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1.
Fungal oxidative exo-enzymes lacking substrate specificity play a central role in the cycling of soil organic matter. Due to their broad ecological impact and available knowledge of their gene structure, laccases appeared to be appropriate markers to monitor fungi with this kind of oxidative potential in soils. A degenerate PCR-primer pair Cu1F/Cu2R, specific for basidiomycetes, was designed to assess directly the diversity of laccase genes in soils. PCR amplification of mycelial cultures and fruit-bodies of a wide spectrum of basidiomycetes, covering all functional groups (saprophytes, symbionts, and pathogens), produced multiple DNA fragments around 200 bp. A neighbor-joining tree analysis of the PCR-amplified laccase sequences showed a clear species-specificity, but also revealed that most fungal taxa possess several laccase genes showing a large sequence divergence. This sequence diversity precluded the systematic attribution of amplified laccase of unknown origin to specific taxa. Amplification of laccase sequences from DNA, extracted from a brown (moder) forest soil, showed a specific distribution of laccase genes and of the corresponding fungal species in the various soil horizons (Oh, Ah, Bv). The most organic Oh-horizon displayed the highest gene diversity. Saprophytic fungi appeared to be less widespread through the soil horizons and displayed a higher diversity of laccase genes than the mycorrhizal ones. 相似文献
2.
Laccases of fungal origin have been intensively studied due to their importance in various biotechnological applications. There is a constant demand for new laccases with improved properties such as stability at higher temperatures or at an alkaline pH. Growing molecular evidence suggests that laccases may also be widespread in bacteria. While only a handful of bacterial laccases have been purified and characterized, several novel traits have already been discovered (e.g. pH-stability and 2-domain organization of the enzyme as opposed to the usual 3-domain structure of fungal laccases). The aim of this study was to examine the diversity of bacterial laccase-like genes in two types of high-organic peat soil using a cloning and sequencing approach. Gene libraries prepared of small fragments (150 base pairs) revealed an amazing diversity of bacterial laccases. The fragments clustered in 11 major lineages, and one third of the 241 sequences resembled laccase-like genes of Acidobacteria. Additionally, a new primer was used to retrieve several larger fragments of the putative bacterial laccase genes that spanned all four copper-binding sites. Both “conventional” 3-domain laccases and the recently described 2-domain small laccases have been obtained using this approach, demonstrating the potential of the primer. The present study thus contributes to the understanding of the diversity of bacterial laccases and provides a new tool for finding laccase-like sequences in bacterial strains and soil samples. 相似文献
3.
Sabina Christ Tesfaye Wubet Susanne Theuerl François Buscot 《Soil biology & biochemistry》2011,43(6):1292-1299
The soil fungal diversity and community partitioning between the bulk soil and stone compartments was investigated using PCR based approaches targeting the barcoding internal transcribed spacer (ITS) of rDNA and the laccase encoding functional gene as genetic markers. Soil samples were collected from the B-horizon of spruce and beech forests at the Hainich Biodiversity Exploratory, central Germany. The targeted markers were amplified from the respective DNA extracts using general fungal primers and basidiomycete laccase gene specific primers, cloned and sequenced. Differences in the fungal community composition between the two forest types and the soil compartments were indicated by both markers. When the effects of ecological factors were considered, the two markers produced different patterns of results. The ITS rDNA marker revealed communities principally influenced by forest type, while those detected with the functional marker were mainly affected by soil pH. The fungal communities detected by the functional marker in particular, differed significantly between soils and stones, indicating that laccase-producing fungi are specifically adapted to degrade organic matter in soils rather than weathering of stones. The study underlines the fact that coherent and complementary results may be obtained with both genetic markers used. 相似文献
4.
