Potential gene exchange between Bacillus thuringiensis subsp. kurstaki and Bacillus spp. in soil in situ

The possible transfer of genes from Bacillus thuringiensis subsp. kurstaki (Btk) to indigenous Bacillus spp. was investigated in soil samples from stands of cork oak in Orotelli (Sardinia, Italy) collected 5 years after spraying of the stands with a commercial insecticidal preparation (FORAY 48B) of Btk. Two colonies with a morphology different from that of Btk were isolated and identified as Bacillus mycoides by morphological and physiological characteristics and by 16S rDNA analysis. Amplification by the polymerase chain reaction (PCR) of the DNA of the two isolated B. mycoides colonies with primers used for the identification of the Btk cry genes showed the presence of a fragment of 238 bp of the cry1Ab9 gene that had a similarity of 100% with the sequence of the cry1Ab9 gene present in GenBank, indicating that the isolates of B. mycoides acquired part of the sequence of this gene from Btk. No cells of Btk or B. mycoides carrying the 238-bp fragment of the cry1Ab9 gene were isolated from samples of unsprayed control soil. However, the isolates of B. mycoides were not able to express the partial Cry1Ab protein. Hybridization with probes for IS231 and the cry1Ab9 gene suggested that the inverted repeated sequence, IS231, was probably involved in the transfer of the 238-bp fragment from Btk to B. mycoides. These results indicate that transfer of genes between introduced Btk and indigenous Bacillus spp. can occur in soil under field conditions.     

Interaction between the soil yeast Rhodotorula mucilaginosa and the arbuscular mycorrhizal fungi Glomus mosseae and Gigaspora rosea

The effect of the soil yeast, Rhodotorula mucilaginosa LBA, on Glomus mosseae (BEG n°12) and Gigaspora rosea (BEG n°9) was studied in vitro and in greenhouse trials. Hyphal length of G. mosseae and G. rosea spores increased significantly in the presence of R. mucilaginosa. Exudates from R. mucilaginosa stimulated hyphal growth of G. mosseae and G. rosea spores. Increase in hyphal length of G. mosseae coincided with an increase in R. mucilaginosa exudates. No stimulation of G. rosea hyphal growth was detected when 0.3 and 0.5 ml per petri dish of yeast exudates was applied. Percentage root length colonization by G. mosseae in soybean (Glycine max L. Merill) and by G. rosea in red clover (Trifolium pratense L. cv. Huia) was increased only when the soil yeast was inoculated before G. mosseae or G. rosea was introduced. Beneficial effects of R. mucilaginosa on arbuscular mycorrhizal (AM) colonization were found when the soil yeast was inoculated either as a thin agar slice or as a volume of 5 and 10 ml of an aqueous solution. R. mucilaginosa exudates (20 ml per pots) applied to soil increased significantly the percentage of AM colonization of soybean and red clover.     

