Nanodissection of Selected Viral Particles by Scanning Transmission Electron Microscopy/Focused Ion Beam for Genetic Identification.

This study presents the development of a new correlative workflow to bridge the gap between electron microscopy imaging and genetic analysis of viruses. The workflow enables the assignment of genetic information to a specific biological entity by harnessing the nanodissection capability of focused ion beam (FIB). This correlative workflow is based on scanning transmission electron microscopy (STEM) and FIB followed by a polymerase chain reaction (PCR). For this purpose, we studied the tomato brown rugose fruit virus (ToBRFV) and the adenovirus that have significant impacts on plant integrity and human health, respectively. STEM imaging was used for the identification and localization of virus particles on a transmission electron microscopy (TEM) grid followed by FIB milling of the desired region of interest. The final-milled product was subjected to genetic analysis by the PCR. The results prove that the FIB-milling process maintains the integrity of the genetic material as confirmed by the PCR. We demonstrate the identification of RNA and DNA viruses extracted from a few micrometers of an FIB-milled TEM grid. This workflow enables the genetic analysis of specifically imaged viral particles directly from heterogeneous clinical samples. In addition to viral diagnostics, the ability to isolate and to genetically identify specific submicrometer structures may prove valuable in additional fields, including subcellular organelle and granule research.

Interactions of Microorganisms with Lipid Langmuir Layers.

Preventing microbial contamination of aquatic environments is crucial for the proper supply of drinking water. Hence, understanding the interactions that govern bacterial and virus adsorption to surfaces is crucial to prevent infection transmittance. Here, we describe a new approach for studying the organization and interactions of various microorganisms, namely, Escherichia coli (E. coli) bacteria, E. coli-specific bacteriophage T4, and plant cucumber green mottle mosaic viruses (CGMMV), at the air/water interface using the Langmuir-Blodgett (LB) technique. CGMMV were found as applicable candidates for further studying their interactions with Langmuir lipid monolayers. The zwitterionic, positively, and negatively charged LB lipid monolayers with adsorbed viruses were deposited onto solid supports and characterized by atomic force microscopy. Using polymerase chain reaction, we indicated that the adsorption of CGMMV onto the LB monolayer is a result of electrostatic interactions. These insights are useful in engineering membrane filters that prevent biofouling for efficient purification systems.

Antiviral Polymers Based on N-Halamine Polyurea.

N-halamines are a commonly applied class of antimicrobial agents used for a variety of applications relating to human health. Here, we present the modulation of the common polymers polyurea and polyguanidine with the N-halamine technology. The N-H bonds in either polymer were converted to N-Cl or N-Br bonds capable of releasing Cl+ or Br+ cations to aqueous media as antiviral agents. Controlled release of the oxidizing agents was monitored for a period of 4 weeks. Antiviral activity was evaluated against the T4 bacteriophage as well as against the highly stable plant virus belonging to the Tobamovirus genus, tomato brown rugose fruit virus. The incorporation of the N-halamine technology on commonly used polymers has effectively introduced antiviral functionality for a wide variety of potential applications.

Transmission of a New Polerovirus Infecting Pepper by the Whitefly Bemisia tabaci.

