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Epidemiology and pathogenicity mechanisms of Pseudomonas cichorii, the causal agent of midrib rot in greenhouse-grown butterhead lettuce (Lactuca sativa L.)

Ellen Pauwelyn UGent (2012)
abstract
Pseudomonas cichorii is an important plant pathogenic bacterium, with a wide host range and a worldwide distribution. In Belgium, this pathogen is the causal agent of midrib rot on greenhouse-grown butterhead lettuce and a serious threat for butterhead lettuce production. However, knowledge about the epidemiology and pathogenicity mechanisms of this pathogen is very limited. The first part of this thesis deals with the epidemiology of P. cichorii with the emphasis on disease development in greenhouse-grown butterhead lettuce. Greenhouse experiments revealed that (1) a single irrigation with water containing 102 CFU/mL P. cichorii results in serious midrib rot infections, which established grower’s surface irrigation water as the major inoculum source, (2) lettuce becomes more susceptible for lettuce midrib rot after the onset of head formation, (3) overhead sprinkler irrigation, which is the common practice in Belgian butterhead lettuce production greenhouses, is more conducive to midrib rot compared to sub-plant irrigation, and (4) disease symptom expression is strongly influenced by the lettuce cultivar group, resulting in varnish spot/tar on crisphead lettuce and midrib rot on butterhead or cutting group lettuce. The second and more extended part of this thesis includes our research about pathogenicity factors of P. cichorii. Our first aim was to study its type three secretion system (TTSS), which is encoded by the hrp genes. Many Gram-negative plant pathogenic bacteria require this TTSS to cause disease on susceptible plants and to elicit a hypersensitive reaction (HR) on resistant plants. Therefore, a transposon bank was constructed and mutants were screened for their ability to cause a hypersensitive respons (HR) on tobacco. Although several mutants with an altered ability to cause a HR were retained, no hrc/hrp-mutant was obtained. Consequently, a site-specific hrpL-deletion mutant was constructed. This mutant was slightly reduced in initial multiplication in planta, but retained its pathogenicity on greenhouse-grown butterhead lettuce, indicating that the TTSS is not important for P. cichorii SF1-54 to cause midrib rot in butterhead lettuce. Surprisingly, this hrpL-mutant still induced tissue collapse in tobacco leaves resembling HR, which may explain why no hrc/hrp-mutants were found among the HR--mutants. However, transposon mutants impeded in their ability to induce a HR-like tobacco leaf collapse were less pathogenic on lettuce and grew poorly in planta. Three mutants were studied in more detail, more specifically a mqoB-mutant, a pqqH-mutant and a lysA-mutant. The mqoB gene codes for malate:quinone oxidoreductase, which is an enzyme of the tricarboxylic acid/glyoxylate cycle. Growth experiments revealed this mqoB gene is probably required for the glyoxylate cycle and consequently suggests to the importance of the glyoxylate cycle in for growth and pathogenicity of P. cichorii in butterhead lettuce. The pqqH gene belongs to the pyrroloquinoline quinone (PQQ) biosynthesis gene cluster, pqqFABCDEH, and PqqH is involved in the secretion of PQQ in the periplasm and culture medium. PQQ may function as cofactor or (anti-)oxidant. The lysA gene encodes meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step of the lysine biosynthesis. A site-specific lysA-deletion mutant confirmed avirulence and reduced in planta growth of the lysA-transposon mutant. Moreover, duplications of genes of the lysine biosynthesis pathway have occurred in the genome of P. cichorii. Based on these findings, we suggest lysine must be importance for this pathogen. Another remarkable finding was that both lysA-mutants were unable to spread in lettuce tissue. Additionally and surprisingly, the genome of P. cichorii contains lodA and lodB genes, involved in L-lysine-ε-oxidase activity and required for spreading from biofilms for certain marine bacteria. Thus, the hypothesis was set that LodA and LodB might require lysine to spread in the plant and consequently for full pathogenicity. A lodA-deletion mutant, however, was not reduced in pathogenicity, and spread in planta even more aggressively than the wild type, disproving our hypothesis. These finding suggest that P. cichorii posseses TTSS-independent pathogenicity factors, requiring the TCA/glyoxylate cycle and amino acids, which reminded us about toxins, such as cyclic lipopeptides (CLP), which contribute to the virulence of several plant pathogenic pseudomonads. Bioassays revealed surfactant, antimicrobial and phytotoxic activity of P. cichorii cells, activities typical for CLPs, which could be retained in the culture filtrate. Bioassay-guided purification revealed that P. cichorii strain SF1-54 produces a complex mixture of 7 bioactive compounds with lipopeptide characteristics. A combination of chemical analysis and genome mining was used to elucidate the structure and encoding genes of these compounds. Compound A has a molecular weight of 1604 Da and surfactant and antimicrobial activity against B. megaterium, but the lipopeptidic nature of this compound is doubtful due to its colour and poor fragmentation. Additionally, P. cichorii SF1-54 produces cyclic lipopeptides, which are expected to be similar or highly related to pseudomycin C and C’, based on their molecular weight, their antimicrobial activity and the sequence of their biosynthesis genes. Furthermore, P. cichorii SF1-54 produces two new phytotoxic CLPs, cichopeptin A and B. They have a molecular weight of 2066 Da and 2052 Da, respectively and consist both of 22 amino acids, N-acylated with an unsaturated C12-fatty acid. They are expected to be members of the tolaasin group and structurally resemble corpeptin B. Cichopeptin B, which was obtained in smaller amounts compared to cichopeptin A, is expected to differ from cichopeptin A only by the C-terminal amino acid residue. Finally, P. cichorii SF1-54 produces cichofactin A and B, which are two related linear lipopeptides with a molecular weight of 1137 Da and 1109 Da, respectively. They consist of a 8-amino acid peptide moiety and a decanoic lipid chain in cichofactin A, and dodecanoic lipid chain in cichofactin B. They both constitute new members of the syringafactin lipopeptide family. To obtain knowledge about the function of the cichofactins, a cichofactin-deficient mutant was constructed in P. cichorii SF1-54. Disruption of the cichofactin biosynthesis genes, cifA and cifB, abolished cichofactin production and surprisingly enhanced the production of the phytotoxic cichopeptins. The cichofactin-deficient mutant was impaired in swarming motility and enhanced in biofilm formation. Based on these findings, we can conclude that P. cichorii SF1-54 is a strong lipopeptide producer and that lipopeptides appear to be very important in the ecology and pathogenicity of this bacterium.
