Stimulation of tentoxin synthesis by aged-culture filtrates and continued synthesis in the presence of protein inhibitors.

Tentoxin, a cyclic tetrapeptide produced by Alternaria alternata (Fries) Keissler, induces chlorosis in certain seedling plants. It can be extracted from culture filtrates of the fungus. Tentoxin production is stimulated and increased by using a mixture of aged culture filtrates and modified Richards solution. Aged culture filtrates can be obtained from 3-week-old or older cultures of A. alternata in modified Richards solution or Pratts solution. A mixture of aged culture filtrate and fresh medium in the ratio 2:3 gives the maximal enhancement of tentoxin production. This growth system provided us with a model for studying the effects of protein synthesis inhibitors on tentoxin production. Two antibiotics which inhibit protein synthesis at the ribosomal level were tested on growth, protein synthesis, and tentoxin production in A. alternata cultures. Cycloheximide at concentrations of 500 mug/ml or emetine at concentrations of 250 mug/ml did not inhibit tentoxin synthesis, although they stopped mycelial growth and protein synthesis of the fungus at the logarithmic growth stage in the enhancement medium. These results led us to conclude that tentoxin, like certain other bioactive cyclic peptides, is synthesized by a nonribosomal peptide synthesis mechanism.