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The ‘wasting disease’ and the effect of abiotic factors (light intensity, temperature, salinity) and infection with Labyrinthula zosterae on the phenolic content of Zostera marina shoots
Aquatic botany ELSEVIER
Aquatic Botany 52 (1995) 35-44
The 'wasting disease' and the effect of abiotic factors (light intensity, temperature, salinity) and infection with Labyrinthula zosterae on the phenolic content of Zostera marina shoots L.H.T. Vergeer*, T.L. Aarts, J.D. de Groot Laboratory of Aquatic Ecology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands Accepted 22 March 1995
Abstract The cause of the 'wasting disease' epidemic that struck Zostera marina L. in the eady 1930s still remains unresolved. Many researchers have pointed out that some adverse environmental circumstances, prevailing in those years, may have resulted in a weakening of eelgrass, making it more susceptible to Labyrinthula zosterae Porter & Muehlstein, the biological agent of the disease. In this paper, the influence of light intensity, temperature and salinity on the production of phenolic compounds in eelgrass shoots was determined. These compounds are well known to play a role in the chemical defence of a plant against invading microorganisms. Plants grown under a high light intensity showed higher levels of phenolic compounds than those grown under a low light intensity. An increase of the water temperature led to a decrease in phenolic content, salinity showed no influence on the phenolic content. However, infection with Labyrinthula itself proves also to have pronounced effects on the production of phenolic compounds.
1. Introduction A satisfactory explanation of the massive destruction in the early 1930s o f Zostera marina L. in the North Atlantic region, due to the so-called 'wasting disease' is still lacking. Recently, Labyrinthula zosterae Porter & Muehlstein, a marine slimemould-like protist has been identified as the biological agent responsible for the disease (Muehlstein et al., 1988, 1991 ). However, other investigations have demonstrated the common occurrence of this * Corresponding author. 0304-3770/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD10304-3770(95)00480-7
36
L.H.T. Vergeeret al. /Aquatic Botany 52 (1995) 35--44
microorganism in many eelgrass stands today, without any apparent decline of the eelgrass populations (Short et al., 1988; Vergeer and Den Hartog, 1994). Furthermore, a study of herbarium material collected long before the epidemic of the 1930s revealed plants with the characteristic black/brown lesions of the wasting disease on their leaves. In these years no significant disappearance of Z. marina has been reported (Den Hartog, 1989). These findings suggest that L. zosterae could be an organism normally occurring in eelgrass beds, perhaps functioning as a secondary decomposer of aged plant material, as proposed by Den Hartog (1987). If this is true, the question remains what event caused Labyrinthula to act as a strong parasite on such a large scale in the 1930s. Many authors (e.g. Renn, 1937; Tutin, 1938; Young, 1938) have pointed out that some adverse environmental circumstances, prevailing in those years, may have resulted in a weakening of eelgrass, making it more susceptible to Labyrinthula. For the Dutch Wadden Sea, Giesen et al. (1990a,b) found a clear correlation between a deficit in sunshine during successive growing seasons in the early 1930s and the decline of Z. mar/na in this area. Rasmussen (1977) found that the decline in Danish waters was associated with a period of warm summers and exceptionally mild winters, resulting in a raise of the water temperature. Autumn 1932 more than 90% of the Atlantic coasteelgrass had disappeared. The only remaining eelgrass plants were in low saline areas (Young, 1938). In general, the local populations that were beginning to recover were those in brackish water, while other more saline areas remained barren of eelgrass (Cottam, 1935). An important way in which plants can protect themselves against invading microorganisms is by producing defensive secondary metabolites. Among these, phenolic compounds are widespread and well known to play a role in disease resistance (Levin, 1976; Friend, 1979). Extracts of eelgrass leaves were found to inhibit the growth of several microorganisms (Harrison and Chan, 1980; Harrison, 1982; Harrison and Durance, 1985). This could be due to the many different types of phenolic compounds present in these extracts (Harborne and Williams, 1976; Zapata and McMillan, 1979; McMillan et al., 1980). The production of phenolics requires energy and primary metabolites, so it is likely that suboptimal growing conditions will reduce the ability of a plant to build up a sufficient chemical de fence. In this paper we present results of culture experiments in which we measured the influence of light intensity, temperature and salinity on the phenolic content of eelgrass shoots. From a pilot experiment, conducted in Roscoff (France), in which artificially infected plants were grown under different light intensities, came indications that not only the light intensity had influence on the phenolic content but also an infection with Labyrinthula itself. Induced resistance, i.e. an increase of secondary compounds as a result of infection, is characteristic for the use of phenolics in defence and has been observed in numerous plants (Levin, 1971, 1976; Friend, 1979). Whether an infection of Z. marina with Labyrinthula zosterae also results in an accumulation of phenolics, was investigated by measuring the phenolic content at various distances from a wasting disease lesion in an affected leaf. To avoid the intervening factor of 'induced resistance' in the culture experiments, we conducted an experimental set-up in which we used young, growing eelgrass plants without wasting disease lesions that were collected at the beginning of the growing season.
