THE EFFECT OF MECHANICAL FORCES ON THE EXUDATION OF ORGANIC SUBSTANCES BY THE ROOTS OF CEREAL PLANTS GROWN UNDER STERILE CONDITIONS

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1 New Phytol. (1974) 73, THE EFFECT OF MECHANICAL FORCES ON THE EXUDATION OF ORGANIC SUBSTANCES BY THE ROOTS OF CEREAL PLANTS GROWN UNDER STERILE CONDITIONS BY D. A. BARBER AND K. B. GUNN Agricultural Research Council, Letcombe Laboratory, Wantage, Berks. {Received 25 July 1973) SUMMARY Cereal plants were grown for 3 weeks under sterile conditions in solution culture with their roots either unrestricted or growing between glass ballotini. The exudation of both amino acids and carbohydrates was increased when the roots were grown through a solid medium and the total quantity exuded was equivalent to about 9% of the dry matter increment of the roots. INTRODUCTION There is ample evidence that a wide range of organic substances is exuded by the roots of both herbaceous plants and trees (Rovira, 1965; Smith, 1969). Although it is now well established that this process is largely responsible for the concentration of microorganisms on and immediately around the roots in the rhizoplane and rhizosphere (Clark, 1949; Katznelson, Lochhead and Timonin, 1948), there is less certainty about its influence on the overall microbial activity of soils (Rovira, 1965; Harley, 1973), on the absorption of inorganic nutrients by plants (Quirk, 1967), on soil structure (Allison, 1968) and on allelopathic interactions between plants (Whittaker and Feeny, 1971). Since the exudates can be rapidly utilized by micro-organisms quantitative estimates of their production can be made only if plants are grown under sterile conditions. This can be most simply achieved in solution culture, a system which also facilitates the collection of the exudates. The range of substances which may be released are conveniently examined under these conditions. However, a number of observations indicate that the quantities of exudates found in such experiments are unlikely to be similar to those produced in soil. Factors which have been shown to enhance exudation include contact with a solid medium (Boulter, Jeremy and Wilding, 1966; Ayers and Thornton, 1968), anaerobiosis (Grineva, 1961), moisture stress (Katznelson, Rouatt and Payne, 1954; Vancura and Garcia, 1969), low temperature (Vancura, 1967) and herbicides (Kaiser and Reber, 1970). Moreover, indications both that the pattern of exudation can vary along root axes and with plant age (Rovira, 1965) suggest that representative information cannot be obtained from very young seedlings. Accordingly, in the present work plants were grown for 3 weeks under sterile conditions, either in contact with glass ballotini or, for comparison, in solution culture. Ballotini had two advantages for this purpose: they are available in a range of uniform 39

