Epiphytic Bacteria Associated with the Bryophyte

Original Paper

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Epiphytic Bacteria Associated with the Bryophyte Funaria hygrometrica: Effects of Methylobacterium Strains on Protonema Development M. Hornschuh, R. Grotha, and U. Kutschera Fachbereich Biologie, Universität Kassel, Kassel, Germany Received: September 5, 2002; Accepted: December 3, 2002

Abstract: Bacteria represent a substantial fraction of the micro-

organisms that inhabit leaf surfaces. We collected samples of the moss Funaria hygrometrica (L.) in the field and analysed the epiphytes on the gametophyte by the agar impression method and scanning electron/fluorescence microscopy. On the phylloid surface numerous bacteria were detected, notably in the grooves between adjacent lamina cells. Methanol-ammonium salts agar surfaces impressed with isolated phylloids of green gametophytes resulted in the growth of methylotrophic colonies. Two Methylobacterium strains (M. mesophilicum and M. sp., isolated from the Funaria phylloids) were found to simulate the wellknown effect of cytokinin application on bud formation in Funaria protonemata. In addition, Methylobacterium inoculation promoted the growth of protonemal filaments. The significance of this novel Methylobacterium±land plant interaction is discussed.

Key words: Epiphytic bacteria, Funaria, Methylobacterium, protonema development. Abbreviations:

PPFMs: Pink-pigmented facultatively methylotrophic bacteria

Introduction The bryophytes (liverworts, hornworts and mosses) are a diverse group of land plants that usually colonize moist habitats, such as forests. Less familiar species have been found in the Arctic tundra on desert boulders and on mountain rocks. They represent the simplest extant land plants and, due to their basal phylogenetic position, have been classified by prominent bryologists as ªliving fossilsº (Frahm, 1994[5]; Reski, 1998[16]). The life cycle of a typical moss, such as Funaria hygrometrica, is comprised of two phases. A haploid photosynthetically active gametophyte stage produces gametes. Pairs of these cells fuse to produce a zygote that develops into the diploid sporophyte. This second, largely heterotrophic generation consists of a small stalk that carries a spore capsule (Bopp and Atzorn, 1992[3]; Reski, 1998[16]). After spore germination a filamentous

Plant biol. 4 (2002) 682 ± 687 Georg Thieme Verlag Stuttgart ´ New York ISSN 1435-8603

stage, the protonema, develops. The Funaria protonemata contain two types of cells, chloronema and caulonema. During the first days of gametophyte development, only chloronema cells that are densely packed with chloroplasts are detectable. After about 8 days the pattern of growth changes and other cells with fewer spindle-shaped chloroplasts occur. These caulonema cells begin to form buds about 15 days after the start of spore germination. The buds develop and form the leafy shoot of the Funaria gametophyte. The addition of cytokinins, such as benzyladenine, causes bud formation before natural budding occurs along the caulonema cells. This rapid induction of shoot buds is a specific and quantitative bioassay for cytokinin activity (Bopp and Atzorn, 1992[3]; Reski, 1998[16]). Despite the fact that cytokinin biosynthesis in plants has been studied over several decades, the entire de novo cytokinin biosynthetic pathway has not yet been elucidated and is a matter of ongoing debate (Kakimoto, 2001[10]; Takei et al., 2001[18]). However, biosynthesis of cytokinins is documented in pinkpigmented facultatively methylotrophic bacteria (PPFMs) of the genus Methylobacterium (Ashby, 2000[1]; Koenig et al., 2002[11]; Trotsenko et al., 2001[19]). Several years ago it was shown that the gametophyte of the liverwort Scapania nemorosa is colonized by epiphytic bacteria (Pseudomonas extorquens, syn. Methylobacterium sp.). The contaminated gametophytes grew larger than control samples raised in axenic cultures (Basile et al., 1969[2]). These classical experiments indicate that certain Methylobacterium strains may release growth-promoting substances and cause an acceleration of development in liverworts. However, the possible effect of Methylobacterium contamination on protonema development in the moss Funaria hygrometrica has not yet been studied. In this report we document that PPFMs are localized in specific regions on Funaria phylloids and exert positive effects on growth and differentiation of the protonemata.

