Plate-trapped Antigen Research Articles

A new single-chain variable fragment (scFv) antibody provides sensitive and specific detection of citrus tristeza virus

Citrus tristeza virus (CTV) is the most economically important virus disease of citrus worldwide. To develop a specific serological assay for CTV, a Tomlinson phage display antibody library of single chain variable fragments (scFv) was screened with a recombinant CTV coat protein (CTV-CP) heterologously expressed in Escherichia coli. The phage clones were checked by ELISA to identify clones with high specificity for CTV-CP. Eight clones were strongly reactive with CTV-CP. Nucleotide sequencing of these clones revealed that all of them contained the same sequence. Thus, the phage-displayed scFv antibody was termed scFvF10. Evaluation of scFvF10 binding to CTV-CP by plate-trapped antigen ELISA (PTA-ELISA) and immunoblotting, showed that it was specific and allowed sensitive detection of CTV-CP. Homology-based molecular modeling and docking analysis confirmed that the interaction between CTV-CP and scFvF10, with a binding energy of -738 kj mol-1, occurred mainly by 12 intermolecular hydrogen bonds. Moreover, triple-antibody sandwich (TAS)-ELISA using scFvF10 as second antibody showed high sensitivity in the detection of CTV infected samples. The CTV detection limit of scFvF10 by PTA-ELISA and TAS-ELISA were 0.05 and 0.01 μg CP/mL, respectively. Our results with different diagnostic assays demonstrated that scFvF10 has the potential to be used as an efficient tool for CTV-infected plant diagnosis.

Molecular characterization and identification of economically important Potyviruses in Cucurbitaceae family from Gujranwala division of Punjab, Pakistan

Cucurbits belongs to Cucurbitaceae family and they are main source of energy, vitamins and minerals. The plant viruses in general and potyviruses in particular are considered to hamper the successful production of cucurbits in Punjab province of Pakistan. The present study was undertaken to determine the current status of major potyviruses infecting cucurbits in Gujranwala division of Punjab. Overall, 528 samples of cucurbit species were collected from 46 fields of six districts (Narowal, Sialkot, Gujranwala, Gujrat, Mandi Baha Uddin and Hafizabad) in Gujranwala division. All disease samples were screened for the presence of potyvirus infection by indirect plate-trapped antigen (PTA)-ELISA using “Poty-group test” kit (Bioreba AG, Switzerland). These samples were further tested for Papaya ring spot virus (PRSV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV) (Bioreba AG) using virus specific DAS-ELISA. Zucchini yellow mosaic virus (ZYMV) was also further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). ZYMV was found as the most predominant virus of cucurbits in Gujranwala division with an average incidence of 20.8%, followed by PRSV (7.2%) and WMV (1.7%). Maximum mixed double infection was observed in combination of ZYMV+PRSV (1.3%) followed by ZYMV+WMV (0.4%) and WMV+PRSV (0.8%). Overall highest disease incidence of potyviruses was in Gujranwala (40.86%) followed by Narowal (34.65%), Gujrat (28.6%), Mandi Baha Uddin (26.56%), Hafizabad (25%) and Sialkot (23.21%). Likewise, the maximum ZYMV incidence was recorded in Angel Gourd whereas no viral infection was observed in snap melon and ash gourd in Gujranwala division. All cucurbits vegetables were found to be highly prone to ZYMV followed by PRSV and WMV. The sequence of CI region of ZYMV AABRM-CI ridge gourd isolate was deposited in GenBank with an accession no. MN897101, which showed nucleotide identities from 91.501-98.401% with other isolates of ZYMV available at database. Highest nucleotide identity 98.401% was found with the sequence of ZYMV South Korean isolate BR1 (MH042024) followed by 98.301% with ZYMV isolate Y23 from Turkey (KP828425). The results of the current study would be useful in devising management strategies to mitigate potyvirus losses in cucurbits from Pakistan.

