Identification of Bremia lactucae races on Lactuca sativa in Kagawa Prefecture and assessment of host range in wild Asteraceae in Japan

Identification of Bremia lactucae races on Lactuca sativa in Kagawa Prefecture and assessment of host range in wild Asteraceae in Japan | 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 Identification of Bremia lactucae races on Lactuca sativa in Kagawa Prefecture and assessment of host range in wild Asteraceae in Japan Fumihiro Nishimura, Takahiro Katayama, Mamoru Satou, Kenichi Ikeda This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7766982/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Feb, 2026 Read the published version in Journal of General Plant Pathology → Version 1 posted 4 You are reading this latest preprint version Abstract Two Bremia lactucae isolates were obtained from infected lettuce ( Lactuca sativa ) plants cultivated in fields in Kagawa Prefecture, Japan. The two isolates exhibited distinct reaction phenotypes compared to those of races previously characterized by the International Bremia Evaluation Board and detected in Hyogo Prefecture, suggesting the presence of previously unreported races in Japan. Commercial lettuce cultivars with resistance genes showed minimal sporulation, confirming their effectiveness for disease control. Cross-inoculation experiments with B. lactucae on wild Lactuca serriola collected from three regions reveled sporulation only on plants from Hiroshima Prefecture. Furthermore, L. sativa cotyledons inoculated with conidia from L. serriola also developed sporulation, providing evidence of cross-infection of B. lactucae between L. sativa and L. serriola in Japan. The detection of new B. lactucae races, as well as the regional susceptibility of L. serriola to B. lactucae , may be influenced by factors such as weed management and crop rotation practices, including paddy rice cultivation in Japan, compared to field crops in other countries. Lettuce downy mildew Lactuca serriola host-range race weed Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Lettuce ( Lactuca sativa ) is one of the most widely cultivated vegetables worldwide. In Japan, annual production exceeds 550,000 tons (E-stat 2023). Bremia lactucae , the causal agent of lettuce downy mildew, is a major pathogen in both field-grown and greenhouse lettuce globally. Bremia lactucae consists of numerous physiological races, which are classified based on their virulence patterns against differential lettuce genotypes carrying known dominant resistance ( Dm ) genes. To date, 51 Dm genes have been reported (Parra et al. 2016 ). The International Bremia Evaluation Board (IBEB), a collaborative effort among lettuce breeding companies in several countries, works to identify new B. lactucae races that pose significant threats to the lettuce industry and to standardized race nomenclature worldwide (IBEB 2025 ). While lettuce is the primary host, B. lactucae can infect many Asteraceae species, with over 230 host species reported globally (Morgan 1981 ). In Brazil, for example, it has been found on Cynara scolymus (Mendes et al. 1998 ) and on Sonchus asper and Sonchus oleraceus (Vieira and Barreto 2006 ). In Europe, 17 Lactuca species are known to be susceptible, though only seven are considered natural hosts (Lebeda et al. 2001 , 2002 ). Among these, Lactuca serriola is the most common wild host in countries such as the Netherlands (Hooftman et al. 2006 ) and the Czech Republic (Petrželová and Lebeda 2004 ; Lebeda et al. 2007 , 2008 ). Natural populations of L. serriola in Europe exhibit high variation in race-specific resistance to B. lactucae , with diverse resistance phenotypes across regions (Lebeda et al. 2008 ; Petrželová and Lebeda 2011 ). In Japan, Nishiguchi and Futai ( 2010 ) described several races of B. lactucae in Hyogo Prefecture, but detailed studies of wild hosts have not been conducted. In this study, we investigated B. lactucae races affecting L. sativa cultivated in Kagawa Prefecture, Japan, during 2016 and 2017, and isolated two new races. We also assessed the resistance of commercial lettuce cultivars to these races and examined the host range in wild Asteraceae species. These findings provide new insights into the occurrence and distribution of B. lactucae on L. serriola in Japan. Materials and methods Isolates and maintenance of host plants Two isolates of B. lactucae were collected from infected lettuce ( Lactuca sativa cv. Ciscoviva) plants grown in fields in Kagawa Prefecture in 2016 and 2017. The isolates were designated KKB001 (2016) and KKB002 (2017), with each isolate obtained from a single infected leaf. The isolates were maintained and propagated on seedlings of L. sativa cv. Ciscoviva. Seeds were sown in a commercial potting soil (Yosaku N-150; Jcam Agri Co., Ltd., Tokyo, Japan) in15-cm terracotta pots. The pots were placed in a growth chamber (Biotron LH-220S, Nippon Medical & Chemical Instruments Co., Ltd., Osaka, Japan) set at 20 ℃ under a 16:8-h L:D photoperiod and watered regularly to maintain optimal moisture levels. Fifteen-day-old seedlings (with fully expanded cotyledons) were inoculated by spraying with a conidial was were placed in a plastic bag containing 30 mL of tap water and shaken manually. After shaking, the leaf was removed and the resulting suspension was adjusted to a concentration of 1.0 × 10 5 conidia/mL. This suspension was then sprayed evenly onto the seedlings. Following inoculation, the seedlings were incubated overnight at 15°C and 100% relative humidity in darkness for 8 h to allow infection. Thereafter, they were transferred to under a 16:8-h photoperiod and natural humidity conditions at 10°C for 20 days. During this period, the seedlings were sprayed with tap water and incubated overnight at 15°C and 100% relative humidity. Unfortunately, the KKB001 isolate was broken during the above maintenance, so the used isolates vary by test. Morphology and nucleotide sequence analyses The isolates were morphologically identified as B. lactucae by microscopic examination of conidiophores and conidia using a BX51 microscope (Olympus Corporation, Tokyo, Japan). For molecular identification, genomic DNA was extracted from diseased leaves using a DNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA). Polymerase chain reaction (PCR) amplification was performed in 20-µL reaction mixtures containing 10 µL of polymerase mix (AmpliTaq Gold 360 Master Mix; Thermo Fisher Scientific, Waltham, MA, USA), 0.2 µM of each primer, and 25 ng/µL of template DNA. Two DNA gene regions were amplified: (1) the large subunit ribosomal RNA (LSU rDNA) region using primers LR0R (Moncalvo et al. 1995 ) and LR6-O (Riethmuller et al. 2002 ), and (2) the cytochrome c oxidase subunit 2 (cox2) region using Cox2-F and Cox2-R (Hudspeth et al. 2000 ). PCR was performed using a Takara PCR Thermal Cycler Dice Touch (Takara Bio Inc., Kusatsu, Japan) under the following cycling conditions: initial polymerase activation at 95°C for 9 min; 36 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 40 s, and extension at 72°C for 1 min; followed by final extension at 72°C for 5 min. The amplified PCR products were purified using a MonoFas DNA Purification Kit I (Animos Inc., Kawaguchi, Japan) and