Leveraging legumes to combat poverty, hunger, malnutrition and environmental degradation

PRODUCT LINE 4: High nitrogen fixing chickpea, common bean, faba bean and soybean

By Team PL 4

Date: 5th Oct 2016http://grainlegumes.cgiar.org

Review Meeting Grain Legumes Phase 1 & Extension Phase

Background Issues and R4D Challenges Rhizobial inoculants available in market are poor quality. Inoculants: No contaminants, rhizobia population (8x109

g) and longer shelf life. Regain confidence on the farmers: On-farm demos. Identify highly nodulating & N2 fixing rhizobia. Variability in particular crop for nodulation. SNF potential limited by abiotic stresses including low P. Standardize mass-multiplication & carrier that suits

rhizobia. Rhizobia as PGP: for P solubilization, Fe mobilization,

phyto-hormone production and biocontrol etc.

5th October 2016

Objectives (2012-16)Goal: Germplasm with high SNF potential and rhizobia,

under stress conditions identified, demonstrated on-farm and made available to the stakeholders.

Outcomes: Breeders access sources of SNF with multiple desirable

traits. Cultivars with enhanced SNF capacity under stress

conditions available.Mass production technologies available to inoculum

producers. Rhizobium producers incorporate superior strains in

commercial products. Farmers have access to improved rhizobial strains.

5th October 2016

Success Stories

1. Variability in nodulation behaviour in particular crop

2. On-farm demonstration of potential of SNF in chickpea

3. Plant growth-promoting (PGP) rhizobia in chickpea

4. Influence of rhizobia, variety and P-fertilizer on grain

yield in soybean and climbing beans

5. Quantification of SNF by 15N natural abundance

method

5th October 2016

1. Variability for nodulation in particular crop (s)

5th October 2016

Induction of EMS Mutations in 1237 M3 lines derived from Ghab 5

Identification SNP of M3 lines which carrying nodulation induced hyper-nodulation mutant. Two SNP were identified in

two bulks (confirmation in progress)

Response to Nitrogen Fixing Bacteria in Chickpea

Super nodulation line

IGgenotype

Terbolrainfa

llTerbol

irrigatedKafardanrainfall

Kafardanirrigated

114795 0.051 0.165 0.520 0.95170270 0.027 0.064 0.641 0.58570278 0.022 0.140 0.361 0.50570293 0.071 0.065 0.333 0.32870764 0.039 0.384 0.315 0.494Average 0.022 0.060 0.270 0.300

114795

115380115390117696

117697

117698117699117700117703117708

117714117718117728125018

125021125028128430

128434

128465128506131983 131985

132032

134532

134564

6035

6041

60506057

6058

61096111

6113

6446

646869605 69620702467024870249

7025270253

70255

7026270265

70269

70270

70272

70273

7027570277

70278

70280

7028570286

7029070291

7029370294

70297

70299

70304

70305

7030870309

70311

7031270313 70316

70328703307033270334

70335

70336

7033870340

7034270345

70346 70347703487034970350

703517035270355

7035770359

70361

703637036970370

70371

7037470375

70377

7037970381

7038370384

703887038970390

703937039470398

7039970401

704027040870409

704107041370416

704197042170427

7042870429

70430

70432

7043470556

7075570760707617076270763

70764

70766 70767

70768 70770

70772707737077470775

707767077770778

7077970780 70781

70782

7078370784707857078770788

7078970822

71832

7336973381

73382

73386 7338873390

7339474021

74929

74940

7497974994

74995

75029

75360

75406

77147717

77227758

8256

8447

891489859088

92399244

9413

94259430

9431

9433

94349435

9436943794389439 9440

9441

9442

9584 9586

9627

96299630

Genesis090

pbapistol

0

0.5

1

1.5

2

2.5

-0.1 0.4 0.9 1.4

Based on the average in each location, among 201 genotypes tested in two irrigation managements & 2 locations, 5 genotypes were superior in nodulation.

