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Reliable detection and identification of plant pathogens are essential for disease control strategies. Diagnostic methods commonly used to detect plant pathogens have limitations such as requirement of prior knowledge of the genome sequence, low sensitivity and a restricted ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled determination of total nucleic acid content in biological samples. The possibility of using the single‐molecule sequencing platform of Oxford Nanopore as a general method for diagnosis of plant diseases was examined. It was tested by sequencing DNA or RNA isolated from tissues with symptoms from plants of several families inoculated with known pathogens (e.g. bacteria, viruses, fungi, phytoplasma). Additionally, samples of groups of 200 seeds containing one infected seed of each of two or three pathogens, as well as samples with symptoms but unidentified pathogens were tested. Sequencing results were analysed with Nanopore data analysis tools. In all the inoculated plants, pathogens were identified in real time within 1–2 h of running the Nanopore sequencer and were classified to the species or genus level. DNA sequencing or direct RNA sequencing of samples with unidentified disease agents were validated by conventional diagnostic procedures (e.g. PCR, ELISA, Koch test), which supported the results obtained by Nanopore sequencing. The advantages of this technology include: long read lengths, fast run times, portability, low cost and the possibility of use in every laboratory. This study indicates that adoption of the Nanopore platform will be greatly advantageous for routine laboratory diagnosis. [ABSTRACT FROM AUTHOR]

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.

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)., 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 © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Philosoph, Dombrovsky, Luria, Sela, Elad and Frenkel.)

Knowledge on symbiotic microorganisms of insects has increased dramatically in recent years, yet relatively little data are available regarding non-pathogenic viruses. Here we studied the virome of the parasitoid wasp Anagyrus vladimiri Triapitsyn (Hymenoptera: Encyrtidae), a biocontrol agent of mealybugs. By high-throughput sequencing of viral nucleic acids, we revealed three novel viruses, belonging to the families Reoviridae [provisionally termed AnvRV (Anagyrus vladimiri reovirus)], Iflaviridae (AnvIFV) and Dicistroviridae (AnvDV). Phylogenetic analysis further classified AnvRV in the genus Idnoreovirus , and AnvDV in the genus Triatovirus . The genome of AnvRV comprises 10 distinct genomic segments ranging in length from 1.5 to 4.2 kb, but only two out of the 10 ORFs have a known function. AnvIFV and AnvDV each have one polypeptide ORF, which is typical of iflaviruses but very un-common among dicistroviruses. Five conserved domains were found along both the ORFs of those two viruses. AnvRV was found to be fixed in an A. vladimiri population that was obtained from a mass rearing facility, whereas its prevalence in field-collected A. vladimiri was ~15 %. Similarly, the prevalence of AnvIFV and AnvDV was much higher in the mass rearing population than in the field population. The presence of AnvDV was positively correlated with the presence of Wolbachia in the same individuals. Transmission electron micrographs of females' ovaries revealed clusters and viroplasms of reovirus-like particles in follicle cells, suggesting that AnvRV is vertically transmitted from mother to offspring. AnvRV was not detected in the mealybugs, supporting the assumption that this virus is truly associated with the wasps. The possible effects of these viruses on A. vladimiri 's biology, and on biocontrol agents in general, are discussed. Our findings identify RNA viruses as potentially involved in the multitrophic system of mealybugs, their parasitoids and other members of the holobiont.

The tobamovirus tomato brown rugose fruit virus (ToBRFV) infects tomato plants harboring the Tm-2 2 resistance allele, which corresponds with tobamoviruses' avirulence ( Avr ) gene encoding the movement protein to activate a resistance-associated hypersensitive response (HR). ToBRFV has caused severe damage to tomato crops worldwide. Unlike tomato plants, pepper plants harboring the L resistance alleles, which correspond with the tobamovirus Avr gene encoding the coat protein, have shown HR manifestations upon ToBRFV infection. We have found that ToBRFV inoculation of a wide range of undefined pepper plant varieties could cause a "hypersensitive-like cell death" response, which was associated with ToBRFV transient systemic infection dissociated from disease symptom manifestations on fruits. Susceptibility of pepper plants harboring L 1 , L 3 , or L 4 resistance alleles to ToBRFV infection following HRs was similarly transient and dissociated from disease symptom manifestations on fruits. Interestingly, ToBRFV stable infection of a pepper cultivar not harboring the L gene was also not associated with disease symptoms on fruits, although ToBRFV was localized in the seed epidermis, parenchyma, and endothelium, which borders the endosperm, indicating that a stable infection of maternal origin of these tissues occurred. Pepper plants with systemic ToBRFV infection could constitute an inoculum source for adjacently grown tomato plants.

