© The Authors, 2024, Published by the Universidad del Zulia*Corresponding author: manel.djouama@univ-biskra.dz
Keywords:
Rhizobia
Morpho-physiological and biochemical
characterization
Phenotypic and symbiotic characterization of bacteria nodulating Genista saharae in the arid
region of Algeria
Caracterización fenotípica y simbiótica de las bacterias que nodulan Genista saharae en la región
árida de Argelia
Caracterização fenotípica e simbiôntica de bactérias noduladoras de Genista saharae na região árida
da Argélia
Manel Djouama
1,3*
Abdelhamid Foughalia
2
Farida Boulila
3
Adel Chala
4
Rev. Fac. Agron. (LUZ). 2024, 41(4): e244238
ISSN 2477-9407
DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v41.n4.07
Crop production
Associate editor: Dra. Lilia Urdaneta
University of Zulia, Faculty of Agronomy
Bolivarian Republic of Venezuela
1
Department of Nature and Life Sciences, University
Mohamed Khider, BP 145 RP, Biskra 07000, Algeria.
2
Biotechnology Laboratory, Scientic and Technical
Research Center on Arid Regions. (CRSTRA.Biskra),
Algeria.
3
Laboratoire d’Ecologie Microbienne, Faculté des Sciences
de la Nature et de la vie, Université de Bejaia,06000 Bejaia,
Algeria.
4
Laboratory of Applied Mathematics. University of
Mohemed Khider. P O Box 145, Biskra 07000, Algeria.
Received: 15-08-2024
Accepted: 11-10-2024
Published: 04-11-2024
Abstract
Twenty bacterial strains had been isolated from root nodules
of Genista saharae that grew wild in Biskra and El Oued city
(Northeastern Algerian Sahara). This study focused on obtaining
isolates of legume nodule bacteria (LNB) from the plant G. saharae
and evaluated their eectiveness in forming a symbiotic relationship
with the legume species Vigna unguiculata through cross-
inoculation. Additionally, the study aimed to identify the successful
cross-inoculation group of LNB strains based on their phenotypic
characteristics. The growth capacity of isolates under varying
salinity conditions [NaCl] and pH levels was investigated using a
spectrophotometer (96-microplate reader). The API 20NE and API
20E systems were used to identify the biochemical characteristics of
the isolates. In addition, the rhizospheric soil samples from the two
study sites were analyzed using standard analytical techniques of soil.
All isolates established eective symbioses with Vigna unguiculata,
were Gram-negative rods and were fast-growing. The optimal
growth temperature was between 28 °C and 37 °C; some isolates
were thermophiles and specically withstood extreme heat between
45-50 °C. Furthermore, they demonstrated a wide tolerance range
to pH (5–10) with salt tolerance ranging from 100 mM to 500 mM.
Biochemical results revealed that the isolates assimilated various
sources of carbon and nitrogen and displayed numerous enzyme
activities. Physicochemical analysis revealed that all the soils were
decient in nutrients and had an alkaline pH. This study enabled us
to identify the eective stress-tolerant strains, which could be used in
the future to inoculate plants for environmental applications.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2024, 41(4): e244138 October-December. ISSN 2477-9407.
2-8 |
Resumen
Se aislaron veinte cepas bacterianas de los nódulos radiculares de
Genista saharae que crecían de forma silvestre en Biskra y El Oued,
en el noreste del Sahara argelino. Este estudio se centró en obtener
aislados de bacterias nodulares de leguminosas (BNL) de G. saharae
y evaluar su ecacia en formar una simbiosis con Vigna unguiculata
mediante inoculación cruzada. Además, se identicaron las cepas
BNL exitosas según sus características fenotípicas. La capacidad de
crecimiento de los aislados bajo diferentes condiciones de salinidad
[NaCl] y niveles de pH se investigó utilizando un espectrofotómetro
(lector de microplacas de 96 pocillos). Los sistemas API 20NE y API
20E se emplearon para identicar las características bioquímicas de
los aislados. Se analizaron muestras de suelo rizosférico de ambos
sitios utilizando técnicas analíticas estándar. Todos los aislados
establecieron simbiosis efectivas con Vigna unguiculata, eran bacilos
Gram-negativos y de rápido crecimiento. La temperatura óptima de
crecimiento fue entre 28 °C y 37 °C; algunos aislados resistieron
calor extremo (45-50 °C). Además, mostraron amplia tolerancia al
pH (5-10) y a la sal (100 mM a 500 mM). Los resultados bioquímicos
indicaron que los aislados asimilaron diversas fuentes de carbono y
nitrógeno, exhibiendo numerosas actividades enzimáticas. El análisis
sicoquímico reveló suelos decientes en nutrientes y con pH
alcalino. Este estudio identicó cepas tolerantes al estrés, útiles para
futuras aplicaciones ambientales.
Palabras clave: rizobios, caracterización morfosiológica y
bioquímica.
