Am. J. Pot Res (2009) 86:504–512 DOI 10.1007/s12230-009-9107-x

Identification of Verticillium Wilt Resistance in U.S. Breeding Programs

Shelley H. Jansky

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

Abstract Verticillium wilt (VW), caused mainly by the principales son susceptibles a VW. Se evaluó la resistencia soil-borne fungus V. dahliae, is a persistent and serious a VW en 14 clones avanzados de programas de mejor- problem in potato production. Host-plant resistance offers amiento de papa de E.U. y 11 variedades. Los dos objetivos an attractive control strategy, but most major cultivars are de este estudio fueron 1) determinar hasta que punto existe susceptible to VW. Resistance to VW was evaluated in 14 resistencia a VW en las selecciones avanzadas de pro- advanced clones from U.S. potato breeding programs and gramas de mejoramiento de papa de E.U. y 2) probar una 11 cultivars. The two objectives of this study were to 1) estrategia de selección basada en múltiples medidas de determine the extent to which VW resistance exists in resistencia. Las tres medidas de resistencia usadas en este advanced selections in U.S. potato breeding programs and estudio fueron la expresión de síntomas en el campo, 2) test a selection strategy based on multiple measures of colonización de la savia del tallo, y el número de resistance. The three measures of resistance used in this propágulos en tallos senescentes. Los clones resistentes study were symptom expression in the field, colonization of tuvieron registros bajos, pero los susceptibles fueron stem sap, and numbers of propagules in senescent stems. altamente variables para las tres medidas. Se puede emplear Resistant clones had low scores, but susceptible clones una estrategia de selección efectiva utilizando las tres were highly variable for all three measures. An effective medidas de evaluación para separar la verdadera resistencia selection strategy utilizing all three measures of assessment de la tolerancia de la siguiente manera: primero, identificar can be used to separate true resistance from tolerance as clones con baja expresión de síntomas, después medir la follows: first, identify clones with low symptom expression, colonización de la savia, y terminar con el establecimiento then measure sap colonization, and finish by establishing de los niveles de propágulos en tallos senescentes. Con propagule levels in senescent stems. Based on this base en este enfoque, los mejoradores estarán en capacidad approach, breeders will be able to identify VW resistant, para identificar germoplasma con resistencia a VW, y no and not just VW tolerant, germplasm for future breeding solamente tolerancia, en esfuerzos de mejoramiento a efforts. futuro.

Resumen La marchites por Veticillium (VW), causada Keywords Solanum tuberosum . Verticillium dahliae . principalmente por el hongo del suelo V. dahliae,esun Resistance breeding 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 Introduction

