Extraction of extraradical arbuscular mycorrhizal mycelium from compartments filled with soil and glass beads

This study presents a novel method for the extraction and quantification of extraradical mycelium (ERM) of arbuscular mycorrhizal fungi (AMF) from a substrate that simulates soil better than previously used artificial growth media. Fungal compartments were constructed from small net pots with a latticed wall and filled with a mixture of glass beads and 40 m wet sieved soil. The net pots were surrounded by a 30-m mesh membrane through which hyphae but not roots could grow. They were inserted into soil where a Glomus intraradices (BEG 110) colonized potato plant was growing. The ERM that had grown out from roots through the membrane was successfully collected and quantified after harvest by washing out the soil/glass bead mixture through a sieve with a mesh width of 40 m. Concentrations of P, Zn, Cu and Mn in the AMF ERM were analysed.     

The spatial distribution of soil hyphae in structured spruce-forest soils

The external mycelium forms the major part of the absorbing surface of mycorrhized tree roots. Because the macro pore space of acid forest soils is selectively depleted of mobile nutrient cations, it is ecologically important, whether soil hyphae grow into the soil aggregates or not. Seedlings of Norway Spruce (Picea abies (L.) Karst.) with defined mycorrhiza were grown in unsterilized soil cores taken from the A and B-horizon of a limed and an unlimed cambisol on triassic sandstone in the Northern Black Forest, Germany. Water-tension treatments were 10, 30, 160 and 900 hPa. On ground and polished vertical cuts stained with acridine orange, we identified and measured the location of hyphae and characterized their micropedological environment using an image analyzing system. Mean length density varied between 17 m/cm³ and 100 m/cm³ and was independent of aeration parameters. The percentage of hyphae completely embedded in the soil matrix varied between 30% and 8% and decreased significantly with increasing CO₂ concentration in the soil air. Of the hyphae in the soil matrix, 70% were located in a 50 m shell around the macro pores. Pair correlation functions show, that the majority of soil hyphae occur in clusters with diameters below 100 m. Between 60% and 80% of randomly chosen circles with 250 m diameters were completely devoid of hyphae. The inefficient opening up of the intra-aggregate space by soil hyphae is explained by the very slow oxygen diffusion between air-filled macro pores and the intra-aggregate space and mechanical restrictions for hyphae growth.     

Polyamines and proline are affected by cadmium stress in nodules and roots of soybean plants

The effect of moderate (50 M) and high (200 M) doses of Cd were studied in relation to polyamine (Pas) metabolism, proline level and the glutamine synthetase/glutamate synthase system (GS/GOGAT) activity in nodules and roots of soybean plants during 6 days of treatment. The lower Cd concentration increased putrescine (Put) in both nodules and roots, while 200 M Cd increased Spm only in nodules and Put in roots. Spermidine (Spd) decreased in roots under both Cd concentrations. Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were both involved in Put biosynthesis in roots. In nodules, Put formation could mainly be attributed to ODC activity. Diamine oxidase (DAO) activity was severely reduced by 50 and 200 M Cd either in nodules or roots. The GS/GOGAT system activity was depressed either with 50 or 200 M Cd, but most significantly with the highest metal concentration. Under 200 M Cd, GS activity decayed to 25% or 60% of the control in nodules and roots, respectively, while GOGAT decreased 85% in nodules and 79% in roots by day 4 of treatment. Ammonium increased greatly in nodules (200% over the controls) and roots (100%) under 200 M Cd. Proline concentration increased significantly in nodules and roots under both Cd treatments, more markedly under 200 M Cd. The relationship between Pas and proline accumulation and nitrogen assimilation is discussed.     

