Lisianthus enation leaf curl virus, a newly invaded begomovirus into Japan, is more virulent than the prevalent tomato yellow leaf curl virus in Ty-gene-mediated resistant tomato cultivars

Abstract

In 2020, tomato plants showing yellow leaf curl disease (TYLCD) were tested for begomovirus infection in Okinawa prefecture, Japan. Amplification and sequence analysis of circular DNA in the diseased samples showed that, some of the tomato plants carrying a Ty-3a resistance gene to tomato yellow leaf curl virus (TYLCV) were co-infected with TYLCV and lisianthus enation leaf curl virus (LELCV). LELCV infection was also detected in pepper plants in Okinawa by PCR-based diagnosis, suggesting that the virus is widespread in the region. To characterize the interactions of LELCV, TYLCV-IL and TYLCV-Mld with tomato cultivars that carry different Ty resistance genes, we conducted agroinoculation of the viruses. The resistance conferred by Ty-2 was effective against TYLCV-IL but not effective against TYLCV-Mld and LELCV. In contrast, Ty-3a conferred resistance was effective against TYLCV strains but not fully effective against LELCV. Furthermore, mixed infection of TYLCV-IL and LELCV in tomato plants bearing Ty-3a induced even more significant TYLCD symptoms and growth inhibition than did LELCV singly infected plants. Our study demonstrated that the newly invaded LELCV is a more virulent begomovirus species than the prevalent TYLCV isolates in resistant tomato cultivars.

Introduction

Begomovirus, one of the seven genera of the family Geminiviridae, has the largest diversity of 445 species (ICTV 2021). The ssDNA genomes of begomovirus are encapsulated in twinned-icosahedral particles, and this virus is transmitted from plant to plant by an insect vector whitefly (Bemisia tabaci). Tomato yellow leaf curl disease (TYLCD), caused by some begomoviral species, is one of the most devastating viral diseases affecting tomato (Solanum lycopersicum L.) plants worldwide (Moriones and Navas-Castillo 2000). Tomato yellow leaf curl virus (TYLCV) is a monopartite begomovirus that infects and causes TYLCD. The global dissemination of Israel (IL) and mild (Mld) strains started in the 1980s (Mabvakure et al. 2016). TYLCV infects tomato plants and at least 49 different species belonging to 16 families (Papayiannis et al. 2011).

TYLCD symptoms comprise more or less prominent upward curling of leaf margins, a reduction in leaflet area, yellowing of young leaves, stunting, and flower abortion. TYLCV infections induce a general decrease in plant growth and reduced yields, and production is almost entirely lost if susceptible plants are infected during early growth. Since genetic resistance against TYLCV in tomato is one of the effective ways to control TYLCD, resistance loci Ty-1 to Ty-6 have been identified from tomato wild relatives (Agrama and Scott 2006; Anbinder et al. 2009; Hutton and Scott 2014; Ji et al. 2009a, b; Zamir et al. 1994). The introgression of allelic loci Ty-1/Ty-3/Ty-3a or another locus Ty-2 into commercial tomato cultivars has been the major focus of resistance breeding globally, including in Japan (Saito et al. 2008; Yan et al. 2018).

