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Nagal threatened to spoil Rune's party by taking the second set after the second round match, which was suspended due to rain on Wednesday, resumed. However, he lost his serve twice in the final set.
Nagal became the first Indian player to win a singles main draw match at an ATP Masters event on clay.
Nagal is the first Indian to feature in singles top 100 since Prajnesh Gunneswaran made the cut in 2019.
Nagal began in the same dominating fashion but the 18-year-old wild card entrant got better as the game progressed to walk out a winner.
Nagal's win on Tuesday was the first time in 35 years that an Indian beat a seeded player in a Grand Slam.
The 26-year-old has qualified for the main draw of a Grand Slam event for the first time since 2021 and fourth overall.
The two players have conveyed their unavailability for the World Group I play-off tie to be played in February but have not specified the reason behind their decision.
Up against top seed and home favourite Zhizhen Zhang, it was always going to be a Herculean task for Nagal.
To stay and play on the ATP Tour, Nagal has invested all his prize money, his salary from Indian Oil Corporation Limited and the support he gets from Maha Tennis Foundation.
Sasikumar Mukund conceded the opening singles when the score was 7-6 (4) 5-7 1-4.