Detection of GFP-labeled Paenibacillus polymyxa in autofluorescing pine seedling tissues

Paenibacillus polymyxa P2b-2R is a bacterium that originated from internal lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedling stem tissue and fixes nitrogen (N) in association with pine and western red cedar (Thuja plicata Donn.). To evaluate endophytic colonization by this microorganism, we generated P. polymyxa P2b-2Rgfp, a green fluorescent protein (GFP)-labeled derivative of P2b-2R, and grew pine seedlings that were inoculated with the marked strain in a N-limited soil. Tissue disintegration during sample preparation precluded examination of needles for the GFP-labeled endophyte but GFP was detected on roots and in stems of 2- to 14-week-old pine seedlings using confocal laser scanning microscopy. Due to excessive autofluorescence of seedling tissues, labeled bacteria were clearly discernible only in stem tissues of 4- and 6-week-old seedlings. P2b-2Rgfp colonized the root surface extensively and was detected inside the stem cortex, primarily intracellularly. Some labeled bacteria appeared to contain endospores and none were detected in vascular tissues. We conclude that P. polymyxa P2b-2R is capable of endophytic colonization of pine seedlings with specific colonization sites that include the stem cortex but that GFP labeling is of limited value for localization of endophytic bacteria in pine seedling tissues.     

Can a diazotrophic endophyte originally isolated from lodgepole pine colonize an agricultural crop (corn) and promote its growth?

Several diazotrophic Paenibacillus strains were isolated from extracts of surface-sterilized lodgepole pine seedling and tree tissues. One strain, Paenibacillus polymyxa P2b-2R, was found to fix high amounts of nitrogen when reintroduced to the gymnosperms, lodgepole pine and western red cedar. We wanted to determine if P. polymyxa P2b-2R could colonize rhizosphere and internal tissues, fix N and promote growth of corn (Zea mays L), an important agricultural crop. We inoculated corn seeds with P. polymyxa strain P2b-2R and grew seedlings for 30 days. Corn seedlings were harvested 10, 20 and 30 days after inoculation for evaluation of endophytic and rhizospheric colonization as well as nitrogen fixation and growth promotion. P2b-2R successfully colonized the rhizosphere as well as internal root tissues of corn (i.e., endophytically) with population densities near 10⁶ cfu. Corn seedling growth was promoted significantly by inoculation with P2b-2R with an increase of up to 35% in length and up to 30% in biomass after 30 days of inoculation. Seedlings inoculated with P2b-2R derived up to 20% of foliar nitrogen from atmosphere after 30 days of inoculation, which is significant considering the fact that this was a short growth trial. These results suggest that P. polymyxa P2b-2R might have a broad range of plant hosts and is able to fix N and promote the growth of at least one important agricultural crop i.e. Corn.     

15N foliar dilution of western red cedar in response to seed inoculation with diazotrophic Paenibacillus polymyxa

Diazotrophic bacteria previously isolated from internal tissues of naturally regenerating lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedlings were tested for their ability to fix N in association with western red cedar (Thuja plicata Donn.). Surface-sterilized cedar seed was sown in glass tubes containing an autoclaved sand/montmorillonite clay mixture that contained a N-free nutrient solution labeled with ¹⁵N as 0.35?mM Ca(¹⁵NO₃)₂ (5?% ¹⁵N label). After sowing, seed was inoculated with one of three bacterial strains: Paenibacillus polymyxa P2b-2R, P. polymyxa P18b-2R, or Dyadobacter fermentans P19a-2R. At the end of the 27-week plant growth period, P2b-2R was the only strain detected in the cedar rhizosphere. No bacteria were found inside plant tissues. Cedar foliar N concentrations were significantly enhanced (26–33?%, P?<?0.05) in response to inoculation with all three bacterial strains but ¹⁵N dilution (23?%, P?<?0.05) was observed only in seedlings treated with strain P2b-2R. This strain also reduced seedling dry weight (27?%, P?<?0.05). We observed similar trends in a second experiment with slight modifications to the protocol, but the magnitude of foliar ¹⁵N dilution was greater (56?%, P?<?0.05). Based on our results, we conclude that cedar seedlings inoculated with strain P2b-2R derived 23 and 56?% of their foliar N from bacterial N fixation in two seedling growth experiments.     

