Complete Genome Sequence of Rice Tungro Bacilliform Virus Infecting Asian Rice (Oryza sativa) in Malaysia

Rice tungro disease was discovered in Malaysia in the 1930s. The first and only genome of Rice tungro bacilliform virus (RTBV) isolated from rice in Malaysia was sequenced in 1999.

R ice tungro disease (RTD) is known to be one of the most economically important viral diseases of rice (1). High incidences of RTD have been reported across South and Southeast Asia (2,3). Every year, RTD causes losses of approximately $1.5 billion in rice yield (4). The disease results from an infection by two distinct viruses, Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) (5,6). The severity of tungro symptoms is driven by RTBV (7), while RTSV mainly plays a role as a helper virus in vector transmission of RTBV (8).
Despite the importance of tungro viruses, less research work has been conducted on RTBV in Malaysia (19). The only genome of RTBV in Malaysia, RTBV-Serdang (GenBank accession no. AF076470), was sequenced in 1999 (18). Since then, no RTBV genome sequence has been reported in the past 19 years, although the disease is still endemic in Malaysia (9). In this study, we report the complete genomic sequence of an RTBV isolate obtained from an infected field. This will enable further studies regarding RTBV evolution and genome variability in Malaysia.
Rice plants exhibiting RTD symptoms were collected from a paddy field in Seberang Perai, Malaysia. The cetyltrimethylammonium bromide (CTAB) method was utilized in the extraction of DNA from infected leaves of rice plants (20). Designation of five overlapping primer pairs was done (Table 1) based on the aligned complete genome sequences of 13 RTBV isolates derived from GenBank (https://www.ncbi.nlm.nih.gov/ nuccore/?termϭriceϩtungroϩbacilliformϩvirusϩcompleteϩgenome). PCR amplification of those five fragments covering the entire genome from the RTBV DNA template was carried out using PCRBIO HiFi polymerase, and the products were electrophoresed on a 1% agarose gel. Once the band size was confirmed, the products were purified from the gel using a QIAquick gel extraction kit (Qiagen, Malaysia) and cloned into pJET1.2 vector. A minimum of two recombinant plasmids for every fragment were sent to the First BASE Laboratories Sdn Bhd company for sequencing in forward and reverse directions using the Sanger sequencing method.
A total of 10 bidirectional reads were obtained. The program Clustal Omega version 1.2.4 with default parameters was used to align all obtained reads in pairs to generate respective consensus nucleotide sequences of five fragments (21). Adjacent segments with overlapping sequences were joined in order to generate the complete genome of RTBV.
The full-length genome sequence of RTBV isolated from Seberang Perai (RTBV-SP) was thus obtained and found to be 8,000 nucleotides in length, with a GϩC content of 33.3%. Searches through BLASTN revealed that the nucleotide sequence of the RTBV-SP isolate is 81.45% to 95.44% identical to those of other RTBV complete genomes available in the GenBank database. The highest nucleotide similarity (95.44%) was observed with the Serdang isolate. Interestingly, the RTBV-SP genome is shorter than that of the Serdang isolate by 16 nucleotides.
This study revealed that the genetic makeup of RTBV-SP has remained stable despite a time gap of approximately 20 years between genome sequencing of the SP isolate and the Serdang isolate. Availability of the data on RTBV variability in Malaysia would be helpful in determining the resistance strategies against tungro.
Data availability. The complete genomic sequence of the RTBV-SP isolate was submitted to NCBI GenBank with the accession no. MK552377.