Abstract Understanding the chronological

changes in soil microbial and biochemical prop-

erties of tea orchard ecosystems after wasteland

has been reclaimed is important from ecological,

environmental, and management perspectives. In

this study, we determined microbial biomass, net

N mineralization, and nitrification, enzyme

(invertase, urease, proteinase, and acid phospha-

tase) activities, microbial community diversity

assessed by denaturing gradient gel electropho-

resis (DGGE) of 16S rDNA polymerase chain

reaction (PCR) products, and related ecological

factors in three tea orchard systems (8-, 50-, and

90-year-old tea orchards), adjacent wasteland and

90-year-old forest. Soil microbial biomass C

(Cmic) and activity, i.e., soil basal respiration

(Rmic), microbial biomass C as a percent of soil

organic C (Cmic/Corg), N mineralization, invert-

ase, urease, proteinase, and acid phosphatase,

significantly increased after wasteland was re-

claimed; however, with the succeeding develop-

ment of tea orchard ecosystems, a decreasing

trend from the 50- to 90-year-old tea orchard

became apparent. Soil net nitrification showed an

increasing trend from the 8- to 50-year-old tea

orchard and then a decreasing trend from the 50-

to 90-year-old tea orchard, and was significantly

higher in the tea orchards compared to the

wasteland and forest. Urea application signifi-

cantly stimulated soil net nitrification, indicating

nitrogen fertilizer application may be an impor-

tant factor leading to high-nitrification rates in tea

orchard soils. The Shannon’s diversity index (H)

and richness (S) based on DGGE profiles of 16S

rRNA genes were obviously lower in all three tea

orchards than those in the wasteland; neverthe-

less, they were significantly higher in all three tea

orchards than those in the forest. As for the three

tea orchard soils, comparatively higher commu-

nity diversity was found in the 50-year-old tea

orchard.

Keywords Microbial biomass Æ Mineralization ÆNitrification Æ Enzyme activities Æ 16S rDNA-

DGGE Æ Tea orchard soil

Introduction

Tea (Camellia sinensis) is an important economic

crop and is planted widely on acid red soils in the

tropical and subtropical zones in China. To im-

prove yield and quality of tea leaves, nitrogen

fertilizer was applied to tea orchards and its use

has increased year after year. However, heavy

D. Xue Æ H. Yao (&) Æ C. HuangDepartment of Soil Science, College ofEnvironmental and Resource Sciences, ZhejiangUniversity, Huajiachi Campus, Hangzhou 310029,Chinae-mail: huaiyingyao@zju.edu.cn

Plant Soil (2006) 288:319–331

DOI 10.1007/s11104-006-9123-2

123

ORIGINAL PAPER

Microbial Biomass, N Mineralization and Nitrification,Enzyme Activities, and Microbial Community Diversityin Tea Orchard Soils

Dong Xue Æ Huaiying Yao Æ Changyong Huang

Received: 27 February 2006 / Accepted: 1 September 2006 / Published online: 27 September 2006� Springer Science+Business Media B.V. 2006

nitrogen fertilizer application stimulates primary

productivity of tea orchards and, at the same

time, also causes tea orchard soil acidification

(Konishi 1991; Chenery 1955; Tachibana et al.

1995) and affects nitrification rates (Chantigny

et al. 1996) with subsequent environmental im-

pacts such as nitrate leaching to groundwater and

release of greenhouse gases into the atmosphere

(Tokuda and Hayatsu 2001, 2004). In addition,

long-term tea cultivation practices can result in

Al, hydroxybenzene accumulation in soil ecosys-

tems due to the input of tea leaf litter, root exu-

dates and pH decrease (Nioh et al. 1993; Yao

et al. 2006).

Soil microorganisms play a crucial role in the

cycling of almost all major plant nutrients and

the energy flow of either natural or anthropo-

genically altered soils (Smith and Paul 1990).

Population size and community structure of soil

microorganisms are sensitive to changes in soil

chemical properties (Bauhus and Khanna 1994;

Pansombat et al. 1997; Tokuda and Hayatsu

2002). Soil microbial biomass (Cmic) is both a

labile nutrient pool and an agent of transfor-

mation and cycling of organic matter and plant

nutrients in soils; so, it is one of the most

important microbiological properties. Several

studies indicated Cmic responds more rapidly to

changes resulting from forest management

activities than soil organic matter and, conse-

quently, may be an early and sensitive indicator

of soil quality change (Wolters and Joergensen

1991; Sparling 1992; Bosatta and Agren 1993).

The proportion of microbial biomass C relative

to soil organic C (Cmic/Corg ) has been used as

an indicator for C availability (Anderson and

Domsch 1986b; Insam and Domsch 1988; Yan

et al. 2003) and can provide an effective early

warning of the deterioration of soil quality

(Powlson et al. 1987; Wardle 1992). Soil basal

respiration mainly comes from microbial respi-

ration, which is the major product of aerobic

catabolic processes in the C cycle, and is used as

an indicator of total soil microbial activity. The

metabolic quotient (qCO2), which is the ratio of

basal respiration to microbial biomass, was used

in several investigations as an ecophysiological

measure for ecosystem succession or acid stress

(Insam 1990; Anderson and Domsch 1993).

Several studies (Anderson and Domsch 1993;

Wardle and Ghani 1995) demonstrated higher

values of qCO2 under unfavorable conditions

rather than favorable conditions.

Soil enzymes are present in important cycles,

such as carbon (invertase), nitrogen (urease

and protease), and phosphorus (phosphatase)

cycles. Soil enzyme activities are used as indi-

ces of microbial activity (Bergstrom et al. 1998)

and react quickly to environment change

(Sparling 1997). Net N mineralization and net

nitrification have frequently been used to assess

the ability of soils to supply N in forest eco-

systems and are increasingly being used as

indicators of other important ecosystem pro-

cesses and ecosystem susceptibility to degrada-

tion (Aber et al. 1989).

Soil microbial diversity is a crucial measure

of sustainable soil ecosystems. Tea orchard soils

are usually managed as a monoculture, which

may result in reduced soil microbial diversity.

