Hot Acidified Cupric Acetate Soaks for Eradication of

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1 APPLID AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1980, p /80/ /05$02.00/0 Vol. 39, No. 4 Hot Acidified Cupric Acetate Soaks for Eradication of Xanthomonas campestris from Crucifer Seeds N. W. SCHAAD,'* R. L. GABRIELSON,2 AND M. W. MULANAX2 Department of Plant Pathology, University of Georgia, Georgia Experiment Station, Experiment, Georgia 30212,' and Washington State University, Western Washington Research and Extension Center, Puyallup, Washington Acidified cupric acetate soaks were tested for eradication of Xanthomonas campestris from naturally infected crucifer seeds. The pathogen was eradicated from seeds by soaking in 0.5% cupric acetate dissolved in N acetic acid for 20 min at 35, 40, 45, and 500C but not 250C. Moreover, normal bacterial flora of crucifer seeds and the seed-borne Phoma lingam and Alternaria spp. were reduced by 95, 92, and 81%, respectively, after the cupric acetate treatment at 400C. The seed germination percentage was generally reduced, but the amount of reduction depended upon the treatment temperature and plant cultivar. At 500C, less than 50% of the seed of all 12 cultivars tested germinated, whereas at 40 C more than 50% of the seeds of most cultivars germinated. Treating seeds in cupric acetate at 400C should prove useful for eradicating X. campestris from seeds of breeding lines and stock seed used for hybrid seed production. Furthermore, a significant reduction in total bacterial flora and seed-borne fungi suggests the usefulness of the treatment for other microorganisms associated with other seeds or foodstuffs. Black rot, caused by Xanthomonas campestris, continues to be a serious seed-borne disease of cruciferous plants throughout the world. Techniques have been developed recently to detect diseased seeds (14, 18), but no method has been proven to eradicate the bacterium after it has been detected. Several methods of eradication have been recommended, including hot water soaks (5), organic mercurials (5), streptomycin (11), and hot water-chlorotetracycline soaks (13). The organic mercurials cannot be used because of Environmental Protection Agency regulations, and the other methods reduce but do not eradicate seed-borne X. campestris (11, 20). Copper is a proven bactericide (3, 12, 19) and is a relatively safe pesticide that can be easily registered for agricultural use. Copper is commonly used on plants for controlling leaf-spotting bacteria, but it is not used as a seed treatment. Hot water, on the other hand, is used with limited success as a seed treatment. Therefore, a study of the effect of hot acidified cupric acetate soaks on the eradication ofx. campestris from seeds was carried out. Naturally infected seeds were used because the pathogen invades the seed coat through the vascular system of the funiculus (6). To determine the effects of the treatment on other microorganisms, we also assayed for other bacteria and two seed-borne fungi, Phoma lingam and Alternaria spp. MATERLALS AND METHODS Preparation of test solutions and test procedures. Stock solutions of cupric acetate were prepared by dissolving 1 g of cupric acetate (J. T. Baker Chemical Co., Phillipsburg, N.J.; analyzed reagent grade) in 100 ml of 0.01 N acetic acid, ph Stock solutions were stored at room temperature. Just before use, the solution was diluted 1:1 with distilled water and brought to the desired temperature in a water bath. Ten milliliters of the test solution and 1 drop of the nonionic detergent Tween 20 (a sorbitan monolaurate polyoxyal xylene derivative) per 100 seeds tested were added to a 250-ml beaker. The beaker was then immediately placed in a water bath for 20 min at the desired temperature. After treatment, the seeds were rinsed once with sterile distilled water and dried in a laminar-flow hood. All tests were conducted as described above unless stated otherwise. Seed. All commercial cabbage seeds used for controls were shown to be free of X. campestris by plating five 10,000-seed samples onto SX