Transformation of C-labelled lignin and humic substances in forest soil by the saprobic basidiomycetes Gymnopus erythropus and Hypholoma fasciculare

Litter decomposing basidiomycetous fungi produce ligninolytic oxidases and peroxidases which are involved in the transformation of lignin, as well as humic and fulvic acids. The aim of this work was to evaluate their importance in lignin transformation in forest litter. Two litter decomposing basidiomycete species differing in their abilities to degrade lignin - Hypholoma fasciculare, and Gymnopus erythropus - were cultured on sterile or non-sterile oak litter and their transformation of a ¹⁴C-labelled synthetic lignin (dehydrogenation polymer ¹⁴C-DHP) was compared with that of the indigenous litter microflora. Both in sterile and non-sterile litter, colonisation by basidiomycetes led to higher titres of lignocellulose-degrading enzymes, in particular of laccase and Mn-peroxidase (MnP). The titres of the latter were 6 to 40-fold increased in the presence of basidiomycetes compared to non-sterile litter. During 10 weeks, G. erythropus mineralised over 31% of ¹⁴C-DHP in sterile litter and 23% in non-sterile litter compared to 14% in the non-sterile control. Lignin mineralization by H. fasciculare was comparable to the non-sterile control, 12% in sterile litter and 16% in the non-sterile litter. The largest part of ¹⁴C from ¹⁴C-DHP was transformed into humic compounds during litter treatment with both fungi as well as in the control. In addition to the fast lignin mineralization, microcosms containing G. erythropus also showed a lower final content of unaltered lignin and 23-28% of the lignin was converted into water-soluble compounds with relatively low molecular mass (<5 kDa). Both G. erythropus and H. fasciculare were also able to further mineralise humic compounds. During a 10-week fungal treatment of an artificial ¹⁴C-humic acid (¹⁴C-HA) supplemented to the natural humic material of a forest soil, the fungi mineralised 42% and 19% of the labelled material, respectively, under sterile conditions. The ¹⁴C-HA mineralization by introduced basidiomycetes in microcosms containing non-sterile humic material, however, did not significantly differ from that of a non-sterile control and was around 12%. Altogether the results show that saprobic basidiomycetes can considerably differ in their rates of lignin and humic substance conversion. Furthermore, lignin degradation in forest soil can rather slow down by interspecific competition than it is accelerated by cooperation of different microorganisms occupying specific nutritional niches. Therefore, the overall contribution of saprobic basidiomycetes depends on their particular eco-physiological status and the competitive pressure, and may be often lower than initially expected. Significant lignin transformation including partial mineralization is seemingly not exclusively dependent on exceptional high titres of ligninolytic enzymes but also on so far unknown factors. Higher endocellulase production and subsequent weight loss was found in microcosms where saprobic basidiomycetes were combined with indigenous microbes. Potentially, lignin degradation by the basidiomycetes may have increased cellulose availability to the indigenous microbes.     

The biochemical transformation of oak (Quercus robur) leaf litter consumed by the pill millipede (Glomeris marginata)

Soil macrofauna play an essential role in the initial comminution and degradation of organic matter entering the soil environment and yet the chemical effects of digestion on leaf litter are poorly understood at the molecular level. This study was undertaken to assess the selective chemical transformations that saprophagous soil invertebrates mediate in consumed leaf litter. A number of pill millipedes (Glomeris marginata) were fed oak leaves (Quercus robur) after which the biomolecular compositions (lipids and macromolecular components) of the leaves and millipede faeces were compared using a series of wet chemical techniques and subsequent analysis by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). It was found that the concentrations of short chain (<C₂₀) n-alkanoic acids, sterols and triacylglycerols reduced dramatically in the millipede faeces relative to the leaf litter. Hydrolysable carbohydrates and proteins both decreased in concentration in the faeces, whereas similar yields of phenolic components were observed for the cupric oxidation products of lignin, although the oxygenated functionalities were affected by passage through the millipede gut, yielding a more highly condensed state for lignin. This shows that the chemical composition of fresh organic matter entering the soil is directly controlled by invertebrates feeding upon the leaf litter and as such that they are key contributors to the early stages of diagenesis in terrestrial soils.     