nif gene sequence and arrangement in the endophytic diazotroph Paenibacillus polymyxa strain P2b-2R

We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazotroph that can provide up to 79 % of foliar N in lodgepole pine through biological nitrogen fixation. We amplified a 388-bp internal nifH gene fragment from P2b-2R using the single specific primer–polymerase chain reaction (SSP-PCR) and performed a Southern blot analysis of Pst I/HindIII-digested genomic DNA to evaluate the sequence, copy number, and location in the nif gene operon. This strain was found to possess a single copy of the nifH gene with nifB located directly upstream of nifH and D. Phylogenetic analyses of the full nifH, partial nifB, and nifD, and 16s rDNA (rrs) gene sequences indicated that P2b-2R was part of a monophyletic cluster with other members of the genus Paenibacillus and suggest that nifH is transmitted horizontally while nifD is transmitted vertically. Our results provide the first full nifH sequence from a P. polymyxa strain and indicate that arrangement of genes in the P2b-2R nif operon is consistent with those observed in other species of the genus Paenibacillus.     

Short-term uptake of <Superscript>15</Superscript>NH<Subscript>4</Subscript><Superscript>+</Superscript> into soil microbes and seedlings of pine,spruce and birch in potted soils

Short-term competition between soil microbes and seedlings of Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies (L.) Karst.) and silver birch (Betula pendula Roth) for N was assessed in a pot study using (¹⁵NH₄)₂SO₄ as a tracer. Seedlings were grown in organic and mineral soil, collected from a podsol soil; 3.18 mg (¹⁵NH₄)₂SO₄ per pot were injected into the soil, corresponding to 4 µg ¹⁵N g^-1 d.m. (dry matter) mineral soil and 17 µg ¹⁵N g^-1 d.m. organic soil. The amounts of N and ¹⁵N in the seedlings and in microbial biomass derived from fumigation-extraction were measured 48 h after addition of ¹⁵N. In the mineral soil, 19–30% of the added ¹⁵N was found in the plants and 14–20% in the microbial biomass. There were no statistically significant differences between the tree species. In the organic soil, 74% of the added ¹⁵N was recovered in the microbial biomass in birch soil, compared to 26% and 17% in pine and spruce soils, respectively. Correspondingly, about 70% of the ¹⁵N was recovered in pine and spruce seedlings, and only 23% in birch seedlings. In conclusion, plants generally competed more successfully for added ¹⁵NH₄ ⁺ than soil microbes did. An exception was birch growing in organic soil, where the greater amount of available C from birch root exudates perhaps enabled micro-organisms to utilise more N.     

Cropping history affects nodulation and symbiotic efficiency of distinct hairy vetch (Vicia villosa Roth.) genotypes with resident soil rhizobia

Compatible rhizobia strains are essential for nodulation and biological nitrogen fixation (BNF) of hairy vetch (Vicia villosa Roth, HV). We evaluated how past HV cultivation affected nodulation and BNF across host genotypes. Five groups of similar HV genotypes were inoculated with soil dilutions from six paired fields, three with 10-year HV cultivation history (HV+) and three with no history (HV?), and used to determine efficiency of rhizobia nodulation and BNF. Nodulation was equated to nodule number and mass, BNF to plant N and Rhizobium leguminosarum biovar viceae (Rlv) soil cell counts using qPCR to generate an amplicon of targeted Rlv nodD genes. Both HV cultivation history and genotype affected BNF parameters. Plants inoculated with HV+ soil dilutions averaged 60 and 70 % greater nodule number and mass, respectively. Such plants also had greater biomass and tissue N than those inoculated with HV? soil. Plant biomass and tissue N were strongly correlated to nodule mass (r ²?=?0.80 and 0.50, respectively), while correlations to nodule number were low (r ²?=?0.50 and 0.31, respectively). Although hairy vetch rhizobia occur naturally in soils, past cultivation of HV was shown in this study to enhance nodulation gene-carrying Rlv population size and/or efficiency of rhizobia capable of nodulation and N fixation.     