subsequently sequenced by a commercial sequencing service (Eurofins Scientific SE, Luxembourg, Luxembourg). Sequence analyses were conducted using MEGA v. 10.0 (Kumar et al. 2018 ). Determination of B. lactucae races Race determination was conducted following the methods of Nishiguchi and Futai ( 2010 ) with minor modifications. Each of the two isolates (KKB001 and KKB002) was inoculated onto lettuce plants, with two replicate tests performed for each isolate. The differential set of L. sativa seeds used for race testing was kindly provided by Professor Richard W. Michelmore (University of California, Davis, CA, USA). Seeds of 25 differential cultivars were sown individually in Petri dishes (Asnol Petri Dish φ55 × 17 mm; AS ONE Corporation, Osaka, Japan) lined with two layers of filter paper (φ55 mm; Advantec Toyo Roshi Kaisha, Ltd., Tokyo, Japan), to which 2 mL of tap water containing 1 ppm iprodione (Rovral WP; Byer Crop Science K.K., Leverkusen, Germany) and a 2000-fold dilution of liquid fertilizer (Hyponex; Hyponex Japan, Ltd., Osaka, Japan) was added. For each cultivar, approximately 30 seeds were sown per Petri dish. The seeds were incubated for 10 days at 15 ℃ in a growth chamber under a 16:8-h L:D photoperiod. The 10-day-old seedlings were inoculated by spraying with a B. lactucae suspension (1.0 × 10 5 conidia/mL). After inoculation, the Petri dishes were covered with their lids and incubated for an additional 10 days under the same conditions. Disease reactions were assessed according to the criteria described by Nishiguchi and Futai ( 2010 ) and classified into four categories: sporulation, sporulation with necrosis, no sporulation with necrosis, and no sporulation. Evaluation of resistance and susceptibility in commercial lettuce This experiment was conducted using the KKB001 isolate and was repeated twice for each variety. At that time, since we had not yet obtained the KKB002 isolate, we only evaluated the KKB001 isolate. Eighteen commercial lettuce varieties were sown in a commercial potting soil (Yosaku N-150) in 200-well seedling trays (1 seed/well× 10; each well: 25 mm diameter × 45 mm depth; Yanmar Holdings Co., Ltd., Osaka, Japan) and grown for 7 days in a growth chamber at 20°C under a 16:8-h L:D photoperiod. The seedlings were then sprayed with a B. lactucae suspension (1.0 × 10 5 conidia/mL) until runoff and incubated overnight in a covered clear plastic box (Passetite TW-50, 250 mm × 356 mm × 100 mm; Gifu Plastic Industry Co., Ltd., Gifu, Japan) at 15 ℃ and 100% relative humidity in darkness for 8 h to facilitate infection. After incubation, the lid was removed and the seedlings were maintained at 15 ℃ and 70% relative humidity under under a 12:12-h L:D photoperiod for an additional 6 days. After this period, the seedlings were sprayed with tap water and incubated overnight at 15 ℃ and 100% relative humidity to promote sporulation. Disease incidence was assessed by evaluating each cotyledon or leaf for the presence of sporulation. Incidence was calculated as (number of sporulation plants / number of grown plants) × 100 (Fig. 1 ). Host range of B. lactucae in natural populations of Asteraceae plants In total, three Asteraceae species representing six populations were tested for susceptibility to B. lactucae . After the above test, the KKB001 isolate was broken during the maintenance. Therefore, we only tested the KKB002 isolate. Seed samples of three L. serriola populations were collected in 2024 from Akita, Fukushima, and Hiroshima Prefectures. Seed samples from two populations of Lactuca indica and one population of S. oleraceus were collected in 2024 from Kagawa Prefecture. The inoculation procedure followed the method used for the determination of B. lactucae races, as described above. Disease reactions were evaluated by assessing sporulation on cotyledons. Additionally, 20 seeds of L. serriola collected from Hiroshima Prefecture were sown in commercial potting soil (Yosaku N-150) contained in 1/5000 Wagner pots and grown for 3 weeks in a greenhouse. The 3-week-old seedlings were then sprayed until runoff with a B. lactucae suspension (1.0 × 10 5 conidia/mL). Inoculated seedlings were incubated overnight in a clear plastic box (Passetite TW-200, 250 mm × 356 mm × 325 mm) at 15 ℃ and 100% relative humidity in darkness for 8 h to facilitate infection. After incubation, the lid was removed, and the seedlings were maintained at 15 ℃ and 70% relative humidity, under a 12:12-h L:D photoperiod for 10 days. During this period, the seedlings were sprayed with tap water and incubated overnight at 15 ℃ and 100% relative humidity to promote sporulation. Conidia obtained from these inoculations were then used to infect new seedlings of L. sativa cv. Ciscoviva following the same method. Sporulation was evaluated on both cotyledons and true leaves. Results Morphology and taxonomy Both isolates exhibited conidiophores that were straight with tree-like dichotomous branching, and candida that were hyaline and spherical (Table 1; Fig. 2 ). In isolate KKB002, conidiophores measured 207–476 × 9.8–13.8 µm, and conidia had a diameter of 16.2–17.8 µm. These morphological characteristics were consistent with previous descriptions (Kochman and Majewski 1970 ; Lee et al. 2021 ; Vieira and Barreto 2006 ). For molecular characterization, nucleotide sequences of the LSU and cox2 fragments of both isolates were deposited in the DNA Data Bank of Japan (KKB001: LC778228 for LSU, LC778226 for cox2; KKB002: LC778229 for LSU, LC778227 for cox2). The LSU sequences of both isolates exhibited high homology with MF693907 (LSU of B. lactucae ), and the cox2 sequences of both isolates were 100% identical to NC040179 (cox2 of B. lactucae ) (data not shown). Determination of races and evaluation of resistance to commercial lettuce cultivars to B. lactucae Twenty-five differential lettuce cultivars were examined for their susceptibility to the two B. lactucae isolates (Table 2). For isolate KKB001, 19 cultivars were susceptible, while six were not (R4T57D ( Dm4 ), Nun Dm17 ( Dm17 ), Argeles ( Dm38 ), RYZ2164 ( Dm25 ), Balesta, and Bellisimo). For isolate KKB002, only seven cultivars were susceptible (Green Towers (no R-gene), Valmaine ( Dm5/8 ), LSE57/15 ( Dm7 ), UC DM10 ( Dm10 ), Capitan ( Dm11 ), Hilde II (R12), UC DM14 ( Dm14 )). The resistance/susceptibility profiles of these two isolates differed markedly from those of races previously reported by the IBEB ( 2025 ) and Nishiguchi and Futai ( 2010 ) (Table 2), indicating that the present isolates represent new races of B. lactucae . For KKB001, nine commercial cultivars exhibited sporulation on cotyledons or leaves. The remaining nine resistant cultivars showed little or no sporulation on cotyledons or leaves (Fig. 3 ). Host range of B. lactucae in Asteraceae plants in the wild One cultivar of L. sativa , two Lactuca species, and one Sonchus species were inoculated with isolate KKB002. Sporulation was observed on the cotyledons of L. sativa cv. Ciscoviva and L. serriola collected from Hiroshima Prefecture. Sporulation on L. serriola seedlings was detected even in 1/5000 Wagner pots (Fig. 4 a–c). Conidia obtained from these L. serriola seedlings were subsequently used to inoculate new seedlings of L. sativa