Periodic Reporting for period 2 - HerbaRice (Herbicide Resistant Rice Development For European Continental Project)
Reporting period: 2022-11-16 to 2023-11-15
Rice, serving as a staple food for over half of the global population, faces significant challenges within its farming system. The most notable issues are weed management, disease control, biotic-abiotic stress, and climatic change. Among these challenges, the issue of weeds, particularly weedy rice (Oryza sativa L., red rice), stands out as a crucial problem, reducing both rice grain yield and quality. The conventional approach of applying herbicides is the common method for weed control, however, it is increasingly losing efficacy due to various factors year by year. This chemical-based weed control method is not only labor-intensive and expensive but also raises environmental concerns when applied repeatedly. On the other hand, since weedy rice is taxonomically classified as the same species as cultivated rice, its chemical control is very difficult. The development of herbicide-resistant rice systems such as Clearfield®, Provisia®, and Roxy® RPS helps manage weedy rice. However, Clearfield® began losing effectiveness soon after its introduction due to gene flow and misuse. Provisia® was introduced in 2018 but few countries utilized it up to now. Roxy® RPS is not yet available in the EU. Recognizing the potential deterioration of these systems, it becomes imperative to establish new resistance resources for deployment by European countries. Our objective was to create a novel herbicide-resistant source of rice and make it available to European farmers in the future. We developed low-dose resistant rice materials but did not achieve high-dose resistance at the commercial level. The project involved launching a new cross-breeding program intended for use in EU countries. This program focused on cross-breeding low-dose clethodim-resistant base material with various EU national rice varieties. The objective of this new breeding program is to contribute to effective weed management in rice fields for rice-growing countries within the European Union.
The project also attempted to model the potential lifetime of Clearfield® technology. For this purpose, surviving weedy rice samples were collected from fields where Clearfield® rice varieties were produced, and as a result, it was determined that there was a 22% gene flow from Clearfield varieties to weedy rice. This point, reached in a 10-year period of use in Türkiye, where is considered as a model country, reveals that the technology may become dysfunctional in the near future. These results clearly revealed the importance of the subject which new herbicide-resistant rice technologies are needed to create a diverse weedy rice management method available for rice production and move towards sustainable rice farming.
The project also attempted to model the potential lifetime of Clearfield® technology. For this purpose, surviving weedy rice samples were collected from fields where Clearfield® rice varieties were produced, and as a result, it was determined that there was a 22% gene flow from Clearfield varieties to weedy rice. This point, reached in a 10-year period of use in Türkiye, where is considered as a model country, reveals that the technology may become dysfunctional in the near future. These results clearly revealed the importance of the subject which new herbicide-resistant rice technologies are needed to create a diverse weedy rice management method available for rice production and move towards sustainable rice farming.
In order to develop clethodim herbicide-resistant rice base material, we initiated a comprehensive process involving mutation application, low-dose resistance study, high-dose resistance study, and mutation point determination study. The mutation generation study involved establishing a standard protocol for EMS mutagenesis application in rice, aiming to obtain a sufficient number of mutant plants rather than inducing severe mutations in a few plants. This protocol included a 12-hour presoaking, a 0.5% dose EMS application for six hours, a final six-hour washing, and a 72-hour drying period.
Following the creation of the protocol, chemical EMS mutation was applied to five rice varieties, and a low-dose clethodim resistance study was initiated. Seeds from chemically mutated plants were planted in a growth chamber. Clethodim was applied at 75 g ai ha-1 (½ × of the labeled rate) dose to M1 plants in the low-dose experiment. Visual injury ratings performed 28 days after treatment revealed that 30 of the plants survived with the frequency was 0.06%. The mutation determination process aimed to identify the clethodim herbicide binding point on the ACCase carboxyl transferase region on the 5th chromosome in rice. Despite finding low-dose resistant plants, the original sequence in the target region remained unchanged.
Moving on to the high-dose resistance study, mutation was applied to 100,000 seeds of five varieties following the established protocol. The M1 generation was planted, and generation advancement was achieved from M1 to M2 plants. In the second year, the 3 million M2 generation seeds were grown in the field, and the herbicide was applied at 150 g ai ha-1 (1×) dose twice on mutated plants for high-dose experiments at the 3-4 leaf stage. A total of 816 individual plants survived the application in the field. These plants were harvested and their progeny were grown in the greenhouse and high-dose herbicide were sprayed again. Five of 816 surviving plants demonstrated resistance in greenhouse conditions. Further analysis revealed that these resistant genotypes did not exhibit resistance exceeding ½ × dose in the herbicide dose-response study, and no mutations were detected in the ACCase gene region according to DNA sequencing. In conclusion, while 30 low-dose resistant genotypes were developed, no material exhibited high-dose resistance suitable for commercial use.
