Fluorescence microscopy for visualization of soil microorganisms—a review

Direct microscopic observation of microorganisms is an important tool in many microbial studies. Such observations have been reported for Protozoa, fungi, inoculated bacteria, and rhizosphere microorganisms but few studies have focused on indigenous bacteria and their spatial relationship within various microhabitats. Principles and applications of epifluorescence microscopy and confocal laser scanning microscopy for visualization of soil microorganisms in situ are reviewed. Both cationic and anionic dyes (also commonly referred to as fluorochromes if they are fluorescent) have been used based on their ability to bind to specific cellular components of microbial cells. Common fluorochromes used for imaging of microbial cells include acridine orange, ethidium bromide, fluorescein isothiocyanate, 5-(4,6-dichlorotriazinyl) aminofluorescein, 4,6-diamidino-2-phenylindole, europium chelate, magnesium salt of 8-anilino-1-naphthalene sulfonic acid, and calcofluor white M2R. Combining fluorescence staining techniques with soil thin section technology allows one to obtain images of microorganisms in situ. Soil texture and the procedures used for resin embedding are important factors affecting the quality of stained soil thin sections. Indeed, general limitations of applying fluorescence microscopy to soil ecological studies are the non-specific binding of dyes to the soil matrix and the autofluorescence of some soil components. The development of fluorescent in situ hybridization and confocal laser scanning microscopy techniques provides new potential for microbial distribution studies.     

Grazing of a common species of soil protozoa (Acanthamoeba castellanii) affects rhizosphere bacterial community composition and root architecture of rice (Oryza sativa L.)

We performed a controlled experiment with rice seedlings (Oryza sativa L.) growing in Petri dishes on homogeneous nutrient agar containing a simple rhizosphere food web consisting of a diverse bacterial community and a common soil protozoa, Acanthamoeba castellanii, as bacterial grazer. Presence of amoebae increased bacterial activity and significantly changed the community composition and spatial distribution of bacteria in the rhizosphere. In particular, Betaproteobacteria did benefit from protozoan grazing. We hypothesize that the changes in bacterial community composition affected the root architecture of rice plants. These effects on root architecture affect a fundamental aspect of plant productivity. Root systems in presence of protozoa were characterized by high numbers of elongated (L-type) laterals, those laterals that are a prerequisite for the construction of branched root systems. This was in sharp contrast to root system development in absence of protozoa, where high numbers of lateral root primordia and short (S-type) laterals occurred which did not grow out of the rhizosphere region of the axile root. As a consequence of nutrient release from grazed bacteria and changes in root architecture, the nitrogen content of rice shoots increased by 45% in presence of protozoa. Our study illustrates that interactions over three trophic levels, i.e. between plants, bacteria and protozoa significantly modify root architecture and nutrient uptake by plants.     

Localization of bacteria in lichens from Alpine soil crusts by fluorescence in situ hybridization

Lichens are prominent components of many biological soil crusts. Owing to their persistence, lichen thalli create microhabitats for other microbes. Here, the structure of bacterial communities at the thallus–soil interface in lichen soil crusts was studied by using fluorescence in situ hybridization (FISH), confocal laser scanning microscopy (CLSM) and 3D image reconstruction. Terricolous lichen thalli above the tree-line in open habitats of the Austrian Alps were sampled. We selected six lichen species associated with green algal photobionts: Arthrorhaphis citrinella, Baeomyces placophyllus, B. rufus, Icmadophila ericetorum, Psora decipiens and Trapeliopsis granulosa. Alphaproteobacteria and Acidobacteria are predominant in these soil crust lichens, where the latter are frequently present in the lower part of lichen thalli and in the hypothallosphere. In the inconspicuous thallus structures of Arthrorhaphis citrinella, Baeomyces rufus, Icmadophila ericetorum and Trapeliopsis granulosa we observed association of bacteria with algal cells in soil particles and on the outer surface of the mycobiont–photobiont aggregates. We found bacterial cells intermixed with photobiont cells in the lower part of the lichen thalli and as small colonies on the surface of the squamules of Baeomyces placophyllus and Psora decipiens. Moreover, technical issues of performing FISH and confocal microscopy with biological soil crusts are discussed.     

