Genome-wide association study and genomic prediction of white rust resistance in USDA GRIN spinach germplasm.

White rust, caused by Albugo occidentalis, is one of the major yield-limiting diseases of spinach (Spinacia oleracea) in some major commercial production areas, particularly in southern Texas in the United States. The use of host resistance is the most economical and environment-friendly approach to managing white rust in spinach production. The objectives of this study were to conduct a genome-wide associating study (GWAS), to identify single nucleotide polymorphism (SNP) markers associated with white rust resistance in spinach, and to perform genomic prediction (GP) to estimate the prediction accuracy (PA). A GWAS panel of 346 USDA (US Dept. of Agriculture) germplasm accessions was phenotyped for white rust resistance under field conditions and GWAS was performed using 13 235 whole-genome resequencing (WGR) generated SNPs. Nine SNPs, chr2_53 049 132, chr3_58 479 501, chr3_95 114 909, chr4_9 176 069, chr4_17 807 168, chr4_83 938 338, chr4_87 601 768, chr6_1 877 096, and chr6_31 287 118, located on chromosomes 2, 3, 4, and 6 were associated with white rust resistance in this GWAS panel. Four scenarios were tested for PA using Pearson's correlation coefficient (r) between the genomic estimation breeding value (GEBV) and the observed values: (1) different ratios between the training set and testing set (fold), (2) different GP models, (3) different SNP numbers in three different SNP sets, and (4) the use of GWAS-derived significant SNP markers. The results indicated that a 2- to 10-fold difference in the various GP models had similar, although not identical, averaged r values in each SNP set; using GWAS-derived significant SNP markers would increase PA with a high r-value up to 0.84. The SNP markers and the high PA can provide valuable information for breeders to improve spinach by marker-assisted selection (MAS) and genomic selection (GS).

Three New Fungal Leaf Spot Diseases of Spinach in the United States and the Evaluation of Fungicide Efficacy for Disease Management.

Leaf spot diseases of spinach, caused by Colletotrichum spinaciae, has become a major production constraint in several production areas, including Texas, in recent years. Leaf spot symptoms were observed in several fields in Texas in 2016 and 2017, with typical anthracnose-like symptoms and leaves with small, circular, and sunken lesions that appeared similar to injury from windblown sand. The lesions were plated on potato dextrose agar, from which fungal cultures were recovered. The fungi were identified based on morphology and sequence analysis of the introns of glutamate synthetase and glyceraldehyde-3-phosphate dehydrogenase (for isolates determined to be Colletotrichum spp.) and the internal transcribed spacer ribosomal DNA (for isolates determined to be Myrothecium spp.). Based on foliar symptoms, fungal colony and spore morphology, pathogenicity tests of fungal isolates on the spinach cultivar 'Viroflay', and DNA sequence analysis of the isolates, the symptoms on spinach leaves for two sets of samples were caused by Colletotrichum coccodes and Colletotrichum truncatum, and leaf spots resembling damage from windblown sand were caused by Myrothecium verrucaria. This is the first report of spinach leaf spot diseases caused by C. coccodes, C. truncatum, and M. verrucaria in the United States. C. coccodes and C. truncatum caused severe symptoms on the spinach cultivar 'Viroflay', whereas M. verrucaria caused symptoms of intermediate severity. Fungicide efficacy tests demonstrated that chlorothalonil, mancozeb, pyraclostrobin, fluxapyroxad + pyraclostrobin, and penthiopyrad were completely effective at preventing leaf spots caused by any of these pathogens when applied 24 h before inoculation of 'Viroflay' plants in greenhouse trials.

Characterization of Leaf Spot Pathogens from Several Spinach Production Areas in the United States.

Leaf spot diseases have become a major concern in spinach production in the United States. Determining the causal agents of leaf spots on spinach, their prevalence and pathogenicity, and fungicide efficacy against these pathogens is vital for effective disease management. Spinach leaves with leaf spots were collected from Texas, California, Arizona, and South Carolina from 2016 to 2018, incubated in a moist chamber, and plated on potato dextrose and tryptic soy agar media. Fungal and bacterial colonies recovered were identified based on morphology and sequence analysis of the internal transcribed spacer rDNA and 16S rRNA, respectively. Two predominant genera were isolated: (i) Colletotrichum spp., which were identified to species based on sequences of both introns of the glutamate synthetase (GS-I) and glyceraldehyde-3-phosphate dehydrogenase (gapdh-I) genes; and (ii) Stemphylium spp., identified to species based on sequences of the gapdh and calmodulin (cmdA) genes. Anthracnose (Colletotrichum spinaciae) and Stemphylium leaf spot (Stemphylium vesicarium and S. beticola) were the predominant diseases. Additional fungi recovered at very limited frequencies that were also pathogenic to spinach included Colletotrichum coccodes, C. truncatum, Cercospora beticola, and Myrothecium verrucaria. All of the bacterial isolates were not pathogenic on spinach. Pathogenicity tests showed that C. spinaciae, S. vesicarium, and S. beticola caused significant leaf damage. The fungicides Bravo WeatherStik (chlorothalonil), Dithane F-45 (mancozeb), Cabrio (pyraclostrobin), and Merivon (fluxapyroxad and pyraclostrobin) were highly effective at reducing leaf spot severity caused by an isolate of each of C. spinaciae and S. vesicarium, when inoculated individually and in combination.

