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Michigan State ‘r‘f‘. um This is to certify that the dissertation entitled SEED TREATMENT FOR ERADICATION 0F COMMON AND FUSCOUS BLIGHT BACTERIA FROM NAVY BEAN SEED ; presented by Mintars ih Ad imihard j a has been accepted towards fulfillment of the requirements for Ph.D. degreein Crop & Soil Sciences for Major professor Date 1/M82 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 w: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from c1 rculetton records 34-45 _ V\ " yie'vllll ‘ \ «\\ ml 4 SEED TREATMENT FOR ERADICATION OF COMMON AND FUSCOUS BLIGHT BACTERIA FROM NAVY BEAN SEED By Mintarsih Adimihardja A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of CrOp and Soil Sciences 1981 ABSTRACT SEED TREATMENT FOR ERADICATION OF COMMON AND FUSCOUS BLIGHT BACTERIA FROM NAVY BEAN SEED By Mintarsih Adimihardja Common and fuscous bacterial blights are important diseases of dry edible beans. In Michigan, sources of primary infection for the diseases consist primarily of internally and externally contaminated seeds. There is no known acceptable method for eradicating internally borne blight bacteria. The objective of this study was to determine whether common and fuscous blight bacteria could be eradicated from contaminated navy bean seed by treatment in antibiotic/solvent combinations or other chemi- cals. The study was conducted in five steps: (1) effect of solvent on antibiotic activity; (2) ability of solvent to infuse antibiotic into the seed; (3) efficient method for contaminating bean seed with blight bacteria; (4) effect of treatment on eradication of blight bacteria from seed; and (5) effect of treatment on seed germination and field emergence . Mintarsih Adimihardja Effect of solvent on antibiotic activity, as assayed in Xantho- gangs seeded agar plates, depended on specific chemical/solvent combi- nation and bacterial isolate. All solvents, except dimethyl sulfoxide, were able to move anti- biotics into the seed when assayed in Bacillus subtilis seeded agar. Artificial inoculation with blight bacteria was more efficient in providing adequate amounts of infected seed for this study than field and greenhouse inoculations. Hot, acidified cupric acetate (0.1, 0.25, 0.5%) soaks at 500C for 20 minutes eradicated blight bacteria, provided that all seeds took up the chemical. However, such treatments greatly reduced seed germina- tion and field emergence. A treatment with tetracycline lKIl (800 ppm) :hi methanol under partial vacuum for two minutes eradicated seedborne bacteria completely from the seed and greatly reduced seed germination of two-year—old but not one-year-old seed. Therefore, the treatment may be useful for seeds less than (nu: year old. Seed immersion in 400 and 800 ppm of aqueous tetracycline and aureomycin, while giving good eradication, drastically reduced seed germination. A subsequent sodium hypochlorite rinse decreased seed viability beyond that caused by the antibiotic alone. Seed hmmersion in tetracycline HCl and aureomycin (800 ppm) for 30 minutes decreased the number of seed containing bacteria without impairing seed germination, and therefore, can be recommended for bean seed treatment. in memory of my mother: Mrs. Rd. HS. A. Adimihardja and to my sons: Ari and Adji ii ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation and grati— tude to the followings: Dr. Lawrence 0. Copeland, who served as chairperson of the Guidance Committee, for his helpful counsel in conducting the experi- ments, and his invaluable criticisms in the preparation of this manu- script. Dr. Alfred W. Saettler, who served as member of the Guidance Committee, for the opportunity to work with him, his supervision and guidance throughout the research study, and invaluable criticisms in the preparation of this manuscript. Drs. Donald Penner and Alvin J. M. Smucker, for their kind service in the Guidance Committee, helpful counsel in conducting the experi- ments, understanding and critical review of this manuscript. Dr. Charles Cress, for his ‘kindness and invaluable counsel in analysing the data. Linda, Mary, Connie, Sally, and Sidney for technical assistance. Dr. Sitanala Arsyad, Rector of Unversity of Lampung, for his kindness and encouragement throughout her graduate study. The government of the Republic of Indonesia, for the study-leave granted and scholarship given to study in the United States of America. iii Ir The Adimihardjas and Baruses, for their love and spiritual support. Eli, her husband, and Ari and Adji, her sons, for their help, love and understanding. iv TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . Names and Classification of Common and Fuscous Blight Bacteria . . . . . . . . . . Life Cycle of Common and Fuscous Blight Bacteria . . . . . . . . . . . . . . Control Strategies for Common and Fuscous Blights . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . . . Effect of Solvent on Antibiotic Activity . . . Ability of Solvent to Infuse Chemical into the Seed . . . . . . . . . . . . . . . . . Seed Contamination with Blight Bacteria to Provide Sufficient Amounts of Infected Seed Treatments for Eradication of Blight Bacteria . Effect of Treatments on Seed Germination and Field Performance . . . . . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . . . . . . . Effect of Solvent on Antibiotic Activity . . . Ability of Solvent to Infuse Antibiotic into the Seed . . . . . . . . . . . . . . . . . Seed Contamination with Blight Bacteria to Provide Sufficient Amounts of Infected Seed Effect of Treatments on Eradication of Blight Bacteria and on Seed Germination and Field Emergence . . . . . . . . . . . . . . . . SUMMARY AND CONCLUSION . . . . . . . . . . . . . . . LIST OF REFERENCES . . . . . . . . . . . . . . . . . Page vi 13 13 l4 17 21 23 25 25 28 37 46 71 75 l_ LIST OF TABLES Table Page 1. Sensitivity of g. phaseoli and g, phaseoli var. fuscans isolates to various chemical/solvent combinations 0 O O O O C O O O O O O O O O O O O O O O O 26 Detection of antibiotic activity in cotyledons and embryo axes of navy bean seed infused by various solvent using partial vacuum technique, as assayed on Bacillus subtilis seeded agar plates . . . . . . . . 29 Zones of inhibition in Bacillus subtilis seeded agar plates around cotyledons of navy bean seed soaked in three different antibiotic/methanol combinations at three concentrations, and three different immersion times . . . . . . . . . . . . . . . 30 Zones of inhibition in Bacillus subtilis agar plates around embryo axes of navy bean seed soaked in three different antibiotics/methanol combinations, at three levels of concentration, and for three different lengths of immersion time . . . . . . . . . . . . . . . 31 Zones of inhibition in E. phaseoli var. fuscans R10 seeded agar plates around the cut surfaces of navy bean seed soaked in three levels of two different antibiotic /methanol combinations, for three different lengths of immersion time . . . . . . . . . . . . . . . 33 Zones of inhibition in B. subtilis agar plates around the cut surfaces of Seafarer and Tuscola navy bean cultivars seed soaked in three levels of tetracycline hydrochloride/methanol combination for three different lengths of immersion time . . . . . . . . . . . . . . . 34 Number of seeds that gave small or no zones of inhibition, compiled from all experiments . . . . . . . 35 vi h—v Table Page 8. Infestation of navy bean seed with R. phaseoli var. fuscans (isolates white variant and R10) by partial vacuum technique using nutrient broth plus 1% dimethyl sulfoxide . . . . . . . . . . . . . . 39 \O o Infestation of navy bean seed with E. phaseoli var. fuscans white variant as a result of partial vacuum technique using three different immersing solutions . . . . . . . . . . . . . . . . . . 40 10. Recovery of R. phaseoli var. fuscans R10 from artificially infected seed incubated in three kinds of liquid media . . . . . . . . . . . . . . . . . 41 ll Presence of symtoms and recovery of §. phaseoli var. fuscans R10 from seedlings grown from artificially infected seed . . . . . . . . . . . . . . . 41 12 Presence of symptoms and recovery of E. phaseoli var. fuscans R10 from seedlings grown from artificially infected seed . . . . . . . . . . . . . . . 42 13. Recovery of R. phaseoli var. fuscans R10 from seedlings grown from seed artificially infected with different concentrations of bacteria inoculum . . 42 14. Populations of g. phaseoli var. fuscans R10 inside seed artificially inoculated with different bacteria concentrations . . . . . . . . . . . 44 15. Effect of artificial inoculation on seed germination and field emergence of Seafarer navy bean . . . . . . . . . . . . . . . . . . . 45 16. Detection of g. phaseoli var. fuscans white variant 1n the naturally infected seed after hot acidified cupric acetate treatment . . . . . . . . . 47 17. Detection of g. phaseoli var. fuscans white variant in artificially infected seed treated with 5000 ppm acidified cupric acetate containing 500 ppm Tween 80 . . . . . . . . . . . . . . 48 18. Effect of hot acidified cupric acetate treatment on seed germination of uninoculated Seafarer navy bean . . . . . . . . . . . . . . . . . . . . . . . 49 Table 19. Detection of X. phaseoli var. fuscans white variant in artificially infected seeds treated with hot (50 C) acidified cupric acetate containing sodium dodecyl sulfate . . . . . . . . . . . . . . . . . 20. Number of uncolored segds treated with hot acidified cupric acetate at 50 C for 20 minutes . . . . . . 21. Detection of X. phaseoli var. fuscans white variant in artificially infected seeds treated with hot (50 oC) acidified cupric acetate and 10 ,000 ppm Tween 20 . . . . . . . . . . . . . . . 22. Detection of X. phaseoli var. fuscans white variant in uncolored seeds resulting from artificially infected seeds treated with hot (50 OC) acidified cupric acetate and 10,000 ppm Tween 20 . . . . . . 