The major Nod factor of Bradyrhizobium japonicum promotes early growth of soybean and corn

Greenhouse experiments were conducted to evaluate the effect of Nod factor Nod Bj-V (C18:1, MeFuc) of Badyrhizobium japonicum on the growth of soybean and corn. Three-day-old seedlings of soybean and corn were grown in hydroponic solutions containing four concentrations (0, 10(-7), 10(-9) or 10(-11) M) of Nod factor. After 7 d of treatment, Nod factor enhanced soybean and corn biomass. Nod factor elicited profound effects on root growth resulting in 34-44% longer roots in soybean. More detailed analyses of the roots, using a scanner based image analysis system, revealed that Nod factor increased the total length, projected area and surface area of the roots and decreased the diameter of soybean roots, while it increased the total length of corn roots. Stem injection of soybean plants with 10(-7) M Nod factor resulted in increased dry matter accumulation. These results suggest that Nod factor, besides mediating early stages of nodulation, has more general plant growth-promoting effects.     

Nod factor induces soybean resistance to powdery mildew.

Plants possess highly sensitive perception systems by which microbial signal molecules are recognized. In the Bradyrhizobium-soybean (Glycine max (L.) Merr.) symbiosis, recognition is initiated through exchange of signal molecules, generally flavonoids from soybean and lipo-chitooligosaccharides (Nod factors) from the microsymbiont. Application of the Nod factor Nod Bj-V (C18:1, MeFuc) induced soybean resistance to powdery mildew caused by Microsphaera diffusa. Addition of Nod factor (concentrations ranging from 10(-6) to 10(-10) M) to soybean root systems led to reductions in disease incidence. The lowest disease incidence was caused by Nod factor treatment at 10(-6) M. The effect of Nod factor application on fungal growth and development was measured at 4, 12, 48, and 96 h after inoculation. Colony diameter and number of germ tubes per conidium were decreased by 10(-6) M Nod factor. Phenylalanine ammonia lyase (PAL, EC.4.3.1.1.) is the first enzyme of the phenyl propanoid pathway, and is commonly activated as part of plant responses to disease. Treatment of soybean seedlings with Nod factor, through stem wounds, induced PAL activity; the most rapid increase followed treatment with 10(-6) M Nod factor. These data show that soybean plants are able to detect root applied LCO and respond by increased disease resistance.     

Nod factor enhances calcium uptake by soybean

Inoculation with rhizobia or application of Nod factors (lipo-chitooligosaccharides, LCOs) causes transient increases in cytosolic calcium concentration in root hairs of legume plants. We conducted experiments to evaluate whether application of LCO and inoculation with rhizobia improved (45)CaCl(2) uptake into soybean (Glycine max [L.] Merr.) leaves. Roots of soybean seedlings with one developing trifoliolate were immersed in Murashige and Skoog (MS) basal liquid medium containing treatment solutions and (45)CaCl(2), and the plants were incubated under continuous light. After 24 h, leaf samples were taken, and their radioactivity levels were determined. Addition of NodBj-V (C18:1 MeFuc) at a concentration of 10(-7) M increased (45)Ca(2+) uptake. Inoculation with genistein-induced Bradyrhizobium japonicum strain 532C and USDA3 also increased (45)Ca(2+) uptake; whereas, inoculation with strain Bj-168, a nodC-mutant incapable of producing LCO, did not. Rhizobia that do not normally nodulate soybean, i.e. Rhizobium leguminosarum, and Sinorhizobium meliloti did not affect calcium uptake, nor did the tetramer or pentamer of chitosan, or lumichrome. Surprisingly, Rhizobium sp. NGR234, which can nodulate some types of soybean, although without effective N(2)-fixation, also did not affect calcium uptake. This work suggests that the rhizobial symbiosis, in addition to its known role in provision of nitrogen fixation, also improves early calcium uptake into soybean plants.     

Gas exchange characteristics and dry matter accumulation of soybean treated with Nod factors

The effect of Nod factors on gas exchange characteristics and growth of soybean plants was studied. Soybean responded positively to some concentrations of Nod factors. Photosynthesis was increased up to 13% over the control, and this was accompanied by increases in stomatal conductance; plant dry weight was also increased. Near-fully expanded leaves responded more strongly to Nod factors than fully expanded leaves. Only Nod factor NodBj-V (C18:1, MeFuc) had significant effects on soybean. The finding suggests that Nod factors can affect photosynthesis, possibly indirectly, by stimulating sink strength.     

