Antimycin A-producing nonphytopathogenic Streptomyces turgidiscabies from potato

Authors


Mirja Salkinoja-Salonen, Department of Applied Chemistry and Microbiology, PO Box 56, FIN-00014 University of Helsinki, Finland. E-mail: mirja.salkinoja-salonen@helsinki.fi

Abstract

Aim:  To detect if substances with mammalian cell toxicity are produced by Streptomyces turgidiscabies and Streptomyces scabiei isolated from potato scab lesions.

Methods and Results: In vitro cultures of phytopathogenic and nonphytopathogenic strains of S. scabiei and S. turgidiscabies, isolated from scab lesions of potato tubers originating from nine different cultivars from Finland and Sweden, were tested for toxicity using the rapid spermatozoan motility inhibition assay, previously shown useful in the detection of many different Streptomyces toxins and antimicrobial compounds. Purified toxins were used as reference. Three nonphytopathogenic strains of S. turgidiscabies were found to produce antimycin A when cultured on solid medium.

Conclusions:  Boar sperm-motility-inhibiting substances are produced by strains of S. turgidiscabies and S. scabiei. The most powerful inhibitory substance, produced by three nonphytopathogenic S. turgidiscabies strains, was identified as antimycin A. The phytotoxic compounds thaxtomin A and concanamycin A did not inhibit sperm motility even at high doses.

Significance and Impact of the Study:  The presence of antimycin A-producing Streptomyces strains, nonpathogenic to potato, was unexpected but important, considering the high mammalian toxicity of this cytochrome bc-blocking antibiotic.

Introduction

One of the most important diseases of potato in all potato-growing areas is common scab caused by Streptomyces spp. (Loria et al. 1997). The symptoms are characterized by superficial, raised or deep-pitted lesions on potato tubers that reduce the marketable yield (Loria 2001). The most widely spread causal agent of common scab is Streptomyces scabiei (Thaxter) (Lambert and Loria 1989; Goyer et al. 1996). A recently described species, Streptomyces turgidiscabies (Takeuchi), produces symptoms on potato similar to those of S. scabiei in some areas, such as northern Scandinavia (Kreuze et al. 1999; Lehtonen et al. 2004; Hiltunen et al. 2005), Japan (Takeuchi et al. 1996; Miyajima et al. 1998) and probably many other areas where it has not been studied in detail (Wilson 2004). Both species produce phytotoxic compounds, thaxtomins (Bukhalid et al. 1998; Ylhäinen 2001; Hiltunen et al. 2006), which is essential for the induction of the characteristic scab symptoms on potato tubers (King et al. 1991).

Potato scab-associated Streptomyces species also produce other secondary metabolites, including vacuolar ATP inhibitors, such as concanamycins and bafilomycins (Natsume et al. 1996, 1998, 2001, 2005; Myers et al. 2003) toxic to mammalian cells (Dröse and Altendorf 1997; Hoornstra et al. 2004). The aim of this study was to investigate if metabolites emitted by Streptomyces strains isolated from scab lesions of potato can be detected by the boar spermatozoan motility test. This test can be executed within a day and was shown to be sensitive to many mitochondriotoxic substances from streptomyces species, both peptides and macrolides, including bafilomycin (Andersson et al. 1998; Hoornstra et al. 2003, 2004; Teplova et al. 2007). We focussed on the recently described species, S. turgidiscabies, which contains phytopathogenic strains highly virulent on potato (Takeuchi et al. 1996; Miyajima et al. 1998; Kreuze et al. 1999; Hiltunen et al. 2005) in addition to many strains that are avirulent on potato (Lindholm et al. 1997).