S. H. Jansky (*) Verticillium wilt (VW) is a persistent and serious problem USDA-ARS and Department of Horticulture, in potato production. It is caused by the soil-borne fungi University of Wisconsin-Madison, V. dahliae Kleb. in warm production areas and V. albo- 1575 Linden Drive, Madison, WI 53706, USA atrum Reinke & Berthold in cooler regions (Rowe and e-mail: [email protected] Powelson 2002). The disease is typically controlled through Am. J. Pot Res (2009) 86:504–512 505 the use of fumigants, but concerns about the economic and of resistance scoring techniques. One such strategy could environmental costs of this strategy bring its sustainability include an initial selection for resistance based on symptom into question (Rowe and Powelson 2002). Host plant expression. Then, sap would be collected and plated from resistance offers an alternative long-term control method, clones with low symptom expression. Finally, dried stems but historically, major cultivars have been susceptible. would be collected and plated from clones with low However, the 2007 U.S. fall crop included two moderately symptom and sap scores. Clones with low scores for all resistant clones, ranked third () and ninth three resistance measures would be considered resistant. (Goldrush) in production area. More importantly, two The two objectives of this study were to 1) determine the recently released cultivars with some resistance were extent to which VW resistance exists in advanced selections included in the top ten, based on production area. Alturas in U.S. potato breeding programs and 2) test a selection was released in 2003 and ranked eighth, while Western strategy based on multiple measures of resistance. 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 Materials and Methods clones. A survey of U.S. potato cultivars in 1974 found that many are closely related to each other (Mendoza and On 2 May, 2006, three replications of nine-hill plots of each Haynes 1974). In recent decades, however, the introduction clone were planted in a randomized complete block design of diverse germplasm into breeding programs in North in a V. dahliae-infested field at the Hancock, Wisconsin, America and Europe has increased opportunities for the Agricultural Experiment Station. This field was fumigated introgression of valuable genes and genetic diversity into in 2004 and then inoculated with V. dahliae. It contains new cultivars (Bradshaw et al. 2006). High levels of approximately 20 cfu V. dahliae per gram of soil. The resistance to VW have been identified in exotic germplasm, entries included 14 advanced clones from U.S. breeding providing the potential to develop resistant cultivars programs and the cultivars Atlantic, Boulder, Dakota (Concibido et al. 1994; Corsini et al. 1988; Jansky and Diamond, Freedom Russet, , Ranger Russet, Rouse 2000; Lynch et al. 1997; Mohan et al. 1990). , , Russet Norkotah, Superior, The ability to identify resistant clones may be a more and White Pearl, which represent a range of resistance serious challenge than the availability of suitable germ- levels. Russet Norkotah is the most susceptible of the plasm. Early attempts to breed for VW resistance focused standard cultivars, while Ranger Russet is the most on symptom expression in plants grown in infested fields resistant. Best management practices were used throughout (Akeley et al. 1956; Hunter et al. 1968). While this strategy the growing season and the field was irrigated using an is easy to employ on a large scale, it does not distinguish overhead sprinkler every other day (Binning et al. 2002). In between tolerance and resistance. In recent decades, each year, nitrogen was applied three times during the resistance screening has been improved through the use of growing season. In 2006 and 2007, 234 and 210 lb. of methods to quantify pathogen populations in host tissue nitrogen was applied, respectively. Daily maximum and (Davis et al. 1983; Hoyos et al. 1991). Estimates of minimum temperatures were recorded at the experiment V. dahliae population sizes in planta are more effective station. Plots were scored visually for percent foliage with than visual disease assessment in explaining variability in VW symptoms on 17 July and 7, 23, and 30 August. Basal yield loss due to VW (Frost et al. 2007). However, segments (7–10 cm) of main stems from four plants per plot considerable variability among stems of the same clone were collected on 8 August for analysis of sap for stem and even the same plant is commonly observed (Frost et al. colonization. Basal segments (7–10 cm) of main stems from 2007; Jansky and Rouse 2000; Slattery 1981). Character- an additional four plants were collected in early September izing clones for VW resistance in different years or at for analysis of dried tissue for stem colonization. At this different times within a year may be difficult if using only time, plants were senescing or dead. An identical trial was disease symptom expression or quantitative assays of carried out on the same field in 2007, with planting on 3 V. dahliae population sizes (Frost et al. 2007). Interactions May, symptom expression recorded on 9, 18, 25, and 30 among levels of symptom expression, pathogen populations July as well as 7 and 21 August, stems for sap were in stems, and production environment are not well collected on 30 July, and stems for dried tissue collected in understood. early September. In order for potato breeders to make progress toward the For each sample collected for sap, all fresh stems from a production of VW resistant cultivars, a selection strategy plant were rinsed in distilled water, soaked in 1% NaOCl must be developed that provides a reliable characterization for 30 s, rinsed three times in sterile distilled water, placed of the host-pathogen interaction in large segregating into a plastic bag, and squeezed with a vice. A 100 ul populations. This evaluation should include a combination aliquot of sap was plated on NPX medium (Butterfield and 506 Am. J. Pot Res (2009) 86:504–512

