260 D.A. Klein, M.W. Paschke Applied Soil Ecology 14 2000 257–268
FE extractable organic carbon, without expression as microbial biomass which would require the use of
conversion factors. All FE analyses were initiated within 12 h of sample acquisition.
2.4. Microscopic analyses The microscopic analyses were completed by Soil
Food Web, Inc., Corvallis, OR, using procedures de- scribed by Klein et al. 1998. In these analyses, one
slide was prepared per soil sample, and three replicate readings of 40 fields were used per soil from each
transect. The bacterial biovolume was determined by the use of soil suspensions stained with fluorescein
isothiocyanate FITC and filtered onto Nucleopore, black stained membrane filters as described by Babiuk
and Paul 1970. The corresponding active bacterial biovolume assays were carried out by measuring
iodonitrotetrazolium INT chloride-responsive bacte- ria as described by Stamatiadis et al. 1990. For the
purposes of this intial study, all bacterial cells were assumed to be spherical. The total and active fungal
hyphal lengths and hyphal diameter measurements were carried out using agar film soil suspensions with
fluorescein diacetate FDA and a combination of epifluorescent and phase contrast-differential interfer-
ence contrast DIC microscopy Ingham and Klein, 1984a,b; Stamatiadis et al., 1990; Lodge and Ingham,
1991. Hyphal lengths and average hyphal diameters were used in these initial analyses. All microscop-
ically determined total and active bacterial values were log transformed before statistical analyses, and
all data were expressed on a dry weight basis. The microscopic analyses were completed within 24 h
after soil sampling.
2.5. TAAFB measures of filamentous fungal–bacterial development
The essence of this approach is to estimate the biovolumes in four parts of the microscopically de-
termined fungal–bacterial community: 1 the fungal total biovolume FT; 2 the active fungal biovolume
FA; 3 the bacterial total biovolume BT and 4 the active bacterial biovolume BA. It should be noted
that the term ‘total,’ used in this context, represents the maximum values obtained with the microscopic
procedures used in this study. The following equations were used:
FT = π r
2
× total hyphal length
FA = π r
2
× total hyphal length × FDA active
BT = 4
3 π r
3
× total bacteria
BA = 4
3 π r
3
× active bacteria
The TAAFB biovolumes ratio was then calculated. As noted in Section 1, the rationale for this approach
was the observation that with lower nutrient availabil- ity and more heterogeneous resources the filamentous
fungi will allocate more resources to hyphal extension at the expense of cytoplasm synthesis. In addition, as
noted by Klein et al. 1998, in more mature systems the active cytoplasm will be more bacterial than fun-
gal dominated. In addition, with externally imposed physical and chemical changes physical disturbance,
metals, N additions, the hyphal lengths often will be decreased. The TAAFB biovolumes ratio value is sug-
gested to provide an integrated index of filamentous fungal–bacterial development, which will be increased
in more successionally developed soil systems, and which will be decreased, on a comparative basis, when
these soils are impacted by externally imposed physi- cal and chemical changes.
2.6. Comparative analyses and statistics The bacterial TAAFB biovolumes ratio values
for the summer and autumn of 1995 were compared with FE-carbon data completed by Klein et al. 1998
and with plant community and soil characteristics for these sites as described by Paschke et al. 2000. The
TAAFB and FE-carbon data were correlated with individual soil sample characteristics using the SAS
System CORR procedure SAS Institute, Cary, NC. The TAAFB calculations given in Tables 1–4 were
based on treatment means to demonstrate the method and therefore do not exactly match the TAAFB mean
values presented in Fig. 1.
