Microbes of the genus Methylobacterium are regularly associated with leaves and fruits of plants. In experimental tests, they promote the growth of germ-free liverworts and mosses, which are ancient land plants. In this study we analyzed the effect of M. mesophilicum, a bacterium that has been isolated from the organs of numerous plant species, including gymnosperms and angiosperms, on the development of sterile embryos of Ginkgo biloba L., a unique living fossil among the embryophyta. In addition, germ-free seeds of Pinus sylvestris were inoculated with the same strain of methylobacteria. In G. biloba seedlings that were raised in a 12 h dark/light regime, a promotion of root development was recorded in samples treated with the methylobacteria. A fresh mass increase of + 25 % occurred within 6 weeks of inoculation with bacteria, compared to the aseptic control. In contrast, shoot development of the same plants was not significantly affected by these bacteria. In Pinus seedlings, organ development was unaffected by the presence of methylobacteria. Our results document a differential sensitivity of the root system versus the shoot towards these ubiquitously distributed plant-associated bacteria. The data are discussed with reference to the isolated taxonomic position of Ginkgo biloba, one of the most primitive gymnosperms in the biosphere that is economically important as a medicinal plant

Hellmuth, J.

Kutschera, U.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Journal of Applied Botany and Food Quality 82, 26 - 29 (2008)Institute of Biology, University of Kassel, GermanyThe effect of growth-promoting methylobacteria on seedling development in Ginkgo biloba L.J. Hellmuth, U. Kutschera(Received March 14, 2008)SummaryMicrobes of the genus Methylobacterium are regularly associatedwith leaves and fruits of plants. In experimental tests, they promotethe growth of germ-free liverworts and mosses, which are ancientland plants. In this study we analyzed the effect of M.  mesophilicum,a bacterium that has been isolated from the organs of numerousplant species, including gymnosperms and angiosperms, on the de-velopment of sterile embryos of Ginkgo biloba L., a unique livingfossil among the embryophyta. In addition, germ-free seeds of Pinussylvestris were inoculated with the same strain of methylobacteria.In G. biloba seedlings that were raised in a 12 h dark/light regime, apromotion of root development was recorded in samples treated withthe methylobacteria. A fresh mass increase of + 25 % occurred within6 weeks of inoculation with bacteria, compared to the aseptic control.In contrast, shoot development of the same plants was not sig-nificantly affected by these bacteria. In Pinus seedlings, organdevelopment was unaffected by the presence of methylobacteria. Ourresults document a differential sensitivity of the root system versusthe shoot towards these ubiquitously distributed plant-associatedbacteria. The data are discussed with reference to the isolatedtaxonomic position of Ginkgo biloba, one of the most primitivegymnosperms in the biosphere that is economically important as amedicinal plant.IntroductionNearly every botanical garden or arboretum in temperate andsubtropical climate regions of the world has at least one Ginkgo(Maidenhair) tree. Due to veneration of this "sacred tree of the East",this unique organism, represented by only one extant species (Ginkgobiloba L. 1771), has been planted since ancient times in China andJapan in temple gardens near shrines and castles. The Ginkgo tree isthe symbol of the Tokyo Metropolitan area; splendid individuals canbe seen in the capital of Japan in many parks and along avenues.Moreover, in China, Korea and Japan grilled Ginkgo nuts are eaten;they are also used in porridges and soups or mixed with rice, stir-fried vegetables and mushrooms. Leaves of Ginkgo trees are used inChinese medicine to treat bronchitis, asthma, stomach pain and otherailments. In Europe and the United