Plant Cell, Tissue and Organ Culture 36: 169-175, 1994. (~) 1994 Kluwer Academic Publishers. Printed in the Netherlands.

An evaluation of antibiotics for the elimination of Xanthomonas campestris pv. pelargonii (Brown) from Pelargonium x domesticum cv. 'Grand Slam'

explants in vitro

Carol Barrett & Alan C. Cassells Department of Plant Science, University College, Cork, Ireland

Received 4 September 1992; accepted in revised form 10 September 1993

Key words: bacterial contamination, cefotaxime, tissue culture

Abstract

A range of antibiotics was evaluated for activity against Xanthomonas campestris pv. pelargonii (Xcp) on diagnostic sensitivity testing and plant tissue culture media. Many of the antibiotics showed reduced or no activity on the latter. Tetracycline and cefotaxime, chosen for further investigation, were screened for light stability under plant culture regimes. Tetracycline was inactivated in photosynthetic photon fluxes of 22 ~tmol m -2 s-1 and above. The minimum bacteriocidal concentration of cefotaxime was determined in bacteriological and plant tissue culture media. Cefotaxime was further tested for phytoxicity and ability to eliminate Xcp from deliberately infected explants. Cefotaxime was shown to eliminate contamination and stimulate the growth of the plant tissue cultures up 500 mg 1-1.

Abbreviations: DST - diagnostic sensitivity testing medium, MBC - minimum bacteriocidal concen- tration, TCM -half-strength Murashige & Skoog (1962) basal plant tissue culture medium, Xcp - Xanthomonas campestris pv. pelargonii, BA - benzyladenine, 2,4-D - 2,4-dichlorophenoxyacetic acid, OD - optical density, PPF - photosynthetic photon flux

Introduction

Serious losses have been reported in tissue cul- tures due to the presence of latent bacteria (e.g. Leifert & Waites 1990; Boxus & Terzi 1987; Cas- sells 1986). These organisms have been shown to affect multiplication rates of plant cultures (Long et al. 1988; Leifert 1990) and, since they multi- ply within the plant tissue (Leifert et al. 1991), they may provide a source of inoculum in the progeny microplants. Xanthomonas campestris pv. pelargonii, the causal agent of bacterial leaf spot and stem rot in Pelargonium, may remain latent in micropropagation due to inhibition by

high salt, sucrose concentration or pH. Xan- thomonas may then be rapidly expressed on trans- fer to media where salt and sucrose concentration is reduced, resulting in severe losses of affected cultures (Cassells et al. 1988).

Meristem culture, followed by rigorous screening is widely used as part of disease- free stock management programmes (Cassells 1991a,b); however, should this procedure not be employed or fail, it is desirable to have alterna- tive strategies to rescue infected cultures. Many attempts have been made to suppress or eliminate endogenous bacteria from cultures with antibi- otics with varying degrees of success (Falkin-

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er 1990). However, not all studies included identification or antibiotic sensitivity testing of bacterial contaminants before incorporation of antibiotics into the culture medium (Leifert et al. 1991). Problems of phytotoxicity have also been observed (Cornu & Michel 1987; Dodds & Roberts 1981; Thurston et al. 1979).

Here, a number of antibiotics were evaluat- ed for their potential to eliminate Xanthomonas campestris pv. pelargonii from infected Pelargo- nium x domesticum explants on adventitious regeneration medium.

Materials and methods

Plant material

Pelargonium x domesticum cv 'Grand Slam' plants to be used as explant donors were main- tained in an isolated glasshouse and raised under stage 0 conditions as outlined by Debergh & Maene (1981).

Tissue culture procedures

Explants for adventitious regeneration were derived from meristems established and multi- plied following the procedure of Menard et al. (1985). Explants were culture-indexed for bac- teria, as below, on each subculture to ensure freedom from cultivable bacteria including Xcp. Petioles explants were cultured using a procedure modified from Cassells & Carney (1987), who described a two-stage adventitious regeneration procedure. Stage 1, induction medium, contained 2.35 g 1-1 of Murashige & Skoog (1962) basal medium half-strength (organics and inorganics, cat no 26-100-24: Flow Laboratories, Irvine, UK) with 4.4 laM BA, 0.45 laM 2,4-D, 30 g 1 - l sucrose and 6 g 1-1 agar (Cat. no. A-7002: Sigma Chemical Co., Poole, Dorset, UK). The stage 2, differentiation medium half-strength (basal medi- um as above), contained 4.4 laM BA as the sole growth regulator with 30 g 1-1 sucrose and 6.0 g 1-1 agar, pH 5.8.

