LC-MS/MS analysis of root exudates
Analysis of root exudates was conducted on 13 genotypes (9 SAPlgs1 natural mutants, 2 LGS1 lines), alongside resistant and susceptible checks (SRN39 and Shanqui Red). Seeds of sorghum were surface sterilized in 50 % sodium hypochlorite and germinated in vitro on petri plates lined with moistened filter papers. One-week-old sorghum seedlings were subjected to a hydroponic system containing normal Hoaglands nutrient solution (with phosphate). After one week, the sorghum seedlings were fed with nutrient solution devoid of phosphate (-Pi) for 7 days. On the day of root exudates collection, seedlings were first fed with fresh –Pi solution for 6 h, and then root exudates were collected for LC-MS/MS analysis and Striga bioassays.
SL analysis of the root exudates was done according to (Wang et al. 2021). Briefly, root exudates spiked with 2 ng of rac -GR24 and passed through C18-Fast Reversed-SPE column (500 mg/3 mL), and the SLs eluted in 5 mL of acetone. SLs were extracted in ethyl acetate, vacuum dried and the final extract was dissolved in acetonitrile:water (25:75). SLs were quantified by LC-MS/MS using UHPLC-Triple-Stage-Quadrupole Mass Spectrometer. Strigolactones were identified by comparing the MS/MS spectrum and retention time with standards including 5-deoxystrigol, orobanchol and GR24. The characteristic Multiple Reaction Monitoring (MRM) transitions (precursor ion → product ion) were 331.15→216.0, 331.15→234.1, 331.15→97.02 for 5-deoxystrigol; 347.14→329.14, 347.14→233.12, 347.14→ 205.12, 347.14→97.02 for orobanchol; 299.09→158.06, 299.09→157.06, 299.09→97.02 for GR24.
LCMS quantification of abscisic acid (ABA) followed the procedure by Wang et al. (2021). ABA was analyzed using LC-MS/MS using UHPLC-Triple-Stage Quadrupole Mass Spectrometer. The characteristic Multiple Reaction Monitoring (MRM) transitions (precursor ion → product ion) was 263.2 → 153.1, 263.3 → 204.1, 263.3 → 219.1 for ABA