Pigment Produced by Glycine-Stimulated Macrophomina Phaseolina Is a (-)-Botryodiplodin Reaction Product and the Basis for an In-Culture Assay for (-)-Botryodiplodin Production

Abstract

An isolate of Macrophomina phaseolina from muskmelons (Cucumis melo)was reported by Dunlap and Bruton to produce 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 seven to eight 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 un-supplemented 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.

Keywords: (−)-Botryodiplodin; Macrophomina phaseolina; amino acid; charcoal rot disease; in-culture bioassay; kojic acid; moniliformin; mycotoxin; natural product; phytotoxin; pigments; secondary metabolite; soybean.

Figure 1

Cultures of three pathogenic M. phaseolina isolates that illustrate the three levels of pigment production observed within a collection of 42 isolates from soybean plants exhibiting symptoms of charcoal rot, specifically high levels of pigment production by isolate Mp 230 (A), moderate levels of pigment production by isolate Mp 264 (B), and no detectable pigment by isolate Mp 009 (C).

Figure 2

Effect of glycine concentration added to Czapek-Dox agar medium on pigment formation by selected M. phaseolina isolates. Pigment levels were measured in triplicate in extracts from agar plugs cut in the culture medium at the indicated times determined as OD₅₂₀. Isolates Mp 183, Mp 230, Mp 264 and Mp 312 produced significantly (ANOVA, p < 0.001) greater amounts of pigment than Mp 009 and Mp 239 at all glycine concentrations greater that 1 g/L.

Figure 3

Time course of the production of pigment (solid lines) and of (−)-botryodiplodin (dashed lines) on Czapek Dox agar medium supplemented with glycine (10 g/L) by selected isolates of M. phaseolina, including two high level pigment producers, Mp 183 (●) and Mp 230 (■); two moderate level pigment producers, Mp 264 (♦) and Mp 312 (▲) and two pigment non-producers, Mp 009 (x) and Mp 239 (∗). Pigment and (−)-botryodiplodin levels were measured in extracts from agar plugs cut in the culture medium at the indicated times. Pigment levels were determined as OD₅₂₀ of pooled triplicate aqueous extracts and (−)-botryodiplodin levels in pooled triplicate ethyl acetate extracts by LC-MS. The rate of pigment production by the selected M. phaseolina isolates correlated with the rate of (−)-botryodiplodin production in a highly significant manner (r = 0.96, p ≤ 0.0001, regression analysis).

Figure 4

Effect of adding sucrose to modified Czapek-Dox agar medium prepared without sucrose on red pigment production by four selected pigment-producing M. phaseolina isolates, Mp 183 (-●-), Mp 230 (-■-), Mp 264 (-♦-) and Mp 312 (-▲-). The optimum sucrose concentration varied with M. phaseolina isolate, but all exhibited optimum red pigment production at the sucrose concentration used in conventional Czapek-Dox agar medium (30 g/L).

Figure 5

Pigment formation by reaction of (±)-botryodiplodin solution with various amines in the presence of air. Pigment production by reaction of (±)-botryodiplodin (0.5 mg/mL) and the following amines at the indicated initial concentrations and at serial 2-fold, 2-fold then 2.5-fold dilutions in the column below it: column 1: concentrated ammonium hydroxide, 20% (v/v); column 2: ammonium chloride, 10 mg/mL; column 3: poly-L-lysine hydrobromide, 1 mg/mL; column 4: bovine serum albumin, 1 mg/mL; column 5: chitosan hydrochloride, 1 mg/mL; column 6: ethylamine, 10 mg/mL; column 7: hexylamine, 10 mg/mL; column 8: ethylenediamine, 10 mg/mL; column 9: ethanolamine, 10 mg/mL; column 10: methylamine hydrochloride, 10 mg/mL; column 11: L-lysine dihydrochloride, 10 mg/mL; column 12: glycine 10 mg/mL.

Figure 6

(−)-Botryodiplodin production (µg/g) by selected M. phaseolina isolates cultured on Czapek-Dox agar medium (means ± standard deviation). Values with the same letter are not significantly different (ANOVA, p < 0.05).

Figure 7

Ultraviolet-visible absorption spectra of pigments formed by reaction of glycine with synthetic (±)-botryodiplodin and with the sucrose-stimulated mycotoxin released from selected Macrophomina phaseolina (Mp) isolates.

Figure 8

Ultraviolet-visible absorption spectra of pigments formed by reaction of β-alanine with synthetic (±)-botryodiplodin and with the sucrose-stimulated mycotoxin released from selected Macrophomina phaseolina (Mp) isolates.

Figure 9

Production by the indicated pathogenic isolates of M. phaseolina from soybean plants expressing symptoms of charcoal rot disease of (i) red pigment (blue bars on the left) measured as light absorbance at 520 nm in aqueous extracts from Czapek-Dox agar culture medium supplemented with glycine (10 g/L); and (ii) (−)-botryodiplodin (orange bars on the right) measured by LC-MS (µg/g) in ethyl acetate extracts from parallel cultures on Czapek-Dox agar medium with no glycine. Red pigment production by the M. phaseolina isolates in the collection cultured on glycine-supplemented medium exhibited a highly significant positive correlation (rs = 0.705, p ≤ 0.0001, Spearman’s rank correlation test) with (−)-botryodiplodin production on the same medium without glycine.