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Phenotypic and genotypic diversity of
Rhizobia nodulating lentil and chickpea
in Morocco and their utilization.
I.THAMI AL...
Outline
Introduction & Objective
Activites
Results
Conclusion
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 l...
Objective
Project aims to:
limit the use of chemical fertilizers Priority to fertilizers based on beneficial
microorganism...
Project activities
- Sampling of chickpea & lentil nodulating Rhizobia in main areas of production
- Examination of phenot...
Prospection & collection of Rhizobium
Collection of Rhizobium: Total number of Rhizobium sampled:
207 of chickpea
206 of l...
Nodulation test: Count of 123 nodulated chickpea rhizobium,
& 112 lentil rhizobium
Confirmed by nod C
Infectivity test, pr...
Phenotyping: chickpea
Cluster 1
fast growing bacteria, tolerants to high
temperature (36°C), to salinity (855mM),
sensitiv...
Phenotyping: lentil
Dendrogram showing phenotypic diversity among rhizobia nodulating lentil in Morocco
Cluster5:
fast gro...
DNA extraction was perfermed using CTAB method (fig5)
rep PCR was perfermed using Rep1 (5' IIIICGICGICATCIGGC 3' )and Rep2...
Characteristics of strains
Chickpea
P72 Sgb, tolerant to high temperature (44°C), salinity
(1198mM), and water stress (-0,...
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
% C...
The experiment was conducted in two sites: Merchouch and Ain Sbit
in complete random bloc with four replications for each ...
Results: effect of lentil inoculation
0
5
10
15
20
25
30
35
N0 L3 L43 N120
Nodules number/plant
Effect of inoculation of l...
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...
Results: effect of chickpea inoculation
0
10
20
30
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N0 P72 P100 N120
Nodules number/plant
b
b
a a
Effect of ino...
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...
conclusion
Substantial phenotypic and genotypic diversity is present in rhizobia
nodulating chickpea & lentil in Morocco;
...
Thank you
Acknowledgement
The authors thank INRA & Hassan II Academy of Science and Technology for their
financial support...
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Phenotypic and genotypic diversity of Rhizobia nodulating lentil and chickpea in Morocco and their utilization

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Oral Presentation 12 by I. Thami Alami at the International Conference on Pulses in Marrakesh, Morocco, 18-20 April 2016

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Phenotypic and genotypic diversity of Rhizobia nodulating lentil and chickpea in Morocco and their utilization

  1. 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. 2. Outline Introduction & Objective Activites Results Conclusion
  3. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 19. Thank you Acknowledgement The authors thank INRA & Hassan II Academy of Science and Technology for their financial support. (BIOFERT project)

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