Population dynamics of QoI-resistant rice blast isolates in Hyogo Prefecture associated with the use and discontinuation of QoI fungicides

Population dynamics of QoI-resistant rice blast isolates in Hyogo Prefecture associated with the use and discontinuation of QoI fungicides | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Population dynamics of QoI-resistant rice blast isolates in Hyogo Prefecture associated with the use and discontinuation of QoI fungicides Kaichi Uchihashi, Fumihiko Suzuki, Soichiro Asuke, Kenichi Ikeda, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9175517/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract In 2013 QoI-resistant isolates of the rice blast fungus (MoO) emerged suddenly and spread in Hyogo Prefecture. To estimate factors associated with their spread and subsequent survival, the population structure of MoO isolates was monitored from 2013 through 2023 using DNA fingerprinting with Pot2 . Most of the QoI-resistant isolates collected in 2013 were classified into haplotype HY1, irrespective of areas of collection in Hyogo Prefecture. This result suggests that the sudden emergence and spread of the QoI-resistant isolates in 2013 were caused by almost the single haplotype HY1 probably through seed transmission. After discontinuance of the QoI fungicide application in 2014, however, the QoI-resistant isolates decreased over the course of several years, suggesting that genetic alternations conferring the QoI resistance may have caused a decrease in fitness. Conversely, QoI-sensitive isolates increased after 2015, but predominant haplotypes changed across years of collection. We suggest that it could be possible to re-use the QoI-fungicide if the disappearance of the resistant strains is confirmed by careful monitoring of QoI resistance throughout the prefecture, especially in and around fields for seed production. Pyricularia oryzae Magnaporthe oryzae rice blast QoI resistance orysastrobin fitness Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Rice blast, caused by Pyricularia oryzae (syn. Magnaporthe oryzae ) pathotype Oryza (MoO), is the most serious disease affecting rice production in paddy fields. Infected seeds are the main inoculum source for the primary infection, although rice straws and husks around seedbeds also serve as inoculum sources (Ishikawa et al. 2024 ). Fungicides applied to nursery boxes remain effective from transplanting to almost the heading stage. Orysastrobin was introduced as a new QoI fungicide in 2004 for nursery box treatment (Stammler et al. 2007 ). Since it is effective not only against rice blast but also against sheath blight, it had been widely used in rice cultivation in Hyogo Prefecture since 2007. QoI-resistant MoO isolates were first reported in Yamaguchi Prefecture in 2012 and have gradually spread to other prefectures, primarily in western Japan (Ishii 2014 ). In 2013 QoI-resistant MoO isolates emerged across a wide area of Hyogo Prefecture. In response to this sudden outbreak, the use of the QoI fungicide in rice cultivation was discontinued in the following years (Hyogo Prefectural Plant Protection Station 2013 ). Since QoI fungicides are crucial in rice cultivation (Gisi et al. 2002 ), it is necessary to identify factors contributing to the spread of QoI-resistant MoO isolates, analyze the fitness of the isolates, and assess the possibility of reintroducing QoI fungicides. In the recent rice cultivation with nursery boxes, seed transmission is the main route for the primary infection of MoO as mentioned above. Fungicides applied to nursery boxes may select fungicide-resistant isolates colonizing rice seeds, potentially facilitating their seed transmission (Arai et al. 2009 ; Takahashi et al. 2010 ). Strobilurin fungicides represented by orysastrobin possess a single point of action that inhibits respiration by disrupting electron transport at the Qo site of the cytochrome bc1 enzyme complex in mitochondria (Araki et al. 2005 ; Gisi et al. 2002 ). The enzyme complex is encoded by the mitochondrial cytochrome b gene. QoI resistance is mainly conferred by a non-synonymous mutation in which glycine at codon 143 of the cytochrome b gene in mitochondrial DNA is replaced by alanine (Ishii et al. 2007 ). This mutation typically enhances proliferation of the resistant isolates in the presence of the fungicides, but may impose a fitness cost as an evolutionary trade-off (Hawkins and Fraaije 2018 ). The fitness reduction in QoI-resistant M. oryzae isolates was observed in perennial ryegrass blast (Ma et al. 2009 ) but not in barley blast (Avilia-Adame and Köller 2003). However, these investigations were conducted in laboratories. To evaluate the fitness of QoI-resistant MoO isolates in the agroecosystem, long-term field investigations are necessary (Hawkins and Fraaije 2018 ). The present study was performed with two objectives: (i) to estimate factors contributing to, or associated with, the spread of QoI-resistant MoO isolates in Hyogo Prefecture, and (ii) to evaluate the fitness of the resistant MoO isolates at the field level. To attain these objectives, we analyzed dynamics of the population structure of MoO from 2013, when the QoI-resistant MoO isolates first occurred, through 2023 using the Pot2 rep-PCR method (Suzuki et al. 2006 ), which has previously been used to infer the dynamics of MBI-D-resistant MoO isolates (Suzuki et al. 2007 ; Suzuki et al. 2012 ). Materials and Methods Fungal materials A total of 471 MoO isolates were collected from 113 paddy fields in Hyogo Prefecture during July–August in 2013–2017 and 2023. Rice plants in these fields had been transplanted in May–June. In 2013 diseased leaves were collected from paddy fields where orysastrobin was widely applied. From 2014 to 2017 and 2023, diseased leaves were all derived from areas with no orysastrobin application to nursery boxes in that year because it was discontinued in 2014 as described later. One leaf was collected from each of up to 10 plants exhibiting typical symptoms of rice blast per field (Suzuki et al. 2012 ). These leaves with lesions were washed with tap water for one minute, dried with Kimwipes, placed on a moistened filter paper inside a plastic box, and kept at 25°C for 48 h under BLB irradiation to induce conidial formation. Conidia were scraped and spread on 2% water agar (WA) in 9 cm Petri dishes using 10 µl of disposable loops. After incubation in the dark for 24 h, germinated conidia were transferred to PDA plates, resulting in 1 to 10 monoconidial MoO isolates per field. DNA isolation Mycelial tips of the MoO isolates grown for 7–10 days on PDA petri dishes were cut into 5 mm squares and suspended in 5 ml of potato dextrose broth (Difco Laboratories, Detroit, MI, USA) in a petri dish and incubated at 25°C in the dark for 4 days. Using a sterile toothpick, the mycelia in the plate were transferred to the Maxwell® Nucleic Acid Isolation Kit AS1620 (Promega, Madison, WI, USA), and DNA was extracted according to the manufacturer's protocol. Diagnosis of QoI resistance The G143A mutation in QoI-resistant MoO isolates was detected using the PCR-RFLP method (Ogasawara et al. 2016 ). The PCR reaction mixture contained 5 µl of TaKaRa Ex Taq (Takara Bio, Shiga, Japan), 1 µl each of 2 µM KES415 (5'-CTTACCTTATCGATGCGTCACAACC-3') and KES-416 (5'-GCAGTATCATGAAGTGCAATTAAGTGC-3') primers, 2 µl of PCR-grade water, and 30 ng of genomic DNA. Cycling parameters were 95°C for 10 min, followed by 40 cycles of 94°C for 15 sec, 60°C for 30 sec, 72°C for 30 sec, and a final extension of 5 min at 72°C. To digest the amplicon, 2.5 µl of PCR product, 0.25 µl of Fnu4HI (10 U/µl) (New England