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

Abstract

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 by 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. . B l a n e y e t a l .

Alkaloid production by isolates of the sorghum ergot pathogen (Claviceps africana) from Australia and other countries

“…This raised questions of whether Australian isolates of C. africana are more toxigenic than those from other continents. The profile of alkaloids in collections of Australian sphacelia/sclerotia was predominantly DHES (Blaney et al 2003), which is consistent with some African isolates (Frederickson et al 1991). However, the concentrations found in Australian sclerotia varied widely, which could account for variable toxicity.…”

“…The production of all 3 alkaloids was always higher in mycelia than in broth, and there was a clear correspondence between alkaloids in mycelium and broth. The general pattern of alkaloid production observed in sorghum ergot sclerotia from Queensland (Blaney et al 2003) was also observed in these cultures: DHES predominated, with smaller amounts of DHEL and FEST. Alkaloids were not detected in the spent broths from the first incubation in Mantle's medium (<0.01 mg/L), whereas they were present in broth after the second incubation as shown in Table 3.…”

“…However, alkaloids are present in honeydew (or spores contained in honeydew) at concentrations up to 10 mg DHES/kg (Blaney et al 2003) from the early stages of infection and long before sclerotial tissue develops. Ergot bodies composed mainly of sphacelial tissues tend to contain 10-200 mg DHES/kg, but concentrations in fully developed sclerotia can rise to 10 000 mg DHES/kg.…”

“…Methylene chloride extracts were reduced to dryness under an air stream, dissolved in 1 mL of methanol, and filtered into a sealed vial. These were assayed using a Shimadzu LC-10 liquid chromatograph equipped with UV (SPD-M10A diode array) and fluorescence (RF-10A XL) detectors, as described by Blaney et al (2003). Separation was on a Novopac (Waters) C18 column, operated isocratically at 40 • C, using acetonitrile : methanol : 0.1% ammonium acetate (31 : 20 : 50).…”

“…Australian isolates have been delineated into 2 different clusters, but both appear to have originated on the Indian subcontinent (Komolong et al 2002) rather than Africa. Isolates from Australia have been identified as C. africana, based on morphology (Ryley and Henzell 1997), molecular analysis (Komolong et al 2002), and production of DHES (Blaney et al 2003).…”