Detección de patógenos asociados a la enfermedad punta morada en los cultivos de papa y tomate en Guatemala / Detection of pathogens associated to potato purple top disease in potato and tomato crops in Guatemala

La punta morada es una enfermedad que afecta la producción de algunas especies de solanáceas como la papa y el tomate, causando enrollamiento en las puntas de las hojas con una marcada coloración morada, decaimiento temprano de la planta y en la papa se observa tuberización aérea. Como patógenos asociados a la enfermedad se consideran al fitoplasma BLTVA y la bacteria Candidatus Liberibacter solanacearum. Dada la similitud en la sin-tomatología foliar que generan ambos patógenos, es difícil precisar cuál de ellos está implicado en la enfermedad. En Guatemala, existen reportes de la sintomatología típica de punta morada en las principales zonas productoras de papa y tomate, desconociéndose el agente asociado. La investigación determinó cuál de los dos patógenos reportados está asociados a la enfermedad en 12 municipios productores de papa y/o tomate en el país. Se realizaron ampli-ficaciones de ADN con cebadores específicos para cada patógeno asociado a la enfermedad. Por la alta incidencia del fitoplasma BLTVA en las muestras de papa (73.9%), en comparación a C. Liberibacter solanacearum (26%), este es considerado como el patógeno asociado más importante en papa. En las muestras de tomate, la incidencia del fitoplasma BLTVA (29.8%) y C. Liberibacter solanacearum del (27.6%) fue similar. Además, sobresale el primer reporte de la detección del fitoplasma BLTVA afectando el cultivo de tomate en Guatemala. Se sugiere un monitoreo constante, mediante métodos moleculares, para un diagnóstico certero y establecer medidas de manejo de la enfermedad para evitar su diseminación hacia zonas aún no afectadas.

The potato purple top is a disease that affects the production of some solanaceous species such as potatoes and tomatoes, causing curl at the tips of the leaves with a marked purple coloration, early decay of the plant, and aerial tuberization is observed in the potato. BLTVA phytoplasma and Candidatus Liberibacter solanacearum are considered as pathogens associated with the disease. Given the similarity in foliar symptoms generated by both pathogens, it is difficult to determine which one is involved in the disease. There are reports of the typical potato purple top symptoms in the main potato and tomato producing areas in Guatemala, being unknown the associated agent. The research determined which of the two reported pathogens is associated with the disease in 12 potatoes and/or tomato producing areas in the country. We performed DNA amplification with specific primers for each disease-associated pathogen. Due to the high incidence of BLTVA phytoplasma in potato samples (73.9%), com-pared to C. liberibacter solanacearum (26%), this is considered the most important associated pathogen in potatoes. In tomato samples, the incidence of BLTVA phytoplasma (29.8%) and C. liberibacter solanacearum (27.6%) was similar. Besides, the first report of the detection of the BLTVA phytoplasma affecting tomato cultivation in Gua-temala stands out. Using molecular methods, constant monitoring is suggested for an accurate diagnosis and to establish management measures for the disease to prevent its spread to areas not yet affected.

Biological and Genomic Characterization of a Novel Tobamovirus Infecting Hoya spp.

Foliar symptoms suggestive of virus infection were observed on the ornamental plant hoya (Hoya spp.; commonly known as waxflower) in Florida. An agent that reacted with commercially available tobamovirus detection reagents was mechanically transmitted to Chenopodium quinoa and Nicotiana benthamiana. Rod-shaped particles ∼300 nm in length and typical of tobamoviruses were observed in partially purified virion preparations by electron microscopy. An experimental host range was determined by mechanical inoculation with virions, and systemic infections were observed in plants in the Asclepiadaceae, Apocynaceae, and Solanaceae families. Some species in the Solanaceae and Chenopodiaceae families allowed virus replication only in inoculated leaves, and were thus only local hosts for the virus. Tested plants in the Amaranthaceae, Apiaceae, Brassicaceae, Cucurbitaceae, Fabaceae, and Malvaceae did not support either local or systemic virus infection. The complete genome for the virus was sequenced and shown to have a typical tobamovirus organization. Comparisons of genome nucleotide sequence and individual gene deduced amino acid sequences indicate that it is a novel tobamovirus sharing the highest level of sequence identity with Streptocarpus flower break virus and members of the Brassicaceae-infecting subgroup of tobamoviruses. The virus, for which the name Hoya chlorotic spot virus (HoCSV) is proposed, was detected in multiple hoya plants from different locations in Florida.

