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Effect of extracellular polysaccharides of rhizosphere bacteria on the concentration of molybdenum in plants
SolI Biol. Biochem. Vol. 9. pp. 41 I to 415. Pergamon
Press 1977. Printed m Great Britam.
EFFECT OF EXTRACELLULAR POLYSACCHARIDES OF RHIZOSPHERE BACTERIA ON THE CONCENTRATION OF MOLYBDENUM IN PLANTS TAN ENG LEE and MARGARETW. LOUTIT Microbiology Department, University of Otago, Dunedin, New Zealand (Accepted
15 March
1977)
Summary-Extracellular polysaccharides produced by bacteria have been shown to bind molybdenum (MO) with the consequence that less MO entered plants. The polysaccharides are produced by a variety of bacteria but those which contain uranic acids appear to be reponsible for binding the MO. The
amount of MO bound is affected by pH.
INTRODUCTION Tan and Loutit (1976) reported that some rhizosphere bacteria grown in pure culture bound MO in their extra-cellular polysaccharides. Such an interaction between polysaccharides and MO offers a possible explanation of the observation that rhizosphere bacteria prevent entry of MO to plants under certain conditions (Loutit et al., 1968; Loutit and Brooks, 1970). This paper reports results of experiments designed to investigate this suggestion.
cells washed twice in HA. The Gram-negative isolates (Pseudomonas, Achromobacter and Flavobacterium) were pooled as were the Gram-positive isolates (Nocardia, Arthrobacter and Bacillus). Each mixture was suspended in HA to give a suspension of lo6 mixed cells ml- l. Estimation of bacteria around plant roots The method et al., 1968).
previously
described
was used (Loutit
Preparation of extra-cellular polysaccharide for use in pot experiments
MATERIALS AND METHODS All glassware and Ballotini spheres were washed in aqua regia and rinsed to neutral pH, in doubly distilled water. Autoclaving was at 121°C for 15-20 min. Bacteria Strains previously described (Tan and Loutit, 1976) were used and in addition Pseudomonas aeruginosa OT 15 was obtained from Professor J. S. Loutit of this department. Media and solutions Soil extract, yeast extract, liquid medium @EYE); soil extract (Liihnis, 1920) lOOOm1, yeast extract (Difco) 1 g. Brain heart infusion (BHI) (BBL). Slimepromoting medium (SP) of Goto et al. (1973) solidified with 1.5% agar (Davis). Hoagland and Arnon solution pH 6.8 (HA) (Hoagland and Arnon, 1938) sometimes supplemented with 10 g glucose (BDH) and 1 g yeast extract (Difco) per litre (HAS). Molybdenum solution; a stock solution of Na,MoO,. 2H20 (BDH) Analar grade containing 1 mg MO ml-‘. McIlvaine’s buffer (McIlvaine, 1921). Preparation of inocula Strains were grown in SEYE for 40 h at 28°C except for Pseudomonas which was grown in BHI at 28°C overnight. Approximately 10’ cells ml-’ were inoculated into lOOm1 HAS and incubated at 28°C for 48 h in a shaking water bath. After incubation the flask contents were centrifuged and the deposited 411
Volumes (2.0ml) of P. aeruginosa OT 15 incubated in BHI overnight were spread over the surface of 250 ml solidified SP medium in polypropylene instrument trays (140 x 160mm) with glass spreaders. The trays were incubated for 5 days at 28°C. Cells and slime were scraped off with a glass slide and suspended in 50 ml sterile saline. The cells and slime were mixed vigorously in a blender for 30 s and cells deposited by centrifugation at 1OOOg for 20 min at 4°C. Slime was precipitated from the supernatant by the addition of an equal volume of ethanol and left for 12 h at 4°C. The supernatant was decanted and the remaining slime redissolved in a minimum quantity of doubly-distilled water. The dissolved slime was dialysed against 2 1 doubly-distilled water at 4°C for 24 h with continuous stirring. The dialysed slime was precipitated with an equal volume of ethanol and the slime removed with a glass rod. After washing with 90% ethanol (v/v) and acetone, the slime was dried over PzO, in mcuo, weighed and stored in a desiccator over P*O,. Plant experiments Ballotini spheres (35 g Jencons No. 19 40 pm dia) were placed in test tubes (32 x 200mm) covered with Peep see-through cooking film (Seto Bags Ltd., Wellington, N.Z.) and autoclaved. HA solution pH 6.0 was added to bring the beads to 60% of their water holding capacity (w.h.c.). MO was incorporated into some tubes by adding MO from the stock solution to HA to give a final concentration of 10 fig MO ml-‘. Where extracted slime was to be added, 300 mg dried
112
TAN ENG L
EE and MARGARETW. LOUTIT
preparation was dissolved in lOOmI of the HA solution before use. In experiments requiring addition of a mixed inoculum of Gram-negative or Gram-positive bacteria, washed pooled cells were suspended in the HA solution to give a final concentration of 10e6 mixed cells ml-’ and the HA used to bring the spheres to 609, w.h.c. In some experiments sterile soil was used in place of spheres. The methods for soil sterilization, seed surface sterilization, germination, planting and the conditions for growth have been described (Loutit et al., 1968, Loutit and Brooks, 1979; Loutit et al. 1972). Atomic
&sorption
spectrophotometry
(AA)
The procedure for estimation of MO in plant material and slime was as described for bacterial cells (Tan and Loutit, 1976). Extraction
qf mono-
and polysaccharide
from
soil
