A Microscopical Study of the Development of a Spore-Like Stage in the Life Cycle of the Root-Nodule Endophyte of Alnus glutinosa (L.) Gaertn

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<ul><li><p>A Microscopical Study of the Development of a Spore-Like Stage in the Life Cycle of theRoot-Nodule Endophyte of Alnus glutinosa (L.) GaertnAuthor(s): C. Van Dijk and E. MerkusSource: New Phytologist, Vol. 77, No. 1 (Jul., 1976), pp. 73-91Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2433642 .Accessed: 13/06/2014 02:29</p><p>Your use of the JSTOR archive indicates your acceptance of the Terms &amp; Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp</p><p> .JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact support@jstor.org.</p><p> .</p><p>Wiley and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to NewPhytologist.</p><p>http://www.jstor.org </p><p>This content downloaded from 194.29.185.216 on Fri, 13 Jun 2014 02:29:08 AMAll use subject to JSTOR Terms and Conditions</p><p>http://www.jstor.org/action/showPublisher?publisherCode=blackhttp://www.jstor.org/action/showPublisher?publisherCode=npthttp://www.jstor.org/stable/2433642?origin=JSTOR-pdfhttp://www.jstor.org/page/info/about/policies/terms.jsphttp://www.jstor.org/page/info/about/policies/terms.jsp</p></li><li><p>New Phytol. (I 976) 77, 73-9 I . </p><p>A MICROSCOPICAL STUDY OF THE DEVELOPMENT OF A SPORE-LIKE STAGE IN THE LIFE CYCLE OF </p><p>THE ROOT-NODULE ENDOPHYTE OF ALNUS GLUTINOSA (L.) GAERTN. </p><p>BY C. VAN DIJK* AND E. MERKUSt </p><p>Department of Botany, Research Group on Biological Nitrogen Fixation, University of Leiden, Leiden, The Netherlands </p><p>(Received 25 October I974) </p><p>SUMMARY </p><p>A light- and electron-microscopical study of the root-nodule endophyte of Alnus glutinosa (L.) Gaertn. was carried out to investigate the development of a spore-like stage, here called the granule, in the life cycle of the endophyte. Comparison of granule-rich and granule-free root nodules showed that granule formation takes place via local transverse growth of thick endo- phytic hyphae, giving rise to multicellular 'granulated bodies' differing in shape and size. Subse- quently, the cells of these granulated bodies are transformed into granules by cell separation and ultrastructural changes, the most striking of which are cell-wall thickening, reduction of the number of mesosomes, and increased density of the cytoplasm. Granule development takes place both intracellularly and intercellularly. Intracellularly produced granules are eventually liberated by the death of the host cell. Mature granules show a strong resemblance to spores of free-living actinomycetes in their ultrastructure and behaviour. </p><p>It is concluded that, in view of the terminology currently used in the description of members of the Actinomycetales, the term granule should be replaced by spore and thus the term granu- lated body by sporogenous body. </p><p>INTRODUCTION </p><p>Morphological and cytological studies of the root-nodule endophyte of Alnus glutinosa (L.) Gaertn. by Peklo (I9IO), Schaede (I933), Kappel and Wartenberg (I958), Pommer (I956), Becking, de Boer and Houwink (i964) and Gardner (i965) revealed the actino- mycetal character of the endophyte. Intracellular hyphae with a close resemblance to the hyphae of free-living actinomycetes (Becking et al., I964) represent the basic appear- ance of the endophyte. The development of intracellular spherical vesicles is easily deduced from their attachment to and continuity with the tips of hyphal branches. Although the possible function(s) of these complex vesicles has long been uncertain, Akkermans (in press) recently found nitrogen fixation activity in isolated clusters of vesicles. A third morphologically distinct stage in the life cycle of the endophyte, mentioned by Peklo as early as I9IO, has been described as characterized by polyhedral cells about I-2 ,um in diameter, usually lying tightly packed in intercellular spaces, and dead host cells. Most authors have called these particles bacteroids, a term in general use </p><p>* Present address: Institute for Ecological Research, Weevers' Duin Biological Station, Oostvoorne, The Netherlands. </p><p>t Present address: National Institute of Public Health, Bilthoven, The Netherlands. </p><p>73 </p><p>This content downloaded from 194.29.185.216 on Fri, 13 Jun 2014 02:29:08 AMAll use subject to JSTOR Terms and Conditions</p><p>http://www.jstor.org/page/info/about/policies/terms.jsp</p></li><li><p>74 C. VAN DIJK AND E. MERKUS </p><p>since the descriptions given by Schaede (I933). Becking et al. (I964) introduced the name bacteria-like cells, which was later changed to polyhedral-shaped cells (Becking, I970). </p><p>Arcularius (I928) suggested that 'bacteroids' might represent a quite different organ- ism, but Schaede (I933) concluded that they were mainly produced by fragmentation of endophytic hyphae. In some cases, however, he observed the disintegration of spherical vesicles, leading to the formation of bacteroids. Electron-microscopical studies of