However, little information is available about

the microbial diversity in tea orchard soil eco-

systems. New molecular techniques, targeting

small subunit rRNA sequences by PCR ampli-

fication, coupled with the rDNA-fragment

analyses by denaturing gradient gel electro-

phoresis (DGGE) (Krave et al. 2002; Rønn

et al. 2002), have recently been developed for

rapidly studying soil microbial diversity. DNA

from the community of soil microorganisms is

amplified using polymerase chain reaction

(PCR), and the fragments are separated in a

gel matrix based on length or sequence poly-

morphism to produce a visual fingerprint of the

microbial community.

Tea orchard ecosystems of different ages rep-

resent a practical system, which can evaluate

temporal changes in soil microbiological and

biochemical properties. Inclusion of adjacent

wasteland, from which tea orchards and forests

were constructed, would facilitate assessing eco-

logical sustainability of the tea orchard ecosystem

and the relative importance of tea orchard man-

agement versus land-use changes in soil microbial

community structure. Our objective was to eval-

uate the changes in soil microbial biomass,

activity, and community diversity given tea orch-

ard age and land-use history.

320 Plant Soil (2006) 288:319–331

123

Materials And Methods

Site description

Soil samples for the study were collected from the

Meijiawu tea area (30�11¢N, 120�05¢E), one of the

original regions of Longjing Tea production, lo-

cated in the West Lake district of Hangzhou,

Zhejiang Province in Southeast China, which is

famous for its Longjing Tea. The area is charac-

terized by a subtropical wet monsoon climate with

mean annual temperatures of 15�C and mean an-

nual rainfall of about 1,500 mm. To assess the

effect of tea orchard age on soil biochemistry and

microbial properties, three tea orchards were se-

lected as study sites. The tea orchards were con-

structed on wasteland in 1914, 1954, and 1996 and

were 90, 50, and 8 years old, respectively, when

soil samples were taken. Each tea orchard was

made up of many plots separated by a foot path.

All tea orchard soils received two or three appli-

cations of N per year, averaging ~450 kg N ha–1

year–1. To evaluate soil biochemistry and micro-

bial properties as a function of land-use change

and management practice, neighboring wasteland

and forest were also chosen as study sites. The

wasteland in this red soil area was covered with

sparse grasses. The 90-year-old forest, established

on wasteland in 1914, was a mixed-conifer forest.

All soils investigated were classified as red soils by

the China Classification System (Ultisols in US

soil taxonomy) and were derived from the same

parent material, namely quartzose sandstone int-

erbedded with shale.

Sample collection and preparation

Soils were collected from three sampling plots

randomly chosen within the 8-year-old tea orch-

ard, 50-year-old tea orchard, 90-year-old tea

orchard, wasteland, and 90-year-old forest in

September 2004. Twenty cores (5 cm diame-

ter · 20 cm length) were taken from each sam-

pling plot and mixed.

The 15 bulked samples were transported on ice

to the laboratory where they were sieved through

a 2-mm mesh to remove plant debris and soil

fauna. Each of the 15 bulked samples was sepa-

rated into three portions. The first portion was air

dried for chemical analysis except that mineral-N

was immediately analyzed. The second portion

was stored at 4�C for microbiological analyses.

The third portion was stored at –20�C until DNA

extractions could be performed.

Soil chemical analysis

Soil pH was measured by a combination glass

electrode (soil : water, 1:2.5). Total nitrogen was

determined by Kjeldahl digestion (Keeney and

Nelson 1982) and quantified using a continuous

flow analyzer (Skalar, Delft, The Netherlands);

and the total Organic C was determined by di-

chromate oxidation (Nelson and Sommers 1982).

Available phosphorus analysis was undertaken by

the method used by Olsen and Sommers (1982).

Inorganic N (NH4+–N and NO3

- –N) was extracted

with 2 M KCl by shaking (1 h, 200 rpm) and fil-

tering through a 0.45 lm polysulfone membrane.

The KCl-extracted N was determined colorimet-

rically in a continuous flow analyzer (Skalar).

Microbial biomass and basal respiration

Microbial biomass C (Cmic) was determined by

the chloroform fumigation–extraction method

(Vance et al. 1987). The K2SO4-extracted C of

both fumigated and unfumigated samples was

analyzed using a total organic C analyzer (Shim-

azu, TOC–500, Chiba, Japan), and a KEC value of

0.45 was used to convert the measured flush of C

to Cmic (Yao et al. 2003). Basal respiration (Rmic)

was determined by measuring CO2 evolution.

Twenty gram (oven-dry basis) of field-moist soil

was incubated in 250 ml airtight glass vessels at

25�C for 1 day. The CO2 produced from the soil

was absorbed in NaOH and determined by titra-

tion with HCl.

The metabolic quotient (qCO2) is defined as

the ratio of basal respiration (Rmic) to microbial

biomass (Cmic), i.e., the amount of CO2–C pro-

duced per unit of microbial biomass carbon

(Anderson and Domsch 1986a).

Net N mineralization and nitrification

A 45-day incubation experiment was carried out

to determine net N mineralization and nitrifica-

Plant Soil (2006) 288:319–331 321

123

tion. Soil samples pre-incubated at room tem-

perature for 1 week were put in sterile polyeth-

yleneglycol bottles (500 ml) and adjusted to the

moisture content of 45% water holding capacity,

and incubated under constant temperature of

25�C and relative humidity of 95%. To avoid loss

of water, all bottles were tightly closed with caps,

but briefly opened once every three days to en-

sure aerobic conditions. Three replicate samples

were extracted with 2 M KCl at day 0, 5, 15, 35,

and 45 of the incubation period. The KCl extracts

were used directly in chemical analysis for inor-

ganic N (NH4+–N and NO3

- –N). Soil inorganic N

on day 0 was determined on pre-incubated soils.

Net soil N mineralization was calculated by sub-

tracting day 0 inorganic N from that measured at

the end of the incubation period. Net nitrification

was calculated in the same manner. At the same

time, fertilizer (400 mg urea-N kg–1 soil) was ap-

plied to assess the effects of nitrogen fertilizer

application on N mineralization and nitrification.

Enzyme activities

Most enzymes are also contained in plant roots.

Therefore, all visible plant fragments were care-

fully removed from the soil sample prior to the

enzyme assays. Enzyme activities were deter-

mined in triplicate field moist samples and are

reported on a dry weight basis.

Invertase activity was determined with sucrose

as substrate; reducing sugars were analyzed as

described by Schinner and von Mersi (1990).

Urease activity was determined according to

Gianfreda et al. (1994) where the product, NH4+,

was measured colorimetrically using the indo-

phenol blue method (Keeney and Nelson 1982).