Xylem transport and shoot accumulation of lumichrome, a newly recognized rhizobial signal, alters root respiration, stomatal conductance, leaf transpiration and photosynthetic rates in legumes and cereals

* Root respiration, stomatal conductance, leaf transpiration and photosynthetic rates were measured in phytotron and field-grown plants following the application of 5 or 10 nM lumichrome, 10 nM ABA (abscisic acid) and 10 ml of 0.2 OD600 infective rhizobial cells. * Providing soybean and cowpea roots with their respective homologous rhizobia and/or purified lumichrome increased the concentration of this molecule in xylem sap and leaf extracts. Relative to control, rhizobial inoculation and lumichrome application significantly increased root respiration in maize, decreased it in lupin, but had no effect on the other test species. * Applying either lumichrome (10 nM), infective rhizobial cells or ABA to roots of plants for 44 h in growth chambers altered leaf stomatal conductance and transpiration in cowpea, lupin, soybean, Bambara groundnut and maize, but not in pea or sorghum. Where stomatal conductance was increased by lumichrome application or rhizobial inoculation, it resulted in increased leaf transpiration relative to control plants. Treating roots of field plants of cowpea with this metabolite up to 63 d after planting showed decreased stomatal conductance, which affected CO2 intake and reduction by Rubisco. * The effect of rhizobial inoculation closely mirrored that of lumichrome application to roots, indicating that rhizobial effects on these physiological activities were most likely due to lumichrome released into the rhizosphere.     

A host-specific bacteria-to-plant signal molecule (Nod factor) enhances germination and early growth of diverse crop plants

Lipo-chitooligosaccharides (LCOs), or Nod factors, are host-specific bacteria-to-plant signal molecules essential for the establishment of a successful N(2)-fixing legume-rhizobia symbiosis. At submicromolar concentrations Nod factors induce physiological changes in host and non-host plants. Here we show that the Nod factor Nod Bj V(C18:1,MeFuc) of Bradyrhizobium japonicum 532C enhances germination of a variety of economically important plants belonging to diverse botanical families: Zea mays, Oryza sativa (Poaceae), Beta vulgaris (Chenopodaceae), Glycine max, Phaseolus vulgaris (Fabaceae), and Gossypium hirsutum (Malvaceae), under laboratory, greenhouse and field conditions. Similar increases in germination were observed for filtrates of genistein-induced cultures of B. japonicum 532C, while non-induced B. japonicum, induced Bj 168 (a nodC mutant of B. japonicum deficient in Nod factor synthesis) or the pentamer of chitin did not invoke such responses, demonstrating the role of Nod factor in the observed effects. In addition, three out of four synthetic LCOs evaluated also promoted germination of corn, soybean and Arabidopsis thaliana seeds. LCO also enhanced the early growth of corn seedlings under greenhouse conditions. These findings suggest the possible use of LCOs for improved crop production.     

The pleiotropic effects of extract containing rhizobial Nod factors on pea growth and yield

Rhizobial lipochitooligosacharides (Nod factors) influence the development of legume roots, including growth stimulation, nodule induction and root hair curling. However, their effect on the green parts of plants is less known, therefore we evaluated seed and foliar application of an extract containing Nod factors on pea growth and yield. Pea plants were examined from emergence to full maturity, including growth dynamics and morphological (nodule number and weight, the quantity and surface area of leaves) or physiological (photosynthesis and transpiration intensity, chlorophyll and nitrogen content) parameters. The foliar application Nod factor extract, or seed dressing followed by foliar application, resulted in the best outcomes. The number and weight of root nodules, the chlorophyll content in leaves, and the intensity of net photosynthesis were all elevated. As a consequence of Nod factor treatment, the dynamics of dry mass accumulation of pea organs were improved and the pod number was increased. A significant increase in pea yield was observed after Nod factor application. Increase of nodule and pod numbers and improved growth of roots appear to be amongst the beneficial effects of Nod factor extract on the activation of secondary plant meristems.     

