Histochemical and ultrastructural characterization of vacuoles and spherosomes as components of the lytic system in hyphae of the fungus Botrytis cinerea

An integrated approach to acid phosphatase (EC 3.1.3.2) histochemistry by t he azo-dye and lead-capture ('Gomori') methods in phosphate-starved hyphae of the fungus Botrytis cinerea revealed strikingly different patterns of lo calization of activity staining. Reaction product formed with the azo-dye m ethod was found in numerous small organelles (< 0.5 mu m diameter), which a lso accumulated the lipophilic dye Nile Red and mislocalized the formazan i ndicating mitochondrial succinate dehydrogenase activity. Such small organe lles were stained only weakly and sporadically with the lead-capture method ; instead, lead phosphate deposits were produced mainly in large vacuoles ( up to 2.5 mu m diam.), similar to those accumulating the vital dye Neutral Red. Additionally, acid phosphatase activity was detected in apical secreto ry vesicles with the lead-capture method but not with the azo-dye method. U ltrastructural studies by transmission electron microscopy confirmed the pr esence of large vacuoles which showed evidence of autophagic activity, and of small moderately osmiophilic organelles. The latter are considered to be spherosomes rather than lysosomes because of their weak reaction with the lead-capture method and their high lipid content. It is suggested that thei r apparently strong reaction with the azo-dye method is caused partly by fa lse localization due to the lipophilic nature of the reaction product.