An infectious clone of citrus yellow mosaic virus (CYMV) was constructed to lay a foundation for further study of its molecular characteristics and pathogenic mechanism.

The amplified CYMV genomic sequence segments were recombined with the ternary expression vector pCY using In-Fusion homologous recombination technology, and a 1.4-fold full-length genomic DNA clone of the virus was constructed and sequenced. The obtained clones were inoculated onto seedlings of Eureka lemon by Agrobacterium-mediated vacuum infiltration (AVI), and their infectivity was identified by molecular detection, presentation of symptoms and virus particles, and re-grafting determination. The obtained infectious clones were used to inoculate different citrus varieties and herbs, and the infection rate was determined by RT-PCR and the various symptoms of different citrus varieties after infection were observed. Mutants of the infectious clones with ORFⅠand ORFⅡ replaced by green fluorescent protein gene (gfp), respectively were constructed, and subjected to infectivity analysis.

Seven clones of 1.4-fold CYMV genome-length DNA were obtained by In-Fusion homologous recombination technology. Among them, CYMV-3 was 90%-100% identical to the corresponding genomic nucleotide sequences of nine CYMV isolates registered in GenBank, and had the highest homology with CYMV-SO isolate (AF347695) and was clustered on the same branch of the genetic evolutionary tree. The results of infectivity identification showed that four out of the 14 Eureka lemon plants inoculated with CYMV-3 were positive by RT-PCR detection, and punctate chlorotic symptoms were observed. DNA was extracted from the new systemic leaves of the infected plant and the expected specific fragments covering the full length of the CYMV genome were obtained by PCR amplification. CYMV-3 infected plants with symptoms were used as the virus source to graft and inoculate rough lemon seedlings, and CYMV specific bands could be detected by RT-PCR at 90 dpi. New leaves of CYMV-3 infected plants with symptoms were taken for fixation and sectioning for electron microscopy and baculovirus particles of about 130 nm × 30 nm were observed, indicating that CYMV-3 was a CYMV infectious clone. Ten and five citrus varieties were inoculated by CYMV-3 via Agrobacterium-mediated vacuum infiltration and injection, respectively, and were tested by RT-PCR. The results showed that the infection rate of the former was 11.11%-100.00% and the latter was 63.16%-90.00%. Some citrus varieties appeared CYMV symptoms such as yellowing, mosaic and yellowing patches. Nicotiana benthamiana, Zea mays, Vigna unguiculata and Sorghum bicolor were inoculated by CYMV-3 via Agrobacterium -mediated injection, and only S. bicolor plants were detected to be positive in RT-PCR detection, and chlorotic strips appeared along the veins of the systemic leaves, indicating that CYMV can infect S. bicolor. The mutants of CYMV-3 with ORFⅠand ORFⅡ replaced by gfp, respectively, were constructed and inoculated onto rough lemons by Agrobacterium-mediated injection. All the inoculated plants were negative by RT-PCR detection and showed no obvious symptoms.

The infectious clone of CYMV was successfully constructed and could infected citrus plants efficiently via Agrobacterium-mediated injection, resulting different symptoms in different varieties.

Citrus yellow mosaic virus (CYMV) is a member of badnaviruses that was first discovered in India and caused serious damage to its citrus industry. At present, CYMV has been listed as a quarantine pest in the United States, Japan, New Zealand, Europe and the Mediterranean region, which has the potential risk of being introduced into China.

The objective of this study is to establish a real-time quantitative PCR (qPCR) detection system for CYMV, screen sensitive citrus varieties to CYMV, elucidate the spatial and temporal distribution of CYMV in host plant, and to provide technical support for monitoring of the virus.

According to the conserved sequences of CYMV coat protein (CP) gene in NCBI, eight pairs of qPCR primers were designed using software Primer Premier 5, and were screened by conventional PCR for primer pairs with good amplification effect and high specificity. The concentration and annealing temperature of the primers were optimized to establish the CYMV qPCR detection system. The specificity of the system was evaluated by detecting different citrus pathogens. Conventional PCR and qPCR were performed in parallel with 1.98×10⁹-1.98 copies/μL of plasmid standards diluted in a gradient as template to evaluate the sensitivity of the established system. The field citrus samples were collected randomly and detected using conventional PCR and qPCR in parallel to evaluate the applicability of the method. Fifteen citrus varieties including Yuhuanyou (Citrus grandis), Chandler pomelo (C. grandis), No.22 karatachi (Poncirus trifoliata), etc., were inoculated with CYMV for symptom observation and molecular detection to screen sensitive indicator plants. Samples from different tissues of Madam Vinous orange (C. sinensis) plants were collected at different times post inoculation, and qPCR assays were performed with C. sinensis elongation factor 1 alpha as an internal reference gene to clarify the spatial and temporal distribution patterns of CYMV in the host plants.

The qPCR detection system for CYMV was established with the optimal primer of CYMV-qF7/R7, the optimal concentration 200 nmol·L^-1 and the optimal annealing temperature 63 ℃. The results of the evaluation experiments showed that the specificity of the established qPCR system is strong, and the detection sensitivity is 1 000 times that of the conventional PCR. The detection results of 660 field citrus samples from different regions showed that the qPCR assay was consistent with the conventional PCR assay, and no CYMV-positive plants were detected except for the positive control. Among the 15 citrus varieties in the inoculation experiment, Yuhuanyou and Chandler pomelo showed strong and typical yellowing mosaic symptoms of CYMV infection at the earliest, suggesting that both could be used as sensitive indicator plants. The detection results of qPCR showed that the titer of CYMV in old leaves was the highest, the titer of virus in old cortex and root was higher, and the titer in tender cortex was lower. The relative content of CYMV in plants was highest from July to September, then decreased month by month, and reached the lowest value in January of the next year. From February of the next year, the relative content of CYMV began to rise, which was consistent with the change trend of the environmental temperature.

A qPCR assay for CYMV detection with high specificity and sensitivity was established, and the spatial and temporal distribution pattern of CYMV in the host plants was determined. In addition, Yuhuanyou and Chandler pomelo can be used as CYMV-sensitive indicator plants.