Isolation of newly invaded monopartite begomovirus LELCV from Okinawa
In 2020, we collected tomato leaves with virus-like symptoms, including typical yellowing caused by begomovirus, in Okinawa Prefecture. TYLCV detection from 18 out of 28 samples by initial PCR-based diagnosis using the TYLCV IL Mld uni F and R primers showed that TYLCV is widely disseminated in Okinawa Prefecture (Table 1). When we genotyped these symptomatic tomato plants for Ty-2 and Ty-3/Ty-3a resistance loci using the molecular markers, 10 tomato plants possessed the Ty-3a gene in a heterozygous state (Table 1).
A Ty-3a-bearing resistant tomato plant cultivated in Tomigusuku City in Okinawa Island showed TYLCD symptoms and was positive for TYLCV infection (Fig. 1b). Sequencing the RCA amplified begomoviral full-length sequence and pairwise comparisons clarified that the obtained sequence showed 98.9% similarity with LELCV-[BG9] (LC091539) infecting lisianthus (E. grandiflorum L.) in Taiwan. The phylogenetic analysis revealed that LELCV sequences separated into three clusters: isolates identified from tomato and pumpkin plants (Cluster 1), isolates identified from tomato and lisianthus plants (Cluster 2), and isolates identified from tomato plants clustering with tomato yellow leaf curl Thailand virus (Cluster 3). LELCV-[JR:Tom:T326] (LC642632) isolated from Okinawa clustered with isolates from Taiwan in the Cluster 2 (Fig. 2a). We also collected leaves from a susceptible tomato plant at Itoman City in Okinawa Island (Fig. 1c) and isolated TYLCV-IL-[JR:Ito:T278] (LC642631), which showed high similarity to the previously reported isolates from Okinawa and Kochi prefectures (Fig. 2b). TYLCV sequences isolated from Japan were separated into two clusters in the phylogenetic tree (Fig. 2b); the cluster 1 comprised TYLCV-IL isolates identified in the Western region, and the cluster 2 comprised TYLCV-Mld isolates identified in the Eastern region of Japan, including the Kansai district.
Because the previously used primer set could not distinguish LELCV from TYLCV, we developed specific primers for LELCV, TYLCV-IL, and TYLCV-Mld according to the newly obtained sequences from this study and those from the GenBank database. The leaf samples of various horticultural crops, including tomatoes, peppers, eggplants, and common beans, with viral-like symptoms were used for PCR-based diagnosis (Table 1). A total of 171 samples were collected at 32 sites in Ishigaki, Tarama, Miyako, and Okinawa islands (Fig. 1a). LELCV was detected in four tomato plants and 17 pepper plants collected from Ishigaki and Okinawa (Yaese, Itoman, Tomigusuku, and Nakagusuku) islands. TYLCV-IL was detected in tomato, pepper, eggplant, and common bean plants collected from four islands. In contrast, TYLCV-Mld was not detected in any of the collected samples from Okinawa Prefecture. Among 22 samples positive for LELCV infection, 6 samples were mixed infected with TYLCV-IL. Sequencing the PCR amplicons of 22 samples positive for LELCV clarified that 1087 bp partial sequences showed similarity between 99.6 to 100% with LELCV-[JR:Tom:T326] (Fig. 2c). These results revealed that TYLCV-IL and LELCV are the predominant begomoviruses infecting horticultural crops, such as tomato plants, in Okinawa.
