RESUMEN
Impatiens necrotic spot virus (INSV) (Orthotospovirus impatiensnecromaculae) is a virus in the Order Bunyavirales and Family Tospoviridae. The virus is vectored by several species of thrips and is a serious pathogen of ornamentals and lettuce in the United States (Hasegawa & Del Pozo-Valdivia 2023; Daughtrey, M. L., et al. 1997; Webster, C. G., et al. 2015). In January 2023, tomato plants (Solanum lycopersicum,'Big Dena') with viral symptoms of reduced vigor, wilting, necrotic spots on leaves, and sunken lesions on the stem were observed in one greenhouse in Guilford County, North Carolina (NC) (Figure 1A-C). Disease incidence was low (2%), with only three symptomatic plants in the single greenhouse. Affected plants also had signs of thrips feeding (dead thrips, frass, and feeding scars) present across the whole plant (Figure 1D). Samples were submitted to the NC State Plant Disease and Insect Clinic and tested positive for INSV, but negative for TSWV, using Agdia ImmunoStrips®. RNA was extracted from symptomatic leaf tissue using the IBI Total RNA Mini kit (Plant), and complementary DNA (cDNA) was generated using the ThermoFisher Verso cDNA synthesis kit. A reverse transcriptase (RT)-PCR with INSV nucleocapsid (N) primers (F:5'-ATGAACAAAGCAAAGATTACC-3' and R:5'- TTAAATAGAATCATTTTTCCC-3') was used to confirm INSV presence (Hassani-Mehraban et al. 2016). Full length N cDNA amplicon sequencing [GenBank No. PP658213] revealed 99.62% nucleotide identity to NCBI GenBank accessions KF926828 (orchid in California), MH453554.1 (hosta from NY), and MH453552.1 (foxglove from NY), all of which are INSV N sequences. The infected leaf samples were used to mechanically inoculate Emilia sonchifolia and tomato (cv.'Moneymaker') using standard virological methods. We successfully infected E. sonchifolia with INSV (confirmed with visual mosaic symptoms and positive INSV ImmunoStrip). However, mechanical inoculation of the tomato plants proved unsuccessful. Using the INSV infected E. sonchifolia leaves as an inoculum source, we generated a viruliferous Frankliniella occidentalis (Western flower thrips) cohort and challenged three week old tomatoes using thrips mediated inoculation (adapted from Aramburu et al. 2009 and Rotenberg et al., 2009). Twenty days post-inoculation, tomatoes with thrips feeding scars were symptomatic for INSV infection with chlorotic and necrotic spots, stunting, and reduced vigor. INSV infection of these tomato plants was verified with a positive INSV ImmunoStrip® result, two-step RT-PCR amplification of N, and Sanger sequencing of N. Samples from thrips-inoculated tomato plants did not test positive for TSWV. Sequence alignment showed that the recovered virus sequence was 99.85% identical to the original INSV sequence from the diagnostic sample (a single nucleotide difference). To the best of our knowledge, this is the first instance of INSV infecting tomato in NC production systems. Although TSWV is more common in vegetable production in NC (253 cases of TSWV compared to 1 case of INSV in vegetable crops based on NC State Plant Disease and Insect Clinic records since 2008), INSV incursion into tomato producing areas is concerning and should be closely monitored, especially at the transplant stage. This report also underscores the importance of using thrips vectors to transmit virus in screening for susceptibility to orthotospoviruses.
RESUMEN
Tomato spotted wilt virus (TSWV) and related thrips-borne orthotospoviruses are a threat to food and ornamental crops. Orthotospoviruses have the capacity for rapid genetic change by genome segment reassortment and mutation. Genetic resistance is one of the most effective strategies for managing orthotospoviruses, but there are multiple examples of resistance gene breakdown. Our goal was to develop effective multigenic, broad-spectrum resistance to TSWV and other orthotospoviruses. The most conserved sequences for each open reading frame (ORF) of the TSWV genome were identified and comparison to other orthotospoviruses revealed sequence conservation within virus clades and some overlapped with domains with well-documented biological functions. We made six hairpin constructs, each of which incorporated sequences matching portions of all five ORFs. Tomato plants expressing the hairpin transgene were challenged with TSWV by thrips and leaf-rub inoculation and four constructs provided strong protection against TSWV in foliage and fruit. To determine if the hairpin constructs provided protection against other emerging orthotospoviruses, we challenged the plants with tomato chlorotic spot virus and resistance-breaking TSWV (RB-TSWV) and found that the same constructs also provided resistance to these related viruses. Antiviral hairpin constructs are an effective way to protect plants from multiple orthotospoviruses and are an important strategy in the fight against RB-TSWV and emerging viruses. Targeting of all five viral ORFs is expected to increase the durability of resistance and combining them with other resistance genes could further extend the utility of this disease control strategy.