Effective plant virus enrichment using carbon nanotubes and microfluidics.

Plant virus detection and identification in crops is a pillar for disease management, import of crop material, production of clean stock plants and basic plant virology studies. In this report, we present a platform for the enrichment and isolation of known or unknown viruses. This platform is based on carbon nanotube arrays inside a microfluidic device that can be a solution for the identification of low titer viruses from plants. Using our microfluidic devices, we achieved enrichment of two economically important viruses, the orthotospovirus, tomato spotted wilt orthotospovirus (TSWV) and the potyvirus, zucchini yellow mosaic virus (ZYMV). The carbon nanotube arrays integrated in these microfluidic devices are capable of trapping viruses discriminated by their size; the virus rich arrays can be then analyzed by common downstream techniques including immunoassays, PCR, HTS and electron microscopy. This procedure offers a simple to operate and portable sample preparation device capable of trapping viruses from raw plant extracts while reducing the host contamination.

A natural substitution of a conserved amino acid in eIF4E confers resistance against multiple potyviruses.

Eukaryotic translation initiation factor 4E (eIF4E), which plays a pivotal role in initiating translation in eukaryotic organisms, is often hijacked by the viral genome-linked protein to facilitate the infection of potyviruses. In this study, we found that the naturally occurring amino acid substitution D71G in eIF4E is widely present in potyvirus-resistant watermelon accessions and disrupts the interaction between watermelon eIF4E and viral genome-linked protein of papaya ringspot virus-watermelon strain, zucchini yellow mosaic virus or watermelon mosaic virus. Multiple sequence alignment and protein modelling showed that the amino acid residue D71 located in the cap-binding pocket of eIF4E is strictly conserved in many plant species. The mutation D71G in watermelon eIF4E conferred resistance against papaya ringspot virus-watermelon strain and zucchini yellow mosaic virus, and the equivalent mutation D55G in tobacco eIF4E conferred resistance to potato virus Y. Therefore, our finding provides a potential precise target for breeding plants resistant to multiple potyviruses.

Efficient artificial microRNA-mediated resistance against zucchini yellow mosaic virus in zucchini via agroinfiltration.

Zucchini yellow mosaic virus (ZYMV) infects cucurbits causing yellow mosaic in leaves, malformations in fruits, and degradation of the product quality. RNA interference (RNAi) is a cellular mechanism in eukaryotes and it is exploited to protect them against viruses. The artificial micro RNA (amiRNA) mediated approach was employed to develop resistance against ZYMV. Four amiRNAs, amiZYMV_HC-115s and amiZYMV_HC-1162s (sense), amiZYMV_HC-182as and amiZYMV_HC-196as (antisense), were computationally designed and introduced into the AtMIR390a backbone. At four days post agroinfiltration (dpa) of zucchini cotyledons the corresponding pre- and the mature amiRNAs were identified in local tissue. Upon ZYMV inoculation of zucchini, ZYMV titer was significantly lower where amiZYMV_HCs were applied in relation to control starting at two days post inoculation (dpi). Control zucchini plants exhibited symptoms at 5-8 dpi, whereas the amiZYMV_HC-treated zucchini had symptoms at 14 dpi; at 21 dpi treated zucchini exhibited a 16 %, 19 %, 32 %, and 42.5 % protection, respectively. For luffa, we observed a lower protection (0 %, 17 %, 22.5 %, and 31 % at 21 dpi). Nicotiana benthamiana DCL4 knock-down mutants were infected by ZYMV, whereas when the amiZYMV_HC-196as was agroinfiltrated ZYMV was not detected by RT-PCR. These results indicate that amiRNA-mediated resistance could be applied against ZYMV in zucchini.

Knockout of elF4E using CRISPR/Cas9 for large-scale production of resistant cucumber cultivar against WMV, ZYMV, and PRSV.

CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling precise and efficient modifications in a genome. This technology is being used for the manipulation of target genes in a host to develop resistance against the plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key role in viral infection during interaction with potyvirus viral proteins genome linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in C. sativus is to be clarified in elF4E-VPg interaction. In addition, there are entanglements in the massive production of pathogen-resistant cultivars suitable for commercial production using CRISPR/Cas9 technology. Therefore, we targeted different positions of the elF4E in G27 and G247 inbred lines, using specific gRNA1 and gRNA2 for the first and third exons, respectively, and 1,221 transgene-free plants were selected in segregated T1 generation, where 192 G27 and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1DEL or elF4E_3DEL) and double (elF4E_1-3DEL) mutants. Disease symptoms of watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1 plants, and we did not observe any symptom in homozygous elF4E_1-3DEL and elF4E_1DEL mutants. However, homozygous elF4E_3DEL was positive in reverse transcription polymerase chain reaction (RT-PCR), even if there were no significant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated lower viral accumulation in homozygous elF4E_3DEL than heterozygous and non-edited plants. Regeneration and transformation protocols were also optimized comprehensively for both the genotypes. The average number of shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0, respectively. We could not detect any distinguishing difference between the non-edited and edited F1 plants for yield and morphology. Our results demonstrate an effective route for mass production of viral resistant cultivars of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant cultivars could be generated to reduce the losses caused by these pathogens in cucumber production.

Zucchini Yellow Mosaic Virus (ZYMV) as a Serious Biotic Stress to Cucurbits: Prevalence, Diversity, and Its Implications for Crop Sustainability.

Zucchini yellow mosaic virus (ZYMV) is a severe threat to cucurbit crops worldwide, including Pakistan. This study was pursued to evaluate the prevalence, geographic distribution, and molecular diversity of ZYMV isolates infecting cucurbits in Pakistan's Pothwar region. Almost all the plant viruses act as a biotic stress on the host plants, which results in a yield loss. These viruses cause losses in single-infection or in mixed-infection cucurbit crops, and we have found a number of mixed-infected samples belonging to the Curubitaceae family. Serological detection of the tested potyviruses in the collected cucurbit samples revealed that ZYMV was the most prevalent virus, with a disease incidence (DI) at 35.2%, followed by Papaya ringspot virus (PRSV) with an incidence of 2.2%, and Watermelon mosaic virus (WMV) having an incidence as little as 0.5% in 2016. In the year 2017, a relatively higher disease incidence of 39.7%, 2.4%, and 0.3% for ZYMV, WMV, and PRSV, respectively, was recorded. ZYMV was the most prevalent virus with the highest incidence in Attock, Rawalpindi, and Islamabad, while PRSV was observed to be the highest in Islamabad and Jhelum. WMV infection was observed only in Rawalpindi and Chakwal. Newly detected Pakistani ZYMV isolates shared 95.8-97.0% nucleotide identities among themselves and 77.1-97.8% with other isolates retrieved from GenBank. Phylogenetic relationships obtained using different ZYMV isolates retrieved from GenBank and validated by in silico restriction analysis revealed that four Pakistani isolates clustered with other ZYMV isolates in group IIb with Chinese, Italian, Polish, and French isolates, while another isolate (MK848239) formed a separate minor clade within IIb. The isolate MK8482490, reported to infect bitter gourd in Pakistan, shared a minor clade with a Chinese isolate (KX884570). Recombination analysis revealed that the recently found ZYMV isolate (MK848239) is most likely a recombinant of Pakistani (MK848237) and Italian (MK956829) isolates, with a recombinant breakpoint between 266 and 814 nucleotide positions. Local isolate comparison and recombination detection may aid in the development of a breeding program that identifies resistant sources against recombinant isolates because the ZYMV is prevalent in a few cucurbit species grown in the surveyed areas and causes heavy losses and economic damage to the agricultural community.

Copper oxide nanostructures as a potential method for control of zucchini yellow mosaic virus in squash.

