Long-term Blast Control in High Eating Quality Rice Using Multilines

doi:10.21203/rs.3.rs-1249069/v1

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Long-term Blast Control in High Eating Quality Rice Using Multilines

https://doi.org/10.21203/rs.3.rs-1249069/v1

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Combining genetic heterogeneity and crop homogeneity serves a dual purpose: disease control and maintaining harvest quality. Multilines, which consist of a genetically uniform mixture of plants, have the potential to suppress disease while maintaining eating quality, yet practical methods that facilitate commercial use over large geographical areas are lacking. Here, we describe effective rice multiline management based on seed mixture composition changes informed by monitoring blast virulent races in Niigata prefecture, Japan. The most elite non-glutinous cultivar Koshihikari (KO) was changed to the multiline, KO BL (blast resistant lines), in 94,000 ha in 2005. The most destructive rice disease, blast, was 79.4 % and 81.8 % less severe in leaves and panicles, respectively, during the 2005–2019 period compared to the year 2004. In addition, fungicidal application was reduced by two-thirds after introduction of BL. Our results suggest that seed mixture diversification and rotation of resistant BL provides long-term disease control by avoiding virulent race evolution.

Blast caused by the fungus, Pyricularia oryzae Cavara, is the most devastating disease in rice (Oryza sativa L.). Rice is a staple cereal crop for over half the world’s population¹. Blast infects rice seedlings in nurseries, and seedling blast leads to leaf blast in paddy fields. This results in panicle blast that causes the seed to become infected during maturity. The infected carrier seeds act as primary infection sources in its life cycle. P. oryzae avirulence in relation to race-specific rice resistance follows a gene-for-gene coevolution pathway^2–7 that has pathogenic variations in relation to host genotype. This arms race between rice resistance and blast race can cause dramatical change of newly emerged virulent races within a few years when monogenic resistant rice is introduced in paddy fields.

Multiline^8–10, which consist of a genetically uniform mixture of monogenic crops is an environment-friendly strategy for stable food production^11,12. The heterogenic theory⁸ offers pathogen population dilution¹¹ with fewer fungicidal applications for sustainable rice culture. However, breeding characteristically uniform rice lines and understanding blast population dynamics over large geographical areas are administrative burdens. The use of genetically heterogeneous genotype or cultivar mixtures have been shown in commercial fields in cereals^13–16. However, the mixture of heterogeneous seeds with identical characters equivalent to monogenic cultivars has not been introduced until now and is needed to maintain eating quality^17–19 for consumers and farmers under commercial crop production. To solve this problem, a multiline; KO BL²⁰ with a high eating quality, was introduced in 2005 in Niigata pref. (Japan). In this study, we demonstrate that a 15-year history of composition change of the BL by monitoring of blast race changes. In addition, we believe that our simulation analysis for blast race dynamics in multiline can support to decide composition change of the BL in the future.

Our field tests were carried out in Niigata pref. (Japan) which has a wet season favorable for blast development during the rice growing season. The top-brand non-glutinous rice variety ‘KO’ which has a high palatability is extremely susceptible to blast. Therefore, farmers apply fungicides over four times during the rice production season. As KO is sold by the Niigata brand, KO has been traditionally viewed as having a high eating quality in Japan, and because of this, both farmers and consumers have requested that the multiline, BL, be tested to check if it is equivalent to KO before its introduction. Trials comparing KO and BL were carried out in 2003 and 2004 in 594 and 622 fields covering 236 and 315 ha, respectively. These trials evaluated plant homogeneity, eating quality, and blast suppression using fewer fungicidal sprays. Following favorable results, in 2005, all KO were converted to the BL multiline covering an area of 94,000 ha. In addition, seed use and cultivation were restricted to the Niigata area to distinguish from BL and KO grown in other prefectures.

Seed production and mixture processes are managed with precision by each prefectural official member (Fig. 1a). Original isogenic lines (IL) were backcrossed five to seven times followed by a three-year line selection for BL which was separately produced from original stock in original strain fields by Niigata Prefectural government. Using a precise mixture machine, the mixture was then blended by weight in 2,000 kg volumes composed of four ILs, all multiplied by ten (giving a total volume of 20 t). Original production fields and commercial fields all used blended seeds that had authorized by seed production and commercial farmers in the 2003 and 2004 trials. Thus, it takes two years for seed production at original strain field followed by original production field for preparation of commercial fields; seed mixture composition needs to be determined at least two years before introduction. Susceptible and resistant ILs were mixed at a ratio of 3:7 from 2005 to 2019 (Fig. 1b). Susceptible ILs, possessing Pia and Pii genes, were always mixed at a ratio of 1:2, but the composition of resistant ILs, containing Pita-2, Piz, Pib, Piz-t, and Pit genes, were changed every two to three years to avoid breakdown of the resistances. These changes were determined by annually monitoring blast race distributions.