Materials and Methods

Plant material and growth conditions Adult gametophytes of the moss Funaria hygrometrica were collected in forests of the natural reserve ªDöncheº in Kassel, Germany. Entire plantlets or excised phylloids were analysed as described below. Single spores (capsules collected in the greenhouse of the University of Kassel) were transferred

Epiphytic Bacteria and Protonema Development

to sterile Knop agar that was composed as follows: 4 mM Ca(NO3)2; 1.8 mM KH2PO4; 3.4 mM KCl; 1 mM MgSO4; 1 mM ZnSO4; 14 mM MnCl2; 70 mM H3BO3; 10 nM CoCI2; 0.5 mM CuSO4; 0.2 mM Na2MoO4; 50 mM FeNa-EDTA; Agar concentration: 15 g/l. Aseptic cultures from single protonemata (clones) were maintained in petri dishes covered with cellophane discs (12 h light, 25 8C/12 h darkness, 20 8C; 100 % relative humidity, photon fluence: 110 mmol m±2 s±1). In some experiments, bacteria (Methylobacterium mesophilicum ATCC 29983, obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany and M. sp., isolated from Funaria phylloids) were added to the sterile cultures.

Isolation of methylotrophic bacteria The agar impression method as described by Corpe (1985[4]) was used in order to reveal the presence of PPFMs. Buffered ammonium salts solution supplemented with methanol as the sole carbon source was employed. Basal salts medium (3 mM K2HPO4; 4 mM KH2PO4; 10 mM NH4Cl; 1.8 mM CaCI2; 4 mM MgSO4; 26 mM FeSO4; 0.6 mM ZnSO4; 0.2 mM MnCl2; 5 mM H3BO3; 0.8 mM CoCI2; 60 nM CuCI2; 84 nM NiCI2; 0.2 mM Na2MoO4; Agar concentration: 15 g/l) was supplemented with methanol to give a final concentration of 1 % (v/v). To prevent the development of fungi, 50 mg/l cycloheximide was added. Immediately after harvest, the plant material was stored under aseptic conditions. Entire plantlets (without rhizoids) or single phylloids were pressed onto the surface of the plates and removed after 16 h. Thereafter, the petri dishes were incubated for 7 d in darkness (27 8C). The plates were photographed for documentation of the results. In the course of the isolation process, colonies were grown on basal salts medium and on glycerol±peptone agar as described by Kutschera (2002[12]).

Microscopy and digital imaging In order to localize epiphytic microorganisms, single phylloids were investigated by fluorescence microscopy. The DNA of the epiphytes was stained for 10 min with DAPI (4¢,6-diamidino-2phenyl-indole, purchased from Sigma, St. Louis, Mo). The stock solution (1 mg DAPI/1 ml H2O) was diluted with phosphate buffer (50 mM, pH 7) to a final concentration of 0.002 %. The samples were viewed in a conventional fluorescence microscope (Zeiss, Oberkochen, Germany). Images were taken and processed with a digital camera (Olympus DP 10, Olympus, Hannover, Germany) as described by Scherp et al. (2001[17]). For scanning electron microscopy, leafy gametophytes (gametophores) or protonemata ( Methylobacterium inoculation) were fixed for 12 h in glutaraldehyde/phosphate buffer (2 %, 0.1 M, pH 7.2). In the next step, the samples were post-fixed in 2 % glutaraldehyde buffer as described above. After removal of the fixative by washing in phosphate buffer, the samples were dehydrated, critical-point dried and coated with platinum as described (Kutschera et al., 1990[14]; Kutschera, 2002[12]). The samples were observed with a scanning electron microscope (HITACHI S-4000, Japan) at 10 kV.

