Actinomycetologica (2006) 20:72–81 VOL. 20, NO. 2

Award Lecture

Endophytic Actinomycetes and Their Interactions with Host Plants

Sachiko Hasegawa1, Akane Meguro1, Masafumi Shimizu2, Tomio Nishimura1* and Hitoshi Kunoh1

1Institute for Biological Process Research, Akatsuka Garden Co. Ltd. 1868-3, Takanoo-cho, Tsu-city, 514-2293, Japan 2Graduate School of Bioresources, Mie University, Kurimamachiya-cho, Tsu-city, 514-8507, Japan

(Received Oct. 27, 2006 / Accepted Oct. 27, 2006)

Endophytic microbes including bacteria, actinomycetes consideration is also confusing because non- or mildly path- and fungi are ubiquitous in most plant species, especially in ogenic microbes are known to induce systemic or localized field-grown plants. As of 1997, bacteria isolated from the resistance of host plants4–9). In such a case it is difficult to internal plant tissue of healthy-looking plants were com- distinguish symbionts from pathogenic parasites. Hallmann prised of over 129 species representing over 54 genera1). et al.1) defined any bacterium as an endophyte if it does not Although some of the endophytes are pathogenic to host visibly harm the plant and it can be isolated from surface- plants and can locally or systemically colonize plant tissues, disinfected plant tissues or extracted from inside the plant. others latently reside in the internal tissues of nonsympto- Their definition includes internal colonies with apparently matic plants without causing any adverse effects to the neutral behavior as well as symbionts. Rosenblueth and plants. Consequently, intimate associations between endo- Martinez-Romero10) proposed that criteria to recognize “true” phytes and host plants can be formed without harming the endophytic bacteria require not only the isolation from sur- plant. Endophytes have been demonstrated to improve and face-disinfected tissues but also microscopic evidence to promote growth of host plants as well as to reduce disease visualize “tagged” bacteria inside plant tissues. True endo- symptoms caused by plant pathogens and/or various envi- phytes may be also be recognized by their capacity to rein- ronmental stresses. The low stress tolerance of axenic plants fect disinfected seedlings. They suggest that bacteria not is commonly believed to result partly from the absence of validated microscopically are “putative” endophytes. Ac- endophytic microbes1). Management of beneficial microbial cording to their definition, most endophytes reported to date communities to favor plant growth could be realized by a are “putative” endophytes, because their microscopic evi- deeper understanding of the physiological and molecular dence has not been obtained. Although their definition is interactions between microbes and plants . This research also logical in a strict sense, enough evidence has not been accu- may have broader economic and environmental impacts. mulated to discuss in planta localization and behavior of endophytic actinomycetes. Therefore, in this review we fol- Definition of endophytes low the above definition by Hallmann et al.1). Before beginning this review, a definition of endophytes should be provided. There are several definitions of endo- Isolation of endophytic actinomycetes phytes, which in general describe fungi and bacteria in- The majority of actinomycetes (or actinobacteria) were cluding actinomycetes. Because this review focuses on once believed to survive in environments such as the soil, actinomycetes, fungal endophytes are excluded here. Kado2) rhizosphere, and pond and lake sediments as saprophytes by defined endophytes as: “bacteria that reside within living degrading organic materials for nutrition. However, in the plant tissues without doing substantive harm or gaining ben- 19th century some actinomycetes were proved to be closely efit other than securing residency.” However, in his defini- associated with living plants, giving beneficial and/or ad- tion, “substantive harm” is quite confusing because there are verse effects to the host plants. As reviewed by Okazaki11), many cases that even pathogenic bacteria do not always Brunchorst isolated the genus Frankia from non-legume root cause symptoms depending on the population density in their nodules in 1886, suggesting its role of nitrogen fixation in host plants. On the other hand, Quispel3) considered endo- roots, similar to Rhizobium in legume plants; also, Thaxter phytes only as bacteria that establish an endosymbiotic rela- first reported pathogenic actinomycete, Streptomyces sca- tionship with the plant, whereby the plant receives an eco- bies, a causal pathogen of potato scab in 1890. This serious logical benefit from the presence of the symbionts, such as disease can be found in all potato-growing areas throughout increased stress tolerance or plant growth promotion. His the world and the scab organism sometimes occurs in soils

