The biological control of plant diseases by application of antagonistic microorganisms to

the plant phyllosphere is an alternative strategy to prevent the frequent treatment of plants

by pesticides. Microbiological antagonists can firstly interact directly against the pathogen

by releasing antimicrobial compounds and/or secondly induce the plant resistance of the

host plant by expression of pathogenesis-related proteins (PR proteins). The focus of our

study is on the interaction of the non-pathogenic bacterium Pseudomonas fluorescens Bk3

to the plant phyllosphere of Malus domestica cv. Holsteiner Cox. After application of P.

fluorescens Bk3 to the phyllosphere of M. domestica cv. Holsteiner Cox we observed

dramatic changes in the protein composition of the apoplast of the host plant. Sequencing

of the induced proteins by ESI-Q-ToF mass spectrometry and homology search identified

these additional proteins as pathogenesis related proteins (PR) like ß-1,3- glucanase,

thaumatin-like protein, chitinase and hevein-like protein. To confirm these findings, a

suppressive subtractive hybridization with total RNA from leaves before and after

inoculation of P. fluorescens Bk3 to the leaves of the host plant was performed. It revealed

an increased expression level of many PR and stress related genes.

The induction of PR proteins and plant defence genes in host plants after application of

non-pathogenic bacterial antagonists to the plant phylloshere can presumably prevent or

reduce successful infections by plant pathogens

Gau, A. E.

Kürkcüoglu, S.

Schmoock, S.