Growth and nitrogen uptake of jack pine seedlings in response to exponential fertilization and weed control in reclaimed soil

We evaluated the impact of exponential fertilization in nursery and weed removal in the field on growth and nitrogen (N) retranslocation and uptake from the soil of jack pine (Pinus banksiana Lamb.) seedlings planted on an oil sands reclaimed soil. Exponential fertilization is a method of supplying nutrients at an exponential rate to achieve constant internal nutrient concentrations in seedlings without changing their size during their growth in the nursery. The N retranslocation in seedlings was traced using ¹⁵N isotope labeling. Exponential fertilization increased nutrient reserve in the seedling in nursery production, and increased height (P = 0.003), root collar diameter (P < 0.001), total biomass (P < 0.001), and N content (P < 0.001) of seedlings at the end of first growing season in the field growth. Conventionally fertilized seedlings allocated a greater percent of biomass to roots than to current-year needles. The ¹⁵N isotope analysis showed that 59 to 82% of total N demand of new growth was met by retranslocation from old tissues. Exponential fertilization increased N retranslocation by 147% (P < 0.001) and N uptake from the soil by 175% (P = 0.012). Weed removal marginally increased (P = 0.077) N uptake from the soil but decreased (P = 0.046) N retranslocation with no net effect on total N content in new tissues. We conclude that exponential fertilization improves the early growth of jack pine and can help improve revegetation in reclaiming disturbed oil sands sites.     

Nitrogen and phosphorus transformations in the rhizospheres of three tree species in a nutrient-poor sandy soil

We examined acid phosphatase activity (APA), N mineralization and nitrification rates, available N and P, and microbial biomass C, N and P in rhizosphere and bulk soils of 18-year-old Siberian elm (Ulmus pumila), Simon poplar (Populus simonii) and Mongolian pine (Pinus sylvestris var. mongolica) plantations on a nutrient-poor sandy soil in Northeast China. The main objective was to compare the rhizosphere effects of different tree species on N and P cycling under nutrient-deficient conditions. All tree species had the similar pattern but considerably different magnitude of rhizosphere effects. The APA, potential net N mineralization and nitrification rates increased significantly (by 27–60%, 110–188% and 106–142% respectively across the three species) in rhizosphere soil compared to bulk soil. This led to significantly higher Olsen-P and NH₄⁺-N concentrations in rhizosphere soil, whereas NO₃⁻-N concentration was significantly lower in rhizosphere soil owing to increased microbial immobilization and root uptake. Microbial biomass C and N generally increased while microbial biomass P remained constant in rhizosphere soil relative to bulk soil, indicating the N-limited rather than P-limited microbial growth. Rhizosphere effects on P transformation were most pronounced for Siberian elm, while rhizosphere effects on N transformation were most pronounced for Mongolian pine, implying the different capacities of these species to acquire nutrients.     

Evidence for enhanced N availability during switchgrass establishment and seeding year production following inoculation with rhizosphere endophytes

Improvement in sustainable production of switchgrass (SG, Panicum virgatum L), as a purpose-grown biomass feedstock crop, could be realized through investigation of plant–microbe interactions associated with plant growth promoting rhizobacteria (PGPR), capable of biological nitrogen fixation (BNF). The objective of this study is to increase establishment year production of SG biofuels by inoculation with a mixed PGPR inoculum. We isolated pure strains of N₂-fixing, and other PGPR, from SG rhizomes. The bacteria were identified as Paenibacillus polymyxa, an N₂-fixing bacterium, and other PGPR capable of solubilizing phosphate and/or producing auxins. Field trials utilizing these strains in a mixed PGPR inoculum showed that inoculated plants contained more N in tillers during anthesis but not at senescence, suggesting that more N could be cycled to belowground roots and rhizomes for winter storage. The amount of N removal in biomass and recovery of fertilizer N were also greater for inoculated than uninoculated plants. PGPR inoculation also resulted in positive N balances, suggesting improved access to N from non-fertilizer N sources, possibly through BNF and improved soil N uptake. Overall, inoculation of SG with PGPR enhanced N acquisition and could be an effective strategy to increase the establishment year production of this crop.     

Effects of weed control and fertilization at early establishment on tree nitrogen and water use in an exotic F1 hybrid pine of subtropical Australia

Purpose

We investigated the effects of weed control and fertilization at early establishment on foliar stable carbon (δ¹³C) and nitrogen (N) isotope (δ¹⁵N) compositions, foliar N concentration, tree growth and biomass, relative weed cover and other physiological traits in a 2-year old F₁ hybrid (Pinus elliottii var. elliottii (Engelm) × Pinus caribaea var. hondurensis (Barr. ex Golf.)) plantation grown on a yellow earth in southeast Queensland of subtropical Australia.

