Alexei Popyrin on Sunday became the latest Australian player to withdraw from the US Open, following compatriots Ashleigh Barty and Nick Kyrgios.
The decision by Popyrin, the 20-year-old world number 103, means that former champion Andy Murray moves into the main draw as of right having already been awarded a wild card by the organisers.
Women's world number one Barty and Kyrgios, one of the sport's major drawcards despite his ranking of 40, had both said they would not make the trip to New York for the Grand Slam event.
They cited serious health concerns over the coronavirus for their decision.
The reason for Popyrin's withdrawal was not given.
Four-time champion Rafael Nadal has also opted not to take part as have women's top 10 players Elina Svitolina and Kiki Bertens.
Murray, currently ranked 129 after a lengthy period battling a hip injury, narrowly missed direct entry into the US Open which starts on 31 August.
The 33-year-old won the first of his three Grand Slam titles at the tournament in 2012, becoming the first British male to win a major since Fred Perry in 1936.
Mitchell Krueger of the United States will receive the wild card entry previously awarded to Murray.