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Aug 14, 2009 - J. Creighton Miller, Jr., Kathleen Haynes, and Richard Novy. This research was funded in part by the Wisconsin Potato and. Vegetable Growers ...
Am. J. Pot Res (2009) 86:504–512 DOI 10.1007/s12230-009-9107-x

Identification of Verticillium Wilt Resistance in U.S. Potato Breeding Programs Shelley H. Jansky

Published online: 14 August 2009 # Potato Association of America 2009

Abstract Verticillium wilt (VW), caused mainly by the soil-borne fungus V. dahliae, is a persistent and serious problem in potato production. Host-plant resistance offers an attractive control strategy, but most major cultivars are susceptible to VW. Resistance to VW was evaluated in 14 advanced clones from U.S. potato breeding programs and 11 cultivars. The two objectives of this study were to 1) determine the extent to which VW resistance exists in advanced selections in U.S. potato breeding programs and 2) test a selection strategy based on multiple measures of resistance. The three measures of resistance used in this study were symptom expression in the field, colonization of stem sap, and numbers of propagules in senescent stems. Resistant clones had low scores, but susceptible clones were highly variable for all three measures. An effective selection strategy utilizing all three measures of assessment can be used to separate true resistance from tolerance as follows: first, identify clones with low symptom expression, then measure sap colonization, and finish by establishing propagule levels in senescent stems. Based on this approach, breeders will be able to identify VW resistant, and not just VW tolerant, germplasm for future breeding efforts.

principales son susceptibles a VW. Se evaluó la resistencia a VW en 14 clones avanzados de programas de mejoramiento de papa de E.U. y 11 variedades. Los dos objetivos de este estudio fueron 1) determinar hasta que punto existe resistencia a VW en las selecciones avanzadas de programas de mejoramiento de papa de E.U. y 2) probar una estrategia de selección basada en múltiples medidas de resistencia. Las tres medidas de resistencia usadas en este estudio fueron la expresión de síntomas en el campo, colonización de la savia del tallo, y el número de propágulos en tallos senescentes. Los clones resistentes tuvieron registros bajos, pero los susceptibles fueron altamente variables para las tres medidas. Se puede emplear una estrategia de selección efectiva utilizando las tres medidas de evaluación para separar la verdadera resistencia de la tolerancia de la siguiente manera: primero, identificar clones con baja expresión de síntomas, después medir la colonización de la savia, y terminar con el establecimiento de los niveles de propágulos en tallos senescentes. Con base en este enfoque, los mejoradores estarán en capacidad para identificar germoplasma con resistencia a VW, y no solamente tolerancia, en esfuerzos de mejoramiento a futuro.

Resumen La marchites por Veticillium (VW), causada principalmente por el hongo del suelo V. dahliae, es un problema persistente y serio en la producción de papa. La resistencia de la planta hospedera es una estrategia de control atractiva, pero la mayoría de las variedades

Keywords Solanum tuberosum . Verticillium dahliae . Resistance breeding

S. H. Jansky (*) USDA-ARS and Department of Horticulture, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI 53706, USA e-mail: [email protected]

Verticillium wilt (VW) is a persistent and serious problem in potato production. It is caused by the soil-borne fungi V. dahliae Kleb. in warm production areas and V. alboatrum Reinke & Berthold in cooler regions (Rowe and Powelson 2002). The disease is typically controlled through

