south african rhizobium culture collection …south african rhizobium culture collection...
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South African Rhizobium Culture Collection
(SARCC),WDCM 968
Agricultural Research Council, Plant Protection Research Institute
Private bag X134, Queens wood 0121, Pretoria, South Africa
AI Hassen (PhD)
Agricultural Research Council (ARC)
• Principal Agricultural research institution in South Africa.
• Conduct research; derive research & development.
• Technology & development transfer.
• Facilitate/ensure natural resources conservation.
• Alleviate poverty.
• Since 1960s, ARC-Plant Protection Research Institute
• South African Rhizobium Culture Collection (SARCC)
• Several hundreds of rhizobia (>700 strains)
• Long term projects, field collections, client samples,
international institutions
• Rhizobium, Bradyrhizobium, Sinorhizobium, Mesorhizobium
• Routinely maintained (viability, purity )
• WFCC (http://www.wfcc.info/ccinfo/collection/by_id/968)
The SARCC
• The Nitrogen fixing bacterial symbionts of legumes
• The most important soil microorganisms in agriculture
• Ability of fixing atmospheric nitrogen (N2)
• Biological Nitrogen Fixation (BNF) by Rhizobia spp.
Flavonoids (Legumes) Nod Factors (Rhizobium) Root nodules Bacteroides
• N2 + 3H2 2NH3 (Cost effective) Nitrogenase
Enzyme
Biological Nitrogen Fixation (BNF)
Screw cap
Nodules
Cotton wool
Desiccant/
Silica gel
Soybean (Glycine max )
nodules ( V-15 growth stage)
Preservation of
nodules in desicant
SARCC- major step in isolation of rhizobia
• Imbibe nodules in water 1hr.
• Surface sterilize nodules
• Transfer into 100µl sterile water
• Crush with sterile rod & mix
• Plate/streak 30µl suspension
• Yeast Mannitol Agar (YMA)
Streaking on YMA-CR agar
Isolation of Rhizobia from nodules
a b c
Bradyrhizobium (a) and Rhizobium (b) species
usually form white or transparent colonies on
YMA-CR medium (do not absorb congo red dye)
Azosprillum absorbs the
congo red dye and forms
red colonies on YMA-CR
• Gram negative, rod shaped ( 1.2 - 3µm )
• Motile (single polar/2-6 peritrichous fla.
• No endospore
Isolation and preliminary characterization
Isolation and characterization
Preservation of SRACC cultures
Frozen 20% Glycerol Lyophilization
• Fix atmospheric N, promote growth & yield
• Source of protein for humans and grazing animals
• Improve soil fertility through BNF
• Rhizobia for soybeans, cowpea, beans & chickpea
• Rhizobia for Indigofera spicata var. spicata, Macrotyloma
axillarae, Vigna unguiculata (Indigenous to South Africa)
• Stylosanthus gracilis, Desmodium uncinatum, Indigofera
hirusta, Cyamopsis tetragonoloba, Onchobrychis vicifolia
(exotic species)
Rhizobium holdings at SARCC
• Glasshouse Koch’s expt. & field trails
• Leonard jar (Hoagland's soln.)
Screening for Effective Rhizobium spp.
