An all-out assault on a dominant resistance gene: Local emergence, establishment, and spread of strains of tomato spotted wilt orthotospovirus (TSWV) that overcome Sw-5b-mediated resistance in fresh market and processing tomatoes in California.

Tomato spotted wilt orthotospovirus (TSWV) causes substantial economic loss to tomato production, and the Sw-5b resistance gene is widely deployed for management. Here, we show (i) the emergence of resistance-breaking (RB) TSWV strains in processing and fresh market tomato production in California over the past ten years, and (ii) evolutionary relationships with RB strains from other areas. A specific RT-PCR test was used to show the C118Y RB strain that emerged in Fresno County in 2016 quickly became predominant in the central production area and remained so through this study. In 2021, the C118Y strain was detected in the Northern production area, and was predominant in 2022. However, in 2023, the C118Y strain was unexpectedly detected in fewer spotted wilt samples from resistant varieties. This was due to emergence of the T120N RB strain, previously known to occur in Spain. A specific RT-PCR test was developed and used to show that the T120N RB strain was predominant in Colusa and Sutter counties (detected in 75-80% of samples), and detected in ~50% of samples from Yolo County. Pathogenicity tests confirmed California isolates of the T120N strain infected Sw-5b tomato varieties and induced severe symptoms. Another RB strain, C118F, was associated with spotted wilt samples of Sw-5 varieties from fresh market tomato production in southern California. Phylogenetic analyses with complete NSm sequences revealed that the C118Y and T120N RB strains infecting resistant processing tomato in California emerged locally, whereas those from fresh market production were more closely related to isolates from Mexico. Thus, widespread deployment of this single dominant resistance gene in California has driven the local emergence of multiple RB strains in different tomato production areas and types. These results further emphasize the need for ongoing monitoring for RB strains, and identification of sources of resistance to these strains.

Unusual outbreaks of curly top disease in processing tomato fields in northern California in 2021 and 2022 were caused by a rare strain of beet curly top virus and facilitated by extreme weather events.

In the western United States, curly top disease (CTD) is caused by beet curly top virus (BCTV). In California, CTD causes economic loss to processing tomato production in central and southern areas but, historically, not in the north. Here, we document unusual CTD outbreaks in processing tomato fields in the northern production area in 2021 and 2022, and show that these were caused by the rare spinach curly top strain (BCTV-SpCT). These outbreaks were associated with proximity of fields to foothills and unusually hot, dry, and windy spring weather conditions, possibly by altering migrations of the beet leafhopper (BLH) vector from locations with BCTV-SpCT reservoirs. Support for this hypothesis came from the failure to observe CTD outbreaks and BLH migrations in 2023, when spring weather conditions were cool and wet. Our results show the climate-induced emergence of a rare plant virus strain to cause an economically important disease in a new crop and location.

First Report of Watermelon Mosaic Virus Naturally Infecting Coriander (Coriandrum sativum) and causing a leaf mottling disease in California.