Biolabs, Ipswich, MA, USA), 0.5 µl of rCutSmart Buffer, and 4.25 µl of PCR-grade water (Promega, Madison, WI, USA) were mixed and incubated at 37°C for 4 h. Restriction fragments were resolved by electrophoresis in a 1.5% agarose gel (type 2, high EEO, Sigma-Aldrich, St. Louis, MO, USA), stained with Midori Green Extra (NIPPON genetics, Tokyo, Japan) in Tris-acetate-EDTA (TAE) buffer, and visualized under UV light. DNA fingerprinting of MoO isolates using rep-PCR DNA fingerprinting of the MoO isolates was performed using the Pot2 rep-PCR method (Suzuki et al. 2006 ) with a single primer Pot2 -TIR (5'-ACAGGGGGTACGCAACGTTA-3') designed from the terminal inverted repeat sequence in the repetitive element Pot2 in P. oryzae . The 20-µl reaction mixture contained 30 ng of genomic DNA, 0.2 µM Pot2-TIR, and 10 µl of Quick Taq (Toyobo, Osaka, Japan). The PCR reaction was performed using TP650 (Takara Bio, Shiga, Japan). Cycling parameters were a 2-min heat denaturation at 94°C, followed by 35 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 6 min, and a final extension at 72°C for 15 min. PCR products were separated by electrophoresis on a 1% agarose gel (type 2, high EEO, Sigma-Aldrich, St. Louis, MO, USA) in 1× Tris-Borate-EDTA (TBE) buffer (NACALAI TESQUE, Kyoto, Japan) for 1.5 h. The amplicon was stained with Midori Green Extra (NIPPON Genetics, Tokyo, Japan) in Tris-borate-EDTA (TBE) buffer. Rep-PCR polymorphic bands ranging from 0.2 to 5 kb in length were scored manually. The presence or absence of bands in each isolate was converted into binary data. Statistical analysis The analysis was performed using the Minimum Spanning Tree method with the statistical software R ver. 4.4.2 (R Core Team 2024 ). Results Association between orysastrobin application and the proportion of QoI-resistant isolates According to the government data in 2012, orysastrobin was applied to nursery boxes in 24.3% of rice paddy fields in Hyogo Prefecture (Fig. 1 a). In 2013 QoI-resistant MoO isolates were isolated from fields at a remarkably high rate of 92.6% (Fig. 1 b). After 2014 when the use of QoI fungicides including orysastrobin was discontinued, however, the frequency of resistant isolates decreased gradually, then dropped sharply in 2016, diminished to less than 5% in 2017, and eventually reached to undetectable level by 2023. Geographic distribution of the HY1 haplotype resistant to QoI We scored the presence or absence of bands generated from pot2 Rep-PCR at 21 positions on the gel within the 0.2 to 5 kb range (Fig. 2 ). Consequently, 52 distinct fingerprint haplotypes were identified in 471 MoO isolates (Fig. 2 , Table S1 , Table S2). In 2013 most of the QoI-resistant MoO isolates belonged to the HY1 haplotype (Table S1 ). The QoI-resistant HY1 isolates were collected from 25 out of 28 fields in Hyogo Prefecture (Fig. 3 ). Despite the short sampling period from mid-July to early August, they were distributed across a wide area measuring 66.4 km east-west and 79.7 km north-south, with the most distant fields separated by 85.8 km. Dynamics of the population structure of MoO isolates after the discontinuation of QoI fungicides The 194 resistant isolates were divided into 8 haplotypes (Table S1 ), among which HY1, HY2, and HY4 were shared by those found in sensitive MoO isolates. Among the resistant isolates, HY1 accounted for the majority (86.4–100%) in each year with a total of 170 isolates (87.6%) over the six-year period (Fig. 4 a, Table S1 ). In contrast, the 277 sensitive isolates were divided into 47 haplotypes (Fig. 4 b, Table S2). Among them HY10 accounted for a large proportion ranging from 22.0% to 47.5% in every year except 2014 with a lower number of samples. The total number of HY10 isolates over six years was 98, representing 35.4% (Fig. 4 b, Table S2). HY1, HY2, and HY4 observed in the resistant isolates were also detected in the sensitive isolates, but their proportions were only 4.3%, 8.7%, and 1.4%, respectively. The frequency of haplotypes unique to a year increased from 5.8% in 2013 to 17.8% in 2023, with an average of 16.2%. The dynamics of haplotypes was chronologically analyzed in detail. In 2013 closely related QoI-resistant haplotypes such as HY1, HY2, and HY4 were predominant (Fig. 5 , Table S1 , S2). In 2014 the resistant HY1 haplotype maintained its dominance, but the sensitive HY1 haplotype emerged. Furthermore, representative sensitive haplotypes such as HY8 and HY21 were also detected. In 2015, the resistant HY1 and sensitive HY10 and HY21 were still predominant, but HY41-45, genetically unique, novel haplotypes occurred. In 2016 the resistant haplotype HY1 decreased while the sensitive haplotypes HY10 and HY21 became dominant. In addition, ten unique haplotypes HY61-71 appeared. In 2017 the resistant HY1 haplotype was still detected, but in a very small number. Twelve unique haplotypes of HY80-91 emerged, which tended to be located on outer branches of the tree. In 2023 HY10 became predominant. MoO isolates with the HY1 haplotype were detected but they were all QoI-sensitive. Additionally, nine unique haplotypes, HY101-109, were observed, which were located outside the phylogenetic tree. No resistant MoO isolates were detected in 2023. Overall, the sharp decline of resistant MoO isolates from 2013 to 2023 could be explained by the decrease of resistant HY1. Conversely, HY10 increased among the sensitive isolates and became predominant after 2015. Discussion A comprehensive understanding of mechanisms underlying the spread of QoI-resistant MoO isolates is imperative for designing effective control strategies. To reveal factors driving the spread of the resistant isolates, we analyzed the population structure of MoO isolates collected from various locations in Hyogo Prefecture immediately after their first emergence from 2013 to 2023. Our results showed that the outbreak in 2013 was caused by almost a single haplotype, HY1, with QoI resistance. In addition, this haplotype was widely distributed in Hyogo Prefecture even in the first year, 2013 (Fig. 1 , Fig. 3 ). In the secondary infection cycles, MoO isolates are estimated to spread approximately 1 km per cycle (Ishiguro et al. 1998 ). It should be noted that the QoI-resistant HY1 was detected at locations 85.8 km apart across the Akashi Strait (Fig. 3 ) in July to August in 2013, only one to two months after transplanting in June. This finding suggests that the observed spread may not have resulted from the secondary infection but rather from the primary infection through seed transmission. Suzuki et al. ( 2017 ) suggested that the risk of the emergence of QoI-resistant isolates would become apparent when orysastrobin was continuously used for ~ 6 years in more than 10% of paddy fields in a prefecture. The application of this QoI fungicide in Hyogo prefecture from 2007 to 2013 exceeded this threshold (Fig. 1 a), suggesting that there had been a high risk of the emergence of resistant isolates in this prefecture. Until 2013, orysastrobin had been already incorporated into the disease control schedules of many branches of Japan Agricultural Cooperatives in Hyogo Prefecture, leading to its widespread utilization. Although it has not been utilized in seed production fields, they are adjacent to farmers’ fields in which orysastrobin was applied. This may have led to the introduction of QoI-resistant isolates from the adjacent fields into the seed production fields. If some seeds sown to nursery boxes harbor fungicide-resistant MoO isolates, they will be selected by the fungicide and spread more easily (Takahashi et al. 2010 ).The nursery box method, in which dense seedlings are grown under conditions with