A Tomato-Free Period Delays and Reduces Begomovirus Disease in Processing Tomato Fields in a Complex Agroecosystem in Central Brazil.

A mandatory tomato-free period (TFP) was implemented in the state of Goiás, Brazil, in 2007 to help manage diseases caused by whitefly-transmitted begomoviruses. The impact of the TFP was examined in five locations across three states in Central Brazil from 2013 to 2016. Surveys revealed significant differences in begomovirus disease incidence among locations, i.e., low in Guaíra-TFP and Patos de Minas-TFP; moderate-high in Itaberaí-TFP and Morrinhos-TFP; and high in the non-TFP (NTFP) control, Cristalina-NTFP. PCR tests and DNA sequencing were used to validate the symptoms and showed that all collected symptomatic plant samples were infected with tomato severe rugose virus (ToSRV), a common indigenous bipartite begomovirus. Early season surveys (20 to 40 days after transplants [DAT]) in Itaberaí-TFP and Morrinhos-TFP revealed significantly less begomovirus disease in fields established sooner after the TFP (0 to 2 months) compared with incidences in (i) equivalent early planted fields in the Cristalina-NTFP control and (ii) fields established longer after the end of the TFP (>2 to 5 months). Whitefly infestation of crops was detected year-round in all locations and years, and all tested adults were classified in the Bemisia tabaci MEAM1 cryptic species. Infestation levels were significantly higher during the summer but did not vary significantly among locations. Results of monthly monitoring of adult whiteflies for general begomovirus and ToSRV were positively correlated and were indicators of disease incidence in the field. Notably, ToSRV was not detected in whiteflies collected from nontomato plants during the TFP, and there was a longer lag period before detection in whiteflies collected from processing tomatoes for Itaberaí-TFP and Morrinhos-TFP compared with Cristalina-NTFP. Taken together with the low levels of ToSRV infection detected in potential nontomato reservoir hosts at all locations, our results revealed low levels of primary inoculum during the TFP. Thus, even in a complex agroecosystem with year-round whitefly infestation of crops, the TFP was beneficial due to delayed and reduced begomovirus disease pressure during a critical stage of plant development (first month) and for favoring low levels of primary inoculum. Thus, we concluded that the TFP should be part of a regional integrated pest management (IPM) program targeting ToSRV in Brazil.

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.

Tomato golden net virus and tomato yellow net virus: two novel New World begomoviruses with monopartite genomes.

Two novel tomato-infecting begomoviruses were discovered via high-throughput sequencing in Brazil. Both viruses were also Sanger-sequenced and displayed DNA-A components phylogenetically related to New World bipartite begomoviruses. The names tomato golden net virus (ToGNV) and tomato yellow net virus (ToYNV) were proposed. The majority of the New World begomoviruses has bipartite genomes. However, extensive analyses revealed that ToGNV and ToYNV have monopartite genomes, because no cognate DNA-B components were detected. Hence, they may comprise a unique group of monopartite New World begomoviruses, which have enormous biological, molecular, and plant breeding interest.

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.

Complete Genome Sequence Resource of a Strain of Curtobacterium flaccumfaciens pv. flaccumfaciens, the Causal Agent of Bacterial Wilt of Common Bean, from Turkey.

Curtobacterium flaccumfaciens pv. flaccumfaciens is the causal agent of bacterial wilt of common bean (Phaseolus vulgaris), a disease that can reduce yields of this economically important crop worldwide. Current genomics resources for this bacterial pathogen are limited. Therefore, long-read sequencing was used to determine the complete genome sequence of a pathogenic C. flaccumfaciens pv. flaccumfaciens strain isolated from common bean leaves showing irregular necrotic lesions with yellow borders collected in a commercial field in Turkey in 2015.

Comparative Genomics to Develop a Specific Multiplex PCR Assay for Detection of Clavibacter michiganensis.

