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Review

Investigating the Mechanisms Underlying the Durability and Sustainable Use of Pb1 Gene-Mediated High Field Resistance to Rice Panicle Blast

by
Kiyoshi Fujii
1,2,
Taro Suzuki
2,3,
Mitsuru Nakamura
2,3,
Tomofumi Yoshida
3,
Yoshinori Uchikawa
4,
Haruka Suwazono
4,
Nagao Hayashi
5,
Yasukazu Kanda
5,* and
Haruhiko Inoue
4,5,*
1
Rice and Wheat Promotion Association of Aichi, Nagoya 460-003, Japan
2
Aichi Agricultural Research Centre, Nagakute 480-1193, Japan
3
Mountainous Region Agricultural Institute, Aichi Agricultural Research Centre, Toyota 441-2513, Japan
4
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda 278-8510, Japan
5
National Agriculture and Food Research Organization (NARO), Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan
*
Authors to whom correspondence should be addressed.
Agronomy 2023, 13(7), 1751; https://doi.org/10.3390/agronomy13071751
Submission received: 17 May 2023 / Revised: 20 June 2023 / Accepted: 27 June 2023 / Published: 28 June 2023
(This article belongs to the Special Issue Advances in Rice Physioecology and Sustainable Cultivation)
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Abstract

:
Rice blast, caused by Magnaporthe oryzae, poses a significant threat to rice production. Rice blast susceptibility has been observed in Japanese rice varieties with excellent eating quality. Enhancing blast resistance is essential to ensure minimal use of agricultural chemicals. Two types of blast resistance are observed: True resistance, which is a type of qualitative resistance expressed by a major gene, and field resistance, which is a type of quantitative resistance expressed by multiple micro-acting genes. ‘Resistance collapse’, in which a variety with a true resistance gene becomes diseased by blast fungus races compatible with the resistance gene, has been observed. Varieties carrying blast-resistance genes, such as Pb1 (panicle blast resistance 1), have been developed through DNA marker-assisted selection. In this review, we focus on the Pb1, which expresses strong quantitative resistance to panicle blast and has been widely used in Japan without showing ‘resistance collapse’ for 40 years. Pb1 is an ‘adult plant resistance gene’ that does not exert strong selection pressure on the blast population during the leaf blast stage, thus preventing the selective multiplication of Pb1-compatible blast strains. This epidemiological mechanism prevents ‘resistance collapse’. Interdisciplinary research and breeding are required to sustainably use genes that induce high field resistance.

1. Introduction

Rice blast is a major disease caused by Magnaporthe oryzae in rice-growing regions worldwide. Rice panicle blast is of particular concern because it is directly correlated with yield loss and quality decline. In Japan, rice production focuses on high-eating-quality varieties, such as ‘Koshihikari’, ‘Hitomebore’, ‘Hinohikari’, ‘Akitakomachi’, and ‘Nanatsuboshi’. However, these varieties have poor resistance to blast, which contributes to disease occurrence and increases the difficulty of controlling the disease [1,2,3,4]. Despite the use of fungicides as prophylactics to prevent damage, panicle blast damage persists (Figure 1A). Breeding resistant varieties is an effective, economical, and environmentally friendly integrated pest management (IPM) technology that can reduce rice blast damage and pesticide use, contributing to the achievement of sustainable development goals (SDGs) [5,6,7]. In Japan, two methods have been used for rice blast-resistant breeding: Introducing true resistance, which is a type of qualitative resistance expressed by a major gene from foreign rice varieties, and introducing field resistance, which is a type of quantitative resistance expressed by a minor gene or polygene from upland rice varieties. Although true resistance is relatively easy to introduce, it is unstable because resistance often collapses within a few years after the dissemination of true-resistant varieties. Moreover, field resistance is generally more stable and less susceptible to resistance collapse. However, it is challenging to introduce and integrate all field resistance genes into one commercial variety while eliminating defective traits, such as poor eating quality associated with upland rice, using conventional breeding techniques [8,9,10].
The discovery of genes that express quantitative resistance (herein referred to as ‘field resistance’) and the development of DNA markers closely linked to these genes started in the 1990s [11,12,13]. DNA marker-assisted selection (MAS) of genes that express high field resistance, such as Pb1 (panicle blast resistance 1), Pi21, Pi35, and Pi39, has been increasingly utilized in breeding [14,15,16,17]. A recent example is the ‘Mineasahi SBL’ variety, which was bred by Mountainous Region Agricultural Institute, Aichi Agricultural Research Center, Japan (Figure 1B). Mineasahi SBL is highly resistant to rice blast and contains both the Pb1 and Pi39 genes, which is widely distributed in the mid-mountainous areas of Aichi Prefecture (Figure 2) [18]. In this review, we discuss the long-term durability of high field resistance to rice blast expressed by the Pb1 gene, which has remained effective in a wide range of rice paddy fields in Japan for over 40 years. This discussion is based on findings from breeding, genetics, molecular genetics, and epidemiological studies. We summarize the results obtained from these fields of study and discuss the sustainable use of major genes expressing high field resistance to rice blast.