Protease activity (casein-protease) was assayed

according to the method used by Hamm and Fe-

ger (1996). The calibration was done according to

tyrosine standards. Acid phosphatase activity was

assayed by Hoffman’s method (Alef and Nanni-

pieri 1995).

DNA Extraction, PCR, and DGGE

DNA extraction was based on the method of

Zhou et al. (1996) by bead-beating with slight

modifications. The crude DNA was dissolved in

100 ll of TE (10 mM Tris–HCl; 1 mM EDTA;

pH 8.0) and purified by using Sephadex G–200

(New Haven, CT, USA), as described previously

by Cahyani et al. (2003), based on the method

used by Jackson et al. (1997). To assess DNA

yield and quality (average molecular size), the

soil DNA was run on 0.8% (w/v) agarose gels

with a molecular size marker (1-kb ladder) as the

reference. DNA purity was assessed using ampl-

ifiability by PCR as the criterion.

The universal bacterial primers, PRBA338f

and PRUN518r, located at the V3 region of the

16S rRNA genes of bacterioplankton (Øvreas

et al. 1997), were used to amplify the variable V3

region of 16S rDNA. A GC-rich clamp attached

to the forward primer prevented the complete

melting of the PCR products during subsequent

separation in DGGE. PCR mixtures were pre-

pared with 1 ll purified DNA template (10 ng),

5 ll 10 · PCR buffer, 2.25 mM MgCl2, 0.8 mM

dNTPs, 0.5 lM of each primer, and 2.5 U Taq

DNA polymerase, and sterile filtered milliQ wa-

ter to a final volume of 50 ll. The PCR cycles

included a 4 min initial denaturation at 94�C,

30 cycles at 94�C for 1 min, 55�C for 30 s, 72�C

for 1 min, followed by 7 min at 72�C for final

extension, and, finally, the PCR samples were

held at 4�C until removal from the thermal cycler.

Denaturing gradient gel electrophoresis was

performed with the DcodeTM

Universal Mutation

Detection System (Bio-Rad Laboratories, Her-

cules, CA, USA). PCR products were loaded

onto a 10% (w/v) polyacrylamide gel with a 35–

60% denaturing gradient, where 100% denatur-

ant contains 7.0 M urea and 40% deionized

formamide. The electrophoresis was run at 70 V

for 16 h at 60�C in 1 · TAE buffer (40 mM Tris

[pH 7.4], 20 mM sodium acetate, 1 mM EDTA).

After electrophoresis, the gel was stained using a

silver staining procedure (Bassam et al. 1991) and

visualized with a Bio-Rad Gel Doc documenta-

tion system.

Statistics

All values reported are the arithmetic means of the

three determinations expressed on an oven-dried

soil basis (105�C). Statistical procedures, except

DGGE gel imaging, were carried out with the

322 Plant Soil (2006) 288:319–331

123

software package SPSS for Windows. Means, least

significant differences (LSD) of 5% level, were

calculated by a one-way ANOVA. Clustering

analysis was performed by hierarchical cluster.

Bio-Rad’s Quantity One� software, used for

DGGE gel image acquisition, was also employed

for analysis. Following removal of background

intensity from lanes, the software performs a

density profile through lanes, detects individual

bands, and matches bands occupying the same

position in different lanes. The genetic diversity

of soil microbial communities was analyzed by

Richness (S), Shannon indices (H), and Evenness

(EH) methods according to the following equa-

tions (Zak et al. 1994).

H ¼ �Xs

i¼1

pi ln pi

EH ¼ H=Hmax ¼ H= ln S

Here pi is the ratio between specific band

intensity and total intensity of all bands in a lane

sample and S is the total number of bands in each

lane sample.

Results

Soil chemical properties

The pH of the tea orchard soils decreased grad-

ually with increasing age after wasteland was re-

claimed as tea orchard (Table 1). The pH in the

90-year-old tea orchard was even lower than that

in the 90-year-old forest.

Soil C and N in the 8-year-old tea orchard were

1.88 and 1.59 times higher than those of the

wasteland, respectively. Both soil C and N accu-

mulation increased with increasing tea orchard

age. Soil N in the 90-year-old tea orchard was

significantly (p < 0.05) higher than that in the 90-

year-old forest. Although no significant difference

concerning soil organic C was observed between

the 90-year-old tea orchard and the 90-year-old

forest, the soil C to N ratio in the 90-year-old tea

orchard was lower than that in the 90-year-old

forest.

The pool size of soil inorganic N was signifi-

cantly (p < 0.05) larger in the three tea orchards

than in either the wasteland or the 90-year-old

forest (Table 1). The majority of the inorganic N

in the three tea orchards was in the form of NO3- –

N, which was significantly (p < 0.05) higher than

that in the wasteland and the 90-year-old forest.

Interestingly, the accumulation content of NO3- –N

in the 50-year-old tea orchard was higher than

that in the 8- and 90-year-old tea orchards. The

available P also showed an increasing trend with

the age of the three tea orchards.

Microbial biomass C and basal respiration

Soil microbial biomass C (Cmic) increased sig-

nificantly from 69 lg g–1 in the wasteland to

358 lg g–1 in the 50-year-old tea orchard; then,

it decreased to about 225 lg g–1 in the 90-year-

old tea orchard, with the greatest amount found

in the 50-year-old tea orchard compared to the

other soils (Table 2). The ratio of microbial

biomass C to soil organic C (Cmic/Corg) was

higher in the 8- and 50-year-old tea orchards

than in the 90-year-old tea orchard as well as

the soils in the wasteland and the 90-year-old

forest.

Similarly, soil basal respiration showed an

increasing trend from the wasteland to the 50-year-

old tea orchard; then, a decreasing trend from

the 50-year-old tea orchard to the 90-year-old

Table 1 Basic physico-chemical properties of the soils

Land use history pH(H2O)

Organic C(g kg–1)

Total N(g kg–1)

C/N Available P(mg kg–1)

NH4+–N

(mg kg–1)NO3

- –N(mg kg–1)

Wasteland 5.16 7.4 0.85 8.7 1.7 5.88 6.6Tea orchard–8 years 4.22 13.9 1.35 10.3 17.8 8.02 46.6Tea orchard–50 years 4.01 22.2 2.05 10.8 19.5 7.05 56.1Tea orchard–90 years 3.71 26.3 2.29 11.5 54.5 4.41 40.3Forest–90 years 3.94 27.5 1.75 15.7 23.9 9.20 13.5LSD0.05 0.02 1.3 0.09 0.6 0.1 0.10 0.1

Plant Soil (2006) 288:319–331 323

123

tea orchard was observed. The metabolic quo-

tient (qCO2) was lower in the three tea orch-

ards and forest than that in the wasteland.