DeVay 1977). Four plates were made for each plot (one cant, except for RAUDPC and August 7 symptom from each plant). Stems collected at the end of the season expression in 2007. were allowed to air dry in paper bags on a bench top at When comparing clones across years, significant effects of room temperature for one month before plating. All stems year and the clone by year interaction were observed for from one plant were ground together in a Wiley mill using a RAUDPC, the August 7 symptom score, sap stem coloniza- 40 mesh screen and a 50 mg sample was plated. Between tion means, and dried stem colonization means (Table 2). samples, debris was removed with a brush. Four plates (one Symptom expression, based on both RAUDPC and the from each plant) were made for each plot. Plates were August 7 score date, was significantly higher in 2007 than incubated in the dark for two weeks before counting in 2006 (Table 3). However, sap counts were higher in colonies. Plant debris was washed off dried stem plates 2007, while dried stem colony counts were higher in 2006. before they were observed microscopically at 100× magni- Disease symptom scores for RAUDPC and the August 7 fication and colonies were counted. score date were significantly correlated between 2006 and Analysis of variance was carried out using the General 2007, based on both clonal mean scores (Pearson Correla- Linear Model in SAS with fixed clone effects and random tion) and ranks (Spearman Rank Correlation) (Table 4). replication effects in the individual year analyses. In the There was no association between years for sap or dried ANOVA across years, rep(year) was considered a random stem means. The correlation between RAUDPC and the effect and was used as the error term for year. Sap and dried August 7 symptom scores was high and significant in both stem colony counts were transformed using log(x+1) prior years. RAUDPC and August 7 symptom scores also to analysis. This resulted in more homogeneous error correlated with dried stem scores in 2006. variances than before transformation. Relative area under Clones were selected for VW resistance by first the disease progress curve (RAUDPC) was calculated in identifying those that were not significantly different from each year by dividing AUDPC by the number of days the moderately resistant cultivar Ranger Russet for symp- across which scores were collected. In addition, symptom tom expression on August 7, based on the LSD test. expression on a single score date (August 7 in both years) However, early maturing clones were at a disadvantage was included in the analyses. A plot of residuals indicated because they were being evaluated at a later physiological that error variances across years for each variable were age than mid- and late-season clones. Consequently, early homogenous, so a combined analysis was carried out to maturing clones (less than 2.0 on a scale of 1 = early, 2 = evaluate genotype by environment interactions. Pearson’s mid-season, 3 = late), based on breeders’ evaluations, were correlation and Spearman’s rank correlation were used to not discarded due to high symptom scores on August 7. In analyze relationships between resistance parameters and 2006, 23 of the 25 clones in the trial were retained production years based on clonal means. A protected Least (Table 5); in 2007, 19 clones were retained (Table 6);. Significant difference (LSD) test was carried out to Then, among the remaining clones, those with mean sap compare August 7 symptom expression scores. That date counts of less than 200 cfu/100 ul sap were retained. In a was chosen because it was near the mid-point of the rating previous study, Jansky and Rouse (2000) suggested a period and an early August score date was suggested by threshold of 100 cfu/g in highly resistant diploid germ- Jansky and Rouse (2000). plasm. That number was doubled for this study to reduce A triage method was used to identify resistant clones. the stringency when selecting in advanced breeding clones. First, mid- to late-maturing clones were discarded if their After this second selection step, 16 clones remained in 2006 disease symptom score on August 7 was greater than that of and 15 clones remained in 2007 (Tables 5 and 6). Finally, the moderately resistant cultivar Ranger Russet. Any of the remaining clones, those with dried stem means less remaining clones with sap colonization scores greater than than 200 cfu/50 mg stem were considered to be resistant. In 200 cfu/100 ul sap were then discarded. Finally, clones 2006, six clones were scored as resistant, while in 2007, 14 remaining after the first two steps were discarded if their were resistant. All six clones identified as resistant in 2006 mean dried stem score was greater than 200 cfu/50 mg were among those selected in 2007. They include MSJ461- dried stem material. 1 (Michigan State University, recently named Missaukee), CO9403515-Rus and CO95051-7 W (Colorado State University), W2133-1 and Megachip (University of Results Wisconsin) and AOND95249-1 (North Dakota State Uni- versity). The moderately resistant control cultivar Ranger In each year, there was a significant effect of clone for Russet was not selected in 2006 because its dried stem RAUDPC, August 7 symptom expression, colonization of counts exceeded the threshold, but it was selected in 2007. stem sap, and colonization of senescent stems (except for The susceptible control cultivar, Russet Norkotah, was 2007) (Table 1). Replication differences were not signifi- discarded in both years. Am. J. Pot Res (2009) 86:504–512 507

Table 1 ANOVA by year for symptom expression across the Data set Source DF Type III SS Mean square F value Pr > F season (RAUDPC), symptom expression on August 7 (Aug 7 RAUDPC 2006 clone 24 4135.28 172.30 22.68 <0.0001 symp), fungal propagules in sap rep 2 32.97 16.48 2.17 0.1253 (sap), and fungal propagules in error 48 364.63 7.60 senescent stems (dry) RAUDPC 2007 clone 24 125933.35 5247.22 24.68 <0.0001 rep 2 3595.84 1797.92 8.46 0.0007 error 48 10205.66 212.62 Aug 7 symp 2006 clone 24 61321.30 2555.05 30.07 <0.0001 rep 2 221.06 110.53 1.3 0.2818 error 48 4078.94 84.98 Aug 7 symp 2007 clone 24 55885.33 2328.56 15.75 <0.0001 rep 2 2384.67 1192.33 8.06 0.001 error 48 7098.67 147.89 Sap 2006 clone 24 28.46 1.19 3.63 <0.0001 rep 2 0.12 0.06 0.18 0.8378 error 48 15.70 0.33 Sap 2007 clone 24 31.43 1.31 5.51 <0.0001 rep 2 0.34 0.17 0.72 0.4907 error 43 10.22 0.24 Dry 2006 clone 24 31.93 1.33 9.25 <0.0001 rep 2 0.78 0.39 2.73 0.0754 error 48 6.90 0.14 Dry 2007 clone 24 8.52 0.35 1.55 0.0991 rep 2 0.09 0.05 0.21 0.8147 error 46 10.52 0.23