Materials and Methods

Bacterial strains

The strains of S. scabiei and S. turgidiscabies were isolated from scab lesions on potato tubers (Fig. 1) obtained from different cultivars grown in Finland or northern Sweden (Table 1), as described (Lindholm et al. 1997; Lehtonen et al. 2004). They were identified to the species level based on morphological and physiological characteristics, such as melanin production (S. scabiei), the shape of sporophores (spiral or flexuous in S. scabiei and S. turgidiscabies, respectively), utilization of the ISP (International Streptomyces Project) sugars (Lambert and Loria 1989; Lindholm et al. 1997; Miyajima et al. 1998) and also the 16S rRNA gene sequences (Kreuze et al. 1999; Lehtonen et al. 2004). Strain 364 of S. scabiei (DSMZ 41744) and strains 287 (DSMZ 41745) and 300 (DSMZ 41747) of S. turgidiscabies are the Finnish type strains of the species (Kreuze et al. 1999) available from the Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany. The type strain 53 of S. scabiei [American Type Culture Collection (ATCC) 49173], thaxtomin A and concanamycin A producer (Natsume et al. 1998, 2001, 2005), was obtained from the ATCC (Manassas, VA, USA). The commercially available preparation of Streptomyces griseoviridis, strain K61, which was originally isolated from peat soil in Finland (Tahvonen 1982) and is used as a plant growth-promoting agent (MYCOSTOP) (Kortemaa et al. 1994; Minuto et al. 2006) was purchased from Verdera Ltd (Espoo, Finland). The bacteria were maintained as pure cultures on potato dextrose agar (PDA).

Figure 1.

 Typical common scab lesions on potato tubers caused by pathogenic Streptomyces scabiei and by Streptomyces turgidiscabies strains. Scab lesions are often colonized by several Streptomyces species, including nonpathogenic strains. The S. scabiei and S. turgidiscabies strains used in this study were isolated from potato scab lesions, as shown.

Table 1.   The origins and potato pathogenicity of potato-scab-associated Streptomyces strains analysed for boar spermatozoa toxicity
Streptomyces (S.) speciesStrain codePotato cultivarSampling sitePotato patho-genicityReferences
  1. Pathogenicity (+) is defined as causing scab lesions on potato tubers (see Fig. 1). Nonpathogenic (−) indicates that the strain did not cause scab symptoms on the tubers.

S. scabiei271KardalTyrnävä, FinlandLindholm et al. 1997
289FelsinaMikkeli, Finland+Lindholm et al. 1997
364 DSMZ 41744FamboTyrnävä, Finland+Lindholm et al. 1997
14BintjeRasmyran, Sweden+Lehtonen et al. 2004
53 ATCC 49173TKatahdinNew York, USA+Lambert and Loria 1989
S. turgidi- scabies250Van GoghApukka, FinlandLindholm et al. 1997
266SatuApukka, FinlandLindholm et al. 1997
286FamboMikkeli, FinlandLindholm et al. 1997
65BintjeRobertsfors, Sweden+Lehtonen et al. 2004
287 DSMZ 41745FamboMikkeli, Finland+Lindholm et al. 1997
300 DSMZ 41747RocketMikkeli, Finland+Lindholm et al. 1997
304MatildaJuva, FinlandLindholm et al. 1997

The pathogenicity of the Streptomyces strains was tested by inoculation of small immature potato tubers in vitro (Lindholm et al. 1997) and/or inoculation of the bacteria to soil used for potato growing in the greenhouse (Kreuze et al. 1999; Ylhäinen 2001; Hiltunen et al. 2005). Strains 14, 53, 289, 364 (S. scabiei), 287 and 300 (S. turgidiscabies) cause typical common scab lesions on tubers, whereas the strains 271 (S. scabiei), 250, 266 and 286 (S. turgidiscabies) are avirulent on potato.

Preparation of Streptomyces extracts

The bacterial strains were cultivated on tryptic soy agar (TSA) (Scharlau Chemie, Barcelona, Spain) and PDA at room temperature for 14–15 days. Bacterial mass (300–600 mg) was suspended in 10 volumes (v/w) of methanol in a glass bottle, vortexed for 1 min and incubated at room temperature overnight. The suspension was moved to a screw-capped plastic tube and centrifuged at 1500 g (Hettich Rotofix 32, Tuttlingen, Germany) for 5 min. The clear supernatant was transferred to a preweighed glass vial. Methanol was evaporated with nitrogen gas. The solid residue was weighed, resuspended in methanol (HPLC-grade; Mallinckrodt Baker, Deventer, Holland) and the concentration adjusted to 10-mg dry substance per ml. After incubation in boiling water for 15 min, the samples were further diluted with methanol to obtain the concentrations of 1 and 0·1 mg ml−1.