high temperature and humidity, has been demonstrated to promote the emergence and rapid spread of MBI-D-resistant MoO isolates (Suzuki et al. 2007 ). Since orysastrobin maintains its efficacy over a long period, its application to nursery boxes with seeds harboring resistant MoO isolates could facilitate the selection and proliferation of resistant isolates more efficiently. Indeed, severe outbreaks of leaf blast were observed in 2013 from the early growth stage in actual fields where orysastrobin was applied at sowing in nursery boxes. The mutation for gaining QoI resistance is associated with dysfunctional mitochondria, which exhibit a reduced electron transport flow within the cytochrome bc1 complex. Therefore, it has been hypothesized that QoI-resistant isolates may exhibit diminished fitness (Köller et al. 2001 ). Indeed, the fitness of QoI-resistant M. oryzae isolates in perennial ryegrass (Ma et al. 2009 ) and MoO isolates (D’Ávila et al. 2022 ) were reported to be lower than those of sensitive isolates. The present study showed that, after the discontinuation of orysastrobin utilization in 2014, the QoI-resistant MoO isolates decreased rapidly (Fig. 1 , 2 ). This is in accordance with the previous hypothesis and results from the laboratory experiments mentioned above. The MoO isolates complete their life cycle four to five times per season. Consequently, even if the fitness cost exerted in a single cycle is relatively low, sensitive isolates may be able to overwhelm resistant isolates in the asexual life cycles (Suzuki et al. 2010 ). Taken together, we deduce that (i) the fitness of the QoI-resistant MoO isolate is lower than that of the sensitive isolate, and that (ii) the reduction of fitness may, even if it is trivial, result in a large reduction of survival rate through repeated asexual reproduction. As the frequency of resistant MoO isolates decreased, unique sensitive haplotypes emerged, many of which were replaced by new haplotypes over time (Fig. 4 , Table S1 , S2, Fig. 5 ). The phylogenetic tree showed that HY10 and HY21 were located at the center, but that unique haplotypes were positioned at a genetic distance from the center (Excoffier et al. 1992 ). Consistent with prior studies with MBI-D-resistant MoO isolates (Suzuki, 2012), the diversity of sensitive isolates exceeded that of resistant isolates (Table S1 , S2). These results could be explained by assuming that QoI-sensitive MoO isolates diversified in the process of proliferation and domination over resistant MoO isolates in the seed production fields. The seed renewal rate among producers in Hyogo Prefecture exceeds 80%. Consequently, MoO isolates in farmers’ fields may have been replaced in each year depending on predominant haplotypes in the seed production fields. What is the origin of the QoI-resistant MoO isolates? As previously described, orysastrobin had been widely used in Hyogo Prefecture from 2007 to 2013. One hypothesis is that indigenous MoO isolates in the prefecture have undergone mutations, thereby acquiring resistance to QoI fungicides. Another possibility is that seeds introduced from external sources beyond the geographical boundaries of the prefecture were contaminated with QoI-resistant isolates. After discontinuing the QoI fungicides utilization, QoI-sensitive HY1 and HY2 were detected although their frequencies were much lower in comparison with those of HY10 and HY21. This raises another question whether the QoI-sensitive HY1 emerged as the QoI-resistant HY1 lost their resistance or was indigenous to Hyogo Prefecture. In experiments involving multiple cultures on artificial media without QoI fungicides, QoI resistance decreased in a report (Markoglou et al. 2006 ) but did not in other reports (Forcelini et al. 2018 ; Ding et al. 2019 ). Further studies are needed to answer these questions. Over ten years have passed after the first occurrence of the QoI-resistant isolates in Hyogo Prefecture. We are now attempting to reuse QoI fungicides in rice paddy fields. There have been some examples of reuse of QoI fungicides in prefectures where resistant isolates have occurred. According to Suzuki et al. ( 2007 ), however, fungicide application to nursery boxes poses a higher risk of driving the emergence of resistant isolates than application to fields due to its long-lasting residual effects. It has been reported that resistance was reacquired through cultivation on media supplemented with QoI fungicides (Markoglou et al. 2006 ). Therefore, the reuse of QoI fungicides should be decided with caution based on meticulous monitoring of the development and disappearance of resistance. Also, the effective management of resistant isolates in seed production fields is imperative for ensuring the stability of reuse. It is essential to comply the QoI Agent Usage Guidelines (Research Committee on Fungicide Resistance, 2008) which proscribes the application of QoI fungicides to seed production fields. Additionally, a comprehensive risk management must be thoroughly implemented in these fields. Declarations Acknowledgements We thank Michiyo Sugano for assisting the experiments. We also thank the staff of Hyogo prefectural agricultural extension center for collecting diseased rice leaves, and Katsunari Matsuura, Shinji Nishiguchi, and Yutaka Iwamoto for isolating the blast fungus. Special thanks are due to Takeshi Kanto for valuable suggestions. Author contributions KU conceived the study and performed the experiments. FS, SA, KI, HN, and YT analyzed the data. The first draft of the manuscript was written by KU and revised by YT. All authors commented on the draft and approved the final manuscript. Funding No funding was received for conducting this study. Conflict of interest The authors declare that they have no conflict of interest. Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. 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Plant Dis 94: 283-382. https://doi.org/10.1094/PDIS-94-3-0329 Suzuki F, Mitsunaga T, Ashizawa T (2017) Statistical analysis of factors affecting resistance development of rice blast fungus to QoI fungicides based on pesticide shipment data (in Japanese). Ann Rept of the Kanto-Tosan Plant Prot Soc 64:6-9. https://doi.org/10.11337/ktpps.2017.6 Suzuki H, Suzuki F, Kusaba M, Tosa Y (2012) Population structure of rice blast isolates resistant to scytalone dehydratase inhibitors in Mie Prefecture and implications for their origin. J Gen Plant Pathol 78: 106–114. DOI 10.1007/s10327-012-0365-y Takahashi N, Tominaga T, Fujisawa Y, Iwadate Y (2010) Analysis of factors for occurrence and predominance of dehydratase inhibitors in melanin biosynthesis (MBI-D) fungicide resistant isolates of Pyricularia oryzae in Iwate prefecture (in Japanese). Ann Rept Plant Prot North Japan 61:9-13. https://doi.org/10.11455/kitanihon.2010.61_9 Supplementary Files TablesS1S2.pdf List of supplementary files Table S1, S2 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 26 Mar, 2026 Reviewers invited by journal 26 Mar, 2026 Editor assigned by journal 26 Mar, 2026 First submitted to journal 19 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9175517","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":612456064,"identity":"fa74c0a1-1cad-41f4-8dc3-ea174c27c1cd","order_by":0,"name":"Kaichi Uchihashi","email":"","orcid":"","institution":"Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries","correspondingAuthor":false,"prefix":"","firstName":"Kaichi","middleName":"","lastName":"Uchihashi","suffix":""},{"id":612456065,"identity":"43183d13-6707-40fa-b4d9-20118945b6cc","order_by":1,"name":"Fumihiko Suzuki","email":"","orcid":"","institution":"Hokkaido Agricultural