Genomic Confirmation of Resistance Genes for Blast, Bacterial Leaf Blight, Rice Tungro Spherical Virus, and Brown Planthopper in Tropically Adapted Temperate Japonica Rice Varieties

Abstract

The Rural Development Administration (RDA) of the Republic of Korea, in collaboration with International Rice Research Institute (IRRI), developed six temperate japonica rice varieties—MS11, Japonica 1, 2, 6, 7, and Cordillera 4—which were officially approved for release in tropical environments. These varieties offer improved eating quality, enhanced lodging resistance, and increased market value. Although initial evaluations indicated that the varieties were resistant to moderately resistant to major biotic stresses, recent field trials revealed a gradual increase in susceptibility over time. To address this, we conducted comprehensive evaluations of these varieties against rice blast under both greenhouse and field conditions and assessed their responses to bacterial leaf blight (BLB), rice tungro spherical virus (RTSV), and brown planthopper (BPH) under controlled environments. Additionally, whole-genome sequencing was employed to confirm the presence of known resistance alleles. Our findings revealed variable resistance profiles across the six varieties. Japonica 1 exhibited the most stable resistance to blast, supported by the presence of the Pi5 allele. Japonica 7 showed strong resistance to key BLB isolates and moderate resistance to a broader range of Xoo races, supported by the resistant Xa25/OsSWEET13 haplotype. In addition, Japonica 7, along with Japonica 6, carried the tsv1 gene for RTSV resistance. However, none of the six varieties possessed other major resistance genes for BPH. These results highlight the urgent need to introgress durable resistance genes into tropical japonica rice to enhance resilience and broaden the spectrum of biotic stress resistance—critical traits for sustainable rice production in tropical environments.

1. Introduction

Temperate Japonica rice has been revered for its distinct sticky texture and high grain quality. Unfortunately, due to its weak resilience in tropical environments, production and market access are limited to farmers, business owners, and direct consumers. There have been instances wherein temperate japonica rice was cultivated in tropical countries, but they were mostly limited in the cool and elevated regions [1]. This leads to higher market prices for japonica rice locally and globally [2]. Therefore, there is a need to develop japonica cultivars adaptable to tropical regions to meet the increasing demand and diversify available varieties. In line with this, the Rural Development Administration (RDA), Republic of Korea, has initiated a collaborative project with the International Rice Research Institute (IRRI) to develop japonica rice varieties adapted to the tropical environment. Six temperate japonica rice varieties—MS11, Japonica 1, 2, 6, 7, and Cordillera 4—were released through the IRRI-RDA collaboration to address the need for japonica rice adapted to tropical environments. These varieties overcame initial challenges such as early heading, low yield, heat sensitivity, stunted growth, spikelet sterility, and poor grain quality [3]. Prior to their release, field trials indicated that these varieties exhibited resistance to moderate resistance against major biotic stresses. However, despite initial reports of resistance, recent field observations suggest an increasing susceptibility of these tropically adapted japonica varieties to major biotic stresses. Among the most significant biotic constraints to rice production in the tropics are rice blast, bacterial leaf blight (BLB), rice tungro spherical virus (RTSV), and brown planthopper (BPH).

Rice blast caused by the fungus Pyricularia oryzae (syn. Magnaporthe oryzae) is widely recognized as the most destructive rice disease worldwide. In tropical rice-growing countries, including the Philippines, it frequently reduces yields by 10–30% under endemic conditions and can cause up to 70–80% yield loss during severe epidemics, resulting in substantial economic damage and threatening food security in rice-dependent communities [4]. BLB caused by the Gram-negative bacterium Xanthomonas oryzae pv. Oryzae (Xoo) infects rice plant through natural openings and wounds, block the water-conducting system of the plant causing the leaves to wilt, dry up, and die. In tropical Asia, this disease has been reported to reduce yields by 20–40% under endemic conditions and up to 74% during severe epidemics, resulting in substantial economic losses for farmers’ livelihoods [5]. Likewise, RTSV that is transmitted by the green leafhopper was found to cause a worldwide annual loss in rice production of approximately US $1.5 billion and 5% to 10% reduction in rice yields in South and Southeast Asia. The rice plants exhibited stunting, twisting of leaves and yellow to orange discoloration of the leaves when infected with RTSV [6]. More than 100 insect species are known to target rice, with 15–20 causing economically significant damage. Among them, BPH (Nilaparvata lugens) causes serious damage not only by feeding on phloem sap and ovipositing in plant tissues but also by transmitting viral diseases such as grassy stunt virus and ragged stunt virus, frequently causing severe yield losses and substantial economic damage to production systems [7]. Overall, these diseases and pests not only threaten yield stability but also undermine the sustainability of rice farming systems in vulnerable regions.

Various control measures have been employed to mitigate the impact of biotic stresses in rice, including chemical and biological control, optimized fertilizer management, adjustment of planting dates, and the use of disease forecasting systems [8]. But the most effective way to sustainably reduce the impact of the disease is through genetic improvement by incorporating major resistance genes into elite rice varieties [9]. In recent years, extensive efforts have led to the identification of numerous resistance genes effective against major biotic stresses in rice, including blast [10], BLB [11], BPH [12,13], and RTSV [14]. Notably, many genes have been validated under tropical field conditions, where they confer broad and durable resistance. Pi9, Pita2, Pizt, and Pi50 are examples of blast major resistance (R) genes which were found to be effective in many tropical areas [15]. Among the major R genes for BLB, Xa5 remains effective in many countries, while Xa13 has been overcome in regions such as China and Thailand. In contrast, Xa10, Xa7, and Xa21 continue to confer strong and durable resistance [16,17]. Likewise, tsv1 gene for rice RTSV resistance [14] and BPH32 resistance gene [18] were both found to be effective in tropical countries. Rice breeders have been utilizing the identified genes by introducing them into elite rice varieties to improve their resistance to diseases through marker-assisted selection (MAS). MAS aims to incorporate one or more desired genes or genomic regions into rice varieties to achieve durable, broad-spectrum resistance against diseases.