Net N mineralization and nitrification and the

effects of heavy nitrogen fertilizer on net N

mineralization and nitrification

The conversion from wasteland to tea orchard

resulted in a significant increase in both net N

mineralization and nitrification (Table 3). How-

ever, with the increase in tea orchard age, net N

mineralization and nitrification showed a

decreasing trend from the 50- to 90-year-old tea

orchard, so that net N mineralization and nitrifi-

cation in the 50-year-old tea orchard were highest

compared to the other soils. The percentage of

mineralized N to soil total N (Nmin/Ntotal) was

higher in the 8- and 50-year-old tea orchards than

that of the other samples.

Based on a 45-day laboratory incubation, al-

most all the applied urea in the three tea orchard

soils was completely transformed into NH4+–N in

the first 5 days, and then NH4+–N significantly

declined with incubation time. However, the urea

applied in the wasteland and the 90-year old

forest may not be entirely transformed into NH4+–

N in the first 5 days since soil NH4+–N concen-

tration continued to increase rapidly after 5-day

incubation (data not shown). With respect to the

effects of urea application on net N mineraliza-

tion based on a 45-day incubation, no obvious

differences between the soils were observed.

However, there was a great contrast between

conditions before and after the application of

urea. The increased NO3- –N content in the three

tea orchards was 305, 374, and 261 mg kg–1,

respectively (Table 3), with the highest value in

the 50-year-old tea orchard. The average of the

increased NO3- –N content in the three tea orchard

soils was 4 and 35 times higher than that of the

forest and wasteland soils, respectively.

Enzyme activities

Invertase activity of the three tea orchard soils

was significantly higher than that of the wasteland

soil and far lower than that of the 90-year-old

Table 2 Microbialbiomass C and basalrespiration of the soils

Land use history Cmic

(lg C g–1

soil)

Cmic/Corg

(%)Rmic

(lg CO2-C g–1

soil h–1 )

qCO2

(lg CO2-C mg–1

Cmic h–1)

Wasteland 69 0.94 0.55 7.96Tea orchard–8 years 176 1.27 1.18 6.71Tea orchard-50 years 358 1.61 1.67 4.67Tea orchard-90 years 225 0.86 1.32 5.88Forest-90 years 261 0.95 1.42 5.44LSD0.05 8 0.05 0.19 0.34

Table 3 N mineralization and nitrification and effect of urea on mineralization and nitrification

Land use history Nmina Increased

Nminb

Nnitc Increased

Nnitd

Nmin/Ntotal

(%)

Wasteland 8.6 351 3.8 9 1.01Tea orchard-8 years 29.7 385 28.3 305 2.20Tea orchard-50 years 46.3 381 43.3 374 2.26Tea orchard-90 years 42.0 361 37.9 261 1.83Forest-90 years 25.7 348 17.2 77 1.47LSD0.05 4.0 38 4.4 9 0.27

a Net N mineralization (mg inorganic N kg-1 soil)b Increased mineralized N by nitrogen fertilizer application (mg inorganic N kg–1 soil)c Net N nitrification (mg NO3

- –N kg–1 soil)d Increased nitrified N by nitrogen fertilizer application (mg NO3

- –N kg–1 soil)

324 Plant Soil (2006) 288:319–331

123

forest soil. In addition, the 50-year-old tea orch-

ard soil had a comparatively higher invertase

activity compared to the 8- and 90-year-old tea

orchard soil. Urease, proteinase, and acid phos-

phatase activities took on a common characteris-

tic, i.e., the three enzyme activities in the three

tea orchard soils were significantly higher than

those in the wasteland and forest soil, with the

largest value occurring in the 50-year-old tea

orchard soil (Table 4).

DGGE

DGGE banding patterns revealed obvious dif-

ferences concerning soil microbial diversity be-

tween tea orchards and other soil ecosystems

(wasteland and forest). Both of the two strong

dominating bands appearing across all of the

samples and a greater number of fainter, well-

resolved and unresolved bands in the background

indicated that the structure of microbial commu-

nities of all tested soils was rather complex. Some

faint bands disappeared, and some bands became

strong in DGGE profiles from wasteland to tea

orchards. Moreover, some of the DGGE bands

were unique to tea orchard soils of different ages.

The numbers of detectable bands in the profiles of

the three tea orchards (24, 27, and 24) were sig-

nificantly lower than those of wasteland (37);

nevertheless, they were higher compared to the

90-year-old forest (16).

Among samples in the Shannon’s diversity in-

dex (H) and richness (S) based on DGGE anal-

ysis of 16S rRNA gene fragments (Table 5), some

differences were detected. The Shannon’s diver-

sity index (H) and richness (S) based on DGGE

profile were obviously lower in all three tea

orchard soils than in the wasteland soil; never-

theless, they were significantly higher in all three

tea orchard soils than in the forest soil. As for the

three tea orchard soils, no differences in com-

munity diversity between the 8- and 90-year-old

tea orchard were observed; and comparatively

higher community diversity was found in the 50-

year-old tea orchard. However, no significant

difference for species evenness was detected.

Cluster analysis of microbial activity and

genetic diversity

Using such properties as basal respiration, N

mineralization, invertase, urease, proteinase, and

acid phosphatase, cluster analysis (Fig. 1a) dem-

onstrated that the three tea orchards formed a

separate cluster compared to the wasteland and

forest.

