Plate-trapped Antigen Enzyme-linked Immunosorbent Assay Research Articles

First report of groundnut ringspot virus infecting Zinnia sp. in Brazil.

Zinnia sp. is a genus belonging to Asteraceae family, originated in Mexico and adapted to a warm-hot climate (Hemmati and Mehrnoosh, 2017). Several types of zinnias with different flower color and forms are cultivated in Brazil (Min et al., 2020 and Souza Jr. et al., 2020). Characteristic symptoms of infection caused by orthotospovirus, including chlorotic spots and concentric rings on the leaves, were observed in two plants of Zinnia sp. of a florist located in the city of Piracicaba, State of São Paulo, Brazil. Orthotospovirus-like particles were observed by transmission electron microscope in leaf extracts from both plants, stained negatively with 1% uranyl acetate. By analyzing ultrathin sections of infected leaf tissues, particles of 80-100 nm in diameter were found in the lumen of the endoplasmic reticulum and nucleocapsid aggregates in the cytoplasm. Total RNA extracted separately from the leaves of both samples, using the Purelink Viral DNA / RNA kit (Thermo Fisher Scientific), was used to detect the virus by reverse transcription polymerase chain reaction (RT-PCR), using the universal primers for orthotospovirus BR60, complementary to the 3' end of the non-translated region of the S RNA (position 1 to 15 nt), and BR65, matching the nucleocapsid gene (N) (position 433 to 453 nt), generating and amplicon of 453 nt (Eiras et al., 2001). Amplicons of the expected size were obtained for the two samples. An amplicon was purified with the Wizard SV Gel and PCR Clean-Up System kit (Promega) and sequenced in both directions at Macrogen Inc (South Korea). The nucleotide sequence (GenBank MW629018) showed 99.29-99.76% identity with nucleotide sequences of the orthotospovirus groundnut ringspot virus (GRSV) isolates (GenBank MH686229 and KY400110). Leaf extracts from symptomatic plants were also analyzed by plate-trapped antigen-enzyme-linked immunosorbent assay (PTA-ELISA), using polyclonal antiserum produced against the GRSV nucleocapsid protein (Esquivel et al., 2019). The absorbance values obtained for the extracts of the two symptomatic plants of Zinnia sp. (1.3 and 1.7) were twice as high as the value obtained for the healthy plant extract (0.5). Leaf extract of symptomatic Zinnia sp. was inoculated mechanically onto leaves of healthy plants of Zinnia sp., Capsicum annuum cv. Dara, Cucumis sativus, Cucurbita pepo cv. Caserta, Chenopodium amaranticolor, Datura stramonium, Nicotiana tabacum cv. Turkish and Solanum lycopersicum cv. Compack. At 5 days post inoculation (dpi), inoculated leaves of D. stramonium reacted with local lesions, and at 9 dpi, newly developed leaves of inoculated S. lycopersicum plants showed necrotic spot and concentric ring symptoms, whereas C. annuum exhibited concentric rings at 10 dpi. Inoculated zinnia plants showed systemic chlorotic spot and concentric ring symptoms at 20 dpi, indistinguishable from those observed under natural infection. The other inoculated plant species were not symptomatic, nor the virus was detected. PTA-ELISA and RT-PCR confirmed infection with GRSV in symptomatic plants. The amplicons generated by RT-PCR of total RNA extracted from an experimentally infected plant of C. annuum and D. stramonium, and two plants of Zinnia sp. were sent for nucleotide sequencing. The obtained nucleotide sequences (MW629019, MW629020, MW629021, MW629022) shares 100% identity with the nucleotide sequence corresponding to the original GRSV isolate (MW629018) identified in Zinnia sp. This is the first report of the natural occurrence of GRSV in Zinnia sp. in Brazil. Studies on incidence and damage are needed to recommend alternatives for management.

