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Efficient method of determining tungro virus resistance in rice (Oryza sativa L.)
195
I
Efficient method of determining tungro virus resistance in rice (Oryza sativa L.)* M. S h a h j a h a n t , A. H. Zakri~, T. Imbed, B. S. J a l a n i t and T. O m u r a §
tDepartment of Genetics, Universiti Kebangsaan Malaysia, Bangi, ++TropicalAgriculture Research Centre, Tsukuba, Japan and §Institute of Virus Research, Tsukuba Science City, Japan
Abstract
Keywords
To determine the level of rice seedlings with rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV), the seedlings were either mass inoculated with viruliferous green leafhopper (Glh), Nephotettix virescens, or individual seedlings were caged with the viruliferous GIh (forced inoculation). The plant samples were analysed by enzyme-linked immunosorbent assay (ELISA) to detect the RTBV and RTSV particles, and by visual assessment. In mass inoculation 17.4% of the seedlings escaped viral infection compared with 1.4% for forced inoculation. A close correlation was found to exist between the assessment by ELISA and visual scoring. Tungro virus; screening; forced inoculation; mass inoculation; ELISA
Introduction
Viral diseases are a major problem in rice production (Ou, 1984). Rice tungro virus (RTV) disease is one of the most widespread and destructive rice virus diseases in South and Southeast Asia (Hibino et al., 1988). The disease has caused enormous losses of the crop in the past. The RTV disease is attributed to the rice tungro bacilliform (RTBV) and rice tungro spherical (RTSV) viruses, where RTBV causes the disease and RTSV intensifies it (Hibino, Roechan and Sudarisman, 1978). The viruses are chiefly transmitted (non-persistent nature) (Ling, 1979) by the green leafhopper, Nephotettix virescens. T h e vector is unable to transmit RTBV without the prior acquisition of RTSV (Hibino, 1983). One approach to control of the disease is by planting virus-resistant varieties (Khush, 1980). Availability of different sources of resistance is the prerequisite for evolving resistant varieties. The search for such genes from the germplasm collections can be made more efficient if better screening methods are developed. At present, virus inoculation for screening of germplasm collections and other elite lines is being done by mass inoculation in either the field (Suriachandraselvan et al., 1986) or the greenhouse (Ling, 1979). Another method is the inoculation of the virus to the test material by caging viruliferous vectors with individual seedlings (forced inoculation) (Shahjahan et al., 1990). This study was undertaken to compare the efficiency of mass inoculation to forced inoculation, and to
"Part of PhD dissertation submitted by the senior author to Universiti Kebangsaan Malaysia, Bangi; correspondence to Professor A H. Zakri, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, 43600 UKM BangioSelangor Darul Ehsan. Malaysia
ascertain the accuracy of visual assessment of virusinfected seedlings by using ELISA.
Materials and methods
Screening for viral resistance was done on 160 F 3 families of the K a t a r i b h o g x T N l cross and 159 families of Pankhari 203 × TN1 cross by mass inoculation. T N I is highly susceptible to RTSV and RTBV, and Kataribhog and Pankhari 203 are resistant to RTSV but tolerant to RTBV. Ten days after sowing, the F 3 generations were screened by caging 40 viruliferous Glh with an individual family of 20 seedlings. After 48 h the insects were killed with carbofuran granules. Another batch of 565 seedlings of F2 populations from the same cross-combinations were used to assess the presence of virus by the forced inoculation method, in which viruses were inoculated by caging two viruliferous Gih (after an acquisition access period of 24 h) with individual seedlings. The parent materials and their F~ progenies were also used in both experiments. After 3 weeks, the seedlings that had been mass inoculated were assessed visually. Seedlings which showed severe stunting, with yellow-orange discoloration of the foliage, were scored as + + and those only slightly stunted as +. Seedlings showing no symptoms were scored as H (healthy). After visual assessment the seedlings were tested for the presence of RTBV and RTSV using ELISA. In the forced inoculation experiment, ELISA was used to detect the RTBV and RTSV 3 weeks after virus inoculation. Pankhari 203 is also resistant to GIh (Shahjahan et al., 1988): therefore, for tests involving Pankhari 203, the Pankhari 203 colony of vectors was used to minimize the Glh resistance factor. Antisera for RTBV and RTSV were
0261/2194/91/03/0195- 04 .~'~ 1991 Butterworth-Heinemann Ltd CROP PROTECTION Vol. 10 June 1991
196
Tungro virus resistance in rice: M. Shahlahan e t a l .
