RTSV transmission test using its vector N. virescens was performed as a response to reports of widespread tungro-like symptoms on rice cultivated in Panca Rijang Subdistrict, Sidenreng Rappang (Sidrap) District, Sulawesi Selatan Province and Muntilan Subdistrict, Magelang District, Jawa Tengah Province of Indonesia in 2011 (Fig. 1).

Fig. (1). Rice plants on fields in (A). Magelang, Jawa Tengah, and (B). Sindrap, Sulawesi Selatan showed severe Tungro symptoms: leaf yellowing and stunting.

The test procedures were similar to those of another study [9], with some modifications. Seeds of rice variant TN1 were sown on soil in pots and then grown for 10 days after germination. Each selected healthy seedling was then put in a small glass test tube filled with distilled water up to 1 cm and sealed with cotton. Ten tubes with rice seedlings were then brought to each sampling location. In each location, one small clump of rice plants with clear symptoms of tungro: yellowing and stunting was selected, and then cleared from N. virescens, and then covered with an insect screen. N. virescens collected from the surrounding area were allowed an acquisition period of 5 hours on the selected clump of symptomatic rice plants. After that, two adult N. virescens were taken and immediately brought to each healthy seedling in a sealed glass test tube and allowed an inoculation period of 24 hours. N. virescens were removed from the inoculated seedlings, and each seedling was replanted into a pot (18 cm in diameter). The 10 inoculated and one uninoculated control seedlings treated in Sidrap District were kept in an insect proof greenhouse in Loka Penelitian Penyakit Tungro, Lanrang, Sidrap, Sulawesi Selatan while 10 inoculated and one uninoculated control seedlings treated in Magelang District were kept in an insect proof greenhouse in Faculty of Agriculture, Universitas Gadjah Mada, Daerah Istimewa Yogyakarta. The plants were observed daily for 21 days for the appearance of Tungro symptoms.

RT-PCR assay was performed to confirm RTSV infection in 20 inoculated rice plants. Total RNA was extracted from leaf samples taken 21 days post inoculation (dpi), using Isogen RNA Extraction Kit (Amersham Pharmacia, UK) following manufacturer instructions except for the use of liquid nitrogen before grinding the samples. The obtained RNA was then used in the cDNA synthesis using oligo d(T) primer and First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) following the standard procedures.

PCR mix was prepared in a volume of 23 μl: 20 μl pureTaq Ready -to-Go PCR Bead (GE Healthcare, USA), 1 μl forward primer (5’-AAACGGTCATTGTGGGGAGGT-3’), 1 μl reverse primer (5’-CAGGCCCAGCAACGACATAA-3’) [10], and 1 μl cDNA sample. The PCR program was as follows Predenaturation: 94 ^oC for 5 min; 35 cycles of 94 ^oC for 50 s, 53 ^oC for 1 min, and 72 ^oC for 50 s; and Final extention: 72 ^oC for 5 min.

PCR products were mixed with loading buffer (ThermoFisher Scientific, USA), loaded into 2% agarose gel, and then run in an electrophoresis apparatus at 75 V, 25 mA for 1.5 h. The agarose gel was then stained with 0.1% ethidium bromide for 10 min, and the appearance of the targeted specific band was visualized under a UV transilluminator. Amplicons of randomly selected two RTSV positive samples (one from each sampling location) was purified using ExoSAP-IT reagent (USB Corporation, USA) and then shipped to Integrated Laboratory of Universitas Islam Negeri Sunan Kalijaga, Indonesia to be sequenced bidirectionally using the Sanger method.

On 7 February 2023, the complete genome sequences of six RTSV isolates were downloaded from NCBI GenBank database and aligned using the ClustalW algorithm available in MEGA X v.10.2.4 program [11]. The 5’ and 3’-UTR regions were removed to construct a ‘Full ORFs’ alignment (10,413 bp). Separately, to assess the phylogeny of each ORF, the alignment was also trimmed according to ORF1/Hypothetical protein (1923 bp), ORF2/CP1 (624 bp), ORF3/CP2 (609 bp), ORF4/CP3 (876 bp), ORF5/NTP-binding domain (3537 bp), ORF6/3C-like protease (978 bp), and ORF7/RdRp (1866 bp) sequences, respectively. The complete sequences of ORFS of other isolates in GenBank and complete sequences of CP1 gene of two novel isolates obtained in this study were also included in their respective ORF alignment (Table S1).

