Phytotoxin Produced by Streptomyces cheloniumii Causing Potato

Chapter 22

Phytotoxin Produced by Streptomyces cheloniumii Causing Potato Russet Scab 1

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Masahiro Natsume, Mayumi Komiya , Fumie Koyanagi , Hiroshi Kawaide , Nobuya Tashiro , and Hiroshi Abe 1

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Downloaded by FUDAN UNIV on April 10, 2017 | http://pubs.acs.org Publication Date: November 23, 2004 | doi: 10.1021/bk-2005-0892.ch022

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Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Saga Fruit Tree Experiment Station, Ogi-cho, Saga 845-0014, Japan

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The russet scab phytotoxin was isolatedfromStreptomyces chelonium ii MAFF 304020 and identified as an 18-membered macrolide, FD-891. It induced necrosis m potato slices at 50 μg/disk, which was 1/100th the activity of thaxtomin A. The phytotoxin was produced by other pathogenic strains, Streptomyces spp. MAFF 225003, 225005 and MAFF 225006, which indicates that this phytotoxin is a pathotoxin.

Studies of phytotoxins produced by phytopathogenic microorganisms are needed not only to elucidate diseases mechanisms, but also to find new targets for the development of herbicides. Potato tuber diseases caused by actinomycetes can be classified into two groups: common scab, which is characterized by corky erumpent or pitted symptoms, and russet or netted scab, which shows superficial reticulations (7). The phytotoxin thaxtomin A (1) was isolated from Streptomyces scabies, the pathogen of common scab, by King et al{2) Thaxtomin A production has been shown to be a pathogenicity factor in common scab because there is a positive correlation between the ability to produce thaxtomin A in various S. scabies isolates and their pathogenicity (5, 4). Outer pathogens of common scab,

© 2005 American Chemical Society Clark and Ohkawa; New Discoveries in Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

239


240 such as S. acidiscabies (J, 4, 5) and S. turgidiscabies (6), also produce thaxtomin A. We showed that 5. scabies produces another phytotoxins, concanamycin A and Β (2X7), and that their production is specific to S. scabies (5). We detected concanamycin A and Β in common scab lesions. The diversity of symptoms in common scab such as erumpent or pitted type may be attributed to the production of concanamycins, although their contribution to pathogenicity has yet to be clarified. On the other hand, there have not been any reports with regard to phytotoxins produced by pathogens that induce superficial symptoms* such as russet or netted scab. O n M et al (8) and Suzui et al (9) showed that the pathogen of russet scab is a Streptomyces sp. and is distinguished from Streptomyces spp. that cause common scab by its negative production of melanin and the spiny structure of its spore surface and they named it S. cheloniumii. They also showed that S. cheloniumii induced russet scab symptoms and not common scab symptoms (8). These results indicate that the pathogen of russet scab produces a phytotoxin other than thaxtomin A and that the phytotoxin is a causal agent for russet scab. We therefore searched for the phytotoxin produced by S. cheloniumii.

Figure I. Structures ofphytotoxins produced by Streptomyces species causing potato scab.

Clark and Ohkawa; New Discoveries in Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

241 We describe here the isolation and identification of a phytotoxin produced by S. cheloniumii and by other Streptomyces strains causing russet scab.


Materials and Methods Bacterial strains The bacterial strains used in this study are listed in Table 1. S. cheloniumii MAFF 304020 and Streptomyces spp. MAFF 225003 and 225004 were collected in Chiba prefecture, Japan, Streptomyces sp. MAFF 225005 in Hokkaido, and Streptomyces sp. MAFF 225006 in Ibaraki prefecture. For isolation of the russet scab toxin, S. cheloniumii MAFF 304020 was used. Pathogenicity assay in greenhouse Each strain was inoculated into a yeast extract-malt extract medium from the agar slant culture and cultivated at 28°C for 5 days on a reciprocal shaker. The cultured material (5 ml) was inoculated in 500 g of soil-bran medium in a 1 liter Erlenmeyer flask and mixed well. The flask was incubated at 23°C for 5 months. The cultured material was mixed with sterilized soil to adjust the population density of pathogens to 52 - 8.3 χ 10 cfu/g soil. The soil used for dilution of the infected soil and for the control experiment was adjusted to pH 6.6 with line. Seed potato (cv. Dansyaku) was planted in the infected soil and cultivated for 3 months under short-day conditions in a greenhouse. Three replicate pots of seed pieces were used for each strain. 4