Dry weight(g) – Drought stress

Dry

wei

ght(g

) -irr

igat

ed

Screening for BNF in chickpea under diverse conditions

Influence of pink-pigmented facultative methylotrophic bacteria PPFMs and Rhizobium inoculation on BNF in Faba bean

DW_Nod DW_Plant N% N_ContentDW_Plant 0.42*** -N% 0.49*** 0.20* -N_Content 0.60*** 0.75*** 0.79*** -Nod_plant 0.99*** 0.42*** 0.47*** 0.59***

• Genotype “45/018F87307/06A“ and “Sakha 1” showed the best plant N contents (mg)/plant comparing to other genotypes.

Nitrogen fixation in faba bean

• Resource poor farmers cultivate pulses on soils of low fertility status.

• P-fertilizer use efficiency is low due to high P-fixing capacity of soils.

• High genotypic dependent variations in SNF and P use efficiency• P-deficiency not only reduce SNF contribution but also results in

decline in grain yield and its quality

Assessment of symbiotic nitrogen fixing potential and ‘P’ use efficiency in chickpea

in-vivo Experiment in-situ ExperimentMesorhizobium ciceri (IC-76) under sterile conditions Locations: ICRISAT & IIPRDesign: Completely Randomized Design (CRD)Sampling points:

Vegetative stage (45 DAS)

Assessment of inherent nodulation potential of chickpeaaccessions under field conditions

Locations: IARI & IIPRDesign: Augmented DesignSampling points: Vegetative stage (45 DAS), Flowering stage (75 DAS), Harvest stage

Chickpea accessions used in this study: 211 mini-core lines, 68 land races, 10 wild accessions and 2 checks (BG-256 and RSG-888)Observations: Nodule No. /plant, Nodule dry weight/plant, biomass/plant , N & P uptake

Genotypic differences in nodulation in chickpea inoculated with M. ciceri under pot experiments

(Total No. of entries 274)

Nodulation variants of min-core lines found common at both IIPR and ICRISAT (In-vivo study )

Nodulation rating* Accession numbers

1 ICC-6294, ICC-90022 ICC-2065, ICC-9590, ICC-11764

3

ICC-2072, ICC-4363, ICC-6293, ICC-6537, ICC-9643, ICC-9712, ICC-12324, ICC-12492, ICC-13764, ICC-15248, ICC-15435, ICC-15612

4

ICC-2969, ICC-3325, ICC-4533, ICC-4639,ICC-5337, ICC-6279, ICC-6306, ICC-7272,ICC-10018, ICC-13719, ICC-15294

5 ICC-2580, ICC-2990, ICC-3421, RSG-888*Ratings used for IIPR: 0 = No nodule; 1= 0.1 to 6 nodules; 2 = 7 to 12 nodules; 3 = 13 to 18 nodules; 4 = 19 to 24 nodules; 5= >24 nodules; *Ratings used for ICRISAT: 0 = No nodule; 1 = 0.1 to 1 nodule; 2 = 1 to 2 nodules; 3 = 2 to 4 nodules; 4 = 4 to 6 nodules; 5 = >6 nodules

High biomass accumulating accessions

Low biomass accumulating accessions

Accessions Nodule dry weight

(mg/plant)

Shoot weight(mg/plant)

Accessions Nodule dry weight

(mg/plant)

Shoot weight

(mg/plant)

ICC15762 38.0 1240 ICC 2210 9.0 190ICC13357 16.5 1115 ICC 6571 7.5 190ICC12328 16.0 1085 IG6154 6.5 185ICC12947 12.5 1085 ICC2679 6.0 180ICC 9137 20.5 1080 ICC 1352 6.0 160ICC12028 23.5 1035 ICC 7184 7.5 155ICC13523 19.5 1035 ICC2737 2.0 150ICC16654 54.0 1025 ICC7052 5.5 145ICC11584 15.5 1010 ICC12654 5.0 125ICC3410 26.0 1005 ICC15610 17.0 70

Therefore, under P-limitation, chickpea acc. with better PAE will supporthigh SNF activity and N2 fixation.

Nodulation behavior of some of the selected genotypes at IIPR & ICRISAT

High variability due to location. Similar genotype and strain of M. ciceri but grown in two different soils.

