Patricia S. McManus1, Virginia O. Stockwell2 George W

12 Jul 2002 10:4 AR AR165-PY40-16.tex AR165-PY40-16.SGM LaTeX2e(2002/01/18)P1: GJB10.1146/annurev.phyto.40.120301.093927

Annu. Rev. Phytopathol. 2002. 40:443–65doi: 10.1146/annurev.phyto.40.120301.093927

Copyright c© 2002 by Annual Reviews. All rights reserved

ANTIBIOTIC USE IN PLANT AGRICULTURE

Patricia S. McManus1, Virginia O. Stockwell2,George W. Sundin3, and Alan L. Jones41Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin53706-1598; e-mail: [email protected];2Department of Botany and PlantPathology, Oregon State University, Corvallis, Oregon 97330;e-mail: [email protected];3Department of Plant Pathology and Microbiology,Texas A&M University, College Station, Texas 77843-2132; e-mail: [email protected];4Department of Plant Pathology, Michigan State University, East Lansing, Michigan48824-1312; e-mail: [email protected]

Key Words antibiotic resistance,Erwinia amylovora, Pseudomonas,Xanthomonas, streptomycin, tetracycline, Tn5393

■ Abstract Antibiotics have been used since the 1950s to control certain bacterialdiseases of high-value fruit, vegetable, and ornamental plants. Today, the antibioticsmost commonly used on plants are oxytetracycline and streptomycin. In the USA, an-tibiotics applied to plants account for less than 0.5% of total antibiotic use. Resistance ofplant pathogens to oxytetracycline is rare, but the emergence of streptomycin-resistantstrains ofErwinia amylovora,Pseudomonasspp., andXanthomonas campestrishas im-peded the control of several important diseases. A fraction of streptomycin-resistancegenes in plant-associated bacteria are similar to those found in bacteria isolated fromhumans, animals, and soil, and are associated with transfer-proficient elements. How-ever, the most common vehicles of streptomycin-resistance genes in human and plantpathogens are genetically distinct. Nonetheless, the role of antibiotic use on plants inthe antibiotic-resistance crisis in human medicine is the subject of debate.

INTRODUCTION

The introduction of antibiotics for the treatment of bacterial diseases nearly sixdecades ago revolutionized human medicine. The ability of antibiotics to curevictims of debilitating and fatal diseases was regarded as nothing short of aston-ishing. Plant pathologists quickly recognized the potential of antibiotics for thetreatment of plant diseases, especially those caused by bacteria. During the 1950s,approximately 40 antibiotics of bacterial or fungal origin were screened for plantdisease control (18). Potency at low doses and negligible toxicity toward plantsdistinguished antibiotics from the metal-based bactericides available to farmersin the 1950s and 1960s. The primary targets of antibiotics were various bacterialdiseases of vegetables and fire blight of apple and pear. Unfortunately, just as theemergence of bacterial strains resistant to antibiotics has limited the effectiveness

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of these drugs in human medicine, streptomycin-resistant plant pathogens havethwarted plant disease control (45).

When the topic of bactericide resistance in plant pathogenic bacteria was lastcovered in 1990 in theAnnual Review of Phytopathology(11), the primary fo-cus was on resistance to copper, while antibiotic resistance garnered just over apage of text. Since the early 1990s, however, events in the laboratory, field, andhuman community have occurred that warrant our revisiting the topic of antibi-otic use on plants. First, great strides have been made in our understanding of thegenetic mechanisms of antibiotic resistance in plant-associated bacteria. In partic-ular, the discovery of homologous resistance determinants in bacteria associatedwith plants, soil, animals, and humans has led to intriguing hypotheses regardingthe origins of resistance (24, 80). Second, over the past 12 years epidemics of fireblight of apple and pear, caused by the bacteriumErwinia amylovora, have height-ened growers’ dependency on antibiotics to control this pernicious disease. Fireblight epidemics have also prompted surveillance for antibiotic-resistant strainsof E. amylovora, fostering new awareness of resistance. Third, the emergence ofantibiotic resistance in the clinical setting has outpaced the development of newantibiotics. The antibiotic-resistance crisis in human medicine has been widelypublicized and is now recognized as a major threat to controlling bacterial diseasesand infections worldwide (13, 33). Efforts to conserve the efficacy of antibiotics inhuman medicine have drawn scrutiny to all antibiotic use, including use on plants.

Our objectives in this review are to describe the current status of antibiotic use inplant agriculture, the nature of antibiotic-resistant plant pathogens, and the ecologyof antibiotic-resistance determinants in plant agricultural systems. We discuss thedifficulties that have arisen as regulators, consumer advocates, and growers attemptto weigh the repercussions of antibiotic use on plants on human health. Finally,we speculate on the future role of antibiotics in plant agriculture.

ANTIBIOTICS USED ON PLANTS

The classic definition of an antibiotic is a substance produced by one microorgan-ism that inhibits or kills other microorganisms (36). Over the years, the definitionhas expanded to include certain synthetic and semisynthetic antibacterial agents.Some antifungal compounds of microbial origin, such as blastomycin S and cy-cloheximide, which have been used to control rice blast and various other fungaldiseases of plants (87), also have been classified as antibiotics. Here our focus ison antibiotics that are currently used to manage plant diseases caused by bacteriaand phytoplasmas.

The number of antibiotics used in plant agriculture is modest relative to appli-cations in human and veterinary medicine and in animal production, where morethan 30 different drugs from at least 14 distinct classes are used in the USA alone(59). Plant-grade antibiotics are typically formulated as powders consisting of 17%to 20% active ingredient. The powders are dissolved or suspended in water to con-centrations of 50 to 300 ppm and then applied as a fine mist to the susceptible aerial


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plant organs. Because they are relatively expensive, antibiotics are used primarilyon high-value fruit and vegetable crops and ornamental plants where their cost ismost likely to be recouped. The use of antibiotics and other chemicals specificallyfor the control of fire blight was reviewed recently (56).