2 40 D. A. BARBER AND K. B. GUNN sizes allowing reproducible mechanical contact to be achieved and both roots and soluble exudates could be readily recovered. EXPERIMENTAL METHODS Growth of plants Barley or maize plants were grown under rigidly sterile conditions in enclosed Pyrex containers by the methods developed for solution culture studies (Barber, 1967). The procedure for plant culture in the present work differed from that previously described in that whereas some root chambers (volume approximately 900 ml) contained nutrient solution only, glass ballotini, i or 3 mm in diameter, were placed in others. The culture solution contained Ca(NO3)2, 3 m-equiv./l; KNO3, 5; KH2PO4, i; MgS04, 3; NaNOj, 2; with H3BO3, CuSO^, FeEDTA, MnSO4, (NH4)^Mo7O24, ZnS04, and KCl, to give the following concentrations (in ppm): B, o.i, Cu, 0.01, Fe, 0.5, Mn, 0.2, Mo, o.oi, Zn, 0.05, Cl, 0.5. Table i. Sterility check: samples of solutions were transferred to potato dextrose and nutrient agars. Any containinated replicates were subsequently discarded. Culture solution Drained off into bottle containing 5 ml chloroform. Ballotini well washed by vigorous shaking with four successive loo-m! aliquots of deionized water saturated with chloroform. Rinses added to original solutions. Solutions and rinses concentrated by rotar\' evaporation at 38^ C. Filtered through 0.22 //m pore diameter millipore membranes. Made up to 10 ml with deionized water and stored in a deep freeze until analysed. Plants Roots carefully separated from ballotini and mucigel removed by transferring them to 250- ml beakers containing 100 ml 0.2 mm CaCl2 which are stood for 5 min in sonic cleaning bath. CaCl2 solution placed in bottle with chloroform. Then treated similarly to culture solutions. Roots and shoots were separated and plunged into 100 ml boiling 70",, ethanol, kept at boiling point for 4 min and then 24 h at 5 C. Rinsed with two successive 50 ml lots of 20% ethanol. Solutions concentrated and deep frozen. Shoots dried and weighed. Roots transferred to 50 ml 0.5",,.\zoblack in 70",^, ethanol and stored till lengths and diameters measured. Finally dried and weighed. One Sterile seedling was planted in each vessel, 6 days after germination and allowed to grow for 3 weeks at 22^ C with a day length of 16 h at a light intensity of 15,000 lux. The plants were harvested in a sterile cabinet and treated as outlined in Table i. This enabled measurements to be made of the amino acids and carbohydrates exuded into the culture solutions, those present on the surface of the roots in the mucigel (Jenny and Grossenbacher, 1963; Brams, 1969) and those present at harvest in a soluble form in the roots and shoots. The effect of treatment in the sonic cleaning bath on the roots was examined. Treatment for 5 min released over 90";, of the total quantity of material that could be removed from roots after three successive 5-min exposures; microscopic examination showed that root hairs remained intact and that protoplasmic streaming could be observed in them; there was no effect on the uptake and translocation of phosphate or rubidium, either immediately after treatment or 24 h later; growth rate when measured daily over a period

3 Mechanical forces and root exudation 41 of 3 weeks was unchanged. These observations were considered to justify the conclusion that sonic cleaning did not damage the roots and was, therefore, a valid procedure for removing exudate from the root surface. Carlson and Belcher (1970) reached a similar conclusion. Assay of amino acids The total quantities of soluble amino acids and ammonium present in the samples were estimated by reaction with ninhydrin and measurement of the resulting optical density at 570 nm. Ammonium was then determined by the method of Horn and Squire (1967). Known amounts of wor-leucine were treated similarly and, after correction for the ammonium content, the amino acids present in the experimental samples were expressed in terms of the equivalent quantity of «or-leucine (/imol). In a limited number of experiments, individual amino acids were estimated after separation on a cation exchange resin using a lithium citrate buffer gradient (Benson, Garden and Patterson, 1967). Assay of soluble carbohydrates The total soluble carbohydrates in the various fractions were estimated by an automated technique similar to that described by Kesker (1965). The solutions were hydrolysed with 70% sulphuric acid, reacted with orcinol and the resulting coloured compound measured at an optical density of 440 nm. Known amounts of glucose were estimated by the same procedure and the total quantities of soluble carbohydrates present were expressed as milligrams of glucose. Measurement of roots The roots were dyed by treatment with o.>"o Azo black in 7O o ethanol, rinsed free of excess dye and floated in shallow trays of water over centimetre-squared graph paper. The seminal and nodal roots were separated and their number and individual lengths noted, together with the distance from the tip to the first visible lateral. The total root system was then displayed on a glass plate and its length determined by a modification (Drew et ah, 1973) of the method described by Rowse and Phillips (1974). The dry weight of the roots was then determined. This was subsequently corrected for the amounts of soluble amino acids and carbohydrates extracted. RESULTS Growth in ballotini modified the form of the root system causing the seminal roots to be shorter and to bear more laterals; the effects were more pronounced in the i-mm- than the 3-mm-diameter ballotini (Table 2). The overall weight and length of the root systems, however, were not significantly affected. The dr}' matter production of the shoots was similarly unaffected. Table 3 shows that growth of barley roots between ballotini caused the quantities of both amino acids and carbohydrates exuded into the culture solutions to be increased; this effect was greater with the i-mm than the 3-mm ballotini. During 3 weeks, the total amount of exudate produced by plants in i-mm ballotini was equivalent to 9"o of the increase in the dry matter content of the roots over the same period compared with 5^0 by plants in culture solution alone while the 3-mm ballotini gave intermediate values. This difference in exudation was accounted for entirely by material present in solution.

