Mineralization Capacity of Bacteria and Fungi from the Rhizosphere ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1980, p. 113-117 0099-2240/80/01-0113/05$02.00/0

Vol. 39, No. 1

Mineralization Capacity of Bacteria and Fungi from the Rhizosphere-Rhizoplane of a Semiarid Grassland J. P. NAKASt* AND D. A. KLEIN Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523

A radiotracer glucose mineralization assay was used with streptomycin and actidione to monitor the relative seasonal contributions of bacteria and fungi to mineralization processes in soils derived from the rhizosphere-rhizoplane zone of plants from a shortgrass prairie ecosystem. Bacteria played a major role in glucose mineralization in both the rhizosphere and rhizoplane. These results indicate that the bacteria may play a greater role in glucose mineralization processes in the rhizosphere and rhizoplane zones of a semiarid grassland than would be assumed, based on available biomass estimates. This technique appears to be valuable for determining bacterial versus fungal contributions to glucose mineralization in the rhizosphere and rhizoplane and may be useful for measuring the decomposition of other more complex substances in this zone of intense microbial activity.

The estimation of relative contributions of bacteria and fun,gi to mineralization processes has remained a major problem in soil microbiology. Although selective inhibition techniques have been used in enumeration studies (7, 15), these efforts were often hampered by the use of inhibitors which did not possess the desired specificity, especially for fungal inhibition. However, Williams and Davies (19) demonstrated the usefulness of actidione (cycloheximnide) in natural systems as a potent fungal inhibitor which had little effect on bacterial or actinomycete populations. These investigators also used streptomycin to inhibit bacterial and actinomycete populations and found a negligible effect on fungal populations in various soil systems. Subsequently, these same antibiotics were employed by Anderson and Domsch (1-3), who investigated the selective inhibition of respiratory activity in forest and agricultural soils. The use of broad-spectrum bacterial and fungal antibiotics to determine the relative contribution of each microbial community to mineralization processes has several disadvantages which have been outlined by Parkinson et al. (16). These problems include the removal of antibiotics by adsorption to soil particles, degradation by unaffected microbiota, development of resistant forms, and the removal of competitors. However, with the use of relatively short incubation periods in a radiolabel mineralization assay, the problems of degradation, resistance development, and adsorption can be minimized. Concerning antibiotic adsorption onto clays and t Present address: Department of Environmental and Forest Biology, State University of New York College of Environ-

mental Science and Forestry, Syracuse, NY 13210.

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humic materials, this can be overcome by applying antibiotics in sufficient concentration so as to maintain desired levels of inhibition. This communication describes a radiotracer method for the evaluation of seasonal changes in bacterial and fungal contributions to glucose mineralizAition processes in soils from the rhizosphererhizoplane zone of a semiarid grassland. MATERIALS AND METHODS Mineralization studies. Soil cores (16.5 cm long, 10 cm in diameter) were taken from plot 26 of the Central Plains Experiment Range in northeastern Colorado (administered by the U.S. Department of Agriculture Science and Education Administration-Federal Research) and stored in plastic bags overnight at 20°C. The following day, core samples were separated into rhizosphere and rhizoplane soil fractions by the procedure of Louw and Webley (13), using 0.05 M potassium phosphate buffer, pH 7.4, as a diluent. At appropriate time intervals, 1.0-ml portions of rhizosphere and 5-ml portions of rhizoplane suspensions (weight of soil in each portion, ca. 50 mg) were placed into 60-ml serum bottles. Each serum bottle then received 0.3 uCi of [U-14C]glucose (198 mCi/ mmol; ICN, Irvine, Calif.) in 100 Ml of sterile water and 16.6 yg of unlabeled glucose in 1 ml of sterile phosphate buffer. After the serum bottles were capped withstoppers containing '4CO2 absorption towers, incubation was allowed to proceed for 12 h at 25°C. The released 4C02 was trapped in phenethylamine and counted as previously described by Wright and Hobbie (20) and Harrison et al. (10). Results for the rhizosphere and rhizoplane are reported as the percent contribution of bacteria and fungi to glucose mineralization by subtracting the counts mineralized from the counts originally added. Cycloheximide (actidione), 3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide, (Sigma Chemical Co., St. Louis, Mo.) and streptomycin sulfate

