A presentation by Dr. Sidney Stürmer

1. Project: Alternatives to slash and burning
ICRAF/TSBF
Sampling date: April, 24 to May 7, 1997
Location Sites (see map):
Isolation and selection of rhizobia PP PP TH
TH
strains, from soils of the western Rondônia State - Theobroma (TH), Ji-Paraná (JP) RECA JP
RECA JP
Acre State - RECA, Pedro Peixoto (PP)
Amazon, to produce inoculants
Fatima M.S.; Messias J.B.Andrade, Adão M. Lacerda, Adriana
i S i J.B.Andrade, dã
d d Lacerda, d i
d
S.Lima (UFLA), João P.A.R.Pereira, André L.L.Soares,
P.A.R.Pereira, L.L.Soares,
Paulo A.A. Ferreira, Bruno L.Soares Land Use Systems(LUS) studied at three sites each:
Disturbed Forest (DFOR) - TH, RECA, PP
Fallow (FAL) - TH1, TH2, PP
Crop (CROP) - TH1 (beans), TH2 (cassava), PP (rice)
Agroforestry (AGF) - JP1 (coffee & Schizolobium amazonicum),
JP2 (coffee & Hevea brasiliensis), RECA (Theobroma grandiflorum &
Bactris gassipaes & Bertolethia excelsa)
Pasture (PAST) - TH, JP, PP
GENERAL HYPOTHESIS
It is a general hypothesis that agricultural intensification results in a
reduction in soil biodiversity leading to a loss of function
detrimental to resilience and sustained productivity. Therefore, the
evaluation of the biodiversity of important functional groups of soil
organisms across scales of intensification in land use systems must
Forest Transition pasture/forest Hevea brasiliensis & Coffea arabica
provide valuable information on how to conserve and manage soil (Theobroma,RO) (Theobroma, RO) (agroforest system)(Ji-Paraná, RO)
system)(Ji-
sustainability.
t i bilit
OBJECTIVE
Verify the effect of different Land Use Systems, across a scale of
agricultural intensification on: 1) the labile component of the soil
organic fraction: the microbial biomass and 2) the biodiversity of
Phaseolus vulgaris
the following functional groups of soil organisms: macrofauna, after slash and burning
mycorrhiza, nematodes, rhizobia. (Theobroma, RO) Pasture (Ji-Paraná,RO)
(Ji- Fallow (Theobroma,RO)
Leguminosae nodulating bacteria SOIL CHEMICAL & PHYSICAL CHARACTERISTICS
Spatial Sampling Strategy: PAST CROP
LAND USE SYSTEMS
FAL DFOR AGF
10
23.5
20 sub-samples / transect 4 x 25 m Al (cmolc/dm3 ) 0.09L*C ** 0.90M B 0.24 L C 1.83H A 0.28L C
Ca (cmolc/dm3 ) 1.9M B 2.2 M AB 2.9M A 0.8L C 2.5 M AB
In each site
9
Mg (cmolc/dm3 ) 0.8M AB 0.7 M AB 0.9M A 0.5L B 0.9 M A
1 composite sample (20 sub-samples) K (mg/dm3 )
P Mehlich (mg/dm3 )
82H A
2.3 LB
66M AB
5.7 L A
50 M B
3.1L AB
59M AB
1.7L B
61M AB
2.8 L B
8
18.5 pH ( H 2 O 1:2.5) 5.8Mac A 5.2 Mac B 5.3 Mac B 4.4 Hac C 5.7 Mac A
Depth: 0-20 cm H+Al (cmolc/dm3 ) 2.4L C 4.2 M B 3.3M BC 7.2H A 2.5 M C
7
Sum of bases (cmolc/dm3 ) 2.9M A 3.1 M A 4.0M A 1.5M B 3.6 M A
CEC ef (cmolc/dm3 ) 3.0M B 4.0 M A 4.2M A 3.3M AB 3.9 M AB
13.5 CEC pH 7.0 (cmolc/dm3) 5.3M C 7.3 M B 7.3M B 8.7M A 6.1 M C
6
Al saturation (%) 4LC 29 M B 11 L C 59 H A 6LC
Bases saturation (%)
ases satu at o 55M AB 43L B
3 52 M AB
5 16VL C
6 63M A
