Experimental Reproduction of Field Symptoms in ‘Shatangju’ Mandarin ( <i>Citrus reticulata</i> ) Confirms Citrus Yellow Vein Clearing Virus as Causal Agent of Spring Shoot Leaf Curl Disease

Citrus yellow vein clearing virus (CYVCV), the causal agent of citrus yellow vein clearing disease, has been posing an emerging threat to citrus production in China. In this study, we confirm that CYVCV is the causal agent of spring shoot leaf curl disease in 'Shatangju' mandarin (<i>Citrus reticulata</i>), a major commercial citrus cultivar in Guangxi Province. Field surveys revealed a high incidence of typical viral symptoms, including leaf curling and vein clearing, with a CYVCV detection rate of 95.65% in the symptomatic samples. To verify the association between CYVCV and field symptoms, we inoculated 'Shatangju' plants using a previously constructed full-length infectious cDNA clone of CYVCV derived from the CYVCV-FL-16 isolate. Graft-inoculated plants developed symptoms resembling those observed in the field, including vein clearing, leaf rolling, chlorosis, stunting, and internode shortening. RT-PCR and Western blot assays confirmed the viral systemic infection. Chlorophyll content was significantly reduced in infected leaves, and virus accumulation showed tissue- and season-specific distribution. These results fulfill Koch's postulates for CYVCV in 'Shatangju' and provide experimental evidence linking CYVCV infection to field symptoms, laying the foundation for disease management and virus-free seedling certification.