Optimization of the Extraction Process and Application of Bacterial Extracts in the Control of Brown Spot and Leaf Blast in Rice Culture

Optimization of the Extraction Process and Application of Bacterial Extracts in the Control of Brown Spot and Leaf Blast in Rice Culture | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimization of the Extraction Process and Application of Bacterial Extracts in the Control of Brown Spot and Leaf Blast in Rice Culture Marina Teixeira Arriel-Elias, Gabriel Carlos Teixeira Freire Arriel, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1087379/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The objective of this work was to optimize the extraction process and application of bacterial extracts of Bacillus sp. and Serratia sp. in leaf blast control ( Magnapothe oryzae ) and brown spot ( Bipolaris oryzae ) in rice culture. The work was divided into three stages: 1) Bacterial obtaining extracts through liquid-liquid extraction 2) Antagonistic capacity of bacterial extracts to M. oryaze and B. oryae 3) Suppression of brown spot (A1) and leaf blast (A2) in greenhouse. The bacterial isolates in present study were identified as Bacillus sp. (BRM32110) and Serratia marcescens (BRM32113). The crude extract of both isolates at different extraction times 6, 16, 24, 48 and 72 hours reduced the growth of colonies of M. oryzae and B. oryzae by up to 92% and 28%, respectively. The extracts that showed highest inhibition of colony growth were those obtained after 6 and 16h of incubation and were selected for subsequent assays. These, for both isolates were able to reduce conidia germination by up to 91% and apressorium formation of M. oryzae by up to 93%. In green house, A1 the treatment that stood out was the extract of Bacillus sp. (16h) with 6.7% of leaf area affected and in A2 the treatment S. marcescens extract (16h) stood out with only 7.6% of leaf area affected with brusone when compared to control. The use of extracts of Bacillus sp. and Serratia marcescens was efficient in reducing the severity of brown spot and leaf blast in rice crop. Environmental Engineering Metabolites Natural products Sustainability Chemistry Alternative control Rice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The warning about the need to increase food production is recurrent, and rice ( Oryza sativa L.) stands out for its strategic role in both social and economic development in Brazil and in the world (CONAB 2020). The average consumption in the world is 57 kg person-1 year-1 (Nascente et al. 2019), and in Brazil consumption is on average 45 kg of rice person-1 year-1 (Embrapa 2020). World rice production was approximately 495.23 million tons, and has the highest potential for increased production among other cereals, supplying 20% of calories and 15% of protein consumed in the diet (Sosbai 2012). The production of this cereal has been showing a drop in profitability, according to the increase in production costs, caused by the excessive number of fungicides and fertilizers, to ensure the production and quality of grains threatened by stresses (Guimarães et al. 2014; Ahmad et al. 2016), as the occurrence of brusone and grain spot. Brusone caused by the fungus Magnaporthe oryzae B. Couch ranks first in economic importance (Silva-Lobo et al. 2012; Dean et al. 2005), is difficult to control and can cause up to 100% losses in rice yield (Prabhu et al. 2009). Brown spot ( Bipolaris oryzae ) causes grain yield losses (90%), particularly when the leaf spot stage assumes epiphytic proportions (Sunder et al. 2014). The cost of pesticides in rice farming has increased by about 180% since the 2007/08 crop (CONAB 2021; Sharma et al. 2019) and fungicides account for up to 45% of production costs for both irrigated and upland systems (DEPEC 2019). In addition, the most commercially used molecules present specific site of action as triazoles and strobilurins, which facilitates the selection of isolates resistant to the most used molecules (Souza 2012; D'Ávila et al. 2021). On the other hand, the market for biological inputs has been growing in recent years, The global agricultural biologicals market reached a value of US$ 8.1 Billion in 2020 and is estimated to register a compound annual growth rate of 9.4% from 2020 to 2027. worldwide due to the trend of an increasing demand in integrated pest and disease management (IPM) (Grand