A. Alfaro-Fernández

Seed Transmission of Pepino mosaic virus and Efficacy of Tomato Seed Disinfection Treatments

Rates of seed transmission for Pepino mosaic virus (PepMV) were estimated in seedlings grown from seeds obtained from symptomatic tomato (Lycopersicon esculentum) fruits of plants naturally infected with the virus. The proportion of seeds infected with PepMV was at least 25% as estimated from enzyme-linked immunosorbent assay (ELISA) analysis of grouped seeds. The seeds from symptomatic fruits were planted, and seedlings at the cotyledon and transplant stage were assayed for PepMV by ELISA. Three of 168 seedlings grown from infected seeds were PepMV-positive, corresponding to a seed-to-seedling transmission rate of 1.84%. Various tomato seed treatments were evaluated for their ability to prevent seed transmission of PepMV. This virus was largely eradicated by immersing the seeds in 10% trisodium phosphate for 3 h. Although heat treatments of 24 h at 80°C and 48 h at 74°C eliminated PepMV in seedlings, these treatments did not eradicate the virus in whole seeds. The three treatments did not adversely affect seed germination. The results suggest that trisodium phosphate can be used to eradicate PepMV in tomato seed without hindering germination.

A new haplotype of "Candidatus Liberibacter solanacearum" identified in the Mediterranean region

Abstract“Candidatus Liberibacter solanacearum”, a phloem-limited and Gram-negative bacterium that is spread from infected to healthy plants by psyllid insect vectors, is an economically important pathogen of solanaceous and carrot crops in the Americas, New Zealand and Europe. Three haplotypes of “Ca. L. solanacearum” have previously been described, two (LsoA and LsoB) in relation to solanaceous crops in the Americas and New Zealand and the third (LsoC) to carrots in Finland. Herein, we describe a fourth haplotype of this ‘Candidatus Liberibacter’ species (LsoD), also associated with carrots, but from Spain and the Canary Islands and vectored by the psyllid Bactericera trigonica. In addition, LsoC was confirmed in carrot and psyllid samples recently collected from Sweden and Norway. Phylogenetic analysis of the 16S rRNA gene suggests that two of the haplotypes, one in the Americas and the other in northern Europe are closer to each other in spite of a large geographic separation and host differences. Furthermore, during this study, potatoes with symptoms of zebra chip disease recently observed in potato crops in Idaho, Oregon and Washington states were analyzed for haplotype and were found to be positive for LsoA. This liberibacter haplotype was found in psyllids associated with the diseased potato crops as well. This finding contrasts with an earlier report of LsoB from psyllids in Washington which came from a laboratory colony originally collected in Texas.

First Report of Tomato torrado virus Infecting Tomato in Hungary

During the growing seasons of 2007 and 2008, in commercial greenhouses of tomato crops (Solanum lycopersicum L.) located in Szeged, Öcsöd, and Csongrád (southeastern regions of Hungary), unusual disease symptoms were observed, including necrotic spots in defined areas at the base of the leaflet, necrosis in the stems, and necrotic lines on the fruits surface. Affected plants appeared inside the greenhouses with a random distribution and the incidence recorded was at least 40%. These symptoms resembled those described for Tomato torrado virus (ToTV) infection in Spain (1) and Poland (3). To verify the identity of the disease, three symptomatic plants from commercial greenhouses of each geographic location were selected and analyzed by double-antibody sandwich-ELISA using polyclonal antibodies specific to Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany), and Pepino mosaic virus (PepMV) (DSMZ, Braunschweig, Germany). Total RNA was extracted and tested by reverse transcription (RT)-PCR with three pair of specific primers: one pair used to amplify the coat protein (CP) gene of PepMV (2) and the other two pairs specific to ToTV that amplify 580 bp of the polyprotein (4) and a fragment of 574 bp in the CP Vp23 (3). Nonisotopic dot-blot hybridization using a digoxygenin-labeled RNA probe complementary to the aforementioned fragment of the polyprotein was also performed. Tomato samples were negative for all the viruses tested by serological analysis and for PepMV by RT-PCR. However, all three samples were positive for ToTV by molecular hybridization and RT-PCR. RT-PCR products were purified and directly sequenced. The amplified fragments of the three Hungarian isolates, ToTV-H1, ToTV-H2, and ToTV-H3, for the polyprotein (GenBank Accession Nos. EU835496, FJ616995, and FJ616994, respectively) and the CP Vp23 (GenBank Accession Nos. FJ616996, FJ616997, and FJ616998, respectively) showed 99 to 98% nt identity with the polyprotein and the coat protein regions of ToTV from Spain and Poland (GenBank Accession Nos. DQ3888880 and EU563947, respectively). Whiteflies, commonly found in Hungarian greenhouses, have been reported to transmit ToTV (3), although the efficiency of transmission is unknown. To our knowledge, this is the first report of ToTV in Hungary. References: (1) A. Alfaro-Fernández et al. Plant Dis. 91:1060, 2007. (2) I. Pagán et al. Phytopathology 96:274, 2006. (3) H. Pospieszny et al. Plant Dis. 91:1364, 2007. (4) J. Van der Heuvel et al. Plant Virus Designated Tomato Torrado Virus. Online publication. World Intellectual Property Organization. WO/2006/085749, 2006.

