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Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
1
NATIVE MYCORRHIZAE FROM ETHIOPIA IMPROVE TREE
GROWTH AND SEEDLINGS SURVIVAL CONTRIBUTING TO THE
GREEN LEGACY PROGRAM
†
[MICORRIZAS NATIVAS DE ETIOPÍA MEJORAN EL CRECIMIENTO
DE ÁRBOLES Y LA SUPERVIVENCIA DE LAS PLÁNTULAS
CONTRIBUYENDO AL PROGRAMA LEGADO VERDE]
Yonatan Legesse1, Mulissa Jida2, Mariana Laura Puente3,
Fernanda Covacevich4 and Zerihun Belay1*
1Department of Applied Biology, Adama Science and Technology University, P.
O. Box: 1888, Adama, Ethiopia. Email: zebelay2009@gmail.com
2Emerging Technology Institute, Ras Biru Street, Near TemenJa Yaj Addis
Ababa P. O. Box: - 5954 A.A, Ethiopia. Email: mulaeabageda@gmail.com
3 Instituto Nacional de Tecnología Agropecuaria (INTA), Instituto de
Microbiología y Zoología Agrícola, Nicolás Repetto y de las Cabañas s/n,
Buenos Aires, Zip Code 1618. Argentina. Email: puente.mariana@inta.gob.ar
4Instituto de Investigaciones en Biodiversidad y Biotecnología-Consejo
Nacional de Investigaciones Científicas y Técnicas, Vieytes 3103, Mar del
Plata, Zip Code 7600. Argentina. Email: covacevich.fernanda@inta.gob.ar
*Corresponding author
SUMMARY
Background: A rapid production of tree seedlings in nurseries with a high survival rate after transplanting is
important to respond to the current demand for programs of restoration of arid environments by forestation. The
low level of seedlings’ survival and establishment, caused by low moisture and nutrient content of soils, has been
a bottleneck to reaching the target of the forest national restoration in Ethiopia of last years. It is suggested that,
inoculation with root-associated plant growth promoting microorganisms could help to ameliorate this scenario
and also respond to the Ethiopia's Green Legacy Program. Objective: To assess the potential of inoculation with
arbuscular mycorrhizal fungi (AMF) native to Ethiopia to improve the survival and growth of trees that could be
used in afforestation programs in Ethiopia. Methodology: The study was carried out in three stages: (1) soil
samples associated with roots of selected acacia species (T1-AMF of A. abyssinica, T2-AMF of A. seyal, T3-AMF
of A. tortilis and T4-Control) were collected of highland and lowland areas from Ethiopia, (2) Sorghum (Sorghum
bicolor (L.), provided by the Melkasa Agricultural Research Center-(MARC) served as a trap plant for the AMF
consortium multiplication and (3) plant growth promotion by AMF was assessed throughout inoculations of
seedlings of Delonix regia (Hook.) Raf., Sesbania grandiflora (L.), Cassia fistula L., and Azadirachta indica A.
Juss., trees. Results: All inoculated seedlings showed significantly greater responses in all growth and mycorrhizal
parameters over the non-inoculated trees. Consortium T2-AMF associated to roots of A. seyal from lowlands of
Batu, showed significantly greater responses in all plant growth and mycorrhizal parameters over the AMF
inoculums associated to other tree species evaluated. Significant and positive correlations were found between
mycorrhizae and plant-growth parameters. Implications: Our results suggest that inoculation with native
arbuscular mycorrhizal fungi indigenous from Ethiopia has the potential to significantly enhance survival and
growth rates of tree seedlings. This could thereby advance national reforestation goals and addressing challenges
in seedling establishment in arid environments. Conclusion: The potential for growth promotion and
establishment of tree seedlings evidenced, implies that further efforts should be directed towards the in-mass
production of AMF-based inoculants, particularly associated with A. seyal roots.
Keywords: Acacia trees; mycorrhizal fungi; forestation program; inoculation; seedlings
RESUMEN
Antecedentes: Una producción rápida de plántulas de árboles en viveros con una alta tasa de supervivencia tras
el trasplante es importante para responder a la demanda actual de programas de restauración de ambientes áridos
por forestación. El bajo nivel de supervivencia y establecimiento de las plántulas, causado por el bajo contenido
de humedad y nutrientes de los suelos, ha sido el factor limitante para alcanzar el objetivo de la restauración
forestal nacional en Etiopía de los últimos años. Se sugiere que la inoculación con microorganismos promotores
†
Submitted December 27, 2023 – Accepted August 15, 2024. http://doi.org/10.56369/tsaes.5373
Copyright © the authors. Work licensed under a CC-BY 4.0 License. https://creativecommons.org/licenses/by/4.0/
ISSN: 1870-0462.
Zerihun Bealy: https://orcid.org/0000-0002-5176-3580
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
2
de crecimiento vegetal asociados a las raíces de las plantas, podría contribuir a mejorar esta situación y también
responder al Programa Legado Verde de Etiopía. Objetivo: Evaluar el potencial de la inoculación con hongos
micorrícicos arbusculares (AMF) nativos de Etiopía, para mejorar la supervivencia y el crecimiento de plántulas
que podrían ser utilizadas en programas de forestación en Etiopía. Metodología: El estudio se llevó a cabo en tres
etapas: (1) colecta de muestras de suelo asociadas a raíces de acacia (T1-AMF de A. abyssinica, T2-AMF de A.
seyal, T3-AMF de A. tortilis y T4-Control) de zonas de tierras altas y bajas de Etiopia, (2) multiplicación de
consorcios de AMF nativos en raíces de sorgo (Sorghum bicolor L.), proporcionado por el Centro de Investigación
Agrícola de Melkasa (MARC), como planta trampa, y (3) evaluación de la promoción del crecimiento en plántulas
de Delonix regia (Hook.) Raf, Sesbania grandiflora (L.), Cassia fistula L. y Azadirachta indica A. Juss., por la
inoculación con los consorcios con AMF. Resultados: Todas las plántulas inoculadas mostraron respuestas
significativamente mayores en todos los parámetros de crecimiento y micorrización que las no inoculadas. El
consorcio T2-AMF asociado a raíces de A. seyal autóctono de tierras bajas de Batu, mostró respuestas
significativamente mayores en todos los parámetros de crecimiento y micorrización de las plantas, que los
inóculos asociados a otras especies arbóreas evaluadas. Se encontraron correlaciones significativas y positivas
entre los parámetros de micorrización y crecimiento de las plantas. Implicaciones: Nuestros resultados sugieren
que la inoculación con hongos micorrícicos arbusculares autóctonos de Etiopía tiene el potencial de mejorar
significativamente las tasas de supervivencia y crecimiento de las plántulas de árboles. De este modo se podría
avanzar en los objetivos nacionales de reforestación, y hacer frente a los retos que plantea el establecimiento de
plántulas en entornos áridos. Conclusiones: El potencial para la promoción del crecimiento y el establecimiento
de plántulas de árboles evidenciado, implica que los futuros esfuerzos se deben dirigir hacia la producción en
masa de inoculantes basados en AMF, particularmente asociados a las raíces de A. seyal.