Evidence of seed transmission of Banana bract mosaic virus in Musa synthetic diploid H-201, a possible threat to banana breeding

Banana bract mosaic virus (BBrMV) is a serious constraint in Musa spp. and Musa textilis causing enormous economic losses to growers. The distribution of the virus in various parts of the seeds, flower and leaf tissues of seedlings from banana plants of a synthetic diploid, H-201, naturally infected in the field was determined by Plate-trapped antigen coating enzyme-linked immunosorbent assay (PTA-ELISA), reverse transcriptase (RT) polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR) analysis and sequencing. RT-PCR and qRT-PCR results revealed the presence of BBrMV in embryo, endosperm and seed coat of seeds from infected plants. Nucleotide sequencing of the RT-PCR amplicons confirmed the BBrMV infection in seed. Quantification of BBrMV in various parts of seed by qRT-PCR using SYBR Green chemistry revealed that the viral load was maximal in the endosperm followed by seed coat and embryo. In a seed growth test, thirteen seedlings, that were raised from embryos of infected seeds in vitro and twenty seedlings raised from infected seeds in pots, expressed typical symptoms of bract mosaic virus disease and were confirmed RT-PCR positive for the virus. This finding will have significant implications in banana breeding programmes.

Molecular and partial biological characterization of the coat protein sequences of Iranian alfalfa mosaic virus isolates

Alfalfa mosaic virus (AMV) is one of the most important viruses that cause significant losses to alfalfa worldwide. A total of 1066 symptomatic alfalfa, 143 weeds and other secondary hosts were collected and assayed for AMV infection. The virus was detected in 360 samples (33.8% incidence) of alfalfa by plate-trapped antigen (PTA)-ELISA. Among the weed species and the other plants tested, potato, pepper and seven weed species were infected with AMV. The coat protein (CP) gene sequences from 59 Iranian isolates were PCR amplified, cloned, sequenced, and compared with the sequences available in GenBank. Sequence analysis of the amplified regions showed that 55 of the Iranian CP sequences share >98% pairwise identity with each other. The remaining four share between 89 and 96% identity. Our host studies indicated that the Iranian AMV isolates were classified into two groups. Only four Iranian isolates were clustered in the symptom-group B and caused relatively milder symptoms compared to those found in symptom-group A.

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.

Nucleotide sequence analyses of coat protein gene of peanut stunt virus isolates from alfalfa and different hosts show a new tentative subgroup from Iran.

Alfalfa cultivars grown in 14 provinces in Iran were surveyed for the relative incidence of peanut stunt virus (PSV) during 2013-2016. PSV were detected in 41.89% of symptomatic alfalfa samples and a few alternate hosts by plate-trapped antigen ELISA. Among other hosts tested only Chenopodium album, Robinia pseudoacacia and Arachis hypogaea were found naturally infected with PSV. Twenty five isolates of PSV were chosen for biological and molecular characterizations based on their geographical distributions. There was not any differences in experimental host range of these isolates; however, variation in systemic symptoms observed on Nicotiana glutinosa. Total RNA from 25 of viral isolates were subjected to reverse transcription polymerase chain reaction analysis using primers directed against coat protein (CP) gene. The CP genes of 25 Iranian PSV isolates were either 651 or 666 nucleotides long. The nucleotide and amino acid identities for CP gene among Iranian PSV isolates were 79.3-99.7 and 72-100%, respectively. They also shared between 67.4 and 82.4% pairwise nucleotide identity with other PSV isolates reported elsewhere in the world. Phylogenetic analyses of CP gene sequences showed formation of a new subgroup comprising only the Iranian isolates. Natural infection of a few alternate hosts with PSV is reported for the first time from Iran.

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.

A Simple Kit of Plate-Trapped Antigen Enzyme-Linked Immunosorbent Assay for Identification of Plant Viruses

Many methods can be used for arriving in a correct virus disease diagnose, and the serological techniques are the most used methods for plant virus identification. The indirect enzyme-linked immunosorbent assay (Indirect-ELISA) or plate-trapped antigen ELISA (PTA-ELISA) has been useful for detection of viruses in a wide range of situations, especially to test a large number of samples in a relatively short period of time. Immune-biological Companies have developed practical kits for direct ELISA or double antibody sandwich (DAS-ELISA), but neither Company has developed kits for PTA-ELISA. As a single universal antibody-conjugate is used for detection of a wide range of plant viruses, the PTA-ELISA technique is more economical, practical and suitable for virus detection. Considering also the great problem of including infectious plant viruses in DAS-ELISA kits, a simple kit for PTA-ELISA was developed for plant virus identification. Extracts from infected plant tissues were added into the ELISA plate wells, which were sealed with plastics and maintained in the refrigerator and Laboratory conditions for different periods of time. The plates were tested by the regular PTA-ELISA and after 20 months of incubation, the plate showed excellent results when used for detection of six virus species from the genera Comovirus, Cucumovirus, Potyvirus and Sobemovirus in infected plant tissues. The ELISA plate trapped virus together with its specific antiserum could constitute a simple kit, which will permit the exchange of antisera among virologists without transferring infectious viruses from one laboratory to another to be used as control.