Lignin and cellulose degradation and nitrogen dynamics during decomposition of three leaf litter species in a Mediterranean ecosystem

Cellulose and lignin degradation dynamics was monitored during the leaf litter decomposition of three typical species of the Mediterranean area, Cistus incanus L., Myrtus communis L. and Quercus ilex L., using the litter bag method. Total N and its distribution among lignin, cellulose and acid-detergent-soluble fractions were measured and related to the overall decay process. The litter organic substance of Cistus and Myrtus decomposed more rapidly than that of Quercus. The decay constants were 0.47 year⁻¹, 0.75 year⁻¹ and 0.30 year⁻¹ for Cistus, Myrtus and Quercus, respectively. Lignin and cellulose contents were different as were their relative amounts (34 and 18%, 15 and 37%, 37 and 39% of the overall litter organic matter before exposure, for Cistus, Myrtus and Quercus, respectively). Lignin began to decrease after 6 and 8 months of exposure in Cistus and Myrtus, respectively, while it did not change significantly during the entire study period in Quercus. The holocellulose, in contrast, began to decompose in Cistus after 1 year, while in Quercus and Myrtus immediately. Nitrogen was strongly immobilized in all the litters in the early period of decay. Its release began after the first year in Cistus and Myrtus and after 2 years of decomposition in Quercus. These litters still contained about 60, 20 and 90% of the initial nitrogen at the end of the experiment (3 years). Prior to litter exposure nitrogen associated with the lignin fraction was 65, 54 and 37% in Cistus, Myrtus and Quercus, while that associated with the cellulose fraction was 30, 24 and 28%. Although most of the nitrogen was not lost from litters, its distribution among the litter components changed significantly during decomposition. In Cistus and Myrtus the nitrogen associated with lignin began to decrease just 4 months after exposure. In Quercus this process was slowed and after 3 years of decomposition 8% of the nitrogen remained associated with lignin or lignin-like substances. The nitrogen associated with cellulose or cellulose-like substances, in contrast, began to decrease from the beginning of cellulose decomposition in all three species. At the end of the study period most of the nitrogen was not associated to the lignocellulose fraction but to the acid-detergent-soluble substance (87, 88 and 84% of the remaining litter nitrogen).     

Elevated UV-B radiation modifies the extractability of carbohydrates from leaf litter of Quercus robur

We examined the influence of elevated UV-B radiation on the extractability of carbohydrates from leaf litter of Quercus robur. Saplings were exposed to a 30% elevation above the ambient level of erythemally weighted UV-B (280-315 nm) radiation for eight months at an outdoor facility. UV-B radiation was applied under arrays of fluorescent lamps filtered with cellulose diacetate, which transmitted both UV-B and UV-A (315-400 nm) radiation. Saplings were also exposed to elevated UV-A radiation under arrays of polyester-filtered lamps and to ambient radiation under arrays of non-energised lamps. Abscised leaves were collected, ground and sequentially treated with seven solvents in order to fractionate extractable carbohydrates based on the way in which they are held in the cell wall. Elevated UV-B radiation reduced the extractability of carbohydrates from cell walls of Q. robur. Sodium phosphate buffer at pH 7 extracted 10% less total carbohydrate from leaf material exposed during growth to elevated UV-B radiation under cellulose diacetate-filtered lamps than from leaf material grown under polyester-filtered and non-energised lamps. The cumulative amount of carbohydrate released by sequential extraction with phosphate buffer, CDTA, urea and sodium carbonate was between 5.1% and 7.8% lower from leaf material grown under cellulose diacetate-filtered lamps relative to that from leaves grown under non-energised lamps. Abscised leaves were also digested with Driselase, an enzyme mixture extracted from a basidiomycete fungus. No effects of elevated UV radiation were recorded on the amount of carbohydrate released by Driselase digestion. Regression analyses, using data from a previous field decomposition study, suggested that reduced availability of carbohydrates enhanced the colonisation of Q. robur litter by basidiomycete fungi, which then accelerated the decomposition rate of the litter in soil. We recommend that future studies into the effects of UV-B radiation on plant litter decomposition measure not only the concentrations of chemical constituents of litter, but also determine the availability of litter carbon sources to soil microbes, using methods similar to those used here.     

