Aggressiveness of Streptomyces on Four Potato Cultivarsand Implications for Common Scab Resistance Breeding

Leslie A. Wanner & Kathleen G. Haynes

Published online: 19 May 2009# Potato Association of America 2009

Abstract Common scab (CS), caused by several species ofStreptomyces, is a serious problem for potato growers.Although the mechanism of pathogenicity, based on thephytotoxin thaxtomin, is presumably conserved in allpathogenic species, Streptomyces isolates vary in aggres-siveness, and regional patterns in Streptomyces speciesdistribution have recently emerged. We demonstrate thatStreptomyces isolates differ significantly in aggressiveness,and there are specific plant genotype-pathogen isolateinteractions in four contrasting potato cultivars treated withStreptomyces isolates belonging to different species andmolecular types. There were significant differences betweenexperiments, among isolates, among cultivars, and some ofthe two-way interactions were significant. The sum of allmain and interaction effects among plant, pathogen, andexperiment accounted for only 50–55% of total variation inCS disease symptoms. More information on specific plant-pathogen interactions combined with knowledge of thedistribution of CS-causing species could form the basis forsuccessful recommendations of suitable potato cultivars,and will contribute to more accurate and reproduciblephenotyping required for genetic studies of CS resistance inpotato.

Resumen La roña común (CS), causada por varias espe-cies de Streptomyces, es un problema grave para loscultivadores de papa. Aunque el mecanismo de patogeni-

cidad, basado en la fitotoxina taxtomina, se conservapresumiblemente en todas las especies patógenas, losaislamientos de Streptomyces varían en agresividad, y hansurgido recientemente patrones regionales en la distribuciónde las especies de Streptomyces. Demostramos que losaislamientos de Streptomyces difieren en agresividad deforma significativa, y hay interacciones especificas entregenotipo de planta-aislamiento del patógeno en cuatrodiferentes cultivares de papa tratadas con aislamientos deStreptomyces pertenecientes a diferentes especies y tiposmoleculares. Hubo diferencias significativas entre exper-imentos, entre aislamientos, entre cultivares, y algunas delas interacciones de dos vías fueron significativas. La sumade todos los efectos principales y de interacción entreplantas, patógenos, y experimentos representaron sólo el50–55% del total de la variación en los síntomas de laenfermedad CS. Más información sobre las interaccionesplanta-patógeno especificas combinado con el conoci-miento de la distribución de las especies que causan CSpodría constituir la base para recomendaciones exitosas decultivares adecuados de papa, y contribuirá a la más exactay reproducible fenotipificación necesaria para los estudiosgenéticos de resistencia a CS en la papa.

Keywords Plant-pathogen-disease triangle .

Resistance breeding

AbbreviationsCS common scabMA mean areatAI transformed mean areaML mean lesionPAI pathogenicity island

Am. J. Pot Res (2009) 86:335–346DOI 10.1007/s12230-009-9088-9

L. A. Wanner (*) :K. G. HaynesUnited States Dept. of Agriculture, Agricultural Research Service,Genetic Improvement of Fruits and Vegetables Laboratory,10300 Baltimore Ave.,Beltsville, MD 20705, USAe-mail: Leslie.Wanner@ars.usda.gov

Introduction

Common scab, caused by soil-inhabiting Streptomycesspecies, is a serious problem for potato growers worldwide.Losses to common scab (CS) in Canada are probablytypical; 82% of growers reported CS, and average losseswere estimated at $90 to $102 (CDN) per hectare (Hill andLazarovits 2005). Costs of common scab to the potatoindustry in Tasmania are estimated at 4% of total crop value(Wilson 2004). Although CS has been described in theliterature for more than one hundred years, effectivecontrols are still lacking. Like many plant diseases, themost desirable method of control would be disease-resistantplant material. There is a relatively wide variation in potatocultivar susceptibility, but most commercially successfulcultivars are highly CS-susceptible, showing a range ofsymptoms from superficial dark-colored scaly lesions todiscrete or coalescing raised, wart-like lesions or sunkenpits. Symptom variation is partly determined by the potatocultivar, with more susceptible cultivars typically display-ing warty or pitted lesions. Progress in breeding for CSresistance is hampered by limitations in resistance screen-ing. The only way of determining CS resistance has beenyears of field trials, with variable results from year to yearand place to place. Regional and season-to-season differ-ences in both disease severity (disease pressure) andrankings of potato cultivar susceptibility to CS have beenreported in North America (Haynes et al. 1997; Wanner etal. 2006).

CS is caused by soil-inhabiting bacteria in the genusStreptomyces. Streptomycetes are abundant in soils, thoughonly a small percentage of these, carrying genes forbiosynthesis of a phytotoxin thaxtomin, cause CS (Loriaet al. 2006). Genes for biosynthesis of thaxtomin are carriedon a distinct chromosomal region with the hallmarks of apathogenicity island (PAI) that can be transferred horizon-tally among streptomycetes (Loria et al. 2006). CS-causingStreptomyces isolates vary in aggressiveness (Bramwell etal. 1998; McKee 1958; Mygind 1962; Pasco et al. 2005;Wanner 2004). Since the mechanism of pathogenicity basedon thaxtomin seems to be conserved, the biological basisfor variation in aggressiveness is assumed to be differencesin the production of thaxtomin, and genetically distinctStreptomyces isolates differing in thaxtomin production invitro have been described (Beausejour et al. 1999; Kinkel etal. 1998; Loria et al. 1995). The taxonomy of plant patho-genic streptomycetes has become more complicated by therecognition that the species Streptomyces scabies is actuallya small group of different species (Bouchek-Mechiche et al.2000a; Bukhalid et al. 2002; Kreuze et al. 1999; Lehtonenet al. 2004; Loria et al. 2006; Wanner 2006). Wanner (2006)reported finding four Streptomyces species or strains thatcause CS in the northern USA S. scabies was apparently

