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APPENDIX B POSS IBLE H~ MATH EFFECTS OF SUBTHERAPEUTIC ANTIMICROBIAL USE AS PESTICIDES Robert N. Goodman1 The need for antibiotics to control bacterial diseases is greater for perennials than it is for annuals because annual plants (e.g., wheat, soybeans, tomatoes, etc.) can be bred for resistance to diseases more quickly. Consider how few new varieties of apples have appeared on the market during the past 25 years. However, if antibiotics are used to control a disease, the emergence of resistance to that antibiotic is greater when a species remains in place for decades and is treated annually for the same bacterial disease. Therefore, the problem of re- sistance to antibiotics must be evaluated in these circumstances. As early as the 1950's, investigators were aware that treat- ment of plants with antibiotics could result in the emergence of antibiotic-resistant organisms and allergenic effects in people who applied the pesticides as well as in the consumers of the treated plants (Logue _ al., 1958~. Among the earliest studies on the mode of uptake of antibiotics by plants and their mode of action against the organisms causing plant disease were those of Goodman and Dowler (1958) and Goodman and Goldberg (1960~. Subsequently, the ease with which resistance to streptomycin might be developed In vitro by the target organism Erwinia amylovora was studied and the resulting antibiot~-resistant organisms were used as markers to study pathogenesis of plant disease (Ayers et al., 1979; Goodman, 1963~. In further studies, antibiotics were used to develop selective media to isolate specific bacterial species (Crosse and Goodman, 1973~. ANTIBIOTICS AS PESTICIDES It is difficult to determine the types and quantities of antibiotics used as pesticides since exact production figures are regarded as confidential information by industry. Nonetheless, some estimates can be provided. The primary antibiotic used to control plant disease is streptomycin. Throughout the United States, approximately 10,000 kg of streptomycin is used in a typical year for various diseases, Department of Plant Pathology, University of Missouri, Columbia. 79

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80 predominantly fire blight in apples and pears. The use of strep- tomycin against bacterial spot of tomatoes and peppers, soft rot of potatoes, bacterial blight of celery, and tobacco pathogens seems to be increasing as does its application to beans to con- trol halo blight. Approximately 1,000 kg of tetracycline is used annually to control fire blight of apples and pears (when resistance to streptomycin is a problem) nationwide and bacterial spot of peaches in the Eastern United States. This amount also includes administration of tetracycline by infusion to control the infec- tion of palm and other ornamental species by mycoplasmas. These amounts are approximate and vary, often substantially, from year to year with disease prevalence (Pfizer, Inc., personal communi- cation, 1979~. THE USE OF PENICILLIN TO CONTROL PLANT PATHOGENS There is no evidence to indicate that penicillin is used to any extent to control plant disease. Most plant pathogens are Gram negative, and only members of the genus Corynebacterium, which comprise comparatively few plant pathogenic species, are Gram-variable and sensitive to penicillin. Hence, the value of the penicillins as therapeutic agents for plant diseases is extremely low. This antibiotic might have limited future application since some pathological disorders in plants that were previously believed to be of viral origin have recently been shown to be caused by rickettsia-like organisms (RLO) and to be unexpectedly sensitive to penicillin (Markham et al., 1975; Ulrychova' et al., 1975~. Where penicillin therapy is being tested experimentally, the most common route of administration is infusion. THE DISPOSITION OF ANTIBIOTICS USED AS PESTICIDES Antibiotics applied to plants have a relatively short half-life and undergo a tremendous dilution factor when applied either as a foliar spray or through infusion (Ulrychova' et al., 1975~. Streptomycin may persist in plant tissues for more than a year (Goodman, 1962, 1963~; however, once these tissues are exposed to natural decay processes, antibiotics are quickly degraded.