Table 1. Mass screening of test varieties assessed by ELISA for RTBV and RTSV Infected seedlings b Total seedlings tested (no.)
(no.) B+ S
B
Infection (%)
S
Healthy seedlings (no.)
B+S
B
S
Escape (%)
Cross combination
Population tested"
Pankhari 203 x TN1
Pan TNI F1 F3
72 80 32 3148
0 50 23 1866
49 2 0 406
0 13 7 258
23 15 2 618
0.0 62.5 71.8 59.2
68.0 2.5 0.0 12.9
0.0 16.2 21.8 8.1
31.9 18.7 6.2 19.8
Kataribhog x TNI
Kat TNt Fl F3
77 78 28 3149
0 55 21 1910
70 3 I 416
0 7 2 107
7 13 4 716
0.0 70.5 75.0 60.6
90.9 3.8 3.5 13.2
0.0 9.1 7.1 3.4
9.0 16.6 14.2 22.8
"Pan, Pankhari 203: Kat, Kataribhog, ~B, RTBV; S. RTSV Table 2. Screening of test varieties by caging individual seedlings assessed by ELISA for RTBV and RTSV Infection
Infected seedlingsh
(%)
(no.) Cross combination
Population tested b
Total seedlings tested (no.)
B+ S
B
S
Healthy seedlings (no.)
B+S
B
S
Escape (%)
Pankhari 203 x TN I
Pan TN 1 F1 F2
15 30 35 228
0 21 35 190
14 9 0 31
0 0 0 0
I 0 0 7
0.0 70.0 100.0 85.9
93.3 30.0 0.0 14.0
0.0 0.0 0.0 0.0
6.7 0.0 0.0 3.1
Kataribhog x TN I
Kat TNI F1 F2
30 30 24 337
0 30 24 320
30 0 0 II
0 0 0 0
0 0 0 6
0.0 0.0 I00.0 94.9
100.0 0.0 0.0 3.2
0.0 0.0 0.0 0.0
0.0 0.0 0.0 1.8
"l'As in Table I
kindly supplied by Dr T. Omura, Institute of Virus Research, Tsukuba, Japan.
F2 seedlings from both cross combinations were not infected either by RTBV or RTSV.
Results
Visual assessment and ELISA
Mass and forced inoculation Data from screening of test seedlings by mass inoculation and forced inoculation are presented in Tables 1 and 2. Of the 6664 seedlings subjected to mass inoculation, 17.4% were not infected by either RTBV or RTSV. All the parents were infected at least by RTBV; it was expected, therefore, that the seedlings of both experiments should at least be infected by RTBV. In the mass inoculation, 17.6% o f T N 1 (average), 31.9% of Pankhari 203, and 9.0% of Kataribhog were not infected with either virus. In the Ft and F 3 populations, an average of 18.5% seedlings from Kataribhog x TNI and 13.0% seedlings from Pankhari 2 0 3 x T N I were not infected with either virus (Table 1). On the other hand, in forced inoculation 100% seedlings of the TN 1, Kataribhog and F~ progenies were infected with either RTBV or RTSV (Table2). In this method only 6.7% of the Pankhari 203 seedlings and an average of 2.4%
CROP PROTECTION Vol. 10 June 1991
In ELISA, four categories of results were observed, as follows (the seedlings of the fourth category were regarded as having escaped viruliferous insect feeding): (l) doubly infected with RTBV and RTSV; (2) infected with RTBV alone; (3) infected with RTSV alone, and (4) negative reactions for both RTBV and RTSV. In visual scoring, + + was equivalent to seedlings doubly infected by RTBV and RTSV as determined by ELISA; + was equivalent to seedlings singly infected by either RTBV or RTSV as determined by ELISA, and H was equivalent to seedlings giving a negative reaction for both RTBV and RTSV in ELISA. Data on the two assessments (visual and ELISA) are presented in Table 3. Of the 6297 seedlings assessed, 3681 were scored + + in visual assessment compared with 3765 seedlings which were found by ELISA to be infected doubly with RTBV and RTSV. Only 84 seedlings (1.3%), therefore, could not be scored properly out of 6297 seedlings in visual assessment. A significant correlation
Tungro virus resistance in rice: M. Shahjahan et al.
