Method for observing Beauveria bassiana colonization in plants

Abstract

Microbes interact in a multitude of ways with host plants, can dwell as endophytes within plants causing no apparent disease, and often provide benefits to their host. Observing microorganism distribution and colonization is a prerequisite for interactive research. To this end, we describe use of fluorescent staining for microorganism labeling and highlight its simplicity, and efficiency. Fluorescein can quickly bind to Beauveria bassiana spores, producing bright green fluorescence that can be observed even inside plant tissues. This method provides an intuitive visual image that can be utilised for subsequent data acquisition and statistical analysis.•Our protocol depends on binding of fluorescein diacetate (FDA) specifically to microorganisms. The fungus hydrolyses and metabolises FDA in cells to produce bright green fluorescent products. This fluorescent signal can easily penetrate plant epidermis and be detected by fluorescence microscopy.•FDA, which itself does not emit light, will emit a fluorescent signal only when combined with B. bassiana. Concomitant genetic testing of the fungal ITS confirmed the high level of the fluorescent staining method for detection of B. bassiana.•Compared with the previous green fluorescent protein (GFP) labeling methods, this protocol improved the labeling efficiency of microorganisms and simplifies the process.