States, G. biloba has also beenrecognized as a medicinal plant (HORI et al., 1997; ZHOU and ZHENG,2003).In a series of studies we have shown that gametophyte developmentin liverworts and mosses is enhanced by methylotrophic bacteriaof the genus Methylobacterium (HORNSCHUH et al., 2002, 2006;KOOPMANN and KUTSCHERA, 2005). However, no such positiveeffect was measured when germ-free seedlings of higher plants(sunflower, maize) were treated with methylobacteria (KUTSCHERA,2007). Bryophytes are, like Ginkgo biloba, organisms that display aphenotype reminiscent to that of ancient forms of life within thekingdom Plantae ("living fossil plants"). Therefore, we investigatedwhether the development of germ-free Ginkgo-seedlings is promotedby added methylobacteria. Taxonomists have classified the speciesG. biloba as a member of the Coniferopsida (HORI et al., 1997;SCHNEIDER et al., 2002; FROHLICH and CHASE, 2007). Hence, wehave included a representative conifer species (Pinus sylvestris L.)in this experimental study for comparison.Materials and methodsSeeds of Ginkgo biloba L. and Pinus sylvestris L. were purchasedfrom a commercial supplier (Carmens Bonsai Garten, Memmingen,Germany). The seeds (in the case of G. biloba after the removal ofthe sarcotesta, see Fig. 1 A) were sterilized as follows. Batches of5 to 20 pre-selected individuals were washed for 12 h in 300 ml ofH2O (aqua bidest). In the next step, the seeds were incubated for20 min in a half concentrated sodium hypochlorite solution (NaOCl,available chlorine ca. 13 %) and thereafter washed five times withgerm-free water. Half of the aseptic seeds were submerged for 24 hin a bacterial solution that contained 108 colony forming units perml of methylobacteria (M. mesophilicum, ATCC 29983, obtainedfrom the Deutsche Sammlung von Mikroorganismen und Zell-kulturen, Braunschweig, Germany); control seeds were treated withthe same amount of distilled water. The seeds (± methylobacteria)were raised under aseptic conditions as follows.Glass jars (750 ml), covered with a lid, were autoclaved (temperature121 °C, pressure: 1.1 kg/cm2) for 15 min and filled to a height of5 cm with sterile vermiculite. Single seeds (G. biloba) or batches of4 individuals (P. sylvestris) were planted under germ-free conditions.The Ginkgo- and Pinus seedlings were raised in a 12 h white light/dark cycle at 99.5 % relative humidity (25 ± 1 °C).Six weeks after sowing, the sterile (or inoculated) plants wereharvested. After careful excision, the length of the washed/dried rootsystems and the corresponding shoots were measured with a ruler.Thereafter, fresh masses of both organs were determined with a micro-balance. In order to determine the extent to which the sterilized seedsand the resulting seedlings may nevertheless have been contaminatedwith bacteria the agar impression method of CORPE (1985) wasused as described in detail in previous reports (KUTSCHERA, 2002;KUTSCHERA et al., 2002). All experiments were repeated at least fourtimes with similar results.ResultsFig. 1 A shows a representative Ginkgo seed, with the outer fleshycoating removed. After removal of the sarcotesta (the soft yellow-brown seed coat), the hard sclerotesta, which encloses the embryo,becomes visible. In all experiments, seeds without sarcotesta (i.e.,embryos with attached endosperm) were used (Fig. 1 B).In order to determine to what extent the smooth outer surface of theembryo is contaminated with spores of microorganisms non-sterileembryos were pressed for 1 day to the surface of petri plates thatcontained glycerol-peptone agar (Fig. 2 A). Seven days later, denselayers of bacteria and, occasionally, colonies of fungi had developedon the surface of the agar (Fig. 2 B). These results show that theembryo of Ginkgo seeds, which is in contact with the surroundingair via a terminal hole in the sarcotesta, is heavily contaminated by aFig. 1: Representative seed of Ginkgo biloba with sarcotesta removed to show the outer surface of the