外来植物紫茎泽兰入侵对菌根菌群落的影响

为了研究紫茎泽兰(Ageratina adenophora)入侵对土壤菌根真菌(mycorrhizal fungi, MF)群落的影响,采用嵌套PCR 技术分析了外来植物紫茎泽兰入侵生境内土著植物群落、土著植物与紫茎泽兰混生群落、紫茎泽兰单优群落中, 侵染紫茎泽兰及土著植物的MF 群落结构, 及紫茎泽兰与土著植物根围土壤中MF 群落结构。结果表明, 紫茎泽兰不同入侵进程MF 群落结构存在差异, 其中, 从土著植物群落的植物根内检测到内养球囊霉(Glomus intraradices)型克隆; 从土著植物与紫茎泽兰混生群落的紫茎泽兰根内也检测到内养球囊霉型克隆, 而在土著植物根内检测到1 个球囊霉属(Glomus sp 2)型克隆; 从紫茎泽兰单优群落的紫茎泽兰根内未检测到MF, 但从其根围土壤中检测到2 个球囊霉属(Glomus sp 1 和Glomus sp 2)型克隆。在土著植物与紫茎泽兰混生群落中, 从紫茎泽兰根围土壤中检测到4 个克隆型, 分别为毛舌菌阔孢(Trichoglossum hirsutum)、皂味口磨(Tricholoma saponaceum)、亚盖趋本菌(Xylobolus subpileatus)和翘鳞肉齿菌(Sarcodon imbricatus), 从土著植物根围土壤中也检测到4 个克隆型, 分别为小皮伞(Camarophyllopsis hymenocephala)、肉色香蘑(Lepista irina)、皂味口磨及亚侧耳(Panellus serotinus)型克隆; 在土著植物群落中, 从根围土壤只检测到皂味口磨型克隆。紫茎泽兰入侵改变了土著MF 群落结构, 其中在土著植物占据的土壤中以外生菌根真菌为主, 而外来植物紫茎泽兰则更多地积累了丛枝菌根真菌。文中讨论了紫茎泽兰改变入侵地土壤菌根菌群落及其可能对紫茎泽兰入侵的反馈。     

The importance of secondary forest blocks for terrestrial mammals within an Acacia/secondary forest matrix in Sarawak, Malaysia

With the rapid conversion of tropical forests in Asia to plantation forests for timber or biofuel production there is a need to determine if these forests serve any viable role in wildlife conservation. We used infra-red trip cameras to survey for large terrestrial mammals within an Acacia/secondary forest matrix being created for pulpwood production in Sarawak, Malaysia. We detected at least 27 species of mammals within the matrix and 18 species were detected in both Acacia and secondary forest. Using occupancy modeling to determine important covariates for seven mammal species within the project area, six of these species were sensitive to forest type, and most were sensitive to the amount of secondary forest within 1 km of the sample point. For four species (sun bear, Helarctos melayanus; common porcupine, Hystrix brachyura; mousedeer, Tragulus spp.; and pig-tailed macaque, Macaca nemestrina), the mean distance of detection from large secondary forest blocks was significantly closer than expected from the distribution of sample points. Most species used Acacia forest less frequently than secondary forest, possibly for transit or foraging, with the exception of bearded pigs (Sus barbatus), sambar (Rucervus unicolor), and civets which were more common in Acacia stands. The amount of secondary forest preserved within forest plantations seems to be the best measure of conservation potential for these industrial forests. Forest plantations can provide a conservation value if managed properly and those retaining significant amounts of secondary forest should be eligible for a base level of certification as they comprise a significant portion of the landscape in this region.     

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.     

Comparison of g23 gene sequence diversity between Novosphingobium and Sphingomonas phages and phage communities in the floodwater of a Japanese paddy field

Our previous study indicated that the diversity of the major capsid gene (g23) of T4-type bacteriophages (phages) of Novosphingobium and Sphingomonas strains isolated from the floodwater of a Japanese paddy field is comparable to those of the clones obtained from other Japanese paddy fields. For more strict comparison of the diversity, this study examined g23 sequences between Novosphingobium and Sphingomonas phages and phage communities in the identical floodwater of a Japanese paddy field. The clones were obtained by applying g23-specific primers to DNA extracted from the floodwaters. Many 23 clones in the floodwater were grouped into the same clusters of Paddy Groups I-VI with g23 genes of Novosphingobium/Sphingomonas phages with some clones belonging to an additional cluster. In addition, the remaining clones belonged to the clusters of marine clones and T4-type enterophages. These findings indicate that the g23 genes in the floodwater are more diversified than those of Novosphingobium/Sphingomonas phages including g23 genes closely related to the genes of enterophages and marine origins.     

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.     

