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Kathryn C. Taylor and Xi He

Citrus blight is a decline disorder of unknown etiology. Its earliest symptom is redistribution of Zn within the tree. The canopy exhibits symptoms of Zn deficiency while Zn accumulates in trunk phloem just above the bud union. Phloem Zn was associated with complexing agents. Zn-binding proteins (ZBPs) were isolated from phloem tissue extracts of mature healthy and blight citrus trees. After purification by ion exchange chromatography (IEC) and size exclusion chromatography, ZBP's from healthy and blight-affected citrus trees {`Valencia' sweet orange [Citrus sinensis (L.) Osbeck] on rough lemon [C. jambhiri (L.)]) were compared for relative abundance. Size exclusion chromatography indicated that the proteins were and 19.5 kD Mr. IEC demonstrated that the 5 kD ZBP was slightly anionic and that the 19.5 kD ZBP was highly anionic. There were 2.5 × times more of the 5 kD ZBP in blight material than in healthy (purified on an equal prot basis). Levels 19.5 kD ZBP were equivalent in blight and healthy phloem tissue extracts, on both fresh weight and total protein bases. The amino acid composition of the 5 kD ZBP was much more complex than would be expected for phytochelatins. Altered phloem structure also was apparent in blight versus healthy trees, with phloem fresh and dry weights increased in the blight-affected citrus. Phloem specific expression of the ZBPs was also apparent.

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Danielle R. Ellis and Kathryn C. Taylor

A partial cDNA (cvzbp-1) was cloned based on the N-terminal sequence of a citrus (Citrus L.) vascular Zn-binding protein (CVZBP) previously isolated from vascular tissue (Taylor et al., 2002). CVZBP has homology to the Kunitz soybean proteinase inhibitor (KSPI) family. Recombinant protein produced using the cDNA clone inhibited the cysteine proteinase, papain. Metal binding capacity has not been reported for any other member of this family. CVZBP was present in leaves, stems, and roots but not seeds of all citrus species examined. However, CVZBP was present in germinating seeds after the cotyledons had turned green. Within four hrs after wounding, CVZBP was undetectable in the wounded leaf and adjacent leaves. It has been suggested that many members of the KSPI family serve a function in defense. However, the expression of the CVZBP is in direct contrast with those of KSPI members that were implicated in defense response. Though systemically regulated during wounding, we suggest that CVZBP is not a defense protein but rather may function in vascular development.

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Kathryn C. Taylor and Danielle R. Elli

A 22-kDa Zn-binding protein (ZBP) was isolated from the phloem tissue and evacuated xylem sap of `Valencia' sweet orange [Citrus sinensis (L.) Osbeck] on rough lemon [C. jambhiri (L.)], as well as Valencia on Rangpur lime [Citrus limonia Osbeck]. Phloem and xylem Zn was associated with the 22 kDa ZBP. The Mr value of this ZBP was estimated to be 19,500 by size exclusion chromatography and 22,800 by SDS-PAGE. This protein was isolated with an isoelectric point of 7.5. Ion exchange chromatography demonstrated that 22-kDa ZBP was highly anionic, requiring 0.43 M NaCl for elution from QAE Sepharose. The 22-kDa ZBP appears unique to citrus, having no cross reaction with protein from several tissues from a range of plant species. Accumulation decreased under Zn-deficient conditions, was enhanced by osmotic stress, and the protein completely disappeared with wounding. Amino acid composition demonstrated that the protein was rich in aspartate, and glutamate; and contained 6 cysteine, and 4 histidine residues. These amino acids may be involved in metal binding. N-terminal amino acid sequencing demonstrated that the 22-kDa ZBP had identity with sporamin A&B precursors, Kunitz-type trypsin inhibitors, and miraculin. It is suggested that the genes that encode these proteins are derived from a common ancestral gene.

