Applied Soil Ecology 46 (2010) 230239

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Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

How effective are Effective microorganisms (EM)? Results from a eld study in
temperate climate
Jochen Mayer a, , Susanne Scheid a , Franco Widmer a , Andreas Fliebach b , Hans-Rudolf Oberholzer a
a
Agroscope Reckenholz-Tnikon Research Station ART, Reckenholzstrasse 191, 8046 Zurich, Switzerland
b
Research Institute of Organic Agriculture FiBL, Ackerstrasse, 5070 Frick, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Effective microorganisms (EM) is a microbial inoculant promoted to stimulate plant growth and soil
Received 19 April 2010 fertility in agriculture. In our study we investigated the effects of EM on crop yields and soil microbial
Received in revised form 23 July 2010 parameters in a 4-year eld experiment under organic management (20032006) in Zurich, Switzerland.
Accepted 6 August 2010
Treatments of the EM preparations (i) the spraying agent EMA, (ii) EMA with the EM enriched organic
substrate Bokashi and (iii) EMA with Bokashi and farmyard manure were applied in each year. As controls
Keywords:
to treatments (i)(iii) the same treatments were included with sterilised EM preparations and a control
Effective microorganisms
without EM application. Crop yields in each year and the soil microbiological parameters soil respiration,
Plant growth-promoting microorganisms
Soil microbiology
microbial biomass (SIR, CFE), dehydrogenase activity and microbial community structure (RISA, CLSU)
Crop yield were determined in spring and autumn 2005 and spring 2006. In laboratory incubation experiments
N mineralisation cellulose degradation, N mineralisation potential and N mineralisation from added substrate were deter-
mined. The EMA application as spraying agent alone (treatment (i)) showed no signicant differences to
the untreated control (treatment without EM application) for any of the investigated parameters. Sig-
nicant differences to the untreated control for crop yields and soil microbial parameters were found if
Bokashi was applied in addition to EMA ((ii) and (iii)). However, these differences were not consistent
throughout the parameters and sampling times. Treatments with living EM compared with its sterilised
control treatments showed no differences on any of the parameters. This indicates that the small effects
observed were not caused by the EM microorganisms but rather by the nutrient inputs derived from
Bokashi. The sampling time showed stronger effects on soil microbial biomass, soil respiration and micro-
bial community structure when compared to the effects of the treatments. We conclude from our results
that Effective microorganisms did not improve yields and soil quality during 4 years of application in
this eld experiment under the temperate climatic conditions of Central Europe.
2010 Elsevier B.V. All rights reserved.

1. Introduction availability to crops (Khan et al., 2007), the prevention of infections

Management strategies in sustainable agricultural systems are
aimed at reducing the inputs of chemical fertilisers, pesticides and
energy demand in general. Sustainable agricultural systems are
characterised by diversied crop rotations, the integration of ani-
mal and crop production, a larger share of leguminous plants as
seed crops, catch crops or intercrops in the rotation, organic fertili-
sation and an integrated pest and disease management (Lichtfouse
et al., 2009). Microbial inoculants of plant growth-promoting
microorganisms (PGPM) used as biofertilisers or biopesticides are
attracting growing interest in such management strategies (Vessey,
2003; Banerjee et al., 2005), because of their expected potential for
improving efciency of N2 -xation (Kennedy et al., 2004), nutrient
Corresponding author. Tel.: +41 44 377 72 14; fax: +41 44 377 72 01.
E-mail address: Jochen.mayer@art.admin.ch (J. Mayer).
by phytopathogens (Compant et al., 2005) and their possible use
as soil conditioners. However, the complexity of the interactions
among PGPM and possible synergistic effects are only poorly under-
stood. Most research on benecial effects of PGPM has focused
on inocula with single microbial species or strains (Vessey, 2003),
but there is evidence that interactions of different microorganisms
may be complementary, and that microbial diversity may inuence
plant growth (van der Heijden et al., 2008).
Many different microbial biofertilisers are available on the mar-
ket for agricultural use. The products claim to enhance plant growth
and yields and to improve soil fertility, but often the microbial com-
position is not specied in detail, making it difcult for the users
to evaluate the product and for scientists to prove its effectiveness
(Schenck zu Schweinsberg-Mickan and Mller, 2009).
One biofertiliser that has received a lot of attention is the so-
called microbial inoculum Effective microorganisms (EM), the
detailed composition of which is kept secret. It was developed by

