First Confirmed Report of Tobacco ringspot virus in Cucurbits Crops in Oklahoma.

Cucurbits are major cash crops of vegetable growers in Oklahoma, particularly watermelon, which is the official state vegetable. In 2010, during a survey for cucurbit viruses (1), symptomatic leaf samples of cucumber (Cucumis sativus), cantaloupe (Cucumis melo), pumpkin, (Cucurbita pepo), squash (Cucumis maxima), and watermelon (Citrullus lanatus) showing mild to severe mosaic, mottling, and chlorotic spots were collected in Atoka, Blaine, Jefferson, and Tulsa counties. A total of 161 samples were tested by dot-immunobinding assay (DIBA) (2) using Tobacco ringspot virus (TRSV; genus Nepovirus, family Comoviridae) specific antiserum. Fourteen samples of cantaloupe, pumpkin, and watermelon from Blaine, Jefferson, and Tulsa counties were positive serologically to TRSV. At least one to two samples from each representative cucurbit collected in the field above were used as a source for mechanical inoculation. Sap was extracted from symptomatic leaves using 0.1 M K2HPO4 buffer (pH 7.2) and rub-inoculated to two squash (cv Elite) seedlings at cotyledonary stage pre-dusted with Carborundum. Seven to 10 days post-inoculation, all inoculated plants produced typical TRSV symptoms including chlorotic spots, systemic ringspot, severe leaf deformation, mottling, and stunting. Sap and total RNA was extracted from 10 mechanically inoculated squash seedlings and tested by DIBA and reverse transcription (RT)-PCR using specific TRSV primers (F: 5'-TACAGTGAGGATGCATG-3' and R: 5'-AGTAGCTGCGACAAGCCA-3'). All of the tested samples were positive by DIBA except the negative control. Similarly, all samples from mechanically inoculated plants were also positive by PCR showing the expected 1,039-bp PCR product when analyzed by agarose gel electrophoresis. Total RNA obtained from mock-inoculated squash seedlings used as a control was negative by PCR. Amplified PCR product (1,039 bp) was directly sequenced from three infected squash seedlings. Sequence analysis confirmed that the virus shared 90 to 92% nucleotide and 94% amino acid identities with RNA2 of TRSV isolate from the U.S. (Accession No AY363727) available in the GenBank database. Total RNA extracted from original tissues of 14 DIBA positive samples collected from field were also positive by RT-PCR. The presence of TRSV could pose a serious threat to many vegetable crops, particularly cucurbits and other agricultural crops, due to its wide host range (3). This report confirms the suspected occurrence of TRSV in 1956 from watermelon in Oklahoma (4). References: (1) Ali et al. Plant Dis. 96:243, 2012 (2) A. Ali and J. W. Randles. Plant Dis. 81:343, 1997 (3) M. J. Adams and J. F. Antoniw. Outlooks Pest Manage. 16:268, 2005 (4) R. J. Shephered and F. B. Struble. Phytopathology 46:358, 1956.

First Report of Fusarium Wilt Caused by Fusarium oxysporum f. sp. niveum Race 2 in Georgia Watermelons.