Soil (chemical and physical analyses): S-SO 4 (mg/dm3) 9.9M B 26.2H A 14.4 H B 17.2 H AB 9.2 M B
5
B (mg/dm3 ) 0.30 M 0.25 M 0.28 M 0.23 M 0.40 M
3 composite samples (6, 6 and 4 sub- Cu DTPA (mg/dm3 )
Zn DTPA (mg/dm3 )
1.6H AB 1.4 H B 1.7 AB 2.0H A 1.4 H B
8.5 4.4H 3.5 H 3.7H 4.0H 4.4 H
4
samples). Mn DTPA (mg/dm3 )
Fe DTPA (mg/dm3 )
40.9H A
85H B
17.7H B
116H B
34.0 H A
282H B
11.8H B
544H A
35.9H A
118H B
3
% P2O 5 total (H2 SO 4) 3D 7A 5B 7A 4C
% P2 O 5 total clay (H 2SO 4) 13 14 14 13 14
3.5 Sand 58 A 36 D 46 C 28 E 63 B
2
Other functional groups studied: microbial Silt 16 A 12 B 14 B 15 AB 12 B
biomass, arbuscular mycorrhyzal fungi,
Meter
Clay 27 E 52 B 41 C 56 A 35 D
1
nematodes, fauna ** Numbers followed by different letters in the same line differ at P=0,05 (Tuckey´s test)
* respectively- VL=very low, L=low, M=medium, H=high levels
3
2
1
0
Meter
3 countries: Brazil, Indonésia, Cameroon
1

2. Density and diversity of rhizobia isolated from Amazonian soil
under different land use systems
(Pereira, 2000)
Phenotypic diversity (total protein profiles by SDS-PAGE) of
Trap species:
Bradyrhizobium strains isolated from Amazonian soils under
Macroptilium atropurpureum (siratro)
different land use systems by using siratro as trap species
Vigna unguiculata (cowpea)
(Lima , 2003, Lima et al. 2005, PAB)
plastic pouches
inoculated with soil supensions dilluted
from 5 L d U systems
f Land Use
(Forest, fallow,pasture,
agroforest, crop)
Symbiotic efficiency withVigna unguiculata of
Bradyrhizobium strains isolated from Amazonian soils
by using siratro as trap species
(Lima , 2003)
1 2 3 4 5 6 7 8 9
Crop
Fallow
Pasture
Forest
Agroforest
Reference strains
Cluster 6
SEMIA 5019/ BR29-
UFLA04-8118
B.elkanii
UFLA04-28
UFLA04-85
UFLA03 84
UFLAO4-321
ATCC10324T
UFLA04-339
UFLA04-110
INPA 0311 B
UFLA04 214
UFLA04-1332
10 11 INPA0311B- .e.
UFLA3-84 B. sp.
ATCC10234T-B.japonicum
)
Leonard jars inoculated with strains trapped from soil with siratro
Strain selection- cowpea
selection-
Cowpea-
Cowpea-trap rhizobia from soil Nodules collected Rhizobia isolation
FLA041001
Sho Total N (mg plant -1)
FLA03 84
FLA04 314
600
FLA04 1417
FLA04 1020
plus N H O3
-N 4N
FLA04 321
U
U
FLA04 546
500
U
FLA04 559
Crop
U
FLA04 885
FLA04 1309
FLA03 170
U
FLA04 110
FLA04 1332
FLA04 1272
400 Fallow
U
U
FLA04 849
U
FLA04 1413
FLA04 1521
PA03 11B
U
Pasture
U
FLA04 401
FLA04 917
U
U
FLA04 204
300
FLA04 640
U
FLA04 151
U
U
Forest
FLA 926
MIA 6145
FLA04 230
U
IN
min mineral N
U
U
U
200
U
Agroforest
U
U
U
SE
U
Controls
oot
100
nus
Reference strains
0 Pots with soil Leonard jars
Shoot total N of cowpea inculated with strains isolated from soil under diverse LUS
by using siratro as trap species (Leonard jars)
FIELD EXPERIMENTS
2

3. Cowpea inoculated with selected Bradyrhizobium strains Cowpea cultivar BR14-Mulato grain yields (kg.ha-1 ) in
1500
isolated from Amazon region : Grain Yields Kg/ha Perdões Municipality (MG) inoculated with selected Bradyrhizobium
1400
A (Lacerda et al., 2004, Ceres
strains isolated from Amazon region.
