Antibiotic Resistance of Agrobacterium Strains Isolated in Japan

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1 Antibiotic Resistance of Agrobacterium Strains Isolated in Japan Abstract To elucidate any of antibiotics usable for selecting bacterial cells harboring a binary vector to be employed for genetic manipulation of plants, we devised a spreading and dilution method on PDA plates for examining antibiotic resistance of Agrobacterium sp. The resistance of 46 strains of Agrobacterium mainly isolated in Japan were examined on 8 antibiotics, ampicillin, carbenicillin, chloramphenicol, kanamycin, rifampicin, streptomycin, tetracycline and vancomycin by the spreading and dilution method. No strains examined here were resistant to tetracycline. Almost all the strains were resistant to ampicillin and kanamycin and were sensitive or weakly resistant to rifampicin. While the strains belonging to biovar 1 except for two strains exhibited high resistance to vancomycin, the strains belonging to biovar 2 except for groups 3 and 4 exhibited high resistance to carbenicillin but sensitivity or weak resistance to vancomycin. On the other hand, each of the strains exhibited various level of resistance to chloramphenicol and streptomycin even if they belonged to the same biovar. We therefore conclude that tetracycline is the best antibiotic for selecting cells of Agrobacterium, examined in the present study, harboring binary vectors. Moreover, we suggest that the results of antibiotic resistance in each biovar are usable for a supplementary classification of Agrobacterium. (Received February 13, 1995; Accepted May 31, 1995) Key words: Agrobacterium, antibiotic resistance. method and the co-cultivation method, plant tissues, INTRODUCTION Soil-borne bacteria, Agrobacterium tumefaciens and A. rhizogenes are phytopathogenic agents of neoplastic plant diseases called crown gall and hairy roots, respectively, damaging crops such as flowers and fruit trees. These diseases are caused by large plasmids harbored in Agrobacteria, the former is tumor-inducing plasmid (pti), and the latter root-inducing plasmid (pri). Each plasmid possesses 15 to 25kb of transferred-dna (T- DNA) that is excised and integrated into the infected plant genome5). Thus, this infection system is utilized for transformation in a plant gene manipulation technique. In order to utilize Agrobacteria for the host-vector system, so called gagrobacterium-pti host-vector system, h the antibiotic resistance of the bacteria needs to be investigated, because the bacteria harboring binary vectors such as pbi 1211) can be selected by antibiotic resistance accompanied with the vector. Especially, ampicillin, chloramphenicol, kanamycin, streptomycin and tetracycline are usually used for selecting bacteria cells and/or protoplasts inoculated with Agrobacterium harboring binary vectors need to be removed from their inocula for establishing aseptic-tissue-culture lines. An addition of antibiotics such as carbenicillin, rifampicin and/or vancomycin into a plant tissue culture medium is indispensable for removing the inocula6). Thus, an examination of antibiotic resistance degree of each strain is one of the most important matters for plant genetic engineering using gagrobacterium-pti host-vector system. h Although the disease symptoms15), phenotypic characteristics14), fatty acid methyl ester profiles18) and serological specificity17) on a lot of Agrobacterium strains isolated in Japan have been investigated, little attention has been given to investigate their antibiotic resistance except for the strain MAFF of A. rhizogenes20). In this article, we report the antibiotic resistance of Japanese Agrobacterium strains easily obtainable from microbe stock centers established in Japan. harboring vectors. On the other hand, in in vitro transformation technique such as the leaf disc inoculation

2 Ann. Phytopathol. Soc. Jpn. 61 (5). October, Table 1. Strains of bacteria used in this study MATERIALS AND METHODS Bacterial strains. Agrobacterium IFO or MAFF strains mainly isolated in Japan were obtained from Institute for Fermentation, Osaka, Japan or Center for Gene Resource, National Institute of Agrobiological Resources, Tsukuba, Japan, respectively. ATCC strains were obtained from American Type Culture Collection. These strains are listed in Table 1. Media and antibiotics. Agrobacterium growth medium (1.0% polypeptone, 0.2% yeast extract, 0.1%

