Molecular analysis of a Spanish isolate of chili pepper mild mottle virus and evaluation of seed transmission and resistance genes

An isolate of chili pepper mild mottle virus (CPMMV-Sp; GenBank OQ920979) with a 99% identity to CPMMV (GenBank MN164455.1) was found in symptomatic pepper plants in Spain. RACE analysis, performed using a stem-loop primer developed in this study to prime at the end of the introduced poly(A)/(U) tail, revealed the presence of an extra 22 nt at the 5' end, starting with a cytosine, which were essential to generate infectious clones. However, the 5' terminal cytosine was dispensable for initiating the infection. The design of two specific digoxigenin riboprobes targeting the more divergent area of CPMMV-Sp, compared to the closely related bell pepper mottle virus (BPeMV) (identity percentage of 80.6% and 75.8%, respectively), showed that both probes specifically detected CPMMV-Sp when the hybridization was performed at 68ºC and 60ºC, respectively. However, the BPeMV probe, targeting a region with an 89.4% identity percentage to CPMMV-Sp, showed cross-hybridization at 60ºC but not at 68ºC. The comparison of the detection limits between molecular hybridization and RT-PCR techniques revealed that the former was 125 times less sensitive than RT-PCR. The analysis of the vertical transmission of CPMMV-Sp using seeds from naturally or mechanically infected pepper plants revealed a transmission percentage ranging from 0.9% to 8.5%. Finally, the analysis of the resistance of capsicum species carrying different alleles of the L gene (L1, L2, L3, and L4) revealed that varieties with the L1 gene were infected by CPMMV-Sp (20–40% of inoculated plants), while varieties with the L2, L3, and L4 genes were resistant.