METABOLIC ACTIVITY OF BACTERIAL ISOLATES FROM WHEAT RHIZOSPHERE AND CONTROL SOIL'

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1 METABOLIC ACTIVITY OF BACTERIAL ISOLATES FROM WHEAT RHIZOSPHERE AND CONTROL SOIL' A. C. ZAGALLO2 AND H. KATZNELSON Bacteriology Division, Science Service, Canada Department of Agriculture, Ottawa Received for publication December 14, 1956 The recognition that roots of plants support a microbial population which differs qualitatively and quantitatively from that of soil a short distance away has stimulated great interest among soil microbiologists and plant physiologists alike. This is understandable in view of the critical importance of this zone, called the rhizosphere, for the well being and nutrition of the plants themselves (Katznelson et al., 1948; Clark, 1949; Norman, 1951). From a microbiological point of view the nature of this microflora is of great interest and warrants study, not only because of its possible practical importance, but also on account of its ecological significance. In this connection it has been shown by Lochhead and his associates (West and Lochhead, 194; Lochhead and Thexton, 1947; Lochhead and Rouatt, 1955) that bacteria requiring amino acids for optimal growth are preferentially stimulated in this zone. It has been reported also that the rhizosphere supports a greater proportion of bacteria which are capable of growing rapidly, and of physiologically active types than does nonrhizosphere soil (Lochhead, 194; Rovira 1956a, 1956b; Rouatt and Katznelson, 1957; Katznelson and Rouatt, 1957a). These and related observations (Katznelson and Rouatt, 1957b) suggest that the rhizosphere microflora may be of greater importance to the plants, directly or indirectly, than originally envisaged. The results presented in this paper support those previously reported concerning the metabolic activity of rhizosphere bacteria. It remains to be determined whether such activity is entirely beneficial or whether it may be detrimental to the plant under certain circumstances. ' Contribution No Canada Department of Agriculture Postdoctorate Fellow for Research in the Biological Sciences. Present address: Department of Bacteriology, Oregon State College, Corvallis, Oregon. MATERIAL AND METHODS The wheat plants used in this investigation were grown in pots in a greenhouse and were 26 days old at the time of sampling; the control soil was taken from pots without plants. The rhizosphere and nonrhizosphere soil samples were taken according to the procedure of Lochhead (194). Suitable dilutions of these samples were prepared and plated with soil extract agar; the plates were incubated for 14 days at 26 C. After being counted, the colonies from an entire plate were picked into soil extract semisolid agar; in one case all the colonies from a sector of a plate were taken. One hundred and thirty colonies from rhizosphere soil and 12 from control soil were thus obtained. The cultures were classified according to their nutritional requirements following methods previously described (Lochhead and Chase, 1943; Rouatt and Lochhead, 1955). The semisolid agar tubes were kept in a cold room (1-3 C) as stock cultures for further studies. From these, 1 cultures (5 of rhizosphere origin and 5 of nonrhizosphere origin) were selected at random and used in the experiments to be described. Cells for manometric experiments were grown in the media described below for hr at 26 C, harvested and washed twice with distilled water. After centrifugation (8,-9, rpm) for 1 min, they were resuspended in phosphate buffer (.7 M and ph 7.) and adjusted to a turbidity of 4 in a Klett-Summerson colorimeter with a 6 m,u filter. Seven substrates (glucose, alanine, sucrose, xylose, acetate, pyruvate and succinate) were used in preliminary trials, and the following four were selected for further tests: glucose, alanine, acetate and succinate. Each Warburg vessel contained 2. ml of the cell suspension in the main compartment,.2 ml of KOH (2 per cent) in the center well, and.1 ml.5 M substrate in the side arm. A period of 45 to 6 min was allowed for equilibration before the substrates were tipped. Oxygen uptake 76 Downloaded from on December 12, 218 by guest