In Niigata prefecture, predominant 5 blast races distributed from 1994 to 2004 were 001.0 (virulent to KO [Pik-s]), 003.0 (virulent to Pik-s and Pia), 005.0 (virulent to Pik-s and Pii), 007.0 (virulent to Pik-s, Pia, and Pii), and 037.1 (virulent to Pik-s, Pia, Pii, and Pik) (Fig. 2a). In the 2005 release year, the predominant blast races, 001.0 (virulent to Pik-s) and 003.0 (virulent to Pia), drastically decreased in distribution from 41.8–22.3% and 27.6–17.3%, respectively (Fig. 2a). Interestingly, races 001.0 and 003.0 rapidly decreased by 5.4% and 1.3% in 2006, respectively, even though especially Pia, which can be infected by the race 003.0, was used in the BL composition. In contrast, race 007.0 (virulent to Pik-s, Pia, and Pii) and 037.1 (virulent to Pik-s, Pia, Pii, and Pik) predominated from 2005 to 2019. The higher rate of race 037.1 detection was affected by a number of factors: the high susceptibility of a minor cultivar that had Pii and Pik, the mosaic configuration of fields typical to Niigata, and the air-borne spread of race 037.1. To maintain consensus on BL cultivation based on total blast suppression in Niigata, rarely detected virulent races to resistant ILs in commercial fields are strictly supervised in prefectural government to avoid unnecessary confusion in Niigata residents.

In 2008, to support the BL composition change mathematically, we developed a simulation software to estimate long-term blast race dynamics in multilines using a plant-pathogen coevolution system²¹. The model calculated the persistence of resistant ILs to determine the optimal timing of changes to multiline compositions. To simulate race dynamics in BL, we set five currently investigated races (001.0, 003.0, 005.0, 007.0, and 037.1) and their rates in 2004, including five emerging races (043.0, 303.0, 003.2, 403.0, and 003.4; see Fig. 2b) against five newly introduced respective resistant BLs (see Fig. 1b) and annual KO BL compositions from 2005 to 2019. The worst case (severe epidemic) simulation result (Fig. 2b) showed that race 007.0 (virulent to susceptible Pia and Pii BL) became the predominant race, and race 037.1 remained at a low frequency until the fifteenth year (corresponding to the year 2019). In addition, super-race virulent to all ILs did not emerge in this simulation. These results indicated that almost all the epidemics analyzed reflected actual race dynamics without affecting other trapped races from other susceptible cultivars grown in Niigata, especially up to 2011. Thus, our decision support system provides an evaluation of BL persistence, and indicates the BL composition changes needed for blast race population control in large areas. In addition, our simulation model may be useful for evaluation of future BL composition changes.

Blast occurrence drastically decreased after 2005 (Fig. 3a). The average occurrence of leaf and panicle blast was 46.1% and 52.9% during the 1995–2004 period, respectively, and 9.5% and 9.6% during the 2005–2019 period, respectively. Current seed production fields are rarely contaminated with virulent races against resistant BLs. This suggests that seed sanitation contributes to the suppression of virulent pathogen epidemics in multilines. In addition, induced resistance^22,23 may have no effect on the practical use of multilines. Rice plants were found to induce a resistance response when inoculated with avirulent races of blast (that stimulate protective responses to virulent race attacks). As detection of several races in one area is rare and blast occurrence tends to be low, conditions that induce resistance in field situations do not occur. Fungicide applications to control blast in BL and other minor cultivars decreased by approximately one-third during the 2005–2019 period compared with 2004 (Fig. 3b). Thus, the commercial scale use of crop diversity is clearly effective in environmentally friendly control of air-borne diseases.

The optimum long-term solution for pathogen population control using genetic diversity includes multilines. Blast occurrence in the BL introduced in Niigata, and the theoretical value of blast suppression in BL tested in small scales, were reduced by approximately 10% compared to that of monoculture plots^24–26. Thirty percent of susceptible ILs in BL have the potential to improve compatible races with susceptible ILs and become predominant in large areas. This would contribute to the suppression of rapid increases in newly virulent races emerging in the blast population. To maintain consensus on BL cultivation based on total blast suppression in Niigata, rarely detected virulent races to resistant ILs in commercial fields are strictly monitored by the prefectural government. Educating Niigata farmers ensures the long-term use of BL. In fact, lower blast occurrence has been attributed to careful BL cultivation and seed management.