Resumo
Foram isoladas vinte estirpes bacterianas de nódulos radiculares
de Genista saharae que cresciam espontaneamente em Biskra
e El Oued, no nordeste do Saara argelino. Este estudo focou na
obtenção de isolados de bactérias nodulares de leguminosas (BNL)
de G. saharae e avaliou sua ecácia na formação de uma relação
simbiótica com Vigna unguiculata por meio de inoculação cruzada.
Além disso, identicaram-se as estirpes BNL de sucesso com base
em suas características fenotípicas. A capacidade de crescimento
dos isolados sob diferentes condições de salinidade [NaCl] e
níveis de pH foi investigada com um espectrofotômetro (leitor de
microplacas de 96 poços). Os sistemas API 20NE e API 20E foram
usados para identicar as características bioquímicas dos isolados.
Adicionalmente, as amostras de solo rizosférico dos dois locais de
estudo foram analisadas com técnicas analíticas padrão. Todos os
isolados estabeleceram simbioses ecazes com Vigna unguiculata,
eram bastonetes Gram-negativos e de rápido crescimento. A
temperatura ótima de crescimento foi entre 28 °C e 37 °C; alguns
isolados eram termólos e resistiram ao calor extremo (45-50 °C).
Além disso, mostraram ampla tolerância ao pH (5-10) e à salinidade
(100 mM a 500 mM). Os resultados bioquímicos revelaram que
os isolados assimilaram diversas fontes de carbono e nitrogênio e
exibiram várias atividades enzimáticas. A análise físico-química
mostrou que todos os solos eram pobres em nutrientes e tinham pH
alcalino. Este estudo identicou estirpes tolerantes ao estresse, úteis
para futuras aplicações ambientais.
Palavras-chave: rizóbios, caracterização morfosiológica e
bioquímica.
Introduction
The Genisteae is one of the largest tribes within the legume
family (Fabaceae), comprising 618 species across 25 genera (Cardoso
et al., 2013). Genista saharae Coss & Dur is a spontaneous shrub
legume thriving in the Sahara. However, as a legume, it is able to
x atmospheric nitrogen (N
2
) via symbiotic association with bacteria
termed rhizobia in its root nodules, contributing to soil fertility
(Mahdhi et al., 2007). The use of these bacteria as bio-inoculants
increases the availability of nutrient elements in soil, helps to
minimize the chemical fertilizer application, reduces environmental
pollution, and promotes sustainable agriculture. Several studies aim
to identify new symbiotic strains resistant to extreme environmental
conditions, such as the research conducted by Zakhia et al. (2004)
nds that two strains isolated from Genista microcephala Coss. &
Durieu, grown in an infra-arid region of Tunisia, are identied as
Rhizobium. Mahdhi et al. (2007) reveal that novel isolates nodulating
G. saharae originating from the infra-arid region of Tunisia are linked
to various species of rhizobia belonging to the genera Ensifer (75 %)
and Rhizobium (10 %). Chaïch et al. (2017) show that the majority
of isolates nodulating
G. saharae, a plant thriving in the hyper-arid
zone of the northern Algerian Sahara, are attributed to the genus
Ensifer meliloti (formerly known as Sinorhizobium). Furthermore,
they identify Neorhizobium, comprising three distinct species: N.
alkalisoli, N. galegae, and N. huautlense along with Mesorhizobium,
represented by the species M. camelthorni. In contrast to fast-
growing strains, the genus Bradyrhizobium (slow-growing strains)
appears to be a predominant group that nodulates the majority of
Genisteae species in Northeastern Algeria (Boulila et al., 2009; Ahnia
et al., 2018; Boudehouche et al., 2020). However, previous studies
(González‐Andrés et al., 1998) nd that other Genista species such as
G. tinctoria growing in Poland (16 strains), Ukraine (17 strains), and
England (10 strains), as well as G. monspessulana and G. linifolia in
Spain, are nodulated by only Bradyrhizobium spp. However, there
is no information available about the microsymbionts associated
with the legume G. saharae growing wild in two distinct geographic
locations in Biskra and El Oued city (Northeastern Algerian Sahara).
The current study, therefore, aims to: (i) investigate the diversity
of symbiotic bacteria associated with G. saharae by phenotypic
methods; including morphological, physiological, and biochemical
characterization, (ii) evaluate their symbiotic eectiveness in order to
select the ecient stress-tolerant strains.
Materials and methods
Study sites
In this study, soil samples, root nodules were collected from G.
saharae plants growing at two dierent sites. The rst site is situated
in the Bouchagroune region (34.80°N / 5.73°E) (Biskra city). The
second site, located at Oued El alanda (Erg Chegamet) (33° 14’ N
/ 6° 14’ E) (El Oued city). El Oued and Biskra are located in the
northeastern part of the Algerian Sahara.
Physico-chemical analysis of rhizospheric soils
Soil sampling
Soil samples were collected from the rhizospheres of G. saharae
and were analyzed for organic carbon (OC) content, total nitrogen
(N), available P, pH, texture, electric conductivity (EC) according to
(Aubert, 1978; Mathieu et al., 2003).