FIRST REPORT OF CHILLI VEINAL MOTTLE VIRUS IN TOMATO IN PAKISTAN

Chilli veinal mottle virus (ChiVMV, genus Potyvirus ) infects several solanaceous species worldwide (Zhao et al ., 2014). In April 2014, a total of 20 tomato leaf samples with symptoms of mosaic, necrosis and mottling were collected from the research farm of PMAS-Arid Agriculture University Rawalpindi. All disease samples were screened for the presence of potyvirus infection by indirect plate-trapped antigen (PTA)-ELISA using “Poty group test” kit (Bioreba AG, Switzerland). Only 12 samples were positive for potyvirus infection. These samples were further tested for Potato virus Y (PVY), Pepper veinal mottle virus (PVMV) (Bioreba AG) and ChiVMV (Loewe Biochemicals, Germany) using virus specific DAS- ELISA. Of 12 potyvirus confirmed samples, two were positive for ChiVMV and remaining ten were positive for PVY. No reaction was observed with antibodies against PVMV. The ChiVMV ELISA-positive samples were further confirmed by RT-PCR using ChiVMV specific primers Poty3 and Oligo(dT) (Tsai et al ., 2008). Both PCR amplicons were purified using QIAquick® PCR purification kit (Qiagen) and subsequently sequenced in both orientations . A product 759 nucleotides in size (partial CP gene including 3’UTR) was obtained (both clones were 100% identical) and the sequence of ChiVMV isolate tomato (AARTPK) was deposited in GenBank (Accession No. KT876048). BLASTn revealed >90% sequence identity with ChiVMV isolates from India (JN624776, JN692501), China (HQ218936, KF738253, KC711055, JX088636) and Pakistan (KJ472764). This destructive virus has been previously reported from chilli pepper in Pakistan (Shah et al ., 2009). To the best of our knowledge, this is the first confirmed report of ChiVMV from tomato in Pakistan.

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.

First Report of Tomato apical stunt viroid in Tomato in Italy.

During a survey conducted in October 2013 in tomato greenhouses in Diano Marina (Imola Province, northwest Italy), in a single greenhouse, unusual disease symptoms were observed in four out 1,400 (~0.3%) of tomato plants cv. Ingrid, grafted on 'Beaufort' rootstock. Symptoms including shortened apical internodes associated with tiny, deformed, and brittle chlorotic leaves, while ripe fruits appeared reduced in size and pale red. Samples of leaves from the four plants were collected and examined using commercial antisera (Bioreba AG, Reinach, Switzerland) by double antibody sandwich (DAS)-ELISA against Tomato spotted wilt virus, Cucumber mosaic virus, Alfalfa mosaic virus, Tomato/Tobacco mosaic viruses, and by indirect plate trapped antigen (PTA)-ELISA against potyviruses (potygroup test). None of the tested viruses were detected in the four leaf samples. In addition, PCR tests for begomoviruses and phytoplasmas were also negative. In a host range study, the original symptoms, consisting mainly of stunting and chlorosis, were reproduced within ~10 days in tomato seedlings (Momor line), mechanically inoculated at two true leaves stage with sap extract obtained from the four symptomatic tomato plants, whereas no symptoms were observed in Chenopodium amaranticolor, C. quinoa, Nicotiana tabacum (cv. Xanthi nc), N. glutinosa, or Phaseolus vulgaris (cv. Borlotto rosso). Total RNAs extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from symptomatic samples were tested in RT-PCR using pospiviroids generic primers PSTVd-32/33 (1), designed to amplify the whole genome of Potato spindle tuber viroid (PSTVd), Tomato apical stunt viroid (TASVd), and Columnea latent viroid (CLVd), and CEVd-FW/RE primers, designed to amplify the whole genome of Citrus exocortis viroid (CEVd) and TASVd (3). Each of the four samples yielded amplicons of the same size (364 bp) with both primer combinations. The identity of the viroid was then determined by sequencing, on both strands, amplicons obtained from the four symptomatic plants at MWG (Ebersberg, Germany). Sequences obtained were identical, showing the highest nucleotide identity (99.7%) with the TASVd isolate Sj1 (AM777161), identified in Germany on Solanum jasminoides. The sequence was deposited in GenBank (Accession No. HG916812) and the field isolate named To1-IT. Two other cases of pospiviroid infection in tomato in Italy have been reported so far and the viroid species detected were PSTVd (2) and CLVd (4), respectively. To our knowledge, this is the first report of TASVd infecting tomato in Italy. The origin of this infection is still unclear, although based on the biological properties and sequence similarity, the To1-IT isolate probably originated from an ornamental species, most likely S. jasminoides, as recently reported for other tomato TASVd isolates, according to their biological and genetic features (5).