Potato virus Y recombinant coat protein production in Escherichia coli: Applications in antibody production and virus detection

AbstractThe coat protein coding region of Potato virus Y (PVY) was cloned into pGEM‐T, sub‐cloned into plasmid pET‐28a (+) then transformed into Escherichia coli strain BL21 (DE3) pLysS to express the protein. Induction of the recombinant protein was made with isopropyl‐β‐d‐thiogalactopyranoside (IPTG), which has been tested in a variety of concentrations and for varying periods of time. The highest expression level of the protein was achieved with 1 mM IPTG for 4 hr. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western blotting has been used to confirm the identity of the expressed protein. The PVY coat protein (30 kDa) was purified under denaturing conditions by affinity chromatography and identity reconfirmed by Western blotting. The expressed protein was used as a recombinant antigen to raise polyclonal antibodies in mice. Purified anti‐PVYCP immunoglobulin (IgG) reacted positively with PVY‐infected leaf samples and the purified expressed CP in plate‐trapped antigen enzyme‐linked immunosorbent assay (PTA‐ELISA). The currently produced antiserum detects the more prevailing strains in the region: PVY N, NTN and Wilga.

Incidence of cymbidium mosaic, odontoglossum ringspot, and orchid fleck virus in orchids in Minnesota and production of antibodies for use in ELISA to detect orchid fleck virus

Ninety-six symptomatic orchids representing 36 genera from seven orchid collections, including two conservatories, in Minnesota, USA were tested for cymbidium mosaic virus (CymMV), odontoglossum ringspot virus (ORSV), and orchid fleck virus (OFV) using dipsticks (CymMV and ORSV), RT-PCR (OFV), and transmission electron microscopy (all viruses). CymMV was identified in 22% of the samples, OFV was identified in 17% of the samples, and ORSV was detected in 6% of the samples. Five percent of samples were infected with both CymMV and ORSV and 1% of samples were infected with both OFV and ORSV. Characteristic orchid virus symptoms of chlorotic and necrotic patterns were observed for the majority of infected orchids. Polyclonal antibodies were produced against Escherichia coli expressed OFV phosphoprotein (OFV P) and evaluated for use in plate trapped antigen-enzyme linked immunosorbent assay (PTA-ELISA). After an overnight incubation with PNPP the OFV P polyclonal antisera diluted from 1:1000–1:20,000 (v/v) readily differentiated between fresh healthy and OFV infected orchid (Phalaenopsis hybrid) tissue diluted from 1:5–1:20 (w/v). The high incidence of viruses detected in orchids (50%) suggests limited use of certified virus-free propagation stock and highlights the importance of sanitation to prevent transmission between plants.

Occurrence and distribution of tospovirus and thrips species infecting tomato crops in Thailand

Tospoviruses are single-stranded RNA viruses that cause severe damage to various economically important crops in Thailand and worldwide. Tospoviruses are transmitted by several species of thrips. In Thailand, four tospovirus species including Capsicum chlorosis virus (CaCV), Melon yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV) and Watermelon silver mottle virus (WSMoV) have been identified in tomato, pepper and cucurbit production fields so far. In this study, we conducted a survey to determine the occurrence and distribution of tospoviruses and their thrips vectors collected from tomato fields in several regions of Thailand during December 2011 to April 2015. Tomato plant samples exhibiting tospovirus-like symptoms (n=385) were screened for tospoviruses using a plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA). RT-PCR was performed on ELISA-positive samples (n=121) using primers specific to the nucleocapsid protein (N) genes of CaCV, MYSV, TNRV and WSMoV. N gene sequencing was conducted in some samples for confirmation. Thrips collected from tomato fields were identified to the species level based on morphological characteristics (n=700). Our results showed that TNRV (44.6%) and CaCV (35.5%) were the two major tospoviruses detected in the examined tomato crops, while a low incidence of WSMoV infection (1.7%) was found. Mixed infections of CaCV and TNRV (16.6%), CaCV and MYSV (0.8%), and CaCV, TNRV and WSMoV (0.8%) were also observed. All thrips found in the tomato fields were identified as Ceratothripoides claratris. Thrips transmission assay confirmed that C. claratris was able to transmit CaCV and TNRV with efficiencies of 70 and 12%, respectively.

