Halophyte Spartina Alterniflora Research Articles

Spatio-temporal pattern of cross-continental invasion: Evidence of climatic niche shift and predicted range expansion provide management insights for smooth cordgrass

Introduction and spread of the halophyte Spartina alterniflora is one of the largest continental-scale biological invasion events in Asia and the Americas. Rapid globalization and broad environmental tolerance of the species increase the chance of novel invasions. Thus, we aimed to identify susceptible regions to inform prevention and control activities.A comprehensive global occurrence dataset and corresponding bioclimatic variables were used to characterize the species' climatic niche and predict current and future potential distributions. Conservatism of climatic niche between native and non-native ranges was tested, and climatic niche dynamics were analysed at spatial and temporal scales. The ensemble of eight species distribution models and eight climate change models was used to map the potential distribution of S. alterniflora under current and future climate conditions. We investigated the susceptibility of threatened ecosystems like mangroves and protected areas to S. alterniflora invasion to better inform management decisions.Our study revealed wide climatic tolerance and significant niche expansion of the species from humid regions of its native range to dry and arid environments of its non-native range with a very short lag period. With a marginal increase in temperature and precipitation in the future, range expansion was predicted towards higher latitude and more inland areas. The mangroves area, salt marshes, and protected areas that are at risk of ongoing and future invasions were identified. Given the invasion potential of S. alterniflora, the areas identified as climatically susceptible for the species’ establishment, both in current and future climates, should be prioritized for management actions.

Photosynthetic and transcriptomic responses of two C4 grass species with different NaCl tolerance

This report reveals the effects of salt on the photosynthetic electron transport and transcriptome of the glycophyte Setaria viridis (S. viridis) and its salt-tolerant close relative halophyte Spartina alterniflora (S. alterniflora). S. viridis was unable to survive exposed to sodium chloride (NaCl) levels higher than 100 mM, in contrast, S. alterniflora could tolerate NaCl up to 550 mM, with negligible effect on gas exchange related parameters and conductance of electrons transport chain (gETC). Under salt, the prompt fluorescence (OJIP-curves) exhibits an increase in the O- and J-steps in S. viridis and much less for S. alterniflora. Flowing NaCl stress, a dramatic decline in the photosystem II (PSII) primary photochemistry was observed for S. viridis, as reflected by the drastic drop in Fv/Fm, Fv/Fo and ΦPSII; however, no substantial change was recorded for these parameters in S. alterniflora. Interestingly, we found an increase in the primary PSII photochemistry (ΦPSII) for S. alterniflora with increasing either NaCl concentration or NaCl treatment duration. The NPQ magnitude was strongly enhanced for S. viridis even at a low NaCl (50 mM); however, it remains unchangeable or slightly increased for S. alterniflora at NaCl levels above 400 mM. After NaCl treatment, we found an increase in both the proportion of oxidized P700 and the amount of active P700 in S. viridis and almost no change for S. alterniflora. Under salt, the net photosynthetic rate (A) and stomatal conductance (gs) measurements demonstrate that A decreases earlier in S. viridis, even after one week exposure to only 50 mM NaCl; in contrast, in S. alterniflora, the effect of NaCl on A and gs was minor even after exposure for two weeks to high NaCl levels. For S. viridis exposed to 50 mM NaCl for 12 d, carbon dioxide (CO2) at a concentration of 2000 μL L−1 could not fully restore A to the control (Ctrl) level. Conversely, in S. alterniflora, high CO2 can fully restore A for all NaCl treatments except at 550 mM. RNA-seq data shows a major impact of NaCl on metabolic pathways in S. viridis and we found a number of transcription factors potentially related to NaCl responses. For S. alterniflora, no major changes in the transcriptomic levels were recorded under NaCl stress. To confirm our data analysis of RNA-seq, we performed quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis for randomly selected four genes for each species (8 genes in total) and we found that our results (up- and/or down-regulated genes) are fully consistent and match well our RNA-seq data. Overall, this study showed drastically different photosynthetic and transcriptomic responses of a salt-tolerant C4 grass species and one salt-sensitive C4 grass species to NaCl stress, which suggests that S. alterniflora could be used as a promising model species to study salt tolerance in C4 or monocot species.

Contrasting Responses of Plastid Terminal Oxidase Activity Under Salt Stress in Two C4 Species With Different Salt Tolerance.