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.

Araujia sericifera New Host of Alfalfa mosaic virus in Italy.

Araujia sericifera Brot. (Fam. Apocynaceae) is an evergreen climbing plant native of South America, originally introduced in Europe as an ornamental. In spring 2012, virus-like symptoms including bright yellow mosaic of calico-type and leaf distortion were observed in three A. sericifera plants growing in an abandoned field located in Pomigliano d'Arco (Campania region, Italy). Leaves from the three plants were collected and examined using commercial antisera (Bioreba AG, Reinach, Switzerland) by double antibody sandwich (DAS)-ELISA against Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), and by indirect plate trapped antigen (PTA)-ELISA against potyviruses (Potygroup test). Only AMV was detected serologically in the three A. sericifera samples. The virus was mechanically transmitted from the ELISA-positive samples to four plants each of Chenopodium quinoa, C. amaranticolor, tobacco (Nicotiana tabacum cv. Xanthi nc), cowpea (Vigna unguiculata, cv. Black eyes), basil (Ocimum basilicum, cv. Gigante), and tomato (Solanum lycopersicum cv. San Marzano), using chilled 0.03 M sodium phosphate buffer, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of Carborundum (600 mesh). Inoculated plants were kept in an insect-proof greenhouse with natural illumination and temperatures of 24 and 18°C day/night. Under these conditions, plants showed the following symptoms after 1 to 3 weeks, consistent with symptoms caused by AMV (1): chlorotic local lesions following by mosaic in C. quinoa and C. amaranticolor, reddish local lesions following by mosaic in cowpea, necrotic local lesions followed by systemic necrosis in tomato, bright yellow mosaic (calico type) in basil, and mosaic and strong deformation of the apical leaves in tobacco. The presence of AMV in ELISA-positive A. sericifera and host plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using primers for the coat protein gene (CP) previously used for the molecular characterization of AMV isolates (2). An Italian isolate of AMV from Lavandula stoechas (GenBank Accession No. FN667967) and RNA extracted from a healthy A. sericifera plant were used as positive and negative controls, respectively. An amplicon of the correct predicted size (∼750 bp) was obtained from each of the infected plants assayed, and that derived from A. sericifera isolate Ars2 was purified (QIAqick PCR Purification Kit, Qiagen), cloned in pGEMT easy vector (Promega, Fitchburg, WI) and sequenced (HF570950). Sequence analysis of the CP gene, conducted with MEGA5 software, revealed the highest nucleotide identity of 98% (99% amino acid identity) with the AMV isolate Tef-1 (FR854391), an isolate belonging to subgroup I (3). To our knowledge, this is the first report of AMV infecting A. sericifera in Italy. Since A. sericifera is considered an invasive plant, in continuous expansion to new areas in Italy and in other European countries, particular attention should be paid to the possibility that this species may play a role in the epidemiology of aphid-transmitted viruses such as AMV and CMV, representing a threat to susceptible crops growing nearby.

First Report of Banana mild mosaic virus Infecting Plantain in Ivory Coast.

