Effect of PGPR (Plant Growth Promoting Rhizobacteria) on in vitro bread wheat
(Triticum aestivum L.) growth parameters and biological control mechanisms
Laid Benderradji1, Kamel Kellou2, Walid Saibi3 and Faiçal Brini3*
1
Natural and life sciences department, Faculty of sciences, Mohamed BOUDIAF University M’sila, PO Box 166, Ishbilia Street-28000, Algeria. 2Genetics, Biochemistry & Plant
biotechnology laboratory, Mentouri University - Constantine, Ain El Bey Street-25000,
Algeria. 3Plant Protection and Improvement Laboratory, Centre of Biotechnologiy of Sfax
(CBS), Km 6 Sidi Mansour, PO BOX “1177” 3018, Sfax- Tunisia.
*Corresponding author: Faiçal BRINI, Email: [email protected]
Abstract
Isolation of actinomycetes isolates was made from rhizosphere of three endemic plants in
Algeria (Astragalus gombo Coss. & Dur., Daucus sahariensis Murb., Ononis angustissima
Lam.). Ninety three isolates have been screened to evaluate their antagonism properties
against phytopathogenic microorganisms and to determine their bio-control properties against
Fusarium culmorum, especially responsible for several cereal diseases like font’s seedlings,
rust and burn of ears. Four coded strains (D2, D8, D5 and AST1) have been tested for in vitro
effect of PGPR characters and bio-control of Hidhab (HD1220) variety seeds of bread wheat
(Triticum aestivum L.), valued on the Murashigue and Skoog (MS) culture medium.
Evaluation of antagonistic isolates of pathogenic fungi and fungicides that affect at different
degrees on the growth parameters showed clearly that these 4 strains have significant effects
on percentage of seeds germination and seedling growth. So, these PGPR (Plant Growth
Promoting rhizobacteria), play a role in protecting off pathogenic fungus (Fusarium
culmorum). However, results argue that these isolates show a very interesting activity
compared with commercial fungicide. While these actinomycetes isolates can be used as
biocontrol agents against Fusarium wilt disease of wheat, which have both a beneficial effect
on growth.
Key words: Endemic plants, PGPR, Bread wheat, Fusarium culmorum, Biological control.
1. Introduction
Pathogenic soil fungi are difficult to be controlled, because their survival for a long periods in
the soil (Tschen, 1985). To fight against these pathogens via unlimited application of
pesticides in soil can cause environmental pollution. In addition, the effectiveness of chemical
fungicides is often compromised by the emergence of resistant pathogens. Due to the
worsening problems in fungal diseases control, further research is needed to identify
alternative methods of protecting plants less dependent on chemicals and more respectful to
the environment (Prapagdee et al., 2008). However, biological control is a promising method
that involves antagonistic microorganisms (actinomycetes) uses that are deemed to apply best
form of living cells because of their ability to colonize plant roots and their ability to control
microorganisms plant pathogens and form spores adapted to the formation of stable products
(Xiao et al., 2002; Bressan, 2003). Actinomycetes are an important part of microbial
community in soil dispersion surface; allow it the ability to colonize the rhizosphere through
their antagonists and competitive characters, and their production of many secondary
metabolites that having chemical structures and a wide variety of biological activities
(Gundliffe, 2006). More than a thousand secondary metabolites are produced by
actinomycetes witch made 45% of microbial metabolites (Berdy, 2005). Streptomyces genus
1
has the capacity to produce plant growth regulator like Indole Acetic Acid (IAA), antibiotics
and lytic enzymes as a biocontrol agents against (Fusarium culmorum) responsible for various
symptoms like damping-off root and stems and spikelet Fusariose in many broadleaf and
monocotyledons plants such as cereals. These diseases have been controlled by synthetic
fungicides (chemical compounds). However, chemical control generates enormous negative
impacts on the environment, and the repeated use of these chemical molecules causes
resistance among these pathogens. This study focuse on the search for biologically
actinomycetes able to fight against these pathogens. They are often isolated from samples of
different ecosystems for producing substances inhibitory activities against plant pathogens,
using suitable culture media and then select the successful stem through the identification of
their in vitro antagonistic activity.
