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The most important worldwide problem in citrus production is the bacterial disease Huanglongbing (HLB; citrus greening) caused by a phloem-limited bacterium Candidatus Liberibacter asiaticus. The earliest visible symptoms of HLB on leaves are vein yellowing and an asymmetrical chlorosis referred to as ‘‘blotchy mottle,’’ thought to be the result of starch accumulation. We tested the hypothesis that such visible symptoms are not unique to HLB by stem girdling 2-year-old seedlings of two citrus rootstocks with and without drought stress in the greenhouse. After 31 days, girdling had little effect on shoot growth but girdling increased the relative growth rate of shoots in drought-stressed trees. Starch content in woody roots of non-girdled trees was three to 19 times higher than in girdled trees. In non-girdled trees, drought stress induced some starch accumulation in roots, but there were no effects of drought stress on root starch or sucrose in girdled trees. Girdling induced a 4-fold greater starch content in leaves on well-watered trees but leaf sucrose content was unaffected. Girdling reduced leaf transpiration in well-watered trees but net assimilation of CO2 was unaffected by girdling or leaf starch accumulation. Leaves on girdled trees clearly had visible blotchy mottle symptoms but no symptoms developed on non-girdled trees. The increase in leaf starch, up to 50% dry weight (DW), resulted in an increase in leaf DW per leaf area (LA) and an artificial reduction of many leaf nutrients on a DW basis. Most of these differences disappeared when expressed on a LA basis. Leaf boron (B), however, was inversely related to leaf starch when both were expressed on a LA basis. In the absence of HLB, girdling increased leaf starch, decreased root starch, and duplicated the asymmetric blotchy mottled visual leaf symptoms that have been associated with HLB-infected trees. This supports our contention that such symptoms generally attributed to HLB are not uniquely related to HLB infection, but rather are directly related to starch accumulation and secondarily to nutrient deficiencies in leaves. Currently, the most important problem in citrus production worldwide is the bacterial disease HLB (syn. citrus greening). HLB is presumably caused by the bacterium Candidatus Liberibacter asiaticus (Clas), a fastidious Gram-negative, obligate parasite, phloem-limited a-proteobacterium (Garnier and Bove, 1983; Jagoueix et al., 1994). When vectored into a citrus tree by phloem-feeding psyllids, Clas triggers a cascade of events causing phloem dysfunction, cellular collapse, and overaccumulation of carbohydrates in leaves (Bove, 2006; Garnier and Bove, 1983). The earliest visible symptoms of HLB in leaves are vein yellowing and asymmetrical chlorosis referred to as ‘‘blotchy mottle,’’ thought to be the result of starch accumulation (Etxeberria et al., 2009). At later stages, leaves become small and upright, common drought stress-like symptoms. Leaves can also develop a variety of chlorotic patterns that often resemble mineral deficiencies such as those of zinc, iron, and manganese deficiency. Such symptoms, however, are likely secondary symptoms resulting from loss of roots from carbohydrate starvation and subsequent drought stress (Jagoueix et al., 1994). At later stages, HLB-affected trees show twig dieback, causing a decline in production, and culminating in tree death within a few years. Root systems of infected trees are often poorly developed and new root growth may be suppressed (Etxeberria et al., 2009). Root loss or root growth restriction can lead to drought stress and changes in mineral nutrition in the shoot (Spann and Schumann, 2009). Thus, secondary symptoms resulting from mineral deficiency often complicate the diagnosis of HLB symptoms (related to starch accumulation in leaves; Etxeberria et al., 2009) from those caused by mineral nutrient imbalances (Spann and Schumann, 2009). Stem girdling has been widely used in citrus, grape, peach, and other fruit tree crops, mainly to increase flowering, fruit set, and fruit size (Goren et al., 2004). Girdling is the removal of a strip of bark with included phloem tissue from the trunk or major limbs of a fruit tree, thereby blocking the downward translocation of photosynthetic carbohydrates and metabolites through the phloem. The best known effects of girdling are the accumulation of assimilates, sugars, and starch above the girdle area (Goren et al., 2004). Tree parts below the girdle, on the other hand, can suffer from a shortage of assimilates. After trunk girdling, the roots are gradually depleted of their carbohydrate reserves, sometimes reaching serious root starvation (Weaver and McCune, 1959). Cytokinin and gibberellins content of shoots can also be disturbed by girdling (Cutting and Lyne, 1993). However, the precise biochemical changes resulting from girdling have been studied only in a few particular cases (Beruter and Feusi, 1997), and a detailed interpretation of the physiological effects of girdling is