sclerotesta (A). The propagule is oriented with themicropyle downward. Broken sclerotesta, viewed from the inside (left) and isolated embryo with attached endosperm (right), oriented with theradicle downward (B). s = sclerotesta, e = embryo. Bar = 1cm.Fig. 2: Petri dish with two non-sterile embryos of Ginkgo biloba pressed onto the surface of sterile glycerol-peptone agar (A). After 7 days of incubation ofthe agar plate, bacteria (b) and fungi (f) developed around the places where the tissue was in contact with the agar (B). Bar = 1cm.variety of microorganisms and their propagules. The correspondingexperiment carried out with sterilized Ginkgo embryos, from whichthe sarcotesta had been removed, is shown in Fig. 3 A, B. It is ap-parent that the sterilization procedure employed here yielded asep-tic propagules. The experiments shown in Fig. 2 and 3 were repeatedwith seeds of Pinus sylvestris. We obtained the same qualitative resultsas those depicted here for Ginkgo biloba (data not shown).In a series of experiments with germ-free Ginkgo embryos (and Pinusseeds) we determined the quantitative effect of Methylobacteriummesophilicum on seedling mass. Sterile embryos were either inocu-lated for 24 h in germ-free water or in a suspension of M. meso-philicum. Thereafter, the seeds were planted into vermiculite andraised under aseptic conditions. A representative pair of Ginkgo-seedlings (± methylobacteria) is shown in Fig. 4 A, B. This photo-graph indicates that root development is promoted by the methylo-bacteria, but shoot growth is unaffected by these microbes.The effect of M. mesophilicum on organ development was quantifiedby determining the fresh- and dry masses of sterile and inoculatedseedlings (Fig. 5). On average, root fresh mass was 25 % higher inseedlings raised in the presence of methylobacteria compared withthe germ-free controls. In the shoots of the same seedlings, no positiveeffect was measured. Hence, M. mesophilicum selectively promotesthe growth of the root system but has no effect on the above-groundorgans. To determine whether or not our seedlings were in fact sterilein the control and covered by methylobacteria in the inoculatedsamples, we used the agar-impression method to examine bacterialgrowth. The agar plate assays documented that our controls weregerm-free and that added methylobacteria had colonized the entireSeedling development in Ginkgo 2728 J. Hellmuth, U. KutscheraFig. 5: Effect of methylobacteria on the development of root and shoot inGinkgo biloba. Sterile embryos were either incubated in germ-freewater or in a suspension of Methylobacterium mesophilicum andthereafter planted into moist sterile vermiculite. Fresh mass (A)and dry mass (B) (open and dark bars, respectively) was determined42 days after start of the experiments (n = 15, ± s.e.m.).Fig. 4: Photographs of 6 week-old seedlings of Ginkgo biloba that wereeither raised in the absence (A) or presence (B) of methylobacteria.Sterile seeds were either soaked in germ-free water or a suspensionof Methylobacterium mesophilicum and thereafter planted in sterilevermiculite. Note that the root system in the inoculated sample (B)is larger than that of the control (A). The onset of the root is indicatedby arrowheads. Bar = 2cm.Fig. 3: Petri dish with two sterilized embryos of Ginkgo biloba, pressed onto the surface of glycerol-peptone agar (A). After 7 days of incubation of theplates the agar was still sterile, i.e., no microorganisms had developed in these germ-free samples (B). Bar = 1cm.Ginkgo seedling (results not shown).The corresponding experiments with Pinus seeds yielded the fol-lowing results. Average fresh mass of the shoots: sterile / inoculated(M. mesophilicum): 35 ± 2 / 34 ± 3 mg (n = 25); fresh mass rootsystem: 7.0 ± 0.5 / 7.3 ± 0.4 mg (n = 25). The measurements werecarried out 42 days after inoculation. These data document that inseedlings of Pinus sylvestris methylobacteria exert no positive effecton organ development under the