a membrane filter (mixed cellulose ester, pore size 0.2µm,. Advantec, Tokyo, Japan) by the method of Polsinelli and. Mazza (1984). A total of 82 strains (MBR-30 ...
J Gen Plant Pathol (2006) 72:351–354 DOI 10.1007/s10327-006-0305-9
FUNGAL DISEASES
© The Phytopathological Society of Japan and Springer 2006
Short communication
Masafumi Shimizu · Akane Meguro · Sachiko Hasegawa Tomio Nishimura · Hitoshi Kunoh
Disease resistance induced by nonantagonistic endophytic Streptomyces spp. on tissue-cultured seedlings of rhododendron
Received: January 12, 2006 / Accepted: May 12, 2006
Abstract Of 82 strains of endophytic actinomycetes isolated from rhododendron plants, 12 were not antagonistic against Pestalotiopsis sydowiana, which is the causal agent of Pestalotia disease. Of these 12, MBR-37 and MBR-38 (identified as Streptomyces spp.) grew on IMA-2 medium. Tissue-cultured seedlings of rhododendron treated with these nonantagonistic strains showed less wilting and/or smaller lesions to P. sydowiana, although the degree of resistance was a little lower than that conferred by antagonistic Streptomyces galbus strain R-5. These seedlings accumulated the anthocyanin(s), suggesting that resistance induced by these strains could depend on activated defense responses associated with the phenylpropanoid pathway rather than with antibiosis. Key words Endophytic Streptomyces · Disease resistance · Tissue culture · Antagonistic activity · Rhododendron
level required to inhibit mycelial growth of P. sydowiana on agar medium (Shimizu et al. 2001b). Many researchers reported that secondary metabolites produced by endophytic microbes and rhizobacteria in or on their host plants often altered the physiology of the host plants, rendering them resistant to pathogenic microbes (Audenaert et al. 2002; De Mayer and Höfte 1997; Tsuda et al. 2001; van Loon et al. 1998). Therefore, we hypothesized that antifungal antibiotic(s) produced by MBR-5 in the seedlings might be involved in the disease resistance that was induced in rhododendron seedlings by inoculation of this strain. However, tissue-cultured seedlings treated with commercial actinomycin and/or polyene macrolide (an analog of fungichromin) did not prominently induce resistance against inoculated P. sydowiana. Therefore, the issue of whether other strains of endophytic Streptomyces spp. lacking antibiotic production could induce similar systemic resistance of tissue-cultured seedlings was examined in this study.
M. Shimizu (*) Laboratory of Crop Production and Ecology, Faculty of Bioresources, Mie University, Tsu 514-8507, Japan Tel. +81-59-231-9496; Fax +81-59-231-9495 e-mail: shimizu@bio.mie-u.ac.jp
Isolation of endophytic actinomycetes from rhododendron. Endophytic actinomycetes were isolated from field-grown rhododendron plants that were harvested from Akatsuka Garden Co. (Tsu, Mie Prefecture, Japan) by the method described in our previous report (Shimizu et al. 2000). Small pieces of leaves, stems and roots were rinsed with 0.1% Tween 20 for a few seconds and then surface-sterilized with 1% sodium hypochlorite for 3 min and 70% ethanol for 1 min. These samples were incubated on IMA-2 agar medium supplemented with antibacterial and antifungal antibiotics at 30°C for about 1 month. Actinomycetous colonies that grew from the samples were isolated and purified with a membrane filter (mixed cellulose ester, pore size 0.2 µm, Advantec, Tokyo, Japan) by the method of Polsinelli and Mazza (1984). A total of 82 strains (MBR-30 to MBR-111) were successfully obtained without contamination. Their spores were suspended in 10% glycerol solution amended with 10% dimethyl sulfoxide and then maintained at −20°C until use.
