Evaluation of the Potential for Systemic Infection of Carrot Seed Crops by Xanthomonas campestris pv. carotae. Fred Crowe and Rhonda Simmons

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1 Evaluation of the Potential for Systemic Infection of Carrot Seed Crops by Xanthomonas campestris pv. carotae Fred Crowe and Rhonda Simmons Introduction Bacterial blight of carrot, caused by Xanthomonas campestris pv. carotae (Xcc), is commonly found in carrot seed production fields in Washington and Oregon and in commercial fields in California. Oregon and Washington produce most of the world s carrot seed, on about 3,000 acres per state, with much of this seed used in California. Seed lots from Washington and Oregon are commonly infested with Xcc, incurring additional expenses to growers and seed companies in the form of careful seed testing, selection and handling, and seed treatment. Further, there is a lingering question whether traces of Xcc might escape seed treatment (or be carried with seed containers and equipment) and serve as primary inoculum for bacterial blight outbreaks in commercial and seed fields, in spite of no apparent recovery of Xcc from treated seed lots. As we pointed out after the 2002 season, a similar question applies to steckling carrots being brought into seed areas from commercial areas i.e., steckling carrots may be carriers of Xcc. The overall objective of our research is to find methods to lower seed-borne levels of Xcc and other pathogens in carrot seed leaving seed production regions, but we also wish to lower bacterial blight incidence in seed fields. This proposal addressed the possibility that Xcc may move systemically within carrot plants, and ultimately within seed. Movement of Xcc in the xylem could provide access to stems, leaves, and umbels, and potentially lead to infection of some developing seed even a few such seeds could be important if they are less likely to be impacted by seed treatments. Systemic infection by Xcc may be more difficult to control than localized infections, as there are no systemic bactericides currently available for control of bacterial blight. Preliminary data in 2002 suggested that Xcc could become systemic, could move within the plant, and multiply in plants (Crowe and Bafus 2003). This preliminary data suggested movement might occur both up and down within the plant; if so, then movement might occur more than simply in xylem. In 2003, we hoped to verify and extend these preliminary findings, using more precise tools that might be good enough to verify whether seed could become infected internally. Literature Review The epidemiology of Xcc in carrot seed fields is poorly understood. Lesions appear on stems, petioles, nodes, leaves, umbels, and flowers, and bacterial ooze may form in these lesions. Potential sources of inoculum include seed, debris of previous carrot crops, neighboring infected carrot crops or other Umbelliferous hosts (weeds/crops) of Xcc, irrigation water, equipment 1

2 moved from field to field, and insects (including pollinators such as bees and flies, as well as lygus bugs and other insect pests). Once established in a field, Xcc theoretically may be dispersed within the field by splashing and formation of aerosol droplets from rain or sprinkler irrigation, by insects, and by equipment such as rolling wheel lines or tractors. However, while survey research conducted by L. Du Toit, F. Crowe and others in indicated that Xcc increased on foliage during the season, this survey also suggested that increase of Xcc in carrot seed fields was comparable for both furrow- and sprinkler-irrigated crops, was somewhat lower on drip-irrigated crops, and that bees were not found to carry measurable Xcc. Thus, the mode of spread within fields remains unclear. Xcc was found on some seed used to plant seed fields, was found in some transplanted steckling, and may be found in the dust blowing from combines, and at least some Xcc may blow onto nearby newly planted seed fields where new plantings are close to old plantings. Seed surface infestation probably occurs throughout seed development in the umbel and during harvest operations. Deeper seed coat infestation or true infection of living seed tissues (if this occurs) likely would occur earlier in seed development, perhaps while the immature seed still has living connections to the mother plant or via pollen or nectar. Xanthomonas campestris pv. vesicatoria has been demonstrated to infect pepper plants via dead flower parts, resulting in contamination of seeds in the absence of any external symptoms (Bashan and Okun 1986). Hot water treatments are superior to surface disinfestations by chorine in eradication of seed borne Xcc (Pscheidt and Ocamb 2001). Because chlorine bleach does not penetrate easily into plant tissues, the question remains just how deeply embedded bacteria might be into or beneath the carrot seed surface. It is also possible Xcc may move systemically through carrot plants without producing internal symptoms of infection. Systemic movement in plants is well documented for several pathovars of X. campestris, including X. campestris pv. campestris (black rot of cabbage) (Cook et al. 1952) and X. campestris pv. phaseoli (common blight of bean) (Weller and Saettler 1980). However, the mechanisms of systemic movement of bacteria through plants and into seed remains unclear. Note our preliminary report from 2002 that suggested both upward and downward movement (Crowe and Bafus 2003). Pfleger et al. (1974) examined the potential for systemic movement of Xcc in carrots, but their results were inconclusive. Vascular tissues in the roots were colonized by Xcc when plants were inoculated by immersing the roots in an aerated suspension of Xcc for 48 hours. In several of these plants the vascular tissue rotted severely and Xcc was recovered from leaves that developed after inoculation. However, the authors did not consider this as adequate demonstration of systemic movement of Xcc. Our own preliminary data from 2002 strongly suggested that Xcc did sometimes move within carrots when roots were wound inoculated, or if Xcc was applied to foliage, and that Xcc multiplied during or after movement (Crowe and Bafus 2003). Thus, we both duplicated and extended the studies by Pfleger, et al (1974), but (as they did) wish for stronger evidence of systemic movement and reproduction as common and natural phenomenon. Research Objectives 1. Verify whether Xcc may move systemically and reproduce within carrot plants. 2

