Investigating the formation of dikaryotic spores between closely related arbuscular mycorrhizal fungi in soil

Arbuscular mycorrhizal fungi (AMF) form important symbiotic relationships with plants, where they provide nutrients to the plant in exchange for carbon. Since this often results in enhanced plant growth, AMF are commercially produced as bioinoculants to reduce the need for chemical fertilizers. AMF contain thousands of nuclei that flow throughout a continuous hyphal network. They can be homokaryotic, where all nuclei are the same genotype, or dikaryotic, where nuclei of two distinct genotypes co-exist. It is believed that dikaryotic AMF are formed by hyphal fusion (anastomosis) and nuclear exchange between two genetically compatible strains; however, this has only been investigated in vitro and subsequent formation of stable dikaryons has yet to be observed. To address whether dikaryons can form between homokaryotic strains in soil, a greenhouse study was conducted using two strains of Rhizophagus irregularis (DAOM 197198, a strain used as a bioinoculant, and DAOM 229455). Strains were inoculated alone and together and grown in pots (n = 27) with a host for three to six months. Spores were individually analyzed to determine if they were homokaryotic or dikaryotic. Out of ~300 spores analyzed, 94% were classified as DAOM 197198, 5% were classified as dikaryotic, and only 1% were classified as DAOM 229455, showing a strong dominance of DAOM 197198. Within the dikaryotic spores, the average relative nucleotype abundance was higher for DAOM 197198 (3:1). The abundance of fungi in the roots was also determined. When grown alone, both strains were equally abundant. When co-inoculated, DAOM 197198 was dominant and, in most cases, was the only strain detected in the roots. Despite this competition, there was no effect on the host, with consistent mycorrhizal growth and phosphorus responses across treatments. Nonetheless, the production of dikaryotic spores, even with high competition, suggests that anastomosis and genetic exchange can occur between R. irregularis strains in soil, although this dikaryotic state is likely temporary. Given that applying competitive, commercialized fungi to novel environments may present an invasion risk, future work should investigate whether anastomosis could be a gateway for foreign nuclei to “cheat” into pre-established mycelial networks and disrupt native mycelial dynamics.