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Djouama et al. Rev. Fac. Agron. (LUZ). 2024 41(4): e244138
3-8 |
Nodule collection and Isolation of bacteria
Nodules were collected from G. saharae roots at depths ranging
from 30 cm to 1 meter. They were carefully detached and stored in
tubes containing silica gel (Somasegaran and Hoben, 1985). The
bacterial isolation from nodules was conducted in accordance with
the protocol followed by Vincent (1970), Somasegaran and Hoben
(1985). Nodules were rehydrated overnight at 4 °C by immersion
in distilled water and then surface-sterilized for 30 seconds in 96
% ethanol and for 5 minutes in a 3.5 % (v/v) sodium hypochlorite
solution. They were then washed ve to ten times using sterile
distilled water. Each nodule was crushed in an Eppendorf tube, and
the resultant suspension was streaked on Yeast Extract Mannitol
Agar (YMA) media plates. The plates were then incubated for 24
to 48 hours at 28 °C. Individual colonies were checked for purity
by repeated streaking on fresh YMA medium and by microscopic
examination of cellular morphology and Gram-stain reaction.
Puried strains were conserved at 4 °C on YMA slopes containing
0.3 % CaCO₃ for short-term use and were cryogenically preserved at
-20 °C on Yeast Extract Mannitol Broth (YMB) with 20 % glycerol
(v/v) for long-term storage.
Plant Nodulation and symbiotic eectiveness test
The isolates were tested for cross-nodulation on Vigna unguiculata
(L.) Walp plants, which is characterized by its high promiscuity
(Pongslip, 2012) and rapid growth. To perform this test, we utilized
the Leonardo’s jar technique (Vincent, 1970). Plastic water bottles
were cut in half, cleaned with detergent, disinfected with ethanol,
and connected with sterilized medical compresses. The bottom part
was lled with a nutrient solution (CaCl₂ (0.10 g.L
-1
), MgSO₄·7H₂O
(0.12 g.L
-1
), KH₂PO₄ (0.10 g.L
-1
), Na₂HPO₄·2H₂O (0.15 g.L
-1
),
Ferric citrate (0.005 g.L
-1
), Trace elements (1 mL). Trace elements:
(H₃BO₄ (2.86 g.L
-1
), MnSO₄·4H₂O (2.03 g.L
-1
), ZnSO₄·7H₂O (0.22
g.L
-1
), CuSO₄·5H₂O (0.08 g.L
-1
), Na₂MoO₄·2H₂O (0.14 g.L
-1
)). The
pH was adjusted to 6.8, and sterilization was performed at 120 °C
for 20 minutes, and the upper part was lled with sterilized sand.
The medical compress cord connecting the two parts allowed for the
moistening of the sand with the nutrient solution. The seed surface
was sterilized in 96 % ethanol for 5 to 10 seconds, left for 2 hours in
sterile distilled water (SDW) and germinated on sterile cotton soaked
in sterile distilled water, in darkness at 25 °C. The seeds underwent
a germination period of 24 hours at 23 °C. The germinated seeds
were then planted at a density of three seedlings per plastic pot,
each containing 300 g of sterilized sand (120 °C for 20 minutes) and
inoculated with 2 mL of an early stationary-phase bacterial culture
cultivated at 28 °C in YMB broth. The plants were grown in a growth
chamber with a 16/8 h light/dark photoperiod at 25 °C/18 °C day/
night. Three replicates were performed per isolate with uninoculated
negative controls (N-free). Six weeks post-inoculation, the plantlets
were harvested and examined for the presence of root nodules.
Phenotypic characterization
One hundred eighty (180) µL of YMB medium containing the
corresponding NaCl concentrations in millimolar (mM) (100, 200,
300, 400, 500, and 1.7 mM which served as control) and 180 µL
of YMB containing pH variations (pH 4, pH 5, pH 6, pH 7, pH 8,
pH9, pH 10
and pH 6.8 which served as control) were distributed
into the wells of a microplate. [NaCl] concentration 1.7 mM (0,1
g.L
-1
) and pH 6,8 were involved in the formulation of the medium
YMB broth medium. Then, 20 µL of inoculum (1.10
9
CFU.mL
-1
)
for each bacterial isolate were added. Each test was conducted with
three repetitions, and wells containing uninoculated YMB served
as negative controls. The microplates were incubated at 28 °C with
orbital shaking (150 rpm) for 48 to 72 hours. The growth of the
bacterial cultures was monitored by measuring the optical density
(OD) at 600 nm using a microplate spectrophotometer (96-microplate
reader Thermoscientic Multiskan Sky) (Lindström and Lehtomäki,
1988). To assess the maximum and optimal growth temperatures, the
isolates were inoculated onto YMA medium, with three repetitions
for the test, and incubated at various temperatures: 26 °C, 32 °C, 37
°C, 44 °C, 50 °C and 28 °C which served as control chosen by Zakhia
et al. (2004). Readings were taken after 24 to 48 hours of incubation.
To identify the biochemical characteristics of the isolates, API 20NE
and API 20E strips were used. The strips were inoculated following
the instructions provided by BioMérieux (BIOMERIEUX REF 20050
Api 20NE) and (BIOMERIEUX REF 20100/20160 Api 20E).
