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Physiological and Molecular Plant Pathology 74 (2009) 76–83

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Physiological and Molecular Plant Pathology

journal homepage: www.elsevier .com/locate/pmpp

Anatomical distribution of abnormally high levels of starchin HLB-affected Valencia orange trees

Ed Etxeberria*, Pedro Gonzalez, Diann Achor, Gene AlbrigoHorticultural Sciences Department, University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA

a r t i c l e i n f o

Article history:Accepted 8 September 2009

Keywords:Candidatus LiberibacterCitrus greeningPhloem collapse

* Corresponding author. Tel.: þ1 863 956 1151; faxE-mail addresses: eetxeber@ulf.edu (E. Etxeberria)

dsar@ufl.edu (D. Achor), albrigo@ufl.edu (G. Albrigo).

0885-5765/$ – see front matter � 2009 Published bydoi:10.1016/j.pmpp.2009.09.004

a b s t r a c t

The citrus disease Huanglongbing (HLB or citrus greening) is characterized, among other symptoms, byextraordinary levels of starch accumulation in leaves. This condition denotes imbalances in carbohydratesource sink relationship which in turn may have direct implications in the overall health of HLB-trees andin future strategies to manage the disease. Using light, scanning, and transmission electron microscopywe investigated the extent of carbohydrate partitioning imbalances throughout the tree. In all aerialtissues, starch accumulation in HLB-affected trees far exceeded that of HLB-negative control trees. Starchaccumulated extensively in photosynthetic cells as well as phloem elements and vascular parenchyma inleaves and petioles. In stems, starch was commonly observed in xylem parenchyma and in the phello-derm of HLB-affected trees but absent from control samples. In contrast, roots from HLB-affected treeswere depleted of starch whereas roots from control trees contain substantial starch deposits. The datasupports the notion that the substantial changes in carbohydrate partitioning observed throughout thecitrus tree may not only be a result of HLB infection, but in itself, a cause for the rapid decline and deathof infected trees.

� 2009 Published by Elsevier Ltd.

1. Introduction

Citrus huanglongbing (HLB, or citrus greening) is a highlydestructive, fast spreading disease of citrus. The disease is linked toa fastidious, gram-negative, phloem-limited bacterium (CandidatusLiberibacter spp.) [9,13] not yet culturable, although recent attemptshave succeeded in partially culturing the organism [6]. Two types ofHLB were commonly known: heat-sensitive African form causedby (Candidatus Liberibacter africanus) and heat-tolerant Asian formby (Candidatus Liberibacter asiaticus). A third type (CandidatusLiberibacter americanus) was recently identified in Brazil [27]. InFlorida, only Ca. L. asiaticus has been detected [15] and is trans-mitted by Diaphorina citri, a psyllid vector also found in Louisiana,Texas and in Southern California. The HLB associated bacteria caninfect most citrus cultivars, species and hybrids [12] with mostsweet oranges, mandarins, and mandarin hybrids severely affected,whereas lime and lemons show less severe symptoms. The bacte-rium is hosted by a variety of other common ornamental plants [12]making its eradication even a more difficult task and managementof the disease more crucial.

: þ1 863 956 4631., pcgo@ufl.edu (P. Gonzalez),

Elsevier Ltd.

Although long established in Eastern Asia and South Africa [2,5],recent findings of HLB in Brazil [4,27] and Florida [11,1] have broughtrenewed interest on this disease given its devastating potential totheir citrus industry. Citrus juice production between Florida andBrazil accounts for over one-third of the world’s output [15]. InFlorida, the disease has spread quickly and is now established in allcitrus growing counties. In some grove blocks located in the southernpart of the state, as much as 80% of trees have been infected with HLB(Mark Colbert, personal communication). Under the presentcircumstances and pathogen distribution, the disease threatens todecimate all 640,000 remaining acres of the Florida Citrus Industry.

