Bacterial Colonization of Sunflower Cotyledons ...

Journal of Applied Botany - Angewandte Botanik 76, 96 - 98 (2002), O 2002, Vereinigung fur Angewandte Botanik, Giittingen

Fachbereich Biologie, Abteilung Pflanzenphysiologie, Universi tat Kassel

Bacterial Colonization of Sunflower Cotyledons during Seed Germination Urn Kutschera (Received February 19, 2002)

Summary The cotyledon S of heal thy sunflower (Hcliar~thusannuus L.) seedlings that were raised in moist vermiculite in closed (aerated) boxes are rapidly colonized by epiphytic bacteria. The agar impression method and scanning electron microscopy were used to analyse this biological invasion. The outer surface of the seed coat (i. e., pericarp of the sunflower achene) was heavily colonized with microorganisms. In contrast, on the enclosed embryo (i. e., at the outer surface of the cotyledons) no bacteria were detected. The results show that the outer surface of the pericarp is the source of the bacteria that colonize the developing sunflow er plant. No seed-transmitted microorganisms were detected.

Introduction The annual sunflower (Helianthus annuus L.) probably originated in North America or Mexico. Today, it is one of the major oilseed crops in Europe and is cultivated on almost all the continents. The edible sunflower oil is extracted from the achenes (seeds) and used for human consumption (margarine, soaps etc.). In addition, it serves as a raw material for the oleochemistry. The residual oilcake is an energyrich material that has been used as cattle-food. In some countries, sunflower seeds are eaten raw or roasted and are fed to cage-birds and poultry (FRANKE, 1985).

tilled water. The seedlings were either kept in darkness or were exposed to continuous white light (100 prnol. . s-' photosynthetically active radiation, PAR, at plant level). The temperature was 25 0,5 ' C and the relative humidity of the air s 95 %.

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For scanning electron microscopy, isolated embryos or excised cotyledons of l . 2 and 5-day old seedlings were fixed for 12 h in 2 % glutaraldehyde in phosphate buffer (0,l M; pH 7,Z). Thereafter, the samples were post-fixed in 2 % glut araldehyde/buffer as described above. The fixative was removed by washing the samples in phosphate buffer. The cotyledons were then dehydrated, critical-point-dried and coated with platin as described by KUTSCHERA et al. (1990). The samples were observed with a scanning electron microscope (HITACHI S-4000) at 10 KV. In order to reveal the presence (or absence) of epiphytic microorganisms, petri dishes were filled 1/3 with sterile glycerol peptone agar medium as described by GREEN and BOUSFIELD (1982). Under sterile conditions, samples (i. e., isolated embryos, the outer surface of the pericarp, entire seedlings or pieces of dry vermiculite) were plated by pressing firmly to the surface of the agar (CORPE,1985). After incubating for 24 h (darkness, 27 "C), the vermiculite or plant material was carefully removed. The growth of the epiphytic microorganisms occurred at 27 "C.After 1 - 3 days the petri dishes were examined and photographed (Fig. I C).

Sunflower seedlings are sturdy juvenile plants that grow rapidly. They have been used as a model organism for analysis of a variety of physiological processes such as photomorphogenesis, cell elongation and gravitropism (KUTSCHERA, 2000). It is common practice for germination to imbibe the achenes for several hours in water and then place the seeds into moist vermiculi te. This sterile material has been .widely used as substrate for the growth of seedlings of higher plants. Moist vermiculite has a water potential of about - 0,QlMPa. In closed (aerated) containers, seedling growth occurs at rates of 1-2 mm h-' (KUTSCHERA, 1990,2000; KRAMER and BOYER,1995).

In this report it is shown that the cotyledons of sunflower seedlings that were raised under the standard conditions summarized above are rapidly colonized by epiphytic bacteria. The implications of this finding are discussed.

Material and Methods Achenes of sunilower (Helianthus annuus L. 'Giganteus') were harvested in October 2000 from an open field within the Botanical Garden of the University of Kassel (Germany). The achenes were Fig. 1: stored in open boxes in a cold room (2 - 4 "C). Six month later, undamaged seeds of average size (20 per batch) were soaked for 1 11 in sterile double distilled water and thereafter planted in verrniculite in closed plastic boxes that were sterilized with 98% ethanol before use (KUTSCHERA, I 990). The vermiculi te was moistened with double dis-

Sunflower achene (A) and isolated embryo (B), removed from the agar plate. The petri dish contained glycerollpeptone-agar, a nonseIective medium. After 24 h of incubation, colonies of microorganisms (m) were detected (achene, left sample). No bacteria developed around the impression that was created by the embryo, pressed to the surface of the plate (right sample) (C).Bar = 2 cm.