*Corresponding author. Phone: +81-59-230-1234, Fax: +81-59-230-7501, E-mail: [email protected] 72 ACTINOMYCETOLOGICA VOL. 20, NO. 2 where potatoes have never been cultivated. Therefore, this In Planta Localization of Endophytic Actinomycetes species is both soil-inhabiting and endophytic12). Hasegawa Although Rosenblueth and Martinez-Romero10) empha- et al.13) isolated a new genus of actinomycete, Actinosynnema, sized importance of in planta evidence of colonization for from a grass blade. As far as we know, this was the first report true endophytes, the number of detailed reports on localiza- describing an actinomycete of plant origin in Japan, although tion of endophytic actinomycetes are still limited. Strepto- its association with host plant was indefinite. Subsequently, myces scabies, a causal pathogen of potato scab, usually Matsukuma et al.14), Okazaki et al.15) and Matsumoto et al.16) penetrates through natural openings (lenticels, stomata), reported that a variety of actinomycetes inhabit a wide range mechanical or insect wounds, or occurs under conditions in of plants as symbionts, parasites or saprophytes, most of which tubers are enlarging rapidly. Following penetration them belong to the genera, Streptomyces and Microbispora. the pathogen grows between or through a few layers of cells, Coombs and Franco17) isolated 38 strains belonging to the cells then die and this pathogen feeds on them as a sapro- Streptomyces, Microbispora, Micromonospora and phyte12). Sardi et al.21) showed hyphae of Streptomyces sp. in Nocardia from surface-sterilized root tissues of healthy cortical tissues of tomato roots by SEM but were unable to wheat plants. Likewise Okazaki11) reported a total of 246 resolve the detail of the interaction with the host cells. In strains of actinomycetes of plant origin: 97 strains belonged melon, Yoshida and Yamaguchi22) also used SEM to find to Streptomyces, 57 Microbispora, 23 Nocardia, 18 Micro- mycelia of a pathogenic Streptomyces sp. on the surfaces of monospora, 4 Actinomadura and several others. Similarly, the epidermis and the endodermal layers exposed when Takahashi and Omura18) successfully isolated 32 strains of branch roots were erupted through the root cortex. Kamiunten Streptomyces, 33 Microbispora and 10 other rare actino- and Suga23) observed by transmission electron microscopy mycetes from fallen leaves of 9 genera of higher plants. (TEM) cells of Streptomyces sp. in root tumor cells of melon. Rosenblueth and Martinez-Romero10) listed 8 genera of Furthermore, Okazaki11) showed by light microscopy and plant-associated actinomycetes including Arthrobacter, SEM the presence of sporophores of Microbispora sp. on the Curtobacterium, Kocuria, Nocardia, Streptomyces and so on surface and the inside of epidermal cells of a plant. in their review. Coombs and Franco24) coated germinating seeds of wheat As far as we have experienced, almost all vascular plants with GFP-expressing endophytic Streptomyces sp. and har- examined to date were found to harbor several endophytic vested every 24 hr for detection of its localization in the seeds actinomycetes. Commonly, several strains of endophytic using fluorescent microscopy. They detected the strain only actinomycetes can be isolated from a single plant and they in the embryo and around the break in the seed husk where are often especially rich in its root. These reports evidently the embryo emerged from the seed 24 hr after incubation. show that plant-associated actinomycetes are quite common They confirmed that the strain grew preferentially in close in nature. No one knows if microbial communities inside proximity to the plant cell walls during intercellular growth. plants interact; it has been speculated that beneficial and/or After 3 days, GFP-expressing microcolonies of the strain harmful effects are the combined effect of their activities10). were seen more frequently in the embryo tissue of the seed This is one of the most attractive and significant research than at 24 hr, indicating its active growth in the plant tissue. themes for plant-microbe interactions. Tokala et al.19) ob- Such colonies were seen also in the emerging radicle