The present study reveals contrasting responses of photosynthesis to salt stress in two C4 species: a glycophyte Setaria viridis (SV) and a halophyte Spartina alterniflora (SA). Specifically, the effect of short-term salt stress treatment on the photosynthetic CO2 uptake and electron transport were investigated in SV and its salt-tolerant close relative SA. In this experiment, at the beginning, plants were grown in soil then were exposed to salt stress under hydroponic conditions for two weeks. SV demonstrated a much higher susceptibility to salt stress than SA; while, SV was incapable to survive subjected to about 100 mM, SA can tolerate salt concentrations up to 550 mM with slight effect on photosynthetic CO2 uptake rates and electrons transport chain conductance (gETC). Regardless the oxygen concentration used, our results show an enhancement in the P700 oxidation with increasing O2 concentration for SV following NaCl treatment and almost no change for SA. We also observed an activation of the cyclic NDH-dependent pathway in SV by about 2.36 times upon exposure to 50 mM NaCl for 12 days (d); however, its activity in SA drops by about 25% compared to the control without salt treatment. Using PTOX inhibitor (n-PG) and that of the Qo-binding site of Cytb6/f (DBMIB), at two O2 levels (2 and 21%), to restrict electrons flow towards PSI, we successfully revealed the presence of a possible PTOX activity under salt stress for SA but not for SV. However, by q-PCR and western-blot analysis, we showed an increase in PTOX amount by about 3–4 times for SA under salt stress but not or very less for SV. Overall, this study provides strong proof for the existence of PTOX as an alternative electron pathway in C4 species (SA), which might play more than a photoprotective role under salt stress.

Potential use of Spartina alterniflora as forage for dairy cattle

This study evaluates the potential use of halophyte Spartina alterniflora (SA) as forage for ruminants by detecting the effect of plant maturity on organic and mineral compositions as well as the effect of increasing dietary inclusion of S. alterniflora on rumen fermentation in dairy cattle. Crude protein (CP), ash content, and in vitro dry matter (DM) digestion of S. alterniflora exhibited a negative correlation with increasing maturity; however, biomass, DM, neutral detergent fiber (NDF) content, and acid detergent fiber (ADF) content showed a positive correlation. Mineral composition drastically changed with increasing plant maturity. Compared with conventional diets, S. alterniflora mixed diets showed higher ash, NDF, ADF, ether extract, and Na, Ca, and P contents, and similar organic matter (OM), CP, and gross energy contents. DM and water intake of dairy cattle linearly increased from 0% to 25% SA in the diet. The mean values of the concentration of total volatile fatty acids (TVFA); the molar percentage of acetate, propionate, butyrate, and isobutyrate; and the acetate-to-propionate ratio did not significantly change at 12:00h with increasing inclusion level of S. alterniflora in the diet. The initial findings obtained from the feed and rumen fermentation analysis of dairy cattle demonstrate that S. alterniflora can be utilized as potential forage for dairy cattle at less than 25% of the animal’s diet. Overall, S. alterniflora could be provided with the potential to improve the sustainability of ruminant production as alternative feeds to replace partially common roughages, alleviate feeding cost and partially control the rapid spread of the invasive plant in salt marsh of China.

Ectopic expression of ADP ribosylation factor 1 (SaARF1) from smooth cordgrass (Spartina alterniflora Loisel) confers drought and salt tolerance in transgenic rice and Arabidopsis

Salinity and drought are two very important abiotic stressors that negatively impact the growth and yield of all sensitive crop plants. Genes from halophytes have been shown to be useful to engineer crop plants that can survive under adverse soil and water conditions. The present report establishes, for the first time, the physiological role of a class one ADP ribosylation factor gene (SaARF1) from the halophyte Spartina alterniflora (smooth cordgrass) in imparting salinity and drought stress tolerance when expressed in both monocot (rice) and dicot (Arabidopsis) systems. The Arabidopsis and rice plants overexpressing ARF1 are many-fold more tolerant to salt and drought than wild-type (WT) plants. The transgenics exhibited improved growth and productivity relative to WT through tissue tolerance by maintaining higher relative water content and membrane stability, and higher photosynthetic yield by retaining higher chlorophyll concentration and fluorescence under stress conditions compared to WT. These findings indicated that genes from halophyte resources can be useful to engineer and improve salt and drought stress tolerance in both monocot and dicot plants.