Plantain (Musa sp., genomic group AAB) is an important crop for millions of the world's poorest people. In Ivory Coast, it is the second most consumed food and an important source of income for farmers. Between 2010 and 2011, a survey for viruses infecting plantain (AAB) was conducted in 10 major plantain-growing regions located in eastern (Abengourou), middle-western (Bouaflé, Daloa, Issia, Oumé, Sinfra, Zuenoula), central (Yamoussoukro), and southern (Aboisso, Gagnoa) Ivory Coast. Leaf samples showing yellow streaks or mild chlorotic streaks were collected and dried on CaCl2 for storage. A representative sample from each location was selected and tested for the presence of Cucumber mosaic virus (CMV, genus Cucumovirus), Banana streak virus (BSV, genus Badnavirus), Banana mild mosaic virus (BanMMV, family Flexiviridae), and Banana bract mosaic virus (BBrMV, genus Potyvirus). Immunocapture (IC)-PCR was used for the detection of BSV while reverse transcription (RT)-PCR was used for the detection of CMV, BanMMV, and BBrMV. The following primers sets were used: BSV cl1 and BSV cl2 (1), CMV 3' and CMV 5' (3), BanMMV BanCP1 and BanCP2 (4), BBrMV Bract N2 and Bract NR (2). BanMMV was detected as mixed infections with BSV in the 10 tested samples, one of which also contained CMV. To confirm the identity of the amplification products from the BanMMV primers, one cDNA fragment was directly sequenced in the forward direction (Macrogen Inc., Seoul, South Korea). BLAST search in GenBank revealed that the partial coat protein (CP) sequence of the Ivorian isolate shared 80 to 88% nucleotides and 81 to 92% deduced amino acid similarities with BanMMV isolates. In contrast, partial CP sequence of the Ivorian isolate had less than 40% deduced amino acid sequence identity with other Flexiviridae CP sequence. The partial CP sequence of the Ivorian BanMMV isolate was deposited in GenBank under Accession No. JX014304. To further confirm the identification, all the samples were tested by plate trapped antigen (PTA)-ELISA with rabbit polyclonal antiserum specific to BanMMV (obtained from B. E. Lockhart, University of Minnesota, U.S.A.) and anti-rabbit IgG (Sigma-Aldrich, Belgium/A3687). The 10 samples reacted positive for BanMMV by ELISA. CMV and BSV have been reported in Ivory Coast, but to our knowledge, this is the first report of BanMMV in the country. The detection of BanMMV in association with BSV or CMV in mixed infection in 10 locations which are important plantain growing areas is a first step in the evaluation of the impact of virus diseases on plantain production in this country.

Signal amplification of microarray-based immunoassay by optimization of nanoliposome formulations

The use of microarray-based immunoassay is often limited by its sensitivity. To increase the sensitivities of such an immunoassay, liposome encapsulation was explored. Two different liposome formations and several preparation methods were examined to optimize encapsulation and signal-enhancing efficacy for enzyme-linked immunosorbent assay (ELISA) and antibody array. The signal amplification by liposome encapsulation was demonstrated through a detection for foodborne pathogenic Listeria. In plate-trapped antigen (PTA) ELISA, horseradish peroxidase (HRP)-loaded liposome increased signal 9-fold more than the control. Limits of detection (LODs) of HRP-encapsulated liposome were 6.4×105 and 5.5×106CFU/ml in sandwich ELISA and antibody array, respectively. Furthermore, when chromogenic 4-chloro-1-naphthol (4-CN) substrate was used for signal development in the antibody array, the signal could be detected with the naked eye. These results suggest that the liposome encapsulation technique can have great potential for signal amplification and, therefore, for increasing assay sensitivity for various formats of immunoassay, especially microarray-based format.

Serological and molecular detection of Macrophomina phaseolina, causing root rot of Citrus reticulata

Polyclonal antibodies (PAbs) were raised against mycelial antigens of Macrophomina phaseolina a causal organism of root rot disease of mandarin plants. IgG was purified and further packaged into immunological formats such as immuno diffusion, Plate trapped antigen (PTA)-ELISA, dot immunobinding assay, Western blot analysis and indirect immunofluorescence for quick and accurate detection of pathogen from soil. Indirect staining of mycelia and sclerotia of M. phaseolina with homologous PAb and labeling with goat antirabbit IgG conjugated with FITC developed strong fluorescence in young hyphal tips and sclerotia of M. phaseolina. Genomic DNA prepared from mycelia of M. phaseolina was purified and PCR amplification of 18S rDNA was done using ITS region specific primer pair. The amplified DNA was sequenced and aligned against ex-type strain sequences from NCBI GenBank using BLAST and phylogenetic analysis was obtained using MEGA4 software. Amplification of ITSI region of the rDNA can be considered as a rapid technique for identifying pathogens successfully in all cases.

