Withdrawal from the EU could jeopardise jobs and investment in British agriculture, with farmers losing as much as £330 million on lamb and beef exports alone, David Cameron has claimed.
The Prime Minister warned that British farmers could "suffer enormously" if the UK votes to leave the EU in the referendum he has called for June 23.
Mr Cameron was putting agriculture at the centre of his latest appeal for voters to remain part of the 28-nation bloc as he visited a farm in north Wales. He was later due to address the Welsh Conservative conference.
He said that more than 90% of UK lamb and beef exports - worth around £605 million - currently go to the EU. The farming sector contributes £9.9 billion to the UK economy and employs almost half a million people.
If farmers had to rely on World Trade Organisation rules, rather than EU membership, to secure access to the market of 500 million people in Europe, they could be faced with tariffs costing £240 million a year for beef and £90 million for lamb, he said.
"British farmers and food producers rely on the single market," said Mr Cameron.
"It gives them access to 500 million consumers, to whom they can sell their goods on an open, unrestricted basis. No tariffs, no barriers, no bogus health and safety rules designed to keep our products out.
"If we left this single market and - as some suggest - relied on World Trade Organisation rules, the extra costs of exporting British beef would be £240 million a year. An extra £90 million would be added to the cost of British lamb exports.
"British agriculture, British farmers and British jobs could suffer enormously if we were to leave the single market."
His comments came as speculation continued over the source of a newspaper story suggesting that the Queen voiced concerns over the UK's EU membership in a private lunch with former deputy prime minister Nick Clegg.
Buckingham Palace has lodged a formal complaint with the Independent Press Standards Organisation (Ipso) about the report in The Sun which appeared under the headline "Queen Backs Brexit".
But Mr Cameron rejected calls for an official investigation into the leak of the reported comments, insisting that the matter was being dealt with by the Independent Press Standards Organisation and there was no need for a further inquiry.
Speaking on BBC Sussex, Mr Cameron said: "The Palace has made a very clear statement, the former deputy prime minister has made a very clear statement saying that this didn't happen and I think we should leave it at that.
"There is obviously a proper investigation now being held by the press complaints commission and I think we should let them do their work."
His comments came as Michael Gove - an ardent Leave campaigner - emerged as the prime suspect for the source of the leak.
The Justice Secretary was one of just four ministers, including Mr Clegg, present at a meeting of the Privy Council held at Windsor Castle in April 2011 when it is thought the conversation with the Queen took place.
A spokeswoman for Mr Gove has refused to comment publicly on the claims, however Mr Cameron appeared to accept that he was not involved.
"These are very serious matters but as far as I can see Michael Gove has made clear that he has no idea where this story came from either," he told BBC Oxford.
The editor of The Sun Tony Gallagher has strongly defended his paper's reporting and the "Queen Backs Brexit" headline - even though she did not explicitly advocate leaving the EU in either of the reported conversations referred to in the story.
"Multiple sources - two sources to be precise - came to us with information about the Queen and her views on the EU and we would have been derelict in our duty if we didn't put them in the paper. It's as simple as that," he told the BBC Radio 4 Today programme.
Labour MP Wes Streeting has written to the Cabinet Secretary Sir Jeremy Heywood urging him to investigate the leak, saying it would be an "extremely serious breach" of Privy Council rules if it came from one of their meetings.