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Preliminary analysis of genetic variationof rice tungro bacilliform virus in twoprovinces of the PhilippinesM. Arboleda, F. Sta. Cruz, and O. AzzamA basic understanding of tungro virus populations is a prerequisite for anydeployment strategy of conventional or transgenic virus resistance. In the1996 and 1997 wet seasons, the genetic variability of rice tungro bacilliformvirus (RTBV) field populations was monitored in lsabela and NorthCotabato provinces of the Philippines. Based on restricted genome DNAprofiles and Pearson’s correlation coefficient analyses, heterogeneous anddistinct RTBV populations were identified in the two provinces. Althoughmembers of the populations reoccurred in some sites, the combination ofgenotypes differed significantly over time, suggesting a rapid evolution ofthe virus population. This study shows that changes in virus populationsneed to be continuously monitored to better understand and predict tungrooutbreaks and to prolong the life of deployed resistance genes.In highly intensive irrigated rice ecosystems in Southeast Asia. tungro disease causesconsiderable yield losses. The rice tungro disease complex is associated with ricetungro bacilliform virus and rice tungro spherical virus. On its own. RTBV causesyellowing and stunting symptoms but it cannot be transmitted by leafhoppers unlessRTSV is present. RTBV is a dsDNA belonging to the pararetrovirus group. a group ofplant DNA viruses that replicate through an RNA template. A DNA hybridizationtechnique was developed to differentiate the viral genomic DNA of four biologicalvariants 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 populationsof RTBV in tungro-endemic areas of the Philippines. Genomic DNA profiles representingsingle infections based on molecular weight estimates were selected and comparedamong the surveyed sites.Materials and methodsRandom samples were collected from the tungro hot spot provinces of Isabela andNorth Cotabato (30–50 samples per field and 4-6 fields per province; Fig. 1 ). Thesesamples were then assayed by enzyme-linked immunosorbent assay (ELISA) againstRTBV and RTSV antisera (Cabauatan et al 1995). Following the procedure developedby Cabauatan et a1 (1998), total DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method.The pelleted DNA was resuspended in 50 m L of sterile distilled water. Three tofive micrograms of total plant DNA was then digested with 30-50 units of EcoRVand 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 nylonmembrane using 2 x SSC. Blots were baked at 80 °C for 2 h prior to hybridizationwith the full-length RTBV-Ic clone. Chemiluminescence ECL (Amersham) was usedfor detection following the manufacturer's instructions.
Fig. 1. Sampling sites for the study in lsabela and North Cotabato provinces in the Philippines.After hybridization, restriction fragment length polymorphisms (RFLP) of differentgenotypes were used to determine genotype frequencies across locations andtimes. A dendrogram was also generated using Pearson’s correlation coefficient todetermine the geographic distribution of the different genotypes.ResultsFollowing digestion with EcoRV of total DNA extracts from 731 isolates and DNAhybridization, 24 distinct DNA genotypes were identified from Isabela and NorthCotabato (Figs. 1 and 2).High genetic variation was observed among fields and between provinces. Geneticvariation was also significantly different over time based on the bulk Chi squareanalysis of major, minor, and mixed genotypes (Fig. 3, Table 1). Results showed that2 to 9 distinct genotypes occurred per field and a few dominated at one time. Genotypes3 and 5 were detected in all sites of Isabela and genotypes 11 and 12 wereobserved in all sites of North Cotabato. Although some genotypes were commonamong sites, each site had its unique set of genotypes and genotype frequencies. Datagathered from North Cotabato in the 1997 dry season (DS) exhibited a spatial distributionpattern similar to that of North Cotabato in the 1997 wet season (WS). Betweensites, fields sampled in Isabela in the 1996 WS were similar to each other intheir set of genotype frequencies, whereas Ilagan and Cauayan in the 1997 WS differedsignificantly in their set of genotype frequencies.12 Arboleda et al
Page 2 and 3: RiceTungro DiseaseManagementEdited
Page 4 and 5: ContentsRice tungro disease in the
Page 6 and 7: ForewordThe intensification of rice
Page 8 and 9: PrefaceProviding farmers with optio
Page 10 and 11: Rice tungro disease in the Philippi
Page 12 and 13: Cotabato, and Laguna) during 1995-9
Page 14 and 15: seed growers and farmers to increas
Page 16 and 17: Fig. 2. Incidence of rice tungro di
