First report of strawberry polerovirus 1 in strawberry in Italy.

Strawberry (Fragaria × ananassa Duch.) was introduced in Sicily (Italy) in the 1930s in the small town of Maletto, on the slopes of Etna volcan, where it's currently cultivated in a total area of 30 ha. The French cv. 'Madame Moutot', appreciated for its unique flavor and intense fragrance, was there propagated vegetatively and after decades, the distinctive 'Etna ecotype' originated by adaptation to the peculiar environmental conditions of the area (Milella et al., 2006). In May 2023, in a 0.5 ha "Etna ecotype" strawberry field, virus-like symptoms were observed in approximately 50% of the plants. Symptoms included severe dwarfing, leaf cupping and chlorotic spotting which lead to decline of infected plants. To investigate the etiology of the disease, leaf samples were collected from eight symptomatic plants for analysis by High-Throughput Sequencing (HTS). To this aim, total RNAs were extracted by using the RNeasy PowerPlant Kit (Qiagen, Germany). The RNAs were pooled, depleted of ribosomal RNA (QIAseq FastSelect; Qiagen), and a library was prepared according to the Illumina DNA Prep Kit. Sequencing on a NextSeq2000 instrument at Leibniz Institute DSMZ (Braunschweig, Germany) generated 31,149,784 of paired-end reads (150 nt), which were further analyzed in Geneious Prime version 2023.2 (Biomatters) using a custom workflow for virus discovery and genome assembly. Analysis of the assembled contigs by local BLASTn and BLASTp alignments against a custom plant virus database of NCBI nuclear-core (NC) reference sequences assigned a number of contigs to accession NC_025435, strawberry polerovirus 1 (SPV-1). Reconstruction of the virus genome by assembly of contigs and reads alignment resulted in a nearly complete genome sequence of SPV-1 (GenBank Acc. No. OR989958) showing by BLASTn 98.69% identity to the SPV-1 NC reference sequence, and 98.99 % identity with an isolate from the Czech Republic (GenBank Acc. OL421571). To confirm the presence of SPV-1 in each sample, RT-PCR using specific primers designed in this study SPV-1-CP-1F (5'-TCGAGATACGTCTAGAACTGCAA-3') and SPV-1-CP-1R (5'-GAGAGGCCCCTTCTACCTATTTG-3') targeting the entire 623 bp coat protein (CP) gene was performed. Amplicons of the expected size were obtained in five samples and Sanger-sequenced. The resulting sequences shared 99.85% - 100% of identity to the HTS - derived sequence (GenBank Acc. No. OR989958) through BLASTn analysis. Strawberry mottle virus (SMoV), strawberry mild yellow edge virus (SMYEV) and strawberry crinkle virus (SCV) were detected in the same library in addition to SPV-1 and then confirmed by RT-PCR using specific primers (Martin & Tzanetakis 2013). Strawberry polerovirus 1, related to the genus Polerovirus in the family Solemoviridae, was first reported in strawberries in Canada (Xiang et al. 2015) and was thereafter detected in the United States (Thekke-Veetil & Tzanetakis 2016), Argentina (Luciani et al. 2016), and Nepal (Kuwak et al. 2022). To date, the virus has been reported in Europe only in the Czech Republic (Franova et al. 2021). To our knowledge, this is the first report of SPV-1 in strawberry plants in Italy. Although the correlation between SPV-1 and strawberry decline (SD) is still uncertain (Xiang et al. 2015) transmission of the virus via aphids has recently been demonstrated (Franova et al. 2021). Our report let to hypothesize that its dissemination in Europe can be considered as increasing.

First report of Convolvulus arvensis and Polycarpon tetraphyllum as natural hosts of tomato brown rugose fruit virus.