197
Table 3. Comparison between visual assessment and ELISA
ELISA Varieties Kataribhog x TNI. F3
Visual scoring
Total seedlings (no.)
RTBV + RTSV
RTBV
3149
1910
416
RTSV
E"
+ +
+
H
107
716
1832
933
384
258
618
1849
735
564
1334
3681
1668
948
523 ^
Pankhari 203 x TNI. F3 Grand total
3148 6297
1855 3765
406 664 t' 1187
"E, negative reaction for RTBV and RTSV; ~'these totals were compared with +
Table 4. Correlations among the categories of assessment
Visual scoring ++ + 1t
ELISA RTBV+ RTSV" (RTBV)+(RTSV)~' E' 0.4203** - 0.0257 - 0.4752** -0.1686"* - 0.0164 0.2593** - 0.3685** 0.0676 0.3735"*
**p < 0.01 ; "seedlings infected by both RTBV and RTSV; %eedlings infected with either RTBV or RTSV; "escaped infection
was observed between these two assessments (r = 0.4203, Table4). In the other category of scoring, 1668 seedlings were scored +, against 1187 seedlings found to be infected singly either by RTBV or RTSV (7.6% seedlings assessed inaccurately through visual means compared with ELISA). Furthermore, 948 seedlings were assessed as having no symptoms (H) in visual scoring compared with 1334 seedlings giving a negative reaction in ELISA for both RTBV and RTSV (6.1% seedlings scored inaccurately visually compared with ELISA, Table3). These results indicate that it is difficult to assess seedlings visually if they are infected singly by either RTBV or RTSV, but visual assessment of doubly infected seedlings is more reliable.
Discussion Screening of rice varieties for tungro virus disease resistance by using a mass inoculation method is a normal practice. IRRI (1985) and other research institutes use this method for screening their huge collections to identify the sources of resistance to pests and diseases. Some researchers screen test materials during disease outbreaks in the field (Suriachandraselvan et al., 1986). Mass inoculation is a convenient method and a large number of entries can be screened in a shorter period if the proper diagnostic materials for screening are available. The mass inoculation method, however, has its limitations. For example, some seedlings may escape feeding vectors, and such uninfected seedlings could be assumed to be resistant whereas, in fact, they may be susceptible. This method therefore, is not accurate in evaluating each and every seedling o f a n entry, thus confusing the assessment of resistance. In this study > 17.0% of the seedlings escaped infection when screened by mass inoculation with RTBV and RTSV
(Table 1). All the parental varieties involved in the present study were susceptible to RTBV; therefore, all the seedlings of the F1, F2 and F 3 generations were theoretically susceptible to infection at least by the RTBV. However, > 17.0% of the seedlings gave a negative reaction to both RTBV and RTSV (Table4). This means that some seedlings remained uninoculated by the viruliferous vectors in the process of screening by mass inoculation. On the other hand, when screened by caging vectors with individual seedlings (forced inoculation), 98.7% of the seedlings were infected by both RTBV and RTSV or singly by RTBV or RTSV (Table2). In this method only 1.3% of the tested seedlings showed a negative reaction both for RTBV and RTSV, which may be attributable either to the inability of the vectors to transmit the virus or to death of the vectors because of poor handling. These results suggest that it is appropriate to inoculate disease-causing agents by caging them with individual seedlings (forced inoculation) for valid screening of test materials, identification of the gene source and genetic investigation for the resistance of pests and diseases of rice, with special emphasis on viral disease. Although this method is tedious compared with mass inoculation, the outcome of the experiment is more reliable. In screening of a germplasm collection, normally 30 seeds are sufficient for testing each entry. The result of screening 30 seedlings by forced inoculation will be more fruitful than mass screening a huge number of seedlings. Although a close correlation exists between the assessment by ELISA and visual scoring (Tabh,4), it would not be advisable to use visual assessment for genetic studies for this disease because of the possibility of misclassification of some seedlings as resistant or susceptible. When dealing with the genetic ratio where more than one gene is involved, classification of tested individuals into groups is crucial and misassessment based on a few individuals may lead to inaccurate conclusions. As genetic experimentation involves an extensive outlay of materials and time, it should be conducted as efficiently as possible. This study suggests that a more sensitive means of detecting tungro viruses can be obtained by screening the test materials by forced inoculation followed by ELISA, if the facilities and diagnostic materials are available. By using specific antisera to detect a specific virus in ELISA tests, it is possible to differentiate between seedlings infected by morphologically different viruses such as RTBV and RTSV.