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is the number one specialty crop grown in Georgia, a state that ranks fourth nationally in watermelon production. In the last 5 years, Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) has been the greatest yield-limiting disease of watermelon in Georgia. In 2004, a seedless-watermelon field of 'Regency' and 'Tri-X 313' in Berrien County, GA exhibited approximately 40% of wilted plants. Affected plants also exhibited strong discoloration in the crown xylem. Plant samples (cultivars unknown) from a similarly affected field were also tested from Crisp County, GA. Xylem tissue was excised from the main stem of eight diseased plants in the area between the second and third internode, surface sterilized for 1 min in 1% NaOCl, rinsed with 80% ethanol, and plated onto water agar amended with 100 µg/liter of streptomycin sulfate. Fungi with the morphological characteristics of Fusarium oxysporum (4) were consistently recovered from the diseased tissue of all eight plants. The isolates were hyphal tipped and maintained in vials of sterile artificial potting mix until ready for use (1). Isolates were grown on Esposito and Fletcher medium (2) for 10 days, filtered through cheesecloth, and adjusted to 1 × 106 spores/ml. Reference isolates of race 1 and 2 were used as comparisons for race determination of the unknowns. In each of four studies, plants at the two-leaf stage were removed from potting mix, washed gently, and their roots were uniformly trimmed to 2.5 cm. Before repotting, the seedlings were subjected to a 2-min root-dip in the respective spore-containing media. In each study, approximately 40 plants of each watermelon differential were inoculated with the respective isolates. In disease scoring, each plant was considered a rep. 'Black Diamond' is susceptible to races 0, 1, and 2; 'Charleston Gray' is resistant to race 0; 'Calhoun Gray' is resistant to races 0 and 1, and PI-296341-FR (3) is resistant to races 0, 1, and 2 of Fon. Four plants were planted per 15-cm plastic pot, maintained in an air-conditioned headhouse for 24 h, and then placed in the greenhouse in a randomized complete block design. After 30 days, all plants were rated as to healthy, wilted, or dead plants. From eight isolates tested, one isolate from each county was determined to be Fon race 2 on the basis of its ability to wilt/kill a high percentage of the race 1 resistant differential, i.e., 'Calhoun Gray'. Mean disease percentages for the isolates from each of the two counties on the watermelon differentials were 95 and 100% on 'Black Diamond', 68 and 80% on 'Charleston Gray', and 70 and 86% on 'Calhoun Gray.' Because of apparent genetic drift within our PI-296341-FR population, we determined that these data were not useful for identifying race 2. In fact, we observed a range of 17 to 80% wilt/death in the PI-296341-FR over a total of four studies that included a known race 2 isolate (Calg 13(15); E. Vivoda). To our knowledge, this is the first report of race 2 in Georgia and it increases the number of states to seven in which race 2 has been identified. Five of the top 10 watermelon-producing states have now reported race 2 of Fon for which there is no genetic resistance within commercial cultivars. References: (1) B. D. Bruton et al. Plant Dis. 84:907, 2000. (2) R. Esposito and A. Fletcher. Arch. Biochem. Biophys. 93:369, 1961. (3) R. D. Martyn and D. Netzer. HortScience 26:429, 1991. (4) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, 1983.

Rapid method for total carotenoid detection in canary yellow-fleshed watermelon.

Yellow-fleshed watermelons (Citrullus lanatus[Thunb.] Matsum. and Nakai) contain many different carotenoids, all in low to trace amounts. Since there is not 1 predominant carotenoid in yellow-fleshed watermelon, testing the total carotenoid content among watermelon lines is important in determining the antioxidant potential and thus potential health benefits of different varieties. Unfortunately, current methods to assay total carotenoid content are time consuming and require organic solvents. This report describes a rapid and reliable light absorption method to assay total carotenoid content for yellow-fleshed watermelon that does not require organic solvents. Light absorption of 78 watermelon flesh purees was measured with a diode array xenon flash spectrophotometer that can measure actual light absorption from opaque samples; results were compared with a hexane extraction method. The puree absorbance method gave a linear relationship (R(2)= 0.88) to total carotenoid content and was independent of watermelon variety within the total carotenoid concentration range measured (0 to 7 mug/g fresh weight).

First Report of Verticillium Wilt of Watermelon in the Texas High Plains.

