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England's batting big gun was deceived by off-spinner Stephen O'Keefe and was bowled off-stump for five as the tourists made hard work of chasing down Australia A's 230.
At tea, England were 147 for five with Paul Collingwood on seven and Ian Bell six not out.
Australia A, featuring the cream of the country's young talent, struck a big morale blow to the tourists ahead of next week's Ashes opener in Brisbane with the prized wicket of Pietersen.
Pietersen, who averages 50.73 in Ashes Tests against Australia, lost some valuable batting time ahead of the first Test only lasting 22 balls.
Alastair Cook scored 60 off 109 balls before he got a leading edge to O'Keefe in the 38th over and was taken by Clint McKay at deep mid-on after putting on 87 runs for the third wicket with Jonathan Trott.
Trott fell in the 41st over for 41 when he got a leading edge while attempting to pull paceman Mark Cameron, and Usman Khawaja took an excellent running catch at mid-off.
At the interval, O'Keefe, who topscored with 66 in Wednesday's first innings, had 2-23 off 9.1 overs.
Play was frequently interrupted by showers and only 7.2 overs were bowled in the morning session.
McKay claimed the wicket of nightwatchman Monty Panesar, who top-edged a pull shot to Cameron at deep backward square for 13.
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