In Vitro CeO. Dev. Bioi. 31:58-64. january 1995e 1995 Society roc In Vitro Biology1054-5476/95 $02.50+0.00

CHARACTERIZATION AND IDENTIFICATION OF BACTERIA ISOLA TED FROMMICROPROPAGATED MINT PLANTS

PATRICIA M. BUCKLEY, TRACI N. DeWILDE, AND BARBARA M. REED I

USDA-ARS, National Clonal Gennplasm Repository, 33447 Peoria Road, CoroaUU, Oregon 97333-2521

(Received 6 April 1994; accepted 30 September 1994; editor T. A. Thorpe)

SUMMARY

Bacterial isolates from contaminated mint shoot cultures were characterized and identified as a preliminary step indetermining an elimination treatment. The 22 bacteria were characterized using biochemical and morphological tests andsubjected to sensitivity tests with four antibiotics. The isolates were compared with known organisms and assigned togenera according to similarities in characteristics. Seven isolates were analyzed by fatty acid analysis carried out by acommercial laboratory. Six were classified as Agrobacterium radiobacter; eight as XanthomonaoS; one each as P$eudo-mona.s fluore$cens, Micrococcu.s spp., Corynebacterium spp., and Curtobacterium spp.; four could not be assigned togenera. Inhibition of growth of the bacteria by most antibiotics was best at pH 7.5. Minimal inhibitory concentration andminimal bactericidal concentrations of gentamicin, rifampicin, streptomycin sulfate, and Timentin varied with genotype.

Key word.s: Agrobacterium; antibiotic; bacterial contamination; Mentha; P$eudomona.s; Xanthomona.s.

plant pathogens, evidence of virulence may be limited by aspects ofdisease resistance in the host such as release or stimulation of natu-rally present inhibitors and the accumulation of agglutinins or phy-toalexins (Billing, 1982; Bastiaens, 1983). None of these is suffi-cient to eliminate either saprophytic or latent pathogenic bacteriathat may be present in any plant part used for micropropagation.

One of the recommended solutions to contamination problems isto discard all infected cultures and begin with clean material. Ensur-ing that endophytic contamination is absent from stock plants re-quires that the method used for detection be dependable and rapid(Debergh and Vanderschaeghe, 1988; Reed et al., in press). Manybacteria grow slowly or not at all in the media used in plant culture,thus escaping detection until considerable time and materials havebeen expended (Viss et al., 1991). A popular solution is to useantibacterial substances (e.g., antibiotics), but this approach hasmet with varying degrees of success (Cornu and Michel, 1987;Falkiner, 1990; Leifert et al., 1991a). In some cases, antibiotictreatments appear bacteriostatic, resulting in reduced rather thaneliminated contamination (Phillips et al., 1981; Bastiaens et al.,1983; Young et al., 1984; Kneifel and Leonhardt, 1992). In othercases, the phytotoxicity of the antibiotics has precluded their use ata level high enough to destroy all contaminants (Falkiner, 1990;Leifert et al., 1991a,1992). Selecting drug-resistant strains of bac-teria from long-term exposure to antibiotics is also of concern(Cornu and Michel, 1988; Kneifel and Leonhardt, 1992). The lackof descriptive information and antibiotic susceptibilities of a largenumber of plant-associated bacteria further complicates the use ofantibiotics (Leifert et al., 1989). Better characterization and identi-fication of plant-associated bacteria should lead to more successfulantibacterial therapies (Cornu and Michel, 1987; Cassells, 1991;Leifert et al., 1991b). Falkiner (1988) reviewed the desirable fea-tures of antimicrobial substances for use in plant tissue culture and

INTRODUC11oN

Plant tissue culture contamination by microorganisms, especiallybacteria, continues to obstruct successful experimentation and theexchange of plant material that is free of detectable microbes. Lei-fert et al. (1991 b) consider it the most important reason for lossesin micropropagation operations. Kneifel and Leonhardt (1992) rec-ognize contamination by endophytic bacteria as "one of the mostserious problems in tissue culture." Increasing attention has beendirected to this problem by workers seeking to ascertain sources ofcontamination and develop procedures for eliminating them byavoidance, rigorous manipulation of environmental and nutritionalfactors, or treatment with antibiotics (DeFossard and DeFossard,1988; Debergh and Zimmerman, 1991). Cassells (1991) pointedout that plants may develop endophytic microbial colonizationthrough wounds and natural openings and via the root system. Bothpathogenic and saprophytic bacteria have been isolated from plantsused for micropropagation (Bastiaens, 1983). The most commonlyisolated bacteria belong to the genera Xanthomonas, Corynebacte-

rium, Erwinia, Bacillus, Pseudomonas, Micrococcus, Agrobacte-rium, Arthrobacter, and Enterobacter (Bastiaens, 1983).

