Biuret Toxicity Symptoms in Citrus Leaves Mimics Cell Senescence Rather Than Nutritional Deficiency Chlorosis

Permanent chlorosis of leaves on plants fertilized with urea containing high levels of the contaminant biuret has been observed in several crops including citrus. Little has been reported as to the cellular changes that result from such chlorosis. Branches from ʻRuby Redʼ grapefruit (Citrus paradisi Macfadyn) and ʻHamlinʼ orange (C. sinensis (L.) Osbeck) were sprayed with urea solutions containing 1.05% biuret. As visible symptoms developed, leaf tissue samples were prepared for transmission electron microscopy. For comparison purposes, leaves from similar trees showing chlorosis from age-related senescence and Zn defi ciency were also sampled. The progressive development of chlorosis in biuret-affected leaves was characterized by: the loss of starch, thylakoidal and granal membranes in chloroplasts along with the enlargement and increase in number of plastoglobuli or lipid bodies. The lipid bodies were liberated alone or in association with membrane vesicles to the cytoplasm and vacuoles. The number and volume of the individual chloroplasts became smaller. Concurrent loss of cytoplasmic content and the enlargement of the vacu- olar space were also observed in the biuret affected leaf tissue. Similar fi ndings were observed in the cells of senescent leaves. In cells of leaves showing nutritional defi ciency, losses in cytoplasmic content and vacuolar enlargement were observed but there was neither complete loss of thylakoidal or granal membranes nor the release of lipids from the plastids. It was concluded that 1) the cytological characteristics of the biuret-affected samples were more similar to age-related senescent samples than to chlorosis from Zn defi ciency and 2) that complete loss of the lipid bodies from the chromoplasts to the cytoplasm and vacuole in the biuret-affected samples and in age-related senescence in citrus leaves was responsible for the permanent nature of the chlorosis. Sprays containing foliar urea as a nutritional supplement have become common in the Florida citrus industry (Albrigo, 1999, 2002). More than 50,000 ha have been sprayed in some years. Biuret is a natural by-product contaminant in the production of urea and occurs at higher concentrations as the process is carried out under elevated temperatures to increase the production rate of urea. Chlorosis (yellowing) of leaves treated with urea containing high levels of the contaminant biuret has been observed in several crops including citrus (Albrigo, 2002; Albrigo and Grosser, 1996; Gooding and Davies, 1992; Iyengar et al., 1976; Jones, 1954; Oberbacher, 1954; Sanford et al., 1954). In apple (Malus ×domestica Borkh.) leaves, urea itself con- tributes to leaf damage but urea toxicity is usually expressed as a burn rather than chlorosis (Khemira et al., 2000). Coffee (Coffea arabica L.) can apparently withstand levels of biuret as high as 2.5% w/w (Iyengar et al., 1976) but symptoms in citrus occurred 3 to 15 weeks after application when biuret levels in urea exceeded as little as 0.35% w/w (Oberbacher, 1954). Chlorotic symptoms caused by biuret toxicity fi rst appear at the leaf tip and margin, and spread to more of the leaf area as the biuret content increases. The amount of visible chlorosis symptoms was directly proportional to the amount of biuret content in the urea (Iyengar et al., 1976; Oberbacher, 1954). In later studies, biuret toxicity symptoms in citrus leaves usually did not appear earlier than 30 to 45 d after spray application (Albrigo, 2002), but included leaf deformation when rates of biuret were near 2% w/w of urea and sprayed on developing foliage. Once biuret-induced chlorosis occurred in citrus, the affected area of the leaf did not recover a green color (Albrigo, 2002; Oberbacher, 1954).