A simple and convenient method for antimicrobial susceptibility testing of plant pathogenic bacteria.

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Mizue Tsuji, Takashi Fujikawa, Yashiro Inoue, Yuichi Takikawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6241655/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Jun, 2025 Read the published version in Journal of General Plant Pathology → Version 1 posted 4 You are reading this latest preprint version Abstract Antimicrobial resistant (AMR) bacteria is the common threat in clinical, stockbreeding, aquaculture, and agricultural fields. To prepare for new AMR bacteria, we developed a simple and convenient method for antimicrobial susceptibility testing (AST) of plant pathogenic bacteria. The new recipe for media preparation and bacterial inoculation was proposed with understandable instructions, based on Goto and Takikawa (1984). Nutrient agar with 1% glucose were equivalent to the original medium. Inoculation using cotton swabs allow users to observe clear inhibition circles. The AST method for plant pathogenic bacteria was not standardized so far, but new method enables comparison and accumulation of data. antimicrobial resistance (AMR) antimicrobial susceptibility testing (AST) bacteria plant pathogen media standardization Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Plant pathogenic bacteria have harmed various crops including grains, vegetables and fruits. They spread rapidly in fields and cause huge losses under the favorite circumstances. Due to recent global warming, the occurrence and distribution of bacterial diseases are increasing worldwide (Garrett et al 2021; Lahlali et al 2024), though effective bactericides to control them are limited now. Continuous use of same compounds is however feared to bring the occurrence of antimicrobial resistance (AMR) or spread of antimicrobial resistance genes. AMR bacteria is difficult to control by conventional methods in the field, because they selectively grow and cause more severe damage on the production. AMR bacteria is one of the grave threats to human health nowadays, due to the depletion of efficient antimicrobial agents all over the world. In 2015, World Health Organization (WHO) adopt the AMR action plan, and governmental or public organizations in many countries follow it to prevent the development and spread of AMR bacteria including plant pathogens. Field spraying is the most appropriate method to confirm the efficacy of a bactericides. However, when AMR bacteria emerge or are suspected to have emerged, rapid confirmation is necessary for the efficacy of the bactericide through comparison between AMR strains and wild type (susceptible) ones. There, the antimicrobial susceptibility testing (AST) on media is a rapid and effective method to assess the efficacy of a bactericides. For AST, disk diffusion methods and minimal inhibitory concentration (MIC) testing methods on media are frequently used in the phytopathological field, as well as clinical or stockbreeding fields. The disk diffusion methods are conducted using paper disks impregnated with antimicrobial agents placed on agar media that inoculated bacteria. Antimicrobial agents inhibit the growth of bacteria around paper disks and produce clear circles, if inoculated strains are sensitive to the antimicrobial agents tested. MIC are estimated by the bacterial growth in broth or on agar media including antimicrobial agents serially diluted. In clinical fields, testing methods and antimicrobial breakpoints are standardized by the Clinical and Laboratory Standards Institute (CLSI) guidelines (Cockerill et al 2012) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST Disk Diffusion Test Methodology 2025; Matuschek et al 2014), though they do not provide enough information for plant pathogenic bacteria in the agricultural field. Therefore, many results of former research in the field cannot be compared directly, because they were obtained using independent media or inoculation methods in each study. In the phytopathological field in Japan, the disk diffusion method described by Goto and Takikawa (1984) is the most popular method for AST of plant pathogenic bacteria. However, their method is somewhat difficult for beginners and those with little experience to understand and conduct examinations practically, because of its small description about the composition of the medium, bacterial inoculations (mixing to agar medium) and determination of results in Japanese. Meanwhile, MIC testing methods are accurate and reproducible, but take much time and effort to be adopted on the emergence of an AMR bacteria. Anyway, results of both methods are influenced by trivial differences in experimental protocols, because they are usually conducted by manual works. For clinical use and for partial aquaculture and stockbreeding applications, paper disks and MIC plates including antimicrobial agents are commercially available, though not for agricultural bactericides. In the present study, we aimed a simple and convenient method for AST of plant pathogenic bacteria, which can be the standardized method in the phytopathological field, through selection of media, bacterial inoculations, preparation of antimicrobial agents, and tests for usability by cooperators of beginners. We choose the disk diffusion method as the standard because this method is already popular as described in Goto and Takikawa (1984), and widely used also in clinical and stockbreeding fields. Our method will contribute to the accumulation of data of AMR bacteria in fields, leading to speedy and effective on-site protection. Materials and Methods Bacterial inoculation to the medium In Goto and Takikawa (1984), bacterial inoculation was explained as follows; take several drops of bacterial suspension of high concentration on petri dish and pour 10ml of medium cooled to 50 ˚C, then mix enough. This explanation may make beginners confused with the concentration or the amount of bacterial suspension, or how to measure temperature of the medium. Therefore, to select a user-friendly method, their method was compared to streaking with cotton swabs, popular in clinical and stockbreeding fields, and spreading with common bacteria spreaders. The bacterial strain used was Burkholderia cepacia TS17-JN07 (Tsuji and Kadota 2020), incubated on Potato-Peptone-Glucose Agar (PPGA) (Nishiyama and Ezuka 1977, Table 1) plates at 28 ˚C for 24–48 h. Then the bacterial growth was suspended in 5 ml of API® NaCl 0.85% Medium (bioMérieux, obtained from Shimadzu Diagnostics Corp., Tokyo, Japan) and density was adjusted to ca. 10 8 cfu/ml. In mixture methods, 100 µl of bacterial suspension was added to 10 ml of the medium described in Goto and Takikawa (1984), according to procedures above. In streaking or spreading methods, 100 µl of bacterial suspension adjusted at ca. 0.5 McFarland standard turbidity was inoculated using a sterilized cotton swab with Φ2.8 mm cotton ball (J.C.B. Industry Limited, Tokyo, Japan) or a sterilized 37 mm width bacteria spreader (AS ONE Corp., Osaka, Japan), respectively, on the dried surface of medium in a petri dish. The antimicrobial agent used was oxolinic acid (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) at 0, 10, 100, 1000 ppm. The antimicrobial was sterilized using a 0.45 µM syringe filter, and 25 µl drop was applied onto sterilized Φ8mm paper disks (thick type, ADVANTEC®, Tokyo, Japan) placed on the surface of the medium. All plates were incubated at 28 ˚C for 48 h with lids upside, then the diameter of the circle produced by the inhibition of bacterial growth was measured using a caliper. When the inhibition zone was oval, the shortest diameter of an oval was measured. The clarity of inhibition zones was noted together. Media optimal for AST of plant pathogenic bacteria Composition of media optimal for AST of plant pathogenic bacteria was investigated. The CLSI manual (M02) recommend the use of Mueller-Hinton agar (Mueller and Hinton 1941, MHA, BD Difco, USA, Table 1) for