Overexpression of an adenosine diphosphate-ribosylation factor gene from the halophytic grass Spartina alterniflora confers salinity and drought tolerance in transgenic Arabidopsis

Adenosine diphosphate-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that play an important role in intracellular protein trafficking necessary for undertaking multiple physiological functions in plant growth and developmental processes. However, little is known about the mechanism of ARF functioning at the molecular level, as well as its involvement in abiotic stress tolerance. In this study, we demonstrated the direct involvement of an ARF gene SaARF from a grass halophyte Spartina alterniflora in abiotic stress adaptation for the first time. SaARF, which encodes a protein with predicted molecular mass of 21 kDa, revealed highest identity with ARF of Oryza sativa. The SaARF gene is transcriptionally regulated by salt, drought, cold, and ABA in the leaves and roots of S. alterniflora. Arabidopsis plants overexpressing SaARF showed improved seed germination and survival of seedlings under salinity stress. Similarly, SaARF transgenic Arabidopsis plants were more tolerant to drought stress, compared to wild-type plants, by maintaining chlorophyll synthesis, increasing osmolyte synthesis, and stabilizing membrane integrity. Oxidative damage due to moisture stress in transgenic Arabidopsis was also reduced possibly by activating antioxidant genes, AtSOD1 and AtCAT. Our results suggest that enhanced drought and salinity tolerance conferred by the SaARF gene may be due to its role in mediating multiple abiotic stress tolerance mechanisms.

Ammonium nutrition in the halophyte Spartina alterniflora under salt stress: evidence for a priming effect of ammonium?

The effects of salt stress on the salt marsh halophyte Spartina alterniflora have been well documented. However, plant responses to combined salinity and ammonium toxicity and the underlying mechanisms are relatively unknown. The aim of the present investigation was to study the effects of both salinity (0, 200 and 500 mM NaCl) and nitrogen form (NO3 −, NH4 + or NH4NO3) on S. alterniflora. Plants were cultivated in sandy soil under greenhouse conditions for 3 months. At harvest, growth parameters were measured and leaf samples were analysed for oxidative stress parameters (malondialdehyde, MDA; electrolyte leakage, EL; and hydrogen peroxide, H2O2 concentration) and the activity of antioxidant enzymes (glutathione reductase, GR; superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX and Guaiacol peroxidase, GPX). In the absence of NaCl, plant growth rate was the highest in the medium containing both nitrogen forms, and the lowest in the medium containing only nitrate. Irrespective of the nitrogen form, plant growth was generally higher at 200 mM NaCl than without salinity. Ammonium-fed plants showed better growth than nitrate-fed plants under high salinity. In the absence of salinity, ammonium-fed plants showed higher SOD, APX, GR, CAT, and GPX activities than nitrate-fed ones. The antioxidant enzymes exhibited higher activity in saline-treated plants. The considerable advantage of NH4 + nutrition to S. alterniflora under saline conditions was associated with high antioxidant enzyme activities, together with low MDA content, EL, and H2O2 concentration. These data clearly demonstrate that NH4 + is more favourable for the growth of S. alterniflora under high salinity than NO3 −. It is suggested that NH4 + nutrition improves the plant’s capacity to limit oxidative damage by stimulating the activities of the major antioxidant enzymes.

Arabidopsis plants constitutively overexpressing a myo-inositol 1-phosphate synthase gene (SaINO1) from the halophyte smooth cordgrass exhibits enhanced level of tolerance to salt stress

Salinity is one of the most important environmental constraints limiting agricultural productivity. Considering the importance of the accumulation of osmolytes, myo-inositol in particular, in halophytic plant's adaptive response to salinity, an effort was made to overexpress the SaINO1 gene from the grass halophyte Spartina alterniflora encoding myo-inositol 1-phosphate synthase (MIPS) in Arabidopsis thaliana. We demonstrated that SaINO1 is a stress-responsive gene and its constitutive over expression in Arabidopsis provides significantly improved tolerance to salt stress during germination and seedling growth and development. The transgenics retained more chlorophyll and carotenoid by protecting the photosystem II. The low level of stress-induced cellular damage in the transgenics was clearly evident by lower accumulation of proline in comparison to WT. Our results indicated that possible overaccumulation of MIPS enzyme in the cytosol protected the transgenic Arabidopsis plants overexpressing SaINO1 from the toxic effect of Na(+) under salt stress by reducing cellular damage and chlorophyll loss.