Harald Kellner Patricia Luis Bettina Zimdars Bärbel Kiesel François Buscot 《Soil biology & biochemistry》2008,40(3):638-648
Laccases- or laccase-like multicopper oxidases (LMCO) catalyze the oxidation of various substrates, such as phenols, diamines and metals, coupled with the reduction of molecular oxygen to water. Compared to studies on function and diversity of LMCO in plants and fungi, little is known about this enzyme type in bacteria and especially on their possible implication in degradation of organic matter in soils. This study presents a molecular investigation of the diversity and distribution of bacterial LMCO genes among three upper horizons of a forest Cambisol and in a grassland Cambisol. Some culture strains of soil bacteria were also analyzed at the molecular level and for their capability to oxidize naturally occurring 2,6-dimethoxyphenol, a LMCO substrate. A high LMCO gene diversity was found in the Cambisol soil samples with 16 distinct sequence type clades, of which approximately one half was not matching with any reference sequence of known bacteria. The highest richness of bacterial LMCO genes was observed in the organic horizon of the forest soil, which is concomitant with a previous analysis of the diversity of fungal laccase genes and corresponding soil laccase activity. Some clusters of sequence types showed a specific distribution in one of the soils or in horizons, while others appeared more ubiquist. Multiple bacterial LMCO genes were described in Agromyces salentinus and Sinorhizobium morelense, what so far was only known from fungi. 相似文献
5.
Alexander Lisov Anna Zavarzina Alexey Zavarzin Vladimir Demin Alexey Leontievsky 《Soil biology & biochemistry》2012
Lichens form the dominant plant cover in extreme environments and participate in mineral weathering, fine-earth stabilization and primary accumulation of soil organic matter. However, biochemical role of lichens in soil processes has never been investigated. Recently, laccases and tyrosinases have been discovered in representatives of the order Peltigerales (Laufer et al., 2006a, b; Zavarzina and Zavarzin, 2006). Laccases from most species had unusually large molecular weights (Laufer et al., 2009). Together with oligomeric laccases, we have found monomeric enzymes in Solorina crocea and Peltigera aphthosa (Lisov et al., 2007). In the present work we have purified homodimeric (large) and monomeric (small) laccases of the soil-stabilizing lichen S. crocea, determined their physico-chemical and catalytic properties and studied their reactions with soil humic acids. Our results suggest that oligomeric nature of lichen laccases can be artifactual, because homodimeric laccase was transformed into the monomeric form following hydrophobic interaction chromatography. We hypothesize that large laccase consists of two monomeric enzymes, each of which is bound with additional hydrophobic component(s). Small laccase is similar in its properties to the laccases of basidiomycetes. It is more resistant to elevated temperature and storage than the large form, showed a higher oxidation potential, had different pH-optima in oxidizing substrates and was less inhibited by humic acids. Despite these differences, both laccases depolymerized and decolorized humic acids from soils at comparable rates, with small laccase being slightly more effective. This finding suggests that lichens have a potential to participate in transformation of soil organic matter. 相似文献
6.
Yucheng Wu Ying Teng Zhengao Li Xuewei Liao Yongming Luo 《Soil biology & biochemistry》2008,40(3):789-796
The transformation of PAHs by fungal laccases has been reported but there are no published studies on the direct application of free laccase in the remediation of PAHs-contaminated soil. Here we report a study in which the transformation of PAHs by a fungal laccase was studied both in reaction mixtures and in soil. Anthracene and benzo(a)pyrene were the most degradable of the 15 US Environmental Protection Agency (EPA) priority PAHs tested. Use of a redox mediator greatly enhanced the oxidation of several PAHs in reaction mixtures and the main intermediates were identified as anthraquinone for anthracene and benzo(a)pyrenyl acetate for benzo(a)pyrene as determined by GC–MS analysis. No significant correlation was found between oxidation and ionization potentials of individual PAHs. Soil microcosms were set up to test the potential of laccase to remediate an aged PAHs-contaminated soil. The laccase transformed the PAHs immediately after it was added to the soil and significant dissimilation of benzo(a)pyrene and toxic-equivalent concentration based on benzo(a)pyrene was observed after incubation for 14 days, indicating the potential of laccase to detoxify the soil. Moreover, extractable laccase activity was completely lost and the biomass of the indigenous microorganisms remained constant in the microcosms at the end of the incubation period, suggesting that the enzyme may have potential as an agent for the efficient and safe cleanup of soil contaminated with PAHs. 相似文献
7.