Streptomycin

Streptomycin, an aminoglycoside antibiotic, is formulated as either streptomycinsulfate or streptomycin nitrate. It is or has been marketed under the trade namesAgrept, Agri-mycin, Agri-strep, Fructocin, and Plantomycin. Since its introductionfor crop protection in 1955, streptomycin has been the major antibiotic used onplants in the USA, primarily for control of fire blight. In Europe, where fire blighthas been spreading from country to country since the late 1950s, streptomycin iseither not permitted, used on an emergency basis, or is used regularly, dependingon the country. More recently, fire blight has emerged in the Middle East, andstreptomycin is used to control this disease in Israel. Other targeted pathogens andtheir hosts includePectobacteriumspp. (formerlyErwinia spp. ), which cause softrot diseases of cut flowers and potato seed pieces;Pseudomonas cichorii, whichcauses bacterial blight of celery; various pathovars ofPseudomonas syringae,which cause fruit-spotting or blossom-blast symptoms on apple, pear, and relatedlandscape trees;Xanthomonas campestrispv. vesicatoria, which causes bacterialspot of pepper and tomato; andAgrobacterium tumefaciens, which causes crowngall of rose. Streptomycin is also permitted for use as a dust on pears in the westernUSA for control of fire blight and on tobacco for control of wildfire (Pseudomonassyringaepv. tabaci) and blue mold (Peronospora tabacina) diseases.P. tabacinais a water mold and the only eukaryotic plant pathogen targeted by streptomycin.

Oxytetracycline

Oxytetracycline, a tetracycline antibiotic, is formulated either as an oxytetracycline-calcium complex or oxytetracycline hydrochloride and is or has been marketedunder the trade names Biostat, Glomycin, Mycoshield, Terrafungine, and Ter-ramycin. In the USA, it is registered on pear for control ofE. amylovoraand onpeach and nectarine for control ofXanthomonas arboricola, which causes bac-terial spot. In states whereE. amylovorahas become resistant to streptomycin,oxytetracycline has been used on apple under special registration. Oxytetracyclinealso is used to controlE. amylovoraon apple in Mexico andPseudomonasspp.andXanthomonasspp. on several vegetable crops in Latin American countries. Aminor use of oxytetracycline is injection into the trunks of palm and elm trees tomitigate symptoms of lethal yellows diseases caused by phytoplasmas (40).

Gentamicin

Gentamicin, an aminoglycoside antibiotic, is formulated as gentamicin sulfate. Itis marketed under the trade name Agry-gent. Gentamicin is sometimes mixed withoxytetracycline and marketed as Agry-gent Plus or Bactrol. Gentamicin is used


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in Mexico to control fire blight of apple and pear and in several Latin Americancountries to control various bacterial diseases of vegetable crops caused by speciesof Erwinia, Pectobacterium, Pseudomonas, Ralstonia, andXanthomonas.

Oxolinic Acid

Oxolinic acid, a synthetic quinolone antibiotic, is marketed under the trade nameStarner. It currently is used on a commercial basis only in Israel to manage fireblight of apple, pear, and related plants, especially in areas whereE. amylovorahas become resistant to streptomycin (69).

AMOUNTS OF ANTIBIOTICS USED

In the USA, the total amount of antibiotics (i.e., oxytetracycline and streptomycin)applied to plants annually has been estimated at 19,550 to 65,227 kg. The discrep-ancy in these figures is due to the different methods used by government agenciesto obtain and summarize pesticide use data. The lower estimate is given by theNational Agricultural Statistics Survey (NASS) (50). It reports actual amounts anduse patterns of antibiotics, but the data are compiled solely from states that aremajor producers of commodities that may be treated with antibiotics. The higherestimate is given by the United States Geological Survey (86) as part of its na-tional assessment of pesticides in streams, rivers, and groundwater conducted inthe early 1990s. It estimated actual use but also extrapolated data from pesticideuse rates compiled by the National Center for Food and Agricultural Policy andcrop-production statistics reported in the 1992 Census of Agriculture. By eitherestimate, the amount of antibiotics used on plants in the USA is less than 0.5% ofthe approximately 22,680,000 kg of antibiotics produced each year (33).

Antibiotic Use on Tree-Fruit Crops

Historically, the most significant use of antibiotics on plants worldwide has beento control fire blight of apple and pear. Because of its relatively high efficacy andlow phytotoxicity, streptomycin has been the antibiotic of choice in most regions.In regions of the USA where streptomycin-resistant strains ofE. amylovoraareestablished, growers use oxytetracycline, either alone or in combination with strep-tomycin. Oxytetracycline is less effective than streptomycin when the pathogenis sensitive to both antibiotics (42, 53) but more effective than streptomycin whenthe pathogen is resistant to this antibiotic (42). Streptomycin killsE. amylovora,whereas oxytetracycline merely inhibits its growth. Thus, when residues of oxyte-tracycline decrease on plant surfaces, bacterial populations rebound. Nonetheless,fire blight can be managed with well-timed applications of oxytetracycline duringbloom.

The use of oxytetracycline and streptomycin on tree-fruit crops in the USA issummarized by the NASS on a biennial basis; data for the 1990s are presentedin Table 1. During this period, the total area surveyed for apple, pear, and peach



TABLE 1 Use of antibiotics on tree-fruit crops in the USA, 1991–1999a

Oxytetracycline Streptomycin

Active ActiveArea Average ingredient Area Average ingredient

Crop Area (Ha) treated number of applied treated number of appliedand year surveyedb (%) applications (kg) (%) applications (kg)

Apple1991 141,538 —c — — 10 2.3 9,4801993 134,534 1 1.1 136 9 1.8 5,8511995 139,757 3 1.4 998 16 1.9 7,5301997 142,024 5 1.1 1,225 25 1.9 11,0681999 148,866 5 1.4 1,315 19 1.5 6,985

Pear1991 18,259 33 2.8 2,858 28 1.8 3,6741993 27,085 53 3.8 5,851 41 2.4 5,3981995 27,470 35 3.3 4,536 38 2.6 3,8101997 27,490 38 5.4 7,757 34 3.9 6,9851999 26,862 41 2.9 5,398 30 2.3 2,722

Peach1991 35,992 13 4.9 3,175 NRd

1993 42,227 5 3.9 1,1791995 58,219 4 2.2 6801997 55,020 8 4.0 3,1751999 57,105 8 4.3 3,130

aSummarized from data of the National Agricultural Statistics Service (50).bIncludes only states with major production of the commodity. In most years this was ten states for apple, six states for pear,and five states for peach.cData not reported because of infrequent use.dStreptomycin not registered for use on peach.

