Molecular detection and b-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese Cabbage seedlings

Aim: To detect L-form bacteria in developing Chinese cabbage seedlings.

Methods and Results: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding beta -glucuronidase). Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium. Histochemical staining of beta-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. beta-Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems. Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues. Stable L-form bacteria were non-culturable after their association with plant material.

Conclusions: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B. subtilis L-form bacteria in plant material.

Significance and Impact of the Study: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association.