A. Meguro · S. Hasegawa · T. Nishimura · H. Kunoh Institute for Biological Process Research, Akatsuka Garden Co., Ltd., Tsu, Japan
Antagonistic activity of isolated actinomycetes. To select nonantagonistic actinomycetous strains, the antagonistic
As reported previously (Shimizu et al. 2000), an endophytic strain of Streptomyces galbus, R-5 (renamed MBR-5), was selected as a candidate biocontrol agent based on its broad and intense antagonistic activity against fungal pathogens of rhododendron. When tissue-cultured seedlings of rhododendron were treated with a mycelial suspension of MBR5 in glass flasks, the seedlings were very resistant to Pestalotia disease caused by Pestalotiopsis sydowiana (Bresadola) Sutton (Shimizu et al. 2001a). MBR-5 produces two known antibiotics, actinomycin X2 and fungichromin, in liquid culture (Shimizu et al. 2004). Furthermore, in seedlings treated with MBR-5, the concentration of accumulated actinomycin was higher than the
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activity of each strain against P. sydowiana (Bresadola) Sutton, a causal agent of rhododendron Pestalotia disease (provided by the National Institute of Agrobiological Resources, stock no. MAFF 305755), was tested. P. sydowiana was grown on potato dextrose agar (PDA) at 25°C for 7 days. Mycelial disks (diameter 6 mm) were punched from the colony margin with a cork borer for use as inocula. A spore suspension (10 µl) of each strain was streaked onto the surface of IMA-2 agar medium (Shimizu et al. 2000). Then, a mycelial disk of P. sydowiana was placed on the medium 3.5 cm away from each strain and incubated at 25°C for 14 days. The strains that inhibited mycelial growth of P. sydowiana were considered to produce an antibiotic(s) that interferes with the growth of the test pathogen. Nearly half of the 82 strains more or less antagonized P. sydowiana; only 12 strains were not antagonistic (Table 1). Of these 12, only MBR-37 and MBR-38 grew actively on IMA-2 agar medium (Table 1). Therefore, MBR-37 and MBR-38 were used for the following experiments. Identification of MBR-37 and MBR-38. Strains MBR-37 and MBR-38 were identified based on morphological characteristics using scanning electron microscopy and on the
Table 1. Growth of nonantagonistic strains of endophytic actinomycetes on IMA-2 agar medium Strain
Antagonism against Pestalotiopsis sydowiana
Growth of strains on IMA-2 agarb
MBR-30 MBR-31 MBR-33 MBR-37 MBR-38 MBR-53 MBR-60 MBR-75 MBR-92 MBR-103 MBR-106 MBR-111
Nonea None None None None None None None None None None None
Scantb Scant Scant Abundantc Abundant Scant Scant Scant Scant Scant Scant Scant
a
No zone of growth inhibition of Pestalotiopsis sydowiana was observed around the actinomycetous colony Poor mycelial growth and sporulation c Active mycelial growth and sporulation b
Fig. 1A, B. Scanning electron micrographs of cells of A MBR37 and B MBR-38
type of diaminopimelic acid (A2pm) isomers in the cell wall peptidoglycan using thin-layer chromatography (TLC) (Society for Actinomycetes Japan 2001). As shown in Fig. 1, spore chains of MBR-37 and MBR-38 were rectiflexibiles type (consisted of more than 50 spores with a smooth surface). In addition, TLC analysis revealed that both strains contained ll-type A2pm in their peptidoglycan (data not shown). From these results, MBR-37 and MBR-38 were identified as Streptomyces spp. Evaluation of disease severity on seedlings treated with antagonistic and nonantagonistic actinomycetes. As described, Streptomyces spp. MBR-37 and MBR-38 were nonantagonistic to P. sydowiana on IMA-2 agar medium. Spore suspensions (500 µl each) of MBR-37, MBR-38, and an antagonistic MBR-5 were each added to 100 ml of IMA2 liquid medium and incubated at 30°C on a shaker (200 rpm) for 24 h. The mycelial suspension (2 ml of 3–4 × 106 cfu/ml) was dropped and spread on the surface of the multiplication medium in a glass flask supporting tissuecultured seedlings of rhododendron (hybrid cultivar Sakigake), followed by incubation in a growth chamber conditioned at 25°C with 12 h of illumination per day at 11.8 Wm−2 for 14 days. Mycelial disks of P. sydowiana were placed as challenge inoculum on the fourth leaf from the tip of the seedlings and incubated for a further 14 days in the same chamber, followed by observation with the naked eye. For the control, seedlings untreated with a Streptomyces strain were challenge-inoculated and incubated in the same way. The seedlings were categorized by disease severity as follows: healthy (no lesions), slight (only challengeinoculated leaves brownish), middle (stem and upper/lower and challenge-inoculated leaves brownish), heavy (seedlings wilted). In the control, lesions developed extensively from challenge-inoculated leaves to other parts of the seedlings within 14 days after the start of incubation, and 5% of the seedlings were categorized in the middle group and 55% of the seedlings were dead (rated as heavy) (Figs. 2B and 3). On the other hand, development of the brownish lesions was strongly suppressed in the antagonistic MBR-5 treatment as reported previously (Shimizu et al. 2001a). In this treatment, about 70% of the seedlings were categorized in the slight group and only 4% in the middle group (Figs. 2C
A
B
3.6 µm
2.0µm