Effect of 2-hydroxybenzoate on the rate of naphthalene mineralization in soil

The effect of 2-hydroxybenzoate (2-OHB, salicylate) on the mineralization rate of [¹⁴C]naphthalene, the population density of naphthalene-degrading bacteria, and the concentration of genes encoding for naphthalene dioxygenase in a soil bacterial community was investigated. Six different concentrations of 2-OHB (10, 20, 50, 100, 150 and 200 g g^–1 soil) were tested in 100-g portions of soil. The addition of 10, 20 or 50 g 2-OHB g^–1 soil produced a general increase in total soil bacterial population density, whereas the addition of 100 g or 200 g 2-OHB g^–1 soil specifically increased the proportion of naphthalene degraders relative to the total population. The addition of 50 g 2-OHB g^–1 soil produced a fourfold increase (the maximum observed) in the rate of naphthalene mineralization relative to the rate in unamended soil. The concentration of 2-OHB ( 100 g/g) added to soil correlated with the population density of naphthalene degraders (r=0.961). Addition of up to 200 g 2-OHB g^–1 correlated with the abundance of DNA sequences homologous to known naphthalene dioxygenase genes (nahAB) (r=0.958). However, mineralization of [¹⁴C]naphthalene was stimulated significantly only by the addition of 50 g 2-OHB g^–1 soil. Results of the mineralization experiments were supported by the detection of nahAB mRNA extracted directly from soil. The specificity of the effect of 2-OHB on naphthalene biodegradation was confirmed in a control experiment using equivalent concentrations of 4-OHB which repressed naphthalene mineralization by about 50%. Addition of ammonium nitrate to the soil also increased the rate of naphthalene mineralization. Ammonium nitrate added together with 2-OHB reduced the mineralization enhancement effect of either compound alone. The study confirmed that specific induction of biodegradative genes can enhance chemical pollutant removal in situ. Correspondence to: O. A. Ogunseitan     

Contribution by two arbuscular mycorrhizal fungi to P uptake by cucumber (Cucumis sativus L.) from32P-labelled organic matter during mineralization in soil

An experiment was set up to investigate the role of arbuscular mycorrhiza (AM) in utilization of P from organic matter during mineralization in soil. Cucumber (Cucumis sativus L.) inoculated with one of two AM fungi or left uninoculated were grown for 30 days in cross-shaped PVC pots. One of two horizontal compartments contained 100 g soil (quartz sand: clay loam, 1:1) with 0.5 g ground clover leaves labelled with³²P. The labelled soil received microbial inoculum without AM fungi to ensure mineralization of the added organic matter. The labelling compartment was separated from a central root compartment by either 37 m or 700 m nylon mesh giving only hyphae or both roots and hyphae, respectively, access to the labelled soil. The recovery of³²P from the hyphal compartment was 5.5 and 8.6% for plants colonized withGlomus sp. andG. caledonium, respectively, but only 0.6 % for the non-mycorrhizal controls. Interfungal differences were not related to root colonization or hyphal length densities, which were lowest forG. caledonium. Both fungi depleted the labelled soil of NaHCO₃-extractable P and³²P compared to controls. A 15–25% recovery of³²P by roots was not enhanced in the presence of mycorrhizas, probably due to high root densities in the labelled soil. The experiment confirms that AM fungi differ in P uptake characteristics, and that mycorrhizal hyphae can intercept some P immobilization by other microorganisms and P-sorbing clay minerals.     

Contribution of the VA mycorrhizal hyphae in acquisition of phosphorus and zinc by maize grown in a calcareous soil

An investigation was carried out to test whether the mechanism of increased zinc (Zn) uptake by mycorrhizal plants is similar to that of increased phosphorus (P) acquisition. Maize (Zea mays L.) was grown in pots containing sterilised calcareous soil either inoculated with a mycorrhizal fungus Glomus mosseae (Nicol. and Gerd.) Gerdemann and Trappe or with a mixture of mycorrhizal fungi, or remaining non-inoculated as non-mycorrhizal control. The pots had three compartments, a central one for root growth and two outer ones for hyphal growth. The compartmentalization was done using a 30-m nylon net. The root compartment received low or high levels of P (50 or 100 mg kg^–1 soil) in combination with low or high levels of P and micronutrients (2 or 10 mg kg^–1 Fe, Zn and Cu) in the hyphal compartments.Mycorrhizal fungus inoculation did not influence shoot dry weight, but reduced root dry weight when low P levels were supplied to the root compartment. Irrespective of the P levels in the root compartment, shoots and roots of mycorrhizal plants had on average 95 and 115% higher P concentrations, and 164 and 22% higher Zn concentrations, respectively, compared to non-mycorrhizal plants. These higher concentrations could be attributed to a substantial translocation of P and Zn from hyphal compartments to the plant via the mycorrhizal hyphae. Mycorrhizal inoculation also enhanced copper concentration in roots (135%) but not in shoots. In contrast, manganese (Mn) concentrations in shoots and roots of mycorrhizal plants were distinctly lower, especially in plants inoculated with the mixture of mycorrhizal fungi.The results demonstrate that VA mycorrhizal hyphae uptake and translocation to the host is an important component of increased acquisition of P and Zn by mycorrhizal plants. The minimal hyphae contribution (delivery by the hyphae from the outer compartments) to the total plant acquisition ranged from 13 to 20% for P and from 16 to 25% for Zn.     

