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Omnipresence of Labyrinthulaceae in seagrasses
Aquatic botany ELSEV I ER
Aquatic Botany 48 (1994) 1-20
Omnipresence of Labyrinthulaceae in seagrasses L.H.T. Vergeer*, C. den Hartog Laboratory of Aquatic Ecology, Catholic University, Toernooiveld, 6525 ED Nijmegen, The Netherlands (Accepted 17 December 1993)
Abstract
Seagrass species collected in different parts of the world have been checked for the presence of Labyrinthula spp. Isolations succeeded from all seagrasses studied, indicating a worldwide association between the two groups of organisms. The isolates have been described and compared. Infection experiments have shown that isolates are genus-specific.
1. Introduction
Species of the family Labyrinthulaceae, usually referred to as marine slime moulds, are widely distributed in coastal areas around the word. They have been isolated from a wide range of marine habitats, including organic detritus, diatoms, macro-algae and marine vascular plants. Labyrinthula spp. are unique in having an ectoplasmatic network in which their fusiform, vegetative cells move (Pokorny, 1967 ). There has been much confusion at the species level in the past, partly because two of the characteristics mostly used in species delimitation (viz. cell size and growth pattern of a colony) are strongly dependent on culture conditions. The life cycle of most Labyrinthula spp. is still obscure. Nowadays, eight to nine 'true' species of Labyrinthula are recognised (Porter, 1990). Much attention has been focussed on Labyrinthula after the 'wasting disease' epidemic of Zostera marina L. (eelgrass) in the early 1930s. The massive destruction of eelgrass beds along the Atlantic coasts of Europe and North America was almost certainly mediated by a Labyrinthula species (Renn, 1936). In the past, this species has been identified as Labyrinthula macrocystis Cienk., but re*Corresponding author. 0304-3770/94/$07.00 © 1994 Elsevier Science Publishers B.V. All rights reserved SSD10304-3770 (94)00376-W
2
L.H.T. Vergeer, C. den Hartog/Aquatic Botany 48 (1994) 1-20
cently it has been recognised as a new species, Labyrinthula zosterae Porter & Muehlstein (Muehlstein et al., 1991 ). A satisfactory explanation for the strong pathogenicity of L. zosterae in the 1930s has still not been presented (Den Hartog, 1987). Labyrinthula spp. seem to flourish on decomposing plant material rather than behaving as parasites. Today, L. zosterae appears to be commonly associated with eelgrass; recent declines of eelgrass along the North American coast have been associated with this species (Short et al., 1987 ). Along the Atlantic coast of Europe, L. zosterae is common, apparently without causing much damage to the populations of eelgrass (Vergeer and Den Hartog, unpublished observations). The symptoms of 'wasting disease' can be easily recognised. In the leaves lesions develop which cause some air lacunae to fill up with water. Small brown spots and stripes develop in these lesions, which spread longitudinally and become darker. They may cover the whole leaf after a few weeks. In truly diseased plants, these phenomena are apparent even in the youngest leaves; in most populations of Z. marina these phenomena are restricted to the oldest leaves. Recently, specimens of a related species, Zostera noltii Hornem., were also found with lesions similar to those produced by wasting disease on their leaves. The same Labyrinthula species could be isolated from these lesions as from affected Z. marina; further Z. noltii could be infected by the Labyrinthula from Z. marina (Vergeer and Den Hartog, 1991 ). On further investigation it turned out that lesions similar to those produced by wasting disease were not only present on the leaves of the Zostera species mentioned, but could be found on almost every seagrass species investigated. To elucidate the universality of the relationship between Labyrinthula and seagrasses, we studied a number of these species and checked them for the presence of Labyrinthula. The isolates of Labyrinthula obtained are described and compared. Recently, host or substrate specificity has been proposed as a potential characteristic in species identification in Labyrinthula (Muehlstein et al., 1991 ). In a few infection experiments, the degree of host specificity of some of the isolated Labyrinthula spp. has been tested. 2. Materials and methods
Seagrasses with lesions similar to those produced by wasting disease were collected in Europe, East Africa, Western Australia and in the Caribbean (Table 1 ). After being rinsed with fresh water, parts of the leaves containing the lesions were surface sterilised with sodium hypochlorite (Newell and Fell, 1982) and plated on an agar layer, 2-3 mm thick, in Petri dishes. The agar was prepared according to Porter (1990): to 1.2% agar on a sterilised seawater basis (artificial seawater 3%; Wimex, Wiegandt, Krefeld, Germany), chicken albumen (1%) and 250 mg 1- ~of streptomycin and penicillin were added. To inhibit the growth of diatoms, 3 mg 1-~ germanium dioxide was added. The parafilm-sealed Petri dishes were stored in the dark at room temperature. Mostly within 24-48 h after plating, colonies of Labyrinthula appeared on the
L.H.T. Vergeer, C. den Hartog /Aquatic Botany 48 (1994) 1-20
3
Table 1 The site ofcoUection and dimensions of vegetative cells ofLabyrinthula spp. isolated from different seagrasses Seagrass species
Na
Cell size (am) Pattern length × width
Potamogetonaceae Subfamily Zosteroideae Zostera marina L. Exmouth, England
50
Zostera mucronata den Hartog Swan River, Perth, Western Australia
25
Heterozostera tasmanica (Martens ex Aschers. ) den Hartog Whitfords area, Mullaloo pl. Western Australia
25
Subfamily Posidonioideae Posidoniaoceanica (L.) Delile Gallipoli, Italy
50
Subfamily Cymodoceoideae Halodule uninervis ( Forssk. ) Aschers. Mombasa, Kenya
Clumping of cells at the margins of a colony; bushy growth in the agar 8-12 X 2-4 Growing only in the agar; bushy growth with even margins; at some places tree-like outgrowths 9-12 X 2- 5 Growing only in the agar; resembling strongly the growth ofZ. mucronata
17-24×4-6
10-15×3-5
(15)-(5)
Mostly growing in the agar; on the agar a few patches of cells with no clumping; in the agar a transparent growth of separate threads of cells Growing only in the agar; bushy growth; cells more tightly packed at the edge; even margins Growing mostly in the agar; bushy growth; cells tightly packed in the middle; becoming looser towards the margin Growing on the agar in a circle away from the centre, with lobate outgrowths On the agar finger-like growth of tightly packed cells; dense and bushy growth in the agar
Cymodocea nodosa ( Ucria ) Aschers. Taranto, Italy
50
9-14×2-5
Syringodium isoetifolium (Aschers.) Dandy Mombasa, Kenya
50
11-14X3-5
Thalassodendron ciliatum (Forssk.) den Hartog Mombasa, Kenya
50
15-18X3-5
15
9-15×3-5
On the agar an open reticulate structure; in the agar an open bushy growth
50
10-14×3-5
Mostly growing on the agar in densely packed aggregates
50
5-8 X 2-4
On the agar individual cells are randomly dispersed; no aggregations
Subfamily Ruppioideae Ruppia cirrhosa (Petagna) Grande The Fleet, England Hydrocharitaceae Subfamily Thalassioideae Thalassia testudinum Banks ex KSnig Curacao, Netherlands Antilles Subfamily Halophiloideae Halophila ovalis (R.Br.) Hook. f. Whitfords area, MuUaloo pl. Western Australia "Number of cells measured.