353 Fig. 2A–E. Brownish lesions caused by Pestalotiopsis sydowiana on tissue-cultured seedlings of rhododendron pretreated and untreated with endophytic Streptomyces spp. A Untreated uninoculated seedlings, B untreated seedlings, C pretreated with MBR5, D pretreated with MBR-37, E pretreated with MBR-38. Anthocyanin(s) accumulated in stems and leaves of seedlings pretreated with actinomycetes
100
Percentage of seedlings
90 80 70
Healthy Slight Middle Heavy
60 50 40 30 20 10 0 None(Control)
MBR-5
MBR-37
MBR-38
Treatment
Fig. 3. Development of brownish necrotic lesions on tissue-cultured seedlings of rhododendron treated with antagonistic and nonantagonistic Streptomyces spp. Categories of lesions described in the text
and 3). Nonantagonistic MBR-37 and MBR-38 also suppressed development of the lesions: brownish lesions developed in stems and leaves in about 30% of MBR-37and 35% of MBR-38-treated seedlings (middle), and 7% of MBR-38-treated seedlings wilted (heavy) (Figs. 2D, E and 3). The slightly lower suppressive effects of MBR-37 and MBR-38 in comparison with MBR-5 suggest that resistance elicited by endophytic Streptomyces might be associated with some mechanisms other than in planta antibiotic production. Suzuki et al. (2005) used electron microscopy to prove that wall appositions formed inside epidermal and mesophyll cells of rhododendron leaves to which MBR-5 cells were attached. Similarly, other researchers (Benhamou et al. 1999; Ramamoorthy et al. 2001) reported that a variety of endophytic bacteria and plant growth-
promoting rhizobacteria modified the structure of host cell walls through the formation of wall appositions. Thus, a mechanical barrier such as a wall apposition can be considered as a resistance reaction induced by the colonizing endophytic Streptomyces. Furthermore, Shimizu et al. (2005) reported that by northern blot analysis using Arabidopsis, another host plant of MBR-5, expression of PDF1.2 markedly increased in the MBR-5-treated plants, while PAL and PR-1 were not expressed. In addition, they also proved that production of camalexin, a phytoalexin, was also enhanced in such plants. These results suggest that the presence of endophytic Streptomyces also induces disease resistance by activating the ethylene/jasmonate-associated expression of defense genes and the synthesis of phytoalexins. As described in a previous article (Shimizu et al. 2001a), anthocyanin(s) accumulated markedly in the seedlings within 1 week after the MBR-5 treatment. Interestingly, the seedlings treated with MBR-37 and MBR-38 also accumulated anthocyanin(s) in leaves and stems (Fig. 2A, D, E). This characteristic phenomenon suggests a change in the physiology of the seedlings in response to infection with endophytic Streptomyces spp. Anthocyanin pigments synthesized through the phenylpropanoid pathway are associated with disease resistance of some plants (Chalker-Scott 1999). For example, Snyder and Nicholson (1990) determined that the anthocyanin pigments, apigeninine and luteonidinine, are phytoalexins of sorghum. Therefore, the anthocyanin accumulation in rhododendron seedlings might also be a sign of induction of systemic resistance associated with the phenylpropanoid pathway. However, the possibility still remains that production of antifungal metabolites in or on the seedlings might also be involved in this resistance, because the resistance induced by the nonantagonistic Streptomyces spp. was slightly less than that by the antagonistic Streptomyces strain MBR-5
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(Fig. 3). As mentioned earlier, actinomycin X2 and fungichromin are produced by MBR-5 in liquid culture (Shimizu et al. 2004). Antagonistic activity of MBR-5 against P. sydowiana may be associated with fungichromin, a strong antifungal antibiotic. However, fungichromin was not detected in the seedlings treated with this strain, although a high level of actinomycin was (Shimizu et al. 2001b). According to Compant et al. (2005), environmental conditions such as pH and the availability of nutrients influence antibiotic synthesis by microbes. Therefore, fungichromin, which effectively suppresses P. sydowiana growth, might not be synthesized by MBR-5 in rhododendron seedlings, although in planta production of actinomycin was already proved (Shimizu et al. 2001b). Conversely, in the seedlings, MBR-37 and MBR-38 might produce antibiotics and/or other secondary metabolites that were not detected in the culture media. Until these possibilities are further investigated, antifungal metabolites cannot be ruled out to explain the mechanism of systemic resistance in tissue-cultured seedlings conferred by endophytic Streptomyces spp. The present study, however, does not reject our previous hypothesis that in planta colonization of endophytic Streptomyces might be responsible for induced resistance through defense responses such as wall apposition formation, defense gene expression related to the ethylene/ jasmonate pathway, and accumulation of anthocyanin(s) via the phenylpropanoid pathway. Acknowledgments Contribution No. 33 from Laboratory of Crop Production and Ecology, Faculty of Bioresources, Mie University. The work was partially supported by a Grant-in-Aid for Young Scientists (B)(17780034) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to Masafumi Shimizu.
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