Ecophysiological needs of the potential biomass crop ... · Ecophysiological needs of the potential biomass crop Spartina ... (control) to 100% seawater ... in a “quick check”

KOYRO & HUCHZERMEYER 123

Tropical Ecology 45(1): 123-139, 2004 ISSN 0564-3295 © International Society for Tropical Ecology

Ecophysiological needs of the potential biomass crop Spartina townsendii Grov.

HANS-WERNER KOYRO1 & BERNHARD HUCHZERMEYER2

1Institute for Plant Ecology, Justus Liebig University Gießen, Heinrich–Buff–Ring 26-32, D-35392, Giessen; 2Botany Institute, Hannover University, Herrenhäuser Str. 2, D-30 419 Hannover

Abstract: The aim of this study was to determine the level of salinity tolerance of Spartina townsendii and to study the mechanisms by which this species survives salinity and the specific conditions at the intertidal zone. Therefore, Spartina was irrigated with five differ-ent saline levels ranging from nutrient solution (control) to 100% seawater (480 mM NaCl) in a “quick check” system (QCS). The salinity tolerance was measured on the basis of the two pa-rameters maximum yield, and gas exchange. a) The maximum yield of Spartina was reduced by 50% (C50-value) at 480 mM NaCl salinity. b) Gas exchange parameters such as net photo-synthesis (µmol * m-2 * s-1), stomatal conductance (mol * m-2 * s-1), transpiration (mol * m-2 * s-1), and water use efficiency of the photosynthesis (µmol CO2 * mmol-1 H2O) were lowest at 500 mM NaCl-salinity. The strategies employed by Townsend´s cordgrass for avoiding salt injury de-pend on adaptation to low water potential and high Na and Cl concentrations. Spartina has sufficient adjustment mechanisms even at high NaCl salinity suggesting that there was no reason for growth reduction by water deficit. The low external water potential was balanced e.g. by a decrease of the osmotic potential. Especially Na and Cl were accumulated in high con-centrations in the root and shoot. The main defence of Spartina townsendii to elevated salinity regimes is the activation of salt glands. These salt glands are working highly selective and eliminate relatively large quantities of salt by secretion to the leaf surface. However, the salt glands were neither able to balance the high burden especially of Na in the leaf tissues at the high salinity treatment, nor useful in maintaining a constant supply of essential elements such as K, Mg and Ca. The gap between a sufficient nutrient supply and NaCl accumulation grows with increasing salinity and seems to limit the salinity tolerance of Spartina by nutrient im-balance and/or ion toxicity. The high salinity tolerance and the ecological engineering of Spartina townsendii can produce various ecological (e.g. highly effective nutrient cycling of N, Fe and S) and economical (e.g. biomass crop) benefits. The QCS offers a reliable basis to define this species as a potentially useful cash crop halophyte.

Resumen: El propósito de este estudio fue determinar el nivel de tolerancia a la salinidad

de Spartina townsendii y estudiar los mecanismos que permiten a esta especie sobrevivir a la salinidad y a las condiciones específicas de la zona intermareal. Para ello, Spartina fue regada con cinco diferentes niveles salinos, variando desde solución de nutrientes (control) hasta agua de mar al 100% (480 mM NaCl) en un sistema de verificación rápida (QCS por sus siglas en in-glés). Las estrategias utilizadas por esta planta (conocida como ‘pasto cuerda de Townsend’) pa-ra evitar el daño por la sal dependen de la adaptación a un bajo potencial hídrico y a altas con-centraciones de Na y Cl. Spartina tiene suficientes mecanismos de ajuste, incluso con una alta salinidad de NaCl, lo que sugiere que no existe ninguna razón para una reducción del creci-miento debida a un déficit hídrico El bajo potencial hídrico externo estuvo balanceado, p.ej. por un decremento del. potencial osmótico. En especial, el Na y el Cl se acumularon en grandes concentraciones tanto en la raíz como en la parte aérea. La principal defensa de Spartina town-sendii contra regímenes de alta salinidad es la activación de glándulas de sal que eliminan can-

124 ECOPHYSIOLOGY OF SPARTINA TOWNSENDII

tidades relativamente grandes de sal por secreción hacia la superficie foliar. La brecha entre una suficiente disponibilidad de nutrientes y la acumulación de NaCl crece conforme aumenta la salinidad y parece limitar la tolerancia a la salinidad de Spartina por un desequilibrio nutri-cional y/o por toxicidad de iones. La alta tolerancia a la salinidad y la ingeniería ecológica de Spartina townsendii pueden producir varios beneficios ecológicos (p.ej. un reciclaje muy eficien-te de N, Fe y S) y económicos (p.ej. cosecha de biomasa). El QCS ofrece una base confiable para definir a esta especie como una halófita potencialmente útil como cultivo comercial.

Resumo: O objectivo deste estudo foi o de determinar o nível de tolerância da Spartina

townsendii ao sal e estudar os mecanismos pelos quais esta espécie sobrevive à salinidade e as condições específicas na zona entre marés. Nesse sentido a Spartina foi irrigada com cinco níveis diferentes de solução salina num intervalo que variou entre uma solução nutriente (con-trolo) a 100% de água do mar (480 mM NaCl) utilizando um sistema de “avaliação rápida” (QCS). As estratégias empregues por “Townsend’s cordgrass” para evitar os danos do sal de-pende na adaptação ao baixo potencial hídrico e elevada concentração em Na e Cl. A Spartina tem mecanismos de adaptação suficientes, mesmo para valores elevados de NaCl, sugerindo que não há razão para a redução do crescimento pelo deficit hídrico. O baixo potencial hídrico externo foi balanceado, p.e. por um decréscimo do potencial osmótico. O Na e o Cl foram espe-cialmente acumulados em elevadas concentrações nas raízes e nos lançamentos. A defesa prin-cipal da Spartina townsendii para regiões salinas elevadas é a activação das glândulas de sal as quais eliminam quantidades relativamente elevadas de sal por secreção da superfície das folhas. A diferença entre uma oferta nutritiva suficiente e a acumulação de NaCl cresce com o acréscimo de salinidade e parece limitar a tolerância à salinidade da Spartina por um dese-quilíbrio nos nutrientes e/ou toxicidade iónica. A elevada tolerância ao sal e o “engineering” ecológico da Spartina townsendii pode produzir vários benefícios ecológicos (p.e. reciclagem al-tamente efectiva de nutrientes quanto ao N, Fe e S) e económicos (p.e. produção de biomassa). O QCS oferece uma base fiável para definir esta espécie como uma cultura halófita potencial-mente útil.

Key words: Spartina townsendii, cash crop halophytes, gas exchange, NaCl salinity, nutrients, “quick

check” system, salinity tolerance, water relations, water potential.

Introduction

Background information The economic basis of the existence of more

than 1 billion of people in 100 countries is threat-ened by the expansion of the deserts. Every year 6 million ha arable land are lost for agricultural use in developing countries. Reasons for the desertifica-tion are climatic changes, intensive pasture, defor-estation and unsuited irrigation practices. The ex-tension of irrigated agriculture and the intensive use of water resources combined with high rates of evaporation in arid and semiarid regions, have in-evitably given rise to the problems of salinity in the soil and in underground water. Among the most pressing problems for the growing mankind are the

increasing shortage of living space and availability of freshwater. It is, therefore, necessary to develop sustainable systems in presently waste or deserted areas. Besides the naturally occurring salt-affected soils, the extent of man-made salinized soils as a consequence of improper irrigation management is significant (Choukr-Allah 1996). It should be noted that salinity problems are liable to spread as irriga-tion is intensified and irrigated areas are extended (Choukr-Allah & Harrouni 1996). Therefore, a new concept of saline irrigation had to be developed and the research into management practices with salt resistant species became increasingly essential. The technology is available now to use unconventional water resources or habitats for salinity-tolerant plant production systems for many purposes.


