Biocontrol potential of actinobacteria against Pantoea ananatis, the causal agent of maize white spot disease

Abstract Pantoea ananatis is the causal agent of maize white spot, a foliar disease responsible for significant maize yield reduction worldwide, especially in Brazil. In general, the maize foliar diseases control involves the adoption of resistant genotypes and pesticides application. However, the use of agrochemicals can significantly cause increase production costs, damage to human health and negative environmental impacts. In this sense, the use of biological control agents has been considered among the most promising eco-friendly technologies for sustainable agriculture. Actinobacteria, particularly of Streptomyces genus, has been widely recognized as agroindustrially important microorganism due to its potential in producing diverse range of secondary metabolites, including antibiotics and enzymes. Thus, the aim of this work is to characterize and to evaluate the potential of soil actinobacteria for P. ananatis control. We observed that 59 actinobacteria strains (85%) exhibited proteolytic or chitinolytic activity. Only the strains Streptomyces pseudovenezuelae ACSL 470, that also exhibited high proteolytic activity, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35 demonstrated high or moderate antagonist activity in vitro against P. ananatis. Temporal analysis of metabolites produced by these strains growth in different liquid media indicated greater antibacterial activity at 72 h. In this condition, chromatographic and mass spectrometry analysis revealed that S. pseudovenezuelae ACSL 470 strain produced neomycin, an aminoglycoside antibiotic that displayed high bactericidal activity in vitro against P. ananatis. This is the first report of actinobacteria acting as potential microbial antagonists for P. ananatis control. Further studies are needed to determine the control efficacy of maize white spot disease by Streptomyces strains or their metabolites in greenhouse and field conditions.

necessary for bacterial and fungal growth.Furthermore, they act directly on structures such as cell membrane and ribosomes, leading to cell damage, cell shrinkage, cytosolic loss and microorganism death (Alekhya and Gopalakrishnan, 2014;Gopalakrishnan et al., 2020;Sharma and Thakur, 2020).Thus, the aim of this work is to evaluate in vitro the potential of Brazilian soil actinobacteria strains and the mechanisms involved in the control of P. ananatis, the causal agent of maize white spot disease.

Evaluation of proteolytic and chitinolytic activity
We evaluated proteolytic and chitinolytic activity of all actinobacteria strains.Chitinase production was verified in chitin-yeast-salt extract (CYS) (Kavitha and Vijayalakshmi, 2011) and proteases in agar-gelatin-milk (AGL) (Sarmento et al., 2021).The formation of a clear halo surrounding the colony indicates proteolytic or chitinolytic activity.

Evaluation of the antibacterial activity of actinobacteria against Pantoea ananatis
In order to select actinobacterial strains with antibacterial activity against P. ananatis, we performed a primary selection in a solid medium.Aliquots containing 10 5 cells.mL - of the sixty-nine actinobacteria strains were individually inoculated, in the form of spots, in Petri dishes containing AGA medium and incubated for 14 days at 28 °C.After incubation, the plates were treated with and without ultraviolet (UV) radiation for 15 min for inactivation of actinobacteria vegetative cells.Then, the colonies were covered with 5 mL of Tryptone Soya Broth (TSB) medium containing P. ananatis cells (Williams et al., 1983), followed by incubation at 28 °C for 48 h.After this period, the inhibition halos indicative of antimicrobial activity against P. ananatis were measured.Antimicrobial activity was considered absent (halo = 0 mm), low (halo: 7 -10 mm), moderate (halo: 11 -14 mm) and high (halo > 14 mm) (Silva et al., 2020) (Figure 1).The actinobacteria with moderate and high antibacterial activity against P. ananatis CMPC 40 strain were selected for secondary antibacterial activity assay.

