Detection and occurrence of airborne Xanthomonas hortorum pv. carotae, causal agent of carrot bacterial blight, in carrot seed production systems

Detection and occurrence of airborne Xanthomonas hortorum pv. carotae, causal agent of carrot bacterial blight, in carrot seed production systems | 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 Detection and occurrence of airborne Xanthomonas hortorum pv. carotae, causal agent of carrot bacterial blight, in carrot seed production systems Jeness Scott, Jeremiah Dung This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1631313/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacterial blight of carrot, caused by the plant pathogenic bacterium Xanthomonas hortorum pv. carotae ( Xhc ), is an important seedborne disease of carrot. Previous research demonstrated that airborne Xhc could be detected up to 1.6 km downwind of carrot seed fields being harvested, but more information is needed to identify dispersal periods of the pathogen during other stages of carrot seed production. The objectives of this research were to: 1) quantify and characterize temporal dynamics of airborne Xhc during carrot seed production; and 2) identify weather factors that contribute to airborne Xhc levels in carrot seed fields. During the 2018–2019 season, air samples were collected on a total of 273 and 289 days in two fields and Xhc was detected on 223 (81.7%) and 245 (84.8%) of sampling days. On average, 2.6 x 10 3 and 7.0 x 10 3 Xhc /day were detected. Principle component analysis identified three components associated with airborne Xhc levels. Temperature variables (daily mean, maximum, and minimum air and soil temperatures) accounted for 53.2% of the total variance. The second component was associated with wind (wind speed, direction, and run) and 19.1% of the total variance. Factors related to moisture (daily precipitation, relative humidity, and dew point) were associated with the third component and accounted for 13.0% of the total variance. Together, these data can be used to build predictive models to inform growers of inoculum risk and improve control through better-timed bactericide applications, modified cultural practices, and increased knowledge of bacterial blight epidemiology in carrot seed crops. aerobiology bacteria epidemiology epiphyte phytopathology plant disease Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction The state of Oregon, and specifically central Oregon, is a major global producer of hybrid carrot ( Daucus carota L.) seed (Hart and Butler 2004 ). From 2010 to 2020, central Oregon grew an average of 3,715 acres of hybrid carrot seed annually (Anonymous 2020 ) with an average estimated value of over $ 16.2 million annually (Butler and Simmons 2013 ). Carrot seed is also an important crop in the U.S. Pacific Northwest states of Washington and Idaho. The majority of the seed produced in the Pacific Northwest is used to produce carrot root crops in the United States, which were grown on over 28,000 hectares and valued at over $ 765 million in 2020 (U.S. Department of Agriculture National Agricultural Statistics 2021 ) Hybrid carrot seed production utilizes in-bred parental lines that are planted in alternating beds within a field, with one parental line serving as the pollen donor and the other, a male-sterile line, producing the seed that is harvested (Rubatzky et al. 1999 ). Carrots are a biennial plant and require a vernalization or a chilling period in order to bolt, flower, and produce seed. Most hybrid carrot seed crops are direct seeded, whereby carrot seeds of in-bred parental lines are planted into the field in early fall (usually August) and overwinter in the field as young plants. Pollen donor lines are destroyed after pollination by rolling or chopping to prevent off-types in the final seed lot. When the seed of the male-sterile lines reach a desired moisture the plants are cut, windrowed, and dried in the field prior to combining. The hybrid seed crop is typically harvested in September or October, resulting in an overlapping period between current and future seed crops. Bacterial blight of carrot, caused by the plant pathogenic bacterium Xanthomonas hortorum pv. carotae , is a common seedborne disease of carrot (Gilbertson 2002 ). Symptoms of bacterial blight include small, irregular, chlorotic areas on leaves, petioles, and stems that can manifest into water-soaked, necrotic lesions and blighted leaves. Floral infections can result in blighted umbels, reduced seed yield, reduced germination rates of harvested seed, and infested seed lots. Individual seeds can be infested at levels reaching 10 7 CFU/seed (Scott and Dung 2020 ). When carrot seed that is heavily infested with Xhc is planted, the Xhc present on the seed can contribute to the development of bacterial blight of carrot within the carrot crop. The seedborne nature of the disease is of concern for both carrot seed and carrot root production wherever these crops are grown (du Toit et al. 2005 ; Scott and Dung 2020 ). Plant surfaces can serve as reservoirs for a wide variety of microorganisms, including both phytopathogenic and nonpathogenic bacteria (Hirano and Upper 1983 ). Epiphytic bacteria, defined as bacteria which can survive and multiply on plant surfaces (Leben 1965 ), are frequently found on the surfaces of leaves, stems, buds and flower parts. Several species of Xanthomonas , including Xhc , are epiphytic in nature (Bernal and Berger 1996 ; Dia et al. 2022 ; du Toit et al. 2005 ; Gilbertson 2002 ). Xhc can persist and reproduce on carrot leaves epiphytically and asymptomatically, only causing disease symptoms when large populations are attained on foliage (> 10 6 CFU/g leaf tissue) (Gilbertson 2002 ; Umesh et al. 1998 ). Epiphytic bacteria, including species of Xanthomonas , are subject to aerial dispersal (Bernal and Berger 1996 ; Bock et al. 2005 ; Kuan et al. 1986 ; McInnes 1988 ). In the case of Xhc , the bacterium has been documented in air sampled during seed harvest, demonstrating the potential for airborne movement of the pathogen (du Toit et al. 2005 ). Using truck-mounted air samplers and a semi-selective medium, du Toit et al. ( 2005 ) were able to detect viable, airborne X. hortorum pv. carotae up to 1.6 km downwind of carrot seed crops being actively harvested. Their study provided some important initial insights into a critical period of Xhc epidemiology and dispersal associated with harvesting practices, but more information is needed to identify dispersal periods of the pathogen during other times of the year. The objectives of this research were to: 1) quantify and characterize temporal dynamics of airborne X. hortorum pv. carotae throughout the carrot seed production season and 2) identify weather factors that contribute to airborne X. hortorum pv. carotae in carrot seed fields. 