An agar medium-based method for screening somatic incompatibility in Agaricus bisporus

To visualize the nonself recognition reaction in the cultivated mushroom Agaricus bisporus, we developed a method using the azo dye Evans blue. The use of Evans blue highlights dead mycelial sections, which are produced following nonself recognition in the interaction zone between two individuals. This method can differentiate between distinct heterokaryons, as well as between closely related heterokaryons constructed from siblings. As it is known that co-cultivation of mixed individuals leads to reduced yield, we compared small-scale cultivation experiments to the results of our laboratory assay. Co-cultivation of strains whose interaction produced noticeable Evans blue staining also produced low yield when mixed. However, a combination that did not produce noticeable Evans blue staining still produced an incompatible-like phenotype (reduced yield) when mixed under cultivation conditions. Together, these results suggest that while our Evans blue assay can discriminate between self and nonself pairings, it alone does not encompass all aspects of this interaction. However, this method can facilitate future research into the genetics and physiology of the incompatibility phenotype in this economically important fungus.