Alkaloids of the sorghum ergot pathogen (Claviceps africana): assay methods for grain and feed and variation between sclerotia/sphacelia

Abstract

Assay methods for the alkaloids of sorghum ergot (Claviceps africana) are described and compared. Sorghum ergot bodies (sclerotia/sphacelia) from various regions of Queensland and New South Wales were collected in 1997 and 2001 and assayed by spectrophotometry, thin layer chromatography, or high performance liquid chromatography (HPLC). All contained dihydroergosine (DHES) as the main alkaloid component (about 80%), with smaller amounts of dihydroelymoclavine and festuclavine. The preferred method of assay for infected sorghum and mixed feeds involved extraction into dichloromethane:methanol:ethyl acetate:ammonium hydroxide (50:5:25:1) using an ultrasonic bath. After solvent removal, the extract was dissolved in diethyl ether and partitioned into 0.5 M hydrochloric acid. After adjusting the pH to 8–10 with ammonium hydroxide, the alkaloids were extracted into dichloromethane, the solvent evaporated, and the residue dissolved in methanol. HPLC separation was on a C18 column, 150 × 3.9 mm, run isocratically at 40°C, with acetonitrile:0.1% ammonium acetate:methanol (31:50:20) as the mobile phase. Detection was either by UV at 280 nm or by fluorescence with excitation at 235 nm and absorbance at 340 nm. Levels of quantitation for DHES in sorghum approached 0.1 mg/kg (UV) and 0.01 mg/kg (fluorescence). Method recoveries for DHES in the range of 0.025–7 mg/kg averaged 75%. The total alkaloid content of ergot bodies (sclerotia/sphacelia) from different batches of grain varied from 100 to 7900 mg/kg (0.79%). Within batches, there was much less variation in the alkaloid content of ergot bodies, but larger ergots tended to contain more alkaloid than smaller ergots, and those infected with Cerebella species contained even less; this probably related to the ratio of sclerotial/sphacelial tissue present. Honeydew also contained DHES (1–10 mg/kg) and might contaminate clean grain at significant levels. Tests on 4 farms showed that substantial amounts of ergot bodies and alkaloids were removed during grain harvesting.