Bacterial colonization of sunflower cotyledons

All observations described here were repeated at least six times with similar results. Representative pictures are reproduced for illustration of the most important findings.

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radicle ca. 1 cm) was carefully removed from the cotyledons (Fig. 2 A). The epidermal cells are covered by epicuticular waxes, but no microorganisms were detected (Fig. 2 B).

In a second set of experiments, 5-day-old de-etiolated seedlings were Results According to CORE(1 985), the agar impression method is suitable not only for the detection of bacteria but also for a study of their distribution on the surfaces of leaves and dead materials. The outer surface of sunflower achenes (Fig. 1 A) and that of the enclosed embryo (Fig. 1 B), isolated under sterile conditions, was pressed to the surface of petri plates that contained glycerol peptone (GP) agar (left and right sample, respectively). After one day the plant materials were carefully removed and the dishes incubated at 27 ' C for another 1 - 3 days. The result of one representative experiment is shown in Fig. 1 C. After 24 h, a thick yellow layer of bacterial growth was detected from the pericarp (left), but on impressions of isolated, undamaged embryos no colonies developed (right sample). This experiment was repeated 40 times with the same result: the outer surface of the pericarp was always found to be heavily contaminated by microorganisms, whereas the enclosed embryo was sterile.

used (Fig. 3 A). At this stage of development the apical hook had opened and the cotyledons unfolded (KUTSCHERA, 2000). Impressions of entire seedlings revealed that the plant surface was covered with microorganisms (Fig. 3 B). The cotyledons of these juvenile plants were examined by scanning electron microscopy. In those regions of the organ surface where the epicuticular wax layer was thin, numerous bacteria were detected. Contamination was extensive in the furrows between adjacent epidermal cells (Fig. 4 A). Examination of these regions at larger magnification revealed that the epiphytic bacteria of the sunflower cotyledons were sometimes covered by the cuticle (Fig. 4 B). No fungi and other eukaryotic microorganisms were observed in these preparations.

Finally, pieces of dry vermiculite were pressed to the surface of agar plates, removed from the dishes after 24 h and incubated as described above. No bacterial growth was detected, i. e., the substrate used for the cultivation of the sunflower seedlings was sterile (results not shown).

The finding that no bacteria are present on the epidermal cells (cuticle) of the sunflower embryo as long as the testa is attached was verified by scanning electron microscopy. Representative pictures are shown in Fig. 2. The seed coat of a Zday-old seedling (length of the

Fig. 3: Five-day-old de-etiolated sunflower seedling (A). Imprint of the same seedling after 24 h of incubation. Colonies of microorganisms (m) had developed on those regions of the agar where the plant was pressed onto the surface of the petri plate (B). Bar = 2 cm.

Discussion

Fig. 2: Low-magnification scanning electron micrograph of the cotyledons prepared from a 2-day-old sunflower seedling. The seed coat was carefully removed (A). At larger magnification the epidermal cells, COvered by epicuticular waxes, can be seen. No bacteria were detected (B). Bars = 1 mm (A), 50 pm (B).

BEWLEY and BLACK(1978) have pointed out that in many 'seeds' the outer coat is not the testa but the pericarp. These 'seeds' are therefore fruits. Sunflower and lettuce (Lactucn sativa) are both a special type of achenes, i. e., testa and pericarp are fused. It is no surprise that the rugged outer surface of the sunflower pericarp is a habitat for numerous bacteria and bacterial spores. However, the fact that the outer surface (cuticle) of the enclosed embryo is not contaminated by any (1997) has shown that in microorganisms is surprising. HOLLAND soybean plants epiphytic bacteria are transmitted via the seeds (i. e., the embryo). In the natural environment in which Heliantl~usplants develop and flower, microbes or microbial propagules are abundant. The mechanisms by which the adult sunflower plant keeps its interior parts of the seed (embryos) sterile during flower and fruit development is largely unknown,

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U. Kutschera

Numerous studies have shown that in field-grown plants, the leaf surface (phyllosphere) is a habitat for a variety of microorganisms (HIRANOand UPPER, 2000). These epiphytic bacteria exploit the few resources that the sun- and rain-exposed surface offers. Under natural conditions, microorganisms (or bacterial spores) usual l y arrive on the leaf via airborne or vector deposition. Some of these immigrants are capable to adapt to their environment and thereafter multiply by cell divisions. Organic molecules such as amino acids or carbohydrates passively leak from the rnetabolically active leaf cells. These nutrients are used for growth by the phyllobacteria residing on the leaf (LEVEAU and LINDOW, 200 1).

