INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

The effects of land management practices on bacterial diversity are not known. It has been suggested that agriculture creates highly selective and homogeneous environments that reduce bacterial diversity (21), and it has been shown that artificial fertilizer application in previously unfertilized soil led to decreased rhizobial diversity in symbiotic nodules (2). Conversely, it has been argued that cultivation results in more diverse populations, and greater diversity of substrate utilization by total microbial communities from cultivated fields than from pastures has been reported previously (13). Lower rates of plasmid transfer have been inferred in natural pastures in comparison with other studies in arable fields (32). A study of Rhizobium leguminosarum populations from a grazed pasture and an ungrazed open woodland revealed similar levels of diversity (28).

Our study compares the diversity of pea-nodulating R. leguminosarum biovar viciae in arable and grassland soils. Arable soils are frequently disturbed by plowing, monoculture rotation, and harvest by denudation. Grass soils harbor a relatively stable plant population which is grazed or mowed. Arable soils are also subject to higher levels of soil amendments, fertilizers, herbicides, and pesticides than are grass soils. It is known that rhizobial numbers are affected by soil amendments, such as manure, lime, and phosphate (see reference 19 for a review), and by levels of fertility (10).

We have used replicate pairs at four locations, sites within each pair being as close to each other as possible. The replication allows us to assess the difference in rhizobial diversity between arable and grass management systems while taking into account the inevitable variation from site to site. R. leguminosarum genotypes are not evenly distributed across the United Kingdom (7, 36). The soil type can affect microbial activity (6), possibly due to differences in the distribution of suitable niches (22) and predation pressure by protozoa (3). Cultivation of the host plant has been shown to have a homogenizing effect on rhizobial populations (10), but none of the arable sites in this study had cropped peas within the past 5 years and host plants of R. leguminosarum biovar viciae were very uncommon at the pasture sites.

Peas (Pisum sativum cv. Kelvedon Wonder) were used as a uniform trap host to sample the rhizobial populations at each site. It has been demonstrated that peas are nodulated by a wide range of R. leguminosarum genotypes (e.g., see reference 34), and we have data demonstrating that peas and wild Vicia and Lathyrus species select a similar range of genotypes from a natural population (L. A. Mutch and J. P. W. Young, unpublished data).

The chromosomal portion of the R. leguminosarum genome was characterized using the internal transcribed spacer (ITS) between the 16S and 23S ribosomal DNA (rDNA) genes. The ITS can be interpreted as an indicator of chromosomal variation: rrn genes have been located only on the chromosome in R. leguminosarum (12). Restriction fragment length polymorphism analysis provides a level of resolution of intraspecific diversity in R. leguminosarum that is comparable to that of other DNA methods (17). The plasmid portion of the genome has been characterized using six sequence groups of the plasmid replication gene, repC, which are postulated to belong to different incompatibility groups (24, 30; K. M. Palmer, S. L. Turner, and J. P. W. Young, unpublished data). This method is independent of the traits borne by the plasmids and has the potential to characterize more than one plasmid in a strain. As well as comparing diversity measures between each pair of arable and grass sites, we tested the response of heterogeneity indices in multiple regression analyses with the soil factors analyzed at each site.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Collection of soil samples. Pairs of sites were selected from four farms in Yorkshire with good records of their management regimens, the grass sites being permanent or semipermanent. The sites from each location were of the same soil type, with the exception of the Bishop Burton sites. All of the arable sites received herbicides, pesticides, and treatments necessary for their crop. High Mowthorpe is situated on chalk uplands. The arable site (with winter wheat) was directly above the grass site, a permanent dairy pasture (grid reference SE898685). Manure from the dairy unit was spread on the arable field in the previous fall. Bishop Burton is situated on loam over chalk. The arable site (winter wheat, SE977406) was 1.5 km distant from the grass site, a horse paddock for several years (SE986415). Piggery manure was spread on the arable site in the previous fall. Headley Hall is situated on calcareous loam over clay. The arable site (potatoes) and the grass site (permanent dairy pasture) were situated on slopes facing each other (SE440420). Askham Bryan is situated on noncalcareous water gley. The arable site (potatoes) was subject to a phosphorus fertilizer trial and 0.2 km distant from the grass site, a permanent hay and sheep grazing meadow (SE547467).