Infectivity of Deinbollia mosaic virus, a novel weed-infecting begomovirus in East Africa.

Weed-infecting begomoviruses play an important role in the epidemiology of crop diseases because they can potentially infect crops and contribute to the genetic diversity of crop-infecting begomoviruses. Despite the important epidemiological role that weed-infecting begomoviruses play, they remain insufficiently studied in Africa. Recently, we identified Deinbollia mosaic virus (DMV), a distinct begomovirus found naturally infecting the weed host Deinbollia borbonica (Sapindaceae) in Kenya and Tanzania. In this study, we investigated the capacity of DMV to infect a restricted host range of Solanaceae and Euphorbiaceae species. Biolistic inoculation of Nicotiana benthamiana with concatemeric DNAs resulted in systemic infection associated with yellow mosaic symptoms, while DNA partial dimers caused asymptomatic systemic infection. DMV was not infectious to cassava (Manihot esculenta Crantz), suggesting host resistance to the virus. Here, we demonstrate the first experimental infectivity analysis of DMV in N. benthamiana and cassava.

A pathogenicity determinant maps to the N-terminal coat protein region of the Pepino mosaic virus genome.

Pepino mosaic virus (PepMV) poses a worldwide threat to the tomato industry. Considerable differences at the genetic level allow for the distinction of four main genotypic clusters; however, the basis of the phenotypic outcome is difficult to elucidate. This work reports the generation of wild-type PepMV infectious clones of both EU (mild) and CH2 (aggressive) genotypes, from which chimeric infectious clones were created. Phenotypic analysis in three solanaceous hosts, Nicotiana benthamiana, Datura stramonium and Solanum lycopersicum, indicated that a PepMV pathogenicity determinant mapped to the 3'-terminal region of the genome. Increased aggression was only observed in N. benthamiana, showing that this factor is host specific. The determinant was localized to amino acids 11-26 of the N-terminal coat protein (CP) region; this is the first report of this region functioning as a virulence factor in PepMV.

Development of reverse transcription loop-mediated isothermal amplification assay for rapid detection of an emerging potyvirus: tomato necrotic stunt virus.

Tomato necrotic stunt virus (ToNStV) is an emerging potyvirus that causes severe stunting to infected tomato plants. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for sensitive detection of ToNStV. The sensitivity of RT-LAMP was comparable to that of conventional RT-PCR, with detection of ToNStV in a reaction containing only 8 pg of total tomato RNA or with 1:20,000 dilution of crude tissue extract. This assay was able to detect ToNStV in a broad range of solanaceous plant species. The RT-LAMP for ToNStV was specific with no cross-reactivity to other potyviruses (i.e. Potato virus Y and Tobacco etch virus), as well as several other common tomato viruses. RT-LAMP should complement RT-PCR and real-time RT-PCR assays reported previously, with a potential to provide a simple, rapid, and sensitive field diagnostic method for ToNStV.

Yellow tailflower mild mottle virus: a new tobamovirus described from Anthocercis littorea (Solanaceae) in Western Australia.

The complete genome sequence of a tobamovirus was determined from a wild plant of yellow tailflower (Anthocercis littorea, family Solanaceae) that exhibited mild mottling and chlorosis on the leaves. The virus induced severe symptoms including systemic necrosis when inoculated to plants of three other solanaceous species. The viral genome was resequenced after passage in Nicotiana benthamiana. The two genomes were 6379 nucleotides in length, and they differed by three nucleotides. Phylogenetic analysis and the deduced architecture of the genome place the virus, provisionally named yellow tailflower mild mottle virus, with other tobamoviruses that infect solanaceous hosts.

Complete genome sequence of a South Korean isolate of Brugmansia mosaic virus.