4 D. A. BARBER AND K. B. GUNN Table 2. Characteristics of barley plants groum in presence and absence of ballotini ROOTS Dr>' weight (mg) Total length (cm) Seminals Number Total length (cm) Root system (%) Distance tip to first lateral (cm) Nodals Number Total length (cm) SHOOTS Dry weight (mg) Environment of roots Culture solution alone 3-mm ballotini i-mm balllotini 34-4± ± ! ± ! 2.8 3S-S± I9.8± ± ± ± B.2 + i6.6± 34.1 ± ± Table 3. Effect of the rooting medium on the exudation of amino acids and carbohydrates by barley plants grown for 3 weeks under sterile conditions together with the quantities present in the roots and shoots at harvest Increase in dr\' weight (mg per plant) Roots Shoots Exudates Amino acids (//mol per plant) Total (mg per plant) Carbohydrates (mg per plant) Total quantity of material exuded Milligrams per plant Percentage weight roots Percentage total plant weight Root content.amino acids (//mol'plant) Carbohydrates (mg plant) Shoot content Amino acids (/imol/plant) Carbohydrates (//mol/plant) Culture solution alone o.84±o.i ± I.22±O ± En \ ironment of roots> 3-mm ballotini : I-mm ballotini ± ± ± ± It is of interest also that the distribution of alcohol soluble compounds in the plants at harvest was affected by the rooting medium; the shoots of plants growing in ballotini contained significantly less amino acids and carbohydrates. Although the ballotini caused an increase in the total quantity of amino acids exuded the relative amounts of the individual acids present, either in solution or on the surface of the roots, appeared to be little affected (Table 4). Exudation by maize roots was similarly enhanced in the presence of ballotini (Table 5). In this experiment, the quantities of amino acids and carbohydrates in the roots were determined in macerates of the fresh root tissue; the shoots were not harvested.

5 Mechanical forces and root exudation 43 Table 4. The relative quantities of amino acids exuded by the roots of barley plants grown for 3 weeks in solution culture under sterile conditions in the presence and absence of i -mmdiameter glass ballotini {the amount of each acid {fimol) is expressed as a percentage of the total quantity present) Amino acid Aspartic acid Threonine Serine Asparagine Glutamine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine 4-NH2 butyric acid Lysine Histidine Arginine Ballotini Present II II Absent /, Trace, not measurable. Ballotini Present Absent O O j O.I I.I Table 5. Effect of the rooting medium on the exudation of amino acids and carbohydrates by maize plants grown for 3 weeks under sterile conditions Fresh weight of roots (g per plant) Exudates Amino acids (//mol/plant) Carbohydrates (mg/plant) Root content Amino acids (//mol/plant) Carbohydrates (mg/plant) Culture solution alone Environment of root 6-mm Ballotini 3-mm I.I I-mm s DISCUSSION The results presented shov^^ that when the roots of barley and maize plants in solution culture grow in the interstices between glass ballotini, the exudation of amino acids and carbohydrates is enhanced compared with that of the roots of plants growing in the absence of ballotini. It should be emphasized, however, that the pressures imposed are probably small in relation to those on roots growing in soil, since the ballotini were not rigidly restrained (Goss and Drew, 1972). Thus, quite apart from the fact that amino acids and carbohydrates only were determined, the finding that the exudation of these compounds into i-mm ballotini was equivalent in quantity to 9% of the increase in the dry matter content of the roots is likely to provide a minimum estimate of the amount of material released into the soil by growing crops.

