DISCUSSION PAPER SERIES

No. 24 1998

Evaluating Two Mass Screening Methods for Tungro Disease Resistance

E. L. Coloquio • E.R. Tiongco • R.C. Cabunagan • O. Azzam

IRRI

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Suggested citation:

Coloquio E, Tiongco ER, Cabunagan RC, Azzam O. 1998. Evaluating two mass screening methods for tungro disease resistance. IRRI Discussion Paper Series No. 24. Manila (Philippines): International Rice Research Institute.

ISBN 971-22-0113-9 ISSN 0117-8180

DISCUSSION PAPER SERIES

No. 24 1998

Evaluating Two Mass Screening Methods for Tungro Disease Resistance

E. L. Coloquio • E.R. Tiongco • R.C. Cabunagan • O. Azzam

IRRI

!\lTERf\ATIONAL RICE RESEARCII!\STITUTE

Evaluating two mass screening methods for tungro disease resistance

E. Coloquio, E. R. Tiongco, R. C. Cabunagan, and O. Azzam International Rice Research Institute, P.O. Box 933, Manila, Philippines

Tungro is one of the most destructive viral diseases of rice

in South and Southeast Asia. It is associated with two viruses-rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV) (Hibino et al 1978). Both viruses are transmitted by the green leafhopper (GLH) Nephotettix virescens (Ling 1979). However, prior acquisition of RTSV is required for the transmission of RTBV alone (Hibino 1983). Plants infected with both viruses show severe stunting and yellowing. Those infected with RTBV alone show mild stunting but no leaf discoloration whereas those infected with RTSV alone do not show any apparent symptoms (Hibino et al 1978).

Since the late 1960s, tungro has been mainly managed through varietal resistance (Khush 1989). The instability of resistant varieties in the field (Dahal et al 1990) led to a reexamination of the nature of the incorporated sources of resistance and to the adoption of more precise and more accurate screening methods.

At present, selection of natural sources of resistance to tungro at IRRI involves four steps: (1) initial mass screening of the germplasm collection, (2) forced tube inoculation and serological indexing for resistance to RTBV and RTSV,

(3) screening for vector resistance, and (4) further screening of selected lines for resistance to virus strains. However, initial mass screening of rice germplasm with a visual score assessment for percentage infection and disease index, based on the Standard evaluation system for rice (SES) CIRRI 1996), remains the basic screening method used by most national rice programs throughout the region. At IRRI, mass screening for rice tungro was developed in 1964; since then, different methods have been improved and adopted (Ling 1974, Hasanuddin et al 1988, Saxena et al 1991). Since 1992, the water tray method and the caged-pot method have been used interchangeably, but little is known about their effectiveness and reliability.

To standardize the mass screening methods and to enable a reliable exchange of information on varietal resistance to tungro among national rice programs and between national and international programs, we compared the inoculation efficiency, consistency, and accuracy of the water tray and caged-pot methods using 16 rice varieties with a known reaction to rice tungro viruses and their vector.

MATERIALS AND METHODS Selected varieties

Sixteen varieties were selected based on their previously confirmed reaction to either tungro or the vector GLH N. virescens (Table 1). The first six varieties were GLH-resistant (SES scores between 1 and 3) and tungro-resistant because of their low %RTD infection « 30%, in spite of their variable symptom severity scores (between 2 and 8). Pankhari 203 showed a similar score for both GLH and tungro resistance. The last nine varieties were GLH-susceptible (scores between 7 and 9) but they varied in their reaction to tungro.

Virus and insect sources

The GLH colony used in this study was collected from fields within 40 km of IRRI and was reared for several generations on 30-45-d-old TN1 plants inside insect cages. The tungro isolates used were the IRRI-maintained isolates on the TN1 plants.

Mass screening methods

Figure 1 describes in detail the six-step procedure for the water tray and caged-pot methods.

