different sites in 24 counties of Ohio in 1992 and 1993. Both years, nodulated roots were taken from three locations at each field site. The number of roots taken ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1994, p. 2939-2943
Vol. 60, No. 8
0099-2240/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Factors Influencing the Synthesis of Polysaccharide by Bradyrhizobium japonicum Bacteroids in Field-Grown Soybean Nodules JOHN G. STREETER,* SEPPO 0. SALMINEN, JAMES E. BEUERLEIN, AND WALTER H. SCHMIDT Department of Agronomy, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691 Received 11 March 1994/Accepted 8 June 1994
Certain strains of Bradyrhizobiumjaponicum produce large quantities of polysaccharide in soybean (Glycine max (L.) Merr.) nodules, and nodule polysaccharide (NPS) is different from that produced in culture. A previous survey of field-grown plants showed highly variable levels of NPS among field sites. To obtain clues about the possible function of NPS, we conducted two additional surveys of field-grown plants. The amount of polysaccharide in bulk samples of nodules was not associated with soil type, texture, slope, drainage, or any of the measured soil chemical properties except pH and [Ca]. Correlations with pH and [Ca] were positive and highly significant for two independent surveys involving a total of 77 sites in two years. In a preliminary comparison of high and low levels of Ca supplied to soybean plants grown in silica sand in a greenhouse, a high level of Ca (200 mg of Ca liter') increased the NPS level and increased the Ca content of the polysaccharide fraction. B. japonicum isolates from 450 nodules collected at 10 field sites in 1993 were used to form nodules on soybean plants grown in sand culture in a greenhouse in order to examine bacterial phenotype under controlled conditions. Results showed that the NPS level in the bulk nodule sample from any given site was a function of the proportion of nodule occupants that were capable of NPS synthesis. Thus, a higher soil pH and/or [Ca] may positively influence the survival of B. japonicum capable of synthesis of the nodule-specific polysaccharide.
Certain strains of Bradyrhizobium japonicum and B. elkanii produce large quantities of polysaccharide in soybean (Glycine max (L.) Merr.) nodules (13, 15). These two bacterial species produce different nodule polysaccharides (NPS), and type I NPS (B. japonicum) and type II NPS (B. elkanii) can be distinguished in the laboratory on the basis of their reactivities to standard uronic acid analysis (13). In samples of soybean nodules from Ohio fields and other sites in the Midwest, the B. elkanii type of NPS is rarely found (13). In this report, only the B. japonicum type of NPS is considered. B. japonicum NPS is different in composition from the extracellular polysaccharide produced by B. japonicum in culture (15). This suggests that NPS may have some useful function in the symbiotic nodule where NPS deposition in the symbiosome space is so pronounced that it is the main feature in micrographs of infected cells (14, 15). As one approach to understanding the function of NPS, we studied the conditions which favor the deposition of NPS in field-grown plants. In the initial survey, NPS was found in all samples, but there was a 20-fold difference in the amount of NPS in nodule samples from different field sites (13). It is important to emphasize that this result was obtained with 4- to 6-g samples of nodules; thus, we could not discern whether the large variation among samples was due to different proportions of individual NPS+ and NPS- nodules or to a uniform level of NPS in all nodules in a sample coupled with a large variation among samples. In the initial field survey, our notes on site characteristics and plant growth stage did not provide any clues to the reasons
for the large variation in NPS levels of bulk samples of soybean nodules. We report here the results of two additional field surveys which included more thorough characterization of field sites. We also report the results of analysis of the NPS phenotypes of B. japonicum occupants of 450 individual nodules from 10 diverse field sites and the results of a preliminary experiment in which the Ca level in the nutrient solution supplied to greenhouse-grown plants was employed to alter NPS levels in nodules. MATERUILS AND METHODS Collection of samples in the field. This report involved a total of 77 samples of soybean nodules obtained from 77 different sites in 24 counties of Ohio in 1992 and 1993. Both years, nodulated roots were taken from three locations at each field site. The number of roots taken varied, depending on nodulation, but was generally 20 to 30 roots per field. Roots were dug with a shovel, so nodule samples represent mainly tap root nodules. A single soil sample 16 to 20 cm deep was taken with a bucket auger type of soil probe at each of the three locations in the field. Notes were taken at each site on the exact location of the field, soil type, slope, drainage, previous crop (based on residue), plant growth stage (number of nodes and reproductive stage), and soil conditions at the time of sampling (mainly wetness). An attempt was made to sample approximately 70 days after planting when plants were in mid- to late bloom. Thus, both years, sampling was done in mid- to late July and there was little difference in plant growth stage among sites or between years. Shoots were cut off in the field and discarded. Soil and root samples were placed in plastic bags for transport and storage. Nodulated roots were stored at 2°C until they could be analyzed, usually less than 1 week. Soil samples were air dried
* Corresponding author. Mailing address: Department of Agronomy, The Ohio State University, Ohio Agricultural Research and Development Center, 1680 Madison Ave., Wooster, OH 44691-4096. Phone: (216) 263-3883. Fax: (216) 263-3658.