In citrus trees, specific HLB symptoms are difficult to charac-terize, but leaf blotchy mottle is very specific for the disease.Symptoms such as yellow shoots, leaf blotchy mottle, and lopsidedfruits with color inversion and aborted seeds, are all characteristic,but they do not always occur together in the same tree. They can bedistorted or masked by symptoms of other diseases, or in somecases, induced by conditions unrelated to HLB [2].

Amongst other HLB-induced characteristics, Schneider [20]noted massive starch accumulation in citrus leaves presumably theresult of necrotic phloem pockets scattered throughout the vascularsystem in the leaf petioles. In his analysis, Schneider [20] theorizedthat these phloem blockages create a photoassimilate back-logresulting in starch levels ubiquitously observed in leaves ofHLB-affected trees. In fact, the excessive starch build-up causing

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disintegration of the chloroplast thylakoid system is believed toproduce the yellowing leaf mottle symptom. Leaf yellowingresulting from above normal starch accumulation and thyakoiddestruction can be artificially induced by branch girdling [19].

Starch content of HLB-affected leaves can be 20 times higherthan leaves from control trees [26]. Based on the ability of iodine tobind starch (resulting in a blue/purple-colored solution; [16]), thecontrasting levels of starch accumulation in citrus leaves have beenused as visual indicator of HLB presence in many locations [17,26]including Florida [8]. The enormous discrepancies in starch contentbetween control and HLB-affected leaves reflect significant varia-tions in the natural balance of carbon source/sink relations.Whether the imbalance in carbon metabolism brought about byHLB infection is restricted to leaf blades or is widespreadthroughout the plant, may have direct implications in the overallhealth decline observed in HLB-affected trees and in future strat-egies to manage the disease. In this report, we analyze in greaterdetail the tissue distribution of starch in leaves from trees inadvance stage of HLB infection. In addition, we further determinestarch distribution in other plant parts compared to control trees.

2. Materials and methods

2.1. Plant material

Leaves, young (green) stems, bark and roots of HLB-confirmed10 year old Valencia orange (Citrus sinensis) trees were collected

Fig. 1. Light micrographs of cross sections of citrus leaves from (A) HLB-affected Valencia treand size, and collected from similar canopy location. C and D are close-ups of secondary vascvascular parenchyma and the phloem collapse (arrowhead) characteristic of HLB-affected tretissue; * ¼ phloem collapse.

from groves near Dover, FL. Overall, sampled trees showed foliarHLB symptoms in approximately 70% of the canopy indicating anadvanced stage of disease infection (PCR ¼ 19.7, 20.9 and 22.3cycles). Corresponding samples from HLB-negative Valencia treesobtained from trees grown at the Citrus Research and EducationCenter in Lake Alfred FL, were used as control.

2.2. Iodine staining

For starch staining, leaves, petioles, stems, bark and roots werecut perpendicular to the long-axis with a sharp razor blade andimmersed for 2 min in a solution of 2% iodine [16] at roomtemperature. Tissue samples were rinsed in water and immediatelyobserved under a 40/14 Wild Heerbrugg stereoscope. Images werecaptured with a Canon PowerShot S3 IS equipped with MM99adapter (Martin Microscope Co.).

2.3. Light microscopy (LM) and transmissionelectron microscopy (TEM)

Leaves, petioles, stems, bark and roots were sampled from bothHLB-affected and control Valencia trees. The samples were fixed in3% glutaraldehyde in 0.1 M potassium phosphate buffer, pH 7.2, for4 h at room temperature to overnight in the refrigerator. They werethen washed in the same buffer and postfixed 4 h at roomtemperature in 2% osmium tetroxide in the above buffer. Thesamples were then dehydrated in an acetone series and embedded

e and (B) non-infected control Valencia tree. Mature leaves were of comparable in ageular bundles from A and B, respectively, showing the accumulation of starch within thees (C) compared to a vascular bundle of a control tree (D). X ¼ xylem tissue; P ¼ phloem

Fig. 2. Cut edge section of a leaf from an HLB-affected Valencia tree (A) and leaf froma control Valencia tree (B). Leaves were cut with a razor blade and immersed for 2 minin a 2% iodine solution. The leaf was mounted on a stand and observed usinga stereomicroscope.