CROP PROTECTION Vol. 10 June 1991
198 Tungro virus resistance in rice: M. Shahjahan etaL
Acknowledgements
IRRI (1985). Annual Report. International Rice Research Institute. pp. 50-52
The authors express their sincere thanks to M r H a b i b u d din Hashim o f M A R D I , B u m b o n g Lima, for supplying Glh populations and virus source plants a n d also for his valuable suggestions. The senior a u t h o r also acknowledges the award o f a postgraduate studentship from Universiti K e b a n g s a a n Malaysia, Bangi.
Khush. G. S. (1980) Breeding rice for multiple disease and insect resistance. In: Rice Improvement in China and other Asian Countries, pp. 219-238, IRRI, Los Bafios, Philippines
References
Ling, K. C. (1979) Rice Virus Disease, pp. 102-105, IRR1, Los Bafios. Philippines On, S. H. (1984). Rice Disease. 2nd edn, pp. 33-43, Commonwealth Mycological Institute, Kew Shahjahan, M., Zakri, A. H., Jalani, B. S. and Otlunan, O. (1988) The correlation of green leafhopper, Nephotettix virescens, (Homoptera: Cicadellidae)resistanceand tungro virus infection in some rice varieties. SABRAO J. 20, 91-99
Hibino, H., Daquioag, R. D., Cabauatan, P. Q. and Dahal, G. (1988) Resistance to rice tungro spherical virus in rice. Plant Dis. 72, 843
Shahjahan, M., Jalani, B. S., Zakri, A. H., Imbe, T. and Othman, O. (I 990) Inheritance of tolerance to rice tungro bacilliform virus (RTBV) in rice (Orvza sativa L.). Theoret. Appl. Genet., 80, 513-517
Hibino, H. (1983) Relations of rice tungro bacilliform virus and rice tungro spherical virus with their vector Nephotettix virescens. Ann. Phytopathol. Soc. Jpn 49, 545-553.
Suriachandraselvan,M., Saroja, R., Vivekanandan,F., Venkatakrishna, J. and Nilakantapillai,K. (1986). Field evaluation office for tungro virus (RTV) resistance. IRRN 1I, 8
Hibino, H., Roechan, M. and Sndarisman, S. (1978) Association of two types of virus particules with penyakit habang (tungro disease) of rice in Indonesia. Phytopathology 68, 1412-1416
Received 25 July 1990 Accepted 29 November 1990
CROP PROTECTION Vol. 10 June 1991
I
Efficient method of determining tungro virus resistance in rice (Oryza sativa L.)* M. S h a h j a h a n t , A. H. Zakri~, T. Imbed, B. S. J a l a n i t and T. O m u r a §
tDepartment of Genetics, Universiti Kebangsaan Malaysia, Bangi, ++TropicalAgriculture Research Centre, Tsukuba, Japan and §Institute of Virus Research, Tsukuba Science City, Japan
Abstract
Keywords
To determine the level of rice seedlings with rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV), the seedlings were either mass inoculated with viruliferous green leafhopper (Glh), Nephotettix virescens, or individual seedlings were caged with the viruliferous GIh (forced inoculation). The plant samples were analysed by enzyme-linked immunosorbent assay (ELISA) to detect the RTBV and RTSV particles, and by visual assessment. In mass inoculation 17.4% of the seedlings escaped viral infection compared with 1.4% for forced inoculation. A close correlation was found to exist between the assessment by ELISA and visual scoring. Tungro virus; screening; forced inoculation; mass inoculation; ELISA
Introduction
Viral diseases are a major problem in rice production (Ou, 1984). Rice tungro virus (RTV) disease is one of the most widespread and destructive rice virus diseases in South and Southeast Asia (Hibino et al., 1988). The disease has caused enormous losses of the crop in the past. The RTV disease is attributed to the rice tungro bacilliform (RTBV) and rice tungro spherical (RTSV) viruses, where RTBV causes the disease and RTSV intensifies it (Hibino, Roechan and Sudarisman, 1978). The viruses are chiefly transmitted (non-persistent nature) (Ling, 1979) by the green leafhopper, Nephotettix virescens. T h e vector is unable to transmit RTBV without the prior acquisition of RTSV (Hibino, 1983). One approach to control of the disease is by planting virus-resistant varieties (Khush, 1980). Availability of different sources of resistance is the prerequisite for evolving resistant varieties. The search for such genes from the germplasm collections can be made more efficient if better screening methods are developed. At present, virus inoculation for screening of germplasm collections and other elite lines is being done by mass inoculation in either the field (Suriachandraselvan et al., 1986) or the greenhouse (Ling, 1979). Another method is the inoculation of the virus to the test material by caging viruliferous vectors with individual seedlings (forced inoculation) (Shahjahan et al., 1990). This study was undertaken to compare the efficiency of mass inoculation to forced inoculation, and to
"Part of PhD dissertation submitted by the senior author to Universiti Kebangsaan Malaysia, Bangi; correspondence to Professor A H. Zakri, Faculty of Life Sciences, Universiti Kebangsaan Malaysia, 43600 UKM BangioSelangor Darul Ehsan. Malaysia
ascertain the accuracy of visual assessment of virusinfected seedlings by using ELISA.