Previous studies have reported the introgression of resistance genes into japonica varieties via MAS breeding strategy. For blast resistance, advanced breeding lines derived from a cross between two japonica rice cultivars and indica rice carrying the Pi40 gene were evaluated in the field for their reaction to field blast isolates, showing only 3% diseased leaf area compared with over 50% in susceptible cultivars [19]. For BLB resistance, three BLB resistance genes (Xa4 + xa5 + Xa21) were transferred from an indica donor IRBB57 into a BLB-susceptible elite japonica rice cultivar that is high-yielding and has good grain quality. Pyramiding these resistance genes provided a higher resistance to Xanthomonas oryzae pv. oryzae (Xoo) than the introduction of the individual resistance genes [20]. For RTSV resistance, the tsv1 gene was introgressed into Japonica 1, tropically adapted japonica varieties [21]. This led to the development of 22 advanced lines that combined RTSV resistance, photoperiod insensitivity, and good grain quality. These lines outperformed Japonica1 and MS11 in yield, reaching up to 5.78 t/ha compared to 3.50 t/ha and 3.55 t/ha, respectively. For BPH resistance, the japonica variety Junambyeo, which is highly susceptible to BPH, gained improved resistance through the introgression of the Bph18 gene [22]. Combining resistance with multiple biotic stresses while maintaining high yield potential and preferred grain quality has been achieved through gene pyramiding. For example, Pi40, Xa4, xa5, Xa21, and Bph18 were combined in Jinbubyeo [23], and japonica rice introgression lines with multiple resistance genes (Bph18  +  qSTV11SG  +  Pib  +  Pik) together with Xa40 exhibited stable resistance to BPH, BLB, blast, and RTSV in all bioassays [24].

The six IRRI-RDA developed temperate japonica varieties were initially classified as moderately resistant based on field observations rather than molecular validation. Given the increasing pressure from tropical pathogens and pests, the disease resilience of these varieties has shown signs of decline under tropical field conditions. Here, the resistance profiles of these varieties against rice blast, BLB, RTSV, and BPH were reassessed to verify their actual resistance capacity under tropical conditions. To further elucidate the genetic basis of resistance, whole-genome sequencing was performed to identify the presence or absence of known resistance alleles, thereby providing insights to guide the improvement of disease resilience in temperate japonica rice.

2. Materials and Methods

2.1. Plant Materials

Six tropically adapted temperate japonica rice varieties developed by the project were used in this study for insect and disease screening, as well as for determining the presence of major biotic resistance genes (

Table 1. Basic information about tropically adapted six temperate japonica rice varieties.

Variety	Release Name	IRRI Designation	Parentage	Year Registered
MS11	NSIC Rc170 SR	IRRI 142	Jinmibyeo/Cheolweon 46	2008
Japonica 1	NSIC Rc 220 SR	IRRI 152	IR77863-95-2-3/HR15490-34	2009
Japonica 2	NSIC Rc 242 SR	IRRI 157	IR80091-46-2-1/IR71663-14-2-3-5	2012
Japonica 6	NSIC Rc 484 SR	IRRI 202	MS 11/IR86743-2B-1-4	2017
Cordillera 4	NSIC Rc 566 SR	IRRI 232	Jinmibyeo/SR18977-2-7-2-TB-1	2019
Japonica 7	NSIC Rc 584 SR	IRRI 236	Japonica 2/IR11K233	2019

). To aid in identifying effective resistance genes under both controlled and field conditions, 23 monogenic differential genotypes (MDGs) with known blast resistance genes and 9 MDGs with known bacterial leaf blight (BLB) resistance genes were also planted alongside the japonica varieties. These MDGs served as reference lines to determine which specific resistance genes were effective against the prevailing pathogen populations (

Table 2. Basic information about monogenic differential genotypes (MDGs) with known resistance genes for blast and BLB.

MDGs (Resistance Genes for Blast and BLB)
23 MDGs in LTH genetic background with known blast resistance genes:
IRBLzt-T(Pizt), IRBLz5-CA(Piz5), IRBLz-Fu(Piz), IRBL9-W(Pi9), IRBL7-M(Pi7), IRBLkp-K60(Pikp), IRBLk-Ka(Pik), IRBL20-IR24(Pi20), IRBL19-A(Pi19), IRBLta-K1(Pita), IRBL12-M(Pi12), IRBLta2-Re(Pita2), IRBL3-CP4(Pi3), IRBLi-F5(Pii), IRBLa-A(Pia), IRBLt-K59(Pit), IRBLb-B(Pib), IRBLsh-B(Pish), IRBLkh-K3(Pikh), IRBL1-CL(Pi1), IRBLkm-Ts(Pikm), IRBLks-F5(Piks), IRBL5-M(Pi5)
9 MDGs in IR24 genetic background with known BB resistance genes
IRBB1(Xa1), IRBB4(Xa4), IRBB5(xa5), IRBB7(Xa7), IRBB10(Xa10), IRBB13(Xa13), IRBB14(Xa14), IRBB21(Xa21), IRBB23(Xa23)

). The seeds of MDGs for blast and BLB screening were provided by the Plant Pathology team of IRRI.

2.2. Evaluation of Blast Resistance

The six developed japonica rice and 23 MDGs were evaluated against rice blast through a blast nursery (field evaluation) and in the greenhouse by isolate inoculation. Screening in a blast nursery was performed during the dry season from February to April 2023 in Benguet, La Trinidad, Philippines, which is located in a cool climate zone where only cold-tolerant rice varieties can grow. In all, 50 to 60 seedlings of test genotypes were planted in a complete randomized block design (CRBD) with three replicates. Border rows with a mixture of susceptible cultivars were set up around each replicate to sustain a diverse pathogen population. The Lesion type was scored on a scale of 1 (R) to 9 (S) in accordance with the standard evaluation method of IRRI Standard Evaluation System (SES) [25].