increased by 5%, 47%, and 59%, respectively. The percentage of apple hectarestreated with antibiotics increased during the 1990s as plantings of varieties that arepopular with consumers, but also highly susceptible to fire blight, increased. Al-though the number of hectares planted to susceptible varieties increased, antibioticapplication frequency remained steady at once or twice per year throughout thedecade. This in part can be attributed to use of fire blight risk assessment systemsand models (2) that help growers pinpoint periods of high risk of infection. Afterthe discovery ofE. amylovorastrains resistant to streptomycin in Michigan in theearly 1990s (7, 42), use of oxytetracycline on apple in that state was permittedand then slowly expanded in subsequent years. Generally, 30% to 40% of pearhectares were treated with oxytetracycline or streptomycin or both antibiotics twoto four times per year. In most years, less than 10% of peach hectares were treatedwith oxytetracycline. In general, the peach orchards treated were in eastern and


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southeastern states where bacterial spot disease pressure is high. On average, peachorchards were treated at least four times in most years.

REGULATION OF ANTIBIOTICS USED ON PLANTS

The United States Environmental Protection Agency (U.S. EPA) regulates the useof oxytetracycline and streptomycin on plants in the USA. Similar governmentalagencies have this responsibility in other countries, although the criteria for reg-istration vary among nations. The registration process for pesticides in the USAincludes evaluating the potential impact of the chemical on human health. Strepto-mycin and oxytetracycline have been assigned the lowest toxicity category by USEPA, and carcinogenic or mutagenic activities have not been observed for eitherantibiotic. Product labels, which are legally binding, state that an applicator orother handler of streptomycin or oxytetracycline must wear a long-sleeved shirt,long pants, waterproof gloves, shoes plus socks, and a dust/mist filtering respira-tor (streptomycin only) or protective eyewear (oxytetracycline only). Workers arenot permitted to enter a treated area until 12 hours after either antibiotic has beenapplied unless they are properly protected. Product labels also specify a preharvestinterval (PHI), which is the period of time between the last permitted applicationand harvest. PHIs are established to ensure that pesticide residues do not exceed apredetermined limit or “tolerance. ” The PHIs for oxytetracycline and streptomycinrange from 21 to 60 days, depending on the crop and antibiotic. Also, US EPA hasestablished residue tolerances of 0.35 ppm and 0.25 ppm, for oxytetracycline andstreptomycin, respectively.

STREPTOMYCIN-RESISTANT PLANT PATHOGENS

The emergence of streptomycin-resistant (SmR) plant pathogens has complicatedthe control of bacterial diseases of plants. For example, in the USA, streptomycinis permitted on tomato and pepper for control ofX. campestrispv. vesicatoria,but it is rarely used for this purpose because resistant strains, first discoveredin Florida in the early 1960s (72), are now widespread (47). Resistance inE. amylovora, the fire blight pathogen, has had widespread economic and po-litical implications, as discussed in later sections. Other phytopathogenic bacteriain which SmR has been reported includePectobacterium(formerlyErwinia) caro-tovora (17), Pseudomonas chichorii(55), Pseudomonas lachrymans(88), Pseu-domonas syringaepv. papulans(4, 23, 52),Pseudomonas syringaepv. syringae(14, 21, 61, 71, 77, 90), andXanthomonas dieffenbachiae(26). Surveillance forantibiotic-resistant plant pathogens has been sporadic and often in response to adisease control failure in the field. Many surveys for antibiotic-resistant pathogenshave yielded negative results and are not reported. Considering that streptomycinhas been used as the sole antibiotic in many commercial operations, the emergenceof resistant pathogens has been surprisingly infrequent.



Erwinia amylovora

SmR strains ofE. amylovorawere first detected on pear in California in 1971 (46)and then in Washington and Oregon in 1972 (12). SmR strains ofE. amylovoraarenow widespread and common in apple and pear orchards of the western UnitedStates and British Columbia in Canada (35, 48, 74, 68, 85). In 1985, resistance wasreported in Missouri (67); and in the early 1990s, in Michigan (7, 42). Outside theUSA, SmR strains ofE. amylovorahave been reported in New Zealand (84), Israel(38), and Lebanon (60). In Lebanon, SmR strains ofE. amylovorawere detectedin regions where streptomycin had never been used, because fire blight did notoccur in those regions until recently (A.T. Saad, personal communication). It ispossible that the initial outbreaks of fire blight in Lebanon were caused by strainsfrom neighboring Israel, where SmR strains ofE. amylovorahad been detected afew years earlier (38).

GENETICS OF RESISTANCE Two resistance phenotypes have been identified amongSmR field strains ofE. amylovora: Highly resistant strains have streptomycin mini-mal inhibitory concentrations (MICs) of 2000µg ml−1, and moderately resistantstrains have MICs of 500 to 750µg ml−1 (9, 12, 42). This suggests at least twomechanisms for resistance, and in the 1990s, molecular genetic studies shed lighton two distinct mechanisms.

MUTATION-DETERMINED RESISTANCE Early studies on the genetics of copper andantibiotic resistance in plant pathogenic bacteria indicated that resistance evolvedmost often through acquisition of plasmid-borne resistance genes (11). However,in E. amylovora, mutation of a chromosomal gene is the most common mecha-nism for streptomycin resistance. After performing fluctuation tests to determinethe mutation rate and level of resistance, Schroth et al. (65) predicted that high-level resistance to streptomycin inE. amylovoraprobably originated in the fieldfrom a single-step mutation. More evidence supporting the hypothesis that resis-tance to streptomycin inE. amylovoraarose through mutation was the findingthat highly resistant strains, unlike moderately resistant strains, also are insensi-tive to myomycin, an antibiotic with the same target site as streptomycin (10).Characterization of the specific mutations for resistance in field strains followed.