0929-1393/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2010.08.007
 J. Mayer et al. / Applied Soil Ecology 46 (2010) 230239
Teruo Higa at the University of Ryukyus, Okinawa, Japan (Higa,
1991) and has been described as a combination of about 80 coex-
isting benecial microorganisms, which were selected from more
than 2000 species isolated from various environments (Higa, 1993;
Higa and Parr, 1995). EM preparations are reported to include pop-
ulations of lactic acid bacteria and yeasts and smaller numbers of
phototrophic bacteria, lamentous fungi and Actinomycetes (Higa
and Parr, 1995). These species may coexist in a liquid media and
are reported to be abundant in the carrier solution at a pH below
3.5 (Higa, 2001). The main species included in EM preparations
are lactic acid bacteria Lactobacillus plantarum, Lactobacillus casei,
Streptococcus lactis, photosynthetic bacteria Rhodopseudomonas
palustris and Rhodobacter sphaeroides, the yeasts Saccharomyces
cerevisiae and Candida utilis, the Actinomycetes Streptomyces albus
and Streptomyces griseus, and nally the fungi Aspergillus oryzae
and Mucor hiemalis (Xu, 2000). It is not clear wether EM con-
tains microorganisms that can act as PGPM. But Higa (1993, 2001,
2003) suggested a very broad application range of EM preparations
and has reported benecial effects in different environments such
as soils, plants and water. It has been proposed that EM prepa-
rations help to produce substances acting as antioxidants, such
as inositol, ubiquinone, saponin, low-molecular polysaccharides,
polyphenols and chelating agents. These substances may inhibit
harmful microbial species; enhance the proliferation of benecial
microorganisms and detoxify harmful substances simultaneously
(Higa, 1993, 2001, 2003). EM is commercially available as concen-
trated EM-1 suspension. This is used as a basic formulation of
different EM preparations produced by anaerobic fermentation of
sugar cane molasses and water (EMA), or with sugar cane molasses,
water, ethanol and vinegar (EM5), or with sugar cane molasses and
a fermentable organic substrate (Bokashi). EMA and EM5 are used
as spraying agents to enhance soil and manure quality or as a dis-
ease control agent. Combined application of EM preparations with
manure or organic substrates is recommended to improve the ef-
ciency of EM (www.EMschweiz.ch/; date of release 01.12.2009).
Only small and often contradictory effects of the application
of EM preparations on crop yields and plant development have
been reported (Iwaishi, 2000; Xu, 2000; Bajwa, 2005; Priyadi et
al., 2005; Javaid, 2006; Khaliq et al., 2006; van Vliet et al., 2006;
Okorski et al., 2008). EM-applications have also been found to have
no effects (Priyadi et al., 2005; Khaliq et al., 2006; van Vliet et
al., 2006). Only few studies exist on the effects of EM on decom-
position of crop residues in soil and on soil quality in general.
Schenck zu Schweinsberg-Mickan and Mller (2009) found no
effects of EM on N mineralisation and microbial biomass C and
N in soil without amendments and with application of wheat
straw and coarse meal of yellow lupins. Small observed effects
have been related to the EM carrier substrate molasses. Okorski
and Majchrzak (2007) observed the highest soil fungal diversity
if EM was applied together with pesticides in a eld experiment
under peas. However, the experiment lacked sterilised EM con-
trol treatments, so possible effects of the carrier substrate were not
accounted for. There is also a lack of eld scale studies, especially in
temperate climates such as in Central Europe. Despite the limited
number of studies and contradictory results, EM is promoted with
the promise of benets like . . . maximizes conversion of organic
matter into soil humus and . . . increases benecial native micro-
bial populations (http://www.emamerica.com/; date of release
15.08.2009).
The focus of this study was to evaluate the effects of EM prepa-
rations on the eld scale under organic farming conditions. The
objectives of our eld experiment were to evaluate the effects of dif-
ferent preparations of EM applied during four consecutive years (i)
on crop yields, (ii) on microbial parameters characterising biomass,
structure and activity of the soil microbial community, and (iii) on
the decomposition of organic substrates.
231
2. Materials and methods
2.1. Experimental site
A eld experiment was established at Agroscope Reckenholz-
Tnikon Research Station ART in Zurich, Switzerland
(681720/253100; Swiss grid). The soil is a medium eutric Regosol
with the topsoil (020 cm) characteristics of 16% clay, 24% silt and
60% sand, a pH (0.01 M CaCl2 ) of 7.2, an organic carbon content of
1.1% and available nutrients of 5 mg kg1 P (CO2 saturated water),
35 mg kg1 K (CO2 saturated water) and 60 mg kg1 Mg (0.0125 M
CaCl2 ). Annual mean air temperature was 8.5 C and average
annual precipitation was 1042 mm year1 (Table 1).
2.2. Experimental treatments and design
Two commercial preparations of Effective Microorganisms
(EM) were applied. Both were produced from the basic EM con-
centrate EM-1 by the manufacturer (Bionova Hygiene GmbH,
Stans, Switzerland). According to the manufacturers specications,
they contained 1.3 107 colony forming units (cfu) of lactic acid
bacteria ml1 , 3.3 104 cfu of photosynthetic bacteria ml1 and
1.3 104 cfu of yeasts ml1 . EMA (activated EM) was applied as
a spraying agent, and was processed from EM-1 by fermentation
under anaerobic conditions in water with sugar cane molasses
for 7 days until pH was below 3.5. Bokashi, produced from EM-
1 concentrate by fermentation with wheat bran and sugar cane
molasses was applied as an organic fertiliser and a soil condi-
tioner. These two preparations were applied and combined in
3 treatments of the eld trial (i) EMA spraying only (sp), (ii)
EMA spraying combined with Bokashi (sp + bok), and (iii) EMA
spraying combined with Bokashi and cattle manure (sp + bok + m;
Table 2). To compare the effects of EM to those of their carrier
substrates, each of the three treatments was related to a par-
allel control with sterilised EMA and Bokashi by autoclaving at
120 C for 20 min (sp au, sp + bok au, sp + bok + m au, Table 2). In
an untreated control treatment, water was applied as a spraying
agent (Table 2). Bokashi contained 401 kg N, 16 kg P, 33 kg K and
7 kg Mg ha1 a1 and manure contained 49 kg N, 14 kg P, 55 kg K and
8 kg Mg ha1 a1 . The eld experiment was run as a complete ran-
domised block design with four replicates and a plot size of 28 m2
(4 m 7 m).
2.3. Cultivation and eld samplings
During the experimental period from 2003 to 2006, the eld
was cultivated according to organic farming practices. Potatoes
(Solanum tuberosum, cv. Appell) were grown in 2003 (planted on
March 15, 2003), followed by winter barley (Hordeum vulgare, cv.
Lyric) in 2004 (sown on September 30, 2003), alfalfa (Medicago
sativa, cv. Sandit) in 2005 (sown after the harvest of barley on
August 9, 2004) and nally winter wheat (Triticum aestivum, cv.
Titlis) in 2006 (sown on October 31, 2005). An additional fertili-
sation of 34 m3 cattle slurry ha1 was applied to winter barley on
April 14, 2004 in all treatments.
The rst EMA spraying, the Bokashi preparation and manure
were applied to the soil after tillage and before seedbed prepara-
tion and sowing of the main crops (Table 2). The following EMA
sprayings were applied during the vegetation period until ower-
ing and after the cutting of alfalfa. In total 110 l EMA ha1 a1
and 2.9 t Bokashi (fresh weight) ha1 a1 were applied
(Table 2).
Soil samples were taken from 0 to 20 cm depth with an auger
(diameter 2.5 cm, 15 cores per plot) on March 18, 2005, on October
26, 2005 (autumn 2005 I) and on November 07, 2005 (autumn 2005
II) immediately before and after sowing of winter wheat and on
232 J. Mayer et al. / Applied Soil Ecology 46 (2010) 230239

Table 1
Temperatures and rainfall at the experimental site from 2003 to 2006 at Agroscope Reckenholz-Tnikon Research Station, Zurich, Switzerland.

