First report of Tomato brown rugose fruit virus in tomato in Spain.

In October 2019, tomato (Solanum lycopersicum L.) plants showing chlorosis and brown necrosis in apical leaflets and rugose surface in fruits were observed in a greenhouse in Vicar, Almería, Spain. A total of 0.5% of the tomato plants in the greenhouse (1,38 ha) showed these symptoms. The presence of tomato brown rugose fruit virus (ToBRFV) was suspected. A total of 5 symptomatic and 2 symptomless leaf samples were collected and analyzed by double-antibody sandwich (DAS)-ELISA with antibodies for ToBRFV, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) (Loewe Biochemica, Germany). Symptomatic samples tested positive by DAS-ELISA only for ToBRFV. Therefore, one sample was selected and analyzed by reverse transcription (RT)-PCR with specific primers ToBRFV-F5722/ToBRFV-R6179 for ToBRFV (Panno et al. 2019a) which amplified a 458 bp fragment of the coat protein gene. The sequence obtained by Sanger sequencing from the amplicon showed 99.7% nt identity with ToBRFV isolate from United Kingdom (Acc. No. MN182533) and was deposited in the GenBank database under the accession number MT211630. Further surveys were performed in Vicar and El Egido (Almería, Spain) on plants showing viral-like symptoms. A total of 50 tomato and two pepper leaf samples from 28 greenhouses were collected and analyzed by DAS-ELISA for ToBRFV, TMV, ToMV, pepino mosaic virus (PepMV) , tomato spotted wilt virus (TSWV) and tomato yellow leaf curl virus (TYLCV) with respective antibodies (Loewe Biochemica, Germany). Five tomato plants (four from Vicar and one from El Ejido) tested positive for ToBRFV. PepMV, ToMV and TYLCV were also detected in one, three and five tomato plants, respectively. The ELISA positive results for ToBRFV were confirmed by end point RT-PCR with primer pairs ToBRFV-F5722/ToBRFV-R6179 and ToBRFV-F/ToBRFV-R (Alkouni et al., 2019) and by real-time RT-PCR with Taqman probe ToB-probe and specific primers ToB5520F/ToB5598R (Panno et al., 2019b). Tomato seeds used for plantation in these greenhouses were also analyzed, but ToBRFV was not detected. Eradication measures have been undertaken to prevent the virus spread and to control this outbreak. Official seed analysis by DAS-ELISA and real time RT-PCR (ISF, 2019) are being conducted on the tomato and pepper imported seeds to prevent the appearance of new sources of ToBRFV inoculum in Spain. References: Alkowni, R., et al. 2019. J. Plant Pathol. 101: 719. doi: 10.1007/s42161-019-00240-7. ISF, 2019. International Seed Federation. Version 1.3, September 2019. Available at https://www.worldseed.org/wp-content/uploads/2019/09/Tomato-ToBRFV_2019.09.pdf Panno, S., et al. 2019a. Plant Dis. 103: 1443. https://doi.org/10.1094/PDIS-12-18-2254-PDN Panno, S. et al. 2019b. PeerJ 7:e7928 DOI 10.7717/peerj.7928.

Genetic variability and evolutionary analysis of parietaria mottle virus: role of selection and genetic exchange.

The genetic variability and evolution of parietaria mottle virus (PMoV) of the genus Ilarvirus was studied by analyzing nucleotide sequences of 2b and CP genes from isolates collected in different countries. Phylogenetic analysis showed that PMoV isolates clustered in different clades: one (clade I) composed of only Italian isolates and three clades (clades II-IV) including the Spanish isolates. The Greek isolate GrT-1 used in this study was in clade IV for the CP phylogenetic tree whereas it formed a separate branch in the 2b phylogenetic tree. The nucleotide sequence diversity of both the 2b and CP genes was low (0.062 ± 0.006 and 0.063 ± 0.006 for 2b and CP, respectively) but higher than those of other ilarviruses. Distribution of synonymous and nonsynonymous substitutions revealed that 2b and CP proteins are under purifying selection, with some positions under diversifying selection. Genetic exchange among Spanish isolates was also detected.

Association of 'Candidatus Liberibacter solanacearum' with a vegetative disorder of celery in Spain and development of a real-time PCR method for its detection.

A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with 'Candidatus Liberibacter solanacearum' and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect 'Ca. L. solanacearum'. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of 'Ca. Liberibacter' were observed using electron microscopy in celery plant tissues. A fifth haplotype of 'Ca. L. solanacearum', named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

Genetic Variation and Evolutionary Analysis of Eggplant Mottled Dwarf Virus Isolates from Spain.