Introduction
Maize (Zea mays L.) is one of the most economically important cereals worldwide, extensively used for food, feed and in high-tech industries (Langner et al., 2019).Although Brazil occupies the third position on world maize production, this crop is susceptible to various foliar diseases (Paccola, 2002;FAO, 2020).Furthermore, due to changes in cultivation strategies, climate, and the extensive use of susceptible hybrids, the occurrence and incidence of maize diseases has increased considerably (Faria et al., 2016;Mueller et al., 2020).
Maize white spot, caused by the bacterium Pantoea ananatis, emerged as one of the most important foliar maize disease widely distributed in America (Kistner et al., 2021), Africa (Goszczynska et al., 2007), Europe (Krawczyk et al., 2010) and Asia (Cui et al., 2022), and is considered a potential threat to maize production in regions where high humidity and low night time temperatures are prevalent during the growing season.Symptoms are initially expressed as aqueous lesions in the basal leaves, rapidly spreading to the plant with a chlorotic appearance at more advanced stages of the disease, causing drastically reducing cycle and photosynthetic area of the affected plants (Derera et al., 2007).In Brazil, yield loss was as high as 60% due to the reduction in grain size and weight (Escanferla et al., 2018;Sun et al., 2020).
The adoption of maize resistant hybrids and pesticides application have been employed for maize white spot disease management.The use of disease-resistant genotypes has been the most efficient strategy to reduce yield losses and collaborate with environmental preservation, although it presents limitations and increases the cost of production (Mueller et al., 2020;Kistner et al., 2021).In the other side, the indiscriminate use of pesticides can lead to negative environmental impacts, damage to human health and the selection of resistant pathogens (Bastos et al., 2019;Lopes-Ferreira et al., 2022).Then, there is an urgent demand for ecologically compatible and efficient strategies to suppress pathogens in both conventional and organic agriculture.
In this scenario, the use of biological control by microorganisms as natural agents can be a viable, promising and ecologically alternative for plant diseases control.In recent decades, the selection of microorganisms with potential use in plant disease biocontrol has become one of the main targets of agricultural research (Chanthasena and Nantapong, 2016;Kaur et al., 2019).The adoption of biocontrol agents can increase productivity, improve the phytosanitary status of plants, stimulate more sustainable food production, decrease the application of pesticides and reduce the costs involved in the process (Zou et al., 2021).Several studies have identified efficient antagonist microorganisms for biological control (Dornelas et al., 2017;Melo et al., 2021).
Actinobacteria, especially of Streptomyces genus, have been widely recognized as agroindustrially important microorganism due to its potential in producing diverse range of secondary metabolites, including antibiotics and enzymes, with potential use in biocontrol of phytopathogenic microorganisms (Kaur et al., 2019).The actinobacteria selected were initially grown in Yeast Malt (ISP-2) medium under constant agitation at 28 °C for 48 h.Then, aliquots of each isolate (10 5 cells.mL - ) were transferred to tubes containing ISP-2 or M1 minimal medium (M1) (Shirling and Gottlieb, 1966).The actinobacteria strains growth in both culture medium was carried out under constant agitation for 120 h at 28 °C.Every 24 h, 5 mL of the samples were centrifuged at 4,000 rpm for 15 minutes and the supernatant was stored at -80 °C.
Paper discs of 6 mm of diameter with 20 µL of the supernatants (metabolites) were transferred to plates individually covered with P. ananatis strains, CMPC 40 and CMPC 105.The plates were incubated at 30 °C for 48 h and inhibition halos was evaluated (Bauer et al., 1966;Rodrigues et al., 2019).After determining the culture medium and time of growth in which there was greater antibacterial activity, the samples containing the metabolites produced by actinobacteria strains were lyophilized and resuspended in 5 mL of sterile deionized water.

Analysis of antibacterial agents produced by actinobacteria strains by ultra-high performance liquid chromatography coupled to mass spectrometry
Based on the results of primary and secondary selection for antibacterial activity against P. ananatis, we evaluated the antibiotic production by the actinobacteria strains Streptomyces pseudovenezuelae ACSL 470, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35.Initially, 1 mL of the concentrated supernatant containing the metabolites from each sample was transferred to conical tubes containing 9 mL of 5% (w/v) aqueous trichloroacetic acid (TCA) solution.The mixture was stirred for 5 min and 2 mL of the mixture was transferred to microtubes and centrifuged at 14,000 rpm for 12 min at 4 °C.The extracts were filtered through a nylon membrane with a 0.22 µm pore and a diameter of 13 mm in glass vials for an automatic injector and analyzed by ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS/MS) with a source of electrospray ionization (ESI in positive mode).The UHPLC-MS/MS system consisted of a binary pump with an automatic injector coupled to a triple quadrupole mass spectrometer with ESI source, RRLC 1200 (Agilent Techonologies, EUA) and API 5000 system (Applied Biosystems, EUA).Data acquisition was performed using Analyst® software version 1.5.1 (Agilent Techonologies, EUA).The analyzes of the extracts were carried out according to the following chromatographic conditions: Zorbax Eclipse XDB C18 column, 50 x 4.6 mm, 1.8 µm; injection volume of 10 µL; column temperature of 35 °C; mobile phase A -0.2% (v/v) of heptafluorobutyric acid in deionized water; mobile phase B -acetronitrile; flow of 0.6 mL/min and gradient system (initial time -90% A; 2.0 min -50% A; 2.5 min -50% A; 3.0 min -90% A and 6 min -90% A).The conditions used in mass spectrometry were: electrospray + ionization mode, 650 °C source temperature, 5500 V ion spray voltage (IS), collision gas (CAD) 6, curtain gas (CUR) 20, ion source gas 1 (GS1) 50 and ion source gas 2 (GS2) 50.
The analyzed metabolytes were from the group of aminoglycosides (spectinomycin, streptomycin, dihydroestreptomycin, amikacin, hygromycin, apramycin, gentamicin, neomycin, tobramycin, kanamycin) and tetracyclines (oxytetracycline, tetracycline, clortetracycline and fluoxoxin and fluoxycline).Positive control (culture medium fortified with a standard solution of the analytes in a concentration varying between 15.0 and 500.0µg/L) and a negative control (culture medium only) were included.
Pantoea ananatis strains were grown in PDA at 30 °C for 24 h.The inoculum was spread over the entire surface of the Petri dishes containing PDA medium.The disks containing individual antibacterials (norfloxacin, ciprofloxacin, nalidixic acid, levofloxacin, tetracycline, chloramphenicol, cefoxitin, sulfazotrim, amikacin, kanamycin, neomycin, tobramycin, gentamycin, clindamycin, sulphonamide, ampicillin, cefazolin, vancomycin, oxacillin, cefotaxime, erythromycin, penicillin G, nitrofurantoin, ceftriaxone and cephalothin) were placed equidistant and the plates incubated at 30 °C for 48 h.The diameter of the inhibition halos was measured for determination of the susceptibility of P. ananatis strains against commercial antibacterial agents ( Mamede et al., 2022).

