Preliminary analysis of genetic variation of rice tungro spherical virus in the Philippines

K.M.L. Umadhay, M.L.M. Yambao, and O. Azzam

Abstract

This study investigates the genetic variability of rice tungro spherical virus (RTSV) in nine tungro-endemic sites of the Philippines and over the 1996 wet season and 1997 wet and dry seasons. Based on a reverse transcriptase- polymerase chain reaction technique (RT-PCR) followed by restriction enzyme analysis, six distinct genotypes of RTSV were identified and their frequency across all sites determined. Results showed that more than one genotype could exist in a plant and at least two RTSV genotypes are present at one site. Although RTSV population did not change during the sampling, the presence of mixed infections and minor genotypes suggest that the structure and composition of the virus population is not stable. it is essential to continue monitoring these populations over an extended period to identify factors that lead to virus outbreaks or extinction of the current prevailing populations. This approach is critical in achieving durable virus resistance.

Introduction

In the tungro disease complex, rice tungro spherical virus assists in the semi-persistent transmission of rice tungro bacilliform virus (RTBV), the other component which causes tungro symptoms. Earlier studies showed that rice varieties react differently to RTSV variants. Recently, polymorphic molecular markers were developed to differentiate two variants of RTSV, RTSV-A and RTSV-Vt6 (Yambao et al 1998). These variants are serologically indistinguishable. The molecular markers are based on the amplification of coat protein regions 1 and 2 (CP1 and CP2) of the viral genome using reverse transcriptase-polyinerase chain reaction (RT-PCR) followed by a restriction analysis of the PCR product. Using this method, the genetic variation of RTSV natural populations in tungro-endemic regions of the Philippines was investigated in 1996 and 1997.

Methods

Nine tungro-endemic sites were sampled during the 1996 wet season (96 WS) and 1997 dry and wet seasons (97 DS and WS). Figure 1 shows the sampling sites. All samples were tested by enzyme-linked immunosorbent assay (ELISA) against both RTBV and RTSV antisera and ELISA-positive samples for RTSV were processed by KT-PCR and restriction analysis. The RT-PCR of CP regions 1 and 2 included (1) extraction of total RNA using trizol reagent. (2) cDNA synthesis using Superscript II, and (3) PCR using the cDNA as a template mixed with a cocktail of the following reagents: Taq DNA polymerase primers. MgC12, dNTP mixture, and buffer. The reaction conditions were initial denaturation at 95 °C for 3 minutes denaturation at 95 °C for 1 minute annealing temperature of 50 °C for 1 minute extension of68 °C for 5 minutes, and final extension of 72 °C for 7 minutes. The generated PCR products were then digested with Hind III and Bst Y I restriction enzymes and run on 1% agarose gel stained with ethidium bromide (Table 1).

Results and discussion

Six distinct coat protein genotypes were identified in the initial 302 analyzed samples from North Cotabato, Nueva Ecija, and Bicol provinces during the 1996 WS and DS, and 1997 WS. The frequency of each coat protein genotype was used as a potential indicator of virus variation per location (Fig. 2). In Nueva Ecija and Bicol, coat protein genotypes II, III, and VI were observed. In North Cotabato, coat protein genotypes II, III, V, and VI were found in the 1997 DS while coat protein genotypes I, II, III, V, and VI were present in the 1997 WS (Fig. 2). In both the 1996 and

1997 cropping seasons, pattern II dominated the RTSV population in the three locations but minor genotypes and mixed infections were also observed in all locations (Table 2).

Mixed infections were observed mainly in North Cotabato and Bicol. Coat protein genotype II predominated in both IRRI and local varieties. IR74 and IR64 were susceptible to RTSV coat protein genotypes II and III. On the other hand, local varieties were susceptible to a wide range of RTSV coat protein genotypes (Table 3).

These results suggested that RTSV populations in the field are heterogeneous and more than one RTSV variant could exist in one plant, that at least two variants could coexist at one location, and that the dominant variant may persist over time depending on the selection pressures present in that location. During the sampling, RTSV populations did not change and one genotype seemed to dominate in the Philippines under current conditions. In addition, IRRI varieties were similar to other varieties and did not seem to exert special selection pressure on current RTSV populations.

Because of the presence of mixed infections and minor genotypes, however, our data suggest that the structure and composition of the virus population may change if a different selection pressure such as a host or environmental shift occurs. It is therefore essential to monitor virus populations when virus-resistant varieties are deployed.

Reference

Yambao MLM, Cabauatan PQ, Azzam O. 1998. Differentiation of rice tungro spherical virus variants by RT-PCR and RFLP. International Rice Research Notes 23(2):22-24.

Notes

Authors' address: K.M.L. Umadhay, M.L.M. Yambao, and O. Azzam. International RiceResearch Institute, MCPO Box 3127, Makati City 1271, Philippines.

Citation: Umadhay, K.M.L., M.L.M. Yambao, and O. Azzam. 1999. Preliminary analysis of genetic variation of rice tungro spherical virus in the Philippines. p. 17-21. In: Chancellor TCB, Azzam O. Heong KL (editors). Rice tungro disease management. Proceedings of the International Workshop on Tungro Disease Management, 9-11 November 1998, International Rice Research Institute, Los Baños, Philippines, 166 p.