Italy midfielder Luigi Di Biagio will miss their deciding group G match against Mexico on Thursday after failing to recover from a calf injury.
"Di Biagio will certainly not play," said team doctor Andrea Ferretti. "It's not going to be a short recovery. I believe that it is an injury that will require at least 15 days to recover."
That would rule the midfielder out of action until at least the semifinals starting June 25, should Italy get that far in the tournament.
Di Biagio limped out of training on Monday with the injury to his right calf and could not finish the session.
Five players sat out Tuesday's training match as coach Giovanni Trapattoni's injury problems mounted, but Di Biagio is the only player definitely out of the game against Mexico that Italy must win to be sure of a place in the second round.
Cristiano Zanetti and Alessandro Nesta (both with a bruised foot) missed the practice match, while captain Paolo Maldini and defender Christian Panucci jogged around the training ground before the training game but did not take part in the action.
However, Ferretti said Maldini and Panucci had minor knocks and would play against the Mexicans, while Nesta and Zanetti should also be fit.
"Nesta is recovering slowly. I hope he will be able to practise tomorrow (Wednesday). If he can train tomorrow he will be available (for the game).
"Zanetti is recovering and should train tomorrow and I believe he will be available to the coach (for the match).
The unfortunate Di Biagio missed Italy's 2-1 defeat to Croatia on Saturday due to a neck injury and had struggled with pain from an ingrowing toenail throughout training last week.
Mexico lead group G with six points followed by Italy and Croatia on three, while bottom-placed Ecuador have lost both their games.