of the served a remarkable degree of preferential colonization of embryo and in the endosperm. Their observations show that pea nodules relative to roots by Streptomyces lydicus the endophytic actinomycetes are able to associate with its WYEC108. The observation indicated that colonization was host at a very early stage in the development of the plant. In initially nodule specific and that hyphae of the strain ex- more detail, Minamiyama et al.25) demonstrated by SEM that tended actively from the nodule onto the root at the base of when Streptomyces galbus was spread on the surface of the the nodule. Once on the nodule and root surface, the hyphae tissue-culture medium in which rhododendron seedlings spread and actually penetrated the surface cell layer. These were growing, its mycelia grew on leaf surfaces and entered observations did not provide direct evidence of a close rela- leaf tissues via stomata, and the internal mycelia grew out of tionship between nodule and this strain but suggested that the stomata after internal multiplication within host leaves. association of a specific actinomycete with nodule bac- Subsequently, Suzuki et al.26) examined the infection mode teroids or other rhizosphere microbes may influence the of S. galbus in the leaves of tissue-cultured rhododendron activity of the microbe as well as physiological activity of seedlings and confirmed that mycelia invaded the host the host plant. through stomatal openings but not through other sites. Takahashi and Omura18) emphasized that diversity of iso- Within host leaves, hyphae were observed individually or in lated actinomycetes was largely dependent on the isolation colonies in intercellular spaces but not inside epidermal or methods. According to Conn and Franco20), the 16S RNA mesophyll cells. More interestingly, mycelia colonizing leaf analyses of endophytes isolated from wheat roots revealed a surfaces, growing through stomatal openings, and coloniz- larger diversity than that obtained by culturing endophytic ing in intercellular spaces were embedded in electron-dense, actinomycetes. Therefore, it is probably not an exaggeration thick, mucilage-like material, while single hyphae were not. to say that the actual number and diversity of actinomycetes These observations were consistent with the SEM obser- still remains unknown. vation by Minamiyama et al.25) and Sardi et al 21). They

73 ACTINOMYCETOLOGICA VOL. 20, NO. 2 hypothesized that such materials were probably required for medicinal lore of native people38). Gurney and Mantle39) massive colonization of mycelial cells, smooth passage of detected 1-N-methylalbonoursin of antifungal activity in mycelial cells in the stomatal openings and adherence of culture of an endophytic Streptomyces sp. isolated from mycelial cells to the surface of the host cell facing the inter- perennial ryegrass. On the other hand, Streptomyces sp. cellular space. This observation raised interesting questions NRRL30562 obtained from snakevine plant was proved to about which factors trigger formation of the materials and produce novel peptide antibiotics, designated as munum- how they are formed when needed. Minamiyama et al.25) and bicins A-D40). These antibiotics possessed a wide-spectrum Suzuki et al.26) proved that, on the leaf surface, the wax that activity against many human and phytopathogenic fungi and covered the undulating cuticular layer was degraded beneath bacteria. Castillo et al.41) found that Streptomyces sp. the growing mycelia. This observation was supported by the NRRL30566, which was isolated from a fern-leaved grevil- presence of indigo blue crystals that formed as hydrolytic lea (Grevillea pteridifolia) tree growing in the northern ter- products of mycelial nonspecific esterase along the mycelia ritory of Australia, produced novel wide-spectrum antibi- on fresh leaves26). otics named kakadumycins that were chemically related to echinomycin. Coronamycin, a novel peptide antibiotics Actinomycetous metabolites that affect plant’s life complex, was isolated from culture broth of Streptomyces sp. Endophytes colonizing inside plants usually get nutrition MSU-2110 recovered from the stems of Monstera sp42). This and protection from the host plants. In return, they confer antibiotic exhibited activity against phytopathogenic profoundly enhanced fitness to the host plants by producing Pythium spp. and is a novel fungicide candidate. a variety of bioactive metabolites. Growth stimulation of plants by endophytes can be a consequence of nitrogen fix- 