Materials and methods
Screening for viral resistance was done on 160 F 3 families of the K a t a r i b h o g x T N l cross and 159 families of Pankhari 203 × TN1 cross by mass inoculation. T N I is highly susceptible to RTSV and RTBV, and Kataribhog and Pankhari 203 are resistant to RTSV but tolerant to RTBV. Ten days after sowing, the F 3 generations were screened by caging 40 viruliferous Glh with an individual family of 20 seedlings. After 48 h the insects were killed with carbofuran granules. Another batch of 565 seedlings of F2 populations from the same cross-combinations were used to assess the presence of virus by the forced inoculation method, in which viruses were inoculated by caging two viruliferous Gih (after an acquisition access period of 24 h) with individual seedlings. The parent materials and their F~ progenies were also used in both experiments. After 3 weeks, the seedlings that had been mass inoculated were assessed visually. Seedlings which showed severe stunting, with yellow-orange discoloration of the foliage, were scored as + + and those only slightly stunted as +. Seedlings showing no symptoms were scored as H (healthy). After visual assessment the seedlings were tested for the presence of RTBV and RTSV using ELISA. In the forced inoculation experiment, ELISA was used to detect the RTBV and RTSV 3 weeks after virus inoculation. Pankhari 203 is also resistant to GIh (Shahjahan et al., 1988): therefore, for tests involving Pankhari 203, the Pankhari 203 colony of vectors was used to minimize the Glh resistance factor. Antisera for RTBV and RTSV were
0261/2194/91/03/0195- 04 .~'~ 1991 Butterworth-Heinemann Ltd CROP PROTECTION Vol. 10 June 1991
196
Tungro virus resistance in rice: M. Shahlahan e t a l .
Table 1. Mass screening of test varieties assessed by ELISA for RTBV and RTSV Infected seedlings b Total seedlings tested (no.)
(no.) B+ S
B
Infection (%)
S
Healthy seedlings (no.)
B+S
B
S
Escape (%)
Cross combination
Population tested"
Pankhari 203 x TN1
Pan TNI F1 F3
72 80 32 3148
0 50 23 1866
49 2 0 406
0 13 7 258
23 15 2 618
0.0 62.5 71.8 59.2
68.0 2.5 0.0 12.9
0.0 16.2 21.8 8.1
31.9 18.7 6.2 19.8
Kataribhog x TNI
Kat TNt Fl F3
77 78 28 3149
0 55 21 1910
70 3 I 416
0 7 2 107
7 13 4 716
0.0 70.5 75.0 60.6
90.9 3.8 3.5 13.2
0.0 9.1 7.1 3.4
9.0 16.6 14.2 22.8
"Pan, Pankhari 203: Kat, Kataribhog, ~B, RTBV; S. RTSV Table 2. Screening of test varieties by caging individual seedlings assessed by ELISA for RTBV and RTSV Infection
Infected seedlingsh
(%)
(no.) Cross combination
Population tested b
Total seedlings tested (no.)
B+ S
B
S
Healthy seedlings (no.)
