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

Crinipellis perniciosa is the cause of witches' broom disease of cocoa, a s erious problem in South America. The aim of the project was to develop a me asure of fungal biomass in cocoa tissue infected with C. perniciosa using c hitin as a marker. Axenic cultures of primary and secondary mycelium of C, perniciosa were exposed to strong hydrochloric acid to hydrolyse the chitin . The glucosamine so formed was converted to hexamitol by sodium borohydrid e and the acetylated and derivatized products were separated by gas chromat ography and identified by mass spectrophotometry. As a proportion of the my celial D.W., chitin was found to be 14% in primary phase mycelium and 17% i n the secondary phase. An average figure was used to calculate the amount o f fungal biomass from estimates of chitin in dried and fresh cocoa broom ti ssue infected with C. perniciosa. The green brooms, which represented an ea rly stage of infection contained 81 mu g fungal biomass mg(-1) D.W., wherea s brown brooms from a late stage in infection contained 161 mu g fungal bio mass mg(-1) D.W. of broom. In a detailed analysis of a single fresh broom m ost fungal biomass was found at the base (215 mu g mg(-1) D.W.) but decline d towards the shoot tip (10 mu g mg(-1) D.W.). The assay was sufficiently s ensitive to measure a minimum fungal biomass in cocoa of 40 ng mg(-1) D.W. of plant tissue. The method developed was specific to chitin, and significa ntly more sensitive than previously described methods.