Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia

Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia | 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 Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia Ryunosuke Yamada, Nobumitsu SASAKI, Ken Komatsu, Kouji Mashimo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4685066/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Sep, 2024 Read the published version in Journal of General Plant Pathology → Version 1 posted 4 You are reading this latest preprint version Abstract Infection of Zizania latifolia by Ustilago esculenta causes edible stem galls called makomotake. The development of stem galls may be influenced by the fungal colonization level at the seedling stage. To evaluate this possibility, we attempted to develop a real-time loop-mediated isothermal amplification (LAMP) method with toothpick sampling for U. esculenta detection, which allows us to examine non-destructively the relationship between fungal levels in seedlings and makomotake production from the same seedlings. Our method detected U. esculenta efficiently in seedlings that eventually produced makomotake, but did not detect it in those that failed to develop stem galls. Figures Figure 1 Figure 2 Figure 3 Introduction Zizania latifolia (Manchurian wild rice) belonging to the family Gramineae is cultivated mainly in Southeast Asian countries, including China, Korea and Japan (Guo et al. 2007 ). Since Z. latifolia plants do not usually produce ears or form seeds when they are infected with the basidiomycete Ustilago esculenta , this fungus is considered to be pathogenic for wild rice. Meanwhile, U. esculenta infection in Z. latifolia results in enlargement of the stem, which forms edible stem galls, called “jiaobai” in China (Zhang et al., 2017 ) and "makomotake" in Japan (Kawagishi et al., 2006 ). To cultivate such edible stem galls, these Z. latifolia plants infected by the fungus have been maintained not by seeds but by vegetative propagation using tillers or new buds emerging from the rhizome (runner). In this way, U. esculenta is transmitted to progeny Z. latifolia plants. In Japan, Z. latifolia is receiving significant interest as a potential crop that can be easily grown in fallow rice fields (Anazawa et al. 2007 ; Guo et al. 2007 ), because machinery for rice cultivation in paddy fields can be used, the planting and harvesting seasons are different from those of rice cultivation, and makomotake has higher commercial value than rice (Chigira et al. 2023 ). In Z. latifolia cultivation, the challenge of makomotake production is the decrease in makomotake yield caused by the occasional appearance of plants without stem gall formation. In our paddy fields in 2019, in addition to the “normal” Z. latifolia plants that form makomotake during the harvesting period, we identified two types of variant plants that neither produced ears nor galls, or produced ears but failed to form galls (Yamato et al. 2024 ). We designated the former type as the "non-gall-forming type". Meanwhile, the latter type was designated as the "male-like type", as in China, Z. latifolia plants that retain the ability to produce ears due to the disappearance of U. esculenta infection are referred to as "male" plants (Guo et al. 2007 ). Recently, our quantitative PCR (qPCR) analyses showed that fungal colonization levels remained significantly low in both the non-gall-forming and male-like types from the seedling to harvesting stages (Yamato et al. 2024 ). In contrast, normal plants that developed stem galls showed higher fungal colonization levels than the two types at the seedling stage. In addition, the normal plants also showed an increasing pattern of the fungal levels in the culm and leaf during vegetative growth and stem gall formation (Yamato et al. 2024 ). The findings indicate that the stem gall development in Z. latifolia may be influenced by the colonization level of the fungus at the seedling stage. However, it is difficult to analyze fungal levels in the same individual plants from the seedling to harvest stages over time in a non-destructive manner. To fully understand the relationship between the colonization level of U. esculenta and makomotake production, it is necessary to investigate the temporal changes in the colonization level of U. esculenta from the seedling to harvesting stages in the same individual. To this end, as an alternative to qPCR, which usually requires DNA isolation in a destructive manner, we take advantage of a real-time loop-mediated isothermal amplification (LAMP) method (Notomi et al. 2000 ; Fukuta et al. 2005 ). LAMP method with toothpick sampling has been shown to be feasible for detection of plant pathogens including viruses and mycoviruses directly from lesions of diseased plants (Kobayashi et al. 2023 ; Komatsu et al. 2016 ; Urayama et al. 2015 ; Wilisiani et al. 2019 ). In this study, we attempted to apply the simple, real-time LAMP method with toothpick sampling to quantify U. esculenta levels in Z. latifolia seedlings and subsequently investigate makomotake production from the same individuals. Materials and methods Preparation of seedlings and cultivation conditions Zizania latifolia (cv. Shirakawa) seedlings were prepared from tillers of the plants grown in 2022 in the paddy fields of Tokyo University of Agriculture and Technology (Honmachi, Fuchu, Tokyo, Japan). The basal part of the seedlings was exposed to running water for about 1 week after collection to promote root development, and expanded leaves were excised to reduce transpiration. Cultivation was conducted in the same field in 2023, with transplanting in late June and maintaining waterlogged conditions until early November. DNA preparation DNA was extracted from the colony of U. esculenta , Athelia rolfsii or Fusarium oxysporum f.sp. conglutinans using the cetyltrimethylammonium bromide (CTAB) method essentially as described previously (Yamato et al. 2024 ) except that the centrifugation speed was changed from 1,900 g to 10,000 g . The colony of the MAT-2 haploid strain, Sn-1-(1)-F1, was grown on YEPS agar medium as described previously (Chigira et al. 2023 ). The extracted DNA was 10-fold serially diluted from 1.0 × 10 2 to 1.0 × 10 − 4 ng/µL to evaluate the detection limit. qPCR analysis THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) was used for qPCR analysis, according to the manufacturer’s instructions. PCR was performed using a program of 1 cycle of 95°C for 30 s for denaturation, 40 cycles of 95°C 5 s, 60°C 30 s, and 72°C 30 s for amplification, and 1cycle of 95°C 15 s, 60°C 30 s, and 95°C 15 s for melting curve analysis. A primer pair (5'-AACCAGTCCTTCTGGGAGGT-3' and 5'-AGCGAAGAACGCTGGATAAA-3') targeting the UeLAM16A gene, encoding β-1,3-glucanase (AB691944), was used based on the report of Zhang et al. ( 2021 ). C t values of amplification curves were determined by the second derivative maximum method in the Thermal Cycler Dice Real Time System Software Ver. 3.00D (Takara Bio, Shiga, Japan). LAMP reaction Six LAMP primers (F3, B3, FIP, BIP, LF, and LB) specific for the UeLAM16A gene (AB691944) were designed with Primer Explorer V5 software ( http://primerexplorer.jp/e/ ). Among the proposed primer sets, two primer sets with high GC content were selected (Table 1 ). A Genie II instrument (OptiGene, Horsham, UK) was used to monitor LAMP reactions by real-time fluorescence detection. LAMP reaction was performed as described previously (Sugawara et al. 2012 ), using Fluorescent Detection Reagent (Eiken Kagaku, Tokyo, Japan). The mixture was incubated at 65°C for 40 min for DNA amplification and then incubated at 80°C for 5 min to inactivate DNA polymerase. In the case of toothpick sampling, scalpel incisions were made at three equally spaced positions around the base of the plant to expose part of the second node of the culm. After wiping the culm surface with 70% ethanol-soaked Kimwipes, a sterile toothpick was pricked into the second node of the culm at a depth of 1 mm, 2 mm, or 3 mm and rotated to attach plant tissue to the tip. Instead of DNA template, 1 µL of sterile RO water was added to the reaction solution, and the tip of the toothpick was stirred in the reaction solution prior to the LAMP reaction. Results and discussion Two LAMP primer sets used for real-time LAMP, ID7 and ID53 (Table 1 ), were designed to target the UeLAM16A gene of U. esculenta . To test the specificity and efficiency of the primer sets, real-time LAMP reaction was performed using U. esculenta genomic DNA as a template. Because LAMP reaction using ID7 resulted in more efficient amplification than that using ID53 (Fig. 1 A), we decided to use the ID7 primer set in further experiments. In addition, the ID7 primer set amplified DNA from U. esculenta but not from two distantly related fungi, the basidiomycete A. rolfsii or the ascomycete F. oxysporum (Fig. 1 B). This result indicated that the ID7 primer set can be used for the specific detection of U. esculenta . Furthermore, to evaluate the detection limit of real-time LAMP using the ID7 primer set, both LAMP and qPCR assays were performed using dilution series of the genomic DNA of U. esculenta ranging from 1.0×10 − 4 to 1.0×10 2 ng/µL. A primer set specific for the UeLAM16A gene of U. esculenta was designed for qPCR. As shown in Fig. 2 A and 2 B, the detection limits of real-time LAMP and qPCR were 1.0 × 10 − 2 ng/µL and 1.0 × 10 − 3 ng/µL, respectively, indicating that the detection sensitivity of real-time LAMP is only 10 times lower than that of qPCR. Similar to our result, Cao et al. ( 2017 ) reported that when testing the genomic DNA of closely related U. maydis , real-time LAMP was 10 times less sensitive than qPCR. In qPCR, the intervals between C t values of amplification curves of the serially diluted samples were nearly equal (4.2 ± 0.4). Meanwhile, the LAMP assay shows variable intervals between curves, which become wider as the DNA concentration in the sample decreases. This indicates that real-time LAMP is not suitable for accurate quantification of a target DNA. However, our data show that the real-time LAMP using the ID7 primer set can be used as a rapid method for comparing the DNA levels of U. esculenta between different samples. Based on previous reports that the hyphae of U. esculenta were observed near the top of the culm of Z. latifolia throughout the growing season (Yang and Leu 1978 ; Zhang et al. 2012 ), we attempted to detect U. esculenta in the culm tissue of Z. latifolia seedlings by real-time LAMP using toothpicks in a non-destructive manner. For this assay, we used seedlings from Shirakawa cultivar grown in our paddy fields. For DNA sampling for the LAMP reaction, a toothpick tip was inserted at three different positions around the second node of the culm, at a depth of 1, 2, or 3 mm (Fig. 3 A). Three seedling samples per depth were examined. As shown in Fig. 3 B, all seedling samples, regardless of the depth of insertion of the toothpick into the culm, showed similar patterns of increased fluorescence starting from mostly similar time points. The results indicate that this real-time LAMP method can detect U. esculenta in Z. latifolia efficiently regardless of sampling depth. To analyze whether the toothpick sampling affects growth of Z. latifolia plants and production of makomotake, the 1 mm toothpick-pricked seedlings were transplanted to paddy fields and compared with intact control seedlings. As a result, the production of makomotake was similar between the toothpick-pricked and control seedlings (Supplementary Figs. 1 and 2), although statistically significant differences in plant height, the number of tillers, and soil and plant analysis development (SPAD) value were observed between experimental groups at some survey time points (i.e., August 15 for tiller number and plant height and September 14 and 30 for SPAD value). The results indicate that toothpick sampling does not affect stem gall development, while it may have partial effects on plant growth. However, due to the small sample size of three plants per toothpick treatment in this study, further experiments with larger sample sizes will be necessary to determine how much the damage caused by toothpick treatment affects plant growth and development. Furthermore, we tested whether the real-time LAMP method that we developed could be used to find plants that would not produce makomotake. For this purpose, we applied this method to seedlings from