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Revista mexicana de fitopatología

versión On-line ISSN 2007-8080versión impresa ISSN 0185-3309

Rev. mex. fitopatol vol.41 no.2 Texcoco may. 2023  Epub 11-Ago-2023 

Artículos científicos

Antagonistic bacteria against Fusarium spp. isolated from sclerotia of Claviceps gigantea in maize (Zea mays)

Ana María Ayala-Torres1 

Sergio Aranda-Ocampo*  1 

Carlos De León-García de Alba1 

Cristian Nava-Díaz1 

Jesús Ricardo Sánchez-Pale2 

1 Postgrado en Fitopatología, Colegio de Postgraduados, Campus Montecillo, Texcoco, Estado de México, CP 56230;

2 Universidad Autónoma del Estado de México, Campus El Cerrillo carretera Toluca-Ixtlahuaca km. 15.5, Piedras Blancas 50200, Toluca de Lerdo, México.


Fungal sclerotia house native bacterial populations of interest for biological control of plant pathogenic fungi. The objectives of this research were: i) to explore the bacterial populations associated with Claviceps gigantea sclerotia in maize from six locations in the State of Mexico, ii) to evaluate the in vitro antagonism of these bacterial populations against three species of Fusarium phytopathogens of maize, iii) to molecularly identify the more efficient antagonists and characterize the in vitro production of metabolites. Bacterial populations in the sclerotia were calculated by direct plate count; in vitro antagonism against Fusarium graminearum, F. sublgutinans, and F. verticillioides was evaluated by confrontation in Waksman agar medium. Antagonistic bacteria were identified by partial sequencing of 16S rRNA gene and evaluated in vitro for the production of indole-3 acetic acid, siderophores, lipolytic and proteolytic activity, and mineral phosphate solubilization. The bacterial density ranged from 2.023 to 2.397 Log10UFC g-1 of sclerotia. Twenty-two strains showed in vitro antagonism against at least one Fusarium species and were identified as members of the genera Bacillus, Delftia, Micromonospora, Pseudomonas, Sphingobacterium, Staphylococcus, and Stenotrophomonas. The 22 antagonists showed in vitro lipolytic, proteolytic, siderophore-producing and phosphate-solubilizing activity; only 12 (55%) produced indole-3-acetic acid. Bacillus subtilis (BA1), Pseudomonas syringae (BA2), and Bacillus amyloliquefaciens (BA18) strains were antagonists against the three Fusarium species and produced all the metabolites evaluated.

Key words: Ear rot; Bacillus; Pseudomonas; inhibition; metabolites


Los esclerocios de algunos hongos albergan bacterias nativas para el control biológico de hongos fitopatógenos. Los objetivos de esta investigación fueron: i) explorar poblaciones bacterianas asociadas a esclerocios de Claviceps gigantea en maíces de seis localidades del Estado de México, ii) evaluar el antagonismo in vitro de estas poblaciones bacterianas contra tres especies de Fusarium fitopatógenos de maíz, iii) identificar molecularmente los antagonistas más eficientes y caracterizar la producción de metabolitos in vitro. Poblaciones bacterianas en los esclerocios se calcularon por conteo directo en placa; el antagonismo in vitro contra Fusarium graminearum, F. sublgutinans y F. verticillioides se evaluó por confrontación en medio Waksman agar. Las bacterias antagonistas se identificaron por secuenciación parcial del gen 16S rRNA y se evaluaron in vitro para la producción de ácido indol-3-acético, sideróforos, actividad lipolítica, proteolítica y solubilización de fosfato mineral. La densidad bacteriana estuvo entre 2.023 a 2.397 Log10UFC g-1 de esclerocio. Veintidos cepas mostraron antagonismo in vitro contra al menos una especie de Fusarium. Se identificaron como miembros de los géneros Bacillus, Delftia, Micromonospora, Pseudomonas, Sphingobacterium, Staphylococcus y Stenotrophomonas. Los 22 antagonistas mostraron in vitro actividad lipolítica, proteolítica, produjeron sideróforos y solubilizaron fosfato; únicamente 12 (55%) produjeron ácido indol-3-acético. Las cepas Bacillus subtilis (BA1), Pseudomonas syringae (BA2) y Bacillus amyloliquefaciens (BA18) fueron antagonistas contra las tres especies de Fusarium y produjeron todos los metabolitos evaluados.

Palabras clave: Pudrición de mazorca; Bacillus; Pseudomonas; inhibición; metabolitos

Zea mays, commonly known as corn, is the most widely produced cereal crop worldwide, with wheat and rice following as close contenders (CIMMYT, 2019). It is worth noting that Mexico is considered the center of origin for corn (Matsuoka et al., 2002) and has the largest annual planted area and per capita consumption of this crop (García-López and Giraldo, 2021; Padrón et al., 2013). However, Mexico’s corn production and grain quality are often compromised by several fungal diseases, among which corn ear rot diseases are particularly prevalent (Moreno-Limón et al., 2011).

Fusarium fungi cause corn ear rot, which poses a significant limitation on the production and safety of corn worldwide. It is reported that most corn hybrids cultivated globally are susceptible to more than 10 Fusarium species causing ear rot (Mesterhazy et al., 2012). These species typically produce mycotoxins that affect not only animal health but also human health (Holf, 2020; Mielniczuk and Skwaryło-Bednarz, 2020). In Mexico, F. verticillioides is the most widely distributed and important species causing ear rot in the states of Guanajuato, Guerrero, Hidalgo, Jalisco, Puebla, and Nuevo Leon. Additionally, F. graminearum and F. subglutinans have also been reported to cause ear rot in the states of Mexico, Michoacan, and Yucatan (Zenteno-Zevada, 1963).

