Distribution and characterization of Streptomyces species causing potato common scab in Germany

Authors

  • J. Leiminger,

    Corresponding author
    1. Bayerische Landesanstalt für Landwirtschaft, Institut für Pflanzenbau und Pflanzenzüchtung, Am Gereuth 8, D-85354 Freising-Weihenstephan
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  • M. Frank,

    1. Bayerische Landesanstalt für Landwirtschaft, Institut für Pflanzenbau und Pflanzenzüchtung, Am Gereuth 8, D-85354 Freising-Weihenstephan
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  • C. Wenk,

    1. Bayerische Landesanstalt für Landwirtschaft, Institut für Pflanzenschutz, Lange Point 10, D-85354 Freising-Weihenstephan, Germany
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  • G. Poschenrieder,

    1. Bayerische Landesanstalt für Landwirtschaft, Institut für Pflanzenschutz, Lange Point 10, D-85354 Freising-Weihenstephan, Germany
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  • A. Kellermann,

    1. Bayerische Landesanstalt für Landwirtschaft, Institut für Pflanzenbau und Pflanzenzüchtung, Am Gereuth 8, D-85354 Freising-Weihenstephan
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  • A. Schwarzfischer

    1. Bayerische Landesanstalt für Landwirtschaft, Institut für Pflanzenbau und Pflanzenzüchtung, Am Gereuth 8, D-85354 Freising-Weihenstephan
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E-mail: juergen.leiminger@lfl.bayern.de

Abstract

Field-grown potatoes showing scab infections were sampled in two successive years and analysed for prevailing Streptomyces strains. In 2008 and 2009, 293 Streptomyces isolates were collected in Germany and analysed for morphology, pathogenicity and strain type. Isolates varied in mycelium colour, sporulation and pigmentation. Based on their morphology, no clear differentiation of species was possible. At the genetic level, sampled isolates, as well as a number of type strains from culture collections, were characterized by PCR using 16S rRNA-specific primers and PCR-RFLP of the 16S–23S internal transcribed spacer (ITS) region with Hpy99I. Using this fingerprinting approach, Streptomyces species could be differentiated genotypically. The data from this study show that diversity among scab-causing species in Germany is much higher than previously thought. Isolates belonged to various Streptomyces spp. previously associated with common scab. This is apparently the first report of pathogenic strains of S. europaeiscabiei, S. stelliscabiei, S. acidiscabiei, S. turgidiscabiei and S. bottropensis within Germany. Streptomyces europaeiscabiei was the predominant species found. Other scab-causing species were identified, but their local distribution was uneven. For most of the isolates, the presence of the txtAB gene was demonstrated, indicating pathogenicity. This analysis is one of the first reports to examine the distribution of common scab-causing species in Germany.

Introduction

Common scab (CS) is a serious bacterial disease that limits successful potato production worldwide (Loria et al., 1997). Potato scab is caused by actinobacteria belonging to the genus Streptomyces (Loria et al., 2006). Streptomycetes are soil-inhabiting and subsist mainly saprophytically, although a small number of species can live as phytopathogens. Once this pathogen is introduced into the soil, it persists for long periods, even in the absence of potatoes (Kritzman & Grinstein, 1991). Scab appears as shallow or deep corky blemishes that disfigure the potato skin and necessitates excessive peeling (Hoffmann & Schmutterer, 1999). If tubers are heavily infected, scab lesions may cover most of the tuber surface. In most European countries, it is common practice to wash ware potatoes before sale (Fiers et al., 2010), whereby superficial blemishes, such as CS, are more obvious. Thus, crop value can decrease considerably as scab infections lead to an aesthetically less appealing potato (Hoffmann & Schmutterer, 1999; Hiltunen et al., 2005; Tagawa et al., 2008). Streptomyces scabiei, which is recognized as the main causal agent of common scab, is distributed worldwide. Unlike S. scabiei, S. acidiscabiei, an acid-tolerant species, can induce potato scab symptoms in acid soils (Lambert & Loria, 1989). However, other causative and genotypically diverse species, such as S. turgidiscabiei (Miyajima et al., 1998), S. europaeiscabiei or S. stelliscabiei (Bouchek-Mechiche et al., 2000; St-Onge et al., 2008) have also been reported to cause scab symptoms (Goyer et al., 1996). The incidence of scab is dependent on the initial inoculum level in the soil (Keinath & Loria, 1991). However, prevailing scab symptoms can differ according to cultivar susceptibility, environmental conditions, infection date or virulence of the Streptomyces species (Cullen & Lees, 2007). The term ‘common scab’ is used for a wide range of symptoms but is generally used to describe lesions which are erumpent with some form of cratering (Cullen & Lees, 2007). As well as potato, scab symptoms can also be found on a number of other root crops, including carrot, radish, beet, parsnip and turnip (Loria et al., 1997). Immature lenticels (breathing pores) are the main site of entry for infection (Adams & Lapwood, 1978). However, direct penetration has also been reported by the pathogen through the periderm. After penetration, the pathogen is believed to grow in between or through a few layers of cells. Induction of cell hypertrophy leads to cell collapse and cell death, thus enabling the pathogen to absorb nutrients from the infected host. Infection takes place during active expansion of plant tissue. In response to infection, tissue surrounding the lesion (wound periderm) divides rapidly, producing a cork wound tissue layer in order to wall off the infected portions (Tegg, 2006). The production of newly suberized layers is facilitated, leaving raised or pitted scab lesions on the tuber surface as a result of inhibited cellulose biosynthesis (Scheible et al., 2003). Scab lesions do not continue to develop on the tuber once expansion has ceased (Loria et al., 2006).