Introduction of halophytes The standard approach to this problem would

be to increase salinity salt tolerance of conven-tional crop plants, but the gained yield is generally low (Koyro & Huchzermeyer 1999b). The alterna-tive approach is to make use of the plants that al-ready have the required level of salt tolerance, and are still productive at high external salinity levels: the halophytes. Halophytes are plants, able to complete their life cycle in a substrate rich in NaCl (Lieth 1999; Schimper 1891). This substrate offers advantages for obligate halophytes in the competi-tion with salt sensitive-plants (glycophytes).

Some of 2600 species of halophytes occur in sa-line coastal environment and inland deserts. In-creasing attention has been paid to research and development of halophytes and several authors proposed utilising undiluted seawater on a large scale for irrigation (Koyro & Huchzermeyer 1999a; Lieth & Al Masoom 1993). The sustainable use of halophytic plants is a promising approach to valor-ize strongly salinised zones unsuitable for conven-tional agriculture and mediocre waters (Boer & Gliddon 1998; Choukr-Allah 1996; Lieth et al. 1999; Pasternak 1990). There are already many halophytic species used for economic interests (human food, fodder) or ecological reasons (soil de-salinisation, dune fixation, CO2-sequestration).

One plant species with a high potential to be-come a cash crop halophyte is Spartina townsendii. Townsend´s Cordgrass was first found at the end of the 19th century on intertidal mud and sand flats at the coasts of the English Channel. It was bigger than the native Spartina alternifolia and colonised these sites immediately. Halophytes such as Spartina alterniflora (Cordgrass) and Spartina townsendii (Townsend´s Cordgrass) introduce the intertidal zone of temperate estuaries and lagoons as a sustainable system for cash crop halophytes (Beale et al. 1999). Salt marshes dominated by Spartina species are among the most productive ecosystems known, despite nitrogen limitation (Bagwell & Lovell 2000). Spartina ecological engi-neering can produce various ecological and eco-nomic benefits (Qin et al. 1998):

Salt marshes dominated by Spartina species: - help to reduce atmospheric CO2 enrichment

(Matamala & Drake 1998), - have a low vulnerability against sea level

change and protect the estuaries against the ef-fects of global changes, are ‘therefore’ an important

component of new coastal management practices and useful in developing strategies for the stabili-zation of deteriorating marshes (in marsh restora-tion projects; Lieth 1999; Miller et al. 2001; Pezeshki & DeLaune 1997; Simas et al. 2001),

- can tolerate oil spills (its growth is even stimulated by crude oils) and are hosts of microbial degraders promoting oil spill cleanup in coastal wetlands (Lin et al. 1999; Lindau et al. 1999; Ny-man 1999; Pezeshki et al. 2001; Smith & Proffitt 1999),

- support biodiversity and the production of marsh fauna (e.g. fishes, benthic invertebrates; Angradi et al. 2001; Connolly 1999; Riera et al. 1999; San Leon et al. 1999; Waide et al. 1999; Weinstein et al. 2000),

- support bioremediation of recalcitrant com-plex carbohydrate biopolymers by marine bacteria (Ensor et al. 1999). Spartina itself - is a potential biomass crop (e.g. grown for fodder; Beale et al. 1999; Lieth 1999) in poor soil conditions,

- is highly effective in nutrient cycling (e.g. N-fixation, Fe-reduction, sulfate-reduction, sulfide-oxidation, Se-biotransformation to DMSeP, Si-reservoir; Ansede et al. 2001; de Bakker et al. 1999; Hines et al. 1999; Lee 1999; Norris & Hack-ney 1999),

- reduces toxic metal bioavailability (e.g. Cd, Pb, Cu, Cr, Hg and Zn), by sequestering a larger proportion of its metal burden in its belowground tissues which are likely to be permanently buried (Burke et al. 2000; Heller & Weber 1998; Klap et al. 1999; Patra & Sharma 2000; Windham et al. 2001),

- is a biomonitor for environmental toxicants from municipal and industrial wastes, agricultural runoff, recreational boating, shipping and coastal development (Lewis et al. 2001; Lytle & Lytle 2001; Padinha et al. 2000),

- is used as an indicator for estuarine sediment quality (Lewis et al. 2001),

Biomineral liquids extracted from Spartina culms have a number of health functions (e.g. car-diotonic, enhance of life span), the total flavonoids of Spartina can be separated and used to resist blood coagulation and encephalon thrombus (Qin et al. 1998).

The C-4 perennial grass Spartina townsendii and other species of this genus are potential bio-


mass crops. However, it is necessary to produce utilisable biomass with unconventional irrigation (up to seawater salinity) in a way that is ecologi-cally sustainable and economically feasible. Spartina spec. is also a perennial grass and dreaded for its impact on estuarine flora and fauna. Spartina can rapidly colonize the intertidal zone of temperate estuaries and lagoons. Conse-quently beside all economic interests there is con-siderable concern about its impact on estuarine flora and fauna especially in regions where its not an indigenous species (Hedge & Kriwoken 2000). Therefore, detailed information are essential for the controlled establishment especially in regions where its not a native species. Current theories differ in their prediction concerning the effects of interspecific interaction on species growth and dis-tribution along environmental gradients (La Peyre et al. 2001). The goal of this research is to provide a reliable, ecophysiological basis as the first step for the development of a ecological sustainable and economically feasible cash crop Spartina town-sendii.

Reproducable experimental growth and substrate conditions

There are major problems to be overcome in determining and selecting the best species and ecotypes to be used in the vast areas of degraded salt-affected soil or other saline habitats around the world. A precondition for a sustainable utilisa-tion of suitable halophytes is the precise knowl-edge about their salinity tolerance and the various mechanisms enabling a plant to grow at (their natural) saline habitats (Koyro & Huchzermeyer 1997; Marcum 1999; Warne et al. 1999; Weber & D´Antonio 1999; Winter et al. 1999).

The study in the natural habitat represents a mean behaviour but the major constraints can vary this much that a precise definition of the sa-linity tolerance of a species (and a selection of use-ful plants) is not possible. Only artificial conditions in sea water irrigation systems in a growth cabinet under photoperiodic conditions offers the possibil-ity to study potentially useful halophytes under reproducible experimental growth and substrate conditions (Koyro & Huchzermeyer 1999a). The supply of different degrees of sea water salinity [0%, 25%, 50%, 75%, 100% (and if necessary higher) sea water salinity] to the roots in separate systems under otherwise identical conditions gives

the necessary preconditions for a reliable quick check system (QCS).

The threshold of salinity tolerance Generally, classification of the salinity toler-

ance (or sensitivity) of crop species is based on the threshold EC (electrical conductivity) and the per-centage of yield decrease beyond threshold (Greenway & Munns 1980; Marschner 1995). The substrate concentration leading to a growth de-pression of 50% (reference to fresh weight, in com-parison to plants without salinity) is widely used by ecophysiologists as a definition for the thresh-old of salinity tolerance (Kinzel 1982), because it is as difficult to fix the upper limit of salinity toler-ance. The agreement to the above-mentioned growth depression is comparatively arbitrary, but it leads to a precise specification of a comparative value for halophytic species and is especially ex-pressive for applied aspects such as economic po-tentials of suitable halophytes.