23. Detection of X. phaseoli var. fuscans R10 in artificially infected seeds treated with 1000 ppm of hot (50 C) acidified cupric acetate and 5000 ppm of surface~active agent for 20 minutes . . . . . . . . . . . . . . . . . . 24. Effect of hot acidified cupric acetate treatment on germination and field emergence of artifically infected Seafarer seed with X. phaseoli var. fuscans R10 . . . . . . . . . . . . . . . . . . 25. Detection of X. phaseoli var. fuscans white varient in artificially infected seeds immersed in chemical/solvent combinations with partial vacuum technique . . . . . . . . . . 26. Detection of X. phaseoli var. fuscans white variant in artificially and naturally infected seeds immersed in tetracycline HCl/methanol with partial vacuum technique . . . . 27. Effect of partial vacuum treatment on eradication of blight bacteria from artificially infected seed . . . . . . . . . . . . 28. Effect of partial vacuum treatment on germination and field emergence of Seafarer navy bean seed artificially infected with with X. phaseoli var. fuscans white variant . . . . . . . . . . . . . . viii Page 50 52 53 55 56 57 59 60 61 63 Table Page 29. Effect of 30 minute soak in chemical/solvent combinations on eradication of blight bacteria from seed artifically infected with X. phaseoli var. fuscans R10 . . . . . . . . . . . . . . . . . . . . 65 30. Effect of soaking time in antibiotic/methanol combinations on eradication of blight bacteria from seed artificially infected with X. phaseoli var. fuscans R10 and seed germination of uninfected Seafarer navy bean . . . . . . . . . . . . . . . . . . . 67 31. Effect of 30—minute soak in 400 ppm of aqueous antibiotic on germination of uninfected Seafarer navy bean seed . . . . . . . . . . . . . . . . . . . . . 68 32. Effect of 500 ppm antibiotic followed by 5000 ppm sodium hypochlorite treatment on seed germination 0f seafarer navy bean O O I O O O C O O O O O O O O O 0 7O ix .7 INTRODUCTION Common and fuscous bacterial blights are two of the most serious seedborne diseases of dry edible beans (Phaseolus vulgaris L.) in many production areas of the world (64, 72, 85). In lesser—developed countries, losses are especially severe due to inadequate control practices. The diseases historically have been important in the United States, especially in Michigan, where approximately 35% of all dry edible beans and 85% of all U.S. navy beans are grown (66). In 1918, 75% of the fields in New York were affected by bacterial blights and serious crop losses resulted. In 1919, crop losses ranged from 40% to 60% of the crop in Colorado. The most severe losses due to common blight in 1921 were in Michigan where yields were reduced by 25% (83). In 1928 there were heavy losses from bacterial blights in several western and southern states (36). In some fields, the crop was almost a complete loss. Calculated on a weight basis, the loss of dry beans in the United States in 1936 was 34,700,000 pounds. Loss of 25 to 30% of field beans occured in Weld County, Colorado in 1937 (84). Andersen (3) estimated that bacterial blight caused a $3,500,000 loss to growers in three Michigan Counties in 1951. Yield reductions in Michigan in 1967 were 10 to 20% (53). In 1962, 60% of the navy bean fields in southwest Ontario, Canada, were infected with fuscous blight (72). 2 Sources of primary infection for the two diseases in Michigan consist primarily of internally and externally contaminated seeds (64). External contamination is easily controlled with streptomycin sulfate (66) and sodium hypochlorite (64). However there are no known acceptable methods for eradicating internally borne blight bacteria from the seed. The objective of this study was to determine whether common and fuscous blight bacteria could be eradicated from. navy bean seed by treatment of seeds in antibiotic/solvent combinations or' with other chemical treatments. An effective eradicative treatment would permit the development of foundation seed stocks or seed for breeding programs free from bacterial blight infestation. It was hypothesized that certain chemicals may eradicate internal common. and fuscous blight infections as long as the chemicals are bactericides which could penetrate the seed uniformly without affecting the viabiblity of the seed itself. if " : 'i'li I ' '- I '1 - 'l ' II I . ' _' " . I I _ '_.‘-) -. '-.- ":1: i u "|'[,|.'1a-,‘:'-. '- ' liM-"u'l': '1'" "1' I : ' -"= '1'" I1? " '- ‘I .-'_41_'('I - ' ‘. -: m .i '- - -.'t.r. -- - - ' .|'.-‘-.‘\ 'i'rr-' loo -- I- J (:3?) I f,‘ l I .l ' l 'I .0. l I . -u'.-=.'. -- ‘ - -.-:. :- :s-- - - -,- ' ' .. “4 -'.- .' I" ' I ' ' ' ' ' I f j'j' .3' I I ' ' I" . . ' i . ':". '-‘ I ' . I'I' . ‘ ' - U ', ' ‘_l'. " ' ' ' . . ‘J ' . " I" . Ii I' :..l ' ' ' ' “u: -!r-' - ' ’u. - - ' ' .I" 'JI . ' .- _, ., :1 ' n . .: . '0" q- - ' .. - ' .., --- ' 'n :‘ "I ' . "I . " I . ‘I - LITERATURE REVIEW Names and Classification of Common and Fuscous Blight Bacteria Common blight is incited by Xanthomonas phaseoli (E. F. Smith) Dowson anui fuscous blight by Xanthomonas phaseoli var. fuscans (Burk) Starr & Burk. In 1897 the common. blight organisar was named Bacillus phaseoli E. F. Smith (68), transferred into genus Pseudomonas in 1901 (69) and then into genus Bacterium in 1905 (70). Bergey's Manual ed. II (9) renamed the blight bacterium Phytomonas phaseoli (E. F. Smith) Bergey et_ El: and Dowson (23) placed the bacterium in the currently accepted genus of Xanthomonas. Xanthomonas phaseoli is presently considered ea pathovar of X. campestris (15, 24). According to Bergey's Manual 8th ed., Xanthomonas belongs to the new kingdom Prokaryotae, Division Scotobacteria, Class Bacteria and family Pseudomonadaceae (10). The bacteria are obligately-aerobic, gram negative, straight rods (0.4 to 1.0 by 1.2 to 3 micrometers), which generally produce a yellow nondiffusable pigment. The cells are Imatile in; a single polar fla- gellum. In addition, Xanthomonas phaseoli var. fuscans produces a brown, water soluble diffusable pigment (10). 4 Life Cycle of Common and Fuscous Blight Bacteria Primary infection is usually established through the planting of contaminated seed. Seedlings which develop from. infected seeds bear lesions on the cotyledons or primary leaves; bacterial ooze may accumu~ late during ‘humid conditions. Such infected plants serve an; primary sources for secondary disease spread to surrounding plants (85). Blight bacteria are located under the seedcoat and do not enter the cotyledons until germination. Imbibition of water swells the seed, resulting in rupture of the epidermal cells of cotyledons. Bacteria then enter the epidermal rifts and multiply in intercellular spaces; lesions eventually develop on the cotyledons (14). The bacteria may move sytemically in an infected plant. An im- portant feature of systemic infection is the ability of the bacteria to move from the stem xylem through the pod and then pass into the funicu- lus, and through the raphe to finally be harbored under the seedcoat with no trace of pod infection (12). Seeds with visible symptoms always are associated with visibly infected pods (81). The seeds :hi severely infected pods do not develop normally, and frequently remain shriveled and of no commercial value. In less severe disease situations, the seed develops normally and either develops no symptoms or becomes slightly wrinkled; in white seeded bean varieties, infected seeds may take on a. yellowish discoloration (14, 18). In Michigan, hilum-spotting is Una most important symptom of internally infected seeds (80). The bacterial blight orgnisms may persist as long as ten years in the seed (85). Aside from overwintering in seed, some investigators believe that the bacteria survive from one crop to another in infected plant refuse (18). Control Strategies for Common and Fuscous Blights Seed certification programs Most bean producing states maintain certification programs which oversee the quality of commercial seed. The Michigan Crop Improvement Association operates under authority delegated by the Michigan Depart- ment of Agriculture. The first step in certified seed production is the planting of foundation seed supplied by the Michigan Foundation Seed Association; such seed is usually grown in isolated areas of Michigan or in the semi-arid or arid west where conditions are unfavorable for seedborne diseases. Seed from fields which pass visual inspection for the presence of common and fuscous blights, and which show no internal contamination in laboratory tests, can be sold as certified (54). By using certified seed a grower minimizes the possibility of blight in his crop. Cultural practices Crop rotation, plowing under bean refuse after harvest, sanitiz- ing planting equipment and storage facilities, weed control, and staying out of fields when plants are wet, are recommended practices to minimize blight contamination (4). Disease resistant varieties Most field bean varieties grown in Michigan are tolerant to halo blight infection (85); unfortunately none is tolerant to common and fuscous blight. However, there are degrees of susceptibility among cultivars (26, 85), which suggests the possibility for breeding resistant varieties. But, Zaumeyer and Thomas reported that under condi- tions very favorable for blight development, all snap and field beans grown in USA are severely damaged by common and fuscous blights (85). Chemical control Field control Bordeaux mixture (11, 25), 5% puratized spray and cuprox dust (copper oxychloride) (10) have been evaluated for disease control but none gave complete control. Sprays of copper hydroxide (50%) and 40% potassium (hydroxymethyl) methyl dithiocarbamate was reported to be effective in controlling blight on the leaves, but not on pods (79); disease control was not accompanied by increase in yield. Seed treatment Bean seed treatment with slurry containing a bactericide such as streptomycin sulfate (66) or rinsing seed