Many animal and plant viruses depend on arthropods for their transmission. Virus-vector interactions are highly specific, and only one vector or one of a group of vectors from the same family is able to transmit a given virus. Poleroviruses (Luteoviridae) are phloem-restricted RNA plant viruses that are exclusively transmitted by aphids. Multiple aphid-transmitted polerovirus species commonly infect pepper, causing vein yellowing, leaf rolling, and fruit discoloration. Despite low aphid populations, a recent outbreak with such severe symptoms in many bell pepper farms in Israel led to reinvestigation of the disease and its insect vector. Here we report that this outbreak was caused by a new whitefly (Bemisia tabaci)-transmitted polerovirus, which we named Pepper whitefly-borne vein yellows virus (PeWBVYV). PeWBVYV is highly (>95%) homologous to Pepper vein yellows virus (PeVYV) from Israel and Greece on its 5' end half, while it is homologous to African eggplant yellows virus (AeYV) on its 3' half. Koch's postulates were proven by constructing a PeWBVYV infectious clone causing the pepper disease, which was in turn transmitted to test pepper plants by B. tabaci but not by aphids. PeWBVYV represents the first report of a whitefly-transmitted polerovirus.IMPORTANCE The high specificity of virus-vector interactions limits the possibility of a given virus changing vectors. Our report describes a new virus from a family of viruses strictly transmitted by aphids which is now transmitted by whiteflies (Bemisia tabaci) and not by aphids. This report presents the first description of polerovirus transmission by whiteflies. Whiteflies are highly resistant to insecticides and disperse over long distances, carrying virus inoculum. Thus, the report of such unusual polerovirus transmission by a supervector has extensive implications for the epidemiology of the virus disease, with ramifications concerning the international trade of agricultural commodities.

Isolation and characterization of a novel cripavirus, the first Dicistroviridae family member infecting the cotton mealybug Phenacoccus solenopsis.

A new virus belonging to the family Dicistroviridae was identified in the hibiscus-infesting cotton mealybug Phenacoccus solenopsis. Using high-throughput sequencing (HTS) on an Illumina HiSeq platform, a single contig of the complete genome sequence was assembled. The authenticity of the sequence obtained by HTS was validated by RT-PCR and Sanger sequencing of the amplicons, which was also employed for the 3' untranslated region (UTR). The 5' UTR was sequenced using a rapid amplification of cDNA ends kit. A large segment encompassing the whole genome was amplified by RT-PCR using viral RNA extracted from mealybugs. A whole-genome nucleotide sequence comparison showed 89% sequence identity to aphid lethal paralysis virus (ALPV), covering a short segment of 44 bp. Pairwise amino acid sequence comparisons of the protein encoded by open reading frame (ORF) 2 with its counterparts in the GenBank database, showed less than 40% identity to several members of the genus Cripavirus, including ALPV. Phylogenetic analysis based on the deduced amino acid sequence of the ORF 2 protein showed that the new virus grouped with members of the genus Cripavirus. The intergenic region (IGR) internal ribosome entry site (IRES) showed the conserved nucleotides of a type I IGR IRES and had two bulge sites, three pseudoknots, and two stem-loops. Virus morphology visualized by transmission electron microscopy demonstrated spherical particles with a diameter of ~30 nm. This virus was the only arthropod virus identified in the sampled mealybugs, and the purified virus was able to infect cotton mealybugs. To the best of our knowledge, this is the first report of a Dicistroviridae family member infecting P. solenopsis, and we have tentatively named this virus Phenacoccus solenopsis virus (PhSoV).

Insight Into Late Wilting Disease of Cucumber Demonstrates the Complexity of the Phenomenon in Fluctuating Environments.

Some diseases are caused by coinfection of several pathogens in the same plant. However, studies on the complexity of these coinfection events under different environmental conditions are scarce. Our ongoing research involves late wilting disease of cucumber caused by coinfection of Cucumber green mottle mosaic virus (CGMMV) and Pythium spp. We specifically investigated the role of various temperatures (18, 25, 32°C) on the coinfection by CGMMV and two predominant Pythium species occurring in cucumber greenhouses under Middle Eastern climatic conditions. During the summer months, Pythium aphanidermatum was most common, whereas P. spinosum predominated during the winter-spring period. P. aphanidermatum preferred higher temperatures while P. spinosum preferred low temperatures and caused very low levels of disease at 32°C when the 6-day-old seedlings were infected with P. spinosum alone. Nevertheless, after applying a later coinfection with CGMMV on the 14-day-old plants, a synergistic effect was detected for both Pythium species at optimal and suboptimal temperatures, with P. spinosum causing high mortality incidence even at 32°C. The symptoms caused by CGMMV infection appeared earlier as the temperature increased. However, within each temperature, no significant influence of the combined infection was detected. Our results demonstrate the complexity of coinfection in changing environmental conditions and indicate its involvement in disease development and severity as compared with infection by each of the pathogens alone.

