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Rhizobium isolates nodulating common bean: A pathway to control Fusarium sp, the causative 
agent of damping-off of seedlings   

 

 

 Ngo Nkot 
Laurette1+ 

 Youagang Gougueu 
Harris Stephane1 

 Semboung Lang 
Firmin1 
Mezatio Ariane Sandra1 

1Plant Biology Department, Faculty of Science, the University of Douala, 
Douala, Cameroon. 
1Email: laurettengonkot@gmail.com  
1Email: hyouagang@yahoo.fr  
1Email: langsemb@yahoo.fr  
1Email: mezatioariana@gmail.com  

 
(+ Corresponding author) 

 ABSTRACT 
 
Article History 
Received: 10 February 2025 
Revised: 24 April 2025 
Accepted: 6 May 2025 
Published: 21 May 2025 
 

Keywords 
Antagonism 
Biocontrol 
Damping-off of seedlings   
Fusarium sp 
Lytic activities 
Phaseolus vulgaris 
Rhizobium. 

 
Obtaining a high-performing inoculant requires combining diversified traits that can 
strongly contribute to promoting plant growth and protection. This work aims to 
contribute to developing biological control of Fusarium sp., the causative agent of 
damping-off using rhizobia. Eight rhizobial isolates from the Laboratory of Plant Biology 
were used. A search for some enzymes (cellulase, proteases, chitinases, and phosphatases) 
produced by rhizobia and involved in biological control was carried out. The rhizobia 
isolates were then tested for their ability to biocontrol Fusarium sp. in direct and indirect 
confrontation. Finally, the effect of inoculation with the isolates on the growth of bean 
plants and the severity of Fusarium sp. was assessed. The results show that all the 
bacterial isolates can synthesize lytic enzymes in varying proportions, while 50% could 
produce phosphatases. The direct antagonism test against Fusarium sp. showed that the 
isolates exert inhibitions ranging from 20.55% to 65.15% and 30% to 30.8% in indirect 
confrontation. The results of the effect of inoculation of infected plants on plant growth 
and pathogen severity indicate that the tested Rhizobium isolates promote the growth of 
infected plants by causing a decrease in disease as well as significant improvement in the 
height of the stems compared to non-inoculated plants. These results, therefore, indicate 
that the rhizobia isolates used considerably reduce the growth of Fusarium sp. using 
enzymes and antagonistic substances, leading to improved growth of bean plants.  
 

Contribution/Originality: The study presents a sustainable method to control the damping-off of seedlings in 

common beans, which is a significant hindrance to bean cultivation. 

 

1. INTRODUCTION 

The common bean (Phaseolus vulgaris L.) is one of the most consumed legumes in the world. Its seeds are rich in 

proteins, folic acid, dietary fiber, minerals, carbohydrates, and vitamins necessary for human and animal nutrition 

[1]. In eastern, central, and southern Africa, dry beans are the most important grain legumes, both in terms of 

cultivated area and consumption [2]. African smallholders cultivate more than 4 million hectares of beans each year, 

the harvest of which is used as a source of food for more than 100 million Africans [3]. Rural populations of Cameroon 

in bean production areas cultivate several varieties of common beans for food and commercial purposes. National, 

sub-regional, and international demand for common beans continues to grow over the years. However, national and 

African production remains low due to the low availability of nitrogen in the soil, as well as the effects of abiotic 

Current Research in Agricultural Sciences 
2025 Vol. 12, No. 1, pp. 63-78 
ISSN(e): 2312-6418 
ISSN(p): 2313-3716 
DOI: 10.18488/cras.v12i1.4214 
© 2025 Conscientia Beam. All Rights Reserved. 

 
 
 

 
 
 
 

 

 
 
 
 

https://orcid.org/0000-0001-8581-6540
mailto:laurettengonkot@gmail.com
mailto:hyouagang@yahoo.fr
mailto:langsemb@yahoo.fr
mailto:mezatioariana@gmail.com
https://orcid.org/0000-0002-9046-8365
https://orcid.org/0000-0001-8883-8374
https://www.doi.org/10.18488/cras.v12i1.4214


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factors (salinity, acidity, drought, etc.) and parasitic diseases [4]. Thus, more than approximately 84% of production 

is lost each year in tropical Africa because of these constraints [4]. Damping-off disease, caused by Fusarium sp. and 

Pythium sp., is one of the most important pathological constraints [5]. Yield losses due to this disease have been 

estimated at 80% in Cameroon [6]. The control strategy based on chemical pesticides is one of the causes of 

environmental pollution when use is poorly controlled. In addition, the high cost of these chemicals is a hindrance for 

farmers [7]. The use of resistant varieties is another improvement strategy; however, in Cameroon, resistant varieties 

are insufficient compared to the needs of producers. Furthermore, the development of resistant genotypes remains 

difficult due to variations in fungal pathogenicity [8]. It is important to limit the damage caused by damping-off in 

order to maximize bean production yield sustainably. Thus, conventional agriculture should move towards more 

sustainable and environmentally friendly cultivation systems. The provision of rhizobial inocula capable of promoting 

plant growth could be exploited for their inhibitory and protective properties in the biological control of 

phytopathogens responsible for the damping-off of common bean seedlings [9]. The main objective is to evaluate the 

ability of rhizobia to biocontrol Fusarium sp. 

