The white mango scale, Aulacaspis tubercularis Newstead (Hemiptera: Diaspididae) is an exotic pest that causes important economic damage in the Spanish mango crops. Prospecting for potential natural enemies, the parasitic wasp Arrhenophagus chionaspidis Aurivillius (Hymenoptera: Encyrtidae) was first detected attacking immature male stages of A. tubercularis in Tenerife (Canary Islands, Spain) in 2022. Still, its influence on pest populations was not evaluated. This endoparasitoid was identified by its morphological characteristics and characterized for the first time by DNA barcoding of adult females using partial (651 bp) mitochondrial cytochrome oxidase 1 (mtCOI) gene (Genbank accession number OQ646824). Its potential utilization as biological control agent of A. tubercularis in mango crops is discussed. Therefore, further studies need to be conducted about its biology, population dynamics and rate of natural parasitism for the development of effective biological control strategies of A. tubercularis in mango.

The control of emerging viruses in current agriculture confronts many challenges, including the appearance of new strains or the arrival of new species, requiring a multidisciplinary approach. The usual methods for the control of these pathogens are based on management practices, vector control, seed control, etc., and the introgression of genetic resistance in cultivars. But the development of resistances by classical genetic methods is a costly and time-consuming process. A promising alternative is the control of these pathogens by means of the so-called SIGS (Spray-Induced Gene Silencing) that consists of topical treatments with specific molecules derived from the viruses as double-stranded RNA (dsRNA), triggering the plant defense mechanisms. In this work, we show the control of two viruses, the potyvirus turnip mosaic virus (TuMV) and the curtovirus beet curly top virus (BCTV) by dsRNA nanocomposites with carbon dots or mesoporous silica nanoparticles in Nicotiana benthamiana. In the case of TuMV, the disease was significantly reduced in terms of plant photosynthetic capacity and viral titers, in the dsRNA treated plants. Moreover, when the treatments were carried out with dsRNAs as nanocomposites, the differences were even more noticeable with respect to untreated inoculated plants. In the case of BCTV, a significant delay in symptoms appearance was observed after the treatments with the dsRNA nanocomposites, but not with the naked dsRNAs. Viral titers were reduced either with the naked or the nanoparticle-delivered dsRNAs. The increased efficiency of dsRNA for virus control when supplied with the nanoparticles can be related with the enhanced dsRNA delivery reported in this work. ### Competing Interest Statement The authors have declared no competing interest.

Andalusia is a Spanish region that is home to numerous minority varieties due to its diversity and territorial extension, offering the local viticulture the possibility of diversifying its wine production. The genotypic characterization of 98 specimens from six areas with a winemaking tradition in Andalusia was carried out between the years 2020 and 2022, by means of thirteen microsatellite markers, including the nine recommended by the OIV. A total of 33 different genotypes were obtained, 20 of which corresponded to profiles of already described varieties (11 of them are of 6 minority cultivars in Andalusia: ‘Rojal Tinto’, ‘Beba’, ‘Zurieles’, ‘Rome’, ‘Hebén’, ‘Mollar Cano’, ‘Listán Prieto’, ‘Listán del Condado’, ‘Jarrosuelto’, ‘Negra Dorada’, and ‘Mantúo de Pilas’), while the other 12 profiles did not match with previously identified varieties. These profiles were registered in the database of the IFAPA “Rancho de la Merced” Germplasm Bank. The eco-geographical groups of the new identified genotypes were determined through an analysis of genetic diversity. The presence of grapevine fanleaf virus, grapevine fleck virus, and grapevine leafroll-associated viruses was also determined due to the requirement of healthy clones of the new varieties for their potential interest to be authorized for cultivation in Spain.