in sodium hypochlorite (64) effectively eliminates external contamination. The antibiotics aureo— mycin (2, 16, 40, 76) streptomycin (2, 7, 21), circulin (21), terra- mycin, polymixin (40), carbenicillin, chloromycetin, erythromycin, kanamycin and tetracycline (70) have shown activity against Xantho— monas phaseoli in vitro. Upon planting of treated seeds, 7 dihydrostreptomycin and streptomycin sulfate were apparently absorbed by the stem of bean seedlings and translocated upward to the primary leaves. Within three to four days the chemical accumulated in suffi- cient amounts to inhibit the growth and develOpment of halo and common blight organisms (50, 51, 52). Marlatt (46) found that streptomycin used as a spray or dust, and seed treatments failed to control common blight under field conditions in New Mexico. Gray (31, 32) showed that addition of glycerin to streptomycin sprays caused a marked increase in effectiveness of the antibiotic against common blight in greenhouse tests. Kreitlow (43) reduced blight incidence from 23.6% to 0.2%, and increased yields two to seven times by using: (1) 0.2% mercuric chloride in diethyl ether; (2) 0.005% brilliant green in 50% ethyl alcohol plus 3% acetic acid; (3) 0.2% mercuric chloride in 70% ethyl alcohol plus 3% acetic acid; and (4) 0.005% gentian violet in 50% ethyl alcohol plus 3% acetic acid as disinfectants. Seed treatment to eradicate internally borne blight bacteria have been recommended by several investigators, but such treatments are not in general use. Edgerton and Moreland (25) treated seeds for 18 to 20 minutes in a solution of either 20% benetol or 0.1% corrosive sub- limate. Person and Edgerton (61) immersed blighted seed in a solution of 0.2% mercuric chloride in 70% ethyl alcohol plus 2% acetic acid. Treatment of seed with dry heat at 80°C for 35 minutes followed by New Improved Ceresan for 24 hours was effective in reducing the number of diseased plants arising from infected seed (11). Prior to 1950, hot water treatment was the most commonly recommend- ed physical method to eliminate internally borne pathogens (35). The 8 standard method for eradicating black rot, caused by Xanthomonas campestris, from crucifer seed is a hot water soak at 500C for twenty to thirty minutes (20). In practice, such methods often fail to control the disease (67). The use of antibiotics such as streptomycin (42), and hot chlortetracycline soak (45) eradicated ‘X;_ campestris from crucifer seed, but often result in reduced germination and seedling vigor (38). Humaydan 33 El (39) overcame this disadvantage with a thirty minute soak in 0.5 (w/v) sodium hypochlorite following one hour soak in 500 ppm of either aureomycin, terramycin, or strepto- mycin. Organic mercurials have also been effective for eradication. of X; campestris from cabbage seed (20). Since the use of organic mercurials is no longer permitted by Environmental ProtectionAgency regulations, and since chlortetracycline is an antibiotic of choice for several human infections (8), Schaad _e_t_ _a_l (67) developed an alter— nate method for eradication of black rot bacteria from crucifer seed. The method involved soaking seed in 0.5% w/v cupric acetate dissolved in 0.005 N acetic acid for twenty minutes at 35, 40, 45, or 50°C. The treatments were equally efficient in eradication of the bacteria, but at 400C seed germination was the best whereas at higher temperatures, germination was much reduced. In recent time, attempts have been made to apply chemicals to dry seed by means of organic solvents. Meyer and Mayer (48) showed that the growth inhibitor, coumarin, could be applied to lettuce seed in organic solvent, and emphasized the possible significance of the approach to practical problems. Using radioautographic techniques, Anderson (5) showed that coumarin reached the seed only superficially when applied 9 as a solution in dichloromethane. Khan g£_ El (41) were able to introduce gibberelic acid and abscisic acid into lettuce seed using acetone and dichloromethane. Seed treated with gibberelic acid germinat- ed nearly 100% in the dark, while seed treated. with abscisic acid failed to germinate in the light. With the same solvents, Tao e_t a_1_ (74) were successful in maintaining seed quality of pea treated with pentachloronitrobenzene, lima bean treated with chlorpyrifos, and lettuce with chloramphenicol and puromycin. By means of 14C-IAA, Tao and Khan (73) showed that acetone carried chemicals through the seedcoat to the embryo during a two hour soak. The amount of chemical penetrating the seed depends on the seed type, permeation time, and concentration of the solution. In most cases, chemicals penetrating the embryo remained biologically active. Royse gE a; (63) reported antibiotic activity in soybean seed after' a. thirty’ minute soak in dichloromethane containing potassium penicillin G. Inhibition zones in bioassay tests of treated seed increased in size as soaking time and penicillin G concentration increased. In ea similar study, Ellis .EE El (28) reported that carboxin, but not captan and thiram, lost fungicidal activity when mixed with dichloromethane. Dichloromethane facilitated the movement of methyl 2-benzimidazolecarbamate and thiaben- dazole, but not of captan and thiram, into the soybean seed. Using acetone and dichloromethane containing 21 dye, it was found that the seedcoat of cottonseed is permeable to solvents and the dye; however the nucellar layer surrounding the embryo is impermeable, at least to the dye. It was suggested that the two solvents ‘may be suitable for surface application of chemicals to sound cotton seed, but not for introduction of chemical into the embryo (33). A similar study 10 was reported by Muchovej and Dhingra (55) on soybean seed. Benzene and ethanol allowed entry of benomyl and thiabendazole, but not of PCNB or chlorothalonil, into the seedcoat. None of the fungicides were carried into the cotyledons. Contrary to the findings by Tao 35 31 (74) and Royse e_t 11 (63), Muchovej and Dhingra (57) found that the quantity of the chemicals in the seedcoat did not increase with increased expo- sure time. These authors also reported (56) that benomyl and methyl thiophanate in dichloromethane were able to penetrate the cotyledons only of mechanically damaged soybean seed. On the other hand, Hepperly and Sinclair (37) reported that polyethylene glycol successfully carried potassium penicillin G and streptomycin sulfate into the seed- coat, hypocotyl, epicotyl, and cotyledons of soybean seed. Papavizas and Lewis (58) reported good introduction of the systemic fungicide pyrochlor into soybean seed with acetone; such treat- ment gave greater control of damping—off and root rot pathogens than when the chemical was applied in a water slurry. However, both systemic and nonsystemic fungicides applied to cotton seed using dichloromethane gave no greater disease control than when applied as wettable powders (32). Subsequent studies by Papavizas and Lewis (59) and Papavizas e_t a; (60) on organic solvent infusion method with acetone resulted in equal or better reduction in soil borne disease of cotton, pea, bean, and soybean, than when seeds were treated with direct application. Ralph (62) attempted to eradicate Pseudomonas phaseolicola from been seed by infusing dichloromethane containing erythromycin into infected seed. While the antibiotic was detected in both embryo and cotyledons, seedborne infection was not controlled. Muchovej and Dhingra (57) found that ethanol, acetone and benzene were equally ..'I.1" ':f'.'.; ,. .'_I . . \ ' ..'p ‘I ‘l ,1. ."I‘. 11 efficient :hi carrying benomyl and thiabendazole into the seedcoat of snap and dry bean seeds; poor results were obtained with thiOphanate methyl and fenopronil. The authors also found that dichloromethane was significantly more effective than trichloromethane and carbon tetrachloride in moving fungicides into the seedcoat. Significant interaction between seed type and solvent, seed type, solvent and fungicide in the infusion of fungicides into the seedcoat was reported (22). Most reports indicate that organic solvent infusion technique does not impair seed germination (37, 44, 49, 57, 63). Muchovej and Dhingra even stated that soaking soybean seed in ethanol and benzene signifi- cantly hnproved emergence over the control (57). Triplett auui Haber (75) suggested that the solvent itself was injurious when allowed to reach the embryo, which was then confirmed by Halloin (31) who showed that excised embryos and gin-damaged cotton seed were killed by organic solvents due 11) disruption.0m.NN cma.mH mmN.0 :00\0aom oaxaenmmz .mn m00.Ha eumm.s 00m.m~ um.00.wn xflnmz.0 0N0\0museOuaopum .ma xms0.0fi x0m.0a N00.0m 3mm.sw Mumm0.0 nonmeuwe\chsaowr=< .AH “00.0n nemn.0 www.0m smn.qN mm000.0 0 m\caus200.s< .0H Hm0.0a sflmz.0a som.mm 0A00.mz m0000.0 Hocmenms\amumEmupoe .ma mmm.an mm~.0 xmn.0m umt00.wm 000.m 0m20\cMUAEm..me .0H www.0H maeomm.m >0m.~N >m0.NN 0mm00.0 0Nm\caos20urms .ME m00.0n xmma.0m som.mm 0Q00.mm 0Mma.s acumenma\aamnosomtume .NH xmm.0a memz.0 som.sm xma.kN sflmom.0 0 m\mcnmusumpume .HH 900.5 mmN.0 amz.0N Hm0.0a m0m.0 Hocmeum\caEHnumeumz .0m onma.0 m00.0 >ma.NN xm00.0n 0mm.N 0020\cnsnaom0uwz .0 % 00m.0z m00.0 3>ma.m~ “00.NH mmN.0 0Nm\cnznnoaeumz .0 00.0 00.0 00.0 00.0 00.0 Hosanna: .A 00.0 00.0 00.0 00.0 00.0 Hosanna .0 00.0 00.0 00.0 00.0 00.0 accuwoa .m 00.0 00.0 00.0 00.0 00.0 Aomzov msmxomst Assamese .0 00.0 00.0 00.0 00.0 00.0 A2000 mamausaoroaeono .m 00.0 00.0 00.0 00.0 00.0 000 .N 00.0 00.0 00.0 00.0 00.0 Horuaoo .H owmum>< ofimMax >3mmx NH ax max sewumcflnfioo AEEV coaumnmLcM mo mGON ucm>H0m\HMU«Em£o mtOmumcfipEoo uco>HOm\Hmo«Ew£o wsoflum> cu mmuwHOmm wamomnm .um> HHoomwnm .M can Maoommsm .M mo xum>mufiwcmm .H manna 27 ummoq %n poSuo some Scum ucmquMHp haucmowmmcwwm uo: mc~.m m0.o u AH0.0Vnmn smm.mfi axmmm.HH 0mN.0H 0000~.m un00.m omo.e n0m.w mnmfi.w mm~.m mmm.o m0~.sm meomD.m owmuo>< ofimmmx o0k.sm 00¢.na 00.H 300.0m 000.00 summ.0m xmma.0a 0n.00.0a sHmH.HH u0m~.AH «n00.0 oam0.NH 000.0 300.0N atom.sfi >3max NH ax mmm.m ca00.