agar (16) and nutrient-starch-cyclohexinide agar, as described previously (14). Green Comet broccoli seeds from X. campestrisinoculated seed plants were obtained from H. Humaydan, Harris Seed Co., Rochester, N.Y., and naturally infected Savoy King cabbage seeds from Japan were obtained from R. MacDonald, Alf Christianson Seed Co., Mt. Vernon, Wash. Ten percent of the broccoli seeds and 0.6% of the cabbage seeds were infected as determined by plating five 10,000-seed samples onto SX agar (14). Healthy seeds were washed in sterile distilled water to reduce the amount of fungicide present and dried in an open 15-cm petri dish in a laminarflow hood. 803

2 804 SCHAAD, GABRIELSON, AND MULANAX Tests with X. campestris. Twenty Green Comet broccoli seeds were added to 80 healthy cabbage seeds in a sterile 80-ml beaker. Savoy King cabbage seeds were used undiluted; 500 seeds were placed in a 250- ml beaker. After treatment, seeds were assayed for X. campestris by direct plating onto nutrient-starch-cycloheximide agar (14) or by sowing seeds in steamsterilized soil or by both. The seeds were germinated in a growth chamber with day and night temperatures of 26 and 22 C, respectively, and the resulting plants were moved to a growth chamber at 30 C or a greenhouse with day and night temperatures of 28 to 31 and 21 C, respectively. Symptoms of black rot were recorded 3 weeks later. Tissue samples of suspected black rot lesions were comminuted in a droplet of water and streaked onto yeast extract-dextrose-caco3 (23) and SX agars. Several colonies typical of X. campestris (16) were retained and tested for pathogenicity (17). The effect of the treatments on other bacteria was determined by recording the number of seeds with other bacteria by using the nutrient-starch-cycloheximide agar plating test. The efficacy of cupric acetate soaks at 400C was further tested under natural field conditions. A commercial experimental hybrid cabbage seed lot containing approximately 90,000 seeds was determined to be infected with X. campestris (14). All but 1,000 seeds were treated in a 4-liter beaker with 1 liter of cupric acetate and 2 ml of Tween 20 at 400C as described above. Treated and untreated seeds were tested for germination, and 11,000 treated seeds were tested for X. campestris. The remainder of the seed lot was returned to the seed company for their field plant quality evaluations at four different locations along with other nontreated lines. Seed germination. The effect of treatments on germination of seeds was analyzed by germination tests with four replicates of 100 seeds each by the Association of Official Seed Analysts wet blotter method (1). Tests with fungi. The effects of cupric acetate soaks on commercial cabbage seed samples naturally infected with P. lingam and Alternaria spp. were evaluated by an International Seed Testing Association method (9). Four replications of 100 seeds from each treatment were plated onto 0.2% 2,4-dichlorophenoxyacetic acid (2,4-D)-saturated filter papers in petri plates. After 11 to 14 days of incubation at 200C under cool white fluorescent lights, seeds were observed for sporulation of P. lingam and Alternaria spp. RESULTS Tests with X. campestris. In preliminary experiments, X. campestris was reduced but not eradicated from infected seeds soaked in water at 500C for 20 min or in acidified cupric acetate at 250C. Therefore, we tested the two treatments in combination. X. campestris was successfully eradicated from infected broccoli seeds by soaking them in acidified cupric acetate at 500C but not by soaking them in water for 20 min at 25 or 500C APPL. ENVIRON. MICROBIOL. (Table 1). However, the 50 C cupric acetate treatment resulted in poor germination of seeds of all varieties tested (Table 2). In further tests at lower temperatures, cupric acetate soaks eradicated the pathogen at 40 and 35 C but not at 25 C (Table 3), and the germination of seeds at the lowest temperatures was significantly improved over that at 50 C (Table 2). All colonies typical of X. campestris on agar media and tested for pathogenicity were