Hebeloma crustuliniforme modifies root hydraulic responses of trembling aspen (Populus tremuloides) seedlings to changes in external pH

The main objective of the study was to compare the effects of short-duration pH treatments on root hydraulic properties in trembling aspen (Populus tremuloides) seedlings that were either inoculated with the ectomycorrhizal fungus Hebeloma crustuliniforme or remained non-inoculated (control). Inoculated and non-inoculated plants were exposed in solution culture to the root zone pH ranging from 4 to 9 and their root hydraulic conductivity was examined using the hydrostatic method and after subjecting the plants to treatments with 100 ??M HgCl₂ (aquaporin blocker) and 0.02% trisodium 3-hydroxy-5,8,10-pyrenetrisulfonic acid (apoplastic transport tracer). In a separate experiment, pure cultures of H. crustuliniforme were also grown on a slid medium with the pH ranging from 4 to 9 to determine their pH growth optimum and changes in medium pH over time in the presence and absence of 8 mM NH₄NO₃. When grown in pure culture, H. crustuliniforme demonstrated maximum growth at pH 7?C8 and was capable of modifying the pH of its growth media, especially in the presence of NH₄NO₃. The plants that were inoculated with H. crustuliniforme had a maximum root hydraulic conductivity at pH 7. At this pH, root hydraulic conductivity was significantly higher compared with non-inoculated plants and showed greater sensitivity of root water transport to pH changes relative to non-inoculated seedlings. Relative apoplastic flux was largely unaffected by pH in inoculated seedlings. Fungal inoculation modified the response of root hydraulic conductivity to pH. The increased root hydraulic conductivity in inoculated seedlings was likely due to an increase in aquaporin-mediated cell-to-cell water transport, particularly at the higher pH. A possible role of fungal aquaporins in the root hydraulic conductivity responses of mycorrhizzal plants should be examined.     

Response of onion plants to arbuscular mycorrhizae

Onion (Allium cepa) plants were grown in pots in two types of irradiated soil, mineral and organic. Onion development was observed under two or three levels of P fertilization, and three methods of arbuscular mycorrhizal fungus inoculation with two fungus species. In mineral soil, preinoculated onion plants had a higher biomass than non-inoculated control plants or plants inoculated with either colonized root segments or spores. Fungus species had no differential effect on dry biomass or final bulb diameter. Preinoculated onion plants reached marketable size (>25 mm bulb diameter) 2-3 weeks earlier than those inoculated by either of the other two methods. Non-inoculated onion plants remained stunted. Bulbs of onions inoculated with Glomus versiforme were firmer than those inoculated with G. intraradices. Increasing P fertilizer rates had a significant positive linear effect on the P tissue concentration of plants inoculated with G. intraradices or G. versiforme, but no effect on bulb firmness. The P tissue concentration of inoculated plants was significantly higher than that of non-inoculated controls, and in inoculated plants, it differed among inoculation methods. The P tissue concentration was higher in onion plants inoculated with G. versiforme than in those inoculated with G. intraradices. In organic soil, the dry biomass of preinoculated plants was higher than that of plants inoculated by root segments. The highest root colonization levels were obtained under a low soil P level with G. intraradices, and with the root segment method of inoculation with G. versiforme.     

Effects of pH on NaCl tolerance of American elm (<Emphasis Type="Italic">Ulmus americana</Emphasis>) seedlings inoculated with <Emphasis Type="Italic">Hebeloma crustuliniforme</Emphasis> and <Emphasis Type="Italic">Laccaria bicolor</Emphasis>