First Report of Cowpea aphid-borne mosaic virus on Sesame in Paraguay.

Sesame (Sesamum indicum L.) is cultivated mainly in the central region of the Departamento de San Pedro in Paraguay from October to February and the seed are exported to Asia. The crop is grown on 100,000 ha annually and Escoba blanca is the most common cultivar. The crop plays an important socioeconomical role since it is cultivated mostly by small growers. A disease characterized by yellowing and curling down leaves and shortening of the internodes has been observed in almost all sesame-growing areas. It is referred to locally as "ka'are" because the affected sesame plant resembles Chenopodium ambrosioides L. This disease occurred occasionally and was of marginal importance prior to 2005, but during the last five growing seasons the disease incidence has increased substantially, with some growers losing the entire crop. To determine the causal agent, symptomatic leaf samples were collected from five commercial fields near Colonia San Pedro and Choré, Departamento San Pedro in December 2009. Preliminary transmission electron microscopy (TEM; Zeiss EM900) of extracts from symptomatic leaves revealed the presence of elongated flexible particles resembling a potyvirus. Mechanical transmission assays resulted in chlorotic local lesions on C. quinoa and C. amaranticolor, mosaic on Vigna unguiculata and Nicotiana benthamiana, and symptoms on sesame that are similar to those observed in the field. The disease could also be reproduced in sesame by aphid (Myzus persicae) transmission in a nonpersistent manner. TEM examination of leaf sections of these naturally or experimentally infected plants showed the presence of the type I cylindrical inclusions and masses of filamentous particles. Leaf extracts of naturally or experimentally infected sesame and test plants were positive for Cowpea aphid-borne mosaic virus (CABMV) on the basis of plate-trapped antigen (PTA)-ELISA. CABMV as the causal agent of "ka'are" disease of sesame in Paraguay was further confirmed by analyzing part of the nucleotide sequence of CABMV coat protein and 3' nontranslated region that were obtained directly from reverse transcription-PCR product amplified with PV1-antisense primer (5'-gatttaggtgacactatagt17-3') and WCIEN-sense primer (5'-atggtttggtgyatygaraat-3') (1,2). Comparisons of the 676-bp nucleotide sequence of two sesame virus isolates (GenBank Accession Nos. HQ336402 and HQ336403) revealed 92% identity with the corresponding nucleotide sequence of CABMV available in the GenBank (Accession No. AF348210). Thus, all the assays indicated that the "ka'are" disease of sesame in Paraguay is caused by an isolate of CABMV. Several cowpea fields, nearby sesame diseased crops, also contained plants exhibiting mosaic symptoms. Transmission assays, electron microscopy, PTA-ELISA, and nucleotide sequence analysis indicated that they were also infected by CABMV and may play an important role in the epidemiology of this disease on sesame. CABMV isolates from passion fruit and cowpea from Brazil were mechanically transmitted to sesame but induced milder symptoms. CABMV-infected sesame was described in the United States (3), but to our knowledge, this is the first report of a severe disease on sesame caused by this virus in Paraguay.

First Report of Turnip mosaic virus on Watercress in Brazil.

