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.

The Dissemination of Metarhizium brunneum Conidia by Females of the Red Palm Weevil, Rhynchophorus ferrugineus, Suggests a New Mechanism for Prevention Practices.

Direct contact between the conidia of entomopathogenic fungi (EPF) and their host is a prerequisite to successful infection; the host can, therefore, be infected by both direct treatment and by transmission of fungal inoculum from infested surfaces. This unique characteristic makes EPF especially relevant for the control of cryptic insects. In the case of the red palm weevil (RPW) Rhynchophorus ferrugineus, the eggs and larvae are almost inaccessible to direct-contact treatment. The objective of the present study was to investigate the mechanism of conidia transmission from a treated surface to host eggs and larvae. Foam pieces infested with Metarhizium brunneum conidial powder, conidial suspension, or distilled water were used as a laying surface for RPW females. The number of eggs laid was not affected by the EPF treatments and ranged from 2 to 14 eggs per female. However, hatching rate and larval survival were significantly reduced in the conidial powder treatment, resulted in 1.5% hatching and no live larvae. In the conidial suspension treatment, 21% of laid eggs hatched, compared to 72% in the control treatment. In both M. brunneum treatments, females' proboscis, front legs and ovipositor were covered with conidia. The females transferred conidia in both treatments to the laying holes, reaching up to 15 mm in depth. This resulted in reduced egg-hatching rate and significant larval mortality due to fungal infection. The stronger effect on egg and larval survival using dry conidia seemed to result from better conidial adhesion to the female weevil in this formulation. In future studies, this dissemination mechanism will be examined as a prevention strategy in date plantations.

The SWC4 subunit of the SWR1 chromatin remodeling complex is involved in varying virulence of Metarhizium brunneum isolates offering role of epigenetic regulation of pathogenicity.

The host - pathogen interaction is a multifactorial process subject to a co-evolutionary arms race consisting of rapid changes in both host and pathogen, controlled at the genetic and epigenetic levels. Previously, we showed intra-species variation in disease progression and pathogenicity in aphids for Metarhizium brunneum isolates MbK and Mb7. Herein, we compared genomic, epigenetic, and metabolomic variations between these isolates and their effects on pathogenicity. Genomic variation could not completely explain the observed differences between the isolates. However, differential N6-adenine methylation (6 mA) and its correlation to reduced expression of the essential SWC4 subunit of SWR1 chromatin-remodelling complex (SWR1-C) led us to hypothesize a role for swc4 in the varying pathogenicity. Mutagenesis of the essential swc4 gene in MbKisolate resulted in reduction of secondary-metabolite (SM) secretion and impaired virulence in Galleria mellonella. Our results suggest the role of SWC4 in the regulation of SMs and the role of both SWC4 and SWR1-C in virulence of M. brunneum isolates. A better understanding of epigenetic regulation of SM production and secretion in entomopathogenic fungi may enable theirmanipulation for better biocontrol performance, and expand possibilities for environmentally friendly pest control.

Not Only a Formulation: The Effects of Pickering Emulsion on the Entomopathogenic Action of Metarhizium brunneum.

Growing global population and environmental concerns necessitate the transition from chemical to eco-friendly pest management. Entomopathogenic fungi (EPF) are rising candidates for this task due to their ease of growing, broad host range and unique disease process, allowing EPF to infect hosts directly through its cuticle. However, EPF's requirement for high humidity negates their integration into conventional agriculture. To mitigate this problem, we formulated Metarhizium brunneum conidia in an oil-in-water Pickering emulsion. Conidia in aqueous and emulsion formulations were sprayed on Ricinus communis leaves, and Spodoptera littoralis larvae were introduced under low or high humidity. The following were examined: conidial dispersion on leaf, larval mortality, conidial acquisition by larvae, effects on larval growth and feeding, and dynamic of disease progression. Emulsion was found to disperse conidia more efficiently and caused two-fold more adhesion of conidia to host cuticle. Mortality from conidia in emulsion was significantly higher than other treatments reaching 86.5% under high humidity. Emulsion was also found to significantly reduce larval growth and feeding, while conferring faster fungal growth in-host. Results suggest that a Pickering emulsion is able to improve physical interactions between the conidia and their surroundings, while weakening the host through a plethora of mechanisms, increasing the chance of an acute infection.

Intraspecies variation of Metarhizium brunneum against the green peach aphid, Myzus persicae, provides insight into the complexity of disease progression.

BACKGROUND: Intensive application of chemical insecticides is required for aphid pest control. Among the biorational alternatives, entomopathogenic fungi are the most sustainable biocontrol agents; those of the order Hypocreales attack and cause fungal disease in arthropod hosts, with variations in host susceptibility attributed to both fungal and host characteristics. We evaluated inter- and intraspecies variations in Metarhizium spp. virulence and differences in fungal disease progression on adult and nymph stages of the green peach aphid, Myzus persicae (Sulzer), a parthenogenetically reproducing insect species. RESULTS: Minor interspecies diversity was detected between the generalist Metarhizium species examined. Interestingly, significant intraspecies diversity was observed between Metarhizium brunneum isolates Mb7 and MbK. Infected adult aphids demonstrated similar disease progression for both isolates, mortality rates of more than 80% and fivefold reduction in fecundity. However, nymph mortality was detected only following MbK infection, with 50% mortality and significant reduction in molting rates. Confocal laser scanning microscopy demonstrated the variation in the disease stages of conidial adhesion and hemocoel colonization on each examined day post inoculation for each isolate. Significantly faster disease progression was observed in MbK-infected versus Mb7-infected nymphs, the latter demonstrating a higher percentage of uninfected nymphs accompanied with aphid molting. CONCLUSIONS: The observed intraspecies variation suggests that altered conidial adhesion to the nymph cuticle is a major factor affecting virulence. We prove the role of nymph ecdysis as a defense mechanism disrupting fungal infection. Because significant differences were observed between closely related isolates, this study emphasizes the importance of appropriate isolate selection for biological control. © 2021 Society of Chemical Industry.

A New Israeli Tobamovirus Isolate Infects Tomato Plants Harboring Tm-22 Resistance Genes.

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.

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.

Ipomovirus--an atypical genus in the family Potyviridae transmitted by whiteflies.

Ipomoviruses (genus Ipomovirus) are whitefly-transmitted viruses assigned to the family Potyviridae. They are characterised by filamentous flexible particles and a positive-sense single-stranded RNA (+ssRNA) genome. The viral genome is translated into a polyprotein precursor, which is processed into mature proteins and a short overlapping open reading frame. The genus Ipomovirus contains four accepted species and one unapproved species, and two other tentative members have recently been characterised. Ipomoviruses cause serious economic losses in many important crops, including cassava, sweet potato, cucurbits, tomato and aubergine. These viruses are transmitted by whiteflies in a non-circulative, semi-persistent manner, the virions being retained on the external surface of the vectors' mouthparts for a few days or weeks. Comparison of the available complete genome sequences of different ipomoviruses revealed differences in their genome organisation and a considerable variation in their proteins and conserved motifs that may reflect functional differences. This review summarises the current knowledge of the members within the genus Ipomovirus, focusing on genome organisation, taxonomic classification and the mechanism by which they are transmitted.

Full-genome sequence of hibiscus chlorotic ringspot virus from Israel.

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.

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.