6 44 D. A, BARBER AND K. B, GUNN Since infection by many root pathogens is known to require the prior stimulation by exudates from the host plants (Schroth and Hildebrand, 1964) the present findings are of particular interest in regard to the observations of Burke and his colleagues (Burke et al., 1972; Burke, Holmes and Barker, 1972). They have shown that the incidence and effect of the root rot of field beans (Phaseohis vulgaris) induced by (Fusarium solani f. sp. phaseoli) is greatly increased in compacted soils, whether in pot experiments or in the field and that fungal infection is decreased by cultivation treatments which reduce soil compaction. An analogous situation has been described by Papavizas and Kovaks (1972) who showed that fatty acids exuded by roots are necessary for the germination of Thielaviopsis basicola. However, during 8 days in solution culture under sterile conditions, no substances were exuded by bean seedlings which, when the solutions were subsequently tested, stimulated the germination of the fungus, whereas in sterile sand culture, 0.60, 0.48 and 0.55 ^g/plant respectively of palmitic, stearic and oleic acids were exuded and induced the germination of fungal spores. The possible extent to which exudates may serve as substrates for soil respiration has been discussed by several workers (Gray and Williams, 1971; Harley, 1973) and it is, therefore, of interest to consider whether exudation at the rate reported in the present paper might make an appreciable contribution to soil respiration in arable land. On the basis of estimates that the respiration of bare soil may be in the range ^ CO2 per m^ per day (Clark, 1967; Hawkins, 1962), and that the total weight of roots of a barley crop may approach 100 g per m^ (Goedewaagen and Schuurman, 1950) it can be calculated that exudation would provide substrates for only a small fraction of the total soil respiration. A similar conclusion may be drawn from the work of Nilovskaya, Kovalenko and Laptev (1970) who, by a '*C-labelIing technique, showed that between 9 and 12% of the CO2 fixed in photosynthesis is released in respiration by micro-organisms associated with the roots of cabbage or beet grown in pots of soil. The uncertainty of such studies is obvious, but pending further information it appears likely that exudates are the substrate of only a small fraction of the total microfiora in soil, presumably mainly rhizosphere micro-organisms. ACKNOWLEDGMENTS We thank Dr R. Scott Russell for his stimulating interest in this work and Miss S. K. Allen and Mr R. G. J. Ryding for their valuable assistance. REFERENCES ALLISON, F. E. (1968). Soil aggregation some facts and fallacies as seen by a microbiologist. Soil Sci., 106, 136. AYERS, W. A. & THORNTON, R. M. (1968). Exudation of amino acids by intact plants and damaged roots of wheat and peas. PL Soil, 28,193. B.^RBER, D. A. (1967). The effect of micro-organisms on the absorption of inorganic nutrients by intact plants. I. Apparatus and culture technique. X exp. Bot., l8, 163. BENSON, J. V., GARDEN, M. J. & PATTERSON, J. A. (1967). Accelerated chromatographic analysis for amino acids in physiological fluids containing glutamine and asparagine. Analyt. Biochem., 18, 228. BOULTER, D., JEREMY, J. J. & WILDING, M. (1966). Amino acids liberated into the culture medium by pea seedling roots. PL Soil, 24, 121. BRAMS, E. (1969). The mucilagenous layer of citrus roots its delineation in the rhizosphere and removal from roots. PL Soil, 30, 105. BURKE, D. W., MILLER, D. E., HOLMES, L. D. & B.ARKER, A. W. (1972). Counteracting bean root rot by loosening tbe soil. Phytopathology, 62, 306. BURKE, D. W., HOLMES, L. D. & B.ARKER, A. W. (1972). Distribution of Fusarium solani f. sp. phaseoli and bean roots in relation to tillage and soil compaction. Phytopathology, 62, 550.

Mechanical forces and root exudation 45 CARLSON, L. W. & BELCHER, J. (1970). Effect of ultrasonic root cleaning on subsequent growth of caragana seedlings. Biomed. Res. Notes, z6, 27. CLARK, F. E. (1949)- Soil micro-organisms and plant roots.