Verticillium dahliae (Kleb.) is known worldwide as a destructive soilborne pathogen with a wide host range (2). Reports of V. dahliae attacking cucurbits are generally limited to 'Casaba' and 'Persian' type melons. During August and September of 2004 to 2006, fields of seedless watermelon (Citrullus lanatus [Thunb.] Matsum. & Nak.) and pollinators in Yoakum County, Texas, exhibited severe symptoms of vine decline. There was no apparent difference between diploid and triploid watermelon cultivars. Night-time temperatures during July, August, and September averaged 20°C or less. Losses were estimated in excess of one-half million dollars. Symptoms consisted of leaf yellowing, wilting, and gradual death of the leaves, but stems generally remained green. The xylem exhibited a uniform tan-to-light brown discoloration that often extended throughout the vine. Dead plants had numerous microsclerotia embedded throughout the root and crown. Crown and root sections (1 cm long) from triploid plants were surface disinfected in 0.5% NaOCl for 30 s, transferred to water agar with 100 ppm of streptomycin sulfate, and incubated at 25°C. Slow-growing colonies were transferred to potato dextrose agar after approximately 72 h. V. dahliae was identified on the basis of morphology (3). Pathogenicity of four selected isolates was determined on the watermelon cultivars used to identify races of Fusarium oxysporum f. sp. niveum (Fon). Flasks containing 100 ml of medium (1) were inoculated with a 1-ml spore suspension at 1 × 105 spores/ml for each isolate and placed on an orbital shaker for 6 days at 100 rpm with continuous near-UV/fluorescent lighting at 25°C. Roots of approximately 40 plants of each of five watermelon cultivars (1 to 2 true-leaf stage) were trimmed to 2 cm long and root dipped for 2 min in the spore suspension (1 × 106/ml) of each isolate. Each cultivar/isolate combination and controls were transplanted into 10 pots (1.5 liter) with four plants per pot. The pots were transferred to the greenhouse where soil temperatures ranged between 15 and 25°C and were fertilized (Jack's fertilizer solution) every 7 days. Plants were rated at the end of 28 days as 1 = healthy, 2 = stunting (≤50% of controls), 3 = wilting, and 4 = dead. Initial wilting was observed within 7 to 10 days postinoculation. All four isolates caused varying degrees of vascular discoloration, stunting, wilting, and plant death. The pathogen was reisolated from symptomatic plants but not the controls. Mean disease ratings for the most virulent Texas isolate (28-040215) on 'Black Diamond', 'Charleston Gray', 'Dixie Lee', 'Calhoun Gray', and 'PI 296341 FR' were 2.7, 3.0, 3.0, 2.9, and 2.9, respectively. All watermelon Fon differentials were equally susceptible to V. dahliae in these studies. Historically, Verticillium wilt has been a problem in this area, which has been in cotton production for approximately 100 years. In the past decade, watermelon production has increased substantially to approximately 3,600 ha in the Texas High Plains. To our knowledge, this is the first known report of Verticillium wilt on watermelon in Texas. References: (1) R. G. Esposito and A. M. Fletcher. Arch. Biochem. Biophys. 93:369, 1961. (2) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, New York, 2002. (3) H. C. Smith. N. Z. J. Agr. Res. 8:450, 1965.

Spinal cord injury and snowboarding--the British Columbia experience.

Snowboarding is an alpine sport growing in popularity, particularly among male youth. This study of 10 consecutive cases admitted to the Vancouver Hospital and Health Sciences Centre Acute Spinal Cord Injury Unit, over the 1997 to 1998 winter season, analyzes the epidemiology of snowboarding spinal injury. Information was collected retrospectively on the mechanism, location, and pattern of injury, and personal details of the individuals who suffered the injuries. The average age at time of injury was 22.4 years, with a range of 16 to 29. All but 1 of the cases were self-acknowledged expert-level snowboarders, with an average of 6.25 years experience. Nine of the injured were male. There was only 1 cervical injury, with the majority being low thoracolumbar, and 4 incidences of L-1 fracture. Compression and burst fracture were the predominant vertebral fracture patterns and there was a 50% incidence of significant neurologic injury and deficit. The most common mechanism of injury was axial loading following a failed jump or fall from a height, varying from 2 to 25 feet. The lack of associated injuries in 9 of the cases suggests a limited ability of the extremities to offset such falls. Contributing factors included the inherent riskiness of the sport, participant characteristics, lack of formal instruction, and self-constructed jumps. Poor weather conditions, unfamiliarity with a run, collisions, and consumption of alcohol also played lesser roles. The frequent association between spinal fracture and significant neurologic deficit in this group has not previously been reported in other studies.