8.
长期施肥对农田土壤真菌的影响 总被引：4，自引：0，他引：4
不合理施肥所引发的土壤环境问题逐渐成为制约我国农业可持续发展的重要因素之一,而土壤真菌作为一类重要的土壤微生物,研究施肥措施对真菌群落的影响对促进农业生产具有重要意义。本研究以有20年历史的长期定位试验田为研究对象,利用末端限制性片段长度多态性分析技术,对长期定位施肥农田生态系统中不同施肥方式对土壤真菌群落的影响以及时间变化规律进行了系统研究。长期施肥定位试验包括EM堆肥(EM)、传统堆肥(OF)、化肥(CF)和不施肥(CK)处理。主要研究结果如下:在0~20 cm土层,施肥处理对土壤真菌多样性有显著影响,Shannon-Weiner多样性指数为2.64~3.53,Simpson集中性指数为0.03~0.08;EM和OF处理的Shannon-Weiner多样性指数均显著高于CF和CK;在3月、6月和10月,EM和OF处理与CF和CK处理相比,有较高的真菌多样性;Simpson集中性指数最高的是3月的CK处理,最低的是10月的EM和OF处理。冗余分析结果表明,土壤pH、有机质、总氮、有效磷和有效钾等对真菌影响显著。因此,长期施用有机肥与化肥相比可以提高土壤真菌多样性,改变其群落结构;与化肥处理相比,施用EM堆肥,不仅可以保持土壤可持续利用性,同时改善0~20 cm土层土壤真菌的生存环境;3种施肥处理对土壤真菌群落结构影响程度由强到弱:EMOFCF。 相似文献
9.
Linke D Bouws H Peters T Nimtz M Berger RG Zorn H 《Journal of agricultural and food chemistry》2005,53(24):9498-9505
Peanut shells, a major waste stream of food processing, served as a renewable substrate for inducing the production of laccases by basidiomycetes. Of 46 surface cultures examined, 29 showed laccase activity under the experimental conditions. The edible fungus Pleurotus sapidus was selected as the most active producer, immobilized on the shells, and cultivated in the fed-batch mode. A continuous rise in laccase activity was found, indicating the inducibility of laccase secretion by the peanut shells and the reusability of the mycelium. Two laccase isoenzymes were purified by decoupled 2-D electrophoresis, and amino acid sequence information was obtained by electrospray ionization tandem mass spectrometry. cDNAs of the corresponding gene and another laccase were cloned and sequenced using a PCR-based screening of a synthesized P. sapidus cDNA library. Data bank searches against public databases returned laccases of P. ostreatus and P. sajor-caju as the best hits. The potential use of laccases by the food industry is discussed. 相似文献
10.
Margarita Ros Jose Antonio Pascual Ma Teresa Hernández Carlos García 《Biology and Fertility of Soils》2009,45(4):435-441
A stable plant cover is essential in the semi-arid soils of the Mediterranean area to maintain their fertility and functionality.
In a semi-arid area, we have studied abundance, structure, and presence of active species of fungal communities of a devegetated
soil (disturbed soil) and vegetated soil (undisturbed soil). Disturbed soil was covered by small spontaneous vegetation (5–10%)
compared to undisturbed soils (70%), and this decreased the content of the total organic C, microbial biomass, microbial activity
(adenosine triphosphate), and fungal counts. The composition and activities of fungal communities were also investigated by
direct extraction of DNA and RNA from soil. Denaturing gradient gel electrophoresis analysis of 18S ribosomal DNA and 18S
ribosomal RNA profiles indicated that total and active fungal communities were changed after vegetation removal. 相似文献
11.