The efficiency of epitope-based vaccination (subunit vaccines) is tightly correlated with heterogeneity and the high density of epitope presentation, which maximizes the potential antigenic determinants. Here, we developed a two-mode platform for intensifying the epitope presentation of subunit vaccines. The two-mode epitope presentation enhancement includes a covalent attachment of high concentrations of SARS-CoV-2-S1 peptide epitope to the surface of virus-like-particles (VLPs) and the subsequent assembly of VLP/epitope conjugates on the oil droplet surface at an oil/water interface of an emulsion as Pickering stabilizers. The resultant emulsions were stable for weeks in ambient conditions, and our platform was challenged using the epitope of the SARS-CoV-2-S1 peptide that served as a model epitope in this study. In vivo assays showed that the αSARS-CoV-2-S1 immunoglobulin G (IgG) titers of the studied mouse antisera, developed against the SARS-CoV-2-S1 peptide under different epitope preparation conditions, showed an order of magnitude higher IgG titers in the studied VLP-based emulsions than epitopes dissolved in water and epitopes administered with an adjuvant, thereby confirming the efficacy of the formulation. This VLP-based Pickering emulsion platform is a fully synthetic approach that can be readily applied for vaccine development to a wide range of pathogens.

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.

Studies of early stages of cucumber green mottle mosaic virus (CGMMV) disease have been recently focused on plant molecular responses. However, extreme diurnal environmental temperatures, characteristic of global climate changes, could affect plant susceptibility and disease phenotype progression. Our studies of CGMMV disease progression, under simulated extreme temperature waves, have revealed two new disease initiation phenotypes that developed gradually, preceding severe symptom manifestations of post-recovery CGMMV systemic infections. 'Early post-recovery stage' bright yellow islands (BYIs) with defined boundaries amid asymptomatic leaf blades were first emerging followed by 'late post-recovery stage' BYIs with diffused boundaries. A deduced CGMMV disease progression scheme, postulating BYI symptom occurrence time-windows, revealed BYIs in field grown cucumber plants exposed to extreme diurnal temperatures. Profiling ontology of cucumber differentially expressed genes in BYIs vs the associated dark-green surrounding tissues disclosed activation of jasmonic acid (JA) pathway in 'early post-recovery stage' BYIs. JA signaling was inactivated in 'late post-recovery stage' BYIs concomitant with increasing expressions of JA signaling inhibitors and downregulation of JA responsive phenylpropanoid pathway. Our results disclosed a new phenotypic description of CGMMV disease initiation, characteristic of cucumbers grown under extreme environmental temperature fluctuations. The BYI phenotypes could define a time-window for CGMMV disease management applications., (© 2021. The Author(s).)

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).

The tobamovirus tomato brown rugose fruit virus (ToBRFV), a major threat to tomato production worldwide, has recently been documented in mixed infections with the potexvirus pepino mosaic virus (PepMV) CH2 strain in traded tomatoes in Israel. A study of greenhouse tomato plants in Israel revealed severe new viral disease symptoms including open unripe fruits and yellow patched leaves. PepMV was only detected in mixed infections with ToBRFV in all 104 tested sites, using serological and molecular analyses. Six PepMV isolates were identified, all had predicted amino acids characteristic of CH2 mild strains excluding an isoleucine at amino acid position 995 of the replicase. High-throughput sequencing of viral RNA extracted from four selected symptomatic plants showed solely the ToBRFV and PepMV, with total aligned read ratios of 40.61% and 11.73%, respectively, indicating prevalence of the viruses. Analyses of interactions between the co-infecting viruses by sequential and mixed viral inoculations of tomato plants, at various temperatures, showed a prominent increase in PepMV titers in ToBRFV pre-inoculated plants and in mixed-infected plants at 18-25 °C, compared to PepMV-single inoculations, as analyzed by Western blot and quantitative RT-PCR tests. These results suggest that Israeli mild PepMV isolate infections, preceded by ToBRFV, could induce symptoms characteristic of PepMV aggressive strains.