2. Classification of Rice Resistance to Blast Disease: True Resistance and Field Resistance

Rice blast resistance can be classified into two types: True/complete/qualitative resistance, which is race-specific and expressed by a major resistance (R) gene, and field /partial/quantitative resistance, which is race-non-specific and expressed by multiple microgenes. True resistance shows a hypersensitive response (HR) accompanied by hypersensitive cell death, whereas field resistance does not show this HR [19,20]. Although true resistance is highly effective against blast disease, it is unstable owing to the potential for resistance breakdown. In contrast, field resistance involving multiple polygenes is generally more stable and less prone to collapse. This type of resistance is called horizontal resistance [21,22,23], and it has been extensively studied in upland rice. So far, more than 140 blast-resistance genes have been identified [24]. Most of them belong to true resistance (R) genes, and 11 genes, including Pb1, Pb2, Pi21, Pi34, Pi35(t), Pi39, Pi63, Pisa(t), Pikur1, Pikur2, and Pif, belong to field resistance genes.
Blast field resistance has traditionally been considered quantitative resistance, in which multiple microgenes are expressed as polygenes. However, recent progress in rice genome research has led to the identification of individual rice QTLs that express quantitative resistance with strong disease-suppressive effects, such as Pb1, Pi21, Pi34, Pi35, Pi39, and Pi63, among blast field resistance genes [14,15,16,17,25,26]. Notably, Pi21, Pb1, and Pi63 have been isolated, and their protein structures have been determined [27,28,29]. Consequently, a single major gene has been shown to express strong quantitative resistance (field resistance) to blast, demonstrating the existence of quantitative resistance governed by these major genes. The traditional definition of field resistance as that ‘expressed by the combined action of multiple microgene sets’ no longer explains the whole picture of field resistance, highlighting the need to reconstruct the concept of field resistance.
In 1989, Shindo and Asaga proposed a redefinition of quantitative resistance to blast disease based on the discovery of Pif, a major gene that expresses strong quantitative resistance [30]. They divided quantitative resistance into two types: One dominated by major acting genes and the other dominated by minor acting genes. The new definition of quantitative resistance dominated by the major acting gene was termed ‘highly potent field resistance’ or ‘high field resistance’, whereas conventional field resistance, which is a quantitative resistance expressed by multiple micro-acting genes, was redefined as ‘field resistance in a narrow sense’. Asaga proposed that both types of quantitative resistance be defined together as ‘field resistance in a broad sense’ (Table 1), while their new proposal was not widely accepted at that time [31].
Although Shindo and Asaga’s (1989) redefinition of field resistance to blast disease is tentative and should be re-evaluated in light of the rapid developments in resistance gene research, this review considers quantitative and strong resistance expressed by a major gene as a type of field resistance known as ‘high field resistance’. This paper reviews the durability of ‘high field resistance’ to rice blast disease based on previous findings and discusses the sustainable use of this resistance.

3. True Resistance and Its Breeding Application in Rice Blast Disease Management

The NBS-LRR and R genes, which encode proteins with similar structures, exhibit specificity for the blast race [32,33,34], which exerts strong ‘selection pressure’ on the blast population that does not have an affinity for the true resistance gene. This pressure results in the development of ‘complete resistance’, which means that susceptible lesions do not occur on rice leaves in areas where true resistance varieties are cultivated. However, true resistance is limited to a specific blast race [6,35,36]. Thus, the emergence of a blast race compatible with true resistance may lead to the proliferation and rapid spread of the blast race in the blast population. This phenomenon is known as ‘resistance collapse’ or ‘breakdown of resistance’, and it has been observed repeatedly in rice varieties carrying true blast resistance genes, where a blast race with an affinity for the true resistance gene spreads in a rice-growing region only a few years after its dissemination, resulting in severe damage from rice blast [37,38,39].
Several attempts have been made to accumulate multiple true blast-resistance genes in one variety to avoid resistance collapse [40,41,42]. However, a severe outbreak of panicle blast was observed in ‘Hama-Asahi,’ a rice cultivar that integrates four true resistance genes (Pib, Pik, Pii, and Pia), in the first year after its introduction in the panicle blast test plot at the Mountainous Region Agricultural Institute, Aichi Prefectural Agricultural Research Center (Figure 1B), which provided an experimental field suitable for the growth of the blast fungus (Fujii, personal communication). The ‘Unification’ series of Japonica-Indica varieties that were jointly developed by Korea and IRRI accumulated multiple true blast resistance genes, had high yields, and were initially popular in South Korea [43]. However, these varieties were severely damaged by rice blast years after their dissemination, and their cultivation ceased [43].
In Niigata and other prefectures, efforts are being made to use multiline varieties with true blast resistance to avoid the breakdown of resistance [44,45]. The ‘Koshihikari Niigata BL’ group of Koshihikari homologous multilines includes the Pia, Pii, Pita-2, Piz, Pik, Pik-m, Piz-t, Pib, Pib+Pia, Pib+Pii, Piz-t+Pii, Piz-t+Pik, and Pit lines [45,46,47]. This multiline is produced by mixing two susceptible (30%) and two resistant lines (70%), and it has been grown throughout Niigata Prefecture since 2005. It has decreased the blast damage, reduced the application of blast control chemicals by 70%, and avoided the breakdown of true resistance [46].
Multiline rice varieties have been proposed as a solution for blast control. However, to effectively utilize multiline varieties, strict management of the maintenance, such as seed production and breeding of multiple homogeneous lines is required. This includes confirming the true resistance genotypes using DNA markers and purity tests. Furthermore, the continuous and systematic monitoring of race distribution of the blast fungus is necessary. These issues must be addressed to fully realize the potential of multiline varieties for blast control. Additionally, integrating blast field resistance into multiline varieties remains a challenge to be addressed.