Possible recombination events in each alignment were scanned using Recombination Detection Program (RDP) v.5.30 [12] applied different algorithms: RDP, Chimaera, MaxChi, 3Seq, GENECONV, Siscan, and Bootscan, with default parameters. Phylogenetic anomalies supported by fewer than five algorithms with a Bonferroni-corrected P-value < 0.05 were not considered significant.

Maximum Likelihood (ML) phylogenetic trees based on each alignment were built using the best-fit Tamura-Nei-parameter model [13] within MEGA X software, plus uniform rates among sites with 1,000 bootstrap replicates to determine the statistical significance of isolated clusters. The percentage identities between major phylogroups tested at the nucleotide (nt) and amino acid (aa) levels were determined by Sequence Demarcation Tool (SDT) v1.2 freeware [14].

At the end of observation (21 dpi), all 20 inoculated plants showed Tungro-like symptoms such as leaf yellowing and stunted growth with different severity while the two control plants did not show any clear symptoms of viral infection.

RT-PCR assay determined that eight inoculated plants, three from Magelang (No. 1, 3, and 7) and five from Sidrap (No. 2, 3, 4, 7, and 9), were tested positive for RTSV while 12 others inoculated and two controls were negative. Since they were assumed from the same inoculum sources, one sample was randomly selected from each location: no. 7 from Magelang and no. 9 from Sindrap are to be sequenced.

RDP5 analysis on the alignment of ‘Full ORFs’ and seven other RTSV genome regions: Hypothetical proteins, CP1, CP2, CP3, NTP-binding domain, 3C-like protease, and RdRp found six significant recombination signals along the full genome sequences of four isolates: Orissa (AM234048), West Bengal (AM234049), Vt6 (AB064963), and AP (KC794785), and a single recombination event on the Phil.1 (U71440) genome sequence was only partially known (Table 1).

Phylogenetic analysis based on ‘Full ORFs’ deduced that the only available 6 complete isolates belonged to two major lineages that were geographically separated. The Malaysian ‘Seberang Perai’ (MK655459) and Filipinos NC_001632 isolates were positioned in a phylogroup that was proposed to be appropriately named South East Asia (SEA). In the second group, one Japanese ‘Vt6’ (AB064963) was clustered together with three Indian isolates: ‘Orissa’ (AM234048), ‘West Bengal’ (AM234049), and ‘AP’ (KC794785) thus this group was suggested to be called South Asia (SA) (Fig. 2). We then tested the ‘Full ORFs’ phylogrouping against the alignments of seven other RTSV genome regions.

After removal of unclear nt readings, the sequencing analysis recovered 624 bp of nt from each of the two samples, covering the complete CP1 gene. Sequences of the two isolates, named Magelang-7 and Sindrap-9, were registered in NCBI GenBank with accession nos. OQ426495 and OQ426496, respectively. They were then aligned with all other 39 RTSV isolates which complete CP1 gene sequences were registered in GenBank. Phylogenetic analysis based on CP1 gene deduced that the compared 41 isolates were mostly also separated into SEA and SA groups. Magelang-7 and Sindrap-9, together with 19 other Indonesian isolates, formed a subcluster which in turn shared a basal node with three Filipino isolates in the SEA phylogroup. In the second cluster, one Malaysian, one Japanese, and six Filipino isolates were positioned together with seven Indian isolates in the SA phylogroup. Interestingly, MK655459 that was a SEA isolate was shown to be closely related to SA isolates in CP1 tree (Fig. 2C). The 21 Indonesian isolates had 92 – 100% and 94 – 100% identity among them at nt and aa levels, respectively. They also shared 91 – 97% nt and 93 – 100% aa identities with three Filipinos isolates belonging to SEA group, and 85 – 95% nt and 93 – 100% aa identities with 15 isolates in SA group according to SDT matrix analysis.

Further observation showed that two other complete SA isolates: KC794785 and AB064963 were grouped together with SEA isolates for the analysis of different genome regions. Similarly, two partially sequenced isolates: U71440 and ‘Malaysian’ (U70989) that belonged to SA group in CP1 tree were found to be clustered in SEA group in other trees, which indicated the presence of recombination events in the CP1 region of U70989 that were not yet observed by RDP5 software probably due to the lack of known parental isolates (Fig. 2). Therefore, the results of both recombination and phylogenetic analyses confirmed frequent recombination among RTSV isolates.