Phloem Production in Huanglongbing- Affected Citrus Trees
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DISEASE AND PEST MANAGEMENT HORTSCIENCE 49(1):59–64. 2014. 1968). Callose plugging of the phloem and phloem degeneration are common symptoms of other phloem-specific diseases such as Phloem Production in Huanglongbing- yellow vine disease of cucurbits (Serratia marcescens; Bruton et al., 2003) and phloem affected Citrus Trees canker disease of walnut (Erwinia rubifaciens; 1 Schaad and Wilson, 1970), whereas elevated Craig Brodersen , Cody Narciso, Mary Reed, and Ed Etxeberria starch levels have been reported in grape- University of Florida, Department of Horticultural Sciences, Citrus Research vines suffering from Bois Noir, a phloem- and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850 specific phytoplasma disease (Santi et al., 2013). Additional index words. citrus, citrus greening, HLB, Huanglongbing, orange, phloem Despite the apparent total collapse of obstruction, ‘Valencia’ phloem elements in HLB-affected leaves Abstract. Citrus greening disease [Huanglongbing (HLB)] is the most significant and and limbs, new vegetative growth continues widespread threat to the citrus industry in recent history. A bacterium [Candidatus to develop for several seasons. In fact, HLB- Liberibacter asiaticus (CLas)] vectored by the Asian citrus psyllid is the presumed causal affected branches with evident HLB symp- agent of the disease, which results in the collapse of phloem tissue, leading to decreased toms and seemingly obstructed phloem are productivity, chlorotic leaves, and bitter, misshapen fruit. Once infected, trees never fully capable of maintaining fruit production for recover and there currently is no cure, although foliar nutrient sprays and intensive some time (Ikpechukwu et al., 2011) (Fig. 1), irrigation appear to slow tree decline in some situations. Despite phloem necrosis in older although succeeding crops are progressively tissue, new vegetative and reproductive growth occurs. Our current understanding of smaller and of lower quality. Furthermore, phloem collapse in citrus resulting from HLB is based on anatomical reports of trees in parenchyma cells in woody stems and bark different stages of decline and does not explain the persistence of growth. Here, we tissue downstream from the leaf canopy (i.e., in present data that show new phloem cells are produced during the periodic flushes of the basipetal direction) also accumulate copious vegetative growth and their subsequent collapse and plugging over a 6-month period. amounts of starch despite the evident dysfunc- Cellular activity within the cambium and the ray parenchyma was diminished in HLB- tionality of the phloem tissue (Exteberria et al., affected petioles, suggesting an important link in the carbohydrate transport pathway is 2009). If photoassimilate transport were totally missing. Because of the short window of time during which the phloem appears healthy, blocked at the leaf/petiole level, there would be the weeks immediately before and after the spring and summer flush are of critical insufficient carbohydrate flow to sustain the importance for the management of citrus health. synthesis of starch in a basipetal direction and to support the growth of developing twigs, lower branches, and fruit. A possible source for the carbohydrate Citrus HLB (synonym citrus greening) is 2010). This swelling is followed by the accumulation in the bark and wood could be a highly destructive, fast-spreading disease of deposition of callose plugs both in lateral the result of temporary phloem transport in citrus. Its presumed pathological agent, Can- pit fields (Koh et al., 2012) as well as in and new foliage. In citrus trees, new growth didatus Liberibacter spp., is a fastidious around sieve plates (Folimonova and Achor, emerges several times throughout the year Gram-negative, obligate parasite, phloem- 2010; Koh et al., 2012). Although otherwise with the most substantial flushes occurring limited a-proteobacterium (Garnier and still functional at this early stage, the walls in the spring and late summer (Reed and Bove´, 1983; Jagoueix et al., 1994). Although begin to collapse concurrently with increased MacDougal, 1938; Syvertsen et al., 1981). not yet cultured to purity, recent attempts to deposition of callose and P-protein plugs at Often, new leaves emerging from trees with do so have resulted in partial or mixed the sieve plates. The presence of callose a recent CLas infection appear healthy, al- cultures of the organism (Davis et al., 2008; plugs and phloem collapse is believed to though some leaves can be either severely Sechler et al., 2009). Of the several species hinder the transport of photoassimilates (ni- deformed by psyllid feeding or eventually identified worldwide (Kim et al., 2009), CLas