Nodulation of Shepherdia ×utahensis ‘Torrey’ and the Diversity of Symbiotic Frankia Strains

Abstract

Shepherdia ×utahensis ‘Torrey’ (hybrid buffaloberry) is an actinorhizal plant that can form symbiotic nodules with the actinobacterial genus Frankia. However, little research has been conducted to investigate the presence of Frankia in their nodules and the effects on plant growth. In this study, plants were grown in a Metro-Mix® 820 substrate and inoculated with soils collected from Mohave County, AZ, or in a low organic-matter substrate inoculated with soils from North Logan, UT. The presence of Frankia was quantified using PolF/PolR primers to amplify their nitrogenase (nifH) gene sequences. In the Metro-Mix 820 substrate, plants irrigated with nitrogen (N)-free Hoagland’s solution at pH 6.5 formed nodules at week 12 after experiment initiation, whereas those receiving the same solution with 2 mm ammonium nitrate (NH4NO3) appeared healthy, but no nodules formed. In the low organic-matter substrate, nodules formed in 5 weeks when plants were irrigated with N-free Hoagland’s solution at pH 7.5. Four 300-bp fragments of query sequences (SU1, SU2, SU3, and SU4) were obtained from nodules. When compared with nifH gene sequences reported in the literature using the Basic Local Alignment Search Tool (BLAST), more than 90% similarity to the nifH of Frankia spp. was obtained. The Frankia strains in the nodules shared nifH sequences similar to those of the same host-specific group of Shepherdia. Furthermore, Frankia strains with similar nifH genes have been reported in nodules of Shepherdia argentea (silver buffaloberry). Additionally, Frankia strains belonging to cluster 3 infective strains consisting of Elaeagnaceae and Rhamnaceae infective Frankia showed high similarity to the query sequences. This research demonstrates that nodulation of S. ×utahensis is inhibited at 2 mm NH4NO3. Apart from N, nodule formation may be associated with the substrate type and pH of the nutrient solution. Based on nifH gene sequence amplification, Frankia strains in the root nodules may have the potential to fix atmospheric nitrogen (N2). These Frankia strains have signature gene sequence characteristics of Elaeagnaceae-infective Frankia, suggesting that S. ×utahensis shares Frankia strains similar to its parents.