Research Center","correspondingAuthor":false,"prefix":"","firstName":"Fumihiko","middleName":"","lastName":"Suzuki","suffix":""},{"id":612456066,"identity":"5608ec29-85b7-4af1-85d0-52610d0ff47a","order_by":2,"name":"Soichiro Asuke","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Soichiro","middleName":"","lastName":"Asuke","suffix":""},{"id":612456067,"identity":"0bc39b95-1a9f-4499-bd6f-9f9bf458fe96","order_by":3,"name":"Kenichi Ikeda","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Kenichi","middleName":"","lastName":"Ikeda","suffix":""},{"id":612456068,"identity":"eb9f705c-6f26-413c-b926-021bae7e1cca","order_by":4,"name":"Hitoshi Nakayashiki","email":"","orcid":"","institution":"Kobe University: Kobe Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Hitoshi","middleName":"","lastName":"Nakayashiki","suffix":""},{"id":612456069,"identity":"b69e58a9-059a-4e37-ba2d-c02676a2f808","order_by":5,"name":"Yukio TOSA","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3QuwrCMBiG4U8FXf7StVLRW2gJBMTBW6kIdnEQBHF0apeCawcvRijUJerqqHR1EFwERYyCIg4pboJ5pyTkIQdAp/vdPDIJ5dfUUe2lJ6lG3xI44o0oa1fC3dEIejUmknR7QAIznGM4UJ1CgtlG0Ce+DHw3lsQSHlisIlYfkoyJr4nbxjUBNgAjJfGz852wqSQXeUojn3j8cTHHiLgNSZxcIgRvzlY9skTquxF8ckVnonxLJQyzzX7UbZtRN92e0KrXF0nKVD92r/h+DTkuBCxHoHD6WChleUSn0+n+qhvznkFYYDLlgwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-4067-5016","institution":"Kobe University","correspondingAuthor":true,"prefix":"","firstName":"Yukio","middleName":"","lastName":"TOSA","suffix":""}],"badges":[],"createdAt":"2026-03-20 06:24:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9175517/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9175517/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105574023,"identity":"32f2a8aa-4697-47bc-b604-d69c24495e49","added_by":"auto","created_at":"2026-03-27 13:33:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20420,"visible":true,"origin":"","legend":"\u003cp\u003eTransition of orysastrobin application and the development of QoI resistance in Hyogo prefecture from 2007 to 2023. (a) Percentage of areas of paddy fields treated with orysastrobin; (b) Percentage of QoI-resistant blast isolates.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9175517/v1/04fad1b53a133ac90aaac2e2.png"},{"id":105573040,"identity":"118ebea5-ec5d-4b53-b475-44b3d6dc9432","added_by":"auto","created_at":"2026-03-27 13:30:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":163022,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative fingerprint patterns of blast isolates collected in Hyogo prefecture. M: Molecular Maker\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9175517/v1/6d982376d639e575b9998cee.png"},{"id":105572730,"identity":"b93899e7-df69-4365-a54d-1b677f403b0e","added_by":"auto","created_at":"2026-03-27 13:29:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":33663,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical distribution of the QoI-resistant HY1 haplotype in 2013 in Hyogo prefecture. ▲, field with HY1; ■, field with QoI-resistant haplotypes other than HY1 but no HY1; □, field with QoI-sensitive haplotypes alone.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9175517/v1/f53668a09826631380acf977.png"},{"id":105573516,"identity":"564caf4a-de1d-4f16-be17-0387586e2e8e","added_by":"auto","created_at":"2026-03-27 13:31:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16100,"visible":true,"origin":"","legend":"\u003cp\u003eTransition of population structures of the rice blast fungus in Hyogo prefecture. (a) QoI-resistant haplotypes; (b) QoI-sensitive haplotypes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9175517/v1/f79090a4eea0f31ae9ec5906.png"},{"id":105572473,"identity":"d3684669-cd9e-4ff5-bb30-3c1acb6c8250","added_by":"auto","created_at":"2026-03-27 13:26:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":171704,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics of haplotypes of rice blast isolates in Hyogo prefecture from 2013 through 2023. A minimum spanning tree of the haplotypes was constructed using the hamming distance. In each year undetected haplotypes are indicated by circles with dotted lines while detected haplotypes are painted with colors indicating the year of their first detection. The size of the circles is proportional to the haplotype frequency. Haplotypes containing QoI-resistant isolates alone, QoI-sensitive isolates alone, and both are described in red, black, and purple, respectively.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9175517/v1/f6080183c7f6201aa8c9afcf.png"},{"id":105575349,"identity":"868d3213-a92f-4ab5-9457-2966135cdeb0","added_by":"auto","created_at":"2026-03-27 13:38:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":684696,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9175517/v1/2d9a451e-b2d8-4bd3-88fd-7ff1a059804c.pdf"},{"id":105572471,"identity":"8ca4cfad-e356-4901-a514-91e4fb2ecbf5","added_by":"auto","created_at":"2026-03-27 13:26:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":222601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eList of supplementary files\u003c/strong\u003e Table S1, S2\u003c/p\u003e","description":"","filename":"TablesS1S2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9175517/v1/314dd1f89c7c83e47fc98e46.pdf"}],"financialInterests":"","formattedTitle":"Population dynamics of QoI-resistant rice blast isolates in Hyogo Prefecture associated with the use and discontinuation of QoI fungicides","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice blast, caused by \u003cem\u003ePyricularia oryzae\u003c/em\u003e (syn. \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e) pathotype \u003cem\u003eOryza\u003c/em\u003e (MoO), is the most serious disease affecting rice production in paddy fields. Infected seeds are the main inoculum source for the primary infection, although rice straws and husks around seedbeds also serve as inoculum sources (Ishikawa et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Fungicides applied to nursery boxes remain effective from transplanting to almost the heading stage. Orysastrobin was introduced as a new QoI fungicide in 2004 for nursery box treatment (Stammler et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Since it is effective not only against rice blast but also against sheath blight, it had been widely used in rice cultivation in Hyogo Prefecture since 2007.\u003c/p\u003e \u003cp\u003eQoI-resistant MoO isolates were first reported in Yamaguchi Prefecture in 2012 and have gradually spread to other prefectures, primarily in western Japan (Ishii \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In 2013 QoI-resistant MoO isolates emerged across a wide area of Hyogo Prefecture. In response to this sudden outbreak, the use of the QoI fungicide in rice cultivation was discontinued in the following years (Hyogo Prefectural Plant Protection Station \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Since QoI fungicides are crucial in rice cultivation (Gisi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), it is necessary to identify factors contributing to the spread of QoI-resistant MoO isolates, analyze the fitness of the isolates, and assess the possibility of reintroducing QoI fungicides. In the recent rice cultivation with nursery boxes, seed transmission is the main route for the primary infection of MoO as mentioned above. Fungicides applied to nursery boxes may select fungicide-resistant isolates colonizing rice seeds, potentially facilitating their seed transmission (Arai et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Takahashi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStrobilurin fungicides represented by