a Shirakawa-derived variant designated as the male-like type. Recently, we have shown by qPCR that seedlings from this type of plants are colonized by U. esculenta at very low levels and did not produce makomotake (Yamato et al. 2024 ). The LAMP reaction did not show an increase in fluorescence in seedlings of male-like types that were pricked with toothpicks at depths of 1, 2, and 3 mm (Fig. 3 C). The seedlings were subsequently cultivated in our paddy fields and underwent only vegetative growth without producing makomotake (data not shown). These results suggest that this non-destructive LAMP assay with toothpick sampling can be utilized to find out and eliminate Z. latifolia seedlings that are infected by U. esculenta below the detection threshold and potentially result in no or little production of makomotake. In this study, we have developed the real-time LAMP method in combination with toothpick sampling to estimate the colonization levels of U. esculenta in the culm of Z. latifolia seedlings. This rapid and easy toothpick sampling that can be applied to any part of the plant during the growing season will help elucidate the relationship between the spatiotemporal colonization level of U. esculenta and stem gall development, and the future improvement of the real-time LAMP method with toothpick sampling may enable stable production of makomotake by efficiently selecting seedlings with high colonization levels of the fungus. Declarations Acknowledgements We thank the Institute of Global Innovation Research at Tokyo University of Agriculture and Technology (GIR-TUAT) for the Special Research Fund. Author contributions NS, KK, and MT conceived and designed the study. RY and KM prepared materials and performed the experiments. RY, NS, KK, and MT analyzed the data. RY and NS wrote the manuscript with edits and additional text from KK and MT. Conflict of interest The authors declare no conflicts of interest. Human and animal rights This article does not contain any studies involving human participants or experimental animals. References Anazawa T, Yoshida T, Kurita H (2007) Growth and yielding ability in wild rice (in Japanese with English abstract). Jpn J Crop Sci 76:52–58 Cao Y, Wang L, Duan L, Li J, Ma J, Xie S, Shi L, Li H. (2017) Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis . Scientific Reports 7:13394 https://doi.org/10.1038/s41598-017-13881-4 Chigira Y, Sasaki N, Komatsu K, Mashimo K, Tanaka S, Numamoto M, Moriyama H, Motobayashi T. (2023) Mating types of Ustilago esculenta infecting Zizania latifolia cultivars in Japan are biased towards MAT-2 and MAT-3. Microbes Environ. 38:ME23034. https://doi.org/10.1264/jsme2.ME23034 Fukuta S, Anai N, Kato M, Yoshimura Y, Fukaya M, Yabe K, Oya T, Kanbe M. (2005) Simplification of template DNA preparation for the loop-mediated isothermal amplification (LAMP) method to detect tomato yellow leaf curl virus (in Japanese with English abstract). Ann Rep Kansai Plant Prot 47:37–41 https://doi.org/10.4165/kapps.47.37 Guo HB, Li SM, Peng J, Ke WD (2007) Zizania latifolia Turcz. cultivated in China. Genet Resour Crop Evol 54:1211–1217 https://doi.org/10.1007/s10722-006-9102-8 Kawagishi H, Hota K, Masuda K, Yamaguchi K, Yazawa K, Shibata K, Uzuka N, Matahira Y (2006) Osteoclast-forming suppressive compounds from Makomotake, Zizania latifolia infected with Ustilago esculenta. Biosci Biotechnol Biochem 70:2800–2802. https://doi.org/10.1271/bbb.60376 Kobayashi M, Mashiko T, Wilisiani F, Hartono S, Nishigawa H, Natsuaki T, Neriya Y. (2023) Development of a RT-LAMP assay for real-time detection of criniviruses infecting tomato. J Virol Methods. 312:114662 https://doi.org/10.1016/j.jviromet.2022.114662 Komatsu K, Urayama S, Katoh Y, Fuji S, Hase S, Fukuhara T, Arie T, Teraoka T, Moriyama. (2016) Detection of Magnaporthe oryzae chrysovirus 1 in Japan and establishment of a rapid, sensitive and direct diagnostic method based on reverse transcription loop-mediated isothermal amplification. Arch Virol 161:317–326 https://doi.org/10.1007/s00705-015-2666-x Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63 https://doi.org/10.1093/nar/28.12.e63 Sugawara K, Himeno M, Keima T, Kitazawa Y, Maejima K, Oshima K, Namba S (2012) Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene. J Gen Plant Pathol 78:389–397 https://doi.org/10.1007/s10327-012-0403-9 Urayama S, Katoh Y, Fukuhara T, Arie T, Moriyama H, Teraoka T (2015) Rapid detection of Magnaporthe oryzae chrysovirus1-A from fungal colonies on agar plates and lesions of rice blast. J Gen Plant Pathol 81:97–102 https://doi.org/10.1007/s10327-014-0567-6 Wilisiani F, Tomiyama A, Katoh H, Hartono S, Neriya Y, Nishigawa H, Natsuaki T. (2019) Development of a LAMP assay with a portable device for real-time detection of begomoviruses under field conditions. J Virol Methods 265:71–76 https://doi.org/10.1016/j.jviromet.2018.10.005 Yamato S, Sasaki N, Komatsu K, Mashimo K, Motobayashi T. (2024) Temporal changes in colonization levels of Ustilago esculenta in different parts of Zizania latifolia plants competent and incompetent for stem gall formation. J Gen Plant Pathol 90:14–22 https://doi.org/10.1007/s10327-023-01152-2 Yang HC, Leu LS (1978) Formation and histopathology of galls induced by Ustilago esculenta in Zizania latifolia . Phytopathology 68:1572–1576 https://doi.org/10.1094/phyto-68-1572 Zhang Y, Cao Q, Hu P, Cui H, Yu X, Ye Z. (2017) Investigation on the differentiation of two Ustilago esculenta strains —implications of a relationship with the host phenotypes appearing in the fields. BMC Microbiol 17: 228. https://doi.org/10.1186/s12866-017-1138-8 Zhang JZ, Chu FQ, Guo DP, Hyde KD, Xie GL (2012) Cytology and ultrastructure of interactions between Ustilago esculenta and Zizania latifolia . Mycol Prog 11:499–508 https://doi.org/10.1007/s11557-011-0765-y Zhang Z, Xu S, Kong M, Dai H, Liu Y, Miao M (2021) Isolation, identification and artificial inoculation of Ustilago esculenta on Zizania latifolia . Hortic Plant J 7:347–358 https://doi.org/10.1016/j.hpj.2020.08.004 Table 1 Table 1. LAMP primers specific for the UeLAM16A gene Supplementary Files FigureS1.pdf FigureS2.