Bacterial cultures

Isolates of Xcp were obtained from diseased Pelargonium plants and the identity confirmed by fatty acid profiling (Stead 1988). Cultures of Xcp were grown and maintained on YGC medium (Table 1). Colonies were subcultured every 4 weeks and incubated at 27°C. Liquid cul- tures were grown in NGB (Table 1) and incubat- ed at 27°C in a shaking waterbath. Isolates of Pseudomonas aeruginasa were cultured on NGA medium (Table 1).

Indexing methods

Plant cultures were indexed for the presence of bacteria by placing petiole, leaf or stem segments, or callus, directly onto non-selective bacteriologi- cal media (Table 1). Plates were incubated at 27°C for 2-7 days and examined for bacterial growth.

Antibiotic sensitivity tests

Antibiotic sensitivity tests were carried out on Oxoid DST medium (Oxoid, Basingstoke, Hants., UK) following the procedure of Bondi et al. (1947) and repeated on TCM (Table 1). The plates were inoculated with a control organism, Pseu- domonas aeruginosa, ATCC strain no. 27853 (ATCC, Rockville, Maryland, USA) and the test organism, Xcp. Absorbance readings were taken at 580 nm of 24 h bacterial suspension cultures and the concentrations adjusted to OD 0.395 by dilution with NGB medium (Table 1). The plates were inoculated by the spread plate method using a sterile cotton wipe impregnated with bacteri- al suspension of the control or test organism, as appropriate. The cultures used as inoculum were in the early exponential growth stage. Antibiotic discs were positioned on the medium as shown in Fig. 1. Ten antibiotics were chosen on the advice of Dr. E Falkiner. The antibiotics used and quantity per disc were as follows: 5 lag ery- thromycin, 25 lag ampicillin, 30 lag cefuroxime, 70 lag piperacillin, 10 lag ampicillin, 100 lag car- benicillin, 10 lag gentamicin, 30 lag cefotaxime

Table 1. Media used for bacterial culture based on & Scherr (1958).

Schaad (1980); DST after Bechte

Medium Composition g 1-1

NGA nutrient glucose agar Beef extract 3.0 Peptone 5.0 Glucose 10.0 Agar (Sigma A-7002) 15.0 pH 7.2

NGB nutrient glucose broth (as above without agar)

YGC yeast extract, glucose, calcium carbonate

DST diagnostic sensitivity testing agar

Yeast extract 10.0 Glucose 10.0 CaCO3 20.0 Agar (Sigma A-7002) 15.0 pH 7.0

Proteose peptone 10.0 veal infusion solids t0.0 glucose 2.0 sodium chloride 2.0 sodium acetate 1.0 disodium phosphate 2.0 adenine sulphate 0.01 guanine hydochloride 0.01 aneurine 0.0002 agar no. 1 (Oxoid) 12.0 pH 7.4

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sodium, 30 Bg chloramphenicol, 10 Bg tetracy- cline, 1.5 units penicillin G.

The antibiotic test plated, incubated at 27°C, were examined for inhibition zones over a period of 2-7 days. Inhibition zones produced by the test organism larger than the control indicated sen- sitivity to the antibiotic tested. The tests were replicated on pure cultures of Xcp.

MBC determination

The MBC was determined using a method out- lined by Falkiner (1988). A concentration of 1- 100 pg m1-1 of cefotaxime sodium was tested in NGB and TCM (Table 1).

Light sensitivity tests

Due to the long exposure of tissue cultures to light, a test for antibiotic light sensitivity was designed. Cefotaxime and tetracycline were tested over a period of 0-22 days as follows. A concentration of three times the MBC for each antibiotic was incor- porated into YGC medium for test plates and no antibiotic was added to control plates. Antibiotic test and control (-antibiotic) plates were incubat- ed in a growth room at 22 Bmol m -2 s -1 and 53 pmol m -2 s -1 , for from 0 to 22 days. Plates were inoculated daily with Xcp, incubated in the dark at 27°C and monitored for bacterial growth. Antibi- otic control plates were incubated in the dark for the respective period before daily inoculation.