The Effects of a Crosslinked, Modified Hyaluronic Acid (xCMHA-S) Gel on Equine Tendon Healing.

OBJECTIVE: To assess the effects of a crosslinked, modified hyaluronic acid (xCMHA-S) gel on equine tendon healing using an in vivo surgical model. STUDY DESIGN: In vivo experimental study. ANIMALS: Adult horses (n = 5). METHODS: Full thickness bilateral forelimb window tenectomies were surgically created in both forelimb superficial digital flexor tendons and xCMHA-S gel was implanted intraoperatively into the right forelimb lesion of each horse whereas the left forelimb served as the untreated control. Healing was monitored by serial ultrasound examinations every 14 days over the course of the 84 day study. In addition, gross pathology, scanning electron microscopy for fiber diameter, and histological scoring were completed on tendon samples harvested after euthanasia at 84 days. RESULTS: Ultrasound assessment demonstrated a significant decrease in mean lesion size of treated (0%) compared to control (30%) tendons at 84 days. Mean (±SD) cumulative histologic tendon scores for control tendons (17.7 ± 2.7) were significantly higher than treated tendons (13.6 ± 1.9), indicating less advanced healing in the control group. Tendon cell density was increased and neovascularization, intensity of inflammation, and uniformity of fiber diameter were increased in control compared to treated tendons. There were no differences in fibroblast shape, levels of intralesional hemorrhage, linearity of collagen fibers, or collagen fiber diameter or distribution between treated and control tendons. CONCLUSION: Tendons treated with xCMHA-S gel at the time of model induction had superior histologic healing scores and sonographically smaller lesions compared to controls, suggesting that xCMHA-S gel may aid the natural healing process.

The role of ciprofloxacin in prolonging polyethylene biliary stent patency: a multicenter, double-blinded effectiveness study.

Plastic stents are the mainstay of the palliation of malignant jaundice but are complicated by recurrent obstruction. Previous trials have failed to demonstrate any improvement in patency with the use of antibiotics. Patients with malignant jaundice were randomized in a double-blind fashion, after polyethylene stent insertion, to receive ciprofloxacin or placebo. After successful stent decompression, there were 50 patients in the treatment arm and 44 in the placebo. There were 14 (33%) episodes of stent occlusion in the ciprofloxacin group versus 23 (49%) in placebo (chi(2) test, P=0.115). There was no significant difference in patency (log-rank test, P=0.17). There were significantly fewer episodes of cholangitis with ciprofloxacin: 10 (23%) versus 21 (42%) in the placebo (P=0.047). The ciprofloxacin group also demonstrated a significant improvement in the Social Function domain of the SF-36 Quality of Life Survey at 1 month (paired T test, P=0.03). The other domains of the SF-36 were not different, nor was survival (log rank, P=0.80). There is insufficient evidence to show that prophylactic ciprofloxacin can prolong plastic biliary stent patency. The observed trends suggest that ciprofloxacin significantly decreases the incidence of cholangitis and results in improvements in certain aspects of quality of life.

Quantitative effect of tenorrhaphy on intrinsic vasculature of the equine superficial digital flexor tendon.

OBJECTIVE: To compare effects of the locking-loop suture pattern (LLP) and 3-loop pulley (3LP) suture pattern for tenorrhaphy on the intrinsic vasculature of the superficial digital flexor tendon (SDFT) of horses in vitro after surgery. SAMPLE POPULATION: 16 forelimbs obtained from 8 mature horses. PROCEDURE: Tenotomy and subsequent tenorrhaphy was performed in anesthetized horses. Following systemic administration of heparin, horses were euthanatized and the limbs were removed and placed under tension to load the flexor tendons. The intrinsic vasculature was then perfused with a mixture of barium sulfate and water. Four-millimeter sections of the SDFT were prepared for microangiographic analysis. Mean vessel density was calculated for each section by use of a grid consisting of 1.5-mm2 vascular assessment squares (VAS). Comparisons were made among the control, LLP and 3LP groups. RESULTS: Mean +/- SD vessel density was 3.11 +/- 0.38, 1.47 +/- 0.47, and 2.01 +/- 0.63 perfused vessels/1.5 mm2 for control, LLP and 3LP groups, respectively. Significant differences in vascular density were detected between the control and 3LP groups, control and LLP groups, and LLP and 3LP groups. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the LLP and 3LP pattern has deleterious effects in vitro on the intrinsic vasculature of the SDFT. However, the 3LP pattern was less disruptive to the intrinsic vasculature, compared with the effects for the LLP. Use of the 3LP tenorrhaphy suture pattern in clinical situations may result in less damage to the intrinsic vasculature of the SDFT of horses during convalescence.