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Sep, 2024 Read the published version in Journal of General Plant Pathology → Version 1 posted Reviewers agreed at journal 08 Jul, 2024 Reviewers invited by journal 08 Jul, 2024 Editor assigned by journal 05 Jul, 2024 First submitted to journal 04 Jul, 2024 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-4685066","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":324092748,"identity":"a3b92757-d8f1-40d0-9042-22f1de9a9a58","order_by":0,"name":"Ryunosuke Yamada","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology: Tokyo Noko Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Ryunosuke","middleName":"","lastName":"Yamada","suffix":""},{"id":324092749,"identity":"47b003d1-cad3-4728-bbd6-8daa3d3948d3","order_by":1,"name":"Nobumitsu SASAKI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYDACCQZmhoQKFKEDxGg5A2SwwRUTo4WxDUULAcA/u/mxwcN5dvIM8j2Gnz8wABmMZ/HrlLhzzDghcVuyYQMbj7HEAQYgg+FcAl4tBhIJxgcStx1IYGDjMQBqYQYqP2NAQEv65wOJc8BajH8cYKgnRksO0GENYC1mQFsOE9YicSOn2CDhWLJhG1tamcUZg+OGbYT8wj8jfbPkjxo7eX7mw5tvVFRUy/NLEAgxOGCDuBPIkDhDnA5ki3tI1jIKRsEoGAXDGwAABvQ/tNwjv20AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9202-5149","institution":"Tokyo University of Agriculture and Technology: Tokyo Noko Daigaku","correspondingAuthor":true,"prefix":"","firstName":"Nobumitsu","middleName":"","lastName":"SASAKI","suffix":""},{"id":324092750,"identity":"ef75e43d-a5c9-438c-ba18-4272b82b32da","order_by":2,"name":"Ken Komatsu","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology: Tokyo Noko Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Ken","middleName":"","lastName":"Komatsu","suffix":""},{"id":324092751,"identity":"93f97fcf-57d0-4ceb-a631-e07a1806cde6","order_by":3,"name":"Kouji Mashimo","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology: Tokyo Noko Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Kouji","middleName":"","lastName":"Mashimo","suffix":""},{"id":324092752,"identity":"9b4d2728-ce91-41a7-889d-5466ad1e5978","order_by":4,"name":"Takashi Motobayashi","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology: Tokyo Noko Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Motobayashi","suffix":""}],"badges":[],"createdAt":"2024-07-04 08:41:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4685066/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4685066/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10327-024-01201-4","type":"published","date":"2024-09-17T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61487086,"identity":"da023a36-5df3-4a6a-bc08-6704e34fa88d","added_by":"auto","created_at":"2024-07-31 09:56:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82274,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelection of a LAMP primer set specific for the UeLAM16A gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eTwo LAMP primer sets (ID7 and ID53) designed to amplify the \u003cem\u003eUeLAM16A\u003c/em\u003e gene were used to compare the detection sensitivity to \u003cem\u003eUstilago esculenta \u003c/em\u003egenomic DNA. \u003cstrong\u003eb\u003c/strong\u003e Target specificity of the ID7 primer set to \u003cem\u003eU. esculenta \u003c/em\u003egenomic DNA was confirmed by comparison with LAMP reactions using the genomic DNA of \u003cem\u003eAthelia rolfsii \u003c/em\u003eand \u003cem\u003eFusarium oxyspornm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4685066/v1/7f977280a734ff24948e1659.png"},{"id":61487087,"identity":"ff0e66aa-9488-4e1f-b1c1-5e28f2c59eef","added_by":"auto","created_at":"2024-07-31 09:56:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":125951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of detection sensitivity between real-time LAMP and qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amplification curves of fluorescence detected during reactions of the real-time LAMP (upper) and qPCR (bottom) are shown. Serial 10-fold dilutions of \u003cem\u003eUstilago esculenta \u003c/em\u003egenomic DNA from 1 × 10\u003csup\u003e2\u003c/sup\u003e to 1 × 10\u003csup\u003e-4 \u003c/sup\u003eng/µL were used as the DNA template. No fluorescence was detected for 1 ×10\u003csup\u003e-3\u003c/sup\u003e and 1 × 10\u003csup\u003e-4 \u003c/sup\u003eng/µL in the real-time LAMP reactions and 1 x 10\u003csup\u003e-4\u003c/sup\u003e ng/µL in the qPCR reaction.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4685066/v1/063126a47635732243aa94f2.png"},{"id":61487931,"identity":"b2d538ff-ba93-4f0e-9b5a-94b003b1b1c5","added_by":"auto","created_at":"2024-07-31 10:04:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":754473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNon-destructive real-time LAMP with toothpick sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003eToothpick sampling of a \u003cem\u003eZizania latifolia\u003c/em\u003e seedling. The base of the plant was excised to expose part of the culm with a scalpel (left panel). A sterile toothpick was pricked around the second node of the culm at a depth of 1 mm, 2 mm, or 3 mm. \u003cstrong\u003eb and c \u003c/strong\u003eThe amplification curves of fluorescence in the real-time LAMP reactions after toothpick sampling from the normal type plant (b) and the male-like type plant (c) are shown. Three samples were tested per toothpick insertion depth.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4685066/v1/a2dcaf5e3ba7f269789e87f6.png"},{"id":65104052,"identity":"98e27dfb-8c48-44c1-b704-5efd07d17f1b","added_by":"auto","created_at":"2024-09-23 16:11:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1699307,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4685066/v1/4180eb24-e343-4efa-b91e-66d278cbe903.pdf"},{"id":61487093,"identity":"d5ecca88-6b1f-409e-926e-8e88e8df5f31","added_by":"auto","created_at":"2024-07-31 09:56:06","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":98376,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4685066/v1/ec37f4c8a6212fa95cb5a201.pdf"},{"id":61487091,"identity":"125aa6c9-c916-4a9b-b6a7-0b72bdf88445","added_by":"auto","created_at":"2024-07-31 09:56:06","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1944836,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4685066/v1/1ef341c7e1534cc12ece58f2.pdf"}],"financialInterests":"","formattedTitle":"Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eZizania