Effect of NaCl on growth and Cd accumulation of halophyte Spartina alterniflora under CdCl2 stress

A study quantifying the effect of NaCl on growth and Cd accumulation of Spartina alterniflora subjected to Cd stress was conducted. Seedlings were cultivated in the presence of 1 or 3mM Cd alone, or combined with NaCl (50 or 100mM). The results showed that NaCl magnified the phytotoxicity of moderate Cd stress (1mM Cd) on plants due to reduced levels of plant biomass, plant height, and chlorophyll a+b, while no synergistic effects were recorded under severe Cd stress (3mM Cd). Proline and Ca2+ accumulated along with additional NaCl under moderate Cd stress, instead of reduced or unchanged levels under severe Cd stress owing to different adoption strategies caused by NaCl under different Cd stresses. NaCl reduced the oxidative stress in Cd-treated plants through increasing levels of antioxidative enzymes (catalase (CAT) and peroxidase (POD)) under moderate Cd stress. With NaCl addition, Cd2+ contents in S. alterniflora increased and reduced under moderate and severe Cd stress, respectively. However, total Cd2+ amounts increased with increasing NaCl concentration due to biological dilution. NaCl improved the increase of Cd2+ translocation factor (TF) under moderate Cd stress, indicating that NaCl might improve Cd2+ uptake and translocation from roots to shoots, and enhance the phytoextraction of S. alterniflora on Cd; while phytostabilization of Cd under severe Cd stress may be possible due to the reduced TF. Thus, NaCl alleviated phytotoxicity caused by Cd stress through improved management of osmotic solutes and oxidative status, and affected Cd accumulations in S. alterniflora differently under moderate and severe Cd stresses.

Overexpression of a nascent polypeptide associated complex gene (SaβNAC) of Spartina alterniflora improves tolerance to salinity and drought in transgenic Arabidopsis

Salinity and drought are the most important environmental constraints limiting crop growth and productivity. Here, we have characterized a gene ‘SaβNAC’ encoding the β subunit of nascent polypeptide associated complex from a halophyte Spartina alterniflora and investigated its role toward abiotic stress regulation. Expression of SaβNAC was differentially regulated by abiotic stresses, including salinity, drought, cold, and ABA in leaves and roots of S. alterniflora. Constitutive over-expression of SaβNAC in Arabidopsis exhibited normal growth under non-stress conditions but enhanced tolerance to salt and drought stresses. Transgenic SaβNAC Arabidopsis retained more chlorophyll, proline, and showed improved ionic homeostasis with less damage under stress conditions compared to WT plants. This is a first report to demonstrate the involvement of βNAC in imparting abiotic stress tolerance which might be due to protection of the newly synthesized polypeptides involved in various stress tolerance mechanisms from abiotic stress induced damage and inhibition of cell death in plant.

Interactive effects of salt and alkali stresses on seed germination, germination recovery, and seedling growth of a halophyte Spartina alterniflora (Poaceae)

Soil salinization and alkalinization frequently co-occur in nature, but there is little information on the interactive effects of salt and alkali stresses on plants. Seed germination and early seedling growth are crucial stages for plant establishment. We investigated the interactive effects of salt and alkali stresses on seed germination, germination recovery and seedling growth of a halophyte Spartina alterniflora. Seed germination percentage was not significantly reduced at low salinity (≤200mM) at pH 6.63–9.95, but decreased with increased salinity and pH. Ungerminated seeds germinated well after transfer to distilled water from treatment solutions, indicating that seeds can remain viable in high salt–alkaline habits. Shoot growth was stimulated at low salinity and pH, but decreased with increased salinity and pH. Radicle elongation decreased sharply with increased salinity and pH, and was significantly inhibited when pH≥9.0, indicating that the radicles are very sensitive to salt–alkaline stress. The deleterious effects of salinity or high pH alone were less than when combined. A reciprocal enhancement of salt and alkali stresses is a characteristic feature for salt–alkaline stress. Stepwise regression analysis indicates that salinity is the dominant factor, while pH and buffer capacity are secondary for salt–alkaline mixed stress.