土壤细菌趋化性研究进展

土壤是地球生态系统中最具生命活性的组成部分,特有的孔隙结构承载着生物圈中最丰富多样的微生物生命形态,为动植物提供了大量的调控功能。土壤是一个不断演变和发展的生态系统,而微生物是土壤生态系统的核心,也是驱动碳、氮等元素以及能量循环的关键因子。趋化性是细菌在长期进化过程中形成的帮助其寻找食物或趋利避害的本能,结合其他的内在生理特征,细菌能够迅速适应动态变化的环境。营养物、异源污染物和水分条件等的不均匀分布致使土壤中细菌趋化现象普遍存在,并且时刻影响土壤微生物的群落组成及其时空分布。近年来,土壤细菌趋化性已成为国内外土壤微生物学研究的热点和重点。本文分析了土壤细菌趋化性研究的前沿问题和主要进展,阐述了细菌趋化行为模式、趋化信号传导通路和趋化性数学模型,探讨了土壤中普遍存在的细菌趋化现象及相关的主要研究技术手段(荧光原位杂交技术、微流控技术和光学显微技术),并对土壤细菌趋化性研究的发展趋势进行了展望,旨在为今后的相关研究和实际应用提供参考。     

Soil carbon sequestration and changes in fungal and bacterial biomass following incorporation of forest residues

Sequestering carbon (C) in forest soils can benefit site fertility and help offset greenhouse gas emissions. However, identifying soil conditions and forest management practices which best promote C accumulation remains a challenging task. We tested whether soil incorporation of masticated woody residues alters short-term C storage at forested sites in western and southeastern USA. Our hypothesis was that woody residues would preferentially stimulate soil fungal biomass, resulting in improved C use efficiency and greater soil C storage. Harvest slash at loblolly pine sites in South Carolina was masticated (chipped) and either (1) retained on the soil surface, (2) tilled to a soil depth of 40 cm, or (3) tilled using at least twice the mass of organics. At comparative sites in California, live woody fuels in ponderosa pine stands were (1) masticated and surface applied, (2) masticated and tilled, or (3) left untreated. Sites with clayey and sandy soils were compared in each region, with residue additions ranging from 20 to 207 Mg ha⁻¹. Total and active fungal biomass were not strongly affected by residue incorporation despite the high input of organics. Limited response was also found for total and active bacterial biomass. As a consequence, fungal:bacterial (F:B) biomass ratios were similar among treatments at each site. Total soil C was elevated at one California site following residue incorporation, yet was significantly lower compared to surface-applied residues at both loblolly pine sites, presumably due to the oxidative effects of tilling on soil organic matter. The findings demonstrated an inconsequential effect of residue incorporation on fungal and bacterial biomass and suggest a limited potential of such practices to enhance long-term soil C storage in these forests.     

Identification and localization of food-source microbial nucleic acids inside soil nematodes

Microorganisms (e.g., prokaryotes, fungi) are food sources for soil nematodes, but they can also be potential mutualists or pathogens. Understanding the linkages between microorganism and invertebrate diversity in soils requires the ability to distinguish between these microbial roles. We tested the potential of a taxon-specific fluorescent in situ hybridization (FISH) procedure for identifying and localizing microbial rRNA within the bodies of soil nematodes. Our objective was to determine whether the rate of digestion permitted detection and identification of food-source nucleic acids within the nematode digestive system (i.e., pharynges, intestines) before their breakdown. First, using laboratory cultures of Caenorhabditis elegans maintained on Escherichia coli, we were able to localize bacterial rRNA throughout the nematode pharynx with the universal bacterial-probe EUB338, although never in the intestines. Second, we applied the fungal rRNA probe FR1 to Aphelenchus avenae cultured on the fungus Rhizoctonia solani. We were unable to detect fungal rRNA within these nematodes, and it appears that this material may be digested rapidly. Next, we applied our technique to nematodes extracted directly from soils. We were able to localize bacterial rRNA within the pharynges of bacterial-feeding species of nematodes from desert soils. We also localized archaeal rRNA using the probe ARC344. Finally, application of EUB338 to desert soil nematodes revealed the presence of bacteria in the intestines of some nematodes and within the ovary of a single nematode. This technique has great potential for use in understanding the feeding behavior of bacterial-feeding soil nematodes and in studies of nematode:bacterial relationships.     

