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Plant viruses are ubiquitous throughout plant communities, but research on viral impacts largely focuses on crops. Little is known about how viruses influence wild plants in their native habitats. To address this gap, we examined virus interactions with wild drought-tolerant perennials in California desert natural areas encroached upon by agriculture. We used metagenomics, targeted diagnostics, and phylogenetics to assess virus diversity and clade relationships, and experiments to investigate viral influence on hosts. We focused on three herbaceous perennials ( Cucurbita foetidissima, C. palmata, and Datura wrightii) and tested the hypothesis that these wild species accumulate virus infections typically found in crops and transmitted by polyphagous insects. We predicted that such infections might be retained across seasons and potentially impair plant performance. Virome profiling revealed a rich community of previously characterized virus species (12 total), with virus community structure varying by site and host species. The dominant viruses in the wild hosts were non-native crop pathogens, including cucurbit aphid-borne yellows virus (CABYV) and cucurbit yellow stunting disorder virus (CYSDV). Targeted testing revealed that CABYV infected as many as 88% of sampled wild Cucurbita individuals, with dual CABYV–CYSDV infections common in natural areas adjacent to desert agriculture. CABYV infections reduced shoot and root production in greenhouse experiments with the two wild Cucurbita species. Phylogenetic analyses suggest that CABYV was introduced to California multiple times from other continents. Our findings provide concerning evidence of ways in which human activities can alter virus pressure on wild plants and potentially contribute to plant decline.

Plant viruses are ubiquitous throughout plant communities, but research on viral impacts largely focuses on crops. Little is known about how viruses influence wild plants in their native habitats. To address this gap, we examined virus interactions with wild drought-tolerant perennials in California desert natural areas encroached upon by agriculture. We used metagenomics, targeted diagnostics, and phylogenetics to assess virus diversity and clade relationships, and experiments to investigate viral influence on hosts. We focused on three herbaceous perennials (Cucurbita foetidissima, C. palmata, and Datura wrightii) and tested the hypothesis that these wild species accumulate virus infections typically found in crops and transmitted by polyphagous insects. We predicted that such infections might be retained across seasons and potentially impair plant performance. Virome profiling revealed a rich community of previously characterized virus species (12 total), with virus community structure varying by site and host species. The dominant viruses in the wild hosts were non-native crop pathogens, including cucurbit aphid-borne yellows virus (CABYV) and cucurbit yellow stunting disorder virus (CYSDV). Targeted testing revealed that CABYV infected as many as 88% of sampled wild Cucurbita individuals, with dual CABYV–CYSDV infections common in natural areas adjacent to desert agriculture. CABYV infections reduced shoot and root production in greenhouse experiments with the two wild Cucurbita species. Phylogenetic analyses suggest that CABYV was introduced to California multiple times from other continents. Our findings provide concerning evidence of ways in which human activities can alter virus pressure on wild plants and potentially contribute to plant decline.

Plant virus management is mostly achieved through control of insect vectors using insecticides. However, insecticides are only marginally effective for preventing virus transmission. Furthermore, it is well established that symptoms of virus infections often encourage vector visitation to infected hosts, which exacerbates secondary spread. Plant defense elicitors, phytohormone analogs that prime the plant immune system against attack, may be a viable approach for virus control that complements insecticide use by disrupting pathologies that attract vectors. To explore this, we tested the effect of a commercial plant elicitor, acibenzolar-S-methyl (ASM), on infection rates, virus titers, and symptom development in melon plants inoculated with one of two virus species, Cucumber mosaic virus (CMV) and Cucurbit yellow stunting disorder virus (CYSDV). We also conducted behavioral assays to assess the effect of ASM treatment and virus inoculation on vector behavior. For both pathogens, ASM treatment reduced symptom severity and delayed disease progression. For CYSDV, this resulted in the attenuation of symptoms that encourage vector visitation and virion uptake. We did observe slight trade-offs in growth vs. defense following ASM treatment, but these effects did not translate into reduced yields or plant performance in the field. Our results suggest that immunity priming may be a valuable tool for improving management of insect-transmitted plant viruses.

