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David M. Eissenstat, James H. Graham, James P. Syvertsen, and Diana L. Drouillard

The effects of phosphorus (P) and of the mycorrhizal (M) fungus, Glomus intraradix, on the carbon (C) economy of sour orange (citrus aurantium L.) were determined during and following active M colonization. There were four treatments: mycorrhizal seedlings grown at standard-strength P (M1) and nonmycorrhizal (NM) plants grown at 1, 2 and 5 times standard-strength P (NM1, NM2 and NM5). Mycorrhizal colonization, tissue dry mass, P content, root length, leaf area, 14C partitioning and rate of c assimilation (A) were determined in five whole-plant harvests from 6 to 15 wks of age. In contrast to the effects of P nutrition on C economy in sour orange, M effects were generally subtle. Mycorrhizae increased the root biomass fraction, the root length/leaf area ratio, and the percent of 14C recovered from belowground components. Mycorrhizal plants had a higher percentage of belowground 14C in the respiration and soil fractions than did NM plants of equivalent P status. Mycorrhizal plants tended to have enhanced A at 8 wks but not at 7 or 12 wks. This temporarily enhanced A of M plants did not fully compensate for their greater belowground C expenditure, as suggested by apparently lower relative growth rates of M than NM plants of equivalent P status. Problems of interpreting the dynamic effects of mycorrhizae on C economy that are independent of p nutrition are discussed.

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John E. Fucik and Dariusz Swietlik

Water extracts of cocklebur,CBX (Xanthium spinosa L.) and velvetleaf,VLX (Abutilon theophrasti Medic.) shoots and Mexican ash,AshX (Fraxinus Berlandieriana A.DC.) roots were added to 9 month-old sour orange Citrus aurantium L.) seedlings(SOs) in water culture. Final extract concentrations represented either 50 or 12.5 g. of plant material liter-1 of culture solution, i.e. 1/20 or 1/80 dilutions. Leaf water potential(ψ); stomatal conductance(gs);transpiration(T) and growth responses were measured for 13 days. After 1 day, SOs in AshX and CBX had lower ψ than controls. After 11 days SOs in CBX had higher ψ than the others. ψ responded similarly to both extract concs.. Thru day 5, AshX decreased gs vs. the controls and VLX. By day 11, gs of SOs in AshX was less than for VLX but not the others. On days 1 and 5, gs for VLX at 1/20 was lower than controls but at 1/80, gs's were the highest of all treatments. These results supported by the T rates, growth responses and others findings suggest AshX and VLX induce water stress by reducing water absorption and/or transport in addition to possibly disrupting normal root/shoot communications

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Amit Vikram, G.K. Jayaprakasha, and Bhimanagouda S. Patil

Our recent studies have shown that certain citrus limonoids protect from colon cancer based on cell and animal studies. Animal studies also suggest that citrus juice protects from osteoporosis. To understand the structure–function relationship through animal studies requires a large amount of purified limonoids. Since certain limonoids are present in low concentration, it is a challenge to obtain the required quantity of different limonoids. In this context, we report the purification of limonin 17-ß-D glucopyranosides (LG), and deacetylnomilinic acid 17-ß-D glucopyranoside (DNAG). However, DNAG was isolated in relatively large amount from sour orange (Citrus aurantium L.) seeds. Defatted seed powder was extracted with methanol and purified using column chromatography to obtain multigrams of DNAG. While LG was found in lower concentration, a large concentration of hesperidin was also purified in this process. This project is based upon work supported by the USDA-CSREES under Agreement USDA IFAFS #2001 52102 02294 and USDA #2005-34402-14401 “Designing Foods for Health” through the Vegetable & Fruit Improvement Center.

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M.A. Maurer, F.S. Davies, and D.A. Graetz

A field study was conducted on mature `Redblush' grapefruit trees (Citrus paradisi Macf.) on sour orange (Citrus aurantium L.) rootstock from 1991 to 1993 near Vero Beach, Fla. on poorly drained (flatwoods) soil to determine the effects of reclaimed water on leaf, soil and shallow well-water nutrients. Treatments consisted of a canal water applied based on soil moisture depletion, and reclaimed water applied at 23.1, 30.7 and 36.6 mm/wk. Reclaimed water treatments received supplemental fertilization in addition to the N present in the water. All treatments received about 130 kg/ha/yr N. Leaf tissue N, P, K, Ca, Mg and Na concentrations were similar for all treatments, but B concentrations were significantly higher for the reclaimed water treatments in 1991 and 1993. Soil P and Na concentrations also increased in the reclaimed water treatments. Water samples taken from shallow depth wells showed that reclaimed water treatments had lower levels of NO, compared to the control possibly due to leaching. Reclaimed water contained only trace or undetectable levels of heavy metals.