Similarity in symptoms and host range of Streptomyces species suggests a common mechanism of pathogenicity, even though they are morphologically and genetically distinct (Healy et al., 2000). According to King et al. (1991) the production of the phytotoxin thaxtomin A is positively correlated with pathogenicity within species associated with common scab. Thaxtomin-like toxins are modified dipeptide molecules belonging to a family of nitrated dipeptides, and are formed by bacteria as secondary metabolites (King & Lawrence, 1996). Strains which are able to produce thaxtomin A are considered to be pathogenic, whilst strains without thaxtomin secretion are unable to induce symptoms and are therefore non-pathogenic (King & Lawrence, 1996; Healy et al., 2000). The secretion of thaxtomin A leads to cell hypertrophy and disturbs the formation of tuber periderm and the development of the primary cell wall (Duval et al., 2005). As both monocot and dicot plants are affected by the toxin, it appears to have a conserved target in plant cells (Loria et al., 1997). According to Kers et al. (2005), pathogenicity and virulence genes are clustered and located within a mobile pathogenicity island (PAI), which can be horizontally transferred among genetically distinct strains (Healy et al., 2000), thus leading to emergence of new pathogenic species. Kers et al. (2005) showed that for the species S. turgidiscabiei, multiple virulence-associated genes are conserved on a 325–660 kb PAI, including the thaxtomin biosynthetic genes (txtA, txtB), nec1, as well as other virulence genes. By contrast, Lerat et al. (2009) revealed that the txtAB sequences of S. turgidiscabiei can differ from those of S. scabiei. Typical PAI genes within strain 87.22 of S. scabiei, the genome of which has been sequenced, were found in two remote regions of the bacterial chromosome (Lerat et al., 2009); all genes involved in thaxtomin biosynthesis (txtAB, txtC, nos and txtR) were found in the first section of the PAI, whilst other genes, such as nec1 and tomA, which are not essential for pathogenicity but contribute to virulence (Wanner, 2009), were located on a distant segment of the PAI.

Diagnosis of pathogenic strains demands specific and sensitive methods that enable accurate and unambiguous characterization. Morphological, physiological and also molecular characteristics have been used for the identification of Streptomyces spp. (Shirling & Gottlieb, 1966; Bouchek-Mechiche et al., 2000; Wanner, 2009). DNA sequencing of genes of taxonomical and functional interest now offers a quick and robust way to perform species identification (St-Onge et al., 2008). According to some investigations, 16S rRNA gene sequences were successfully used for identification of Streptomyces strains (Lehtonen et al., 2004; Wanner, 2006; Tagawa et al., 2008).

The objective of this research was to isolate and map the distribution of CS-causing Streptomyces species within German potato-growing areas. It is thought that no broad monitoring of Streptomyces spp. in Germany has been carried out before. The diversity of Streptomyces spp. was investigated by systematic isolation followed by identification by sequencing the 16S rRNA. Further aims were to identify the main causal agents of CS on potatoes by means of molecular techniques. Thorough investigations of the distribution and coexistence of CS-causing Streptomyces spp. are important requirements for the development of successful control strategies.

Material and methods

Sampling and characterization of symptoms

Naturally scab-infected potato tubers were collected from different potato-growing areas within Germany in 2008 and 2009. Potatoes from 41 locations and 48 cultivars were sampled (Fig. 1). Tubers were evaluated for disease severity on a rating scale (0 = no visible symptoms; 1–4 = up to 10, 40, 60 and 90% of tuber surface infected, respectively; 5 = heavy infections with >90% of tuber surface infected). If tubers showed multiple lesions, the most severe lesion type was assigned. The appearance of lesions was rated shallow (superficial corky tissue), erumpent (lesions higher than surrounding tissue) or pitted (crater-like symptoms).

Figure 1.

 Map of Germany indicating locations from which common scab-infected potato tubers were collected in 2008 and 2009.

Isolation and morphological characterization of Streptomyces strains

Strains of Streptomyces were isolated from scab lesions of field-grown potato tubers. Tubers were washed, surface-sterilized with sodium hypochlorite (NaOCl, 2·8% active substance) for 1 min and washed again with sterile water. Scab lesions were excised using a scalpel, put in a reaction vessel (2 mL) and homogenized with a pestle. Vessels were filled with 1 mL Tris buffer (pH 7·2) and incubated at 50°C with shaking for 10 min using a thermomix (Eppendorf). Fifty microlitres of the solution was transferred into a new reaction tube, mixed with 900 μL Tris (pH 7·2) and vortexed for 10 s. An aliquot of 100 μL was streaked onto yeast dextrose chalk (YDC) medium (Lelliott & Stead, 1987) and incubated in the dark at 28°C for 3 days. YDC medium was compared to yeast malt extract (YME) medium, which was recommended by the Streptomyces project (ISP). YDC medium facilitated the formation of characteristic colonies, similar to YME medium. The production of dark diffusible pigments was monitored using peptone–yeast extract–iron agar (PYI, Difco) according to Shirling & Gottlieb (1966). Single colonies were separated and transferred onto fresh YDC. After incubation at 28°C for 21 days, bacterial growth and mycelium and spore colour were examined, as well as the production of soluble pigments. Different strains of Streptomyces spp. were used as references for morphological characterization and molecular characteristics (Table 1).