The threshold of salinity tolerance according to Kinzel (1982) is used in the quick check system (QCS) for halophytes as an objective parameter for the description of the actual condition of a plant (Ashraf & O´Leary 1996; Koyro 2000). There are now reliable informations available about studies with several species such as Plantago cf. coronopus, Beta vulgaris ssp. maritima, Laguncu-laria racemosa and Batis maritima (Koyro & Huchzermeyer 1997, 1999a; Koyro et al. 1999; Koyro 2000). The substrate concentration leading to a growth depression of 50% (refer to fresh-weight) is easy to calculate with the QCS (by ex-trapolation of the data) and it leads to a precise specification of a comparative value for halophytic species. It will be used for the reasons listed above to compare the threshold of salinty tolerance of the experimental plants.

Ecophysiological parameters of the QCS The threshold of salinity tolerance describes

the limitations of productivity, but not the physio-logical mechanisms of salinity-induced growth re-duction. Therefore, a selection of parameters with a close connection to the four major constraints of plant growth on saline substrates is necessary: a) water deficit, b) restriction of CO2-uptake, c) ion toxicity d) nutrient imbalance. Plants growing in saline habitats face the problem of encountering a


low water potential in the soil solution and high concentrations of potentially toxic ions such as chloride and sodium (Marschner 1995). Salt exclu-sion minimizes ion toxicity but accelerates water deficit and diminishes indirectly the CO2 uptake. Salt absorption (includer) facilitates osmotic ad-justment but can lead to toxicity and nutritional imbalance. The QCS of potential crop halophytes comprises general scientific ecophysiological data and some special physiological examinations (Koyro 2000). The collected data comprise informa-tion about morphology, photosynthesis (incl. gas exchange), water relations (leaf water potential, osmotic potential), mineral content, content of os-motically active organic substances (such as car-bohydrates and amino acids) and growth.

Aim of the study The aim of this study is to provide reliable,

ecophysiological parameters of Spartina town-sendii to get a survey of the mechanisms leading e.g. to the salinity tolerance of this species. It will be shown that the salinity-induced multiplicity (network) of structural and functional changes constitutes a group of indicators for the salinity tolerance and growth potential of this species and gives an impression of its potential as a cash crop halophyte.

Material and methods

Plant material and culture conditions Spartina townsendii is an intertidal, estuarine

saltmarsh grass which can grow up to 130 cm in height. Its leaves are narrow, usually 45 cm long and 1.5 cm wide. It is a perennial grass spreading by underground stems. Cuttings of Spartina town-sendii (Townsends Cordgrass, original habitat We-ser marshes in Germany) were transplanted into a soilless (gravel/hydroponics) culture quick check system (Fig. 1, Koyro & Huchzermeyer 1999a). The free surface of the substrate was covered with a black foil to hinder the spattering of the plants with the nutrient solutions. The plants were irri-gated with a basic nutrient solution as modified by Epstein (1972) under photoperiodic conditions (16 h light/ 8 h dark) in an environment controlled greenhouse. Temperatures were 25 ± 2 °C during the day and 15 ± 2 °C during the night. Relative humidity ranged from 45 % to 70 %. Light inten-

sity was in the range of 1500 µmol * m-2 * s-1 at plant level.

The stepwise addition of NaCl to the basic nu-trient solution began after a period of 6 months by raising salinity of the solution in steps of 40 mM NaCl each day. There were altogether six treat-ments: Control, 1, 40, 240, 360 and 480 mM NaCl (equivalent to 0, 2, 8, 50, 75 and 100 % NaCl). The highest salinity treatment was reached after 12 days. The “quick check” system was programmed by a timer to water the plants every 4 hours for 30 min starting at midnight, 4 a.m., 8 a.m., 12 noon, 16 p.m. and 20 p.m. daily and allow the saline so-lutions to drain freely from the pots. Solutions were recycled and changed every 2 weeks to avoid nutrient depletion. The experiment was performed for a total period of twelve months. Three weeks before the harvest, the leaves were washed prop-erly with distilled water and not touched anymore until the end of the experiment.

Growth parameters and quantitative chemical analysis

The number, the LMA (leaf mass per area ra-tio defined as weight per surface area) of the leaves and the weight of plants, leaves and roots (main and adventitious roots) were noted. A piece of 4 ± 1 mm (width) and 80 mm (length) was cut out of the middle region of an adult leaf. The ad-axial and abaxial leaf surface was rinsed with 10 ml distilled water and stored in a refrigerator until the quantitative chemical analysis (see below).

The plant was devided into two parts (under and above ground). Subsamples were taken for (a) the determination of the dry weight and water content and for (b) the quantitative chemical analysis (QCA).

To (a) The samples were weighed and dried for 48 h at 90 °C in an oven.

To (b) The samples for the QCA were washed separately for 1 minute in ice-cooled 0.2 mol . m-3 CaSO4 solution, 1 min in distilled water and blot-ted carefully with tissue paper. Representative parts of the leaves and roots were weighed and extracted with 0.5% HNO3 in a water bath (80°C) for 12 h. Na, K, Mg and Ca were determined di-rectly from the extract with an atomic absorption spectrophotometer (Perkin Elmer PE3300) and Cl- by electrochemical titration (AMINCO COTLOVE chloride titration).


Chlorophyll, total carbohydrate and total protein

Leaf discs (0.78 cm2) of fresh material were ex-tracted in 80% ethanol at 80 °C (carbohydrates) or in 80% acetone (for the determination of chloro-phyll a and b). Carbohydrates were measured spectrophotometrically with the Molisch reaction (Wild 1999), chlorophyll a and b were determined

spectrophotometrically after Lichtenthaler & Wellburn (1983).

Freeze dried sample (100 mg) was homoge-nized, extracted in 6 ml phosphate buffer (pH 7.4), and centrifuged at 4°C for 30 min at 30 000 x g. The soluble proteins in the supernatant were pre-cipitated with 400 µl TCA [10% (w/v), for 1 h on ice]. The sediment was resolved with 100 µl 1 N NaOH (for 24 h). The soluble protein was meas-

Fig. 1. (a) Hydroponic quick check system (QCS) of Spartina townsendii under photoperiodic conditions in a growth cabinet; (b) Leaves of the control plants (1 mM NaCl); (c) Salt crystals on the leaves of the high salinity treatment (480 mM NaCl).


ured photometrically after Bradford (1976) against a standard with bovine serum albumin.

CO2-gas exchange The closed photosynthesis measurement

system LI-COR 6200 (LI-COR, Lincoln, NE, USA) was used to determine the response of photosynthesis to substrate salinity (Von Willert et al. 1995). An initial concentration of 400 + 14 ppm was adjusted using a gas-tight Hamilton syringe filled with pure CO2. Photosynthesis reduced the concentration in the closed loop until the compensation point was reached. Calculation of the gas exchange parameters was corrected for leaks as described by LI-COR Inc. Net photosynthesis (µmol * m-2 * s-1) stomatal conductance (mol * m-2 * s-1), transpiration (mol * m-2 * s-1) and water-use efficiency (µmol * mmol-1) were measured on youngest, fully emerged leaf blades. All measurements were taken at light saturation (PPFD = 1500 µmol * m-2 * s-1) and 24-26 °C.

Osmotic potential Leaf samples were frozen with liquid nitrogen

and homogenized in a mortar. After thawing the samples were centrifuged (at 4 °C, for 5 min at 3000 x g). The osmotic potential was determined in the supernatant (leaf sap) with the freeze-point depression method by an cryo-osmometer (GONOTEC).

