Tropical Plant Pathology

, Volume 43, Issue 4, pp 289–296 | Cite as

Low-molecular-weight metabolites produced by Pseudomonas aeruginosa as an alternative to control Huanglongbing in Citrus sinensis cv. Valencia

  • Juliana F. Pistori
  • Ane S. Simionato
  • Miguel O. P. Navarro
  • Matheus F. L. Andreata
  • Igor M. O. Santos
  • Luciana Meneguim
  • Rui P. Leite Junior
  • Admilton G. Oliveira
  • Galdino AndradeEmail author
Original Article


Huanglongbing (HLB) is the most destructive disease of citrus worldwide and an efficient management strategy to control it has not yet been established. The potential of pseudomonads to suppress plant pathogens is well known and the secondary metabolites they produce represent new alternatives of compounds to control plant diseases. The main challenge is to find new compounds that show strong antibiotic activity, low toxicity to plants and little or no harm to the environment. The objectives of the present study were to determine the potential of the F4A fraction from Pseudomonas aeruginosa to control HLB and to induce systemic resistance. Low molecular weight compounds with antimicrobial activity were purified with organic solvent, thin layer chromatography (TLC) and normal and reverse phase chromatography. Compounds present in the F4A fraction were mainly obtained by thin-layer chromatography (TLC) and Preparative-High Performance Liquid Chromatography (HPLC-prep). To assess their biological activities, conventional and quantitative polymerase chain reaction were usxed. The F4A was sprayed on citrus trees infected with the causal agent of HLB, ‘Candidatus Liberibacter asiaticus’ under greenhouse conditions. The bacterial titers were reduced and defense genes were induced in leaves of trees treated with F4A, as assed by PCR analysis. The results showed that F4A (pseudomonads secondary metabolites) may provide a useful tool for the management of HLB.


Natural products Systemic acquired resistance (SAR) Citrus Secondary metabolites 



The authors thank CAPES Foundation for scholarships, the National Research Council (CNPq) for grants that allow to carry out the studies and FUNDECITRUS for technical support.

Supplementary material

40858_2018_231_MOESM1_ESM.docx (5.7 mb)
ESM 1 (DOCX 5838 kb)