cv. Ciscoviva, resulting in sporulation on cotyledons (Fig. 4 d, e). This represents the first recorded instance of cross-infection of B. lactucae between L. sativa and L. serriola in Japan. Discussion The genome of L. sativa has been sequenced and assembled, covering 2.4 Gb of the total 2.7 Gb, with these sequences organized into chromosomal pseudomolecules (Reyes-Chin-Wo et al. 2017 ). To date, 51 genes and 15 quantitative trait loci for resistance have been reported (Parra et al. 2016 ), and 27 Dm genes are located within major resistance clusters containing nucleotide-binding leucine-rich repeat genes (Christopoulou et al. 2015a , b ). In addition, 11 Dm genes were reported more recently (Parra et al. 2021 ). The gene-for-gene interaction between L. sativa and B. lactucae is now one of the best-characterized plant–pathogen systems (Hulbert and Michelmore 1985 ; Farrara et al. 1987 ; Ilott et al. 1987 , 1989 ). Races of B. lactucae are defined based on their reactions on differential cultivars carrying specific resistance genes. The newly identified races from Japan were distinct from those recorded by the IBEB and from races previously reported in Hyogo Prefecture. The IBEB primarily evaluates races collected from northwestern Europe and the western United States (IBEB 2025 ). The present findings indicate that B. lactucae races not considered by the IBEB are emerging in Japan. One possible explanation is seed transmission. Although seed-borne B. lactucae has not been reported, infected seeds are known to serve as a major source of primary inoculum for other downy mildew pathogens, including Peronospora effusa (spinach) (Inaba et al. 1983 ), Peronospora manshurica (soybean) (Roongruangsree et al. 1988 ), Peronospora halstedii (sunflower) (Cohen and Sackston 1974 ), Peronospora cristata (opium poppy) (Montes-Borrego et al. 2009 ) and Pseudoperonospora cubensis (cucumber) (Cohen et al. 2014 ). Many commercial lettuce seeds supplied to Japan have been harvested from regions outside of the areas surveyed by the IBEB (Fig. 3 ), which could contribute to the emergence of novel races. A second possible explanation is transmission via L. serriola . Wild populations of B. lactucae appear to play a major role in the epidemiology of downy mildew on cultivated lettuce, as B. lactucae isolates from L. sativa are thought to have emerged through several host shifts from L. serriola (Runge et al. 2021 ). Sexual reproduction is recognized as the primary mechanism generating new virulence phenotypes in B. lactucae (Michelmore and Ingram 1981 ); however, variation can also arise through asexual events such as recombination via anastomosis (Van Hese et al. 2016 ). These observations suggest that new races of B. lactucae are likely being produced on L. serriola . The timing of the Invasion of L. serriola , the most dominant wild host, into Japan is unknown. Nonetheless, considerable variation in resistance to B. lactucae has been reported both among and within countries (Petrželová and Lebeda 2011 ). In the present study, sporulation was observed on the inoculated cotyledons of L. sativa cv. Ciscoviva and L. serriola (collected in Hiroshima Prefecture). Factors contributing to this variation include the ephemeral nature of this annual weed host, local ecological conditions, and agronomic management and weed control practices, which may influence the spatial structure of populations by affecting the survival of specific resistance-gene genotypes in the landscape (Petrželová and Lebeda 2011 ). Thus, L. serriola may serve as the primary source of B. lactucae transmission in Japan. However, regular weeding is conducted throughout the country, including along field edges, suggesting that there are limited opportunities for encounters between L. serriola and cultivated L. sativa . Additionally, crop rotation, including with paddy rice, is common in lettuce fields in Kagawa Prefecture. Transmission by oospores is also possible, but oospore survival of Peronospora destructor is reduced when paddy rice is cultivated (Nishimura et al. 2022 ), implying that B. lactucae oospores may similarly be affected. Consequently, lettuce downy mildew may be less prevalent in Japan than in other countries. Nevertheless, continued evaluation of B. lactucae races in Japan remains important to anticipate the potential emergence of new races. Finally, the resistant cultivars exhibited little or no sporulation on the cotyledons or leaves; thus, the use of resistant cultivars likely remains the most effective strategy for controlling B. lactucae . However, variety determining priorities are often driven by market demands for quality such as head form and cultivation suitability, as well as resistance to other major diseases, including Mirafiori lettuce big-vein ophiovirus and Fusarium oxysporum f. sp. lactucae . Declarations The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies performed with human participants or animals. Acknowledgments We express our gratitude to Prof. Richard W. Michelmore, UC Davis, for providing the lettuce seeds, and Dr. Motoaki Asai and Ms. Rio Takama-Nishikata, National Agriculture Research Organization, for providing the L. serriola seeds. We thank Mr. Shinji Nishiguchi, Hyogo Prefecture Technology Center for Agriculture, Forestry and Fishing, for providing us with information about this disease. We also thank Mr. Yoshifumi Ogawara and Ms. Takako Yamada for their help on this work. References Christopoulou M, McHale LK, Kozik A, Reyes-Chin-Wo S, Wroblewski T, Michelmore RW (2015a) Dissection of two complex clusters of resistance genes in lettuce ( Lactuca sativa ). MPMI 28:751–765 Christopoulou M, Wo SRC, Kozik A, McHale LK, Truco MJ, Wroblewski T, Michelmore RW (2015b) Genome-wide architecture of disease resistance genes in lettuce. G3 5:2655–2669 Cohen Y, Sackston WE (1974) Seed infection and latent infection of sunflowers by Plasmopara halstedii . 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Eur J Plant Pathol 144:431–441 Vieira BS, Barreto RW (2006) First record of Bremia lactucae infecting Sonchus oleraceus and Sonchus asper in Brazil and its infectivity to lettuce. J Phytopathol 154:84–87 Tables Table 1 to 3 are available in the Supplementary Files section. Supplementary Files 251002NishimuraTables.xlsx Cite Share Download PDF Status: Published Journal Publication published 05 Feb, 2026 Read the published version in Journal of General Plant Pathology → Version 1 posted Reviewers agreed at journal 05 Oct, 2025 Reviewers invited by journal 05 Oct, 2025 Editor assigned by journal 02 Oct, 2025 First submitted to journal 02 Oct, 2025 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. 