Non-sterile Peat Research Articles

Biotechnology of biological bacterial preparations used in resource-saving farming

The article presents materials on the use of selenium compounds in the production of biological fertilizers, the main active component of which being artificially cultivated nodule bacteria. The objects of research in this work were various types of bacterial organisms used to prepare bacterial fertilizers: symbiotic nitrogen-fixing bacteria (rhizoagrin, rhizotorfin for galega and pea) and free-living agrobacteria (agrophil). Sodium selenate (Na2SeO4) was used as a source of selenium. The most optimal concentration of selenium ions in the liquid culture of microorganisms was 10–4 g/kg. A positive effect of selenium on the titer of finished preparations was observed: on average for the study period Rhizobiumpisum (rhizotorfin for peas) and Agrobacter 10 (agrophil) on the variant with selenium exceeded that on the control variant by 35.2 and 26.6%, respectively. The absolute titer values of the finished preparations Rhizobiumpisum (pea rhizotorfin) and Agrobacter 10 (agrophil) on non-sterile peat turned out to be higher than on sterile peat, although the trend remained. The inoculation of pea seeds with bacterial preparations increased the number of nodules in the flowering phase on the roots of pea plants by 1.8–2.1 times compared with the control. The treatment of pea seeds with rhizotorfin activated by selenium compounds during its manufacture increased the grain yield by 0.51 t/ha (17.1%) compared with the control, and by 0.30 t/ha (10.0%) when inoculated with rhizotorfin.

Сontrolled breeding of the psychrophilic strain G-034 VIZR of Trichoderma asperellum for fast crop residues’ polymers utilization and soil enhancement

Genus Trichoderma strains as the natural plant residues’ biodestructors, highly active antagonists of soil phytopathogens and phytoregulators with the widest range of optimum conditions for their development, are widely used in biologics development. Of particular importance in Russia’s northern regions, especially in winter crop cultivation, is the ability of a microorganism’s strain used in agro-technologies to maintain viability and target biological activity at low temperatures. In this connection, this work purpose is to select a psychrotolerant strain of T. asperellum for the rapid crop residues’ polymer utilization and soil enhancement at low temperature, as well as to evaluate its activity under laboratory and field conditions. In the work process, the following tasks were addressed: selecting psychrotolerant strains of T. asperellum with high cellulolytic activity; further controlled breeding of psychrophilic strains capable of rapid growth, active colonization of plant substrates and high sporulation at 4–8 °C; evaluating the target activity of the selected psychrophilic strain as a cellulolytic as well as antagonistic activity against cereal pathogens; obtaining laboratory samples of bioformulations by deep-surface cultivation on non-sterile peat and multirecycled wastes from the edible mushrooms production and assessing their efficacy in field small-plot trials. The methods for inoculum cultivation, sporulation capacity determination, modified wet chamber, estimating antagonistic activity and biologics’ quality, field small-plot trials management, quantitative estimates of biomass losses, cellulose and lignin content were used in the work. The active psychrophilic strain for the rapid crop residues’ polymer utilization and soil enhancement controlled breeding was selected during a four-step screening of 29 T. asperellum strains from All-Russian Research Institute of Plant Protection (VIZR) State Microorganisms’ Collection with high cellulolytic and antagonistic activities. In terms of linear growth rate, antagonistic and hyperparasitic activities at 4–8 °С, a high rate of wheat and maize stubble residues’ colonization, a perspective psychrophilic strain G-034 of T. asperellum was selected for developing the laboratory samples of biologics and for running field trials. In small-plot trials, the active maize crop residues’ decomposition under the T. asperellum G-034 influence was revealed, resulting in the complete loss of plant intact state in 12 months due to more than 80 % cellulose and 20 % lignin biodestruction. The maximum loss of maize crop residues biomass for 12 months was more than 70 %. The T. asperellum strain G-034 was active after field hibernation in an amount of ×104 cfu/g, resulting in a titer increase with seasonal temperature rising and the trophic base bioavailability growth.