 

2. MATERIALS AND METHODS 

2.1. Materials 

2.1.1. Plant Material 

The plant material consists of common bean (Phaseolus vulgaris) grains of the GLP-190 variety for the 

greenhouse inoculation test and bean plants affected by damping-off collected in the field for Fusarium sp. isolation. 

 

2.1.2. Microbial Inoculant 

The microbial material consists of eight rhizobial isolates from the Laboratory of Plant Biology at the University 

of Douala. The isolates used (Figure 1) were obtained from bean nodules collected in Mbouda and Foumbot in the 

West Region, and Njombe and Nkongsamba in the Littoral Region of Cameroon, along with the reference strain 

ISRA 352 from the LCM (Laboratoire Commun de Microbiologie) in Dakar, Senegal. 

 

 
Figure 1. Macroscopic aspect of isolates used. a: PvNj5; b: PvFt3; c: PvMb1; d: PvMb5; e: PvNj8; f: PvNk7; g: PvNk8; h: ISRA 352. 

 

The microbial material also consists of Fusarium sp. isolated from diseased common bean plants collected in 

Foumbot, West Cameroon. 

 

 

 



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2.1.3. Soil Sample 

The soil sample comes from the fallows of the experimental fields of the Institute of Agricultural Research for 

Development (IRAD) in Foumbot. 

 

2.2. Methods 

2.2.1. Assessing Some Lytic Activities of Rhizobial Isolates 

The detection of certain lytic enzymes in bean rhizobia isolates includes the production of cellulases, proteases, 

chitinases, and phosphatases. 

 

2.2.1.1. Cellulase Production  

The search for the production of extracellular cellulases by bacteria is conducted in Petri dishes containing agar 

media supplemented with CMC (carboxymethyl cellulose) as the sole carbon source, according to the method of 

Johnsen and Krause [10]. The composition of the medium in g/L distilled water is as follows: Na2HPO4: 6; KH2PO4: 

3; NaCl: 0.5; NH4Cl: 1; MgSO4: 0.5; CaCl2: 0.015; glucose: 2; CMC: 10 g; yeast extract: 1.2; pH: 7.2 [11]. The Petri 

dishes are then incubated at 28°C for 8 days, and the presence of cellulase is checked by flooding the surface of the 

dish with a 0.5% solution of Congo red for 30 minutes, followed by washing with distilled water. A second wash is 

then carried out with a 1M NaCl solution. A bright yellow halo around the bacterial colony indicates the cellulolytic 

activity of the bacterial isolate tested. 

 

2.2.1.2. Protease Production 

The search for proteolytic activity in the different isolates of rhizobia is determined by seeding the bacteria on 

the surface of the Skimmed Milk Agar medium [12], the composition of which per litre of distilled water is as follows: 

Peptone: 5g; Yeast extract: 2.5g; Glucose: 1g; Skimmed milk: 7%; Agar Agar: 15g; pH 7.2. The presence of a clear 

zone surrounding the bacterial colonies reflects proteolytic activity (caseinase) in the culture medium, which is 

determined after 5 days of incubation at 28°C. Three replicates were performed for each isolate. The diameter of the 

light zone (halo) as well as that of the colonies was measured. 

 

2.2.1.3. Chitinase Production 

The composition of the medium in g/L used to screen rhizobia isolates for chitinase activity is as follows: chitin: 

5.0; yeast extract: 0.5; K2HPO4: 0.5; MgSO4·7H2O: 0.2; NaCl: 0.1; Agar: 20. The pH of the medium was adjusted to 

7.0, and the medium was sterilized by autoclaving at 120°C for 20 min. After cooling, the medium is transferred to 

sterile Petri dishes. Each rhizobia isolate is inoculated into the center of the plate and examined for a clear zone 

around the colony after incubation at 28°C for up to 7 days. The growth of an isolate on this medium reflects its 

ability to hydrolyze chitin. 