Physiological responses to acid stress of an acid-soil tolerant and an acid-soil sensitive strain of Rhizobium leguminosarum biovar trifolii

Physiological responses to acid stress in two strains of Rhizobium leguminosarum bv trifolii of differing acid-soil tolerance were compared. Acidity affected the size and morphology of the acid-tolerant strain, WSM409, but not of the acid-sensitive strain, TA1. Acid grown cells of WSM409 and TA1 had less cell-associated Ca and Mg and more P than cells grown at pH 7.0. Potassium content was lower in acid grown cells; WSM409 was less affected by pH than that in TA1. WSM409 was more tolerant of pH shock at pH 3.5 when grown at pH 4.8 than when grown at pH 7.0. TA1 was more sensitive to pH shock when grown at pH 4.8 than when grown at pH 7.0. WSM409 shows a characteristic adaptive acid tolerance response, whereas TA1 shows an acid sensitive response.     

Humus macro-morphology and soil microbial community changes along a 130-yr-old Fagus sylvatica chronosequence

We aimed to characterize humus macro-morphology and the associated soil microbial community within the unmodified litter (OL), the fragmented and humified layers (FH) and the organo-mineral (A) layer along a beech (Fagus sylvatica L.) forest chronosequence with four stand age-classes (15-, 65-, 95-, 130-yr-old) in Normandy, France. Humus macro-morphology was described with 36 quantitative and semi-quantitative variables. We measured microbial biomass N (N_mic), microbial N quotient (N_mic-to-N_t), fungal ergosterol, bacterial and fungal DNA using 16S and 18S rDNA real-time qPCR and evaluated the potential metabolic profile of heterotrophic bacteria within each soil layer and stand age-class. The log-transform ergosterol/fungal DNA ratio (EFR index) was used as an indicator related to active fungal biomass and the fungal/bacterial (F/B) ratio was calculated from qPCR results. There was a shift from mull (mainly dysmull) to moder humus forms along the chronosequence. While the N_mic did not change significantly, the N_mic-to-N_t decreased along the chronosequence in the OL layer. Ergosterol content increased in FH and A layers and the F/B ratio increased in the FH layer with increasing beech forest age. The EFR index was significantly higher in the OL and A layers of the oldest stands, whereas the highest EFR index in the FH layer occurred in the 15-yr-old stands. The functional diversity of heterotrophic bacteria was greater within OL and FH layers of 130-yr-old stands, but highest in the A layer of 15-yr-old stands while the Average Well Color Development remained stable for all soil layers. We found significant correlations between macro-morphology and microbial variables, especially between FH-based morphology and fungal biomass. Our main results are that beech forest maturation is accompanied by (1) an increase in fungal biomass in the FH layers and, (2) an increase in heterotrophic bacteria functional diversity in the organic layers. We have identified key macro-morphology variables that are good predictors of the structural and functional profile of the soil microbial community during beech forest development.     

Temporal shifts in diversity and quantity of nirS and nirK in a rice paddy field soil

Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.     

No evidence that nitrogen enrichment affect fungal communities of Vaccinium roots in two contrasting boreal forest types

In boreal forests ericaceous shrubs often dominate the forest floor vegetation. Nitrogen enrichment has been shown to decrease shrub abundance and in this study we explored whether it also affects the root associated fungal communities. Fine roots of Vaccinium myrtillus were collected in a Norway spruce dominated forest and of Vaccinium vitis-idaea in a Scots pine dominated forest. In both forests, nitrogen enrichment was experimentally induced by adding 12.5 and 50 kg N ha⁻¹ yr⁻¹ for 12 (spruce forest) and four (pine forest) years. Based on terminal restriction fragment length polymorphisms, subcloning and sequencing analyses, the root associated fungal communities were examined. We found 93 fungal species including Asco-, Basidio- and Zygo-mycota. In general, the Rhizoscyphus ericae aggregate was the most dominant and this was followed by Herpotrichiellaceae and Sebacina. Ordination analysis revealed that nitrogen enrichment did not change species composition of the fungal communities in neither the spruce nor the pine forest, while fungal community structures were clearly discriminated between the dominant shrub species in each forest. Similarly, no fungal species showed a significant response to nitrogen enrichment. Therefore, nitrogen enrichment appears to have no effect on root associated fungi of understorey dwarf shrubs in boreal forests, while it is clear that spruce and pine forests harbor distinctive communities of these fungi.     

