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White yam (Dioscorea rotundata) plants collected from farmers' fields and planted at the Areka Agricultural Research Center, Southern Ethiopia, displayed mosaic, mottling, and chlorosis symptoms. To determine the presence of viral pathogens, an investigation for virome characterization was conducted by Illumina high-throughput sequencing. The bioinformatics analysis allowed the assembly of five viral genomes, which according to the ICTV criteria were assigned to a novel potyvirus (3 genome sequences) and a novel crinivirus (2 genome sequences). The potyvirus showed ~ 66% nucleotide (nt) identity in the polyprotein sequence to yam mosaic virus (NC004752), clearly below the demarcation criteria of 76% identity. For the crinivirus, the RNA 1 and RNA 2 shared the highest sequence identity to lettuce chlorosis virus, and alignment of the aa sequence of the RdRp, CP and HSP70h (~ 49%, 45% and 76% identity), considered for the demarcation criteria, revealed the finding of a novel virus species. The names Ethiopian yam virus (EYV) and Yam virus 1 (YV-1) are proposed for the two tentative new virus species.

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Tomato chlorosis virus (ToCV) is an emerging pathogen that cause severe yellow leaf disorder syndrome in tomato plants. In this study, we aimed to generate a recombinant ToCV tagged with green fluorescent protein (GFP) to enable real-time monitoring of viral infection in living plants. Transformation of the full-length cDNA construct of ToCV RNA1 into Escherichia coli resulted in instability issues, which were successfully overcome by inserting a plant intron into RNA1. Subsequently, a GFP tag was engineered into a cDNA construct of ToCV RNA2. The resulting recombinant ToCV-GFP could systemically infect Nicotiana benthamiana plants, and GFP expression was observed along the major veins. Utilizing ToCV-GFP, we also showed that ToCV engages in antagonistic relationships with two different tomato-infecting viruses in mixed infections in N. benthamiana. This study demonstrates the potential of ToCV-GFP as a valuable tool for the visual tracking of infection and movement of criniviruses in living plants.

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Plant viruses produce particular suppressors to antagonize the host defense response of RNA silencing to establish infection. Cucurbit chlorotic yellows virus (CCYV), a member of the genus Crinivirus of the family Closteroviridae, severely damages the production of economically essential cucurbits worldwide. Here, we used the attenuated zucchini yellow mosaic virus (ZYMV) vector ZAC to express individual coding sequences, including CP, CPm, P25, and P22, of a Taiwan CCYV isolate (CCYV-TW) to identify their possible roles as pathogenicity determinants. ZAC is an HC-Pro function mutant that lacks the ability of local lesion induction on Chenopodium quinoa leaves and induces mild mottling followed by recovery on its natural host zucchini squash plants. Only the recombinant expressing CCYV-TW P22 complemented the effect of ZAC HC-Pro dysfunction, causing more severe symptoms on zucchini squash plants and restoring lesion formation on C. quinoa leaves, with lesions forming faster than those generated by the wild-type ZYMV. This suggests that CCYV-TW P22 is a virulence enhancer. Sequence analysis of criniviral P22s revealed the presence of four conserved leucine residues (L10, L17, L84, and L127) and one conserved lysine residue (K185). The five P22 residues conserved among the CCYV isolates and the P22 orthologs of two other criniviruses were each substituted with alanine in CCYV-TW P22 to investigate its ability to suppress RNA silencing and pathogenicity. The results provide new insights into CCYV-P22, showing that the L127 residue of P22 is indispensable for maintaining its stability in RNA silencing suppression and essential for virulence enhancement.

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In the years 2021 and 2022, lettuce plants showing blistering, chlorosis, mosaic, rosetting/ excess proliferation, and stunting symptoms were subjected to leaf-dip transmission electron microscopy, RT-PCR followed by sequence analysis and bio-assay to unfold the identity of associated virus(es). The association of long filamentous virions (~ 850 nm in length) as seen through leaf-dip transmission electron microscopy suggested the possible infection by a potyvirus or crinivirus, either singly or in combination. RT-PCR assays using generic primers targeting the RdRp region of criniviruses and the NIb region of potyviruses revealed the association of both a crinivirus as well as a potyvirus. The gel-purified RT-PCR products derived from the RdRp region of criniviruses upon cloning, sequencing, and NCBI BLAST analysis indicated the associated crinivirus as cucurbit chlorotic yellows virus (CCYV). Further, RT-PCR assays using specific primers targeting CP and CP minor genes of CCYV followed by cloning and sequencing confirmed its association with the diseased lettuce plants. Besides, the bioassay based on whitefly-mediated virus transmission followed by RT-PCR confirmed the infectivity of CCYV from diseased to healthy lettuce plants. The results of this study confirmed the natural infection of CCYV in lettuce host for the first time in the world indicating its distribution across the crop families.

