Abraham Nova Soil Sciences
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Diversity of Bacteria Isolated from Root Nodules of Agroforestry legumes in Southern Ethiopia
Advisors: Endalkachew Wolde-Meskel Kristina Lindstrom Leena Rasanen
03.07.09
Abraham Mikru, University of Helsinki
1
Introduction
Over 500 indigenous legume species known in Ethiopia, 48 of which are endemic Most previous studies focus on agronomy/agroforestry aspects of legumes Less attention given to the associated microsymbionts Could the diversity in legumes be accompanied by a corrosponding diversity in microsymbionts?
03.07.09
Abraham Mikru, University of Helsinki
2
2.Objectives General objectives To unravel diversity of microsymbionts with focus on: Unexplored geographical sites Less studied legumes Use of new/better Methods Specific aims Taxonomy Diversity Phylogeny Specificity/promiscuity/host range 03.07.09
Abraham Mikru, University of Helsinki
3
Target legumes 1. Acacia abyssinica Hochst. Ex. Benth. (1846) Uses (Hunde and Thulin 1989) • • •
Fuel wood Shade Forage
Associated rhizobia •
Sinorhizobium saheli, Rhizobium giardinii, Mesorhizobium plurifarium & Agrobacterium tumefaciens (Wolde-Meskel et al. 2005)
03.07.09
Abraham Mikru, University of Helsinki
4
Albizia gummifera Durazz (1772) Trees up to 30 m long Uses Bee nectar, Fuel wood Timber, Shade & medicinal (Analgesic) Associated rhizobia Rhizobium leguminosarum, Bradyrhizobium japonicum and Bradyrhizobium liaoningense (Wolde-meskel et al. 2005); Messorhizobium albiziae (Wang et al. 2007) new species
03.07.09
Abraham Mikru, University of Helsinki
5
Millettia Wright and Arn. (1834) A single endemic sp in Ethiopia • Trees up to 30 m long Uses • Shade, Timber, & Narcotic to stun fish Associated rhizobia • Bradyrhizobium elkani, & Rhizobium etli (Wolde-meskel et al 2005) 03.07.09
Abraham Mikru, University of Helsinki
6
Indigofera L. (1753) • More than 78 Indigofera spp • Shrubs up to 2 m Uses • Indigo dye • Erosion control • Medicinal (Analgesic, anti-inflammatory) Associated rhizobia Rhizobium indigoferae (Wei et al. 2004) 03.07.09
Abraham Mikru, University of Helsinki
7
Grain legumes • • • • • • •
Cow pea (Vigna unguiculata), Pigeon pea (Cajanus cajan), Chick pea (Cicer arietenum), Ground nut (Aarachis hypogeae), Soy bean (Glycine max L), Mung bean (Vigna radiata), and Haricot bean (Phaseolus vulgaris).
03.07.09
Abraham Mikru, University of Helsinki
8
Study locations
Range of differences in soil physicochemical parameters pH =4.7-7.9 Total nitrogen= 0.14% - 0.623% Organic carbon=1.23% - 6.63% Altitude and agroecological zones 1480 – 2800 m a.s.l Semi-arid to subhumid
03.07.09
Abraham Mikru, University of Helsinki
9
Methods Nodule collection July 2007- January 2008 • Excavation • Trapping Bacterial Isolation and purification • Surface sterilized root nodules homogenized and streaked • Yeast Manitol agar with congo red • Tween buffer dilution and repeated subculturing DNA isolation • Enzymatic and detergent cell lysis • Phenol-chloroform extraction Molecular study • AFLP fingerprinting • Partial 16S rDNA sequence 03.07.09
Abraham Mikru, University of Helsinki
10
Results and Discussion 17 32 9 Rhizobia and Agrobacteria Enterobacter Klebsiella Bacillus sp 25
