Sep 24, 1990 - Department ofSoil Science, University ofCalifornia, Berkeley, California 94720. Received 24 September .... Placerville, Calif. Two-year-old ...
APPLIED
AND
ENVIRONMENTAL MICROBIOLOGY, Apr. 1991,
p.
Vol. 57, No. 4
1161-1167
0099-2240/91/041161-07$02.00/0 Copyright ©3 1991, American Society for Microbiology
Metabolic Status of Bacteria and Fungi in the Rhizosphere of Ponderosa Pine Seedlings JEANETTE M. NORTON* AND MARY K. FIRESTONE
Department of Soil Science, University of California, Berkeley, California 94720 Received 24 September 1990/Accepted 22 January 1991
We determined the quantity and metabolic status of bacteria and fungi in rhizosphere and nonrhizosphere from microcosms containing ponderosa pine seedlings. Rhizosphere soil was sampled adjacent to coarse, fine, or young roots. The biovolume and metabolic status of bacterial and fungal cells was determined microscopically and converted to total and active biomass values. Cells were considered active if they possessed the ability to reduce the artificial electron acceptor 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) to visible intracellular deposits of INT formazan. A colorimetric assay of INT formazan production was also used to assess dehydrogenase activity. INT-active microorganisms made up 44 to 55% of the microbial biomass in the soils studied. The proportion of fungal biomass that exhibited INT-reducing activity (40 to 50%) was higher than previous estimates of the active proportion of soil fungi determined by using fluorescein diacetate. Comparison between soils from different root zones revealed that the highest total and INT-active fungal biomass was adjacent to fine mycorrhizal roots, whereas the highest total and active bacterial biomass was adjacent to the young growing root tips. These observations suggest that fungi are enhanced adjacent to the fine roots compared with the nonrhizosphere soil, whereas bacteria are more responsive than fungi to labile carbon inputs in the young root zone. Colorimetric dehydrogenase assays detected gross differences between bulk and rhizosphere soil activity but were unable to detect more subtle differences due to root types. Determination of total and INT-active biomass has increased our understanding of the role of spatial compartmentalization of bacteria and fungi in rhizosphere carbon flow.
soil
often referred to as dehydrogenase assays, although they do not measure the results of one specific enzyme reaction but rather measure the ability of a soil to reduce a tetrazolium salt under defined conditions (36). This study compares dehydrogenase assay results with results of the more specific determinations of active microbial biomass by direct microscopic observation of intracellular INT reduction. The objective of this research was to determine the quantity and metabolic status of the microflora in soil adjacent to different morphological root types of ponderosa pine. Although some information on the quantity of microorganisms in the rhizosphere of agronomic crops is available (20), the rhizosphere of mycorrhizal forest trees is relatively uncharacterized in terms of the quantity of bacteria and fungi. Even less direct information exists on the metabolic status of the forest soil microflora in relation to the substrate supply. We chose an experimental system in which the C flow from the different root types has been determined so that the quantity of active bacteria and fungi could be conceptually related to the rate of C input from the root. We believe that the relationship between C inputs and the maintenance of active microorganisms is an integral component in evaluating the quantitative importance of the rhizosphere in below-ground carbon cycling.
The soil microbial biomass plays a central role in the cycling of carbon from primary production into the soil organic matter. Models of carbon in the below-ground ecosystem (5, 30, 39, 41) include the microbial biomass both as a pool of relatively available soil C and as the mediator of soil organic matter transformations. Although the total soil microbial biomass is important as a C source and sink, it is primarily the respiring biomass which acts as a catalyst of cycling reactions and utilizes substrates for maintenance. We have examined the quantity and metabolic status of the soil microbial biomass in a ponderosa pine experimental system in which the C flow in the rhizosphere has been characterized (21). Microorganisms which have a functional electron transport system capable of oxidizing NADH or NADPH meet one of the basic requirements for metabolic activity (18). We have used the reduction of the artificial electron acceptor 2(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) to visible intracellular deposits of INT formazan in soil suspensions with increased electron donor availability to distinguish metabolically active from inactive soil microorganisms.
Direct microscopic observation of INT reduction has been used previously to differentiate active from inactive microorganisms in both aquatic (19, 35) and terrestrial (18, 31, 33) environments. INT has been used as an indicator of active bacterial or fungal cells from soil, but the biovolume determinations of active cells that are necessary for estimation of the active microbial biomass have not been previously reported. Tetrazolium salts are also useful in colorimetric assays for general microbial activity in soils (38). These assays are
*
MATERIALS AND METHODS Soils and plants. A sandy loam soil (ultic haploxeralf) was collected from the 0- to 10-cm depth of a young mixedconifer site in the foothills of the Sierra Nevada, Calif. The site had been clear-cut in 1980 and replanted in 1981. The soil was sieved (4-mm sieve) but otherwise untreated before use. Plexiglas chambers (27 by 27 by 2.2 cm) (microcosms) were used for plant growth (14). The microcosms were filled to achieve the bulk density of the field soil (1.06 g cm-3).