We determined the complete genome sequence of a South Korean (SK) isolate of Brugmansia mosaic virus (BruMV), which has recently been proposed to be a member of a new species in the genus Potyvirus. The genomic RNA of BruMV isolate SK is 9781 nucleotides in length (excluding the 3'-terminal poly (A)) and shares complete nucleotide and polyprotein amino acid sequence identities of 85.6 % and 93.1 %, respectively, with the type isolate (D-437) of BruMV described in the USA. To our knowledge, this is the first report providing evidence of considerable sequence variation in BruMV.

A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia.

A suspected virus disease was identified from an arborescent Brugmansia x candida Pers. (syn. Datura candida Pers.) tree. The causal agent was aphid transmissible at low rates. Viral particles were purified from infected tobacco tissue, analyzed, and purified virions were inoculated into healthy tobacco plants to recreate the symptoms. The virions had a mean length of 720-729 nm, and infected cells contained inclusion bodies typical of potyvirus infections. Analysis of infected tissues and purified virions with a panel of potyvirus-specific antibodies confirmed identification as a potyvirus. Viral host range, dilution end point, thermal tolerance and aphid transmission characteristics were examined. The viral genome (9761 nt) is typical of potyviruses, with the closest related potyvirus being pepper mottle virus, at 72 % nt sequence identity. Based on conventions for naming novel potyviruses, the virus was determined to be a member of a previously undescribed species, tentatively named "Brugmansia mosaic virus" (BruMV).

Molecular evolution and phylogeography of potato virus Y based on the CP gene.

Potato virus Y (PVY) is an important plant pathogen with a wide host range that includes, among others, potato, tobacco, tomato and pepper. The coat protein (CP) of PVY has been commonly used in phylogenetic studies for strain classification. In this study, we used a pool of 292 CP sequences from isolates collected worldwide. After detecting and removing recombinant sequences, we applied Bayesian techniques to study the influence of geography and host species in CP population structure and dynamics. Finally, we performed selection and covariation analyses to identify specific amino acids involved in adaptation. Our results show that PVY CP diversification is significantly accounted for by both geographical and host-driven adaptations. Amino acid positions detected as positively selected concentrate in the N-terminal region of the protein. Some of these selected positions may discriminate among strains, and to a much lesser extent, between potato and non-potato isolates.

Leaf curl diseases of two solanaceous species in Southwest Arabia are caused by a monopartite begomovirus evolutionarily most closely related to a species from the Nile Basin and unique suite of betasatellites.

The complete genome of 2780 bases was amplified using rolling circle amplification, and cloned, and sequenced for two distinct strains of the monopartite begomovirus Tomato leaf curl Sudan virus (ToLCSDV). The two strains shared 86-91% identity with the previously described ToLCSDV from the Nile Basin, and 90-91% identity with one another. One strain was cloned from symptomatic tomato plants from Tihamah (ToLCSDV-YE[YE:Tih:05]) while the other was cloned from symptomatic tobacco plants collected from Wadi Hadramaut (ToLCSDV-YE[YE:Had:89]). A distinct full-length betasatellite molecule (1352 bases) was cloned from the respective field-infected tomato and tobacco plants. Agro-inoculation of tomato and Nicotiana benthamiana plants with cloned partial tandem repeats of ToLCSDV-YE[YE:Tih11:05]) and the associated betasatellite, Tomato leaf curl Yemen betasatellite (ToLCYEB-[Tih:tom:137:05]), resulted in the reproduction of leaf curl disease symptoms in test plants like those observed in the field-infected plants. The betasatellite contributed to symptom severity in N. benthamiana test plants when it was co-inoculated with ToLCSDV-YE, compared to the milder symptoms that were observed in tobacco plants infected with the helper virus alone.

Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species.