For isolate inoculation set-up in the greenhouse, the test materials were screened against 27 individual M. grisea isolates by spray following Bonman et al. method [26]. A single-race spore suspension concentration was adjusted to 1.5 × 10⁵ spores/mL. Seedlings (21 days old) were inoculated with 20 mL of spore suspension by a spray method and were kept for 24 h in darkness inside a dew growth chamber at 25 °C and then transferred to a greenhouse with a 12 h-day, 12 h-night photoperiod at 90% relative humidity for 7 days. Disease reactions were scored 7 days post inoculation using a numerical system [27]. All inoculations and disease evaluations were conducted in the greenhouse of the Plant Pathology team of IRRI.

2.3. Bacterial Leaf Blight (BLB) Resistance Screening

The test materials were grown in the glasshouse of the Plant Pathology team of IRRI. At the maximum tillering stage, the plants were inoculated with the 14 representative Xoo isolates covering all the pathotypes found in the Philippines using the leaf clipping method [28]. Plant reaction to the disease was scored 14 days after inoculation by measuring lesion length (cm). The reaction of resistance was expressed in lesion length (resistant: <3 cm, moderately resistant: 3–5 cm, susceptible: >5 cm).

2.4. Rice Tungro Spherical Virus (RTSV) Resistance Screening

RTSV screening was conducted over four consecutive seasons (2023 DS, 2023 WS, 2024 DS, and 2024 WS) by the Cross-Cutting Services—Biotic Stress Resistance Evaluation Center (CCO-BSREC) at IRRI. Test entries, two check varieties, TN1 (susceptible) and TW16 (resistant), were seeded in trays for standardized evaluation. The screening process began with the acquisition of rice tungro virus (RTV) by mass-reared green leafhoppers (GLH), which were allowed to feed on RTV-infected source plants 30 days after sowing to become viruliferous. At 5 days after sowing (DAS), test seedlings were exposed to these viruliferous GLHs for 4–5 days. After a 9–10-day incubation period, the seedlings were trimmed and transferred to seed boxes placed inside water trays enclosed by screen cages. Viruliferous GLHs were then released into the cages at a density of 6–7 insects per seedling, allowing inoculation access for 2–3 h. Following inoculation, the seedlings were moved to concrete benches outside the greenhouse to allow disease progression until the tillering stage, approximately 21 days after inoculation (DAI). Disease scoring was performed at 21 DAI or earlier if TN1 exhibited typical tungro symptoms, using the IRRI Standard Evaluation System [25].

2.5. Brown Planthopper (BPH) Resistance Screening

Screening for BPH resistance was also conducted over four consecutive seasons (described above) by CCO-BSREC at IRRI. Entries were seeded in seed boxes measuring 110 × 65 × 5 cm, each including two check varieties: IR62 (resistant) and TN1 (susceptible). After seeding, the boxes were placed in a germination room for 7 days to ensure uniform seedling emergence. At 7 DAS, the seed boxes were transferred to the resistance screening room, where plants were infested with second and third instar BPH nymphs. The infestation density was approximately 10 nymphs per seedling for BPH. Damage progression was monitored regularly, and additional insects were introduced if no visible damage was observed on the susceptible check (TN1) within 3 days of infestation. Plant damage was scored using the IRRI Standard Evaluation System [25], allowing for consistent assessment of resistance across all entries.

2.6. Whole Genome Sequencing

Genomic DNAs were extracted from seedling leaves of the six japonica rice varieties (MS11, Cordillera 4, Japonica 1, 2, 6, and 7), respectively, by using the modified CTAB method [29]. PCR-free library was constructed, and the samples were sequenced by the BGI DNBseq NGS platform. Number of 150 bp Paired-End reads ranged from 80,077,301 (24,023,190,300 bp) to 80,261,886 (24,078,565,800 bp), resulting in ~63.1× coverage of the rice reference genome (O. sativa ssp. japonica var. Nipponbare = ~381 Mbp) in each sample (

Table 3. Summary of NGS results and de novo sequence assembly.

Entries	NGS Results		De Novo Sequence Assembly
	Read Count	Total Bases (bp)	n	N50	E-Size (bp)	Max (bp)
MS 11	80,261,886	24,078,565,800	451,900	22,351	29,473	186,259
Japonica 1	80,233,025	24,069,907,500	574,499	20,068	26,851	186,312
Japonica 2	80,178,258	24,053,477,400	538,383	21,674	28,225	186,266
Japonica 6	80,077,301	24,023,190,300	779,768	17,025	22,069	141,647
Cordillera 4	80,194,377	24,058,313,100	422,982	20,023	26,557	185,804
Japonica 7	80,215,226	24,064,567,800	486,943	20,863	27,599	158,615

).

2.7. NGS Analysis and Allele Typing of the Biotic Stress Resistance Genes

The whole-genome sequence data was analyzed by using the embedded NGS analysis software and computing resources at the Galaxy EU server (https://usegalaxy.eu, accessed on 6 November 2025) [30]. De Novo sequence assembly was conducted by using ABySS software (Word size: 70, filter out < 500 bp, and default parameters). Average sizes of scaffolds were 22,069–29,473 bp. The assembled scaffold sequences were made as nucleotide blast database by using ‘Make BLAST DB’ tool. Homologous sequences of the biotic stress resistance genes were extracted from the BLAST DB containing scaffold sequences from the six japonica rice varieties by using the BLAST tool embedded in the NCBI Genome Workbench software packages with the default parameters (Word size: 40–80). After extraction of the target biotic stress resistance genes in each variety, multiple sequence alignment was conducted together with the donor allele sequence and the corresponding genes from the rice reference genome IRGSP1.0 (https://rapdb.dna.affrc.go.jp/index.html, accessed on 1 August 2023) by using BioEdit version 7.7 [31]. Based on the sequence comparisons, susceptible and resistant alleles for the target genes were defined.