4. Field Resistance and Its Use in Breeding for Rice Blast Resistance

As mentioned in Section 2, conventional field resistance in rice is controlled by multiple microgenes expressing quantitative resistance as a ‘polygene’ [48], and it is generally non-specific to various blast races and has been considered a durable resistance type [49,50] that does not collapse over time due to the absence of specificity to a blast race/strain/isolate that attacks field resistance [22]. However, owing to the complexity of multiple micro-acting genes (quantitative trait loci: QTLs) that express field resistance, it is challenging to identify individual QTLs through differences in disease severity in field tests. To effectively breed rice for blast resistance using field resistance, it is necessary to identify the major gene that controls high field resistance and exerts strong suppressive effects against rice blast, develop a DNA marker closely linked to the high field resistance gene, and establish a breeding method using MAS. Until these advancements are made, breeding efforts for rice blast resistance using field resistance will be limited to a few specialized institutes, such as the Tohoku Agricultural Research Center of the National Agricultural Research Organization and the Mountainous Regional Agricultural Institute of Aichi Prefectural Agricultural Research Center, which have favorable weather conditions for leaf and panicle blast development [51,52].
In practical breeding, the use of MAS for rice blast resistance breeding based on the finding of blast high field resistance governed by major gene(s) is rapidly progressing in Japan [53,54,55,56]. However, to ensure specificity to the blast fungus race/strain/isolate and the stability and persistence of resistance expression, it is necessary to distinguish between conventional field resistance and high field resistance expressed by a major gene. The latter is considered a quantitative resistance, in which a major gene is expressed with high potency.

5. Durability of High Field Resistance Conferred by Major Gene, Which Is Highly Effective against Rice Blast Disease

It has been reported that strain specificity occurs during Pif gene-mediated high field resistance to blast in the indica rice variety Modan as well as during the leaf blast field resistance of ‘Chubu 32’ derived from upland rice. In the case of ‘Chubu 32’, the major gene expressing high field resistance to blast Pi34 shows the obvious strain specificity, which is similar to the R gene expressing true resistance [57,58,59]. These findings suggest that resistance genes expressing quantitative resistance may also establish a gene-for-gene relationship with respect to strain specificity. In addition, it has been reported that field resistance is also strain-specific for ‘Koganenishiki’ and ‘Yamabiko’, which are Japanese rice varieties [60]. Furthermore, the high field resistance gene Pi63 carried by the upland rice variety ‘Jiapei’ encodes a CC-NBS-LRR protein and has clear strain specificity. There are further reports on rice cultivars, QTLs, or R genes conferring field resistance (partial resistance) with specificity to the blast fungus [61,62,63]. These results suggest that field resistance alone does not guarantee non-specificity for various blast strains or durability of the expressed resistance, even for high field resistance genes. Similar reports on race specificity have been published for several resistance genes that quantitatively express resistance to rust in wheat [49,52,59,60,64].

6. Pb1 Gene: A Partially Mutated R Gene Structure Conferring Durable High Filed Resistance to Panicle Blast in Rice

The Pb1 gene, which was derived from the indica rice variety ‘Modan’ and introduced into Japanese rice cultivars via the rice stripe virus-resistant intermediate maternal line ‘St No. 1’, confers strong field resistance to panicle blast but only moderate field resistance to leaf blast [65,66]. The Pb1 gene is located on the long arm of rice chromosome 11, which is also the location of the rice stripe virus resistance gene Stvb-i, and the two genes are linked with a recombination value of 5.2% [14,66,67].
Hayashi et al. revealed that the Pb1 gene encodes a partial structural variant of R gene with an ‘atypical’ CC-NBS-LRR structural protein [28]. The P-loop in NBS is missing, and other structures have been reported to be partially altered. Surprisingly, the Pb1 gene was found to have a partially mutated R gene structure, making it the principal active gene. The molecular genetic mechanism by which Pb1, originally a ‘non-expressed gene’, led to blast resistance has been revealed to be the duplication of a specific genomic region (Figure 3) [28]. The duplication of a 60-kb region resulted in two Pb1 genes being placed in series, and the Pb1 gene copied backwards became an ‘expressed gene’ by the action of a promoter present behind the forward Pb1 gene, which led to the development of high field resistance to blast (Figure 4). This study elucidated the ‘gene evolution’ associated with field resistance [28].

7. Mechanism of Non-Specific and Adult Plant Resistance to Rice Blast Conferred by the Pb1 Gene and Involvement of WRKY45