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(Eli Lilly & Co., Indianapolis, Ind.) were used at concentrations of 3.0 and 1.0 mg/ml, respectively. These compounds were added to the serum bottles as aqueous solutions before the introduction of rhizosphere or rhizoplane soil samples. Enumeration studies. To evaluate the selective inhibition patterns of streptomycin and actidione, both antibiotics were incorporated into culture media. Martin medium (15) was chosen to enumerate fungi, and sodium caseinate agar (12) was used to enumerate bacteria and actinomycetes. Both antibiotics were added to the cooled (45°C) media as aqueous solutions after being sterilized by passage through 0.22-,um membrane filters (Millipore Corp., Bedford, Mass.) before the preparation of spread plates. Serial 10-fold dilutions of Pawnee Grassland soil were made, using 0.05 M phosphate buffer, pH 7.2, as a diluent, and 0.1-ml samples were spread (in triplicate) onto the surface of rose bengal and sodium caseinate agar plates. Plates containing between 50 to 200 colonies were counted, and these data are expressed as the means of triplicate samples. The standard deviations are also provided. Inhibition of fungal isolates. Eight representative fungal cultures were isolated from plot 26 of the Pawnee National Grassland and identified as members of the following taxonomic groups: Aspergillus, Mucor, Penicillium, Geotrichum, and Mycelia Sterilia. Initial isolation was from colonies grQwn on rose bengal agar, and cultures were maintained on Sabouraud dextrose (Difco) slants. These cultures were grown at 25°C in Erlenmeyer flasks containing 100 ml of nutrient broth (Difco) plus 1.0% (wt/vol) yeast extract (Difco) and 1.8% (wt/vol) glucose for 48 h while being shaken on a G-2 Gyrotory shaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 150 rpm. After 48 h of growth, the fungal cultures were harvested aseptically by centrifugation (4,080 x g) and washed twice and resuspended in 50 ml of sterile phosphate buffer (0.05 M, pH 7.4). Sterile glass beads (15 g) were then added to the fungal suspensions, and the mixtures were gently shaken for 5 to 10 min to disperse the packed hyphae. Microscopic examination revealed negligible mycelial rupture. Serum bottles were then prepared containing the following: unlabeled glucose, 1.0 mg/ml; [U-'4C]glucose (198 mCi/mmol), 0.1 ytCi; cycloheximide, 3.0 mg/ml; and fungal suspension, 1.0 ml. Addition of the fungal cultures was accomplished, using an automatic pipettor fitted with a wide-bore plastic pipette and removing samples from a magnetically stirred culture. This method of sample addition was chosen because it resulted in values (counts per minute) which were consistently within ±5% for all time periods sampled. Inhibition of microbial populations in soil. The effectiveness of streptomycin and actidione as inhibitors of bacterial and fungal activities in the soil was demonstrated by mineralization studies using [U'4C]glucose. A mixture of rhizosphere and rhizoplane microorganisms was prepared by removing all roots from a random soil core (ca. 250 g) and washing the roots in the presence of glass beads (13). Subsequently, the beads and roots were removed, and the remaining suspension was assumed to contain both rhizosphere and rhizoplane microorganisms. Mineralization reactions

were

initiated with the addition of 1-ml rhizo-

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sphere-rhizoplane portions to 60-ml-capacity serum bottles containing the following: 4 ml of 0.05 M phosphate buffer, pH 7.2, 1 ml of unlabeled glucose (1 mg/ ml), 0.3 jtCi of [U-'4C]glucose (198 mCi/mmol) in 100 p1 of deionized water, and 1 ml of either streptomycin (1 mg/ml) or actidione (3 mg/nil). Control bottles received 1 ml of buffer instead of antibiotic. All bottles were incubated at 25°C for a total of 24 h and 14C02 was trapped in phenethylamine as described by Harrison et al. (10). All data have been adjusted by using the external standard ratio and are expressed as disintegrations per minute. 100 mg-' (dry weight) of soil.

