholar
  5. Bottalico A, Perrone G (2002) Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain cereals in Europe. Eur J Plant Pathol 108:611–624CrossRefGoogle Scholar
  6. Bulgarelli D, Schlaeppi K, Spaepen S, Ver Loren van Themaat E, Schulze-Lefert P (2013) Structure and functions of the bacterial microbiota of plants. Annu Rev Plant Biol 4:807–838. CrossRefGoogle Scholar
  7. Bundessortenamt (2019) Beschreibende Sortenliste – Getreide, Mais, Öl- und Faserpflanzen, Leguminosen, Rüben, Zwischenfrüchte. Bundessortenamt (Ed.), ISSN 2190-6130Google Scholar
  8. Cock MJW, van Lenteren JC, Brodeur J, Barratt BIP, Bigler F, Bolckmans K, Cônsoli FL, Haas F, Mason PG, Parra JRP (2010) Do new access and benefit sharing procedures under the convention on biological diversity threaten the future of biological control? BioControl 55:199–218. CrossRefGoogle Scholar
  9. European Commission (EC) (2006) Commission Regulation (EC) no. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Union L364:5–24Google Scholar
  10. Figueroa M, Hammond-Kosack KE, Solomon PS (2018) A review of wheat diseases - a field perspective. Mol Plant Pathol 19:1523–1536. CrossRefPubMedGoogle Scholar
  11. García-Valdés E, Lalucat J (2016) Pseudomonas: molecular phylogeny and current taxonomy. In: Kahlon RS (ed) Pseudomonas: Molecular and Applied Biology. Springer International Publishing, Cham, pp 1–23Google Scholar
  12. Gopal M, Gupta A (2016) Microbiome selection could spur next-generation Plant Breeding Strategies. Front Microbiol 7:1971. CrossRefPubMedPubMedCentralGoogle Scholar
  13. He J, Boland GJ, Zhou T (2009) Concurrent selection for microbial suppression of Fusarium graminearum, Fusarium head blight and deoxynivalenol in wheat. J Appl Microbiol 106:1805–1817. CrossRefPubMedGoogle Scholar
  14. Hirano SS, Upper CD (1993) Dynamics, spread, and persistence of single genotype of Pseudomonas syringae relative to those of its conspecifics on populations of snap bean leaflets. Appl Environ Microbiol 59:1082–1091PubMedPubMedCentralGoogle Scholar
  15. Kahl SM, Ulrich A, Kirichenko AA, Müller MEH (2016) Phenotypic and phylogenetic segregation of Alternaria infectoria from small-spored Alternaria species isolated from wheat in Germany and Russia. J Appl Microbiol 119:1637–1650. CrossRefGoogle Scholar
  16. Kinkel LL, Wilson M, Lindow SE (1996) Utility of microcosm studies for predicting phylloplane bacterium population sizes in the field. Appl Environ Microbiol 62:3413–3423PubMedPubMedCentralGoogle Scholar
  17. Korn U, Müller T, Ulrich A, Müller MEH (2011) Impact of aggressiveness of Fusarium graminearum and F. culmorum isolates on yield parameters and mycotoxin production in wheat. Mycotoxin Res 27:195–206. CrossRefPubMedGoogle Scholar
  18. Lally RD, Galbally P, Moreira AS, Spink J, Ryan D, Germaine KJ, Dowling DN (2017) Application of endophytic Pseudomonas fluorescens and a bacterial consortium to Brassica napus can increase plant height and biomass under greenhouse and field conditions. Front Plant Sci 8:2193. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lancashire PD, Bleiholder H, Van den Boom T, Langelüddeke P, Stauss R, Weber E, Witzenberger A (1991) A uniform decimal code for growth stages of crops and weeds. Ann Appl Biol 119:561–601. CrossRefGoogle Scholar
  20. Logrieco A, Moretti A, Solfrizzo M (2009) Alternaria toxins and plant diseases: an overview of origin, occurrence and risks. World Mycotoxin J 2:129–140. CrossRefGoogle Scholar
  21. Magan N, Lacey J (1986) The phylloplane microflora of ripening wheat and effect of late fungicide applications. Ann Appl Biol 109:117–128. CrossRefGoogle Scholar
  22. Massart S, Perazzolli M, Höfte M, Pertot I, Jijakli MH (2015) Impact of the omic technologies for understanding the modes of action of biological control agents against plant pathogens. BioControl 60:725–746. CrossRefGoogle Scholar
  23. McMullen M, Bergstrom G, de Wolf E, Dill-Macky R, Hershman D, Shaner G, van Sanford D (2012) A united effort to fight an enemy of wheat and barley: Fusarium head blight. Plant Dis 96:1712–1728. CrossRefPubMedGoogle Scholar
  24. McSpadden Gardener BB (2007) Diversity and ecology of biocontrol Pseudomonas spp. in agricultural systems. Phytopathology 97:221–226. CrossRefPubMedGoogle Scholar
  25. Müller MEH, Korn U (2013) Alternaria mycotoxins in wheat - a 10 years survey in the Northeast of Germany. Food Control 34:191–197. CrossRefGoogle Scholar
  26. Müller MEH, Brenning A, Verch G, Koszinski S, Sommer M (2010) Multifactorial spatial analysis of mycotoxin contamination of winter wheat at the field and landscape scale. Agric Ecosyst Environ 139:245–254. CrossRefGoogle Scholar
  27. Müller MEH, Steier I, Köppen R, Siegel D, Proske M, Korn U, Koch M (2012) Cocultivation of phytopathogenic Fusarium and Alternaria strains affects fungal growth and mycotoxin production. J Appl Microbiol 113:874–887. CrossRefPubMedGoogle Scholar