In the present study, we investigated the effects of pH treatments on NaCl tolerance in mycorrhizal and non-mycorrhizal American elm. American elm (Ulmus americana) seedlings were inoculated with Hebeloma crustuliniforme, Laccaria bicolor or with both mycorrhizal fungi and subsequently subjected to different pH solutions (pH 3, 6 and 9) containing 0 mM (control) and 60 mM NaCl for 4 weeks. Inoculation with the mycorrhizal fungi did not have a large effect on seedling dry weights when the pH and NaCl treatments were considered independently. However, when the inoculated seedlings were treated with 60 mM NaCl at pH 3 or 6, shoot to root ratios and root hydraulic conductivity were higher compared with non-inoculated plants, likely reflecting changes in seedling water flow properties. At pH 6, transpiration rates were about twofold lower in non-inoculated plants treated with NaCl compared with non-treated controls. For NaCl-treated H. crustuliniforme- and L. bicolor-inoculated plants, the greatest reduction of transpiration rates was at pH 9. Treatment with 60 mM NaCl reduced leaf chlorophyll concentrations more in non-inoculated compared with inoculated plants, with the greatest, twofold, decrease occurring at pH 6. At pH 3, root Na concentrations were higher in inoculated than non-inoculated seedlings; however, there was no effect of inoculation on root Na concentrations at pH 6 and 9. Contrary to the roots, the leaves of inoculated plants had lower Na concentrations at pH 6 and 9, but not at pH 3. The results point to an interaction between ECM fungi and root zone pH for salt tolerance of American elm.     

Chromate resistantBacillus cereus augments sunflower growth by reducing toxicity of Cr (VI)

Impact of four chromium resistant bacterial strains (S3, S4, S6, and S7) was studied on the different growth parameters of sunflower (Helianthus annuus var SF-187) in chromium free or under chromium stress. Strains used exhibited very high-level resistance to chromate (up to 50 mg ml^-1 on nutrient agar and 1-2 mg ml^-1 in minimal medium). Application of Cr(VI) salt adversely affected the seed germination, root and shoot length, and fresh weight of seedlings. Bacterial inoculations improved the growth parameters. The effects of Cr(VI) on the different biochemical parameters were also very severe but seedlings inoculated with bacteria showed much improvements as compared to non-inoculated controls. Uptake of Cr(VI) was higher than Cr(III) by the seedlings. Inoculated seedlings contained less chromium than non-inoculated seedlings. Much improvement in the internal region of root and shoot was observed in inoculated plants especially in guard cells.     

Effects of Glomus fasciculatum and isolated rhizosphere microorganisms on growth and phosphate uptake of Plantago major ssp. pleiosperma

An experiment was set up in order to study 1) the relationship between net P uptake and dry matter production in mycorrhizal and non-mycorrhizal plants and 2) the effects of isolated rhizosphere bacteria and fungi on net P uptake and growth of P. major ssp. pleiosperma. A similar relationship between net P uptake and dry matter production was found for both mycorrhizal and non-mycorrhizal plants, although the regression lines differed in intercept.Compared to non-inoculated treatments, inoculation with bacteria slightly decreased dry matter production and P uptake of P. major, whereas inoculation with fungi or bacteria + fungi showed no effect. The results are discussed in terms of competition for available P and host photosynthates between host plant and rhizosphere microorganisms.     

Cytokinin producing bacteria enhance plant growth in drying soil

Cytokinins can promote stomatal opening, stimulate shoot growth and decrease root growth. When soil is drying, natural cytokinin concentrations decrease in association with stomatal closure and a redirection of growth away from the shoots to the roots. We asked if decreased cytokinin concentrations mediate these adaptive responses by lessening water loss and promoting root growth thereby favouring exploration for soil water. Our approach was to follow the consequences for 12-d-old lettuce seedlings of inoculating the growing medium with cytokinin-producing bacteria under conditions of water sufficiency and deficit. Inoculation increased shoot cytokinins as assessed by immunoassay and mass spectrometry. Inoculation also promoted the accumulation of shoot mass and shortened roots while having a smaller effect on root mass. Inoculation did not raise stomatal conductance. The possible promoting effect of these cytokinins on stomatal conductance was seemingly hampered by increases in shoot ABA that inoculation also induced. Inoculation lowered root/shoot ratios by stimulating shoot growth. The effect was greater in non-droughted plants but remained sufficiently strong for shoot mass of inoculated droughted plants to exceed that of well-watered non-inoculated plants. We conclude that compensating for the loss of natural cytokinins in droughted plants interferes with the suppression of shoot growth and the enhancement of root elongation normally seen in droughted plants.     