Statistical analysis
The inuence of [NaCl] concentration and pH levels on the growth
of isolates was investigated using a one-way Analysis of Variance
(ANOVA) in SPSS, version 2020, followed by the Tukey post hoc test.
The phenotypic character results were coded numerically in a binary
table where “1” represented a positive result and “0” a negative result,
using PAST software (version 4.03, 2020, folk.uio.no/ohammer/past).
Hierarchical clusters were generated in the UPGMA algorithm using
1000 bootstrapped Jaccard similarity (Sj).
Results and discussion
Physicochemical characteristics of rhizosphere soils
The rhizosphere soil varied in its physicochemical properties.
Bouchagroune soil had a sandy texture with a high sand content 78.79
% while the clay and silt content were 0 %, and it was characterized
by alkaline pH (8.20). Oued El Alanda soil also had a sandy loam
texture. The clay and silt content were 0 % and 19.06 %, respectively
and it was characterized by alkaline pH (8.03). The texture of the
two dierent sites indicated low water retention. The electrical
conductivity (EC) values of Bouchagroune and Oued El Alanda
sites were 0.2 ms.cm
-1
and 0.3 ms.cm
-1
, respectively. The soils were
classied as non-saline according to the agronomic scale (Scianna et
al., 2007). The nitrogen and available phosphorus levels were: Site
01; (N) 0.056 %, (P) 51.20 ppm and Site 02; (N) 0 %, (P) 52.08 ppm.
The organic matter percentages from the two studied sites were both
less than 1 %. So they characterized by low content of organic matter.
Chemical analysis revealed that all the soils were poor in nutrients,
specically available (N) and (P). Plant Pi levels played a crucial
role in shaping the plant-associated microbiota and regulating the
endosymbiosis with soil microbes (Míguez-Montero et al., 2020).
When legumes encountered low Pi levels, they exhibited a decrease
in nodule formation and a reduction in the nitrogen-xing capacity
within the developed nodules (Ma and Chen, 2021).
Rhizobial isolation and symbiotic eectiveness
A collection of twenty (20) legume nodule bacteria (LNB) was
selected from the root nodules of G. saharae (table 1). All isolates
grew rapidly on YMA medium. Colonies appeared mucoid, white,
and creamy in color. They were Gram-negative rods. All strains
were able to induce nodulation on the roots of Vigna unguiculata.
In contrast, the non-inoculated control plants did not produce any
nodules, conrming the infectivity of the tested strains. All strains
induced the formation of ineective white nodules, meaning they did
not x nitrogen (Ahnia et al., 2018), as well as eective red nodules,
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2024, 41(4): e244138 October-December. ISSN 2477-9407.
4-8 |
Phenotypic
characteristics
Strains isolated from Genista saharae
Bouchagroune site Oued El alanda site
Bg57 Bg51 Bg40 Bg53 Bg05 Bg17 Bg28 Bg39 Bg20 Bg50 Bg79 Bg34 Bg80 Bg38 Bg42 Bg60 Os02 Os05 Os07 Os10
Nodule
number
06 11 05 24 23 08 14 11 16 12 17 13 06 04 07 08 18 10 02 02
Temperature
26 °C
+ + + + + + + + + + + + + + + + + + + +
28 °C ©
+ + + + + + + + + + + + + + + + + + + +
32 °C
+ + + + + + + + + + + + + - + + + + + +
37 °C
+ + - + + + + + + + + + + + + + + + + +
44 °C
- - - - + - - NR + - + + - - - - - - + +
50 °C
- - - - + - - - + - + + - - - - - - + +
pH
4
+ + + + + + + + + + + + + + + + + + + +
5
+ + + + + + + + + + + + + + + + + + + +
6.8 ©
+ + + + + + + + + + + + + + + + + + + +
8
+ + + + + + + + + + + + + + + + + + + +
9
+ + + + + + + + + + + + + + + + + + + +
10
+ + + + + + + + + + + + + + + + + + + +
NaCl mM
1.7 ©
+ + + + + + + + + + + + + + + + + + + +
100
+ + + + + + + + + + + + + + + + + + + +
200
+ + + + + + + + + + + + + + + + + + + +
300
+ + + + + + + + + + + + + + + + + + + +
400
+ + + + + + + + + + + + + + + + + + + +
500
+ + + + + + + + + + + + + + + + + + + +
leghemoglobin accumulation, a hemoprotein exclusive to nitrogen-
xing nodules (Downie, 2005). The development of symbiotic nodules
on legume roots was controlled by a host genetic program (Tsyganov
and Tsyganova, 2020). Several hosts, such as V. unguiculata (cowpea),
were known to be highly promiscuous (Pongslip, 2012). The absence
of nodules depended on several extrinsic and intrinsic factors.