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Cucurbits are economically important crops that are widely cultivated in many parts of the world, including the southern US. In recent years, higher temperatures have favored the rapid build-up of whiteflies in the fall-grown cucurbits in this region. As a result, whitefly-transmitted viruses (WTVs) have severely impacted the marketable yield of cucurbits. In this review, we discuss three major groups of WTVs negatively impacting cucurbit cultivation in the southern US, including begomoviruses, criniviruses, and ipomoviruses. Here, we discuss the available information on the biology, epidemiology and advances made toward detecting and managing these viruses, including sources of resistance and cultural practices.

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Whitefly, Bemisia tabaci Gennadius (B cryptic species), transmits cucurbit leaf crumple virus (CuLCrV) in a persistent fashion. CuLCrV affects several crops such as squash and snap bean in the southeastern United States. CuLCrV is often found as a mixed infection with whitefly transmitted criniviruses, such as cucurbit yellow stunting disorder virus (CYSDV) in hosts such as squash, or as a single infection in hosts such as snap bean. The implications of different host plants (inoculum sources) with varying infection status on CuLCrV transmission/epidemics is not clear. This study conducted a series of whitefly mediated CuLCrV transmission experiments. In the first experiment, three plants species: squash, snap bean, and tobacco were inoculated by whiteflies feeding on field-collected mixed-infected squash plants. In the second experiment, three plant species, namely squash, snap bean, and tobacco with varying infection status (squash infected with CuLCrV and CYSDV and snap bean and tobacco infected with CuLCrV), were used as inoculum sources. In the third experiment, squash plants with differential CuLCrV accumulation levels and infection status (either singly infected with CuLCrV or mixed infected with CuLCrV and CYSDV) were used as inoculum sources. Irrespective of plant species and its infection status, CuLCrV accumulation in whiteflies was dependent upon the CuLCrV accumulation in the inoculum source plants. Furthermore, differential CuLCrV accumulation in whiteflies resulted in differential transmission, CuLCrV accumulation, and disease phenotype in the recipient squash plants. Overall, results demonstrate that whitefly mediated CuLCrV transmission between host plants follows a virus density dependent phenomenon with implications for epidemics.

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Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses.

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The cucurbit vegetable chieh-qua (Benincasa hispida var. chieh-qua How) is an important crop in South China and southeast Asian countries. Viral diseases cause substantial loss of chieh-qua yield. To identify the viruses that affect chieh-qua in China, ribosomal RNA-depleted total RNA sequencing was performed using chieh-qua leaf samples with typical viral symptoms. The virome of chieh-qua comprises four known viruses (melon yellow spot virus (MYSV), cucurbit chlorotic yellows virus (CCYV), papaya ringspot virus (PRSV) and watermelon silver mottle virus (WSMoV) and two novel viruses: cucurbit chlorotic virus (CuCV) in the genus Crinivirus and chieh-qua endornavirus (CqEV) in the genus Alphaendornavirus. The complete genomes of the two novel viruses in chieh-qua and three other isolates of CuCV in pumpkin, watermelon and cucumber were determined and the recombination signals of pumpkin and watermelon isolates of CuCV were detected. A reverse transcriptase PCR indicated that the dominant viruses of chieh-qua in Hainan are MYSV (66.67%) and CCYV (55.56%), followed by CuCV (27.41%), WSMoV (7.41%), cucumber mosaic virus (8.15%), zucchini yellow mosaic virus (6.67%), PRSV (6.67%) and CqEV (35.56%). Our findings support diagnostic and prevalence studies of viruses infecting chieh-qua in China, enabling sustainable control strategies for cucurbit viruses worldwide.

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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.