Boar spermatozoa toxicity assay

The toxicity assays were carried out in three independent experiments as described by Hoornstra et al. (2003). In brief, extracts that were diluted in methanol were added in 20 μl aliquots to 2 ml (1% v/v) of extended boar semen (obtained from an artificial insemination center, Jalostuspalvelu, Kaarina, Finland) to reach the exposure concentrations of 100, 75, 50, 25, 15, 10, 5, 2·5, 1 and 0·5 μg day s ml−1, and the spermatozoa were incubated at room temperature for up to 4 days. In 30-min exposures, the extracts were added in a 4-μl volume to 200 μl (2% v/v) of extended boar semen and the exposure concentrations were 200, 100, 20, 10 and 5 μg ml−1.

The motility of the spermatozoa was inspected under a light microscope after 1 and 4 days of exposure to the bacterial extract. Effects on the transmembrane electric potentials (Δψ) of the sperm cells were visualized by staining with JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenz-imidazolyl carbocyanine iodide) as described by Hoornstra et al. (2003). In the assay, high membrane potential (−140 mV) is revealed by orange fluorescence, whereas depolarization of the membrane results in a shift of fluorescence to the green wavelength. Viability (live dead) staining was done with calcein acetoxymethyl (CAM) and propidium iodide (PI) as described by Hoornstra et al. (2003). CAM permeates the plasma membrane and is hydrolysed by cellular cytoplasmic esterases, which results in green fluorescence in the viable cells. PI does not permeate intact cell membranes but enters the cell only following damage to the plasma membrane and binds to DNA yielding red fluorescence of the nuclei. The stained spermatozoa were viewed using an epifluorescence microscope (Axiovert 200M; Carl Zeiss, Göttingen, Germany). The results of the sperm-toxicity test are presented as the lowest observable adverse affect level (LOAEL) of the methanol extracts of the Streptomyces strains or reference substances (μg dry substance per ml semen) causing motility loss and/or membrane damages to boar sperm cells.

HPLC purification and mass spectrometry (MS) analysis

The HPLC column used was an Atlantis C18 4·6 i.d. × 150 mm, 3 μm (Waters, Milford, MA, USA) and isocratic elution was done with 85 : 15; methanol : 0·1% formic acid (v/v) (flow rate 1·0 ml min−1). The bacterial methanol extracts were diluted to 85% methanol by adding aqueous 0·1% formic acid before injection to HPLC. Electrospray ionization ion trap MS analysis (ESI-IT-MS) was performed using an MSD-Trap-XCT plus ion-trap mass spectrometer equipped with 1100 series LC (Agilent Technology, Wilmington, DE, USA). The ESI-IT-MS was performed using positive mode in the mass range of 100–2200 m/z.

The HPLC fractions were screened for toxicity by using the boar sperm motility inhibition assay (Andersson et al. 2004). The toxic peaks (7·40 and 9·25 min with the S. turgidiscabies strain 250) were analysed by HPLC-ESI-IT-MS. The peaks of the corresponding retention times from the S. turgidiscabies strains 266, 286 and 287 were analysed similarly. The mass peaks obtained were further fragmented by MS (MS/MS). Antimycin A (mixture of A1–A4 compounds) was used as reference substance in HPLC-MS analysis. The calculated monoisotopic mass ions [M+H]+ and [M+Na]+ of antimycin A are m/z 507·2 and 529·2 (A4), m/z 521·2 and 543·2 (A3), m/z 535·3 and 557·2 (A2) and m/z 549·3 and 571·3 (A1), respectively.