Corresponding author. 1161
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Field-grown ponderosa pine seedlings (Pinus ponderosa Laws.) were supplied by the U.S. Forest Service Nursery, Placerville, Calif. Two-year-old mycorrhizal seedlings were transplanted into the microcosms and allowed to grow for 6 to 9 months in the greenhouse before sampling. During the growth period, the microcosms were positioned at 45°C to promote root growth along the lower face (11). Four replicate microcosms were used for the determination of total and active biomass in root zone soils, and an additional five microcosms were used for dehydrogenase assays.
Sampling and dissection. The front panel of the microcosm was removed for dissection of soil adjacent to roots. A minimum of four individual segments of coarse woody roots (diameter, >2 mm), fine roots (bearing mycorrhizae and fine laterals; diameter, 5 mm from any root. b Values followed by the same letter are not significantly different; no letters are given for quantities whose analysis of variance did not show a significant effect of root type (P > 0.05).
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APPL. ENVIRON. MICROBIOL.
NORTON AND FIRESTONE
M total
Fungal Length
400
IVIIK.I
300
S active
b
b
Il C
DIUI ao> II
b
%. b
'0)300
200
C0)
2 200 z
E
y 100
100
300
B:
Fungal: Bacterial Ratio 10 l
° 200
08 m
cn
m
10) 100
2 2
coarse
tine
young
none
root zone FIG. 2. Effect of root type on active and total fungal length and biomass in root zone soils. Differences between means are indicated by group letters (a, b, and c for total and x, y, and z for active).
size classes, respectively. This comparison also illustrates the importance of size class determinations for accurate estimation of active biomass. Fungal length and biomass estimates are shown in Fig. 2. Soil adjacent to fine mycorrhizal roots and coarse roots contained the greatest fungal length and the largest amount of biomass (Fig. 2). Fungal length and biomass in the young-root-zone soil were not significantly higher than estimates of those from soil not adjacent to roots. Active fungal biomass was greatest adjacent to fine roots, intermediate in the coarse and young root zones, and lowest in soil not adjacent to roots (Fig. 2). The proportion of fungal biomass that was INT active (43 to 51%) was not dependent on the root type (Table 1). The combined fungal and bacterial biomass is shown in Fig. 3. Microbial biomass determined by microscopy ranged from 190 to 280 ,ug of C g of soil-' and made up approximately 0.5% of the soil organic C. The microbial biomass was greatest adjacent to fine roots, although estimates from young and coarse root zones were higher than estimates from soil not adjacent to roots. The soil adjacent to fine and young roots contained the highest active microbial biomass, coarse-root-zone soil had intermediate values, and soil not adjacent to roots contained the lowest active microbial biomass. Root type did not have a significant effect on the proportion of active biomass, which ranged from 44 to 55% active (Table 1). The ratio of fungal to bacterial biomass C (Fig. 3B) reflects the relative importance of bacteria and fungi in the different soil zones. Although the biomass is clearly dominated by fungi in this forest soil, bacteria are of greater relative importance in the young-root-zone soil than in soils adjacent to fine or coarse roots. The fungal/bacterial ratio is consistently lower for the active than for the total biomass, which is indicative of a slightly higher active percentage for the bacterial biomass. Dehydrogenase activity. Dehydrogenase enzyme assays on
0
I,,,,
InIUnet yuungy root zone FIG. 3. Effect of root type on active and total microbial biomass and fungal-to-bacterial ratios (all values from microscopic determinations). Differences between means are indicated by group letters (a, b, and c for total and x, y, and z for active).