Japan is an island country with a long archipelago, and its climate ranges from subtropical in the south to subarctic in the north. The first report of TYLCD caused by TYLCV was from Shizuoka and Aichi prefectures in the Tokai district in central Japan and Nagasaki Prefecture in the Kyushu district in southwestern Japan in 1996 (Kato et al. 1998). Further studies isolated additional full-length genome sequences of TYLCV-Mld strain from Shizuoka, Aichi, and Mie prefectures in the Tokai district and Osaka Prefecture in the Kansai district, whereas sequences of TYLCV-IL strain were isolated from Nagasaki and Miyazaki prefectures of the Kyushu district and Kochi Prefecture of the Shikoku district, indicating that TYLCV-IL isolates are disseminated mainly in Western Japan, and TYLCV-Mld isolates are disseminated in Central and Eastern Japan (Koeda et al. 2015; Onuki et al 2004; Ueda et al. 2004, 2005). Okinawa Prefecture, which is located at the southwestern tip of the Japanese archipelago, constitutes a chain of 160 islands surrounded by Kuroshio warm current and has a subtropical climate that differs from the temperate climate of the Japanese main islands. A study conducted in Okinawa Island identified TYLCV-IL isolates that genetically resemble the isolates from Kochi Prefecture (Ueda et al. 2009). Moreover, the isolates of monopartite begomovirus ageratum yellow vein virus (AYVV) were identified from ageratum, tomato, and common bean plants in Ishigaki Island, which is only 230 km from Taiwan (Andou et al. 2010; Tomitaka et al. 2020). Since various tomato-infecting begomoviral species have been reported in Southeast Asia (Kenyon et al. 2014), Okinawa Prefecture has the highest risk of invasion by begomovirus species from Southeast Asia into Japan, considering its geographic location.

In the present study, we observed typical yellowing symptoms on TYLCV-resistant tomato plants possessing Ty-3a in Okinawa Prefecture and isolated newly invaded monopartite begomovirus lisianthus elation leaf curl virus (LELCV). LELCV was first isolated from diseased lisianthus (Eustoma grandiflorum L.) from central Taiwan in 2015 (Accession No. LC091538 and LC091539). More recently, the full-length viral sequences of LELCV were isolated from tomato and pumpkin plants from Taiwan in 2016 and 2019. Although multiple sequences of LELCV have been reposited to GenBank, fundamental information such as LELCV caused disease symptoms, and biological characterization has not been yet reported. In the present study, we agroinoculated LELCV along with TYLCV-IL and TYLCV-Mld to tomato plants possessing Ty-2 or Ty-3a. This is the first study to characterize LELCV and evaluate the effectiveness of Ty resistance genes.

Materials And Methods

Field sampling, PCR diagnosis, and isolation of full-length viral sequences

Naturally infected tomato (S. lycopersicum), pepper (Capsicum annuum L., Capsicum frutescens L., and Capsicum baccatum L.), eggplant (Solanum melongena L.), and common bean (Phaseolus vulgaris L.) plants with virus-like symptoms, such as typical leaf yellowing caused by begomovirus, were collected at Ishigaki, Tarama, Miyako, and Okinawa islands on March 2020 (Fig. 1a). DNA was extracted from the collected samples using the Nucleon PhytoPure kit (Cytiva, MA, USA), and TYLCV-IL and TYLCV-Mld were initially detected by conventional PCR using TYLCV IL Mld uni F and R primers. PCR reactions were conducted using EmeraldAmp PCR Master Mix (Takara Bio, Shiga, Japan), and the mixtures were initially denatured at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, terminating with 3 min of extension at 72°C. The amplified PCR products were subjected to electrophoresis using a 1.0 % (w/v) agarose gel. The presence of Ty-2 and Ty-3/Ty-3a resistance genes in the collected tomato plants was confirmed using molecular markers following the method reported by Koeda et al. (2020).

The full-length genome sequences of begomoviruses infecting susceptible and resistant tomato plants were amplified using a rolling-circle amplification (RCA)-based TempliPhi DNA Amplification Kit (Cytiva), according to Koeda et al. (2015). Amplified products were cut into monomers using the restriction enzyme and cloned to sequence. Phylogenetic analysis of the obtained sequences was conducted following Koeda et al. (2021).

From the isolated sequences in this study and sequences obtained from GenBank, specific primers were designed for each begomovirus species and strain: LELCV F and R primers, TYLCV-IL F and R primers, and TYLCV-Mld F and R primers. PCR reactions were conducted using KOD Plus neo (Toyobo, Osaka, Japan), and the mixtures were initially denatured at 94°C for 2 min, followed by 35 cycles at 98°C for 10 s, 64°C for 30 s, and 68°C for 1 min, terminating with 3 min of extension at 68°C. The amplified PCR products were subjected to electrophoresis using a 1.0 % (w/v) agarose gel. The PCR amplicons of samples positive for LELCV were used for direct sequencing after treating with ExoSAP-IT Express (Thermofisher, MA, USA). Supplementary Table S1 shows the primer sequences used for viral DNA detection.