4
L.H.T. Vergeer,C. den Hartog/Aquatic Botany 48 (1994) 1-20
agar. After a week, the dimensions of the vegetative cells were measured and the growth forms of the colonies described. Photographs were taken directly from the plates, using a Nikon light microscope. Infection experiments were carded out by fastening a small piece of an infected leaf of the same or another species to the leaves of an apparently healthy plant. This method was first described by Renn (1936). The experiments were carded out in 15 1 aquaria, containing a bottom layer of coarse river sand. Artificial seawater with a concentration of 3% was pumped through each aquarium separately at a turnover rate of once every 2 days. The water temperature was kept at 24 ° C. The aquaria were illuminated with 178 g E m -2 s-1, using daylight simulation lamps (Philips, 400 W, type HPI-t 150), with a 16 h light:8 h dark cycle. Air was bubbled continuously through the aquaria. The plants were then monitored over a 3 week period for the development of wasting disease-like lesions. 3. Results
As shown in Table 1, Labyrinthula species could be isolated from all seagrass species collected. All isolations were from lesions on the leaves, except for Halophila ovalis, where a "Labyrinthula' sp. was isolated from green, healthy tissue. For the Labyrinthula sp. isolated from Halodule uninervis, no reliable cell dimensions can be given; the colonies grew only in the agar with just a few cells located in a horizontal plane. 3.1. Descriptions o f the Labyrinthula spp. isolated
Host Zostera marina: vegetative cells fusiform; 17-24 gm X 4-6/tm; pale yellow to brown in mass in agar culture; cells usually containing three large vacuoles and numerous lipid droplets (Fig. la); cells growing out of the colony in massive lobes (Fig. 2a) with cells clumping at the end of the lobes; lobes sometimes segregating from behind; in the middle of a colony thick strands of cells with small interconnected branches (Fig. 2b); cells growing sporadically in the agar (black spots), forming a dense and bushy pattern (Fig. 2c). Cell size and growth pattern are in agreement with the description of L. zosterae given by Muehlstein et al. (1991). Host Zostera mucronata: vegetative cells fusiform; 8-12 gm X 2-4 gm; brown in mass in agar culture; cells only observed growing in the agar (Fig. 1b); bushy growth with even margins; at some places cells growing out of the margins in a tree-like manner, with branches originating from a single base (Fig. 2d). Host Heterozostera tasmanica: vegetative cells fusiform; 9 - 1 2 / t m X 2 - 5 pm; brown in mass in agar culture; cells only observed growing in the agar (Fig. Ic); resembling the growth-form of Labyrinthula from Z. mucronata: growth bushy in the agar with even margins (Fig. 2e) and at some places with tree-like outgrowths. Host Posidonia oceanica: vegetative cells fusiform; 10-15 #In× 3-5 #m; pale yellow in mass in agar culture; cells with one or two large vacuoles (Fig. ld); a
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(a)
(b)
Fig. 1, Vegetative cells ofLabyrinthula spp., isolated from: (a) Zostera marina; (b) Zostera mucronata; (c) Heterozostera tasmanica; (d) Posidonia oceanica; ( e ) Cymodocea nodosa; (f) Syringodium isoetifolium; (g) Thalassodendron ciliatum; ( h ) R uppia cirrhosa; ( i ) Thalassia testudinum; (j) Halophila ovalis. Magnification 750 ×.
.
Ci
J
(p)
~
(~) )~-I (P66I) gP Xuvtog oltvnbv / goJ.~vH uap "D '~aag.laA "Z"H'7
9
r~
%
h
8
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(i)
O)
Fig. 1 (continued).
9
10
L.H.T. Vergeer, C. den Hartog /Aquatic Botany 48 (1994) 1-20
(a)
(b)
Fig. 2. Growth ofLabyrinthula spp. on agar plates, isolated from: ( a ) - ( c ) Zostera marina; (d) Zostera mucronata; (e) Heterozostera tasmanica; (f) Posidonia oceanica; (g) Halodule uninervis; (h) Cymodocea nodosa; (i) Syringodium isoetifolium; (j) Thalassia testudinum; (k) and (1) Thalassodendron ciliatum; (m) and (n) Ruppia cirrhosa. Magnification 75 X.
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(c)
(d)
11
12
(e)
(f)
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(g)
(h)
Fig. 2 (continued).
13
14
(i)
(J)
L.H.T. Vergeer, C. den Hartog /Aquatic Botany 48 (1994) 1-20
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(k)
0)
Fig. 2 (continued).
15
16
L.H.T. Vergeer,C. den Hartog / Aquatic Botany 48 (1994) 1-20
(m)
(n)
Fig. 2 (continued).
