Isolation and characterisation of novel Bacillus thuringiensis strains with nematicidal activity

This study aimed to isolate and characterize Bt strains that produce nematicidal toxins, with the ultimate purpose of expressing their toxin-genes in bacteria that live in the ruminant digestive tract. The expression of toxins in the rumen, large intestine, and faeces of ruminants is designed to kill nematode eggs and larvae and break the parasitic cycle. A total of 66 strains of spore and crystal-forming bacteria were isolated from natural environments in Western Australia, using the acetate/heating method to select Btlike organisms. Parasporal crystals were isolated using Ludox gradient centrifugation, but to obtain highly purified crystals a novel re-germination process was developed. An agar microtitre-plate method, for hatching nematode eggs and culturing larvae, was tested, modified, and used to measure ovicidal or larvicidal effects of the crystals from the 66 Bt strains. 16 strains showed ovicidal effects. The most toxic crystals disrupted nematode eggs within the first 12 h of incubation, reducing the hatching rate to 4%, compared with a control rate of 96%. Toxicity toward nematode larvae was also observed for 16 isolates, of which 9 were ovicidal strains, reducing larval worm survival to 8% - 45%, compared with 98% in the controls. The most toxic strain, 4-1, appeared to contain 2 plasmids, totalling 48 - 49 kb of DNA. Attempts to clone restriction fragments from these plasmids were of very limited success. Only two or three small DNA fragments were represented among a large number of recombinants, suggesting that larger fragments or other regions of the DNA caused instability in E. coli plasmids. Sequence of the 16S rDNA of isolate 4- 1 was identical with the matching 1503 bp of sequence from two strains of B. cereus, and differed by only a single base-pair from one B. thuringiensis strain and three B. anthracis strains. This supported the recently published conclusion that B. cereus, B. thuringiensis. and B. anthracis are a single species, acquiring their distinguishing phenotypic features from plasmid-borne genes