Sybr Green- and TaqMan-Based Quantitative PCR Approaches Allow Assessment of the Abundance and Relative Distribution of Frankia Clusters in Soils

Abstract

The nodule-forming actinobacterial genus Frankia can generally be divided into 4 taxonomic clusters, with clusters 1, 2, and 3 representing nitrogen-fixing strains of different host infection groups and cluster 4 representing atypical, generally non-nitrogen-fixing strains. Recently, quantitative PCR (qPCR)-based quantification methods have been developed for frankiae of clusters 1 and 3; however, similar approaches for clusters 2 and 4 were missing. We amended a database of partial 23S rRNA gene sequences of Frankia strains belonging to clusters 1 and 3 with sequences of frankiae representing clusters 2 and 4. The alignment allowed us to design primers and probes for the specific detection and quantification of these Frankia clusters by either Sybr Green-or TaqMan-based qPCR. Analyses of frankiae in different soils, all obtained from the same region in Illinois, USA, provided similar results, independent of the qPCR method applied, with abundance estimates of 10 ϫ 10 5 to 15 ϫ 10 5 cells (g soil) Ϫ1 depending on the soil. Diversity was higher in prairie soils (native, restored, and cultivated), with frankiae of all 4 clusters detected and those of cluster 4 dominating, while diversity in soils under Alnus glutinosa, a host plant for cluster 1 frankiae, or Betula nigra, a related nonhost plant, was restricted to cluster 1 and 3 frankiae and generally members of subgroup 1b were dominating. These results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees. IMPORTANCE Root nodule formation by the actinobacterium Frankia is host plant specific and largely, but not exclusively, correlates with assignments of strains to specific clusters within the genus. Due to the lack of adequate detection and quantification tools, studies on Frankia have been limited to clusters 1 and 3 and generally excluded clusters 2 and 4. We have developed tools for the detection and quantification of clusters 2 and 4, which can now be used in combination with those developed for clusters 1 and 3 to retrieve information on the ecology of all clusters delineated within the genus Frankia. Our initial results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.

Frankia Diversity in Host Plant Root Nodules Is Independent of Abundance or Relative Diversity of Frankia Populations in Corresponding Rhizosphere Soils

“…The diversity of Frankia populations, however, was relatively low, with frankiae of clusters 1b, 1e, and 3 present in nonvegetated controls, and cluster 1e and 3 frankiae generally were present in vegetated microcosms only. Previous studies reported on the low diversity of Frankia populations in different soil environments, e.g., the presence of cluster 1b only in wet or even water-logged soils in natural stands of A. glutinosa (23), clusters 1b and 3 in forest soils with A. glutinosa or nonhost plants, including Betula nigra (21,24), clusters 1a, 1b, and 3 in forest soils with A. glutinosa (21), clusters 1a and 3 in microcosms with A. glutinosa or C. equisetifolia (30), or clusters 1b, 2, 3, and 4 in prairie soils with Ceanothus species as potential host plants (24). These studies suggest soil-specific environmental effects on Frankia populations in soils, with frankiae of clusters 1b and 3 being quite ubiquitous and independent of the presence of host plant species.…”

“…A single nodule was found on C. equisetifolia, with a Frankia population representing cluster 1e. Casuarina species usually produce nodules with Frankia populations of cluster 1c (24), meant to represent a group of highly specialized frankiae that depend on cointroduction with their exotic host plant species outside their native range (28,29). Consequently, Casuarina-infective Frankia populations were not expected to form nodules in soils that have never supported Casuarina spp.…”

“…Prior to European settlement of the study region in the early nineteenth century, the landscape encompassing the site was tallgrass prairie and oak savanna, having the native actinorhizal shrub Ceanothus americanus L. as a common component. Existing native prairie remnants contain C. americanus and the corresponding, detectable soil frankiae specific to cluster 2 (24). It has been speculated that cluster 2 frankiae depend on their host plants for growth and establishment (24,37), and thus, the conversion of native vegetation almost entirely to agricultural and urban uses, primarily the cultivation of maize, legume hay crops, and, in recent decades, soybeans may have resulted in the loss of cluster 2 frankiae at this site and other cultivated sites in this region.…”

“…The case is different for highly complex and diverse environments, such as soil with Frankia populations present in small numbers within a large and complex microbial community (21), for which even molecular tools such as gene clone library analyses provide limited information (14,17). The development of Frankia-specific quantitative PCR (qPCR) (22)(23)(24) or targeted Illumina sequencing (25) analyses now provides increasingly sophisticated molecular tools for the analyses of Frankia populations in soil.…”

“…The case is different for highly complex and diverse environments, such as soil with Frankia populations present in small numbers within a large and complex microbial community (21), for which even molecular tools such as gene clone library analyses provide limited information (14,17). The development of Frankia-specific quantitative PCR (qPCR) (22)(23)(24) or targeted Illumina sequencing (25) analyses now provides increasingly sophisticated molecular tools for the analyses of Frankia populations in soil.The aim of this study was to assess Frankia diversity in root nodules of different host plant species growing in the same soil and to relate this diversity to the abundance and relative distribution of indigenous frankiae in rhizosphere soils. Different actinorhizal plant species, including Alnus glutinosa, Alnus cordata, Alnus viridis, Casuarina equisetifolia, Shepherdia argentea, and Hippophaë rhamnoides, as well as the nonactinorhizal plant species Betula pendula, were grown in soil microcosms that, together with a nonvegetated control, were analyzed by Frankia-specific qPCR and Illumina sequencing in order to assess the abundance and relative distribution of indigenous frankiae in rhizosphere soils.…”