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
17
few lipid droplets; cells mostly growing in the agar; on the agar just a few patches of cells with no clumping; in the agar a transparent, even growth, consisting of separate threads of cells (Fig. 2f ); margins even. Host Halodule uninervis: vegetative cells fusiform; dimensions could not be measured exactly, but size in the order of 15 gm X 5 gm; dark brown in mass in agar culture; cells only observed growing in the agar, forming a bushy pattern (Fig. 2g); cells becoming more tightly packed towards the edge of the colony; margins even. Host Cymodocea nodosa: vegetative cells fusiform; 9-14 gm × 2-5 gm; greenbrown in mass in agar culture; cells with one or two large vacuoles (Fig. 1e) and many lipid droplets; cells mostly growing in the agar with in the middle of a colony a few close aggregations; growth bushy in the agar (Fig. 2h); cells tightly packed in the middle, becoming looser towards the margin. Host Syringodium isoetifolium: vegetative cells fusiform; 1 1-14/tm× 3-5/~m; pale yellow in mass in agar culture; cells containing one or two large vacuoles (Fig. I f ) and a few lipid droplets; cells only observed growing on the agar; cells growing in a circle away from the centre; lobe-shaped outgrowths from this circle with clumping of cells at the margin (Fig. 2i ); behind the front with a loose network of branching and anastomosing threads. Host Thalassodendron ciliatum: vegetative cells fusiform; 15-18/tm × 3-5 gm; yellow in mass in agar culture; cells mostly containing one large vacuole (Fig. lg) and many lipid droplets; growth on the agar finger-like (Fig. 2j ) or hand-shaped (Fig. 2k), consisting of tightly packed cells; at the edge small patches of cells growing in the agar; dense and bushy cell aggregates in the agar. Host Ruppia cirrhosa: vegetative cells fusiform (Fig. I h); 9-15 gm × 3-5 gm; yellow in mass in agar culture; cells containing no visible vacuole or lipid droplets; colonies on the agar making a fragile appearance with beautiful tree-like outgrowths at the margin (Fig. 21) with an open reticulate structure of threads of cells in the middle of a colony (Fig. 2m) and in the agar an open bushy pattern of cells. Host Thalassia testudinum: vegetative cells fusiform; 10-14 gm × 3-5/tin; pale yellow in mass in agar culture; cells containing a few vacuoles and lipid droplets (Fig. I i ); cells on the agar forming densely packed aggregates (Fig. 2n ); those in the agar confined to small separate patches (black spots in Fig. 2n). Host Halophila ovalis: vegetative cells ovoid; 5 - 8 / a n × 2-4 gm; cells sometimes containing one large vacuole; one to three lipid droplets (Fig. lj ); individual cells are randomly dispersed on the agar without any tendency to aggregate.
18
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
Table 2 The occurrence of infection a in the leaves of a healthy 'target' plant following contact with an infected 'source' plant Target
Source
N
Infection
Ruppia cirrhosa Zostera marina Thalassia testudinum Zostera marina Zostera marina Zostera noltii Ruppia cirrhosa Posidonia oceanica
Ruppia cirrhosa Zostera marina Thalassia testudinum Halodule uninervis Thalassodendron ciliatum Zostera marina Zostera marina Cymodocea nodosa
5 80 2 4 3 94 7 2
Yes Yes b Yes No No Yes b No No
aPlants were monitored over a 3 week period. bFrom Vergeer and Den Hartog ( 1991 ).
It is clear that all seagrass species seem to have their own Labyrinthula species. Only the species isolated from the Western Australian Z. mucronata and Heterozostera tasmanica seem to be morphologically identical; the measurements are not significantly different and the growth forms on agar are very similar. A Labyrinthula sp. isolated by Armiger (1964) from a New Zealand Zostera has the same cell dimensions ( 10 gm × 3/zm) but, because of the different culture methods used, cannot be otherwise compared. The Labyrinthula sp. isolated from Halophila ovalis is the most aberrant and may represent another genus.
3.2. Infection experiments Table 2 gives the results of the infection experiments. Only a limited number of combinations of seagrasses could be tested, owing to a lack of material. It appears from Table 2 that the infection of healthy seagrasses is only possible if the affected leaf part of the same species or genus is used. Cross infection did not succeed, with the exception of the closely related Z. marina and Z. noltii (Vergeer and Den Hartog, 1991 ). 4. Discussion We did not attempt to describe and name the isolated Labyrinthula taxa forreally as this seems premature in the present stage of research. Except for Z. marina and Z. noltii, only one population of the other seagrass species could be investigated. Further research on other populations of these species is necessary to check whether the characteristics of the taxa distinguished are of a wider validity. We agree with Muehlstein et al. ( 1991 ) that standardised culture conditions are essential in identifying and comparing the different Labyrinthula species. For this purpose we used the agar composition proposed by Porter (1990). Although it is often stated that Labyrinthula can be isolated from marine vascular plants, specific information concerning the plant species is rarely given. Labyrinthula species have been recorded from brackish water species such as Zan-
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (I 994) 1-20
19
