The main of the present study was to prepare chitosan (Chit) and chitosan hydroxyapatite nanoparticle (Chit-nHap) to assess their activities against two major diseases affecting tomato crop: pepino ...mosaic virus (PepMV) and verticillium wilt caused by the fungus Verticillium dahliae. The synthesized compounds were characterized using X-rays diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electronic microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). In vitro, the application of various concentrations of Chit and Chit-nHap showed significant inhibition of radial mycelial growth against V. dahliae. However, Chit-nHap was found to be more effective than Chit. In greenhouse trials, the disease severity was significantly reduced when Chit and Chit-nHap were sprayed to the leaves at 150 and 250 μg mL−1. The highest concentration of Chit and Chit-nHap reduced leaf alteration index by more than 81% and browning index by 96%. Both compounds also reduced PepMV disease severity and virus accumulation. Protection afforded against PepMV was associated with differential accumulation of H2O2 and with potentiation of the activity of antioxidant enzymes such as catalase, ascorbate peroxidase, guaiacol peroxidase and polyphenol oxidase.
•Chitosan nanohydroxyapatite was synthetized and characterized.•Chitosan nanohydroxyapatite reduced Verticillium wilt and PepMV severity in tomato.•Protection against PepMV was associated with modulation of antioxidant defenses.
Pepino mosaic virus (PepMV) is a highly infectious potexvirus, which has presently become a major pathogen for tomato crops worldwide. Since there is no effective method control for PepMV, strict ...preventive hygienic measures and cross‐protection have been established. However, the use of mild isolates to protect against aggressive isolates provides opportunities for interaction with other microorganisms, which under certain conditions may worsen disease symptoms. Thus, alternative control methods are needed. In this study we aimed at the development of chemical control against PepMV based on the use of heterocyclic compounds including symmetrical 2,5‐disubstituted 1,3,4‐oxadiazoles, symmetrical 2,5‐disubstituted 1,3,4‐thiadiazole and dihydrotetrazine derivatives. Results showed that spray application of three 1,3,4‐oxadiazole derivatives (OH‐Oxa, CH3‐Oxa, NO2‐Oxa) and of the derivative of 1,3,4‐thiadiazole (OH‐Thia) resulted in reducing the main symptoms of PepMV in tomato leaves, conversely to the dihydrotetrazine carboxylic acid. Double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) revealed that PepMV was not detected from tomato plants pre‐treated with the derivatives conferring elevated protection. Moreover, the protective ability of OH‐Oxa, CH3‐Oxa, NO2‐Oxa and OH‐Thia was closely related to the enhancement of the activity of antioxidant enzymes upon infection with PepMV.
The pepino (Solanum muricatum) is an Andean vegetable crop closely related to tomato. In the last decades, it has been introduced in the Mediterranean region and other parts of the world as a ...potential new crop. However, several tomato major pathogens may threaten the expansion of pepino cultivation. We identified Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), pepino mosaic virus (PepMV) and tomato mosaic virus (ToMV) as four of the most likely pathogens to cause damage to pepino crops in Mediterranean climates. In order to evaluate the response of the pepino genepool against these pathogens, as well as to identify sources of tolerance, we inoculated six accessions of cultivated pepino, nine accessions of seven pepino wild relatives, and one interspecific hybrid with FOL, VE, PepMV and ToMV and followed its symptomatology for 30 days (FOL and VE) or 60 days (PepMV and ToMV). ELISA tests were also performed for PepMV and ToMV. Susceptible tomato materials were used as controls. The pepino genepool displayed fewer symptoms than susceptible tomato controls after inoculation with FOL, with most accessions being tolerant or resistant. Regarding VE, a wide variation of values for the symptoms index (SI) was observed, with three cultivated pepino accessions displaying tolerance. For PepMV a wide variation for SI was also observed, with one accession of S. caripense being resistant, and several accessions of pepino and other wild relatives displaying different degrees of tolerance. PepMV absorbance values obtained by ELISA tests followed a pattern similar to that of SI. For ToMV no resistances were found, although two wild accessions and the interspecific hybrid displayed low values for the SI and were considered as moderately tolerant. ELISA tests against ToMV revealed that the virus replicated well in all materials. None of the accessions evaluated displayed resistance or high levels of tolerance to the four pathogens, but some of them were complementary for resistance or high levels of tolerance. Although the interspecific hybrid tested was not resistant to any of the pathogens, it was tolerant to FOL and PepMV and moderately tolerant to VE and ToMV. A multivariate hierarchical clustering revealed similar patterns among accessions in the response to the two fungal diseases (FOL and VE) on one side and to the two viral ones (PepMV and ToMV) on the other. The information generated in this study has allowed identifying materials within the pepino genepool for the development of multi‐resistant pepino cultivars to major diseases threatening its expansion in the Mediterranean region.