RNA silencing is a crucial mechanism of the antiviral immunity system in plants. Small RNAs guide Argonaut proteins to target viral RNA or DNA, preventing virus accumulation. Small RNA profiles in Cucurbita pepo line PI 420328 with tolerance to cucurbit yellow stunting disorder virus (CYSDV) were compared with those in Gold Star, a susceptible cultivar. The lower CYSDV symptom severity in PI 420328 correlated with lower virus titers and fewer sRNAs derived from CYSDV (vsRNA) compared to Gold Star. Elevated levels of 21- and 22-nucleotide (nt) size class vsRNAs were observed in PI 420328, indicating more robust and efficient RNA silencing in PI 420328. The distribution of vsRNA hotspots along the CYSDV genome was similar in both PI 420328 and Gold Star. However, the 3' UTRs, CPm, and p26 were targeted at a higher frequency in PI 420328. [ABSTRACT FROM AUTHOR]

Summer squash (Cucurbita pepo L.) is a major vegetable crop produced in Georgia and Florida during the fall season. This production is vulnerable to whitefly (Bemisisia tabaci Genn.)-transmitted viruses that lead to severe yield losses. Over the past several years, whitefly populations have increased during the fall, thus leading to an increase in whitefly-transmitted viruses such as Cucurbit leaf crumple virus (CuLCrV) and Cucurbit yellow stunting disorder virus (CYSDV). Whitefly management for summer squash relies on the use of insecticides and can be costly without providing adequate management of the viruses. Deployment of host resistance to whiteflies and their transmitted viruses (CuLCrV and CYSDV) is the best strategy for mitigating yield loss of summer squash; however, no resistant cultivars are commercially available. In the current study, resistance or tolerance to whiteflies, CuLCrV, and CYSDV was determined for squash germplasm from the U.S. Department of Agriculture (USDA) Germplasm Resources Information Network (GRIN), university breeding programs, and commercial companies in Georgia and Florida across 2 years. In both locations and years, visual virus symptom severity scores were collected and a quantitative polymerase chain reaction (qPCR) was used to determine the CuLCrV viral load and CYSDV presence in Georgia. Whitefly-induced feeding damage was evaluated by directly assessing the intensity of silverleaf symptoms and visual counts of whitefly adults on the foliage in the field or in photographs. Virus symptom severity was lower in C. moschata Duchesne ex Poir. genotypes, namely, PI 550689, PI 550692, PI 550694, PI 653064, and Squash Betternut 900, than in other evaluated genotypes. Two C. pepo accessions were common between both locations for viral severity (PI 442294) or viral severity and viral load (PI 171625). Lower CuLCrV loads were identified in C. ecuadorensis Cutler & Whitaker (PI 540895), and C. okeechobeensis (Small) L.H.Bailey (PI 540900) than other evaluated genotypes. Four genotypes tested negative for CYSDV during both years: C. pepo (PI 507882), C. moschata (PI 483345), C. ecuadorensis (PI 390455), and C. okeechobeensis (PI 540900); they are potential sources of resistance. Six C. moschata accessions (PI 211999, PI 550690, PI 550692, PI 550694, PI 634982, and PI 653064) showed high tolerance to silverleaf disorder and had the lowest adult whitefly counts. Collectively, the accessions identified in the current study are potential sources of resistance or tolerance to whitefly and whitefly-transmitted viruses (CuLCrV and CYSDV).

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing-mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA-dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus-derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non-transgenic N. benthamiana plants as well as transgene-derived siRNAs (t-siRNAs) derived from the immune-transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t-siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t-siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t-siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24-nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24-nucleotide t-siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24-nucleotide t-siRNAs is associated with crinivirus immunity in transgenic plants.

RNA silencing is a crucial mechanism of the antiviral immunity system in plants. Small RNAs guide Argonaut proteins to target viral RNA or DNA, preventing virus accumulation. Small RNA profiles in Cucurbita pepo line PI 420328 with tolerance to cucurbit yellow stunting disorder virus (CYSDV) were compared with those in Gold Star, a susceptible cultivar. The lower CYSDV symptom severity in PI 420328 correlated with lower virus titers and fewer sRNAs derived from CYSDV (vsRNA) compared to Gold Star. Elevated levels of 21- and 22-nucleotide (nt) size class vsRNAs were observed in PI 420328, indicating more robust and efficient RNA silencing in PI 420328. The distribution of vsRNA hotspots along the CYSDV genome was similar in both PI 420328 and Gold Star. However, the 3’ UTRs, CPm, and p26 were targeted at a higher frequency in PI 420328.