Controlled ectomycorrhization of an exotic legume tree species Acacia holosericea affects the structure of root nodule bacteria community and their symbiotic effectiveness on Faidherbia albida, a native Sahelian Acacia

Many fast growing tree species have been introduced to promote biodiversity rehabilitation on degraded tropical lands. Although it has been shown that plant productivity and stability are dependent on the composition and functionalities of soil microbial communities, more particularly on the abundance and diversity of soil symbiotic micro-organisms (mycorrhizal fungi and rhizobia), the impact of tree introduction on soil microbiota has been scarcely studied. This research has been carried in a field plantation of Acacia holosericea (Australian Acacia species) inoculated or not with an ectomycorrhizal fungus isolate, Pisolithus albus IR100. After 7 year's plantation, the diversity and the symbiotic properties of Bradyrhizobia isolated from the plantation soil or from the surrounding area (Faidherbia albida (Del.) a. Chev. parkland) and able to nodulate F. albida, a native Sahelian Acacia species, have been studied. Results clearly showed that A. holosericea modified the structure of Bradyrhizobia populations and their effectiveness on F. albida growth. This negative effect was counterbalanced by the introduction of an ectomycorrhizal fungus, P. albus, on A. holosericea root systems.In conclusion, this study shows that exotic plant species can drastically affect genotypic and symbiotic effectiveness of native Bradyrhizobia populations that could limit the natural regeneration of endemic plant species such as F. albida. This effect could be counterbalanced by controlled ectomycorrhization with P. albus. These results have to be considered when exotic tree species are used in afforestation programs that target preservation of native plants and soil ecosystem rehabilitation.     

Overwintering management on upland pasture causes shifts in an abundance of denitrifying microbial communities, their activity and N2O-reducing ability

Pasture soils used for cattle overwintering may represent significant sources of N₂O emissions from soils. Therefore, the long-term effect of cattle overwintering on the abundance and activity of a denitrifying community was explored. The study was performed at a cattle overwintering area in South Bohemia (Czech Republic), where three sites differing in the degree of animal impact were selected: severely impacted (SI) and moderately impacted (MI), as well as a control site with no impact (NI). N₂O flux measurement and soil sampling were performed in spring and fall of 2005. The activity was measured in terms of potential denitrification activity. Bacterial nirK, nirS and nosZ genes were used as functional markers of the denitrifying communities; abundance was analyzed using a real-time PCR assay. Surprisingly, in situ N₂O emissions were the highest in spring at MI and significantly differed from those at SI and NI, while in autumn, rates of emissions generally decreased. In contrast potential denitrification rates were highest at SI, followed by MI, and the lowest at NI. An overall significant shift in N₂O/N₂ molar ratio was shown in cattle impacted sites. The highest abundance of all genes measured at both sampling times was found at site SI, whereas at site MI increased numbers were observed only in spring. Our results indicate a strong influence of cattle on the abundance as well as the activity of microbes involved in denitrification.     

Symbiotic relationships and root nodule ultrastructure of the pasture legume Biserrula pelecinus L.—a new legume in agriculture