3 Table 2. Antibiotics and their concentrations used in this study MgSO4 E7H2O, ph 7.0) recommended by Institute for Fermentation, Osaka was used for the bacterial preculture. Potato dextrose agar (PDA) medium purchased from Nissui Pharmaceutical Co., Ltd. was used as a culture medium to examine antibiotic resistance of each strain. Antibiotics, their abbreviations and their concentrations used in the present study are listed in Table 2. Examination of antibiotic resistance. The strains of Agrobacterium were inoculated to 5ml of Agrobacterium growth medium in test tubes (20cm in length and 2cm in diameter) and precultured on a reciprocal shaker at 30 Ž for 16-32hr until their stationary phase. The method for examining resistance degrees of each Agrobacterium strain on PDA plates containing adequate concentrations of antibiotics was shown in Fig. 1. At the start, a platinum loop sterilized by flaming was dipped into the cultured-bacterial suspension. A loopful of bacterial culture was streaked with 11 strokes for spreading in a trapezoid region of ca. 4cm ~1cm on a PDA plate containing an antibiotic (primary spreading), as shown in Fig. 1-a. Next, the plate was rotated through ca. 70 and then was streaked with 11 strokes with same loop by passing across the right edge of the primarily spread area into a fresh trapezoid region of ca. 4cm ~1 cm of the medium (secondary spreading, Fig. 1-b). This manipulation was repeated 3 times more in order that the bacterial culture might be stepwise diluted on the plate by drawing pentagon as shown in Fig. 1-c to -e. Triplicate plates of each antibiotic were inoculated with Fig. 1. The inoculation method, spreading and dilution method, for examining antibiotic resistance of Agrobacterium strains. The circle demonstrates an outward form of a petri dish. The zigzag line demonstrates a trace of a platinum loop for spreading bacteria. The shaded trapezoid demonstrates the area spread by bacteria. The characters of a to e exhibit each step of inoculation. The numbers demonstrate degrees of antibiotic resistance. each strain in the same manner and were incubated in an inverted position at 30 Ž for 2 days. Degree of an antibiotic resistance of each strain was judged from the extent of the colony formation. In the present study, we determined five resistance degrees based on the extent of colony formation; for example, when the colonies were formed in the overall area of the secondary spreading and in the partial area of the tertiary spreading, we estimated it degree 2. If the colony formation was under degree 1, the spontaneous mutation frequency against the antibiotic was examined; two day-cultured bacterial suspension was adequately diluted and spread on triplicate PDA plates containing the antibiotic; the plates were incubated in an inverted position at 30 Ž for 2 days and the number of colonies formed were counted. RESULTS AND DISCUSSION Spreading and dilution method for examining antibiotic resistance To elucidate any of antibiotics usable for selecting cells of each Agrobacterium strain transformed with a binary vector carrying one or more antibiotic resistance genes, we need to find out their antibiotic resistance in detail. In other words, if the strain shows sensitivity, it is usable for a recipient of the binary vector; if the strain shows high resistance, antibiotic-sensitive mutants are obtainable by irradiation of UV or treatment of mutagens such as nitrosoguanidine and ethyl methansulfonate. On the contrary, if the strain showing weak

4 Ann. Phytopathol. Soc. Jpn. 61 (5). October, resistance is used as a recipient of the binary vector, both of the bacterial cells harboring the vector and the spontaneous mutants (probably caused by selection by using the antibiotic and/or by transfer of R factor) show antibiotic resistance; thus, it is difficult to select the colonies harboring the vector. Moreover, antibioticsensitive mutants may be difficult to be obtained because of the spontaneous mutation. In general, for examining antibiotic resistance of bacteria, a minimal inhibitory concentration (MIC) test2) or a disk diffusion test11) is used to estimate an effective concentration or a titer of antibiotics. These methods are excellent in quantitation, but it is not suitable for estimating the degree of antibiotic resistance of each strain on standard antibiotic concentration used for selecting the bacteria harboring a vector. Thus, a method allowing us to estimate antibiotic resistance for selecting the bacteria harboring a vector has been Table 3. Degrees of antibiotic resistance and frequencies of spontaneous mutation of Agrobacterium strains The integral numbers indicate the degrees of resistance. The decimal ~10-x indicates the frequency of spontaneous mutation.