Phenotypic characterization and numerical taxonomy
Phenotypically, all isolates were able to grow at pH levels
between 5 and 10 (gure 1). However, pH 4 had a negative impact
on bacterial growth (Tukey HSD, P < 0.05), as seen in gures 1
and 1.2. All isolates were able to grow at pH 6.8 with signicant
dierences observed between isolates (Tukey HSD, P < 0.05), where
Bg38 showed the best growth at pH 6.8, while OS07 exhibited the
lowest growth at this pH, as seen in gure 1.1. The optimum pH for
growth was between 6.8 and 8 (gure 1). Most of the tested isolates
tolerated NaCl concentrations from 100 mM to 500 mM (Tukey HSD,
P<0.05), as shown in table 1. According to the Tukey HSD test, the
best bacterial growth was recorded at 100 mM and 200 mM and
the lowest growth was recorded at 500 mM (gure 2). The Tukey
model showed that the increase in NaCl concentration from 100 mM
to 500 mM resulted in a signicant decrease (P<0.05) in the growth
rate of all tested isolates (gure 2). All the selected isolates were
able to grow at 1.7 mM (Tukey HSD, P < 0.05). Bg38 showed the
best growth at 1.7 mM, while OS07 exhibited the lowest growth at
this concentration (gure 2.1). Concerning the isolates’ response to
dierent temperatures. The results obtained showed that the majority
of strains from two dierent sites were able to grow at 26 °C, 28 °C, 32°C
and 37 °C. Approximately 25 % (two strains from Oued El Alanda
site and four strains from Bouchagroune site) were able to withstand
temperatures ranging from 44 °C to 50 °C (table 1). Mahdhi et al.
(2007) demonstrated that the majority of the isolates of G. saharae
from Tunisian soil were capable of growth within a pH range of
6 to 12. However, none of the isolates were able to grow at pH 4,
while Dekak et al. (2018) demonstrated that the isolates of Genista
microcephala and Argyrolobium uniorum (Genisteae) were able to
grow in acidic and alkaline pH values ranging from pH 3.5 to pH
10. Extreme pH levels aected nodulation by reducing infection by
rhizobia. Highly acidic soils and highly alkaline soils aected the
survival and growth of both partners, thus reducing nitrogen xation
(Bordeleau and Prévost, 1994). Succinoglycan might have played a
role in adaptation to low pH (Hawkins et al., 2017) and contributed to
survival in nodules (Maillet et al., 2020). Most of the isolated strains
of Genista saharae in the Algerian Sahara tolerated up to 4 % NaCl
and grew at 45 °C (Chaïch et al., 2017).
Mahdhi et al. (2007) conrmed that all of the isolates were resistant
to high temperature and most of them continued to grow at 40 °C. In
addition, the majority of the isolates were able to grow in high NaCl
concentrations (3 %). This may have been a specic adaptation to the high
soil temperatures and salinity in arid regions. These results conrmed
those obtained by Mahdhi et al. (2007) and Chaïch et al. (2017).
Table 1. Phenotypic characteristics of bacterial strains isolates the root nodules of Genista saharae.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Djouama et al. Rev. Fac. Agron. (LUZ). 2024 41(4): e244138
5-8 |
Figure 1.1. pH eect on the growth of Genista saharae
microsymbiont isolates.
Figure 1.2. pH eect on the growth of Genista saharae
microsymbiont isolates.
Figure 1. pH eect on the growth of Genista saharae microsymbiont
isolates (Tukey HSD, P < 0.05).
Figure 2. [NaCl] eect on the growth of Genista saharae
microsymbiont isolates (Tukey HSD, P < 0.05).
Figure 2.1. NaCl eect on the growth of Genista saharae
microsymbiont isolates.
Figure 2.2. NaCl eect on the growth of Genista saharae
microsymbiont isolates.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2024, 41(4): e244138 October-December. ISSN 2477-9407.
6-8 |
Table 2. Biochemical characteristics of bacterial strains isolates the root nodules of Genista saharae.