Multiplex PCR assay for the simultaneous detection and differentiation of Olpidium bornovanus, O. brassicae, and O. virulentus

A multiplex PCR method has been developed to detect, differentiate, and confirm the morphological identification of three root infecting Olpidium spp.: O. bornovanus, O. brassicae, and O. virulentus. Of the 132 root samples examined, 101 samples were infected by Olpidium spp.. Based on the morphology of resting spores, the presence of O. bornovanus was confirmed in 20.5% of the samples, whereas species identity could not be determined for the remaining samples because they failed to reproduce sexually. With multiplex PCR, it was possible to determine the Olpidium identity of all the infected samples, even when resting spores were not formed. This method was also effective for detecting Olpidium spp. in water samples. In addition, the specificity and sensitivity of multiplex PCR were evaluated. The multiplex PCR method was validated with samples of 9 different crops from 11 countries of America, Europe, and Africa.

Molecular identification of 16SrII-D subgroup phytoplasmas associated with chickpea and faba bean in Sudan

In January 2011, symptomatic chickpea and faba bean plants were observed in fields located in the Gezira state (Sudan). Faba bean plants showed yellowing and stunting, whereas chickpea plants presented yellowing, reddening and little leaves. The disease etiology was investigated using nested polymerase chain reaction (PCR) with phytoplasma-specific primers which amplify a fragment of the 16S rRNA gene. Sequencing and restriction fragment length polymorphism (RFLP) analyses revealed that the tested phytoplasmas belonged to the group 16SrII. Phylogenetic analyses of the 16S rRNA gene of the obtained sequences indicated that the chickpea and faba bean phytoplasmas from Sudan were more closely related to the phytoplasmas subgroup 16SrII-D. To our knowledge, this is the first report of phytoplasmas from the group 16SrII-D infecting chickpea in Sudan, and faba bean worldwide.

Genetic variability and evolutionary analyses of the coat protein gene of Tomato mosaic virus.

Tomato mosaic virus (ToMV), a member of the genus Tobamovirus, infects several ornamental and horticultural crops worldwide. In this study, the nucleotide sequences of the coat protein gene of worldwide ToMV isolates were analyzed to estimate the genetic structure and diversity of this virus and the involved evolutionary forces. The phylogenetic analysis showed three clades with high bootstrap support: Clade I contained three ToMV isolates from Brazil collected from pepper, Clade II comprised one Brazilian ToMV isolate from pepper, and Clade III was composed of ToMV isolates collected from different plant hosts (pepper, tomato, eggplant, lilac, camellia, dogwood, red spruce, etc.) and water (from melting ice, lakes and streams) from different countries: USA, Brazil, Korea, Germany, Spain, Denmark (Greenland), China, Taiwan, Malaysia, Iran, and Kazakhstan. With the exception of Brazil, nucleotide diversity within and between different geographic regions was very low, although statistical analyses suggested some gene flow between most of these regions. Our analyses also suggested a strong negative selection which could have contributed to the genetic stability of ToMV.

FIRST REPORT OF SOUTHERN TOMATO VIRUS IN TOMATO IN THE CANARY ISLANDS, SPAIN

In October 2006, tomato plants with torrado disease were sampled in Spain. In a sample of cv. Mariana, originating from Gran Canaria, Tomato torrado virus (ToTV, genus Torradovirus) was detected (isolate GCN06; Alfaro-Fernandez et al., 2010). In 2013, the sample was further analysed using next-generation sequencing (NGS). Total RNA, extracted from ca. 150 mg of leaf tissues, was used for an RNA library suitable for Illumina HiSeq paired end sequencing. A de novo assembly (using CLC Genomic Workbench v.6.5) resulted in 1380 sequence contigs. BLAST searches revealed the presence of ToTV and Tomato chlorosis virus (ToCV, genus Crinivirus). One contig (3427 nts, 8901 reads), showed 99.85% identity with Southern tomato virus (STV, genus Amalgavirus), isolate Mexico-1 (GenBank accession No. EF442780), a dsRNA seed-transmitted virus (Sabanadzovic et al., 2009). Reference assembly with the Mexico-1 sequence resulted in a partial sequence of 3420 nts (GenBank KJ174690). The presence of STV in sample GCN06 was verified in RT-PCR using the specific primers STV-fw and STV-rev (Candresse et al., 2013) and sequencing of the obtained amplicon. A new sampling in May 2014 in Gran Canaria revealed the presence of STV in six out of seven tomato samples of cv Mariana. All these samples were co-infected with Pepino mosaic virus (PepMV, genus Potexvirus), Tomato spotted wilt virus (TSWV, genus Tospovirus), and Tomato yellow leaf curl virus (TYLCV, genus Begomovirus). Because STV was generally found in samples co-infected with other viruses, its symptomatology in tomato remains unclear. To our knowledge, this is the first report of STV in the Canary Islands and Spain.

