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Microorganisms from corn stigma with biocontrol potential of Fusarium verticillioides

Microrganismos do estigma do milho com potencial de biocontrole de Fusarium verticillioides

Abstract

The mycotoxigenic fungus Fusarium verticillioides is the primary maize pathogen and causes the maize stalk and ear rot diseases with significant economic losses. Furthermore, the excessive use of fungicides to control F. verticillioides constitutes threats to the environment and human health. Thus, sustainable alternatives such as biological control are needed to minimize the hazards associated with the current method. Although much is known about the vulnerability of the maize silks as a gateway for several fungal pathogens invading the developing grains, studies on the chemical properties of silk extracts and their resident microbiota are scarce. This study isolated and characterized bacteria and fungi that colonize the maize stigma to assess new potential biocontrol agents. The samples were collected from maize fields in the Brazilian localities of Sete Lagoas-MG, Sidrolândia-MS, Sertaneja-PR, and Goiânia-GO. One hundred sixty-seven microorganisms were isolated, 46% endophytic and 54% epiphytic. First, the antagonist activity was evaluated by the agar disc diffusion method performed in triplicate, and 83% of the isolates showed antagonist activity against F. verticillioides. Then, the 42 most efficient isolates were identified based on the partial sequencing of the bacterial 16S rRNA gene and fungi ITS region. The bacteria belong to the genera Bacillus (57.1%), Burkholderia (23.8%), Achromobacter (7.1%), Pseudomonas (2.4%), and Serratia (2.4%), while the fungi are Penicillium (2.4%), Candida (2.4), and Aspergillus (2.4%). The results showed that microorganisms from maize stigma might represent new promising agents for F. verticillioides control.

Keywords:
microorganisms; antagonists; Fusarium moniliforme; biofungicide; phytopathogen; maize

Resumo

O fungo micotoxigênico Fusarium verticillioides é o principal patógeno do milho e causa doenças do colmo e da podridão da espiga com perdas econômicas significativas. Além disso, o uso excessivo de fungicidas no controle de F. verticillioides constitui uma ameaça ao meio ambiente e à saúde humana. Assim, alternativas sustentáveis, como o controle biológico, são necessárias para minimizar os riscos associados ao método atual. Este estudo isolou e caracterizou bactérias e fungos que colonizam o estigma do milho para avaliar novos agentes de biocontrole em potencial. As amostras foram coletadas em campos de milho nas localidades brasileiras de Sete Lagoas-MG, Sidrolândia-MS, Sertaneja-PR e Goiânia-GO. Cento e sessenta e sete microrganismos foram isolados, 46% endofíticos e 54% epifíticos. O teste de antagonismo empregando a técnica de disco de difusão em meio sólido, mostrou que 83% dos isolados apresentaram atividade antagonista contra F. verticillioides. Em seguida, 42 isolados mais eficientes foram identificados a partir do sequenciamento parcial do gene 16S rRNA bacteriano e da região ITS de fungos. Os isolados bacterianos pertencem ao gênero Bacillus (57,1%), Burkholderia (23,8%), Achromobacter (7,1%), Pseudomonas (2,4%) e Serratia (2,4%), enquanto os fungos são Penicillium (2,4%), Candida (2.4), e Aspergillus (2,4%). Os resultados mostraram que microrganismos do estigma do milho podem representar novos agentes promissores para o controle de F. verticillioides.

Palavras-chave:
microrganismos; antagonistas; Fusarium moniliforme; biofungicida; fitopatógeno; milho

1. Introduction

Fusarium verticillioides (Sacc.) Nirenberg (sinônimo, Fusarium moniliforme, Sheldon) is considered one of the primary pathogens of maize, being found both in temperate and humid tropical to subtropical climate regions (Gomes et al., 2020GOMES, A.A.M., DE MELO, M.P., TESSMANN, D.J. and LIMA, C.S., 2020. Sexual reproduction parameters in Fusarium verticillioides populations from maize in Brazil. European Journal of Plant Pathology, vol. 156, no. 1, pp. 317-323. http://dx.doi.org/10.1007/s10658-019-01881-1.
http://dx.doi.org/10.1007/s10658-019-018...
). This pathogen can cause disease symptoms at all stages of a plant’s development, causing seed losses, seedling death, stem rot, root and ear rot, and damages to stored grains (Samsudin and Magan, 2016SAMSUDIN, N.I.P. and MAGAN, N., 2016. Efficacy of potential biocontrol agents for control of Fusarium verticillioides and fumonisin B1 under different environmental conditions. World Mycotoxin Journal, vol. 9, no. 2, pp. 205-213. http://dx.doi.org/10.3920/WMJ2015.1886.
http://dx.doi.org/10.3920/WMJ2015.1886...
). In addition, F. verticillioides produces fumonisins, mainly fumonisin B1, which can cause various health problems for humans and animals (Blacutt et al., 2018BLACUTT, A.A., GOLD, S.E., VOSS, K.A., GAO, M. and GLENN, A.E., 2018. Fusarium verticillioides: advancements in understanding the toxicity, virulence, and niche adaptations of a model mycotoxigenic pathogen of maize. Phytopathology, vol. 108, no. 3, pp. 312-326. http://dx.doi.org/10.1094/PHYTO-06-17-0203-RVW. PMid:28971734.
http://dx.doi.org/10.1094/PHYTO-06-17-02...
). Fusarium verticillioides can infect maize through the seeds, plant wounds, or stigma (Munkvold et al., 1997MUNKVOLD, G.P., MCGEE, D.C. and CARLTON, W.M., 1997. Importance of different pathways for maize kernel infection by Fusarium moniliforme. Phytopathology, vol. 87, no. 2, pp. 209-217. http://dx.doi.org/10.1094/PHYTO.1997.87.2.209. PMid:18945144.
http://dx.doi.org/10.1094/PHYTO.1997.87....
).

In maize, the style is called silk to refer to the female maize flowers (Rosli et al., 2008ROSLI, W.I., NURHANAN, A.R., MOHSIN, S.S.J. and FARID, C.G., 2008. Aqueous, alcoholic treated and proximate analysis of maydis stigma (Zea mays hairs). Annals of Microscopy, vol. 8, pp. 66-72.). Therefore, the silk emerging at the tips of corn cobs is critically important, not only for seed establishment but also because it is the gateway of entrance of carcinogenic mycotoxin-producing fungal pathogens that devastate millions of maize farmers (Johnson et al., 2007JOHNSON, E.T., BERHOW, M.A. and DOWD, P.F., 2007. Expression of a maize Myb transcription factor driven by a putative silk-specific promoter significantly enhances resistance to Helicoverpa zea in transgenic maize. Journal of Agricultural and Food Chemistry, vol. 55, no. 8, pp. 2998-3003. http://dx.doi.org/10.1021/jf0633600. PMid:17385885.
http://dx.doi.org/10.1021/jf0633600...
; Thompson and Raizada, 2018THOMPSON, M.E.H. and RAIZADA, M.N., 2018. Fungal pathogens of maize gaining free passage along the silk road. Pathogens, vol. 7, no. 4, pp. 81. http://dx.doi.org/10.3390/pathogens7040081. PMid:30314351.
http://dx.doi.org/10.3390/pathogens70400...
). Thus, the maize stigma has been described as the most efficient form of plant contamination by fungal pathogens invading the developing grain using the silk channels and causing disruption of the seeds and severe ear rot (Warfield and Davis, 1996WARFIELD, C.Y. and DAVIS, R.M., 1996. Importance of the husk covering on the susceptibility of corn hybrids to Fusarium ear rot. Plant Disease, vol. 80, no. 2, pp. 208-210. http://dx.doi.org/10.1094/PD-80-0208.
http://dx.doi.org/10.1094/PD-80-0208...
; Khalaf et al., 2021KHALAF, E.M., SHRESTHA, A., RINNE, J., LYNCH, M.D.J., SHEARER, C.R., LIMAY-RIOS, V., REID, L.M. and RAIZADA, M.N., 2021. Transmitting silks of maize have a complex and dynamic microbiome. Scientific Reports, vol. 11, no. 1, pp. 13215. http://dx.doi.org/10.1038/s41598-021-92648-4. PMid:34168223.
http://dx.doi.org/10.1038/s41598-021-926...
).

