First report of Chili pepper mild mottle virus in calibrachoa in the United States

Abstract

Beginning in January 2022, plants of the popular solanaceous ornamental calibrachoa (Calibrachoa spp.) from commercial producers in the United States were observed with various viral disease symptoms including chlorosis, purple mottling and tip necrosis of leaves, and colour-break of flowers (Figures 1, 2). Seventy cuttings of the affected plants were submitted to Agdia Testing Services (Agdia Inc., USA) for analysis and produced positive results with Tobacco mosaic virus ImmunoStrip® (lateral flow immunoassay) and ELISA, Pepper mild mottle virus ImmunoStrip® and ELISA, and Tomato mosaic virus ELISA suggesting the presence of a tobamovirus. To confirm and extend these serological results, total RNA was isolated from seven samples using a modified RNeasy protocol (Qiagen, Germany) and tested using a tobamovirus group RT-PCR (Maroon & Zavriev, 2002). The sequence of the RT-PCR amplicons indicated the presence of the recently described tobamovirus, Chili pepper mild mottle virus (CPMMoV; Vélez-Olmedo et al., 2021). For further characterisation, total RNA from four calibrachoa samples (two each from two different greenhouses located in two different states) representing four different calibrachoa varieties were selected for additional RT-PCR testing at USDA-ARS with degenerate tobamovirus primers. Amplicons of c. 200 bp were observed from all samples with the tob4404v and tob4593 primers (Adkins et al., 2018) designed to amplify a conserved portion of the replicase gene. Similarly, amplicons of c. 800 bp were observed from all samples with the SolACPv and SolACPvc primers (Chellemi et al., 2011) designed to amplify the 3′ terminus of the movement protein gene, the entire coat protein (CP) gene, and part of the 3′ UTR. Amplicons were directly sequenced (GenBank Accession Nos. ON381946-ON381953) and those from each primer set shared 99–100% nucleotide identity with each other, and 99–100% nucleotide identity with the corresponding regions of CPMMoV isolate Oxampampa4 (MN164455) confirming the presence of this tobamovirus. CPMMoV-specific CP gene primers (Vélez-Olmedo et al., 2021) also produced amplicons of the expected size, c. 1,100 bp. No other tobamovirus sequences were detected with any of these primer sets. We additionally tested the same four samples with degenerate potyvirus (Gibbs & Mackenzie, 1997) and orthotospovirus (Chu et al., 2001) primers; no amplicons were observed. CPMMoV was first reported in 2021 from field-grown pepper in Peru (Vélez-Olmedo et al., 2021). To the best of our knowledge, this is the first report of CPMMoV in calibrachoa or any other greenhouse-grown ornamental host. Further research is necessary to determine the prevalence of CPMMoV in ornamental and vegetable crop hosts, and the potential impact this tobamovirus may have on these crops. Salvador Lopez and Carrie Vanderspool are acknowledged for their excellent technical assistance.