still lacking. Progress in tree research during the past decade has led to the understanding that, beyond their nutritional and energetic contribution, sugars can play regulatory roles, which involve upand downregulation of gene expression (Jang and Sheen, 1997; Koch and Zeng, 2002; Smeekens, 2000). Gonzalez et al. (2012) showed that girdled ‘Valencia’ citrus trees accumulated large amounts of starch producing symptoms analogous to those characteristic of HLBaffected tissues. Starch grains from both HLB and girdled trees were morphologically similar with only small biochemical difference in glucan chain lengths. This similarity prompted us to investigate the increase in starch concentration through time using girdled citrus trees of Cleopatra and Swingle rootstocks exposed to water deficit. The aim of this study was to determine which HLBlike symptoms were related to starch accumulation and/or drought stress in the absence of any HLB infection. Girdling of the stem bark was intended to mimic the decreased carbohydrate transport caused by the phloem dysfunction in HLB-affected trees. We hypothesized that both girdling and drought stress should lead to measurable changes in tree growth, starch allocation, and mineral nutrient composition that are similar to those reported for HLB. Understanding these responses will contribute to our knowledge of symptom development in HLB because some symptoms may be the result of secondary responses of carbohydrate accumulation and, thus, only indirectly related to HLB. Received for publication 4 Apr. 2013. Accepted for publication 9 May 2013. To whom reprint requests should be addressed; e-mail jmsn@ufl.edu. 920 HORTSCIENCE VOL. 48(7) JULY 2013 Materials and Methods Tree material and experimental design. Forty-eight uniform, well-nourished 2-yearold seedlings of Swingle citrumelo (Citrus paradise Macf. · Poncirus trifoliata L.) with trifoliate leaves and Cleopatra mandarin (C. reticulata Blanco) with entire leaves were purchased from a local nursery certified free of HLB. Seedling trees were 180 cm tall and grown in 2.25-L pots filled with a soilless media consisting of peat/sponge-rock/ vermiculite (3:1:1). Trees were grown in an unshaded greenhouse from 29 May to 29 June 2012 at the University of Florida/IFAS Citrus Research and Education Center, Lake Alfred, FL (lat. 28 N, long. 82 W, elevation 51 m). Maximum photosynthetically active radiation (PAR) at tree level was 1200 mmol·m·s (LI-170; LICOR, Inc., Lincoln, NE). Average day/night temperature was 40/30 C and relative humidity (RH) varied diurnally from 40% to 100%. Each cultivar was divided into four treatment groups (with six trees per treatment). The treatments were: 1) non-girdled and wellwatered (control); 2) non-girdled and droughtstressed; 3) girdled and well-watered; and 4) girdled and drought-stressed. Well-watered trees were thoroughly irrigated three times a week, whereas droughtstressed trees were irrigated only when wilting symptoms appeared, which averaged approximately once per week. All trees were fertilized once a week with 600 mL of complete fertilizer solution containing chelated iron (Fe; 1 mL·L), 7N–3P–7K, and all micronutrients. No psyllids were ever observed but for preventative pest control, trees were sprayed once a week with either 1% agricultural spray oil or 1% soap solution. Total stem length and basal stem diameter, above and below the girdled area and at a similar height for non-girdled trees, were measured at time zero and again at the end of the experiment, 32 d later. Stem relative growth rate (RGR) was calculated using the difference between total stem length at the beginning and the end of the experiment divided by the number of elapsed days between the two measurements (cm·m·d). Girdling was performed on 29 May 2012 on the main stem 20 cm above the pot. A ring of stem bark tissue ( 4 mm in width) was carefully removed (Fig. 1A) using a pair of razor blades glued together with a space of 4 mm between them. The girdled strip was renewed every 9 d to prevent healing and phloem repair. Preliminary experiment. A preliminary experiment was conducted with 16 additional trees of the same two rootstocks, using two replicate trees per treatment, from 24 Apr. to 15 May 2012. Six leaf disk samples per tree (three disks per leaf/two leaves per tree) were sampled and used to evaluate starch, sucrose, and DW per area (DW/LA) on the day of girdling (time zero) and at 3, 7, 10, 13, 14, and 21 d after girdling. The analyses did not show any difference in starch and sucrose content between disk samples from the same leaf. Starch content was still increasing at 21 d after girdling (data not shown). Based on these results, we decided to reduce the number of samples per leaf and extend experimental time up to 31 d to determine any changes in the rate of carbohydrate accumulation. Two weeks after girdling, a few new shoots developed directly below the girdle in

Carbohydrate and Nutritional Responses to Stem Girdling and Drought Stress with Respect to Understanding Symptoms of Huanglongbing in Citrus