Dan Xiang Baodong Chen 《Acta Agriculturae Scandinavica, Section B - Plant Soil Science》2016,66(4):333-339
An open question with regard to the community ecology of arbuscular mycorrhizal fungi (AMF) concerns how to best amplify AMF in the soil, which contains a large proportion of DNA from AM extra-radical mycelium and spores. However, to date, a direct comparison of AMF primers for soil samples, which would systematically assess their amplification efficiency, is still missing. In our present study, we compared and characterized four widely used primer sets targeting AMF 18S rDNA or SSU-ITS-LSU rDNA from three soil samples as follows: (1) SSUmAf/LSUmAr?+?SSUmCf/LSUmBr, (2) GeoA2/Geo11?+?NS31/AM1, (3) AML1/AML2?+?NS31/AM1 and (4) AMV4.5NF/AMDGR. These primer sets were compared in terms of the proportion of Glomeromycota detected, AMF diversity and community composition. Our data revealed that the newly combined primer set 3 was the most suitable one for amplifying AMF from soil samples. It yielded the highest AMF alpha diversity, and was very specific to Glomeromycota. Primer set 2 was unable to amplify Claroideoglomus from soil 1, which was the dominant AMF clade as proved by other three primer sets. Primer set 4 demonstrated its instability among different soil samples, since the proportion of AMF in total sequences varied from 5% to 83%. Although primer set 1 showed the highest proportion of AMF (95–100%) in the soil samples, it captured the lowest AMF diversity, and the operational taxonomic units obtained by this primer set were only 36.4% of that by primer set 4. Taken together, our data suggested that AMF diversity in soil samples could be underestimated by primer set 1, 2 and 4. Our result confirmed the important role of the choice of AMF primers for analyzing AMF communities in soil and explored the most suitable one for amplifying AMF from soil samples. 相似文献
12.
?eněk Novotný Kate?ina Svobodová Tomáš Cajthaml Elke Lang 《Soil biology & biochemistry》2004,36(10):1545-1551
Ligninolytic fungi can be used for remediation of pollutants in water and soil. Extracellular peroxidases and laccases have been shown to oxidize recalcitrant compounds in vitro but the likely significance of individual enzyme levels in vivo remains unclear. This study documents the amounts and activities of Mn-dependent peroxidase (MnP), lignin peroxidase and laccase (LAC) in various species of ligninolytic fungi grown in liquid medium and soil and their effect on degradation of polycyclic aromatic hydrocarbons (anthracene and pyrene), a polychlorinated biphenyl mixture (Delor 106) and a number of synthetic dyes. Stationary cultures of a highly degradative strain Irpex lacteus exhibited 380-fold and 2-fold increase in production of MnP and LAC, respectively, compared to submerged cultures. Addition of Tween 80 to the submerged culture increased MnP levels 260-fold. High levels of MnP correlated with efficient decolorization of Reactive Orange 16 azo dye but not of Remazol Brilliant Blue R anthraquinone dye. Degradation of anthracene and pyrene in spiked soil by straw-grown explorative mycelium of Phanerochaete chrysosporium, Trametes versicolor and Pleurotus ostreatus showed the importance of MnP and LAC levels secreted into the soil. The importance of high fungal enzyme levels for efficient degradation of recalcitrant compounds was better demonstrated in liquid media compared to the same strains growing in soil. 相似文献
13.
Anna Andreetta Cristina Macci Maria Teresa Ceccherini Guia Cecchini Graziana Masciandaro Giacomo Pietramellara Stefano Carnicelli 《Biology and Fertility of Soils》2012,48(3):259-270
There is a growing interest in the links between humus forms and soil biota, and little is known about these links in Mediterranean
ecosystems. Culture-independent techniques, such as DNA extraction followed by DGGE and enzyme activities, allowed us to compare
microbial communities in two horizons of a forest soil in different seasonal conditions. Direct in situ lysis was applied
for extraction of DNA from soil; intracellular DNA was separated from extracellular and used to represent the composition
of microflora. The aims were to describe how biochemical and microbiological parameters correlate with topsoil properties
in typical Mediterranean Moder humus. Changes in bacterial and fungal community composition were evident from DGGE profiles.
