File Name: methods of soil analysis part 2 microbiological and biochemical properties .zip
The responses of extracellular enzyme activities and the microbial community diversity determined by phospholipid fatty acid analysis [PLFA] and denaturing gradient gel electrophoresis [DGGE] were measured. The abundances of gram-negative bacterial and fungal PLFAs probably effective in improving fertility of low-yield albic paddy soil because of their significant influence on DGGE profile. Fertilization is an important agricultural measure that affects soil quality and sustainable utilization of soils.
The responses of extracellular enzyme activities and the microbial community diversity determined by phospholipid fatty acid analysis [PLFA] and denaturing gradient gel electrophoresis [DGGE] were measured.
The abundances of gram-negative bacterial and fungal PLFAs probably effective in improving fertility of low-yield albic paddy soil because of their significant influence on DGGE profile. Fertilization is an important agricultural measure that affects soil quality and sustainable utilization of soils. Low inputs of organic materials and excessive use of mineral fertilizers have contributed to a general reduction in soil organic carbon SOC contents, with a consequent decline in agricultural soil quality.
A considerable number of studies have focused on the effects of organic manures on soil physicochemical properties, such as SOC [ 3 , 4 ]. However, increases in the SOC content following the addition of organic matter may take considerable time.
Consequently, changes in SOC cannot fully and sensitively reflect the influence that the complexity of the organic compounds may have on the microbiological processes controlling nutrient availability. To make up for these defects, other soil indicators to study the effects of organic manure applications have been proposed [ 5 ]. The biochemical parameters include variables directly related to microbial activity microbial biomass carbon, soil respiration etc.
Most organic manures added into soil contain polymeric compounds, and thus the decomposition of these organic matters depends on the microbial production of extracellular enzymes and their break down should occur before taking up of low molecular weight organic molecules by microbial cells [ 7 , 8 , 9 ]. Because the growth and activity of microorganisms are sensitive functions of soil properties including nutrition, texture, pH, temperature, and water content, dynamic changes of microbial community can represent the improving effects of different types and amounts of organic materials on soil quality [ 10 , 11 ].
A variety of microbial parameters have the potential for use as indicators of soil quality such as microbial biomass and microbial diversity [ 12 , 13 , 14 ]. The determination of phospholipid fatty acid analysis PLFA profiles reveals the structural characteristics of the microbial community and also provides estimate of the abundance about various microbial groups [ 15 , 16 ]. The microbial community, size and activity showed by PLFA vary with different fertilizer managements, and therefore affect soil fertility and productivity [ 17 ].
Other studies have shown increases in PLFA biomarkers for arbuscular mycorrhizal fungi AMF in response to composted green waste as well as long-term organic management [ 23 , 24 , 25 ]. Analysis with PCR-DGGE has been used to isolate microbial DNA and identify predominant microbial populations in a variety of systems to reflect the genetic diversity of the microbial community [ 26 , 27 ]. There is an abundance of studies about the effects of different fertilizations on rice yield and soil fertility [ 28 , 29 , 30 ].
Changes under different tillage managements and different fertilizer regimes and sampling times have been investigated by the combination of PLFA and DGGE profiles [ 31 , 32 ] which reflect both the community and genetic diversity of microorganisms.
However, direct comparisons between different organic manures have not been fully explored by the combination of PLFA and DGGE profiles, especially under albic paddy soil which is a typical low-yield soil in the Anhui and Jiangsu provinces of China and covers an area of 1 million hectares.
Because of rain wash, loss of clay particles and deposition of silt and sand particles, albic paddy soil has an obvious condition of poor nutrition, a shallow plough layer and bad structure [ 33 ]. Long-term fertilizer experiment of quaternary red clay showed that most microbial parameters were mainly correlated with SOC and N, P which did not directly affect microbial parameters but did so indirectly by increasing crop yields, thus promoting the accumulation of soil organic matter [ 34 , 35 ].
Experiment under a typic haplustoll silt loam in Argentina showed total PLFA content was lowest in soil under reduced tillage and soybeen monoculture and DGGE analysis estimated fungal communities were influenced by combined effects of crop rotation and tillage system [ 31 ]. So far, organic amendments to albic soil have focused on returning straw as well as livestock manure and green manure additions, of which single measures are preferred.