Table 1. Streptomyces reference strains used in this study
SpeciesStrain
  1. CFBP, Collection Francaise des Bactéries Phytopathogènes; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen; wildtype USA, provided by Professor Rosemary Loria, Cornell University, Ithaca, New York, USA.

S. scabiei DSMZ 41658
CFBP 4517
S. europaeiscabiei DSMZ 41802
CFBP 4497
S. turgidiscabiei DSMZ 41838
S. aureofaciens DSMZ 40127
S. acidiscabiei DSMZ 41668
wildtype USA
S. stelliscabiei DSMZ 41803
S. bottropensis DSMZ 40262
DSMZ 40800
txtAB-positive isolatesDSMZ 41658
DSMZ 41838
CFBP 4517
wildtype USA

DNA isolation

For DNA isolation, cultures of Streptomyces spp. were grown on liquid nutrient saccharose (NS) bouillon on a rotary shaker at 28°C for 2 days. NS bouillon consisted of 1·5 g meat extract (Sigma-Aldrich), 2·5 g peptone no. 3 (Difco), 25 g saccharose and 500 mL distilled water. Incubation for 2 days led to bacterial enrichment before mycelium was spun down at 5000 g for 10 min. DNA was extracted using cetyl trimethylammonium bromide (CTAB) according to Saghai-Maroof et al. (1984). Quantity and quality of DNA was checked spectrophotometrically (NanoDrop). This method yielded approximately 1 μμL−1 DNA.

PCR amplification

Analysis for the presence of genes characteristic of the respective species was carried out by the polymerase chain reaction (PCR). Previously published primer sets for species-specific 16S rRNA gene sequences were used (Edwards et al., 1989; Lehtonen et al., 2004; Wanner, 2006; Flores-González et al., 2008; Tagawa et al., 2008). PCR conditions, accessions numbers and origins of primer pairs are listed in Table 2. Eight previously described strains of different Streptomyces spp. were included as reference strains (Table 1) for phenotyping and genotyping studies and were used for optimization of PCR conditions. Universal primers developed by Edwards et al. (1989) were used for the general identification of Streptomyces spp. PCR analyses were carried out in 25-μL reaction volumes each containing 2·5 μL buffer (MgCl2), 0·125 μL Taq DNA polymerase (Thermoprime Plus, Thermo Fisher Scientific), 2·5 μL 2 mm dNTPs (Fermentas), 1·5 μL each primer, 1 μL DNA template and 16 μL H2O. Amplification was carried out in an Eppendorf Mastercycler pro S. PCR programmes were chosen according to the investigated species. Amplified DNA fragments were separated on 1·5% agarose gels and stained with ethidium bromide. All pairs of primers were used on all strains in this study. To ensure that DNA extracts contained Streptomyces DNA, all isolates were first tested with the primers for universal amplification of 16S rDNA. Specific primer pairs were used to distinguish scab-causing species (S. scabiei, S. europaeiscabiei, S. turgidiscabiei, S. acidiscabiei, S. stelliscabiei, S. bottropensis and S. aureofaciens).

Table 2. Primer pairs and conditions used in PCR detection of Streptomyces isolates
Primer specificity (GenBank accession no.)Primer name and sequenceAmplicon size (bp)DenaturationAnnealingElongationNumber of cyclesPositive DNA templateReference
Streptomyces universal 16S rRNAUniF 5′-AGA GTT TGA TCC TGG CTC AG-3′
UniR 5′-AAG GAG GTG ATC CAG CCG CA-3′
151494°C, 30 s57°C, 30 s72°C, 90 s34 Edwards et al.,
1989
S. scabiei 16S rRNA
(Y15500)
ScabF 5′-CAA CAC TCT CGG GCA TCC GA-3′
ScabR 5′-TTC GAC AGC TCC CTC CCT TAC-3′
127894°C, 30 s57°C, 30 s72°C, 90 s34DSMZ 41658,
CFBP 4517
Lehtonen et al., 2004
S. europaeiscabiei 16S rRNA (AB026208)SeurF 5′-CGA CAC TCT CGG GCA TCC GA-3′
ScabR 5′-TTC GAC AGC TCC CTC CCT TAC-3′
128094°C, 30 s57°C, 30 s72°C, 90 s34DSMZ 41802, CFBP 4497Wanner, 2006
S. turgidiscabiei 16S rRNA (AB026221)SturF 5′-CGG AAA CAT CCA GAG ATG GGT G-3′
SturR 5′-CTT CAC CGC TTC CCT CAT CG-3′
72394°C, 30 s57°C, 30 s72°C, 90 s34DSMZ 41838Tagawa et al., 2008
S. aureofaciens 16S rRNA (Y15504)SaurF 5′-TCC GCA TGG GGG TTG GTG-3′
SaurR 5′-TGT GAG TCC CCG ACA TTA CT-3′
94395°C, 30 s59°C, 30 s72°C, 80 s35DSMZ 40127Lehtonen et al., 2004
S. acidiscabiei 16S rRNA (AB026220)SaciF 5′-ATA TCA CTC CTG CCT GCA TGG-3′
SaciR 5′-CCT ACC GAA CTC TAG CCT GC-3′
46894°C, 30 s57°C, 30 s72°C, 90 s34DSMZ 41668, wildtype USTagawa et al., 2008
S. stelliscabiei 16S rRNA (gi526074, AB026212)SsteF 5′-GAA AGC ATC AGA GAT GGT GCC-3′
SsteR 5′-CGA CAG CTC CCT CCC CGT AAG-3′
47695°C, 20 s60°C, 30 s72°C, 120 s40DSMZ 41803Wanner, 2006
S. bottropensis 16S rRNA (gi971133, AB026215)SsteF 5′-GAA AGC ATC AGA GAT GGT GCC-3′
SbotR 5′-CGA CAG CTC CCT CCC ACA AG-3′
47595°C, 20 s60°C, 30 s72°C, 120 s40DSMZ 40262, DSMZ 40800Wanner, 2006
Thaxtomin A
(AF255732)
TxtF 5′-GTG GAC CGT GGA GCA TCT-3′
TxtR 5′-CAG TTC GGC GTA ACT CAG C-3′
40295°C, 30 s60°C, 45 s72°C, 60 s35DSMZ 41658, DSMZ 41838, CFBP 4517,Flores-González et al., 2008
16S–23S rRNA ITS
(AB026199)
ITSF 5′-AAACTTGGCCACAGATGCTG-3′
ITSR 5′-GTCAAGTCATCATGCCCCTT-3′
65094°C, 60 s58°C, 60 s72°C, 120 s35DSMZ 41658,
DSMZ 41802
Song et al., 2004