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Tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato crops in warm regions of the world, and is associated with infections of several whitefly (Bemisia tabaci)-transmitted single-stranded (ss)DNA begomoviruses (genus Begomovirus, family Geminiviridae). The most widespread begomovirus isolates associated with TYLCD are those of the type strain of the Tomato yellow leaf curl virus species, known as Israel (TYLCV-IL). The Ty-1 gene is widely used in commercial tomato cultivars to control TYLCV-IL damage, providing resistance to the virus by restricting viral accumulation and tolerance to TYLCD by inhibiting disease symptoms. However, several reports suggest that TYLCV-IL-like isolates are adapting to the Ty-1 gene and are causes of concern for possibly overcoming the provided control. This is the case with TYLCV-IL IS76-like recombinants that have a small genome fragment acquired by genetic exchange from an isolate of Tomato yellow leaf curl Sardinia virus, another begomovirus species associated with TYLCD. Here we show that TYLCV-IL IS76-like isolates partially break down the TYLCD-tolerance provided by the Ty-1 gene and that virulence differences might exist between isolates. Interestingly, we demonstrate that mixed infections with an isolate of the crinivirus (genus Crinivirus, family Closteroviridae) species Tomato chlorosis virus (ToCV), an ssRNA virus also transmitted by B. tabaci and emerging worldwide in tomato crops, boosts the breakdown of the TYLCD-tolerance provided by the Ty-1 gene either with TYLCV-IL IS76-like or canonical TYLCV-IL isolates. Moreover, we demonstrate the incorporation of the Ty-2 gene in Ty-1-commercial tomatoes to restrict (no virus or virus traces, no symptoms) systemic infections of recombinant TYLCV-IL IS76-like and canonical TYLCV-IL isolates, even in the presence of ToCV infections, which provides more robust and durable control of TYLCD.

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Specificity and efficiency of plant virus transmission depend largely on protein-protein interactions of vectors and viruses. Cucurbit chlorotic yellows virus (CCYV), transmitted specifically by tobacco whitefly, Bemisia tabaci, in a semi-persistent manner, has caused serious damage on cucurbit and vegetable crops around the world. However, the molecular mechanism of interaction during CCYV retention and transmission are still lacking. CCYV was proven to bind particularly to the whitefly foregut, and here, we confirmed that the minor coat protein (CPm) of CCYV is participated in the interaction with the vector. In order to identify proteins of B. tabaci that interact directly with CPm of CCYV, the immunoprecipitation (IP) assay and DUALmembrane cDNA library screening technology were applied. The cytochrome c oxidase subunit 5A (COX), tubulin beta chain (TUB) and keratin, type I cytoskeletal 9-like (KRT) of B. tabaci shown strong interactions with CPm and are closely associated with the retention within the vector and transmission of CCYV. These findings on whitefly protein-CCYV CPm interactions are crucial for a much better understanding the mechanism of semi-persistent plant virus transmission by insect vectors, as well as for implement new strategies for effective management of plant viruses and their vector insects.

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Yellowing symptoms caused by tomato chlorosis virus (ToCV) and tomato infectious chlorosis virus (TICV), both assigned to the genus Crinivirus, resemble nutrient deficiencies. Therefore, early diagnosis of infections will prevent crop damage and the spread of the viruses. In this study, we established a rapid detection method for ToCV and TICV by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). We first designed primer sets for RT-LAMP specific for ToCV and TICV. Next, by selecting the optimum primer set and determining the optimum conditions for the RT-LAMP reaction, each virus was detected within 50 min by piercing the diseased area of a tomato leaf with a toothpick, immersing the toothpick in the reaction solution, and conducting the RT-LAMP reaction. To verify the accuracy of the procedure, 61 tomato leaf samples showing disease symptoms were collected from five regions of Indonesia, and the RT-LAMP results for the samples were identical to those obtained with the commonly used reverse transcription-polymerase chain reaction.

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The viromic profile of Polyscias balfouriana cv. Marginata, a perennial woody and ornamental plant, was determined using ribosomal RNA-depleted total RNA (rRNA-depleted totRNA) sequencing. Five viruses (i.e., polyscias mosaic virus, PoMV; one potential novel rhabdovirus; and three novel viruses of Betaflexiviridae and Closteroviridae) were detected and prevalence-surveyed in Hainan province, China. The genomes of polyscias capillovirus 1 (PCaV-1) and polyscias citrivirus 1 (PCiV-1) of family Betaflexiviridae were completed, and the genomes of polyscias crinivirus 1 (PCrV-1) of Closteroviridae were nearly completed lacking the 5' and 3' termini. PCaV-1 shares 68% genome nucleotide (nt) identity and 66% replicase (Rep) amino acid (aa) identity with homologues in apple stem grooving virus (ASGV). PCiV-1 shares 65% genome nt identity and 64% Rep aa identity with homologs in citrus leaf blotch virus (CLBV). Meeting the species demarcation criteria, PCaV-1 and PCiV-1 were considered to be new species in genera Capillovirus and Citrivirus, respectively. PCrV-1 shares high genome nt identity (62%), heat shock protein 70-like protein (HSP70h) and RNA-dependent RNA polymerase (RdRp) aa identity (78−80%) with homologues in tomato chlorosis virus (ToCV). We tentatively consider PCrV-1 to be an unclassified member of the Crinivirus genus. PoMV, PCaV-1, PCiV-1, and PCrV-1 are the prevalent viruses with >73% occurrence in the Xinglong Tropical Botanical Garden, Hainan, China.