Analysis for thaxtomin A, concanamycins and russet scab toxin Each strain was cultured on an oatmeal agar medium (10) at 28°C for 14 days. Thaxtomin A and concanamycins were determined with an HPLC equipped with a photodiode array detector. The analytical procedure was described in detail previously (5). Conditions for analysis of the russet scab toxin were the same as those for the concanamycins except for the detection wavelength (266 nm). Isolation ofrusset scab toxin produced by S. cheloniumii S. cheloniumii MAFF 304020 was cultured on an oatmeal agar medium at 28°C for 14 days. The cultured material was macerated with acetone, and the acetone extract was purified as outlined in Figure 2. Tuber slice assays (4) were used for fractionation guides. Results Symptoms induced by pathogenicity assay First, it was determined whether or not the symptoms caused by the russet scab pathogens differed from those by common scab pathogens. All of the 5. cheloniumii MAFF 304020 and Streptomyces spp. MAFF 225003, 225004, 225005 and 225006 strains induced superficial symptoms, which were rough in


242 texture and had shallow cracks such as tortoise shell (Table 1). Lesions caused by S. scabies, S. acidiscabies, or S. turgidiscabies are necrotic and sunken and are entirely different from those caused by russet scab pathogens. There was no clear difference among symptoms caused by S. scabies, S. acidiscabies and 51 turgidiscabies.

Table 1. Results of Pathogenicity Assay and Production of Phytotoxins by Streptomyces spp.


species

strain

S. cheloniumii MAFF 304020 Streptomyces sp. MAFF 225003 Streptomyces sp. MAFF 225004 Streptomyces sp. MAFF 225005 Streptomyces sp. MAFF 225006 S. scabies JCM 7914 S. acidiscabies JCM 7913 S. turgidiscabies IFO16080 1

2 3 4

symptoms induced superficial superficial superficial superficial superficial necrotic, sunken necrotic, sunken necrotic, sunken 1

production'' of Txt(l) Con ( 2 ) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 2.50 0.086 n.d. 5.31 2.37 n.d. 3

3

4

Results of pathogenicity assay. unit: ug/ ml cultured material Txt (1): thaxtomin A; Con (2): cncanamycins A plus Β n.d. : not detected

Production of thaxtomin A and concanamycins Production of the known phytotoxins was examined in an agar culture. None of the pathogens that caused russet scab produced thaxtomin A or concanamycins (Table 1). S. scabies produced both thaxtomin A and concanamycins, as described previously (5). S. acidiscabies and S. turgidiscabies produced only thaxtomin A. These results indicate that the pathogens of russet scab produce a new phytotoxin. Isolation and identification of the russet scab phytotoxin The procedure for purifying the russet scab toxin is outlined in Figure 2. Chloroform extract was fractionated by silica gel column chromatography. Only the 10% MeOH - CHC1 fraction induced necrosis by the tuber slice assay. This fraction was rechromatographed using a different solvent system. The active fraction was then purified by preparative TLC and separated into five fractions 3


243 with the guidance of U V absorption. The active fraction with R 0.38-0.47 was finally purified by ODS-HPLC and afforded a single peak. f

S. cheloniumii MAFF 304020 cultured material J acetone extraction acetone extract

J

CHCI3

extraction at pH 3

CHCb extract si lb a gel column chromatography (MeOH-CHCI ) Downloaded by FUDAN UNIV on April 10, 2017 | http://pubs.acs.org Publication Date: November 23, 2004 | doi: 10.1021/bk-2005-0892.ch022

3

— 0%

ι — ι — ι — ι 1 10% 20% 40% 60% 100% silica gel column chromatography (Et Ο Ac - n-hexane) — 50%

ι — 70%

ι — 90%

I 100% slica gel preparative TLC (10% Me OH h CHCI3)

o.O.OO- 0.13- 0.14- 0.38' 0.13 0.14 0.38 0.47 f

0.471.00

ODS-HPLC (70% aq. CH CN, UV220 nm) 3

active compound (fo 14.6 min) Figure 2. Purification procedure for russet scab toxin.

+

The phytotoxin showed a quasi molecular ion at m/z 579 (M + H) with FAB-MS (matrix: glycerol), the molecular formula of which was determined to be C33H55O8 based on the results of high resolution FAB-MS (m/z 579.3867, calcd. 579.3897). The UV spectrum of the toxin showed maxima at Xmax 205 and 267 nm. In the 'H-NMR spectrum (500 MHz, CDC1 ), 5 doublet methyl, 2 olefinic or acetyl methyl, 1 methoxy methyl and 6 olefinic proton signals were observed as characteristic peaks. Searching a database based on the molecular formula and narrowing down candidates from the features of the *H-NMR spectrum, the russet scab toxin was presumed to be the 18-membered macrolide, FD-891 (3) (//, 12, 13). Comparison of the retention time and UV spectrum in ODS-HPLC and of the H-NMR spectrum of the isolated toxin with those of an authentic sample confirmed that the phyototxin was FD-891. 3

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244 Necrosis-inducing activity of the russet scab toxin The russet scab toxin induced weak necrosis at 50 μg/disk and clear activity at 100 Hg/disk (Figure 3). Thaxtomin A showed similar activity at 1 μg/disk.