L.H.T. Vergeeret al. /Aquatic Botany52 (1995)35-44
37
2. Materials and methods 2.1. Pilot experiment (Roscoff, France)
Eelgrass plants without visible lesions were collected on 23 April 1989 in Roscoff (France). They were placed in six plastic containers (0.65 m X 0.40 m × 0.20 m) at three different light conditions: 27, 78 and 110/.rE m -2 s -1 with a 16 h light: 8 h dark cycle. According to Dennison (1987) the light saturation point of Z. marina is approximately 85 /zE m - 2 s - i. The containers had a bottom layer of coarse sand. Sea water with a temperature of about 10°C was pumped through the containers continuously. The plants in one container of each illumination class were infected artificially by clamping a small surface-sterilised piece of leaf with a wasting disease lesion, to the oldest leaf of each plant (Renn, 1936; Vergeer and Den Hartog, 1991). To the plants in the other containers (controls), green, healthy leaf pieces were attached, in order to check whether the handling of the plants in using this method had itself any influence on the development of wasting disease lesions. After 4 weeks, 25 plants from each container were checked for the occurrence of wasting disease lesions; from ten plants the phenolic content of the shoots was determined, using the method of Hagerman and Butler (1978), as described in Mole and Waterman (1987). Plants were freeze-dried overnight and ground with the use of liquid nitrogen. Ten milligrams of the dried plant material were extracted with 5 ml 80% ethanol for 10 min at 80°C. One millilitre of the extract was mixed with 2 ml sodium dodecyl sulphate (SDS) and 1 ml FeC13.The absorption was measured at 510 nm using a Schimadzu spectrophotometer (UV-120-01). Tannic acid (Sigma Chemical Company) was used as standard. 2.2. Induced resistance
On 18 May 1989, eelgrass leaves with one, clear wasting disease lesion were collected in Roscoff. The leaf blade between the leaf sheath and the lesion was divided into parts of 2 cm length. From these parts, the phenolic content was measured as described above. To get enough material for extraction, 15 parts of 2 cm, located at the same distance from a lesion, were pooled for each measurement. 2.3. Culture experiments
For the three separate culture experiments, young plants showing no wasting disease lesions were collected in Lake Grevelingen (The Netherlands) in spring 1991 (light intensity), 1992 (temperature) and 1993 (salinity). The plants were placed in 18 aquaria (0.25 m X 0.25 m × 0.30 m), with 25 plants in each aquarium. The aquaria contained a bottom layer of sand, also collected in Lake Grevelingen. The aquaria were placed in a waterbath, connected to a cool flow system that kept the water temperature at 15°C. The aquaria were illuminated with 178 ~E m -z s -1 using daylight simulation lamps (Philips, 400W, type HPI-tI50) with a 16 h light: 8 h dark cycle. Artificial sea water (Wimex, Wiegandt GmbH, Krefeld, Germany) with a salinity of 30%o was pumped through the aquaria at a turnover rate of once every 2 days. Each aquarium
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L.H.T. Vergeer et al. /Aquatic Botany 52 (1995) 35-44
was replenished from its own stock container. Air was bubbled continuously through the aquaria. The plants acclimated in this 'standard' situation for 1 week, after which the conditions were changed as follows. Light intensity: above six aquaria the light intensity was diminished to 110/~E m - 2 s - 1 and above six other aquaria to 37 ~E m - 2 S-1 with the aid of mesh cloth. The remaining six aquaria stayed at 178/xE m -2 s- 1. Temperature: using heating elements the temperature in six aquaria was raised to 20°C and in six other aquaria to 25°C. The remaining six aquaria stayed at 15°C. Salinity: six aquaria were provided with seawater with a salinity of 10%o, six aquaria with 200/oo,and six remained at 30%0. After 4 weeks ten plants were harvested from each aquarium and the phenolic content of the shoots determined.
2.4. Statistical procedures No statistics were applied to the results of the pilot experiment because only one container with plants was used for each condition. In the culture experiments the Proc NParlWay procedure of the Statistical Analysis System Institute Inc. (1989) was used to test the differences of the parameters between the conditions.
3. Results
3.1. Pilot experiment After placing the plants in the experimental set-up, their condition was monitored from day to day. Within 24 h some of the artificially infected plants showed wasting disease lesions at the place where the diseased piece of leaf had been attached. After 3 days, ten Infection rate (%)
72
110
78 27 Light intensity ~ E m -2 s -1)
Fig. 1. Rate of infection with wasting disease of untreated (shaded) and aRiflcially infected (black) Z. marina plants, grown for 4 weeks under three different light intensities ( for every column, N = 25).