The maize silk is an herb used traditionally as a therapeutic remedy to treat various diseases in many parts of the world (Alam, 2011ALAM, E.A., 2011. Evaluation of antioxidant and antibacterial activities of Egyptian Maydis stigma (Zea mays hairs) rich in some bioactive constituents. The Journal of American Science, vol. 7, no. 4, pp. 726-729.; Hasanudin et al., 2012HASANUDIN, K., HASHIM, P. and MUSTAFA, S., 2012. Corn silk (Stigma maydis) in healthcare: a phytochemical and pharmacological review. Molecules, vol. 17, no. 8, pp. 9697-9715. http://dx.doi.org/10.3390/molecules17089697. PMid:22890173.
http://dx.doi.org/10.3390/molecules17089...
). The potential antioxidant and healthcare applications of maize silk are due to the properties of its bioactive constituents, such as flavonoids and terpenoids. In addition, many microorganisms and natural compounds derived from plants have fungistatic or fungicidal effects and can be used to manage plant diseases (Dias et al., 2020DIAS, A.L.B., SOUSA, W.C., BATISTA, H.R.F., ALVES, C.C.F., SOUCHIE, E.L., SILVA, F.G., PEREIRA, P.S., SPERANDIO, E.M., CAZAL, C.M., FORIM, M.R. and MIRANDA, M.L.D., 2020. Chemical composition and in vitro inhibitory effects of essential oils from fruit peel of three Citrus species and limonene on mycelial growth of Sclerotinia sclerotiorum. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 80, no. 2, pp. 460-464. http://dx.doi.org/10.1590/1519-6984.216848. PMid:31291410.
http://dx.doi.org/10.1590/1519-6984.2168...
; Ahmadu et al., 2021AHMADU, T., AHMAD, K., ISMAIL, S.I., RASHED, O., ASIB, N. and OMAR, D., 2021. Antifungal efficacy of Moringa oleifera leaf and seed extracts against Botrytis cinerea causing gray mold disease of tomato (Solanum lycopersicum L.). Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 81, no. 4, pp. 1007-1022. http://dx.doi.org/10.1590/1519-6984.233173. PMid:33175006.
http://dx.doi.org/10.1590/1519-6984.2331...
; Matrose et al., 2021MATROSE, N.A., OBIKEZE, K., BELAY, Z.A. and CALEB, O.J., 2021. Plant extracts and other natural compounds as alternatives for post-harvest management of fruit fungal pathogens: a review. Food Bioscience, vol. 41, pp. 100840. http://dx.doi.org/10.1016/j.fbio.2020.100840.
http://dx.doi.org/10.1016/j.fbio.2020.10...
). However, despite the importance of maize silks to both reproduction and global grain disease, there are few studies in the literature about the effect of maize silk extracts and their microbiome for controlling plant diseases (Khalaf et al., 2021KHALAF, E.M., SHRESTHA, A., RINNE, J., LYNCH, M.D.J., SHEARER, C.R., LIMAY-RIOS, V., REID, L.M. and RAIZADA, M.N., 2021. Transmitting silks of maize have a complex and dynamic microbiome. Scientific Reports, vol. 11, no. 1, pp. 13215. http://dx.doi.org/10.1038/s41598-021-92648-4. PMid:34168223.
http://dx.doi.org/10.1038/s41598-021-926...
). Alam (2011)ALAM, E.A., 2011. Evaluation of antioxidant and antibacterial activities of Egyptian Maydis stigma (Zea mays hairs) rich in some bioactive constituents. The Journal of American Science, vol. 7, no. 4, pp. 726-729., evaluating the antioxidant and antibacterial activities of ethanolic extract of Egyptian Maydis stigma against human pathogenic bacterial species, found an antibacterial activity of ethanolic extract of both upper and lower parts of maize silk. Also, Feng et al. (2012)FENG, X., WANG, L., TAO, M.L., ZHOU, Q. and ZHONG, Z.H., 2012. Studies on antimicrobial activity of ethanolic extract of maize silk. African Journal of Microbiological Research, vol. 6, no. 2, pp. 335-338. http://dx.doi.org/10.5897/AJMR11.974.
http://dx.doi.org/10.5897/AJMR11.974...
reported that the antimicrobial activity of ethanolic extract of maize silk could inhibit the microbial activity of several human pathogenic species of bacteria and fungi.

Recent data from scientific literature highlight the potential of bacteria and fungi isolated from maize silks as biocontrol agents for plant pathogens. In the first report on maize silk-associated microbial communities, Diniz et al. (2017)DINIZ, G.F.D., AGUIAR, F.M., SANTOS, K.K.B., RIBEIRO, V.P., GOMES, E.A., COTA, L.V., CURY, J.C., MARRIEL, I.E. and OLIVEIRA, C.A., 2017 [accessed 29 March 2022]. Antagonistic potential of microorganisms isolated from corn against Fusarium verticillioides. In: Anais do 29º Congresso Brasileiro de Microbiologia [online], 22-25 October 2017, Foz do Iguaçu, Paraná, Brasil. Brasília: Embrapa. Available from: https://www.alice.cnptia.embrapa.br/alice/bitstream/doc/1078763/1/Antagonisticpotential.pdf
https://www.alice.cnptia.embrapa.br/alic...
found that the antagonistic activity of 174 bacteria and fungi against F. verticillioides was highly variable with 20% to up 60% activity. Fifty seven isolates reduced the radial growth of Fusarium verticillioides between 40% and 60%, and twelve isolates stood out for antagonistic activity by reducing above 60% the growth of the pathogen.

In another study, Diniz et al. (2021)DINIZ, G.F.D., COTA, L.V., FIGUEIREDO, J.E.F., AGUIAR, F.M., SILVA, D.D., LANA, U.G.P., SANTOS, V.L., MARRIEL, I.E. and OLIVEIRA-PAIVA, C.A., 2021. Antifungal activity of bacterial strains from maize silks against Fusarium verticillioides. Archives of Microbiology, vol. 204, no. 1, pp. 89. http://dx.doi.org/10.1007/s00203-021-02726-4. PMid:34962587.
http://dx.doi.org/10.1007/s00203-021-027...
tested four endophytic and epiphytic bacteria from maize silks in vitro and greenhouse against F. verticillioides and hydrolytic enzyme production. The authors found that all strains showed antifungal activity in vitro with inhibition values from 58.5 to 100%; they changed hyphae morphology and inhibited conidial germination up to 100% (strain IPR45) by producing at least one enzyme with antifungal activity. In addition, the microbiolized seeds reduced the fungal development in stored grains and stalk rot severity in the greenhouse by approximately 73% (strain ISD04).

Khalaf et al. (2021)KHALAF, E.M., SHRESTHA, A., RINNE, J., LYNCH, M.D.J., SHEARER, C.R., LIMAY-RIOS, V., REID, L.M. and RAIZADA, M.N., 2021. Transmitting silks of maize have a complex and dynamic microbiome. Scientific Reports, vol. 11, no. 1, pp. 13215. http://dx.doi.org/10.1038/s41598-021-92648-4. PMid:34168223.
http://dx.doi.org/10.1038/s41598-021-926...
, applying MiSeq 16S rRNA gene sequencing of 328 open-pollinated silk samples (healthy/fungi-infected), reported that maize silks possess complex and dynamic microbiomes with more than 5,000 taxa spanning the prokaryotic tree of life (47 phyla/1,300 genera).