Uji Aktivitas Lakase dan Selulase pada Lignoselulosa Gambut dengan Berbagai Kadar Air

The degradation of lignocellulose as the main constituents of peat is catalyzed by enzymes such as laccase or cellulase. The purpose of this research was to study the pattern of laccase and cellulase activities on sterile and non-sterile peat materials at three different water contents (125-175, 225-275, and 325-375%). The peat soil used was collected from oil palm rhizosphere in Riau Province. This research used the descriptive method by comparing the mean values between treatments. The results showed that enzymes activities on sterile and non-sterile peat added with laccase or cellulase were higher compared to those without enzyme addition. The highest laccase activity was at the first day of incubation, while that for selulase was at the 10 th days of incubation. The activity of enzyme then decreased with the increase in the respected incubation time. The difference in decreasing of lignin and cellulose content at 125-175% water content was higher than at the other water contents. The decreases in lignin and cellulose contents were higher by addition of respected enzymes both in sterile and non-sterile peat. It could be concluded that at higher water content, laccase and cellulase activities were depressed both on sterile and non sterile peat, especially after the addition of enzyme.

Rhizobium cellulosilyticum as a co-inoculant enhances Phaseolus vulgaris grain yield under greenhouse conditions

The Rhizobium-legume symbiosis is a complex partnership with many factors, with initial bacterial colonization of the plant root surface and primary infection as key early stages. Two molecules are strongly involved in these processes: the structural carbohydrate cellulose and the enzyme cellulase, which breaks down the former and allows rhizobia to infect the roots. Here, we report the effect on common bean (Phaseolus vulgaris L.) after co-inoculation of the non-nodulating, cellulase-overproducing strain Rhizobium cellulosilyticum ALA10B2T and the P. vulgaris-nodulating R. leguminosarum strain TPV08. In order to elucidate the effect of combined inoculation with both strains, we designed greenhouse assays, including single inoculation with strain TPV08, co-inoculation with both strains and an uninoculated treatment in non-sterile peat. Chemical fertilizers were not added. Chlorophyll content in the leaves was measured after the flowering stage by spectrophotometry and was considered to be indicative of the nutrient status of the plants. Nodule formation was observed on roots of the inoculated plants, while no nodulation was observed on roots of the uninoculated plants. The results indicate a synergistic effect between the two Rhizobium strains. Co-inoculated plants exhibited significant increases in seed yield and nitrogen content in comparison with the uninoculated control plants and with plants inoculated with a single strain. It is suggested that co-inoculation with strain ALA10B2T greatly increased the efficiency of N fixation by strain TPV08.

Quantitative detection of agar-cultivated and rhizotron-grown Piloderma croceum Erikss. & Hjortst. by ITS1-based fluorescent PCR.

A real-time quantitative TaqMan-PCR was established for the absolute quantification of extramatrical hyphal biomass of the ectomycorrhizal fungus Piloderma croceum in pure cultures as well as in rhizotron samples with non-sterile peat substrate. After cloning and sequencing of internal transcribed spacer (ITS) sequences ITS1/ITS2 and the 5.8S rRNA gene from several fungi, including Tomentellopsis submollis, Paxillus involutus, and Cortinarius obtusus, species-specific primers and a dual-labelled fluorogenic probe were designed for Piloderma croceum. The dynamic range of the TaqMan assay spans seven orders of magnitude, producing an online-detectable fluorescence signal during the cycling run that is directly related to the starting number of ITS copies present. To test the confidence of the PCR-based quantification results, the hyphal length of Piloderma croceum was counted under the microscope to determine the recovery from two defined but different amounts of agar-cultivated mycelia. Inspection of the registered Ct values (defined as that cycle number at which a statistically significant increase in the reporter fluorescence can first be detected) in a 10-fold dilution series of template DNA represents a suitable and stringent quality control standard for exclusion of false PCR-based quantification results. The fast real-time PCR approach enables high throughput of samples, making this method well suited for quantitative analysis of ectomycorrhizal fungi in communities of natural and artificial ecosystems, so long as applicable DNA extraction protocols exist for different types of soil.