3. Results

3.1. Blast Resistance at Natural Blast Nursery and 27 M. grisea Isolate Inoculation

The six released, tropically adapted temperate japonica rice varieties, along with 24 MDLs carrying characterized blast R genes, were evaluated under natural blast nursery conditions. For the japonica varieties, Japonica 1 consistently exhibited stable resistance (Figure 1). Japonica 2 and Japonica 7 recorded scores of 4 and 5, respectively. MS 11 and Cordillera 4 recorded median scores of 6. In contrast, Japonica 6 as rated was susceptible among the Japonica group. Among the MDGs, the two lines carrying the Pita2 gene, IRBLta2-Re and IRBLta2-Pi, exhibited strong resistance to leaf blast (Figure 1). Likewise, monogenic lines harboring the Pi9 gene (IRBL9-W) or Pi12 gene (IRBL12-M) displayed moderate resistance. In case of Pita gene, IRBLta-K1 line showed more moderate resistance phenotype than IRBLta-CP1 line. In addition, several lines carrying Piz-5, Pit, Pi12, Pikm, and Piks also showed moderate resistance, whereas those carrying Piz-t, Piz, Pi7, Pikh, Pikp, Pi1, Pi20, Pi19, Pi3, Pii, Pia, Pib, Pish, and Pi5 were susceptible under the same conditions.

To verify these field-based observations and assess isolate-specific reactions, the same japonica entries and MDGs were subjected to artificial inoculation using 27 P. oryzae isolates. The japonica varieties exhibited high resistance frequencies ranging from 65% to 85%. Japonica 2 achieved the highest artificial inoculation resistance frequency among the japonica varieties, showing strong resistance to 23 isolates, moderate to three, and weak to one (

Table 4. Summary of the disease resistance frequencies of Japonica rice varieties and monogenic lines against 27 Magnaporthe oryzae isolates.

Entries	R Genes	Total Number of Resistant Entries to 27 Blast Races	Percentage of Resistant Monogenic Lines
MS 11	-	21	77.78
Japonica 1	-	21	77.78
Japonica 2	-	23	85.19
Japonica 6	-	17	62.96
Cordillera 4	-	22	81.48
Japonica 7	-	19	70.37
IRBLa-C	Pia	2	7.41
IRBLks-F5	Piks	3	11.11
IRBLk-Ka	Pik	13	48.15
IRBLkp-K60	Pikp	14	51.85
IRBLkh-K3	Pikh	14	51.85
IRBL7-M	Pi7	13	48.15
IRBL1-CL	Pi1	14	51.85
IRBLz-Fu	Piz	23	85.19
IRBLz5-CA	Piz-5	23	85.19
IRBLzt-T	Piz-t	12	44.44
IRBLta-CT2	Pita	9	33.33
IRBLb-B	Pib	7	25.93
IRBLt-K59	Pit	2	7.41
IRBLsh-S	Pish	16	59.26
IRBLi-F5	Pii	16	59.26
IRBL3-CP4	Pi3	18	66.67
IRBL5-M	Pi5	18	66.67
IRBL9-W	Pi9	25	92.59
IRBL12-M	Pi12	24	88.89
IRBL19-A	Pi19	1	3.70
IRBL20-IR24	Pi20	9	33.33
IRBLta2-Pi	Pita-2	22	81.48
IRBL11-Zh	Pi11	11	40.74
LTH	-	-	-

R: resistance (1~3), MR: moderate resistance (4~6), S: susceptible (7~9).

, Supplementary Table S1). Notably, Japonica 1, despite showing the strongest resistance in the natural blast nursery, recorded a slightly narrower resistance spectrum under artificial inoculation compared to Japonica 2. Japonica 1 demonstrated strong resistance to 21 isolates, moderate resistance to three isolates, and weak resistance to three isolates, maintaining robust responses to highly virulent isolates such as M36-1-3-10-1 and BN111. However, moderate reactions to PO6-6 and M64-1-3-9-1 indicated potential gaps in its resistance coverage. MS11 and Japonica 7 also maintained relatively broad spectra, each showing strong resistance to more than 19 isolates but with moderate susceptibility to several. Japonica 6 exhibited much narrower resistance profiles, with strong resistance to 17 isolates. Among the MDGs, IRBL9-W (Pi9), which had exhibited strong field resistance, showed the highest resistance frequency (92%) in the isolate inoculation test, displaying strong resistance to 25 isolates, moderate resistance to two isolates, and no weak reactions (Table 4, Supplementary Table S1). IRBLta2-Pi, which had exhibited strong field resistance, showed high strong-resistance frequencies but exhibited moderate reactions to a few isolates. Among the lines that had shown moderate field resistance, those carrying Pi12 (89%) and Piz/Piz-5 (85%) ranked next in resistance frequency.

3.2. BLB Resistance by 14 Representative Xoo Isolates Inoculation

The screening of the six developed japonica rice varieties for BLB resistance was conducted using 14 representative Xoo isolates under controlled screenhouse conditions (

Table 5. Summary of BLB resistance frequencies of Japonica rice varieties and NILs against 14 Xanthomonas oryzae pv. oryzae isolates in the Philippines.

Entry	R Gene	Number of Resistant Reactions (R-MR)	Resistance Frequency (%)
MS11	-	2	14.3
Japonica 1	-	4	28.6
Japonica 2	-	6	42.9
Japonica 6	-	6	42.9
Cordillera 4	-	6	42.9
Japonica 7	-	9	64.3
IRBB1	Xa1	5	35.7
IRBB4	Xa4	6	42.9
IRBB5	xa5	12	85.7
IRBB7	Xa7	10	71.4
IRBB10	Xa10	5	35.7
IRBB13	Xa13	1	7.1
IRBB14	Xa14	4	28.6
IRBB21	Xa21	12	85.7
IRBB23	Xa23	14	100
IR24	-	0	0

R: resistance (<3 cm), MR: moderate resistance (3~5 cm), S: susceptible (>5 cm).

