XXVIII. Greenhouse Gas Mitigation Potentials of Water Saving Technologies for Rice Fields in Central
XXIX. Genetic Diversity of RTBV and RTSV Isolates from Different Rice Tungro-infected Areas in the
Philippines and Screen the Reaction of a Different Set of Varieties
XGI Caguiat
There are two viruses, rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV) that could occur singly or combined to cause the rice tungro disease (RTD). RTD has been known to cause devastating impact in rice industry worldwide. It is important to know that different strains of these two viruses could affect different high-yielding varieties.
However, in order to determine which strains affects which varieties, identification of the different strains of the viruses and their genetics should be considered.
In the long run, knowledge on the genetic variation present in these organisms will help in disease management and development of rice tungro disease-resistant varieties. Through this study, exploring the differences in nucleotide and protein variation of the tungro viruses will provide information on how much genetic differentiation exists in the pathogen population in the country, and may provide clues as to why the disease is more severe in some places than in others. This would help researchers to determine the right type of resistance genes for the different virus strains to be used in developing new rice varieties. By planting the right variety, farmers will no longer suffer the destructive disease, and they will produce enough rice which may lead to sustainable development. Another thing is that the composition and structure of virus population is not stable, and its genotype differ significantly over time, so by continuously monitoring virus populations, we can better understand and identify the factors why there is tungro outbreak or extinction of the current prevailing tungro virus populations and by this, it may lead for us to achieve durable virus resistance genes and varieties of rice. In bridging the gap of using nucleotide sequence data of each isolates, differential varietal responses on different RTBV and RTSV strains in a particular region would aim in selecting possible accession that could be as source of resistance gene per isolate per location.
The study aims to determine the genetic variability of RTBV and RTSV isolates from different rice tungro-infected areas in the Philippines through the use of molecular markers and screen the reaction of a differential set of rice varieties.
Highlights:
• 200 tungro-infected plants were obtained from five rice- growing provinces: Isabela, Nueva Ecija, Negros Occidental, North Cotabato, Camarines Sur and Laguna (Figure 62). PCR Analysis confirms CP1 amplification (Figures 63)
• 4 conserved regions were found in Isabela (regions 319-326, 337-355, 361—377 and 448-462) while there was none from the other provinces indicating stability of RTBV isolates in Isabela.
• Selection pressure on the RTBV CP1 gene at the province and national level could be absent as indicative of non-significant Tajima’s D values. The differences in the patterns of nucleotide diversity in RTBV across geographic regions may imply that the factors affecting nucleotide diversity could also vary within provinces. This information will be used in conjunction with varietal reaction patterns, spatial and genomic diversity monitoring to understand RTBV evolution
• At the national level, nucleotide diversity levels of RTBV (π=0.0662, π=0.0712) were slightly higher than RTSV (π=0.0599, π=0.0632). Among the different provinces, nucleotide diversity of RTBV was highest in ISA (π=0.0564, π=0.0588) and NOC (π=0.0553, π=0.0637), and lowest in COT (π=0.0297, π=0.0265). For RTSV, the nucleotide diversity levels were generally lower in magnitude among the different provinces, except in NOC (π=0.0720, π=0.0711).
The high levels of nucleotide diversity of both RTBV and RTSV in NOC may imply that the extent of the diversity of tungro viruses across the country is present in this particular province because of different environmental factors present in the particular area which affect the genome. There was no indication of selection pressure acting on the coat protein genes at the province and national levels, as the Tajima’s D values (Tajima, 1989) were all not statistically significant. The differences in the patterns of nucleotide diversity between RTBV and RTSV across geographic regions may imply that the factors affecting nucleotide diversity are affecting the two viruses independently.
• The degree of genetic differentiation among provinces was generally lower in RTBV than in RTSV. In RTBV, pairwise FST (fixation index) tended to be lower for NOC with other provinces (0.2073 to 0.2878), except between ISA and NEC (0.1261). In RTSV, the same trend was observed for NOC
with other provinces: FST ranged from 0.2422 to 0.4728. In contrast, genetic differentiation was very high for the other provinces (0.7703 to 0.9214). Apparently, the amount of distance between any two provinces seemed to have the general effect of increasing the divergence of isolates between them. Two distinct clusters of RTBV isolates were reported by Cabauatan et al. (1999), whereas three lineages of RTSV isolates from Indonesia and the Philippines were inferred by Azzam and Chancellor (2000).
• The amount of heterogeneity among isolates from the same province and the degree of genetic differentiation between provinces were reflected in the phylogenetic trees. In both RTBV and RTSV, the NOC isolates tended to form a less tight cluster compared to other provinces. The star-like topology of the RTBV dendrogram (Figure 65A) was due to higher nucleotide diversity levels within provinces and less genetic differentiation of RTBV CP1 sequences among different provinces. Having the highest FST values and lower nucleotide diversity levels within provinces, the RTSV dendrogram (Figure 65B) was deeply bifurcated with very tightly clustered branches that corresponded to provinces.
Figure 62. Geo-tagged of provinces where tungro-infected plants were collected. These areas included sites of outbreak reported in the past.
Figure 63. Representative gel showing amplification of RTBV CP1 gene at 618 bp.
Figure 64. Sample BLAST result showing position of the CP1 from a representative isolate in the complete RTBV genome (GenBank accession
D10774.1).
Figure 65. (A) Rooted maximum likelihood tree of RTBV isolates based on Tamura-Nei distance. (B) Rooted maximum likelihood tree of RTSV isolates
based on Kimura two-parameter distance.