L.H.T. Vergeer et al. /Aquatic Botany 52 (1995) 35-44
Phenolic concentration (mg gDW "1) -
110
78
39
-
27
Light intensity (liE m "2 s "1)
Fig. 2. Total phenoliccontent (mg g- 1dry weight) of untreated (shaded) and artificiallyinfected (black) shoots of Z. marina, grown for 4 weeks under three differentlight intensities (for everycolumn,N= 10). Phenolic concentration (rag gDW "1) -
2
4
6
-
8 10 Distance to lesion (cm)
Fig. 3. Total phenolic content (mg g - t dry weight) of infected Z. marina leaves at various distances (cm) from
a wasting disease lesion. plants on average appeared to be infected, while among the controls no visible lesions occurred. In the second week, however, dark spots appeared also on the leaves of the controls, but not at the places where a piece of leaf had been attached. The number of infected plants after 4 weeks, expressed as a percentage of the total number of plants checked, is shown in Fig. 1. The infection rate of the artificially infected plants dropped with increasing light intensity. Unfortunately, the plants from the controls had also become infected, although the infection rates observed here were considerably lower than among the artificially infected plants. Fig. 2 shows the total phenolic content of the shoots after 4 weeks under three different light intensities. The concentration of phenolic compounds was higher in plants grown at a higher light intensity. Within one illumination class, the concentration of the artificially infected plants was higher than that of the controls.
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L.H.T. Vergeer et al. /Aquatic Botany 52 (1995) 35--44
Phenolic concentration (rag gOW "1)
(a)
178
110 37 Light.inten~tjaE m"2 s-l)
(b)
15
20
1
25 Temperature
Phenolic concentration (mg gDW"1) -
(C)
30
20
{°C)
-
10 Salinity (%.)
Fig. 4. Total phenolic content (mg g - i dry weight) o f Z . marina shoots, grown for 4 weeks under three different light intensities (a), temperatures (b) and salinities (c). For every column, N = 60.
L.H.T. Vergeer et al./Aquatic Botany 52 (1995) 35-44
41
3.2. Induced resistance The phenolic content of infected leaves at various distances from the wasting disease lesion is shown in Fig. 3. The concentration increased dramatically towards the point where the lesion occurs (Pearson's r = 0.83; P < 0.01 ). 3.3. Culture experiment The plants in the experimental set-up were monitored during a period of 4 weeks. No wasting disease lesions occurred on the leaves of the plants. The leaves of plants grown under a low light intensity turned brown and the plants died. The phenolic content of the shoots in the different culture experiments is given in Fig. 4. A decrease of the light intensity (Fig. 4a) as well as an increase of the water temperature (Fig. 4b) resulted in a significant decrease of the phenolic content (P < 0.05). A change in the salinity (Fig. 4c) of the water hardly had any effect on the concentrations of phenolics.
4. Discussion
The results of both the pilot experiment and the culture experiment indicate a positive effect of light intensity on the phenolic biosynthesis in the leaves of Z. marina. Plants grown for 4 weeks at a high light intensity showed a significantly higher phenolic content than plants grown under a low light intensity (Figs. 1 and 4a). This is a phenomenon which was also demonstrated in terrestrial plants. Waterman et al. (1984) found that individuals of Barteria fistulosa Mast. growing in high light intensity responded by producing greater amounts of phenolics than individuals in a more shaded position. Similar results were obtained by Cooper-Driver et al. (1977), working with Pteridium aquilinum (L.) and Kuhn and Woodhead (1981), working with Sorghum bicolor (L.) Moench. A variety of mechanisms has been proposed to account for the increase of phenolics with increased light intensity and it is likely that some of these mechanisms operate simultaneously. One of the proposed mechanisms is the availability of minerals and especially nitrogen. The photosynthetic activity of a plant at high light intensity may be so high in relation to the quantity of nitrogen available that, once its limited supply has been used in the production of primary metabolites (amino acids, proteins), the remaining carbohydrates can only be used to produce nitrogen free molecules such as phenolics (Waterman et al., 1984). In this way phenolics are regarded as storage compounds of carbohydrates, which are only produced at times when the plants can not convert the carbohydrates into growth. Such an inverse relation between nitrogen availability and phenolic content was also found in Z. marina (Buchsbaum et al., 1990). In the experiment the amount of nitrogen available to the plants was not measured. However, the higher biomass of the plants grown under the highest light intensity and the presence of newly formed leaves at the end of the experiment, makes it unlikely that there were growth-limiting circumstances in the containers with the highest light intensity. Another explanation for the increased phenolic content is the known stimulation by light of most of the enzymes involved in phenolic biosynthesis of plants. In particular, the activity
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L.H.T. Vergeeret al. /Aquatic Botany 52 (1995) 35--44