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Deepak Dandekar, G. K. Jayaprakasha, and Bhimanagouda Patil

Citrus consumption has been shown to promote human health due to presence of several bioactive compounds. In the process of understanding the health benefits of citrus, we need to isolate and characterize these compounds. Limonoids are one of such prominent, but lesser-known phytonutrients that have been shown to prevent cancers of the mouth, skin, lung, breast, and colon. With the growing interest in the health-promoting properties of citrus limonoids, the demand for these bioactives has significantly increased. It has been critical to explore environment-friendly extraction methods rather than using hazardous organic solvents. A water-based hydrotropic extraction of limonoid aglycones from sour orange (Citrus aurantium L.) seeds was developed. Two hydrotropes, sodium salicylate (Na-Sal) and sodium cumene sulfonate (Na-CuS), were studied for extraction efficiency using the Box Behnken experiment design method. The extraction efficiency of prominent aglycone limonin was observed depending on hydrotrope concentration, extraction temperature, and percentage of raw material loading. Response Surface Analysis (RSA) of data predicted the optimum conditions for maximum yield. Recovery of aglycones from filtered extract is also easily achieved by mere dilution using water at pH 3 or 7 or by partitioning the extract with dichloromethane. At optimum conditions, limonin yield of 0.46 mg/g seeds in the case of Na-Sal extraction and 0.65 mg/g seeds in the case of Na-CuS extraction was achieved. The results demonstrated that the hydrotropic extraction process of limonoid aglycones has practical commercial importance. This project is based upon work supported by the USDA–CSREES IFAFS #2001 52102 02294 and USDA–CSREES #2005-34402-14401 “Designing Foods for Health” through the Vegetable and Fruit Improvement Center.

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Frederick S. Davies, Craig A. Campbell, and Matthew W. Fidelibus

It is desirable to mix gibberellic acid (GA3) with other commonly applied materials to reduce application cost. However, applying GA3 with some compounds can reduce its efficacy or cause phytotoxicity. We conducted experiments in 1997-98 and 1998-99 to determine if GA3 (ProGibb) can be tank-mixed with fosetyl-Al (Aliette), or avermectin (Agri-Mek) and oil, without reducing GA3 efficacy. In addition, we compared Silwet and Kinetic adjuvants for enhancement of GA3 efficacy. Five tank mixes were tested along with a nonsprayed control. These included 1) GA3; 2) GA3 and Silwet; 3) GA3 and Kinetic; 4) GA3 Silwet, and fosetyl-Al; and 5) GA3, Silwet, avermectin, and oil. All compounds were applied at recommended concentrations. In September 1997 or October 1998, about 2.5 gal (9.5 L) of each tank mix was applied with a hand sprayer to 14- or 15-year-old `Hamlin' orange (Citrus sinensis) trees on sour orange (Citrus aurantium) rootstock. Peel puncture resistance (PPR), color, and juice yield (% juice weight) were evaluated monthly between December 1997 and March 1998, and December 1998 and January 1999. In both years, fruit of treated trees usually had higher PPR and were less yellow in color than fruit from control trees. There were tank mix effects on juice yield in January of both seasons and February 1998. Gibberellic acid was most effective at enhancing juice yield when applied singly or with avermectin and oil. In both seasons there were dates when GA3 applied singly was superior at enhancing juice yield than a tank mix of GA3, Silwet and fosetyl-Al, indicating that GA3 was incompatible with fosetyl-Al. Neither Kinetic nor Silwet adjuvants consistently enhanced GA3 effects on peel quality or juice yield over GA3 alone.

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David M. Eissenstat, James P. Syvertsen, Thomas J. Dean, Jon D. Johnson, and George Yelenosky

The combined effects of O3 and acid rain on freeze resistance, growth, and mineral nutrition were studied using broadleaf-evergreen citrus and avocado trees. Using a factorial design, `Ruby red' grapefruit (Citrus paradisi L.) trees on either Volkamer lemon (Citrus volkameriana Ten. & Pasq.) or sour orange (Citrus aurantium L.) rootstock and `Pancho' avocado trees (Persea americana Mill.) on `Waldin' rootstock were exposed to O3 and acid rain for 8 months in open-top chambers under field conditions. The O3 treatments were one-third ambient (0.3X), ambient (1X), twice ambient (2X), or thrice ambient (3X). Ambient O3 concentrations averaged 39.1 nl·liter-3 over a 12-hour day. The acid rain treatments had a pH of 3.3, 4.3, or 5.3 and were applied to simulate long-term rainfall averages. In general, the effects of acid rain on growth and freeze resistance were small. Rain of high acidity (pH = 3.3) offset the negative effects of O3 on growth (total leaf mass) in avocado and grapefruit/Volkamer lemon trees. In contrast, rain of high acidity magnified the detrimental effects of O3 on electrolyte leakage of leaf disks at subzero temperatures, especially for citrus. Freeze resistance, determined by stem and whole-plant survival following freezing temperatures, was lower in the most rapidly growing trees. Consequently, for trees exposed to a combination of O3 and acidic rain, leaf electrolyte leakage did not correlate significantly with stem survival of freezing temperatures. We conclude that the danger of acid rain to citrus and avocado in Florida is rather slight and would only present a potential problem in the presence of extremely high O3.

