Introduction

Undaria pinnatifida (Harvey) Suringar 1873 (Laminariales, Phaeophyceae) is a kelp that show large (ca. 0.5–3.0 m), annual sporophytes with a lobed lamina with evident midrib, attached by root-like haptera to rock, stone, wood, shells, tunicates or sometimes plants and also to most artificial substrates. U. pinnatifida is widely distributed in Europe (Floc’h et al. 1991; Fletcher and Manfredi 1995; Hardy and Guiry 2003); Asia (Tseng 1984; Yoshida et al. 1990; Lee and Yoon 1998); North America (Aguilar-Rosas et al. 2004); Australia and New Zealand (Hay and Luckens 1987; Adams 1994; Nelson 1999; Womersley 2003).

In December 1992, U. pinnatifida was detected for the first time in Argentina close to the international harbor of Puerto Madryn (Piriz and Casas 1994). Thalli were normally colonized by the microscopic brown epi-endophyte Laminariocolax aecidioides (Streblonema aecidioides sensu Yoshida and Akiyama (1979) Ectocarpales, Phaeophyceae), whose individuals formed a hairy cover on the host surface.

The main goals of the present study are: (1) to describe the bona fide nature and the morphology of the symbiosis between L. aecidioides and U. pinnatifida in Argentinian populations; (2) to describe for the first time the morphology of L. aecidioides under culture conditions; and (3) to find clues to determine the state of health of the Argentinian U. pinnatifida community.

In general, most of the morphological studies on epiphytism refer to infections on red algal hosts of commercial interest, mainly by green epiphytes (Correa and McLachlan 1991, 1992, 1994; Correa et al. 1988, 1993, 1994). Comprehensive morphological studies on epi-endophytes on brown seaweeds are very scarce.

Thalli of another species of Undaria have been observed in Europe infected with filaments of L. aecidioides by Veiga et al. (1997) and the relationship between Undaria sp. and Streblonema aecidioides (=L. aecidioides) populations has been described on Japanese coasts in the north-west Pacific by Yoshida and Akiyama (1979).

Reports indicate that L. aecidioides filaments also infect blades of Laminaria saccharina from Greenland (Pedersen 1981), the western Baltic (Burkhardt and Peters 1998) and the north coast of Spain (South and Tittley 1986; Veiga et al. 1997), and also blades of Laminaria hyperborea from the coasts of Helgoland (Lein et al. 1991). Some populations of L. aecidioides found on Hedophyllum sessile have been mentioned as S. aecidioides by Abbott and Hollenberg (1976).

Materials and methods

Fronds of Undaria pinnatifida were obtained from intertidal and subtidal populations along the coast of Puerto Madryn, in the province of Chubut, Argentina (42°47′S, 65°02′W) during December 2004.

After being collected, fronds of U. pinnatifida were kept on ice and retained in labelled plastic bags until they were examined in the laboratory, usually within 5 h after collection. Fronds were then brushed and rinsed under running tap water. Small portions of infected fronds were sectioned, then immersed in fresh 0.5% solution of sodium hypochlorite for 30 s, and finally rinsed three times, 5 min each, in sterile seawater. A 2-min sonication was subsequently applied to 5 × 5 mm portions in sterile seawater, renewing the seawater after each burst. This cleaning procedure was followed in order to remove diatoms as well as other epiphytes.

Crude cultures of L. aecidioides were initiated by inoculating portions of cleaned fronds U. pinnatifida in plastic Petri dishes containing PES medium (Provasoli 1968). Cultures were maintained at 21 ± 1° C with an illumination regime of 12:12 h LD, with a photon flux density of 15 μmol photons m−2 s−1. Vegetative endophytic filaments, and also spores from both plurilocular and unilocular sporangia, were isolated using the hanging-drop technique. To avoid further diatoms contamination, a 2.5% germanium dioxide solution was added to the culture during the first week (Lewin 1966; Christensen 1982).

Clonal cultures were established by pipetting single germlings or thallus fragments in advance of first signs of maturity in the cultures. Strains were maintained for 4 weeks. L. aecidioides filaments formed small 0.1-mm spherical masses inside excised host tissue after 4 weeks in culture. No other algae or microorganisms were present. The filaments were removed and cultured separately for study.