nichellia palustris L. and Ruppia maritima L. (Young, 1937) and from the saltmarsh grass Spartina alterniflora Loisel. (e.g. Amon, 1978 ). Isolations from true seagrasses are mainly restricted to species of the genus Zostera. Molisch ( 1926 ) isolated Labyrinthula vitellina Cienk. and Labyrinthula minuta Watson & Raper from Z. marina, and Tutin (1938 ) isolated Labyrinthula macrocystis from the same species. Labyrinthula macrocystis has also been isolated from Zostera hornemanniana Tutin (a narrow-leaved morph of Z. marina). Giesen ( 1990 ) mentions the isolation of a Labyrinthula sp. from Z. marina with cells approximately 8-10/zm long and 3-4/zm wide. These dimensions correspond to those given by Muehlstein et al. ( 1988 ) for Labyrinthula sp. 'S', also obtained from Z. marina. Further, Armiger (1964) isolated a Labyrinthula species from a Zostera species in New Zealand (probably Zostera novazelandica Setchell), and Muehlstein et al. (1991 ) mention a single isolation of L. zosterae from Zostera japonica Aschers. & Graebn. Apart from Zostera, a Labyrinthula species has also been recorded from Thalassia by Pokorny ( 1967 ), more specifically from Thalassia testudinum (Porter and Muehlstein, 1989), and recently from Syringodium filiforme Kiitz. (Muehlstein, 1992 ). The occurrence of Labyrinthulaceae in 11 seagrass species belonging to nine genera as well as in the brackish water species Ruppia cirrhosa, shows that there is a close association between these slime moulds and the marine phanerogams. The omnipresence of Labyrinthulaceae in seagrasses may point to a functional relationship. In all species investigated, with the notable exception of Halophila ovalis, the occurrence of Labyrinthula was restricted to the wasting disease-like lesions in the oldest leaves. Therefore, it is very likely that Labyrinthula normally does play a part in the senescence of the leaves. This would support the view that environmental factors played a major role in the outbreak of the wasting disease epidemic in the 1930s, by either increasing the susceptibility of Z. marina or stimulating the growth of Labyrinthula. Labyrinthula-linked declines have been recorded from Z. marina, the New Zealand Zostera (Armiger, 1964) and recently from Thalassia testudinum (Porter and Muehlstein, 1989 ). In the latter case, the Labyrinthula species involved is not described, apart from the statement that it differs from L. zosterae. In these cases, Labyrinthula appeared to damage also the youngest leaves, hampering the normal functioning of the plants. Apart from these observations, there are no indications that under normal conditions Labyrinthula species cause any harm to the seagrasses. All beds from which our material was collected looked lush and healthy. Along the west coast of Europe, where Z. marina has been monitored since 1986 with attention focussed on the possible build-up of disease, no indication has been found that Labyrinthula is harmful. Where decline was observed, this could be linked with environmental parameters, such as fouling by epiphytic algae, erosion, and overlayering with blankets of Enteromorpha sp. The results of the infection experiments indicate that host specificity is likely to occur in Labyrinthula. Cross infections did not produce any lesions, with the exception of Z. marina and Z. noltii (Vergeer and Den Hartog, 1991 ). Muehl-
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L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
stein et al. ( 1991 ) have also isolated L. zosterae from Z. japonica. Host specificity, therefore, does not seem to be confined to one species, but rather the species of a genus. The same holds true for the Western Australian Labyrinthula of Z. mucronata and Heterozostera tasmanica, which belong to the same subfamily; in view of the morphological data presented, the Labyrinthula specimens isolated seem to represent the same species. No cross infections have been carried out between members of the Cymodoceoideae. 5. Acknowledgements This research was partly supported by the Foundation for Biological Research (BION). Grant 437.142 made it possible to study the seagrass populations along the coast of Kenya. We thank Ivan Nagelkerken for collecting seagrasses in Italy and the Netherlands Antilles, and Dr. H. Kirkman from the CSIRO Division of Fisheries, North Beach, Western Australia for sending us the material from Australia. Professor dr. W. Gams and Professor dr. G. van der Velde provided critical reviews of the manuscript. References Amon, J.P., 1978. A method for obtaining sporulating Labyrinthula. Mycologia, 70:1297-1299. Armiger, L.C., 1964. An occurrence of Labyrinthula in New Zealand Zostera. N.Z.J. Bot., 2: 3-9. Den Hartog, C., 1987. 'Wasting disease' and other dynamic phenomena in Zostera beds. Aquat. Bot., 27: 3-14. Giesen, W.B.J.T., 1990. Wasting disease and present eelgrass condition. Laboratory of Aquatic Ecology, Catholic University of Nijmegen, The Netherlands, 138 pp. Molisch, H., 1926. Pseudoplasmodium aurantiacum n.g. et n.sp., eine neue Acriasiee aus Japan. Sci. Rep. Tohoku Univ. Ser. 4, 1: 119. Muehlstein, L.K., 1992. The host-pathogen interaction in the wasting disease of eelgrass, Zostera marina. Can. J. Bot., 70: 2081-2088. Muehlstein, L.K., Porter, D. and Short, F.T., 1988. Labyrinthula sp., a marine slime mold producing the symptoms of wasting disease in eelgrass, Zostera marina. Mar. Biol., 99: 465-472. Muehlstein, L.K., Porter, D. and Short, F.T., 1991. Labyrinthula zosterae sp. nov., the causative agent of wasting disease of eelgrass, Zostera marina. Mycologia, 83:180-191. Newell, S.Y. and Fell, J.W., 1982. Surface sterilization and the active mycoflora of leaves of seagrass. Bot. Mar., 25: 339-346. Pokorny, K.S., 1967. Labyrinthula. J. Protozool., 14: 697-708. Porter, D., 1990. Labyrinthulomyceta.In: L. Margulis, J.O. Corliss, M. Melkonian and D.J. Chapman (Editors), Handbook of Protoctista. Jones and Barlett, Boston, MA, pp. 388-398. Porter, D. and Muehlstein, L.K., 1989. A species of Labyrinthula is the prime suspect as the cause of a massive die offofthe seagrass Thalassia testudinum in Florida Bay. Mycol. Soc. Am. Newsl., 40: 43. Renn, C.E., 1936. The wasting disease of Zostera marina. I. A phytological investigation of the diseased plant. Biol. Bull., 70: 148-158. Short, F.T., Muehlstein, L.K. and Porter, D., 1987. Eelgrass wasting disease: cause and recurrence of a marine epidemic. Biol. Bull., 173: 557-562. Tutin, T.G., 1938. The autecology of Zostera marina in relation to its wasting disease. New Phytol., 37: 50-71. Vergeer, L.H.T. and Den Hartog, C., 1991. Occurrence of wasting disease in Zostera noltii. Aquat. Bot., 40: 155-163. Young, E.L., 1937. Recent investigations on the eel-grass problem: preliminary report. Bull. Mt. Desert Isl. Biol. Lab., 1937: 33-35.