,ne
soils dissected from different root zones could not detect an effect of root type (P = 0.32) on the amount of INT reduced to formazan during the 4-h incubation; the mean INT formazan production for all soil zones was 18.8 ,ug of INT formazan h-1. Rhizosphere soil (operationally defined) had a significantly higher production of INT formazan and biomass C as determined by CFIM than did the bulk soil (Table 2). The reduction of INT per unit of biomass C was not significantly different for rhizosphere and bulk soils. DISCUSSION Few previous attempts have been made to quantify the populations of soil bacteria with active electron transport. Our estimate for the proportion of bacterial numbers that was INT active (41 to 57%) is within the range found in soil aggregate films (11 to 46%) (18) and in aquatic samples (25 to 94%) (35). Conversion of INT-active bacterial numbers to biomass was reported in a previous study of soil from agroecosystems (31), but the extremely low values reported TABLE 2. Dehydrogenase activity in rhizosphere and bulk soilsa Soil type'
Rhizosphere Bulk
Microbial biomass (,ug of C/g of soil)c
Dehydrogenase activity (,ug of INT-F/g of soil)d
DH/MBC (jig of INT-F/mg
566 a 314 b
16.8 a 8.7 b
31.0 a 27.5 a
of MBC)Q
a The letters following the numbers are explained in Table 1, footnote b. b Rhizosphere and bulk soils are defined operationally (see text). Microbial biomass C was determined by CFIM. d Dehydrogenase activity, INT formazan (INT-F) produced during a 4-h incubation. e DH/MBC, Dehydrogenase activity per unit of microbial biomass C.
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(0.07 ,um3 were culturable on laboratory media. The large discrepancy between total microscopic and viable counts when using plate-counting techniques has been repeatedly noted (23, 24); our results indicate that this discrepancy can be explained only partially by the presence of inactive cells. Many cells with electron transport activity may not be culturable on currently used media. These results are consistent with previous observations that a substantial proportion of the cells in soil have maintained a minimum integrity as indicated by an intact genome (4). Our results indicate that 40 to 50% of the soil hyphae are capable of reducing INT. We could locate only one previous reference to the use of INT to determine the active hyphal length (33). Sylvia (33) found 32 and 90% of the hyphal length to be INT active in the soil and on the root surface, respectively, in a pasteurized soil and grass system inoculated with vesicular arbuscular mycorrhizae. Several alternative techniques have been used to quantify the active portion of fungal hyphae, and earlier work has been reviewed (16). Estimates from the surface mineral horizon and litter layers of a forest soil found that 27% and 23 to 80% of the hyphae present contained cytoplasm, respectively. Methods based on the uptake and enzymatic cleavage of fluorescein diacetate have generally found that less than 10% of the hyphal length was active (16). In a microcosm experiment with soil from a lodgepole pine forest, the total hyphal lengths (75 to 250 m g-') were comparable in magnitude to values from nonrhizosphere samples in our study (214 m g-'); however, the fluorescein diacetate-active length was less than 5% of the total length (12). If fluorescein diacetate is hydrolyzed only in actively growing tips or very young hyphae (24), INT-active hyphae may be a better estimate of the hyphae capable of utilizating C for respiration. Our results suggest that fungal and bacterial biomass respond differently to labile C inputs from roots. Bacteria were more responsive to the C inputs from the young-roottip region, whereas fungal biomass was enhanced in the soil adjacent to the fine mycorrhizal roots (Fig. 3B). Newman (20) previously suggested that rhizosphere bacteria were more dependent on soluble exudates than were fungi; we observed the highest active bacterial biomass in the soil adjacent to the young root, where we previously found that the concentration of rhizodeposited C was the highest (21). The quantity of hyphae which are mycorrhizal in the soil adjacent to the fine roots may be a significant portion of the total. Mycorrhizal hyphae have been observed to play a major role in C translocation into the soil in this system (21), and their importance would be greatest directly adjacent to the fine roots. Our observations of increased bacterial biomass adjacent to the young root tip and higher fungal biomass adjacent to the fine mycorrhizal roots in comparison with the nonrhizosphere soil substantiate the theoretical spatial and temporal sequence of events along roots proposed by Elliott et al. (6). The use of direct microscopic determinations of active bacteria and fungi is extremely time and labor intensive, and enzymatic methods of estimating electron transport activity
TABLE 3. Carbon inputs and maintenance requirements in root zone soils Root zonea
Coarse Fine Young None
C inputs (pg of C g of soil-,
Maintenance requirement
(p.g of C g of soil-' day-l)c
day-1)b
Total biomass
Active biomass
38.0 50.0 85.6 6.3
19.8 22.8 18.9 14.8
8.5 11.1 10.0 6.8
a Root zone soil is soil within 2 mm of root surface; none is soil >5 mm from any root. b C inputs are calculated from the data of Norton et al. (21), assuming that the specific activity of rhizodeposits is equivalent to the specific activity of young roots. c The maintenance requirement is calculated by using a specific maintenance rate of 0.002 h-1 and an efficiency of 0.60 (5, 30) and the total or active biomass data from this study.
