nodulating Bradyrhizobium japonicum strains ...

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Predominant populations of indigenous soybeannodulating Bradyrhizobium japonicum strains obtained from organic farming systems in Minnesota M. Wongphatcharachai1, C. Staley1, P. Wang1, K.M. Moncada2, C.C. Sheaffer2 and M.J. Sadowsky1 1 Department of Soil, Water and Climate, BioTechnology Institute, University of Minnesota, St. Paul, MN, USA 2 Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN, USA

Keywords Bradyrhizobium japnicum, genotyping, indigenous population, organic farms, serotyping, soybean-bradyrhizobia. Correspondence Michael J. Sadowsky, BioTechnology Institute, University of Minnesota, 140 Gortner Lab, 1479 Gortner Ave, St. Paul, MN 55108, USA. E-mail: [email protected] 2014/2436: received 26 November 2014, revised 27 January 2015 and accepted 29 January 2015 doi:10.1111/jam.12771

Abstract Aims: Bradyrhizobium from organic fields in Minnesota were isolated and genotyped to assess diversity of soybean-bradyrhizobia in organic farming systems that can be used to improve soybean productivity. Methods and Results: Soil samples were collected from 25 organic fields in Minnesota during May to July 2012. Soybean (cv. Lambert) was used as a host to trap indigenous bradyrhizobia in each sample. Genetic diversity of Bradyrhizobium strains (n = 733) was determined using the horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (HFERP) DNA fingerprinting technique and the soybean-bradyrhizobia were classified into 79 different genotypes. Of these, 15 dominant genotypes were found and were highly similar (>92% fingerprint similarity) to serotypes USDA 127 (404%), USDA 4 (318%) and USDA 123 (155%), which were the three main populations of soybean-bradyrhizobia in organic fields. Conclusions: Bradyrhizobium japonicum serogroup USDA 4 strains were found to make up a previously unrecognized, predominant rhizobial population in the organic farming soils examined. The relative abundance of strain USDA 4 was negatively correlated with that of USDA 127 and this relationship may be influenced by the levels of NO3-N and other soil edaphic factors. Significance and Impact of the Study: The local community of bradyrhizobia can be affected by applying inoculant bacteria to organic fields. Based on these results, soybean production in organic farms may be improved by displacing strains similar to USDA 4 with those better at nitrogen fixation and competitive ability than indigenous strains.

Introduction Soybean (Glycine max) cultivation is of principal agronomic importance in the United States. In 2009, Minnesota was among the three top soybean producing states (USDA 2010). To date, however, ~93% of soybean crops grown in the U.S. are comprised of genetically modified organisms (GMOs) (USDA 2013). In conventional farms, synthetic fertilizers, pesticides and uniform high-yield hybrid crops are used to achieve enhanced crop productivity. The idea of organic farming systems places a focus on the health of the environment, soils and the ecosystem, 1152

as well as humans, and does not use synthetic fertilizers, pesticides or GMO crops for production. Organic farming methods are regulated at the national and international levels. Based on information from USDA-accredited state and private organic certifiers, Minnesota ranks first in certified organic soybean acres (USDA 2011). According to the USDA’s National Organic Program, one of the most challenging problems for organic farming systems is the limitation of soil nitrogen, since synthetic N compounds cannot be used for production (http:// www.ams.usda.gov/AMSv1.0/nop). Over-use of synthetic fertilizers has been shown to cause serious water

Journal of Applied Microbiology 118, 1152--1164 © 2015 The Society for Applied Microbiology

M. Wongphatcharachai et al.

pollution problems (Comly 1987; Smith et al. 1987; Randall and Mulla 2001; Elmi et al. 2002; WHO 2011). Biological N2-fixation remains a viable option for providing a nitrogen source in organic farming systems for legumes, in place of synthetic fertilizers. This can be achieved by applying Rhizobium or Bradyrhizobium spp. strains, the nitrogen-fixing root- and -stem nodule symbionts of legumes (e.g. soybeans, alfalfa, beans and peas) (Zahran 1999). In the U.S. soybean bradyrhizobia are comprised mainly of two species, Bradyrhizobium japonicum and B. elkanii, with strains of the former being more prevalent in northern states and strains of the latter in the south (Keyser et al. 1984; Dobert et al. 1994; Shiro et al. 2013). The genotypes of soybean bradyrhizobia found in any given soil vary, in part due to the previous inoculant used, soil edaphic factors and to symbiotic interactions with specific soybean varieties (Singleton and Tavares 1986; Shiro et al. 2013). However, relatively few strains have been shown to be highly competitive and effective at carrying-out N2 fixation in most soils (Triplett and Sadowsky 1992). These strains are frequently the ones most often found in nodules from nitrogen-sufficient, highly productive, soybeans found in the field. It has been welldocumented that introduced rhizobia often poorly compete with indigenous strains, as demonstrated by low recovery rates in field crops (Ham et al. 1971; Kvien et al. 1981; Streeter 1994). Moreover, the application of genetically engineered rhizobia or GMO crops is prohibited for use in organic systems. In conventional farms, several studies reported that strains in B. japonicum serocluster 123 are the major indigenous competitors of soybean-bradyrhizobia in the upper Midwest U.S., including Minnesota (Ham et al. 1971; Ellis et al. 1984; Keyser et al. 1984; Schmidt et al. 1986; Cregan et al. 1989). These strains, however, are less effective for N2-fixation than some less competitive strains, such as USDA 110 (Caldwell and Vest 1970; Ellis et al. 1984; Moawad et al. 1984). In contrast, B. elkanii serogroup 31 and 76 strains are predominant in the southeastern United States (Caldwell and Hartwig 1970; Keyser et al. 1984). Thus, the selection and use of highly competitive, efficient nitrogen-fixing, indigenous bradyrhizobial strains can be a useful method to improve soybean productivity on organic farms. Despite the use of legume inoculants on conventional farms and an understanding of indigenous bradyrhizobia in U.S. soils, little work has been done to determine the numbers and types of Bradyrhizobium strains in organic farm soils and whether new inoculants specifically selected for organic systems could be useful for organic soybean systems. The selection and use of highly competitive, efficient nitrogen-fixing, indigenous bradyrhizobial

