Preliminary analysis of genetic variation of rice tungro bacilliform virusin two provinces of the Philippines

M. Arboleda, F. Santa Cruz, and O. Azzam

Abstract

A basic understanding of tungro virus populations is a prerequisite for any deployment strategy of conventional or transgenic virus resistance. In the 1996 and 1997 wet seasons, the genetic variability of rice tungro bacilliform virus (RTBV) field populations was monitored in lsabela and North Cotabato provinces of the Philippines. Based on restricted genome DNA profiles and Pearson’s correlation coefficient analyses, heterogeneous and distinct RTBV populations were identified in the two provinces. Although members of the populations reoccurred in some sites, the combination of genotypes differed significantly over time, suggesting a rapid evolution of the virus population. This study shows that changes in virus populations need to be continuously monitored to better understand and predict tungro outbreaks and to prolong the life of deployed resistance genes.

Introduction

In highly intensive irrigated rice ecosystems in Southeast Asia, tungro disease causes considerable yield losses. The rice tungro disease complex is associated with rice tungro bacilliform virus and rice tungro spherical virus. On its own. RTBV causes yellowing and stunting symptoms but it cannot be transmitted by leafhoppers unless RTSV is present. RTBV is a dsDNA belonging to the para-retrovirus group, a group of plant DNA viruses that replicate through an RNA template. A DNA hybridization technique was developed to differentiate the viral genomic DNA of four biological variants of RTBV using total DNA extracts of infected plants (Cabauatan et al 1998). The technique was also applied to examine the variability of natural field populations of RTBV in tungro-endemic areas of the Philippines. Genomic DNA profiles representing single infections based on molecular weight estimates were selected and compared among the surveyed sites.

Materials and methods

Random samples were collected from the tungro hot spot provinces of Isabela and North Cotabato (30–50 samples per field and 4-6 fields per province; (Fig. 1). These samples were then assayed by enzyme-linked immunosorbent assay (ELISA) against RTBV and RTSV antisera (Cabauatan et al 1995). Following the procedure developed by Cabauatan et al. (1998), total DNA was extracted using a modified cetyl-trimethylammonium bromide (CTAB) method. The pelleted DNA was re-suspended in 50 m L of sterile distilled water. Three to five micrograms of total plant DNA was then digested with 30-50 units of EcoRV and incubated overnight at 37 °C. The samples were then electrophoresed in 0.8% agarose gel for 5 h at 100 V in 1 x TBE buffer and blotted onto Hybond-N nylon membrane using 2 x SSC. Blots were baked at 80 °C for 2 h prior to hybridization with the full-length RTBV-Ic clone. Chemiluminescence ECL (Amersham) was used for detection following the manufacturer's instructions.

After hybridization, restriction fragment length polymorphisms (RFLP) of different genotypes were used to determine genotype frequencies across locations and times. A dendrogram was also generated using Pearson’s correlation coefficient to determine the geographic distribution of the different genotypes.

Results

Following digestion with EcoRV of total DNA extracts from 731 isolates and DNA hybridization, 24 distinct DNA genotypes were identified from Isabela and North Cotabato (Figs. 1 and 2). High genetic variation was observed among fields and between provinces. Genetic variation was also significantly different over time based on the bulk Chi square analysis of major, minor, and mixed genotypes (Fig. 3, Table 1). Results showed that 2 to 9 distinct genotypes occurred per field and a few dominated at one time. Genotypes 3 and 5 were detected in all sites of Isabela and genotypes 11 and 12 were observed in all sites of North Cotabato. Although some genotypes were common among sites, each site had its unique set of genotypes and genotype frequencies. Data gathered from North Cotabato in the 1997 dry season (DS) exhibited a spatial distribution pattern similar to that of North Cotabato in the 1997 wet season (WS). Between sites, fields sampled in Isabela in the 1996 WS were similar to each other in their set of genotype frequencies, whereas Ilagan and Cauayan in the 1997 WS differed significantly in their set of genotype frequencies.

RTBV mixed infections (based on the total number of DNA fragments detected >16 kb) were common and accounted for 10–54% of the total sample population. During the 1997 DS, their frequency was even higher than for a single infection in North Cotabato (Fig. 3, Table 1). Based on the frequency of the genotypes using Pearson’s correlation coefficient, RTBV genotypes were distributed geographically (Fig. 4). This showed that the genotype frequencies between Isabela and North Cotabato were significantly different. In Isabela, 10 distinct genotypes (1, 2, 3, 5, 9, 11, 17, 16, 19, and 35) were identified during the 1996 WS and in the 1997 WS. Only five genotypes (3, 5, 9, 19, and 25) were identical to those detected earlier from the same province. Genotypes 3 and 5 generally dominated in the 1996 WS, but these genotypes were augmented with genotypes 9 and 25 in the 1997 WS. A similar pattern was observed in North Cotabato during the 1997 DS and WS. In the 1997 DS, 13 distinct genotypes were observed (2, 3, 10–16, 18–20, and 24) and, in the following season (1997 WS), 11 genotypes were identified (10–16, 18–20, and 24). Genotypes 10–13 were the most frequent in the 1997 DS and remained dominant in the 1997 WS.

Conclusion

Based on the EcoRV genome profile assay, RTBV populations are genetically variable in the field. The data showed that more than one RTBV isolate could be found in one location. Even with just two planting seasons studied, results showed that RTBV populations exist as a diverse group of variants and are continuously evolving. This study showed that single isolates from a given location are not necessarily representative of or specific to that location. Virus populations need to be monitored continuously to ensure that varietal screening for deployed rice varieties uses appropriate virus isolates in the area.

References

Cabauatan P, Cabunagan R, Koganezawa H. 1995. Biological variants of rice tungro viruses in the Philippines. Phytopathology 85:77–81.

Cabauatan P, Arboleda M, Azzam O. 1998. Differentiation of rice tungro bacilliform virus strains by restriction analysis and DNA hybridization. Journal of Virological Methods 76:121–126.

Notes

Authors’ address: M. Arboleda, E Santa Cruz, and O. Azzam. International Rice Research Institute, MCPO Box 3127, Makati City 1271, Philippines.

Citation: Arboleda M, E Santa Cruz, and O. Azzam. 1999. Preliminary analysis of genetic variation of rice tungro bacilliform virus in two provinces of the Philippines. p. 11-16. 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.