Table 2 ANOVA across years for symptom expression across Source DF Type III SS Mean square F value Pr > F the season (RAUDPC), symp- tom expression on August 7 RAUDPC clone 24 83091.57 3462.15 31.44 <0.0001 (Aug 7 symp), fungal propa- year 1 60507.33 60507.33 66.7 0.0012 gules in sap (sap), and fungal clone*year 24 46977.06 1957.38 17.78 <0.0001 propagules in senescent stems (dry) rep(year) 4 3628.81 907.20 8.24 <0.0001 error 96 10570.29 110.11 Aug 7 symp clone 24 104479.26 4353.30 37.39 <0.0001 year 1 13093.18 13093.18 20.1 0.011 clone*year 24 12789.38 532.89 4.58 <0.0001 rep(year) 4 2605.73 651.43 5.59 0.0004 error 96 11177.60 116.43 Sap clone 24 42.83 1.78 6.27 <0.0001 year 1 8.11 8.11 70.52 0.0011 clone*year 24 17.32 0.72 2.53 0.0008 rep(year) 4 0.46 0.12 0.4 0.8054 error 91 25.92 0.28 Dry clone 24 21.27 0.89 4.78 <0.0001 year 1 50.19 50.19 228.36 0.0001 clone*year 24 19.19 0.80 4.31 <0.0001 rep(year) 4 0.88 0.22 1.19 0.3221 error 94 17.42 0.19 508 Am. J. Pot Res (2009) 86:504–512

Table 3 Means across years for symptom expression across the measurable progress toward VW resistance is apparent in season (RAUDPC), percent disease symptoms on August 7 (Aug 7 U.S. potato breeding programs symp), log cfu per 100 ul in sap from living stems (sap), and log cfu per 50 mg dried stem tissue (dry). Means across years followed by Among the susceptible clones, though, scores varied different letters are different from each other at P>0.05 widely across years and among screening methods. The range in mean levels of conidia in sap and microsclerotia in Year N Mean senescent stems was large in susceptible clones, a finding RAUDPC 2006 75 15.8a similar to previous reports (Dan et al. 2001; Frost et al. 2007 75 56.0b 2007; Jansky and Rouse 2000). In addition, in this study Aug 7 symp 2006 75 33.8a and previous ones, variability in sap and dried stem colony 2007 75 52.5b counts was observed among stems from the same clone and Sap 2006 75 1.08a even from the same plant (Frost et al. 2007; Jansky and 2007 70 1.58b Rouse 2000; Slattery 1981). Consequently, mean colony Dry 2006 75 2.12b count scores may not effectively distinguish among clones, 2007 73 0.94a especially if the clones are not highly resistant. Fungal propagule levels may fluctuate widely in the host environ- ment. Perhaps quantification of fungal biomass in stems using real-time PCR analyses would provide a more Discussion consistent measure of infection (Atallah et al. 2007; Dan et al. 2001). Efforts are underway to evaluate this The six clones identified as resistant in both years had low alternative measure of disease evaluation. scores for all three measures of resistance (Tables 5 and 6). Disease symptom expression is the most common Five of them are breeding clones (AOND95249-1, method to assess VW resistance, but it cannot distinguish CO94035-15RU, MSJ461-1, CO95051-7 W, and between resistant and tolerant clones. For example, W2133-1) and the sixth (Megachip) is a recent release. W2324-1 and W2683-2r appear to be tolerant, with low These clones were more consistently resistant than the levels of symptom expression, but substantial pathogen moderately resistant cultivar standard Ranger Russet. Thus, populations were sometimes observed in sap and dried