most common throughout much of the region, but two otherpathogenic species or strains, S. stelliscabiei and temporaryspecies/strain IdahoX (Wanner 2007), were characteristic ofspecific regions or field locations. S. acidiscabies, a speciestolerant of acidic soil conditions, has also been describedfrom the northeastern US and Canada (Faucher et al. 1992;Lambert and Loria 1989; Lambert et al. 2006). At leastthree, and perhaps as many as 10, CS-causing Streptomycesspecies or strains have now been delineated in NorthAmerica (Goyer et al. 1996; St-Onge et al. 2008; Wanner2009). All of these species or strains cause a similarspectrum of CS symptoms on potatoes. Though a growingbody of evidence indicates that different Streptomycesspecies or strains are characteristic of geographic areas(St-Onge et al. 2008; Wanner 2006; Wanner 2009),Streptomyces pathogenicity groups specific to potatocultivars have not been recognized in North America.

The CS situation in Europe is apparently more complex.There seem to be two partially overlapping potato scabdiseases caused by plant pathogenic Streptomyces species(Bouchek-Mechiche et al. 2006; Bouchek-Mechiche et al.2000b; Pasco et al. 2005). CS affects tubers (andunderground stems) on all potato cultivars to differingdegrees, and is caused primarily by S. europaeiscabieiin northern Europe (Bouchek-Mechiche et al. 2000a;Flores-Gonzalez et al. 2008) or S. scabies and S. turgid-iscabies in Scandinavia (Kreuze et al. 1999). A seconddisease, netted scab, affects tubers only superficially, butdamages roots, thereby affecting yields. Netted scab islimited to very few potato cultivars, and is caused by S.reticuliscabiei or S. europaeiscabiei (Bouchek-Mechiche etal. 2000b; Pasco et al. 2005). In a third variation, somestrains of S. europaeiscabiei can cause either common ornetted scab, depending on soil temperature (Bouchek-Mechiche et al. 2000b; Pasco et al. 2005). The netted scabpathogen S. reticuliscabiei is genetically very closelyrelated to another species causing CS, S. turgidiscabies(Bouchek-Mechiche et al. 2006). A netted scab-like diseasereferred to as russet scab has also been described in NorthAmerica (Faucher et al. 1993; Harrison 1962).

Our objective in this study was to compare CS diseasephenotypes on four potato cultivars in response to genet-ically different Streptomyces isolates. Streptomyces isolateswere chosen to represent several plant pathogenic speciesfound in North America. Two of the potato genotypeschosen, Superior and Pike, are considered to be relativelyresistant to CS, while Chippewa and Green Mountain arehighly susceptible. The lineages of these four potatocultivars are quite distinct. Information on the responsesof individual potato cultivars to Streptomyces species andstrains combined with an understanding of prevalentpathogen populations in North America is required toanalyze the genetics of plant resistance to Streptomyces,

336 Am. J. Pot Res (2009) 86:335–346

and could be useful in understanding the CS diseasepotential in different regions.

Materials and Methods

Origins, Subculture, Storage of Streptomyces Isolates

Streptomyces isolates were cultured directly from scabbylesions on potato tissue as described (Wanner 2006; Wanner2007) and maintained at −80°C as spore suspensions in20% glycerol or/and plugs cut from YME agar plates ofconfluent sporulating cultures (Wanner 2006). Isolates arenamed according to the state or province from which theycame and the harvest year of the scabby potatoes (first twoletters and two numbers, respectively). Details on theorigins of isolates are given in Table 1.

Molecular Characterization of Isolates by PCR

DNA extraction and PCR characterization of variableregions of the 16s ribosomal RNA genes (used to assignStreptomyces species) and presence or absence of genes inthe Streptomyces pathogenicity island was carried out asdescribed (Wanner 2009).

Anchored PCR was carried out using the primer TRR-s(5’-ACTGTCGGATGCGGTCAT-3’), a derivative of aprimer designed to an open reading frame with homologyto transposases, located adjacent to the nec1 gene in S.scabies and S. turgidiscabies (Bukhalid et al. 1998),coupled with random primers Sac15A (5’- CGCCGAGCTCTGCG-3’) and Sph15A (5’-CACCGCATGCGCAC-3’).

Fifty microliter PCR reactions contained 1X Green GoTaqFlexi buffer (Promega Corp., Madison, WI), 2.0 mMMgCl2,200 µM each dNTP, 25 pmol each primer, 1.25 U GoTaqDNA polymerase (Promega Corp.), approximately 5 ngDNA template (1 µl of a 1:9 dilution) and MilliQ water.Amplification was carried out in a Perkin-Elmer ThermalCycler 480 programmed for one initial denaturation step at95°C for 5 min, followed by 40 cycles of denaturing at 95°Cfor 20 s, annealing at 46°C for 30 s and extension at 72°Cfor 2 min, and ending with a 4°C hold.