3. Results
Illustrative examples of the calculations used to generate the TAAFB biovolumes ratio values are
D.A. Klein, M.W. Paschke Applied Soil Ecology 14 2000 257–268 261
Table 1 Biovolumes in microscopically determined bacteria and fungi for the summer sampling of an uncultivated late successional LS site,
northeastern Colorado, 1995, used in TAAFB calculations to demonstrate method
a
Biovolume Units
Soil treatments None
N FT Fungi total
Length cmg 5601.715
8,013.25 Diameter
m
m 2.5
2.5 Volume
m
m
3
274,978,704 393,356,874
FA Fungal active Length cm g
52.7 109.78
Volume
m
m
3
2,586,937 5,338,915
BT Bacteria total log g
− 1
7.95 7.79
Diameter 1.0
m
m 0.5236
m
m
3
per cell 46,665,899
32,284,914 BA Bacteria active
log g
− 1
7.16 7.18
Volume
m
g
3
7,568,322 7,925,007
Calculation TA=FT+BTFA+BA
321,644,60310,155,259=31.67 425,641,78813,313,922=31.97
AFB=FABA 2,586,9377,568,322=0.34
5,338,9157,925,007=0.68 TABAFB
31.670.34=93.15 31.970.68=47.01
a
For statistical analyses see Klein et al. 1998. The TAAFB calculation procedure and soil nitrogen treatment are described in the text. Table 2
Biovolumes in microscopically determined bacteria and fungi for the summer sampling of early successional ES sites, northeastern Colorado, l995, used in TAAFB calculations to demonstrate method
a
Biovolume Units
Soil treatments None
N FT Fungi total
Length cmg 3239.50
3179.63 Diameter
m
m 2.5
2.5 Volume
m
m
3
159,020,576 156,084,283
FA Fungal active Length cm g
107.68 147.73
Volume
m
m
3
5,285,795 7,251,816
BT Bacteria total log g
− 1
7.72 7.77
Diameter 1.0
m
m 0.5236
m
m
3
per cell 27,478,918
30,831,854 BA Bacteria active
log g
− 1
6.88 6.90
Volume
m
g
3
3,971,912 4,159,103
Calculation TA=FT+BTFA+B
186,499,4949,257,707=20.15 186,916,13711,410,919=16.38
AFB=FABA 5,285,7953,971,912=1.33
7,251,8164,159,103=1.74 TABAFB
20.151.33=15.15 16.831.74=9.41
a
For statistical analyses see Klein et al. 1998. The TAAFB calculation procedure and soil nitrogen treatment are described in the text.
presented in Tables 1–4. These summarize the micro- scopically determined fungal total and fungal active
biovolume hyphal lengths per gram dry weight of soil and total and active bacterial log numbers per
gram biovolumes and the calculated TAAFB values for the 1995 summer sampling of the uncultivated
late successional LS and the early successional ES site, for the unamended and N-treated subplots. As
shown in Tables 1 and 2, TAAFB values for the summer samplings from these sites showed decreases
262 D.A. Klein, M.W. Paschke Applied Soil Ecology 14 2000 257–268
Table 3 Biovolumes in microscopically determined bacteria and fungi for the autumn sampling of an uncultivated late successional LS site,
northeastern Colorado, 1995, used in TAAFB calculations to demonstrate method
a
Biovolume Units
Soil treatments None
N FT Fungi total
Length cmg 5,288.65
4,404.54 Diameter
m
m 2.0
2.0 Volume
m
m
3
166,148,228 138,373,029
FA Fungal active Length cm g
107.25 124.01
Volume
m
m
3
3,369,366 3,895,898
BT Bacteria total log g
− 1
8.16 8.17
Diameter 1.0
m
m 0.5236
m
m
3
per cell 75,683,226
77,446,115 BA Bacteria active
log g
− 1
7.54 7.54
Volume
m
g
3
18,155,141 18,155,142
Calculation TA=FT+BTFA+BA
241,831,45421,524,507=11.24 215,819,14422,051,039=9.79
AFB=FABA 3,369,36618,155,141=0.19
3,895,89818,155,141=0.21 TABAFB
11.240.19=59.16 9.790.21=46.62
a
For statistical analyses see Klein et al. 1998. The TAAFB calculation procedure and soil nitrogen treatment are described in the text.
with soil disturbance, from 93.15 to 59.16 which were directly related to a major decrease in the total fungal
hyphal lengths. Similar data for the autumn sampling of the of the
uncultivated LS and the ES site, for the unamended and N-treated subplots, are summarized in Tables 3 and 4.