Streptomycin blocks protein synthesis by binding to the 30S subunit of the bac-terial ribosome. The site of mutation for resistance to streptomycin inE. amylovoraand numerous other bacteria is the highly conservedrpsLgene, which encodes ri-bosomal protein S12 (10). Sensitive strains ofE. amylovorawere made resistantby complementation with a plasmid carrying the mutantrpsL gene from highlyresistant strains; conversely, resistant strains were made sensitive by complemen-tation with a high-copy-number plasmid carrying the wild-type gene (10). Specifi-cally, high-level resistance to streptomycin is conferred by a point mutation thatalters codon 43 of therpsL gene (10). In the majority of highly resistant fieldstrains, lysine-43 (AAA) is changed to arginine (AGA), but in other strains, it is


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changed to asparagine (AAT or AAC) or threonine (ACA). These mutations allrenderE. amylovorahighly resistant to streptomycin. The fact that SmR mutants ofE. amylovorafrom several areas of North America and New Zealand vary in pointmutations inrpsL(10) and also in ribotype fingerprints (44) suggests that resistancehas arisen independently and been selected for multiple times.

PLASMID/TRANSPOSON-DETERMINED RESISTANCE Although mutation ofrpsL isthe primary mechanism of SmR detected inE. amylovora, most SmR strains iso-lated in Michigan have a resistance system unrelated to mutation. These strainscarry a 34-kb self-transmissible plasmid, pEa34 (Figure 1), which confers a mod-erate level of resistance to streptomycin when transferred into sensitive strains (7).The involvement of a functional SmR transposon on pEa34 was suspected whenresistance was detected by entrapment on two recipient replicons inEscherichiacoli (8). Further analysis revealed a novel 6.7-kb, Tn3-type transposon, Tn5393,containing linkedstrA-strBgenes that encode streptomycin-inactivating phospho-transferases (8, 9) (Figure 2).

Although pEa34 is the most common vehicle for Tn5393 in E. amylovora,some moderately SmR strains carry Tn5393 on the nontransmissible plasmidpEA29 (41, 42) (Figure 1). Plasmid pEA29, without Tn5393, is ubiquitous in

Figure 1 Antibiotic-resistance plasmids found inErwinia amylovorashowing thelocations of the SmR genesstrA andstrB, which are carried by Tn5393in pEa34 andpEA29. pEa8.7 also carries thesulII gene for resistance to sulfonamide antibiotics.The sizes of self-transmissible plasmid pEa34, broad-host-range plasmid pEa8.7, andnontransferable plasmid pEA29 with Tn5393are 34 kb, 8.7 kb, and 35 kb, respectively.



nearly all field strains ofE. amylovora(15, 30); it has been completely sequenced(41) and is unrelated to pEa34. In five SmR strains ofE. amylovora, Tn5393was inserted at five different positions in pEA29 (41; G.C. Foster, G. McGhee &A.L. Jones, unpublished data). In other strains, Tn5393was integrated into thechromosome (42). Taken together, these recent discoveries of insertion of Tn5393in different genomic locations suggest that we are tracking evolution as it occurs.

In addition to pEa34 and pEA29, an 8.7-kb SmR plasmid, pEa8.7 (Figure 1),was detected in isolates ofE. amylovorafrom an apple orchard in California(54). Plasmid pEa8.7 confers resistance to both streptomycin and sulfonamideantibiotics, encoded bystrA-strBandsulII genes, respectively, and the plasmid isprobably identical to the broad-host-range plasmid RSF1010 (54). Plasmids of thistype commonly are found in a variety of clinically important bacteria of humanand animal origin (80). Although not self-transmissible, RSF1010 is mobilized byother plasmids. The origin of pEa8.7 remains unknown; however, the evolution ofRSF1010-like plasmids and Tn5393may be linked, as we describe below.

Pseudomonas and Xanthomonas

The first discoveries of SmR in P. syringaewere in Canada and New Zealand inthe late 1970s (14, 90). Interest in the genetics of SmR was initially stimulated by areport on the conjugative transfer of resistance associated with a conserved 108-kbplasmid in the apple blister spot pathogen,P. syringaepv. papulans, isolated inNew York (4). Minsavage et al. (47), working withX. campestrispv. vesicato-ria, cloned a SmR locus from the Argentinian isolate BV5-4a and demonstratedthat the probe hybridized to DNA of SmR strains ofX. campestrispv. vesica-toria from Florida andP. syringaepv. papulansfrom New York. Norelli et al.(52) concurrently cloned the SmR determinant fromP. syringaepv.papulans, con-structed a DNA probe termed SMP3, and demonstrated that the probe hybridized toDNA from SmR but not streptomycin-sensitive strains ofP. syringaepv.papulans.The SMP3 probe also hybridized to plasmids of various sizes in SmR strains ofP. syringaepv. papulansfrom Michigan (23) and to pEa34, the SmR plasmid ofE. amylovoradescribed above (7).

Hybridization of SMP3 to pEa34 and the subsequent cloning and sequencingof Tn5393(8) provided a definitive answer as to the determinant responsible forthe widespread distribution of homologous SmR sequences in plant pathogenicbacteria. ThestrA-strBgene pair was later identified inP. syringaepv. syringaestrains from Oklahoma, and further analysis showed that variants of Tn5393werepresent in these strains andX. campestrispv. vesicatoria(77, 79) (Figure 2). Themultifunctionalressite appears to have played an important role in the evolution ofalternate strategies for expressingstrA-strBin different organisms. As with the re-lated Tn3, theressite of Tn5393contains promoter sequences in either direction forthe transcription of the transposase (tnpA) and resolvase (tnpR) genes;resalso con-tains three binding sites for TnpR, which, upon binding, repress transcription fromboth promoters (20). Therespromoter sequence is a strong constitutive promoter in


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P. syringaepv.syringae; however, repression by TnpR limits the expression ofstrA-strB. As a consequence, the streptomycin MIC in this organism is only 75µg ml−1

(79). The higher streptomycin MICs inE. amylovoraandX. campestrispv. vesi-catoria (250–1000µg ml−1) are attributed to additional promoter sequences forstrA-strBprovided by IS1133and IS6100, respectively (8, 79).

Persistence of Streptomycin-Resistant Pathogens

Whether conferred by a chromosomal mutation or by variants of Tn5393, SmR ap-pears to be a stable trait in plant pathogenic bacteria. SmR isolates ofE. amylovorawere detected in a pear orchard in California 10 years after applications of theantibiotic were halted (48). Similarly, SmR strains ofE. amylovoraremain wide-spread in Washington state (V.O. Stockwell, unpublished data), even though strep-tomycin use decreased dramatically in the 1990s after it was reported that 88% ofthe surveyed orchards harbored resistant strains (35). A survey of celery beds inFlorida in the early 1990s revealed widespread resistance to streptomycin amongisolates ofP. cichorii, even though streptomycin had not been used since the late1960s (55).