Stimulation of Nitrate and Nitrite Efflux by Ammonium in Barley (Hordeum vulgare L.) Seedlings

The inhibitory effect of NH4+ on net NO3- uptake has been attributed to an enhancement of efflux and, recently, to an inhibition of influx. To study this controversy, we devised treatments to distinguish the effects of NH4+ on these two processes. Roots of intact barley (Hordeum vulgare L.) seedlings, uninduced or induced with NO3- or NO2-, were used. Net uptake and efflux, respectively, were determined by following the depletion and accumulation in the external solutions. In roots of both uninduced and NO2- -induced seedlings, NO3- efflux was negligible; hence, the initial uptake rates were equivalent to influx. Under these conditions, NH4+ had little effect on NO3- uptake (influx) rates by either the low- or high-Km uptake systems. In contrast, in plants preloaded with NO3-, NH4+ and its analog CH3NH3+ decreased net uptake, presumably by enhancing NO3- efflux. The stimulatory effect of NH4+ on NO3- efflux was a function of external NH4+ and internal NO3- concentration. These results were corroborated by the absence of any effect of NH4+ on NO2- uptake unless the roots were preloaded with NO2-. In this case NH4+ increased efflux and decreased net uptake. Hence, the main effect of NH4+ on net NO3- and NO2- uptake appears to be due to enhancement of efflux and not to inhibition of influx.     

A comparison of ammonium, nitrate and proton net fluxes along seedling roots of Douglas-fir and lodgepole pine grown and measured with different inorganic nitrogen sources

Significant spatial variability in NH4+, NO3- and H+ net fluxes was measured in roots of young seedlings of Douglas-fir (Pseudotsuga menziesii) and lodgepole pine (Pinus contorta) with ion-selective microelectrodes. Seedlings were grown with NH4+, NO3-, NH4NO3 or no nitrogen (N), and were measured in solutions containing one or both N ions, or no N in a full factorial design. Net NO3- and NH4+ uptake and H+ efflux were greater in Douglas-fir than lodgepole pine and in roots not exposed to N in pretreatment. In general, the rates of net NH4+ uptake were the same in the presence or absence of NO3-, and vice versa. The highest NO3- influx occurred 0-30 mm from the root apex in Douglas-fir and 0-10 mm from the apex in lodgepole pine. Net NH4+ flux was zero or negative (efflux) at Douglas-fir root tips, and the highest NH4+ influx occurred 5-20 mm from the root tip. Lodgepole pine had some NH4+ influx at the root tips, and the maximum net uptake 5 mm from the root tip. Net H+ efflux was greatest in the first 10 mm of roots of both species. This study demonstrates that nutrient uptake by conifer roots can vary significantly across different regions of the root, and indicates that ion flux profiles along the roots may be influenced by rates of root growth and maturation.     

Effect of associative bacteria on element composition of barley seedlings grown in solution culture at toxic cadmium concentrations

The response of barley seedlings to inoculation with associative rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10 and Flavobacterium sp. L30 was studied in hydroponic and quartz sand cultures in the presence of 50 microM CdCl2. Cadmium caused severe inhibition in the growth and uptake of nutrient elements by the plants. Inoculation with the bacteria slightly stimulated root length and biomass of hydroponically grown Cd-treated seedlings. The bacteria increased the content of nutrients such as P, Mg, Ca, Fe, Mn and Na in roots and or shoots of the plants grown in the absence of Cd. Positive changes in the element composition caused by the bacteria were less pronounced in Cd-treated plants, whereas the total amount of nutrients taken by the inoculated plants was generally increased significantly. The content of Cd in the inoculated plants was unchanged, except increased in roots upon addition of A. lipoferum 137. Inoculation did not affect the activity of peroxidase, alpha-mannosidase, phosphodiesterae, alpha-galactosidase, and concentration of sulfhydryl compounds used as biochemical markers of stress in plant roots. The results showed that associative bacteria were capable of decreasing partially the toxicity of Cd for the barley plants through the improvement in uptake of nutrient elements.     

Enhanced Mineral Uptake by Zea mays and Sorghum bicolor Roots Inoculated with Azospirillum brasilense

Inoculation of corn (Zea mays) seeds with Azospirillum brasilense strain Cd or Sp 7 significantly enhanced (30 to 50% over controls) the uptake of NO₃⁻, K⁺, and H₂PO₄⁻ into 3- to 4-day- and 2-week-old root segments. No gross changes in root morphology were observed; altered cell arrangement in the outer four or five layers of the cortex was seen in photomicrographs of cross sections of inoculated corn roots. The surface activity involved in ion uptake probably increased, as shown by the darker staining by methylene blue of the affected area. Shoot dry weight increased 20 to 30% in inoculated plants after 3 weeks, presumably by enhancement of mineral uptake. Corn and sorghum plants grown to maturity on limiting nutrients in the greenhouse showed improved growth from inoculation approaching that of plants grown on normal nutrient concentrations. Enhanced ion uptake may be a significant factor in the crop yield enhancement reported for Azospirillum inoculation.     