1 2
TJ14
SR4
SAC2
Rhizobium rhizogenes 163C
UD5
Rhizobium tropici strain LNW10
Rhizobium tropici strain LNP6
XCR6
RAK1
Sinorhizobium meliloti QTX 8-2
Sinorhizobium meliloti Qtx-8-1
RF14
Rhizobium taeanense strain PSB 2-6
XAP2
Mesorhizobium amorphae
XEL1
Mesorhizobium sp. SOD33
Azospirillum brasilense REC3
Azospirillum brasilense strain 13-2C
Bradyrhizobium elkanii DASA35013
XHX1
WB74
Bradyrhizobium japonicum USDA 110
Bradyrhizobium japonicum DASA38025
XHT1
XBQ5
Sinorhizobium fredii USDA 194
Mesorhizobium loti
Bradyrhizobium sp. USDA 4424
Bradyrhizobium sp. AP28 FN566882.1 89
68
98
72
58
56
54
100
90
79
99
98
60
94
99 91 90
81 89
98
78
94
99
0.1
• fD1(5’AGAGTTTGATCCTGGCTCAG3)
• rD1(5’AAGGAGGTGATCCAGCC3’)
• Sequencing
• Bioedit, Chromas Lite
• MAFFT
• MEGA5
M a b c d e f g h i j k l m n o p q r s t u nc
16S rRNA
Identification of Effective Strains
B. japonicum strain SEMIA 6439
SA59-2A
XBD2
Bradyrhizobium sp. CCBAU 05327
XBV7
XFH3
SA59-2F
XBT2
B. liaoningen se CCBAU 13045
Bradyrhizobium genosp.TUXTLAS-16
XHG1
SA59-2E
B. japonicum CCBAU 53152
XHF12
XHF14
XBU4
XCG2
XGE1
Bradyrhizobium elkanii (AB231916.1)
XGA1
XBQ5
SA59-2C
XGO1
XHH1
Rhizobium sp. Qtx 14 2
Sinorhizobium meliloti (FM178848.1)
XFH1
Rhizobium sp. CCBAU 15278(GU552897.1)
Rhizobium etli CFN 42 (U28916.1)
Rhizobium leguminosarum WSM1325
Rhizobium tropici (GQ287633.1)
Methylobacterium cerastii (FR733885.1)
Methylobacterium nodulans ORS2060
100
64 54
91
58
50
10
100
67
99
100
78 86
76
67
68 56
81
61
0.01
- Onchobrychis vicifolia
- Vigna sp.
- Indigofera hirusta
- Cyamopsis tetragonoloba
- Vigna unguiculata
- Macrotyloma axilarae
- Stylosanthus gracilis
- Desmodium uncinatum
Identification of Effective Strains
Glycine max
(Soybean)
10 20 30 40 50....|....| ....|....| ....|....| ....|....| ....|....|
WB74 ATGCAAGTCG AGCGGGCGTA GCAATACGTC AGCGGCAGAC GGGTGAGTAA 50
B.japonicum strain CCBAU -------TCG AGCGGGCGTA GCAATACGTC AGCGGCAGAC GGGTGAGTAA 43
60 70 80 90 100....|....| ....|....| ....|....| ....|....| ....|....|
WB74 CGCGTGCGAA CGTACCTTTT GGTTCGGAAC AACACAGGGA AACTTGTGCT 100
B.japonicum strain CCBAU CGCGTGGGAA CGTACCTTTT GGTTCGGAAC AACACAGGGA AACTTGTGCT 93
110 120 130 140 150....|....| ....|....| ....|....| ....|....| ....|....|
WB74 AATACCGGAT AAGCCCTTAC GGGGAAAGAT TTATCGCCGA AAGATCGGCC 150
B.japonicum strain CCBAU AATACCGGAT AAGCCCTTAC GGGGAAAGAT TTATCGCCGA AAGATCGGCC 143
160 170 180 190 200....|....| ....|....| ....|....| ....|....| ....|....|
WB74 CGCGTCTGAT TAGCTAGTTG GTAGGGTAAC GGCCTACCAA GGCGACGATC 200