Sources of resistance in accessions of Cucurbita spp. to virus species from the genus Potyvirus

ABSTRACT The identification of source of resistance in cultivated cucurbits species is very important for the development of resistant cultivars to control diseases caused by virus from the genus Potyvirus. The present research had the objective to evaluate the phenotypic reactions and the behavior of pumpkin (Cucurbita spp.) accessions to the virus species from the genus Potyvirus: Papaya ringspot virus type Watermelon (PRSV-W), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV). Twenty-eight accessions of pumpkin from the Cucurbit Germplasm Bank from Embrapa Semiarido, Petrolina, PE, Brazil were evaluated. Twelve young plants from each pumpkin accession were inoculated with each one of the virus species and were maintained at greenhouse for their symptom reaction evaluations. All possible virus infections or absence of infection were confirmed by plate-trapped antigen enzyme linked immunosorbent assay (PTA-ELISA) against antisera specific to PRSV-W, ZYMV and WMV at the Plant Virus Laboratory, Universidade Federal do Ceara. Three pumpkin accessions showed extreme resistant to WMV and eight accessions presented mild mosaic when inoculated with PRSV-W. The most severe symptoms were observed on those accessions inoculated with ZYMV and 50% of the accessions showed to be highly susceptible. On the other hand, the pumpkin accessions inoculated with WMV presented the mildest symptoms, indicating that 39% of them were resistant, and 39% were tolerant. The Cucurbita spp. accessions BGC 518, BGC 530, BGC 567, and BGC 683 that showed resistance to one or more than one virus species constitute promising sources of resistance for developing virus resistant pumpkin cultivars or hybrids.

Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli.

The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

Screening Sugarcane Wild Accessions for Resistance to Sugarcane Mosaic Virus (SCMV)

In order to identify new sources of resistance for future introgression crosses, wild sugarcane germplasm was screened for resistance to Sugarcane mosaic virus (SCMV) under high inoculum pressure. Evaluation of symptoms by a grade scale associated with serological diagnostic by plate-trapped antigen-enzyme-linked immunosorbent assay (PTA-ELISA) with a specific antiserum were performed in 43 accessions, encompassing Saccharum officinarum, S. barberi, S. spontaneum and S. robustum species and hybrids. Differential patterns of resistance and susceptibility were observed among the Saccharum species, with S. officinarum followed by S. robustum accessions showing higher susceptibility, whereas S. barberi and S. spontaneum had the most resistant accessions. The results indicate that accessions IS76-155, IJ76-418 red, NG57-50, Ceram red, Badilla, Sac.off 8276, Fiji19, IJ76-313, US57-141-5, Krakatau, IN84-58, IN84-88, IN84-82, Gandacheni, and Chin, possibly consist in good sources of resistance to mosaic.

Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors

Rice dwarf virus (RDV) causes Rice dwarf disease, which leads to considerable losses in rice production in Asia. Purified RDV virions were used as the immunogen to prepare monoclonal antibodies (mAbs). Three murine mAbs against RDV were prepared. Plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and immunocapture-RT-PCR (IC-RT-PCR) were then developed for sensitive, specific, and rapid detection of RDV in rice and leafhopper samples obtained in the field using the mAbs. The PTA-ELISA, dot-ELISA and IC-RT-PCR detected the virus in infected tissue crude extracts diluted at 1:81,920, 1:10,240 and 1:655,360 (w/v, gmL−1), in individual viruliferous rice green leafhopper crude extracts diluted at 1:25,600, 1:6400 and 1:3,276,800 (individual leafhopper/μL), respectively. 878 rice field samples and 531 leafhopper field samples from ten provinces of China were screened for the presence of RDV using the two serological assays and the IC-RT-PCR and the results indicated that 37 of the 878 rice samples and 22 of the 531 leafhopper samples were infected by RDV. All positive samples were from Yunnan Province, indicating that RDV is prevalent in this province, but not in the other nine provinces. The dot-ELISA is suitable for routine detection of large-scale rice and leafhopper samples in field surveys.