clinical pathogens of bacteria (Bauer et al 1966), though growth of some strains of plant pathogenic bacteria was weaker than others in preliminary tests. The medium for AST described in Goto and Takikawa (1984) (GTM, Table 1) is reliable and conventionally used for plant pathogenic bacteria, but it needs to be manually mixed using individual reagents. On the emergence of AMR bacteria, easy and rapid acquisition and preparation are needed. Therefore, the newly developed medium is devised from well-known and commercially available medium and modified, through the comparison with MHA, GTM and PPGA that is usually used for incubation of plant pathogenic bacteria. Candidates of the base of the new medium are nutrient agar (ENA, EIKEN CHEMICAL Co., Ltd., Tokyo, Japan) and standard agar (ESA, EIKEN CHEMICAL) (Table 1). Information for the recipe of each media was listed in Table 1. Using these media, the clarity of inhibition zones was compared between the inoculation of B. cepacia (TS17-JN07), Xanthomonas campestris pv. campestris (MAFF106692), Pectobacterium carotovorum subsp. carotovorum (TS20-127, Accession No. PV232584 and PV232586), Pantoea ananatis (TS17-KP24, Accession No. PV232583 and PV232585), Pseudomonas marginalis pv. marginalis (MAFF301674). Inoculations of bacteria were conducted moving cotton balls from side to side, as filling the surface of plates (Fig. 4 , S1 ). Application of antimicrobial agents and incubation were conducted as same as above. Antimicrobial agents used were oxolinic acid and oxytetracycline (both from FUJIFILM Wako Pure Chemical) at 0, 10, 100, 1000 ppm. All experiments were repeated twice. In the manual (M02), the amount of medium is not indicated intelligibly because the size of plates recommended are various, depending on numbers of strains tested. In Goto and Takikawa (1984), the amount of medium per a petri dish was instructed as 10 ml with several drops of bacterial suspension, because Φ90mm ones are popular in Japan. Hence, the amount of medium suitable to streaking with cotton swabs in Φ90mm petri dishes was examined, at 10 ml, 15 ml and 20 ml. Density of inoculums Density of inoculums appropriate for streaking with cotton swabs was examined. In the CLSI manual (M02), 0.5 McFarland standard is recommended for clinical pathogens of bacteria, for both gram positive and negative pathogens, while majority of plant pathogenic bacteria are gram negative but belonging to various genera, showing various morphological characteristics. Because of colony colors, extracellular polysaccharide (EPS) or water-soluble pigment productions, it is difficult to guess the exact density of cells based on turbidity of bacterial suspension. Thus, appropriate turbidity of inoculum was investigated. Using BaSO 4 turbidity standard, bacterial suspensions equivalent to 0.5, 2, and 4 in McFarland standard turbidity were prepared. Inoculations of bacteria, application of antimicrobial agents, and incubation were conducted as same as above. The antimicrobial agent used was oxolinic acid. All experiments were repeated twice on ENA with 1% glucose (NAG) medium. Additionally, the numbers of bacterial cells recovered from a bacterial suspension on a plate media were counted, because the appearance of plant pathogenic bacteria varies by their various morphological characteristics. Bacterial suspensions of five species indicated above were adjusted to 2 McFarland standard, then serially diluted 10-fold. A 50 µl aliquot of each dilution was spread on plates of Yeast-Peptone agar (YPA, Tsuji and Takikawa 2018), PPGA, GTM, NAG, and ENA, and the numbers of colonies were counted. All experiments were repeated twice, and the average of colony numbers was calculated. Verification using more strains and species of bacteria As mentioned above, the usability of the newly developed method was verified using four strains of Pectobacterium carotovorum , 35 strains of Erwinia persicina , five strains of Erwinia rhapontici , seven strains of Pantoea ananatis , four strains of Pantoea agglomerans , five strains of Burkholderia cenocepacia , eight strains of Burkholderia cepacia , five strains of Burkholderia gladioli , 74 strains of Burkholderia glumae , 35 strains of Burkholderia plantarii , three strains of Pseudomonas marginalis pv. marginalis , two strains of Pseudomonas palleroniana , two strains of Pseudomonas viridiflava , and three strains of Xanthomonas campestris pv. campestris , with oxolinic acid, oxytetracycline hydrochloride, streptomycin sulfate, and kasugamycin hydrochloride monohydrate (these are available from FUJIFILM Wako Pure Chemical) at 0, 10, 100, 1000 ppm. Strains used were obtained from Shizuoka University Plant Pathogen (SUPP) collection, Research Center of Genetic Resources of NARO (MAFF collection), and isolated from fields in Japan (Table S1). Some strains originally isolated in this study were identified through preliminary bacteriological characterization, PCR tests using primers for species specific detection, and sequence analysis of gyrB and rpoD genes. Results Bacterial inoculation to the medium When comparing the methods of bacterial inoculation to the medium, streaking with cotton swabs was most appropriate, considered from the points of easy handlings and clarity of inhibition zones (Fig. 1 ). These inhibition zones were observed more clearly, when the bacterial suspensions were mixed to the medium. Spreading with bacteria spreaders is a common way, though it was difficult to spread the bacterial suspension equally without a turn-table. By contrast, clear inhibition zones were stably observed by streaking with cotton swabs. The CLSI manual (M02) instruct to streak the swab over the entire sterile agar surface, rotating the plate approximately 60° two more times to ensure an even distribution of inoculum. But when rotating the plate approximately 120° two times, the bacterial suspension was evenly distributed on the surface of the medium. Media optimal for plant pathogenic bacteria When comparing the types of media used for AST of plant pathogenic bacteria, inhibition zones were observed on all media tested, though the growth of bacteria depends on bacterial species and components of medium (Fig. 2 ). On GTM, all strains grew vigorously and made the clearest inhibition zones. On MHA, recommended in clinical, aquaculture or stockbreeding fields worldwide, some of plant pathogenic bacteria showed weak growth. On PPGA, bacteria grew well, but mass EPS such as produced by X. campestris pv. campestris disturbed the observation of inhibition zones. For premix media, ENA showed clearer inhibition zones than ESA, nearly equal to GTM. The major difference between GTM and ENA is presence or absence of glucose, therefore, equal amount of glucose to GTM (1%) was added to ENA. Thus, strains of five species tested grew well, and clear inhibition zones as same as on GTM were observed. On ENA without glucose, the growth of some species looked somewhat weak (Fig. 3 ). Therefore, ENA with 1% glucose (NAG) was considered optimal for simple and convenient AST of plant pathogenic bacteria. About the amount of medium needed, 10 ml media failed to grow some of bacterial species enough to observe clear inhibition zones and were often destroyed by the friction of cotton swabs when beginners tried. On 20 ml media, clear inhibition zones were observed, but 20 ml media is too much for thin type petri dishes and caused frequent condensation on lids. 15ml media showed clear inhibition zones as same as 20 ml, therefore, 15 ml was considered as appropriate amount for Φ90mm petri dishes and streaking with cotton swabs. Density of inoculums We investigated the effect of different densities of inoculums on AST, and unexpectedly, clarity of inhibition zones produced by tested strains were almost same between inoculum at 0.5 and 4 McFarland standard. Using inoculum at 0.5 McFarland standard, Pe. carotovorum subsp. carotovorum showed slightly weaker growth than others. Using inoculum at 4 McFarland standard, B. cepacia and X. campestris