Iain Flannigan and Callum Crane were both on target for Alloa as they dented Ayr's title hopes with a 2-1 home win.
Flannigan and Crane gave Alloa a two-goal half-time lead and although Craig Moore pulled one back for the visitors early in the second period, the Ayr striker missed a 77th-minute penalty.
Alloa's play-off place was already guaranteed following Stranraer's home defeat to Raith on Saturday, while Ayr, who slipped to back-to-back defeats, would have leapfrogged leaders Raith into top spot with victory.
Ayr can still snatch top spot and automatic promotion if they win their final game next week at home to Albion and Raith fail to beat Alloa, who moved level on points with third-placed Arbroath.
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