offer a rapid alternative. However, dehydrogenase assays do not supply information on the composition of the biomass or the proportion that is active. In our study, dehydrogenase assays were capable of detecting gross differences in electron transport activity between rhizosphere and bulk soils (Table 3) but were unable to detect more subtle differences due to root types. Dehydrogenase activity per unit of biomass C was not significantly different for rhizosphere and bulk soils; this is consistent with the microscopic information that the proportion of the total biomass that was active was not dependent on root type, although the amount of active biomass was different for different soil zones. The definition of active and inactive microorganisms is complex and must be approached within the limits of the technique used. With our technique, dispersion of the soil would have released physically protected organic matter and decreased diffusional constraints, thereby increasing the microbial supply of available carbon substrate useful as electron donors. NADH and NADPH were also added as electron donors because of their central role in microbial metabolism (9) and because of results of a previous method (18) which showed that increased concentrations of these electron donors increased the proportions of active cells. Our study did not determine the proportion of soil organisms which were capable of utilizing reducing equivalents from exogenous NADH or NADPH. However, the utilization of exogenous pyridine nucleotides has been demonstrated for mutants of Escherichia coli and Salmonella typhimurium which are deficient in de novo synthesis of NAD (8). These enteric organisms would be expected to have more restrictive permeability than many soil organisms. We have estimated the bacterial and fungal biomass C which has the capability to use exogenous and/or endogenous electron donors [i.e., soil C, added NAD(P)H, or internal reserves] to reduce the artificial electron acceptor INT. This is one of the most basic indicators of metabolic activity available; it does not require that the organism have existing energy reserves, utilize a specific C source, or be in a growing state. Our technique does not distinguish organisms that are active in situ from those that have potential activity if supplied with available substrate. Our results suggest that a large proportion of the soil microbial biomass can respond relatively rapidly to increased substrate availability because it maintains functional electron transport systems. Investigations into starvation survival of aquatic organisms suggest that the
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energy-yielding mechanisms may remain intact in starved cells (22). In the energy-limited soil environment, the maintenance requirements of the metabolically active microorganisms are a major sink for soil C inputs (30). Modeling of the microbial maintenance C requirements has previously been based on steady-state analysis of C inputs and losses (5), product formation (CO2 production) (2, 27, 28), or total biomass determinations and in vitro values of specific maintenance rates (3). Results from these models indicate that when the total biomass is used as a basis for C requirements, either there is insufficient C input to maintain the observed biomass or specific maintenance rates in soil are several orders of
magnitude lower than the in vitro values (28). In this study we observed that a significant portion of the biomass had electron transport activity below the level of detection. This inactive biomass has previously been referred to as necromass; although it may be important as a substrate for the process of cryptic growth (5), it would not be expected to utilize significant C for maintenance. We believe that the definition of active biomass used in this study would most closely approximate the fraction of the biomass which utilizes C for maintenance. The substrate required to maintain this smaller active biomass would therefore be lower than
previous estimates based on total biomass, which often equaled or exceeded the C inputs to the system (3, 27, 29, 30). Estimates of maintenance requirements based on the total versus the active biomass and C input values from this experimental system are given in Table 3. The maintenance C requirement based on the total biomass C exceeds the C input value for the soil farther than 5 mm from any root; however, when based on the active biomass, the requirement is of the same magnitude as the input value, indicating that sufficient C inputs exist to support the observed active biomass. Little or no growth would be expected in this region. The root zone soils all show higher C inputs than maintenance C requirements, indicating that these compartments are potential regions of microbial growth. Although there is uncertainty about the choice of a specific maintenance rate and the calculation of C input values, using total biomass C as the basis for the maintenance requirement will clearly result in an overestimation of the C required to support the biomass. The growth of the microbial biomass is theoretically controlled by the C supply above that required for maintenance (29). In our system, soils adjacent to young roots have the highest potential for microbial growth and support a biomass enriched in bacteria compared with the other soil zones. Increases in the bacterial biomass with a relatively low C/N ratio would be predicted to result in N immobilization into the biomass. This hypothesis is an example of the implications of our study for models of nutrient cycling in the below-ground ecosystem. Further studies are in progress which will investigate the interactions of C and N flows through the rhizosphere microbial biomass. We have attempted to investigate one of the long-standing questions of soil microbiology, "What is the metabolic status of microbial cells in the energy-limited soil environment?" Our observations suggest that potentially active microorganisms are present in relatively large quantities in both rhizosphere and nonrhizosphere soil, but it is only in the C-rich environment adjacent to the plant root that these microorganisms have C substrate in excess of their requirement for maintenance.