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Cucurbit chlorotic yellows virus (CCYV) belongs to the genus Crinivirus and is part of a complex of whitefly-transmitted viruses that cause yellowing disease in cucurbits. In the southeastern USA, heavy incidences of CCYV have been observed on all cucurbits grown in the fall. CCYV was detected from wild radish (Raphanus raphanistrum L.), a common weed that grows in the southeastern USA by high-throughput sequencing as well as RT-PCR. CCYV sequence from wild radish was 99.90% and 99.95%, identical to RNA 1 and RNA 2 of cucurbit isolates of CCYV from the region. Transmission assays using whiteflies demonstrated that wild radish is a good host for CCYV. Whiteflies were also able to acquire CCYV from wild radish and transmit the virus to cucurbit hosts, which developed typical symptoms associated with CCYV. Using quantitative PCR, the titer of CCYV in wild radish was also estimated to be on par with that of cucurbit hosts of the virus. Whitefly bioassays revealed that wild radish is an acceptable feeding and reproductive host plant. These results indicate that wild radish could serve as a reservoir host for CCYV in the USA and other parts of the world where similar conditions exist.

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Plant viruses can change the phenotypes and defense pathways of the host plants and the performance of their vectors to facilitate their transmission. Cucurbit chlorotic yellows virus (CCYV) (Crinivirus), a newly reported virus occurring on cucurbit plants and many other plant species, is transmitted specifically by Bemisia tabaci MEAM1 (B biotype) and MED (Q biotype) cryptic species in a semipersistent manner. This study evaluated the impacts of CCYV on B. tabaci to better understand the plant-virus-vector interactions. By using CCYV-B. tabaci MED-cucumber as the model, we investigated whether or how a semipersistent plant virus impacts the biology of its whitefly vector. CCYV mRNAs were detectable in nymphs from first to fourth instars and adults of B. tabaci with different titers. Nymph instar durations and adult longevity of female whiteflies greatly extended on CCYV-infected plants, but nymph instar durations and adult longevity of male whiteflies were not significantly influenced. In addition, the body length and oviposition increased in adults feeding on CCYV-infected plants, but the hatching rates of eggs and survival rates of different stages were not affected. Most interestingly, the sex ratio (male:female) significantly reduced to 0.5:1 in whitefly populations on CCYV-infected plants, while the ratio remained about 1:1 on healthy plants. These results indicated that CCYV can significantly impact the biological characteristics of its vector B. tabaci. It is speculated that CCYV and B. tabaci have established a typical mutualist relationship mediated by host plants.

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Transmission of the crinivirus, lettuce infectious yellows virus (LIYV), is determined by a minor coat protein (CPm)-mediated virion retention mechanism located in the foregut of its whitefly vector. To better understand the functions of LIYV CPm, chimeric CPm mutants engineered with different lengths of the LIYV CPm amino acid sequence and that of the crinivirus, lettuce chlorosis virus (LCV), were constructed based on bioinformatics and sequence alignment data. The 485 amino acid-long chimeric CPm of LIYV mutant, CPmP-1, contains 60 % (from position 3 to 294) of LCV CPm amino acids. The chimeric CPm of mutants CPmP-2, CPmP-3 and CPmP-4 contains 46 (position 3 to 208), 51 (position 3 to 238) and 41 % (position 261 to 442) of LCV CPm amino acids, respectively. All four mutants moved systemically, expressed the chimeric CPm and formed virus particles. However, following acquisition feeding of the virus preparations, only CPmP-1 was retained in the foreguts of a significant number of vectors and transmitted. In immuno-gold labelling transmission electron microscopy (IGL-TEM) analysis, CPmP-1 particles were distinctly labelled by antibodies directed against the LCV but not LIYV CPm. In contrast, CPmP-4 particles were not labelled by antibodies directed against the LCV or LIYV CPm, while CPmP-2 and -3 particles were weakly labelled by anti-LIYV CPm but not anti-LCV CPm antibodies. The unique antibody recognition and binding pattern of CPmP-1 was also displayed in the foreguts of whitefly vectors that fed on CPmP-1 virions. These results are consistent with the hypothesis that the chimeric CPm of CPmP-1 is incorporated into functional virions, with the LCV CPm region being potentially exposed on the surface and accessible to anti-LCV CPm antibodies.