Pseudomonas spp Others
38 16
A total of 137 isolates identified into five major genera Fig 2 Proportion of five major bacterial genera Identified based on partial 16S rDNA sequence comprison. Non-rhizobia were predominant 03.07.09
Abraham Mikru, University of Helsinki
11
Dice (Opt:0.85%) (Tol 0.9%-0.9%) (H>0.0% S>0.0%) [0.0%-100.0%]
AFLP
Origin
Legume host
l . Rhizobium
44AG20b
Ethiop. WolayitaSodo
Albizia gummera
l . Rhizobium
45AG22a
Ethiop. Dilla
Albizia gummefera
l . Rhizobium
41AG14b
Ethiop. WolayitaSodo
Albizia gummifera
l . Rhizobium
42AG17b
Ethiop. Durame
Albizia gummifera
l . Rhizobium
43AG19b
Ethiop. Shone
Albizia gummifera
l . /Rhizobium
38AG11a
Ethiop. WolayitaSodo
Albizia gummifera
N . Rhizobium
83CW 55a
Ethiop. Dilla
Cowpea
l . Rhizobium
52AG4a
Ethiop. Shone
Albizia gummifera
l . Rhizobium
56AG28a
Ethiop. Dilla
Albizia gummifera
l . Rhizobium
32AG6B
Ethiop. Shone
Albizia gummifera
« . Rhizobium
7MF23B
Ethiop. Durame
Milletia ferruginea
0
« . Rhizobium
10MF26A
Ethiop. Bule
Milletia ferruginea
100
« . Rhizobium
16MF30G
Ethiop. Dilla
Milletia ferruginea
« . Rhizobium
9MF25A
Ethiop. Yirgalem
Milletia ferruginea
O . Rhizobium
142IF16a
Ethiop. Durame
Indigofera arrecta
« . Agrobacterium/Rhi.
15MF-30c
Ethiop. Dila
Milletia feruginea
t . Agrobacterium
79HB87a
Ethiop. Wondogenet
Haricotbean
¤ . Agrobacterium
106PP143a
Ethiop. Leku
Pigeonpea
t . Agrobacterium/Rhi.
138HB76a
Ethiop. Wolyta-Sodo
Haricot bean
n . Agrobacterium/Rhi.
140Ac1g
Ethiop. Durame
Acacia abyssinica
l . Agrobacterium/Rhi.
135AG96a
Ethiop. Wondogenet
Albizia gummifera
¤ . Bradyrhizobium sp.
103PP127a
Ethiop. Dilla
Pigeonpea
¤ . Bradyrhizobium sp.
107PP141a
Ethiop. Leku
Pigeonpea
t . Rhizobium
80HB100a
Ethiop. Shone
Haricotbean
O . Bradyrhizobium sp.
143IF106a
Ethiop. Durame
Indigofera arrecta
l . Rhizobium
33AG7A
Ethiop. WolayitaSodo
Albizia gummifera
l . Rhizobium
34AG8A
Ethiop. Arsinegele
Albizia gummifera
l . Rhizobium
36AG10A
Ethiop. WolyitaSodo
Albizia gummifera
100
100 100 89
0
98
75
100
86
92
100
92 100
93
Strain
100
80
60
40
Genus
100
91 100 90 92 89
100
91 100
100 50
Dendrogram based on AFLP fingerprinting of 30 rhizobial isolates revealed their genetic diversity. 7 AFLP clusters and one unclustered strain at 70% similarity 03.07.09
Abraham Mikru, University of Helsinki
12
Rhizobium =20
Agrobacterium =7
Bradyrhizobium =3
Phylogram based on partial16S rDNA sequence(414bp) comparison showed 20/30 of the rhizobiales were related to Rhizobium spp. 7 to Agrobacterium and 3 to Bradyrhizobium. (Isolates from 4 woody and 3 crop legumes) 03.07.09
Abraham Mikru, University of Helsinki
13
Conclusion and Recommendations • Several non-rhizobial genera may coexist with rhizobia in the root nodules • This compromise efficiency of recovery methods for rhizobia • More sampling and better recovery methods needed • The role of the non-rhizobia in plant growth modulation should be assessed • Characterize the rhizobia for nodulation, physiological and biochemical features • Assess the rhizobia further with regard to symbiotic and house keeping genes to get better overview of phylogeny
03.07.09
Abraham Mikru, University of Helsinki
14
Thank you!