2. Plant growth promoters ation or the production of phytohormones, biocontrol of Igarashi et al.43) isolated pteridic acids A and B from the phytopathogens through production of antibiotics or sider- fermentation broth of an endophytic Streptomyces hygro- ophores, nutrient competition, and induction of systemic dis- scopicus TP-A045 (isolated from Pteridium aquilinum) as ease resistance7,27–31). Some of these metabolites directly plant growth promoters with auxin-like activity. These affect physiology of the host plants but others do so indirectly compounds accelerated formation of adventitious roots in by affecting the microbe population by antibiosis and/or hypocotyls of kidney beans at 1 nM as effectively as indole competition. acetic acid. Several species of Streptomyces including S. vio- laceus, S. scabies, S. griseus, S. exfoliates, S. coelicolor and 1. Antibiotics S. lividans were reported to secrete indole-3-acetic acid Igarashi et al.32) isolated 398 actinomycete strains from (IAA) when fed with L-tryptophan44), although their produc- leaves, stems and roots of cultivated or wild plants. About tion within plant tissues was not proved. Recently, Meguro 10~20% of the n-butanol extracts of their fermentation et al.45) reported a strain of Streptomyces sp. MBR-52 that broths showed antagonistic activity against phytopathogenic accelerated emergence and elongation of plant adventitious fungi and bacteria. One of the extracts from Streptomyces sp. roots. When tissue-cultured seedlings of rhododendron were collected from Allium fistulosum had a potential to suppress treated with MBR-52 in flasks, this strain colonized within infection of Alternaria brassicicola on Chinese cabbage the seedlings and survived there even after their transplan- seedlings. Igarashi et al.33) found that such an effect was ing in soil. Emergence of adventitious roots was prominently dependent on a novel plant protective compound named fis- accelerated in the MBR-52-treated tissue-cultured seedlings, tupyrone. Their results suggest that a wide range of endo- suggesting that this strain might release some type of root- phytic actinomycetes have a potential to produce antimicro- ing-promoting plant hormone(s). This strain certainly gives bial compounds which probably contribute to competition a great practical advantage to shorten the acclimatization with other microbes in host plants. Furumai and col- period of tissue-cultured seedlings in warm and humid envi- leagues34,35) identified several new bioactive compounds ronments, thereby decreasing the disease-infection risk of produced by actinomycetes isolated from live plants. Two the seedlings. new novobiocin analogs were produced by Streptomyces sp. collected off Aucuba japonica 34), and cedarmycins by Strep- 3. Plant growth inhibitors tomyces sp. collected from Cryptomeria japonica 35) and According to Okazaki11), some actinomycete strains of were determined to be antimicrobial metabolites. A new plant origin produce herbicidal antibiotics. Herbicidin H is a naphthoquinone antibiotic, alnumycin, was also reported in metabolite of Streptomyces sp. strain SANK 63997 that was Streptomyces sp. isolated from root nodules of Alnus gluti- isolated from leaves of Setaria viridis var. pachystachys. nosa collected in Germany36). Shimizu et al.37) proved that Another strain SANK 62597 of Microbispora sp. recovered

Streptomyces galbus strain R-5 produced actinomycin X2 from Carex kobomugi produced g-glutamylmethionine sul- and fungichromin that could account for the in vitro anti-bac- foximine in culture broth. He speculated that this metabolite terial and -fungal activities of R-5. with no in vitro herbicidal activity could be converted into A search for specific endophytes that may produce antibi- methionine sulfoxide with strong activity in vivo. Further- otics can have its origin in ethnobotany, which utilizes the more, strain SANK 61299 of Dactylosporangium sp. iso-