UNLABELLED: Virus-induced gene silencing (VIGS) is a robust technique for identifying the functions of plant genes. Tobacco rattle virus (TRV)-mediated VIGS has been commonly used in many plants. In order to overcome the limitations of existing agroinoculation methods, we report an easy and effective method of agroinoculation for virus-induced gene silencing-sprout vacuum-infiltration (SVI). Using sprout vacuum-infiltration, we have successfully silenced the expression of phytoene desaturase and Mg-protoporphyrin chelatase genes in four important solanaceous crops, including tomato, eggplant, pepper, and Nicotiana benthamiana. The gene-silenced phenotypes are conspicuous in 1-week-old plants. The method is simple, low cost and rapid compared to other techniques such as leaf infiltration or agrodrench. It may be more practical for studying gene function in the early stages of plant growth. An important aspect of SVI is that it will be used for high-throughput VIGS screens in the future. SVI will be an effective tool to overcome the limitations of current inoculation methods and to facilitate large-scale VIGS analysis of cDNA libraries. KEY MESSAGE: SVI is a simple, low cost agroinoculation method for VIGS. It is practical for studying the function of genes expressed in early stages of plant growth and high-throughput VIGS screens.

The 3'-proximal part of the Plum pox virus P1 gene determinates the symptom expression in two herbaceous host plants.

Three major strains of the Plum pox virus (PPV) are the most important in Europe: PPV-D, PPV-M, and PPV-Rec. By combining the genomes of two different strains of PPV (PPV-D with PPV-Rec; PPV-D with PPV-M), 20 inter-strain chimeric infectious clones (CICPPV) were constructed. Biological properties of CICPPV were tested by inoculating them on different herbaceous host species susceptible to PPV. Four of the seven species tested, exhibited visible symptoms. In Nicotiana benthamiana all CICPPV induced systemic mosaic and leaf malformation. Pisum sativum showed a broad range of symptom severity (systemic chlorotic and necrotic lesions) but neither qualitative nor quantitative aspects of symptomatology were related to a single PPV genome locus. Nicotiana occidentalis and Nicandra physaloides proved to be suitable for symptom-based differentiation. Depending on the virus strain/chimera, N. occidentalis showed two types of symptoms: mild systemic chlorotic spots or local necrotic lesions/systemic vein necroses. N. physaloides reacted to the PPV infection either symptomless or by local necrotic lesions. Our results demonstrated that the P1/HC-pro region of the PPV genome appears to be the determinant of the symptom manifestation in these host plants. In silico analysis mapped it to the 3'-proximal part of the P1 gene.

Multihost experimental evolution of a plant RNA virus reveals local adaptation and host-specific mutations.

For multihost pathogens, adaptation to multiple hosts has important implications for both applied and basic research. At the applied level, it is one of the main factors determining the probability and the severity of emerging disease outbreaks. At the basic level, it is thought to be a key mechanism for the maintenance of genetic diversity both in host and pathogen species. Using Tobacco etch potyvirus (TEV) and four natural hosts, we have designed an evolution experiment whose strength and novelty are the use of complex multicellular host organism as hosts and a high level of replication of different evolutionary histories and lineages. A pattern of local adaptation, characterized by a higher infectivity and virulence on host(s) encountered during the experimental evolution was found. Local adaptation only had a cost in terms of performance on other hosts in some cases. We could not verify the existence of a cost for generalists, as expected to arise from antagonistic pleiotropy and other genetic mechanisms generating a fitness trade-off between hosts. This observation confirms that this classical theoretical prediction lacks empirical support. We discuss the reasons for this discrepancy between theory and experiment in the light of our results. The analysis of full genome consensus sequences of the evolved lineages established that all mutations shared between lineages were host specific. A low degree of parallel evolution was observed, possibly reflecting the various adaptive pathways available for TEV in each host. Altogether, these results reveal a strong adaptive potential of TEV to new hosts without severe evolutionary constraints.

A cryohistological protocol for preparation of large plant tissue sections for screening intracellular fluorescent protein expression.

In this study, we have developed a robust cryohistological method that allows imaging of virtually any type of plant cell or tissue while preserving fluorescent protein signals and maintaining excellent cellular and subcellular morphology. This method involves modified fixation of plant tissues (i.e., leaves, stems, and petioles), infiltration in a sucrose gradient, freezing, and collection of cryosections directly onto a cryoadhesive tape. Using this method followed by microscopic analysis, we demonstrated a localized accumulation of green fluorescent protein (GFP) in Nicotiana benthamiana plants agroinfiltrated with the movement-incompetent tobacco mosaic virus-based vector and systemic accumulation of GFP in plants infiltrated with the movement-competent vector. Overall, this simple cryohistological procedure reduced sample preparation time and allowed processing of tissue sections for high-resolution imaging of targeted fluorescent proteins in all plant tissues.

