Gloucestershire Warriors were denied a place in the second round of the Powergen Challenge Cup after losing 36-20 at home to Blackbrook.
It was the first time in 110 years that a team from the county had hosted a match in the competition.
But the St Helens-based Blackbrook, who play in the North West Counties Premier Division, proved too strong.
Normanton Knights, who were featured in BBC Sport's Road to Wembley, also bowed out, losing 12-8 to Skirlaugh.
There were mixed fortunes for the armed forces.
The Army claimed a second victory in consecutive seasons over West Hull, winning 38-18, while Royal Navy lost 38-16 to Wigan St Patricks.
Powergen Challenge Cup first round results/fixtures:
Barrow Island 32-18 Stanley Rangers
Bradford Dudley Hill 42-12 Gateshead
Bramley 30-12 Thatto Heath
Clayton 24-32 Warrington Wizards
Crosfields 20-28 Oulton Raiders
East Hull 46-0 Castleford Lock Lane
East Leeds 28-12 Milford Marlins
Eccles & Salford 10-12 Hunslet Warriors
Gloucestershire Warriors 20-36 Blackbrook
Leigh East 86-10 Askam
Leigh MR 76-0 Bransholme North
Ovenden 31-28 Bank Quay Bulls
Royal Air Force 13-42 Siddal
Royal Navy 16-38 Wigan St Patricks
Saddleworth Rangers 33-32 Hull Dockers
Seaton Rangers 34-4 Rochdale Mayfield
Shaw Cross Sharks 32-22 Oldham St Annes
Skirlaugh 12-8 Normanton Knights
The Army 38-18 West Hull
Thornhill 30-12 Eastmoor Dragons
Walney Central v Ideal Isberg
Wath Brow Hornets v Castleford Panthers
West Bowling 15-14 Halton Simms Cross
Widnes St Maries 40-12 St Albans
Wigan St Judes 12-33 Kells
York Acorn 29-22 Ince Rose Bridge