Statistical analysis
All tests were arranged in completely randomized design with three replications per sample.The antibacterial activity and proteolytic and chitinolytic activity assays were analyzed according to a 2 x 69 factorial scheme.The temporal production of metabolites with antibacterial action in liquid medium was analyzed according to a 2 x 3 x 5 factorial scheme.The antibiogram assays were analyzed according to a 2 x 25 factorial scheme.The results of the antibacterial and enzymatic activity assays were analyzed individually and, when significant differences occurred by the F test (p ≤ 0, 05), the data were subjected to analysis of variance and the means compared by the Scott-Knott test, at 5% probability.For the temporal evaluation, the analysis was performed by the Tukey's test, at 5% probability.Statistical analysis was performed using the SISVAR 5.3 program (Ferreira, 2010).All assays were performed at least twice and the results were reproducible.

Proteolytic and chitinolytic activity of actinobacteria
Enzymatic activity assays of 69 actinobacteria strains, belonged to Streptomyces, Amycolatopsis and Kitasatospora genera, revealed that 59 (85%) strains exhibited proteolytic or chitinolytic activity.Streptomyces longwoodensis ACSL 18A exhibited the highest proteolytic or chitinolytic activity when compared to the other strains.Streptomyces hygroscopicus ACSL 6 exhibited higher chitinase activity, but lower protease activity.In contrast, S. pseudovenezuelae ACSL 470 exhibited higher protease activity, but lower chitinase activity.Taken together, the strains S. longwoodensis ACSL 18A, S. hygroscopicus ACSL 6 and S. pseudovenezuelae ACSL 470 showed the best results for proteolytic or chitinolytic activity (Table 1).Biocontrol of actinobacteria against Pantoea ananatis P. ananatis.Thus, the strains S. pseudovenezuelae ACSL 470, S. novaecaesareae ACSL 432 and S. laculatispora ACP35 were selected for analysis of temporal changes in metabolites production in two different liquid media against two phytopathogenic P. ananatis strains.

Temporal evaluation of antibacterial activity of metabolites produced in liquid mediums by actinobacteria strains against Pantoea ananatis strains
We also evaluated the temporal production of metabolites by actinobacteria S. pseudovenezuelae ACSL 470, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35 grown for until 120 h in ISP-2 and M1 liquid media.The metabolites produced in at 72 h in both media exhibited greater inhibition of two phytopathogenic P. ananatis strains, CMPC 40 and CMPC 105 (Table 3).

Primary selection of actinobacteria strains in solid medium against Pantoea ananatis
We performed the primary selection of all sixty-nine actinobacteria strains in solid medium against phytopathogenic P. ananatis CMPC 40 strain.The actinobacteria strains were also exposure to UV radiation to eliminated vegetative cell and to verify only the effect of their metabolites against P. ananatis.
Only nine actinobacteria strains (13%) showed antimicrobial activity against P. ananatis CMPC 40 strain (Table 2).Of then, three isolates (4.4%), belong to Streptomyces genus, showed moderate or high antimicrobial activity when exposed or not to UV radiation.The maintenance of antimicrobial activity even after exposure to UV radiation suggests the production of stable metabolites by Streptomyces against  and cephalothin did not exhibit any antibacterial activity against P. ananatis strains (Table 4).

