ISOLATION AND IDENTIFICATION OF BROAD-SPECTRUM ANTAGONIST BACTERIA AGAINST PATHOGENIC FUNGI OF MAIZE CROP

– Fungal diseases may cause significant damage to crops worldwide, generating yield losses, poor grain quality, and health risks to humans and animals. Biological control using antagonistic bacteria offers innovative solutions for sustainable management aiming at plant protection. However, beneficial plant-microorganism interactions are particular, and few antagonists with broad-spectrum activity have been reported. In this work, two bacteria isolated from sorghum seeds were identified by partial sequencing of the 16S rRNA gene and tested in vitro for their capacity to control six important pathogenic fungi of maize: Fusarium verticillioides , Macrophomina phaseolina , Stenocarpella sp., Fusarium graminearum , Colletotrichum graminicola , and Bipolaris sp. The molecular identification revealed that the bacterial isolates belong to the genera Bacillus (strain LIS05) and Paenibacillus (strain LIS04). Both bacterial isolates inhibited the growth of all six phytopathogens by at least 49%. The isolate LIS05 showed the most significant antagonistic potential against the fungal pathogens tested, at an average of 73% inhibition. The highest antagonist activity (86.1% inhibition) was observed in the confrontation test between the isolate LIS05 and C . graminicola . In addition to the mycelial growth inhibition, the isolate LIS04 blocked the production of dark pigments by Bipolaris sp. This study showed that LIS05 and LIS04 are promising alternatives for developing integrated management strategies to control fungal diseases in maize and sorghum.

Fusarium verticillioides, Fusarium graminearum, Stenocarpella maydis, and Colletotrichum graminicola are currently considered the most important diseases of maize due to the damages, yield losses, and reduction of grain size and quality they cause to the crop (Reis & Casa, 1996;Wordell Filho & Casa, 2010;Sabato & Fernandes, 2014;Pfordt et al., 2020).In addition, these phytopathogens also cause ear rot with the formation of burnt kernels, resulting in a reduction of the product's value in the market since a percentage referring to the presence of these grains is deducted from the sale value.(Silva et al., 2015).
In addition, burned grains have a high reduction in their nutritional value, besides lighter than healthy grains, leading to a reduction in productivity (Costa et al., 2011).

Fusarium
verticillioides and F.
graminearum cause seedling death and ear-, rootand stem rot, leading to loss of productivity and grain quality worldwide (Munkvold, 2003).Both species produce mycotoxins, toxic metabolites harmful to humans and animals that consume the contaminated grains (Deepa & Sreenivasa, 2017;Blacutt et al., 2018).Fumonisins are the most common mycotoxins associated with F.
verticillioides (Lanza et al., 2014;Pitt, 2014;Blacutt et al., 2018), while deoxynivalenol (DON) and nivalenol (NIV) of the type B trichothecene group are frequent in F. graminearum (Munkvold & Desjardins, 1997).Grain contamination by mycotoxins may constitute an obstacle to international trade due to phytosanitary barriers imposed by consumer countries with strict control Maize is the second most important crop in the Brazilian agricultural scenario, surpassed by soybean (CONAB, 2022).Although Brazil is one of the world's largest maize producers, its average productivity is still lower than the leading producer countries.This scenario has been attributed to factors affecting maize yield, including soil type, low fertility, water stress, sowing period, hybrid yield potential, weeds competition, insect attack, and diseases (Barroso et al., 2017).Crop damages and yield losses caused by fungal diseases have raised concerns for producers and technicians (Lanza et al., 2012), affecting the quantity and quality of the grains produced (Nguyen et al., 2017).
It is estimated that more than 15% of all losses in agricultural production worldwide are due to plant diseases, with more than 70% being caused by fungi (Dobrzyński et al., 2023;Asad, 2022;Liu et al., 2017) Reis & Casa, 1996;Sabato & Fernandes, 2014;White, 1999;).When the plant is lodged, the ear comes into direct contact with the soil, favoring contamination by storing fungi and compromising grain quality (Casa et al., 2006).
Macrophomina phaseolina is also a critical phytopathogen of maize causing the disease known as macrophomina rot or gray rot, whose symptoms are characterized by black lesions on the roots and stems of infected plants, which can progress to wilting, falling plants and until an early death.When death does not occur, the plants wilt, drop leaves and reduce productivity.(Singh et al., 1990, Costa et al., 2019).Colletotrichum graminicola is another permanent fungus species in the maize crop, causing the stem rot known as stem anthracnose.This phytopathogen can infect all plant parts, resulting in different symptoms on the leaves, stem, ear, roots, and tassel (White, 1999).The fungus colonizes the stem tissues, reducing the absorption of water and nutrients, thus reducing weight and compromising the regular filling of the grains, which results in significant yield losses (Parreira et al., 2016).Bipolaris maydis affect maize crops in hot and humid regions around the world, causing the foliar disease known as bipolaris spot (Costa et al., 2014).The increase of lesions on the leaf surface consequently impairs the photosynthetic area of the plants, generating losses or reductions in crop productivity (Agrios, 2005).Losses were more significant than 70% reported in temperate regions and hot and humid tropical areas, where bipolaris spot occurs more severely.In Brazil, high disease severity was detected in some states, such as Rondônia, Mato Grosso, Goiás, and Tocantins (Costa et al., 2014).
Biological control involves using organisms that interfere with developing pathogens and plant pests.Biological control is a natural tool and an eco-friendly alternative to overcome the hazards of chemical methods for plant protection (Saito et al., 2009;Brito et al., 2013).Thus, microorganisms with antagonistic activity may depict a promising approach for fungal disease management besides their simple cultivation, environmental safety, and low-cost compared to conventional methods (Silva et al., 2003;Brito et al., 2013).Therefore, in this work, epiphytic bacteria isolated from sorghum seeds without apparent fungal growth were selected for in vitro tests against F. verticillioides since it constitutes one of the primary source of grain contamination.