Light (LM), transmission- (TEM) and scanning electron microscopy (SEM)

Leaf sections were fixed for 3 h with 4% glu-taraldehyde in 50 mM Pipes-buffer (pH 7). After washing the leaf sections were rinsed three times in buffer and subsequently postfixed for 4 hours with a 2% OsO4 solution in buffer. After a few washings in distilled water, the leaf sections were dehydrated in acetone, imbedded in Durcupan ACM (Fluka) or ERL—4206 (Spurr) and polymer-ized at 70 °C for 8 h. The embedded material was sectioned for the light microscopy with a glass knife on a LKB ultramicrotome. The sections were stained with methylene blue (in 2% ethanol) on a heating plate. For a closer examination of the leaf surface by scanning electron microscopy (Philips SEM XL20), dehydrated leaf sections (see above) were dried with CO2 at high pressure (supercriti-

cal fluid) after the critical point method and a thin gold layer was sputtered on the surface to enhance the electrical conductivity (Robinson et al. 1985).

Statistical treatment Data (n > 5) were subjected to one-way

analysis of variance using pc-stat computer software. An analysis of variance was conducted, and least significant differences (LSD) were determined with F test (p=0.05).

Results

Morphological adaptation Spartina townsendii reduces the salt

concentrations of the active photosynthetic tissue by various mechanisms such as the secretion by salt glands (Figs. 1 & 2). The leaf anatomy of Spartina shows some additional adjustment to its natural habitat - the changing tide. The adaxial leaf surface has longitudinal incisions giving evidence for a structural adaptation to drought (typical for gramineae such as Bouteloua curtipendula, Oryzopsis canadiensis and Stipa tenacissima, Eschrich 1995). The stomates are located near the bottom of these incisions (Sutherland & Eastwood 1916) and can be protected against water loss by rolling up of the leaf longitudinally. The salt glands are inserted into the leaf just beyond the laminar edges, and NaCl salinity leads to an expansion of the lateral papillas enabling the plant to use it as a zip and to control influx and efflux of substances between the longitudinal incisions and the atmosphere (or seawater). This seems to be extremely important when the tide gets higher than the plant and the water attempts to flood the longitudinal incisions of the leaf. The laminar edges can be used to control the stream of water into this region and to prevent a flooding of the stomatas. This has two advantages: a) Transpiration can be diminished and photosynthesis can continue without significant water loss. b) The salt excreted from the salt glands can still be washed off the surface.

Growth In contrast to glycophytes, Spartina town-

sendii was able to complete its life cycle at high salt concentrations (Fig. 3). Spartina showed the overall growth response (transient increase) to ele-


vated salinity typical for halophytes or halophytic crops (Greenway & Munns 1980). However, the halophyte Spartina townsendii has a physiological requirement for salt in a range of 1 mM NaCl (see squares in Fig. 3). The plants showed negative growth without NaCl addition after 6 months of culture. The salinity threshold (initial significant reduction in the expected maximum yield, Shan-non & Grieve 1999) of Spartina townsendii was reached at 50% seawater salinity, the growth was reduced by NaCl treatment to 50% at 480 mM seawater salinity.

Leaf water potential, osmotic potential and mineral content

The leaf water potentials (Ψ) of Spartina townsendii plants were generally lower than the water potentials in the accessory five seawater di-lutions (Koyro 2002). The reduction of the leaf wa-ter potential was mainly reached by an increase of the concentrations of osmotically active solutes (Fig. 4). The osmolality in adult leaves was gener-ally higher than in juvenile leaves. However, there was only a minor increase of the osmolality in

Fig. 2. Adaxial leaf surface of Spartina townsendii (Gramineae). (a), (b) and (c) SEM micrograph of the adaxial surfaces of a leaf of plants grown at 0, 40 and 240 mM NaCl salinity. Elevated salinity led to an expansion of the lateral papilles (please compare a, b and c); (d) SEM micrograph of the salt glands (Sg) and stomates (St) on the adaxial surface of a control leaf (see arrows); (e) Cross sections of a leaf of Spartina townsendii (240 mM NaCl). Saltglands are inserted into the leaf beyond the laminar edges but above the stomates; (f) SEM micrograph of a trichome on the abaxial surface of a leaf. La = laminar edge, Sg = saltgland, St = stomate


adult leaves between 50 and 100 % seawater salin-ity (240 and 480 mM NaCl).

Potassium and magnesium were the major cations in the root tissues of plants at 0.2% sea-water salinity (Control, Fig. 5b). However, it was much more conspicuous that even the Na and Cl concentrations in the root tissue under these cir-

cumstances were much higher than in the nutrient solution. Elevated salinity led to a transient in-crease of the potassium and to an increase of the magnesium, calcium, sodium and chloride concen-trations. The sodium and the chloride concentra-tions in the root tissues were in the same range and always above the salinity level in the nutrient solutions. Chloride, potassium, sodium, but also magnesium and calcium, were important osmoti-cally active substances in the shoots of control plants (1 mM NaCl, Fig. 5b). However, especially the potassium and calcium concentrations in the shoot were significantly higher than in the root tissues. With the increase of the NaCl salinity the potassium, calcium and magnesium concentrations declined and especially the sodium, but also the chloride concentrations reached levels far above those present in the nutrient solutions or in the root tissues. However, sodium was accumulated to a much higher level in the leaf tissues than chlo-ride. The difference between sodium and chloride concentration in leaves reached a value of nearly 40 % at 480 mM NaCl salinity. It corresponds to the osmolalities in adult and juvenile leaves that these changes were much more pronounced in the latter ones (results not shown). The difference be-tween chloride and sodium accumulation in the leaves of the high salinity treatment cannot be ex-plained by differences in the excretion. There was only a minor increase of the sodium and chloride

0

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20

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0 100 200 300 400 500NaCl-concentration in the nutrient solution

Fres

hwei

ght i

n [g

]

Fig. 3. Development of the plant fresh weight [wholeplant ( ) and ( ); shoot ( ) and ( ); root ( ) and ( )]at treatments with different percentages of sea watersalinity. The squared symbols document the freshweight of plants grown without additional NaCl supply.The crossover of the dotted and the black line reflectsthe NaCl salinity where the growth depression fallsdown to 50% of the control plant.

0

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tip base tip base

Osm

otic

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ue [m

Osm

ol]

Juvenile leaf Adult leaf

Fig. 4. Osmotic values (mOsmol) in the tip or basis of juvenile and adult leaves of Spartina townsendii.


excretion between 50 % and 100 % seawater salin-ity (Fig. 5c).

The chloride accumulation seems to be bal-anced even at high salinity level with the existing mechanisms (a) selective uptake in the root, (b) selective translocation from the root to the shoot, and (c) via excretion through the salt gland. This is not the case for sodium. The sodium concentration in the shoot in the high salinity treatment reaches a level twice as high as in the nutrient solution. This overflooding with sodium cannot be balanced by the excretion through the salt glands. Beside evidence for sodium toxicity there are also some

indications of ion imbalance: Elevated salinity promotes the excretion of essential elements such as K, Ca and Mg and these values are still re-markable at the high salinity treatment.

Dry matter, protein and carbohydrate content The reduction in fresh weight (Fig. 3) at

elevated salinity was partially compensated in root and shoot tissues by an increase of the dry matter (in % fresh weight, Fig. 6a; ratio Control/100% seawater salinity in the root 1.5 and in the shoot 1.3) and of the protein content of the shoot (in % dry matter, Fig. 6b; ratio Control/100% seawater salinity 1.2). Spartina townsendii showed an obvious shift from storage of carbohydrates (Fig. 6c) to the synthesis of proteins.

Photosynthesis and chlorophyll content NaCl salinity affected [beside changes of the

metabolism (such as increases of dry matter and protein content)] also the chlorophyll content. The enhancement of the NaCl salinity led in Spartina to an increase of the chlorophyll a/b ratio in the juvenile and adult leaves (Fig. 6d) but also to a decrease of the chlorophyll a and even more to a decrease of the chlorophyll b content. The CO2-net assimilation rate showed a similar curve as the carbohydrate content (Table 1 and Fig. 6c). An (insignificant) increase at low salinity was followed by a steep decrease at higher salinities. Additionally, a steep decrease between 240 and 480 mM NaCl was a common reaction of growth, carbohydrate content in the leaves CO2-net assimilation rate, stomatal conductance and transpiration. The water-use efficiency of the photosynthesis decreased with elevated salinity from 5.24 (control) to 3.68 (100 % seawater salinity) µmol CO2 * m mol-1 H2O.