Construction of infectious clones for the isolates LELCV-[JR:Tom:T326], TYLCV-IL-[Jr:Ito:T278], and TYLCV-Mld-[JR:Tak]

Infectious clones for LELCV-[JR:Tom:T326] (LC642632), TYLCV-IL-[Jr:Ito:T278] (LC642631), and TYLCV-Mld-[JR:Tak] (AB921568) were obtained following the method reported by Koeda et al. (2017). Supplementary Table S1 shows the primer sequences. The obtained constructs were named pGreenII-p35S-LELCV, pGreenII-p35S-TYLCV-IL, and pGreenII-p35S-TYLCV-Mld.

Inoculation of tomato plants with begomoviruses

The tomato cultivars Momotaro (M) (susceptible to begomovirus), Momotaro Sakura (MS) (Ty-2/ty-2), and Momotaro Peace (MP) (Ty-3a/ty-3) (Takii Seed, Kyoto, Japan) were utilized for agroinoculation. Momotaro is one of the most popular fresh market tomato cultivars in Japan, and MS and MP share a Momotaro genetic background (Koeda et al. 2020). All plants were kept in a growth room at a temperature range of 23°C–30°C with 13 h photoperiods.

Single inoculation experiments with LELCV, TYLCV-IL, or TYLCV-Mld were conducted using Rhizobium radiobacter (EHA105) transformed with pGreenII-p35S-LELCV, pGreenII-p35S-TYLCV-IL, or pGreenII-p35S-TYLCV-Mld using a colony inoculation procedure (Koeda et al. 2017). For mixed inoculations, the first inoculation contained TYCLV-IL, and LELCV was later introduced approximately seven days after the second inoculation. The disease severity index score for evaluating the symptoms ranged from 0 to 4 as follows: 0, no symptoms; 1, very mild symptoms with slight yellowing; 2, yellowing; 3, small leaves with yellowing; 4, small and distorted leaves with yellowing. Plant height (cm) was measured as the distance from the cotyledonary node to the shoot tip. Statistical analysis was performed using the Bonferroni–Dunn test of Excel Toukei ver. 7.0, and a p value < 0.05 was considered statistically significant.

Quantification of virus titer by real-time PCR

The DNAs of LELCV, TYLCV-IL, and TYLCV-Mld were quantified by qPCR according to Koeda et al. (2021). The LELCV SP Real F and R primers, TYLCV-IL SP Real F and TYLCV-IL-Mld Real R primers, and TYLCV-Mld SP Real F and TYLCV-IL-Mld Real R primers listed in Supplementary Table S1 were used to detect LELCV, TYLCV-IL, and TYLCV-Mld, respectively. The 25S ribosomal RNA gene of tomato genome DNA was amplified using 25S-rRNA 2F and 2R (Koeda et al. 2020) for data normalization. Statistical analysis was performed using the Tukey–Kramer test of Excel Toukei ver. 7.0, and a p value < 0.05 was considered statistically significant.

Results

Discussion

TYLCD caused by begomoviruses is one of the most devastating viral diseases affecting tomato plants worldwide (Moriones and Navas-Castillo 2000). The first evidence of TYLCD caused by TYLCV in Japan was reported in 1996, and the virus has disseminated widely throughout the country after several independent invasions from abroad (Kato et al. 1998; Koeda et al. 2015; Onuki et al. 2000; Ueda et al. 2004, 2005). Considerable efforts have been made to introgress genetic resistance from wild relatives to cultivate tomato cultivars, which induced successful control of TYLCD in Japan. However, the invasion of a new begomovirus species has been a continuous threat. In this study, we report on the isolation of LELCV for the first time in Okinawa Prefecture, Japan.

