Serena Williams is examined by medical staff for a knee injury before retiring against Jamila Wolfe during the Hopman Cup tennis tournament in Perth, on January 5, 2016. [Photo/Agencies]

PERTH, AUSTRALIA - The Australian Open preparations of Serena Williams suffered a potentially significant blow when she was forced to retire with a knee injury during the United States' 3-0 Hopman Cup loss to Australia Gold on Tuesday evening.

The world number one retired due to soreness in her left knee during her singles match against Jarmila Wolfe (nee Gajdosova) when trailing 7-5, 2-1.

Veteran Lleyton Hewitt then secured the tie for Australia Gold with a 7-5, 6-4 win over Jack Sock, and it became a clean sweep for the home side when the mixed doubles was a walkover due to Williams' absence.

The result meant the US bowed out of contention in the tournament. But of more concern to their camp was the fitness of the defending Australian Open champion.

Williams had already missed the United States’ opening Hopman Cup tie on Monday due to inflammation of her left knee and was keen to gain some valuable match practice against Wolfe.

However, she appeared extremely restricted in her movements, particularly on her left side.

The ailment appeared to cast significant doubt over her title defence in Melbourne, where she will aim for a seventh singles title later this month.

Williams admitted she retired because she was being hampered by the knee and could not move around the court as she wanted.

However, the 21-times Grand Slam singles champion was confident it was only a minor problem and even refused to rule herself out of Thursday’s Hopman Cup tie against the Czech Republic.

"I just have some inflammation that's been going away very slowly," she explained.

"It's still there, it's going away, but just needs a little more time. A little rest, a little treatment.

"I've been training really hard during the off-season and really pushing myself beyond the limits, I just think a day off or two will make a world of difference."

Williams said her 2015 campaign, which saw the 34-year-old on target for a calendar Grand Slam until she was beaten by Italian Roberta Vinci in the US Open final in early September, had taken a heavy physical toll.

She barely played after the US Open, although she returned in the International Premier Tennis League in Manila last month.

Williams’ coach Patrick Mouratoglou conceded Williams' knees were troubling her in November.

However, Williams said she remained confident of another successful Australian Open campaign.

"Speed is my strength so I definitely think I will be able to get it right for Australia," she said.

"I only think positive and I know I’ll be okay."

Earlier in the day, Alexandr Dolgopolov put Ukraine within reach of their second Hopman Cup final.

In Ukraine's first appearance at the event since being runners-up in 1995, world number 36 Dolgopolov gave his country two wins from as many ties with his victory over the Czech Republic's Jiri Vesely.

After the 19th-ranked Elina Svitolina upset world number 11 Karolina Pliskova in straight sets in the women's singles, Dolgopolov clinched the tie when he beat Vesely in similar fashion 7-5, 7-6 (7/3).

In the dead mixed doubles rubber, the Czechs kept their final hopes alive with a 6-3, 6-1 win.

Having already beaten the USA on Monday, the result puts Ukraine in pole position to reach Saturday's final.

As the only unbeaten team in their group, Dolgopolov and Svitolina simply need to beat Australia Gold in their last round-robin match Thursday to reach the final.

In Ukraine's last appearance at the Hopman Cup, siblings Andrei Medvedev and Natalia Medvedeva were beaten in the final by Germany's Boris Becker and Anke Huber.