Isolation and partial amino acid sequence of three subunit species of porcine spleen ferritin: evidence of multiple H subunits.

A partial amino acid sequence for three different subunits of the iron storage protein, ferritin, has been determined. Ferritin (Mr approximately 480,000) was isolated from porcine spleen and dissociated into its component subunits (Mr approximately 20,000). The subunits, in turn, were separated into three fractions by reversed-phase HPLC. The fractions appeared to be of equal size by sedimentation velocity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and size-exclusion chromatography in 6 M guanidinium chloride. All three fractions were shown to be monomeric and to have no covalently attached carbohydrate (J. F. Collawn et al. (1984) Arch. Biochem. Biophys. 233, 260-266). Determination of the amino acid sequence of the C-terminal 70-80 residues from each of the fractions demonstrated three different sequences. Comparison with human liver H and L subunit sequences indicates that two of the porcine ferritin subunits are H-type subunits and one is an L-type subunit. Application of the Chou-Fasman algorithm on the three partial sequences suggests that these respective regions from each of the three subunits would probably adopt the same conformation.

Galactosyl-binding lectins from the tunicate Didemnum candidum. Purification and physicochemical characterization.

The plasma of the ascidian Didemnum candidum possesses lectin activity directed toward galactosyl moieties. We report the purification by affinity chromatography, the physicochemical properties, amino acid composition, and partial N-terminal amino acid sequence of two galactosyl-binding lectins D. candidum lectins I and II (DCL-I and DCL-II) from the plasma of this protochordate species. Both lectins were purified by affinity chromatography (on acid-treated Sepharose 4B and asialofetuin conjugated to Sepharose 4B) to homogeneity as judged by immunoelectrophoresis, size exclusion chromatography on high performance liquid chromatography, and polyacrylamide gel electrophoresis. Isoelectric focusing in polyacrylamide gels revealed that DCL-I focuses as a family of bands at pH 3.8-5.2, while DCL-II focuses at pH 9.2-10.2. Gas chromatography analyses of alditol acetate derivatives indicated that no carbohydrate components are associated with the lectins. Approximate subunit molecular weights estimated by polyacrylamide gel electrophoresis and size exclusion chromatography on high performance liquid chromatography in 6 M guanidine HCl under reducing conditions were 13,400-14,500 for DCL-I and 14,500-15,500 for DCL-II. Native molecular weights estimated by sedimentation equilibrium were 56,600 (DCL-I) and 57,500 (DCL-II), indicating that both species are constituted by four equal-sized subunits. Frictional ratios suggested that both lectins are globular proteins. Using rabbit antisera, the two molecules appeared serologically distinct. The extinction coefficient for DCL-I was E280 mg/ml = 2.52 ml mg-1 cm-1. Circular dichroism analyses of DCL-I suggested 29% alpha-helix and 37% beta-structure in the protein. Excitation/emission fluorescence spectra for DCL-I yielded maximum excitation and emission wavelengths at 288 and 330 nm, respectively. Amino acid compositions of DCL-I and DCL-II differed mainly in the proportions of aspartic and glutamic acids, serine, alanine, cysteine, valine, phenylalanine, and histidine. Amino acid compositions of DCL-I and DCL-II were compared to each other and to immunoglobulins and putative recognition molecules by the parameter S delta Q. DCL-I exhibited similarities in amino acid composition to lectins from the tunicate Halocynthia pyriformis, the lamprey Petromyzon marinus, and the horseshoe crab Carcinoscorpius rotundicauda, rabbit C-reactive protein, and lamprey and carp immunoglobulin mu chains. DCL-II showed amino acid composition and similarities with several fish immunoglobulin light chains, immunoglobulin-related molecules isolated from mouse and marmoset T cells, and carp and goldfish immunoglobulin heavy chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that H-enriched human placental ferritin is structurally similar to L-enriched ferritins of other tissues.

