Peach (Prunus persica) is one of the most popular stone fruit in the world. In April 2017, three flat peach and eleven nectarine samples showing leaf yellowing and mosaic symptoms were collected in Pinggu district in Beijing, Peoples Republic of China. To identify viruses that infect peach trees, symptomatic leaves from three flat peach trees (cv. Rui Pan 19) were mixed well into one pool (designated as RP19) for high-throughput sequencing (HTS). Total RNA of leaf samples was extracted using the TRIzol reagent (Tiangen Biotech Co., Ltd, Beijing) following manufacturer’s instructions. The cDNA library was constructed after the removal of ribosomal RNA by Epicentre Ribo-zeroTM rRNA Removal Kit (Epicentre, USA). Sequencing was performed on an Illumina Hiseq 4000 platform and generated 150 bp paired-end reads. Approximately 32.3 million reads were obtained after trimming, and the resulting reads (after removal of host reads) were assembled using Trinity platform (Grabherr et al. 2013). The obtained 9,206 cont...

Mei (Prunus mume Sieb. et Zucc.), widely distributed in East Asian countries for both fruiting- and flowering-purposes, is susceptible to viral infections (Marais et al. 2018). Infection by plum bark necrosis stem pitting-associated virus (PBNSPaV) or little cherry virus 2 (LChV-2) possibly caused overall yield loss in mei in Japan due to incomplete flower development, low fruit bearing rate, and interveinal chlorosis (Numaguchi et al. 2019). Virus-like disease showing mosaic, interveinal chlorosis, vein clearing, or necrotic spot on leaf was observed in mei trees in Beijing, Wuhan, Wuxi, and Nanjing in spring and early summer from 2017 to 2018. Symptomatic leaves collected from the four regions were pooled as two samples for RNA-sequencing (RNA-seq) analysis. After ribosomal RNA (rRNA)-depletion, total RNA extracted by TRNzol reagent (TIANGEN, China) was subjected to library construction using NEBNext Ultra RNA Library Prep Kit (NEB, MA, USA) and sequenced on an Illumina Hiseq 4000 (Novogene, China). Sequencing data was filtered, screened, and assembled as described previously (Zhou et al. 2020) to generate contigs, following by BLAST-x/n search in viral genomes in GenBank. We identified >300 contigs (208-10756 nt) homologous to Asian prunus virus 1 and Asian prunus virus 2 (APV1 and 2), mume virus A (MuVA), PBNSPaV, and peach leaf pitting-associated virus (PLPaV), with 71-100% of nucleotide sequence identity values. APV1 and 2 have been reported in mei in China (Wang et al. 2018), here, we focused on the other three viruses. Contigs homologous to these three viruses were further assembled into three scaffolds of 14,224 nt, 1107 nt, and 753 nt for PBNSPaV, MuVA, and PLPaV, respectively. The scaffold of PBNSPaV (MW217574) nearly covered the whole genome of the isolate VIC3 from Australia (LC523039.1) (Kinoti et al. 2020) with 92.30% of sequence identity; the scalffold of MuVA (MW217572) covered 14.50% of the genome of the isolate pm14 from Japan (NC 040568.1) (Marais et al.2018) with 98.47% sequence identity; the scaffold of PLPaV (MW217573) covered 15.26% of the genome of the isolate XJ-6 from peach (KY867750.1) (He et al. 2017) with 85.23% sequence identity. Presence of the three viruses were verified by RT-PCR detection using designed specific primers for PBNSPaV (Forward: 5'-CAACAAAACTCCCACAGCGG-3 [positions 4014-4033, NC_009992.1] / Reverse: 5'-GCCAAAAGAAGTGCTGGTGG-3' [positions 4659-4640, NC_009992.1]), MuVA (Forward: 5'-AAGAGAATTACTTCAATGCCCTC-3' [positions 171-194, NC_040568.1] / Reverse: 5'-GATATCCAAGATACGATTAGCCAG-3' [positions 533-510, NC_040568.1]), and PLPaV (Forward: 5'-GCTATATCTCAACAACTGCAAGAA-3 [positions 5798-5821, KY867750.1] / Reverse: 5'- GAGTGATACATAGTCCACAGAGAT-3'[ positions 6045-6022, KY867750.1]). The amplified 626, 350 and 251 bp fragments of PBNSPaV, MuVA and PLPaV had 91.47%, 98.07% and 81.89% sequence identity to their respective reference sequences. This is the first report of PBNSPaV and MuVA infecting mei in China, and more importantly, the first report of a new host for PLPaV. In addition, 30 collected leaf samples from Nanjing and Wuhan were analyzed by RT-PCR and 15, 6, and 5 samples tested positive to PLPaV, PBNSPaV, and MuVA, respectively. Although it is difficult to link a particular virus with the observed symptoms due to mixed infections, the symptoms were significantly associated with viral infection because almost all symptomatic leaf samples were virus(es)-positive. Further studies would be required to determine the distribution and impact of these viruses on mei trees and other stone fruits species and to understand the possibility that mei trees may play a role in PLPaV epidemiology.