R. leguminosarum bv. viciae isolation. Pea seeds (Kelvedon Wonder) were surface sterilized in 2% hypochlorite, rinsed twice in sterile water, and incubated on solidified TY (1) at 26°C for 3 days. Pots (12-cm diameter) were sterilized in 0.1% sodium hypochlorite, rinsed in sterile water, and autoclaved. Autoclaved Terragreen soil conditioner, a calcined attapulgite clay (Oil Dri UK Ltd.), was placed in the bottom 4 cm, followed by a 5-cm layer of fresh sample soil (stored at 4°C for 1 to 3 days) into which were placed sterile, germinated peas. The soil surface was covered with a layer of sterile Terragreen to prevent splash-over during watering. Two pots for each soil sample were each seeded with four peas, placed on an open mesh in a greenhouse, and watered with sterile water. All of the plants were equally well nodulated. Nodules were collected 4 to 5 weeks later, and isolates were obtained from sterilized (cleaned in Tween, sterilized in 0.1% sodium hypochlorite for 15 min, and rinsed twice in sterile water) and crushed nodules on TY agar plates. After incubation at 26°C for 3 days, a well-separated colony from each nodule was subcultured onto fresh TY plates. Isolates were stored at 80°C in TY broths containing 20% glycerol. Ten isolates were collected from each plant and coded as follows: High Mowthorpe (M), Bishop Burton (B), Headley Hall (H), or Askham Bryan (A); arable (A) or grass (G); pot (1 or 2); plant (a, b, c, or d); and number (1 to 10). A total of 285 isolates were characterized: 47 from MA, 50 from MG, 44 from BA, 49 from BG, 35 from HA, 14 from HG, 20 from AA, and 26 from AG.

PCR and restriction digest conditions. PCRs were performed using Taq polymerase (Promega) with total DNA extracts or fresh cells as template. 16S-23S rDNA ITS PCR used the primers FGPS1490 and FGPL132' and conditions previously described (17). HaeIII (Promega) digestions in buffer C at 37°C were performed after ammonium acetate-ethanol precipitation and resuspension. Conditions for PCR amplification of repC1 to repC6 sequence groups have been described previously (30; K. M. Palmer, S. L. Turner, and J. P. W. Young, unpublished data). The presence (+) or absence () of each repC group was scored for each isolate. A repC profile of does not mean that the isolate is barren in plasmids but shows that there are no plasmids bearing repC sequences from any of the six sequence groups. Other repC sequences have been found in R. leguminosarum strains (K. M. Palmer, S. L. Turner, and J. P. W. Young, unpublished data).

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Diversity measures. The estimated numbers of types in each site were calculated by rarefaction with an estimation of the variance using Simberloff's (26) computer program modified by Krebs (14). The use of rarefaction allows comparison of the number of types in samples of different sizes by limiting the sample to the smallest size in the set of populations and calculating the richness in types. We have used 2  variance as an estimate of the 95% confidence interval. Excel worksheet formulae (Microsoft) were used to calculate the Shannon-Wiener index, H' =  p_i · lnp_i, where p_i is proportion of the ith type in the population (20), and Simpson's index of diversity with Pielou's estimator for finite populations, 1  D = 1  [n_i(n_i  1)/N(N  1)], where n_i is number of the ith type and N is number of individuals in the population (23). The Shannon-Wiener index calculates the uncertainty of predicting the type of another isolate from that population, and Simpson's index of diversity is based on the probability of picking two different individuals. Both the Shannon-Wiener and Simpson indices measure heterogeneity by incorporating both richness and distribution (evenness) of types, but the Simpson index reflects differences in the dominant types more than the Shannon-Wiener index does.