pv. campestris showed somewhat vigorous growth, and clarity of inhibition zones slightly declined. The appropriate density of inoculum was therefore considered approximately 2 McFarland standard (also, the density around this range is acceptable) for strains tested on NAG plates. As the result of colony counts, bacterial suspension of Pe. carotovorum subsp. carotovorum at approximately 2 McFarland standard corresponds to 9.0–14.1×10 8 cfu/ml, Pa. ananatis to 8.8–11.8×10 8 cfu/ml, Ps. marginalis pv. marginalis to 4.5–9.7×10 8 cfu/ml, B. cepacia to 6.3–9.2×10 8 cfu/ml, X. campestris pv. campestris 3.9–5.3×10 8 cfu/ml, respectively. Table 2 shows the recovery rate on each media. Verification using more strains and species of bacteria All strains used for verification showed the usefulness of the newly developed method. Table 3 and Table S1 show detail results of AST. Two strain of E. persicina and two of B. glumae showed AMR to oxolinic acid. Six of B. plantarii was AMR to Kasgamycin. On these strains, the experiment was repeated, and same results were obtained. Instruction for the newly developed AST method of plant pathogenic bacteria is in Fig. 4 . Discussion For rapid and effective controls of plant pathogenic bacteria, also for reduction of use of chemically synthesized agrochemicals, bacterial sensitivity to bactericides including antimicrobials needs to be investigated in advance. Some species of bacteria are not sensitive by nature and others acquires the resistance in the natural environment. When the efficacy of a bactericide become weak, suggesting the occurrence of AMR bacteria, we must select a substitute bactericide in hurry to control the disease. Until now, AST method for bacteria was not standardized in the phytopathological field, because the most popular method of Goto and Takikawa (1984) is a specialized and skillful one. ASTs have been therefore done under various conditions (Petriccione et al 2017; Takeuchi 1995; Valenzuela et al 2019; Wakimoto and Mukoo 1963; Yamaguchi et al 2014), and the results were difficult to compare each other. New method was developed with modification of the original method of Goto and Takikawa (1984), in the purpose of developing a simple and convenient method especially for beginners. NAG medium using commercially premixed media showed vigorous growth of bacterial strains tested, same as on the original medium GTM. Of course, the vigor of bacterial growth varied among bacterial species, the circles produced by inhibition of bacterial growth were able to be observed at the same time on NAG, for 192 strains used for verification. In preliminary tests, we planned to adopt Luria-Bertani (LB) agar, popular as routine media for bacteria, with 1% glucose. All strains tested grew well on it. But confusingly, composition of LB agar is sometimes different between manufacturers or laboratories. This is one of the reasons that we used premixed media in the present study, to accumulate standardized results in the future. About the inoculation of bacteria, the density of inoculums is appropriate at approximately 2 McFarland standard for plant pathogenic bacteria, for the use on NAG plates. Shown by the results of colony count, density of bacterial suspensions seemed to be affected by EPS or dead cells, though clear inhibition zones were stably observed for strains used in this study. For inoculation tools, cotton swabs were most user-friendly in the view of a beginner’s handling, as well as its availability. Many of beginners may have difficulty in mixing the bacterial suspension into the medium. If the temperature of the medium was not properly controlled, it resulted in uneven coloration and made observation of the inhibition zones difficult. In more severe cases, the inoculated bacterial strain died due to excessive heat. Many beginners seemed to need in-advance preparation of media as solid plates, therefore, streaking with cotton swabs on solid plates media is convenient way of inoculation for beginners. Additionally, it is necessary to procure tools and reagents for AST as quickly as possible on the emergence of AMR bacteria. From these reasons, we prioritized to use domestically produced or stably provided chemicals and tools. The usefulness of newly developed method was verified. Some beginners kindly cooperating our experiment could execute all procedures well. When skilled one conduct the experiment, actual working time was really shortened, because of no waiting time to adjust the temperature of medium repeatedly, needed in the original method. It made possible to test more strains and obtain more results of AST of plant pathogenic bacteria. 192 strains of 14 species were used for verification and AMR strains of E. persicina , B. glumae and B. plntarii were found. Reduction of antimicrobial susceptibility was also observed on several strains tested. All strains of B. cepacia and B. cenocepacia were insusceptible to all antimicrobials except oxolinic acid. The disk diffusion method suited for this prompt detection, because difference of diameters of inhibition zones were observed enough between sensitive, nonsensitive and low susceptible strains. Compared to MIC methods, the precision of the susceptibility leads by disk diffusion methods are surely limited. However, in real cultivation fields where the concentration of agrochemicals is stipulated, disk diffusion methods give enough useful data by comparison between AMR bacteria and sensitive ones. We used paper disks includes antimicrobials at 0, 10, 100, 1000 ppm. Bacterial growth is however naturally different between on nutrient-rich medium and in the natural environment. Further studies are needed on correlation of antimicrobial susceptibility between in vitro and in situ . Only one point to be careful is that antimicrobial susceptibility on medium may not directly affect to the concentration of bactericide splayed in agricultural fields. Spread and standardization of new method enables comparison and accumulation of the data of antimicrobial susceptibility of plant pathogenic bacteria. These records will help the detection of AMR bacteria and the selection of bactericides in the future. Declarations Funding This research was supported by grants from the project of the Ministry of Agriculture, Forestry and Fisheries of JAPAN (Research program on development of comprehensive regulatory science for providing safe crops, livestock products, and marine products). Compliance with Ethical Standards Conflicts of interest The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals. References Bauer AW, Kirby WMM, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45: 493-496. Cockerill FR, Hindler JA, Wikler MA, Patel JB, Alder J, Powell M, Dudley MN, Swenson JM, Eliopoulos GM, Thomson RB Jr., Ferraro MJ, Traczewski MM, Hardy DJ, Turnidge JD, Hecht DW, Weinstein MP, Zimmer BL (2012) Clinical and laboratory standards institute M02 Performance Standards for Antimicrobial Disk Susceptibility Tests, 11th Edition. Clinical and laboratory standards institute, Wayne, PA. EUCAST Disk Diffusion Test Methodology, Antimicrobial susceptibility testing EUCAST disk diffusion method Version 13.0 (2025) European Committee on Antimicrobial Susceptibility Testing. https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_test_documents/2025_manuals/Manual_v_13.0_EUCAST_Disk_Test_2025.pdf Garrett KA, Nita M, De Wolf ED, Esker PD, Gomez-Montano L, Sparks AH (2021). Plant pathogens as indicators of climate change. Climate change pp. 499-513. Elsevier. Goto M, Takikawa Y (1984) The methods to investigate bacteriological characteristics of plant pathogenic bacteria to identify them (4). (in Japanese). Plant protection 38:479–484 Lahlali R, Taoussi M, Laasli SE, Gachara G, Ezzouggari R, Belabess Z, Aberkani K, Assouguem A, Meddich A, Jarroudi ME, Barka EA (2024) Effects of climate change on plant pathogens and host-pathogen interactions. Crop and Environ 3:159-170. 