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Nishimura","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9509-0536","institution":"Kagawa Prefecture: Kagawa-ken","correspondingAuthor":true,"prefix":"","firstName":"Fumihiro","middleName":"","lastName":"Nishimura","suffix":""},{"id":525029381,"identity":"33b9afa4-cc42-4484-a924-6067240613fd","order_by":1,"name":"Takahiro Katayama","email":"","orcid":"","institution":"Kagawa Prefecture: Kagawa-ken","correspondingAuthor":false,"prefix":"","firstName":"Takahiro","middleName":"","lastName":"Katayama","suffix":""},{"id":525029382,"identity":"aa8fbebf-1ee9-4a98-8370-ae30c4da1a8f","order_by":2,"name":"Mamoru Satou","email":"","orcid":"","institution":"NARO/WARC: Nogyo Shokuhin Sangyo Gijutsu Sogo Kenkyu Kiko Nishinihon Nogyo Kenkyu Center Shikoku Kenkyu Center","correspondingAuthor":false,"prefix":"","firstName":"Mamoru","middleName":"","lastName":"Satou","suffix":""},{"id":525029383,"identity":"64481dc6-842e-4145-b60a-2d05986f2a3a","order_by":3,"name":"Kenichi Ikeda","email":"","orcid":"","institution":"Kobe University: Kobe Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Kenichi","middleName":"","lastName":"Ikeda","suffix":""}],"badges":[],"createdAt":"2025-10-02 13:49:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7766982/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7766982/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10327-026-01272-5","type":"published","date":"2026-02-05T15:58:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93694479,"identity":"d10b45f8-64e6-445f-8df8-6f74e898f1d2","added_by":"auto","created_at":"2025-10-16 14:29:31","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7772,"visible":true,"origin":"","legend":"","description":"","filename":"jgppJGPPD2500317.xml","url":"https://assets-eu.researchsquare.com/files/rs-7766982/v1/29d12b16d992aa2b64fbe54b.xml"},{"id":93694484,"identity":"4edf7227-df37-4211-9c7d-b738044f152a","added_by":"auto","created_at":"2025-10-16 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14:29:31","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82768,"visible":true,"origin":"","legend":"","description":"","filename":"JGPPD25003170structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7766982/v1/29c906c5b44f998eb5756c11.xml"},{"id":93695019,"identity":"6488695c-e821-49b0-a944-4deee33baa1e","added_by":"auto","created_at":"2025-10-16 14:37:31","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91930,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7766982/v1/d7b36940010ec18e21b39a6e.html"},{"id":93694998,"identity":"fd864599-2bc4-4608-9f26-f355082b28ad","added_by":"auto","created_at":"2025-10-16 14:37:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114597,"visible":true,"origin":"","legend":"\u003cp\u003eMethodology used to investigate the resistance and susceptibility of commercial varieties of \u003cem\u003eLactuca sativa\u003c/em\u003eto \u003cem\u003eBremia lactucae\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-7766982/v1/235f7716992b8835186646a2.png"},{"id":93694477,"identity":"e7d6b7ff-0d58-46cc-ac7b-b00befbbb20f","added_by":"auto","created_at":"2025-10-16 14:29:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":536543,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs showing downy mildew caused by \u003cem\u003eBremia lactucae\u003c/em\u003e on \u003cem\u003eLactuca sativa\u003c/em\u003e collected from Kagawa Prefecture, Japan. (a) Sporulation on the leaf. (b) Conidiophore. (c) Germinating conidia with germ tube. Scale bars, 50 μm.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-7766982/v1/66cb46c3275dc675f262312c.png"},{"id":93694473,"identity":"0279ed81-f1d3-487b-aa06-f1d02545fb70","added_by":"auto","created_at":"2025-10-16 14:29:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24707,"visible":true,"origin":"","legend":"\u003cp\u003eResistance of 18 commercial lettuce cultivars to a \u003cem\u003eBremia lactucae\u003c/em\u003e isolate collected from Kagawa Prefecture, Japan (KKB001). Parentheses indicate the country of origin of the seeds. Asterisks indicate cultivars that the seed company’s website lists as resistant to downy mildew. Error bar indicates SD.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-7766982/v1/fa24b65166efff492b583479.png"},{"id":93694475,"identity":"e1946912-644b-49d6-a119-ae1b7054e61d","added_by":"auto","created_at":"2025-10-16 14:29:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":717350,"visible":true,"origin":"","legend":"\u003cp\u003eSymptoms and morphology of \u003cem\u003eBremia lactucae\u003c/em\u003e(KKB002) on \u003cem\u003eLactuca serriola\u003c/em\u003e, and cross-infection between \u003cem\u003eL. serriola\u003c/em\u003eand \u003cem\u003eLactuca sativa\u003c/em\u003e. \u003cem\u003eLactuca serriola\u003c/em\u003e was collected from Hiroshima Prefecture, Japan. (a) Whole \u003cem\u003eL. serriola\u003c/em\u003e plant. (b) Leaf (abaxial side) showing necrosis and sporulation. (c) Conidiophore of \u003cem\u003eB. lactucae\u003c/em\u003e isolated from the \u003cem\u003eL. serriola\u003c/em\u003e leaf shown in panel b. (d) Sporangia on the \u003cem\u003eL. sativa\u003c/em\u003ecotyledon inoculated by panel b’s sporangia. (e) Conidiophore of \u003cem\u003eB. lactucae\u003c/em\u003efrom \u003cem\u003eL. sativa\u003c/em\u003e cotyledon. Scale bars, 50μm.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-7766982/v1/efe7fc2d3393c1a1e5c506e6.png"},{"id":102234331,"identity":"fd645a2f-7baf-40e9-916d-d728c7f76d8e","added_by":"auto","created_at":"2026-02-09 16:10:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2208652,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7766982/v1/4e9d9154-6d65-4e7d-970a-cfa19c4493f3.pdf"},{"id":93694476,"identity":"3bcdcb1e-ea15-46f7-b53f-5f41befc6a05","added_by":"auto","created_at":"2025-10-16 14:29:31","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21211,"visible":true,"origin":"","legend":"","description":"","filename":"251002NishimuraTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7766982/v1/c062a81c164c3293a25aa663.xlsx"}],"financialInterests":"","formattedTitle":"Identification of Bremia lactucae races on Lactuca sativa in Kagawa Prefecture and assessment of host range in wild Asteraceae in Japan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e) is one of the most widely cultivated vegetables worldwide. In Japan, annual production exceeds 550,000 tons (E-stat 2023). \u003cem\u003eBremia lactucae\u003c/em\u003e, the causal agent of lettuce downy mildew, is a major pathogen in both field-grown and greenhouse lettuce globally. \u003cem\u003eBremia lactucae\u003c/em\u003e consists of numerous physiological races, which are classified based on their virulence patterns against differential lettuce genotypes carrying known dominant resistance (\u003cem\u003eDm\u003c/em\u003e) genes. To date, 51 \u003cem\u003eDm\u003c/em\u003e genes have been reported (Parra et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The International Bremia Evaluation Board (IBEB), a collaborative effort among lettuce breeding companies in several countries, works to identify new \u003cem\u003eB. lactucae\u003c/em\u003e races that pose significant threats to the lettuce industry and to standardized race nomenclature worldwide (IBEB \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile lettuce is the primary host, \u003cem\u003eB. lactucae\u003c/em\u003e can infect many Asteraceae species, with over 230 host species reported globally (Morgan \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). In Brazil, for example, it has been found on \u003cem\u003eCynara scolymus\u003c/em\u003e (Mendes et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) and on \u003cem\u003eSonchus asper\u003c/em\u003e and \u003cem\u003eSonchus oleraceus\u003c/em\u003e (Vieira and Barreto \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In Europe, 17 \u003cem\u003eLactuca\u003c/em\u003e species are known to be susceptible, though only seven are considered natural hosts (Lebeda et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Among these, \u003cem\u003eLactuca serriola\u003c/em\u003e is the most common wild host in countries such as the Netherlands (Hooftman et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and the Czech Republic (Petrželov\u0026aacute; and Lebeda \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Lebeda et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Natural populations of \u003cem\u003eL. serriola\u003c/em\u003e in Europe exhibit high variation in race-specific resistance to \u003cem\u003eB. lactucae\u003c/em\u003e, with diverse resistance phenotypes across regions (Lebeda et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Petrželov\u0026aacute; and Lebeda \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In Japan, Nishiguchi and Futai (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) described several races of \u003cem\u003eB. lactucae\u003c/em\u003e in Hyogo Prefecture, but detailed studies of wild hosts have not been conducted.\u003c/p\u003e\u003cp\u003eIn this study, we investigated \u003cem\u003eB. lactucae\u003c/em\u003e races affecting \u003cem\u003eL. sativa\u003c/em\u003e cultivated in Kagawa Prefecture, Japan, during 2016 and 2017, and isolated two new races. We also assessed the resistance of commercial lettuce cultivars to these races and examined the host range in wild Asteraceae species. These findings provide new insights into the occurrence and distribution of \u003cem\u003eB. lactucae\u003c/em\u003e on \u003cem\u003eL. serriola\u003c/em\u003e in Japan.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIsolates and maintenance of host plants\u003c/h2\u003e\u003cp\u003eTwo isolates of \u003cem\u003eB. lactucae\u003c/em\u003e were collected from infected lettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e cv. Ciscoviva) plants grown in fields in Kagawa Prefecture in 2016 and 2017. The isolates were designated KKB001 (2016) and KKB002 (2017), with each isolate obtained from a single infected leaf.\u003c/p\u003e\u003cp\u003eThe isolates were maintained and propagated on seedlings of \u003cem\u003eL. sativa\u003c/em\u003e cv. Ciscoviva. Seeds were sown in a commercial potting soil (Yosaku N-150; Jcam Agri Co., Ltd., Tokyo, Japan) in15-cm terracotta pots. The pots were placed in a growth chamber (Biotron LH-220S, Nippon Medical \u0026amp; Chemical Instruments Co., Ltd., Osaka, Japan) set at 20 ℃ under a 16:8-h L:D photoperiod and watered regularly to maintain optimal moisture levels.\u003c/p\u003e\u003cp\u003eFifteen-day-old seedlings (with fully expanded cotyledons) were inoculated by spraying with a conidial was were placed in a plastic bag containing 30 mL of tap water and shaken manually. After shaking, the leaf was removed and the resulting suspension was adjusted to a concentration of 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e conidia/mL. This suspension was then sprayed evenly onto the seedlings.\u003c/p\u003e\u003cp\u003eFollowing inoculation, the seedlings were incubated overnight at 15\u0026deg;C and 100% relative humidity in darkness for 8 h to allow infection. Thereafter, they were transferred to under a 16:8-h photoperiod and natural humidity conditions at 10\u0026deg;C for 20 days. During this period, the seedlings were sprayed with tap water and incubated overnight at 15\u0026deg;C and 100% relative humidity. Unfortunately, the KKB001 isolate was broken during the above maintenance, so the used isolates vary by test.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMorphology and nucleotide sequence analyses\u003c/h3\u003e\n\u003cp\u003eThe isolates were morphologically identified as \u003cem\u003eB. lactucae\u003c/em\u003e by microscopic examination of conidiophores and conidia using a BX51 microscope (Olympus Corporation, Tokyo, Japan).\u003c/p\u003e\u003cp\u003eFor molecular identification, genomic DNA was extracted from diseased leaves using a DNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA). Polymerase chain reaction (PCR) amplification was performed in 20-\u0026micro;L reaction mixtures containing 10 \u0026micro;L of polymerase mix (AmpliTaq Gold 360 Master Mix; Thermo Fisher Scientific, Waltham, MA, USA), 0.2 \u0026micro;M of each primer, and 25 ng/\u0026micro;L of template DNA.\u003c/p\u003e\u003cp\u003eTwo DNA gene regions were amplified: (1) the large subunit ribosomal RNA (LSU rDNA) region using primers LR0R (Moncalvo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and LR6-O (Riethmuller et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and (2) the cytochrome c oxidase subunit 2 (cox2) region using Cox2-F and Cox2-R (Hudspeth et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). PCR was performed using a Takara PCR Thermal Cycler Dice Touch (Takara Bio Inc., Kusatsu, Japan) under the following cycling conditions: initial polymerase activation at 95\u0026deg;C for 9 min; 36 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 55\u0026deg;C for 40 s, and extension at 72\u0026deg;C for 1 min; followed by final extension at 72\u0026deg;C for 5 min.\u003c/p\u003e\u003cp\u003eThe amplified PCR products were purified using a MonoFas DNA Purification Kit I (Animos Inc., Kawaguchi, Japan) and subsequently sequenced by a commercial sequencing service (Eurofins Scientific SE, Luxembourg, Luxembourg). Sequence analyses were conducted using MEGA v. 10.0 (Kumar et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of\u003c/b\u003e \u003cb\u003eB. lactucae\u003c/b\u003e \u003cb\u003eraces\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRace determination was conducted following the methods of Nishiguchi and Futai (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) with minor modifications. Each of the two isolates (KKB001 and KKB002) was inoculated onto lettuce plants, with two replicate tests performed for each isolate.\u003c/p\u003e\u003cp\u003eThe differential set of \u003cem\u003eL. sativa\u003c/em\u003e seeds used for race testing was kindly provided by Professor Richard W. Michelmore (University of California, Davis, CA, USA). Seeds of 25 differential cultivars were sown individually in Petri dishes (Asnol Petri Dish φ55 \u0026times; 17 mm; AS ONE Corporation, Osaka, Japan) lined with two layers of filter paper (φ55 mm; Advantec Toyo Roshi Kaisha, Ltd., Tokyo, Japan), to which 2 mL of tap water containing 1 ppm iprodione (Rovral WP; Byer Crop Science K.K., Leverkusen, Germany) and a 2000-fold dilution of liquid fertilizer (Hyponex; Hyponex Japan, Ltd., Osaka, Japan) was added. For each cultivar, approximately 30 seeds were sown per Petri dish. The seeds were incubated for 10 days at 15 ℃ in a growth chamber under a 16:8-h L:D photoperiod. The 10-day-old seedlings were inoculated by spraying with a \u003cem\u003eB. lactucae\u003c/em\u003e suspension (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e conidia/mL). After inoculation, the Petri dishes were covered with their lids and incubated for an additional 10 days under the same conditions.