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Watermelon (Citrullus lanatus) is a high nutrient crop, high in vitamins and very popular in the U.S and globally. The crop was harvested from 101,800 acres with a value of $560 million in the U.S (USDA-NASS, 2020). California, Florida, Georgia and Texas are the four-leading watermelon-producing states in the U.S. During the fall season of 2020, plants in two North Florida watermelon fields, one in Levy County (~20 acres) and one in Suwannee County (~80 acres) with varieties Talca and Troubadour, respectively, exhibited viral-like symptoms. The fields had 100% disease incidence that led to fruit quality issues and yield losses of 80% and above. Symptoms observed in the watermelon samples included leaf crumpling, yellowing and curling, and vein yellowing similar to that of single/and or mixed infection of cucurbit leaf crumple virus (CuLCrV; genus: Begomovirus, family: Geminiviridae), cucurbit yellow stunting disorder virus (CYSDV; genus: Crinivirus, family: Closteroviridae) and squash vein yellowing virus (SqVYV; genus: Ipomovirus, family: Potyviridae), although the vine decline symptoms often associated with SqVYV infection of watermelon were not observed. All three viruses are vectored by whiteflies and previously described in Florida (Akad et al., 2008; Polston et al., 2008; Adkins et al., 2009). To confirm the presence of these viruses, RNA was isolated from 20 symptomatic samples using the RNeasy Plant Mini Kit (Qiagen, USA) as per protocol. This was followed by RT-PCR (NEB, USA) using gene-specific primers described for CuLCrV, CYSDV and SqVYV (Adkins et al., 2009). Amplicons of expected sizes were obtained for all the viruses with the infection of CuLCrV in 17/20, CYSDV in 16/20, and SqVYV in 8/20 samples. In addition, the presence of cucurbit chlorotic yellows virus (CCYV; genus: Crinivirus, family: Closteroviridae) in mixed infection was confirmed in 4/20 samples (3 leaves and 1 fruit) by RT-PCR with primers specific to the CCYV coat protein (CP), heat shock protein 70 homolog (HSP70h) and RNA dependent RNA polymerase (RdRp) designed based on the available CCYV sequences (Sup Table. 1). The RT-PCR amplification was performed using a symptomatic watermelon sample and the amplicons of RdRp, HSP70h and CP were directly sequenced by Sanger method, and the sequences of the amplicons were deposited in GenBank under the accession number: MW527462 (RdRp, 952 bp), MW527461 (HSP70h, 583 bp) and MW527460 (CP, 852 bp). BLASTn analysis demonstrated that the sequences exhibited an identity of 99% to 100% (RdRp and HSP70h, 100%; and CP, 99%) with the corresponding regions of the CCYV isolate Shanghai from China (accession number: KY400636 and KY400633). The presence of CCYV was further confirmed in the watermelon samples by ELISA (Loewe, Germany) using crude sap extracted from the RT-PCR-positive, symptomatic watermelon samples. CCYV was first identified in Kumamoto, Japan in 2004 on melon plants (Gyoutoku et al. 2009). The CCYV was previously reported on melon from Imperial Valley, California (Wintermantel et al., 2019), and more recently on squash in Tifton, Georgia (Kavalappara et al., 2021) and cantaloupe in Cameron, Texas (Hernandez et al., 2021). To our knowledge, this is the first report of CCYV on field watermelon production in the U.S. Continued monitoring of the CCYV in spring and fall watermelon crop, and cucurbit volunteers and weeds will be critical toward understanding the spread of this virus and its potential risk to watermelon in Florida and other regions of the U.S.

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Lettuce infectious yellows virus is the first crinivirus for which the retention of purified virions ingested into the whitefly (Bemisia tabaci New World (NW)) vector's foregut, has been demonstrated to be a requisite for successful virus transmission. This key finding supports the hypothesis that the determinant of foregut retention and transmission is present on the virion itself. However, whether this is also true for other criniviruses has not been established. Here, we provide evidence that lettuce chlorosis virus (LCV) acquired from plants is retained in the foreguts of both the B. tabaci NW and Middle East-Asia Minor 1 (MEAM1) vector species and transmitted upon inoculation feeding. An association between foregut retention and transmission by NW vectors is also observed following the acquisition and inoculation feeding of LCV virions purified using a standard procedure involving 2% or 4% (v/v) Triton™ X-100 (TX-100). However, while virions purified with 2% or 4% TX-100 are also retained in the foreguts of MEAM1 vectors, transmission is observed with the 4% TX-100-purified virions or when more vectors are used for acquisition and inoculation feeding. These results suggest that an intrinsic difference exists between NW and MEAM1 vectors in their interactions with, and transmission of, LCV virions.

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