Diversity of soybean-bradyrhizobia from organic farms

strains can be a useful method to improve soybean productivity on organic farms (Keyser and Li 1992). In this study, we evaluate the serological and genetic diversities of soybean-bradyrhizobia isolated from organic field soils in Minnesota using horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (HFERP) DNA fingerprinting to determine how these populations vary from those previously reported to be present in soils from conventional farming systems (Schmidt et al. 1986; Shiro et al. 2013). In addition, soil nutrient and physicochemical parameters were evaluated to assess relationships between these factors and bradyrhizobial diversity. Results of this study provide novel insights into the bradyrhizobial populations in organic fields and suggest possible means by which to increase organic soybean yields. Materials and methods Soil sampling and isolation of indigenous bradyrhizobia from organic fields Twenty-five soil samples were collected from organic fields at the 0–10 cm depth. Fields were located in 13 counties in Minnesota and in one county in Wisconsin (close to Washington County, Minnesota) during May to July in 2012 (Fig. 1 and Table 1). All fields were certified and were practicing organic farming for at least 4 years before sampling. Soil samples were analysed for the concentration of soybean-bradyrhizobia by using the most probable number (MPN)-growth pouch technique with soybean cv. Lambert as trap host (Somasegaran and Hoben 1985). The remaining soil samples (210 g) were submitted to the Soil Testing Laboratory, University of Minnesota-St. Paul for analysis of soil chemical properties (Table S1). Soybean seeds were surface sterilized as previously described (Somasegaran and Hoben 1985). Briefly, seeds were rinsed with 95% ethanol for 10 s, and immersed in 20% Chlorox bleach for 10 min. Seeds were rinsed exhaustively with sterile water and submerged in water for 30 min at room temperature. Sterile seeds were pregerminated by plating on 1% water agar. Growth pouches were prepared by adding 50 ml of 059 Hoagland’s solution (Hoagland and Arnon 1950) and autoclaved for 30 min. Germinated seeds were placed in sterilized growth pouches, and inoculated with 1 ml of each soil dilution, from 101 through 106, using four replications per dilution. Seeds were incubated in growth chambers at 23°C with 70% humidity and a 16 h photoperiod. Root nodules were harvested after 4 weeks and the concentration of bradyrhizobia in each soil sample, per host plant, was calculated and presented as the MPN per


1153


Be

Bj 94 11 0


Group 1 (9 fields: S1, S2, S5–S11)

(a)

Bj

Bj 4

Bj 127

(b) Kittson

Group 2 (2 fields: S3, S4)

Roseau

Lake of the Woods

Marshall Koochiching Beltrami

Pennington

Bj 4 Bj 1 35

Be 94 (2·0% ) Bj 1 35 ( 3·8% Bj ) 11 0( 5·1 %)

Bj 4 (31·8%)

Cook

Reda Lake

Bj 123

Polk

Lake

Clearwater

Itasca

St.Louis

Norman

Bj 127

Mahnomen

Hubbard

Cass

Becker

Clay

Bj 123 ) (15·8%

Otter Tail (S5, S6)

Morrison

Douglas

Grant

Kanabec Benton

Bj 13 5

Traverse

Bj 4

Stevens

Stearns

Pope

Mi ssis Sherburne sip pi R Wright iver

Big Stone

29

1 Bj

Pine

Mille Lacs

Todd

Bj 129 (1·3%)

Carlton

Aitkin

Crow Wing

Wadena

Wilkin

Bj 127 (40·4%)

3

12

Swift

Lac qui Parle

Mi

Chippewa

nn McLeod esoRenville ta Riv Sibley er

Yellow Medicine

Bj 127 Lincoln Lyon

Redwood

0

Isanti

Bj

Chisago Anoka

Kandiyohi Meeker

Washington

Hennepin

Ramsey

Polk (WI)

Bj 1

23

Carver Scott

Dakota

Bj 127

Goodhue

Nicollet Le Sueur Rice

Wabasha

Brown

Group 3 (8 fields: S16–S23)

Pipestone

Nobles

Cottonwood Watonwam

Jsckson

Martin

Blue Earth Waseca

Faribault

Steele Dodge

Olmsted

Mower

Freeborn

Winona

Group 4 (4 fields: S12–S15)

Fillmore Houston

Bj 110

Rock

Murray

11

Bj 4

Bj 123 Bj 4 Bj 127

Group 5 (2 fields: S24, S25)

Figure. 1 Distribution of serotypes of indigenous soybean-bradyrhizobia isolated from organic farms in Minnesota. (a) Composite distribution among all sites. (b) Sampling locations were grouped based on best management practices (BMP’s) in Minnesota to show the population ratio of indigenous soybean-bradyrhizobia in each area. Group 1, Irrigated and non-irrigated sandy soils; Group 2, Northwestern; Group 3, Southwestern and West Central; Group 4, South Central; Group 5, Southeastern. Red triangles indicate the locations where soybean-bradyrhizobia were taken and yellow triangles indicate the locations where bradyrhizobia could not be obtained in this study. Abbreviation: Bj, Bradyrhizobium japonicum; Be, B. etli. Legends; ( ) Bj USDA 4; ( ) Be USDA 94; ( ) Bj USDA 110; ( ) Bj USDA 123; ( ) Bj USDA 127; ( ) Bj USDA 129 and ( ) Bj USDA 135.

gram of dry soil. Root nodules were used for isolation of indigenous bradyrhizobia. Nodules were rinsed with 95% ethanol for 10 s and surface sterilized with 5% bleach for 10 min, with vigorous shaking. Surface-sterilized nodules were washed with eight changes of sterile, distilled water and each nodule was placed individually into 100 ll of 085% NaCl in wells of microtitre plates. Nodules were crushed with a sterile loop and streaked onto the surface of arabinose-gluconate (AG) agar plates (Sadowsky et al. 1987). Nodule bacteria were purified by streaking onto the same medium. HFERP DNA genotyping of indigenous soybeanbradyrhizobia DNA fingerprints from eight reference strains (Table 2) and about 30 Bradyrhizobium isolates per sample, per host plant, were analysed by using a modification of the HFERP DNA fingerprinting technique as described previously (Johnson et al. 2004). Escherichia coli strain 1154

Pig294, isolated in our laboratory, was used as a control, and an internal ROX-labelled molecular weight marker was included as a control for each sample for inter-gel comparisons (Dombek et al. 2000; Johnson et al. 2004). Briefly, each Bradyrhizobium strain was streaked onto AG medium for single colony isolation. A portion of a single colony of each sample was picked using a 1-ll sterile loop (Fisher Scientific, Waltham, MA) and suspended in 100 ll of 50 mmol l1 NaOH in low 96-well clear PCR plates (Bio-Rad Laboratories, Hercules, CA). The partial colony was suspended vigorously and heated at 95°C for 15 min. Samples were centrifuged at 4°C for 10 min at 640 rev min1 (82 g). A 2-ll aliquot of supernatant for each sample was subjected to HFERP DNA fingerprinting as previously described (Johnson et al. 2004) and PCR was performed using the following conditions: 95°C for 2 min, 30 cycles of 94°C for 3 s, 92°C for 30 s, 50°C for 1 min, 65°C for 8 min and a final extension at 65°C for 8 min. Amplicons were visualized via gel electrophoresis at 70V for 16 h at 4°C




Table 1 Farm locations, soil characteristics, crop rotation practices and nodule number and size of indigenous Bradyrhizobium japonicum strains from 25 organic farms in Minnesota

Application of inoculants

Crops prior to sampling

Nodule number/plant (at 1 : 100 dilution)†

Yes Yes Yes

Kidney bean Kidney bean Soybean

115 125 138 153 158 66

31 30 45

Yes

Soybean

285 82

53

Otter Tail

I: Sandy loam, neutral I: Sandy loam, neutral II: Borup loam, Colvin silty clay loam, slightly basic III: Northcote clay, Fargo clay, slightly basic I: Sandy loam, neutral

Yes

14 June 2012

Otter Tail

IV: Clay loam, neutral

S7 S8 S9 S10 S11 S12

19 08 08 18 18 18

Mille Lacs Morrison Morrison Polk (WI) Polk (WI) Dakota

Green bean Soybean Soybean Green bean Dry bean Soybean

S13

18 May 2012

Dakota

No

Dry bean

S14

18 May 2012

Dakota

No

S15

23 May 2012

Dakota

S16

09 May 2012

S17

30 May 2012

S18 S19 S20

09 May 2012 23 May 2012 30 May 2012

Lac qui Parle Yellow Med Lincoln Murray Redwood

IV: Clay loam, slightly acid V: Loam, neutral VI: Loamy sand, slightly acid VII: Santiago silt loam, neutral VII: Santiago silt loam, neutral VII: Lester loam, Kennebec silt loam, moderately acid) VII: Lester loam, Kennebec silt loam, slightly acid VII: Lester loam, Kennebec silt loam, slightly acid VII: Lester loam, Blooming silt loam, moderately acid VII: Silty clay, neutral

No information No§ Yes Yes Yes Yes No

Dry bean, soybean Green bean

S21 S22 S23 S24

09 09 09 22

Redwood Redwood Redwood Sibley

S25

12 May 2012

Field no.