For the differentiation of S. scabiei and S. europaeiscabiei, methods described by Flores-González et al. (2008) were used. As S. scabiei and S. europaeiscabiei have nearly identical 16S rDNA sequences, the two species can be distinguished only on the basis of differences in the sequence of the 16S–23S ribosomal internal transcribed spacer (ITS) –S. scabiei has an Hpy99I site in the 16S–23S rDNA ITS (positions 1629–1633 of the S. scabiei ITS region sequence), which is missing in S. europaeiscabiei. The ribosomal intergenic spacer was amplified using primers ITS-F and ITS-R and the ITS amplicon was subsequently digested with the restriction enzyme Hpy99I (New England Biolabs) for 2–3 h at 37°C in the buffer recommended by the manufacturer. Differences in restriction patterns of PCR-amplified 16S–23S rDNA amplicons cut with Hpy99I were visualized by electrophoresis.

Characterization of pathogenic Streptomyces isolates

In order to characterize the sampled Streptomyces in terms of pathogenicity, the presence or absence of the txtAB gene required for the synthesis of thaxtomin A was determined by PCR. Aliquots of yielded DNA samples were tested with primers developed by Flores-González et al. (2008). PCR conditions consisted of an initial denaturation step at 95°C for 5 min, followed by 35 reaction cycles consisting of denaturation at 95°C for 30 s, primer annealing at 60°C for 45 s and DNA extension at 72°C for 1 min, followed by a final extension step at 72°C for 10 min. The amplified DNA was run on 1·5% agarose gels and stained with ethidium bromide.

Pathogenicity test

A selection of 22 Streptomyces isolates obtained from scab lesions and with different morphological characteristics were evaluated for pathogenicity. Streptomyces cultures were cultivated in liquid oat bran broth according to Wanner (2006) with minor modifications. Investigated strains were either txtAB-positive or txtAB-negative and belonged to different Streptomyces species. Potatoes were planted into a soil/sand mixture infested with single Streptomyces isolates at a known density. In each experiment, five pots not inoculated with any bacterial strain were included as controls. Reference cultures of each species were included as positive controls. Potato plants were grown in a greenhouse under controlled conditions at 22°C in natural daylight. Trials were adapted to local conditions. Tubers were harvested 3 months after planting and symptom appearance was recorded. From each species-specific test, reisolations were made from all lesions to confirm Koch’s postulates.

Results

Common scab (CS) pathogens were isolated from scab lesions on potato tubers sampled at different locations in Germany. Samples were obtained from a wide geographic range as well as from multiple locations in close proximity. CS-causing Streptomyces species were characterized according to their morphological and molecular properties. In 2008 and 2009, a combined total of 293 isolates was collected from 41 potato fields and 48 cultivars. CS symptoms were diverse, ranging from superficial to raised or pitted lesions. Disease incidence of the investigated tuber samples was quite varied, including samples with just one or a few infected tubers and samples with heavy scab infections on almost all tubers. Disease severity was assessed on a scale of 0–5. Symptom appearance and disease severity seemed to be correlated with location, as tubers of the same cultivar, but sampled at different locations, varied in disease incidence and symptom development. For example, cv. Marabel, which was sampled at five different locations, showed superficial, raised and pitted symptoms, ranging in severity from 2 up to 5.