  1. Andrade G (2008) Processo de produção, purificação e obtenção de substâncias com atividades antibióticas para o controle de doenças causadas por bactérias em plantas (Process of production, purification and obtaining substances with antibiotic activity to control disease caused by bacteria in plants), #PI0803350–1 A2. Available at
  2. Audenaert K, De Meyer G, Hofte M (2002b) Abscisic acid determines basal susceptibility of tomato to Botrytis cinerea and suppresses salicylic acid-dependent signaling mechanisms. Plant Physiology 128:491–501CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bassanezi RB, Montesino LH, Gimenes-Fernandes N, Yamamoto PT, Gottwald TR, Amorim L, Filho AB (2013) Efficacy of area-wide inoculum reduction and vector control on temporal progress of Huanglongbing in young sweet orange plantings. Plant Disease 97:789–796Google Scholar
  4. Belasque J, Bergamin A, Bassanezi RB, Barbosa JC, Fernandes NG, Yamamoto PT, Lopes SA, Machado MA, Leite RP, Ayres AJ, Massari CA (2009) Base científica para a erradicação de plantas sintomáticas e assintomáticas de Huanglongbing (HLB, Greening) visando o controle efetivo da doença. Tropical Plant Pathology 34:137–145Google Scholar
  5. Belasque J, Bassanesi RB, Yamamoto PT, Ayres AJ, Tachibana A, Violante AR, Tank A, Di Giorgi F, Tersi FEA, Menezes GM, Dragone J, Jank RH, Bové JM (2010) Lessons from Huanglongbing management in São Paulo state, Brazil. Journal of Plant Pathology 92:285–302Google Scholar
  6. Bigirimana J, Hofte M (2002) Induction of systemic resistance to Colletotrichum lindemuthianum in bean by a benzothiadiazole derivative and rhizobacteria. Phytoparasitica 30:159–168CrossRefGoogle Scholar
  7. Bové JM (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology 88:7–37Google Scholar
  8. Cardozo VF, de Oliveira AG, Nishio EK, Perugini MRE, Andrade CGTJ, Silveira WD, Durán N, Andrade G, Kobayashi RKT, Nakazato G (2013) Antibacterial activity of extracellular compounds produced by Pseudomonas strain against methicillin-resistant Staphylococcus aureus (MRSA) strains. Annals of Clinical Microbiology and Antimicrobials 12:1–8Google Scholar
  9. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162:156–159CrossRefPubMedGoogle Scholar
  10. Compant S, Reiter B, Sessitsch A, Nowak J, Clement C, Ait Barka E (2005) Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Applied and Environmental Microbiology 71:1685–1693CrossRefPubMedPubMedCentralGoogle Scholar
  11. Dhanasekaran S, Doherty TM, Kenneth J (2010) Comparison of different standards for real-time PCR-based absolute quantification. Journal of Immunological Methods 354:34–39CrossRefPubMedGoogle Scholar
  12. FAO (1994) Soil map of the world. Revised legend with corrections. FAO-UNESCO ISRIC, RomeGoogle Scholar
  13. Francis MI, Redondo A, Burns JK, Graham JH (2009) Soil application of imidacloprid and related SAR-inducing compounds produces effective and persistent control of citrus canker. European Journal of Plant Pathology 124:283–292CrossRefGoogle Scholar
  14. Gottwald TR, Graham JH, Irey MS, McCollum TG, Wood BW (2012) Inconsequential effect of nutritional treatments on Huanglongbing control, fruit quality, bacterial titer and disease progress. Crop Protection 36:73–82CrossRefGoogle Scholar
  15. Hammond-Kosack KE, Parker JE (2003) Deciphering plant-pathogen communication: fresh perspectives for molecular resistance breeding. Current Opinion in Biotechnology 14:177–193CrossRefPubMedGoogle Scholar
  16. Heil M, Bostock RM (2002) Induced Systemic Resistance (ISR) Against Pathogens in the Context of Induced Plant Defences. Annals Botany 89:503–512Google Scholar
  17. Hocquellet A, Toorawa P, Bové JM, Garnier M (1999) Detection and identification of the two Candidatus Liberobacter species associated with citrus Huanglongbing by PCR amplification of ribosomal protein genes of the b operon. Molecular and Cellular Probes 13:373–379CrossRefPubMedGoogle Scholar
  18. ivak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408Google Scholar
  19. Kerbauy G, Vivan ACP, Simoes GC, Simionato AS, Pelisson M, Vespero EC, Costa SF, Andrade CGTJ, Barbieri DM, Mello JCP, Morey AT, Lioni LMY, Ogatta SFY, Oliveira AG, Andrade G (2016) Effect of a metalloantibiotic produced by Pseudomonas aeruginosa on Klebsiella pneumoniae carbapenemase (KPC)-producing. Current Pharmacology and Biotechnology 17:389–397Google Scholar
  20. Li W, Hartung JS, Levy L (2006) Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus Huanglongbing. Journal Microbiological Methods 66:104–115CrossRefGoogle Scholar
  21. Lopes LP, Oliveira Jr AG, Beranger JPO, Góis CG, Vasconcellos FCS, Martin JABS, Andrade CGTJ, Mello JCP, Andrade G (2012) Activity of extracellular compounds of Pseudomonas sp. against Xanthomonas axonopodis in vitro and bacterial leaf blight in eucalyptus. Tropical Plant Pathology 37:233–238Google Scholar
  22. Louws FJ, Wilson M, Campbell HL (2001) Field control of bacterial spot and bacterial speck of tomato using a plant activator. Plant Disease 85:481–488CrossRefGoogle Scholar