Mass inoculation

For the water tray method, the 16 varieties were planted in a soil-filled seedbox with twenty 9-10-d-old seedlings variety"

Table 1. Selected varieties for mass inoculation using water tray and caged-pot methods and their previously con-
firmed reaction to rice tungro disease (RTD) and green leafhopper (GLH).a
Code IRGC no. Variety Origin GLH SSb RTD%
1 731 Gampai 30-12-15 Thailand 3 (R) 2 (R) 28 (R)
2 21473 ARCl1554 India 3 (R) 2 (R) 12 (R)
3 37748 Laki 1120 Bangladesh 3 (R) 4 (MR) 26 (R)
4 26306 Bhaira Nota Bangladesh 1 (R) 7 (5) 21 (R)
5 26475 Jatra Matuk Bangladesh 1 (R) 75) 17 (R)
6 26492 Khoiamotor Bangladesh 1 (R) 8 (5) 21 (R)
7 5999 Pankhari 203 India 5 (MR) 4 (MR) 16 (R)
8 26793 Sham Rush Bangladesh 9 (5) 7 (5) 32 (MR)
9 27870 Chahora Pakistan 7 (5) 8 (5) 30 (R)
10 21383 ARCl1427 India 9 (5) 9 (5) 63 (5)
11 16684 Utri Rajapan Indonesia 7 (5) 2 (R) 21 (R)
12 237 TKM6 India 7 (5) 7 (5) 72 (5)
13 17204 Balimau Putih Indonesia 9 (5) 8 (5) 16 (R)
14 16602 Tjempo Tijik Indonesia 7 (5) 6 (5) 19 (R)
15 742 FK135 Philippines 9 (5) 9 (5) 93 (5)
16 105 TN1 Taiwan 9 (5) 9 (5) 92 (5)
aEvaluated at IRRI based on rice virus database. bSS = symptom severity, R = resistant, MR = moderately resistant, S = susceptible. row:', confined in a water tray (inoculation cage) and covered with a screen cage. Viruliferous adult GLH were released inside the inoculation cage for 2.S h (6-10 GLH seedling:'). The tray was then filled with water. As the water rose, the insects moved from the inoculated plants to the upper screen cage and seedlings became fully submerged. The seedbox was then gently pulled out, and a second batch of varieties was placed in the water tray for a second mass inoculation for 2.5 h. The process was repeated to inoculate a third batch. After the third batch, the GLH were placed on the source plants to effect virus reacquisition overnight and they were used again the following day. This daily inoculation procedure was repeated for 4 d (Fig. 1).

For the caged-pot method, 16 pots with 20 seedlings (9- 10-d-old) each were confined in a cage with 2000-3000 adult viruliferous GLH. The inoculation access period was 2.S h, after which the pots were removed and the disease source plants introduced in the cage to allow the GLHs to reacquire the viruses for another 2.5 h. The virus source was then removed and a new set of pots was placed in the cage for the second inoculation. The viruliferous insects used were about 6-10 GLH seedling:'. Vector mortality was observed during inoculation and reserve viruliferous GLHs were added, when needed, to maintain the population needed for mass inoculation. After the second inoculation, the GLH were placed on the source plants to acquire the viruses overnight and were used for the next inoculation the following day.

2 Evaluating two mass screening methods for tungro disease resistance

Scoring

Three to 4 wk after inoculation, the 20 seedlings were scored for symptom severity and disease index. Symptom severity, based on the SES,Jollowed a scale of 1-9: 1 = healthy looking, 3 = 1-10% height reduction with no distinct yellow or yellow-orange leaf discoloration, S = 11-30% height reduction with no distinct yellow or yellow-orange leaf discoloration, 7 = 31-S0% height reduction with distinct yellow or yellow-orange leaf discoloration, and 9 = more than SO% height reduction with distinct yellow or yelloworange leaf discoloration. Percentage visual infection was calculated as the total number of plants showing symptom severity scores above 3 divided by the total number of inoculated plants. Disease index was calculated as nO) + n(S) + n(7) + n(9) /tn where n(3), n(S), n(7), and n(9) are the respective number of plants showing a reaction score of 3, S, 7, and 9, and tn is the total number of plants scored. The experiment was repeated three times and in the third trial, varieties that showed variable reactions using the two methods were tested by enzyme-linked immunosorbent assay (ELISA), a serological test.