-1
A (Soares et al., 2006, RBCS) populations: 23 cel.g soil
Native
1300
A A Origin of strains: a
1000 a a
1200 INPA3-11B-
INPA3-11B- Centrosema sp./AM 900
UFLA3-
UFLA3-36 – fallow/RO
Y
800 b b
1100
UFLA 3-84 - pasture/RO
3- I
UFLA3-129-
UFLA3-129-pasture/AC 700
B E 600
1000 L c
500 cc
D 400
900
CONTROL INPA03-11b UFLA03-36 UFLA03-84 N-fertilizer
300
Soil pH = 5.5 200
without Low input P+ K 100
Inoculation with
inoculation 0
Bradyrhizobium strains Test. BR UFLA UFLA UFLA INPA Test.
Cowpeas are most consumed in Brazil’s north and northeast regions (most poor S/ N 2001 03-36 03-129 03-84 03- C/ N
regions), where it is cultivated by small farmers that don’t use this biotechnology 11B Soil pH = 4,9
(inoculation with nitrogen-fixing bacteria), because they don’t know it.
nitrogen- N-Uréia 70 Kg.ha-1
Means with same letters are equal, Scott Knott test, 5% de probability Low input P+ K
Phylogenetic affinity of the partial 16SrDNA sequences of Bradyrhizobium
Grain yields (kg.ha-1) strains isolated from diverse forest Leguminosae species For each isolate
the most similar published sequence at GenBank is shown.
Cultivar Poços de Caldas, Iguatama Municipality (MG), inoculated with Strain Most similar Accession Identities
published bp/bp (%)
Bradyrhizobium Strains Isolated from Amazon region. sequence
BRFL27 B.elkanii. AF208510 723/729 (99%)
(Pereira et al., 2004) Native populations: 48 cel.g-1 soil BR3617 B.elkanii AF208510 669/685 (97%)
BR3901 B.elkanii AF208510 679/708 (95%)
1200 a a BR5004 B.elkanii AF208510 692/710 (97%)
Y a BR5202 B. sp BSPZ94812 629/641 (98%)
BR5301 B.elkanii AF293379 713/726 (98%)
1000 BR5611 B.elkanii AF208510 614/667 (92%)
i b BR6009 B.sp AF384136 703/706 (99%)
800 c BR8404 B.elkanii AF208510 680/692 (98%)
e
d c BR8601 B.elkanii AF208510 704/714 (98%)
600 INPA311B B.elkanii AF208510 703/711 (98%)
INPA10A B.elkanii AF208510 712/728 (97%)
l INPA14A B.elkanii AF208510 726/734 (98%)
400 INPA33A B.sp AF384136 723/723 (100%)
d INPA54B B.sp AF193818 721/725 (99%)
200 INPA86A B.japonicum BJU69638 452/465 (97%)
INPA104A B.sp AF384136 676/691 (97%)
0 INPA125A B.elkanii AF208510 706/720 (98%)
Test. S/ UFLA BR UFLA UFLA Test. C/ INPA INPA170B B.sp AF384136 726/730 (99%)
INPA237B B.sp AF384136 728/737 (98%)
N 03-36 2001 03-129 03-84 N 03-11B INPA553A B.elkanii AF293379 704/722 (97%)
UFLA1-457 B. sp AF384136 733/738 (99%)
New cowpea
N- Uréia 80Kg.ha-1 UFLA 1-473 B.sp AF384136 345/365 (94%) inoculant strains!
Soil pH 5,9
UFLA3-84 B.sp AF384136 722/737 (97%)
Means with the same letter are equal Scott Knott, 5%probability
Low input P+ K Prefix BR isolated from Atlantic forest, INPA isolated from Amazônia,
UFLA isolated from heavy metal contaminated soil pot experiments
N 240 mg L-1
Phaseolus vulgaris shoot dry matter inoculated with diverse rhizobia
strains isolated from field nodules-
nodules-
UFLA02 127
Leonard jars (Pereira et al., 1998) TSBF report
Shoot dry matter (g)
BR322/ CIAT899
UFLA02 68
UFLA02 86
UFLA02 100
UFLA02 54
UFLA02 104
UFLA02 87
UFLA02 116
UFLA02 84
UFLA02 66
UFLA02 97
UFLA02 102
UFLA02 63
ATCC 10004 R.l
USDA 205 S.f
CFN 42 R.e.
UFLA02 60
Control
U
C
A
e o seu assunto qual é?