74 ACTINOMYCETOLOGICA VOL. 20, NO. 2 lated from Cucubalus sp. was found to produce streptol and ported by the presence of indigo blue crystals that formed as two plant growth inhibitors that inhibit germination of hydrolytic products of mycelial nonspecific estrerase along Brassica rapa. Furumai et al.46) and Igarashi et al.47) found the mycelia on fresh leaves. a novel inhibitor of pollen tube growth named clethramycin in the fermentation broth of Streptomyces hygroscopicus that Agricultural Use of Endophytic Actinomycetes was isolated from roots of Clethra barbinervis. This linear polyene antibiotic showed in vitro antifungal activity against 1. Biocontrol Agents Candida sp. and Aspergillus sp. but weak activity to bacte- Pathogenic microorganisms affecting plant health are a ria. By a growth assay of pollen tube of the Japanese pear, major and chronic threat to food production and ecosystem they found that clethramycin inhibited the pollen tube stability worldwide. Agriculture is becoming increasingly growth similarly as the kinase inhibitors staurosporine and dependent on agrochemicals to help insure reliable crop herbimycin suggesting that such an effect could be associated protection and operational economic stability. However, with inhibition of MAP kinase, which is supposed to control increasing use of chemical inputs causes several negative the pollen germination. Gebhardt et al.48) discovered new effects, such as development of pathogen resistance to the phenazine compounds, endophenazines A-D, produced by applied agents and their nontarget environmental impacts58). Streptomyces anulatus in fermentation broth. These com- Because of such circumstances, biological control has been pounds showed herbicidal activity against Lemna minor considered an alternative or a supplemental way of reducing (duckweed). On the other hand, taxol, a highly functionalized the use of agrochemicals in food production. deterpenoid was found in yew (Taxus) species38). Caruso et There has been a large body of literature describing po- al.49) isolated 71 actinomycetes from woody and herbaceous tential uses of plant associated bacteria (including actino- tissues of Taxus species growing in Italy and found that 10 mycetes) as agents to stimulate plant growth and for out of 71 isolated strains (Actinomadura, Actinoplanes, management of soil and plant health, as reviewed by Com- Kitasatospora, Micromonospora, Nocardioforme and pant et al.58), Hallmann et al.1), Rosenblueth and Martinez- Streptomyces spp.) produced taxanes including taxol. Since Romero10) and Winding et al.59). Most of these bacteria and these terpenoids are known as the inhibitors of microtubule actinomycetes were isolated from the soil, rhizosphere and polymerization, if endophytic production occurred, it could phylloplane. Their anti-pathogen potentials were demon- interfere positively or negatively with microtubule-asso- strated in the laboratory but their direct association with ciated activities of host cells such as cell division, inter- and plants was not definitively demonstrated. Smith60) success- intra-cellular transportation. fully isolated Micromonospora sp. from tissue section of an Only a few actinomycetes are known to produce phy- apparently healthy tomato plant and found that this actino- totoxins. The phytotoxins, named thaxtomins, are produced mycete showed a strong inhibitory effect to Fusarium oxys- by S. scabies and related species12). The symptoms elicited porum f. sp. lycopersici. He reisolated the same strain from in aseptically cultured potato tubers by these species did not 4 of 20 tomato plants previously inoculated with this strain, differ from those produced by thaxtomins, suggesting that suggesting its endophytic residence in tomato. As far as we these compounds might be responsible for pathogenicity of know, this is one of the earliest reports describing biocontrol this species on potato tubers. effects of endophytic actinomycetes. In parallel with phar- maceutical concern of endophytic actinomycetes as a source 4. Enzymes of novel antibiotics, some microbiologists have considered Some Streptomyces strains are known to produce hydro- the use of endophytic actinomycetes as biocontrol agents lytic cell wall-degrading enzymes such as cellulases, hemi- over last two decades. cellulases, chitinases, amylases and glucanases50–53). Other Taechowisan and Lumyong 61) isolated 59 endophytic strains are also known for their ability to produce enzymes actinomycetes from the roots of Zingiber officinale and that degrade the lignin, cellulose and hemicellulose of higher Alpinia galanga and tested their antifungal activity against plants54–56). However, these strains were isolated from soil, Candida albicans and phytopathogenic fungi. Among them sediments or rhizosphere. Only a few strains producing Streptomyces aureofaciens showed the most intense antago- hydrolytic enzymes were of plant origin. Streptosporangium nism against these test organisms. Similarly, Tian et al.62) sp. isolated from leaves of maize was reported to produce isolated 274 actinomycete strains from