Cacti are succulent plants within the family
Cactaceae and are often used as ornamental plants. Cactus plants can be infected by various viruses, such as cactus mild mottle virus (CMMoV), detected from
Gymnocalycium mihanovichii in South Korea; cactus virus 2 (CV2), detected from
Mammillaria elongata f.
cristata in Ukraine; cactus virus X (CVX), detected from
Zygocactus sp. in Yugoslavia,
Platyopuntia chlorotica in the USA,
Cereus sp.,
Echinocererus sp.,
Lobibia sp.,
Nopalea cochenillifera,
Mamillaria sp.,
Opuntia sp.,
Pereskia sp., and
Zygocactus truncatum in Brazil, and
Hylocereus spp. in South Korea, China, and Taiwan; opuntia virus X (OpVX), detected from
Opuntia sp. in the USA; rattail cactus necrosis-associated virus (RCNaV), detected from
Aporcactus flagelliformis in South Korea; sammon’s Opuntia virus (SOV), detected from
Opuntia sp. in the USA; saguaro cactus virus (SCV), detected from
Carnegiea gigantea in the USA and
Hylocereus trigonus in South Korea; schlumbergera virus X (SchVX), detected from
Schlumbergera bridgesii in Yugoslavia; tomato spotted wilt virus (TSWV), detected from
Opuntia sp. and
Schlumbergera trancata in the USA; and zygocactus virus X (ZyVX), detected from
Zygocactus sp. in Yugoslavia (
Aragao et al., 1993;
Hausbeck et al., 1992;
Kim et al., 2012;
Koenig et al., 2004;
Lim et al., 2016;
Liou et al., 2001;
Maliarenko and Mudrak, 2013;
Milbrath and Melson et al., 1972;
Milbrath et al., 1973;
Min et al., 2006;
Suaste-Dzul et al., 2012). Pitaya virus X (PiVX), isolated from
Hylocereus sp. (pitaya) in Taiwan, was recently reported in the NCBI nucleotide database. The viruses mentioned above can be divided into five genera:
Tobamovirus, including CMMoV, RCNaV, and SOV;
Carlavirus including CV2;
Potexvirus, including CVX, OpVX, SchVX, PiVX, and ZyVX;
Carmovirus, including SCV; and
Tospovirus, including TSWV.
Co-infections of some cactus-infecting viruses were reported in
Platyopuntia chlorotica,
Mammillaria elonga f.
cristata, and
Opuntia basillaris cacti and the viruses exhibited particles corresponding to tobamovirus (SOV)-like and potexvirus-like structures (CVX or ZyVX) under electron microscopy (
Chessin and Lesemann, 1972;
Maliareko and Mudrak, 2013;
Milbrath et al., 1973).
Based on these findings, we collected 84 cacti samples from cactus farms and fields in four provinces (Gyeonggi, Gangwon, Chungcheong and Jeju) in South Korea and investigated the co-infection of cactus plants with various viruses by electron microscopy and RT-PCR analysis from previously unpublished studies. In the investigated cactus plants, only
Notocactus leninghausii f.
cristatus, obtained from a cactus farm in Gyeonggi province, was co-infected with various viruses. In this study, we identified the viral co-infections in
N. leninghausii f.
cristatus. The
N. leninghausii f.
cristatus did not exhibit the typical virus symptoms of mosaic and mottle, chlorotic spots, necrosis, stunting, or distortion. Electron microscopy analysis was performed using a method modified from that described by
Min et al. (2006) at NISEM (Seoul National University,
http://www.nicem.snu.ac.kr/). Here, crude cactus sap was used for analysis rather than purified virus as described in the original method. In the sap of asymptomatic
N. leninghausii f.
cristatus (
Fig. 1A), we were able to observe virus particles, such as rod-shaped and filamentous virions (
Fig. 1B) typical of tobamovirus (320 nm in length and 18 nm in width) and potexvirus (580 nm in length and 13 nm in width), respectively, as previously reported (
Maliareko and Mudrak, 2013;
Milbrath et al., 1973;
Min et al., 2006).
To confirm co-infection with tobamovirus and potexvirus in
N. leninghausii f.
cristatus, specific primers for detecting the gene encoding the coat proteins (CPs) of two tobamoviruses (CMMoV and RCNaV) and five potexviruses (CVX, OpVX, PiVX, SchVX and ZyVX) were designed using the NCBI nucleotide database (NC_011803, NC_002815, LC128411, JF937699, NC_006060, NC_024458, NC_016442, KU854932, NC_011659, KU854929, KP090203, NC_006059, JF930326) with Lasergene software (DNA Star Inc., Wl, USA) (
Table 1). Total RNA of the cactus plants was used for RT-PCR analysis. Total RNA was purified using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, followed by a reverse transcription (RT) reaction using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Pittsburgh, PA, USA). PCR products were amplified using a thermocycler (MyCycler, Bio-Rad, Hercules, CA, USA) with the following program: initial denaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 30 s, annealing at 55-58°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 10 min. The annealing temperatures used were determined according to
Table 1.
Following RT-PCR analysis, two tobamoviruses (CMMoV and RCNaV) and four potexviruses (CVX, PiVX, SchVX, and ZyVX) were detected in the
N. leninghausii f.
cristatus sample; OpVX was not detected (
Fig. 1C). RT-PCR products for detection of CMMoV and RCNaV were observed at the expected target sizes of 486 and 513 bp, respectively, as were those for detection of CVX (678 bp), PiVX (678 bp), SchVX (678 bp), and ZyVX (681 bp) (
Table 1,
Fig. 1C). Additionally, roughly spherical cactus-infecting viruses, such as TSWV and SCV, were not observed via electron microscopy analysis and were not detected via RT-PCR using specific TSWV CP and SCV CP primers (data not shown).
