Over 400 bacterial strains, isolated from leaf surfaces of Curcuma alismatifolia Gagnep. and hot springs in the Chiang Mai province of northern Thailand, were screened in vitro for antagonistic activity against Colletotrichum musae, an anthracnose fungus. Three isolates provided greater than 75% growth inhibition of the fungus in vitro and were identified as Bacillus licheniformis, B. amyloliquefaciens and B. subtilis. Using in planta tests, B. amyloliquefaciens and B. subtilis were shown to efficiently colonize the curcuma bracts, provide a statistically significant growth suppression of C. musae over that of B. licheniformis, and all three isolates could provide 100% inhibition of conidial fungal germination. When B. licheniformis was co-inoculated in combination with either of the other two bacteria, the ability of B. amyloliquefaciens and B. subtilis to suppress the fungal disease was dramatically reduced. Both B. amyloliquefaciens and B. subtilis were found to contain an isoform of iturin A with antifungal activity against C. musae. As a preventative measure to control the spread of C. musae and reduce the severity of fungal infections, B. amyloliquefaciens could be used to inoculate curcuma flowers cost effectively and reduce the need for the toxic synthetic fungicides currently in use.

A protocol for regeneration and genetic transformation was established for Curcuma alismatifolia Gagnep. 'Chiang Mai Pink' using retarded shoots as explants. In vitro retarded shoots were cut into 0.5ϫ0.5ϫ0.5 cm blocks and cocultivated with Agrobacterium tumefaciens strain AGLO harboring the binary vector, pBI121 or pBI121-Ca-ACS1. The explants were incubated in the bacteria suspension for 30 min. The explants and bacteria were cultured on MS medium for 2 days in darkness at 25°C for co-cultivation. Then, the explants were transferred onto MS medium containing 0.1 mg l Ϫ1 IAA, 4 mg l Ϫ1 IMA, 0.5 mg l Ϫ1 TDZ, 50 mg l Ϫ1 kanamycin and 500 mg l Ϫ1 vancomycin. The explants were subcultured every 2 weeks. After 4 weeks in culture, the explants with small shoot buds were transferred onto MS medium containing 50 mg l Ϫ1 kanamycin. Within 4 weeks, the shoots were separated and subcultured every 2 weeks on MS medium containing 0.1 mg l Ϫ1 IAA and 50 mg l Ϫ1 kanamycin. PCR analysis, histochemical GUS assay and Southern blotting of the regenerated plants confirmed transformation events. We obtained transformed plants within 3 months after co-cultivation with the bacteria and the transformation frequency exceeded 14%, which is suitable for practical use.

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