During 2019, tomato fruits showing viral-like symptoms of marbled yellow spots were abundant in Israel. The new symptoms were distinctive from those typical of tomato brown rugose fruit virus (ToBRFV) infection but resembled symptoms of pepino mosaic virus (PepMV) infection. RT-PCR analysis and the serological tests (enzyme linked immunosorbent assay, western blot and in situ immunofluorescence) revealed and confirmed the presence of both the tobamovirus ToBRFV and the potexvirus PepMV in the symptomatic fruits. A mixture of rod-like and filamentous particles, characteristic of viruses belonging to tobamovirus and potexvirus genera, was visualized by transmission electron microscopy of the tomato fruit viral extract. Sanger sequencing of amplified PepMV-coat protein gene segments showed ~98% sequence identity to the Chilean (CH2)-strain. In a biological assay testing the contribution of traded infected tomatoes to the establishment of tomato plant disease, we applied direct and indirect inoculation modes using Tm-2 2 -resistant tomato plants. The results, assessed by disease symptom development along with serological and molecular analyses, showed that the ToBRFV and PepMV co-infected fruits were an effective inoculum source for disease spread only when fruits were damaged. Importantly, intact fruits did not spread the viral disease. These results added a new factor to disease epidemiology of these viruses.

In a survey conducted in Cannabis sativa L. (cannabis) authorized farms in Israel, plants showed disease symptoms characteristic of nutrition deprivation. Interveinal chlorosis, brittleness, and occasional necrosis were observed in older leaves. Next generation sequencing analysis of RNA extracted from symptomatic leaves revealed the presence of lettuce chlorosis virus (LCV), a crinivirus that belongs to the Closteroviridae family. The complete viral genome sequence was obtained using RT-PCR and Rapid Amplification of cDNA Ends (RACE) PCR followed by Sanger sequencing. The two LCV RNA genome segments shared 85-99% nucleotide sequence identity with LCV isolates from GenBank database. The whitefly Bemisia tabaci Middle Eastern Asia Minor1 (MEAM1) biotype transmitted the disease from symptomatic cannabis plants to un-infected 'healthy' cannabis, Lactuca sativa , and Catharanthus roseus plants. Shoots from symptomatic cannabis plants, used for plant propagation, constituted a primary inoculum of the disease. To the best of our knowledge, this is the first report of cannabis plant disease caused by LCV.

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., (Copyright © 2019 American Society for Microbiology.)

Cucumber green mottle mosaic virus (CGMMV), genus Tobamovirus, is a major pathogen of cucurbits that primarily affects cucumber, melon, and watermelon crops. The aim of this study was to reveal the contribution of CGMMV-infected female flowers to disease spread. Using a fluorescent in situ hybridization (FISH) technique, we show that ovaries and ovules of CGMMV-infected cucumber and melon plants showed a CGMMV-specific fluorescence signal prior to and following anthesis. The fluorescence signal was prominent but sporadic. Ripe fruits of infected melon plants showed strong signals in the funiculus, the seed stalk, which connects the developing seed to the interior ovary wall. Importantly, in seeds, a strong fluorescence signal was observed in the perisperm-endosperm (PE) envelope, which underlies the seed coat and surrounds the embryo. Interestingly, the fluorescence signal was not uniformly distributed in the PE envelope but was localized to a specific envelope layer. These results have important epidemiological implications for CGMMV management and commercial seed production, particularly regarding the improvement of seed disinfection methods that will contribute to limit the global distribution of the virus.

The bumblebee Bombus terrestris is a beneficial pollinator extensively used in tomato production. Our hypothesis was that bumblebee hives collected from a Tomato brown rugose fruit virus (ToBRFV) infected tomato greenhouse, preserve an infectious primary inoculum. Placing a bumblebee hive collected from a ToBRFV contaminated greenhouse, in a glass-/net-house containing only uninfected healthy tomato plants, spread ToBRFV disease. Control uninfected tomato plants grown in a glass-/net-house devoid of any beehive remained uninfected. ToBRFV-contaminated hives carried infectious viral particles as demonstrated in a biological assay on laboratory test plants of virus extracted from hive components. Viral particles isolated from a contaminated hive had a typical tobamovirus morphology observed in transmission electron microscopy. Assembly of ToBRFV genome was achieved by next generation sequencing analysis of RNA adhering to the bumblebee body. Bumblebee dissection showed that ToBRFV was mostly present in the abdomen suggesting viral disease spread via buzz pollination. These results demonstrate that bumblebee hives collected from ToBRFV-contaminated greenhouses carry a primary inoculum that reflects the status of viruses in the growing area. This new mode of ToBRFV spread by pollinators opens an avenue for detection of viruses in a growing area through analysis of the pollinators, as well as emphasizes the need to reevaluate the appropriate disease management protocols., Competing Interests: The authors have declared that no competing interests exist.