Field comparison of pre-inoculated alfalfa seed and traditional seed inoculation with inoculant prepared in sterile or non-sterile peat

Rhizobium inoculant products in North America are often prepared using non-sterile peat and may contain more contaminants than rhizobia. The effectiveness of sterile and non-sterile peat-based inoculants applied to alfalfa (Medicago sativa) seed using either traditional seed inoculation just before planting, or pre-inoculated and commercially coated, were evaluated for three years (eight harvests) at two sites in northwestern Alberta. Seeds inoculated just before planting had more rhizobia at planting time than pre-inoculated seed. When results from all the three years were combined, inoculation with Rhizobium, either seed-applied or pre-inoculated, significantly (P<0.05) increased nodule dry matter (DM) at both sites. However, the response of forage N and DM yields to inoculation were significantly higher with seed application than with seed coating at one site, and vice–versa at the other site. Differences resulting from the use of non-sterile peat or sterile peat were not significant.

Rates and quantities of carbon flux to ectomycorrhizal mycelium following 14C pulse labeling of Pinus sylvestris seedlings: effects of litter patches and interaction with a wood-decomposer fungus.

We used a novel digital autoradiographic technique that enabled, for the first time, simultaneous visualization and quantification of spatial and temporal changes in carbon allocation patterns in ectomycorrhizal mycelia. Mycorrhizal plants of Pinus sylvestris L. were grown in microcosms containing non-sterile peat. The time course and spatial distribution of carbon allocation by P. sylvestris to mycelia of its mycorrhizal partners, Paxillus involutus (Batsch) Fr. and Suillus bovinus (L.): Kuntze, were quantified following 14C pulse labeling of the plants. Litter patches were used to investigate the effects of nutrient resource quality on carbon allocation. The wood-decomposer fungus Phanerochaete velutina (D.C.: Pers.) Parmasto was introduced to evaluate competitive and territorial interactions between its mycelial cords and the mycelial system of S. bovinus. Growth of ectomycorrhizal mycelium was stimulated in the litter patches. Nearly 60% of the C transferred from host plant to external mycorrhizal mycelium (> 2 mm from root surfaces) was allocated to mycelium in the patches, which comprised only 12% of the soil area available for mycelial colonization. Mycelia in the litter patch most recently colonized by mycorrhizal mycelium received the largest investment of carbon, amounting to 27 to 50% of the total 14C in external mycorrhizal mycelium. The amount of C transfer to external mycelium of S. bovinus following pulse labeling was reduced from a maximum of 167 nmol in systems with no saprotroph to a maximum of 61 nmol in systems interacting with P. velutina. The 14C content of S. bovinus mycelium reached a maximum 24-36 h after labeling in control microcosms, but allocation did not reach a peak until 56 h after labeling, when S. bovinus interacted with mycelium of P. velutina. The mycelium of S. bovinus contained 9% of the total 14C in the plants (including mycorrhizae) at the end of the experiment, but this was reduced to 4% in the presence of P. velutina. The results demonstrate the dynamic manner in which mycorrhizal mycelia deploy C when foraging for nutrients. The inhibitory effect of the wood-decomposer fungus P. velutina on C allocation to external mycorrhizal mycelium has important implications for nutrient cycling in forest ecosystems.

Plant and fungal responses to elevated atmospheric carbon dioxide in mycorrhizal seedlings of Pinus sylvestris