Please use this url to cite or link to this publication:
author
promoter
UGent
organization
alternative title
Epidemiologie en pathogeniciteitsmechanismen van Pseudomonas cichorii, de verwekker van nerfrot in groene botersla onder glas (Lactuca sativa L.)
year
type
dissertation (monograph)
subject
keyword
midrib rot, Pseudomonas cichorii, lettuce, Pseudomonas, Lactuca sativa L.
pages
218 pages
publisher
Ghent University. Faculty of Bioscience Engineering
place of publication
Ghent, Belgium
defense location
Gent : Faculteit Bio-ingenieurswetenschappen (A0.030)
defense date
2012-02-02 17:00
ISBN
9789059894952
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
2004520
handle
http://hdl.handle.net/1854/LU-2004520
date created
2012-01-26 11:22:48
date last changed
2012-01-30 08:36:16
@phdthesis{2004520,
  abstract     = {Pseudomonas cichorii is an important plant pathogenic bacterium, with a wide host range and a worldwide distribution. In Belgium, this pathogen is the causal agent of midrib rot on greenhouse-grown butterhead lettuce and a serious threat for butterhead lettuce production. However, knowledge about the epidemiology and pathogenicity mechanisms of this pathogen is very limited. 
The first part of this thesis deals with the epidemiology of P. cichorii with the emphasis on disease development in greenhouse-grown butterhead lettuce. Greenhouse experiments revealed that (1) a single irrigation with water containing 102 CFU/mL P. cichorii results in serious midrib rot infections, which established grower{\textquoteright}s surface irrigation water as the major inoculum source, (2) lettuce becomes more susceptible for lettuce midrib rot after the onset of head formation, (3) overhead sprinkler irrigation, which is the common practice in Belgian butterhead lettuce production greenhouses, is more conducive to midrib rot compared to sub-plant irrigation, and (4) disease symptom expression is strongly influenced by the lettuce cultivar group, resulting in varnish spot/tar on crisphead lettuce and midrib rot on butterhead or cutting group lettuce. 
The second and more extended part of this thesis includes our research about pathogenicity factors of P. cichorii. Our first aim was to study its type three secretion system (TTSS), which is encoded by the hrp genes. Many Gram-negative plant pathogenic bacteria require this TTSS to cause disease on susceptible plants and to elicit a hypersensitive reaction (HR) on resistant plants. Therefore, a transposon bank was constructed and mutants were screened for their ability to cause a hypersensitive respons (HR) on tobacco. Although several mutants with an altered ability to cause a HR were retained, no hrc/hrp-mutant was obtained. Consequently, a site-specific hrpL-deletion mutant was constructed. This mutant was slightly reduced in initial multiplication in planta, but retained its pathogenicity on greenhouse-grown butterhead lettuce, indicating that the TTSS is not important for P. cichorii SF1-54 to cause midrib rot in butterhead lettuce. Surprisingly, this hrpL-mutant still induced tissue collapse in tobacco leaves resembling HR, which may explain why no hrc/hrp-mutants were found among the HR--mutants. 