Following the creation of the protocol, chemical EMS mutation was applied to five rice varieties, and a low-dose clethodim resistance study was initiated. Seeds from chemically mutated plants were planted in a growth chamber. Clethodim was applied at 75 g ai ha-1 (½ × of the labeled rate) dose to M1 plants in the low-dose experiment. Visual injury ratings performed 28 days after treatment revealed that 30 of the plants survived with the frequency was 0.06%. The mutation determination process aimed to identify the clethodim herbicide binding point on the ACCase carboxyl transferase region on the 5th chromosome in rice. Despite finding low-dose resistant plants, the original sequence in the target region remained unchanged.
Moving on to the high-dose resistance study, mutation was applied to 100,000 seeds of five varieties following the established protocol. The M1 generation was planted, and generation advancement was achieved from M1 to M2 plants. In the second year, the 3 million M2 generation seeds were grown in the field, and the herbicide was applied at 150 g ai ha-1 (1×) dose twice on mutated plants for high-dose experiments at the 3-4 leaf stage. A total of 816 individual plants survived the application in the field. These plants were harvested and their progeny were grown in the greenhouse and high-dose herbicide were sprayed again. Five of 816 surviving plants demonstrated resistance in greenhouse conditions. Further analysis revealed that these resistant genotypes did not exhibit resistance exceeding ½ × dose in the herbicide dose-response study, and no mutations were detected in the ACCase gene region according to DNA sequencing. In conclusion, while 30 low-dose resistant genotypes were developed, no material exhibited high-dose resistance suitable for commercial use.
The state-of-the-art objective of the project is to develop a non-GMO resistant clethodim herbicide for rice. A total 30 low-dose clethodim-resistant plants were selected but no one high-dose resistant plant was developed. In the project, an optimized method for EMS mutation in rice was developed. This method has implications beyond the state-of-the-art objective because it can be utilized by other researchers not only for herbicide resistance but also to develop some other biotic and abiotic stress tolerance. Furthermore, low-dose clethodim-resistant base materials were developed. A crossbreeding program was initiated between these materials and conventional cultivars. Using classical breeding methods will enable the development of non-GMO cultivars resistant to low doses of clethodim herbicide.
Spot spray application was developed as an alternative solution to the red rice problem in conventional rice cultivation areas. The effectiveness of this application was found to be 96%. Clethodim dispersion in a water-seeded rice field was determined to not be of concern. These results revealed that clethodim has great potential as a spot treatment for the control of weedy rice and weedy grasses.
The sustainability of Clearfield® technology, which is widely used in Europe for red rice control, was also surveyed. In the case study, the S653N point is the same substitution point that serves as the origin of IMI-resistant rice, and it was identified in the red rice biotypes as well. The high rate of gene flow illustrates that the technology can reduce its efficacy in the near future and further imply the need for additional weed management tools.
The scientific publications accomplished in this project encompassed a range of investigations, including studies on clethodim applications, dose-response experiments involving rice, red rice, and weeds, as well as examinations of application timing and methods. A dedicated website was launched (www.herbarice.org) with subsequent updates aimed at enhancing the project's visibility. The project has yielded significant outcomes, including the generation of scientific literature, the establishment of chemical usage standards, and the promotion of awareness regarding weed management in rice farming systems.
Spot spray application was developed as an alternative solution to the red rice problem in conventional rice cultivation areas. The effectiveness of this application was found to be 96%. Clethodim dispersion in a water-seeded rice field was determined to not be of concern. These results revealed that clethodim has great potential as a spot treatment for the control of weedy rice and weedy grasses.
The sustainability of Clearfield® technology, which is widely used in Europe for red rice control, was also surveyed. In the case study, the S653N point is the same substitution point that serves as the origin of IMI-resistant rice, and it was identified in the red rice biotypes as well. The high rate of gene flow illustrates that the technology can reduce its efficacy in the near future and further imply the need for additional weed management tools.
The scientific publications accomplished in this project encompassed a range of investigations, including studies on clethodim applications, dose-response experiments involving rice, red rice, and weeds, as well as examinations of application timing and methods. A dedicated website was launched (www.herbarice.org) with subsequent updates aimed at enhancing the project's visibility. The project has yielded significant outcomes, including the generation of scientific literature, the establishment of chemical usage standards, and the promotion of awareness regarding weed management in rice farming systems.