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-2 2 -resistant tomato plants with the PaMMV-IL and Tomato brown rugose fruit virus (ToBRFV) resulted in accelerated viral expression in the plants.

Background: The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi-SlPH tomato lines was sequenced and gene-expression patterns were compared., Results: C. gloeosporioides inoculation of the RNAi-SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi-SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi-SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi-SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi-SlPH tomato, it mainly upregulates the nitrate metabolic process., Conclusions: Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi-SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi-SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.

The GATA transcription factor AreA is a global nitrogen regulator that restricts the utilization of complex and poor nitrogen sources in the presence of good nitrogen sources in microorganisms. In this study, we report the biological function of an AreA homolog (the CgareA gene) in the fruit postharvest pathogen Colletotrichum gloeosporioides. Targeted gene deletion mutants of areA exhibited significant reductions in vegetative growth, increases in conidia production, and slight decreases in conidial germination rates. Quantitative RT-PCR (qRT-PCR) analysis revealed that the expression of AreA was highly induced under nitrogen-limiting conditions. Moreover, compared to wild-type and complemented strains, nitrogen metabolism-related genes were misregulated in ΔareA mutant strains. Pathogenicity assays indicated that the virulence of ΔareA mutant strains were affected by the nitrogen content, but not the carbon content, of fruit hosts. Taken together, our results indicate that CgareA plays a critical role in fungal development, conidia production, regulation of nitrogen metabolism and virulence in Colletotrichum gloeosporioides., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An outbreak of a new disease infecting tomatoes occurred in October-November 2014 at the Ohad village in Southern Israel. Symptomatic plants showed a mosaic pattern on leaves accompanied occasionally by narrowing of leaves and yellow spotted fruit. The disease spread mechanically and rapidly reminiscent of tobamovirus infection. Epidemiological studies showed the spread of the disease in various growing areas, in the South and towards the Southeast and Northern parts of the country within a year. Transmission electron microscope (TEM) analysis showed a single rod-like form characteristic to the Tobamovirus genus. We confirmed Koch's postulates for the disease followed by partial host range determination and revealed that tomato cultivars certified to harbor the Tm-22 resistance gene are susceptible to the new viral disease. We further characterized the viral source of the disease using a range of antisera for serological detection and analyzed various virus genera and families for cross-reactivity with the virus. In addition, next generation sequencing of total small RNA was performed on two cultivars grown in two different locations. In samples collected from commercial cultivars across Israel, we found a single virus that caused the disease. The complete genome sequence of the new Israeli tobamovirus showed high sequence identity to the Jordanian isolate of tomato brown rugose fruit virus., Competing Interests: The authors have declared that no competing interests exist.

Growth of Colletotrichum gloeosporioides in the presence of cation salts NaCl and KCl inhibited fungal growth and anthracnose symptom of colonization. Previous reports indicate that adaptation of Aspergillus nidulans to salt- and osmotic-stress conditions revealed the role of zinc-finger transcription factors SltA and CrzA in cation homeostasis. Homologs of A. nidulans SltA and CrzA were identified in C. gloeosporioides. The C. gloeosporioides CrzA homolog is a 682-amino acid protein, which contains a C2H2 zinc finger DNA-binding domain that is highly conserved among CrzA proteins from yeast and filamentous fungi. The C. gloeosporioides SltA homolog encodes a 775-amino acid protein with strong similarity to A. nidulans SltA and Trichoderma reesei ACE1, and highest conservation in the three zinc-finger regions with almost no changes compared to ACE1 sequences. Knockout of C. gloeosporioides crzA (ΔcrzA) resulted in a phenotype with inhibited growth, sporulation, germination and appressorium formation, indicating the importance of this calciu006D-activated transcription factor in regulating these morphogenetic processes. In contrast, knockout of C. gloeosporioides sltA (ΔsltA) mainly inhibited appressorium formation. Both mutants had reduced pathogenicity on mango and avocado fruit. Inhibition of the different morphogenetic stages in the ΔcrzA mutant was accompanied by drastic inhibition of chitin synthase A and B and glucan synthase, which was partially restored with Ca2+ supplementation. Inhibition of appressorium formation in ΔsltA mutants was accompanied by downregulation of the MAP kinase pmk1 and carnitine acetyl transferase (cat1), genes involved in appressorium formation and colonization, which was restored by Ca2+ supplementation. Furthermore, exposure of C. gloeosporioides ΔcrzA or ΔsltA mutants to cations such as Na+, K+ and Li+ at concentrations that the wild type C. gloeosporioides is not affected had further adverse morphogenetic effects on C. gloeosporioides which were partially or fully restored by Ca2+. Overall results suggest that both genes modulating alkali cation homeostasis have significant morphogenetic effects that reduce C. gloeosporioides colonization., Competing Interests: The authors have declared that no competing interests exist.