The implementation of genetically diversified homogeneous seed mixtures, rotations with resistant BL, restricted BL cultivation, and pathogen monitoring allowed rice quality to be maintained, diseases to be suppressed, and environmentally-sound agriculture to be economically viable in Niigata. Collaboration between prefectural officers, farmers, and consumers in Niigata has resulted in safer rice production with good agricultural practices (GAP) that meet sustainable development goals (SDGs). In addition, DNA tests differentiate BL from the original KO for buyers, thereby inhibiting illegal distribution. The multiline has been used in small areas at two different prefectures. For example, in Miyagi pref., Sasanishiki BL consisted of Pik, Pik-m, and Piz at ratios of 4:3:3 and 3:3:4 in 1995 and 1996, respectively. This composition was changed to Pik, Pik-m, Piz, and Piz-t at a ratio of 1:1:4:4, from 1997 to 2007 to prevent increase of the race 037.1 (virulent to the BL: Pik and Pik-m). In addition, an equal mixture of seven BL (Pib, Pik, Pik-m, Piz, Piz-t, Pita, and Pita-2) was cultivated in 300 ha areas (maximum 4000 ha) from 2008 to 2014 without any outbreaks observed. In Toyama pref., the KO Toyama BL which consists of resistant ILs, Pita-2, Pik, and Pik-p (at a ratio of 3:3:4) is cultivated in an area of 300 ha and required 50% less chemical inputs from 2003 up to the present. Our model also calculated a greater than 50-year persistence in terms of the small area effect in both prefectural cases. This depends on insufficient pathogen population increase for virulent mutations against resistant ILs (data not shown). In this way, practical use of a multiline provides control without the need for as much fungicide with or without a periodic change in IL composition. Our results demonstrate that management of crop and pathogen co-evolution can control disease at large scales and, thereby, contribute to global food security.

Rice plants

Koshihikari BL (KO BL) is a rice (Oryza sativa L.) variety, which is a staple food crop around the world including Japan. KO BL²⁰ used in this study including 7 registered lines: Pia, Pii, Pita-2, Piz, Pib, Piz-t, and Pit, and protected by the Plant Variety Protection and Seed Act (Japanese Law) by the Ministry of Agriculture, Forestry and Fisheries in Japanese Government. Seeds of original stock field were obtained from paddy fields in Niigata Agricultural Research Institute under Niigata Prefectural Major Crop Seed Law. Seeds of original strain field and original production field were produced by designated farmers under the same law. Rice of commercial field were produced by general farmers restricted in Niigata Prefecture under Agricultural Products Inspection Act (Japanese Law) by the Ministry of Agriculture, Forestry and Fisheries in Japanese Government.

Blast infected rice leaves and panicles were collected by Niigata prefectural officers performed under Plant Protection Law by the Ministry of Agriculture, Forestry and Fisheries in Japanese Government, and permissions were previously obtained from all farmers before samplings. The plant protection committees of all the Niigata municipalities were also authorized for these samplings.

Race investigation

To investigate blast race distributions, the Niigata pref. area was divided into 5 km² grid squares. If leaf and panicle blast lesions were found in each grid square, infected leaf and panicle samples were collected by Niigata prefectural officers, and plant pathological research was performed using the single spore isolation method. The pathogenicity of isolated M. oryzae were determined by rice seedling inoculation using the race differentiation method^27,28. Officers from the plant protection committees of the various Niigata municipalities also collaborated in the blast lesion sampling.

Disease assessments

Before each cropping season started, a representative field from each 15 km² grid square was selected to monitor disease and predict pest occurrence. After rice seedlings were planted in paddy fields, general epidemics, which initially started as a small number of leaf blasts, were observed in June. The subsequent development of leaf blasts (in July) and panicle blasts (in August and September) were evaluated as leaf blast lesion area per hill and diseased grains, respectively.

BL composition determination

Once a year, the Niigata prefectural committee assessed proposals for altering resistant IL composition in BL for the following two years using data on monitored race frequency and blast severity.

Simulation model

Gene for gene system¹⁹ between rice genotypes and blast races was used to develop a simulation software. For Niigata simulation, the parameters set were: actual races and their rates investigated in 2004 (001.0 [0.423], 003.0 [0.245], 005.0 [0.038], 007.0 [0.196], 037.1 [0.049]), actual proportion of susceptible and resistant ILs used from 2005–2019 (Fig. 2), mutation rate (10^− 5)²⁹, overwintering probability (0.01), favorable weather condition (10), maximum lesion number (10,000,000), number of simulated years (15), number of simulation trials (1,000), and variables (lesion number of each race). Races on each IL increased exponentially until maximum lesion number, i.e., 10,000,000×10 (100,000,000) at favorable weather condition in every year. Mutated races emerged at 10^− 5 rate and randomly selected races were overwintered, including mutated race from 10,000,000×10×0.01 (1,000,000) lesions. The 1,000 trial results of the lesion number increased on each race were averaged in each year and transferred to the rates to show race dynamics.

Data availability

All the data used in the present study are included in this manuscript and supplementary information files, however, we will provide data of simulation results requested reasonable combination of races and rice resistance genes to all the interested scientists.

Acknowledgements

The breeding and introduction of the KO BL, as well as the investigations of blast race and their occurrences were done by Niigata Prefectural officers supported by prefectural funding. The simulations and analysis work were supported by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan (Research and development projects for application in promoting new policy of Agriculture Forestry and Fisheries).

Contributions

T. A. designed the project for the simulation and analysis work. K.I., T.K., T.H., D.I., and S.M. performed race investigations. J.N. and A.S. performed the simulations. T.A. analyzed the data and wrote the manuscript. All authors reviewed, edited and approved the final version of the manuscript.

Ethics declarations

Competing interests

The authors declare no competing interests. This study was performed in accordance with relevant rules, guidelines and regulations. There are no human subjects in this study.

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No competing interests reported.

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Long-term Blast Control in High Eating Quality Rice Using Multilines

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