Similar numbers despite the locationdifferences

0.0

5.0

10.0

15.0

20.0

25.0

30.0

T1 T2 T3 T4 T1 T2 T3 T4 T1 T2 T3 T4

JAKI 9218 Shubra JG 11

Nodule No/pl Nodule wt (mg/pl)

0

10

20

30

40

50

60

T1 T2 T3 T4 T1 T2 T3 T4 T1 T2 T3 T4

JAKI 9218 Shubra PUSA 372

Nodule No/pl

Nodule wt (mg/pl)IARI

T1 - Absolute control (No DAP, Inoculation) T2 - Control (100 % RDF) T3 - M Ciceri T4 - M ciceri + DAP

ICRISAT

Evaluation of inoculation effect and P fertilization on symbiotic potential of chickpea genotypes at 45 DAS under field conditions

Shubra higher nodulation under low P at ICRISAT and IIPR

0

200

400

600

800

1000

1200

1400

T1 T2 T3 T4 T1 T2 T3 T4 T1 T2 T3 T4

JAKI 9218 Shubra PUSA 372

Shoo

t wei

ght (

mg/

plan

t)

T1 - Absolute control (No DAP, Inoculation) T2 - Control (100 % RDF) T3 - M ciceri T4 - M ciceri + DAP

JAKI 9218 showed higher biomass under P deficient conditions at IARI and ICRISAT

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

T1 T2 T3 T4 T1 T2 T3 T4 T1 T2 T3 T4

JAKI 9218 Shubra JG 11

Shoo

t wei

ght (

g/pl

ant)

ICRISAT

IARI

Experiment 2: Evaluation of inoculation effect and P fertilization on symbiotic biomass accumulation of chickpea genotypes at 45 DAS under field conditions

2. i. Quality assessment & characterization

of inoculants

ii. On-farm demonstration of potential of SNF

5th October 2016

2.1. Quality assessment of rhizobialinoculants available in Indian market

28 rhizobial inoculants collected from market: Odisha, Chhattisgarh, Madhya Pradesh, Jharkhand and Andhra Pradesh During Sept-Oct. i.e. prior to chickpea sowing season. These inoculants were stored at room temperatures in

shops. “Lignite” was used as carrier materials (all samples). Samples checked for its pH, moisture %, rhizobial count,

contamination count and nodulation. These were compared with ICRISAT rhizobial inoculants

5th October 2016

pH of the rhizobial inoculants procured

*= 1 to 4 from Orissa, 5 to 11 from Chhattisgarh, 12 to 13 from Jharkhand, 14 to 26 from MP, 27 and 28 from Telengana. Sample numbers 29 and 30 are from ICRISAT.

Optimum pH: 7

Highly acidic (pH 2.1-5.8) : 13Alkaline (pH 8.2-9.4): 7

Only 8 were found fit for growing rhizobia.

Moisture content of rhizobial inoculants

5th October 2016

*= 1 to 4 from Orissa, 5 to 11 from Chhattisgarh, 12 to 13 from Jharkhand, 14 to 26 from MP, 27 and 28 from Telengana. Sample numbers 29 and 30 are from ICRISAT.

Optimum moisture% for growing rhizobia : 30%.

5 contained < 15% &6 hadcontained >40%.

Chickpea rhizobial inoculants quality assessment

Of the 28 samples, only

15 contained rhizobia-like colonies in the media

6 had contamination.

4 nodulated but had poor count

Rhizobial quality assessment

Conclusion• Quality of rhizobia available in market is very poor.• Need to identify the formulation that suits better for

rhizobia.• Demonstrate on-farm so that farmers will get

confidence on SNF.

Effect of rhizobia (IC 76) on yield traits: on-farm study

• Under on-farm condition, IC-76 not only significantly

enhanced grain and stover yields but also other yield traits

including seed number, seed weight and pod weight.

• It is concluded that rhizobia not only fix atmospheric N2 but

also promote plant growth and yield traits

Effect of IC-76 on PGP traits of chickpea at 35 DAS: on-farm study

IC-76 enhanced not only nodule number and weight

but also shoot weight

Formulation for rhizobia Selection of formulation is very crucial as it can

determine the success or failure SNF by rhizobia. A good carrier material : should be able to deliver the

right number of viable cells in good physiological conditions, easy to use and economically affordable by the farmers. Five carrier materials: Peat, Talc, Talc + Starch, Charcoal

and Charcoal + Sugarcane stalk powder. Longevity studies conducted with 2 chickpea rhizobia,

IC-76 and IC-59 for 10 months.

