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Fruit colour is a critical determinant for the appearance quality and commercial value of apple fruits. Viroid-induced dapple symptom severely affects the fruit coloration, however, the underlying mechanism remains unknown. In this study, we identified an apple dimple fruit viroid (ADFVd)-derived small interfering RNA, named vsiR693, which targeted the mRNA coding for a bHLH transcription factor MdPIF1 (PHYTOCHROME-INTERACTING FACTOR 1) to regulate anthocyanin biosynthesis in apple. 5' RLM-RACE and artificial microRNA transient expression system proved that vsiR693 directly targeted the mRNA of MdPIF1 for cleavage. MdPIF1 positively regulated anthocyanin biosynthesis in both apple calli and fruits, and it directly bound to G-box element in the promoter of MdPAL and MdF3H, two anthocyanin biosynthetic genes, to promote their transcription. Expression of vsiR693 negatively regulated anthocyanin biosynthesis in both apple calli and fruits. Furthermore, co-expression of vsiR693 and MdPIF1 suppressed MdPIF1-promoted anthocyanin biosynthesis in apple fruits. Infiltration of ADFVd infectious clone suppressed coloration surrounding the injection sites in apple fruits, while a mutated version of ADFVd, in which the vsiR693 producing region was mutated, failed to repress fruit coloration around the injection sites. These data provide evidence that a viroid-derived small interfering RNA targets host transcription factor to regulate anthocyanin biosynthesis in apple., (© 2024 John Wiley & Sons Ltd.)

Commercially cultivated Prunus species are commonly grown in adjacent or mixed orchards and can be infected with unique or commonly shared viruses. Apple ( Malus domestica ), another member of the Rosacea and distantly related to Prunus , can share the same growing regions and common pathogens. Pollen can be a major route for virus transmission, and analysis of the pollen virome in tree fruit orchards can provide insights into these virus pathogen complexes from mixed production sites. Commercial honey bee ( Apis mellifera ) pollination is essential for improved fruit sets and yields in tree fruit production systems. To better understand the pollen-associated virome in tree fruits, metagenomics-based detection of plant viruses was employed on bee and pollen samples collected at four time points during the peak bloom period of apricot, cherry, peach, and apple trees at one orchard site. Twenty-one unique viruses were detected in samples collected during tree fruit blooms, including prune dwarf virus (PDV) and prunus necrotic ringspot virus (PNRSV) (Genus Ilarvirus , family Bromoviridae ), Secoviridae family members tomato ringspot virus (genus Nepovirus ), tobacco ringspot virus (genus Nepovirus ), prunus virus F (genus Fabavirus ), and Betaflexiviridae family member cherry virus A (CVA; genus Capillovirus ). Viruses were also identified in composite leaf and flower samples to compare the pollen virome with the virome associated with vegetative tissues. At all four time points, a greater diversity of viruses was detected in the bee and pollen samples. Finally, the nucleotide sequence diversity of the coat protein regions of CVA, PDV, and PNRSV was profiled from this site, demonstrating a wide range of sequence diversity in pollen samples from this site. These results demonstrate the benefits of area-wide monitoring through bee pollination activities and provide new insights into the diversity of viruses in tree fruit pollination ecosystems.

A novel disease affecting small immature fruits has surfaced in 'Saiwaihong' apples ( Malus pumila ), a recently developed variety extensively cultivated across more than 20,000 ha in China. In an effort to pinpoint the causal agent(s) responsible for this ailment, RNA sequencing analysis was conducted on four symptomatic and four asymptomatic apple samples. The results revealed a diverse range of viruses and viroids, indicating mixed viral infection in diseased samples. However, a more focused examination involving 152 symptomatic and 122 asymptomatic fruit samples, using RT-PCR and dot-blotting hybridization techniques, highlighted a close association between the disease and the presence of apple scar skin viroid (ASSVd). Among the ASSVd variants obtained from diseased 'Saiwaihong' apples, 20 were identified, and they were either identical or closely related to isolates from various apple varieties cultivated in different regions and countries. This suggests that ASSVd isolates in 'Saiwaihong' might have been introduced from other apple varieties. Furthermore, the analysis indicates the possibility of two separate introductions, as the ASSVd 'Saiwaihong' isolates exhibited two distinct phylogenetic groups. These insights provide valuable guidance for disease control strategies and emphasize the significance of ongoing monitoring for ASSVd, both in its familiar forms and potential new variants., Competing Interests: The author(s) declare no conflict of interest.

This study assessed the SARS-CoV-2 surrogate bacteriophage φ6 cross-contamination between high-density polyethylene or polyvinyl chloride gloves and fruits (tomato and cucumber) using different inoculum levels (6.0 and 4.0 log PFU/sample). Bacteriophage φ6 survival on contaminated gloves was assessed over 9 days at 25 °C. The effectiveness of photodynamic treatment using curcumin as a photosensitizer to inactivate φ6 on fruits was determined. The fruit type and the glove material influenced the φ6 transfer. Longer contact times resulted in greater φ6 transfer. The highest φ6 transfer occurred from tomato to HDPE glove (0.8% or -1.1 log % transfer) after 30 s of contact at the higher inoculum level. Bacteriophage φ6 was detected on cross-contaminated HDPE gloves for up to 6 days. Bacteriophage φ6 survived better on vinyl gloves cross-contaminated by cucumber vs. tomato (detected up to 6 vs 3 days). Photodynamic inactivation of φ6 was time-dependent and varied with the tested fruit but was not influenced by viral starting concentration. Photodynamic treatment decreased the φ6 titer by 3.0 and 2.2 log PFU/sample in tomato and cucumber, respectively. Transmission electronic microscopy showed that photodynamic treatment changed the structure of the φ6 capsid. These findings may help in the management of SARS-CoV-2 contamination risks in fruit handling. They may also help in the establishment of effective measures to manage cross-contamination risk., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Plant defenses are conserved among closely related species, but domestication can alter host genotypes through artificial selection with potential losses in host defenses. Therefore, both domestication and host phylogenetic structure may influence plant virus infection outcomes. Here, we examined the association of phylogeny and domestication with the fitness of infected plants. We inoculated three pairs of domesticated and wild/noncultivated squash ( Cucurbita spp.) with a combination of two viruses commonly found to coinfect cucurbits, zucchini yellow mosaic virus and squash mosaic virus, and recorded fitness traits related to flowers, pollination, fruit, and seed viability in the field over 2 separate years. In an additional field experiment, we recorded the relative abundance of both viruses via RT-qPCR. We found a gradient of susceptibility across the six tested lineages, and phylogenetic structure, but not domestication, contributed to differences in infection outcomes and impacts on several fitness traits, including fruit number, fruit weight, and germination. Plant virus infection also impacted the quantity and quality of floral rewards and visitation rates of specialist bee pollinators. There were no detectable differences in viral load between the six host taxa for either virus individually or the ratio of zucchini yellow mosaic virus to squash mosaic virus. Our results highlight the importance of phylogenetic structure in predicting host susceptibility to disease across wild and domesticated plants and the ability of several hosts to maintain fitness in the field despite infection. Broader consequences of plant pathogens for beneficial insects, such as pollinators, should also be considered in future research., Competing Interests: The author(s) declare no conflict of interest.