The sweetpotato whitefly, Bemisia tabaci MEAM1 Gennadius (Hemiptera: Aleyrodidae), and the complex of viruses it transmits are major limiting factors to squash production in the southeastern United States. At this time, insecticides are extensively relied upon for the management of whiteflies and, indirectly, whitefly-transmitted viruses. The development of a multi-faceted, integrated pest management (IPM) program is needed to increase the sustainability and profitability of squash production. Experiments in 2018 and 2019 evaluated the effects of insect exclusion netting (IEN) in combination with selected pesticides on whitefly population dynamics and virus incidence in greenhouse-grown squash seedlings. Field experiments from 2018 to 2021 evaluated the effects of mulch type (UV-reflective mulch, live mulch, and white plastic mulch), row covers, and insecticides on whitefly population dynamics, silver leaf disorder (SSL) intensity, virus symptom severity, and marketable yield. IEN significantly reduced whiteflies and virus incidence on squash seedlings in the greenhouse study. In the field mulch study, lower whitefly abundance and SSL intensity, as well as reduced virus symptom severity, were observed in plots with reflective mulch compared with white plastic or live mulch. In the insecticide/row cover study, whitefly abundance, SSL intensity, and virus symptom severity were lowest in the row cover and cyantraniliprole- and flupyradifurone-treated plots. Field plots with row covers and those with UV-reflective mulch consistently produced the greatest marketable yields. These findings demonstrate that growers can reduce whitefly and virus pressure and preserve yields in squash production in the southeastern United States by combining cultural and chemical tactics, including row covers, UV-reflective mulch, and select insecticides. [ABSTRACT FROM AUTHOR]

Abstract Background Cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus, Closteroviridae) is transmitted in a semipersistent manner by the whitefly, Bemisia tabaci, and is efficiently transmitted by the widely prevalent B. tabaci cryptic species, MEAM1. In this study, we compared transcriptome profiles of B. tabaci MEAM1, after 24 h, 72 h and 7 days of acquisition feeding on melon plants infected with CYSDV (CYSDV-whiteflies) with those fed on virus-free melon, using RNA-Seq technology. We also compared transcriptome profiles with whiteflies fed on tomato plants separately infected with Tomato chlorosis virus (ToCV), a crinivirus closely related to CYSDV, and Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, which has a distinctly different mode of transmission and their respective virus-free controls, to find common gene expression changes among viruliferous whiteflies feeding on different host plants infected with distinct (TYLCV) and related (CYSDV and ToCV) viruses. Results A total of 275 differentially expressed genes (DEGs) were identified in CYSDV-whiteflies, with 3 DEGs at 24 h, 221 DEGs at 72 h, and 51 DEGs at 7 days of virus acquisition. Changes in genes encoding orphan genes (54 genes), phosphatidylethanolamine-binding proteins (PEBP) (20 genes), and AAA-ATPase domain containing proteins (10 genes) were associated with the 72 h time point. Several more orphan genes (20 genes) were differentially expressed at 7 days. A total of 59 common DEGs were found between CYSDV-whiteflies and ToCV-whiteflies, which included 20 orphan genes and 6 lysosomal genes. A comparison of DEGs across the three different virus-host systems revealed 14 common DEGs, among which, eight showed similar and significant up-regulation in CYSDV-whiteflies at 72 h and TYLCV-whiteflies at 24 h, while down-regulation of the same genes was observed in ToCV-whiteflies at 72 h. Conclusions Dynamic gene expression changes occurred in CYSDV-whiteflies after 72 h feeding, with decreased gene expression changes associated with 7 days of CYSDV acquisition. Similarities in gene expression changes among CYSDV-whiteflies, ToCV-whiteflies and TYLCV-whiteflies suggest the possible involvement of common genes or pathways for virus acquisition and transmission by whiteflies, even for viruses with distinctly different modes of transmission.