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81 INGESTION OF ANTIBIOTIC RESIDUES BY ANIMALS AND HUMANS Both humans and animals could be exposed to antibiotics used as pesticides as a result of ingesting apples, pears, and peaches; however, the levels in pears at harvest are below those detectable by sensitive bioassay (Goodman, 1962~. Antibiotic bioassay procedures were developed for assaying antibiotic residues in fruit and vegetable tissue (Goodman et al., 1958; Morgan et al., 1955~. The residual levels determined by these procedures were subsequently used as the basis for feeding trials with volunteers (Goldberg et al., 1961~. Ingestion by ani- mals is not a real concern at present since plants used as animal feeds are not being treated with antibiotics for disease control. There has been a great deal of experimentation to disinfest and disinfect seeds assumed to be contaminated or infected by bac- terial pathogens (Lockhart et al., 1976;- Sutton and Bell, 1954~. Although this type of antibiotic therapy has not been used widely in general practice, this technique is fairly promising and should not contribute a significant hazard to the health of animals or humans. It appears to be the ideal process for controlling plant diseases, particularly when resultant antibiotic residues would be at low levels. The dilution of antibiotic concentrations in the germinating seedlings would continue through growth and maturity of the plant. At harvest, concentrations of the antibiotic in tissue would be beyond detection (Goodman, 1962~. BACTERIAL RESISTANCE The emergence of resistance in target organisms resulting from prolonged use of antibiotics under field conditions has concerned plant pathologists for some time. In the spring of 1953 streptomycin was first used in Missouri to control Erwinia amylovora, a Gram-negative member of Enterobacteriaceae that causes blight in apples and pears (Murneek, 1952~. Since that time, streptomycin has been used regularly in the fruit-growing regions of Missouri and other states. There has been no evidence of the emergence of resistant strains of E. amylovora in Missouri, although there have been frequent efforts to detect resistance in that state and in New York where streptomycin has been used for almost three decades (Beer and Norelli, 1976~. However, in 1971 California investigators found considerable evidence that resist- ance to streptomycin has emerged in E. amylovora (Moller et al.,

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82 1972), possibly because there have been significantly more appli- cations of streptomycin per growing season in orchards in the West than in the East. In apple orchards in Missouri and in the Eastern States strep- tomycin is used to control fire blight an average of 3 to 4 times (a maximum of 6 times) during the growing season. However, the anti- biotic has been applied as many as 13 to 15 times in pear orchards in the Far West where the disease develops continuously over the longer growing season. The selective pressure of 15 applications of streptomycin clearly uncovered those members of the E. amylovora population that were resistant to streptomycin, in some cases in concentrations as high as 500 ~g/ml (Moller et al., 1972~. This has never occurred in orchards in the East. Furthermore, laboratory attempts to develop streptomycin-resistant mutants of E. amylovora are successful only with the greatest difficulty (Shaffer and Goodman, 1962). Using the gradient plate technique, restreaking an inoculum on plates containing streptomycin in increasing concentrations from O to 10 Vigil, from O to 100 ~g/1, and, finally, from O to 1,000 g/ml eventually produces mutants with resistance to 1,000 ~g/ml after 35 to 40 days. Apparently, the frequency of mutations with significant resistance to streptomycin in this organism is extremely low. By far the largest number of species of bacterial plant patho- gens with resistance to antibiotics are in the genus Pseudomonas, and the second largest number reside in the genus Xanthomonas (yellow Pseudomonadaceae). Antibiotics have been used for many years in attempts to control diseases caused by these bacterial species. However, after only two or three applications it became apparent that large proportions of resident populations were not susceptible to concentrations of antibiotics well over 500 ~g/ml (Cox and Hayslip, 1957; Crossan and Krupka, 1955~. Hence, the efficacy of antibiotics for the control of bacterial diseases has been narrowed to precious few species. HISTORY OF ANTIBIOTIC USE AS PESTICIDES The first report of an antibiotic used to control a plant bacterial pathogen appeared as a note in Phytopathology in 1952. The antibiotic was thiolutin, and the researcher was Murneek (1952~. Since that time, almost all antibiotics with either antibacterial or antifungal activity have been screened at one time or another against a wide spectrum of