Apple and citrus are perennial tree fruit crops that are vital for nutritional security and agricultural economy and to achieve the Sustainable Development Goals of the United Nations. Apple scab and fire blight, along with Huanglongbing, canker, and tristeza virus, stand out as their most notorious diseases and annually destabilize fruit supply. An environmentally sound approach to managing these diseases is improving tree resistance through breeding and biotechnology. Perennial fruit tree germplasm collections are distributed globally and offer untapped potential as sources of resistance. However, long juvenility, specific pollination and flowering habits, and extensive outcrossing hinder apple and citrus breeding. Advances in breeding approaches include trans - and cis- genesis, genome editing, and rapid-cycle breeding, which, in addition to conventional crossbreeding, can all facilitate accelerated integration of resistance into elite germplasm. In addition, the global pool of available sources of resistance can be characterized by the existing genetic mapping and gene expression studies for accurate discovery of associated loci, genes, and markers to efficiently include these sources in breeding efforts. We discuss and propose a multitude of approaches to overcome the challenges of breeding for resistance in woody perennials and outline a technical path to reduce the time required for the ultimate deployment of disease-resistant cultivars.

Men who have sex with men (MSM) are at high risk of acquiring hepatitis A virus (HAV) and in recent years several HAV outbreaks mostly affecting MSM have been described. These outbreaks were caused by subtype IA strains circulating in this high-risk population. After years of low incidence, an outbreak among MSM in Hungary caused a significant increase in reported HAV infections in 2022. Samples from 224 HAV IgM-positive patients diagnosed in 2022 were tested for HAV RNA and positive samples were genotyped by sequencing. In 171 patients a unique subtype IB virus was detected with 99.8-100% sequence identity in the VP1/P2A junction. It was distinct from previously published strains, but most closely related to an Egyptian isolate. Sequence analysis revealed one dominant and three minor variants based on VP1/P2A. Whole genome sequencing revealed limited variation among these variants, suggesting a recent common origin. Epidemiological data indicated that sexual transmission was driving the outbreak for most of the year, suggested by the high male to female ratio and the large number of coinfections with HIV and other sexually transmitted infections among the patients. The outbreak was also associated with a restaurant cluster, in which one of the variants was detected and frozen berries were implicated as the source of infections. The outbreak strain was also detected in other countries around Europe and remained frequently detectable in Hungary in 2023. This study provides insights into the molecular and epidemiological characteristics of the described HAV outbreak. The results show that sequencing is not only useful in connecting cases to an outbreak, but also helps to clarify the relatedness of detected variants. Prevention strategies focusing on vulnerable communities may reduce the burden of HAV infections in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Grapevine Pinot gris virus (GPGV) infects grapevines worldwide and causes symptoms such as chlorotic mottling and deformations on leaves, stunted shoots and short panicles, or none of these symptoms if it appears as latent infection. So far, the consequences of GPGV infections for winegrowers are difficult to assess since important information such as plant performance at different GPGV infection levels and symptom expression are not fully clarified., Methods: In order to investigate the course of GPGV spread, annual visual evaluations and ELISA tests were conducted over 3-4 consecutive years in four GPGV-infected vineyards in southern Germany: GEM, HEC, NIM, and REI. The program PATCHY was used to analyze spatial disease patterns. Sanger sequencing was used to determine virus isolates in vines at different GPGV infection levels, to test their respective influence on symptom expression. Yield and GrapeScan (FTIR) analyses were conducted to test the impact of different GPGV infection levels and isolates on fruit quantity and quality., Results: GPGV infections significantly increased in all four vineyards (GEM 22-32%, HEC 50-99%, NIM 83-90%, REI 56-76%) with significant spreading patterns across and along rows. Specific symptom progression patterns were not observed. According to our results, the virus isolate has an influence on whether symptoms develop during a GPGV infection. While yield analyses revealed that yield losses only occur in symptomatic vines and range from 13 to 96% depending on the severity of symptoms, latent infections have no impact on grape production. No relevant effects of GPGV infections on must quality were observed., Conclusions: Secondary spread of GPGV was observed in all vineyards monitored, indicating vector-borne transmission that is likely to be accelerated by human viticultural management. GPGV should be further monitored to prevent the accumulation of detrimental symptomatic isolates. The results of this study can be used to assess the risk of GPGV to viticulture and should be considered when developing management strategies against the virus., (© 2024. The Author(s).)

In the main cactus pear ( Opuntia ficus-indica )-producing region in the State of Mexico, fruit production occupies the largest cultivated area with 15,800 ha, while 900 ha are cultivated for edible young Opuntia pads ("nopalitos") which are consumed as vegetables. Two composite samples consisting of cladodes of plants for fruit production ( n = 6) and another of "nopalitos" ( n = 6) showing virus-like symptoms were collected. Both sample sets were subjected to high-throughput sequencing (HTS) to identify the viruses and viroids. The HTS results were verified using RT-PCR and Sanger sequencing. Subsequently, 86 samples including cladodes from "nopalitos", plants for fruit production, xoconostles, and some wild Opuntia were analyzed via RT-PCR with specific primers for the viruses and viroids previously detected via HTS. Three viruses were discovered [Opuntia virus 2 (OV2), cactus carlavirus 1 (CCV-1), and Opuntia potexvirus A (OPV-A)], along with a previously reported viroid [Opuntia viroid 1 (OVd-1)]. Additionally, two new viroids were identified, provisionally named the Mexican opuntia viroid (MOVd, genus Pospiviroid ) and Opuntia viroid 2 (OVd-2, genus Apscaviroid ). A phylogenetic analysis, pairwise identity comparison, and conserved structural elements analysis confirmed the classification of these two viroids as new species within the Pospiviroidae family. This is the first report of a pospiviroid and two apscaviroids infecting cactus pears in the world. Overall, this study enhances our understanding of the virome associated with cactus pears in Mexico.

There is limited information on the compared performances of biological, serological. and molecular assays with high-throughput sequencing (HTS) for viral indexing in temperate fruit crops. Here, using a range of samples of predetermined virological status, we compared two performance criteria (inclusivity and analytical sensitivity) of enzyme-linked immunosorbent assay (ELISA), molecular hybridization, reverse transcription (RT)-PCR, and double-stranded RNA (dsRNA) HTS for the detection of a total of 14 viruses (10 genera) and four viroids (three genera). When undiluted samples from individual plants were used, ELISA had the lowest performance, with an overall detection rate of 68.7%, followed by RT-PCR (82.5%) and HTS (90.7%; 100% if considering only viruses). The lower performance of RT-PCR reflected the inability to amplify some isolates as a consequence of point mutations affecting primer-binding sites. In addition, HTS identified viruses that had not been identified by other assays in nearly two-thirds of the samples. Analysis of serial dilutions of fruit tree samples allowed comparison of analytical sensitivities for various viruses. ELISA showed the lowest analytical sensitivity, but RT-PCR showed higher analytical sensitivity than HTS for most of the samples. Overall, these results confirm the superiority of HTS over biological indexing in terms of speed and inclusivity and show that while the absolute analytical sensitivity of RT-PCR tends to be higher than that of HTS, PCR inclusivity is affected by viral genetic diversity. Taken together, these results make a strong case for the implementation of HTS-based approaches in fruit tree viral testing protocols supporting quarantine and certification programs., Competing Interests: The author(s) declare no conflict of interest.