Risk Assessment Methods for the Ringspot Pathogen Mycosphaerella brassicicola in Vegetable Brassica Crops.

Mycosphaerella brassicicola causes ringspot on Brussels sprouts, which can result in substantial yield loss in commercial production. Brussels sprout buttons are downgraded if this pathogen occurs on them. In this study, the effect of temperature and wetness duration was investigated on infection of Brussels sprouts using controlled environments (CE). The effect of temperature and wetness duration on inoculum production and ascospore discharge was also investigated. Infection by M. brassicicola was described using a mathematical model and was compared to estimates of ascospore availability obtained via a volumetric air sampler and immunofluorescence (IF). Infection of M. brassicicola was correlated (r = 0.92) with temperature during leaf wetness periods. The relationship between temperature and time to discharge of 5 and 50% of the cumulative total number of ascospores from ringspot lesions was r = 0.99 and 0.98, respectively (P < 0.001). In field experiments, an optimal wind run (the product of the average wind speed and the period over which that average speed was measured) of 250 to 500 km day-1 was required for the dissemination of ringspot inoculum to field bait plants. Quantification of M. brassicicola inoculum in collected field aerosols was possible using a monoclonal antibody in a plate-trapped antigen enzyme-linked immunosorbent assay. Precoating of the air sampler wells with sodium azide prevented trapped spores from germinating. Ringspot inoculum could be detected and quantified in air samples from commercial crops of Brussels sprouts in the United Kingdom. Low levels of ringspot inoculum measured within crops did not lead to disease development.

First Report of Anthracnose Caused by Colletotrichum acutatum on Strawberry in the Czech Republic.

Colletotrichum acutatum J. H. Simmond is the causal agent of anthracnose rot and strawberry blackspot. This pathogen is listed by the EPPO as a regulated (formerly quarantined) organism for all European countries and is widely distributed throughout Europe (e.g., the United Kingdom, France, and Germany) (2). In the autumn of 2005, typical symptoms of anthracnose caused by C. acutatum (circular, dark, and sunken spots on fruit, dark, sunken lesions on petioles, and withering of the leaf, buds, and flowers) were repeatedly observed on field-grown strawberry plants in the Mělník Region of central Bohemia and Břeclav Region in southern Moravia, Czech Republic. Strawberry fruits and petioles showing typical symptoms were surface sterilized (30 s in 70% ethanol, 1 min in 10% NaOCl, and 15 s in 70% ethanol), rinsed in sterile water, dissected under aseptic conditions, and plated on 2% malt extract agar or placed in wet chambers and incubated at room temperature (18 to 20°C) for 10 days. All isolated strains were independently identified by morphological characteristics, plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) (1), and PCR with the C. acutatum-specific primers ITS4 and CaInt2 (3). Morphological studies of C. acutatum were carried out on potato dextrose agar (4). The colonies were white, cream, grayish, or rose-orange before sporulation and the colony reverse was cream to orange with brown spots. The mycelial growth rate was 7.5 mm per day at 25°C. The conidia were one-celled, hyaline, cylindrical, 11.3 to 19.7 × 3.6 to 5.5 μm, and the majority of conidia were pointed at either or both ends. The appresoria were brown, globose to ellipsoidal, 5.0 to 7.5 × 5.0 to 6.2 μm, and the sclerotia were absent. Ten strawberry plants with green fruits of each cultivar Elsanta and Kama were sprayed with 500 ml of suspension of C. acutatum conidia (104 conidia per ml). This test was carried out in the glasshouse under quarantine conditions at 20 to 25°C. C. acutatum caused withering of the flowers or dark brown spots on green fruits on five plants of cv. Elsanta and on four plants of cv. Kama after a 6-week incubation period. Isolation and identification of the pathogen from the diseased tissues were done as described above. C. acutatum was reisolated from three fruits, four leaf blades, and four petioles from five plants of cv. Elsanta and four fruits, four leaf blades, and two petioles from four plants of cv. Kama. The fungus was not reisolated from the control strawberry plants. In three cases, the pathogen was detected in the crown of plants of cv. Elsanta by PCR and ELISA.