Discussion

Photosynthesis In the present study, there was evidence for a

correlation between salinity tolerance and the pho-tosynthesis and growth responses of Spartina townsendii. Similar results were found also for Spartina patens, S. maritima and S. densiflora populations collected in Lousiana Gulf Coast marshes or on the coasts of SW Europe (Nieva et al. 1999; Pezeshki & DeLaune 1997). The sensitiv-

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Fig. 5. Chloride-, potassium- , sodium- , calcium andmagnesium concentrations in mM in root (a), shoottissues (b) and excreted salt crystals (c) of Spartinatownsendii treated with 1 ( ), 40 ( ), 240 ( ) or 480( ) mM NaCl-salinity.

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Fig. 5. Chloride-, potassium- , sodium- , calcium andmagnesium concentrations in mM in root (a), shoottissues (b) and excreted salt crystals (c) of Spartinatownsendii treated with 1 ( ), 40 ( ), 240 ( ) or 480( ) mM NaCl-salinity.


ity in the response of the net photosynthetic rate is an ideal indicator of salt tolerance for Spartina townsendii and seawater salinity irrigation is as-sociated with a decrease in the net photosynthesis rate, the transpiration rate and the stomatal con-

ductance in this and in many other species (Pearcy & Ustin 1984; Rozema & van Diggelen 1991).

In agreement with the results presented for Spartina townsendii in this paper, other species of the genus Spartina, such as S. foliosa, S. maritima and S. densiflora, also present high water use efficiency (WUE) at low salinity, with a decline at higher salinity (Mahall & Park 1976; Nieva et al. 1999). However, the WUE of all these C4 plants mentioned above at elevated salinity were still high in comparison with other halophytic C3 species (Nieva et al. 1999).

The WUE of photosynthesis decreased in Spartina townsendii leaves with elevated salinity and in response to low salinity levels mainly because of increasing stomatal conductance and waterloss. It is not clear whether the water-use efficiency of photosynthesis would be similar for Spartina townsendii in its natural habitat (the intertidal zone under various flooding-salinity combinations). The gas exchange of Spartina maritima was independent of elevation of salinity as were its chlorophyll fluorescence parameters (Castillo et al. 2000). In contrast, in S. densiflora the rate of CO2 uptake declined, and stress to photosystem II increased at lower salinities. It is not known whether transpiration is diminished in S. townsendii during flooding enabling the photosynthesis to operate without significant water loss. Further investigations are necessary to examine the differences in physiological and growth response to distinct flooding-salinity combinations. The influence of flooding on gas exchange could be also an explanation for considerable variation in the performance among certain populations of Spartina in response to salinity regimes (Pezeshki et al. 1993).

In Spartina townsendii the lower net photo-synthesis rate under salinity is not solely due to a decrease in the contribution of CO2 by reduction in the stomatal conductance (see Table 1). Non-stomatal limitations of CO2-assimilation capacity were shown also for Spartina maritima and S. densiflora (Nieva et al. 1999). All three Spartina species maintained higher intercellular CO2 con-centrations under saline irrigation (results not shown for Spartina townsendii). The variation in the net photosynthesis rate could be related with biochemical changes due to ion toxicity that affect carboxylase activity of the ribulose-1,5-bisphosphate carboxylase/oxygenase and the size

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Fig. 6. Dry matter ( : root, : shoot) protein content(shoot), total leaf carbohydrate content (TCh, :juvenile, : adult) and chlorophyll a/b ratio in leaves ( :juvenile, : adult) of Spartina townsendii.


of the phosphate triose pool (Wyn Jones & Pollard 1983; Ziska et al. 1990; Antolin & Sánchez-Díaz 1993; Nieva et al. 1999).

NaCl salinity had an significant effect on the chlorophyll a and b content and the chlorophyll b/a ratio of Spartina townsendii and on chlorophyll fluorescence in S. densiflora (Castillo et al. 2000; Nieva et al. 1999) with significantly higher values in freshwater irrigation, probably indicating a greater size of the PSII electron acceptor pool, which would lead to a decrease in non-photochemical quenching.

Leaf water potential Data of the leaf water potentials demonstrated

clearly that Spartina townsendii, S. matritima and S. densiflora (Koyro 2002; Nieva et al. 1999) have sufficient adjustment mechanisms even at high salinity, suggesting that there was no reason for growth reduction by water deficit. Thus, if the rate of supply of water to the shoot is not restricted, the depression of the shoot growth shown in Fig. 3 is likely to depend mainly on mineral nutrition. It seems to be only a matter of controversy as to whether a decrease in the amounts of nutrients or unfavourable nutrient ratios (e.g. Na+/K+) are important factors for impaired leaf elongation (Lynch et al. 1988; Munns et al. 1989).

Nutrients The osmotic adjustment in the root and shoot

tissues in our experiment mainly based on the ac-cumulation of Na and Cl (Figs. 5a & b). It is, there-fore, not astonishing that there was also a correla-tion between salinity tolerance and ion composi-tion in the shoot. The main defence of Spartina

townsendii to elevated salinity regimes is the acti-vation of salt glands. It was shown in the previous study that these salt glands are working highly selective and eliminate relatively large quantities of salt by secretion to the leaf surface, where it can be washed off by seawater, rain or dew (Marcum et al. 1998). However, the salt glands were neither able to balance the high burden especially of Na in the leaf tissues at the high salinity treatment, nor useful in maintaining a constant supply of essen-tial elements such as K, Mg and Ca. The gap be-tween a sufficient nutrient supply and NaCl accu-mulation grows with increasing salinity and seems to limit the salinity tolerance of Spartina town-sendi by nutrient imbalance and/or ion toxicity.

This hypothesis was also confirmed and specified by studies of the element compositions (with EDXA analysis of bulk frozen samples in a scanning electron microscope) in the cytoplasm of single epidermal leaf cells of Spartina townsendii (Koyro 2002). NaCl at 100 % seawater salinity led only to a minor decrease of the major elements K and P (in comparison with the control) but to a significant increase of the Na and Cl concentrations in this compartment. The author discussed these findings as a beginning Na and Cl toxicity in the cytoplasm supporting the hypothesis that a major reason for the limitation of salinity tolerance was ion toxicity. It was recommended to reduce the ion toxicity by the supply of sufficient fertilizers at high NaCl salinities (Koyro 2002).

Nitrogen metabolism The rhizosphere sediments of cordgrasses are

generally a site of intense nitrogen fixation activity and a major source of biologically available nitro-gen providing a significant source of nitrogen (e.g.