2 Methods 2.1 Air sampling and Xhc quantification Hirst-type (Hirst 1952 ) Burkard 7-day volumetric spore samplers (Burkard Scientific Ltd., Uxbridge, Middlesex, UK) were used to sample air continuously in two carrot seed-to-seed fields (Field A and Field B), which were located approximately 6.4 km apart and intended for harvest in 2019. The volumetric spore samplers were placed at commercial carrot seed production fields either 2 (Field A) or 3 m (Field B) from the field edge and the sampling orifice positioned 60 cm above the ground. The spore samplers were calibrated weekly to maintain an inflow of 10 liters of air per minute. Airborne particulates were impacted onto clear Melinex tape coated with silicone grease (Ted Brown Associates, Los Altos, CA). Air samples were collected from the beginning of stand establishment (August 29, 2018) through November 30, 2018 and from April 12, 2019 (Field A) or April 1, 2019 (Field B) through harvest (October 7, 2019 and October 14, 2019 for Field A and Field B, respectively). Fields were not sampled from December 2018 through March 2019 due to the inability of accessing and maintaining the spore samplers located at each field site. Tape samples from Burkard spore traps were separated into daily segments for DNA extraction. To each tube containing half of one daily segment, 25 µl volume of acid washed glass beads 0.1 mm in diameter (Scientific Industries, Inc., Bohemia, New York, USA) and 150 µl InstaGene™ Matrix (Bio-Rad, Hercules, CA, USA) were added. The manufacturer’s instructions for DNA isolation from bacterial cells were followed, except that the first and second vortex steps (10 s on high) were each replaced with an agitation step of 30s at 6.0 m/s in a FastPrep machine (MP Biomedical LLC, Irvine, CA, USA). Equal volumes from both tubes corresponding to a full daily sample were pooled. The DNA of Xhc was quantified using 2 µl of this extraction as the template in a previously published quantitative polymerase chain reaction (qPCR) assay with a fluorescently labeled oligo probe specific to Xhc as described by Temple et al. ( 2013 ). Cycle threshold values obtained from the qPCR assay were converted to Xhc genomes/day based on a standard curve ranging from 10.1 fg DNA (2 genomes per reaction) to 10.1 ng DNA (2,000,000 genomes per reaction) (Kimbrel et al. 2011 ). 2.2 Assays for the quantification of Xhc on field leaf and harvested seed samples Leaf samples (100 leaves) were randomly collected from different plants on November 16, 2018 and on May 24, June 7, and July 3, 2019 to quantify Xhc populations in each field. Leaf samples were cut into 1–2 cm 2 pieces and subjected to a phosphate buffer wash assay (Scott and Dung 2020 ). One ml aliquots were sampled from each leaf wash assay and treated with a DNA binding dye propidium monoazide (PMAxx™, Biotium, Fremont, CA, USA) (Temple et al. 2013 ). Following the PMAxx™ treatment, the samples were centrifuged for 3 min at 12,500 rcf after which the supernatant was discarded. A preparation of genomic DNA was obtained from this pelleted material through an InstaGene™ Matrix extraction (150 µl) according to the manufacturer’s instructions. Two µl of this DNA preparation was used as the template in the Xhc specific qPCR assay described above. The leaf tissue was retained from the leaf wash assay, dried at 35°C for 5–7 days and subsequently weighed. The level of Xhc in the field was calculated as Xhc per gram leaf tissue dry weight based on the leaves that were sampled at each time point. Similarly, three 10-g samples of harvested and conditioned seed from each field were also subjected to a seed wash in a phosphaete buffer, followed by PMAxx™ treatment, DNA extraction with InstaGene™ Matrix (200 ul), and qPCR quantification of Xhc as described by Temple et al. ( 2013 ). 2.3 Identifying weather factors associated with airborne Xhc To identify factors correlated with airborne levels of Xhc , principal component analyses (Jolliffe 1973 ) was conducted using weather data collected by the MRSO AgriMet Weather station located at the Central Oregon Agricultural Research and Extension Center in Madras, OR. Several meteorological variables were selected to investigate potential associations between environmental conditions and airborne Xhc in each of the carrot seed production fields, including daily minimum, maximum, and mean air and soil temperatures, solar irradiation, mean relative humidity, mean dew point, daily precipitation, wind speed, wind direction, and wind run. Data for each field were analyzed and visualized separately using PROC PRINCOMP in SAS ver. 9.2 (SAS Institute, Cary, NC). Scree plots of eigenvalues were used to help identify meaningful components (Cattell 1966 ) and components with eigenvalues > 1 were considered informative (Kaiser 1960 ). 3 Results 3.1 Air sampling and Xhc quantification Air samples were collected on a total of 273 and 289 days in fields A and B, respectively, and Xhc was detected on 223 days (81.7%) and 245 (84.8%) of those days (Fig. 1 ). The total number of Xhc detected during the entire sampling period was 7.0 x 10 5 in field A and 2.0 x 10 6 in field B. On average, 2.6 x 10 3 and 7.0 x 10 3 Xhc /day were detected in field A and B, respectively, with the maximum number of Xhc detected on a given day being 1.6 x 10 5 (field A) and 7.3 x 10 5 (field B). Over the course of the experiment, the mean number of Xhc genomes detected were 0.179 and 0.478 genomes/liter of air. In field A, Xhc was detected in the highest total numbers during April and May 2019, followed by September 2018 and July 2019; in contrast, the highest amounts of Xhc were observed in Field B in the months of November 2018 and September and October 2019 (Fig. 2 ). The months of May, July, and September 2019 had the most detection events in field A (97, 87, and 87% of days, respectively), while in field B the pathogen was detected on 100, 97, and 94% of days in September, August, and May 2019 (Fig. 3 ). 3.2 Assays for the quantification of Xhc on field leaf and harvested seed samples Leaf samples collected November 2018 from fields A and B showed Xhc populations were 2.7 x 10 3 and 4.3 x 10 3 bacteria/g leaf tissue, respectively, demonstrating that considerable Xhc populations can be established on seed-to-seed carrot fields prior to overwintering. Xhc was not detected in leaf samples collected from field A in May 2019, but the pathogen was recovered in June (1.7 x 10 3 bacteria/g) and July (4.0 x 10 3 bacteria/g). The pathogen was recovered at high levels from leaf samples collected from field B in May (1.2 x 10 7 bacteria/g), June (1.4 x 10 8 bacteria/g), and July (1.1 x 10 8 bacteria/g) of 2019. Xhc levels in the harvested seed were 2.7 x 10 5 and 3.5 x 10 6 in fields A and B, respectively. 3.3 Identifying weather factors associated with airborne Xhc For each field, principle component analysis identified three components with eigenvalues greater than one (Table 1 ) and the scree plot also indicated that only the first three components were informative (data not shown). The first component was associated with temperature variables (daily mean, maximum, and minimum air and soil temperatures) and accounted for approximately 53% of the total variance in each field. The second component was associated with wind (wind speed, direction, and run) and approximately 19% of the total variance. Factors related to moisture (daily precipitation, relative humidity, and dew point) were associated with the third component and accounted for approximately 13% of the total variance. Results were consistent for both fields, so data were combined for visualization in biplots (Fig. 4 ). Discussion The first objective of this research was to quantify and characterize the temporal dynamics of airborne X . hortorum pv. carotae , the causal agent of bacterial blight of carrot. In this study, Xhc was detected in air samples at a high frequency in the two fields sampled during the carrot seed cropping season (81.7 to and 84.8% of sampling days), suggesting a high potential for aerial dispersal of the pathogen within carrot seed production systems. The amounts of airborne Xhc detected in each field (2.6 to 7.0 x 10 3 Xhc /day) were similar to the average bacterial immigration estimates of 10 4 cells/month described by Lindow ( 1996 ). Plant canopies can be