Biochemical
characteristics
Strains isolated from Genista saharae
Bouchagroune site Oued El alanda site
Bg57 Bg51 Bg40 Bg53 Bg05 Bg17 Bg28 Bg39 Bg20 Bg50 Bg79 Bg34 Bg80 Bg38 Bg42 Bg60 Os02 Os05 Os07 Os10
API Test
NO3
+ + - + + + + + + - + - - + - - - + - +
TRP
- - - - - - - - - - - - - - - - - - - -
TDA
- - - - - - - - - - - - - - - - - - - -
IND
- - - - - - - - - - - - - - - - - - - -
ADH
+ + - - - + + + - - - - - + - - - + -
+
LDC
- - - - - - - - - - - - - - - - - - - -
ODC
+ + - - - + + + - - + - - + - - - - - -
URE
+ - - - - + + + - - - - - - - - - + - +
ESC
+ + + + + + + + + + + + + + + + + + + +
GEL
+ + + + + - + - - + - + + - + + + + + -
H2S
- - - - - - - - - - - - - - - - - - - -
PNPG
+ + + + + +
+ + + + + + + - + + + + + +
ONPG
+ + + + + + + + + + + + + - + + + + + +
VP
- - - - - - + + - - - - - - - - - - - -
GLU
f
+ + - - - + + + + - + - - + - - - + - +
GLU
a
+ + + + + + + + + + + + + + + + + + + +
ARA
+ + + + + + + + + + + + + + + + + + + +
MNE
+ + + - + + + + + + + +
+ + + + + + + +
MAN
+ + + + + + + + + + + + + + + + + + + +
NAG
+ + + + + + + + + + + + + - + + + + + +
MAL
+ + - + - + + + + + + - + - - - + + - +
GNT
+ + + + + + + + + + + + + + + + + + + +
CAP
- - - - - - - - - - - - - + - - - - - -
ADI
- - - + - - - - - - - - - - - - + - - -
MLT
+ + + - + + + + + + + + + + + + + + + +
CIT
+ + + + + + + + + + + + + + + + + + + +
PAC
+ + - - - + + + + - + - - - - - - + - +
INO
- - - - - - - - + - - - - - - - - - - -
SOR
+ + - - - + + + - - + - - + - - - - - -
RHA
+ + - - - + + + - - + - - + - - - - - -
SAC
+ + - - - + +
+ - - + - - + - - - - - -
MEL
+ + - - - + + + - - + - - + - - - - - -
AMY
+ + - - - + + + - - + - - + - + - - - -
(+): positive, (-): negative, NR: growth but not resistant, ©: control
ONPG (β-galactosidase), ADH (Arginine Dihydrolase), LDC (Lysine Decarboxylase), ODC (Ornithine Decarboxylase), CIT (Citrate utilization), H2S (Hydrogen sulde
production), URE (Urease), TDA (Tryptophan Deaminase), IND (Indole production), VP (Voges-Proskauer - Acetoin production), GEL (Gelatinase), GLU (Glucose
fermentation/oxidation), MAN (Mannitol fermentation/oxidation), INO (Inositol fermentation/oxidation), SOR (Sorbitol fermentation/oxidation), RHA (Rhamnose
fermentation/oxidation), SAC (Sucrose fermentation/oxidation), MEL (Melibiose fermentation/oxidation), AMY (Amygdalin fermentation/oxidation), ARA (Arabinose
fermentation/oxidation).NO3 (Nitrate reductase), TRP (Indol production), GLU
a
(Glucose assimilation), GLU
f
(Glucose fermentation), ARA (Arabinose assimilation),
MNE (Mannose assimilation), MAN (Mannitol assimilation), NAG (N-acetyl-glucosamine assimilation), MAL (Maltose assimilation), GNT (Gluconate assimilation),
CAP (Caprique acid assimilation), ADI (Adipate assimilation), MLT (Malate assimilation), PAC (Phenylacetic acid assimilation), PNPG ( (β-galactosidase para-
Nitrophenyl-β-D-galactopyranoside), ESC (Esculin hydrolysis).
The ability to survive in such extreme environments was conferred
by adaptation mechanisms that enabled them to withstand salt stress
(Miller and Wood, 1996). Some studies reported that dierent
species of Rhizobium presented adaptation mechanisms related
to the production of Heat Shock Proteins (HSPs) at temperatures
beyond their normal growth range, as noted by Michiels et al. (1994).
This conrmed the resistance of some strains in our study to high
temperatures, ranging from 44 °C to 50 °C. The isolates demonstrated
variability in assimilation of carbon and utilization of nitrogen
sources, as well as enzymatic activity. Numerical taxonomic analysis
based on the 51 variable features (tables 1 and 2) revealed two distinct
classes (I and II) at a Jaccard similarity coecient level of 0.84, as
shown in (gure 3), because bacterial isolates were obtained from
two dierent regions, Biskra and El Oued. The two dierent classes
generated ve dierent clusters. The rst cluster included the strains
(Bg28, Bg 57, Bg 51, Bg 17, Bg 39, and Bg 79) from Genista saharea
(Bouchagroune). For example, the subgroup (Bg17 and Bg39)
showed 100 % similarity, used all carbon sources tested except for
inositol, adipic acid, and capric acid. They assimilated and fermented
glucose as a source of energy. They used ornithine and arginine as
nitrogen sources but did not use tryptophan and lysine because they
did not have tryptophan desaminase and lysine decarboxylase. They
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Djouama et al. Rev. Fac. Agron. (LUZ). 2024 41(4): e244138
7-8 |
had the nitrate reductase enzyme, which reduced nitrate to nitrite.
The strains were considered as aero-anaerobic lithotrophs. They
produced esculin, urease, β-galactosidase, and did not have gelatinase
and sulfate reductase, so they could not produce hydrogen sulde,
because they were aerobic bacteria. Both strains could not withstand
44 °C. Cluster II included Bg38. The most particular characteristic of
strain Bg38 was that it could not grow at 32 °C and its optimal growth
occurred at a salinity of 1.7 mM (Tukey HSD, P<0.05) (gure 2.1).
It did not produce indole, lysine decarboxylase, urease, gelatinase,
and β-galactosidase. It could not use N-acetyl-glucosamine, maltose,
adipic acid, phenylacetic acid, and inositol as sources of carbon.