Chemicals and culture media

Concanamycin A (folimycin from Streptomyces sp.; Calbiochem-Novabiochem Corp., San Diego, CA, USA), valinomycin and antimycin A (Sigma-Aldrich, St Louis, MO, USA) were dissolved in methanol. JC-1, CAM and PI were from molecular probes (Eugene, OR, USA). HPLC-purified thaxtomin A (stock solution 500 ng ml−1 ethanol) was obtained from the liquid cultures of phytopathogenic strains of S. scabiei and S. turgidiscabies grown in oat meal broth, as previously described (Hiltunen et al. 2006). TSA was from Scharlau Chemie S.A. (Barcelona, Spain) and PDA from Biokar Diagnostics (Allonne, France).

Results

Seven S. turgidiscabies strains and five S. scabiei strains, isolated from potato scab lesions and representing phytopathogenic and nonpathogenic strains, and the biocontrol strain S. griseoviridis K61 (Table 1), were investigated by using the boar spermatozoan motility test. The strains were grown on solid culture media and the obtained biomass was extracted with methanol. The extracts were initially tested for toxicity using the boar sperm motility inhibition assay. When motility inhibition was found, the effect of the substances on mitochondrial and the plasma membrane potentials of sperm cells were studied using JC-1 staining and CAM/PI staining. Based on the data, the LOAEL of the Streptomyces methanol extracts (μg dry substance per ml semen) was determined in terms of motility loss and damages detected in the sperm cells.

Toxic substances were obtained from three nonphytopathogenic strains of S. turgidiscabies (250, 266 and 286), one phytopathogenic and one nonpathogenic strain of S. scabiei (strains 289 and 271, respectively), and the S. griseoviridis strain K61 (Table 2a). The remaining potato pathogenic strains, four of S. turgidiscabies (65, 287, 300 and 304) and two of S. scabiei (14, 53) exhibited no detectable toxicity on the sperm cells in terms of motility inhibition (Table 2a). Hence, toxicity was found, but it was not correlated with phytopathogenicity of the strains.

Table 2.   Toxicity of substances from Streptomyces strains causing or not causing potato scab
 StrainCulture mediaLOAEL (μg d.s. per ml)
Motility lossΔΨm lossViability loss
30 min1 day4 days1 day4 days4 days
(a) Streptomyces (S.) species
S. scabiei271PDAn10075n100n
 TSAn5025n50n
289PDAnnn***
 TSAn7550n75n
364, 14, 53PDAnnn***
 TSAnnn***
S. turgidiscabies250PDA511501n
 TSA201010n10n
266PDA52·52·51002·5n
 TSA52·52·51002·5n
286PDA52·52·51002·5n
 TSA201010n10n
65, 287, 300, 304PDAnnn***
 TSAnnn***
S. griseoviridisK61PDAn5025nnn
 TSAnnn***
Vehicle controls
Methanol  nnnnnn
Ethanol  nnnnnn
SubstanceBiological activityLOAEL (μg d.s. per ml)
Motility lossΔΨm lossViability loss
30 min1 day4 days1 day4 days4 days
  1. The toxicity was assayed by exposing boar sperm cells to methanol extracts prepared from plate cultures of the Streptomyces strains (a) and compared with those provoked by reference substances (b). The lowest observable adverse effect levels (LOAEL) are indicated for motility loss, the loss of ΔΨm and viability loss. All values for the Streptomyces extracts are expressed as dry weight (d.s.) of methanol-soluble substance extracted from cultures harvested from agar plates.

  2. S., Streptomyces; n, no adverse effect observed at the highest tested concentration (200 μg d.s. per ml); PDA, potato dextrose agar; TSA, tryptic soy agar.

  3. *Analysis not done.

  4. Natsume et al. 2005.

  5. Dröse and Altendorf 1997.

  6. §Teplova et al. 2007.

  7. **Andersson et al. 1998.

  8. ††Crofts 2004.