positive. Other bacteria were not eradicated, but their numbers were significantly reduced in seeds treated with hot acidified cupric acetate (Table 3). X. campestris was not detected in a seed assay (14) of the commercial experimental hybrid seeds after treatment in cupric acetate at 400C. Although germination was only 71% for the treated seeds as compared with 94% for the untreated seeds, enough plants were obtained in the field trials for breeding evaluation purposes. No black rot was observed in plants in the four different field plantings, although a few infected plants were observed in several surrounding plots planted with nontreated seeds of other cultivars at two of the locations. Tests with fungi. The 400C cupric acetate soaks significantly reduced but did not eradicate P. lingam or Alternaria spp. (Table 3). DISCUSSION The presence of X. campestris in crucifer seeds is well documented (6, 22). Using an improved technique (unpublished data), N.W.S. TABLE 1. Reduction of seed-borne X. campestris with hot water and hot acidified cupric acetate soaksa Germina- No. of seedlings' Treatment tionb (%) Healthy Black rot Cupric acetate, 55xd 633w ox 500C Water, 50 C 80Y 924Y 3Y Water, 25 C 90Z 720Z 24Z a Twenty Green Comet broccoli seeds from a lot of seed determined (14) to be 109%o naturally infested with X. campestris were added to 80 healthy cabbage seeds. Seeds were soaked for 20 min, washed in sterile distilled water, dried, and sown in steam-sterilized soil in a growth chamber with day and night temperatures of 26 and 22 C, respectively, for 10 days and then at 30 C for 3 days (14). All data are averages of 12 replications of 100 seeds. b Recorded 10 days after treatment. 'Recorded 28 days after treatment. d Values in each column followed by the same letter are not significantly different according to Duncan's multiple-range test; P c 0.05.

3 VOL. 39, 1980 TABLE 2. ERADICATION OF X. CAMPESTRIS IN SEED 805 Effect of cupric acetate treatments on seed germination of Brassica cultivars Seeds gemiinated' (%) Collard Cabbage Broccoli Brussels Treatment (Georgia Early Rio Red A&C Savoy Market Ruby (Spartan (PJroduts LS) Round Dan- A&C avoy Che-Snibel Early) Dutch Verde no. ish 5 King tain Prize Ball Cross) Water, 25'C 88X 97X 95X 86' 96X 94X 93X 96' 92X 95X 47X 90X Cupric acetate 250C NDc 84Y 79Y ND 82Y 69Y 52` 21Y 21"' 29" 28Y 2(Yz 350C ND 89Y 79Y ND 78Y 77Y 74Y 19YZ 15Z 32Y 22Y 18Z 400C ND 87Y 72Y ND 80Y 71Y 67"' 34Y 23Y 35Y 25Y 25Y 450C 84X 81Y 81Y 81' 77Y 64Y 50zz 28Y 11Z 2Z 137 3ZZ 500C 47Y 36Z ND 26Y 34Z ND 0"Z' 0Y' 0"7 0Z 0 1 ' Numbers are means of four replications of 100 seeds each by the American Association of Official Seed Analysts wet blotter method (1). b Values in each column followed by the same letter are not significantly different according to Duncan's multiple-range test; P c ND, Not determined. TABLE 3. Eradication of seed-borne X. campestris and reduction of seed-borne P. lingam and Alternaria spp. with cupric acetate treatments at 25, 35, and 400C Treatment No. of black rot No. of seeds with: srateno.ofhags rot X. campestrisb Bacteria" P. lingam' Alternaria spp.' Water, 250C 8.0' 3.0' 205.0x 12.7x 84.3x Cupric acetate 25 C 0.25Y 0.0y 29.4y 4.0y 51.2y 350C 0.0y O.OY 11.0Z 2.8y 32.2' 400C 0.0Y 0.0y 9.6z 1.0' 16.1"7 'Savoy King cabbage seeds naturally infected with X. campestris were treated and sown in steam-sterilized soil, incubated in a growth chamber with day and night temperatures of 26 and 220C, respectively, for 10 days and then in a greenhouse with day and night temperatures of 28 to 31 and 210C for 3 weeks. Means of eight replications of 500 seeds. b Savoy King cabbage seeds naturally infected with X. campestris were treated and plated onto 15-cmdiameter plates (250 seeds each) of nutrient-starch-cycloheximide agar (14). Means of four replications of 500 seeds. C Plated 100 Langedijker Autumn cabbage and 100 Tuffy cabbage seeds naturally infected with P. lingam and Alternaria spp., respectively, onto 2,4-D-soaked blotters, as described previously (9). Means of three individual experiments with four replications of 