First Report of Pepper Chlorotic Spot Virus in Bidens pilosa in Yunnan, China

Bidens pilosa (common name Spanish needle) is a species of wild flowering plant in the Asteraceae family. It is native to the Americas and has become a noxious invasive weed of Yunnan province, China. In July 2018, as part of a survey for orthotospoviruses (order Bunyavirales, family Tospoviridae) in flue-cured tobacco, orthotospovirus-like symptoms, including chlorotic spots on leaves and death of growing tips, were observed on B. pilosa around one tobacco field in Yuxi, Yunnan province. The presence of thrips on B. pilosa indicated that the causal agent could be an orthotospovirus. When sap extracts from three symptomatic B. pilosa samples were mechanically inoculated onto 12 Nicotiana benthamiana plants separately, typical orthotospovirus symptoms including chlorotic spots on leaves and death of growing tips were observed beginning from 10 days postinoculation on all 12 N. benthamiana plants. Although tomato spotted wilt virus (TSWV) and tomato zonate spot virus (TZSV) are two common orthotospoviruses in flue-cured tobacco fields in Yunnan, no reaction was observed from all three symptomatic B. pilosa and 12 N. benthamiana samples in an ELISA using TSWV and TZSV antibodies (Dong et al. 2008; Wu et al. 2009). To identify the causal agent of these symptomatic B. pilosa samples, total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and then subjected to reverse transcription using a random primer (Takara, Dalian, China). These cDNAs were then used as a template in a universal polymerase chain reaction (PCR) assay using a recently reported degenerate primer pair dTospo-F2 (5′-GATCAATCNAARTGGTCDGCWTC-3′) and dTospo-R2 (5′-CATDGCACAAGARTGRTAVACWGA-3′), which was designed to amplify the conserved regions of the orthotospoviral L RNA sequence (Huang et al. 2018). A single band of the predicted size (around 300 bp) was obtained from symptomatic B. pilosa plants but not from healthy control B. pilosa plants. PCR amplicons, sequenced in both directions, from the three samples were identical (accession no. MK070341) and showed 96% nucleotide identity with the corresponding sequence of pepper chlorotic spot virus (PCSV) TwPep3 isolate (accession no. KY315809) from Capsicum annuum (Cheng et al. 2014). To further confirm the identity of PCSV, a specific primers pair, PCSVNF (5′-ATGTCTAACGTCAGGAAAACTTTGT-3′) and PCSVNR (5′-TTACAGCGATATGCTTCCTCTCT-3′), was designed to amplify the complete nucleocapsid (N) protein on the S segment of PCSV. An 0.85-kb DNA fragment was amplified from three B. pilosa samples. The derived sequences of the N gene from three samples were identical (accession no. MK070340) and had 94% nucleotide identity with PCSV TwPep3 isolate. These combined results provide further confirmation of PCSV in B. pilosa. B. pilosa is an invasive perennial weed and has become widely distributed in Yunnan province with the potential to serve as a reservoir for both orthotospovirus and thrips (Huang et al. 2016). Therefore, further studies will be necessary to determine the prevalence of PCSV in crops and B. pilosa. To our knowledge, this is the first report of the occurrence of PCSV in B. pilosa.