, Supplementary Table S2). All six japonica rice varieties demonstrated a strong resistance response against PXO 339 race. Beyond this common response, however, the varieties displayed a differential spectrum of resistance across the other isolates, highlighting both shared and unique resistance characteristics. Among the six entries, Japonica 7 consistently exhibited the broadest resistance spectrum, showing resistance to moderately resistant reactions against 9 out of the 10 tested PXO isolates. In contrast, the other japonica varieties revealed more selective responses. For instance, Japonica 2 was resistant to PXO 79, showed resistant to moderately resistant reactions against PXO 340, and maintained moderate resistance to PXO 349. Meanwhile, Japonica 6 expressed only moderate resistance to PXO 79, indicating a narrower resistance profile compared to Japonica 7 and Japonica 2. Japonica 1 and MS11 showed intermediate responses, with moderate resistance observed against certain isolates but susceptibility to others. Cordillera 4, however, displayed relatively weaker resistance across most isolates tested, making it more vulnerable compared to the other entries.

The NILs carrying single R genes were also evaluated with the same set of isolates, thereby providing a reference framework to interpret the resistance mechanisms in the japonica varieties (Table 5, Supplementary Table S2). Notably, lines harboring xa5, Xa7, Xa21, and Xa23 consistently showed resistance across representative Xoo isolates. When compared to the japonica varieties, it is evident that certain japonica entries, particularly Japonica 7 and Japonica 2, may share functional resistance pathways similar to those mediated by Xa21 or Xa23, given their broad-spectrum responses.

3.3. RTSV and BPH Resistance

The resistance screening against RTSV was performed across four consecutive seasons using the same japonica entries (

Table 6. RTV and BPH resistance evaluation across cropping seasons.

MS 11	MR	R	MR	MR	RTV Disease Index Rating
Japonica 1	MR	R	MR	MR
Japonica 2	MR	R	MR	MR
Japonica 6	MR	R	MR	MR
Cordillera 4	MR	R	MR	MR
Japonica 7	R	R	MR	MR
	2023 DS	2023 WS	2024 DS	2024 WS
MS 11	S	S	MR	S	BPH Scores
Japonica 1	S	S	MR	S
Japonica 2	MR	S	MR	S
Japonica 6	MR	S	S	MR
Cordillera 4	MR	S	MR	R
Japonica 7	MR	S	MR	R

R: resistance (1~3), MR: moderate resistance (4~6), S: susceptible (7~9).

). The results revealed a consistent but seasonally influenced pattern of resistance expression. In the 2023 dry season (DS), five entries, MS 11, Japonica 1, Japonica 2, Japonica 6, and Cordillera 4, were rated moderately resistant (MR), while Japonica 7 showed a stronger reaction, being rated resistant (R). By contrast, in the 2023 wet season (WS), all six entries exhibited an R rating, showing complete resistance across the japonica panel. In the following year, however, resistance expression decreased. During 2024 DS and 2024 WS, all six entries were consistently rated MR. Despite these seasonal shifts, no entry was rated Susceptible (S) in any season.

Resistance to BPH was evaluated in the same six japonica entries across the same four seasons (Table 6). In contrast to the RTSV results, BPH resistance showed greater variation both across seasons and among varieties, with no uniformly stable resistant type observed. Cordillera 4 and Japonica 7 exhibited the strongest resistance profiles among the panel. Cordillera 4 was rated MR in 2023 DS and 2024 DS, and notably R in 2024 WS, while Japonica 7 showed a similar trend, being rated MR in 2023 DS and 2024 DS and R in 2024 WS. In contrast, Japonica 1 and MS 11 were the most vulnerable, receiving three S ratings and only one MR rating each across the four seasons. Japonica 2 and Japonica 6 displayed intermediate but unstable resistance, with both varieties recording two MR and two S ratings.

3.4. Allele Typing of the Major Biotic Stress Resistance Genes by Whole Genome Sequencing

DNA sequence variation directly affects protein functionality and the expression of defense-related genes, thereby determining the susceptibility or resistance of host plants to pathogens and pests. To elucidate the genetic basis of resistance within the japonica rice panel, whole genome sequencing of the six varieties was performed, followed by de novo sequence assembly (Table 3). Using BLAST-based approaches, scaffolds containing key resistance (R) genes were extracted from each genome and aligned against both known donor resistance alleles and the rice reference genome (IRGSP-1.0). The analysis specifically targeted four major biotic stresses: blast, BLB, RTSV, and BPH.

For blast resistance, three major genes (Pi9, Pita2, and Pi5) were selected for detailed analysis. These loci were prioritized because they represent some of the most widely deployed and well-characterized resistance sources in global rice breeding programs. Pi9 and its related haplotypes (Pi2, Piz-t, and Pi50) are clustered within a ~10.4 Mb region on chromosome 6 that harbors a repeated array of NLR (nucleotide-binding leucine-rich repeat) genes (2–13 repeats), and multiple studies have confirmed that they correspond to the same locus exhibiting different resistance haplotypes [32]. The corresponding NLR cluster sequences were extracted from the six japonica varieties and compared with donor resistance haplotypes. The results revealed that Japonica 6 carried a sequence identical to Nipponbare (a known susceptible allele), while the remaining five japonica varieties possessed a uniform haplotype distinct from both Nipponbare and the resistant donor haplotypes. Importantly, none of the sequences matched the functional Pi9/Pi2/Piz-t/Pi50 resistant haplotypes, strongly indicating that all six japonica rice varieties harbor only susceptible alleles at this locus (

Table 7. Allele typing of the major biotic stress resistance genes by using NGS analysis.