100 seeds each. d Values in each column followed by the same letter are not significantly different according to Duncan's multiple-range test; P c assayed 152 commercial seed lots for X. campestris from July 1976 to March 1977 and found 8 lots infected. Because a single infected seed in 10,000 is capable of initiating a relatively high level of disease incidence in transplant beds (15), complete eradication of the pathogen from seeds is most likely required for control of black rot in transplant beds. Although water soaks at 500C have been recommended for eradicating X. campestris from crucifer seeds (4, 5, 8), there has been no agreement upon the time of exposure. The original work reported by Clayton (5) was not definitive with respect to time; he simply suggested a 15- to 30-min exposure. Recommended exposure times have varied from 20 min for cauliflower and broccoli (4) to 25 min for cabbage and Brussels sprouts (4) and 30 min for all crucifers (21). Because we had only a limited amount of naturally infected seeds, we chose to use a 20-min exposure for our control hot water treatment. Our results agree with those of others (11, 18) in that X. campestris is not always eradicated from crucifer seeds by soaking seeds in water at 500C for 20 min. Chlorotetracycline soaks at room temperature (20) and at 500C for 25 min (13) have been shown to eradicate X. campestris from seeds of rutabaga (20) and Brussels sprouts

4 806 SCHAAD, GABRIELSON, AND MULANAX (13) soaked in suspensions of X. campestris. The latter authors concluded that the chlorotetracycline-hot water treatment also eradicated X. campestris from naturally infected seeds. However, their failure to isolate X. campestris on medium D-5 (10), a medium upon which many strains ofx. campestris fail to grow (16), and the statement that 1% of the plants resulting from chlorotetracycline-treated seed were infected were not convincing evidence of efficacy. We chose not to test the chlorotetracycline-hot water treatment because chlorotetracycline is an antibiotic of choice for several human infections and therefore should not be recommended for treating plants (2). Copper, on the other hand, is an effective bactericide (3, 12, 19) and is not used for human infections. Copper is an effective bactericidal agent because of its action on cellular proteins, but it is a poor surface-active agent (12). The most likely reason for the apparent synergistic effect of heat and copper that we observed (preliminary experiments and Table 3) is a membrane permeability increase of the seeds or bacterium or both (12). An increased permeability would allow the ionic copper to come into closer contact with membrane proteins of cells of X. campestris. Since the toxicity of copper toward Klebsiella aerogenes is influenced by the presence of its cell wall lipopolysaccharide (3), the apparent synergistic effect may be due to the alteration by heat of the secondary structure of lipopolysaccharide or proteins of cell membranes of X. campestris. The synergistic effect may also be due to the increased temperatures causing a decrease in the number of disinfectant molecules required for reaction with receptor molecules of the bacterial cells (12). It would be desirable if a single treatment could eradicate important seed-borne fungi as well as X. campestris. Although numbers of P. lingam- and Alternaria spp.-infected seeds were significantly reduced by the 400C cupric acetate treatment, the pathogens were not eradicated. Therefore, if seeds are severely infected with P. lingam or Alternaria spp. or both, they should be treated separately with benomyl-thiriam (7). Results showing a significant reduction in numbers of bacteria and the two seed-borne fungi -suggest the possible usefulness of the hot acidified cupric acetate treatment for microorganisms of other seeds or foodstuffs. Although X. campestris was successfully eradicated from crucifer seeds by the acidified cupric acetate treatment at 400C, such a treatment should not be used routinely because of the adverse effect on germination of some cultivars. Instead, only seed