strong sources of airborne bacteria (Lindemann et al. 1982 ; Lindemann and Upper 1985 ), including epiphytic bacteria like Xanthomonas , and it is likely that the high frequency of Xhc detection was due in part because of the close proximity of the air samplers to the crop canopy (2 to 3 m); however, the relative contribution of carrot debris or soil as sources of airborne Xhc was not determined. Sampling was not performed between December and March, so the frequency and amount of Xhc in the air during the overwintering and vernalization period is still not known. The protocols used in this study relied on molecular methods to quantify Xhc genomes and did not quantify viable bacteria using a semi-selective medium. Quantitative PCR used in this study for several reasons: 1) the uncertainty of bacterial viability on spore trap tape during the one week sampling interval; 2) the large number and amount of non-target organisms that are also able to grow on various semi-selective media for Xhc ; and 3) increased sensitivity and specificity of qPCR compared to plating, especially in the presence of non-target organisms. Regardless, others have isolated viable Xhc cultures in air samples (du Toit et al. 2005 ); the authors have similarly collected Xhc isolates from air samples that are pathogenic on carrots ( data unpublished ). More research is needed to determine the relative proportions of viable and non-viable Xhc cells in aerosols and airborne debris. Carrot seed is a biennial crop, with seed-to-seed production fields are typically planted in August and harvested in September or October of the following year. Harvesting practices often generate plumes of dust and plant material that can contain viable bacteria (Lighthart 1984 ), including Xhc (du Toit et al. 2005 ), which can potentially be dispersed and deposited in newly planted fields. We detected large numbers of airborne Xhc in September and October during both years of the study, which were likely due to fields being harvested during these months. Additionally, leaf samples taken in November 2018 and in other studies (du Toit et al. 2005 ) show that considerable Xhc populations can become established on seed-to-seed carrot fields prior to winter. These results lend further support for a green bridge effect that contributes to the annual and seemingly endemic presence of Xhc in the region. There are other cultural practices associated with carrot seed production that have the potential to generate airborne Xhc , including plant thinning, mechanical weed management, applying pesticides, and the destruction of pollen donor lines prior to harvest. McInnes et al. (McInnes 1988 ) observed more significantly more X. campestris pv. vesicatoria in the air above tomato transplants after clipping and harvest. In carrot seed production, some of these activities (e.g. mechanical weed management, pesticide applications) occur multiple times throughout the year, while other practices only occur once per season (e.g. plant thinning, destruction of pollen donor lines), and the exact timing of these activities will vary depending on cultivar, weather conditions, and logistics. In this study, we detected airborne Xhc during the majority of sampling days and on days when field activities were not occurring, including immediately after seedling emergence, suggesting that the pathogen was either originating from neighboring fields or there are other mechanisms for Xhc to become airborne from carrot seed crop canopies. Another objective of this research was to identify weather factors that contribute to airborne X. hortorum pv. carotae . In this study, principle component analysis identified temperature, wind (speed, direction and run) and factors related to moisture as the primary factors accounting for the variance. Previous studies have also demonstrated that the airborne dispersal of bacteria in aerosols is dependent on temperature, wind, and moisture. Lindemann and Upper (Lindemann and Upper 1985 ) reported that upward aerial fluxes of bacteria from bean phyllospheres occurred during the middle of dry, warm days and not in the evening or when moisture was present on the leaves in the form of rain or dew. In their study, bacterial numbers were stronger after precipitation events compared to when soils were dry, and wind speed was positively correlated with aerosol strength. In carrot seed crops, strong winds may promote the release and dispersal of Xhc leaf-to-leaf contact within and between individual plant canopies, which could potentially dislodge bacterial cells from epidermal surfaces or biofilms (Morris and Monier 2003 ). The central Oregon growing season is typified by sunny and dry days, windy afternoons, and cool nights, which may contribute towards the production of aerial fluxes of Xhc in and around carrot seed fields. Diurnal fluctuations of bacterial aerosols are known to occur in climates similar to the high desert of central Oregon, with greater upward fluxes occurring during the afternoon on warm sunny days and after rain or irrigation events (Lindemann and Upper 1985 ); it would be reasonable to hypothesize that similar temporal patterns might occur with Xhc aerosols from carrot seed crop canopies. Declarations Acknowledgements The researchers thank the grower-cooperators for allowing this work to be performed in their fields. The technical support of Tiffany Belvoir, Shaelynn Downing, and Kelley Duggan was greatly appreciated. Funding: This work was supported by the USDA National Institute of Food and Agriculture, through the Specialty Crops Research Initiative Project grant no. 2020-51181-32154 and the Western Integrated Pest Management Center project ID 1616. Additional funding was also received from the Agricultural Research Foundation. Author Contributions: All authors contributed to the study conception and design. Material preparation and data collection were performed by Jeness Scott and Jeremiah Dung. Data analysis was performed by Jeremiah Dung. The first draft of the manuscript was written by Jeremiah Dung and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Conflicts of interest/Competing interests: The authors have no conflicts of interest to declare that are relevant to the content of this article. References Anonymous. (2020). North Unit Irrigation District 2020 Crop Report . Madras, OR. Bernal, R. F., & Berger, R. D. (1996). The spread of epiphytic populations of Xanthomonas campestris pv. vesicatoria on pepper in the field. Journal of Phytopathology , 144 (9–10), 479–484. Bock, C. H., Parker, P. E., & Gottwald, T. R. (2005). 