B+S
B
S
Escape (%)
Pankhari 203 x TN I
Pan TN 1 F1 F2
15 30 35 228
0 21 35 190
14 9 0 31
0 0 0 0
I 0 0 7
0.0 70.0 100.0 85.9
93.3 30.0 0.0 14.0
0.0 0.0 0.0 0.0
6.7 0.0 0.0 3.1
Kataribhog x TN I
Kat TNI F1 F2
30 30 24 337
0 30 24 320
30 0 0 II
0 0 0 0
0 0 0 6
0.0 0.0 I00.0 94.9
100.0 0.0 0.0 3.2
0.0 0.0 0.0 0.0
0.0 0.0 0.0 1.8
"l'As in Table I
kindly supplied by Dr T. Omura, Institute of Virus Research, Tsukuba, Japan.
F2 seedlings from both cross combinations were not infected either by RTBV or RTSV.
Results
Visual assessment and ELISA
Mass and forced inoculation Data from screening of test seedlings by mass inoculation and forced inoculation are presented in Tables 1 and 2. Of the 6664 seedlings subjected to mass inoculation, 17.4% were not infected by either RTBV or RTSV. All the parents were infected at least by RTBV; it was expected, therefore, that the seedlings of both experiments should at least be infected by RTBV. In the mass inoculation, 17.6% o f T N 1 (average), 31.9% of Pankhari 203, and 9.0% of Kataribhog were not infected with either virus. In the Ft and F 3 populations, an average of 18.5% seedlings from Kataribhog x TNI and 13.0% seedlings from Pankhari 2 0 3 x T N I were not infected with either virus (Table 1). On the other hand, in forced inoculation 100% seedlings of the TN 1, Kataribhog and F~ progenies were infected with either RTBV or RTSV (Table2). In this method only 6.7% of the Pankhari 203 seedlings and an average of 2.4%
CROP PROTECTION Vol. 10 June 1991
In ELISA, four categories of results were observed, as follows (the seedlings of the fourth category were regarded as having escaped viruliferous insect feeding): (l) doubly infected with RTBV and RTSV; (2) infected with RTBV alone; (3) infected with RTSV alone, and (4) negative reactions for both RTBV and RTSV. In visual scoring, + + was equivalent to seedlings doubly infected by RTBV and RTSV as determined by ELISA; + was equivalent to seedlings singly infected by either RTBV or RTSV as determined by ELISA, and H was equivalent to seedlings giving a negative reaction for both RTBV and RTSV in ELISA. Data on the two assessments (visual and ELISA) are presented in Table 3. Of the 6297 seedlings assessed, 3681 were scored + + in visual assessment compared with 3765 seedlings which were found by ELISA to be infected doubly with RTBV and RTSV. Only 84 seedlings (1.3%), therefore, could not be scored properly out of 6297 seedlings in visual assessment. A significant correlation
Tungro virus resistance in rice: M. Shahjahan et al.
197
Table 3. Comparison between visual assessment and ELISA
ELISA Varieties Kataribhog x TNI. F3
Visual scoring
Total seedlings (no.)
RTBV + RTSV
RTBV
3149
1910
416
RTSV
E"
+ +
+
H
107
716
1832
933
384
258
618
1849
735
564
1334
3681
1668
948
523 ^
Pankhari 203 x TNI. F3 Grand total
3148 6297
1855 3765
406 664 t' 1187
"E, negative reaction for RTBV and RTSV; ~'these totals were compared with +
Table 4. Correlations among the categories of assessment
Visual scoring ++ + 1t
ELISA RTBV+ RTSV" (RTBV)+(RTSV)~' E' 0.4203** - 0.0257 - 0.4752** -0.1686"* - 0.0164 0.2593** - 0.3685** 0.0676 0.3735"*
**p < 0.01 ; "seedlings infected by both RTBV and RTSV; %eedlings infected with either RTBV or RTSV; "escaped infection
was observed between these two assessments (r = 0.4203, Table4). In the other category of scoring, 1668 seedlings were scored +, against 1187 seedlings found to be infected singly either by RTBV or RTSV (7.6% seedlings assessed inaccurately through visual means compared with ELISA). Furthermore, 948 seedlings were assessed as having no symptoms (H) in visual scoring compared with 1334 seedlings giving a negative reaction in ELISA for both RTBV and RTSV (6.1% seedlings scored inaccurately visually compared with ELISA, Table3). These results indicate that it is difficult to assess seedlings visually if they are infected singly by either RTBV or RTSV, but visual assessment of doubly infected seedlings is more reliable.