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in Spurr’s resin [24]. For light microscopy 1 mm sections were cutwith glass knives and stained with methylene blue/azure A, post-stained in basic fuchsin [21]. Light micrographs were taken ona Leitz Laborlux S compound microscope (Germany) with a CanonPowershot S31S digital camera (Tokyo, Japan). For TEM, the sameblocks were thin sectioned (90–100 nm) with a diamond knife,collected on 200 mesh copper grids and stained with 2% aq. uranylacetate and poststained with lead citrate [21]. Micrographs weremade with an AMT (Advanced Microscopy Techniques Corp., Dan-vers, MA) digital camera on a Morgagni 268 (FEI Company, Hills-boro, OR) transmission electron microscope.

2.4. Scanning electron microscopy (SEM)

Leaf tissue from HLB-affected and control Valencia was takenand fixed using the above procedure. The samples were thendehydrated in acetone and critical point dried using a Ladd criticalpoint dryer (Ladd Research Industries, Burlington, VT). The sampleswere then carefully re-cut with a razor blade down the long-axis ofthe leaf, mounted on stubs and coated with gold/palladium usinga Ladd sputter coater (Ladd Research Industries, Burlington, VT).The samples were viewed on a Hitachi S530 scanning electronmicroscope (Tokyo, Japan) and photographed using a Canon EOSRebel XT digital camera (Tokyo, Japan).

3. Results

3.1. Foliar tissues

To determine the extent by which HLB-induced imbalances incarbohydrate metabolism (reflected as starch accumulation) aredistributed throughout the leaf, we sampled autotrophic as well asheterotrophic foliar tissues. Micrographs of cross sectioned leavesfrom HLB-affected compared to controls trees confirmed earlyobservations by Schneider [20] that HLB infection results inabnormally high levels of starch accumulation (Fig. 1A versus 1B). Inlight micrographs, starch grains are visible throughout both pali-sade and spongy mesophyll cells (Fig. 1A). Furthermore, sizablestarch grains are also evident in epidermal cells (Fig. 1A), phloemelements, phloem parenchyma as well as the xylem parenchyma(Fig. 1C). Leaves from nonsymptomatic HLB-negative trees werealmost completely devoid of starch grains (Fig. 1B, D), althoughoccasional grains were observed in palisade and/or spongy cells butnever within the vascular tissue as in HLB leaves. In fact, thedisparity in starch content between no symptomatic HLB negative-and HLB-affected leaves were substantial enough to be visible atthe lowest magnification when leaves are stained with 1.2% iodine(Fig. 2). In addition to the starch accumulation, there was evidenceof phloem collapse in Fig. 1C. This can be seen as non-oval celloutlines (crushed) and opaque inner cell areas (arrows).

The intensity of starch accumulation and details of grain size andintracellular distribution within leaf cells are better appreciated inelectron micrographs (Figs. 3 and 4). Multiple starch grains per chlo-roplast are visible in HLB-affected leaf palisade cells (Fig. 3A, C),whereas control-leaf chloroplasts contained only a small number oflipid inclusions and infrequent smaller starch grains (Fig. 3B, D). Inaddition, lipid inclusions in HLB-affected leaf cells (Fig. 3C, arrowheads)were more numerous and visibly larger than in controls (Fig. 3D,arrowheads). Starch grains in epidermal cells were less numerous thanin mesophyll and palisade cells, however their sizes were considerablylarger and in combination occupied larger portion of the cell’s volumecompared to palisade cells of affected leaves (Fig. 3A).