The white mango scale (WMS), Aulacaspis tubercularis, is the main pest of mango crops in Southern Spain. Field surveys were carried out from July 2019 to May 2022 to investigate the identity, seasonal abundance and active parasitism rates of A. tubercularis primary parasitoid species, using yellow sticky traps and periodic leaf sampling. The combination of morphological characters and DNA sequences of the mitochondrial cytochrome c oxidase subunit 1 (mtCOI) and 28S ribosomal (28S rDNA) genes allowed the identification of only two parasitoid species belonging to the genus Encarsia F & ouml;rster (Hymenoptera: Aphelinidae). The most prevalent, E. citrina (Crawford), is a widespread species and proved to be the dominant species in the sampled mango orchards. Next, Encarsia sp., is an unidentified species genetically close to E. lounsburyi Berlese but significantly differs in the sequences of the mtCOI and 28S rDNA regions. Population abundance and active parasitism rates of Encarsia parasitoids depended on the presence of susceptible WMS stages for parasitism and on the abiotic conditions. The seasonal abundance of these parasitoid species fluctuated around the year, being lower during the winter but it increased in late spring, reaching the highest values at the end of summer. The average percentage of active parasitism was 12.48% for female scales and 13.97% for male scales but reached maximum values of 38.56 and 43.52%, respectively at specific seasons in the year. The highest rates of parasitism for female scales were observed in summer, while for male scales were observed in winter. The natural occurrence of parasitoids was unable to control the WMS populations and, consequently, complementary measures should be implemented to reduce its density levels.

Debido a su diversidad y extensión territorial, Andalucía es una región española que alberga numerosas variedades minoritarias, ofreciendo a la vitivinicultura local la posibilidad de diversificar su producción vitivinícola. Desde 2020, se ha realizado la identificación molecular de 98 ejemplares procedentes de seis zonas con tradición vitivinícola: la Alpujarra (9 individuos) y el Altiplano (2) en Granada; Moguer (12) en Huelva; el Valle de los Pedroches (36) y Montilla-Moriles (32) en Córdoba; el Pago Burujena (7)en Cádiz. Se han empleado trece marcadores microsatélites, entre ellos los nueve recomendados por la OIV. Se han obtenido 31 genotipos distintos, 20 se corresponden a perfiles de variedades ya descritas, los otros 11 no se han identificado previamente. Los perfiles se han integrado en el database del banco de germoplasma de vid “Rancho de la Merced”. Mediante análisis de diversidad genética, se ha determinado en cuales grupos eco-geográficos se colocan los nuevos genotipos identificados. Al tratarse de nuevas variedades con un interés potencial en ser autorizadas, se quiere disponer de clones certificables; por ello, se ha analizado la presencia de los virus del enrollado, del entrenudo corto infeccioso y del jaspeado de la vid, mediante test ELISA.

In this work, we obtained carbon dots from glucose or saccharose as the nucleation source and passivated them with branched polyethylenimines for developing dsRNA nanocomposites. The CDs were fully characterized using hydrodynamic analyses, transmission electron microscopy, X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The ζ potential determined that the CDs had positive charges, good electrophoretic mobility and conductivity, and were suitable for obtaining dsRNA nanocomposites. DsRNA naked or coated with the CDs were delivered to leaves of cucumber plants by spraying. Quantitation of the dsRNA that entered the leaves showed that when coated with the CDs, 50-fold more dsRNA was detected than when naked dsRNA. Moreover, specific siRNAs derived from the sprayed dsRNAs were 13 times more abundant when the dsRNA was coated with the CDs. Systemic dsRNAs were determined in distal leaves and showed a dramatic increase in concentration when delivered as a nanocomposite. Similarly, systemic siRNAs were significantly more abundant in distal leaves when spraying with the CD-dsRNA nanocomposite. Furthermore, FITC-labeled dsRNA was shown to accumulate in the apoplast and increase its entry into the plant when coated with CDs. These results indicate that CDs obtained by hydrothermal synthesis are suitable for dsRNA foliar delivery in RNAi plant applications.