2 1957] METABOLIC ACTIVITY OF BACTERIAL ISOLATES 761 TABLE 1 Oxygen uptake (jil) by bacteria from rhizosphere and control soil on different substrates* Source of o Ca Bacteria c 2 2 Rhizosphere soil Control soil * Average oxygen uptake by 1 rhizosphere and 1 control soil isolates. ia 6 4 r Y 2 16 Z 2 6 > o x 4 *..... oo * RHIZOSPHERE BACTERIA 3 - CONTROL SOIL BACTERIA ** * - noo _, ~ oooc O. )~ * 36 _ 5 Figure 1. Oxygen uptake with glucose by bacteria was measured at 28 C over a period of 3 hr only, even if the substrate was not completely utilized. Endogenous respiration varied considerably among the cultures, with no consistent pattern emerging. All results are reported with endogenous values subtracted. EXPERIMENTAL RESULTS The numbers of microorganisms (bacteria and actinomycetes) in wheat rhizosphere soil and control soil, were respectively 2,47 and 45 millions per gram of soil, calculated on an oven dry basis. This shows a rhizosphere to soil ratio (R:S) of 55:1 and indicates a distinct "rhizosphere effect." This effect was also evident from the nutritional evaluation of the isolates, which showed a preferential stimulation in the rhizosphere soil of organisms requiring amino acids for optimal growth. A number of tests were carried out at first to 4 36 j _ 24 2 IL 16 Z x 4. RHIZOSPHERE BACTERIA - CONTROL SOIL BACTERIA so_ * oooooooooo _ ~ ~ ~ ~~f * 1234 s Figure 2. Oxygen uptake with alanine by bacteria " r a o 4 36 D ~~~~~~~~~~~ D P * - RHIZOSPHERE BACTERIA. - CONTROL SOIL BACTERIA W 24 I-- oo. (L ~ ~ ~ ~ ~ *o* so ooooo 4 ooo.o _ s Figure S. Oxygen uptake with acetate by bacteria select a medium which would support the best growth of the greatest number of isolates. Fluid and solid media were compared in Fernbach flasks (shaken and stationary) and in Roux bottles, respectively; "penassay" base agar (Difco) was selected as the most suitable medium for the purpose. Manometric experiments were then carried out with 2 isolates with the 7 substrates mentioned earlier. In table 1 are recorded the results obtained with 1 rhizosphere and 1 soil cultures. It may be observed that the greatest differences, in favor of the rhizosphere bacteria, occurred with sucrose, glucose, alanine, acetate and succinate as substrates. The latter 4 Downloaded from on December 12, 218 by guest

3 762 ZAGALLO AND KATZNELSON [VOL. 73 I& 36 w 32 - O O _ were then selected for study with the remaining rhizosphere and soil cultures. In figures 1 to 4 are presented over-all summaries of the metabolic activity of all the bacterial isolates examined, on the 4 substrates. With glucose and alanine, greater activity is clearly demonstrated by rhizosphere cultures. This is generally true also with succinate. Glucose appears to be used most rapidly by the rhizosphere cultures and succinate the least. Alanine and acetate are intermediate. For the soil cultures, glucose and acetate may be somewhat better than alanine and succinate. A comparison was then made of the oxygen uptake with these 4 substrates of organisms belonging to specific nutritional groups (Loch- 2-. F- 16 _ RHIZOSPHERE BACTERIA CONTROL SOIL - BACTERIA ~~~~~~~~~~~~@ D12 _ x 4 <o...,,,,... l lw ~ R S Figure 4. Oxygen uptake with succinate by bacteria TABLE 2 A comparison of oxygen uptake with different substrates by bacteria of different nutritional requirements* Source Souce off Bcteia Bacteria Bacteria All Group "A" Group "AG"' Group "cys" Glucose Rhizosphere soil Control soil Alanine Rhizosphere soil Control soil Acetate Rhizosphere soil Control soil Succinate Rhizosphere