Preliminary Studies on the Development of Monoclonal Antibodies Against Mycelia of Ganoderma boninense, the Causal Pathogen of Basal Stem Rot of Oil Palm

This study aimed to raise specific MAbs against G. boninense , the causal pathogen of basal stem rot (BSR) of oil palm. Crude mycelium extract of G. boninense was used as immunogen to generate MAbs. Mycelium was harvested from liquid culture and freeze-dried followed by re-suspension in phosphate buffer saline (PBS). Two 10-week old BALB-C mice were immunized with the mycelial extract. The mice were boosted once before harvesting their spleens for fusion. The MAbs were fused with myeloma cells from BALB-C mice. Initial screening was carried out using plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) with mycelial immunogen of G. boninense . The MAbs with positive signals were verified via secondary screening and cloned for cross-reactivity test. Cross-reactivity testing was carried out with 2 other fungi namely; Trichoderma and Botrytis along with 2 different species of Ganoderma commonly found in oil palm plantations namely; G. zonatum , and G. miniatocinctum . This study found that the MAbs raised against G. boninense were not specific as the MAbs gave positive signals through the cross-reactivity test with all fungi tested in the cross-reactivity. Future work would be using these MAbs in a co-immunization program whereby the generated Ganoderma sp generic monoclonal antibody will be pre-mixed with the G. boninense mycelium immunogen to allow reduction in the potential cross-reactivity of newly generated antibodies with Ganoderma sp. Our efforts are also currently directed at optimizing the immunogen preparation for the production of MAbs specific to G. boninense .

Reliability of diagnostic techniques forErwinia amylovora, the causative agent of fire blight disease

A total of 20 putative strains of Erwinia amylovora originating from 11 samples of host plants with symptoms of fire blight were analyzed in detail using commercial polyclonal antibodies in immunochemical tests. Fourteen strains reacted negatively in all tests; 6 strains reacted positively with a polyclonal antibody for PTA-ELISA (plate-trapped antigen-enzyme linked immunosorbent assay) at a concentration corresponding to A620 = 0.1, while at A620 readings of 0.01 and 0.001 the results were negative. Five strains reacted positively with a polyclonal antibody for indirect immunofluorescence test at all tested concentrations. Three of those strains were positive in the PCR test with AMSbL and AMSbR primers designed for detection of E. amylovora. In hypersensitivity test in tobacco and in immature pear fruit assay, all putative strains were negative while a known reference strain of E. amylovora gave a typical hypersensitive-reaction response. On a medium with 5% sucrose the reference strain of E. amylovora produced levan while putative strains did not. After modification of the PCR protocol, 3 putative strains reacted as negatives. Optimization of PCR test was achieved by finding the optimum annealing temperature and time for primers. The recommended annealing temperature (49 degrees C) for these primers was increased to 55 degrees C and the annealing time was reduced from 2 min to 30 s. Using the microbial identification system Biolog those 3 strains were identified as Pantoea dispersa (1 strain) and Pantoea agglomerans (2 strains). The strains are supposed to be white variants of the species P. dispersa and P. agglomerans occurring less frequently than the yellow variants. Since there were positive reactions in our immunochemical tests these strains could cause false positives in routine screening of plant samples.