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Plant protection 49:403–404 Tsuji M, Kadota I (2020) Identification and phylogenetic analysis of Burkholderia cepacia complex bacteria isolated from rot of onion bulbs in Tohoku region of Japan. J Gen Plant Pathol 86:376–386 Tsuji M, Takikawa Y (2018) Pseudomonas syringae pv. alliifistulosi pv. nov., the causal agent of bacterial leaf spot of onions. J Gen Plant Pathol 84:343–358 Valenzuela M, Méndez V, Montenegro I, Besoain X, Seeger M (2019) Streptomycin resistance in Clavibacter michiganensis subsp. michiganensis strains from Chile is related to an rpsL gene mutation. Plant Pathol 68:426–433 Wakimoto S, Mukoo H (1963) Natural occurrences of streptomycin resistant Xanthomonas oryzae , the causal bacteria of leaf blight disease of rice. (in Japanese). Jpn J Phytopathol 28:153–158 Yamaguchi S, Iyama K, Tagata K, Natsuaki K, Negishi H, Shinohara H (2014) Bactericide susceptibility of bacterial shot hole bacteria of Peach (mainly Xanthomonas arboricola pv. pruni ). J Agric Sci Tokyo Univ Agric 59:74–80 Tables Tables 1 to 3 are available in the Supplementary Files section Supplementary Files Tables.docx SupplementalFigure1.docx Figure S1 Inoculations of bacteria using cotton swabs. (a) Dipping a cotton swab into a bacterial suspension. (b) bacterial inoculation conducted moving cotton balls from side to side as filling the surface of plates (inside a clean bench). (c) bacterial inoculation (outside a clean bench) SupplementoryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 18 Jun, 2025 Read the published version in Journal of General Plant Pathology → Version 1 posted Reviewers agreed at journal 23 Mar, 2025 Reviewers invited by journal 22 Mar, 2025 Editor assigned by journal 18 Mar, 2025 First submitted to journal 16 Mar, 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. We do this by developing innovative software and high quality services for the global research community. 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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-6241655","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432579921,"identity":"c04b2fd8-af88-4f3e-9f83-4815d4d21ad3","order_by":0,"name":"Mizue Tsuji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIie3RrwoCMRzA8d8xuCs/sE4OvFfYWLBp9DUmB6YLJxabA0GLYD0fw2IyTASv3AMYlQOzRpNORcQyjYL7to192D8Al+tHYxIupJkrOPafM7bl+CAkgEJ7WfEtMZEKbOWLWGtWCpbuEz/0ZuVerZYNiFoKeqltF5ow1l6gIGGHK32IgRcaRGYleCM09kNZL49aA88kCLQRczBD2GBSzU9Kf0XgfjBJKEV+JxH9RLad1BBNGCZdQ2JkuFLWuwTT9YKfbyTI54Y0atF4uBG2FzP5bx+HTHsjYRdAdm/DSAEpPxCXy+X6r66ny018gq4rEQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6161-8136","institution":"National Agriculture and Food Research Organization Tohoku Agricultural Research Center","correspondingAuthor":true,"prefix":"","firstName":"Mizue","middleName":"","lastName":"Tsuji","suffix":""},{"id":432579922,"identity":"f1bdad1e-04eb-4d65-bc64-cf48df998b0b","order_by":1,"name":"Takashi Fujikawa","email":"","orcid":"","institution":"National Agriculture and Food Research Organization Institute for Plant Protection","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Fujikawa","suffix":""},{"id":432579923,"identity":"35570631-9e26-4bfd-bd84-208d87bb174e","order_by":2,"name":"Yashiro Inoue","email":"","orcid":"","institution":"National Agriculture and food Research Organization Institute for Plant Protection","correspondingAuthor":false,"prefix":"","firstName":"Yashiro","middleName":"","lastName":"Inoue","suffix":""},{"id":432579924,"identity":"0ac528f4-672e-4efd-ac8e-77074d5576a1","order_by":3,"name":"Yuichi Takikawa","email":"","orcid":"","institution":"Shizuoka University Faculty of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Yuichi","middleName":"","lastName":"Takikawa","suffix":""}],"badges":[],"createdAt":"2025-03-17 06:49:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6241655/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6241655/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10327-025-01234-3","type":"published","date":"2025-06-18T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79662668,"identity":"a197e7c8-5576-4806-ba9b-b24cb8219c11","added_by":"auto","created_at":"2025-04-01 09:52:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":174410,"visible":true,"origin":"","legend":"\u003cp\u003eResults of bacterial inoculation. (a) Bacterial suspension was mixed into medium. (b)Bacterial suspension was streaked using a cotton swab on the surface of the medium. (c) Bacterial suspension was spread using bacteria spreader on the surface of the medium.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6241655/v1/48e01e735e6980f4f270ad1c.png"},{"id":79662665,"identity":"82743a32-c6ac-4e94-a9d8-b015df022e8d","added_by":"auto","created_at":"2025-04-01 09:52:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":623417,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of clarity of circles produced by the inhibition of bacterial growth on (a) the medium for AST (Goto and Takikawa 1984), (b) potato-peptone-glucose agar medium (Nishiyama and Ezuka 1977),(c) Mueller-Hinton agar (BD Difco), (d) nutrient agar (EIKEN CHEMICAL), (e) standard agar (EIKEN CHEMICAL). Pictures on the left side are results of oxolinic acid, and the right side are oxytetracycline hydrochloride. Bacterial strains inoculated were \u003cem\u003eBurkholderia cepacia\u003c/em\u003e TS17-JN07 (upper left), \u003cem\u003eXanthomonas campestris\u003c/em\u003e pv. \u003cem\u003ecampestris \u003c/em\u003eMAFF106692 (upper right), \u003cem\u003ePectobacterium carotovorum\u003c/em\u003e subsp. \u003cem\u003ecarotovorum\u003c/em\u003e TS20-127 (lower left), \u003cem\u003ePantoea ananatis\u003c/em\u003e TS17-KP24 (lower center), \u003cem\u003ePseudomonas marginalis\u003c/em\u003e pv.\u003cem\u003emarginalis\u003c/em\u003e MAFF301674 (lower right) in each picture.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6241655/v1/7922286402be5abd6edde204.png"},{"id":79662661,"identity":"5cdde25f-5119-4a71-8228-4fc3eb0ec0c2","added_by":"auto","created_at":"2025-04-01 09:52:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":275835,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of clarity of circles produced by the inhibition of bacterial growth on (a) nutrient agar (EIKEN CHEMICAL), (b) nutrient agar (EIKEN CHEMICAL) added 1% glucose, (c) the medium for AST (Goto and Takikawa 1984). Antimicrobial agent used was oxolinic acid. Bacterial strains inoculated were \u003cem\u003ePectobacterium carotovorum\u003c/em\u003esubsp. \u003cem\u003ecarotovorum\u003c/em\u003e TS20-127 (upper left), \u003cem\u003ePantoea ananatis\u003c/em\u003e TS17-KP24 (upper right), \u003cem\u003ePseudomonas marginalis\u003c/em\u003e pv.\u003cem\u003e marginalis\u003c/em\u003e MAFF301674 (lower left), \u003cem\u003eBurkholderia cepacia\u003c/em\u003e TS17-JN07 (lower center), \u003cem\u003eXanthomonas campestris\u003c/em\u003e pv. \u003cem\u003ecampestris \u003c/em\u003eMAFF106692 (lower right) in each picture.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6241655/v1/0ad1ccd46af02749579f9ebf.png"},{"id":79662689,"identity":"8f69a8b6-5701-43d2-8f3c-53882a4877aa","added_by":"auto","created_at":"2025-04-01 09:52:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":335288,"visible":true,"origin":"","legend":"\u003cp\u003eInstruction for the newly developed AST method of Plant pathogenic bacteria.