The method was a modification of that of Wagner and Tang (1975). After 4 weeks the plants were removed from the tubes and the bulk of the soil removed from the roots. The plants with adhering soil were then shaken vigorously and the soil freed and collected into sterile containers. Quantities (10 g) were placed in 250ml Erlenmeyer flasks. Further quantities were placed in weighed glass Petri dishes and heated at 80°C for 16 h and reweighed to allow calculation of moisture content of the soil. To each flask was added 50ml 707: (v/v) ethanol. The flask was covered and placed in a shaking water bath at 45°C for 4 h. The suspension was filtered through a sintered glass filter (20-30 pm). The deposit on the filter was retained for estimation of polysaccharide and monosaccharide estimation was carried out on the filtrate. The filtrate was dried under vacuum, lOm1 doubly-distilled water added, and the mixture shaken at 20°C for 1 h. Total monosaccharide was determined as glucose equivalents by using anthrone reagent and the results expressed as pg glucose g -i dry weight of soil. Total uranic acid was determined as glucuronic acid equivalents using carbazole (Bitter and Muir, 1962). To the deposit on each filter 50ml N H-SO4 was added, the mixture poured into a 250ml flask and the whole shaken at 20°C for 16 h. The suspensions were filtered through sintered glass filters (20-30pm) and 50ml doubly-distilled water used to wash the residue. The total filtrate was then concentrated in vacua at 50°C. To the dried extract was added 20ml doubly-distilled water and the solution centrifuged at 48,250 g in a Sorvall RC2B for 10 min. The precipitate was discarded and the supernatant dialysed against 2 1 doubly-distilled water for 16 h. The solution in the dialysis sac was then lyophilized and the dried polysaccharide hydrolysed in 2 ml 0.5 N H,S04 in a sealed ampoule at 100°C for 16 h Total carbohydrate and uranic acid estimation were then carried out on the hydrolysate. Analysis of extracellular rhizosphere bacteria
polysaccharides
produced
by
Each bacterial strain was grown in triplicate in 250 ml Basks containing 100 ml Hoagland and Arnon solution containing glucose and yeast extract (HAS).
After 5 days in a shaking water bath at 28 c‘ the cultures were centrifuged at 32009 for 20min. The cells were resuspended in 50ml sterile saline and extracellular slime was treated as described for Pseudomonas slime. In addition the slime was recovered from the culture solution by first concentrating the sohition to IO’/” of its original volume irz racuo. To this concentrate was added an equal volume of absolute ethanol. The slime that precipitated out was pooled together with that obtained from the cells. Molecules with a molecular weight of less than SOOOdaltons were removed by dialysis against six changes of 2-l volumes doubly-distilled water. Proteins in excess of this size were removed by precipitation with CHCl, by adding 0.25 volume CHCI, and 0.1 volume butyl alcohol to 1 volume of slime in 1 volume doubly-distilled water. This mixture was shaken and centrifuged at 5000 g for 20 min by which time two layers had formed, a lower stable proteinCHCl, gel which was discarded and an upper aqueous layer containing polysaccharide which was decanted. Excess CHCI, collected at the bottom of the centrifuge tube. Polysaccharide was precipitated by adding acetone in the ratio of four volumes acetone to one aqueous polysaccharide solution and the mixture left overnight at 4’C. The precipitate was redissolved in a minimal quantity of doubly-distilled water. One ml of this solution was tested (Schacterle and Pollack, 1973) to ensure no protein was present. The remaining solution was then lyophilized. The purified polysaccharide was hydrolysed by dissolving 20 mg in 2.0 ml 0.5 N H2S0, and placing the mixture in an ampoule, sealing and leaving the whole at 1OO’Cfor 16 h. The hydrolysate was neutralized with excess CaCO,. CaS04 formed was removed by centrifugation and the neutralized hydrolysate lyophilized. From the lyophilized hydrolysate trimethylsiiate (TMS) derivatives were prepared by a modified method (Wurst et al., 1974). Quantities of the hydrolysate (10mg) were dissolved in 1 ml anhydrous pyridine and left at 37°C for 4 h. When all of the lyophilized hydrolysate had dissolved 0.1 ml hexamethyl disilizone and 0.5 ml methylchlorosilane was added. The precipitate formed was removed by centrifugation at 10,000 g for 20 min and the supernatant transferred to a clean dry screw cap tube (10 x 127 mm). TMS derivatives were analysed by g.1.c. in a PYE series 104 chromatograph using a 2 m column (3 mm dia) packed with 21 g Chromosorb Q 100/120 mesh impregnated with 3”/1JXR (Applied Science Lab. Inc.). The TMS derivatives of the monosaccharides were separated isothermally at 185°C with the temperature of the detector and sample injector at 24’C. Samples (1 ~1) were injected with 1 ~1 syringe (Hamilton, New York, U.S.A.). The carrier gas was N, at a flow rate of 20 ml mini’ and the flow rates of H, and air in the flame ionization detector were 40 and 4OOml respectively. A Phillips 8000 electronic recorder was used to record emission peaks. Identification of peaks was made by comparing retention time of TMS derivatives of pure monosaccharides with those from the hydrolysed extracellular polysaccharide. Standards were o-glucose (Mallinckrodt), D-glucuronic acid, D-fructose, D-mannuronic, D-lactose and D-galacturonic (Sigma). Six estimations were made on each hydrolysed sample.