Materials and methods

Treatments included routine weed control, luxury weed control, intermediate weed control, mechanical weed control, nil weed control, and routine and luxury fertilization in a randomised complete block design. Initial soil nutrition and soil fertility parameters included (hot water extractable organic carbon (C) and total nitrogen (N), total C and N, C/N ratio, labile N pools (nitrate (NO₃ ^?) and ammonium (NH₄ ⁺)), extractable potassium (K⁺)), soil δ¹⁵N and δ¹³C. Relative weed cover, foliar N concentrations, tree growth rate and physiological parameters including photosynthesis, stomatal conductance, photosynthetic nitrogen use efficiency, foliar δ¹⁵N and foliar δ¹³C were also measured at early establishment.

Results and discussion

Foliar N concentration at 1.25 years was significantly different amongst the weed control treatments and was negatively correlated to the relative weed cover at 1.1 years. Foliar N concentration was also positively correlated to foliar δ¹⁵N and foliar δ¹³C, tree height, height growth rates and tree biomass. Foliar δ¹⁵N was negatively correlated to the relative weed cover at 0.8 and 1.1 years. The physiological measurements indicated that luxury fertilization and increasing weed competition on these soils decreased leaf xylem pressure potential (Ψ_xpp) when compared to the other treatments.

Conclusions

These results indicate how increasing N resources and weed competition have implications for tree N and water use at establishment in F₁ hybrid plantations of southeast Queensland, Australia. These results suggest the desirability of weed control, in the inter-planting row, in the first year to maximise site N and water resources available for seedling growth. It also showed the need to avoid over-fertilisation, which interfered with the balance between available N and water on these soils.     

Foliar phosphorus fertilization of common bean complementing phosphate fertilization at sowing

The objective of this study was to evaluate the response of common bean plants to phosphorus (P) applied to the leaves at different growth stages, as a complement to phosphate fertilization at sowing. The experiment followed the 2 × 7-factor randomized block design (RBD) with four replicates. The first factor was fertilization with P at the base with 60 and 120 kg ha^?1 of phosphorus pentoxide (P₂O₅). The second factor was the times and doses of P applied to the leaves: 0.5 or 1 kg ha^?1 of P₂O₅, at the V3 phenological stage; parceled in the following manner: 20% in V3, another 40% in R5 and 40% in R6, or 50% in R5 and 50% in R6; and one treatment with no P₂O₅ application to the leaves. P-based foliar fertilization increased P content in the grains of plants grown in soils with greater P availability. The lower common bean biomass production under scarce P availability was minimized by the foliar fertilization, as well as its productivity when 1 kg ha^?1 of P₂O₅ was applied to the leaves, 50% in the R5 phenological stage and 50% in R6. The highest productivity was obtained with the application of 0.5 kg ha^?1 of P₂O₅ to the leaves in the V3 phenological stage with the 120 kg ha^?1 dose at the base.     

Carbon Isotope Composition, Macronutrient Concentrations, and Carboxylating Enzymes in Relation to the Growth of Pinus halepensis Mill. When Subject to Ozone Stress

We present here the effects of ambient ozone (O₃)-induced decline in carbon availability, accelerated foliar senescence, and a decrease in aboveground biomass accumulation in the Aleppo pine (Pinus halepensis Mill.). Aleppo pine seedlings were continuously exposed in open-top chambers for 39 months to three different types of O₃ treatments, which are as follows: charcoal-filtered air, nonfiltered air (NFA), and nonfiltered air supplemented with 40 ppb O₃ (NFA+). Stable carbon isotope discrimination (??) and derived time-integrated c _i/c _a ratios were reduced after an accumulated ozone exposure over a threshold of 40 ppb (AOT40) value from April to September of around 20,000 ppb·h. An AOT40 of above 67,000 ppb·h induced reductions in ribulose-1,5-biphosphate carboxylase/oxygenase activity, aboveground C and needle N and K concentrations, the C/N ratio, Ca concentrations in twigs under 3 mm, and the aerial biomass, as well as increases in needle P concentrations and phosphoenolpyruvate carboxylase (PEPC) activity and the N and K concentrations in twigs under 3 mm. Macronutrients losses, the limitations placed on carbon uptake, and increases in catabolic processes may be the causes of carbon gain diminution in leaves which was reflected as a reduction in aboveground biomass at tree level. Stimulation of PEPC activity, the consequent decreased ??, and compensation processes in nutrient distribution may increase O₃ tolerance and might be interpreted as part of Aleppo pine acclimation response to O₃.     