In March 2019, the Finnish Institute for Health and Welfare and Finnish Food Authority started an outbreak investigation after a notification of food business operators' recall of frozen bilberries due to a norovirus finding. A retrospective search was conducted in the food and waterborne outbreak notification system to identify the notifications linked to norovirus and consumption of bilberries in January-March 2019. Five outbreaks were found in which norovirus GII or GII.17 had been detected in patient samples. A pooled retrospective cohort study was performed for those four in which a questionnaire study had been done. A case was defined as a person with diarrhoea or vomiting within 2 days after consuming a meal studied at one of the outbreak locations. Of 79 participants, 45 (57%) cases were identified. Persons that had consumed foods containing unheated bilberries were three times more likely to get ill than those who had not consumed them (RR 3.1, CI 95% 1.2-8.1, p = 0.02). Norovirus GII.17 was found in 16/17 patient samples sent for further typing. Identical norovirus GII.17 was detected in frozen Finnish bilberries and patient samples. At the berry packaging premises, signs of norovirus GII contamination were found in packaging lines. A new procedure for extracting viral nucleic acid from food and environmental samples was used during the outbreak investigation. Consumption of industrially packed frozen berries as heated would be one of the means to prevent norovirus infections., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Accurate detection, identification, and subsequent confirmation of pathogens causing foodborne illness are essential for the prevention and investigation of foodborne outbreaks. This is particularly true when the causative agent is an enteric virus that has a very low infectious dose and is likely to be present at or near the limit of detection. In this study, whole-genome sequencing (WGS) was combined with either of two non-targeted pre-amplification methods (SPIA and SISPA) to investigate their utility as a confirmatory method for RT-qPCR positive results of foods contaminated with enteric viruses. Frozen berries (raspberries, strawberries, and blackberries) were chosen as the food matrix of interest due to their association with numerous outbreaks of foodborne illness. The hepatitis A virus (HAV) and human norovirus (HuNoV) were used as the contaminating agents. The non-targeted WGS strategy employed in this study could detect and confirm HuNoV and HAV at genomic copy numbers in the single digit range, and in a few cases, identified viruses present in samples that had been found negative by RT-qPCR analyses. However, some RT-qPCR-positive samples could not be confirmed using the WGS method, and in cases with very high Ct values, only a few viral reads and short sequences were recovered from the samples. WGS techniques show great potential for confirmation and identification of virally contaminated food items. The approaches described here should be further optimized for routine application to confirm the viral contamination in berries., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Tomato fruit blotch virus (ToFBV) ( Blunervirus solani , family Kitaviridae ) was firstly identified in Italy in 2018 in tomato plants that showed the uneven, blotchy ripening and dimpling of fruits. Subsequent High-Throughput Sequencing (HTS) analysis allowed ToFBV to be identified in samples collected in Australia, Brazil, and several European countries, and its presence in tomato crops was dated back to 2012. In 2023, the virus was found to be associated with two outbreaks in Italy and Belgium, and it was included in the EPPO Alert list as a potential new threat for tomato fruit production. Many epidemiologic features of ToFBV need to be still clarified, including transmission. Aculops lycopersici Massee (Acariformes: Eriophyoidea), the tomato russet mite (TRM), is a likely candidate vector, since high population densities were found in most of the ToFBV-infected tomato cultivations worldwide. Real-time RT-PCR tests for ToFBV detection and TRM identification were developed, also as a duplex assay. The optimized tests were then transferred to an RT-ddPCR assay and validated according to the EPPO Standard PM 7/98 (5). Such sensitive, reliable, and validated tests provide an important diagnostic tool in view of the probable threat posed by this virus-vector system to solanaceous crops worldwide and can contribute to epidemiological studies by simplifying the efficiency of research. To our knowledge, these are the first molecular methods developed for the simultaneous detection and identification of ToFBV and TRM.

Grapevine leafroll disease is a viral disease that affects grapevines (Vitis vinifera L.) and has a severe economic impact on viticulture. In this study, the effect of grapevine leafroll-associated viruses (GLRaV) on berry quality was investigated in clones of cultivar cv. Crimson Seedless table grapes infected with GLRaV. RT-PCR confirmed the identity of the clones: clone 3236, infected only with GLRaV-3 (termed single); clone 3215, infected with GLRaV-3, GLRaV-4 strain 9 and grapevine virus A (termed mixed); and a viral free clone of the same genetic background of the infected clones (termed control). The berry quality indices of size, sugar, acidity and anthocyanin content were measured at harvest maturity. RT-qPCR was used to determine the viral load. The study was repeated over 2 year. A two-way, multivariate analysis of variance was applied with clone and year as independent variables and the measured berry quality parameters as a dependent variable. All dependent variables were significantly affected by viral infection (Wilks, λ, (2,33) = 0.033895, P-value <0.001), while only titratable acidity was affected by year. The average berry dry mass decreased (P-value <0.001). The water content of both infected clones was greater than that of the control (P-value <0.001). Both infected clones displayed reduced sugar content as a fraction of the berry dry mass (P-value <0.001). The anthocyanin and the phenol content of the infected clones were significantly reduced compared with the control clone (P < 0.001, P < 0.05, clone 3236 and clone 3215, respectively). Finally, the viral load was highly variable, and no quantitative relationship between viral load and berry composition was found., (© The Author(s) 2024. Published by Oxford University Press.)

Plant virus-based sgRNA delivery strategy has been widely applied for efficient genome editing across various plant species, leveraging its significant advantages in the rapid expression and expansion of sgRNA through virus replication and movement. However, the efficacy of the virus-induced gene editing (VIGE) tool in tomato has yet to be explored. In this paper, we established a TRV-mediated CRISPR/Cas9 genome editing system in the somatic cells of tomato, reporting the validation of VIGE and evaluating the mutagenesis efficiency in both tomato leaves and fruits using high-throughput sequencing. The results demonstrated an approximate 65% efficiency of VIGE in tomato leaves for the selected target genes, with VIGE efficiency reaching up to 50% in tomato fruits. This research not only introduces an efficient tool for reverse genetics but also reveals substantial potential of VIGE in surpassing traditional tissue culture techniques for creating heritable mutations in tomato., (© 2024 Wiley‐VCH GmbH.)

Sustainable production of pome fruit crops is dependent upon having virus-free planting materials. The production and distribution of plants derived from virus- and viroid-negative sources is necessary not only to control pome fruit viral diseases but also for sustainable breeding activities, as well as the safe movement of plant materials across borders. With variable success rates, different in vitro-based techniques, including shoot tip culture, micrografting, thermotherapy, chemotherapy, and shoot tip cryotherapy, have been employed to eliminate viruses from pome fruits. Higher pathogen eradication efficiencies have been achieved by combining two or more of these techniques. An accurate diagnosis that confirms complete viral elimination is crucial for developing effective management strategies. In recent years, considerable efforts have resulted in new reliable and efficient virus detection methods. This comprehensive review documents the development and recent advances in biotechnological methods that produce healthy pome fruit plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Viruses pose major global challenges to crop production as infections reduce the yield and quality of harvested products, hinder germplasm exchange, increase financial inputs, and threaten food security. Small island or archipelago habitat conditions such as those in the Caribbean are particularly susceptible as the region is characterized by high rainfall and uniform, warm temperatures throughout the year. Moreover, Caribbean islands are continuously exposed to disease risks because of their location at the intersection of transcontinental trade between North and South America and their role as central hubs for regional and global agricultural commodity trade. This review provides a summary of virus disease epidemics that originated in the Caribbean and those that were introduced and spread throughout the islands. Epidemic-associated factors that impact disease development are also discussed. Understanding virus disease epidemiology, adoption of new diagnostic technologies, implementation of biosafety protocols, and widespread acceptance of biotechnology solutions to counter the effects of cultivar susceptibility remain important challenges to the region. Effective integrated disease management requires a comprehensive approach that should include upgraded phytosanitary measures and continuous surveillance with rapid and appropriate responses.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample., (Copyright © 2021. Published by Elsevier Ltd.)