latifolia\u003c/em\u003e (Manchurian wild rice) belonging to the family Gramineae is cultivated mainly in Southeast Asian countries, including China, Korea and Japan (Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Since \u003cem\u003eZ. latifolia\u003c/em\u003e plants do not usually produce ears or form seeds when they are infected with the basidiomycete \u003cem\u003eUstilago esculenta\u003c/em\u003e, this fungus is considered to be pathogenic for wild rice. Meanwhile, \u003cem\u003eU. esculenta\u003c/em\u003e infection in \u003cem\u003eZ. latifolia\u003c/em\u003e results in enlargement of the stem, which forms edible stem galls, called \u0026ldquo;jiaobai\u0026rdquo; in China (Zhang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and \"makomotake\" in Japan (Kawagishi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). To cultivate such edible stem galls, these \u003cem\u003eZ. latifolia\u003c/em\u003e plants infected by the fungus have been maintained not by seeds but by vegetative propagation using tillers or new buds emerging from the rhizome (runner). In this way, \u003cem\u003eU. esculenta\u003c/em\u003e is transmitted to progeny \u003cem\u003eZ. latifolia\u003c/em\u003e plants. In Japan, \u003cem\u003eZ. latifolia\u003c/em\u003e is receiving significant interest as a potential crop that can be easily grown in fallow rice fields (Anazawa et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), because machinery for rice cultivation in paddy fields can be used, the planting and harvesting seasons are different from those of rice cultivation, and makomotake has higher commercial value than rice (Chigira et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eZ. latifolia\u003c/em\u003e cultivation, the challenge of makomotake production is the decrease in makomotake yield caused by the occasional appearance of plants without stem gall formation. In our paddy fields in 2019, in addition to the \u0026ldquo;normal\u0026rdquo; \u003cem\u003eZ. latifolia\u003c/em\u003e plants that form makomotake during the harvesting period, we identified two types of variant plants that neither produced ears nor galls, or produced ears but failed to form galls (Yamato et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We designated the former type as the \"non-gall-forming type\". Meanwhile, the latter type was designated as the \"male-like type\", as in China, \u003cem\u003eZ. latifolia\u003c/em\u003e plants that retain the ability to produce ears due to the disappearance of \u003cem\u003eU. esculenta\u003c/em\u003e infection are referred to as \"male\" plants (Guo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Recently, our quantitative PCR (qPCR) analyses showed that fungal colonization levels remained significantly low in both the non-gall-forming and male-like types from the seedling to harvesting stages (Yamato et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In contrast, normal plants that developed stem galls showed higher fungal colonization levels than the two types at the seedling stage. In addition, the normal plants also showed an increasing pattern of the fungal levels in the culm and leaf during vegetative growth and stem gall formation (Yamato et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The findings indicate that the stem gall development in \u003cem\u003eZ. latifolia\u003c/em\u003e may be influenced by the colonization level of the fungus at the seedling stage. However, it is difficult to analyze fungal levels in the same individual plants from the seedling to harvest stages over time in a non-destructive manner.\u003c/p\u003e \u003cp\u003eTo fully understand the relationship between the colonization level of \u003cem\u003eU. esculenta\u003c/em\u003e and makomotake production, it is necessary to investigate the temporal changes in the colonization level of \u003cem\u003eU. esculenta\u003c/em\u003e from the seedling to harvesting stages in the same individual. To this end, as an alternative to qPCR, which usually requires DNA isolation in a destructive manner, we take advantage of a real-time loop-mediated isothermal amplification (LAMP) method (Notomi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Fukuta et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). LAMP method with toothpick sampling has been shown to be feasible for detection of plant pathogens including viruses and mycoviruses directly from lesions of diseased plants (Kobayashi et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Komatsu et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Urayama et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wilisiani et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, we attempted to apply the simple, real-time LAMP method with toothpick sampling to quantify \u003cem\u003eU. esculenta\u003c/em\u003e levels in \u003cem\u003eZ. latifolia\u003c/em\u003e seedlings and subsequently investigate makomotake production from the same individuals.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of seedlings and cultivation conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eZizania latifolia\u003c/em\u003e (cv. Shirakawa) seedlings were prepared from tillers of the plants grown in 2022 in the paddy fields of Tokyo University of Agriculture and Technology (Honmachi, Fuchu, Tokyo, Japan). The basal part of the seedlings was exposed to running water for about 1 week after collection to promote root development, and expanded leaves were excised to reduce transpiration. Cultivation was conducted in the same field in 2023, with transplanting in late June and maintaining waterlogged conditions until early November.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA preparation\u003c/h2\u003e \u003cp\u003eDNA was extracted from the colony of \u003cem\u003eU. esculenta\u003c/em\u003e, \u003cem\u003eAthelia rolfsii\u003c/em\u003e or \u003cem\u003eFusarium oxysporum\u003c/em\u003e f.sp. \u003cem\u003econglutinans\u003c/em\u003e using the cetyltrimethylammonium bromide (CTAB) method essentially as described previously (Yamato et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) except that the centrifugation speed was changed from 1,900 \u003cem\u003eg\u003c/em\u003e to 10,000 \u003cem\u003eg\u003c/em\u003e. The colony of the MAT-2 haploid strain, Sn-1-(1)-F1, was grown on YEPS agar medium as described previously (Chigira et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The extracted DNA was 10-fold serially diluted from 1.0 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e to 1.