Introduction

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the use of fumigants, but concerns about the economic and environmental costs of this strategy bring its sustainability into question (Rowe and Powelson 2002). Host plant resistance offers an alternative long-term control method, but historically, major cultivars have been susceptible. However, the 2007 U.S. fall crop included two moderately resistant clones, ranked third (Ranger Russet) and ninth (Goldrush) in production area. More importantly, two recently released cultivars with some resistance were included in the top ten, based on production area. Alturas was released in 2003 and ranked eighth, while Western Russet, a 2006 release, ranked tenth in production. Breeding programs depend on both access to germplasm carrying resistance genes and the ability to identify resistant clones. A survey of U.S. potato cultivars in 1974 found that many are closely related to each other (Mendoza and Haynes 1974). In recent decades, however, the introduction of diverse germplasm into breeding programs in North America and Europe has increased opportunities for the introgression of valuable genes and genetic diversity into new cultivars (Bradshaw et al. 2006). High levels of resistance to VW have been identified in exotic germplasm, providing the potential to develop resistant cultivars (Concibido et al. 1994; Corsini et al. 1988; Jansky and Rouse 2000; Lynch et al. 1997; Mohan et al. 1990). The ability to identify resistant clones may be a more serious challenge than the availability of suitable germplasm. Early attempts to breed for VW resistance focused on symptom expression in plants grown in infested fields (Akeley et al. 1956; Hunter et al. 1968). While this strategy is easy to employ on a large scale, it does not distinguish between tolerance and resistance. In recent decades, resistance screening has been improved through the use of methods to quantify pathogen populations in host tissue (Davis et al. 1983; Hoyos et al. 1991). Estimates of V. dahliae population sizes in planta are more effective than visual disease assessment in explaining variability in yield loss due to VW (Frost et al. 2007). However, considerable variability among stems of the same clone and even the same plant is commonly observed (Frost et al. 2007; Jansky and Rouse 2000; Slattery 1981). Characterizing clones for VW resistance in different years or at different times within a year may be difficult if using only disease symptom expression or quantitative assays of V. dahliae population sizes (Frost et al. 2007). Interactions among levels of symptom expression, pathogen populations in stems, and production environment are not well understood. In order for potato breeders to make progress toward the production of VW resistant cultivars, a selection strategy must be developed that provides a reliable characterization of the host-pathogen interaction in large segregating populations. This evaluation should include a combination

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of resistance scoring techniques. One such strategy could include an initial selection for resistance based on symptom expression. Then, sap would be collected and plated from clones with low symptom expression. Finally, dried stems would be collected and plated from clones with low symptom and sap scores. Clones with low scores for all three resistance measures would be considered resistant. The two objectives of this study were to 1) determine the extent to which VW resistance exists in advanced selections in U.S. potato breeding programs and 2) test a selection strategy based on multiple measures of resistance.

Materials and Methods On 2 May, 2006, three replications of nine-hill plots of each clone were planted in a randomized complete block design in a V. dahliae-infested field at the Hancock, Wisconsin, Agricultural Experiment Station. This field was fumigated in 2004 and then inoculated with V. dahliae. It contains approximately 20 cfu V. dahliae per gram of soil. The entries included 14 advanced clones from U.S. breeding programs and the cultivars Atlantic, Boulder, Dakota Diamond, Freedom Russet, Megachip, Ranger Russet, Red Norland, Russet Burbank, Russet Norkotah, Superior, and White Pearl, which represent a range of resistance levels. Russet Norkotah is the most susceptible of the standard cultivars, while Ranger Russet is the most resistant. Best management practices were used throughout the growing season and the field was irrigated using an overhead sprinkler every other day (Binning et al. 2002). In each year, nitrogen was applied three times during the growing season. In 2006 and 2007, 234 and 210 lb. of nitrogen was applied, respectively. Daily maximum and minimum temperatures were recorded at the experiment station. Plots were scored visually for percent foliage with VW symptoms on 17 July and 7, 23, and 30 August. Basal segments (7–10 cm) of main stems from four plants per plot were collected on 8 August for analysis of sap for stem colonization. Basal segments (7–10 cm) of main stems from an additional four plants were collected in early September for analysis of dried tissue for stem colonization. At this time, plants were senescing or dead. An identical trial was carried out on the same field in 2007, with planting on 3 May, symptom expression recorded on 9, 18, 25, and 30 July as well as 7 and 21 August, stems for sap were collected on 30 July, and stems for dried tissue collected in early September. For each sample collected for sap, all fresh stems from a plant were rinsed in distilled water, soaked in 1% NaOCl for 30 s, rinsed three times in sterile distilled water, placed into a plastic bag, and squeezed with a vice. A 100 ul aliquot of sap was plated on NPX medium (Butterfield and