 

2.2.1.4. Production of Phosphatases 

The study of the capacity of bean-nodulating rhizobia isolates to solubilize phosphate was carried out using a 

qualitative test. The isolates were inoculated using a sterile loop per touch at the rate of three repetitions per Petri 

dish containing Pikovskaya medium (PVK) composed as follows (g/L): Yeast extract: 0.5; Glucose: 10; Ca3(PO4)2: 5; 

(NH4) SO4: 0.5; KCl: 0.2; MnSO4 7H2O: 0.1; FeSO4: 0.0001; Agar: 15; Distilled water: 1L; pH: 7.2. The seeded Petri 

dishes were then incubated at 28°C for 14 days. Naked-eye observations of the Petri dishes were made from the 3rd 

day. The presence of a yellow halo around the colonies indicates the production of phosphate-dissolving substances 

[13]. 

 

 



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2.2.2. Evaluation of the Capacity of Rhizobia Isolates to Biocontrol Fusarium sp. in Vitro 

The evaluation of the ability of bean-nodulating rhizobia isolates to control Fusarium sp. started with the isolation 

of the pathogen, followed by its identification. A leaf disc pathogenicity test was performed. The ability of rhizobia to 

control Fusarium sp. was assessed in direct and indirect confrontations. 

 

2.2.2.1. Sampling of Common Bean Plants and Soil 

Numerous surveys carried out in the region of West Cameroon (Foumbot) made it possible to collect several 

samples of plants showing characteristic symptoms of damping-off (crown rot, wilting, and yellowing of leaves). The 

samples were taken to the laboratory for the isolation of the phytopathogen using a steel auger previously sterilized 

by flaming with 90° alcohol. Soil is taken randomly from the diagonals of the 5m x 5m plot at a depth between 0 and 

20 cm. Within the plot, 10 randomly collected samples are carefully mixed to form a composite sample. The composite 

sample is used for the greenhouse experiment.  

 

2.2.2.2. Sanitization and Isolation of Fusarium 

Leaves and stems of bean plants showing symptoms of disease are used for this test. The various organs, cut with 

sterile pruning shears, are superficially disinfected with ethanol (10%) for 10 seconds and then with 5% sodium 

hypochlorite for 30 minutes. These samples are rinsed with sterile distilled water to remove all traces of bleach and 

then dried on sterile filter paper close to the Bunsen burner flame under a laminar flow hood. After disinfection, the 

organ fragments obtained from diseased bean plants are inoculated (2 to 3 fragments) on potato dextrose agar (PDA) 

medium autoclaved at 120 °C and poured into Petri dishes with the following composition per litre of solution: apple: 

20 g; dextrose: 20 g; agar: 18 g. These dishes are incubated for 8 days at 28°C. Subsequent subcultures on other Petri 

dishes containing the PDA medium are carried out under aseptic conditions using a sterile loop [14] for purification. 

 

2.2.2.3. Fungi Identification 

The identification of fungi is mainly based on macroscopic characteristics such as growth rate, appearance of 

aerial mycelium, and color of the underside of the colony. Microscopic identification consists of observing under a 

light microscope the presence of septate mycelium, microconidia, macroconidia, and chlamydospores [15]. 

 

2.2.2.4. Pathogenicity Test 

The bean leaves are gently rinsed with tap water and then placed in Petri dishes, each containing a cotton pad 

soaked in sterile water. A 5 mm fragment of the fungal isolate is then applied to each leaf. No fungal isolate fragment 

is applied to the control. Three replicates are carried out. The presence or absence of symptoms (yellowing or wilting 

of the leaves) is observed from the 3rd day. The fungi, isolated and purified on new media, are then examined by the 

naked eye. The identification of the fungi is based on macroscopic (cultural) and microscopic criteria. 

 

2.2.2.5. In Vitro Antagonism of Rhizobia Isolates Against Fusarium sp. 

The study of the antagonistic effects of bean-nodulating rhizobial isolates on Fusarium sp. was carried out using 

direct and indirect confrontation techniques.  

 

2.2.2.5.1. Direct Confrontation Test   

The direct confrontation technique makes it possible to demonstrate the production of diffusible substances by 

the rhizobia. The method of Inam-ul-Haq et al. [16] was used to evaluate the inhibitory effect of rhizobial isolates on 

the mycelial growth of Fusarium sp. A 5 mm diameter explant from a fungal culture is placed in Petri dishes containing 

12 mL of nutrient agar medium on one side and bacterial streaks on the other. Controls consist of isolates of Fusarium 

sp. in Petri dishes in the absence of antagonists. Mycelial growth of the isolates is assessed by measuring the radius 



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of the colonies after 7 days of incubation. The inhibition of fungal growth was evaluated by calculating the percentage 

reduction in mycelial growth according to the following Whipps formula [17]: 

𝐼% =  ((𝑅1 − 𝑅2) / 𝑅1)  × 100 

R1 = Radial distance (in mm) of mycelial growth in the absence of the antagonistic strain (control); R2 = Radial 

distance (in mm) of mycelial growth towards the site of inoculation by bacteria. 