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F.S. Davies, M.W. Fidelibus, and C.A. Campbell

An experiment was conducted to determine if gibberellic acid (GA; ProGibb, Abbott Labs) can be mixed with Aliette or Agri-Mek and oil to reduce application costs, without reducing GA efficacy, and if Silwet and Kinetic adjuvants enhance GA efficacy. Five tank mixes were tested along with a nonsprayed control. The tank mixes included: 1) GA, 2) GA + Silwet, 3) GA + Kinetic, 4) GA + Silwet + Aliette, and 5) GA + Silwet + Agri-Mek + oil. All compounds were applied at recommended concentrations. In September, ≈24 L of each tank mix was applied with a hand sprayer to mature `Hamlin' orange trees [Citrus sinensis (L.) Osb.] on sour orange (Citrus aurantium L.) rootstock. Peel puncture resistance (PPR), peel color, and juice yield (percent juice weight) were evaluated monthly between Dec. 1997 and Mar. 1998. On most sampling dates the fruit of treated trees had higher PPR and were less yellow in color than fruit from control trees. However, in Jan., fruit treated with GA + Silwet and GA + Kinetic had greater PPR than other treatments. In Feb., fruit treated with GA + Silwet + Agri-Mek + oil had the lowest PPR. The effect of the different tank mixes on juice yield was usually similar to the effect of the tank mixes on PPR and peel color. On 8 Jan. 1998, fruit from trees treated with GA alone yielded significantly more juice than fruit from control trees. On 24 Feb. 1998, fruit from trees treated with GA alone yielded more juice than fruit from the other treatments. Thus, GA efficacy is generally not reduced by these tank mixes, nor improved by adjuvants.

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B.L. Tan, N. Reddy, V. Sarafis, G.A.C. Beattie, and R. Spooner-Hart

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to detect petroleum-derived spray oils (PDSOs) in citrus seedlings and trees. The NMR spectrum of the phantom containing 10% (v/v) of a nC24 agricultural mineral oil (AMO) showed the resonance of the water protons at δ ≈ 5 ppm, while the resonance of the oil protons at δ = 1.3 to 1.7 ppm. The peak resolution and the chemical shift difference of more than 3.3 ppm between water and oil protons effectively differentiated water and the oil. Chemical shift selective imaging (CSSI) was performed to localize the AMO within the stems of Citrus trifoliata L. seedlings after the application of a 4% (v/v) spray. The chemical shift selective images of the oil were acquired by excitation at δ = 1.5 ppm by averaging over 400 transients in each phase-encoding step. Oil was mainly detected in the outer cortex of stems within 10 d of spray application; some oil was also observed in the inner vascular bundle and pith of the stems at this point. CSSI was also applied to investigate the persistence of oil deposits in sprayed mature Washington navel orange (Citrus ×aurantium L.) trees in an orchard. The trees were treated with either fourteen 0.25%, fourteen 0.5%, four 1.75%, or single 7% sprays of a nC23 horticultural mineral oil (HMO) 12 to 16 months before examination of plant tissues by CSSI, and were still showing symptoms of chronic phytotoxicity largely manifested as reduced yield. The oil deposits were detected in stems of sprayed flushes and unsprayed flushes produced 4 to 5 months after the last spray was applied, suggesting a potential movement of the oil via phloem and a correlation of the persistence of oil deposit in plants and the phytotoxicity. The results demonstrate that MRI is an effective method to probe the uptake and localization of PDSOs and other xenobiotics in vivo in plants noninvasively and nondestructively.

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Bingru Huang and David M. Eissenstat

In Citrus L. sp., specific root length of whole root systems has been correlated positively with root hydraulic conductivity, but there is little mechanistic understanding of the causes for this association. The hydraulic conductivity of individual roots in relation to root anatomical characteristics in seedlings of three citrus rootstocks [sour orange (SO) (Citrus aurantium L.), trifoliate orange (TO) (Poncirus trifoliate (L.) Raf.), and Swingle citrumelo (SC) (C. paradisi Macf. × P. trifoliata)] that vary widely in specific root length (SRL) was measured. Among fibrous roots, first-order and secondorder laterals were examined. Relative differences among rootstocks in the overall hydraulic conductivity (LP) and radial conductivity (LR) for individual 1-month-old and 6-month-old second- and first-order roots generally were consistent with hydraulic conductivity determined previously for entire root systems. There were no significant differences in axial conductance per unit pressure (Kh) in either first- or second-order roots among the rootstocks. This was consistent with the similarity in number and diameter of xylem vessels. One-month-old second-order roots had no suberized exodermis but varied in cortical radius. Six-month-old second-order roots of TO, however, had more nonsuberized cells (passage cells) in the exodermis than roots of SC and SO, although the cortical radius of SC and SO roots were not different. Compared to 6-month-old second-order roots, 1-month-old second-order roots had much higher LP and LR but lower Kh. Differences in overall root hydraulic conductivity among the citrus rootstocks were mainly related to structural differences in the radial pathway for water movement, suggesting that radial hydraulic conductivity was the primary determining factor of water uptake in citrus rootstocks.