Diversity of rhizobia associated with root nodulation in
pigeonpea (Cajanus cajan) from South Africa
F L Bopape
ARC-PLANT HEALTH AND PROTECTION SCHOOL OF AGRICULTURE
DEPARTMENT OF PLANT PRODUCTION
Dr A.I. Hassen Prof. E.T. Gwata
Prof. G.R.A. Mchau
BIMF-FBIP 2018
Introduction
♦ Cajanus cajan
♦ Indian 3,500 yrs ago
♦ short lived perennial
(5 yrs) legume
♦ 1.0 - 4.0 m height Fig 1: Pigeonpea plant, flower and seeds
(Sources: http://www.dreamstime.com)
Introduction cont’d Major Uses and Importance:
(a) Human food
♦ rich in protein (25% in grain); vitamins and minerals (Amatefalo, 2000)
(b) Animal feeds (e.g. dairy cattle)
(c) Soil fertility improvement
♦ symbiosis with rhizobia (166-235 kg N2 / ha) (Peoples et al.,1995)
♦ crop rotation / intercropping, smallholder systems
(d) Highly tolerant to drought
therefore suitable in semi-arid areas (LP/EC)
Introduction cont’d
Fig 3: Pigeonpea Production areas in South Africa. (Source:
https://www.google.com/maps).
Objectives To collect rhizobial strains associated with root nodulation in pigeonpea;
Molecular characterization of the rhizobial isolates from diverse locations
in South Africa associated with pigeonpea
Materials and methods ♦ 40 soil samples (approx. 200.0 g each) x 2 sachets from each
Province across SA transect
♦ soil used for inoculating pigeonpea seeds in greenhouse at ARC-
PHP in order to isolate specific RS
♦ 5 pigeonpea genotype seed used (2 exotic and 3 local)
♦Planted in Leonard jars containing sand + Hg/land’s solution; inoculated with soil from each location (40 samples)
♦ rhizobia isolated from nodules by streaking the extracts on YMA
(Somasagaren & Hoben, 1994)
♦ DNA extracted pure bacterial colonies, PCR and sequencing 16S RNA
Results New pigeonpea rhizobia isolates collected
Deposited strains to the SARCC
In seven province natural rhizobia compatible with pigeonpea
Diverse rhizobia : Rhizobium, Bradyrhizobium, Paraburkholderia
and Phyllobacterium
Results
Fig 4: Pigeonpea effective with soil from location 16, presence of nodules and rhizobial
colonies isolated
Results
Fig 6: Pigeonpea plant inoculated with soil compared to uninoculated plants
Fig 5: Five pigeonpea genotypes / cultivars inoculated with soil from same location
Results
Fig 7: Various nodules shapes of the pigeonpea when inoculated with different soil
from locality 1-40
Results
1
10
100
Number of Isolates per Province
Limpopo Mpumalanga North West Gauteng KwaZulu Natal
Free State Northern Cape Eastern Cape Western Cape
Fig 9: Distribution of Pigeonpea rhizobial isolates per Province
18a.pp3.MP
29a.pp1.NW
17a.pp1.MP
15a.pp3.MP
15a.pp1.MP
36a.pp5.LP
31b.pp4.LP
13b1.pp4.GP
26a2.pp5.NW
34a2.pp5.LP
22a.pp5.NC
32b2.pp3.LP
35b.pp1.LP
A. tumefaciens (X67223.1)
R. tropici (LMG 9503)
29a2.pp2.NW
31b2.pp3.LP
R. phaseoli (NR 044112.1)
R. etli (LMG 17827)
R. tropici (NR 102511.1)
14a1.pp5.GP
17a1.pp3.MP
26b.pp3.NW
29a1.pp2b.NW
31b1.pp3.LP
35a.pp5.LP
37a.pp4.LP
39a3.pp3.LP
23a.pp5.NC
Phyllo leguminum (AY785337.1)
B. japonicum (NR 036865.1)
B. liaoningense (NR 041785.1)
B. japonicum (LMG 6138)
27b2.pp5.NW
11a2.pp3.GP
18a.pp4.MP
11b2.pp5.GP
13b.pp3.GP
15b.pp3.MP
15b.pp5.MP
19a1.pp3.FS
19b.pp5.FS
32b1.pp5.LP
33a.pp4.LP
B. elkanii (LMG 6134)
B. elkanii (NR 036953.1)
31b1.pp5.LP
31b3.pp3.LP
Paraburk phytofirmans (NR 102845.1)
30a2.pp3a.NW
Paraburk kirstenboschensis (NR 146352.1)
33a.pp2.LP
Ps. koreensis (KY910143.1)
Ps. koreensis (KY910143.1)
14b.pp5.GP
30b2.pp4. NW
8b2.pp1.KZN
5b2.pp1.KZN
10a.pp3.GP
6b.pp3.KZN
7a2.pp3.KZN
8a2.pp3.KZN99
88
62
100
100
82
3
7
99
12
98
9
5
49
69
87
11
92
57
59
27
8
46
81
Bradyrhizobium
Rhizobium
Paraburkholderia
Phyllobacterium
Fig10: 16S RNA neighbor joining tree using Mega 7
Key references
(i) Silva et al., (2012). Genetic diversity of rhizobia isolates from Amazon
soils using cowpea (Vigna unguiculata) as trap plant. Braz. J. Microbiol.
43(2): 682–691.
(ii) Martens et al., (2008). Advantages of multilocus sequence analysis for
taxonomic studies: a case study using 10 housekeeping genes in the genus
Ensifer (including former Sinorhizobium). Int. J. Syst. Evol. Microbiol. 58:
200-214.
(iii) Rai et al.,(2012). Phenotypic and molecular characterization of
indigenous rhizobia nodulaing chickpea in India. Indian J Exp. Biol. 50 (5):
340-350.
(iv) Chen et al., (2015). Genetic and heterosis analysis using Hayman’s six-
generation model for grain yield and yield componenents in maize. Crop Sci.
55: 1006-1016