(b) Reference substances
 Thaxtomin APhytotoxin†*nn***
 Concanamycin APhytotoxin†, inhibitor of V- and P-ATPases‡*nnnn*
 Bafilomycin A1Inhibitor of V- and P-ATPases‡ and K+ ionophore§*0·020·0025*0·0025*
 ValinomycinPotassium ionophore§,**0·0050·00250·00250·00250·0025n
 Antimycin AInhibitor of electron transfer at complex III††0·0050·0050·0050·250·005n

The extracts prepared from the cultures of strains 250 and 286 grown on PDA were more toxic (i.e. lower LOAEL) than those obtained from these bacteria grown on TSA, whereas the contrary was found with strain 271 (Table 2a). Strains 289 and K61 exhibited toxicity only when grown on TSA and PDA, respectively.

The extracts from S. turgidiscabies strains 250, 266 and 286 exhibited their full toxic effect rapidly. They inhibited sperm cell motility completely at low concentrations [5–20 μg dry weight (d.s.) per ml] already following 30 min of exposure (Table 2a). Even lower concentrations (1–10 μg d.s. per ml) inhibited motility following an exposure for 1–4 days. In contrast, extracts from S. scabiei strains 271 and 289 and S. griseoviridis K61 brought on toxicity but slow acting. No adverse effects were observed after exposure of 30 min up to 200 μg d.s. per ml1, but after 1 or 4 days, a LOAEL of 25–75 μg d.s. per ml was reached (Table 2a). The mode of sperm motility inhibition of the two S. scabiei extracts also differed from those of the toxic S. turgidicabies extracts by greatly slowing down the tail whipping without completely blocking the motility at the LOAEL exposure concentrations.

The extracts that impaired sperm cell motility were tested for effects on the transmembrane electric potentials of sperm cells, as visualized by JC-1 staining (Fig. 2). When the lowest sperm motility inhibiting concentrations of extracts from S. turgidiscabies strains 250, 266 and 286 were used, 4 days was required for an observable loss of Δψm (Table 2a; Fig. 2c,d). None of the extracts that caused motility inhibition or changes visible in JC-1 staining, caused loss of viability detectable by the live dead staining up to doses of 100 μg d.s. per ml and exposures for up to 4 days (Table 2a).

Figure 2.

 Epifluorescence micrographs of boar sperm cells exposed to methanol-soluble substances from Streptomyces turgidiscabies strains and reference substances, stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenz-imidazolo carbocyanine iodide (JC-1), a dye that changes emission wavelength in response to decreasing membrane potential by a shift from orange (high) to green (low). The amount of methanol-soluble dry matter per 1 ml of the semen dilution and the exposure time in days are displayed on the panels. (a) Sperm cells exposed to methanol (vehicle); the mitochondria in the spermatozoan midpiece fluoresce bright orange, indicating high Δψm. (b) Sperm cells exposed to the extract from strain 287. (c, d). Exposure to motility inhibiting dose of the extract of strain 250 for 1 and 4 days, respectively. The results for strains 266 and 286 were similar to that of strain 250. (e, f) Sperm cells exposed to antimycin A, 0·005 μg ml−1 for 1 and 4 days, respectively. (g) Cells exposed to valinomycin, 0·0025 μg ml−1 for 1 day.

Thaxtomin A and concanamycin A are phytotoxins contributing to the phytopathogenicity of S. scabiei. These substances had no effect on sperm cell motility at concentrations up to 1·0 μg ml−1 and 2·5 μg ml−1, respectively, during an exposure for up to 4 days (Table 2b). Furthermore, the scab pathogenic reference strain S. scabiei 53 (ATCC 49173), a known producer of thaxtomin A and concanamycin A (Table 1), yielded an extract with no toxicity on the sperm cells up to concentrations of 100 μg (d.wt.) per ml (Table 2a). These results show that the substances toxic to sperm cells were different from those causing the pathogenicity on potato. No mitochondriotoxicity was detected in the extracts of the biocontrol strain S. griseoviridis K61 (Table 2a).

