Identification of blast-resistant advanced rice breeding lines through uniform blast nursery screening

Introduction

Rice (Oryza sativa L.) is one of the most important cereal crops worldwide and serves as the primary staple food for more than half of the global population. Rice plays a critical role in global food and nutritional security (Thapa & Bhusal, 2020) [1] . At present, global demand for rice continues to rise due to population growth and improved living standards. Projections indicate that rice production must increase by nearly 30% by 2030 to meet future food requirements (Devanna et al., 2022) [3] . Both abiotic and biotic stresses significantly affect rice production. Among them, rice blast caused by Magnaporthe oryzae is the most destructive and economically significant disease, resulting in yield losses of 70% to 80% under severe conditions (Miah et al., 2013) [2] . Although fungicides can reduce disease severity, their application is often uneconomical and environmentally unsafe, particularly in developing countries. Consequently, the development of blast-resistant rice cultivars remains the most effective, cost-efficient and environmentally sustainable strategy for managing rice blast and ensuring stable rice production. The pathogen shows high genomic variability, driven by transposable elements and the rapid evolution of effector genes. This dynamic nature allows the pathogen to adapt and overcome resistance genes introduced into cultivated varieties quickly. As a result, there is a constant need to discover and incorporate new resistance (R) genes to combat emerging pathogenic races effectively. In view of this challenge, the present study emphasized phenotypic screening of advanced breeding lines to identify genotypes with stable and effective resistance to blast disease, thereby contributing to the development of durable resistance in rice.

Materials and Methods

Near-isogenic lines derived from RNR 15048 × MTU1010 NIL that are available at Rice Research Unit, ARI, Hyderabad were used in the present investigation. These NILs possess a long slender grain type carrying blast resistance gene Pi54.

Two control genotypes, TN1 and NLR 34449, were used as susceptible and resistant checks, respectively, during phenotypic screening in the Uniform Blast Nursery. A total of 40 advanced breeding lines at F6 generation derived from the cross between RNR 15048 (Telangana Sona) and MTU1010 NIL (Pi54 donor) was selected for phenotypic evaluation during Rabi 2025-26.RNR 15048, an elite, shortduration (125 days), high-yielding variety with a low glycemic index (~51.7) with desirable cooking quality, served as the female parent, while MTU1010 NIL was used as the male parent for introgression of blast resistance. The entire study, including screening and evaluation, was undertaken at the Rice Research Unit, Agricultural Research Institute, Rajendranagar, Hyderabad.

Isolation and maintenance of blast culture Scraping Method

Rice leaves showing blast symptoms were collected and cut into small pieces. These pieces were surface-sterilized with 0.1% mercuric chloride and washed 3-4 times with sterile distilled water. The cleaned pieces were then placed on leaf extract agar in sterile Petri dishes under aseptic conditions and incubated at 27 °C for 3-4 days, until fungal growth was visible. After appearance of fungal growth, a small portion of the mycelium was carefully picked and transferred onto fresh agar plates under aseptic conditions. These plates were then incubated at 27 °C to promote further growth. Once the culture became well established, it was sub-cultured onto agar slants in test tubes for storage and routine maintenance.

Mass Multiplication on Oat Meal Agar (OMA)

A seven-day-old actively growing culture was aseptically transferred to sterile oatmeal agar plates. These plates are incubated at 27-28 °C for 8-10 days to allow sufficient fungal growth and sporulation. The pathogen produces simple, grey conidiophores bearing terminal, pear-shaped and 2-septate conidia.

Uniform Blast Nursery Method for Screening

Screening for blast resistance was conducted using the Uniform Blast Nursery (UBN) method. The nursery consists of raised beds measuring 10 m × 1 m, which were prepared with farm yard manure (FYM) with the recommended dose of fertilizers. The test entries were sown in rows about 50 cm long, with a spacing of 10 cm between rows. To maintain enough disease pressure, the highly susceptible variety, TN1, was planted as border rows around the nursery and also after every ten test entries. This arrangement will facilitate the uniform spread of the pathogen. The test lines were sown perpendicular to the border rows to enhance exposure to inoculum. Relative humidity was maintained using regular water sprinkling. To further enhance moisture levels and increase disease pressure on the test entries, the nursery beds were covered with polythene sheets during night.

Pathogen Inoculation and Disease Scoring

A spore suspension of Magnaporthe oryzae is prepared from a 7-to 10-day-old culture grown on oatmeal agar at 25-28 °C. The culture plates were washed with sterile distilled water, and the fungal growth, including mycelium and conidia, was gently scraped. The suspension was shaken for a few minutes to release spores and then filtered through muslin/cheese cloth to remove debris. Spore concentration was adjusted to approximately 1 × 10⁵ conidia ml⁻¹ using a haemocytometer, and a small amount of Tween-20 was added to the spore suspension to ensure uniform spreading. The prepared inoculum was evenly sprayed onto 15-21-dayold seedlings in the Uniform Blast Nursery using a hand sprayer in the evening, ensuring complete foliage wetting. High humidity, which essential for disease development, was maintained by regular sprinkler irrigation during the day, taking care not to wash off the inoculum immediately after spraying. Disease development was monitored, and scoring was carried out 15 days after inoculation using the Standard Evaluation System (SES, 2013). Based on lesion severity, the entries were grouped into highly resistant (0-1), resistant (1.1-3), moderately resistant (3.1-5), moderately susceptible (5.1-6), susceptible (6.1-8.9) and highly susceptible (9).