RESULTS The inhibition of Pawnee Grassland microbial populations caused by increasing concentrations of actidione and streptomycin is presented in Table 1. Streptomycin caused complete inhibition of bacterial and actinomycete growth at all concentrations tested and demonstrated little effect on the expression of fungi. Conversely, actidione concentrations of 3 mg/ml were necessary to achieve a 10-fold reduction in fungal numbers and caused no observable effect on bacterial and actinomycete populations. Increasing the actidione concentration beyond 3 mg/ml did not result in a further reduction in fungal numbers. As an additional means of evaluating the inhibition of fungal populations by actidione, eight fungal cultures were isolated and tested for their ability to mineralize glucose in the presence of actidione (Fig. 1). Approximately 50% inhibition occurred within 6 h for seven of the eight cultures tested, and greater than 80% inhibition occurred withii; 12 h for six of the eight cultures tested. After '!4 h, the level of inhibition decreased markedly. In addition, the satisfactory inhibition of fungal and bacterial glucose mineralization was TABLE 1. Effect of actidione and streptomycin on bacterial, fungal, and actinomycete populations in Pawnee Grassland soil as determined by the spread plate method Microbial populations (g-' dry wt)

Antibiotic

Concn

(,ug/ml) Bacteria (X106)

Actidione Actidione Actidione Actidione Actidione Actidione

Fungi

(X102)

Actmo-

mycetes

(X106)

2.0 ± 0.3 30.0 ± 2.0 2.3 ± 0.3 5.0 + 0.1 2.3 ± 0.3 1.8 + 0.1 1.5 ± 0.1 5.0 + 0.2 2.1 + 0.2 1.6 + 0.4 7.0 ± 0.3 2.2 ± 0.4 1.9 + 0.2 6.3 + 0.2 2.5 + 0.3 1,000 3,000 1.8 + 0.2 3.2 + 0.3 2.1 ± 0.4 0 2.0 _ 0.3 21.1 + 0.5 2.3 _ 0.3 Streptomycin 250 20.0 + 1.1 Streptomycin _ 500 16.2 + 0.6 Streptomycin 18.3 + 0.5 Streptomycin 1,000 aNo growth visible after 6 days of incubation at 25°C. 0 125 250 500

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At the beginning of September and through October, the bacterial population again was responsible for a greater portion of the combined bacterial and fungal glucose mineralization activity. When the soil moisture content increased significantly in November due to early snowfall moisture, the fungal contribution to glucose mineralization increased in both the rhizosphere and

12

TIME ( hr)

FIG. 1. Second-order polynomial regression curve depicting the time course of inhibition of actidione (3 mg/ml) on eight fungal cultures isolated from plot 26 of the Pawnee National Grassland. Penicillum sp. (A, A), Mycelia Sterilia (x), Mucor sp. (0, 5). Geotrichum sp. (U), and Aspergillus sp. (V, 0). Each value is the average of samples run in triplicate.

noted when these inhibitiors were used with a combination of rhizosphere and rhizoplane microbial populations. The degree of inhibition of glucose mineralization caused by streptomycin and actidione in this combined soil fraction is presented in Fig. 2 and illustrates the time-dependent variability of the microbial responses to the presence of these antibiotics. Both bacteria and fungi were substantially inhibited after 12 h, and the additive effect of each antibiotic treatment (88%) was within 10% of the combined use of both antibiotics. The remaining 12% of glucose mineralization activity most likely represents a portion of the microbial population unaffected by the use of either antibiotic. However, the degree of inhibition imposed by the separate use of each antibiotic decreased at 18 and 24 h. Streptomycin-treated soil resulted in glucose mineralization activity which was very near control values at these later incubation times. The 1977 seasonal analyses of fungal and bacterial contributions to mineralization processes (as judged by the mineralization of [U-'4C]glucose) in the rhizosphere and rhizoplane zones are summarized in Fig. 3A. In the rhizosphere, the bacterial population accounted for approximately 40 to 80% of the glucose mineralization activity. During the spring, with the availability of sufficient soil moisture (Fig. 3B), a gradual increase in the fungal contributions to glucose mineralization occurred. This trend was markedly reinforced at the end of July when the fungal contribution became dominant after an intensive precipitation event (15.2 cm in 3 days).