Gene	Chr.	Location (bp)	Encoding Protein	Haplotype (S = Susceptible/R = Resistant Allele)
				MS11	Japonica1	Japonica2	Japonica6	Cordillera4	Japonica7
Pi9/Pi2/ Piz-t/Pi50	6	10,387,509	NBS-LRR	GUVA1 (S)	GUVA1 (S)	GUVA1 (S)	Nip (S)	GUVA1 (S)	GUVA1 (S)
Pita2 = Ptr	12	10,822,534	Armadillo repeats protein	Nip (S)	Nip (S)	Nip (S)	Nip (S)	Nip (S)	Nip (S)
Pi5	9	9,681,913	NBS-LRR	(S)	(R)	(S)	(S)	(S)	(S)
xa5	5	437,043	Transcription factor IIA gamma subunit	Nip (S)	Nip (S)	GUVA1 (S)	Nip (S)	Nip (S)	GUVA1 (S)
Xa7	6	28,015,259	Executor R protein	(S)	(S)	(S)	(S)	(S)	(S)
Xa21	11	21,277,443	LRR receptor kinase-like protein	Nip (S)	GUVA1 (S)	Nip (S)	Nip (S)	Nip (S)	Nip (S)
Xa23	11	22,204,131	Executor R protein	Nip (S)	GUVA1 (S)	Nip (S)	Nip (S)	Nip (S)	GUVA2 (S)
Xa25/OsSWEET13	12	17,302,127	SWEET-type protein	Nip (R)	Nip (R)	Nip (R)	Nip (R)	Nip (R)	GUVA1 (R?)
BPH32 = BPH3	6	1,223,069	Unknown short consensus repeat (SCR) domain-containing protein	Nip (S)	Nip (S)	Nip (S)	Nip (S)	Nip (S)	Nip (S)
tsv1	7	22,114,961	Eukaryotic translation initiation factor 4G (eIF4G)	Nip (S)	Nip (S)	Nip (S)	Kinmaze (R)	Nip (S)	Kinmaze (R)

Nip, Nipponbare allele; GUVA, new alleles not belonging to Nip or the resistance donor alleles; Kinmaze, RTSV resistance allele possessing.

).

Analysis of the Pita2/Ptr gene on chromosome 12 further supported this trend. All six japonica varieties, along with Nipponbare, shared identical sequences, none of which were identical to the functional resistant allele reported from Katy [33]. Thus, despite the importance of Pita2 as a resistance source in indica breeding, the japonica panel does not appear to carry this allele. The Pi5 locus, however, revealed a contrasting result. Pi5-mediated resistance requires the functional cooperation of two NBS-LRR genes, Pi5-1 and Pi5-2 [34]. Sequence analysis showed that only Japonica 1 contained alleles identical to the resistant donor sequences of both Pi5-1 and Pi5-2, whereas the other five japonica varieties carried non-functional or susceptible haplotypes.

With similar approaches, allelotypes of five major BLB resistance genes (xa5, Xa7, Xa21, Xa23, and xa25) were analyzed across the six-japonica rice varieties (Table 7). The xa5 locus on chromosome 5 encodes the transcription factor IIA γ-subunit. Resistance is defined by a critical nonsynonymous SNP at the 39th amino acid position, where the susceptible allele carries GTC (valine), while the resistant allele carries GAG (glutamic acid) [35]. All six-japonica varieties carried the same coding sequence as the Nipponbare allele, containing the GTC codon, indicating susceptibility. However, Japonica 2 and Japonica 7 contained a few SNPs within the final intron of xa5, though these variations are unlikely to affect gene function. This finding confirms that xa5-mediated resistance is absent in the -panel. The Xa7 gene, encoding an executor-type R protein [36], was also investigated. BLAST-based searches using donor Xa7 sequences failed to identify a homologous sequence in any of the six-japonica varieties, suggesting that none of the entries carry the functional Xa7 resistance allele. This result is consistent with phenotypic observations, where the japonica varieties showed susceptibility to several Xoo isolates typically defeated by Xa7 (Table 5). The Xa21 locus, a well-known introgression from Oryza longistaminata, encodes a leucine-rich repeat (LRR) receptor kinase that mediates broad-spectrum BLB resistance [37].

Multiple sequence alignments demonstrated that none of the six japonica varieties carried sequences identical to the functional Xa21 donor allele found in IRBB21. Instead, all were aligned with susceptible haplotypes, implying that Xa21-mediated broad-spectrum resistance is also absent from the panel. The Xa23 gene encodes another executor-type R protein, whose resistance is triggered when the bacterial effector AvrXa23 binds to the effector-binding element (EBE^AvrXa23) in its promoter [38]. Sequence analysis revealed three haplotypes—Xa23-Nip, Xa23-GUVA1, and Xa23-GUVA2—across the panel. However, none of these carried the EBE^AvrXa23 motif, confirming that all six japonica varieties harbor only susceptible Xa23 alleles. This provides a genetic explanation for their limited resistance spectrum against Xoo races that are controlled by Xa23. Interestingly, analysis of xa25 (OsSWEET13) provided a contrasting result. Resistance at this locus is conferred by mutations in the PthXo2 promoter region, which prevent transcriptional activation of OsSWEET13 by Xoo TAL effectors [39]. All six japonica varieties, except for Japonica 7, carried resistant-type alleles like Nipponbare and Kitaake, both of which contain an adenine (A) at the sixth nucleotide position in the EBE motif of the PthXo2 promoter (Figure 2). Japonica 7 harbored a novel promoter haplotype, but functional inference suggested that it also conferred resistance due to the same critical deletion. Thus, xa25/OsSWEET13 represents the only major BLB resistance locus retained across the japonica panel, albeit with limited functional diversity.

