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Applied and Environmental Microbiology, February 2009, p. 915-924, Vol. 75, No. 4
0099-2240/09/$08.00+0     doi:10.1128/AEM.01296-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Linking Sequence to Function in Soil Bacteria: Sequence-Directed Isolation of Novel Bacteria Contributing to Soilborne Plant Disease Suppression{triangledown} ,{dagger}

María-Soledad Benítez and Brian B. McSpadden Gardener*

Department of Plant Pathology, The Ohio State University, OARDC, 1680 Madison Avenue, Wooster, Ohio 44691

Received 10 June 2008/ Accepted 9 December 2008

Microbial community profiling of samples differing in a specific ecological function, i.e., soilborne plant disease suppression, can be used to mark, recover, and ultimately identify the bacteria responsible for that specific function. Previously, several terminal restriction fragments (TRF) of 16S rRNA genes were statistically associated with damping-off disease suppression. This work presents the development of sequence-based TRF length polymorphism (T-RFLP)-derived molecular markers to direct the identification and isolation of novel bacteria involved in damping-off pathogen suppression. Multiple sequences matching TRF M139 and M141 were cloned and displayed identity to multiple database entries in the genera incertae sedis of the Burkholderiales. Sequences matching TRF M148, in contrast, displayed greater sequence diversity. A sequence-directed culturing strategy was developed using M139- and M141-derived markers and media reported to be selective for the genera identified within this group. Using this approach, we isolated and identified novel Mitsuaria and Burkholderia species with high levels of sequence similarity to the targeted M139 and M141 TRF, respectively. As predicted, these Mitsuaria and Burkholderia isolates displayed the targeted function by reducing fungal and oomycete plant pathogen growth in vitro and reducing disease severity in infected tomato and soybean seedlings. This work represents the first successful example of the use of T-RFLP-derived markers to direct the isolation of microbes with pathogen-suppressing activities, and it establishes the power of low-cost molecular screening to identify and direct the recovery of functionally important microbes, such as these novel biocontrol strains.

* Corresponding author. Mailing address: Department of Plant Pathology, The Ohio State University, OARDC, 1680 Madison Avenue, Wooster, OH 44691. Phone: (330) 202-3565. Fax: (330) 263-3841. E-mail: mcspadden-garden.1{at}osu.edu

{triangledown} Published ahead of print on 16 December 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, February 2009, p. 915-924, Vol. 75, No. 4
0099-2240/09/$08.00+0     doi:10.1128/AEM.01296-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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