Many strategies have been proposed
to control these phytopathogens (Khan et al., 2017), including a promising alternative to biological control (Rahman, 2018).This strategy has the advantages of being environmentally safe and inexpensive compared to conventional methods for controlling fungal diseases (Brito et al., 2013).
Among the bacterial genera used as

Microorganisms and culture conditions
In the study, the phytopathogenic strain concentration of 20 ng/μL.

16S rRNA gene amplification
PCR reactions were made with the universal primers 8F and 1492R (Lane et al., 1985;Turner et al., 1999)

DNA sequencing
The amplified DNA fragments were purified with 4 μL of Exo-Sap enzyme (GE HealthCare, USA) in a 15 μL product of the PCR reaction.

Identification of bacteria isolated from sorghum seeds and antifungal activity
During an experiment aiming to isolate pathogenic fungi from sorghum seeds, the observation of seeds completely colonized by fungi and seeds without fungi but colonized by bacteria indicated that those bacteria were antagonists of seed-colonizing or saprophytic fungi.Thus, the seeds without fungi were selected for further experiments.After the bacterial colonies grew in the medium, two colonies were replicated until pure colonies isolation.
The two bacterial isolates showed a broad spectrum and high growth inhibition activity against the six phytopathogenic fungi relevant to the maize crop: Fusarium verticillioides, Bipolaris sp., C. graminicola, F. graminearum, M. phaseolina and Stenorcapella maydis (Figure 2, Table 1.).Both bacterial isolates showed inhibition values above 49% (Table 1).As expected, the inhibition zone was not observed in plates used as a negative control, where fungal mycelia covered the entire surface of the culture medium.The Bacillus isolate LIS05 statistically showed the highest percentages of inhibition of the five phytopathogens (overall average of 73% inhibition).The highest overall inhibition percentage was also observed with the isolate (LIS05) against the fungus C. graminicola (86.1%), which causes anthracnose disease in maize.
Plants face constant challenges against fungal diseases in their natural environment, and several diseases caused by different phytopathogens may occur in the same crop throughout its life cycle (Fernandes et al., 2021).
A notable effect observed in this study was the inhibition of the production of dark pigments by the hyphae of Bipolaris sp. by metabolites secreted by the isolate (LIS04) of Paenibacillus sp.(Figure 3).
Melanized fungi are more resistant to lysis caused by cell wall-degrading enzymes released by antagonistic microorganisms, especially in soil.As demonstrated by Butler et al. (1989), the cellular integrity of a melanized fungus was maintained in the presence of high concentrations of lytic enzymes for several days, while its albino mutants (without melanin) were destroyed in a matter of minutes.Controlling a pathogenic fungus by interfering with its infection mechanism is an exciting strategy, as it does not involve interference in biological processes common to other organisms and causes fewer effects on the host plant.(Jennings et al., 2000).
Thus, the inhibition of melanin synthesis by LIS04 is a vital control mechanism that must be considered when selecting antagonists to develop new biofungicides.
Diseases caused by fungi in maize crops have been a serious problem worldwide, and different strategies have been proposed to control these pathogens (Khan et al., 2017).
Biological control has been presented as a promising alternative in this case (Rahman, 2018), as it has the advantages of being safe for the environment and low cost compared to conventional treatment methods (Brito et al., 2013).In this work, the in vitro selection method made it possible to evaluate bacteria of the genera Antagonist organisms can prevent the growth of phytopathogenic fungi through different modes of action, such as releasing toxic substances into the environment, such as antibiotics and extracellular hydrolytic enzymes (chitinases, cellulases, glucanases, proteases, and lipases (Mabood et al., 2014).In addition, many microbial biological control agents induce the plant defense mechanisms against pathogens by acting as elicitors that induce a signal to stimulate the plant defense mechanism against pathogens (Zehra et al., 2021), or controlling phytopathogens by mycoparasitism (Deacon, 1980).Currently, biological control using antagonistic microorganisms has been consolidating as a viable alternative to control the development of phytopathogens in different crops (Tariq et al., 2020).Antagonistic microorganisms can be isolated from soil (rhizosphere) or different parts of plants (epiphytic and endophytic) of different species.On the other hand, isolating native microorganisms increases the efficiency of biological control, as they present adaptations to compete and survive in their original environment (Figueroa-López et al., 2016).