Results and Discussion

The study aims to identify rice genotypes with improved resistance to blast disease through screening under Uniform Blast Nursery (UBN) conditions. A total of 40 advanced breeding lines, along with parental lines RNR 15048, MTU1010 NILs, and the check varieties TN1 (susceptible) and NLR 34449 (resistant), were evaluated. Disease severity was assessed using the IRRI Standard Evaluation System (SES, 2013) [12] on a 0-9 scale. Adequate disease pressure was confirmed by the high susceptibility of TN1, which scored around 9, indicating the reliability of the screening conditions. Based on SES scoring, the breeding lines are grouped into different resistance classes. Of the 40 advanced lines evaluated, 10 showed high resistance (score 0-1), 20 were classified as resistant (1.1-3.0) and 10 exhibited moderate resistance (3.1-5.0). All 40 advanced breeding lines, along with the parental genotypes, exhibited highly resistant to moderately resistant reactions against blast disease, with scores ranging from 1 and 5 on the IRRI 0-9 SES scale. Among them, 10 lines CDR-7, CDR-9, CDR-10, CDR-13, CDR-14, CDR-16, CDR-18, CDR-22, CDR-32 and CDR-37 exhibited strong resistance with a score of 1 and were categorized as highly resistant. A total of 20 lines CDR-1, CDR-2, CDR-3, CDR-5, CDR-6, CDR-8, CDR-11, CDR-12, CDR-17, CDR-19, CDR-20, CDR-23, CDR-24, CDR-28, CDR-29, CDR-30, CDR-31, CDR-33, CDR-35 and CDR-40 recorded a score of 3 and were classified as resistant, while 10 lines CDR-4, CDR-15, CDR-21, CDR-25, CDR-26, CDR-27, CDR-34, CDR-36, CDR-38 and CDR-39 showed moderate resistance with a score of 5. The parental lines, RNR 15048 and MTU1010 NIL, showed moderate resistance and resistance with scores of 5 and 3, respectively. [4] , Umamahesh et al. (2024) [5] and Sai Akhila et al. (2025) [8] , who reported comparable findings in their respective studies.

Conclusion

In the present study, the major dominant blast resistance gene Pi54 gene was successfully introgressed into RNR 15048 (Telangana Sona) through marker-assisted pedigree breeding. The developed breeding lines carrying Pi54 gene demonstrated a high level of resistance to blast under station conditions. Due to broad-spectrum resistance of Pi54 gene against prevalent races of Magnaporthe oryzae in India, these lines are also expected to perform consistently across different regions of Telangana and other parts of South India. The improved lines retain the key characteristics of the recurrent parent RNR 15048, including its short duration, high yield potential and preferred short slender grain type with good cooking quality, while incorporating durable resistance to blast disease.

Acknowledgement I gratefully acknowledge Professor Jayashankar Telangana

Agricultural University for the financial support that enabled this research.

References

1. Thapa R, Bhusal N. Designing rice for the 22nd century: Towards a rice with an enhanced productivity and efficient photosynthetic pathway. Turkish Journal of Agriculture-Food Science and Technology. 2020;8(12):2623-2634.
2. Miah G, Rafii MY, Ismail MR, Puteh AB, Rahim HA, Asfaliza R, et al. Blast resistance in rice: A review of conventional breeding to molecular approaches. Molecular Biology Reports. 2013;40(3):2369-2388.
3. Devanna BN, Jain P, Solanke AU, Das A, Thakur S, Singh PK, et al. Understanding the dynamics of blast resistance in rice-Magnaporthe oryzae interactions. Journal of Fungi. 2022;8(6):584.
4. Sadhana P, Rao P, Prasanna BL, Babu TK, Sundaram RM, Balram N. Identification of blast resistant breeding lines through uniform blast nursery in rice (Oryza sativa L.). International Journal of Environment and Climate Change. 2023;13(9):1059-1065.
5. Umamahesh P, Nayak PG, Prasanna BL, Goud TY. Phenotypic screening of advanced breeding lines for blast resistance in rice (Oryza sativa L.). Journal of Experimental Agriculture International. 2024;46(8):415-420.
6. Sharma TR, Rai AK, Gupta SK, Singh NK. Broad spectrum blast resistance gene Pi-kh cloned from rice line Tetep designated as Pi54. Journal of Plant Biochemistry and Biotechnology. 2010;19(1):87-89.
7. Arun M, Seshu G, Srinivas B, Goud TY. Identification of superior rice (Oryza sativa L.) genotypes for blast International Journal of Advanced Biochemistry Research https://www.biochemjournal.com resistance through UBN method (Uniform Blast Nursery). The Pharma Innovation Journal. 2022;11(7):542-545.
8. Sai Akhila J, Laxmi Prasanna BL, Pushpavalli SNCVL, Kiran Babu T. Genetic improvement of RNR 15048 for blast resistance by using UBN phenotypic screening method. International Journal of Advanced Biochemistry Research. 2025;9(7):1794-1796.
9. Dangi KS, CH DR, Jagadeeshwar R, Rekha G, Sinha P, Aleena D, et al. Marker assisted pedigree breeding based improvement of the Indian mega variety of rice MTU1010 for resistance against bacterial blight and blast and tolerance to low soil phosphorus. PLOS ONE. 2022;17(1):e0260535.
10. Saikiran V, Krishna L, Jagadeeshwar R, Mohan YC, Shankar VG. Identification of superior hybrids and parents for blast resistance in rice (Oryza sativa L.). Journal of Pharmacognosy and Phytochemistry. 2019;8(3):3010-3013.
11. Jeevan B, Hosahatti R, Koti PS, Devappa VH, Ngangkham U, Devanna P, et al. Phenotypic and genotypic screening of fifty-two rice (Oryza sativa L.) genotypes for desirable cultivars against blast disease. PLOS ONE. 2023;18(3):e0280762.
12. International Rice Research Institute (IRRI). Standard evaluation system (SES) for rice. 5th ed. Manila (Philippines): International Rice Research Institute; 2013.