  23. Ma Z, Hua GKH, Ongena M, Höfte M (2016) Role of phenazines and cyclic lipopeptides produced by Pseudomonas sp. CMR12a in induced systemic resistance on rice and bean. Environmental Microbiology Reports 8:896–904CrossRefPubMedGoogle Scholar
  24. Maurhofer M, Hase C, Meuwly P, Métraux J-P, Défago G (1994) Induction of systemic resistance of tobacco to tobacco necrosis virus by the root-colonizing Pseudomonas fluorescens strain CHA0: influence of the gacA gene and of pyoverdine production. Phytopathology 84:139–146Google Scholar
  25. de Meyer G, Capiau K, Audenaert K, Buchala A (1999b) Nanogram amounts of salicylic acid produced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 activate the systemic acquired resistance pathway on bean. Molecular Plant-Microbe Interactions 12:450–459CrossRefPubMedGoogle Scholar
  26. de Meyer G, Hofte M (1997) Salicylic acid produced by the rhizobacterium Pseudomonas aeruginosa 7SNK2 induces resistance to leaf infection by Botrytis cinerea on bean. Phytopathology 87:588–593CrossRefPubMedGoogle Scholar
  27. Morrison CK, Arseneault T, Novinscak A, Filion M (2017) Phenazine-1-carboxylic acid production by Pseudomonas fluorescens LBUM636 alters Phytophthora infestans growth and late blight development. Biological Control 107:273–279Google Scholar
  28. Munhoz LD, Fonteque JP, Santos IMO, Navarro MOP, Simionato AS, Goya ET, Rezende MI, Balbi-Peña MI, De Oliveira AG, Andrade G (2017) Control of bacterial stem rot on tomato by extracellular bioactive compounds produced by Pseudomonas aeruginosa LV strain. Congent Food and Agriculture 3:1–16Google Scholar
  29. Murray HG, Thompson WF (1980) Rapid isolation of high molecular weight DNA. Nucleic Acids Research 8:4321–4325CrossRefPubMedPubMedCentralGoogle Scholar
  30. de Oliveira AG, Spago FR, Simionato AS, Navarro MOP, Silva CS, Barazetti AR, Cely MVT, Tischer CA, San Martin JAB, Andrade CGTJ, Novello CR, Mello JCP, Andrade G (2016) Bioactive organocopper compound from Pseudomonas aeruginosa inhibits the growth of Xanthomonas citri subsp. Citri. Frontiers in Microbiology 7:1–12Google Scholar
  31. de Oliveira Júnior AG, Murate LS, Souza PB, Spago FR, Lopes LP Beranger JPO, San Martin JAB, Nogueira MA, Mello JCP, Andrade CGTJ, Andrade G (2011) Evaluation of the antibiotic activity of extracellular compounds produced by the Pseudomonas strain against the Xanthomonas citri pv. citri 306 strain. Biological Control 56:125–131Google Scholar
  32. Pierson III LS, Pierson EA (2010) Metabolism and function of bacteria in the environment and biotechnological processes. Applied Microbiology and Biotechnology 86:1659–1670CrossRefPubMedPubMedCentralGoogle Scholar
  33. Pozo MJ, Jung SC, López-Ráez JA, Azcón-Aguilar C (2010) Impact of arbuscular mycorrhizal symbiosis on plant response to biotic stress: the role of plant defense mechanisms. In: Koltai H, Kapulnik Y (eds) Arbuscular mycorrhizas: physiology and function. Springer, New York, pp 3–31Google Scholar
  34. Rampazo, LGL (2004) Evaluation of the Effect of Biological Agents and Their Products into the Incidence of Citrus Canker Lesions. Master’s, Universidade Estadual de LondrinaGoogle Scholar
  35. Schlumbaum A, Mauch F, Vogeli U, Boller T (1986) Plant chitinases are potent inhibitors of fungal growth. Nature 324:365–367CrossRefGoogle Scholar
  36. Simionato AS, Navarro MOP, Jesus MLA, Barazetti AR, Silva CS, Simões GC, Balbi-Peña MI, Mello JCP, Panagio LA, Almeida RSC, Andrade G, De Oliveira AG (2017) The effect of phenazine-1-carboxylic acid on mycelial growth of Botrytis cinerea produced by Pseudomonas aeruginosa LV strain. Frontiers in Microbiology 8:1–9Google Scholar
  37. Spago FR, Mauro CSI, Oliveira AG, Beranger JPO, Cely MVT, Stanganelli MM, Simionato AS, San Martin JAB, Andrade CGTJ, Mello JCP, Andrade G (2014) Pseudomonas aeruginosa produces secondary metabolites that have biological activity against plant pathogenic Xanthomonas species. Crop Protection 62:46–54Google Scholar
  38. van Loon LC, van Strien EA (1999) The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology 55:85–97CrossRefGoogle Scholar
  39. Vasconcellos FCS, de Oliveira AG, Lopes-Santos L, Beranger JPO, Cely MVT, Simionato AS, Pistori JF, Spago FR; Mello JCP, San Martin JAB, Andrade CGTJ, Andrade G (2014) Evaluation of antibiotic activity produced by Pseudomonas aeruginosa LV strain against Xanthomonas arboricola pv. pruni. Agricultural Sciences 5:71–76Google Scholar
  40. Wang C, El-Shetehy M, Shine MB, Yu K, Navarre D, Wendehenne D, Kachroo A, Kachroo P (2014). Free radicals mediate systemic acquired resistance. Cell Report 24:348–55Google Scholar

Copyright information

© Sociedade Brasileira de Fitopatologia 2018

Authors and Affiliations

  • Juliana F. Pistori
    • 1
    • 2
  • Ane S. Simionato
    • 1
  • Miguel O. P. Navarro
    • 1
  • Matheus F. L. Andreata
    • 1
  • Igor M. O. Santos
    • 1
  • Luciana Meneguim
    • 2
  • Rui P. Leite Junior
    • 2
  • Admilton G. Oliveira
    • 1
  • Galdino Andrade
    • 1
    Email author
  1. 1.CCB, Department of Microbiology, Microbial Ecology LaboratoryUniversidade Estadual de LondrinaLondrinaBrazil
  2. 2.Bacteriology LaboratoryInstituto Agronômico do ParanáLondrinaBrazil

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