Degrees of similarity and clustering correlation coefficients showed that the seasonal conditions may affect the composition
and activity of bacterial and fungal communities in the OH horizon, while in the E horizon the two communities were hardly
modified. In the same season, OH and E horizons showed a different composition of bacterial and fungal communities and different
enzyme activities, suggesting similar behaviour of eubacteria and fungi relatively to all the variables analysed. Evidently,
different organic carbon content in soil horizons influenced microflora composition and microbial activities involved in the
P and N cycles. 相似文献
14.
Jun Zhou Li Kang Hong-Wei Wang Teng Yang 《Acta Agriculturae Scandinavica, Section B - Plant Soil Science》2013,63(8):683-693
The accumulation of phenolic acids in soil is one of the main problems associated with continuous cropping of peanut. Although laccases secreted by fungi can efficiently transform phenolic acids, there are few reports on the use of these enzymes to bioremediate continuous cropping soil. Food waste and wheat straw are waste products; however, they could be used productively as resources for laccase production by the endophytic fungus Phomopsis liquidambari B3. We cultured Phomopsis liquidambari B3 in medium containing food waste as the main nitrogen source and wheat straw as the main carbon source. In order to study the effects of fermentation liquid on phenolic acid degradation, rhizosphere soil microbial communities and peanut seedling growth, the fermentation product, which had high laccase activity, was added to continuously cropped soil of peanut. The concentration of 4-hydroxybenzoic acid, vanillic acid, and coumaric acid in soil had decreased by 57.4, 52.5, and 49.4%, respectively, compared with no-treatment control during 28 days. Analysis of denaturing gradient gel electrophoresis profiles showed that the bacterial and fungal community structures in rhizosphere soil were affected by changes in the phenolic acids concentration. The biomass of peanut plants and the number of root nodules were increased 68.3% and 5.9-fold, respectively. These results showed that the laccase product reduced the accumulation of phenolic acids in soil, the decrease in phenolic acids concentration and the increase in certain dominant microorganisms promoted peanut seedling growth and nodulation. This technology provides a new strategy for bioremediation of continuous cropping soil, while simultaneously reducing waste and protecting the environment. 相似文献
15.
S. Papa E. Cembrola A. Pellegrino A. Fuggi A. Fioretto 《European Journal of Soil Science》2014,65(2):274-285
Forest soils contain a large amount of organic matter (OM) and therefore represent a considerable carbon reserve. The amount of OM sequestered in the soil is dependent on annual input of litter and its quality. The aim of this study was to investigate the quantity and quality of OM, the microbial capacity to degrade it and its recalcitrance to further degradation, by considering some extracellular enzyme activities in a beech (Fagus sylvatica L.) forest in south Italy (Mediterranean area). Our attention was focused on the decomposition continuum of the litter horizon and upper soil layer. Because fungi are the major decomposers of plant material, fungal biomass was also measured and its relationship with enzyme activities was tested. The results showed that: (i) the litter horizon and the upper soil layer differed in chemical characteristics and biological activities; (ii) within the litter horizon, the three layers detected for their different degree of degradation (L, recently fallen, not decomposed and not compressed material; F, partially decomposed and fragmented but macroscopically recognizable material; H, compressed and strongly fragmented) differed more in chemical characteristics than in biological activities; (iii) the enzyme activities and fungal biomass changed during the study period but a clear relationship with succession of seasons was evident only for cellulase, laccase, peroxidase and fungal biomass; and (iv) the upper soil layer included 42% OM and less than 50% of that was susceptible to further decomposition. This percentage was 30% in the OM of L. 相似文献
16.