~H mm0000.0 mmm.a nm~.0 ama.m xm00.0m Hm>oH NH um oocmquMMm unmowmwcwfim mum umuumfi mama osu kn mmBoHHom mcwoz Aqm.0ufimo.0vamqv mwmpm>< n ma .omm\fioomz .0N NH ma .0 m\Hoomz .mN Oman\oumuoom ofiumso .qN Omm\mumumom afiumso .mN OmZQ\cmoucmuamouufiz .NN Hoamnuo\fioomaonmamuofiso .HN cemumcwaaoo ucm>H0m\Hmofiao£o .A.0.uaoov H magma 28 activity but increased the activities of terramycin and aureomycin. Chloramphenicol had higher activity in ethanol than when applied in acetone. Antibiotic combinations giving greatest inhibition were tetra- cycline in water, aureomycin in methanol, aureomycin in water, tetracycline in methanol, and terramycin in methanol. Activities of certain antibiotics dissolved in water agreed with Trujillo's findings (76), but not with those of Katznelson and Sutton (40). Use of differ— ent bacterial blight isolates and assay techniques could account for the discrepancies (82). The use of acidified water significantly increased bactericidal activity of sodium hypochlorite. At pH 5, the solution had the highest concentration of HOCl, which is responsible for the actual oxidizing power (1). Available chlorine decreased as pH increased. The results could also have been affected by the solubility of antibiotics in water which affected the mobility in the agar midium. Ability of Solvent to Infuse Antibiotic into the Seed All solvents except DMSO, were able to move antibiotics into the seed. Antibiotic activity was found both in cotyledons and embryo axis of seeds treated with a partial vacuum technique (Table 2). There was little or no inhibiting activity associated with the embryo axis if the seed was soaked 30 minutes or less (Table 4). The magnitude of antibiotic activity against Bacillus subtilis both in cotyledons and embryo varied significantly with antibiotic/sol— vent combinations (Table 2). A twenty minute soak in tetracycline/5% Table 2. 29 antibiotic of navy bean Detection of embryo axes seed solvent using partial vacuum technique, Bacillus subtilis seeded agar plates activity in cotyledons infused by various as assayed on and ) Inhibition zone (mm)2) Chemical/solvent Cotyledon Embryo Average Axis 1. Untreated 00.00 00.00 00.00a 2. Tetracycline/5% methanol, 20 minute soak 10.25 10.75 10.50f 3. Chloramphenicol/ethanol 2.80 5.30 4.05bc 4. Methicillin/ethanol 8.13 6.75 7.44e 5. Nitrofurantoin/dimethyl 00.00 00.00 00.00a sulfoxide 6. Nalidixic acid/dichloromethane 3.05 5.38 4.21e 7. Tetracycline/methanol 4.50 5.30 5.78d 8. Streptomycin/5% methanol 5.75 5.80 5.78d 9. Streptomycin/5% methanol 3.25 3.50 3.78b Average 4.19 4.75 LSD(0.01) = 0.98 l) 800 ppm 2) Mean of two replications of ten seeds Values followed by the same letter are not significantly different by LSD at 1% level 30 Table 3. Zones of inhibition in Bacillus subtilis seeded agar plates around cotyledons of navy bean seed soaked in three different antibiotic/methanol combinations at three concentrations, and three different immersion times ---__‘-_"__‘_-""‘—---_5--T—'-—“_-_-""—'_T—_T-T-T___-—-—I) 5555 Immer31on Zone of inhibition (mm) Antibiotic Time ——————————— --- (minutes) 200 ppm' 400 ppm' 800 ppm' Tetracycline HCl 10 1.80bcde 2.05defg 2.05defg 20 2.90ij 2.70hi 2.50gh 30 2.75hi 2.65hi 2.80hi Terramycin 10 1.5abe 2.00def 2.30fghi 20 1.5abe 1.9cde 2.5fghij 30 2.5gh 2.15efg 2.25efgh Aureomycin 10 1.30a 1.40ab 1.65abcd 20 2.35fghij 2.35fghij 2.50ghijk 30 2.25efgh 2.00def 2.40ghij LSD (0.01) 0.48 1) Mean of two replications of 10 seeds Values followed by the same letter are not significantly different by LSD at 1% level 31 Table 4. Zones of inhibition. in. Bacillus subtilis agar" plates around embryo axes of navy bean seed soaked in three different antibiotics/methanol combinations, at three levels of concentration, and for three different lengths of immersion time Immersion Zone of inhibition (mm) Antibiotic time --- (minutes) 200 ppm 400 ppm 800 ppm Tetracycline HCl 10 Terramycin 10 Aureomycin 10 —_——-—---——--—_—_-——-—--———_--—_--——-—————-——————-—c—————-——_-————-_-_ IF 32 methanol was superior to all other antibiotic/solvent combinations with the partial vacuum technique. The magnitude of inhibiting activity was less with a soak of up to 30 minutes if the antibiotic was dissolved in 95% to 100% organic solvent (Tables 2, 3). Zones of inhibition generally increased with: (a) increased time of immersion up to two hours, and (b) antibiotic concentration up to 1600 ppm (Tables 3, 5, 6). This is in agreement with observations by Royse (63), who used dichloromethane to infuse penicillin into soybean seed, and those of Tao and Khan (73) who used acetone to move radio- actively labelled growth subtances into vegetable seeds. There was significant difference between Tuscola and Seafarer in magnitude of inhibition zones of seeds soaked in tetracycline HCl/me- thanol up to 1600 ppm for 30 minutes. Methanol was more effective in infusing aureomycin than terramycin into the seed, as assayed in pr R10 seeded agar plates. It was interesting that there were always a few seeds that gave little or no inhibition zones (Table 7). The number of such seeds seemed to depend more on antibiotic/solvent combinations than on time or concentration. How the seeds were processed and stored prior to treatment may influence this phenomenon. Bilayer Xanthomonas seeded agar plates apparently were not sufficiently sensitive to detect inhibition zones around cut parts of treated seeds. The largest inhibition zone from seed cuts was almost always around the hilum, suggesting that the hilum as the primary site of solvent entry. 33 Table 5. Zones of inhibition in .H‘ phaseoli var. fuscans R10 seeded agar plates around the cut surfaces of navy bean seed soaked in three levels of two different antibiotic /methanol combinations, for three different lengths of immersion time Time of Inhibition zone (mm)1) Antibiotic immersion (minutes) 400 ppm 800 ppm 1600 ppm Terramycin 30 8.36a 10.15abc 16.35gh 60 10.10abc 11.95cde 14.8 efg 120 8.82ab 10.65bcd 13.00def Aureomycin 30 13.50ef 16.50gh 19.00ij 60 15.05fg 18.05hi 20.251j 120 14.70efg 19.30ij 23.40k LSD (0.01) 2.37 1) Mean of two replications of 10 seeds Values followed by the same letter are not significantly different by LSD at 1% level 34 Table 6. Zones of inhibition in B. subtilis agar plates around the cut surfaces of Seafarer and Tuscola navy bean culti— vars seed soaked in three levels of tetracycline hydro- chloride/methanol combination for three different lengths of immersion time 1) Length of Inhibition zone (mm) Cultivar immersion (minutes) 400 ppm 800 ppm 1600 ppm Average Tuscola 30 3.69 6.00 6.29 5.32a 60 4.50 6.90 8.50 6.63bc 120 6.33 7.88 8.66 7.62cd Average 4.84a 6.93bc 7.82cd Seafarer 30 5.38 5.88 7.65 6.30ab 60 6.75 6.50 8.84 7.36de 120 6.75 8.25 9.32 8.11d Average 6.29b 6.88bc 8.60d LSD (0.05) for average values 1.25 l) . . Mean of two replications of 10 seeds each Values followed by the same letter are not significantly different by LSD at 1% level 35 Table 7. Number of seeds that gave small or no zones of inhibition, compiled from all experiments -—_——-—_—*-——---———_————_—_——nun—_unaunn—e—nnun—-nun--—_-—_-—_c—-———---_—-——--‘-—-‘~—_ Treatment No. of seeds ) out of 10 seeds Untreated 10 Seafarer, 400 ppm chloramphenicol/ethanol, vacuumed 4 Seafarer, 400 ppm methicillin/ethanol, vaccumed 2 Seafarer, 400 ppm nitrofurantoin/DMSO, vacuumed 10 2 1 5 Seafarer, 400 ppm nalidixic acid/DOM, vacuumed Seafarer, 400 ppm tetracycline/methanol, vacuumed Seafarer, 400 ppm tetracycline/5% methanol, vacuumed Seafarer, 400 ppm tetracycline/5% methanol, 20 minute soak 9. Seafarer, 400 ppm streptomycin sulfate/5% methanol, vacuumed 10. Seafarer, 200 ppm tetracycline/methanol, 10 minute soak 11. Seafarer, 400 ppm tetracycline/methanol, 10 minute soak 12. Seafarer, 800 ppm tetracycline/methanol, 10 minute soak 13. Seafarer, 800 ppm tetracycline/methanol, 20 minute soak l4. Seafarer, 400 ppm tetracycline/methanol, 20 minute soak 15. Seafarer, 800 ppm tetracycline/methanol, 20 minute soak 16. Seafarer, 200 ppm tetracycline/methanol, 30 minute soak l7. Seafarer, 400 ppm tetracycline/methanol, 30 minute soak 18. Seafarer, 800 ppm tetracycline/methanol, 30 minute soak l9. Seafarer, 200 ppm terramycin/methanol, 10 minute soak 20. Seafarer, 400 ppm terramycin/methanol, 10 minute soak 21. Seafarer, 800 ppm terramycin/methanol, 10 minute soak 22. Seafarer, 200 ppm terramycin/methanol, 20 minute soak 23. Seafarer, 400 ppm terramycin/methanol, 20 minute soak 24. Seafarer, 800 ppm terramycin/methanol, 20 minute soak 25. Seafarer, 200 ppm terramycin/methanol, 30 minute soak 26. Seafarer, 400 ppm terramycin/methanol, 30 minute soak 27. Seafarer, 800 ppm terramycin/methanol, 30 minute soak 28. Seafarer, 200 ppm aureomycin/methanol, 10 minute soak 29. Seafarer, 400 ppm aureomycin/methanol, 10 minute soak 30. Seafarer, 800 ppm aureomycin/methanol, 10 minute soak 31. Seafarer, 200 ppm aureomycin/methanol, 20 minute soak 32. Seafarer, 400 ppm aureomycin/methanol, 20 minute soak 33. Seafarer, 800 ppm aureomycin/methanol, 20 minute soak 34. Seafarer, 200 ppm aureomycin/methanol, 30 minute soak 35. Seafarer, 400 ppm aureomycin/methanol, 30 minute soak 36. Seafarer, 800 ppm aureomycin/methanol, 30 minute soak 37. Seafarer, 1600 ppm terramycin/methanol, 30 minute soak 38. Seafarer, 400 ppm terramycin/methanol, 60 minute soak 39. Seafarer, 800 ppm terramycin/methanol, 60 minute soak 40. Seafarer, 1600 ppm terramycin/methanol, 60 minute soak mNChU'I-PWNH O N 0 U1 0 U1 U1 0 U1 U1 0 U1 0 U1 U1 OOl-‘Ol—‘t—‘Nl—iHwawle—‘l—‘WOOHHt—it—IONOOHHW-b U1 U1 0 U1 U1 J E Table 7 (Cont'd.). 36 Treatment No. of seeds ) out of 10 seeds 41. 42. 43. 44 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56 57. 58. 59. 60. 61. 62 63 64. 