Perception of Bradyrhizobium japonicum Nod factor by soybean [Glycine max (L.) Merr.] root hairs under abiotic stress conditions

Suboptimal growth conditions, such as low rhizosphere temperature, high salinity, and low pH can negatively affect the rhizobia-legume symbioses, resulting in poor nodulation and lower amounts of nitrogen fixed. Early stages of the Bradyrhizobium japonicum-soybean [Glycine max (L.) Merr.] symbiosis, such as excretion of genistein (the plant-to-bacteria signal) and infection initiation can be inhibited by abiotic stresses; however, the effect on early events modulated by Nod factors (bacteria-to-plant signalling), particularly root hair deformations is unknown. Thus, the objective of this study was to evaluate the perception of Nod factor by soybean root hairs under three stress conditions: low temperature, low pH, and high salinity. Three experiments were conducted using a 1:1 ratio of Nod Bj-V (C(18:1), MeFuc) and Nod Bj-V (Ac, C(16:0), MeFuc). Nod factor induced four types of root hair deformation (HAD), wiggling, bulging, curling, and branching. Under optimal experimental conditions root hair response to the three levels of Nod factor tested (10(-6), 10(-8), and 10(-10) M) was dose-dependent. The highest frequency of root hair deformations was elicited by the 10(-6) M level. Root hair deformation decreased with temperature (25, 17, and 15 degrees C), low pH, and high salinity. Nod factor concentration did not interact with either low temperature or pH. However, salinity strongly inhibited HAD responses to increases in Nod factor concentration. Thus, the addition of higher levels of Nod factor is able to overcome the effects of low pH and temperature stress, but not salinity.     

Effect of Foliar Application of Chitin and Chitosan Oligosaccharides on Photosynthesis of Maize and Soybean

On the first day after foliar application, chitosan pentamer (CH5) and chitin pentamer (CHIT5) decreased net photosynthetic rate (P _N) of soybean and maize, however, on subsequent days there was an increase in P _N in some treatments. CH5 caused an increase in maize P _N on day 3 at 10^–5 and 10^–7 M; the increases were 18 and 10 % over the control plants. This increase was correlated with increases in stomatal conductance (g _s) and transpiration rate (E), while the intercellular CO₂ concentration (C _i) was not different from the control plants. P _N of soybean plants did not differ from the control plants except for treatment CH5 (10^–7 M) which caused an 8 % increase on day 2, along with increased g _s, E, and C _i. On days 5 and 6 the CHIT5 treatment caused a 6–8 % increase in P _N of maize, which was accompanied by increases in g _s, E, and C _i. However, there was no such increase for soybean plants treated with CHIT5. In general, foliar application of high molecular mass chitin (CHH) resulted in decreased P _N, particularly for 0.010 % treated plants, both in maize and soybean. Foliar applications of chitosan and chitin oligomers did not affect (p > 0.05) maize or soybean height, root length, leaf area, shoot or root or total dry mass.     

Allelopathy in black walnut (Juglans nigraL.) alley cropping. II. Effects of juglone on hydroponically grown corn (Zea maysL.) and soybean (Glycine maxL. Merr.) growth and physiology

We conducted an experiment to investigate the effects of juglone (5-hydroxy-1, 4-napthoquinone) on the growth and physiology of hydroponically grown corn (Zea mays L.) and soybean (Glycine max L. Merr.) seedlings. Three different concentrations of juglone (10^-6 M, 10^-5 M, and 10^-4 M) along with a control were applied. Within 3 days, juglone exhibited significant inhibitory effects on all measured variables including shoot and root relative growth rates (RGR_s and RGR_r), leaf photosynthesis (P_net), transpiration (E), stomatal conductance (g_s), and leaf and root respiration. In general, soybean was found to be more sensitive to juglone than corn. RGR_r was the most inhibited variable for both species, and reductions of 86.5 and 99% were observed in corn and soybean, respectively, with 10_-4 M juglone concentrations. Among the physiological variables measured, P_net showed the greatest impact of toxicity though the other physiological parameters were also impacted. We conclude that both corn and soybean are sensitive to juglone and observed growth reductions in corn and soybean in black walnut alley cropping may partly be due to juglone phytotoxicity. Determination of actual phytotoxicity will require quantification of soil solution juglone levels, particularly in areas where soil solid-phase levels are high in close proximity to trees.     

