Enzyme digestion of HLB-infected tissues: a better approach to study phloem anatomy

Naveen Kumar, Craig Brodersen, Cody Narciso, Ed Etxeberria


At anatomical level citrus greening (HLB; Huanglongbing) is characterized by excessive deposition of starch, p-proteins, and callose in phloem tissue of various plant parts. The presence of these polysaccharides and proteins often obstruct observations of key anatomical details of phloem tissue. In the present investigation, we evaluated the effect of carbohydrate and protein metabolizing enzymes on leaf tissue aimed at studying ultra-structural details of phloem that remain hidden in HLB-infected phloem tissue. The HLB-affected ‘Valencia’ orange leaf samples were collected from the groves of CREC, Lake Alfred, Florida. Leaf petioles were detached, immediately stored in liquid nitrogen and transported to the laboratory. The petioles were kept in superchilled ethanol (100%) and stored in –20°C freezer for two days. Petiole samples were warmed to room temperature for two hours in distilled water and thereafter thoroughly washed. Samples were thin sectioned using a razor blade and again washed with distilled water. Petiole sections were incubated in combination and individually with 0.1% Proteinase K and 0.1% Amylase at various time intervals. After enzymatic treatments sections were washed with 100% ethanol, vacuum dried and prepared for scanning electron microscopy. Enzymatic treatments of samples revealed clear images of phloem tissue with visible pit fields on side walls, sieve tubes, and sieve plates. However, none of these structures were clearly visible in untreated HLB-affected petiole samples. The data fails to explain any possible lateral transmission of Candidatus Liberibacter asiaticus (CLas).


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Proc. Fla. State Hort. Soc.     ISSN 0886-7283