The third cluster regrouped (Bg60, Bg42, Bg40, Bg80, Bg50, Os02,
Os07, Bg34, Bg05). The strain of Os02 from G. saharea (Oued El
Alanda) formed a subgroup with Bg50 and Bg80. It diered from
them in its inability to use adipic acid as a source of energy. Cluster
IV included one isolate, Bg53, from G. saharea (Bouchagroune).
It was characterized by its inability to grow beyond 40 °C, non-
production of indole, ornithine decarboxylase, lysine decarboxylase,
and urease. It produced nitrate reductase, gelatinase, esculin, and
β-galactosidase. It assimilated glucose, arabinose, mannitol, N-acetyl-
glucosamine, maltose, potassium gluconate, adipic acid, and citrate.
Cluster V consisted of three strains (Os10, Bg20, Os05): these
strains used ornithine as a nitrogen source and could not use sorbitol,
rhamnose, sucrose, melibiose, and amygdalin as sources of energy.
They could withstand up to 50 °C, except for Os05, for which the
maximum resistance was 40 °C. An important biochemical diversity
was observed. The isolates demonstrated variability in enzymatic
activity; some isolates were chemo-heterotrophs, capable of
fermentation and exhibit facultative aero-anaerobic respiration. This
extraordinary metabolic exibility allowed the bacteria nodulating
legumes to possess great adaptive capacities in response to extreme
environmental conditions.
Conclusion
In general, phenotypic studies show a large physiological and
biochemical diversity of selected isolates, exhibiting the basic
characteristics of rhizobia. In addition, the isolates show variable
tolerance to dierent stress factors (temperature, pH, salinity).
The present study enables us to identify the most eective strains
nodulating Genista saharae growing in arid soils of the Northeastern
Algerian Sahara, which we can use in the future to inoculate plants
for environmental applications such as the restoration of degraded
and poor soils. However, the work needs to be completed by studying
genotypic biodiversity to identify new eective genospecies.
Literature cited
Ahnia, H., Bourebaba, Y., Durán, D., Boulila, F., Palacios, J. M., Rey, L.,
Ruiz-Argüeso, T., Boulila, A., & Imperial, J. (2018). Bradyrhizobium
algeriense sp. nov., a novel species isolated from eective nodules
of Retama sphaerocarpa from Northeastern Algeria. Systematic and
Applied Microbiology, 41(4), 333-339. https://doi.org/10.1016/j.
syapm.2018.03.004
Aubert, G. (1978). Méthodes d’analyses des sols. Centre national de documentation
pédagogique,Centre régional de Documentation Pédagogique, CRDP
Marseille, 191 p. https://search.worldcat.org/fr/title/methode-danalyses-
des-sols-documents-de-travail/oclc/461689985
Bordeleau, L., & Prévost, D. (1994). Nodulation and nitrogen xation in extreme
environments. Plant and Soil, 161, 115-125. https://doi.org/10.1007/
BF02183092
BioMérieux. (S.d.). API 20NE. Systéme d’identication des bacilles a Gram
négatif
non Enterobactérie e non fastidieux. https://microbiologiemedicale.fr/wp-
content/uploads/2019/02/API-20-NE.pdf
BioMérieux. (S.d.). API 20E. Systéme d’identication des Enterobacteriaceae
et autres bacilles à Gram
négatif non fastidieux. https://
microbiologiemedicale.fr/wp-content/uploads/2019/02/Fiche-technique-
API-20E.pdf
Boudehouche, W., Parker, M. A., & Boulila, F. (2020). Relationships of
Bradyrhizobium strains nodulating three Algerian Genista species.
Systematic and Applied Microbiology, 43(3), 126074. https://doi.
org/10.1016/j.syapm.2020.126074
Boulila, F., Depret, G., Boulila, A., Belhadi, Dj., Benallaoua, S., & Laguerre, G.
(2009). Retama species growing in dierent ecological-climatic areas
of northeastern Algeria have a narrow range of rhizobia that form a
novel phylogenetic clade within the Bradyrhizobium
genus. Systematic
and Applied Microbiology
, 32(4), 245-255. https://doi.org/10.1016/j.
syapm.2009.01.005
Cardoso, D., Pennington, R. T., de Queiroz, L. P., Boatwright, J. S., Van Wyk,
B.-E., Wojciechowski, M., & Lavin, M. (2013). Reconstructing the
deep-branching relationships of the papilionoid legumes. South African
Journal of Botany, 89, 58-75. https://doi.org/10.1016/j.sajb.2013.05.001
Chaïch, K., Bekki, A., Bouras, N., Holtz, M. D., Soussou, S., Mauré, L.,
Brunel, B., de Lajudie, P., & Cleyet-Marel, J. C. (2017). Rhizobial
diversity associated with the spontaneous legume Genista saharae in
the northeastern Algerian Sahara. Symbiosis, 71, 111-120. https://doi.