Figure 1 .
photoperiod.Seed with visible bacterial mass without mycelial growth were used for bacterial isolation (Figure1).Samples of bacterial mass were collected using sterile platinum loops and inoculated through streaks on the surface of plates containing TSA Soy Triptone Agar culture medium(Kasvi, Brazil).Colonies with different macromorphological characteristics were

, 0. 5 μL
Big Dye V3.1 (Applied Biosystems, USA), 1.75 μL of reaction buffer (Applied Biosystems, USA) and 2.75 μL of ultrapure water.Then, the plates were incubated in a thermocycler under the following conditions: 96 °C for 1 minute, 96 °C for 2 seconds, 50 °C for 15 seconds, 60 °C for 4 minutes, repeated 30 times.After the end of the reaction, 60 μL of absolute ethanol plus 5 μL of EDTA (125 mM) were added.Then, the samples were homogenized and incubated in the dark for 15 minutes at room temperature.After this time, the samples were centrifuged at 4.000 rpm for 45 minutes.The supernatant was discarded, and 100 μL of 70% (v/v) ethanol was added to each sample.Again, the samples were centrifuged at 4.000 rpm for 10 minutes.Then, the ethanol was discarded, and the samples were centrifuged upside down at 300 rpm with low acceleration and deceleration.Subsequently, they were placed in an oven at 65 °C for 3 minutes or until complete drying.Finally, 10 μL of HI-DI formamide (Applied Biosystems, USA) were added to each sample, followed by denaturation in a thermocycler at 95 °C for 5 minutes.The samples were analyzed in the DNA sequencer model ABI PRISM 3500xL Genetic Analyzer (Applied Biosystem, USA), and the sequences obtained were aligned using the Sequencher 4.1.4program (Genes Codes Corporation).The isolates were identified by sequence comparison to the National Center for Biotechnology Information (NCBI) database using the BLASTN program (http://blast.ncbi.nlm.nih.gov).The sequences were deposited in Genbank and received the following access codes: OR542589.1 and OR548005.1.

Bacillus
and Paenibacillus with broad-spectrum antagonistic potential more quickly and at a lower cost.Furthermore, it allowed us to identify another possible mechanism of action used by antagonists: the inhibition of the production of dark pigments by the phytopathogen.These results open new perspectives for using LIS04 and LIS05 strains to develop multifunctional biofungicides.

Figure 3 .
Figure 3. Inhibition of melanin production by the phytopathogen Bipolaris sp. by the antagonist bacterium LIS04 of Paenibacillus sp.Growth on the control plate of the phytopathogen Bipolaris sp. with the production of dark pigments (A).Inhibition of fungus and pigment production by Paenibacillus sp.LIS04 (B).

Table 1 .
In vitro Inhibition Zone (IZ %) of the growth of six phytopathogens by antagonist bacteria compared to control plates containing only a phytopathogen.The Inhibition Zone was calculated by the formula IZ % = (N1-N2) x 100/N1, where N1= radius of the mycelium in the absence of the antagonist; N2= radius of the mycelium in the presence of the antagonist.Means followed by the same letters in the column (lower case letters) or row (upper case letters) do not differ statistically by the Scott-Knott Test at 5% probability (p <0.05).