Enormous rhizobial diversity 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

THE GLOBAL NITROGEN CYCLE NO 2 - N2N2 NH 4 + NO 3 - Nitrogen fixation Biomass NH NO 2 - → N 2 + 2H 2 O ANAMMOX NO 2 - Nitrification Denitrification NO N2ON2O Assimilatory NO 3 - reduction or DNRA The first step in Nitrogen cycle

Rhizobial niches Symbiotic Mutualists Inside the nodule Endophytes of legumes Saprophytic Endophytes of non- legumes Root cortex

Nitrogen fixation 255 ×10 6 t N is fixed annually through Three ways ★ Atmospheric Nitrogen fixation ★ Industrial Nitrogen fixation ★ Biological Nitrogen fixation (BNF)  BNF contributes 139 to 170 ×10 6 t N / yr

Comparatively:  Less than the total N reserves (105, 000 ×10 6 t N), but  2 to 3 times greater than inputs of N from fertilizers (65 ×10 6 t N/yr).

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 Pulses:- Eragrositis teff

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

Our own earlier research in Ethiopia, HwU  As many as 16 different agroclimatic zones  Among the eight important primary gene centers of the world (Vavilov, 1951)  Centre of origin for many leguminous crop plants  Despite this, investigations of rhizobia in Ethiopian soils are scarce

19 sites ● 21 Legume spp. ● Alt.  1190 – 2800m a.s.l. ● Tº  ºC (min – max) ● RF  450 – 1350 (mm) ● pH  4.8 – 9.6 Strains isolated form Ethiopian soils  Addis Ababa  Awassa  Abergele  Alemaya  Arba- minch  B orena-negele 3N3N 48  E 6N6N 9N9N 12  N 15  N 33  E36  E40  E44  E  Moyale  Nazret  Ziway  Yavelo Sampling Route The beauty of diversity study is when you have large number of samples from different Legumes & Agroecology

Techniques Collecting nodules Trapping

Authentication of rhizobial isolates on homologous host

Neighbour-joining Phylogeny estimated from partial 16S rRNA (203 unnamed test strains) Steadily growing number of rhizobia in our biobank more than 500 strains

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

Symbiotic effectiveness of indigenous rhizobia and P fertilizer … Haricot bean (Phaseolus vulgaris L.) at Boricha, S. Ethiopia (Tarekegn 2010) Nodule Leaf Treatmentnumberdry wt.area plant -1 (g)(cm 2 ) Inoc. HB de 0.54 c 2.29 cd HB bc 0.69 b 2.62 cb HB ef 0.50 c 2.19 d HB b 0.69 b 2.78 b HB a 0.78 a 3.28 a HB 92b62.00 ab 0.72 ab 2.51 bc HB de 0.67 b 2.35 cd 23 kg N ha bc 0.60 b 2.77 b - Inoc. & -N33.00 f 0.47 c 1.78 e P (Kgha -1 ) c 0.54 b c b 0.67 a b a 0.70 a a 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) Same letter(s) in a column are not significant, p< 0.05 a b cd f bc de bc cd ab

Number HundredGrainStrawHarvest Treatmentof podsof seedseedyield index plant -1 pod -1 weight (g)(ton ha -1 ) (%) Inoc. HB c 2.91 b cd 1.34 de 2.75 bc 35.0 bc HB bc 3.36 a b 2.03 ab 3.26 ab 39.2 ab HB c 2.78 b cd 1.54 cd 2.68 c 37.8 ab HB ab 3.38 a ab 1.81 bc 2.80 bc 40.2 ab HB a 3.42 a a 2.36 a 3.38 a 41.6 a HB 92b9.20 bc 3.02 ab ab 1.92 b 3.14 ab 38.3 ab HB bc 2.85 b c 1.74 bc 2.76 bc 39.6 ab 23 kg N/ha10.50 ab 3.03 ab ab 1.92 b 2.88 bc 40.5 ab No Inoc. & -N7.47 c 2.36 c d 1.17 e 2.76 bc 31.2 c P (Kgha -1 ) c 2.78 b c 1.13 b 2.53 b 32.8 b b 3.05 a b 2.01 a 3.11 a 40.5 a a 3.21 a a 2.13 a 3.16 a 41.2 a Table: Effect of rhizobium inoculation and P fertilizer on grain yield, yield components and harvest index on haricot bean (P. vulgaris) (Tarekegn, 2010) Same letter(s) in a column are not significant, p< 0.05 a b bc e cd b de ab

Table: Plant and soil residual N contents as influenced by Inoculation and P fertilization on P. vulgaris, Boricha, S. Ethiopia (Tarekegn, 2010) Nitrogen Residual Treatmentcontent in nitrogen straw (%)grain (%)(%) Inoc. HB c 0.14 bc HB abc 0.15 ab HB bc 0.13 bcd HB bc 0.14 bc HB a 0.17 a HB 92b ab 0.15 ab HB bc 0.14 bc 23 kg N ha bc 0.11 cd No Inoc. & -N d 0.11 cd P (Kgha -1 ) b 0.12 b a 0.14 ab a 0.15 a a abc bc d b c ab

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

In general From genetic and symbiotic characterization, Ethiopia represent a hotspot Cross-inoculation experiments (few + target hosts (Haricot bean, soyabean, chickpea, cowpea, lentils etc.) demonstrated variations in performance Thus, great potential for selecting elite strains for prompting sustainable agriculture and to benefit small holder farmers

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

Thank you