Breeding for rice tungro disease resistance at PhilRiceL.S. Sebastian, E.R. Tiongco, D.A. Tabanao, G.V. Maramara, S. Abdula, and E.B. Tabelin

Abstract

Breeding for resistance to rice tungro disease is a major objective of the rice breeding program for irrigated lowland and rainfed ecosystems at the Philippine Rice Research Institute. Tungro-resistant donors are identified using visual screening and serological assays and are then crossed with selected high-yielding modern genotypes with good grain quality. Field evaluation of advanced breeding lines is carried out at experimental stations in Nueva Ecija and in North Cotabato. Methods for generation advance and resistance screening are described. As of the 1998 wet season, several F8 lines from the following crosses showed good resistance to tungro: PSB Rc4 x TI-11-8, BPI Ri10 x TI-11-8, IR64 x TI-11-8. Molecular markers are currently being used to map resistance genes to tungro from Utri Merah and Utri Rajapan and molecular techniques are being developed to characterize strains of rice tungro spherical virus.

Introduction

The development of tungro-resistant varieties remains a primary concern in the Philippines because tungro is still the most destructive rice virus disease in the country. Almost all resistant varieties so far developed and released are resistant to the insect vector Nephottetix virescens only. A few varieties have resistance to rice tungro spherical virus (RTSV). Chemical control against the vector is very costly and the disease can still occur even if the vector population is not high. Furthermore, there is no effective chemical control measure against tungro viruses. As such, the development of varieties resistant to this disease is a priority objective of the Philippine rice breeding program.

Rice tungro disease concerns at PhilRice

Breeding for yield and grain quality

The breeding program for irrigated lowland (transplanted and direct-seeded) and rainfed rice ecosystems in the Philippine Rice Research Institute (PhilRice) has always included tungro resistance as an objective. Selection for desirable plant morphology and grain structure starts at F3. Segregating generations are advanced with continuous selection for yield, grain quality, and other important traits. Resistance to important insect pests and diseases is incorporated by crossing selections with high yield and good grain quality with breeding lines and traditional accessions carrying the desired resistance traits. For tungro resistance, the most widely used materials are TI-11-8 (a BC4Fn derived from ARC11554 × TN1) and Utri Merah. To screen for rice tungro disease (RTD) resistance specifically, lines advanced from these crosses are shuttled to Midsayap, North Cotabato, where tungro incidence is consistently high even during the dry season. Each entry is observed for visual symptoms caused by the disease. Lines found to have some amount of resistance are included in multilocation preliminary and advanced yield trials for further and final-stage tests. This method of selection for tungro resistance, however, usually results in the selection of lines that are resistant only to the vector.

Developing breeding lines with RTD resistance

In 1994, a special breeding project began with tungro resistance as the primary trait of interest. The project aims to develop lines with resistance to tungro using various donor sources. By backcrossing and/or selection in the segregating population, resistance genes are transferred to

a modem genetic background. The project seeks to produce breeding materials with durable resistance to either RTSV or rice tungro bacilliform virus (RTBV) or to both viruses, with or without resistance to the vector.

With the availability of these breeding lines, the efforts of breeders working mainly on high yield and good grain quality will become easier and faster. This prevents the occurrence of undesirable recombinants that arise when landraces are used as sources of resistance. It also disrupts the progress in generation advance by reintroducing many undesirable traits into lines that have been previously considered promising for other traits. Thus, instead of, for instance, using Utri Merah directly, the resistance gene can be transferred into promising selections by way of breeding lines containing the resistance gene but with improved characteristics.

For most of the activities under this project, workers at the PhilRice Central Experimental Station (CES) in Muñoz, Nueva Ecija, work closely with researchers at the PhilRice Midsayap Experiment Station (MES) in North Cotabato. CES acts mainly as a source of materials while conducting field tests on its own and performs the more sophisticated laboratory procedures. MES serves as a satellite station for field tests and as a second source of leaf samples to be analyzed by enzyme-linked immunosorbent assay (ELISA), which is the responsibility of CES. Midsayap is strategically located because of the constantly high insect and disease pressure in the area notably including tungro. With the strengthening of networks, the research group at MES has started performing its own crossing work and is now also equipped with a green leafhopper rearing nursery for virus strain maintenance and test tube inoculation.