surface-sterilized glucoamylase, which is expected to improve industrial proc- roots and leaves of field-grown rice plants and found that esses of starch degradation57). about 50% of these strains showed antagonism to some fun- Minamiyama et al.25) demonstrated by electron micro- gal pathogens, including the rice blast fungus. However, both scopy that leaf surface of rhododendron was degraded by authors did not show any evidence of biocontrol effects in enzymes released from contacting hyphae of Streptomyces field experiments. Detailed biocontrol effects of endophytic galbus. Furthermore, Suzuki et al.26) presented ultrastruc- actinomycetes both in vitro and in planta were given by tural evidence that the wax that covered the undulating cutic- Coombs et al.63). They isolated 38 strains of endophytic ular layer of rhododendron leaves was degraded beneath the actinomycetes from surface-sterilized wheat and barley roots growing mycelia of S. galbus. This observation was sup- and tested their antagonistic activity to Gaeumannomyces

75 ACTINOMYCETOLOGICA VOL. 20, NO. 2 graminis, Rhizoctonia solani and Pythium spp., which are all and finally killed them within 2 weeks. However, when the pathogenic to wheat roots. They demonstrated that 17 of the medium surface was treated R-5 to allow it to reside in the isolates displayed statistically significant activity in planta seedlings, mycelia of P. sydowiana stayed on the inoculated against G. graminis and some of them were able to control leaves alone without visible growth on the neighboring the development of disease symptoms in treated wheat plants leaves. Consequently, all leaves, aside from the inoculated exposed to the former two pathogens in field soil. leaves, remained green for 2 weeks. Kunoh67) noted that the seedlings treated with R-5 as above in flasks showed resis- 2. Use of endophytic actinomycetes to produce disease- tance to Phytophthora cinnamomi (a causal fungus of root resistant tissue-cultured seedlings rot) after they were transplanted to soil in plug-trays. The As reviewed so far, the endophytic presence of actino- subsequent in vivo test using seedlings transplanted in soil mycetes may play important roles in plant development and demonstrated that R-5 was effective at protecting the health because they affect plant growth either by nutrient seedlings from soil-born diseases and that this strain was an assimilation or by secondary metabolite production. As excellent biocontrol agent for producing disease-resistant Coombs et al.63) reported, when applied in actual fields, seedlings of flower pot plants for practical purposes. Using antagonistic endophytic actinomycetes do not always show a similar technique, Meguro et al.68) produced disease-resis- biocontrol effects as expected due to their ability to colonize tant tissue-cultured seedlings of mountain laurel. They de- the internal tissues of the target host plant. To use endophytic monstrated that the treatment of seedlings with S. padanus actinomycetes in practical agronomical production, reliable strain AOK-30 significantly slowed the expansion of root rot and practical techniques of inoculum delivery must be de- caused by Rhizoctonia sp. in seedlings in plug trays. veloped. A variety of techniques have been used for the application of endophytic microbes at the experimental level, 3. Mechanism of disease resistance of tissue-cultured ranging from several variations of seed treatments, soil seedlings induced by inoculated endophytic actino- drenches, stem injections and foliar sprays of microbial sus- mycetes pension1). Recent progress and identification of antimicrobial Considering such circumstances, Shimizu and colleagues64,65) metabolites have led to the hypothesis that plant protection assumed that if a useful endophytic actinomycete isolated against virulent pathogens relied, at least partially, on the from a field-grown plant can successfully colonize tissue- production of antibiotics69). Considering the mechanism of cultured seedlings of a plant, the seedlings could become disease resistance in tissue-cultured seedlings induced by S. resistant to various plant pathogens. Because tissue-culture galbus R-5, Shimizu et al.65) first assumed that the endo- flasks are usually axenic, such a novel technique should phytic production of antibiotics by R-5 could account for this allow this actinomycete to colonize its host plant without disease resistance. Shimizu et al.37) found two antibiotics, competition and/or antagonism by any other microbes in soil. actinomycin