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Carrots and Related Vegetable Umbelliferae (Vol. 10). New York. Scott, J. C., & Dung, J. K. (2020). Distribution of Xanthomonas hortorum pv. carotae populations in naturally-infested carrot seed lots. Plant Disease , in print. https://doi.org/10.1094/PDIS-12-19-2674-RE. https://doi.org/10.1094/PDIS-12-19-2674-RE Temple, T. N., du Toit, L. J., Derie, M. L., & Johnson, K. B. (2013). Quantitative molecular detection of Xanthomonas hortorum pv. carotae in carrot seed before and after hot-water treatment. Plant Disease, 97 (12), 1585–1592. Umesh, K. C., Davis, R. M., & Gilbertson, R. L. (1998). Seed contamination thresholds for development of carrot bacterial blight caused by Xanthomonas campestris pv. carotae . Plant Disease, 82 (11), 1271–1275. U.S. Department of Agriculture National Agricultural Statistics, U. (2021). Vegetables 2020 Summary . https://downloads.usda.library.cornell.edu/usda-esmis/files/02870v86p/j6731x86f/9306tr664/vegean21.pdf . Accessed 19 April 2022 Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Posted Version 1 posted 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-1631313","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":106587651,"identity":"b8fd721d-784d-49f3-bf7a-c10860551c1f","order_by":0,"name":"Jeness Scott","email":"","orcid":"","institution":"Oregon State University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jeness","middleName":"","lastName":"Scott","suffix":""},{"id":106587654,"identity":"f895069c-9c00-4795-b11b-209670b5f454","order_by":1,"name":"Jeremiah Dung","email":"data:image/png;base64,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","orcid":"","institution":"Oregon State University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jeremiah","middleName":"","lastName":"Dung","suffix":""}],"badges":[],"createdAt":"2022-05-06 22:44:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1631313/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1631313/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":21677334,"identity":"27e2e129-0e32-4e3c-8e4f-8a5e1c089258","added_by":"auto","created_at":"2022-05-19 17:36:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":141602,"visible":true,"origin":"","legend":"\u003cp\u003eDaily levels of airborne \u003cem\u003eXanthomonas\u003c/em\u003e \u003cem\u003ehortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e bacteria captured in carrot seed field A (top) and carrot seed field B (bottom) using Burkard air samplers; fields were not sampled from December 2018 through March 2019\u003c/p\u003e\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1631313/v1/c03a62418a99e03f3fc4e9f3.png"},{"id":21676377,"identity":"71ec5366-5730-401d-803c-95e0c7481140","added_by":"auto","created_at":"2022-05-19 17:26:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116102,"visible":true,"origin":"","legend":"\u003cp\u003eMean daily (lines) and total monthly (bars) \u003cem\u003eXhc\u003c/em\u003e genomes detected during the study\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1631313/v1/ffb71b65da1d0290317193d9.png"},{"id":21676376,"identity":"cfe2b2d3-e663-4379-a8d8-9065d9a7f9c3","added_by":"auto","created_at":"2022-05-19 17:26:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63094,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency of daily \u003cem\u003eXhc\u003c/em\u003e detection during each month of the study\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1631313/v1/1b3b3b5b67ef7cfeb4ed9c84.png"},{"id":21676886,"identity":"57f47c74-341b-490a-86dc-f5006295c68e","added_by":"auto","created_at":"2022-05-19 17:31:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":110300,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis of weather variables for airborne \u003cem\u003eXanthomonas\u003c/em\u003e \u003cem\u003ehortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e in Field A (a and b) and Field B (c and d) shown as biplots\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-1631313/v1/ab1f0be405e902ef1aa33668.png"},{"id":31981229,"identity":"335f44f6-3bfe-4a69-b79f-9f3906e96a94","added_by":"auto","created_at":"2023-01-24 06:59:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1090539,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1631313/v1/c40fe264-6f0a-476f-bd4d-bc2d8fd6b34b.pdf"},{"id":21676373,"identity":"e482eefc-de2e-456c-80a9-e0349de491fd","added_by":"auto","created_at":"2022-05-19 17:26:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15328,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-1631313/v1/8d53be390899a2cc5ab9be77.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Detection and occurrence of airborne Xanthomonas hortorum pv. carotae, causal agent of carrot bacterial blight, in carrot seed production systems","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe state of Oregon, and specifically central Oregon, is a major global producer of hybrid carrot (\u003cem\u003eDaucus carota\u003c/em\u003e L.) seed (Hart and Butler \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). From 2010 to 2020, central Oregon grew an average of 3,715 acres of hybrid carrot seed annually (Anonymous \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with an average estimated value of over \u003cspan\u003e$\u003c/span\u003e16.2\u0026nbsp;million annually (Butler and Simmons \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Carrot seed is also an important crop in the U.S. Pacific Northwest states of Washington and Idaho. The majority of the seed produced in the Pacific Northwest is used to produce carrot root crops in the United States, which were grown on over 28,000 hectares and valued at over \u003cspan\u003e$\u003c/span\u003e765\u0026nbsp;million in 2020 (U.S. Department of Agriculture National Agricultural Statistics \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eHybrid carrot seed production utilizes in-bred parental lines that are planted in alternating beds within a field, with one parental line serving as the pollen donor and the other, a male-sterile line, producing the seed that is harvested (Rubatzky et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Carrots are a biennial plant and require a vernalization or a chilling period in order to bolt, flower, and produce seed. Most hybrid carrot seed crops are direct seeded, whereby carrot seeds of in-bred parental lines are planted into the field in early fall (usually August) and overwinter in the field as young plants. Pollen donor lines are destroyed after pollination by rolling or chopping to prevent off-types in the final seed lot. When the seed of the male-sterile lines reach a desired moisture the plants are cut, windrowed, and dried in the field prior to combining. The hybrid seed crop is typically harvested in September or October, resulting in an overlapping period between current and future seed crops.\u003c/p\u003e \u003cp\u003eBacterial blight of carrot, caused by the plant pathogenic bacterium \u003cem\u003eXanthomonas hortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e, is a common seedborne disease of carrot (Gilbertson \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Symptoms of bacterial blight include small, irregular, chlorotic areas on leaves, petioles, and stems that can manifest into water-soaked, necrotic lesions and blighted leaves. Floral infections can result in blighted umbels, reduced seed yield, reduced germination rates of harvested seed, and infested seed lots. Individual seeds can be infested at levels reaching 10\u003csup\u003e7\u003c/sup\u003e CFU/seed (Scott and Dung \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). When carrot seed that is heavily infested with \u003cem\u003eXhc\u003c/em\u003e is planted, the \u003cem\u003eXhc\u003c/em\u003e present on the seed can contribute to the development of bacterial blight of carrot within the carrot crop. The seedborne nature of the disease is of concern for both carrot seed and carrot root production wherever these crops are grown (du Toit et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Scott and Dung \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlant surfaces can serve as reservoirs for a wide variety of microorganisms, including both phytopathogenic and nonpathogenic bacteria (Hirano and Upper \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Epiphytic bacteria, defined as bacteria which can survive and multiply on plant surfaces (Leben \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1965\u003c/span\u003e), are frequently found on the surfaces of leaves, stems, buds