Palabras clave: Acacia; hongos micorrícicos; programa de forestación; inoculación; establecimiento de plántulas.
INTRODUCTION
The Government of Ethiopia has been working to
rehabilitate degraded lands and restore forests
through national tree planting campaigns since this
millennium. These development activities have been
accomplished under the country’s National Green
Development program, to reduce climate change
and environmental degradation. The Ethiopian
government announced a larger goal of planting 20
billion trees during a four-year period (2019-2023).
However, according to reports of Adama Woreda
Agriculture and Natural Resources Office
(AWANRO) during 2020 and 2021, the survival rate
of seedlings was lower than expected and ranged
from 40-50% respectively (AWANRO, 2021). So
that, the original projection is not being achieved. In
order to meet the objective of promoting the
contribution of forestry to agriculture, water and
energy, it is proposed to cover 22 million hectares of
degraded land in Ethiopia with forests by 2030
(Kassa, 2018; Mekonnen, 2018).
The low level of seedlings’ survival and
establishment in Ethiopia’s fields have been a
bottleneck for the achievement of the objectives of
the national restoration target (Eba, 2017; Asmelash
et al., 2019). In fact, it caused loss of a huge amount
of money allocated for this purpose. Poor survival
and establishment of trees after transplanting in
fields were caused by low moisture and nutrient
content of soils (Mahari, 2014; Asmelash et al.,
2019). In addition, the selection of suitable trees for
each particular site, providing the necessary
aftercare and the use of relevant technologies to
improve the moisture and nutrient balance of the
seedlings should have been considered. Since some
soil micro-organisms can contribute to the mineral
nutrition of plants and to the improvement of soil
quality, biological inoculation could be a promising
alternative. One of available alternative could be the
inoculation of tree seedlings with arbuscular
mycorrhizal fungi (AMF) (Asmelash et al., 2016),
which are considered as natural growth regulators of
a majority of terrestrial flora. The AMF establish a
mutualistic symbiotic association with roots of their
host plants and, as results of mycorrhizae formation,
increases in water and nutrient uptake are usually
detected both under nursery and field conditions
(Diagne et al., 2020) that are mediated by increases
in the volume of soil explored by the root system and
external AMF mycelium (dos Santos et al., 2017).
They also confer the plant’s resistance against roots
pathogens and improvement in water relations
(Poveda et al., 2020). In addition to the benefits on
host plants, ecosystem services on the soil by the
formation of mycorrhizae have been detected. For
example, higher stable aggregate content in soils
with higher AMF presence has been detected and
associated with the release of binding glycoproteins
known as glomalins (Smith and Read, 2008). In
addition, the contribution of soil macroaggregate
trapping by the external mycelium of AMFs has
been evidenced (Syamsiyah et al., 2018). Therefore,
the use of inoculation with AMF to improve crop
performance and soil quality is advised (Barrow,
2012; Kuila and Ghosh, 2022).
Tran et al., (2019) examined the growth and
nutritional responses of agriculturally important
plant species, with and without inoculation with
AMF, and found that mycorrhizal colonization of
roots, plant growth, and plant nutrient responses was
increased after inoculation. Ortega et al., (2004)
reported that at afforestation sites devoid of
mycorrhizal propagules, seedlings never develop
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
3
fully and often die. Thus, the selection and
inoculation of mycorrhizal fungi with beneficial
functions can contribute to the establishment and
plantlet survival by enhancing nutrient and water
acquisition and also by increasing tolerance to
transplant stress (dos Santos et al., 2017). In spite of
their potential and benefits, the large-scale use of
AMFs is still limited, mainly due to the lack of
availability of inoculant in high quantities, at low
cost and high quality (IJdo et al., 2011). In addition,
studies on the role of AMF in rehabilitation and
regeneration of degraded lands are very limited or
nil. Therefore, in view of the urgent need based on
low seedling growth and survival, and also in
response to national revegetation programs, a
promising alternative should take into consideration
the inoculation with indigenous AMF. The first step
is then to select the inoculum (AMF consortium)
that could be most efficient for the survival and
establishment of trees considered in the reforestation
plan. Subsequent confirmation of their potential in
the field, will contribute to reduce costs of chemical
fertilizers and to produce seedlings with good vigor
which would translate into high survival and growth
at the field.
Therefore, aimed to answer the goal of the Green
Legacy Program of Ethiopia, the objective of this
study was to evaluate the potential of native AMF
inoculums isolated from highland and lowland
areas, to improve the growth and survival of tree
seedlings.
MATERIALS AND METHODS
Description of the study area
The study was performed in selected lowland areas
of central Rift Valley and highland regions of
Ethiopia. Selection of areas was based on prior
studies of AMF diversity and abundance associated
with acacia trees under different land use systems
(Belay et al., 2013). The Central Rift Valley is a
lowland area of Ethiopia, in which Batu and
Bishoftu have woody grassland where naturally
different acacia tree species are dominant. The high
land area is Sululta, the tertiary of Addis Ababa.
Bishoftu and Batu are located in the East Showa
zone, Oromia Regional State, at an altitude of 1600
to 1960 above sea level. The zone extends between
7o33’N-9o08’N and from 38o24’E- 40o05’E, with a
total area coverage of approximately 13765.7 km2.
