Mekdela Amba University

College of Agriculture and Natural Resource

Isolation and Evaluation of Biofertilizer Inoculants for Improved

Productivity of Leguminous Crops and Tree Species in
Northcentral Highlands of Ethiopia

Research proposal

Presenter:
Mohammed Ahmed

June, 2024
Gimba, Ethiopia
 Researchers profile
S/N Participants Specialization Role Responsibility
1 Mohammed Agroforestry & soil PI Soil analysis, soil-plant-water systems,
Ahmed (MSc) management seedling inoculation experiment

2 Birhanu Agronomy CI Legume crop experiment, soil analysis,

Gebeyehu agronomy, crop productivity analysis
(MSc)

3 Getachew Industrial CI Microbial isolation, characterization,

Alamnie biotechnology efficient strain selection, biofertilizer
(PhD) preparation

4 Abayeneh Microbiology CI Laboratory administration, microbial

Girma (MSc) culturing and maintenance

5 Yesuf Forest CI Nursery supervision, experiments on

Mohammed management & seedling inoculation, seedling growth
(MSc) climate change analysis
 Presentation outline

 Biofertilizer (introduction)

 Research gaps and objectives

 Materials and methods

 Workplan

 Financial plan
 1. INTRODUCTION

1.1 Background and Justification

 Microbes play key functions in the rhizosphere and soil systems.
 solubility and availability of plant nutrients,
 enhancing uptake of soil nutrients,
 amelioration of abiotic stresses(e.g. drought),
 suppress plant pathogens (Raimi et al., 2021).
 Some of the most known of these microbes are;
 Nitrogen-fixing bacteria (Rhizobium, Frankia), 40-200kg/ha
 Phosphate-solubilizing bacteria, 30-50kg/ha
 P-mobilizing mycorrhizal fungi (Aloo et al., 2021).

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 INTRODUCTION…Cont’d

 Biofertilizers are formulations comprising of beneficial microbes to

improve plant nutrition (Aloo et al., 2021).
 It is a cost-effective and eco-friendly way for soil fertility
improvement (Chaudhary et al., 2022).
 Thus, it becomes important in enhancing crop productivity,
rehabilitation of degraded ecosystems and establishment of forest
plantations (Araujo et al., 2012).
 The presence of an efficient and large population of soil microbes
greatly enhances the productivity of leguminous crops (Araujo et al.,
2012). Up to 50%.

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 INTRODUCTION…Cont’d

 Forest tree species (Pinus and Casuarina) are also reliant on

mycorrhizal fungi for their growth (Dyshko et al., 2024).
 However, disturbed agricultural soils, degraded lands, and non-
native planting sites lack or have lower inoculum of soil
microorganisms (Wang et al., 2022).
 Thus, introducing and maintaining a high level of soil microbial
community is a key strategy to improve the productivity of
leguminous crops (Chaudhary, 2022).

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 1.2 Problem/research gaps

 The cost and adverse environmental impacts of chemical fertilizers

suggest the need for alternative options for soil fertility improvement
(Aloo et al., 2021).
 The promising alternative, biofertilizer, is not widely applied in
Africa, including Ethiopia (Raimi., 2021).
 The reasons are low accessibility to farmers, inadequate research
for efficient microbial strains and low skill for biofertilizer
production (Keneni et al., 2021).
 Therefore, the study aims to fill research gaps in selection of
efficient microbial strains of biofertilizer for legume crops and
exotic tree species.

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 1.3 Objective of the study

 Overall objective
 To isolate efficient microbial strains and evaluate biofertilizer
inoculants for improved productivity of leguminous crops and tree
species.
 Specific objectives
• Isolate and select efficient microbial strains of nitrogen-fixing and
phosphate-solubilizing/mobilizing microbes
• Evaluate effect of biofertilizer inoculants on growth and yield of
leguminous crops
• Investigate seedling growth responses of Pinus patula and
Casuarina equisetifolia to biofertilizer inoculation.