BOX-PCR was carried out using the BOX1AR primer(Clark et al. 1998) in a 50 µl PCR reaction containing 1XGreen GoTaq Flexi buffer (Promega Corp., Madison, WI),4.0 mM MgCl2, 300 µM each dNTP, 25 pmol primerBOX1AR, 1.25 U GoTaq DNA polymerase (PromegaCorp.), approx. 5 ng DNA template and MilliQ water.Amplification was carried out in a Perkin-Elmer ThermalCycler 480 programmed for initial denaturation at 95°Cfor 5 min, followed by 40 cycles of denaturing at 95°C for30 s, annealing at 53°C for 40 s and extension at 72°C for3 min, a final extension at 72°C for 7 min and ending with a4°C hold. All PCR amplification experiments were repeatedat least twice for each DNA template.

Potato Cultivars and Sources

Potato cultivar ‘Chippewa’ was originally obtained ascertified seed from Jewel Brothers (Monticello, ME), andmaintained in the greenhouse at USDA-ARS in Beltsville,MD. ‘Green Mountain’ and ‘Superior’ were obtained ascertified seed from Bradstreet Family Farm (Bridgewater,ME) and ‘Pike’ was obtained as certified seed from Dan

Table 1 Characteristics of Streptomyces isolates compared in this study

Species and isolate #a Isolate name originb Melc PAI Genotype d

S. europaeiscabiei

1 ID-01-16c ID, 2001, cv. not noted – TNT

2 ID-02-12 eastern WA, 2001, cv. Desiree + Tnt

S. sp. Idaho X

3 ID-01-6.2a American Falls, ID 2001 cv. Shepody + TnT

4 ID-03-3A Aberdeen, ID, 2003 cv. Ranger Russet + Tnt

5 ID-05-11E Aberdeen, ID, 2005 cv. Russet Burbank + TnT

S. stelliscabiei

6 NY-02-3a Freeville, NY, 2003; cv. not noted + TNT

a Isolate numbers are the same as those used on gel lanes in Fig. 1b place, year, potato cv. of originc mel, production of melanin when grown on peptone-yeast extract-iron agard presence (upper case) or absence (lower case) of genes characteristic of the Streptomyces scabies pathogenicity island (PAI), as determined usingthe methods of Wanner (2009, in press) First letter, presence of the txtAB operon; second letter, presence or absence of the nec1 gene; third letter,presence or absence of the tomA gene

Am. J. Pot Res (2009) 86:335–346 337

Corey, Monticello ME. Potato cultivars utilized represent arange of genetic heritage in US cultivars. Chippewa is around, white, smooth-skinned chipping cultivar, relativelyearly in maturity. It is highly susceptible to CS, typicallydeveloping reddish-brown or blue-black inky deep pits.Chippewa is not widely grown presently, but is related toKatahdin, a cultivar grown widely in the northeastern US.Green Mountain is a late maturing white, netted skinnedcultivar, introduced in 1885. It is still valued as a specialtycultivar in Maine, but is highly susceptible to CS, typicallydeveloping corky pits. Superior is a medium maturity,lightly netted cultivar introduced from Wisconsin in 1961,and is relatively CS-resistant, rarely developing pits. It isstill grown widely in Maine, Wisconsin, Minnesota, andNew York. Pike is a recently introduced CS-resistant white,netted skinned cultivar from New York. Katahdin was agreat-grandparent of Pike. Pike is widely cultivated inIdaho, Maine, Michigan, Nebraska, New York, Washing-ton, and Wisconsin as a chipping cultivar.

Culture of Streptomyces Isolates for Soil Inoculation

Original −80°C stored cultures of Streptomyces isolateswere plated on YME agar, and grown at 28°C for 2–3 weeks until uniformly sporulating. Spores were harvestedby gently scraping water-flooded plates. Harvested sporeswere counted using a microscope and hemocytometer slide,and equal numbers were used to directly inoculate sterilevermiculite in plastic bags. Vermiculite cultures were grownfor 14–18 days and then sampled to estimate CFU per cm−3

vermiculite.

Pathogenicity/Aggressiveness Assays on Potato Cultivars

For assays of Streptomyces pathogenicity and aggressive-ness on potato, 500 cm−3 un-infested 1:1 sand:soil-lesspotting mix (ProMix PGX, produced by Premier Horticul-ture, Ltd. Dorval, Quebec) was placed in the bottom of15 cm diameter pots, and then 1,000 cm−3 of sand: pottingmix (1:1) containing 1010 CFU of Streptomyces vermiculiteculture was added (final pathogen concentration, 6.6×106

CFU/ cm−3 soil mixture). A single uniform mixture of sand:soil:inoculum was made for each Streptomyces isolate anddistributed into pots. Potato tubers were surface-disinfestedin 1.5% commercial bleach (0.09% sodium hypochlorite)containing a few drops of commercial dishwashing liquidfor 2 minutes and rinsed thoroughly in reverse-osmosispurified tap water before cutting into ca. 28 g seed piecesand planting in pots containing prepared mixture ofStreptomyces isolate. Three seed pieces were planted foreach cultivar for each isolate in a complete randomizeddesign. Pots were distributed into three growth chambers(one pot of each treatment per chamber). Plants were grown

under controlled conditions of 24°C constant temperature,14 h photoperiod with 200–220 µmol-m2-s−1 light intensityfrom Phillips Natural Daylight fluorescent bulbs. Plantswere watered when the soil became dry, and fertilizedevery three weeks with1.5 cm−3 Vigoro All Purpose PlantFood 12-5-7 (Spectrum Group Division of United Indus-tries Corporation, St. Louis, MO). The experiment wasconducted twice. Pots were harvested individually after3 ½ months, when plants were beginning to naturallysenesce.

