(1) Background: Cultivating resistant rootstocks is an effective way to mitigate apple replant disease (ARD), and we developed superior apple rootstock line 12-2 (self-named), which shows improved ARD resistance. (2) Methods: We used ARD-associated pathogen Fusarium proliferatum MR5 (MR5) to test the fungal infection in the 12-2 line. Seedlings of the 12-2, T337, and M26 rootstock lines were planted in a substrate with potato dextrose broth and MR5 spore solution, and aboveground physiological indicators were measured. (3) Results: MR5 had the greatest effect on the leaf growth of T337 and M26. The incidence rates of infectious symptoms in the T337 and M26 lines were 68 and 100%, respectively. MR5 significantly affected the leaf chlorophyll content, ETR, and NPQ of T337 and M26, as well as P-n and T-r of M26. MR5 tended to reduce the leaf photosynthetic parameters of T337, but the decreases were not significant. The leaf reactive-oxygen-species levels of T337 and M26, the leaf antioxidant-enzyme activities of M26, and the superoxide-dismutase activity of T337 were significantly affected by MR5. MR5 also had a significant effect on the leaf malondialdehyde, proline, and soluble-sugar contents of T337 and M26. None of these aboveground physiological indicators were affected by MR5 in the 12-2 rootstock. (4) Conclusions: The 12-2 rootstock was more resistant to ARD-associated MR5 and could serve as an important test material for resistant-apple-rootstock breeding in China.
