Biological Control of Crown Gall : Construction and Testing of New Biocontrol Agents

Shim, J.-S., Farrand, S. K., and Kerr, A. 1987. Biological control of crown gall: Construction and testing of new biocontrol agents. Phytopathology 77:463-466. Transfer deficient (Tra-) Tn5 insertion mutants of the agrocin 84 plasmid strain K84 in controlling crown gall. Strains of Agrobacterium radiobacter pAgK84 were transformed into two chromosomal backgrounds and the which contained a high copy number mutant of pAgK84 and overproduced transformants tested as agents for biological control of crown gall on agrocin 84 were no more effective, and in one case, significantly less almonds and for colonization of almond seedling roots. Efficiency of root effective in controlling crown gall than strains containing the normal copy colonization and of biological control was influenced by the chromosomal number of pAgK84. background. Two Tra strains were as effective as the commercial Tra÷ Additional key word: Agrobacterium tumefaciens. The biological control of crown gall on stone fruit and rose by MATERIALS AND METHODS strain K84 of Agrobacterium radiobacter (Beijerinck & van Delden 1902) Conn 1942 is used commercially in many countries, Bacterial strains. The bacterial strains used are listed in Table 1. including Australia, Greece, Israel, Italy, Japan, New Zealand, Media and buffers. The following media were used: CitrateSouth Africa, Spain, and the United States. Strain K84 produces glutamate-glucose (CGG) medium (18); selective media IA and 2E agrocin 84, a novel nucleotide antibiotic (26) that inhibits the (13) for biovars I and 2, respectively; Bergerson's medium (2); causal organism, Agrobacterium tumefaciens (Smith & Townsend Stonier's medium (24), L-broth (15); nutrient agar (NA) (Difco, 1907) Conn 1942 if the latter contains a nopaline-type tumorDetroit, MI); yeast extract broth (YEB) (27), and yeast mannitol inducing (Ti) plasmid (7,14). By this means, infection of susceptible agar (YM) (5). plants by such a strain is prevented. The synthesis of agrocin 84 is The following buffers were used: buffered saline (BS) (0.02 M encoded by a 47.7 kb (kilobase) plasmid, pAgK84 (5,23). sodium phosphate 0.85% NaCl, pH 7.3), LTE (8), and TEB (89 Although this method of control has generally proved very mM Tris, 2.5 mM EDTA, 89 mM boric acid). efficient, there have been some reports that control is not always Standard bioassay for agrocin production. The method of effective (1,9,17,19). Not all strains of A. tumefaciens are sensitive Stonier (24) as modified by Kerr and Htay (12) was used. Strain to agrocin 84, especially strains isolated from grapevine, apple, K198 was used as the indicator. chrysanthemum, and blackberry, but biological control of crown Quantitative bioassay for agrocin production. Fresh YM agar gall by strain K84 has never been recommended for these crops. cultures were suspended in sterile distilled water (SDW) and the Even on stone fruit where biological control is usually very A 680nm values adjusted to 0.45; 100-.tl aliquots were inoculated into effective, some isolates are resistant to agrocin 84 (13). These 20 ml of CGG medium in 100-ml flasks and incubated at 26 C on a innately resistant strains probably account for many reports of rotary shaker. At appropriate times A 680nm was measured, 1-ml unsatisfactory control. Some pathogenic strains become resistant samples removed and centrifuged, and 150 1l of supernatant added to agrocin 84 after field use of strain 84 (19; M. Lopez, personal to 9-mm-diameter wells in plates of Stonier's agar. Three replicates communication). This is due to transfer of pAgK84 from strain of each culture were used. The plates were treated with chloroform K84 to A. tumefaciens (4) and poses a serious threat to the to kill bacteria and left for 3 hr to allow agrocin 84 to diffuse into continued success of biological control of crown gall. This threat the agar before being overlaid with the indicator strain K 198. could be markedly reduced if pAgK84 were made transfer deficient Isolation of an agrocin 84 overproducing mutant. A purified (Tra-). preparation of the Trainsertion derivative, pAgK84A I was The transfer region of pAgK84 has been located (8), and this prepared from strain Al and mutagenized in vitro with paper reports that a Trainsertion mutant in a suitable hydroxylamine essentially as described (15). The treated DNA was chromosomal background is an efficient agent of biological precipitated with ethanol, the pellet washed four times with 70% control. In addition, mutants that overproduce agrocin 84. are ethanol, and redissolved in 50 AI of LTE. A 0-/1 aliquot was used tested as biological control agents. to transform strain NT-1. Kanamycin-resistant transformants were picked to master NA+Km plates and also to Stonier's plates, which were incubated for 3 hr at 26 C. This is the minimum time necessary to detect agrocin 84 production by strain A 1. After this incubation, the Stonier's plates were exposed to chloroform and The publication costs of this article were defrayed in part by page charge payment. This overlayed with the sensitive indicator strain, C58. Colonies giving article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. § 1734 solely to indicate this fact. zones of inhibition larger than that of the parent strain, A 1, were recovered from the master plates and reassayed for increased levels © 1987 The American Phytopathological Society of agrocin 84 production. Vol. 77, No. 3, 1987 463 TABLE 1. Bacterial strains