Photosynthetic responses of corn and soybean to foliar application of salicylates

Salicylic acid (SA) and related phenolic compounds can exert control over stomatal opening and previous work in our laboratory has shown that chronic injection of SA increases the photosynthetic rate of corn. The work reported in this paper investigated the effects of foliar applied SA, acetyl salicylic acid (ASA) and gentisic acid (GTA) on photosynthetic rates and growth of soybean (a C3 plant) and corn (a C4 plant) under greenhouse conditions. In general, the tested compounds enhanced photosynthetic rates in both soybean and corn. Stomatal conductance and transpiration were also increased. These compounds do not alter chlorophyll content. In some cases treatment with these compounds resulted in increased leaf areas and plant dry mass, however, plant height and root length were not affected.     

Nod factor inhibition of reactive oxygen efflux in a host legume

Hydrogen peroxide (H(2)O(2)) efflux was measured from Medicago truncatula root segments exposed to purified Nod factor and to poly-GalUA (PGA) heptamers. Nod factor, at concentrations > 100 pM, reduced H(2)O(2) efflux rates to 60% of baseline levels beginning 20 to 30 min after exposure, whereas the PGA elicitor, at > 75 nM, caused a rapid increase in H(2)O(2) efflux to >200% of baseline rates. Pretreatment of plants with Nod factor alters the effect of PGA by limiting the maximum H(2)O(2) efflux rate to 125% of that observed for untreated plants. Two Nod factor-related compounds showed no ability to modulate peroxide efflux, and tomato (Lycopersicon esculentum), a nonlegume, showed no response to 1 nM Nod factor. Seven M. truncatula mutants, lacking the ability to make nodules, were tested for Nod factor effects on H(2)O(2) efflux. The nfp mutant was blocked for suppression of peroxide efflux, whereas the dmi1 and dmi2 mutants, previously shown to be blocked for early Nod factor responses, showed a wild-type peroxide efflux modulation. These data demonstrate that exposure to Nod factor suppresses the activity of the reactive oxygen-generating system used for plant defense responses.     

The rhizosphere signal molecule lumichrome alters seedling development in both legumes and cereals

The stimulatory role of lumichrome, a rhizosphere metabolite, was assessed on the growth of legume and cereal seedlings. At a very low nanomolar concentration (5 nm), lumichrome elicited growth promotion in cowpea, soybean, sorghum, millet and maize, but not in common bean, Bambara groundnut and Sudan grass. In soybean and cowpea only, 5 nm lumichrome caused early initiation of trifoliate leaf development, expansion in unifoliate and trifoliate leaves, increased stem elongation and, as a result, an increase in shoot and plant total biomass relative to control. Lumichrome (5 nm) also increased leaf area in maize and sorghum, and thus raised shoot and total biomass but there was no effect on the leaf area of the other cereals. Root growth was also stimulated in sorghum and millet by the supply of 5 nm lumichrome. By contrast, the application of a higher dose of lumichrome (50 nm) depressed development of unifoliate leaves in soybean, the second trifoliate leaf in cowpea, and shoot biomass in soybean. The 50 nm concentration also consistently decreased root development in cowpea and millet, but had no effect on the other species. These data show that lumichrome is a rhizosphere signal molecule that affects seedling development in both monocots and dicots.     

Cry3Bb1 protein from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in root exudates and biomass of transgenic corn does not persist in soil

The Cry3Bb1 protein, insecticidal to the corn rootworm complex (Diabrotica spp.), of Bacillus thuringiensis (Bt) subsp. kumamotoensis was released in root exudates of transgenic Bt corn (event MON863) in sterile hydroponic culture (7.5 +/- 1.12 ng/ml after 28 days of growth) and in nonsterile soil throughout growth of the plants (2.2 +/- 0.62 ng/g after 63 days of growth). Kitchawan soil, which contains predominantly kaolinite (K) but not montmorillonite (M), was amended to 3 or 6% (vol./vol.) with K (3K and 6K soils) or M (3M and 6M soils) and with 1, 3, 5, or 10% (wt./wt.) of ground biomass of Bt corn expressing the Cry3Bb1 protein and incubated at 25 +/- 2 degrees C at the -33-kPa water tension for 60 days. Soils were analyzed for the presence of the protein every 7 to 10 days with a western blot assay (ImmunoStrip) and verified by ELISA. Persistence of the protein varied with the type and amount of clay mineral and the pH of the soils and increased as the concentration of K was increased but decreased as the concentration of M was increased. Persistence decreased when the pH of the K-amended soils was increased from ca. 5 to ca. 7 with CaCO(3): the protein was not detected after 14 and 21 days in the pH-adjusted 3K and 6K soils, respectively, whereas it was detected after 40 days in the 3K and 6K soils not adjusted to pH 7. The protein was detected for only 21 days in the 3M soil and for 14 days in the 6M soil, which were not adjusted in pH. These results indicate that the Cry3Bb1 protein does not persist or accumulate in soil and is degraded rapidly.     