\u003c/p\u003e\u003cp\u003eDisease reactions were assessed according to the criteria described by Nishiguchi and Futai (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and classified into four categories: sporulation, sporulation with necrosis, no sporulation with necrosis, and no sporulation.\u003c/p\u003e\n\u003ch3\u003eEvaluation of resistance and susceptibility in commercial lettuce\u003c/h3\u003e\n\u003cp\u003eThis experiment was conducted using the KKB001 isolate and was repeated twice for each variety. At that time, since we had not yet obtained the KKB002 isolate, we only evaluated the KKB001 isolate. Eighteen commercial lettuce varieties were sown in a commercial potting soil (Yosaku N-150) in 200-well seedling trays (1 seed/well\u0026times; 10; each well: 25 mm diameter \u0026times; 45 mm depth; Yanmar Holdings Co., Ltd., Osaka, Japan) and grown for 7 days in a growth chamber at 20\u0026deg;C under a 16:8-h L:D photoperiod. The seedlings were then sprayed with a \u003cem\u003eB. lactucae\u003c/em\u003e suspension (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e conidia/mL) until runoff and incubated overnight in a covered clear plastic box (Passetite TW-50, 250 mm \u0026times; 356 mm \u0026times; 100 mm; Gifu Plastic Industry Co., Ltd., Gifu, Japan) at 15 ℃ and 100% relative humidity in darkness for 8 h to facilitate infection.\u003c/p\u003e\u003cp\u003eAfter incubation, the lid was removed and the seedlings were maintained at 15 ℃ and 70% relative humidity under under a 12:12-h L:D photoperiod for an additional 6 days. After this period, the seedlings were sprayed with tap water and incubated overnight at 15 ℃ and 100% relative humidity to promote sporulation.\u003c/p\u003e\u003cp\u003eDisease incidence was assessed by evaluating each cotyledon or leaf for the presence of sporulation. Incidence was calculated as (number of sporulation plants / number of grown plants) \u0026times; 100 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHost range of\u003c/b\u003e \u003cb\u003eB. lactucae\u003c/b\u003e \u003cb\u003ein natural populations of Asteraceae plants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn total, three Asteraceae species representing six populations were tested for susceptibility to \u003cem\u003eB. lactucae\u003c/em\u003e. After the above test, the KKB001 isolate was broken during the maintenance. Therefore, we only tested the KKB002 isolate. Seed samples of three \u003cem\u003eL. serriola\u003c/em\u003e populations were collected in 2024 from Akita, Fukushima, and Hiroshima Prefectures. Seed samples from two populations of \u003cem\u003eLactuca indica\u003c/em\u003e and one population of \u003cem\u003eS. oleraceus\u003c/em\u003e were collected in 2024 from Kagawa Prefecture.\u003c/p\u003e\u003cp\u003eThe inoculation procedure followed the method used for the determination of \u003cem\u003eB. lactucae\u003c/em\u003e races, as described above. Disease reactions were evaluated by assessing sporulation on cotyledons.\u003c/p\u003e\u003cp\u003eAdditionally, 20 seeds of \u003cem\u003eL. serriola\u003c/em\u003e collected from Hiroshima Prefecture were sown in commercial potting soil (Yosaku N-150) contained in 1/5000 Wagner pots and grown for 3 weeks in a greenhouse. The 3-week-old seedlings were then sprayed until runoff with a \u003cem\u003eB. lactucae\u003c/em\u003e suspension (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e conidia/mL). Inoculated seedlings were incubated overnight in a clear plastic box (Passetite TW-200, 250 mm \u0026times; 356 mm \u0026times; 325 mm) at 15 ℃ and 100% relative humidity in darkness for 8 h to facilitate infection. After incubation, the lid was removed, and the seedlings were maintained at 15 ℃ and 70% relative humidity, under a 12:12-h L:D photoperiod for 10 days. During this period, the seedlings were sprayed with tap water and incubated overnight at 15 ℃ and 100% relative humidity to promote sporulation. Conidia obtained from these inoculations were then used to infect new seedlings of \u003cem\u003eL. sativa\u003c/em\u003e cv. Ciscoviva following the same method. Sporulation was evaluated on both cotyledons and true leaves.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eMorphology and taxonomy\u003c/h2\u003e\u003cp\u003eBoth isolates exhibited conidiophores that were straight with tree-like dichotomous branching, and candida that were hyaline and spherical (Table\u0026nbsp;1; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In isolate KKB002, conidiophores measured 207\u0026ndash;476 \u0026times; 9.8\u0026ndash;13.8 \u0026micro;m, and conidia had a diameter of 16.2\u0026ndash;17.8 \u0026micro;m. These morphological characteristics were consistent with previous descriptions (Kochman and Majewski \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Vieira and Barreto \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor molecular characterization, nucleotide sequences of the LSU and cox2 fragments of both isolates were deposited in the DNA Data Bank of Japan (KKB001: LC778228 for LSU, LC778226 for cox2; KKB002: LC778229 for LSU, LC778227 for cox2). The LSU sequences of both isolates exhibited high homology with MF693907 (LSU of \u003cem\u003eB. lactucae\u003c/em\u003e), and the cox2 sequences of both isolates were 100% identical to NC040179 (cox2 of \u003cem\u003eB. lactucae\u003c/em\u003e) (data not shown).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of races and evaluation of resistance to commercial lettuce cultivars to\u003c/b\u003e \u003cb\u003eB. lactucae\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTwenty-five differential lettuce cultivars were examined for their susceptibility to the two \u003cem\u003eB. lactucae\u003c/em\u003e isolates (Table\u0026nbsp;2). For isolate KKB001, 19 cultivars were susceptible, while six were not (R4T57D (\u003cem\u003eDm4\u003c/em\u003e), Nun Dm17 (\u003cem\u003eDm17\u003c/em\u003e), Argeles (\u003cem\u003eDm38\u003c/em\u003e), RYZ2164 (\u003cem\u003eDm25\u003c/em\u003e), Balesta, and Bellisimo). For isolate KKB002, only seven cultivars were susceptible (Green Towers (no R-gene), Valmaine (\u003cem\u003eDm5/8\u003c/em\u003e), LSE57/15 (\u003cem\u003eDm7\u003c/em\u003e), UC DM10 (\u003cem\u003eDm10\u003c/em\u003e), Capitan (\u003cem\u003eDm11\u003c/em\u003e), Hilde II (R12), UC DM14 (\u003cem\u003eDm14\u003c/em\u003e)). The resistance/susceptibility profiles of these two isolates differed markedly from those of races previously reported by the IBEB (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and Nishiguchi and Futai (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) (Table\u0026nbsp;2), indicating that the present isolates represent new races of \u003cem\u003eB. lactucae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eFor KKB001, nine commercial cultivars exhibited sporulation on cotyledons or leaves. The remaining nine resistant cultivars showed little or no sporulation on cotyledons or leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHost range of\u003c/b\u003e \u003cb\u003eB. lactucae\u003c/b\u003e \u003cb\u003ein Asteraceae plants in the wild\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOne cultivar of \u003cem\u003eL. sativa\u003c/em\u003e, two \u003cem\u003eLactuca\u003c/em\u003e species, and one \u003cem\u003eSonchus\u003c/em\u003e species were inoculated with isolate KKB002. Sporulation was observed on the cotyledons of \u003cem\u003eL. sativa\u003c/em\u003e cv. Ciscoviva and \u003cem\u003eL. serriola\u003c/em\u003e collected from Hiroshima Prefecture. Sporulation on \u003cem\u003eL. serriola\u003c/em\u003e seedlings was detected even in 1/5000 Wagner pots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026ndash;c). Conidia obtained from these \u003cem\u003eL. serriola\u003c/em\u003e seedlings were subsequently used to inoculate new seedlings of \u003cem\u003eL. sativa\u003c/em\u003e cv. Ciscoviva, resulting in sporulation on cotyledons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). This represents the first recorded instance of cross-infection of \u003cem\u003eB. lactucae\u003c/em\u003e between \u003cem\u003eL. sativa\u003c/em\u003e and \u003cem\u003eL. serriola\u003c/em\u003e in Japan.