Biocontrol of plant diseases with antagonistic microorganisms is a useful strategy to reduce the use of pesticides in agriculture and lower production costs and environmental impact (Dimkić et al., 2022; Guzmán-Guzmán and Santoyo 2022; Luo et al., 2022; Singh et al., 2022). Previous studies have shown that exudates from the sclerotia of phytopathogenic fungi such as Sclerotium rolfsii and Sclerotinia sclerotiorum stimulate the growth of specific bacterial populations in the same agroecosystem with greater antifungal activity against these same fungi, compared to bacterial strains isolated from other ecological niches (Abdullah et al., 2008; Coley-Smith and Dickenson, 1971; Gilbert and Linderman, 1971; Hou et al., 2006). Bacterial populations that coexist in specific habitats with limited nutrients, such as sclerotia, promote the production of metabolites that increase their ability to compete against other microorganisms. Therefore, sclerotia of fungi are considered potential natural reservoirs of efficient antagonists for the biological control of phytopathogens (Hou et al., 2006; Zachow et al., 2011).

Claviceps gigantea, an ascomycete, causes the disease commonly known as “horse’s tooth” due to the shape of its sclerotia. This fungus produces several alkaloids, mainly ergoline, festuclavine, dihydroelymoclavine, chanoclavine, and elimoclavine, which are also toxic to both animals and humans (Agurell et al., 1963; Solano-Báez et al., 2018; Mielniczuk and Skwaryło-Bednarz, 2020; Bragg et al., 2017; Hof, 2020). C. gigantea is an endemic fungus limited geographically to locations in Mexico with altitudes above 1800 masl and relative humidity ≥ 60% (Fucikovsky and Moreno, 1976; Fuentes et al., 1964; Ullstrup, 1973). In the high valleys of Mexico (>2000 masl), maize is grown in environments with temperate climates, where ear rots caused by Fusarium spp. are common, often occurring simultaneously with C. gigantea, causing losses of up to 100% (CIMMYT, 2004). In this study, we hypothesize that C. gigantea sclerotia harbor bacterial populations with efficient antagonism against Fusarium species causing maize ear rot in Mexico. Our objectives were to: i) explore the bacterial populations associated with C. gigantea sclerotia in the State of Mexico, ii) evaluate the in vitro antagonism of these bacterial populations against Fusarium graminearum, F. subglutinans, and F. verticillioides, iii) molecularly identify the most efficient antagonists and characterize the in vitro production of their metabolites.

Materials and methods

Collecting Claviceps gigantea sclerotia

Sclerotia of C. gigantea were collected from six localities in the State of Mexico, which showed severe history under natural conditions of “horse tooth” disease and ear rot caused by Fusarium spp. (see Table 1 and Figure 1). For each locality, sclerotia were collected from C. gigantea-infected ears without symptoms of rot caused by Fusarium spp.

Table 1 Collection of C. gigantea sclerotia in localities of the State of Mexico. 

zLocalidad Latitud (N) Longitud (O) Altitud (msnm)
Almoloya de Juárez 19° 14´ 10” 90° 42´ 07” 2600
Atlacomulco 19° 43´ 37” 99° 42´ 12” 2700
Calimaya 19° 10´ 25” 99° 32´ 12” 2680
Mina México 19° 40´ 35” 99° 40´ 10” 2580
Toluca 18° 59´ 00” 99° 40´ 58” 2600
Villa Victoria 19° 26´ 00” 100° 00´ 00” 2570

z Sample size of 10 sclerotia per locality estimated with Cochran’s model (1982).

Figure 1 A) Corn ears with simultaneous infection of Claviceps gigantea and Fusarium spp. B) Claviceps gigantea sclerotia on corn ears. 

Isolation of bacteria from Claviceps gigantea sclerotia

Ten sclerotia of Claviceps gigantea were collected from each of the six locations. The sample size for the number of sclerotia was determined using the model described by Cochran (1982). The sclerotia (n=10) from each location were ground separately in a sterile mortar. One gram of ground sclerotia was diluted in 100 mL of sterile distilled water to perform serial dilutions up to 10-4. From each dilution, 100 µL was plated on Petri dishes with R2A culture medium (Difco) in triplicate and incubated at 28 °C for 24 h. Bacterial growth was quantified using the direct plate count method, and microbial density was expressed in Log10 CFU g-1 of sclerotium (Peng et al., 2009). A total of 129 bacterial isolates associated with sclerotia, exhibiting different colony morphology observed under a stereoscopic microscope (American Optical AO), were selected from the six sampled locations for further study of in vitro antagonism.

In vitro antagonism against Fusarium spp.

The in vitro antagonism of 129 bacterial strains was evaluated against three Fusarium species (F. graminearum, F. subglutinans, and F. verticillioides) responsible for ear rot in the sampled locations. The pathogenicity of these strains was experimentally verified in seedlings of three native populations of corn in the State of Mexico by inoculating the fungus in the substrate. The internal regions ITS of the rRNA 18S-5.8S and 5.8S-28S genes were molecularly identified through amplification, provided by Dr. Dolores Briones Reyes from the Graduate Program of Genetic Resources and Productivity of the Colegio de Postgraduados. In vitro antagonism was evaluated by dual confrontation on square Petri dishes (120 x 120 mm) with Waksman agar culture medium, which was selected from King’s B media, R2A, nutrient agar, and potato-dextrose agar, as it allowed optimal growth of both Fusarium species and bacterial isolates. The initial inoculation of Fusarium species strains was carried out by extension with an L-digralsky loop on the culture medium surface, and the inoculated Petri dishes were kept at room temperature for 60 min. Bacteria (n=129) were subsequently inoculated with a multi-point inoculator (Boekel®, 96-point microplate replicator) and incubated at 28 °C for 7 days. Bacterial strains that showed antagonism (inhibition halo of fungal growth) against one or more Fusarium species were selected. In another assay, antagonistic bacterial strains were individually inoculated by puncturing the bacterial mass with a sterile stick under the same experimental conditions as the previous assay. Only bacterial strains that showed antagonism by forming an inhibition halo ≥ 5mm of mycelial growth against one, two, or all three evaluated Fusarium species were selected. Daily observations were made, and the assay was repeated three times. Controls consisted of Petri dishes with growth of Fusarium species in the absence of antagonistic bacteria.

