Analysis of Thaxtomin Production

Thaxtomins were extracted from S. scabiei OBBC cultures and were detected by reverse phase HPLC as described before (Li et al., 2019a). Briefly, each strain was cultured in triplicate, and in the case of the MLP overexpression strains, two different isolates per strain were cultured in triplicate for a total of six cultures. Culture extracts were prepared by extracting the culture supernatants with ethyl acetate, drying the extracts by evaporation, and resuspending the residual material in 100% v/v HPLC-grade methanol. The extracts were analyzed using an Agilent 1260 Infinity Quaternary LC system (Agilent Technologies Canada Inc.) with a Poroshell 120 EC-C18 column (4.6 × 50 mm, 2.7 μm particle size; Agilent Technologies Canada, Inc.) held at a constant temperature of 40°C. An isocratic mobile phase consisting of 30% acetonitrile and 70% water at a constant flow rate of 1.0 mL/min was used for metabolite separation, and metabolites were monitored using a detection wavelength of 380 nm. The normalized total thaxtomin production level for each culture was determined by summing the measured peak area for thaxtomin A, thaxtomin B, and thaxtomin D and then dividing the total area by the measured dry cell weight of the culture. The results for each strain were then averaged among the replicate samples and were reported as the percent thaxtomin production relative to wild-type S. scabiei 87.22. Statistical analysis of the results was conducted in Minitab 19 using one-way ANOVAs with a posteriori multiple comparisons of least squared means performed using the Tukey test. P values ≤ 0.05 were denoted as statistically significant in all analyses.