Viability and longevity of rhizobia on five different formulations

Colony Forming Unit (CFU/ml) of different carrier materials in months0 1st 8th 9th 10th

Peat 3.9x10⁹ 4.3x109 5.1x108 4.3x108 1.2x108

Talc 3.3x109 1.8x109 3.4x107 2.1x107 1.1x107

Talc+Starch 5.0x10⁹ 2.0x109 4.9x107 3.1x107 1.2x107

Charcoal 7.0x108 4.0x108 8.1x106 6.1x106 3.2x106

Charcoal+ Sugarcane stem powder 2.3x10⁹ 1.1x109 9.3x106 3.7x106 2.3x106

Of the five different formulations studied, viability and longevity were found more in peat (3.9 x 109 to 1.2 x 108)even after 10 months

On-farm demonstration of SNF

• On-farm demo (35 farmers) done at Mahbubnagar(Telangana), Raichur and Gulbarga (Karnataka), andLudhiana (Punjab)

• Rhizobia specific to chickpea, IC-76 (ICRISAT product)was evaluated.

• Chickpea varieties : JG 11, PBG 5

Demonstrations at farmers’ fields

Application of biofertilizer (cost @ US$ 1.5 ha-1) at farmer’s field improved

Yield by 1.9 to 7.7% in Punjab.

13-21 % in Telangana and Karnataka.

Promotion of biofertilizer technology required to ensure full benefits to resource poor farmers.

Plant growth-promoting (PGP) rhizobia

Plant growth-promoting (PGP) rhizobia

30

PGP bacteria are known to enhance plant growth when inoculated : soil or seeds. PGP bacteria affect plant growth through fixation of

atmospheric N2, solubilization of minerals, synthesis of enzymes & phyto-hormones and inhibition of pathogens. Rhizobia have also been looked for its PGP properties, as

rhizobia can provide additional plant and soil health benefits besides fixing nitrogen. Hence, while selecting a rhizobia one needs to consider its other PGP traits also. In the present study, IC-76 (rhizobia) was characterized

for its PGP traits on chickpea under field conditions.

Mechanism of PGP traits (IC-76) Indole acetic acid (IAA) production

Control Positive

IAA accelerates plant growthand development by improvingroot/shoot growth and seedlingvigor through its involvement incell division, differentiation andvascular bundle formation.

IAA production by IC-76 =327±7.3 µg mL-1

Mechanism of PGP traits (IC-76 ):Siderophore production

Siderophore production

Low molecular weightcompounds with high affinitytowards Fe3+, hence involvedin sequestering Fe3+ and thusmakes the iron available forplants.

Siderophore production byIC-76 = 44.7±0.2 Units

Mechanism of PGP traits: Cellulase production

Cellulase production

Cellulase degrades cellulose in the plantcell wall, thus destroying the adhesion ofthe pathogen to the root surface of theplant.

Cellulase production by IC-76 = 2(Cellulase production is estimatedthrough the halo zone around themicrobial colonies on a 0−3 rating scaleas follows: 0 = no change; 1 =positive; 2 =halo zone of 1−3 mm; 3 = halo zone of4−6 mm and 4 = halo zone of 7 mm andabove

Mechanism of PGP traits (IC-76 ):β-1,3-glucanase production

Control Positive

β-1,3-glucanase degradesthe 1,3-glucan layer ofmany pathogens thusinhibiting the pathogeninvasion.

β-1,3-glucanaseproduction by IC-76 =0.9±0.003 Units

Mechanism of PGP traits (IC-76) :Hydrocyanic acid (HCN)

Control Positive Low molecular weight antimicrobial metabolite which inhibits

electron transport and hence disruption of energy supply tothe cells.