Morphological characteristics and melanin production

The development of bacterial colonies was more rapid on YDC than YME medium. As colony size, development of mycelium and spore colour were similar on both media, YDC was used as the standard medium in this survey. In total, 293 isolates were obtained and attributed to Streptomyces spp. according to their colony form. Appearance, mycelium and spore colour varied among isolates. Most produced caramel-brown mycelium and grey spores. However, in others mycelium colour varied from clear light gold to dark brown. Bordeaux-red and pink mycelium colours were also observed. Graduations in the production of pigments and spore colour were also evident. Production of a melanoid pigment was noted in most of the isolates and was observed as brownish halos around colonies. The ability to produce diffusible pigments differed considerably within species. Thirty-four out of 293 isolates were completely free of pigmentation. Interestingly, many sampled isolates which belonged to different species (such as S. turgidiscabiei, S. acidiscabiei, S. bottropensis and S. aureofaciens) did not produce melanin or other diffusible pigments, although some of them tested positive for pathogenicity. Melanoid pigments were produced by both pathogenic and non-pathogenic isolates.

Molecular characterization of isolates

PCR fingerprinting was carried out to assess the genomic diversity of isolates and to determine scab-causing Streptomyces spp. All recovered isolates (293) tested positive for the genus Streptomyces. Further characterization of scab-related species (S. scabiei, S. europaeiscabiei, S. turgidiscabiei, S. acidiscabiei, S. stelliscabiei, S. bottropensis and S. aureofaciens) was carried out using species-specific primers (Fig. 2). All except six isolates were assigned to one of these species and produced amplicons with at least one of these primer sets. As S. scabiei is described as the predominant causal agent within scab pathogens, all isolates were first tested for this species. However, most of the isolations amplified segments characteristic of S. europaeiscabiei. As S. scabiei and S. europaeiscabiei are nearly identical in their 16S rRNA gene sequence, these species were distinguished based on sequence differences in the 16S–23S ITS, and S. europaeiscabiei was found to be the prevalent species among the sampled Streptomyces isolates; a total of 239 isolates were characterized as S. europaeiscabiei (82%). This species was identified at almost all investigated locations, demonstrating that S. europaeiscabiei is widely distributed within German potato-growing areas (Table 3). However, other species were identified as well, although their distributions were quite irregular. Of all the investigated isolates, 20 (7%) belonged to the species S. stelliscabiei and were identified at 12 different locations. In addition 13 isolates (4%) were identified as S. turgidiscabiei, from seven different locations. Eleven isolates were positive for S. bottropensis, and four for S. acidiscabiei. None of the tested isolates produced an amplicon specific for S. aureofaciens. Six out of the 293 isolates (2%) did not correspond to any of the tested species (Fig. 3). However, all isolates were confirmed as Streptomyces spp., as positive amplicons were produced with the universal primer set. An overview of sampled isolates and isolate characteristics is presented in Table 4.

Figure 2.

 Electrophoresis of PCR amplification products obtained with different 16S rRNA-specific primers (target species on right) from DNA samples of Streptomyces reference strains (Table 1) and field isolates. Reference isolates belonged to S. scabiei (DSMZ 41658, CFBP 4517), S. europaeiscabiei (DSMZ 41802, CFBP 4497), S. turgidiscabiei (DSMZ 41838), S. acidiscabiei (DSMZ 41668), S. bottropensis (DSMZ 40262), S. stelliscabiei (DSMZ 41803) and S. aureofaciens (DSMZ 40127).

Table 3. Overview of locations, disease severity, symptom specification and types of Streptomyces isolates
OriginSymptom characteristicsSpecies detection by PCRaITS PCR-RFLP analysisbVirulence gene (PCR)
SiteLocationPotato cultivarDisease severity (0–5)DescriptionSelected isolates (n) Streptomyces spp. S. europaeiscabiei S. turgidiscabiei S. acidiscabiei S. bottropensis S. stelliscabiei Hpy99INo. of txtAB-positive isolates
  1. aNo isolates of S. scabei and S. aureofaciens were found.

  2. bPCR-RFLP analysis of 16S–23S ribosomal ITS sequences to differentiate S. europaeiscabiei and S. scabiei. – , absence of Hyp99I site (S. europaeiscabiei).