The IFAPA research center “Rancho de la Merced” (Jerez, Spain) hosts one of the oldest and most diverse grapevine germplasm repositories in Europe, and is aimed at providing feasible solutions to deal with any agronomic trait by exploring its genetic variability and by means of association and Deoxyribonucleic Acid (DNA) editing studies. In this work, we focused on a wine and dual-use grapevine subcollection that consists of 930 accessions. Genetic analysis allowed to identify 521 unique genotypes. After comparing them with several databases, matches were found for 476 genetic profiles while the remaining 45 have not been previously described. Combination with clustering analysis suggested a total pool of 481 Vitis vinifera accessions that included some table cultivars. Several synonymies, homonymies and mislabeling have also been detected. Structure analysis allowed identifying six clusters according to eco-geographic cultivation areas and one additional group including non-vinifera accessions. Diversity analysis pointed out that Spanish Mediterranean varieties are genetically closer to oriental genotypes than to European varieties typical of oceanic and continental climates. The origin of Spanish varieties is discussed in depth considering our data and previous studies. Analysis of molecular variance partition confirmed a well-structured germplasm, although differentiation among groups had a much lower effect on genetic variability than differences within groups, which are strongly related to a very high heterozygosity. A core collection that covers all allele richness is proposed. It is constituted of about 13% of total accessions, and each cluster inferred by structure analysis is represented.

The recurrent emergence of viral diseases in intensive horticultural crops requires alternative control strategies. The topical application of double-stranded RNA (dsRNA) molecules homologous to pathogens has been proposed as a tool for virus control in plants. These dsRNAs induce the silencing mechanism, the RNA interference (RNAi), that degrades homologous dsRNAs. Cucumber green mottle mosaic virus (CGMMV) represents a serious threat to cucurbit crops. Since genetic resistance to the virus is not yet available in commercial varieties, we aimed to control this virus by RNAi. For this purpose, we obtained constructions both for expressing dsRNA in bacteria to treat cucumber plants by topical application and for agroinoculation in experiments done in the growth chamber. Besides, greenhouse tests were performed in spring and in summer when plants were challenged with the virus, and differences in several parameters were investigated, including the severity of symptoms, dry weight, total height, virus accumulation, and virus-derived small interfering RNAs (vsiRNAs). Spraying of plants with dsRNA reduced significatively CGMMV symptoms in the plants in growth chamber tests. Agroinfiltration experiments done under identical conditions were also effective in limiting the progress of CGMMV disease. In the greenhouse assay performed in spring, symptoms were significantly reduced in dsRNA-sprayed plants, and the development of the plants improved with respect to non-treated plants. Virus titers and vsiRNAs were clearly reduced in dsRNA-treated plants. The effect of protection of the dsRNA was less evident in the greenhouse assay carried out in the summer. Besides, we investigated the mobility of long (ds)RNA derived from spraying or agroinfiltrated dsRNA and found that it could be detected in local, close distal, and far distal points from the site of application. VsiRNAs were also detected in local and distal points and the differences in accumulation were compared. In parallel, we investigated the capacity of dsRNAs derived from genes of tomato leaf curl New Delhi virus (ToLCNDV), another economically important virus in cucurbits, to limit the disease in zucchini, both by agroinfiltration or by direct spraying, but found no protective effect. In view of the results, the topical application of dsRNAs is postulated as a promising strategy for CGMMV control in the cucumber.

Droughts and high temperatures deeply affect crop production. The use of desiccation-tolerant (or xerotolerant) microorganisms able to protect plants from droughts represents a promising alternative. These xerotolerant microorganisms have previously been used to modulate plant responses and improve their tolerance to drought. In addition, these microorganisms could be stored and used in dry formats, which would improve their viability and resilience at a much lower cost than current market alternatives. In the present study we analyze the possibility of using strains of xerotolerant Actinobacteria in encapsulated format on seeds. Under this formulation, we carried out greenhouse with farming soil with maize plants. Under greenhouse conditions, the plants showed greater resistance to drought, as well as increased growth and production yield, but not as well in field trials. This alternative could represent a useful tool to improve water efficiency in crops for drought-affected areas or affected by water scarcity.