Breeding and screening strategies

Modern varieties, landraces, introductions, and breeding lines are first evaluated for their response to tungro disease using visual screening and ELISA. Selected modern genotypes are then crossed with resistance donors. Table 1 lists materials used in the crossing work conducted from 1994 to 1996. F1 and F2 plants are reared in CES. Individual plant selection begins with F2 plants, which are space-planted in the field. During the wet season (WS), when disease pressure is high, selection is primarily for resistance (visual screening). During the dry season (DS), when disease pressure is insignificant, selection is primarily for morphology and grain structure. Starting at F3, dual-location testing is carried out at both CES and MES. During wet seasons, generation advance and field screening are conducted at both CES and MES. Only generation advance activities are conducted at CES during dry seasons, however, because of very low occurrence or nonoccurrence of tungro in Central Luzon at this time of the year.

The F3 materials are planted in small (2.2-2.8 × 1.2-1.6 m), unreplicated plots. Selection is done within plots and seeds are bulked in each plot to constitute the next generation. At F4 and F5, the small plots are replicated thrice. Selection is still within plots and seeds are also bulked, Lines that are consistently damaged by tungro in all three replicates are dropped, whereas lines that show a high degree of resistance in all three replicates are considered as the best selections. At F6 through F8, lines are planted in unreplicated but bigger plots (5.0 × 5.0 m) to minimize escape and/or preference of insects, which is especially the case when many genotypes are arrayed in succession in the field. At this stage, selections within each plot can be separated into distinct sublines, but each subline can still be composed of several bulked plants. Visual screening is also coupled with ELISA at this stage. Promising selections are then tested in preliminary yield trials.

The ELISA technique is carried out as outlined by Bajet et al. (1985). Leaves are sampled at 30 and 60 days after transplanting (DAT). Absorbance is set at 405 nm in a MicroELISA reader. For the field screening, spreader rows consisting of IR64 or PSB Rc14 are planted 1 month ahead to ensure a sufficient source of virus. Disease incidence (% infection) is observed at 30 and 60 DAT.

Rice tungro disease observation nursery

This part of the project aims to (1) evaluate selected rice collections and advanced lines for resistance to rice tungro disease and (2) identify potential parents for hybridization work. An initial screening of 56 commercial varieties and breeding lines was conducted at CES and MES in the 1995 WS. The entries were assigned to 1.0 × 2.5- m plots each in three replications. Visual scoring of tungro incidence and leaf sampling for ELISA were done at 30 and 60 DAT.

In CES, 28 entries had low tungro incidence (0–23%). while in MES all entries had high tungro incidence (47-100%). In both locations, TI-11-8 showed low virus titers of combined RTBV and RTSV (0%), RTBV (8% in CES, 0% in MES), and RTSV (0% in CES, 13% in MES), indicating a high level of resistance to tungro. In CES, IR74, PSB Rcl8, and PSB Rc34 showed low infections with combined RTBV and RTSV (0–4%), with RTBV (0- 4%), and with RTSV (0–25%). In MES, LS519, LS551, TK298, and TK300 had low combined RTBV-RTSV (8-25%), RTBV (4–25%). and RTSV (4–13%) infection.

In 1996, the observation nursery in CES handled 399 entries, most of which were F4 lines. Sixty-seven lines showed a high level of resistance to tungro. In MES, only one entry, IR69705-1-1-3-2-1, exhibited high resistance against tungro out of more than 900 entries evaluated, the bulk of which were F4 and F3 lines.

Generating RTD-resistant advanced lines

This task involves the following procedures: (1) advancing early generations of crosses between tungro-resistant genotypes and selected rice cultivars, (2) screening for RTD resistance in early and advanced generations, and (3) purifying and increasing seed of selected lines that are resistant to RTD. Figure 1 shows the scheme for generation advance and resistance screening.