65. Seafarer, 400 Seafarer, 800 Seafarer, 1600 Seafarer, 1600 Seafarer, 400 Seafarer, 800 Seafarer, 1600 Seafarer, 400 Seafarer, 800 Seafarer, 1600 Tuscola, 400 Tuscola, 800 Tuscola, 1600 Tuscola, 400 Tuscola, 800 Tuscola, 400 Tuscola, 800 Tuscola, 1600 Seafarer, 1600 Seafarer, 400 Seafarer, 800 Seafarer, 1600 Seafarer, 400 Seafarer, 800 Seafarer, 1600 Ppm ppm ppm Ppm Ppm ppm Ppm ppm Ppm Ppm Ppm: ppm, Ppm: PPm; Ppm: ppm, Ppm, ppm, ppm, Ppm: PPm: ppm, Ppm) ppm, ppm, terramycin/methanol, 120 minute soak terramycin/methanol, 120 minute soak terramycin/methanol, 120 minute soak aureomycin/methanol, 30 minute soak aureomycin/methanol, 60 minute soak aureomycin/methanol, 60 minute soak aureomycin/methanol, 60 minute soak aureomycin/methanol, 120 minute soak aureomycin/methanol, 120 minute soak aureomycin/methanol, 120 minute soak 30 minute soak in tetracycline HCl 30 minute soak in tetracycline HCl 30 minute soak in tetracycline HCl 60 minute soak in tetracycline HCl 60 minute soak in tetracycline H01 120 minute soak in tetracycline H01 120 minute soak in tetracycline HCl 120 minute soak in tetracycline HCl 30 minute soak in tetracycline HCl 60 minute soak in tetracycline HCl 60 minute soak in tetracycline HCl 60 minute soak in tetracycline HCl 120 minute soak in tetracycline HCl 120 minute soak in tetracycline HCl 120 minute soak in tetracycline HCl a U1 0 U1 u k." U1 - U1anl HOP—NHt-‘Ol—‘t—‘NNNHOOOOOOOD—‘OOP—‘b U1 U1U1 1) Mean of 2 replications 36 Table 7 (cont'd.). Treatment No. of seeds out of 10 seeds 41. Seafarer, 400 ppm terramycin/methanol, 120 minute soak 42. Seafarer, 800 ppm terramycin/methanol, 120 minute soak 43. Seafarer, 1600 ppm terramycin/methanol, 120 minute soak 44. Seafarer, 1600 ppm aureomycin/methanol, 30 minute soak 45. Seafarer, 400 ppm aureomycin/methanol, 60 minute soak 46. Seafarer, 800 ppm aureomycin/methanol, 60 minute soak 47. Seafarer, 1600 ppm aureomycin/methanol, 60 minute soak 48. Seafarer, 400 ppm aureomycin/methanol, 120 minute soak 49. Seafarer, 800 ppm aureomycin/methanol, 120 minute soak 50. Seafarer, 1600 ppm aureomycin/methanol, 120 minute soak 51. Tuscola, 400 ppm, 30 minute soak in tetracycline HCl 52. Tuscola, 800 ppm, 30 minute soak in tetracycline HCl 53. Tuscola, 1600 ppm, 30 minute soak in tetracycline HCl 54. Tuscola, 400 ppm, 60 minute soak in tetracycline HCl 55. Tuscola, 800 ppm, 60 minute soak in tetracycline HCl 56. Tuscola, 400 ppm, 120 minute soak in tetracycline HCl 57. Tuscola, 800 ppm, 120 minute soak in tetracycline HCl 58. Tuscola, 1600 ppm, 120 minute soak in tetracycline HCl 59. Seafarer, 1600 ppm, 30 minute soak in tetracycline HCl 60. Seafarer, 400 ppm, 60 minute soak in tetracycline HCl 61. Seafarer, 800 ppm, 60 minute soak in tetracycline HCl 62. Seafarer, 1600 ppm, 60 minute soak in tetracycline HCl 63. Seafarer, 400 ppm, 120 minute soak in tetracycline HCl 64. Seafarer, 800 ppm, 120 minute soak in tetracycline HCl 65. Seafarer, 1600 ppm, 120 minute soak in tetracycline HCl 0 U1 0 U1 0 U1U1 HOHNHHOHHNNNHOOOOOOOHOOH-b mmm U1U1 Mean of 2 replications 37 Seed Contamination with Blight Bacteria to Provide Sufficient Amounts of Infected Seed Field inoculation Less than 2% cu? all pods inoculated with blight bacteria yielded seeds with disease symptoms. This was due ‘mostly' to ‘hot and rainy weather during the growing season. The scratched pods dried quickly and this may have affected the viability and mobility of the pathogenic bacteria. The difficulty of finding the right stage of pod development to inoculate, tended to result in symptomless seed or shrivelled pods. Not all of the diseased seeds collected from the field yielded the correct bacteria when reisolated. Cafati and Saettler (15) recovered R 15-1 mutant of X_._ phaseoli from 70% of seeds contained in inoculat— ed pods of Tuscola variety. Greenhouse inoculation Almost 100% (If the inoculated pods contained seeds with visible symptoms, showing the advantge of greenhouse inoculation, where the environment can in; more easily controlled. The high relative humidity was maintained by wrapping the pods in a plastic bag immediately after inoculation, enabling the bacteria to survive longer and move into the seed through the funiculus. One disadvantage of greenhouse inoculation is the limited space, which limited the number of harvested seeds. The bacteria isolated from greenhouse seeds almost always yielded the original pathogen used in inoculating the pods, often in pure culture. Cafati and Saettler (15) recovered the R15—l pathogen from 68% of infected seeds contained in scratched, inoculated pods of Seafarer. 38 Artificial inoculation of seeds In a preliminary experiment it was found that 70% to 100% of the individually tested seeds were infected with the pathogen. Goth (29) stated that the level of infestation with the pathogen in this tech— nique depended upon the escape of bubbles from the funiculus; therefore the previous history of seed processing could result in different degrees of infestation. When seed was oven-dried after surface sterili- zation, levels of infestation were less than when dried at room tempera- ture (Table 8). This may be due to the so-called 'casehardening' when seed was force-dried, especially from high moisture contents. Drying seed at room temperature in thin layers was used for the remainder of this study. The use of 0.01 M phosphate buffer as immersing solution was not effective (Table 9). The use of nutrient broth plus 1% DMSO or BYE liquid medium gave similar results. The use of high and low concentra- tions of bacterial inoculum was not different for qualitative detec- tion of individual seed infection (Table 9). The use of SSM as an incubation medium did not completely inhibit all contaminants. In fact, similar results were obtained by using cycloheximide BYE or modified cycloheximide BYE (Table 10). Seventy-eight percent of seedlings grown from inoculated seeds developed symptoms in the mist chamber. This compares favorably with Goth's work (29) in which infected seedlings ranged from 60 to 85%. He noted no difference in percentage of infected plants between varieties. Pathogenic bacteria were recovered from all seedling parts (Tables 11, 12, 13), even parts with no symptoms. Stem lesions were the most prevalent of all symptoms in plants grown in the mist chamber. In 39 Table 8. Infestation of ‘navy ‘bean seed 'with. X. phaseoli 'var. fuscans (isolates white variant and R10) by partial vacuum technique using nutrient broth plus 1% dimethyl sulfoxide Number of seeds containing bacterial) "'- 2) _ 33__-_-_____-_-: ______ Pretreatment prwv prRlO Average % oven-dried4) 11.0 12.0 11.5 57.5 room temperatures) 16.5 18 17.5 87.5 Average 13.75 15 Mean of 2 replications of 20 seeds each incubated in 5 ml SSM/seed, streaked on YCA incubated in 5 ml rifampin—cycloheximide BYE liquid medium/seed, streaked on RAM 4) before inogulation seed were surfaced sterilized and dried in oven of 31 C 5 . . . . . ) before inoculation the seeds were surface sterilized and dried at room temperature 40 Table 9. Infestation of navy bean seed with X. phaseoli var. fuscans white variant as a result of partial vacuum technique using three different immersing solutions % seeds Immersing solution containing .1) bacteria PhOSphate buffer, 0.01 M, pH 7.2 0 Nutrient broth plus 1% DMSO (0.85 A620 of bacterial suspension) 85% BYE liquid medium with 0.85 A620 of bacterial suspension 90% BYE liquid medium with 0.32 A620 of bacterial suspension 85% BYE liquid medium with 0.03 A620 of bacterial suspension 85% mean of two replications of 20 seeds each 41 Table 10. Recovery of X. phaseoli var. fuscans R10 from artificially infected seed incubated in three kinds of liquid media Incubating medium % seeds containing X f wvl) IQISEQIQEEQE ___________________________ 9o Cycloheximide reduced-BYE 90 Semi Selective Medium 82.5 mean of 4 replications of 10 seeds Table 11. Presence of symtoms and recovery of X. phaseoli var. fuscans R10 from seedlings grown from artificially infected seed % seedlings with prR10 recovery2 . 1) -_- stem 1e31ons --— - --------------- stem around stem near lst leaves decayed cotyledons lst leaves no obvious plants symptoms 78 ++ ++ ++ ++ 1) mean of two replications of 100 seeds streaking from two sample of 5 plants except for decayed plants ++ high numbers of bacteria colonies on RAM plates I. ' l r ._, v. I. -» .. l . I . . .’ ‘ I "lfiBT — I- l . I I ‘ III. III I I‘ I , ' I -'. l . . "‘n: . L I’I ‘: .- ..—,.—-[ ‘ n’ ‘_. .13” - I 4 I. I ‘l II'I‘I', I | 'il. I - I ‘ ~i I \ I O I ' e‘ll' - ,. . ., ., . I . U I . . J .. ‘ I .l I I J . II. l {-1 , ‘ ‘ l ' l . . 42 Table 12. Presence of symptoms and recovery of X} phaseoli var. fuscans R10 from seedlings grown from artificially infect- ed seed ‘pr R10 recovery A620 0f EEE R10 % seedlings with 1 --------- ___ . inoculum cotyledon lesions cotyledons symptomless with symptoms 0.75 88 ++ + 0.45 78 ++ + 0.36 66 ++ + grown in the mist chamber ++ high of bacteria colonies on the RAM plates + low of bacteria colonies on the RAM plates Table 13. Recovery of X. phaseoli var. fuscans R10 from seedlings grown from seed artificially infected with different concentrations of bacteria inoculum A620 of Decayed plantsl) leaves with symptomless pr R10 --------------------------- symptomsl) leavesl) inoculum No. of plants R10 recovery 0.75 5 ++ ++ + 0.45 3 ++ ++ + 0.36 3 ++ ++ + .