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DeVay 1977). Four plates were made for each plot (one from each plant). Stems collected at the end of the season were allowed to air dry in paper bags on a bench top at room temperature for one month before plating. All stems from one plant were ground together in a Wiley mill using a 40 mesh screen and a 50 mg sample was plated. Between samples, debris was removed with a brush. Four plates (one from each plant) were made for each plot. Plates were incubated in the dark for two weeks before counting colonies. Plant debris was washed off dried stem plates before they were observed microscopically at 100× magnification and colonies were counted. Analysis of variance was carried out using the General Linear Model in SAS with fixed clone effects and random replication effects in the individual year analyses. In the ANOVA across years, rep(year) was considered a random effect and was used as the error term for year. Sap and dried stem colony counts were transformed using log(x+1) prior to analysis. This resulted in more homogeneous error variances than before transformation. Relative area under the disease progress curve (RAUDPC) was calculated in each year by dividing AUDPC by the number of days across which scores were collected. In addition, symptom expression on a single score date (August 7 in both years) was included in the analyses. A plot of residuals indicated that error variances across years for each variable were homogenous, so a combined analysis was carried out to evaluate genotype by environment interactions. Pearson’s correlation and Spearman’s rank correlation were used to analyze relationships between resistance parameters and production years based on clonal means. A protected Least Significant difference (LSD) test was carried out to compare August 7 symptom expression scores. That date was chosen because it was near the mid-point of the rating period and an early August score date was suggested by Jansky and Rouse (2000). A triage method was used to identify resistant clones. First, mid- to late-maturing clones were discarded if their disease symptom score on August 7 was greater than that of the moderately resistant cultivar Ranger Russet. Any remaining clones with sap colonization scores greater than 200 cfu/100 ul sap were then discarded. Finally, clones remaining after the first two steps were discarded if their mean dried stem score was greater than 200 cfu/50 mg dried stem material.

Results In each year, there was a significant effect of clone for RAUDPC, August 7 symptom expression, colonization of stem sap, and colonization of senescent stems (except for 2007) (Table 1). Replication differences were not signifi-

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cant, except for RAUDPC and August 7 symptom expression in 2007. When comparing clones across years, significant effects of year and the clone by year interaction were observed for RAUDPC, the August 7 symptom score, sap stem colonization means, and dried stem colonization means (Table 2). Symptom expression, based on both RAUDPC and the August 7 score date, was significantly higher in 2007 than in 2006 (Table 3). However, sap counts were higher in 2007, while dried stem colony counts were higher in 2006. Disease symptom scores for RAUDPC and the August 7 score date were significantly correlated between 2006 and 2007, based on both clonal mean scores (Pearson Correlation) and ranks (Spearman Rank Correlation) (Table 4). There was no association between years for sap or dried stem means. The correlation between RAUDPC and the August 7 symptom scores was high and significant in both years. RAUDPC and August 7 symptom scores also correlated with dried stem scores in 2006. Clones were selected for VW resistance by first identifying those that were not significantly different from the moderately resistant cultivar Ranger Russet for symptom expression on August 7, based on the LSD test. However, early maturing clones were at a disadvantage because they were being evaluated at a later physiological age than mid- and late-season clones. Consequently, early maturing clones (less than 2.0 on a scale of 1 = early, 2 = mid-season, 3 = late), based on breeders’ evaluations, were not discarded due to high symptom scores on August 7. In 2006, 23 of the 25 clones in the trial were retained (Table 5); in 2007, 19 clones were retained (Table 6);. Then, among the remaining clones, those with mean sap counts of less than 200 cfu/100 ul sap were retained. In a previous study, Jansky and Rouse (2000) suggested a threshold of 100 cfu/g in highly resistant diploid germplasm. That number was doubled for this study to reduce the stringency when selecting in advanced breeding clones. After this second selection step, 16 clones remained in 2006 and 15 clones remained in 2007 (Tables 5 and 6). Finally, of the remaining clones, those with dried stem means less than 200 cfu/50 mg stem were considered to be resistant. In 2006, six clones were scored as resistant, while in 2007, 14 were resistant. All six clones identified as resistant in 2006 were among those selected in 2007. They include MSJ4611 (Michigan State University, recently named Missaukee), CO9403515-Rus and CO95051-7 W (Colorado State University), W2133-1 and Megachip (University of Wisconsin) and AOND95249-1 (North Dakota State University). The moderately resistant control cultivar Ranger Russet was not selected in 2006 because its dried stem counts exceeded the threshold, but it was selected in 2007. The susceptible control cultivar, Russet Norkotah, was discarded in both years.

Am. J. Pot Res (2009) 86:504–512 Table 1 ANOVA by year for symptom expression across the season (RAUDPC), symptom expression on August 7 (Aug 7 symp), fungal propagules in sap (sap), and fungal propagules in senescent stems (dry)

507 Data set

Source

DF

Type III SS

Mean square

F value

RAUDPC 2006

clone rep error clone rep error clone rep error clone rep error clone rep error

24 2 48 24 2 48 24 2 48 24 2 48 24 2 48

4135.28 32.97 364.63 125933.35 3595.84 10205.66 61321.30 221.06 4078.94 55885.33 2384.67 7098.67 28.46 0.12 15.70

172.30 16.48 7.60 5247.22 1797.92 212.62 2555.05 110.53 84.98 2328.56 1192.33 147.89 1.19 0.06 0.33

22.68 2.17