of phenylalanine ammonia-lyase (PAL), a key enzyme in the synthetic pathway of phenolics, increases under the influence of light (Bidwell, 1974). This phenomenon has been observed in plants, as well as in plant cell cultures (DiCosmo and Towers, 1984), where no deficiency of nutrients occurred. The increase of the temperature results in a decrease of the phenolic content of eelgrass shoots. A clear explanation for this phenomenon is hard to give. Temperature influences photosynthesis as well as respiration, and in this way, the C/N balance in the plant. But also, temperature has a direct impact on the enzymes of the phenolic biosynthesis, in which every enzyme can have its own optimum. Salinity proved to have no effect on the phenolic content of eelgrass shoots. Zostera marina is euryhaline, so the survival of populations in brackish waters in the 1930s epidemic is more explained by the fact that a low salinity is detrimental to Labyrinthula. In the culture experiments we only determined the impact of abiotic factors on the host plant, but a specific factor has, of course, influence on the microorganism and on the relationship between the two. Among the infected plants in the pilot experiment, a higher light intensity was not only accompanied by an increased level of phenolic compounds, but also by a decrease in the infection rate of Z. marina with wasting disease. A correlation between a high phenolic content of Z. marina and a low infection rate was also found by Buchsbaum et al. (1990). The differences in infection rate among the infected plants were rather small, however, and among the controls no relation between light intensity/phenolic content and the infection rate could be shown, owing to a low infection rate among the plants of the middle illumination class (Fig. 1). Thus, the results only partly support the hypothesis of Giesen et al. (1990a,b) that a deficit of sunshine results in Z. marina being more susceptible to Labyrinthula, and the suggestion that this may be caused by a reduced level of phenolic defence compounds. An assessment of the effect of light intensity on the phenolic content together with the infection rate, is in the pilot experiment obscured by the interfering influence of infection itself on the phenolic content. As shown in Fig. 2, the infected plants had higher levels of phenolic compounds than the controls. This same problem was also raised by Woodhead (1981). In order to determine the influence of light alone on the phenolic content of Z. marina, the experiment was repeated. An increase in phenolics as a result of infection is often correlated with resistance to the disease. As shown in Fig. 4, an infection of Z. marina with Labyrinthula indeed leads to a drastic increase in phenolic biosynthesis. In what way the interaction between host and invading microorganism induces the production of phenolics is unclear, but, as with the light intensity, inoculation of plants with pathogenic microorganisms, often leads to an increase in PAL activity associated with the production of specific phenolic compounds. Increases in phenolase and peroxidase activity have been reported in many plants (Levin, 1976; Friend, 1979). Phenolics are oxidized by these enzymes into quinones, which are highly reactive compounds and therefore good bactericides and fungicides. Activity of these enzymes produces enzymatic browning, a phenomenon also observed after infection of Z. marina with Labyrinthula. Whether the increase of phenolic compounds forms a defence against Labyrinthula has yet to be established. As resistance to a microorganism may depend more upon the quality
L.H.T. Vergeer et al. /Aquatic Botany 52 (1995) 35-44
43
of the phenolics than upon their quantity, further research will be directed to the separation of the different phenolic compounds present in Z. marina and an assessment of their specific growth-limiting properties with respect to the microorganism.
Acknowledgements This research was supported by the Foundation for Biological Research (BION) of the Netherlands Organisation for Scientific Research (NWO). Part of the research was carried out at the Station d'Oceanologie et de Biologie Marine in Roscoff (France), thanks to a subvention of the Royal Dutch Academy of Sciences. We wish to thank the Academy and Dr. P. Lasserre, Director of the station, for this opportunity. Professor Dr. C. den Hartog and Professor Dr. G. van der Velde are acknowledged for their critical comments on earlier drafts of this manuscript.
References Bidwell, R.G.S., 1974. Plant Physiology. Macmillan, New York, 643 pp. Buchsbaum, R.N., Short, F.T. and Cheney, D.P., 1990. Phenolic-nitrogen interactions in eelgrass, Zostera marina L.: possible implications for disease resistance. Aquat. Bot., 37:291-297. Cooper-Driver, G., Finch, S., Swain, T. and Bernays, E., 1977. Seasonal variation in secondary plant compounds in relation to palatability ofPteridium aquilinum. Biochem. Syst. Ecol., 5: 177-183. Den Hartog, C., 1987. 'Wasting disease' and other dynamic phenomena in Zostera beds. Aquat. Bot., 27: 3-14. Den Hartog, C., 1989. Early records of wasting-disease-like damage patterns in eelgrass Zostera marina. Dis. Aquat. Org., 7: 223-226. Dennison, W.C., 1987. Effects of light on seagrass photosynthesis, growth and depth distribution. Aquat. Bot., 27: 15-26. DiCosmo, F. and Towers, G.H.N., 1984. Stress and secondary metabolism in cultured plant cells. In: B.N. Timmerman, C. Stellink and F.A. Loewus (Editors), Phytochemical Adaptation to Stress. Recent advances in Phytochemistry, Vol. 18. Plenum, New York, pp. 97-180. Friend, J., 1979. Phenolic substances and plant disease. Rec. Adv. Phytochem., 12: 557-588. Giesen, W.B.J.T., van Katwijk, M.M. and den Hartog, C., 1990a. Temperature, salinity, insolation and wasting disease of eelgrass (Zostera marina L.) in the Dutch Wadden Sea in the 1930's. Neth. J. Sea Res., 25: 395404. Giesen, W.B.J.T., van Katwijk, M.M. and den Hartog, C., 1990b. Eelgrass condition and turbidity in the Dutch Wadden Sea. Aquat. Bot., 37: 71-85. Hagerman, A.E. and Butler, L.G., 1978. Protein precipitation method for the quantitative determination of tannins. L Agric. Food Chem., 26: 809-812. Harborne, J.B. and Williams, C., 1976. Occurrence of sulfated flavones and caffeic acid esters in members of the Fluviales. Biochem. Syst. Ecol., 4: 37-41. Harrison, P.G., 1982. Control of microbial growth and of amphipod grazing by water-soluble compounds from leaves of Zostera marina. Mar. Biol., 67: 225-230. Harrison, P.G. and Chan, A.T., 1980. Inhibition of the growth of micro-algae and bacteria by extracts of eelgrass (Zostera marina) leaves. Mar. Biol., 61: 21-26. Harrison, P.G. and Durance, C.D., 1985. Reductions in photosynthetic carbon uptake in epiphytic diatoms by water-soluble extracts of leaves of Zostera marina. Mar. Biol., 90:117-119. Levin, D .A., 1971. Plant phenolics: an ecological perspective. Am. Nat., 105: 157-181. Levin, D.A., 1976. The chemical defenses of plants to pathogens and herbivores. Ann. Rev. Ecol. Syst., 7:211259.