and flower parts. Several species of \u003cem\u003eXanthomonas\u003c/em\u003e, including \u003cem\u003eXhc\u003c/em\u003e, are epiphytic in nature (Bernal and Berger \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Dia et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; du Toit et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gilbertson \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). \u003cem\u003eXhc\u003c/em\u003e can persist and reproduce on carrot leaves epiphytically and asymptomatically, only causing disease symptoms when large populations are attained on foliage (\u0026gt;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e CFU/g leaf tissue) (Gilbertson \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Umesh et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEpiphytic bacteria, including species of \u003cem\u003eXanthomonas\u003c/em\u003e, are subject to aerial dispersal (Bernal and Berger \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Bock et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kuan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; McInnes \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). In the case of \u003cem\u003eXhc\u003c/em\u003e, the bacterium has been documented in air sampled during seed harvest, demonstrating the potential for airborne movement of the pathogen (du Toit et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Using truck-mounted air samplers and a semi-selective medium, du Toit et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) were able to detect viable, airborne \u003cem\u003eX. hortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e up to 1.6 km downwind of carrot seed crops being actively harvested. Their study provided some important initial insights into a critical period of \u003cem\u003eXhc\u003c/em\u003e epidemiology and dispersal associated with harvesting practices, but more information is needed to identify dispersal periods of the pathogen during other times of the year. The objectives of this research were to: 1) quantify and characterize temporal dynamics of airborne \u003cem\u003eX. hortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e throughout the carrot seed production season and 2) identify weather factors that contribute to airborne \u003cem\u003eX. hortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e in carrot seed fields.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Air sampling and \u003cem\u003eXhc\u003c/em\u003e quantification\u003c/h2\u003e \u003cp\u003eHirst-type (Hirst \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1952\u003c/span\u003e) Burkard 7-day volumetric spore samplers (Burkard Scientific Ltd., Uxbridge, Middlesex, UK) were used to sample air continuously in two carrot seed-to-seed fields (Field A and Field B), which were located approximately 6.4 km apart and intended for harvest in 2019. The volumetric spore samplers were placed at commercial carrot seed production fields either 2 (Field A) or 3 m (Field B) from the field edge and the sampling orifice positioned 60 cm above the ground. The spore samplers were calibrated weekly to maintain an inflow of 10 liters of air per minute. Airborne particulates were impacted onto clear Melinex tape coated with silicone grease (Ted Brown Associates, Los Altos, CA). Air samples were collected from the beginning of stand establishment (August 29, 2018) through November 30, 2018 and from April 12, 2019 (Field A) or April 1, 2019 (Field B) through harvest (October 7, 2019 and October 14, 2019 for Field A and Field B, respectively). Fields were not sampled from December 2018 through March 2019 due to the inability of accessing and maintaining the spore samplers located at each field site.\u003c/p\u003e \u003cp\u003eTape samples from Burkard spore traps were separated into daily segments for DNA extraction. To each tube containing half of one daily segment, 25 \u0026micro;l volume of acid washed glass beads 0.1 mm in diameter (Scientific Industries, Inc., Bohemia, New York, USA) and 150 \u0026micro;l InstaGene\u0026trade; Matrix (Bio-Rad, Hercules, CA, USA) were added. The manufacturer\u0026rsquo;s instructions for DNA isolation from bacterial cells were followed, except that the first and second vortex steps (10 s on high) were each replaced with an agitation step of 30s at 6.0 m/s in a FastPrep machine (MP Biomedical LLC, Irvine, CA, USA). Equal volumes from both tubes corresponding to a full daily sample were pooled. The DNA of \u003cem\u003eXhc\u003c/em\u003e was quantified using 2 \u0026micro;l of this extraction as the template in a previously published quantitative polymerase chain reaction (qPCR) assay with a fluorescently labeled oligo probe specific to \u003cem\u003eXhc\u003c/em\u003e as described by Temple et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Cycle threshold values obtained from the qPCR assay were converted to \u003cem\u003eXhc\u003c/em\u003e genomes/day based on a standard curve ranging from 10.1 fg DNA (2 genomes per reaction) to 10.1 ng DNA (2,000,000 genomes per reaction) (Kimbrel et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Assays for the quantification of \u003cem\u003eXhc\u003c/em\u003e on field leaf and harvested seed samples\u003c/h2\u003e \u003cp\u003eLeaf samples (100 leaves) were randomly collected from different plants on November 16, 2018 and on May 24, June 7, and July 3, 2019 to quantify \u003cem\u003eXhc\u003c/em\u003e populations in each field. Leaf samples were cut into 1\u0026ndash;2 cm\u003csup\u003e2\u003c/sup\u003e pieces and subjected to a phosphate buffer wash assay (Scott and Dung \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). One ml aliquots were sampled from each leaf wash assay and treated with a DNA binding dye propidium monoazide (PMAxx\u0026trade;, Biotium, Fremont, CA, USA) (Temple et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Following the PMAxx\u0026trade; treatment, the samples were centrifuged for 3 min at 12,500 rcf after which the supernatant was discarded. A preparation of genomic DNA was obtained from this pelleted material through an InstaGene\u0026trade; Matrix extraction (150 \u0026micro;l) according to the manufacturer\u0026rsquo;s instructions. Two \u0026micro;l of this DNA preparation was used as the template in the Xhc specific qPCR assay described above. The leaf tissue was retained from the leaf wash assay, dried at 35\u0026deg;C for 5\u0026ndash;7 days and subsequently weighed. The level of Xhc in the field was calculated as Xhc per gram leaf tissue dry weight based on the leaves that were sampled at each time point. Similarly, three 10-g samples of harvested and conditioned seed from each field were also subjected to a seed wash in a phosphaete buffer, followed by PMAxx\u0026trade; treatment, DNA extraction with InstaGene\u0026trade; Matrix (200 ul), and qPCR quantification of \u003cem\u003eXhc\u003c/em\u003e as described by Temple et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Identifying weather factors associated with airborne \u003cem\u003eXhc\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo identify factors correlated with airborne levels of \u003cem\u003eXhc\u003c/em\u003e, principal component analyses (Jolliffe \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1973\u003c/span\u003e) was conducted using weather data collected by the MRSO AgriMet Weather station located at the Central Oregon Agricultural Research and Extension Center in Madras, OR. Several meteorological variables were selected to investigate potential associations between