Tomato brown rugose fruit virus (ToBRFV) is a tobamovirus recently identified on tomatoes in Jordan (Salem et al. 2016). New infections were rapidly reported all over the world becoming a serious threat to tomato production. About 40 species belonging to four families (Amaranthaceae, Apocynaceae, Asteraceae, and Solanaceae) have been reported as experimental hosts (Salem et al. 2023). Tomato and pepper have been reported as natural hosts of ToBRFV but recently Salem and coworkers (2022) detected the presence of the virus in 12 wild species. To identify potential natural hosts of the virus, 10 plants of bindweed (Convolvulus arvensis L.) and 7 of fourleaf allseed (Polycarpon tetraphyllum L.) were collected in the summer 2023 in a tomato greenhouse located in Pachino, Siracuse province (Sicily, Italy), with high-rate infection of ToBRFV. These two species were chosen because predominant among the spontaneous weeds inside the greenhouse. No symptoms ascribable to ToBRFV were observed on bindweed and fourleaf allseed during the surveys. All leaf samples were analyzed for ToBRFV infections by DAS-ELISA with a commercial antiserum (LOEWE Biochemica, Germany), including tomato positive and negative controls. Eight C. arvensis and seven P. tetraphyllum samples out of the total tested positive to ToBRFV. To confirm virus presence, total RNA was extracted from all samples using the RNeasy Plant Mini Kit (QIAGEN) and used as template for RT-PCR with ToBRFV-specific primers (Alkowni et al. 2019). RT-PCR products of the expected size (560bp) confirmed DAS-ELISA results. Amplicons from two isolates of each plant species (Conv-01, Conv-02, Poly-01, and Poly-02) were gel-purified and sequenced in both directions. Sequences were edited and deposited in GenBank (Acc. Num: Conv-01, OP150933; Conv-02, OP193999; Poly-01, OP150934; Poly-02, OP194000). According to sequence analysis, the four isolates shared 100% identity among them and 98.82% identity with the ToBRFV reference sequence (GenBank Accession No. KT383474). To our knowledge, this is the first report of ToBRFV natural infections in C. arvensis and P. tetraphyllum. Since these weeds are common in our tomato production areas, they could act as ToBRFV reservoirs.

Soil and Soilless Tomato Cultivation Promote Different Microbial Communities That Provide New Models for Future Crop Interventions.

The cultivation of soilless tomato in greenhouses has increased considerably, but little is known about the assembly of the root microbiome compared to plants grown in soil. To obtain such information, we constructed an assay in which we traced the bacterial and fungal communities by amplicon-based metagenomics during the cultivation chain from nursery to greenhouse. In the greenhouse, the plants were transplanted either into agricultural soil or into coconut fiber bags (soilless). At the phylum level, bacterial and fungal communities were primarily constituted in all microhabitats by Proteobacteria and Ascomycota, respectively. The results showed that the tomato rhizosphere microbiome was shaped by the substrate or soil in which the plants were grown. The microbiome was different particularly in terms of the bacterial communities. In agriculture, enrichment has been observed in putative biological control bacteria of the genera Pseudomonas and Bacillus and in potential phytopathogenic fungi. Overall, the study describes the different shaping of microbial communities in the two cultivation methods.

Can Biological Control Agents Reduce Multiple Fungal Infections Causing Decline of Milkwort in Ornamental Nursery?

This research evaluates biological control agents (BCAs) and fungicide alone and in combination for the management of decline caused by multiple fungi on milkwort (Polygala myrtifolia). Four experiments were performed in a greenhouse within a nursery located in Catania province (southern Italy). The activity of fungicides and biological control agents was evaluated by calculating the plant mortality (%) and recovery frequency (%) of different fungi associated with symptomatic tissue. Comprehensively, boscalid + pyraclostrobin and fosetyl-Al showed the best results in managing disease complex on milkwort. Biological control agents provided, on average, the lowest performances; nevertheless, in most cases, they were able to significantly reduce multiple infections and sometimes when combined with fungicide enhanced the effectiveness. The molecular analysis of 86 isolates obtained from symptomatic tissue allowed to identify the fungi involved in the disease as Calonectriapauciramosa, C. pseudomexicana, Fusariumoxysporum, Neocosmospora solani (syn. F. solani) and binucleate Rhizoctonia AG-R. Calonectriapseudomexicana never reported on milkwort and in Europe was inoculated on P. myrtifolia potted healthy cuttings and produced crown and root rot after 40 days. Our findings represent the first worldwide report about disease complex of milkwort caused by several fungi (Calonectria spp., Fusarium spp. and binucleate Rhizoctonia) and on the effects of integrated control strategies to manage this disease in the nursery.

Polyphenols: Plant Sources and Food Industry Applications.

BACKGROUND: Numerous epidemiological studies have demonstrated the association between (poly)phenol-rich foods and human health. Growing consumer knowledge of the relationship between diet, good health and disease prevention stimulated manufacturing opportunities. Functional foods and their synergistic health benefits, beyond just being a source of individual nutrients, are highly demanded by consumers. OBJECTIVE: Therefore, the aim of this study was to review available literature focusing on the nutraceutical role of (poly)phenols, and role in functional foods production, with a specific focus on (poly)phenols derived from byproducts. RESULTS: The review of the literature showed that antioxidant extracted from residual sources could prevent lipid peroxidation and protect against oxidative damage by scavenging oxygen radicals, and therefore contribute to the longer stability of foods, be used for increasing the stability of foods. These effects may also translate to humans, improving the effects on health of certain foods. CONCLUSION: However, careful monitoring of production is needed in order to maximize the (poly)phenol content of plant foods.