In strains ofP. syringaepv. syringaeisolated from ornamental tree nurseries inOklahoma, Tn5393a was carried by a group of related plasmids that were stableand did not impair host fitness in the absence of streptomycin selection (78, 81).The stability of the resistance phenotype might be explained by the fact that theseplasmids belong to the pPT23A plasmid family, a group that is highly conservedamongP. syringaepathovars where they impart a variety of functions importantin host-pathogen interactions, including pathogenicity and virulence (66). As de-scribed above, the SmR transposon Tn5393has been detected on stable geneticelements (i.e., an indigenous plasmid and the chromosome) ofE. amylovora. Thesecases of stable SmR in plant pathogenic bacteria parallel the stable antibiotic re-sistance in clinically important species in which resistance genes acquired frombacterial cohorts take up residence on indigenous plasmids in their new host (32).

In E. coli, mutation of the S12 ribosomal protein to high-level SmR has anegative effect on fitness. However, after the initial decrease in fitness, antibiotic-resistant bacterial pathogens apparently accumulate beneficial mutations that ame-liorate the fitness costs (64). The end result of such “compensatory” mutations isthat antibiotic-resistance genes persist in future generations. Although this phe-nomenon has not been investigated in SmR E. amylovora, compensatory mutationsmight be expected, considering the similarity of mutations in the S12 protein inE. amylovoraandE. coli (10).

TETRACYCLINE-RESISTANT BACTERIA IN ORCHARDS

In regions where the emergence of SmR bacterial pathogens of fruit trees has ham-pered disease control, oxytetracycline has been applied. A survey of pear orchardsin Oregon and Washington during 1989 to 1992 yielded strains ofP. syringaepv. syringaeresistant to 50, 100, 250, and 500µg oxytetracycline per ml from



six, three, two, and one of six orchards, respectively (71). However, the frequencyof tetracycline-resistant (TcR) strains at these sites was generally low; more than70% and 90% of the strains in all six orchards were sensitive to 50 and 250µgoxytetracycline per ml, respectively. Of the 320 strains tested, 3 were resistantto oxytetracycline, streptomycin, and copper, although linkage of the resistancegenes was not determined.

Oxytetracycline is the antibiotic of choice on peach and nectarine for con-trol of bacterial spot caused byX. arboricolaand on apple and pear where SmR

E. amylovorahas been documented. Surveys have not revealed TcR strains ofX.arboricola (D. Ritchie, G. Schnabel, personal communication). Several TcR de-terminants in nonpathogenic bacteria from apple orchards in Michigan have beencharacterized (63). TcR was attributed to the presence oftetB in Pantoea agglo-meransand other members of the family Enterobacteriacae andtetA, tetC, or tetGin most isolates ofPseudomonas. The cause of TcR was not identified in 16% ofthe isolates. The TcR genes were almost always found on large plasmids that alsocarried the SmR transposon Tn5393, discussed above. Entrapment studies revealedthat tetA, tetB, andtetCwere carried on transposons identical or similar to trans-posons described in TcR bacteria of environmental, animal, or human origin (58).Despite the carriage of TcR genes on mobile genetic elements in nonpathogenicorchard bacteria, TcR strains ofE. amylovorahave not been detected (35, 63; V.O.Stockwell, unpublished data).

ECOLOGY OF ANTIBIOTIC RESISTANCEDETERMINANTS

How Did Plant Pathogens AcquireStreptomycin-Resistance Genes?

Several studies have confirmed the presence ofstrA-strB, often within a variant ofTn5393, in gram-negative, nonpathogenic plant epiphytes and soilborne bacteriafrom nurseries and orchards where streptomycin was used and also from adja-cent areas where the antibiotic was not used (5, 21, 52, 82). These results implicatenontarget organisms as the initial source of Tn5393that, through a chain of hori-zontal transfer events, eventually was acquired by plant pathogens. The detectionof pEa34 with Tn5393 in isolates ofE. amylovorain Michigan but not in otherregions suggests a relatively recent acquisition of the plasmid in this species. Plas-mid pEa34 is not common in populations ofE. amylovora, and it was not detectedin sensitive strains ofE. amylovorafound among resistant strains in Michigan or-chards. How didE. amylovoraacquire pEa34? Among the many bacterial speciesthat are reservoirs ofstrA-strBin Michigan apple orchards (63, 70),Pantoea ag-glomerans(formerlyErwinia herbicola) is the most likely donor of pEa34. Strainsof this species were detected with pEa34 carrying Tn5393with IS1133inserted, asis found inE. amylovora. Plasmid pEa34 was transferred in vitro at high frequencyfrom P. agglomeransto E. amylovora(8, 24). BecauseP. agglomeransshares anecological niche (i.e., apple blossoms and bark) withE. amylovora(57), there


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should be ample opportunity for exchange of genetic information between thesespecies. A scenario by whichE. amylovoramight have initially acquired SmR

genes, and the subsequent build up of SmR populations ofE. amylovora, are il-lustrated in Figure 3. Likewise, the selection for SmR strains with a chromosomalmutation is illustrated.

Horizontal Transfer of Streptomycin-ResistanceDeterminants among Diverse Bacteria

Although antibiotics have been used in human medicine, animal husbandry, andin plant protection since the 1950s, the extensive occurrence of resistance in manybacteria has involved surprisingly few genetic mechanisms. The association of

Figure 3 Streptomycin resistance inErwinia amylovoracan occur as a result of chromoso-mal mutation or through gene acquisition; the use of streptomycin for disease control reducesthe population of streptomycin-sensitive bacteria while favoring the increase of SmR bacte-ria. Mutation is the more common of the two forms of streptomycin resistance detected inE. amylovora. All mutations for resistance detected thus far are point mutations in therpsLgene, which alter ribosomal protein S12. Acquired resistance has occurred when an SmR

plasmid, such as pEa34 carrying transposon Tn5393with tandemstrA-strB, is transferred byconjugation toE. amylovora.



antibiotic-resistance genes with transfer-proficient elements such as plasmids,transposons, and integrons, coupled with selection by antibiotics, has facilitatedthe widespread distribution of resistance determinants (13). Integrons are discretegenetic elements that can “capture” antibiotic-resistance genes by site-specific inte-gration, through the action of a DNA integrase (16). Integrons have been critical inthe evolution of strains of human pathogens that are resistant to multiple antibio-tics (13). The human pathogensPseudomonas aeruginosaandShigella flexneriharborstrA-strBwithin Class 1 integrons (49, 76). However, integrons probablyare not important in the dissemination ofstrA-strB, because many integrons al-ready carry a SmR gene (aadA; 16); carriage of additional SmR genes would notbe of great advantage to the bacterial host.