B.japonicum strain CCBAU CGCGTCTGAT TAGCTAGTTG GTAGGGTAAC GGCCTACCAA GGCGACGATC 193
210 220 230 240 250....|....| ....|....| ....|....| ....|....| ....|....|
WB74 AGTAGCTGGT CTGAGAGGAT GATCAGCCAC ATTGGGACTG AGACACGGCC 250
B.japonicum strain CCBAU AGTAGCTGGT CTGAGAGGAT GATCAGCCAC ATTGGGACTG AGACACGGCC 243
260 270 280 290 300....|....| ....|....| ....|....| ....|....| ....|....|
WB74 CAAACTCCTA CGGGAGGCAG CAGTGGGGAA TATTGGACAA TGGGGGCAAC 300
B.japonicum strain CCBAU CAAACTCCTA CGGGAGGCAG CAGTGGGGAA TATTGGACAA TGGGGGCAAC 293
310 320 330 340 350....|....| ....|....| ....|....| ....|....| ....|....|
WB74 CCTGATCCAG CCATGCCGCG TGAGTGATGA AGGCCCTAGG GTTGTAAAGC 350
B.japonicum strain CCBAU CCTGATCCAG CCATGCCGCG TGAGTGATGA AGGCCCTAGG GTTGTAAAGC 343
360 370 380 390 400....|....| ....|....| ....|....| ....|....| ....|....|
WB74 TCTTTTGTGC GGGAAGATAA TGACGGTACC GCAAGAATAA GCCCCGGCTA 400
B.japonicum strain CCBAU TCTTTTGTGC GGGAAGATAA TGACGGTACC GCAAGAATAA GCCCCGGCTA 393
410 420 430 440 450....|....| ....|....| ....|....| ....|....| ....|....|
WB74 ACTTCGTGCC AGCAGCCGCG GTAATACGAA GGGGGCTAGC GTTGCTCGGA 450
B.japonicum strain CCBAU ACTTCGTGCC AGCAGCCGCG GTAATACGAA GGGGGCTAGC GTTGCTCGGA 443
460 470 480 490 500....|....| ....|....| ....|....| ....|....| ....|....|
WB74 ATCACTGGGC GTAAAGGGTG CGTAGGCGGG TCTTTAAGTC AGGGGTGAAA 500
B.japonicum strain CCBAU ATCACTGGGC GTAAAGGGTG CGTAGGCGGG TCTTTAAGTC AGGGGTGAAA 493
510 520 530 540 550....|....| ....|....| ....|....| ....|....| ....|....|
WB74 TCCTGGAGCT CAACTCCAGA ACTGCCTTTG ATACTGAAGA TCTTGAGTTC 550
B.japonicum strain CCBAU TCCTGGAGCT CAACTCCAGA ACTGCCTTTG ATACTGAAGA TCTTGAGTTC 543
560 570 580 590 600....|....| ....|....| ....|....| ....|....| ....|....|
WB74 GGGAGAGGTG AGTGGAACTG CGAGTGTAGA GGTGAAATTC GTAGATATTC 600
B.japonicum strain CCBAU GGGAGAGGTG AGTGGAACTG CGAGTGTAGA GGTGAAATTC GTAGATATTC 593
610 620 630 640 650....|....| ....|....| ....|....| ....|....| ....|....|
WB74 GCAAGAACAC CAGTGGCGAA GGCGGCTCAC TGGCCCGATA CTGACGCTGA 650
B.japonicum strain CCBAU GCAAGAACAC CAGTGGCGAA GGCGGCTCAC TGGCCCGATA CTGACGCTGA 643
660 670 680 690 700....|....| ....|....| ....|....| ....|....| ....|....|
WB74 GGCACGAAAG CGTGGGGAGC AAACAGGATT AGATACCCTG GTAGTCCACG 700
B.japonicum strain CCBAU GGCACGAAAG CGTGGGGAGC AAACAGGATT AGATACCCTG GTAGTCCACG 693
710 720 730 740 750....|....| ....|....| ....|....| ....|....| ....|....|
WB74 CCGTAAACGA TGAATGCCAG CCGTTAGTGG GTTTACTCAC TAGTGGCGCA 750
B.japonicum strain CCBAU CCGTAAACGA TGAATGCCAG CCGTTAGTGG GTTTACTCAC TAGTGGCGCA 743
Glycine max: SARCC-WB74 = 100% Bradyrhizobium spp.