--————-_‘_—‘~——_~————--_-e—---“~——“-——-~——-—~—-~-——--———-—_————-_--—— seedsowere germinated in the mist chamber, the pots then removed to 30 C growth chamber + low numbers of bacterial colonies on RAM plates ++ high number of bacterial colonies on RAM plates 43 plants grown in the 30°C growth chamber after germination in the mist chamber, leaf lesions were the most prominent. The leaf lesions were first seen about five days following transfer or three days after the polyethylene bags were opened. The symptoms were more prominent one month after planting. Some of the seedlings decayed soon after emergence of the first leaves and they yielded large numbers of blight bacteria. The symptoms appeared similar to those described previously for bean bacterial blight (12, 13, 14, 84), thus the technique appeared to duplicate the natural sequence of infection. The recovery of pr R10 from diseased and symptom—free cotyledons, hypocotyls, first nodes, and first leaves, supports Weller's conclusion that all above— and below-ground parts of seedlings from infected seeds are colonilized by blight bacteria soon after germination (78). Numbers of bacteria inside the seed by artificial inoculation ranged between 5 x 103 to 4.8 x 105 cells (Table 14). The concentra- tion of bacteria in the suspension did not seem to affect these numbers appreciable, except for 0.2 at A620 inoculum. However, it seemed to influence the number of seedlings having blight symptoms (Table 12). Weller and Saettler (81) found that the lowest number of bacteria in hilum spotted seed was 1.8 x 105. The number produced by artificial inoculation was similar to that from natural infection. Artificial inoculation did not affect laboratory or field germina- tion (Table 15). Even in the field, plants grown from artificially infected seeds grew more vigorously than the control seeds. In this case, the treatment was similar to presoaking which has been shown to accelerate germination of several grass species (17). The technique described in this study could be an answer to one of L"? . -. -., . . .I I ., . ‘JI‘ I 'I' 51ncls ., . - ., I, . I . . 'I . .. . 44 UmumCHewud—OU .HO wmhwuumfi GMGHCOU HOG @H—v 1 AH --wmmu ...... H ........ H ........ “----mem---flw---Hww.m...mn...-u...am---wmwmNEH ..... mm- 00Hx~.~ . I u . .00Hx0.a .m00x0.m .00me.m .momxm.m .00Hx0.0 ma.0 m0ax0.a - I - .00me .m0Hx0.H .0000H .00Hxn .000x0.m .monx00.m m0.0 m0000.H I u . .00me.m .00Hxn.m .00me.a .monxm.a .moaxh.~ .m0ax0.n 0.0 m0Hx~.m . . .000x0.0 .m0H00.m .monxm.a .00Hx0.0 .00Hx0 .monxa.m .moaxm.0 00.0 --..mmm ...... ... ........ H ........ ”----Hsmmhmam:new---“...mwmmfimHmmmm ..... mm- cum: 0 0 A 0 m 0 m N a --..-..--....---..------------------..EmWm.Manama-“mmwflmww .................... as mcofiumuuooocoo wwuouomn ucwuowmwv sums wouwasoocw mfifimmowwfiuum comm memmcw cam manomam .um> wfioommnm .m.wo mnemumfioaom .qH mfian 45 Table 15. Effect of artificial inoculation on seed germination and field emergence of Seafarer navy bean Treatment % germination Laboratery testl) Field—emergence _- ESQZEQI IIIIIIIIIIIIIIIIIIIIIIIII 95.3 575 IIIIIIII Artificial infected seed 92 55.5 mean of 3 replications of 100 seeds mean of 4 replications of 100 seeds 46 the obstacles described by Guthrie (31) to the development of a suc- cessful routine identification method. He stated that the first obsta- che was to obtain a source of seed with a constant and reliable level of seedborne bacteria. Effect of Treatments on Eradication of Blight Bacteria and on Seed Germination and Field Emergence Hot acidified cupric acetate treatments Results of eradication of blight bacteria using naturally infected seeds were inconsistent (Table 16). This was believed partly due to contaminating bacteria that overgrew blight bacteria and partly to uninfected seeds which appeared to have the bacterial symptoms. The use of artificially infected seed appears to be a better procedure for the study, even though contaminating bacteria still persisted. Semi- selective medium did not appear to work well (Table 17). Seed soak treatments of 5000 ppm of cupric acetate in 0.005 N aceth: acid for 20 minutes seemed to work well at 5000 (Table 17) but germinathmn was greatly reduced (Table 18). There were no differences (Tables 19, 21) between the use of SDS and Tween 20; however with Tween m) the blue color on treated seeds appeared more uniform. SDS, a pro- tein denaturing agent, was expected to work better alone or in combina- tion with cupric acetate, but when used alone did not eradicate blight bacteria at any temperature or soaking time. This may have been due to poor uptake of the chemical by a few seeds. The inconsistent uptake 47 Table 16. Detection of X. fuscans ‘white variant phaseoli var. in the naturally‘ infected seed-_Eff2fr hot acidified cupric acetate treatment I IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII if; IIIIII {JQIIQEIIII Time of Temperature tubes Plates soak of Treatment showing containing minutes solution turbidity pr.wv IIISIIIIIIIIZSQ IIIIII 63.133173};2;;IIIIIIIIIIIISIIIIIIIIII IIIIII 5 room 5000 ppm Cupric acetate 15 - 35°C 5000 ppm Cupric acetate 16 — 400C 5000 ppm Cupric acetate 13 l 450C 5000 ppm Cupric acetate 17 - 500C 5000 ppm Cupric acetate 17 - 10 room 5000 ppm Cupric acetate 14 l 350C 5000 ppm Cupric acetate 19 1 400C 5000 ppm Cupric acetate 19 1 450C 5000 ppm Cupric acetate 15 ~ 500C 5000 ppm Cupric acetate 19 - 15 room 5000 ppm Cupric acetate 19 1 350C 5000 ppm Cupric acetate 14 - 400C 5000 ppm Cupric acetate 16 1 450C 5000 ppm Cupric acetate 20 - 500C 5000 ppm Cupric acetate 18 - 20 room 5000 ppm Cupric acetate 15 1 350C 5000 ppm Cupric acetate 16 1 400C 5000 ppm Cupric acetate 10 1 450C 5000 ppm Cupric acetate 7 - 500C 5000 ppm Cupric acetate 13 - I) IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Out of 20 seeds 48 Table 17. Detection of X. phaseoli var. fuscans white variant in artificially infected seed treated with 5000 ppm acidified cupric acetate containing 500 ppm Tween 80 Soaking Soaking No. of tubes/seeds time temperature containing (minutes) (0C) bacterial) 0 0 17 5 40 8 45 6 50 4 10 40 4 45 4 50 4 15 40 7 45 4 50 3 20 40 5 45 2 50 0 1) Out of 20 seeds 49 Table 18. Effect of hot acidified cupric acetate treatment on seed germination of uninoculated Seafarer navy bean % Germinationl) 00.1925 IIIII 5000 ppm cupric acetate at 45°C for 15 minutes 35 5000 ppm cupric acetate at 500C for 5 minutes 82.5 5000 ppm cupric acetate at 50°C for 10 minutes 47.5 5000 ppm cupric acetate at 500C for 15 minutes 19.5 1) Mean of 4 replications of 50 seeds . , . v ‘1" I ,y . .' I I:- f n 1103' 50 Table 19. Detection of X. phaseoli var. fuscans white variant in artificially infected seeds treated with hot (50 OC) acidified cupric acetate containing sodium dodecyl sulfate Soaking Cupric SDS Uniformity Presence time acetate of seed of 1) (minutes) (ppm) (ppm) blue color pr.wv 0 0 0 - + 5 0 1000 — + 1000 1000 poor + 1000 5000 poor + 1000 10000 poor + 2500 1000 poor + 2500 5000 poor + 2500 10000 poor + 5000 1000 poor + 5000 5000 poor + 5000 10000 poor + 0 5000 - + 0 10000 - + 10 0 1000 - + 0 5000 - + 0 10000 - + 1000 1000 poor + 1000 5000 poor + 1000 10000 poor + 2500 1000 poor + 2500 5000 poor + 2500 10000 poor + 1000 1000 poor + 2500 5000 poor + 2) 5000 10000 poor + 20 0 1000 - + 0 5000 - + 10000 - + 1000 1000 poor c 51 Table 19 (cont'd.) Uniformity of seed blue color good good good good good good good good °f 1) Soaking Cupric SDS time acetate (minutes) (ppm) (ppm) 20 1000 5000 1000 10000 2500 1000 2500 5000 2500 10000 5000 1000 5000 5000 5000 10000 1) 2) Mean of three flasks containing 20 grams of infected seeds + at least one flask contained pr wv; detected on YCA plates - none of the flasks contained pr wv; detected on YCA plates c all flasks were contaminated Colored and uncolored seeds were separated, assayed separately 52 Table 20. Number of uncolored seeds treated with hot acidified cupric acetate at 50 C for 20 minutes Cupric acetate SDS No. of Detection of (ppm) (ppm) uncolored seeds ) pr wvz) 1000 1000 10 + 1000 5000 7 + 1000 10000 4 + 2500 1000 13 + 2500 5000 9 c 2500 10000 1 - 1) Out of 60 seeds 2) Mean of three replications of three flasks containing uncolored seeds; + at least one flask contained pr wv; detected on YCA plates - none of the flasks contained pr wv; detected on YCA plates c all flasks were contaminated 53 Table 21. Detection of X. phaseoli var. fuscans white variant in O O U ? m. o art1f1c1ally infected seeds treated w1th hot (50 C) acidified cupric acetate and 10,000 ppm Tween 20 Soaking time Cupric acetate Uniformity of Presence of (minutes) (ppm) seed color pr wvl) 5 1000 poor c 2500 poor - 1000, vacuumed good + 10 1000 good - c 2500 good - c 20 1000 good - c 2500 good c c 0 0, untreated poor + 1) Conclusion from three replications of 20-gram sample of infected seeds + at least one flask contained pr wv; detected on YCA plates - none of the flasks contained pr wv; detected on YCA plates c all flasks were contaminated - c only one replication did not contain pr wv; the two others were contaminated 54 phenomenon was very clear with cupric acetate treatments (Tables 21, 22, 23). When colored and uncolored seeds were assayed separately, the colored seeds did not contain blight bacteria, while uncolored seeds did. This accounts for the inconsistent results. The proportion of uncolored seeds decreased with increasing SDS concentration (Table 20), time (Table 22) and temperature (Table 24). However, uncolored seeds still occured after treatment in 2500 ppm of cupric acetate containing 10,000 ppm SDS at 50°C for 20 minutes (Table 20). Serological and plant injection tests for detection of blight bacteria did not perform as expected (Table 23). Although bacteria were detected by individual seed assays, they could not be detected by serological and plant injection tests. The reason for this failure was not clear. On the other hand, the pod injection test was successful. One disadvantage of this test is unpredictable availability of freshly harvested pods. The use of rifampin resistant M R10 bacteria was more efficient than Xpi wv for bacterial detection, since it allowed better control of contamination. Decreasing the concentration of cupric acetate to 1000 ppm did not improve seed germination (Table 24) and it seemed that decreasing the number of uncolored seeds also decreased germination. At 1000 ppm cupric acetate concentration and 500C, bacteria were effectively killed. Field emergence of the treated seed was also very poor. SDS alone at 50°C for 