Quantitative bioassay for cytokinin activity The isolates of F. hygrometrica used in our experiments were maintained on agar plates supplied with Knop agar supplemented with the anti-auxin parachloroisobutyric acid

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(0.1 mM) under low light conditions (35 mmol photons m±2 s±1). Pieces of protonemata, 1.5 mm in diameter, were cut under sterile conditions and pre-incubated for 8 days on cellophane-covered Knop agar (photon fluence: 110 mmol m±2 s±1). Thereafter, the protonemata (diameter ~ 4 ± 5 mm) were transferred via the cellophane discs onto fresh media (8 protonemata per plate). Sterile control plants were kept on Knop agar that contained 0, 5, 10, 50, 100 nM benzyladenine (BA). To investigate the influence of Methylobacterium strains on budding, protonemata on Knop agar were inoculated with 10 ml of a suspension (about 108 cells per ml) of Methylobacterium mesophilicum or M. sp. (isolated from phylloids). After 6 days of incubation (see above) the buds that had developed on each individual colony were counted. All experiments were repeated at least 4 times with similar results. Standard errors of the means ( SE) were included where appropriate.

Results

Bacteria associated with the surface of moss phylloids In the first set of experiments we collected healthy Funaria hygrometrica plantlets of average size in the field and investigated whether or not bacteria are common residents on the surface of the leaf-like organs. A representative gametophore with stalk and spore capsule is depicted in Fig. 1 A. Impressions of isolated entire plantlets (without rhizoids) or phylloids on selective agar (methanol±ammonium salts) were prepared and incubated for up to 7 days in darkness (27 8C). Pink colonies of bacteria could be seen within 3 days. This result demonstrates that PPFMs of the genus Methylobacterium occupy the surface of the adult Funaria plant (Fig. 1 A). In order to explore the specific sites of the microhabitat where the bacteria reside, two microscopic techniques were employed. Fluorescence micrographs (DAPI-stained bacteria) revealed that the epiphytes prefer to occur along the grooves of adjacent epidermal cells (Fig. 1 B). In a separate set of experiments, using scanning electron microscopy, it was found that the most common habitat of the methylobacteria are regions where the cuticle appears to be thin (Fig. 2 A). In the grooves between adjacent epidermal cells numerous bacteria can be detected that are in part covered by the cuticle (Fig. 2 B). It is obvious that the microscopic examinations of moss phylloids collected in the field do not reveal the exact localization of the bacterial sub-population that grows on methanol (i.e., the PPFMs, Fig. 1 A). However, since all phyllobacteria prefer the same specific microhabitat, it follows that members of the genus Methylobacterium behave as depicted in Figs. 1 B and 2 B.

Association of Methylobacterium with protonema cells In order to explore whether the Funaria-associated Methylobacterium sp. strain binds to the protonemata of this bryophyte species, the following experiments were carried out. Single spores of Funaria hygrometrica were germinated under sterile conditions on agar plates. In one batch of samples a suspension of Methylobacterium sp. was added; control plates were kept under aseptic conditions. The bacteria were found on the surface of the cellophane plane and in association with the protonemal filaments (Figs. 3 A, B). Since our Methylobacterium strains contain motile organisms, we suggest that

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Fig. 1 Adult Funaria hygrometrica: green gametophyte and stalk-like sporophyte. Imprints of two plantlets after 7 d of incubation. Colonies of pink-pigmented facultatively methylotrophic bacteria (PPFMs) had developed on those regions of the agar where the plants were pressed onto the surface of the petri plate (A). Fluorescence micrograph of lamina cells of a Funaria phylloid. Numerous epiphytic bacteria (blue dots) are present, notably in the grooves between the cells (B). Bars = 1 cm (A); 20 mm (B).

M. Hornschuh, R. Grotha, and U. Kutschera

Fig. 2 Scanning electron micrograph of the phylloid of an adult Funaria hygrometrica gametophyte (A). Bacteria can be detected as white dots (arrow head). In the grooves between certain lamina cells rod-shaped epiphytic bacteria are present that are in part covered by the cuticle (B). Bars = 20 mm (A), 2 mm (B).

the microbes actively colonize the living cells of the protonemata.

Fig. 3 Phase-contrast micrographs of 7-d-old branching protonema cells of Funaria hygrometrica that were grown in the absence (A) or presence (B) of a Methylobacterium strain isolated from the phylloid (see Fig. 1 A). Note that bacteria are associated with the surface of the cells (B). Bars = 20 mm.

