INTRODUCTION

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Common scab of potato is caused by several Streptomyces species (12, 28) and is widely distributed in potato-growing areas. Common scab is also occasionally found on other crops, such as carrot, beet, radish, parsnip, and turnip (12). Superficial or deep corky lesions on tubers or roots, which affect the quality of the vegetables and thus reduce their marketable yields, characterize the disease.

Streptomyces scabies, a soilborne actinomycete, is considered the principal causal agent of potato scab (26). Lambert and Loria (20) characterized S. scabies as an organism that has smooth gray spores borne in spiral chains, produces melanin, and is able to utilize L-arabinose, D-fructose, D-glucose, D-mannitol, D-xylose, raffinose, rhamnose, or sucrose as a carbon source.

Lawrence et al. (21) elucidated an important aspect of the pathogenicity of S. scabies. These authors demonstrated that phytotoxins called thaxtomins are produced by this pathogen. Thaxtomins are unique 4-nitroindol-3-yl-containing 2,5-dioxopiperazines. These toxins induce the development of necrotic lesions on aseptically cultured potato minitubers (21). King et al. (16) also demonstrated that pathogenicity was positively correlated with the ability of S. scabies strains to produce thaxtomin A on potato slices. Goyer et al. (13) showed that mutants of S. scabies that exhibited reduced production of thaxtomin A were less virulent than a wild strain, which emphasized the potential importance of thaxtomins in pathogenesis. Even though it has been demonstrated that thaxtomin A does not affect the growth of microorganisms (11), the effect of thaxtomin production on the microbial ecology of the rhizosphere has not been studied extensively yet.

The methods used to control common scab include chemical treatment of seed potato tubers (6), irrigation (1), and soil amendments (40). Biological control of potato scab by nonpathogenic streptomycetes has also been described (24, 34). It has been suggested that these antagonistic streptomycetes control common scab by producing antibiotics that inhibit the growth of the pathogens (7). Control methods that interfere with thaxtomin synthesis or control the negative effects of thaxtomin on plant tissues have not been proposed yet. In this work, we hypothesized that thaxtomin A-utilizing microbes may protect potato tubers against potato scab.

The purposes of this study were (i) to identify microorganisms capable of degrading thaxtomin A and (ii) to evaluate the potential of these thaxtomin A-utilizing microorganisms as biocontrol agents.

	MATERIALS AND METHODS

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Thaxtomin purification. Thaxtomin A was purified from culture supernatant of S. scabies EF-35 (30) grown in oat bran medium (13) for 6 days at 30°C as previously described (13). Briefly, the culture supernatant was extracted twice with an equal volume of ethyl acetate. The ethyl acetate extract was concentrated by evaporation and applied to a Silica Gel 60 thin-layer chromatography plate. The yellow spot having an R_f of 0.27 (thaxtomin A) was scraped off the plate, eluted with chloroform-methanol (7:3), and air dried. Thaxtomin A was purified further on Whatman KC₁₈F plates.

Screening for thaxtomin A-utilizing microorganisms. A total of 186 isolates of nonpathogenic actinomycetes isolated from potato tubers (9) were examined for the ability to utilize thaxtomin A as a sole nitrogen or carbon source. Utilization of thaxtomin A as a sole carbon source was analyzed in MSB medium, a minimal medium containing (per liter of water) 0.5 g of K₂HPO₄, 0.2 g of MgSO₄ · 7H₂O, 2 g of (NH₄)₂SO₄, and 0.01 g of FeSO₄ · 7H₂O, supplemented with 20 mM thaxtomin A (molecular weight, 438). Utilization of thaxtomin A as a sole nitrogen source was analyzed in MSB medium lacking (NH₄)₂SO₄ and supplemented with 0.5% glycerol and 2.0 mM thaxtomin A.

Characterization and identification of thaxtomin A-utilizing microbes. Fungal isolates were associated with a genus by using the taxonomic key proposed by Malloch (29). The gram-negative isolate was identified by using the Biolog System, release 3 (Biolog Inc., Hayward, Calif.) as recommended by the manufacturer.