Mean Temperature ( C) Rainfall (mm year1 )

2003 2004 2005 2006 2003 2004 2005 2006

JanuaryDecember 10.2 9.7 9.6 10.3 750 976 901 1140

MarchMay 10.6 8.7 9.7 9.0 141 205 276 488
JuneAugust 21.6 17.9 18.1 18.9 236 335 372 301

Table 2
Treatments of the eld experiment.

No. Treatmenta EMA sprayingb EM-Bokashi Manure

1 control 3 H2 O
2 sp 3c
3 sp au 3 auc
4 sp + bok 3 2.9 t ha1
5 sp + bok au 3 au 2.9 t ha1 au
6 sp + bok + m 3 2.9 t ha1 10 t ha1
7 sp + bok + m au 3 au 2.9 t ha1 au 10 t ha1
a
sp: EMA spraying; bok: Bokashi application, m: manure application, au: autoclaved treatments.
b
110 l EMA ha1 per application.
c
in 2003 additional pickling of potato seed stock with EMA.

March 27, 2006. Soil parameters were analysed on the respective where EN = (Nt in fumigated samples Nt in control samples) and
sampling dates (Table 3). kEN = 0.54 (Jrgensen and Mller, 1996).

2.4. Analytical methods 2.4.2. Basal soil respiration

2.4.1. Soil microbial biomass
2.4.1.1. Substrate-induced respiration. Soil microbial biomass C
(Cmic ) was estimated by substrate-induced respiration (SIR) accord-
ing to Anderson and Domsch (1978) after a pre-incubation period
of 7 days at 22 C. Sieved soil samples (2 mm mesh) were mixed
with 150 mg glucose 50 g1 soil (dry weight equivalent). The initial
CO2 evolution response of each of the four replicates was measured
with an infrared gas analyzer (IRGA) according to Heinemeyer et
al. (1989). Soil microbial biomass Cmic -SIR (g g1 soil) was calcu-
lated from the initial respiration rates as 30.0 l CO2 g1 soil h1 ,
equivalent to 1 g Cmic g1 (Kaiser et al., 1992).
2.4.1.2. Chloroform fumigation extraction. Microbial biomass car-
bon (Cmic ) and nitrogen (Nmic ) were measured according to Vance
et al. (1987) by chloroform fumigation extraction (CFE). CFE was
performed in triplicate on 20 g (dry matter) subsamples that were
extracted with 80 ml of a 0.5 M K2 SO4 solution. Total organic carbon
(TOC) was determined by infrared spectrometry after combus-
tion at 850 C (DIMA-TOC 100, Dimatec, Essen, Germany). Total
nitrogen was subsequently measured in the same sample and gas
stream by chemoluminescence (TNb , Dimatec, Essen, Germany).
Soil microbial biomass C and N was then calculated as follows:
Cmic = EC /kEC , where EC = (TOC in fumigated samples TOC in con-
trol samples) and kEC = 0.45 (Jrgensen, 1996), and Nmic = EN /kEN ,
Basal soil respiration (BSR) was measured in pre-incubated (7
days at 22 C) samples by determining CO2 evolution over a period
of 72 h (FAL, 1996). Soil samples (20 g dry weight equivalent) were
weighed into perforated centrifuge tubes and placed into a screw
bottle (Schott, 250 ml) containing 0.025 N NaOH as a CO2 -trap for a
24 h pre-incubation period in the bottle. The actual measurement
started by adding exactly 20 ml of 0.025 N NaOH. After 72 h the
alkali was titrated with 0.025 N HCl.
2.4.3. Soil dehydrogenase activity
Dehydrogenase activity was measured according to Tabatabai
(1982) in 5 g soil samples (dry weight equivalent) that were
incubated at 30 C for 24 h in the presence of triphenyltetrazoli-
umchloride as an alternative electron acceptor. The red coloured
product triphenylformazan (TPF) was extracted with acetone and
measured in a spectrophotometer at 546 nm.
2.4.4. Cellulose degradation
Cellulose degradation potential (Jggi 2005, pers. communica-
tion) in soil samples was determined by placing a cellulose lter
disk, (diameter 55 mm; Schleicher & Schuell 2668) in a petri dish
(94 mm 16 mm), covered with a nylon mesh (1 mm) and wetted.
Fifty grams (dry weight equivalent) moist, 2 mm sieved soil was
added. The soil was compacted on a vibration plate weighted with
a metal plate of 500 g to obtain a close contact between the soil and

Table 3
Time of soil sampling in the eld experiment and determined soil microbiological parameters at the respective sampling date.