In tropical Africa and Latin America, silk-invaders include the mycotoxin-producing fungal pathogens Aspergillus flavus and Fusarium verticillioides that devastate tens of millions of maize farmers, where they produce carcinogens (Patriarca and Pinto, 2017PATRIARCA, A. and PINTO, F.V., 2017. Prevalence of mycotoxins in foods and decontamination. Current Opinion in Food Science, vol. 14, pp. 50-60. http://dx.doi.org/10.1016/j.cofs.2017.01.011.
http://dx.doi.org/10.1016/j.cofs.2017.01...
; Thompson and Raizada, 2018THOMPSON, M.E.H. and RAIZADA, M.N., 2018. Fungal pathogens of maize gaining free passage along the silk road. Pathogens, vol. 7, no. 4, pp. 81. http://dx.doi.org/10.3390/pathogens7040081. PMid:30314351.
http://dx.doi.org/10.3390/pathogens70400...
). These fungi produce mycotoxins with toxic effects on gastrointestinal, hematic, and reproductive systems, affecting livestock and human health (Patriarca and Pinto, 2017PATRIARCA, A. and PINTO, F.V., 2017. Prevalence of mycotoxins in foods and decontamination. Current Opinion in Food Science, vol. 14, pp. 50-60. http://dx.doi.org/10.1016/j.cofs.2017.01.011.
http://dx.doi.org/10.1016/j.cofs.2017.01...
). Altered temperature, rainfall, and humidity associated with climate change are expected to increase the fungus-based mycotoxins in maize (Magan and Medina, 2016MAGAN, N. and MEDINA, A., 2016. Integrating gene expression, ecology and mycotoxin production by Fusarium and Aspergillus species in relation to interacting environmental factors. World Mycotoxin Journal, vol. 9, no. 5, pp. 673-684. http://dx.doi.org/10.3920/WMJ2016.2076.
http://dx.doi.org/10.3920/WMJ2016.2076...
).

Alternative control methods of disease control for minimizing the harmful effects of chemical fungicides and increasing food production and quality have been studied and tested since the early twentieth century (Baker, 1987BAKER, K.F., 1987. Evolving concepts of biological control of plant pathogens. Annual Review of Phytopathology, vol. 25, no. 1, pp. 67-85. http://dx.doi.org/10.1146/annurev.py.25.090187.000435.
http://dx.doi.org/10.1146/annurev.py.25....
; Adetunji et al., 2019ADETUNJI, C.O., KUMAR, D., RAINA, M., AROGUNDADE, O. and SARIN, N.B., 2019. Endophytic microorganisms as biological control agents for plant pathogens: A panacea for sustainable agriculture. In: A. VARMA, S. TRIPATHI and R. PRASAD, eds. Plant biotic interactions. Cham: Springer. http://dx.doi.org/10.1007/978-3-030-26657-8_1.
http://dx.doi.org/10.1007/978-3-030-2665...
; Ons et al., 2020ONS, L., BYLEMANS, D., THEVISSEN, K. and CAMMUE, B.P.A., 2020. Combining biocontrol agents with chemical fungicides for integrated plant fungal disease control. Microorganisms, vol. 8, no. 12, pp. 1930. http://dx.doi.org/10.3390/microorganisms8121930. PMid:33291811.
http://dx.doi.org/10.3390/microorganisms...
; Awuchi et al., 2021AWUCHI, C.G., ONDARI, E.N., OGBONNA, C.U., UPADHYAY, A.K., BARAN, K., OKPALA, C.O.R., KORZENIOWSKA, M. and GUINÉ, R.P.F., 2021. Mycotoxins affecting animals, foods, humans, and plants: Types, occurrence, toxicities, action mechanisms, prevention, and detoxification strategies: a revisit. Foods, vol. 10, no. 6, pp. 1279. http://dx.doi.org/10.3390/foods10061279. PMid:34205122.
http://dx.doi.org/10.3390/foods10061279...
). Among the methods, the use of microorganisms with potential to control plant pathogens has been increasingly recognized as a promising environmentally friendly and low-cost approach compared to conventional methods (van Lenteren et al., 2018VAN LENTEREN, J.C., BOLCKMANS, K., KÖHL, J., RAVENSBERG, W.J. and URBANEJA, A., 2018. Biological control using invertebrates and microorganisms: plenty of new opportunities. BioControl, vol. 63, no. 1, pp. 39-59. http://dx.doi.org/10.1007/s10526-017-9801-4.
http://dx.doi.org/10.1007/s10526-017-980...
). Thus, biological control using antagonistic microorganisms may be a powerful alternative to inhibit the development of phytopathogens in different cultures (Tariq et al., 2020TARIQ, M., KHAN, A., ASIF, M., KHAN, F., ANSARI, T., SHARIQ, M. and SIDDIQUI, M.A., 2020. Biological control: a sustainable and practical approach for plant disease management. Acta Agriculturæ Scandinavica. Section B, Soil and Plant Science, vol. 70, no. 6, pp. 507-524. http://dx.doi.org/10.1080/09064710.2020.1784262.
http://dx.doi.org/10.1080/09064710.2020....
). Furthermore, the isolation and characterization of native microorganisms increase the efficiency of the biological control since they have a great capacity to compete and survive in their original environment (Figueroa-López et al., 2016FIGUEROA-LÓPEZ, A.M., CORDERO-RAMÍREZ, J.D., MARTÍNEZ-ÁLVAREZ, J.C., LÓPEZ-MEYER, M., LIZÁRRAGA-SÁNCHEZ, G.J., FÉLIX-GASTÉLUM, R., CASTRO-MARTÍNEZ, C. and MALDONADO-MENDOZA, I.E., 2016. Rhizospheric bacteria of maize with potential for biocontrol of Fusarium verticillioides. SpringerPlus, vol. 5, no. 1, pp. 330. http://dx.doi.org/10.1186/s40064-016-1780-x. PMid:27066355.
http://dx.doi.org/10.1186/s40064-016-178...
). Therefore, in this work, native endophytic and epiphytic microorganisms were isolated from the maize stigma collected from different locations aiming to obtain greater diversity and efficiency for F. verticillioides control. The direct isolation of microorganisms from the maize stigma, the main entry points for F. verticillioides into the plants, was carried out for the first time and may contribute to providing information about the microbial diversity in this organ of the maize plant and lead to the discovery of new species with biotechnological potential.

2. Material and Methods

2.1. Sample collection and isolation of microorganisms

Stigma of maize plant (Zea mays) cultivated in the cities of Sete Lagoas-MG (Embrapa Maize and Sorghum, experimental plots), Sidrolândia-MS (field maize), Goiânia-GO (Embrapa Rice and bean, experimental plots) and Sertaneja-PR (field maize) in the 2016 harvest were randomly collected.

Endophytic microorganisms candidates for biocontrol were isolated from corn stigmas washed and disinfected as Bulgari et al. (2009)BULGARI, D., CASATI, P., BRUSETTI, L., QUAGLINO, F., BRASCA, M., DAFFONCHIO, D. and BIANCO, P.A., 2009. Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR. Journal of Microbiology, vol. 47, no. 4, pp. 393-401. http://dx.doi.org/10.1007/s12275-009-0082-1. PMid:19763412.
http://dx.doi.org/10.1007/s12275-009-008...
. Then, they were macerated with the aid of a pistil and suspended in sterile saline solution (0.9%) to obtain dilutions (10-4 to 10−6) that were plated on Potato Dextrose Agar (BDA) medium. In half of the plates, a streptomycin solution and the other half of cycloheximide (20mg/L) were added to select fungi and bacteria, respectively. Epiphytic microorganisms were isolated using the same technique but without initial disinfestation of stigmas. The plates were incubated for seven days at 28 °C and visualized every 48 h for the isolation of microorganisms based on morphological differences such as colony shape and appearance, size, color, border type, and growth rate. The 167 isolated morphotypes were preserved in glycerol (20%) and stored at -4 °C (bacteria) and eight 8 °C (fungi) for further studies.

2.2. Selection of biocontrol agents against Fusarium verticillioides

One hundred and sixty-seven isolates were tested for their potential to inhibit the growth of F. verticillioides by the confrontation test of cultures in a solid medium. The F. verticillioides isolate (CML2743) used in the antagonism tests was obtained from the Phytopathology Laboratory of Embrapa Milho e Sorghum in Sete Lagoas-MG.

