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 2: World major Production areas of pigeonpea. (Source: FAOSTAT, 2007).

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

Materials and methods

10

5

1

3 3

4 6

4

3

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

Fig 8: Diverse rhizobial colonies from soil of different provinces of South Africa

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

Acknowledgements

I would like to express my sincere gratitude to the supervisors

and mentors for advice, guidance and encouragement.

BIMF-FBIP organizers for funding

Thank you!!!