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83 pathogens affecting plants (Goodman, 1959). Antifungal antibio- tics have been successful only upon rare occasions, the most effective being cycloheximide (Whiffin, 1950~. However, with the advent of systemic as well as prophylactic organic fungicides, interest in antifungal antibiotics to control fungal plant dis- eases waned precipitiously. The use of streptomycin has persisted for approximately 26 years and is now an accepted practice in the production of apples and pears and in many parts of this country. Recently, there has been renewed interest in the use of the tetra- cyclines to control infections of a number of ornamental plants, especially southern palms, by mycoplasma-like organisms (MOO). Experiments have shown that infusion of 1 to 1.5 g of oxytetracy- cline in 10 to 20 ml of water into a single palm tree causes remission of the disease for more than a year (Arai et al., 1967; Bowyer and Calavan, 1974; McCoy and Gwin, 1977; Rosenberger and Jones, 1977~. As mentioned previously, MLO disorders of plants, formerly thought to be viral in origin, seem to be adequately con- trolled by penicillin (Markham et al., 1975; Ulrychova et al., 1975). EPIDEMIOLOGICAL STUDIES Perhaps the single most important question that should be asked in epidemiological studies is whether the transfer of anti- biotic resistance genes from the ubiquitous colifonms to plant bacterial pathogens, or vice versa, has resulted from the use of antibiotics to control plant diseases. Experiments that have been conducted In vitro clearly show that resistance genes can be transferred from Escherichia cold and Shigella flexneri to Erwinia amylovora under laboratory conditions (Chatterjee and Starr, 1973a) and that reciprocal transfer of resistant genes from E. amylovora to E. cold and other human and plant pathogens also occur _ (Chatterjee and Starr, 1973b). One must ask whether the levels of antibiotic titer achieved in the control of plant diseases, either by infusion or spray application, are sufficiently high to support the emergence of the resistant forms of pathogens or epiphytic saprophytic species (U.S. Environmental Protection Agency, 1978~. Rollins _ al. (1975) reported that in a 44-day feeding ex- periment more than 2 but less than 10 fig of oxytetracycline per gram of diet is required to increase significantly the proportion of resistant colifonm bacteria in the intestine of a dog. The infusion of pear trees with oxytetracycline results in residues of approximately 0.0043 ~g/ml oxytetracycline in plant tissue, less than 1/500 of the amount known to select for resistant microflora when administered daily. However, higher levels of streptomycin

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84 have been observed (Goodman, 1959~. Hence, it is reasonably clear that the treatment of plant tissue with antibiotics to control plant disease can and does result in comprehensive residual levels of these substances. Concentrations likely to be found in plant tissue can, after prolonged feeding periods, give rise to the development of resistant coliforms in the gut flora of laboratory animals (Goldberg et al., 1958, 1959) and humans (Goldberg et al., 1961~. This was observed with both streptomycin and oxytetracycline. The investigators also noted that the resistant organisms were transient in the gut flora of the test subjects. Unfortunately, comprehension of R plasmids at that time was nil, and further study of specific antibiotic-resistant isolates was not attempted. Recent studies have addressed the emergence of tetracycline resistance in humans exposed to subtherapeutic levels of this antibiotic (Graber et al., 1979~. The resulting data are not particularly conclusive (Goldberg et al., 1959) because the study was not as comprehensive as it might have been. Future studies should use larger populations. Perhaps then more meaningful com- parisons could be made. The experiences with oxytetracycline or tetracyclines as pesticides have not been sufficiently long nor have they been sufficiently widespread to provide enough test subjects for com- prehensive epidemiological studies among agriculturalists. This is not the case, however, for streptomycin. As indicated earlier in this report, streptomycin has been used in apple orchards for the control of E. amylovora on a regular and routine basis since 1953 (Goodman, 1953~. The applicators of the antibiotic, at least in Missouri, represent a stable population that can be tested for both the presence of resistant coliforms in their gut flora and inordinate allergenic responses to this antibiotic. The studies of Chatterjee and Starr (1973a, b) clearly show that R factors from coliforms can be transferred to E. amylovora and other enter- obacteria and that the reverse is also possible. The extent to which resistant coliforms occur in the applicators of streptomycin in Missouri might be reinvestigated (Goldberg et al., 1958, 1960~. Research should be conducted to determine categorically that strep- tomycin does not have some adverse physiological affects on agri- cultural workers exposed to this drug. The applicator population in Missouri could be examined for an inordinate number demonstrat- ing the clinical symptoms involved. There is little doubt that the examinations could be conducted on between 20 and 25 adult males who have applied streptomycin in apple orchards annually for at