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STREETER ET AL.
on benches in the greenhouse and then ground and sieved to remove coarse debris before analysis (1). Analysis of NPS. Soil was washed from nodulated roots with tap water. Nodule samples consisted of both tap and lateral root nodules obtained from all or nearly all of the roots collected at a field site; sample size was between 5 and 9 g
(fresh weight) and consisted of several hundred individual nodules. The methods used for analysis of NPS levels have been described previously (15). Briefly, nodules were extracted in ethanol-water to give a final ethanol concentration of 36% (vol/vol). Debris was removed by centrifugation at 48,000 x g, and the combined supernatants were made up to a precise volume equal to five times the initial fresh weight. The viscosity of this extract was determined by placing the extract in a glass tube 1.2 m in length and timing the passage of a stainless steel ball through the extract (15). Following viscosity analysis, the extract was returned to a small flask and some nodule protein was precipitated by the addition of absolute ethanol to give a concentration of 50% ethanol (vol/vol). Protein was removed by centrifugation, and the NPS sample was precipitated by bringing the ethanol concentration of the supernatant up to 87% (vol/vol) in the cold. Precipitate was collected by centrifugation and freezedrying. Although this ethanol-insoluble precipitate contains some plant proteins and polysaccharides, it consists mainly of NPS (15) and is hereafter referred to as the NPS fraction for convenience. Weighed samples of the dried NPS fraction were dissolved in water and analyzed for glucuronic acid concentration (2). B. japonicum NPS contains 2-O-methylglucuronic acid, so the method is sensitive to NPS but insensitive to many other polysaccharides. Although low viscosity is most diagnostic for the absence of NPS, glucuronic acid analysis of the NPS fraction provides the most accurate quantitative estimate of the NPS level (13, 14). The accuracy of this variable can be improved even more by subtracting the background uronic acid levels present in NPS- samples (see reference 14 for examples); this background uronic acid level of about 400 ,ug g-' (fresh weight) of nodule was subtracted for the analyses shown in Tables 2 and 3. Analysis of NPS formation by individual rhizobial isolates. Samples obtained in 1993 were first analyzed as described above, i.e., viscosity analysis of a bulk sample of nodules and glucuronic acid analysis of the NPS fraction. Data were arranged in rank order by viscosity of the bulk nodule sample, and mean and median values were determined. We then selected 10 sites, 5 below the median and 5 above the median, for analysis of the NPS phenotype of the bacterial residents in individual nodules. The sites selected represented the highest and lowest NPS levels available and eight sites with intermediate NPS levels. Forty-five nodules, including at least one nodule from every root in the sample, were pulled from tap roots from each of the 10 selected sites. Nodules were surface sterilized by gently swirling in 95% ethanol in a 150-ml beaker for 10 s, placing nodules in a solution of 5% H202, swirling gently for a few seconds, and then allowing the mixture to stand at room temperature for 5 min. Nodules were then thoroughly washed in deionized water, and individual nodules were placed in disposable test tubes. By using a sterile glass rod, each nodule was crushed in 3 ml of 0.9% NaCl containing 0.01% Triton X-100. Nodule homogenate was further diluted with 9 ml of the NaCl-Triton X-100 solution and mixed once on a vortex mixer. Three seeds of Glycine max (L.) Merr. cv. Ripley were planted in approximately 1 dm3 of sterile silica sand in a paper
APPL. ENVIRON. MICROBIOL.