Drought decreases soil enzyme activity in a Mediterranean Quercus ilex L. forest

Longer and more severe drought periods are expected in the near future for Mediterranean ecosystems. Soil enzymes play an essential role in the nutrient mineralization and their activity is an exceptional sensor in predicting the capacity of nutrient supply to plants. We conducted an experiment of water availability manipulation in evergreen oak mountain stands with the aim to study the effects of enhanced drought on the activity of five soil enzymes. The drought treatment consisted of runoff exclusion by a ditch along the entire top edge of the upper part of treatment plots and partial rain exclusion by suspending PVC strips and funnels. The reduction of 10% of soil moisture produced by runoff exclusion decreased urease activity by 10-67%, protease activity by 15-66% and β-glucosidase activity by 10-80%, depending on annual period and soil depth. The reduction of 21% of soil moisture produced by runoff and rainfall exclusion together reduced urease activity by 42-60%, protease activity by 35-45%, β-glucosidase activity by 35-83% and acid phosphatase activity by 31-40%. No significant effects were observed on alkaline phosphatase activity. The activities of the enzymes involved in the nitrogen cycle, protease and urease, were the most affected by drought. In all cases, the activities of these enzymes strongly decreased with soil depth and they were greater in spring than in autumn. These results show the link between drought and a slower nutrient turn-over, which decreases the nutrient supply to plants.     

Laccase and peroxidase isoenzymes during leaf litter decomposition of Quercus ilex in a Mediterranean ecosystem

The dynamics of leaf litter decomposition of Quercus ilex (L.) were investigated over a 2 year period by determining the activities and isoenzyme distribution of laccases and peroxidases. The analysis of isoenzymes was performed by isoelectric focusing on high stability pH gradients with high resolving power. The preparation of zymograms was carried out using the leaf litter extract without previous concentration. During litter decomposition, laccase and peroxidase activities changed as well as the type and number of enzyme isoforms. The activities of both enzymes were low (≤0.017 and ≤0.031 mmol o-tolidine oxidized h⁻¹ g⁻¹ d.w. for laccase and peroxidase, respectively) in first year and increased in October-January of the second year of litter decay. The highest activities measured after 15-18 months of litter exposure (0.37±0.03 and 0.19±0.02 mmol o-tolidine oxidized h⁻¹ g⁻¹ d.w. for laccase and peroxidase, respectively), showed that litter chemical composition affected the growth of ligninolytic microbial community. The activation energy for laccase and peroxidase reactions also changed during decomposition: the highest values (55±6 kJ mol⁻¹ for laccase and 60±6 kJ mol⁻¹ for peroxidase) occurred in autumn-winter, even if spatial changes were evidenced. Some enzyme isoforms (pI=5.3 and 5.5 for laccase and pI=5.0 and 5.1 for peroxidase, respectively), contributed more than others to the overall laccase and peroxidase activity, suggesting that some ligninolytic species bloomed in particular seasons of the year, even if other species with similar functional activities colonized the litter.     

Mineralization of forest litter nutrients by heat and combustion

Temperature dependant mineralization dynamics during fire of litter species characteristic of the New Jersey pine barrens was determined. Senescent leaf material of pitch pine (Pinus rigida), white oak (Quercus alba) and black huckleberry (Gaylusssacia baccata) were collected at the time of abscission; sorted, ground and oven-dried at 70 °C. Replicate samples were then heated for 2 h at: 70, 100, 200, 300, 400, and 550 °C. Mass loss and total nitrogen and total phosphorus concentration of the heated material were determined. Additional samples of the residual material were extracted with deionized water, and the filtrate was assayed for the anions: , , ; and cations: , K⁺, Mg⁺⁺, and Ca⁺⁺.By heating leaf litter over a range of temperatures, to simulate the heterogeneous nature of forest litter burning, we identified patterns of nutrient mineralization characteristic of specific temperatures, some of which were common to all three litter species and others unique to individual species. In general, it appears that black huckleberry leaf litter was the most nutrient rich and the most labile. In huckleberry litter, there was a large reserve of soluble nitrogen, sulfur, phosphate, calcium and magnesium that became available upon heating to 200 °C. Pitch pine litter was the most nutrient poor, and the rates of nutrient mineralization were also generally the lowest of the three species studied. White oak litter nutrient concentration and rates of mineralization along the temperature gradient were intermediate. For all three litter species examined organic and inorganic nitrogen losses due to volatilization were >99% upon heating to 550 °C, and soluble magnesium concentrations declined significantly at temperatures of 300 °C, despite having a volatilization temperature greater than 1100 °C. Under the temperature range employed, heating of leaf litter resulted in little volatilization loss of phosphorus; however, the amount of soluble phosphate phosphorus was much lower in all three litter types at temperatures of 300 °C and above. With increasing temperatures, inorganic phosphate ions presumably became bound to cations in the ash, forming insoluble metal phosphates. The dramatic increase of the ratio of total phosphorus to soluble inorganic phosphate at higher temperatures, the loss of soluble magnesium above 300 °C, and the near complete loss of nitrogen at 550 °C suggests that after intense fires availability of these minerals may be dramatically reduced.     