Some microbial nitrogen (N) cycling processes continue under low soil moisture levels in drought-adapted ecosystems. These processes are of particular importance in winter cropping systems, where N availability during autumn sowing informs fertilizer practices and impacts crop productivity. We evaluated the organic and inorganic N-cycling communities in a key cropping soil (Vertosol), using a controlled-environment incubation study that was designed to model the autumn break in south Australian grain growing regions. Soils from wheat, lucerne, and green manure (disced-in vetch) rotations of the Sustainable Cropping Rotations in Mediterranean Environments trial (Victoria, Australia) were collected during the summer when soil moisture was low. Microbial community structure and functional capacity were measured both before and after wetting (21, 49, and 77 days post-wetting) using terminal restriction fragment length polymorphism measures of bacterial and fungal communities, and quantitative PCR of nitrogen cycling genes. Quantified genes included those associated with organic matter decomposition (laccase, cellobiohydrolase), mineralization of N from organic matter (peptidases) and nitrification (bacterial and archaeal ammonia monooxygenase and nitrite oxidoreductase). In general, the N cycling functional capacity decreased with soil wetting, and there was an apparent shift from organic-N cycling dominance to autotrophic mineral-N cycling dominance. Soil nitrate levels were best predicted by laccase and neutral peptidase genes under drought conditions, but by neutral peptidase and bacterial ammonia monooxygenase genes under moist conditions. Rotation history affected both the structural and functional resilience of the soil microbial communities to changing soil moisture. Discing in green manure (vetch) residues promoted a resilient microbial community, with a high organic-N cycling capacity in dry soils. Although this was a small-scale microcosm study, our results suggest that management strategies could be developed to control microbial organic-N processing during the summer fallow period and thus improve crop-available N levels at sowing. 相似文献
17.
18.
Fungal denitrification in soils is receiving considerable attention as one of the dominant N2O production processes. However, because of the lack of a methodology to detect fungal denitrification-related genes, the diversity and ecological behavior of denitrifying fungi in soil remains unknown. Thus, we designed a primer set to detect the fungal nitrite reductase gene (nirK) and validated its sensitivity and specificity. Through clone library analyses, we identified congruence between phylogenies of the 18S rRNA gene and nirK of denitrifying fungal isolates obtained from the surface-fertilized cropland soil and showed that fungi belonging to Eurotiales, Hypocreales, and Sordariales were primarily responsible for N2O emissions in the soil. 相似文献
19.
Petr Baldrian Věra Merhautová Tomáš Cajthaml Mirka Petránková Jaroslav Šnajdr 《Biology and Fertility of Soils》2010,46(7):717-726
The small-scale distribution of activities of extracellular laccase, Mn-peroxidase, endoglucanase, cellobiohydrolase, β-glucosidase,
endoxylanase, β-xylosidase, chitinase, and acid phosphatase were studied in the litter (L) and organic (H) horizons of Quercus petraea forest soil and related to the distribution of microbial biomass. Geostatistical analysis showed that the spatial autocorrelation
of the enzyme activities and soil microbial biomass measured as phospholipid fatty acids (PLFA) and ergosterol content occurred
at similar scales, typically in the range of tens of centimeters. The size of the spatial structures differed between the
L and H horizons; for most of the studied enzymatic processes, litter exhibited a higher spatial variability (smaller autocorrelation
distances). The distribution of several enzymes, including laccase, Mn-peroxidase, and some hydrolases, reflected the distribution
of fungal biomass. Polysaccharide hydrolases exhibited similar spatial distribution patterns in the L horizon, and their activity coincided with a high fungal/bacterial biomass ratio. 相似文献
20.
通过特定的引物,以土壤宏基因组DNA为模板,采用PCR技术扩增获得3．3kb的DNA片段,该片段连接于pSE380载体构建重组质粒pSE380-dhaB12用于测序和表达。序列分析表明,该DNA片段编码的氨基酸序列与已报导的丁酸梭菌不依赖辅酶B12甘油脱水酶的相似性达99％。通过IPTG诱导,dhaB12基因在大肠杆菌中表达成功,SDS—PAGE分析表明有88kD和34kD两条蛋白质条带,在没有辅酶B12存在的情况下,所表达的酶具有明显的甘油脱水酶活性。 相似文献