Watermelon mosaic virus (WMV, genus Potyvirus, family Potyviridae) is a species of considerable economic importance to cucurbit crops worldwide (Keinath et al. 2017). This virus has a wide host range that includes more than 170 plant species from 27 families (Dong et al. 2017; Lecoq et al. 2011). In 2018, leaves of coriander (Coriandrum sativum) plants in a student garden (C-SG) at UC Davis, and in a home garden in Davis, CA (C-Pet) (~1.1 miles apart) showed symptoms of light green mottling and crumpling. Symptomatic leaves from each location were weakly positive with the general potyvirus immunostrip test (Agdia, Elkhart, IN). In RT-PCR tests with total RNA extracts (RNeasy Plant Mini Kit Qiagen, Germantown, MD) of these leaves and the potyvirus degenerate primer pair CIFor/CIRev (Ha et al. 2008), the expected-size ~0.7 kb fragment was amplified. These fragments were gel-purified and sequenced, and a BLASTn search revealed highest identities of 91.6% (C-SG) and 97.9% (C-Pet) with the sequence of an isolate of WMV from watermelon in the U.S. (TX29, KU246036). Thus, these isolates are designated WMV-C-SG-18 and WMV-C-Pet-18. Mechanical inoculation experiments were next performed with sap prepared with symptomatic coriander leaf tissue in ice-cold 0.01 M phosphate buffer (pH 7.0) in a 1:4 wt/vol ratio. First, to obtain pure isolates, sap was inoculated onto celite-dusted leaves of Chenopodium quinoa plants (3-4 leaf stage). As expected for WMV, leaves inoculated with sap of each isolate developed chlorotic local lesions ~9 d post-inoculation (dpi) (Moreno et al. 2004). One lesion for each isolate was excised, ground in phosphate buffer, and the sap was mechanically inoculated onto leaves of Nicotiana benthamiana plants. By ~14 dpi, newly emerged leaves showed mild mottling and crumpling, and were weakly positive with the potyvirus immunostrip test. To confirm that these plants were only infected with WMV, total RNA was extracted from symptomatic leaves and used for high throughput sequencing (HTS) (Soltani et al. 2021) at the Foundation Plant Services at UC Davis. The HTS analyses revealed infection with only WMV, i.e., no other viral contigs were identified, and allowed for determination of the complete sequences (~10,000 nt) of WMV [US-CA-C-SG-18] and WMV [US-CA-C-Pet-18] with GenBank accession numbers: OM746964 and OM746965, respectively. Whole genome sequence comparisons revealed that the sequences are 99.0% identical, and 97.3% identical to the sequence of WMV TX29. Sap from symptomatic N. benthamiana leaves infected with each isolate was mechanical inoculated onto leaves of coriander plants (30-35 d old). Newly emerged leaves developed epinasty, crumpling and light green mottling by 14 dpi, and WMV infection was confirmed by RT-PCR with the WMV-specific primer pair WMV-UNI-1F and WMV-UNI-1R (Kim et al. 2019). Thus, Koch's postulates were fulfilled for this leaf mottling disease of coriander. Furthermore, the isolates from coriander induced stunting and distortion and mosaic in leaves of melon, pumpkin and squash plants by 7 dpi, whereas watermelon plants developed stunting and small leaves with mild mottling by 20 dpi. Similar results were obtained with sap prepared from infected coriander leaves. Thus, infected coriander plants are a potential inoculum source for cucurbits via several aphid vectors (Keinath et al. 2017). This is the first report of a mottle disease of coriander caused by WMV, and adds to the wide host range of the virus.

How To Be a Successful Monopartite Begomovirus in a Bipartite-Dominated World: Emergence and Spread of Tomato Mottle Leaf Curl Virus in Brazil.

Begomoviruses are members of the family Geminiviridae, a large and diverse group of plant viruses characterized by a small circular single-stranded DNA genome encapsidated in twinned quasi-icosahedral virions. Cultivated tomato (Solanum lycopersicum L.) is particularly susceptible and is infected by >100 bipartite and monopartite begomoviruses worldwide. In Brazil, 25 tomato-infecting begomoviruses have been described, most of which are bipartite. Tomato mottle leaf curl virus (ToMoLCV) is one of the most important of these and was first described in the late 1990s but has not been fully characterized. Here, we show that ToMoLCV is a monopartite begomovirus with a genomic DNA similar in size and genome organization to those of DNA-A components of New World (NW) begomoviruses. Tomato plants agroinoculated with the cloned ToMoLCV genomic DNA developed typical tomato mottle leaf curl disease symptoms, thereby fulfilling Koch's postulates and confirming the monopartite nature of the ToMoLCV genome. We further show that ToMoLCV is transmitted by whiteflies, but not mechanically. Phylogenetic analyses placed ToMoLCV in a distinct and strongly supported clade with other begomoviruses from northeastern Brazil, designated the ToMoLCV lineage. Genetic analyses of the complete sequences of 87 ToMoLCV isolates revealed substantial genetic diversity, including five strain groups and seven subpopulations, consistent with a long evolutionary history. Phylogeographic models generated with partial or complete sequences predicted that the ToMoLCV emerged in northeastern Brazil >700 years ago, diversifying locally and then spreading widely in the country. Thus, ToMoLCV emerged well before the introduction of MEAM1 whiteflies, suggesting that the evolution of NW monopartite begomoviruses was facilitated by local whitefly populations and the highly susceptible tomato host. IMPORTANCE Worldwide, diseases of tomato caused by whitefly-transmitted geminiviruses (begomoviruses) cause substantial economic losses and a reliance on insecticides for management. Here, we describe the molecular and biological properties of tomato mottle leaf curl virus (ToMoLCV) from Brazil and establish that it is a NW monopartite begomovirus indigenous to northeastern Brazil. This answered a long-standing question regarding the genome of this virus, and it is part of an emerging group of these viruses in Latin America. This appears to be driven by widespread planting of the highly susceptible tomato and by local and exotic whiteflies. Our extensive phylogenetic studies placed ToMoLCV in a distinct strongly supported clade with other begomoviruses from northeastern Brazil and revealed new insights into the origin of Brazilian begomoviruses. The novel phylogeographic analysis indicated that ToMoLCV has had a long evolutionary history, emerging in northeastern Brazil >700 years ago. Finally, the tools used here (agroinoculation system and ToMoLCV-specific PCR test) and information on the biology of the virus (host range and whitefly transmission) will be useful in developing and implementing integrated pest management (IPM) programs targeting ToMoLCV.

