Netherlands Journal of Plant Pathology

, Volume 95, Issue 5, pp 281–292 | Cite as

Pseudomonas for biological control of dutch elm disease. I. Labeling, detection and identification of pseudomonas isolates injected into elms; comparison of various methods

  • R. J. Scheffer
  • D. M. Elgersma
  • Letty A. De Weger
  • G. A. Strobel
Article

Abstract

To understand the mechanisms involved in biological control of Dutch elm disease byPseudomonas, data were needed on the distribution of the introduced bacteria within elm and on the development of the bacterial population over a period of time.

As traditional biochemical identification techniques are not suitable for distinguishment between individualPseudomonas isolates, three alternative approaches were compared.
  1. 1)

    Chemotaxonomy, using lipopolysaccharide pattern, cell envelope protein pattern or DNA restriction fragment pattern. These techniques were reliable, but tedious.

     
  2. 2)

    Labeling bacteria with a transposon (Tn903) or a plasmid construct (pMON5003) with a metabolic marker (Lac ZY, coding for β-galactosidase and lactose permease) allowed for a reliable identification of reisolates. However, populations of transposon-labeled bacteria in elms declined much faster than populations of the unlabeled wild type. The plasmid carrying the metabolic marker disappeared from the bacterial populations over time. Apparently both the transposon and the plasmid were a disadvantage to the bacteria compared with the wild type parent strains.

     
  3. 3)

    Immunoagglutination of representative reisolates with an antiserum against theP. fluorescens isolate in use proved to be specific and fast. For routine purposes the immunoagglutination test therefore was the best method of the various ones employed.

     

Studies on the distribution of aPseudomonas isolate in elm twigs showed that a stable bacterial population developed in the twigs within three months, but that the bacteria in general did not escape from the xylem vessels in which they were introduced.

Samenvatting

Voor een beter begrip van de mechanismen die ten grondslag liggen aan de biologische bestrijding van de iepeziekte doorPseudomonas spp. zijn gegevens over de verspreiding van de bacteriën binnen de iep en over het verloop van de bacteriedichtheid in de tijd nodig.

Omdat klassieke biochemisch-taxonomische technieken niet geschikt zijn voor het identificeren van individuelePseudomonas-isolaten zijn drie alternatieve benaderingen vergeleken. Chemotaxonomie gebasserd op lipopolysaccharidepatronen, celenvelop eiwitpatronen of DNA-restrictiepatronen bleek betrouwbaar, maar arbeidsintensief. Merken van bacteriën met een transposon (Tn903) of een zogenaamde ‘metabolic marker’ (het LacZY gen, dat codeert voor β-galactosidase en lactose permease) maakte een betrouwbare identificatie van herisolaten mogelijk. Het bleek echter dat de populatiedichtheid van transposon-gemerkte bacteriën in de iep sneller afnam dan de dichtheid van wildtype populaties. Ook bleek het plasmide met het LacZY gen uit de bacterie-populaties te verdwijnen. Blijkbaar had zowel het transposon als het plasmide een negatief effekt op de bacteriën, wat deze methode onbetrouwbaar maakt omdat de verkregen gegevens niet geëxtrapoleerd mogen worden naar het bijbehorende wild-type. Identificatie met immuno-agglutinatie met een antiserum bereid tegen het betreffendePseudomonas-isolaat bleek specifiek en snel. Immuno-agglutinatie bleek daarom de beste methode voor routinewerk.

Studie naar het verloop van dePseudomonas populatie in twijgen van iepen liet zien dat zich binnen drie maanden een stabiele bacteriepopulatie instelde (circa 7×104 bacteriën per twijgmonster), maar dat de bacteriën zich mogelijk niet naar hoger gelegen xyleemelementen konden verspreiden vanuit de houtvaten waar zij bij inoculatie waren ingebracht.

Additional keywords

Ophiostoma ulmi plasmid construct (pMON5003) metabolic marker transposon Tn903 immuno-agglutination lipopolysaccharide pattern cell envelope protein pattern DNA restriction fragments 

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Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 1989

Authors and Affiliations

  • R. J. Scheffer
    • 1
  • D. M. Elgersma
    • 1
  • Letty A. De Weger
    • 2
  • G. A. Strobel
    • 3
  1. 1.Willie Commelin Scholten Phytopathological LaboratoryBaarnthe Netherlands
  2. 2.Department of Plant Molecular BiologyUniversity of LeidenLeidenthe Netherlands
  3. 3.Department of Plant PathologyMontana State UniversityBozemanUSA

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