First report of Iris yellow spot virus in commercial leek (Allium porrum) in Spain.

Iris yellow spot virus (IYSV; family Bunyaviridae, genus Tospovirus) has a wide host range, with onion (Allium cepa L.) being one of the most economically important hosts. The first report of IYSV in Spain was from Albacete in 2003 (1) followed by the Canary Islands in 2005. In November of 2006, disease symptoms suspected to be caused by IYSV were observed on the central and outer leaves of commercial leeks plants (cvs. Asthow, Edison, and Shelton) from Alicante, Spain. Symptoms consisted of dry, white-to-straw-colored, spindle-shaped, irregular chlorotic and necrotic lesions on the leaves. Tissue from symptomatic leaves was sampled and analyzed by a double-antibody sandwich (DAS)-ELISA with specific polyclonal antibodies against Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV) (Biorad Phyto-Diagnostics, Marnes-La Coquette, France), IYSV, and Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany). Five of seven leek samples belonging to the three cultivars tested were positive for IYSV. All samples were negative for the other viruses tested. The presence of IYSV was verified in the positive samples by reverse transcription (RT)-PCR using primers derived from the nucleocapsid (N) gene of IYSV (1). RT-PCR gave a PCR amplicon of expected size (approximately 790 bp) from symptomatic leek plants. The product of one of the positive leek samples was purified and sequenced (GenBank Accession No. EF427447). Nucleotide sequence analysis confirmed the identity of the amplicon as that of the IYSV N gene. Sequence comparisons showed 99% identity with the sequence of the IYSV Spanish isolate available in GenBank (Accession No. EF419888). Thrips tabaci is the primary vector of IYSV. Although the vector is present in Spain, the efficiency of the Mediterranean ecotype in transmitting the virus is not known. Leek has been reported to be a host of IYSV in countries such as the Netherlands, Reunion Island, Australia, and the United States (2). To our knowledge, this is the first report of natural infection of leek with IYSV in Spain. References: (1) C. Córdoba-Sellés et al. Plant Dis. 89:1243, 2005. (2) H. F. Schwartz et al. Plant Dis. 91:113, 2007.

Fast detection of Southern tomato virus by one-step transcription loop-mediated isothermal amplification (RT-LAMP)

Southern tomato virus (STV) is a double stranded RNA (dsRNA) virus belonging to genus Amalgavirus (family Amalgamaviridae) which has been detected in tomato plants showing stunting, fruit discoloration and size reduction. A one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of STV in total RNA or sap extracts (obtained just by grinding in buffer) from STV-infected tomato plants by using a set of three primers pairs which were designed to the sequence of the STV putative coat protein. Amplification products were visualized by gel electrophoresis or direct staining of DNA. The sensitivity of RT-LAMP was identical to that of the conventional RT-PCR and less affected by the presence of polymerase inhibitors. STV was detected by RT-LAMP in different tomato tissues, i.e. leaves, roots, fruits and seeds. Also the virus was successfully detected by RT-LAMP from sap extracts obtained from field tomato plants whereas conventional RT-PCR did not. Results of this work show that RT-LAMP is a specific, rapid and cheap procedure to detect STV and it could be implemented on field surveys and sanitation programs.

Detection, characterization and host range studies of Pepino mosaic virus in Cyprus

Pepino mosaic virus (PepMV, Genus Potexvirus, Family Flexiviridae) is a mechanically transmitted viral disease that has emerged as a significant problem of greenhouse tomato crops in Europe and around the world. Although previous studies in Cyprus suggested that the virus was not present on the island, in 2009 tomato fruits from two major tomato production areas exhibited symptoms of yellow mosaic and discolouration, similar to those induced by PepMV. Consequently, an extensive survey was conducted in all tomato producing areas of the country to identify the incidence and prevalence of PepMV in protected and open field tomato crops. Analysis of 3500 leaf samples from tomato plants and weeds with DAS-ELISA and real-time RT-PCR showed that PepMV was present in all tomato growing areas of the island. The virus was detected in both protected and open field tomato plants, as well as in 20 weed species in the families of Amaranthaceae, Chenopodiaceae, Compositae, Convolvulaceae, Malvaceae, Plantaginaceae and Solanaceae. All Cypriot isolates assayed belonged to the CH2 genotype. Biological assays with two Cypriot isolates showed that they could infect cultivated and weed species including Vigna unguiculata, Solanum melongena, Nicotiana tabacum, Malva parviflora, Sonchus oleraceus, Solanum nigrum, Convolvulus arvensis, Chrysanthemum segetum and Calendula arvensis. To our knowledge, this is the first study to report Chrysanthemum segetum and Calendula arvensis as hosts of PepMV.