20 minutes decreased germination drastically, while H20 under similar soaking time and temperature conditions only slight- ly decreased germination. The use of partial vacuum at 500C was help- ful in infiltrating copper uniformly, but such treatment also killed 55 Table 22. Detection of .X. phaseoli var. fuscans *white ‘variant in uncolored seeds resulting from artificially infected seeds treated with hot (50°C) acidified cupric acetate and 10,000 ppm Tween 20 Soaking time Cupric acetate No. of Presence of (minutes) (ppm) Uncolored seedsl) pr wv2) 5 1000 25 + 2500 28 + 10 1000 18 + 2500 18 + 20 1000 5 + 2500 4 + 1) Out of 60 seeds 2) + at least one flask contained pr wv; detected on YCA plate 56 Table 23. Detection of 'X. phaseoli var. fuscans R10 in .arti- ficially infected seeds treated with 1000 ppm of hot (50°C) acidified cupric acetate and 5000 ppm of surface-active agent for 20 minutes Presence of‘pr R10 as detected by1 Surface- ------------ -—-- active Individual Manitou plant Serology Flask Pod agent test injection plated pathogenicity (pathogenicity) test SDS, colored seeds — — - — SDS, un- colored + — — + + Tween 20, colored seeds Tween 20, uncolored + - - + + 1) Data from 4 replications; + detected - not detected 57 Table 24. Effect of hot acidified cupric acetate treatment on germination and field emergence of artifically infected Seafarer seed with X. phaseoli var. fuscans R10 Number of Warm Field Treatment uncolored germination emergence seeds, % test, % % Control 94.5 57.5 H20, 500C, 20 minutes 87 - 1000 ppm cupric agetate, 5000 ppm SDS, 50 C, 20 minutes 11.5 18 0.25 1000 ppm cupric acetatg, 5000 ppm Tween 20, 50 C, 20 minutes 10.5 16.5 1.0 1000 ppm cupric acetatg, 5000 ppm Tween 80, 50 C, vacuum - 3.2 - 1008 ppm cupric acetate, 50 C, 20 minutes 15.5 19 - 1000 ppm cupric acetate, room temperature, 20 minutes 74.5 87 - SDS 5000 ppm, 500C, 20 minutes - 18 - Mean of four replications of 100 seeds - was not conducted 58 the seed. As Schaad e_t 3.1. (67) pointed out, the synergistic effect of heat is probably due to increased membrane permeability of seeds or bacteria or both. Since seed germination. was greatly reduced, this effect apparently applied to both. The authors further stated that increased permeability allowed cepper ions to come into closer contact with the membrane protein of bacterial cells. They suggested the possibility that the synergism was due to heat alteration of secondary structure of lipopolysaccharide component of Xanthomonas campestris cell membrane. Effectiveness (If the treatments may also be due to the increased temperature, causing a decrease in number of disinfectant molecules required for reaction with receptor molecules of bacterial cells. The killing of the seed also may be due to the vulnerable position of the radicle in the bean seed. Consequently, this treatment cannot be recommended for bean seed treatment. Organic solvent infusion technique 1. Partial vacuum technique Five treatments were promising (Table 25), but in individual seed assays, only tetraccline/methanol gave 1K) evidence of bacterial growth after a ten day incubation (Table 27). Four hundred ppm of tetracycline in methanol worked well for 40 grams of artificially infected seeds, but not for lO-seed samples of naturally infected seeds (Table 26). The latter was probably due to lack of absorption of the chemical by some seeds. The success of tetracycline in methanol with partial vacuum in eradicating seedborne blight bacteria from the seed could be [.- 59 Table 25. Detection of X. phaseoli var. artificially infected seeds immersed combinations with partial vacuum technique in fuscans white variant in chemical/solvent Detection of Chemical/solvent Concentration prwv on 1) YCA plates Control + Acetone + Chloramphenicol/acetone 400 ppm + Chloramphenicol/acetone 800 ppm + Streptomycin in 2 ml H O/acetone 400 ppm + Streptomycin in 2 ml HZO/acetone 800 ppm + Ethanol Chloramphenicol/acetone 400 ppm + Chloramphenicol/acetone 800 ppm + Methicillin/ethanol 400 ppm + Methicillin/ethanol 800 ppm -c Dichloromethane + Nalidixic acid/dichloromethane 400 ppm + Nalidixic acid/dichloromethane 800 ppm + Dhmethyl sulfoxide (DMSO) + Nitrofurantoin/dimethyl sulfoxide 400 ppm + Nitrofurantoin/dimethyl sulfoxide 800 ppm + Methicillin/dimethyl sulfoxide 400 ppm + Methicillin/dimethyl sulfoxide 800 ppm -c Cupric acetate/DMSO 5000 ppm -c Terramycin/DMSO 400 ppm + Terramycin/DMSO 800 ppm —c Methanol + Tetracycline HCl/methanol 400 ppm — Tetracycline HCl/methanol 800 ppm - Tetracycline HC1/5% methanol 800 ppm + Streptomycin, Tween 80/H O 800 ppm + Terramycin, Tween 80/H20 800 ppm + 1) Data from two replications; + at least one flask contained pr wv - none of the flasks contained pr wv c one flask was contaminated; the other did not contain pr wv 60 Table 26. Detection of X. phaseoli var. fuscans white variant in artificially and naturally infected seeds immersed in tetracycline HCl/methanol with partial vacuum technique Detection of pr wvl) Concentration of tetracycline artificial natural (ppm) infection infection 0 (control) + + 50 + + 100 + + 200 + + 400 - + 800 - - 1) Data from three replications of 20 gram sample of artificially infected seeds and of 20—naturally—infected—seed samples + at least one flask contained pr wv - none of the flasks contained pr wv 61 wouooumv mus OHM WNW + wouoouov no: mma OHM w x I mcofiumomammu know Eouw mung 3 + - - + 0.00 Hoppcoo + I u + m.m 000 0020\capssmppa + I I + mn.o cow Omza\a«HH«oHnuoz + - I + m~.H 000m 0020\0000000 00.000 0.0 oow Hocmzuoe msoosvm\ocHHomomuuoH I . I I I 0 com Hocmsuoa\oc«aozomuuwa I I I I m.N oom Hocweuo\fioowcmnmamuofino mpwom ON mo coauooncH coauoonca wdmumfim uso uwou Ema coHumcanEoo AH . vow awofloHOHom AH . unmfim AH xmmam Hmsufl>fiecH ocoo uco>HowVHmowszo 3 meow wouoowcm xfiamfiomwfluum Scum emuouomn unwman mo cemumomkuw co ucoaumwnu Essum> ammunma mo uommmm .NN oHpmH 62 explained by any combination of the following: (a) With the escape of air from the seed and the pressure of air coming into the suction flask, methanol carried tetracycline HCl into the seed uniformly. All bacteria were therefore exposed to the antibiotic. (b) All seeds took up the antibiotic. (c v Seed(s) that did not take up tetracycline HCl, did not contain bacteria. (d) The incubation period was not long enough to allow bacterial multiplication. The length of incubation and the amount of the liquid medium to give the best result in detecting bacteria in small numbers needs further study. (e V Tetracycline HCl is a potent bactericide. Individual seed assays and plating appeared to be the Inost sensitive means for detecting internally borne bacteria. Mass incubation assays could be confused by the overgrowth of contami- nants from one or a few seeds in the sample. In individual seed assays, the overgrowing bacterial contaminants only interfered with certain tubes. The remaining tubes were still able to show possible growth of blight bacteria. Contaminating bacteria generally grew much faster than blight bacteria, and overtook the space and the nutrient supply for bacterial growth. Pod injection was sensitive in detecting bacterial pathogenicity but, as has been discussed previously, had severe disadvantage. Introduction by partial vacuum (of antibiotic/organic solvent into the seed reduced germination severely (Table 28). Such treatments also reduced field emergence more than germination as 63 mpoom ooH mo mGOmumoflamou know we cmoz UQUUDUfiOU HOG wm3 l A0 0.00 0k.0 0.00 0k.00 00 200 000\0020\0000Empp00 0.00 0 0.00 00 0.50 500 000\0020\00000000002 0.0a 0.0 H.N0 0k.k0 . E00 0000\0020\0pmpppm papaso m mn.¢ ¢.Nm m.m¢ mm Ema oow\HOGM£umE\oa0Hu%omuuoH 00.~ 50.0 0“ 0N 0.00 900 000\Hopmepp\HoomcmeaampoHeo I 0.N0 I 00 00 0000000 poem moon vac comm vac vacuumomlm poem UHOIumomIN Inmmhlm Inmm%IH Houucoo mo oocowuoeo mo IIIIIIIIIIIIIIIIIII cofiumHDCwoaoo\ucm>HOm\Hmomawno um>o N vfimmm N Houucoo uo>o N GOMumcfiEpow EHmB N I unswuw> 600:3 mcmomsm .um> HHoommnm .x 5003 wouoomcw zfiamwomwmuum comm coon h>mc noummmom mo wocmwumam vfiwfiw mam acmumcwaumw co unmauwmuu Essom> Hmduuma mo mowmmm .wN wapmH 64 indicated by the percentage of seedling emergence of treated seeds over control. Warm germination of one—year-old seeds treated with antibiotic/organic solvent using partial vacuum technique was much better. Huber and Gould (38) found that hot water treatment of high vigor cabbage seeds resulted in no injury but three-year-old seed of certain varieties was definitely injured. It was probably due to the loss of semipermeability of the membrane of cells surrounding the embryo of the aging seeds. Antibiotic activity was found in the embryo, when antibiotic/organic solvent was applied with partial vacuum. The reduced germination was probably due to the penetration of antibiotic thrOugh the membrane of cells around embryo axis. Since the treatment only slightly decreased seed germination of one year old bean seeds, it can be suggested for such seeds. Two years of field trial under weather conditions favorable for bacteri— al blight epidemy should be performed before recommendation of the treatments . Non-vacuum immersion Immersion of artificially infected seeds in aqueous tetra- cycline and aureomycin solution gave complete eradication. (Table 29), provided that all seeds imbibed the liquid as indicated by a wrinkled seedcoat. For different lots of seed, penetration time was different. Klisiewiecz and Pound (42) reported only partial control of X; campestris in naturally infected cabbage and broccoli seeds even with concentrations as high as 3000 ppm of terramycin, agri-mycin, streptomycin, aureomycin, achromycin, and mercuric .. . ... .. h .. . I . . u r. I .. . J . n I— .l. . . . I .I I . . u . _ n . t . . . ... I u. n .1 n u p . I u p) .. .. .. . I II. . u 65 Table 29. Effect of 30 minute soak in chemical/solvent combina- tions on eradication of blight bacteria from seed arti- fically infected with g. Ehaseoli var. fuscans R10 Chemical Concentration Solvent Presence of ppm M R101) 53;; _____________________________________________________ I ______ Nalidixic acid 800 Dichloromethane + Methicillin 800 Ethanol + Chloramphenicol 800 Ethanol + Nitrofurantoin 800 Dimethyl sulfoxide + NaOCl 10,000 H20, pH 5 + NaOCl 10,000 H20, pH 12 + Tetracycline 800 methanol + 2) Tetracycline 800 H20, 5% methanol — Tetracycline 400 H20, 5% methanol - Aureomycin 800 methanol + 2) Aureomycin 400 H20, 5% methanol — Terramycin 800 methanol + Terramycin 800 Dimethyl sulfoxide + Cupric acetate 5000 Dimethyl sulfoxide + 1) Data from three replications, each consisting of 20 grams sample of infected seeds + at least one flask contained XBf R10 - none of the flasks contained XBf R10 2) The bacteria in the liquid medium grew slower than the others. The medium turned turbid after 6 days of incubation. 