Spread and survival of a genetically modified Pseudomonas aureofaciens in the phytosphere of wheat and in soil

An isolate of Pseudomonas aureofaciens indigenous to the UK, which was chromosomally modified by the insertion of two gene cassettes (xylE in combination with the gene for kanamycin resistance and lacZY) for sensitive monitoring purposes was provided by IVEM (Oxford). Growth of this organism and the non-modified wildtype strain were not significantly different in sterile tryptic soy broth. Spread and survival of the marked bacterium in the phytosphere of spring wheat and in soil was quantified in a number of glasshouse and laboratory experiments. Six days after vacuum infiltration of wheat seeds (growth stage (GS) 11; seedling) with bacteria, most (approximately 99%) of the bacteria stayed associated with the seed, while a small proportion was recovered from the roots and leaves (0.8% and 0.2%, respectively). When seeds were inoculated with 5 x 108 recombinant colony forming units (cfu) per seed the bacterium was able to survive up to levels of 105 cfu g(-1) root during the whole of the plant cycle (4 months), but the bacterium established on the first emerging leaves only in low numbers (approximately 1000 cfu g(-1) tissue). The bacterium could be recovered from roots extracted from a depth of 60 cm, but most (> 95%) of the bacteria stayed associated with roots in the top 15 cm of the soil profile. When bacteria were sprayed onto wheat plants at GS 22 (tillering), they could be recovered 1 month later (GS 69; flowering) from all leaves at levels greater than 105 cfu g(-1) tissue, but not from the ear. Seventy days after spray application (GS 92; ripening) most of the bacteria had disappeared from the phylloplane. After incorporation in soil at rates of 106 recombinant cfu g(-1) soil, bacterial numbers of P. aureofaciens declined by 0.028 log units/d at a temperature of 5 C and by 0.07 log units per day at 18 C. The isolate of P. aureofaciens used in this study was able to survive desiccation at 60% r. h. for 1 month, but was unable to survive for more than 1 month when exposed to high levels of microbial competition at 100% r. h.