The strA-strBgene pair has been disseminated among gram-negative bacte-ria that inhabit diverse ecosystems on broad-host-range nonconjugative plasmidssuch as the IncQ plasmid RSF1010 and on Tn5393-type transposons (80). Hostsof RSF1010 include commensal and pathogenic bacteria from animals and hu-mans (80) and as indicated earlier,E. amylovora(54). Comparative analysis of thegenetic context ofstrA-strBon SmR determinants RSF1010 and Tn5393showsthat the 81-base-pair right inverted repeat of Tn5393 is present downstream ofstrB in RSF1010 (8) (Figure 4). This suggests that a Tn5393-type element orig-inally deliveredstrA-strB to an RSF1010 ancestral plasmid. ThestrA-strBgenepair is currently linked with the sulfonamide-resistance genesulII on RSF1010(62) (Figure 4). It is not known ifsulII or other antibiotic-resistance genes linkedto strA-strBfound in clinical and animal pathogens (examples shown in Figure 4)were originally located on Tn5393-type transposons. Full-length Tn5393elementsare distributed among isolates of the fish pathogenAeromonas salmonicidafromNorway (27). An intact Tn5393sequence, which is interrupted in two places byadditional insertions, was discovered on a large plasmid from a soil isolate ofthe gram-positive opportunistic human pathogenCorynebacterium striatum(83)(Figure 4). Truncated variants of Tn5393, which usually containstrA-strBbut havelost part or all of thetnpRand tnpA genes, have also been identified in clinicalisolates ofP. aeruginosafrom France andYersinia pestisfrom Madagascar (19, 49)(Figure 4).

A critical deficiency in the current knowledge ofstrA-strBecology is the po-tential for transfer among broadly defined bacterial groups (e.g., human and plantpathogens). Studies demonstrating direct transfer ofstrA-strBbetween pathogensof humans and plants have not been reported. Many reports indicate that both hu-man and plant pathogens dwell in habitats with commensal organisms that func-tion as a reservoir providing the pathogens with antibiotic-resistance determinants(1, 13, 24, 80). Comparative sequence analyses from 15 sources have identified dis-tinct base-pair variants that distinguish thestrA alleles carried on nonconjugativeplasmids and integrons fromstrAalleles carried on Tn5393(75, 76). The observa-tion that these lineages are nonoverlapping suggests that the original transpositionof a Tn5393-type element to an RSF1010 ancestor occurred relatively early, prob-ably during the initial emergence of bacterial streptomycin resistance. Thus, the


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most common genetic vehicles ofstrA-strB in human and plant pathogens areeffectively distinguished and isolated from recent gene transfer events. However,two exceptions, the Tn5393-associatedstrA-strB in Y. pestis, and the RSF1010–like pEa8.7 inE. amylovora, illustrate the occurrence of commonstrA-strBvectorswithin the human and plant pathogen groups. A potential shared route of transfer ofTn5393and RSF1010-like plasmids among bacteria from animals and plants mayinvolve soilborne bacteria acting as intermediates or reservoirs for these elements.

CONTROVERSIAL ASPECTS OF ANTIBIOTICUSE ON PLANTS

Does antibiotic use in agriculture contribute to the development of antibiotic re-sistance in human pathogens? This question has been the subject of contentiousdebate in recent years, especially in Europe and North America. In agriculture,most censure has been directed toward antibiotic use in animal husbandry andavian production, which accounts for about 40% of total antibiotic use in the USA(33). Concerns have extended to antibiotic use in plant agriculture, although theamount and spectrum of antibiotics used on plants is very small. Critics contendthat spraying antibiotics in the open and over large expanses of land may increasethe frequency of antibiotic resistance in the gene pool and in the food chain (e.g., viaantibiotic-resistant bacteria on fresh produce). This would include not only genesfor resistance to the antibiotic applied but any other resistance genes that might belinked, thereby increasing the risk of these genes finding their way into clinicallyimportant bacteria. Growers and their advocates defend the practice as being solimited in scope as to be inconsequential to the emergence of antibiotic-resistantbacteria in hospitals and communities. They maintain that antibiotics have beenused on crop plants for nearly 50 years without reports of adverse effects on hu-mans. At stake for pome-fruit growers is an effective weapon in the battle againstfire blight, a disease that has emerged as the greatest threat to apple and pear pro-duction in many parts of the world. Eliminating the use of streptomycin and oxyte-tracycline on plants would not ameliorate the antibiotic-resistance crisis in humanmedicine, but it certainly would be detrimental to the apple and pear industries.

The body of knowledge regarding the genetics and ecology of antibiotic resis-tance in plant-associated bacteria, including comparisons with human-associatedbacteria, was discussed previously. However, research has not directly addressedwhether bacterial populations that inhabit farmworkers or consumers of fresh pro-duce are influenced by the application of antibiotics to crops. In laboratory stud-ies, transfer of a TcR plasmid among enteric bacteria of human and plant origin(E. coli,Erwiniaspp.,Salmonella typhimurium, andShigella dysentariae) occurredat various frequencies (6), and several subsequent studies confirmed the in plantatransfer and stability of antibiotic-resistance plasmids among plant-associated bac-teria (28, 29, 37). However, the broad-host-range resistance plasmids used in thosestudies originated in bacteria isolated from humans, not plants. If there were



transfers of antibiotic-resistance genes from bacteria in the environment to bac-teria that inhabit humans, would those genes be expressed in their new bacterialhost? This question was addressed by Schnabel & Jones (63), who found thatstreptomycin- and tetracycline-resistance determinants were often linked on largeplasmids in orchard bacteria (primarilyP. agglomeransand a species ofPseu-domonas). However, attempts to transfer the resistance plasmids by conjugationfrom bacteria isolated from orchards toE. coli failed. When introduced intoE. coliby electroporation, the plasmids were maintained, but the new host was resistantonly to tetracycline and not to streptomycin. Studies in this area have been few;clearly, additional research is needed to clarify the relevance of antibiotic use inplant agriculture to the overall resistance problem in human medicine.