Table 4 Pearson and Spearman rank correlation coefficients be- Comparison Trait/year Pearson correlation (P) Spearman rank correlation (P) tween years and measures of Verticillium wilt resistance on 2006 vs. 2007 potato clones grown in a field at RAUDPCa 0.79 (<0.01) 0.81 (<0.01) Hancock, Wisconsin Aug 7 0.78 (<0.01) 0.70 (<0.01) sap 0.07 (0.76) 0.35 (0.09) dried stems −0.12 (0.55) −0.14 (0.51) RAUDPC vs. Aug 7 2006 0.96 (<0.01) 0.84 (<0.01) 2007 0.81 (<0.01) 0.90 (<0.01) RAUDPC vs. sap 2006 0.08 (0.70) 0.15 (0.47) 2007 0.06 (0.79) 0.40 (0.05) RAUDPC vs. dried stems 2006 0.65 (<0.01) 0.67 (<0.01) 2007 0.44 (0.03) −0.28 (0.182) Aug 7 vs. sap 2006 0.03 (0.89) 0.03 (0.91) a RAUDPC relative area under 2007 0.28 (0.18) 0.39 (0.06) the disease progress curve, Aug Aug 7 vs. dried stems 7 = percent vine area with 2006 0.60 (<0.01) 0.54 (<0.01) Verticillium wilt symptoms on 2007 0.13 (0.53) −0.25 (0.24) August 7, sap = mean colony forming units in 100 ul sap, sap vs. dried stems dried stems = mean colony 2006 0.37 (0.07) 0.43 (0.03) forming units in 50 mg senes- 2007 −0.19 (0.37) −0.21 (0.32) cent stem Am. J. Pot Res (2009) 86:504–512 509

Table 5 Selection for Verticillium wilt resistance in 2006. 2006. First First clones clones werewere selected selected if symptoms if their mean on August sap count 7 were (cfu/100 no greater ul sap) than was those less of than the moderatelywere selected resistant if symptoms clone Ranger on August Russet. 7 were Early no maturing greater than clones those (<2.0 of on a200. scale Finally, of 1 = early, the remaining 2 = mid-season, clones 3 were = late) selected clones if were their not mean discarded dried basedthe moderately on symptom resistant expression. clone RangerThen, the Russet. remaining Early clones maturing were clones selected ifstem their count mean (cfu/50 sap count mg (cfu/100 stem) was ul less sap) than was 200. less than Discarded 200. Finally, clones theare remaining(<2.0 on a scaleclones of were 1 = early, selected 2 = mid-season,if their mean 3 =dried late) stem clones count were (cfu/50 not mgindicated stem) with was a less strikethrough than 200. Discarded clones are indicated with a strikethroughdiscarded based on symptom expression. Then, the remaining clones Clone Aug 7 Clone Sap Clone Dry AOND95249-1 0.0 CO95051-7W 0.4 AOND95249-1 2.5 Boulder 6.7 MSJ461-1 3.4 CO94035-15RU 15.3 CO94035-15RU 8.3 Red Norland 3.8 MSJ461-1 17.7 MSJ461-1 10.0 ATTX961014-1R/Y 5.5 CO95051-7W 60.1 Freedom Russet 15.0 AOND95249-1 5.9 W2133-1 96.7 Russet Burbank 15.0 B2327-2 13.4 Megachip 124.8 W2324-1 16.7 W2310-3 20.4 Boulder 144.3 Atlantic 18.3 CO94035-15RU 21.6 A95109-1 224.8 CO95051-7W 20.0 Megachip 27.0 Russet Norkotah 254.2 W2133-1 20.0 W2133-1 34.8 Ranger Russet 259.3 W2310-3 20.0 W2683-2r 54.6 Dakota Diamond 292.1 B2327-2 21.7 Atlantic 56.5 B2327-2 297.4 Dakota Diamond 21.7 ND7818-1 61.4 W2683-2r 306.7 ND5002-3 21.7 Russet Norkotah 75.9 ATTX961014-1R/Y 353.0 Ranger Russet 21.7 A95109-1 91.5 W2310-3 391.7 