In the next step, the association between the bacteria and the Funaria protonemata was investigated by scanning electron microscopy. In the aseptic control, no microorganisms were observed (Fig. 4 A). In the inoculated samples, bacteria associated with the surface of the protonemata were detected on numerous cells (Fig. 4 B). However, the abundance and density was lower than in the in situ preparations shown in Fig. 3 B. This may be due to the fact that during fixation of the samples for scanning electron microscopy the majority of microbes had been removed. Nevertheless, a significant proportion of the methylobacteria were tightly associated to the surface so that we could study further details of this association.


Fig. 4 Scanning electron micrographs of the surface of 7-d-old protonema cells that were either grown in the absence (A) or presence (B) of a Methylobacterium strain isolated from the phylloid. Some bacteria are attached to the cells. Bar = 2 mm.

In some regions of the Funaria cells, notably where the protonemata branch out, clusters of Methylobacterium were observed (Fig. 5 A). At larger magnification, the surface of the Funaria-associated methylobacteria became visible (Fig. 5 B). The wall of these bacteria is characterized by numerous short fimbriae-like structures that also appear as white dots on the micrograph shown in Fig. 4 B.

Effects of Methylobacterium on bud formation and growth Koenig et al. (2002[11]) have shown that Methylobacterium strains that were isolated from plant leaves produce the cytokinin trans-zeatin and excrete this phytohormone into the culture medium. We analysed whether bud formation in Funaria protonemata, which is a quantitative bioassay for cytokinin activity, can be elicited by our Methylobacterium strains. In axenic cultures, exposure to the synthetic cytokinin benzyladenine (BA) resulted in an approximately log-linear dose-dependent increase in the number of buds (from 5 to 100 nM BA, Fig. 6). This effect of BA could be simulated by the addition of the Methylobacterium strain isolated from Funaria phylloids. A benzyladenine concentration of about 10±8 M was equivalent


Fig. 5 Scanning electron micrographs of 7-d-old branching caulonema cells that were grown in the presence of a Methylobacterium strain that was isolated from the phylloids. Clusters of rod-shaped bacteria are attached to the Funaria cells (A). At larger magnification, on the surface of the epiphytic methylobacteria numerous fimbriaelike structures (i.e., protrusions of the cell wall) can be detected (B). Bars = 20 mm (A), 1 mm (B).

to the corresponding effect of the bacteria. In addition, a second strain (M. mesophilicum ATCC 29983) was used in this part of our investigation. The effect of this defined Methylobacterium species was indistinguishable from that described above, i.e., both microbe strains caused an acceleration of bud formation in the pre-cultured protonemata of Funaria. We observed that the growth of single protonemata was stimulated by our Methylobacterium strains. In Figs. 7 A, B representative protonemata, 5 days after sowing of the spores, are depicted ( Methylobacterium). In the control, the average number of cells per protonema was 74 3; in the presence of our phylloid-associated Methylobacterium strain 126 4 and in the M. mesophilicum inoculated samples 134 4 cells were counted (means SEM, n = 20 each). Hence, cell division activity of Funaria protonemata was stimulated by both Methylobacterium strains.

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Fig. 6 Dose±response curve of applied benzyladenine (BA) on bud formation in protonemata of Funaria hygrometrica. The effects of one Methylobacterium strain (M. sp. isolated from the phylloid) are indicated by an arrow. Data points represent means ( S.E.) of 6 protonemata.