2. Materials and Methods
Plant material
Seeds of the wheat (Triticum aestivum L.) variety Hidhab (HD1220) treated or not
by a fungicide were used as negative control (C-) or positive control (C+)
respectively.
Techniques of isolation, seeding and recognition of actinomycetes strains
Several solid media, namely: ISP2, GYEA and GYME (Table 1) with added antibiotics
(Streptomycin, Penicillin and Actidione) were used to isolate and retain the strains of
actinomycetes from selected soil samples.
Culture media were autoclaved at 120°C for 2h; the glassware is sterilized at 180°C in
ventilated oven for 30min. Microbiological manipulations are performed in a laminar flow
hood around a Bunsen burner. Using the method of Pochon and Tardieux (1962), 3 samples of
100g to 5cm of the top soil away from roots have been taken from the rhizosphere of 3
endemic plants zones (Daucus sahariensis Murb., Astragalus gombo Coss. & Dur., and
Ononis anguistissima Lam.), located in Boussaâda region at M’sila- State in Algeria (Fig.
1A); then put in plastic bags and analyzed directly or stored at 4°C for 6h. 10g of each sample
was ground in a sterile mortar and then sieved. 5g are processed by 0,02g and introduced into
a container containing 50ml of sterile distilled water. After stirring and homogenization of the
solution, a series of dilutions of 10-1 to 10-5 was prepared and 0.1ml from the last 3 dilutions
was collected and seeded depositing 0.1ml of each sample corresponding to the dilution 10-3,
10-4 and 10-5 to the spread surface of the corresponding culture medium (GYME, GYEA,
ISP2) in Petri dish which were incubated at 28°C for 7 to 14 days, while the boxes containing
only ISP2 backgrounds, GYEA, GYME serve as control and were incubated in the same
previous conditions (Kitouni et al., 2007). Actinomycetes colonies appeared were observed
under an optical microscope (x40). They are marked by their macroscopic and microscopic
appearance like hard colonies, small size with a round shape surrounded by micro-filaments.
Crop diversity is thus carefully analyzed and investigated.
Purification, conservation and antifungal activity of actinomycetes
Colonies are separated on Petri dish containing (ISP2), purified and subcultures streaked on
the same medium. After incubation at 28°C, several morphotypes have been observed and
strains were conserved at 4°C in test tubes containing incline agar for 4 weeks for frequency
subculture. So, actinomycetes stayed for 14 days in a liquid medium which are added to
sterile glycerol (15%), and immediately frozen for long term storage. Antifungal activity of
isolates was underscored by cross streaks method on ISP2 environment, agar-cylinder method
and direct confrontation method in Petri dish (Badji et al., 2005). The target seeds used are
filamentous fungi and pathogenic bacteria.
2
Cross streaks techniques, direct confrontation in Petri dish method and agar disc
Antimicrobial activity of actinomycetes isolates is evaluated by cross-striations method on
ISP2. It consists in seeding actinomycetes isolates in a single line on the surface of solid
medium. After incubation at 30°C for 7 days, target strains are seeded perpendicular to the
actinomycetes and results were obtained after 36 to 48 hours by measuring the distance
between the edges of inhibition of target strain and actinomycetes isolates. Actinomycetes are
seeded, on PDA (Potato Dextrose Agar), the device of the Petri dish (away from the edge of
the box by 3mm), with a streak of 6mm wide. These cultures are incubated at 28°C for 5 days.