The genetic variation and population structure of gene N (nucleocapsid) and part of gene L (replicase) from 13 eggplant mottle dwarf virus (EMDV) isolates from Spain were evaluated and compared with sequences of EMDV isolates from other countries retrieved from GenBank. Phylogenetic inference of part of gene L showed three main clades, one containing an EMDV isolate from Australia and the other two containing isolates from Iran and Europe, as well as four subclades. EMDV isolates from Spain were genetically very similar and grouped in a subclade together with one isolate from Germany and one from the UK. No new recombination events were detected in addition to one recombination previously reported, suggesting that recombination is rare for EMDV. The comparison of synonymous and non-synonymous rates showed that negative selection played an important role, and only two codons were under positive selection. Genetic differentiation (Fst test), phylogenetic and nucleotide diversity analyses suggest a unique introduction of EMDV to Spain and low gene flow with other countries. In contrast, Greece and Italy showed diverse populations with high gene flow between both.

Detection and absolute quantitation of watermelon mosaic virus by real-time RT-PCR with a TaqMan probe.

Watermelon mosaic virus (WMV) causes serious damage to several crops worldwide, mainly cucurbits. Disease control is based on preventing spread and search for natural resistances for plant breeding, which requires tools for sensitive detection and precise quantitation. We developed a procedure based on reverse transcription followed by real-time quantitative polymerase chain reaction (RT-qPCR) with a primer pair and a TaqMan® probe specific for WMV. The primers and probe were designed from conserved sequence stretches to target a wide range of WMV isolates. A standard curve performed with transcripts enabled estimation of WMV RNA copies per ng of total RNA, with a wide dynamic range and sensitivity (104 to 1011). This RT-qPCR was assayed with field samples from different cucurbits and used to evaluate the temporal accumulation in pumpkin plants.

Assessment of Multilocus Sequence Analysis (MLSA) for Identification of Candidatus Liberibacter Solanacearum from Different Host Plants in Spain.

Liberibacter is a bacterial group causing different diseases and disorders in plants. Among liberibacters, Candidatus Liberibacter solanaceraum (CLso) produces disorders in several species mainly within Apiaceae and Solanaceae families. CLso isolates are usually grouped in defined haplotypes according to single nucleotide polymorphisms in genes associated with ribosomal elements. In order to characterize more precisely isolates of CLso identified in potato in Spain, a Multilocus Sequence Analysis (MLSA) was applied. This methodology was validated by a complete analysis of ten housekeeping genes that showed an absence of positive selection and a nearly neutral mechanism for their evolution. Most of the analysis performed with single housekeeping genes, as well as MLSA, grouped together isolates of CLso detected in potato crops in Spain within the haplotype E, undistinguishable from those infecting carrots, parsnips or celery. Moreover, the information from these housekeeping genes was used to estimate the evolutionary divergence among the different CLso by using the concatenated sequences of the genes assayed. Data obtained on the divergence among CLso haplotypes support the hypothesis of evolutionary events connected with different hosts, in different geographic areas, and possibly associated with different vectors. Our results demonstrate the absence in Spain of CLso isolates molecularly classified as haplotypes A and B, traditionally considered causal agents of zebra chip in potato, as well as the uncertain possibility of the present haplotype to produce major disease outbreaks in potato that may depend on many factors that should be further evaluated in future works.

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.

Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with other techniques.

BACKGROUND: Tomato brown rugose fruit virus (ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (Solanum lycopersicum L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin. METHODS: Molecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan minor groove binder probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extraction, direct crude extract and leaf-disk crude extract) were evaluated. RESULTS: The Sicilian isolate ToB-SIC01/19 (6,391 nt) showed a strong sequence identity with the isolates TBRFV-P12-3H and TBRFV-P12-3G from Germany, Tom1-Jo from Jordan and TBRFV-IL from Israel. The ToB-SIC01/19 isolate was successfully transmitted by mechanical inoculations in S. lycopersicum L. and Capsicum annuum L., but no transmission occurred in S. melongena L. The developed real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the open reading frames3, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus Tobamovirus, minimising false-negative results. Using immunocapture and total RNA extraction procedures, the real-time RT-PCR and end-point RT-PCR gave the same comparable results. Using direct crude extracts and leaf-disk crude extracts, the end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive real-time RT-PCR assay will be a particularly valuable tool for early ToBRFV diagnosis, optimising procedures in terms of costs and time.

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.

Detection and absolute quantitation of Tomato torrado virus (ToTV) by real time RT-PCR.

Tomato torrado virus (ToTV) causes serious damage to the tomato industry and significant economic losses. A quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) method using primers and a specific TaqMan(®) MGB probe for ToTV was developed for sensitive detection and quantitation of different ToTV isolates. A standard curve using RNA transcripts enabled absolute quantitation, with a dynamic range from 10(4) to 10(10) ToTV RNA copies/ng of total RNA. The specificity of the RT-qPCR was tested with twenty-three ToTV isolates from tomato (Solanum lycopersicum L.), and black nightshade (Solanum nigrum L.) collected in Spain, Australia, Hungary and France, which covered the genetic variation range of this virus. This new RT-qPCR assay enables a reproducible, sensitive and specific detection and quantitation of ToTV, which can be a valuable tool in disease management programs and epidemiological studies.