Bacterial
polysaccharides
and molybdenum
Table 1. Effect of bacteria and extracted extracellular bacterial polysaccharide on MO concentration (ngg-’ dry weight) in Raphanus satiuus L. White Icicle grown under controlled conditions in Ballotini spheres and Hoagland and Arnon solution with and without MO and at different pH values* MO ng g- 1 dry weight pH 6.0 pH 8.0 pH 6.8
Treatment No MO MO Polysaccharide MO plus polysaccharide Gram-positive cells Gram-positive cells plus MO Gram-negative Gram-negative plus MO
cells cells
8.2 582.9 2.2
5.2 351.2 2.6
3.5 337.9 2.4
222.7 5.4 403.4
255.1 3.0 276.3
243.9 2.8 238.8
1.2 113.8
1.7 167.5
1.5 236.1
* HA solution added to all tubes. MO add where appropriate to give a final concentration of 10~gmll’.
RESULTS
Although extracellular bacteria1 polysaccharides have been shown to complex with MO in pure culture their effect around the roots of plants on MO concentration in the plants has not been established. A series of experiments was therefore undertaken to investigate this effect. Radish plants were grown under controlled conditions in Ballotini spheres to which HA solution had been added. In some treatments MO was incorporated in the HA solution as was either extracted bacterial extracellular polysaccharide or an inoculum of bacteria1 cells. The tubes containing the spheres and the various additives were held in the climate cabinet for 24 h before adding the germinated seeds. After 4 weeks the plants were harvested, the MO in the tops estimated and the number of bacteria in the rhizosphere estimated. Gram-positive cells were estimated at lo5 g-’ dry weight and Gram-negative cells at lo6 g-l. The results (Table 1) indicate that addition of Gram-negative and Gram-positive cells resulted in a lower concentration of MO in plants and the effect of the Gram-negative cells was greater. Extracted polysaccharide also resulted in a marked reduction in the MO concentration in the plants. In addition to studying the effect of bacteria and polysaccharide Table
2. Composition
of
extracellular
413
in plants
on MO concentration, the effect of pH was also studied. The results (Table 1) indicated that at pH 6.0, MO concentration in the plants was greatest and least at pH 8.0. The addition of bacteria and polysaccharide at different pH values showed that the effect of Gram-negative bacteria was greatest at pH 6.0 and Gram-positive cells had the least effect on MO concentration in plants at pH 6.0. pH did not greatly influence the effect of the polysaccharide although at pH 6.0 a slightly lower concentration of MO was found in plants than at pH 6.8 and pH 8.0. The question arose as to whether the quantity of polysaccharide produced by bacteria varied with pH and P. aeruginosa OT15 was grown on SP medium at pH 6.0, 6.8 and 8.0, harvested, and the polysaccharide extracted and weighed. There was little or no difference in the quantity produced; at pH 6.0, 24.8 mg dry weight was produced, at pH 6.8, 24.3 mg and at pH 8.0, 24.6 mg. The possibility that pH could affect the binding ability of the polysaccharide was then investigated. Lyophilized slime (10 mg quantities) from P. aeruginosa was placed in 5 ml volumes of McIlvaine’s buffer at a variety of pH values and MO added to give 100 pg ml-’ of MO final concentration. The mixtures were left overnight at room temperature. Absolute ethanol (5ml) was added to each tube and the polysaccharide precipitated out, harvested and washed twice with 9O’A(v/v) and with acetone. The washed slime was dried and ashed at 500°C overnight and subjected to AA for estimation of MO. The results (an average of three estimations) showed that at pH 5.0, 3430 pg MO g-i dry weight were bound to the slime; at pH 6.0, 3253 pg MO gg’; at pH 7.0, 1134pg Mog-’ and at pH 8.0, 455 pg MO gg’ were bound. MO thus appears to be bound to the extracellular polysaccharide to a greater extent at pH 5 and 6 than at pH 7 or 8. Tan and Loutit (1976) suggested that uranic acids in extracellular polysaccharides are responsible for binding the MO. Extracellular polysaccharides were therefore isolated from a variety of rhizosphere isolates and analysed after silation by g.1.c. (Table 2). It is clear from these results that of the strains tested, the Gram-negative bacteria all contain at least one uranic acid while only one Gram-positive strain contained any uranic acid at all. It could be argued that polysaccharide exudates from the plants and not from bacteria could be responsible for binding MO and experiments were therefore set up to find the origin of polysaccharides in
polysaccharides from chromatography
bacterial
rhizosphere
isolates
analysed
by
gas
Bacterium Compound a D-ghCOSe
D-fructose D-mannose D-glucuronic acid D-galactouronic acid p-mannuronic acid ND = None
detected.
Pseudomonas
Flavobacterium
Achromobacter
Nocardia
Bacillus
Arthrobacter
+
+
+
+
+
+
+
+
+ +
ND ND ND
ND ND ND
+ +
ND
ND
ND +
I& +
ND ND
i’& ND
414 Table
TAN ENG LEE and MARGARET W. LOUTIT 3. Quantity
of mono- and polysaccharide extracted from soil to which bacterial cultures and MO had been Raphams saticus had been grown. MO in pg g-’ dry weight in the plants at the end of the experiment is also given
added and in which
glucose equivalent (fig g- ’ dry wt soil) MonoPolysaccharide saccharide
Treatment Sterile Hastings soil (SH) Sterile Napier soil (SN) Gram-positive cells added to Gram-positive cells added to Gram-negative cells added to Gram-negative cells added to
glucuronic acid equivalent dry wt soil) (pgg-’ MonoPolysaccharide saccharide
Mo pg g-’ No MO added
dry wt MO* added
134.8
20.9
2.4
6.7
2
261
143.1
14.4
1.6
5.9
14
581
48.5
162.0
3.8
56.2
ND
159
39.6
127.0
3.0
28.0
I
497
35.5
398.4
1.5
224.8
ND
53
36.5
140.5
4.1
35.5
ND
304
SH SN SH SN
* MO added to give final conccentration ND = None detected.