Real-time quantitative PCR (RT-qPCR) is a powerful tool to detect and quantify transcription abundance, and the stability of the reference gene determines its success. However, the most suitable reference gene for different genotypes and tobacco rattle virus (TRV) infected fruits was unclear in peach ( Prunus persica L. Batsch). In this study, 10 reference genes were selected and gene expression was characterized by RT-qPCR across all samples, including different genotypes and TRV-infected fruits during ripening. Four statistical algorithms (geNorm, NormFinder, BestKeeper, and RefFinder) were used to calculate the stability of 10 reference genes. The geNorm analysis indicated that two suitable reference genes should be used for gene expression normalization. In general, the best combination of reference genes was CYP2 and Tua5 for TRV-infected fruits and CYP2 and Tub1 for different genotypes. In 18S , GADPH, and TEF2 , there is an unacceptable variability of gene expression in all experimental conditions. Furthermore, to confirm the validity of the reference genes, the expression levels of PpACO1 , PpEIN2 , and PpPL were normalized at different fruit storage periods. In summary, our results provide guidelines for selecting reliable reference genes in different genotypes and TRV-infected fruits and lay the foundation for accurate evaluation of gene expression for RT-qPCR analysis in peach.

Marburg virus (MARV), the causative agent of Marburg virus disease, emerges sporadically in sub-Saharan Africa and is often fatal in humas. The natural reservoir for this zoonotic virus is the frugivorous Egyptian rousette bat ( Rousettus aegyptiacus ) that when infected, sheds virus in the highest amounts in oral secretions and urine. Being fruit bats, these animals forage nightly for ripened fruit throughout the year, including those types often preferred by humans. During feeding, they continually discard partially eaten fruit on the ground that could then be consumed by other Marburg virus susceptible animals or humans. In this study, using qRT-PCR and virus isolation, we tested fruit discarded by Egyptian rousette bats experimentally infected with a natural bat isolate of Marburg virus. We then separately tested viral persistence on fruit varieties commonly cultivated in sub-Saharan Africa using a recombinant Marburg virus expressing the fluorescent ZsGreen1. Marburg virus RNA was repeatedly detected on fruit in the food bowls of the infected bats and viable MARV was recovered from inoculated fruit for up to 6 h.

The adhesion of noroviruses to strawberry, turkey slices, ham, and cheddar cheese was studied using murine norovirus 1 (MNV-1) as a surrogate for human norovirus (NoV). Based on plaque assay, the recovery and adhesion of MNV-1 depended on the food type (turkey versus strawberry), pH of the initial suspension buffer (pH 4 versus pH 7), and food fat composition (C 8 versus C 18 ). Recovery of infectious particles from turkey was 68% compared to 9.4% from strawberry. On turkey, adhesion of MNV-1 was lower at pH 7 (pH of fecal matter), and virus particles adhered to this pH were recovered more easily (33,875 PFU) than at pH 4 (pH of vomitus). The presence of fat and the composition of fatty acids seemed to increase MNV-1 recovery and adherent viral particles recovered but did not affect adhesion (68% on fat-free turkey and regular turkey). Adherent MNV-1 particles recovered from stainless steel coated with saturated fatty acid (C 8 , C 14 , C 18 ) increased significantly with chain length ( P <  0.05), but adhesion did not seem to change. Using liquid droplet contact angle to measure surface energy, it was deduced that hydrophobic interactions contribute considerably to the adhesion of MNV-1 to stainless steel, polyvinyl chloride, and high-density polyethylene. IMPORTANCE Ready-to-eat (RTE) foods are major vehicles of transmission of foodborne viral pathogens, including NoV. The high incidence of gastroenteritis caused by viruses is due largely to their persistence in the environment and adhesion to different kinds of surfaces in the food industry, including the foods themselves. Compared with bacteria, adhesion of viruses to surfaces is poorly understood. Better knowledge of the physicochemical parameters involved in the adhesion of NoV to ready-to-eat foods is essential to devising effective strategies for reducing the persistence and, thus, the transmission of this virus.

Virus infection of plants can result in various degrees of detrimental impacts and disparate symptom types and severities. Although great strides have been made in our understanding of the virus-host interactions in herbaceous model plants, the mechanisms underlying symptom development are poorly understood in perennial fruit crops. Grapevine fanleaf virus (GFLV) causes variable symptoms in most vineyards worldwide. To better understand GFLV-grapevine interactions in relation to symptom development, field and greenhouse trials were conducted with a grapevine genotype that exhibits distinct symptoms in response to a severe and a mild strain of GFLV. After validation of the infection status of the experimental vines by high-throughput sequencing, the transcriptomic and metabolomic profiles in plants infected with the two viral strains were tested and compared by RNA-Seq and LC-MS, respectively, in the differentiating grapevine genotype. In vines infected with the severe GFLV strain, 1023 genes, among which some are implicated in the regulation of the hypersensitive-type response, were specifically deregulated, and a higher accumulation of resveratrol and phytohormones was observed. Interestingly, some experimental vines restricted the virus to the rootstock and remained symptomless. Our results suggest that GFLV induces a strain- and cultivar-specific defense reaction similar to a hypersensitive reaction. This type of defense leads to a severe stunting phenotype in some grapevines, whereas others are resistant. This work is the first evidence of a hypersensitive-like reaction in grapevine during virus infection.

Bacterial kiwifruit vine disease ( Pseudomonas &nbsp; syringae pv. actinidiae , Psa) and halo blight of bean ( P. syringae pv. phaseolicola , Pph) are routinely treated with copper, leading to environmental pollution and bacterial copper resistance. An alternative sustainable control method could be based on bacteriophages, as phage biocontrol offers high specificity and does not result in the spread of toxic residues into the environment or the food chain. In this research, specific phages suitable for phage-based biocontrol strategies effective against Psa and Pph were isolated and characterized. In total, sixteen lytic Pph phage isolates and seven lytic Psa phage isolates were isolated from soil in Piedmont and Veneto in northern Italy. Genome characterization of fifteen selected phages revealed that the isolated Pph phages were highly similar and could be considered as isolates of a novel species, whereas the isolated Psa phages grouped into four distinct clades, two of which represent putative novel species. No lysogeny-, virulence- or toxin-related genes were found in four phages, making them suitable for potential biocontrol purposes. A partial biological characterization including a host range analysis was performed on a representative subset of these isolates. This analysis was a prerequisite to assess their efficacy in greenhouse and in field trials, using different delivery strategies.

Eriophyid mites are commonly found on the leaf surface of different plant species. In the present study, a novel virus associated with an eriophyid mite species was detected using high-throughput sequencing (HTS) of total RNA from fruit tree leaves, primarily growing under greenhouse conditions. The complete genome sequence was characterized using rapid amplification of cDNA ends followed by Sanger sequencing, revealing a genome of 8885 nucleotides in length. The single positive-stranded RNA genome was predicted to encode typical conserved domains of members of the genus Iflavirus in the family Iflaviridae. Phylogenetic analysis showed this virus to be closely related to the unclassified iflavirus tomato matilda associated virus (TMaV), with a maximum amino acid sequence identity of 59% in the RNA-dependent RNA polymerase domain. This low identity value justifies the recognition of the novel virus as a potential novel iflavirus. In addition to a lack of graft-transmissibility evidence, RT-PCR and HTS detection of this virus in the putative host plants were not consistent through different years and growing seasons, raising the possibility that rather than a plant virus, this was a virus infecting an organism associated with fruit tree leaves. Identification of Tetra pinnatifidae HTS-derived contigs in all fruit tree samples carrying the novel virus suggested this mite as the most likely host of the new virus (p-value < 1e-11), which is tentatively named "eriophyid mite-associated virus" (EMaV). This study highlights the importance of a careful biological study before assigning a new virus to a particular plant host when using metagenomics data., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Human coronaviruses (HCoVs) are mainly associated with respiratory infections. However, there is evidence that highly pathogenic HCoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome (MERS-CoV), infect the gastrointestinal (GI) tract and are shed in the fecal matter of the infected individuals. These observations have raised questions regarding the possibility of fecal-oral route as well as foodborne transmission of SARS-CoV-2 and MERS-CoV. Studies regarding the survival of HCoVs on inanimate surfaces demonstrate that these viruses can remain infectious for hours to days, however, there is limited data regarding the viral survival on fresh produce, which is usually consumed raw or with minimal heat processing. To address this knowledge gap, we examined the persistence of HCoV-229E, as a surrogate for highly pathogenic HCoVs, on the surface of commonly consumed fresh produce, including: apples, tomatoes, cucumbers and lettuce. Herein, we demonstrated that viral infectivity declines within a few hours post-inoculation (p.i) on apples and tomatoes, and no infectious virus was detected at 24h p.i, while the virus persists in infectious form for 72h p.i on cucumbers and lettuce. The stability of viral RNA was examined by droplet-digital RT-PCR (ddRT-PCR), and it was observed that there is no considerable reduction in viral RNA within 72h p.i., (Copyright © 2021. Published by Elsevier Ltd.)