Interactive effects of various salt and alkali stresses on growth, organic solutes, and cation accumulation in a halophyte Spartina alterniflora (Poaceae)

Soil salinization and alkalinization frequently co-occur in nature, but very few studies focus on the interactive effects of various salt and alkali stresses on plants. A study quantifying the effects of various salt and alkali stresses on growth, organic solutes, and cation accumulation in a halophyte Spartina alterniflora was conducted. The experiment consisted of six levels of salinity (100, 200, 300, 400, 500, and 600 mM) in each of six pH levels: A (pH 7.1 ± 0.02), B (pH 8.1 ± 0.17), C (pH 8.8 ± 0.14), D (pH 9.8 ± 0.14), E (pH 10.4 ± 0.08), and F (pH 10.8 ± 0.10). Survival rate, relative growth rate (RGR), tiller rate, rhizome number, leaf chlorophyll content, root activity, electrolyte leakage rate (ELR), and content of proline, soluble carbohydrates, Na +, and K + were determined. The results showed that S. alterniflora was capable of surviving all treatments under low pH (pH ≤8.30) regardless of the levels of salinity. However, high pH in conjunction with salinity sharply reduced survival rate. As expected, the interactive effects of salinity and high pH on RGR, tiller rate, rhizome number, leaf chlorophyll content, and relative root activity were additive. Moreover, ELR and Na +/K + ratio increased with increasing salinity and pH. The content of Na + increased, while K + decreased with increasing salinity and pH, suggesting competitive inhibition between absorptions of Na + and K +. The content of proline and soluble carbohydrates both increased with increasing salinity and pH, suggesting that alkali stress may also cause accumulation of these organic solutes. The deleterious effects of high pH or salinity alone were significantly less than those of combined high pH and salinity. For salt–alkali mixed stress, it is concluded that reciprocal enhancement between salt and alkali stress is a characteristic feature. We also constructed models to elucidate this feature of mixed salt–alkali stress. Multiple linear regressions found strong correlations between all physiological indices and the three stress factors. However, the effects of the three stress factors on the physiological indices were different in magnitude. Overall, salinity is the dominant factor, while pH and buffer capacity are secondary. The combined action of salinity, pH, and buffer capacity should be considered when evaluating the strengths of salt–alkali mixed stress.

Primary responses to salt stress in a halophyte, smooth cordgrass (Spartina alterniflora Loisel.)

The response of a grass halophyte Spartina alterniflora at early stages of salt stress was investigated through generation and systematic analysis of expressed sequence tags (ESTs) from both leaf and root tissues. Random EST sequencing produced 1,227 quality ESTs, which were clustered into 127 contigs, and 368 were singletons. Of the 495 unigenes, 27% represented genes for stress response. Comparison of the 368 singletons against the Oryza sativa gene index showed that >85% of these genes had similarity with the rice unigenes. Moreover, the phylogenetic analysis of an EST similar to myo-inositol 1-phosphate synthase of Spartina and some selected grasses and halophytes showed closeness of Spartina with maize and rice. Transcript abundance analysis involving eight known genes of various metabolic pathways and nine transcription factor genes showed temporal and tissue-dependent variation in expression under salinity. Reverse northern analysis of a few selected unknown and ribosomal genes exhibited much higher abundance of transcripts in response to salt stress. The results provide evidence that, in addition to several unknown genes discovered in this study, genes involved in ion transport, osmolyte production, and house-keeping functions may play an important role in the primary responses to salt stress in this grass halophyte.

Threshhold for recovery in the marsh halophyte Spartina alterniflora grown under the combined effects of salinity and soil drying

A study quantifying the physiological threshhold at which Spartina alterniflora plants are able to tolerate the interactive effects of salinity and soil drying was conducted in a climate controlled greenhouse. The experiment consisted of two levels of salinity (3-5 ppt, L and 35-38 ppt, H) as well as four dynamic water levels: flooding (water level maintained 3-5 cm above the soil surface at high tide and 10 cm below the soil surface at low tide for entire study duration, F), 8-day drought (water level maintained at least 20 cm below the soil surface at high tide for 8 days then flooded, 8 days), 16-day drought (water level maintained at least 20 cm below the soil surface at high tide for 16 days then flooded, 16 days), and 24-day drought (water level maintained at least 20 cm below the soil surface at high tide for 24 days then flooded, 24 days). Plant gas exchange and growth responses were measured along with soil conditions of redox potential and water potential. Significant decreases were seen in plant gas exchange and growth in response to increases in salinity and soil drying. Survival was 100% for all flooded treatments while increased salinity combined with soil drying decreased survival to 86% in both low salt/24-day drought plants (LD24) and high salt/16-day drought plants (HD16). The lowest survival rate was seen in the high salt/24-day drought treatment (HD24) at 29%. Therefore, it appears that the critical time for recovery from the combined effects of increased salinity and soil drying may greatly diminish after two weeks from the onset of stress conditions. Consequently, if salinity continues to increase along the MRDP, marshes dominated by S. alterniflora may be more susceptible to short-term drought and likewise large-scale marsh browning.

