Abstract
A strict relationship exists between the Sirex noctilio and the Amylostereum areolatum, which is carried and spread by its partner. The growth and development of this symbiotic fungus is key to complete the life history of the Sirex woodwasp. Real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure gene expression in samples of A. areolatum at different growth stages and explore the key genes and pathways involved in the growth and development of this symbiotic fungus. To obtain accurate RT-qPCR data, target genes need to be normalized by reference genes that are stably expressed under specific experimental conditions. In our study, the stability of 10 candidate reference genes in symbiotic fungal samples at different growth and development stages was evaluated using geNorm, NormFinder, BestKeeper, delta Ct methods, and RefFinder. Meanwhile, laccase1 was used to validate the stability of the selected reference gene. Under the experimental conditions of this study, p450, CYP, and γ-TUB were identified as suitable reference genes. This work is the first to systematically evaluate the reference genes for RT-qPCR results normalization during the growth of this symbiotic fungus, which lays a foundation for further gene expression experiments and understanding the symbiotic relationship and mechanism between S. noctilio and A. areolatum.
Highlights
Sirex noctilio Fabricius (Hymenoptera; Symphyta; and Siricidae) is an important forest woodborer, which mainly damages Pinus species (Slippers et al, 2012) worldwide
There is a strict mutualism between S. noctilio and A. areolatum (Slippers et al, 2012; Biedermann and Vega, 2020)
The growth and development of this symbiotic fungus in host trees play an important role in the life history of the woodwasp (Li et al, 2015)
Summary
Sirex noctilio Fabricius (Hymenoptera; Symphyta; and Siricidae) is an important forest woodborer, which mainly damages Pinus species (Slippers et al, 2012) worldwide. S. noctilio carries the oidia and mycelia of A. areolatum through the specialized mycangium, which disperses and introduces the fungus into a new host during oviposition (Slippers et al, 2012; Li et al, 2015). A. areolatum digests and degrades large molecular carbohydrates to small ones in the xylem of pine wood by secreting extracellular enzymes, which provides necessary nutritional for the incubation of woodwasp eggs and larval growth and development (Talbot, 1977; Madden and Coutts, 1979; Hajek et al, 2013; Wang et al, 2019). Before the third or fourth instar stages, the woodwasp larvae obtain nutrition by directly feeding on the mycelia of the symbiotic fungus, and on the xylem infected by A. areolatum (Madden and Coutts, 1979; Thompson et al, 2014). We believe that the growth and development of S. noctilio larvae are directly affected by the growth of A. areolatum
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