Supplementary data
for
Relationships among bulk soil physicochemical,
biochemical and microbiological parameters in
an organic alfalfa-rice rotation system
Lopes ARa,c, Bello Db, Prieto-Fernández Ab, Trasar-Cepeda Cb, Manaia CMc and Nunes
OCa*
a
LEPABE, Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia,
Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal
b
Dpto Bioquímica del Suelo, IIAG-CSIC, Apartado 122, E-15780 Santiago de Compostela, Spain
c
CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de
Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino Almeida,
4200-072 Porto, Portugal
Tables 2
Figure 1
* Corresponding author phone: +351 225081917; fax: +351 225081449; e-mail:
opnunes@fe.up.pt
Table S1 List of selective liquid media and incubation conditions used in this study to enumerate MPN of different cultivable microbial
populations
Aerobic heterotrophs
1
25
Selective media
(references)
Kidd et al., 2008
Anaerobic heterotrophs
2
28
Kidd et al., 2008
Aerobic ammonifiers
3
25
Kidd et al., 2008
Anaerobic ammonifiers
3
28
Kidd et al., 2008
Denitrifiers
3
30
Nogales et al., 2002
3
30
Cote and Gherna, 1994
3
28
Lin et al., 2008
Microbial populations
Incubation time (week) Temperature (ºC)
Aerobic diazotrophs
Anaerobic diazotrophs
*
*
The medium was supplemented with Na2CO3 (20 mg l-1)
References
Cote RJ, Gherna RL (1994) Nutrition and media. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (ed) Methods for general and molecular
bacteriology. American Society for Microbiology, Washington, DC, pp 173.
Kidd PS, Prieto-Fernandez A, Monterroso C, Acea MJ (2008) Rhizosphere microbial community and hexachlorocyclohexane degradative
potential in contrasting plant species. Plant Soil 302: 233-247.
Lin X, Zhao Y, Fu Q, Umashankara ML, Feng Z (2008) Analysis of culturable and unculturable microbial community in bensulfuron-methyl
contaminated paddy soils. Journal of Environmental Sciences 20: 1494-1500.
Lopes AR, Manaia CM, Nunes OC (2014) Bacterial community variations in an alfalfa-rice rotation system revealed by 16S rRNA gene 454pyrosequencing. FEMS Microbiol Ecol 87: 650-663.
Nogales B, Timmis KN, Nedwell DB, Osborn AM (2002) Detection and diversity of expressed denitrification genes in estuarine sediments after
reverse transcription-PCR amplification from mRNA. Appl Environ Microb 68: 5017-5025.
2
Table S2 Affiliation, abundance and frequency of OTUs with a fraction of variance higher than 0.4/1 with axis 1, i.e., with abundance
correlated with total C content or abundance of aerobic heterotrophs, and with axis 2, i.e., with abundance correlated with BAA-protease
activity or abundance of anaerobic diazotrophs, of the CCA analysis of the 16S rRNA gene pyrosequencing data (Lopes et al. 2014)
Domain
Bacteria
Phylum
Acidobacteria
Bacteroidetes
Class
Acidobacteria
Order
Genus
ASep
BApr
BSep
2
2
2
2
Total C content
Candidatus Koribacter
4
5
6
6
(% C)
Candidatus Solibacter
6
11
11
11
Acidobacteriales
"Solibacteres"
"Solibacterales"
Bacteroidia
Bacteroidales
2
2
2
2
Sphingobacteriia
Sphingobacteriales
1
1
1
1
2
3
5
5
2
5
8
8
0
1
1
1
0
0
1
1
8
7
5
4
Aerobic heterotrophs
Candidatus Koribacter
2
2
2
1
(aer Het)
Candidatus Solibacter
10
6
1
2
8
4
2
4
1
0
0
0
7
1
1
1
Cellulomonas
1
0
0
0
Demequina
1
0
0
0
Pilimelia
1
0
0
0
Flavobacterium
4
3
1
2
43
17
10
5
1
0
0
0
Anaerolineae
Anaerolineales
Anaerolinea
Bacteria
Acidobacteria
Actinobacteria