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e ng/\u0026micro;L to evaluate the detection limit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eqPCR analysis\u003c/h2\u003e \u003cp\u003eTHUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) was used for qPCR analysis, according to the manufacturer\u0026rsquo;s instructions. PCR was performed using a program of 1 cycle of 95\u0026deg;C for 30 s for denaturation, 40 cycles of 95\u0026deg;C 5 s, 60\u0026deg;C 30 s, and 72\u0026deg;C 30 s for amplification, and 1cycle of 95\u0026deg;C 15 s, 60\u0026deg;C 30 s, and 95\u0026deg;C 15 s for melting curve analysis. A primer pair (5'-AACCAGTCCTTCTGGGAGGT-3' and 5'-AGCGAAGAACGCTGGATAAA-3') targeting the \u003cem\u003eUeLAM16A\u003c/em\u003e gene, encoding β-1,3-glucanase (AB691944), was used based on the report of Zhang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eC\u003c/em\u003et values of amplification curves were determined by the second derivative maximum method in the Thermal Cycler Dice Real Time System Software Ver. 3.00D (Takara Bio, Shiga, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLAMP reaction\u003c/h2\u003e \u003cp\u003eSix LAMP primers (F3, B3, FIP, BIP, LF, and LB) specific for the \u003cem\u003eUeLAM16A\u003c/em\u003e gene (AB691944) were designed with Primer Explorer V5 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://primerexplorer.jp/e/\u003c/span\u003e\u003cspan address=\"http://primerexplorer.jp/e/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Among the proposed primer sets, two primer sets with high GC content were selected (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A Genie II instrument (OptiGene, Horsham, UK) was used to monitor LAMP reactions by real-time fluorescence detection. LAMP reaction was performed as described previously (Sugawara et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), using Fluorescent Detection Reagent (Eiken Kagaku, Tokyo, Japan). The mixture was incubated at 65\u0026deg;C for 40 min for DNA amplification and then incubated at 80\u0026deg;C for 5 min to inactivate DNA polymerase.\u003c/p\u003e \u003cp\u003eIn the case of toothpick sampling, scalpel incisions were made at three equally spaced positions around the base of the plant to expose part of the second node of the culm. After wiping the culm surface with 70% ethanol-soaked Kimwipes, a sterile toothpick was pricked into the second node of the culm at a depth of 1 mm, 2 mm, or 3 mm and rotated to attach plant tissue to the tip. Instead of DNA template, 1 \u0026micro;L of sterile RO water was added to the reaction solution, and the tip of the toothpick was stirred in the reaction solution prior to the LAMP reaction.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eTwo LAMP primer sets used for real-time LAMP, ID7 and ID53 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), were designed to target the \u003cem\u003eUeLAM16A\u003c/em\u003e gene of \u003cem\u003eU. esculenta\u003c/em\u003e. To test the specificity and efficiency of the primer sets, real-time LAMP reaction was performed using \u003cem\u003eU. esculenta\u003c/em\u003e genomic DNA as a template. Because LAMP reaction using ID7 resulted in more efficient amplification than that using ID53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), we decided to use the ID7 primer set in further experiments. In addition, the ID7 primer set amplified DNA from \u003cem\u003eU. esculenta\u003c/em\u003e but not from two distantly related fungi, the basidiomycete \u003cem\u003eA. rolfsii\u003c/em\u003e or the ascomycete \u003cem\u003eF. oxysporum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This result indicated that the ID7 primer set can be used for the specific detection of \u003cem\u003eU. esculenta\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, to evaluate the detection limit of real-time LAMP using the ID7 primer set, both LAMP and qPCR assays were performed using dilution series of the genomic DNA of \u003cem\u003eU. esculenta\u003c/em\u003e ranging from 1.0\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e to 1.0\u0026times;10\u003csup\u003e2\u003c/sup\u003e ng/\u0026micro;L. A primer set specific for the \u003cem\u003eUeLAM16A\u003c/em\u003e gene of \u003cem\u003eU. esculenta\u003c/em\u003e was designed for qPCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the detection limits of real-time LAMP and qPCR were 1.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e ng/\u0026micro;L and 1.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ng/\u0026micro;L, respectively, indicating that the detection sensitivity of real-time LAMP is only 10 times lower than that of qPCR. Similar to our result, Cao et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported that when testing the genomic DNA of closely related \u003cem\u003eU. maydis\u003c/em\u003e, real-time LAMP was 10 times less sensitive than qPCR. In qPCR, the intervals between \u003cem\u003eC\u003c/em\u003et values of amplification curves of the serially diluted samples were nearly equal (4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4). Meanwhile, the LAMP assay shows variable intervals between curves, which become wider as the DNA concentration in the sample decreases. This indicates that real-time LAMP is not suitable for accurate quantification of a target DNA. However, our data show that the real-time LAMP using the ID7 primer set can be used as a rapid method for comparing the DNA levels of \u003cem\u003eU. esculenta\u003c/em\u003e between different samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on previous reports that the hyphae of \u003cem\u003eU. esculenta\u003c/em\u003e were observed near the top of the culm of \u003cem\u003eZ. latifolia\u003c/em\u003e throughout the growing season (Yang and Leu \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), we attempted to detect \u003cem\u003eU. esculenta\u003c/em\u003e