The sweetpotato whitefly, Bemisia tabaci MEAM1 Gennadius (Hemiptera: Aleyrodidae), and the complex of viruses it transmits are major limiting factors to squash production in the southeastern United States. At this time, insecticides are extensively relied upon for the management of whiteflies and, indirectly, whitefly-transmitted viruses. The development of a multi-faceted, integrated pest management (IPM) program is needed to increase the sustainability and profitability of squash production. Experiments in 2018 and 2019 evaluated the effects of insect exclusion netting (IEN) in combination with selected pesticides on whitefly population dynamics and virus incidence in greenhouse-grown squash seedlings. Field experiments from 2018 to 2021 evaluated the effects of mulch type (UV-reflective mulch, live mulch, and white plastic mulch), row covers, and insecticides on whitefly population dynamics, silver leaf disorder (SSL) intensity, virus symptom severity, and marketable yield. IEN significantly reduced whiteflies and virus incidence on squash seedlings in the greenhouse study. In the field mulch study, lower whitefly abundance and SSL intensity, as well as reduced virus symptom severity, were observed in plots with reflective mulch compared with white plastic or live mulch. In the insecticide/row cover study, whitefly abundance, SSL intensity, and virus symptom severity were lowest in the row cover and cyantraniliprole- and flupyradifurone-treated plots. Field plots with row covers and those with UV-reflective mulch consistently produced the greatest marketable yields. These findings demonstrate that growers can reduce whitefly and virus pressure and preserve yields in squash production in the southeastern United States by combining cultural and chemical tactics, including row covers, UV-reflective mulch, and select insecticides.

During the spring season of 2014, watermelon leaves were exhibiting yellowing, mottling and stunting symptoms in Western and Southwestern regions (Jeddah, Al-Lith, Jezan, Asfan, Ghonfada, Tofeel, Wadi Baish, Abu Arish), Saudi Arabia. One hundred thirty-nine samples were collected from symptomatic and asymptomatic watermelon plants. DASELISA was performed to detect Cucurbit yellow stunting disorder virus (CYSDV) and other suspected cucurbit viruses. The obtained results revealed that out of 139 watermelon samples, nine and 98 samples were found to be positive with CYSDV and WmCSV, respectively and 22 samples were found to have mixed infection for both viruses. While no reaction was detected with, any of the other antisera tested against. Approximately 465 bp product were amplified by RT-PCR when CYSDV-Partial heat shock protein (HSP70) specific primer were used with ELISA positive symptomatic watermelon samples. Dot blot hybridization was also performed to detect CYSDV in the samples using CYSDV specific cDNA probe. Asymptomatic plant did not react when RT-PCR and dot blot hybridization assay were used. Partial HSP70 sequences of four CYSDV isolates were determined. When these HSP70 sequences were compared with other CYSDV isolates reported worldwide, the highest identity was between CYSDV KSA isolates (33-CYSDVKSA, 36- CYSDVKSA) and isolate from Portugal. Lowest identity was found between (34-CYSDVKSA, 35-CYSDVKSA), four Spanish isolates and one isolate from each USA and Lebanon. This is the first comprehensive study involving this virus in different regions in Saudi Arabia. [ABSTRACT FROM AUTHOR]

Viruses transmitted by the sweet potato whitefly (Bemisia tabaci) have been detrimental to the sustainable production of cucurbits in the southeastern USA. Surveys were conducted in the fall of 2019 and 2020 in Georgia, a major cucurbit-producing state of the USA, to identify the viruses infecting cucurbits and their distribution. Symptomatic samples were collected and small RNA libraries were prepared and sequenced from three cantaloupes, four cucumbers, and two yellow squash samples. An analysis of the sequences revealed the presence of the criniviruses cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and the begomovirus cucurbit leaf crumple virus (CuLCrV). CuLCrV was detected in 76%, CCYV in 60%, and CYSDV in 43% of the total samples (n = 820) tested. The level of mixed infections was high in all the cucurbits, with most plants tested being infected with at least two of these viruses. Near-complete genome sequences of two criniviruses, CCYV and CYSDV, were assembled from the small RNA sequences. An analysis of the coding regions showed low genetic variability among isolates from different hosts. In phylogenetic analysis, the CCYV isolates from Georgia clustered with Asian isolates, while CYSDV isolates clustered with European and USA isolates. This work enhances our understanding of the distribution of viruses on cucurbits in South Georgia and will be useful to develop strategies for managing the complex of whitefly-transmitted viruses in the region.

Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5'- and 3'-UTRs plus the 3'-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8? will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants. [ABSTRACT FROM AUTHOR]

In the past two decades, viruses in the genera Crinivirus and Begomovirus, transmitted by whiteflies, have emerged as threatening diseases to cucurbit cultivation. The criniviruses Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) occur in mixed infection at high rates in cucumber greenhouses in Lebanon. The begomovirus Squash leaf curl virus (SLCV) is also present in the country infecting cucumber but at a lower incidence. The effect of single, dual or triple virus infections on cucumber was studied. Single infection by SLCV did not lead to any symptoms or yield reduction. CYSDV or CCYV infections led to development of characteristic yellowing symptoms. In single infections, CYSDV caused the greatest reductions in height and yield that ranged from 25 to 62 % and 52 to 63 %, respectively, depending on season and variety. CCYV induced less severe reduction in height, 10 to 33 %, and in fruit number, 10 to 12 %. Yellowing symptoms induced by dual infections in any combination were similar to those of singly-infected plants. Triple infections resulted in substantial reductions in plant height and yield as compared to single or dual infections. This is the first report that characterizes a significant increase in disease severity upon co-infection between cucurbit-infecting criniviruses and begomoviruses. The implications of these observations on integrated disease management strategies are discussed. [ABSTRACT FROM AUTHOR]

Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.

Intensive vegetable production is constantly facing the emergence of new viral diseases. Apart from the intrinsic features of viruses as plant pathogens, the highly dynamic turnover of cultivars and cultural practices, and the global trade of seeds and products characteristic of intensive vegetable production may favour the emergence of new viruses, as well as the expansion of the geographical range of vectors responsible for their dissemination. Indeed, the efficient transmission of viruses plays a major role in the impact and outcome of viral epidemics. Whiteflies ( Hemiptera: Aleyrodidae) that belong to the genera Bemisia and Trialeurodes are efficient virus vectors. Whiteflies transmit viruses of at least four genera, one of DNA viruses, the genus Begomovirus, and three of RNA viruses, Crinivirus, Ipomovirus and Torradovirus. Begomoviruses have been the subject of recent reviews. In this article we review the genome structure, epidemiology and control of whitefly-transmitted RNA viruses that belong to the genera Crinivirus, Ipomovirus and Torradovirus, with an extended discussion on the particular viruses within these genera that are currently causing important outbreaks, such are Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Tomato chlorosis virus (ToCV) and Tomato torrado virus (ToTV). [ABSTRACT FROM AUTHOR]

Theory indicates that landscape composition affects transmission of vector-borne crop diseases, but few empirical studies have investigated how landscape composition affects plant disease epidemiology. Since 2006, Bemisia tabad (Gennadius) has vectored the cucurbit yellow stunting disorder virus (CYSDV) to cantaloupe and honeydew melons (Cucumis me/o L.) in the southwestern United States and northern Mexico, causing significant reductions in yield of fall melons and increased use of insecticides. Here, we show that a landscape-based approach allowing simultaneous assessment of impacts of local (i.e., planting date) and regional (i.e., landscape composition) factors provides valuable insights on how to reduce crop disease risks. Specifically, we found that planting fall melon fields early in the growing season, eliminating plants germinating from seeds produced by spring melons after harvest, and planting fall melon fields away from cotton and spring melon fields may significantly reduce the incidence of CYSDV infection in fall melons. Because the largest scale of significance of the positive association between abundance of cotton and spring melon fields and CYSDV incidence was 1,750 and 3,000 m, respectively, reducing areas of cotton and spring melon fields within these distances from fall melon fields may decrease CYSDV incidence. Our results indicate that landscape-based studies will be fruitful to alleviate limitations imposed on crop production by vector-borne diseases. [ABSTRACT FROM AUTHOR]

Highlights: [•] Multiplex RT-PCR was developed to differentiate between CCYV and CYSDV in mixed infection. [•] RT-qPCR assay was developed for specific detection of CCYV and CYSDV. [•] RT-qPCR was 100–1000 times more sensitive than RT-PCR. [•] An internal plant gene was used for normalization in quantitation of both viruses in mixed infection. [ABSTRACT FROM AUTHOR]