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Fig. 1. Testing of antibiotics on DST against Xcp. The central area of the plate was inoculated with Pseudomonas aeroginosa (control), the outer regions with Xcp. The antibiotics evaluated here were (from top right, clockwise) chloramphenicol, cefotaxime and tetracycline. Note P. aeroginosa is resistant to chloramphenicol and tetracycline: resistance to cefotaxime is seen to be developing.

Phytotoxicity tests

Cefotaxime was incorporated into TCM at con- centrations of 0 -500 pg ml - l . Petiole explants and in vitro adventitious shoots were cultured on antibiotic-containing medium and examined for growth inhibition and other indications of toxicity.

Bacterial elimination tests

Plantlets from meristem cultures that culture indexed negative for bacteria were used for these tests. A 1 x 10 - 4 dilution of a stan- dardised Xcp culture (OD 0.528 value at 580 nm) served as a source of inoculum. Plantlets were inoculated by dipping the base of the stems into the Xcp inoculum for 15 sec (Long et al. 1988).

After 4 days petioles from inoculated plants were excised and cut longitudinally. Half was used for culture indexing. Plantlets were indexed from the base to the tip to confirm that Xcp was systemic. The second half of the petiole was cultured on antibiotic-containing TCM. Explants were cultured for 8 days on induction medium and then were transferred to differentiation medi- um. Both media contained cefotaxime at 300 ~tg 1-1. Explants were subcultured on the lat- ter to encourage adventitious shoot formation. After 12-15 weeks the original explant and peti- oles from the adventitious shoots derived from it were culture-indexed for Xcp. Contaminated cultures grown without antibiotic were used as controls.

Table 2. Comparative sensitivity of Xan- thomonas campestris pv. pelargonii to antibi- otics on DST and TCM media.

Antibiotic (~tg/disc) Inhibition

DST TCM

Penicillin G (1.5 units)

Ampicillin (10) ++ +

Ampicill in (25) ++ +

Piperacillin (75) ++

Carbenicillin (100) ++ +

Cefuroxime (30) + +

Cefotaxime sodium (30) ++ ++

Chloramphenicol (30) ++ ++

Gentamicin (10) ++ -

Tetracycline (10) ++ ++

Erythromycin (5) ++

- not sensitive(0-3 mm diam.);

+ sensitive (4-6 mm diam.);

++ very sensitive (7-16 mm diam.)

Results

Antibiotic sensitivity tests

Table 2 shows that many of the antibiotics were affected by TCM with a decrease in activity with three becoming inactive. Of those antibiotics that retained activity on TCM, chloramphenicol was rejected since it has been reported to have adverse effects in plant tissue cultures (Bastaiens et al. 1983). Tetracycline and cefotaxime were chosen for further tests.

MBC determination

The MBC of cefotaxime sodium in NGB was 60 ~tg ml - l and in TCM 50 ~tg ml - t. Following con- vention, a concentration of 2-4 times the MBC, that is, 240 ~tg ml - 1 was chosen for light sensitiv- ity test. The MBC of tetracycline was 2 ~tg ml-1 in both media; accordingly, a concentration of 8 ~tg ml-1 was selected for tests.

173

10 y= .017x+ 1.849 R-squared: ,862

8

~ 8. z

z

N •

• , . , . , . , . , . , . , . , . , . ,

50 100 150 200 250 300 350 400 450 500

C E F O T A X I M E i t g / m l

Fig. 2. The relationship between cefotaxime concentra- tion and the mean number of shoots produced per container from petiole explants ofPelargonium × domesticum 'Grand Slam' after 8 weeks. (Number of replicates per antibiotic concentration = 20).

Light sensitivity tests

A PPF of 53 ~tmol m -2 s -1 did not affect antibi- otic activity of cefotaxime against Xcp over a 22 day incubation period as confirmed by daily inoc- ulation of plates exposed to light for 0 to 22 days. Tetracycline, however, lost all activity after 3 days at 22 ~tmol m -2 s - t , while plates in the dark retained antibiotic activity after 22 days. Cefo- taxime therefore was chosen for further study.

Phytotoxicity tests

Petioles were examined for adventitious shoot production after 12-15 weeks on cefotaxime- containing TCM. Fig. 2 shows the relationship between the mean shoot number produced per container and cefotaxime concentration. A pos- itive correlation is evident with the correlation coefficient, 0.928, significant at p < 0.05.