Bastiaens (1983) emphasized the need for sound screening ofdonor plants and for effective decontamination followed by meticu-lously practiced asepsis in the handling of tissue cultures in thelaboratory. Persistent endophytic microorganisms may be trans-ferred unknowingly at each subculture and may become apparentweeks or months after the material was initiated in vitro (Leifert etal., 1989). Contaminants may be latent and fail to produce overtdisease symptoms (Bastiaens, 1983). The role of endophytic organ-isms in the metabolism of the host plant is unknown. In the case of

I To whom correspondence should be addressed.

58

BACTERIAL IDENTIFICATION 59

outlined the testing necessary to establish antibiotic concentrationsand effective combinations for treatment of infected tissues.

Our experience with the subcultivation and long-term cold stor-age of shoot-tip explants, especially from numerous (>400) speci-mens of mint, led to the observation that endophytic bacteria fre-quently persisted in the absence of obvious damage to the plants.Repeated subcultivation of tips from these cryptically infectedplants showed characteristic haloes of bacterial growth in the plantgrowth medium, despite vigorous disinfection and aseptic handlingof the explant tissues during and after their collection from pot-grown screenhouse plants. Often the contamination became appar-ent only after several subcultures.

This report describes a) the characterization and identification ofbacteria isolated from persistently contaminated mint cultures usingdiagnostic biochemical tests, as well as tests of nutritional and envi-ronmental factors and b) the susceptibility of these bacteria to fourantibiotics.

B49C), Enterooocter cloacae (EcCT -50 I), Erwinia herbicola (952), Pseu-domonasj/uorescens (Pf-5), and P. putida (AI2). These strains were usedas standards to make comparisons and aid in the characterization and pre-sumptive identification of the bacterial strains isolated in this study.

Antibiotic $usceptibility. Streptomycin sulfate, gentamicin, rifampicin(Sigma), and Timentin (SmithKIine Beecham Pharmaceuticals, Philadel-phia, PA) were prepared as wt/vol or vol/vol solutions in deionized water,filter sterilized, and added in appropriate concentrations to nutrient agar(pH 6.8-6.9). Drops (50 Ill) of bacterial suspensions were inoculated ontothe plates that were incubated at 250 C for 4-7 d and observed for totalinhibition of growth.

Minimal inhibitory concentrations. Minimal inhibitory concentrations(MIC) of the antibiotics were estimated by a tube dilution method for bothstandard bacterial cultures and bacterial isolates from infected mints. Liq-uid plant medium 0.5X at pH 6.9 provided a suitable substrate for the testin which a series of tubes was set up containing doubling serial dilutions ofantibiotic in a final volume of 2 ml per tube. Each tube was inoculated with50 p.I of 72 h broth culture of bacteria and incubated at 250 C for 4-7 d.Cell growth was assayed by measuring turbidity; a clear tube implied totalinhibition of growth. Very slight turbidity was often difficult to detect, andled to imprecise determinations of MIC. To overcome this, we sampled oneconcentration below and three or four at and above the last clear tube, forplating onto sectors of the minimal bactericidal concentration (MBC) detec-tor plates. Also included was a control sample that contained bacteria but noantibiotic, as well as one that had been incubated with diluent alone. MIC isrecognized to be a preliminary test, indicating the lowest concentration of anantibiotic that will prevent bacterial growth and multiplication; it does notdetect whether viable organisms remain.

A second test was used to determine the minimal bactericidal concentra-tion (MBC). MBC, usually reported in ILg/ml, was set up by inoculating 50p.l droplets from the MIC tubes (as described above) onto marked sectors ofa nutrient agar plate (lacking antibiotic). Plates were incubated for 4-7 dbefore examining for colony formation. The MIC tube with the lowest con-centration of antibiotic that resulted in complete absence of growth on a testplate was considered to be the minimal bactericidal concentration.