environmental conditions and airborne \u003cem\u003eXhc\u003c/em\u003e in each of the carrot seed production fields, including daily minimum, maximum, and mean air and soil temperatures, solar irradiation, mean relative humidity, mean dew point, daily precipitation, wind speed, wind direction, and wind run. Data for each field were analyzed and visualized separately using PROC PRINCOMP in SAS ver. 9.2 (SAS Institute, Cary, NC). Scree plots of eigenvalues were used to help identify meaningful components (Cattell \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1966\u003c/span\u003e) and components with eigenvalues\u0026thinsp;\u0026gt;\u0026thinsp;1 were considered informative (Kaiser \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1960\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Air sampling and \u003cem\u003eXhc\u003c/em\u003e quantification\u003c/h2\u003e \u003cp\u003eAir samples were collected on a total of 273 and 289 days in fields A and B, respectively, and \u003cem\u003eXhc\u003c/em\u003e was detected on 223 days (81.7%) and 245 (84.8%) of those days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The total number of \u003cem\u003eXhc\u003c/em\u003e detected during the entire sampling period was 7.0 x 10\u003csup\u003e5\u003c/sup\u003e in field A and 2.0 x 10\u003csup\u003e6\u003c/sup\u003e in field B. On average, 2.6 x 10\u003csup\u003e3\u003c/sup\u003e and 7.0 x 10\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eXhc\u003c/em\u003e/day were detected in field A and B, respectively, with the maximum number of \u003cem\u003eXhc\u003c/em\u003e detected on a given day being 1.6 x 10\u003csup\u003e5\u003c/sup\u003e (field A) and 7.3 x 10\u003csup\u003e5\u003c/sup\u003e (field B). Over the course of the experiment, the mean number of \u003cem\u003eXhc\u003c/em\u003e genomes detected were 0.179 and 0.478 genomes/liter of air.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn field A, \u003cem\u003eXhc\u003c/em\u003e was detected in the highest total numbers during April and May 2019, followed by September 2018 and July 2019; in contrast, the highest amounts of \u003cem\u003eXhc\u003c/em\u003e were observed in Field B in the months of November 2018 and September and October 2019 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The months of May, July, and September 2019 had the most detection events in field A (97, 87, and 87% of days, respectively), while in field B the pathogen was detected on 100, 97, and 94% of days in September, August, and May 2019 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Assays for the quantification of \u003cem\u003eXhc\u003c/em\u003e on field leaf and harvested seed samples\u003c/h2\u003e \u003cp\u003eLeaf samples collected November 2018 from fields A and B showed \u003cem\u003eXhc\u003c/em\u003e populations were 2.7 x 10\u003csup\u003e3\u003c/sup\u003e and 4.3 x 10\u003csup\u003e3\u003c/sup\u003e bacteria/g leaf tissue, respectively, demonstrating that considerable \u003cem\u003eXhc\u003c/em\u003e populations can be established on seed-to-seed carrot fields prior to overwintering. \u003cem\u003eXhc\u003c/em\u003e was not detected in leaf samples collected from field A in May 2019, but the pathogen was recovered in June (1.7 x 10\u003csup\u003e3\u003c/sup\u003e bacteria/g) and July (4.0 x 10\u003csup\u003e3\u003c/sup\u003e bacteria/g). The pathogen was recovered at high levels from leaf samples collected from field B in May (1.2 x 10\u003csup\u003e7\u003c/sup\u003e bacteria/g), June (1.4 x 10\u003csup\u003e8\u003c/sup\u003e bacteria/g), and July (1.1 x 10\u003csup\u003e8\u003c/sup\u003e bacteria/g) of 2019. \u003cem\u003eXhc\u003c/em\u003e levels in the harvested seed were 2.7 x 10\u003csup\u003e5\u003c/sup\u003e and 3.5 x 10\u003csup\u003e6\u003c/sup\u003e in fields A and B, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Identifying weather factors associated with airborne \u003cem\u003eXhc\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eFor each field, principle component analysis identified three components with eigenvalues greater than one (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the scree plot also indicated that only the first three components were informative (data not shown). The first component was associated with temperature variables (daily mean, maximum, and minimum air and soil temperatures) and accounted for approximately 53% of the total variance in each field. The second component was associated with wind (wind speed, direction, and run) and approximately 19% of the total variance. Factors related to moisture (daily precipitation, relative humidity, and dew point) were associated with the third component and accounted for approximately 13% of the total variance. Results were consistent for both fields, so data were combined for visualization in biplots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003eThe first objective of this research was to quantify and characterize the temporal dynamics of airborne \u003cem\u003eX\u003c/em\u003e. \u003cem\u003ehortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e, the causal agent of bacterial blight of carrot. In this study, \u003cem\u003eXhc\u003c/em\u003e was detected in air samples at a high frequency in the two fields sampled during the carrot seed cropping season (81.7 to and 84.8% of sampling days), suggesting a high potential for aerial dispersal of the pathogen within carrot seed production systems. The amounts of airborne \u003cem\u003eXhc\u003c/em\u003e detected in each field (2.6 to 7.0 x 10\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eXhc\u003c/em\u003e/day) were similar to the average bacterial immigration estimates of 10\u003csup\u003e4\u003c/sup\u003e cells/month described by Lindow (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlant canopies can be strong sources of airborne bacteria (Lindemann et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Lindemann and Upper \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), including epiphytic bacteria like \u003cem\u003eXanthomonas\u003c/em\u003e, and it is likely that the high frequency of \u003cem\u003eXhc\u003c/em\u003e detection was due in part because of the close proximity of the air samplers to the crop canopy (2 to 3 m); however, the relative contribution of carrot debris or soil as sources of airborne \u003cem\u003eXhc\u003c/em\u003e was not determined. Sampling was not performed between December and March, so the frequency and amount of \u003cem\u003eXhc\u003c/em\u003e in the air during the overwintering and vernalization period is still not known.\u003c/p\u003e \u003cp\u003eThe protocols used in this study relied on molecular methods to quantify \u003cem\u003eXhc\u003c/em\u003e genomes and did not quantify viable bacteria using a semi-selective medium. Quantitative PCR used in this study for several reasons: 1) the uncertainty of bacterial viability on spore trap tape during the one week sampling interval; 2) the large number and amount of non-target organisms that are also able to grow on various semi-selective media for \u003cem\u003eXhc\u003c/em\u003e; and 3) increased sensitivity and specificity of qPCR compared to plating, especially in the presence of non-target organisms. Regardless, others have isolated viable \u003cem\u003eXhc\u003c/em\u003e cultures in air samples (du Toit et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e); the authors have similarly collected \u003cem\u003eXhc\u003c/em\u003e isolates from air samples that are pathogenic on carrots (\u003cem\u003edata unpublished\u003c/em\u003e). More research is needed to determine the relative proportions of viable and non-viable \u003cem\u003eXhc\u003c/em\u003e cells in aerosols and airborne debris.