A disk of 7mm diameter mushroom cultivation 8 days old is deposited share of actinomycetes
culture. Distance between mushroom and disc edge of the box is 15mm. For each fungal
isolate is provided a control treatment, with disc deposited on PDA medium without
actinomycetes. Diameter of fungus colonies is measured for all treatments including the
control (Soares et al. 2006). According to Williams and Willis (1962); Aghighi et al. (2004),
agar discs (6mm ϕ) are collected from an actinomycetes culture of 14 days then put them into
a Petri dish containing (PDA) medium. A washer (8mm ϕ) of Fusarium sp of 10 days old is
then deposited in the center of the box and at a distance of 3cm agar discs away. Petri dishes
were then incubated at 25°C for 5 days. Control sample contains only a washer of Fusarium
sp. A daily reading is made with a respect to control sample culture.
Application of antifungal activity
The following protocol showed the antifungal activity and biological control against plant
diseases by actinomycetes isolates that produce antibiotics (Fig. 1B).
In vivo antagonistic isolates potential of actinomycetes on Fusarium wither of wheat
seeds and disease expression
We have studied actinomycetes action on Fusarium mycelium growth to assess the
effectiveness of actinomycetes suspensions on Hidhab (HD1220) variety seeds. Spores of
phytopathogenic fungus was obtained by flooding a culture of 14 days on (PDA) medium
incubated at 25°C with 10ml of sterile distilled water, conidia are dislodged by scraping the
medium surface with sterile Pasteur pipette. The resulting liquid is filtered through 4 layers of
cotton to remove debris from the mycelium. Filtrate obtained containing spores washed twice
with sterile distilled water and the spore suspension was centrifuged at 1000rpm for 5min.
Thus, pre-identified isolates of actinomycetes are streaked on Petri dish containing nutrient
agar medium and incubated 24h at 30°C. The cans are then flooded with 10m1 of sterile
distilled water and scraped with a sterile Pasteur pipette; the recovered suspension is
homogenized by stirring. Spore concentration is estimated using a Malassez cell and adjusted
to the required concentration (108 spores/ml) by adding it to sterile distilled water (Barakat et
al., 2002).
It is noted that we have used in this study the MS medium (Murashigue and Skoog 1962)
supplemented with 0.5g/l of 2.4, Dichlorophenoxyacetic acid (Table 2). Wheat seeds are
disinfected by 70% alcohol for 70 seconds, and then they are soaked in water for 15 minutes
bleach followed by 3 successive rinses with sterile distilled water.
Antifungal activity of actinomycetes and pathogen for treated and untreated seeds by
fungicide
Both of 8 treated and untreated seeds of Hidhab (HD1220) variety with the fungicide were
seeded on MS medium supplemented with 3ml of Fusarium suspension and 3ml of
actinomycetes suspension.
Measures and statistical analysis
Measurements relate to the enumeration of isolated actinomycetes and their behavior towards
the culture medium used and macro-morphological characterizations and their antagonistic
activities are mentioned. Data are processed by the analysis of variance, using Cropstat 7.2.3
3
software (2009), and then Newman-Keuls test at 5% probability, is used to compare treatment
means.
3. Results
Growth parameters effects
 Seed germination percentage
Results of seeds germination capacity of bread wheat (Triticum aestivum L.), variety Hidhab
(HD1220), after inoculation by actinomycetes isolates in presence of Fusarium culmorum are
represented by the figure 4A. In general, percentage of both germinated seeds treated and no
by fungicide exceeds 85%, so there is a significant effect of treatment by fungicide. These
results, confirm that the treatment by fungicide has a depressive effect on the pathogen and
therefore a positive effect on the germination rate (Fig. 2A).
 Number of leaves
Number of leaves is very high for the seedlings obtained from seeds treated with fungicide
and inoculated with actinomycetes strains (4-6 leaves), but the number of sheets remains
limited to seedlings in untreated seeds (2-5 leaves) (Fig. 2B).