The annual rainfall distribution ranges from 600 mm
to 1000 mm with average of 816 mm, with the
temperature range from 10ºC in the uplands to over
30ºC in the depressions of the Rift Valley with a
mean temperature of 20 ºC (CSA, 2020/21). Sululta
is located in the central part of Ethiopia, in the
Oromia Special zone, 23 km from Addis Ababa to
the north. Geographically, the study area extends
from 9o30'00"N to 9o12'15"N latitude and 38o42'0"E
to 38o46'45" E longitude with the altitude of 2589 m
above sea level. The administrative area of the town
is about 4471 ha. Sululta has similar climatological
characteristics to Addis Ababa. Globally, it is a part
of a tropical humid climatic region, which is
characterized by warm temperatures and high
rainfall (the maximum annual rainfall is 1447 mm
with a mean of 1140 mm and a minimum of 834
mm). The soils of the area are derived from
Mesozoic sedimentary and volcanic rocks. The
major soil types of the Sululta area are Chromic
Luvisols (Gelan, 2021).
Soil sampling
Soil samples were collected in February 2021.
Triplicate samples were taken randomly from each
site within a 100 m2 (10m x 10m) quadrant. Soil
associated with the roots of Acacia abyssinica from
Sululta (the high land area) and of A. seyal and A.
tortilis from Batu and Bishoftu (the low land areas)
was collected (Table 1). Within each site, three
replicates of each acacia tree species were randomly
selected, and about 3 kg of soil were taken (0-30 cm
depth). After collection, samples were pooled into a
composite sample (9 kg from each sample location
for each species). As result, 45 kg of soil samples
were collected in sterilized plastic bags and stored at
room temperature until use.
Establishment of trap cultures for AMF
inoculum production
In order to obtain many healthy and infective AMF
propagules for inoculation, a trap culture was
established in the greenhouse at Adama Science and
Technology University (ASTU) for four months
(March to June 2021), following the protocol of
INVAM (http://invam.caf.wvu.edu). Trap cultures
were set up in triplicates for the five collected soil
samples (A. seyal and A. tortilis each from Bishoftu
and Batu and A. abyssinica from Sululta). According
to Belay et al. (2013), the dominant AMF species
associated with these acacia trees based on the
relative abundance and frequency of spores were:
Claroideoglomus claroideum, Claroideoglomus
etunicatum, Claroideoglomus luteum,
Funneliformis geosporus, and Glomus aggregatum.
To set up trap cultures, each collected soil sample
was thoroughly mixed (1:1 v/v) with washed and
autoclaved (121 ºC, 1 hour, twice with intervals of
24 h) sand, and about 3 kg of each mixed substrate
was transferred to 100 cm3 plastic pots that were
irrigated for three days prior to seeding. Seeds
(80/pot) of sorghum [Sorghum bicolor (L.), Melkam
cv.], selected as trap plants for its mycotrophic
capacity and to induce high spore multiplication
(INVAM, http://invam.caf.wvu.edu), were provided
by the Melkasa Agricultural Research Center
(MARC). Seeds were surface sterilized by soaking
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
4
for 15 min in a 0.5 % sodium hypochlorite solution
and washing with sterile water. Seeds were sown at
a 2 cm depth in each plastic pot and covered with
sterilized sand. All seedlings were grown in the
greenhouse under natural light and temperature
conditions during four months. Plants were irrigated
daily as needed, and no fertilizer was applied. To
induce spore production, watering was reduced
during the last week of the trap cultures growth. At
the end of trap cultures growth, aerial part of trap
plants were cut near the base, and roots, substrate
and accompanying microflora were air-dried and
stored in zipped plastic bags at room temperature for
30 days before inoculation (INVAM,
http://invam.caf.wvu.edu).
Assessment of root colonization and spore
density of trap culture
Roots of seedlings of each trap culture were washed
several times in tap water and cut into segments of
about 1–2 cm long. To confirm the quality of each
inoculum, mycorrhizal colonization on the roots of
the trap plants and spore density in the substrate
were quantified. Briefly, about 0.5 g of root
segments were cleared in 10 % (w/v) KOH at 90 oC
for 1 hour in water bath. Roots were further bleached
with alkaline hydrogen peroxide (10 % H2O2) for 3
min at room temperature. Thereafter, the roots were
treated with 2 % HCl (v/v) for 15-20 min at room
temperature and finally stained in 0.05 % w/v trypan
blue in lactoglycerol (1:1:1 lactic acid, glycerol and
water) at 90 °C for one hour in water bath (Brundrett
et al., 1996). Samples were washed thoroughly with
distilled water at the end of every step except HCl
treatment. The samples were left in distaining
solution (lactoglycerol) for more than two days in a
dark room. Finally, roots were mounted on
microscopic slides and covered with 24×24 mm
coverslips. AMF colonization was assessed
according to the method of Mc Gonigle et al. (1990).
A total of 150 intersections were taken for each
subsample to estimate percent AM root colonization
under a compound microscope at a magnification of
100X. The presence of arbuscular colonization (AC)
and vesicular colonization (VC) were calculated by
dividing the count for the ‘arbuscules’ and ‘vesicles’
categories, respectively by the total number of
intersections. Total colonization (TC) was
calculated a proportion of non-negative
intersections. The assessment of AMF spore density
was performed by wet sieving and decanting method
(Gerdemann and Nicolson, 1963), followed by
centrifugation in water and in a 50 % sucrose
solution (Brundrett et al., 1996). Spore density (SD)
was quantified according to INVAM
(http://invam.caf.wvu.edu).
Experimental design and inoculation of AMF on
selected trees
In order to assess the efficiency of each inoculum in
poor fertile soil, a soil with low pH and low
phosphorus levels was collected from surrounding
of Holeta Town (Ethiopia). The physical and
chemical characteristics of the test soil were as
reported by Belay and Assefa (2011), as described:
P (6.44 ppm), N (1.66), organic matter content
(1.549 %), pH (4.75), and EC (0.059 ds/m) . Plastic
pots (capacity 3 kg, diameter of 15 cm, and 20 cm
depth) were filled with 2.7 kg of the unsterilized
poor fertile soil and 0.3 kg of each inoculum (where
appropriate according to the inoculation treatments
that will be described below).
For the growth promotion experiment, healthy
selected seeds of Azodichta indica (Neem), Delonix
regia, Sesbania grandiflora, and Cassia fistula
provided by Adama Woreda Agriculture and
Natural Resources office, were surface disinfected
(0.5 % sodium hypochlorite solution for 15 min,
washing with sterilized water) and allowed to
germinate on a 0.75 % (w/v) water agar for 48 h at
25 °C before planting or sowing. Then, two
germinated seeds were sown at 2 cm depth in each
plastic pot.