Sample collection date

County

Soil characteristics*

S1 S2 S3

17 July 2012 17 July 2012 16 May 2012

Hubbard Hubbard Clay

S4

16 May 2012

Clay

S5

14 June 2012

S6

May 2012 June 2012 June 2012 May 2012 May 2012 May 2012

May May May May

2012 2012 2012 2012

Steele

Log10 no. of Bradyrhizobia‡

12 10N

23

02 05N

09

35 40 35 30 28 28

157 105 68 90 88 210

101 95 73 72 63 121

92 44

40

Green bean

108 73

35

Never¶

Dry bean

100 22

38

Never

Soybean

175 176

41

IV: Canisteo clay loam, neutral

Never

Soybean

120 52

39

I: Sandy loam, neutral V: La Prairie loam, slightly acid V: Ves loam/Normania loam, slightly acid IV: Webster clay loam, neutral V: Ves loam, slightly acid V: Normania loam, neutral V: Loam, mix; Crippen, Cordova-Rolfe, slightly acid V: Hayden loam, slightly acid

Never Never No

Soybean Soybean Soybean

45 41 165 31 198 86

34 51 46

Yes No No Never

Dry bean Soybean Soybean Soybean

38 55 50 122

21 43 26 35

No

Soybean Average

130 90 113 73

39 48 60 96

42 43 10

*Soil characteristics were grouped based on USDA textural soil classification. Soil pH descriptions: moderately acid, 55–59; slightly acid, 60–65; neutral, 66–75; slightly basic, 76–80). †Nodule number per plant was reported as x̅ S.D. ‡Population size (log10 MPN g1 dry soil) estimated by the MPN technique using soybean cv. Lambert as trap host. §No; farmers did not use inoculant for the target bean crop and there is no information on past inoculant use. ¶Never; farmers did not use inoculant for the target bean crop and that they have never used inoculant in the past.

with a pump attached for buffer recirculation. Gel images were captured using a Typhoon 8600 scanner (Molecular Dynamics/Amersham Biosciences, Sunnyvale, CA) with a fluorescence acquisition mode. Gel images were analysed using BIONUMERICS ver. 2.5 (Applied Maths, Austin, TX). Phylogenetic analysis was conducted using

the unweighted pair group method with arithmetic means (UPGMA). Genetic diversity was evaluated based on the HFERP DNA fingerprint profiles using SPADE (Species Prediction And Diversity Estimation) software (Chao and Shen 2010) to calculate strain richness (using the abundance-based


1155



coverage estimator, ACE and Shannon and Simpson diversity indices (Shannon 1948; Simpson 1949). 16S rRNA gene sequencing To identify strains of soybean-bradyrhizobia in group XIII, XIV and XV (Fig. 2), some representatives from Table 2 Reference strains used in this study

each group were chosen for 16S rRNA gene sequence analysis using PCR primers; Bac27F (50 -AGA GTT TGA TCM TGG CTC AG-30 ) and Univ1492R (50 -CGG TTA CCT TGT TAC GAC TT-30 (Lane 1991). PCR amplicons were submitted to the University of Minnesota Genomics Center (UMGC, St. Paul, MN) for DNA sequencing using the Sanger sequencing method and an ABI PRISMTM 3730xl DNA Analyser.

Species

Strains

Sources

Serological analyses using immuno dot-blot assays

Bradyrhizobium japonicum

USDA 4, USDA 110, USDA 127, USDA 135 USDA 122, USDA 123, USDA 129, USDA 76, USDA 94

1

Two representative strains of indigenous soybean-bradyrhizobia in each of 15 genotypic group (n = 30) were selected for immuno dot-blot assays for serological identification as previously described (Cregan et al. 1989; Wongphatcharachai et al. 2013). The representative indigenous isolates and reference Bradyrhizobium strains were grown in AG medium for 2 days to OD600 = 08–12.

(b) B. elkani USDA76 B. elkani USDA94

100

90

80

% Relative similarity 70

20

100

90

80

70

60

50

40

30

20

% Relative similarity

60

(a)

50

Sources: 1, Reference strains belonging to this laboratory. 2, Obtained from the Rhizobium culture collection, United States Department of Agriculture (USDA)-ARS, Beltsville, MD.

40

2 1

30

Bradyrhizobium elkanii

Groups

No. of isolates

% population

No. of farma

I

12

1·95

4

USDA 94

II

23

3·75

6

USDA 135

III IV V VI VII

18 11 12 11 13

2·93 1·79 1·95 1·79 3·12

4 5 3 6 7

USDA 110 USDA 110 USDA 129 USDA 123 USDA 123

VIII

21

3·42

9

USDA 127

IX

71

11·56

18

USDA 123

X

32

5·21

13

USDA 127

XI

35

5·70

12

USDA 127

XII

160

26·06

20

USDA 127

XIII XIV

14 12

2·28 1·95

8 6

†ND †ND

XV

169

27·52

16

‡ND (USDA 4)

Total

614

100

23

Serotype

B. japonicum USDA135


B. japonicum USDA122 B. japonicum USDA129



B. japonicum USDA4

Figure. 2 Dendrograms based on HFERP DNA fingerprinting and serotypes of soybean- nodulating bradyrhizobia isolated from soils of 25 organic farms. (a) all isolates were submitted showing a whole picture of genetic diversity of bradyrhizobia in organic farms. (b) samples which cannot be classified into any groups or fewer than 10 isolates per group were excluded to show the 15 predominant genotypic groups. The black lines ( ) are used to show the reference strains that did not group with any sample and the black triangles ( ) are used to show the reference strains which grouped with samples. Legend: ND = no specific antibody to determine serogroup, †Based on the results of 16S DNA sequencing, ‡Based on the results of HFERP fingerprinting.