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 2. MATERIALS AND METHODS

2.1 Study area description

 Target areas
 Potential agroecosystems of leguminous crops (Faba bean,
Field pea and Lentil)
 Degraded highland areas with potential for forest plantation.
 Specific location: Borena district, north-central Ethiopia.
 Climate: areas of sub-tropical (Woyna Dega) and temperate (Dega)
climatic conditions.
 Research station: MAU research sites

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 Methods…cont’d

2.2 Field sample collection

 Nodule sample from 10 plants from four (4) potential areas of each
legume crop (10x4x3)
 10 composite samples of root and rhizosphere soils (10x5).
 Sampling depth15-30cm for crops & 40-60cm for forest trees.
Microbial type Sample Reference
Symbiotic nitrogen- Root nodule for legume crops & Raimi et al.,
fixing bacteria tree species 2021
Phosphate solubilizing Rhizosphere soils for crops Guardiola et
bacteria al., 2023
Mycorrhizal fungi (P- Fine roots & Rhizosphere soils Dyshko et al.,
mobilizing) for tree species 2024

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 Methods…cont’d

2.3 Laboratory analysis

1) Microbial isolation
Microbial type Isolation technique Reference
Symbiotic nitrogen- Needle isolation and streak Raimi et al.,
fixing bacteria plating on yeast extract mannitol 2021
agar (YEMA) media
Phosphate Serial dilution culturing on YEMA Guardiola et al.,
solubilizing bacteria with Congo rod absorption 2023
And Pikovskaya’s broth (PKB)
Mycorrhizal fungi (P- Wet sieving of fungal spores and Dyshko et al.,
mobilizing) trypan blue staining 2024

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 Methods…cont’d

2) Characterization of microbial isolates

 Characterization and evaluation is essential for selection of efficient
microbial strains (Tam et al., 2020).
 The study will apply morphological, functional and molecular
characterization techniques.
 Morphological characterization

Techniques Parameters
 Gram staining  gram reaction,
 KOH string test  cellular form,
 Spore staining  shape, size, pigmentation
 Phenotypic methods  elevation, opacity, texture,
 margin, surface of colonies

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 Methods…cont’d

 Functional characterization
 Selection of microbial isolates based on colony formation and
quantitative analysis N-fixing and P solubilization rate.
 Detect and quantify the targeted ability of microbial strains
(Guardiola et al., 2023).
 Parameters: carbon source, growth rate, doubling time, optical
density (OD) with spectrophotometer quantify N-fixation and P-
solubilization.
 Molecular characterization
 Advanced technique only for three promising microbial isolates
 It uses 16S rDNA sequence analysis (Oo et al., 2020).

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 Methods…cont’d

3) Biofertilizer inoculant production

 The formulation type is liquid biofertilizer
 Mother cultures are inoculated to broth medium for three promising
microbial isolates
 Biofertilizer inoculants incubation on sterilized shakers.

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 Methods…cont’d

4) Quality control measures

 Quality control analysis and measures
 Ensure quality in every steps (mother culture test, broth test,
mixing & storage)
 Lessen contamination levels (autoclave sterilization)
 Periodic viability evaluation (each month)
 Microbial density analysis (number of cells per milliliter)

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 Methods…cont’d

2.4 Field experiment on promising strains

# Crop experiment
 Experiment on rainfed cropping systems
 Seed inoculation technique of diluted liquid biofertilizers.
 RCBD experimental design (best for field experiments)
 3 replication
 4 treatments
N-fixing bacteria P-solubilizing bacteria
Control NFB0 Control PSB0
Biofertilizer NFB1 Biofertilizer PSB1
Biofertilizer NFB2 Biofertilizer PSB2
Biofertilizer NFB3 Biofertilizer PSB3

