First report of Cotton leaf curl Multan virus of Spinach in China
Authors: Gao Guolong , Zhang Xingwang , Du Yejuan 
Published: September 2024

Cotton leaf curl Multan virus(CLCuMuV; Begomovirus gossypimultanese, family Geminiviridea) is a single-stranded circular DNA virus, with a genome size of about 2.7 kb, CLCuMuV, which is commonly associated with its satellite DNA, Cotton leaf curl Multan betasatellite (CLCuMuB) (Mansoor et al., 2003), is a serious threat in cotton production causing cotton leaf curl disease (CLCuD) (Briddon et al., 2000). The spread of CLCuMuV is closely linked to its insect vector, whitefly (Bemisia tabaci), which is the exclusive vector species for CLCuMuV transmission (Pan et al., 2018). In May 2019, two spinach (Spinacia oleracea L) samples (XJBC01, XJBC02) showing upward curling of the leaf margins, vein thickening, and enation, symptoms were collected in Shihezi City, Xinjiang, China (Fig. 1B). A 570 bp fragment was amplified from the two symptomatic spinach samples using Begomovirus universal primer pair AV494 (5'-GCCYATRTAYAGRAAGCCMAG-3') and COPR (5'-GANGSATGHTRCADGCCAT ATA-3'), Sequences generated from these amplicons shared 99% nucleotide sequence identities with CLCuMuV DNA-A sequences, suggesting CLCuMuV infection in spinach. To our knowledge CLCuMuV has not been reported in spinach previously. The complete sequences of CLCuMuV and CLCuMuB were then sequenced using CLCuMuV-specific primers GD37-F (5'-GGATCCATTGTTAAACGAATTTCC-3') and GD37-R (5'-GGATCCCACATGTTTGAATTTGA-3') (Gu et al., 2015), as well as betasatellite universal primers β01 (5'-GGTACCACTACGCTACGCAGCAGCC-3') and β02 (5'-GGTACCTACCCTCCCAGGGGTACAC-3') (Zhou et al.,2003). The full length CLCuMuV DNA-A in spinach spans 2737 nt (GenBank accession number: MW561346), while CLCuMuB in spinach covers 1343 nt (GenBank accession number: MW561347). The 2737 nt full length CLCuMuV DNA-A and the associated 1343 nt CLCuMuB genome sequences generated from spinach samples were deposited in the GenBank with accession numbers MW561346 and MW561347. The MW561346 shared 99.5% sequence identity with CLCuMV GD37 from Hibiscus rosasinensis. Whereas the MW561347 shared 98.4% sequence identity with CLCuMuB GD37β. Therefore, we used infectious clones of CLCuMuV (GD37) and CLCuMuB (GD37β), provided by Xueping Zhou (Gu et al., 2015), to inoculate healthy spinach via Agrobacterium. Infected plants showed typical symptoms 14 days post-inoculation, including leaf edge curling, shrinkage, and vein enlargement, which is consistent with symptoms observed in infected spinach plants in the field (Fig. 1C). The expected 570 bp fragments were amplified in the uninoculated upper leaves of spinach showing symptoms, while not detected in the control spinach, indicating that the symptoms on spinach plants were caused by CLCuMuV associated with CLCuMuB. The transmission efficiency of CLCuMuV to spinach was assessed using two whitefly species, MEAM1 and MED, which were fed on h. rosasinensis infected with CLCuMuV. To compare the transmission efficiency between the two species, 14 spinach plants were inoculated with MEAM1, and 11 spinach plants were inoculated with MED. Each spinach plant was inoculated by releasing 10 whiteflies. After 30 days, MEAM1 transmitted CLCuMuV to spinach inducing typical symptoms (Fig. 1D), with a 78.57% (11/14) transmission efficiency. Similarly, MED also transmitted CLCuMuV to spinach but with a lower efficiency of 54.54% (6/11). These results suggested both MEAM1 and MED could transmit CLCuMuV to spinach, with MEAM1 demonstrating higher efficiency than MED. To the best of our knowledge, this study marks the first report of CLCuMuV infecting spinach, indicating an expanded host range for the virus.