Donald A. Klein

Soil fungi: relationships between hyphal activity and staining with fluorescein diacetate

Abstract The relationship between fungal activity in soil and staining with fluorescein diacetate (FDA) was investigated using Penicillium citrinum and Rhizoctonia solani inoculated into autoclaved and non-sterilized soil, with or without nutrient amendment. Correlations of fungal activity with FDA staining allowed a quantitative relationship between FDA-staining and fungal CO 2 -evolution to be calculated. Results suggest that where nutrient fluxes occur in the soil, correlation between FDA-staining and CO 2 evolution may be useful in assessing fungal contributions to carbon transformations.

Trophic interactions in soils as they affect energy and nutrient dynamics. I. Introduction.

The dynamics of nutrient transformations at the soil-root interface are complex but amenable to controlled experimental study. Using a conceptual model we introduce a series of papers which ascertain the role of microfloral-faunal trophic interactions in carbon, nitrogen, and phosphorus transformations in soil microcosms.

Filamentous fungi: the indeterminate lifestyle and microbial ecology.

The filamentous fungi have dynamic and variable hyphal structures within which cytoplasm can be moved, synthesized, and degraded, in response to changes in environmental conditions, resource availability, and resource distribution. Their study has gone through several phases. In the first phase, direct observation was emphasized without undue concern for interior structures or in the presence of cytoplasm. By the mid-1970s, single biochemical proxies (ergosterol, marker fatty acids, chitin derivatives, etc.) were being used increasingly. The use of these surrogate single measurements continues, in spite of their inability to provide information on the physical structure of the filamentous fungi. Molecular approaches also are being used, primarily through the use of bulk nucleic acid extraction and cloning. Because the sources of the nucleic acids used in such studies usually are not known, taxonomic and phylogenetic information derived by this approach cannot be linked to specific fungal structures. Recently, a greater emphasis has been placed on assessing physical aspects of indeterminate fungal growth, involving the assessment of cytoplasm-filled and evacuated (empty) hyphae. Both of these parameters are important for describing filamentous fungal growth and function. The use of phase contrast microscopy and varied general stains, as well as fluorogenic substrates with observation by epifluorescence microscopy, has made it possible to provide estimates of cytoplasm-filled hyphal lengths. Using this approach, it has been possible to evaluate the responses of the indeterminate fungal community to changes in environmental conditions, including soil management. It is now possible to obtain molecular information from individual bacteria and fungal structures (hyphae, spores, fruiting bodies) recovered from environments, making it possible to link individual fungal structures with their taxonomic and phylogenetic information. In addition, this information can be considered in the context of the indeterminate filamentous fungal lifestyle, involving the dynamics of resource allocation to hyphal structural development and synthesis of cytoplasm. Use of this approach should make it possible to gain a greater appreciation of the indeterminate filamentous fungal lifestyle, particularly in the context of microbial ecology.

Carbon and nitrogen losses through root exudation by Agropyron cristatum, A smithii and Bouteloua gracilis

Abstract C and N released in root exudates throughout a growing season were estimated in Bouteloua gracilis and Agropyron smithii (dominant species in the shortgrass steppe ecosystem) and A. cristatum (dominant species in a 40-yr-old crested wheatgrass ecosystem). The release of C and N exudate was measured with or without the presence of a rhizosphere microflora. These values were used to estimate the potential contribution of root exudates to the N cycle of both ecosystems. Total C released through root exudates by A. cristatum , A. smithii and vB. gracilis was estimated at 8, 17 and 15%, respectively, of C fixed. The contributions of root exudates to the N cycle were estimated to be 11 g N m −2 in the shortgrass steppe ecosystem and 6 g N m −2 in the crested wheatgrass ecosystem. The presence of rhizosphere microorganisms resulted in a significant increase in root exudates of A. cristatum and A. smithii . A. cristatum and A. smithii plants grown under axenic conditions released 60 and 17%, respectively, of the C released via root exudates by inoculated plants. No differences between inoculated and non-inoculated plants were found in vB. gracilis , which indicated that it may be inherently insensitive to the presence of a rhizosphere microflora. These data suggested that an introduced plant species may be markedly different from native species in the shortgrass steppe in terms of exudate releases. These differences may have a significant influence upon the N cycle dynamics of both ecosystems.

Rhizosphere microorganism effects on soluble amino acids, sugars and organic acids in the root zone ofAgropyron cristatum, A. smithii andBouteloua gracilis

Three axenic and rhizosphere microorganism-inoculated shortgrass steppe plant species were evaluated for possible differences in residual organic carbon and nitrogen present as sugars, organic acids and amino acids. IntroducedAgropyron cristatum was compared toA. smithii andBouteloua gracilis, which are dominant species in the native shortgrass steppe. These plants, grown for 90 days in root growth chambers, showed differences in residual organic carbon and nitrogen per gram of root, and rhizosphere microbe presence resulted in additional changes in these compounds. The root biomass ofB. gracilis was significantly increased with microbes present. TheAgropyron species had significantly lower amino acid levels with microbes present, while under the same conditions, theB. gracilis showed significant decreases in residual sugars. Based on the amino acids, sugars and organic acids, the C/N ratio of the sterileA. cristatum was higher than forB. gracilis. Rhizosphere microbe presence did not result in changes in these C/N ratios. These results suggest thatA. cristatum, with microbes present, will have lower levels of amino acids present, whileB. gracilis, with a lower C/N ratio, will have sugars used to a greater extent by the rhizosphere microbes. This resulted in the higher levels of residual soluble organic C and N in the rhizosphere ofB. gracilis, in comparison with the introducedA. cristatum. These differences may be critical in influencing the course of nutrient accumulation and plant competition in short-grass steppe communities, and in understanding basic aspects of plant-rhizosphere microorganism interactions.