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

Seed transmission is an important factor in the epidemiology of plant pathogens. Geminiviruses are serious pests spread in tropical and subtropical regions. They are transmitted by hemipteran insects, but a few cases of transmission through seeds were recently reported. Here, we investigated the tomato seed transmissibility of the begomovirus tomato yellow leaf curl Sardinia virus (TYLCSV), one of the agents inducing the tomato yellow leaf curl disease, heavily affecting tomato crops in the Mediterranean area. None of the 180 seedlings originating from TYLCSV-infected plants showed any phenotypic alteration typical of virus infection. Moreover, whole viral genomic molecules could not be detected in their cotyledons and true leaves, neither by membrane hybridization nor by rolling-circle amplification followed by PCR, indicating that TYLCSV is not a seed-transmissible pathogen for tomato. Examining the localization of TYLCSV DNA in progenitor plants, we detected the virus genome by PCR in all vegetative and reproductive tissues, but viral genomic and replicative forms were found only in leaves, flowers and fruit flesh, not in seeds and embryos. Closer investigations allowed us to discover for the first time that these embryos were superficially contaminated by TYLCSV DNA but whole genomic molecules were not detectable. Therefore, the inability of TYLCSV genomic molecules to colonize tomato embryos during infection justifies the lack of seed transmissibility observed in this host.

Southern highbush blueberry (interspecific hybrids of Vaccinium corymbosum L.) is cultivated near wild V. corymbosum as well as closely related species in Florida, USA. The expansion of blueberry cultivation into new areas in Florida and deployment of new cultivars containing viruses can potentially increase the diversity of viruses in wild and cultivated V. corymbosum . In this study, viral diversity in wild and cultivated blueberries ( V. corymbosum ) is described using a metagenomic approach. RNA viromes from V. corymbosum plants collected from six locations (two cultivated and four wild) in North Central Florida were generated by high throughput sequencing (HTS) and analyzed using a bioinformatic analysis pipeline. De novo assembled contigs obtained from viromes of both commercial and wild sites produced sequences with similarities to plant virus species from a diverse range of families ( Amalgaviridae , Caulimoviridae , Endornaviridae , Ophioviridae , Phenuiviridae , and Virgaviridae ). In addition, this study has enabled the identification of blueberry latent virus (BlLV) and blueberry mosaic associated ophiovirus (BlMaV) for the first time in Florida, as well as a tentative novel tepovirus (blueberry virus T) (BlVT) in blueberry. To the best of our knowledge, this is the first study that compares viral diversity in wild and cultivated blueberry using a metagenomic approach.

Red pepper ( Capsicum annuum , L.), is one of the most important spice plants in Korea. Overwintering pepper fruits are a reservoir of various microbial pepper diseases. Here, we conducted metagenomics (DNA sequencing) and metatranscriptomics (RNA sequencing) using samples collected from three different fields. We compared two different library types and three different analytical methods for the identification of microbiomes in overwintering pepper fruits. Our results demonstrated that DNA sequencing might be useful for the identification of bacteria and DNA viruses such as bacteriophages, while mRNA sequencing might be beneficial for the identification of fungi and RNA viruses. Among three analytical methods, KRAKEN2 with raw data reads (KRAKEN2&#95;R) might be superior for the identification of microbial species to other analytical methods. However, some microbial species with a low number of reads were wrongly assigned at the species level by KRAKEN2&#95;R. Moreover, we found that the databases for bacteria and viruses were better established as compared to the fungal database with limited genome data. In summary, we carefully suggest that different library types and analytical methods with proper databases should be applied for the purpose of microbiome study.

Human noroviruses (HuNoV) are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several HuNoV food-related outbreaks. However, the extraction of HuNoV RNA from frozen raspberries remains challenging. Recovery yields are low, and real-time quantitative reverse transcriptase PCR (RT-qPCR) inhibitors limit the sensitivity of the detection methodologies. A new approach using fine magnetic silica beads was developed for the extraction of HuNoV spiked on frozen raspberries. Relatively low recovery yields were observed with both the magnetic silica bead and the reference ISO 15216-1:2017 methods. High RT-qPCR inhibition was observed with the ISO 15216-1:2017 recommended amplification kit but could be reduced by using an alternative kit. Reducing RT-qPCR inhibition is important to limit the number of inconclusive HuNoV assays thus increasing the capacity to assess the HuNoV prevalence in frozen raspberries.

The objective of this study was to use high-energy electron beam (HEEB) treatments to find surrogate microorganisms for enteric viruses and to use the selected surrogates as proof of concept to investigate low-energy electron beam (LEEB) treatments for enteric virus inactivation at industrial scale on frozen blueberries. Six food matrices inoculated with HAV (hepatitis A virus), MNV S99 (murine norovirus), bacteriophages MS2 and Qβ, and Geobacillus stearothermophilus spores were treated with HEEB at 10 MeV using 4, 8 and 16 kGy doses. G. stearothermophilus spores showed the highest inactivation on all matrices except on raisins, with a dose-dependent effect. HAV reached the maximum measurable log 10 reduction (> 3.2 log 10 ) when treated at 16 kGy on raisins. MNV showed the highest resistance of all tested microorganisms, independent of the dose, except on frozen blueberries. On frozen blueberries, freeze-dried raspberries, sesame seeds and black peppercorns, HAV showed a mean inactivation level in between those of MS2 and G. stearothermophilus. Based on this, we selected both surrogate organisms as first approximation to estimate HAV inactivation on frozen blueberries during LEEB treatment at 250 keV using 16 kGy. Reductions of 3.1 and 1.3 log 10 were measured for G. stearothermophilus spores and MS2, respectively, suggesting that a minimum reduction of 1.4 log 10 can be expected for HAV under the same conditions.

Seeking a means of sanitizing berries, the effectiveness of steady state levels of gaseous chlorine dioxide (ClO 2 ) against hepatitis A virus (HAV) on laboratory-contaminated berries was determined. The generated ClO 2 was maintained with 1 or 2 mg/l air inside a 269-l glove box to treat 50 g batches of blueberries, raspberries, and blackberries, and 100 g batches of strawberries that were immersion coated with HAV. Normalized data for ClO 2 (ppm-h/g product) is reported as a function of ClO 2 concentration, treatment time, and weight of treated product. Treatments of ClO 2 ranging from 1.00 to 6.27 ppm-h/g berry were evaluated. When compared to untreated HAV-contaminated berries, log reductions of HAV were > 2.1 for all berry types and conditions tested indicating the gaseous ClO 2 was effective. The average log reduction with strawberries, raspberries, blueberries and blackberries treated with 1.00 ppm-h/g, the lowest ClO 2 treatment tested, were 2.44, 2.49, 3.23, and 3.45, respectively. The highest treatment of 6.27 ppm-h/g was applied at two different gas concentrations of 1 mg/l and 2 mg/l. Average log reductions for blueberries and strawberries treated with 6.27 ppm-h/g were 4.34 and 4.42, and 4.03 and 3.51, applied at 1 mg/l and 2 mg/l, respectively. For blackberries and raspberries 3.20 and 3.24, and 3.23 and 3.97 log reductions were observed for 6.27 ppm-h/g treatments applied at 1 mg/l and 2 mg/l, respectively. Results indicate that HAV contamination of berries can be substantially reduced by gaseous ClO 2 and offer industry a waterless means of sanitizing berries against HAV.

