D.A. Klein, M.W. Paschke Applied Soil Ecology 14 2000 257–268 259
better exploit these more heterogeneous and lignified substrates. This response, as observed by Holland and
Coleman 1987, is central to the filamentous fungal growth strategy in soils. The increased allocation of
available resources to hyphal extension in such more mature plant–soil systems can result in a decrease
in the cytoplasm maintained in the fungal structure, as discussed by Paustian 1985 and Paustian and
Schnürer 1987. In disturbed and earlier successional plant communities, in comparison, less-lignified sub-
strates are released from these earlier successional plants Frederick and Klein, 1994. This can result in
an increased allocation of carbon to cytoplasm synthe- sis, at the expense of hyphal development, based on
concepts presented by Paustian and Schnürer 1987.
The filamentous fungi also can be affected by environmental factors which are often observed to
decrease the hyphal lengths that occur in a soil. The filamentous fungi are sensitive to physical disturbance
Gupta and Germida, 1988; Dick, 1992; McGonigle and Miller, 1996, mineral nitrogen Klein et al., 1989;
Arnolds, 1991; Berg et al., 1998, pesticides Ander- son et al., 1981; Duah-Yentumi and Johnson, 1986;
Beare et al., 1993, earthworm activity McLean and Parkinson, 1997; Zhu and Carreiro, 1999, and heavy
metals Nordgren et al., 1983.
To document the total and active biovolumes of fila- mentous fungi and bacteria in the soil, and to provide a
normalized index of this filamentous fungal–bacterial structural development, the totalactiveactive fun-
galbacterial TAAFB biovolumes ratio method is presented in this communication. This approach has
been tested with data from a disturbed and adjacent undisturbed native shortgrass steppe system sam-
pled during the summer of 1995, using control and nitrogen-amended subplots. This TAAFB biovol-
umes ratio method has been compared with results for the FE method, as well as with plant community
and soil characteristics for these sites.
2. Materials and methods
2.1. Study sites The study sites used in this experiment have been
described by Klein et al. 1998 and by Paschke et al. 2000. The recently disturbed early successional ES
site and the uncultivated late successional LS site are located in northeastern Colorado at and near the
Central Plains Experimental Range, 50 km northeast of Fort Collins, CO. The purpose of the experiment
is to assess the effects of soil N availability on the recovery characteristics of the shortgrass steppe after
disturbance.
The ES site was last cultivated in 1989. By 1995, it was dominated by exotic weedy annuals. Major
species were prickly lettuce Lactuca scariola L. and cheatgrass Bromus tectorum L.. The uncultivated LS
site, in comparison, was dominated by native perennial plants. Major species observed included blue grama
Bouteloua gracilis Willd., buffalo grass Buchloe dactyloides Nutt. Engl. and prickly pear Opuntia
polyacantha Haw..
At each of these sites, the experiment was arranged as a randomized block design. Twelve 10 m×10 m
plots with 2 m buffer zones were established at each of the sites in 1993. The plots were arranged in four
blocks of three plots each. Control plots received no nitrogen amendments. The nitrogen treatments, which
were randomly assigned to one of the three plots in each block, were first applied in the summer of 1993
and continued annually through 1995. The nitrogen plots received ammonium nitrate at a rate of 100 kg
N ha
− 1
per year. The N was hand broadcast in three equal increments annually April, June and August.
2.2. Field sampling procedures The individual blocks within plots were sampled us-
ing randomly generated coordinates. Two composited samples were taken from each block by combining
three replicate 0–10 cm depth samples for each sample. The samples were immediately cooled and
samples were sieved using a 2.0 mm mesh screen.
2.3. Fumigation–extraction FE analyses As described in Klein et al. 1998, the FE analysis
was carried out using the basic procedures of Vance et al. 1987 and Tate et al. 1988. Zero-time con-
trols were extracted immediately without chloroform treatment. Carbon analyses were completed by wet
oxidation–diffusion using the procedure of Snyder and Trofymow 1984. The results were expressed as
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