Biocontrol assay. Thaxtomin A-utilizing bacteria were tested for the ability to protect potato tubers against S. scabies infection. An S. scabies inoculum was prepared by growing strain EF-35 for 2 weeks at 30°C in 50-ml tubes containing vermiculite saturated with a Say solution containing (per liter of water) 20 g of sucrose, 1.2 g of L-asparagine, 0.6 g of K₂HPO₄, and 10 g of yeast extract (18). Whole potato tubers (cultivar Kennebec) that were free from common scab were soaked twice for 15 min each time in a 0.5% sodium hypochloride solution and rinsed in sterile water for 15 min. The potato tubers were then soaked for 5 min in sterile TSB (controls) or in a 3-day-old TSB culture of a thaxtomin A-utilizing bacterium. Potato tubers, including the controls, were then planted in 12.5-cm-diameter pots containing sterile sand mixed with an inoculum containing S. scabies EF-35. Five replicates of pots were randomly dispersed in a growth chamber. Potatoes were grown at 25°C with a 16-h photoperiod and were harvested after 3 months. The progeny tubers were then examined for symptoms of scab infection (13).

Nucleotide sequence accession number. The 16S ribosomal DNA sequence of Streptomyces sp. strain EF-73 has been deposited in the GenBank database under accession no. AF076309.

	RESULTS

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Selection of thaxtomin A-degrading microorganisms. A total of 186 nonpathogenic actinomycetes previously isolated from potato tubers (9) were examined in order to identify thaxtomin A-utilizing bacteria. Of these 186 isolates, 2 strains (EF-50 and EF-73) exhibited slight growth in minimal medium containing thaxtomin A as the sole nitrogen source. None of the actinomycetes grew when thaxtomin A was added to minimal medium as the sole carbon source. Growth of strains EF-50 and EF-73 in a medium containing thaxtomin A as the nitrogen source resulted in significant decreases in the OD₄₀₀ culture supernatants (Table 1). Since thaxtomin A is a yellow compound, a decrease in the OD₄₀₀ of a culture supernatant might reflect some microbial degradation of thaxtomin A. Furthermore, HPLC quantification of residual thaxtomin A amounts in culture supernatant extracts revealed that strains EF-50 and EF-73 removed about 9% of the thaxtomin A originally present in the culture medium (Table 1).

Identification and characterization of thaxtomin A-utilizing microorganisms. Gram-negative bacterial strain S-2016 was identified as a Burkholderia pickettii strain with a probability of 85.6% by using the Biolog System. Below, we refer to this strain as Ralstonia pickettii S-2016 since Yabuuchi et al. (43) proposed that B. pickettii should be transferred to the genus Ralstonia as R. pickettii.

Biocontrol assay. Inoculation of potato tubers with EF-50, EF-73, or S-2016 resulted in protection of progeny potato tubers against common scab. In control treatments, more than 70% of the potato tubers harvested had common scab lesions, while the percentage of infected potato tubers was less than 40% when potato tubers were soaked in cultures of thaxtomin A-utilizing bacteria before planting. In addition, soaking the potato tubers in a medium containing thaxtomin A-utilizing bacteria significantly reduced the severity of the symptoms. The area of scab lesions on the infected potato tubers accounted for less than 5% of the surface area when potato tubers were treated with thaxtomin A-utilizing bacteria, compared to 25% of the surface area for the control treatment (Table 1). In contrast, the fungal strains Penicillium sp. strain CL-8 and Trichoderma sp. strain CL-22 were not able to control potato scab. The percentage of infected tubers, as well as the area of lesions on infected tubers, did not differ significantly in control treatments and fungal treatments (Table 1).

	DISCUSSION

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We showed that various microorganisms, including gram-positive and gram-negative bacteria, as well as fungi, can degrade thaxtomin A, a phytotoxin produced by S. scabies. Five thaxtomin A-utilizing strains were identified. Growth of these strains in thaxtomin A-containing media could not be attributed to the presence of contaminating substances since the amount of thaxtomin A in the media significantly decreased after microbial growth, as shown by HPLC analysis (Table 1). As an alternative to HPLC analysis, we established that thaxtomin A biodegradation could also be estimated by measuring the OD₄₀₀ of culture supernatants (Table 1).