For RTSV resistance, only one major gene, tsv1, has been reported to date. TSV1 encodes the eukaryotic translation initiation factor 4G (eIF4G). Resistance is associated with nonsynonymous mutations at amino acid positions 1056–1063, which alter host–virus interactions and prevent successful viral replication [40,41]. Sequence analysis revealed that among the japonica rice panel, Japonica 6 and Japonica 7 carried the Kinmaze-derived resistance allele (tsv1-Kin), while the remaining four varieties carried the Nipponbare-type susceptible allele (tsv1-Nip). The resistant tsv1-Kin allele includes specific amino acid substitutions in eIF4G, which have been demonstrated to confer robust RTSV resistance in japonica backgrounds. We further examined allelotypes of one major brown planthopper (BPH) resistance gene and the only reported rice tungro spherical virus (RTSV) resistance gene. The Bph32 (allelic to Bph3) locus on chromosome 6 encodes a protein associated with antibiosis and antixenosis effects against BPH feeding [42]. Functional alleles of Bph32, introgressed from indica sources, have conferred stable resistance in multiple genetic backgrounds. Sequence comparison revealed that all six japonica rice varieties carried the identical sequence of the Nipponbare (Bph32-Nip) allele, which is known to be susceptible. This indicates that none of the varieties possess functional Bph32 resistance alleles, consistent with phenotypic screening results showing universal susceptibility to BPH infestation. The absence of functional Bph32 emphasizes the vulnerability of japonica varieties to hopperburn and virus transmission mediated by BPH, suggesting that targeted introgression of BPH resistance loci—such as Bph32, Bph17, or Bph18—from indica germplasm could be beneficial for future breeding.

4. Discussion

The six temperate japonica rice varieties, released through the IRRI-RDA project and adapted for tropical environments, were initially characterized as resistant to moderately resistant to most major biotic stresses at the time of their release [3]. However, as commonly seen in agricultural ecosystems, plant pathogens rapidly evolve under strong selection pressure [43]. Consequently, recent field observations and systematic re-evaluations have revealed that these japonica varieties now exhibit varying levels of susceptibility, particularly to blast, BLB, RTSV, and BPH. These findings highlight the dynamic nature of host pathogen interactions and the importance of continuous monitoring and genetic reinforcement of resistance in released varieties.

Field screening in natural blast nurseries provided a clear example of this phenomenon. While all six japonica varieties showed tolerance to multiple P. oryzae isolates collected from Bohol, a hot and humid lowland field conditions, the response under cooler highland conditions such as Benguet differed markedly. In particular, only Japonica 1 maintained strong and stable resistance across environments. The unique presence of Pi5 donor alleles in Japonica 1 is consistent with its relatively stronger blast resistance phenotype observed under both natural nursery and artificial inoculation conditions, thereby providing strong genetic evidence linking the phenotypic resistance to the underlying functional alleles. The Cordillera 4, previously classified as cold-tolerant with intermediate blast resistance, exhibited clear susceptibility under the colder nursery conditions (Figure 1). Molecular analyses supported these phenotypic observations. While Japonica 1 alone carries the functional Pi5 allele, the absence of other major resistance genes such as Pi9 and Pita2 in the remaining japonica lines highlights a critical gap in their genetic defense against blast. This finding aligns with broader evidence from field screening in Benguet, where Pi9 and Pita2 genes were shown to confer high levels of resistance under cooler conditions (Table 3). Both genes encode novel proteins that have been widely reported to provide durable and broad-spectrum blast resistance across diverse environments [44,45]. Differences between field and greenhouse resistance responses observed in this study can be explained by the contrasting pathogen population structures and environmental modulation of host defense. In field blast nurseries, genetically diverse and mixed P. oryzae populations interact with fluctuating temperature and humidity, which can suppress or destabilize NLR-mediated immunity, resulting in reduced resistance expression in several japonica varieties under cooler highland conditions. In contrast, greenhouse inoculations use single isolates under controlled conditions, often allowing quantitative or minor-effect resistance loci to express more clearly.

These findings highlight the potential value of incorporating Pi9 and Pita2 into japonica backgrounds, particularly for production areas in the tropical highlands or regions with cool climates where blast epidemics are frequently more severe. Durability of blast resistance is consistently greater when R genes are pyramided rather than used singly. For example, pyramiding Pi9 with Pita/Pita2 improved resistance in Basmati/japonica derivatives, while combining Piz-t and Pi54 enhanced panicle blast resistance, although some combinations (e.g., Pi9 + Pi54) showed variable outcomes, emphasizing the need for empirical validation [46]. At the breeding scale, MAS-enabled pyramiding of Pi1, Pi2, Pi9, Piz-t, and Pi54 has generated broad-spectrum, more stable resistance in japonica [47]. Reviews confirm that pyramiding (Pi1, Pi2, Pi9, Pi54, Pigm, Piz-t) remains the most reliable approach under heterogeneous pathogen populations. Population surveys reinforce this strategy: japonica varieties carrying R-gene sets such as Pik, Pi9, Piz-t, and Pita, or broader stacks (Pib + Pita + Pi5 + Pikh + Pik + Pi9), exhibited consistently higher tolerance against diverse P. oryzae races [48].

Furthermore, cases in which resistance was detected despite the absence of known major-effect genes suggest that background-dependent quantitative resistance or epistatic interactions may contribute to the phenotype; however, such resistance is inherently less stable and more vulnerable to shifts in pathogen virulence or environmental stress. Taken together, these observations highlight a structural vulnerability in the current resistance architecture of the japonica varieties and reinforce the need to introduce major broad-spectrum resistance genes—such as Pi9, Pita2/Ptr, Pi54, or Pigm for blast, to achieve durable and environment-stable resistance across diverse rice-growing conditions.

In the context of BLB, Japonica 7 emerged as a key entry, exhibiting strong resistance to the representative Xoo isolates PXO 339 and moderate resistance to a broader spectrum of Xoo races. These phenotypic observations are supported by whole-genome sequencing results, which revealed that all six japonica varieties carry the resistant haplotype of Xa25/OsSWEET13. Previous study reported that a substantial portion of Japonica germplasm already harbors resistant Xa25 alleles, suggesting that the presence of this allele in Japonica lines likely originated from their parental Japonica backgrounds rather than from donor introgression [39]. The resistant allele at the Xa25/OsSWEET13 locus is known to confer defense against Xoo strains carrying TAL effectors such as PthXo2 or AvrXa7. Although functional alleles of other major BLB resistance genes (Xa5, Xa7, Xa21, and Xa23) were absent in the japonica varieties, the presence of Xa25 may contribute to the partial or unstable resistance responses observed during inoculation trials. To further explore the potential of these resistance genes, screening of near-isogenic lines (NILs) was conducted, revealing that xa5, Xa7, Xa21, and Xa23 conferred reliable resistance against representative Xoo isolates. These genes are well-recognized and have been widely adopted in global breeding programs to broaden resistance spectra against BLB [49,50]. Their effectiveness in NILs emphasizes their importance for japonica improvement, as introgression of these genes could stabilize BLB resistance under fluctuating pathogen pressures. The stacking of Xa7 and Xa21, for instance, has been proven to confer long-lasting field resistance in tropical rice-growing areas, and similar strategies could be employed for developed japonica lines to enhance yield stability under disease pressure.