Biserrula pelecinus is a pasture legume species new to Australian agriculture. The potential N benefit from B. pelecinus pastures in agricultural systems may not be realised if its symbiotic interactions with Mesorhizobium spp. are not well understood. This study evaluated the symbiotic interactions of four strains of Biserrula root-nodule bacteria (WSM1271, WSM1283, WSM1284, WSM1497) with four genotypes of B. pelecinus (cv. Casbah, 93GRC4, 93ITA33, IFBI1) and with a range of related legumes, including species known to be nodulated by strains of Mesorhizobium loti and other Mesorhizobium spp. Structures of root nodules were studied using light and electron microscopy enabling the ultrastructure of effective and ineffective nodules to be compared. B. pelecinus always formed typical indeterminate, finger-like nodules. The number of bacteroids inside symbiosomes varied between host×strain combinations, however, nodules formed by ineffective associations had well developed peribacteroid membranes and abundant bacteroids. Considerable variation was found in N₂-fixing effectiveness of strains isolated from B. pelecinus on the four B. pelecinus genotypes. Strains WSM1271, WSM1284 and WSM1497 nodulated Astragalus membranaceus, only strains WSM1284 and WSM1497 nodulated Astragalus adsurgens. Strain WSM1284 also nodulated Dorycnium rectum, Dorycnium hirsutum, Glycyrrhiza uralensis, Leucaena leucocephala, Lotus edulis, Lotus glaber, Lotus maroccanus, Lotus ornithopodioides, Lotus pedunculatus, Lotus peregrinus, Lotus subbiflorus and Ornithopus sativus. The four strains from B. pelecinus did not nodulate Amorpha fruticosa, Astragalus sinicus, Cicer arietinum, Hedysarum spinosissimum, Lotus parviflorus, Macroptilium atropurpureum or Trifolium lupinaster. M. loti strain SU343 nodulated all four genotypes of B. pelecinus. However, M. loti strain CC829 only nodulated B. pelecinus genotypes 93ITA33 and IFBI1 and the nodules were ineffective. The root nodule isolates from H. spinosissimum (E13 and H4) nodulated B. pelecinus cv. Casbah whereas the commercial inoculant strain for Cicer (CC1192) could not nodulate any genotype of B. pelecinus. These results indicate that strains WSM1271, WSM1283 and WSM1497 isolated originally from B. pelecinus have a specific host range while strain WSM1284 is promiscuous in its capacity to nodulate with a broad range of related species. As B. pelecinus can be nodulated by Mesorhizobium spp. from other agricultural legumes, particularly Lotus, there is an opportunity to utilise this trait in cultivar development.     

Brassica napus seed meal soil amendment modifies microbial community structure, nitric oxide production and incidence of Rhizoctonia root rot

A low glucosinolate content (21.8 μmol g⁻¹) Brassica napus seed meal (RSM) applied to orchard soils altered communities of both pathogenic and saprophytic soil micro-organisms. RSM amendment reduced infection by native and introduced isolates of Rhizoctonia spp. and recovery of Pratylenchus spp. from apple roots. Root infection by Rhizoctonia solani AG-5 was also suppressed in split-root assays where a portion of the root system was cultivated in RSM-amended soils and the remainder grown in the presence of the pathogen but lacking RSM. R. solani hyphal growth was not inhibited by RSM amendment. Suppression of Pratylenchus was attained to an equivalent extent by amending soils with either RSM or soybean meal (SM) when applied to provide a similar N content. Thus, glucosinolate hydrolysis products did not appear to have a significant role in the suppression of Rhizoctonia spp. or Pratylenchus spp. obtained via RSM amendment. RSM amendment elevated populations of Pythium spp. and of ammonia-oxidizing bacteria that release nitric oxide but suppressed fluorescent pseudomonad numbers. Streptomyces spp. soil populations increased significantly in response to RSM but not SM amendment. The vast majority of Streptomyces spp. recovered from the apple rhizosphere produced nitric oxide and possessed a nitric oxide synthase homolog. We propose that transformations in the bacterial community structure are associated with the observed control of Rhizoctonia root rot, with NO production by soil bacteria potentially having a role in the induction of plant systemic resistance.     