The distributional changes and role of microtubules in Nod factor-challenged<Emphasis Type="Italic"> Medicago sativa</Emphasis> root hairs

The normal tip-growing pattern exhibited by root hairs of legumes is disrupted when the hair is exposed to Nod factors generated by compatible bacteria capable of inducing nodule formation. Since microtubules (MTs) play an important role in regulating directionality and stability of apical growth in root hairs [T.N. Bibikova et al. (1999) Plant J 17:657–665], we examined the possibility that Nod factors might affect the MT distribution patterns in root hairs of Medicago sativa L. We observed that Nod factor application caused rapid changes in the pattern of MTs starting as early as 3 min after perfusion. Within 3 to 10 min after Nod factor application, first endoplasmic and then cortical MTs depolymerised, initially at the proximal ends of cells. Twenty minutes after exposure to Nod factors, a transverse band of microtubules was seen behind the tip, while almost all other MTs had depolymerised. By 30 min, very few MTs remained in the root hair and yet by 1 h the MT cytoskeleton re-formed. When Nod factors were applied in the presence of 10 M oryzalin or 5 M taxol, the MTs appeared disintegrated while the morphological effects, such as bulging and branching, became enhanced. Compared to the treatments with oryzalin or taxol alone, the combinatory treatments exhibited higher growth rates. Since microtubule reorganization is one of the earliest measurable events following Nod factor application we conclude that microtubules have an important role in the early phases of the signalling cascade. Microtubule involvement could be direct or a consequence of Nod factor-induced changes in ion levels.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-003-1097-1Abbreviations BNM buffered nodulation medium - CLSM confocal laser scanning microscopy - MT microtubule     

Activation of the cell cycle machinery and the isoflavonoid biosynthesis pathway by active Rhizobium meliloti Nod signal molecules in Medicago microcallus suspensions.

We have shown that treatment of Medicago microcallus suspensions with the cognate Rhizobium meliloti Nod signal molecule NodRm-IV(C16:2,S) can modify gene expression both qualitatively and quantitatively. At concentrations of 10(-6) - 10(-9) M, this host specific plant morphogen but not the inactive non-sulfated molecule stimulated cell cycle progression as indicated by the significantly enhanced thymidine incorporation, elevated number of S phase cells, increase in kinase activity of the p34cdc2-related complexes and enhancement of the level of expression of several cell cycle marker genes, the histone H3-1, the cdc2Ms and the cyclin cycMs2. The presented data suggest that at least part of the physiological role of the Nod factor may be linked to molecular events involved in the control of the plant cell division cycle. In situ hybridization experiments with antisense H3-1 RNA probe indicated that only certain cells of the calli were able to respond to the Nod factor. High (10(-6) M) but not low (10(-9) M) concentrations of the active Nod factors induced the expression of the isoflavone reductase gene (IFR), a marker gene of the isoflavonoid biosynthesis pathway in most callus cells. Our results indicate that Medicago cell responses to the Nod signal molecules can be investigated in suspension cultures.     

Influence of brick air scrubber by-product on growth and development of corn and hybrid poplar