English

Organic Eprints

306________________________________________________________________ 1Institute  of  Botany,  Leibniz  University  Hannover,  Herrenhäuserstr.  2,  D-30419  Hannnover,  e-mail: achim.gau@botanik.uni-hannover.de Biological control of apple scab and fire blight by the application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3  to the leaf surface S. Schmoock1, S. Kürkcüoglu1 and A. E. Gau1AbstractThe biological control of plant diseases by application of antagonistic microorganisms to the plant phyllosphere is an alternative strategy to prevent the frequent treatment of plants by pesticides. Microbiological antagonists can firstly interact directly against the pathogen by releasing antimicrobial compounds and/or secondly induce the plant resistance of the host plant by expression of pathogenesis-related proteins (PR proteins). The focus of our study is on the interaction of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the plant phyllosphere of Malus domestica cv. Holsteiner Cox. After application of P.fluorescens  Bk3  to  the  phyllosphere  of  M.  domestica  cv.  Holsteiner  Cox  we  observed dramatic changes in the protein composition of the apoplast of the host plant. Sequencing of the induced proteins by ESI-Q-ToF mass spectrometry and homology search identified these  additional  proteins  as  pathogenesis  related  proteins  (PR)  like  ß-1,3-  glucanase, thaumatin-like  protein,  chitinase  and  hevein-like  protein.  To  confirm  these  findings,  a suppressive  subtractive  hybridization  with  total  RNA  from  leaves  before  and  after inoculation of P. fluorescens Bk3 to the leaves of the host plant was performed. It revealed an increased expression level of many PR and stress related genes.The induction of PR proteins and plant defence genes in host plants after application of non-pathogenic bacterial antagonists to the plant phylloshere can presumably prevent or reduce successful infections by plant pathogens. Keywords: biological control, bioluminescence, Erwinia amylovora, pathogenesis related proteins, Venturia inaequalisIntroductionApple  scab,  caused  by  the  fungus  Venturia  inaequalis  and  fire  blight,  caused  by  the bacterium Erwinia amylovora, are the most important diseases of apples worldwide, and they  very  likely  occur  in  every  country  where  apples  (Malus  ×  domestica  Borkh.)  are grown.  Without  any  control,  the  pathogens  can  cause  extensive  production  losses  in regions with humid, cool weather during the spring months.In some circumstances, the losses from apple scab can be 70% or more of the total fruit value (Agrios, 2005). Despite the tremendous amount of research on scab management (MacHardy,  1996),  its  control  still  requires  up  to  20  fungicide  applications  annually. Independent of the cost of chemical control, seasonal plasticity of fungicide sensitivities (Köller et al., 1995) is known and the long-term efficacy of fungicides is questionable. The only promising treatment against fire blight is the application of antibiotics which causes the risk of the establishment of antibiotic resistances. Therefore  the  application  of  natural  microbial  antagonists  to  control  microbial  plant diseases could be a promising alternative to the application of synthetic pesticides. Spurr and Knudsen (1985) concluded that biological control of leaf diseases by management of bacteria on the phylloplane is a strategy with great potential and is needed to relieve thepressures and failings of other control measures. Archived at http://orgprints.org/13736/307________________________________________________________________ 1Institute  of  Botany,  Leibniz  University  Hannover,  Herrenhäuserstr.  2,  D-30419  Hannnover,  e-mail: achim.gau@botanik.uni-hannover.de Biological control of apple scab and fire blight by the application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3  to the leaf surface S. Schmoock1, S. Kürkcüoglu1 and A. E. Gau1AbstractThe biological control of plant diseases by application of antagonistic microorganisms to the plant phyllosphere is an alternative strategy to prevent the frequent treatment of plants by pesticides. Microbiological antagonists can firstly interact directly against the pathogen by releasing antimicrobial compounds and/or secondly induce the plant resistance of the host plant by expression of pathogenesis-related proteins (PR proteins). The focus of our study is on the interaction of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the plant phyllosphere of Malus domestica cv. Holsteiner Cox. After application of P.fluorescens  Bk3  to  the  phyllosphere  of  M.  domestica  cv.  Holsteiner  Cox  we  observed dramatic changes in the protein composition of the apoplast of the host plant. Sequencing of the induced proteins by ESI-Q-ToF mass spectrometry and homology search identified these  additional  proteins  as  pathogenesis  related  proteins  (PR)  like  ß-1,3-  glucanase, thaumatin-like  protein,  chitinase  and  hevein-like  protein.  