This document summarizes a study on the phenotypic and genotypic diversity of rhizobia that nodulate lentils and chickpeas in Morocco. Rhizobia samples were collected from various production areas and tested for traits like tolerance to temperature, salinity, heavy metals, and water stress. Selected high-performing strains were tested in pot and field trials, where they increased nodulation, plant growth, grain yield, and nitrogen content compared to uninoculated controls. The study found substantial diversity among the rhizobia populations and that inoculation with efficient strains is an effective way to increase legume productivity and reduce chemical fertilizer use.

1. Phenotypic and genotypic diversity of
Rhizobia nodulating lentil and chickpea
in Morocco and their utilization.
I.THAMI ALAMI, I. BENJELLOUNS, .M. UDUPA and A.DOUIRA

2. Outline
Introduction & Objective
Activites
Results
Conclusion

3. 0
100
200
300
400
500
600
700
800
900
1000
Yield (Kg/ha) Area (1000 ha)
Year
Figure 1. Trend in area and productivity of legumes (1962-2014)
Assessment of legumes situation in Morocco
This decrease is mainly due to:
the severe bioclimatic conditions
&
devpt of more profitable alternative crops
Development of monocultures
Intensive use of chemical fertilizers
Disastrous impacts on the environment
Food legumes play a substantial socio-economic role and important component in
Moroccan diet. However yield remains low.
Chickpea (Cicer arietenum) and lentil (lens culinaris) rank second in Morocco, after
faba bean
Reintroducing legumes in crop systems using biofertilizers is essential to respond
to the increased demand of consumers and to preserve the quality of soils and
sustainability of agroecosystemes

4. Objective
Project aims to:
limit the use of chemical fertilizers Priority to fertilizers based on beneficial
microorganisms (Biofertilizers)
Advantage of Biofertilizers contributing to sustainable agriculture that respects
the environment.
mainly nitrogen fixing bacteria in symbiosis with legumes
& soil bacteria solubilizing phosphates

5. Project activities
- Sampling of chickpea & lentil nodulating Rhizobia in main areas of production
- Examination of phenotypic and genotypic diversity for tolerance to environmental
stresses of rhizobia nodulating chickpea and lentil
- Test of the more infective, effective and tolerant strains as biofertilizers under
controlled conditions and field conditions

6. Prospection & collection of Rhizobium
Collection of Rhizobium: Total number of Rhizobium sampled:
207 of chickpea
206 of lentil
Number of farm’s field surveyed:
Lentil 14
Chickpea 15
Nodulation was observed in all the
fields except in waterlogged soils
No functional nodules were also
observed in some fields (Benslimane,
Ain Sbit).
Prospection of 29 sites of lentil & chickpea
Results

7. Nodulation test: Count of 123 nodulated chickpea rhizobium,
& 112 lentil rhizobium
Confirmed by nod C
Infectivity test, presence of nodC
Phenotyping
The phenotyping tests were carried-out on Petri dishes with YEM agar medium.
Phenotypic markers studied
Resistance to heavy metals (2%- 100%)
Resistance to antibiotics (30%-100%)
Tolerance to high temperature (60%-70%)
Tolerance to salinity (4%- 11%)
Tolerance to water stress (4% to 5%)
Assessment of Growth and symbiotic traits
Results
nod C gene amplification

8. Phenotyping: chickpea
Cluster 1
fast growing bacteria, tolerants to high
temperature (36°C), to salinity (855mM),
sensitive to water stress , tolerant to
heavy metals except ZnCl2 and HgCl2
Cluster2:
Fast growing bacteria, tolerant to
high temperature (40°C), salinity
(1198mM), water stress (-0,25
Mpa) and heavy metals
Cluster 3:
slow growing bacteria, tolerante
to high temperature (40°C), to
salinity ( 684mM), sensitive to
water stress and to heavy
metals except HgCl2
Cluster4:
fast growing bacteria, tolerante
to high temperature (36°C),
sensitive to salinity (171mM),
to water stress, to heavy metals
except ZnCl2
Cluster5:
Slow growing bacteria, tolerant to high
temperature (44°C), salinity (1198mM),
and water stress (-0,25 Mpa), tolerant to
heavy metals except ZnCl2 and HgCl2
Cluster6:
fast growing bacteria, tolerante
to high temperature (36°C), to
salinity (513mM), sensitive to
water stress, to HgCl2
cluster7
fast growing bacteria, tolerante to high
temperature (40°C-44°C), to salinity
(684mM), to water stress (-0,15Mpa),
sensitive to heavy metals except MnCl2
Dendrogram showing phenotypic diversity among the 123 rhizobia nodulating chickpea in
Morocco