Effect of host species on the distribution of mutational fitness effects for an RNA virus.

Knowledge about the distribution of mutational fitness effects (DMFE) is essential for many evolutionary models. In recent years, the properties of the DMFE have been carefully described for some microorganisms. In most cases, however, this information has been obtained only for a single environment, and very few studies have explored the effect that environmental variation may have on the DMFE. Environmental effects are particularly relevant for the evolution of multi-host parasites and thus for the emergence of new pathogens. Here we characterize the DMFE for a collection of twenty single-nucleotide substitution mutants of Tobacco etch potyvirus (TEV) across a set of eight host environments. Five of these host species were naturally infected by TEV, all belonging to family Solanaceae, whereas the other three were partially susceptible hosts belonging to three other plant families. First, we found a significant virus genotype-by-host species interaction, which was sustained by differences in genetic variance for fitness and the pleiotropic effect of mutations among hosts. Second, we found that the DMFEs were markedly different between Solanaceae and non-Solanaceae hosts. Exposure of TEV genotypes to non-Solanaceae hosts led to a large reduction of mean viral fitness, while the variance remained constant and skewness increased towards the right tail. Within the Solanaceae hosts, the distribution contained an excess of deleterious mutations, whereas for the non-Solanaceae the fraction of beneficial mutations was significantly larger. All together, this result suggests that TEV may easily broaden its host range and improve fitness in new hosts, and that knowledge about the DMFE in the natural host does not allow for making predictions about its properties in an alternative host.

What can we learn from tobacco and other Solanaceae about horizontal DNA transfer?

In eukaryotic organisms, horizontal gene transfer (HGT) is regarded as an important though infrequent source of reticulate evolution. Many confirmed instances of natural HGT involving multicellular eukaryotes come from flowering plants. This review intends to provide a synthesis of present knowledge regarding HGT in higher plants, with an emphasis on tobacco and other species in the Solanaceae family because there are numerous detailed reports concerning natural HGT events, involving various donors, in this family. Moreover, in-depth experimental studies using transgenic tobacco are of great importance for understanding this process. Valuable insights are offered concerning the mechanisms of HGT, the adaptive role and regulation of natural transgenes, and new routes for gene trafficking. With an increasing amount of data on HGT, a synthetic view is beginning to emerge.

Formation of protein complexes containing plant virus movement protein TGBp3 is necessary for its intracellular trafficking.

Cell-to-cell movement of Poa semilatent virus (genus Hordeivirus) in infected plants is mediated by three viral 'triple gene block' (TGB) proteins. One of those termed TGBp3 is an integral membrane protein essential for intracellular transport of other TGB proteins and viral genomic RNA to plasmodesmata. TGBp3 targeting to plasmodesmata-associated sites is believed to involve an unconventional mechanism which does not employ endoplasmic reticulum-derived transport vesicles. Previously TGBp3 has been shown to contain a composite transport signal consisting of the central hydrophilic protein region which includes a conserved pentapeptide YQDLN and the C-terminal transmembrane segment. This study demonstrates that these TGBp3 structural elements have distinct functions in protein transport. The YQDLN-containing region is essential for TGBp3 incorporation into high-molecular-mass protein complexes. In transient expression assay formation of such complexes is necessary for entering the TGBp3-specific pathway of intracellular transport and protein delivery to plasmodesmata-associated sites. In virus-infected plants TGBp3 is also found predominantly in the form of high-molecular-mass complexes. When the complex-formation function of YQDLN-containing region is disabled by a mutation, targeting to plasmodesmata-associated sites can be complemented by a heterologous peptide capable of formation multimeric complexes. The C-terminal transmembrane segment is found to be an essential signal of TGBp3 intracellular transport to peripheral sites.

A broad-spectrum, efficient and nontransgenic approach to control plant viruses by application of salicylic acid and jasmonic acid.