Using genomic analysis to identify tomato Tm-2 resistance-breaking mutations and their underlying evolutionary path in a new and emerging tobamovirus.

In September 2014, a new tobamovirus was discovered in Israel that was able to break Tm-2-mediated resistance in tomato that had lasted 55 years. The virus was isolated, and sequencing of its genome showed it to be tomato brown rugose fruit virus (ToBRFV), a new tobamovirus recently identified in Jordan. Previous studies on mutant viruses that cause resistance breaking, including Tm-2-mediated resistance, demonstrated that this phenotype had resulted from only a few mutations. Identification of important residues in resistance breakers is hindered by significant background variation, with 9-15% variability in the genomic sequences of known isolates. To understand the evolutionary path leading to the emergence of this resistance breaker, we performed a comprehensive phylogenetic analysis and genomic comparison of different tobamoviruses, followed by molecular modeling of the viral helicase. The phylogenetic location of the resistance-breaking genes was found to be among host-shifting clades, and this, together with the observation of a relatively low mutation rate, suggests that a host shift contributed to the emergence of this new virus. Our comparative genomic analysis identified twelve potential resistance-breaking mutations in the viral movement protein (MP), the primary target of the related Tm-2 resistance, and nine in its replicase. Finally, molecular modeling of the helicase enabled the identification of three additional potential resistance-breaking mutations.

A local strain of Paprika mild mottle virus breaks L3 resistance in peppers and is accelerated in Tomato brown rugose fruit virus-infected Tm-22-resistant tomatoes.

During October 2014, unfamiliar mild mosaic and mottling symptoms were identified on leaves of pepper (Capsicum chinense cv. Habanero) seedlings grown in the Arava valley in Israel 2-3 weeks post planting. Symptomatic plants were tested positive by ELISA using laboratory-produced antisera for tobamovirus species. Typical tobamovirus rod-shaped morphology was observed by transmission electron microscopy (TEM) analysis of purified virion preparation that was used for mechanical inoculation of laboratory test plants for the completion of Koch's postulates. The complete viral genome was sequenced from small interfering RNA purified from symptomatic pepper leaves and fruits by next-generation sequencing (NGS) using Illumina MiSeq platform. The contigs generated by the assembly covered 80% of the viral genome. RT-PCR amplification and Sanger sequencing were employed in order to validate the sequence generated by NGS technology. The nucleotide sequence of the complete viral genome was 99% identical to the complete genome of Paprika mild mottle virus isolate from Japan (PaMMV-J), and the deduced amino acid sequence was 99% identical to PaMMV-J protein. Amplicons from seed RNA showed 100% identity to the viral isolate from the collected symptomatic pepper plants. Partial host range analysis revealed a slow development of systemic infection in inoculated tomato plants (Lycopersicon esculentum). Interestingly, double inoculation of susceptible wild-type tomato plants and Tm-22-resistant tomato plants with the PaMMV-IL and Tomato brown rugose fruit virus (ToBRFV) resulted in accelerated viral expression in the plants.

Combined Infection with Cucumber green mottle mosaic virus and Pythium Species Causes Extensive Collapse in Cucumber Plants.

In the last decade, the phenomenon of late-wilting has increased in cucumber greenhouses during Cucumber green mottle mosaic virus (CGMMV) epidemics. Because the wilting appears in defined patches accompanied by root rot, it was hypothesized that the phenomenon is caused by coinfection of soilborne pathogens and CGMMV. A field survey showed that 69% of the collapsed plants were infected with both Pythium spp. and CGMMV, whereas only 20 and 6.6% were singly infected with Pythium spp. or CGMMV, respectively. Artificial inoculations in controlled-environmental growth chambers and glasshouse experiments showed that coinfection with Pythium spinosum and CGMMV leads to a strong synergistic wilting effect and reduces growth parameters. The synergy values of the wilting effect were not influenced by the time interval between P. spinosum and CGMMV infection. However, dry mass synergy values were decreased with longer intervals between infections. The results obtained in this study support the complexity of the wilting phenomenon described in commercial cucumber grown in protected structures during infection of Pythium spp. on the background of a vast CGMMV epidemic. They encourage a wider perspective of the complexity of agricultural diseases to apply the most suitable disease management.

