Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (17): 3451-3460.doi: 10.3864/j.issn.0578-1752.2025.17.007

• PLANT PROTECTION • Previous Articles     Next Articles

Construction and Infectivity Identification of Genome-Length cDNA of Citrus Psorosis Virus

LI YaYu(), WANG XinLiang(), ZHOU JinHuan, LI ChuXin, LI JiaXin, TIAN XuBin, SONG Zhen*()   

  1. Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712
  • Received:2025-05-28 Accepted:2025-07-07 Online:2025-09-02 Published:2025-09-02
  • Contact: SONG Zhen

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Abstract:

【Objective】Citrus psorosis virus (CPsV) is a tripartite, negative-sense single-stranded RNA virus of the genus Ophiovirus in the family Aspiviridae, which can cause cracking of citrus trunks and even the death of the whole plant, seriously threatening the safety of the citrus industry. The construction of reverse genetics systems for negative-strand RNA viruses is challenging. This study aims to establish a full-length cDNA clone of CPsV genome and determine its infectivity, with the expectation of laying a foundation for research on its pathogenic mechanism and other aspects.【Method】The primers were designed using software Primer 5, and the total nucleic acid of CPsV-infected plants was used as a template for RT-PCR amplification of the three strands of CPsV, RNA1, RNA2 and RNA3, respectively. Based on the dual-expression vector pXT1, cDNA clones of three RNA strands were constructed by In-Fusion homologous recombination technology, which were verified by enzyme digestion and sequencing analysis. The cDNA clones of the three strands of CPsV were screened by the Nicotiana benthamiana inoculation system, and further inoculated into the herb host Gomphrena globosa by Agrobacterium-mediated injection, and then inoculated into different citrus varieties by vacuum infiltration to observe their symptoms and perform molecular detection.【Result】A total of two CPsV RNA1 full-length cDNA clones, two RNA2 full-length cDNA clones, and two RNA3 full-length cDNA clones were obtained, respectively. One full-length cDNA clone of RNA1, RNA2 and RNA3 was randomly selected and combined as a full-length cDNA clone of CPsV genome, eight RNA1, RNA2 and RNA3 cDNA clones were selected and inoculated on N. benthamiana through Agrobacterium-mediated inoculation and RT-PCR detection. Among the eight combinations, the positive rate of CPsV-122 was the highest (62.50%). Sequence analysis showed that CPsV-122 had the highest sequence identity with Spanish isolate P-121, and the corresponding sequence identities of RNA1, RNA2 and RNA3 were 98.06%, 97.10% and 99.32%, respectively. In a phylogenetic tree based on the amino acid sequence of coat protein, CPsV-122 and P-121 were clustered in the same clade and five isolates from China, Tunisia and Italy. CPsV-122 was inoculated by Agrobacterium-mediated inoculation to G. globosa, Citrus limon and C. paradise for symptom observation and RT-PCR test. The results showed that at 7 dpi, the positive rate of G. globosa was 16.67% (2/12), and at 25 dpi, the positive plants showed obvious reddish-brown blight, local necrosis of leaves and other CPsV infection symptoms. The RT-PCR results of C. paradise and C. limon were negative except for the positive control, but at 90 dpi, 13 out of 20 C. limon plants inoculated with CPsV-122 showed obvious CPsV infection symptoms such as dwarfing, yellowing, wilting shoots and shoot gelatinization, while no specific symptoms were observed in the empty vector group and the healthy control group.【Conclusion】CPsV-122 is a full-length cDNA clone of the CPsV genome, which can systematically infect G. globosa and cause typical CPsV infection symptoms on citrus, such as dwarfing and withering of shoots.

Key words: citrus psorosis virus (CPsV), full-length cDNA clone, Agrobacterium-mediated inoculation, infectivity identification, citrus virus

Table 1

Information of the primers used in this study"

引物名称Primer name 引物序列Primer sequence (5′-3′) 用途Usage
CPsV1(354)-F1 ctaaggaaggtgtcagaatgg CPsV-RNA1的2054 nt片段扩增
2054 nt fragment amplification of CPsV-RNA1
CPsV1(2409)-R1 accaacaagaggaaatgcc
CPsV1(2251)-F2 gaataagaaatgcgcccaca CPsV-RNA1的2894 nt片段扩增
2894 nt fragment amplification of CPsV-RNA1
CPsV1(5125)-R2 tcaaggaggcaaaagg
CPsV1(5040)-F3 ccatcatgtcatccaacatca CPsV-RNA1的3078 nt片段扩增
3078 nt fragment amplification of CPsV-RNA1
CPsV1(8118)-R3 acttacacaaatggggattgg
CPV-RNA1(JC)-F1 aagaccagagggctattgaga CPsV-RNA1检测
CPsV-RNA1 detection
CPV-RNA1(JC)-R1 ggatcttgaattgtcattgtgctg
CPV-RNA2(JC)-F1 tatctgtcacttcatcgaaagg CPsV-RNA2检测
CPsV-RNA2 detection
CPV-RNA2(JC)-R1 gtcagcagtatttccatccttt
CPV-RNA3(JC)-F1 tccaaccacgtcttcaaagcaa CPsV-RNA3检测
CPsV-RNA3 detection
CPV-RNA3(JC)-R1 tatcagttatcttaatgttgctgg
CPsV-RNA1-24K-F aagtaagggttgaaaACTTACACAAATGGGGATTGGA CPsV-RNA1全长序列扩增
CPsV-RNA1 full-length sequence amplification
CPsV-RNA1-RdRp-R atcttgaacccctaggaaacttctgAAGCCTTCAGACAATATG
CPsV-RNA2-F3 tcacgttgacaggttatcac CPsV-RNA2全长序列扩增
CPsV-RNA2 full-length sequence amplification
CPsV-RNA2-R3 tcatcctggtgacatttccac
CPsV-RNA3-F2 agtctcacagctaatcgatgCAAAAGCAAAATGGGAAAGCG CPsV-RNA3全长序列扩增
CPsV-RNA3 full-length sequence amplification
CPsV-RNA3-R2 catagtcgatgctaccccaAAGATACCTTTGAGTCCAT

Fig. 1

Construction scheme of CPsV clone"

Fig. 2

RT-PCR amplification of CPsV genome and colony PCR detection of cDNA clones"

Fig. 3

Verification of CPsV RNA1 full-length cDNA clones by enzyme digestion and PCR"

Fig. 4

RT-PCR detection of tobacco inoculated with CPsV-121, CPsV-122, and CPsV-221"

Fig. 5

Phylogenetic tree of CPsV-122 and other known CPsV isolates"

Fig. 6

RT-PCR detection of G. globosa inoculated with CPsV-122 at 7 dpi"

Fig. 7

Symptoms of G. globosa inoculated with CPsV-122 at 25 dpi"

Fig. 8

Symptoms of C. limon inoculated with CPsV-122"

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