Changes in chemical composition of Pinus sylvestris needle litter during decomposition along a European coniferous forest climatic transect

The objective of this investigation was to assess the changes in chemical composition (lignin, cellulose, hemicelluloses, non-structural compounds, N, and ash) of decomposing litter. Standard Pinus sylvestris needle litter, originating from southern Sweden, was incubated in litterbags at 15 sites selected from the Netherlands to south Spain. The changes in chemical composition of this litter were determined using near infrared reflectance spectroscopy. The hypothesis was that standard (chemically uniform) litter decomposing under a range of climates would show different dynamics of accumulation and loss of C-fractions, N, and ash, relative to mass loss. It was shown that, for a given mass-loss value (10, 20, 30, 40, or 50%), the proportion of lignin, cellulose, hemicelluloses, non-structural compounds, N, and ash in the decomposing pine needles differed between sites. Lignin concentration in the litter residue at 50% mass loss ranged from approximately 26 to 43%, cellulose from 19 to 27%, hemicelluloses from 7 to 11%, non-structural compounds from 19 to 25%, N from 0.7 to 1.3%, and ash content from 1.4 to 10.1%. Lignin concentrations showed the highest range of variation. Lignin concentrations during decomposition were positively related to moisture factors as significant correlations were found with actual evapotranspiration and were improved in multiple regressions by the mean annual precipitation or the water surplus. Cellulose was degraded further at sites with high precipitation whereas hemicellulose degradation was related to temperature. This leads to the conclusion that the remaining organic matter produced by standard litter decomposition within the studied climatic range of variations tended to be more recalcitrant under wet and warm climatic conditions than under cold or dry climate.     

Enzymatic activities of three ectomycorrhizal types of Quercus robur L. in relation to tree decline and thinning

In a declining oak forest, a thinning treatment was performed in August 2004, targeting trees belonging to two decline classes. The whole ectomycorrhizal (EM) community was dominated by the fungal symbionts Clavulina cinerea, Tomentella sublilacina and Russula spp. The potential activities of eight secreted enzymes, involved in mobilizing nutrients (N, P) from soil organic matter, were measured on these three EM types in winter and spring 2006 using multiwell microplate photometric and fluorogenic methods. The enzymatic activities recorded in winter were generally significantly higher than in spring. Most of the enzyme activities studied, and particularly phosphatase and β-glucosidase, changed according to both decline class and sylvicultural treatment. In spring, each anatomotype displayed different enzymatic profile according to the decline class. These results suggest that the potential enzymatic activity of ectomycorrhizae adapts to the changes resulting from the sylvicultural treatment and reacts to the anthropic disturbance by adjusting to the new resource structure.     

Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere

The capability of native bacterial strains isolated from Lolium perenne rhizosphere to behave as plant growth promoting bacteria and /or biocontrol agents was investigated. One strain (BNM 0357) over 13 isolates from the root tips of L. perenne resulted proved to be nitrogenase positive (ARA test) and an IAA producer. Conventional tests and the API 20E diagnostic kit indicated that BNM 0357 behaves to the Enterobacteriaceae family and to the Enterobacter genus. Molecular identification by 16S rRNA sequence analysis indicated that BNM 0357 had the highest similarity to Enterobacter ludwigii (EN-119). Isolate BNM 0357 had the capability to solubilize calcium triphosphate and to antagonize Fusarium solani mycelial growth and spore germination. Strain BNM 0357 also showed the ability to improve the development of the root system of L. perenne. This study disclosed features of E. ludwigii BNM 0357 that deserve further studies aimed at confirming its putative importance as a PGPR.     