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe genome of \u003cem\u003eL. sativa\u003c/em\u003e has been sequenced and assembled, covering 2.4 Gb of the total 2.7 Gb, with these sequences organized into chromosomal pseudomolecules (Reyes-Chin-Wo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). To date, 51 genes and 15 quantitative trait loci for resistance have been reported (Parra et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and 27 \u003cem\u003eDm\u003c/em\u003e genes are located within major resistance clusters containing nucleotide-binding leucine-rich repeat genes (Christopoulou et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003eb\u003c/span\u003e). In addition, 11 \u003cem\u003eDm\u003c/em\u003e genes were reported more recently (Parra et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe gene-for-gene interaction between \u003cem\u003eL. sativa\u003c/em\u003e and \u003cem\u003eB. lactucae\u003c/em\u003e is now one of the best-characterized plant\u0026ndash;pathogen systems (Hulbert and Michelmore \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Farrara et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Ilott et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1987\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Races of \u003cem\u003eB. lactucae\u003c/em\u003e are defined based on their reactions on differential cultivars carrying specific resistance genes. The newly identified races from Japan were distinct from those recorded by the IBEB and from races previously reported in Hyogo Prefecture. The IBEB primarily evaluates races collected from northwestern Europe and the western United States (IBEB \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The present findings indicate that \u003cem\u003eB. lactucae\u003c/em\u003e races not considered by the IBEB are emerging in Japan.\u003c/p\u003e\u003cp\u003eOne possible explanation is seed transmission. Although seed-borne \u003cem\u003eB. lactucae\u003c/em\u003e has not been reported, infected seeds are known to serve as a major source of primary inoculum for other downy mildew pathogens, including \u003cem\u003ePeronospora effusa\u003c/em\u003e (spinach) (Inaba et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), \u003cem\u003ePeronospora manshurica\u003c/em\u003e (soybean) (Roongruangsree et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1988\u003c/span\u003e), \u003cem\u003ePeronospora halstedii\u003c/em\u003e (sunflower) (Cohen and Sackston \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1974\u003c/span\u003e), \u003cem\u003ePeronospora cristata\u003c/em\u003e (opium poppy) (Montes-Borrego et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and \u003cem\u003ePseudoperonospora cubensis\u003c/em\u003e (cucumber) (Cohen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Many commercial lettuce seeds supplied to Japan have been harvested from regions outside of the areas surveyed by the IBEB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which could contribute to the emergence of novel races.\u003c/p\u003e\u003cp\u003eA second possible explanation is transmission via \u003cem\u003eL. serriola\u003c/em\u003e. Wild populations of \u003cem\u003eB. lactucae\u003c/em\u003e appear to play a major role in the epidemiology of downy mildew on cultivated lettuce, as \u003cem\u003eB. lactucae\u003c/em\u003e isolates from \u003cem\u003eL. sativa\u003c/em\u003e are thought to have emerged through several host shifts from \u003cem\u003eL. serriola\u003c/em\u003e (Runge et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Sexual reproduction is recognized as the primary mechanism generating new virulence phenotypes in \u003cem\u003eB. lactucae\u003c/em\u003e (Michelmore and Ingram \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1981\u003c/span\u003e); however, variation can also arise through asexual events such as recombination via anastomosis (Van Hese et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These observations suggest that new races of \u003cem\u003eB. lactucae\u003c/em\u003e are likely being produced on \u003cem\u003eL. serriola\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe timing of the Invasion of \u003cem\u003eL. serriola\u003c/em\u003e, the most dominant wild host, into Japan is unknown. Nonetheless, considerable variation in resistance to \u003cem\u003eB. lactucae\u003c/em\u003e has been reported both among and within countries (Petrželov\u0026aacute; and Lebeda \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the present study, sporulation was observed on the inoculated cotyledons of \u003cem\u003eL. sativa\u003c/em\u003e cv. Ciscoviva and \u003cem\u003eL. serriola\u003c/em\u003e (collected in Hiroshima Prefecture). Factors contributing to this variation include the ephemeral nature of this annual weed host, local ecological conditions, and agronomic management and weed control practices, which may influence the spatial structure of populations by affecting the survival of specific resistance-gene genotypes in the landscape (Petrželov\u0026aacute; and Lebeda \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThus, \u003cem\u003eL. serriola\u003c/em\u003e may serve as the primary source of \u003cem\u003eB. lactucae\u003c/em\u003e transmission in Japan. However, regular weeding is conducted throughout the country, including along field edges, suggesting that there are limited opportunities for encounters between \u003cem\u003eL. serriola\u003c/em\u003e and cultivated \u003cem\u003eL. sativa\u003c/em\u003e. Additionally, crop rotation, including with paddy rice, is common in lettuce fields in Kagawa Prefecture. Transmission by oospores is also possible, but oospore survival of \u003cem\u003ePeronospora destructor\u003c/em\u003e is reduced when paddy rice is cultivated (Nishimura et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), implying that \u003cem\u003eB. lactucae\u003c/em\u003e oospores may similarly be affected. Consequently, lettuce downy mildew may be less prevalent in Japan than in other countries. Nevertheless, continued evaluation of \u003cem\u003eB. lactucae\u003c/em\u003e races in Japan remains important to anticipate the potential emergence of new races.