Bacteria in this study are designated by the local identification numbersof the mint accessions from which they were isolated.

RESULTS AND DISCUSSION

Characterization tests. Bacteria were grouped into tentative ge-neric categories by comparisons of diagnostic tests and characteris-tics of known strains (Tables 1-4). Colony characteristics werebased on growth on NA. Pigmentation developed on NA but was notpresent on the plant growth medium.

Xanthomonas group. Seven yellow pigmented forms weregrouped as Xanthomonas species based on colony appearance,diagnostic tests, and genus descriptions found in Bergey's Manual(Krieg and Holt, 1984). The three known strains of Xanthomonasproduced mucoidfbutyrous, yellow colonies; exhibited starch hydro-lysis and motility typical of xanthomonads; and gave typical re-sponses to oxidase, gelatinase, and oxidation\fermentation (O\F)tests (Table 1). The known X. campestris strain digested potatoslices, a characteristic associated with pathovars of the X. campestrisgroup, but none of the mint isolates did. Isolates from mint culturesgenerally matched the test results of one or more of the knownstrains and three were identified by fatty acid analysis as related toX.

campestris celebensis GC subgroup B (M26, SI 0.5; M59, SI0.48; M363b, SI 0.36).

Isolates M128 and M214 deviated from the known strains espe-cially by their fermentive activity; however. the xanthomonads are adiverse group, shown by the results for the known X. vesicatoriastrain (Table 1). For example, X. ampelina grows poorly on NA andlacks characteristic xanthomonadins in its pigment compared withX.

campestris pathovars. The X. ampelina group also differs from X.campestris in urease activity and failure to hydrolyze starch or gela-

MATERIALS AND METHODS

Plant material wed. The 35 infected mint accessions used were:Mentha spicata L. (10 accessions), (3 rugose form, M. cordifolia Opiz exFresen); M. suaveolens Ehrh. (4); M. spicala var. Crispa (Benth.) Danhert(3); M. canadensis L. (2); M. longifolia (L.) Hudson (1); M. suaveo/ensvariegala Ehrh. (1); Mentha X piperila, sspp. citrala (Ehrh.) Briq. (4); M. Xsmithiana R. H. Graham (1); M. X villosa Hudson (1); and the followinghybrids: M. suaveo/ens Ehrh. X M. longifolia (L.) Hudson (3), M. citralaEhrh. X M. aquatica L. (2), M. aquatica L. X M. spicala L. (1), M. cana-densis L. X M. spicala L. (1), and M. spicala L. X M. suaveolens Ehrh. (1).The plants were grown in pots in a screenhouse at the USDA-ARS NationalClonal Germplasm Repository, Corvallis, OR. Explants were disinfested byimmersion in 10% household bleach (5.25% sodium hypochlorite; Clorox,Oakland, CA) solution containing 1 % Tween-20 (Sigma Chemical Co., St.Louis, MO) for 10 min, rinsed twice in sterile deionized water, and grown inindividual 16 X 100 mm tubes containing Murashige and Skoog (MS)medium (Murashige and Skoog, 1962), with 0.5 mg ° liter-I N6-benzyl-

adenine, 3 go liter-I agar (Bitec, Difco, Detroit, Ml) and 1.25 goliter-1Gelrite (Schweizerhall, South Plainfield, NJ), pH 5.7, at 26-300 C with 16h of light (25 ILmolo m-2. S-I).

Detection and isolation of bacteria. Bacterial growth appeared as acloudy zone in the agar medium around the shoot base or as turbidity inliquid plant medium containing infected material (Reed et aI., in press).Contaminants were transferred with a cotton swab to 0.8% nutrient broth(Difco; Sigma) with glucose 1% and yeast extract 0.5% at pH 6.9 andincubated at 250 C until visibly turbid. Bacteria were purified by repetitivestreaking and subsequently maintained on nutrient agar (NA). Single colo-nies were transferred to NA slants for short-term storage and to plates of thesame agar and grown to provide inocula for the various tests.