66 chloride. The authors stated that the phenomenon was due to lack of antibiotic absorption into the seed, bacteriostatic action of tetra— cycline, and resistance of 5; campestris to streptomycin. Infected seeds immersed in tetracycline and aureomycin in methanol produced turbidity more slowly than other treatments, which could have been due to low concentration of bacteria in the treated seeds. In individual seed assays (Table 30), the number of seeds which contained bacteria did not correspond to the number of seedlings with antibiotic toxicity symptom. Toxicity was indicated by discoloration of the cotyledon in the area of hilum, with or without chlorosis on the first leaves. The area of discoloration seemed to increase with increased time of immersion. This supports Klisiewiecz and Pound's findings (42) that severity of toxicity caused by antibiotics increased with increased concentration and length of treatment. In the non—vacuum treatments, methanol may not have surrounded the seed completely, and consequently the bacteria may not have been killed entirely, but only for cells in contact with the antibiotics. Germination of seed after a thirty—minute soak in either tetra- cycline or aureomycin in methanol was not altered (Table 30). Longer periods of immersion reduced germination drastically. Germi- nation of seeds soaked in aqueous tetracycline and aureomycin for 30 minutes was greatly reduced (Table 31). Monobasic sodium, potassium phosphate (42), and sodium hypo- chlorite (34) were reported to be effective in reducing injury caused by antibiotics in crucifer seeds. Apparently, this is not true for navy bean seed. The use of 5000 ppm of sodium hypochlorite 67 Table 30. Effect of soaking time in antibiotic/methanol combina- tions on eradication of blight bacteria from seed arti- ficially infected with .g. phaseoli. var. fuscans 'R10 and seed germination of uninfected Seafarer navy bean Germination Soaking Individual —- ------- Antibiotic time seed assay % seedlings /800 ppm (minutes) Detection of % without toxicity R10 on RAMl) sympton Control 0 18 98 Tetracycline 3O 10 94 4 60 8 22 3 120 7 5 3 360 8 4 3 Aureomycin 3O 7 96 5 6O 6 25 4 120 5 6 3 360 5 3 1 Methanol 30 - 98 - 120 - 98 - 1) 2) Mean of three replications of 20 artifically infected seeds Mean of two replications of 50 uninfected seeds - was not conducted/detected 68 Table 31. Effect of 30-minute soak in 400 ppm of aqueous antibiotic on germination of uninfected Seafarer navy bean seed Antibiotics Germinationl) Growth of fungiz) Aureomycin 34 ++ Tetracycline 37 ++ Control 98 - 1) Mean of two replications of 50 seeds 2) ++ grew vigorously; appeared similar to seed borne fungi 7:} 3"- : .' (I U] 1151‘ 69 following antibiotic treatment even increased the death of the seeds without reducing toxicity symptoms in the seedlings (Table 32). Thirty-minute soaks can be suggested for bean seed treatments, based on the facts that: (a) the treatments decreased the number of infected seeds; and (b) some seeds that gave positive results in individual seed tests may have low numbers of bacteria which cannot result in field infections. Since tetracycline and aureomycin do not provide protection against seed and soilborne fungal pathogens, any treatment should accompanied by fungicide treatment (42). 70 Table 32. Effect of 500 ppm antibiotic followed by 5000 ppm sodium hypochlorite treatment on seed germination of Seafarer navy bean Time of Soaking Germina— Toxicity Antibiotic soak in time in tion, % symptoms on in H O antibiotic NaOCl seedlingsz) 2 . . (minutes) (minutes) Control 0 0 86.50 Terramycin 15 0 78.50 - 15 10 30 + 15 30 7.5 + 30 0 56 + 30 10 17.5 + 30 30 6.5 + 60 0 17 + 60 10 6.5 + 60 30 l 5 + Streptomycin sulfate 15 O 76.5 - 15 10 46 + 15 30 18 + 30 0 26.5 + 30 10 10 + 30 30 9 + 60 O 3 + 60 10 2.5 + 60 30 3 + Methicillin 15 O 57 + 15 10 16.5 + 15 30 10 + 30 O 28.5 + 30 10 15 + 30 30 5.5 + 60 0 21 + 60 10 2 + 60 30 4.5 + 1) Mean of two replications of 100 seeds 2) . - no obv1ous symptom + stunted, discoloration SUMMARY AND CONCLUS ION A study was conducted on the eradication of internally—borne blight bacteria from bean seed using hot acidified cupric acetate soaks and bactericide infusion by organic solvents or water. The magnitude of inhibition zones around impregnated filter paper discs in Xanthomonas seeded agar was significantly affected by chemical/solvent combinations and by bacterial isolates; both factors interacted significantly. Antibiotic/solvent combinations giving greatest inhibition were tetracycline/water, aureomycin/methanol, aureomycin/water, tetracycline/methanol, and terramycin/methanol. There was no significant difference between applications of cupric acetate in dimethyl sulfoxide and in water. Use of acidified water down to pH 5 significantly increased the activity of sodium hypochlorite by increas- ing available chlorine. Averaged over all chemical/solvent combina— tions, 52a and gpf R10 were the most resistant, §2. 12 intermedi- ate, and épf wv least resistant, which seems to correspond with their relative virulence. All solvents except DMSO, were able to move antibiotics into the seed. Antibiotic activity was detected in cotyledons and embryos of seeds treated with a partial vacuum, but little or no inhibiting activi- ty was associated with the embryo if seed was soaked for 30 minutes or less. Zones of inhibition generally increased with (a) increased time 71 72 of soaking up to two hours, and (b) increased antibiotic concontration up to 1600 ppm. Magnitudes of inhibition zones also varied with anti— biotic/solvent combination, bacterial isolate, and navy bean variety. Methanol was more efficient at infusing aureomycin into the seed than terramycin, as assayed in 52f R10 seeded agar plates. There were always a few seeds that gave little or no zone of inhibition, and this seemed to depend more on antibiotic/solvent combinations than on immer- sion time or antibiotic concentration. Artificial inoculation with blight bacteria was more efficient in providing adequate amounts of infected seed for this study than field and greenhouse inoculations. Artificially infected seed duplicated natural infection in all perspectives. Partial vacuum technique did not impair seed germination or field emergence, and even gave better plant stands in the field. Hot acidified cupric acetate (0.1, 0.25, 0.5%) soaks at 50°C for 20 minutes eradicated blight bacteria, provided that all seed took up the chemical as indicated by blue colored seedcoats. Such treatments greatly reduced seed viability and field emergence. The killing effect of the treatment was apparently due to heat—induced increased permeabil- ity of seed and bacterial membranes. Increased permeability could then allow copper ions to come into closer contact with the membrane pro— teins of bacterial and embryo axis cells. Killing of the seed could also have been due to the vulnerable position of the radicle in bean seed. Obviously, this treatment cannot be recommended as a practical bean seed treatment. Treatment with tetracycline HCl (800 ppm) in methanol under partial vacuum eradicated seedborne blight bacteria completely from 73 bean seed. However, the treatment also reduced germination and field emergence of two-year-old seed drastically, while germination of one— year-old seed was only slightly affected. The reduced germination was probably associated with the presence of antibiotic on embryo axes, which could lead to penetration of the membrane of cells around the embryo axis. The penetration could be enabled by the loss of membrane semipermeability due to aging. The altered semipermeability apparently had not occured in one-year—old bean seed. The treatment, therefore, may be useful for seeds less than one year old on a small scale. Field trials for several different cultivars and for at least two years with weather favorable for common and fusc0us blight infestations, are recommended. Seed immersion in 400 and 800 ppm of aqueous tetracycline and aureomycin, while giving good eradication, cannot be recommended due to greatly reduced seed germination. A sodium hypochlorite rinse following antibiotic treatments actually increased the deleterious effect on seed germination. Seed immersion in 1% sodium hypochlorite at pH 5, as well as at pH 12, for 30 minutes did not eradicate the blight bacteria. Seed immersion in 800 ppm of tetracycline HCl and aureomycin in methanol for 30 minutes can be recommended for large scale bean treat- ments. Soaks greater than 30 minutes resulted in reduced germination as a result of antibiotic phytotoxicity. At least two years of field trials are needed before commercial recommendation. The treatment cannot eradicate blight bacteria completely because of the limited penetration of antibiotic/methanol and lack of chemical absorption by some seeds. However, the treatment can help by reducing numbers of 74 bacteria in the seed to a number that cannot result in field infection. 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