Growth responses of tropical forage plant species to vesicular-arbuscular mycorrhizae

Summary Growth and mineral uptake of twenty-four tropical forage legumes and grasses were compared under glasshouse conditions in a sterile low P oxisol, one part inoculated and the other not inoculated with mycorrhizal fungi. Shoot and root dry weights and total uptake of P, N, K, Ca, and Mg of all the test plants were significantly increased by mycorrhizal inoculation. Mycorrhizal inoculation, with few exceptions, decreased the root/shoot ratio. Non-mycorrhizal plants contained always lower quantities of mineral elements than mycorrhizal plants. Plant species showed differences in percentage mycorrhizal root length and there was no correlation between percentage mycorrhizal infection and plant growth parameters. A great variation in dependence on mycorrhiza was observed among forage species. Total uptake of all elements by non-mycorrhizal legumes and uptake of P, N and K by non-mycorrhizal grasses correlated inversely with mycorrhizal dependency. Mycorrhizal plants of all species used significantly greater quantities of soil P than the nonmycorrhizal plants. Utilization of soil P by non-mycorrhizal plants was correlated inversely with mycorrhizal dependency.     

Phyto-Extraction of Nickel by Linum usitatissimum in Association with Glomus intraradices

Plants show enhanced phytoremediation of heavy metal contaminated soils particularly in response to fungal inoculation. Present study was conducted to find out the influence of Nickel (Ni) toxicity on plant biomass, growth, chlorophyll content, proline production and metal accumulation by L. usitatissimum (flax) in the presence of Glomus intraradices. Flax seedlings of both inoculated with G. intraradices and non-inoculated were exposed to different concentrations i.e., 250, 350 and 500 ppm of Ni at different time intervals. Analysis of physiological parameters revealed that Ni depressed the growth and photosynthetic activity of plants. However, the inoculation of plants with arbuscular mycorrhizae (G. intraradices) partially helped in the alleviation of Ni toxicity as indicated by improved plant growth under Ni stress. Ni uptake of non- mycorrhizal flax plants was increased by 98% as compared to control conditions whereas inoculated plants showed 19% more uptake when compared with the non-inoculated plants. Mycorrhizal plants exhibited increasing capacity to remediate contaminated soils along with improved growth. Thus, AM assisted phytoremediation helps in the accumulation of Ni in plants to reclaim Ni toxic soils. Based on our findings, it can be concluded that the role of flax plants and mycorrhizal fungi is extremely important in phytoremediation.     

The influence of the ectomycorrhizal fungus Rhizopogon subareolatus on growth and nutrient element localisation in two varieties of Douglas fir (Pseudotsuga menziesii var. menziesii and var. glauca) in response to manganese stress

Acidification of forest ecosystems leads to increased plant availability of the micronutrient manganese (Mn), which is toxic when taken up in excess. To investigate whether ectomycorrhizas protect against excessive Mn by improving plant growth and nutrition or by retention of excess Mn in the hyphal mantle, seedlings of two populations of Douglas fir (Pseudotsuga menziesii), two varieties, one being menziesii (DFM) and the other being glauca (DFG), were inoculated with the ectomycorrhizal fungus Rhizopogon subareolatus in sand cultures. Five months after inoculation, half of the inoculated and non-inoculated seedlings were exposed to excess Mn in the nutrient solution for further 5 months. At the end of this period, plant productivity, nutrient concentrations, Mn uptake and subcellular compartmentalisation were evaluated. Non-inoculated, non-stressed DFM plants produced about 2.5 times more biomass than similarly treated DFG. Excess Mn in the nutrient solution led to high accumulation of Mn in needles and roots but only to marginal loss in biomass. Colonisation with R. subareolatus slightly suppressed DFM growth but strongly reduced that of DFG (-50%) despite positive effects of mycorrhizas on plant phosphorus nutrition. Growth reductions of inoculated Douglas fir seedlings were unexpected since the degree of mycorrhization was not high, i.e. ca. 30% in DFM and 8% in DFG. Accumulation of high Mn was not prevented in inoculated seedlings. The hyphal mantle of mycorrhizal root tips accumulated divalent cations such as Ca, but not Mn, thus not providing a barrier against excessive Mn uptake into the plants associated with R. subareolatus.     

