Chitin as a measure of biomass of <e1>Crinipellis perniciosa</e1>, causal agent of witches' broom disease of <e1>Theobroma cacao</e1>

<e1>Crinipellis perniciosa</e1> is the cause of witches' broom disease of cocoa, a serious problem in South America. The aim of the project was to develop a measure of fungal biomass in cocoa tissue infected with <e1>C. perniciosa</e1> using chitin as a marker. Axenic cultures of primary and secondary mycelium of <e1>C. perniciosa</e1> were exposed to strong hydrochloric acid to hydrolyse the chitin. The glucosamine so formed was converted to hexamitol by sodium borohydride and the acetylated and derivatized products were separated by gas chromatography and identified by mass spectrophotometry. As a proportion of the mycelial <e5>D.W.</e5>, chitin was found to be 14% in primary phase mycelium and 17% in the secondary phase. An average figure was used to calculate the amount of fungal biomass from estimates of chitin in dried and fresh cocoa broom tissue infected with <e1>C. perniciosa</e1>. The green brooms, which represented an early stage of infection, contained 81 μg fungal biomass mg⁻¹ <e5>D.W.</e5>, whereas brown brooms from a late stage in infection contained 161 μg fungal biomass mg⁻¹ <e5>D.W.</e5> of broom. In a detailed analysis of a single fresh broom most fungal biomass was found at the base (215 μg mg⁻¹ <e5>D.W.</e5>) but declined towards the shoot tip (10 μg mg⁻¹ <e5>D.W.</e5>). The assay was sufficiently sensitive to measure a minimum fungal biomass in cocoa of 40 ng mg⁻¹ <e5>D.W.</e5> of plant tissue. The method developed was specific to chitin, and significantly more sensitive than previously described methods.