Tulu Degefu (PhD )

1. Enormous rhizobialdiversity resident in Ethiopian soils: A potential hotspot to realize the benefits of BNF Tulu Degefu (PhD) Hawassa University, College of Natural and Computational Sciences, Department of Biology Hawassa, Ethiopia

2. THE GLOBAL NITROGEN CYCLE Biomass Nitrogen fixation N2 NH4+ The first step in Nitrogen cycle NH4+ + NO2- → N2 + 2H2O N2O ANAMMOX Assimilatory NO3- reduction or DNRA NO2- Nitrification NO NO2- Denitrification NO3-

3. Rhizobial niches Root cortex Endophytesof non-legumes Symbiotic Mutualists Inside thenodule Endophytesoflegumes Saprophytic

4. Nitrogen fixation • 255 ×106t N is fixedannuallythrough • Three ways ★ Atmospheric Nitrogen fixation ★ Industrial Nitrogen fixation ★Biological Nitrogen fixation (BNF) • BNF contributes 139 to 170 ×106 t N / yr

5. Comparatively: • Less thanthe total N reserves (105, 000 ×106 t N), but • 2 to 3 times greaterthan inputs of N from fertilizers (65 ×106 t N/yr).

6. 50% of the biologically fixed N is fixed through Legume-rhizobial association • In areas of arable agriculture, legumes contribute >80% • 25-30% protein intake world-wide Eragrositisteff Pulses:-

7. Pre-requisites for BNFmax • Symbiotic N fixation is fully realized only if legumes are nodulated with effective rhizobia • Genotype  Environment  Management [(L  R)  E M]

8. Our own earlier research in Ethiopia, HwU • As many as 16 different agroclimaticzones • Amongtheeightimportantprimary gene centersoftheworld (Vavilov, 1951) • Centre of origin for many leguminous crop plants • Despite this, investigations of rhizobia in Ethiopian soils are scarce

9. Sampling Route Strains isolated form Ethiopian soils • 19 sites • ● 21 Legume spp. • ● Alt.  1190 – 2800m a.s.l. • ● Tº 9 - 32 ºC (min – max) • ● RF  450 – 1350 (mm) • ●pH  4.8 – 9.6 15N  Abergele 12N 9N  Addis Ababa  Alemaya  Nazret  Ziway The beauty of diversity study is when you have large number of samples from different Legumes & Agroecology Awassa 6N  Arba- minch  Borena-negele Yavelo  Moyale 3N 33E 36E 40E 44E 48E

10. Techniques Collectingnodules Trapping

11. Authentication of rhizobial isolates on homologous host

12. Rhizobium sp. (SDW024) AC56b AC4d 0.1 I (2) 95 AC87k1 77 R. etli AC86a II 71 A. rhizogenes R. tropici 98 R. leguminosarum AC100a AC82b III (2) AC100b R. hainanense AC86c1 IV R. indigoferae R. sullae R. yanglingense Rhizobium R. gallicum 86 AC90b 70 R. mongolense Rhizobium sp. (X59) 71 AC90e1 V (2) AC93e AC91e AC26e VI R. giardinii 97 Rhizobium sp. (SDW058) 56 VII AC88a AC87k3 VIII AC73d R. huautlense 92 R. galegae 100 AC51e IX (2) R. loessense A. vitis 100 74 I AC97c1 A. undicola 89 AC77b 94 AC11a II A. albertimagni Agrobacterium A. larrymoorei A. rubi A. tumefaciens 93 III AC42c 96 A. radiobacter IV AC79c1 S. arboris AC50e I(2) 89 S meliloti AC52c S. medicae II AC28a AC38b2 AC10a1 58 AC1b III (3) S. saheli S. terangae Ensifer S. kostiense AC18a AC20b AC10d S. americanus IV AC27e S. fredii 99 S. xinjiangensis AC22d S. kummerowiae 82 AC47d V AC47a 91 S. adhaerens S. morelense AC39d 100e 93 AC98a I (7) AC21c2 AC21a2 83 AC99d AC39a 94 Mesorhizobium 39c1 M. plurifarium M. huakuii M. amorphae M. loti 80 M. ciceri M. mediterraneum 98 90 M. chacoense II AC88c M. tianshanense AC86b2 AC92c 100 AC101b AC92d I (2) AC97a AC70c AC79a 60 AC29c B. liaoningense AC87L 72 II AC87n Bradyrhizobium B. japonicum 61 AC64a AC64b III (3) AC65c B. yuanmingense AC64c IV AC82d V AC62a VI AC104a VII (2) AC104c1 Blastobacter dinitrificans 100 AC94a AC87b1 85 VIII B. elkanii 83 AC107e Methylobacterium 93 Met. organophylum ● 94 96 Met. nodulans AC72a 99 Beijerinckia indica ● Xanthobacter autotrophicus ● Azorhizobium caulinodans Devosia neptuniae Steadily growing number of rhizobia in our biobank more than 500 strains Neighbour-joining Phylogeny estimated from partial 16S rRNA (203 unnamed test strains)

13. Total nitrogen accumulation in pigeon pea varieties inoculated with bradyrhizobia isolates • Results from similar investigations greenhouse • Haricot bean (P. vulgaris) • Soybean (G. max) • Chickpea (Cicerarietinum) • Lentil (Lens culinaris) • Cowpea • 13 strains Mung bean varieties vs R.strains • 23 strains

14. Symbiotic effectiveness of indigenous rhizobia and P fertilizer … Haricot bean (Phaseolus vulgaris L.) at Boricha, S. Ethiopia (Tarekegn 2010) Table: Effect of rhizobium inoculation and P fertilizer on nodulation and growth of haricot bean at 50% flowering stage, field trial at Boricha(Tarekegn, 2010) a ab b bc bc cd cd de f Same letter(s) in a column are not significant, p< 0.05

15. Table: Effect of rhizobium inoculation and P fertilizer on grain yield, yield components and harvest index on haricot bean (P. vulgaris) (Tarekegn, 2010) a ab b b bc bc cd de e Same letter(s) in a column are not significant, p< 0.05

16. Table: Plant and soil residual N contents as influenced by Inoculation and Pfertilization on P. vulgaris, Boricha, S. Ethiopia (Tarekegn, 2010) a ab abc bc bc b bc c d

17. Our lab HwU • A technician working on pure rhizobial strains in the laminar flow chamber in the soil microbiology laboratory at HwU • Investigating purity and Gram staining under microscope

18. In general • From genetic and symbiotic characterization, Ethiopia represent a hotspot • Cross-inoculationexperiments (few + target hosts (Haricotbean, soyabean, chickpea, cowpea, lentilsetc.) demonstrated variations in performance • Thus, great potential for selecting elite strains for prompting sustainable agriculture and to benefit small holder farmers

19. But!!! • Handling, preparation and application of these strains as legume seed inoculants • Improve crop yield • Soil fertility and • Nutritional quality (protein content) of the legume crops

20. Thank you