 

2.2.2.5.2. Indirect Confrontation Test 

This technique consists of placing in the center of each Petri dish containing the nutrient agar medium an explant 

of approximately 5 mm in diameter taken from young colonies of the pathogen and in another dish bacterial streaks. 

The lids of the two dishes are removed, and the bottoms are sealed with a strip of parafilm to prevent the loss of 

volatile substances. The whole setup is incubated at 28 °C for 7 days. As a control, a Petri dish containing only the 

nutrient agar medium is inoculated with the pathogen [18]. The rate of inhibition of fungal growth is determined 

according to Whipps' method [17]. 

 

2.2.3. Evaluation of the Effect of Inoculation with Rhizobia Isolates on the Growth of Plants Infected with Fusarium and the 

Severity of the Disease 

The isolates with the best results in enzymatic tests and the ISRA 352 strain are used for greenhouse inoculation. 

 

2.2.3.1. Sanitizing and Pre-Germination of Seeds 

Phaseolus vulgaris seeds are treated with 3% sodium hypochlorite for 3 minutes and then thoroughly rinsed with 

sterile distilled water. They are then soaked in 70% alcohol for 2 minutes and rinsed several times with sterile distilled 

water. The disinfected seeds are germinated in sterile plastic trays containing sterile filter paper soaked in sterile 

distilled water for 24 hours in the dark. 

 

2.2.3.2. Sowing of Seeds 

Pre-germinated seeds with short radicles are aseptically transferred into plastic bags using sterile tweezers at a 

rate of one seed per bag. After sowing, the bags are first soaked with sterile distilled water. 

 

2.2.3.3. Preparation of Bacterial and Fungal Inocula  

The bacterial isolates to be tested were cultured on Yeast Extract Mannitol (YEM) medium with the following 

composition per litre of distilled water: Mannitol: 10 g; K2HPO4: 0.5 g; MgSO4·7H2O: 0.22 g; NaCl: 0.1 g; yeast 

extract: 0.5 g. The pH of the medium is adjusted to 6.5. The cultures are placed in a thermostatically controlled shaker 

set at 125 rpm at 28 °C for 2 days. The spore suspension is prepared by flooding the agar surface with 5 ml of sterile 

distilled water. The fungal culture is then scraped off with a sterile spatula. The conidial suspension obtained is stirred 

for 30 s and then filtered through Whatman No. 1 paper to remove mycelial debris. The spore concentration is 

adjusted to 1,106 spores/ml using a Malassez cell [19]. 

 

2.2.3.4. Inoculation of Plants  

Seedlings were obtained from seeds sown in polystyrene bags containing sterile soil. 30 mL of a fungal suspension 

and 30 mL of a bacterial suspension of each isolate tested, previously prepared and adjusted to 108 and 109 CFU/mL, 

respectively, are placed at the base of the stem of each seedling [20]. The control batches consist of a set of seedlings 

treated with sterile distilled water and a set of seedlings grown in soil infected with Fusarium sp. To allow better 

contamination, regular watering with sterile distilled water is carried out to maintain a humidity level sufficient for 

the growth of the phytopathogen. The bean plants are assessed for growth and susceptibility to disease. The plant 



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growth parameters measured are stem length and leaf area [21]. Disease severity was scored on a 1-5 scale developed 

by CIAT(International Center for Tropical Agriculture) [22].  

 

2.2.4. Statistical Analysis  

The data collected were subjected to an analysis of variance (ANOVA) using SPSS 12.0, and means were 

separated by the Duncan test at the 5% probability level. 

 

3. RESULTS 

3.1. Production of Lytic Enzymes by Bacterial Isolates 

The production of lytic enzymes by bacterial isolates is shown in Figure 2. 

 

 
Figure 2. Production of lytic enzymes by bean nodulating rhizobia isolates (a. cellulase; b. protease; c. chitinase; d. phosphatase). 

 

3.1.1. Production of Cellulases 

Figure 3  shows the diameters of the halos produced by cellulase-producing rhizobial isolates on agar 

supplemented with CMC. All rhizobial isolates (100%) had the ability to produce halos on specific agar media, 

although the diameters varied between isolates. The diameter of the halos varied from 0.5 to 3.2 cm. Cellulase 

production is significantly higher for isolate PvFt3 (3.2 cm) and significantly lower for PvNj8 (0.55 cm).  

Isolates PvMb1 (2.55 cm), PvNk7 (2.65 cm), and PvNj5 (2.55 cm) do not show any significant difference. These 

isolates had significantly larger halo diameters than isolates PvMb5 (1.1 cm) and PvNk8 (1.25 cm), which in turn had 

significantly smaller halo diameters than the reference strain ISRA 352 (1.2 cm). 

 

 
Figure 3. Diameters of halos obtained by cellulase-producing rhizobial isolates. 