Association between Burkholderia species and arbuscular mycorrhizal fungus spores in soil

Burkholderia pseudomallei, the bacterial cause of the potentially fatal infection known as melioidosis, has a facultative intracellular lifestyle. The intracellular presence of B. pseudomallei in various eukaryotes including arbuscular mycorrhizal fungus (AMF) spores can be demonstrated in vitro. AMF spores were isolated from soils in a melioidosis-endemic area. B. pseudomallei and other Burkholderia spp. DNA was detected in these AMF spore samples, confirming an AMF spore-Burkholderia spp. association in soils which did not yield Burkholderia spp. by culture. This association may explain the environmental persistence, difficulty of recovery and dispersal of Burkholderia spp. in specific environments.     

Hybridization mechanisms between the endangered marble trout (Salmo marmoratus) and the brown trout (Salmo trutta) as revealed by in-stream experiments

Populations of the marble trout (Salmo marmoratus) are critically declining due to introgression by brown trout (S. trutta) strains. Hybrids between the two forms are fertile and presently predominant in most rivers of the species’ range.The involved hybridization mechanisms have been studied through two large scale in-stream experiments (Driselpoh and Stopnikarca) as 50% of each species have been stocked at the age of one year in fishless streams, each fish being individually marked. Diagnostic molecular markers were applied to test a partial reproductive isolation between the two species. Emphasis was put on survival and growth patterns of stocked fish (parental generation) as well as on fish hatched within the stream (F₁ generation).No evidence of a partial reproductive barrier between the two species was observed. Survival of the parental generation depended on the year in both streams, as well as on the species in Stopnikarca, but was identical for both species in Driselpoh. In both streams survival of instream hatched individuals measured from 0+ to 2+ was lower for brown trout. In Driselpoh, F₁ 0+, 1+ and 2+ hybrids were larger than pure individuals. Larger hybrids were only observed in Stopnikarca when analyses focused on individuals in inter-specific competition suggesting that heterosis and stress effects may explain the observed size differences.Our results point out important ecological differences between marble and brown trout and have shown that hybridization can easily take place. The findings indicate that high F₁ hybrid presence, survival and heterosis effects may impede marble trout rehabilitation in the area.     

Factors affecting the behavioural responses of whooper swans (Cygnus c. cygnus) to various human activities

The effects of human activity on bird behaviour and distribution have been studied extensively in recent years, but variation in their response to disturbance is still poorly understood. Here, we analyse variation in the behaviour of wintering whooper swans Cygnus c. cygnus, to determine whether their susceptibility to human activity changes with time, location and the type of disturbance involved. Overall, the swans' feeding activity varied within and between years, and in relation to feeding site, but there was less variation in the amount of time spent alert. Disturbance frequency resulting from human activity was lower with increasing flock size and with increased distance to the nearest road or track. Distances that humans could approach before alerting the birds similarly varied with field characteristics (e.g. size and proximity to roads or tracks), and also with the type of disturbance involved. The distance at which >5% of the flock became alert because of human activity decreased with the number of previous disturbance incidents in the day, indicating that swans become less sensitive to disturbance events if daily disturbance frequency is high, but there was no evidence that habituation to disturbance persisted over longer periods. The time taken for the birds to resume undisturbed behaviour varied with the duration of the disturbance event, which in turn depended on the type of disturbance involved, with pedestrians alerting the birds for longer periods than vehicles and aircraft. Recovery rates following disturbance were also associated with field size, flock size and the proportion of the flock alerted. Feeding activity was influenced by a range of variables, including year, season, field location, crop type and the number of days that the flock had used the field (32.9% of variance in the data explained by these variables), with disturbance factors explaining an additional 4.9% of variance in the proportion feeding per hour. Conversely, alert activity was influenced mainly by disturbance events. The range of factors influencing the swans' feeding behaviour, and variability in their response to human activity, has implications for management programmes and for attempts to predict the effects of human activity on the birds at a local and larger scale.     