Control Downloaded by FUDAN UNIV on April 10, 2017 | http://pubs.acs.org Publication Date: November 23, 2004 | doi: 10.1021/bk-2005-0892.ch022

^g

russet scab toxin 10 μg 50 μg

thaxtomin A 100 μg 1 m

Figure 3. Necrosis inducing-activity of the russet scab toxin and thaxtomin A.

Production of the russet scab toxin by other strains Whether production of the russet scab toxin would be common to other russet scab pathogens was examined in two strains isolated in different regions. The results showed that Streptomyces spp. MAFF 225003, 225005 and 225006 produced a phytotoxin with the same retention time and UV spectrum in HPLC analysis. Discussion We showed that the russet scab pathogens produced a phytotoxin other than thaxtomin A or concanamycins and isolated it. The phytotoxin was identified as the 18-membered macrolide, FD-891, and its effect in inducing necrosis on potato tuber disks was about 1/100th that of thaxtomin A. The russet scab toxin was produced by the other three pathogenic strains that were isolated in different regions. These results strongly suggest that the phytotoxin is a pathotoxin. To confirm this possibility, we are now trying to detect the phytotoxin in lesions. FD-891 has been reported as a cytocidal compound for mammal cells (77), and this is the first report on its phytotoxicity. FD-891 has the same ring size as concanamycins and both have a quite similar structure. Phytotoxic activity of concanamycins is based on the inhibition of V-ATPase (14). FD-891 does not show inhibitory activity in mammal cells (15), though the first report described its inhibitoiy activity (77). The action mechanism of the toxin remains an interesting problem. Acknowledgment We are grateful to Professors Katsumi Kakinuma and Tadashi Eguchi, Tokyo Institute of Technology for providing authentic FD-891 and its NMR spectra.



245 References (1) Loria, R.; Bukhalid, R. Α.; Fry, Β. Α.; King, R. R. Plant Dis. 1997, 81, 836846. (2) King, R. R.; Lawrence, C. H.; Clark, M. C.; Calhoun, L. A. J. Chem. Soc., Chem. Commun. 1989, 849-850. (3) King, R. R.; Lawrence, C. H.; Clark, M. C. Am. Potato J. 1991, 68, 675-680. (4) Loria, R.; Bukhalid, R. Α.; Creath, R. Α.; Leiner, R. H.; Oliver, M.; Steffens, J. C. Phytopathology 1995, 85, 537-541. (5) Natsume, M.; Yamada, Α.; Tashiro, N . ; Abe, H. Ann. Phytopathol. Soc. Jpn. 1998, 64, 202-204. (6) Toth, L.; Akino, S.; Kobayashi, K.; Doi, Α.; Tanaka, F.; Ogoshi, A. Soil Microorganisms 1998, 51, 29-34. (7) Natsume, M.; Ryu, R.; Abe, H. Ann. Phytopathol. Soc. Jpn. 1996, 62, 411413. (8) Oniki, M.; Suzui, T.; Araki, T.; Sonoda, R.; Chiba, T.; Takeda, T.Bull.Natl. Inst. Agro-Environ. Sci. 1986, 2,45-59 (in Japanese with English summary). (9) Suzui, T.; Miyashita, Κ.; Tashiro, Ν. In Abstracts of Papers,5thInt. Cong. Plant Pathology; Kyoto, Japan, 1988; p 177. (10) Babcock, M. J.; Eckwall, E. C.; Schottel, J. L. J. Gen. Microbiol. 1993, 139, 1579-1586. (11) Seki-Asano, M.; Okazaki, T.; Yamagishi, M.; Sakai, N . ; Hanada, K.; Mizoue, K. J. Antibiotics 1994, 47, 1226-1233. (12) Seki-Asano, M.; Tsuchida, Y.; Hanada, K.; Mizoue, K. J. Antibiotics 1994, 47, 1234-1241. (13) Eguchi, T.; Kobayashi, K.; Uekusa, H.; Ohashi, Y . ; Mizoue, K.; Matsushima, Y.; Kakinuma, K. Org. Lett. 2002, 4, 3383-3386. (14) Dröse, S.; Bindseil, Κ. U.; Bowman, E. J.; Siebers, Α.; Zeeck, Α.; Altendorf, K. Biochemistry 1993, 32, 3902-3906. (15) Kataoka, T.; Yamada, Α.; Bando, M.; Honma, T.; Mizoue, K.; Nagai, K. Immunology 2000, 100, 170-177.


Phytotoxin Produced by Streptomyces cheloniumii Causing Potato

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