A 5 mm disk from the edge of the phytopathogen culture grown in PDA medium for five days at 28°C was removed and transferred to the center of a new plate containing the same culture medium. A 5mm mycelial disk of each fungal isolate or 10 µL of bacterial suspension (108 CFU/mL) was added at four equidistant points. Incubation was carried out at 28°C and 12 h photoperiod. In the negative control, measurements were performed after the entire growth of the pathogen in the plate.

Percent inhibition was calculated by PI= [(radius of mycelium in control - radius of mycelium with antagonist) / radius of mycelium in control)] × 100.

The analysis of variance (ANOVA) was used to check the obtained data, followed by the Scott-Knott means comparison test at p <0.05. All treatments were performed in triplicate and the results were expressed as mean of the repetitions.

2.3. Molecular identification of the most efficient isolates

Forty-two isolates that showed greater antagonist activities were identified through partial sequencing of the 16S rRNA gene and the ITS region. DNA extraction from the isolated bacteria and fungi was performed using the Wizard® Genomic DNA Purification Kit (Promega, USA) and the UltraClean™ Microbial DNA Isolation Kit (MoBio, USA) respectively. PCR amplification of the 16S rRNA region of bacterial isolates was performed with 8F primers: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3 (Turner et al., 1999TURNER, S., PRYER, K.M., MIAO, V.P.W. and PALMER, J.D., 1999. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. The Journal of Eukaryotic Microbiology, vol. 46, no. 4, pp. 327-338. http://dx.doi.org/10.1111/j.1550-7408.1999.tb04612.x. PMid:10461381.
http://dx.doi.org/10.1111/j.1550-7408.19...
). For the ITS region of the isolated fungi, the primers ITS4R:5’-TCCTCCGCTTATTGATATGC-3’ and ITS5F: 5’-GGAAGTAAAAGTCGTAACAAGG-3’ were used (White et al., 1990WHITE, T.J., BRUNS, T.D., LEE, S.B. and TAYLOR, J.W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M.A. INNIS, GELFAND, D.H., SNINSKY, J.J. and T.J. WHITE, eds. PCR protocols: a guide to methods and applications and applications. New York: Academic Press, pp. 315-322. http://dx.doi.org/10.1016/B978-0-12-372180-8.50042-1
http://dx.doi.org/10.1016/B978-0-12-3721...
). Products were analyzed by 1.0% agarose gel electrophoresis and documented using a Gel Logic 200 system (KODAK Company, USA). The PCR products were sequenced on the ABI PRISM 3500xL Genetic Analyzer sequencer (Applied Biosystem, USA) and the sequences obtained aligned with the Sequencer 4.1.4 program (Genes Codes Corporation). To identify the isolates, the alignment search tool (BLAST) was used to find similarities with sequences deposited in the NCBI database (http://www.ncbi.nlm.nih.gov/).

3. Results

3.1. Isolation and selection of biocontrol agents against Fusarium verticillioides

Based on their morphological characteristics, 167 microorganisms were isolated from maize stigmas collected in different producing regions of Brazil, being 46 isolated from the cities of Goiânia, 43 from Sidrolândia, 42 from Sertaneja, and 36 from Sete Lagoas. Endophytic and epiphytic microorganisms represented 46% and 54% of the total isolates, respectively.

Variations in the inhibition capacity of the isolates were observed according to the region of each microorganism (Figure 1). The most significant number of isolates that inhibited the growth of F. verticillioides (0 and 20%) were collected in Sete Lagoas. The inhibition index between 20.01 and 40% was obtained mainly by the isolates from Goiânia-GO. The isolates from Sidrolândia-MS showed the highest inhibition potential among all the microorganisms evaluated. In this region, the most significant number of microorganisms showed inhibition potential ranging from 40.01 to 60%, and it was the only region that contained an isolate that inhibited 100% of the phytopathogen growth (ISD04). The development of new, sustainable technologies based on the search aims for genetic resistance of plants, chemical substances, and biological control to reduce environmental and health problems generated by fungicides in agriculture. In this work, the search for microorganisms from different regions aimed to obtain a more significant number of isolates that could have potential as biocontrol agents. The antagonist agent selection test showed that in all four regions, microorganisms were found that showed great potential for inhibiting F. verticillioides.

Figure 1
Percentage of mycelial growth inhibition of Fusarium verticillioides by endophytic and epiphytic microorganisms from maize silks collected in different Brazilian regions.

All isolates were tested for antagonist activity by measuring the growth of F. verticillioides in the direct challenge test. Of these, 28 showed no difference in growth reduction compared to the control (Group A, Table 1). Another 139 isolates that significantly reduced the growth of F. verticillioides were separated into fifteen groups according to the statistical test concerning the percentage of inhibition. The most significant number of isolates (group K) showed an inhibition potential that ranged from 48.7 to 52.1% (Table 1).

Table 1
Groupings of isolates concerning the percentage of inhibition of F. verticillioides growth in a direct challenge test.

3.2. Molecular identification of selected isolates

Forty-two isolates with inhibition values of F. verticillioides growth ​​above 48.7% were identified based on partial sequencing of the 16S rRNA bacterial gene and ITS region of fungi. The molecular identification showed that these isolates belong to eight different genera: Bacillus sp. (57.1%), Burkholderia sp. (23.8%), Achromobacter sp. (7.1%), Pseudomonas sp. (2.4%), Serratia sp. (2.4%), Penicillium sp. (2.4%), Candida sp. (2.4%) and Aspergillus sp. (2.4%).

The most efficient bacteria were Bacillus subtilis, Bacillus velezensis, Burkholderia cepacia, Pseudomonas aeruginosa, Serratia nematodiphila, Achromobacter insuavis, Achromobacter xylosoxidans, and Burkholderia gladioli. The fungi that stood out in the control of F. verticillioides in vitro were Aspergillus flavus (IPR24) and Penicillium sp. (CT01) (Table 2).

Table 2
Isolates, collection site, type (lifestyle), percentage of inhibition, molecular identification and Genbank accession number of the 42 isolates that showed inhibition of the growth of Fusarium verticillioides ≥ 48.7%.

4. Discussion

Screening is a critical step in biological control, as the success of all subsequent steps depends on the ability of a procedure to identify candidates with antagonist potential (Cavaglieri et al., 2004CAVAGLIERI, L., PASSONE, A. and ETCHEVERRY, M., 2004. Screening procedures for selecting rhizobacteria with biocontrol effects upon Fusarium verticillioides growth and fumonisin B1 production. Research in Microbiology, vol. 155, no. 9, pp. 747-754. http://dx.doi.org/10.1016/j.resmic.2004.06.001. PMid:15501652.
http://dx.doi.org/10.1016/j.resmic.2004....
). In this work, the in vitro confrontation test was appropriate to evaluate many antagonistic microorganisms faster at a low cost. Of all tested isolates, 83% showed inhibitory activity against F. verticillioides. The microorganisms were isolated from various Brazilian maize-producing areas to obtain isolates to screen their potential as biocontrol agents against Fusarium verticillioides.