Cytomorphometry was carried out using a stereoscopic microscope Wild-Herbrugg and an inverted microscope Nikon Eclipse TE 300, with anoptral phase contrast and differential interference contrast (DIC) and with an incorporated camera Nikon FDX 35. The presence or absence of epi-endophyte filaments was determined under light microscope in semi-thin sections of thalli of U. pinnatifida obtained with glass knives on a Reicher Ultracut OM U2 ultramicrotome. In order to obtain semi-thin sections, thalli were fixed in 2.5% glutaraldehyde in seawater for 2 h at 4°C, and postfixed in 1% OsO4 in sea water for 2 h at 4°C. The material was dehydrated in a graded acetone series and embedded in Spurr´s low viscosity resin. The resin was removed using a metallic sodium, benzene and methylic alcohol solution (Hayat 1986). Sections were stained with a combination of colorants, namely haematoxiline-malachite green-basic fucsine (1:1:1) (Berkowitz et al. 1968).

Chromosome counts were carried out using unialgal cultures of L. aecidioides, derived from biflagellate zoospores. Thalli were fixed either in 1:3 mixture glacial acetic acid/absolute ethanol or in 6:3:1 mixture formaldehyde-absolute ethanol-glacial acetic acid at 5°C during a period of 2–24 h. Postfixation was carried out with 70% ethyl alcohol. The material was subsequently hydrolyzed for 30 min in 1 N HCL at room temperature and stained with Schiff stain in darkness for 2 h (Johansen 1940). It was bleached during 20 min in a 1:3:3 mixture of sodium metasulphite: 1 N HCL: distilled water, washed with distilled water for 30 min, and finally mounted in a drop of 2% acetic acid solution of ferric haematoxylin with added iron acetate (Núñez 1968).

Scanning electron microscopy

Filaments of L. aecidiodes and thallus of U. pinnatifida were fixed in 2.5% glutaraldehide-seawater at 5°C in cacodilate buffer for 2 h. They were then mounted on slides covered with 0.5% poly-D-lysine and dehydrated in a graded acetone series. Samples were finally critical point dried during 1 h, coated with gold, and observed with a Jeol 35 CF scanning electron microscope (SEM).

Results

Morphology of thalli Laminariocolax aecidioides in nature

Infected fronds of Undaria pinnatifida exhibited brown spots near the mucilage ducts (Fig. 1a). SEM images revealed the spots were formed by thalli of L. aecidioides partially covering the surface of the fronds (Fig. 1b, arrows). Cross-sections of infected U. pinnatifida tallus showed that thalli L. aecidioides were localized principally on the cortical zone of the hosts. Thalli of L. aecidioides were filamentous, uniseriate and branched (Figs. 1c and 1d). They formed a postrate system that reached the medulla of the host but did not penetrate host cells, thus no necrotic tissues were present in their vicinity. Filaments exhibited cells 5–15 μm in diameter and 10–40 μm in length, with one to several chloroplasts, discoid or band-shaped.

Fig. 1
figure 1

Undaria pinnatifida infected by Laminariocolax aecidioides. (a) Dark spots observed on the surface of a blade of U. pinnatifida (white arrows), symptom of the infection of L. aecidioides. (b) SEM image showing several thalli of L. aecidioides (arrows) covering partially the surface of a sporophyte of frond of U. pinnatifida. (c) Semi-thin transverse sections through an infected frond of U. pinnatifida to see the location of the L. aecidioides filaments; prostrate filament of L. aecidioides on the surface of hosts. A plurilocular sporangium is observed (arrow). (d) Semi-thin transverse sections through an infected frond of U. pinnatifida to see the location of the L. aecidioides filaments. A filament of L. aecidioides is observed, which has penetrated into the cortical zone through intercellular spaces (arrow). (e) SEM showing a thallus of L. aecidioides with reproductive structures formed in the erect system (arrows). (f) SEM, detail of the Fig. 1e image, to show vegetative cells and reproductive structures located in the erect system. (g) SEM detail of an empty L. aecidioides unilocular sporangium exhibiting basal and apical dehiscence, respectively. (h) SEM detail of an empty L. aecidioides unilocular sporangium exhibiting basal and apical dehiscence, respectively. (i) A semi-thin transverse section of a U. pinnatifida fronds to see a L. aecidioides filaments. Note a unilocular sporangium in the prostrate system (arrow). (j) Detail of a perforation of U. pinnatifida frond caused by the presence of the epi-endophyte. The central part of the perforation is occupied by reproductive structures of L. aecidioides. (k) SEM detail of the surface of an infected frond of U. pinnatifida to show numerous epiphytic bacteria normally present