 Leaf area
Leaves of treated and untreated seeds by fungicide and inoculated with 2 isolates (D8 and D5)
in the presence of the pathogen, have a moderately developed leaf area (5 and 5.5cm2); (5.4
and 14cm2) respectively). So, for the seeds inoculated by AST1isolate untreated by fungicide,
we note that leaf area affected (17cm2), while the leaf area of treated seeds by fungicide and
inoculated by AST1 isolate is reduced to (8cm2). For the D2 strain, leaf area is 17cm2 for
treated seeds and 22cm2 for untreated seeds (Fig. 2C).
 Length of roots
Measurement carried out show that seeds treated and untreated with fungicide having lengths
of roots between 5 and 6,5 cm, for all 3 actinomycetes isolates (D2, D5 and D8), while the
seeds inoculated with the isolate AST1 treated with fungicide have a length of very small
roots (3cm only) (Fig. 2D).
 Chlorophyll content
Seedlings from seed inoculated by 3 isolates D5, D8 and AST1 treated or not with fungicide
present a major chlorophyll content (1, 2, 1.1 and 1, 18 Spad Unit respectively), while
chlorophyll content is reduced for treated seeds of the plantlets inoculated with the same
isolates (1.01; 0.9 and 0.95 Spad Unit respectively). So, for D2 isolate, chlorophyll content is
1 and 1.15 respectively for both treated and untreated seeds with fungicide (Fig. 3).
4. Discussion
Generally, the results of our study are consistent with the work of many researchers confirmed
the role of microorganisms in biological control of plants against diseases. It showed that the
use of microorganisms to fight against the enemy culture (bio-pesticides) is a plant protection
means respecting the environment. Thus, Yekkour et al. (2015) applied the biocontrol
seedling blight through saharian actinomycetes. Indeed, most parasites, fungi or weeds of the
plants have one or more natural microbial enemies, but to ensure the efficacy of a biopesticide, we must know the pathogenic fungi development cycle and conditions of antagonist
activity. Streptomycetes soil have been reported that they produce hydroxamate-type
siderophore that could inhibit the growth of plant pathogens and thus reduce their action by
competing phenomenon for iron (Muller et al. 1984; Muller and Raymond 1984; Tokala et al.
2002) or by stimulation of the defenses of the plant (Franceschi et al. 2005; Vassilev et al.
2006; Prévost et al. 2006; Lehr et al. 2007). Thus the results obtained in this study on
Fusarium soft wheat will contribute positively to the improvement of biocontrol process
against these diseases. Analysis of the results of the effect of PGPR actinomycetes isolates on
4
growth parameters show that the isolates have a significant effect on the germination rate for
the treated and untreated seeds of the same degree by a fungicide, and then Newman-Keuls
test at 5% reached 8 homogeneous groups (Table 3a and 3b).
Analysis of variance revealed a very highly significant effect on germination rate, highly
significant for foliage and significant for the sheet number and root length; while the
chlorophyll content, the effect is not significant and that for all the treated seeds or not by the
fungicide inoculated by actinomycetes isolates and it is the same for the interaction (treatment
x actinomycetes isolates fungicide) (Table 4).
As against the number of sheets is differential between treated seeds by a fungicide with a
high number of sheets especially for seeds inoculated with D2 and D8 strains, while for non
treated with fungicide seed number is reduced for both isolates, in contrast to strain AST1. D5
isolate has a high number of sheets for the seeds treated or not treated with fungicide,
whereas, this isolate has a beneficial effect on the development of leaf area in addition;
analysis results show that D5 isolate has a remarkable influence on the root length. Seed
treatment by actinomycetes isolates from different areas of the rhizosphere of endemic plants
gave important results of PGPR effects, resulting in improved growth parameters studied.