Diagnostic tests for bacterial characterization. The foUowing tests wereperformed according to the methods described by Klement et aI. (1990):Gram stain, KOH, motility, oxidase, oxidative/fermentive (O/F), starchhydrolysis, gelatinase, urease, phosphatase, arginine dihydrolase, and po-tato slice digestion. Also employed were D-series selective media preparedas described by Kado and Heskett (1970) for the detection of agrobacteria,xanthomonads, and pseudomonads. Standard methods were used to evalu-ate bacterial response to temperature and pH. Preparations from brothcultures were negatively stained with sodium phosphotungstate and exam-ined for flageUa by transmission electron microscopy.

FaUy acid methyl ester analysis. Selected bacterial strains were sent toMicrocheck, Inc. (Northfield, VT) to be identified by gas chromatographicfatty acid methyl ester analysis. Strains with a similarity index (51) of ~0.5were identified to species, those with 51 <0.5 and ~O.lto genus, and thosewith an SI <0.1 to a group (Boehm el aI., 1993).

Standard bacterial cultures. Cultures of the foUowing bacterial strainswere provided by Ms. Marilyn Canfield, Department of Botany and PlantPathology, Oregon State University: Xanthomonos campestris, X. incanae,and X. vesicatoria; and by Dr. Joyce Loper, USDA-ARS, HorticulturalCrops Laboratory, Corvallis, OR: Agrobact"rium tumefaci"ns (Slrain

60 BUCKLEY ET AL.

TABLE 1

CULTURAL CHARACTERISTICS OF GRAM-NEGATIVE BACTERIA FROM MENTHA IN VITRO CULTURESTENTATIVELY IDENTlnED AS XANTHOMONAS SPECIES AND KNOWN STRAINS OF X. CAMPESTRIS, X. INCANAE,

AND X. VESICATORIA GROWN ON NUTRIENT AGAR

x. ...icatoriaCharacteristic/Organism 26" 45 58 59" 77 128 214 363b" X. campestriJ X. incanae

.Identified as related to X. campestris celebensis GC subgroup B by fatty acid analysis (M26, 51 0.5; M59, 51 0.48; M363b, 51 0.36).nd = not done; +/- = present/absent.

tin (Krieg and Holt, 1984). Some of the mint isolates grouped withthe xanthomonads possessed the latter two characteristics, but werepigmented and grew readily on the NA. Xanthomonad selectivemedium 0-5 (Kado and Heskett. 1970) failed to favor the growth ofour xanthomonad isolates and instead supported growth of severalother groups.

Agrobacterium group. Six isolates with beige pigmentation asso-ciated with highly mucoid, moist colonies were categorized as Agro-bacterium species (Table 2). They were able to grow on Agrobacte-rium selective medium 0-1 (Kado and Heskett. 1970). All six wereoxidase positive, OfF negative and most were starch hydrolysisnegative and urease positive typical of Agrobacterium. Four wereidentified further by fatty acid analysis as A. radiobacter (M4 7, SI0.9; M76, SI 0.9; M 2348, SI 0.9; M591, SI 0.6) and two otherswith similar characteristics were included among the agrobacteria,based on biochemical and morphological data.

Other Gram-negative isolates. The remaining four Gram-negativeisolates varied in their characteristics (fable 3). The known P.fluorescens

strain was oxidase positive, starch hydrolysis negative,arginine dihydrolase positive and showed fluorescence on King's 8medium (Klement et al., 1990) under ultraviolet light. Isolate 363aclosely matched the known P.jluorescens strain in biochemical reac-tions and showed strong fluorescence (fable 3). Medium 0-4 (Kadoand Heskett, 1970), selective for pseudomonads, not only favoredtheir growth but also supported the growth of several other genera.

Isolates M221 and M234a may belong to the genus Flavobacte-

rium (Table 3). Both isolates produced small, round, convex, moist,butyrous, yellow colonies with entire edges, conforming to the col-ony characteristics described in Bergey's Manual (Krieg and Holt,1984). Flavobacterium strains are reported to resist many antimi-crobials; this was not the case with mint isolate M221 but M234ashowed resistance to streptomycin and Timentin at all pH levelstested but not to gentamicin. These isolates also resemble Alcali-genes paradoxus. Fatty acid analysis was inconclusive for M234a.

Gram-positive isolates. Gram-positive bacteria were assigned toseveral groups, based on diagnostic tests and comparison with char-acteristics of genera containing nonsporeformers, especially thoseassociated with or isolated from plants (Table 4). Isolate M363cwas similar in cell morphology and colony characteristics to Micro-coccus species, however, results of the oxidase, OfF, arginine dihy-drolase, and urease tests differed from those expected for manyMicrococcus species.