HCN production by IC-76 = 2(HCN production is estimated on a 0−3 rating scale based onthe intensity of the reddish brown color as follows: 0 = nocolor change; 1 = light reddish brown; 2 = medium reddishbrown and 3 = dark reddish brown)

IARI

• Of 243 Mesorhizobium isolates of chickpea (Ludhiana, Pantnagar), 8 were found promising with PGP and BNF traits. Identified potential isolates of Mesorhizobium needs to be characterized on molecular basis and evaluated for BNF and yield under field conditions (min. 3 years) for harnessing their potential for chickpea and chickpea based cropping systems.

• Few non-rhizobial bacteria also noticed in the nodules which should be deciphered for their exact role in chickpea.

Identification of promising Mesorhizobium with PGP traits

3. Influence of rhizobia, variety and P-fertilizer on grain yield in soybean and climbing beans

Response of promiscuous and specific soybean varieties to inoculation in Kenya

0.0

1.0

2.0

3.0

4.0

5.0

EAI 3

600

Nam

soy

4m

TGx

1740

-2F

TGx

1895

-33F

TGx

1835

-10F

EAI 3

600

Nam

soy

4m

TGx

1740

-2F

TGx

1895

-33F

TGx

1835

-10F

ARDAP George Kivanda

Soyb

ean

grai

n yi

eld

(t/h

a)- Inoc

+ Inoc

Stronger inoculation response significantly evident in some varieties

Effect of P-fertilizer and/or Inoculant on soybean grain yield (t ha-1) in Nigeria

P and rhizobialinoculant plays key role in grain yield. Highest yield was obtained when applied both.

Cumulative probability of soybean grain yield (t ha-1) in Nigeria

50% of farmers gets grain yield > 1 t ha-1 in the treatment without inputs. With P + Inoculant, 50% gets >1.8 t ha-1 yield

Cumulative probability of relative increase (%) in soybean grain yield compared to control in Nigeria

Inoculation makes P economic!!

40% of farmers got 100% more yield with P + Inoculant whereas 50% of farmers gets a yield increase of about 40% or more with P or I only.

Soybean on problem soils in ZimbabweSingle Super Phosphate (SSP), Compound L (N:P205:K20:S + B -5:17:10:8:0.25) and Dolomitic lime (500 kg/ha)

P in combination with rhizobia yielded more. When lime added with P & rhizobia, yield was higher

Effect of herbicides on rhizobium nodulation

• Metribuzin and Imizathapyr postemergence treatment hasnegative effects on Rhizobiumnodulation in Faba bean

• The accession IG100096 collectedfrom morocco showed highnodulation number underherbicides treatments

0

2

4

6

8

10

12

[5.5

6-11

.95]

[13.

42-1

3.66

]

>37.

5

Number of genotypes

NNP

Current and future suitability (left and right) of climbing beans

Analyzing demand and setting research priorities

Grain Yield & Total Ndfa of promising climbing bean genotypes: Field evaluation at 2 locations in ColombiaDarien: 68.3

MAC 27: high grain yield & good SNF ability

ENF 235

Darien: 91.2 Popayan: 51.2

CGA 10

Darien: 90.3 Popayan: 42.3

Grain yield (kg ha -1) Total Ndfa (kgN ha -1)Genotype Darien Popayan Darien Popayán

Prom

isin

g fo

r SN

F ENF 235 3224 2969 91.7 51.2

CGA 10 4572 3294 90.4 42.3ENF 21 4264 3357 71.7 54.6

ENF 28 3362 3229 68.1 50.2

ENF 234 3241 3632 62.0 51.5

High

yie

ldin

g

ENF 83 5975 3909 47.3 46.7

MBC 117 5192 3615 48.8 39.4

MAC 57 4628 4584 50.2 50.3

Controls

MAC 27 6827 4460 56.6 56.8GLORIABAMBA NN 2043 2557 0.0 0.0

Range100 lines 6827- 1777 4643 - 1930 91.7 - 0.2 60.1 - 0.3average 3592 3172 49.2 42.4Sig. diff *** *** *** ***

Gloriabamba NN: a non-nodulating check that yielded less, indicating N deficiency at each site. SNF ability combined with high grain yield, NUE, NPI and Canopy Biomass.