5 demanded. Our spreading and dilution method provides us with detailed information on antibiotic resistance of each of Agrobacterium strains. In our method, it seems reasonable to conclude that the bacterial culture could be stepwise diluted by each spreading: i.e., the bacteria inoculated could form colonies until the overall area of the final spreading on antibiotic-free PDA medium after 2-day incubation; further, a bacterial strain with high antibiotic resistance formed colonies in the final spread area, while an other strain with weak resistance formed colonies in the primary- or secondary-spread area. In spite of the simple manner, the difference of antibiotic resistance among the strains was clearly displayed and the reproducibility was high. YEB (yeast extract-beef extract), LB (Luria broth), or NB (nutrient broth) is generally employed for Agrobacterium culture6). In our previous study, LB agar plates were used for examining antibiotic resistance of A. rhizogenes strain MAFF ). Since this strain produced a little polysaccharide on the LB plates, the formed colonies could not be enough countable on the plates containing antibiotics, especially Tc, after 36-hr culture. In the present study, since all the Agrobacterium strains formed white colonies with more amount of polysaccharide on PDA plates, we could easily confirm them and could judge the resistance degree of each strain after 36-hr culture. Judging from the above, we concluded that the spreading and dilution method using PDA plates which we devised is available for estimating antibiotic resistance of each of Agrobacterium strains. Antibiotic resistance of Agrobacterium strains The levels of antibiotic resistance of each strain are listed in Table 3. All the Agrobacterium strains examined are highly sensitive to Tc. In our previous study, A. rhizogenes strain MAFF exhibited weak resistance to Tc on LB plates 36hr after inoculation, being obtained the resistant colonies with the spontaneous mutation frequency of 3.5 ~10-420). These mutants, however, gave no continuous or a very slow growth after transferring on a fresh LB plate containing Tc. On the other hand, this strain could form no colony on PDA and exhibited the definite sensitivity to Tc. A similar result was also obtained from examining Km resistance of strain MAFF363001; the strain showed weak resistance on LB, but definite sensitivity on PDA. The difference as described above may be caused by the Table 4. Characteristics of antibiotic resistance in each biovar of Agrobacterium Abbreviations: R, high resistance (resistance degree=2, 3, 4 and 5); WR, weak resistance (0<resistance degree 1); S, sensitivity (resistance degree=0); V, variation (0 resistance degree 5). a) Unknown biovar.

6 Ann. Phytopathol. Soc. Jpn. 61 (5). October, difference of components between LB and PDA. All the Agrobacterium strains except for strains MAFF and MAFF ) exhibited high resistance to Am and Km. Twenty eight strains exhibited sensitivity to Ri and the rest strains exhibited weak resistance to Ri. Thus, it is likely that a general Agrobacterium strain exhibits high Am and Km resistance, no or weak Ri resistance and high Tc sensitivity on PDA. In the cases of Cb, Cm, Sm and Vm, each of the strains examined exhibited various degrees of resistance. Characteristics of antibiotic resistance in each biovar We analyzed the characteristics of antibiotic resistance existing in each biovar, because the characteristics may provide us a supplementary classification method of Agrobacterium strains. We attempted to find out any antibiotic resistance characteristics from all the results of each strain, but Cm and Sm resistance was not adequate factors for division, because of resistance variations existing among the strains closely relating in their phenotype and serological specificity. Thus, Cm and Sm resistance was taken into no consideration for the classification. To avoid excessively small grouping, the resistance was divided into two groups: weak resistance (WR, 0<resistance degree 1) and high resistance (R, resistance degree=2, 