Bergey's Manual (Krieg and Holt, 1984) describes severalgroups of Gram-positive, irregular, nonsporeforming rods, whichinclude organisms isolated from plants and soil. Among these areCorynebacterium, Aureobacterium, Curtobacterium, and Arthro-bacter. Reliable differentiation of the several Corynebacteriumgroups rests on the composition of cell wall material, including thetype of peptidoglycan and the major peptidoglycan diamino acid.Based chiefly on morphological similarities, isolate M 119 wasplaced with the coryneform group.

M557b was identified by fatty acid analysis as most closely re-

TABLE 2

CULTURAL CHARACTERISTICS OF GRAM-NEGATIVE BACTERIA FROM MENTHA IN VITRO CULTURESTENTATIVELY IDENTIFIED AS AGROBACTERIUM SPECIES AND A KNOWN STRAIN OF A. TUMEFACIENS

234bO 470 591'Characleristic/ organism 47' 76" 228 A lum.facie.u

+++

-/-++

-++

-/-+

-++/-+

-++-/-

+

+

-++

-/-+

+

-++

-1-+

++

++

-/-+

++

Yellow pigment

Mucoid/moistOxidase

OfF (glucose)MotilityStarch hydrolysisUreaseGrowth @ pH 4.4

"Identified by fallY acid analysis as A. radiobacll!r (M47, 51 0.9; M76, 51 0.9; M 2348, 51 0.9; M59l, 51 0.6). +{present/absent.

61BACTERIAL IDENTIFICATION

TABLE 3

CHARACTERISTICS OF GRAM-NEGATIVE BACTERIA FROM MENTHA IN VITRO CULTURES TENTATIVELYIDENTIFIED AS PSEUDOMONAS OR OTHER SPECIES AND KNOWN STRAINS

OR DESCRIPTIONS OF RELATED SPECIES

Raised. Spreading, Entire

E. h.rbico/a Alcaligenes'Colony form

Characteristic/Organism

++

-/-

+++/-

++

-/-

+++

v/-

v

+

+-/-

++++

+

+-/-++++

+

+-/-+

++

++

+/++

+

+-/+.+

+/-+

na

Moist/butyrousYellow pigmentOxidase

OfF (glucose)MotilityGelatinaseGrowth at 50 C

Arginine dihydrolase

.Showed fluorescence on King's B medium under uv light.b Description from Bergey's Manual (Krieg and Holt, 1984)., Alcaligenes paradoxus, moist, slimy, yellow colonies. v = species may vary; na = information not available; + /- = present/absent.

lated to Curtobacteriumflaccumfaciens betae (SI 0.4). Members ofthe genus Curtobacterium, containing saprophytes and plant patho-gens, exhibit morphology very similar to organisms of Corynebacte-rium species. The group C. flaccumfaciens is regarded as phyto-pathogenic. The main characteristic for differentiating Curtobacte-rium from Aureobacterium, Cellulomonas, and Microbacterium isthe structure of the peptidoglycan (Krieg and Holt, 1984). We didnot analyze for peptidoglycan structure.

Leifert et aI. (1989) isolated 190 strains of bacteria from micro-propagated plant cultures of which 30% were identified as belong-ing to the genus Pseudomonas. They found that about 70% of con-taminants isolated after plants had been propagated for more than12 months were Gram-positive bacteria belonging to the generaStaphylococcus, Micrococcus, and Lactobacillus, commonly foundamong human flora. These isolates were thought to have been intro-duced by the workers, while others including 57 Pseudomonas spp.were believed to have been introduced from infected stock plants orinadequate sterilization of media or equipment (Leifert et aI.,1989). Bacteria isolated from mint cultures, including the Gram-

positive isolates, were from groups generally recognized as soil orplant associated organisms.