Grape and environmental mycoflora monitoring in old, traditionally cultivated vineyards on Mount Etna, southern Italy.

BACKGROUND: Grape contamination by several fungal species occurs during a vineyard's preharvest and harvest. Agronomic management and microclimatic conditions can affect fungi occurrence and epidemiology, thus explaining qualitative differences in mycoflora composition, including the presence of phytopathogenic or mycotoxigenic fungi. In this study a two-year grape, air and soil mycoflora monitoring programme was undertaken in vineyards on Mount Etna (eastern Sicily, Italy). The mycoflora composition was investigated at pea berry and veraison phenological phases from air and soil and at ripening from sample grapes. RESULTS: Mycoflora in air and soil varied according to the phenological stage. In the air samples, penicillia were dominant over aspergilli at the pea berry phase, but their ratio was inverted at early veraison. Black aspergilli (BA) were isolated from the vine environment and grape samples, where BA were represented mainly by Aspergillus niger aggregate, which showed no or low ochratoxin A (OTA) production. Aspergillus carbonarius was either not identified or identified at low frequency, although most of the isolates produced OTA. CONCLUSION: Monitoring focused on the environmental mycoflora composition and highlighted the good health profile of various Sicilian autochthonous grape cultivars. In addition, data suggest that the lower relative humidity occurring at the highest altitudes reduces BA incidence. © 2016 Society of Chemical Industry.

The genetic diversity of Citrus dwarfing viroid populations is mainly dependent on the infected host species.

As with viruses, viroids infect their hosts as polymorphic populations of variants. Identifying possible sources of genetic variability is significant in the case of the species Citrus dwarfing viroid (CDVd) which has been proposed as a dwarfing agent for high-density citrus plantings. Here, a natural CDVd isolate (CMC) was used as an inoculum source for long-term (25 years) and short-term (1 year) bioassays in different citrus host species. Characterization of progenies indicated that the genetic stability of CDVd populations was high in certain hosts (trifoliate orange, Troyer citrange, Etrog citron, Navelina sweet orange), which preserve viroid populations similar to the original CMC isolate even after 25 years. By contrast, CDVd variant populations in Interdonato lemon and Volkamer lemon were completely different to those in the inoculated sources, highlighting how influential the host is on the genetic variability of CDVd populations. Implications for risk assessment of CDVd as a dwarfing agent are discussed. The GenBank/EMBL/DDBJ accession numbers for the complete sequences of the Citrus dwarfing viroid variants are JF970266.1 forH2-2, JF970267.1 for H2-7, EU938647.1 for H6-2, EU938651.1 forH6-10, JF970268.1 for H10-7, EU938652.1 for H14-13, EU938653.1for H14-14, JF970269.1 for H14-16, EU938648.1 for H15-9,EU938649.1 for H16-2, JF970265.1 for H16-9, EU938654.1 forH16-13, EU938650.1 for H20-3, JF970270.1 for H20-7, EU938641.1for PR-1, EU938642.1 for PR-3, EU938643.1 for PR-7, EU938644.1for CR-1, EU938639.1 for VR-4, JF12070.1 for VR-15, JF812069.1LS-4, EU938640.1 for LS-10 and JF970264.1 for LS-11.

Differential display analysis of gene expression in Etrog citron leaves infected by Citrus viroid III.

Citrus are natural hosts of several viroids, which are plant pathogens composed exclusively of a non-protein-coding, small single-stranded circular RNA that is able to replicate autonomously in susceptible hosts. They are responsible for symptoms such as stunting, leaf epinasty, and chlorosis. Citrus viroid III (CVd-III) has been long regarded as a possible dwarfing agent of citrus grafted on trifoliate orange and its hybrids. To investigate molecular mechanisms involved in pathogenesis, the messenger RNA (mRNA) differential display technique was here applied to identify genes whose transcription was significantly altered in leaves of Etrog citron (Citrus medica) infected by CVd-III (variant b). Of eighteen genes identified, thirteen were up-regulated by viroid infection, while five were down-regulated. Except for two genes that encode proteins of unknown function, the remaining genes are mainly involved in plant defence/stress responses, signal transduction, amino acid transport, and cell wall structure. Among the up-regulated genes, it is noteworthy a suppressor of RNA silencing that might be involved in viroid and virus pathogenicity. The functions of these genes are discussed.