standard conditions employed here.DiscussionIn a series of studies it has been shown that plant-associated microbesof the genus Methylobacterium, which are probably distributedubiquitously in nature, produce and secrete phytohormones such asauxins and cytokinines (HORNSCHUH et al., 2002, 2006; SCHAUERand KUTSCHERA, 2008). It is now definitively clear that methanol,emitted from the stomata of plants, is consumed by these epiphytes.Since the growth of axenically raised gametophytes of bryophytes,but not the shoots of sterile higher plants (sunflower, maize) isSeedling development in Ginkgo 29promoted in response to the addition of methylobacteria, these micro-organisms have been classified as phytosymbionts (KUTSCHERA,2007). It should be noted that ABANDA-NKPWATT et al. (2006)analyzed the effect of the species Methylobacterium extorquens onthe germination of surface-sterilized seeds of 10 species of angio-sperms (wheat, barley, maize, carrot, pea, bean, tomato, strawberry,tobacco, and mustard). In the first six species of this list, no positiveeffect was measured, but in the last four a slight promotion of organgrowth occurred. However, the authors did not distinguish betweenthe roots and the above-ground phytosphere. Hence, it is not knownwhether these effects occur in the shoot or root of the correspondingseedlings.In this study we report that in G. biloba methylobacteria exert aspecific effect on root development, but the growth of organs abovethe surface of the earth are unaffected by these microbes. This findinghas two implications: 1. The root system in this "living fossil plant"appears to be more sensitive to the addition of growth-promotingmethylobacteria than the shoot, and 2. germination and subsequentseedling development can occur in the absence of any epiphyticmicrobes. SCHAUER and KUTSCHERA (2008) have shown that the rootsof adult, field-grown sunflower plants are more heavily contaminatedby methylobacteria than the leaves. This may be due to the fact thatthe zone surrounding the roots, the rhizosphere, provides moreprotection than the phyllosphere from light stress, high temperature,and desiccation. In addition, sources of mineral salts and organicsubstances are more abundant in the rhizosphere. Sugars, amino acids,organic acids and many other metabolites compose, together withsmall amounts of polysaccharides and proteins, the majority of theroot exudates (BAIS et al., 2006). Since our Ginkgo seedlings (Fig. 4)were raised under moist, low-light conditions, the protective effectof the rhizosphere provided in the sun-exposed field is negligible inour experimental system. Hence, it is conceivable that the developingroots of our Ginkgo seedlings may be more densely populated bymethylobacteria than the shoot; this  may result in a promotion ofroot growth caused by phytohormones secreted by the epiphyticbacteria (KUTSCHERA, 2007). However, in seedlings of Pinus syl-vestris that were treated the same as our Ginkgo samples no suchdifferential effect of methylobacteria on root versus shoot develop-ment was measured. These data indicate that the root cells of Ginkgoseedlings may be more sensitive to the growth hormones secreted bymethylobacteria than those of the shoot. However, direct proof insupport of this hypothesis is currently lacking. At any rate, our datashow that the "living fossil" Ginkgo biloba, which has extinct relativesthat appeared in the Early Jurassic at a time when the first large-sized dinosaurs evolved (FROHLICH and CHASE, 2007), displays abehaviour that is analogous to that of mosses and liverworts(HORNSCHUH et al., 2002, 2006; KUTSCHERA and KOOPMANN, 2005).These ancient, morphologically primitive lower plants live insymbiosis with growth-promoting methylobacteria that consumewaste products, such as methanol, that were released by the plantcells (KUTSCHERA, 2007). We suggest that the juvenile developingroot system of Ginkgo biloba (Fig. 4) is likewise dependent on theseubiquitous, naturally