The influence of organic matter on soil physical properties depends upon the amount, type and size of the added organic materials [ 36 ]. To explore suitable measures for improving albic paddy soil, three different organic manure carbon sources livestock manure, straw and green manure were investigated. Eight extracellular enzyme activities and PLFA and PCR-DGGE profiles were determined to evaluate the effects of different organic manures on extracellular enzyme activities and the microbial community and genetic diversity in a low yield albic paddy soil.
The results would explore changes of soil fertility under different organic manure amendments by measuring soil biochemical and microbiological properties and thus provide strong evidence for improving the quality of low productivity albic paddy soils. The site is located in the warm temperate and subtropical transition zone and has a warm humid climate with an annual average temperature and precipitation of Furthermore, this study was carried out on private land and the owner of the land gave permission to conduct the study on this site on the basis of a certain rent.
This work was approved by Institute of Soil and Fertilizer, Anhui Academy of Agricultural Sciences who is in charge of the communication with the owners.
At the start of the experiment, the soil had a pH H 2 O of 5. The experiment was a wheat-rice rotation in a completely randomized design of four treatments with three replicates in 20 m 2 plots. The four treatments were investigated as follows:. The amounts of organic manures applied were based on the farmers' practice and recommendation from the local academy of agricultural sciences.
The livestock manure had 5. The straw had 3. The green manure had 5. The C content of three organic manures was approximately equal. Seeds were sown on May 10, , transplanting was June 13 and harvest was September Rice planting density and cultivation management was consistent with local farmers. Soil samples 0—20 cm , consisting of five cores, were randomly collected from every plot on September 22, at harvest stage, when the activities of soil microorganisms were relatively stable.
The five sub-samples were mixed into one sample, representing each replicate of the four treatments. The samples were immediately transported to the laboratory. Soil pH was measured with a compound electrode PB, Sartorius, Germany using a soil to water ratio of Soil organic C was determined by dichromate oxidation [ 37 ] and total N by Kjeldahl digestion [ 38 ].
Available P was determined by the Olsen method [ 40 ] and available K was analyzed by ammonium displacement of the exchangeable cations [ 11 ]. The activities of all tested extracellular enzymes except urease and phenol oxidase were measured using MUB-linked or AMC-linked model substrates yielding the highly fluorescent cleavage products 4-methylumbelliferyl MUB or 7-aminomethylcoumarin AMC upon hydrolysis [ 41 , 42 , 43 ] Table 1.
The method is very sensitive and allowed a high throughput analysis of enzymatic activities [ 43 ]. Specifically, each equivalent of 1.
An additional 50 mL of acetate buffer washed the centrifuge tube and was poured into the same beaker. A magnetic stirrer was used to maintain a uniform suspension.
The activities were expressed in units of nmol h -1 g The non-fluorometric enzyme, phenol oxidase, was measured spectrophotometrically in the clear well microplate using the substrate of L-3, 4-dihydroxyphenylalanine L-DOPA. Urease activities was determined using urea as the substrate as described by Lu [ 44 ]. The determination was based on the product of ammonium, which was determined by kjeldahl apparatus.
Differences in the microbial community and microbial biomass among the various nutrients managements were determined by phospholipid fatty acid PLFA analysis following the procedure described by Wu et al.
Briefly, three-gram freeze-dried soil samples were used to extract the PLFAs with a single-phase mixture of chloroform: methanol: citrate buffer The extracted fatty acids in the chloroform were fractionated into neutral lipids, glycolipids, and polar lipids using a silica-bonded phase column SPE-Si, Supelco, Poole, UK with chloroform, acetone and methanol, respectively.
The recovered polar lipids were transesterified to the fatty acid methyl esters FAMES by a mild alkaline methanolysis. Nonadecanoic acid methyl ester was added as the internal standard. Concentrations of PLFAs were expressed in units of nmol g Soil total DNA was extracted from 0. PCR mixtures consisted of Enzyme activities were calculated by Origin 8 and MS Excel , respectively. Redundancy analysis RDA with the Monte Carlo permutation test permutations was performed to determine if soil microbial community composition and structure were correlated with variation on enzyme activities and physic-chemical parameters, as implemented in Canoco for Windows version 4.
Some other complemental calculations were carried out using MS Excel Simple correlation analysis was applied to detect the relations among soil pH, nutrient concentrations and enzyme activities in soils that were treated with different fertilization. The data on rice yield and albic soil physicochemical parameters were given in Table 2.