1156



Three replicate 15-ll aliquots of each culture were spotted onto the surface of nitrocellulose membranes and left to air dry for 10 min. The membrane was blocked with 5% fish gelatin (Sigma–Aldrich, St. Louis, MO) in 19 PBST (005% Tween20) at 4°C for 2 h. The membrane was washed once with 19 PBST and incubated for 30 min in a 1 : 400 diluted serotype-specific antibodies with 19 PBST as previously described (Madrzak et al. 1995). The membrane was washed five times with 19 PBST, in 5 min intervals, and incubated for 30 min in a 1 : 5000 diluted goat anti-rabbit antibody-HRP conjugate (Bio-Rad Laboratories) with 19 PBST. The membrane was washed once with 19 PBST, and immunological reactions were detected by incubating with reagents A and B (Pierceâ ECL Plus Western Blotting Substrate, Thermo Scientific) at room temperature for 3 min. The membrane spots were captured by using LABWORKS 4.5 software (UVP products, Upland, CA). Statistical analyses For statistical comparisons of genetic diversity indices, outliers and fields at which bradyrhizobia could not be isolated were excluded from further analyses. Nonparametric unpaired t-test (Mann-Whitney) and Kruskal-Wallis test were used to determine significant differences in comparisons. Spearman rank correlations were also calculated to determine relationships between inoculant application categories, edaphic properties, and bradyrhizobial abundance and diversity. All statistical analyses were performed using SPSS ver. 19 software (SPSS Inc., Chicago, IL) at a = 005. Results Concentrations of bradyrhizobia in soils The concentration of soybean-bradyrhizobia in soils from the 25 farms examined varied from log10 09–53 bradyrhizobia g1 dry soil (Table 1). On average, soils contained log10 43 bradyrhizobia g1 dry soil. Five of the farms in three counties (S3, S4 in Clay; S19 in Murray; S20, S22 in Redwood) contained populations of soybeanbradyrhizobia greater than the average, with log10 45, 53, 51, 46 and 43 bradyrhizobia g1dry soil, respectively, and the greatest concentration of bradyrhizobia was found in Clay County (S4). Bradyrhizobial concentrations did not differ significantly due to inoculant application practices (P > 005). However, concentrations were significantly greater when soybeans were planted prior to sampling (P = 0008, Table 3). The average number of nodules per plant was 113 73 nodules at the 100-fold-diluted soil suspensions


(Table 1). The number of nodules observed was significantly and positively correlated with the bradyrhizobial concentration in the soil (r = 0509, P = 0018). The greatest number of nodules per plant was found in soils from site S4 in Clay County, with 285 82 nodules per plant. Of note, soybean nodules from two fields in Otter Tail County (sites S5 and S6) were small in size and bradyrhizobia could not be obtained after surface sterilization. These were likely ineffective nodules. The number of nodules observed did not differ significantly by crop prior to sampling or inoculant application practices (P = 0129 and 0871, respectively). Genetic diversity of soybean-bradyrhizobia from organic farms Evaluation of genetic diversity of all the isolates was determined by using the HFERP DNA fingerprinting technique. Results of this analysis revealed that the soybean-bradyrhizobia (n = 733) could be classified into 79 different genotypes at >92% similarity (Fig. 2a). This similarity value was previously shown to represent clonal isolates (Johnson et al. 2004). Genetic diversity and richness of soybean-bradyrhizobia were not significantly different as a result of crop planting prior to sampling, inoculant application practices, or locations (P > 005; Table 3). After excluding the ungrouped unique strains (n = 119, samples which could not be classified into any groups or contained fewer than 10 isolates per group), soybean-bradyrhizobia were classified into 15 predominant genotypic groups (I - XV) based on HFERP DNA fingerprints (Fig. 2a,b). Soils from organic farming systems in Minnesota contained three main populations. Group IX accounted for 116% of isolates typed and was found in soils at 18 farms. This group also included the reference strain B. japonicum USDA 123. Group XII accounted for 261% of isolates typed and was present at 20 farms in all counties, except in Otter Tail county where soybean-bradyrhizobia could not be obtained in this study. Group XV accounted for 275% of isolates and also included the reference B. japonicum strain USDA 4. This genotype was obtained from 16 farms in 10 counties, except from Mille Lacs, Lac qui Parle and Yellow Medicine farms (Table 4). Soil chemical properties and genetic diversity Correlation analysis of soil chemical properties and genetic diversity revealed that the percentage of soil organic matter was significantly and negatively correlated with the Shannon diversity index (r = 0512,


1157

1158

plant

bradyrhizobia

135 70

(n = 14)

93 45

(n = 10)

P = 0129

(n = 14)

30 09

(n = 10)

P = 0008

Common

beans

P value

(n = 2)

P = 0100

(n = 2)

P = 0617

(n = 4)

(n = 4)

(n = 4)

126 05

(n = 8) 59 02

(n = 8)

128 55

(n = 8)

35 05

(n = 4)

(n = 2) 66 05

(n = 2)

106 67

(n = 2)

37 10

39 05

20 05

78 05

221 90

P < 0001

(n = 2)

61 02 P = 0761

(n = 2)

19 08

(n = 8)

16 04

(n = 2)

18 01

(n = 7)

49 05

17 08

(n = 9)

(n = 9)

(n = 9)

86 52

29 09

67 03

(n = 6)

P = 0440

(n = 2)

191 174

(n = 4)

254 120

(n = 8)

123 106

(n = 2)

145 50

(n = 7)

212 134

P = 0095

(n = 6) P = 0247

ND

(n = 6)

P = 0871

(n = 6)

P = 0346

(n = 8) 116 54

(n = 8)

250 153

(n = 8)

18 04

184 78

(n = 8)

P = 0644

18 04

P = 0684

(n = 9)

196 140

17 07 (n = 9)

(n = 14)

171 111

ACE

(n = 14)

18 05

H’

16 02

ND

(n = 8)

ND

121 47

125 60

37 07

ND§

(n = 8)

(n = 10)

(n = 10)

40 06

110 75

34 10

P = 0770

(n = 10)

66 04

(n = 14)

66 02

pH (soil)

P = 0151

(n = 2)

85 35

(n = 4)

240 194

(n = 8)

93 67

(n = 2)

82 05

(n = 7)

224 141

P = 0242

(n = 6)

837 62

(n = 8)

202 178

(n = 8)

176 104

P = 0116

(n = 9)

211 174

(n = 14)

122 85

strains

unidentified

Percent of

P = 0185

(n = 2)

50 14

(n = 4)

128 45

(n = 8)

71 36

(n = 2)

95 21

(n = 7)

100 52

P = 0329

(n = 6)

77 16

(n = 8)

110 58

(n = 8)

99 29

P = 0315

(n = 9)

97 97

(n = 14)

86 41

No. of strains

P = 0428

(n = 2)

529 119

(n = 4)

440 339

(n = 8)

245 234

(n = 2)

29 40

(n = 7)

298 374

P = 0857

(n = 6)

360 269

(n = 8)

291 306

(n = 8)

271 333

P = 0590

(n = 9)

365 337

(n = 14)

260 272

USDA4

(n = 9)

(n = 9)

(n = 8)

(n = 8) (n = 6)

P = 0394

0 (n = 2)

(n = 4)

44 88

P = 0280

(n = 2)

37 52

(n = 4)

183 297

0 (n = 8)

0 (n = 2)

0 (n = 2) 0 (n = 8)

(n = 7)

50 88

P = 0638

(n = 7)

47 67

P = 0521

19 45

87 217

28 62 0 (n = 6)

(n = 8)

39 79

P = 0087

(n = 8)

31 62

P = 0105

111 207

(n = 14)

43 73

11 27

(n = 14)

USDA110

08 31

USDA94

Relative abundance of serotypes

Bold texts indicate significant difference at 005 levels. *The mean difference between groups was analysed by Mann-Whitney Test (a = 005 level). †The mean difference between groups was analysed by one way-ANOVA, Kruskal-Wallis Test (a = 005 level). ‡Locations were grouped as shown in Fig. 1. §ND, not determined. ¶No; farmers did not use inoculant for the target bean crop and there is no information on past inoculants use. **Never; farmers did not use inoculant for the target bean crop and that they have never used inoculants in the past.