Effect of pH on antibiotic susceptibility. Gram-negative bacteriawere tested for antibiotic susceptibility on NA plates at three pHlevels (Table 5). Antibiotic activity usually is affected by the pH ofthe carrier medium (Falkiner, 1988). Gentamicin (50 JLg/ml) inhib-ited growth of xanthomonad isolates and known strains at pH 7.5but five isolates grew at pH 6.5 and seven at pH 5.5. Susceptibilityof the Agrobacterium group to gentamicin varied from total at pH7.5 and variable at pH 6.5, to completely resistant at pH 5.5. Theother Gram-negative isolates and known strains were variable intheir responses to gentamicin at lower pH levels but all were inhib-ited at pH 7.5 (Table 5).

Rifampicin (25 JLg/ml) inhibited the growth of all known strainsand isolates at all pH levels except for A. tumefaciens and P. putida,which were totally resistant (data not shown). For most organisms,the bactericidal level of rifampicin exceeded the phytotoxic level formint plants, limiting its use (Reed et al., in press).

Streptomycin results were variable; one isolate (M591, A. radio-

TABLE 4

CHARACTERISTICS OF GRAM-POSITIVE BACTERIA ISOLATED FROM MENTHA IN VITRO CULTURESAND DESCRIPTIONS OF OTHER GRAM-POSITIVE BACTERIA

Organism or Group

Feature M363c Ml19 M557b'Micrococcus" Corynebacteria'

Colony fonn roundsmallcreamcoccus

+/+

+

roundsmallvariouscoccus

++/v-, v

-, v

-', v

roundsmall

gray-whiterod

roundsmallpinkrod

+/-

-,V+

round. raisedsmallyellowishrod

round, entiresmallvariousrod

PigmentCell morphology

MotilityOxidase

OfFStarchGelatinaseUrease

-

+/+

;...

-

+/+

-na

+/--, usually-,

usuallyv

.Description from Bergey's Manual (Krieg and Holt. 1984).b Identified as related to Culobaclerium jlaccumfaciens balae by fallY acid analysis (51 0.4)., Only Micrococcus varians and M. nishinomiyaensis are positive for urease. v = species may vary; na = information nol available;

+/- = present/absent.

62 BUCKLEY ET AL. I

TABLE 5

GROWTH OF GRAM-NEGATIVE BACTERIA AS AFFECTED BY pH AND GENTAMICIN AT 50 p.g/mlOR STREPTOMYCIN OR TIMENTIN AT 500 p.g/ml IN NUTRIENT AGAR

-

Timen!in

pH 5.5 7.5 5.5 7.5 5.5 7.5

Xanthomonas groupX. campestrisX. incanaeX. vesicatoriaM26"M45M57M58M59"M128M214M363b"

++

++

nd+++

+

+

+

++

-

nd

-

+++

nd++

-

++

nd+

-

++

nd+

-

+

+

-

:t

:t

-

:t

++++++

+

+

++

+-+

++

- -

+

- --

+

Agrobaclerium groupA. lumefaciensM47b

M76b

M234bbM470M59lb

Other Gram-negative rodsEnlerobacler cloacaeErwinia herbicolaP. j/uorescens'P. pulida'M363a'M234a

+

++++

-

+

+

- -

+

-

+

-

+

-

++++

-

++++

-

+++:t

.Identified as X. campestris celebensis GC subgroup B by fatty acid analysis (M26, 51 0.5; M59, 51 0.48; M363b, 51 0.36).b Identified as A. radiobacter by fatty acid analysis (M4 7, 51 0.9; M76, 51 0.9; M234b, 51 0.9; M591, 51 0.6).'Fluorescent on King's B medium, under ultraviolet light. nd = not done; +/- = bacterial growth/no growth.

bacter) was totally resistant at any pH while the known A. tumefa-ciens strain was susceptible, but only at pH 7.5. The other isolateswere either inhibited at all pH levels or only at pH 6.5 and 7.5.Streptomycin (500 Jlg/ml) inhibited growth of seven xanthomonadsat pH 6.5 and 7.5 but only five at pH 5.5. Four other isolates wereresistant to streptomycin at all pH levels (Table 5).

Timentin, composed of the broad-spectrum antibiotic ticarcillinand a {3-lactamase inhibitor, clavulanic acid, at 500 Jlg/ml inhibitedgrowth in 15 of 23 organisms at three pH levels and four otherisolates at least at one pH level (Table 5). Four others were resistantat all three pH levels while most agrobacteria and xanthomonadswere inhibited at all three. Two isolates, xanthomonads M45 andM128, were inhibited by Timentin at pH 7.5 but not at lower pHlevels; and in two other isolates, corynebacter M 119 and agrobacterM470, inhibition was present at lower but not higher pH levels.

