Linking sequence to function in soil bacteria: sequence-directed isolation of novel bacteria contributing to soilborne plant disease suppression

Abstract

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.

FIG. 1.

Classification of M139-associated isolates (⋄) as Mitsuaria sp. based on 16S rRNA gene sequence analyses. Included in the dendrogram are the sequences of the type strains representative of other species of genera incertae sedis of the order Burkholderiales. The phylogenetic relationships among taxa were inferred from ∼1,200 bp of the 16S rRNA gene by using the neighbor-joining method from distances computed with the maximum composite likelihood algorithm. Bootstrap values of >60% (1,000 replicates) are shown next to the branches. GenBank accession numbers for each sequence are shown in parentheses. Scale bar shows number of base substitutions per site.

FIG. 2.

Classification of M141-associated isolates (⋄) as representing a novel Burkholderia sp. based on 16S rRNA gene sequence analyses. Included in the dendrogram are the sequences of the other 22 named Burkholderia species. The phylogenetic relationship among taxa was inferred from ∼1,300 bp of the 16S rRNA gene by using the neighbor-joining method from distances computed with the maximum composite likelihood algorithm. Bootstrap values of >60% (1,000 replicates) are shown next to the branches. GenBank accession numbers for each sequence are shown in parentheses. Scale bar shows number of base substitutions per site. *, “Candidatus Burkholderia” species with no cultured isolate.

FIG. 3.

Frequency of positive in vitro inhibition activity of Mitsuaria (A) and Burkholderia (B) isolates identified in this study against multiple fungal and oomycete tomato and soybean pathogens. In vitro inhibition activity was tested for eight isolates of each genus on three different media and was scored as positive or negative.