BACKGROUND: Zucchini yellow mosaic virus (ZYMV) infects cucurbits and has been identified as a major limiting factor in their production. The purpose of this study was to create copper oxide nanostructures (CONS) to control ZYMV in squash plants. Protection of squash against ZYMV was assessed in terms of virus severity, ZYMV concentration, transcription of pathogenesis-related genes and growth enhancement of treated squash. RESULTS: The findings revealed that squash plants treated with CONS had a significant reduction in disease severity when compared with untreated plants. In squash plants treated with CONS, defense genes associated with the salicylic acid signaling pathway were strongly expressed compared with untreated plants. The structural characteristics of CONS, such as their small size and appropriate shape, added to their excellent anti-ZYMV efficacy. CONS-treated squash plants show significantly improved growth traits compared with untreated plants. CONCLUSION: Based on the results of this study, CONS may be a new strategy for the control of ZYMV in squash. This represents an unconventional solution to control this virus, particularly as no chemical pesticides can control viral diseases. © 2022 Society of Chemical Industry.

A New Benzothiadiazole Derivative with Systemic Acquired Resistance Activity in the Protection of Zucchini (Cucurbita pepo convar. giromontiina) against Viral and Fungal Pathogens.

The ability of plant resistance inducers to provide protection against viral diseases is one of their main advantages over conventional pesticides. In the case of viral diseases that cannot be controlled directly with pesticides, insecticides are used to control the vectors of viruses. However, the effectiveness of such treatments is strictly dependent on the time of application. The plant response to the application of systemic acquired resistance (SAR) inducers, as a result of the stimulating action of these substances, does not depend on the time of application as it triggers the plant's natural defence mechanism. The best-recognised substance showing SAR inducer activity is acibenzolar-S-methyl ester (ASM, BTH). As its activity against different plant pathogens of crops has been well documented, the current research is concentrated on the search for novel substances of the type. The tested substance, N-methoxy-N-methylbenzo(1,2,3)thiadiazole-7-carboxamide (BTHWA), is an amide derivative of benzothiadiazole, showing plant resistance-inducing activity. This article presents the activity of BTHWA that has led to increased resistance of zucchini (Cucurbita pepo convar. giromontiina) towards viral infections. In addition, since the occurrence of the fungal pathogen, powdery mildew, was also observed during the two-year field experiments, the activity of BTHWA related to the reduction of infection with this fungus was also investigated. The substance was applied in two different variants either four or eight times, over the whole vegetation season. Surprisingly, the variant of four applications performed at the beginning of the vegetation season proved more effective in protection against viruses and fungus. A possible explanation may be the occurrence of the growth-immunity trade-off phenomenon that is known in the literature. Disturbance in plant metabolism resulting from eight applications may lead to lower yields of plants treated with SAR inducers. Perhaps such overstimulation of the plants we treated eight times may not have brought the optimum increase in plant resistance.

Analysis of the molecular and biological variability of Zucchini yellow mosaic virus isolates from Iran and Iraq.

During the growing season of 2018, several field-grown cucurbit plants in different parts of Iraq and Iran were surveyed for the presence of zucchini yellow mosaic virus (ZYMV), using two degenerate primer pairs (CIF/Rev and NIb2F/3R) targeting the two separated partial regions of the potyvirus genome (CI and NIb respectively). 7 out of 20 samples were confirmed to be infected with ZYMV. Phylogenetic analyses based on the CI gene grouped all Iranian and two Iraqi (ZYMV1 and ZYMV2) isolates together with isolates from the Middle East in the subgroup (AI), whereas the other Iraqi (ZYMV3 and ZYMV4) isolates were clustered in the subgroup (DI), which was only consisted of American isolates. The highest and lowest identity between the studied isolates and the GenBank isolates showed that the two genes (CI, NIb) of each isolate particularly the Iraqi isolates were more similar to a specific and geographically scattered mosaic of worldwide isolates, suggestive of mixed infection might have occurred between different worldwide isolates in Iraq. Furthermore, the first complete nucleotide sequence of an Iraqi ZYMV (ZYMV-Iq) isolate was done, using the Illumina sequencing technique. The complete nucleotide sequence of ZYMV-Iq isolate was 9650 nt, excluding the 3'poly (A) tail. ZYMV-Iq isolate shared the highest nt identity of 98.8% with an American (KC665630) isolate. Phylogenetic analysis based on the full genome sequence placed ZYMV-Iq in subgroup A of group I alongside 18 isolates from the US and two isolates from Australia. In addition, recombination analysis detected lone significant recombination between ZYMV-Iq and South Korean (AY279000) isolate. Moreover, the results showed that symptom intensity was varied across experimental host plants.