W2683-2r 21.7 Dakota Diamond 107.1 ND5002-3 424.3 A95109-1* 23.3 Ranger Russet 116.0 Russet Burbank 430.3 Megachip 23.3 White Pearl 123.3 Freedom Russet 475.1 White Pearl 45.0 ND5002-3 200.2 NDTX4271-5R 482.8 Superior* 68.3 NDTX4271-5R 312.8 Red Norland 644.1 ND7818-1* 78.3 W2324-1 341.0 W2324-1 813.7 Red Norland* 80.0 Boulder 346.3 ND7818-1 814.0 ATTX961014-1R/Y 86.7 Russet Burbank 381.7 Superior 884.0 Russet Norkotah* 90.0 Freedom Russet 526.1 Atlantic 897.2 NDTX4271-5R* 91.7 Superior 533.6 White Pearl 967.6 stems (Tables 5 and 6). Stem residue left behind during the clones. VW symptom expression increases with increasing harvesting operation will introduce inoculum into the soil, air temperatures (Nnodu and Harrison 1979). The mean contaminating it for future crops. Estimates indicate that a July temperature in 2006 was 25.3°C, while that in 2007 single infected stem can introduce up to 90,000 micro- was 28.3°C. Perhaps symptom expression is a good sclerotia into the soil (Slattery 1981). These propagules can indicator of resistance in years that favor pathogen growth. survive for ten years or more (Lacy and Horner 1966). A large effect of year on symptom expression has been Breeding programs that rely on symptom expression to previously reported (Frost et al. 2007; Hoyos et al. 1991; identify clones with VW resistance may inadvertently select Jansky and Rouse 2000). for tolerant clones along with resistant ones and build up It is interesting to note that the correlation between soil inoculum. Consequently, it is important to consider AUDPC and symptom expression on a single date (August both symptom expression and stem colonization when 7) was very strong. Consequently, a breeder could save identifying resistant clones. resources by scoring on a date in early August rather than The three measures of VW resistance in this study throughout the season. Jansky and Rouse (14) also found that evaluate different parameters of disease. Symptom expres- a single score date could be effective, as long as it was not too sion measures the effect of the pathogen on the host plant early in the season. A single score date early in August would phenotype. It may be confounded by maturity and other identify potentially resistant clones and still allow time to disease symptoms. Classic VW symptoms, such as unilat- collect sap from those clones for additional evaluation. eral wilting and chlorosis, were prevalent in 2007. Active Colony counts from sap presumably measure asexual growth of the fungus in plant stems and its consequent reproduction rate as conidia produced in the xylem of effects likely led to strong symptom expression. In contrast, growing plants are plated onto selective medium. The symptom expression was poor in 2006, leading to difficul- triggers for conidia production in potato sap are not well ties in distinguishing between resistant and susceptible understood, but air temperature and soil moisture levels are 510 Am. J. Pot Res (2009) 86:504–512

