Due to the possibility that bacteria could be involved in the clearance of paralytic shellfish toxins (PST) from bivalve molluscs, investigations into which, if any, bacteria were able to grow at the expense of PST focused on several common shellfish species. These species were blue mussels, oysters, razor fish, cockles, and queen and king scallops. Bacteria associated with these shellfish were isolated on marine agar 2216 and characterized by their carbon utilization profiles (BIOLOG). Selected isolates from groups demonstrating 90% similarity were screened for their ability to metabolize a range of PST (gonyautoxins 1 and 4 [GTX 1/4], GTX 2/3, GTX 5, saxitoxin, and neosaxitoxin) using a novel screening method and confirming its results by high-performance liquid chromatography. Results suggest that molluscan bacteria have different capacities to utilize and transform PST analogues. For example, isolates M12 and R65 were able to reductively transform GTX 1/4 with concomitant production of GTX 2/3, while isolate Q5 apparently degraded GTX 1/4 without the appearance of other GTXs. Other observed possible mechanisms of PST transformations include decarbamoylation by isolate M12 and sulfation of GTXs by isolates Q5, R65, M12, and C3. These findings raise questions as to the possible role of bacteria resident in the shellfish food transport system. Some researchers have suggested that the microflora play a role in supplying nutritional requirements of the host. This study demonstrates that bacteria may also be involved in PST transformation and elimination in molluscan species.Paralytic shellfish toxins (PST) are potent neurotoxins produced by some strains of dinoflagellates, such as Alexandrium spp., Pyrodinium bahamense var. compressum, and Gymnodinium catenatum (7). When passed through the marine food web, these toxins can lead to human disease through consumption of contaminated shellfish. For example, filter-feeding bivalves, such as mussels, cockles, oysters, and scallops, feed on dinoflagellates, transferring them from the gills to digestive organs where the toxins become concentrated (6, 21).The incidence of human PST poisoning has increased markedly since the early 1970s, with approximately 2,000 cases reported annually worldwide (13). This represents a serious health risk, as mortality rates in humans are reported to be between 10 and 20% (14), and although molluscan shellfish are themselves relatively unaffected by PST, outbreaks of PST poisoning in humans result in detrimental economic impacts on both the fish and shellfish industries.The structures of PST are based on a tetrahydropurine skeleton with two permanent guanidinium functions. Substitutions at four distinct positions around the basic PST structure categorize different PST analogues (Fig. 1). Currently, there are 22 known PST derivatives split into three categories: carbamate (with saxitoxin [STX] generally being considered the most potent), N-sulfocarbamoyl, and decarbamoyl (10, 23). Although PST analogues have different toxicities, they have ...

Aims: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L-form bacteria and to detect these in plants injected with L-form bacteria. Methods and Results: A sandwich ELISA was developed and its speci®city was investigated using L-forms and cell-walled forms of B. subtilis, different Bacillus species and a range of bacteria isolated from glasshouse-grown strawberry plants. The detection limits of the ELISA were approximately 10 3 viable cells ml À1 for L-forms compared with 10 7 viable cells ml À1 for cell-walled forms. Results showed that L-forms survived and moved within strawberry tissues injected with L-form bacteria. Conclusion: An ELISA that selectively detects B. subtilis L-form bacteria was developed and shown to con®rm the presence of L-forms in plants. Signi®cance and Impact of the Study: This will be a valuable rapid method to further studies on L-form plant interactions.

There is increasing evidence that bacterial-algal interactions play a role in Harmful Algal Bloom (HAB) ecology. Bacteria that are associated with bloom-forming algal species, specifically toxic dinoflagellate algae, have been implicated in the production and biotransformation of paralytic shellfish toxins (PSTs). To clarify the role that these bacteria may play in the production of PSTs, it is desirable to identify and localize the bacteria associated with the dinoflagellates and enumerate them during the course of the algal blooms that the toxic dinoflagellates produce. Because 16S rRNA-targeted probes offer the possibility of both, we previously made and tested probes for some putatively toxigenic bacteria isolated from cultures of the PSP-related dinoflagellates Alexandrium tamarense, A. affine and A. lusitanicum. The bacteria isolated from the dinoflagellates belong primarily to the alpha-proteobacterial group of Roseobacter and the gamma-proteobacterial group of Alteromonas. Here, we report the successful application of these probes to Lugol's-fixed seawater samples. We detected these bacteria in high numbers in the water column when Alexandrium spp. were both present and absent, and during periods when mussels contained PSTs.

Aims: To establish whether germination of Botrytis cinerea was affected by the symbiosis of Bacillus subtilis L-form bacteria with Chinese cabbage. Methods and Results: Germinating seeds of Chinese cabbage were co-cultivated with either L-forms of Bacillus subtilis or 5% (w/v) mannitol by soaking for 3 h. Seeds were then washed in sterile water, sown on a minimal medium and incubated in controlled conditions. L-form symbiosis was detected over a time course by ELISA. Conidial germination of Botrytis cinerea was signi®cantly reduced on cotyledonous leaves of L-form-treated plants compared with controls. Conclusions: Symbiosis of B. subtilis L-form bacteria during seed germination of Chinese cabbage inhibits conidial germination in plants on subsequent exposure to Botrytis cinerea. Signi®cance and Impact of the Study: This is the ®rst account of plant symbiosis with L-form bacteria showing antagonism to a fungal plant pathogen. This has promising implications for the use of this L-form as a biocontrol agent.

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