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Kathryn C. Taylor and Danielle R. Ellis

A 5-kDa zinc-binding protein (ZBP) accumulates in the phloem above the graft union in citrus trees affected by two citrus decline disorders, citrus blight and Macrophylla decline. Citrus blight and Macrophylla decline are decline disorders of unknown etiology. Citrus blight has historically been restricted to humid production regions, and Macrophylla decline occurs in arid regions. A causal agent has not been identified for either disorder. Levels of the 5 kDa ZBP are increased 2.5- and 1.5-fold, respectively, in the phloems above the graft union in trees with citrus blight and Macrophylla decline. The protein was purified from citrus phloem and a partial N-terminal sequence was obtained. The protein has homology to the cystein-rich chitin binding domain of several plant chitinases and hevein. Hevein and class I chitinases, which have this N-terminal chitin binding domain, have antifungal activity and antibacterial activity and are induced by wounding and several other stresses. Oligonucleotides corresponding to the greatest conserved region in the N-terminal domain of the ZBP were produced. They were used as primers for PCR reactions against a citrus leaf cDNA library. Four PCR products were obtained from these reactions. The products are being subcloned and sequenced. They will be used to probe the citrus cDNA library to obtain the cDNA clone for the 5 kDa ZBP.

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Kathryn C. Taylor and Parshall B. Bush

To discern how the packing process influences pesticide residue loads on peach (Prunus persica L. Batsch) fruit; postharvest, post hydrocooled, and post brushed fruit were assessed for levels of several pesticides. The packing house process reduced pesticide residue levels on fresh peaches to levels that were generally below detection limits of our assays in 1998. Carbaryl and captan residues from field packed fruit were 32.2× and 21.9×, respectively, of that found in the peel of fruit processed in the packing house in 1998. Carbaryl levels were not reduced by hydrocooling but postharvest brushing reduced pesticide residues up to 94% in fruit peel. Across processing operations and cultivars assessed in 1999, hydrocooling, hydrocooling plus brushing, and brushing alone removed 37%, 62%, and 53%, respectively, of the encapsulated methyl parathion residues from fruit peel. Hydrocooling had the greatest impact on phosmet removal from peel, reducing levels by 72.5%. After hydrocooling, phosmet was 5.7× following brushing in one-half of the subsequent samples. This increase occurred at all three farms, suggesting that periodic cleaning of brushes may be necessary to prevent later contamination of peach peel with pesticides. In the only example in which propiconazole residue remained on peaches at picking, it was removed most effectively (69%) by the brushing operation. Nearly 31% of the propiconazole was removed in the hydrocooler. The packing process before shipment to retail outlets was generally effective in the removal of pesticides that may be present on peel at the time of harvest. Assessment of pesticide residue levels in peach flesh was uniformly below the levels of detection in our assays, suggesting that the classes of pesticide analyzed in peaches were not transepidermal.

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Glenn C. Wright, William B. McCloskey, and Kathryn C. Taylor

Several orchard floor management strategies were evaluated beginning in Fall 1993 in a `Limoneira 8A Lisbon' lemon (Citrus limon) grove on the Yuma Mesa in Yuma, Ariz. and in a `Valencia' orange (Citrus sinensis) grove at the University of Arizona Citrus Agricultural Center, Waddell, Ariz. At Yuma, disking provided acceptable weed control except underneath the tree canopies where bermudagrass (Cynodon dactylon), purple nutsedge (Cyperus rotundus), and other weed species survived. Mowing the orchard floor suppressed broadleaf weed species allowing the spread of grasses, primarily bermudagrass. Preemergence (norflurazon and oryzalin) and postemergence (glyphosate and sethoxydim) herbicides were used to control weeds in the clean culture treatment in Yuma. After three harvest seasons (1994-95 through 1996-97), the cumulative yield of the clean culture treatment was 385 kg (848.8 lb) per tree, which was significantly greater than the 332 kg (731.9 lb) and 320 kg (705.5 lb) per tree harvested in the disking and mowing treatments, respectively. In addition, the clean culture treatment had a significantly greater percentage of fruit in the 115 and larger size category at the first harvest of the 1995-96 season than either the disk or mow treatments. At Waddell, the management strategies compared were clean culture (at this location only postemergence herbicides were used), mowing of resident weeds with a vegetation-free strip in the tree row, and a `Salina' strawberry clover (Trifolium fragiferum) cover crop with a vegetation-free strip. The cumulative 3-year yield (1994-95 through 1996-97) of the clean culture treatment was 131 kg (288.8 lb) per tree, which was significantly greater then the 110 kg (242.5 lb) per tree yield of the mowed resident weed treatment. The yield of the strawberry clover treatment, 115 kg (253.5 lb) of oranges per tree, was not significantly different from the other two treatments. The presence of cover crops or weeds on the orchard floor was found to have beneficial effects on soil nitrogen and soil organic matter content, but no effect on orange leaf nutrient content. The decrease in yield in the disked or mowed resident weed treatments compared to the clean culture treatment in both locations was attributed to competition for water.