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Plurilocular sporangia were solitary, ovoid and uniseriate, 15–20 μm diameter (Fig. 1c, arrows). Unilocular sporangia were also solitary, ovoid, 7–12 μm in diameter, 20–40 μm in length (Figs. 1e–1h and 1i); they showed either basal or apical dehiscence.

Infection

Infected sporophytes did not present their characteristic healthy brown colour, being smooth and flexible, in contrast to the non-infected ones, which were darker, rough, less flexible and curved. The presence of L. aecidioides also produced perforations in the tissues that were eventually occupied by vegetative and reproductive structures of the endophyte (Fig. 1j).

Semi-thin sections of infected thalli showed that U. pinnatifida cortical cells did not exhibit vertical compression but rather a lateral compression owing to the presence of L. aecidioides filaments among them.

Also, unidentified epiphytic bacteria distributed throughout all the surface of hosts were normally present in infected thalli (Fig. 1k).

Characteristics of Laminariocolax aecidioides developing in culture

Vegetative morphology

Thalli consisted on branched filaments with diffuse growth. They did not develop hyaline hairs, like those observed in natural conditions. Cells were 25.56 μm (11–55 μm; SD: 5; n = 32) in length and 14.8 μm (10–19 μm; SD: 2.44; n = 32) in diameter (Fig. 2a). Short, erect filaments formed by cells of smaller dimensions were also observed; their cells reached 6.28 μm (3.36–11.2 μm; SD: 2.42; n = 30) in diameter and 9.70 μm (4.5–15.7 μm; SD: 3.92; n = 30) in length (Fig. 2b). All cells normally contained 2 discoid plastids (Fig. 2b).

Fig. 2
figure 2

Laminariocolax aecidioides in culture. (a) A thallus of L. aecidioides after 4 weeks in culture. (b) Short, erect filaments of L. aecidioides, exhibiting doliiforms cells. All cells normally contained 2 discoid plastids. (c) A cylindrical, terminal unilocular sporangium (arrow), formed in an erect filament. (d) A cylindrical, intercalar unilocular sporangium (arrow), formed in an erect filament. (e) A zoosporangium with unispores (arrow). (f) Detail of a biflagellate unispores. Note, it shows a laterally arranged plastid and eyespot. (g) Plurilocular sporangium with biflagellated pluripores. Note, it is cylindrical, uniseriate and that it exhibits an apical pore (arrow). (h) Gametophyte filament whose cells posses 2-4 discoid plastids (white arrows), and conspicuous pyrenoids (black arrows). (i) Intercalar, unilocular immature gametangia in a gametophyte filaments. (j) Release of isogametes through a single apical pore in each gametangium (arrows). (k) Haploid metaphase plate with 8 chromosomes. (l) Haploid metaphase plate with 8 chromosomes. Graphical representation of the metaphase plate

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Reproduction

After a week under culture conditions, filaments developed terminal and intercalar, cylindrical, unilocular sporangia (Figs. 2c and 2d). Each unilocular sporangium was 30 μm (12–42 μm; SD: 8.5; n = 15) in length and 14.3 μm (11–18 μm; SD: 2.45; n = 15) in diameter. Unilocular sporangia formed spherical, biflagellate zoospores, 5-6 μm in diameter, with a lateral chloroplast with an eyespot (Figs. 2e and 2f). Filaments also formed cylindrical, plurilocular sporangia, 49 μm (30–81 μm; SD: 12. 3; n = 13) in length and 14.4 μm (7–20; SD: 3.66; n = 13) in diameter. Plurilocular sporangia produced biflagellate zoospores, which were liberated through an apical porus at maturity (Fig. 2g). Zoospores, which were negatively phototactic, were pear-shaped to ovoid, 5–7 μm × 6-5 μm , and possessed one or two chloroplasts and a single red eyespot.