The effects of elevated CO 2 concentration upon the mycorrhizal relationships of Scots pine ( Pinus sylvestris) seedlings were investigated. Plants were grown for 4 months with their shoots exposed to ambient ( C AMB=360 μl l −1) or elevated ( C ELEV=700 μl l −1) CO 2 environments while their root systems, either colonised by the mycorrhizal fungi Paxillus involutus or Suillus bovinus, or left in the non-mycorrhizal condition, were maintained in sealed dishes. In one series of these plants the effects of C ELEV upon the extent of mycorrhizal development and upon their growth and nutrition were determined, while another series were transferred from the dishes after 1 month, to transparent observation chambers before being returned to the two CO 2 environments. In these chambers, the effects of C ELEV upon development of the external mycelial systems of the two mycorrhizal fungi was determined by measuring the advance of the hyphal fronts of the mycorrhizal fungi across non-sterile peat from the colonised plants. The dry mass and number of mycorrhizal tips were significantly higher in C ELEV than in the C AMB condition in plants colonised by both fungi in the dishes. Yields of whole plants and of shoots were higher in the C ELEV treatment whether or not they were grown in the mycorrhizal condition, but the greater yields were not associated in these sealed systems with enhanced nutrient gain. The dry mass of non-mycorrhizal plants was greater than that of those colonised by mycorrhizal fungi under elevated CO 2. This is thought to be attributable to the energetic cost of production of the larger mycorrhizal systems in this treatment. The extent of development of the mycorrhizal mycelial systems of both fungi was greatly increased in C ELEV relative to that in C AMB environments. It is hypothesised that increased allocation of carbon to mycorrhizal root systems and their associated mycelia would provide the potential for enhancement of nutrient acquisition in open systems of greater fertility.

Levels and identities of nonrhizobial microorganisms found in commercial legume inoculant made with nonsterile peat carrier

Sixty samples of commercial North American legume inoculants manufactured for sale in 1994 using nonsterile peat as carrier were tested for Rhizobium (or Bradyrhizobium) content and non-Rhizobium biological contaminant load. Products of three major producers of such inoculants for sale in Canada were examined. Viable Rhizobium content varied from 5.6 x 10(5) to 8.1 x 10(9) cells/g, while the contaminant load varied from 1.8 x 10(8) to 5.5 x 10(10) cfu/g. Most of the inoculants contained more nonrhizobial organisms than they did rhizobia. Identifications were made of the most numerous nonrhizobial bacteria occurring in 100 samples of inoculants collected in 1993 and 1994. The most commonly identified contaminant was Xanthomonas maltophilia. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloacae were also found at high levels in some products. Contaminant organisms capable of inhibiting rhizobial growth in plate culture were found in the products of all three manufacturers.

Biological contaminants in North American legume inoculants

A survey of 40 commercial North American rhizobial inoculants was made to determine the number of contaminant cells relative to rhizobial cells. All of the inoculants were manufactured for sale during the 1993 season and were made using non-sterile peat as carrier. Products from three major North American manufacturing companies were examined. All of the inoculant samples were collected as part of the Canadian Legume Inoculant and Pre-Inoculated Seed Product Testing Program. Only one of the inoculants contained more rhizobia ( Rhizobium or Bradyrhizobium spp) than contaminants. Three others contained 1–2 times more contaminants than rhizobia, 21 of the samples contained 2–10 times more contaminants than rhizobia, 8 contained 10–100 times more contaminants than rhizobia, and 3 contained more than 1000 times more contaminants than rhizobia. Four of the samples contained too few rhizobia to be detected in the standard most-probable-number (MPN) test used by the Canadian regulatory inoculant testing program. The most common contaminants were bacteria (10 9–10 10 g −1), actinomycetes (10 8–10 9 g −1), and fungi (10 5–10 7 g −1). Contaminant bacteria capable of inhibiting growth of R. leguminosarum bv. trifolii or R. meliloti were isolated from inoculant products of all three manufacturers.