High-throughput sequencing from symptomatic tomato and pepper plants collected in Panama rendered the complete genome of the southern tomato virus (isolate STV_Panama) and bell pepper endornavirus (isolate BPEV_Panama), and almost-complete genomes of three other BPEV isolates. Tomato chlorosis virus, tomato mosaic virus, and impatiens necrotic spot virus were also detected. Analysis of the complete genome of STV and BPEV worldwide isolates revealed nucleotide diversities of 0.004246 and 0.070523, respectively. Bayesian phylogenetic analysis showed two main groups for each virus (I and II), and several subgroups for BPEV (IA, IB, IC, IIA and IIB). Isolate STV_Panama clustered with NC_12-03-08 from USA and Tom3-T from France (99.97% nucleotide identity) in Group I and BPEV_Panama was close to the Canadian isolate BPEV_Ontario (99.66% nucleotide identity) in Subgroup IB. No correlation was observed between geographic and genetic distances for both viruses. Panamanian BPEV isolates were divergent, belonging to Groups I and II (nucleotide identities > 87.33%). Evolutionary analysis showed purifying selection in all encoding regions of both viruses, being stronger in the overlapping region of both STV genes. Finally, recombination was detected in BPEV but not in STV. This is the first report of STV and BPEV in Panama.

Synthetic dsRNA are valuable tools for reverse genetics research and virus silencing applications. Its synthesis can be performed both in vivo or in vitro. Whilst the latter presents the drawback of high production cost, the former has the advantage of being less expensive and suitable for scalable production. In general, dsRNAs are obtained in vivo from Escherichia coli heterologous systems that require the gene for the T7 RNA polymerase inducible by IPTG. The (ds)RNAs for gene of interest are then synthesized under the T7 promoter. In this work, we present a reliable vector system that includes the insulated promoter proD for the constitutive expression of dsRNA in E. coli that does not require any inducer and that renders elevated dsRNA yield. In tandem, the T7 and proD promoters render the highest dsRNA yield. The accumulation of dsRNA in this system entails a high metabolic cost for the cell. Bacterial RNA extractions that included dsRNAs homologous to the m5GFPer gene and derived from both the synthetic and constitutive promoters induce silencing of GFP expression in Nicotiana benthamiana 16c. Key points • A vector system that includes a constitutive promoter and a T7 promoter in tandem for maximizing dsRNA synthesis. • The metabolic cost for bacteria is maximum when the two promoters are operating simultaneously and results from the accumulation of dsRNA. • Bacterial RNA extractions from both the induced and constitutive systems that include a mGFP5er-derived dsRNA are capable of silencing the GFP expression in Nicotiana benthamiana 16c plants

Biological indexing is the method generally recognized for the certification of propagative grapevines in many countries, and it is mandatory in the European Union. It consists of the evaluation of the plant material after grafting on indicators that are inspected for symptom development. This is a lengthy process that requires well-trained workers, testing field, etc. Alternative diagnostic methods such as serology and RT-qPCR have been discarded for certification because of their intrinsic drawbacks. In turn, high-throughput sequencing (HTS) of plant RNA has been proposed as a plausible alternative to bioassay, but before it is accepted, different aspects of this process must be evaluated. We have compared the HTS of small RNAs with bioassays and other diagnostic methods from a set of 40 grapevine plants submitted for certification. The results allowed the authors the identification of numerous grapevine viruses in the samples, as well as different variants. Besides, relationships between symptom expression and viromes were investigated, in particular leafroll-associated viruses. We compared HTS results using analytical and bioinformatics approaches in order to define minimum acceptable quality standards for certification schemes, resulting in a pipeline proposal. Finally, the comparison between HTS and bioassay resulted favorable for the former in terms of reliability, cost, and timing.