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6241655/v1/5fb124830be0b4753d6c5835.png"},{"id":85231338,"identity":"7d526b5b-9da5-4379-97ef-2fb1adb638f2","added_by":"auto","created_at":"2025-06-23 16:06:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1900640,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6241655/v1/ec8deed2-6435-4efe-93ab-0249eecba1dd.pdf"},{"id":79662657,"identity":"68c389d3-8193-450e-8d02-c75123df2630","added_by":"auto","created_at":"2025-04-01 09:52:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37190,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6241655/v1/319fa6ef16852c6ca451aca0.docx"},{"id":79662697,"identity":"98d7e915-bec4-4568-a048-253fc4d973ed","added_by":"auto","created_at":"2025-04-01 09:52:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":93091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1\u003c/strong\u003e Inoculations of bacteria using cotton swabs. (a) Dipping a cotton swab into a bacterial suspension. (b) bacterial inoculation conducted moving cotton balls from side to side as filling the surface of plates (inside a clean bench). (c) bacterial inoculation (outside a clean bench)\u003c/p\u003e","description":"","filename":"SupplementalFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6241655/v1/46916a020f1bd717f61a4bfb.docx"},{"id":79662662,"identity":"4c4dd0b5-120a-4f7c-b096-c16bc6905b2e","added_by":"auto","created_at":"2025-04-01 09:52:44","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":126264,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementoryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6241655/v1/2da4b220da8262f9bf98141d.docx"}],"financialInterests":"","formattedTitle":"A simple and convenient method for antimicrobial susceptibility testing of plant pathogenic bacteria.","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant pathogenic bacteria have harmed various crops including grains, vegetables and fruits. They spread rapidly in fields and cause huge losses under the favorite circumstances. Due to recent global warming, the occurrence and distribution of bacterial diseases are increasing worldwide (Garrett et al 2021; Lahlali et al 2024), though effective bactericides to control them are limited now. Continuous use of same compounds is however feared to bring the occurrence of antimicrobial resistance (AMR) or spread of antimicrobial resistance genes. AMR bacteria is difficult to control by conventional methods in the field, because they selectively grow and cause more severe damage on the production. AMR bacteria is one of the grave threats to human health nowadays, due to the depletion of efficient antimicrobial agents all over the world. In 2015, World Health Organization (WHO) adopt the AMR action plan, and governmental or public organizations in many countries follow it to prevent the development and spread of AMR bacteria including plant pathogens.\u003c/p\u003e \u003cp\u003eField spraying is the most appropriate method to confirm the efficacy of a bactericides. However, when AMR bacteria emerge or are suspected to have emerged, rapid confirmation is necessary for the efficacy of the bactericide through comparison between AMR strains and wild type (susceptible) ones. There, the antimicrobial susceptibility testing (AST) on media is a rapid and effective method to assess the efficacy of a bactericides. For AST, disk diffusion methods and minimal inhibitory concentration (MIC) testing methods on media are frequently used in the phytopathological field, as well as clinical or stockbreeding fields. The disk diffusion methods are conducted using paper disks impregnated with antimicrobial agents placed on agar media that inoculated bacteria. Antimicrobial agents inhibit the growth of bacteria around paper disks and produce clear circles, if inoculated strains are sensitive to the antimicrobial agents tested. MIC are estimated by the bacterial growth in broth or on agar media including antimicrobial agents serially diluted. In clinical fields, testing methods and antimicrobial breakpoints are standardized by the Clinical and Laboratory Standards Institute (CLSI) guidelines (Cockerill et al 2012) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST Disk Diffusion Test Methodology 2025; Matuschek et al 2014), though they do not provide enough information for plant pathogenic bacteria in the agricultural field. Therefore, many results of former research in the field cannot be compared directly, because they were obtained using independent media or inoculation methods in each study. In the phytopathological field in Japan, the disk diffusion method described by Goto and Takikawa (1984) is the most popular method for AST of plant pathogenic bacteria. However, their method is somewhat difficult for beginners and those with little experience to understand and conduct examinations practically, because of its small description about the composition of the medium, bacterial inoculations (mixing to agar medium) and determination of results in Japanese. Meanwhile, MIC testing methods are accurate and reproducible, but take much time and effort to be adopted on the emergence of an AMR bacteria. Anyway, results of both methods are influenced by trivial differences in experimental protocols, because they are usually conducted by manual works. For clinical use and for partial aquaculture and stockbreeding applications, paper disks and MIC plates including antimicrobial agents are commercially available, though not for agricultural bactericides.\u003c/p\u003e \u003cp\u003eIn the present study, we aimed a simple and convenient method for AST of plant pathogenic bacteria, which can be the standardized method in the phytopathological field, through selection of media, bacterial inoculations, preparation of antimicrobial agents, and tests for usability by cooperators of beginners. We choose the disk diffusion method as the standard because this method is already popular as described in Goto and Takikawa (1984), and widely used also in clinical and stockbreeding fields. Our method will contribute to the accumulation of data of AMR bacteria in fields, leading to speedy and effective on-site protection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eBacterial inoculation to the medium\u003c/p\u003e \u003cp\u003eIn Goto and Takikawa (1984), bacterial inoculation was explained as follows; take several drops of bacterial suspension of high concentration on petri dish and pour 10ml of medium cooled to 50 ˚C, then mix enough. This explanation may make beginners confused with the concentration or the amount of bacterial suspension, or how to measure temperature of the medium. Therefore, to select a user-friendly method, their method was compared to streaking with cotton swabs, popular in clinical and stockbreeding fields, and spreading with common bacteria spreaders. The bacterial strain used was \u003cem\u003eBurkholderia cepacia\u003c/em\u003e TS17-JN07 (Tsuji and Kadota 2020), incubated on Potato-Peptone-Glucose Agar (PPGA) (Nishiyama and Ezuka 1977, Table\u0026nbsp;1) plates at 28 ˚C for 24\u0026ndash;48 h. Then the bacterial growth was suspended in 5 ml of API\u0026reg; NaCl 0.85% Medium (bioM\u0026eacute;rieux, obtained from Shimadzu Diagnostics Corp., Tokyo, Japan) and density was adjusted to ca. 10\u003csup\u003e8\u003c/sup\u003ecfu/ml. In mixture methods, 100 \u0026micro;l of bacterial suspension was added to 10 ml of the medium described in Goto and Takikawa (1984), according to procedures above. In streaking or spreading methods, 100 \u0026micro;l of bacterial suspension adjusted at ca. 0.5 McFarland standard turbidity was inoculated using a sterilized cotton swab with Φ2.8 mm cotton ball (J.C.B. Industry Limited, Tokyo, Japan) or a sterilized 37 mm width bacteria spreader (AS ONE Corp., Osaka, Japan), respectively, on the dried surface of medium in a petri dish. The antimicrobial agent used was oxolinic acid (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) at 0, 10, 100, 1000 ppm. The antimicrobial was sterilized using a 0.45 \u0026micro;M syringe filter, and 25 \u0026micro;l drop was applied onto sterilized Φ8mm paper disks (thick type, ADVANTEC\u0026reg;, Tokyo, Japan) placed on the surface of the medium. All plates were incubated at 28 ˚C for 48 h with lids upside, then the diameter of the circle produced by the inhibition of bacterial growth was measured using a caliper. When the inhibition zone was oval, the shortest diameter of an oval was measured. The clarity of inhibition zones was noted together.