Aquatic Botany 48 (1994) 1-20
Omnipresence of Labyrinthulaceae in seagrasses L.H.T. Vergeer*, C. den Hartog Laboratory of Aquatic Ecology, Catholic University, Toernooiveld, 6525 ED Nijmegen, The Netherlands (Accepted 17 December 1993)
Abstract
Seagrass species collected in different parts of the world have been checked for the presence of Labyrinthula spp. Isolations succeeded from all seagrasses studied, indicating a worldwide association between the two groups of organisms. The isolates have been described and compared. Infection experiments have shown that isolates are genus-specific.
1. Introduction
Species of the family Labyrinthulaceae, usually referred to as marine slime moulds, are widely distributed in coastal areas around the word. They have been isolated from a wide range of marine habitats, including organic detritus, diatoms, macro-algae and marine vascular plants. Labyrinthula spp. are unique in having an ectoplasmatic network in which their fusiform, vegetative cells move (Pokorny, 1967 ). There has been much confusion at the species level in the past, partly because two of the characteristics mostly used in species delimitation (viz. cell size and growth pattern of a colony) are strongly dependent on culture conditions. The life cycle of most Labyrinthula spp. is still obscure. Nowadays, eight to nine 'true' species of Labyrinthula are recognised (Porter, 1990). Much attention has been focussed on Labyrinthula after the 'wasting disease' epidemic of Zostera marina L. (eelgrass) in the early 1930s. The massive destruction of eelgrass beds along the Atlantic coasts of Europe and North America was almost certainly mediated by a Labyrinthula species (Renn, 1936). In the past, this species has been identified as Labyrinthula macrocystis Cienk., but re*Corresponding author. 0304-3770/94/$07.00 © 1994 Elsevier Science Publishers B.V. All rights reserved SSD10304-3770 (94)00376-W
2
L.H.T. Vergeer, C. den Hartog/Aquatic Botany 48 (1994) 1-20
cently it has been recognised as a new species, Labyrinthula zosterae Porter & Muehlstein (Muehlstein et al., 1991 ). A satisfactory explanation for the strong pathogenicity of L. zosterae in the 1930s has still not been presented (Den Hartog, 1987). Labyrinthula spp. seem to flourish on decomposing plant material rather than behaving as parasites. Today, L. zosterae appears to be commonly associated with eelgrass; recent declines of eelgrass along the North American coast have been associated with this species (Short et al., 1987 ). Along the Atlantic coast of Europe, L. zosterae is common, apparently without causing much damage to the populations of eelgrass (Vergeer and Den Hartog, unpublished observations). The symptoms of 'wasting disease' can be easily recognised. In the leaves lesions develop which cause some air lacunae to fill up with water. Small brown spots and stripes develop in these lesions, which spread longitudinally and become darker. They may cover the whole leaf after a few weeks. In truly diseased plants, these phenomena are apparent even in the youngest leaves; in most populations of Z. marina these phenomena are restricted to the oldest leaves. Recently, specimens of a related species, Zostera noltii Hornem., were also found with lesions similar to those produced by wasting disease on their leaves. The same Labyrinthula species could be isolated from these lesions as from affected Z. marina; further Z. noltii could be infected by the Labyrinthula from Z. marina (Vergeer and Den Hartog, 1991 ). On further investigation it turned out that lesions similar to those produced by wasting disease were not only present on the leaves of the Zostera species mentioned, but could be found on almost every seagrass species investigated. To elucidate the universality of the relationship between Labyrinthula and seagrasses, we studied a number of these species and checked them for the presence of Labyrinthula. The isolates of Labyrinthula obtained are described and compared. Recently, host or substrate specificity has been proposed as a potential characteristic in species identification in Labyrinthula (Muehlstein et al., 1991 ). In a few infection experiments, the degree of host specificity of some of the isolated Labyrinthula spp. has been tested. 2. Materials and methods
Seagrasses with lesions similar to those produced by wasting disease were collected in Europe, East Africa, Western Australia and in the Caribbean (Table 1 ). After being rinsed with fresh water, parts of the leaves containing the lesions were surface sterilised with sodium hypochlorite (Newell and Fell, 1982) and plated on an agar layer, 2-3 mm thick, in Petri dishes. The agar was prepared according to Porter (1990): to 1.2% agar on a sterilised seawater basis (artificial seawater 3%; Wimex, Wiegandt, Krefeld, Germany), chicken albumen (1%) and 250 mg 1- ~of streptomycin and penicillin were added. To inhibit the growth of diatoms, 3 mg 1-~ germanium dioxide was added. The parafilm-sealed Petri dishes were stored in the dark at room temperature. Mostly within 24-48 h after plating, colonies of Labyrinthula appeared on the
L.H.T. Vergeer, C. den Hartog /Aquatic Botany 48 (1994) 1-20
3
Table 1 The site ofcoUection and dimensions of vegetative cells ofLabyrinthula spp. isolated from different seagrasses Seagrass species
Na
Cell size (am) Pattern length × width
Potamogetonaceae Subfamily Zosteroideae Zostera marina L. Exmouth, England
50
Zostera mucronata den Hartog Swan River, Perth, Western Australia
25
Heterozostera tasmanica (Martens ex Aschers. ) den Hartog Whitfords area, Mullaloo pl. Western Australia
25
Subfamily Posidonioideae Posidoniaoceanica (L.) Delile Gallipoli, Italy
50
Subfamily Cymodoceoideae Halodule uninervis ( Forssk. ) Aschers. Mombasa, Kenya
Clumping of cells at the margins of a colony; bushy growth in the agar 8-12 X 2-4 Growing only in the agar; bushy growth with even margins; at some places tree-like outgrowths 9-12 X 2- 5 Growing only in the agar; resembling strongly the growth ofZ. mucronata
17-24×4-6
10-15×3-5
(15)-(5)
Mostly growing in the agar; on the agar a few patches of cells with no clumping; in the agar a transparent growth of separate threads of cells Growing only in the agar; bushy growth; cells more tightly packed at the edge; even margins Growing mostly in the agar; bushy growth; cells tightly packed in the middle; becoming looser towards the margin Growing on the agar in a circle away from the centre, with lobate outgrowths On the agar finger-like growth of tightly packed cells; dense and bushy growth in the agar
Cymodocea nodosa ( Ucria ) Aschers. Taranto, Italy
50
9-14×2-5
Syringodium isoetifolium (Aschers.) Dandy Mombasa, Kenya
50
11-14X3-5
Thalassodendron ciliatum (Forssk.) den Hartog Mombasa, Kenya
50
15-18X3-5
15
9-15×3-5
On the agar an open reticulate structure; in the agar an open bushy growth
50
10-14×3-5
Mostly growing on the agar in densely packed aggregates
50
5-8 X 2-4
On the agar individual cells are randomly dispersed; no aggregations
Subfamily Ruppioideae Ruppia cirrhosa (Petagna) Grande The Fleet, England Hydrocharitaceae Subfamily Thalassioideae Thalassia testudinum Banks ex KSnig Curacao, Netherlands Antilles Subfamily Halophiloideae Halophila ovalis (R.Br.) Hook. f. Whitfords area, MuUaloo pl. Western Australia "Number of cells measured.