Production of lignocellulose-degrading enzymes and degradation of leaf litter by saprotrophic basidiomycetes isolated from a Quercus petraea forest

Due to the production of lignocellulose-degrading enzymes, saprotrophic basidiomycetes can significantly contribute to the turnover of soil organic matter. The production of lignin- and polysaccharide-degrading enzymes and changes of the chemical composition of litter were studied with three isolates from a Quercus petraea forest. These isolates were capable of fresh litter degradation and were identified as Gymnopus sp., Hypholoma fasciculare and Rhodocollybia butyracea. Within 12 weeks of incubation, H. fasciculare decomposed 23%, R. butyracea 32% and Gymnopus sp. 38% of the substrate dry mass. All fungi produced laccase and Mn-peroxidase (MnP) and none of them produced lignin peroxidase or other Mn-independent peroxidases. There was a clear distinction in the enzyme production pattern between R. butyracea or H. fasciculare compared to Gymnopus sp. The two former species caused the fastest mass loss during the initial phase of litter degradation, accompanied by the temporary production of laccase (and MnP in H. fasciculare) and also high production of hydrolytic enzymes that later decreased. In contrast, Gymnopus sp. showed a stable rate of litter mass loss over the whole incubation period with a later onset of ligninolytic enzyme production and a longer lasting production of both lignin and cellulose-degrading enzymes. The activity of endo-cleaving polysaccharide hydrolases in this fungus was relatively low but it produced the most cellobiose hydrolase. All fungi decreased the C/N ratio of the litter from 24 to 15-19 and Gymnopus sp. also caused a substantial decrease in the lignin content. Analytical pyrolysis mass spectrometry of litter decomposed by this fungus showed changes in the litter composition similar to those caused by white-rot fungi during wood decay. These changes were less pronounced in the case of H. fasciculare and R. butyracea. All fungi also changed the mean masses of humic acid and fulvic acid fractions isolated from degraded litter. The humic acid fraction after degradation by all three fungi contained more lignin and less carbohydrates. Compared to the decomposition by saprotrophic basidiomycetes, litter degradation in situ on the site of fungal isolation resulted in the relative enrichment of lignin and differences in lignin composition revealed by analytical pyrolysis. It can most probably be explained by the participation of non-basidiomycetous fungi and bacteria during natural litter decomposition.     

Soil nitrogen conservation mechanisms in a pristine south Chilean Nothofagus forest ecosystem

A ¹⁵N tracing study was carried out to identify microbial and abiotic nitrogen (N) transformations in a south Chilean Nothofagus betuloides forest soil which is characterized by low N inputs and absence of human disturbance. Gross N transformation rates were quantified with a ¹⁵N tracing model in combination with a Markov chain Monte Carlo sampling algorithm for parameter estimation. The ¹⁵N tracing model included five different N pools (ammonium (NH₄⁺), nitrate (NO₃⁻), labile (N_lab) and recalcitrant (N_rec) soil organic matter and adsorbed NH₄⁺), and ten gross N transformation rates. The N dynamics in the N. betuloides ecosystem are characterized by low net but high gross mineralization rates. Mineralization in this soil was dominated by turnover of N_lab, while immobilization of NH₄⁺ predominantly entered the N_rec pool. A fast exchange between the NH₄⁺ and the adsorbed NH₄⁺ pool was observed, possibly via physical adsorption on and release from clay lattices, providing an effective buffer for NH₄⁺. Moreover, high NH₄⁺ immobilization rates into the N_rec pool ensure a sustained ecosystem productivity. Nitrate, the most mobile form of N in the system, is characterized by a slow turnover and was produced in roughly equal amounts from NH₄⁺ oxidation and organic N oxidation. More than 86% of the NO₃⁻ produced was immediately consumed again. This study showed for the first time that dissimilatory nitrate reduction to ammonium (DNRA) was almost exclusively (>99%) responsible for NO₃⁻ consumption. DNRA rather than NO₃⁻ immobilization ensures that NO₃⁻ is transformed into another available N form. DNRA may therefore be a widespread N retention mechanism in ecosystems that are N-limited and receive high rainfalls.