For each tree species, the following AMF
inoculation treatments were established in a
completely randomized design replicated three
times: T1-AMF of A. abyssinica from Sululta site,
T2-AMF of A. seyal, T3-AMF of A. tortilis from Batu
site and T4-Control (Non-AMF inoculated, which
received a mixture of the three inoculums in equal
proportion, but sterilized). The AMF inocula for
each experimental treatments (with respect to the
site of origin) were selected based on the AMF spore
density and percentage of the root colonization
obtained during trap culture establishment (Table 1).
The plants were watered 4 days a week and grown
in a greenhouse (with natural light and temperature)
for 4 months.
Evaluation of growth promotion and
mycorrhizal colonization
The measurement of plant growth parameters was
conducted twice at 60 and 120 days intervals after
sowing. One seedling from each pot was uprooted at
the end of 60 days and the remaining seedling from
each pot was uprooted at the end of the growth
season (120 days) for the measurement of growth
parameters. At each harvest, shoot and root height
(SH, RH, respectively) of plants, total leaves
number (NL), root length (RL) fresh shoot mass
(FSM) and fresh root mass (FRM) were recorded.
After drying plant material at 70 0C in an oven for
48 h, dry shoot and root mass (SDM and RDM,
respectively) also were recorded. Roots were
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
5
processed to quantify mycorrhizal colonization as
described above. Similarly, spore density in the
substrate at 120 of plant growth (final harvest) was
quantified as described above.
For each plant species and inoculation treatment, the
mycorrhizal dependency (MD) were calculated as a
percentage as follows (Plenchette, 1983):
,
In which DWM represents dry weight of
mycorrhizal (inoculated) plants and DWNM non-
mycorrhizal (control) plants.
Statistical analysis
The statistical analysis of the data was performed
using the SPSS software package (version 26.0).
AMF spore density and percentage of root
colonization and growth responses (plant shoot
height, root height, number of leaves, dry and fresh
weight of root and shoot) of the test plants data were
subjected to one-way analysis of variance
(ANOVA) using the treatments as factor. Tukey’s
honestly significant difference (HSD) post hoc test
was used for pair-wise multiple mean comparisons
tests. Pearson’s correlation coefficients among
parameters were analyzed. All the tests of statistical
significance were decided at p<0.05.
RESULTS
AMF root colonization and spore density of the
trap culture
In order to obtain more infective and abundant AMF
spores and propagules for inoculum production, the
comparison of the inocula associated to roots of the
same host plant but from different sampling
locations (Batu and Bishoftu), and the establishment
of trap culture under greenhouse conditions for
multiplication, was required. Accordingly, five soil
samples (associated to roots of A. seyal and A.
tortilis each from Bishoftu and Batu and A.
abyssinica from Sululta) were collected, AMF and
accompanying microflora were multiplied and AMF
root colonization and spore abundance were
examined.
The density of AMF spores of the trap cultures
ranged from 18.7 to 32.4 g-1 (Table 1). There were
significant differences in AMF spore density among
the areas of sampling and the acacia species. Highest
AMF spore density was found associated to roots of
A. seyal from Batu and the lowest was recorded at
the highland soil associated to the roots of A.
abyssinica. All seedlings of sorghum in trap culture
were colonized by AMF. The percentage of AMF
root colonization significantly differed between the
tree species in which multiplied soil was associated
(p<0.05) (Table 1). Average of total colonization
ranged between 56.9 % and 94.9 %. The highest
percentage of root colonization of the trap plants
was found in multiplied soil associated with A. seyal
from lowland Batu, and the lowest colonization in
the roots of multiplied soil from A. abyssinica.
Growth response of the trees to AMF inocula
Azadirachta indica A. Juss.
At 60 and 120 days-growth, all plants inoculated
with AMF showed significantly greater responses at
all growth parameters over the non-inoculated
control plants (T4). Inoculation of A. indica (Neem)
with AMF inocula of A. abyssinica (T1) from Sululta
and A. seyal (T2) from Batu showed, in general,
significantly greater responses in growth parameters
in relation to those inoculated with AMF of A.
tortilis (T3) from Batu (Table 2). At 120 days-
growth, plants inoculated with AMF of A. seyal (T2)
showed significantly greater responses at all
parameters over the T1 and T3 inoculated plants.
Plants inoculated with AMF from A. abyssinica (T1)
and AMF from A. tortilis (T3) showed comparable
responses in SH, NL, FRM and DRM parameters at
120 days-growth.
Delonix regia (Hook.) Raf.
At 60 and 120 days-growth, all plants inoculated
with AMF showed significantly greater responses at
all growth parameters over the non-inoculated
control plants (T4). After 60 days of growth, plants
of D. regia inoculated with AMF of A. seyal (T2) and
plants inoculated with AMF of A. abyssinica (T1)
showed significant increases of all parameters over
non-inoculated control plants (Table 3). However,
plants inoculated with AMF from Batu’s A. seyal
(T2) showed greater responses over the seedlings
than inoculated with AMF of A. abyssinica (T1) in
RH, NL and FRM. After 120 days-growth, plants
inoculated with AMF of A. seyal (T2) showed
significantly greater responses over T1 and T3
inoculated plants.
Sesbania grandiflora (L.)
At 60 days-growth, plants inoculated with AMF of
all acacia species A. abyssinica (T1) from Sululta, A.
seyal (T2) and A. tortilis (T3) from Batu, showed
significant greater responses in all growth
parameters over the control (T4), except in DSM
(Table 4). Seedlings inoculated with AMF of A.
seyal (T2) showed significantly greater growth
responses in FSM and DRM than T1 and T3
inoculated plants. Similarly, seedlings inoculated
with AMF of A. seyal (T2) showed significantly
greater growth responses in RH and DRM than both
T1 and T3 respectively.
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
6
At 120 days, the growth parameters showed
variability among the treatments (Table 4).
Accordingly, the results of all growth parameters of
the seedlings inoculated with AMF of A. seyal (T2)
showed significantly greater responses over
seedlings that were the control (T4) and other
inoculated group (T1 and T3). Secondly, seedlings
inoculated with AMF of A. abyssinica (T1) and A.
Table 1. Spore density and percentage of root colonization with arbuscules, vesicles and hyphae of
arbuscular mycorrhizal fungi recorded at the trap cultures.