Considering that the maize silk is one of the main entry points of F. verticillioides into the plant (Munkvold et al., 1997MUNKVOLD, G.P., MCGEE, D.C. and CARLTON, W.M., 1997. Importance of different pathways for maize kernel infection by Fusarium moniliforme. Phytopathology, vol. 87, no. 2, pp. 209-217. http://dx.doi.org/10.1094/PHYTO.1997.87.2.209. PMid:18945144.
http://dx.doi.org/10.1094/PHYTO.1997.87....
), isolating native microorganisms from the maize silk was the main criteria used in this work. Due to the isolates' adaptation, they are presumably more suitable for biological control of F. verticillioides entering thorough maize stigma. Furthermore, adopting this strategy may allow the identification of microorganisms coexisting with the natural microbiota. Thus, they may present more competitive advantages than introduced exotic species (Figueroa-López et al., 2016FIGUEROA-LÓPEZ, A.M., CORDERO-RAMÍREZ, J.D., MARTÍNEZ-ÁLVAREZ, J.C., LÓPEZ-MEYER, M., LIZÁRRAGA-SÁNCHEZ, G.J., FÉLIX-GASTÉLUM, R., CASTRO-MARTÍNEZ, C. and MALDONADO-MENDOZA, I.E., 2016. Rhizospheric bacteria of maize with potential for biocontrol of Fusarium verticillioides. SpringerPlus, vol. 5, no. 1, pp. 330. http://dx.doi.org/10.1186/s40064-016-1780-x. PMid:27066355.
http://dx.doi.org/10.1186/s40064-016-178...
). The tests of antagonistic activity against F. verticillioides showed that the isolates from the four regions showed great potential for inhibiting F. verticillioides. In addition, these microorganisms, more adapted to their original site, may have a more remarkable ability to survive under field conditions and colonize the maize plants, which may increase the chances of successful biological control. Despite the widespread use of maize silk in traditional medicine and the knowledge of its role as one of the main entries of diseases in maize plants, studies on its chemical constituents and microbiota are scarce (Alam, 2011ALAM, E.A., 2011. Evaluation of antioxidant and antibacterial activities of Egyptian Maydis stigma (Zea mays hairs) rich in some bioactive constituents. The Journal of American Science, vol. 7, no. 4, pp. 726-729.; Hasanudin et al., 2012HASANUDIN, K., HASHIM, P. and MUSTAFA, S., 2012. Corn silk (Stigma maydis) in healthcare: a phytochemical and pharmacological review. Molecules, vol. 17, no. 8, pp. 9697-9715. http://dx.doi.org/10.3390/molecules17089697. PMid:22890173.
http://dx.doi.org/10.3390/molecules17089...
; Žilić et al., 2016ŽILIĆ, S., JANKOVIĆ, M., BASIĆ, B., VANČETOVIĆ, J. and MAKSIMOVIĆ, V., 2016. Antioxidant activity, phenolic profile, chlorophyll and mineral matter content of corn silk (Zea mays L): comparison with medicinal herbs. Journal of Cereal Science, vol. 69, pp. 363-370. http://dx.doi.org/10.1016/j.jcs.2016.05.003.
http://dx.doi.org/10.1016/j.jcs.2016.05....
; Thompson and Raizada, 2018THOMPSON, M.E.H. and RAIZADA, M.N., 2018. Fungal pathogens of maize gaining free passage along the silk road. Pathogens, vol. 7, no. 4, pp. 81. http://dx.doi.org/10.3390/pathogens7040081. PMid:30314351.
http://dx.doi.org/10.3390/pathogens70400...
).

The maize silk is an herb used traditionally as a therapeutic remedy to treat various diseases in many parts of the world (Alam, 2011ALAM, E.A., 2011. Evaluation of antioxidant and antibacterial activities of Egyptian Maydis stigma (Zea mays hairs) rich in some bioactive constituents. The Journal of American Science, vol. 7, no. 4, pp. 726-729.; Hasanudin et al., 2012HASANUDIN, K., HASHIM, P. and MUSTAFA, S., 2012. Corn silk (Stigma maydis) in healthcare: a phytochemical and pharmacological review. Molecules, vol. 17, no. 8, pp. 9697-9715. http://dx.doi.org/10.3390/molecules17089697. PMid:22890173.
http://dx.doi.org/10.3390/molecules17089...
). Furthermore, the maize stigma has been described as the most efficient form of plant contamination by fungal pathogens invading the developing grain using the silk channels and causing disruption of the seeds and severe ear rot (Warfield and Davis, 1996WARFIELD, C.Y. and DAVIS, R.M., 1996. Importance of the husk covering on the susceptibility of corn hybrids to Fusarium ear rot. Plant Disease, vol. 80, no. 2, pp. 208-210. http://dx.doi.org/10.1094/PD-80-0208.
http://dx.doi.org/10.1094/PD-80-0208...
; Khalaf et al., 2021KHALAF, E.M., SHRESTHA, A., RINNE, J., LYNCH, M.D.J., SHEARER, C.R., LIMAY-RIOS, V., REID, L.M. and RAIZADA, M.N., 2021. Transmitting silks of maize have a complex and dynamic microbiome. Scientific Reports, vol. 11, no. 1, pp. 13215. http://dx.doi.org/10.1038/s41598-021-92648-4. PMid:34168223.
http://dx.doi.org/10.1038/s41598-021-926...
). Thus, despite the widespread use of maize silk in traditional medicine and the knowledge of its role as one of the main entries of diseases in maize plants, studies on its chemical constituents and microbiota are scarce recent (Alam, 2011ALAM, E.A., 2011. Evaluation of antioxidant and antibacterial activities of Egyptian Maydis stigma (Zea mays hairs) rich in some bioactive constituents. The Journal of American Science, vol. 7, no. 4, pp. 726-729.; Hasanudin et al., 2012HASANUDIN, K., HASHIM, P. and MUSTAFA, S., 2012. Corn silk (Stigma maydis) in healthcare: a phytochemical and pharmacological review. Molecules, vol. 17, no. 8, pp. 9697-9715. http://dx.doi.org/10.3390/molecules17089697. PMid:22890173.
http://dx.doi.org/10.3390/molecules17089...
; Žilić et al., 2016ŽILIĆ, S., JANKOVIĆ, M., BASIĆ, B., VANČETOVIĆ, J. and MAKSIMOVIĆ, V., 2016. Antioxidant activity, phenolic profile, chlorophyll and mineral matter content of corn silk (Zea mays L): comparison with medicinal herbs. Journal of Cereal Science, vol. 69, pp. 363-370. http://dx.doi.org/10.1016/j.jcs.2016.05.003.
http://dx.doi.org/10.1016/j.jcs.2016.05....
; Thompson and Raizada, 2018THOMPSON, M.E.H. and RAIZADA, M.N., 2018. Fungal pathogens of maize gaining free passage along the silk road. Pathogens, vol. 7, no. 4, pp. 81. http://dx.doi.org/10.3390/pathogens7040081. PMid:30314351.
http://dx.doi.org/10.3390/pathogens70400...
). The potential antioxidant and healthcare applications of maize silk are due to the properties of its bioactive constituents, such as flavonoids and terpenoids (Alam, 2011ALAM, E.A., 2011. Evaluation of antioxidant and antibacterial activities of Egyptian Maydis stigma (Zea mays hairs) rich in some bioactive constituents. The Journal of American Science, vol. 7, no. 4, pp. 726-729.). In addition, many microorganisms and natural compounds derived from plants have fungistatic or fungicidal effects and can be used to manage plant diseases (Dias et al., 2020DIAS, A.L.B., SOUSA, W.C., BATISTA, H.R.F., ALVES, C.C.F., SOUCHIE, E.L., SILVA, F.G., PEREIRA, P.S., SPERANDIO, E.M., CAZAL, C.M., FORIM, M.R. and MIRANDA, M.L.D., 2020. Chemical composition and in vitro inhibitory effects of essential oils from fruit peel of three Citrus species and limonene on mycelial growth of Sclerotinia sclerotiorum. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 80, no. 2, pp. 460-464. http://dx.doi.org/10.1590/1519-6984.216848. PMid:31291410.
http://dx.doi.org/10.1590/1519-6984.2168...
; Ahmadu et al., 2021AHMADU, T., AHMAD, K., ISMAIL, S.I., RASHED, O., ASIB, N. and OMAR, D., 2021. Antifungal efficacy of Moringa oleifera leaf and seed extracts against Botrytis cinerea causing gray mold disease of tomato (Solanum lycopersicum L.). Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 81, no. 4, pp. 1007-1022. http://dx.doi.org/10.1590/1519-6984.233173. PMid:33175006.
http://dx.doi.org/10.1590/1519-6984.2331...
; Matrose et al., 2021MATROSE, N.A., OBIKEZE, K., BELAY, Z.A. and CALEB, O.J., 2021. Plant extracts and other natural compounds as alternatives for post-harvest management of fruit fungal pathogens: a review. Food Bioscience, vol. 41, pp. 100840. http://dx.doi.org/10.1016/j.fbio.2020.100840.
http://dx.doi.org/10.1016/j.fbio.2020.10...
). Feng et al. (2012)FENG, X., WANG, L., TAO, M.L., ZHOU, Q. and ZHONG, Z.H., 2012. Studies on antimicrobial activity of ethanolic extract of maize silk. African Journal of Microbiological Research, vol. 6, no. 2, pp. 335-338. http://dx.doi.org/10.5897/AJMR11.974.
http://dx.doi.org/10.5897/AJMR11.974...
reported that the ethanolic extract of maize silk could inhibit the microbial activity of many human pathogenic bacteria and fungi.