Disease Scoring

CS was rated as two components. Tubers were ratedindividually for the amount of surface area covered on apercentage basis (visual estimate). For each pot, the meanpercentage area covered (MA) was calculated from alltubers larger than 0.75 cm in diameter.

The second measurement was the type of lesion present(lesion severity). The scoring scheme used was:

0.25–0.5 = Rough scaly or cracked surface, dark incolor and distinct from russeting1 = Discrete superficial lesions of small size (<0.5 cm indiameter)2 = Coalescing superficial lesions (>0.5 cm in diameter)3 = Discrete raised (warty-looking) lesions (<0.5 cm indiameter)4 = Coalescing raised (warty-looking) lesions (>0.5 cmin diameter)5 = Pitted lesions of any size or depth.

Intermediate values were assigned when most lesionswere of one type, but a second lesion type was also found.For each pot, a mean lesion severity (ML) was calculatedfrom all tubers larger than 0.75 cm in diameter.

Data Analysis

MA and ML from the two experiments were analyzedseparately by the general linear models procedure in SASversion 9.1 (SAS Institute, Cary, NC). Since the errorvariances were homogeneous, the data were combined overexperiments and similarly analyzed. Residuals from MLand the square root transformation of MA (tMA) werenormally distributed between and across experiments. Leastsquares means for isolate, cultivar, and individual cultivar-isolate pairs were compared using the pdiff option.Correlations between tMA and ML were calculated overexperiments and for clones over experiments using thecorrelations procedure in SAS. Sums of squares from thesources of variation were used to determine the contributionmade by each component to the host plant-disease-experiment disease triangle.

338 Am. J. Pot Res (2009) 86:335–346

Results

Streptomyces Isolates Used for Comparison

We compared six Streptomyces isolates representing threeStreptomyces species (all common in North America) and atleast five genotypes. Origins and molecular characteristicsfor the six Streptomyces isolates are presented in Table 1and Fig. 1. All six isolates had genes encoding biosynthesisof the pathogenicity determinant thaxtomin, but three

isolates were missing the PAI marker gene nec1 and onewas missing both the nec1 and tomA PAI marker genes. Thetwo isolates of S. europaeiscabiei are different genetic typesor strains, as they differ in PAI markers (Table 1), repetitivesequence element (BOX) PCR patterns (Fig. 1a) andbanding patterns obtained by a RAPD method using oneprimer anchored in a transposase sequence (Fig. 1b and c).The three Idaho X (IdX) isolates also fell into at least twogroups based on repetitive sequence (BOX and transposon-anchored) PCR patterns (Table 1 and Fig. 1). Virulence andaggressiveness of all of the isolates except ID05-11E hadpreviously been examined in radish, where all werepathogenic (Wanner 2004) and highly aggressive.

Disease Severity Measurements

Aggressiveness of isolates on potato cultivars was mea-sured as two components. The first component was themean area (MA), which was a simple percentage of surfacearea showing CS symptoms at potato tuber maturity. Thesecond component was the mean lesion rating (ML). Smallsuperficial lesions were ranked as least serious, raisedlesions as more serious and pitted lesions as most serious.These two measures of CS severity were positivelycorrelated overall (r=0.30, P=0.0005), and in each exper-iment (0.27 < r <0.29). However, the correlations betweenthe two measures differed greatly depending on the level ofsusceptibility in the cultivars. In the two highly susceptiblecultivars, correlations between these two measures of CSseverity were highly significant (Chippewa, r=0.78, P<0.0001; Green Mountain, r=0.53, P=0.004). In the resistantcultivars, there was no correlation between these two mea-sures (Pike, r=0.02, P=0.90; Superior, r=−0.05, P=0.78).

Despite standardization of the initial Streptomycesinoculum density and growth conditions within andbetween experiments, there were significant differencesbetween the two experiments for tMA and ML (Table 2).Both tMA and ML were significantly greater in experimentA than in experiment B. In experiment A 15 pots (out of72) failed to produce any tubers, whereas in the secondreplicate (experiment B) tubers were produced in all thepots. Variance in symptoms within and between pots in atreatment was also high (Figs. 2a, 3a). This variable diseasephenotype is reminiscent of the variability seen from fieldto field and year to year in field testing.