1

3

12

6

18

24

TIME, hours FIG. 2. Effect of streptomycin (I mg/ml) and actidione (3 mg/ml), singly and in combination, on glucose mineralization in the rhizosphere-rhizoplane region of Bouteloua gracilis (blue grama). Control system with no antibiotic added (0); added streptomycin (A); added actidione (O); and both antibiotics (5). Each value is the average of samples run in triplicate, and standard deviations are indicated. 0

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FIG. 3. Seasonal changes in bacterial contributions to glucose mineralization in Pawnee Grassland soil expressed as a percentage of total bacterial and fungal contributions: (A) bacterial contribution (%) to glucose mineralization in the rhizosphere (0) and rhizoplane (A). Fungal contributions to glucose mineralization can be obtained by substracting indicated values from 10X%. (B) moisture content of biweekly soil samples taken from the Pawnee National Grassland from April to December 1977.

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rhizoplane. Bacterial and fungal contributions to glucose mineralization were almost equivalent in samples from the rhizosphere at this time. Microbial contributions to glucose mineralization processes in the rhizoplane (Fig. 3A) were clearly dominated by the bacteria. The bacterial component exhibited wide fluctuations from September to December, which probably reflects changing weather conditions, especially temperature. Also, the fungal component did not demonstrate an increased glucose mineralization potential coincident with the July precipitation event, which suggests that the fungal population in the rhizoplane regjon is less sensitive to soil moisture variations. DISCUSSION The use of the antibiotics streptomycin and actidione for the selective inhibition of bacterial and fungal activities, as described by Anderson and Domsch (1-3), appears to be suitable for use in a rapid radioactive glucose mineralization assay. Based on this approach, it appears possible to evaluate the relative fungal and bacterial activities in soils from the rhizosphere and rhizoplane zones of plants growing under field conditions. In various ecosystems, as reviewed by Clark and Paul (6), the relative biomass of bacteria and fungi has been calculated. Jackson (11) noted that the calculated fungal biomass was somewhat less than that of the bacteria, whereas data from the Matador site, a high plains grassland environment, indicate that the fungal biomass exceeds that of the bacteria by at least a factor of two (5, 6, 17). These calculations must be considered in light of estimates of the functional fungal biomass which have been made and which suggest that at any particular time, only a relatively small portion of the measured fungal biomass may be in an active physiological state. Soderstrom (18) noted that only 1 to 5% of the hyphal material examined for the ability to hydrolyze fluorescein diacetate could be considered to be functional. Harley and Waid (9) found that only 23% of fungal fragments isolated from soil could be considered active, based on growth criteria. In our own studies carried out during 1977 at the Pawnee site during an extremely dry season, the calculated fungal biomass estimates were equal to or less than those of the bacteria and actinomycetes based on procedures and assumptions used by Clark and Paul (6). During more optimal plant growth conditions, the fungal biomass has been noted to increase more rapidly than that of the bacteria. This phenomenon has also been observed in some soil microcosm studies carried out in our laboratory.

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Ares (4) observed that soil dessication can result in a 30 to 60% decomposition of newly formed roots, thus providing an additional source of utilizable carbon, an effect also noted by Martin (14). Prolonged drought conditions during most of the summer and early fall of 1977 can be assumed to have made plant-derived materials available for use by the rhizosphererhizoplane microbiota. With the precipitation events which occurred in early November, increased mineralization by the fungi occurred in the rhizosphere and rhizoplane. It should be recognized that glucose represents only one of many substrates which may be utilized by the soil microorganisms, and it may not provide an estimate of the total relative contributions of bacteria and fungi towards mineralization processes. Glucose, together with other sugars, water-soluble nitrogenous compounds, and organic acids, are considered to represent substrates which are readily availabl* during the initial phase in plant litter decomposition (8). Based upon the use of glucose as the assay substrate, it appears that bacteria are somewhat more efficient in the utilization of low-molecular-weight organics from the root zone of blue grama (Fig. 3A). However, the utilization of other plant-derived polymeric substrates such as starch, hemicelluloses, and cellulose may be accentuated by the synthesis of extracellular enzymes by the fungal population. Cleavage of these biopolymers may then result in an increase in fungal biomass and therefore in the mineralization potential. Although the bacteria may be more efficient in the utilization of glucose and other related monomers, the fungi may be more successful in the overall competition for growth space on plant tissue simply because they are more efficient in the initial polymeric cleavage (8). Based on these studies, it appears to be possible to combine selective antibiotic inhibition with a rapid radioactive mineralization assay to monitor the relative contributions of bacteria and fungi to mineralization processes in the plant root zone. By use of this procedure, it should be possible to study the decomposition of a wide range of purified substrates as well as plant-related substrates. Further development of this technique may lead to an optimization of conditions which will allow shorter incubation times and analyses with larger soil samples. Current studies in our laboratory are directed toward elucidating the contributions of bacteria and fungi to the mineralization of more complex substrates including starch, cellulose, and lignocellulose complexes. This should improve our understanding of microbial substrate utilization

