Pigment produced by glycine-stimulated macrophomina phaseolina is a (-)-botryodiplodin reaction product and the basis for an in-culture assay for (-)-botryodiplodin production

Authors: ALAM, SAHIB - University Of Minnesota; Abbas, Hamed; SULYOK, MICHAEL - University Of Natural Resources And Life Sciences, Vienna; KHAMBHATI, VIVEK - Oak Ridge Institute For Science And Education (ORISE); OKUNOWO, WAHAB - University Of Lagos; SHIER, THOMAS - University Of Minnesota

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/17/2022
Publication Date: 2/22/2022
Citation: Alam, S., Abbas, H.K., Sulyok, M., Khambhati, V.H., Okunowo, W.O., Shier, T.W. 2022. Pigment produced by glycine-stimulated macrophomina phaseolina is a (-)-botryodiplodin reaction product and the basis for an in-culture assay for (-)-botryodiplodin production. Pathogens. 11(3):280. https://doi.org/10.3390/pathogens11030280.
DOI: https://doi.org/10.3390/pathogens11030280

Interpretive Summary: (-)-Botryodiplodin is a toxin produced by the fungus Macrophomina phaseolina, the causal agent of charcoal rot in soybean and other economically important crops, and it plays an important role in the infection process of the fungus. Currently, there are no inexpensive and easy methods to detect this toxin. This paper develops an inexpensive and simple method to detect (-)-botryodiplodin under natural conditions, and due to the simplicity of the method, a large number of samples can be simultaneously tested for botryodiplodin. Furthermore, the method does not require specialized technical expertise or equipment, so the method can be utilized by individuals or laboratories with limited resources. The findings from this paper will be useful for growers, industry, and scientists working in this area of research.

Technical Abstract: Dunlap and Bruton reported that a pathogenic Macrophomina phaseolina isolate from muskmelons (Cucumis melo) produced red pigment(s) in melons and in culture in the presence of added glycine, alanine, leucine or asparagine in the medium, but not with some other amino acids and nitrogen-containing compounds. We explored the generality and mechanism of this pigment production response using pathogenic M. phaseolina isolates from soybean plants expressing symptoms of charcoal rot disease. A survey of 42 M. phaseolina isolates growing on Czapek-Dox agar medium supplemented with glycine confirmed pigment production by 71% of isolates at the optimal glycine concentration (10 g/L). Studies in this laboratory have demonstrated that some pathogenic isolates of M. phaseolina produce the mycotoxin (-)-botryodiplodin, which has been reported to react with amino acids, proteins and other amines to produce red pigments. Time course studies showed a significant positive correlation between pigment and (-)-botryodiplodin production by selected M. phaseolina isolates with maximum production at 7-8 days. Pigments produced in agar culture medium supplemented with glycine, beta-alanine or other amines exhibited similar UV-vis adsorption spectra as did pigments produced by (±)-botryodiplodin reacting in the same agar medium. In a separate study of 39 M. phaseolina isolates, red pigment production (OD520) on 10 g/L glycine-supplemented Czapek-Dox agar medium correlated significantly with (-)-botryodiplodin production (LC/MS analysis of culture filtrates) in parallel cultures on unsupplemented medium. These results support pigment production on glycine-supplemented agar medium as a simple and inexpensive in culture method for detecting (-)-botryodiplodin production by M. phaseolina isolates.