First Report of Impatiens Necrotic Spot Virus (INSV) Infecting Lettuce in Mexico

Impatiens necrotic spot virus (INSV; Order Bunyavirales, Family Tospoviridae) is transmitted by several thrips species and has emerged as an important pathogen of lettuce (Lactuca sativa) in several countries (Beris et al., 2020 and Hasegawa & Del Pozo-Valdivia, 2023). In 2023, a total of 22,092 hectares of lettuce were planted in Mexico, with a total production of 523,739 tons (Agri-Food and Fisheries Information Service, 2024). In the last several years, increased outbreaks of a disease showing virus-like symptoms have emerged in lettuce in central Mexico. In all cases, plants exhibited symptoms of yellowing, brown necrotic spots and ringspots on the leaves and midribs, and reduced growth. In fall 2023, symptoms were observed in 12 iceberg lettuce fields, with incidences between 5-70% in the municipality of Tenango del Valle, in the state of Mexico. 18 plants collected from 6 fields were initially tested for the presence of two thrips-transmitted viruses, INSV and tomato spotted wilt virus (TSWV) using ImmunoStrips (Agdia Inc., Elkhart, Indiana), which indicated positive results for INSV in 16 plants and TSWV in 2 plants. None of the plants tested positive for both viruses. Follow up sampling was conducted, which included a total of 17 symptomatic lettuce plants from three fields in October 2023, while an additional 11 lettuce plants were collected from Tepeaca, in the neighboring state of Puebla in March 2024. DAS-ELISA confirmed the presence of INSV in 13/17 plants and TSWV in 2/17 plants from Tenango del Valle, while only INSV was detected in the 11 plants from Tepeaca. Based on ELISA, none of the lettuce samples were co-infected by INSV and TSWV, which was similarly observed in California and Greece (Koike et al., 2008 and Beris et al., 2020). Initially, RNA was purified from 3 plants (two from Tenango del Valle, one from Tepeaca) reverse-transcribed, and PCR amplified with primers to the N gene of the INSV S RNA, as previously described (Hasegawa et al., 2022). All three reactions produced a single expected amplicon of 524 bp and were confirmed by bi-directional Sanger sequencing (MCLab, South San Francisco, CA). To obtain the full-length sequences for the N and NSm genes, RNA from a fourth sample (Tenango del Valle) was amplified with primers (Kuo et al., 2014), and Sanger sequenced. The 789 bp N gene (PP726902) shared >99% nucleotide and amino acid identity to the corresponding region of the INSV isolate from orchid in California (KF926828), while the 912 bp NSm gene (PP726901) shared >98% nucleotide and amino acid identity to the INSV isolate from basil in Washington (KX790322). Additionally, all four samples showed >99.5% similarity to one another. INSV was previously reported to affect other crops in Mexico, including tomatillo (Physalis ixocarpa) and pepper (Capsicum spp.) (González-Pacheco and Silva-Rosales, 2013), while TSWV has been reported to infect lettuce (Moreno et al., 2016). To our knowledge, this is the first report of INSV infecting lettuce in Mexico and was the dominant orthotospovirus in lettuce samples that were tested. INSV should be closely monitored throughout central Mexico, where majority of lettuce production occurs. Additional studies are warranted to identify the thrips vector species that are present, and to understand the role of crop and non-crop hosts in the epidemiology of INSV throughout the region.