We report here the sequence of a new benyvirus found through mango cultivar Shelly RNA-seq analysis in Israel. This is the first virus strain reported in mango trees., (Copyright © 2016 Sela et al.)

Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens-Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum-secrete small pH-affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC-dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non-preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high-carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host-dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization., (© 2015 BSPP and John Wiley & Sons Ltd.)

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.

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".

Gene expression regulation by pH in filamentous fungi and yeasts is controlled by the PACC/RIM101 transcription factor. In Colletotrichum gloeosporioides, PACC is known to act as positive regulator of alkaline-expressed genes, and this regulation was shown to contribute to fungal pathogenicity. PACC is also a negative regulator of acid-expressed genes, however; the mechanism of downregulation of acid-expressed genes by PACC and their contribution to C. gloeosporioides pathogenicity is not well understood. RNA sequencing data analysis was employed to demonstrate that PACC transcription factor binding sites (TFBS) are significantly overrepresented in the promoter of PACC-upregulated, alkaline-expressed genes. In contrast, they are not overrepresented in the PACC-downregulated, acid-expressed genes. Instead, acid-expressed genes showed overrepresentation of AREB GATA TFBS in C. gloeosporioides and in homologs of five other ascomycetes genomes. The areB promoter contains PACC TFBS; its transcript was upregulated at pH 7 and repressed in ΔpacC. Furthermore, acid-expressed genes were found to be constitutively upregulated in ΔareB during alkalizing conditions. The areB mutants showed significantly reduced ammonia secretion and pathogenicity on tomato fruit. Present results indicate that PACC activates areB expression, thereby conditionally repressing acid-expressed genes and contributing critically to C. gloeosporioides pathogenicity.

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.

Background: The mango belongs to the genus Mangifera, consisting of numerous tropical fruiting trees in the flowering plant family, Anacardiaceae. Postharvest treatment by hot water brushing (HWB) for 15-20 s was introduced commercially to improve fruit quality and reduce postharvest disease. This treatment enabled successful storage for 3-4 weeks at 12°C, with improved color and reduced disease development, but it enhanced lenticel discoloration on the fruit peel. We investigated global gene expression induced in fruit peel by HWB treatment, and identified key genes involved in mechanisms potentially associated with fruit resistance to pathogens, peel color improvement, and development of lenticel discoloration; this might explain the fruit's phenotypic responses., Results: The mango transcriptome assembly was created and characterized by application of RNA-seq to fruit-peel samples. RNA-seq-based gene-expression profiling identified three main groups of genes associated with HWB treatment: 1) genes involved with biotic and abiotic stress responses and pathogen-defense mechanisms, which were highly expressed; 2) genes associated with chlorophyll degradation and photosynthesis, which showed transient and low expression; and 3) genes involved with sugar and flavonoid metabolism, which were highly expressed., Conclusions: We describe a new transcriptome of mango fruit peel of cultivar Shelly. The existence of three main groups of genes that were differentially expressed following HWB treatment suggests a molecular basis for the biochemical and physiological consequences of the postharvest HWB treatment, including resistance to pathogens, improved color development, and occurrence of lenticel discoloration.

Hibiscus rosa-sinensis is one of the most prevalent ornamental plants grown in private and public gardens. Hibiscus chlorotic ringspot virus (HCRSV) is a member of the Carmovirus genus, with a positive single-strand RNA that putatively encodes seven proteins. The complete genome of the first Israeli isolate of HCRSV, HCRSV-IL, comprises 3,908 nucleotides and shows 93% nucleotide sequence identity to the Singapore isolate and 87% identity to the Taiwanese isolate.

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.

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.

IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.