Sampling area
Source (host plant)
of soil inoculum
SD (g-1)
RC %
AC %
VC %
TC %
Sululta
A. abyssinica
19±0.4e
25.2±0.0e
14.6±0.1d
56.9±0.2e
A. seyal
32±0.2a
45.7±0.8a
25.6±0.4a
94.9±0.4a
A. tortilis
22±0.06d
34.4±0.4c
19.9±0.4c
62.9±1d
A. seyal
28±0.1b
41.7±0.4b
27.3±0.4a
79.1±1.1b
A. tortilis
22±0.1d
28.4±0.4d
22.5±0.4b
61±0.5d
Note- SD- spore density, RC- root colonization, AC- arbuscular colonization, VC – vesicular colonization, and
TC-Total colonization. Different lowercase letters in the same column represent significant differences at 0.05
level; Mean values followed by the same letter are not significantly different at P< 0.05. Mean ± Standard error.
Table 2. Effect of arbuscular mycorrhizal inoculation on growth characteristics of Azadirachta indica.
60 days-growth
Parameters
SH (cm)
RH (cm)
NL
FSM (g)
FRM (g)
DSM (g)
DRM (g)
Treatment
T1
14.1±0.4a
7.6±0.0b
35±1a
1.2±0.1b
2.0±0.0a
0.5±0b
0.9±0a
T2
14.5±0.2a
7.8±0.4a
29±1b
1.3±0.0a
1.7±0.0b
0.7±0a
0.7±0b
T3
11.3±0.1b
6.5±0.1c
22±1c
1.0±0.0c
1.0±0.0c
0.4±0c
0.3±0c
T4
9.5±0.0c
5.4±0.1d
17±1d
0.6±0.0d
0.7±0.0d
0.3±0d
0.1±0d
120 days-growth
T1
29.3±0.2b
21.5±0.2b
65±2b
3.9±0.1b
2.4±0.0b
1.8±0b
1.0+0b
T2
32.3±0.1a
22.8±0.1a
77±2a
4.6±0.9a
4.1±0.0a
2.0±0a
1.7+0a
T3
29.3±0.4b
19.8±0.1c
65±2b
3.0±0.0c
2.4±0.1b
1.1±0c
1.0+0b
T4
24.1±0.2c
16.3±0.1d
55±1c
1.9±0.0d
1.7±0.0c
0.8±0d
0.8+0c
Note: SH-Shoot Height, RH-Root Height, NL-Number of Leaves, FSM-Fresh Mass of Shoot, FRM-Fresh Mass
of Root, DSM-Dry Mass of Shoot, DRM-Dry Mass of Root. T1-A. abyssinica of AMF, T2-A. seyal AMF, T3-A.
tortilis and T4 -Control (Non-Inoculum). At each column, and for each sampling date, same letter of rows indicates
not significant differences in values among inoculation treatments (Tukey’s test, P<0.05). Mean ± Standard error
Table 3. Effect of arbuscular mycorrhizal inoculation on growth characteristics of Delonix regia.
60 days-growth
Parameters
SH (cm)
RH (cm)
NL
FSM (g)
FRM (g)
DSM (g)
DRM (g)
Treatment
T1
14.3±0.1a
13.0±0.2b
181±6b
2.0±0.0a
1.3±0.0b
1.0±0.0a
0.7±0a
T2
15.0±0.2a
13.8±0.1a
204±2a
2.1±0.0a
1.6±0.0a
1.0±0.0ab
0.7±0a
T3
14.0±0.2a
13.2±0.1ab
164±7b
2.0±0.1a
1.2±0.0b
0.9±0.0b
0.5±0a
T4
11.6±0.1b
12.0±0.0c
120±1c
1.4±0.0b
0.9±0.0c
0.7±0.0c
0.3±0b
120 days-growth
T1
19.3±0.1b
18.0±0.2b
242±1b
3.5±0.1b
3.0±0.0b
2.1±0.0b
1.9±0b
T2
23.5±0.2a
20.5±0.2a
296±3a
4.6±0.1a
3.6±0.1a
2.7±0.1a
2.2±0a
T3
17.8±0.1c
16.1±0.1c
231±1c
3.9±0.2b
3.0±0.0b
2.2±0.0b
1.8±0b
T4
15.2±0.1d
12.5±0.2d
187±1d
2.5±0.0c
2.1±0.0c
1.6±0.0c
1.1±0c
Note: SH-Shoot Height, RH-Root Height, NL-Number of Leaves, FSM-Fresh Mass of Shoot, FRM-Fresh Mass
of Root, DSM-Dry Mass of Shoot, DRM-Dry Mass of Root. T1-A. abyssinica of AMF, T2-A. seyal AMF, T3-A.
tortilis and T4 -Control (Non-inoculated). At each column, and for each sampling date, same letter of rows
indicates not significant differences in values among inoculation treatments (Tukey’s test, P<0.05). Mean ±
Standard error
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
7
Table 4. Effect of microbial inoculums on growth characteristics of Sesbania grandiflora.
60 days-growth
Parameters
SH (cm)
RH (cm)
NL
FSM (g)
FRM (g)
DSM (g)
DRM (g)
Treatment
T1
37.3±0.4a
14.2±0.1b
243±3.0a
4.1±0.0b
1.1±0.0b
2.9±0.5ab
0.6±0b
T2
38.5±0.2a
15.8±0.3a
250±10.0a
4.4±0.0a
1.4±0.0a
4.1±0.1a
0.8±0a
T3
37.6±0.4a
15.3±0.1a
226±4.0a
4.0±0.0b
1.3±0.1ab
2.4±0bc
0.6±0b
T4
30.6±0.6b
12.6±0.0c
163±9.0b
3.6±0.0c
0.7±0.0c
1.3±0.1c
0.3±0c
120 days-growth
T1
99.5±0.5b
20.8±0.4b
946±3.0b
27.3±0.3b
6.7±0.1b
6.3±0.1b
2.1±0b
T2
118.0±2.0a
26.3±0.6a
1024±9.2a
32.2±0.6a
8.0±0.0a
7.4±0.2a
2.7±0a
T3
100.4±0.3b
23.0±0.5b
950±1.1b
28.0±0.5b
6.9±0.0b
6.8±0.0ab
2.2±0b
T4
85.0±3.5c
17.9±0.2c
733±8.8c
23.0±0.5c
5.2±0.0c
4.5±0.1c
1.6±0c
Note: SH-Shoot Height, RH-Root Height, NL-Number of Leaves, FSM-Fresh Mass of Shoot, FRM-Fresh Mass
of Root, DSM-Dry Mass of Shoot, DRM-Dry Mass of Root. T1-A. abyssinica of AMF, T2-A. seyal AMF, T3-A.
tortilis and T4 -Control (Non-Inocululated). At each column, and for each sampling date, same letter of rows
indicates not significant differences in values among inoculation treatments (Tukey’s test, P<0.05). Mean ±
Standard error
tortilis (T3) also showed significantly greater growth
response in all parameters than the control (T4)
seedlings (p<0.05).
