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Title: Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp.

item GUERMACHE, F - European Biological Control Laboratory (EBCL)
item RODIER-GOUD, M - European Biological Control Laboratory (EBCL)
item Caesar, Anthony
item HÉRAUD, C - European Biological Control Laboratory (EBCL)
item CLAUDE-BON, M - European Biological Control Laboratory (EBCL)

Submitted to: Letters in Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/7/2012
Publication Date: 6/1/2012
Citation: Guermache, F., Rodier-Goud, M., Caesar, A.J., Héraud, C., Claude-Bon, M. 2012. Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp.. Letters in Applied Microbiology. 54(6): 568–571.

Interpretive Summary: The initial step in the identification of isolates of the soilborne, often plant pathogenic fungus Rhizoctonia is determination of the number of nuclei in each cell (which are separated by cross walls at intervals along the tubular, microscopic filaments) of the fungal filaments (hyphae) that collectively comprise the vegetative body of the fungus (mycelium). Red fluorescence induced by green light allowed individual cells to be seen more clearly since the bright red outline of individual cells were thus made more distinct. When this induced red fluorescence is novelly combined with a stain which makes each nucleus distinct as bright blue, called DAPI, counting the number of nuclei is made more accurate. Once the number of nuclei per fungal cell is determined, those with more than two nuclei per cell enter a different second round of identifying them to subspecies or anastomosis groupings (AG) than those with 1-2 nuclei. The classification to AG provides clues about the pathogenicity and typical host range of individual isolates of the Rhizoctonia fungus. This also aids study of distinctions in the ecology of Rhizoctonia species, all of which are important for their use as biological control agents of weeds or their ability to cause synergistic effects with insects released for weed biological control, for example.

Technical Abstract: Aims: To simplify the determination of the nuclear condition of the pathogenic Rhizoctonia, which currently needs to be performed either using two fluorescent dyes, thus is more costly and time-consuming, or using only one fluorescent dye, and thus less accurate. Methods and Results: A red primary fluorescence (autofluorescence) of the hyphal cell walls and septa of Rhizoctonia spp. with green excitation is evident in Rhizoctonia spp. This property is exploited and combined for the first time with conventional DAPI fluorescence to accurately determine the nuclear condition of Rhizoctonia. This bi-fluorescence imaging strategy depicted the nuclear condition in Rhizoctonia spp. more accurately than the conventional DAPI fluorescence used alone and was validated against isolates previously genotyped by DNA sequencing. Conclusions: We demonstrated that the bi-fluorescence imaging strategy was safe, accurate and simple to perform and interpret. Significance and Impact of the Study: This novel bi-fluorescence imaging strategy provides a sensitive tool for determining the nuclear condition of Rhizoctonia isolates. Its accurate simplicity is a key advantage when there are numerous cultures to be examined.