orysastrobin possess a single point of action that inhibits respiration by disrupting electron transport at the Qo site of the cytochrome bc1 enzyme complex in mitochondria (Araki et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gisi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The enzyme complex is encoded by the mitochondrial cytochrome b gene. QoI resistance is mainly conferred by a non-synonymous mutation in which glycine at codon 143 of the cytochrome b gene in mitochondrial DNA is replaced by alanine (Ishii et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This mutation typically enhances proliferation of the resistant isolates in the presence of the fungicides, but may impose a fitness cost as an evolutionary trade-off (Hawkins and Fraaije \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The fitness reduction in QoI-resistant \u003cem\u003eM. oryzae\u003c/em\u003e isolates was observed in perennial ryegrass blast (Ma et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) but not in barley blast (Avilia-Adame and K\u0026ouml;ller 2003). However, these investigations were conducted in laboratories. To evaluate the fitness of QoI-resistant MoO isolates in the agroecosystem, long-term field investigations are necessary (Hawkins and Fraaije \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study was performed with two objectives: (i) to estimate factors contributing to, or associated with, the spread of QoI-resistant MoO isolates in Hyogo Prefecture, and (ii) to evaluate the fitness of the resistant MoO isolates at the field level. To attain these objectives, we analyzed dynamics of the population structure of MoO from 2013, when the QoI-resistant MoO isolates first occurred, through 2023 using the \u003cem\u003ePot2\u003c/em\u003e rep-PCR method (Suzuki et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), which has previously been used to infer the dynamics of MBI-D-resistant MoO isolates (Suzuki et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Suzuki et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eFungal materials\u003c/p\u003e \u003cp\u003eA total of 471 MoO isolates were collected from 113 paddy fields in Hyogo Prefecture during July\u0026ndash;August in 2013\u0026ndash;2017 and 2023. Rice plants in these fields had been transplanted in May\u0026ndash;June. In 2013 diseased leaves were collected from paddy fields where orysastrobin was widely applied. From 2014 to 2017 and 2023, diseased leaves were all derived from areas with no orysastrobin application to nursery boxes in that year because it was discontinued in 2014 as described later. One leaf was collected from each of up to 10 plants exhibiting typical symptoms of rice blast per field (Suzuki et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These leaves with lesions were washed with tap water for one minute, dried with Kimwipes, placed on a moistened filter paper inside a plastic box, and kept at 25\u0026deg;C for 48 h under BLB irradiation to induce conidial formation. Conidia were scraped and spread on 2% water agar (WA) in 9 cm Petri dishes using 10 \u0026micro;l of disposable loops. After incubation in the dark for 24 h, germinated conidia were transferred to PDA plates, resulting in 1 to 10 monoconidial MoO isolates per field.\u003c/p\u003e \u003cp\u003eDNA isolation\u003c/p\u003e \u003cp\u003eMycelial tips of the MoO isolates grown for 7\u0026ndash;10 days on PDA petri dishes were cut into 5 mm squares and suspended in 5 ml of potato dextrose broth (Difco Laboratories, Detroit, MI, USA) in a petri dish and incubated at 25\u0026deg;C in the dark for 4 days. Using a sterile toothpick, the mycelia in the plate were transferred to the Maxwell\u0026reg; Nucleic Acid Isolation Kit AS1620 (Promega, Madison, WI, USA), and DNA was extracted according to the manufacturer's protocol.\u003c/p\u003e \u003cp\u003eDiagnosis of QoI resistance\u003c/p\u003e \u003cp\u003eThe G143A mutation in QoI-resistant MoO isolates was detected using the PCR-RFLP method (Ogasawara et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The PCR reaction mixture contained 5 \u0026micro;l of TaKaRa Ex Taq (Takara Bio, Shiga, Japan), 1 \u0026micro;l each of 2 \u0026micro;M KES415 (5'-CTTACCTTATCGATGCGTCACAACC-3') and KES-416 (5'-GCAGTATCATGAAGTGCAATTAAGTGC-3') primers, 2 \u0026micro;l of PCR-grade water, and 30 ng of genomic DNA. Cycling parameters were 95\u0026deg;C for 10 min, followed by 40 cycles of 94\u0026deg;C for 15 sec, 60\u0026deg;C for 30 sec, 72\u0026deg;C for 30 sec, and a final extension of 5 min at 72\u0026deg;C. To digest the amplicon, 2.5 \u0026micro;l of PCR product, 0.25 \u0026micro;l of Fnu4HI (10 U/\u0026micro;l) (New England Biolabs, Ipswich, MA, USA), 0.5 \u0026micro;l of rCutSmart Buffer, and 4.25 \u0026micro;l of PCR-grade water (Promega, Madison, WI, USA) were mixed and incubated at 37\u0026deg;C for 4 h. Restriction fragments were resolved by electrophoresis in a 1.5% agarose gel (type 2, high EEO, Sigma-Aldrich, St. Louis, MO, USA), stained with Midori Green Extra (NIPPON genetics, Tokyo, Japan) in Tris-acetate-EDTA (TAE) buffer, and visualized under UV light.\u003c/p\u003e \u003cp\u003eDNA fingerprinting of MoO isolates using rep-PCR\u003c/p\u003e \u003cp\u003eDNA fingerprinting of the MoO isolates was performed using the \u003cem\u003ePot2\u003c/em\u003e rep-PCR method (Suzuki et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) with a single primer \u003cem\u003ePot2\u003c/em\u003e-TIR (5'-ACAGGGGGTACGCAACGTTA-3') designed from the terminal inverted repeat sequence in the repetitive element \u003cem\u003ePot2\u003c/em\u003e in \u003cem\u003eP. oryzae\u003c/em\u003e. The 20-\u0026micro;l reaction mixture contained 30 ng of genomic DNA, 0.2 \u0026micro;M Pot2-TIR, and 10 \u0026micro;l of Quick Taq (Toyobo, Osaka, Japan). The PCR reaction was performed using TP650 (Takara Bio, Shiga, Japan). Cycling parameters were a 2-min heat denaturation at 94\u0026deg;C, followed by 35 cycles of 94\u0026deg;C for 1 min, 62\u0026deg;C for 1 min, and 72\u0026deg;C for 6 min, and a final extension at 72\u0026deg;C for 15 min. PCR products were separated by electrophoresis on a 1% agarose gel (type 2, high EEO, Sigma-Aldrich, St. Louis, MO, USA) in 1\u0026times; Tris-Borate-EDTA (TBE) buffer (NACALAI TESQUE, Kyoto, Japan) for 1.5 h. The amplicon was stained with Midori Green Extra (NIPPON Genetics, Tokyo, Japan) in Tris-borate-EDTA (TBE) buffer. Rep-PCR polymorphic bands ranging from 0.2 to 5 kb in length were scored manually. The presence or absence of bands in each isolate was converted into binary data.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe analysis was performed using the Minimum Spanning Tree method with the statistical software R ver. 4.4.2 (R Core Team \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAssociation between orysastrobin application and the proportion of QoI-resistant isolates\u003c/p\u003e \u003cp\u003eAccording to the government data in 2012, orysastrobin was applied to nursery boxes in 24.3% of rice paddy fields in Hyogo Prefecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In 2013 QoI-resistant MoO isolates were isolated from fields at a remarkably high rate of 92.6% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). After 2014 when the use of QoI fungicides including orysastrobin was discontinued, however, the frequency of resistant isolates decreased gradually, then dropped sharply in 2016, diminished to less than 5% in 2017, and eventually reached to undetectable level by 2023.