Higher magnification electron micrographs of spongy palisadecells revealed distinctive cytological differences between controland HLB-affected leaf cells. In cells from HLB-affected leaves, starch

granules-containing chloroplasts comprise a larger portion of thecell’s volume (Fig. 4A, B), the cytosol contained a larger diversity ofmembranous organelles, and mitochondria, aside from being morenumerous, tend to congregate around the starch-filled chloroplasts(Fig. 4C, arrowheads). The larger number of mitochondria and theirclose proximity to the chloroplasts in HLB-cells was evident in allpreparations made and indicate a higher rate of metabolic activityassociated with starch accumulation. Another prominent charac-teristic was the more granular and denser appearance of thecontrol-leaf vacuole (Fig. 4B, D). It is noteworthy that control leavesoften contained starch grains, however, these were usuallyconsiderably smaller (Fig. 4D, arrows) than those in leaf cells ofHLB-affected trees (Fig. 4C, arrow). The differential starch contentand vacuolar texture were not only evident in transmission electronmicrographs, but scanning electron views confirmed these obser-vations (Fig. 5). Clusters of starch grains were evident in HLB-affected leaf cells likely originating from separate chloroplasts(Fig. 5A) whereas these were absent from control cells (Fig. 5B). Thegranular texture of the vacuole in control leaves was seen asa filamentous network forming a web within the cell (Fig. 5B). Thisfilamentous network was absent from the cellular space likelyoccupied by the vacuole in HLB-infected leaves (Fig. 5A).

Chloroplasts from HLB-affected leaf cells became virtuallyunrecognizable (Fig. 6A) as the result of the enormous starch grains.

Fig. 3. Electron micrographs of leaf palisade tissue from (A) HLB-affected and (B) control citrus trees. Leaves from HLB-affected trees had the typical corky vein pattern associatedwith HLB infection. C and D are close-ups of A and B, respectively, showing the discrepancies in starch grains and lipid content. Arrowheads indicate lipid bodies.

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The thylakoid system, with no distinctive granna, was visible onlybetween the large starch granules or pressed against the chloro-plast double membrane envelope (Fig. 6A). In contrast, chloroplastsfrom control leaves showed the typical oblong morphology withwell developed granna and containing small starch grains (Fig. 6B,arrows). Another distinguishing characteristic of photosyntheticcells from HLB-affected tree was the visibly thicker cell walls (Figs.4 and 6), which give the leaves their cardboard, corky texture.

Following a similar pattern of starch distribution, petioles ofHLB-affected trees contained large amounts of starch grains inparenchyma cells both within the vascular bundle (Fig. 7A) andsurrounding parenchymatous tissue (Fig. 7C) compared to petiolesfrom control leaves (Fig. 7B, D, respectively). In all cases, grainswere visible in the xylem vascular rays and in phloem elements(Fig. 7A). The latter are often concealed by the collapsed phloemcells (Fig. 7A, arrowhead), an intrinsic characteristic of greeningdisease [20]. In contrast, petioles from control trees showeda normal, active phloem with little or no starch accumulation(Fig. 7B). Scanning-EM micrographs confirmed the disparity instarch accumulation in petiole parenchyma (Fig. 7C, D). Whereaspetiole cells from control leaves contained an occasional starchgrain (Fig. 7D), HLB-affected petiole parenchyma containedsubstantial amounts of starch grains all throughout (Fig. 7C).

3.2. Stem and bark

The distribution of starch in stem and bark tissue was analogousto leaves in that accumulation of starch in HLB-affected tissues farexceeded control ones (Fig. 8). In stems of HLB-affected trees, xylemparenchyma contained copious amounts of starch. Whereas starchaccumulated conspicuously in xylem parenchyma cells, phloemcollapse allowed only scattered accumulation in the phloem tissue.In addition, the cambial zone and phloem appear obliterated inHLB-affected stems with no distinctive cellular organization as theresult of phloem collapse. Xylem tissue in HLB-affected stems wasalso distinctive in that all cell types had thinner cell walls whencompared to healthy controls.

Starch grains were also highly abundant in the exterior phello-derm of HLB-affected trees (Fig. 9A), in agreement with theremaining observations of aerial parts. However, our observationswere also consistent in that starch also accumulates in the bark ofcontrol trees, but to a lesser extent (Fig. 9B).