Qualitative detection of metabolite production

The antagonistic bacteria were qualitatively characterized for their ability to produce metabolites in vitro, such as indole-3-acetic acid, using soy tryptone broth (TSB) as the culture medium. Bacterial strains that produced a reddish coloration in the medium were considered positive (Frey-Klett et al., 2005), while siderophores were assessed using the universal medium chrome azurol S (CAS), with bacterial strains that evidenced a yellow halo around the colony being considered positive (Schwyn and Neilands, 1987). Lipolytic and proteolytic activities were evaluated using nutrient agar supplemented with Tween 80 and TSB medium supplemented with skimmed milk, respectively. Bacterial strains that evidenced an opaque and clear halo around the colony were considered positive for lipolytic and proteolytic activities (Hantsis-Zacharov and Halpern, 2007). Mineral phosphate solubilization was assessed using TCP medium, with bacterial strains that evidenced an opaque halo around the colony being considered positive (El-Yazeid et al., 2007). All evaluations were performed in triplicate.

Molecular identification of antagonistic bacteria

The DNA of the potential antagonists was obtained from individual colonies with 72 h of growth at 28 °C on Waksman agar medium using the PureLink Genomic DNA kit (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol. Partial amplification of the 16S rRNA gene was carried out using the primers 8F (5’ AGAGTTTGATCCTGGCTCAG 3’) and 1492R (3’ GGTTACCTTGTTACGACTT 5’) and PCR conditions described by Baker et al. (2003). The amplified products were sequenced (Sanger sequencing) at Macrogen Inc. (Seoul, Korea) (; the sequences were compared in the gene bank (GenBank) of the International Center for Biotechnology Information (NCBI) ( using the Blastn (Basic Local Alignment Search Tool) 2.2.29+ algorithm (Altschul et al., 1990).


Bacterial population density in Claviceps gigantea sclerotia

The study revealed bacterial growth in R2A culture medium obtained from sclerotia at all sampling locations. Colony counting was performed to determine bacterial density in C. gigantea sclerotia, which varied from 2.023 to 2.397 Log10CFU g-1 of sclerotium across the six sampling sites (Table 2). Notably, the highest bacterial density was found in Atlacomulco lots (2.397) and the lowest in Villa Victoria (2.023) Log10CFU g-1 of sclerotium. These findings imply the presence of bacterial populations associated with C. gigantea sclerotia exudates. Nevertheless, further research is required to investigate factors that influence bacterial density and structure in this ecological niche.

Table 2 Bacterial density (Log10UFC g-1) in C. gigantea sclerotia by sampling location. 

Localidad Número de colonias Log10UFC g-1 de esclerocio
Almoloya de Juárez z204.1 2.309
Atlacomulco 249.9 2.397
Calimaya 188.8 2.276
Mina México 200.4 2.301
Toluca 133.6 2.125
Villa Victoria 105.6 2.023

Z Average number of colonies isolated from 10 C. gigantea sclerotia per location sampled by direct plate count on R2A medium with three replicates

In vitro antagonism against Fusarium spp.

Out of 129 morphologically different bacterial strains isolated from sclerotia, 22 (17%) were identified by their in vitro antagonism (inhibition halo of fungal growth ≥ 5 mm) against one or more species of Fusarium (Table 3). Among these, 13 bacterial strains (59%) were isolated from the Atlacomulco location, and three strains (13%) from each of the Calimaya, Mina, and Toluca locations. Among the antagonists (n=22), 10 (45%) strains were antagonistic to F. verticillioides, 14 (63%) to F. subglutinans, and 17 (81%) to F. graminearum. Among the antagonistic bacteria, the strains Bacillus subtilis (BA1), Pseudomonas syringae (BA2), and Bacillus amyloliquefaciens (BA18) stood out, which were antagonistic against the three evaluated species of Fusarium. These three strains originated from the Atlacomulco location (Table 3).

Table 3 Molecular identification, origin, antagonism against Fusarium spp. and metabolite production of 22 antagonistic strains isolated from sclerotia of Claviceps gigantea. 

Antagonismo in vitro Producción de metabolitos in vitro
ID cepa Localidad Identificación por secuenciación parcial del gen 16S rRNA No. acceso de secuencias tipo de la especie (NCBI) % de identidad zFg Fs Fv AIA LIP PRO SID SFM
BA1 Atlacomulco Bacillus subtilis KF021537.1 99.39 + + + + + + + +
BA2 Atlacomulco Pseudomonas syringae NR_043716.1 99.28 + + + + + + + +
BA3 Atlacomulco Delftia lacustris KF054933.1 99.46 + - + - + + + +
BA4 Mina Stenotrophomonas sp. AM913974.1 99.40 + - + - + + + +
BA5 Calimaya Delftia acidovorans EF564190.1 99.38 + - + - + + + +
BA6 Calimaya Sphingobacterium sp. KF777439.1 99.41 + - - - + + + +
BA7 Atlacomulco Pseudomonas geniculata JX042457.1 99.44 + - - - + + + +
BA8 Mina Micromonospora sp. KY015111.1 99.44 - + - - + + + +
BA9 Mina Stenotrophomonas maltophilia FJ859699.1 99.37 - + - - + + + +
BA10 Calimaya Staphylococcus aureus LN929738.1 99.49 + + - - + + + +
BA11 Atlacomulco Pseudomonas putida KC582298.1 99.05 + + - + + + + +
BA12 Toluca Bacillus sp. HM032893.1 99.48 + + - + + + + +
BA13 Atlacomulco Pseudomonas fluorescens GU198115.1 99.47 + + - + + + + +
BA14 Atlacomulco Bacillus subtilis KF527828.1 99.35 + + - - + + + +
BA15 Atlacomulco Bacillus amyloliquefaciens KC494392.1 99.02 + + - + + + + +
BA16 Atlacomulco Pseudomonas putida KC582298.1 99.16 + + - + + + + +
BA17 Atlacomulco Pseudomonas fluorescens GU198113.1 99.40 - + - + + + + +
BA18 Atlacomulco Bacillus amyloliquefaciens MH781489.1 99.02 + + + + + + + +
BA19 Atlacomulco Pseudomonas sp. DQ991143.2 99.05 - + + - + + + +
BA20 Toluca Bacillus amyloliquefaciens MF765339.1 99.48 + - + + + + + +
BA21 Atlacomulco Pseudomonas putida JX120503.1 99.15 + - + + + + + +
BA22 Toluca Bacillus sp. MF510169.1 99.48 - - + + + + + +

z Fg= Fusarium graminearum; Fs=Fusarium subglutinans; Fv=Fusarium verticillioides; IAA=indole-3-acetic acid production; LIP= lipolytic activity; PRO= proteolytic activity; SID= siderophore production; SFM= mineral phosphate solubilization.

Molecular identification of antagonists

The identification of the 22 antagonist strains was made possible through partial amplification of the 16S rRNA gene, which showed a similarity range of 99.02% to 99.49% when aligned against the NCBI gene bank (refer to Table 3). These strains belonged to various genera, including Pseudomonas (36.3%), Bacillus (31.8%), Delftia (9.09%), Stenotrophomonas (9.09%), Micromonospora (4.5%), Sphingobacterium (4.5%), and Staphylococcus (4.5%). Notably, P. putida (37.5%), P. fluorescens (25%), P. geniculata, and P. syringae (12.5%) were identified among the Pseudomonas species, while B. amyloliquefaciens (57.1%) and B. subtilis (28.5%) were the most common among the Bacillus species. It is worth mentioning that the majority of the antagonists (n=13) were isolated from Atlacomulco (59%), while the remaining strains (n=3) (14.3%) were obtained in equal proportions from Calimaya, Mina, and Toluca, respectively (refer to Table 2). Taxonomically, these 22 strains were distributed across four phyla and six orders, with the Firmicutes (Gram-positive) and Proteobacteria (Gram-negative) phyla, as well as the Bacillales and Pseudomonadales orders, having a higher abundance of Bacillus and Pseudomonas genera. Conversely, the Micromonospora, Sphingobacterium, and Staphylococcus genera were less abundant, with the Firmicutes (Gram-positive) and Bacteroidetes (Gram-negative) phyla, as well as the Actinomycetales, Sphingobacteriales, and Bacillales orders, having the least number of strains (refer to Table 4).

Table 4 Taxonomic distribution of 22 bacterial strains antagonistic in vitro against Fusarium spp. isolated from sclerotia of C. gigantea

Phylum Clase Orden Familia Género Frecuencia %
Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas 36.3
Firmicutes Bacilli Bacillales Bacillaceae Bacillus 31.8
Proteobacteria Beta Proteobacteria Burkholderiales Comamonadaceae Delftia 9.1
Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae Stenotrophomonas 9.1
Bacteroidetes Sphingobacteria Sphingobacteriales Sphingobacteriaceae Sphingobacterium 4.5
Actinobacteria Actinobacteria Actinomycetales Micromonosporaceae Micromonospora 4.5
Firmicutes Bacilli Bacillales Staphylococcaceae Staphylococcus 4.5

Qualitative in vitro production of metabolites

The 22 antagonists showed in vitro lipolytic and proteolytic activity, produced siderophores, and solubilized mineral phosphate; however, only 12 (55%) produced indole-3-acetic acid. Only the strains Bacillus subtilis (BA1), Pseudomonas syringae (BA2), and Bacillus amyloliquefaciens (BA18) were antagonistic against the three species of Fusarium and produced all the evaluated metabolites (Figure 2, Table 3).

Figure 2 In vitro antagonism. A) Sclerotia of C. gigantea, B) bacterial growth from 1 g of sclerotia on R2A medium, C) in vitro antagonism of bacteria in Waksman agar culture medium inoculated with a multipoint inoculator (Boekel®, 96-point microplate replicator 96 points) against Fusarium spp. D) selection of antagonistic bacterial strains showing inhibition zones ≥5 mm of fungal growth of Fusarium spp. In vitro production of metabolites by antagonistic strains. E) production of indole-3-acetic acid, F) production of siderophores, G) proteolytic activity, H) mineral phosphate solubilization, I) lipolytic activity. Strains that showed reddish coloration of the medium, yellow, clear and opaque halo around the colony, respectively, were considered positive. 


In this study, we estimated bacterial density in C. gigantea sclerotia and evaluated their in vitro antagonism against three Fusarium species causing ear rot in maize. Our results showed varying bacterial densities associated with C. gigantea sclerotia across the different sampled locations. The highest bacterial density was recorded in the lots of Atlacomulco (2.397), while the lowest was observed in Villa Victoria (2.023) Log10CFU g-1 of sclerotium. This variation may be attributed to the use of agrochemical products and/or residues as part of the agronomic management of the crop, which may have affected the diversity and density of microorganisms associated with maize cultivation (FAO, 2010). Thus, there is a need for further research to examine the impact of agrochemical residues used in the sampled sites on the structure and diversity of bacterial communities in sclerotia. Additionally, it has also been shown that bacterial density is closely related to the sources of carbon contained in sclerotia exudates (Coley-Smith and Dickenson, 1971). Hence, future research should focus on identifying the sources of carbon present in C. gigantea sclerotia exudates and their quantity. Previous studies have shown that bacterial populations are associated with sclerotia. Coley-Smith and Dickenson (1971) found that the Sclerotium cepivorum fungus sclerotia favor the growth of specific bacteria due to the exudates containing carbon sources such as ethanol, trehalose, glucose, and mannitol, and that this microenvironment constitutes a specific ecological niche. Similarly, Gilbert and Linderman (1971) reported qualitative changes and increased microbial activity in soil near Sclerotium rolfsii sclerotia attributed to the exudates in the sclerotia of this pathogen. These authors coined the term “mycosphere” to describe the portion of soil influenced by sclerotia, which harbors bacterial populations with a greater representation of antagonistic species against S. rolfsii than those obtained from other ecological niches.

In this study, 129 bacterial strains were isolated from sclerotia in various sampled locations. Of these, 22 (17%) showed in vitro antagonism against one or more Fusarium species that cause maize ear rot, indicated by a fungal growth inhibition halo of ≥ 5 mm. Through partial sequencing of the 16S rRNA gene, it was possible to identify the 22 antagonistic bacterial strains as belonging to the genera Bacillus, Delftia, Micromonospora, Pseudomonas, Sphingobacterium, Staphylococcus, and Stenotrophomonas.

The most abundant antagonists in C. gigantea sclerotia belonged to the genera Pseudomonas (Pseudomonadaceae) (36.3%) and Bacillus (Bacillaceae) (31.8%). In both genera, their mechanisms for phytopathogen biocontrol mediated by antibiosis, competition for nutrients and space, promotion of growth, and induction of resistance in plants have been elucidated (Cui et al., 2019; Dimkić et al., 2022; Fira et al., 2018; Guzmán-Guzmán and Santoyo, 2022; Luo et al., 2022; Singh et al., 2022).

The findings of this investigation align with those of previous studies, which have highlighted the prevalence of both Pseudomonas (Pseudomonadaceae) (36.3%) and Bacillus (Bacillaceae) (31.8%) genera colonizing Rhizoctonia solani sclerotia (Zachow et al., 2011) and Sclerotium cepivorum (Backhouse and Stewart, 1989; Utkhede and Rahe, 1980), demonstrating their strong antagonism against these pathogens. Stenetrophomonas genus was also identified in this research. Wong and Hughes (1986) have shown that Bacillus species constitute 80% of antagonists isolated from soil and S. cepivorum sclerotia samples. This genus includes a group of Gram-positive bacteria with high phenotypic and genetic heterogeneity, considered among the most common colonizers in various ecological niches (Abriouel et al., 2011). Although most of the identified strains in this study were in vitro antagonistic against one or two Fusarium species, Bacillus amyloliquefaciens (BA18), B. subtilis (BA1), and Pseudomonas syringae (BA2) strains were antagonistic against all three evaluated Fusarium species (F. graminearum, F. subglutinas, and F. verticillioides) causing maize ear rot.

In other studies, B. amyloliquefaciens has been reported as an antagonist with high potential for the biocontrol of other Fusarium species in spinach (Spinacia oleracea) (Zhao et al., 2014), banana (Musa sp.) (Tian et al., 2021), tomato (Solanum lycopersicum) (Elanchezhiyan et al., 2018; Proca et al., 2020), and wheat (Triticum sp.) (Ursan et al., 2019). Some strains of B. amyloliquefaciens have been reported as antagonists and efficient biocontrol agents against F. graminearum (de Ángel et al., 2021; Gu et al., 2017; Liu et al., 2019) and F. verticillioides (Xu et al., 2021).

The prevalence and antagonism of B. subtilis isolated from sclerotia of C. gigantea coincides with other studies. Utkhede and Rahe (1980) showed that there is a greater prevalence of B. subtilis in S. cepivorum sclerotia collected from various parts of the world and that most of these strains significantly protected onion (Allium cepa) from white rot caused by this pathogen when inoculated into seed at the time of planting. These authors attributed the prevalence of B. subtilis to specific carbohydrates contained in the exudates of S. cepivorum sclerotia. Also, strains of B. subtilis have shown antagonism and efficient biocontrol against F. graminearum and F. verticillioides in wheat and maize, respectively (Cavaglieri et al., 2005; Guimarães et al., 2021; Wang et al., 2020; Yu et al., 2021).

The genus Pseudomonas includes Gram-negative species which have been widely studied as an alternative in the biological control of phytopathogens and promotion of plant growth (Dimkić et al., 2022; Guzmán-Guzmán and Santoyo, 2022; Singh et al., 2022). Pseudomonas syringae, identified in this study as an antagonist against the three species of Fusarium, belongs to the P. fluorescens species complex and has been described as an important biocontrol agent along with P. aeruginosa, P. aureofaciens, P. chlororaphis, P. fluorescens, and P. putida (Panpatte et al., 2016). Pseudomonas syringae is a species complex that includes phytopathogenic strains on a wide range of host plants (Baltrus et al., 2017). However, non-phytopathogenic strains of P. syringae have been identified whose genome harbors an extensive group of genes related to the biocontrol of phytopathogens, promotion of growth, and induction of resistance in plants (Passera et al., 2019). Yu et al. (2017) showed that the Pseudomonas syringae BAF.1 strain completely inhibited the germination of conidia and affected the structure of the mycelium of Fusarium oxysporum, proposing it as a promising biocontrol agent against this pathogen.

In this research, Bacillus subtilis (BA1), Pseudomonas syringae (BA2), and Bacillus amyloliquefaciens (BA18) strains were antagonistic against all three Fusarium species and produced in vitro all evaluated metabolites (indole-3-acetic acid production, lipolytic and proteolytic enzymes, siderophores, and mineral phosphate solubilization) (Table 3). The production of these metabolites has been shown to play an important role in the biocontrol of phytopathogenic fungi and promotion of plant growth (Sagar et al., 2022). Proteolytic and lipolytic enzymes produced by Bacillus and Pseudomonas species cause cellular lysis of fungi; siderophores are antimicrobial compounds that facilitate iron mobilization and solubilization of nutrients not available to plants (Admassie et al., 2022). The production of plant growth regulators such as indole-3-acetic acid stimulates the development of the plant root system and induces resistance to pathogens; while mineral solubilization such as phosphorus promotes plant development, induces resistance to pathogens, and improves water and nutrient uptake (Mahapatra et al., 2022; Sagar et al., 2022).

The ability of Bacillus and Pseudomonas species to produce a wide range of secondary metabolites encoded by various gene clusters has been suggested to result in disease suppression, growth promotion, and induction of resistance in plants (Andrić et al., 2020; Dimkić et al., 2022; Luo et al., 2022). However, it is important to note that the proportion of genes involved in the synthesis of antimicrobial compounds and other bioactive secondary metabolites varies depending on the species and strain (Devi et al., 2019). Therefore, prior to registering their 16S rRNA sequences in the NCBI GenBank, it is recommended to further investigate the genome characteristics of the three antagonistic strains identified in this research. Previous studies have shown that inoculation of Bacillus and Pseudomonas spp. strains promotes growth and induces resistance in maize plants (Egamberdiyeva et al., 2007).

Species of the genus Bacillus are promising biological control agents due to their genetic characteristics, high heat and desiccation resistance through the formation of endospores (Luo et al., 2022). These strains are considered to be safe for human health, hence there are no restrictions on their use as biocontrol agents according to the United States Environmental Protection Agency (Dimkić et al., 2022; Hou et al., 2006). Additionally, commercial formulations of P. syringae are approved for the management of Fusarium spp. in post-harvest in the USA and Canada (Al-Mughrabi et al., 2013). Therefore, the Bacillus subtilis (BA1), Pseudomonas syringae (BA2), and B. amyloliquefaciens (BA18) strains identified in this research, isolated from sclerotia of C. gigantea that were antagonistic in vitro against three Fusarium species causing corn ear rot, and multifunctional in the production of secondary metabolites, represent an important biotechnological resource for future investigations as biocontrol agents against this pathogen in corn cultivation in Mexico.


The findings of this study suggest that the sclerotia of C. gigantea contain varying densities of bacterial populations, with those from the Atlacomulco locality exhibiting the highest density. Among these bacterial populations are strains that exhibit antagonism against at least one species of Fusarium, known to cause corn ear rot and produce metabolites that may promote plant growth. Through sequencing of the 16S rRNA gene, Bacillus and Pseudomonas genera were identified as the most abundant antagonistic bacteria in C. gigantea sclerotia. In vitro experiments demonstrated that Bacillus subtilis (BA1), Pseudomonas syringae (BA2), and Bacillus amyloliquefaciens (BA18) strains were effective antagonists against all three species of Fusarium evaluated, with Fusarium graminaerum being the most susceptible. These results suggest that these strains may represent a viable option for biocontrol of Fusarium species in maize cultivation in Mexico.


To the National Council for Science and Technology (CONACyT). To Dr. Dolores Briones Reyes from the Postgraduate Program in Genetic Resources and Productivity at the College of Postgraduates Montecillo campus for donating the Fusarium spp. strains used in this research.

Cited literature

Abdullah MT, Ali NY and Suleman P. 2008. Biological control of Sclerotinia sclerotiorum (Lib.) de Bary with Trichoderma harzianum and Bacillus amyloliquefaciens. Crop Protection 27: 1354-1359. [ Links ]

Admassie M, Woldehawariat Y and Alemu T. 2022. In vitro evaluation of extracellular enzyme activity and Iits biocontrol efficacy of bacterial isolates from pepper plants for the management of Phytophthora capsici. BioMed Research International 2022. [ Links ]

Agurell SL, Ramstad E and Ullstrup AJ. 1963. The alkaloids of maize ergot. Part V of biogenetic studies in ergot; Partt IV: Svensk Farmaceutisk Tidskrift 66(741). Planta Medika 11:392-398. [ Links ]

Al-Mughrabi KI, Vikram A, Peters RD, Howard RJ, Grant L, Barasubiye T and Jayasuriya KE. 2013. Efficacy of Pseudomonas syringae in the management of potato tuber diseases in storage. Biological Control 64:315-322. [ Links ]

Altschul SF, Gish W, Miller W, Myers EW and Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215:403-410. [ Links ]

Andrić S, Meyer T and Ongena M. 2020. Bacillus responses to plant-associated fungal and bacterial communities. Frontiers in Microbiology 11:1350. [ Links ]

Backhouse D and Stewart A. 1989. Interactions between Bacillus species and sclerotia of Sclerotium cepivorum. Soil Biology and Biochemistry 21:173-176. [ Links ]

Baker GC, Smith JJ and Cowan DA. 2003. Review and re-analysis of domain-specific 16S primers. Journal of Microbiological Methods 55:541-555. [ Links ]

Baltrus DA, McCann HC and Guttman DS. 2017. Evolution, genomics and epidemiology of Pseudomonas syringae: challenges in bacterial molecular plant pathology. Molecular Plant Pathology 18:152-168. [ Links ]

Bragg PE, Maust MD and Panaccione DG. 2017. Ergot alkaloid biosynthesis in the maize (Zea mays) ergot fungus Claviceps gigantea. Journal of Agricultural and Food Chemistry 65(49): 10703-10710. [ Links ]

Cavaglieri L, Orlando JR, Rodriguez MI, Chulze S and Etcheverry M. 2005. Biocontrol of Bacillus subtilis against Fusarium verticillioides in vitro and at the maize root level. Research in Microbiology 156:748-754. [ Links ]

CIMMYT. 2004. Enfermedades del maíz: Una Guía para su identificación en el campo. Cuarta edición. México, D.F. CIMMYT. 112 p. [ Links ]

CIMMYT. 2019. Maíz para México. Plan Estratégico 2030. ]

Cochran WG. 1982. Técnicas de muestreo. Compañía Editorial Continental. México. 513 p. [ Links ]

Coley-Smith JR and Dickinson DJ. 1971. Effects of sclerotia of Sclerotium cepivorum Berk. on soil bacteria. The nature of substances exuded by sclerotia. Soil Biology and Biochemistry 3(1):27-32. [ Links ]

Cui W, He P, Munir S, He P, Li X, Li Y, Wu J, Yang L, He P and He Y. 2019. Efficacy of plant growth promoting bacteria Bacillus amyloliquefaciens B9601-Y2 for biocontrol of southern corn leaf blight. Biological Control 139:104080. [ Links ]

de Ángel EC, Hernández-Castillo FD, Gallegos-Morales G, and Ochoa-Fuentes YM. 2021. Actividad antifúngica de bacterias endófitas para el control de Fusarium verticillioides en maíz. Ecosistemas y Recursos Agropecuarios 8(2): e2790. [ Links ]

Devi S, Kiesewalter HT, Kovács R, Frisvad JC, Weber T, Larsen TO, Kováks AT and Ding L. 2019. Depiction of secondary metabolites and antifungal activity of Bacillus velezensis DTU001. Synthetic and Systems Biotechnology 4(3):142-149. [ Links ]

Dimkić I, Janakiev T, Petrović M, Degrassi G and Fira D. 2022. Plant-associated Bacillus and Pseudomonas antimicrobial activities in plant disease suppression via biological control mechanisms-A review. Physiological and Molecular Plant Pathology 117:101754. [ Links ]

Egamberdiyeva D. 2007. The effect of plant growth promoting bacteria on growth and nutrient uptake of maize in two different soils. Applied Soil Ecology 36:184-189. [ Links ]

Elanchezhiyan K, Keerthana U, Nagendran K, Prabhukarthikeyan SR, Prabakar K, Raguchander T and Karthikeyan G. 2018. Multifaceted benefits of Bacillus amyloliquefaciens strain FBZ24 in the management of wilt disease in tomato caused by Fusarium oxysporum f. sp. lycopersici. Physiological and Molecular Plant Pathology 103:92-101. [ Links ]

El-Yazeid AA, Abou-Aly HA, Mady MA and Moussa SAM. 2007 Enhancing growth, productivity and quality of squash plants using phosphate dissolving microorganisms (bio phosphor) combined with boron foliar spray. Research Journal of Agriculture and Biological Sciences 3:274-286. [ Links ]

Fira D, Dimkić I, Berić T, Lozo J and Stanković S. 2018. Biological control of plant pathogens by Bacillus species. Journal of Biotechnology 285:44-55. [ Links ]

Fucikovsky L and Moreno M. 1976. Distribution of Claviceps gigantea and its percent attack on two lines of corn in the state of México, México. Plant Disease Reporter 55:231-233. [ Links ]

Fuentes SF, De la Isa ML, Ullstrup AJ and Rodríguez AE. 1964. Claviceps gigantea, a new pathogen of Maize in Mexico. Phytopathology 54(4):379-381. [ Links ]

García-López V and Giraldo OF. 2021. Redes y estrategias para la defensa del maíz en México. Revista Mexicana de Sociología 83(2):297-329. [ Links ]

Guzmán-Guzmán P and Santoyo G. 2022. Action mechanisms, biodiversity, and omics approaches in biocontrol and plant growth-promotingPseudomonas: an updated review. Biocontrol Science and Technology 32(5):527-550. [ Links ]

Gilbert RG and Linderman RG. 1971. Increased activity of soil microorganisms near sclerotia of Sclerotium rolfsii in soil. Canadian Journal of Microbiology 17(4):557-562. [ Links ]

Gu Q, Yang Y, Yuan Q, Shi G, Wu L, Lou Z, Huo R, Wu H, Borriss R and Gao X. 2017. Bacillomycin D produced by Bacillus amyloliquefaciens is involved in the antagonistic interaction with the plantpathogenic fungus Fusarium graminearum. Applied Environmental Microbiology 83:e01075-17. https:// [ Links ]

Guimarães RA, Zanotto E, Perrony PEP, Zanotto LAS, da Silva LJ, Machado JDC and de Medeiros FHV. 2021. Integrating a chemical fungicide and Bacillus subtilis BIOUFLA2 ensures leaf protection and reduces ear rot (Fusarium verticillioides) and fumonisin content in maize. Journal of Phytopathology 169(3):139-148. [ Links ]

Hantsis-Zacharov E and Halpern M. 2007. Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lipolytic traits. Applied and Environmental Microbiology73(22):7162-7168. [ Links ]

Hof H. 2020. The medical relevance of Fusarium spp. Journal of Fungi 6(3): 117. [ Links ]

Hou X, Boyetchko SM, Brkic M, Olson D, Ross A and Hegedus D. 2006. Characterization of the anti-fungal activity of a Bacillus spp. associated with sclerotia from Sclerotinia sclerotiorum. Applied Microbiology and Biotechnology 72(4):644-653. [ Links ]

Liu Y, Lu J, Sun J, Lu F, Bie X and Lu Z. 2019. Membrane disruption and DNA binding of Fusarium graminearum cell induced by C16-Fengycin A produced by Bacillus amyloliquefaciens. Food Control 102:206-213. [ Links ]

Luo L, Zhao C, Wang E, Raza A and Yin C. 2022. Bacillus amyloliquefaciens as an excellent agent for biofertilizer and biocontrol in agriculture: An overview for its mechanisms. Microbiological Research 259:127016. [ Links ]

Mahapatra S, Yadav R and Ramakrishna W. 2022. Bacillus subtilis impact on plant growth, soil health and environment: Dr. Jekyll and Mr. Hyde. Journal of Applied Microbiology 132(5): 3543-3562. [ Links ]

Matsuoka Y, Vigouroux Y, Goodman MM, Sanchez J, Buckler E and Doebley J. 2002. A single domestication for maize shown by multilocus microsatellite genotyping. Proceedings of the National Academy of Sciences 99(9):6080-6084. [ Links ]

Mesterhazy A, Lemmens M and Reid LM. 2012. Breeding for resistance to ear rots caused by Fusarium spp. in maize-a review. Plant Breeding 131(1):1-19. [ Links ]

Mielniczuk E and Skwaryło-Bednarz B. 2020. Fusarium head blight, mycotoxins and strategies for their reduction. Agronomy 10(4):509. [ Links ]

Moreno-Limón S, González-Solís LN, Salcedo-Martínez SM, Cárdenas-Ávila ML and Perales-Ramírez A. 2011. Efecto antifúngico de extractos de gobernadora (Larrea tridentata L.) sobre la inhibición in vitro de Aspergillus flavus y Penicillium sp. Polibotánica 32:193-205. [ Links ]

Padrón HYM, Delgado SH, Méndez CAR and Carrillo GV. 2013. El género Aspergillus y sus micotoxinas en maíz en México: problemática y perspectivas. Revista Mexicana de Fitopatología 31(2): 126-146. [ Links ]