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGeographic distribution of the HY1 haplotype resistant to QoI\u003c/p\u003e \u003cp\u003eWe scored the presence or absence of bands generated from \u003cem\u003epot2\u003c/em\u003e Rep-PCR at 21 positions on the gel within the 0.2 to 5 kb range (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Consequently, 52 distinct fingerprint haplotypes were identified in 471 MoO isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Table S2). In 2013 most of the QoI-resistant MoO isolates belonged to the HY1 haplotype (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The QoI-resistant HY1 isolates were collected from 25 out of 28 fields in Hyogo Prefecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Despite the short sampling period from mid-July to early August, they were distributed across a wide area measuring 66.4 km east-west and 79.7 km north-south, with the most distant fields separated by 85.8 km.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDynamics of the population structure of MoO isolates after the discontinuation of QoI fungicides\u003c/p\u003e \u003cp\u003eThe 194 resistant isolates were divided into 8 haplotypes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), among which HY1, HY2, and HY4 were shared by those found in sensitive MoO isolates. Among the resistant isolates, HY1 accounted for the majority (86.4\u0026ndash;100%) in each year with a total of 170 isolates (87.6%) over the six-year period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In contrast, the 277 sensitive isolates were divided into 47 haplotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Table S2). Among them HY10 accounted for a large proportion ranging from 22.0% to 47.5% in every year except 2014 with a lower number of samples. The total number of HY10 isolates over six years was 98, representing 35.4% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Table S2). HY1, HY2, and HY4 observed in the resistant isolates were also detected in the sensitive isolates, but their proportions were only 4.3%, 8.7%, and 1.4%, respectively. The frequency of haplotypes unique to a year increased from 5.8% in 2013 to 17.8% in 2023, with an average of 16.2%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe dynamics of haplotypes was chronologically analyzed in detail. In 2013 closely related QoI-resistant haplotypes such as HY1, HY2, and HY4 were predominant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2). In 2014 the resistant HY1 haplotype maintained its dominance, but the sensitive HY1 haplotype emerged. Furthermore, representative sensitive haplotypes such as HY8 and HY21 were also detected. In 2015, the resistant HY1 and sensitive HY10 and HY21 were still predominant, but HY41-45, genetically unique, novel haplotypes occurred. In 2016 the resistant haplotype HY1 decreased while the sensitive haplotypes HY10 and HY21 became dominant. In addition, ten unique haplotypes HY61-71 appeared. In 2017 the resistant HY1 haplotype was still detected, but in a very small number. Twelve unique haplotypes of HY80-91 emerged, which tended to be located on outer branches of the tree. In 2023 HY10 became predominant. MoO isolates with the HY1 haplotype were detected but they were all QoI-sensitive. Additionally, nine unique haplotypes, HY101-109, were observed, which were located outside the phylogenetic tree. No resistant MoO isolates were detected in 2023. Overall, the sharp decline of resistant MoO isolates from 2013 to 2023 could be explained by the decrease of resistant HY1. Conversely, HY10 increased among the sensitive isolates and became predominant after 2015.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eA comprehensive understanding of mechanisms underlying the spread of QoI-resistant MoO isolates is imperative for designing effective control strategies. To reveal factors driving the spread of the resistant isolates, we analyzed the population structure of MoO isolates collected from various locations in Hyogo Prefecture immediately after their first emergence from 2013 to 2023. Our results showed that the outbreak in 2013 was caused by almost a single haplotype, HY1, with QoI resistance. In addition, this haplotype was widely distributed in Hyogo Prefecture even in the first year, 2013 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the secondary infection cycles, MoO isolates are estimated to spread approximately 1 km per cycle (Ishiguro et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). It should be noted that the QoI-resistant HY1 was detected at locations 85.8 km apart across the Akashi Strait (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) in July to August in 2013, only one to two months after transplanting in June. This finding suggests that the observed spread may not have resulted from the secondary infection but rather from the primary infection through seed transmission.\u003c/p\u003e \u003cp\u003eSuzuki et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) suggested that the risk of the emergence of QoI-resistant isolates would become apparent when orysastrobin was continuously used for ~\u0026thinsp;6 years in more than 10% of paddy fields in a prefecture. The application of this QoI fungicide in Hyogo prefecture from 2007 to 2013 exceeded this threshold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), suggesting that there had been a high risk of the emergence of resistant isolates in this prefecture. Until 2013, orysastrobin had been already incorporated into the disease control schedules of many branches of Japan Agricultural Cooperatives in Hyogo Prefecture, leading to its widespread utilization. Although it has not been utilized in seed production fields, they are adjacent to farmers\u0026rsquo; fields in which orysastrobin was applied. This may have led to the introduction of QoI-resistant isolates from the adjacent fields into the seed production fields.\u003c/p\u003e \u003cp\u003eIf some seeds sown to nursery boxes harbor fungicide-resistant MoO isolates, they will be selected by the fungicide and spread more easily (Takahashi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).The nursery box method, in which dense seedlings are grown under conditions with high temperature and humidity, has been demonstrated to promote the emergence and rapid spread of MBI-D-resistant MoO isolates (Suzuki et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Since orysastrobin maintains its efficacy over a long period, its application to nursery boxes with seeds harboring resistant MoO isolates could facilitate the selection and proliferation of resistant isolates more efficiently. Indeed, severe outbreaks of leaf blast were observed in 2013 from the early growth stage in actual fields where orysastrobin was applied at sowing in nursery boxes.\u003c/p\u003e \u003cp\u003eThe mutation for gaining QoI resistance is associated with dysfunctional mitochondria, which exhibit a reduced electron transport flow within the cytochrome bc1 complex. Therefore, it has been hypothesized that QoI-resistant isolates may exhibit diminished fitness (K\u0026ouml;ller et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Indeed, the fitness of QoI-resistant \u003cem\u003eM. oryzae\u003c/em\u003e isolates in perennial ryegrass (Ma et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and MoO isolates (D\u0026rsquo;\u0026Aacute;vila et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) were reported to be lower than those of sensitive isolates. The present study showed that, after the discontinuation of orysastrobin utilization in 2014, the QoI-resistant MoO isolates decreased rapidly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This is in accordance with the previous hypothesis and results from the laboratory experiments mentioned above. The MoO isolates complete their life cycle four to five times per season. Consequently, even if the fitness cost exerted in a single cycle is relatively low, sensitive isolates may be able to overwhelm resistant isolates in the asexual life cycles (Suzuki et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Taken together, we deduce that (i) the fitness of the QoI-resistant MoO isolate is lower than that of the sensitive isolate, and that (ii) the reduction of fitness may, even if it is trivial, result in a large reduction of survival rate through repeated asexual reproduction.