Note: Bars with the same letter (a, b, c, d, e) are not significantly different by Duncan's test with a mean probability of 5%. 



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3.1.2. Production of Proteases 

The production diameter (Figure 4) ranges from 1.08 cm to 2.5 cm. Protease production is significantly higher 

for isolates PvNj5 (1.8 cm) and PvMb5 (1.7 cm) and significantly lower for isolates PvNj8, ISRA 352, and PvNK8 

with values of 1.21 cm and 1.08 cm, respectively. The ANOVA test shows that there is no significant difference 

between isolates PvNj5 and PvMb5 (1.19 cm). No significant difference was found between the reference strain ISRA 

352 (1.2 cm) and isolates PvNj8 (1.21 cm) and PvNk8 (1.3 cm). 

     

 
Figure 4. Production of proteases by rhizobia isolates. 

Note: Bars with the same letters (a, b) are not significantly different at 5% average probability by the Duncan test. 

 

3.1.3. Production of Chitinases 

The production of chitinases by the different bacterial isolates is presented in Table 1. Isolates PvFt3 and PvNj5 

presented distinct colonies and good growth compared to ISRA 352, PvMb1, PvMb5 and PvNk8, which represent 

50%. PvNj8 and PvNk7 had weak growth. 

 

Table 1. Production of chitinases by rhizobia. 

Isolates of rhizobia Chitinases 

PvFt3 +++ 

PvMb1 ++ 
PvNj8 + 
PvMb5 ++ 
ISRA 352 ++ 
PvNk8 ++ 
PvNj5 +++ 
PvNk7 + 
Note: -: No growth; +: Low growth; ++: average growth; +++: Strong growth. 

 

3.1.4. Production of Phosphatases 

Table 2 shows the results of the phosphatase production test. Good production of phosphatases, reflected by the 

formation of the yellow halo around the colony, was recorded by isolates PvMb5 and PvNk7 (25%) compared to all 



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isolates. ISRA 352, the reference strain from Senegal, and PvMb1 (25%) showed low phosphatase production, while 

isolates PvFt3, PvNj8, PvNk8, and PvNj5 (50%) did not produce a solubilization halo. 

 

Table 2. Production of phosphatases by rhizobia. 

Isolates  Phosphatases 

PvFt3 - 
 PvMb1 + 
PvNj8 - 
PvMb5 ++ 
ISRA 352 + 
PvNk8 - 
PvNj5 - 
PvNk7 ++ 
Note: -: No production; +: Low production; ++: Good production. 

 

3.2. Ability of Rhizobia Isolates to Biocontrol Fusarium sp. in Vitro 

To evaluate the biopesticidal capacity of rhizobial isolates, the isolation of the phytopathogen was carried out 

using plants affected by damping-off of common bean and then identified. A pathogenicity test was conducted, and 

finally, direct and indirect confrontation tests were performed against Fusarium sp. 

 

3.2.1. Appearance of the Phytopathogen Isolate Obtained from Diseased Bean Plants 

Samples of diseased stems and leaves used to isolate the plant pathogen resulted in a pure Fusarium isolate. 

Macroscopic observation of the colonies shows that they grow rapidly. On PDA (Figure 5A), the colony is pale with 

well-developed mycelium. Microscopic observation of Fusarium sp. revealed the presence of septate filaments 

accompanied by microconidia and macroconidia (Figure 5B), straight or slightly curved, small, rifle-shaped. 

 

 
Figure 5. Macroscopic (A) and microscopic (X40) (B) appearance of the Fusarium isolate. 

 

3.2.2. Ability of Bean Nodulating Rhizobia Isolates to Control Fusarium sp. in Direct Confrontation 

The results show that 62.5% of the isolates have antagonistic activity. The most significant inhibitions were 

obtained with isolates PvMb1 (Figure 6a) and PvFt3 (Figure 6b), which caused a significant reduction in the diameter 



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of Fusarium sp. compared to all isolates. Isolate PvNk7 (Figure 6c) caused weak inhibition of the phytopathogen 

compared to the diameter of the Fusarium sp. control (Figure 6d), which completely invaded the dishes 8 days after 

sowing. Isolates PvNJ8 and ISRA 352 showed no inhibition of Fusarium sp. 

 

 
Figure 6. Direct confrontation test of bean nodulating rhizobia - Fusarium sp: a) inhibition of Fusarium by PvMb1; 
b: inhibition of Fusarium by PvFt3; c: No inhibition of Fusarium by PvNk7; d: Control dish containing only 
Fusarium. 