Thalli bearing both unilocular and plurilocular zoosporangia were placed in hanging-drop cultures. After 2 weeks of cultivation, new plurilocular sporangia of the same type as those on the parent thallus were formed. The zoids released were identical to those described above. Settlement and germination of zoospores was observed. They developed into thalli with both unilocular and plurilocular sporangia.

The settlement and early germination of unispores was not observed. They eventually developed into gametophytes that were morphologically different from the sporophytes. Adult gametophytes were branched filaments with diffuse growth that did not develop hyaline hairs. Filaments exhibited cells 14 μm (5–31 μm; SD: 5.6; n = 32) in length and 8 μm (5–16; SD: 2.62; n = 32) in diameter (Fig. 2h). Cells contained 2–4 discoid chloroplasts, each one with a conspicuous pyrenoid (Fig. 2h). Gametophytes also developed branched prostrate filaments constituted by doliiform cells, 5.26 μm (4–8 μm; SD: 1.16; n = 15) in length and 4.7 μm (4–7 μm; SD: 0.85; n = 15) in diameter. These cells generally contained 2 discoid plastids with only one pyrenoid (Fig. 2h) At maturity, many of the cells of the erect filaments were transformed into cylindrical (and both terminal and intercalar plurilocular) gametangia, 21.9 μm (5–50 μm; SD: 18.5; n = 10) in length and 8.9 μm (5–13 μm; DS: 2.23; n = 10) in diameter (Fig. 2i). Eventually, 4–6 μm ×  3–4 μm negatively phototactic isogametes were released by an apical opening (Fig. 2j).

Caryology

Chromosome counts were made in both sporophytic and gametophytic thalli. Small, cane-shaped chromosomes were observed placed in very tight prometaphase plates. In the sporophytes, diploid assemblages of 16 chromosomes were counted whereas in haploid gametophytes plates of 8 chromosomes were found (Figs. 2k and 2l).

Discussion

Infections by microscopic endophytic brown algae are common diseases of kelp species. They are known from various host species in different parts of the world, such as Japan (Yoshida and Akiyama 1979; Kawai and Tokuyama 1995), Pacific North America (Andrews 1977; Apt 1988), Europe (Dangeard 1931; Russell 1983a, b; Lein et al. 1991; Peters and Ellertsdóttir 1996; Peters and Schaffelke 1996; Heesch and Peters 1999), and Pacific South America (Peters 1991).

In this study, the only endophyte identified was L. aecidioides. Except for the strictly parasitic Herpodiscus durvillaeae (Lindauer) South (South 1974; Peters 1990) on Durvillea antartica, all brown endophytes so far described are pigmented (Burkhardt and Peters 1998). In general, and as in the present case, the epi-endophytes are included in the “simple brown algae” that is, the “Ectocarpales sensu lato” (Fritsch 1945; Burkhardt and Peters 1998). Many were originally identified as Ectocarpus Lyngbye and are now commonly included in Streblonema (Derbès et Solier in Castagne (Derbès and Solier 1851). Several other genera, however, have also been proposed as the identity of these organisms. These are Entonema Reinsch (1875), Phycocelis Stroemfelf (Kuckuck 1894), Myrionema Greville (Sauvageau 1898), Pilocladus Kuckuck (1854), Gononema Kuckuck et Skottsberg in Skottsberg (Pedersen 1981), Microspongium Reinke (Pedersen 1984), Laminarionema Kawai et Tokuyama (Kawai and Tokuyama 1995) and Laminariocolax Kylin (Kylin 1947). Regarding the Argentinian coasts, the present study is the first made on an epi-endophyte of a brown algal host; thus, we are not able to made comparations with the relationships of L. aecidioides with thalli of some other kelp species different from U. pinnatifida. Moreover, in studies on red algae hosts from Argentina, such as Rhodymenia sp., Hymenena falklandica (Gauna 2005), and Gracilaria gracilis (Martin et al. 2007), L. aecidioides has not been mentioned.

Although dramatic morphological effects were observed in infected hosts of U. pinnatifida, like galls or necrotic tissues, as a result of the infection, the presence of L. aecidioides did not prevent the normal maturation of the sorus, the production of zoospores and thus the reproductive capacity and biomass augment in U. pinnatifida. In consequence, we can assume that the effects of the epi-endophytic infection of L. aecidioides on U. pinnatifida are neither severe nor deleterious.

