Endophytic Actinomycetes and Their Interactions with Host Plants

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

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