Extended phylogenetic analysis of a new Israeli isolate of Brevicoryne brassicae virus (BrBV-IL) suggests taxonomic revision of the genus Iflavirus.

BACKGROUND: Brevicoryne brassicae virus (BrBV) is a positive-strand genomic RNA virus which is unassigned tentative member of the genus Iflavirus. BrBv was first identified and characterized in the late 90's in the cabbage aphid in the United Kingdom (UK) (J Gen Virol 88:2590-2595, 2007) and was fully sequenced, using random amplification of encapsidated RNA. No other reports have been published demonstrating detection of this virus outside the UK. FINDINGS: A new isolate of the cabbage aphid virus Brevicoryne brassicae virus was identified from Brevicoryne brassicae aphids growing on wild mustard plants (Sinapis arvensis) in northern Israel. The virus genome was partially assembled from purified siRNA using the Illumina MiSeq Sequencing System with limited success. Combining classical viral RNA purification and RT-PCR amplification followed by traditional Sanger sequencing enabled obtaining the complete genomic sequence. The Israeli strain of BrBV shared 95 % nucleotide sequence identity with the BrBV found in the United Kingdom. CONCLUSIONS: The detection of BrBV in Israel indicates a broader geographical distribution of the virus".

Watermelon Fruit Rot Disease in Israel is Caused by a Distinct Squash vein yellowing virus (SqVYV) Strain.

In spring 2014, unfamiliar watermelon disease symptoms were observed on 1,000 ha of watermelon plants (Citrullus lanatus Thunb.) growing in open fields in Jordan and Beit-She'an Valleys, Israel. These represented systemic wilt and yellowing of leaves with necrosis on leaves and stems, in some cases leading to plant dieback, fruit exocarp deterioration, and rotting of the fleshy mesocarp, leading to unmarketable fruit. Virus purification was carried out from watermelon exocarp and necrotic leaves, and transmission electron microscopy revealed viral particles with flexible filamentous morphology. The disease was transmitted by mechanical inoculation from symptomatic fruit and by the silverleaf whitefly Bemisia tabaci from symptomatic to healthy cucurbits. A reverse-transcription polymerase chain reaction (RT-PCR) test was conducted on purified virus preparation of Squash vein yellowing virus (SqVYV) using specific primers targeting the capsid protein gene revealing the expected amplicon size. The complete viral genome was sequenced and assembled by next-generation sequencing (NGS) Illumina MiSeq of small interfering RNA purified from symptomatic watermelon fruit, producing 92% genome coverage, and RT-PCR amplification and Sanger sequencing to close the genome gaps, validating the NGS sequence. The complete SqVYV-IL genome sequence shared 84% nucleotide sequence identity with the two complete genomes of SqVYV isolates from Florida, and 91% identity with the deduced amino acid sequence of the viral polyprotein.

Adenine methylation may contribute to endosymbiont selection in a clonal aphid population.

