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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.

SUBMITTER: Wei QY

PROVIDER: S-EPMC8374352 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Entomopathogenic fungi from the genus Beauveria (Vuillemin) play an important role in controlling insect populations and have been increasingly utilized for the biological control of insect pests. Various studies have reported that Beauveria bassiana (Bals.), Vuill. also has the ability to colonize a broad range of plant hosts as endophytes without causing disease but while still maintaining the capacity to infect insects. Beauveria is often applied as an inundative spore application, but little research has considered how plant colonization may alter the ability to persist in the environment. The aim of this study was to investigate potential interactions between B. bassiana and Zea mays L. (maize) in the rhizosphere following inoculation, in order to understand the factors that may affect environmental persistence of the fungi. The hypothesis was that different isolates of B. bassiana have the ability to colonize maize roots and/or rhizosphere soil, resulting in effects to the plant microbiome. To test this hypothesis, a two-step nested PCR protocol was developed to find and amplify Beauveria in planta or in soil; based on the translation elongation factor 1-alpha (ef1α) gene. The nested protocol was also designed to enable Beauveria species differentiation by sequence analysis. The impact of three selected B. bassiana isolates applied topically to roots on the rhizosphere soil community structure and function were consequently assessed using denaturing gradient gel electrophoresis (DGGE) and MicroRespTM techniques. The microbial community structure and function were not significantly affected by the presence of the isolates, however, retention of the inocula in the rhizosphere at 30 days after inoculation was enhanced when plants were subjected to intensive wounding of foliage to crudely simulate herbivory. The plant defense response likely changed under wound stress resulting in the apparent recruitment of Beauveria in the rhizosphere, which may be an indirect defensive strategy against herbivory and/or the result of induced systemic susceptibility in maize enabling plant colonization.

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Method for observing Beauveria bassiana colonization in plants.

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