Hybridization work began in 1994, producing 14 crosses. In the succeeding years, many materials were handled in both the dry and wet seasons. In 1995, 70 crosses, 256 F1 plants from 42 crosses, and 17 F2 populations were advanced. In 1996, 116 crosses, 270 F1 plants from 42 crosses, 16 F2 populations. 1.047 F3 lines from 24 crosses, and 387 F4 lines from 4 crosses were advanced. In 1997, 14 crosses, 18 F2 populations, 334 F3 lines from 8 crosses, 464 F4 lines from 16 crosses, 262 F5 lines from 14 crosses, and 46 F6 lines from 4 crosses were advanced. In 1998, 6 F3 crosses, 64 F4 lines from 8 crosses, 226 F5 lines from 8 crosses, 276 F6

lines from 16 crosses, 198 F7 lines from 14 crosses, and 24 F8 lines from 4 crosses were advanced.

As of the 1998 WS, several F 8 lines from four crosses have been considered as the best tungro-resistant selections. These include four lines from PSB Rc4 × TI-11-8, three lines from BPI Ri10 × TI-11-8, and one line from IR64 × TI-11-8. Table 2 shows the other crosses with promising lines.

Biotechnology

In support of breeding activities, molecular markers are currently being used to map genes with resistance to tungro from Utri Merah and Utri Rajapan. The molecular markers being used are restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and randomly amplified polymorphic DNA (RAPD). Resistance from ARC11554 was mapped earlier (Fig. 2, Sebastian et al. 1996a, b). Molecular markers tightly linked to the RTSV resistance gene are now being tested for possible use in marker-aided selection (MAS). Cloning of the RTSV resistance gene is also under way (G.O. Romero, personal communication, 1999).

Furthermore, molecular techniques are also being tested in differentiating RTSV strains in Muñoz, Nueva Ecija, and Midsayap, North Cotabato. This study uses eight differential varieties and detects variation in RTSV strains using the reverse transcriptase- polymerase chain reaction technique.

Tungro resistance has also been introgressed into IR64 from Oryza rufipogon as part of a wide hybridization project aiming to transfer insect and disease resistance genes from wild species. Several BC 4 lines with resistance to RTSV and RTBV have already been generated.

Future outlook

The development of varieties resistant to tungro will remain a challenge to various breeding programs because of the complexity of the disease. It is hoped, however, that, with new tools and knowledge from concerted research and development efforts, the development of tungro-resistant varieties will become a reality. In PhilRice, promising F8 lines will be evaluated in preliminary yield trials for grain yield potential and yield component characteristics. Even materials with below optimum yields will be maintained, purified, and characterized as long as

they have resistance to tungro. Backcrossing to the modern parent may be done to recover as many desirable traits as possible while maintaining the resistance to tungro at the same time.The development of a marker-aided selection procedure for tungro resistance using the resistance gene from ARC11554 will continue to be undertaken because MAS, as a technique, has become an integral part of other breeding programs for pest and disease resistance. Molecular mapping for resistance genes and characterization of RTSV strains constitute the other thrusts in RTD research, both of which are relevant to the development of strategies for resistance breeding.

References

Bajet NB, Daquioag RD, Hibino H. 1985. Enzyme-linked immunosorbent assay to diagnose rice tungro. Journal of Plant Protection in the Tropics 2:125–129.

Sebastian LS, Ikeda R, Huang N, Imbe T, Coffman WR, McCouch SR. 1996a. Molecular mapping of resistance to rice tungro spherical virus (RTSV) and green leafhopper (GLH) in rice. Phytopathology 86(1):25–30.

Sebastian LS, Ikeda R, Huang N, Imbe T. Coffman WR, Yano M, Sasaki T, McCouch SR. 1996b. Genetic mapping of resistance to rice tungro spherical virus (RTSV) and green leafhopper (GLH) in ARCl1554. In: Khush GS, editor. Rice genetics III. Proceedings of the 3rd lnternational Rice Genetics Symposium, Manila. Philippines. p 560–563.

NotesAuthors’ address : L.S. Sebastian, E.R. Tiongco, D.A. Tabanao, and G.V. Maramara, Philippine Rice Research Institute (PhilRice), Central Experiment Station, Muñoz, Nueva Ecija, Philippines; S. Abdula and E.B. Tabelin, PhilRice, Midsayap Experiment Station, North Cotabato. Philippines.

Citation: Sebastian L.S., E.R. Tiongco, D.A. Tabanao, G.V. Maramara, S. Abdula, and E.B. Tabelin. 1999. Breeding for rice tungro disease resistance at PhilRice. p. 22-29. 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.