There were significant differences in mean aggressive-ness among isolates (Tables 2, 3, Fig. 3). The mostaggressive isolate in terms of area covered (tMA) wasNY02-3A, while NY02-3A and ID01-6.2 were the mostaggressive isolates as measured by lesion severity (ML).The two S. europaeiscabiei isolates ID02-12, ID01-16c,and an IdX isolate ID05-11E were the least aggressiveisolates, as measured by either tMA or ML, on all potato

M1 1 4 2 3 M25 6

europ Id ste

BOX1AR

TRR-Sac

TRR-Sph

Fig. 1 Repetitive element PCR profiles differ in Streptomyces isolatesused in this study. a BOX1AR primer. b TRR/Sph15A primer pair. cTRR/Sac15A primer pair. Lane M1, Promega pGEM marker; fromtop: 2,645, 1,605, 1,198, 676, 517, 460, 396, 350 bp bands visible;lanes 1-2, S. europaeiscabiei isolates: lane1, ID01-16c; lane 2, ID02-12; lanes 3-5, S. species Idaho X isolates: lane 3, ID01-6.2a, lane 4,ID03-3A: lane 5, ID05-11E; lane 6, S. stelliscabiei isolate NY02-3A;Lane M2, Promega PCR markers; from top, 1,000, 750, 500, and300 bp bands are visible

Am. J. Pot Res (2009) 86:335–346 339

cultivars. These results contrast to results in radish whereID01-6.2A was most aggressive and NY02-3A was leastaggressive (Wanner 2004).

As expected, there were significant differences amongcultivars (Tables 2, 4). However, unexpectedly, in terms ofarea covered, tMAwas greater in the resistant cultivars thanin the susceptible cultivars. As expected, though, lesionseverity (ML) was greater in the susceptible cultivars thanin resistant cultivars (Fig. 3, Table 4).

The isolate x cultivar interaction was significant for ML,but not for tMA (Table 2). However, the experiment xisolate interaction was significant for tMA, but not for ML.Although the initial soil inoculum density was adjusted tobe the same in both experiments for all isolates, thesesignificant differences are a reflection of some isolate-specific difference in disease pressure between experimentsamong some cultivars (Figs. 2, 3). The three wayinteraction involving experiment, isolate, and cultivar wasnot significant for either tMA or ML.

Differences in Isolate Aggressiveness on the Four PotatoCultivars

Streptomyces isolates varied in mean aggressiveness onpotato cultivars (Table 3); we were also interested inwhether the response of potato cultivars to individualisolates varied. Aggressiveness of each isolate on eachcultivar is shown in Fig. 2a (tMA) and b (ML). In general,the range in tMA between treatments was narrow, withmean surface area affected by CS symptoms rangingbetween 5% and 20% (Fig. 2a). Isolates ID01-16c (onSuperior vs. Green Mountain) and isolate ID05-11E (onChippewa vs. Superior) were the only isolate-cultivar pairsshowing significant differences in tMA (Fig. 2c). Incontrast, there was a wide spread in types of CS lesions(ML) observed in response to different isolates, with mostisolates capable of producing all types of CS lesions onmost potato cultivars (Fig. 2b). Notable exceptions to this

were that no pitted lesions were observed on cultivars Pike andSuperior in response to isolates ID01-16c, on cultivars GreenMountain, Pike and Superior in response to isolate ID02-12,or on cultivars Green Mountain and Superior in response toisolate ID03-3A, while pitted lesions were the typical responseof cultivar Chippewa to isolate NY02-3A (Fig. 2b). Even withthe wide variation in symptoms indicated in large interquar-tile ranges (Fig. 2), comparisons of least squares meansrevealed some significant specific differences in cultivarresponse to individual isolates (Fig. 2c). Notably, significantdifferences in ML were seen between cultivars Pike andChippewa in response to isolate ID01-6.2A and betweenChippewa and Green Mountain vs. Pike and Superior inresponse to isolate NY02-3A (Fig. 2c).

Differences in Cultivar Response to Individual IsolatesMean lesion types seen on potato cultivars in response toindividual Streptomyces isolates are displayed in Fig. 3a.Variability in response to different isolates is apparent amongcultivars. For example, while isolates NY02-3A and ID01-6.2A were most severe on Chippewa and ID03-3A was leastsevere, isolate ID03-3A was most severe on cultivar Pike,and isolate NY02-3A was one of the least severe isolates onthis cultivar. Responses to nearly all individual isolates weredistinct on the most susceptible cultivar Chippewa (Fig. 3b).Only in cultivar Superior was there no difference in responseto any isolates significant at P<0.05 (Fig. 3b).

Discussion

The mechanism of pathogenicity, based on the phytotoxinthaxtomin, is presumably conserved in all plant pathogenicStreptomyces, but there are well-documented and consistentregional differences in CS severity in North America(Haynes et al. 1997; Haynes et al. 2006; Hill and Lazarovits2005), and year-to-year variation in CS severity is alsoregularly noted. It has long been recognized that potato

Source Mean squares

Experiment A Experiment B Both experiments

d.f tMAa MLa d.f. tMA ML d.f. tMA ML

Experiment 1 11.92*a 8.73**

Pathogen isolate 5 4.16 1.69 5 10.35** 5.97** 5 7.15* 5.21**

Potato cultivar 3 2.96 1.49 3 13.54** 4.52** 3 9.17* 4.75*

Isolate x cultivar 15 2.27 1.26 15 3.73 2.63** 15 2.95 2.34*

Experiment x isolate 5 6.77* 1.69

Experiment x cultivar 3 6.28 0.73

Expt x isolate x cultivar 15 3.30 1.59

Error 31 2.39 1.63 48 3.00 0.93 79 2.76 1.21

Table 2 Analysis of varianceon the square root of the meanarea (tMA) affected by commonscab and the mean common scablesion type (ML) in replicatedexperiments and over bothexperiments

a tMA, square-root transformedmean percentage area affectedby common scab; ML, meancommon scab lesion type asdescribed in Materials andMethods