\u003c/p\u003e \u003cp\u003eAs the frequency of resistant MoO isolates decreased, unique sensitive haplotypes emerged, many of which were replaced by new haplotypes over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The phylogenetic tree showed that HY10 and HY21 were located at the center, but that unique haplotypes were positioned at a genetic distance from the center (Excoffier et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Consistent with prior studies with MBI-D-resistant MoO isolates (Suzuki, 2012), the diversity of sensitive isolates exceeded that of resistant isolates (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2). These results could be explained by assuming that QoI-sensitive MoO isolates diversified in the process of proliferation and domination over resistant MoO isolates in the seed production fields. The seed renewal rate among producers in Hyogo Prefecture exceeds 80%. Consequently, MoO isolates in farmers\u0026rsquo; fields may have been replaced in each year depending on predominant haplotypes in the seed production fields.\u003c/p\u003e \u003cp\u003eWhat is the origin of the QoI-resistant MoO isolates? As previously described, orysastrobin had been widely used in Hyogo Prefecture from 2007 to 2013. One hypothesis is that indigenous MoO isolates in the prefecture have undergone mutations, thereby acquiring resistance to QoI fungicides. Another possibility is that seeds introduced from external sources beyond the geographical boundaries of the prefecture were contaminated with QoI-resistant isolates. After discontinuing the QoI fungicides utilization, QoI-sensitive HY1 and HY2 were detected although their frequencies were much lower in comparison with those of HY10 and HY21. This raises another question whether the QoI-sensitive HY1 emerged as the QoI-resistant HY1 lost their resistance or was indigenous to Hyogo Prefecture. In experiments involving multiple cultures on artificial media without QoI fungicides, QoI resistance decreased in a report (Markoglou et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) but did not in other reports (Forcelini et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ding et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Further studies are needed to answer these questions.\u003c/p\u003e \u003cp\u003eOver ten years have passed after the first occurrence of the QoI-resistant isolates in Hyogo Prefecture. We are now attempting to reuse QoI fungicides in rice paddy fields. There have been some examples of reuse of QoI fungicides in prefectures where resistant isolates have occurred. According to Suzuki et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), however, fungicide application to nursery boxes poses a higher risk of driving the emergence of resistant isolates than application to fields due to its long-lasting residual effects. It has been reported that resistance was reacquired through cultivation on media supplemented with QoI fungicides (Markoglou et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Therefore, the reuse of QoI fungicides should be decided with caution based on meticulous monitoring of the development and disappearance of resistance. Also, the effective management of resistant isolates in seed production fields is imperative for ensuring the stability of reuse. It is essential to comply the QoI Agent Usage Guidelines (Research Committee on Fungicide Resistance, 2008) which proscribes the application of QoI fungicides to seed production fields. Additionally, a comprehensive risk management must be thoroughly implemented in these fields.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e We thank Michiyo Sugano for assisting the experiments. We also thank the staff of Hyogo prefectural agricultural extension center for collecting diseased rice leaves, and Katsunari Matsuura, Shinji Nishiguchi, and Yutaka Iwamoto for isolating the blast fungus. Special thanks are due to Takeshi Kanto for valuable suggestions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e KU conceived the study and performed the experiments. FS, SA, KI, HN, and YT analyzed the data. The first draft of the manuscript was written by KU and revised by YT. All authors commented on the draft and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp; \u0026nbsp;No funding was received for conducting this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e This article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArai M, Suzuki F, Koba A (2009) An epidemiological survey of MBI-D-resistant isolates in the rice blast fungus (\u003cem\u003ePyricularia grisea\u003c/em\u003e) in Kyushu, 2003 (in Japanese). Kyushu Pl Prot Res 55:7-12. https://doi.org/10.4241/kyubyochu.55.7\u003c/li\u003e\n \u003cli\u003eAraki Y, Sugihara M, Sawada H, Fujimoto H, Masuko M (2005) Monitoring of the sensitivity of \u003cem\u003eMagnaporthe grisea\u003c/em\u003e to metominostrobin 2001\u0026ndash;2003: no emergence of resistant strains and no mutations at codon 143 or 129 of the cytochrome \u003cem\u003eb\u003c/em\u003e gene. J Pestic Sci 30:203-208. https://doi.org/10.1584/jpestics.30.203\u003c/li\u003e\n \u003cli\u003eAvila-Adame C, K\u0026ouml;ller W (2003) Characterization of spontaneous mutants of \u003cem\u003eMagnaporthe grisea\u003c/em\u003e expressing stable resistance to the Qo-inhibiting fungicide azoxystrobin. Curr Genet 42:332-338. 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DOI: 10.1093/genetics/131.2.479\u003c/li\u003e\n \u003cli\u003eForcelini BB, Rebello CS, Wang NY, Peres NA (2018) Fitness, competitive ability, and mutation stability of isolates of \u003cem\u003eColletotrichum acutatum\u003c/em\u003e from strawberry resistant to QoI fungicides. Phytopathology 108:462-468. https://doi.org/10.1094/PHYTO-09-17-0296-R\u003c/li\u003e\n \u003cli\u003eGisi U, Sierotzki H, Cook A, McCaffery A (2002) Mechanisms influencing the evolution of resistance to Qo inhibitor fungicides. Pest Manag Sci 58:859-867. DOI: 10.1002/ps.565\u003c/li\u003e\n \u003cli\u003eHawkins NJ, Fraaije BA (2018) Fitness penalties in the evolution of fungicide resistance. Annu Rev Phytopathol 56:339-360. DOI: 10.1146/annurev-phyto-080417-0500\u003c/li\u003e\n \u003cli\u003eHyogo prefectural plant protection station (2013) Pest and disease forecasting technical information No. 1 (QoI-resistant rice blast diseases report) (in Japanese). https://web1.jppn.ne.jp/cgi-bin/bbs/cgi/p5-2-2.cgi?IDX=7b084b3a7e0b4e3a7c15177d6e1a5e296e184129701b\u0026amp;choice=1\u003c/li\u003e\n \u003cli\u003eIshiguro K, Kobayashi T, Nakajima T, Kanematsu S (1998) Disease gradients at the hundreds meters level from initial disease foci at the beginning of general epidemics of rice leaf blast (in Japanese). Jpn J Phytopathol 64:613-614. https://doi.org/10.3186/jjphytopath.64.605\u003c/li\u003e\n \u003cli\u003eIshii H (2014) Situation of QoI fungicide resistance in rice blast isolate and countermeasures (in Japanese). Plant Protection 68:274-279. ISSN: 0037-4091\u003c/li\u003e\n \u003cli\u003eIshii H, Yano K, Date