The study was designed to develop a sensitive one-step duplex reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) to detect norovirus genogroup I and II (NoV GI and GII) in lettuce and strawberry. The specificity, sensitivity, repeatability and robustness of the assay was compared with RT-qPCR. The lowest concentration detected by RT-ddPCR for NoV GI and NoV GII were 4.68 and 8.47 copies/μL respectively, much lower than that of RT-qPCR with a number of 46.8 and 84.7 copies/μL, respectively. Lettuce and strawberry samples were artificially contaminated with NoV GI and GII suspensions, with inoculum size of 3.00 × 10 6 to 1.70 × 10 8 copies and 4.80 × 10 5 to 2.50 × 10 7 copies, respectively. Strawberry spiked with low inoculum size revealed positive results by RT-ddPCR, while recorded negative by RT-qPCR. Meanwhile, RT-ddPCR also showed a higher average recovery rate for NoV in lettuce and strawberry than RT-qPCR.The limit of detection (LoDs) of RT-ddPCR for NoVs in lettuce was 2.32 × 10 4 copies/25g (NoV GI) and 2.36 × 10 4 ciopies/25g (NoV GII), and that in strawberry was 2.56 × 10 4 copies/25g (NoV GI) and 2.64 × 10 4 ciopies/25g (NoV GII), which were 10 folds lower than that of RT-qPCR. The developed duplex RT-ddPCR assay exhibited stability and increased capacity to resist inhibitors in food samples with low concentration of NoV, making it a reliable method to avoid false negative result as opposed to RT-qPCR. In conclusion, one-step RT-ddPCR method developed in this study is pertinent in detecting foodborne virus such as NoV., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Grapevine leafroll is one of the most widespread and highly destructive grapevine diseases that is responsible for great economic losses to the grape and wine industries throughout the world. Six distinct viruses have been implicated in this disease complex. They belong to three genera, all in the family Closteroviridae. For the sake of convenience, these viruses are named as grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, and -13). However, their etiological role in the disease has yet to be established. Furthermore, how infections with each GLRaV induce the characteristic disease symptoms remains unresolved. Here, we first provide a brief overview on each of these GLRaVs with a focus on genome structure, expression strategies and gene functions, where available. We then provide a review on the effects of GLRaV infection on the physiology, fruit quality, fruit chemical composition, and gene expression of grapevine based on the limited information so far reported in the literature. We outline key methodologies that have been used to study how GLRaV infections alter gene expression in the grapevine host at the transcriptomic level. Finally, we present a working model as an initial attempt to explain how infections with GLRaVs lead to the characteristic symptoms of grapevine leafroll disease: leaf discoloration and downward rolling. It is our hope that this review will serve as a starting point for grapevine virology and the related research community to tackle this vastly important and yet virtually uncharted territory in virus-host interactions involving woody and perennial fruit crops.

Among the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD 95 was 10 2 genome copies/g for HAV, HEV and norovirus GII and 10 3 genome copies/g for norovirus GI. The LOQ was 2.90 × 10 4 , 1.40 × 10 3 , 1.60 × 10 4 and 1.30 × 10 4 genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Strawberries are often consumed fresh or only receive minimal processing, inducing a significant health risk to the consumer if contamination occurs anywhere from farm to fork. Outbreaks of foodborne illness associated with strawberries often involve a broad range of microbiological agents, from viruses (human norovirus) to bacteria (Salmonella spp. and Listeria monocytogenes). The addition of sanitizers to water washes is one of the most commonly studied strategies to remove or inactivate pathogens on berries as well as avoid cross contamination due to reuse of process wash water. The risk posed with the safety issues of by-products from chlorine disinfection in the fruit industry has led to a search for alternative sanitizers. We evaluated the applicability of different chemical sanitizers (peracetic acid (PA), hydrogen peroxide (H 2 O 2 ), citric acid (CA), lactic acid (LA) and acetic acid (AA)) for the inactivation of S. enterica, L. monocytogenes and murine norovirus (MNV-1) on strawberries. A control treatment with chlorine (NaClO) (100 ppm) was included. For each sanitizer, different doses (40, 80 and 120 ppm for PA and 1, 2.5 and 5% for H 2 O 2 , LA, AA and CA) and time (2 and 5 min) were studied in order to optimize the decontamination washing step. The best concentrations were 80 ppm for PA, 5% for H 2 O 2 and 2.5% for organic acids (LA, AA and CA) after 2 min treatment. Results indicate that the sanitizers selected may be a feasible alternative to chlorine (100 ppm) for removing selected pathogenic microorganisms (P > 0.05), with reductions about ≥2 log for bacterial strains and ≥ 1.7 log for MNV-1. As the washing water may also increase the microbial counts by cross-contamination, we observed that no pathogenic bacteria were found in wash water after 5% H 2 O 2 and 80 ppm PA after 2 min treatment. On the other hand, we also reported reductions about total aerobic mesophyll (TAM) (0.0-1.4 log CFU/g) and molds and yeasts (M&Y) (0.3-1.8 log CFU/g) with all alternative sanitizers tested. Strawberries treated did not shown significant differences about physio-chemical parameters compared to the untreated samples (initial). For this study, the optimal sanitizer selected was PA, due to the low concentration and cost needed and its microbiocidal effect in wash water and fruit. Notwithstanding the results obtained, the effect of PA in combination with other non-thermal technologies such as water-assisted ultraviolet (UV-C) light should be studied in future research to improve the disinfection of strawberries., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The leading causes of foodborne viral disease outbreaks are human norovirus and hepatitis A virus (HAV). Their environmental persistence enables contamination of kitchen surfaces and crops often consumed raw, such as berries. Many decontamination procedures are inefficient and unsuitable for surfaces of industrial kitchen environments and soft fruits. In this study, we investigated the efficiency of a novel surface decontamination technology, combining steam and ultrasound (steam-ultrasound). Plastic, steel or raspberry surfaces were spiked with the norovirus surrogate, murine norovirus (MNV), and HAV, and steam-ultrasound treated at 85, 90 and 95 °C for 0-5 s. Post treatment viruses were titrated for survival by plaque assay and for genome stability by real-time quantitative PCR (RT-qPCR) of nucleic acid extracts. Survival of viruses were estimated in a log-linear model and the treatment time requirements for each decimal reduction (D value) in viral survival were calculated. The estimated D values of MNV or HAV were 0.4-0.2 or 1.1-0.8 s on plastic, 0.9-0.7 or 1.4-0.8 s on steel and 1.6-1.7 or 3.2-4.7 s on raspberries. No clear trend of genome reduction was observed with tested treatment parameters. Raspberries treated up to 4 s retained its natural texture and visual appeal similar to untreated controls whilst monitored for 7 days. In conclusion, steam-ultrasound treatment can within seconds reduce the titre of foodborne viruses on surfaces of plastic, steel and raspberries. This may particularly benefit industrial scale production of soft fruits for raw consumption and for swift non-hazardous decontamination of industrial kitchen surfaces.