According to the genetic diversity of the

microbial community (Fig. 1b), cluster analysis

results showed a similar separation among the

different land uses. All three tea orchard soils

presented microbial profiles strictly linked to-

gether at a low-Euclidean distance (2.5), whereas

Table 4 Enzyme activities of the soils

Land use history Invertase(mg glucose kg–1

soil h–1)

Urease(mg NH4

+ kg–1

soil h–1)

Proteinase(mg tyrosine kg–1

soil h–1)

Acid phosphatase(mg phenol kg–1

soil h–1)

Wasteland 210 10.3 21.3 41.9Tea orchard-8 years 691 29.0 69.2 52.2Tea orchard-50 years 712 47.0 86.7 62.3Tea orchard-90 years 623 40.9 64.2 54.2Forest-90 years 1314 8.4 25.1 38.8LSD0.05 20 1.6 1.5 1.4

Table 5 Diversity indices are based on DGGE analysis of16S rRNA gene fragments

Land use history Shannon’sdiversityindex (H)

Richness(S)

Evenness(EH)

Wasteland 3.60 37 0.998Tea orchard-8 years 3.17 24 0.997Tea orchard-50 years 3.28 27 0.997Tea orchard-90 years 3.16 24 0.995Forest-90 years 2.76 16 0.997LSD0.05 0.06 1 0.005

Plant Soil (2006) 288:319–331 325

123

the wasteland and forest soils had different

microbial communities.

Discussion

It is generally accepted that the accumulation of

soil organic C enhances both the Cmic and the

proportion of Cmic relative to soil organic C

(Jenkinson and Ladd 1981). In addition, Ander-

son and Domsch (1989) and Sparling (1992) ob-

served that soil Cmic generally accounts for 1–4%

of total soil organic C. Our study showed that the

percentage of Cmic to soil organic C (Cmic/Corg)

(Table 2) was almost within the range of 1–4%. It

is often assumed that a reasonably close, linear,

and positive relationship exists between the or-

ganic C and biomass C contents. However, we

found that Cmic in the 90-year-old tea orchard was

lower than that in the 50-year-old tea orchard,

although the 90-year-old tea orchard had higher

organic C. This indicates that, with the succeeding

development of tea orchard systems, soil organic

C available for microbes did not increase pro-

portionately to the accumulation of soil organic

matter. The reasons may be as follows: on one

hand, the microbes present under the severely

acidic soil of the 90-year-old tea orchard metab-

olize very slowly, which leads to the extraordi-

narily low capacity of organic C mineralization

(Wardle, 1992; Anderson and Domsch 1993; Yao

et al. 2000). On the other hand, decomposition of

tea leaf litter results in a large amount of organic

C accumulated in the 90-year-old tea orchard soil

and, consequently, lower Cmic/Corg in this soil.

Soil respiration indicates the capacity of total

carbon turnover and soil total microbial activities.

Our results (Table 2) showed that soil respiration

followed the same trend of the Cmic and Cmic/Corg,

which further indicated that, with the chronolog-

ical development of tea orchard systems, the 90-

year-old tea orchard soil organic C that was

available for microorganism activity was signifi-

cantly lower than that from the 50-year-old tea

orchard.

Pansombat et al. (1997) found high concen-

trations of NO3–N in tea orchards and suggested

that nitrification could occur in pH range 3.2–3.8.

Our results also convincingly showed strong

nitrification in acid (pH range, 3.71–4.22) tea

orchard soils. Several factors may be involved.

First, tea orchard soil ecosystems are intensively

Fig. 1 Cluster analysis(a) According to basalrespiration, Nmineralization, invertase,urease, proteinase, andacid phosphatase;(b) According to geneticdiversity indices based onDGGE analysis of 16SrRNA gene fragments.Scale indicates Euclideandistance. Soil no. 1:wasteland, 2: 8-year-oldtea orchard, 3: 50-year-oldtea orchard, 4: 90-year-oldtea orchard, 5: 90-year-oldforest

326 Plant Soil (2006) 288:319–331

123

managed. In particular, frequent nitrogen

fertilizer application stimulated soil nitrification.

Previous studies showed nitrifiers and nitrification

rates are limited by the supply of ammonium

(Robertson and Vitousek 1981; Wedin and Til-

man 1990), and fertilized soils commonly display

higher nitrifying activity due to stimulation of

nitrification by fertilizer ammonium (Martikainen

1985; Aarnio and Martikainen 1996; Mendum

et al. 1999). Our results also showed that urea

application is stimulatory to soil nitrification.

Second, there are probably some acid-tolerant

nitrifiers present in acid tea orchard soils. For

example, Walker and Wickramasinghe (1979)

isolated in pure culture acid-tolerant strains of

Nitrosospira spp from acid tea orchard soils (pH

range, 4.0–4.5) in Bangladesh. As for the mecha-

nism of nitrifiers present at low pH, it is not clear

at present whether acid-tolerant nitrifiers are

comprised of different species or if they are acid-

sensitive strains that have adapted to low-pH

conditions. Lastly, perhaps the soil environment

provides pH-neutral micro-sites for nitrifiers (De

Boer and Kowalchuk 2001).

Tea orchard soils are very particular soil eco-

systems compared to other soils. Long-term hea-

vy nitrogen application, root exudates and leaf

litter have positive and negative effects on nitri-

fier and nitrification rate. On one hand, they can

increase organic matter content and N supplying

capacity. On the other hand, they also can result

in the decline of pH and the accumulation of Al

toxicity and antimicrobial substances (Pandey and

Palni 1996). The higher nitrification in the 50-

year-old tea orchard soil than that in the 8- and

90-year-old tea orchard soil was possibly a result

of a combination reflection of positive and nega-

tive aspects in tea orchard soil ecosystems.

Both tea orchard soils and forest soils are

typical acid soils with similar soil pH. However,

the data showed that soil nitrification in tea

orchards was significantly (p < 0.05) higher than

that in forests. Except for long-term N fertilizer

application in the tea orchard soils, the difference

in soil microbial community between the tea

orchard and the forest soils also likely affects

nitrifier activity and nitrification rate. In addition,

microbial assimilation of nitrate may be one of

the factors resulting in lower net nitrification rates

in forest soil. Many studies (Vitousek and

Andariese 1986; Zak et al. 1990; Davidson et al.

1992; Stark and Hart 1997) showed that a sub-

stantive part of the nitrate that is transformed

from ammonium by nitrification is assimilated by

microorganisms in forest ecosystems.

In natural soil, all kinds of enzyme activities

are positively related to organic matter content

(Guan 1986). However, our results indicated that

only invertase and organic matter are positively

related, and no correlations between urease,

proteinase and organic matter were observed.

This suggests that an aggregate of multi-enzyme

activity may be better correlated with soil fertility

than a single enzyme (Dick and Tabatabai 1992).

Particularly, urease and proteinase activities in

three tea orchards were markedly higher than

those in the wasteland and the 90-year-old forest.

It is well known that urease and proteinase are

mainly involved in the transformation of soil N.