soil Control soil * Average oxygen uptake CsL). head and Chase, 1943) i.e.: "A" group, requiring amino acids for maximum growth; "AG" group, requiring amino acids and known growth factors; "YS", requiring various substances present in yeast and soil extracts. The data are given in table 2. Rhizosphere cultures falling into the "YS" group showed consistently and distinctly higher averages in all substrates than soil isolates (figure 5). This effect may be noted also (though perhaps not to such an extent) with "AG" bacteria. No consistent pattern was obtained with "A" group organisms. Qo2 values (,ul of oxygen uptake per hr, per mg of dry weight of cells) were also calculated. Their averages are presented in table 3. Again there is evidence of a rhizosphere effect: higher average Qo2 values with rhizosphere isolates than with soil isolates, the difference becoming narrower from glucose to succinate, following a decrease of the Qo2 values of rhizosphere bacteria. These results are in accord with those presented in figures 1 to 4 and table GLUCOSE W 32 -i 28 ; U 24 1,; 2 _ < 16 D3 DL 12 _ * z 83 - (O X _ v. RHIZOSPHERE BACTERIA - CONTROL SOIL BACTERIJA _- ALANINE ACETATE SUCCINATE.. 1oooo oe!....i... Oo... Figure 5. Metabolic activity, in different substrates, of bacteria from wheat rhizosphere and control soil, requiring yeast and soil extracts for optimal growth (after 3 hr). TABLE 3 Average Qo2 values of bacteria from rhizosphere and control soil* Source of Bacteria Substrate Glucose Alanine Acetate Suci cinate Rhizosphere soil Control soil * Fifty bacteria from wheat rhizosphere and 5 from control soil. *- Downloaded from on December 12, 218 by guest

4 19571 METABOLIC ACTIVITY OF BACTERIAL ISOLATES 763 DISCUSSION Studies such as those recorded above are complicated by the fact that large numbers of cultures must be tested before satisfactory conclusions can be drawn. This is especially true of such a complex system as soil, with its extremely diversified bacterial flora. Consequently, although the results presented are suggestive of a rhizosphere effect, it would be highly desirable to conduct such tests with larger numbers of organisms. Nevertheless, when considered in relation to work along similar lines reported previously (Lochhead, 194; Rovira, 1956a, 1956b; Rouatt and Katznelson 1957; Katznelson and Rouatt, 1957a, 1957b) the data are of interest and, by and large, support the conclusion that a selective effect is exerted in the rhizosphere favoring organisms which are more active physiologically than those from corresponding control soil. The presence of such an active microbial population at the root-soil interface may be of great significance to the growing plant (Katznelson et al., 1948; Clark, 1949; Wadleigh, 1954). The effects may be direct or indirect, harmful or beneficial. Active ammonification can provide the plant with ammonium salts as a source of nitrogen, and active carbon dioxide evolution may aid in rendering soluble inorganic nutrients. Production of various intermediates in the breakdown of carbohydrates or proteins may also exert an effect which may be either useful or harmful. The synthetic activities of rhizosphere bacteria with respect to the production of amino compounds, vitamins, auxins and other growth factors may be considered of some significance, depending on whether or not evidence is presented that plants require such substances for good growth (Schmidt, 1951). At the same time, however, the rhizosphere microflora may compete with the plant for nutrients or render them unavailable for the plant as has been noted for example in studies on the grey-speck disease of oats (Gerretsen, 1937; Timonin, 1947). It would appear therefore that if the microbial equilibrium is such that beneficial forms are predominant, the plant may be benefited; if not, the plant may suffer. By suitable treatment it