3.3. Roots

Whereas starch accumulated disproportionally in cells of allaerial parts in HLB-affected trees (Figs. 1–9), starch in root cells

Fig. 4. Electron micrographs of single palisade cells from HLB-affected (A) and control (B) citrus leaves. Chloroplasts in HLB-affected leaves are not easily recognizable due to thelarge size of starch grains which obliterate chloroplast morphology compared to control-leaf chloroplasts. C and D are close-ups of chloroplasts from A and B respectively. Note thelarge number of mitochondria at the periphery of the chloroplast from the HLB-affected leaf (C). V ¼ vacuole. Arrowheads ¼ mitochondria. Arrows ¼ starch grains.

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presented a contrasting situation. Iodine staining of HLB-affectedtree roots revealed a complete absence of starch from root cells(Fig. 10A, C). A similar size root from a control tree appeared tocontain starch in virtually all living cells (Fig. 10B, D). The completedepletion of root starch may have severe implications to the survivalpotential of HLB-infected trees compared to control-tree roots.

4. Discussion

Starch is a natural product of photosynthetic CO2 fixation ingreen tissues. Formed by a-1,4 glucose linkages, starch exists in 2forms, the soluble, small linear chain amylose and the highly

Fig. 5. Scanning electron micrographs of leaf palisade cells from (A) HLB-affected and (B) conature of the control cell.

branched insoluble amylopectin [22,28]. In green cells, starchaccumulates during light hours and mobilized at night (and othertimes of low photosynthetic activity) to maintain a constant carbonsupply to heterotrophic tissues. Citrus leaves, however, normallyaccumulate very low levels of starch at any time [29] and consid-erable amounts are accumulated only as a result of zinc deficiency[23] or girdling [19]. Once accumulated, starch in citrus leaves is notdegraded [10] even during the night cycles and remains in theleaves indefinitely.

In the current study we demonstrate that the accumulation ofabnormally high levels of starch resulting from HLB infection isnot restricted to photosynthetic leaf cells as widely documented

ntrol citrus leaves. Note the starch grains in the cell of HLB-infected leaf and the fibrous

Fig. 6. Transmission electron micrographs of chloroplasts from (A) HLB-affected and (B) control citrus leaves. Note the thylakoid membrane pressed between starch grains andagainst the chloroplast membrane in samples from HLB-affected leaves (A). Thylakoid and granna stacks are evident in samples from control leaves (B).

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[17,20,25,26], but in fact, this characteristic is extended to virtuallyall aerial tissues. In our samples, starch grains were commonlyobserved throughout all photosynthetic cells of the leaf blade andpetiole, in parenchyma cells of the pith and vascular tissue and evenin the phloem elements (Figs. 1–9). That carbohydrate metabolismis significantly altered by HLB beyond leaf tissue is unequivocallysupported by additional data presented here. Starch accumulationwas sharply different between control and HLB-affected trees in alltissues investigated. Whereas starch in stem vascular parenchymaand phelloderm of HLB trees also accumulated at levels muchhigher than in control trees, roots were practically depleted of anystarch reserves. This observation contrasts with control trees whereaccumulation of starch in leaves and other aerial parts was minimal

Fig. 7. Cross section light micrographs of petiole midveins from HLB-affected (A) and controthe pith and vascular parenchyma. C and D are scanning electron micrographs of cortex paP ¼ phloem tissue; * ¼ phloem collapse.

(Figs. 1–9), yet roots were starch laden (Fig. 10). The absence ofstarch reserves in roots of HLB-affected trees (Fig. 10A, C) is likelythe result of the diminished transport of photoassimilates to theroots and the ensuing usage of starch reserves to sustain their ownmetabolic activities. Final starch depletion likely leads to starvationand to lowering the survival potential when compared to controltrees (Fig. 10B, D).

Given that our observations were made from mature trees ata relatively advanced stage of HLB decline (estimated by thewidespread symptoms), it is difficult to reconcile a chronologicalprogression of events that lead to starch accumulation in leavesand other plant parts. Nevertheless, several conclusions can bedrawn with a high degree of certainty. First, according to

l (B) citrus leaves. (A) Vascular bundle showing the abundance of starch grains both inrenchyma of HLB-affected (C) and control (D) Valencia orange trees. X ¼ xylem tissue;

Fig. 8. Light micrographs of stem sections from (A) HLB-affected and (B) controlValencia orange trees. Starch grains and phloem collapse (bluish inner area of phloem)are evident in the stem sample from HLB-affected trees (A) and absent in the controltree sample (B). X ¼ xylem tissue; P ¼ phloem tissue; * ¼ phloem collapse.

