Sequence of Anatomical Symptom Observations in Citrus Affected with Huanglongbing Disease

This study was undertaken to develop a better understanding of the relationship between symptom development and the nature of the citrus disease, huanglongbing. The most characteristic symptom of huanglongbing (HLB) is the non-symmetrical mottled chlorosis of leaf blades. Starch accumulation and phloem collapse have been associated with symptom development in this disease presumed to be caused by Candidatus Liberibacter asiaticus. Several hypotheses regarding phloem disruption to starch accumulation to chlorosis evolved concerning symptom development. These were tested using light and Transmission Electron Microscopy (TEM). Samples collected and fixed for TEM with various stages of HLB symptoms, revealed the following: starch accumulation occurred after phloem plugging and cell collapse and therefore, localized carbohydrate deficiency may be a factor. Starch packing of chloroplasts did not rupture the outer membranes, but the inner grana structure was disrupted thus, leading to chlorosis. This occurred only in parts of the leaf where phloem plugging occurred. Sieve elements were obstructed by both amorphous and filamentous materials and both occurred in readily observed amounts, while bacteria were insufficient to directly cause plugging. The amorphous material was positively identified as callose by immunoassay with gold labeling. Phloem protein 2 was identified in the filamentous plugging material using immunoassay with gold labeling. This information supports the development of HLB symptoms in the following sequence: phloem plugging and necrosis with cell wall swelling of sieve elements and companion cells followed by some phloem cell collapse, presumed sugar backup in localized leaf blade areas leading to starch accumulation until chloroplast structure is disrupted with resulting chlorosis.