Healing characteristics of deep digital flexor tenorrhaphy within the digital sheath of horses.

OBJECTIVE: To describe the healing characteristics of deep digital flexor tenorrhaphy within the digital sheath. STUDY DESIGN: Experimental study. ANIMALS: Five mature horses. METHODS: Right thoracic limb, deep digital flexor tenorrhaphy was performed within the digital sheath. Limbs were cast in partial flexion using a short limb cast for 6 weeks. Next, extended heel shoes were used for limb support for 14 weeks. Healing was evaluated by sequential ultrasonographic examinations, and limb use was evaluated by force plate analysis. At 26 weeks, mechanical strength and morphologic characteristics of the repair site were evaluated. RESULTS: Gap (mean, 0.93 cm.) formation was evident in unloaded limbs at 3 weeks. This increased markedly by 6 weeks and was 5 cm at 26 weeks. Demarcation between the deep and superficial flexor tendons decreased as the transected ends adhered to the dorsal surface of the superficial flexor tendon. The intrathecal space was reduced by fibrous tissue. Mean maximum load to failure of the repair tissue was 4,616 +/- 3,556 N, with a mean stress of 12.99 +/- 2.78 MPa. The repair consistently failed at the adhesion between the transected tendon and the superficial flexor tendon. CONCLUSIONS: Intrathecal tenorrhaphy with external coaptation (in partial limb flexion) for 6 weeks resulted in gap healing, fibrous adhesion between the deep and superficial flexor tendons, fibrous tissue reduction of the intrathecal space, and a pasture-sound horse at 26 weeks. CLINICAL RELEVANCE: Without improved methods for immobilizing the deep digital flexor tendon, intrathecal tenorrhaphy is unlikely to result in first intention tendon healing.

An evaluation of two autologous tendon grafting techniques in ponies.

OBJECTIVE: To compare the healing and mechanical strength of a multiple split autologous tendon graft (MG) to a whole autologous tendon graft (WG) in the deep digital flexor tendon of ponies. STUDY DESIGN: In vitro evaluation of two different tendon-grafting techniques. ANIMALS OR SAMPLE POPULATION: Six ponies of mixed gender and age. METHODS: Tenotomies performed in forelimb deep digital flexor tendons (DDFT) distal to the insertion of the accessory ligament (AL-DDFT) were repaired with free autologous grafts from the hindlimb lateral digital extensor tendon (LDET). Grafts were either whole (WG) or split into three longitudinal strips (multiple graft, MG). Tendons and graft sites were collected and loaded (2.54 cm/s) to failure at either 4 or 8 weeks after surgery. Cross-sectional area was determined by both impression cast (IC) and an inkblot (IB) method. Tissue maturity and inflammation were evaluated by microscopy. RESULTS: Gap formation was a consistent finding in all repair sites. No statistical differences were found in healing or mechanical variables between MG and WG techniques. The failure stress for the 8-week repairs (15.51 +/- 3.1 MPa IB and 11.73 +/- .77 MPa IC, 16.13 +/- 2.2 MPa IB and 10.22 +/- .76 MPa IC for MG and WG, respectively) were significantly greater (P <.0005) than for 4-week repairs (3.71 +/- 1.7 MPa IB and 2.68 +/- 1.44 MPa IC, 2.81 +/- 1.46 MPa IB and 2.3 +/- 1.7 MPa IC for MG and WG, respectively). The repair tissue was more mature (P <.05) at 8 weeks than at 4 weeks, but there was no significant difference in inflammatory responses at 4 and 8 weeks. CONCLUSIONS: There was a sixfold increase in strength between 4 and 8 weeks of healing, but no significant difference in healing or strength between the MG and WG techniques. CLINICAL RELEVANCE: In ponies, autologous tendon grafting contributes to a strong repair during the early convalescent period, but splitting a tendon graft seemingly offers no appreciable advantage over use of a whole graft.