\u003c/p\u003e \u003cp\u003eCarrot seed is a biennial crop, with seed-to-seed production fields are typically planted in August and harvested in September or October of the following year. Harvesting practices often generate plumes of dust and plant material that can contain viable bacteria (Lighthart \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), including \u003cem\u003eXhc\u003c/em\u003e (du Toit et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), which can potentially be dispersed and deposited in newly planted fields. We detected large numbers of airborne \u003cem\u003eXhc\u003c/em\u003e in September and October during both years of the study, which were likely due to fields being harvested during these months. Additionally, leaf samples taken in November 2018 and in other studies (du Toit et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) show that considerable \u003cem\u003eXhc\u003c/em\u003e populations can become established on seed-to-seed carrot fields prior to winter. These results lend further support for a green bridge effect that contributes to the annual and seemingly endemic presence of \u003cem\u003eXhc\u003c/em\u003e in the region.\u003c/p\u003e \u003cp\u003eThere are other cultural practices associated with carrot seed production that have the potential to generate airborne \u003cem\u003eXhc\u003c/em\u003e, including plant thinning, mechanical weed management, applying pesticides, and the destruction of pollen donor lines prior to harvest. McInnes et al. (McInnes \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) observed more significantly more \u003cem\u003eX. campestris\u003c/em\u003e pv. \u003cem\u003evesicatoria\u003c/em\u003e in the air above tomato transplants after clipping and harvest. In carrot seed production, some of these activities (e.g. mechanical weed management, pesticide applications) occur multiple times throughout the year, while other practices only occur once per season (e.g. plant thinning, destruction of pollen donor lines), and the exact timing of these activities will vary depending on cultivar, weather conditions, and logistics. In this study, we detected airborne \u003cem\u003eXhc\u003c/em\u003e during the majority of sampling days and on days when field activities were not occurring, including immediately after seedling emergence, suggesting that the pathogen was either originating from neighboring fields or there are other mechanisms for \u003cem\u003eXhc\u003c/em\u003e to become airborne from carrot seed crop canopies.\u003c/p\u003e \u003cp\u003eAnother objective of this research was to identify weather factors that contribute to airborne \u003cem\u003eX. hortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e. In this study, principle component analysis identified temperature, wind (speed, direction and run) and factors related to moisture as the primary factors accounting for the variance. Previous studies have also demonstrated that the airborne dispersal of bacteria in aerosols is dependent on temperature, wind, and moisture. Lindemann and Upper (Lindemann and Upper \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) reported that upward aerial fluxes of bacteria from bean phyllospheres occurred during the middle of dry, warm days and not in the evening or when moisture was present on the leaves in the form of rain or dew. In their study, bacterial numbers were stronger after precipitation events compared to when soils were dry, and wind speed was positively correlated with aerosol strength. In carrot seed crops, strong winds may promote the release and dispersal of \u003cem\u003eXhc\u003c/em\u003e leaf-to-leaf contact within and between individual plant canopies, which could potentially dislodge bacterial cells from epidermal surfaces or biofilms (Morris and Monier \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The central Oregon growing season is typified by sunny and dry days, windy afternoons, and cool nights, which may contribute towards the production of aerial fluxes of \u003cem\u003eXhc\u003c/em\u003e in and around carrot seed fields. Diurnal fluctuations of bacterial aerosols are known to occur in climates similar to the high desert of central Oregon, with greater upward fluxes occurring during the afternoon on warm sunny days and after rain or irrigation events (Lindemann and Upper \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1985\u003c/span\u003e); it would be reasonable to hypothesize that similar temporal patterns might occur with \u003cem\u003eXhc\u003c/em\u003e aerosols from carrot seed crop canopies.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe researchers thank the grower-cooperators for allowing this work to be performed in their fields. The technical support of Tiffany Belvoir, Shaelynn Downing, and Kelley Duggan was greatly appreciated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the USDA National Institute of Food and Agriculture, through the Specialty Crops Research Initiative Project grant no. 2020-51181-32154 and the Western Integrated Pest Management Center project ID 1616. Additional funding was also received from the Agricultural Research Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAll authors contributed to the study conception and design. Material preparation and data collection were performed by Jeness Scott and Jeremiah Dung. Data analysis was performed by Jeremiah Dung. The first draft of the manuscript was written by Jeremiah Dung and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests:\u003c/strong\u003e The authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnonymous. (2020). \u003cem\u003eNorth Unit Irrigation District 2020 Crop Report\u003c/em\u003e. Madras, OR.\u003c/li\u003e\n\u003cli\u003eBernal, R. F., \u0026amp; Berger, R. D. (1996). The spread of epiphytic populations of \u003cem\u003eXanthomonas campestris\u003c/em\u003e pv. \u003cem\u003evesicatoria\u003c/em\u003e on pepper in the field. \u003cem\u003eJournal of Phytopathology\u003c/em\u003e, \u003cem\u003e144\u003c/em\u003e(9\u0026ndash;10), 479\u0026ndash;484.\u003c/li\u003e\n\u003cli\u003eBock, C. H., Parker, P. E., \u0026amp; Gottwald, T. R. (2005). Effect of simulated wind-driven rain on duration and distance of dispersal of \u003cem\u003eXanthomonas axonopodis\u003c/em\u003e pv. \u003cem\u003ecitri\u003c/em\u003e from canker-infected citrus trees. Plant Disease, \u003cem\u003e89\u003c/em\u003e(1), 71\u0026ndash;80.\u003c/li\u003e\n\u003cli\u003eButler, M., \u0026amp; Simmons, R. (2013). \u003cem\u003e2013 Agricultural Statistics: Central Oregon\u003c/em\u003e. Corvallis, OR: Oregon State University.\u003c/li\u003e\n\u003cli\u003eCattell, R. B. (1966). The scree test for the number of factors. Multivariate Behavioral Research, \u003cem\u003e1\u003c/em\u003e(2), 245\u0026ndash;276.\u003c/li\u003e\n\u003cli\u003eDia, N. C., Morini\u0026egrave;re, L., Cottyn, B., Bernal, E., Jacobs, J. M., Koebnik, R., et al. (2022). \u003cem\u003eXanthomonas hortorum\u003c/em\u003e \u0026ndash; beyond gardens: Current taxonomy, genomics, and virulence repertoires. 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Educational and Psychological Measurement, \u003cem\u003e20\u003c/em\u003e(1), 141\u0026ndash;151.