03.07.09
Abraham Mikru, University of Helsinki
15
Advisors: Endalkachew Wolde-Meskel Kristina Lindstrom Leena Rasanen
03.07.09
Abraham Mikru, University of Helsinki
1
Introduction
Over 500 indigenous legume species known in Ethiopia, 48 of which are endemic Most previous studies focus on agronomy/agroforestry aspects of legumes Less attention given to the associated microsymbionts Could the diversity in legumes be accompanied by a corrosponding diversity in microsymbionts?
03.07.09
Abraham Mikru, University of Helsinki
2
2.Objectives General objectives To unravel diversity of microsymbionts with focus on: Unexplored geographical sites Less studied legumes Use of new/better Methods Specific aims Taxonomy Diversity Phylogeny Specificity/promiscuity/host range 03.07.09
Abraham Mikru, University of Helsinki
3
Target legumes 1. Acacia abyssinica Hochst. Ex. Benth. (1846) Uses (Hunde and Thulin 1989) • • •
Fuel wood Shade Forage
Associated rhizobia •
Sinorhizobium saheli, Rhizobium giardinii, Mesorhizobium plurifarium & Agrobacterium tumefaciens (Wolde-Meskel et al. 2005)
03.07.09
Abraham Mikru, University of Helsinki
4
Albizia gummifera Durazz (1772) Trees up to 30 m long Uses Bee nectar, Fuel wood Timber, Shade & medicinal (Analgesic) Associated rhizobia Rhizobium leguminosarum, Bradyrhizobium japonicum and Bradyrhizobium liaoningense (Wolde-meskel et al. 2005); Messorhizobium albiziae (Wang et al. 2007) new species
03.07.09
Abraham Mikru, University of Helsinki
5
Millettia Wright and Arn. (1834) A single endemic sp in Ethiopia • Trees up to 30 m long Uses • Shade, Timber, & Narcotic to stun fish Associated rhizobia • Bradyrhizobium elkani, & Rhizobium etli (Wolde-meskel et al 2005) 03.07.09
Abraham Mikru, University of Helsinki
6
Indigofera L. (1753) • More than 78 Indigofera spp • Shrubs up to 2 m Uses • Indigo dye • Erosion control • Medicinal (Analgesic, anti-inflammatory) Associated rhizobia Rhizobium indigoferae (Wei et al. 2004) 03.07.09
Abraham Mikru, University of Helsinki
7
Grain legumes • • • • • • •
Cow pea (Vigna unguiculata), Pigeon pea (Cajanus cajan), Chick pea (Cicer arietenum), Ground nut (Aarachis hypogeae), Soy bean (Glycine max L), Mung bean (Vigna radiata), and Haricot bean (Phaseolus vulgaris).
03.07.09
Abraham Mikru, University of Helsinki
8
Study locations
Range of differences in soil physicochemical parameters pH =4.7-7.9 Total nitrogen= 0.14% - 0.623% Organic carbon=1.23% - 6.63% Altitude and agroecological zones 1480 – 2800 m a.s.l Semi-arid to subhumid
03.07.09
Abraham Mikru, University of Helsinki
9
Methods Nodule collection July 2007- January 2008 • Excavation • Trapping Bacterial Isolation and purification • Surface sterilized root nodules homogenized and streaked • Yeast Manitol agar with congo red • Tween buffer dilution and repeated subculturing DNA isolation • Enzymatic and detergent cell lysis • Phenol-chloroform extraction Molecular study • AFLP fingerprinting • Partial 16S rDNA sequence 03.07.09
Abraham Mikru, University of Helsinki
10
Results and Discussion 17 32 9 Rhizobia and Agrobacteria Enterobacter Klebsiella Bacillus sp 25
Pseudomonas spp Others
38 16
