Osaka hit 37 unforced errors as she lost to Pegula in one hour and 27 minutes
Japanese world number two Naomi Osaka suffered her second straight defeat on clay by losing to Jessica Pegula in the Italian Open second round.
Osaka, who received a first-round bye, lost 7-6 (7-2) 6-2 to the American world number 31 in Rome.
Osaka, 23, has often struggled on clay and negative body language showed her discomfort against a confident Pegula.
After a tight opener where she missed three set points, Osaka's level dropped in the second and Pegula took control.
Pegula, 27, kept her focus and discipline to take advantage of Osaka's increasingly wayward hitting, breaking for 4-2 when Osaka walloped a wild volley wide and going on to seal the biggest win of her career.
For Osaka, this defeat followed a second-round loss at the Madrid Open earlier this month and comes two and a half weeks before the start of the French Open.
Serena Williams plays the 1,000th match of her career later on Wednesday when she faces Argentina's Nadia Podoroska, a surprise French Open semi-finalist last year.
The 39-year-old American, a 23-time Grand Slam champion, returns to competitive action for the first time since her Australian Open semi-final defeat by Osaka on 18 February.
Meanwhile, Australian world number one Ashleigh Barty reached the third round by beating Kazakhstan's Yaroslava Shvedova 6-4 6-1.
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