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.

Passiflora mottle virus (PaMoV), an aphid-borne potyvirus, is the primary causal virus of the devastating passionfruit woodiness disease in Vietnam. Here we generated a non-pathogenic, attenuated PaMoV strain for disease control by cross protection. A full-length genomic cDNA of PaMoV strain DN4 from Vietnam was constructed to generate an infectious clone. The green fluorescent protein was tagged at the N-terminal region of the coat protein gene to monitor in planta the severe PaMoV-DN4. Two amino acids within the conserved motifs of HC-Pro of PaMoV-DN4 were mutated individually or in combination as K53E or/and R181I. Mutants PaMoV-E53 and PaMoV-I181 induced local lesions in Chenopodium quinoa plants, while PaMoV-E53I181 caused infection without apparent symptoms. In passionfruit plants, PaMoV-E53 elicited severe leaf mosaic, PaMoV-I181 induced leaf mottling, while PaMoV-E53I181 caused transient mottling followed by symptomless recovery. PaMoV- E53I181 was stable after six serial passages in yellow passionfruit plants. Its temporal accumulation levels were lower than those of the wild type, with a zigzag accumulation pattern, typical of a beneficial protective virus. An RNA silencing suppression (RSS) assay revealed that all three mutated HC-Pros are defective in RSS. Triplicated cross-protection experiments with a total of 45 plants showed that the attenuated mutant PaMoV-E53I181 provided a high protection rate (91%) against the homologous wild-type virus in passionfruit plants. This work revealed that PaMoV-E53I181 can be used as a protective virus to control PaMoV by cross protection.

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.