Identification of Suitable Reference Genes for RT-qPCR Normalization in Amylostereum areolatum Cultured on Pinus sylvestris var. mongholica Wood Powder

Sirex noctilio and Amylostereum areolatum form a highly specific mutualistic symbiosis. The growth and host-degrading activities of the symbiotic fungus are critical to the woodwasps, which directly influence the larval survival rate and adult body size of the woodwasps. Gene expression analysis has been extensively employed to decrypt the intricate growth patterns of symbiotic fungi and identify the associated functional genes underpinning their degradation pathways. Appropriate reference genes are crucial for enhancing the accuracy of studies on gene expression. In an effort to refine gene expression analysis in A. areolatum, our study cultivated the symbiotic fungi on the wood powder medium of Pinus sylvestris var. mongolica, aligning closer to its natural growth conditions. Thirteen reference genes underwent meticulous evaluation via algorithms such as delta Ct, geNorm, BestKeeper, RefFinder, and NormFinder, depending on their stability amidst diverse growth and developmental epochs of A. areolatum. α-TUB, P450, and the combination (α-TUB + P450) were distinguished as the most stable candidates for RT-qPCR analysis, confirmed through AaLac1 expression validation. These findings contribute significantly to the investigation of gene expression in A. areolatum and facilitate a deeper understanding of its symbiotic relationship with S. noctilio.