Ferritin was purified from normal full-term placenta, and the native structure and subunit composition were characterized. Reversed-phase high-performance liquid chromatographic analysis of the placental ferritin subunits suggested the presence of three subunit types. Using acid urea gel electrophoresis and amino acid analysis, these subunits were tentatively identified as two H-type and one L-type. The relative proportions of the subunit types were approx. 23% H-1, 33% H-2 and 44% L. The native structure of placental ferritin as judged by circular dichroism and fluorescence spectroscopy was quite similar to that of ferritin isolated from horse spleen, a source that is composed predominantly of L subunits. These results are consistent with a ferritin tetracosameric structure whose H and L subunits fit into 24 equivalent sites interchangeably because the secondary and tertiary structures of the two subunit types are very similar.

Arginine residues are critical for the heparin-cofactor activity of antithrombin III.

A dilution/quench technique was used to monitor the time course of chemical modification on the heparin-cofactor (a) and progressive thrombin-inhibitory (b) activities of human antithrombin III. Treatment of antithrombin III (AT III) with 2,4,6-trinitrobenzenesulphonate at pH 8.3 and 25 degrees C leads to the loss of (a) at 60-fold more rapid rate than the loss of (b). This is consistent with previous reports [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Pecon & Blackburn (1984) J. Biol. Chem. 259, 935-938] that lysine residues are involved in the binding of heparin to AT III, but not in thrombin binding. Treatment of AT III with phenylglyoxal at pH 8.3 and 25 degrees C again leads to a more rapid loss of (a) than of (b), with the loss of the former proceeding at a 4-fold faster rate. The presence of heparin during modification with phenylglyoxal significantly decreases the rate of loss of (a). Full loss of (a) correlates with the modification of seven arginine residues per inhibitor molecule, whereas loss of (b) does not commence until approximately four arginine residues are modified and is complete upon the modification of approximately eleven arginine residues per inhibitor molecule. This suggests that (the) arginine residue(s) in AT III are involved in the binding of heparin in addition to the known role of Arg-393 at the thrombin-recognition site [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Jörnvall, Fish & Björk (1979) FEBS Lett. 106, 358-362].

Hydrogen-tritium exchange by apoferritin and ferritin.

The out-exchange kinetics of tritium from apoferritin, ferritin of various iron contents, and apoferritin subunits were examined. The exchange kinetics indicated no detectable conformational differences in the tetracosamer with and without hydrous ferric oxide in the internal cavity of the molecule. The data for apoferritin subunits were markedly different from those for the tetracosameric state. The exchange kinetics for apoferritin were consistent with a rapid exchange of water between the internal cavity of the protein and the bulk solvent outside the protein shell.

Denaturation behavior of antithrombin in guanidinium chloride. Irreversibility of unfolding caused by aggregation.

The structural stability of the protease inhibitor antithrombin from bovine plasma was examined as a function of the concentration of guanidinium chloride (GdmCl). A biphasic unfolding curve at pH 7.4, with midpoints for the two phases at 0.8 and 2.8 M GdmCl, was measured by far-ultraviolet circular dichroism. Spectroscopic and hydrodynamic analyses suggest that the intermediate state which exists at 1.5 M GdmCl involves a partial unfolding of the antithrombin molecule that exposes regions of the polypeptide chain through which slow, intermolecular association subsequently takes place. The partially unfolded molecule can be reversed to its fully functional state only before the aggregation occurs. Upon return of the aggregated state to dilute buffer, the partially unfolded antithrombin remains aggregated and does not regain the spectroscopic properties, thrombin-inhibitory activity, or heparin affinity of the native inhibitor. This behavior indicates that the loss of the functional properties of the proteins is caused by the macromolecular association. Comparative experiments gave similar results for the human inhibitor. Analyses of bovine antithrombin in 6 M GdmCl indicated that the second transition reflects the total unfolding of the protein to a disulfide-cross-linked random coil. This transition is spectroscopically reversible; however, on further reversal to dilute buffer, the molecules apparently are trapped in the partially unfolded, aggregated, intermediate state. The results are consistent with the existence of two separate domains in antithrombin which unfold at different concentrations of GdmCl but do not support the contention that the thrombin-binding and heparin-binding regions of the protein are located in different domains [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053].

