ELISA detection of Bacillus subtilis L-forms in strawberry
An Enzyme-Linked Immunosorbent Assay (ELISA) was developed for the detection of Bacillus subtilis L-form bacteria in pure culture and in strawberry plant material. Investigations led to the use of an assay where the enzyme, alkaline phosphatase was bound to streptavidin and was one of the components of the avidin-biotin amplification step. The polyclonal antibodies used were shown to be selective for Bacillus subtilis L-forms, with the cell-walled parent and other Bacillus species also being detected, but only at much higher population numbers. Detection limits for the biotin-ELISA were established by preparing standard curves of L-forms from pure culture suspended in plant extracts from micropropagated strawberry tissues and mature strawberry plants. These were 10 3 viable cells ml-1 in 10% mannitol, 104 ml-1 in extracts of micropropagated shoots and 106 ml-1 in mature plant extracts. This enabled the L-form status of micropropagated strawberry shoots and mature, glasshouse-grown plants to be monitored during the development of appropriate association techniques. Detailed investigations showed that L-forms, introduced to micropropagated strawberry shoot explants, were maintained throughout their growth (9 weeks), as well as after subculture i.e. 19wk after L-form treatment. L-form bacteria sprayed directly into the surface of micropropagated strawberry plants were antagonistic towards the fungal plant pathogen Botrytis cinerea. Glasshouse-grown strawberry plants were injected with L-forms and ELISA was used to monitor the distribution of L-forms in different plant parts up to 14d after treatment. L-form bacteria were randomly distributed throughout the plants within 4 days and detected in leaves, petioles, runners and crowns. A large experiment undertaken in a polythene tunnel monitored the presence of L-forms, at t = 1 and 11wk, in plants treated with B. subtilis L-form bacteria (test) , or 10% mannitol (control). The ELISA showed L-form presence in all test plants (n = 50) and, although post-harvest spoilage was shown to be delayed, there was no correlation between L-form presence and reduced port-harvest spoilage. This is discussed along with other aspects concerning the symbiosis(ses) of L-form bacteria with strawberry plants.