Table 6 Selection for Verticillium wilt resistance in 2007. 2007. First First clones clones wereexpression. selected if Then, symptoms the remaining on August clones 7 were were no selected greater thanif their those mean of sap the moderatelywere selected resistant if symptoms clone Ranger on August Russet. 7 were Early no maturing greater than clones those (<2.0 of on acount scale (cfu/100 of 1 = early, ul sap) 2 = was mid-season, less than 3 200. = late) Finally, clones the are remaining indicated clones by an asteriskthe moderately and were resistant not discarded clone Ranger based on Russet. symptom Early expression. maturing Then, clones the remainingwere selected clones if their were mean selected dried if stemtheir meancount sap(cfu/50 count mg (cfu/100 stem) was ul sap) less was(<2.0 less on than a scale 200. of Finally, 1 = early, theremaining 2 = mid-season, clones 3were = late) selected clones if their are meanthan dried 200. stemDiscarded count clones (cfu/50 are mg indicated stem) was with less a strikethrough. than 200. Discarded Clones clonesindicated are by indicated an asterisk with and a strikethrough. were not discarded Clones that based were on selected symptom in 2006that are were indicated selected by in a 2006 plus sign are indicated by a plus sign

Clone Aug 7 Clone Sap Clone Dry AOND95249-1 15.0 MSJ461-1 2.8 White Pearl 1.4 B2327-2 20.0 CO94035-15RU 6.2 Red Norland 2.1 CO94035-15RU 20.0 Russet Norkotah 20.0 Ranger Russet 7.7 W2683-2r 21.7 W2324-1 24.6 Superior 8.9 Dakota Diamond 21.7 W2133-1 45.3 W2683-2r 9.0 W2324-1 21.7 Atlantic 71.7 A95109-1 10.0 MSJ461-1 25.0 MegaChip 90.4 ATTX961014-1R/Y 11.4 Boulder 26.7 Dakota Diamond 119.3 Russet Burbank 14.0 CO95051-7W 26.7 B2327-2 121.5 Atlantic 14.2 MegaChip 26.7 CO95051-7W 126.9 + CO95051-7W 14.7 Ranger Russet 31.7 Red Norland 131.3 + MegaChip 15.2 Freedom Russet 35.0 Ranger Russet 131.7 + AOND95249-1 15.8 W2133-1 38.3 AOND95249-1 149.5 + MSJ461-1 18.5 ND5002-3 51.7 Superior 155.1 + CO94035-15RU 24.2 ND7818-1* 60.0 NDTX4271-5R 159.8 B2327-2 25.7 Atlantic 61.7 Freedom Russet 197.3 ND5002-3 31.7 W2310-3 66.7 W2683-2r 242.0 + W2133-1 35.4 White Pearl 73.3 A95109-1 291.9 Freedom Russet 37.0 Russet Burbank 75.0 W2310-3 299.9 ND7818-1 37.8 A95109-1* 76.7 ND5002-3 325.7 W2310-3 39.9 ATTX961014-1R/Y 76.7 Boulder 365.8 Dakota Diamond 45.1 NDTX4271-5R* 93.3 ATTX961014-1R/Y 385.9 NDTX4271-5R 65.0 Superior* 95.0 Russet Burbank 403.9 W2324-1 89.0 Red Norland* 98.3 ND7818-1 869.8 Boulder 104.7 Russet Norkotah* 100.0 White Pearl 949.7 Russet Norkotah 208.3

known to influence disease severity (Rowe and Powelson in the host-pathogen interaction. Therefore, both parameters 2002). As with disease symptom data, higher sap counts should be considered when evaluating VW resistance. were recorded in 2007, which was warmer in July than The clone by year interaction for both RAUDPC and 2006. Hoyos et al. (1991) also reported a positive symptom expression on August 7 was significant (Table 2). correlation between colony counts from sap and mean Some clones exhibited a much larger increase in symptom summer field temperature. However, differences in soil expression in 2007 than in 2006. The magnitude of the inoculum levels and sample dates across years may also increase was greater in more susceptible clones such as have contributed to the higher sap counts in 2007. Russet Norkotah, Red Norland, and Superior. Perhaps these The dried stem colony counts in this study measured the clones were particularly vulnerable to the physiological production of resting structures (microsclerotia) by the effects of the fungus when it was present in large quantities pathogen during host senescence at the end of the growing in stems. The clone by year interaction for sap colony counts season. This method has been previously used to measure was large and significant (Table 2). Again, some clones (e.g. stem colonization differences among cultivars (Slattery White Pearl, ND7818-1 and ATTX961014-R/Y) exhibited a 1981). As with conidial production, the host plant con- much greater increase in colony counts in 2007 than other ditions that support the production of microsclerotia in clones. In addition, some clones (e.g. W2324-1 and Freedom dying potato stems are not clearly understood. The lack of Russet) had lower sap colony counts in 2007 than in 2006. A correlation between sap and dried stem counts has been large clone by year interaction was also observed for dried previously reported (Jansky et al. 2004; Jansky and Rouse stem colony counts (Table 2). There was a dramatic decrease 2002). Although both measures of resistance quantify stem in 2007 in some clones (W2324-1, Superior, Atlantic, colonization by the pathogen, they evaluate different stages ND7818-1, and White Pearl). Am. J. Pot Res (2009) 86:504–512 511