Huanglongbing (HLB) is a devastating citrus disease caused by Candidatus Liberibacter asiaticus (CLas). Since its initial outbreak in Guangdong Province, China, it has spread to 10 provinces and caused significant economic losses. Hence, assessing CLas genetic diversity and demographic history is crucial for HLB epidemic prevention and control. In this study, we collected 500 leaf samples of CLas-infected plants from 10 provinces. We performed multi-loci sequence analysis on four gene fragments (omp, DnaA, GroEL, and SDE1) to explore the genetic differentiation and diversity of CLas in China. Our results indicated low nucleotide diversity (0.00005 ± 0.00001) in CLas, with the absence of significant systematic geographic structure in its distribution. Molecular variance analysis revealed predominant (81.7%) genetic variations within the population, with a minor variation (18.3%) occurring between populations as well as Yunnan provinces. In the Fujian population, significant gene exchange occurred with the other nine populations. Significant negative values in Tajima’s D and Fu’s F_S neutrality tests indicated historical population expansions. The nucleotide mismatch distribution curve exhibits a single peak pattern, further supporting the expansion events. Our findings hold potential for advancing epidemiological research and providing suggestions for effective strategies to mitigate the spread of CLas and control HLB.

Peach latent mosaic viroid (PLMVd) infects peach trees in China and induces a conspicuous albino phenotype (peach calico, PC) that is closely associated with variants containing a 12-to-14 nucleotide hairpin insertion capped by a U-rich loop. Initially, PC disease distribution was limited to parts of Italy, and it was first detected in the field in China in 2019. To explore the molecular and biological characteristics of PLMVd PC isolates in peach in China, we conducted a comprehensive analysis of disease phenotype development and investigated the data-associated pathogenicity and in vivo dynamics of the Chinese isolate PC-A2 using slash-inoculation into GF-305 peach seedlings. Inoculated seedlings displayed PC symptoms much earlier following topping treatment, and PLMVd infectivity was further assessed using bioassay and semiquantitative RT-PCR experiments. Evolutionary analysis showed that the PC isolate and its progeny variants clustered into a single phylogroup distinct from reference PC-C40 isolates from Italy and PC-K1 and PC-K2 from South Korea. Some PC-A2 progeny variants from green leaves of PC-expressing seedlings showed unbalanced point mutations in hairpin stems compared with the PC-C40 reference sequence and constituted a new stem insertion type. The results reveal associations between the recessive phenotypes of peach albino symptoms and base variation in hairpin stem insertions relative to the PC-C40/chloroplastic heat shock protein 90 reference sequence.

Citrus huanglongbing (HLB) is one of the most severe diseases affecting the citrus industry, with Diaphorina citri (Hemiptera: Liviidae) serving as its primary natural vector. To understand the genetic diversity and population structure of D. citri in the context of HLB diffusion, we analyzed 13 populations from the HLB diffusion frontier and 25 populations from epidemic areas in China. The HLB diffusion frontier areas refer to the peripheral regions of HLB distribution in China, including the western Zhejiang, southern Jiangsu, northern Jiangxi, northern Hunan, and eastern Sichuan provinces. In contrast, the HLB epidemic areas represent regions in China where HLB is actively widespread and causing significant impacts. We utilized mitochondrial genes (COI, ND5, and Cytb) of D. citri and housekeeping genes (dnaQ, rpoC, and argH) of its endosymbiont Candidatus Carsonella ruddii (Ca. C. ruddii) for this analysis. Our findings revealed that the D. citri and Ca. C. ruddii in different regions showed low haplotype diversity and nucleotide diversity. While the genetic variation in D. citri populations primarily occurred within populations, the endosymbiont showed contrasting patterns in the HLB epidemic areas. We identified three dispersal paths: (1) migration of the Yunnan population to Sichuan, Guizhou, and Guangxi; (2) movement of the Guangdong population to Fujian, Jiangxi, and Zhejiang; and (3) dispersal of the Guangdong population to Hunan and Guangxi. Our study suggests that D. citri populations at the HLB diffusion frontier are predominantly transmitted from neighboring epidemic areas.

Citrus tristeza virus (CTV) is an important pathogen threatening the global citrus industry, but its evolution and transmission mechanism in wild citrus has not been clarified. Most of the existing studies are based on CTV-specific gene fragments, lacking genome-wide analysis. There is especially a lack of understanding of CTV transmission dynamics in wild citrus, which needs further investigation. In this study, wild citrus samples from three provinces of China were collected, virus genome data were obtained by high-throughput sequencing (HTS) technology and combined with public database data, and Bayesian phylogeographic inference was used to analyze virus composition characteristics in wild citrus, as well as the population genetic structure, temporal dynamic evolution, and spatial transmission mode of CTV. The results showed that Yunnan wild citrus samples contained the most abundant virus components, including CTV, Citrus Exocortis Viroid (CEVd), Citrus associated Ampelovirus 1 (CaAV-1), and Citrus Virus B (CiVB), while Jiangxi and Hunan samples only contained CTV and CEVd, with all samples showing mixed infection. Phylogenetic analysis showed that nine wild citrus CTV isolates were scattered in different evolutionary clades, and only 9.27% of genetic variation existed between the populations, while 90.72% of genetic variation existed within the populations, indicating little effect of geographic isolation on gene flow. The time to the most recent common ancestor (tMRCA) of CTV was estimated at 1360 CE, with subsequent divergence into two lineages, with population size stabilizing after a rapid increase in 1980–1990. Asia has been identified as the central source of CTV’s global spread, with key migration events including Asia to North America (1746), Asia to Oceania (1829), and Asia to South America (1965), coinciding with global maritime trade and the expansion of the citrus industry.