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85 least 10 years. The concentrations of the antibiotic sprays (at 150 ~g/ml) applied by these men are extremely high. The applica- tors could be examined in winter prior to the short spraying season, which begins in m~d-April and ends in June, and at the termination of the spraying season. Residual levels of streptomy- cin could be sought in both blood and stool samples, and the latter could also be examined for the presence of resistant coliforms. Patch tests or other procedures for allergenic responses could be performed (Goldberg et al., 1960~. RESISTANT MYCOPLASt~S There is no evidence to suggest that administration of tetra- cyclines to plants has resulted in the emergence of antibiotic- resistant mycoplasmas. Failure to detect resistance, however, may be the result of technical problems related to the culturing of mycoplasmas or MLO's found in plants In vitro. Consequently, whether resistance, if it occurs, is due to plasmids cannot be determined. The manner in which oxytetracycline is used to control MLO disorders, mainly by infusion or injection procedures (Rosenberger and Jones, 1977), appears to present little risk of hazardous exposure either to the general population or to the applicators. Simple procedures such as the use of rubber gloves and masks should protect the appli- cator from contamination. The low volatility and small concentra- tions of these antibiotics in plant tissue suggest that secondary contamination of humans would also be rather rare. Under unusual circumstances, however, such as when animals ingest treated plant tissue in large quantities, resistant microorganisms might be found in their gut flora. POSSIBLE HEALTH HAZARDS TO HUMANS FROM R-FACTOR TRANSFER Another point that might be discussed more fully concerns the development of resistance to streptomycin in Erwinia amylovora, a species that is currently combatted routinely with this antibiotic. Brief mention was made earlier in this report that the emergence of resistance to streptomycin by this species is rare in nature but when it has occurred it has been widespread. Laboratory studies have shown that streptomycin-resistant mutants of Erwinia amylovora are extremely rare (Shaffer and Goodman, 1962~. Following are some features of the streptomycin-resistant mutants that have been re- covered. First, many are not as well adapted to the natural environ- ment as the wild type virulent forms. They are often completely

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86 avirulent, lacking extracellular polysaccharide (EPS) (Ayers et al., 1979), which some investigators believe to be necessary, _ priori, for pathogenicity. It also appears that the production of EPS is not controlled by plasmids. Antibiotic usage to suppress or eradicate bacterial infec- tion in plant cuttings, which are used in vegetative propagation, will probably be expanded. This type of chemotherapy can offer minimal opportunity for the emergence of significant levels of antibiotic resistance in the gut flora of the applicator or of the eventual consumer of the plant. However, plant pathogens carrying the R factor may emerge. The possible increase in the environment of plant pathogens carrying R factors and their transfer to a ubiquitous plant sapro- phyte such as Erwinia herbicola and vice versa may be possible. Furthermore, human clinical strains of E. herbicola appear to - possess similar plasmid transfer capabilities (Chatterjee et al., 1978~. However, the amount of antibiotics contributed to the environment via plant pesticides must be comparatively small. Unfortunately, it-factor transfer In the field from plant pathogens to saprophytes on human or animal pathogens has not been adequately assessed (Cho et al., 1975~. A few well conceived field experi- ments conducted regionally could provide the necessary answers and the likely assurances that antibiotics as plant pesticides do not constitute a significant hazard to human health. CONCLUSIONS Although exceptional progress has been made in the control of fungal diseases of plants, the control of bacterial disorders in plants has been only minimally successful. In general, the use of antibiotics as pesticides represents an extremely small fraction of the total use of antibiotics in therapy of humans and animals and in the production of animal tissues as food. In the few instances when antibiotics have been successful there appear to have been no obvious deleterious effects to either the applicator or the consumer. Experimentation exploring new and improved anti- biotics to control bacterial diseases of plants should be synchro- nized with efforts in laboratories that can adequately test the safety of the new procedures.