In the first report on maize silk-associated microbial communities, Diniz et al. (2017)DINIZ, G.F.D., AGUIAR, F.M., SANTOS, K.K.B., RIBEIRO, V.P., GOMES, E.A., COTA, L.V., CURY, J.C., MARRIEL, I.E. and OLIVEIRA, C.A., 2017 [accessed 29 March 2022]. Antagonistic potential of microorganisms isolated from corn against Fusarium verticillioides. In: Anais do 29º Congresso Brasileiro de Microbiologia [online], 22-25 October 2017, Foz do Iguaçu, Paraná, Brasil. Brasília: Embrapa. Available from: https://www.alice.cnptia.embrapa.br/alice/bitstream/doc/1078763/1/Antagonisticpotential.pdf
https://www.alice.cnptia.embrapa.br/alic...
found that the antagonistic activity of 174 bacteria and fungi against F. verticillioides was highly variable with 20% to up 60% activity.

The isolates from Sidrolândia-MS showed the highest inhibition potential among all the microorganisms evaluated. Probably, this find may be related to the wetter climate of this region, which is more favorable to the development of Fusarium. Also, this result may be due to higher disease pressure since the area in which the isolates were collected correspond to a large commercial farming area (Sidrolândia) and not a restricted experimental area, as in SL and Goiânia. However, these factors need to be investigated. Nevertheless, this result revealed that sampling at different sites is more efficient than sampling at a single site when searching for more antagonistic microorganisms in the control of Fusarium verticillioides.

In another study, Diniz et al. (2021)DINIZ, G.F.D., COTA, L.V., FIGUEIREDO, J.E.F., AGUIAR, F.M., SILVA, D.D., LANA, U.G.P., SANTOS, V.L., MARRIEL, I.E. and OLIVEIRA-PAIVA, C.A., 2021. Antifungal activity of bacterial strains from maize silks against Fusarium verticillioides. Archives of Microbiology, vol. 204, no. 1, pp. 89. http://dx.doi.org/10.1007/s00203-021-02726-4. PMid:34962587.
http://dx.doi.org/10.1007/s00203-021-027...
, analyzing the in vitro antifungal activity of four endophytic (ISD04 and IPR45) and epiphytic (CT02 and IM14) bacterial strains from maize silks, found inhibition values from 58.5 to 100%. In addition, the four isolates changed the hyphae morphology and inhibited the conidial germination by up to 100% (strain IPR45). Also, the four strains produced at least one enzyme with antifungal activity, and the microbiolized seeds reduced the fungal development in stored grains and stalk rot severity in the greenhouse by 72.6% (strain ISD04). Recently, Khalaf et al. (2021)KHALAF, E.M., SHRESTHA, A., RINNE, J., LYNCH, M.D.J., SHEARER, C.R., LIMAY-RIOS, V., REID, L.M. and RAIZADA, M.N., 2021. Transmitting silks of maize have a complex and dynamic microbiome. Scientific Reports, vol. 11, no. 1, pp. 13215. http://dx.doi.org/10.1038/s41598-021-92648-4. PMid:34168223.
http://dx.doi.org/10.1038/s41598-021-926...
, applying MiSeq 16S rRNA gene sequencing of 328 open-pollinated silk samples (healthy/fungi-infected), reported that maize silks possess complex and dynamic microbiomes with more than 5,000 taxa spanning the prokaryotic tree of life (47 phyla/1,300 genera). These results highlight the high complexity and the antifungal activity potential of silk-associated microbial communities as new biocontrol agents against F. verticillioides entering the maize silks.

Endophytic microorganisms live, at least part of their life cycle, within plant tissues, such as leaves, stems (Johnston-Monje and Raizada, 2011JOHNSTON-MONJE, D. and RAIZADA, M.N., 2011. Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. PLoS One, vol. 6, no. 6, pp. e20396. http://dx.doi.org/10.1371/journal.pone.0020396. PMid:21673982.
http://dx.doi.org/10.1371/journal.pone.0...
), and mainly roots (Miliute et al., 2015MILIUTE, I., BUZAITE, O., BANIULIS, D. and STANYS, V., 2015. Bacterial endophytes in agricultural crops and their role in stress tolerance: a review. Zemdirbyste-Agriculture, vol. 102, no. 4, pp. 465-478. http://dx.doi.org/10.13080/z-a.2015.102.060.
http://dx.doi.org/10.13080/z-a.2015.102....
). However, microorganisms can enter the plant tissue through emergence sites of lateral roots, lesions, natural openings such as stomata, and germinating radicles. Endophytic microorganisms are asymptomatic and, unlike pathogenic microorganisms, do not cause damage to the host plant. Furthermore, they can protect plants against phytopathogens by producing antimicrobial compounds, competition for space and nutrients, and induction of host plant resistance (Souza, 2001SOUZA, M.L., 2001. Utilização de microrganismos na agricultura. Biotecnologia, Ciência e Desenvolvimento, vol. 4, no. 21, pp. 28-31.).

The great diversity of endophytic and epiphytic microorganisms in the maize silks (Khalaf et al., 2021KHALAF, E.M., SHRESTHA, A., RINNE, J., LYNCH, M.D.J., SHEARER, C.R., LIMAY-RIOS, V., REID, L.M. and RAIZADA, M.N., 2021. Transmitting silks of maize have a complex and dynamic microbiome. Scientific Reports, vol. 11, no. 1, pp. 13215. http://dx.doi.org/10.1038/s41598-021-92648-4. PMid:34168223.
http://dx.doi.org/10.1038/s41598-021-926...
) and the high silk vulnerability as one of the main gateway for Fusarium verticillioides entering into the maize plants and colonizing the grains (Thompson and Raizada, 2018THOMPSON, M.E.H. and RAIZADA, M.N., 2018. Fungal pathogens of maize gaining free passage along the silk road. Pathogens, vol. 7, no. 4, pp. 81. http://dx.doi.org/10.3390/pathogens7040081. PMid:30314351.
http://dx.doi.org/10.3390/pathogens70400...
) point out the existence of intense niche competition for space and food between species (Senghor et al., 2020SENGHOR, L.A., ORTEGA-BELTRAN, A., ATEHNKENG, J., CALLICOTT, K.A., COTTY, P.J. and BANDYOPADHYAY, R., 2020. The atoxigenic biocontrol product aflasafe SN01 is a valuable tool to mitigate aflatoxin contamination of both maize and groundnut cultivated in Senegal. Plant Disease, vol. 104, no. 2, pp. 510-520. http://dx.doi.org/10.1094/PDIS-03-19-0575-RE. PMid:31790640.
http://dx.doi.org/10.1094/PDIS-03-19-057...
). Thus, it is reasonable to presume the occurrence of potential Fusarium antagonists among the maize-friendly microorganisms.