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Charles C. Reilly, Michael W. Hotchkiss, and Kathryn C. Taylor

Pesticide application in peach (Prunus persica) orchards with a commercial airblast sprayer was compared to that of an air assisted rotary atomizer (AARA), low-volume sprayer during the 2000 through 2003 seasons. The two technologies were employed during early season petal fall applications, shuck split applications and standard cover sprays using phosmet, sulfur, propiconazole, chlorothalonil, azoxystrobin and captan. Ripe fruit, picked 1 day prior to first harvest each season were rated for peach scab (Cladosporium carpophilum), brown rot (Monilinia fructicola), insect (Hemipteran) damage (cat facing), and blemishes. Differences in brown rot, insect damage, and blemish ratings were not detected between the treatments for each of the four seasons. Differences were detected during the 2000 and 2001 seasons for peach scab, with the AARA sprayer plots having a higher incidence. Spray coverage was quantitatively evaluated with Rhodamine B dye by leaf rinses that indicated there was equivalent coverage for each application method. Phosmet residue detection on trees of the treated rows was also equivalent from each method. Phosmet off-target spray movement (drift) was reduced 59% one row away from the treated row and 93% in the fifth row from the treated row by the AARA sprayer compared to airblast sprayer drift.

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Mohammad R. Karim, Glenn C. Wright, and Kathryn C. Taylor

A field trial conducted at Yuma, Ariz., examined the effect of foliar boron application on fruit yield and quality of Citrus sinensis cv. Hamlin. Boron was applied to 5-year-old trees at five treatment levels (0, 500, 1000, 2000, and 3000 ppm) before or after flowering in a split plot design. At harvest, fruit number, size and quality were determined. Yield (P = 0.01) and average fruit number per tree (P = 0.02) were different among treatments. The highest yield was obtained with the 500 ppm treatment. In this first year of the trial there was no difference in average fruit weight, fruit pH, titratable acidity, peel thickness, juice volume, or soluble solid content of fruit between the treatments. Previous studies indicate that boron influenced in vivo and in vitro pollen germination in many crops. Increased fruit yield may have occurred because boron was transported to the flowers where it exerted its influence on increased fruit set through an effect on pollen viability or pollen tube growth. Further investigation of this hypothesis is underway.

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Kathryn C. Taylor, Danielle R. Ellis, and Luciano V. Paiva

Zinc in xylem and phloem of the citrus rootstock, rough lemon [Citrus jambhiri (L.)] was associated with a Zn-binding protein, designated citrus vascular Zn-binding protein (CVZBP). The apparent molecular mass of the CVZBP was 19.5 kDa after nondenaturing size exclusion chromatography and 21.8 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion exchange chromatography demonstrated that CVZBP was anionic, requiring 0.43 n NaCl for elution from quaternary aminoethyl Sepharose. Antiserum to the protein cross-reacted more with total protein extracts from leaf midveins than with total protein from the rest of the leaf lamina, further suggesting a vascular location of the Zn-binding protein. Quantitative analysis indicated that ≈2 to 3 mol of Zn were associated with 1 mol of native protein. Binding studies with the partially purified CVZBP demonstrated a capacity to bind several divalent cations: Cd, Ni, Pb, and Zn. Reaction with Ellman's reagent suggested that the protein has significant sulfhydryl group content that may be involved in metal binding. N-terminal sequencing demonstrates identity with papaya latex trypsin inhibitor, sporamin, or other Kunitz soybean proteinase inhibitors.