Therefore, our work further demonstrates that

long-term heavy nitrogen fertilizer application

with continuous development of tea orchard soil

ecosystems results in high-enzyme activities in-

volved in soil N cycles (such as urease and pro-

teinase). In three tea orchards, urease, and

proteinase showed the same trend as other

microbial activity properties (such as Rmic, Cmic/

Corg), i.e., urease and proteinase in the 90-year-

old tea orchard were lower than that in the 50-

year-old tea orchard. This may be due to low-

microbial biomass, low pH and high content of

antimicrobial substances in the 90-year-old tea

orchard. These results also indicated that enzyme

activities can be used as indices of microbial

activity (Bergstrom et al. 1998).

According to the banding number and the

varying distances that the PCR products migrated

in the gradient gel, the combined application of

PCR amplification of 16S rDNA sequences and

DGGE could be used to distinguish differences in

microbial community diversity among wasteland,

tea orchards and forest. These differences in

microbial community diversity may be due to the

soils being covered with different plant types

(Waid 1999; Grayston et al. 2001; Marschner et al.

2001). In the present experiment, some faint

bands disappeared, and some bands became

strong in DGGE profile from wasteland to tea

Plant Soil (2006) 288:319–331 327

123

orchards. Additionally, some of the DGGE bands

were unique to tea orchard soils of different ages,

indicating with the tea orchard ecosystems

established after wasteland was reclaimed, a

remarkable finding of change in the soil microbial

community diversity. As some microbial species

can not adapt to the unfavourable stresses of tea

orchard soils and therefore disappeared, never-

theless, some unique microbial species can adapt

to the changed environment and do exist in the

stressed habitat. Some bands with common dis-

tances that migrated in the DGGE profile from

different soils may result from the same micro-

organisms in different soils. Of course, these

bands may also consist of several different

microbial species having the same GC content in

the amplified region.

Many studies have documented that soil

microbial community structure responds to soil

management practices as well as several envi-

ronmental factors. The studies of Bossio et al.

(2005) demonstrated that DGGE analysis of

amplified 16S rDNA fragments revealed differ-

ences in the microbial community structure in

relation to differing soil management practices.

Yang et al. (2000) observed that the application of

NH4HCO3 resulted in a decrease in DNA se-

quence diversity of the soil microbial community.

Waid (1999) pointed out that vegetation type,

quantity and chemical composing may be impor-

tant factors in determining soil microbial com-

munity diversity. Pandey and Palni (1996)

observed that the roots of tea bushes are able to

restrain microorganisms. In addition, the tea bush

leaf litter can also contain antimicrobial sub-

stances, and the low pH can result in Al toxicity,

as a result, leading to a decrease in microbial

species. Low-soil pH has been identified as the

major constraint on soil microorganisms within

study sites of oak, acidophilous beech and spruce–

fir–beech forests (Hackl et al. 2000). Based on the

studies mentioned above, it is possible that per-

sistent input of leaf litter, root exudates and se-

vere acidification lead to a decrease in soil

microbial community diversity in tea orchards

and forests. In addition, the application of long-

term heavy nitrogen fertilizer may partly induce

the change in soil microbial community diversity

in tea orchards.

The cluster analysis figures (Fig. 1) took on a

common characteristic: namely, tea orchards and

wasteland had significant differences in microbial

biomass, activity and genetic diversity. This may

be attributed to the positive relationship between

microbial biomass and organic matter content

(Vance and Nadkarni 1990; Sparling 1992; Spar-

ling et al. 1994). Large amounts of organic matter

that accumulated due to the planting of tea bu-

shes after wasteland was reclaimed resulted in

markedly larger microbial biomass in tea orch-

ards than in wasteland. This was consistent with

higher microbial activity in tea orchards than in

wasteland. However, tea orchard soils exist

amidst so many potentially inhibitory factors,

such as severe acidification, Al toxicity, root ex-

udates and heavy nitrogen fertilizer application.

As a result, obviously lower microbial genetic

diversity was detected in tea orchards than in

wasteland.

Our cluster analysis indicated that land use

type could considerably affect the soil microbial

community. All of the three tea orchard soils

presented microbial profiles strictly linked to-

gether at a low-Euclidean distance, although the

three tea orchard soils seemed to have a sig-

nificant difference in soil organic C or microbial

biomass. The result suggested that a change in

the microbial biomass is not always accompa-

nied by a change in the microbial community

diversity and activity. Most studies demon-

strated that vegetation type is an important

factor in determining soil microbial community

diversity (Grayston et al. 2001; Marschner et al.

2001). Consequently, shifts in soil microbial

community diversity were attributed primarily to

the change in plant type rather than organic

matter content.

Conclusions

With the chronological development of tea orch-

ard ecosystems, soil C and N gradually increased;

however, Cmic, microbial activity properties, and

microbial community diversity showed a common

characteristic: namely, they showed an increasing

trend from the 8- to 50-year-old tea orchard and

then a decreasing trend from the 50- to 90-year-old

328 Plant Soil (2006) 288:319–331

123

tea orchard. These results suggested that fertiliza-

tion, root exudates and leaf litter in tea orchard

ecosystems have positive and negative effects on

the microbial community. They may increase

organic matter content and N supplying capacity;

therefore, the conversion from wasteland to tea

orchard may afford increased microbial biomass

and activity accordingly. However, they may also

result in the decline of pH, increased Al toxicity

and increased production of antimicrobial

substances, which consequently lead to the decline

of microbial biomass and activity from the 50- to

90-year-old tea orchard.

Higher net N nitrification in the tea orchards

compared to the wasteland and the forest sug-

gested that nitrogen fertilizer application can af-

fect nitrifiers and nitrification rates. In addition,

our results demonstrated that microbial commu-

nity diversity was distinctly different in the three

different soil ecosystems, indicating that the shifts

in soil microbial community diversity were

attributed primarily to the change in vegetation

type rather than in organic matter content.

Acknowledgments This work was financially supportedby the National Science Foundation of China (No.30671207 and 40371063). The authors greatly appreciatethe field support provided by Professor Wenyan Han.