\u003c/li\u003e\n\u003cli\u003eKimbrel, J. A., Givan, S. A., Temple, T. N., Johnson, K. B., \u0026amp; Chang, J. H. (2011). Genome sequencing and comparative analysis of the carrot bacterial blight pathogen, \u003cem\u003eXanthomonas hortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e M081, for insights into pathogenicity and applications in molecular diagnostics. \u003cem\u003eMolecular Plant Pathology\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(6), 580\u0026ndash;594.\u003c/li\u003e\n\u003cli\u003eKuan, T.-L., Minsavage, G. V., \u0026amp; Schaad, N. W. (1986). Aerial dispersal of \u003cem\u003eXanthomonas campestris\u003c/em\u003e pv. \u003cem\u003ecampestris\u003c/em\u003e from naturally infected \u003cem\u003eBrassica campestris\u003c/em\u003e. \u003cem\u003ePlant Disease\u003c/em\u003e, \u003cem\u003e70\u003c/em\u003e(5), 409\u0026ndash;413.\u003c/li\u003e\n\u003cli\u003eLeben, C. (1965). Epiphytic microorganisms in relation to plant disease. Annual Review of Phytopathology, \u003cem\u003e3\u003c/em\u003e(1), 209\u0026ndash;230.\u003c/li\u003e\n\u003cli\u003eLighthart, B. (1984). Microbial aerosols: Estimated contribution of combine harvesting to an airshed. Applied and Environmental Microbiology, \u003cem\u003e47\u003c/em\u003e(2), 430\u0026ndash;432. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aem.47.2.430-432.1984\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eLindemann, J., Constantinidou, H. A., Barchet, W. R., \u0026amp; Upper, C. D. (1982). Plants as sources of airborne bacteria, including ice nucleation-active bacteria. Applied and Environmental Microbiology, \u003cem\u003e44\u003c/em\u003e(5), 1059\u0026ndash;1063. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aem.44.5.1059-1063.1982\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eLindemann, J., \u0026amp; Upper, C. D. (1985). Aerial dispersal of epiphytic bacteria over bean plants. Applied and Environmental Microbiology, \u003cem\u003e50\u003c/em\u003e(5), 1229\u0026ndash;1232.\u003c/li\u003e\n\u003cli\u003eLindow, S. E. (1996). Role of Immigration and Other Processes in Determining Epiphytic Bacterial Populations. In C. E. Morris, P. C. Nicot, \u0026amp; C. Nguyen-The (Eds.), \u003cem\u003eAerial Plant Surface Microbiology\u003c/em\u003e (pp.\u0026nbsp;155\u0026ndash;168). Boston, MA: Springer US. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-0-585-34164-4_10\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eMcInnes, T. B. (1988). Airborne dispersal of bacteria in tomato and pepper transplant fields. Plant Disease, \u003cem\u003e72\u003c/em\u003e(7), 575. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PD-72-0575\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eMorris, C. E., \u0026amp; Monier, J.-M. (2003). The ecological significance of biofilm formation by plant-asssociated bacteria. Annual Review of Phytopathology, \u003cem\u003e41\u003c/em\u003e(1), 429\u0026ndash;453. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev.phyto.41.022103.134521\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eRubatzky, V. E., Quiros, C. F., \u0026amp; Simon, P. W. (1999). \u003cem\u003eCarrots and Related Vegetable Umbelliferae\u003c/em\u003e (Vol. 10). New York.\u003c/li\u003e\n\u003cli\u003eScott, J. C., \u0026amp; Dung, J. K. (2020). Distribution of \u003cem\u003eXanthomonas hortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e populations in naturally-infested carrot seed lots. \u003cem\u003ePlant Disease\u003c/em\u003e, in print.\n\u003cdiv class=\"ExternalRefDOI\"\u003ehttps://doi.org/10.1094/PDIS-12-19-2674-RE. https://doi.org/10.1094/PDIS-12-19-2674-RE\u003c/div\u003e\n\u003c/li\u003e\n\u003cli\u003eTemple, T. N., du Toit, L. J., Derie, M. L., \u0026amp; Johnson, K. B. (2013). Quantitative molecular detection of \u003cem\u003eXanthomonas hortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e in carrot seed before and after hot-water treatment. Plant Disease, \u003cem\u003e97\u003c/em\u003e(12), 1585\u0026ndash;1592.\u003c/li\u003e\n\u003cli\u003eUmesh, K. C., Davis, R. M., \u0026amp; Gilbertson, R. L. (1998). Seed contamination thresholds for development of carrot bacterial blight caused by \u003cem\u003eXanthomonas campestris\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e. Plant Disease, \u003cem\u003e82\u003c/em\u003e(11), 1271\u0026ndash;1275.\u003c/li\u003e\n\u003cli\u003eU.S. Department of Agriculture National Agricultural Statistics, U. (2021). \u003cem\u003eVegetables 2020 Summary\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://downloads.usda.library.cornell.edu/usda-esmis/files/02870v86p/j6731x86f/9306tr664/vegean21.pdf\u003c/span\u003e\u003c/span\u003e. Accessed 19 April 2022\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"Table 1 is available in the Supplementary Files section."}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"aerobiology, bacteria, epidemiology, epiphyte, phytopathology, plant disease","lastPublishedDoi":"10.21203/rs.3.rs-1631313/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1631313/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacterial blight of carrot, caused by the plant pathogenic bacterium \u003cem\u003eXanthomonas hortorum\u003c/em\u003e pv. \u003cem\u003ecarotae\u003c/em\u003e (\u003cem\u003eXhc\u003c/em\u003e), is an important seedborne disease of carrot. Previous research demonstrated that airborne \u003cem\u003eXhc\u003c/em\u003e could be detected up to 1.6 km downwind of carrot seed fields being harvested, but more information is needed to identify dispersal periods of the pathogen during other stages of carrot seed production. The objectives of this research were to: 1) quantify and characterize temporal dynamics of airborne \u003cem\u003eXhc\u003c/em\u003e during carrot seed production; and 2) identify weather factors that contribute to airborne \u003cem\u003eXhc\u003c/em\u003e levels in carrot seed fields. During the 2018\u0026ndash;2019 season, air samples were collected on a total of 273 and 289 days in two fields and \u003cem\u003eXhc\u003c/em\u003e was detected on 223 (81.7%) and 245 (84.8%) of sampling days. On average, 2.6 x 10\u003csup\u003e3\u003c/sup\u003e and 7.0 x 10\u003csup\u003e3\u003c/sup\u003e \u003cem\u003eXhc\u003c/em\u003e/day were detected. Principle component analysis identified three components associated with airborne \u003cem\u003eXhc\u003c/em\u003e levels. Temperature variables (daily mean, maximum, and minimum air and soil temperatures) accounted for 53.2% of the total variance. The second component was associated with wind (wind speed, direction, and run) and 19.1% of the total variance. Factors related to moisture (daily precipitation, relative humidity, and dew point) were associated with the third component and accounted for 13.0% of the total variance. Together, these data can be used to build predictive models to inform growers of inoculum risk and improve control through better-timed bactericide applications, modified cultural practices, and increased knowledge of bacterial blight epidemiology in carrot seed crops.\u003c/p\u003e","manuscriptTitle":"Detection and occurrence of airborne Xanthomonas hortorum pv. carotae, causal agent of carrot bacterial blight, in carrot seed production systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-19 17:26:40","doi":"10.21203/rs.3.rs-1631313/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd240864-3af8-4620-a862-0a4ee336b90a","owner":[],"postedDate":"May 19th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-24T06:59:30+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-19 17:26:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1631313","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1631313","identity":"rs-1631313","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}