Fig. 9. Light micrographs of stem bark collected from (A) HLB-affected and (B) controlValencia orange trees. Bark was collected at a height of 30 cm above the graft line.Starch grains are present on both samples, although more abundant in HLB-infectedtrees (A).

Fig. 10. Hand cut section from root tissue from HLB-affected (A) and control (B) trees. Root tissue in A and B were stained with 2% iodine for 2 min and observed under 10Xstereomicroscope. C and D show sections of corresponding root tissue under light microscopy showing the lack of starch reserves in roots from HLB-affected trees (C) and theabundance of starch in roots from control trees (D). X ¼ xylem tissue; P ¼ phloem tissue.

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Schneider [20], starch accumulation in foliar tissues results froma photoassimilate transport blockade prompted by HLB-inducedphloem necrosis. However, our observations of high starch contentin stems and bark tissues further down the photoassimilatepathway argue against this hypothesis. If photoassimilate trans-port were to be blocked at the petiole level, no starch would beexpected to accumulate in stems and bark below the leaf canopy.It is noteworthy that the bark samples collected just above thegraft line from HLB-affected trees contained visibly higher levels ofstarch than controls (Fig. 9) without any sign of phloem blockage(data not shown). Furthermore, we often collected asymptomaticleaves located amongst highly symptomatic, starch-loaded leaves,a condition that argues against any type of phloem blockade.Alternatively, element plugging may not be entirely uniformallowing some passage of sugars and starch accumulation beforephloem becomes completely necrotic. In this form, some sugarsreach the lower stem but not the roots where little or no sugar isavailable for starch synthesis.

Second, the difficulties encountered in finding secondary rootsfrom HLB-affected trees, and the total depletion of starch observedin samples when found (Fig. 10 A, C) are good indication ofcarbohydrate starvation in the root system resulting from a physi-ological re-distribution of reserve carbon. Under normal circum-stances, the root system accumulates substantial amounts of starchpresumably utilized during vegetative and reproductive flushes[10]. However, with carbohydrates sequestered in the aerial parts ofthe tree triggered by HLB, it is likely that the roots utilize thesereserves to sustain metabolic activity until depletion results in rootdepletion or degradation.

Since starch accumulation in pepper [14] and potato [18], andphloem disintegration in barley [7], and plugging in cucurbits [3]have been observed in response to a variety of phloem-limitedpathological conditions, we also examined the possibility of starchhyper-accumulation in citrus leaves from conditions such as blight,tristeza, zinc deficiency and branch fracture (girdling). Only underZn deficiency and girdled branches did we observe above averagelevels of starch accumulation in leaves. However, in neither casewere starch levels comparable to those observed in HLB-infectedtrees.

Although anatomical in nature, the data shown in thiscommunication represent clear evidence of the systemic and farreaching influence of HLB on citrus tree carbohydrate metabolism.The data supports the notion that the substantial changes incarbohydrate partitioning observed throughout the citrus tree maynot only be a result of HLB infection, but in itself, a cause for therapid decline and death of infected trees as carbohydrates arechannelled towards the synthesis and accumulation of starch in theaerial parts resulting in root starvation. Starch accumulation wasobserved in practically all living cells of the aerial parts of the treedenoting a clear redirection of carbohydrate transport. Whether thewidespread starch accumulation in aerial parts resulted at theexpense of root starch mobilization or whether the capacity ofcarbon fixation is increased as a consequence of HLB infection is yetto be demonstrated. However, upward movement of reserve starchcarbon is highly unlikely with a plugged phloem system. Mostlikely, root starch is consumed to sustain root metabolic activitieswhen little sugar is translocated down from the leaves resulting inroot death and eventually tree decline.

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