3, 4 and 5). Based on this criterion, the characteristics of antibiotic resistance on each biovar were concluded as presented in Table 4. A characteristic of the strains belonging to biovar 1 except for 2 strains is highly resistant to Vm. Based on the difference of Cb resistance among the strains, most of them were further divided into two main groups. A. tumefaciens chrysanthemum and marguerite strains and a rose strain isolated in Shizuoka7,12) and Osaka Prefectures, respectively, fell into group 1. These strains, however, exhibited the various degrees of Cm and Sm resistance (Table 3). In the cases of A. tumefaciens strain IFO3058 and A. sp. strain IFO137143, each of the antibiotic resistance degrees was very closed to each other (Table 3), classified into group 1. The origin of the former strain is uncertain and the latter was isolated as a strain of Alcaligenes faecalis from soil in Osaka Prefecture, but these strains may be the similar one. A. radiobacter strains IFO13532 and ATCC13332 also belong to group 1. In the case of A. rhizogenes melon strains MAFF to isolated from melon plants in Chiba Prefecture19), they are characterized by high-cb and -Sm resistance, fell into group 2. These strains seem to be different from A. rhizogenes strains IFO15189 to , though all of them were isolated from hairy root-diseased melon plants. The strains IFO15188 and were unclassified into both groups 1 and 2, exhibiting slightly different Ri- and Vmresistance degree (see Table 3), but the antibootic resistance patterns of these strains resembled to those of the strains in group 1. In spite of a member of biovar 1, A. tumefaciens strain MAFF isolated from a diseased cherry plant exhibited different antibiotic resistance pattern from the other strains in biovar 1 examined in the present study (Table 3), classified into neither group 1 nor 2. The strain probably has a different genetic background from the other strains in biovar 1. All the strains of A. tumefaciens belonging to biovar 2 were isolated from the diseased rose plants cultivated in Shizuoka Prefecture and were reported to be very closed each other8,12). These strains would be divided into 4 groups by their antibiotic resistance patterns. In the case of group 2, the characteristic of antibiotic resistance was same as that of group 2 in biovar 1. On the other hand, A. tumefaciens strain MAFF and A. rhizogenes strain ATCC fell into group 4, characterized by weak resistance or sensitivity to the most of antibiotics except for Km. A strain of A. tumefaciens MAFF belonging to biovar 3 and A. rubi strain ATCC13335 exhibited fairly different antibiotic resistance patterns from most of the strains in biovars 1 and 2. They may have different genetic backgrounds from the strains in biovars 1 and 2. Although we cannot say so definitely, the general tendency of antibiotic resistance of Agrobacterium strains belonging to each biovar would be understood as follows; the strains in biovar 1 exhibit high resistance to Vm; and the ones in biovar 2 exhibit high resistance to Cb and sensitivity or weak resistance to Vm. Though more examinations must be done on a lot of Agrobacterium strains belonging to each biovar because of some exceptions to the tendency, we suggest that the present results of antibiotic resistance are usable for a supplementary classification of Agrobacterium. Practical use of antibiotic sensitivity on Agrobacterium strains Recently, genetic manipulation in plant field is rapidly proceeding and the genetically improved crops have already come into the market. For obtaining transformants with high frequency, a host-vector system employing both Agrobacterium and pti vectors is one of the most powerful tool. At present, a system employing a binary vector pbi 1211) with a Km resistance gene and a disarmed A. tumefaciens strain LBA44044) is a widespread host-vector system for Agrobacterium-mediated transformation. The strain LBA 4404 is not, however, virulent to all the plant genuses. Therefore, to establish much better host-vector system, further efforts have been made for obtaining each strain of Agrobacterium infectious to plants belonging to each genus. For example, Wabiko et al. (1989) have isolated a good strain of A. tumefaciens from crown galls of Populus plants22). In the present study, however, a lot of Agrobacterium strains isolated in Japan exhibit Km resistance. Thus, Km-resistant binary vector is never used for these strains unless a Km-resistant mutant is obtained. On the other hand, since all the strains examined in the present study exhibited Tc sensitivity, we propose that Tcresistant binary vector such as pgah10) can be used for