RESULTS

Consistency of percentage visual infection per inoculation time

Three us two sets oj inoculation day] In the water tray method, the same set of viruliferous insects was used successively to inoculate the three sets of 16 rice varieties day:' without reacquisition on a virus source. In the caged-

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pot method, the viruliferous insects used were allowed to feed again on the virus source after each inoculation set. Percentage visual infection was calculated after each inoculation set for each of the methods. Both methods showed little variability in percentage visual infection variety? inoculation set! day:' (Fig. 2) and both were consistent. In the water tray method, visual infection of 10 out of 16 varieties did not change much between inoculation sets 1 and 2; for the other six varieties, it decreased by 10% in the second inoculation set. For the third inoculation set, percentage infection of 11 varieties was similar to that of previous inoculations, even increasing by 5-10% in some. It decreased by 10% in the remaining five varieties. In the caged-pot method, visual infection of nine varieties was similar or even increased by 5-10% between the first two inoculation sets, but for the remaining seven varieties, it decreased by 10%.

Among 4 inoculation days. In the water tray method, during the second inoculation day, percentage visual infection of four varieties increased by 10% and that of eight varieties decreased by 10-15% (Fig. 2). No variability was shown in percentage visual infection of the remaining four varieties. In general, visual infection was similar on day 1, day 3, and day 4 in the water tray method. In the caged-pot method, variability in percent visual infection ranged between 20 and 30% among the 4 inoculation days and across different varieties. Only four out of 16 varieties showed consistency in percentage visual infection in the 4 inoculation days. When the data were plotted using disease index instead of percentage visual infection, similar trends were seen within the same inoculation day or across the 4 days (data not shown).

Inoculation efficiency of the two methods. To examine the inoculation efficiency of both methods, we used average data from the first inoculation set since at that time both methods had the same variables-i.e., the same ratio of viruliferous insects plant! and the same acquisition and inoculation time. Averages of the three trials and the 4 days of inoculation were plotted against either disease index or percentage visual infection. Results show that across the 4 inoculation days, eight varieties showed a higher percentage visual infection (Fig 3a) and disease index (Fig. 3b) in the water tray method than in the caged-pot method. For those varieties that exhibited different visual reactions in both methods, the water tray method had a consistently higher inoculation efficiency than the caged-pot method (Fig. 3). For the varieties that expressed complete susceptibility (TNI and FK135) or high level of resistance (Gampai 30-12-15 and

ARC11554), inoculation efficiency was similar in both methods (data not shown). Similar results were obtained when average data from the last inoculation set were compared (data not shown}

Accuracy of both methods. During the third trial, 20 plants from both the first and last sets of inoculation were selected for each of these rice varieties: Laki 112 (3), Bhaira Nota (4), jatra Matuk (5), Khoiamotor (6), Pankhari 203 (7), Chahora (9), and Tjempo Tijik (14) (Table 1). Most of these varieties showed differential visual scores in the two methods (Fig. 3), They were therefore indexed visually and serologically using ELISA. Figure 4 shows the percentage infection based on the ELISA results. Percentage infection by RTBV was higher in the water tray method than in the caged-pot method for six out of seven varieties tested

(Fig. 4). Percentage infection by RTSV was similar in both methods. Percentage infection of Tjempo Tijik by RTBV and RTSV varied in both methods. In addition, percentage infection of the same variety was variable between the different inoculation days in the two methods (Fig. 4).

DISCUSSION

In this study, we have demonstrated that the water tray method, as a greenhouse mass screening method, has several advantages over the caged-pot method. It has a higher inoculation efficiency and, based on ELISA results, it is more accurate and reliable in predicting the status of both RTBV and RTSV infection, especially in varieties that show moderate to high symptom severity scores but low percentage visual infection.