of lO~gml_‘.
soil in which plants were grown and to which bacteria had been added. As there had been marked differences in MO concentration in the same variety of plant grown under the same conditions in two similar soils with the same total MO, the following experiments were carried out using these soils (Loutit et al., 1968). Plants were grown in gamma-irradiated soils adjusted to 60% w.h.c. Inocula of mixed Grampositive or Gram-negative cells were added to the soils and the tubes left for 24 h before adding the seedlings. After 4 weeks the plants were gently removed and rhizosphere soil shaken into sterile Petri dishes. From this soil mono-and polysaccharide was extracted and the results expressed as glucose equivalents and the uranic acid estimated and expressed as glucuronic acid equivalents (Table 3). It appears that in the absence of microorganisms monosaccharide is excreted from the plants and appears in the rhizosphere soil. Little uranic acid is associated with this exudate. If bacteria are introduced the amount of monosaccharide that can be extracted diminishes considerably, presumably because the bacteria use the monosaccharides as C sources. On the other hand the amount of polysaccharide exuded by the plants is small by comparison with that that can be extracted from the Hastings soil to which Gram-negative cells have been added and the quantity of uranic acid was also greatest from this soil. DISCUSSION
Previous work both in ai~o (Ludwig et a!., 1962) and in vitro (Loutit et al., 1968; Loutit and Brooks, 1970; Loutit et al., 1972a, b) showed that when the same plants were grown under the same conditions in two soils of similar origin, containing the same total MO but differing in pH value, Gram-negative bacteria predominated in the rhizosphere of plants grown in the soil at pH 6.0 and these bacteria apparently affected the MO concentration in these plants.
In seeking to explain how bacteria might mediate this effect it was postulated that bacteria might concentrate MO in their cells and so prevent entry to the plant. Pure culture experiments showed that MO was taken up mostly by a passive process by extracellular polysaccharide and bound to uranic acid (Tan and Loutit, 1976). The present results indicate that extracellular polysaccharide can aflect the concentration of MO in plants and the polysaccharide produced by the bacteria and not material exuded by the plant is of prime importance in this interaction. The fact that binding of MO to the extracellular polysaccharide is affected by pH is also of considerable interest. It is clear that in addition to chemical and physical factors that affect MO entry to plants, the rhizosphere population may also affect entry, particularly the polysaccharides produced by these populations. It might be argued that any polysaccharide produced will be rapidly broken down by soil organisms and the MO released. There is evidence, however, that when metals are bound to polysaccharides these polysaccharides become resistant to degradation (Martin et al., 1966). Emphasis has been placed on the effect of the polysaccharides at the root surface and while soil bacteria away from the roots are able to bind some MO (Loutit et al., 1967) there is no doubt that the greatest binding occurs at the root surface. Since the greatest numbers of Gram-negative bacteria are usually found at the root surface it is likely to be the site of the greatest binding of MO. Acknowledgement-This a DSIR grant obtained Biochemistry.
work was supported through the Division
in part by of Applied
REFERENCES BITTER T. and MUIR H. M. (1962) A modified uranic carbazole reaction. Analyt. Biochem. 4, 330-334.
acid
Bacterial polysaccharides and molybdenum in plants GOTO S., MURAKAWAT. and KUMHARAS. (1973) Slime production by Pseudomonas aeruginosa. J. Micro&of. Japan 17, 45-51. HOAGLANDD. R. and ARNOND. I. (1938) The water culture method for growing plants without soil. Circ. Univ. Calif. Coil. Agric. 347, pp. 3637. L~~HNIS F. (1920) Landwartschaftfich-bakteriologisches. Praktikum 2 Aufl. Gebriider. Born-traeger. Berlin. LOUTIT M. W. and BROOKSR. R. (1970) Rhizosphere organisms and molybdenum concentration in plants. Soil Biol. Biochem. 2, 131-135. LOUTITM. W., LOUTITJ. S. and BROOKSR. R. (1967) Differences in molybdenum uptake by microorganisms from the rhizosphere of Raphanus satiuus L. grown in two soils of similar origin. PI. Soil 27, 335-346. LOUTITM. W., MALTHUSR. S. and LOUTITJ. S. (1968) The effect of soil microorganisms on the concentration of molybdenum in the radish (Raphanus satiuus L.) variety “White Icicle” N.Z. JI. agric. Res. 11, 420-434. LOUTIT M. W., HILLAS J. and SPEARSG. F. S. (1972a) Studies on rhizosphere organisms and molybdenum concentration in plants. 1. Identification of rhizosphere isolates to generic level. Soil Biol. Biochem. 4, 261-265. LOUTIT M. W., HILLAS J. and SPEARSG. F. S. (1972b) Studies on rhizosphere organisms and molybdenum concentration in plants. 2. Comparison of isolates from the
415
rhizospheres of plants grown in two soils under the same conditions. Soil Biol. Biochem. 4, 267-270. LUDWIGT. G., HEALYW. B. and MALTHUSR. S. (1962) Dental caries prevalence in specific soil areas at Napier and Hastings. Trans. Int. Sot. Soil Sci. Commissions IV and V. 3-11. MARTINJ. P., ERVIN J. 0. and SHEPHERDR. A. (1966) Decomposition of the iron, aluminium, zinc and copper salts or complexes of some microbial and plant polysaccharides in soil. Proc. Soil Sci. Sot. Am. 30, 196-200. MCILVAINET. C. (1921) A buffer solution for calorimetric comparison. J. Biol. Chem. 49, 183-187. TAN E. L. and LOUTIT M. W. (1976) Concentration of molybdenum by extracellular material produced by bacteria. Soil Biol. Biochem. 8, 461464. SCHACTERLE G. R. and POLLACKR. L. (1973) A simplified method for the qualitative assay of small amounts of protein in biological material. Analyt. Biochem. 51, 654-655.