The pH of the medium is important for the optimum activity ofmost antibiotics and, therefore, for the success of antibiotic treat-ment (Falkiner, 1988). Standard tissue culture media range in pHfrom 4.8 to 5.9 and are optimized for plant growth (Wolfe et al.,1986). Inhibition of bacteria from cultured mint shoots by gentami-cin, and in some cases streptomycin, was ineffectual at pH 5.5, apH usual for plant growth media, and most effective at pH 6.5 and7.5 where plant growth could be suboptimal. Results from the testswith Timentin at 500 Jlg/ml showed that its inhibitory activity is lessaffected by differing pH levels than that of gentamicin or streptomy-

cin. In preliminary tests with additional bacterial contaminants (P.Buckley and B. Reed, unpublished data), Timentin was stronglyinhibitory to 26 of 56 bacteria at three pH levels, at concentrationsof 250 and 500 JLg/ml, and to II more at one or more pH levels.However, others, notably pseudomonads, were resistant at all threepH levels and at both concentrations used. Timentin at less than1000 JLg/ml was not phytotoxic to micropropagated mint shoots.The consistent pattern of resistance exhibited by Pseudomonas spp.when exposed to Timentin suggests that the drug might be useful inmedia selective for this genus.

Results from tests with these four antibiotics illustrate the need toevaluate not only the concentrations but also the effect of pH on theactivity of any antibiotics considered for treatment. Falkiner (1988)is among the few workers emphasizing the importance of pH amongthe other factors in treatment design.

Minimal bactericidal concentrations (MBCs). Gentamicin wasbactericidal at 40 JLg/ml or less to all of the organisms in Tables1-4 with the exception of M76 (A. radiobacter) (Table 6). Effectivetreatment of plant tissues may require gentamicin levels of up to 80JLg/ml; however, the risk of phytotoxicity increases at concentra-tions greater than 50 JLg/ml (Reed et al., in press).

The MBCs for rifampicin varied from :;,4 (M363c) to ?IOOJLg/ml (M47, A. radiobacter, A. tumefaciens), with most of theknown strains requiring more than 60 JLg/ml for killing. Of theGram-positive bacteria, isolate M557b (Curtobacterium) had an

BACTERIAL IDENTIFICATION 63

TABLE 6

MINIMAL BACTERICIDAL CONCENTRA nONS IN lJ.g/mlDETERMINED AT pH 6.9 FOR FOUR ANTIBIOTICS AGAINST

KNOWN BACTERIA AND ISOLATES FROM INFECT~DMINT CULTURES

otics, routinely successful treatment of plant materials may be forth-coming (Quesnel and Russell, 1983).

It was not surprising to find that the kinds of bacteria isolatedfrom contaminated micropropagated mint shoots were similar tothose described by workers with other plants (Bastiaens, 1983;Oebergh and Vanderschaeghe, 1988). Nor was it surprising to dis-cover a preponderance of Gram-negative bacteria that varied intheir response to different antibiotics. Of the procedures that wereused to characterize and identify the microorganisms, the fatty acidprofile analysis is the most definitive, but the lack of definite identi-fication for 4 of the 11 cultures analyzed implies that a more exten-sive inventory of plant-associated bacteria is needed for compari-son. It is also possible that these unidentified bacteria have neverbeen described or characterized. The standard laboratory tests thatwere most helpful in categorizing the bacterial isolates were theGram stain, colony morphology and appearance, and the oxidase,0 IF, starch hydrolysis, motility, and gelatinase tests. The selectivemedium 0-1 (agrobacteria) provided fairly satisfactory selection,but the media 0-4 (pseudomonads) and 0-5 (xanthomonads) gaveless clearcut results.