4
L.H.T. Vergeer,C. den Hartog/Aquatic Botany 48 (1994) 1-20
agar. After a week, the dimensions of the vegetative cells were measured and the growth forms of the colonies described. Photographs were taken directly from the plates, using a Nikon light microscope. Infection experiments were carded out by fastening a small piece of an infected leaf of the same or another species to the leaves of an apparently healthy plant. This method was first described by Renn (1936). The experiments were carded out in 15 1 aquaria, containing a bottom layer of coarse river sand. Artificial seawater with a concentration of 3% was pumped through each aquarium separately at a turnover rate of once every 2 days. The water temperature was kept at 24 ° C. The aquaria were illuminated with 178 g E m -2 s-1, using daylight simulation lamps (Philips, 400 W, type HPI-t 150), with a 16 h light:8 h dark cycle. Air was bubbled continuously through the aquaria. The plants were then monitored over a 3 week period for the development of wasting disease-like lesions. 3. Results
As shown in Table 1, Labyrinthula species could be isolated from all seagrass species collected. All isolations were from lesions on the leaves, except for Halophila ovalis, where a "Labyrinthula' sp. was isolated from green, healthy tissue. For the Labyrinthula sp. isolated from Halodule uninervis, no reliable cell dimensions can be given; the colonies grew only in the agar with just a few cells located in a horizontal plane. 3.1. Descriptions o f the Labyrinthula spp. isolated
Host Zostera marina: vegetative cells fusiform; 17-24 gm X 4-6/tm; pale yellow to brown in mass in agar culture; cells usually containing three large vacuoles and numerous lipid droplets (Fig. la); cells growing out of the colony in massive lobes (Fig. 2a) with cells clumping at the end of the lobes; lobes sometimes segregating from behind; in the middle of a colony thick strands of cells with small interconnected branches (Fig. 2b); cells growing sporadically in the agar (black spots), forming a dense and bushy pattern (Fig. 2c). Cell size and growth pattern are in agreement with the description of L. zosterae given by Muehlstein et al. (1991). Host Zostera mucronata: vegetative cells fusiform; 8-12 gm X 2-4 gm; brown in mass in agar culture; cells only observed growing in the agar (Fig. 1b); bushy growth with even margins; at some places cells growing out of the margins in a tree-like manner, with branches originating from a single base (Fig. 2d). Host Heterozostera tasmanica: vegetative cells fusiform; 9 - 1 2 / t m X 2 - 5 pm; brown in mass in agar culture; cells only observed growing in the agar (Fig. Ic); resembling the growth-form of Labyrinthula from Z. mucronata: growth bushy in the agar with even margins (Fig. 2e) and at some places with tree-like outgrowths. Host Posidonia oceanica: vegetative cells fusiform; 10-15 #In× 3-5 #m; pale yellow in mass in agar culture; cells with one or two large vacuoles (Fig. ld); a
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(a)
(b)
Fig. 1, Vegetative cells ofLabyrinthula spp., isolated from: (a) Zostera marina; (b) Zostera mucronata; (c) Heterozostera tasmanica; (d) Posidonia oceanica; ( e ) Cymodocea nodosa; (f) Syringodium isoetifolium; (g) Thalassodendron ciliatum; ( h ) R uppia cirrhosa; ( i ) Thalassia testudinum; (j) Halophila ovalis. Magnification 750 ×.
.
Ci
J
(p)
~
(~) )~-I (P66I) gP Xuvtog oltvnbv / goJ.~vH uap "D '~aag.laA "Z"H'7
9
r~
%
h
8
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(i)
O)
Fig. 1 (continued).
9
10
L.H.T. Vergeer, C. den Hartog /Aquatic Botany 48 (1994) 1-20
(a)
(b)
Fig. 2. Growth ofLabyrinthula spp. on agar plates, isolated from: ( a ) - ( c ) Zostera marina; (d) Zostera mucronata; (e) Heterozostera tasmanica; (f) Posidonia oceanica; (g) Halodule uninervis; (h) Cymodocea nodosa; (i) Syringodium isoetifolium; (j) Thalassia testudinum; (k) and (1) Thalassodendron ciliatum; (m) and (n) Ruppia cirrhosa. Magnification 75 X.
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(c)
(d)
11
12
(e)
(f)
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(g)
(h)
Fig. 2 (continued).
13
14
(i)
(J)
L.H.T. Vergeer, C. den Hartog /Aquatic Botany 48 (1994) 1-20
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
(k)
0)
Fig. 2 (continued).
15
16
L.H.T. Vergeer,C. den Hartog / Aquatic Botany 48 (1994) 1-20
(m)
(n)
Fig. 2 (continued).