CDNA-AFLP analysis reveals differential gene expression in response to salt stress in a halophyte Spartina alterniflora Loisel

Salinity impedes the growth and productivity of various crop species. Genome-wide expression analysis has identified a number of salinity induced up- and/or down-regulated genes that are also common during dehydration and temperature stress in the glycophyte like Arabidopsis. To date, Spartina alterniflora L. (smooth cordgrass) has not been exploited to understand the molecular basis of its unusually high level of salt tolerance. We used cDNA-AFLP as an RNA imaging technique to identify transcripts that are strongly accumulated and are induced de novo in response to salinity stress. Among 213 isolated, we have identified 28 cDNAs that belong to different groups of genes involved in ion transport and compartmentalization, cell division, metabolism, and protein synthesis, etc. At different stages of salt stress the expression patterns of 14 such genes through RT-PCR were analyzed, and their direct and indirect relationship with salinity tolerance mechanism is discussed.

Chronic Low Level Hydrocarbon Amendments Stimulate Plant Growth and Microbial Activity in Salt-Marsh Microcosms

(1) Salt-marsh microcosms containing the halophyte Spartina alterniflora were maintained in a greenhouse for 2 years. Microbial and plant activity were monitored monthly in microcosms given daily dosages of a hydrocarbon mixture containing hexane, heptane, octane, nonane, benzene, toluene, ethylbenzene and xylene that comprises about 15% of crude oil. This mixture was added at 3 33 (1 x treatment) and 33 3 g C m-2 dayI (10 x treatment) for 10 months. (2) Aboveand below-ground plant biomass and total respiration rates were significantly increased by the 1 x hydrocarbon additions, and the differences persisted for at least 7 months after the hydrocarbon additions ended. Growth, total sediment organic matter and macro-organic matter were significantly reduced in the 10 x treatment. (3) Soil microbial activities were monitored using non-invasive gas-exchange techniques. CO2 production, methanogenesis, N2 fixation and denitrification were stimulated by the 1 x hydrocarbon treatment but inhibited by the 10 x treatment. (4) Unlike the linear dose-response relationship assumed in many toxicological studies, both plant and microbial activities showed a non-linear response to hydrocarbon concentration in this chronic exposure study, with stimulation of activity at the 1 x level and inhibition at 10 x . The stimulation of microbial activity by the 1 x treatment suggests that the microbes were carbon-limited, while the enhanced plant growth seen at 1 x may have resulted from increased N2 fixation and/or nitrogen mineralization.

Factors affecting nitrogenase activity (acetylene-reducing activity) associated with excised roots of the emergent halophyte Spartina alterniflora Loisel

In other studies, excised roots of some marine and aquatic angiosperms have been found to exhibit high nitrogenase activity (acetylene-reducing activity) during long term (24 h or more), but not during short term (2 h) incubations with acetylene. In this study, the effects of oxygen, acetylene, and washing of roots on ARA (acetylene-reducing activity) associated with excised roots of Spartina alterniflora Loisel. were examined. ARA occurred only after a lag period of 12–24 h. This ARA was both “oxygen sensitive” (suppressed by high pO 2) and “oxygen-dependent”, reaching a maximum at low pO 2. When excised roots exhibiting ARA were shaken in air, and then re-exposed to C 2H 2, ARA ceased for 8 h and then resumend, suggesting that the initial lag period may result from exposure of roots to high pO 2 during sampling. However, handling of samples in the absence of O 2 failed to eliminate the lag. High ARA was observed for some rhizosphere soil samples lacking roots, and also occurred after a lag phase. The apparent K m for C 2H 2 reduction decreased with increasing lenght of exposure to C 2H 2. Some inhibitory effects of C 2H 2 on ARA in the excised root systems were noted. It is concluded that, while the maximum ARA observed when roots are incubated for 1–2 days under 5% initial O 2 and 5% C 2H 2 may give an indication of the “potential for nitrogenase activity”, this is not a reliable indication of the in situ nitrogenase activity because of uncertainty concerning the cause of the lag phase, and because the in situ pO 2 may differ from that in the excised root systems.