Rosellinia necatrbc is responsible for the white rot root disease of avocado in Southern Spain. Entoleuca sp. is a fungus isolated from roots of these same trees, but it is not pathogenic in avocado. Here, we describe two new species of partitiviruses detected in isolates of the avocado syrnpatric fungi Entoleuca sp. and R. necatrix, termed Entoleuca partitivirus 1 (EnPV1), genus Alphapartitivirus, and Entoleuca partitivirus 2 (EnPV2), genus Betapartitivirus. For both R. necatrix and Entoleuca sp., the dsRNA of the RdRp genomic segment of EnPV1 accumulates at a higher rate than the CP dsRNA, except for a set of Entoleuca sp. isolates where titers of the CP dsRNA are 35-50 times higher than those of the RdRp dsRNA and between 250-380 times higher than the CP dsRNA titers found in the rest of Entoleuca sp. and R. necatrix isolates. For EnPV2, the accumulation rates of the RdRp dsRNA in Entoleuca sp., is in most of the cases, higher than the CP dsRNA. In contrast, in R. necatrix isolates, EnPV2 dsRNA2 generally accumulates at a higher rate. Genetic analysis of the partitiviruses revealed that there is no apparent variation in the nucleotide sequences among the strains. RNA silencing of the partitiviruses appears to be limited in Entoleuca sp., as shown by small RNA sequencing. Finally, the investigation of the presence of these partitiviruses in a fungal collection revealed that they have no role in the pathogenicity of R. necatrix in avocado or in the avirulence of Entoleuca sp. in this host.

The epidemiological dynamics followed by viruses in protected horticultural crops in the Mediterranean Arc of Spain has evolved from a majority of those transmitted by aphids to the predominance of whitefly-transmitted ones. Later, due to the shift towards an integrated control that has been quite successful in the control of aleyrodids, not so much in the control of aphids, aphid-borne viruses are having a significant revival in open field cultivation. Another threat is the continuous emergence of new species or variants of mechanically transmitted viruses. Thus, the number of viruses affecting these crops is constantly increasing and their control demands dynamic actions. The main measures that have managed to limit the damage of these diseases have been the improvement in the physical barriers that limit the spread of vectors and the introduction of resistances in the germplasm. Recently, the increased movement of plant materials and of people, the popularity of growing local crop varieties that lack natural resistances against pathogens, and the prospects of global climate change, may well have boosted the frequency of diseases and pests. Faced with this picture, strategies must be addressed from a multidisciplinary approach. The need for in-field diagnostics tools, easy access to information, novel breeding technologies and alternatives to control of these viruses are discussed.

Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem.

Lettuce chlorosis virus-SP (LCV-SP) (family Closteroviridae, genus Crinivirus), is a new strain of LCV which is able to infect green bean plants and incapable of infecting lettuce crops. In the present study, high throughput and Sanger sequencing of RNA was used to obtain the LCV-SP full-length sequence. The LCV-SP genome comprises 8825 nt and 8672 nt equivalent with RNA1 and RNA2 respectively. RNA1 of LCV-SP contains four ORFs, the proteins encoded by the ORF1a and ORF1b are closely related to LCV RNA1 from California (FJ380118) whereas the 39 end encodes proteins which share high amino acid sequence identity with RNA1 of BnYDV (EU191904). The genomic sequence of RNA2 consists of 8 ORFs, instead of 10 ORFs contained in LCV-California isolate. The distribution of vsiRNA (virus-derived small interfering RNA) along the LCV-SP genome suggested the presence of subgenomic RNAs corresponding with HSP70, P6.4 and P60. Results of the analysis using RDP4 and Simplot programs are the proof of the evidence that LCV-SP is the first recombinant of the family Closteroviridae by crossover recombination of intact ORFs, being the LCV RNA1 (FJ380118) and BnYDV RNA1 (EU191904) the origin of the new LCV strain. Genetic diversity values of virus isolates in the recombinant region obtained after sampling LCV-SP infected green bean between 2011 and 2017 might suggest that the recombinant virus event occurred in the area before this period. The presence of LCV-SP shows the role of recombination as a driving force of evolution within the genus Crinivirus, a globally distributed, emergent genus.