\u003c/p\u003e \u003cp\u003eMedia optimal for AST of plant pathogenic bacteria\u003c/p\u003e \u003cp\u003eComposition of media optimal for AST of plant pathogenic bacteria was investigated. The CLSI manual (M02) recommend the use of Mueller-Hinton agar (Mueller and Hinton 1941, MHA, BD Difco, USA, Table\u0026nbsp;1) for clinical pathogens of bacteria (Bauer et al 1966), though growth of some strains of plant pathogenic bacteria was weaker than others in preliminary tests. The medium for AST described in Goto and Takikawa (1984) (GTM, Table\u0026nbsp;1) is reliable and conventionally used for plant pathogenic bacteria, but it needs to be manually mixed using individual reagents. On the emergence of AMR bacteria, easy and rapid acquisition and preparation are needed. Therefore, the newly developed medium is devised from well-known and commercially available medium and modified, through the comparison with MHA, GTM and PPGA that is usually used for incubation of plant pathogenic bacteria. Candidates of the base of the new medium are nutrient agar (ENA, EIKEN CHEMICAL Co., Ltd., Tokyo, Japan) and standard agar (ESA, EIKEN CHEMICAL) (Table\u0026nbsp;1). Information for the recipe of each media was listed in Table\u0026nbsp;1. Using these media, the clarity of inhibition zones was compared between the inoculation of \u003cem\u003eB. cepacia\u003c/em\u003e (TS17-JN07), \u003cem\u003eXanthomonas campestris\u003c/em\u003e pv. \u003cem\u003ecampestris\u003c/em\u003e (MAFF106692), \u003cem\u003ePectobacterium carotovorum\u003c/em\u003e subsp. \u003cem\u003ecarotovorum\u003c/em\u003e (TS20-127, Accession No. PV232584 and PV232586), \u003cem\u003ePantoea ananatis\u003c/em\u003e (TS17-KP24, Accession No. PV232583 and PV232585), \u003cem\u003ePseudomonas marginalis\u003c/em\u003e pv. \u003cem\u003emarginalis\u003c/em\u003e (MAFF301674). Inoculations of bacteria were conducted moving cotton balls from side to side, as filling the surface of plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Application of antimicrobial agents and incubation were conducted as same as above. Antimicrobial agents used were oxolinic acid and oxytetracycline (both from FUJIFILM Wako Pure Chemical) at 0, 10, 100, 1000 ppm. All experiments were repeated twice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the manual (M02), the amount of medium is not indicated intelligibly because the size of plates recommended are various, depending on numbers of strains tested. In Goto and Takikawa (1984), the amount of medium per a petri dish was instructed as 10 ml with several drops of bacterial suspension, because Φ90mm ones are popular in Japan. Hence, the amount of medium suitable to streaking with cotton swabs in Φ90mm petri dishes was examined, at 10 ml, 15 ml and 20 ml.\u003c/p\u003e \u003cp\u003eDensity of inoculums\u003c/p\u003e \u003cp\u003eDensity of inoculums appropriate for streaking with cotton swabs was examined. In the CLSI manual (M02), 0.5 McFarland standard is recommended for clinical pathogens of bacteria, for both gram positive and negative pathogens, while majority of plant pathogenic bacteria are gram negative but belonging to various genera, showing various morphological characteristics. Because of colony colors, extracellular polysaccharide (EPS) or water-soluble pigment productions, it is difficult to guess the exact density of cells based on turbidity of bacterial suspension. Thus, appropriate turbidity of inoculum was investigated. Using BaSO\u003csub\u003e4\u003c/sub\u003e turbidity standard, bacterial suspensions equivalent to 0.5, 2, and 4 in McFarland standard turbidity were prepared. Inoculations of bacteria, application of antimicrobial agents, and incubation were conducted as same as above. The antimicrobial agent used was oxolinic acid. All experiments were repeated twice on ENA with 1% glucose (NAG) medium. Additionally, the numbers of bacterial cells recovered from a bacterial suspension on a plate media were counted, because the appearance of plant pathogenic bacteria varies by their various morphological characteristics. Bacterial suspensions of five species indicated above were adjusted to 2 McFarland standard, then serially diluted 10-fold. A 50 \u0026micro;l aliquot of each dilution was spread on plates of Yeast-Peptone agar (YPA, Tsuji and Takikawa 2018), PPGA, GTM, NAG, and ENA, and the numbers of colonies were counted. All experiments were repeated twice, and the average of colony numbers was calculated.\u003c/p\u003e \u003cp\u003eVerification using more strains and species of bacteria\u003c/p\u003e \u003cp\u003eAs mentioned above, the usability of the newly developed method was verified using four strains of \u003cem\u003ePectobacterium carotovorum\u003c/em\u003e, 35 strains of \u003cem\u003eErwinia persicina\u003c/em\u003e, five strains of \u003cem\u003eErwinia rhapontici\u003c/em\u003e, seven strains of \u003cem\u003ePantoea ananatis\u003c/em\u003e, four strains of \u003cem\u003ePantoea agglomerans\u003c/em\u003e, five strains of \u003cem\u003eBurkholderia cenocepacia\u003c/em\u003e, eight strains of \u003cem\u003eBurkholderia cepacia\u003c/em\u003e, five strains of \u003cem\u003eBurkholderia gladioli\u003c/em\u003e, 74 strains of \u003cem\u003eBurkholderia glumae\u003c/em\u003e, 35 strains of \u003cem\u003eBurkholderia plantarii\u003c/em\u003e, three strains of \u003cem\u003ePseudomonas marginalis\u003c/em\u003e pv. \u003cem\u003emarginalis\u003c/em\u003e, two strains of \u003cem\u003ePseudomonas palleroniana\u003c/em\u003e, two strains of \u003cem\u003ePseudomonas viridiflava\u003c/em\u003e, and three strains of \u003cem\u003eXanthomonas campestris\u003c/em\u003e pv. \u003cem\u003ecampestris\u003c/em\u003e, with oxolinic acid, oxytetracycline hydrochloride, streptomycin sulfate, and kasugamycin hydrochloride monohydrate (these are available from FUJIFILM Wako Pure Chemical) at 0, 10, 100, 1000 ppm. Strains used were obtained from Shizuoka University Plant Pathogen (SUPP) collection, Research Center of Genetic Resources of NARO (MAFF collection), and isolated from fields in Japan (Table S1). Some strains originally isolated in this study were identified through preliminary bacteriological characterization, PCR tests using primers for species specific detection, and sequence analysis of \u003cem\u003egyrB\u003c/em\u003e and \u003cem\u003erpoD\u003c/em\u003e genes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eBacterial inoculation to the medium\u003c/p\u003e \u003cp\u003eWhen comparing the methods of bacterial inoculation to the medium, streaking with cotton swabs was most appropriate, considered from the points of easy handlings and clarity of inhibition zones (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These inhibition zones were observed more clearly, when the bacterial suspensions were mixed to the medium. Spreading with bacteria spreaders is a common way, though it was difficult to spread the bacterial suspension equally without a turn-table. By contrast, clear inhibition zones were stably observed by streaking with cotton swabs. The CLSI manual (M02) instruct to streak the swab over the entire sterile agar surface, rotating the plate approximately 60\u0026deg; two more times to ensure an even distribution of inoculum. But when rotating the plate approximately 120\u0026deg; two times, the bacterial suspension was evenly distributed on the surface of the medium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMedia optimal for plant pathogenic bacteria\u003c/p\u003e \u003cp\u003eWhen comparing the types of media used for AST of plant pathogenic bacteria, inhibition zones were observed on all media tested, though the growth of bacteria depends on bacterial species and components of medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On GTM, all strains grew vigorously and made the clearest inhibition zones. On MHA, recommended in clinical, aquaculture or stockbreeding fields worldwide, some of plant pathogenic bacteria showed weak growth. On PPGA, bacteria grew well, but mass EPS such as produced by \u003cem\u003eX. campestris\u003c/em\u003e pv. \u003cem\u003ecampestris\u003c/em\u003e disturbed the observation of inhibition zones. For premix media, ENA showed clearer inhibition zones than ESA, nearly equal to GTM. The major difference between GTM and ENA is presence or absence of glucose, therefore, equal amount of glucose to GTM (1%) was added to ENA. Thus, strains of five species tested grew well, and clear inhibition zones as same as on GTM were observed. On ENA without glucose, the growth of some species looked somewhat weak (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, ENA with 1% glucose (NAG) was considered optimal for simple and convenient AST of plant pathogenic bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAbout the amount of medium needed, 10 ml media failed to grow some of bacterial species enough to observe clear inhibition zones and were often destroyed by the friction of cotton swabs when beginners tried. On 20 ml media, clear inhibition zones were observed, but 20 ml media is too much for thin type petri dishes and caused frequent condensation on lids. 15ml media showed clear inhibition zones as same as 20 ml, therefore, 15 ml was considered as appropriate amount for Φ90mm petri dishes and streaking with cotton swabs.