* P<0.05; ** P<0.01

340 Am. J. Pot Res (2009) 86:335–346

cultivars differ in CS susceptibility (Caligari and Wastie1985; Haynes et al. 1997; McKee 1958). Recently, it wasrecognized that Streptomyces causing CS in North Americabelong to several species, and some evidence suggests thatcertain species and/or molecular types are characteristic ofparticular regions (Faucher et al. 1992; St-Onge et al. 2008;Wanner 2006; Wanner 2009) Although the existence andsignificance of physiological specialization between Strep-tomyces species/isolates and potato cultivars has beencontroversial (Bjor and Roer 1980; Lambert et al. 2006;McKee 1958), in Europe it has clearly been shown that twodifferent diseases, common scab and netted scab, are

caused by Streptomyces species, and that potato cultivarsrespond differentially to individual pathogenic species/strains (Bouchek-Mechiche et al. 2000b; Pasco et al.2005). We therefore thought that closer scrutiny of specificplant-pathogen interactions was warranted. Using sixisolates representing some of the Streptomyces species andmolecular genetic groups found in the USA, we obtainedevidence for significant differences in susceptibility to CSamong four potato cultivars in growth chamber experi-ments. Specific interactions between individual Streptomy-ces isolates and individual potato cultivars were found forthe tuber surface area affected by CS lesions (tMA) and for

0

2

4

6

8

10

ID01-16c ID02- 12 ID01-6.2A ID03- 3A ID05-11E NY02-3A

C G P S . C G P S . C G P S . C G P S . C G P S . C G P S

29 17 12 22 . 26 16 17 13 . 36 17 13 7 . 38 12 17 17 . 36 21 18 17 . 33 17 16 21

tMA

min median mean max

0

1

2

3

4

5

ID01-16c ID02- 12 ID01-6.2a ID03- 3A ID05-11E NY02-3A

C G P S . C G P S . C G P S . C G P S . C G P S . C G P S

ML (le

sio

n type)

min median mean max

a

b

Fig. 2 Differences in aggressiveness of 6 Streptomyces isolates onfour potato cultivars, arranged by isolate. a Box plots showing square-root transformed tuber surface area (tMA) affected by common scab. bBox plots showing differences in type of scab lesion (ML) rated asdescribed in the Material and Methods. Inter-quartile range, mean,median, and maxima and minima are shown. Potato cultivars areindicated as letters: C, Chippewa; G, Green Mountain; P, Pike; S,

Superior. Numbers beneath cultivars in A are total numbers of tubersscored. c Matrix showing the probability of significant differences inmean response of potato cultivars to each of the Streptomyces isolatesstudied. Combinations with P<0.05 are highlighted. In all six tables,differences in lesion type (ML) are displayed above the diagonal anddifferences in surface area affected (tMA) are displayed below thediagonal

Am. J. Pot Res (2009) 86:335–346 341

the types of CS lesions (ML). Our results demonstrate thatdifferences in Streptomyces strain or species prevalencecould contribute to regional differences in CS diseaseseverity.

To clarify the genetics behind CS resistance, it isimportant to have a robust and reproducible phenotypeassay. Phenotyping CS resistance is difficult because CSsymptoms are so variable, typically ranging from superfi-cial cracks and scales to raised warty lesions to shallow ordeep pits. Frequently, more than one of these lesion typesoccurs on a single tuber. Lesions may be discrete or theymay coalesce to cover large areas of the tuber surface, andthe proportion of the tuber surface area affected by lesionsvaries. Symptoms are highly variable on tubers within andbetween replicate experiments, as is illustrated in Figs. 2and 3. Often, the within-pot symptom variation equals thebetween-pot and between-replicate experiment variation.The biological reasons for this large variation in symptomsare not clear, but it complicates data analysis. We haveattempted to reduce the variation in symptoms by usingsingle, well-characterized Streptomyces isolates, carefullycontrolling inoculum density, and conducting experimentsin controlled growth chamber environments. Disappoint-ingly, carefully controlled conditions did not reduce thesymptom variation within or between experiments.

Some investigators have assessed CS simply based onincidence of any symptoms, or on tuber surface areaaffected by symptoms (Caligari and Wastie 1985; Goyer

et al. 1998; Kinkel et al. 1998), although the variability inCS symptoms has inspired most investigators to assess boththe type of lesion and the area affected. We assessed CSsymptoms as two separate components, using ML as ameasure of the degree of disfigurement of potato tubers andMA to express the extent of symptoms. Both componentsare important in the appearance of potato tubers for freshmarket sale, but non-superficial disfigurement in the formof warts or deep pits adds an additional economic concern,as pitted lesions in particular are not easily removed bypeeling during processing. This is the reason we and othershave ranked pitted lesions as more severe. Others havegenerally reported a significantly positive correlationbetween affected area and the severity of lesions (Bjorand Roer 1980; Goth et al. 1993; Lambert et al. 2006),although correlation coefficients were sometimes low andvariable among years (Bjor and Roer 1980). We found apositive correlation between the two measures only for thetwo susceptible potato cultivars, and no correlation for theresistant cultivars. Furthermore, we found that while MLwas higher for susceptible cultivars Chippewa and GreenMountain as expected, the mean surface area affected byCS was greater in the resistant cultivars. Disconnection ofthese two measures of CS in susceptible versus resistantpotato cultivars suggests there are multiple plant geneticcomponents to CS disease response. We also note that theuse of either incidence or MA data alone would give adifferent assessment of CS disease severity than ML, less