P value

Group 5

Group 4

Group 3

Group 2

Group 1

Locations†,‡

P value

Never**

No¶

Yes

Inoculants application practices†

Soybean

40 08

Crop prior to sampling*

nodules per

No. of

Conc. of

Genetic diversity

P = 0858

(n = 2)

210 69

(n = 4)

127 122

(n = 8)

162 142

(n = 2)

248 174

(n = 7)

132 182

P = 0468

(n = 6)

137 73

(n = 8)

210 194

(n = 8)

1273 127

P = 0486

(n = 9)

14. 4 168

(n = 14)

168 130

USDA123

(n = 4)

P = 0042

(n = 2) P = 0060

0 (n = 2)

(n = 4) 224 03

64 74

0 (n = 8)

P = 0395

0 (n = 2)

0 (n = 4)

(n = 8)

107 181

(n = 2)

75 26

(n = 7) 0 (n = 2)

06 16 (n = 7)

P = 0734

(n = 6)

60 100

(n = 8)

62 177

(n = 8)

21 35

P = 0028

0 (n = 9)

(n = 14)

75 140

USDA135

13 23

P = 0097

0 (n = 6)

(n = 8)

38 58

(n = 8)

05 14

P = 0086

(n = 9)

34 56

(n = 14)

03 11

USDA129

142 84

(n = 8)

486 128

(n = 2)

648 189

(n = 7)

454 316

P = 0167

(n = 6)

426 221

(n = 8)

283 152

(n = 8)

506 292

P = 0038

(n = 9)

303 243

(n = 14)

475 183

USDA127

Table 3 Comparison of Bradyrhizobium concentrations, number of nodules and population diversity of bradyrhizobia in relation to crop prior to sampling, inoculant application and location of the farm

Diversity of soybean-bradyrhizobia from organic farms M. Wongphatcharachai et al.


36

29

Hubbard Clay

Clay Otter Tail

Otter Tail Mille Lacs

Morrison Morrison

Polk Polk

Dakota Dakota

Dakota Dakota

Lac qui Parle

Yellow

Med Lincoln

Murray Redwood

Redwood Redwood

Redwood Sibley

Steele

S2 S3

S4 S5

S6 S7

S8 S9

S10 S11

S12 S13

S14 S15

S16

S17

S18

S19 S20

S21 S22

S23 S24

S25 Total


4

1 18

– –

– –

– –

–

–

–

9 3

– –

5 –

– –

– –

– –

– –

–

III

5

– 11

– –

– –

– –

–

–

–

4 –

– 2

– –

1 –

– 3

– –

– –

1

IV

3

1 12

– –

– –

– –

–

–

–

9 –

– –

– –

– 2

– –

– –

– –

–

V

6

– 11

– –

2 4

– –

–

1

–

– 1

– 2

– –

– –

– 1

– –

– –

–

VI

7

2 13

– 3

– –

– 1

–

1

–

2 1

– –

– –

– –

– –

– –

– 3

–

VII

9

2 21

– –

– –

– 1

–

–

–

– –

– –

2 1

1 2

– –

8 –

– 1

3

VIII

18

5 71

– 2

4 7

2 1

4

7

2

1 1

– 3

– –

5 4

– 7

4 –

– 10

2

IX

13

1 32

– –

1 –

2 1

1

1

3

2 –

2 –

– –

3 10

– 3

– –

– 2

–

X

12

2 35

– –

– –

4 4

–

3

–

– 1

– –

– 1

6 3

– 4

4 –

– 2

1

XI 1

20

1 160

7 7

12 11

16 –

10

17

11

2 2

6 1

7 16

6 –

– 1

13 –

– 13

XII

6

1 14

– –

– –

1 2

4

–

–

– –

5 –

– –

1 –

– –

– –

– –

–

XIII

6

4 12

– 1

– 1

– 2

–

–

3

– –

1 –

– –

– –

– –

– –

– –

–

XIV

16

7 169

– 18

8 2

9 12

9

–

–

– 18

17 6

7 1

– 2

– –

– –

33 2

18

XV

23

27 614

14 31

27 25

34 24

28

34

25

29 27

31 17

21 19

24 26

0 20

32 0

33 35

31

Total

4 119

0 2

3 5

1 5

1

2

5

2 5

9 18

11 5

10 7

– 14

3 –

1 3

3

unidentified strains§

Diversity from all 25 organic fields‡‡

11 15

2 6

5 5

6 8

5

7

5

7 7

5 6

4 4

8 7

0 7

5 0

1 8

7

No. of groups‡

No. of

315 1894

–

2 67

91 75

93 361

75

18

91

124 19

312 39

89 265

249 268

ND 403

109 ND††

–1 18

219

Richness (ACE)

288

242 –

069 132

158 168

149 205

151

162

179

185 159

195 273

189 119

236 23

ND 245

172 ND

013 192

162

Shannon index

Genetic diversity¶

012

012 –

05 036

026 023

03 021

026

028

021

02 034

023 008

017 047

012 014

ND 011

023 ND

094 021

032

Simpson’s index

*Total number of indigenous soybean–nodulating bradyrhizobia of each farm submitted for HFERP DNA fingerprinting. †Number of indigenous soybean-nodulating bradyrhizobia in each group and field after excluding the ungroup strains. ‡No. of predominant genetic groups in each farm based on HFERP DNA fingerprinting at 92% relative similarity. §Not all of the unidentified strains were singletons. Several of the unidentified strains shared the same genotype but were represented by fewer than 10 isolates. ¶Genetic diversities were calculated based on a total number of bacterial entries submitted to HFERP DNA fingerprinting. **Number of different genotypes in each field before excluding the ungrouped strains. ††ND, not determined. ‡‡Diversity from all 25 organic fields was calculated based on HFERP fingerprinting before excluding the ungrouped strains (Fig. 2a).

6

4

14 33

30 30

35 29

Number of fields in each group

4

–

– 23

6

–

– 12

– –

– –

31 733

– –

– 3

7 –

– –

– –

– –

1 –

– 3

– –

– –

– 1

– –

3 –

– –

– –

– 2

– –

–

–

5

II

– –

I

–

30

31 32

40 35

32 24

34 33

0 34

35 0

34 38

34

Hubbard

S1

Total entries*

County

Field no.