BACKGROUND: The pea aphid Acyrthosiphon pisum has two modes of reproduction: parthenogenetic during the spring and summer and sexual in autumn. This ability to alternate between reproductive modes and the emergence of clonal populations under favorable conditions make this organism an interesting model for genetic and epigenetic studies. The pea aphid hosts different types of endosymbiotic bacteria within bacteriocytes which help the aphids survive and adapt to new environmental conditions and habitats. The obligate endosymbiont Buchnera aphidicola has a drastically reduced and stable genome, whereas facultative endosymbionts such as Regiella insecticola have large and dynamic genomes due to phages, mobile elements and high levels of genetic recombination. In previous work, selection toward cold adaptation resulted in the appearance of parthenogenetic A. pisum individuals characterized by heavier weights and remarkable green pigmentation. RESULTS: Six adenine-methylated DNA fragments were isolated from genomic DNA (gDNA) extracted from the cold-induced green variant of A. pisum using deoxyadenosine methylase (Dam) by digesting the gDNA with the restriction enzymes DpnI and DpnII, which recognize the methylated and unmethylated GATC sites, respectively. The six resultant fragments did not match any sequence in the A. pisum or Buchnera genomes, implying that they came from facultative endosymbionts. The A1 fragment encoding a putative transposase and the A6 fragment encoding a putative helicase were selected for further comparison between the two A. pisum variants (green and orange) based on Dam analysis followed by PCR amplification. An association between adenine methylation and the two A. pisum variants was demonstrated by higher adenine methylation levels on both genes in the green variant as compared to the orange one. CONCLUSION: Temperature selection may affect the secondary endosymbiont and the sensitive Dam involved in the survival and adaptation of aphids to cold temperatures. There is a high degree of adenine methylation at the GATC sites of the endosymbiont genes at 8°C, an effect that disappears at 22°C. We suggest that endosymbionts can be modified or selected to increase host fitness under unfavorable climatic conditions, and that the phenotype of the newly adapted aphids can be inherited.

A Distinct Arabidopsis Latent Virus 1 Isolate Was Found in Wild Brassica hirta Plants and Bees, Suggesting the Potential Involvement of Pollinators in Virus Spread.

During our search for aphid-pathogenic viruses, a comovirus was isolated from wild asymptomatic Brassica hirta (white mustard) plants harboring a dense population of Brevicoryne brassicae aphids. The transmission-electron-microscopy visualization of purified virions revealed icosahedral particles. The virus was mechanically transmitted to plants belonging to Brassicaceae, Solanaceae, Amaranthaceae, and Fabaceae families, showing unique ringspot symptoms only on B. rapa var. perviridis plants. The complete viral genome, comprised of two RNA segments, was sequenced. RNA1 and RNA2 contained 5921 and 3457 nucleotides, respectively, excluding the 3' terminal poly-adenylated tails. RNA1 and RNA2 each had one open-reading frame encoding a polyprotein of 1850 and 1050 amino acids, respectively. The deduced amino acids at the Pro-Pol region, delineated between a conserved CG motif of 3C-like proteinase and a GDD motif of RNA-dependent RNA polymerase, shared a 96.5% and 90% identity with the newly identified Apis mellifera-associated comovirus and Arabidopsis latent virus 1 (ArLV1), respectively. Because ArLV1 was identified early in 2018, the B. hirta comovirus was designated as ArLV1-IL-Bh. A high-throughput-sequencing-analyses of the extracted RNA from managed honeybees and three abundant wild bee genera, mining bees, long-horned bees, and masked bees, sampled while co-foraging in a Mediterranean ecosystem, allowed the assembly of ArLV1-IL-Bh, suggesting pollinators' involvement in comovirus spread in weeds.

Genome assembly of bell pepper endornavirus from small RNA.

The family Endornaviridae infects diverse hosts, including plants, fungi, and oomycetes. Here we report for the first time the assembly of bell pepper endornavirus by next-generation sequencing of viral small RNA. Such a population of small RNA indicates the activation of the viral immunity silencing machinery by this cryptic virus, which probably encodes a novel silencing suppressor.

The Nerium oleander aphid Aphis nerii is tolerant to a local isolate of Aphid lethal paralysis virus (ALPV).