7 Agrobacterium-mediated transformation system. Literature cited 1. Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987). GUS fusions: Ĉ-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: Goto, M. (1983). In Shokubutu Byourigaku Jikkenhou (Sato, S., et al. eds.), Koudansha Scientific, Tokyo, pp (in Japanese). 3. Harada, T., Yoshimura, T., Hidaka, H. and Koreeda, A. (1965). Production of a new acidic polysaccharide, succinoglucan by Alcaligenes faecalis var. myxogenes. Agric. Biol. Chem. 29: Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. (1983). A binary plant vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: Hooykaas, P.J.J. and Schilperoort, R.A. (1992). Agrobacterium and plant genetic engineering. Plant. Mol. Biol. 19: Lichtenstein, C. and Draper, J. (1985). In DNA Cloning, Volume II (Glover, D.M. ed.), IRL Press, Oxford, Washington, D.C., pp Makino, T. (1991). Biological control of crown gall on chrysanthemum and Fotina glabra with Agrobacterium radiobacter K84. Bull. Shizuoka Agr. Exp. Stn. 36: (in Japanese). 8. Makino, T. and Morita, H. (1985). Biovar and sensitivity to Agrocin 84 of Agrobacterium tumefaciens isolated from roses in Shizuoka Prefecture. Bull. Shizuoka Agr. 15. Sawada, H., Ieki, H., Kobayashi, S. and Oiyama, I. (1992). Grouping of tumorigenic Agrobacterium spp. based on Ti plasmid-related phenotypes. Ann. Phytopathol. Soc. Jpn. 58: Sawada, H., Ieki, H. and Takikawa, Y. (1990). Identification of grapevine crown gall bacteria isolated in Japan. Ann. Phytopathol. Soc. Jpn. 56: Sawada, H., Imada, J. and Ieki, H. (1992). Serogroups of Agrobacterium tumefaciens biovar 3 determined using somatic antigens. Ann. Phytopathol. Soc. Jpn. 58: Sawada, H., Takikawa, Y. and Ieki, H. (1992). Fatty acid methyl ester profiles of the genus Agrobacterium. Ann. Phytopathol. Soc. Jpn. 58: Shiomi, T., Shirakawa, T., Takeuchi, S., Oizumi, T. and Uematsu, S. (1987). Hairy root of melon caused by Agrobacterium rhizogenes biovar 1. Ann. Phytopathol. Soc. Jpn. 53: (in Japanese). 20. Tanaka, N., Takao, M., Matsumoto, T. and Machida, Y. (1993). Transformation of Agrobacterium rhizogenes strain MAFF by electroporation. Ann. Phytopathol. Soc. Jpn. 59: (in Japanese). 21. Yokota, A. and Sakane, T. (1991). Taxonomic significance of fatty acid compositions in whole cells and lipopolysaccharides in Rhizobiaceae. IFO Res. Comm. 15: Wabiko, H. (1989). Isolation and characterization of diverse Ti plasmids of Agrobacterium tumefaciens from Japan. Chem. Regul. Plants 24: (in Japanese). Exp. Stn. 30: (in Japanese). 9. Makino, T. and Osawa, T. (1987). Observation of hairy root (a new disease) in muskmelon and identification of the causal bacterium. Bull. Shizuoka Agr. Exp. Stn. 32: (in Japanese). 10. Matsumoto, S., Machida, C. and Machida, Y. (1989). Toransujenikku syokubutu no sakuseihou. Cell Technol. 8: (in Japanese). 11. Nakamura, H. and Maejima, Y. (1983). In Shokubutu Byourigaku Jikkenhou (Sato, S., et al. eds.), Koudansha Scientific, Tokyo, pp (in Japanese). 12. Ohta, K. and Nishiyama, K. (1984). Studies on the crown gall disease of flower crops. I. Occurrence of the disease and the characterization of the causal bacterium. Ann. Phytopathol. Soc. Jpn. 50: (in Japanese). 13. Ricker, A.J., Banfield, W.M., Wright, W.H., Keitt, G.W. and Sagen, H.E. (1930). Study on infectious hairy root of nursery apple trees. J. Agric. Res. 41: Sawada, H. and Ieki, H. (1992). Phenotypic characterization of the genus Agrobacterium. Ann. Phytopathol. Soc. Jpn. 58: