







































 

 

 
123 

© 2025 Conscientia Beam. All Rights Reserved. 

Effect of selected microbial isolates from maize rhizosphere on disease incidence and severity 
of maize common smut disease caused by Ustilago maydis   

 

 

 Rosemary Osendi 
Opwondi1 

 David Mutisya 
Musyimi2 

 Phoebe Anyango 
Sikuku3+ 

1,2,3Department of Botany, School of Physical and Biological Sciences, Maseno 
University, Private Bag, Maseno, Kenya. 
1Email: rosemaryopwondi@gmail.com 
2Email: dmusyimi@maseno.ac.ke 
3Email: sphoebe@maseno.ac.ke  

(+ Corresponding author) 

 ABSTRACT 
 
Article History 
Received: 11 May 2025 
Revised: 7 July 2025 
Accepted: 16 July 2025 
Published: 21 July 2025 
 

Keywords 
Antagonistic 
Biocontrol 
Growth inhibition 
Incidence 
Maize 
Rhizosphere 
Severity 
Ustilago maydis. 

 
The study was conducted to evaluate the effect of selected microbial isolates from the 
maize rhizosphere on the incidence and severity of maize common smut disease, which 
causes a decline in maize production. The rhizosphere contains microorganisms with 
potential antagonistic activities against microbial diseases. However, there is limited 
information regarding their use as biocontrol agents. The maize varieties Dk 8033 and 
Duma 43 were used because they have a short maturity period, are recently released, 
and are highly preferred by farmers. In-vitro screening of microbial isolates against 
Ustilago maydis was conducted using the dual culture method in a completely 
randomized design, with three replicates. Greenhouse experiments followed a 
completely randomized design with ten treatments, also in triplicate. Data on growth 
inhibition, disease incidence, and severity were collected. Significant differences (P ≤ 
0.05) were observed among isolates. The highest in-vitro inhibition was recorded by 
fungal isolate MF14 (22.0 mm, unidentified), followed by Serratia sp. (19.0 mm), Bacillus 
sp. (16.0 mm), and Aspergillus sp. (15.0 mm). In greenhouse trials, MF14 showed the 
lowest disease incidence (49.3%), while Serratia sp. had the highest (84.3%). These 
findings highlight the potential of selected microbial isolates, especially MF14, as 
promising biocontrol agents for managing maize common smut. 
 

Contribution/Originality: There is limited information regarding maize rhizospheric microflora, which may be 

important in identifying biocontrol potential against Ustilago maydis, a fungal pathogen of maize. Therefore, this 

study aims to evaluate disease incidence and severity using microbial isolates from the maize rhizosphere as 

biocontrol agents to identify potential biocontrol agents against Ustilago maydis, with the goal of increasing maize 

production. 

 

1. INTRODUCTION 

The rhizosphere refers to the soil zone immediately surrounding plant roots, characterized by increased 

microbial activity [1]. Studies have identified this area as a hotspot of microbial diversity, with plant roots 

supporting a wide range of microbial communities [2, 3]. The release of nutrients through root exudates 

contributes to the development of a dynamic, nutrient-enriched environment in the rhizosphere. In maize, seeds are 

known to release various compounds, including amino acids, sugars, and weak organic acids that influence the 

chemical properties of the adjacent soil [4]. 

Current Research in Agricultural Sciences 
2025 Vol. 12, No. 2, pp. 123-134 
ISSN(e): 2312-6418 
ISSN(p): 2313-3716 
DOI: 10.18488/cras.v12i2.4308 
© 2025 Conscientia Beam. All Rights Reserved. 

 
 
 

 
 
 
 

 

 
 
 
 

mailto:rosemaryopwondi@gmail.com
mailto:dmusyimi@maseno.ac.ke
mailto:sphoebe@maseno.ac.ke
https://orcid.org/0009-0007-8506-4757
https://orcid.org/0000-0002-8729-5870
https://orcid.org/0009-0005-7166-6904
https://www.doi.org/10.18488/cras.v12i2.4308


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124 

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The rhizosphere plays a vital role in protecting plant roots from pathogenic attacks by serving as a frontline 

defense zone [5]. Microorganisms that inhabit this region have demonstrated potential as effective biocontrol 

agents. According to Hong et al. [6] soil microbes can influence plant health by either stimulating, inhibiting, or 

completely suppressing the growth of soil-borne pathogens. This antagonistic activity has been well documented in 

bacterial genera such as Pseudomonas, Burkholderia, and Bacillus, as well as fungal genera like Trichoderma. 

Pathogens are subject to microbial antagonism during both the initial and later stages of infection [7]. However, 

there is limited research on the antagonistic capabilities of maize rhizosphere microorganisms, particularly in 

Kenya, against plant pathogens such as Ustilago maydis. 

Biological control offers a promising approach to managing plant pathogens, but its effectiveness depends on 

selecting microbial agents that are adapted to the local environments where maize is cultivated [8]. The large-scale 

introduction of non-native microbes can disturb native microbial communities and potentially alter the ecological 

balance within the rhizosphere [8]. In some cases, these introduced organisms may unintentionally affect non-

target species, including beneficial microbes, alongside the intended pathogens. This highlights the importance of 

exploring native microbial populations associated with maize roots to identify environmentally compatible and 

effective biocontrol candidates. 

Plant disease incidence (percentage of infected plants), often based on visual symptoms of disease, and disease 

severity (percentage of leaf covered or affected) are important in the development of disease control methods. 

Therefore, in this study, there was a need to quantify disease incidence and severity while using microbial 

antagonists from the rhizosphere of maize varieties DK 8033 and Duma 43 as biocontrol agents against Ustilago 

maydis. 

Ustilago maydis is a fungal pathogen specific to maize and the causal agent of the maize disease, common smut 

[9, 10]. The continued cultivation of maize on the same land without rotation encourages the persistence and 

spread of this disease, as the pathogen's teliospores can survive in soil and crop residues for several years. This 

study highlights the potential of microbial isolates as biocontrol agents against U. maydis, offering promising 

prospects for improving maize production and supporting the country’s agricultural economy. Employing these 

microbial antagonists presents an eco-friendly and sustainable alternative to existing disease management 

strategies. 

 

2. MATERIALS AND METHODS 

2.1. Study Site 

The experiment was conducted between April 2023 and July 2024 at Maseno University, utilizing the Botany 

Department’s microbiology laboratory, greenhouse, and the university research farm. The greenhouse conditions 

included daytime temperatures of 25℃–40℃, nighttime temperatures of 20℃–30℃, a 14/10-hour photoperiod, 

and relative humidity ranging from 70% to 90%. Maseno was selected to ensure uniform environmental conditions 

and to safeguard crop growth. Geographically, the site is situated at 0°10′0″ S, 34°36′0″ E along the Kisumu–Busia 

Road, at an elevation of 1,503 meters (4,934 feet) above sea level. The region experiences both long and short rainy 

seasons, with an annual average rainfall of 1,750 mm and a mean temperature of 28.7℃ [11]. 

 

2.2. Pathogen Isolation  

A collection of maize smut gall samples was randomly conducted from naturally infected maize plants at the 

end of the growing season at Maseno University farm in 2023. Potato dextrose agar and 20% carrot solution were 

used to obtain pure cultures of Ustilago maydis and for propagation of sporidia (basidiospores), respectively. The 

galls were chopped, and teliospores were separated from the gall tissues by sieving through a tea strainer. The 

teliospores were surface-sterilized by immersion in 1% copper sulfate solution for 20-60 seconds and filtered 

through two layers of sterile cheesecloth, which prevented the teliospores from passing through. Subsequently, the 



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teliospores on the cheesecloth were washed in three changes of sterile distilled water and dried on sterile filter 

paper, then transferred onto PDA supplemented with antibiotics (streptomycin sulfate) in petri dishes. The dishes 

were incubated at 25°C for 4-5 days until sporidia of U. maydis emerged. When the sporidia reached about a pinhead 

size, they were taken from cultures and transferred into 500 ml Erlenmeyer flasks containing 20% sterile carrot 

solution, then incubated at 25°C for 7 days to facilitate sporidia multiplication. The flasks were shaken once or twice 

a week. For inoculum preparation, basidiospore suspensions in the Erlenmeyer flasks were stirred to achieve a 

homogeneous solution, and basidiospores were counted using a hemocytometer. The suspensions were diluted to 

appropriate concentrations with sterile carrot solutions and adjusted to 4 × 106 sporidia per ml. Similarly, teliospore 

suspensions were prepared at 1×106 teliospores per ml and added to the basidiospore suspensions [12].  