9. Phenotyping: lentil
Dendrogram showing phenotypic diversity among rhizobia nodulating lentil in Morocco
Cluster5:
fast growing bacteria, tolerante
to high temperature (40°C),
sensitive to salinity ), to water
stress, to heavy metals except
ZnCl2
Cluster3:
fast growing bacteria, tolerants to high
temperature (40°C), to salinity (855mM),
to water stress (-0,15) and to heavy metals
except ZnCl2 and HgCl2
Cluster 2:
fast growing
bacteria, tolerante
to high temperature
(40°C), to salinity),
sensitive to water
stress and tolerant
to heavy metals
except HgCl2
Cluster1:
Fast growing bacteria, tolerant to
high temperature (40°C), salinity),
water stress (-0,15Mpa)
Cluster4:
fast growing bacteria, tolerante
to high temperature (36°C), to
salinity (513mM), sensitive to
water stress, to HgCl2
Cluster 6
fast growing bacteria, tolerant to high
temperature (44°C), salinity (1198mM),
and water stress (-0,25 Mpa)

10. DNA extraction was perfermed using CTAB method (fig5)
rep PCR was perfermed using Rep1 (5' IIIICGICGICATCIGGC 3' )and Rep2
(5' ICGICTTATCIGGCCTAC 3‘) primers according to Versalovic et al. (1991)
methodology
Analysis of Rep profiles classify isolated strains from lentil into 22 cluster, and
those of chickpea into 38 cluster. rhizobia of each cluster are genetically
different than those of other clusters.
UPGMA dendrogram showing genetic diversity among
rhizobia nodulating chickpea in Morocco
Isolates genotyping
181
192
193
194
195
196
197
198
190
191
391
395374388386396397401176182
183
184
185
187
188
189
153
140
141
142
143
146
177
179
180
147
355
362
394
151
352
390
392
393
148
344
309
310152356358359361377378384122123
124
144
422
430
431
432
433
434
435
444
408
436
165
337
8
224
335
230
209
212
222
227
228
229
256
343
234
238
239
240244245246250252253254255
236
242
251
100
216
235
237
403412417
421
201
247
402
407
410
437
438
442
443
445
171
399
441
446
423
425
426
427
398
385
381
383
382
72
351
447
448
449450452453454455456457
458
459
460
0.00
0.05
0.10
0.15
0.20
Shared allel based neighbor joining tree showing genetic
diversity among rhizobia isolated from lentil nodules

11. Characteristics of strains
Chickpea
P72 Sgb, tolerant to high temperature (44°C), salinity
(1198mM), and water stress (-0,25 Mpa), tolerant
to heavy metals except ZnCl2 and HgCl2, M
Infective & efficient
P100 fgb, tolerante to high temperature (40°C-44°C), to
salinity (684mM), to water stress (-0,15Mpa),
sensitive to heavy metals except MnCl2, AS
Infective & efficient
Lentil
L3 fgb, tolerant to high temperature (44°C), salinity
(1198mM), and water stress (-0,25 Mpa), tolerant
to heavy metals, M
Infective & efficient
L43 fast growing bacteria, tolerants to high
temperature (40°C), to salinity (855mM), to
water stress (-0,15) and to heavy metals except
ZnCl2 and HgCl2, HB
Infective & efficient
Chickpea & lentill Strains selected for field trials
Field trials: Two sites for chickpea: Mercouch & Ain Sbit
& Two sites for lentil: Merchouch & Ain sbit

12. Site details for the field trials
Site Merchouch Ain Sbit
Location N 33°36.719’
W 006°42.534
N 33°32.384’
W 006°30.938
% Clay
(<2 mm)
47,6 42
% Silt
(2-60 mm)
41 25,3
% sand
(60-2000 mm)
11,4 32,7
Soil pH
(Kcl 1N)
7 6,25
OM (g kg-1) 2,7 2,9
P2O5(ppm) 15,39 20,24
K2O(ppm) 228 165

13. The experiment was conducted in two sites: Merchouch and Ain Sbit
in complete random bloc with four replications for each treatment
Methodology
Treatments: for chickpea: N0 ; P72; P100 ; N120 kg/ha
for lentil: N0 ; L3; L43 ; N120 kg/ha
Observations:
flowering stage:
• Chlorophyll concentration
• Nodulation (location, number, weight and color)
• Dry matter weight of root and shoot
At harvest stage:
• Grain yield
• Straw yield
• 1000 seed weight
• Quantity of nitrogen content
• Quantity of exported nitrogen