Three-year growth responses of Pinus taeda L. to simulated rain chemistry,soil magnesium status,and ozone

Height, diameter, and biomass were measured for loblolly pine (Pinus taeda L) seedlings grown in soil containing 15 or 35 Μg Mg g^?1 and exposed from May to October in 1987, 1988, and 1989 to three O₃ concentrations (sub-ambient, ambient, or twice-ambient) and to rain pH levels of 3.8 or 5.2. Reduction in biomass accumulation in seedlings exposed to twice-ambient O₃ vs sub-ambient O₃ was 14% (P = 0.03) in 1987, 11.4% (P = 0.002) by 1988, and 8% (P = 0.15) by 1989. The greatest height growth occurred in seedlings exposed to twice-ambient O₃, and the greatest stem diameter growth occurred in seedlings exposed to sub-ambient O₃. A comparison of stem volume (d²h) with stem biomass suggested that tissue density was reduced by elevated O₃. Biomass accumulation response to rainfall chemistry was small (5.5% reduction in the low pH treatment in 1989) and not statistically significant for most plant tissues. Growth response to soil Mg status was not significant. Hoewever, in 1989 treatment interactions between rainfall chemistry and soil Mg status were observed. Height was 5% greater (P = 0.02) and biomass was 6% greater (P = 0.10) in seedlings grown in higher-Mg soil and receiving higher-pH rainfall than seedlings grown in any of the other pH-Mg treatment combinations. The data suggest direct adverse effects of near ambient O₃ and indirect, slower acting and interacting adverse effects of rainfall chemistry and soil nutrient status on growth of loblolly pine.     

Arbuscular mycorrhizae affect the N and C economy of nodulated Phaseolus vulgaris (L.) during NH4 nutrition

In the symbiosis between nodulated legume roots and arbuscular mycorrhizal (AM) fungi, the C and N economy can be influenced by the source of N-supply from either AM-derived NH₄⁺ uptake or nodule-derived biological nitrogen fixation (BNF). This relationship was investigated in terms of NH₄⁺ supply and BNF by the two symbionts. Nodulated Phaseolus vulgaris seedlings with and without AM, were hydroponically grown with either 0 N or 1 mM NH₄⁺ supply. Plants were harvested at 30 days after emergence and measurements were taken for biomass, N₂ fixation, photosynthesis, CO₂ and O₂ root respiration, calculated C and N economy. AM roots had higher NH₄⁺ uptake and this was associated with the suppression of BNF and nodule growth. The higher NH₄⁺ uptake in AM roots occurred with lower root maintenance respiration, compared to when N was derived from BNF. There was also an increase in the below-ground sink strength of NH₄⁺ fed AM roots compared to NH₄⁺ fed non-AM roots, as evidenced by the increases in root CO₂ and O₂ respiration and photosynthetic stimulation. These results indicate that although the AM root had higher total below-ground respiratory costs during NH₄⁺ nutrition, there were lower respiratory C costs associated with N derived from AM symbionts in comparison to N from BNF.     

Nutrient uptake and utilization in Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr.) seedlings exposed to a combination of light-emitting diode spectra and exponential fertilization

ABSTRACT

Exponential fertilization (EF) can cause seedlings to load more nutrients than they need to grow to establish reserves. Lighting spectrum adjustment may strengthen the growth and nutrient utilization of seedlings, which may modify seedling response to EF. In this study, containerized Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr.) seedlings were cultured in commercial substrates (275.59 mg nitrogen [N] and 60.05 mg phosphorus [P] per plant) and received EF at 0 (control), low (88 mg N and 36 mg P per plant), and high (130 mg N and 54 mg P per plant) doses under continuous lighting for 18 h daily. Two light-emitting diode (LED) spectra with different red (R), green (G), and blue (B) ratios were used as R-tinted (R7BG1) and G + B-tinted (R3BG10) colours. Under the R7BG1 spectrum, seedlings receiving low-dose EF had the best growth in height of 29 cm (P = 0.0100) and root-collar diameter (RCD) of 4.7 mm (P < 0.0001) and the highest N (170 mg plant^?1; P < 0.0001) and P contents (154 mg plant^?1; P < 0.0001). These seedlings also had the greatest biomass of leaves (P = 0.0005), stems (P = 0.0062), and roots (P = 0.0016) in the high-dose EF treatment. This combined effect resulted in the highest N uptake efficiency of nearly 40%. High-dose EF increased the chlorophyll-a and -b contents, while light spectra modified leaf protein content. Therefore, the LED spectra had an interactive effect with the EF dose on nutrient uptake and utilization in Prince Rupprecht’s larch seedlings with a recommended regime of EF at 88 mg N and 36 mg P per plant under the R7BG1 LED spectrum.     

Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp.

 Four experiments were performed under gnotobiotic conditions to select strains of the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. as inocula of rice plants. Eighty strains of H. seropedicae originally isolated from rice, sorghum and maize plants, were tested in test tube cultures with N-free agar as the substrate. Rice plants showed medium and high increases in their fresh weight in response to inoculation with nineteen strains. These strains were tested again, and six strains were then selected to evaluate their contribution to the N of the plant via biological N₂ fixation (BNF) using an agar growth medium containing 5 mg N l^–1of ¹⁵N-labelled (NH₄)₂SO₄. The contribution of the strains to plant N via BNF varied from 54% when rice plants were inoculated with strain ZAE94, to 31% when strain ZAE67 was used. These results were confirmed in the fourth gnotobiotic experiment, which also included strains of the new N-fixing bacteria belonging to the genus Burkholderia, isolated from rice, as well as a strain of Burkholderia vietnamiensis, isolated from rice rhizosphere. Burkholderia spp. strains showed similar effects to those observed for H. seropedicae strains, while B. vietnamiensis fixed only 19% of plant total N. The best four strains were tested in a pot experiment where pre-germinated, inoculated rice seedlings were grown in soil labelled with ¹⁵N. The results confirmed the gnotobiotic experiments, although the levels of N in the rice plants derived from BNF of the selected H. seropedicae and Burkholderia spp. strains were lower. Nevertheless, there was an increase in N content in grains of inoculated plants, and the results showed that the method used for strain selection is very useful and can be applied to other strains of N₂-fixing bacteria and plants. Received: 4 May 1999     

Variable impacts of enchytraeid worms and ectomycorrhizal fungi on plant growth in raw humus soil treated with wood ash

An increasing amount of evidence shows the context dependent nature of various biotic interactions across terrestrial and aquatic ecosystems. We established a laboratory experiment to study whether the effects of Cognettia sphagnetorum (Enchytraeidae) and ectomycorrhizal fungi on Scots pine (Pinus sylvestris) seedling growth are influenced by wood ash application. Acidic coniferous forest soil was treated with wood ash at 5000 kg ha⁻¹ or left as ash-free control and inoculated with soil saprotrophic microbes and nematodes. The microcosms were destructively sampled 26 and 51 weeks after initiation of the experiment. We measured enchytraeid and pine seedling biomass, abundance of nematodes and leaching of NH₄⁺-N and NO₃⁻-N at both samplings, and root length and N concentration of pine needles at the end of the experiment. On average enchytraeids and mycorrhizal fungi enhanced pine biomass production in the ash-free control soils, however, their impact was most pronounced when these organisms were alone in the systems. In fact, mycorrhizas tended to have a negative impact on the seedlings in the presence of enchytraeids. Wood ash had a clear negative impact on enchytraeid populations. Wood ash decreased pine growth when enchytraeids and EM-fungi were alone in the systems, but when together they apparently compensated for the negative effects of wood ash on the seedlings. It is concluded that interactions between soil fauna, mycorrhizal fungi and plants are context dependent, thereby rendering predictions of the outcome of species interactions in soil food webs a demanding task.     