 

2.3. Pathogenicity Test of Ustilago Maydis Isolate 

Pathogenicity assays were conducted under greenhouse conditions at Maseno University using a completely 

randomized design with three replicates, following a modified protocol adapted from Aydoğdu and Boyraz [12]. 

Two seeds of each maize variety, DK 8033 and Duma 43, were manually planted in 15 cm diameter pots filled with 

sterilized topsoil collected from the university’s research farm. Di-ammonium phosphate (DAP) fertilizer was 

applied at planting at a rate of 1.5 g per pot. Top dressing was carried out at the second leaf stage using calcium 

ammonium nitrate (CAN) at 2.5 g per pot. When plants reached a height of 40–60 cm, 2 ml of prepared inoculum 

comprising 4 × 10⁴ sporidia/ml and 1 × 10⁶ teliospores/ml was injected into the apical node using a sterile 

hypodermic syringe. Disease development was assessed by monitoring the appearance of characteristic U. maydis 

symptoms 15 days post-inoculation. 

 

2.4. In-Vitro Antagonism Assays to Evaluate the Ability of Microbial Isolates from the Rhizosphere to Inhibit Growth of 

Ustilago Maydis  

These experiments were conducted in the Botany Department laboratory at Maseno University. Fungal 

isolates were tested using a dual culture assay on PDA medium, following the methods outlined in Alwathnani and 

Perveen [13] and Dhanya et al. [14]. Plugs (5 mm in diameter) cut from five-day-old fungal cultures of both the 

pathogen (U. maydis) and the test fungi (potential antagonists) were excised using a sterile cork borer. Antagonist 

plugs were positioned approximately 2 cm inward from the edge of the Petri dish, while a second 5 mm plug of U. 

maydis was inoculated on the same plate. Plates were incubated at 25 ± 2 °C, and pathogen-only plates served as 

controls. The experiment was arranged in a completely randomized design, replicated three times. Inhibition zones 

were measured after seven days of incubation. 

For bacterial isolates, the antagonists were line-streaked 5 cm away from the fungal pathogen plug on the same 

PDA plate, using a sterile loop, following the method of Dhanya et al. [14]. This experiment also followed a 

completely randomized design, replicated three times. After incubation at 28 ± 2 °C for seven days, inhibition was 

recorded as the distance between bacterial growth and the pathogen. Based on inhibition results, two fungal and 

two bacterial isolates with the strongest antagonistic effects were selected for further greenhouse experiments. 

 

2.5. In- Vivo Greenhouse Experiment to Evaluate the Effect of Selected Microbial Isolates on Disease Incidence and Severity of 

Maize Common Smut Disease  

2.5.1. Experimental Design and Set-Up 

The experiment was conducted following the procedures described in Frommer et al. [15] and Juma et al. 

[16]. The microbial isolates selected for evaluation included two bacterial strains, MB2 (Serratia sp.) and NB16 

(Bacillus sp.) and two fungal isolates, MF13 (Aspergillus sp.) and MF14 (unidentified). These isolates were chosen 

based on their demonstrated antagonistic efficacy against Ustilago maydis in preliminary in vitro assays. Plastic pots 

(30 × 30 cm, 20 L capacity) with drainage holes were sterilized using a sodium hypochlorite solution and filled with 



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steam-sterilized topsoil collected from the Maseno University farm. Seeds of the maize varieties DK 8033 and 

Duma 43 were sown manually at a depth of 5 cm. Di-ammonium phosphate (DAP) fertilizer was applied at planting 

at a rate of 1.5 g per pot. Each pot received two seeds, which were later thinned to one seedling. The pots were 

spaced 1 m apart and arranged in a completely randomized design, with treatments replicated three times for both 

maize varieties. Top dressing was performed at the second leaf stage using calcium ammonium nitrate (CAN) at a 

rate of 2.5 g per pot. Manual weeding was conducted twice during the crop growth period. 

 

2.5.2. Inoculation and Pathogenicity Tests 

The experiment was conducted based on the procedures described in Juma et al. [16] and Frommer et al. [15] 

One month after planting, maize plants were inoculated with Ustilago maydis by injecting 2 ml of spore suspension 

near the leaf whorls. Antagonistic cultures Serratia sp., Bacillus sp., Aspergillus sp., and MF14 were cultured on 

potato dextrose agar (PDA) at 25 °C for 14 days (for fungi) and on nutrient agar (NA) at the same temperature for 

48 hours (for bacteria). The biomass from these cultures was aseptically scraped using a sterilized spatula, 

suspended in sterile distilled water, and filtered through nylon mesh. Spore and bacterial concentrations were 

adjusted to 1.0 × 10⁶ spores/ml and 1.0 × 10⁴ colony-forming units (cfu)/ml, respectively, using a hemocytometer. 

Suspensions were pipetted into hemocytometer chambers using sterile tips, and spore or cell counts were made 

under a microscope using standard grid volumes (0.1 µl) to estimate concentration per milliliter. 

Infected maize plants were then treated by injecting the respective microbial suspensions, while control plants 

received sterile distilled water. All treatments were replicated three times and arranged in a completely randomized 

design. Plants were observed daily for disease progression, with particular attention to gall formation, which 

typically became visible three weeks post-inoculation. Soil was watered daily to maintain adequate moisture 

throughout the experimental period. 

Table 1 presents the layout of the research, which consists of ten treatments (i.e., DK 8033 seedling inoculated 

with Serratia sp. (MB2) + Ustilago maydis, DK 8033 seedling inoculated with Bacillus sp. (NB16) + Ustilago 

maydis, Duma 43 seedling inoculated with Serratia sp. (MB2) + Ustilago maydis, Duma 43 seedling inoculated with 

Bacillus sp. (NB16) + Ustilago maydis, DK 8033 seedling inoculated with Aspergillus sp. (MF13) + Ustilago 

maydis, DK 8033 seedling inoculated with MF14 (fungal isolate) + Ustilago maydis, Duma 43 seedling inoculated 

with Aspergillus sp. (MF13) + Ustilago maydis, Duma 43 seedling inoculated with MF14 (fungal isolate) + 

Ustilago maydis, DK 8033 seedling inoculated with sterile distilled water + Ustilago maydis (control), and Duma 

43 seedling inoculated with sterile distilled water + Ustilago maydis (control). Each treatment was replicated three 

times. 

 

Table 1. Treatments in the greenhouse experiment. 

Treatment  Description  

N + MB2 DK 8033 inoculated with Serratia sp. (MB2) + Ustilago maydis 
N + NB16 DK 8033 inoculated with Bacillus sp. (NB16) + Ustilago maydis 

M + MB2 Duma 43 inoculated with Serratia sp. (MB2) + Ustilago maydis 
M + NB16 Duma 43 inoculated with Bacillus sp. (NB16) + Ustilago maydis 
N + MF13 DK 8033 inoculated with Aspergillus sp (MF13) + Ustilago maydis 
N + MF14 DK 8033 inoculated with MF14 (fungal isolate) + Ustilago maydis 
M + MF13 Duma 43 inoculated with Aspergillus sp. (MF13) + Ustilago maydis 
M + MF14 Duma 43 inoculated with MF14 (Fungal isolate) + Ustilago maydis 
N + Control DK 8033 inoculated with sterile distilled water + Ustilago maydis (Control) 
M + Control Duma 43 inoculated with sterile distilled water + Ustilago maydis (Control) 

 

2.5.3. Disease Incidence 

Fifteen days post-inoculation, the incidence of smut disease (%) was assessed. Data collection involved 

evaluating all treatments and the control by recording the number of symptomatic plants. Disease symptoms 



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included leaf chlorosis, shoot stunting, dwarfism, morphological deformities such as plant slanting, the formation of 

small galls on leaves, stems, and basal regions, tassel galls, and plant mortality [17, 18]. 

DI     =          
Total no.of diseased plants

Total no.of observed plants
× 100 

Where DI is disease incidence. 

 

2.5.4. Disease Severity 

Severity of common smut symptoms was evaluated using a rating scale below, based on the percentage of 

plants exhibiting galls or typical symptoms of Ustilago maydis, as described by Frommer et al. [15]. 

1: No symptom. 

2:1-3% of the plants are infected (One leaf has symptoms). 

3: 4-10% of the plants are infected (Two leaves have symptoms). 

4: 11-25% of the plants are infected (Three leaves have symptoms). 

5: 25-50% of the plants are infected (Four leaves have symptoms). 

6: 51-75% of the plants are infected (Four leaves and tassel have symptoms). 

7: 76-100% of the plants are infected (Five leaves and tassel have symptoms). 

 

2.6. Data Analysis 

The collected data were analyzed statistically using SAS version 9.1 software to evaluate the effects of fungal 

and bacterial antagonist treatments on the inhibition zone of Ustilago maydis, as well as smut disease incidence and 

severity under greenhouse conditions. Treatment means were compared using the Least Significant Difference 

(LSD) test at a significance level of P ≤ 0.05. 

 

3. RESULTS  

3.1. In-Vitro Antagonism Assays to Evaluate the Ability of Microbial Isolates from the Rhizosphere to Inhibit Growth of 

Ustilago Maydis 

3.1.1. Bacteria  

The bacterial antagonists tested exhibited varying degrees of inhibition against Ustilago maydis, as indicated by 

the different sizes of growth inhibition zones (Table 2). Isolate MB2 (Serratia sp.) produced the largest inhibition 

zone (19.0 mm), followed by NB16 (Bacillus sp.) with 16.0 mm. MB2 demonstrated a statistically significant 

difference from all other bacterial isolates (P ≤ 0.05). Additionally, isolates MB2, NB16, and NB17 showed 

significantly greater inhibition compared to NB22, NB25, MB12, MB3, MB6, NB18, NB23, MB9, MB4, and MB1. 

However, no significant differences were observed among NB16, NB17, NB19, NB24, and MB8. The smallest 

inhibition zones were recorded for isolates NB23, MB9, and MB4, with no significant differences among them. 

 

Table 2. Zones of growth inhibition (mm) of bacterial isolates from the rhizosphere of maize against Ustilago maydis. 

Bacterial isolate + Pathogen Zone of inhibition (mm) 

MB2 19.00ᵃ 
NB16 16.00ᵇ 
NB17 16.00ᵇ 
NB19 15.67ᵇᶜ 
NB24 15.00ᵇᶜ 
MB8 14.33ᵇᶜᵈ 
NB22 14.00ᶜᵈ 
NB25 14.00ᶜᵈ 
MB12 14.00ᶜᵈ 
MB3 14.00ᶜᵈ 



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Bacterial isolate + Pathogen Zone of inhibition (mm) 

MB6 14.00ᶜᵈ 
NB18 13.00ᵈᵉ 
NB23 12.00ᵉ 
MB9 12.00ᵉ 
MB4 11.67ᵉ 
MB1 5.00ᶠ 
Mean 9.15 
LSD 1.93 
P. value < 0.0001 
% C.V. 12.81 
Note: a,b,c,d,e,f, means that in a column having the same superscript letter(s) do not differ significantly at p ≤ 0.05. Means followed by different superscript 

letters along the column are significantly different at p ≤ .05. Means with more than one letter down the columns are intermediates. Values are means 
of three replications. C.V: Coefficient of variation, LSD: Least significant difference, P. value: Probability value, M: Duma 43 Maize variety, N: DK 
8033 Maize variety, B: Bacteria. 

 

3.1.2. Fungi 

The fungal isolates demonstrated varying antagonistic activity against Ustilago maydis. Among these, MF14 

(unidentified) and MF13 (Aspergillus sp.) were the most effective, producing inhibition zones of 22.0 mm and 15.0 

mm, respectively. Both isolates were significantly more inhibitory than the others (P ≤ 0.05) (Table 3). Isolates 

MF6, MF3, and NF21 did not differ significantly in their suppressive effects on the pathogen. Similarly, MF3, 

NF21, MF17, MF1, and MF12 also showed no statistically significant differences among themselves. 

 

Table 3. Zone of growth inhibition (mm) of fungal isolates from the rhizosphere of maize against Ustilago maydis 

Fungal isolate and pathogen Zone of inhibition (mm) 

MF14 22.000ᵃ 
MF13 15.000ᵇ 
MF5 8.667ᶜ 
MF3 7.000ᶜᵈ 
NF21 6.000ᶜᵈᵉ 
MF17 4.000ᵈᵉ 
MF1 2.667ᵉ 
MF11 2.333ᵉ 
Mean 8.46 
LSD 4.11 
P value < 0.0001 
%C. V 28.04 
Note: a,b,c,d,e, means in a column having same superscript letter(s) do not differ significantly at p ≤ 0.05. Means followed by different superscript letters 

down the column indicate significant differences at p ≤ .05. Means with more than one letter down the columns are intermediates. Values are means of 
three replications. C.V: Coefficient of variation, LSD: Least significant difference, P. value: Probability value, M: Duma 43 Maize variety, N: DK 8033 
Maize variety, F- Fungi. 

 

3.2. In- Vivo Greenhouse Experiment to Evaluate the Effect of Selected Microbial Isolates on Disease Incidence and Severity of 

Maize Common Smut Disease 

3.2.1. Disease Incidence 

A greenhouse experiment was conducted to assess the effect of selected microbial isolates on the incidence of 

common smut disease (Ustilago maydis) in maize. Four microbial antagonists, Bacillus sp., Serratia sp., Aspergillus sp., 

and MF14 (an unidentified fungal isolate) were evaluated for their biocontrol potential on two maize varieties: 

Duma 43 (coded as "M") and DK 8033 (coded as "N") (Table 4). Among the treatments, the fungal isolate MF14 

applied to the DK 8033 variety (N-MF14) resulted in the lowest disease incidence (49.3%), indicating the highest 

level of disease suppression. This was followed by M-MF14 (53.03%), demonstrating that MF14 was effective in 

both maize varieties, with slightly better performance in DK 8033. The bacterial isolate, Bacillus sp., showed 

moderate biocontrol efficacy, with disease incidences of 81.1% (N-NB16) and 84.3% (M-NB16). These values, 

although significantly lower than the controls, indicate a less potent suppression effect compared to MF14. 



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Aspergillus and Serratia sp. also showed moderate disease suppression, with incidences ranging from 63.93% to 

67.80%, and no significant differences between the two maize varieties. Notably, no significant difference was 

observed among treatments N-MF13, N-MB2, M-MF13, and M-MB2, suggesting comparable biocontrol potential 

for these isolates (Table 4).  

 

Table 4. Disease incidence in maize plants following treatment with selected microbial isolates from the rhizosphere of DK 8033 and Duma 43 
maize varieties. 

Treatment Disease incidence (%) 

N- DD (Control) 100.00ᵃ 
N- NB16 81.10ᶜ 
N- MF13 65.53ᵈᵉ 
N- MF14 49.30ᵍ 
N- MB2 66.13ᵈᵉ 
M- DD (Control) 100.00ᵃ 
M- NB16 84.30ᵇ 
M- MF13 63.93ᵉ 
M- MF14 53.03ᶠ 
M- MB2 67.80ᵈ 
Mean  73.113 
LSD 2.733 
P .value <0.0001 
% C. V 2.2 
Note: a,b,c,d,e,f,g means in a column having same superscript letter(s) do not differ significantly at p ≤ 0.05. Means with more than one letter down the 

columns are intermediates. Values are means of three replications. C.V: Coefficient of variation, LSD: Least significant difference, P. value: Probability 
value, DD: Distilled water, M: Duma 43 maize variety, N: DK 8033 maize variety, NB16: Bacillus sp., MF13: Aspergillus sp., MF14: unidentified 
isolate, MB2: Serratia sp.    

 

3.2.2. Disease Severity 

The control treatments (M-DD and N-DD), which involved no microbial intervention, recorded the highest 

disease severity (100%), confirming the full susceptibility of both maize varieties in the absence of biological control. 

All microbial treatments significantly reduced disease severity (p < 0.05) compared to the controls. Among the 

microbial antagonists, the fungal isolate MF14 demonstrated the highest efficacy, particularly in the DK 8033 

variety (N-MF14), where it reduced disease severity to 49.30%, followed closely by M-MF14 at 53.03%. These 

values represent a substantial suppression of disease symptoms and highlight MF14 as the most promising 

biocontrol agent in this study. The bacterial isolate NB16 (Bacillus sp.) showed moderate disease suppression, with 

disease severity of 81.10% (N-NB16) and 84.30% (M-NB16). While these treatments significantly outperformed the 

controls, they were less effective compared to the MF14 isolate. The bacterial isolate MB2 (Serratia sp.) and the 

fungal isolate MF13 (Aspergillus sp.) produced intermediate levels of disease suppression. Their severity values 

ranged between 63.93% and 67.80%, with no significant difference among N-MB2, M-MB2, N-MF13, and M-MF13 

treatments. This suggests comparable biocontrol potential for these two antagonists across both maize varieties 

(Table 5). 

 

Table 5. Disease severity in maize plants after treatment with selected microbial isolates from the rhizosphere of Duma 43 and DK 8033 maize 
varieties. 