the endophyte (Becking et al., I964; Gardner, I965) did not contribute to a satisfactory reconstruction of the development of the bacteroids. Becking et al. (I964) suggested that they are only formed by the fragmentation of tightly interlaced hyphae in dead cells, whereas Gardner (I965) came to the conclusion that they are only formed by the frag- mentation of spherical vesicles with complete septa. Kappel and Wartenberg (I958) con- cluded from light-microscopical observations that bacteroids originate from hyphae via an intermediate multicellular stage they called the Spindel and which they supposed to be identical to the Schlauchen and Blasen described by Schaede (I933). </p><p>Despite the divergent views with respect to the development of bacteroids or bacteria- like cells, it is generally believed that they represent some kind of a resting stage, capable of survival and nodulation. But although Kappel and Wartenberg (I958) were able to obtain nodulation of alder plants with crude isolates of them, strict proof of the infective capacity of bacteroids has not yet been given. </p><p>To avoid any premature implicit conclusion concerning morphological and functional relationships with other organisms, the names bacteroid and bacteria-like cell will be replaced here by the more neutral term granule, since we prefer to wait until sufficient knowledge allows a definite use of the term spore. This paper presents the results of a light- and electron microscopical study of the development of bacteroids (further to be called granules) performed to contribute to the understanding of a probably important part of the life cycle of the endophyte of A. glutinosa root-nodules. Unless otherwise stated the root-nodule endophyte of A. glutinosa will be referred to in this paper as the endophyte. The name Frankia alni, introduced by Becking (I970), will not be used, because the present study was restricted to the endophyte of only one Alnus species and there is no certainty that the results apply in all details to the life cycles of the root-nodule endophytes of other Alnus species. </p><p>MATERIAL AND METHODS </p><p>Collection of root-nodules Root-nodules of Alnus glutinosa were collected in the vicinity of Leiden from several </p><p>alder shrubs bearing granule-rich or granule-free root-nodules. Samples of both granule types were also obtained from alder plants cultivated on a liquid medium. For this purpose, plants were raised from seed collected from native trees. The seeds were washed, surface sterilized with a o.i1% bromine solution, and left to germinate at z2zC in the dark in a glass vessel provided with wet gravel. After io days, a modified Hoagland solution (Quispel, I954) reduced to half strength was added to the gravel and the seedlings were exposed to 220C, 70-80% relative humidity, and I7 h illumination by 30,000 Lux Philips TL 65/33 alternating with 7 h darkness. After 3 weeks, plants with two exposed leaves were transferred to jars filled with Crone's solution containing KNO3 (Bond, I95i), Fe-EDTA, and trace elements, according to Allen and Arnon (1955). One week before inoculation, the nutrient solution was replaced by Crone's solution without </p><p>This content downloaded from 194.29.185.216 on Fri, 13 Jun 2014 02:29:08 AMAll use subject to JSTOR Terms and Conditions</p><p>http://www.jstor.org/page/info/about/policies/terms.jsp</p></li><li><p>Endophytes of Alnus 75 </p><p>nitrogen. Root-nodule formation was initiated by bringing 500 mg (fresh-weight) crushed lobes of granule-rich or granule-free field nodules into 400-ml jars containing twelve alder plants on nitrogen-free Crone's solution. Nutrient solution was refreshed weekly and when necessary plants were transferred to larger jars. Cultivated nodules were collected over a period of 4 weeks to 6 months after inoculation of the plants. </p><p>Preparation of nodule sections for light microscopy Peripheral lobes of cultured and field-sampled root nodules were fixed in FAPA </p><p>(50 ml 40?0 formaldehyde, 250 ml 96% ethanol, I5 ml propionic acid, and I5 ml glacial acetic acid) and embedded in diglycol stearate via an ethanol-xylol series, after which 6-pum sections were cut on a rotary microtome and stuck to object slides with Haupt adhesive (Johansen, I940). Staining was carried out with Toluidine Blue (Feder and O'Brien, i968) or Schiff's reagent. Epon sections 2 /um thick, prepared as described for electron microscopy, were fixed to glass slides on a hot plate and stained with Toluidine Blue I % in a I % borax solution for light-microscopical observation. Fresh sections cut about 20 ,um thick with a hand microtome were transferred to a drop of water on a microscope slide, and a small drop of Fabil reagent (Noel, i964) was added as staining and clearing reagent. For optimal colour differentiation, immediate evaluation was necessary. </p><p>Preparation of ultrathin sections Tissue blocks up to 2 mm in diameter were cut from the cortical region of fresh </p><p>nodule lobes (Fig. i, regions C and D) and transferred to I0% OS04 in buffer (pH 6.o) according to