The molecular identification of the isolates revealed that both endophytic and epiphytic Bacillus spp. were found in more significant numbers. The prevalence of the genus Bacillus in different environments can be explained by the ability of this genus to form endospores that give them advantages over other microorganisms, such as greater tolerance to temperature fluctuations and nutrient shortages, among other environmental stresses (Sonenshein et al., 2002SONENSHEIN, A.L., LOSICK, R. and HOCK, J.A., 2002. Bacillus subtilis and its closest relatives: from genes to cells. Washington: American Society for Microbiology Press, pp. 451-475. https://doi.org/10.1128/9781555817992.
https://doi.org/10.1128/9781555817992...
). In addition, Bacillus isolates stood out as F. verticillioides control agents. This result agrees with other works that have shown the potential of this genus in the control of different fungal phytopathogens, including Fusarium (Figueroa-López et al. 2016FIGUEROA-LÓPEZ, A.M., CORDERO-RAMÍREZ, J.D., MARTÍNEZ-ÁLVAREZ, J.C., LÓPEZ-MEYER, M., LIZÁRRAGA-SÁNCHEZ, G.J., FÉLIX-GASTÉLUM, R., CASTRO-MARTÍNEZ, C. and MALDONADO-MENDOZA, I.E., 2016. Rhizospheric bacteria of maize with potential for biocontrol of Fusarium verticillioides. SpringerPlus, vol. 5, no. 1, pp. 330. http://dx.doi.org/10.1186/s40064-016-1780-x. PMid:27066355.
http://dx.doi.org/10.1186/s40064-016-178...
; Raaijmakers and Mazzola, 2012RAAIJMAKERS, J.M. and MAZZOLA, M., 2012. Diversity and natural functions of antibiotics produced by beneficial and plant pathogenic bacteria. Annual Review of Phytopathology, vol. 50, no. 1, pp. 403-424. http://dx.doi.org/10.1146/annurev-phyto-081211-172908. PMid:22681451.
http://dx.doi.org/10.1146/annurev-phyto-...
).

In a study by Passari et al. (2018)PASSARI, A.K., LALSIAMTHARI, P.C., ZOTHANPUIA., LEO, V.V., MISHRA, V.K., YADAV, M.K., GUPTA, V.K. and SINGH, B.P., 2018. Biocontrol of Fusarium wilt of Capsicum annuum by rhizospheric bacteria isolated from turmeric endowed with plant growth promotion and disease suppression potential. European Journal of Plant Pathology, vol. 150, no. 4, pp. 831-846. http://dx.doi.org/10.1007/s10658-017-1325-3.
http://dx.doi.org/10.1007/s10658-017-132...
, Bacillus was also the dominant genus among rhizosphere isolates with the potential to inhibit mainly Fusarium species. This genus represented 56.2% of the total of isolates, followed by Staphylococcus (15.6%), Pseudomonas (12.5%), Sphingomonas (9.3%), and Achromobacter (3.1%). The prevalence of Bacillus species was also found in the maize rhizosphere by Figueroa-López et al. (2016)FIGUEROA-LÓPEZ, A.M., CORDERO-RAMÍREZ, J.D., MARTÍNEZ-ÁLVAREZ, J.C., LÓPEZ-MEYER, M., LIZÁRRAGA-SÁNCHEZ, G.J., FÉLIX-GASTÉLUM, R., CASTRO-MARTÍNEZ, C. and MALDONADO-MENDOZA, I.E., 2016. Rhizospheric bacteria of maize with potential for biocontrol of Fusarium verticillioides. SpringerPlus, vol. 5, no. 1, pp. 330. http://dx.doi.org/10.1186/s40064-016-1780-x. PMid:27066355.
http://dx.doi.org/10.1186/s40064-016-178...
, looking for native microorganisms with the potential to control F. verticillioides. These authors identified as the primary bacterial genera Bacillus (341 isolated), Enterobacter (38), Pseudomonas (23), and Lysinibacillus (13), which exhibited percentage of inhibition ranging from 53 to 99%.

In the present study Bacillus represented 56.2% of the total of isolates, followed by Staphylococcus (15.6%), Pseudomonas (12.5%), Sphingomonas (9.3%), and Achromobacter (3.1%). The high prevalence of Bacillus species (82.17%) with the potential to control F. verticillioides was also found in the maize rhizosphere by Figueroa-López et al. (2016)FIGUEROA-LÓPEZ, A.M., CORDERO-RAMÍREZ, J.D., MARTÍNEZ-ÁLVAREZ, J.C., LÓPEZ-MEYER, M., LIZÁRRAGA-SÁNCHEZ, G.J., FÉLIX-GASTÉLUM, R., CASTRO-MARTÍNEZ, C. and MALDONADO-MENDOZA, I.E., 2016. Rhizospheric bacteria of maize with potential for biocontrol of Fusarium verticillioides. SpringerPlus, vol. 5, no. 1, pp. 330. http://dx.doi.org/10.1186/s40064-016-1780-x. PMid:27066355.
http://dx.doi.org/10.1186/s40064-016-178...
. The Bacillus species in this work have been described as biocontrol agents against different phytopathogens of important crops. Bacillus subtilis and Bacillus velezensis, are two closely related species (Dunlap et al., 2016DUNLAP, C.A., KIM, S.J., KWON, S.W. and ROONEY, A.P., 2016. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp. plantarum and ‘Bacillus oryzicola’ are later heterotypic synonyms of Bacillus velezensis based on phylogenomics. International Journal of Systematic and Evolutionary Microbiology, vol. 66, no. 3, pp. 1212-1217. http://dx.doi.org/10.1099/ijsem.0.000858. PMid:26702995.
http://dx.doi.org/10.1099/ijsem.0.000858...
). Indeed, recently many B. subtilis was reclassified as B. velezensis based in phylogenetic analysis (Palazzini et al., 2016PALAZZINI, J.M., DUNLAP, C.A., BOWMAN, M.J. and CHULZE, S.N., 2016. Bacillus velezensis RC 218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation: genome sequencing and secondary metabolite cluster profiles. Microbiological Research, vol. 192, pp. 30-36. http://dx.doi.org/10.1016/j.micres.2016.06.002. PMid:27664721.
http://dx.doi.org/10.1016/j.micres.2016....
). These authors also demonstrated the antagonist activity of B. velezensis against Fusarium graminearum, by reducing disease severity and decreasing mycotoxin accumulation under field conditions. The antifungal activity was attributed to antimicrobial lipopeptides such as surfactin, phengycin, and iturine, produced by B. velezensis (Palazzini et al., 2016PALAZZINI, J.M., DUNLAP, C.A., BOWMAN, M.J. and CHULZE, S.N., 2016. Bacillus velezensis RC 218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation: genome sequencing and secondary metabolite cluster profiles. Microbiological Research, vol. 192, pp. 30-36. http://dx.doi.org/10.1016/j.micres.2016.06.002. PMid:27664721.
http://dx.doi.org/10.1016/j.micres.2016....
). Martínez-Raudales et al. (2017)MARTÍNEZ-RAUDALES, I., DE LA CRUZ-RODRÍGUEZ, Y., ALVARADO-GUTIÉRREZ, A., VEGA-ARREGUÍN, J., FRAIRE-MAYORGA, A., ALVARADO-RODRÍGUEZ, M., BALDERAS-HERNÁNDEZ, V. and FRAIRE-VELÁZQUEZ, S., 2017. Draft genome sequence of Bacillus velezensis 2A-2B strain: a rhizospheric inhabitant of Sporobolus airoides (Torr.) Torr., with antifungal activity against root rot causing phytopathogens. Standards in Genomic Sciences, vol. 12, no. 73, pp. 73. http://dx.doi.org/10.1186/s40793-017-0289-4. PMid:29225729.
http://dx.doi.org/10.1186/s40793-017-028...
, analyzing the genome of B. velezensis, found several putative genes involved in the biosynthesis of antifungal compounds with toxic effects on fungi cell growth and morphology that resulted in cell death, and genes related to systemic induced resistance in plants. Chung et al. (2016)CHUNG, J.H., SONG, G.C. and RYU, C.M., 2016. Sweet scents from good bacteria: case studies on bacterial volatile compounds for plant growth and immunity. Plant Molecular Biology, vol. 90, no. 6, pp. 677-687. http://dx.doi.org/10.1007/s11103-015-0344-8. PMid:26177913.
http://dx.doi.org/10.1007/s11103-015-034...
demonstrated that species of the genus Bacillus, as well as species from Paenibacillus, Serratia, and Pseudomonas produce volatile compounds (e.g., 2,3-butanediol) that elicit the host immune response. These results may explains the characteristics of this species as fungal antagonist agent (Khalaf et al., 2021KHALAF, E.M., SHRESTHA, A., RINNE, J., LYNCH, M.D.J., SHEARER, C.R., LIMAY-RIOS, V., REID, L.M. and RAIZADA, M.N., 2021. Transmitting silks of maize have a complex and dynamic microbiome. Scientific Reports, vol. 11, no. 1, pp. 13215. http://dx.doi.org/10.1038/s41598-021-92648-4. PMid:34168223.
http://dx.doi.org/10.1038/s41598-021-926...
).