7 Mechanical forces and root exudation 45 CARLSON, L. W. & BELCHER, J. (1970). Effect of ultrasonic root cleaning on subsequent growth of caragana seedlings. Biomed. Res. Notes, z6, 27. CLARK, F. E. (1949)- Soil micro-organisms and plant roots. Adv. Agron., i, 241. CLARK, F. E. (1967)- Bacteria in soil. In: Soil Biology (Ed. by N. A. Burges & F. Raw), p. 15. Academic Press, New York. DREW, M. C, SAKER, L. R., LAY, P. M., HARRIS, W. & ELSWORTH, M. J. (1973). An automatic method for measuring root length. Rep. agric. Res. Coun. radiobiol. Lab. (1972), p. 37. GoEDEWAAGEN, M. A. J. & SCHUURMAN, J. J. (1950). Wortelproductie op bouw en grasland als bron van organische stof in de grond. Landb. Tijdschr., 62, 469. Goss, M. J. & DREW, M. C. (1972). Effect of mechanical impedance on growth of seedlings. Rep. agric. Res. Coun. radiobiol. Lab. (1971), p. 35. GRAY, T. R. G. & WILLIAMS, S. T. (1971). Microbial productivity in soil. In: Microbes and biological productivity (Ed. by D. E. Hughes & A. H. Rose), p Cambridge University Press, London. GRINEV'A, G. M. (1961). Excretion by plant roots during brief periods of anaerobiosis. Soviet PL PhysioL, 8, 549- HARLEY, J. L. (1973). Symbiosis in the ecosystem. J. Nat. Sci. Council, Sri Lanka, i, 31. HAWKINS, J. C. (1962). The effects of cultivation on aeration, drainage, and other soil factors important in plant growth. X Sci. Fd Agric., 13, 386. HORN, D. B. & SQUIRE, C. R. (1967). An improved method for the extraction of ammonia in blood plasma. Clin. Chim. Acta, 17, 99. JENNY, H. & GROSSENBACHER, K. (1963). Root-soil boundary zones as seen in the electron microscope. Proc. Soil Sci. Soc. Am., 27, 273. KAISER, P. & REBER, H. (1970). Interactions entre la simazine herbicide et les microorganisms de la rhizosphere du mais. Meded. Rijufas Landb. Wet Gent., 35, 689. KATZNELSON, H., LOCHHEAD, A. G. & TIMONIK, M. I. (1948). Soil micro-organisms and the rhizosphere. Bot. Rev., 14, 543. KATZNELSON, H., ROUATT, J. W. & PAYNE, T. M. B. (1954). Liberation of amino acids by plant roots in relation to desiccation. Nature, Lond., 174, mo. KESKER, R. B. (1965). Ion exchange chromatography of sugars in wood pulp and paper hydrolysates. Automation in Analytical Chemistry, Technicon Symposium, p NiLOVSKAYA, N. T., KovALENKO, V. K. & L.\PTE\, V. V. (1970). Uptake and liberation of carbon dioxide by plants under artificial environmental conditions. Soviet PL PhysioL, 17, 567. PAPAVIZAS, G. C. & KOVAKS, M. F. JR. (1972). Stimulation of spore germination of Thielaviopsis basicola by fatty acids from rhizosphere soil. Phytopathology, 62, 688. QUIRK, J. P. (1967). Aspects of nutrient-absorption by plants from soils. Aust. Soc. Soil Sci., Canberra, Publication No. 4. RoviRA, A. D. (1965). Plant root exudates and their influence upon soil micro-organisms. In: Ecology of Soil-borne Plant Pathogens Prelude to Biological Control (Ed. by K. F. Baker & W. C. Snyder), p University of California, U.S.A. RowsE, H. R. & PHILLIPS, D. A. (1974). An instrument for estimating the total length of roots in a sample. y. appl. EcoL, II (in press). SCHROTH, M. N. & HiLDEBR.'VND, D.C. (1964). Influence of plant exudates on root-infecting fungi. A. Rev. PhytopathoL, 2, ioi. SMITH, W. H. (1969). Release of organic material from the roots of tree seedlings. Forest Sci., 15, 138. V.ANCURA, V. (1967). Root exudates of plants. III. Effect of temperature and 'cold-shock' on the exudation of various compounds from seeds and seedlings of maize and cucumber. PL Soil, 27, 319. VANCURA, V. & GARCIA, J. L. (1969). Root exudates of reversibly wilted millet plants {Paricum miliaceum L). Ecol. Plant, 4, 93. WHITTAKER, R. H. & FEENY, P. P. (1971). AUelochemies. Chemical interactions between species. Science, N.Y.,

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

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