Bacterial elimination

Culture indexing of petioles of deliberately inocu- lated microshoots 4 days after inoculation showed petioles at all positions to be contaminated with Xcp. After two subcultures (16 weeks) explants from the inoculated donor shoots on cefotaxime- containing TCM were again culture-indexed. Apart from one plate, which had fungal contami- nation, no bacterial contamination was observed.

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Control cultures indexed positive and rapidly died. It should be emphasised that previous stud- ies using DNA-probes and ELISA confirmed that Xcp is consistently expressed on the bacterio- logical media listed in Table 1 (Cassells et al. 1988).

Discussion

Sensitivity tests gave a general indication of the sensitivity of Xcp to particular antibiotics con- firming and extending earlier studies (van den Mooter et al. 1981). DST medium is used because none of its components are known to affect the action of antibiotics. It also contain agar no. 1 as a gelling agent, which permits superior dif- fusion of large antibiotic molecules (Bechte & Scherr 1958). One of the difficulties in using antibiotics in plant tissue culture is that sensi- tivity may be reduced in complex plant tissue culture media, which is evident from the present results.

The size of the inhibition zone may be affected by the molecular weight of the antibiotic. Peni- cillin, for example, has a low molecular weight and diffuses rapidly whereas polymyxins have a high molecular weight and diffuse at a slower rate (Garrod et al. 1973). Some antibiotics are sensi- tive to pH, e.g. lincomycin and cephaloridine are more active in alkaline conditions, tetracycline and methicillin in acid ones (Sabath et al. 1968). Therefore, it is essential that antibiotic sensitivity tests should be carried out on the appropriate plant tissue culture media to confirm results obtained on DST medium.

MBC determination is a necessary step to decide on the lowest concentration of antibiotic required to kill the bacterium. Once this has been determined it is possible to decide on a functional concentration, arbitrarily 2-4 times the MBC of the chosen antibiotic, in this case cefotaxime, to be used for elimination studies. Light sensitivity tests were carried out because of exposure of the antibiotic to light for the relatively long periods involved in the culture of plant tissues. Tetracy- cline, while active against Xcp, was eliminated

on this criterion. Tetracycline has previously been used in tissue culture (Pollock et al. 1983; Young et al. 1984; Cornu & Michel 1987); however, the results of this study indicate that it is not suitable for use due to high sensitivity to light.

Phytotoxicity tests are necessary before using antibiotics in the culture medium. Several reports of phytotoxicity have been recorded. Dodds & Roberts (1981) found that gentamicin strong- ly inhibits the differentiation of tracheary ele- ments in cultured explants of pith parenchyma from heads of romaine lettuce. Bastaiens et al. (1983) reported that chloroamphenicol inhibit- ed organogenesis in various plants. Polymyx- in B was found to be highly toxic to apple, rhododendron and Douglas fir (Young et al. 1984). Our results indicated, however, that cefotaxime is not phytotoxic; indeed it has growth promoting activity, confirming reports by Mathias & Mukasa (1987), Mathias & Boyd (1986) and Young et al. (1984). Cefotaxime is a ¢3-1actam antibiotic that inhibits cell wall synthesis in dividing bacterial cells. /3-1actams bind to peptidoglycan transpeptidases, which are involved in the final stages of the cell wall synthesis, and results in cell lysis (Sel- wyn 1980). Therefore, cefotaxime is unlikely to affect plant metabolism, which is supported by the widespread use of cefotaxime, at up to 500 ~tg m1-1, in controlling Agrobacterium in trans- formation studies (see e.g. Severin & Schoeffi 1990).

In order to use antibiotics with any measure of success, a good protocol is essential. In addi- tion to the points investigated here, there are oth- er considerations that must not be overlooked. More than one type of bacterium, for example, may be present within the plant tissue. There- fore, two or more antibiotics may need to be used and then the problem of incompatibility must be taken inot account (Falkiner 1990). The compli- cation of bacteria acquiring resistance must also be considered (Falkiner 1988). Finally, the risk of toxicity to humans must be addressed (Falkiner 1990). Only when all of the above factors have been considered may the use of antibiotics in vitro be successful.

Acknowledgement

The authors are grateful to Dr. F. Fa lk ine r for

advice and for his generous gift of Pseudomonas

aeruginosa.

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