The transmission of the apscaviroid tentatively named apple chlorotic fruit spot viroid (ACFSVd) was investigated using a one-step reverse-transcription (RT) droplet digital PCR assay for absolute quantification of the viroid, followed by quantification of relative standard curves by RT-qPCR. Our results indicate that ACFSVd is effectively transmitted by grafting, budding and seeds. No transmission has yet been observed to the viroid-inoculated pome fruit species Pyrus sp. and Cydonia sp. ACFSVd was detected in viruliferous aphids (Myzus persicae, Dysaphis plantaginea) and in codling moths (Cydia pomonella). The viroid was also detected systemically in the infected hemiparasitic plant Viscum album subsp. album (mistletoe).

We modeled the stability of SARS-CoV-2 on apples, tomatoes, and jalapeño peppers at two temperatures following a low-dose aerosol exposure designed to simulate an airborne transmission event involving droplet nuclei. Infectious virus was not recovered postexposure.

Norovirus (NoV) is the leading cause of epidemic and sporadic gastroenteritis worldwide; a high number of those cases are attributed to the consumption of contaminated food. Crop producers have used several strategies to inactivate the virus present in these products and thus stop the NoV transmission chain. Physical methods such as gamma radiation show excellent results in the inactivation of bacteria, but its effect on NoV has been little studied. This study aimed to evaluate the effect of gamma radiation for NoV inactivation, and over the surface topographic characteristics of strawberry cells, as a prototype of soft fruit. A 10% suspension of GII norovirus-positive stool samples were treated with either 200 mg/L of sodium hypochlorite (NaClO) or gamma-irradiated at doses of 5, 10, 15 and 20 kilograys (kGy). Viral inactivation was determined by measuring the integrity of viral capsid using RNase A alone or in combination with proteinase K followed by RT-qPCR. The effect over cellular surface topology characteristics of the fruit was measured by atomic force microscopy (AFM) and confocal microscopy. High doses of radiation (20 kGy) were necessary to detect a significant (p < 0.05) decrease of up to 1.26 log 10 viral copy number. This dose significantly (p < 0.05) raises the root means square roughness (Rq), which affects directly the quality and texture of the product. The gamma irradiation doses tested in this study were not enough to inactivate NoV. The allowed gamma irradiation doses for fresh produce does not alter the surface topology of the fruit, but they affect the content of fluorescent compounds, responsible for the antioxidant activity of the fruit., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Virus detection in food requires appropriate elution and concentration techniques which need to be adapted for different food matrices. ISO/TS-15216-1:2017 and ISO/TS-15216-2:2019 describe standard methods for hepatitis A virus (HAV) research in some food only. Milk-based products containing one or more types of fruit are not covered by ISO procedures, even though they can be contaminated by fruit added to these products or by the food handlers. The aim of this work was to identify an efficient method for the detection of HAV in milk-based products. Four methods were tested to recover HAV from artificially contaminated milk, yoghurt and ice cream containing soft fruits. Results showed that the efficiency of the tested methods depends on the analyzed matrix. In milk we obtained a mean recovery from 13.4% to 1.9%; method based on high speed centrifuge gave the best values. The average recovery in yoghurt was between 3.3% and 114.4%, the latter value achieved by method with beef extract at 3% as eluent. Finally, two methods gave the best results in ice cream with similar recoveries: 29.1% and 27.7% respectively. The first method used glycine as eluent while the other one was based on high speed centrifugation. The ISO method has never proved to be the most efficient in the matrices studied. Therefore, based on the results obtained, a complete rethinking of the ISO method may be necessary to improve its recovery for some products such as milk, while only small changes would be sufficient for other products, such as yoghurt and ice cream., Competing Interests: Declaration of Competing Interest The authors declare there are no conflicts of interest regarding the study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Following outbreaks linked to frozen strawberries in Sweden and Austria in 2018, 65 cases linked to the same hepatitis A virus strain were detected in Germany between October 2018 and January 2020, presenting in two waves. Two case-control studies and a comparison of cases' consumption frequencies with purchase data from a large consumer panel provided strong evidence for frozen strawberry cake as the main vehicle of transmission. Of 46 cases interviewed, 27 reported consuming frozen strawberry cake and 25 of these identified cake(s) from brand A spontaneously or in product picture-assisted recall. Trace back investigations revealed that the Polish producer involved in the previous outbreaks in Sweden and Austria had received frozen strawberries from Egypt via a wholesaler that also delivered frozen strawberries to manufacturer of brand A. Phylogenetic analyses linked the outbreak strain to similar strains formerly isolated from sewage, stool and strawberries in Egypt. Complete trace back and timely recall of products with strong evidence of contamination is important to control an outbreak and prevent later resurgence, particularly for food items with a long shelf life. Continued molecular surveillance of hepatitis A is needed to identify outbreaks and monitor the success of food safety interventions.

Detection of norovirus (NoV) and hepatitis A virus (HAV) on fruits and vegetables using current standard methodologies can be inefficient. Method optimisation focussing on ease, rapidity and increased viral RNA recovery is needed for efficient reverse transcription (RT)-qPCR detection of viruses. A simple and quick direct lysis method for RNA extraction was optimised (method A) to achieve increased viral RNA recovery and minimised RT-qPCR inhibition by increasing the volume of lysis buffer and inclusion of pectinase, Plant RNA Isolation Aid and OneStep PCR Inhibitor Removal Kit. Method A and an internal method structurally comparable to the ISO 15216 standard (method B) were compared for their efficiencies to recover viral RNA from the process controls, mengovirus (MC 0 ) and murine norovirus (MNV), spiked in 13 types of fruits, vegetables, compound foods or seeds/nuts. All extracts (> 61) were also analysed for RT-qPCR inhibition and for natural contamination of NoV and HAV. The overall mean extraction efficiencies of MC 0 and MNV were 36 ± 31 and 44 ± 38%, respectively, for method A and 9 ± 16 and 5 ± 11%, respectively, for method B. Inhibition of RT-qPCR amplification of RNA from NoV genogroup (G)I, NoV GII, and HAV ranged from 5 ± 10 to 13 ± 14% for method A and 34 ± 36 to 48 ± 40% for method B. NoV GII was detected in samples of strawberries and seaweed processed by both methods. In conclusion, the new direct lysis method showed an overall better performance compared to the modified ISO 15216 standard and should be validated for implementation in analysis of viruses in foods of plant origin.

Plum pox virus (PPV) is a worldwide threat to stone fruit production. Its woody perennial hosts provide a dynamic environment for virus evolution over multiple growing seasons. To investigate the impact seasonal host development plays in PPV population structure, next generation sequencing of ribosome associated viral genomes, termed translatome, was used to assess PPV variants derived from phloem or whole leaf tissues over a range of plum leaf and bud developmental stages. Results show that translatome PPV variants occur at proportionately higher levels in bud and newly developing leaf tissues that have low infection levels while more mature tissues with high infection levels display proportionately lower numbers of viral variants. Additional variant analysis identified distinct groups based on population frequency as well as sets of phloem and whole tissue specific variants. Combined, these results indicate PPV population dynamics are impacted by the tissue type and developmental stage of their host., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Region-specific local landraces represent a germplasm diversity adapted and acclimatized to local conditions, and are ideal to breed for targeted market niches while maintaining the variability of heirloom traits. A collection of 180 pepper accessions, collected from 62 diverse locations across six Balkan countries, were characterized and evaluated for phenotypic and biochemical variation during a multi-year environment. An assortment of 32 agro-morphological, fruit quality, and virus resistance traits were evaluated, and the top 10% accessions were identified. A wide range of trait variation concerning plant architecture, inflorescence and fruit traits, yield and fruit quality was observed, and appreciable variation was noticed. According to hierarchical clustering, six distinct clusters were established based on pre-defined varietal groups. Divergence among accessions for phenotypic and fruit compositional variability was analyzed, and eight principal components were identified that contributed ~71% of the variation, with fruit shape, width, wall thickness, weight, and fruit quality traits being the most discriminant. Evaluation of the response to tobacco mosaic virus (TMV) and pepper mild mottle mosaic virus (PMMoV) showed that 24 and 1 accession were resistant, respectively while no tomato spotted wilt virus (TSWV) resistance was found. Considerable diversity for agro-bio-morphological traits indicates the Balkan pepper collection as good gene sources for pre-breeding and cultivar development that are locally adapted., Competing Interests: All authors declare that there is no conflict of interest.

