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

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

Genus Strain Origin Legume host

100
40

60

80
l . Rhizobium 44AG20b Ethiop. WolayitaSodo Albizia gummera
100
l . Rhizobium 45AG22a Ethiop. Dilla Albizia gummefera
100 l . Rhizobium 41AG14b Ethiop. WolayitaSodo Albizia gummifera
100

0
l . Rhizobium 42AG17b Ethiop. Durame Albizia gummifera
89
l . Rhizobium 43AG19b Ethiop. Shone Albizia gummifera
98
l . /Rhizobium 38AG11a Ethiop. WolayitaSodo Albizia gummifera
75 N . Rhizobium 83CW 55a Ethiop. Dilla Cowpea
l . Rhizobium 52AG4a Ethiop. Shone Albizia gummifera
86 100
l . Rhizobium 56AG28a Ethiop. Dilla Albizia gummifera
l . Rhizobium 32AG6B Ethiop. Shone Albizia gummifera

92
« . Rhizobium 7MF23B Ethiop. Durame Milletia ferruginea
100

0
« . Rhizobium 10MF26A Ethiop. Bule Milletia ferruginea
100 « . Rhizobium 16MF30G Ethiop. Dilla Milletia ferruginea
92
100 « . Rhizobium 9MF25A Ethiop. Yirgalem Milletia ferruginea
O . Rhizobium 142IF16a Ethiop. Durame Indigofera arrecta
« . Agrobacterium/Rhi. 15MF-30c Ethiop. Dila Milletia feruginea
93 t . Agrobacterium 79HB87a Ethiop. Wondogenet Haricotbean
100
¤ . Agrobacterium 106PP143a Ethiop. Leku Pigeonpea
91
t . Agrobacterium/Rhi. 138HB76a Ethiop. Wolyta-Sodo Haricot bean
100

90
n . Agrobacterium/Rhi. 140Ac1g Ethiop. Durame Acacia abyssinica
92 89
l . Agrobacterium/Rhi. 135AG96a Ethiop. Wondogenet Albizia gummifera
¤ . Bradyrhizobium sp. 103PP127a Ethiop. Dilla Pigeonpea
100
¤ . Bradyrhizobium sp. 107PP141a Ethiop. Leku Pigeonpea
91 t . Rhizobium 80HB100a Ethiop. Shone Haricotbean
100
O . Bradyrhizobium sp. 143IF106a Ethiop. Durame Indigofera arrecta
l . Rhizobium 33AG7A Ethiop. WolayitaSodo Albizia gummifera
100

50
l . Rhizobium 34AG8A Ethiop. Arsinegele Albizia gummifera
l . Rhizobium 36AG10A Ethiop. WolyitaSodo Albizia gummifera

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