First Report of Tomato Chlorosis Virus Infecting Tomato in Pakistan

Abstract

Tomato chlorosis virus (ToCV) is a bipartite crinivirus (Closteroviridae) consisting of two positive-sense single-stranded RNAs: RNA1 and RNA2 (Fiallo-Olive and Navas-Castillo 2019). It is a phloem-limited virus transmitted by whiteflies (Bemisia tabaci and Trialeurodes vaporariorum) in a semipersistent manner (Fiallo-Olive and Navas-Castillo 2019; Lee et al. 2018). In March 2019, a survey of the tomato crop (cv. Richman) was conducted in the Multan district of Pakistan; 35% of plants in the crop showed virus-like symptoms such as interveinal chlorosis, brittleness, and general yellowing in the older and middle leaves. These symptoms were similar to those caused by ToCV and tomato infectious chlorosis virus (TICV) (Wisler et al. 1998). Tomato plants were infested by dense (20/leaf) whitefly (B. tabaci) populations. Thirty-six samples were collected from different plants (30 symptomatic and six asymptomatic) and were tested for ToCV and TICV. Total RNA was extracted from leaf samples using TRIzol reagent and assayed by RT-PCR, targeting the ToCV movement protein gene with primers ToCV-RNA2-IF and ToCV-RNA2-1R (Lee et al. 2018) and the TICV heat shock protein homolog gene with primers TICV-32 and TICV532 (Vaira et al. 2002). RT-PCR was also performed to target the clathrin adaptor complexes medium subunit (CAC), a reference protein gene of tomato, by using the forward primer 5′-CCTCCGTTGTGATGTAACTGG-3′ and the reverse primer 5′-ATTGGTGGAAAGTAACATCATCG-3′ (amplification size: 173 bp) (Gonzalez-Aguilera et al. 2016). Twenty-seven of 30 samples yielded the 827-bp ToCV product, whereas no amplification was obtained in any of the asymptomatic samples. TICV was not detected in any of the analyzed samples. Whiteflies (B. tabaci biotype B) collected from disease-free cotton plants and reared on cucumber plants (nonhost of ToCV) under greenhouse conditions were used in the transmission assay. One hundred nonviruliferous whiteflies were given an acquisition access period (AAP) of 48 h on an infected ToCV tomato plant and then transferred onto 20 healthy tomato seedlings (hybrid Richman, 5 whiteflies/seedling in one cage) for a 48-h inoculation access period (IAP). To ascertain that the whiteflies were nonviruliferous, 100 whiteflies were transferred onto five healthy tomato plants (20 whiteflies/plant) for an IAP of 48 h. Sixteen of 20 inoculated plants showed typical ToCV symptoms 28 days postinoculation, and ToCV was detected by RT-PCR using the ToCV-specific primers. No symptoms were observed on the negative control plants, confirming the nonviruliferous nature of whiteflies. The ToCV-positive tomato plants were used as a source for back inoculation to an indicator host, Nicotiana benthamiana. About 200 nonviruliferous whiteflies were given a 48-h AAP on the ToCV-infected tomatoes and then transferred to four healthy N. benthamiana plants (50 whiteflies/plant) for a 48-h IAP, and two healthy N. benthamiana plants were controls (fed on by nonviruliferous whiteflies). All ToCV-inoculated N. benthamiana plants showed distinctive symptoms of yellowing, interveinal chlorosis, and leaf brittleness; no symptoms were observed on the control plants. RT-PCR confirmed ToCV in symptomatic indicator plants and its absence in the control plants. The amplicons (movement protein gene) of two selected ToCV isolates from the field plants were sequenced in both directions. The resulting sequences were submitted to NCBI (MN385628 and MN385629). BLASTn analysis of the sequences revealed that the newly identified isolates are 99.88% identical to isolates reported from Turkey (KY419528), China (KJ815045), and Greece (EU284744). To our knowledge, this is the first report of ToCV infecting tomato in Pakistan. The wide occurrence of ToCV and its whitefly vectors in tomato crops in the region indicates that effective integrated management measures are needed to minimize losses caused by the virus in tomato.