 

Figure 7 shows the inhibition percentages of Fusarium sp. by isolates of rhizobia nodulating bean and the 

reference strain ISRA 352 in direct confrontation. The inhibition percentages of Fusarium sp. varied from 20.55% for 

the reference strain IRSA 352 to 65.15% for the PvFt3 isolate. Statistical analysis of the results using the ANOVA 

test shows a highly significant effect (p<0.05) of the PvFt3 and PvMb1 isolates with inhibition rates of 65.15% and 

64.44%, respectively on the growth of Fusarium sp. The PvNj8 (41.66%) and PvNj5 (38.66%) isolates show non-

significant inhibition percentages between them but differ from the PvMb5 isolate, which shows an inhibition 

percentage of 31.1%. There is no apparent inhibition for isolates PvNk7 (14.99%) and PvNk8 (16.66%), which have 

inhibition percentages of less than 20%. These percentages are not significantly different from those observed for 

ISRA 352, which shows a slight inhibitory activity. Among the eight rhizobial isolates tested, five (62.5%) inhibited 

the development of Fusarium sp. by more than 20%. 

 

 
Figure 7. Percentage inhibition of Fusarium sp. by rhizobia isolates in direct comparison. 

Note: Bars with the same letters (a, b, c, d, e) are not significantly different at 5% average probability by 
the Duncan test. 

 



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3.2.3. Ability of Bean Nodulating Rhizobia Isolates to Biocontrol Fusarium sp. in Indirect Confrontation   

The indirect confrontation test makes it possible to screen rhizobia for volatile substances capable of inhibiting 

the growth of Fusarium sp. The results show a distant inhibitory effect of rhizobial isolates on the growth of Fusarium 

sp. Figure 8a illustrates the inhibitory effect of the PvFt3 isolate on the growth of the phytopathogen in comparison 

with the control dish (Figure 8b), in which the fungi invaded the entire Petri dish. 

 

 
Figure 8. Indirect rhizobia - Fusarium sp. confrontation test: A) Inhibitory effect of PvFt3 against Fusarium sp.; B: Control. 

 

Figure 9 shows the percentages of inhibition of Fusarium sp. by the isolates tested in indirect confrontation. The 

percentage of inhibition varied between 30% and 30.8% depending on the isolate. Not all isolates are able to produce 

volatile substances that inhibit the growth of the phytopathogen. Isolates PvNj8, IRSRA 352, PvNk8, and PvNj5 did 

not induce inhibition (50%). The highest inhibition rates were recorded by the isolates PvFt3 and PvMb1, with 

inhibition percentages of 30% and 30.8%, respectively (25%). The inhibition percentage of PvFt3 is significantly 

higher, followed by the PvMb1 isolate. PvMb5 (17.18%) and PvNk7 (13%) showed negligible antagonistic effects, 

with inhibition rates below 20%. 

         

 
Figure 1. Percentage inhibition of Fusarium sp. by bean nodulating rhizobia isolates in indirect confrontation. 
Note: Bars with the same letters (a, b, c, d, e)  are not significantly different at 5% average probability by the Duncan test. 

 

3.3. Results of the Effect of Inoculation with Rhizobia Isolates of Plants Infected with Fusarium sp. on their Growth and the 

Severity of the Phytopathogen   

3.3.1. Results of the Effect of Inoculation of Rhizobia Isolates on the Growth of Infected Bean Plants  

Inoculation resulted in a significant improvement in stem height compared to non-inoculated and non-infected 

controls. Infection of common bean plants with Fusarium sp. significantly reduced the height of the plant stems in 



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some cases. The stem length of plants infected with Fusarium sp. is significantly higher for plants inoculated with 

isolates PvFt3, PvMb1, PvNj5, and PvMb5, with values of 24.3 cm, 23 cm, 22.15 cm, and 21.5 cm, respectively and 

significantly lower for strain ISRA 352 (19.75 cm). The stem length of uninfected plants (F-) is significantly higher 

for plants inoculated with isolate PvFt3 (26.1 cm), followed by isolates PvMb1 (25 cm) and PvNj5 (25.75 cm), with 

no significant difference between them, and significantly lower for strain ISRA 352 (24 cm). Inoculation with PvFt3 

improves the stem growth of infected plants (F+). A significant difference in stem length was observed between plants 

infected with Fusarium sp. and inoculated with PvFt3 (24 cm) and uninfected plants (26.1 cm) inoculated with the 

PvFt3 isolate. An improvement of 1.9 cm was observed in the stems of plants inoculated and infected with Fusarium 

sp. compared to infected control plants. The improvements in stem length of isolates PvMb1, PvNj5, and PvMb5 are 

2.8 cm, 3.2 cm, and 3.5 cm, respectively. Strain ISRA 352 shows no significant difference (Figure 10). Inoculation 

with strain ISRA 352 showed no significant difference between infected and uninfected plants. An improvement in 

stem height of 2.4 cm was observed in inoculated and infected plants. 

  

 
Figure 10. Stem height of common bean plants with rhizobia isolates and infected with Fusarium sp. 