Cassia fistula L.
At 60 and 120 days-growth, all plants inoculated
with AMF showed significantly greater responses at
all growth parameters over the non-inoculated
control plants (T4) (Table 5). Plants of C. fistula
inoculated with AMF of A. seyal (T2) showed
significantly greater responses in all parameters over
plants inoculated with AMF of T1 and T3. In general,
similar growth increases were obtained for plants
inoculated with T1 and T3.
AMF root colonization and spore density
Arbuscular mycorrhizal fungal structures were
found associated with all root systems of inoculated
plants. However, although non-sterile soil was used,
no root colonization nor AMF spores were detected
on non-inoculated plants or substrate, respectively.
The percentage of AMF root colonization showed
variability among the treatments and the trees host-
plants (Table 6). In general, highest TC was found
in roots of plants inoculated with AMF of A. seyal
(T2). Furthermore, S. grandiflora was the host plants
which roots formed highest mycorrhizae.
Spore density of AMF recorded in the growth
substrate showed differences among host plants. The
mean SD value recorded in plants of S. grandiflora
was significantly greater than the other host plants.
Furthermore, plants of S. grandiflora inoculated
with AMF of A. abyssinica (T1) resulted in highest
SD in their substrate, plants of Delonix regia
inoculated with AMF of A. seyal (T2) resulted in
highest SD in their substrate, and plants of A. indica
did not show differences of SD among inoculation
treatments (Table 6).
Table 5. Effect of arbuscular mycorrhizal inoculation on growth characteristics of Cassia fistula.
60 days-growth
Parameters
SH (cm)
RH (cm)
NL
FSM (g)
FRM (g)
DSM (g)
DRM (g)
Treatment
T1
18.7±0.1b
12.3±0.3b
83±2b
2.4±0.0b
1.5±0.0a
1.1±0.0b
0.6±0ab
T2
20.3±0.2a
13.3±0.1a
89±2a
2.8±0.0a
1.7±0.1a
1.3±0.0a
0.9±0a
T3
17.8±0.1c
12.0±0.2c
78±1c
2.5±0.0ab
1.4±0.0a
1.1±0.0ab
0.5±0b
T4
12.8±0.1d
10.5±0.2d
50±1d
1.3±0.0c
1.0±0.0b
0.6±0.0c
0.2±0c
120 days-growth
T1
34.3±1.2b
14.3±0.3b
120±6a
10.2± 0.1c
2.9±0.0c
4.7±0.1b
2.1±0b
T2
42.3±1.4a
16.9±0.0a
132±1a
13.2± 0.1a
4.5±0.0a
5.4±0.0a
2.3±0a
T3
39.0±0.5ab
16.8±0.5a
124±2a
11.0± 0.0b
4.1±0.0b
4.8±0.1b
2.0±0c
T4
25.6±0.8c
11.3±0.1c
69±1b
8.9± 0.0d
2.1±0.0d
2.0±0.0c
0.9±0d
Note: SH-Shoot Height, RH-Root Height, NL-Number of Leaves, FSM-Fresh Mass of Shoot, FRM-Fresh Mass
of Root, DSM-Dry Mass of Shoot, DRM-Dry Mass of Root. T1-A. abyssinica of AMF, T2- A.seyal AMF, T3-
A.tortilis and T4-Control (Non-Inoculated). At each column, and for each sampling date, same letter of rows
indicates not significant differences in values among inoculation treatments (Tukey’s test, P<0.05). Mean ±
Standard error
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
8
Table 6. Effect of AMF inoculation on total colonization of A. indica, D. regia, S. grandiflora, and C. fistula
at 60 and 120 days-growth and spore density of plant growth substrate.
Host plant species
Inoculation
Treatment
TC (%)
60 days-growth
TC (%)
120 days-growth
SD
(soil g-1)
Sesbania grandiflora
T1
42.1±3.4b
71.0±1.1a
56.1±0.3a
T2
56.1±0.6a
71.0±1.1a
45.4±0.1b
T3
20.2±3.6c
45.2±3.9b
35.2±0.2c
Mean value
39.5±10B
62.4±1.0A
45.6±10A
Azadirachta indica
T1
30.8±0.0a
37.4±1.1b
35.3±0.3a
T2
33.9±1.2a
43.9±0.7a
39.8±0.2a
T3
18.5±1.8b
30.8±1.0c
36.3±1.7a
Mean value
27.7 ±8.0C
37.4±6.0A
37.1±1.0B
Delonix regia
T1
25.7±0.0b
57.6±0.6b
35.3±0.2b
T2
35.1±1.9a
62.8±0.0a
40.2±0.1a
T3
26.9±0.7b
33.7±0.2c
32.6±0.2c
Mean value
29.3±2.0C
54.4±8.0C
36.0±2.0B
Cassia fistula
T1
45.2±1.0a
53.4±0.4c
29.7±0.2b
T2
49.8±2.2a
61.9±0.2a
33.2±0.3a
T3
45.9±3.1a
58.8±0.7b
29.4±0.3b
Mean value
47.0±1.0A
58.0±2.0B
30.8±1.0C
Note: TC-Total Colonization, SD-Spore Density. At each column, and for each sampling date, different lowercase
letters denote, for the same host plant, differences between inoculation treatments. Different uppercase letters
denote differences, in average values, between host plants. (Tukey’s test, P<0.05).
Mycorrhizal dependency
At 60 days of growth, C. fistula plants showed the
highest mycorrhizal dependence (MD), however,
this response to mycorrhization changed and, at 120
days of growth, the highest MD was recorded in S.
grandiflora plants (Table 7). On the other hand, it
should be noted that in the two growth stages
evaluated, the highest MD was recorded in the plants
inoculated with AMF of A. seyal (T2).
Table 7. Mycorrhizal dependency of tree plants
inoculated with arbuscular mycorrhizal fungi
indigenous from Ethiopia.