As mentioned above, Pb1 has been identified as a major gene that exhibits an altered R gene structure, and its expression is known to confer resistance to rice blast disease. However, a unique property of Pb1 is its ability to confer broad-spectrum resistance, which is largely characterized by its non-specificity towards different blast races and strains. Studies have shown that Pb1-conferred strong field resistance to panicle blast is not only non-specific to different blast races but also exhibits a high level of non-specificity to various strains, including foreign strains [28]. This broad-spectrum resistance is a major feature of the Pb1 gene and is likely due to the ability of the gene to target a conserved component of the pathogen, which is present in all blast races and strains. The non-specificity of Pb1 is a highly desirable trait for rice breeders because it provides durable resistance against a wide range of blast races and strains.
Inoue et al. (2013) demonstrated that the transcription factor WRKY45 plays a critical role in the regulation of blast resistance by the Pb1 gene [68,69]. They found that Pb1 protein interacts with WRKY45 in the coiled coil (CC) region to suppress the regulatory degradation of WRKY45 by the ubiquitin proteasome [70], which, in turn, increases the accumulation of WRKY45 in the cell and enhances blast resistance (Figure 5). In addition, WRKY45 knockdown significantly weakens the blast resistance of Pb1-possessing varieties and Pb1-overexpressing transformants [68]. While proteins with the CC-NBS-LRR structure derived from the five true blast resistance genes Pi36, Pib, Pita, Pit, and Piz-t also bind to WRKY45 in the CC region, the effector-triggered immunity of each of these five genes was reported to be unaffected by WRKY45 [71], indicating that resistance responses mediated by WRKY45 are highly specific.
The Pb1 gene exhibits a modified R gene structure, and its protein has a CC-NBS-LRR structure lacking the R gene-like P-loop without showing a typical HR response [65,66,72], indicating that Pb1 is a major resistance gene that has lost its intrinsic R gene function. The interaction between the Pb1 protein and WRKY45 amplifies blast resistance by suppressing the regulatory degradation of WRKY45, and overexpression of Pb1 and WRKY45 results in pre-invasion resistance and shows similar resistance responses, supporting the idea that Pb1 does not act as an R gene [68,69]. These findings strongly suggest that the non-specificity of blast-resistant varieties carrying Pb1 to various blast strains may be attributed to the indirect expression of blast resistance of the Pb1 gene via WRKY45.
The identification of genes that contribute to rice blast resistance has been a major focus of research. The WRKY45 gene was found to be involved in the regulation of defense genes in response to blast fungus infection [73]. A study of the genetic basis of resistance to rice blast found that resistance to leaf blast was associated with the accumulation of WRKY45 [68].
Although the Pb1 gene was shown to confer strong field resistance to panicle blast, it does not confer strong resistance to leaf blast, suggesting that different types of resistance mechanisms are involved in both cases [28,65]. Field tests using rice NILs for Pb1 and RNA expression studies during different developmental stages of rice plants have shown that Pb1 confers adult plant resistance (APR) to rice blast [1,28]. Recent studies have shown that the field resistance of the glutinous rice variety ‘Miyazakimochi’ to rice blast is also associated with APR, which is similar to Pb1-bearing varieties. However, the qPbm11 QTL, which is responsible for the resistance of ‘Miyazakimochi’ to panicle blast, is different from Pb1 and acts in a WRKY45-independent manner [74,75]. These findings suggest that different genetic mechanisms are involved in resistance to panicle blast and leaf blast in rice and that the regulation of WRKY45 and QTLs may play important roles in the expression of blast resistance.
A detailed look at the difference in the degree of panicle blast disease development between susceptible cultivar ‘Aichinokaori’ without Pb1 and resistant cultivar NIL ‘Aichinokaori SBL’ with Pb1 revealed that the differences in disease severity were relatively small in some rice blast strains [28], indicating that other factors may also play a role in the blast resistance of rice. Therefore, in addition to studying Pb1 and its interaction with WRKY45, careful observations of affinitive strains that specifically attack Pb1 are necessary to better understand the complex mechanisms underlying rice blast resistance. Furthermore, it has been reported that ‘Matsuribare’ [76], which possesses Pb1 and exhibits strong field resistance to panicle blast, does not show resistance to bacterial leaf blight, which is caused by a different pathogen and may develop resistance through the accumulation of WRKY45. Interestingly, ‘Matsuribare’ shows a ‘weak’ disease susceptibility response to bacterial leaf blight, suggesting that the Pb1-mediated blast resistance may also involve other defense mechanisms in addition to WRKY45-mediated pathways. This important issue requires further clarification to fully understand the complex interplay between the different defense mechanisms and their contributions to blast resistance in rice.
The complex interactions between the genetic makeup of rice plants and the specific strains of blast fungi encountered in the field can lead to variations in resistance among varieties carrying the same resistance gene. For instance, although the Pb1 gene has been identified as a key resistance gene against rice panicle blast, some Pb1-carrying varieties have been found to exhibit weak field resistance to panicle blast. To better understand the mechanisms underlying these differences in resistance, several studies have identified the QTLs associated with the expression of Pb1. Inoue et al. (2017) identified four QTLs on chromosomes 7, 8, 9, and 11 that regulate the expression of the Pb1 gene and proposed a set of expression-regulated QTLs that can stably and strongly express Pb1 [77]. These QTLs were found to differ between the Pb1-carrying varieties ‘Satojiman’, which has weaker field resistance to panicle blast, and ‘Koshihikari Aichi SBL’ [78], which has strong field resistance to panicle blast. Similarly, the NRI-bred variety ‘Niji-no -kirameki’, which carries the Pb1 gene from the weaker-resistant variety ‘Satojiman’, has been found to exhibit significantly stronger field resistance to panicle blast compared with ‘Satojiman’. Nagaoka et al. (2020) hypothesized that the improved resistance of ‘Niji-no-kirameki’ may be due to the recombination of the QTLs identified by Inoue et al. (2017) in the genetic background of the Pb1 donor variety. These findings suggest that a better understanding of the genetic factors underlying blast resistance and careful observations of the occurrence of strains compatible with Pb1 are crucial for developing rice varieties with robust resistance to this devastating disease [77,79].