Complete Genome Sequence of Zucchini Yellow Mosaic Virus Strain Kurdistan, Iran.

The complete genome sequence of Zucchini yellow mosaic virus strain Kurdistan (ZYMV-Kurdistan) infecting squash from Iran was determined from 13 overlapping fragments. Excluding the poly (A) tail, ZYMV-Kurdistan genome consisted of 9593 nucleotides (nt), with 138 and 211 nt at the 5' and 3' non-translated regions, respectively. It contained two open-reading frames (ORFs), the large ORF encoding a polyprotein of 3080 amino acids (aa) and the small overlapping ORF encoding a P3N-PIPO protein of 74 aa. This isolate had six unique aa differences compared to other ZYMV isolates and shared 79.6-98.8% identities with other ZYMV genome sequences at the nt level and 90.1-99% identities at the aa level. A phylogenetic tree of ZYMV complete genomic sequences showed that Iranian and Central European isolates are closely related and form a phylogenetically homogenous group. All values in the ratio of substitution rates at non-synonymous and synonymous sites (dN/dS) were below 1, suggestive of strong negative selection forces during ZYMV protein history. This is the first report of complete genome sequence information of the most prevalent virus in the west of Iran. This study helps our understanding of the genetic diversity of ZYMV isolates infecting cucurbit plants in Iran, virus evolution and epidemiology and can assist in designing better diagnostic tools.

Corrigendum: Zucchini Yellow Mosaic Virus Infection Limits Establishment and Severity of Powdery Mildew in Wild Populations of Cucurbita pepo.

[This corrects the article DOI: 10.3389/fpls.2018.00792.].

Zucchini Yellow Mosaic Virus Infection Limits Establishment and Severity of Powdery Mildew in Wild Populations of Cucurbita pepo.

Few studies have examined the combined effect of multiple parasites on host fitness. Previous work in the Cucurbita pepo pathosystem indicates that infection with Zucchini yellow mosaic virus (ZYMV) reduces exposure to a second insect-vectored parasite (Erwinia tracheiphila). In this study, we performed two large-scale field experiments employing wild gourds (Cucurbita pepo ssp. texana), including plants with a highly introgressed transgene conferring resistance to ZYMV, to examine the interaction of ZYMV and powdery mildew, a common fungal disease. We found that ZYMV-infected plants are more resistant to powdery mildew (i.e., less likely to experience powdery mildew infection and when infected with powdery mildew, have reduced severity of powdery mildew symptoms). As a consequence, during widespread viral epidemics, proportionally more transgenic plants get powdery mildew than non-transgenic plants, potentially mitigating the benefits of the transgene. A greenhouse study using ZYMV-inoculated and non-inoculated controls (non-transgenic plants) revealed that ZYMV-infected plants were more resistant to powdery mildew than controls, suggesting that the transgene itself had no direct effect on the powdery mildew resistance in our field study. Additionally, we found evidence of elevated levels of salicylic acid, a phytohormone that mediates anti-pathogen defenses, in ZYMV-infected plants, suggesting that viral infection induces a plant immune response (systemic acquired resistance), thereby reducing plant susceptibility to powdery mildew infection.

Establishment of a Simple and Rapid Gene Delivery System for Cucurbits by Using Engineered of Zucchini yellow mosaic virus.

The infectious full-length cDNA clone of zucchini yellow mosaic virus (ZYMV) isolate PA (pZYMV-PA), which was isolated from pumpkin, was constructed by utilizing viral transcription and processing signals to produce infectious in vivo transcripts. Simple rub-inoculation of plasmid DNAs of pZYMV-PA was successful to cause infection of zucchini plants (Cucurbita pepo L.). We further engineered this infectious cDNA clone of ZYMV as a viral vector for systemic expression of heterologous proteins in cucurbits. We successfully expressed two reporter genes including gfp and bar in zucchini plants by simple rub-inoculation of plasmid DNAs of the ZYMV-based expression constructs. Our method of the ZYMV-based viral vector in association with the simple rub-inoculation provides an easy and rapid approach for introduction and evaluation of heterologous genes in cucurbits.