Discussion Screening of rice varieties for tungro virus disease resistance by using a mass inoculation method is a normal practice. IRRI (1985) and other research institutes use this method for screening their huge collections to identify the sources of resistance to pests and diseases. Some researchers screen test materials during disease outbreaks in the field (Suriachandraselvan et al., 1986). Mass inoculation is a convenient method and a large number of entries can be screened in a shorter period if the proper diagnostic materials for screening are available. The mass inoculation method, however, has its limitations. For example, some seedlings may escape feeding vectors, and such uninfected seedlings could be assumed to be resistant whereas, in fact, they may be susceptible. This method therefore, is not accurate in evaluating each and every seedling o f a n entry, thus confusing the assessment of resistance. In this study > 17.0% of the seedlings escaped infection when screened by mass inoculation with RTBV and RTSV
(Table 1). All the parental varieties involved in the present study were susceptible to RTBV; therefore, all the seedlings of the F1, F2 and F 3 generations were theoretically susceptible to infection at least by the RTBV. However, > 17.0% of the seedlings gave a negative reaction to both RTBV and RTSV (Table4). This means that some seedlings remained uninoculated by the viruliferous vectors in the process of screening by mass inoculation. On the other hand, when screened by caging vectors with individual seedlings (forced inoculation), 98.7% of the seedlings were infected by both RTBV and RTSV or singly by RTBV or RTSV (Table2). In this method only 1.3% of the tested seedlings showed a negative reaction both for RTBV and RTSV, which may be attributable either to the inability of the vectors to transmit the virus or to death of the vectors because of poor handling. These results suggest that it is appropriate to inoculate disease-causing agents by caging them with individual seedlings (forced inoculation) for valid screening of test materials, identification of the gene source and genetic investigation for the resistance of pests and diseases of rice, with special emphasis on viral disease. Although this method is tedious compared with mass inoculation, the outcome of the experiment is more reliable. In screening of a germplasm collection, normally 30 seeds are sufficient for testing each entry. The result of screening 30 seedlings by forced inoculation will be more fruitful than mass screening a huge number of seedlings. Although a close correlation exists between the assessment by ELISA and visual scoring (Tabh,4), it would not be advisable to use visual assessment for genetic studies for this disease because of the possibility of misclassification of some seedlings as resistant or susceptible. When dealing with the genetic ratio where more than one gene is involved, classification of tested individuals into groups is crucial and misassessment based on a few individuals may lead to inaccurate conclusions. As genetic experimentation involves an extensive outlay of materials and time, it should be conducted as efficiently as possible. This study suggests that a more sensitive means of detecting tungro viruses can be obtained by screening the test materials by forced inoculation followed by ELISA, if the facilities and diagnostic materials are available. By using specific antisera to detect a specific virus in ELISA tests, it is possible to differentiate between seedlings infected by morphologically different viruses such as RTBV and RTSV.
CROP PROTECTION Vol. 10 June 1991
198 Tungro virus resistance in rice: M. Shahjahan etaL
Acknowledgements
IRRI (1985). Annual Report. International Rice Research Institute. pp. 50-52
The authors express their sincere thanks to M r H a b i b u d din Hashim o f M A R D I , B u m b o n g Lima, for supplying Glh populations and virus source plants a n d also for his valuable suggestions. The senior a u t h o r also acknowledges the award o f a postgraduate studentship from Universiti K e b a n g s a a n Malaysia, Bangi.
Khush. G. S. (1980) Breeding rice for multiple disease and insect resistance. In: Rice Improvement in China and other Asian Countries, pp. 219-238, IRRI, Los Bafios, Philippines
References
Ling, K. C. (1979) Rice Virus Disease, pp. 102-105, IRR1, Los Bafios. Philippines On, S. H. (1984). Rice Disease. 2nd edn, pp. 33-43, Commonwealth Mycological Institute, Kew Shahjahan, M., Zakri, A. H., Jalani, B. S. and Otlunan, O. (1988) The correlation of green leafhopper, Nephotettix virescens, (Homoptera: Cicadellidae)resistanceand tungro virus infection in some rice varieties. SABRAO J. 20, 91-99
Hibino, H., Daquioag, R. D., Cabauatan, P. Q. and Dahal, G. (1988) Resistance to rice tungro spherical virus in rice. Plant Dis. 72, 843
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Received 25 July 1990 Accepted 29 November 1990
CROP PROTECTION Vol. 10 June 1991