In a survey that was conducted during the year 2011, a local strain of Aphid lethal paralysis virus (ALPV) was identified and isolated from a wild population of Aphis nerii aphids living on Nerium oleander plants located in northern Israel. The new strain was tentatively named (ALPV-An). RNA extracted from the viral particles allowed the amplification and determination of the complete genome sequence. The virus genome is comprised of 9835 nucleotides. In a BLAST search analysis, the ALPV-An sequence showed 89 % nucleotide sequence identity with the whole genome of a South African ALPV and 96 and 94 % amino acid sequence identity with the ORF1 and ORF2 of that strain, respectively. In preliminary experiments, spray-applied, purified ALPV virions were highly pathogenic to the green peach aphid Myzus persicae; 95 % mortality was recorded 4 days post-infection. These preliminary results demonstrate the potential of ALPV for use as a biologic agent for some aphid control. Surprisingly, no visible ALPV pathogenic effects, such as morphological changes or paralysis, were observed in the A. nerii aphids infected with ALPV-An. The absence of clear ALPV symptoms in A. nerii led to the formulation of two hypotheses, which were partially examined in this study. The first hypothesis suggest that A. nerii is resistant or tolerant of ALPV, while the second hypothesis propose that ALPV-An may be a mild strain of ALPV. Currently, our results is in favor with the first hypothesis since ALPV-An is cryptic in A. nerii aphids and can be lethal for M. persicae aphids.

A new cryptic virus belonging to the family Partitiviridae was found in watermelon co-infected with Melon necrotic spot virus.

A novel virus was detected in watermelon plants (Citrullus lanatus Thunb.) infected with Melon necrotic spot virus (MNSV) using SOLiD next-generation sequence analysis. In addition to the expected MSNV genome, two double-stranded RNA (dsRNA) segments of 1,312 and 1,118 bp were also identified and sequenced from the purified virus preparations. These two dsRNA segments encode two putative partitivirus-related proteins, an RNA-dependent RNA polymerase (RdRP) and a capsid protein, which were sequenced. Genomic-sequence analysis and analysis of phylogenetic relationships indicate that these two dsRNAs together make up the genome of a novel Partitivirus. This virus was found to be closely related to the Pepper cryptic virus 1 and Raphanus sativus cryptic virus. It is suggested that this novel virus putatively named Citrullus lanatus cryptic virus be considered as a new member of the family Partitiviridae.

Solanum elaeagnifolium and S. rostratum as potential hosts of the tomato brown rugose fruit virus.

Invasive weeds cause significant crop yield and economic losses in agriculture. The highest indirect impact may be attributed to the role of invasive weeds as virus reservoirs within commercial growing areas. The new tobamovirus tomato brown rugose fruit virus (ToBRFV), first identified in the Middle East, overcame the Tm-22 resistance allele of cultivated tomato varieties and caused severe damage to crops. In this study, we determined the role of invasive weed species as potential hosts of ToBRFV and a mild strain of pepino mosaic virus (PepMV-IL). Of newly tested weed species, only the invasive species Solanum elaeagnifolium and S. rostratum, sap inoculated with ToBRFV, were susceptible to ToBRFV infection. S. rostratum was also susceptible to PepMV-IL infection. No phenotype was observed on ToBRFV-infected S. elaeagnifolium grown in the wild or following ToBRFV sap inoculation. S. rostratum plants inoculated with ToBRFV contained a high ToBRFV titer compared to ToBRFV-infected S. elaeagnifolium plants. Mixed infection with ToBRFV and PepMV-IL of S. rostratum plants, as well as S. nigrum plants (a known host of ToBRFV and PepMV), displayed synergism between the two viruses, manifested by increasing PepMV-IL levels. Additionally, when inoculated with either ToBRFV or PepMV-IL, disease symptoms were apparent in S. rostratum plants and the symptoms were exacerbated upon mixed infections with both viruses. In a bioassay, ToBRFV-inoculated S. elaeagnifolium, S. rostratum and S. nigrum plants infected tomato plants harboring the Tm-22 resistant allele with ToBRFV. The distribution and abundance of these Solanaceae species increase the risks of virus transmission between species.

Characterization of a novel psyllid-transmitted waikavirus in carrots.