Collembolan grazing affects the growth strategy of the cord-forming fungus Hypholoma fasciculare

Mycelia of cord-forming fungi show remarkable patterns of reallocation of biomass and nutrients indicating an important role of these, often extensive, organisms in the spatial translocation of energy and nutrients in forest soils. Despite the rich tradition of interaction studies between soil microarthropods and fungi, the spatial implications of these interactions, due to the potential growth responses of the fungi and to the translocation of energy and nutrients within the mycelial network, have been largely ignored. In this paper we analyse fungal growth responses in two-dimensional model systems composed of compressed soil, the cord-forming fungus Hypholoma fasciculare and three fungivorous Collembolan species. We hypothesised that (i) the highly co-ordinated nature of cord-forming fungi would lead to growth responses following collembolan grazing, and that, (ii) such changes are dependent on grazing intensity, and (iii) changes are dependent on the species grazing. Mycelial extent and hyphal cover decreased with increasing grazing density; at highest grazing density also the fractal dimension of the mycelial border decreased, indicating a less branched foraging front due to the regression of fine hyphae and the development of mycelial cords. Effects differed greatly between collembolan species although they exerted comparable grazing pressure (the smaller species were added in larger numbers according to their allometric size-metabolic rate relationships): while grazing by Folsomia candida resulted in less mycelial extension and hyphal cover, these variables were not affected when Proisotoma minuta and Hypogastrura cf. tullbergi grazed. The effects of a species mix suggested an additive effect of the component species. This shows that fungal mycelia may suffer from damage caused by few but large collembolans, affecting extension as well as coverage of the mycelium, but that fungi may compensate for the biomass loss caused by more but slightly smaller collembolans. In about 20% of the model systems H. fasciculare switched from a growth pattern with a broad contiguous foraging front and uniform growth in all directions to a pattern with fast growing sectors while other sectors stopped growth completely. The switch occurred in grazed systems exclusively; thus we interpret this observation as a fugitive response and as a strategy for quickly escaping from places where grazing pressure is experienced.     

Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.     

Immobilization of avian excreta-derived nutrients and reduced lignin decomposition in needle and twig litter in a temperate coniferous forest

The possible effects of excreta of the Great Cormorant Phalacrocorax carbo on decomposition processes and dynamics of nutrients (N, P, Ca, K, Mg) and organic chemical components (lignin, total carbohydrates) were investigated in a temperate evergreen coniferous forest near Lake Biwa in central Japan. Two-year decomposition processes of needles and twigs of Chamaecyparis obtusa were examined at two sites, control site never colonized by the cormorants (site C) and colonizing site (site 2). Mass loss was faster in needles than in twigs. Mass loss of these litter types was faster at site C than at site 2, which was ascribed to the decreased mass loss rate of acid-insoluble ‘lignin’ at site 2. Net immobilization of N, P, and Ca occurred in needles and twigs at site 2; whereas at site C, mass of these elements decreased without immobilization during decomposition. Duration of immobilization phase of these nutrients at site 2 was estimated to be 1.6 to 2.5 years in needles and 19.6 to 23.5 years in twigs. Immobilization potential (maximum amount of exogenous nutrient immobilized per gram initial material) was similar between needles and twigs for N and Ca but was about 10 times higher in twigs than in needles for P. δ¹³C in needles was relatively constant during the first year and then increased during the second year, whereas δ¹³C in twigs was variable during decomposition. Acid-insoluble fraction was depleted in ¹³C compared to whole needles (1.6-2.1‰) and twigs (2.0-2.5‰). δ¹⁵N of needles and twigs and their acid-insoluble fractions approached to δ¹⁵N of excreta during decomposition at site 2. This result demonstrated the immobilization of excreta-derived N into litter due to the formation of acid-insoluble lignin-like substances complexed with excreta-derived N. No immobilization occurred in K and Mg and their mass decreased during decomposition at both sites. Based on these results of nutrient immobilization during decomposition and on the data of litter fall and excreta amount at site 2, we tentatively calculated stand-level immobilization potential of litter fall and its contribution to total amount of N and P deposited as excreta. Thus, the potential maximum amount immobilized into litter fall (needles and twigs) was estimated to account for 5-7% of total excreta-derived N and P.     