The results of this paper also allowed us to suggest that L. aecidioides uses the thalli of U. pinnatifida as a proper substrate, since their thalli developed their entire life cycle on them.

None of the thalli with dark spots or distorted thalli lacked endophytes, but more than half of the plants that contained endophytes did not show macroscopically visible disease symptoms. The mere presence of endophytes in any part of the host does not seem to be sufficient for disease symptoms to develop, and both endophyte density and distribution in the host may be important. A lag phase between infection and appearance of a pathological disorder is usual in infectious diseases and has also been reported by Peters and Schaffelke (1996) for the endophyte Gononema aecidioides in Laminaria saccharina in the western Baltic.

The young and adult thalli of U. pinnatifida infected by L. aecidioides in Argentina presented equal sintomatology as that described by Yoshida and Akiyama (1979) in Japanese adult populations of the same species. Nevertheless, individuals of the populations of L. aecidioides deciphered by Yoshida and Akiyama presented minor cytological vegetative and reproductive dimensions compared with those of the Argentinian populations.

The information about how endophytes are transmitted and the way of penetration in the host’s is scarce. Apt (1988) has suggested that the epiphytic filaments enter the internal hosts tissues of these organisms through wounds. Nevertheless, the observations made in this study on U. pinnatifida, indicate that L. aecidioides do not require a wound to gain access to internal tissues, as we observed a prevalence of penetrations in regions near the openings of mucilage glands. Kylin (1947) for Lamiraiocolax tomentosoides and Peters and Ellertsdóttir (1996) for Laminarionema elsbetiae suggested that spores are specialized infective agents that attach to and penetrate the healthy host surface, and they also observed that no wounds or other openings are required for a successful invasion of the host.

Chromosomes counts in species of the genus Laminariocolax have not been published so far. For Laminariocolax macrocystis, there is only the mention of chromosomal complements of n = 13-–5. The present cytological studies of L. aecidioides determined that the registered n = 8 is in the lower rank of the proposed haploid chromosomal number for the Ectocarpales (8–13), indicated by Lewis (1996).

The results described here on culture material, in particular the caryological observations, provide good evidence that L. aecidioides possesses sexuality and two different alternating generations. Both generations are microscopic and perhaps live on the same host. The sporophyte of L. aecidioides may directly replicate itself by means of plurispores, but can also produce unispores that develop into gametophytes. The gametophytes may form identical male and female gametes to give a new sporophytic generation.

Taxonomical comments

Up to now, four species of Laminariocolax have been described: L. eckloniae A. F. Peters, L. aecidioides, L. tomentosoides (Fallow) Kylin and L. macrocystis (A. F. Peters) A. F. Peters (Burkhardt and Peters 1998). All these species exhibit the same endophytic habit, but morphological differences exist. However, more significant for the species definition is the dependability of each species to a specific host species, i.e. L. tomentosoides colonises several species of Laminaria digitata, whereas L. macrocystis lives on sporophylls and cauloid of Macrocystis pyrifera and L. eckloniae, in turn colonises thalli of Ecklonia maxima (Osbeck) Papenfuss (Burkhardt and Peters 1998). L. aecidioides was mentioned under their different synonyms in several regions. Although the information is not complete, since references to hosts were generally not indicated, it is mainly distributed in the Northern hemisphere. In this work, the distribution of the species is extended to the Southern hemisphere, being also the first report for the Atlantic Ocean coasts.

With the exception of one report on the occurrence of a species of Laminariocolax on the red alga Grateloupia doryphora (Montagne) Howe (Villalard-Bohnsack and Harlin 2001), members of this genus were mentioned as mainly growing on seaweeds of the order Laminariales. L. aecidioides was also isolated from fronds of L. hyperborea (Gunnerus) Foslie in Britain, Norway (Lein et al. 1991) and Helgoland (Ellertsdóttir and Peters 1997), also from Laminaria digitata frond (Hudson) J. V. Lamouroux in Maine, (Burkhardt and Peters 1998), Undaria sp. (Yoshida and Akiyama 1979), Heldophyllum sessile (Areschoug) Setchell and Gardner (1922) (Abbott and Hollenberg 1976) and recently from Undaria pinnatifida (Gauna 2005).