Fungal ectomycorrhizal community and drought affect root hydraulic properties and soil adherence to roots ofPinus pinaster seedlings

Pinus pinaster seedlings were grown in a sandy dune soil either inoculated withHebeloma cylindrosporum or let to natural colonisation. Six months later, half of the seedlings of both treatments were subjected to a 3-week moderate drought. Root colonisation analysis showed that root tips were colonised to almost 100% independent of the inoculation. DNA determination of the ectomycorrhizal morphotypes showed that inoculated seedlings were extensively mycorrhized byH. cylindrosporum (more than 75%) whereas non-inoculated seedlings were mycorrhized by the exotic speciesThelephora terrestris (50%) andLaccaria bicolor (30%) and to a lesser extent byH. cylindrosporum (20%). Drought did not affect these frequencies. Total plant biomass was not affected by the mycorrhizal status or by drought but the root/shoot biomass ratio as well as the root/leaf surface area ratio were much lower in seedlings extensively colonised byH. cylindrosporum. Root hydraulic conductivity was higher in plants mainly mycorrhized byH. cylindrosporum, showing that this fungus improved the water uptake capacity of the root system as compared toT. terrestris and/orL. bicolor. This positive effect was also found under drought but to a lesser extent.H. cylindrosporum also increased the amount of root-adhering soil as compared to the other fungal symbionts, illustrating the performance of this association in aggregating sandy soil particles and developing the rhizosheath. The origin of the reduced root hydraulic resistance byH. cylindrosporum mycorrhization is discussed for the whole path including soil, soil-root interface and root cortex.     

Effects of bacterial single inoculation and co-inoculation on growth and phytohormone production of sunflower seedlings under water stress

The purpose of the study was to measure shoot and root dry matter (DM) and production of auxins, salicylic acid, abscisic acid, and jasmonic acid in sunflower (Helianthus annuus L.) seedlings cultivated under water stress and singly inoculated or co-inoculated with Achromobacter xylosoxidans (SF2) and Bacillus pumilus (SF3 and SF4) bacterial strains. Shoot DM was higher in non-stressed seedlings than in stressed seedlings for all inoculation treatments. Water stress resulted in decreased relative water content and reduction of shoot DM. Root DM was higher in stressed seedlings than in non-stressed seedlings. Salicylic acid was the most abundant phytohormone in shoots of stressed, singly inoculated and co-inoculated seedlings. High salicylic acid content in stressed seedlings suggests that this hormone plays a key role in abiotic stress. Abscisic acid was higher in stressed and co-inoculated seedlings than in non-stressed seedlings but was lower than that of salicylic acid. Auxin profile was similar to that of abscisic acid in co-inoculated seedlings. Shoot jasmonic acid content was increased in stressed seedlings co-inoculated with SF2/SF3 or SF2/SF4. Shoot hormonal profiles were different from those of root, suggesting a differential effect of bacterial inoculation on these plant organs. Our findings will be useful in future strategies to mitigate drought effects on crop plants through bacterial inoculation treatments.     

Establishment of Desmoncus orthacanthos Martius (Arecaceae): effect of inoculation with arbuscular mycorrhizae

Inoculation with arbuscular mycorrhizal (AM) fungi has often promoted increased growth of plants but very little work has been done in the tropics to evaluate the effects of inoculation on the establishment and development of seedlings in forests. Desmoncus orthacanthos Martius is a scandent palm present both in early and late succession, and consequently can be used in restoration processes. A test was conducted to determine the effect of AM on the establishment of Desmoncus orthacanthos in tropical forest in the Yucatan Peninsula, Mexico. Thirty inoculated and 30 non-inoculated seedlings were introduced in two sites of different successional age, a mature forest and an eight-year old abandoned cornfield (acahual). Survival and growth parameters were evaluated after 12 months. Leaf area and phosphorus, but not height, were greater in inoculated than non-inoculated plants in the forest but not in the acahual. However, mycorrhizae had a clear effect on plant survival in both sites, with a threefold increase in survival of inoculated compared with non-inoculated plants bassed on an odds ratio. The results suggest that inoculation will be important to increase the establishment of this commercially important palm.     