A total of 137 isolates identified into five major genera Fig 2 Proportion of five major bacterial genera Identified based on partial 16S rDNA sequence comprison. Non-rhizobia were predominant 03.07.09
Abraham Mikru, University of Helsinki
11
Dice (Opt:0.85%) (Tol 0.9%-0.9%) (H>0.0% S>0.0%) [0.0%-100.0%]
AFLP
Origin
Legume host
l . Rhizobium
44AG20b
Ethiop. WolayitaSodo
Albizia gummera
l . Rhizobium
45AG22a
Ethiop. Dilla
Albizia gummefera
l . Rhizobium
41AG14b
Ethiop. WolayitaSodo
Albizia gummifera
l . Rhizobium
42AG17b
Ethiop. Durame
Albizia gummifera
l . Rhizobium
43AG19b
Ethiop. Shone
Albizia gummifera
l . /Rhizobium
38AG11a
Ethiop. WolayitaSodo
Albizia gummifera
N . Rhizobium
83CW 55a
Ethiop. Dilla
Cowpea
l . Rhizobium
52AG4a
Ethiop. Shone
Albizia gummifera
l . Rhizobium
56AG28a
Ethiop. Dilla
Albizia gummifera
l . Rhizobium
32AG6B
Ethiop. Shone
Albizia gummifera
« . Rhizobium
7MF23B
Ethiop. Durame
Milletia ferruginea
0
« . Rhizobium
10MF26A
Ethiop. Bule
Milletia ferruginea
100
« . Rhizobium
16MF30G
Ethiop. Dilla
Milletia ferruginea
« . Rhizobium
9MF25A
Ethiop. Yirgalem
Milletia ferruginea
O . Rhizobium
142IF16a
Ethiop. Durame
Indigofera arrecta
« . Agrobacterium/Rhi.
15MF-30c
Ethiop. Dila
Milletia feruginea
t . Agrobacterium
79HB87a
Ethiop. Wondogenet
Haricotbean
¤ . Agrobacterium
106PP143a
Ethiop. Leku
Pigeonpea
t . Agrobacterium/Rhi.
138HB76a
Ethiop. Wolyta-Sodo
Haricot bean
n . Agrobacterium/Rhi.
140Ac1g
Ethiop. Durame
Acacia abyssinica
l . Agrobacterium/Rhi.
135AG96a
Ethiop. Wondogenet
Albizia gummifera
¤ . Bradyrhizobium sp.
103PP127a
Ethiop. Dilla
Pigeonpea
¤ . Bradyrhizobium sp.
107PP141a
Ethiop. Leku
Pigeonpea
t . Rhizobium
80HB100a
Ethiop. Shone
Haricotbean
O . Bradyrhizobium sp.
143IF106a
Ethiop. Durame
Indigofera arrecta
l . Rhizobium
33AG7A
Ethiop. WolayitaSodo
Albizia gummifera
l . Rhizobium
34AG8A
Ethiop. Arsinegele
Albizia gummifera
l . Rhizobium
36AG10A
Ethiop. WolyitaSodo
Albizia gummifera
100
100 100 89
0
98
75
100
86
92
100
92 100
93
Strain
100
80
60
40
Genus
100
91 100 90 92 89
100
91 100
100 50
Dendrogram based on AFLP fingerprinting of 30 rhizobial isolates revealed their genetic diversity. 7 AFLP clusters and one unclustered strain at 70% similarity 03.07.09
Abraham Mikru, University of Helsinki
12
Rhizobium =20
Agrobacterium =7
Bradyrhizobium =3
Phylogram based on partial16S rDNA sequence(414bp) comparison showed 20/30 of the rhizobiales were related to Rhizobium spp. 7 to Agrobacterium and 3 to Bradyrhizobium. (Isolates from 4 woody and 3 crop legumes) 03.07.09
Abraham Mikru, University of Helsinki
13
Conclusion and Recommendations • Several non-rhizobial genera may coexist with rhizobia in the root nodules • This compromise efficiency of recovery methods for rhizobia • More sampling and better recovery methods needed • The role of the non-rhizobia in plant growth modulation should be assessed • Characterize the rhizobia for nodulation, physiological and biochemical features • Assess the rhizobia further with regard to symbiotic and house keeping genes to get better overview of phylogeny
03.07.09
Abraham Mikru, University of Helsinki
14
Thank you!
03.07.09
Abraham Mikru, University of Helsinki
15
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