Treatment Disease severity (%) 

N- DD (Control) 100.00ᵃ 
M- DD (Control) 100.00ᵃ 
M- NB16 84.30ᵇ 
N- NB16 81.10ᶜ 
M- MB2 67.80ᵈ 
N- MB2 66.13ᵈᵉ 
N- MF13 65.53ᵈᵉ 
M- MF13 63.93ᵉ 



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Treatment Disease severity (%) 

M- MF14 53.03ᶠ 
N- MF14 49.30ᵍ 
Mean 73.113 
LSD 2.733 
P. Value < 0.0001 
% C. V 2.2 
Note: a,b,c,d,e,f,g, means followed by different superscript letters along the column are significantly different at P < 0.05. Values represent the mean of three 

replicates. Means with more than one letter down the columns are intermediates.  C.V: Coefficient of variation, LSD: Least significant difference, P. 
value: Probability value, DD: Distilled water, M: Duma 43 Maize variety, N: DK 8033 Maize variety, NB16: Bacillus sp., MF13: Aspergillus sp., MF14: 
unidentified isolate, MB2: Serratia sp.    

 

4. DISCUSSION  

4.1. In-Vitro Antagonism Assays to Evaluate the Ability of Microbial Isolates from the Rhizosphere to Inhibit Growth of 

Ustilago Maydis  

4.1.1. Bacteria  

Bacterial isolates obtained from the rhizosphere of maize exhibited statistically significant antagonistic activity 

against Ustilago maydis, as evidenced by distinct zones of growth inhibition (Table 2). The inhibition zones ranged 

from 5.00 mm to 19.00 mm (p < .0001). Among the isolates, MB2 (Serratia sp.) demonstrated the highest antifungal 

activity (19.00 mm), followed by NB16 and NB17, both with zones of 16.00 mm. 

These findings suggest that the ability to suppress U. maydis varies among isolates, likely due to differences in 

the type and quantity of bioactive metabolites they produce. This observation aligns with earlier studies 

highlighting the biocontrol potential of rhizosphere-associated bacteria. For example, Serratia spp. are known 

producers of chitinases and other antifungal enzymes capable of degrading fungal cell walls [2]. The strong 

inhibition observed for Serratia sp. in this study supports previous reports and highlights its potential as an effective 

biocontrol agent. Additionally, several Bacillus species are recognized for producing antibiotics and bioactive 

compounds that inhibit fungal growth. 

The current findings are consistent with prior research indicating that Bacillus amyloliquefaciens can reduce 

fungal contamination in post-harvest grains and enhance plant resistance when applied as a seed coating [19-21]. 

Other isolates, including NB19, NB24, and MB8, showed moderate antifungal activity, with inhibition zones 

ranging from 14.33 mm to 15.67 mm. In contrast, MB1 (Enterobacter sp.) exhibited minimal activity (5.00 mm), 

significantly lower than the group mean of 9.15 mm. This variation further supports the hypothesis that 

antagonistic efficiency is linked to the metabolic diversity of bacterial isolates. Comparable conclusions have been 

drawn in studies involving Pseudomonas fluorescens and Bacillus spp., where antifungal efficacy differed according to 

each strain's capacity to produce secondary metabolites such as hydrogen cyanide, siderophores, and lipopeptides 

[22, 23]. These results are also consistent with observations by Ab Rahman et al. [24] who reported that bacterial 

biocontrol agents act either directly via antimicrobial secretion or indirectly by inducing systemic resistance in 

plants. 

 

4.1.2. Fungi 

Fungal isolates from the maize rhizosphere exhibited antagonistic activity against Ustilago maydis, with 

inhibition zones ranging from 2.33 mm to 22.00 mm (Table 3). Isolate MF14 demonstrated the strongest inhibition 

(22.00 mm, p < .0001), followed by MF13 (15.00 mm). Moderate activity was observed in MF5 (8.67 mm) and MF3 

(7.00 mm), while MF1 (2.67 mm) and MF11 (2.33 mm) were the least effective, both below the group mean (8.46 

mm). The high LSD (4.11 mm) and coefficient of variation (28.04%) reflect significant differences among isolates. 

The strong activity of MF14 aligns with prior reports on Trichoderma and Aspergillus spp., which produce 

antifungal enzymes such as chitinases and proteases [25, 26]. These findings are consistent with studies showing 

that soil fungi can inhibit plant pathogens [27, 28]. Similar inhibitory effects by Aspergillus and Penicillium species 



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against other fungal pathogens further support the potential of maize rhizosphere fungi as biocontrol agents [13, 

29]. 