Pepino mosaic virus (PepMV, genus Potexvirus) is an emergent and highly infectious pathogen responsible for economically important diseases in tomato crops. An extensive survey of tomato plants ...showing PepMV‐like symptoms was carried out in 2017 to study the PepMV genetic diversity and populations structure in different tomato‐producing areas of Spain and Morocco. Molecular dot‐blot hybridization analysis showed that virus populations from Spain and Morocco were mainly composed of isolates belonging to the Chilean 2 (CH2) strain, although isolates of the European (EU) strain were detected in significant proportions in Spanish populations, mainly in mixed infections. A few isolates of the American (US1) strain were also detected in Tenerife (Canary Islands, Spain) crops. Eighty‐five isolates were randomly selected and sequenced in the genomic region that encodes the triple gene block and capsid protein genes. Our phylogenetic and population genetics analyses confirmed the presence of the CH2, EU and US1 PepMV strains. Despite the high genetic similarity observed within populations, variants were maintained at low frequency under purifying selection, and differentiation among more geographically distant locations was identified, with potential gene flow contributing to the shaping of the PepMV populations structure.
This study provides insights on the occurrence and molecular characterization of PepMV in two major tomato‐producing areas in Spain (south‐eastern Spain and Canary Islands) and Morocco in 2017. Our results show that Occidental Mediterranean PepMV populations are predominantly composed of PepMV isolates belonging to the CH2 strain, although the EU strain was also found predominantly under mixed infections, and the US1 genotype was restricted to a few cases in Tenerife.
In situ hybridization (ISH) is an informative and relatively accessible technique for the localization of viral genomes in plant tissue and cells. However, simultaneous visualization of related plant ...viruses in mixed infections may be limited by the nucleotide similarity in the genomes and the single chromogenic detection over the same sample preparation. To address this issue, we used two Pepino mosaic virus isolates and performed ISH over consecutive serial cross-sections of paraffin-embedded leaf samples of single and mixed infected Nicotiana benthamiana plants. Moreover, the probe design was optimized to reduce cross-hybridisation, and co-localization was based on the overlapping of consecutive cross-sections from mixed infected leaves; thus, our results showed that both Pepino mosaic virus isolates co-localized in the same leaf tissue. In turn, both isolates were localized in the cytoplasm of the same cells. These results provide valuable information for studying mixed infections in plants by using a simple ISH procedure that is accessible to any pathology laboratory.
Tomato (Solanum lycopersicum L.) originated in South America and was brought to Europe by the Spaniards in the sixteenth century following their colonization of Mexico. From Europe, tomato was ...introduced to North America in the eighteenth century. Tomato plants show a wide climatic tolerance and are grown in both tropical and temperate regions around the world. The climatic conditions in the Mediterranean basin favor tomato cultivation, where it is traditionally produced as an open-field plant. However, viral diseases are responsible for heavy yield losses and are one of the reasons that tomato production has shifted to greenhouses. The major tomato viruses endemic to the Mediterranean basin are described in this chapter. These viruses include Tomato yellow leaf curl virus, Tomato torrado virus, Tomato spotted wilt virus, Tomato infectious chlorosis virus, Tomato chlorosis virus, Pepino mosaic virus, and a few minor viruses as well.
