presence in the inoculum. Samples were collected in plastic bags, placed in an ice chest, and finally stored at –4 °C until testing. Samples were analyzed to confirm the presence of IYSV by the ELISA ( Copeland, 1998 ) and RT-PCR ( Cortês et al., 1998 ). In
Mohsen Mohseni-Moghadam, Christopher S. Cramer, Robert L. Steiner and Rebecca Creamer
Parminder S. Multani, Christopher S. Cramer, Robert L. Steiner and Rebecca Creamer
-linked immunosorbent assay (ELISA) optical density with IYSV symptom severity at different times during the crop season. Materials and Methods Plant materials. Eighteen winter-sown onion entries were selected from released New Mexico onion cultivars, breeding lines
C.A. Powell, A. Hadidi and J.M. Halbrendt
The ability of 32P-labeled transcribed cRNA probes to detect tomato ringspot virus (TmRSV) RNA in nucleic acid extracts from roots, bark, and leaves of nectarine (Prunus persica [L.] Batsch) trees with the Prunus stem-pitting disease was assessed and compared with detection of TmRSV antigen by enzyme-linked immunosorbent assay (ELISA) in the same tissues. Neither TmRSV-specific nucleic acid nor antigen was detected in nectarine leaf tissue. ELISA detected TmRSV antigen in root extracts from 71% of the diseased trees, while dot hybridization detected virus-specific nucleic acid in 18% of the same samples. However, ELISA detected TmRSV antigen in only 47% of bark extracts; whereas TmRSV-specific nucleic acid was detected in 100% of the bark extracts from samples collected at or near the soil line. When nucleic acid extracts from bark were prepared from various locations on diseased trees and tested for TmRSV-specific nucleic acid by dot hybridization, there was an almost perfect correlation between the presence of stem-pitting symptoms and the detection of TmRSV nucleic acid. Detection of TmRSV RNA from the bark tissue of rootstock suckers from TmRSV-infected `Delicious'/MM.lO6 apple (Malus × domestica Borkh.) trees was unsuccessful using dot hybridization. The viral RNA, however, was usually detected in either leaf or root tissue of these same trees.
J. Michele Myers, Philipp W. Simon, M.E.N. Fonseca and Leonardo S. Boiteux
Garlic is an asexually propagated crop in which the greatest yield losses are attributed to virus infection. Currently, virus-free garlic is produced through shoot tip culture, and there are no known naturally occurring resistant clones. This study evaluated garlic germplasm (propagated from typical bulbs, not shoot tips) for incidence of two viruses known to infect garlic (onion yellow dwarf virus, OYDV and leek yellow stripe virus, LYSV) using dot blot ELISA. Young leaf tissue was collected from 173 garlic clones. For 118 clones, plants grown in the field from typical bulbs only were evaluated. For 55 clones, plants grown in the greenhouse from both bulbs and topsets (bulbils) were evaluated. Topsets are small bulbs that are produced in the inflorescence of stalking garlic. Each clone was tested at least three times for incidence of both viruses. In field grown bulbs, we found that 70% were infected with OYDV and 85 % were infected with LYSV. In greenhouse grown samples, incidence of OYDV was generally higher in plants from topsets than those from bulbs while no differences were seen for LYSV. Three clones were negative for both viruses and might be a useful source of resistance that can be used in producing virus resistant lines.
Antonio O. Lessa, Luis A.S. de Castro and Gerson R. de L. Fortes
The purpose of this study was to adapt an ELISA test for diagnosing of “Apple Chlorotic Leaf Spot Virus” (CLSV) in apple trees. This work was carried out at Centro de Pesquisa Agropecuária de Clima Temperado–CPACT/EMBRAPA, Pelotas–RS, Brazil, during the 1996 spring season. The application of ADGEN Diagnostic Systems protocol does not give some positive results from diseased apple trees. The procedure modified by FLEGG & CLARK (1979) gives an unsatisfactory result for color reaction in the positive samples. It means it is necessary to adapt this methodology. When the antigen was obtained from leaves grown from the base to the intermediate position in the stem and grounded with extracting buffer—0.02 M, pH 7.4 (1 g tissue: 3 ml extracting buffer) and polyclonal antisera and antibody alkaline phosphatase conjugate was diluted in coating buffer—0.05 M, pH 9.6 (1 μg antisera or antibody: 500 μl coating buffer) the reaction become more intensive and the test was able to diagnosticate the presence of the pathogen in infected leaves of apple trees.
