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.
Chitin is a naturally occurring linear polymer of N-acetylglucosamine and the major structural component of fungal cell walls and exoskeletons of insects and arthropods. Chitinases are the enzymes that breakdown chitin to economically important derivatives and found in a range of organisms including bacteria, insects, crustaceans, invertebrates, some vertebrates and higher plants. In the present study, four Bacillus thuringiensis (Bt) isolates were grown in media supplemented with 0.1% (w/v) regenerated chitin and chitinase inducing medium and screened for chitinolytic activity. All isolates showed notable extracellular chitinase activity with very low levels in cell bound fractions. When the isolates were screened with fluorogenic substrates, Bt HD133, Bt T7002 and Bt IPS78 showed a similar pattern of distribution with the highest production from Bt IPS 78. The lowest activity was detected from the isolate Bt 36-3. Similar to the results obtained with fluorogenic substrates, Bt 36-3 and Bt IPS 78 expressed the lowest and the highest activities with the substrate Carboxymethyl-Chitin Remazol Brilliant Violet 5R (CM-chitin RBV 5R) respectively. When the culture supernatants of isolates grown in nutrient broth supplemented with regenerated chitin were electrophoresed on activity gels, one chitinase band of approximate molecular mass of 36 kDa was obtained from all four sub species. Future experiments can be carried out to find out whether these chitinases have any insecticidal activity aiming, to develop environmental-friendly biopesticides, and in vivo antifungal activities to test against plant pathogenic fungi in growth chambers and under glasshouse condition.
Induction of L-form bacteria from Bacillus thuringiensis was investigated with the long-term view of their use as targeted biological control agents within plants. L-forms were successfully induced from B. thuringiensis subspecies israelensis 36-3 by preparing pour plates of exponential cultures in L-phase medium (LPM) containing 175 g ml -1 cephalosporin C. The induced L-forms were then subcultured and maintained in L-phase medium supplemented with inactivated mycoplasma screened 5% (v/v) horse serum (5HS) with a combination of 225 g ml -1 cephalosporin C and 0.6 mg ml -1 penicillin G, using the push block method. Induced L-forms showed typical features with pleiomorphic cells that contained granular particles and small vacuoles but no parasporal bodies. These L-forms were capable of growing on solidified media but failed to grow in liquid media.
Poplars (Populus spp.) of the Family Salicaceae are extensively cultivated worldwide and are susceptible to a variety of bacterial and fungal diseases. In Populus species, leaf rust disease caused by several species of Melampsora leads to considerable damages in plantations. Melampsora larici-populina is the most devastating and widespread fungal pathogen causing leaf rust disease in poplars. In this study, leaves and young stems of rooted cuttings of two poplar clones were treated with L-form bacteria of Bacillus subtilis NCIMB 8054, ATCC 6633 and then challenged with the spores of rust pathogen M. larici-populina. The development of uredinia was evaluated in the laboratory using the leaf disc assay. The L-forms greatly reduced rust severity in inoculated poplar leaves (local effect), while to a lesser extent in non-inoculated leaves obtained from inoculated plants showing a low systemic effect on pustule development. This plant-L-form symbiosis may have contributed significantly to a quantitative resistance to M. larici-populina indicating a promising implication for the use of L-form bacteria of B. subtilis as a biocontrol agent for poplars against the rust pathogen.
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