Visual Detection of Calcium by GBHA Staining in Bitter Pit-affected Apples

Bitter pit is a physiological disorder of apple fruits apparently caused by a localized calcium deficiency or imbalance in fruits associated with low levels of calcium in the flesh. A new, highly selective method using glyoxal bis(2-hydroxyanil, GBHA) was tested to reveal the presence of calcium within the fruit as a red stain. Water-soluble and insoluble calcium was analyzed by capillary electrophoresis and atomic absorption spectroscopy in pitted regions, adjacent sound areas and pulp from sound apples. Both methods, selective calcium staining and mineral analysis, showed that calcium accumulates in the pitted areas of apples affected by bitter pit. However, in mechanically inflicted wounds, the pulp of the apple was heavily stained but not the corresponding fingerprint, indicating a similar mechanism of insoluble calcium accumulation but a different distribution of soluble calcium compared to the pits.