Time of soil sampling Microbiological parameters

March 18th 2005 Lucerne Microbial biomass,a soil respiration

October 26th 2005 (Autumn 05 I) Lucerne, immediately before tillage and EM application Microbial biomass, soil respiration, cellulose
decomposition, RISAb
November 07th 2005 (Autumns 05 II) After tillage, seedbed preparation, 1. EM spraying and Microbial biomass, soil respiration, cellulose
Bokashi application and sowing of winter wheat decomposition, RISAb
March 27th 2006 Winter wheat Microbial biomass, soil respiration, dehydrogenase
activity, N-mineralisation,c RISA,b CLSUd
a
Chloroform fumigation extraction C and N, substrate-induced respiration C.
b
Ribosomal intergenic spacer analysis.
c
N-mineralisation and substrate N mineralisation determined in a laboratory incubation experiment.
d
Community level substrate utilisation.
 J. Mayer et al. / Applied Soil Ecology 46 (2010) 230239
cellulose paper. Water was added to 55% of maximum water hold-
ing capacity (WHC). After 4 weeks of incubation at 25 C, the soil
was removed and the remaining cellulose was determined as dry
weight minus ash. Results were calculated as mg cellulose degra-
dation cm2 lter disk.
2.4.5. Nitrogen mineralisation
N mineralisation was determined in 50 g (dry weight equiva-
lent) of sieved soil (2 mm mesh) in 500 ml polyethylene bottles.
Samples adjusted to 50% WHC with and without substrate addi-
tion were incubated at 22 C for 56 days. Added substrate was
clover-grass powder (5 g dry weight kg1 soil corresponding to
0.13 g total N kg1 ) that was ground to <1 mm and mixed with the
soil prior to adjusting WHC. After 56 days of incubation, the sam-
ples were extracted with 200 ml 0.5 M K2 SO4 for 30 min. NH4 + N
and NO3 N contents of extracts were analysed on a continuous
ow analyser (Skalar, Sanplus , Netherlands). Net N mineralisation
without substrate (S) addition was calculated as NH4 + N + NO3 N
(Nmin ) after 56 days of incubation (t56 ) minus Nmin at start of
experiment (t0 ); (Nmin t56 S)(Nmin t0 S). Net N mineralisa-
tion with substrate addition was calculated analogously as (Nmin
t56 + S)(Nmin t0 S). Net N mineralisation from added substrate
was calculated as (Nmin t56 + S)(Nmin t56 S), assuming that sub-
strate addition did not cause any soil substrate interactions as
priming effects.
2.4.6. Community level substrate utilisation
Community level substrate utilisation (CLSU) patterns were
determined according to Garland and Mills (1991) using Biolog
eco-plates prelled with 31 different C sources and a tetrazolium
dye (Biolog Inc. Hayward Cal. USA) (Insam, 1997). Soil suspensions
were prepared from 10 g (dry matter) of soil suspended in 90 ml
of a saline solution (0.9% NaCl) on a rotary shaker at 300 min1
for 30 min. Extracts were allowed to settle for 10 min to clear the
supernatant. A tenfold dilution of the suspension (dilution gradi-
ent 102 ) was prepared and 125 l were inoculated to each well of
the eco-plate and incubated at 20 C. Absorbance of each well was
measured at 600 nm (MRX Microplate reader, Dynex Technologies,
Chantilly, VA). Values of CLSU patterns were standardised by water
blank subtraction and transformation by the average well colour
value (Garland, 1996; Widmer et al., 2006). Set points after ll-
ing suspensions into the wells were chosen, when the increase in
colour formation of a plate reached a maximum. This maximum
occurred between 47 and 66 h of incubation and depended on the
soil sample analyzed. Substrate utilisation patterns at these set
points of maximum substrate utilisation were used for comparison
of experimental soils.
2.5. Ribosomal intergenic spacer analysis (RISA)
2.5.1. Extraction and quantication of DNA from soil
Nucleic acids were extracted from 0.5 g fresh soil according to
the protocol of Brgmann et al. (2001) using a FastPrep bead beater
(FP 120, Savant Instruments Inc., Holbrook, NY). Extracted DNA was
quantied with PicoGreens (Molecular Probes, Eugene, OR) on a
luminescence spectrometer (Perkin Elmer, LS 30, Wellesley, MA)
according to Hartmann et al. (2005). In addition to the four separate
eld replicates, a pooled DNA sample of the four corresponding
replicates was prepared.
2.5.2. Genetic proling of soil bacterial populations
Ribosomal intergenic spacer analysis (RISA) was per-
formed as described by Hartmann et al. (2005). PCR for RISA
was performed with bacteria-specic primers bRISAfor (5 -
TGCGGCTGGATCCCCTCCTT-3 , HEX-labelled) and bRISArev
(5 -CCGGGTTTCCCCATTCGG-3 (Normand et al., 1996) on 30 ng
233
DNA, corresponding to 600 mg soil (dry matter), in a total volume
of 25 l. The PCR products were puried with Microcon YM-100
lter columns (Millipore).
2.6. Statistical analysis
Data of microbial biomass (SIR, CFE), soil basal respiration and
cellulose degradation were analysed for variance by a full factorial
two-way ANOVA using the factors treatment and sampling time.
Treatment effects on crop yields, N mineralisation and soil dehy-
drogenase activity were analysed by one-way ANOVA. The effect
of sterilisation of EM preparations was tested by contrast analysis
within the general linear model procedure comparing EM treat-
ments (Nos. 2, 4, 6; Table 2) with sterilised EM treatments (Nos. 3,
5, 7; Table 2). Since the experimental eld showed a slight spatial
inhomogeneity, ANOVAs were calculated with the initial clay con-
tents of the experimental plots as covariates. Differences of means
were compared by a TukeyKramer test at a p-level 0.05. Anal-
yses were carried out with Statistica 7.0 (StatSoft Inc., Tulsa, OK,
USA). Multivariate analyses of community level substrate utilisa-
tion proles were performed with CANOCO 4.5 (Microcomputer
Power, Ithaca, NY, USA).
For RISA data, explorative statistics were performed with Ward
cluster analysis based on Euclidean distances with Statistica ver-
sion 7.0 (StatSoft Inc.). For cluster analysis, the full data set and
additionally the mean of the four replicates were used in order to
reduce dendrogram complexity. The main factors explaining dif-
ferences among genetic proles as well as differences observed in
Ward dendrograms were quantied by applying Monte Carlo per-
mutation testing with CANOCO for Windows 4.5 (Microcomputer
Power) according to ter Braak and Smilauer (2002), followed by
Bonferroni correction (Bland and Altman, 1995). Signicance values
were assigned to nodes in dendrograms.
3. Results
3.1. Crop yields
Yields of potatoes, winter barley and alfalfa in the years 2003,
2004 and 2005 showed no signicant differences (Table 4) between
the treatments. In 2006, winter wheat yields with EMA spraying,
treatments sp and sp au, were not signicantly different from the
control, however, additional Bokashi application increased yields
compared to the control by 13% in the treatment sp + bok + m (not
signicant; n.s.) and 23% in treatment sp + bok + m au (p < 0.009).
Similar trends that were not statistically signicant were observed
for winter barley yields in 2004, with an increase of 23% in treat-
ment sp + bok + m and 36% in treatment sp + bok, as compared to
the control, and the effect was similar with sterilised amendments.
Comparing the treatments living EM with sterilised EM (Treat-
ments Nos. 2, 4, 6 vs. 3, 5, 7) revealed no signicant difference.
3.2. Basal soil respiration and microbial biomass
On none of the individual sampling times was the difference
between the untreated control and the EMA spraying treatments sp
and sp au signicant for BSR (Fig. 1; Table 5), Cmic (SIR or CFE) or Nmic
(CFE) (Fig. 2AC). In individual cases, BSR, Cmic and Nmic showed
an increase in treatments with Bokashi application compared to
the control and EMA spraying (Treatments control, sp, sp au vs.
sp + bok, sp + bok au, sp + bok + m, sp + bok + m au). The treatments
with sole Bokashi application (treatments sp + bok and sp + bok au)
and the ones with Bokashi and manure application (treatments
sp + bok + m, sp + bok + m au) showed no signicant differences.
However, the data evaluation over the four sampling times showed
a signicantly smaller mean of Cmic (SIR and CFE) when sterilised
234 J. Mayer et al. / Applied Soil Ecology 46 (2010) 230239