The physical disturbance, which caused a halving of
Table 4 Biovolumes in microscopically determined bacteria and fungi for the autumn sampling of an early successional ES site, northeastern
Colorado, 1995, northeastern Colorado, 1995, used in TAAFB calculations to demonstrate method
a
Biovolume Units
Soil treatments None
N FT Fungi total
Length cmg 2313.75
2416.88 Diameter
m
m 2.0
2.0 Volume
m
m
3
72,688,770 75,928,702
FA Fungal active Length cm g
190.25 299.42
Volume
m
m
3
5,034,414 9,406,579
BT Bacteria total log g
− 1
8.29 8.18
Diameter 1.0
m
m 0.5236
m
m
3
per cell 102,093,863
79,250,067 BA Bacteria active
log g
− 1
7.52 7.56
Volume
m
g
3
17,338,025 19,010,767
Calculation TA=FT+BTFA+BA
174,782,63322,372,439=7.81 155,178,76928,417,346=5.46
AFB=FABA 5,034,41417,338,025=0.29
9,406,57919,010,767=0.49 TABAFB=
7.810.29=26.93 5.460.49=11.14
a
For statistical analyses see Klein et al. 1998. The TAAFB calculation procedure and soil nitrogen treatment are described in the text.
the fungal lengths on the disturbed site in comparison with the LS site soils, resulted in a distinct decrease
in the TAABF ratio. The nitrogen amendments resulted in slight
decreases in the TAAFB biovolumes ratio values when these were compared with the control plot
values. These TAAFB ratio value changes occurred
D.A. Klein, M.W. Paschke Applied Soil Ecology 14 2000 257–268 263
Fig. 1. TAAFB ratios top and fumigation–extraction FE carbon bottom values for nitrogen-amended and control late successional LS and early successional ES sites from northeastern Colorado, 1995. See Klein et al. 1998 for FE procedural details. Standard error
bars are given.
because: 1 the ratio of TA biovolume decreased in three of four cases and 2 the proportion of the AFB
biovolume increased in all cases, when control and N-amended plot treatment means were compared.
The statistical analyses of the TAAFB biovolumes ratio values, based on the individual soil sample char-
acteristics, and of the corresponding FE-carbon val- ues, are summarized in Fig. 1. As noted in the upper
panel, the decreased TAAFB biovolume ratio val- ues observed at the ES sites, in comparison with the
higher uncultivated LS site values, indicate that the physical disturbance, which occurred 6 years earlier,
was still negatively affecting the TAAFB biovolume ratio values. The data for the comparative N-treated
plots indicate that the N addition caused only slight further decreases in these TAAFB values, a trend
also observed in Tables 1–4, in comparison with the values observed for the untreated plots. In comparison
lower panel, the FE-carbon values for the various unamended sites did not show significant differences,
in spite of the major decreases in total hyphal lengths which had occurred with disturbance. The N-amended
soils did show a trend towards increased FE-carbon values which may reflect the higher active fungal plus
bacterial biovolume values and decreased TAAFB biovolumes ratio values observed for these sites.
Plant community and soil characteristics for these test plots have been summarized by Paschke et al.
264 D.A. Klein, M.W. Paschke Applied Soil Ecology 14 2000 257–268
Table 5 TAAFB ratio and fumigation extraction FE carbon correlations
with site parameters total plant biomass TPB, grass biomass GB, annual grass biomass AGB, soil carbon SC, total ion ex-
change resin IER nitrate NO
3 −
, ammonium ion NH
4 +
, and total IER nitrogen for combined data from combined early succes-
sional and late successional shortgrass steppe sites, northeastern Colorado, 1995
a
Site parameter TAAFB ratio
FE-C Summer
Autumn Summer
Autumn TPB
0.72 0.42
− 0.33
0.0001 0.0198
0.0655 NS
GB 0.74
0.31 −
0.33 0.0001
0.0853 0.0613
NS AGB
0.68 0.30
− 0.32
0.0001 0.0973
0.0771 NS
Soil C −
0.33 0.0680
NS
b
NS NS
IER 0.49
0.53 −
0.53 −
0.50 NO
3 −
0.0042 0.0022
0.0020 0.0038
IER 0.38
0.46 −
0.44 NH
4 +
0.0325 0.0089
NS 0.0118
IER 0.45
0.52 −
0.42 −
0.49 Total N
0.0090 0.0030
0.0165 0.0049
a
See Paschke et al. 2000 for procedural details. Pearson correlation coefficients are given followed by probability.
b
NS=p=0.10 N=32.
2000. The correlations of these results with the TAAFB and FE-carbon values are presented in Table
5. These results indicate that the TAAFB biovolumes ratio values showed strong correlations with criti-
cal indices of plant community structure total plant biomass, grass biomass, and annual grass biomass
and mineral nitrogen measurements. The correlations with the FE-carbon were primarily negative and were
most evident with the summer sampling. These cor- relations suggest that this TAAFB biovolumes ratio
may be useful as an indicator of plant–soil system development.
4. Discussion