3 2. Determine the location of Xcc within plant tissues, including roots, foliage, flower parts and seed. Methods Lab Studies Our proposal depended on using strains of Xcc that had been transformed (genetically engineered) to glow green (or some other color) when illuminated by fluorescence microscopy. Such a tool would allow direct visualization of Xcc in fresh carrot tissues, and possibly in/on seed. Such transformation typically has been relatively easy and straightforward for most bacteria and other organisms. The transformation involves moving genes for expression of green fluorescing protein (GFP), perhaps including genes that promote expression of the GFP, from donor bacteria (typically Escherichia coli) to the target organism by one of several means, either by electroporation or by conjugation mating. Electroporation temporarily alters the cell membranes enough to allow DNA from the E. coli vector to be inserted into the target strain. Mating involves natural transfer of DNA from similar or even different bacteria when such bacteria form mating connections and exchange DNA (Sambrook and Russell 2001). In either case, the stability of the transformed bacterium must be confirmed with respect to the inserted genes e.g., with electroporation, the genes can be lost easily. Similarly, the original character of the strain must be reassessed e.g., with conjugation, DNA might insert anywhere into the bacterial chromosome and cause unwanted mutations. Once the desired type and stability have been determined, the GFP-Xcc could be used as suggested above. Five Xcc strains were obtained from Lindsey Du Toit of Washington State University. They were initially checked for purity on YDC media and then stored at 80 C. All vectors were both received and maintained in the bacterium E. coli. These are shown in Table 1. Table 1. Vectors used in transformation experiments. Vector name GFP type Antibiotic resistance Source pagr2 DsRed Ap R, Gm R Tim Denny, UGA 4 pagc cyanfp Ap R, Gm R Tim Denny, UGA 10 pagy yellowfp Ap R, Gm R Tim Denny, UGA 10 pagy408 GFPuv Ap R, Km R, Gm R gfp + Tim Denny, UGA 10 put mini-tn5gfp GFPmut2 Ap R Tim Denny, UGA 10 pfp6301 GFPuv Km R Mahaffee pfp6158 DsRed Km R BurkPromoter+TIR+ Mahaffee Electrocompetent cells of five Xcc strains were prepared for initial transformation experiments. Cells were grown overnight in SOB medium then transferred to fresh medium, grown until an optical density of 0.8 (A590nm), then centrifuged. The pellet was resuspended in 10 percent glycerol, aliquoted, then snap-frozen in liquid nitrogen and stored at -80 C. Electroporation (4k resistance and 330uF capacitance) was conducted by mixing 5-20ug of vector DNA with 20 ul of 3

4 electrocompetent cells. After electroporation, cells were transferred to SOC medium, shaken (200prm) for 90 min, and then plated onto selective LB agar (Sambrook and Russell 2001). In a follow-up study, electroporation also was attempted with all five Xcc strains with five vectors that contained various variants of the GFP protein all within a minitn5 transposon that had been used with other Xanthomonas strains (other pathovars than carotae). Conjugation matings were attempted to transfer the GFP vectors into Xcc strains (Sambrook and Russell 2001). Methods Field Studies The lab studies above were to be followed by extensive greenhouse tracking of GFP-Xcc in carrots grown in the greenhouse. Because the development of GFP-Xcc became delayed, we attempted to gather at least some field data that might relate to the question of systemic activity of Xcc. In retrospect, it would have been better to initiate such field studies earlier in the season. Two carrot seed fields that were part of our broad field survey were selected based on our having detected Xcc from foliage on about percent of the plants in April and June of Both fields had been planted with true seed from which no Xcc had been recovered. At the next fieldsampling period (mid-august 2003), we collected roots along with foliage from 20 female plants from each field. At this time of year, plants were fully pollinated and seed was developing. Plant sampling is described in the companion report, but was essentially random. Hands, gloves, and tools were sterilized before handling each sampled plant, both in field and lab. Roots were large, frequently cracked, with some decay present in many of them. Foliage was processed and washed as per our companion field survey (see companion report by L. Du Toit, F. Crowe, and others), and roots were processed as per our report from the 2002 season (Crowe and Bafus 2003) the surface was trimmed away from the roots, removing most decay and cracked areas, and roots were surface sterilized for 5 min in 0.5 percent NaOCl. Roots were then chopped and ground, and a sample was collected for dry weight determination and another sample was assayed for Xcc as per foliage, using XCS medium, to determine colony-forming units (CFU) per g dry weight of trimmed root. Results Lab Studies Electroporation: No Xcc:GFP transformants were ever observed in initial and repeated experiments using vectors pfp6301 and pfp6158, while controls using puc19 worked. This indicated that the strains were electrocompetent but that transformation with GFP vectors was at a very low frequency or that these particular constructs were not expressed in the Xcc strains. Follow-up (and repeated) electroporation studies were not successful since we were unable to differentiate Xcc from the E.coli donor strains using various antibiotics including Gentamycin, Tetracyclin, Tobramycin, Amikacin, Streptomycin, Spectinomycin, Kanamycin, and Vancomycin, or YDC or XCS media. XCS is highly selective for Xcc (Weller and Saettler 1980), but unfortunately E. coli seemed to grow on it equally well. 4