Number of soybean-Bradyrhizobium isolates in each genotypic group†

Table 4 Genetic diversity and number of soybean-nodulating bradyrhizobia in each group isolated from soils from 25 organic fields in Minnesota

044

071 –

026 038

046 049

042 061

045

045

053

054 046

053 077

055 038

067 066

ND 07

048 ND

004 053

046

Evenness

79

15 –

2 6

7 7

7 13

6

9

8

9 10

13 19

8 7

14 14

0 16

8 0

2 11

10

No. of genotypes**

M. Wongphatcharachai et al. Diversity of soybean-bradyrhizobia from organic farms

1159



P = 0021), evenness (r = 0556, P = 0011), relative abundance of unidentified strains (r = 0798, P < 0001) and the number of different strains (r = 0510, P = 0022) (Table S2). Distribution of serotypes of soybean-bradyrhizobia in organic fields Serological analyses were performed by using immunospot blot assays to identify serotypes of each predominant genotypic group (Table 5). The B. japonicum USDA 127 strains (part of serocluster 123) made up the largest percentage of the population (404%; group VIII, X, XI and XII) followed by B. japonicum USDA 4 (318%; group XIII, XIV and XV) and USDA 123 strains (158%; group VI, VII and IX). Isolates in groups XIII and XIV were identified by using 16S rRNA gene sequencing (~ 1300 bp) and strains were found to be 100% identical to B. japonicum strain USDA 4, thus samples in those two groups were reported as serotype USDA 4 strains.

This serotype was frequently detected among organic farms in Minnesota (318% of isolates) and was obtained from 18 farms in 11 counties (except Mille Lacs and Yellow Medicine). Application of soybean inoculants did not significantly affect serotype distributions (P > 005). The distribution of serotype USDA 127 and USDA 135 strains were significantly greater at farms where soybean was the crop in the year prior to sampling, compared to farms where common bean was planted as the previous crop at P = 0038 and 0028, respectively. The relative abundance of serotype USDA 127 strains was significantly different among the locations (P = 0042, Table 3). The relative abundances of several serotypes were inter-correlated. For example, the relative abundance of USDA 4 strains was negatively correlated with abundances of USDA 127 and 135 (r = 0641 and 0571; P = 0001 and 0006, respectively), and the abundances of strains in serotypes USDA 127 and 135 were positively correlated (r = 0532, P = 0006).

Table 5 Serological assignment (via immuno dot-blot assays) of 614 isolates of Bradyrhizobium japonicum strains isolated from 25 organic fields in Minnesota Number of isolates and percentage (%) in each serotype Farm no.

Counties

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 Total

Hubbard Hubbard Clay Clay Otter Tail Otter Tail Mille Lacs Morrison Morrison Polk (WI) Polk (WI) Dakota Dakota Dakota Dakota Lac qui Parle Yellow Med Lincoln Murray Redwood Redwood Redwood Redwood Sibley Steele

No. of strain 31 33 35 32 – – 20 24 26 21 19 31 17 29 27 25 34 28 34 24 27 25 14 31 27 614

USDA 4* 18 (581) 33 (100) 2 (57) – – – –

USDA 94 5 (161) – – – – – 1 (50)

1 2 7 1 23 6

(42) (77) (333) (53) (742) (353)

– 18 (667) 3 (120) – 13 10 16 8 3

(464) (294) (667) (296) (120)

– 19 (613) 12 (444) 195 (318)

USDA 110

– 3 (115) – – – 3 (176) – – – – – – – – – – – – 12 (20)

1 (32) – – – – – – – 2 (77) 5 (238) – – – 18 3 – – – – – – – – – 2 31

(621) (111)

(74) (51)

USDA 123 3 – 13 4 – – 10 5 4 – – – 5 3 3 2 9 4 2 2 6 11 – 5 7 97

USDA 127*

(65)

5 (161) –

(371) (125)

18 (514) 25 (781) – –

(500) (208) (154)

(294) (103) (111) (80) (265) (143) (59) (83) (222) (440) (161) (259) (158)

8 16 15 9 18 8 1 4 3 14 21 11 22 6 13 11 7 7 6 248

(400) (667) (577) (429) (947) (258) (59) (138) (111) (560) (618) (393) (647) (250) (482) (440) (500) (226) (222) (404)

USDA 129

USDA 135

– – – – – – 1 1 – – – – 2 4 – – – – – – – – – – – 8

– – 2 (57) 3 (94)

(50) (42)

(118) (138)

– – – 1 (42) – – – – – – – 6 (240) 4 (118) – – – – –

(13)

7 (500) – – 23 (38)

*The relative abundance of USDA 4 was negatively correlated with USDA 127 (r = 0641, P = 0001). Number in parenthesis indicates percentage (%) of serotype in each group.

1160



Soil chemical properties and distribution of serotypes Soil pH was significantly and positively correlated with the relative abundance of USDA 127 strains (r = 0582, P = 0004) and inversely correlated with the relative abundance of USDA 4 (r = 0469, P = 0024). Interestingly, the concentration of nitrate-nitrogen (NO3-N) was significant positively correlated with the relative abundance of strains in serotype USDA127 (r = 0720, P < 0001), and inversely correlated with the relative abundance of serotype USDA 4 (r = 0441, P = 0040; Table S2). Grouping of soil samples based on farm locations (Fig. 1) indicated that the relative abundance of strains in serotype USDA 127 was significantly greater at farms in Group 3, compared to soil samples in Group 4 (P = 0011). Moreover, soil pH and concentration of NO3-N was also significantly greater at farms in Group 3 with P = 0027 and 0007, respectively (Table S3). Discussion DNA fingerprinting methods are commonly used in bacterial biogeography and epidemiology studies (Fajardo-Cavazos and Nicholson 2006). The rep-PCR DNA fingerprinting technique, using the BOX A1R primer, has been used to amplify specific genome regions located between adjacent BOX repetitive elements (154 bp), and is commonly found in a number of gram positive and negative bacterial species (Martin et al. 1992; Ishii and Sadowsky 2009). This technique has proven to be a valuable tool for genotyping due to relative simplicity and reproducibility as well as high discriminatory power for clustering inter- and intra-specific bacterial genotypes (Olive and Bean 1999). In this study, the HFERP DNA fingerprinting technique was modified to include an internal standard to circumvent issues of inter-gel variability (Johnson et al. 2004). Moreover, HFERP fingerprinting output can be easily analysed by computer assisted methods making HFERP fingerprinting a practical tool for use in environmental microbial studies (Johnson et al. 2004). It has been reported that Bradyrhizobium serocluster 123 strains comprise a major component of field populations found in conventional soybean farms in the Midwest U.S. (Schmidt et al. 1986). Based on differences in their somatic antigens (Date and Decker 1965), this serocluster is defined by three serotypes, represented by strains USDA 123, USDA 127 and USDA 129 (Schmidt et al. 1986). Strains in serocluster 123, especially strain USDA 123, have shown exceptional competitive ability. These strains, however, are reported to be less effective for N2-fixation than other strains, such as USDA 110, and may negatively affect soybean productivity (Caldwell