Note: Bars with the same letters (a, b, c, d, e) are not significantly different at 5% average probability by the Duncan test. F-: No 
infection by Fusarium sp.; F+: Infection by Fusarium sp. 

 

The growth of infected and inoculated plants was also investigated by assessing the leaf area of plants infected 

with Fusarium sp. and inoculated with rhizobia. Infection of common bean plants with Fusarium sp. reduced the leaf 

area of infected plants (29.33 cm2) compared to uninfected control plants (33 cm2), but this difference is not 

significant. All rhizobia isolates caused an improvement in leaf area of infected and rhizobia-inoculated plants 

compared to infected and non-inoculated plants, with variation between rhizobia isolates (Figure 11). The leaf area of 

plants infected with Fusarium sp. is highest in plants inoculated with isolate PvFt3 (50.6 cm2), followed by isolates 

PvMb1 (49 cm2), ISRA 352 (42 cm2), PvNj5 (40.66 cm2), and PvMb5 (38 cm2). The leaf area of plants not infected 

with Fusarium sp. is larger for plants inoculated with isolate PvMb1 (55.6 cm2), followed by isolates PvFt3 (50.33 

cm2), IRSA 352 (51 cm2), PvNj5 (43 cm2), and PvMb5 (39 cm2). Slight improvements in leaf area were recorded for 

all isolates, but these improvements were not significant. 

        



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Figure 11. Leaf surface of bean plants infected with Fusarium sp. and inoculated with rhizobia. 

Note: Bars with the same letter (a, b, c, d, e) are not significantly different according to Duncan's test with an average probability 
of 5%. F-: No infection by Fusarium sp.; F+: Infection by Fusarium sp. 

 

3.3.2. Severity of Fusarium sp. on Plants 

The protective effect of the different isolates selected against damping-off disease in common bean plants was 

evaluated by measuring the severity of the disease (Table 3) in seedlings previously inoculated and infected with 

bacterial suspensions. Inoculation with rhizobia (antagonists) has a positive effect on disease reduction compared with 

infected but non-inoculated control plants. No obvious damage was observed in plants inoculated with PvFt3 and 

PvMb1. In other words, these isolates confer resistance to the pathogen. These isolates resulted in a 90% reduction 

in disease. For PvNj5 and PvMb5, 16-25% damage was observed, showing that the isolates reduced disease severity 

by 75%. These isolates are intermediate between resistant and susceptible isolates. For the reference strain ISRA 352, 

26-50% damage was observed. The ISRA 352 strain reduced the disease by 50%. None of the plants showed symptoms 

severe enough to cause yield loss or plant death. 

 

Table 3. Severity of bean plants affected by the damping off of seedlings. 

Isolates Severity range Reduction of the disease (%) 

PvFt3 1 - 5 % 90 

PvMb1 1 - 5 % 90 

PvMb5 16 - 25 % 75 

PvNj5 16 - 25 % 75 

ISRA 352 26 - 50 % 50 

Control 26-50% 50 
Note: 1 - 25%: No visible symptoms; 26 - 50%: Visible symptoms. 

 

4. DISCUSSION 

4.1. In Vitro Biocontrol of Fusarium sp. in Direct and Indirect Confrontation  

Fusarium sp. is a ubiquitous plant pathogen that causes root rot, vascular wilt, and damping-off in a wide range 

of host plants. The isolation steps in the laboratory allowed a fungus to be obtained from the leaves and stems of 

diseased bean plants. Microscopic observations revealed the presence of a septate mycelium with two categories of 

conidia: more or less numerous microconidia and macroconidia. The presence of these two types of spores is 

considered one of the main characteristics for the identification of Fusarium sp. [23]. Fusarium sp. was recovered from 

the stems and leaves of plants showing symptoms of crown rot specific to wilt. Analysis of the morphological 

appearance of the colonies revealed whitish filamentous hyphae. This result is in agreement with the work carried out 



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by Nelson et al. [24] and Hibar et al. [25], who claim that this pigmentation produced by the phytopathogen is 

generally specific to Fusarium oxysporum species. The demonstration of antagonistic activity against bean seedling 

wilt (Fusarium sp.) was carried out in vitro using the direct confrontation technique in Petri dishes containing a PDA 

agar medium. All isolates induced a reduction in pathogenic mycelial growth. The basic mechanisms of such inhibition 

may be due to antibiosis and parasitism. The fact that the fungal colony does not completely colonize the box in the 

presence of the bacteria reflects the ability of the antagonists to produce diffusible substances that limit the mycelial 

growth of the pathogen. According to Nourozian et al. [26], the formation of zones of inhibition between the 

pathogen and the antagonist bacteria in the confrontation test is probably due to the production and diffusion in the 

culture medium of substances with antimicrobial activity produced by the antagonists. In addition, the use of nutrient-

rich media eliminates the possibility of this zone occurring due to a phenomenon of competition for nutrients [20]. 