Table 1. Transpiration (mmol * m-2 * s-1), stomatal conductance (mmol * m-2 * s-1), CO2 net-assimilationrate (µmol * m-2 * s-1) and water use efficiency of the photosynthesis, (µmol CO2 * mmol-1 H2O) of leaves of Spartina townsendii. 0,2% sea water salinity (sws) = 1NaCl, 8% sws = 40NaCl, 50% sws = 240NaCl and 100% sws = 480NaCl

Nutrient solution [mM NaCl]

Transpiration [mmol * cm-2 *s-1]

Stomatal Conductance [mmol * m-2 *s-1]

CO2 Net assimilation rate [µmol * cm-2 *s-1]

Water use efficiency of the photosynthesis

[µmol CO2 * mmol-1 H2O] 1 2.632 ± 0.782 58.100 ± 5.110 13.78 ± 1.27 5.24 ± 0.28

40 3.006 ± 0.383 70.077 ± 4.071 14.88 ± 1.64 4.95 ± 0.02 240 3.388 ± 0.384 75.453 ± 7.015 12.95 ± 1.91 3.82 ± 0.02 480 2.499 ± 0.307 56.671 ± 5.169 9.20 ± 1.69 3.68 ± 0.03


91 % for Spartina maritima) for the growth of the plants (Nielsen et al. 2001; Welsh 2000). Nitrogen fixation in the rhizosphere is regulated by photo-synthetically driven release of oxygen and fixed carbon by the plant roots and rhizomes. In addi-tion, plant-associated nitrogen fixation could sup-ply more than 37% of the nitrogen needed by the sulfate-reducing community. Sucrose stimulated nitrogen fixation and sulfate reduction signifi-cantly in root and rhizome of Spartina maritima (Nielsen et al. 2001). Elevated salinity led to an increased demand for proteins (nitrogen see Fig. 6) in Spartina townsendii, and in other species of this genus also for glycine betaine (nitrogen), proline (nitrogen) and dimethyl-sulphoniopropionate [(DMSP) sulfur], an osmoprotectant accumulated by the cordgrass at elevated salinity (Kocsis & Hanson 2000; Mulholland & Otte 2001). The in-creased demand for S and N can be one explana-tion for the reduction of the total carbohydrate pool in the leaves (Fig. 6). A higher demand for the proteins can lead also to a switch in the metabo-lism from carbohydrate storage to the synthesis of proteins. The increased uptake of S and N into the plant at elevated salinity points to a higher meta-bolic activity in the rhizosphere of Townsend´s Cordgrass. Spartina is a potential biomass crop (e.g. grown for fodder; Beale et al. 1999; Lieth 1999). This effective activation of N and S com-pounds in flooded soils by the mechanisms de-scribed above can be a major reason for the inva-sive growth of Spartina townsendii in the poor nu-trient conditions (especially for N and S) of the salt marsh ecosystem.

Development of cash crop halophytes The results presented in this paper inform

about the eco-physiological needs of the Spartina townsendii at high salinity. However, this study is only the first step for the development of cash crops or other usable plants from existing halo-phytes. It is of major importance for all further investigations that the potential benefits and risks of nonindigenous species are balanced carefully. This OCS offers a reliable base, but not more than the first step, for the selection of economically im-portant cash crop halophytes or other usable plants from existing halophytes. It is necessary to stepwise approach the propagation at promising sites under controlled conditions to establish po-

tentially useful cash crop halophytes (Koyro 2000; Isla et al. 1997).

The results of this study confirm again that the final selection of halophytic species suited for a particular climate and for a particular utilisation, a sustainable production system has to be designed in plantations at coastal areas or at inland sites. A cultivation of Spartina townsendii in foreign salt marshes needs to be accompanied by studies about the interaction between plant and habitat, especially the physiological range, ecological optimum and yield characteristics. Selecting the best adapted species or populations for wetland creation and restoration involves e.g. the matching of the planting stock with the site conditions (Igartua 1995; Rozema 1996). Among the other characters that determines survival and growth are phenology (early or late emergence of sprouts), responses to diseases and herbivory, responses to other chemical and physical factors such as fluctuation of soil salinity, waterlogging, nutrient enrichment, periods of drought and periods of frost (Rozema 1996). The importance of these investigations should not be underestimated. It is well known that e.g. salinity and waterlogging conditions are the major factors underlying salt marsh vegetation zonation and succession and that lower marsh species such as Salicornia and Spartina are well adapted to seawater salinity and waterlogged conditions.

Finally, it is important to balance the potential benefits and risks of nonindigenous species. A precondition for an introduction of nonindigenous species should be the sustainability as described by Ewel et al. (1999) with the eight key areas of consensus.

References

Angradi, T.R., S.M. Hagan & K.W. Able. 2001. Vegetation type and the intertidal macroinvertebrate fauna of a brackish marsh: Phragmites vs. Spartina. Wetlands 21: 75-92.

Ansede, J.H., R. Friedman & D.C. Yoch. 2001. Phylogenetic analysis of culturable dimethyl sulfide-producing bacteria from a Spartina-dominated salt marsh and estuarine water. Applied and Environmental Microbiology 67: 1210-1217.

Antolin, M. C. & M. Sánchez-Díaz. 1993. Effects of temporary drought on photosynthesis of alfalfa plants. Journal of Experimental Botany 44: 1341-1349.


Ashraf, M. & J.W. O´Leary. 1996. Effect of drought

stress on growth, water relations and gas exchange of two lines of sunflower differing in degree of salt tolerance. International Journal of Plant Sciences 157: 729-732.

Bagwell, C.E. & C.R. Lovell. 2000. Persistence of selected Spartina alterniflora rhizoplane diazotrophs exposed to natural and manipulated environmental variability. Applied and Environmental Microbiology 66: 4625-4633.

Beale, C.V., J.I.L. Morison & S.P. Long. 1999. Water use efficiency of C-4 perennial grasses in a temperate climate. Agricultural and Forest Meteorology 96:103-115.

Boer, B & D. Gliddon. 1998 Mapping of coastal ecosystems and halophytes (case study of Abu Dhabi, United Arab Emirates). Marine and Freshwater Research 49: 297-301.

Bradford, M.M. 1976. Rapid and sensitive method for the quantification of microgram of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254.

Burke, D.J., J.S. Weis & P. Weis. 2000. Release of metals by the leaves of the salt marsh grasses Spartina alterniflora and Phragmites australis. Estuarine Coastal and Shelf Science 51: 153-159.

Castillo, J.M., L. Fernandez-Baco, E.M. Castellanos, C. J. Luque, M.E. Figueroa & A. J. Davy. 2000. Lower limits of Spartina densiflora and S. maritima in a Mediterranean salt marsh determined by different ecophysiological tolerances. Journal of Ecology 88: 801-812.

Choukr-Allah, R. 1996. The potential of halophytes in the development and rehabilitation of arid and semi-arid-zones. pp. 3-13. In: R. Choukr-Allah, C.V. Malcolm & A. Hamdy (eds.) Halophytes and Biosaline Agriculture. Marcel Dekker Inc. New York, Basel, Hongkong.

Choukr-Allah, R & M.C. Harrouni. 1996. The potential use of halophytes under saline irrigation in Morocco. Symposium on the Conservation of Mangal Ecosystems. December 1996, Al Ain, United Arab Emirates15-17 (Abstract).

Connolly, R. M. 1999. Saltmarsh as habitat for fish and nektonic crustaceans: Challenges in sampling designs and methods. Australian Journal of Ecology 24: 422-430.

de Bakker, N.V.J., M.A. Hemminga & J. Van Soelen. 1999. The relationship between silicon availability, and growth and silicon concentration of the salt marsh halophyte Spartina anglica. Plant and Soil 215: 19-27.

Greenway, H. & R. Munns. 1980. Mechanisms of salt tolerance in nonhalophytes. Annual Review of Plant Physiology 31: 149-190.

Ensor, L. A., S. K. Stosz & R. M. Weiner. 1999. Expression of multiple insoluble complex polysaccharide degrading enzyme systems by a marine bacterium. Journal of Industrial Microbiology & Biotechnology 23: 123-126.

Epstein, E. 1972. Mineral Nutrition of Plants: Principles and Perspectives. J. Wiley & Sons, New York, London, Sydney, Toronto.

Eschrich, W. 1995. Funktionelle Pflanzenanatomie. Springer Verlag.