4. Quantification of SNF by 15N natural abundance method

Quantification of SNF by 15N natural abundance methodOne of CIAT’s specific objectives was to validate the

measurement of 15N in grain to quantify SNF ability. This observation was similar to bush beans published

this year by Polania et al entitled “Estimation ofphenotypic variability in symbiotic nitrogen fixationability of common bean under drought stress using15Nnatural abundance in grain”. Europ. J. Agronomy 79(2016) 66–73 . This study reaffirms that the use of grain sample is

valid to quantify SNF ability using 15N naturalabundance method. Here again, Glroiabamba NN is anon-nodulating genotype.

3 4 5 6 7 8 91

2

3

4

5

6

7

8

9

shoot 15N Natural abundance (0/00)

Gra

in 1

5N N

atur

al a

bund

ance

(0/0

0)

Mean: 5.9***

Mean: 4.2 **

r= 0.51***GLORIABAMBA NN

ENF 81

ENF 235ENF 28

PO 07AT 49CGA 10

ENF 234ENF 21ENF 159

ENF 82

CGA 11

ENF 213

ENF 207

ENF 117

ENF 64ENF 188

CGA 64ENF 34

CGA 52ENF 24ENF 71

ENF 85ENF 211 ENF 86

CGA 52

Relationship between 15N natural abundance in shoot vs 15N natural abundance in grain to validate the use of grainmethod for breeding

Positive & highly significant correlation. Grain sample can beconsidered as valid to quantify SNF ability using 15 N naturalabundance method.

Impact Ex-post evaluation of climbing bean technology in

Rwanda (DIIVA, 2013) Yield increase compared to bush beans of … 270 kg/ha in poor soil fertility plots 657 kg/ha in better fertility plots

For 10 kg of climbing bean planted… Per capita consumption increases by 5 kg in that

agricultural season Per capita annual expenditure increases by 9% Poverty gap reduces by 3% per year

Lessons Learned/Summary

Quality of rhizobial inoculants need to be ensured. Reliable and quick quality control techniques need to

be developed. Capacity building for inoculants production Influence of variety, high nodulating and nitrogen fixing

rhizobia and phosphorous on SNF and grain yield well established. It is time to pass on this knowledge to farmers. Large scale demonstration need to be conducted on

farmers fields to provide confidence to the farmers.Grain sample can be considered as valid to quantify SNF

ability using 15 N natural abundance method.

Future areas for R4D Identification of high nodulating and nitrogen fixing

indigenous rhizobia, suitable for a region. Rhizobia for problem soils such as acidic/saline soils. Identify the crop variety that supports high SNF.Mass multiplication and easy to handle formulations. Easy and quick quality check technique and

monitoring of supplied chain.On-farm demonstrations to convince farmers. Pinpointing contribution of nitrogen in terms of

nitrogen economy to succeeding crop(s).

Contributing Bilateral Projects

N2 AfricaCRP-GL Supported projects

Indian Council of Agril. Research (ICAR) is going to create 16 Bio-fertilizer and Bio-pesticide Hubs in different states of India for production of Bio-inoculants in 2016

Posters displayed 1. Assessment of inherent nodulation potential among chickpea

accessions belonging to minicore set. (K. Swarnalakshmi, S. Gopalakrishnan, M. Singh, Sushil K Chaturvedi, Abhishek Rathore, Vijay Pooniya, Hari D Upadhyaya and P S Basu).

1. Studies on influence of pink-pigmented facultative methylotrophic bacteria PPFMs and Rhizobium inoculation on BNF in Faba bean and Chickpea genotypes. By Fouad Maalouf, Aladdin Hamwieh, H.A. Taleb and Rehab Abdul Rehman.

2. Assessment of chickpea Mesorhizobium symbiosis for biological Nitrogen fixation and yield enhancement. By Poonam Sharma, Ramesh Chandra, G. Kaur, S. Kaur, N. Pareek and Sarvjeet Singh.

3. Evaluation of genotypic variation in symbiotic nitrogen fixation ability in climbing bean. By Norma Barbosa, Elizabeth P., HectorFabio Buendia, Bodo Raatz, Idupulapati Rao, Stephen Beebe.

Leveraging legumes to combat poverty, hunger, malnutrition and environmental degradation

Thank you very much

Presented on behalf of Team PL 4