Studies were conducted to determine the effects of spent reagent from air pollution control scrubbers used at a brick manufacturing facility on emergence, growth, and physiological responses of corn and hybrid poplar plants. Scrubber by-product was obtained from General Shale Brick, Louisville, KY. Potting substrate was weighed and quantities of scrubber by-product were added to the substrate to obtain treatments of 0%, 6.25%, 12.5%, 25%, 50%, 75%, and 100% scrubber by-product (w:w) for the corn study. Each treatment mix was potted into nine replicate polyethylene pots and four corn seeds were sown per pot. The pots were randomized in a greenhouse at Clemson University and the number of seedlings emerging from each treatment, dark-adapted leaf chlorophyll a fluorescence, and shoot heights were measured at the end of a 21-day growth period. Then, dry shoot biomass was determined for plants from each treatment and plant tissues were analyzed for selected constituents. For the poplar study, nine-inch cuttings of hybrid poplar clone 15-29 (Populus trichocarpa x P. deltoides) and clone OP367 (P. deltoides x P. nigra) were planted in treatments of scrubber by-product-potting soil mixes of 0% , 5% , 10% , and 25% w:w. Leaf chlorophyll a fluorescence was measured over six weeks and cumulative leaf area, dry biomass, and nutrient content of tissues were determined upon harvest. Results of these studies indicate that percent seedling emergence for corn plants decreased with increasing scrubber by-product application rates. Application rates up to 12.5% scrubber by-product w:w had no adverse effect on corn seedling emergence. Shoot elongation, biomass production, and the status of the photosynthetic apparatus of the seedlings were also not severely impaired at applications below this level. A critical value of 58.2% w:w scrubber by-product was estimated to cause 25% inhibition of seedling emergence. Biomass production, cumulative leaf area, and chlorophyll a fluorescence of hybrid poplar plants were not affected by scrubber by-product applications of up to 5% w:w.     

Effects of seismic stress on the vegetative growth of Glycine max (L.) Merr. cv. Wells II

Abstract. Vegetative plants of soybean [ Glycine max (L.) Merr. cv. Wells II] grown in a greenhouse and agitated periodically on a gyratory shaker had shorter stems, less leaf area, and lower leaf and plant dry weight than did undisturbed greenhouse-grown (GG) plants after 16 d of treatment. Outdoor-grown (OG) plants, which were subjected to additional environmental stresses including ultraviolet radiation, wind loading, and uncontrolled temperature and humidity fluctuations, were smaller and had less dry weight than GG controls, but growth was not inhibited further by gyratory shaking. Periodic shaking of GG soybeans resulted in the same plant and leaf dry weight as for OG soybeans. Response of GG plants to mechanical stress depended on light intensity, with minimum growth reduction occurring under full light (FL) level, and maximum growth reduction occurring under lower light levels (24–45% FL). Reduction in dry weight gain due to mechanical stress corresponded to a decrease in relative growth rate (RGR). Decreases in net assimilation rate and leaf area ratio contributed equally to the lower RGR of shaken plants, indicating that seismic stress inhibits dry weight accumulation by decreasing both the photosynthetic efficiency and the assimilatory surface of soybean.     

Effects of drought stress on photosynthetic gas exchange, chlorophyll fluorescence and stem diameter of soybean plants

Changes in plant growth, photosynthetic gas exchange, chlorophyll fluorescence and stem diameter of soybean [Glycine max (L.) Merr.] plants under drought stress were studied. Total plant dry mass was reduced by 30 % compared to well-watered control plants. Leaf water potential was slightly decreased by water stress. Water stress induced daytime shrinkage and reduced night-time expansion of stem. Photosynthetic rate, stomatal conductance and transpiration rate were significantly declined by water stress, while the intercellular CO₂ concentration was changed only slightly at the initiation of stress treatment. The maximum photochemical efficiency of photosystem 2 and apparent photosynthetic electron transport rate were not changed by water stress.     

Endophytic Neotyphodium lolii induced tolerance to Zn stress in Lolium perenne

The effect of Zn on growth, chlorophyll a fluorescence, net photosynthetic rate, gas exchange, water content and mineral concentrations (Zn, Mn and Mg) in ryegrass infected with or free from Neotyphodium lolii was studied by addition of ZnSO₄ (0–20 m M ) to the nutrient solution. Zn induced a decrease in growth of plants at 1, 5 and 10 m M and cessation of growth at 20 m M ZnSO₄. From 1 to 10 m M , the decrease was less pronounced in the presence of the endophytic fungus than in its absence. The growth limitation was due to an accumulation of Zn in leaves. From 8 to 15 days, the presence of the fungus in the plant led to a limitation of the Zn concentration in the leaves (24–32% lower with N. lolii than without). This restriction of Zn concentrations in leaves also had a beneficial effect on photosystem II (PSII) activities, net photosynthetic rate and internal CO₂ concentration. Particularly at 1 and 5 m M , the quantum yield of electron flow throughout PSII was greater in the presence of the fungus than in its absence and at 5 and 10 m M , the internal CO₂ concentration was maintained at a normal level. Compared with the endophyte-free ryegrass, the symbiotic plants showed higher values of total dry weight and tiller number, indicating a tolerance to environmental Zn stress.