Relative Virulence of Agrobacterium Strains on Strawberry

Several strains of Agrobacterium tumefaciens and A. rhizogenes were shown to form tumors on runners of the diploid strawberry species Fragaria vesca L. Tumors, weighing from 0.1 to 8.3 mg, appeared from 2 to 4.5 weeks after infection. The majority of tumors tested for opine synthesis by high-voltage paper electrophoresis analysis showed positive results. These results demonstrate that diploid strawberry plants are susceptible to infection with Agrobacterium and that there are differences in the relative virulence of Agrobacterium strains. Agrobacterium is a genus of bacteria that resides in the soil and causes the common disease crown gall on a variety of dicotyledonous plants. Recently, this organism has gained widespread importance as a vector to transfer genes into plants. Currently, the infection of strawberry by Agrobacterium is not a problem in strawberry nurseries or production fields; therefore, it is not clear whether this plant can be transformed by Agrobacterium. The natural host range of A. tumefaciens and A. rhizogenes can be determined by demonstrating the ability of these bacteria to form crown gall or hairy root disease (De Cleene and De Ley, 1976, 1981). Pathogenicity of Agrobacterium depends on the presence of a Ti or Ri plasmid. During infection, this pathogen transfers a portion of its Ti or Ri plasmid, the T-DNA segment, to the plant’s genome (Chilton et al.,. 1977, 1982). Transcription and translation of genes present in the T-DNA causes crown gall tumors in A. tumefaciens or hairy root disease in A. rhizogenes (Chilton et al., 1977, 1982). Thus, the appearance of tumors or abnormally prolific roots, respectively, is a good indication that transformation has occurred. The T-DNA also contains genes that encode the synthesis of opines. The presence of these compounds in plant tissue extracts is unique to tissue infected with Agrobacterium. DifReceived for publication 6 Nov. 1989. We are grateful to the following for providing us with Agrobacterium strains: M.D. Chilton (A208);. C.I. Kado (C58, Ach5); E.W. Nester (A722, A281, A4, R1000); V. Knauf (K12, K12×167, K12×563E, A4T×178); J. Tempé (15955); S. Rogers (pMON200); and G. Strobel (232). The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1To whom reprint requests should be addressed. 196 ferent strains of Agrobacterium induce the synthesis of different opines; included among these are nopaline (NOP) and octopine (OCT) (Bomhoff et al., 1976), mannopine (MOP), mannopinic acid (MOA), agropine (AGR), and agropinic acid (AGA) (Petit et al., 1983). We have used diploid strawberry F. vesca in these studies to determine its susceptibility Fig. 1. Tumor formation on runners 4 weeks after in size varies even within a given strain of Agrobacte tumefaciens strains A208, C58, A722, A281, K spectively. Lane i shows runners inoculated with inoculated with 523 medium. HOR to infection by Agrobacterium. It is the most extensively distributed species of this genus and can be found in the northern regions of America, Asia, and Europe. The cultivated strawberry is an octaploid and, therefore, genetically more complex than the diploid varieties. Plant material. The plant source used in this investigation was a runner-producing derivative of the F. vesca, alpine clone (2n = 14, x = 7). Plants were grown in a growth chamber at 24C under a light intensity of 50 μmol·s ·m. Runners used for infection were 4 to 6 weeks old. Bacterial strains. Various tumor and rootinducing strains of Agrobacterium were used to infect the strawberry runners. Some characteristics of the Agrobacterium strains used in our experiments are shown in Table 1. Culture medium and growth of bacteria. The Agrobacterium cultures were grown at 26 to 28C in 523 medium (Kado et al., 1972). Kanamycin (100 μg·ml ) and spectinomycin (50 μg·m ) were supplemented in the growth medium as required for bacteria that contained the corresponding resistance gene. Tumor induction. Runners were wounded by cutting through the epidermal tissue with oculation with Agrobacterium strains. Note tumor rium. Lanes a–h show runners inoculated with A. 12×562E, K12×167, B6S3×200, and 15955, reA. rhizogenes strain R1000. Lane j shows runners TSCIENCE, VO L. 26(2), FE B R U A R Y 1 9 9 1 Table 1. Opines produced by Agrobacterium strains used for the infection of strawberry. zOct = octopine, NOP = nopaline, AGR = agropine, AGA = agropinic acid, MOP = mannopine, MOA = mannopinic acid yND = not determined. Table 2. Analysis