WAGNERG.H.~~~TANGD. S. W. (1976)Soilpolysaccharides synthesized during decomposition of glucose dextran and determined by 14C labelling. Soil Sci. 121, 222-226. WURSTM., VANCURAV. and KALACHOVA L. (1974) Analysis of the polysaccharides of some soil bacteria by gas chromatography. J. Chromat. 91, 469-471.
Press 1977. Printed m Great Britam.
EFFECT OF EXTRACELLULAR POLYSACCHARIDES OF RHIZOSPHERE BACTERIA ON THE CONCENTRATION OF MOLYBDENUM IN PLANTS TAN ENG LEE and MARGARETW. LOUTIT Microbiology Department, University of Otago, Dunedin, New Zealand (Accepted
15 March
1977)
Summary-Extracellular polysaccharides produced by bacteria have been shown to bind molybdenum (MO) with the consequence that less MO entered plants. The polysaccharides are produced by a variety of bacteria but those which contain uranic acids appear to be reponsible for binding the MO. The
amount of MO bound is affected by pH.
INTRODUCTION Tan and Loutit (1976) reported that some rhizosphere bacteria grown in pure culture bound MO in their extra-cellular polysaccharides. Such an interaction between polysaccharides and MO offers a possible explanation of the observation that rhizosphere bacteria prevent entry of MO to plants under certain conditions (Loutit et al., 1968; Loutit and Brooks, 1970). This paper reports results of experiments designed to investigate this suggestion.
cells washed twice in HA. The Gram-negative isolates (Pseudomonas, Achromobacter and Flavobacterium) were pooled as were the Gram-positive isolates (Nocardia, Arthrobacter and Bacillus). Each mixture was suspended in HA to give a suspension of lo6 mixed cells ml- l. Estimation of bacteria around plant roots The method et al., 1968).
previously
described
was used (Loutit
Preparation of extra-cellular polysaccharide for use in pot experiments
MATERIALS AND METHODS All glassware and Ballotini spheres were washed in aqua regia and rinsed to neutral pH, in doubly distilled water. Autoclaving was at 121°C for 15-20 min. Bacteria Strains previously described (Tan and Loutit, 1976) were used and in addition Pseudomonas aeruginosa OT 15 was obtained from Professor J. S. Loutit of this department. Media and solutions Soil extract, yeast extract, liquid medium @EYE); soil extract (Liihnis, 1920) lOOOm1, yeast extract (Difco) 1 g. Brain heart infusion (BHI) (BBL). Slimepromoting medium (SP) of Goto et al. (1973) solidified with 1.5% agar (Davis). Hoagland and Arnon solution pH 6.8 (HA) (Hoagland and Arnon, 1938) sometimes supplemented with 10 g glucose (BDH) and 1 g yeast extract (Difco) per litre (HAS). Molybdenum solution; a stock solution of Na,MoO,. 2H20 (BDH) Analar grade containing 1 mg MO ml-‘. McIlvaine’s buffer (McIlvaine, 1921). Preparation of inocula Strains were grown in SEYE for 40 h at 28°C except for Pseudomonas which was grown in BHI at 28°C overnight. Approximately 10’ cells ml-’ were inoculated into lOOm1 HAS and incubated at 28°C for 48 h in a shaking water bath. After incubation the flask contents were centrifuged and the deposited 411
Volumes (2.0ml) of P. aeruginosa OT 15 incubated in BHI overnight were spread over the surface of 250 ml solidified SP medium in polypropylene instrument trays (140 x 160mm) with glass spreaders. The trays were incubated for 5 days at 28°C. Cells and slime were scraped off with a glass slide and suspended in 50 ml sterile saline. The cells and slime were mixed vigorously in a blender for 30 s and cells deposited by centrifugation at 1OOOg for 20 min at 4°C. Slime was precipitated from the supernatant by the addition of an equal volume of ethanol and left for 12 h at 4°C. The supernatant was decanted and the remaining slime redissolved in a minimum quantity of doubly-distilled water. The dissolved slime was dialysed against 2 1 doubly-distilled water at 4°C for 24 h with continuous stirring. The dialysed slime was precipitated with an equal volume of ethanol and the slime removed with a glass rod. After washing with 90% ethanol (v/v) and acetone, the slime was dried over PzO, in mcuo, weighed and stored in a desiccator over P*O,. Plant experiments Ballotini spheres (35 g Jencons No. 19 40 pm dia) were placed in test tubes (32 x 200mm) covered with Peep see-through cooking film (Seto Bags Ltd., Wellington, N.Z.) and autoclaved. HA solution pH 6.0 was added to bring the beads to 60% of their water holding capacity (w.h.c.). MO was incorporated into some tubes by adding MO from the stock solution to HA to give a final concentration of 10 fig MO ml-‘. Where extracted slime was to be added, 300 mg dried
112
TAN ENG L
EE and MARGARETW. LOUTIT
preparation was dissolved in lOOmI of the HA solution before use. In experiments requiring addition of a mixed inoculum of Gram-negative or Gram-positive bacteria, washed pooled cells were suspended in the HA solution to give a final concentration of 10e6 mixed cells ml-’ and the HA used to bring the spheres to 609, w.h.c. In some experiments sterile soil was used in place of spheres. The methods for soil sterilization, seed surface sterilization, germination, planting and the conditions for growth have been described (Loutit et al., 1968, Loutit and Brooks, 1979; Loutit et al. 1972). Atomic
&sorption
spectrophotometry
(AA)
The procedure for estimation of MO in plant material and slime was as described for bacterial cells (Tan and Loutit, 1976). Extraction
qf mono-
and polysaccharide
from
soil