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87 REFERENCE S Arai, S. ~ K. Y. Yuri, A. Kudo, M. Kikuchi, K. Kumagai, and N. Ishida. 1967. Ef feet of antibiotics on the growth of Mycoplasma. J. Antibiot. (Tokyo) Series A 20:246-253. Ayers, A. R., S. B. Ayers, and R. N. Goodman. 1979. Extracellu- lar polysaccharide of Erwinia amylovora: A correlation with virulence. Appl. Environ. Microbial. 38:659-666. Beer, S. V. 1976. Fire blight control with streptomycin sprays and adjuvants at different application volumes. Plant Dis. Rep. 60:541-544. Beer, S. V., and J. L. Norelli. 1976. Streptomycin-resistant Erwinia amylovora not found in western New York pear and apple orchards. Plant Dis. Rep. 60:624-626. Bennett, R. A., and E. Billing. 1975. Development and proper- ties of streptomycin resistant cultures of Erwinia amylovora derived from English isolates. J. Appl. Bacterial. 39:307-315. Bowyer, J. W., and E. C. Calavan. 1974. Antibiotic sensitivity _ vitro of the mycoplasmalike organism associated with citrus stubborn disease. Phytopathology 64:346-349. Chatterjee, A. K., and M. P. Starr. 1973a. Gene transmission among strains of Erwinia amylovora. J. Bacteriol. 116:1100-1106. Chatterjee, A. K., and M. P. Starr. 1973b. Transmission of lac by the sex factor E in Erwinia strains from human clinical sources. Infect. Immun. 8.563-572. Chatterjee, A. K., M. K. Behrens, and M. P. Starr. 1978. Genetic and molecular properties of E-lac , a transmissible plasmid of Erwinia herbicola. Pp. 75-79 in Proceedings of the 4th Inter- national Conference on Plant Pathogenic Bacteriology held August 27-September 2, 1978, Angers, France. Phytobacteriology Section, Institut National de la Recherche Agronomique, Angers, France. Cho, J. J., M. N. Schroth, S. D. Kominos, and S. K. Green. 1975. Ornamental plants as carriers of Pseudomonas aeruginosa. Phytopathology 65:425-431.

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88 Cox, R. S., and N. C. Hayslip. 1957. Recent developments on the control of foliar diseases of tomato in south Florida. Plant Dis. Rep. 41:878-883. Crossan, D. F., and L. R. Krupka. 1955. The use of strepto- mycin on pepper plants for the control of Xanthomonas vesicatoria. Plant Dis. Rep. 39:480-483. Crosse, J. E., and R. N. Goodman. 1973. A selective medium for, and a definitive colony characteristic of, Erwinia amylovora. Phytopathology 63:1425-1426. Goldberg, H. S., B. E. Read, and R. N. Goodman. 1958. Studies on the emergence of streptomycin-resistant bacteria as a result of low-level, long-term feeding of streptomycin. Pp. 144-148 in H. Welch and F. Marti-Ibanez, eds. Antibiotics Annual, 1957- 1958. Medical Encyclopedia, Inc., N.Y. Goldberg, H. S., R. N. Goodman, and B. Lanning. 1959. Low-level, long-term feeding of chlortetracycline and the emergence of antibiotic-resistant enteric bacteria. Pp. 930-934 in H. Welch and F. Marti-Ibanez, eds. Antibiotics Annual, 1958-1959. Medical Encyclopedia, Inc., N.Y. Goldberg, H. S., J. T. Logue, and R. N. Goodman. 1960. Untoward reactions in human beings from application of antibiotics in plant disease control. Pp. 531-535 in H. Welch, Chairman of the Symposium, and F. Marti-Ibanez, ed. Antibiotics Annual, 1959-1960. Antibiotica, Inc., N.Y. Goldberg, H. S., R. N. Goodman, J. T. Logue, and F. P. Handler. 1961. Long-tenm, low-level antibiotics and the emergence of antibiotic-resistant bacteria in human volunteers. Antimicrob. Agents Chemother. 80-88. Goodman, R. N. 1953. Antibiotics. A new weapon for fire blight control. Am. Fruit Grower 73411~:7, 16, 17. Goodman, R. N. 1959. Chapter IV. The influence of antibiotics on plants and plant disease control. Pp. 322-447 in H. S. Goldberg, ed. Antibiotics. Their Chemistry and Non-Medical Uses. D. Van Nostrand Company, Inc., Princeton, N.J.