Clavibacter michiganensis is a Gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial wilt and canker symptoms. Accurate detection is a crucial step in confirming outbreaks of bacterial canker and developing management strategies. A major problem with existing detection methods are false-positive and -negative results. Here, we report the use of comparative genomics of 37 diverse Clavibacter strains, including 21 strains sequenced in this study, to identify specific sequences that are C. michiganensis detection targets. Genome-wide phylogenic analyses revealed additional diversity within the genus Clavibacter. Pathogenic C. michiganensis strains varied in plasmid composition, highlighting the need for detection methods based on chromosomal targets. We utilized sequences of C. michiganensis-specific loci to develop a multiplex PCR-based diagnostic platform using two C. michiganensis chromosomal genes (rhuM and tomA) and an internal control amplifying both bacterial and plant DNA (16s ribosomal RNA). The multiplex PCR assay specifically detected C. michiganensis strains from a panel of 110 additional bacteria, including other Clavibacter spp. and bacterial pathogens of tomato. The assay was adapted to detect the presence of C. michiganensis in seed and tomato plant materials with high sensitivity and specificity. In conclusion, the described method represents a robust, specific tool for detection of C. michiganensis in tomato seed and infected plants.

Sequence analysis of the medium and small RNAs of impatiens necrotic spot virus reveals segment reassortment but not recombination.

The complete sequence of the medium (M) and small (S) RNA genome segments were determined for twelve isolates of impatiens necrotic spot virus from eight plant species. The M- and S-RNAs of these isolates shared 97-99% and 93-98% nucleotide sequence identity, respectively, with the corresponding full-length sequences available in public databases. Phylogenetic analysis based on the M- or S-RNA sequences showed incongruence in the phylogenetic position of some isolates, suggesting intraspecies segment reassortment. The lack of phylogenetic discordance in individual and concatenated sequences of individual genes encoded by M- or S-RNAs suggests that segment reassortment rather than recombination is driving evolution of these INSV isolates.

Nanopore sequencing of a novel bipartite New World begomovirus infecting cowpea.

A new bipartite begomovirus (family Geminiviridae) was detected on cowpea (Vigna unguiculata) plants exhibiting bright golden mosaic symptoms on leaves under field conditions in Brazil. Complete consensus sequences of DNA-A and DNA-B components of an isolate of the virus (PE-088) were obtained by nanopore sequencing and confirmed by Sanger sequencing. The genome components presented the typical genomic organization of New World (NW) begomoviruses. Pairwise sequence comparisons revealed low levels of identity with other begomovirus species previously reported infecting cowpea around the world. Phylogenetic analysis using complete sequences of DNA-A components revealed that the closest relatives of PE-088 (85-87% nucleotide sequence identities) were three legume-infecting begomoviruses from Brazil: bean golden mosaic virus, macroptilium common mosaic virus and macroptilium yellow vein virus. According to the current classification criteria, PE-088 represents a new species in the genus Begomovirus, tentatively named as cowpea bright yellow mosaic virus (CoBYMV).

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.

Rasta Disease of Tomato in Ghana is Caused by the Pospiviroids Potato spindle tuber viroid and Tomato apical stunt viroid.

Rasta is a virus-like disease of unknown etiology affecting tomato (Solanum lycopersicum) plants in Ghana. Symptoms include stunting; epinasty, crumpling, and chlorosis of leaves; and necrosis of leaf veins, petioles, and stems. Leaf samples with rasta symptoms were collected from commercial tomato fields in Ghana in October 2012 and applied to FTA cards, and RNA extracts were prepared. Reverse-transcription polymerase chain reaction (RT-PCR) tests with primers for Columnea latent viroid, which causes rasta-like symptoms in tomato plants in Mali, were negative, whereas tests with degenerate viroid primer pairs were inconclusive. However, tomato seedlings (Early Pak 7) mechanically inoculated with RNA extracts of 10 of 13 samples developed rasta-like symptoms. In RT-PCR tests with RNA from leaves of the 10 symptomatic seedlings and primers for Potato spindle tuber viroid (PSTVd) or Tomato apical stunt viroid (TASVd), the expected size (approximately 360 bp) of DNA fragment was amplified from eight and two seedlings, respectively. Sequence analyses confirmed that these fragments were from PSTVd and TASVd isolates, and revealed a single PSTVd haplotype and two TASVd haplotypes. The PSTVd and TASVd isolates from Ghana had high nucleotide identities (>94%) with isolates from other geographic regions. In a host range study, PSTVd and TASVd isolates from Ghana induced rasta symptoms in the highly susceptible tomato cultivar Early Pak 7 and mild or no symptoms in Glamour, and symptomless infections in a number of other solanaceous species. PSTVd and TASVd isolates were seed associated and possibly seed transmitted.