Methods for the introduction of bacteria into soil: A review

Summary Literature on the use of microbial inoculants to increase crop yields, to control soil-borne plant diseases, or to degrade pollutants has been reviewed. Established inoculant technology based on Rhizobium/peat inoculants has been summarized. Special emphasis has been placed on the use of carrier materials for the delivery of microbial inoculants. Some new developments, e.g., the use of synthetic carriers, have been highlighted. The fact that not only inoculant survival in carrier materials should be studied, but also the ecological consequences of the introduction of bacteria, has been stressed.     

土壤中拟除虫菊酯微生物降解研究

微生物降解是拟除虫菊酯类农药从土壤中消去的主要途径。本文介绍了拟除虫菊酯降解菌的分离鉴定、降解基因的克隆以及微生物降解机理研究的近期成果,综合介绍了拟除虫菊酯异构体选择降解的特征、原因以及可能产生的环境效应,重点分析了农药疏水性、土壤吸附、重金属、土壤养分及长期施肥、共存农药对土壤中拟除虫菊酯微生物降解的影响,最后对土壤微生物修复前景进行了展望。     

Searching for unifying principles in soil ecology

The field of soil ecology has relatively few fundamental unifying principles that can be used to explain and predict patterns and processes in belowground ecosystems. Here we propose that a first step towards developing a more comprehensive set of unifying principles in soil ecology is to identify and understand the characteristics shared by a wide range of soils, the common mechanisms driving soil biogeochemical processes, and the biogeochemical constraints imposed on soil biota regardless of soil type. Very often, soil ecologists focus on the differences between soils when, in fact, many soils share a common set of ecological mechanisms that govern biogeochemical processes. Here we explore evidence for the existence of unifying principles in soil ecology, highlighting some of the similarities in carbon dynamics and soil communities across widely different soil types and examining the various mechanisms that may drive these similarities. Given that soils are extremely complex environments that exhibit substantial spatial and temporal heterogeneity, defining overarching principles is, arguably, more challenging in soil ecology than in other disciplines. However, recent methodological advances hold great promise for testing and formulating unifying principles, particularly when such methods are used consistently, in concert with other interdisciplinary approaches, and across a range of sites. Soils are not identical, but they do exhibit consistent patterns and processes that, if explored more intensively, will affirm the existence of unifying principles in soil ecology.     

薄层黑土微生物生物量碳氮对土壤侵蚀—沉积的响应

研究土壤侵蚀—沉积对土壤微生物生物量的影响可以为科学评估土壤侵蚀的环境效应提供依据。以典型薄层黑土区——黑龙江省宾州河流域为研究区,利用土壤137Cs含量估算侵蚀速率,通过分析流域不同位置和不同坡面部位土壤微生物生物量碳和氮含量以及土壤侵蚀强度的差异,揭示土壤微生物生物量对土壤侵蚀—沉积的响应规律。结果表明:流域不同位置和不同坡面部位土壤微生物生物量的分布存在明显差异,并呈现出与土壤侵蚀—沉积空间分布相反的变化趋势。土壤侵蚀速率在流域的分布为上游中游下游,在坡面的分布为坡中部坡上部坡下部;土壤微生物生物量碳(Microbial biomass carbon,MBC)和微生物生物量氮(Microbial biomass nitrogen,MBN)在流域表现为下游中游上游,在坡面表现为坡下部坡上部坡中部。回归分析表明,MBC、MBN、有机质(Organic matter,OM)和全氮(Total nitrogen,TN)含量随土壤侵蚀强度的增大而减少。土壤侵蚀对土壤微生物生物量的分布有重要影响,土壤侵蚀—沉积过程引起土壤养分的迁移和再分布是导致侵蚀区和沉积区土壤微生物生物量分布产生差异的重要原因。     

