First Report of Phytoplasma Belongs to 16SrV Group Associated with Witches'-broom Disease of Taxillus chinensis in China.

Abstract

Taxillus chinensis (DC.) Danser is a hemiparasitic shrub, widespread in Southern China (Fu et al., 2001). T. chinensis can parasitize a wide range of species (e.g., Camellia spp., Ficus virens and Osmanthus fragrans), which obviously suppressed host growth by robbing nutrient and water through haustorium, causing considerable tree damage. During field visits to Dongguan (22°86'N, 13°97'E) and Guangzhou (23°19'N, 113°31'E), Guangdong Province, in April-July 2021, the typical phytoplasma-suspected symptom manifested as stunting, leaflet, leaf chlorosis and witches'-broom were observed in almost 36% of T. chinensis plants. Leaf samples were collected from six randomly collected plants with symptoms and six symptomless plants (Fig 1). Among them, half of T. chinensis plants parasitized on the host Elaeocarpus sylvestris, the other half on the O. fragrans. No apparent symptoms were observed on both two host plants. Total DNA was extracted from 0.5 g fresh leaf of T. chinensis plants with and without symptoms, as well as two host plants E. sylvestris and O. fragrans, using the CTAB method (Doyle et al., 1990). Nested polymerase chain reactions (PCRs) were performed on DNA extracts of all tested plants with primer pairs of P1/P7 and R16mF2/R16mR1 for 16S rRNA gene (Lee et al., 1993) and rp(v)F1/rpR1 for rp gene (Lee et al., 1998). All amplicons were obtained from symptomatic samples of T. chinensis and host plant E. sylvestris, whereas no such products resulted from DNAs of symptomless plants and O. fragrans. The amplicons were purified and sequenced by Sanger method (Rui Biotech, Guangzhou, China). The amplicon of 16S rRNA and rp genes is 1346 bp and 938 bp, respectively. BLAST comparison of the 16S rRNA (accession no. OL412744) and rp (accession no. OL473789) sequences of the T. chinensis witches'-broom phytoplasma yielded 99.6% sequence identity with those of phytoplasmas of group 16SrV jujube witches'-broom (JWB) phytoplasma (accession no. CP025121 for 16S rRNA gene and AF396941 for rp gene). The 16S rRNA gene sequence of phytoplasma in host plant E. sylvestris (accession no. OM885990) is 99.7% similarity to the 'Elaeocarpus zollingeri' yellows phytoplasma (accession no. LC257960) and 99.4% similarity to the 'Elaeocarpus sylvestris' decline phytoplasma (accession no. MW553140), but 95.8% similarity to the 16S rRNA gene of phytoplasma in T. chinensis. The virtual RFLP tool, iPhyClassifier delineated the T. chinensis phytoplasma (accession no. OL412744) to group 16SrV-B (accession no. AB052876) with the similarity coefficient 1.0 (Fig 2), and phytoplasma in E. sylvestris to group 16Sr group XXXII with the similarity coefficient 0.97. Phylogeny analyses of 16S rRNA and rp genes (MEGA version 7.0.14, USA) using reference phytoplasmas from GenBank confirmed sequencing results and placed the T. chinensis phytoplasma in group 16SrV-B (Fig. 3 and 4). In China, the 16SrV-B phytoplasma group has been reported in Amaranthus retroflexus (Yang et al., 2011), Liriodendron chinense (Li et al., 2012), Prunus salicina (Gao et al., 2020) and sweet potato (Li et al., 2021). This is the first report of a 'Ca. Phytoplasma ziziphi', 16SrV-B related phytoplasma associated with parasitic T. chinensis in China. The results of this study indicate that T. chinensis could be a vector to spread phytoplasmas 16SrV group through parasitism and this can be helpful for related research.