The results obtained show that the inhibition rate varies from 20.55% to 65.15%. These results are in agreement with 

those of Youagang Gougueu [9], who showed that rhizobia can significantly reduce the growth of Fusarium sp. and 

Rhizoctonia, phytopathogens responsible for the damping-off of bean seedlings. The variability in the rate of 

inhibition is probably due to both the concentration of the diffusible substance produced by the antagonist isolate 

[27] and the nature of this substance [28]. Rhizobia inhibit Fusarium sp. in indirect confrontation with inhibition 

rates varying from 30 (PvMb1) to 30.8% (PvFt3). This activity was detected in 25% of the isolates tested. This 

inhibition may be due to antibiosis or to the production of volatile compounds. According to Kai et al. [29], more 

than 200 volatile organic compounds produced by bacteria and molds have been identified. The majority of these 

molecules are involved in multiple interactions and exert antimicrobial or modulatory effects on the growth of 

microorganisms and plants. 

 

4.2. Inoculation Effect of Inhibitory Rhizobial Isolates in Vitro on Bean Growth and Disease Severity 

The results obtained show that the majority of isolates improve bean growth and are able to reduce disease in 

the presence of the pathogen. Disease severity was reduced at rates ranging from 50% to 90%. The isolates PvFt3 

and PvMb1 were the most effective in reducing disease severity, with a reduction of 90%. These results are in 

agreement with those of Asseng et al. [30] and Youagang Gougueu [9] who reported the beneficial effect of 

endophytes such as Rhizobium on plant growth and the reduction in the incidence of fungal diseases. 

The protective activity of isolates is not linked to a single mechanism, but rather to the synergy of several 

parameters [31]. Although the effects of PGPR are divided into two distinct categories: growth-promoting effects 

and growth-promoting biological control effects, there is always a close relationship between the two [32]. PGPRs 

promote overall plant growth, leading to increased disease tolerance in the plant, and conversely, biological control 

of plant diseases by PGPRs can indirectly lead to promotion of plant growth [33]. Numerous studies have shown 

that the protective effect of PGPR is most often the result of the presence of multiple traits that can act directly or 

indirectly on disease development in the plant. 

The reduction in disease severity after treatment with antagonists (rhizobia) is most often accompanied by an 

improvement in plant growth compared to infected controls [34, 35]. The results of the present study confirm these 

observations. A significant improvement (p < 0.05) was observed in the different growth parameters. The stem length 

and leaf area of infected bean seedlings were significantly improved after inoculation with the five selected isolates. 

PvMb1 and PvFt3 isolates showed the best performance compared to the other isolates. In addition to the action of 

antibiotics, volatile or diffusible substances, as the main agents in reducing the growth of pathogens, promoting plant 

growth through the synthesis of growth regulators and making mineral elements available to the plant, can also 

contribute indirectly to preventing the installation of the pathogen in the rhizosphere of the plant [10, 32]. 

 



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5. CONCLUSION  

The use of PGPR for the sustainable improvement of agricultural production is becoming increasingly common 

in agriculture. The aim of this study was to contribute to the development of biological control of Fusarium sp., the 

causal agent of damping-off in common bean, through the use of rhizobia. Eight isolates from the collection of the 

Plant Biology Laboratory were tested for their ability to synthesize lytic enzymes. The results obtained showed that 

all isolates were positive in the cellulase, chitinase, and protease production tests, but 50% of the isolates were positive 

in the phosphatase production test. The in vitro antagonistic activity of the eight bean-nodulating isolates against 

Fusarium sp. showed an inhibitory effect on the growth of the mycelium of Fusarium sp. in direct and indirect 

confrontation on Petri dishes. The inhibition rate varied from 20.55% to 65.15% in direct confrontation and from 30% 

to 30.8% in indirect confrontation. It seems that the most effective mechanism of inhibition of Fusarium sp. by rhizobia 

is the production of diffusible substances. The effect of rhizobial inoculation of infected plants on the growth and 

severity of Fusarium sp. showed a clear improvement in stem length and leaf area of bean plants and a reduction in 

damage caused by the pathogen. The best isolates in the whole study are PvFt3 and PvMb1. 

 

Funding: This study received no specific financial support. 
Institutional Review Board Statement: Not applicable. 
Transparency: The authors state that the manuscript is honest, truthful, and transparent, that no key aspects 
of the investigation have been omitted, and that any differences from the study as planned have been clarified. 
This study followed all writing ethics. 
Competing Interests: The authors declare that they have no competing interests. 
Authors’ Contributions: All authors contributed equally to the conception and design of the study. All 
authors have read and agreed to the published version of the manuscript. 

 

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