H, Furuta A, Sagehashi Y, Yamaguchi T, Sugiyama T, Nishimura K, Hasama W (2007) Molecular characterization and diagnosis of QoI resistance in cucumber and eggplant fungal pathogens. Phytopathology 97:1458-1466. https://doi.org/10.1094/PHYTO-97-11-1458\u003c/li\u003e\n \u003cli\u003eIshikawa K, Kuroda T, Hori T (2024) Effect of \u003cem\u003ePyricularia oryzae\u003c/em\u003e population structure at seed farms on local population structure (in Japanese). Proc Assoc Plant Prot Hokuriku 73:15-21. ISSN: 0388-8053\u003c/li\u003e\n \u003cli\u003eK\u0026ouml;ller W, Avila-Adame C, Olaya G, Zheng D (2001) Resistance to strobilurin fungicides. In: Clark JM and Yamaguchi I (eds). Agrochemical resistance\u0026mdash;extent, mechanism, and detection. American Chemical Society, pp 215-229 ISBN13: \u0026zwj;9780841237230\u003c/li\u003e\n \u003cli\u003eMa B, Uddin W, Olaya G (2009) Baseline and non-baseline sensitivity of \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e isolates from perennial ryegrass to azoxystrobin in the northeastern United States. Can J Plant Pathol 31:57-64. https://doi.org/10.1080/07060660909507572\u003c/li\u003e\n \u003cli\u003eMarkoglou AN, Malandrakis AA, Vitoratos AG, Ziogas BN (2006) Characterization of laboratory mutants of \u003cem\u003eBotrytis cinerea\u003c/em\u003e resistant to QoI fungicides. Eur J Plant Pathol 115:149-162. https://doi.org/10.1007/s10658-006-0008-2\u003c/li\u003e\n \u003cli\u003eOgasawara I, Matsuhashi M, Niiyama T, Kikkawa S, Shiratori R, Sayama A, Fujii N, Saitou T, Fujisawa M, Fuji S (2016) Occurrence of QoI-resistant isolates of rice blast in Akita prefecture in 2015 (in Japanese). Ann Rept Plant Prot North Japan 67:53-56. https://doi.org/10.11455/kitanihon.2016.67_53\u003c/li\u003e\n \u003cli\u003eR Core Team (2024) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Website https://www.R-project.org/ [Accessed 21 December 2024]\u003c/li\u003e\n \u003cli\u003eResearch Committee on Fungicide Resistance, the Phytopathological Society of Japan (2008) Guidelines for managing resistance to QoI and MBI-D fungicides in rice blast disease control (in Japanese). Website http://www.taiseikin.jp/guidelines/\u003c/li\u003e\n \u003cli\u003eStammler G, Itoh M, Hino I, Watanabe A, Kojima K, Motoyoshi M, Koch A, Haden E (2007) Efficacy of orysastrobin against blast and sheath blight in transplanted rice. J Pestic Sci 32:10-15. https://doi.org/10.1584/jpestics.G06-22\u003c/li\u003e\n \u003cli\u003eSuzuki F, Arai M, Yamaguchi J (2006) DNA fingerprinting of \u003cem\u003ePyricularia grisea\u003c/em\u003e by rep-PCR using single primers designed from the terminal inverted repeat of each of the transposable elements \u003cem\u003ePot2\u003c/em\u003e and MGR586. J Gen Plant Pathol 72:314-317. DOI 10.1007/s10327-006-0290-z\u003c/li\u003e\n \u003cli\u003eSuzuki F, Arai M, Yamaguchi J (2007) Genetic analysis of \u003cem\u003ePyricularia grisea\u003c/em\u003e population by rep-PCR during development of resistance to scytalone dehydratase inhibitors of melanin biosynthesis. Plant Dis 91:176-184. https://doi.org/10.1094/PDIS-91-2-0176\u003c/li\u003e\n \u003cli\u003eSuzuki F, Yamaguchi J, Koba A, Nakajima T, Arai M (2010) Changes in fungicide resistance frequency and population structure of \u003cem\u003ePyricularia oryzae\u003c/em\u003e after discontinuance of MBI-D fungicides. Plant Dis 94: 283-382. https://doi.org/10.1094/PDIS-94-3-0329\u003c/li\u003e\n \u003cli\u003eSuzuki F, Mitsunaga T, Ashizawa T (2017) Statistical analysis of factors affecting resistance development of rice blast fungus to QoI fungicides based on pesticide shipment data (in Japanese). Ann Rept of the Kanto-Tosan Plant Prot Soc 64:6-9. https://doi.org/10.11337/ktpps.2017.6\u003c/li\u003e\n \u003cli\u003eSuzuki H, Suzuki F, Kusaba M, Tosa Y (2012) Population structure of rice blast isolates resistant to scytalone dehydratase inhibitors in Mie Prefecture and implications for their origin. J Gen Plant Pathol 78: 106\u0026ndash;114. DOI 10.1007/s10327-012-0365-y\u003c/li\u003e\n \u003cli\u003eTakahashi N, Tominaga T, Fujisawa Y, Iwadate Y (2010) Analysis of factors for occurrence and predominance of dehydratase inhibitors in melanin biosynthesis (MBI-D) fungicide resistant isolates of \u003cem\u003ePyricularia oryzae\u003c/em\u003e in Iwate prefecture (in Japanese). Ann Rept Plant Prot North Japan 61:9-13. https://doi.org/10.11455/kitanihon.2010.61_9\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-general-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jgpp","sideBox":"Learn more about [Journal of General Plant Pathology](http://link.springer.com/journal/10327)","snPcode":"10327","submissionUrl":"https://www.editorialmanager.com/jgpp/default2.aspx","title":"Journal of General Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pyricularia oryzae, Magnaporthe oryzae, rice blast, QoI resistance, orysastrobin, fitness","lastPublishedDoi":"10.21203/rs.3.rs-9175517/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9175517/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn 2013 QoI-resistant isolates of the rice blast fungus (MoO) emerged suddenly and spread in Hyogo Prefecture. To estimate factors associated with their spread and subsequent survival, the population structure of MoO isolates was monitored from 2013 through 2023 using DNA fingerprinting with \u003cem\u003ePot2\u003c/em\u003e. Most of the QoI-resistant isolates collected in 2013 were classified into haplotype HY1, irrespective of areas of collection in Hyogo Prefecture. This result suggests that the sudden emergence and spread of the QoI-resistant isolates in 2013 were caused by almost the single haplotype HY1 probably through seed transmission. After discontinuance of the QoI fungicide application in 2014, however, the QoI-resistant isolates decreased over the course of several years, suggesting that genetic alternations conferring the QoI resistance may have caused a decrease in fitness. Conversely, QoI-sensitive isolates increased after 2015, but predominant haplotypes changed across years of collection. We suggest that it could be possible to re-use the QoI-fungicide if the disappearance of the resistant strains is confirmed by careful monitoring of QoI resistance throughout the prefecture, especially in and around fields for seed production.\u003c/p\u003e","manuscriptTitle":"Population dynamics of QoI-resistant rice blast isolates in Hyogo Prefecture associated with the use and discontinuation of QoI fungicides","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-27 13:00:49","doi":"10.21203/rs.3.rs-9175517/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-03-26T22:50:32+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-26T07:11:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-26T06:49:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of General Plant Pathology","date":"2026-03-20T02:23:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-general-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jgpp","sideBox":"Learn more about [Journal of General Plant Pathology](http://link.springer.com/journal/10327)","snPcode":"10327","submissionUrl":"https://www.editorialmanager.com/jgpp/default2.aspx","title":"Journal of General Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4b85351d-25b1-46f9-8a6d-844ceaef90bc","owner":[],"postedDate":"March 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-27T13:00:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-27 13:00:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9175517","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9175517","identity":"rs-9175517","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}