occurring epiphytes of the genus Methylo-bacterium, but more experimental work is required to further elucidatethis postulated plant root-microbe interaction.HOLLAND (1997, a, b) postulated that higher plants obtain part oftheir phytohormones from attached methylobacteria and hence aredependent on these epiphytes. We have previously shown that shootdevelopment in two representative higher plants (the angiospermsHelianthus annuus and Zea maize) is not promoted by added me-thylobacteria (KUTSCHERA et al., 2002; KUTSCHERA and KOOPMANN,2005; KOOPMANN and KUTSCHERA, 2005). Here we document thattwo gymnosperms (Ginkgo biloba, Pinus sylvestris) can grow over aperiod of six weeks in the absence of any bacteria. Hence, thesephylogenetically much older embryophytes are largely independentof bacterial phytohormones, although the root system of the "livingfossil" G. biloba displays a behaviour that is similar to that of themost ancient land plants on Earth (liverworts).ReferencesABANDA-NKPWATT, D., MÜSCH, M., TSCHIERSCH, J., BOETTNER, M., SCHWAB,W., 2006: Molecular interaction between Methylobacterium extorquensand seedlings: growth promotion, methanol consumption, and localizationof the methanol emission site. J. Exp. Bot. 57, 4025-4032.BAIS, H.P., WEIR, T.L., PERRY, L.G., GILROY, S., VIVANCO, J.M., 2006: Therole of root exudates in rhizosphere interactions with plants and otherorganisms. Annu. Rev. Plant Biol. 57, 233-266.CORPE, W.A., 1985: A method for detecting methylotrophic bacteria on solidsurfaces. J. Microbiol. Methods 3, 215-221.FROHLICH, W., CHASE, M.W., 2007: After a dozen years of progress the originof angiosperms is still a great mystery. Nature 450, 1184-1189.HOLLAND, M.A., 1997a: Methylobacterium and plants. Rec. Res. Dev. PlantPhysiol. 1, 207-213.HOLLAND, M.A., 1997b: Occam's razor applied to hormonology. Arecytokinins produced by plants? Plant Physiol. 115, 865-868.HORI, T., RIDGE, R.W., TULECKE, W., DEL TREDICI, R., TRÉMOUILLAUX-GUILLER, J., TOBE, H. (eds.), 1997: Ginkgo biloba – A Global Treasure.From Biology to Medicine. Springer-Verlag, Tokyo.HORNSCHUH, M., GROTHA, R., KUTSCHERA, U., 2002: Epiphytic bacteriaassociated with the bryophyte Funaria hygrometrica: Effects ofMethylobacterium strains on protonema development. Plant Biol. 4, 682-687.HORNSCHUH, M., GROTHA, R., KUTSCHERA, U., 2006: Moss-associatedmethylobacteria as phytosymbionts: an experimental study. Natur-wissenschaften 93, 480-486.KOOPMANN, V., KUTSCHERA, U., 2005:  In-vitro regeneration of sunflowerplants: Effects of a Methylobacterium strain on organ development. J.Appl. Bot. 79, 59-62.KUTSCHERA U., 2002: Bacterial colonization of sunflower cotyledons duringseed germination. J. Appl. Bot. 76, 96-98.KUTSCHERA U., 2007: Plant-associated methylobacteria as co-evolvedphytosymbionts: a hypothesis.  Plant Signal. Behav. 2, 74-78.KUTSCHERA, U., KOOPMANN, V., 2005: Growth in liverworts of the March-antiales is  promoted by epiphytic methylobacteria. Naturwissen-schaften 92, 347-349.KUTSCHERA, U., KOOPMANN, V., GROTHA, R., 2002: Plant development inthe absence of epiphytic microorganisms. Naturwissenschaften 89,319-321.SCHAUER, S., KUTSCHERA, U., 2008: Methylotrophic bacteria on the surfacesof field-grown sunflower plants: a biogeographic perspective. TheoryBiosci. 127, 23-29.SCHNEIDER, D., WINK, M., SPORER, F., LOUNIBOS, P., 2002: Cycads: theirevolution, toxins, herbivores and insect pollinators. Naturwissen-schaften 89, 281-294.ZHOU, Z., ZHENG, S., 2003: The missing link in Ginkgo evolution. Nature423, 821-822.Address of the author:U. Kutschera, Institute of Biology, University of Kassel, Heinrich-Plett-Straße 40, D-34109 Kassel, Germany.E-mail: kut@uni-kassel.de

The effect of growth-promoting methylobacteria on seedling development in Ginkgo biloba L.

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The effect of growth-promoting methylobacteria on seedling development in Ginkgo biloba L.

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