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
17
few lipid droplets; cells mostly growing in the agar; on the agar just a few patches of cells with no clumping; in the agar a transparent, even growth, consisting of separate threads of cells (Fig. 2f ); margins even. Host Halodule uninervis: vegetative cells fusiform; dimensions could not be measured exactly, but size in the order of 15 gm X 5 gm; dark brown in mass in agar culture; cells only observed growing in the agar, forming a bushy pattern (Fig. 2g); cells becoming more tightly packed towards the edge of the colony; margins even. Host Cymodocea nodosa: vegetative cells fusiform; 9-14 gm × 2-5 gm; greenbrown in mass in agar culture; cells with one or two large vacuoles (Fig. 1e) and many lipid droplets; cells mostly growing in the agar with in the middle of a colony a few close aggregations; growth bushy in the agar (Fig. 2h); cells tightly packed in the middle, becoming looser towards the margin. Host Syringodium isoetifolium: vegetative cells fusiform; 1 1-14/tm× 3-5/~m; pale yellow in mass in agar culture; cells containing one or two large vacuoles (Fig. I f ) and a few lipid droplets; cells only observed growing on the agar; cells growing in a circle away from the centre; lobe-shaped outgrowths from this circle with clumping of cells at the margin (Fig. 2i ); behind the front with a loose network of branching and anastomosing threads. Host Thalassodendron ciliatum: vegetative cells fusiform; 15-18/tm × 3-5 gm; yellow in mass in agar culture; cells mostly containing one large vacuole (Fig. lg) and many lipid droplets; growth on the agar finger-like (Fig. 2j ) or hand-shaped (Fig. 2k), consisting of tightly packed cells; at the edge small patches of cells growing in the agar; dense and bushy cell aggregates in the agar. Host Ruppia cirrhosa: vegetative cells fusiform (Fig. I h); 9-15 gm × 3-5 gm; yellow in mass in agar culture; cells containing no visible vacuole or lipid droplets; colonies on the agar making a fragile appearance with beautiful tree-like outgrowths at the margin (Fig. 21) with an open reticulate structure of threads of cells in the middle of a colony (Fig. 2m) and in the agar an open bushy pattern of cells. Host Thalassia testudinum: vegetative cells fusiform; 10-14 gm × 3-5/tin; pale yellow in mass in agar culture; cells containing a few vacuoles and lipid droplets (Fig. I i ); cells on the agar forming densely packed aggregates (Fig. 2n ); those in the agar confined to small separate patches (black spots in Fig. 2n). Host Halophila ovalis: vegetative cells ovoid; 5 - 8 / a n × 2-4 gm; cells sometimes containing one large vacuole; one to three lipid droplets (Fig. lj ); individual cells are randomly dispersed on the agar without any tendency to aggregate.
18
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
Table 2 The occurrence of infection a in the leaves of a healthy 'target' plant following contact with an infected 'source' plant Target
Source
N
Infection
Ruppia cirrhosa Zostera marina Thalassia testudinum Zostera marina Zostera marina Zostera noltii Ruppia cirrhosa Posidonia oceanica
Ruppia cirrhosa Zostera marina Thalassia testudinum Halodule uninervis Thalassodendron ciliatum Zostera marina Zostera marina Cymodocea nodosa
5 80 2 4 3 94 7 2
Yes Yes b Yes No No Yes b No No
aPlants were monitored over a 3 week period. bFrom Vergeer and Den Hartog ( 1991 ).
It is clear that all seagrass species seem to have their own Labyrinthula species. Only the species isolated from the Western Australian Z. mucronata and Heterozostera tasmanica seem to be morphologically identical; the measurements are not significantly different and the growth forms on agar are very similar. A Labyrinthula sp. isolated by Armiger (1964) from a New Zealand Zostera has the same cell dimensions ( 10 gm × 3/zm) but, because of the different culture methods used, cannot be otherwise compared. The Labyrinthula sp. isolated from Halophila ovalis is the most aberrant and may represent another genus.
3.2. Infection experiments Table 2 gives the results of the infection experiments. Only a limited number of combinations of seagrasses could be tested, owing to a lack of material. It appears from Table 2 that the infection of healthy seagrasses is only possible if the affected leaf part of the same species or genus is used. Cross infection did not succeed, with the exception of the closely related Z. marina and Z. noltii (Vergeer and Den Hartog, 1991 ). 4. Discussion We did not attempt to describe and name the isolated Labyrinthula taxa forreally as this seems premature in the present stage of research. Except for Z. marina and Z. noltii, only one population of the other seagrass species could be investigated. Further research on other populations of these species is necessary to check whether the characteristics of the taxa distinguished are of a wider validity. We agree with Muehlstein et al. ( 1991 ) that standardised culture conditions are essential in identifying and comparing the different Labyrinthula species. For this purpose we used the agar composition proposed by Porter (1990). Although it is often stated that Labyrinthula can be isolated from marine vascular plants, specific information concerning the plant species is rarely given. Labyrinthula species have been recorded from brackish water species such as Zan-
L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (I 994) 1-20
19