 

4.2. In- Vivo Greenhouse Experiment to Evaluate the Effect of Selected Microbial Isolates on Disease Incidence and Severity of 

Maize Common Smut Disease  

4.2.1. Disease Incidence 

In the greenhouse experiment, selected rhizospheric microbial isolates NB16 (Bacillus sp.), MF13 (Aspergillus 

sp.), MF14 (unidentified fungal isolate), and MB2 (Serratia sp.) demonstrated varying levels of effectiveness in 

reducing the incidence of common smut in two maize varieties: DK 8033 and Duma 43 (Table 4). Control plants 

treated with distilled water (N-DD and M-DD) showed 100.00% disease incidence, while all microbial treatments 

significantly reduced disease incidence (p < .0001). Isolate MF14 provided the greatest suppression, reducing 

incidence to 49.30% in DK 8033 and 53.03% in Duma 43 well below the group mean of 73.11%. MF13 followed, 

with reductions to 65.53% and 63.93%, respectively. NB16 and MB2 showed moderate efficacy, with incidence 

levels ranging from 66.13% to 84.30%, depending on the maize variety. 

These results align with previous findings, such as those by Cheng et al. [27] showing fungal isolates 

effectively suppressing smut diseases in cereals. The differential efficacy among isolates likely reflects variations in 

their production of antifungal compounds, including enzymes, siderophores, and secondary metabolites. Prior 

studies by Dutta et al. [30] have reported that rhizospheric microbes employ diverse antagonistic strategies, such 

as rapid root colonization, acidification, and antimicrobial secretion. Additionally, Serratia spp. are known to inhibit 

fungal pathogens through antifungal compound production and biofilm formation, further supporting their role in 

disease management [30]. 

 

4.2.2. Disease Severity  

The selected microbial isolates NB16 (Bacillus sp.), MF13 (Aspergillus sp.), MF14 (unclassified fungal isolate), 

and MB2 (Serratia sp.) significantly reduced disease severity caused by Ustilago maydis under greenhouse conditions 

(Table 5). All treatments demonstrated a significant reduction in severity compared to untreated controls, 

confirming their biocontrol potential. Variations in suppression levels likely reflect differences in the types and 

quantities of bioactive secondary metabolites produced by each isolate. 

These results align with previous studies showing that Bacillus and Aspergillus spp. mitigate fungal disease 

severity through the secretion of hydrolytic enzymes such as chitinases, glucanases, and proteases, which degrade 

fungal cell walls [26, 31]. For instance, Bacillus subtilis strains have been shown to suppress maize pathogens 

through the enzymatic breakdown of pathogenic structures [31] while Aspergillus spp. reduce soilborne diseases 

through both enzymatic and metabolite-mediated mechanisms [26]. Furthermore, the results are consistent with 

findings in other crops where rhizospheric Serratia spp. and fungal isolates reduced disease severity through 

antimicrobial production and induction of systemic resistance [32].  

Collectively, these findings support the role of microbial antagonists as effective and sustainable biocontrol 

agents in maize disease management. 

 

5. CONCLUSION AND RECOMMENDATION  

Maize plant diseases such as common smut remain a significant threat to maize cultivation, affecting both 

smallholder farmers' food security and their income. This study aimed to evaluate the effect of selected microbial 

antagonists on the incidence and severity of maize common smut disease. The fungal and bacterial isolates exhibited 

potential antagonistic activities against the pathogen, Ustilago maydis, from the in vitro experiment, displaying them 

as potential microbial biocontrol agents. In vivo studies to control common smut in the greenhouse revealed that 

the four selected potential antagonists, MF13 (Aspergillus fumigatus), MF14 (unidentified), NB16 (Bacillus sp), and 



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MB3 (Serratia sp), had varying levels of disease control regarding incidence and severity data recorded. MF13, 

MF14, NB16, and MB3 may therefore be recommended for use as potential biocontrol agents against Ustilago 

maydis after further exploring their performance on the best inoculation method, formulation, and ecological fitness. 

Further studies should focus on identifying the most effective antagonistic strain concerning the timing of 

application, inoculation method, and ecological fitness in an effort to control maize smut disease. 

 

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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