Southgate has a thigh injury and will be replaced in the squad by a first senior call-up for Anthony Gardner of Tottenham Hotspur, the Football Association said on Tuesday.
Southgate was himself a stop-gap for the game at Old Trafford, his re-call on Monday being due to the unavailability of Sol Campbell and Jonathan Woodgate.
Beckham, who suffered a back injury in Real Madrid's 4-1 defeat by Sevilla at the weekend, will be assessed by England's medical staff when he reports for duty on Thursday.
Having been advised by club doctors to rest until Wednesday, Beckham has been left out of Real's squad for a friendly against Valladolid on Tuesday.
England's medical staff will speak to their Everton counterparts on Wednesday about Rooney, who missed their defeat by Blackburn Rovers on Monday due to 'flu.