Detection of Phomopsis vaccinii on Blueberry and Cranberry in Europe by Direct Tissue Blot Immunoassay and Plate‐Trapped Antigen ELISA

AbstractUpright dieback, caused by Phomopsis vaccinii Shear (teleomorph Diaporthe vaccinii Shear), is an economically important disease that could threaten blueberry and cranberry production. Diagnosis of the disease by traditional phytopathological methods is difficult. We report the successful detection of P. vaccinii by direct tissue blot immunoassay (DTBIA) and plate‐trapped antigen enzyme‐linked immunosorbent assay (PTA‐ELISA). The infected samples obtained from Lithuania are the findings reported on the fungus in Europe.

A One-Step, Immunochromatographic Lateral Flow Device Specific toRhizoctonia solaniand Certain Related Species, and Its Use to Detect and QuantifyR. solaniin Soil

ABSTRACT A murine hybridoma cell line GD2 secreting an immunoglobulin (Ig)M monoclonal antibody (MAb) was produced against surface antigens from an anastomosis group (AG) 4 isolate of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris). Ascites were produced in mice using GD2 hybridoma cells and used to develop a rapid immunochromatographic lateral flow device (LFD) for the detection of antigens from R. solani and certain related Rhizoctonia spp. The LFD was tested for specificity against surface antigens from related and unrelated soil fungi. Antigens from representative isolates of R. solani AGs 1, 2-1, 2-3, 2-t, 3, 4, 5, 6, 7, 8, 9, 10, 11, and BI gave a positive response in LFD tests, as did antigens from Thanatephorus orchidicola, T. praticola, R. fragariae (teleomorph: Ceratorhiza fragariae), Ceratorhiza goodyerae-repentis, Ceratobasidium cornigerum, and binucleate AGE. Antigens from R. solani AGs 2-2, 2-2IIIB, and 2-2IV and from the related fungi R. carotae, R. cerealis (teleomorph: Ceratobasium cereale), R. crocorum (teleomorph: Helicobasidium brebissonii), R. oryzae (teleomorph Waitea circinata), and R. zeae gave negative responses, as did antigens from a range of unrelated fungi and oomycetes including Fusarium, Gliocladium, Trichoderma, Pythium, and Phytophthora spp. The usefulness of the LFD to detect R. solani was demonstrated in soils naturally infested with R. solani AG3. There was close agreement between results of LFD tests and conventional plate enrichment tests employing selective medium. The specificity of the technique was confirmed by polymerase chain reaction PCR using R. solani AG3-specific primers and by analyses based on sequences of the internal transcribed spacer (ITS)1-5.8S-ITS2 rRNA-encoding regions of unrelated fungi recovered from soil samples. The LFD was used to quantify R. solani AG4 in artificially infested soil samples (chopped potato soil inoculum). Estimates of CFU per gram of soil were derived using a most-probable number technique, which was based on the presence or absence of a detectable signal in the LFD. Estimates of CFU obtained in LFD tests and those obtained in a plate-trapped antigen enzyme-linked immunosorbent assay incorporating MAb GD2 were identical (449 CFU g(-1) of soil).

Estimation of deoxynivalenol (DON) content by symptom rating and exoantigen content for resistance selection in wheat and triticale

Fusarium head blight (FHB) in wheat and triticale leads to contamination of the grain with the mycotoxin deoxynivalenol (DON) that is harmful to animal and man. A fast, low-cost, and reliable method for quantification of the DON content in the grain is essential for selection. We analysed 113 wheat and 55 triticale genotypes for their symptom development on spikes, Fusarium exoantigen (ExAg) and DON content in the grain after artificial inoculation with a highly aggressive isolate of F. culmorum in three (wheat) and six (triticale) location-by-year combinations. Additionally, in triticale the amount of Fusarium damaged kernels (FDK) was assessed. ExAg content was analysed by a newly developed Fusarium-specific plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) and DON content by an immunoassay. A moderate disease severity resulted in an ExAg content of 0.87 optical density (OD) units in wheat and 1.02 OD in triticale. DON content ranged from 12.0 to 105.2 mg kg−1 in wheat and from 24.2 to 74.0 mg kg−1 in triticale. Genotypic and genotype-by-environment interaction variances were significant (P < 0.01). Coefficient of phenotypic correlation between DON content analysed by the immunoassay and ExAg content was r = 0.86 for wheat and r = 0.60 for triticale. The highest correlation between DON content and symptom rating was found by FHB rating in wheat (r = 0.77) and by FDK rating in triticale (r = 0.71). In conclusion, selection for reduced FHB symptoms should lead to a correlated selection response in low fungal biomass and low DON content in the grain.