  28. Müller T, Behrendt U, Ruppel S, von der Waydbrink G, Müller MEH (2016a) Fluorescent pseudomonads in the phyllosphere of wheat: potential antagonists against fungal phytopathogens. Curr Microbiol 72:383–389. CrossRefPubMedGoogle Scholar
  29. Müller MEH, Koszinski S, Bangs DE, Wehrhan M, Ulrich A, Verch G, Brenning A (2016b) Crop biomass and humidity related factors reflect the spatial distribution of phytopathogenic Fusarium fungi and their mycotoxins in heterogeneous fields and landscapes. Precis Agric 17:698–720. CrossRefGoogle Scholar
  30. Müller T, Ruppel S, Behrendt U, Lentzsch P, Müller MEH (2018) Antagonistic potential of fluorescent pseudomonads colonizing wheat heads against mycotoxin producing alternaria and fusaria. Front Microbiol 9:2124. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Nicolaisen M, Justesen AF, Knorr K, Wang J, Pinnschmidt HO (2014) Fungal communities in wheat grain show significant co-existence patterns among species. Fungal Ecol 11:145–153. CrossRefGoogle Scholar
  32. Ostry V (2008) Alternaria mycotoxins: An overview of chemical characterization, producers, toxicity, analysis and occurrence in foodstuffs. World Mycotoxin J 1:175–188. CrossRefGoogle Scholar
  33. Palazzini JM, Ramirez ML, Torres AM, Chulze SN (2007) Potential biocontrol agents for Fusarium head blight and deoxynivalenol production in wheat. Crop Prot 26:1702–1710. CrossRefGoogle Scholar
  34. Palazzini JM, Alberione E, Torres A, Donat C, Köhl J, Chulze SN (2016) Biological control of Fusarium graminearum sensu stricto, causal agent of Fusarium head blight of wheat, using formulated antagonists under field conditions in Argentina. Biol Control 94:56–61. CrossRefGoogle Scholar
  35. Pan D, Mionetto A, Tiscornia S, Bettucci L (2015) Endophytic bacteria from wheat grain as biocontrol agents of Fusarium graminearum and deoxynivalenol production in wheat. Mycotoxin Res 31:137–143. CrossRefPubMedGoogle Scholar
  36. Pretty J (2008) Agricultural sustainability: concepts, principles and evidence. Philos Trans R Soc B 363:447–465. CrossRefGoogle Scholar
  37. Samuel MS, Sivaramakrishna A, Mehta A (2014) Degradation and detoxification of aflatoxin B1 by Pseudomonas putida. Int Biodeterior Biodegradation 86:202–209. CrossRefGoogle Scholar
  38. Schiro G, Verch G, Grimm V, Müller MEH (2018a) Alternaria and Fusarium fungi: differences in distribution and spore deposition in a topographically heterogeneous wheat field. J Fungi 4:63. CrossRefGoogle Scholar
  39. Schiro G, Müller T, Verch G, Sommerfeld T, Mauch T, Koch M, Grimm V, Müller MEH (2018b) The distribution of mycotoxins in a heterogeneous wheat field in relation to microclimate, fungal and bacterial abundance. J Appl Microbiol 126:177–190. CrossRefPubMedGoogle Scholar
  40. Schisler DA, Khan NI, Boehm MJ, Lipps PE, Slininger PJ, Zhang S (2006) Selection and evaluation of the potential of choline-metabolizing microbial strains to reduce Fusarium head blight. Biol Control 39:497–506. CrossRefGoogle Scholar
  41. Schlaeppi K, Bulgarelli D (2015) The plant microbiome at work. Mol Plant-Microbe Interact 28:212–217. CrossRefPubMedGoogle Scholar
  42. Shi C, Yan P, Li J, Wu H, Li Q, Guan S (2014) Biocontrol of Fusarium graminearum growth and deoxynivalenol production in wheat kernels with bacterial antagonists. Int J Environ Res Public Health 11:1094–1105. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Stockwell VO, Stack JP (2007) Using Pseudomonas spp. for integrated biological control. Phytopathology 97:244–249. CrossRefPubMedGoogle Scholar
  44. van Lenteren JC, Bolckmans K, Köhl J, Ravensberg WJ, Urbaneja A (2018) Biological control using invertebrates and microorganisms: plenty of new opportunities. BioControl 63:39–59. CrossRefGoogle Scholar
  45. Vanhoutte I, Audenaert K, De Gelder L (2016) Biodegradation of mycotoxins: tales from known and unexplored worlds. Front Microbiol 7:561. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Vučković JN, Brkljača JS, Bodroža-Solarov MI, Bagi FF, Stojšin VB, Ćulafić JN, Aćimović MG (2012) Alternaria spp. on small grains. Food and Feed Research 39:79–88Google Scholar
  47. Yang F, Jacobsen S, Jørgensen HJL, Collinge DB, Svensson B, Finnie C (2013) Fusarium graminearum and its interactions with cereal heads: studies in the proteomics era. Front Plant Sci 4:37. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Zhao Y, Selvaraj JN, Xing F, Zhou L, Wang Y, Song H, Tan X, Sun L, Sangare L, Folly YME, Liu Y (2014) Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum. PLoS One 9(3):e92486. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Leibniz-Centre for Agricultural Landscape Research (ZALF)MünchebergGermany

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