Ling and the virology group at IRRI developed and modified the caged-pot method over the years (Ling 1967, 1974; Cabunagan et al 1990). Inoculation access time was studied and changed from 24 h to 8 h and then to 2.5 h without any significant reduction in the transmission efficiency of tungro disease to susceptible TNI plants (Ling 1974). By reducing the inoculation access time, two batches of 16 pots instead of a single batch could be inoculated day! and the same viruliferous insects could be used for 2 inoculation days. In 1991, Saxena and coworkers used the same inoculation access period but showed that the seedbox-based or water tray method could accelerate the screening process for tungro resistance by using viruliferous GLH nymphs. Although this method is similar to the water tray method used in this study, three modifications have been introduced: 1) the viruliferous insects used were adults and not nymphs, 2) three inoculation sets were used day! instead of two, and 3) the same viruliferous insects were

Evaluating two mass screening methods for tungro disease resistance 5

A

Water tray Percent visual infection

100 90 80 70 60 50 40 30 20 10

o L_L_ L_ L- ~

lakl1120 Pankhari 203

~:-----....l

1

2

3

Time set of inoculation day!

1

2

3

B

Percent visual infection

.-----::: .. --1. •• ~~1135

ARC 11427

100 - 90 - 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 -

OL_--L_----~------_L------~----~

jIL.....:::::::- .... Sham Rush Pankhari 203

~::;;~::::;;;~~;;;;;:jM;i~~~:S~ ~haira Nota

I >/ Chahora ,..... Jatra Matul<

Khoiamotor

'te'

';"_ """rf\.

Bafimau Putih

01

02

04

03

Inoculation day

Caged-pot

Fig. 2. Percent visual infection of 16 rice varieties using either three (water tray method) or two (caged-pot method) inoculation sets per day (A) or average inoculation per day across the four days (B). Time points represent average of four inoculation days (D1, D2, D3, D4) and three trials (A) or average of three vs two inoculation sets dayl for three trials. X is the mean percentage of visual infection of all varieties.

6 Evaluating two mass screening methods for tungro disease resistance

TN1

~'~---""':==l.. ~~~~~427

TKM6

Sham Rush

laki 1120

..... Pankhari 203

i.iI::~. ~~~~;;::.~.. ~hahora

~ ,l. Jatra Matuk

~

1]empo Tijik

Bhaira Nota Khoiamoto( Gampai 30-12-15 Vtri Rajapan ARC11554

)!Ej[::-------:;(;Ii 8allmau Putih

_l£-~==_""""'_==l'J==-......j •• ~~1135

.. ARC 11427

01

02

03

04

A RTBV Water tray Caged-pot
Percent infection (ELISA)
100 8haifa Nota
90 Tjempo Tijik
Chahora
80 Pankhari 203
Tjempo Tijil< Pankhari 203
70 Laki 1120 lstre Maruk
Bhaifa Nota
60
50 cnerore
30 Laki 1120
20 Khoiamotor
20
10
0
Dl D2 D3 D4 Dl D2 D3 D4
B RTSV Days
Disease index
100
90
80
70
Tjempo TUfk
60
50
30
20 Pankhari 203

20
«notemotor
10
8haifa Nota
Ietrs Matul<
0 Chahora
Dl D2 D3 D4 Laki 1120 Dl D2 D3
Days Fig. 3. Percent visual infection (A) and disease index (B) for selected rice varieties which behaved diHerently in the water tray and the caged-pot screening methods. Time points represent the average of the first inoculation set for three trials and four consecutive inoculation days (D 1, D2, D3, and D4).

Evaluating two mass screening methods for tungro disease resistance 7

Fig. 4. Percent infection, based on ELISA, of selected rice varieties that showed diHerent reactions by two screening methods. Time points represent the average percent infection of 20 plants (per variety) with either rice tungro bacilliform virus (RTBV, A) or rice tungro spherical virus (RTSV, B).

8 Evaluating two mass screening methods for tungro disease resistance

used for 4 consecutive inoculation days. These modifications have allowed us to experiment with the method, interchangeably with the caged-pot method, since 1992 (Cabunagan et al 1993).