The white rot root disease caused by Rosellinia necatrix is a major concern for avocado cultivation in Spain. Healthy escapes of avocado trees surrounded by diseased trees prompted us to hypothesize the presence of hypovirulent R. necatrix due to mycovirus infections. Recently, we reported the presence of another fungal species, Entoleuca sp., belonging to the Xylariaceae, that was also found in healthy avocado trees and frequently co-infecting the same roots than R. necatrix. We investigated the presence of mycoviruses that might explain the hypovirulence. For that, we performed deep sequencing of dsRNAs from two isolates of Entoleuca sp. that revealed the simultaneous infection of several mycoviruses, not described previously. In this work, we report a new member of the Hypoviridae, tentatively named Entoleuca hypovirus 1 (EnHV1). The complete genome sequence was obtained for two EnHV1 strains, which lengths resulted to be 14,958 and 14,984 nt, respectively, excluding the poly(A) tails. The genome shows two ORFs separated by a 32-nt inter-ORF, and both 5'- and 3'-UTRs longer than any other hypovirus reported to date. The analysis of virus-derived siRNA populations obtained from Entoleuca sp. demonstrated antiviral silencing activity in this fungus. We screened a collection of Entoleuca sp. and R. necatrix isolates and found that EnHV1 was present in both fungal species. A genetic population analysis of EnHV1 strains revealed the presence of two main clades, each of them including members from both Entoleuca sp. and R. necatrix, which suggests intra- and interspecific virus transmission in the field. Several attempts failed to cure Entoleuca sp. from EnHV1. However, all Entoleuca sp. isolates collected from avocado, whether harboring the virus or not, showed hypovirulence. Conversely, all R. necatrix isolates were pathogenic to that crop, regardless of being infected by EnHV1.

Tomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) has recently been introduced in western Mediterranean countries. Isolates in Spain constitute a new strain, denominated ToLCNDVES, that is causing losses in commercial zucchini and melon crops; however, it is also, although less often, detected in commercial tomato crops. We developed a tissue-print hybridization test to detect the two genomic components of the virus and a TaqMan quantitative polymerase chain reaction (qPCR) test to estimate the number of genome copies in plants. qPCR was approximately 104 to 106 times more sensitive than tissue-print hybridization to detect viral genomic DNA-A and DNA-B, respectively. It also detected the virus in more experimentally and naturally ToLCNDV-ES-infected zucchini squash and tomato plants. ToLCNDV-ES DNA-A titers were significantly lower in tomato than in zucchini plants, often falling below the detection limits in the hybridization test. In addition, the DNA-B accumulation was impaired in tomato when compared with zucchini. According to the data obtained in this study, the differences in viral titers of both plant species contribute to explain the dissimilarities in symptom expression, capability of detection, and transmission of the virus.

Lettuce chlorosis virus-SP (LCV-SP) (family Closteroviridae, genus Crinivirus), is a new strain of LCV which is able to infect green bean plants but not lettuce. In the present study, high-throughput and Sanger sequencing of RNA was used to obtain the LCV-SP full-length sequence. The LCV-SP genome comprises 8825 nt and 8672 nt long RNA1 and RNA2 respectively. RNA1 of LCV-SP contains four ORFs, the proteins encoded by the ORF1a and ORF1b are closely related to LCV RNA1 from California (FJ380118) whereas the 3´ end encodes proteins which share high amino acid sequence identity with RNA1 of Bean yellow disorder virus (BnYDV; EU191904). The genomic sequence of RNA2 consists of 8 ORFs, instead of 10 ORFs contained in LCV-California isolate. The distribution of vsiRNA (virus-derived small interfering RNA) along the LCV-SP genome suggested the presence of subgenomic RNAs corresponding with HSP70, P6.4 and P60. Results of the analysis using RDP4 and Simplot programs are the proof of the evidence that LCV-SP is the first recombinant of the family Closteroviridae by crossover recombination of intact ORFs, being the LCV RNA1 (FJ380118) and BnYDV RNA1 (EU191904) the origin of the new LCV strain. Genetic diversity values of virus isolates in the recombinant region obtained after sampling LCV-SP infected green bean between 2011 and 2017 might suggest that the recombinant virus event occurred in the area before this period. The presence of LCV-SP shows the role of recombination as a driving force of evolution within the genus Crinivirus, a globally distributed, emergent genus.