milk shake cup. Holes were punched in the bottom of each cup to provide drainage, and sand was washed with tap water prior to placement of the seeds about 2 cm below the sand surface. Nodule homogenate from a single nodule was poured over the three seeds, and the seeds were covered with moist sand. Following emergence, seedlings were thinned to one per pot; thinning allowed selection of a healthy plant and permitted confirmation of nodule formation. Analysis of the NPS phenotypes of 45 nodules from 10 field sites gave a total of 450 samples. Plants were irrigated daily with tap water except for two days per week when a nutrient solution lacking combined nitrogen was provided (14). Nodules on test plants were evaluated 70 days after planting for the presence of NPS by a microviscosity assay as follows. A sample of tap root nodules weighing 150 mg was homogenized in 1.0 ml of 36% ethanol with a 15-ml Potter Elvehjem glass homogenizer. Homogenate was transferred to a 1.7-ml microcentrifuge tube. The glass grinder was rinsed with 0.5 ml of 36% ethanol, and the rinse was added to the microcentrifuge tube. Microcentrifuge tubes were centrifuged at 7,000 x g at room temperature for 1 min. Supernatant from each sample was placed in a 1.0-ml glass syringe attached to an 18-gauge needle 3.8 cm long. Extracts from NPS- nodules flow rapidly through a needle of this size, requiring 3 s or less for the syringe barrel to empty. Extracts from NPS+ nodules flow slowly through the needle; transit time varies, depending on the concentration of polysaccharide, but is generally from 8 to 15 s. Thus, NPS+ and NPS- extracts are almost always clearly delineated, revealing the NPS phenotype of the bacteria in the nodule used as the inoculant. Effects of calcium concentration in nutrient solution. Soybean plants were grown in sand culture as described above except that ceramic pots containing about 6 kg of sand and 10 plants per pot were used. There were four replicate pots for each treatment. The stock nutrient solution contained the following (in mg liter-1): K, 25; P, 10; Mg, 10; Fe, 2; and micronutrients (14). pH was maintained at about 7.5 by the inclusion of 5 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] buffer (9, 12). For treatments with low and high [Ca], Ca was supplied as CaCl2 at 1.0 and 200 mg of Ca liter-1, respectively. Nutrient solution was supplied daily for 5 days, and water was supplied on weekends. Plants were grown in a greenhouse with supplementary light provided by metal halide lamps at about 800 microeinsteins m-2 s-1 at plant level. Nodules were formed by B. japonicum USDA 438, an NPS+ strain (15). There was a single harvest 64 days after planting. Three nodulated roots were assayed for acetylene reduction activity on the basis of four gas samples taken over a 12-min period (14); ethylene formation was linear over time. Nodules were removed from these roots, and fresh weight was determined. Nodules from the other seven plants in each pot were pulled, extracted, and analyzed for NPS concentration as described above. Following uronic acid analysis of the crude NPS fraction, the mineral content of the NPS solution was determined by emission spectroscopy. Shoots and roots were heat dried and weighed, and tissue was ground, dry ashed, and analyzed for mineral content by emission spectroscopy (10). RESULTS In the 1992 survey, 57 samples of nodules were obtained from field-grown soybean plants in 24 counties of Ohio. In 1992, soil type was the only sampling criterion, and emphasis was placed on sampling a wide variety of soils. County soil survey maps were used to identify soil boundaries. In the 1993
BACTERIAL POLYSACCHARIDE IN SOYBEAN NODULES
VOL. 60, 1994
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TABLE 1. Diversity of NPS levels and soil properties related to NPS levels in two surveys of field-grown plants NPSa
Yro.ofsamples)
(no. of samples)
1992 (57) 1993 (20)
(Viscosity
(centipoise)
Soil properties Uronic acid concn g' [fresh wt] of nodule)
(p.g
pHb
Ca
Range
Mean
Range
2,209
500-6,380
2,395
900-3,800
65 56
3-155 23-102
160 132
51-494 52-297
Range
Mean
1,945
416-4,870
2,264
401-4,860
6.9 6.8
4.9-8.1 5.3-7.7
Mean
23.2 21.7
0.8-227 1-113
Fec (g kg-')
(g kg-')
Mean
Mean
Range
Mn
Range
Range
Mean
(g kg-')
a These values are for bulk samples of from 4 to 8 g of nodules; each uronic acid concentration was determined with a crude fraction of NPS (see text). b pH, water pH. c Fe, DTPA-extractable iron.