Prospecting and identification of antibiotics produced by actinobacteria
Based on temporal antibacterial activity of three Streptomyces strains (S. pseudovenezuelae ACSL 470, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35) that showed high inhibition in vitro against P. ananatis, we selected their metabolites produced at 72 h in two different media for the identification of antibacterial substances.The chromatographic analysis coupled with mass spectrometry revealed, in the metabolites produced by S. pseudovenezuelae ACSL 470 in ISP-2 medium, one peak at 3.77 min and one peak with mass/charge (m/z) of 615.3, typical of neomycin, an aminoglycoside antibiotic (Figure 2).

Discussion
Actinobacteria are microorganisms with great biotechnological potential due to their ability to produce metabolites, such a antibiotics, capable of inhibiting and controlling phytopathogenic microorganisms growth (Minotto et al., 2014;Dornelas et al., 2017;Kaur et al., 2019).In this work, we evaluated in vitro the antagonism of actinobacteria strains, isolated from Brazilian soil, against P. ananatis, the causal agent of maize white spot disease that is responsible for significant maize yield reduction worldwide.Only 4.4% of the actinobacteria strains, from Streptomyces genus, exhibited high antimicrobial in vitro activity against P. ananatis.The low frequency of strain with antimicrobial activity against Gram-negative bacteria, such as P. ananatis, may be related to the complex structure of the outer membrane of these microorganisms, which reduces the action of the antibacterial agents (Nithya et al., 2012).
Since actinobacteria strains exhibited antibacterial activity against P. ananatis, we also evaluated whether exposure to UV radiation interferes with the degradation and instability of antimicrobial substances spread in the culture medium.We observed that some actinobacteria strains were able to inhibit the growth of P. ananatis when not exposed to UV radiation.When the same isolates were exposed to radiation, only three strains were able to inhibit P. ananatis, indicating that the UV radiation may have influenced the stability of their metabolites (Cordeiro et al., 2021).Actinobacteria, especially of the genus Streptomyces, are capable of producing several molecules with antimicrobial properties, being responsible for producing 80% of the antimicrobials currently commercialized (Sharma and Thakur, 2020).In vivo and in vitro bioassays demonstrated that Streptomyces spp.showed high effect against some bacterial phytopathogens, including Erwinia (Pantoea) carotovora, the causal agent of potato and cabbage soft rot (Qiu et al., 2011;Salem and Abd El-Shafea, 2018).
As fact, it has been well established that antibiotics producing by Streptomyces strains can be used for combating certain bacterial diseases of many economically important plants (Vidaver, 2002).Antibiotics can provide a protective barrier on the surface of plants to suppress the growth of the pathogens before infection (McManus and Stockwell, 2001).Some synthetic antibacterials derived from Streptomyces have been used to maize white spot disease control.For example, oxytetracycline reduced the number of white spot lesions in maize plants grown in the field by approximately 80-90%, as well inhibited 100% of P. ananatis growth in vitro (Costa et al., 2011;Gonçalves et al., 2013).Furthermore, the combined use of oxytetracycline and streptomycin demonstrated 60% effectiveness in the management of maize white spot disease in the phenological stages V8 (eight fully developed leaves) and pre-flowering (Manerba et al., 2013).
In the present study, Streptomyces pseudovenezuelae ACSL 470, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35 were responsible for the production of antimicrobial agents against P. ananatis.Among them, only S. pseudovenezuelae ACSL 470 exhibited proteolytic and chitinolytic activity.These enzymes can play an important role in the degradation of different compounds present in different microorganisms, assisting in nutrition and, consequently, exhibiting antibacterial, antifungal, insecticidal or nematicidal activity (Edreva, 2005;Alekhya and Gopalakrishnan, 2014).The isolation, screening and characterization of secondary metabolite-producing strains of actinobacteria has become an area of interest for research worldwide (Chaudhary et al., 2013).In addition, the search for Streptomyces genus actinobacteria in poorly studied habitats raises the prospect of discovering natural products that can be developed with the help of biotechnology (Ogunmwonyi et al., 2010).In vitro tests with actinomycetes have shown antagonism against several phytopathogens (Sahilah et al., 2010;Minotto et al., 2014;Yang et al., 2019), revealing the potencial use of these microorganisms for biological control.
In our study, the temporal evaluation of antibacterial activity demonstrated that S. pseudovenezuelae ACSL 470, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35 were able to produce metabolites with activity against P. ananatis, reinforcing the potential of Streptomyces sp.antimicrobial activity (Daskalaki et al., 2018).The best result for the production of bioactive substances obtained with the ISP-2 medium when compared to M1 medium may have been influenced by glucose concentration (Sánchez and Demain, 2002;Cunha et al., 2009).
In the prospection and identification of antibiotics produced by S. pseudovenezuelae ACSL 470, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35, we verified the presence of neomycin in the ISP-2 fermentative medium from the metabolism of S. pseudovenezuelae ACSL 470.As fact, the metabolism of biosynthesis is highly influenced by the availability of nutrients in the medium used, which may be associated with the absence of antimicrobial substances produced in the M1 medium (Charousová et al., 2019).In our study, the antibiogram assay confirmed the high susceptibility of two phytopathogenic P. ananatis strains to neomycin.Similar results based on experiments against plant bacterial pathogens showed that other Pantoea species, P. carotovora, was also sensitive in vitro to neomycin (Ma et al., 2011).Neomycin is an aminoglycoside antibiotic that has activity against most gram-negative aerobes, and inhibits protein synthesis by binding, with high affinity, to the A-site on the 16S ribosomal RNA of the 30S ribosome (Kotra et al., 2000).As a result, the antibiotic promotes error prone protein synthesis, allowing for incorrect amino acids to assemble into a polypeptide that is subsequently released to cause damage to the cell membrane and elsewhere (Krause et al., 2016).Post-inoculation spraying with neomycin from the liquid culture of the actinomycete S. fradiae HTP has antibacterial activity in vitro and in vivo against Pantoea carotovora, Ralstonia solanacearum and Xanthomonas oryzae.Surprisingly, neomycin from the liquid culture reducing disease caused by these phytopathogens ranged from 69 to 78% in greenhouse condition (Tao et al., 2011).
In conclusion, S. pseudovenezuelae ACSL 470 strain exhibited high antibacterial action against P. ananatis and was able to produce hydrolytic enzymes and metabolites contained neomycin, that show high activity against the target microorganism.Therefore, we strongly believe that the use of S. pseudovenezuelae ACSL 470 and/or their metabolites for the biocontrol of P. ananatis is highly promising.
Brazilian Journal of Biology, 2023, vol.83, e268015 9/11 Biocontrol of actinobacteria against Pantoea ananatis This is the first report of actinobacteria acting as potential microbial antagonists for P. ananatis control.However, it is important to evaluate this effectiveness in vivo, carrying out tests in controlled and field conditions.
Antibiotics and enzymes may to inhibit protein synthesis and several enzymes Brazilian Journal of Biology, 2023, vol.83, e268015 3/11 Biocontrol of actinobacteria against Pantoea ananatis