Effect of pH and calcium on short-term NO3- fluxes in roots of barley seedlings

The effect of pH and Ca2+ on net NO3- uptake, influx, and efflux by intact roots of barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with NO3- or NO2-. Net NO3- uptake and efflux, respectively, were determined by following its depletion from, and accumulation in, the external solution. Since roots of both uninduced and NO2(-)-induced seedlings contain little internal NO3- initial net uptake rates are equivalent to influx (M. Aslam, R.L. Travis, R.C. Huffaker [1994] Plant Physiol 106: 1293-1301). NO3-, uptake (influx) by these roots was little affected at acidic pH. In contrast, in NO3(-)-induced roots, which accumulate NO3-, net uptake rates decreased in response to acidic pH. Under these conditions, NO3- efflux was stimulated and was a function of root NO3- concentration. Conversely, at basic pH, NO3- uptake by NO3- and NO2(-)-induced and uninduced roots decreased, apparently because of the inhibition of influx. Calcium had little effect on NO3- uptake (influx) by NO2(-)-induced roots at either pH 3 or 6. However, in NO3(-)-induced roots, lack of Ca2+ at pH 3 significantly decreased net NO3- uptake and stimulated efflux. The results indicate that at acidic pH the decrease in net NO3- uptake is due to the stimulation of efflux, whereas at basic pH, it is due to the inhibition of influx.     

丛枝菌根真菌对枳根净离子流及锌污染下枳苗矿质营养的影响

盆栽实验研究了不同施Zn水平(0、300 mg/kg和600 mg/kg)下,接种丛枝菌根真菌Glomus intraradices对枳苗生长、Zn、Cu、P、K、Ca、Mg分布的影响,并采用非损伤微测技术测定分析了菌根化与非菌根化枳根净Ca²⁺、H⁺、NO₃^-离子流动态。结果表明:(1)在不同施Zn水平下,接种菌根真菌显著提高了枳苗地上部及根部鲜重;随着施Zn水平的提高,菌根侵染率呈降低趋势,枳苗地上部与根部Zn含量呈增加趋势,且接种株根部Zn含量显著高于未接种株。(2)接种株未施Zn处理的地上部Cu、P、K、Mg和根部Cu含量、施600 mg/kg Zn处理的根部Cu及施300 mg/kg Zn处理的根部P含量均显著高于对照,而菌根真菌侵染对枳苗Ca含量并无显著性影响。(3)接种株未施Zn处理的根部距根尖端0 μm和600 μm处净Ca²⁺流出速率、600 μm处净H⁺流入速率、2400 μm处净NO₃^-流入速率均显著高于未接种株。     

Copper-Resistant Bacteria Enhance Plant Growth and Copper Phytoextraction

In this study, we investigated the role of rhizospheric bacteria in solubilizing soil copper (Cu) and promoting plant growth. The Cu-resistant bacterium DGS6 was isolated from a natural Cu-contaminated soil and was identified as Pseudomonas sp. DGS6. This isolate solubilized Cu in Cu-contaminated soil and stimulated root elongation of maize and sunflower. Maize was more sensitive to inoculation with DGS6 than was sunflower and exhibited greater root elongation. In pot experiment, inoculation with DGS6 increased the shoot dry weight of maize by 49% and sunflower by 34%, and increased the root dry weight of maize by 85% and sunflower by 45%. Although the concentrations of Cu in inoculated and non-inoculated seedlings did not differ significantly, the total accumulation of Cu in the plants increased after inoculation. DGS6 showed a high ability to solubilize P and produce iron-chelating siderophores, as well as significantly improved the accumulation of P and Fe in both maize and sunflower shoots. In addition, DGS6 produced indole–3–acetic acid (IAA) and ACC deaminase, which suggests that it may modulate ethylene levels in plants. The bacterial strain DGS6 could be a good candidate for re-vegetation of Cu-contaminated sites.

Supplemental materials are available for this article. Go to the publisher's online edition of International Journal of Phytoremediation to view the supplemental file.