Watercress (Nasturtium officinale L.), a member of the family Brassicaceae, is consumed mainly as salad. Medicinal properties have also been attributed to this species. In Brazil, watercress is grown mainly by very small farmers. The crop is primarily seed propagated and growers can harvest several times per year in an established planting. Very few diseases have been reported in this crop worldwide. In Brazil, watercress infection by Cauliflower mosaic virus (CaMV) (3), Cucumber mosaic virus (CMV) (1), and an unidentified potyvirus (2) were previously reported. In January 2009, 80% of watercress plants, cv. Gigante Redondo, exhibiting severe mosaic, leaf size reduction, and plant stunting were observed in a crop in Marechal Floriano Municipality, State of Espírito Santo, Brazil. Preliminary leaf dip analysis by transmission electron microscopy revealed the presence of potyvirus-like particles. Sap from five infected plants reacted in plate-trapped antigen (PTA)-ELISA with polyclonal antiserum against Turnip mosaic virus (TuMV), but not with antiserum against CMV. Both antisera were produced in the Plant Virology Laboratory, ESALQ/USP. Mechanically inoculated watercress plants developed similar systemic mosaic symptoms. The virus was also transmitted to Nicotiana benthamiana, which exhibited severe mosaic and stunting. The presence of TuMV on these inoculated plants was confirmed by PTA-ELISA and reverse transcription (RT)-PCR. Total RNA extracted from infected and healthy watercress and infected N. benthamiana was analyzed by RT-PCR using specific pairs of primers flanking the coat protein gene of TuMV. Degenerated anti-sense (5'-t/caacccctt/gaacgcca/cagt/ca-3') and sense (5'-gcaggtgaa/gacg/acttgat/ca/gc-3') primers were designed after analysis to an alignment of the nucleotide sequences for five isolates of TuMV available in the GenBank (Accession Nos. NC_002509, D10927, EU680574, AB362513, and D88614). One fragment of 838 bp was amplified from samples in the infected plants, but not in the healthy controls. Two amplicons were purified and directly sequenced in both directions. Comparisons of the 731-bp consensus nucleotide sequence (Accession No. HM008961) to several other isolates of TuMV revealed 94 to 95% identity in the coat protein region. To our knowledge, this is the first report of TuMV in watercress in Brazil. Management of the disease should include propagation by seeds instead of vegetative parts of the plants and rouging of diseased plants to prevent mechanical transmission during successive harvestings.

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 Papaya ringspot virus-Type W and Zucchini yellow mosaic virus Infecting Trichosanthes cucumerina in Brazil.

Trichosanthes cucumerina L., known as snake gourd, is a cucurbitaceous plant that is probably native to and originally domesticated in India. It is cultivated in humid subtropical and tropical countries of Australia, Latin America, and Africa (2). Plants of this species exhibiting symptoms of mosaic and leaf malformation were found during November 2008 near an experimental field of the Departamento de Fitopatologia e Nematologia, Universidade de São Paulo, Piracicaba, State of São Paulo, Brazil. Electron microscopy examination of negatively stained extract of infected tissue showed the presence of filamentous potyvirus-like particles. Sap from these infected plants reacted in plate-trapped antigen (PTA)-ELISA with the antiserum against Papaya ringspot virus-type W (PRSV-W) or Zucchini yellow mosaic virus (ZYMV), but not with the antiserum against Cucumber mosaic virus (CMV) or Zucchini lethal chlorosis virus (ZLCV). PRSV-W and ZYMV were simultaneously transmitted by mechanical inoculation to four plants of Cucurbita pepo cv. Caserta and one plant of T. cucumerina, causing mosaic. In addition, PRSV-W and ZYMV isolates from our virus collection separately infected one plant of T. cucumerina after mechanical inoculation. Infections were confirmed by PTA-ELISA. Total RNA extracted from infected and healthy T. cucumerina was analyzed by reverse transcription (RT)-PCR using a primer pair specific to the coat protein (CP) gene of PRSV-W (4) or ZYMV (3). Fragments of 864 bp and 1,045 bp were amplified with each pair of primers, respectively. Nucleotide sequences directly obtained from purified PCR products were used for further identification of these potyviruses. The nucleotide and deduced amino acid sequences of part of the CP gene (792 nt) of PRSV-W (GenBank Accession No. GU586789) shared 99 and 98% identity, respectively, with that of the Brazilian isolate PRSV-W-C (GenBank Accession No. 4152). The nucleotide and deduced amino acid sequences of the entire CP gene (837 nt) of ZYMV (GenBank Accession No. 6790) shared 91 to 98% and 94 to 100% identity, respectively, with innumerous isolates of ZYMV deposited in the GenBank (e.g., Accession Nos. AB004640, D13914, AB004641, and AJ420019). Natural infection of T. cucumerina by PRSV-W was reported in Nepal (1). To our knowledge, this is the first report of T. cucumerina infected by PRSV-W and ZYMV in Brazil.