Changes in root growth patterns of (Picea abies) spruce roots by inoculation with an ectomycorrhizal fungusPisolithus tinctorius and jasmonic acid treatment

Symbiosis between fungi and plant roots forming a mycorrhiza involves extensive interactions at the molecular level between both partners. The role of plant hormones in the regulation of mycorrhizal infection is not known to involve jasmonates. Their endogenous levels increase during pathogen attack; however, little has been done on their involvement in mycorrhizae. In our recent work, root growth patterns of 2-month-old spruce seedlings after inoculation withPisolithus tinctorius and/or jasmonic acid (JA) treatment were studied using a paper-sandwich technique. Changes in root length, the degree of branching, presence and length of root hairs, and infection parameters were followed using a stereomicroscope. The first mycorrhizal contact of hyphae with roots was significantly accelerated upon treatment with 0.5 M JA. Interactions between root hairs and fungal hyphae were seen by scanning electron microscopy. The multiplication of root hairs of non-mycorrhized seedlings treated with 5.0 M JA and changes of the root surface were observed by the same technique.     

Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effect of explant type, age and plant growth regulators

A callus induction and in vitro plantlet regeneration system for the endangered state flower of Uttaranchal (Saussurea obvallata) was optimized by studying the influence of explant type (root, hypocotyl, cotyledon and leaf), age and different concentrations of plant growth regulators. Explants from 10 to 15-day-old seedlings showed maximum callus induction. Callus formation and shoot differentiation was initiated on Murashige-Skoog (MS) medium containing 6-benzyladenine (BA) and -naphthalene acetic acid (NAA) in all explant types. The best results were obtained using leaf explants: 100% callusing was achieved in MS medium supplemented with 2.5 M BA and 1.0 M NAA, and 100% differentiation along with a multiplication rate of 12 shoots per explant with a combination of 5.0 M BA and 1.0 M NAA. However, the results reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved at an efficiency of 100% in one-half strength MS medium supplemented with 2.5 M indole-3-butyric acid. Application of this protocol has potential for mass multiplication of the target species in a limited time period.     

Influence of nitrogen on the behaviour of nickel in wheat

A glasshouse experiment was conducted to study the effect of Ni on the growth and nutrients concentration in wheat (Triticum aestivum Cv. WH 291) in the presence and absence of applied N as urea. Responses to N application were observed up to 120 g N g^–1 soil. No response to Ni was observed in the dry matter yield of wheat tops (leaves + stem) in the absence of applied N while in the presence of applied N, significant yield increases were obtained at 12.5g Ni g^–1 soil. Nickel was not toxic to wheat up to 50g Ni g^–1 soil in the presence of 120g N g^–1 soil. Nitrogen and Ni concentration in wheat tops and roots increased with increasing levels of applied N and Ni, respectively. Applied Ni had an antagonistic effect on N concentration. Similarly, N reduced the Ni concentration in the wheat tissues. Positive growth responses to Ni were associated with 22 and 15g Ni g^–1 in wheat tops, in the presence of applied N at 60 and 120g N g^–1 soil, while Ni toxicity was associated with 63, 92.5 and 112.5g Ni g^–1 in wheat tops, in the absence and presence of applied N at 60 and 120g N g^–1 soil, respectively.     

The relationship between cell size and viability of soil bacteria

The number of bacterial cells in soil that form colonies on nutrient agar represent a small fraction of the direct microscopic counts (DMC). The colony-forming cells have larger cell dimensions than the very small (dwarf) cells which represent the majority of the DMC. This may indicate that the dwarf cells are species unable to form visible colonies on agar, or that they swell to normal dimensions when growing. Indigenous bacterial cells were separated from soil by density gradient centrifugation and fractionated according to diameter by filtration through polycarbonate filters. Each filtrate was studied with respect to DMC, cell dimensions, colony-forming cells (visible colonies and microcolonies), and cell dimensions during growth on the agar. The calculated average percent viability was only 0.2% for cells with diameters below 0.4m, about 10% for cells with diameters between 0.4 and 0.6m, and 30–40% for cells with diameters above 0.6m. Only 10–20% of the viable cells with diameters <0.4m increased their diameter to >0.4m prior to growth. Thus, size change during starvation and growth cycles did not explain the high numbers of dwarf cells observed by microscopy. The results show that despite the relatively low number of colony-forming bacteria in soil, the species that form colonies may be fairly representative for the medium size and large cells, which constitute a major part of the bacterial biovolume. Thus plate counting could be a useful method to count and isolate the bacteria accounting for much of the biovolume in soil. The origin of the dwarf cells is still unclear, but the low number of small cells that increased in size seems to indicate that the majority of these bacterial cells are not small forms of ordinary sized bacteria.     