However, transposon mutants impeded in their ability to induce a HR-like tobacco leaf collapse were less pathogenic on lettuce and grew poorly in planta. Three mutants were studied in more detail, more specifically a mqoB-mutant, a pqqH-mutant and a lysA-mutant. The mqoB gene codes for malate:quinone oxidoreductase, which is an enzyme of the tricarboxylic acid/glyoxylate cycle. Growth experiments revealed this mqoB gene is probably required for the glyoxylate cycle and consequently suggests to the importance of the glyoxylate cycle in for growth and pathogenicity of P. cichorii in butterhead lettuce. The pqqH gene belongs to the pyrroloquinoline quinone (PQQ) biosynthesis gene cluster, pqqFABCDEH, and PqqH is involved in the secretion of PQQ in the periplasm and culture medium. PQQ may function as cofactor or (anti-)oxidant. The lysA gene encodes meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step of the lysine biosynthesis. A site-specific lysA-deletion mutant confirmed avirulence and reduced in planta growth of the lysA-transposon mutant. Moreover, duplications of genes of the lysine biosynthesis pathway have occurred in the genome of P. cichorii. Based on these findings, we suggest lysine must be importance for this pathogen. Another remarkable finding was that both lysA-mutants were unable to spread in lettuce tissue. Additionally and surprisingly, the genome of P. cichorii contains lodA and lodB genes, involved in L-lysine-\ensuremath{\epsilon}-oxidase activity and required for spreading from biofilms for certain marine bacteria. Thus, the hypothesis was set that LodA and LodB might require lysine to spread in the plant and consequently for full pathogenicity. A lodA-deletion mutant, however, was not reduced in pathogenicity, and spread in planta even more aggressively than the wild type, disproving our hypothesis. 
These finding suggest that P. cichorii posseses TTSS-independent pathogenicity factors, requiring the TCA/glyoxylate cycle and amino acids, which reminded us about toxins, such as cyclic lipopeptides (CLP), which contribute to the virulence of several plant pathogenic pseudomonads. Bioassays revealed surfactant, antimicrobial and phytotoxic activity of P. cichorii cells, activities typical for CLPs, which could be retained in the culture filtrate. Bioassay-guided purification revealed that P. cichorii strain SF1-54 produces a complex mixture of 7 bioactive compounds with lipopeptide characteristics. A combination of chemical analysis and genome mining was used to elucidate the structure and encoding genes of these compounds. Compound A has a molecular weight of 1604 Da and surfactant and antimicrobial activity against B. megaterium, but the lipopeptidic nature of this compound is doubtful due to its colour and poor fragmentation. Additionally, P. cichorii SF1-54 produces cyclic lipopeptides, which are expected to be similar or highly related to pseudomycin C and C{\textquoteright}, based on their molecular weight, their antimicrobial activity and the sequence of their biosynthesis genes. Furthermore, P. cichorii SF1-54 produces two new phytotoxic CLPs, cichopeptin A and B. They have a molecular weight of 2066 Da and 2052 Da, respectively and consist both of 22 amino acids, N-acylated with an unsaturated C12-fatty acid. They are expected to be members of the tolaasin group and structurally resemble corpeptin B. Cichopeptin B, which was obtained in smaller amounts compared to cichopeptin A, is expected to differ from cichopeptin A only by the C-terminal amino acid residue. Finally, P. cichorii SF1-54 produces cichofactin A and B, which are two related linear lipopeptides with a molecular weight of 1137 Da and 1109 Da, respectively. They consist of a 8-amino acid peptide moiety and a decanoic lipid chain in cichofactin A, and dodecanoic lipid chain in cichofactin B. They both constitute new members of the syringafactin lipopeptide family. To obtain knowledge about the function of the cichofactins, a cichofactin-deficient mutant was constructed in P. cichorii SF1-54. Disruption of the cichofactin biosynthesis genes, cifA and cifB, abolished cichofactin production and surprisingly enhanced the production of the phytotoxic cichopeptins. The cichofactin-deficient mutant was impaired in swarming motility and enhanced in biofilm formation. Based on these findings, we can conclude that P. cichorii SF1-54 is a strong lipopeptide producer and that lipopeptides appear to be very important in the ecology and pathogenicity of this bacterium.},
  author       = {Pauwelyn, Ellen},
  isbn         = {9789059894952},
  keyword      = {midrib rot,Pseudomonas cichorii,lettuce,Pseudomonas,Lactuca sativa L.},
  language     = {eng},
  pages        = {218},
  publisher    = {Ghent University. Faculty of Bioscience Engineering},
  school       = {Ghent University},
  title        = {Epidemiology and pathogenicity mechanisms of Pseudomonas cichorii, the causal agent of midrib rot in greenhouse-grown butterhead lettuce (Lactuca sativa L.)},
  year         = {2012},
}

Chicago
Pauwelyn, Ellen. 2012. “Epidemiology and Pathogenicity Mechanisms of Pseudomonas Cichorii, the Causal Agent of Midrib Rot in Greenhouse-grown Butterhead Lettuce (Lactuca Sativa L.)”. Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
APA
Pauwelyn, E. (2012). Epidemiology and pathogenicity mechanisms of Pseudomonas cichorii, the causal agent of midrib rot in greenhouse-grown butterhead lettuce (Lactuca sativa L.). Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Vancouver
1.
Pauwelyn E. Epidemiology and pathogenicity mechanisms of Pseudomonas cichorii, the causal agent of midrib rot in greenhouse-grown butterhead lettuce (Lactuca sativa L.). [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2012.
MLA
Pauwelyn, Ellen. “Epidemiology and Pathogenicity Mechanisms of Pseudomonas Cichorii, the Causal Agent of Midrib Rot in Greenhouse-grown Butterhead Lettuce (Lactuca Sativa L.).” 2012 : n. pag. Print.