Resource utilization capability of bacteria predicts their invasion potential in soil

Biological invasion is governed by intrinsic and extrinsic factors, but few studies have explored the interactive roles of species traits and disturbance in soil microbial invasion. A microcosm experiment was conducted to compare the survival of four non-indigenous bacteria in soil previously subjected to heating disturbance (60 °C, 24 h). The survival of non-indigenous bacteria was positively correlated with their utilization capability of saccharose and glucose 3 days after inoculation, and positively with maltose, saccharose, D-mannitol, glycerol, glucose and amylose 42 days following inoculation. Disturbance increased resource availability and also reduced diversity of the native microbial community. Bacteria survival was significantly increased in disturbed soil, especially for the bacteria with weak resource utilization capability. Bacterial invasion potential was determined by resource utilization capability, with that dependence increased with incubation duration and reduced if soil was initially disturbed.     

Three decades of soil microbial biomass studies in Brazilian ecosystems: Lessons learned about soil quality and indications for improving sustainability

Soil microbial biomass plays important roles in nutrient cycling, plant-pathogen suppression, decomposition of residues and degradation of pollutants; therefore, it is often regarded as a good indicator of soil quality. We reviewed more than a hundred studies in which microbial biomass-C (MB-C), microbial quotient (MB-C/TSOC, total soil organic carbon) and metabolic quotient (qCO₂) were evaluated with the objective of understanding MB-C responses to various soil-management practices in Brazilian ecosystems. These practices included tillage systems, crop rotations, pastures, organic farming, inputs of industrial residues and urban sewage sludge, applications of agrochemicals and burning. With a meta-analysis of 233 data points, we confirmed the benefits of no-tillage in preserving MB-C and reducing qCO₂ in comparison to conventional tillage. A large number of studies described increases in MB-C and MB-C/TSOC due to permanent organic farming, also benefits from crop rotations particularly with several species involved, whereas application of agrochemicals and burning severely disturbed soil microbial communities. The MB-C decreased in overgrazed pastures, but increased in pastures rotated with well-managed crops. Responses of MB-C, MB-C/TSOC and qCO₂ to amendment with organic industrial residues varied with residue type, dose applied and soil texture. In conclusion, MB-C and related parameters were, indeed, useful indicators of soil quality in various Brazilian ecosystems. However, direct relationships between MB-C and nutrient-cycling dynamics, microbial diversity and functionality are still unclear. Further studies are needed to develop strategies to maximize beneficial effects of microbial communities on soil fertility and crop productivity.     

土壤结构定量化研究进展

土壤结构是土壤中各种过程进行的物理框架,土壤结构的定量化对土壤水、气、热和土壤生物、化学过程定量化研究具有重要意义。本文主要讨论了土壤结构研究的内容,介绍了土壤结构研究的主要方法,对分形理论、布尔模型和孔隙网络模型等在土壤结构中的应用作了简要的介绍,并且对未来土壤结构定量化研究进行了展望。     