The method was a modification of that of Wagner and Tang (1975). After 4 weeks the plants were removed from the tubes and the bulk of the soil removed from the roots. The plants with adhering soil were then shaken vigorously and the soil freed and collected into sterile containers. Quantities (10 g) were placed in 250ml Erlenmeyer flasks. Further quantities were placed in weighed glass Petri dishes and heated at 80°C for 16 h and reweighed to allow calculation of moisture content of the soil. To each flask was added 50ml 707: (v/v) ethanol. The flask was covered and placed in a shaking water bath at 45°C for 4 h. The suspension was filtered through a sintered glass filter (20-30 pm). The deposit on the filter was retained for estimation of polysaccharide and monosaccharide estimation was carried out on the filtrate. The filtrate was dried under vacuum, lOm1 doubly-distilled water added, and the mixture shaken at 20°C for 1 h. Total monosaccharide was determined as glucose equivalents by using anthrone reagent and the results expressed as pg glucose g -i dry weight of soil. Total uranic acid was determined as glucuronic acid equivalents using carbazole (Bitter and Muir, 1962). To the deposit on each filter 50ml N H-SO4 was added, the mixture poured into a 250ml flask and the whole shaken at 20°C for 16 h. The suspensions were filtered through sintered glass filters (20-30pm) and 50ml doubly-distilled water used to wash the residue. The total filtrate was then concentrated in vacua at 50°C. To the dried extract was added 20ml doubly-distilled water and the solution centrifuged at 48,250 g in a Sorvall RC2B for 10 min. The precipitate was discarded and the supernatant dialysed against 2 1 doubly-distilled water for 16 h. The solution in the dialysis sac was then lyophilized and the dried polysaccharide hydrolysed in 2 ml 0.5 N H,S04 in a sealed ampoule at 100°C for 16 h Total carbohydrate and uranic acid estimation were then carried out on the hydrolysate. Analysis of extracellular rhizosphere bacteria
polysaccharides
produced
by
Each bacterial strain was grown in triplicate in 250 ml Basks containing 100 ml Hoagland and Arnon solution containing glucose and yeast extract (HAS).
After 5 days in a shaking water bath at 28 c‘ the cultures were centrifuged at 32009 for 20min. The cells were resuspended in 50ml sterile saline and extracellular slime was treated as described for Pseudomonas slime. In addition the slime was recovered from the culture solution by first concentrating the sohition to IO’/” of its original volume irz racuo. To this concentrate was added an equal volume of absolute ethanol. The slime that precipitated out was pooled together with that obtained from the cells. Molecules with a molecular weight of less than SOOOdaltons were removed by dialysis against six changes of 2-l volumes doubly-distilled water. Proteins in excess of this size were removed by precipitation with CHCl, by adding 0.25 volume CHCI, and 0.1 volume butyl alcohol to 1 volume of slime in 1 volume doubly-distilled water. This mixture was shaken and centrifuged at 5000 g for 20 min by which time two layers had formed, a lower stable proteinCHCl, gel which was discarded and an upper aqueous layer containing polysaccharide which was decanted. Excess CHCI, collected at the bottom of the centrifuge tube. Polysaccharide was precipitated by adding acetone in the ratio of four volumes acetone to one aqueous polysaccharide solution and the mixture left overnight at 4’C. The precipitate was redissolved in a minimal quantity of doubly-distilled water. One ml of this solution was tested (Schacterle and Pollack, 1973) to ensure no protein was present. The remaining solution was then lyophilized. The purified polysaccharide was hydrolysed by dissolving 20 mg in 2.0 ml 0.5 N H2S0, and placing the mixture in an ampoule, sealing and leaving the whole at 1OO’Cfor 16 h. The hydrolysate was neutralized with excess CaCO,. CaS04 formed was removed by centrifugation and the neutralized hydrolysate lyophilized. From the lyophilized hydrolysate trimethylsiiate (TMS) derivatives were prepared by a modified method (Wurst et al., 1974). Quantities of the hydrolysate (10mg) were dissolved in 1 ml anhydrous pyridine and left at 37°C for 4 h. When all of the lyophilized hydrolysate had dissolved 0.1 ml hexamethyl disilizone and 0.5 ml methylchlorosilane was added. The precipitate formed was removed by centrifugation at 10,000 g for 20 min and the supernatant transferred to a clean dry screw cap tube (10 x 127 mm). TMS derivatives were analysed by g.1.c. in a PYE series 104 chromatograph using a 2 m column (3 mm dia) packed with 21 g Chromosorb Q 100/120 mesh impregnated with 3”/1JXR (Applied Science Lab. Inc.). The TMS derivatives of the monosaccharides were separated isothermally at 185°C with the temperature of the detector and sample injector at 24’C. Samples (1 ~1) were injected with 1 ~1 syringe (Hamilton, New York, U.S.A.). The carrier gas was N, at a flow rate of 20 ml mini’ and the flow rates of H, and air in the flame ionization detector were 40 and 4OOml respectively. A Phillips 8000 electronic recorder was used to record emission peaks. Identification of peaks was made by comparing retention time of TMS derivatives of pure monosaccharides with those from the hydrolysed extracellular polysaccharide. Standards were o-glucose (Mallinckrodt), D-glucuronic acid, D-fructose, D-mannuronic, D-lactose and D-galacturonic (Sigma). Six estimations were made on each hydrolysed sample.