may be possible to shift this equilibrium in favor of beneficial types and render this root zone inimical to harmful forms such as root pathogens (West and Hildebrand, 1941). ACKNOWLEDGMENTS The authors are grateful to Dr. J. W. Rouatt and to Dr. A. G. Lochhead for helpful suggestions and advice, and to Miss Agnes McIntosh for technical assistance. SUMMARY Metabolic studies were conducted with representative bacteria obtained from the rhizosphere of wheat and from control soil. It was demonstrated that in general, rhizosphere cultures were more active in oxidizing glucose, alanine, acetate, and possibly succinate, than isolates from the control soil. This effect was particularly noteworthy with bacteria with complex nutritional requirements. The results support the conclusion of other investigators that the rhizosphere contains a bacterial flora which is more active physiologically than that of nonrhizosphere soil. REFERENCES CLAR1K, F. E Soil microorganisms and plant roots. Advances in Agron., 1, GERRETSEN, F. C Manganese deficiency of oats and its relation to soil bacteria. Ann. Botany, N.S. I, KATZNELSON, H., LOCHHEAD, A. G. AND TIMONIN, M. I Soil microorganisms and the rhizosphere. Botan. Rev., 14, KATZNELSON, H. AND ROUATT, J. W. 1957a Studies on the incidence of certain physiological groups of bacteria in the rhizosphere. Can. J. Microbiol. 3, KATZNELSON, H. AND ROUATT, J. W. 1957b Manometric studies with rhizosphere and non-rhizosphere soil. Can. J. Microbiol., accepted for publication. LOCHHEAD, A. G. 194 Qualitative studies of soil microorganisms. III. Influence of plant growth on the character of the bacterial flora. Can. J. Res., (C) 18, LOCHHEAD, A. G. AND CHASE, F. E Qualitative studies of soil microorganisms. V. Nutritional requirements of the predominant bacterial flora. Soil Sci., 55, LOCHHEAD, A. G. AND ROUATT, J. W The "rhizosphere effect" on the nutritional groups of soil bacteria. Soil Sci. Soc. Amer. Proc. 19, LOCHHEAD, A. G. AND THEXTON, R. H Qualitative studies of soil microorganisms. VII. The "rhizosphere effect" in relation to amino acid nutrition of bacteria. Can. J. Res. (C), 25, Downloaded from on December 12, 218 by guest

5 764 ZAGALLO AND KATZNELSON [VOL. 73 NORMAN, A. G Role of soil microorganisms in nutrient availability. In Mineral Nutrition of Plants. University of Wisconsin Press ROUATT, J. W. AND KATZNELSON, H The comparative growth of bacterial isolates from rhizosphere and nonrhizosphere soils. Can. J. Microbiol. 3, ROUATr, J. W. AND LOCHHEAD, A. G Qualitative studies of soil microorganisms. XIII. Effect of decomposition of various crop plants on the nutritional groups of soil bacteria. Soil Sci., 8, ROVIRA, A. D. 1956a Plant root excretions in relation to the "rhizosphere effect". II. A study of the properties of root exudate and its effect on the growth of microorganisms isolated from the rhizosphere and control soil. Plant and Soil, 7, ROVIRA, A. D. 1956b Plant and root excretions in relation to the "rhizosphere effect". III. The effect of root exudate on numbers and activity of microorganisms in soil. Plant and Soil, 7, SCHMIDT, E. L Soil microorganisms and plant growth substances. I. Historical. Soil Sci., 71, TIMONIN, M. I Microflora of the rhizosphere in relation to manganese deficiency disease of oats. Soil Sci. Soc. Amer. Proc., 11, WADLEIGH, C. H Mineral nutrition of plants as related to microbial activities in soils. In Soil Microbiology Conference, Purdue University. WEST, P. M. AND HILDEBRAND, A. A The microbiological balance of strawberry root rot soil as related to the rhizosphere and decomposition effects of certain cover crops. Can. J. Res. (C), 19, WEST, P. M. AND LOCHHEAD, A. G. 194 Qualitative studies of soil microorganisms. IV. The rhizosphere in relation to the nutritive requirements of soil bacteria. Can. J. Res. (C), 18, Downloaded from on December 12, 218 by guest