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Applied and Environmental Microbiology, October 2009, p. 6504-6514, Vol. 75, No. 20
0099-2240/09/$08.00+0     doi:10.1128/AEM.01867-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Recovery of Nonpathogenic Mutant Bacteria from Tumors Caused by Several Agrobacterium tumefaciens Strains: a Frequent Event?{triangledown}

Pablo Llop,1 Jesús Murillo,2 Beatriz Lastra,3 and María M. López1*

Centro de Protección Vegetal y Biotecnología Instituto Valenciano de Investigaciones Agrarias, IVIA, 46113 Moncada, Valencia, Spain,1 Departamento de Producción Agraria, Universidad Pública de Navarra, 31006 Pamplona, Spain,2 Departamento de Producción Vegetal, Universidad de Santiago, Campus Universitario, 27002 Lugo, Spain3

Received 12 August 2008/ Accepted 14 August 2009

We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor.

* Corresponding author. Mailing address: Centro de Protección Vegetal y Biotecnología Instituto Valenciano de Investigaciones Agrarias, IVIA, 46113 Moncada, Valencia, Spain. Phone: 34-96-3424000. Fax: 34-96-3424001. E-mail: mlopez{at}ivia.es

{triangledown} Published ahead of print on 21 August 2009.

Applied and Environmental Microbiology, October 2009, p. 6504-6514, Vol. 75, No. 20
0099-2240/09/$08.00+0     doi:10.1128/AEM.01867-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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