trogenous and reduced carbon compounds) develop the typical blotchy mottling and is the only species found in Florida (Wang from photosynthetic source leaves to the yellowing patterns characteristic of the dis- and Trivedi, 2013). CLas is vectored by the remaining heterotrophic sink tissues (Schneider, ease (Fig. 1). In trees known to have been phloem-feeding psyllid Diaphorina citri 1968). As the flow of photoassimilates is infected with HLB for several years, new (Halbert and Manjunath, 2004) and trans- progressively obstructed by phloem necrosis, leaves do not grow to their typical size, are mitted into the phloem of citrus leaves during starch granules begin to accumulate in sieve often upright in orientation, and remain a pale the feeding process. When transmitted into elements and all parenchyma cell types along yellow color, never developing the dark a citrus leaf by the psyllid, CLas unravels the radial and longitudinal transport pathway, green coloring of healthy leaves (Fig. 1). a cascade of physiological and metabolic but especially in photosynthetically active These observations suggest that, for a yet changes that culminate in reduced vigor, cells (Etxeberria et al., 2009; Schneider, undetermined period of time, photoassimilate diminished production, and ultimately tree death (Etxeberria et al., 2009). Citrus trees affected by HLB exhibit a progressive degeneration of the phloem tissue (Achor et al., 2010; Schneider, 1968) that results in partial or total phloem collapse (Folimonova and Achor, 2010). At the ana- tomical level, the first observable indication of CLas infection is the conspicuous swelling of the middle lamella between and surround- ing sieve elements (Folimonova and Achor, Received for publication 13 Sept. 2013. Accepted for publication 8 Nov. 2013. 1To whom reprint requests should be addressed; Fig. 1. Production of healthy fruit by an Huanglongbing (HLB)-affected citrus tree. Subtending branch e-mail [email protected]. shows the yellow, upright leaves with blotchy mottle typical of symptomatic leaves. HORTSCIENCE VOL. 49(1) JANUARY 2014 59 transport takes place in newly developed FDA viability staining for healthy and layer of thin-walled cells between the cam- flush tissue and within the older supporting HLB-infected ‘Valencia’ petioles. To deter- bium and the protophloem (Fig. 2A, white branches and trunk. At some point after leaf mine the viability of cells within the vascular arrow). Differentiation of the metaphloem development, the phloem eventually col- tissue of petioles we used fluorescein dia- outwardly becomes evident with the matura- lapses and becomes dysfunctional. Although cetate (FDA), a cell-permeant vital stain that, tion of sieve cells and companion cells to- previous studies have shown HLB symptoms on hydrolysis by esterase activity, releases ward the periphery. The phloem sieve at extreme stages of the disease, the goal of green fluorescence indicating cell viability elements and their companion cells are some this study was to monitor the progression of and integrity (Ruzin, 1999). Eight transverse of the smallest cells in the plant (Knoblauch phloem production over time in field-grown freehand sections from both healthy and and Oparka, 2012), have thin primary walls, trees to determine anatomically how the trees HLB-infected ‘Valencia’ petioles were placed and in petioles are often found in discrete are capable of sustaining new growth and in labeled vials containing a 1:100 FDA bundles (Fig. 2A–B). Companion cells (Fig. then document the subsequent phloem col- solution. The vials were vacuum infiltrated 2B, black arrow) are easily distinguished lapse. for 15 s, capped with aluminum foil to from their associated sieve elements (Fig. minimize light exposure, and shaken for 40 2B, white arrow) by their smaller size and Materials and Methods min at 1000 rpm on an Eppendorf Thermo- denser cytoplasmic content. Large phloem mixer R (Eppendorf Inc., New York, NY). parenchyma cells were invariably present Plant material. Fully expanded and de- Each section was mounted on a slide and scattered within the tissue and as part of the veloped tissue from HLB-affected trees was viewed with a 10· objective under a fluores- vascular rays and bordering the inner face of collected based on their visual symptoms cence microscope with a lex = 488 nm, and the tracheary ring (Figs. 2A–B and 3A, white (i.e., symptomatic vs. asymptomatic) from a digital image was captured. Sample image arrow). Longitudinally, the sieve elements 5-year-old ‘Valencia’ orange (Citrus sinensis brightness and contrast were adjusted equally can be distinguished by their thin walls and L. Osbeck) trees grown at the Citrus Research for all samples using ImageJ (<http://image- oblique end walls (Fig. 3A, black arrow) as and Education Center in Lake Alfred, FL, j.nih.gov/ij>).