and Vest 1970; Ellis et al. 1984; Moawad et al. 1984). Therefore, displacement of members of serocluster 123 with more effective B. japonicum strains may significantly increase soybean productivity, as shown by using inoculant strains USDA 110, 122 (CB1809) and 138 (Kvien et al. 1981; Kogan et al. 1987). However, these latter strains are more likely to lack competitive ability against indigenous strains, as relatively few were identified in this study. In contrast with what was reported by Shiro et al. (2013), B. elkanii was found in only about 20% of the population in four farms. This result is consistent with other studies that have shown that B. elkanii strains are more prevalent in southern U.S. soils than those in the north (Keyser et al. 1984), at least using modern soybean cultivars. Serological testing using antibodies against USDA 122, 123, 127, and 129 can provide false-positive results due to cross-reactivity, as previously reported (Date and Decker 1965; Schmidt et al. 1986). Therefore, in this study, the results of HFERP DNA fingerprinting profiles together with serological assays using cross-adsorbed, serotype-specific antisera were performed to identify and confirm the serological identity of nodule isolates. It should be noted that only 5 of 32 isolates (156%) from farm S1 in Hubbard County were located in the same group with the reference strain B. japonicum USDA 122 (Fig. 2a). Strain USDA 122 is a parent strain of a commercial inoculant CB1809, and inoculation with this strain has been documented as being effective at promoting N2-fixation with most soybean cultivars (Keyser and Griffin 1987). This is also a member of serocluster 123 (Schmidt et al. 1986). The lack of USDA 122 populations in organic fields in this study is similar to what has been shown for non-organic fields (Keyser et al. 1984). Interestingly, B. japonicum strain USDA 4 was found to comprise a previously unrecognized, predominant population of indigenous soybean-bradyrhizobia in organic farm fields. We hypothesize that elevated abundance of this strain may be due to its highly competitive nature in organic farming systems, in contrast with what has been reported in several studies done at conventional farms (Keyser et al. 1984; Schmidt et al. 1986; Cregan et al. 1989; Shiro et al. 2013). Strain USDA 4, the type strain for this serotype, was originally isolated from a soybean field in Iowa in 1932 (Van Berkum and Fuhrmann 2000) and is not frequently isolated from conventional farm fields. Among all the samples in this study, soil pH was related to the distribution of two of the most abundant serotypes strains, USDA 4 and 127, and it was also significantly associated with inoculant application practice. Serotype strain USDA 4 may represent a previously


1161



unrecognized, dominant and indigenous serotype in these soils that is displaced due to biological and/or chemical changes associated with conventional farm managements. In this study, genetic diversity and relative abundance of serotypes did not differ significantly by inoculant application practices, suggesting that inoculation strategies used by farmers may not displace indigenous bradyrhizobial strains. The results of soil chemical properties revealed that most of the farms contained the essential nutrients, nitrogen (N), phosphorous (P) and potassium (K) at elevated concentrations. The influence of high concentrations of P and K on legume-rhizobium symbiosis also remains unclear. Soil nitrogen concentrations in organic farms are not well-understood, since the amount of nitrogen is dependent upon the previous legume crops, crop rotations, as well as manure history. However, the amount of NO3-N recommended should not exceed 25 ppm and nitrate testing has now evolved as accepted best management practices (BMPs) to determine and improve the accuracy of the amount of nitrogen needed for agriculture farms in Minnesota (Rehm and Schmitt 2002). In contrast, it is well-documented that nitrate is a potent inhibitor of nodulation and nodule activity in soybean. In the presence of nitrate, the number of nodules formed on soybean are often reduced and delayed (Streeter 1988). While nodule trapping used in this study was done under controlled conditions in the laboratory, the diversity of indigenous soybean-bradyrhizobia recovered on soybeans may be different in field conditions using other cultivars. However, the technique does give a good estimate of Bradyrhizobium concentration and rough approximation of diversity in field soils, and is an accepted practice for this type of analysis. That said, our next goal is to measure bradyrhizobial population density and diversity directly at organic farms sites using several different soybean trap cultivars. Result from this study may be useful for strain selection for future studies e.g. N2-fixation assays and field-based yield experiments. Further characterization of the competitive ability of these strains and their efficiency at N2-fixation may also be a useful benefit to organic farmers, since it can provide strategies to improve soybean production without application of synthetic fertilizers. In summary, high levels of B. japonicum USDA 4 strains were found to be predominant populations in organic farms in Minnesota. This may highlight difference in soil edaphic factors in organic vs conventional soybean farms. The relative abundance of USDA 4 strains was negatively correlated with the abundances of strains in serogroup 127 and levels of NO3-N in soils, and these may also contribute to this difference. A long1162

term study for N2-fixation with selected and improved indigenous soybean-bradyrhizobial strains would be important to establish new inoculants for organic farms. Acknowledgements This work was support, in part, from a grant 201151300-30743 from USDA-NIFA. The authors would also like to thank Patrick Elia from USDA-ARS, Beltsville, MD for providing Bradyrhizobium japonicum USDA 122, USDA 123 and USDA 129 reference strains, and the organic farmers in Minnesota and Wisconsin who participated in our study. Authors’ contributions MW and MS designed the study of diversity of soybeanbradyrhizobia in organic farms and MW performed HFERP fingerprinting, serological assay, statistical analysis and drafted the manuscript. CS and PW helped wrote the manuscript and gave valuable suggestions for data analysis, and CS performed statistical analysis. KMM and CCS contacted the farmers, collected soil samples from organic farms, and provided soybean seeds for the MPN method. MJS supervised the study design, data analyses and helped write the manuscript. All authors read and approved the final manuscript. Conflict of interest The author(s) declare that they have no competing interests. References Caldwell, B.E. and Hartwig, E.E. (1970) Serological distribution of soybean root nodule bacteria in soils of southeastern USA. Agron J 62, 621–622. Caldwell, B.E. and Vest, G. (1970) Effects of Rhizobium japonicum strains on soybean yields. Crop Sci 10, 19–21. Chao, A. and Shen, T.-J. (2010) Program SPADE (species prediction and diversity estimation). Program and User’s Guide published at http://chao.stat.nthu.edu.tw. Comly, H.H. (1987) Landmark article Sept 8, 1945: cyanosis in infants caused by nitrates in well-water By Hunter H. Comly. JAMA 257, 2788–2792. Cregan, P.B., Keyser, H.H. and Sadowsky, M.J. (1989) Host plant effects on nodulation and competitiveness of the Bradyrhizobium japonicum serotype strains constituting serocluster 123. Appl Environ Microbiol 55, 2532–2536. Date, R.A. and Decker, A.M. (1965) Minimal antigenic constitution of 28 strains of Rhizobium japonicum. Can J Microbiol 11, 1–8.