Because Okinawa Prefecture is located at the southwestern tip of the Japanese archipelago, which is close to Taiwan and Southeast Asia, the infection of distinct begomovirus species from TYLCV has been reported. A monopartite begomovirus AYVV is found in Southeast and East Asia, mainly in tropical and subtropical regions. In Ishigaki Island, AYVV, which was most probably invaded from Taiwan, causes diseases in tomato, common bean, and ageratum plants (Ageratum conyzoides L.) (Andou et al. 2010; Tomitaka et al. 2020). We also frequently observed symptomatic ageratum plants around the greenhouse in Ishigaki Island during the field survey conducted in this study. Shahid et al. (2014) isolated AYVV from tomato plants cultivated in Tokyo, which has a high genetic similarity with the AYVV isolate from Ishigaki Island. However, since their isolation in 2011, no study has reported AYVV infection in the main island of Japan, suggesting that this virus is not widespread.

Twenty one LELCV isolates from Taiwan are identified and reposited to GenBank. LELCV-[JR:Tom:T326] isolated in Okinawa showed the highest 98.9% similarity with LELCV-[BG-9] (LC091539) recovered from lisianthus (E. grandiflorum) and clustered with isolates infecting to tomato and lisianthus plants in the phylogenetic tree (Fig. 2a). Ueda et al. (2009) isolated full- and partial-length TYLCV-IL sequences from tomato plants cultivated at several sites of Okinawa Island in 2007. A single full-length and six partial sequences of approximately 1400 bp were isolated from Tomigusuku, and additional eight partial sequences from Uruma, Yomitan-son, and Nakijin-son. All these sequences clustered together with TYLCV-IL, especially with the isolate from the Kochi Prefecture in their phylogenetic analysis. Although we could not check the sequence similarity of these un-reposited 14 partial TYLCV-IL sequences with the LELCV sequence obtained from Tomigusuku in 2020, their phylogenetic analysis strongly suggested that LELCV did not infect tomato plants in 2007. The partial genome DNA of LELCVs infecting to 22 samples collected from Okinawa Prefecture showed high similarity (99.6–100%) to LELCV-[JR:Tom:T326] indicates that genetic diversity of LELCV in Okinawa is low compared to Taiwan (Fig. 2c). Based on the results of pairwise comparisons and phylogenetic analyses, and considering that Okinawa is situated close to Taiwan, LELCV probably invaded Okinawa between 2007 and 2020 through the transport of lisianthus, tomato, other plants infected with LELCV, or viruliferous whitefly.

In this study, LELCV was detected in tomato and pepper plants from Ishigaki and Okinawa islands, and TYLCV-IL was detected in tomato, pepper, eggplant, and common bean plants from Ishigaki, Tarama, Miyako, and Okinawa islands (Fig. 1a; Table 1). TYLCV can infect pepper and common bean plants (Kashina et al. 2003; Navas-Castillo et al. 1999; Quiñones et al. 2002; Roye et al. 1999; Salati et al. 2002). Morilla et al. (2005) reported symptomless infection of pepper plants by TYLCV-Mld and IL from Spain, and whitefly transmission occurs at a low frequency from tomato to pepper plants, but not from pepper to tomato plants. Moreover, TYLCV-IL infected C. annuum, C. chinense, and C. frutescens by whitefly, but TYLCV-Mld was unable (Polston et al. 2006). In Okinawa, C. frutescens plants are perennial semidomesticated crops mainly grown in home gardens and small fields and are used for seasoning foods in daily diets (Yamamoto and Nawata 2006). Since LELCV and TYLCV-IL were detected in C. frutescens plants (Table 1), these plants may act as reservoirs. Further study is needed to clarify the infectivity and virulence of LELCV to pepper plants and virus movements between pepper and tomato plants by whitefly. In addition, using the primer sets developed in this study to check LELCV presence in large tomato-producing areas on the main Japanese islands will be beneficial.