\u003c/p\u003e \u003cp\u003eDensity of inoculums\u003c/p\u003e \u003cp\u003eWe investigated the effect of different densities of inoculums on AST, and unexpectedly, clarity of inhibition zones produced by tested strains were almost same between inoculum at 0.5 and 4 McFarland standard. Using inoculum at 0.5 McFarland standard, \u003cem\u003ePe. carotovorum\u003c/em\u003e subsp. \u003cem\u003ecarotovorum\u003c/em\u003e showed slightly weaker growth than others. Using inoculum at 4 McFarland standard, \u003cem\u003eB. cepacia\u003c/em\u003e and \u003cem\u003eX. campestris\u003c/em\u003e pv. \u003cem\u003ecampestris\u003c/em\u003e showed somewhat vigorous growth, and clarity of inhibition zones slightly declined. The appropriate density of inoculum was therefore considered approximately 2 McFarland standard (also, the density around this range is acceptable) for strains tested on NAG plates. As the result of colony counts, bacterial suspension of \u003cem\u003ePe. carotovorum\u003c/em\u003e subsp. \u003cem\u003ecarotovorum\u003c/em\u003e at approximately 2 McFarland standard corresponds to 9.0\u0026ndash;14.1\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu/ml, \u003cem\u003ePa. ananatis\u003c/em\u003e to 8.8\u0026ndash;11.8\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu/ml, \u003cem\u003ePs. marginalis\u003c/em\u003e pv. \u003cem\u003emarginalis\u003c/em\u003e to 4.5\u0026ndash;9.7\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu/ml, \u003cem\u003eB. cepacia\u003c/em\u003e to 6.3\u0026ndash;9.2\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu/ml, \u003cem\u003eX. campestris\u003c/em\u003e pv. \u003cem\u003ecampestris\u003c/em\u003e 3.9\u0026ndash;5.3\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu/ml, respectively. Table\u0026nbsp;2 shows the recovery rate on each media.\u003c/p\u003e \u003cp\u003eVerification using more strains and species of bacteria\u003c/p\u003e \u003cp\u003eAll strains used for verification showed the usefulness of the newly developed method. Table\u0026nbsp;3 and Table S1 show detail results of AST. Two strain of \u003cem\u003eE. persicina\u003c/em\u003e and two of \u003cem\u003eB. glumae\u003c/em\u003e showed AMR to oxolinic acid. Six of \u003cem\u003eB. plantarii\u003c/em\u003e was AMR to Kasgamycin. On these strains, the experiment was repeated, and same results were obtained. Instruction for the newly developed AST method of plant pathogenic bacteria is in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor rapid and effective controls of plant pathogenic bacteria, also for reduction of use of chemically synthesized agrochemicals, bacterial sensitivity to bactericides including antimicrobials needs to be investigated in advance. Some species of bacteria are not sensitive by nature and others acquires the resistance in the natural environment. When the efficacy of a bactericide become weak, suggesting the occurrence of AMR bacteria, we must select a substitute bactericide in hurry to control the disease.\u003c/p\u003e \u003cp\u003eUntil now, AST method for bacteria was not standardized in the phytopathological field, because the most popular method of Goto and Takikawa (1984) is a specialized and skillful one. ASTs have been therefore done under various conditions (Petriccione et al 2017; Takeuchi 1995; Valenzuela et al 2019; Wakimoto and Mukoo 1963; Yamaguchi et al 2014), and the results were difficult to compare each other. New method was developed with modification of the original method of Goto and Takikawa (1984), in the purpose of developing a simple and convenient method especially for beginners. NAG medium using commercially premixed media showed vigorous growth of bacterial strains tested, same as on the original medium GTM. Of course, the vigor of bacterial growth varied among bacterial species, the circles produced by inhibition of bacterial growth were able to be observed at the same time on NAG, for 192 strains used for verification. In preliminary tests, we planned to adopt Luria-Bertani (LB) agar, popular as routine media for bacteria, with 1% glucose. All strains tested grew well on it. But confusingly, composition of LB agar is sometimes different between manufacturers or laboratories. This is one of the reasons that we used premixed media in the present study, to accumulate standardized results in the future.\u003c/p\u003e \u003cp\u003eAbout the inoculation of bacteria, the density of inoculums is appropriate at approximately 2 McFarland standard for plant pathogenic bacteria, for the use on NAG plates. Shown by the results of colony count, density of bacterial suspensions seemed to be affected by EPS or dead cells, though clear inhibition zones were stably observed for strains used in this study. For inoculation tools, cotton swabs were most user-friendly in the view of a beginner\u0026rsquo;s handling, as well as its availability. Many of beginners may have difficulty in mixing the bacterial suspension into the medium. If the temperature of the medium was not properly controlled, it resulted in uneven coloration and made observation of the inhibition zones difficult. In more severe cases, the inoculated bacterial strain died due to excessive heat. Many beginners seemed to need in-advance preparation of media as solid plates, therefore, streaking with cotton swabs on solid plates media is convenient way of inoculation for beginners. Additionally, it is necessary to procure tools and reagents for AST as quickly as possible on the emergence of AMR bacteria. From these reasons, we prioritized to use domestically produced or stably provided chemicals and tools.\u003c/p\u003e \u003cp\u003eThe usefulness of newly developed method was verified. Some beginners kindly cooperating our experiment could execute all procedures well. When skilled one conduct the experiment, actual working time was really shortened, because of no waiting time to adjust the temperature of medium repeatedly, needed in the original method. It made possible to test more strains and obtain more results of AST of plant pathogenic bacteria. 192 strains of 14 species were used for verification and AMR strains of \u003cem\u003eE. persicina\u003c/em\u003e, \u003cem\u003eB. glumae\u003c/em\u003e and \u003cem\u003eB. plntarii\u003c/em\u003e were found. Reduction of antimicrobial susceptibility was also observed on several strains tested. All strains of \u003cem\u003eB. cepacia\u003c/em\u003e and \u003cem\u003eB. cenocepacia\u003c/em\u003e were insusceptible to all antimicrobials except oxolinic acid. The disk diffusion method suited for this prompt detection, because difference of diameters of inhibition zones were observed enough between sensitive, nonsensitive and low susceptible strains. Compared to MIC methods, the precision of the susceptibility leads by disk diffusion methods are surely limited. However, in real cultivation fields where the concentration of agrochemicals is stipulated, disk diffusion methods give enough useful data by comparison between AMR bacteria and sensitive ones.