nichellia palustris L. and Ruppia maritima L. (Young, 1937) and from the saltmarsh grass Spartina alterniflora Loisel. (e.g. Amon, 1978 ). Isolations from true seagrasses are mainly restricted to species of the genus Zostera. Molisch ( 1926 ) isolated Labyrinthula vitellina Cienk. and Labyrinthula minuta Watson & Raper from Z. marina, and Tutin (1938 ) isolated Labyrinthula macrocystis from the same species. Labyrinthula macrocystis has also been isolated from Zostera hornemanniana Tutin (a narrow-leaved morph of Z. marina). Giesen ( 1990 ) mentions the isolation of a Labyrinthula sp. from Z. marina with cells approximately 8-10/zm long and 3-4/zm wide. These dimensions correspond to those given by Muehlstein et al. ( 1988 ) for Labyrinthula sp. 'S', also obtained from Z. marina. Further, Armiger (1964) isolated a Labyrinthula species from a Zostera species in New Zealand (probably Zostera novazelandica Setchell), and Muehlstein et al. (1991 ) mention a single isolation of L. zosterae from Zostera japonica Aschers. & Graebn. Apart from Zostera, a Labyrinthula species has also been recorded from Thalassia by Pokorny ( 1967 ), more specifically from Thalassia testudinum (Porter and Muehlstein, 1989), and recently from Syringodium filiforme Kiitz. (Muehlstein, 1992 ). The occurrence of Labyrinthulaceae in 11 seagrass species belonging to nine genera as well as in the brackish water species Ruppia cirrhosa, shows that there is a close association between these slime moulds and the marine phanerogams. The omnipresence of Labyrinthulaceae in seagrasses may point to a functional relationship. In all species investigated, with the notable exception of Halophila ovalis, the occurrence of Labyrinthula was restricted to the wasting disease-like lesions in the oldest leaves. Therefore, it is very likely that Labyrinthula normally does play a part in the senescence of the leaves. This would support the view that environmental factors played a major role in the outbreak of the wasting disease epidemic in the 1930s, by either increasing the susceptibility of Z. marina or stimulating the growth of Labyrinthula. Labyrinthula-linked declines have been recorded from Z. marina, the New Zealand Zostera (Armiger, 1964) and recently from Thalassia testudinum (Porter and Muehlstein, 1989 ). In the latter case, the Labyrinthula species involved is not described, apart from the statement that it differs from L. zosterae. In these cases, Labyrinthula appeared to damage also the youngest leaves, hampering the normal functioning of the plants. Apart from these observations, there are no indications that under normal conditions Labyrinthula species cause any harm to the seagrasses. All beds from which our material was collected looked lush and healthy. Along the west coast of Europe, where Z. marina has been monitored since 1986 with attention focussed on the possible build-up of disease, no indication has been found that Labyrinthula is harmful. Where decline was observed, this could be linked with environmental parameters, such as fouling by epiphytic algae, erosion, and overlayering with blankets of Enteromorpha sp. The results of the infection experiments indicate that host specificity is likely to occur in Labyrinthula. Cross infections did not produce any lesions, with the exception of Z. marina and Z. noltii (Vergeer and Den Hartog, 1991 ). Muehl-
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L.H.T. Vergeer, C. den Hartog / Aquatic Botany 48 (1994) 1-20
stein et al. ( 1991 ) have also isolated L. zosterae from Z. japonica. Host specificity, therefore, does not seem to be confined to one species, but rather the species of a genus. The same holds true for the Western Australian Labyrinthula of Z. mucronata and Heterozostera tasmanica, which belong to the same subfamily; in view of the morphological data presented, the Labyrinthula specimens isolated seem to represent the same species. No cross infections have been carried out between members of the Cymodoceoideae. 5. Acknowledgements This research was partly supported by the Foundation for Biological Research (BION). Grant 437.142 made it possible to study the seagrass populations along the coast of Kenya. We thank Ivan Nagelkerken for collecting seagrasses in Italy and the Netherlands Antilles, and Dr. H. Kirkman from the CSIRO Division of Fisheries, North Beach, Western Australia for sending us the material from Australia. Professor dr. W. Gams and Professor dr. G. van der Velde provided critical reviews of the manuscript. References Amon, J.P., 1978. A method for obtaining sporulating Labyrinthula. Mycologia, 70:1297-1299. Armiger, L.C., 1964. An occurrence of Labyrinthula in New Zealand Zostera. N.Z.J. Bot., 2: 3-9. Den Hartog, C., 1987. 'Wasting disease' and other dynamic phenomena in Zostera beds. Aquat. Bot., 27: 3-14. Giesen, W.B.J.T., 1990. Wasting disease and present eelgrass condition. Laboratory of Aquatic Ecology, Catholic University of Nijmegen, The Netherlands, 138 pp. Molisch, H., 1926. Pseudoplasmodium aurantiacum n.g. et n.sp., eine neue Acriasiee aus Japan. Sci. Rep. Tohoku Univ. Ser. 4, 1: 119. Muehlstein, L.K., 1992. The host-pathogen interaction in the wasting disease of eelgrass, Zostera marina. Can. J. Bot., 70: 2081-2088. Muehlstein, L.K., Porter, D. and Short, F.T., 1988. Labyrinthula sp., a marine slime mold producing the symptoms of wasting disease in eelgrass, Zostera marina. Mar. Biol., 99: 465-472. Muehlstein, L.K., Porter, D. and Short, F.T., 1991. Labyrinthula zosterae sp. nov., the causative agent of wasting disease of eelgrass, Zostera marina. Mycologia, 83:180-191. Newell, S.Y. and Fell, J.W., 1982. Surface sterilization and the active mycoflora of leaves of seagrass. Bot. Mar., 25: 339-346. Pokorny, K.S., 1967. Labyrinthula. J. Protozool., 14: 697-708. Porter, D., 1990. Labyrinthulomyceta.In: L. Margulis, J.O. Corliss, M. Melkonian and D.J. Chapman (Editors), Handbook of Protoctista. Jones and Barlett, Boston, MA, pp. 388-398. Porter, D. and Muehlstein, L.K., 1989. A species of Labyrinthula is the prime suspect as the cause of a massive die offofthe seagrass Thalassia testudinum in Florida Bay. Mycol. Soc. Am. Newsl., 40: 43. Renn, C.E., 1936. The wasting disease of Zostera marina. I. A phytological investigation of the diseased plant. Biol. Bull., 70: 148-158. Short, F.T., Muehlstein, L.K. and Porter, D., 1987. Eelgrass wasting disease: cause and recurrence of a marine epidemic. Biol. Bull., 173: 557-562. Tutin, T.G., 1938. The autecology of Zostera marina in relation to its wasting disease. New Phytol., 37: 50-71. Vergeer, L.H.T. and Den Hartog, C., 1991. Occurrence of wasting disease in Zostera noltii. Aquat. Bot., 40: 155-163. Young, E.L., 1937. Recent investigations on the eel-grass problem: preliminary report. Bull. Mt. Desert Isl. Biol. Lab., 1937: 33-35.