Acidobacteria
Acidobacteriales
"Solibacteres"
"Solibacterales"
Acidimicrobiia
Acidimicrobiales
Actinobacteria
Actinomycetales
Iamia
Bacteroidetes
Correlated parameter
AApr
Chlorobi
Chloroflexi
Abundance (n) and Frequency
(shadow) of OTUs
Flavobacteriia
Flavobacteriales
Sphingobacteriia
Sphingobacteriales
Emticicia
3
Table S2 Continuation
Domain
Bacteria
Phylum
Bacteroidetes
Class
Sphingobacteriia
Order
Genus
Sphingobacteriales Niastella
Pedobacter
Correlacted parameter
AApr
ASep
BApr
BSep
1
0
0
0
Aerobic heterotrophs
2
0
0
0
(aer Het)
Chloroflexi
"Ellin6529"
10
5
3
3
Gemmatimonadetes
"Gemm-1"
4
2
1
1
Gemmatimonadetes
3
1
1
0
Alphaproteobacteria
10
3
2
2
2
1
0
1
Asticcacaulis
1
0
0
0
Phenylobacterium
2
2
1
1
5
4
1
2
Balneimonas
1
0
0
0
Devosia
1
1
1
1
Kaistia
1
0
0
0
Rhizobium
1
1
1
1
Rhodospirillales
8
5
3
2
Sphingomonadales
11
4
3
4
4
4
1
2
1
2
1
2
Anaerobic diazotrophs
Candidatus Koribacter
1
2
2
2
(anaer N2)
Candidatus Solibacter
3
9
3
7
Proteobacteria
Caulobacterales
Rhizobiales
Novosphingobium
Bacteria
Abundance (n) and Frequency
(shadow) of OTUs
Acidobacteria
Acidobacteria
Acidobacteriales
"Solibacteres"
"Solibacterales"
Actinobacteria
Acidimicrobiia
Acidimicrobiales
1
6
1
4
Bacteroidetes
Sphingobacteriia
Sphingobacteriales
0
4
0
2
1
4
0
2
0
2
0
0
Chloroflexi
Anaerolineae
4
Table S2 Continuation
Domain
Bacteria
Phylum
Chloroflexi
Class
Anaerolineae
Order
Genus
Proteobacteria
Clostridia
ASep
BApr
BSep
0
1
0
0
Anaerobic diazotrophs
1
6
1
3
(anaer N2)
Caloramator
0
1
0
0
Clostridium
0
2
0
0
1
2
1
1
Methylobacterium
0
1
0
1
Rhodoplanes
1
1
1
1
0
3
1
1
0
3
0
1
4
0
4
3
BAA-protease activity
Candidatus Koribacter
1
0
1
0
(BAA)
Candidatus Solibacter
4
2
8
3
Anaerolineales
Clostridiales
Alphaproteobacteria
Rhizobiales
Rhodospirillales
Betaproteobacteria
Bacteria
Acidobacteria
Acidobacteria
Acidobacteriales
"Solibacteres"
"Solibacterales"
Bacteroidia
Bacteroidales
2
0
4
0
Sphingobacteriia
Sphingobacteriales
3
1
5
2
Chlorobi
0
0
2
0
Chloroflexi
1
0
1
0
0
0
2
0
0
0
2
0
1
1
1
1
4
0
4
2
1
0
1
0
Bacteroidetes
Anaerolineae
Anaerolineales
Proteobacteria
Correlacted parameter
AApr
"Ellin6529"
Firmicutes
Abundance (n) and Frequency
(shadow) of OTUs
Anaerolinea
Alphaproteobacteria
Rhizobiales
Afifella
5
frequency* = 0 %
frequency* > 0 and < 0.1 %
frequency* > 0.1 and < 0.5 %
frequency* > 0.5 and < 1 %
frequency* > 1 %
* sum of the percentage (number of reads / total number of reads) of each individual OTU present in the sample; Note: Each OTU referred to in the table is only related to one
explanatory parameter.
6
Fig. S1 Catabolic profiling data - density of microorganisms in soil from paddy A and B growing in the tested substrates before rice seeding and
rice harvesting all the values are means ± standard deviation (n=3)
7
Cyclod: α-cyclodextrin; Glycog: glycogen; Tw80: Tween 80; Tw60: Tween 60; Gluc: α-D-glucose-1-phosphate; PheEthAm: β-phenyl ethylamine; Put: putrescine. Cellob: D(+) cellobiose; GluAm: N-acetyl-D-glucosamine; Lac: α-lactose; Xyl: D-(+) xylose; MetGlu: β-methyl D-glucoside; Malt: maltose; Eryth: i-erythritol; Gly: glyceraldehyde.
Phe: L-phenylalanine; Glu: L-glutamic acid; Thr: L-threonine; Asn: L-asparagine; Arg: L-arginine; Ser: L-serine. Galactur: galacturonic acid; GalLact: D-galactonic acid δlactone; Malon: malonic acid; Mal: malic acid; Ketobut: α-keto butyric acid; o-OHBenz: o-hydroxybenzoic acid; p-OHBenz: p-hydroxybenzoic acid.
8