Treviso and Sambenedettese have been declared bankrupt.
They join Avellino, Pisa and Venezia in going under.
"It is always painful to lose clubs, in this case some very glorious ones," sighed Lega Pro (formerly Serie C) President Macalli.
"I can say that if the clubs coming down from Serie B had remained in that division, they would have continued playing even with 10m Euros of debt.
"It is different for us in Lega Pro, where even 100,000 Euros is enough to send you under."