Page 18 and 19: De los Reyes JB, Cabunagan, RC, Col
Page 22 and 23: Fig. 2. Distinct rice tungro bacill
Page 24 and 25: Fig. 4. Dendogram depicting the rel
Page 26 and 27: Preliminary analysis of genetic var
Page 28 and 29: Table 1. Size characteristics of th
Page 30 and 31: varieties and did not seem to exert
Page 32 and 33: Developing breeding lines with RTD
Page 34 and 35: In CES, 28 entries had low tungro i
Page 36 and 37: Table 2. Best rice tungro disease-r
Page 38 and 39: Breeding for rice tungro virus resi
Page 40 and 41: Table 2. Promising lines resistant
Page 42 and 43: Table 4. Rice cultivars used as hyb
Page 44 and 45: Shahjahan MB, Jalani S, Zakri AH, I
Page 46 and 47: Table 1. Initial 19 transgenic line
Page 48 and 49: Table 4. Percent infection of trans
Page 50 and 51: very low level. In fact, transgene
Page 52 and 53: of Agricultural Research (ICAR). Pr
Page 54 and 55: Fig. 2. Mean infection with rice tu
Page 56 and 57: (RTBV) and rice tungro spherical vi
Page 58 and 59: Table 4. Percent infection a with r
Page 60 and 61: GLH numbers were much lower on IR62
Page 62 and 63: Cabunagan RC, Hibino H, Sama S, Riz
Page 64 and 65: Prospects of virus-resistant variet
Page 66 and 67: Tungro in Bali (1987-97)Tungro infe
Page 68 and 69: Fig. 5. Proportion of varieties gro
Page 70 and 71:
Table 3. Percent incidence of rice
Page 72 and 73:
Evaluating rice germplasm for resis
Page 74 and 75:
Table 2. Percent infection a with r
Page 76 and 77:
Rice tungro disease resistance andm
Page 78 and 79:
Table 1. Percent infection a with r
Page 80 and 81:
ecommended vector-resistant variety
Page 82 and 83:
Materials and methodsBatches of ant
Page 84 and 85:
ReferencesClark MF, Adams AN. 1977.
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(e.g., Sokal and Rohlf 1995, p 213;
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Surveillance scheme for tungro fore
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entrusted with implementing the pro
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Table 1. Number of mobile nursery t
Page 94 and 95:
are implemented immediately. The co
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Farmers’ rice tungro managementpr
Page 98 and 99:
Experience with tungroAlthough both
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Table 5. Farmers (%) reporting tung
Page 102 and 103:
Table 7. Farmers’ reported tungro
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Table 9. Mean number (standard devi
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than rice), so they may be reluctan
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Community-based rice pest managemen
Page 110 and 111:
Establishment of farmers’ indigen
Page 112 and 113:
Meanwhile, insects, diseases, and n
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Table 2. Most important diseases fo
Page 116 and 117:
Farmers’ knowledge of pest contro
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ha -1 and above) had a lower RTD in
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Fig. 4. Three major peaks of green
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RBB hill -1 and 4% WSB (Fig. 6). In
Page 124 and 125:
The influence of varietal resistanc
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Limited data exist on how resistant
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tember plantings, reaching 13% and
Page 130 and 131:
Holt J, Chancellor TCB, Reynolds DR
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glh4, respectively. Planting at the
Page 134 and 135:
Results and discussionMinimum unit
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Fig. 4. Transmission efficiency of
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Table 1. Enzyme-linked immunosorben
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Leafhopper control by insecticides
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Fig. 1. Cartoon used for tungro man
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The role of vector control in rice
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To establish relationships between
Page 148 and 149:
Fig. 1. Influence of antifeedants o
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Fig. 4. Tungro incidence before har
Page 152 and 153:
ReferencesAryawan IGN, Widiarta IN,
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Materials and methodsSeedbed protec
Page 156 and 157:
Table 2. Relationship of RTD incide
Page 158 and 159:
Management of rice tungro disease b
Page 160 and 161:
Results and discussionGLH populatio
Page 162 and 163:
Table 4. Field evaluation of foliar
Page 164 and 165:
3.6 to 2.7 t ha -1 . These yields w
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