org/10.1007/s13199-016-0414-y
Dekak, A., Chabi, R., Menasria, T., & Benhizia, Y. (2018). Phenotypic
characterization of rhizobia nodulating legumes Genista microcephala
and Argyrolobium uniorum growing under arid conditions. Journal of
Advanced Research, 14, 35-42. https://doi.org/10.1016/j.jare.2018.06.001
Downie, J. A. (2005). Legume haemoglobins: symbiotic nitrogen xation needs
bloody nodules. Current Biology, 15(6), R196-R198. DOI: 10.1016/j.
cub.2005.03.006
González‐Andrés, F., & Ortiz, J. M. (1998). Biodiversity of rhizobia nodulating
Genista monspessulana and Genista linifolia in Spain. New Zealand
Journal of Agricultural Research, 41(4), 585-594. https://doi.org/10.1080
/00288233.1998.9513342
Hawkins, J. P., Geddes, B. A., & Oresnik, I. J. (2017). Succinoglycan production
contributes to acidic pH tolerance in Sinorhizobium meliloti Rm1021.
Molecular Plant-Microbe Interactions, 30(12), 1009-1019. https://doi.
org/10.1094/MPMI-07-17-0176-R
Lindström, K., & Lehtomäki, S. (1988). Metabolic properties, maximum growth
temperature and phage sensitivity of Rhizobium sp. (Galega) compared
with other fast-growing rhizobia. FEMS Microbiology Letters, 50(2-3),
277-287. https://doi.org/10.1111/j.1574-6968.1988.tb02951.x
Mahdhi, M., Nzoué, A., Gueye, F., Merabet, C., De Lajudie, P., & Mars, M. (2007).
Phenotypic and genotypic diversity of Genista saharae microsymbionts
from the infra‐arid region of Tunisia. Letters in Applied Microbiology,
45(6), 604-609. https://doi.org/10.1111/j.1472-765X.2007.02233.x
Ma, Y., & Chen, R. (2021). Nitrogen and phosphorus signaling and transport
during legume-rhizobium symbiosis. Frontiers in Plant Science, 12,
683601. https://doi.org/10.3389/fpls.2021.683601
Maillet, F., Fournier, J., Mendis, H. C., Tadege, M., Wen, J., Ratet, P., Mysore, K.
S., Gough, C., & Jones, K. M. (2020). Sinorhizobium meliloti succinylated
high‐molecular‐weight succinoglycan and the Medicago truncatula LysM
receptor‐like kinase MtLYK10 participate independently in symbiotic
infection. The Plant Journal, 102(2), 311-326. https://doi.org/10.1111/
tpj.14625
Mathieu, C., Pieltain, F., & Jeanroy, E. (2003). Analyse chimique des sols:
Méthodes choisies. Tec & doc. Lavoisier, Paris, 408 p.
Miller, K. J., & Wood, J. M. (1996). Osmoadaptation by rhizosphere
bacteria. Annual Review of Microbiology, 50(1), 101-136. https://doi.
org/10.1146/annurev.micro.50.1.101
Figure 3. UPGMA dendrogram showing the similarities based
on phenotypic tests between Genista saharae strains
isolates.
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2024, 41(4): e244138 October-December. ISSN 2477-9407.
8-8 |
Míguez-Montero, M. A., Valentine, A., & Pérez-Fernández, M. A. (2020).
Regulatory eect of phosphorus and nitrogen on nodulation and plant
performance of leguminous shrubs. AoB Plants, 12(1), plz047. https://
doi.org/10.1093/aobpla/plz047
Michiels, J., Verreth, C., & Vanderleyden, J. (1994). Eects of temperature stress
on bean-nodulating Rhizobium strains. Applied and Environmental
Microbiology, 60 (4), 1206-1212. https://doi.org/10.1128/aem.60.4.1206-
1212.1994
Pongslip, N. (2012). Phenotypic and genotypic diversity of rhizobia. Bentham
Science Publishers. DOI:10.2174/97816080546191120101
Somasegaran, P., & Hoben, H. J. (1985). Handbook for rhizobia: methods in
legume-Rhizobium technology. Springer Science & Business Media.
https://www.ctahr.hawaii.edu/bnf/Downloads/Training/Rhizobium%20
technology/Title%20Page.PDF
Scianna, J., Logar, R., & Pick, T. (2007). Testing and interpreting salt aected
soil for tree and shrub plantings. Natural Resources Conservation Service,
Plant Materials Technical Note No. MT-61. https://www.nrcs.usda.gov/
plantmaterials/mtpmctn7580.pdf
Tsyganov, V. E., & Tsyganova, A. V. (2020). Symbiotic regulatory genes
controlling nodule development in Pisum sativum
L. Plants, 9(12), 1741.
https://doi.org/10.3390/plants9121741
Vincent, J. M. (1970). A manual for the practical study of the root-nodule bacteria.
https://doi.org/10.1002/jobm.19720120524
Zakhia, F., Jeder, H., Domergue, O., Willems, A., Cleyet-Marel, J. C., Gillis, M.,
... & De Lajudie, P. (2004). Characterisation of wild legume nodulating
bacteria (LNB) in the infra-arid zone of Tunisia. Systematic and Applied
Microbiology
, 27(3), 380-395. https://doi.org/10.1078/0723-2020-00273