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

Abstract

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 b-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 b-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. b-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.

Induction of Resistance in Poplar to Melampsora larici-populina Using L-form Bacteria

“…Stable L-forms derived from the cell walled form of Bacillus subtilis NCIMB 8054, ATCC 6633 have previously been shown to associate with plants such as Strawberry [14] and Chinese cabbage [15]. Similar to the work done by [14] in this study also leaves and stems of 3 month old Poplar plants were injected with L-forms of B. subtilis and 5% mannitol as treated and control tests and grown under greenhouse conditions.…”

“…There is previous evidence to prove the movement of L-form bacteria of B. subtilis within the plant tissues from the point of their inoculation [14,15] . An ELISA that was selective for the L-forms of Bacillus subtilis showed that Lforms have moved up to 32-42 cm along the stolon tissues of strawberry form the point of injection.…”

Association of L-form Bacteria with Plants and their Application in Biological Control of Phytopathogenic Fungi and Bacteria: A Review

“…In all these associations, either stable or unstable L-form suspensions were prepared from newly grown exponentially growing liquid cultures and they were either used directly or resuspended in mannitol solution (to provide osmotic protection) before being introduced to plants. Those above mentioned associations were mainly done on fresh tissues by imbibing the germinating seeds (radicle emerged) in Lform suspensions [36,38,34,39,35] or by injecting L-form suspensions to different plant parts by hypodermic inoculations [32,33].…”

“…In another study, histochemical localization of B. subtilis P6 gus transformant Lform bacteria in treated Chinese cabbage seedlings was determined by a gene system which was successfully applied to detect the distribution of stable L-forms in different parts of the seedlings [39]. The B. subtilis P6 gus transformant L-form bacteria generated characteristic blue colonies when grown on plates containing L-phase medium and the substrate 5-bromo-4-chloro-3-indolyl-b-Dglucuronide (X-gluc) for βglucuronidase enzyme.…”