X2 and fungichromin, in fermentation broth of Based on such assumptions, they developed a novel tech- R-5. Interestingly, actinomycin(s) was detected in the tissue nique to produce disease resistant seedlings of ornamental culture medium and also in the growing rhododendron flower trees seedlings when R-5 was inoculated into the medium surface Shimizu et al.64) isolated a total of 17 strains of actino- followed by 10 days incubation70). These data indicate that mycetes from field-grown rhododendron plants. To suit their actinomycins produced by R-5 in the medium could move to above-mentioned assumption, the candidate strain must the seedlings and/or that R-5 grown in the seedlings could grow on the growth (multiplication) medium for tissue cul- produce this antibiotic within tissues. Interestingly, they ture of rhododendron and must not adversely affect growth showed that occurrence of Pestalotia disease in the seedlings of the seedlings. In addition, its antagonistic activity must be was suppressed by treatment of the medium with R-5 but not intense against a broad range of microbes. They selected by treatment with analogues of actinomycin X2 and fun- strain R-5 [identified as Streptomyces galbus strain R-537)] gichromin. This finding suggests that endophytic coloniza- that fulfilled these requirements as a candidate for this pur- tion of R-5, an antibiotic producer, could be essential for pose. Similarly Nishimura et al.66) isolated Streptomyces induction of disease resistance in tissue-cultured seedlings padanus strain AOK-30 from potted mountain laurel trees of rhododendron rather than in planta antibiosis. that fulfilled the above requirements. Minamiyama et al.25) and Suzuki et al.26) observed that When the mycelial suspension of R-5 was spread on mul- when R-5 was inoculated to the surface of tissue culture tiplication medium in a glass flask with growing rhododen- medium where seedlings of rhododendron were growing, dron seedlings, mycelia of R-5 grew and sporulated to form mycelia extended on and into the seedlings, without causing white, powdery colonies on the medium surface within 2–3 any adverse effects. The mycelia colonized around stomata days. Their growth never adversely affected the seedlings65). of leaves, entered substomatal cavities through stomatal When untreated leaves of rhododendron seedlings in the openings, and continued to grow in intercellular spaces of multiplication medium were inoculated with Pestalotiopsis the leaves. Contact of R-5 to surfaces of mesophyll cells sydowiana (a causal fungus of Pestalotia disease), whitish induced large wall appositions inside these cells, which mycelia of this fungus actively grew on the entire seedlings block direct invasion of R-5. Their observations apparently

76 ACTINOMYCETOLOGICA VOL. 20, NO. 2 indicate that contact and/or release of signal products by R- proteins by sequence analysis and found that cell walls of the 5 elicit the internal barrier of the host cells. It is most plau- treated seedlings contained malate dehydrogenase which sible that elicitation of wall appositions at least partially was known to bind the apoplast and cell walls of other plants explains the induced resistance of the seedlings. and was thought to be associated with lignification74). Sub- Shimizu et al.71) examined expression of pathogenesis- sequent histochemical observation revealed that cell walls of related genes in Arabidopsis (another host plant of R-5) sieve cells were more intensely lignified in treated seedlings inoculated with R-5 by northern blot analysis. They con- in contrast to weak staining in untreated seedlings. All these firmed that expression of jasmonate-related PDF1.2 was reports show that the drought tolerance of mountain laurel remarkably increased in the R-5 treated plants, while PAL seedlings induced by endophytic colonization of AOK-30 and PR-1 were slightly expressed. In addition, production of could be closely associated with elevation of osmotic pres- camalexin, a phytoalexin of this plant, was also enhanced in sure in cells, accelerated callose accumulation, and lignifi- such plants. In this study, Arabidopsis seedlings were trans- cation in cell walls of sieve cells. planted to sterilized soil supplemented with a mycelial sus- Drought-tolerance of seedlings is significant in practical pension of Colletotrichum higginsianum, an anthracnose tissue culture technology, because decreased watering is pathogen, followed by 9 days incubation. In R-5-untreated desirable for reducing