10 20 30 40 50....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 ---------- ---------- ---------- ---------- -------KAY 3
R.leguminosarum strain CIAM AACGAACGCT GGCGGCAGGC TTAACACATG CAAGTCGAGC GCCCCGC--- 47
60 70 80 90 100....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 AARGGGAGCG GCAGACGGGT GAGTAACGCG TGGGAATCYA CCCTTGACTA 53
R.leguminosarum strain CIAM AAGGGGAGCG GCAGACGGGT GAGTAACGCG TGGGAATCTA CCCTTGACTA 97
110 120 130 140 150....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 CGGAATAACG CAGGGAAACT TGTGCTAATA CCGTATGTGT CCTTCGGGAG 103
R.leguminosarum strain CIAM CGGAATAACG CAGGGAAACT TGTGCTAATA CCGTATGTGT CCTTCGGGAG 147
160 170 180 190 200....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 AAAGATTTAT CGGTCAAGGA TGAGCCCGCG TTGGATTAGC TAGTTGGTGG 153
R.leguminosarum strain CIAM AAAGATTTAT CGGTCAAGGA TGAGCCCGCG TTGGATTAGC TAGTTGGTGG 197
210 220 230 240 250....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 GGTRAAGKCS TAMCARGGMR ACGATCCATA GCTGGTCTGA GAGGATGATC 203
R.leguminosarum strain CIAM GGTAAAGGCC TACCAAGGCG ACGATCCATA GCTGGTCTGA GAGGATGATC 247
260 270 280 290 300....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 AGCCACATTG GGACTGAGAC ACGGCCCAAA CTCCTACGGG AGGCAGCAGT 253
R.leguminosarum strain CIAM AGCCACATTG GGACTGAGAC ACGGCCCAAA CTCCTACGGG AGGCAGCAGT 297
310 320 330 340 350....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 GGGGAATATT GGACAATGGG CGCAAGCCTG ATCCAGCCAT GCCGCGTGAG 303
R.leguminosarum strain CIAM GGGGAATATT GGACAATGGG CGCAAGCCTG ATCCAGCCAT GCCGCGTGAG 347
360 370 380 390 400....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 TGATGAAGGC CCTAGGGTTG TAAAGCTCTT TCACCGGAGA AGATAATGAC 353
R.leguminosarum strain CIAM TGATGAAGGC CCTAGGGTTG TAAAGCTCTT TCACCGGAGA AGATAATGAC 397
410 420 430 440 450....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 GGTATCCGGA GAAGAAGCCC CGGCTAACTT CGTGCCAGCA GCCGCGGTAA 403
R.leguminosarum strain CIAM GGTATCCGGA GAAGAAGCCC CGGCTAACTT CGTGCCAGCA GCCGCGGTAA 447
460 470 480 490 500....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 TACGAAGGGG GCTAGCGTTG TTCGGAATTA CTGGGCGTAA AGCGCACGTA 453
R.leguminosarum strain CIAM TACGAAGGGG GCTAGCGTTG TTCGGAATTA CTGGGCGTAA AGCGCACGTA 497
510 520 530 540 550....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 GGCGGATCGA TCAGTCAGGG GTGAAATCCC AGGGCTCAAC CCTGGAACTG 503
R.leguminosarum strain CIAM GGCGGATCGA TCAGTCAGGG GTGAAATCCC AGGGCTCAAC CCTGGAACTG 547
560 570 580 590 600....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 CCTTTGATAC TGTCGATCTG GAGTATGGAA GAGGTGAGTG GAATTCCGAG 553
R.leguminosarum strain CIAM CCTTTGATAC TGTCGATCTG GAGTATGGAA GAGGTGAGTG GAATTCCGAG 597
610 620 630 640 650....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 TGTAGAGGTG AAATTCGTAG ATATTCGGAG GAACACCAGT GGCGAAGGCG 603
R.leguminosarum strain CIAM TGTAGAGGTG AAATTCGTAG ATATTCGGAG GAACACCAGT GGCGAAGGCG 647
660 670 680 690 700....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 GCTCACTGGT CCATTACTGA CGCTGAGGTG CGAAAGCGTG GGGAGCAAAC 653
R.leguminosarum strain CIAM GCTCACTGGT CCATTACTGA CGCTGAGGTG CGAAAGCGTG GGGAGCAAAC 697
710 720 730 740 750....|....| ....|....| ....|....| ....|....| ....|....|
SAC2 AGGATTAGAT ACCCTGGTAG TCCACGCCGT AAACGATGAA TGTTAGCCGT 703
R.leguminosarum strain CIAM AGGATTAGAT ACCCTGGTAG TCCACGCCGT AAACGATGAA TGTTAGCCGT 747
Trifolium vesiculosum
(Woogenlup clover)
Trifolium sp: SARCC-SAC2 = 100% Bradyrhizobium spp.