Nitrogen amendments reduce the growth of extramatrical ectomycorrhizal mycelium

The effect of three different nitrogen sources on the growth of external ectomycorrhizal mycelium was studied in Perspex micorocosms. Nonsterile peat was used as substrate. Five different fungal isolates growing in symbiosis with pine seedlings were investigated: two isolates of Paxillus involutus, one of Suillus bovinus and two unidentified ectomycorrhizal fungi isolated from ectomycorrhizal root tips. Three different nitrogen sources were used: ammonium as (NH4)2SO4, nitrate as NaNO3 and a complete nutrient solution (Ingestad 1979), and three different nitrogen concentrations, 1, 2 or 4 mg N/g dry wt. of peat. The mycelial growth of all fungi was found to be negatively affected by the nitrogen amendments, although the sensitivity to nitrogen varied between the isolates. One of the unidentified isolates was extremely sensitive and growth was completely inhibited by all nitrogen treatments. In contrast, the growth of one of the P. involutus isolates was only slightly reduced by the nitrogen amendments. The different nitrogen sources all reduced growth, and since no significant difference was found between the nitrogen sources or between the different nitrogen concentrations the results were pooled to give one value that summarized the effect of nitrogen on mycelial growth. Thus, the mycelial growth of one of the two P. involutus isolates was reduced to approximately 80% of the growth in the control, the other P. involutus and one of the unidentified fungi, vgk 2 89.10, were reduced to 40–50% of the control growth, S. bovinus to 30% of the control and the most sensitive fungus, the unidentified isolate vg 1 87.10, was reduced to 3% of the growth in the control treatment. In all experiments, the shoot to root ratio generally increased, mainly as a result of increased shoot growth.

The influence of substrate pH on carbon translocation in ectomycorrhizal and non‐mycorrhizal pine seedlings

The effects of changed substrate pH on translocation and partitioning of 14 C-labeled plant assimilates were examined in laboratory microcosms containing mycorrhizal (unidentified fungal isolate 'Pink FMT 87:2') and non-mycorrhizal seedlings of Pinus sylvestris L. and Pinus contorta Dougl. ex Loud. The mycorrhizal plants had intact mycelial systems at different developmental stages, and microcosms contained non-sterile peat (pH 3.8) or peat adjusted to different pH values with CaO. In systems with mycorrhizal mycelium which had just started to colonize the peat no significant differences in 14 C assimilation were found, either with respect to substrate pH or mycorrhizal status of the plant. Loss of activity from the mycorrhizal plants was more rapid, however, probably mainly as a result of increased respiration from the infected root systems. After 8 wk growth in peat at pH 3.8 and 5.2 shoot weights of all seedlings were the same, whereas non-mycorrhizal plants had root systems twice the size of the mycorrhizal ones. In plants with well developed extramatrical mycelia translocation of labeled carbon to the mycelium growing at pH 3.8 was faster than that to mycelium growing at pH 5.2. After 4 d incubation, however, the percentage of the originally supplied carbon present in the mycelium was 5% regardless of substrate pH. Activity found in the peat surrounding non-mycorrhizal plants rarely exceeded 0.3%.

Kinetics of the Nitrite to Nitric Oxide Reaction in Peat

AbstractNitrite consumption and net NO production rates were measured across a pH range of about 3 to 8 and a temperature range of 5 to 40 or 10 to 70°C in nonsterile and sterile peat, respectively. The NO was sparged from stirred peat/water (1:10) slurries by N2 or Ar carrier gas and analyzed. Addition of small quantities, 1 to 280 µg NO‐2‐N g−1 dry peat, resulted in immediate No production. Biological production of NO exceeded chemical (nonbiological) production in nonsterile peat at pH &gt;5. At pH &gt;6, rates were less than 5 × 10−9 mol min−1 g−1 dry peat with chemical production rates relatively insignificant. Decreasing pH increased rates significantly, particularly the nonenzymatic process, which apparently dominates NO production at pH &lt;4.5. The chemical process was first order with respect to [NO‐2]. The dependence of [H+] was complex, approximating first‐order behavior at [H+] &lt;4 × 10−4 mol g−1 dry peat and more strongly dependent at higher concentrations. The activation energy for the chemical reaction at pH 5.09 was 52.3 kJ mol−1. The average value for nonsterile peat measured at pH 5.45 and 6.26 was 41.4 kJ mol−1. The stoichiometric coefficient, a, for the overall reaction NO‐2 → a No was 2.14 ± 0.39 in sterile peat and 2.00 ± 0.38 in nonsterile peat. In the latter case, where both enzymatic and nonenzymatic reactions occur, the value may also reflect the nonenzymatic reaction. Other nitrogenous gases, N2O, N2, NOx and CH3ONO were not observed under the flow conditions employed, suggesting that NO is the primary product of NO‐2 reduction.