Recent events in the regulatory arena illustrate the very political nature of an-tibiotic use in plant agriculture. In 1994, a company in Mexico applied to US EPAto register a plant-grade formulation of gentamicin sulfate as a pesticide to managefire blight of apple and pear in the USA. Gentamicin sulfate is registered for useon several crops in Mexico and some other Latin American countries and has beenfield-tested on apple in the USA. The Union of Concerned Scientists, the AmericanSociety for Microbiology, the Centers for Disease Control and Prevention, and theFood and Drug Administration were united in their disapproval of the registration,primarily because gentamicin is a critical tool in human medicine, used alone orin combination with other drugs for treatment of various infections. Recognizingthe uphill battle that lay ahead, the company withdrew their application in 1999.This case suggests that attempts to register additional antibiotics for use on plantswill face strong political opposition, particularly if the antibiotic is used in hu-man medicine or is related to clinically important drugs. Furthermore, without astated policy from US EPA regarding the registration of antibiotics for plant use,agrochemical companies are not inclined to develop and test new antibiotics.

An obvious strategy to avoid or delay the onset of antibiotic resistance inE. amylovorawould be to mix oxytetracycline and streptomycin or alternate thetwo antibiotics in a spray program. However, apple producers and their pest man-agement advisers have been frustrated by the fact that US EPA grants specialregistrations of oxytetracycline on a state-by-state basis only after the existence ofSmR E. amylovorahas been confirmed. For example, requests from Utah to useoxytetracycline on apple in the late 1990s were rejected by US EPA, because SmR

E. amylovorahad not been detected in that state. Unfortunately, in 2000 severe fireblight was observed in apple orchards in Utah, and subsequent analyses revealedSmR E. amylovora(85). Perhaps this outbreak and the development of resistancecould have been avoided or mitigated had oxytetracycline been allowed on appleduring the 1990s. Interregional Research Project No. 4 (IR-4), a federal programwhose mission is to assist in the registration of pest management products for useon minor crops, submitted a tolerance petition for oxytetracycline use on appleto US EPA in June 1997. Full, nationwide registration was expected to follow,because the data supporting the petition had been collected in a coordinated ef-fort across the USA and because problems had not arisen from previous use of


458 MCMANUS ET AL.

oxytetracycline through special registrations in several states. However, to datethere has been no formal response from US EPA regarding this petition.

A FUTURE WITHOUT ANTIBIOTICS?

What would be the consequences for agriculture if antibiotics were lost from plantdisease–management programs? Only a small fraction of plant diseases caused bybacteria and phytoplasmas are currently managed with antibiotics; therefore, pro-duction of most crops would be unaffected. Even for crops such as apple, peach,and pear, to which the bulk of plant-grade streptomycin and oxytetracycline are ap-plied, a minority of all hectares is treated (Table 1). This suggests that alternativesto antibiotics are available and, at least to some extent, practical. Indeed, bacte-rial disease management in most cropping systems is based on the integration ofgenetic resistance of the host, sanitation (avoidance or removal of inoculum), andcultural practices that create an environment unfavorable for disease development.Application of copper compounds is effective in reducing populations of some bac-terial plant pathogens, although several species have become resistant to copper(11), and most tree-fruit crops are sensitive to copper injury. For many fruit crops,including apple, peach, and pear, the preference of consumers and processors dic-tates which cultivars will be planted, even if they are susceptible to disease. In thecase of fire blight,E. amylovorabecomes systemic in trees, making the completeremoval of infected tissues problematic. Sanitation efforts also can be hamperedby the introduction of bacterial pathogens on nursery stock (43, 61, 73, 77). Thus,application of antibiotics remains important in the management of certain bacterialdiseases, especially of deciduous fruit and ornamental trees.

Predicted Impacts on Apple and Pear Production

The emergence SmR E. amylovorahas intensified fire blight epidemics in the west-ern USA and Michigan and serves as a presage of what life would be like if antibi-otics became unavailable through loss of registration. As with any pesticide, USEPA periodically reviews the registrations of antibiotics used on plants, and strep-tomycin and oxytetracycline are due for review in 2006. In light of this, we mustconsider the ramifications if registrations of these antibiotics were not renewed.

We asked university extension scientists who advise pear and apple growerson managing fire blight to speculate on the future of pome-fruit production in theabsence of antibiotics. David Sugar (Oregon State University) and Rachel Elkins(University of California-Davis) both noted that pear production in the UnitedStates is largely confined to the interior valleys of western states with cool springsand dry summers, which reduces the probability of severe fire blight epidemics.Elkins predicts that in California, Bartlett pear, which accounts for 55% of totalpear production nationwide, would be particularly vulnerable to losses if antibi-otics were not available. Timothy Smith (Washington State University) reportedthat when antibiotic sprays have been necessary in orchards in Washington but



not applied, severe damage has resulted. Sugar emphasized that losses due to fireblight can inflict critical economic damage to a grower who operates with littleprofit margin. Without antibiotics, pear growers more frequently would experiencefire blight epidemics, and many would likely cease their pear-growing enterprises.Extension scientists who advise apple growers voiced similar concerns. In the1990s apple cultivars that are highly susceptible to fire blight (e.g., Braeburn,Gala, Ginger Gold, and Pink Lady) were planted to satisfy consumer demand.Sherman Thomson (Utah State University) noted that the fire blight threat to applecould be partially attributed to the planting of high-density apple orchards of sus-ceptible cultivars grafted to susceptible rootstocks. Without antibiotics, Thomsonand Mark Longstroth (Michigan State University) expect that certain popular applecultivars and even entire orchards would be abandoned because of excessive lossesfrom fire blight. In fact, between 1997 and 2000 Michigan lost 18% of its applehectares and 15% of its growers, primarily because of fire blight and low appleprices.