To  confirm  these  findings,  a suppressive  subtractive  hybridization  with  total  RNA  from  leaves  before  and  after inoculation of P. fluorescens Bk3 to the leaves of the host plant was performed. It revealed an increased expression level of many PR and stress related genes.The induction of PR proteins and plant defence genes in host plants after application of non-pathogenic bacterial antagonists to the plant phylloshere can presumably prevent or reduce successful infections by plant pathogens. Keywords: biological control, bioluminescence, Erwinia amylovora, pathogenesis related proteins, Venturia inaequalisIntroductionApple  scab,  caused  by  the  fungus  Venturia  inaequalis  and  fire  blight,  caused  by  the bacterium Erwinia amylovora, are the most important diseases of apples worldwide, and they  very  likely  occur  in  every  country  where  apples  (Malus  ×  domestica  Borkh.)  are grown.  Without  any  control,  the  pathogens  can  cause  extensive  production  losses  in regions with humid, cool weather during the spring months.In some circumstances, the losses from apple scab can be 70% or more of the total fruit value (Agrios, 2005). Despite the tremendous amount of research on scab management (MacHardy,  1996),  its  control  still  requires  up  to  20  fungicide  applications  annually. Independent of the cost of chemical control, seasonal plasticity of fungicide sensitivities (Köller et al., 1995) is known and the long-term efficacy of fungicides is questionable. The only promising treatment against fire blight is the application of antibiotics which causes the risk of the establishment of antibiotic resistances. Therefore  the  application  of  natural  microbial  antagonists  to  control  microbial  plant diseases could be a promising alternative to the application of synthetic pesticides. Spurr and Knudsen (1985) concluded that biological control of leaf diseases by management of bacteria on the phylloplane is a strategy with great potential and is needed to relieve thepressures and failings of other control measures. Biological  pest  control  with  epiphytic  living  antagonists  (e.g.,  bacteria,  yeast  and  other fungi) could help to avoid or to reduce the intensive application of pesticides and minimize their  residues  in  the  crop.  However,  despite  intensive  investigations  and  practical approaches (Boland and Kuykendall, 1997; Elad et al., 2001), this type of biological control remains marginally understood. The  investigation  of  the  relationship  between  the  antagonist,  the  host  plant  and  the pathogen  could  offer  a  new  approach  for  understanding  the  mechanism  of  biological control of plant diseases. Materials and Methods Plant propagation, inoculation of non-pathogenic microbial antagonists, and isolation of intercellular washing fluid (IWF) from Malus domestica were done as previously reported by Kürkcüoglu et al. (2004). The non-invasive determination of the microbial antagonists as well as the bacterial pathogen E. amylovora were performed as described by Gau et al. (2002) and Schmoock (2006), respectively. ResultsThe intercellular washing fluid (IWF) of the pathogen susceptible cultivar Holsteiner Cox before and after application of the non-pathogenic bacterium P. fluorescens Bk3 to the leaves was investigated in a comparative manner. For the analyses eight-week-old rooted apple  plants  of  M.  domestica  cv.  Holsteiner  Cox  being genetically  identical  and  grown under sterile conditions were used for isolation of the IWF before and after treatment of the plant leaves with the non-pathogenic P. fluorescens Bk3. The IWF was isolated by the infiltration-centrifugation technique and subsequently separated on SDS PAGE.Some of the proteins being increased or decreased (see Fig. 1) by the treatment of M.domestica  cv.  Holsteiner  Cox  with  P.  fluorescens  Bk3  were  excised  from  the  gel  and digested  with  trypsin.  The  obtained  peptide  fragments  were  submitted  to  de  novosequencing on a quadrupole/time-of-flight hybrid mass spectrometer and were identified as e.g. chitinase, ß-1,3-glucanase, thaumatin-like protein and ribonuclease. All identified proteins belong to the group of pathogensis-related proteins (PR). Fig.  1  Separation  of  apoplastic  fluid  on  SDS-PAGE  from  M.  domestica  cv.  Holstein  Cox  after application of the non-pathogenic bacterium P. fluorescens Bk3 to the leaf surface. C:Control; Bk3 after treatment with P. fluorescens Bk3 . Numbered bands were sequenced by ESI Q ToF mass spectrometry and identified with homology search. Band I: ß-1,3-glucanase, band II: thaumatin-like protein/chitinase, band III: ribonuclease-like PR10b protein, band IV: hevein-like protein, band V: non-specific lipid transfer protein. 