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patterns in the rhizosphere-rhizoplane zone of this ecosystem and help identify the factors which influence decomposition processes in this

environment. ACKNOWLEDGMENTS This study was supported in part by National Science Foundation grant DEB 7518765. Technical assistance was provided by Nicholas Nagle. Consultations with Marianne Clarholm and D. C. Coleman during the initial phases of this study are gratefully acknowledged. LITERATURE CITED 1. Anderson, J. P. E., and K. H. Domsch. 1973. Quantification of bacterial and fungal contributions to soil res-

piration. Arch. Microbiol. 93:113-127. 2. Anderson, J. P. E., and K. H. Domsch. 1973. Selective inhibition as a method for estimation of the relative activities of microbial populations in soils. Bull. Ecol. Res. Comm. (Stockholm). 17:281-282. 3. Anderson, J. P. E., and K. H. Domsch. 1975. Measurement of bacterial and fungal contributions to respiration of selected agricultural and forest soils. Can. J. Microbiol. 21:314-322. 4. Ares, J. 1976. Dynamics of the root system of blue grama. J. Range Manage. 29:208-213. 5. Clark, F. E. 1967. Bacteria in soil, p. 15-49. In A. Burges and F. Raw (ed.), Soil biology. Academic Press Inc., London. 6. Clark, F. E., and E. A. Paul. 1970. The microflora of grassland. Adv. Agron. 22:375-435. 7. Corke, C. T., and F. E. Chase. 1965. The selective enumeration of actinomycetes in the presence of large numbers of fungi. Can. J. Microbiol. 2:12-17. 8. Gyllenberg, HI G., and E. E. Eklund. 1974. Bacteria. pp. 245-268. In C. H. Dickinson and G. J. F. Pugh (ed.), Biology of plant litter decomposition, vol. 2. Academic

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9. Harley, J. L*, and J. S. Waid. 1955. A method of studying active mycelia on living roots and other surfaces in soil. Trans. Br. Mycol. Soc. 2:104-108. 10. Harrison, M. J., W. T. Wright, and R. Y. Morita. 1971. Method for measuring mineralization in lake sediments. Appl. Environ. Microbiol. 21:698-702. 11. Jackson, R. M. 1965. Studies of fungi in pasture soils. II. Fungi associated with plant debris and fungal hyphae in soil. N. Z. J. Agric. Res. 8:865-877. 12. Klein, D. A. 1977. Seasonal carbon flow and decomposer parameter relationships in a semi-arid grassland soil.

Ecology 58:184-190. 13. Louw, H. A., and D. M. Webley. 1959. The bacteriology of the root region of the oat plant grown under controlled pot culture conditions. J. Appl. Bacteriol. 22: 216-226. 14. Martin, J. K. 1977. Effect of soil moisture on the release of organic carbon from wheat roots. Soil Biol. Biochem. 9:303-304. 15. Martin, J. P. 1950. Use of acid, Rose Bengal, and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69:215-232. 16. Parkinson, D., T. R. G. Gray, and S. T. Williams. 1971. IPB handbook no. 19: methods for studying the ecology of soil microorganisms. Blackwell Scientific Publications, Oxford. 17. Shields, J. A., E. A. Paul, W. E. Lowe, and D. Parkinson. 1971. Turnover of microbial tissue in soil under field conditions. Soil Biol. Biochem. 5:753-764. 18. Soderstrom, B. E. 1977. Vital staining of fungi in pure cultures and in soil with fluorescein diacetate. Soil Biol. Biochem. 9:59-3. 19. Williams, S. T., and F. L. Davies. 1965. Use of antibiotics for selective isolation and enumeration of actinomycetes in soil. J. Gen. Microbiol. 38:251-261. 20. Wright, R. T., and J. E. Hobbie. 1966. Use of glucose and acetate by bacteria and algae in aquatic ecosystems. Ecology 47:447-464.