New Tools to Combat Fire Blight

The vulnerability of antibiotics to the emergence of resistance and the potential lossof registration have prompted substantial research to develop new tools to combatfire blight and other bacterial diseases of plants. Through conventional breeding,scientists at Cornell University have developed and released a new series of applerootstocks with resistance to fire blight (31). The Cornell group also has developedtransgenic apple cultivars and rootstocks with enhanced resistance to fire blight(51), but these have not been released for use by growers. Some exciting lines ofresearch are yielding insights into how bacterial diseases might be managed in thefuture, and some new products are now available to growers.

In the mid-1990s, biological control of fire blight withPseudomonas fluorescensstrain A506 (BlightBan A506) was introduced, and other biological controls arebeing developed (22). Large-scale field trials suggest that A506 can significantlyreduce the amount of fire blight, but it is most effective in an integrated programthat includes antibiotics (34). Unfortunately, this product has not been available togrowers every year.

Three other relatively new products do not act directly onE. amylovora, butrather alter the tree’s physiology to diminish the effects of fire blight. The harpinprotein (Messenger, Eden Biosciences) is an inducer of plant defense mechanisms(25), and in some field trials has reduced fire blight. Another activator of plantdefenses is acibenzolar-S-methyl (ASM; Actigard or Bion, Syngenta Crop Pro-tection, Inc.). ASM was recently registered on tomato to prevent symptoms ofbacterial spot and bacterial speck diseases; registration on apple is being consid-ered. In the field, ASM reduced fire blight symptoms on apple blossoms by about50%; however, the long-lasting protection observed on some herbaceous crops fol-lowing ASM treatment was not observed on apple (3, 39). Prohexadione calcium(Apogee, BASF Corp.) is a plant growth regulator that restricts shoot elongation


460 MCMANUS ET AL.

and thereby reduces fire blight symptom development in shoots. Registered inMay 2000, it is the first chemical permitted in the United States for fire blightmanagement since the antibiotics were registered that has been effective in bothresearch trials (89) and commercial orchards.

While these products show promise in reducing the amount of antibiotics usedin plant agriculture, they have only recently been introduced and not used on asufficiently large scale. As such, their role in the long-term management of allstages of fire blight and their potential horticultural “side-effects,” good or bad,are largely unknown. Moreover, the efficacy of these products is low and theircost high compared to antibiotics. Therefore, we expect that the new products willbe used in conjunction with streptomycin and oxytetracycline, but they will notreplace antibiotics entirely.

CONCLUDING REMARKS

Until recently, the use of antibiotics on plants went largely unnoticed by those out-side of the plant agricultural community. However, the alarming rise of antibiotic-resistant bacterial pathogens of humans, and efforts to conserve the efficacy ofantibiotics in human medicine, have drawn attention to nonmedical applicationsof antibiotics. In our review, we approached the topic on four fronts. First, rec-ognizing a potentially diverse readership, we summarized the practical aspects ofantibiotic use on plants, specifically that the diversity and quantity of antibioticsapplied to plants is small relative to uses in human and veterinary medicine. Sec-ond, we assimilated the body of knowledge on the molecular genetics of antibioticresistance in plant-associated bacteria to propose routes of horizontal gene transferthat might explain the widespread occurrence of homologous resistance genes indiverse bacteria. Third, we offered a perspective on the effect on human health ofantibiotic use on plants. Although we concluded that the role of antibiotic use onplants is probably insignificant, we cannot state that there is no risk, as research islacking in this area. Finally, we envisioned a future without antibiotics. Some newproducts for the control of fire blight of apple and pear have the potential to signif-icantly reduce the amount of antibiotics used in plant agriculture. From a practicalstandpoint, however, antibiotic application remains the most effective and econom-ical method for managing fire blight, the major plant disease targeted by antibiotics.

ACKNOWLEDGMENTS

The authors thank Marlene Cameron for preparing Figures 1 and 3; Anne Vi-daver for providing insights and contacts related to antibiotic use in agriculture;and Donald Cooksey, Robert Hollingsworth, Steven Lindow, and Luis Sequeira forthoughtful reviews of the manuscript. The authors’ research on antibiotics was sup-ported in part by USDA/CSREES, the Michigan Agricultural Experiment Station,the Texas Higher Education Coordinating Board Advanced Research Program,and the Winter Pear Control Commission.



The Annual Review of Phytopathologyis online at http://phyto.annualreviews.org

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22 Jul 2002 8:45 AR AR165-16-COLOR.tex AR165-16-COLOR.SGM LaTeX2e(2002/01/18)P1: GDL

Figure 2 Genetic organization of (a) Tn5393from Erwinia amylovora, (b) Tn5393afrom Pseudomonas syringaepv. syringae, and (c) Tn5393b from Xanthomonascampestrispv. vesicatoria. IR, inverted repeat;tnpA, transposase;res, resolution site;tnpR, resolvase; IS1133and IS6100, insertion sequences;strAandstrB, streptomycinphosphotransferases;tnpR*, resolvase interrupted by IS6100. The direction of tran-scription of individual genes is shown.

22 Jul 2002 8:45 AR AR165-16-COLOR.tex AR165-16-COLOR.SGM LaTeX2e(2002/01/18)P1: GDL

Figure 4 Genetic maps of thestrA-strB resistance region and flanking DNA fromnonconjugative plasmids, integrons, and Tn5393-related elements.sulII, sulfonamide-resistant dihydropteroate synthase;strA andstrB, streptomycin phosphotransferases;strAXandstrA*, truncatedstrA genes;dfrIII , dfrIb, anddfr9, trimethoprim-resistantdihydrofolate reductases;intI1, integrase;aacA4, gentamicin acetyltransferase;oxa20,β-lactamase; IS26 and IS1250, insertion sequences; IR, inverted repeat;tnpA, trans-posase;res, resolution site;tnpR, resolvase;tnpR*, truncated resolvases; andstrB*,strB gene with an incomplete sequence. Genomic locations are either the plasmidslisted by name or chromosomal insertions. The location of coding sequences and di-rection of transcription are shown by boxes and arrows. Bacterial hosts are as follows:(a) broad-host-range; (b) Escherichia coli; (c) Campylobacter jejuni; (d) Shigellaflexneri; (e) Pseudomonas aeruginosa; ( f ) Corynebacterium striatum; and (g) Yersiniapestis.

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Patricia S. McManus1, Virginia O. Stockwell2 George W