Distinguish the Autoecious and Heteroecious Forms of Scots Pine Blister Rust by Digital <scp>PCR</scp> and Aeciospore Germination

ABSTRACT Scots pine blister rust is one of the most devastating diseases in the Swedish pine forest. Its causal agent, Cronartium pini , has two forms. The autoecious form only infects pine, while the heteroecious form is macrocyclic and infects pine as the aecial host and many herbaceous plants as telial hosts. Both forms cause similar symptoms on pine and produce aecia with aeciospores, which are morphologically indistinguishable. Distinguishing the two forms relies on inoculation tests, which are time‐consuming and low‐throughput, and microsatellite (SSR) genotyping, which provides limited resolution. In this study, we developed a more efficient identification method using a digital PCR (dPCR)‐based protocol targeting three markers: the housekeeping gene TEF1‐α and two mating‐type genes, bW1‐HD1 and bE1‐HD2 . We applied this protocol to 20 samples representing both autoecious and heteroecious forms and compared the results with those from inoculation tests and SSR genotyping. The accuracy of the dPCR protocol was examined using C. pini samples identified previously. Additionally, we analysed the morphology and nuclear behaviour during the autoecious and heteroecious aeciospores germination. Heteroecious aeciospores have non‐septate germ tubes with two nuclei, while the germ tube of autoecious aeciospores forms a vesicle and septa, producing one to four uninucleate buds. The microscopic observations provide additional insight into the biology of each life cycle form. To the best of our knowledge, this protocol with mating‐type gene markers is the first application of dPCR to differentiate lineages within a single species and provide a simple and cost‐effective method to differentiate the two forms.