Ryter and Kellenberger (1958), for 3 h of fixation, after which the tissue was rinsed and stained with uranyl acetate 0.5 0 in the same buffer for 2 h. Dehydration was carried out via an ethanol series into propylene oxide. Fixation, staining, and dehydra- tion were carried out at 40C. The dehydrated tissue blocks were embedded in Epon 8I2, according to Luft (I96I). Thin sections (about 50 nm) cut with an LKB Ultratome III were fixed on Formvar-coated grids and post-stained with uranyl acetate 7.7%0 and lead citrate according to Reynolds (I963). </p><p>Microscopical observations Electron microscopy was performed in a Philips EM 200 and a Philips EM 300. </p><p>Light microscopy was done with a Wild M20 microscope. Photomicrographs were made on Kodak Panatomic-X film with the latter microscope provided with a Fluotar x 50 oil-immersion objective. </p><p>RESULTS </p><p>Morphologically distinct endophyte stages observed in the nodule tissue The existence of root-nodules with and without endophytic granules (bacteroids, </p><p>polyhedral cells), already mentioned by Schaede (I933), was established by making an inventory of root nodules on Alnus glutinosa trees occurring in various parts of The Netherlands. Moreover, inoculation experiments showed that the presence or absence of granules in nodules is due to what are probably rather stable differences between strains of the endophyte. The type of root nodules containing granules will be called Gr( +) and the type without granules Gr(-). In some cases, apparently Gr(-) nodules were found to contain a very small quantity of granules of the intercellular type. Because </p><p>This content downloaded from 194.29.185.216 on Fri, 13 Jun 2014 02:29:08 AMAll use subject to JSTOR Terms and Conditions</p><p>http://www.jstor.org/page/info/about/policies/terms.jsp</p></li><li><p>76 C. VAN DIJK AND E. MERKUS </p><p>the latter granules are easily overlooked in routine nodule analysis and such cases never showed intracellular granule formation, we continue to classify them as Gr(-). </p><p>A stock of nodules of both the Gr(+) and the Gr(-) types kept on liquid-cultured alder plants was used for light-microscopical investigation of the morphologically distinct endophyte stages. This study was performed to define the endophyte stages involved in granule formation. The location of distinct endophyte stages of both nodule types is roughly indicated in Fig. i. The regions can be characterized as follows: </p><p>Region A. Apical meristematic cells without signs of infection. Region B. Area of young, growing cortex cells invaded by thin, curved hyphae. In </p><p>this region, thick hyphae, often straight and running from cell to cell, are sometimes visible. These thick hyphae show internal division (see Schaede, 1933). </p><p>Region C. Area with an abundance of mature vesicle clusters in living host cells. The spherical vesicles (about 4 um in diameter) are attached to and continuous with the tips of branched hyphae, thus resembling a bunch of grapes. Only a small percentage </p><p>\ D. . . .. . . . . . . C .. .. . . . . . D. ..' </p><p>Fig. I. Distribution of distinct endophyte regions in a longitudinally sectioned nodule lobe; drawing after a light micrograph. S: stele. (See text.) x 30 </p><p>of the original hyphal clusters are not transformed into vesicle clusters. Decay of vesicles and hyphae starts in the oldest part of region C. So far, no differences have been found between Gr( +) and Gr( -) nodules. Beside the stages mentioned, region C of Gr( +) type nodules also contains the granules. Clumps of mature granules are found in dead cells and in intercellular spaces interspersed between cortical cells filled with vesicles (Plate 3, Nos I9 and 2I). The number of these granule clumps increases toward the base of region C but-and this must be stressed-clumps of granules are already present in the youngest part of region C, where first vesicle clusters are also found. </p><p>A remarkable endophytic stage of development, occurring in region C of Gr(+?) nodules, will be called the granulated body. This term is introduced here to indicate a polymorphic stage of the endophyte most easily recognized by the partition of its con- tents into granule-like cells. Many of these granulated bodies could be identified as a Spindel (Eng.: spindle) described by Kaippel and Wartenberg (I958). In these cases the origin from straight endophytic hyphae is clearly shown by the morphological continuity between the spindle poles and the attached hyphae. This type of spool-shaped structure </p><p>This content downloaded from 194.29.185.216 on Fri, 13 Jun 2014 02:29:08 AMAll use subject to JSTOR Terms and Conditions</p><p>http://www.jstor.org/page/info/about/policies/terms.jsp</p></li><li><p>Endophytes of Alnus 77 </p><p>will be called the typical spindle (Plate i, Nos 3-7) to distinguish them from granulated bodies with a less regular shape suggesting an origin from irregularly curved hyphae. The latter type will be called the atypical s...</p></li></ul>

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