Soil analysis. Two replicate samples from each mixture of subsamples were taken for chemical analysis. Fresh soil-water (1:2) mixtures were used to estimate pH (4). All other analyses used air-dried (30°C for 12 h) and sieved (2-mm-pore-size) soil. Conductivity at 25°C (25) and water-extractable phosphate, calcium, and magnesium were measured with 1:2 soil-water mixtures. Phosphate content was determined by the measurement of molybdenum blue by ascorbic acid reduction (0.5 M sulfuric acid, 0.0024% ammonium molybdate, 0.01375% potassium antimony tartrate, 0.53% ascorbic acid), calibrated with known phosphate concentrations (4) at 660 nm. Calcium and magnesium concentrations were determined by atomic absorption spectrometry in the presence of 1% lanthanum (18). Mineral nitrogen (nitrate, nitrite, and ammonium) was extracted in potassium chloride and distilled with a modification of Kjeldahl digestion (5) with Devarda's alloy, collecting the distillate into boric acid indicator solution (50 g of boric acid liter¹, 4.5 µg of methyl red liter¹, and 7.5 µg of bromocresol green liter¹) (H. Vallack, personal communication). The content of organic matter was estimated by loss on ignition at 550°C (4), which also included loss due to heat decomposition of carbonates. As each pair of soils from High Mowthorpe, Headley Hall, and Askham Bryan were the same soil type, differences in this weight loss can be attributed to differences in organic content at these sites. The potential mineral nitrogen in the soil samples (i.e., that in the organic fraction of the soil) was assessed using an incubation technique (4; H. Vallack, personal communication): 20 g of air-dried soil was moistened to 25% water content and inoculated with a drop of garden soil water to provide a bacterial population for the decomposition of organic material. The samples were incubated at 27°C in the dark for 6 weeks, moisture was replenished weekly by weight, the mineral nitrogen content was determined as described above, and the potential nitrogen values were determined by subtraction of the original mineral nitrogen value. The clay composition was measured with a Bouyoucos hydrometer, essentially according to reference 4 using 5% sodium hexametaphosphate and 0.7% sodium carbonate as a dispersal agent. Water-soluble calcium and magnesium concentrations were determined by atomic absorption spectrometry in the presence of 1% lanthanum (18).

Data analysis. All analyses were performed using SPSS 8.0 for Windows. The relationship between the soil factors was determined using principal components analysis. Stepwise multiple regression analyses were performed between the set of analyzed soil parameters and each of the Shannon-Wiener and Simpson diversity indices. The default criterion probabilities of F for inclusion and removal (0.05 and 0.1, respectively) did not allow the inclusion of any parameters in many analyses, so the criterion probability for inclusion was increased to 0.25, and that for exclusion was increased to 0.255.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Genotypes of R. leguminosarum bv. viciae. Table 1 shows the genotypes of the R. leguminosarum bv. viciae isolates at each of the sites, based on the ITS typing of the chromosome and the repC profiles in the plasmid portion of the genome.

Comparison of diversity levels between arable and grass sites. The rarefaction of the samples to 14 isolates with an estimate of the 95% confidence intervals allows comparison of the number of ITS types, repC profiles, and combined genotypes within each pair of sites (Table 2). The arable sites have a significantly higher expected number of types than the grass sites at High Mowthorpe (all three character sets), Headley Hall (repC profiles and combined genotypes), and Askham Bryan (ITS types). These observations are supported by the differences in the Shannon-Wiener and Simpson diversity indices.

Relationship between soil parameters. We analyzed each soil sample in duplicate in order to determine whether there are specific soil parameters that influence rhizobial diversity. Factors were chosen that have been shown to influence rhizobial diversity (pH [7] and magnesium [15]), cell functioning (calcium [31] and magnesium [29]), or bacterial survival (clay [3]) and that can be expected to change according to the land management (conductivity, mineral nitrogen, organic content, potential nitrogen, phosphate, and clay). The results are shown in Table 3. The duplicate results largely agreed with each other. Some consistent differences between the soil factors at the two types of site can be observed. The arable sites contained larger proportions of clay and smaller proportions of organic matter and potential nitrogen than the grass sites.

The response of diversity measures to soil parameters. The general trend of increased diversity in arable soils compared with that in grass soils, described above, has been detected by comparing contrasting pairs of sites. To test the response of the Shannon-Wiener and Simpson indices to the soil parameters, multiple regression analyses were performed (SPSS 8.0 for Windows). The most significant models are shown in Table 5.