Rhizosphere soil microbial index of tree species in a coal mining ecosystem

Microbial characterization of the tree rhizosphere provides important information relating to the screening of tree species for re-vegetation of degraded land. Rhizosphere soil samples collected from a few predominant tree species growing in the coal mining ecosystem of Dhanbad, India, were analyzed for soil organic carbon (SOC), mineralizable N, microbial biomass carbon (MBC), active microbial biomass carbon (AMBC), basal soil respiration (BSR), and soil enzyme activities (dehydrogenase, urease, catalase, phenol oxidase, and peroxidase). Among the tree species studied, Aegle marmelos recorded the highest value for MBC (590 mg kg⁻¹), urease (190.5 μg NH₄⁺-N g⁻¹ h⁻¹), catalase (513 μg H₂O₂ g⁻¹ h⁻¹), dehydrogenase (92.3 μg TPF g⁻¹ h⁻¹), phenol oxidase (0.057 μM g⁻¹ h⁻¹) and BSR/AMBC (0.498 mg CO₂-C mg biomass⁻¹ day⁻¹); Tamarindus indica for mineralizable N (69.5 mg kg⁻¹); Morus alba for catalase (513 μg H₂O₂ g⁻¹ h⁻¹) and phenol oxidase (0.058 μM g⁻¹ h⁻¹); Tectona grandis for peroxidase (0.276 μM g⁻¹ h⁻¹), AMBC/MBC (99.4%), and BSR/MBC (0.108 mg CO₂-C mg biomass⁻¹ day⁻¹); Ficus religiosa for AMBC (128.4 mg kg⁻¹) and BSR (12.85 mg CO₂-C kg⁻¹ day⁻¹); Eugenia jambolana for MBC/SOC (8.03%); Butea monosoperma for AMBC/SOC (1.32%) and Azadirachta indica for BSR/AMBC (0.1134 mg CO₂-C mg biomass⁻¹ day⁻¹). Principal component analysis was employed to derive a rhizosphere soil microbial index (RSMI) and accordingly, dehydrogenase, BSR/MBC, MBC/SOC, EC, phenol oxidase and AMBC were found to be the most critical properties. The observed values for the above properties were converted into a unitless score (0–1.00) and the scores were integrated into RSMI. The tree species could be arranged in decreasing order of the RSMI as: A. marmelos (0.718), A. indica (0.715), Bauhinia bauhinia (0.693), B. monosperma (0.611), E. jambolana (0.601), Moringa oleifera (0.565), Dalbergia sissoo (0.498), T. indica (0.488), Morus alba (0.415), F. religiosa (0.291), Eucalyptus sp. (0.232) and T. grandis (0.181). It was concluded that tree species in coal mining areas had diverse effects on their respective rhizosphere microbial processes, which could directly or indirectly determine the survival and performance of the planted tree species in degraded coal mining areas. Tree species with higher RSMI values could be recommended for re-vegetation of degraded coal mining area.     

Variability of communities and physiological characteristics between free-living bacteria and attached bacteria during the PAH biodegradation in a soil/water system

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) via free-living and attached micro-organisms in soil/water systems was observed in order to examine the variability in the community dynamics and physiological profiles of the micro-organisms. As determined by fluorescence in situ hybridization (FISH), the Domain Bacteria, consisting of three phyla α-, β- and γ-Proteobacteria, reached 41.27–56.05% of all organisms in the soil/water system for PAH biodegradation. Among the free-living species, Proteobacteria, including Brevundimonas (Pseudomonas) diminuta, Caulobacter spp., Mycoplana bullata, Acidovorax spp. and Pseudomonas aeruginosa were found to be dominant—making up 93.51–99.80% of the population—and therefore seem to be associated with PAH biodegradation. Total plate count numbers and the count of Pseudomonas sp. present in the free-living population increased to between 10³ and 10⁶ CFU ml⁻¹ when clay with very low organic matter content was used as the matrix for PAH degradation. However, total plate count microbial numbers increased to only 10¹–10² CFU ml⁻¹ using natural soil from Taichung containing 1.883% organic matter. The soil organic content (SOM) seemed to affect the mass transfer of PAH in soil, leading to the difference in PAH biodegradation. Two different approaches, which included community-level physiological profiling (CLPP) and ectoenzymatic activities, were used to explain the functional diversity between free-living and attached bacteria. The free-living and attached bacterial communities from the clay system showed proportionately greater differences using CLPP. Relatively high levels of esterases, aminopeptidases and some specific glycolysis-gluconeogenesis enzymes gave an identifiable correlation with PAH biodegradation. The differences in bacterial composition, numbers and physiological characteristics show that free-living and attached micro-organisms may play different biochemical roles in PAH degradation in soil.     