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 Methods…cont’d

Crop experiment

Crops type (local Faba bean Field pea Lentil

variety) (Vicia faba) (Pisum sativum) (Lens cultinaris)
Plot size (W x L) 1.2m x 2m 1.2m x 2m 1m x 2m
Row spacing (cm) 40 30 20
Plant spacing (cm) 15 10 5
Plot spacing (m) 1 1 1
# plots 24 24 24

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 Methods…cont’d

# Seedling experiment
N-fixing bacteria P-mobilizing
(Mycorrhiza)
Control NFB0 Control PMF0
Biofertilizer NFB1 Biofertilizer PMF1
Biofertilizer NFB2 Biofertilizer PMF1
Biofertilizer NFB3 Biofertilizer PMF1

Tree species Casuarina equisetifolia Pinus patula

Plot size (W x L) 1m x 1m 1m x1m
Row spacing (cm) 8 8
Plant spacing (cm) 8 8
Plot spacing (m) 1 1
# plots 24 24

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 Methods…cont’d

2.5 Data collection

 Crop data: randomly selected 5 plants from central rows.

 Number, size and color of nodule
(8 weeks after sowing)
 Plant height (cm) at maturity
 Number of branches per plant
 Number of pods per plant
 Number of seeds per pods
 Seed yield per plant (g)
 100-seed weight (g)
 Dry biomass of crops (t/ha)
 Grain yield per plot (t/ha).
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 Methods…cont’d

 Seedling data: randomly selected 10 seedlings

 seedling height (cm)
 collar diameter of seedlings (cm)
 root length of seedlings (cm)
 nodulation (Casuarina equisetifolia)
 Dry biomass (kg)
 Node length

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 Methods…cont’d

2.6 Data analysis

 Descriptive analysis of microbial strains' nitrogen fixation and

phosphate solubilization/mobilization.
 Analysis of variance (ANOVA) on plant growth and yield
parameters.
 LSD multiple comparisons test at 5% level of significance.
 R-programming (version 4.4.1) for the statistical analysis.

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 Methods…cont’d

2.7 Research outcome and beneficiary

 Research outcomes
 Efficient microbial strains for biofertilizer production
 Technical information for large-scale production
 Easily accessible microbial strains

 Beneficiary
 Biofertilizer producers
 Farmers and local community
 Researchers and academicians (agriculture, biotechnology)

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 3. WORKPLAN

Phase I: October-June, 2017 E.C

June
May
Mar
Nov

Apr
Feb
Oct
Sep

Jan
Dec
S/N
Major activities
1 Material procurement 
2 Reconnaissance survey of potential
 
areas of legume crops
3 Nodule, root and soil sample
collection  
4 Lab I: Isolation and screening
laboratory works  
5 Lab II: Molecular characterization of
efficient microbial stains  
6 Lab and pot experimental testing    
7 Field plot preparation and sowing
 

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 Workplan… cont’d

Phase II: September, 2017- June, 2018 E.C

June
May
Mar
Nov

Apr
Feb
Oct
Sep

Jan
Dec
S/N
Major activities
1 Field experimentation of biofertilizer     
inoculants
2 Lab III: Soil and plant nutrient
analysis    
3

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 4. FINANCIAL PLAN

Budget summary (in ETB)

S/N Budget item description Sub-total costs

1 Equipment and consumable 21,300
2 Chemicals and reagents 292,250
3 Labor cost 60,000
4 Technical & professional fee 260,840
5 Laboratory analysis payment 240,000
6 Transport cost 22,700
Grand total 897,090

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Thank you for
attention!
 Notes

Biofertilizer experiments
 Controlled laboratory experiment (test tube experiment) with CRD experimental
layout
 Green house pot experiment CRD
 Field experiment on farm plots. (RCBD)

 Note: The combined inoculation of N fixing, PSB and mycorrhizal fungi could be
more effective than biofertilizer formulations of single microorganisms.

Biofertilizer producer Ethiopia

 National soil testing center (NSTC)
 Menagesha Biotech Industry (MBI)
 Ethiopian Institute of Agricultural Research (EIAR)

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