12 kDa17 kDa30 kDa45 kDa66 kDaI    II   III IV  V1d 2d 5dC C C Bk3 Bk3 Bk312 kDa17 kDa30 kDa45 kDa66 kDaI    II   III IV  V1d 2d 5dC C C Bk3 Bk3 Bk3Archived at http://orgprints.org/13736/308Further investigations on transcript level revealed the up-regulation of 113 EST clones which also belong to the class of PR-proteins, oxidative stress, and transcripts that code for proteins which have  a  crucial  role  at  different  stages  of  pathogen  recognition  and  in  signalling  pathways (Kürkcüoglu et al. 2007). Moreover  applied  investigations  under  field  conditions  revealed  the  reduction  of  apple scab up to 84% when P. fluorescens Bk3 was applied to the leaf surface two days before the artificial infection was done (Kürcüoglu, 2006). In addition to the molecular biological investigations we have developed a method for the non-invasive  determination  of  microbial  antagonists  or  bacterial  pathogens  on  the  leaf surface or inside the host plant by using bioluminescence. Therefore the microorganisms were transformed with the luxCDABE gene cluster that codes for the two structural genes of the luciferase as well as for the genes of the substrate biosynthesis.  To  evaluate  the  protective  function  of  bacterial  antagonists,  the  fire  blight  susceptible cultivar Holsteiner Cox was pre-treated with 1 x 109 cells of P. fluorescens Bk3 two days before  the  artificial  infection  with  E.  amylovora  occurred.  The  measurement  of  the bioluminescence as shown in Fig. 2 revealed that the pre-treated  plants are protected against the infection of E. amylovora in contrast to the untreated control plants.Fig. 2  Determination of E. amylovora 222::Tn5-luxCDABE on M. domestica cv. Holsteiner Cox. Left: Infection of M. domestica cv. Holsteiner Cox with 5 x 106 Zellen  E. amylovora 222::Tn5-luxCDABE. Right: Infektion of M. domestica cv. Holsteiner Cox with 5 x 106 cells of E. amylovora222::Tn5-luxCDABE  two  days  after  pre-treatment  with  1  x  109  cells  of  P.  fluorescens  Bk3. (Schmoock, 2006). E. amylovora is marked with a white ellipse.Moreover, the application of the non-pathogenic bacterium P. fluorescens Bk3 causes no visible morphological change of the plants (data not shown). Archived at http://orgprints.org/13736/309Discussion The  results  presented  here  could  be  interpreted  by  suggesting  that  a  pre-treatment  of apple cultivars with P. fluorescens Bk3 leads to a induced resistance against infections by V. inaequalis or E. amylovora due to the fact that PR proteins are expressed before the infection with the pathogen. Such a pre-treatment of apple cultivars with non-pathogenic bacteria can eventually replace excessive treatment of apple trees with pesticides. Acknowledgements S.K. expresses her gratitude to the Deutsche Bundesstiftung Umwelt for financial support. For sequencing proteins by ESI Q-ToF mass spectrometry we are very thankful to Dr. M. Piotrowski, (Ruhr University Bochum, Germany). References Agrios, G.N. (2005). Plant Pathology. 5th edition. Academic Press Ltd, San Diego, California, USA. Boland, G. J., Kuykendall, L. D. (1997). Plant-microbe interactions and biological control. Dekker, New York. Elad, Y., Freemann, S., Monte, E. (eds.) (2001). Biocontrol Agents: mode of action and interaction with other means control. IOBC/wprs Bulletin 24, 1-376. Gau,  A.E.,  Dietrich,  C.,  Kloppstech,  K.  (2002). Non-invasive  determination  of  plant-associated bacteria in the phyllosphere of plants. Environmental Microbiology 4, 744-753.  Köller,  W.,  Smith,  F.D.,  Reynolds,  K.L.,  Wilcox,  W.F.,  Burr,  J.A.  (1995).  Seasonal  changes  of sensitivities  to  sterol  demethylation  inhibitors  in  Venturia  inaequalis  populations.  Mycol. Res. 99, 689-692. Kürkcüoglu, S., Piotrowski, M., Kloppstech, K., Gau, A.E. (2004). Up-regulation of Pathogenesis-related Proteins in the apoplast of Malus domestica cv. Holsteiner Cox after application of a non-pathogenic  bacterium  Pseudomonas  fluorescens  Bk3  to  the  plant  phyllosphere.  Z. Naturforsch. 59c, 843-848. Kürcüoglü S (2006) Eine Strategie zur biologischen Kontrolle von Pflanzenkrankheiten durch den Einsatz  natürlicher,  nicht  pathogener  mikrobieller  Antagonisten  am  Modellsystem  Malusdomestica (Apfel)/Venturia inaequalis (Apfelschorferreger)/ Pseudomonas fluorescens Bk3(mikrobieller Antagonist) PhD thesis, University Hannover, Germany. Kürkcüoglu, S., Al-Masri A.N., Degenhardt, J., Gau, A.E. (2007). Characterisation of the difference in  the  transcriptional  level  of  Malus  domestica  before  and  after  application  of  the  non-pathogenic bacterium Pseudomonas fluorescens Bk3. J. Exp. Botany 58, 733-41. MacHardy, W.E. (1996). Apple Scab- Biology, epidemiology and management. APS Press, St. USA.Schmoock, S. (2006) Infektion und Ausbreitung des Feuerbranderregers Erwinia amylovora in An- und  Abwesenheit  des  bakteriellen  Antagonisten  Pseudomonas  fluorescens  Bk3  auf verschiedenen Apfelkultivaren. Diploma thesis, University Hannover, Germany. Spurr, H. W., Knudsen, G.R. (1985). Biological control of leaf diseases with bacteria. In: Biological control on the phylloplane (Windels, C. E. Lindow, S. E. Eds.), Am. Phytophathol. Soc. Press, St. Paul, MN, pp. 45-62. Archived at http://orgprints.org/13736/

Biological control of apple scab and fire blight by the application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the leaf surface

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