First report of pelargonium line pattern virus infecting Geranium ( <i>Pelargonium hortorum</i> ) in Tibet, China

Pelargonium line pattern virus (PLPV), belongs to the family Tombusviridae), was first identified in western Germany (Castaño and Hernández, 2005) and later reported in Yugoslavia (Stone, 1980). PLPV induces yellow-green spots and stripe-like lesions on host leaves, severely reducing crop quality (Ivars et al., 2004; Stone and Hollings, 1971, 1976). Geranium (Pelargonium hortorum) is an economically important ornamental plant widely cultivated in China with acknowledged medicinal value. Except tomato spotted wilt virus (TSWV) (Zhu at al.,2017), no other virus was reported from Geranium in China. In June 2024, seven mottled geranium leaf samples were collected from Tibet, China (S Fig. 1A). Total RNA was extracted with the FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China) and subjected to small RNA deep sequencing on the Illumina HiSeq 2000 platform (Sangon Biotech, Shanghai, China). After Trimmomatic-based adapter trimming and low-quality read filtering, 11,199,822 clean reads were obtained; de novo assembled contigs analyzed via BLASTn against the NCBI nonredundant protein database showed the highest identity with PLPV Spanish isolate (EU849616) (S Fig. 1B), confirming PLPV infection in Tibet. No other virus sequence was detected. Five specific primer sets were designed by aligning seven NCBI-registered complete PLPV genomes to identify conserved regions and out of five primer pairs PLPV-F-322/PLPV-R-322 was selected for PLPV detection (S Table. 1). Total RNA was extracted with TRIzol reagent (TaKaRa, Liaoning, China) and reverse-transcribed with PrimeScript RT Kit (TaKaRa) using reverse primer PLPV-R. RT-PCR was carried out in 20 μL reactions. Each reaction contained 1 μL of each primer, 1 μL cDNA, 10 μL 2× Taq Master Mix (Vazyme), and 3 μL nuclease-free water. The thermal profile was as follows: 95 °C for 3 min; 32 cycles of 95 °C/30 s, 56 °C/15 s, 72 °C/8 s; and a final extension at 72 °C for 5 min. Out of seven, five Tibet geranium samples were PLPV-positive (S Fig. 1C). For distribution assessment, 29 geranium samples were collected and further divided into western region (n=7), eastern region (n=9), southern region (n=7), and the northern region (n=6) (S Table. 2). No PLPV was detected in the samples collected from non-Tibet regions, which are over 2000 kilometers away from the location of Tibet (S Fig. 2). The PLPV genome (Acc. No. PZ170267) was amplified with primers PLPV-F-3883/PLPV-R-3883, cloned into pCE2 TA/Blunt Zero (Vazyme), Sanger-sequenced; and assembled using DNAMAN. The PLPV genome shared 96.16% nucleotide identity with Spanish isolate (EU835946). The full-length nucleotide sequence of PLPV shows a similarity of over 93% with the sequences from Europe and American isolates (S Fig. 1D). A CP gene-based phylogenetic tree showed PLPV China 2024 clustered closely with EU849616 Spain 2014 but formed a distinct clade (S Fig. 3). PLPV often establishes systemic, low titer, and asymptomatic infections (Pérez-Cañamás et al., 2017). This study confirms PLPV infection in geranium in Tibet, China. Importantly, this is the first report of PLPV naturally infecting P. hortorum in Tibet, expanding the known geographic distribution of PLPV. This study documented genome sequence variation in the Chinese PLPV isolates, offering valuable insights into the PLPV population and spatial dynamics in geranium.