First Report of Orchid fleck virus in Costa Rica.

Orchid fleck virus (OFV), a tentative member of the family Rhabdoviridae, infects orchids in several countries. The virus is vectored worldwide by the mite Brevipalpus californicus (Banks) (Acari: Tenuipalpidae). Eleven plants of Oncidium spp. and one plant each of the genera Cymbidium and Maxillaria exhibiting numerous yellow flecks and necrotic ringspot lesions on leaves were collected in two private orchid collections in Costa Rica. Presence of OFV was assessed by plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) using an antiserum developed against an OFV isolate in Japan (2), analyses of ultrathin sections of the host cell with transmission electron microscopy (TEM), and reverse transcription-polymerase chain reaction (RT-PCR) amplification using specific primers for the viral nucleocapsid gene (1). Eight of eleven Oncidium samples, and both Cymbidium and Maxillaria samples tested positive for OFV with PTA-ELISA having A405 values ranging from 3.9 to 14.6 times higher than negative controls. Thin sections from individual samples of Cymbidium, Oncidium, and Maxillaria revealed electron-lucent intranuclear viroplasm and short, rodlike particles (40 to 50 × 100 nm) in the nucleus or cytoplasm typical of OFV-infected cells. RT-PCR amplifications from one sample of each genera resulted in PCR-product bands of approximately 800 bp. The Cymbidium RT-PCR product was cloned into a pGEM-T-Easy expression vector and sequenced using an ABI 3700 sequencer. The 619-bp nucleocapsid gene consensus sequence had 98% homology with the OFV isolate 0023 identified in Germany (GenBank Accession No. AF343870) (1). However, it had only approximately 85% nucleocapsid gene homology with other OFV isolates available through GenBank, including those from countries geographically closer to Costa Rica, such as Brazil (1). To our knowledge, this is the first report of OFV infecting orchids in Costa Rica.

Production of Monoclonal Antibodies to Sugarcane Yellow Leaf Virus Using Recombinant Readthrough Protein

AbstractYellow leaf syndrome (YLS) of sugarcane is associated with sugarcane yellow leaf virus (SCYLV), a member of the family Luteoviridae. A fragment of the coat protein and readthrough domain of SCYLV wasexpressed in a bacterial expression system. The resulting protein was purified and used to immunize mice for monoclonal antibody (MAb) production. Two MAbs, 3A2E3 and 2F7H5, were selected following the screening of hybridoma cells using both plate‐trapped antigen enzyme‐linked immunosorbent assay (PTA‐ELISA) and tissue blot immunoassay (TBIA). These MAbs can be incorporated into the TBIA assay currently used for the routine detection of SCYLV but could not be used in triple antibody sandwich enzyme‐linked immunosorbent assay (TAS‐ELISA). The two antibodies selected have slightly different specificities. Antibody 3A2E3 gave equivalent results to a polyclonal antiserum (raised to purified virus) in comparative testing using TBIA. The MAbs produced should provide a widely available, uniform reagent for SCYLV diagnosis with the potential to help manage YLS.