Liping Wang, Guoping Wang, Ni Hong, Rongrong Tang, Xiaoyun Deng and Hong Zhang
Apple stem grooving virus (ASGV) and apple chlorotic leaf spot virus (ACLSV) are two major viruses of pear. In this study, in vitro thermotherapy was carried out at 37°C for 25, 30 and 35 days followed by subculturing of meristem tips of different sizes to eliminate ASGV and ACLSV from pear plants. Virus titers in heat-treated shoot tips were evaluated by ELISA testing of regenerated plants. Results showed that thermotherapy for 35 days significantly decreased the titer of ASGV and ACLSV in cultures regenerated from tips of main and axillary shoots, especially in those from explants 1 mm in length from the tip of meristems. Dot-blot hybridization of biotinylated cDNA probes derived from ACLSV and ASGV was used to detect these viruses in crude tissue extracts of in vitro-grown pear plants. Intense signals were consistently detected in untreated plant samples equivalent to less than 0.5 mg tissue. Comparison of signals from dot-blot hybridization and ELISA absorbance values (A405) confirmed that dot-blot hybridization had a higher sensitivity than PAS-ELISA. Dot-blot hybridization could detect viruses with a titer below the threshold level of ELISA. These results indicate that dot-blot hybridization is a useful tool for large-scale surveys of viruses, which facilitates the production of virus-free propagation materials in certification and sanitation programs. Results of PAS-ELISA and dot-blot hybridization showed that high virus elimination efficiency was achieved by a combination of thermotherapy for 35 days and in vitro culture of 1 mm meristem tips.
J. Kabashima, T.D. Paine and R. Redak
Pesticide use in the landscape has been reduced through the implementation of integrated pest management (IPM) (Holmes and Davidson, 1984, Olkowski et al., 1978; Smith and Raupp, 1986). IPM emphasizes prevention, identifying pests and their symptoms, regular surveying for pests, determining action thresholds and guidelines, and using sound management methods. Monitoring techniques such as pheromone traps, degree-day models, and ELISA kits, in addition to traditional methods, have enabled pest managers to determine accurately when to apply IPM techniques. Examples of serious California landscape insect pests successfully controlled through IPM include the ash whitefly [Siphoninus phillyreae (Halliday)], the Nantucket pine tip moth [Rhyacionia frustrana (Comstock)], and the eucalyptus longhorned borer (Phoracantha semipunctata F.).
M-C Sanchez-Cuevas and S.G.P. Nameth
Double petunia plants expressing virus-like symptoms were collected in greenhouses and garden centers throughout Ohio in Spring 1997 and 1998 in an effort to determine the frequency and distribution of petunia viruses present in the state. Direct antibody-sandwich and indirect enzyme-linked immunosorbent assay (ELISA) were conducted with commercial antisera made against 13 viruses, a potyvirus kit capable of detecting 80 different potyviruses, and our antiserum raised against a tobamo-like virus inducing severe mosaic in double petunia. Viral-associated double-stranded ribonucleic acid (dsRNA) analysis and light microscopy for detection of inclusion bodies were also carried out. ELISA, dsRNA analysis, and light microscopy revealed the presence of tobacco mosaic tobamovirus, an unknown tobamo-like petunia virus, tomato ringspot nepovirus, tobacco streak ilarvirus, and tobacco ringspot nepovirus. Tomato aspermy cucumovirus, tomato spotted wilt tospovirus, impatiens necrotic spot tospovirus, alfalfa mosaic virus, cucumber mosaic cucumovirus, potato virus X potexvirus, and chrysanthemum B carlavirus were not detected. No potyviruses were identified. A number of plants with virus-like symptoms tested negative for all viruses.
Mari Marutani and Veronica Endirveersingham
The effect of shade covers on degradation of insecticide, carbaryl on field-grown pakchoi (Brassica rapa subsp. chinensis) was examined by a commercial enzyme-linked immunosorbent assay (ELISA) kit. Carbaryl at a.i. 10.6 g·L-1 (1.42 oz/gal) was applied to the plants grown under five different shade treatments including control without any coverings. The experiment was arranged in a randomized complete block design with three replications. Pesticide residue on leaf tissues was examined on dates of 1, 3, 5, and 7 days after pesticide application. On all sampling dates, pesticide residue was greater with treatments with higher shade percentage. Both linear and quadratic relationship of shade (independent variable) and the concentration of remained carbaryl (dependent variable) were significant (P < 0.05). The half-life of carbaryl on pakchoi leaves ranged from 2 days for control to 9 days for the heaviest shade (75%) treatment with rain protection.
Neel Kamal and Christopher S. Cramer
(ELISA) using the method developed by Copeland (1998) but with minor changes carried out by Mohseni-Moghadam et al. (2011) and Singh (2013) was used to detect the presence of IYSV. About 0.5 cm of leaf tissue from the 10 plants of each plot was