5 In repeated conjugation mating experiments, there was still no success in transforming any Xcc strains. Biparental matings were then attempted with these five vectors since they are promotorless and not expressed in the E. coli host. After mating, cultures were placed on YDC or XCS to help select for Xcc stains expressing the GFP protein. All fluorescent colonies selected were subsequently identified as E.coli. No Xcc strains were transformed. We concluded that we could not pursue the transformation of Xcc strains further until we had suitable counter-selection (i.e., selective medium or spontaneous antibiotic resistant mutant of an Xcc strain). Thus, we also screened for antibiotic resistant strains, using various concentrations of rifampicin in both YDC and XCS media, similar to that conducted by Weller and Saettler (1980). No resistant isolates were recovered after repeated attempts. No further work was conducted, and we could not proceed with our primary investigations of visualizing Xcc in carrot tissues. Except we did, however, inject GFP-transformed bacteria of other types into carrot tissues and found that there were no complications of autoflourescence of carrot tissues under the microscope at the wavelengths used. Such autoflourescence can be a substantial problem with some plant tissues if fluorescing bacteria are obscured. Results Field Studies Results for the mid-august assay from foliage and roots for two seed carrot fields in central Oregon is shown in Tables 2a and 2b. For Field A (Table 2a), June foliage sampling yielded Xcc from 6 of 20 plants (not shown), whereas mid-august sampling yielded Xcc from 18 of 20 plants. Six of these plants at mid- August yielded abundant Xcc from root tissues. For 5 plants (nos. 4, 7, 16, 17, 20), there were corresponding recoveries of Xcc from foliage and roots. For one plant (no. 13), Xcc was recovered from the root only. For twelve plants, Xcc was recovered only from foliage. Two plants were found free of Xcc. For Field B (Table 2b), June foliage sampling yielded Xcc from 7 of 20 plants (not shown), whereas mid-august sampling yielded Xcc from 14 of 20 plants. Four of these plants at mid- August yielded abundant Xcc from root tissues. For these four plants (nos. 12, 16, 18, 20), there were corresponding recoveries from foliage and roots. No plants were found in which Xcc was recovered from roots only. Xcc was recovered only from foliage of 10 plants. Xcc was not recovered at all from six plants. There was no direct relationship between the CFU from foliage and root recovery. Additionally, as emphasized in our survey report, in 2003 there were no measurable bacterial blight symptoms on surveyed plants, including those listed in Table 2. 5

