Passionfruit plantation in Vietnam increased to 10,000 ha in 2019. However, the outbreaks of passionfruit woodiness disease (PWD) have become a serious threat for the production. In this study, five virus isolates DN1, DN4, NA1, GL1 and GL2 were collected from different areas of Vietnam. Their causal roles for PWD were verified by back inoculation to passionfruit. Analyses of coat protein (CP) and genomic sequences revealed that GL1 isolate is closely related to East Asia Passiflora virus (EAPV) AO strain of Japan (polyprotein nt/aa identities of 98.1% / 98.2%), while GL2 isolate is related to Telosma mosaic virus (TelMV) isolate PasFru, China (polyprotein nt/aa identities of 87.1% / 90.9%). CP comparison, host range and cytological characterization indicated that DN1, DN4 and NA1 are potyviruses, but different from EAPV and TelMV. Phylogenic analyses of their CP and genome sequences indicated that these three isolates and passionfruit severe mottle-associated virus Fujian isolate of China belong to a distinct clade, which does not satisfy the threshold (76% nt identity of polyprotein) to be regarded as any of potyviral species. Thus, a new species name of “Passiflora mottle virus” has been proposed by ICTV. A rabbit antiserum was produced against the CP of DN1 and it can discriminate Passiflora mottle virus (PaMoV) from TelMV and EAPV in western blotting and ELISA without cross reactions. Field surveys of 240 samples by ELISA and RT-PCR disclosed that PWD in Vietnam is mainly caused by PaMoV; followed by EAPV, mixed-infection of PaMoV/EAPV, and rare cases of TelMV.

East Asian Passiflora virus (EAPV) seriously affects passionfruit production in Taiwan and Vietnam. In this study, an infectious clone of EAPV Taiwan strain (EAPV-TW) was constructed, and EAPV-TWnss with an nss-tag attached to its helper component-protease (HC-Pro) was generated for monitoring the virus. Four conserved motifs of EAPV-TW HC-Pro were manipulated to create single mutations of F8I (simplified as I8), R181I (I181), F206L (L206), and E397N (N397); and double mutations of I8I181, I8L206, I8N397, I181L206, I181N397, and L206N397. Four mutants EAPV-I8I181, I8N397, I181L206, and I181N397 infected Nicotiana benthamiana and yellow passionfruit plants without conspicuous symptoms. Mutants EAPV-I181N397 and I8N397 were stable after six passages in yellow passionfruit plants and expressed a zigzag pattern of accumulation dynamic, typical of beneficial protective viruses. Agroinfiltration assay indicated that the RNA-silencing- suppression capabilities of the four double mutated HC-Pros are significantly reduced. Mutant EAPV-I181N397 accumulated the highest level of siRNA at ten days post-inoculation (dpi) in N. benthamiana plants, then dropped to background levels after 15 dpi. In both N. benthamiana and yellow passionfruit plants, EAPV-I181N397 conferred complete cross protection (100%) against severe EAPV-TWnss, as defined by no severe symptoms and absence of the challenge virus checked by western blotting and RT-PCR. Mutant EAPV-I8N397 provided high degrees of complete protection against EAPV-TWnss in yellow passionfruit plants (90%) but not in N. benthamiana plants (0%). Both mutants showed complete protection (100%) against Vietnam’s severe strain EAPV-GL1 in passionfruit plants. Thus, the mutants EAPV-I181N397 and I8N397 have excellent potential for controlling EAPV in Taiwan and Vietnam.

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