Dobert, R.C., Breil, B.T. and Triplett, E.W. (1994) DNA sequence of the common nodulation genes of Bradyrhizobium elkanii and their phylogenetic relationship to those of other nodulating bacteria. Mol Plant Microbe Interact 7, 564–572. Dombek, P.E., Johnson, L.K., Zimmerley, S.T. and Sadowsky, M.J. (2000) Use of repetitive DNA sequences and the PCR To differentiate Escherichia coli isolates from human and animal sources. Appl Environ Microbiol 66, 2572–2577. Ellis, W.R., Ham, G.E. and Schmidt, E.L. (1984) Persistence and recovery of Rhizobium japonicum inoculum in a field soil. Agron J 76, 573–576. Elmi, A.A., Madramootoo, C., Egeh, M., Liu, A. and Hamel, C. (2002) Environmental and agronomic implications of water table and nitrogen fertilization management. J Environ Qual 31, 1858–1867. Fajardo-Cavazos, P. and Nicholson, W. (2006) Bacillus endospores isolated from granite: close molecular relationships to globally distributed Bacillus spp. from endolithic and extreme environments. Appl Environ Microbiol 72, 2856–2863. Ham, G.E., Frederick, L.R. and Anderson, I.C. (1971) Serogroups of Rhizobium japonicum in soybean nodules. Agron J 63, 69–72. Hoagland, D.R. and Arnon, D.I. (1950) The Water-Culture Method for Growing Plants Without Soil.Berkeley, CA: College of Agriculture, University of California. Ishii, S. and Sadowsky, M.J. (2009) Applications of the repPCR DNA fingerprinting technique to study microbial diversity, ecology and evolution. Environ Microbiol 11, 733–740. Johnson, L.K., Brown, M.B., Carruthers, E.A., Ferguson, J.A., Dombek, P.E. and Sadowsky, M.J. (2004) Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution. Appl Environ Microbiol 70, 4478–4485. Keyser, H.H. and Griffin, R.F. (1987) Beltsville Rhizobium Culture Collection Catalog. Beltsville, MD: U.S. Department of Agriculture. ARS-60. Keyser, H.H. and Li, F.D. (1992) Potential for increasing biological nitrogen-fixation in soybean. Plant Soil 141, 119–135. Keyser, H.H., Weber, D.F. and Uratsu, S.L. (1984) Rhizobium japonicum serogroup and hydrogenase phenotype distribution in 12 states. Appl Environ Microbiol 47, 613–615. Kogan, S.C., Doherty, M. and Gitschier, J. (1987) An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. Application to hemophilia A. N Engl J Med 317, 985–990. Kvien, C.S., Ham, G.E. and Lambert, J.W. (1981) Recovery of introduced Rhizobium japonicum strains by soybean genotypes. Agron J 73, 900–905.


Lane, D.J. (1991) 16S/23S rRNA sequencing. In Nucleic Acids Techniques in Bacterial Systematics ed. Stackebrandt, E. and Goodfellow, M. pp. 115–147. Chichester: John Wiley & Sons. Madrzak, C.J., Golinska, B., Kroliczak, J., Pudelko, K., Lazewska, D., Lampka, B. and Sadowsky, M.J. (1995) Diversity among field populations of Bradyrhizobium japonicum in Poland. Appl Environ Microbiol 61, 1194– 1200. Martin, B., Humbert, O., Camara, M., Guenzi, E., Walker, J., Mitchell, T., Andrew, P., Prudhomme, M. et al. (1992) A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. Nucleic Acids Res 20, 3479–3483. Moawad, H.A., Ellis, W.R. and Schmidt, E.L. (1984) Rhizosphere response as a factor in competition among three serogroups of indigenous Rhizobium japonicum for nodulation of field-grown soybeans. Appl Environ Microbiol 47, 607–612. Olive, D.M. and Bean, P. (1999) Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol 37, 1661–1669. Randall, G.W. and Mulla, D.J. (2001) Nitrate nitrogen in surface waters as influenced by climatic conditions and agricultural practices. J Environ Qual 30, 337–344. Rehm, G. and Schmitt, M. (2002) Using the soil nitrate test in Minnesota. University of Minnesota Extension Service. Document retrieved from: http://www.extension.umn.edu/ agriculture/nutrient-management/nitrogen/using-thesoil-nitrate-test-in-mn/index.html. Sadowsky, M.J., Tully, R.E., Cregan, P.B. and Keyser, H.H. (1987) Genetic diversity in Bradyrhizobium Japonicum serogroup 123 and its relation to genotype-specific nodulation of soybean. Appl Environ Microbiol 53, 2624– 2630. Schmidt, E.L., Zidwick, M.J. and Abebe, H.M. (1986) Bradyrhizobium japonicum serocluster 123 and diversity among member isolates. Appl Environ Microbiol 51, 1212– 1215. Shannon, C.E. (1948) A mathematical theory of communication. Bell Syst Tech J 27, 379–423. Shiro, S., Matsuura, S., Saiki, R., Sigua, G.C., Yamamoto, A., Umehara, Y., Hayashi, M. and Saeki, Y. (2013) Genetic diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in the United States. Appl Environ Microbiol 79, 3610–3618. Simpson, E.H. (1949) Measurement of diversity. Nature 163, 688. Singleton, P.W. and Tavares, J.W. (1986) Inoculation response of legumes in relation to the number and effectiveness of indigenous Rhizobium populations. Appl Environ Microbiol 51, 1013–1018. Smith, R.A., Alexander, R.B. and Wolman, M.G. (1987) Water-quality trends in the nation’s rivers. Science 235, 1607–1615.


1163



Somasegaran, P. and Hoben, H.J. and University of Hawaii at Manoa. Niftal Project. (1985) Methods in LegumeRhizobium Technology. Paia, Maui, HI, USA: University of Hawaii NifTAL Project and MIRCEN, Dept. of Agronomy and Soil Science, Hawaii Institute of Tropical Agriculture and Human Resources, College of Tropical Agriculture and Human Resources. Streeter, J. (1988) Inhibition of legume nodule formation and N2 fixation by nitrate. CRC Crit Rev Plant Sci 7, 1–23. Streeter, J. (1994) Failure of inoculant rhizobia to overcome the dominance of indigenous strains for nodule formation. Can J Microbiol 40, 513–522. Triplett, E.W. and Sadowsky, M.J. (1992) Genetics of competition for nodulation of legumes. Annu Rev Microbiol 46, 399–428. USDA (2010) Economic research service: National Agricultural Statistics Service. [Online] Avaible: http://usda.mannlib. cornell.edu/MannUsda/viewDocumentInfo.do? documentID=1251. USDA (2011) Economic research service: organic production. Table 7 Certified organic beans. Acres of soybeans, dry beans, dry peas/lentils by State, 1997 and 2000-11. Last updated: 9/27/2013. [Online] Available: http://www.ers. usda.gov/data-products/organic-production.aspx#. Uml4EZETuDU. USDA (2013) Economic research service: adoption of genetically engineered crops in the U.S. [Online] available: http://www.ers.usda.gov/data-products/adoption-ofgenetically-engineered-crops-in-the-us/recent-trends-inge-adoption.aspx. Van Berkum, P. and Fuhrmann, J.J. (2000) Evolutionary relationships among the soybean bradyrhizobia

1164

reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence. Int J Syst Evol Microbiol 50, 2165–2172. WHO (2011) Nitrate and nitrite in drinking-water. Document retrieved from: http://www.who.int/water_sanitation_ health/dwq/chemicals/nitratesnitrite/en/index.html. Wongphatcharachai, M., Wang, P., Enomoto, S., Webby, R.J., Gramer, M.R., Amonsin, A. and Sreevatsan, S. (2013) Neutralizing DNA aptamers against swine influenza H3N2 viruses. J Clin Microbiol 51, 46–54. Zahran, H.H. (1999) Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol Mol Biol Rev 63, 968–989.

Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Soil chemical properties of organic fields in Minnesota. Table S2 Statistical correlations of soil chemical properties compared with genetic diversity and relative abundance of serotypes. Table S3 Correlations of soil pH, NO3-N concentration, and relative abundance of serotype USDA 127 strains in different locations of organic farms in Minnesota.