LELCV sequences were isolated from Taiwan in 2015, 2016, and 2019, but the biological characterization of this virus has not yet been reported. To understand the possible effects of LELCV on tomato production, we evaluated the effectiveness of widely used Ty-3a and Ty-2 resistance genes against TYLCV-IL, Mld, and LELCV (Fig. 3, 4; Table 2, 3). Our result corresponds to previous studies showing that Ty-2 is fully effective against TYLCV-IL but not effective against TYLCV-Mld and other monopartite and bipartite begomoviruses (Barbieri et al. 2010; Hanson et al. 2000; Ohnishi et al. 2016; Prasanna et al. 2015; Shahid et al. 2013; Tsai et al. 2011; Yamguchi et al. 2018). The Ty-3a resistance gene was effective against both the IL and Mld strains of TYLCV, but a slight symptom of yellowing was observed occasionally when infected with an IL strain. In contrast, Ty-3a-conferred resistance was not fully effective against LELCV, and MP plants showed mild symptoms. Moreover, symptoms and growth inhibition became more prominent when LELCV was mixed infected with TYLCV-IL (Fig. 4a, b). Allelic Ty-1, Ty-3, and Ty-3a encode an RNA-dependent RNA polymerase, and Ty-1 confers resistance to TYLCV by increasing cytosine methylation of viral genomes, suggesting enhanced transcriptional gene silencing (TGS) (Butterbach et al. 2014; Verlaan et al. 2013). However, begomoviruses are able to suppress the host antiviral silencing response (Vanitharani et al. 2005). Since single-infection of LELCV to MP induced mild symptoms and growth inhibition became more prominent when mixed infected (Fig. 4a and b), there is a possibility that LELCV has a suppressor protein against TGS conferred by Ty-3a. However, heavier symptom expression could not be explained solely by the amount of viral DNA accumulation, because there were no significant differences in the accumulating viral DNAs of LELCV and TYLCV-IL between singly and mixed-infection (Fig. 4c). Similar observation is reported by Shafiq et al. (2017). Although a positive correlation between disease severity of cotton (Gossypium arboretum L.) plants and begomovirus DNA accumulation was observed, some plants only showed very mild or no symptoms despite relatively high virus titers, whereas other plants showed severe symptoms but had relatively low virus titers. Further functional characterization of open reading frames of LELCV is needed to clarify this point. Some of the begomovirus species from Southeast Asia are more virulent than TYLCV, and Ty gene-conferred resistance is also not fully effective against these viruses (Koeda et al. 2020). Therefore, from the practical point of view, evaluating and preparing pyramided commercial cultivars with multiple Ty resistance genes are imperative for the Japanese tomato market.

In this study, we isolated LELCV from symptomatic Ty-3a-bearing resistant tomato plants in Okinawa for the first time in Japan and characterized its virulence in comparison with the prevalent TYLCV-IL and -Mld isolates. The invasion of multiple begomovirus species into tomato cultivation areas increases the chance of recombination, which is one of the driving forces in their evolution, extensive host range, and increasing virulence (Lefeuvre and Moriones 2015). Moreover, several studies have inferred that the use of Ty-gene-harboring tomato cultivars may serve as a positive selection of specific begomovirus species or recombinants (Belabess et al. 2015; García-Andrés et al. 2009). Further field study using a larger number of samples collected from multiple regions in Japan is required.

Declarations

Acknowledgements

The authors would like to thank Kohei Teramura, Kozo Gima, Shuto Taba (Okinawa Prefectural Plant Protection Center), and Haruki Sunagawa (Okinawa Churashima Foundation Research Center) for supporting our field study at Okinawa. We thank Takii Seeds (Japan) for suppling M, MS, and MP tomato seeds as research materials. Map of Okinawa region for Fig. 1a was kindly provided from Dr. Nobuhiko Komaki (Aichi University). This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 19H02950 and 21KK0109 to S. Koeda.

Competing interests:

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Tables

Tables 1 to 3 are available in the Supplementary Files section.