 1AbenbergQuarta4superficial3 1   22
Laura4superficial3 3    3
Christa5pitted5 3   23
 2AbensbergKrone3raised2    2 2
 3BöhlendorfEldena3pitted6 6    6
Tomba4pitted5 5    5
Naviga3superficial4 4    5
Producent4pitted3 3    3
Agria4superficial5 5    5
Bellarossa3pitted6 5   15
Finka4superficial5 4  1 4
Melba4raised6 6    5
 4BorsflethAgria2superficial11      0
 5BrunnenAgria5pitted2 2    2
 6BüchenbachKuras4superficial31    2 2
 7DillingenAlbatros4pitted2 2    2
 8FriedrichsgabekoogMarabel3pitted3 2 1  1
 9GachenbachAlbatros5pitted1 1    1
10GundhöringKuras4raised1  1    1
11HaimbuchPriamos5superficial1 1    1
12KaltenbergAmado5raised5 4  1 5
Turdus2superficial5 5    5
Anouschka5pitted3111   2
Marlen1superficial5 4  1 5
Quarta1superficial5 2 1114
Eldena4pitted2 2    2
Marabel3superficial1 1    1
Bellarossa5raised3 3    2
Angela3superficial4 4    4
Kuras4superficial3 3    3
13KarlshuldPonto1raised2 2    2
14KarolinenkoogBellarossa3raised3 3    2
Vineta4superficial3 3    3
15LaberweintingKuras4superficial1  1    1
16Lichtenauunknown3raised2 2    2
17LindholzMarabel5pitted5 5    5
Elfe4pitted6 6    5
Kuras5raised5 5    5
Marlen5raised5 5    5
Eurobona5raised6 6    6
18LüneburgMelba2pitted4 21  13
29Mötzingunknown5pitted3     3 3
20NahburgOpal5pitted514    3
21NatendorfEurobona3raised7 7    7
22NiebüllMarabel2raised1 1    1
23NiederambachPommqueen5pitted3 1  2 3
24NorddeichMelba3superficial4 3  1 3
25PuchLaura2superficial4 4    4
26RainWestamyl3raised2 2    2
27SchmatzinNatascha3superficial3 3    3
Miranda3superficial5 5    5
Satina3superficial4 4    4
28SchrobenhausenPatrona3superficial3 3    3
Ulme3superficial2     2 2
29SchülpCilena2superficial3 3    3
30SchwandorfPanda3superficial1 1    1
31SchwarzenfeldTosca5pitted6 5   16
32SönderbyMiranda4raised10 8 11 8
Sibu5raised7 51   5
33StadeAllians2raised3 3    3
34StolpePanda3superficial4 3   13
Opal4superficial5 5    4
Miranda3superficial4 4    4
Verdi3superficial3 3    3
Ramses2raised5 4   15
35StörensteinFambo4superficial5 5    5
36SünchingKuba4superficial2 2    2
37TarrottAndante5raised5 4 1  4
Christa5pitted6 6    6
Francisca5pitted615    5
38UelzenAgria4superficial4 31   4
Cilena4pitted4 3   14
Marabel2pitted6 32 1 6
Presto2pitted4 3   13
39WaidhofenAlbatros5pitted2 2    2
40WeihenstephanKuras5raised6  5  1 5
41WindsbachRenate4raised1 1    1
  Total29362391341120 265
Figure 3.

 Composition of identified Streptomyces spp. among German isolates in 2008 and 2009. ‘Universal’ indicates isolates which did not correspond to any of the species tested for.

Table 4. Phenotypic and morphological characterization of selected Streptomyces isolates from potato tubers in Germany and reference strains
Species/strainColony colour (YDC agar)Spore colour (YDC agar)Melanin production (YDC agar)Production of diffusible pigment (PYI agar)Presence of txtAB gene
S. scabiei
 DSMZ 41658ochrewhite++
 CFBP 4517ochrewhite+++
S. europaeiscabiei
 DSMZ 41802ochrewhite+
 CFBP 4497ochreno sp.+
 402/1aochrewhite+ (few)++
 403/1abrownwhite++
 1405/1cyellowwhite++
 1406/1ayellowwhite+++
 1604/2abright yellowwhite
 1704/1ayellowwhite+++
 4202/1aBordeaux red to brownno sp.++
 4602/1alight greywhite++
S. turgidiscabiei
 DSMZ 41838ochrewhite+
 1401/1ayellowwhite++
 5603/1ayellowwhite++
 6405/1ayellowwhite++
 10203/1aochrewhite+ (few)+
 10602/1ayellowwhite++
 15801/1atanwhite++
 16403/1aochrewhite+ (few)+
S. acidiscabiei
 DSMZ 41688tanwhite
 US wildtypetanwhite
 905/1abright yellowno sp.++
 1503/3aochrewhite
 1801/1aochrewhite
 5703/1aochreno sp.
S. bottropensis
 DSMZ 40262tangray+++
 DSMZ 40800ochregray+
 1503/2ayellowwhite
 2201/1adark greyyellow++
 2401/1awhite to greywhite
 3301/1apinkwhite
 6302/1ayellowwhite++
 7005/1aochregray
 9302/1ayellowwhite+
 10402/1aochrewhite+++
S. stelliscabiei
 DSMZ 41803ochrewhite++
 2402/1aamberno sp.+ (few)+
 3104/1awhitewhite
 6505/1ayellowno sp.+
 6902/1ayellowwhite+
 7501/1aochrewhite++
 7503/1ayellowwhite++
 8602/1ayellowwhite+++
 13304/1abright yellowwhite++
 13802/1ayellowwhite+++
S. aureofaciens
 DSMZ 40127dark ochreno sp.

Analysis of the presence of pathogenicity-related genes

All Streptomyces isolates were obtained from necrotic lesions of potato tubers. Isolates were classified as putatively pathogenic, i.e. able to induce scab symptoms, if they possessed the genes responsible for the formation of thaxtomin A. Thaxtomin A is the only known pathogenicity determinant in Streptomyces, and the presence or absence of the txtAB gene is 100% correlated with pathogenicity. Of the 293 isolates investigated, 265 (90%) were found to have the txtAB gene by PCR. Pathogenicity testing (see below) revealed that only txtAB-positive isolates were fully pathogenic. Putatively pathogenic isolates belonged to several Streptomyces spp; txtAB-positive strains were found in S. europaeiscabiei, S. turgidiscabiei, S. stelliscabiei and also in S. bottropensis. The largest number of pathogenic isolates was found for S. europaeiscabiei, in which the txtAB gene was found in 205 out of 239 isolates. The highest expression of pathogenicity was seen for S. turgidiscabiei, with all sampled isolates (n = 13) identified as pathogenic. Isolates identified as S. acidiscabiei (n = 4) were all txtAB-negative, while 40% (eight of 20) of isolates belonging to S. stelliscabiei lacked txtAB, as did 36% (four of 11) of S. bottropensis. The presence of txtAB in Streptomyces isolates was equally distributed over all tested locations. There was no location that did not yield at least one isolate positive for txtAB.