Minimal Bactericidal Concentration (ltg/ml)

Organism/Antibiotic Gentamicin Rifampicin Streptomycin

Xanthomonas groupX. campestrisX. vesicatoriaM26M45363b

12.5<6101040

>60>60

2515

>60

1000 5001000 5001000 >1000

>2000 500<125 500

Agrobaclerium groupA. lumefaciensM47M76591

4010

>8020

>100>100>60

15

>2000>1000>2000

500

500500500250

4020<5<5

30>60>60>60

<500 >1000<500 >1000<125 >1000<125 500

Other Gram-negativePseudomona.l fluorescensP. putidaM363aEnterobacter cloacae

Gram-positive groupMl19M363cM557b

10<510

30<460

<125<125<125

1000<67.5500

CONCLUSION

Contaminants of micropropagated mint shoots were mostlyGram-negative bacteria often found associated with. plants and soil.The 22 bacteria were assigned to major groups based on colonycharacteristics and biochemical tests. Six were classified as AgrO-bacterium; eight as Xanthomonas; one each as P. jluorescens, Mi-crococcus spp., Corynebacterium spp., and a Curtobacterium spp.;four could not be assigned to genera. Microorganisms can be accu-rately identified by fatty acid profiles, patterns of carbon compoundutilization, or nucleic acid studies. Those procedures are costly andclassical methods may suffice, depending on the investigator's bud-get and objectives. More known strains need to be added to the database for fatty acid and carbon source utilization to provide a greaterlikelihood of identification.

The best bacterial inhibition by antibiotics occurred at pH levelsof 6.5 and 7.5. Effective antibiotic type and concentration variedwith bacterial type. Gentamicin (50 pg/ml) and streptomycin (500JLg/ml) at pH 7.5 were effective treatments for most of the isolates.Rifampicin (25 JLg/ml) was effective for some xanthomonads andGram-positive isolates but not for agrobacteria or pseudomonads.Timentin (500 JLg/ml) was very effective against most xanthomo-nads and agrobacteria but not pseudomonads.

Ascertaining the Gram reaction, purity of the isolate, colonygrowth, and a few defining biochemical characteristics along withscreening for responses to selected antibiotics should prove helpfulin predicting whether treatment to eliminate a contaminant will suc-ceed.

MBC of 60 JLg/ml, while M363c, a Gram-positive coccus, showedthe least resistance (Table 6). Rifampicin was used in treatment butat no level higher than 30 JLg/ml because of phytotoxicity (Reed etal., in press).

Streptomycin at 1000 JLg/ml or less was bactericidal to most ofthe known strains and the isolates, indicating that this level, whichwas near or below the phytotoxicity level for many of the mintcultures, could be useful in treatment. Exceptions included theknown bacterial strain of A. tumefaciens, and isolates M45 (Xantho-monas), and M76 (A. radiobacter), which survived exposure tostreptomycin at >2000 JLg/ml. Of interest is the observation thatM45 and M76 were among organisms isolated from infected mintsthat remained positive for bacteria after antibiotic treatments andstorage (Reed et al., in press). For both gentamicin and streptomy-cin, MBCs were lower for the Gram-positive than for many of theGram-negative isolates. These results further emphasize the need toobtain pertinent information about contaminants before attemptingtreatment.

Leifert et al. (1991a) attempted to eliminate several Gram-posi-tive and Gram-negative bacteria from micropropagated shoots usingantibiolics. Their studies indicale that certain combinations of antibi-olics can eliminale some conlaminants, but phytotoxicity and highcosts dictale Ihat identificalion and antibiotic sensitivity testing mustprecede Irealment. They cautioned againsl Ihe use of antibacterialcompounds for prophylaxis. Commercial micropropagalors havebeen advised 10 discard contaminated cultures and start from cleandonor plants (Leifert et al., 1989; Cassells, 1991). This is lesspraclicable in cases where plant material is limited. It is encourag-ing 10 nole thai from an armamenlarium of more than 7000 anlibi-

ACKNOWLEDGMENTS

The advice and suggestions given by Dr. William Doughel1y. Depal1menlof Microbiology, Oregon State University, are gratefully acknowledged. Wethank Elzi Yolk, Depal1ment of Botany and Plant Pathology, Oregon StateUniversity, for the gift of Timentin. Known bacterial strains were receivedfrom Ms. Marilyn Canfield, Department of Botany and Plant Pathology.Oregon State University and Dr. Joyce Loper, USDA-ARS, HOl1iculturalCrops Laboratory, Corvallis, OR. T. N. D. gratefully acknowledges thereceipt of a USDA/Agricultural Research Service Research Apprenticeshipto work on this project. The use of trade names in this publication does notimply endorsement by the U.S. Depal1ment of Agriculture.

64 BUCKLEY ET AL.

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