Although unrelated to potato scab pathogenicity, the data compiled in Fig. 2 and Table 2a indicate that three S. turgidiscabies strains (250, 266, 286) produced substances that were highly potent in causing mitochondrial damage in sperm cells. When these effects were compared (Table 2b, Fig. 2) with those of other reference substances, it was found that the effects caused by the three S. turgidiscabies extracts were similar to those by antimycin A (0·005 μg ml−1) – a rapid loss of motility and a delayed loss of Δψm. The effects differed from those of valinomycin and of bafilomycin A1 (Table 2b, Fig. 2g). The toxic effects brought on by the extracts from S. scabiei strains 271 and 289 and S. griseoviridis strain K61 did not resemble those of any of the tested reference substances.

Next, the S. turgidiscabies extracts toxic to sperm cells were further characterized by HPLC-ESI-IT-MS. Two HPLC fractions were identified as toxic in the sperm test, one major toxic peak eluted in all cases at c. 9 min and contained components with mass ions of m/z 549·.5 and 571·5 (for strain 286, see Fig. 3a). The minor toxic peak eluted at c. 7 min and yielded mass ions with m/z 535·5 and 557·5. None of these mass ions were found when nontoxic S. turgidiscabies strain 287 was analysed with HPLC-MS (Fig. 3a). The commercial antimycin A preparation used in the boar sperm toxicity test (Table 2b; Fig. 2e,f), consisting of a mixture of the antimycins A1, A2, A3 and A4, was similarly analysed. It was found that both the retention time (9 min) and the mass ions [M+H]+ at m/z 549·4 and [M+Na]+ at m/z 571·5 (Fig. 3b), of antimycin A1 matched with those of the major toxic peak from the S. turgidiscabies and the retention time of antimycin A2 (7 min), and mass ions [M+H]+ at m/z 535·5 and [M+Na]+ at m/z 557·5 matched with those obtained from the minor toxic HPLC peak. The MS/MS analysis of the toxic strains 250, 266 and 286 using the precursor ion [M+Na]+ at m/z 571·5 yielded eight main product ions with m/z from 186·0 to 408·3 (for the strain 286, see Fig. 3c). All of these product ions were identical to those obtained by MS/MS analysis of the commercial antimycin A1 precursor ion [M+Na]+ at m/z 571·5 (Fig. 3d). These data indicate that the toxic fraction in S. turgidiscabies extracts eluting at c. 9 min was identical to antimycin A1. The HPLC peaks that displayed toxicity did not contain mass fragments with m/z values different from those originating from the antimycin A. This indicates that no other substance (e.g. bafilomycin A or valinomycin) contributed to the sperm cell toxicity of the three S. turgidiscabies strains.

Figure 3.

 The HPLC-MS analysis of the methanol extracts of the toxic Streptomyces turgidiscabies strain 286 (marked with blue colour) and of the nontoxic S. turgidiscabies strain 287 (marked with red colour). (a) The mass spectra of toxic fraction of the methanol extract of S. turgidiscabies strain 286 eluted at 9·01 min and nontoxic S. turgidiscabies strain eluted at 9·31 min. (b) The mass ions [M+H]+ at m/z 549·5 and [M+Na]+ at m/z 571·5 of antimycin A1 eluted at 9·37 min. (c) The MS/MS spectrum of the precursor ion at m/z 571 from toxic compound of S. turgidiscabies strain 286. (d) The MS/MS spectrum of the precursor ion [M+Na]+ at m/z 571 of antimycin A1.

Discussion

We identified substances highly toxic by the boar spermatozoan motility test in strains of S. turgidiscabies isolated from potato scab lesions and grown in vitro. The highest toxicities were detected from three strains nonpathogenic to potato but inhabiting scab lesions caused by pathogenic strains (Lindholm et al. 1997). In addition, one out of the seven tested potato pathogenic biovars was toxic but only at exposure concentrations 25 to 50 times higher. Thaxtomin A and concanamycin A, the known phytotoxins of S. scabies (Natsume et al. 2005), had no observable toxicity in the boar spermatozoan assay.