lots assayed (14) and shown to be infected with X. campestris should be APPL. ENVIRON. MICROBIOL. treated. After treatment, a sample of the treated seeds should be reassayed to confirm that the pathogen was eradicated. Seed companies would like to be able to offer pathogen-free seeds for sale, but to date no consistently effective or ecologically safe seed treatment method has been devised. Although this method is not uniformly safe, it is effective and can be cautiously used by seedsmen to clean stock breeder seeds as a part of an overall sanitation program in producing X. campestris-free seeds. ACKNOWLEDGMENTS The technical assistance of R. C. Donaldson (Georgia Experiment Station) is gratefully acknowledged. We thank NCR- 100 Regional Committee members, H. Humaydan, and Bob MacDonald for supplying infected seeds. This work was supported in part by Hatch project LITERATURE CITED 1. Association of Official Seed Analysts Rules for testing seeds. 60: Bacteriology Committee of American Phytopathological Society Antibiotic use for control of bacterial diseases of plants. Phytopathol. News 12: Bitton, G., and V. Freihofer Influence of lipopolysaccharides on toxicity of copper and cadmium toward Klebsiella aerogenes. Microbiol. Ecol. 4: Chupp, C., and A. F. Sherf Crucifer diseases, p In Vegetable diseases and their control. The Ronald Press Co., New York. 5. Clayton, E. E Vegetable seed treatment with special reference to the use of hot water and organic mercurials. N.Y. Agric. Exp. Stn. (Geneva) Tech. Bull. no Cook, A. A., R. H. Larson, and J. C. Walker Relation of the black rot pathogen to cabbage seed. Phytopathology 42: Gabrielson, R. L., M. W. Mulanax, K. Matsuoka, P. Williams, G. P. Whiteaker, and J. D. Maguire Fungicidal eradication of seedborn Phoma lingam of crucifers. Plant Dis. Rep. 61: Gay, D. J Vegetable seed treatments, p In The Extension Agronomy-Weed Science, Entomology, and Plant Pathology Departments, The Georgia Cooperative Extension Service, Athens, Ga. 9. International Seed Testing Association Cabbage etc. blackleg, dry rot canker. In Proc. Int. Seed Testing Assoc.. 30: Kado, C. I., and M. G. Heskett Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas and Xanthomonas. Phytopathology 60: Klisiewicz, J. M., and G. S. Pound Studies on control of black rot of crucifers by treating seeds with antibiotics. Phytopathology 51: Lamana, C., and F. M. Mallette Physical factors affecting bacteria, p In Basic bacteriology. The Williams & Wilkins Co., Baltimore. 13. Lockhart, C. L., C. 0. Gourley, and E. W. Chapman Control of Xanthomonas campestris in Brussels sprouts with hot water and Aureomycin seed treatment. Can. Plant. Dis. Surv. 56: Schaad, N. W., and R. Kendrick A qualitative method of detecting Xanthomonas campestris in crucifer seed. Phytopathology 65: Schaad, N. W., W. R. Sitterly, and H. Humaydan.

5 VOL. 39, 1980 ERADICATION OF X. CAMPESTRIS IN SEED Relationship of incidence of seed-borne Xanthomonas campestris to black rot of crucifers in the field. Plant Dis. 64: Schaad, N. W., and W. C. White A selective medium for soil isolation and enumeration of Xanthomonas campestris. Phytopathology 64: Schaad, N. W., and W. C. White Survival of Xanthomonas campestris in soil. Phytopathology 64: Srivasan, M. C., P. Neergaard, and S. B. Mathur A technique for detection of Xanthomonas campestris in routine seed health testing of crucifers. Seed Sci. Technol. 1: Starr, T. J., and M. E. Jones The effect of copper on the growth of bacteria isolated from marine environments. Limnol. Oceanogr. 2: Sutton, M. D., and W. Bell The use ofaureomycin as a treatment of swede seed for the control of blackrot (Xanthomonas campestris). Plant Dis. Rep. 38: Walker, J. C Black rot of crucifers, p In Plant pathology, 2nd ed. McGraw-Hill Book Co., New York. 22. Walker, J. C., and W. B. Tisdale Observations on seed transmission of the cabbage black rot organism. Phytopathology 10: Wilson, E. E., F. M. Zeitoun, and D. L. Fredrickson Bacterial phloem canker, a new disease of Persian walnut trees. Phytopathology 57: Downloaded from on August 26, 2018 by guest