Cucurbit yellow stunting disorder virus (CYSDV) (genus, Crinivirus: family, Closteroviridae) is an emerging plant pathogen, transmitted by the sweet potato whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), which infects cucurbit crops causing significant economic losses. A TaqMan® real-time fluorescent, one-step reverse transcription (RT), polymerase chain reaction (PCR) assay for the detection of the virus has been developed and optimized. The assay is over 100-fold more sensitive than conventional RT-PCR and involves template preparation that does not require RNA purification. The assay can be accomplished either by first spotting the sap extract on a positively charged nylon membrane and elution, or by the direct addition of crude plant extract into the real-time reaction cocktail. Several factors affecting the efficiency of the tests were studied, such as the type and amount of reverse transcription (RT) enzymes and the use of different additives on the elution extract. The addition of 5 units of RT enzymes in the real-time PCR cocktail and the use of Tween 20, Triton X and Betaine in the virus release buffer resulted in improved detection efficiency. The applicability of the real-time RT-PCR assay was validated with CYSDV isolates from the USA, Mexico, the Mediterranean Basin, Jordan, and the United Arab Emirates and provides a simple, efficient and accurate detection technique, whereas the membrane preparation techniques can be used for long-term storage of samples allowing the shipment of samples from the field to remote laboratories for testing without compromising the reliability of the test. [ABSTRACT FROM AUTHOR]

Abstract: Post-transcriptional gene silencing (PTGS) degrades RNA in a sequence-specific manner and is utilised by plants as a natural defence mechanism against virus invaders. Two members of the genus Crinivirus have been reported to encode suppressors and counter PTGS: Sweet potato chlorotic stunt virus p22 and Tomato chlorosis virus (ToCV) p22, coat protein and coat protein minor. Using an Agrobacterium-mediated transient assay on Nicotiana benthamiana wildtype and 16c plants, we screened four Cucurbit yellow stunting disorder virus (CYSDV) RNA 1-encoded proteins (papain-like protease, p25, p5.2 and p22) to determine which one possess PTGS suppressor activity. Amongst these proteins, only CYSDV p25 was able to suppress (double- and single-stranded) RNA-induced silencing of the green fluorescent protein (GFP) mRNA. Restoration of GFP expression by CYSDV p25 in both of these experiments had no apparent effect on the accumulation of the small interfering RNAs. The identification of CYSDV p25 adds to the list of suppressors encoded by crinivirus RNA 1 molecules, which are unrelated in terms of amino acid sequence homology suggesting distinct PTGS suppression mechanisms and possible roles in viral replication. [Copyright &y& Elsevier]

Abstract: Reverse transcription followed by real-time PCR assays based on TaqMan® chemistry have been developed for the detection and quantification of Cucumber vein yellowing virus (CVYV) and Cucurbit yellow stunting disorder virus (CYSDV) in individual adults of the whitefly vector Bemisia tabaci. The method includes an internal control for the detection of a gene from B. tabaci to compensate for variations in extraction efficiency. The assays designed were used to estimate proportions of viruliferous whiteflies collected from commercial greenhouse-grown crops in Spain. In a significant number of whiteflies, both viruses were detected and their amounts were estimated. The assays could be used to assist risk assessment of CVYV and CYSDV which constitute limiting factors in cucurbit crops. They are also suited to investigating the epidemiology and plant–virus–vector relationships in these diseases. [Copyright &y& Elsevier]

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA 2, a whitefly ( Bemisia tabaci)-transmitted closterovirus with a bi-partite genome, is reported. CYSDV RNA 2 is 7,281 nucleotides long and contains the closterovirus hallmark gene array with a similar arrangement to the prototype member of the genus Crinivirus, Lettuce infectious yellows virus (LIYV). CYSDV RNA 2 contains open reading frames (ORFs) potentially encoding in a 5′ to 3′ direction for proteins of 5 kDa (ORF 1; hydrophobic protein), 62 kDa (ORF 2; heat shock protein 70 homolog, HSP70h), 59 kDa (ORF 3; protein of unknown function), 9 kDa (ORF 4; protein of unknown function), 28.5 kDa (ORF 5; coat protein, CP), 53 kDa (ORF 6; coat protein minor, CPm), and 26.5 kDa (ORF 7; protein of unknown function). Pairwise comparisons of CYSDV RNA 2-encoded proteins (HSP70h, p59 and CPm) among the closteroviruses showed that CYSDV is closely related to LIYV. Phylogenetic analysis based on the amino acid sequence of the HSP70h, indicated that CYSDV clusters with other members of the genus Crinivirus, and it is related to Little cherry virus-1 (LChV-1), but is distinct from the aphid- or mealybug-transmitted closteroviruses. [ABSTRACT FROM AUTHOR]