Rapid method for the detection and quantification of &lt;i&gt;Botrytis cinerea&lt;/i&gt; in plant tissues

Monoclonal antibody to B cinerea (BC58) was used to develop a platetrapped antigen enzyme linked immunosorbent assay (PTAELISA) to detect and quantify Botrytis antigens in boysenberry flowers The ability of antibody BC58 to detect B cinerea in extracts from artificially infected boysenberry flowers was assessed Results showed that the antigen could be detected in latent infections Antibody BC58 sensitivity to heat treatment of the antigen incubation conditions and the detection limit were also investigated Autoclaving at 121C reduced the sensitivity of the antibody Additionally the incubation of the antigen at 4C overnight produced higher absorbance values at 405 nm than incubation at 37C for 2 h The detection and quantification of B cinerea antigen was reliable within 016 g dried mycelium per ml of PBS buffer and at least 8 x 103 spores

Cyclanthera pedata var. edulis: New Host of Papaya ringspot virus-type W in Brazil.

Stuffing cucumber (Cyclanthera pedata var. edulis Schrad.) is native to the Americas, where it often occurs as an escape. The species is monoecious, with small flowers and large and deeply palmately lobed leaves. Fruits are puffy, partially hollow, and measure 5 × 15 cm long. The plant has soft spines, a tapered neck, and black seeds. The species has long been cultivated in Asia, where fruits are eaten raw as a substitute for cucumber or cooked (1). One plant showing intense mosaic, without leaf malformation, was found near a squash crop (Cucurbita moschata) in Anhembi County, State of São Paulo. Electron microscopic examination of a negatively stained sap preparation from leaves of this plant showed that it contained numerous flexuous rod-shaped particles, approximately 700 × 760 nm long, similar to those of potyviruses. Extracts from symptomatic leaves were rub-inoculated to zucchini squash (Cucurbita pepo), Carica papaya, Chenopodium amaranticolor, C. quinoa, Gomphrena globosa, and Phaseolus vulgaris cv. Black Turtle 2. Only zucchini squash was infected and developed severe mosaic with intense leaf malformation. Extracts from field infected stuffing cucumber and experimentally inoculated zucchini squash were tested by plate-trapped antigen enzyme linked immunosorbent assay (PTA-ELISA) with antisera against the following potyviruses: Papaya ringspot virus-type W (PRSV-W), Zucchini yellow mosaic virus (ZYMV), and Watermelon mosaic virus-2 (WMV-2). Samples were also tested with antisera against a tospovirus (Zucchini lethal chlorosis virus) and a cucumovirus (Cucumber mosaic virus). Both samples were positive in PTA-ELISA only with PRSV-W antiserum. This is the first report of C. pedata var. edulis as a natural host for PRSV-W in Brazil.

Occurrence, Distribution, and Relative Incidence of Five Viruses Infecting Cucurbits in the State of São Paulo, Brazil.

Cucurbits species grown in 38 of 40 agricultural regions in the state of São Paulo, Brazil, were surveyed for the relative incidence of Cucumber mosaic virus (CMV), Papaya ringspot virus-type W (PRSV-W), Watermelon mosaic virus-2 (WMV-2), Zucchini lethal chlorosis virus(ZLCV), and Zucchini yellow mosaic virus (ZYMV) during May 1997 and June 1999. Samples from 621 plants, representing eight cultivated species, six wild species, and one commercial hybrid (Cucurbita moschata × C. maxima), were analyzed by plate trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA). PRSV-W and ZYMV were the most frequently found viruses, accounting for 49.1 and 24.8%, respectively, of 605 samples tested. ZLCV, CMV, and WMV-2 were detected in 7.8, 6.0, and 4.5% of 612, 497, and 423 samples tested, respectively. Double infection was found in 97 samples, and triple infection was found in 10 samples. Quadruple infection was detected in one C. pepo sample. Plants that were symptomatic but negative by PTA-ELISA might be due to abiotic agents, infection by virus for which antiserum was not available, such as Squash mosaic virus, or infection with an as yet uncharacterized virus.