Discussion Bacteria of the genus Methylobacterium are aerobes (or facultative anaerobes) and ubiquitous in nature: they have been found in dust, soils, freshwater, lake sediments and on the surface of leaves. Since these microbes are characterized by a distinctive pink pigmentation and can grow on methanol, they are referred to as PPFMs (pink-pigmented facultative methylotrophs). These rod-shaped bacteria can utilize methanol emitted by the stomata of plants and cause no visible damage to their host. Therefore, they have been classified as commensals (Green, 1992[6]). Experiments designed to initiate cultures of the liverwort Scarpania nemorosa with pre-sterilized field-collected gametophytes led to the discovery that this plant lives in association with bacteria of the genus Pseudomonas (syn. Methylobacterium) (Basile et al., 1969[2]). However, the fact that these epiphytes are capable of producing (and secreting) phytohormones, such as cytokinins (Koenig et al., 2002[11]; Ivanova et al., 2000[8]) and auxins (Ivanova et al., 2001[9]), which may stimulate the growth of the host plant, was not taken into account by these authors. According to Holland (1997[7]) epiphytes of the genus Methylobacterium, which have been found on almost all plants analysed so far, excrete growth substances, such as cytokinins. These external phytohormones are taken up by the epidermal cells and may promote the growth of the sessile organism. Since no unequivocal biochemical pathway for de novo cytokinin biosynthesis in plants has been documented (Kakimoto, 2001[10]) this hypothesis is still debated in the current literature (Ashby, 2000[1]; Trotsenko et al., 2001[19]). With the recent discovery of cytokinin production in Methylobacterium strains, this putative plant-microbe interaction has received additional support (Koenig et al., 2002[11]).

M. Hornschuh, R. Grotha, and U. Kutschera

Fig. 7 Effect of Methylobacterium sp. on the growth of Funaria protonemata. Representative protonemata 5 days after sowing (before bud formation) are depicted that were either grown in the absence (A) or presence (B) of methylobacteria. Note that the axenic protonema is smaller than that raised in the presence of the bacteria. Bar = 0.5 mm.

Experiments with sunflower seedlings have shown that, upon germination, the plant body is rapidly colonized by epiphytic bacteria after germination (Kutschera, 2002[12]). Subsequent studies revealed that germ-free seedlings grow more rapidly than the non-sterilized control (Kutschera et al., 2002[13]). These results do not support the concept discussed above. The aim of the present study was to explore the occurrence of Methylobacterium sp. on the moss Funaria hygrometrica and to test whether these epiphytes modulate the development of the protonemata. Our results demonstrate that the phylloids are colonized by bacteria that prefer the microhabitat created by the furrows of adjacent lamina cells. This distribution is similar to that observed on the cotyledons of sunflower seedlings (Kutschera, 2002[12]). At least part of this natural population of prokaryotic epiphytes consists of microbes that belong to the genus Methylobacterium. In a number of studies it has been postulated that PPFMs may be more than passive inhabitants on leaf surfaces: indications exist that methylobacteria interact with their green host organism (Holland, 1997[7]). In a recent review article, Trotsenko et al. (2001[19]) concluded that methylobacteria can be classified as phytosymbionts. However, these authors pointed out that this putative symbiotic relationship is only poorly understood. Our data may contribute to the elucidation of this problem. We have shown that selected Methylobacterium strains exerted two separate effects on axenically grown Funaria protonemata: an enhancement in the rate of bud formation and a promotion of cell growth. In 5-day-old protonemata the number of cells was almost twice as large compared to the control. We suggest that these effects are attributable to the secretion of phytohormones (cytokinines, auxins) by the methylobacteria. This assumption is supported by the recent report by Koenig et al. (2002[11]) and other studies (Trotsenko et al., 2001[19]). Experiments with the moss Physcomitrella patens (Reutter et al., 1998[15]) have shown that in the filamentous protonema most of the auxin and cytokinin is localized extracellularly. Our data indicate that the extracellular pool of cytokinins (as well as that of auxins) may be enhanced as a result of Methylobacterium colonization. Direct proof in support of this hypothesis is currently lacking.


Bryophytes are ancient land plants that have been classified as living fossils (Frahm, 1994[5]). Most extant species inhabit moist regions around rivers, streams and creeks. These habitats have not changed very much over the past 300 million years. It is conceivable that the Methylobacterium/Funaria interaction described here represents an ancient symbiosis that persists to the present day. More work is required to further elucidate the role of phyllosphere methylobacteria in developing Funaria plants.

Acknowledgements We thank Mr. H. Rühling (Abt. Zellbiologie) for help with the Scanning Electron Microscopy. Supported by the Fonds der Chemischen Industrie (Frankfurt/M.).

References 1

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U. Kutschera Fachbereich Biologie Universität Kassel Heinrich-Plett-Straûe 40 34109 Kassel Germany E-mail: [email protected] Section Editor: G. Thiel

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