Ewel, J. J., D. J. O'Dowd, J. Bergelson, C. C. Daehler, C. M. D'Antonio, D. Gomez, D. R. Gordon, R. J. Hobbs, A. Holt, K.R.Hopper, C. E. Hughes, M. Lahart, R. R. B. Leakey, W. G. Lee, L. L. Loope, D. H. Lorence, S. M. Louda, A. E. Lugo, P. B. Mcevoy, D. M. Richardson & P. M. Vitousek. 1999. Deliberate introductions of species: Research needs — Benefits can be reaped, but risks are high. BioScience 49: 619-630.

Heller, A.A. & J.H. Weber. 1998. Seasonal study of speciation of mercury (II) and monomethylmercury in Spartina alterniflora from the Great Bay Estuary, NH. Science of the Total Environment 221: 181-188.

Hedge, P. & L.K. Kriwoken. 2000. Evidence for effects of Spartina anglica invasion on benthic macrofauna in Little Swanport estuary, Tasmania. Australian Journal of Ecology 25: 150-159.

Hines, M.E., R. S. Evans, B. R. S. Genthner, S. G. Willis, S. Friedman, J. N. Rooney-Varga & R. Devereux. 1999. Molecular phylogenetic and biogeochemical studies of sulfate-reducing bacteria in the rhizosphere of Spartina alterniflora. Applied and Environmental Microbiology 65: 2209-2216.

Igartua, E. 1995. Choice of selection environment for improving crop yields in saline areas. Theoretical and Applied Science 91: 1016-1021.

Isla, R., A. Royo & R. Aragues. 1997. Field screening of barley cultivars to soil salinity using a sprinkler and a drip irrigation. Plant and Soil 197: 105-117.

Kinzel, H. 1982. Pflanzenökologie und Mineralstoffwechsel. Eugen Ulmer Publication, Stuttgart.

Klap, V.A., P. Louchouarn, J.J. Boon, M.A. Hemminga & J. Van Soelen. 1999. Decomposition dynamics of six salt marsh halophytes as determined by cupric oxide oxidation and direct temperature-resolved mass spectrometry. Limnology and Oceanography 44: 1458-1476.

Kocsis, M.G. & A.D. Hanson. 2000. Biochemical evi-dence for two novel enzymes in the biosynthesis of


3-dimethylsulfoniopropionate in Spartina al-terniflora. Plant Physiology 123: 1153-1161.

Koyro, H.-W. & B. Huchzermeyer. 1997. The physiological response of Beta vulgaris ssp. maritima to sea water irrigation. pp. 29-50. In: H. Lieth, A. Hamdy & H.-W. Koyro (eds.) Water Management, Salinity and Pollution Control Towards Sustainable Irrigation in the Mediterranean Region. Salinity Problems and Halophyte Use. Tecnomack, Bari, Italy.

Koyro, H.-W. & B. Huchzermeyer. 1999a. Influence of high NaCl-salinity on growth, water and osmotic relations of the halophyte Beta vulgaris ssp. maritima. Development of a quick check. pp. 87-101. In: H. Lieth, M. Moschenko, M. Lohmann, H.-W. Koyro & A. Hamdy (eds.) Progress in Biometeorology. Volume 13. Backhuys Publishers, Leiden, NL.

Koyro, H.-W. & B. Huchzermeyer. 1999b. Salt and drought stress effects on metabolic regulation in maize. pp. 843-878. In: M. Pessarakli, (ed.) Handbook of Plant and Crop Stress. 2nd Ed. Marcel Dekker Inc., New York, USA.

Koyro, H.-W., L. Wegmann, H. Lehmann & H. Lieth. 1999. Adaptation of the mangrove Laguncularia racemosa to high NaCl salinity. pp .41-62. In: H. Lieth, M. Moschenko, M. Lohmann, H.-W. Koyro & A. Hamdy (eds.) Progress in Biometeorology. Volume 13. Backhuys Publishers, Leiden.

Koyro, H.-W. 2000. Effect of high NaCl-salinity on plant growth, leaf morphology and ion composition in leaf tissues of Beta vulgaris ssp. maritima. Journal of Applied Botany 74: 67-73.

Koyro, H.-W. 2002. Strategies of the halophyte Spartina townsendii to avoid salt injury. pp. 105-120. In: Halophyte Utilisation and Regional Sustainable Development of Agriculture. Xiaojing Liu & Mengyu Liu (eds.) Quxiang Press, Bejing.

La Peyre, M.K.G., J.B. Grace, E. Hahn & I.A. Mendelssohn. 2001. The importance of competition in regulating plant species abundance along a salinity gradient. Ecology 82: 62-69.

Lee, R. W. 1999. Oxidation of sulfide by Spartina alterniflora roots. Limnology and Oceanography 44: 1155-1159.

Lewis, M.A., D.E. Weber, R.S. Stanley & J.C. Moore. 2001. The relevance of rooted vascular plants as indicators of estuarine sediment quality. Archives of Environmental Contamination and Toxicology 40: 25-34.

Lichtenthaler, H.K. & A.R. Wellburn. 1983. Determination of the total carotenoids and chlorophyll a and b of leaf extracts in different solvents. Biochemical Transaction 603: 591-592.

Lieth, H. & A.A. Al Masoom (eds.). 1993. Towards the Rational Use of High Salinity Tolerant Plants. Dordrecht, Boston, London: Kluwer Academic Publisher.

Lieth, H. 1999. Development of crops and other useful plants from halophytes. pp. 1-18. In: H. Lieth, M. Moschenko, M. Lohmann, H.-W. Koyro & A. Hamdy (eds.) Halophytes Uses in different Climates, Ecological and Ecophysiological Studies. Backhuys Publishers, Leiden.

Lieth, H., M. Moschenko, M. Lohmann, H.-W. Koyro & A. Hamdy. 1999. Halophyte uses in different climates I. Ecological and ecophysiological studies. In: Progress in Biometeorology 13. Backhuys Publishers, Leiden.

Lin, Q., I.A. Mendelssohn, C.B. Henry, P.O. Roberts, M. M. Walsh, E.B. Overton & R.J. Portier. 1999. Effects of bioremediation agents on oil degradation in mineral and sandy salt marsh sediments. Environmental Technology 20: 825-837.

Lindau, C.W., R.D. DeLaune, A. Jugsujinda & E. Sajo. 1999. Response of Spartina alterniflora vegetation to oiling and burning of applied oil. Marine Pollution Bulletin 38: 1216-1220.

Lynch, J., G. Thiel & A. Läuchli. 1988. Effects of salinity on the extensibility and Ca availability in the expanding region of growing barley leaves. Botanica Acta 101: 355-361.

Lytle, J.S. & T.F. Lytle. 2001. Use of plants for toxicity assessment of estuarine ecosystems. Environmental Toxicology and Chemistry 20: 68-83.

Mahall, B.E. & R.B. Park. 1976. The ecotone between Spartina foliosa Trin. and Salicornia virginica L. in salt marshes of Northern San Francisco Bay. Soil water and salinity. Journal of Ecology 64: 793-809.

Marcum, K.B., S.J. Anderson & M.C. Engelke. 1998. Salt gland ion secretion: A salinity tolerance mechanism among five zoysiagrass species. Crop Science 38: 806-810.

Marcum, K.B.. 1999. Salinity tolerance mechanisms of grasses in the subfamily Chloridoideae. Crop Science 39: 1153-1160.

Marschner, H. 1995. Mineral Nutrition of Higher Plants. Academic Press, London - New York – San Diego –Boston – Sydney – Tokyo – Toronto.

Matamala, R. & B.G. Drake. 1999. The influence of atmospheric CO2 enrichment on plant-soil nitrogen interactions in a wetland plant community on the Chesapeake Bay. Plant and Soil 210: 93-101.