APPL. ENVIRON. MICROBIOL. ACKNOWLEDGMENTS
This work was supported by National Science Foundation Doctoral Dissertation Improvement Grant no. BSR-8800892 and by a McIntire Stennis Project of the California Agricultural Experiment Station. We thank the staff of the University of California Blodgett Experimental Forest for their help and management of the study site and the U.S. Forest Service Nursery at Placerville for the generous donation of tree seedlings. Jeffrey Smith provided valuable discussion on microbial activity and maintenance energy concepts. REFERENCES 1. Abacus Concepts. 1989. SuperANOVA. Accessible general linear modeling. Abacus Concepts, Inc., Berkeley, Calif. 2. Anderson, T. H., and K. H. Domsch. 1985. Maintenance carbon requirements of actively-metabolizing microbial populations under in situ conditions. Soil Biol. Biochem. 17:197-203. 3. Babiuk, L. A., and E. A. Paul. 1970. The use of fluorescein isothiocyanate in the determination of the bacterial biomass of grassland soil. Can. J. Microbiol. 16:57-62. 4. Bakken, L. R., and R. A. Olsen. 1989. DNA-content of soil bacteria of different cell size. Soil Biol. Biochem. 21:789-793. 5. Chapman, S. J., and T. R. G. Gray. 1986. Importance of cryptic growth, yield factors and maintenance energy in models of microbial growth in soils. Soil Biol. Biochem. 18:1-4. 6. Elliott, E. T., D. C. Coleman, R. E. Ingham, and J. A. Trofymow. 1984. Carbon and energy flow through microflora and microfauna in the soil subsystem of terrestrial ecosystems, p. 424-433. In M. J. Klug and C. A. Reddy (ed.), Current perspectives in microbial ecology. American Society for Microbiology, Washington, D.C. 7. Fisher, R. A. 1949. The design of experiments. Oliver & Boyd, Edinburgh. 8. Foster, J. W., D. M. Kinney, and A. G. Moat. 1979. Pyridine nucleotide cycle of Salmonella typhimurium: isolation and characterization of pncA, pncB, and pncC mutants and utilization of exogenous nicotinamide adenine dinucleotide. J. Bacteriol. 137: 1165-1175. 9. Foster, J. W., and A. G. Moat. 1980. Nicotinamide adenine dinucleotide biosynthesis and pyridine nucleotide cycle metabolism in microbial systems. Microbiol. Rev. 44:83-105. 10. Griffiths, B. S. 1989. Improved extraction of iodonitrotetrazolium-formazan from soil with dimethylformamide. Soil Biol. Biochem. 21:179-180. 11. Hattingh, M. J., L. E. Gray, and J. W. Gerdemann. 1973. Uptake and translocation of 32P-labeled phosphate to onion roots by mycorrhizal fungi. Soil Sci. 116:383-387. 12. Ingham, E. R., C. Cambardelia, and D. C. Coleman. 1986. Manipulation of bacteria, fungi and protozoa by biocides in lodgepole forest soil microcosms: effects on organism interactions and nitrogen mineralization. Can. J. Soil Sci. 66:261-272. 13. Ingham, E. R., and D. A. Klein. 1984. Soil fungi: measurement of hyphal length. Soil Biol. Biochem. 16:279-280. 14. James, B. R., R. J. Bartlett, and J. F. Amadon. 1985. A root observation and sampling chamber (rhizotron) for pot studies. Plant Soil 85:291-293. 15. Jenkinson, D. S., and D. S. Powlson. 1976. The effects of biocidal treatments on metabolism in soil. V. A method for measuring soil biomass. Soil Biol. Biochem. 8:209-213. 16. Kjoller, A., and S. Struwe. 1982. Microfungi in ecosystems: fungal occurrence and activity in litter and soil. Oikos 39:389422. 17. Lundgren, B. 1984. Size classification of soil bacteria: effects on microscopically estimated biovolumes. Soil Biol. Biochem. 16: 283-284. 18. Macdonald, R. M. 1980. Cytochemical demonstration of catabolism in soil micro-organisms. Soil Biol. Biochem. 12:419-423. 19. Maki, J. S., and C. C. Remsen. 1981. Comparison of two direct-count methods for determining metabolizing bacteria in freshwater. Appl. Environ. Microbiol. 41:1132-1138. 20. Newman, E. I. 1985. The rhizosphere: carbon sources and microbial populations, p. 107-121. In A. Fritter, D. Atkinson, D. Read, and M. Usher (ed.), Ecological interactions in soils.
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