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89 Goodman, R. N. 1962. The impact of antibiotics upon plant disease control. Pp. 1-46 in R. L. Metcalf, ed. Advances in Pest Control Research, Volume 5. Interscience Pub- l~shers, A Divi sion of John Wiley & Sons, New York and London. Goodman, R. N. 1963. Systemic effects of antibiotics. Pp. 165-184 in M. Woodbine, ed. Antibiotics in Agriculture. Butterworths, London. Goodman, R. N., and W. M. Dowler. 1958. The absorption of streptomycin by bean plants as influenced by growth regu- lators and humectants. Plant Dis. Rep. 42:122-126. Goodman, R. N., and H. S. Goldberg. 1960. The influence of cation competition, time, and temperature on the uptake of streptomycin by foliage. Phytopathology 50:851-854. Goodman, R. N., M. R. Johnston, and H. S. Goldberg. 1958. Residual quantities of antibiotics detected in treated plant tissue. Pp. 236-240 in H. Welch and F. Ilarti- Ibanez, eds. Antibiotics Annual, 1957-1958. Medical Encyclopedia, Inc., N.Y. Graber, C. D., S. H. Sandifer, W. H. Whitlock, C. B. Loadholt, and B. J. Poore. 1979. Acquired resistance of autochtho- nous E. cold in controls and orchardists engaged in the _ spraying of oxytetracycline. Bull. Environ. Contam. Toxi- col. 22:202-207. Lockhart, C. L., C. 0. Gourley, and E. W. Chipman. 1976. Control of Xanthomonas compestris in Brussels sprouts with hot water and aureomycin seed treatment. Can. Plant Dis. Surv. 56:63-66. Logue, J. T., H. S. Goldberg , and R. N. Goodman. 1958. The public health s ignificance of antibiotic residues in foods. Pp. 333-335 in t1. Welch, ed. Antibiotics Annual, 1957-1958. Medical Encyclopedia , Inc ., N. Y. Markham, P. G., R. Townsend , and K. A. Plaskitt. 1975. A rick- ettsia-like organism associated with diseased white clover. Ann. Appl. Biol. 81:91-93.

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go McCoy, R. E., and G. H. Gwin. 1977. Response of mycoplasmalike organism-infected Pritchardia, Trachycarpus and Veitchia palms to oxytetracycline. Plant Dots. Rep. 61:154-158. Moller, W. J., J. A. Beutel, W. O. Reil, and B. G. Zoller. 1972. Fireblight resistance to streptomycin- in California. Phyto- pathology 62:779. Morgan, B. S., H. S. Goldberg, and R. N. Goodman. 1955. Residual quantities of a streptomycin-oxytetracycline combination, as determined by a simple microbiologic assay. Pp. 536-539 in H. Welch, ed. Antibiotics Annual, 1954-1955. Medical Encyclo- pedia, Inc., N.Y. Murneek, A. E. 1952. Thiolutin as a possible inhibitor of fire blight. Phytopathology 42:57. Rollins, L. D., S. A. Gaines, D. W. Pocurull, and H. D. Mercer. 1975. Animal model for determining the no-effect level of an antimicrobial drug on drug resistance in the lactose-ferment- ing enteric flora. Antimicrob. Agents Chemother. 7:661-665. Rosenberger, D. A., and A. L. Jones. 1977. Symptom remission in x-diseased peach trees as affected by date, method, and rate of application of oxytetracycline-HCl. Plant Dis. Rep. 67: 277-282. Shaffer, W. H., and R. N. Goodman. 1962. Progression In viva, rate of growth In vitro, and resistance to streptomycin, as indices of virulence of Erwinia amylovora. Phytopathology 62:1201-1207. Sutton, M. D., and W. Bell. 1954. The use of aureomycin as a treatment of Swede seed for the control of black rot (Xantho- monas campestris). Plant Dis. Rep. 38:547-552. Ulrychova, M., G. Van ek, M. Jokes, Z. Klobtska, and 0. Kral~k. 1975. Association of rickettsia-like organisms with infec- tious necrosis of grapevines and remission of symptoms after penicillin treatment. Phytopathol. Z. 82:254-265. U.S. Environmental Protection Agency. 1978. Tolerances and exemptions from tolerances for pesticide chemicals in or on raw agricultural commodities. Oxytetracycline. Fed. Regist. 43(154):35309.

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91 Whiffin, A. J. 1950. The activity in vitro of cycloheximide (acti dione) against fungi pathogenic to plants. Mycologia 42: 253- 258. -