Genomic Analysis of Clavibacter michiganensis Reveals Insight Into Virulence Strategies and Genetic Diversity of a Gram-Positive Bacterial Pathogen.

Clavibacter michiganensis subsp. michiganensis is a gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial canker disease. In this study, we sequenced and assembled genomes of 11 C. michiganensis subsp. michiganensis strains isolated from infected tomato fields in California as well as five Clavibacter strains that colonize tomato endophytically but are not pathogenic in this host. The analysis of the C. michiganensis subsp. michiganensis genomes supported the monophyletic nature of this pathogen but revealed genetic diversity among strains, consistent with multiple introduction events. Two tomato endophytes that clustered phylogenetically with C. michiganensis strains capable of infecting wheat and pepper and were also able to cause disease in these plants. Plasmid profiles of the California strains were variable and supported the essential role of the pCM1-like plasmid and the CelA cellulase in virulence, whereas the absence of the pCM2-like plasmid in some pathogenic C. michiganensis subsp. michiganensis strains revealed it is not essential. A large number of secreted C. michiganensis subsp. michiganensis proteins were carbohydrate-active enzymes (CAZymes). Glycome profiling revealed that C. michiganensis subsp. michiganensis but not endophytic Clavibacter strains is able to extensively alter tomato cell-wall composition. Two secreted CAZymes found in all C. michiganensis subsp. michiganensis strains, CelA and PelA1, enhanced pathogenicity on tomato. Collectively, these results provide a deeper understanding of C. michiganensis subsp. michiganensis diversity and virulence strategies.

Preservation of viral genomes in 700-y-old caribou feces from a subarctic ice patch.

Viruses preserved in ancient materials provide snapshots of past viral diversity and a means to trace viral evolution through time. Here, we use a metagenomics approach to identify filterable and nuclease-resistant nucleic acids preserved in 700-y-old caribou feces frozen in a permanent ice patch. We were able to recover and characterize two viruses in replicated experiments performed in two different laboratories: a small circular DNA viral genome (ancient caribou feces associated virus, or aCFV) and a partial RNA viral genome (Ancient Northwest Territories cripavirus, or aNCV). Phylogenetic analysis identifies aCFV as distantly related to the plant-infecting geminiviruses and the fungi-infecting Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 and aNCV as within the insect-infecting Cripavirus genus. We hypothesize that these viruses originate from plant material ingested by caribou or from flying insects and that their preservation can be attributed to protection within viral capsids maintained at cold temperatures. To investigate the tropism of aCFV, we used the geminiviral reverse genetic system and introduced a multimeric clone into the laboratory model plant Nicotiana benthamiana. Evidence for infectivity came from the detection of viral DNA in newly emerged leaves and the precise excision of the viral genome from the multimeric clones in inoculated leaves. Our findings indicate that viral genomes may in some circumstances be protected from degradation for centuries.

Host-Specific Relationship Between Virus Titer and Whitefly Transmission of Cucurbit yellow stunting disorder virus.

Cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus, family Closteroviridae) was identified in the melon (Cucumis melo) production regions of the desert southwestern United States in fall 2006. It is now well established in the region, where it is transmitted efficiently by the sweet potato whitefly, Bemisia tabaci biotype B (MEAM1). In order to evaluate the spread and establishment of the virus, nearly all spring and fall cucurbit fields planted in the Imperial Valley of California from 2007 to 2009 were surveyed and representative plants were tested for CYSDV infection. Incidence of CYSDV in spring melon fields was initially low and limited to a small number of fields in 2007 but increased to 63% of fields by spring 2009. Virus incidence in fall melon fields was 100% in each year. These results suggested that the virus had become established in native vegetation, weeds, and other crop species, and represented an increasing threat to melon production in the southwestern United States. Therefore, a select set of weed and crop species which grow or are cultivated in the Imperial Valley were evaluated as CYSDV reservoir hosts. For each species, we determined the capacity of CYSDV to accumulate, the relationship between virus titer in these source plants and transmission by whiteflies, as well as subsequent accumulation in inoculated cucurbit plants. Among these hosts, there was considerable variation in virus accumulation and transmission rates. Cucurbit hosts had the highest CYSDV titers, were efficient sources for virus acquisition, and showed a positive correlation between titer in source plants and transmission. Noncucurbit hosts had significantly lower CYSDV titers and varied in their capacity to serve as sources for transmission. CYSDV titers in some noncucurbit source plants, specifically common bean (Phaseolus vulgaris) and shepherd's purse (Capsella bursa-pastoris), were not positively correlated with transmission, demonstrating that additional environmental, physical, or biochemical factors were involved. These results demonstrate that multiple factors influence the efficiency with which a host plant species will be a reservoir for vector transmission of virus to crops.

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.

Characterization and Epidemiology of Outbreaks of Impatiens necrotic spot virus on Lettuce in Coastal California.

California is the leading producer of lettuce (Lactuca sativa) for the United States and grows 77% of the country's supply. Prior to 2006, coastal California lettuce was only periodically and incidentally infected by a single tospoviruses species: Tomato spotted wilt virus (TSWV). However, beginning in 2006 and continuing through 2012, severe outbreaks of disease caused by Impatiens necrotic spot virus (INSV) have affected the coastal lettuce crop, though TSWV was also present. In contrast, TSWV was the only tospovirus associated with disease outbreaks in Central Valley lettuce during this period. Disease surveys conducted over two seasons (2008 and 2009) in 10 commercial fields (acreage of 6 to 20 ha) indicated that INSV was the only tospovirus associated with economically damaging disease outbreaks in lettuce in the coastal region, with incidences of 0.5 to 27% (mean = 5.7%). Molecular characterization of INSV isolates associated with these disease outbreaks revealed little genetic diversity and indicated that lettuce-infecting INSV isolates were nearly identical to those previously characterized from ornamental or other hosts from different locations in the United States and the world. Monitoring of thrips revealed moderate to large populations in all surveyed lettuce fields, and the majority of thrips identified from these fields were western flower thrips, Frankliniella occidentalis. There was significant positive correlation (r2 = 0.91, P = 0.003) between thrips populations and INSV incidence in the most commonly encountered type of commercial lettuce (romaine, direct seeded, conventional) included in this study. A reverse-transcription polymerase chain reaction assay developed for detection of INSV in thrips showed promise as a monitoring tool in the field. Surveys for INSV reservoir hosts in the coastal production area revealed that the weeds little mallow (Malva parvifolia) and shepherd's purse (Capsella bursa-pastoris) were commonly infected. M. parvifolia plants infected in the field did not show obvious symptoms, whereas plants of this species inoculated in the laboratory with INSV by sap transmission developed necrotic spots and chlorosis. Eleven other weed species growing in the lettuce production areas were found to be hosts of INSV. Coastal crops found to be infected with INSV included basil (Ocimum basilicum), bell pepper (Capsicum annuum), calla lily (Zantedeschia aethiopica), faba bean (Vicia faba), radicchio (Cichorium intybus), and spinach (Spinacia oleracea). Thus, it is likely that INSV was introduced into coastal California lettuce fields via viruliferous thrips that initially acquired the virus from other local susceptible plant species. Results of this study provide a better understanding of INSV epidemiology in coastal California and may help growers devise appropriate disease management strategies.