Table 4
Crop yields of the main crops in the eld experiment, results from analysis of variance and in between contrast analysis of treatments 2, 4, 6 vs. treatments 3, 5, 7.

No. Treatment Potatoes 2003 Winter barley 2004 Lucerne 2005a Winter wheat 2006
(t FM ha1 ) (t FM ha1 ) (t DM ha1 ) (t FM ha1 )

1 Control 27.4a 2.95a 14.0a 2.97a

2 sp 33.3a 3.30a 14.6a 3.16ab
3 sp au 30.6a 2.88a 13.8a 2.95a
4 sp + bok 27.0a 4.00a 14.5a 3.53ab
5 sp + bok au 26.9a 3.80a 14.4a 3.48ab
6 sp + bok + m 30.3a 3.63a 15.1a 3.36ab
7 sp + bok + m au 29.0a 3.75a 14.7a 3.64b

Analysis of variance

Observed effects df LS p LS p LS p LS p

Treatment 6 n.s. 0.219 n.s 0.151 n.s. 0.344 ** 0.009

Covariate (% clay) 1 n.s. 0.180 ** 0.003 n.s. 0.089 ** 0.006
Contrast analysis 1 n.s. 0.745 n.s 0.555 n.s. 0.557 n.s. 0.272

Differing letters in columns indicate signicant differences (Tukey, p < 0.05). sp, EMA spraying; bok, Bokashi application; m, manure application; au, autoclaved treatments;
FM, fresh matter; DM, dry matter.
a
Summ of 4 cuts; df, degrees of freedom; p: signicance level from F-statistics; LS, level of signicance (n.s., not signicant; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig. 1. Soil respiration at differing sampling dates. Autumn 05 I (before EM application), Autumn 05 II (after EM application). Bars show standard error of mean (n = 4),
differing letters show signicant differences at the respective sampling date (Tukey, p < 0.05). sp, EMA spraying; bok, Bokashi application; m, manure application; au,
autoclaved treatments.

Table 5
Mean of four observed sampling times of soil respiration and microbial biomass carbon and nitrogen. Results from analysis of variance and in between contrast analysis of
treatments 2, 4, 6 vs. treatments 3, 5, 7.

No. Treatment Mean of four sampling times (Spring 05, autumn 05 I, autumn 05 II, spring 06)

SR (g g1 soil h1 ) Cmic (SIR) (g g1 soil) Cmic (CFE) (g g1 soil) Nmic (CFE) (g g1 soil)

1 Control 0.462ab 353a 389ab 64.5ab

2 sp 0.468ab 361a 399b 65.6abc
3 sp au 0.444a 338a 367a 61.3a
4 sp + bok 0.520cd 427b 428c 70.5bc
5 sp + bok au 0.486bc 390c 396b 65.9abc
6 sp + bok + m 0.506cd 404bc 405bc 68.2bc
7 sp + bok + m au 0.525d 429b 425c 70.8c

Analysis of variance

Observed effects df LS p LS p LS p LS p
*** *** *** **
Treatment 6 0.000 0.000 0.000 0.001
*** *** *** ***
Sampling time 3 0.000 0.000 0.000 0.000
Treatment sampling time 18 *
0.038 n.s. 0.064 n.s. 0.849 n.s. 0.847
*** *** *** *
Covariate (% clay) 1 0.000 0.000 0.000 0.036
Contrast analysis 1 n.s. 0.700 n.s. 0.717 n.s. 0.319 n.s. 0.338

Differing letters in columns indicate signicant differences (Tukey, p < 0.05); SR, soil respiration; Cmic , microbial biomass C; Nmic , microbial biomass N; SIR, substrate-induced
respiration; CFE, chloroform fumigation extraction; df, degrees of freedom; p: signicance level from F-statistics; LS, level of signicance (n.s. not signicant; *, p < 0.05;
**, p < 0.01; ***, p < 0.001).
 J. Mayer et al. / Applied Soil Ecology 46 (2010) 230239 235

Fig. 2. Microbial biomass C-SIR (substrate-induced respiration) (A) and C-CFE (chloroform fumigation extraction) (B) and N-CFE (C) at differing sampling dates. Autumn 05 I
(before EM application), Autumn 05 II (after EM application). Bars show standard error of mean (n = 4), differing letters show signicant differences at the respective sampling
date (Tukey, p < 0.05). sp, EMA spraying; bok, Bokashi application; m, manure application; au, autoclaved treatments.

