Synchronous Sporulation of Ascosphaera Proliperda

ABSTRACT

A minimal medium containing glucose, phosphate-buffered saline, and (NH₄)₂SO₄, CaCl₂, MgSO₄, FeSO₄, ZnSO₄, and agar was developed to insure the production of large number of synchronous ascocysts of Ascosphaera proliperda with minimal vegetative growth. This fungus is a larval pathogen of the alfalfa leaf cutting bee, Megachile rotundata. The synchronous production of ascocysts was essential for the isolation of two proteins associated with sexual reproduction of this fungus. To develop this medium, pH, carbon and nitrogen sources, and trace elements were studied for their effects on the differentiation of ascocysts. Over the pH range 5.4-8, pH 6 was the most suitable for production of synchronous ascocysts in abundance. Size of colonies grown on media containing galactose, sucrose, or glucose did not vary significantly; however, more ascocysts were produced with medium containing either sucrose or glucose. Only (NH₄)₂SO₄ at concentration of 0.25% was suitable as a nitrogen source. Other sources such as (NH₄)₂CO₃, NH₄Cl, and NH₄NO₃ were not suitable for fungal growth or production of ascocysts. Medium lacking trace elements (B, Cl, Cu, Fe, Mg, Mn, Mo, S, Zn) did not support fungal growth or ascocyst formation; only Fe, Mg, S and Zn were necessary for sporulation. To maximize synchronous contact between the two mating types, a mixture of hyphal fragments of both mating types was used as an inoculum in the tested media.

Key Words:

• Ascosphaera proliperda
• carbon sources
• minimal medium
• nitrogen sources
• synchronous sporulation
• trace elements