The Al Hilal midfielder, 32, suffered the injury in his side’s 3-1 loss to Mexico in a friendly on Saturday and needs up to three weeks to recover.
Castillo needs 15 to 21 days to recover from the strained ligament, ruling him out of their Group E opener against Switzerland.
He is likely to miss the clash against Honduras but could be available for the June 25 meeting with France.
Stuttgart midfielder Carlos Gruezo, 19, has been included in the squad as additional cover in the centre of the park.
Manchester United winger Antonio Valencia is the star of Reinaldo Rueda’s squad, who face England in a friendly on Wednesday.
Squad in full:
Goalkeepers: Maximo Banguera (Barcelona), Adrian Bone (El Nacional), Alexander Dominguez (LDU)
Defenders: Gabriel Achilier (Emelec), Walter Ayovi (Pachuca), Oscar Bagui (Emelec), Frickson Erazo (Flamengo), Jorge Guagua (Emelec), Juan Carlos Paredes (Barcelona)
Midfielders: Michael Arroyo (Atlante), Segundo Castillo (Al Hilal), Carlos Gruezo (Stuttgart), Renato Ibarra (Vitesse), Fidel Martinez (Tijuana), Edison Mendez (Independiente Santa Fe), Christian Noboa (Dynamo Moscow), Luis Fernando Saritama (Barcelona), Antonio Valencia (Manchester United)
Forwards: Jaime Ayovi (Tijuana), Felipe Caicedo (Al Jazira), Jefferson Montero (Morelia), Joao Rojas (Cruz Azul), Enner Valencia (Pachuca)
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