Many varieties, which were bred as tungro-resistant in the past, had resistance only to GLH and failed to have durable resistance when released and grown in the field for a few seasons (Rapusas et al 1982, Dahal et al 1990). Because virus diagnostic tools are available at IRRI, screening of rice germ plasm for virus resistance is possible and several sources of resistance to or tolerance for both tungro viruses have been identified. But many national rice programs do not have the resources yet to use these tools and screening for tungro resistance continues to rely on mass screening methods and visual assessment of resistance in the field. Using a reliable and consistent greenhouse mass screening method can minimize variability in results and can offer an alternative to initial field screening for tungro resistance. In addition to its reliability and large testing capacity, the water tray method is affordable by most rice institutes. Three batches could be inoculated day' using the same viruliferous insects and without any reacquisition. Moreover, the same insects could be used for 4 inoculation days. Although the average visual infection in both methods was Similar, the visual infection of separately tested varieties in the water tray method was less variable across the 4 inoculation days compared with that in the caged-pot method (Fig. 2).

Although this study shows several advantages of the greenhouse mass screening methods, there are also limitations. Shahjahan et al (1991) showed that> 17% of the seedlings inoculated by the caged-pot method escaped infection by escaping feeding vectors. Similar results were obtained with the water tray method. In addition, though visual assessment and ELISA using the water tray method are correlated, this method cannot reliably differentiate RTBV from RTSV or vector resistance. Therefore, the water tray method is recommended for initial mass screening of germplasm but not for genetic studies of tungro resistance. For such studies, more sensitive methods such as forcedtube inoculation followed by ELISA or nucleic acid hybridization are recommended.

REFERENCES

Cabunagan RC, Daquioag RD, Flores, ZM, Koganezawa H. 1990.

Identifying tolerance for tungro for rice tungro (RTV)associated viruses in rice varieties using severity index scoring and serology. Int. Rice Res. News!. 15(2):13-14.

Cabunagan RC, Flores ZM, Koganezawa H. 1993. Resistance to rice tungro spherical virus (RTSV) in germplasm. Int. Rice Res. Newsl, 18(1):24-25

Dahal G, Hibino H, Cabunagan RC, Tiongco ER, Flores ZM, Aguiero V. 1990. Changes in cultivar reaction to tungro due to changes in "virulence" of the leafhopper vector. Phytopathology 80:659-665.

Hasanuddin A, Daquioag RD, Hibino H. 1988. A method for scoring resistance to tungro (RTV). Int. Rice Res. New!. 13(6): 13-14

Hibino H. 1983. Relations of rice tungro bacilliform and rice tungro spherical viruses with their vector Nephotettix virescens. Ann. Phytopatho!. Soc. ]pn. 49:545-553.

Hibino H, Roechan M, Sudarisman S. 1978. Association of two types of virus particles with penyakit habang (tungro disease) of rice in Indonesia. Phytopathology 68:1412-1416.

[IRRI) International Rice Research Institute. 1996. Standard evaluation system for rice. 4th ed. Manila (Philippines): International Rice Research Institute.

Khush GS. 1989. Multiple disease and insect resistance for increased yield stability in rice. In: Progress in irrigated rice research. Manila (Philippines): International Rice Research Institute.

Ling KC. 1967. A method for testing resistance to tungro disease in rice. In: Abstract, 6th International Congress on Plant Protection. Vienna, Austria. p 129-130.

Ling KC. 1974. An improved mass screening method for testing the resistance of rice varieties to tungro disease in the greenhouse. Philipp. Phytopatho!. 10(1-2):19-30.

Ling KC. 1979. Rice virus diseases. Manila (Philippines): International Rice Research Institute. p 102-105.

Rapusas HR, Heinrich EA. 1982. Plant age and level of resistance to GLH, Nephotettix virescens and tungro virus in rice varieties. Crop Prot. 1 :91-98.

Saxena RC, Medrano FG, Bernal cc. 1991. Rapid screening for rice tungro resistance using viruliferous green leafhopper (GLH) nymphs. Int. Rice Res. New! 16 (1):10-11.

Shahjahan M, Zakri AH, Imbe T, ]alani BS, Omura T 1991.

Efficient method of determining tungro virus resistance in rice (Oryza sativa L.). Crop Prot. 10 (3): 195-198.

Evaluating two mass screening methods for tungro disease resistance 9

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No. 23 Olk D, ed. 1998. Reversing trends of declining productivity in intensive irrigated rice systems.

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