Silicon amelioration of aluminium toxicity in teosinte (Zea mays L. ssp. mexicana)

The influence of different Al concentrations, (0, 60 and 120 M Al) on growth and internal concentrations of Al, Si and selected organic acids was analysed in plants of teosinte (Zea mays L. ssp. mexicana), a wild form of maize from acid soils from Mexico. The plants were grown in nutrient solutions (pH 4.0) with or without 4 M silicon. Analysis with the GEOCHEM speciation program did not reveal differences between free activities of Al³⁺ in solutions with and without 4 M Si, but solutions with Si yielded lower concentrations of monomeric Al species, [Al]_mono, when analysed by a modified aluminon method. Plants grown on solutions with similar [Al]_mono, but differing in silicon, showed highly significant differences in growth and tissue concentrations of Al and organic acids. Silicon prevented growth inhibition at [Al]_mono concentrations as high as 35 M, while plants grown without Si suffered severe growth reductions with 33 M [Al]_mono. In solutions with similar [Al]_mono concentrations plants with Si had lower tissue Al concentrations and higher concentrations of malic acid than plants without Si. In view of both the significant influence of Si on the response of plants to Al toxicity and the fact that some soluble Si is always present in soil solutions, the addition of low Si concentrations to nutrient solutions used for Al-tolerance screening is recommended.     

Aluminum-induced secretion of both citrate and malate in rye

Aluminum (Al)-resistant mechanisms responsible for Al-induced secretion of organic acids are poorly understood. In this study, we characterized the Al-induced secretion of both citrate and malate from rye (Secale cereale L. cv. King). Secretion of organic acids increased with increasing concentration (10, 30 and 50 M) and duration of Al treatments. Neither phosphorous (P) deficiency up to 15 days nor addition of 50M lanthanum, 50 M lead, 10 M cadmium, or 200 M manganese caused secretion of organic acids, suggesting that this secretion was a specific response to Al stress. Aluminum activated citrate synthase, the main enzyme for the synthesis of citrate, but its activation occurred only in the root tip. The elongation of roots of an Al-sensitive cultivar of wheat (Tritium aestivum L. cv. Scout 66) was not inhibited by 50 M Al in the presence of externally applied 50 M citrate or 400 M malate. The secretion of citrate and malate from intact rye roots exposed to 50 M Al corresponded to 31.3 ± 1.7 M and 11.5 ± 2.5 M, respectively, in the rhizosphere based on an assumption of a 2 mm thick unstirred layer around root tips. This result indicated that Al-resistance in rye was achieved by the Al-induced synthesis of citrate in root apices followed by Al-induced specific secretion of citrate from root tips.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

Micropropagation of candelilla,Euphorbia antisyphilitica Zucc

Axillary shoot proliferation was induced in vitro from shoot explants of greenhouse grown candellila (Euphorbia antisyphilitica Zucc). Optimum shoot proliferation was obtained by supplementing a modified Murashige and Skoog [7] medium with 0.13 M naphthalene-acetic acid and 4.44 M 6-benzylaminopurine. Rooting occurred on 100% of shoots transferred to a medium containing half strength salts supplemented with 0.49 M indole-3-butyric acid. Fully rooted plants were transferred to potting soil and established under greenhouse conditions without special acclimatization techniques.     