in the culm tissue of \u003cem\u003eZ. latifolia\u003c/em\u003e seedlings by real-time LAMP using toothpicks in a non-destructive manner. For this assay, we used seedlings from Shirakawa cultivar grown in our paddy fields. For DNA sampling for the LAMP reaction, a toothpick tip was inserted at three different positions around the second node of the culm, at a depth of 1, 2, or 3 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Three seedling samples per depth were examined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, all seedling samples, regardless of the depth of insertion of the toothpick into the culm, showed similar patterns of increased fluorescence starting from mostly similar time points. The results indicate that this real-time LAMP method can detect \u003cem\u003eU. esculenta\u003c/em\u003e in \u003cem\u003eZ. latifolia\u003c/em\u003e efficiently regardless of sampling depth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo analyze whether the toothpick sampling affects growth of \u003cem\u003eZ. latifolia\u003c/em\u003e plants and production of makomotake, the 1 mm toothpick-pricked seedlings were transplanted to paddy fields and compared with intact control seedlings. As a result, the production of makomotake was similar between the toothpick-pricked and control seedlings (Supplementary Figs.\u0026nbsp;1 and 2), although statistically significant differences in plant height, the number of tillers, and soil and plant analysis development (SPAD) value were observed between experimental groups at some survey time points (i.e., August 15 for tiller number and plant height and September 14 and 30 for SPAD value). The results indicate that toothpick sampling does not affect stem gall development, while it may have partial effects on plant growth. However, due to the small sample size of three plants per toothpick treatment in this study, further experiments with larger sample sizes will be necessary to determine how much the damage caused by toothpick treatment affects plant growth and development.\u003c/p\u003e \u003cp\u003eFurthermore, we tested whether the real-time LAMP method that we developed could be used to find plants that would not produce makomotake. For this purpose, we applied this method to seedlings from a Shirakawa-derived variant designated as the male-like type. Recently, we have shown by qPCR that seedlings from this type of plants are colonized by \u003cem\u003eU. esculenta\u003c/em\u003e at very low levels and did not produce makomotake (Yamato et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The LAMP reaction did not show an increase in fluorescence in seedlings of male-like types that were pricked with toothpicks at depths of 1, 2, and 3 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The seedlings were subsequently cultivated in our paddy fields and underwent only vegetative growth without producing makomotake (data not shown). These results suggest that this non-destructive LAMP assay with toothpick sampling can be utilized to find out and eliminate \u003cem\u003eZ. latifolia\u003c/em\u003e seedlings that are infected by \u003cem\u003eU. esculenta\u003c/em\u003e below the detection threshold and potentially result in no or little production of makomotake.\u003c/p\u003e \u003cp\u003eIn this study, we have developed the real-time LAMP method in combination with toothpick sampling to estimate the colonization levels of \u003cem\u003eU. esculenta\u003c/em\u003e in the culm of \u003cem\u003eZ. latifolia\u003c/em\u003e seedlings. This rapid and easy toothpick sampling that can be applied to any part of the plant during the growing season will help elucidate the relationship between the spatiotemporal colonization level of \u003cem\u003eU. esculenta\u003c/em\u003e and stem gall development, and the future improvement of the real-time LAMP method with toothpick sampling may enable stable production of makomotake by efficiently selecting seedlings with high colonization levels of the fungus.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Institute of Global Innovation Research at Tokyo University of Agriculture and Technology (GIR-TUAT) for the Special Research Fund.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNS, KK, and MT conceived and designed the study. RY and KM prepared materials and performed the experiments. RY, NS, KK, and MT analyzed the data. RY and NS wrote the manuscript with edits and additional text from KK and MT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman and animal rights\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies involving human participants or experimental animals.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnazawa T, Yoshida T, Kurita H (2007) Growth and yielding ability in wild rice (in Japanese with English abstract). Jpn J Crop Sci 76:52\u0026ndash;58\u003c/li\u003e\n\u003cli\u003eCao Y, Wang L, Duan L, Li J, Ma J, Xie S, Shi L, Li H. (2017) Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of \u003cem\u003eUstilago maydis\u003c/em\u003e. Scientific Reports 7:13394 https://doi.org/10.1038/s41598-017-13881-4\u003c/li\u003e\n\u003cli\u003eChigira Y, Sasaki N, Komatsu K, Mashimo K, Tanaka S, Numamoto M, Moriyama H, Motobayashi T. (2023) Mating types of \u003cem\u003eUstilago esculenta\u003c/em\u003e infecting \u003cem\u003eZizania latifolia\u003c/em\u003e cultivars in Japan are biased towards MAT-2 and MAT-3. Microbes Environ. 38:ME23034. https://doi.org/10.1264/jsme2.ME23034\u003c/li\u003e\n\u003cli\u003eFukuta S, Anai N, Kato M, Yoshimura Y, Fukaya M, Yabe K, Oya T, Kanbe M. (2005) Simplification of template DNA preparation for the loop-mediated isothermal\u003c/li\u003e\n\u003cli\u003eamplification (LAMP) method to detect tomato yellow leaf curl virus (in Japanese with English abstract). Ann Rep Kansai Plant Prot 47:37\u0026ndash;41 https://doi.org/10.4165/kapps.47.37\u003c/li\u003e\n\u003cli\u003eGuo HB, Li SM, Peng J, Ke WD (2007) \u003cem\u003eZizania latifolia\u003c/em\u003e Turcz. cultivated in China. Genet Resour Crop Evol 54:1211\u0026ndash;1217 https://doi.org/10.1007/s10722-006-9102-8\u003c/li\u003e\n\u003cli\u003eKawagishi H, Hota K, Masuda K, Yamaguchi K, Yazawa K, Shibata K, Uzuka N, Matahira Y (2006) Osteoclast-forming suppressive compounds from Makomotake, \u003cem\u003eZizania latifolia\u003c/em\u003e infected with \u003cem\u003eUstilago esculenta.