An Isozyme Marker for Resistance to Root-Knot Nematode in Sugarbeet

sought for accelerating sugarbeet root-knot nematoderesistance breeding. An isozyme pattern of phosphogluRoot-knot nematode (Meloidogyne spp.) is a destructive pest of comutase (PGM) has been shown to be associated with sugarbeet (Beta vulgaris L.) that reduces production in infested areas root-knot nematode resistance found in the Mi-1 Beta and is difficult to manage. Identification of nematode-resistant plants is a time-consuming process that is subject to genotype–environment germplasm lines. The PGM isozyme marker can be eminteraction. Development of resistant cultivars/hybrids is the most ployed to easily identify individual Mi-1 Beta genotypes effective control. This study was conducted to establish a rapid and with resistance to Meloidogyne spp. effective screening technique to detect a large number of sugarbeet The objective of this study was to establish a rapid genotypes with resistance to Meloidogyne spp. A nematode-resistant and effective screening procedure to detect a large numsugarbeet germplasm line, Mi-1 Beta, was previously developed using ber of sugarbeet genotypes with resistance to root-knot J2 inoculation and screening procedures. Leaf and cotyledon extracnematode. tions were used in diagnosis. Phosphoglucomutase (PGM) was found to be a potentially useful isozyme marker of resistance in Mi-1 Beta MATERIALS AND METHODS and derived lines in starch gel electrophoresis. Seven banding patterns (four resistant and three susceptible) were produced. All susceptible The Beta materials with resistance to Meloidogyne spp. used plants shared the banding pattern of the resistant strains, except for in this research were the Mi-1 germplasm (PI 593237; Yu, a single PGM band. If demonstrated to be tightly linked to nematode 1997), progeny of its hybrid crosses to sugarbeet, and susceptiresistance, this novel PGM isozyme marker will accelerate breeding ble controls (sugarbeet 1783 and 4500) available in the USDAsugarbeet with resistance to root-knot nematode. ARS program. Stecklings (seedlings) of the susceptible sugarbeet lines C37, C78, C80, and R876-89-5NB were obtained from R.T. Lewellen, USDA-ARS, Salinas. Crosses to transfer root-knot nematode resistance from wild beet to sugarbeet T root-knot nematode, Meloidogyne spp., is an were conducted in the greenhouse and laboratory. Test plants economically important sugarbeet (Beta vulgaris L.) were examined for root gall formations 35 to 40 d after inoculapest that is difficult to control in production fields. Pretion of early-stage beet seedlings with 1200 M. incognita J2 viously, soil fumigation was the most reliable technique. per plant (Yu et al., 1999). Individual plants producing none Root-knot nematode is now yield-limiting due to conor fewer than 10 small ( 1 mm) root galls were classified as tinuing loss and restriction of soil fumigants, thereby resistant. All other plants were classified as susceptible. Seeds preventing farmers from growing sugarbeet crops profitwere germinated in steam-sterilized sand. ably. Host-plant resistance to root-knot nematode is, Tissue of cotyledons and expanding young leaves was coltherefore, the most environmentally safe means to allelected from greenhouse or field (Spence farm in Salinas, CA, sandy loam soil classification) plants, from seedling stage to viate sugarbeet production problems and nematicide full maturation for isozyme analysis. A minimum of 55 plants hazards. per germplasm line were examined. Samples, about 5 mm in The wild sea beet [B. vulgaris ssp. maritima (L.) Arsize, were ground in pre-chilled mortars with 0.1-mL extraction cang] source of root-knot nematode resistance is effecbuffer. The extraction buffer consisted of 0.1 M Tris (tristive against multiple species of Meloidogyne spp. (Yu [hydroxymethyl]aminomethane)–HCl, pH 7.0, 2.45 M of glycet al., 1999). Introgression of root-knot nematode resiserol, 1.25 mM of PVP-40T (polyvinylpyrrolidone), 0.5% (v/ tance from wild beet to cultivated sugarbeet has been v) Triton X-100, and 125 mM 2-mercaptoethanol, the latter achieved using second-stage juveniles (J2) inoculation, added just before use (modified from Aicher and Saunders, screening, and pollination procedures in the green1990). Paper wicks (11 by 2 mm; Northfork Products, Syracuse, house. Improved sugarbeet-breeding materials with reIN) were soaked in each sample extract and stored at 4 C. Buffers and the phosphoglucomutase stain were adapted sistance to root-knot nematode were further selected from systems described by Van Geyt and Smed (1984) with a for tap-root conformation and root-yield performance. slight modification. The gel buffer consisted of 5 mM of histiThe process of transferring resistant genes from nondinemonohydrochloride, pH 7.0, and the electrode buffer was cultivated taxa or species to the cultivated genomes, and 0.2 M of trisodium citrate-2 H2O, pH 7.0. The staining solution selecting productive genotypes to become hybrid parents, for the isozyme PGM consisted of 25 mL of 0.1 M Tris–HCl always has been time-consuming and labor-intensive. A buffer, pH 8.0; 5 mM of -D-glucose-1-phosphate (glucosesimpler and more accurate screening procedure has been 1-P); 7 units glucose-6-phosphate dehydrogenase (G-6-PDH); 5 mM of -nicotinamide adenine dinucleotide phosphate (NADP); 2 mL of 0.025 M MgCl2; 1 mM of 3-[4,5-dimethylthiaM.H. Yu and L.M. Pakish, USDA-ARS-PWA, U.S. Agric. Res. Stn., zol-2-yl]-2,5 diphenyltetrazolium bromide (MTT); and 0.13 mM 1636 East Alisal St., Salinas, CA 93905-3018; H. Zhou, Harris Moran of phenazine methosulfate (PMS). Seed Co., 100 Breen Rd., San Juan Bautista, CA 95045. Mention of a trademark, vendor, or proprietary product does not constitute a The gel was prepared using a 1:2:1 mixture of hydrolyzed guarantee or warranty of the product by the USDA and does not starch (Pasteur Merieux Connaught, Charlotte, NC), hyimply its approval to the exclusion of other products or vendors that drolyzed potato starch (Starch-Art, Smithville, TX), and pomay also be suitable. Received 16 Aug. 2000. *Corresponding author ([email protected]). Abbreviations: PGM, phosphoglucomutase; PMS, phenazine methosulfate. Published in Crop Sci. 41:1051–1053 (2001).