Even though symptom expression was more pronounced study, identical results would have been obtained if clones in 2006 than in 2007, there was a strong correlation were discarded because two or more plates (stems) between the two years for this trait (Table 4). Similarly, contained more than 500 colonies. Jansky and Rouse (2000) found significant correlations Eventually it may be possible to use molecular markers between years for VW symptom scores. In contrast, mean to more easily identify resistant clones. Such a marker has colony counts from sap and dried stems were not correlated recently been developed (Bae et al. 2008). However, it may across years (Table 4), which is consistent with previous not be broadly applicable in diverse breeding populations. work (Jansky and Rouse 2000). Hoyos et al. (1991) found Until markers become widely used, it will be necessary to that, although mean sap counts varied by year, clones continue to evaluate host-pathogen interactions in order to generally remained in the same resistance category. Simi- identify clones with resistance to VW. larly, a previous study reported consistent rankings among cultivars between years based on dried stem data (Slattery Acknowledgements The contributions of clones and ideas from the 1981). The inconsistencies in stem colonization data among following potato breeders are gratefully acknowledged: Asunta Thompson, David Holm, David Douches, Felix Navarro, Jiwan Palta, years highlight a need to understand the effect of J. Creighton Miller, Jr., Kathleen Haynes, and Richard Novy. environment on pathogen reproduction in stem tissue. This research was funded in part by the Wisconsin Potato and The only significant correlation between symptom Vegetable Growers Association and USDA-ARS Project No. 3655- expression and colony forming units was observed in dried 21000-035-00D. Statistical assistance from Nicholas Keuler is appreciated. stems in 2006, when large differences in dried stem colony scores were detected. A lack of correlation between symptom expression and dried stem colony counts has been previously reported (Jansky and Rouse 2000), but is in References contrast to early work on this method of resistance evaluation (Davis et al. 1983; Mohan et al. 1990). There Akeley, R.V., F.J. Stevenson, D. Folson, and R. Bonde. 1956. was no significant correlation between symptom expression Breeding varieties of potato resistant to Verticillium wilt in and sap means. The lack of correlation is similar to Maine. American Potato Journal 33: 15–21. previous reports (Frost et al. 2007; Jansky and Rouse Atallah, Z.K., J. Bae, S.H. Jansky, D.I. Rouse, and W.R. Stevenson. 2007. Multiplex real-time quantitative PCR to detect and 2000; Lynch et al. 1997), but it is in contrast to the results quantify Verticillium dahliae colonization in potato lines that of Hoyos et al. (1991). differ in response to Verticillium wilt. Phytopathology 97: 865– All three measures of resistance (symptom expression, 872. levels of conidia in sap, and levels of microsclerotia in Bae, J., D. Halterman, and S. Jansky. 2008. Development of a molecular marker associated with Verticillium wilt resistance in dried stems) provide important contributions to the under- diploid interspecific potato hybrids. Molecular Breeding 22: 61– standing of a clone’s response to V. dahliae. However, it is 69. cost prohibitive to evaluate all measures in all clones of Binning, L.K., C.M. Boerboom, L.G. Bundy, K.A. Delahaut, H.C. interest in the large populations generated in a breeding Harrison, K.A. Kelling, D.L. Mahr, S.E.R. Mahr, B.A. Michaelis, W.R. Stevenson, J.L. Wedberg, and J.A. Wyman. 2002. Madison, program. Consequently, a three-tiered approach was eval- WI: Commercial Vegetable Production in Wisconsin. Coopera- uated for its ability to identify resistant clones. First, clones tive Extension Publishing. were retained if they were early maturing or if they were at Bradshaw, J., G. Bryan, and G. Ramsay. 2006. Genetic resources least as resistant as Ranger Russet based on symptom (Including wild and cultivated Solanum species) and progress in their utilisation in potato breeding. Potato Research 49: 49– scores on a single date (August 7). Then, any remaining 65. clones were discarded if their mean sap colony count score Butterfield, E.J., and J.E. DeVay. 1977. Reassessment of soil assays was greater than 200 cfu/100 ul sap. Finally, clones for Verticillium dahliae. Phytopathology 67: 1073–1078. remaining after the first two steps were discarded if their Concibido, V.C., G.A. Secor, and S.H. Jansky. 1994. Evaluation of resistance to Verticillium wilt in diploid, wild potato interspecific mean dried stem score was greater than 200 cfu/50 mg hybrids. Euphytica 76: 145–152. dried stem material. This method reduces the number of Corsini, D.L., J.J. Pavek, and J.R. Davis. 1988. Verticillium wilt samples processed for costly sap and dried stem assays, resistance in noncultivated tuber-bearing Solanum species. Plant while consistently identifying VW resistant clones. Disease 72: 148–151. Dan, H., S.T. Ali-Khan, and J. Robb. 2001. Use of quantitative PCR Because of the large variability among and within plants diagnostics to identify tolerance and resistance to Verticillium for stem colonization scores, it is important to sample a dahliae in potato. Plant Disease 85: 700–705. large number of stems. This study evaluated 12 stems per Davis, J.R., J.J. Pavek, and D.L. Corsini. 1983. A sensitive method for clone in each year. Counting and recording colony numbers quantifying Verticillium dahliae colonization in plant tissue and evaluating resistance among potato genotypes. Phytopathology on each plate is time-consuming. An alternative strategy 73: 1009–1014. could be to simply look for plates with high colony Frost, K.E., D.I. Rouse, and S.H. Jansky. 2007. Considerations for numbers, without actually counting colonies. In the present Verticillium wilt resistance in potato. Plant Disease 91: 360–367. 512 Am. J. Pot Res (2009) 86:504–512

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