\u003c/p\u003e\u003cp\u003eFinally, the resistant cultivars exhibited little or no sporulation on the cotyledons or leaves; thus, the use of resistant cultivars likely remains the most effective strategy for controlling \u003cem\u003eB. lactucae\u003c/em\u003e. However, variety determining priorities are often driven by market demands for quality such as head form and cultivation suitability, as well as resistance to other major diseases, including Mirafiori lettuce big-vein ophiovirus and \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003elactucae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003cp\u003eThis article does not contain any studies performed with human participants or animals.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eWe express our gratitude to Prof. Richard W. Michelmore, UC Davis, for providing the lettuce seeds, and Dr. Motoaki Asai and Ms. Rio Takama-Nishikata, National Agriculture Research Organization, for providing the \u003cem\u003eL. serriola\u003c/em\u003e seeds. We thank Mr. Shinji Nishiguchi, Hyogo Prefecture Technology Center for Agriculture, Forestry and Fishing, for providing us with information about this disease. We also thank Mr. Yoshifumi Ogawara and Ms. Takako Yamada for their help on this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChristopoulou M, McHale LK, Kozik A, Reyes-Chin-Wo S, Wroblewski T, Michelmore RW (2015a) Dissection of two complex clusters of resistance genes in lettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e). MPMI 28:751\u0026ndash;765\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChristopoulou M, Wo SRC, Kozik A, McHale LK, Truco MJ, Wroblewski T, Michelmore RW (2015b) Genome-wide architecture of disease resistance genes in lettuce. G3 5:2655\u0026ndash;2669\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCohen Y, Sackston WE (1974) Seed infection and latent infection of sunflowers by \u003cem\u003ePlasmopara halstedii\u003c/em\u003e. Can J Bot 52:231\u0026ndash;238\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCohen Y, Rubin AE, Galperin M, Ploch S, Runge F, Thines M (2014) Seed transmission of \u003cem\u003ePseudoperonospora cubensis\u003c/em\u003e. PLoS ONE, 9(10), e109766\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eE-Stat (2023) Crop Statistics Survey: Vegetable production and shipping statistics for the 2023 harvest \u0026ndash; lettuce. 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Mol Biol Evol 35:1547\u0026ndash;1549\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLebeda A, Doležalov\u0026aacute; I, Kř\u0026iacute;stkov\u0026aacute; E, Mieslerov\u0026aacute; B (2001) Biodiversity and ecogeography of wild \u003cem\u003eLactuca\u003c/em\u003e spp. in some European countries. Genet Resour Crop Evol 48:153\u0026ndash;164\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLebeda A, Pink DAC, Astley D (2002) Aspects of the interactions between wild \u003cem\u003eLactuca\u003c/em\u003e spp. and related genera and lettuce downy mildew (\u003cem\u003eBremia lactucae\u003c/em\u003e). In: Spencer-Phillips PTN, Gisi U, Lebeda A (eds) Advances in Downy Mildew Research. Kluwer, Dordrecht, pp 85\u0026ndash;117\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLebeda A, Doležalov\u0026aacute; I, Kř\u0026iacute;stkov\u0026aacute; E, Dehmer KJ, Astley D, van de Wiel CCM, van Treuren R (2007) Acquisition and ecological characterization of \u003cem\u003eLactuca serriola\u003c/em\u003e L. germplasm collected in the Czech Republic, Germany, the Netherlands and United Kingdom. Genet Resour Crop Evol 54:555\u0026ndash;562\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLebeda A, Petrželov\u0026aacute; I, Maryška Z (2008) Structure and variation in the wild-plant pathosystem: \u003cem\u003eLactuca serriola\u0026ndash;Bremia lactucae\u003c/em\u003e. Eur J Plant Pathol 122:127\u0026ndash;146\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee JA, Kim B, Lee DJ, Choi YJ (2021) \u003cem\u003eBremia lactucae\u003c/em\u003e causing downy mildew on \u003cem\u003eLactuca serriola\u003c/em\u003e in Korea. 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J Phytopathol 154:84\u0026ndash;87\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"Lettuce downy mildew, Lactuca serriola, host-range, race, weed","lastPublishedDoi":"10.21203/rs.3.rs-7766982/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7766982/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTwo \u003cem\u003eBremia lactucae\u003c/em\u003e isolates were obtained from infected lettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e) plants cultivated in fields in Kagawa Prefecture, Japan. The two isolates exhibited distinct reaction phenotypes compared to those of races previously characterized by the International Bremia Evaluation Board and detected in Hyogo Prefecture, suggesting the presence of previously unreported races in Japan. Commercial lettuce cultivars with resistance genes showed minimal sporulation, confirming their effectiveness for disease control. Cross-inoculation experiments with \u003cem\u003eB. lactucae\u003c/em\u003e on wild \u003cem\u003eLactuca serriola\u003c/em\u003e collected from three regions reveled sporulation only on plants from Hiroshima Prefecture. Furthermore, \u003cem\u003eL. sativa\u003c/em\u003e cotyledons inoculated with conidia from \u003cem\u003eL. serriola\u003c/em\u003e also developed sporulation, providing evidence of cross-infection of \u003cem\u003eB. lactucae\u003c/em\u003e between \u003cem\u003eL. sativa\u003c/em\u003e and \u003cem\u003eL. serriola\u003c/em\u003e in Japan. The detection of new \u003cem\u003eB. lactucae\u003c/em\u003e races, as well as the regional susceptibility of \u003cem\u003eL. serriola\u003c/em\u003e to \u003cem\u003eB. lactucae\u003c/em\u003e, may be influenced by factors such as weed management and crop rotation practices, including paddy rice cultivation in Japan, compared to field crops in other countries.\u003c/p\u003e","manuscriptTitle":"Identification of Bremia lactucae races on Lactuca sativa in Kagawa Prefecture and assessment of host range in wild Asteraceae in Japan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 14:29:26","doi":"10.21203/rs.3.rs-7766982/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-10-06T00:14:48+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-05T23:26:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T01:08:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of General Plant Pathology","date":"2025-10-02T09:48:12+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":"d04e6d7f-d9b5-4df4-84e6-6602733a47f8","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:06:34+00:00","versionOfRecord":{"articleIdentity":"rs-7766982","link":"https://doi.org/10.1007/s10327-026-01272-5","journal":{"identity":"journal-of-general-plant-pathology","isVorOnly":false,"title":"Journal of General Plant Pathology"},"publishedOn":"2026-02-05 15:58:29","publishedOnDateReadable":"February 5th, 2026"},"versionCreatedAt":"2025-10-16 14:29:26","video":"","vorDoi":"10.1007/s10327-026-01272-5","vorDoiUrl":"https://doi.org/10.1007/s10327-026-01272-5","workflowStages":[]},"version":"v1","identity":"rs-7766982","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7766982","identity":"rs-7766982","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}