Polyphasic characteristics of bradyrhizobia isolated from nodules of peanut (Arachis hypogaea) in China

Peanuts (Arachis hypogaea L.) were introduced to China about 500 years ago. However, the diversity of Rhizobial strains in China that can nodulate peanut was poorly understand. Diversity and phylogeny of 50 slow-growing strains, isolated from root nodules of peanut in different geographical regions of China, were studied using polyphasic techniques. All stains were clustered by phenotypic tests into two distinct groups: Group I: 16S rRNA RFLP genotype 3, and Group II, which divided into 16S rRNA RFLP genotypes 1 and 2. Genotype 1 shares the same genotype with USDA110, USDA122 and USDA127 of Bradyrhizobium japonicum, and genotype 2 solely consisted of extra-slow growing bradyrhizobia isolated from Hongan, China. Results of 16S rRNA sequencing revealed that peanut bradyrhizobia were phylogenetically related to B. japonicum and their sequence divergence was less than 1.1%. Based upon the size of the internally transcribed spacer (ITS) between the16S and 23S RNA genes, strains were classified into ITS-I, ITS-II and ITS-III genotypes. Strains could be further divided into sub-clusters IA, IB, IIa, IIb and IIc five sub-clusters through ITS PCR-RFLP and repetitive extragenic palindromic PCR (REP-PCR) analysis. Host specificity test revealed that all peanut bradyrhizobia tested nodulated Phaseolus vulgaris and strains of clusters IIb and IIc nodulated Glycine soja efficiently. Bradyrhizobia isolated from peanut were related, but still exhibited phylogenetical divergence with B. japonicum.     

Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: the role of laccase and versatile peroxidase

The ability of Pleurotus eryngii, Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus sajor-caju to degrade the aromatic pollutants 2,4-dichorophenol (2,4-DCP) and benzo(a)pyrene [B(a)P] in liquid culture and microcosm (using wheat straw as growth substrate and sea sand as a xenobiotic carrier) was investigated by HPLC and ¹⁴CO₂ release from labeled pollutants. We found that 100 μM 2,4-DCP was very quickly transformed by the four fungi, disappearing 24 h after its addition to the liquid cultures. However, a 2-week incubation period was required to transform 100 μM B(a)P up to 75% by P. eryngii and P. pulmonarius. Whereas the fungi were able to begin degradation of the two pollutants with high transformation rates, their complete degradation (mineralization) rates were very low. Mineralization of B(a)P in liquid cultures was only observed with P. eryngii and P. pulmonarius, although the four Pleurotus species studied were able to mineralize this compound in solid state fermentation (SSF). The ligninolytic enzymes laccase and versatile peroxidase (VP), together with aryl-alcohol oxidase (AAO) providing extracellular H₂O₂, were found in liquid cultures. Except AAO, these enzymes were also detected in SSF experiments. In order to investigate the role of ligninolytic enzymes in the process, their action on both pollutants (50 μM) was studied in vitro in the absence and presence of redox mediators. As observed with the fungal cultures, 2,4-DCP was oxidized faster than B(a)P by both laccase (60% transformation after 6 h) and VP (100% transformation after 1 h). Moreover, laccase oxidation was strongly increased (up to 90% transformation after 3 h), by the presence of the mediators 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT). In the case of B(a)P, the presence of ABTS or HBT was strictly required for oxidation by laccase (25% transformation after 8 h). Degradation of B(a)P was also observed in reactions with VP (40% transformation after 6 h). The results obtained suggest that Pleurotus species can be used in applications focused to the degradation of aromatic pollutants using wheat straw as a growth substrate, and provide the first evidence on the direct transformation of recalcitrant aromatic pollutants by VP.     