Changes in the kinetics of phosphate and potassium absorption in nutrient-deficient barley roots measured by a solution-depletion technique

From measurements of the rates of depletion of labelled ions from solution in the low concentration range, we described the phosphate and potassium uptake characteristics of the roots of intact barley plants in terms of the kinetic parameters, K _m and I _max (the maximum rate of uptake). In relatively young (13 d) and older (42 d) plants, cessation of phosphate supply for 4 d or more caused a marked increase in I _max (up to four times), without concomitant change in K _m, which remained between 5 and 7 M. By contrast, 1 d of potassium starvation with 14-d plants caused a decline in the K _m (i.e. an increased apparent affinity for potassium) from 53 M to 11 M, without alteration to I _max. After longer periods of potassium starvation, I _max increased (about two times) while the K _m remained at the same low value. Growth of shoots and roots were unaffected by these treatments, so that concentrations of ions in the tissues declined after 1 d or more of nutrient starvation, but we could not identify a characteristic endogenous concentration for either nutrient at which changes in kinetic parameters were invariably induced. The possible mechanisms regulating carriermediated transport, and the importance of changes induced in kinetic parameters in ion uptake from solution and soil are discussed.Symbol I _max the maximum rate of absorption at saturating concentrations     

Determination of mannitol in ectomycorrhizal fungi and ectomycorrhizas by enzymatic micro-assays

Two sensitive methods for the enzymatic determination of mannitol are described which were applied to fungal and mycorrhizal extracts. Both methods are based on the oxidation of mannitol by mannitol dehydrogenase from Agaricus hortensis and the fluorometric determination of the NADPH produced in this reaction. The detection limits are 125 pmol for the direct fluorometric assay and 100 fmol, when enzymatic cycling of NADPH is included. The levels of mannitol detected were 123 pmol/g dry wt (mycelia from Cenococcum geophilum, cultivated on malt medium), below 0.3 or about 2.4 pmol/g dry wt (mycelia from Amanita muscaria, dependent on carbon source in the cultivation medium), and between 1.9 and 5.2 pmol/g dry wt in mycorrhizal short roots of Picea abies/Amanita muscaria.     

The nutritional status of the apical meristem of Lactuca sativa as affected by NaCl salinization: An electron-probe microanalytic study

A volume of tissue of lettuce (Lactuca sativa L.) plants extending 2 mm basipetally from the apical meristem and including leaf primordia and young expanding leaves was surveyed using electron-probe microanalysis (EPMA) on both frozen-hydrated and freeze-dried samples. This analysis was carried out either 2 or 5 d following NaCl salinization of the medium from the 10 mol · m^–-3 control level up to 80 mol · m^–-3. The objective was the investigation of possible changes in the nutritional status of the apical meristem that might account for some aspects of salt-induced growth inhibition. Sodium and chloride increased significantly in tissues basal to the apical meristem, while both phosphorus and potassium decreased in the same region. These changes were evident in specimens collected just 2 d after the commencement of salinization (20 h after completion of the salinization) and were not exacerbated by an additional 3 d of treatment; they were present in tissue as close as 100 m to the meristem and extending down to 500 m. The apical 10–50 m were relatively protected from both the increase in sodium and chloride and the decrease in phosphorus and potassium that occurred in more basal regions. Young leaves (up to 1.5 mm in length) appear to control their own mineral nutrient levels when challenged by salinization of the medium, presumably because of altered growth. A decrease in the concentration of total Ca as a result of salinization was significant in cells 500 m basal to the meristem, but was evident as a tendency in the data even within the first 50 m. Using an improved automatic method for the analysis of calcium by EPMA, it was found that total Ca was reduced by salinization, especially in basal regions (500 m below the apex) and also in young leaves (1–1.5 mm in length). We suggest that the nutrition of the shoot apical meristem may be disturbed soon after salinization and that the shoot meristem might be the source of a signal to expanding leaves, as well as exerting its own direct influence over leaf emergence.Abbreviation EPMA electron-probe microanalysis This work was supported by U.S. Department of Agriculture grant 87-CRCR-1-2462.     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

Callus induction and plantlet regeneration in ornamental Alocasia micholitziana

Callus induction from petiole explants has been achieved in Alocasia micholitziana `Green Velvet'. The highest percentage (71%) of explants inducing callus was obtained on MS medium supplemented with 0.5 M 2,4-D and 0.5 M kinetin in the dark after 4 months of culture. Shoots were regenerated at the highest frequency of 33.3% under light condition when 0.5 M BA was added to MS medium with the average of 7.8±2.3 shoots per callus explant. The callus-derived shoots rooted on hormone free MS medium and within 4 weeks the plantlets were ready for acclimatization. The regenerated plants appeared morphologically similar to mother plants.