Thaxtomin A is a nitroaromatic compound, and like degradation of other molecules of this type, degradation of thaxtomin A has been associated with various microorganisms, including Trichoderma sp., Penicillium sp., Streptomyces sp., and R. pickettii. Degradation or biotransformation of other nitroaromatic compounds, such as 2,4,6-trinitrotoluene and p-nitrobenzoate, by Trichoderma sp. (2), Penicillium sp. (3), Streptomyces sp. (10), and R. pickettii (42) has been described previously.

The catabolic pathway for biodegradation of thaxtomin A was not elucidated in this study. Studies are underway to determine if the enzymatic mechanisms involved in thaxtomin A catabolism are similar to those involved in biodegradation of other nitroaromatic compounds. It has been shown that in Penicillium and Trichoderma species nitroaromatic compounds are catabolized via a nonspecific complex of extracellular peroxidases involved in lignin degradation. The level of degradation of lignin by Penicillium chrysogenum was 8%, as determined by spectrophotometry (33), a value which is comparable to the level of degradation of thaxtomin A by Penicillium sp. strain CL-8 (10%).

Catabolism of nitroaromatic compounds is initiated by nitroreductases in R. pickettii (42) and in actinomycete strains (10, 23, 31, 35). It has not been confirmed that nitroreductase synthesis occurs in R. pickettii S-2016 or in Streptomyces sp. strains EF-50 and EF-73. However, the fact that these strains could degrade other nitroaromatic compounds (results not shown) suggests that the enzymes involved in the catabolism of thaxtomin A are not specific to thaxtomin A. Nitroreductases often exhibit activity with diverse nitroaromatic compounds (17).

No strict correlation between the ability to control potato scab and the ability to degrade thaxtomin A was established in this study. The thaxtomin A-degrading fungi Penicillium sp. strain CL-8 and Trichoderma sp. strain CL-22 were ineffective in controlling potato scab, although they were able to grow with thaxtomin A as a carbon and nitrogen source. Worse, common scab lesions on potato tubers treated with both fungi were surrounded by soft rot symptoms, suggesting that these fungi are pathogenic or opportunistic organisms. In contrast, all three thaxtomin A-utilizing bacteria protected potato tubers against common scab. Even though the ability of these bacterial isolates to protect potatoes against common scab has not been linked to their ability to degrade thaxtomin A yet, other workers (22, 37) have reported that detoxification of phytotoxins is a mode of action of some biocontrol agents.

Other strains belonging to the genera Burkholderia and Streptomyces have been reported to control plant diseases (14, 19, 44). Different mechanisms of action, such as production of antibiotics (7), production of lytic enzymes (5, 39), production of plant hormones (38), and production of siderophores (4), have been associated with Streptomyces and Burkholderia biocontrol agents. Thus, mutagenesis of the genes involved in thaxtomin A degradation should be carried out to determine the role of thaxtomin A catabolism in biocontrol.

The use of streptomycetes to control common scab of potato has been described previously by Liu et al. (24, 25). These authors isolated from suppressive soils some Streptomyces strains that were antagonistic to S. scabies, the main agent of potato scab, and they used these strains with success in field experiments to control common scab. In contrast to these suppressive streptomycetes, thaxtomin A-degrading Streptomyces strains EF-50 and EF-73 did not inhibit S. scabies growth (data not shown). This finding suggests that the mechanisms that protect tubers are different in the suppressive and thaxtomin-utilizing actinomycetes. The suppressive Streptomyces strains were identified as Streptomyces diastatochromogenes strains (27), while strains EF-50 and EF-73 were phylogenetically related to S. mirabilis on the basis of their 16S rRNA gene sequences. S. diastatochromogenes, S. mirabilis, and the plant-pathogenic organism S. scabies are members of the Diastatochromogenes group (20, 41); this could explain the sharing of several phenotypic traits by biocontrol agents and pathogens. Thaxtomin A-utilizing strains EF-50 and EF-73 were previously isolated from potato tubers (9), while the suppressive strains identified by Liu et al. (24) were isolated from a potato field. Thus, these biocontrol strains have an ecological niche similar to that of the pathogenic strains. The occupation of a similar ecological niche may also explain the common phenotypic traits observed in biocontrol agents and common scab-inducing pathogens.