\u003c/em\u003e Biosci Biotechnol Biochem 70:2800\u0026ndash;2802. https://doi.org/10.1271/bbb.60376\u003c/li\u003e\n\u003cli\u003eKobayashi M, Mashiko T, Wilisiani F, Hartono S, Nishigawa H, Natsuaki T, Neriya Y. (2023) Development of a RT-LAMP assay for real-time detection of criniviruses infecting tomato. J Virol Methods. 312:114662 https://doi.org/10.1016/j.jviromet.2022.114662\u003c/li\u003e\n\u003cli\u003eKomatsu K, Urayama S, Katoh Y, Fuji S, Hase S, Fukuhara T, Arie T, Teraoka T, Moriyama. (2016) Detection of \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e chrysovirus 1 in Japan and establishment of a rapid, sensitive and direct diagnostic method based on reverse transcription loop-mediated isothermal amplification. Arch Virol 161:317\u0026ndash;326 https://doi.org/10.1007/s00705-015-2666-x\u003c/li\u003e\n\u003cli\u003eNotomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63 https://doi.org/10.1093/nar/28.12.e63\u003c/li\u003e\n\u003cli\u003eSugawara K, Himeno M, Keima T, Kitazawa Y, Maejima K, Oshima K, Namba S (2012) Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene. J Gen Plant Pathol 78:389\u0026ndash;397 https://doi.org/10.1007/s10327-012-0403-9\u003c/li\u003e\n\u003cli\u003eUrayama S, Katoh Y, Fukuhara T, Arie T, Moriyama H, Teraoka T (2015) Rapid detection of Magnaporthe oryzae chrysovirus1-A from fungal colonies on agar plates and lesions of rice blast. J Gen Plant Pathol 81:97\u0026ndash;102 https://doi.org/10.1007/s10327-014-0567-6\u003c/li\u003e\n\u003cli\u003eWilisiani F, Tomiyama A, Katoh H, Hartono S, Neriya Y, Nishigawa H, Natsuaki T. (2019) Development of a LAMP assay with a portable device for real-time detection of begomoviruses under field conditions. J Virol Methods 265:71\u0026ndash;76 https://doi.org/10.1016/j.jviromet.2018.10.005\u003c/li\u003e\n\u003cli\u003eYamato S, Sasaki N, Komatsu K, Mashimo K, Motobayashi T. (2024) Temporal changes in colonization levels of \u003cem\u003eUstilago esculenta\u003c/em\u003e in different parts of \u003cem\u003eZizania latifolia\u003c/em\u003e plants competent and incompetent for stem gall formation. J Gen Plant Pathol 90:14\u0026ndash;22 https://doi.org/10.1007/s10327-023-01152-2\u003c/li\u003e\n\u003cli\u003eYang HC, Leu LS (1978) Formation and histopathology of galls induced by \u003cem\u003eUstilago esculenta\u003c/em\u003e in \u003cem\u003eZizania latifolia\u003c/em\u003e. Phytopathology 68:1572\u0026ndash;1576 https://doi.org/10.1094/phyto-68-1572\u003c/li\u003e\n\u003cli\u003eZhang Y, Cao Q, Hu P, Cui H, Yu X, Ye Z. (2017) Investigation on the differentiation of two Ustilago esculenta strains \u0026mdash;implications of a relationship with the host phenotypes appearing in the fields. BMC Microbiol 17: 228. https://doi.org/10.1186/s12866-017-1138-8\u003c/li\u003e\n\u003cli\u003eZhang JZ, Chu FQ, Guo DP, Hyde KD, Xie GL (2012) Cytology and ultrastructure of interactions between \u003cem\u003eUstilago esculenta\u003c/em\u003e and \u003cem\u003eZizania latifolia\u003c/em\u003e. Mycol Prog 11:499\u0026ndash;508 https://doi.org/10.1007/s11557-011-0765-y\u003c/li\u003e\n\u003cli\u003eZhang Z, Xu S, Kong M, Dai H, Liu Y, Miao M (2021) Isolation, identification and artificial inoculation of \u003cem\u003eUstilago esculenta\u003c/em\u003e on \u003cem\u003eZizania latifolia\u003c/em\u003e. Hortic Plant J 7:347\u0026ndash;358 https://doi.org/10.1016/j.hpj.2020.08.004\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1. LAMP primers specific for the \u003cem\u003eUeLAM16A\u003c/em\u003e gene\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cimg src=\"data:image/png;base64,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\" alt=\"image\" style=\"width: 493px; height: 214.348px;\" width=\"493\" height=\"214.348\"\u003e\u003c/strong\u003e\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":"","lastPublishedDoi":"10.21203/rs.3.rs-4685066/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4685066/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Infection of Zizania latifolia by Ustilago esculenta causes edible stem galls called makomotake. The development of stem galls may be influenced by the fungal colonization level at the seedling stage. To evaluate this possibility, we attempted to develop a real-time loop-mediated isothermal amplification (LAMP) method with toothpick sampling for U. esculenta detection, which allows us to examine non-destructively the relationship between fungal levels in seedlings and makomotake production from the same seedlings. Our method detected U. esculenta efficiently in seedlings that eventually produced makomotake, but did not detect it in those that failed to develop stem galls.","manuscriptTitle":"Development of a real-time loop-mediated isothermal amplification method with toothpick sampling for non-destructive detection of Ustilago esculenta in Zizania latifolia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-31 09:56:01","doi":"10.21203/rs.3.rs-4685066/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-07-09T00:43:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-08T10:30:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-05T06:56:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of General Plant Pathology","date":"2024-07-04T04:41:16+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":"f95da0ba-5cdd-4b21-80d9-6fac07db9d95","owner":[],"postedDate":"July 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:03:28+00:00","versionOfRecord":{"articleIdentity":"rs-4685066","link":"https://doi.org/10.1007/s10327-024-01201-4","journal":{"identity":"journal-of-general-plant-pathology","isVorOnly":false,"title":"Journal of General Plant Pathology"},"publishedOn":"2024-09-17 15:57:42","publishedOnDateReadable":"September 17th, 2024"},"versionCreatedAt":"2024-07-31 09:56:01","video":"","vorDoi":"10.1007/s10327-024-01201-4","vorDoiUrl":"https://doi.org/10.1007/s10327-024-01201-4","workflowStages":[]},"version":"v1","identity":"rs-4685066","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4685066","identity":"rs-4685066","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}