Tomato (
) plants from a commercial glasshouse were identified with symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection. Reverse transcription-PCR and quantitative PCR ...confirmed the presence of ToBRFV. Subsequently, the same RNA sample and a second from tomato plants infected with a similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT). For the targeted detection of ToBRFV, the two libraries were synthesized by using six ToBRFV sequence-specific primers in the reverse transcription step. This innovative target enrichment technology enabled deep coverage sequencing of ToBRFV, with 30% of the total reads mapping to the target virus genome and 57% mapping to the host genome. The same set of primers applied to the ToMMV library generated 5% of the total reads mapping to the latter virus, indicating that sequencing of similar, non-target viral sequences was also allowed. Further, the complete genome of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus suggesting that, even using multiple sequence-specific primers, a low rate of off-target sequencing can usefully provide additional information on unexpected viral species coinfecting the same samples in an individual assay. These results demonstrate that targeted nanopore sequencing can specifically identify viral agents and has sufficient sensitivity towards non-target organisms to provide evidence of mixed virus infections.
Waterborne and seedborne Pepino mosaic virus (PepMV) and Potato spindle tuber viroid (PSTVd) pose serious threats to tomato production due to seed transmission and mechanical transmission, coupled ...with their long‐term stability outside the host plant. Therefore, rapid and sensitive diagnostic procedures are needed to prevent the spread of these quarantine pathogens. In particular, water and seed contamination are difficult to detect and confirm without efficient concentration methods. This study presents procedures that improve detection of PSTVd from tomato seeds and leaf tissue, and PepMV from water and tomato leaf tissue. For efficient concentration of PepMV from water samples, a procedure was optimized using convective interaction media monolithic chromatography columns, which provides concentration by three orders of magnitude. For concentration of PSTVd from seed extracts, an easy‐to‐use and efficient method was developed based on RNA binding to positively charged anion‐exchange resin beads that provides up to 100‐fold more sensitive detection in comparison with procedures without a concentration step. This thus allows confirmation of RT‐qPCR results with sequencing of RT‐PCR products in samples with low viroid levels. In addition, reverse‐transcription loop‐mediated isothermal amplification assays for detection of PSTVd and PepMV were optimized and adapted to both laboratory and on‐site testing requirements. This allows rapid detection of these pathogens in crude leaf homogenates, in under 30 min. These procedures of concentration and detection are shown to be efficient and to fill the gaps in diagnostics of PepMV and PSTVd, especially when these pathogens are present at low levels in difficult matrices such as water and seeds.
Shortage of water availability and awareness of the need for sustainable resource management have generated a significant increase in the use of recycled water for irrigation and processing of crops ...and harvest products, respectively. As a result, irrigation systems face the challenge of neutralizing plant pathogens to reduce the risk of their dispersal and the subsequent occurrence of diseases with potentially high economic impacts. We evaluated the efficacy of an innovative electrolytic disinfection system based on potassium hypochlorite (KCLO) to inactivate major pathogens in hydroponically grown tomatoes: Fusarium oxysporum (Synder and Hans), Rizocthonia solani (Kühn), Tobacco mosaic virus (TMV) and Pepino mosaic virus (PepMV). The electrolytically derived disinfectant was prepared on-site and added to the recirculating fertigation solution once a week for 60 min in an automated manner using sensor technology at a dosage of 0.5 mg of free chlorine/L (fertigation solution at pH 6.0 ± 0.3 and ORP 780 ± 31 mV). Tomato fruit yield and pathogen dispersal were determined for 16 weeks. At the applied dosage, the disinfectant has been shown to inhibit the spread of plant pathogenic fungi and, remarkably, plant viruses in recirculating fertigation solutions. Phytotoxic effects did not occur.
PDF