\u003c/p\u003e \u003cp\u003eWe used paper disks includes antimicrobials at 0, 10, 100, 1000 ppm. Bacterial growth is however naturally different between on nutrient-rich medium and in the natural environment. Further studies are needed on correlation of antimicrobial susceptibility between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein situ\u003c/em\u003e. Only one point to be careful is that antimicrobial susceptibility on medium may not directly affect to the concentration of bactericide splayed in agricultural fields.\u003c/p\u003e \u003cp\u003eSpread and standardization of new method enables comparison and accumulation of the data of antimicrobial susceptibility of plant pathogenic bacteria. These records will help the detection of AMR bacteria and the selection of bactericides in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the project of the Ministry of Agriculture, Forestry and Fisheries of JAPAN (Research program on development of comprehensive regulatory science for providing safe crops, livestock products, and marine products).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflicts of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003eEthical approval\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBauer AW, Kirby WMM, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45: 493-496.\u003c/li\u003e\n \u003cli\u003eCockerill FR, Hindler JA, Wikler MA, Patel JB, Alder J, Powell M, Dudley MN, Swenson JM, Eliopoulos GM, Thomson RB Jr., Ferraro MJ, Traczewski MM, Hardy DJ, Turnidge JD, Hecht DW, Weinstein MP, Zimmer BL (2012) Clinical and laboratory standards institute M02 Performance Standards for Antimicrobial Disk Susceptibility Tests, 11th Edition. Clinical and laboratory standards institute, Wayne, PA.\u003c/li\u003e\n \u003cli\u003eEUCAST Disk Diffusion Test Methodology, Antimicrobial susceptibility testing EUCAST disk diffusion method Version 13.0 (2025) European Committee on Antimicrobial Susceptibility Testing. https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_test_documents/2025_manuals/Manual_v_13.0_EUCAST_Disk_Test_2025.pdf\u003c/li\u003e\n \u003cli\u003eGarrett KA, Nita M, De Wolf ED, Esker PD, Gomez-Montano L, Sparks AH (2021). Plant pathogens as indicators of climate change. Climate change pp. 499-513. Elsevier.\u003c/li\u003e\n \u003cli\u003eGoto M, Takikawa Y (1984) The methods to investigate bacteriological characteristics of plant pathogenic bacteria to identify them (4). (in Japanese). Plant protection 38:479\u0026ndash;484\u003c/li\u003e\n \u003cli\u003eLahlali R, Taoussi M, Laasli SE, Gachara G, Ezzouggari R, Belabess Z, Aberkani K, Assouguem A, Meddich A, Jarroudi ME, Barka EA (2024) Effects of climate change on plant pathogens and host-pathogen interactions. Crop and Environ 3:159-170.\u003c/li\u003e\n \u003cli\u003eMatuschek E, Brown DFJ, Kahlmeter G (2014) Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. Clinical microbiol infection 20: O255-O266.\u003c/li\u003e\n \u003cli\u003eMueller JH, Hinton J (1941) A protein-free medium for primary isolation of the \u003cem\u003eGonococcus\u003c/em\u003e and \u003cem\u003eMeningococcus\u003c/em\u003e. Proc Soc Exp Biol Med 48: 330-333.\u003c/li\u003e\n \u003cli\u003eNishiyama K, Ezuka A (1977) \u003cem\u003ePseudomonas coronafaciens\u003c/em\u003e var. \u003cem\u003eatropurpurea\u003c/em\u003e obtained from diseased leaves of ryegrasses. (in Japanese). Jpn J Phytopathol 43:426\u0026ndash;431\u003c/li\u003e\n \u003cli\u003ePetriccione M, Zampella L, Mastrobuoni F, Scortichini M (2017) Occurrence of copper-resistant \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003esyringae\u003c/em\u003e strains isolated from rain and kiwifruit orchards also infected by \u003cem\u003eP. s.\u003c/em\u003e pv. \u003cem\u003eactinidiae\u003c/em\u003e. Eur J Plant Pathol 149:953\u0026ndash;968\u003c/li\u003e\n \u003cli\u003eTakeuchi T (1995) The manual for antimicrobial susceptibility testing of plant pathogens (21) \u003cem\u003ePseudomonas avenae\u003c/em\u003e. (in Japanese). Plant protection 49:403\u0026ndash;404\u003c/li\u003e\n \u003cli\u003eTsuji M, Kadota I (2020) Identification and phylogenetic analysis of \u003cem\u003eBurkholderia cepacia\u003c/em\u003e complex bacteria isolated from rot of onion bulbs in Tohoku region of Japan. J Gen Plant Pathol 86:376\u0026ndash;386\u003c/li\u003e\n \u003cli\u003eTsuji M, Takikawa Y (2018) \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003ealliifistulosi\u003c/em\u003e pv. nov., the causal agent of bacterial leaf spot of onions. J Gen Plant Pathol 84:343\u0026ndash;358\u003c/li\u003e\n \u003cli\u003eValenzuela M, M\u0026eacute;ndez V, Montenegro I, Besoain X, Seeger M (2019) Streptomycin resistance in \u003cem\u003eClavibacter michiganensis\u003c/em\u003e subsp. \u003cem\u003emichiganensis\u003c/em\u003e strains from Chile is related to an \u003cem\u003erpsL\u003c/em\u003e gene mutation. Plant Pathol 68:426\u0026ndash;433\u003c/li\u003e\n \u003cli\u003eWakimoto S, Mukoo H (1963) Natural occurrences of streptomycin resistant \u003cem\u003eXanthomonas oryzae\u003c/em\u003e, the causal bacteria of leaf blight disease of rice. (in Japanese). Jpn J Phytopathol 28:153\u0026ndash;158\u003c/li\u003e\n \u003cli\u003eYamaguchi S, Iyama K, Tagata K, Natsuaki K, Negishi H, Shinohara H (2014) Bactericide susceptibility of bacterial shot hole bacteria of Peach (mainly \u003cem\u003eXanthomonas arboricola\u003c/em\u003e pv. \u003cem\u003epruni\u003c/em\u003e). J Agric Sci Tokyo Univ Agric 59:74\u0026ndash;80\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 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":"antimicrobial resistance (AMR), antimicrobial susceptibility testing (AST), bacteria, plant pathogen, media, standardization","lastPublishedDoi":"10.21203/rs.3.rs-6241655/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6241655/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial resistant (AMR) bacteria is the common threat in clinical, stockbreeding, aquaculture, and agricultural fields. To prepare for new AMR bacteria, we developed a simple and convenient method for antimicrobial susceptibility testing (AST) of plant pathogenic bacteria. The new recipe for media preparation and bacterial inoculation was proposed with understandable instructions, based on Goto and Takikawa (1984). Nutrient agar with 1% glucose were equivalent to the original medium. Inoculation using cotton swabs allow users to observe clear inhibition circles. The AST method for plant pathogenic bacteria was not standardized so far, but new method enables comparison and accumulation of data.\u003c/p\u003e","manuscriptTitle":"A simple and convenient method for antimicrobial susceptibility testing of plant pathogenic bacteria.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 09:52:37","doi":"10.21203/rs.3.rs-6241655/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-23T04:11:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-23T00:15:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-18T04:49:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of General Plant Pathology","date":"2025-03-17T02:47:25+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":"4e3dbce4-6088-449e-a1a4-09f6ff528fb8","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-23T16:01:16+00:00","versionOfRecord":{"articleIdentity":"rs-6241655","link":"https://doi.org/10.1007/s10327-025-01234-3","journal":{"identity":"journal-of-general-plant-pathology","isVorOnly":false,"title":"Journal of General Plant Pathology"},"publishedOn":"2025-06-18 15:56:55","publishedOnDateReadable":"June 18th, 2025"},"versionCreatedAt":"2025-04-01 09:52:37","video":"","vorDoi":"10.1007/s10327-025-01234-3","vorDoiUrl":"https://doi.org/10.1007/s10327-025-01234-3","workflowStages":[]},"version":"v1","identity":"rs-6241655","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6241655","identity":"rs-6241655","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}