10 20 30 40 50....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 ---------- ---------- ---------- ---------- ---------- 1
R.leguminosarum bv. viciae 384 AGAGTTTGAT CCTGGCTCAG AACGAACGCT GGCGGCAGGC TTAACACATG 50
60 70 80 90 100....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 ---------- ---------- ---------- ---------- -------tgg 3
R.leguminosarum bv. viciae 384 CAAGTCGAGC GCCCCGCAAG GGGAGCGGCA GACGGGTGAG TAACGCGTGG 100
110 120 130 140 150....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 gaatctaccc ttgactacgg aataacgcag ggaaacttgt gctaataccg 53
R.leguminosarum bv. viciae 384 GAATCTACCC TTGACTACGG AATAACGCAG GGAAACTTGT GCTAATACCG 150
160 170 180 190 200....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 tatgtgtcct tcgggagaaa gatttatcgg tcaaggatga gcccgcgttg 103
R.leguminosarum bv. viciae 384 TATGTGTCCT TCGGGAGAAA GATTTATCGG TCAAGGATGA GCCCGCGTTG 200
210 220 230 240 250....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 gattagctag ttggtggggt aaaggcctac caaggcgacg atccatagct 153
R.leguminosarum bv. viciae 384 GATTAGCTAG TTGGTGGGGT AAAGGCCTAC CAAGGCGACG ATCCATAGCT 250
260 270 280 290 300....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 ggtctgagag gatgatcagc cacattggga ctgagacacg gcccaaactc 203
R.leguminosarum bv. viciae 384 GGTCTGAGAG GATGATCAGC CACATTGGGA CTGAGACACG GCCCAAACTC 300
310 320 330 340 350....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 ctacgggagg cagcagtggg gaatattgga caatgggcgc aagcctgatc 253
R.leguminosarum bv. viciae 384 CTACGGGAGG CAGCAGTGGG GAATATTGGA CAATGGGCGC AAGCCTGATC 350
360 370 380 390 400....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 cagccatgcc gcgtgagtga tgaaggccct agggttgtaa agctctttca 303
R.leguminosarum bv. viciae 384 CAGCCATGCC GCGTGAGTGA TGAAGGCCCT AGGGTTGTAA AGCTCTTTCA 400
410 420 430 440 450....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 ccggagaaga taatgacggt atccggagaa gaagccccgg ctaacttcgt 353
R.leguminosarum bv. viciae 384 CCGGAGAAGA TAATGACGGT ATCCGGAGAA GAAGCCCCGG CTAACTTCGT 450
460 470 480 490 500....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 gccagcagcc gcggtaatac gaagggggct agcgttgttc ggaattactg 403
R.leguminosarum bv. viciae 384 GCCAGCAGCC GCGGTAATAC GAAGGGGGCT AGCGTTGTTC GGAATTACTG 500
510 520 530 540 550....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 ggcgtaaagc gcacgtaggc ggatcgatca gtcaggggtg aaatcccagg 453
R.leguminosarum bv. viciae 384 GGCGTAAAGC GCACGTAGGC GGATCGATCA GTCAGGGGTG AAATCCCAGG 550
560 570 580 590 600....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 gctcaaccct ggaactgcct ttgatactgt cgatctggag tatggaagag 503
R.leguminosarum bv. viciae 384 GCTCAACCCT GGAACTGCCT TTGATACTGT CGATCTGGAG TATGGAAGAG 600
610 620 630 640 650....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 gtgagtggaa ttccgagtgt agaggtgaaa ttcgtagata ttcggaggaa 553
R.leguminosarum bv. viciae 384 GTGAGTGGAA TTCCGAGTGT AGAGGTGAAA TTCGTAGATA TTCGGAGGAA 650
660 670 680 690 700....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 caccagtggc gaaggcggct cactggtcca ttactgacgc tgaggtgcga 603
R.leguminosarum bv. viciae 384 CACCAGTGGC GAAGGCGGCT CACTGGTCCA TTACTGACGC TGAGGTGCGA 700
710 720 730 740 750....|....| ....|....| ....|....| ....|....| ....|....|
TJ14 aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 653
R.leguminosarum bv. viciae 384 AAGCGTGGGG AGCAAACAGG ATTAGATACC CTGGTAGTCC ACGCCGTAAA 750
Pisum sativum
Peas
Pisum sativum: SARCC-TJ14 = 100% Bradyrhizobium spp.
SARCC data base – Card System
• SARCC – South Africa’s National Assets (>700 strains)
• Commercial strains used elsewhere in the world
• Most of the rhizobial strains actively nodulating/ N2 fixing
• Lack of skilled man power, modern Lab facilities
• Conventional method of identification
• Recent improvement , new information to SARCC
- Research Legume-Rhizobium symbiosis
- Identification (molecular tools)
• Development of the SARCC
Concluding Summary