8. Sustainability of Pb1-Mediated High Field Resistance to Panicle Blast

Durable resistance, as defined by Johnson (1981, 1984), is characterized by the ability to maintain disease suppression effects under disease-favorable conditions and observation in a wide range of resistant varieties over an extended period of time. To assess whether the resistance gene is durable, it is necessary to evaluate the stability of resistance expression, including the occurrence of resistance breakdown, through demonstration trials conducted over a prolonged period on a large area of farmers’ fields in different regions [49,50]. Although this criterion is time-consuming, it remains a fundamental principle that is still relevant today and has been cited extensively.
Johnson’s criterion is cited below (Johnson 1984). A critical analysis of durable resistance: Durable resistance is a resistance that remains effective during its prolonged and widespread use in an environment favorable to the disease. The test for durable resistance must include two elements, time (long) and area (large). Resistance in a cultivar cannot justifiably be described as durable if the cultivar has been grown only in small-scale experiments, even if such tests are repeated at many locations and over several or many years. Experience shows that resistance that survives such tests may, nevertheless, not survive widespread use under agricultural conditions. Nor can resistance be described as durable if it has only been used briefly, even though on a large area [50].
A series of rice varieties with high field resistance to panicle blast conferred by the Pb1 gene, including ‘Hoshinohikari’, ‘Aoisora’, ‘Tsukinohikari’, ‘Asanohikari’, ‘Matsuribare’, ‘Akanezora’, ‘Asahinoyume’, ‘Aoinokaze’, ‘Daichinokaze’, ‘Aichinokaori SBL’, ‘Sainokagayaki’, ‘Goropikari’, ‘Yumematsuri’, ‘Sainokizuna’, ‘Koshihikari Aichi SBL’ and ‘Niji-no-kirameki’, has been cultivated in farmers’ paddy fields in the Kanto and Tokai regions of Japan for over 40 years [65]. These varieties have been expressing strong and continuous field resistance to panicle blast for more than 40 years and have been used as parent materials for breeding rice varieties with blast resistance in many rice breeding institutions in Japan (Table 2) [53,54]. Furthermore, blast strains with high affinity for the Pb1 gene have not resulted in the breakdown of resistance in farmers’ fields to date, leading to the recognition of durable resistance of this gene in Japan based on Johnson’s criteria. Recently, the Pb1 gene has also been used for blast-resistance breeding in Korea, where it has expressed strong field resistance to panicle blast in the southern part of the country [79].
The durability of the resistance expressed by the Pb1 gene and the occurrence of resistance breakdown in countries other than Japan and Korea have yet to be explored. The worldwide stability and persistence of high-field resistance to panicle blast mediated by Pb1 also require further investigation. Therefore, it is crucial to conduct studies that evaluate the durability of Pb1-mediated resistance in other global regions. This research will enhance our understanding of the stability of Pb1 gene-mediated resistance to panicle blast, which is essential for the development of sustainable strategies for blast disease management.

9. Uncovering the Epidemiological Mechanisms for the Persistent Resistance of Pb1-Conferred High Field Resistance to Panicle Blast

APR has been proposed to be the epidemiological mechanism responsible for the lack of breakdown of the highly potent field resistance to panicle blast expression of the Pb1 gene. The Pb1 gene provides only moderate quantitative resistance to leaf blast and exerts low selection pressure on the blast fungus population at the leaf blast stage. Consequently, the selective growth and spread of blast strains with a specific affinity for the Pb1 gene are hindered because of the low selective pressure for the affinitive strain that specifically attacks Pb1 at the leaf blast stage. This mechanism has resulted in the long-standing non-collapse of resistance to panicle blast conferred by the Pb1 gene over the past 40 years [65,66,67,68,69,70,71,72,73,74,75,76,77,78,79]. This hypothesis is supported by subsequent studies conducted by other investigators [25,28].
In a recent study, Zenbayashi (2007) reported that the number of times the blast fungus can change generations before rice harvest is limited after the panicle emergence stage, which is when the Pb1 gene develops strong quantitative resistance to rice panicle blast. This insufficient number of generations is not conducive to the selective multiplication and spread of affinitive strains that specifically attack Pb1, thus leading to a bottleneck effect. This bottleneck effect, in which the number of Pb1-compatible strains is drastically reduced by seed disinfection the following year, is another epidemiological mechanism underlying the lack of selection and spread of Pb1-specific affinitive strains. Similarly, the wheat disease resistance gene Lr34, which encodes an ABC transporter with a nucleotide-binding domain (NBD) in the plasma membrane, has shown stable and durable resistance against rust and powdery mildew in wheat cultivars worldwide, with no breakdown observed for more than 50 years. Lr34-carrying wheat cultivars have been reported to express APR against these diseases, which is similar to the expression of APR by Pb1, indicating that high-level field resistance without long-term collapse is a common feature of APR expressed by differently structured major genes [80,81,82].
Probenazole, an agrochemical used in rice cultivation for over 45 years, has not caused any rice blast resistance outbreaks to date [83,84,85,86,87]. Despite having no fungicidal activity against the blast fungus, probenazole acts as a plant activator [69] by inducing the biosynthesis of salicylic acid in rice plants, which then induces the expression of WRKY45 and OsNPR1 [87,88,89,90,91]. The Pb1 gene plays a role in suppressing the regulatory degradation of WRKY45 in rice and promoting its accumulation, which results in high field resistance to blast disease [68,69,92]. WRKY45 also regulates the expression of 260 defense genes encoding PR proteins and ABC transporters [90]. The activation of WRKY45 is regulated by the cooperation between MAP kinase-mediated phosphorylation [91]. Therefore, the long-term absence of the breakdown of Pb1-mediated high-field resistance may also be attributed to the indirect mechanism of blast resistance via WRKY45. This Pb1 strategy for indirect control of rice blast is similar to that of probenazole [86]. In conclusion, we confirmed the importance of maintaining the race and strain diversity of rice blast fungus populations, especially during the leaf blast stage, to avoid the breakdown of resistance when using resistance gene(s) for blast disease management in rice cultivation.