14. Results: effect of lentil inoculation
0
5
10
15
20
25
30
35
N0 L3 L43 N120
Nodules number/plant
Effect of inoculation of lentil on nodules nomber in Ain Sbit
0
5
10
15
20
25
30
35
N0 L3 L43 N120
Nodules number /plant
bb
b
a
Effect of inoculation of lentil on nodules nomber in Merchouch
0
200
400
600
800
1000
1200
1400
1600
N0 L3 L43 N120
DW shoot (mg/plant)
DW root(mg/plant)
aa
aaa
b
a
a
Effect of inoculation of lentil on dry matter of shoot and
root in Merchouch
0
200
400
600
800
1000
1200
1400
1600
N0 L3 L43 N120
DW shoot (mg/plant)
DW root(mg/plant)
Effect of inoculation of lentil on dry matter of shoot and root
in Ain Sbit
Ain Sbit Merchouch

15. Results: effect of lentil inoculation
5
8
7 8
0
5
10
15
20
25
30
35
N0 L3 L43 N120
Grain yield (q/ha)
Straw yield (q/ha)
a
ab a
Effect of inoculation of lentil on grain and shoot yield in Ain Sbit
b
19
24 23
23
0
5
10
15
20
25
30
35
N0 L3 L43 N120
Grain yield
(q/ha)
Straw yield (q/ha)
b
a aa
Effect of inoculation of lentil on grain and shoot yield in Merchouch
0
20
40
60
80
100
120
140
N0 L3 L43 N120
% Total nitrogen
Exported nitrogen (kg/ha)
aa
b
ab
b
ab
b
Effect of inoculation of lentil on total nitrogen in grain and
exported nitrogen in Ain Sbit
0
20
40
60
80
100
120
140
N0 L3 L43 N120
%Total nitrogen
Exported nitrogen…
a
a ab
a
b
a
b
Effect of inoculation of lentil on total nitrogen in grain and
exported nitrogen in Merchouch
Ain Sbit

16. Results: effect of chickpea inoculation
0
10
20
30
40
50
60
70
N0 P72 P100 N120
Nodules number/plant
b
b
a a
Effect of inoculation of chickpea on nodules number in
Ain Sbit
0
10
20
30
40
50
60
70
N0 P72 P100 N120
Nodules number/plant
Effect of inoculation of chickpea on nodules number in
Merchouch
0
5000
10000
15000
20000
25000
N0 P72 P100 N120
DW shoot (mg/plant)
DW root (mg/plant)
a
a
a a
a a
a
b
Effect of inoculation of chickpea on shoot and root dry
matter in Merchouch
0
500
1000
1500
2000
2500
3000
3500
4000
N0 P72 P100 N120
DW shoot (mg/plant)
DW root (mg/plant)
ab
ab
ab
a
a
b
bcc
Effect of inoculation of chickpea on shoot and
root dry matter in Ain sbit
MerchouchAin Sbit

17. Results: effect of chickpea inoculation
17 25 22 25
0
10
20
30
40
50
60
N0 P72 P100 N120
Grain yield (q/ha)
Strow yield (q/ha)
b
ab
a
a
ab
a
a
Effect of inoculation of chickpea on grain and shoot yield
in Merchouch
0
10
20
30
40
50
60
N0 P72 P100 N120
Grain yield (q/ha)
Straw yield (q/ha)
Effect of inoculation of chickpea on grain and shoot yield
in Ain Sbit
0
20
40
60
80
100
120
140
N0 P72 P100 N120
%Total nitrogen
Exported nitrogen (kg/ha)
a
Effect of inoculation of chickpea on total nitrogen in
grain and exported nitrogen in Ain Sbit
b
b b
b
b
b
a
0
20
40
60
80
100
120
N0 P72 P100 N120
% Total nitrogen
Exported nitrogen (kg/ha)
a
b b b
b
a
ab
a
Effect of inoculation of chickpea on total nitrogen in
grain and exported nitrogen in Merchouch
Ain Sbit

18. conclusion
Substantial phenotypic and genotypic diversity is present in rhizobia
nodulating chickpea & lentil in Morocco;
Both pot culture & field experiments showed enhanced grain yield
and straw yiel as Nitrogen content
P application along with rhizobium inoculation showed significantly
high performance
Field trial involving inoculation along with other bacteria “PGPR” to
study the phosphate solubilization is in progress.

19. Thank you
Acknowledgement
The authors thank INRA & Hassan II Academy of Science and Technology for their
financial support. (BIOFERT project)