Crown Gall Resistance in Accessions of 20 Prunus Species

Crown gall is an important disease of many fruit and nut crops, but little is known about sources of resistance. We screened germplasm from Prunus armeniaca L., P. angustifolia Marsh., P. argentia L., P. avium L., P. besseyi Bailey, P. bokhariensis Schneid., P. brigantica L., P. cerasifera Ehrh., P. cerasus L., P. dulcis (Mill.) D.A. Webb, P. fruiticosa Pall., P. hortulana Bailey, P. insititia L., P. japonica Thunb., P. mahaleb L., P. persica (L.) Batsch, P. serotina Ehrh., P. simonii Carr., P. sogdiana L., and P. webbii (Spach) Vieh. When either main stems or lateral branches of seedlings were inoculated with strains K12 and C58 of Agrobacterium tumefaciens (Smith and Townsend) Conn., the incidence of resistance was less than 10% except in some accessions of P. mahaleb L. where up to 30% of the plants were resistant. Some resistant plants were identified in other species, with P. insititia L. being the most promising. Symptoms based on presence and size of galls should be allowed to develop for up to 90 days after inoculation to reduce the likelihood of misclassifying plants as resistant when they are slightly susceptible. cytokinin, and Nam et al. (1997) found that one partially resistant A. thaliana ecotype showed impaired transformation and integration of T-DNA into the host genome. Among dicots, A. tumefaciens has a wide host range. Differences in virulence exist among pathogenic strains in, e.g., strawberry (Fragaria vesca L.) (Uratsu et al., 1991), and in most cases there is some specificity between host genotype and pathogen strain [e.g., chrysanthemum (Bush and Pueppke, 1991), pea (Robbs et al., 1991), and soybean (Bailey et al., 1994; Byrne et al., 1987)]. Thus, when screening for resistance, the pathogen strain and conditions surrounding inoculation and disease development should be considered, as well as the host plant genotype. Crown gall often occurs on plants wounded during transplanting and cultivation. However, when bacteria enter the potential host through natural openings and incidental wounds, disease expression may vary even among susceptible plants. To ensure inoculation and enhance uniform disease development for efficient screening, artificial wounding [i.e., inoculation of the cut surface of cuttings (Peirronnet and Salesses, 1996), inoculation of stem incisions, etc.] may be used. Susceptibility can be judged by the occurrence of galls, rate of gall growth, and gall size. Our objectives were to optimize procedures for a disease screen, then to screen seedlings of a range of Prunus accessions for reaction to A. tumefaciens and identify sources of resistance to the pathogen. Materials and Methods Plant materials. To begin a multiyear study, accessions of Prunus sp. were chosen in 1994 as sources of seedling populations, primarily because of their current or potential use as rootstocks. In some cases there was a prior indication of possible resistance or tolerance to crown gall. Seeds were collected from source trees in the germplasm collection at the Univ. of California, Davis (UCD), and some were received from other sources, including David Byrne, Texas A&M Univ., College Station, and the IR-2 Program, Pullman, Wash. After cold stratification under moist conditions for a time sufficient for each species, endocarps were cracked, the seed coats removed, and the embryos placed on the surface of soil contained in plastic cones. The soil surface was kept moist with intermittent misting until after germination, when the plants were moved to a greenhouse with supplemental light. Bacterial inoculum. Four wild-type strains of A. tumefaciens (A208, C58, K12, and ACH5) were used for resistance studies, and a strain of C58 selected for kanamycin resistance (C58Km1) to study the mobility of the bacteria in inoculated branches. Bacterial cultures were prepared as follows: 100 μL of bacterial culture, stored at –80 °C in 15% glycerol, were placed in a test tube containing 15 mL of the growth medium. The LB medium (10 g tryptone, 5 g·L yeast extract, pH 7.1) was used for the wild type strains K12 and C58. The growth medium 523 Received for publication 2 Apr. 1998. Accepted for publication 5 Aug. 1998. This research was supported in part by funding from the California Cherry Advisory Board, California Tree Fruit Agreement, U.S. Dept. of Agriculture Special Grant No. 595302-5-703, Will W. Lester Endowment, and Project No. 5501-RR of the Western Regional Research Project W-6. 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. To whom reprint requests should be addressed. Current address: Univ. of Missouri, Dept. of Biological Sciences, 109 Tucker Hall, Columbia, MO 65211-7400. Crown gall disease incited by A. tumefaciens is an important problem for nursery and field production of many stone fruit and nut crops (Kennedy and Alcorn, 1980). Soil fumigation is effective as a means of control, but because of increasing regulation, continued use of chemicals will probably be limited. Biological agents such as strain K84 of A. radiobacter var. tumefaciens provide some protection against the disease (Pierronnet and Escalettes, 1991; Ryder and Jones, 1991), and crown gall-resistant rootstocks would be an attractive complement. Germplasm assessment is the first step towards determining the feasibility of breeding for resistance and developing a strategy for selecting plants that combine crown gall resistance with other important traits (e.g., resistance to other diseases, size control, etc.). Because evaluation of Prunus germplasm for crown gall reaction has been limited, little is known about the occurrence of resistance (DeCleene and DeLey, 1976; Smith, 1925). Recently, Peirronnet and Salesses (1996) found varying reactions to A. tumefaciens among several species of plum. No resistance was found, and only a few accessions of P. insititia, P. domestica L., P. besseyi Bailey, and some interspecific hybrids were less susceptible, but they concluded that P. domestica might be useful in breeding for low susceptibility. There has been occasional mention of plants that are resistant, e.g., ‘Rubira’ peach [Prunus persica (L.) Batsch] (see Layne, 1987), but the conditions surrounding those observations were not specified. In addition to limited information about sources of crown gall resistance, little is known about the pattern of inheritance in crop plants. In Vitis, Sule et al. (1994) reported differential sensitivity to Agrobacterium strains and some resistance, and Stover et al. (1997a) found different levels of susceptibility but no immunity among species and interspecific hybrids. Segregation analyses have shown phenotypic differences in, e.g., soybean [Glycine max (L.) Merr.] (Owens and Cress, 1985), rose (Rosa sp.) (Boelema, 1969), chrysanthemum (Dendranthema ×grandiflora Kitam.), and raspberry (Rubus ideas L.) (Miller et al., 1975; Zurowski et al., 1985), and have demonstrated that resistance is heritable and expressed quantitatively in, e.g., pea (Pisum sativum L.) (Robbs et al., 1991), soybean (Bailey et al., 1994; Mauro et al., 1995), and grape (Vitis sp.) (Szegedi and Kozma, 1984), but no specific resistance genes were identified. Nam et al. (1997) found differences in susceptibility among Arabidopsis thaliana (L.) ecotypes and segregation at a single locus in one hybrid population, with resistance being recessive. Little is known about the mechanisms of crown gall resistance. Beneddra et al. (1996) reported that genotypic differences in reactions among aspen (Populus sp.) cultivars were related to differences in sensitivity to