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McMillan, C., Zapata, O. and Escobar, L., 1980. Sulfated phenolic compounds in seagrasses. Aquat. Bot., 8: 267278. Mole, S. and Waterman, P.G., 1987. A critical analysis of techniques for measuring tannins in ecolgical studies. I. Techniques for chemically defining tannins. Oecologia, 72: 137-147. 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. Rasmussen, E., 1977. The wasting disease of eelgrass (Zostera marina) and its effects on environmental factors and fauna. In: C.P. McRoy and C. Helfferich (Editors), Seagrass Ecosystems. A Scientific Perspective. Marcel Dekker, New York/Basel, pp. 1-52. Renn, C.E., 1936. The wasting disease of Zostera marina: I. A phytological investigation of the diseased plant. Biol. Bull., 70: 148-158. Renn, C.E., 1937. The eel-grass situation along the middle Atlantic coast. Ecology, 18: 323-325. Short, F.T., Ibelings, B.W. and den Hartog, C., 1988. Comparison of a current eeigrass disease to the wasting disease in the 1930's. Aquat. Bot., 30: 295-304. Statistical Analysis Systems Institute Inc., 1989. SAS/STAT User's Guide. Version 6, 4th edn., Vol. 2. SAS Institute Inc., Cary, NC. 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: 155163. Vergeer, L.H.T. and den Hartog, C., 1994. Omnipresence of Labyrinthulaceae in seagrasses. Aquat. Bot., 48: 120. Waterman, P.G., Ross, J.A.M. and McKey, D.B., 1984. Factors affecting the levels of some phenolic compounds, digestibility, and nitrogen content of mature leaves ofBarteriafistulosa (Passifloracean). J. Chem. Ecol., 10: 387-40 I. Woodhead, S., 1981. Environmental and biotic factors affecting the phenolic content of different cnltivars of Sorghum bicolor. J. Chem. Ecol., 7: 1035-1047. Young, E.L., 1938. Labyrinthula on Pacific coast eelgrass. Can. J. Res., 16:115-117. Zapata, O. and McMillan, C., 1979. Phenolic acids in seagrasses. Aquat. Bot., 7: 307-317.
Aquatic Botany 52 (1995) 35-44
The 'wasting disease' and the effect of abiotic factors (light intensity, temperature, salinity) and infection with Labyrinthula zosterae on the phenolic content of Zostera marina shoots L.H.T. Vergeer*, T.L. Aarts, J.D. de Groot Laboratory of Aquatic Ecology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands Accepted 22 March 1995
Abstract The cause of the 'wasting disease' epidemic that struck Zostera marina L. in the eady 1930s still remains unresolved. Many researchers have pointed out that some adverse environmental circumstances, prevailing in those years, may have resulted in a weakening of eelgrass, making it more susceptible to Labyrinthula zosterae Porter & Muehlstein, the biological agent of the disease. In this paper, the influence of light intensity, temperature and salinity on the production of phenolic compounds in eelgrass shoots was determined. These compounds are well known to play a role in the chemical defence of a plant against invading microorganisms. Plants grown under a high light intensity showed higher levels of phenolic compounds than those grown under a low light intensity. An increase of the water temperature led to a decrease in phenolic content, salinity showed no influence on the phenolic content. However, infection with Labyrinthula itself proves also to have pronounced effects on the production of phenolic compounds.