10. Pyramiding of Major Genes Conferring High Field Resistance for Sustainable Use

Several high field resistance genes to blast have been identified in rice, including Pif derived from the indica rice variety ‘Modan’, Pi34, Pi63, and Pi21 derived from upland rice, and Pi39 derived from a Chinese rice landrace [17,27,29,52,59,92]. Although Pif, Pi34, and Pi63 exhibit blast strain specificity, Pi21 and Pi39 are non-specific to the blast race. Paddy rice cultivars harboring Pi21 or Pi39 alone have been reported to exhibit high field resistance to blast fungus races in rice paddies in various regions of Japan. However, the total cultivated area with these resistance genes remains limited and does not meet the criteria for durable resistance, which requires extensive and continuous cultivation of rice varieties possessing only resistance genes [50]. Additionally, while Pi21 and Pi39 showed significantly stronger quantitative resistance to leaf blast than Pb1, the higher selection pressure on the blast population at the leaf blast stage increased the possibility of selection and spread of a strain with a specific affinity for the resistance gene in the blast fungus population, which may eventually compromise the durability of resistance. Therefore, large-scale cultivation of rice varieties possessing these genes alone and cultivation over a long period in various parts of the country are necessary for the future verification of their resistance durability [50]. Recently, the two novel genes Pb2 and Pb3, which are located on rice chromosome 11 and encode NLR proteins that express panicle blast resistance, were identified by Chinese researchers [93,94]. Therefore, it is important to elucidate the mechanisms of panicle blast resistance conferred by these genes. Furthermore, it is crucial to clarify whether these genes confer durable resistance to rice blast.
Rather than relying solely on the innate durability of individual genes, multiple high-potential field-resistance genes with distinct resistance mechanisms can be incorporated into a single cultivar to improve the stability and sustainability of blast resistance expressed by driver genes [43,95]. These gene combinations include Pb1+Pi21, Pb1+Pi39, and Pb1+Pi21+Pi39 [96]. The pyramiding of multiple high-field resistance genes is anticipated to increase the degree, stability, and persistence of quantitative resistance exhibited by blast high-field resistance varieties. Pb1-carrying varieties have been widely cultivated in various regions of Japan for over 40 years without any breakdown in panicle blast resistance. Therefore, the resistance of Pb1 meets the criteria for durable resistance. Thus, Pb1 is a promising candidate key gene for the pyramiding of blast resistance genes. To this end, rice blast-resistant varieties such as ‘Tachiharuka’ [97], ‘Minenohoshi (Chubu 134)’ [98], and ‘Yawakoimochi (Aichi 126)’ [99] carrying both Pb1 and Pi39 high field resistance genes have already been developed. Furthermore, ‘Chubu-mochi 136′ containing three high field resistance genes to rice blast (Pb1, Pi21, and Pi39) was bred at the Mountains Region Agricultural Institute of Aichi Agricultural Research Center. These varieties also possess the rice stripe virus resistance gene Stvb-i [67].
The new rice variety ‘Mineasahi SBL’ was developed by introducing two high field resistance genes to blast (Pb1 and Pi39) and a rice stripe virus resistance gene (Stvb-i) into the original susceptible variety ‘Mineasahi’ using the MAS breeding method, and it has shown promising results in controlling panicle blast (Figure 2, Table 2). The variety was fully updated in 2021 and has been widely grown on 1500 ha of land in the mid-mountainous areas of Aichi Prefecture. The effectiveness of ‘Mineasahi SBL’ in controlling panicle blast has been observed in farmers’ fields, and it received the highest disease preventive value of 100 for its efficacy in suppressing panicle blast. Chemical pesticides for rice blast have been excluded from rice cultivation in response to the high control efficacy of ‘Mineasahi SBL’. Furthermore, the variety has received a ‘Special A’ rating for its eating quality, indicating that the chromosomal regions carrying the Pb1, Pi39, and Stvb-i genes are free from QTLs associated with poor eating quality.
Continuous large-scale cultivation of highly field-resistant genotypes in an environment conducive to blast fungi, as observed in the case of true resistance-accumulated varieties, is yet to be investigated. Whether it will result in the selection and spread of super-blast isolates with a strong affinity for all high-field resistance genes, which would potentially lead to the collapse of resistance in varieties with high field resistance, is a matter of interest. To increase the durability of resistant varieties, high-field resistance genes (groups) and conventional microarray genes that express field resistance may be accumulated. Zenbayashi (2007) suggested that high-field resistance genes, such as Pi34, which has confirmed strain specificity in a multiline approach, could be applied to continuously suppress blast disease similar to true resistance genes. Although the breeding costs and labor requirements for multiline use remain a challenge, it is considered an effective strategy for preventing the collapse of resistance in strain-specific high field resistance genes.