Promotion of plant growth and in situ degradation of phenol by an engineered Pseudomonas fluorescens strain in different contaminated environments

We show that Pseudomonas fluorescens strain P13, a plant growth-promoting bacterium, enhanced the growth of corn in uncontaminated soil but not in contaminated soil, perhaps because of its inability to reduce phytotoxicity. Another bacterial strain, Pseudomonas aeruginosa strain SZH16, showed in situ phenol-degrading activity and contained a plasmid loaded with a gene encoding for catechol 2, 3-dioxygenase, an important enzyme in the degradation pathway of aromatic compounds. We implanted this biodegradation ability into strain P13, using horizontal gene transfer techniques using strain SZH16 as the donor and P13 as the recipient, to generate a phenol-degrading transconjugant which obtained the effective plasmid from strain SZH16. Introduction of the transconjugant P13 strain into an artificially phenol-spiked soil promoted the growth of corn and in situ phenol degradation, and the increase in plant biomass correlated with the decrease in soil phenol content. Furthermore, the transconjugant P13 strain was also found to stimulate corn growth and reduce phenol concentration in water containing phenol and in historically contaminated field soils, indicating that the transconjugant strain could promote plant growth in both contaminated and uncontaminated environments. The transconjugant P13 strain was more efficient than either strain P13 or SZH16, and shows how plant growth-promoting bacteria which show no, or only limited, ability to degrade organic pollutants may be modified. This technique is attractive for many environmental remediation and agronomic applications.     

Competition for inorganic and organic N by blue oak (Quercus douglasii) seedlings, an annual grass, and soil microorganisms in a pot study

Sources of competition for limited soil resources, such as nitrogen (N), include competitive interactions among different plant species and between plants and soil microorganisms (microbes). To study these competitive interactions, blue oak seedlings (Quercus douglasii) were grown alone or grown together with an annual grass, wild oats (Avena barbata) in pots containing field soil. We injected ¹⁵N-labeled ammonium, nitrate or glycine into the soil of each pot and harvested plants 5 days later. Plant shoots and roots, soil microbial N and soil KCl-extractable N were analyzed for ¹⁵N content. When oak and grass were grown together, ¹⁵N recovery from the inorganic N treatments (NH₄⁺, or NO₃⁻) was 34, 9 and 4% for the grass, microbes and oak seedlings, respectively, and only 1% remained as KCl-extractable N. ¹⁵N recovery from the glycine treatment was 18, 22, 5% for the grass, microbes and oak seedlings, respectively, and 4% remained as KCl-extractable N. When oaks were grown alone, ¹⁵N recovery by soil microbes was 21, 48 and 40% in the NO₃⁻, NH₄⁺ and glycine treatments, respectively. N forms had no effects on ¹⁵N recovery in oak seedlings (7%) and in KCl-extractable N pool (13%). In general, total N recovery by the grass was much greater than by oaks. However, on a fine root surface area or length basis, oaks exhibited higher N uptake than the grass. Our results suggest that the high rooting density and rapid growth rate of the annual grasses such as Avena barbata made them superior competitors for available soil N when compared to blue oak seedlings and to microbes. Soil microbes were better competitors for organic than inorganic N when annual grasses were present, but preferred NH₄⁺ when competing only with oak seedlings.     

Temperature sensitivity of respiration differs among forest floor layers in a Pinus resinosa plantation

Decomposer microorganisms contribute to carbon loss from the forest floor as they metabolize organic substances and respire CO₂. In temperate and boreal forest ecosystems, the temperature of the forest floor can fluctuate significantly on a day-to-night or day-to-day basis. In order to estimate total respiratory CO₂ loss over even relatively short durations, therefore, we need to know the temperature sensitivity (Q₁₀) of microbial respiration. Temperature sensitivity has been calculated for microbes in different soil horizons, soil fractions, and at different depths, but we would suggest that for some forests, other ecologically relative soil portions should be considered to accurately predict the contribution of soil to respiration under warming. The floor of many forests is heterogeneous, consisting of an organic horizon comprising a few more-or-less distinct layers varying in decomposition status. We therefore determined at various measurement temperatures the respiration rates of litter, F-layer, and H-layer collected from a Pinus resinosa plantation, and calculated Q₁₀ values for each layer. Q₁₀ depended on measurement temperature, and was significantly greater in H-layer than in litter or F-layer between 5 and 17 °C. Our results indicate, therefore, that as the temperature of the forest floor rises, the increase in respiration by the H-layer will be disproportionate to the increase by other layers. However, change in respiration by the H-layer associated with change in temperature may contribute minimally or significantly to changes of total forest floor respiration in response to changes in temperature depending on the depth and thickness of the layer in different forest ecosystems.     

Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N₂-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.     

Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed

A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.