A comparison by HPLC of ferritin subunit types in human tissues.

Ferritin was isolated from human liver and spleen. Reversed phase high performance liquid chromatography of the ferritin subunits from each tissue yielded the same three chromatographic fractions. Physical and chemical characterization of the three fractions indicated that they represented at least two, perhaps three, chemically distinct subunits.

Anomalous behavior of goldfish IgM heavy chain in sodium dodecylsulfate polyacrylamide gel electrophoresis.

By sodium dodecylsulfate polyacrylamide gel electrophoresis, the heavy chain of the serum immunoglobulin (IgM) of the goldfish (Carassius auratus) differs not only from other studied vertebrate serum IgM heavy chains, but also from other vertebrate lymphocyte membrane IgM heavy chains including those from the goldfish itself. This difference, an increase in apparent Mr of approximately 5000, was investigated by assessing in comparison with the IgM heavy chain of human and rainbow trout (Salmo gairdneri) the following properties: (1) molecular size by gel filtration in denaturing buffers; (2) carbohydrate content, by direct analysis; (3) intrinsic net charge, by isoelectric focusing; (4) net hydrophobicity, deduced from amino acid analysis; and (5) sodium dodecylsulfate binding by direct measurement. Results indicate that goldfish IgM heavy chain is indistinguishable from other IgM heavy chains in terms of (a) its gel-filtration behavior in denaturing conditions, (b) its carbohydrate content (which is similar to trout IgM heavy chain) and (c) its intrinsic net charge and hydrophobicity. However, goldfish IgM does differ from the other proteins studied in its detergent-binding ability and it is this behavior that is concluded to be the cause of its unusual mobility in sodium dodecylsulfate polyacrylamide gel electrophoresis.

A chemical and physical comparison of ferritin subunit species fractionated by high-performance liquid chromatography.

Selected chemical and physical properties were measured for different forms of ferritin subunits which had been separated by reverse-phase high-performance liquid chromatography. Ferritin subunits from porcine spleen behaved, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as though they were approximately Mr 2000 larger than equine spleen ferritin, whereas no difference in size was observed by gel chromatography in 6 M guanidinium chloride. All subunit species exhibited similar isoelectric focusing properties. In contrast to previous reports, no carbohydrate could be found associated with any of the isolated subunit species. Thus, the aberrant behavior of the porcine ferritin subunits between the two empirical molecular weight estimation methods appears to be the result of factor(s) other than protein intrinsic charge or covalently attached carbohydrate.

The distribution of ferritin subunit types in porcine tissues.

Ferritin was isolated from porcine heart, liver and spleen. Reversed phase high performance liquid chromatography of the ferritin subunits yielded three chromatographic fractions. The relative proportions of the three chromatographic fractions were different for each tissue ferritin. These results support the model which proposes a combination of (at least) two subunit types as the basis for the existence of isoferritins.

Rapid molecular weight estimation and separation of selected immunoglobulin chains by high speed gel filtration.

Rapid and reliable molecular weight estimations of reduced and alkylated immunoglobulin heavy or light chains were performed by high speed gel filtration in 6 M guanidinium chloride using a short (30 cm X 7.5 mm) TSK 3000 SW type column. Molecular weight estimations based on Kav values of eluted polypeptides and glycopolypeptides were generally unaffected by protein bound carbohydrate. Rapid separation of immunoglobulin H and L chains was also achieved during high speed gel filtration.

A single column method to measure hydrogen--tritium exchange by gel chromatography.

Employment of a commercially integrated gel chromatography system together with the utilization of cross-linked polyacrylamide as the chromatographic medium simplifies the methodology of hydrogen--tritium exchange measurements. The system described allows the execution of hydrogen--tritium exchange measurements with as little as 0.5 mg protein per time point and with only a single pass of sample through the column for out-exchange times of less than 1 min to at least 24 h. The accuracy and precision of this system are comparable to those of existing methodologies.