Antiviral activity of Thuja orientalis extracts against watermelon mosaic virus (WMV) on Citrullus lanatus.

Watermelon mosaic potyvirus (WMV) is considered as an important virus infecting watermelon and causing adverse effects on crop productivity. To overcome this problem one of the main objectives of plant breeders is to make these strains less effective in the ability to infect plants by treatment with plant extracts. Due to the advantages of plant tissue culture, in vitro, in the process of the selection of different cultivars under biotic stress, this study was conducted to achieve this aim by evaluating the effect of three concentrations of Thuja extract on the multiplication of WMV in watermelon by measuring callus fresh weight and soluble proteins (mg g(-1) fresh weight) of healthy and infected hypocotyl explants. Also, WMV was isolated from naturally infected watermelon and characterized as potyvirus by serological and molecular analyses. The isolated virus gave a positive reaction with WMV antiserum compared with other antibodies of CMV, ZYMV and SqMV using DAS-ELISA. RT-PCR, with the specific primer for WMV-cp. gene, yielded 825 base pair DNA fragments. The results that belong to soluble protein analysis indicated that infected hypocotyl explants treated with 6 g L(-1) recorded the highest rate in the number of soluble protein bands compared with the rest of treatments. As a conclusion of these results, we can recommend to apply the Thuja extract at 6 g L(-1) as a optimum dosage to decrease the infection caused by watermelon mosaic potyvirus.

Comparative molecular epidemiology provides new insights into Zucchini yellow mosaic virus occurrence in France.

Zucchini yellow mosaic virus (ZYMV, genus Potyvirus) causes important crop losses in cucurbits worldwide. In France, ZYMV epidemics are sporadic but occasionally very severe. This contrasts with Watermelon mosaic virus (WMV, genus Potyvirus) which causes regular and early epidemics. Factors influencing ZYMV epidemiology are still poorly understood. In order to gain new insights on the ecology and epidemiology of this virus, a 5-year multilocation trial was conducted in which ZYMV spread and populations were studied in each of the 20 plot/year combinations and compared with WMV. Search for ZYMV alternative hosts was conducted by testing weeds growing naturally around one plot and also by checking ZYMV natural infections in selected ornamental species. Although similar ZYMV populations were observed occasionally in the same plot in two successive years suggesting the occurrence of overwintering hosts nearby, only two Lamium amplexicaule plants were found to be infected by ZYMV of 3459 weed samples that were tested. The scarcity of ZYMV reservoirs contrasts with the frequent detection of WMV in the same samples. Since ZYMV and WMV have many aphid vectors in common and are transmitted with similar efficiencies, the differences observed in ZYMV and WMV reservoir abundances could be a major explanatory factor for the differences observed in the typology of ZYMV and WMV epidemics in France. Other potential ZYMV alternative hosts have been identified in ornamental species including begonia. Although possible in a few cases, exchanges of populations between different plots located from 500 m to 4 km apart seem uncommon. Therefore, the potential dissemination range of ZYMV by its aphid vectors seems to be rather limited in a fragmented landscape.

Preventive and curative effects of Apple latent spherical virus vectors harboring part of the target virus genome against potyvirus and cucumovirus infections.

Apple latent spherical virus (ALSV)-based vectors experimentally infect a broad range of plant species without causing symptoms and can effectively induce stable virus-induced gene silencing in plants. Here, we show that pre-infection of ALSV vectors harboring part of a target viral genome (we called ALSV vector vaccines here) inhibits the multiplication and spread of the corresponding challenge viruses [Bean yellow mosaic virus, Zucchini yellow mosaic virus (ZYMV), and Cucumber mosaic virus (CMV)] by a homology-dependent resistance. Further, the plants pre-infected with an ALSV vector having genome sequences of both ZYMV and CMV were protected against double inoculation of ZYMV and CMV. More interestingly, a curative effect of an ALSV vector vaccine could also be expected in ZYMV-infected cucumber plants, because the symptoms subsided on subsequent inoculation with an ALSV vector vaccine. This may be due to the invasion of ALSV, but not ZYMV, in the shoot apical meristem of cucumber.