Responses of soil organic matter and greenhouse gas fluxes to soil management and land use changes in a humid temperate region of southern Europe

We studied the effects of soil management and changes of land use on soils of three adjacent plots of cropland, pasture and oak (Quercus robur) forest. The pasture and the forest were established in part of the cropland, respectively, 20 and 40 yr before the study began. Soil organic matter (SOM) dynamics, water-filled pore space (WFPS), soil temperature, inorganic N and microbial C, as well as fluxes of CO₂, CH₄ and N₂O were measured in the plots over 25 months. The transformation of the cropland to mowed pasture slightly increased the soil organic and microbial C contents, whereas afforestation significantly increased these variables. The cropland and pasture soils showed low CH₄ uptake rates (<1 kg C ha⁻¹ yr⁻¹) and, coinciding with WFPS values >70%, episodes of CH₄ emission, which could be favoured by soil compaction. In the forest site, possibly because of the changes in soil structure and microbial activity, the soil always acted as a sink for CH₄ (4.7 kg C ha⁻¹ yr⁻¹). The N₂O releases at the cropland and pasture sites (2.7 and 4.8 kg N₂O-N ha⁻¹ yr⁻¹) were, respectively, 3 and 6 times higher than at the forest site (0.8 kg N₂O-N ha⁻¹ yr⁻¹). The highest N₂O emissions in the cultivated soils were related to fertilisation and slurry application, and always occurred when the WFPS >60%. These results show that the changes in soil properties as a consequence of the transformation of cropfield to intensive grassland do not imply substantial changes in SOM or in the dynamics of CH₄ and N₂O. On the contrary, afforestation resulted in increases in SOM content and CH₄ uptake, as well as decreases in N₂O emissions.     

Effects of long-term litter manipulation on soil carbon,nitrogen, and phosphorus in a temperate deciduous forest

Changes in above-ground litterfall can influence below-ground biogeochemical processes in forests. In order to examine how above-ground litter inputs affect soil carbon (C), nitrogen (N) and phosphorus (P) in a temperate deciduous forest, we studied a 14-year-old small-scale litter manipulation experiment that included control, litter exclusion, and doubled litter addition at a mature Fagus sylvatica L. site. Total organic C (TOC), total N (TN) and total P (TP), total organic P (TOP), bioavailable inorganic P (Pi), microbial C, N and P, soil respiration and fine root biomass were analyzed in the A and in two B horizons. Our results showed that litter manipulation had no significant effect on TOC in the mineral soil. Litter addition increased the bioavailable Pi in the A horizon but had no significant effect on N in the mineral soil. Litter exclusion decreased TN and TP in the B horizon to a depth of 10 cm. In the A horizon of the litter exclusion treatment, TP, TOP and bioavailable Pi were increased, which is most likely due to the higher root biomass in this treatment. The high fine root biomass seems to have counteracted the effects of the excluded aboveground litter. In conclusion, our study indicates that aboveground litter is not an important source for C in the mineral soil and that P recycling from root litter might be more important than from above-ground litter.     

