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Typing of Pantoea agglomerans isolated from colonies of honey bees (Apis mellifera) and culturability of selected strains from honey

Caractérisation de la diversité des bactéries Pantoea agglomerans isolées de colonies d’abeilles domestiques (Apis mellifera) et possibilité de croissance dans le miel de lignées sélectionnées

Typisierungen von Pantoea agglomerans isoliert aus Kolonien von Honig-Bienen (Apis mellifera), sowie Untersuchung der Kultivierbarkeit ausgewählter Stämme in Honig

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Abstract

Pantoea agglomerans is a possible biocontrol agent against fire blight (Erwinia amylovora) and a facultative pathogen of humans. Isolates were gathered from flowers, pollen loads, honey sacs, and freshly stored nectar (FSN). Under artificial inoculation conditions, strains completely lost their culturability at 24 °C after 120 h of incubation in honey and 156 h in honey solution, respectively. None of tested strains could be cultivated from FSN, honey, or honey solution after 48 h at temperatures higher then 28 °C. At different time intervals, culturable population levels at 35 °C and 24 °C were significantly higher in blossom honey or its solution than in blossom and honeydew honey or its solution. Our results indicated that P. agglomerans is widely spread throughout honey bee’s environment. Strains lost culturability after short periods of incubation in honey or honey solution. In samples of honey and royal jelly from test colonies, no culturable P. agglomerans isolates could be detected.

Zusammenfassung

Ziel dieser Studie war die Untersuchung der Diversität von Pantoea agglomerans und seiner Rückverfolgbarkeit von Trachtpflanzen zum Bienenvolk, sowie die Abschätzung der kultivierbaren Keimzahl (CPL) dieses Bakteriums in Honig, Honiglösung und frisch eingelagertem Nektar (FSN). P. agglomerans ist ein möglicher Kandidat zur biologischen Bekämpfung von Feuerbrand (Erwinia amylovora),wurde aber auch als fakultativ human-pathogener Keim beschrieben.

Blüten verschiedener Pflanzen, Pollenhöschen, Honigblaseninhalt und frisch eingelagerter Nektar wurden gesammelt, aufbereitet und das gewonnene Probenmaterial auf Agarplatten inkubiert, um Isolate von P. agglomerans zu gewinnen. Zur Bewertung der Diversität wurden SDS-PAGE, RAPD- and ERIC-PCR eingesetzt. Die Abschätzung der Verwandtschaft der Isolate erfolgte mittels Cluster-Analyse.

Zur Untersuchung der Kultivierbarkeit von P. agglomerans in Honig, Honiglösung und frisch eingelagertem Nektar wurden diese mit einer wässrigen Suspension von 5 ausgewählten Stämmen des Bakteriums künstlich inokuliert und sorgfältig durchmischt. In bestimmten Intervallen wurden Proben entnommen, auf Agarplatten ausplattiert und diese bebrütet. Für jeden Stamm wurde die Zahl der kultivierbaren koloniebildenden Einheiten (KBE) ermittelt.

Von Blüten verschiedener Pflanzenarten, Pollenhöschen, Honigblaseninhalten und frisch eingelagertem Nektar konnten einzelne Isolate von P. agglomerans gewonnen werden, nicht aber aus Honig- und Weiselfuttersaftproben der Testvölker. Auf Basis der Koloniemorphologie, Pigmentierung, biochemischen Eigenschaften und dem Vergleich der Proteinmuster mit Referenzstämmen nach Natriumsulfat-Polyacrylamid Gel Elektrophorese (SDS-PAGE) wurden 301 Isolate ausgewählt. Von diesen zeigten 50 Stämme unterschiedliche Eiweißprofile. Die Analyse mittels RAPD-PCR erbrachte die gleiche Profilanzahl PCR (Abb. 1). Eine Identifizierung der isolierten Stämme erfolgte mit ERIC-PCR (Abb. 2).

Aus den künstlich inokulierten Proben konnte auf PYE Agar bei 24 °C und einer Inkubationsdauer von mehr als 120 h (Honig) bzw. 156 h (Honiglösung) keiner der Teststämme reisoliert werden. Bei Temperaturen über 28 °C war bereits nach 48 h Bebrütungsdauer keine Rückisolation der Teststämme aus Honig, Honiglösung oder frisch eingelagertem Nektar mehr möglich.

Sowohl bei 35 °C als auch bei 24 °C war die kultivierbare Keimzahl der Teststämme zu verschiedenen Zeitintervallen in „Blütenhonig“ (BH) signifikant höher (P < 0,05) als in „Blütenmit Waldhonig“, oder ihren Lösungen (Abb. 4).

Gestützt auf diese Ergebnisse kann geschlossen werden, dass die Zeit, in der die getesteten P. agglomerans Stämme aus Honig kultiviert werden konnten, beträchtlich kürzer ist, als die Zeitspanne, die in der imkerlichen Praxis zwischen dem Sammeln des Nektars und der Ernte des Honigs üblicherweise eingehalten wird. Im Falle eines Einsatzes von P agglomerans als biologisches Mittel zur Feuerbrandbekämpfung in blühenden Obstanlagen kann dies ein wichtiger Punkt sein.

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Loncaric, I., Heigl, H., Licek, E. et al. Typing of Pantoea agglomerans isolated from colonies of honey bees (Apis mellifera) and culturability of selected strains from honey. Apidologie 40, 40–54 (2009). https://doi.org/10.1051/apido/2008062

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