Bokashi was applied in comparison to living Bokashi (Table 5).
Additional manure application caused an increase in BSR and Cmic
(SIR and CFE) in sterilised treatments with Bokashi (sp + bok + au
compared to sp + bok + m au). The comparison of living EM with
sterilised EM (Treatments Nos. 2, 4, 6 vs. 3, 5, 7) revealed no sig-
nicant differences, either for BSR, Cmic (SIR and CFE) or Nmic (CFE)
(Table 5).
Unlike the treatment effects, the sampling date showed clear
effects on BSR, Cmic , and Nmic (Table 5). The parameters differed
signicantly between spring 2005 and autumn 2005 I and autumn
2005 II, but not between autumn 2005 II and spring 2006 (BSR
p < 0.000; Cmic (SIR) p < 0.000; Cmic (CFE) p < 0.000). Nmic (CFE) was
signicantly different between spring 2005 and autumn 2005 I and
also between autumn 2005 II and spring 2006, but not between
autumn 2005 I and II (p < 0.001).
For BSR and Cmic (SIR), signicant interactions were observed
(BSR, p < 0.005; Cmic (SIR), p < 0.016) between the sampling dates
autumn 2005 I and II and the treatments. The parameter values
increased in EM treatments from autumn 2005 I to autumn 2005
II, whereas the control did not show any effects of sampling time
(data not shown).
3.3. Cellulose degradation
Cellulose degradation was signicantly increased by 48%
(p < 0.027) only between treatments sp au and sp + bok + m au at
the sampling point autumn 2005 I (Table 6). After tillage, sowing
of winter wheat, and EM application, however, average cellulose
degradation over all treatments increased by 44% from autumn
2005 I to autumn 2005 II (p < 0.000).
236 J. Mayer et al. / Applied Soil Ecology 46 (2010) 230239

Table 6
Cellulose degradation at sampling autumn 2005 before (I) and after (II) EM application and dehydrogenase activity at sampling spring 2006. Results from analysis of variance
and in between contrast analysis of treatments 2, 4, 6 vs. treatments 3, 5, 7.

No Treatment Cellulose degradation (mg cm2 ) Dehydrogenase activity

(mg TPF g1 soil h1 )
Autumn 05 I Autumn 05 II Spring 2006

1 Control 2.82ab 3.73a 7.89a

2 sp 2.75ab 3.74a 7.57a
3 sp au 2.28a 3.76a 7.23a
4 sp + bok 2.95ab 4.40a 8.01a
5 sp + bok au 2.84ab 4.12a 7.91a
6 sp + bok + m 2.72ab 3.94a 8.12a
7 sp + bok + m au 3.37b 4.68a 8.40a

Analysis of variance df Mean of autumn 05 I and II LS p

Observed effects LS p

Treatment 6 *** 0.000 n.s. 0.207

Sampling time 1 *** 0.000
Treatment sampling time 6 n.s. 0.706
Covariate (% clay) 1 n.s. 0.847 n.s. 0.488
Contrast analysis 1 n.s. 0.403 n.s. 0.972

Differing letters in columns indicate signicant differences (Tukey, p < 0.05); sp, EMA spraying; bok, Bokashi application; m, manure application; au, autoclaved treatments;
TPF, triphenylformazan; df, degrees of freedom; p: signicance level from F-statistics; LS, level of signicance (n.s., not signicant; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

3.4. Nitrogen mineralisation and dehydrogenase activity 3.5. Soil bacterial and fungal community structure

Two EM treatments with Bokashi application (treatments
sp + bok au and sp + bok + m au) showed signicant effects on the
net nitrogen mineralisation potential without substrate addition
of the soils compared to the control treatment and treatment sp
au (Fig. 3). The EM spraying treatments sp and sp au did not
result in signicant differences compared to the control, whereas in
treatments sp + bok au and sp + bok + m au values were 17% higher
compared to the control.
With clover-grass addition to the soil, N mineralisation did
not differ between the treatments control, sp, sp au, sp + bok and
sp + bok au, and the previously found increase in N-mineralisation
in Bokashi treatments without clover-grass disappeared. The treat-
ments sp au and sp + bok mineralised signicantly less nitrogen
compared to treatments sp + bok + m and sp + bok + m au that addi-
tionally received manure (Fig. 3). Net N mineralisation derived from
clover-grass revealed no signicant differences between treat-
ments. Dehydrogenase activity determined in spring 2006 did not
differ signicantly among treatments (Table 6).
The community structure of soil bacteria determined with RISA
showed distinct effects of Bokashi application (p < 0.001) and sam-
pling date (p < 0.001; Table 7). Treatments that received Bokashi
(sp + bok, sp + bok au, sp + bok + m, sp + bok + m au) sampled at
autumn 2005 II (t2) and spring 06 (t3) differed at the highest
branching level (p < 0.001) from treatments receiving no Bokashi
(control, sp, sp au) or treatments that had received Bokashi 15
months before soil sampling in autumn 2004 (treatments sp + bok,
sp + bok au, sp + bok + m at t1, autumn 2005 I, Fig. 4, branches I and
II; Table 7). Signicant time effects also occurred between branches
Ia and Ib (autumn 2005 I and autumn 2005 II). EMA spraying (treat-
ments sp and sp au), additional manure application (treatments
sp + bok + m and sp + bok + m au) or treatments with and with-
out sterilised EM (Nos. 2, 4, 6 vs. 3, 5, 7) revealed no signicant
effects on bacterial community structure (Table 7). Changes of fun-
gal community structure showed the strongest differentiation over
time (Table 7; p < 0.001). Effects of Bokashi were less distinctive
(p < 0.002, Table 7) and could not be detected when the sampling

Fig. 3. N mineralisation potential of soil (tn S)(t0 S), net N mineralisation of soil with clover grass (tn + S)(t0 S) and N mineralisation derived from clover grass
(tn + S)(tn S) after 56 days of incubation. Bars show standard error of mean (n = 4), differing letters show signicant differences at the respective sampling date (Tukey,
p < 0.05). sp, EMA spraying; bok, Bokashi application; m, manure application; au, autoclaved treatments.
 J. Mayer et al. / Applied Soil Ecology 46 (2010) 230239 237

Table 7
Signicance of effects as determined with Monte Carlo permutation testing (p) and percentage of variance in ribosomal intergenic spacer analysis (RISA) and community
substrate utilisation (CLSU) data sets explained by the experimental design, treatment and sampling time factors in the eld experiment.

