Applied Soil Ecology 14 2000 257–268
A soil microbial community structural-functional index: the microscopy-based totalactiveactive fungalbacterial
TAAFB biovolumes ratio
Donald A. Klein
a,∗
, Mark W. Paschke
b
a
Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1677, USA
b
Department of Rangeland Ecosystem Science, Colorado State University, Fort Collins, CO 80523, USA Received 2 June 1999; accepted 17 January 2000
Abstract
In most studies of fungal–bacterial communities in soils, single-value indices such as fumigation–extraction FE of microbe-derived organic carbon, measures of specific microbial cell chemical constituents, or activity-related measures have
been used. These widely used single value indices, however, do not provide information on the physical structure of the filamentous fungal and bacterial community in a soil. The filamentous fungi, considered as indeterminate organisms, have
a variable and changing hyphal network, most of which is devoid of cytoplasm. To meet this need for a direct integrated measure of the physical characteristics of the indeterminate fungi and their associated bacteria, a microscopy-based microbial
biovolumes ratios approach is suggested. To provide this information, the total and active biovolumes of both the filamentous fungi and bacteria are assessed by microscopy. To normalize these responses, the ratio of total to active TA fungal plus
bacterial biovolumes is divided by the ratio of the active fungal to bacterial biovolume AFB, to yield the totalactiveactive fungalbacterial TAAFB biovolumes ratio. This approach has been used to analyze data from recently-cultivated early
successional ES and uncultivated late successional LS sites at a shortgrass steppe of northeastern Colorado, where control plots were compared with those receiving mineral nitrogen amendments, using samples taken during the summer of 1995. The
TAAFB ratio index showed distinct and significant decreases in response to soil disturbance which reflected the decreased hyphal lengths present in these disturbed soils. These changes were not detected by the use of FE-based extractable carbon
measurements. The TAAFB ratio also showed significant positive correlations with indices of plant community development and mineral nitrogen, especially in the plots not amended with N. This TAAFB ratios index should be able to provide infor-
mation on the physical structure of the indeterminate filamentous fungi and associated soil bacteria for use in the assessment of soil quality, health and resiliency. © 2000 Published by Elsevier Science B.V. All rights reserved.
Keywords: Bacteria; Biovolumes ratio; Filamentous fungi; Indeterminate microbes; Microscopy; Nitrogen; Soil health; Soil quality; Soil resilience
∗
Corresponding author. Tel.: +1-970-491-6947; fax: +1-970-491-1815.
E-mail address: dakspklamar.colostate.edu D.A. Klein
1. Introduction
Most studies of fungi and bacteria in soils have been carried out using single value measurements.
Such single-value approaches are often based on chloroform-fumigation followed by extraction FE
of soluble carbon products Vance et al., 1987 and
0929-139300 – see front matter © 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 9 - 1 3 9 3 0 0 0 0 0 6 1 - 5
258 D.A. Klein, M.W. Paschke Applied Soil Ecology 14 2000 257–268
the use of conversion factors to provide biomass val- ues. In addition, substrate-induced respiration SIR
Anderson and Domsch, 1978; Blagodatskaya and Anderson, 1998 can be used, where biomass values
are related to CO
2
evolution after substrate addition. Selective antibiotics Anderson and Domsch, 1973,
1975; Beare et al., 1991; Alphei et al., 1995; Blago- datskaya and Anderson, 1998 also have been used
to assess relative contributions of fungi versus bacte- ria to soil respiration. This approach has often been
suggested to provide information on the relative fun- gal and bacterial biomass in soils Lin and Brookes,
1996; Blagodatskaya and Anderson, 1998. Fungal and bacterial chemical markers also can be applied
to this problem. Chantigny et al. 1997 monitored fungal-derived glucosamine and bacterial-derived
muramic acid to assess fungal–bacterial development in relation to soil aggregation processes. Bardgett
and McAlister 1999 have used phospholipid fatty acids PLFA to assess fungal and bacterial biomass
in control and disturbed pasture soils.
Microbiological aspects of soil quality also can be determined by the use of single value activity measure-
ments, whether these involve respiration Stenberg et al., 1998 or mineralization rates Smith et al., 1993.
In addition, fluorogenic substrates also can provide in- formation related to the fungal soil community. Miller
et al. 1998 compared fluorescent substrates related to chitinase and cellulase activities with levels of two
fungal-specific molecules, PLFA and ergosterol. These single value types of measurements have been used
to assess soil quality Smith et al., 1993; Doran and Jones, 1996; Doran et al., 1996; Rice et al., 1996;
Elliott et al., 1998; Stenberg et al., 1998; Torstensson et al., 1998, soil health Smith et al., 1993; Doran and
Jones, 1996; Doran et al., 1996 and soil resilience Seybold et al., 1999.
As discussed by Klein et al. 1998, such widely used single-value measurements do not provide
information on the structure of the filamentous fungal–bacterial community, particularly related to the
total and active biovolumes of these microbes. The fil- amentous fungi are indeterminate microbes Anderson
and Kohn, 1998; Rayner et al., 1999; Worrall, 1999, which are characterized by their variable boundaries,
where the hyphal network will be extended into areas where nutrients are available. Cytoplasm can
be moved to these new areas of hyphal extension Carlisle, 1994, and older hyphae will be abandoned
as resources in these soil volumes are depleted. In this context, it is particularly critical to assess
the total and active biovolumes of the filamentous fungi as well as of the bacteria present in a soil. At the
present time, filamentous fungal development is best assessed by the use of microscopy Klein et al., 1998,
a technique which can provide information on total hyphal lengths and the portion of the fungal hyphal
network which contains active biovolume. Frankland 1975, Söderström 1979, Ingham et al. 1989,
Norton and Firestone 1991, Carlisle 1994, Klein et al. 1996 and Thorn 1997 have noted that only a
portion of the hyphal lengths will be active. The re- lationship between total and active hyphal lengths in
a soil can be influenced by the location of the fungus in relation to energy sources such as the root Norton
and Firestone, 1991, the presence of added nitrogen Klein et al., 1989, and whether the filamentous fungi
are functioning in early versus later successional soils Klein et al., 1995, as examples. Griffin 1972, p. 17
has emphasized, in this regard, that “Objectively, the length of viable or better of actively growing hyphae
of each species, along with the frequency of spores, are necessary to quantitate the fungal population.” In
a similar vein, Frankland 1990 has noted that the fungal mycelium should “be specified as living, dead
or total.” These critical aspects of filamentous fungal structure will not be documented by the single-value
biomass estimates used in most soil assessments. Microscopy-based studies of soil bacteria also have
been carried out where various size classes have been measured Lundgren, 1984. With biovolume infor-
mation available, biomass values can be generated using specific conversion factors Van Veen and Paul,
1979; Jenkinson and Ladd, 1981.
Information on hyphal lengths in a soil also is im- portant in terms of understanding the development
of soil structure and soil tilth, where the filamentous fungi bind particles to form aggregates through their
hyphal network Lynch, 1984; Brussaard et al., 1997; Chantigny et al., 1997. In addition, as soils become
more mature in the process of plant community suc- cession, available nutrient resources become more
heterogeneous and contain higher amounts of ligni- fied, higher CN ratio compounds. The filamentous
fungi, especially, can play an increasing role in these soils by allocating resources to hyphal development to
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