1. Introduction A satisfactory explanation of the massive destruction in the early 1930s o f Zostera marina L. in the North Atlantic region, due to the so-called 'wasting disease' is still lacking. Recently, Labyrinthula zosterae Porter & Muehlstein, a marine slimemould-like protist has been identified as the biological agent responsible for the disease (Muehlstein et al., 1988, 1991 ). However, other investigations have demonstrated the common occurrence of this * Corresponding author. 0304-3770/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD10304-3770(95)00480-7
36
L.H.T. Vergeeret al. /Aquatic Botany 52 (1995) 35--44
microorganism in many eelgrass stands today, without any apparent decline of the eelgrass populations (Short et al., 1988; Vergeer and Den Hartog, 1994). Furthermore, a study of herbarium material collected long before the epidemic of the 1930s revealed plants with the characteristic black/brown lesions of the wasting disease on their leaves. In these years no significant disappearance of Z. marina has been reported (Den Hartog, 1989). These findings suggest that L. zosterae could be an organism normally occurring in eelgrass beds, perhaps functioning as a secondary decomposer of aged plant material, as proposed by Den Hartog (1987). If this is true, the question remains what event caused Labyrinthula to act as a strong parasite on such a large scale in the 1930s. Many authors (e.g. Renn, 1937; Tutin, 1938; Young, 1938) have pointed out that some adverse environmental circumstances, prevailing in those years, may have resulted in a weakening of eelgrass, making it more susceptible to Labyrinthula. For the Dutch Wadden Sea, Giesen et al. (1990a,b) found a clear correlation between a deficit in sunshine during successive growing seasons in the early 1930s and the decline of Z. mar/na in this area. Rasmussen (1977) found that the decline in Danish waters was associated with a period of warm summers and exceptionally mild winters, resulting in a raise of the water temperature. Autumn 1932 more than 90% of the Atlantic coasteelgrass had disappeared. The only remaining eelgrass plants were in low saline areas (Young, 1938). In general, the local populations that were beginning to recover were those in brackish water, while other more saline areas remained barren of eelgrass (Cottam, 1935). An important way in which plants can protect themselves against invading microorganisms is by producing defensive secondary metabolites. Among these, phenolic compounds are widespread and well known to play a role in disease resistance (Levin, 1976; Friend, 1979). Extracts of eelgrass leaves were found to inhibit the growth of several microorganisms (Harrison and Chan, 1980; Harrison, 1982; Harrison and Durance, 1985). This could be due to the many different types of phenolic compounds present in these extracts (Harborne and Williams, 1976; Zapata and McMillan, 1979; McMillan et al., 1980). The production of phenolics requires energy and primary metabolites, so it is likely that suboptimal growing conditions will reduce the ability of a plant to build up a sufficient chemical de fence. In this paper we present results of culture experiments in which we measured the influence of light intensity, temperature and salinity on the phenolic content of eelgrass shoots. From a pilot experiment, conducted in Roscoff (France), in which artificially infected plants were grown under different light intensities, came indications that not only the light intensity had influence on the phenolic content but also an infection with Labyrinthula itself. Induced resistance, i.e. an increase of secondary compounds as a result of infection, is characteristic for the use of phenolics in defence and has been observed in numerous plants (Levin, 1971, 1976; Friend, 1979). Whether an infection of Z. marina with Labyrinthula zosterae also results in an accumulation of phenolics, was investigated by measuring the phenolic content at various distances from a wasting disease lesion in an affected leaf. To avoid the intervening factor of 'induced resistance' in the culture experiments, we conducted an experimental set-up in which we used young, growing eelgrass plants without wasting disease lesions that were collected at the beginning of the growing season.
L.H.T. Vergeeret al. /Aquatic Botany52 (1995)35-44
37
2. Materials and methods 2.1. Pilot experiment (Roscoff, France)
Eelgrass plants without visible lesions were collected on 23 April 1989 in Roscoff (France). They were placed in six plastic containers (0.65 m X 0.40 m × 0.20 m) at three different light conditions: 27, 78 and 110/.rE m -2 s -1 with a 16 h light: 8 h dark cycle. According to Dennison (1987) the light saturation point of Z. marina is approximately 85 /zE m - 2 s - i. The containers had a bottom layer of coarse sand. Sea water with a temperature of about 10°C was pumped through the containers continuously. The plants in one container of each illumination class were infected artificially by clamping a small surface-sterilised piece of leaf with a wasting disease lesion, to the oldest leaf of each plant (Renn, 1936; Vergeer and Den Hartog, 1991). To the plants in the other containers (controls), green, healthy leaf pieces were attached, in order to check whether the handling of the plants in using this method had itself any influence on the development of wasting disease lesions. After 4 weeks, 25 plants from each container were checked for the occurrence of wasting disease lesions; from ten plants the phenolic content of the shoots was determined, using the method of Hagerman and Butler (1978), as described in Mole and Waterman (1987). Plants were freeze-dried overnight and ground with the use of liquid nitrogen. Ten milligrams of the dried plant material were extracted with 5 ml 80% ethanol for 10 min at 80°C. One millilitre of the extract was mixed with 2 ml sodium dodecyl sulphate (SDS) and 1 ml FeC13.The absorption was measured at 510 nm using a Schimadzu spectrophotometer (UV-120-01). Tannic acid (Sigma Chemical Company) was used as standard. 2.2. Induced resistance
On 18 May 1989, eelgrass leaves with one, clear wasting disease lesion were collected in Roscoff. The leaf blade between the leaf sheath and the lesion was divided into parts of 2 cm length. From these parts, the phenolic content was measured as described above. To get enough material for extraction, 15 parts of 2 cm, located at the same distance from a lesion, were pooled for each measurement. 2.3. Culture experiments
For the three separate culture experiments, young plants showing no wasting disease lesions were collected in Lake Grevelingen (The Netherlands) in spring 1991 (light intensity), 1992 (temperature) and 1993 (salinity). The plants were placed in 18 aquaria (0.25 m X 0.25 m × 0.30 m), with 25 plants in each aquarium. The aquaria contained a bottom layer of sand, also collected in Lake Grevelingen. The aquaria were placed in a waterbath, connected to a cool flow system that kept the water temperature at 15°C. The aquaria were illuminated with 178 ~E m -z s -1 using daylight simulation lamps (Philips, 400W, type HPI-tI50) with a 16 h light: 8 h dark cycle. Artificial sea water (Wimex, Wiegandt GmbH, Krefeld, Germany) with a salinity of 30%o was pumped through the aquaria at a turnover rate of once every 2 days. Each aquarium
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was replenished from its own stock container. Air was bubbled continuously through the aquaria. The plants acclimated in this 'standard' situation for 1 week, after which the conditions were changed as follows. Light intensity: above six aquaria the light intensity was diminished to 110/~E m - 2 s - 1 and above six other aquaria to 37 ~E m - 2 S-1 with the aid of mesh cloth. The remaining six aquaria stayed at 178/xE m -2 s- 1. Temperature: using heating elements the temperature in six aquaria was raised to 20°C and in six other aquaria to 25°C. The remaining six aquaria stayed at 15°C. Salinity: six aquaria were provided with seawater with a salinity of 10%o, six aquaria with 200/oo,and six remained at 30%0. After 4 weeks ten plants were harvested from each aquarium and the phenolic content of the shoots determined.
