Originalarbeit 211 Journal für Kulturpflanzen, 71 (7). S. 211–218, 2019, ISSN 1867-0911, DOI: 10.5073/JfK.2019.07.02 Verlag Eugen Ulmer KG, Stuttgart In situ immunofluorescence localization: A method for rapid detection of Beauveria spp. in the rhizosphere of Quercus robur saplings In-situ -Immunfluoreszenzlokalisierung: Eine Methode zum schnellen Nachweis von Beauveria spp. in der Rhizosphäre von Quercus robur -Setzlingen Marta Matek 1 , Cornelia I. Ullrich 2 , Frank Rabenstein 3 , Eckhard Koch 2 , Regina G. Kleespies 2 Affiliations Croatian Forest Research Institute, Division for Forest Protection and Game Management, Jastrebarsko, Croatia 1 Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Biological Control, Darmstadt, Germany 2 Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Quedlinburg, Germany 3 Correspondence Dr. Regina G. Kleespies, Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Biological Control, Heinrichstraße 243, 64287 Darmstadt, Germany, e-mail: regina.kleespies@julius-kuehn.de Accepted 19 June 2019 Abstract For biological control of plant pests, e.g. cockchafer grubs, in the rhizosphere of oak, apple or pine trees, ento- mopathogenic Beauveria spp. are increasingly applied. For successful use, it is important to monitor the spread and persistence of the inoculated fungi, both qualitati- vely and quantitatively. The determination of both para- meters by plating on selective nutrient media or by mole- cular methods such as PCR of soil samples are quite labo- rious and often do not yield satisfactory results. There- fore, the aim of the present study was to develop a spe- cific in situ method using immunofluorescence labelling of Beauveria spp. growing on young fine roots of three- year old oak saplings. All fine roots investigated were covered with a dense net of soil rhizosphere fungi, as visualized by staining with the nonspecific dye blanko- phor. On non-inoculated roots, polyclonal Beauveria anti- bodies did not label any of these naturally growing fungi. Only samples of roots inoculated with Beauveria brongni- artii displayed specific labelling up to ten months after inoculation. Whereas the natural rhizosphere fungi were detected growing in the intercellular space of the root cortex in an ectomycorrhiza-like manner up to the endo- dermis, hyphae of the inoculated B. brongniartii were never seen within the root tissue but only growing on the surface of the rhizodermis. These observations indicate that B. brongniartii does not grow endophytically, and that the method used allows to discriminate B. brongniar- tii from the resident fungal flora in the oak tree rhizo- sphere. Detection by immunofluorescence labelling employed in the current study may be a useful tool to fol- low B. brongniartii in experiments aimed at establishing the entomopathogen in the rhizosphere and to monitor its fate in long-term control of entomopathogens. Key words: biological pest control, blastospores, entomopathogenic fungi, immuno-fluorescence microscopy, Quercus robur, sustainability. Zusammenfassung Zur biologischen Bekämpfung von Pflanzenschädlingen, z.B. Maikäfer-Engerlingen, in der Rhizosphäre von Eichen, Apfelbäumen oder Kiefern werden zunehmend entomo- pathogene Beauveria-Spezies eingesetzt. Für eine erfolg- reiche Anwendung ist es wichtig, die Ausbreitung und Persistenz der ausgebrachten Pilze qualitativ und quanti- tativ zu erfassen. Die Bestimmung beider Größen durch Ausplattieren auf selektiven Nährmedien oder durch molekulare Methoden wie PCR ist mühsam und oft unge- nügend. Ziel der vorliegenden Studie war daher, eine spezifische In-situ-Methode durch Immunfluoreszenz- markierung von Beauveria spp. zu entwickeln, hier an jungen Feinwurzeln dreijähriger Stieleichen. Durch Anfärben mit dem unspezifischen Farbstoff Blankophor wurde sichtbar, dass alle untersuchten Feinwurzeln ein