Inoculation
Treatment
MD %
60 d-
growth
MD %
120 d-
growth
T1
35.99b
54.90b
T2
43.75a
59.99a
T3
27.92c
42.17c
Host plant species
Sesbania
grandiflora
39.50b
62.40a
Azadirachta indica
27.70c
37.40d
Delonix regia
29.30c
54.40c
Cassia fistula
47.00a
58.00b
Note: MD- mycorrhizal dependency, T1-AMF from
A. abyssinica, T2-A. seyal, T3-A. tortilis. At each
column, and for each sampling date, same lowercase
letter of rows indicates not significant differences in
values among inoculation treatments (Tukey’s test,
P<0.05).
Correlation between the parameters
Pearson's correlation revealed that all plant growth
parameters in each seedling showed significant
strong and positive correlations (Table 8). Likewise,
significantly positive associations between AMF
root colonization and SD and the plant growth
parameters were also found, except in A. indica in
which the association between SD and FRM; SD
and DRM were positive but not significant (P>0.05)
(Table 8).
DISCUSSION
The current study was focused on evaluating the
effects of AMF inoculation isolated from soil
associated to roots of acacia trees growing at
highland and lowland areas of Ethiopia, to improve
the growth and survival of the seedlings of the
selected forest trees (A. indica, D. regia, S.
grandiflora and C. fistula) under low fertility and
acidic soil. After multiplication in trap cultures, the
mycorrhizal propagules associated with roots of A.
seyal, native from Batu and Bishoftu, produced the
greatest multiplication, which was revealed both by
the colonisation of the roots of the trap cultures and
by the abundance of spores in the multiplication
substrate. Similarly, plants inoculated with
mycorrhizal propagules associated with roots of A.
seyal showed, in general, the best performance in all
growth parameters evaluated. This result was also
observed in the study reported by Belay et al. (2013)
who concluded that A. seyal is characterized by
relatively high AMF colonization and AMF
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
9
diversity compared to the other acacia species.
Differences in AMF mycorrhization between the
acacia species could be due to factors such as
climatic and edaphic properties, host-specificity
between fungi and plants, age of the host plants,
disturbance, and differential sporulation ability of
AMF taxa (Song et al., 2019; Vieira et al., 2019; Ma
et al., 2023), that are specific to each site where soil
associated to roots samples for AMF multiplication
were collected. Yang et al. (2012) also reported that
the distribution of AMF showed a pattern of high
endemism at large scales. This pattern indicates high
specificity of AMF for hosts at different scales
(plant taxonomic order and functional group) and
high selectivity from host plants for AMF.
All inoculated plants showed higher performance in
their growth parameters than non-inoculated plants.
This performance was associated with the formation
of mycorrhizae as a result of inoculation. The
absence of mycorrhizal and spore-forming
propagules in the non-inoculated control plants
highlights the need to reinforce microbial
populations, particularly mycorrhizal fungi, in studied
Table 8. Pearson correlation coefficients between the growth parameters, total colonization and spore
density of trees inoculated with arbuscular mycorrhizal fungi.
SH (cm)
RH (cm)
NB
FSM (g)
FRM
(g)
DSM
(g)
DRM
(g)
TC
SD
Azadirachta indica (neem)
SH
1
RH
0.935**
1
NL
0.916**
0.874**
1
FSM
0.925**
0.964**
0.911**
1
FRM
0.881**
0.807**
0.922**
0.862**
1
DSM
0.831**
0.936**
0.814**
0.966**
0.794**
1
DRM
0.833**
0.780**
0.910**
0.842**
0.987**
0.790**
1
HC
0.877**
0.870**
0.707*
0.778**
0.582*
0.686*
0.498
1
SD
0.752**
0.896**
0.629*
0.854**
0.509
0.865**
0.469
0.840**
1
Delonix regia
SH
1
.
RH
0.961**
1
NL
0.991**
0.973**
1
FSM
0.876**
0.887**
0.905**
1
FRM
0.944**
0.967**
0.963**
0.956**
1
DSM
0.930**
0.897**
0.941**
0.951**
0.958**
1
DRM
0.894**
0.936**
0.925**
0.871**
0.942**
0.852**
1
TC
0.707*
0.823**
0.772**
0.809**
0.852**
0.730**
0.911**
1
.
SD
0.795**
0.894**
0.850**
0.889**
0.923**
0.830**
0.941**
0.963**
1
Sesbania grandiflora
SH
1
RH
0.928**
1
NL
0.897**
0.881**
1
.
FSM
0.934**
0.947**
0.912**
1
FRM
0.951**
0.940**
0.961**
0.952**
1
DSM
0.917**
0.923**
0.955**
0.942**
0.960**
1
.
DRM
0.953**
0.968**
0.916**
0.962**
0.971**
0.933**
1
TC
0.743**
0.742**
0.941**
0.806**
0.852**
0.882**
0.765**
1
SD
0.889**
0.852**
0.988**
0.912**
0.952**
0.944**
0.890**
0.954**
1
Cassia fistula
SH
1
RH
0.926**
1
NL
0.930**
0.925**
1
FSM
0.908**
0.849**
0.814**
1
FRM
0.943**
0.939**
0.849**
0.925**
1
DSM
0.915**
0.879**
0.960**
0.821**
0.867**
1
DRM
0.899**
0.865**
0.973**
0.818**
0.817**
0.983**
1
.
TC
0.878**
0.863**
0.977**
0.735**
0.781**
0.972**
0.983**
1
SD
0.920**
0.885**
0.978**
0.834**
0.854**
0.994**
0.993**
0.979**
1
Note: SH-Shoot Height, RH-Root height, NL-Number of leaves, FSM-Fresh Shoot Mass, FRM-Fresh Root Mass,
DSM-Dry Shoot Mass, DRM-Dry Root Mass, TC-Total Colonization, and SD–Spore Density. ** Correlation is
significant at the 0.01 level (2-tailed) and * Correlation is significant at the 0.05 level (2-tailed)
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
10
areas. From the outcome of the experiment, it can be
inferred that AMF inoculation in unsterile soil,
although with low-null active AMF populations,
definitely boosted the growth of the seedlings. The
results obtained, although not surprising, are very
promising considering that they could be used for
reforestation plans in Ethiopia. Decades ago, a
greenhouse experiment was conducted in S.
grandiflora by Habte and Aziz (1985), who found
that nutrient uptake and growth of S. grandiflora in
nonsterile soil was significantly stimulated by
inoculation of soil with AMF. Another study by
Banerjee et al. (2013) in India showed clear
evidence of positive growth responses after AMF
inoculation with AMF associated with roots of A.
indica (Neem). They found that the screening of
efficient AMF for A. indica under nursery
conditions showed that significant increases in the
growth parameters and phosphorous uptake was
found for most of the AMF species against control.