LELCV induces symptoms in tomato plants harboring Ty genes
Single inoculations of LELCV, TYLCV-IL, or TYLCV-Mld were conducted on tomato cultivars M (susceptible), MS (Ty-2/ty-2), and MP (Ty-3a/ty-3) (Table 2). For agroinoculation, we used isolates of LELCV-[JR:Tom:T326] (LC642632) and TYLCV-IL-[JR:Ito:T278] (LC642631) identified in this study, and TYLCV-Mld-[JR:Tak] (AB921568) identified from Osaka Prefecture in our previous study (Koeda et al. 2015). At 20-day post-inoculation (dpi), M plants infected with LELCV, TYLCV-IL, or TYLCV-Mld showed typical TYLCD symptoms of yellowing, curling, and stunting. MS plants infected with LELCV or TYLCV-Mld showed similar symptoms to M plants. In contrast, the infectivity rate of TYLCV-IL was significantly low at 12–30%, and MS-infected plants showed no symptoms. All inoculated MP plants were infected with TYLCV-IL and TYLCV-Mld, and some of the TYLCV-IL-infected plants showed very slight yellowing symptoms, but the TYLCV-Mld-infected plants did not show any viral symptoms. Meanwhile, MP plants infected with LELCV showed mild symptoms of leaf yellowing and curling. Similar results were obtained in an independently conducted experiment. These results indicate that Ty-2-conferred resistance is not effective, and Ty-3a-conferred resistance is not fully effective against LELCV.
To assess the correlation between symptom expression and the accumulation of viral DNA, we conducted relative quantification of begomoviral DNA through qPCR using DNA extracted from upper young leaves collected at 20 dpi from agroinoculated plants (Fig. 3). Significantly lower TYLCV-IL viral DNAs were accumulated in MS (1/14) and MP (1/21) compared to M plants. In addition, TYLCV-Mld viral DNA accumulation was significantly lower in MP (1/24) than in M and MS plants. Moreover, LELCV viral DNA accumulation was also significantly lower in MP (1/6) than in M and MS plants. Although LELCV viral DNA accumulation was lower in MP plants than in M plants (1/6), it was less restricted compared to TYLCV-IL (1/21) and TYLCV-Mld (1/24). Similar results were obtained in an independently conducted experiment. Overall, these results revealed that the repression of LELCV viral DNA accumulation conferred by Ty-3a in the heterozygous state is insufficient for MP to confer complete resistance.
Mixed infection of LELCV and TYLCV-IL inhibits the growth of Ty-3a-bearing resistant tomato plants
Single and mixed inoculations of LELCV and TYLCV-IL were conducted on tomato cultivars M (susceptible), MS (Ty-2/ty-2), and MP (Ty-3a/ty-3) (Fig. 4a; Table 3). Single-infected M and MS plants at 35 dpi showed similar results to the previous experiments, and mixed infection of TYLCV-IL and LELCV also induced heavy symptoms in M and MS plants. In contrast, MP plants singly infected with TYLCV-IL or LELCV showed slight and mild symptoms, respectively, but the observed symptoms became heavier in the mixed-infected plants than in the single-infected plants of either virus.
To evaluate the effect of single and mixed infection of TYLCV-IL and LELCV on tomato plant growth, we measured plant height at 35 dpi (Fig. 4b). No statistically significant differences were observed between mock-inoculated M, MS, and MP plants (Tukey–Kramer test). However, significant stunting was observed in single and mixed-infected M plants, single LELCV-infected and mixed-infected MS plants compared to mock-inoculated plants. Moreover, single infections of TYLCV-IL or LELCV caused significant stunting in MP plants compared to mock-inoculated plants, and mixed infection of the two viruses caused even heavier stunting. Similar results were obtained in independently conducted experiments. These results showed that a single infection with LELCV inhibited the growth of Ty-3a-bearing resistant tomato plants and mixed infection with TYLCV-IL further accelerated the inhibition effect.
To assess the correlation between symptom expression and accumulation of viral DNA, we conducted relative quantification of begomoviral DNA through qPCR using DNA extracted from upper young leaves collected at 35 dpi from single and mixed-infected plants (Fig. 4c). Regardless of single or mixed infection, TYLCV-IL viral DNA accumulation was significantly lower in MS and MP plants than in M plants. In contrast, LELCV viral DNA accumulation was significantly low in the MP plants. However, no significant difference was observed between LELCV-single-infected and mixed-infected MP plants. Overall, these results revealed that the repression of LELCV viral DNA accumulation conferred by Ty-3a in the heterozygous state is insufficient for MP to confer complete resistance.