Factor Bacteria (RISA) Fungi (RISA) CLSU (BiologTM )

p % Var p % Var p % Var

All factors 0.001 39.1 0.001 23.1 0.001 13.8

Replicate 0.001 12.9 0.001 6.9 0.001 6.9
Time 0.001 10.4 0.001 8.9
Bokashi 0.001 10.0 0.002 3.0 0.102 1.9
Manure 0.862 0.4 0.778 0.7 0.406 1.1
EMA spraying 0.938 0.3 0.664 0.8 0.553 1.0
EM sterilisation 0.349 0.9 0.160 1.4 0.570 0.9

was done 15 months after the last application. Overall, effects of
treatments explained 39.1 and 23.1% of variation for bacterial and
fungal community structure, respectively (Table 7).
3.6. Community level substrate utilisation
Soils for CLSU analysis were sampled in spring 2006. CLSU pat-
terns showed signicant effects between two clusters (not shown).
However, these clusters could not be related to any experimental
treatment. Only the factor replicate was signicant (Table 7).
4. Discussion
4.1. Crop yields
The vegetation period of 2003 was extremely dry and hot at
the experimental site. Temperatures from March to May 2003
exceeded the mean temperatures in the following years 2004 to
2006 by 1.5 C and in the period from June to August by 3.3 C. Rain-
fall of 377 mm from March to August in 2003 was almost half of the
mean in the following years of 662 mm (Table 1). This resulted in
reduced potato growth due to water deciency and consequently
in low yields (Table 4). Infection with late blight (Phytophthora
infestans), which regularly occurs in organically managed potatoes
(Bouws and Finckh, 2008), was not observed in 2003, probably due
to dry weather conditions. However, in a eld experiment with
potatoes at the same location at Agroscope Reckenholz-Tnikon
Fig. 4. Cluster analysis based on Euclidean distances of bacterial ribosomal inter-
genic spacer analysis (RISA) data for three sampling dates (t1 = autumn 2005 before
EM application, t2: autumn 2005 after EM application, t3: spring 2006). Asterisks
at nodes indicate signicant branchings as determined with Monte Carlo permuta-
tion testing performed on the four independent replicates (***p < 0.001). Labels on
specic branches refer to information specied in the text. sp, EMA spraying; bok,
Bokashi application; m, manure application; au, autoclaved treatments.
Research Station, EM application (EM 5 preparation) used as a
spraying agent showed no reduction of P. infestans infection com-
pared to the control, but resulted in 24% higher yields compared to
the untreated control (Dorn et al., 2007). In vitro trials showed only
a small inhibiting effect of EM5 on P. infestans, as revealed by inhi-
bition of sporangia germination (17%) and mycelial growth (12%)
(Dorn et al., 2007).
Overall, EMA spraying alone resulted in no yield effects from
2003 to 2006. However, EMA spraying combined with Bokashi
and manure increased wheat yields signicantly in treatment
sp + bok + m au in 2006, as compared to treatments control and
sp. The yield pattern observed in 2006 in wheat was the same
as that observed in 2004 in barley, but was not signicant there.
Yields of alfalfa in 2005 did not differ much between treatments
with signicant nutrient inputs (Bokashi, manure) and almost zero
inputs (control, sole EMA spraying), suggesting that the N2 -xing
system was not limited by nutrient or water supply. Hence, win-
ter wheat yield differences observed in 2006 can be explained
by a limitation of nitrogen availability. This is supported by the
fact that in treatments with Bokashi application (Treatment Nos.
4, 5, 6 and 7, Table 2) large amounts of nutrients, mainly nitro-
gen (401 kg N ha1 a1 ), were applied with the carrier substrate of
Bokashi. Additionally, in treatments sp + bok + m and sp + bok + m
au, moderate amounts of nutrients were applied with manure,
which caused no supplemental yield effects. No signicant differ-
ences in mean crop yields were found between the EM treatments
(Nos. 2, 4, 6, Table 2) and the treatments with sterilised EM
(Nos. 3, 5, 7, Table 2) from 2003 to 2006. These ndings indicate
that EM had no effects on yield, the observed yield effects rather
being attributable to the nutrient inputs by the carrier substrate
of Bokashi. This conclusion is supported by a pot experiment in
which no EM effects were observed on wheat grain yield and plant
biomass in heat sterilised and native soils, additionally fertilised
with farmyard manure or green manure of Trifolium alexandrinum
L. (Javaid et al., 2008). In their study, soil sterilisation increased
yields and plant biomass compared to the native soils, which may
indicate that nutrients are mobilised by heat sterilisation, whereas
EM-effects were missing.
Field studies on EM effects in Asia under tropical or sub-tropical
climate have shown contrasting results. Cotton yield signicantly
increased if concentrated EM spraying agent was applied without
fertilisation, and it improved yields also in combined application
with manure if compared to sole manure application (Khaliq et
al., 2006). In a eld study on fodder maize, EM application did not
improve the yield effects of a commercial compost product (Jilani et
al., 2007). A signicant yield increase was observed, however, if EM
was applied with a 50% reduced mineral nitrogen and phosphorus
fertilisation. Iwaishi (2000) found yield increases of 12% with paddy
rice at low and of 3% at high fertilisation rate, if organic fertilisers
were fermented with EM, although it is not clear whether those dif-
ferences were signicant. Priyadi et al. (2005) observed no effect of
EMA spraying on maize yield. EM application to peas resulted in
decreased grain yields if EM was applied directly to soil or to soil
238 J. Mayer et al. / Applied Soil Ecology 46 (2010) 230239
and leaves. Foliar application alone, however, increased yield com-
pared to the untreated control (Javaid, 2006). Tomato yields were
increased by EM in all treatments combining EM with manure, plant
residues and mineral fertilisers (Xu et al., 2000). In a similar exper-
iment Xu (2000) found effects on sweet maize yields. No effects
of EMA-treated liquid manure were found on nitrogen uptake and
biomass production of Lolium perenne L. (van Vliet et al., 2006) or
shoot dry matter of banana plants fertilised with EM Bokashi of
banana residues (Formowitz et al., 2007).
4.2. Microbial biomass, community structure and activity
Initial effects of EM might rst be indicated by a change of
soil microbial parameters (Dilly and Blume, 1996). Under eld
conditions it may be expected that the inoculated microorgan-
isms establish an indigenous microbial community in the soil after
repeated applications and a longer application period and/or stim-
