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"I'd be very surprised if I was playing in Paris," a subdued Murray said after quitting a second-round start at the Foro Italico, handing a 6-3, 6-7 (5/7) win to Spain's Marcel Gronollers.
Murray, ranked world number two behind Novak Djokovic, had been seen by the trainer for on-court hip and back treatment after the third game of the second set.
The defeat marked the second straight year in Rome that Murray, a 2011 semi-finalist, has gone out early, following a third round exit a year ago.
The French Open begins in Paris on May 26 and even if Murray should recover in time to play in the Grand Slam, he heads to the tournamanet with just three clay wins this season, alongside as many defeats.
Murray was joined on the sidelines on a cloudy day at the Foro Italico by Jo-Wilfried Tsonga, with Polish underdog Jerzy Janowizc ripping off his shirt in celebration seconds after clinching the 6-4, 7-6 (7/5) win over the eighth seed.
Ukranian Alexandr Dolgopolov got a free trip into the third round as Madrid finalist Stanislas Wawrinka quit before their meeting with a thigh injury which also bothered him last week.
In women's play, former Roland Garros winner and fifth seed Li Na won a battle with Chinese countrywoman Zheng Jie 6-3, 6-1 while Australian ninth seed Samantha Stosur, the 2010 beaten finalist in Paris, beat China's Peng Shuai 7-6 (7/5), 6-0.
Russian 12th seed Maria Kirilenko beat Varvara Lepchenko of the United States 6-3, 6-1. The 13th-seeded Roberta Vinci kept her home public happy as she beat compatriot Natassja Burnett 6-1, 6-4. Slovak 14th seed Dominika Cibulkova put out US qualifier Melanie Oudin 5-7, 6-1, 6-3.
Results from the fourth day of the ATP and WTA Rome Open (x denoted seeding):
Men:
Second round:
Jerzy Janowicz (POL) bt Jo-Wilfried Tsonga (FRA x8) 6-4, 7-6 (7/5)
Alexandr Dolgopolov (UKR) bt Stanislas Wawrinka (SUI x15) walkover, thigh injury
Marcel Granollers (ESP) bt Andy Murray (GBR x3) 6-3, 6-7 (5/7), retired, (hip injury)
Benoit Paire (FRA) bt Julien Benneteau (FRA) 6-7 (3/7), 6-4, 7-6 (7/4)
Gilles Simon (FRA) bt Mikhail Youzhny (RUS) 7-5, 6-3
Women:
Second round:
Dominika Cibulkova (SVK x14) bt Melanie Oudin (USA) 5-7, 6-1, 6-3
Roberta Vinci (ITA x13) bt Nastassja Burnett (ITA) 6-1, 6-4
Li Na (CHN x5) bt Zheng Zie (CHN) 6-3, 6-1
Maria Kirilenko (RUS x12) bt Varvara Lepchenko (USA) 6-3, 6-1
Samantha Stosur (AUS x9) bt Peng Shuai (CHN) 7-6 (7/5), 6-0
