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

NO2-

N2 NH4+

NO3-

Nitrogen fixation Biomass

NH4+ + NO2

- → N2 + 2H2OANAMMOX

NO2- Nitrification

Denitrification

NO

N2OAssimilatory

NO3- reduction

orDNRA

The first step in Nitrogen cycle

Rhizobial niches

Symbiotic MutualistsInside the noduleEndophytes of legumes

Saprophytic

Endophytes of non-legumes

Root cortex

Nitrogen fixation 255 ×106 t N is fixed annually through

Three ways ★ Atmospheric Nitrogen fixation ★ Industrial Nitrogen fixation ★ Biological Nitrogen fixation (BNF)

BNF contributes 139 to 170 ×106 t N / yr

Comparatively: Less than the total N reserves (105,

000 ×106 t N), but

2 to 3 times greater than inputs of N from fertilizers (65 ×106 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 BNFmax 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 9 - 32 ºC

(min – max)● RF 450 –

1350 (mm) ● pH 4.8 – 9.6

Strains isolated form Ethiopian soils

Addis Ababa

Awassa

Abergele

Alemaya

Arba- minch Borena-negele

3N48E

6N

9N

12N

15N

33E 36E 40E 44E Moyal

e

Nazret

Ziway

Yavelo

Sampling Route

The beauty of d

iversit

y study is

when yo

u have

large number of sa

mples from diffe

rent

Legumes & Agroecology

Techniques

Collecting nodules Trapping

Authentication of rhizobial isolates on homologous host

0.1

Devosia neptuniae

Beijerinckia indica ●

S. morelense

Blastobacter dinitrificans

AC26e

AC104c1 AC104a

AC62a AC82d

M. tianshanense

AC64c

R. hainanense

B. japonicum

AC86c1

B. liaoningense

M. plurifarium

S. kummerowiae

AC29c

AC39d

AC91e

AC22d

AC79a

S. xinjiangensis

AC86a

AC70c

S. arboris

S. kostiense

S. fredii

AC97a

R. loessense

R. etli

S. terangae

AC27e

AC21a2

AC92d

B. yuanmingense

AC51e

AC107e

AC87k1

AC93e

AC87k3

S. americanus

AC101b

AC21c2

39c1

AC65c

M. mediterraneum

B. elkanii

A. albertimagni

R. galegae

A. undicola

AC88a

AC4d

AC100b

A. rhizogenes

AC90e1

AC90b

AC52c

S. adhaerens

AC72a

AC10d

AC100a AC82b

R. tropici R. leguminosarum

Rhizobium sp. (SDW024)AC56b

R. indigoferaeR. sullae

R. yanglingenseR. gallicum

R. mongolenseRhizobium sp. (X59)

R. giardiniiRhizobium sp. (SDW058)

AC73d R. huautlense

A. vitis AC97c1

AC77b AC11a A. larrymooreiA. rubi

A. tumefaciensAC42c A. radiobacterAC79c1

AC47d AC47a

AC18a AC20b

AC38b2 AC10a1 AC1b S. saheli

S. medicae AC28a

AC50e S meliloti

M. chacoenseAC88c

M. loti M. ciceri

M. huakuii M. amorphae

AC99d AC39a

100e AC98a

Xanthobacter autotrophicus ●Azorhizobium caulinodans

Met. organophylum ●Met. nodulans

AC94a AC87b1

AC64a AC64b

AC87L AC87n

AC86b2 AC92c 100

99

93

9994

83

100

82

9880

89

71

94

86

83

60

71

98

77

89

100

95

92

85

56

58

93

94

74

97

91

96

100

96

90

93

6172

70 Rhizobium

I (2)II

III (2)IV

V (2)VI

VIII VII

IX (2)

Agrobacterium

I

II

III IV

Ensifer

I(2)

II

III (3)

IV

V

Mesorhizobium

I (7)

II

Bradyrhizobium

I (2)

II

III (3) IV V VI

VII (2)

VIII

Methylobacterium

Neighbour-joining Phylogeny estimated from partial 16S rRNA (203 unnamed test strains)