environmental humidity and thereby plants, a majority of inoculated leaves were entirely necrotic, decreasing disease occurrence during the acclimatization while in R-5-treated plants, brown lesions with clear margins process. were restricted to small areas within the sites which the spore suspension contacted the leaves. Their results suggest that Concluding Remarks growth of the symbiotic R-5 strain induces disease resistance As Rosenblueth and Martinez-Romero10) concluded in by activating the expression of defense genes associated with their review, plants seem to be in a close interaction with a jasmonate and the synthesis of phytoalexin. Thus, disease variety of endophytes in nature. As reviewed herein, al- resistance of the R-5-treated seedlings could be caused by a though endophytic actinomycetes seem promising for posi- complex mechanism that includes antibiosis and systemic tive interactions that affect plant growth, their effects and resistance associated with jasmonate and phytoalexin pro- functions are not comprehensively understood. duction by the seedlings. These works propose that one of Hallmann et al.1) pointed out important questions to con- the most promising options in the context of microbially- tinue the development of endophytes as plant growth con- mediated resistance is the use of selected endophytes as trollers: i) how do endophytes bypass plant defense mecha- potential elicitors of plant defense mechanisms. nisms?, ii) what food source do they utilize?, iii) why and how are endophytes linked to plant resistance and tolerance?. 4. Enhanced drought-tolerance of tissue-cultured seedl- The answers to these questions will decide if endophytes are ings by endophytic colonization of actinomycetes of potential use in agriculture practice. Hasegawa et al.72) discovered that tissue-cultured seedl- Recently, Meguro et al.75) reported that tissue-cultured ings of mountain laurel treated with S. padanus strain AOK- seedlings of mountain laurel inoculated with an endophytic 30 were turgid in room conditions even 1 hr after removal strain of Streptomyces padanus showed a high salt-tolerance. from the flasks, while the untreated seedlings wilted within A similar conclusion was obtained in the monocotyledonous 1 hr in the same conditions. They found that osmotic pres- plant barley using the endophytic colonization of Pirifor- sure of protoplasts from the AOK-30-treated seedlings was mospora indica, a plant-root-colonizing basidiomycete fun- higher than those from the untreated seedlings. In these gus9). As reviewed herein, disease resistance, drought toler- experiments, they noticed that wall-degrading enzymes ance and salt tolerance were confirmed in tissue-cultured required for preparation of protoplasts from both seedlings seedlings of flower trees in our laboratories. Some physio- were different, suggesting structural modifications of cell logical, biochemical, cytological and molecular events that walls in the seedlings. Further, Hasegawa et al.73) proved that explain these visible phenomena have been obtained. about 1.5 times more callose accumulated in cell walls of the However, we believe that these events are closely linked with treated seedling than those that were untreated. Their results one another and that the macroscopically visible phenomena are consistent with the observations by Suzuki et al.26) who are not caused by an independent single mechanism (Fig. 1). reported apparent accumulation of wall appositions in the Recognition of the actinomycetes by host plants triggers a tissue-cultured seedlings of rhododendron treated with S. cascade of signal transduction, which causes plants to initi- galbus strain R-5 because a major component of wall appo- ate a variety of defense responses to cope with the stresses sition is known to be callose. Callose deposition near the cell at cell, tissue and organ levels. surface likely strengthens the cell frame and may interfere We hope that further research on endophytic actino- with water loss through cell walls, thereby enhancing os- mycetes will contribute to the development of novel tech- motic pressure in cells of the treated seedlings. Hasegawa et nologies and methodologies in the agricultural, medical and al.73) further analyzed amino acids derived from cell wall pharmaceutical fields.

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Fig.1. Detected events associated with disease resistance and drought tolerance in tissue-cultured seedlings

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