Production of clonal plantlets of tropical pine in tissue culture via axillary shoot activation

A method for the production of multiple clonal plantlets of Pinuscaribaea var. hondurensis Morlet, P. oocarpa Schiede, and P. tecunumanii Equiluz and Perry (P. patula Schiede and Deppe ssp. tecunumanii (Equiluz and Perry) Styles) from juvenile sources is described. The procedure is based on the spontaneous production of axillary shoots following shoot elongation. Much interclonal variation exists with respect to expiant multiplication. The addition of activated charcoal (0.1%) stimulates shoot elongation, especially in recalcitrant clones of P. caribaea. Axillary bud initiation may be stimulated by 6-benzylaminopurine, but levels above 1 μM can result in failure of induced buds to form shoots. Rooting occurs at about 50% in nonsterile peat–perlite medium without auxin pretreatment. Rooted plantlets can be readily hardened and will grow normally in the glasshouse.

In vitro Propagation of Araucaria cunninghamii and Other Species of the Araucariaceae Via Axillary Meristems

Stem segments with 3-5 leaf axils, excised from the upper portion of the mainstem of 2-year-old hoop pine (Araucaria cunninghamii Aiton ex D. Don) seedlings, produced orthotropic buds from the concealed axillary meristems when cultured on a basal medium (BM) of half-strength Murashige and Skoog (MS) inorganic salts, the medium level of growth factors and amino acids of de Fossard, 20 g L sucrose and 6.5 g/L agar. This procedure was also successful with A. balansae, A. bidwillii, A. colurnnaris, A. hunsteinri, A. luxurians, A. montana, A. rulei, A. scopulorum and Agathis robusta and with stem segments from orthotropic coppice shoots of juvenile morphology collected from the stumps of 20-year old hoop pines felled near ground level. The hoop pine explants were highly sensitive to cytokinin; 1 μM and 10 μM 6-benzylaminopurine caused the formation of distorted buds and total inhibition of bud development respectively. Lofier concentrations (0.001-0.1 μM ) did not noticeably influence bud formation or development. A low rate of multiplication was induced by reculturing the stem segments after the excision of the initial shoots. New buds developed in the leaf axils of that part of the initial shoot which remained attached to the primary stem explant. Shoots derived from seedling and coppice cultures of hoop pine and seedling cultures of Agathis robusta rooted in vitro on BM + 0.1-10.0 μM indole-3-butyric acid (IBA), but with only 5-20% success. Up to 80% rooting was obtained if both hoop pine shoot types (i. e. from seedling and coppice cultures) were cultured on modified BM (quarter strength MS salts, 10 μM IBA plus no agar) for 2 weeks, before being transferred to a mixture of non-sterile peat and perlite or vermiculite and perlite, maintained under a high humidity (90-95%). Plantlets were subsequently transferred to normal glasshouse conditions and then to the field with less than 5% mortality. Thus hoop pine can be added to the relatively small number of conifers for which the capacity to micropropagate juvenile and mature plants and successfully establish their clones in the field has been demonstrated.

Determination of the number of root nodule bacteria in the presence of other organisms

Use of the plant-dilution method for the determination of the number of root-nodule bacteria in nonsterile carrier materials has been described for both small and large-seeded legume hosts. Calculation of the most probable number of rhizobia is described using values based on the appropriate tables of Fisher and Yates, and approximate factors of error for 95 per cent fiducial limits are stated. It was found that the plant-dilution method was likely to underestimate the number of rhizobia by a factor not greater than 2, when comparisons were made with pure cultures. However for non-sterile peat the tendency was for the plate count to give the lower estimate. This latter effect was no doubt due to the presence of other organisms and therefore the plant-dilution method is to be preferred in such cases.