ulate the indigenous microorganisms and processes. Crop yields,
microbial biomass (SIR; CFE), soil respiration and N mineralisa-
tion potential showed signicant responses only if Bokashi was
applied. EMA spraying alone never caused signicant effects and
EM treatments were not signicantly different from sterilised con-
trols (Figs. 13). The observed changes may be interpreted as
an effect of organic material added to the soil. Effects of appli-
cation of organic materials on soil microbial parameters have
frequently been reported (Hartmann et al., 2006; Widmer et al.,
2006; Fliebach et al., 2007; Heitkamp et al., 2009). Similar results
with respect to EM effect as in our experiment have been obtained
by Schenck zu Schweinsberg-Mickan and Mller (2009) in a labo-
ratory incubation experiment. EMA application to soil alone and
to soil with straw or lupin meal in some cases increased soil
microbial biomass C and N and soil respiration. EMA caused a
small net N immobilisation if no additional organic substrate was
applied. Straw or lupin meal combined with EMA, sterilised EMA
or molasses caused a small signicant increase in net N mineralisa-
tion when compared to the control. However, between treatments
with EMA, sterilised EMA and molasses alone, differences were not
signicant. The authors concluded that the observed effects were
caused by the carrier substrate molasses which is a component of
EMA. The only exception where we found signicant higher values
for applied living EM Bokashi compared to sterilised Bokashi
(Treatments sp + bok compared to sp + bok au) was for Cmic if the
mean over four sampling times was considered (Table 5). But this
effect disappeared if additionally manure was applied (Treatments
sp + bok + m compared to sp + bok + m au; Table 5). Hence the Cmic
difference could probably related to the amount of microorganisms
applied with living EM Bokashi.
Two signicant effects in bacterial community structure (RISA)
were found, rst between sampling date 1 and sampling date 2 or 3
(groups Ia vs. Ib and II in Fig. 4) for all treatments, second between
treatments with Bokashi application at sampling date 2 and 3 and
treatments without Bokashi application and those with Bokashi
application at sampling date 1. The rst effect was observed within
a very short time in the eld, namely between the end of the alfalfa
crop and the establishment of the wheat crop. But the effect was
observed in all treatments including control; hence it must depend
on normal management practices, probably on soil tillage, leading
to soil loosening, aeration and incorporation of plant residues. The
second effect may be related to the application of organic matter,
because an effect of sterilised EM was not detectable. Fungal com-
munity structure (RISA) showed similar results, but not particularly
clear or consistent. Effects of organic matter addition to soil on
microbial community structure have been reported by Hartmann
et al. (2006) who found the most pronounced effect in a long-term
eld trial in treatments with farmyard manure application. These
ndings are supported by Parham et al. (2003) and Sun et al. (2004).
Effects of plant residues on soil microorganisms measured as colony
forming units of several species have been reported by Pieta and
Kesik (2008). Concerning EM application, Okorski and Majchrzak
(2007) found an increase in the abundance of fungal species in the
rhizosphere and rhizoplane of peas and a similar diversity in a eld
experiment if EM was applied compared to the unamended control.
However, the fungal abundance was the same if only pesticides
were applied without EM. EM application increased the number
of phosphorous solubilising bacteria Bacillus, Pseudomonas and
N-xing bacteria Azotobacter, Azospirillum in the maize rhizo-
sphere under eld conditions (Jilani et al., 2007).
Community level substrate utilisation (CLSU) revealed two sig-
nicantly different groups, but an interpretation of the difference
between the Bokashi treatments sp + bok au and sp + bok + m with
Bokashi treatments sp + bok + m au and sp + bok and all other treat-
ments is not appropriate, as it does not follow the patterns observed
in the other soil biological parameters. And the lack of signi-
cance between treatments with and without Bokashi application is
surprising, because at the same sampling time bacterial commu-
nity structure (RISA) showed clear treatment effects. Signicant
differences in CLSU between eld replicates (Table 7) reect the
heterogeneity of soil properties at the experimental site, which
was observed in other parameters as well. Generally, all three
methods for microbial community structure showed treatment
effects. These effects can be explained by known effects of man-
agement practices and by inputs of organic substrates, and were
in no way caused by microorganisms applied by means of EM
preparations.
5. Conclusions
The 4-year application of two preparations of Effective Microor-
ganisms (EM), EMA and Bokashi, in an arable organic farming
crop rotation in the temperate climate of Central Europe caused
no effects on crop yields, soil microbial biomass, soil microbial
activity parameters, substrate turnover or microbial community
structure in soil, that could be attributed to the microbial inoculum.
The observed effects of Bokashi application could be related to the
nutrient inputs by the carrier substrate of Bokashi. The community
structure of the indigenous soil microbial population could not be
altered by repeated EM applications over 4 years. The observed
substrate effects of EM Bokashi on community structure in soil
monitored by RISA were compensated by the indigenous soil pop-
ulation within less than 1 year. In general, the ecological impact on
soil quality was small; effects of sampling time and differences in
soil properties exceeded effects of EM treatments.
Acknowledgements
We thank Ernst Brack and Robert Richli for managing the eld
experiment. We thank Andrea Bonvicini, Susanne Mller, Yvonne
Hfele, Bruno Nietlispach and Hansruedi Bosshard for carrying out
soil microbial and chemical analysis. We wish to thank Martin Hart-
mann for helping with RISA analysis and Walter Richner for helpful
statistical advice. Thanks to the Bionova-Hygiene AG, Switzerland
for supplying the EM preparations. The nancial support by the Fed-
eral Ofce for the Environment FOEN is gratefully acknowledged.
Last we thank the two anonymous reviewers for helping to improve
the paper.
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