Development of colchicine‐induced tetraploid St. Augustinegrass (Stenotaphrum secundatum) lines

St. Augustinegrass is well suited for lawns and commercial landscapes. While many genotypes are cross‐fertile, all cultivars are propagated vegetatively in sod production. To ensure varietal purity, development of sterile triploid hybrids by crossing tetraploid and diploid genotypes has been successfully used in other warm‐season turfgrasses. Applying this model in St. Augustinegrass would be beneficial to sod producers and turf managers who require purity for certification and uniformity for performance, respectively. This study was conducted to develop colchicine‐induced tetraploid lines of St. Augustinegrass. Seeds of cultivar ‘Raleigh’ were treated with four colchicine concentrations at four exposure times. A non‐treated control was included among the treatments. Seedlings that germinated were screened for genome size changes using flow cytometry. Line DSA 13005 and two progeny lines derived through selfing, DSA 16001 and DSA 16016, were corroborated as tetraploids (2n = 4x = 36) through chromosome counts. These lines will be used in future breeding efforts to attempt development of sterile triploid cultivars of St. Augustinegrass.     

Evaluation of Olive Cultivars for Resistance to Verticillium dahliae

Resistance of 23 important olive cultivars to Verticillium dahliae has been evaluated in four experiments under controlled conditions. Nine-month-old nursery olive plants were inoculated with a cotton non-defoliating (ND) (V4) or a cotton defoliating (D) (V117) isolate of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves. The percentage of plants killed and of those which recovered from the disease were used as additional parameters for including a particular cultivar into a defined category. Most of the evaluated cultivars were susceptible, although at different levels, to both isolates of V. dahliae. All cultivars were more susceptible to the D pathotype than to the ND one. A group of 11 cultivars, including several important Spanish cultivars, were susceptible or extremely susceptible to both pathotypes of V. dahliae. A second group showed differences of resistance depending on the pathotype used. They were susceptible or extremely susceptible to the D pathotype but resistant or moderately susceptible to the ND one. Finally, 'Frantoio', 'Oblonga' and 'Empeltre' were moderately susceptible to the D isolate of V. dahliae and resistant to the ND one. The resistance of 'Empeltre' was evident by the plant ability to recover from infection with either isolates. 'Empeltre' is considered to be a valuable cultivar for inclusion in breeding programmes for resistance to Verticillium wilt.     

Assessing freeze-tolerance in St. Augustinegrass: temperature response and evaluation methods

Winter hardiness is a major-limiting factor for St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] grown in the transitional climatic region of the United States. Lab-based freeze tests that mimic the range of field winter survivability in St. Augustinegrass can contribute to the selection of cold hardy genotypes. This study used a whole container method, four freezing temperatures, and two data collection systems to evaluate the freezing response of nine St. Augustinegrass genotypes ranging in their winter hardiness. Results indicated ?3 and ?4 °C with average regrowth ratings of 33.6 and 17.8% respectively, were more suitable temperatures for evaluating freeze survival in St. Augustinegrass than ?5 and ?6 °C with average regrowth ratings of 0.4 and 0%, respectively. Visual ratings of surviving green tissue and regrowth were generally well correlated when evaluated over a six week period post-freeze with Pearson correlation coefficients ranging of 0.17–0.62  for ?3 °C freeze tests and 0.79–0.93 for ?4 °C freeze tests. Additionally, measurement of percent green cover using digital imaging techniques commonly utilized in turfgrass field studies were significantly correlated (0.66) with visual ratings averaged across weekly post-freeze evaluation measurements for both ?3 and ?4 °C freezing temperatures. These results provide evidence that digital imaging analyses are useful in estimating surviving green tissue and regrowth in lab-based freeze tests. This study provides additional information regarding freezing temperatures, genotype responses, and data collection methods in St. Augustinegrass, which should aid breeders in the improvement of freeze tolerance in the species.     