Steadily growing number o

f rhizo

bia in our biobank

more than 500 str

ains

13 strainsTotal 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

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9 Shoot dry wt. Plant-1 SUNAINAShoot dry wt. Plant-1 MH-97-6Shoot dry wt. Plant-1 SML32Shoot dry wt. Plant-1 SML134

Rhizobial strains

Shoo

t dry

wt.

plan

t-1(g

)

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 Nodule Leaf Treatment number dry wt. area

plant-1 (g) (cm2)Inoc.HB 99 40.78de 0.54c 2.29cd

HB113 52.00bc 0.69b 2.62cb

HB 67 37.44ef 0.50c 2.19d

HB110 59.22b 0.69b 2.78b

HB 429 66.78a 0.78a 3.28a

HB 92b 62.00ab 0.72ab 2.51bc

HB 48 44.22de 0.67b 2.35cd

23 kg N ha-1 57.78bc 0.60b 2.77b

- Inoc. & -N 33.00f 0.47c 1.78e

P (Kgha-1)0 43.96c 0.54b 31.42c

23 48.52b 0.67a 34.79b

46 55.26a 0.70a 38.03a

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

HB 429

HB 92b

HB110

HB113

HB 48

HB 67

HB 99

N+ (23Kg)

N- & R-

01020304050607080

Noulde number as affected by inoculation

Rhizobium Strains

Nod

ule

Num

ber/

plan

t

ab

cd

f

bc

debccd

ab

Number Number Hundred Grain Straw HarvestTreatment of pods of seed seed yield yield index

plant-1 pod-1 weight (g) (ton ha-1) (ton ha-1) (%)Inoc.HB 99 8.56c 2.91b 35.22cd 1.34de 2.75bc 35.0bc

HB113 9.03bc 3.36a 40.30b 2.03ab 3.26ab 39.2ab

HB 67 8.44c 2.78b 35.60cd 1.54cd 2.68c 37.8ab

HB110 10.50ab 3.38a 41.00ab 1.81bc 2.80bc 40.2ab

HB 429 11.25a 3.42a 44.42a 2.36a 3.38a 41.6a

HB 92b 9.20bc 3.02ab 41.00ab 1.92b 3.14ab 38.3ab

HB 48 8.73bc 2.85b 36.50c 1.74bc 2.76bc 39.6ab

23 kg N/ha 10.50ab 3.03ab 41.80ab 1.92b 2.88bc 40.5ab

No Inoc. & -N 7.47c 2.36c 32.60d 1.17e 2.76bc 31.2c

P (Kgha-1)  0 7.72c 2.78b 36.48c 1.13b 2.53b 32.8b

  23 9.60b 3.05a 38.60b 2.01a 3.11a 40.5a

  46 10.58a 3.21a 41.01a 2.13a 3.16a 41.2a

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

HB 429

HB113 HB 92b

HB110 HB 48 HB 67 HB 99 N+ N- & I-0

0.5

1

1.5

2

2.5

Grain yield (t/ha) as affected by inoculation

rhizobiumStrains

Grai

n yi

eld

(t/h

a)

a

bbc

e

bccd

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 Nitrogen ResidualTreatment content in content in nitrogen

straw (%) grain (%) (%)Inoc.HB 99 0.69 2.65c 0.14bc

HB 113 0.73 3.01abc 0.15ab

HB 67 0.69 2.87bc 0.13bcd

HB 110 0.73 2.92bc 0.14bc

HB 429 0.78 3.33a 0.17a

HB 92b 0.73 3.05ab 0.15ab

HB 48 0.70 2.91bc 0.14bc

23 kg N ha-1 0.71 2.94bc 0.11cd

No Inoc. & -N 0.67 2.10d 0.11cd

P (Kgha-1)0 0.69 2.61b 0.12b

23 0.70 2.96a 0.14ab

46 0.78 3.02a 0.15a

HB 429

HB 92b

HB 113

HB 110

HB 48

HB 67

HB 99

N+ N- & R-

00.5

11.5

22.5

33.5

Grain N content (%) as affected by inoculation

Rhizobium Strains

Grai

n N

cont

ent (

%)

aabc bc

d

bc bc b cab

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