The connection between the presence of txtAB genes and pathogenicity was evaluated in plant assays in potatoes. Twenty-two isolates were tested for pathogenicity (data not shown). Fifteen of 22 isolates PCR positive for txtAB were able to induce characteristic CS symptoms. The remaining seven isolates lacking thaxtomin A biosynthesis genes were unable to induce symptoms. In order to determine the causative agent, bacterial isolates were reisolated from infected lesions. Koch’s postulates were confirmed for all isolates that produced symptoms. However, there was no correlation between the inoculated species and the type of evaluated symptoms. Characteristic CS symptoms were evaluated for different Streptomyces spp. (Fig. 4).

Figure 4.

 Appearance of potato tubers after artificial inoculation with different Streptomyces species. Potato tubers were grown in soil inoculated with (a) a txtAB-positive strain of S. turgidiscabiei causing characteristic CS symptoms and (b) a txtAB-negative strain of S. acidiscabiei causing no scab symptoms.

Discussion

Potato scab is a widespread disease, affecting potatoes wherever they are grown. Potato scab is caused by different species of the genus Streptomyces, which are ubiquitous in soil and are known as important plant pathogens. Potato scab has been described from most locations around the world. Because of its wide distribution, S. scabiei has been recognized as the main causal agent of potato scab (Hosaka et al., 2000). However, other CS-causing species have been identified from one or more geographic locations around the world (Takeuchi et al., 1996; Bouchek-Mechiche et al., 2000; Lehtonen et al., 2004; Wanner, 2009). The first reports of CS as an economically important disease in Germany were made by Schlumberger (1927). Until now, no detailed investigations had been carried out to characterize the distribution and presence of CS-causing species in Germany.

This paper reports the characterization of 293 Streptomyces isolates sampled from 48 different potato cultivars at over 40 locations in Germany. The results suggest that Streptomyces causing CS of potato in Germany are genetically diverse. The prevalence of phylogenetically distinct species of Streptomyces able to cause scab symptoms was described by Loria et al. (2006), according to whom, new pathogenic species aside from the widespread S. scabiei have occurred many times in agricultural systems. The results of the present study confirm that different species within the genus Streptomyces can cause CS of potato and demonstrate the presence and prevalence of species other than S. scabiei. None of the isolates investigated here could be characterized as S. scabiei, although it has been described as the most abundant species of scab-causing streptomycete worldwide (Loria et al., 1997). Previously, CS-causing bacteria were mainly designated as S. scabiei based on morphological characteristics. In 1962, Hoffmann concluded that CS in Germany was only attributable to S. scabiei (Schick & Klinkowski, 1962). As the differentiation of proposed species could only be carried out according to symptoms and biological conditions, Hoffmann attributed CS only to S. scabiei. Current molecular methods, including 16S rRNA gene sequencing and DNA:DNA hybridization, are more discriminating, supporting the existence of different and genetically distinct species (Kers et al., 2005). According to Bouchek-Mechiche et al. (2000) the former species S. scabiei, despite its phenotypic uniformity, could be divided into a genetically diverse taxon comprising three different species: S. scabiei, S. europaeiscabiei and S. stelliscabiei. Up to now, S. europaeiscabiei, which was first described in France (Bouchek-Mechiche et al., 2000), has been identified in different European countries (Dees et al., 2012) and North America (Wanner, 2009). According to Flores-González et al. (2008), S. europaeiscabiei is the most common CS-causing species within Europe. The present study confirms that, within Germany, S. europaeiscabiei seems to be among the main species responsible for CS, because it was isolated from almost all locations. The present study also provides evidence for the importance of additional species such as S. turgidiscabiei, S. acidiscabiei, S. stelliscabiei and S. bottropensis, whose distribution in Germany was shown for the first time. Phylogenetically distinct CS-causing species have been reported from various countries. Streptomyces stelliscabiei has been found in France and the USA (Bouchek-Mechiche et al., 2000; Wanner, 2006). Streptomyces turgidiscabiei has been reported from Japan and northern Scandinavia (Miyajima et al., 1998; Lehtonen et al., 2004), and S. acidiscabiei from Maine (USA), Korea and Japan (Lambert & Loria, 1989; Song et al., 2004). The present analysis of investigated locations in Germany illustrates that the composition of species differs not only within locations, but also that different species are able to coexist in the same field and even the same tuber. Six out of 293 isolates were attributed to Streptomyces but did not belong to the seven species investigated here. None of these six strains contained the txtAB gene. These isolates may be related to others of the hundreds of described species within the genus Streptomyces which have not been evaluated during this survey.

Thaxtomin is the only known pathogenicity determinant in Streptomyces, and the presence or absence of txtAB is 100% correlated with pathogenicity of common scab species (Wanner, 2006). The hypothesis that thaxtomin A production is responsible for pathogenicity on potato, as reported by Loria et al. (1997) and King & Lawrence (1996), was confirmed for German CS-causing species in the pathogenicity tests here.