Bacterial
polysaccharides
and molybdenum
Table 1. Effect of bacteria and extracted extracellular bacterial polysaccharide on MO concentration (ngg-’ dry weight) in Raphanus satiuus L. White Icicle grown under controlled conditions in Ballotini spheres and Hoagland and Arnon solution with and without MO and at different pH values* MO ng g- 1 dry weight pH 6.0 pH 8.0 pH 6.8
Treatment No MO MO Polysaccharide MO plus polysaccharide Gram-positive cells Gram-positive cells plus MO Gram-negative Gram-negative plus MO
cells cells
8.2 582.9 2.2
5.2 351.2 2.6
3.5 337.9 2.4
222.7 5.4 403.4
255.1 3.0 276.3
243.9 2.8 238.8
1.2 113.8
1.7 167.5
1.5 236.1
* HA solution added to all tubes. MO add where appropriate to give a final concentration of 10~gmll’.
RESULTS
Although extracellular bacteria1 polysaccharides have been shown to complex with MO in pure culture their effect around the roots of plants on MO concentration in the plants has not been established. A series of experiments was therefore undertaken to investigate this effect. Radish plants were grown under controlled conditions in Ballotini spheres to which HA solution had been added. In some treatments MO was incorporated in the HA solution as was either extracted bacterial extracellular polysaccharide or an inoculum of bacteria1 cells. The tubes containing the spheres and the various additives were held in the climate cabinet for 24 h before adding the germinated seeds. After 4 weeks the plants were harvested, the MO in the tops estimated and the number of bacteria in the rhizosphere estimated. Gram-positive cells were estimated at lo5 g-’ dry weight and Gram-negative cells at lo6 g-l. The results (Table 1) indicate that addition of Gram-negative and Gram-positive cells resulted in a lower concentration of MO in plants and the effect of the Gram-negative cells was greater. Extracted polysaccharide also resulted in a marked reduction in the MO concentration in the plants. In addition to studying the effect of bacteria and polysaccharide Table
2. Composition
of
extracellular
413
in plants
on MO concentration, the effect of pH was also studied. The results (Table 1) indicated that at pH 6.0, MO concentration in the plants was greatest and least at pH 8.0. The addition of bacteria and polysaccharide at different pH values showed that the effect of Gram-negative bacteria was greatest at pH 6.0 and Gram-positive cells had the least effect on MO concentration in plants at pH 6.0. pH did not greatly influence the effect of the polysaccharide although at pH 6.0 a slightly lower concentration of MO was found in plants than at pH 6.8 and pH 8.0. The question arose as to whether the quantity of polysaccharide produced by bacteria varied with pH and P. aeruginosa OT15 was grown on SP medium at pH 6.0, 6.8 and 8.0, harvested, and the polysaccharide extracted and weighed. There was little or no difference in the quantity produced; at pH 6.0, 24.8 mg dry weight was produced, at pH 6.8, 24.3 mg and at pH 8.0, 24.6 mg. The possibility that pH could affect the binding ability of the polysaccharide was then investigated. Lyophilized slime (10 mg quantities) from P. aeruginosa was placed in 5 ml volumes of McIlvaine’s buffer at a variety of pH values and MO added to give 100 pg ml-’ of MO final concentration. The mixtures were left overnight at room temperature. Absolute ethanol (5ml) was added to each tube and the polysaccharide precipitated out, harvested and washed twice with 9O’A(v/v) and with acetone. The washed slime was dried and ashed at 500°C overnight and subjected to AA for estimation of MO. The results (an average of three estimations) showed that at pH 5.0, 3430 pg MO g-i dry weight were bound to the slime; at pH 6.0, 3253 pg MO gg’; at pH 7.0, 1134pg Mog-’ and at pH 8.0, 455 pg MO gg’ were bound. MO thus appears to be bound to the extracellular polysaccharide to a greater extent at pH 5 and 6 than at pH 7 or 8. Tan and Loutit (1976) suggested that uranic acids in extracellular polysaccharides are responsible for binding the MO. Extracellular polysaccharides were therefore isolated from a variety of rhizosphere isolates and analysed after silation by g.1.c. (Table 2). It is clear from these results that of the strains tested, the Gram-negative bacteria all contain at least one uranic acid while only one Gram-positive strain contained any uranic acid at all. It could be argued that polysaccharide exudates from the plants and not from bacteria could be responsible for binding MO and experiments were therefore set up to find the origin of polysaccharides in
polysaccharides from chromatography
bacterial
rhizosphere
isolates
analysed
by
gas
Bacterium Compound a D-ghCOSe
D-fructose D-mannose D-glucuronic acid D-galactouronic acid p-mannuronic acid ND = None
detected.
Pseudomonas
Flavobacterium
Achromobacter
Nocardia
Bacillus
Arthrobacter
+
+
+
+
+
+
+
+
+ +
ND ND ND
ND ND ND
+ +
ND
ND
ND +
I& +
ND ND
i’& ND
414 Table
TAN ENG LEE and MARGARET W. LOUTIT 3. Quantity
of mono- and polysaccharide extracted from soil to which bacterial cultures and MO had been Raphams saticus had been grown. MO in pg g-’ dry weight in the plants at the end of the experiment is also given
added and in which
glucose equivalent (fig g- ’ dry wt soil) MonoPolysaccharide saccharide
Treatment Sterile Hastings soil (SH) Sterile Napier soil (SN) Gram-positive cells added to Gram-positive cells added to Gram-negative cells added to Gram-negative cells added to
glucuronic acid equivalent dry wt soil) (pgg-’ MonoPolysaccharide saccharide
Mo pg g-’ No MO added
dry wt MO* added
134.8
20.9
2.4
6.7
2
261
143.1
14.4
1.6
5.9
14
581
48.5
162.0
3.8
56.2
ND
159
39.6
127.0
3.0
28.0
I
497
35.5
398.4
1.5
224.8
ND
53
36.5
140.5
4.1
35.5
ND
304
SH SN SH SN
* MO added to give final conccentration ND = None detected.