2.4. Statistical procedures No statistics were applied to the results of the pilot experiment because only one container with plants was used for each condition. In the culture experiments the Proc NParlWay procedure of the Statistical Analysis System Institute Inc. (1989) was used to test the differences of the parameters between the conditions.
3. Results
3.1. Pilot experiment After placing the plants in the experimental set-up, their condition was monitored from day to day. Within 24 h some of the artificially infected plants showed wasting disease lesions at the place where the diseased piece of leaf had been attached. After 3 days, ten Infection rate (%)
72
110
78 27 Light intensity ~ E m -2 s -1)
Fig. 1. Rate of infection with wasting disease of untreated (shaded) and aRiflcially infected (black) Z. marina plants, grown for 4 weeks under three different light intensities ( for every column, N = 25).
L.H.T. Vergeer et al. /Aquatic Botany 52 (1995) 35-44
Phenolic concentration (mg gDW "1) -
110
78
39
-
27
Light intensity (liE m "2 s "1)
Fig. 2. Total phenoliccontent (mg g- 1dry weight) of untreated (shaded) and artificiallyinfected (black) shoots of Z. marina, grown for 4 weeks under three differentlight intensities (for everycolumn,N= 10). Phenolic concentration (rag gDW "1) -
2
4
6
-
8 10 Distance to lesion (cm)
Fig. 3. Total phenolic content (mg g - t dry weight) of infected Z. marina leaves at various distances (cm) from
a wasting disease lesion. plants on average appeared to be infected, while among the controls no visible lesions occurred. In the second week, however, dark spots appeared also on the leaves of the controls, but not at the places where a piece of leaf had been attached. The number of infected plants after 4 weeks, expressed as a percentage of the total number of plants checked, is shown in Fig. 1. The infection rate of the artificially infected plants dropped with increasing light intensity. Unfortunately, the plants from the controls had also become infected, although the infection rates observed here were considerably lower than among the artificially infected plants. Fig. 2 shows the total phenolic content of the shoots after 4 weeks under three different light intensities. The concentration of phenolic compounds was higher in plants grown at a higher light intensity. Within one illumination class, the concentration of the artificially infected plants was higher than that of the controls.
40
L.H.T. Vergeer et al. /Aquatic Botany 52 (1995) 35--44
Phenolic concentration (rag gOW "1)
(a)
178
110 37 Light.inten~tjaE m"2 s-l)
(b)
15
20
1
25 Temperature
Phenolic concentration (mg gDW"1) -
(C)
30
20
{°C)
-
10 Salinity (%.)
Fig. 4. Total phenolic content (mg g - i dry weight) o f Z . marina shoots, grown for 4 weeks under three different light intensities (a), temperatures (b) and salinities (c). For every column, N = 60.
L.H.T. Vergeer et al./Aquatic Botany 52 (1995) 35-44
41
3.2. Induced resistance The phenolic content of infected leaves at various distances from the wasting disease lesion is shown in Fig. 3. The concentration increased dramatically towards the point where the lesion occurs (Pearson's r = 0.83; P < 0.01 ). 3.3. Culture experiment The plants in the experimental set-up were monitored during a period of 4 weeks. No wasting disease lesions occurred on the leaves of the plants. The leaves of plants grown under a low light intensity turned brown and the plants died. The phenolic content of the shoots in the different culture experiments is given in Fig. 4. A decrease of the light intensity (Fig. 4a) as well as an increase of the water temperature (Fig. 4b) resulted in a significant decrease of the phenolic content (P < 0.05). A change in the salinity (Fig. 4c) of the water hardly had any effect on the concentrations of phenolics.