Viruses transmitted by the sweet potato whitefly (Bemisia tabaci) have been detrimental to the sustainable production of cucurbits in the southeastern USA. Surveys were conducted in the fall of 2019 and 2020 in Georgia, a major cucurbit-producing state of the USA, to identify the viruses infecting cucurbits and their distribution. Symptomatic samples were collected and small RNA libraries were prepared and sequenced from three cantaloupes, four cucumbers, and two yellow squash samples. An analysis of the sequences revealed the presence of the criniviruses cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and the begomovirus cucurbit leaf crumple virus (CuLCrV). CuLCrV was detected in 76%, CCYV in 60%, and CYSDV in 43% of the total samples (n = 820) tested. The level of mixed infections was high in all the cucurbits, with most plants tested being infected with at least two of these viruses. Near-complete genome sequences of two criniviruses, CCYV and CYSDV, were assembled from the small RNA sequences. An analysis of the coding regions showed low genetic variability among isolates from different hosts. In phylogenetic analysis, the CCYV isolates from Georgia clustered with Asian isolates, while CYSDV isolates clustered with European and USA isolates. This work enhances our understanding of the distribution of viruses on cucurbits in South Georgia and will be useful to develop strategies for managing the complex of whitefly-transmitted viruses in the region. [ABSTRACT FROM AUTHOR]

• This study examined mixed virus infection in a plant host and acquisition of multiple viruses by the vector. • Effects of CuLCrV and/or CYSDV infection in squash on virus load, and whitefly preference and fitness were examined. • Effects of whitefly acquisition of TYLCV and/or CuLCrV on virus load, and whitefly preference and fitness were examined. • Vector-virus-host interactions were sometimes different in the presence of two viruses than one virus. Mixed virus infection in host plants can differentially alter the plant phenotype, influence vector fitness, and affect virus acquisition and inoculation by vectors than single-virus infection. Vector acquisition of multiple viruses from multiple host plants could also differentially affect vector fitness and virus inoculation than acquisition of one virus. Whitefly-virus pathosystems in the southern United States include both the above-stated facets. For the first facet, this study examined the effects of single and mixed infection of cucurbit leaf crumple virus (CuLCrV, a begomovirus) and cucurbit yellow stunting disorder virus (CYSDV, a crinivirus) infecting squash on whitefly (Bemisia tabaci Gennadius MEAM1) host preference and fitness. Mixed infection of CuLCrV and CYSDV in squash plants severely altered their phenotype than single infection. The CYSDV load was reduced in mixed-infected squash plants than in singly-infected plants. Consequently, whiteflies acquired reduced amounts of CYSDV from mixed-infected plants than singly-infected plants. No differences in CuLCrV load were found between singly- and mixed-infected squash plants, and acquisition of CuLCrV by whiteflies did not vary between singly- and mixed-infected squash plants. Both singly- and mixed-infected plants similarly affected whitefly preference, wherein non-viruliferous and viruliferous (CuLCrV and/or CYSDV) whiteflies preferred non-infected plants over infected plants. The fitness study involving viruliferous and non-viruliferous whiteflies revealed no differences in developmental time and fecundity. For the second facet, this study evaluated the effects of individual or combined acquisition of tomato-infecting tomato yellow leaf curl virus (TYLCV, a begomovirus) and squash-infecting CuLCrV on whitefly host preference and fitness. Whiteflies that acquired both CuLCrV and TYLCV had significantly lower CuLCrV load than whiteflies that acquired CuLCrV alone, whereas TYLCV load remained unaltered when acquired individually or in conjunction with CuLCrV. Whitefly preference was not affected following individual or combined virus acquisition. Viruliferous (CuLCrV and/or TYLCV) whiteflies preferred to settle on non-infected tomato and squash plants. The mere presence of CuLCrV and/or TYLCV in whiteflies did not affect their fitness. Taken together, these results indicate that mixed infection of viruses in host plants and acquisition of multiple viruses by the vector could have implications for virus accumulation, virus acquisition, vector preference, and epidemics that sometimes are different from single-virus infection or acquisition. [ABSTRACT FROM AUTHOR]