Plant viruses cause many diseases that lead to significant economic losses. However, most of the approaches to control plant viruses, including transgenic processes or drugs are plant-species-limited or virus-species-limited, and not very effective. We introduce an application of jasmonic acid (JA) and salicylic acid (SA), a broad-spectrum, efficient and nontransgenic method, to improve plant resistance to RNA viruses. Applying 0.06 mM JA and then 0.1 mM SA 24 h later, enhanced resistance to Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV) and Turnip crinkle virus (TCV) in Arabidopsis, tobacco, tomato and hot pepper. The inhibition efficiency to virus replication usually achieved up to 80-90%. The putative molecular mechanism was investigated. Some possible factors affecting the synergism of JA and SA have been defined, including WRKY53, WRKY70, PDF1.2, MPK4, MPK2, MPK3, MPK5, MPK12, MPK14, MKK1, MKK2, and MKK6. All genes involving in the synergism of JA and SA were investigated. This approach is safe to human beings and environmentally friendly and shows potential as a strong tool for crop protection against plant viruses.

Complete genome sequence of Brugmansia suaveolens mottle virus, a potyvirus from an ornamental shrub.

Brugmansia suaveolens mottle virus (BsMoV) was the first potyvirus isolated from "angel trumpet" (Brugmansia suaveolens), described in Brazil. In this study, the complete nucleotide (nt) genome sequence of BsMoV was determined, and the deduced amino acid (aa) sequence was analyzed. The BsMoV RNA genome consists of 9870 nt without a poly-A tail, encoding a putative typical potyviral polyprotein of 3090 aa. Pairwise comparisons of the complete BsMoV genome with those of the most closely related potyviruses revealed a maximum nucleotide identity of 63.7% with pepper mottle virus. These results and phylogenetic analyses based on the complete genome sequence of the most closely related potyviruses confirmed that BsMoV should be considered a member of a distinct species of the genus Potyvirus.

Endogenous pararetroviral sequences in tomato (Solanum lycopersicum) and related species.

BACKGROUND: Endogenous pararetroviral sequences (EPRVs) are a recently discovered class of repetitive sequences that is broadly distributed in the plant kingdom. The potential contribution of EPRVs to plant pathogenicity or, conversely, to virus resistance is just beginning to be explored. Some members of the family Solanaceae are particularly rich in EPRVs. In previous work, EPRVs have been characterized molecularly in various species of Nicotiana including N.tabacum (tobacco) and Solanum tuberosum (potato). Here we describe a family of EPRVs in cultivated tomato (Solanum lycopersicum L.) and a wild relative (S.habrochaites). RESULTS: Molecular cloning and DNA sequence analysis revealed that tomato EPRVs (named LycEPRVs) are most closely related to those in tobacco. The sequence similarity of LycEPRVs in S.lycopersicum and S.habrochaites indicates they are potentially derived from the same pararetrovirus. DNA blot analysis revealed a similar genomic organization in the two species, but also some independent excision or insertion events after species separation, or flanking sequence divergence. LycEPRVs share with the tobacco elements a disrupted genomic structure and frequent association with retrotransposons. Fluorescence in situ hybridization revealed that copies of LycEPRV are dispersed on all chromosomes in predominantly heterochromatic regions. Methylation of LycEPRVs was detected in CHG and asymmetric CHH nucleotide groups. Although normally quiescent EPRVs can be reactivated and produce symptoms of infection in some Nicotiana interspecific hybrids, a similar pathogenicity of LycEPRVs could not be demonstrated in Solanum L. section Lycopersicon [Mill.] hybrids. Even in healthy plants, however, transcripts derived from multiple LycEPRV loci and short RNAs complementary to LycEPRVs were detected and were elevated upon infection with heterologous viruses encoding suppressors of PTGS. CONCLUSION: The analysis of LycEPRVs provides further evidence for the extensive invasion of pararetroviral sequences into the genomes of solanaceous plants. The detection of asymmetric CHH methylation and short RNAs, which are hallmarks of RNAi in plants, suggests that LycEPRVs are controlled by an RNA-mediated silencing mechanism.