However, D5 isolate has a very important effect on growth compared to the effect of
fungicide. This stimulation resulted mainly from better growth for untreated seeds by the
fungicide (Photos 1A & 1B). These results certainly participate in the biocontrol process
(Kleopper et al. 1980). We can say that our strains may be affiliated with PGPR (Plant
Growth Promoting Rhizobacteria). Then, it is known that PGPR may also protect plants
against infections of phytopathogene. This is confirmed by the test isolates by the action
against Fusarium culmorum. Biocontrol results actinomycetes isolates against the pathogenic
Fusarium culmorum agent are reflected in the percentage of germination that is very
important for all seeds inoculated with the 4 isolates (untreated and treated with fungicide),
whereas, so we can said that our isolates have a inhibitory effect against the pathogen, for
untreated seeds by the fungicide and inoculated into both the isolate of actinomycetes and
Fusarium have a highly germination rate; but the number of sheets differs between treated
seed (restricted) and untreated (highly) especially for AST1 isolates, D2 and D5. These results
let us concluded that actinomycetes isolates can reduce Fusarium mycelium growth butter
than commercial fungicide; it does not increase the number of leaves. Similarly to the
parameter of the root length recorded, D2 and AST1 isolates, have a very interesting
effectiveness in protecting the root system against Fusarium culmorum. The hypothesis
proposed is that the fungicide has a detrimental effect on increase leaves number and root
length; while biological control against the pathogen by different actinomycetes isolates,
especially D5 and D8, is more best to flight against diseases in bread wheat (Triticum
aestivum L.), Hidhab (HD1220) variety (Photo 1C, 1D).
In this study we have isolated actinomycetes strains from soil of 3 endemic plants in Algeria,
and assessed their antagonistic activities against Fusarium culmorum which is a causal agent
of Fusarium disease. We have used seeds of bread wheat (Triticum aestivum L.), Hidhab
(HD1220) variety, treated and untreated with fungicide as a negative control (C-) and positive
control (C+) respectively in the test of PGPR / bio-control to make comparison between the
action of a fungicide and the effect of antagonist’s actinomycetes suspensions. Results
obtained showed that PGPR treatment in addition to the fungicide is less effective and it
appeared with the comparison of wheat seeds inoculated only with the suspension D5, D8,
D2, AST1 isolates (without treatment with fungicide) that have better results in terms of
growth parameters, like germination rate, leaf number, roots length, leaf area and chlorophyll
content. Similarly for the bio-control test, isolate D5 has a strong action against Fusarium
suspension but the action of fungicide is very interesting against this pathogen. Depending on
the results, we can say that the fungicide has an inhibitory effect against Fusarium culmorum,
5
but has a harmful effect on growth. While actinomycetes isolates play both a role of
protection against pathogenic agents and improved growth parameters.
Acknowledgements
Thanks for the supporting to this project by ministry of higher education and scientific
research–Algeria. Code project number: Cnepru, F05620110025, entitled: Inventory
valorization and in vitro characterization of useful plants (medicinal, aromatic and fodder) in
the steppe region-El Hodna (M'sila, Algeria).
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Figure Legends
Figure 1: Endemic plants studied in this research
Figure 2: Antifungal Activity protocol
Figure 3: Malassez cell used for spore’s concentration estimation
Figure 4: Actinomycetes isolates effects on germination percentage (A); leaves number (B);
roots length (C) and leaf area (D) within and without Fusarium colmorum on treated seeds by
fungicide (D2, D5, D8 = Daucus sahariensis Murb., isolate number 2, 5 and 8. Ast1 =
Astragalus gombo Coss. & Dur., isolate number1, Fc = Fusarium colmorum). ). Asterisks
indicate statistically significant differences (P < 0.05) level between the WT plants and the
corresponding treatment.
Figure 5: Actinomycetes isolates effects on chlorophyll content within and without Fusarium
colmorum on treated seeds by fungicide (D2, D5, D8 = Daucus sahariensis Murb., isolate
number 2, 5 and 8. Ast1 = Astragalus gombo Coss. & Dur., isolate number1, Fc = Fusarium
colmorum). Asterisks indicate statistically significant differences (P < 0.05) level between the
WT plants and the corresponding treatment.