11. Towards Sustainable Management with Highly Potent Field Resistance Genes

To achieve sustainable and environmentally friendly rice cultivation, it is crucial to recognize the value of high field resistance genes as a ‘common treasure’ bestowed by nature on humankind. A major challenge for the future is to ensure long-term and sustainable use of these genes in controlling rice blast disease while avoiding the collapse of resistance. This achievement is expected to contribute towards achieving an SDG for rice blast disease control [8,100,101]. While utilizing high field resistance genes for controlling rice blast, it is important to consider not only their high disease suppression effect and inherent durability but also their limited lifespan as ‘disposable’ consumables. This entails addressing bottlenecks in seed disinfection, utilizing pest forecasting information, and re-establishing control levels for resistant varieties.
To conclude, it is imperative that researchers from diverse fields, such as plant pathology, epidemiology, ecology, molecular genetics, and breeding, collaborate with responsible breeders for advancements in interdisciplinary research and practical breeding.

Author Contributions

Conceptualization, K.F., Y.K. and H.I.; methodology, T.S., M.N., T.Y. and N.H.; writing—review and editing, Y.U. and H.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by JSPS KAKENHI Grant Number 17K07678, Grant-in-Aid for Scientific Research(B) 20H02953 and Grant-in-Aid for Scientific Research(C) 22K12075.

Data Availability Statement

Not applicable.

Acknowledgments

We acknowledge the support and contributions of several individuals who contributed to this review. First, we are grateful to the late Mabito Iwasaki’s encouragement in writing this review on the persistence of field resistance to blast disease. We also thank Yuriko Hayano-Saito, Senior Research Scientist at the Disease Research Laboratory, Central Agricultural Research Centre, and Kaoru Zenbayashi-Sawata, Director of the Planning Office, Tohoku Agricultural Research Centre, National Agricultural Research Organization, for their helpful discussions, valuable suggestions, and advice during the compilation of this review. Additionally, Makoto Sakai, Director of the Centre for Food and Agricultural Business Promotion, National Institute of Agrobiological Sciences, provided a list of recent Pb1 rice varieties, whereas Akihiro Ikeda, former Director of the Rice Crop Research Laboratory, Mountainous Region Agricultural Institute, Aichi Prefectural Agricultural Research Centre, shared a list of Pb1-possessing rice varieties.

Conflicts of Interest

The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.