survey, a smaller number of samples (20) was obtained from a smaller area (seven counties). Fourteen of the twenty samples were paired samples, i.e., they were from nearby soybean fields, often located across a road from each other. For each pair, the soil type was the same, although soil chemical properties were different. The objective of the 1992 survey of field plants was to sample as wide a variety of soils as possible, and the diversity of the soils sampled is illustrated in Table 1. Not shown in the table are the wide differences in soil texture from sands to clay soils, differences in organic matter from 0.7 to 23%, differences in phosphorus from 1 to 429 g kg-', and differences in cationexchange capacity from 0.06 to 0.50 meq g- The smaller region sampled in 1993 gave us less diversity in soil properties. Analysis of the viscosity of bulk nodule samples and uronic acid concentration of the NPS fraction of nodules also showed a very large range of NPS levels in our field samples (Table 1). NPS was present in all samples for both years, but three samples in 1992 and one sample in 1993 had very low viscosity. In the 1992 survey of 57 diverse field sites, NPS levels in nodule samples were established on the basis of the analysis of bulk nodule samples. Data were then arranged in rank order and compared with the information we had gathered on site characteristics. There was no correspondence between the NPS level in nodules and either the slope, drainage, soil type, previous crop, region of the state, or stage of plant development. In the 1993 survey, field sites which were within 100 m but under different management gave nodule samples which often had very different levels of NPS (data not shown). The combined results of these surveys indicate that the NPS level was not related to soil type or physical properties. On the basis of 1992 observations of very dry conditions at some field sites, we thought that the NPS level might be related to water stress. We conducted a small greenhouse trial in which soybeans inoculated with an NPS+ strain (USDA 438) and grown in sand culture were subjected to daily water stress for 0, 3, or 5 weeks prior to harvest at 65 days after planting. There was no relationship between stress level and NPS concentration in nodules (data not shown). Initial soil analyses in the 1992 field survey included P, K, Ca, and Mg contents; pH; cation-exchange capacity; and percent base saturation by Ca, Mg, and K. When correlations between the NPS level and either pH or Ca content were discovered, soils were also analyzed for Mn, Fe, and organic matter levels. Only those variables that correlated significantly with the NPS level are shown in Table 2. Correlations between NPS levels in nodules and soil Mn and Fe levels in 1992 may be misleading for several reasons. First, pH significantly correlated with soil Fe levels in the 1992 samples (data not shown). Second, numerous studies by others have established that soil Mn and Fe levels are likely to vary with soil pH. Third, a .
relationship between NPS levels in nodules and soil Mn or Fe content was not observed with the 1993 samples. Multiple correlation-regression analyses of the 1992 data did not indicate any statistically significant relationships beyond those shown in Table 2. Correlations between the NPS level and either pH or Ca concentration may also be confounded because pH and Ca concentration significantly correlated with each other in both surveys (data not shown). Thus, from the correlation coefficients alone, one cannot determine which of the two variables might be the more important one in influencing NPS deposition in nodules. Because uronic acid-containing polysaccharides are known to bind large amounts of Ca in plant cell walls and for other reasons discussed later, a preliminary investigation of the effect of the Ca supply on NPS accumulation was conducted. The effectiveness of Ca treatments was monitored by analysis of effluent from pots, and these analyses showed about 175 mg of Ca liter-1 for high-Ca treatment and about 5 mg of Ca liter-1 for low-Ca treatment. Plant growth of high-Ca plants was about double that of low-Ca plants, and the [Ca] in shoots was elevated about fourfold by high levels of Ca (Table 3). Nodule mass per plant was also higher with high levels of Ca, but nitrogenase activity per unit of nodule mass was not significantly influenced. Most importantly, there was an increase in NPS levels for nodules on plants supplied with high levels of Ca, and the NPS fractions in these nodules also contained much more Ca. Mg, K, and Na were the major cations associated with the NPS fraction when the Ca supply was low (Table 3). Although the [Ca] in the NPS fraction was higher in the greenhouse experiment, mineral analysis of crude NPS fractions from 57 samples in the 1992 field survey did not reveal a correlation between NPS [Ca] and NPS concentration in nodules. Analysis of the NPS phenotypes of 450 nodules from 10 field sites in 1993 showed that the NPS level at a given site was a function of the proportion of nodule occupants capable of NPS
TABLE 2. Correlations between soil properties and NPS levels in field-grown plantsa Soil variable
Yr of samples pH
[Ca]
1992
0.415b
1993
0.673b
0.502b 0.437c
[Mn] 0.403b NSd
[Fe] - 0.347b
NSd
a The variable used for the calculation of coefficients was the net uronic acid concentration in the crude NPS fraction. b Significant at a level of confidence of 1%. c Significant at a level of confidence of 5%. d NS, not significant.