Curly Top Disease of Hemp (Cannabis sativa) in California Is Caused by Mild-Type Strains of Beet curly top virus Often in Mixed Infection.

Interest in industrial hemp (Cannabis sativa) as a potential crop led to the establishment of commercial fields in a number of counties in California in 2019 and 2020. Plants in these fields developed different types of virus-like symptoms. The most prevalent type was stunted and bushy plants with distorted, upcurled, and yellowed leaves, which were similar to those associated with curly top disease (CTD) caused by the beet curly top virus (BCTV). This beet leafhopper-vectored virus is endemic in California and can cause economic losses to processing tomato production. Using a multiplex PCR test, BCTV infection was detected in 89% of hemp samples with CTD-like symptoms from Fresno, San Bernardino, and Ventura counties. Other symptom types had low incidence of BCTV infection and were associated with other factors. Hemp plants in California were infected only with the mild-type strains, BCTV-CO and BCTV-Wor, and often in mixed infection (43% of samples). Finally, using an infectious clone of a BCTV-CO isolate from hemp, we demonstrated that agroinoculated hemp plants developed these CTD-like symptoms, thereby fulfilling Koch's postulates for the disease.

The Begomovirus Species Melon Chlorotic Leaf Curl Virus is Composed of Two Highly Divergent Strains that Differ in Their Genetic and Biological Properties.

Since the early 1990s, squash production in Costa Rica has been affected by a whitefly-transmitted disease characterized by stunting and yellow mottling of leaves. The squash yellow mottle disease (SYMoD) was shown to be associated with a bipartite begomovirus, originally named squash yellow mild mottle virus (SYMMoV). It was subsequently established that SYMMoV is a strain of melon chlorotic leaf curl virus (MCLCuV), a bipartite begomovirus that causes a chlorotic leaf curl disease of melons in Guatemala. In the present study, the complete sequences of the DNA-A and DNA-B components of a new isolate of the strain MCLCuV-Costa Rica (MCLCuV-CR) were determined. Comparisons of full-length DNA-A sequences revealed 97% identity with a previously characterized isolate of MCLCuV-CR and identities of 90 to 91% with those of isolates of the strain MCLCuV-Guatemala (MCLCuV-GT), which is below or at the current begomovirus species demarcation threshold of 91%. A more extensive analysis of the MCLCuV-CR and -GT sequences revealed substantial divergence in both components and different histories of recombination for the DNA-A components. The cloned full-length DNA-A and DNA-B components of this new MCLCuV-CR isolate were infectious and induced SYMoD in a range of squashes and in pumpkin, thereby fulfilling Koch's postulates for this disease. However, in contrast to MCLCuV-GT, MCLCuV-CR induced mild symptoms in watermelon and no symptoms in melon and cucumber. Taken together, our results indicate that MCLCuV-CR and -GT have substantially diverged, genetically and biologically, and have evolved to cause distinct diseases of different cucurbit crops. Taxonomically, these viruses are at the strain/species boundary, but retain the designation as strains of Melon chlorotic leaf curl virus under current International Committee on Taxonomy guidelines.

A Bipartite Begomovirus Infecting Boerhavia erecta (Family Nyctaginaceae) in the Dominican Republic Represents a Distinct Phylogenetic Lineage and has a High Degree of Host Specificity.