4. Discussion
The results of both the pilot experiment and the culture experiment indicate a positive effect of light intensity on the phenolic biosynthesis in the leaves of Z. marina. Plants grown for 4 weeks at a high light intensity showed a significantly higher phenolic content than plants grown under a low light intensity (Figs. 1 and 4a). This is a phenomenon which was also demonstrated in terrestrial plants. Waterman et al. (1984) found that individuals of Barteria fistulosa Mast. growing in high light intensity responded by producing greater amounts of phenolics than individuals in a more shaded position. Similar results were obtained by Cooper-Driver et al. (1977), working with Pteridium aquilinum (L.) and Kuhn and Woodhead (1981), working with Sorghum bicolor (L.) Moench. A variety of mechanisms has been proposed to account for the increase of phenolics with increased light intensity and it is likely that some of these mechanisms operate simultaneously. One of the proposed mechanisms is the availability of minerals and especially nitrogen. The photosynthetic activity of a plant at high light intensity may be so high in relation to the quantity of nitrogen available that, once its limited supply has been used in the production of primary metabolites (amino acids, proteins), the remaining carbohydrates can only be used to produce nitrogen free molecules such as phenolics (Waterman et al., 1984). In this way phenolics are regarded as storage compounds of carbohydrates, which are only produced at times when the plants can not convert the carbohydrates into growth. Such an inverse relation between nitrogen availability and phenolic content was also found in Z. marina (Buchsbaum et al., 1990). In the experiment the amount of nitrogen available to the plants was not measured. However, the higher biomass of the plants grown under the highest light intensity and the presence of newly formed leaves at the end of the experiment, makes it unlikely that there were growth-limiting circumstances in the containers with the highest light intensity. Another explanation for the increased phenolic content is the known stimulation by light of most of the enzymes involved in phenolic biosynthesis of plants. In particular, the activity
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L.H.T. Vergeeret al. /Aquatic Botany 52 (1995) 35--44
of phenylalanine ammonia-lyase (PAL), a key enzyme in the synthetic pathway of phenolics, increases under the influence of light (Bidwell, 1974). This phenomenon has been observed in plants, as well as in plant cell cultures (DiCosmo and Towers, 1984), where no deficiency of nutrients occurred. The increase of the temperature results in a decrease of the phenolic content of eelgrass shoots. A clear explanation for this phenomenon is hard to give. Temperature influences photosynthesis as well as respiration, and in this way, the C/N balance in the plant. But also, temperature has a direct impact on the enzymes of the phenolic biosynthesis, in which every enzyme can have its own optimum. Salinity proved to have no effect on the phenolic content of eelgrass shoots. Zostera marina is euryhaline, so the survival of populations in brackish waters in the 1930s epidemic is more explained by the fact that a low salinity is detrimental to Labyrinthula. In the culture experiments we only determined the impact of abiotic factors on the host plant, but a specific factor has, of course, influence on the microorganism and on the relationship between the two. Among the infected plants in the pilot experiment, a higher light intensity was not only accompanied by an increased level of phenolic compounds, but also by a decrease in the infection rate of Z. marina with wasting disease. A correlation between a high phenolic content of Z. marina and a low infection rate was also found by Buchsbaum et al. (1990). The differences in infection rate among the infected plants were rather small, however, and among the controls no relation between light intensity/phenolic content and the infection rate could be shown, owing to a low infection rate among the plants of the middle illumination class (Fig. 1). Thus, the results only partly support the hypothesis of Giesen et al. (1990a,b) that a deficit of sunshine results in Z. marina being more susceptible to Labyrinthula, and the suggestion that this may be caused by a reduced level of phenolic defence compounds. An assessment of the effect of light intensity on the phenolic content together with the infection rate, is in the pilot experiment obscured by the interfering influence of infection itself on the phenolic content. As shown in Fig. 2, the infected plants had higher levels of phenolic compounds than the controls. This same problem was also raised by Woodhead (1981). In order to determine the influence of light alone on the phenolic content of Z. marina, the experiment was repeated. An increase in phenolics as a result of infection is often correlated with resistance to the disease. As shown in Fig. 4, an infection of Z. marina with Labyrinthula indeed leads to a drastic increase in phenolic biosynthesis. In what way the interaction between host and invading microorganism induces the production of phenolics is unclear, but, as with the light intensity, inoculation of plants with pathogenic microorganisms, often leads to an increase in PAL activity associated with the production of specific phenolic compounds. Increases in phenolase and peroxidase activity have been reported in many plants (Levin, 1976; Friend, 1979). Phenolics are oxidized by these enzymes into quinones, which are highly reactive compounds and therefore good bactericides and fungicides. Activity of these enzymes produces enzymatic browning, a phenomenon also observed after infection of Z. marina with Labyrinthula. Whether the increase of phenolic compounds forms a defence against Labyrinthula has yet to be established. As resistance to a microorganism may depend more upon the quality
L.H.T. Vergeer et al. /Aquatic Botany 52 (1995) 35-44
43
of the phenolics than upon their quantity, further research will be directed to the separation of the different phenolic compounds present in Z. marina and an assessment of their specific growth-limiting properties with respect to the microorganism.
Acknowledgements This research was supported by the Foundation for Biological Research (BION) of the Netherlands Organisation for Scientific Research (NWO). Part of the research was carried out at the Station d'Oceanologie et de Biologie Marine in Roscoff (France), thanks to a subvention of the Royal Dutch Academy of Sciences. We wish to thank the Academy and Dr. P. Lasserre, Director of the station, for this opportunity. Professor Dr. C. den Hartog and Professor Dr. G. van der Velde are acknowledged for their critical comments on earlier drafts of this manuscript.
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