Use of dimethylformamide to cryopreserve alpaca semen previously incubated with collagenase

The objective of this study was to evaluate the effect of collagenase and two final dimethylformamide (DMF) concentrations (4% and 7%) on alpaca frozen-thawed sperm quality. A total of 25 ejaculates from 5 alpaca were obtained using electroejaculation. Each individual ejaculate was evaluated and then diluted 4:1 in a solution of 1 mg/ml collagenase in HEPES-TALP medium and incubated for 4 min at 37°C. Subsequently, samples were diluted in TRIS-fructose-citric acid-egg yolk and cooled to 5°C. Then, each sample was divided in two aliquots and DMF at final concentration of 4% or 7% was added, equilibrated for 1 hr at 5°C and frozen over liquid nitrogen vapours. A Kruskal–Wallis test was used to evaluate the sperm morphometry, and Completely Random Block designs were used to analyse sperm motility, viability, membrane function and acrosome status. After collagenase incubation, none of the samples showed thread formation, and sperm parameters were preserved. Non-progressive motile sperm were higher (p < .05) in equilibrated samples (4% DMF: 31.8 ± 8.3% and 7% DMF: 36.3 ± 11.8%) compared to raw (10.1 ± 4.3%) and frozen-thawed semen (4% DMF: 9.7 ± 1.8% and 7% DMF: 7.5 ± 3.2%). Sperm membrane function, membrane integrity and intact acrosomes were higher (p < .05) in raw semen (40.1 ± 12.2%, 94.6 ± 3.2% and 91.3 ± 8.1%) compared to frozen-thawed samples (4% DMF: 19.8 ± 4.7%, 53.2 ± 2.7%, 65.7 ± 8.7% and 7% DMF: 20.4 ± 4.5%, 54.1 ± 1.4%, 64.6 ± 9.1%). Length of the sperm head was lower in frozen-thawed samples, being statistically different with 4% DMF compared to pre-freezing samples. The ratio between acrosome and head areas was greater (p < .05) in frozen-thawed samples. Incubation of raw alpaca semen with collagenase decreased the thread formation without affecting sperm quality. Frozen of collagenase treated alpaca semen with 4% or 7% DMF did not preserve the sperm parameters in thawed samples.     

Effects of gamma radiation on the immature stages ofLiriomyza Trifolii

The effects of γ-radiation on the immature stages ofLiriomyza trifolii Burgess were studied with doses of 2000-25 Gy. The high doses were lethal to all stages. At doses of 750 Gy or less, larvae survived to pupate, but their number was reduced and the majority were either dead or deformed and did not give rise to adults. Eggs and prepupae were more susceptible to radiation than other stages. Larval radiosensitivity decreased with progressing instar, and pupal sensitivity decreased with increasing age. Contribution from the Agricultural Research Organization. No. 2922-E, 1990 series.     

Crude protein fractions in common vetch (Vicia sativa L.) fresh forage during pod filling

Crude protein (CP) of forages can be separated into fractions of differentiated abilities to provide available amino acids in the lower gut of ruminants. This knowledge is critical to develop feeding systems and to predict animal responses. We have measured during two growing seasons (1996 to 1997 and 1997 to 1998) the CP fractions of common vetch fresh forage with the objective being to assess the influence of maturity on concentration of CP fractions (as a percentage of total CP) and fraction yields. Fraction B2, which represents true protein of intermediate ruminal degradation rate, was the largest single fraction in common vetch forage (about 40% of CP across seasons and maturity stages). Soluble fractions (A plus B1) were less than 50% of total CP while the unavailable fraction C ranged from 4 to 8% of total CP. As a result, the remaining fraction B3 (true protein of very low degradation rate) only represented 2 to 9% of total CP. Concentration and yield of fraction B3 increased (P < 0.05) from flowering to pod-filling. Results showed that undegraded dietary protein represented a small proportion of total CP in common vetch forage. Moving the harvesting stage from flowering to the pod filling phase allowed for greater yield of undegraded dietary protein.     

Gene discovery in Eimeria tenella by immunoscreening cDNA expression libraries of sporozoites and schizonts with chicken intestinal antibodies

Specific antibodies were produced ex vivo from intestinal culture of Eimeria tenella infected chickens. The specificity of these intestinal antibodies was tested against different parasite stages. These antibodies were used to immunoscreen first generation schizont and sporozoite cDNA libraries permitting the identification of new E. tenella antigens. We obtained a total of 119 cDNA clones which were subjected to sequence analysis. The sequences coding for the proteins inducing local immune responses were compared with nucleotide or protein databases and with expressed sequence tags (ESTs) databases. We identified new Eimeria genes coding for heat shock proteins, a ribosomal protein, a pyruvate kinase and a pyridoxine kinase. Specific features of other sequences are discussed.     

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.