HOLLAND (1 997) speculated that higher plants depend on certain epiphytic bacteria for the removal of metabolic waste products such as methanol. The corresponding leaf microbes may produce hormones (cytokinins) that facilitate plant growth. According to this hypothesis some phyllobacteria are no accidential visitors but represent CO-evolved symbionts of the plant. The possible role of the epiphytic bacteria described in this report for the development of the sunflower seedling is currently under investigation.

Acknowledgements I thank Mrs. B. Teubert and Mr. H. Ruhling for technical assistance. This work was supported by a grant from the Fonds der Chemischen Indus tti e (FrankfurtlM .).

Zusammenfassung Bakterielle Besiedelung der Cotyledonen der Sonnenblume wahrend der Samenkeirnung

Die Cotyledonen gesunder Sonnenblumenkeimli nge (Heliar~tlzus annuus L.), welche in sterilem Vermiculit angezogen wurden, werden innerhalb weniger Tage von Bakterien besiedelt. Diese biologische Invasion wurde rnit der Agar-Abdruckmethode und der RasterElektronenmikroskopie dokumenti ert. Die AuRenseite der Achane (Perikarp) ist mit Bakterien kontaminiert, wahrend der eingeschlossene Embryo keimfrei ist. Die Ergebnisse zeigen, dass iiber die AuRenfliiche des Perikarps wghrend der Keimung eine bakterielle Besiedelung erfolgt. Es konnten keine Samen-transferierten Mikroorgani smen nachgewi esen werden.

References BEWLEY, J.D. and BLACK,M*, 1978: Pllysiology and biochemistry of seeds in relation to germination. Springer-Verlag, Berlin, Hei del berg, New York. CORPE, W.A., 1985: A method for detecting methylotrophic bacteria on solid surfaces. J. Microbiol. Methods 3,2 1 5-22 1. FRANKE, W,, 1985: Nutzpflanzenkunde. 3. Auflage. G. Thieme-Verlag, Stuttgart, New York. GREEN,P.N. and BOUSFIELD, LJ., 1982: A taxonomic study of some gramnegative facultatively methylotrophic bacteria. I. Gen. Microbiol. 128,623-

Fig. 4: Scanning electron micrographs (lower epidermis) of the cotyledon of a 5-day-old sunflower seedling (see Fig. 3 A). Numerous epiphytic bacteria are present in certain regions of the organ surface (A). At larger magnification, it becomes apparent that some bacteria are covered by the cuticle (B), Bars = 25 p n (A), 5 pm (B).

In the present study, sunflower seedlings were raised in an artificial environment that was sterile (vermiculite, moistened with double distilled water). The major source of bacteria was the contaminated outer surface of the achene (pericarp). Since no seed-transmitted bacteria were detected it is obvious that the colonization of the cotyledons (as well as the other organs of the developing seedlings) occurred via the pericarp. It should be noted that for all observations and experiments healthy embryos and seedlings were used. It follows that the bacteria detected by organ imprints and scanning electron microscopy are nonpathogens. These saprophytic bacteria were transmitted from seed (pericarp) to the seedling. Since they are not pathogenic to sunflower, they did not infect their host and caused no symtoms.

638. HIRANO,S . S . and UPPERC.D., 2000: Baclteria in the leaf ecosystem with emphasis on Pseudomtrnas syringtre - n pathogen, ice nucleus, and epiphyte. Microbial. Mol. Biol. Rev. 64, 624-653. HOLLAND,M. A., 1997: Methylobaoteriurn and plants. Rec. Res. Develop. Plant PhysioI. l:, 207-213. KRAMER,P. J. and BOYER, l. S., 1995, Water relations of plants and soils. Academic Press, San Diego. KUTSCHERA, W.,l 990: Cell-wall synthesis and elongation growth in hypocotyls of NeliuntEzus annuus L. PIcrnta 18 I , 3 1 6-323. KUTSCHERA, U,, 2000: Cell expansion in plant development. Rev. B rasiF .Fisiol . Veg. 12,6595. KUTSCHERA, U,,SIEBERT, C., MASUDA, Y.,and SIEVERS, A., 1 990: Effects of submergence on development and gmvitropism in the coleoptile of 0ryz.a sativa L. PEanta 183, 112-119. L~vmu,J. H.J. and LINDOW, S . E., 2001:Appetite of an epiphyte: Quantitative monitoring of bacterial sugar consumption in the plzyllosphere. Proc. NatI. Acad. Sci. (USA) 98, 346-3453, Address of the author: U. Kutschera, Fachbereich BioIogie, Abt. Pflanzenphysiologie, Universitat Kassel, Heinrich-Plett-StraBe 40, D-34 109 Kassel