Miller, W.D., S.C. Neubauer & I.C. Anderson. 2001. Effects of sea level induced disturbances on high salt marsh metabolism. Estuaries 24: 357-367.

Mulholland, M.M. & M.L. Otte. 2001. The effects of ni-trogen supply and salinity on [DMSP], [glycine be-


taine] and [proline] concentrations in leaves of Spartina anglica. Aquatic Botany 71: 63-70.

Munns, R., P.A. Gardner, M.L. Tonnet & H.M. Rawson. 1989. Growth and development in NaCl-treated plants. II Do Na+ or Cl- concentrations in dividing or expanding tissues determine growth in barley. Australian Journal of Plant Physiology 15: 529-540.

Nielsen, L.B., K. Finster, D.T. Welsh, A. Donelly, R.A. Herbert, R. de Wit & B.A. Lomstein. 2001. Sulphate reduction and nitrogen fixation rates associated with roots, rhizomes and sediments from Zostera noltii and Spartina maritima meadows. Environmental Microbiology 3: 63-71.

Nieva, F.J.J., E.M. Castellanos, M.E. Figueroa & J.W. Gill. 1999. Gas exchange and chlorophyll fluorescence of C3 and C4 saltmarsh species. Photosynthetica 36: 397-406.

Norris, A.R. & C.T. Hackney. 1999. Silica content of a mesohaline tidal marsh in North Carolina. Estuarine Coastal and Shelf Science 49: 597-605.

Nyman, J.A. 1999. Effect of crude oil and chemical additives on metabolic activity of mixed microbial populations in fresh marsh soils. Microbial Ecology 37: 152-162.

Padinha, C., R. Santos & M.T. Brown. 2000. Evaluating environmental contamination in Ria Formosa (Portugal) using stress indexes of Spartina maritima. Marine Environmental Research 49: 67-78.

Pasternak, D. 1990. Fodder Production with Saline Water. The Institute for Applied Research, Ben Gurion University of the Negev. Project report BGUN-ARI-35-90. Beer-Sheva/Israel.

Patra, M. & A. Sharma. 2000. Mercury toxicity in plants. Botanical Review 66: 379-422.

Pearcy, R.W. & S.L. Ustin. 1984. Effects of salinity on growth and photosynthesis of three California tidal marsh species. Oecologia 62: 68-73.

Pezeshki, S.R., H.S. Choi & R.D. DeLaune. 1993. Poupulation differentiation in Spartina patens: Water potential components and bulk modulus of elasticity. Biologia Plantarum 35: 43-51.

Pezeshki, S.R. & R.D. DeLaune. 1997. Population diffrentiation in Spartina patens: Responses of photosynthesis and biomass partitioning to elevated salinity. Botanical Bulletin of Academia Sinica 38: 115-120.

Pezeshki, S.R., R.D. DeLaune & A. Jugsujinda. 2001. The effects of crude oil and the effectiveness of cleaner application following oiling on US Gulf of Mexico coastal marsh plants. Environmental Pollution 112: 483-489.

Qin, P., M. Xie & Y.S. Jiang. 1998. Spartina green food ecological engineering. Ecological Engineering 11: 147-156.

Riera, P., L.J. Stal, J. Nieuwenhuize, P. Richard, G. Blanchard & F. Gentil. 1999. Determination of food sources for benthic invertebrates in a salt marsh (Aiguillon Bay, France) by carbon and nitrogen stable isotopes: importance of locally produced sources. Marine Ecology Progress Series 187: 301-307.

Robinson, D.G., U. Ehlers, R. Herken, B. Herrmann, F. Mayer & F.-W. Schürmann. 1985. Präpara-tionsmethodik in der Elektronenmikroskopie. Springer Verlag.

Rozema, J. & J. van Diggelen. 1991. A comparative study of growth and photosynthesis of four halophytes in response to salinity. Acta Oecologia 12: 673-681.

Rozema, J. 1996. Biology of halophytes. pp. 17-30. In: R. Choukr-Allah, C. V. Malcolm & A. Hamdy (eds.) Halophytes and Biosaline Agriculture. New York, Basel, Hong Kong: Marcel Dekker.

SanLeon, D.G., J. Izco & J.M. Sanchez. 1999. Spartina patens as a weed in Galician saltmarshes (NW Iberian Peninsula). Hydrobiologia 415: 213-222.

Schimper, A.F.W. 1891. Pflanzengeographie auf Physiologischer Grundlag. Fischer Publication, Jena.

Shannon, M.C. & C.M. Grieve. 1999. Tolerance of vegetable crops to salinity. Scientia Horticulturae 78: 5-38.

Simas, T., J.P. Nunes & J.G. Ferreira. 2001. Effects of global climate change on coastal salt marshes. Ecological Modelling 139: 1-15.

Smith, D.L. & C.E. Proffitt. 1999. The effects of crude oil and remediation burning on three clones of smooth cordgrass (Spartina alterniflora Loisel.). Estuaries 22: 616-623.

Sutherland, G.K. & A. Eastwood. 1916. The physio-logical anatomy of Spartina Townsendii. Annals of Botany 30: 333-351.

Von Willert, D.J., R. Matyssek & W. Herppich. 1995. Experimentelle Pflanzenökologie. Georg Thieme, Stuttgart, New York.

Waide, R.B., M.R. Willig, C.F. Steiner, G. Mittelbach, L. Gough, S.I. Dodson, G.P. Juday & R. Parmenter. 1999. The relationship between productivity and species richness. Annual Review of Ecology and Systematics 30: 257-300.

Weinstein, M.P., S.Y. Litvin, K.L. Bosley, C.M. Fuller & S.C. Wainright. 2000. The role of tidal salt marsh as an energy source for marine transient and resident finfishes: A stable isotope approach. Transactions of the American Fisheries Society 129: 797-810.


Windham, L., J.S. Weis & P. Weis. 2001. Lead uptake, distribution and effects in two dominant salt marsh macrophytes, Spartina alterniflora (cordgrass) and Phragmites australis (common reed). Marine Pollution Bulletin 42: 811-816.

Warne, T.R., L.G. Hickok, C.E. Sams & D.L. Vogelien. 1999. Sodium/potassium selectivity and pleiotropy in stl2, a highly salt-tolerant mutation of Ceratopteris richardii. Plant Cell and Environment 22: 1027-1034.

Weber, E. & C.M. D'Antonio. 1999. Germination and growth responses of hybridizing Carpobrotus species (Aizoaceae) from coastal California to soil salinity. American Journal of Botany 86: 1257-1263.

Welsh, D.T. 2000. Nitrogen fixation in seagrass mead-ows: Regulation, plant-bacteria interactions and significance to primary productivity. Ecology Letters 3: 58-71.

Wild, A. 1999. Pflanzenphysiologische Versuche. Biologische Arbeitsbücher Vol. 56. Verlag Quelle und Meyer, Wiebelsheim.

Winter, U., G.O. Kirst, V. Grabowski, U. Heinemann, I. Plettner & S. Wiese. 1999. Salinity tolerance in Nitellopsis obtusa. Australian Journal of Botany 47: 337-346.

Wyn Jones, R.G. & A. Pollard. 1983. Proteins, enzymes and inorganic ions. pp. 528-555. In: A. Läuchli & R.L. Bieleski (eds.) Inorganic Plant Nutrition. Encyclopedia of Plant Physiology. 15b, Springer Verlag,

Ziska, L.H., J.R. Seemann & T.M. DeJong. 1990. Salinity induced limitations on photosynthesis in Prunus salicina, a deciduous tree species. Plant Physiology 93: 864-870.

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Ecophysiological needs of the potential biomass crop ... · Ecophysiological needs of the potential biomass crop Spartina ... (control) to 100% seawater ... in a “quick check”