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Figure 1. Rice blast in paddy field (A) and experimental field for rice blast disease (B). (A) In 2003, rice blast was spread in Japan, and production was affected by the blast. This picture was taken in a paddy field, Miyagi prefecture in Japan. The red part of rice was damaged by the rice blast. (B) Mountainous Region Agricultural Institute, Aichi Agricultural Research Center, Japan, which is developing rice blast resistance breeding. The institute is situated in a mountainous region at an elevation of 505 m, which boasts favorable weather conditions conducive to the development of leaf and panicle blast.
Figure 1. Rice blast in paddy field (A) and experimental field for rice blast disease (B). (A) In 2003, rice blast was spread in Japan, and production was affected by the blast. This picture was taken in a paddy field, Miyagi prefecture in Japan. The red part of rice was damaged by the rice blast. (B) Mountainous Region Agricultural Institute, Aichi Agricultural Research Center, Japan, which is developing rice blast resistance breeding. The institute is situated in a mountainous region at an elevation of 505 m, which boasts favorable weather conditions conducive to the development of leaf and panicle blast.
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Figure 2. Mineasahi SBL harbors two quantitative resistance genes: Pb1 and Pi39. Disease symptoms of Mineasahi SBL (Pb1+Pi39) and near-isogenic Mineasahi (Pb1-, Pi39-) cultivars grown in a rice field in Tsukude, Shinshiro, Aichi, Japan. The altitude was 500 m. The picture was taken approximately two weeks after the full heading stage in 2021. Mineasahi SBL did not show any disease symptoms of panicle blast, whereas Mineasahi did show symptoms.
Figure 2. Mineasahi SBL harbors two quantitative resistance genes: Pb1 and Pi39. Disease symptoms of Mineasahi SBL (Pb1+Pi39) and near-isogenic Mineasahi (Pb1-, Pi39-) cultivars grown in a rice field in Tsukude, Shinshiro, Aichi, Japan. The altitude was 500 m. The picture was taken approximately two weeks after the full heading stage in 2021. Mineasahi SBL did not show any disease symptoms of panicle blast, whereas Mineasahi did show symptoms.
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Figure 3. The sequence of bacterial artificial chromosome (BAC) clone, including Pb1 resistance gene. The BAC clone (clone name: St-h-49J11) contained two fragments of 60 kb sequence (repeat-1: 58,725 bp, repeat-2: 58,720 bp) with tandem repeat. The clone had only two deletion sites (a, d) and three SNPs (b, c, and e) even over 150 kb sequence. The figures indicate the actual position of the deletion and SNPs sites. As a reference, Nipponbare has a similar single sequence to the Pb1 cultivar.
Figure 3. The sequence of bacterial artificial chromosome (BAC) clone, including Pb1 resistance gene. The BAC clone (clone name: St-h-49J11) contained two fragments of 60 kb sequence (repeat-1: 58,725 bp, repeat-2: 58,720 bp) with tandem repeat. The clone had only two deletion sites (a, d) and three SNPs (b, c, and e) even over 150 kb sequence. The figures indicate the actual position of the deletion and SNPs sites. As a reference, Nipponbare has a similar single sequence to the Pb1 cultivar.
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Figure 4. A model: Tandem genome duplication confers the active promoter for Pb1 resistance gene expression. An ancestral Pb1 genomic region was thought to exist, and the region was duplicated as a tandem genome. Although the proto-Pb1 (driven from the sequence of light blue and pink) does not have a strong expression activity, Pb1 acquires a new promoter (the sequence of red to yellow), which enables resistance to the rice blast through tandem genome duplication.
Figure 4. A model: Tandem genome duplication confers the active promoter for Pb1 resistance gene expression. An ancestral Pb1 genomic region was thought to exist, and the region was duplicated as a tandem genome. Although the proto-Pb1 (driven from the sequence of light blue and pink) does not have a strong expression activity, Pb1 acquires a new promoter (the sequence of red to yellow), which enables resistance to the rice blast through tandem genome duplication.
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Figure 5. Predicted mechanisms of resistance against rice blast in varieties with the Pb1 resistance gene. In rice, the accumulation level of WRKY45 protein increases in response to blast infection. However, that level is considered insufficient to confer blast resistance. In Pb1-possessing rice varieties, Pb1 protein protects WRKY45 from proteasome-dependent degradation, resulting in enhanced accumulation of WRKY45 and resistance to rice blast. UPS, ubiquitin-proteasome system.
Figure 5. Predicted mechanisms of resistance against rice blast in varieties with the Pb1 resistance gene. In rice, the accumulation level of WRKY45 protein increases in response to blast infection. However, that level is considered insufficient to confer blast resistance. In Pb1-possessing rice varieties, Pb1 protein protects WRKY45 from proteasome-dependent degradation, resulting in enhanced accumulation of WRKY45 and resistance to rice blast. UPS, ubiquitin-proteasome system.
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Table
Table 1. Classification of rice blast resistance modified by Asaga (1987).
Table 1. Classification of rice blast resistance modified by Asaga (1987).
Oligogenic ResistancePolygenic Resistance
QualitativeTrue resistance-
QuantitativeHigh field resistance Field resistance
Table
Table 2. Rice cultivars harboring the panicle blast resistance gene, Pb1 spreads all over Japan. Each prefectural Agricultural institute and National Agriculture and Food Research, NARO breeds Pb1-including cultivars since 1985.
Table 2. Rice cultivars harboring the panicle blast resistance gene, Pb1 spreads all over Japan. Each prefectural Agricultural institute and National Agriculture and Food Research, NARO breeds Pb1-including cultivars since 1985.
Variety Year of Development Breeder
Tsukinohikari 1985 Aichi pref.
Asanohikari 1987 Aichi pref.
Aoinokaze 1989 Aichi pref.
Yumeminori 1990 Saitama pref.
Akanezora 1991 Aichi pref.
Maturibare 1993 Aichi pref.
Goropikari 1993 Gunma pref.
Koigokoro 1995 NARO
Asahinoyume 1996 Aichi pref.
Daichinokaze 1998 Aichi pref.
AichinokaoriSBL 2000 Aichi pref.
KoshihikariAichiSBL 2002 Aichi pref.
Sainokagayaki 2002 Saitama pref.
Tsuyaotome 2003 Fukuoka pref.
Yumematsuri 2007 Aichi pref.
Kinuhanamochi 2008 Aichi pref.
Yumeginga 2010 Aichi pref.
Koharumochi 2011 Aichi pref.
Harumoni 2011 NARO
Sainokizuna 2012 Saitama pref.
Kiraho 2013 Iwate pref.
Ginganoshizuku 2015 Iwate pref.
MineasahiSBL 2017 Aichi pref.
Yawakoimochi 2018 Aichi pref.
Nijinokirameki 2018 NARO
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Fujii, K.; Suzuki, T.; Nakamura, M.; Yoshida, T.; Uchikawa, Y.; Suwazono, H.; Hayashi, N.; Kanda, Y.; Inoue, H. Investigating the Mechanisms Underlying the Durability and Sustainable Use of Pb1 Gene-Mediated High Field Resistance to Rice Panicle Blast. Agronomy 2023, 13, 1751. https://doi.org/10.3390/agronomy13071751

AMA Style

Fujii K, Suzuki T, Nakamura M, Yoshida T, Uchikawa Y, Suwazono H, Hayashi N, Kanda Y, Inoue H. Investigating the Mechanisms Underlying the Durability and Sustainable Use of Pb1 Gene-Mediated High Field Resistance to Rice Panicle Blast. Agronomy. 2023; 13(7):1751. https://doi.org/10.3390/agronomy13071751

Chicago/Turabian Style

Fujii, Kiyoshi, Taro Suzuki, Mitsuru Nakamura, Tomofumi Yoshida, Yoshinori Uchikawa, Haruka Suwazono, Nagao Hayashi, Yasukazu Kanda, and Haruhiko Inoue. 2023. "Investigating the Mechanisms Underlying the Durability and Sustainable Use of Pb1 Gene-Mediated High Field Resistance to Rice Panicle Blast" Agronomy 13, no. 7: 1751. https://doi.org/10.3390/agronomy13071751

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