However, a recent experiment that carried out in
Ogun State, Nigeria indicated that the interaction of
mycorrhiza and moisture supply had no significant
effect on the seedling growth of C. fistula (Oladipo
et al., 2021). Similarly, Gehring and Connell (2006)
examined the occurrence and levels of AMF
colonization of some common seedling species in a
tropical and a subtropical rain forest site in
Queensland, Australia. They found that seedling
survival was significantly positively associated with
seed biomass but not with AMF colonization.
Sesbania grandiflora was the host plant species that
showed the highest mycotrophic capacity (formation
of highest mycorrhizal colonization) as well as the
highest mycorrhizal dependence at the end of the
period of study. It should be noted that in early
stages (60 days of growth) C. fistula also showed
high mycorrhizal formation ability and also high
mycorrhizal dependence. So that, these two species
could be considered good candidates for
reforestation programmes. Furthermore, the legume
seedlings (S. grandiflora, C. fistula and D. regia)
treated with AMF showed higher percentage of root
colonization by AMF, MD and spore density than A.
indica the non-leguminous tree (Table 6 and 7). This
could be due to the fact that legumes require more P
than non-leguminous plants because the
maintenance of biological N2 fixation process in root
nodules is highly dependent on P (Plenchette, 1983).
In addition, and as mentioned, the microflora
together with the AMF associated with A. seyal was
the inoculum with which the best performance was
achieved in all the growth parameters evaluated in
the trees. Future studies should, at first, confirm the
results obtained in overwintering under field
conditions. Furthermore, the identification (by
classical and/or molecular taxonomy) of both the
AMF and associated microbiota that belong the
consortium associated with A. seyal should be
carried out. The present research also revealed that
all plant growth parameters were positively
correlated with mycorrhizal formation.
Accordingly, significantly and positive associations
between mycorrhizal parameters and the plant
growth parameters such as total dry biomass,
number of leaves, root height, shoot height, fresh
shoot mass, fresh root mass, dry shoot mass, and dry
root mass were obtained (Table 8). This confirms the
effect of mycorrhizae formation enhanced by
inoculation on the growth of the studied trees.
Additionally, very strong positive correlations
(p<0.01) in AMF spore density with all plants’
growth parameters were found. Likewise, except for
D. regia, the AMF root colonization in all plants was
also found to have very high positive relationships
(p<0.01) with the growth parameters in all plant
varieties. This could suggest that the growth benefit
at D. regia was due to the large extracellular hyphae
AMF network, which transports water and nutrients
to roots, increasing their absorption range and
helping plants in the low nutrient soil (Pei et al.,
2020). To confirm this, future studies should
quantify the extent of fungal mycelium on
inoculated plants, as well as its effect on improving
soil structure. AMF root colonization was also found
to have very high positive relationships (p<0.01)
with AMF spore density in the substrate. Other
studies also reported positive correlations between
spore numbers and root colonization (Songachan et
al., 2011; Sivakumar, 2013; Birhane et al., 2020).
However, Salim et al. ( 2020) found that AMF root
colonization had a negative relationship with the
number of spores in the soil during their evaluation
of the status of colonization of the roots of the host
plant in various age classes of revegetation of post-
coal mining land associated with AMF spores
populations and soil fertility in Indonesia. In our
study, this would indicate that AMF colonization
had a favorable impact on the growth parameters of
selected tree seedlings and their mycorrhizal
dependence, as well as on the spore density in the
growth substrate.
Sometimes, AMF propagules in fresh soil (mainly in
degraded soils) consist mainly of segments of AMF
hyphae and colonized roots, rather than viable
spores (Lugo and Cabello, 2002; Troeh and
Loynachan, 2009; Thougnon Islas et al., 2016;
Covacevich et al., 2021) that can be used in
inoculation schemes. In our study, multiplication in
field soil on trap plants yielded AMF spores (and
other propagules) that were able to germinate,
colonize and promote growth of the studied tree host
plants. Future studies should consider the
establishment of large-scale multiplication of the
most promising AMFs to achieve inoculum
quantities needed for field inoculation programs.
Tropical and Subtropical Agroecosystems 27 (2024): Art. No. 131 Legesse et al., 2024
11
CONCLUSIONS
From the above results, it can be concluded that
inoculum with AMF from soil associated to roots of
A. seyal native from lowlands, followed by A.
abyssinica from highland, were found to be the best
sources of inoculum to improve greatly the seedlings
establishment and performance of tree grown in
poor fertile and low pH soil. Therefore, AMFs
associated with these host plant species can be
considered potential candidates for inoculation
programs than can be adopted as regular practices at
different sites of nurseries in Ethiopia in improving
the survival and growth of seedlings in the national
tree seedling planting campaigns. However, future
studies should aim at evaluating (i) the potential of
these inoculants to environmental stress under the
field conditions and, (ii) the competitiveness of
these native inoculums with commercial microbial
inoculants (iii) the genetic identity of AMFs and the
accompanying microbiota in inoculants.
Acknowledgements
The first author is thankful to Adama Science and
Technology University for the M.Sc. scholarship.
The authors are grateful to the Bio and Emerging
Technology Institute for the financial assistance and
Argentina Embassy in Ethiopia for technical
training at Instituto Nacional de Tecnología
Agropecuaria (INTA), Instituto de Investigaciones
en Biodiversidad y Biotecnología (INBIOTEC-
CONICET) that was possible in the framework of
South-South and Triangular Cooperation (FO.AR).
Our gratitude is also extended to Melkassa
Agricultural Research Center for their supply of
selected seed varieties for the research work.
Funding. Bio and Emerging Technology Institute,
Ethiopia.
Conflict of interests. The authors declare that they
have no conflict of interests.
Compliance with ethical standards. The nature of
this work does not require approval by a (bio) ethical
committee.
Data availability. The data that support the findings
of this study are available from the corresponding
author upon the reasonable request.
Author contribution statement (CRediT). Y.
Legesse-investigation, writing original draft, M.L.
Puente-writing-review and editing, F. Covacevich-
writing-review and editing, M. Jida-project
management, writing-review and editing, Z. Belay-
conceptualization, methodology, writing-review
and editing.
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