Although the phytopathogenicity of S. turgidiscabies could not be explained by substances detectable in the spermatozoan assay, the highly toxic substance identified from this species deserves attention. Antimycin A is known to be fungicidal (Klueppel et al. 1970; Gräfe 1992), highly toxic to fish and mammals (Lennon et al. 1970) and cytotoxic in mammalian cells (Campas et al. 2006; Duewelhenke N. and Eysel 2007). Antimycin A has so far been reported from Streptomyces antibioticus (Rehacek et al. 1968; Neft and Farley 1972; Gräfe 1992), in addition to undefined Streptomyces spp. isolated from soil (Klueppel et al. 1970). The results suggest that c. 0·5 weight per cent of the crude, cell-free extract of S. turgidiscabies may have consisted of antimycin A. The present report is to our knowledge the first one to document the presence of an antimycin A producer isolated from a crop plant.

The three S. turgidiscabies strains produced more antimycin A when grown on PDA than on TSA, suggesting that antimycin A production by S. turgidiscabies could be produced during growth on potato tubers and in the scab lesions on tubers. Many soilborne Streptomyces species produce antimicrobial substances on laboratory media, but antimicrobials are seldom found in soil. Further studies should investigate whether antimycin A is produced while the S. turgidiscabies grow in potato scab lesions.

Antimycin A may not be toxic to potatoes owing to the electron-transfer chain of plant mitochondria that contains five additional enzymes compared with mammalian mitochondria (Taiz and Zeiger 2006). One of these enzymes is the alternative oxidase (AOX), which enables plants to survive exposure to inhibitors of the respiratory complexes, such as the cytochrome b inhibitor antimycin A. AOX bypasses cytochrome bc1 complex to deliver electrons directly from ubiquinone to O2 (Vanlerberghe and McIntosh 1992; Wagner and Wagner 1997; Geisler et al. 2004). Mitochondria with AOX, as those in potato plants (Pinheiro et al. 2004) are insensitive to antimycin A. Antimycin A production could be a mechanism for S. turgidiscabies to compete against other microbes inhabiting similar ecological niches, being potentially beneficial for the tubers against aerobic phytopathogenic bacteria, fungi and nematodes. Recent studies show that while many fungi contain the AOX pathway, the phytopathogenic species may not efficiently utilize it during the infection process, which impairs their growth on plants (Avila-Adame and Köller 2002).

The toxic activities of the biocontrol strain S. griseoviridis K61 and of the S. scabiei strains differed from that of the antimycin-producing S. turgidiscabies strains. Thus, the crop-protecting effect of K61 against plant pathogens (Tahvonen 1982; Anon 2003) cannot be based on antimycin A.

Vacuolar organelles are not needed for sperm cell motility, and this may explain why concanamycin A does not affect sperm cells. Concanamycin A is toxic through inhibition of vacuolar H+ ATPase (Dröse and Altendorf 1997). The complete lack of sensitivity of sperm cells to thaxtomin A and concanamycin A and extracts from their producer strain S. scabiei ATCC 49173 (Natsume et al. 2005) indicates that the substance produced by the sperm toxic strains S. scabiei 271 and 289 must be different from concanamycin A or thaxtomin A. The detected level of toxicity towards the sperm cells was however too low to be usable for screening of fractionated extracts as required for purifying the toxic substance from these strains.

Considering the high mammalian toxicity of antimycin A (Lennon et al. 1970), Streptomyces strains which produce antimycin A might be a safety risk if the amounts produced in plant materials used for food and feed reach significant concentrations. However, data to this end are not available and needs to be further studied.

Acknowledgements

Financial support to J.P.T. Valkonen from the Ministry of Agriculture and Forestry (grant 4655/501/2003) and Finnish Technology Agency TEKES (grant 2431/31/04) is gratefully acknowledged. This project was a part of CoE Microbial Resources, supported by the Academy of Finland (grant 53305). We thank the Faculty Instrument Centre and Viikki Science Library for expert support, and Leena Steininger, Hannele Tukiainen and Tuula Suortti for many kinds of help.

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