Deciduous Conifers: High N Deposition and O3 Exposure Effects on Growth and Biomass Allocation in Ponderosa Pine

Ponderosa pines (Pinus ponderosa Dougl. ex. Laws) 21 to 60 yr old were used to assess the relative importance of environmental stressors (O₃, drought) versus an enhancer (N deposition) on foliar retention, components of aboveground growth, and whole tree biomass allocation. Sites were chosen across a well-described gradient in ozone exposure (40 to 80 ppb per h, 24 h basis, 6 month growing season) and nitrogen deposition (5 to 40 kg ha^-1 yr^-1) in the San Bernardino Mountains east of Los Angeles, California. A high level of chlorotic mottle indicated high O₃ injury at sites closest to the pollution source, despite potential for the mitigating effects of N deposition. At the least polluted site, foliar biomass was evenly distributed across three of the five needle-age classes retained. At the most polluted site, 95% of the foliar biomass was found in the current year's growth. High N deposition and O₃ exposure combined to shift biomass allocation in pine to that of a deciduous tree with one overwintering needle age class. Based on whole tree harvests, root biomass was lowest at sites with the highest pollution exposure, confirming previous chamber exposure and field studies. Aboveground growth responses in the high-pollution sites were opposite to those expected for O₃ injury. Needle and lateral branch elongation growth, and measures of wood production increased with increasing proximity to the pollution source. An enhancement of these growth attributes suggested that N deposition dominated the ponderosa pine response despite high O₃ exposure.     

Use of green manures in increasing inputs of biologically fixed nitrogen to sugar cane

In this study the contribution of biological N₂ fixation (BNF) to several green manure crops used at planting of sugar cane, or in the inter-rows of the ratoon crop, was evaluated. The subsequent effects of these legumes on the N supply and yield of the sugar cane crop was also investigated. The experiment was conducted in an extremely sandy N-deficient soil over two consecutive years. In the first year the legumes were planted on the same day as the sugar cane and in the second year they were planted immediately after the harvesting of the cane. The treatments consisted of four legume species (Crotalaria juncea, C. spectabilis, Canavalia ensiformis and Mucuna deeringiana), 100 kg N as urea fertiliser in a split addition, and no fertiliser N addition. The legumes were planted as two rows spaced 40 cm apart between each row of cane. Sorghum (Sorghum vulgare) and maize (Zea mays) were planted along the side of the experiment to act as non-N₂-fixing reference plants for the application of the ¹⁵N natural abundance technique to quantify the BNF input to the legumes and the sugar cane. The soil cover, monitored using an overhead digital camera, showed that Crotalaria juncea was the quickest to establish and cover the soil. At 31 days after planting (DAP) it reached a height of over 60 cm, more than twice that of any of the other green manures, and covered 55% of the soil, 16% more than that of the second most vigorous legume, Canavalia ensiformis. Using the natural abundance ¹⁵N technique it was deduced that all of the legumes had obtained over 60% of their N from BNF. BNF inputs to some of the green-manure legumes were quite significant, ranging from 35 to 55 kg N ha^?1 for Canavalia ensiformis, Crotalaria juncea and Mucuna deeringiana at plant crop, whereas the effect of the presence of these legumes on final cane yield and dry matter and N accumulation was not significant. There was also considerable evidence of negative (allelopathic) effects on cane growth induced by the presence of some of the legumes, especially Crotalaria juncea.     

Iron requirement for soybean [Glycine max (L.) Merrill] inoculated with selected exotic and native isolates of Bradyrhizobium sp. under irrigated conditions

A field and greenhouse experiments were conducted to determine the requirement of Fe nutrient supplied through foliar and soil application in soybean inoculated with different selected isolates of exotic and native Bradyrhizobium spp. in saline soils. Six soybean genotypes and three Bradyrhizobium spp. were used for the greenhouse experiments, whereas only two soybean genotypes, namely TGx-1336424 and GIZA, were selected for further study under field conditions. Two levels of FeSO₄ (0 and 4 mg Fe kg^?1 soil) directly supplied to the soil and three levels of Fe-ethylenediaminetetraacetic acid (0–2% of Fe) through foliar application were used for greenhouse and field experiments, respectively. The results of the greenhouse experiment indicated a non-significant effect of Fe application on nodulation and shoot biomass in soybean. Fe application did not improve the grain yield and total biomass yield in soybean inoculated with UK isolate and local isolate but showed remarkable improvement with TAL-379. High soil native N might be the cause for insignificant effect of Fe applied at 2% in highly effective inoculated plants. Therefore, it can be concluded that the symbiotic effectiveness of Bradyrhizobium sp. and the native soil N would affect the soybean Fe requirement supplied through foliar application.