Figure 1 .
Figure 1.Diagram representing the (A) absent, (B) low, (C) moderate, and (D) high antimicrobial activity of commercial antibacterials against Pantoea ananatis.Antimicrobial activity is based on the absence, presence, and the measurement of halos indicative of microbial growth inhibition.

Figure 2 .
Figure 2. Identification of the antibiotic neomycin produced by Streptomyces pseudovenezuelae ACSL 470 in ISP-2 medium after 72 h.(A) Chromatogram with the neomycin pattern (blue line) and the identification of neomycin (red line; 3.77 min) produced by S. pseudovenezuelae ACSL 470 (red arrow).(B) Mass/charge transitions (m/z) characteristic of neomycin produced by S. pseudovenezuelae ACSL 470 (red box).

Table 1 .
Chitinase and protease enzymatic index (EI) of soil actinobacteria strains.
*Mean values followed by the same letter in each column do not differ by the Scott-Knott test at 5% probability.Brazilian Journal of Biology, 2023, vol.83, e268015 5/11
*Mean values followed by the same letter in each column do not differ by the Scott-Knott test at 5% probability.

Table 2 .
Antibacterial activity of actinobacteria strains grown in solid medium, exposed and no exposure to UV radiation, against Pantoea ananatis CMPC 40 strain.Means followed by the same letter in each column do not differ by the Scott-Knott test at 5% probability. *

Table 3 .
Antibacterial activity of metabolites produced by Streptomyces strains grown in liquid medium against two Pantoea ananatis strains (CMPC 40 and CMPC 105).
*Means followed by the same letter in strain and liquid medium do not differ by the Tukey test at 5% probability.Brazilian Journal of Biology, 2023, vol.83, e268015 7/11 Biocontrol of actinobacteria against Pantoea ananatis
*Means followed by the same letter in each column do not differ by the Scott-Knott test at 5% probability.