Panpatte DG, Jhala YK, Shelat HN and Vyas RV. 2016. Pseudomonas fluorescens: A Promising Biocontrol Agent and PGPR for Sustainable Agriculture. In: Singh, D., Singh, H., Prabha, R. (eds) Microbial Inoculants in Sustainable Agricultural Productivity (pp. 257-270). Springer, New Delhi. [ Links ]

Passera A, Compant S, Casati P, Maturo MG, Battelli G, Quaglino F, Antonielli L, Salerno D, Brasca M, Toffolatti SL, Mantegazza F, Delledonne M and Mitter B. 2019. Not Just a Pathogen? Description of a Plant-Beneficial Pseudomonas syringae Strain. Frontiers in Microbiology 10:1409. doi: 10.3389/fmicb.2019.01409 [ Links ]

Peng S, Zhou Q, Cai Z and Zhang Z. 2009. Phytoremediation of petroleum contaminated soils by Mirabilis Jalapa L. in a greenhouse plot experiment. Journal of Hazardous Materials 168(2-3):1490-1496. [ Links ]

Proca IG, Diguță CF, Cornea CP, Jurcoane S and Matei F. 2020. Halotolerant Bacillus amyloliquefaciens 24.5. Rom Biotechnology Letters 25(4):1744-1753. [ Links ]

Sagar A, Yadav SS, Sayyed RZ, Sharma S and Ramteke PW. 2022. Bacillus subtilis: a multifarious plant growth promoter, biocontrol agent, and bioalleviator of abiotic stress. In Bacilli in Agrobiotechnology: Plant Stress Tolerance, Bioremediation, and Bioprospecting (pp. 561-580). Cham: Springer International Publishing. [ Links ]

Schwyn B, and Neilands JB. 1987. Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry 160(1):47-56. [ Links ]

Singh P, Singh RK, Zhoua Y, Wanga J, Jiangb Y, Shena N, Wanga Y, Yangb L and Mingguo J. 2022. Unlocking the strength of plant growth promoting Pseudomonas in improving crop productivity in normal and challenging environments: a review. Journal of Plant Interactions 17(1):220-238. [ Links ]

Solano-Báez AR, Cuca-García JM, Delgado-Alvarado A, Panaccione D, De León-García de Alba C, Leyva-Mir SG, Sánchez-Pale JR and Hernández-Morales J. 2018. Biological activity of Claviceps gigantea in juvenile New Zealand rabbits. Mycotoxin Research 34:297-305. [ Links ]

Tian D, Song X, Li C, Zhou W, Qin L, Wei L, Di W, Huang S, Li B, Huang Q, Long S, Hew Z and Wei S. 2021. Antifungal mechanism of Bacillus amyloliquefaciens strain GKT04 against Fusarium wilt revealed using genomic and transcriptomic analyses. Microbiology Open 10(3):e1192. [ Links ]

Ullstrup AJ. 1973. Maize ergot: a disease with a restricted ecological niche. PANS Pest Articles & News Summaries 19(3):389-391. [ Links ]

Ursan MD, Boiu-Sicuia OA and Cornea CP. 2019. Bacillus amyloliquefaciens strains with biocontrol potential against Fusarium spp. wheat pathogens. Scientific Papers. Series A. Agronomy: 486-491. [ Links ]

Utkhede RS and Rahe JE. 1980. Biological control of onion white rot. Soil Biology and Biochemistry 12(2):101-104. [ Links ]

Wang S, Sun L, Zhang W, Chi F, Hao X, Bian J and Li Y. 2020. Bacillus velezensis BM21, a potential and efficient biocontrol agent in control of corn stalk rot caused by Fusarium graminearum. Egyptian Journal of Biological Pest Control 30(1):1-10. [ Links ]

Wong WC and Hughes IK. 1986. Sclerotium cepivorum Berk. in onion (Allium cepa L.) crops: isolation and characterization of bacteria antagonistic to the fungus in Queensland. Journal of Applied Bacteriology 60(1):57-60. [ Links ]

Xu S, Wang Y, Hu J, Chen X, Qiu Y., Shi J, Wang G and Xu J. 2021. Isolation and characterization of Bacillus amyloliquefaciens MQ01, a bifunctional biocontrol bacterium with antagonistic activity against Fusarium graminearum and biodegradation capacity of zearalenone. Food Control 130:108259. [ Links ]

Yu C, Liu X, Zhang X, Zhang M, Gu Y, Ali Q, Mohamed MSR, Xu J, Shi J, Gao X, Wu H and Gu Q. 2021. Mycosubtilin produced by Bacillus subtilis ATCC6633 inhibits growth and mycotoxin biosynthesis of Fusarium graminearum and Fusarium verticillioides. Toxins 13(11):791. [ Links ]

Yu S, Teng C, Liang J, Song T, Dong L, Bai, X Jin Y and Qu J. 2017. Characterization of siderophore produced byPseudomonas syringaeBAF.1 and its inhibitory effects on spore germination and mycelium morphology ofFusarium oxysporum. Journal Microbiology 55:877-884. [ Links ]

Zachow C, Grosch R and Berg G. 2011. Impact of biotic and a-biotic parameters on structure and function of microbial communities living on sclerotia of the soil-borne pathogenic fungus Rhizoctonia solani. Applied Soil Ecology 48(2):193-200. [ Links ]

Zenteno-Zevada M, 1963. Estudios sobre hongos parásitos de gramíneas de la República Mexicana. III. Pruebas de inoculación en plántulas de maíz con Gibberella fujikuroi (Saw.) Wr. In Anales del Instituto de Biología, Universidad Nacional Autónoma de México 34: 69-83. [ Links ]

Zhao P, Quan C, Wang Y, Wang J and Fan S. 2014. Bacillus amyloliquefaciens Q‐426 as a potential biocontrol agent against Fusarium oxysporum f. sp. spinaciae. Journal of Basic Microbiology 54(5):448-456. [ Links ]

Received: August 03, 2022; Accepted: March 25, 2023

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