References

Aarnio T, Martikainen PJ (1996) Mineralization of carbonand nitrogen, and nitrification in Scots pine forest soiltreated with fast- and slow-release nitrogen fertilizers.Biol Fertil Soils 22:214–220

Aber JD, Nadlehoffer KJ, Steudler PJ, Melillo JM (1989)Nitrogen saturation in northern forest ecosystems.BioScience 39:378–393

Alef K, Nannipieri P (1995) Methods in applied soilmicrobiology and biochemistry. Academic Press, NewYork, pp335–337

Anderson TH, Domsch KH (1986a) Carbon assimilationand microbial activity in soil. Z PflanzenernahrBodenkd 149:457–486

Anderson TH, Domsch KH (1986b) Carbon link betweenmicrobial biomass and soil organic matter. In: Megu-sar F, Gantar M (eds) Perspectives in microbialecology. Slovene Society for Microbiology, Ljubljana,pp467–471

Anderson TH, Domsch KH (1989) Ratios of microbialbiomass carbon to total organic carbon in arable soils.Soil Biol Biochem 21:471–479

Anderson TH, Domsch KH (1993) The metabolic quotientfor CO2 (qCO2) as a specific activity parameter toassess the effects of environmental conditions, such aspH, on the microbial biomass of forest soils. Soil BiolBiochem 25:393–395

Bassam BJ, Caetano-Anolles G, Gresshoff PM (1991) Fastand sensitive silver staining of DNA in polyacryl-amide gels. Anal Biochem 196:80–83

Bauhus J, Khanna PK (1994) Carbon and nitrogen turn-over in two acid forest soils of southeast Australia asaffected by phosphorus addition and drying and rew-etting. Biol Fertil Soils 17:212–218

Bergstrom DW, Monreal CM, King DJ (1998) Sensitivityof soil enzyme activities to conservation practices. SoilSci Soc Am J 62:1286–1295

Bosatta E, Agren G (1993) Theoretical analysis ofmicrobial biomass dynamics in soils. Soil Biol Bio-chem 26:143–148

Bossio DA, Girvan MS, Verchot L, Bullimore J, Borelli T,Albrecht A, Scow KM, Ball AS, Pretty JN, OsbornAM (2005) Soil microbial community response toland use change in an agricultural landscape of wes-tern Kenya. Microbial Ecol 49:50–62

Cahyani VR, Matsuya K, Asakawa S, Kimura M (2003)Succession and phylogenetic composition of bacterialcommunities responsible for the composting processof rice straw estimated by PCR-DGGE analysis. SoilSci Plant Nutr 49:619–630

Chantigny MH, Prevost D, Angers DA, Vezina LP,Chalifour FP (1996) Microbial biomass and N trans-formations in two soils cropped with annual andperennial species. Biol Fertil Soils 21:239–244

Chenery EM (1955) A preliminary study of aluminium andthe tea bush. Plant Soil 6:174–200

Davidson EA, Hart CS, Firestone MK (1992) Internalcycling of nitrate in soils of a mature coniferous forest.Ecology 73:1148–1156

De Boer W, Kowalchuk GA (2001) Nitrification in acidsoils: micro-organisms and mechanisms. Soil BiolBiochem 33:853–866

Dick WA, Tabatabai MA (1992) Significance and potentialuses of soil enzymes. In: Metting FJB (ed) Soilmicrobial ecology: applications in agriculture andenvironmental management. Marcel Dekker, NewYork, pp95–125

Gianfreda L, Sannino F, Ortefa N, Nannipieri P (1994)Activity of free and immobilized urease in soil: effectsof pesticides. Soil Biol Biochem 26:777–784

Grayston SJ, Griffith GS, Mawdsley CD, Campbell CD,Bardgett RD (2001) Accounting for variability in soilmicrobial communities of temperate upland grasslandecosystems. Soil Biol Biochem 33:533–551

Guan Y (1986) Soil enzyme and research methods. ChinaAgricultural Press, Beijing, pp206–239

Hackl E, Bachmann G, Zechmeister-Boltenstern S (2000)Soil microbial biomass and rhizosphere effects innatural forest stands. Phyton 40:83–90

Hamm D, Feger KH (1996) An optimized method for thedetermination of protease activity in acid forest soils.Z Pflanzenernahr Bodenkd 159:37–39

Plant Soil (2006) 288:319–331 329

123

Insam H (1990) Are the soil microbial biomass and basalrespiration governed by the climate regime? Soil BiolBiochem 22:525–532

Insam H, Domsch KH (1988) Relationship between soilorganic carbon and microbial biomass on chronose-quences of reclamation sites. Microbial Ecol 15:177–188

Jackson CR, Harper JP, Willoughby D, Roden EE,Churchill PF (1997) A simple, efficient method for theseparation of humic substances and DNA from envi-ronmental samples. Appl Environ Microb 63:4993–4995

Jenkinson DS, Ladd JN (1981) Microbial biomass in soil:measurement and turnover. In: Paul EA, Ladd JN(eds) Soil biochemistry. Marcel Dekker, New York,pp415–471

Keeney DR, Nelson DW (1982) Nitrogen-inorganic forms.In: Page AL, Miller RH, Keeney DR (eds) Methodsof soil analysis. American Society of Agronomy,Madison, pp643–698

Konishi S (1991) Chemistry of tea. In: Muramatsu K (eds.),Tea science. Asakura-Shoten, Tokyo, pp21–32

Krave AS, Lin B, Braster M, Laverman AM, Van StraalenNM, Roling WFM, Van Verseveld HW (2002) Strat-ification and seasonal stability of diverse bacterialcommunities in a Pinus merkusii (pine) forest soil incentral Java, Indonesia. Environ Microb 4:361–373

Marschner P, Yang CH, Lieberei R, Crowley DE (2001)Soil and plant specific effects on bacterial communitycomposition in the rhizosphere. Soil Biol Biochem33:1437–1445

Martikainen PJ (1985) Numbers of autotrophic nitrifiersand nitrification in fertilized forest soil. Soil BiolBiochem 17:245–248

Mendum TA, Sockett RE, Hirsch PR (1999) Use ofmolecular and isotopic techniques to monitor the re-sponse of autotrophic ammonia-oxidizing populationsof the b subdivision of the class Proteobacteria inarable soils to nitrogen fertilizer. Appl Environ Mic-rob 66:4155–4162

Nelson DW, Sommers LE (1982) Total carbon, organiccarbon and organic matter. In: Page AL, Miller RH,Keeney DR (eds) Methods of soil analysis. AmericanSociety of Agronomy, Madison, pp539–580