Burkholderia cepacia is also an antagonistic of phytopathogenic fungi, including Fusarium. B. cepacia produce an antifungal compound (CF66I) that inhibit the growth of F. solani (Li et al., 2009LI, X., QUAN, C.S., YU, H.Y., WANG, J.H. and FAN, S.D., 2009. Assessment of antifungal effects of a novel compound from Burkholderia cepacia against Fusarium solani by fluorescent staining. World Journal of Microbiology & Biotechnology, vol. 25, no. 1, pp. 151-154. http://dx.doi.org/10.1007/s11274-008-9861-9.
http://dx.doi.org/10.1007/s11274-008-986...
). High concentrations of CF66I caused the death of the phytopathogen, and low concentrations changed the hyphae structure, such as swelling and abnormal deposition of chitin, which also inhibited the growth of the fungus. Pseudomonas aeruginosa, another species identified in this work, is an efficient biocontrol agent against F. verticillioides (Borah et al., 2016BORAH, S.N., GOSWAMI, D., SARMA, H.K., CAMEOTRA, S.S. and DEKA, S., 2016. Rhamnolipid biosurfactant against Fusarium verticillioides to control stalk and ear rot disease of maize. Frontiers in Microbiology, vol. 7, pp. 1505. http://dx.doi.org/10.3389/fmicb.2016.01505. PMid:27708638.
http://dx.doi.org/10.3389/fmicb.2016.015...
). P. aeruginosa secret biosurfactant, identified as rhamnolipid, with antifungal activity through damaging the mycelial structure of the fungus. In addition, P. aeruginosa supernatants disperse sulfate-reducing bacterium biofilms, by producing four proteins (putative phospholipases) that influence biofilm formation (Wood et al., 2018WOOD, T.L., GONG, T., ZHU, L., MILLER, J., MILLER, D.S., YIN, B. and WOOD, T.K., 2018. Rhamnolipids from Pseudomonas aeruginosa disperse the biofilms of sulfate-reducing bacteria. NPJ Biofilms and Microbiomes, vol. 4, no. 1, pp. 22. http://dx.doi.org/10.1038/s41522-018-0066-1. PMid:30302271.
http://dx.doi.org/10.1038/s41522-018-006...
). Seed treatment with P. aeruginosa efficiently suppressed the disease symptoms and controlled stem and ear rot, increased plant biomass, and improved the fruiting of corn plants.

The species Serratia nematodiphila can inhibit the growth and aflatoxins levels of Aspergillus parasiticus in peanut seeds through chitinase production, which degrades the fungal cell wall (Wang et al., 2013WANG, K., YAN, P., CAO, L., DING, Q., SHAO, C. and ZHAO, T., 2013. Potential of chitinolytic Serratia marcescens strain JPP1 for biological control of Aspergillus parasiticus and aflatoxin. BioMed Research International, vol. 2013, pp. 397142. http://dx.doi.org/10.1155/2013/397142. PMid:23865052.
https://doi.org/10.1155/2013/397142...
). Thus, in addition to its potential as a biological control agent, this species is a plant growth promoter. In work by Khan et al. (2017)KHAN, A.R., PARK, G.-S., ASAF, S., HONG, S.-J., JUNG, B.K. and SHIN, J.-H., 2017. Complete genome analysis of Serratia marcescens RSC-14: A plant growth-promoting bacterium that alleviates cadmium stress in host plants. PLoS One, vol. 12, no. 2, pp. e0171534. http://dx.doi.org/10.1371/journal.pone.0171534. PMid:28187139.
http://dx.doi.org/10.1371/journal.pone.0...
, the complete genome of Serratia nematodiphila revealed the existence of genes involved in the biosynthesis of indoleacetic acid (IAA), phosphate solubilization, and enzymatic genes to oxidative stress. Likewise, Burkholderia gladioli specie is a biological control agent against different phytopathogenic fungi (Elshafie et al., 2012ELSHAFIE, H.S., CAMELE, I., RACIOPPI, R., SCRANO, L., IACOBELLIS, N.S. and BUFO, S.A., 2012. In vitro antifungal activity of Burkholderia gladioli pv. agaricicola against some phytopathogenic fungi. International Journal of Molecular Sciences, vol. 13, no. 12, pp. 16291-16302. http://dx.doi.org/10.3390/ijms131216291. PMid:23208371.
http://dx.doi.org/10.3390/ijms131216291...
). The antifungal activity of B. gladioli is due to extracellular hydrolytic enzymes, such as chitinase, protease, and glucanase, besides diffusible and volatile secondary metabolites that reduced the growth rate of F. oxysporum and Rhizoctonia solani. Burkholderia gladioli were also identified as corn growth promoter due to its activity of phosphorus solubilizer, phytohormone producers such as indoleacetic acid (IAA), gibberellin, and other compounds (Gunjal and Kapadnis, 2013GUNJAL, A.B. and KAPADNIS, B.P., 2013. Burkholderia gladioli, an endophyte with plant growth promoting potential. Journal of Chemo and Biosphere, pp. 43-53.).

Achromobacter xylosoxidans, frequently found in the rhizosphere zone, has been identified as a biocontrol agent of Fusarium spp. in tomato plants due to siderophore and chitinase production, and induction of systemic resistance in plants (Vaidya et al., 2001VAIDYA, R.J., SHAH, I.M., VYAS, P.R. and CHHATPAR, H.S., 2001. Production of chitinase and its optimization from a novel isolate Alcaligenes xylosoxydans: potential in antifungal biocontrol. World Journal of Microbiology & Biotechnology, vol. 17, no. 7, pp. 691-696. http://dx.doi.org/10.1023/A:1012927116756.
http://dx.doi.org/10.1023/A:101292711675...
; Moretti et al., 2008MORETTI, M., GILARDI, G., GULLINO, M.L. and GARIBALDI, A., 2008. Biological control potential of Achromobacter xylosoxydans for suppressing Fusarium wilt of tomato. International Journal of Botany, vol. 4, no. 4, pp. 369-375. http://dx.doi.org/10.3923/ijb.2008.369.375.
http://dx.doi.org/10.3923/ijb.2008.369.3...
).

5. Conclusions

The present study successfully evaluated the antifungal activity of endophytic and epiphytic microbiota associated with maize silks.

The maize silk microbiota is highly variable, highlighting its potential for selecting bacterial antagonists against fungal diseases in maize.

The study highlights the potential of epiphytic and endophytic microorganisms as an effective biocontrol strategy against colonization of maize plants by F. verticillioides.

Sustainable alternatives such as biological control are critical to minimize the hazards associated with the current chemical methods for fungal disease control.

Acknowledgements

The authors would like to thank the CNPq and Embrapa Milho e Sorgo for financial support.

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Publication Dates

  • Publication in this collection
    29 Aug 2022
  • Date of issue
    2022

History

  • Received
    29 Mar 2022
  • Accepted
    01 Aug 2022
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