of tumor formation in strawberry. zNumber of tumors/number of infections. yRange of tumor weights (average weight). a sterile scalpel. One drop (2-5 μl) of a culture of Agrobacterium grown for 16 h and having a cell density of 5 × 10 cells/ml (as measured by absorbance at 420 nm) was placed on each wound. The wounded surfaces were then wrapped with parafilm to prevent drying. Tumors were harvested 4 to 8 weeks after inoculation. Kalanchoe daigremontiana plants were used as positive controls to check for oncogenicity of the various strains. The Kalanchoe plants were wounded by making incisions on the leaf surface and then were inoculated with the various Agrobacterium strains. Opine analysis. Extracts for opine analysis were prepared by placing 1 to 4 mg of tumor tissue in an Eppendorf tube containing 200. μl of distilled water and boiling it for 10 min. The tumor tissue was removed by centrifugation (5000× g, 10 rein). The extract was then dried in a speed vac (Savant, New York) and resuspended in water to 0.5 μl·mg -1 of tissue. One to 2 μl of extract was spotted onto Whatman 3-mm paper along with 1 μl of standards of the NOP/OCT family (1 mg octopine, nopaline, and arginine/ ml) or 1 μl of standards belonging to the H O R TSCIENCE , VO L. 26(2), FEBRUARY 1 9 mannityl opine family (1 mg MOP, MOA, AGP, and AGA/ml; authentic standards of these opines were provided by J. Tempé). Aqueous methyl green solution, which migrates just behind arginine, was used as a visual marker during electrophoresis. The paper was moistened with the appropriate electrophoresis buffer immediately before the current was applied. For analysis of the NOP/ OCT family, the buffer used was 5% formic acid and 15% glacial acetic acid; for the mannityl opines, the buffer was 3% formic acid and 6% glacial acetic acid, Paper electrophoresis was performed at 10 volts·cm for 40 min. The paper was then dried. Nopaline and octopine, which react with the fluorescent compound 9,10-phenanthrenequinone, were assayed as described by Otten and Schilperoort (1978). The detection of agropine, agropinic acid, mannopine, and mannopinic acid (silver nitrate-positive compounds) was performed as described by Petit et al. (1983). Tumors appeared from 2 to 4.5 weeks after inoculation of strawberry runners with Agrobacterium (Fig. 1). Of 14 Agrobacterium strains (10 A. tumefaciens and four A. rhi9 1 zogenes) used to infect strawberry runners, all 14 produced tumors of various sizes (Table 2). Strains containing pTi15955 (15955) and pMON 200 II (B6S3×200) were found to be the most infectious (Table 2), producing tumors in eight of the 10 sites inoculated: The average weight of the tumors incited by these strains was under 2 mg. Strains that contained pTiA6 (A722) or its recombinant derivatives K12×562E and K12×167 produced tumors in more than 60% of infected sites (Table 2); their average weights were also under 2 mg. The highly virulent strain A281 (Hood et al., 1986a, 1986b) produced average-sized tumors in five of 10 sites inoculated. Strains containing the Ti plasmids pTiAch5 and pTiC58 produced the largest tumors but were found to be the least infectious (one of three sites and two of 10 sites, Table 2). The tumor morphology in all cases was hard and compact with no visible differentiated structures (buds, leaves, or roots). No root formation was observed when A. rhizogenes was used to infect strawberry; tumors were found, however, at the infection sites. The A. rhizogenes strains 232 and A4T×178 were the most infectious of the Rhizogenes strains tested (Table 2), and they produced tumors on six of 10 infected sites. All tumors weighed under 2 mg. Control runners inoculated with sterile medium failed to produce any tumors. Opine expression in tumors was verified by analysis of tumors for the presence of opine compounds. The aim of the analysis was to determine the presence of the nopaline/octopine family and/or the mannityl opine family (AGR, AGA, MOP, MOA). Sixtythree tumors were analyzed for opines, 53 of which were analyzed for all six opines. Ten tumors that were too small to be analyzed for both opine families were analyzed only for the presence of the mannityl opine family. The analysis for the presence of both families of opine compounds was necessary because many of the strains were capable of synthesizing more than one class of opine (Table 1). Both agropine and mannopine could be clearly distinguished in our analysis (Fig. 2), but mannopinic acid comigrated with mannopine, and agropinic acid comigrated