Artificial inoculation with txtAB-positive isolates of different Streptomyces species led to the induction of characteristic CS symptoms, provided the strain carried the txtAB gene and was able to produce thaxtomin A. Isolates missing this site were not able to produce symptoms. This clearly supports the role of thaxtomin A as a pathogenicity determinant for Streptomyces causing common scab. Koch’s postulates were confirmed for all isolates that produced symptoms. In total, 265 out of 293 isolates (90%) showed presence of the txtAB gene. In the USA, Wanner (2009) investigated 1471 isolates, of which 73% harboured the txtAB gene and pathogenicity assays carried out for more than 100 isolates revealed that the presence or absence of the txtAB gene was 100% correlated with pathogenicity. Although recent studies showed that the txtAB sequences of different species such as S. turgidiscabiei, S. scabiei and S. europaeiscabiei are rather divergent (Dees et al., 2012), this does not contradict the relevance of the txtAB gene for pathogenicity.

Streptomycetes are soil-inhabiting pathogens, which are normally territorially constant. The horizontal and vertical distribution of soilborne pathogens depends primarily on production practices, as well as the cropping history of the field (Koike et al., 2003). Schick & Klinkowski (1962) made the assumption that CS-infected seed tubers are important sources for the transfer of pathogens and the contamination of fields. Keinath & Loria (1991) reported that once a species has been introduced into the soil, population densities increase in the rhizosphere. As a result of this, infected seed tubers can lead to infections of daughter tubers and contamination of soil (Wilson et al., 1999). Because seed tubers are distributed worldwide, there is a high risk that scab-causing species may be transferred to other fields as well as to other countries, leading to the appearance of new species in German potato-growing areas.

Tuber symptoms were diverse in this study, showing graduations in severity as well as in type, ranging from superficial to raised or pitted lesions. However, there was no correlation between the prevailing species and the type of symptoms. For example, the predominant species S. europaeiscabiei was identified in superficial, raised and pitted scab symptoms. Thus, the type of symptoms is not a useful feature for the characterization of Streptomyces spp. This is in accordance with the finding that scab symptoms caused by pathogenic species were indistinguishable from each other, and with the findings of Dees et al. (2012) that symptoms were not species-specific. Rather, severity of symptoms seems to be influenced by inoculum level in the soil, cultivar susceptibility and/or the virulence of the infecting species (Cullen & Lees, 2007).

Colony colour and pigmentation were noted for isolated colonies. Many isolates produced a tan to caramel-brown mycelium, grey spores and a brownish diffusible pigment and were phenotypically similar to S. scabiei, although they belonged to other species. The production of melanoid pigments was observed both for pathogens and non-pathogens. Schick & Klinkowski (1962) asserted that the formation of pigments is associated with pathogenicity in actinomycetes. Takeuchi et al. (1996) described the occurrence of a new potato scab-causing species, S. turgidiscabiei, which was found in the eastern Hokkaido, Japan, and which did not produce melanin or other diffusible pigments. Similarly, Dees et al. (2012) reported Norwegian scab isolates related to S. turgidiscabiei, which caused symptoms but were unable to produce melanin. According to Lambert & Loria (1989) melanin production is associated with pathogenicity, but it is neither essential (Gregory & Vaisey, 1956) nor invariable. Twenty-two txtAB-positive isolates did not produce melanoid pigments in the present study although they were pathogenic. Regarding the production of thaxtomin A, which is necessary for the formation of symptoms, no uniform consensus on the formation of melanoid pigments was observed over all species. No species could be differentiated from other CS-causing species by visible tuber symptoms or morphological characteristics alone. Also, colony morphology, especially pigmentation, did not correlate with pathogenicity and could not be used as a reliable indicator for the differentiation of pathogenic and non-pathogenic isolates, which was explicitly proven by PCR.

Diagnostic tools such as PCR enable secure and quick characterization of species. Here, sequence information provided a convenient basis for identifying different species. Highly conserved regions have proven useful as primer target sites for reliable detection of different species (Lane et al., 1985). Almost all species could be easily differentiated using PCR, except S. scabiei and S. europaeiscabiei, which were distinguished based on sequence differences in the 16S–23S ribosomal internal transcribed spacer (Song et al., 2004). PCR proved to be a reliable tool to test the distribution and composition of potato scab pathogens.

In this study, the composition of species, as well as the appearance of pathogenic and non-pathogenic strains, was heterogenic. In some cases, the co-occurrence of different pathogenic and/or non-pathogenic isolates in single scab symptoms was observed, confirming Norwegian results (Lehtonen et al., 2004). This heterogeneous composition, as well as the fact that species are ever evolving, may therefore contribute to the emergence of new pathogenic species. This study shows that diversity among scab-causing species in Germany is much higher than previously thought and that CS can be caused by different species of the genus Streptomyces. This is apparently the first report of the occurrence of pathogenic strains of S. europaeiscabiei, S. turgidiscabiei, S. stelliscabiei and S. bottropensis in Germany.

Acknowledgements

We gratefully acknowledge excellent technical assistance and preparation of samples by Melanie Friedrich-Zorn, Bianka Huber, Sigrid Theil and Sigrid Ziegltrum (LfL, Freising, Germany). We thank Rosemary Loria (Ithaca, USA) for providing wildtype isolates and Leslie Wanner (Beltsville, USA) for her helpful suggestions and valuable support. For the critical review of this manuscript and helpful comments we thank Ruth Eichmann (TUM, Freising, Germany).

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