Boerhavia erecta plants in and around agricultural fields in the Azua Valley of the southeastern Dominican Republic often show striking golden mosaic symptoms. Leaf samples from B. erecta plants showing these symptoms were collected in 2012 and 2013, and PCR tests with degenerate primers revealed begomovirus DNA-A and DNA-B components. The complete sequences of the DNA-A and DNA-B components of four isolates show a high degree of sequence identity (>96%) and a genome organization typical of New World (NW) bipartite begomoviruses. Sequence comparisons and phylogenetic analyses revealed that these isolates composed a new phylogenetic lineage of NW bipartite begomoviruses. The most closely related begomovirus is Merremia mosaic virus, a weed-infecting species from Puerto Rico. Because DNA-A sequence identities are well below the 91% threshold, these isolates represent a new begomovirus species, for which the name Boerhavia golden mosaic virus (BoGMV) is proposed. Infectious cloned BoGMV DNA-A and DNA-B components induced golden mosaic symptoms in agroinoculated B. erecta plants, thereby fulfilling Koch's postulates for this disease. Agroinoculation and mechanical transmission experiments revealed that BoGMV has an unusually narrow host range, limited to members of the family Nyctaginaceae and not including the permissive host Nicotiana benthamiana. The inability of BoGMV to infect N. benthamiana was due to a deficiency in cell-to-cell movement but not to a unique amino acid residue in the movement protein.

Molecular and Biological Characterization of Distinct Strains of Jatropha mosaic virus from the Dominican Republic Reveal a Potential to Infect Crop Plants.

In the Dominican Republic (DO), jatropha plants with yellow mosaic symptoms are commonly observed in and around fields of various crop plants. Complete nucleotide sequences of DNA-A and DNA-B components of four bipartite begomovirus isolates associated with symptomatic jatropha plants collected from three geographical locations in the DO were determined. Sequence comparisons revealed highest identities (91 to 92%) with the DNA-A component of an isolate of Jatropha mosaic virus (JMV) from Jamaica, indicating that the bipartite begomovirus isolates from the DO are strains of JMV. When introduced into jatropha seedlings by particle bombardment, the cloned components of the JMV strains from the DO induced stunting and yellow mosaic, indistinguishable from symptoms observed in the field, thereby fulfilling Koch's postulates for the disease. The JMV strains also induced disease symptoms in Nicotiana benthamiana, tobacco, and several cultivars of common bean from the Andean gene pool, including one locally grown in the DO. Asymmetry in the infectivity and symptomatology of pseudorecombinants provided further support for the strain designation of the JMV isolates from the DO. Thus, JMV in the DO is a complex of genetically distinct strains that have undergone local evolution and have the potential to cause disease in crop plants.

Characterization of a new world monopartite begomovirus causing leaf curl disease of tomato in Ecuador and Peru reveals a new direction in geminivirus evolution.

All characterized whitefly-transmitted geminiviruses (begomoviruses) with origins in the New World (NW) have bipartite genomes composed of a DNA-A and DNA-B component. Recently, an NW begomovirus lacking a DNA-B component was associated with tomato leaf curl disease (ToLCD) in Peru, and it was named Tomato leaf deformation virus (ToLDeV). Here, we show that isolates of ToLDeV associated with ToLCD in Ecuador and Peru have a single, genetically diverse genomic DNA that is most closely related to DNA-A components of NW bipartite begomoviruses. Agroinoculation of multimeric clones of the genomic DNA of three ToLDeV genotypes (two variants and a strain) resulted in the development of tomato leaf curl symptoms indistinguishable from those of ToLCD in Ecuador and Peru. Biological properties of these ToLDeV genotypes were similar to those of Old World (OW) monopartite tomato-infecting begomoviruses, including lack of sap transmissibility, phloem limitation, a resistance phenotype in tomato germplasm with the Ty-1 gene, and functional properties of the V1 (capsid protein) and C4 genes. Differences in symptom phenotypes induced by the ToLDeV genotypes in tomato and Nicotiana benthamiana plants were associated with a highly divergent left intergenic region and C4 gene. Together, these results establish that ToLDeV is an emergent NW monopartite begomovirus that is causing ToLCD in Ecuador and Peru. This is the first report of an indigenous NW monopartite begomovirus, and evidence is presented that it emerged from the DNA-A component of a NW bipartite progenitor via convergent evolution and recombination.