To confirm the identification of the detected viruses in the N. leninghausii f. cristatus, DNA products obtained by RT-PCR were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) and sequenced by Bioneer Inc (Daejeon, Korea). Sequences were compared with previously reported nucleotide sequences in the NCBI BLAST database. Nucleotide and amino acid sequences were analyzed using DNAMAN software version 5.1 (Lynnon Biosoft, San Ramon, CA, USA). The viral sequences obtained in this study were given the strain name Nl derived from N. leninghausii and were classified into tobamoviruses and potexviruses.
The tobamoviruses CMMoV and RCNaV were characterized as follows. The CP open reading frame (ORF) of CMMoV-Nl (GenBank accession KY581585) shared 99.8% nucleotide (nt) sequence identity and 99.4% amino acid (aa) identity with CMMoV-Kr (GenBank accession NC_011803). This CMMoV was isolated from
Gymnocalycium mihanovichii in South Korea (
Min et al., 2009). The CP ORF of RCNaV-Nl (GenBank accession KY581586) shared 99.4% and 93.1% nt sequence identity and 99.4% and 97.6% aa identity with RCNaV (GenBank accession NC_016442) isolated from
Aporcactus flagelliformis in South Korea and RCNaV-nopal verdure-2 (GenBank accession KU854932) isolated from
Opuntia ficus-indica in Mexico, respectively. As shown in
Fig. 2A-2B, CMMoV-Nl and RCNaV-Nl are closely related to CMMoV and RCNaV based on a phylogenetic tree of tobamovirus CPs. The CPs of CMMoV-Nl and RCNaV-Nl obtained here shared 60.0% nt identity and 53.5% aa identity.
The CP ORFs of the potexviruses CVX-Nl, PiVX-Nl, SchVX-Nl, and ZyVX-Nl were similarly analyzed. CVX-Nl (GenBank accession KY581587) shared 77.5%, 77.1%, and 74.1% nt sequence identity and 88.9%, 87.1%, and 88.4% aa identity with CVX-Jeju (GenBank accession LC12841) isolated from South Korea and CVX-TW (NC_002815) and CVX-NTU (JF937699) isolated from Taiwan, respectively. The CP of CVX-Jeju was itself 97.0% and 76.3% identical at the nt level and 94.6% and 91.6% identical at the aa level to CVX-TW and CVX-NTU, respectively. The three CVX strains TW, NTU, and Jeju clustered together, forming a subgroup. The previously reported CVX strains were isolated from
Hylocereus undatus (
Kim et al., 2016;
Liou et al., 2004). PiVX-Nl (GenBank accession KY581589) shared 98.4% nt sequence identity and 97.8% aa identity with PiVX-P37 (GenBank accession NC_024458) isolated from
Hylocereus sp. in Taiwan. SchVX-Nl (GenBank accession KY581588) shared 95.7% and 89.1% nt sequence identity and 96.9% aa identity with SchVX-K11 (GenBank accession NC_011659) isolated from
Schlumbergera bridgesii in Yugoslavia and SchVX-nopal verdure 1 (KU854929) isolated from
Opuntia ficus-indica in Mexico, respectively, and it shared 95.4% nt identity and 99.6% aa identity with SchVX-Palma-PE (GenBank KP090203) isolated from
Opuntia cochemillifera in Brazil. ZyVX-Nl (GenBank accession KY581590) shared 97.9% and 95.7% nt sequence identity and 98.2% and 96.9% aa identity with ZyVX-B1 (GenBank accession NC_006059) isolated from
Zygocactus sp. in Yugoslavia and ZyVX-P39 (JF930326) isolated from
Hylocereus sp.in Taiwan, respectively. The nucleotide sequences of the ZyVX-B1 and P39 CPs were themselves 96.3% identical at the nt level and 96.9% identical at the aa level. As shown in
Fig. 2C-D, the four potexviruses detected in this study, CVX-Nl, PiVX-Nl, SchVX-Nl, and ZyVX-Nl, each clustered in different subgroups with their respective previously reported CVX, PiVX, SchVX and ZyVX strains.
Most cactus-infecting viruses, such as CVX, OpVX, SOV, SCV, and TSWV, have been reported in the USA, which is one of the countries that exports cacti into South Korea. CMMoV, CVX, and RCNaV were previously reported in
Gymnocalycium mihanovichii,
Hylocereus spp., and
Aporcactus flagelliformis, respectively, in South Korea (
Kim et al., 2012;
Min et al., 2006). Grafting is generally used to reproduce and propagate cactus plants, but viruses can be transmitted easily through this method (
Bausher, 2013;
Estrada-Luna et al., 2002;
de Medeiros et al., 2006;
Maliareko and Mudrak, 2013;
Min et al., 2006). The
N. leninghausii f.
cristatus collected from the cactus farms in this study was also produced using the grafting method. Consequently, co-infection with various viruses (six species) in
N. leninghausii f.
cristatus may be related to grafting. This study demonstrates that
N. leninghausii f.
cristatus can be co-infected with at least six different viruses (CMMoV, CVX, RCNaV, PiVX, SchVX, and ZyVX).