RTSV resistance screening across four seasons showed that none of the six Japonica entries were susceptible, indicating inherent tolerance within the Japonica background. Japonica 7 consistently exhibited the strongest resistance, while Japonica 6 maintained moderate resistance across all seasons. Complete resistance observed in the 2023 WS across all entries suggests environmental enhancement of tolerance. However, the decline to moderate resistance in 2024 highlights seasonal instability. The seasonal fluctuations observed in RTSV resistance suggest that its expression is influenced by a combination of environmental conditions, vector dynamics, and genetic background interactions. Environmental factors such as temperature, humidity, and rainfall patterns are known to affect both the efficiency of virus transmission by green leafhoppers and the physiological state of the host plant, which in turn can modulate the expression of resistance genes. Climatic irregularities, asynchronous planting schedules, and intensive cropping systems have been associated with heightened viral disease pressure and diminished resistance stability in tropical rice-growing areas [51]. These seasonal patterns in resistance expression are further clarified by molecular evidence, which provides insight into the genetic basis underlying the observed phenotypic responses.

The phenotype results are supported by the whole-genome sequencing analysis which revealed the presence of the RTSV resistance allele (Kinmaze-type resistant allele of tsv1) in two entries, Japonica 6 and Japonica 7. This result supports previous findings indicating that Japonica 7 possesses the resistant haplotype of EIF4G [40]. The presence of the tsv1 resistance allele in the two japonica entries aligns with their relatively improved tolerance to RTSV infection, highlighting the critical role of EIF4G-mediated translation initiation control in conferring viral resistance. Moreover, it provides a molecular explanation for the superior performance of Japonica 7 under RTSV stress compared with its sibling varieties.

In contrast to RTSV, BPH resistance among the six Japonica entries was more variable and less stable across seasons. Cordillera 4 and Japonica 7 showed the highest relative tolerance, while Japonica 1 and MS 11 were consistently susceptible. This susceptibility aligns with whole-genome sequencing results, which confirmed the absence of functional alleles at the BPH32 locus across all entries. The inconsistent performance of japonica varieties further highlights the lack of durable resistance in the panel. Seasonal instability in BPH resistance may be explained by shifts in BPH population dynamics and biotype composition. BPH is known for its rapid adaptation and the emergence of new virulent biotypes that can overcome host resistance, especially when resistance is conferred by a limited number of genes. Resistance conferred by single genes is often short-lived due to the emergence of new BPH biotypes, while environmental stressors such as drought and nutrient imbalances may further weaken plant defense mechanisms [52]. Moreover, BPH populations exhibit complex biotype variation and genetic differentiation across regions, which can affect the durability of resistance genes and contribute to seasonal fluctuations in resistance expression [53]. To address this instability, functional validation studies have highlighted the potential of BPH32 in conferring durable resistance. The functional validation of BPH32, which encodes a novel protein, has also shown its critical role in conferring resistance when transferred into otherwise susceptible backgrounds [42]. The addition of BPH32 into japonica varieties could significantly reduce yield losses associated with hopper burn and virus transmission.

5. Conclusions

From a breeding perspective, the results clearly indicate that despite their initial classification as tolerant to moderately tolerant, the six tropically adapted japonica varieties require significant improvement in their resistance portfolio. The agreement between independent genotyping approaches provides strong validation of their genetic predisposition to major biotic stresses, helping explain the high variability and weak resistance observed in phenotypic screening trials across different environments. The whole-genome sequencing revealed that Japonica 1 carries the Pi5 allele for blast resistance, all six possess the Xa25/OsSWEET13 allele associated with BLB susceptibility, Japonica 6 and Japonica 7 contain the tsv1 allele conferring RTSV resistance, while none carry functional resistance alleles for BPH. These findings are consistent with previous SNP-based allele typing [53], further confirming their genetic vulnerability. This vulnerability, combined with the evolutionary adaptability of pathogens such as Magnaporthe oryzae, Xoo, and BPH, affirms the urgency of pyramiding multiple resistance alleles into new japonica backgrounds. Given the instability of field resistance and the confirmed susceptibility at key loci, intensified efforts to incorporate durable resistance genes through marker-assisted selection or genomic breeding are indispensable. Without such improvement, yield stability will remain at risk, especially under rapidly changing pathogen populations in tropical agro-ecosystems.

Taken together, the genomic insights corroborate the phenotypic screening results and provide a clear genetic basis for the observed differences among varieties, offering significant implications for improving japonica rice in tropical environments. The breakdown of blast resistance in several japonica varieties necessitates urgent incorporation of new resistance sources, particularly those effective under cold stress conditions. Notably, Japonica 1’s consistent resistance across contrasting environments positions it as a key donor for developing blast-resistant varieties suited to both lowland and highland ecosystems. Likewise, the strong and stable performance of Japonica 7 against BLB and RTSV highlights its potential as a valuable contributor for assembling resistance alleles. In contrast, the absence of functional BPH resistance across all entries represents a critical vulnerability, especially given BPH’s dual role as a pest and virus vector. These findings emphasize the need for molecular breeding strategies to strengthen the genetic base of japonica lines. Although initially released as moderately resistant, their current susceptibility reflects the dynamic nature of pathogen and pest populations. Future breeding efforts should prioritize the introgression of validated resistance genes such as Pi9, Pita2, and BPH32 into the japonica rice background to enhance multi-disease resilience and ensure durable protection under evolving tropical rice production systems.