6 Discussion Clearly, things did not proceed smoothly in the lab as we anticipated. No stable transformed isolates of Xcc were derived that fluoresced either green or red, and no rifampicin-antibiotic resistant isolates were developed either. It was unusual and frustrating to encounter these difficulties. We are not sure how to proceed further in developing a fluorescing isolate of Xcc. We had hoped to obtain stable fluorescing isolates by about April, and have them tested for competent to incite bacterial blight of carrots by the end of May. Attempts at both transformation and finding antibiotic resistant isolates continued well into October. It is interesting to note our failure here in relation to some earlier work, previously only presented as a minor report (Parks and Crowe 1999). The central Oregon carrot industry became concerned in the late 1990 s that copper resistance might be present in Xcc populations, following 20 years of use of copper pesticides to ameliorate bacterial blight in central Oregon carrot fields. (Copper applications were never particularly successful on carrot bacterial blight even in early years, so it wasn t necessarily easy to discern the reasons for control failures.) Following the procedure of Scheck et al. (1996) who had found abundant copper tolerance in Psuedomonas syringe in ornamental nurseries in Oregon, we recovered many isolates of Xcc from central Oregon carrot fields and tested them for such tolerance. The tests included Xcc isolates from regions with no history of copper application. We found no evidence for copper tolerance or resistance in over 40 randomly selected isolates. These and our results above make us wonder whether Xcc is less likely to form stable mutants than many other bacteria? Our limited field studies should not be over-interpreted. We would rather have done root sampling throughout the year, and before roots were so mature as they were in August to avoid issues of whether bacteria recovered were strictly from sound, internal tissues. Nevertheless, taken together, our 2002 and 2003 data do provide food for thought. In 2002, inoculation of Xcc onto foliage or roots (either surface contact or via wounding) resulted in high populations of Xcc recovered from root tissue some months later for some but not all roots. The simplest explanation of our limited 2003 field data is that something similar occurred i.e., that Xcc moved either from naturally infested/infected foliage into roots, or perhaps that Xcc washed from foliage onto crown or upper roots and entered roots in this manner. In any case, the CFU/g dry tissue found in roots in both 2002 and 2003 strongly suggest reproduction by Xcc in root tissue, without symptom development. The single plant from which Xcc was recovered from roots but not foliage suggests that these Xcc did not arise from foliage. Our 2002 and 2003 field data, when combined with our companion survey findings of Xcc recovery from some strecklings entering seed fields from source fields in desert California, suggest at least that roots can be substantial carriers of Xcc over long distances. Because our 2002 study also found that Xcc eventually could be recovered from the foliage on some rootinoculated plants, it may be reasonably assumed that such steckling-sourced bacteria reach the foliage and become available for general field transmission. While eradication of Xcc from true seed seems routine, movement of Xcc with stecklings could be an even more substantial and untreatable problem than true seed except that increase in Xcc foliar populations seems delayed 6

7 in steckling fields in the Pacific Northwest in comparison to seed-to-seed fields (see companion report by L. Du Toit, F. Crowe, and others). Our 2002 and 2003 data, taken together with the companion survey data, suggest that Xcc commonly persist in and on carrot tissues without causing damage under conditions that are generally cool in seed production regions. We have provided no information about internal seed infection by Xcc. No further work to verify systemic movement of Xcc is proposed at this time, but we may re-propose such work in the future. Literature Cited Bashan, Y., and Y. Okun Internal and external infections of fruits and seeds of peppers by Xanthomonas campestris pv. vesicatoria. Can. J. Bot. 64: Cook, A.A., R.H, Larson, and J.C. Walker Relation of the black rot pathogen to cabbage seed. Phytopathology 88: Crowe, F., and R. Bafus Evaluation of the potential for systemic infection of carrot seed crops by Xanthomonas campestris pv. carotae. Progr. Rep. to the California Fresh Carrot Advisory Board, 7 pages. Matthysse, A.G, S. Stretton, C. Dandie, N.C. McClure, and A.E. Goodman Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiology Letters145: Parks, R., and F. Crowe Sensitivity of Xanthomonas campestris pv. carotae to copper pesticides in central Oregon carrot seed fields. Final Rep. to the Oregon State Univ. Integrated Plant Protection Cent., 4 pages. Pfleger, F.L., G.E. Harman, and G.A. Marx Bacterial blight of carrots: Interaction of temperature, light and inoculation procedures on disease development of various carrot varieties. Phytopathology 64: Pscheidt, J.W., and C.M. Ocamb Pacific Northwest Disease Management Handbook. Oregon State Univ., Washington State Univ., and Univ. of Idaho. Sambrook, J., and D.W. Russell Molecular Cloning: A Laboratory Manual. Third ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Scheck, H.J., J.W. Pscheidt, and L.W. Moore Copper and streptomycin resistance in strains of Psuedomonas syringe from Pacific Northwest nurseries. Plant Disease 80: Suarez, A, A. Güttler, M. Strätz, L.H. Staendner, K.N. Timmis, and C.A. Guzmán Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications. Gene 196:

8 Weller. D.M., and A.W. Saettler Evaluation of seed borne Xanthomonas phaseoli and X. phaseoli var. fuscans as primary inocula in bean blights. Phytopathology 70: Williford, R.E., and N.W. Schaad Agar medium for selective isolation of Xanthomonas campestris pv. carotae from carrot seeds. Phytopathology 74:1142 (Abstract). 8

9 Table 2a. Recovery of Xcc from foliage and roots from Field A of two seed fields in central Oregon, mid-august Field A Foliage Foliage Roots Roots Plant # CFU/g dry wt Log[CFU/g dry wt] CFU/g dry wt Log[CFU/g dry wt x x x x x x x x x x x x x x x x x x x x x x x

10 Table 2b. Recovery of Xcc from foliage and roots from Field B of two seed fields in central Oregon, mid-august Field B Foliage Foliage Roots Roots Plant # CFU/g dry wt Log[CFU/g dry wt] CFU/g dry wt Log[CFU/g dry wt x x x x x x x x x x x x x x x x x x