Molecular Dissection of Symptom Determinants in Tomato Leaf Curl New Delhi Virus in Zucchini Through Mechanical Transmission.

Among begomovirus species, tomato leaf curl New Delhi virus (ToLCNDV) is significant and stands out as a mechanically transmissible bipartite begomovirus originating from the Old World. However, the mechanisms underlying the mechanical transmission of different ToLCNDV strains remain understudied, as their natural transmission occurs via insect vectors. In this study, we investigated the mechanical transmissibility of two ToLCNDVs, one from Italy and another from Pakistan, in host plants. Several cucurbit species were screened, and symptom differences between the two ToLCNDV clones were observed only in zucchini when subjected to rubbing inoculation. The Italian isolate (ToLCNDV-ES) induced typical disease symptoms such as leaf curling, yellow mosaic, and internode stunting, whereas a normal phenotype was observed in zucchini mechanically infected with ToLCNDV-In (Pakistani isolate). Subsequently, a gene-swapping experiment between the two ToLCNDVs was conducted, and ToLCNDV-ES DNA-B was identified as a crucial factor in mechanical transmission. We then constructed chimeric mutant clones based on the DNA-B sequence and assessed their ability to induce symptoms in zucchini. These results indicated that the nuclear shuttle protein is a determinant of symptom development during ToLCNDV mechanical transmission. Moreover, several defense-related host genes showed significant changes in relative expression in different ToLCNDV clones, indicating their potential role in disease symptom development through the mechanical transmission of ToLCNDV. This is the first report comparing the mechanical transmissibility of two isolates of different ToLCNDV strains from the Mediterranean region and the Indian subcontinent in the same host plant, providing new insights into the virus's pathogenicity across different geographic regions.