Evaluation of qPCR to Detect Shifts in Population Composition of the Rhizobial Symbiont Mesorhizobium japonicum during Serial in Planta Transfers

SIMPLE SUMMARY: Rhizobia species that fix atmospheric nitrogen in nodules that form on the root are very beneficial to plant growth and productivity. Highly efficient nitrogen-fixing rhizobia developed under laboratory conditions often do not function at the same level in natural soils with native microbial communities. The aim of this research is to study how the plant (Lotus japonicus) associates with three rhizobia from the same species (Mesorhizobium japonicum) when they are present together at the same time. These three rhizobia have different nitrogen-fixing abilities, making them highly beneficial (HB), moderately beneficial (MB), and non-beneficial (NM) to the plant. Using a quantitative PCR (qPCR)-based approach, we show that the plant selectively favors the HB rhizobia, such that at the end of the third generation, the plant was able to exclude almost all the MB and NB rhizobia from its nodules. To our surprise, when only the MB and NB rhizobia were present together, the plant could not favor the MB rhizobia and eliminate the NB rhizobia from its nodules. Studying more complex scenarios, with tens and hundreds of different rhizobia together, will help us better understand this interaction between rhizobia and plants and develop highly beneficial rhizobia to increase agricultural productivity. ABSTRACT: Microbial symbionts range from mutualistic to commensal to antagonistic. While these roles are distinct in their outcome, they are also fluid in a changing environment. Here, we used the Lotus japonicus–Mesorhizobium japonicum symbiosis to investigate short-term and long-term shifts in population abundance using an effective, fast, and low-cost tracking methodology for M. japonicum. We use quantitative polymerase chain reaction (qPCR) to track previously generated signature-tagged M. japonicum mutants targeting the Tn5 transposon insertion and the flanking gene. We used a highly beneficial wild type and moderately beneficial and non-beneficial mutants of M. japonicum sp. nov. to demonstrate the specificity of these primers to estimate the relative abundance of each genotype within individual nodules and after serial transfers to new hosts. For the moderate and non-beneficial genotypes, qPCR allowed us to differentiate genotypes that are phenotypically indistinguishable and investigate host control with suboptimal symbionts. We consistently found the wild type increasing in the proportion of the population, but our data suggest a potential reproductive trade-off between the moderate and non-beneficial genotypes. The multi-generation framework we used, coupled with qPCR, can easily be scaled up to track dozens of M. japonicum mutants simultaneously. Moreover, these mutants can be used to explore M. japonicum genotype abundance in the presence of a complex soil community.