Nioh I, Isobe T, Osada M (1993) Microbial biomass andsome biochemical characteristics of a strongly acid teafield soil. Soil Sci Plant Nutr 39:617–626

Olsen SR, Sommers LE (1982) Phosphorus. In: Page AL,Miller RH, Keeney DR (eds) Methods of soil analysis.American Society of Agronomy, Madison, pp539–580

Øvreas L, Forney L, Daae FL, Torsvik T (1997) Distri-bution of bacterioplankton in meromictic Lake Sae-lenvannet, as determined by denaturing gradient gelelectrophoresis of PCR-amplified gene fragmentscoding for 16S rRNA. Appl Environ Microb 63:3367–3373

Pandey A, Palni LMS (1996) The rhizosphere effect of teaon soil microbes in a Himalayan monsoonal location.Biol Fertil Soils 21:131–137

Pansombat K, Kanazawa S, Horiguchi T (1997) Micro-bial ecology in tea soils I. Soil properties andmicrobial populations. Soil Sci Plant Nutr 43:317–327

Powlson DS, Brookes PC, Jenkinson DS (1987) Mea-surement of soil microbial biomass provides an earlyindication of changes in total soil organic matter dueto straw incorporation. Soil Biol Biochem 19:159–164

Robertson GP, Vitousek PM (1981) Nitrification poten-tials in primary and secondary succession. Ecology62:376–386

Rønn R, McCaig AE, Griffiths BS, Prosser JI (2002) Im-pact of protozoan grazing on bacterial communitystructure in soil microcosms. Appl Environ Microb68:6094–6105

Schinner F, Von Mersi W (1990) Xylanase-, CM-cellulase-and invertase activity in soil: an improved method.Soil Biol Biochem 22:511–515

Smith JL, Paul EA (1990) The significance of soil micro-bial biomass estimations. In: Stotzky G, Bollag JM(eds) Soil biochemistry. Marcel Dekker, New York,pp357–396

Sparling GP (1992) Ratios of microbial biomass carbon tosoil organic carbon as a indicator of changes in soilorganic matter. Aust J Soil Res 30:195–207

Sparling GP (1997) Soil microbial biomass, activity andnutrient cycling as indicators of soil health. In: Pank-hurst CE, Doube BM, Gupta VVSR (eds) Biologicalindicators of soil health. CAB International, NewYork, pp97–119

Sparling GP, Hart PBS, August JA, Leslie DM (1994) Acomparison of soil and microbial carbon, nitrogen,and phosphorus contents, and macro-aggregate sta-bility of a soil under native forest and after clearancefor pastures and plantation forest. Biol Fertil Soils17:91–100

Stark JM, Hart SC (1997) High rates of nitrification andnitrate turnover in undisturbed coniferous forests.Nature 385:61–64

Tachibana N, Yoshikawa S, Ikeda K (1995) Influences ofheavy application of nitrogen on soil acidificationand root growth in tea fields. Jpn J Crop Sci 64:516–522

Tokuda S, Hayatsu M (2001) Nitrous oxide emission po-tential of 21 acidic tea field soils in Japan. Soil SciPlant Nutr 47:637–642

Tokuda S, Hayatsu M (2002) Soil microbial biomass andfluorescein diacetate hydrolytic activity in Japaneseacidic tea field soils. Soil Sci Plant Nutr 47:865–869

Tokuda S, Hayatsu M (2004) Nitrous oxide flux from a teafield amended with a large amount of nitrogen fertil-izer and soil environmental factors controlling theflux. Soil Sci Plant Nutr 50:365–374

Vance ED, Brookes PC, Jenkinson DC (1987) Anextraction method for measuring soil microbial bio-mass-C. Soil Biol Biochem 19:703–707

Vance ED, Nadkarni NM (1990) Microbial biomass andactivity in canopy organic matter and the forest floor ofa tropical cloud forest. Soil Biol Biochem 22:677–684

330 Plant Soil (2006) 288:319–331

123

Vitousek PM, Andariese SW (1986) Microbial transfor-mations of labelled nitrogen in a clear-cut pine plan-tation. Oecologia 68:601–605

Waid JS (1999) Does soil biodiversity depend uponmetabiotic activity and influences? Appl Soil Ecol13:151–158

Walker N, Wickramasinghe KN (1979) Nitrification andautotrophic nitrifying bacteria in acid tea soils. SoilBiol Biochem 11:231–236

Wardle DA (1992) A comparative assessment of factorswhich influence microbial biomass carbon and nitro-gen levels in soils. Biol Rev 67:321–358

Wardle DA, Ghani A (1995) A critique of the microbialmetabolic quotient (qCO2) as a bioindicator of dis-turbance and ecosystem development. Soil Biol Bio-chem 27:1601–1610

Wedin DA, Tilman D (1990) Species effects on nitrogencycling: a test with perennial grasses. Oecologia84:433–441

Wolters V, Joergensen RG (1991) Microbial carbonturnover in beach forest soils at different stages ofacidification. Soil Biol Biochem 23:897–902

Yan T, Yang L, Campbell CD (2003) Microbial biomassand metabolic quotient of soils under different landuse in the three Gorges reservoir area. Geoderma115:129–138

Yang YH, Yao J, Hu S, Qi Y (2000) Effects of agriculturalchemicals on DNA sequence diversity of soil micro-bial community: a study with RAPD marker. Micro-bial Ecol. 39:72–79

Yao H, He Z, Wilson MJ, Campbell CD (2000) Microbialbiomass and community structure in a sequence ofsoils with increasing fertility and changing land use.Microbial Ecol 40:223–237

Yao H, Liu Y, Xue D (2006) Influence of tea cultivation onsoil microbial biomass and substrate utilization pat-tern. Commun Soil Sci Plan 37:641–651

Yao H, Xu J, Huang C (2003) Substrate utilization pattern,biomass and activity of microbial communities in asequence of heavy metal—polluted paddy soils.Geoderma 115:139–148

Zak DR, Groffman PM, Pregitzer KS, Christensen S,Tiedje JM (1990) The vernal dam: plant-microbecompetition for nitrogen in northern hardwood for-ests. Ecology 71:651–656

Zak JC, Willig MR, Moorhead DL, Wildman HG (1994)Functional diversity of microbial communities: aquantitative approach. Soil Biol Biochem 26:1101–1108

Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery fromsoils of diverse composition. Appl Environ Microb62:316–322

Plant Soil (2006) 288:319–331 331

123