In general, soil pH ranged from 5. The activities of eight extracellular enzymes were quantified in different fertilizer treatments. There were changes in enzyme activities under the different organic treatments, but not in the same way Fig 1. A total of 60 PLFAs were identified and used as measures of total microbial biomass. Abundance of microbial groups for data analysis to determine the microbial community composition under various fertilizer treatments was calculated Table 3.
The amendment of different organic manures significantly increased the total PLFA abundance. The PLFAs were also used to assess whether the observed changes in microbial composition parameters were accompanied by changes in the composition of microbial communities under different organic manure additions.
Differences were identified using PCA and outlined according to proportions of structural classes, biomarkers and individual fatty acids present under each treatment. The first two principal components PCs accounted for The DGGE patterns from different soil samples were generally similar, although several strong bands were observed in individual treatments, such as those marked with red arrows.
This indicated that the addition of livestock manure enhanced the bacterial biomass of some species and thus affected the microbial community structure. This may indicate that the mechanism of the green manure impact on fugal community was different from the other treatments. It confirmed that different organic manures had a greater influence on fungal than bacterial community.
No significant relationship between biochemical properties and soil pH was observed, while total N and available P were negatively related to soil pH values Table 4 in this experiment. Furthermore, significant correlations were detected between some of the tested enzymes. Soil properties and enzyme activities were used as environmental variables Fig 5a.
The first and second axes accounted for Results of RDA analysis between soil properties and enzyme activities with microbial community structure as indicated by DGGE banding patterns are shown in Fig 5b. It is commonly acknowledged that paddy soils with organic fertilization can maintain soil sustainability and enhance more accumulation of organic C than arable soil relying on chemical fertilizer only [ 4 , 52 , 53 ].
The results of this study indicated that, compared to chemical fertilizer, organic amendments improved rice yield and physic-chemical character of albic paddy soil which also significantly enhanced enzyme activities and altered microbial community at the same time.
Organic fertilizers are believed to stabilize or even increase soil pH [ 11 , 54 ], which we did not observe in this experiment. Although no significant correlation was detected between bacterial communities with soil pH under a typical clay loamy anthrosol [ 32 ], many studies have reported that environmental factors altered soil microbial community, especially the soil pH, which has been demonstrated in several studies to be the strongest factor shaping microbial community structure [ 55 , 56 , 57 ].
This is different from Deng et al. The soil pH values varied significantly from 3 to 9 in those studies allowing insight into the relationship between pH and soil bacterial communities [ 55 , 58 , 59 ].
Thus, we hypothesize that there are important factors other than pH in shaping soil bacterial communities. As reported by Navarrete et al. Therefore, the soil pH may not represent an integrating variable in our study due to the mild variation of soil pH, which suggests that soil characteristics such as soil nutrients, rather than soil pH can shape the microbial community. Furthermore, organic manures increased biological activity [ 62 ]. In this experiment, three organic manures all had positive effects on the enzyme activity of albic paddy soil.
Ramesh et al. Bowen and Harper [ 64 ] also reported that on one hand, straw increased the soil microbial biomass and improved the activity of microorganisms but on the other hand, the increase in soil microbial biomass and microbial activity also accelerated the decay of the straw which brought more nutrients to the soil.
British Wildlife is the leading natural history magazine in the UK, providing essential reading for both enthusiast and professional naturalists and wildlife conservationists. Published eight times a year, British Wildlife bridges the gap between popular writing and scientific literature through a combination of long-form articles, regular columns and reports, book reviews and letters. Conservation Land Management CLM is a quarterly magazine that is widely regarded as essential reading for all who are involved in land management for nature conservation, across the British Isles. CLM includes long-form articles, events listings, publication reviews, new product information and updates, reports of conferences and letters. Part 5. Mineralogical Methods", presents valuable techniques that will enable researchers to analyze mineralogy for a wide variety of applications. An understanding of mineralogical composition provides crucial insight into the fundamental behavior of soils and their response to environmental conditions and management.
One of the primary references on analytical methods in soil science, Part 2 of the Methods of Soil Analysis, Part 2: Microbiological and Biochemical Properties.
Effects of soil management regimes on biochemical properties of a loess soil. Yang 2 , B. Sun 2. A long-term field experiment was conducted over a twenty year period to examine the effects of three different soil management regimes Abandonment, Fallow and Cropping and eight nutrient management regimes under Cropping on soil organic carbon SOC , N and P levels, microbial biomass, and enzymatic activities related to C, N, and P cycling in a loess soil. Abandonment resulted in greater levels of soil microbial biomass than did Cropping but similar levels of enzyme activity were observed under both regimes.
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