Decomposition of bacterial polymers in soil and their influence on soil structure

Summary The adherence of soil particles into stable aggregates increases with the addition of monosaccharides or polysaccharde polymers to soil, either as plant residues, microbial metabolites, or as simple carbohydrates. Microbial polysaccharides are one of the most effective organic agents that promote soil aggregate stability, but the effectiveness of these polymers in stabilizing soil particles varies dramatically between microbial strains, the amount present and the prevailing environmental conditions. We conducted glasshouse and laboratory studies to determine the effectiveness of selected microbial polymers in stabilizing soil aggregates. The addition and thorough mixing of 1.0 mg microbial polymer C g^–1 soil of seven bacteria strains (Arthrobacter viscocus, Azotobacter indicus, Bacillus subtilus, Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas strain I, and Pseudomonas strain II), three deuteromycete strains (Cryptococcus laurentii, Hansenula holstii, and Mucor rouxii), and two reference compounds (hydroxyethyl guar and glucose) to an Arlington coarse-loamy soil resulted in stimulated soil respiration, increased aggregate stability, and decreased soil bulk density and modulus of rupture when incubated from 1 to 12 weeks. The monosaccharides present in the added polymers were rapidly decomposed and the sacchride content of the polymer-treated soil returned to the level of the soil control (with no polymer addition) after 2 weeks of incubation, while the maximum increase in soil aggregate stability was noted during the 3rd and 4th weeks of incubation. Statistical analyses showed that the glucose content of the polymers added was significantly correlated with soil aggregation [weeks 1 (r=0.78^***) and 2 (r=0.61^*)], but the extractable soil saccharides were not significantly correlated with increased aggregate stability or decreased soil bulk density during this study. When microbial extracellular polymers were added to soil only a transient increase in soil stability was measured upon decomposition of the added saccharides. This finding suggests that the stabilization of soil aggregates is a result of other microbial processes or metabolites rather than the direct binding effects of the added polysaccharides.     

Influence of leaf litter types on microbial functions and nutrient status of soil: Ecological suitability of forest trees for afforestation in tropical laterite wastelands

The impact of forest tree leaf litters on microbial activity and nutrient status of red laterite soil was tested for the ecological suitability of Cassia siamea, Shorea robusta, Acacia auriculiformes and Dalbergia sissoo, which are typically used for afforestation of wastelands in eastern India. The objectives were to compare seasonal variation in soil enzyme activity in 30-years old afforested sites, and to study nutrient status and microbial biomass and function during short-term in-situ incubation of litter in decomposition pits. In afforested soils, enzyme activities significantly varied between litters and seasons. All enzyme activity except invertase dominated in the soils containing Dalbergia and Cassia litters compared to the others. The seasonal effect was enzyme-dependent, with amylase and cellulase reaching peaks during the rainy season but invertase activity showed a reverse trend with lowest values in rainy season, except in Acacia soil, and protease activity was lowest in the soil containing Cassia and Dalbergia during the rainy season. Dehydrogenase activity was negligible in the soils containing Shorea and Acacia, but remained high with respect to Dalbergia and Cassia during all seasons. The decomposition pit study showed significant increase of soil nutrients with respect to litter types and intervals, except with respect to electrical conductivity. Cassia and Dalbergia litters enabled notable increase of soil nutrients than Shorea and Acacia. The soil enzyme activity, in general, increased with duration of litter decay, but microbial biomass C (MBC) decreased over time except in Shorea. Therefore, the enzyme rates normalized to the MBC indicated inverse relations for all enzymes, except in the soil containing Shorea. A positive relationship existed between MBC and soil respiration in Cassia, Acacia and Dalbergia. Analysis of variance revealed main effects of litter types for increasing protease, MBC and CO₂ output, and a main effect of intervals for enhancing enzymes other than cellulase. Rates of soil respiration were greater in soils contain Cassia and Dalbergia, and showed significant differences between litters and between intervals. All enzymes were significantly correlated with electrical conductivity, organic carbon and available phosphorus contents, and all enzymes except invertase were correlated with nitrate concentrations. The acidic soil pH did not affect enzyme activities, and soil nutrients exerted only weak effect on MBC and respiration. Our study showed that leaf litters of Cassia and Dalbergia trees improved the nutrient status and microbial activity in soil more so than Shorea and Acacia litters, and therefore, afforestation using Cassia and Dalbergia trees may be particularly suitable for soil restoration in tropical laterite wastelands.