Photo 1: (A) Seeds of wheat (HD) inoculated with the actinomycetes isolates and treated with
a fungicide; (B) Seeds of wheat (HD) inoculated by actinomycetes isolates and not treated
with a fungicide; (C) Seedlings obtained from wheat seeds not treated with fungicide and
inoculated with the suspension of Fusarium culmorum isolate + actinomycetes; (D) Seedlings
obtained from wheat seeds treated with fungicide and inoculated with the suspension of
Fusarium culmorum isolate + actinomycetes.
7
Table1: Media used in isolation, seeding and recognition of actinomycetes isolates
Compounds
ISP2 GLM GYEA
PDA
Malt extract
10
3
Yeast extract
4
3
10
Glucose
4
10
10
Peptone
5
Dextrose
20
Infusât de pomme de terre (ml)
200
Agar
20
20
18
20
Distilled water (ml)
1000
1000
1000
1000
pH
7.3
7.2
6.8
5.4
ISP2= International Streptomyces Project (Shirling and Gottlieb 1966), GYME= Gelose Yeast-Malt Extract
(Kitouni 2007), GYEA= Glucose-Yeast Extract-Agar (Athalye et al. 1981), PDA = Potato dextrose agar.
Table 2: Culture medium compound of Murashigue and Skoog (1962).
MacroInorganic
mg/l Micro-elements mg/l
mg/l
elements
elements
NH4NO3
1650 KI
0.83
FeSO4.7H2O
KNO3
1900 H3BO3
6.2
Na2EDTA.2H2O
CaCl2.2H2O
150
MnSO4.4H2O
22.3
MgSO4.7H2O
250
ZnSO4.7H2O
8.6
KH2PO4
170
Na2MoO4.2H2O
CuSO4.5H2O
CoCl2.6H2O
0.25
0.025
0.025
Organic
elements
Myo27.8
Inositol
Nicotinic
37.3
Acid
HClPyridoxine
HClThiamine
Glycine
mg/l
100
0.5
0.5
0.1
2
8
Figures
Daucus sahariensis Murb
Astragalus gombo Coss. & Dur
Ononis angustissima Lam
Rhizosphere soil of 3 endemic plants
Seeding media (ISP2- GYEA- GLM-PDA)
Actinomycete isolates
Crusaders streaks
Agar discs
Direct confrontation in Petri dish
In vivo test against Fusarium disease of bread wheat
Figure 1
9
Treated seeds by
fungicide
**
*
90
Untreated seeds
by fungicide
80
70
60
50
40
30
(B) 7
**
6
Number of leaves
Percentage of germination (%)
(A) 100
20
**
4
3
2
0
D2+ Fc
D5+ Fc
D8+ Fc
Ast1 + Fc
D2 + Fc
Actinomycetes isolates + Fusarium colmorum
Treated seeds by
fungicide
**
Untreated seeds
by fungicide
6
*
5
**
4
3
D5 + Fc
D8 + Fc
Ast1 + Fc
Actinomycetes isolates + Fusarium culmorum
(D) 25
20
Leaf area (cm2)
(C) 8
Root length (cm)
Untreated seeds
by fungicide
5
1
10
0
7
Treated seeds by
fungicide
*
**
2
Treated seeds by
fungicide
**
Untreated seeds
by fungicide
15
10
**
**
*
5
1
0
0
D2 + Fc
D5 + Fc
D8 + Fc
D2 + Fc
Ast1 + Fc
D5 + Fc
D8 + Fc
Ast1 + Fc
Actinomycetes isolates + Fusarium culmorum
Actinomycètes isolates + Fusarium culmorium
Figure 2
10
Treated seeds by
fungicide
Chlorophyll content
SPAD Unit
1,40
Untreated seeds by
fungicide
1,20
1,00
0,80
0,60
0,40
0,20
0,00
D2 + Fc
D5 + Fc
D8 + Fc
Ast1 + Fc
Actinomycetes isolates + Fusarium culmorum
Figure 3
11
D5-HD
Da8-HD
D2-HD
(A)
(B)
(C)
(D)
Photo 1
12