Carrots collected from the Western Negev region in Israel during the winter of 2019 showed disease symptoms of chlorosis, leaf curling, a loss of apical dominance, and multiple lateral roots that were not associated with known pathogens of the carrot yellows disease. Symptomatic carrots were studied for a possible involvement of plant viruses in disease manifestations using high throughput sequencing analyses. The results revealed the presence of a waikavirus, sharing a ∼70% nucleotide sequence identity with Waikavirus genus members. Virions purified from waikavirus-positive carrots were visualized by transmission electron microscopy, showing icosahedral particle diameter of ∼28 nm. The genome sequence was validated by overlapping amplicons by designed 12 primer sets. A complete genome sequence was achieved by rapid amplification of cDNA ends (RACE) for sequencing the 5' end, and RT-PCR with oligo dT for sequencing the 3' end. The genome encodes a single large ORF, characteristic of waikaviruses. Aligning the waikavirus-deduced amino-acid sequence with other waikavirus species at the Pro-Pol region, a conserved sequence between the putative proteinase and the RNA-dependent RNA polymerase, showed a ∼40% identity, indicating the identification of a new waikavirus species. The amino-acid sequence of the three coat proteins and cleavage sites were experimentally determined by liquid chromatography-mass spectrometry. A phylogenetic analysis based on the Pro-Pol region revealed that the new waikavirus clusters with persimmon waikavirus and actinidia yellowing virus 1. The new waikavirus genome was localized in the phloem of waikavirus-infected carrots. The virus was transmitted to carrot and coriander plants by the psyllid Bactericera trigonica Hodkinson (Hemiptera: Triozidae).

Rapid defense mechanism suppression during viral- oomycete disease complex formation.

Combined infection of the host plant with pathogens involving different parasitic lifestyles may result in synergistic effects that intensify disease symptoms. Understanding the molecular dynamics during concurrent infection provides essential insight into the host response. The transcriptomic pattern of cucumber plants infected with a necrotrophic pathogen, Pythium spinosum, and a biotrophic pathogen, Cucumber green mottle mosaic virus (CGMMV) was studied at different time points, under regimes of single and co-infection. Analysis of CGMMV infection alone revealed a mild influence on host gene expression at the stem base, while the infection by P. spinosum is associated with drastic changes in gene expression. Comparing P. spinosum as a single infecting pathogen with a later co-infection by CGMMV revealed a rapid host response as early as 24 hours post-CGMMV inoculation with a sharp downregulation of genes related to the host defense mechanism against the necrotrophic pathogen. Suppression of the defense mechanism of co-infected plants was followed by severe stress, including 30% plants mortality and an increase of the P. spinosum hyphae. The first evidence of defense recovery against the necrotrophic pathogen only occurred 13 days post-viral infection. These results support the hypothesis that the viral infection of the Pythium pre-infected plants subverted the host defense system and changed the equilibrium obtained with P. spinosum. It also implies a time window in which the plants are most susceptible to P. spinosum after CGMMV infection.

Synthesis and Characterization of Durable Antibiofilm and Antiviral Silane-Phosphonium Thin Coatings for Medical and Agricultural Applications.

Pathogens such as bacteria and viruses cause disease in a range of hosts, from humans to plants. Bacterial biofilms, communities of bacteria, e.g., Staphylococcus aureusand Escherichia coli, attached to the surface, create a protective layer that enhances their survival in harsh environments and resistance to antibiotics and the host's immune system. Biofilms are commonly associated with food spoilage and chronic infections, posing challenges for treatment and prevention. Tomato brown rugose fruit virus (ToBRFV), a newly discovered tobamovirus, infects tomato plants, causing unique symptoms on the fruit, posing a risk for tomato production. The present study focuses on the effectiveness of silane-phosphonium thin coatings on polymeric films, e.g., polypropylene. Phosphonium has significant antibacterial activity and is less susceptible to antibacterial resistance, making it a safer alternative with a reduced environmental impact. We successfully synthesized silane-phosphonium monomers as confirmed by 31P NMR and mass spectrometry. The chemical composition, thickness, morphology, and wetting properties of the coatings were tested by Fourier-transform infrared spectroscopy with attenuated total reflectance, focused ion beam, atomic force microscopy, environmental scanning electron microscope, and contact angle (CA) measurements. The antibiofilm and antibacterial activities of the coatings were tested against S. aureus and E. coli, while the antiviral activity was evaluated against ToBRFV. The significant antibiofilm and antiviral activity suggests applications in various fields including medicine, agriculture, and the food industry.