S
INPA 03-11b UFLA 03-84
Meio 79 Meio 79
SDS-
SDS-
PAGE
Cluster
Protein
profile
D F B B F B B B F B B A B Ref F Ref Ref Ref
3

4. Strain selection for beans
Field nodules or
Grain yields (kg.ha-1) of Phaseolus vulgaris cultivar
Trap species inoculated with soil suspensions Talismã in Perdões Municipality (MG) with selected
rhizobia strains from Amazon region (AM) (Soares et
al., 2006) Native populations: 4,2 X 103 cel.g-1 soil
1200 a
P a a
R 1000 a a
O 800 b
D
U 600 b
Ç 400
Ã
O 200
0
Test. UFLA CIAT UFLA UFLA UFLA Test.
S/N 02-100 899 02-68 02-86 02-127 C/N
Control Low input P +K
N-fertilizer N-Uréia 70 kg.ha-1
Phaseolus vulgaris beans Letras iguais não diferem entre si pelo teste de Scott Knott a 5% de probabilidade
Objective: Super diazotrophic bacteria
Phylogenetic affiliationβ of partial 16SrDNA sequences of
isolates from diverse Land Use Systems in Amazonia.
Isolate γ Most similar Accession Identities
published sequence bp
UFLA 2-68 Rhizobium sp. AF313906 636/709 (89%)
UFLA 2-100 Rhizobium sp. AF313906 718/722 (99%)
UFLA 2-127 Rhizobium RLU89828 392/427 (91%)
leguminosarum
Similarity determined with BLAST (REF). Isolated from Phaseolus vulgaris, nodules in the field Rondônia
Nitrogen fixation symbiotic (high efficiency) and or free-living
free-
AND
Other Plant Growth Promoting processes
AND
Tolerance to soils stresses
CIAT899 10K
10,0 10,0
Efficient strains for Phaseolus vulgaris from Amazonian land use systems 9,5
9,5
LOG UFC, mL-1
LOG UFC, mL-1
9,0
Three most efficient (nitrogen fixation) strains among 18 tested 9,0
8,5
8,5
8,0
pH5,0 y=4,89+4,52(1-exp(-0,17x)) R2:0,96
S 8,0 pH6,0 y=6,91+2,32(1-exp(-0,14x)) R2:0,94
7,5
pH5,0 y=6,67+2,85(1-exp(-0,08x)) R2:0,95
Soil characteristics cultural cultural pH6,9 y=7,64+1,55(1-exp(-0,09x)) R2:0,90
pH6,0 y=4,08+5,38(1-exp(-0,22x)) R2:0,96
pH6,9 y=6,66+2,84(1-exp(-0,08x)) R2:0,95
7,5 7,0
612 24 48 72 96 120 612 24 48 72 96 120 10
pH 6,0
Strains Land use/ pH 5,0 pH 6,9
Al+3 342K
3K
country pH cmolc dm-1 GR1 pH 10,0 10,0
8
EPS production
g de EPS/ g de Células
3K Rhizobium sp. Fallow/AM- 4,0 9,4 9,5 9,5
6
LOG UFC, mL-1
1
LOG UFC, mL-1
1
9,0
Brasil F acid 9,0
8,5 4
10K Rhizobium sp. Crop-AM- 5,1 2,5 8,0
8,5
8,0 2
Brasil F acid 7,5 pH5,0 y=5,01+4,36(1-exp(-0,16x)) R2:0,91
pH6,0 y=7,32 -0,035x+0,52x
0.5
R2:0,84
pH5,0 y=5,76+3,58(1-exp(-0,14x)) R2:0,93
pH6,0 y=6,73 -0,05x+0,73x
0.5
R2:0,89
0.5
pH6,9 y=6,89 -0,04x +0,65x R2:0,91 pH6,9 y=7,39+1,76(1-exp(-0,16x)) R2:0,83
7,0 7,5
342K R. tropici Pasture-AM- 5,2 2,3 612 24 48 72 96 120 612 24 48 72 96 120 0
3K 10K 342K CIAT 899
Brasil F acid TEMPO, h Tratam entos
CIAT899 R. tropici Colômbia ----- --------------- F acid
Growth of strains CIAT899, 10K, 3K e 342K in
CIAT899, 10K, 342K
liquid medium with different pH values.
values.
Cultural characteristic: 1. Growth rate, F (Fast);
characteristic: Fast)
4

5. 5