2942
STREETER ET AL.
o
APPL. ENvIRON. MICROBIOL.
riL 0fX +1 +1-
z
0~~~~~~~~~ Ut4+1 +1 z~~~~~~~
+1 +1 0E 3 o-4 3 __ +1 +1
u
100
lU
0
8
0
(a
0.40 0.96 1.24 1.36 2.07 2.32 2.83 3.82 3.89 4.86
acid concentration (mglg fresh wt nodule) ~~~~~~~~~~~Uronic FIG. 1. Relationship between the level of nodule-specific polysaccharide (as represented by the uronic acid concentration in a bulk
of nodules) and the proportion of bacterial nodule occupants _ o S osample which were polysaccharide formers. Data represent the analysis of 450 bacterial isolates from 10 field sites. Sites are arranged in rank order along the horizontal axis of the figure.
n
Q)+1 +1
or. 3
>
!t
synthesis (Fig. 1). At the site where the NPS level was highest (uronic acid content of 4.86 mg) (Table 1), 96% of the nodule @occupants analyzed were NPS formers. At the site where the NPS level was lowest (uronic acid content of 0.40 mg), only 11% of the nodule occupants were NPS formers. Other levels of NPS in nodules were associated with intermediate proportions of NPS+ nodule residents. The correlation coefficient relating the uronic acid concentration in nodules to the proportion of isolates which were NPS+ was 0.934 (significant at 1%).
D^
Q o oo X0
++
CZ
~~~~~~intermediate
0
(A
0 .
t:> 8 ot N .o ,..
i iD t+1 +1
U0~
DISCUSSION
It is possible for an individual nodule to be occupied by more than one bacterial type (13). If NPS- and NPS+ bacteria were -. both present in a nodule used for analysis of bacterial phenot o 4o4 the nodule would probably have been rated as NPS+, olatype, rA . thus introducing some bias into the data. However, the relaE tionship illustrated in Fig. 1 provided a reasonably clear o0 o indication that the proportion of nodule occupants which were M _ NPS+ varied widely among sites and that this variation was 'r 4m related to the NPS level in the bulk sample. It is now clear that .E ¢ !3 nodule samples from field sites contain some individual nodo % X 00 ules which are NPS+ and some which are NPS- and that the 3_ \0+1 +1 overall NPS level in the bulk sample reflects the proportion of 0. the two nodule types in the sample. At sites where the NPS level in the bulk nodule sample is high, some site characteristic 1>> N ;* oo o6 o s 0has led to the dominance of NPS+ B. japonicum in the soil or somehow favors the successful infection of roots by NPS+ soil +1 +o _ X residents. In analyses of polysaccharide synthesized by B. 0 o 0 japonicum in culture under aerobic conditions, we have never N xo < o adetected NPS (unpublished data). Thus, it seems likely that NPS o o is fisynthesized only in the nodule and unlikely that there 0 =° would be selectivity by the plant for NPS+ bacteria in the +1 ++1 0 z Cn N X Y= 0 F ~~~~rhizosphere. -t > a ( 3The results of the two field surveys were consistent and indicated that among site characteristics measured, only pH and i,!soil [Ca] were significantly associated with the deposition 0 = of bacteroid polysaccharide in soybean nodules. It is possible 5.l o < Z¢ > o = .: w that NPS represents a mechanism to sequester Ca in the