Leafroll viruses are among the most devastating pathogens in viticulture and are responsible for major economic losses in the wine industry. However, the molecular interactions underlying the effects on fruit quality deterioration are not well understood. The few molecular studies conducted on berries from infected vines, associated quality decreases with the repression of key genes in sugar transport and anthocyanin biosynthesis. Sampling protocols in these studies did however not account for berry heterogeneity and potential virus induced phenological shifts, which could have biased the molecular information. In the present study, we adopted an innovative individual berry sampling protocol to produce homogeneous batches for RNA extraction, thereby circumventing berry heterogeneity and compensating for virus induced phenological shifts. This way a characterization of the transcriptomic modulation by viral infections was possible and explain why our results differ significantly from previously reported repression of anthocyanin biosynthesis and sugar metabolism. The present study provides new insights into the berry transcriptome modulation by leafroll infection, highlighting the virus induced upregulation of plant innate immunity as well as an increased responsiveness of the early ripening berry to biotic stressors. The study furthermore emphasizes the importance of sampling protocols in physiological studies on grapevine berry metabolism.

This study investigated the obstacles in detecting enteric viruses from berry fruits, which are on the one hand often associated with outbreaks of viral enteric disease, and on the other hand recognized as a challenging food matrix for molecular detection of enteric viruses. According to the ISO 15216 protocol, for soft fruit samples, virus extraction is by elution with agitation followed by precipitation with polyethylene glycol/NaCl. As a result, first, the phenolic content in the berry eluate was found to be weakly correlated with the detection of coliphage MS2 spiked in the berry samples. Second and more importantly, it was observed that the gel-like pellets formed after precipitation could entrap considerable portions of viruses from being further purified and recovered for detection, suggesting that the low virus detection sensitivity from berries is largely due to the pectin content with complicated chemical structures in the berry fruits. Future research is needed to solve this problem in a targeted way.

Osmotically dehydrated and air dried berry fruits are used as ingredients for the production of yoghurts, chocolates, cereal bars and mixes, ice creams and cakes and these fruits are often subjected to mild thermal treatments only, posing questions around their microbiological safety. As osmotic dehydration methods and parameters vary considerably within the industry and minimally processed high quality fruits are increasingly sought, the scope of this study was to determine which temperatures are required for the inactivation of relevant bacteria and viruses during osmotic dehydration of berries, using blueberries as a model berry in a thawed state to mimic common industrial practices. Additionally, we studied the inactivation of osmotic dehydration at 23 °C, sometimes referred to "cold infusion" followed by air drying at 100 °C to determine the microbiological safety achieved by this combined treatment. Four pathogens (Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes and hepatitis A virus (HAV)) and five surrogates (Enterococcus faecium, Escherichia coli P1, Listeria innocua, murine Norovirus (MNV) and bacteriophage MS2) were inoculated on blueberries and reductions were measured after different treatment combinations. After osmotic dehydration of bacterial strains at 40 °C no survivors were detected on blueberries, with the exception of E. faecium. Inactivation of the viruses at 45 °C showed no survivors for MS2 and mean reductions of 1.5 and 3.4 log 10 median tissue culture infectious dose (TCID 50 )/g for HAV and MNV, respectively. Similarly, in the sugar solution at 40 °C, no survivors were observed, with the exception of E. faecium and the three viruses. The combined process (osmotic dehydration at 23 °C followed by air-drying at 100 °C) achieved an >6 log reduction of all tested bacterial strains and MS2. For HAV and MNV, 2.6 and >3.4 log 10 TCID 50 /g were measured. In summary, the present study shows that osmotic dehydration appears an efficient control measure for the control of L. monocytogenes, S. enterica and E. coli O157:H7 if carried out at 40 °C or at 23 °C and followed by air-drying at 100 °C. Based on the results generated with MNV, the combined treatment is also expected to reduce human Norovirus (NoV) but does not appear to be sufficient to fully control HAV. The results contribute to a better management of the microbial safety of osmotically dehydrated and dried berries and especially the results generated for the viruses emphasize that within a robust food safety management system, safety must be assured through the entire food supply chain and therefore must start at primary production with the implementation of Good Agricultural Practices (GAP)., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Latent fruit tree viruses present economic threat to the industry and nurseries as diseases they cause not only reduce fruit quality and production yield, but can also be spread inadvertently through propagation due to the lack of viral symptoms on an infected mother plant. As a result, these viruses require appropriate detection tools for effective management. In this study we developed RT-qPCR assays for the detection of three latent viruses of pome, apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), and apple mosaic virus (ApMV), using the alignment of representative sequences from the NCBI database. The optimized assays were shown to be specific by successfully amplifying the target from positive controls without showing any detectable amplification in negative and non-target controls, and revealed high sensitivity by reliably detecting as low as 10 1 copies per reaction. The results also demonstrated that both the choice of extraction method and the reagents used for RT-qPCRcould play a critical role in virus detection outcome. These assays were both reliable and robust compared to the extant RT-PCR methods, and they could be a viable tool for making informed management decisions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Strawberry production and exports have been increasing in Spain in recent decades. However, little information is available about their microbiological quality. Due to the growing concern about the microbial safety of these fruits, the objective of this investigation was to study the microbiological quality and the prevalence of the main foodborne pathogens on strawberries sold in Spain. Fresh (n = 152) and frozen (n = 31) samples were obtained from marketplaces and fields in 2017 and 2018. The samples were assayed for total aerobic mesophilic microorganisms (TAM), moulds and yeasts (M&Y), total coliforms (TC), Escherichia coli, Salmonella spp., Listeria monocytogenes as well as Norovirus (NoV) GI and GII. The microbiological counts ranged from <1.70 (detection limit, dl) - 5.89 log 10 CFU/g (mean 3.78 log 10 CFU/g) for TAM; 2.10-5.86 log 10 CFU/g (mean 3.80 log 10 CFU/g) for M&Y; and <0.70 (dl) - 4.91 log 10 CFU/g (mean 2.15 log 10 CFU/g) for TC in fresh strawberries. In frozen strawberries, the counts were <1.70 (dl) - 3.66 log 10 CFU/g (mean 2.30 log 10 CFU/g) for TAM; <1.70 (dl) - 2.76 log 10 CFU/g (mean 1.82 log 10 CFU/g) for M&Y; and <0.70(dl) - 1.74 log 10 CFU/g (mean 0.77 log 10 CFU/g) for TC. All the samples in this study tested negative for Salmonella spp., L. monocytogenes. E. coli and NoV GI and GII genome. A global overview of all the data was executed using Principal Component Analysis (PCA), and the results showed that the scores and loadings according to principal components 1 (PC1) and 2 (PC2) accounted for 75.9% of the total variance, allowing a distinction between fresh and frozen samples. The presence of moulds was significantly higher in the supermarket samples whereas the presence of total coliforms was significantly higher in the field samples (p < 0.05). Although pathogenic microorganisms were not found, preventative measures and prerequisites in the strawberry production chain must be considered in order to avoid possible foodborne diseases related to the microbiological quality of the fruit., (Copyright © 2019. Published by Elsevier B.V.)