Origin and expression of phosphatase activity in a semi-arid grassland soil

Abstract The phosphatase activity of a grassland soil and some of the microorganisms producing phosphatase were characterized. Soil supplemented with casamino acids (1% w/w) had increased alkaline phosphatase activity (pH 8.5) after 2 days of incubation, which decreased to the original level after 24 days. No increase in phosphatase activity was observed at pH values of 5.5 or 6.5. Antibiotics were added to several soil samples in order to suppress either bacterial or fungal activity. Alkaline phosphatase activity did not increase in soils supplemented with casamino acids when the fungi were suppressed. These data may help to explain why correlations between phosphatase activity and crop yield are inconsistent.

A simulation model for the effect of predation on bacteria in continuous culture

A simulation model was developed for the carbon (C), nitrogen (N), and phosphorus (P) content of bacteria and their medium in a chemostat. Cell components distinguished included the structural component, synthetic machinery, building blocks and intermediates, C reserves, ammonium (NH4), orthophosphate (PO4), and polyphosphate. Growth, incorporation of substrates, and production of waste products were related to physiological status, as indicated by the amounts of various cell components. The model was fitted to data from chemostats on the chemical composition of bacteria growing in C-, N-, and P-limiting media and was used to explore the consequences of predation on bacterial populations. In C-limiting media predation (without the return of nutrients to the medium by the predator) increased NH4 uptake in spite of a decrease in bacterial biomass. In N-limiting media predation decreased both biomass and the rate of N uptake. These results were accounted for by the effect of growth rate on bacterial N demand. In C-limiting media the return of NH4 and PO4 by the predator did not change the effect of predation on bacteria. But in N-limiting media the return of nutrients decreased the effect of predation on biomass, and stimulated respiration and NH4 uptake by the bacteria. The effect of growth rate on the chemical composition of bacteria was proposed as a possible explanation of the stimulatory effect of predators on bacteria.

Saprophytic fungal-bacterial biomass variations in successional communities of a semi-arid steppe ecosystem

A major goal in attempting to understand plant succession is to assess the implications of fungal and bacterial biomass changes over time as plant-soil systems develop. In this study, the soil fungal and bacterial biomass of three successional semi-arid steppe communities, sampled 4, 12, and 38 years after cultivation ended, were compared with an uncultivated native plant community using microscopic procedures. In the course of the succession, significant increases in fungal hyphal lengths occurred, reaching a maximum in the oldest successional (38-year) community. Active (cytoplasm filled) hyphae decreased along the chronosequence, with the native plant community having the lowest values. Similar decreases in active bacterial biomass values occurred. In contranst, microscopically determined total bacterial numbers did not differ in soils associated with the 4-year-old and native plant communities. The ratio of active bacterial to fungal biomass, which increased over the chronosequence tested in this study, appears to provide a valuable integrative measure of plant-soil resource system development and ecosystem maturity.

A soil microbial community structural-functional index: the microscopy-based total/active/active fungal/bacterial (TA/AFB) biovolumes ratio.

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 total/active/active fungal/bacterial (TA/AFB) 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 TA/AFB 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 TA/AFB ratio also showed significant positive correlations with indices of plant community development and mineral nitrogen, especially in the plots not amended with N. This TA/AFB ratios index should be able to provide information on the physical structure of the indeterminate filamentous fungi and associated soil bacteria for use in the assessment of soil quality, health and resiliency.

Trophic interactions in soils as they affect energy and nutrient dynamics. II. Physiological responses of selected rhizosphere bacteria

Comparative microbial functions in the plant root zone were studied by evaluating rhizosphere-derivedPseudomonas andArthrobacter growth in chemostat culture and responses to root-exudate-related nutrients after varied starvation periods. These organisms were chosen to represent zymogenous and autochthonous microbes, respectively. In chemostat culture, thePseudomonas isolate showed increased energy charge and decreased populations with higher growth rates, whereas theArthrobacter had lower energy charge and cell population values which did not change appreciably with growth rate. The responses of these two types of organisms also differed with starvation. ThePseudomonas lost its ability to respire efficiently in the presence of several known root exudate components, whereas theArthrobacter isolate, in comparison, maintained a lower but more consistent ability to utilize these nutrients with increased starvation. TheArthrobacter also showed increased utilization of several substrates after starvation, suggesting its potential ability to function under restricted nutrient availability conditions. These results suggest thatPseudomonas-type organisms in the rhizosphere may best function in periods of more intense exudate release, whereas organisms of theArthrobacter- type may be more efficient at nutrient utilization during periods of lesser nutrient flux. Based on these data the rhizosphere-derivedPseudomonas isolate was considered to be an appropriate bacterium to use in more complex rhizosphere microcosm experiments where nutrient flux dynamics would be emphasized.