ID01-16c tMA ID03-3A tMA

Chipp GM Pike Sup Chipp GM Pike Sup

Chipp 0.31 0.27 0.06 Chipp 0.52 0.25 0.14

GM 0.06 0.01 GM 0.62 0.43

Pike 0.27 0.52 Pike 0.23 0.77M

L

Sup 0.38 0.75

ML

Sup 0.73 0.13

ID02-12 tMA ID05-11E tMA

Chipp GM Pike Sup Chipp GM Pike Sup

Chipp 0.81 0.06 0.40 Chipp 0.19 0.35 0.05

GM 0.17 0.63 GM 0.84 0.51

Pike 0.70 0.27 Pike 0.97 0.43

ML

Sup 0.97 0.60

ML

Sup 0.82 0.81

ID01-6.2A tMA NY02-3A tMA

Chipp GM Pike Sup Chipp GM Pike Sup

Chipp 0.48 0.17 0.67 Chipp 0.07 0.71 0.10

GM 0.57 0.79 GM 0.14 0.77

Pike 0.03 0.39 Pike <.01 <0.01 0.20

ML

Sup 0.90 0.41

ML

Sup <.01 0.02

c

0.98

0.73

0.39

0.42

0.19

0.17

0.21

0.34

0.26 0.11

0.53

0.66

0.57

0.43

0.09

0.66

0.94

Fig. 2 (continued)

342 Am. J. Pot Res (2009) 86:335–346

0

1

2

3

4

5

ID01-

16c

ID02-

12

ID01-

6.2A

ID03-

3A

ID05-

11E

NY0

2-3A

ID01-

16c

ID02-

12

ID01-

6.2A

ID03-

3A

ID05-

11E

NY0

2-3A

ID01-

16c

ID02-

12

ID01-

6.2A

ID03-

3A

ID05-

11E

NY0

2-3A

ID01-

16c

ID02-

12

ID01-

6.2A

ID03-

3A

ID05-

11E

NY0

2-3A

Chippewa Green Mt Pike Superior

29 26 36 38 36 33 . 17 16 17 12 21 17 . 12 15 13 17 18 16 . 22 13 7 17 17 21

ML

(le

sio

n t

yp

e)

tMA

Chippewa ID01-

16c

ID02-12 ID01-6.2A ID03-3A ID05-

11E

NY02-

3A

ID01-16c 0.88 0.63 0.75 0.83 <0.01

ID02-12 0.45 0.55 0.88 0.95 <0.01

ID01-6.2A <0.01 0.04 0.43 0.48 0.01

ID03-3A 0.75 <0.01 0.93 <0.01

ID05-11E 0.20 0.08 0.33 <0.01

NY02-3A <0.01 <0.01 <0.01 <0.01

ID01-16c 0.32 0.06 0.21 0.06 0.07

ID02-12 0.41 0.39 0.87 0.42 0.45

ID01-6.2A 0.54 0.43 0.96 0.91

ID03-3A 0.82 0.34 0.46 0.50

ID05-11E 0.77 0.30 0.94 0.96

Green

Mountain

NY02-3A 0.17 0.02 0.07 0.06

ID01-16c 0.53 0.53 0.81 0.84 0.16

ID02-12 0.83 0.99 0.38 0.45 0.40

ID01-6.2A 0.26 0.38 0.45 0.39

ID03-3A 0.03 0.01 0.99 0.10

ID05-11E 0.39 0.94 0.28 0.15

Pike

NY02-3A 0.94 0.26 0.03

ID01-16c 0.26 0.38 0.56 0.96 0.66

ID02-12 0.97 0.85 0.63 0.27 0.49

ID01-6.2A 0.11 0.78 0.38 0.65

ID03-3A 0.69 0.24 0.55 0.87

ID05-11E 0.66 0.26 0.97 0.64

ML

Superior

NY02-3A 0.71 0.20 0.96 0.92

min median mean max

0.38

0.23

b

a

0.14

0.40

0.65

0.65

0.13

0.48

0.53

0.28

0.76

0.10

0.67

0.64

0.69

0.16

Fig. 3 Differences in response of four potato cultivars to 6Streptomyces isolates. a Box plots showing differences in type ofscab lesion (ML), rated as described in the Material and Methods.Interquartile range, mean, median, maxima and minima are shown.Numbers under the cultivar names are the number of tubers rated forthe isolate-cultivar combination. b Matrix showing the probability of

significant differences in response of potato cultivars to each of theStreptomyces isolates studied. Combinations with P<0.05 are high-lighted. Differences in lesion type (ML) are displayed above thediagonal and differences in surface area affected (tMA) are displayedbelow the diagonal

Am. J. Pot Res (2009) 86:335–346 343

accurately gauging acceptability of affected tubers by thepotato processing industry. It therefore seems important toinclude a lesion type component when phenotyping plantsfor CS susceptibility, rather than using an evaluationscheme based only on incidence or affected area.

We did not find evidence for specialized disease typeswith cultivar specificities such as those that characterizescab diseases caused by Streptomyces species in Europe(Bouchek-Mechiche et al. 2006; Bouchek-Mechiche et al.2000b; Pasco et al. 2005). However, in our small selectionof only 6 Streptomyces isolates, different isolates were mostaggressive on particular potato cultivars, and an isolate thatwas most aggressive on one cultivar was least aggressiveon another cultivar. It seems likely that further specificity inwhat isolates or species are most aggressive on individualpotato cultivars will be discovered. Possibly we were ableto discern cultivar x isolate interactions because wedeliberately selected widely divergent plant genetic materialand Streptomyces isolates. The significant cultivar-isolateinteraction for ML and experiment x isolate interaction fortMA we document here have implications for screeningpotato cultivars for CS resistance; isolate(s) used to testcultivars for CS resistance should reflect Streptomycesspecies and types found in the target growing region.

Decomposition of Variation Due to Plant Genotype,Pathogen Genotype and “Environment”

The difficulty in reproducing disease severities betweenexperiments remains an obstacle to statistical power ingenetic analysis, and necessitates multiple years of fieldevaluation of plant cultivars to assess CS resistance in

commercial potato cultivars. Variation in CS phenotype iscommonly attributed to lack of control of the environmentalcomponent of the plant-pathogen-environment diseasetriangle. By doing experiments in growth chambers withcarefully controlled initial pathogen pressure, we attemptedto reduce the contribution of experimental and extraneousvariation, and standardize the pathogen pressure. We wereonly partially successful in doing this. The ANOVA modelfor tMA and ML explained only 50.1 and 55% of the totalvariation, respectively (Fig. 4). Pathogen genotypeexplained the most variation (8.1 and 12.1%), followed bythe plant genotype (6.2 and 6.6%), and then the experiment(4.0 and 2.7%) for tMA and ML, respectively. The twolargest sources of variation, after unexplained variation,were the two-way interaction effects between plant geno-type x pathogen genotype x experiment and plant genotype xpathogen genotype which explained 10.0 and 16.3% of thevariation for tMA and ML, respectively. In contrast, in fieldstudies looking at CS area index and lesion index at twolocations over 2 years, Haynes et al. were able to explain74.2 and 76.9% of the total variation, respectively (Haynes etal. 1997) and computations from their data). We would

Host 6.6%

Experiment 4.0%

Pathogen 12.1%

H x P 16.3%

H x E 1.0%

P x E 3.9%

H x P x E 11.1%

A. Lesion Index (ML)

45.0% of variation is unexplained

Host 6.2%

Experiment 2.7%

Pathogen 8.1%

H x P 10.0%

H x E 4.3%

P x E 7.7%

H x P x E11.2%

49.9% of variation is unexplained

B. Area Index (tAI)

Fig. 4 Decomposition of variation in common scab disease pheno-type, showing that a large amount of variation remains unexplained byhost plant genotype, pathogen genotype, experiment, and theinteractions between these sources of variance. a Host-Pathogen-Experiment disease triangle for mean lesion. b Host-Pathogen-Experiment Disease Triangle for square root transformed mean area

Isolate tMA ML

ID01-16ca 3.14 a 1.31 a

ID02-12 3.33 a 1.21 a

ID01-6.2a 3.79 ab 2.39 b

ID03-3A 3.24 a 1.78 ab

ID05-11E 3.62 a 1.71 ab

NY02-3A 4.71 b 2.39 b

Table 3 Mean tMA and ML byStreptomyces isolate from bothexperiments

a means followed by the sameletter do not differ at P≤0.05

Table 4 Mean tMA and ML by potato cultivar from both experiments

Cultivar tMA ML

Chippewa 3.17 aa 2.27 b

Green Mountain 3.16 a 1.96 ab

Pike 4.22 b 1.46 a

Superior 4.01 b 1.50 a

a means followed by the same letter do not differ at P≤0.05

344 Am. J. Pot Res (2009) 86:335–346

expect that experiments in controlled environments withstandardized inoculum levels would be less variable thanfield studies. This was not the case, indicating that there isstill much we don’t know about the complex interplaybetween pathogen genotype, plant genotype, and thecontext in which they are expressed. To minimize testingrequired to adequately characterize potato germplasm,design efficient breeding strategies and map resistancegenes, ways must be found to reduce the large unexplain-able variation.

In conclusion, we have demonstrated specific differencesin CS phenotypes in response to plant pathogenic Strepto-myces isolates belonging to different species and moleculartypes. We show that isolates differ significantly in aggres-siveness, and there are specific plant genotype-pathogenisolate interactions. The present model accounts for only50–55% of the total variation in CS disease symptoms.Further information on the distribution and prevalence ofCS-causing Streptomyces species together with the recog-nition of specific interactions between pathogenic speciesand potato cultivars could form the basis for successfulrecommendations of suitable potato cultivars, and willcontribute to more accurate and reproducible phenotypingrequired for genetic studies of CS resistance in potato.Research must be undertaken on ways to reduce the amountof unexplained variation if germplasm is to be accuratelyphenotyped and genetic studies are to progress.

Acknowledgments Technical assistance by Stephanie Ray is grate-fully acknowledged. This research was supported by USDA-ARS CRISproject numbers 1275-21220-223 0OD and 1275-21000-176-0OD.

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