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.

Histone H3 interacts and colocalizes with the nuclear shuttle protein and the movement protein of a geminivirus.

Geminiviruses are plant-infecting viruses with small circular single-stranded DNA genomes. These viruses utilize nuclear shuttle proteins (NSPs) and movement proteins (MPs) for trafficking of infectious DNA through the nuclear pore complex and plasmodesmata, respectively. Here, a biochemical approach was used to identify host factors interacting with the NSP and MP of the geminivirus Bean dwarf mosaic virus (BDMV). Based on these studies, we identified and characterized a host nucleoprotein, histone H3, which interacts with both the NSP and MP. The specific nature of the interaction of histone H3 with these viral proteins was established by gel overlay and in vitro and in vivo coimmunoprecipitation (co-IP) assays. The NSP and MP interaction domains were mapped to the N-terminal region of histone H3. These experiments also revealed a direct interaction between the BDMV NSP and MP, as well as interactions between histone H3 and the capsid proteins of various geminiviruses. Transient-expression assays revealed the colocalization of histone H3 and NSP in the nucleus and nucleolus and of histone H3 and MP in the cell periphery and plasmodesmata. Finally, using in vivo co-IP assays with a Myc-tagged histone H3, a complex composed of histone H3, NSP, MP, and viral DNA was recovered. Taken together, these findings implicate the host factor histone H3 in the process by which an infectious geminiviral DNA complex forms within the nucleus for export to the cell periphery and cell-to-cell movement through plasmodesmata.

Two genetically related begomoviruses causing tomato leaf curl disease in Togo and Nigeria differ in virulence and host range but do not require a betasatellite for induction of disease symptoms.

Tomato leaf curl disease (ToLCD) has emerged as a major constraint on tomato production in some parts of West Africa. In this study, begomoviruses associated with ToLCD in Togo and Nigeria were characterized, as well as a betasatellite associated with the disease in Togo. The genome organization of both viruses is typical of Old World monopartite begomoviruses. Sequence analysis revealed that the begomovirus from Togo is a variant of tomato leaf curl Kumasi virus (ToLCKuV) from Ghana, and it is designated ToLCKuV-[Togo:Pagouda:2006] (ToLCKuV-[TG:Pag:06]). The begomovirus from Nigeria has a recombinant genome, composed of sequences of ToLCKuV (major parent) and a cotton leaf curl Gezira virus (CLCuGV)-like virus, and possesses an unusual non-reiterated replication-associated protein (Rep) binding site. Moreover, because the sequence has <89% identity with those of previously characterized begomoviruses, it is a new species and is designated tomato leaf curl Nigeria virus-[Nigeria:Odogbo:2006] (ToLCNGV-[NG:Odo:06]). The cloned DNAs of ToLCKuV-TG and ToLCNGV were infectious and induced leaf curl symptoms in tomato plants, but ToLCNGV was comparatively more virulent. Both viruses also induced stunted growth and leaf curl symptoms in other solanaceous species (various Nicotiana spp. and Datura stramonium), whereas ToLCNGV but not ToLCKuV-TG induced symptoms in common bean plants. The betasatellite associated with ToLCD in Togo is genetically distinct (i.e., <78% nucleotide sequence identity with previously identified betasatellites) and is designated tomato leaf curl Togo betasatellite-[Togo:Pagouda:2006] (ToLCTGB-[TG:Pag:06]). Replication and systemic spread of ToLCTGB in tomato was mediated by ToLCKuV-TG and ToLCNGV; however, the betasatellite had no effect on disease symptoms induced by either begomovirus. In contrast, ToLCTGB increased symptom severity induced by both viruses in Nicotiana spp. and D. stramonium. Thus, although ToLCTGB increased symptom severity in a host-dependent manner, it does not appear to play a role in ToLCD and may have been present with ToLCKuV-TG as a reassortant.