of lO~gml_‘.
soil in which plants were grown and to which bacteria had been added. As there had been marked differences in MO concentration in the same variety of plant grown under the same conditions in two similar soils with the same total MO, the following experiments were carried out using these soils (Loutit et al., 1968). Plants were grown in gamma-irradiated soils adjusted to 60% w.h.c. Inocula of mixed Grampositive or Gram-negative cells were added to the soils and the tubes left for 24 h before adding the seedlings. After 4 weeks the plants were gently removed and rhizosphere soil shaken into sterile Petri dishes. From this soil mono-and polysaccharide was extracted and the results expressed as glucose equivalents and the uranic acid estimated and expressed as glucuronic acid equivalents (Table 3). It appears that in the absence of microorganisms monosaccharide is excreted from the plants and appears in the rhizosphere soil. Little uranic acid is associated with this exudate. If bacteria are introduced the amount of monosaccharide that can be extracted diminishes considerably, presumably because the bacteria use the monosaccharides as C sources. On the other hand the amount of polysaccharide exuded by the plants is small by comparison with that that can be extracted from the Hastings soil to which Gram-negative cells have been added and the quantity of uranic acid was also greatest from this soil. DISCUSSION
Previous work both in ai~o (Ludwig et a!., 1962) and in vitro (Loutit et al., 1968; Loutit and Brooks, 1970; Loutit et al., 1972a, b) showed that when the same plants were grown under the same conditions in two soils of similar origin, containing the same total MO but differing in pH value, Gram-negative bacteria predominated in the rhizosphere of plants grown in the soil at pH 6.0 and these bacteria apparently affected the MO concentration in these plants.
In seeking to explain how bacteria might mediate this effect it was postulated that bacteria might concentrate MO in their cells and so prevent entry to the plant. Pure culture experiments showed that MO was taken up mostly by a passive process by extracellular polysaccharide and bound to uranic acid (Tan and Loutit, 1976). The present results indicate that extracellular polysaccharide can aflect the concentration of MO in plants and the polysaccharide produced by the bacteria and not material exuded by the plant is of prime importance in this interaction. The fact that binding of MO to the extracellular polysaccharide is affected by pH is also of considerable interest. It is clear that in addition to chemical and physical factors that affect MO entry to plants, the rhizosphere population may also affect entry, particularly the polysaccharides produced by these populations. It might be argued that any polysaccharide produced will be rapidly broken down by soil organisms and the MO released. There is evidence, however, that when metals are bound to polysaccharides these polysaccharides become resistant to degradation (Martin et al., 1966). Emphasis has been placed on the effect of the polysaccharides at the root surface and while soil bacteria away from the roots are able to bind some MO (Loutit et al., 1967) there is no doubt that the greatest binding occurs at the root surface. Since the greatest numbers of Gram-negative bacteria are usually found at the root surface it is likely to be the site of the greatest binding of MO. Acknowledgement-This a DSIR grant obtained Biochemistry.
work was supported through the Division
in part by of Applied
REFERENCES BITTER T. and MUIR H. M. (1962) A modified uranic carbazole reaction. Analyt. Biochem. 4, 330-334.
acid
Bacterial polysaccharides and molybdenum in plants GOTO S., MURAKAWAT. and KUMHARAS. (1973) Slime production by Pseudomonas aeruginosa. J. Micro&of. Japan 17, 45-51. HOAGLANDD. R. and ARNOND. I. (1938) The water culture method for growing plants without soil. Circ. Univ. Calif. Coil. Agric. 347, pp. 3637. L~~HNIS F. (1920) Landwartschaftfich-bakteriologisches. Praktikum 2 Aufl. Gebriider. Born-traeger. Berlin. LOUTIT M. W. and BROOKSR. R. (1970) Rhizosphere organisms and molybdenum concentration in plants. Soil Biol. Biochem. 2, 131-135. LOUTITM. W., LOUTITJ. S. and BROOKSR. R. (1967) Differences in molybdenum uptake by microorganisms from the rhizosphere of Raphanus satiuus L. grown in two soils of similar origin. PI. Soil 27, 335-346. LOUTITM. W., MALTHUSR. S. and LOUTITJ. S. (1968) The effect of soil microorganisms on the concentration of molybdenum in the radish (Raphanus satiuus L.) variety “White Icicle” N.Z. JI. agric. Res. 11, 420-434. LOUTIT M. W., HILLAS J. and SPEARSG. F. S. (1972a) Studies on rhizosphere organisms and molybdenum concentration in plants. 1. Identification of rhizosphere isolates to generic level. Soil Biol. Biochem. 4, 261-265. LOUTIT M. W., HILLAS J. and SPEARSG. F. S. (1972b) Studies on rhizosphere organisms and molybdenum concentration in plants. 2. Comparison of isolates from the
415
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WAGNERG.H.~~~TANGD. S. W. (1976)Soilpolysaccharides synthesized during decomposition of glucose dextran and determined by 14C labelling. Soil Sci. 121, 222-226. WURSTM., VANCURAV. and KALACHOVA L. (1974) Analysis of the polysaccharides of some soil bacteria by gas chromatography. J. Chromat. 91, 469-471.