View Research 2020). Despite the fast growing market, the production of a bioproduct for plant health protection faces obstacles such as the development of an efficient and sustainable formulation. The main technical barriers to legalize and use these bioproducts are: lack of stability of the formulated product (low shelf life), low viability of microorganisms after exposure to ultraviolet rays of the sun, difficulty in the application of the product in the field, acceptance of the producers for presenting incompatibility with chemicals used in the management of various crops and species of microorganisms that are close to species pathogenic to humans (Bettiol et al. 2014). In previous studies conducted by our team, six bacterial isolates were shown to be potential biological agents in rice blast suppression. Among them we highlight those of the genus Bacillus and Serratia. (Filippi et al. 2011; Martins 2015; Martins et al. 2020). Bacteria of the genus Bacillus belong to the phylum Firmicutes, family Bacillaceae, can be found in soil, in association with plants, rivers, and estuaries and produce several metabolites of industrial interest such as enzymes, B-exotoxin, thuricin, petrobactim, bacillibactim (Bach et al. 2011; Sansinenea et al. 2011). The species of the genus Serratia stand out in their capacity to produce siderophores, indol acetic acid, phosphate solubilizers and production of antifungal metabolites, such as pyrrolnitrin, carbapenem, prodigiosin, haterumalide and siderophores (Levenfors et al. 2004; Thompson et al. 2002). These natural products, when compared to synthetic compounds, offer greater structural diversity, synergism between molecules and are considered exceptional sources of new agrochemicals (Tremacoldi and Filho 2006). We hypothesized that rhizobacteria produce primary and secondary metabolites that are responsible for their biological characteristics and activities, of potential importance, to be used in agriculture, because, they present a wide chemical diversity and can be a new integrant for the integrated management of diseases in rice culture. Therefore, the objective of this work was to optimize the extraction process and evaluate the efficiency of bacterial extracts of Bacillus sp. and Serratia sp. in the control of leaf blast ( M. oryzae ) and leaf brown spot ( B. oryzae ) in rice culture. Materials And Methods Microorganisms The isolates Serratia sp. (BRM 32113), Bacillus sp. (BRM 32110) M. oryzae (BRM 31295) and B. oryzae (BRM46676) belongs to Embrapa Microrganism Collection, preserved by the Castellani and deep freeze methods. Colonies of bacterial strains were transferred to Petri plates containing Nutrient Agar (AN-Extatro de carne, Peptona, Cloreto de sódio, Ágar), and incubated during 48 hours at 28 °C. M. oryzae isolate was transferred to Petri plates containing Potato Dextrose Agar (PDA), and incubated for seven days, at 24 °C, under light regime for seven days. B. oryzae isolate were transferred to Petri plates containing PDA, and incubated for seven days, at 24 °C, under dark conditions. Bacterial isolates identification The two bacterial isolates had their complete genome sequenced using the Illumina platform, where the Nextera XT kit (shotgun) is used, which contains the adaptor primers required for sequencing. Coverage: 100x (1 million reads). We extracted, for the BRM32110 and BRM32113, the 16S rRNA region from the genomes assembled, using the ContEst16S (Contamination Estimator by 16S) (Lee et al., 2017) in which 16S rRNA gene fragments from the query genome assemblies are screened. The database for both isolates was constructed by species from monophyletic clade of the S. marcescens and Bacillus thuringiensis (Table 1). Sequences in the GenBank database were compared to check for molecular identification through the nucleotide BLAST algorithm (Altschul et al. 1990). MUSCLE was used in the alignments (Edgar 2004), implemented in MEGA v.7 (Kumar et al. 2016). Bayesian inference (BI) analysis was carried out, for each isolate reconstruction, based on the Monte Carlo Markov chain method (MCMC). The MrMODELTEST v. 3.04 (Nylander 2004) was adopted to select the nucleotide substitution model for BI analysis. Likelihood values were calculated, and the model was selected according to the Akaike information criterion (AIC). Evolution models selected for each isolates reconstruction were HKY + I for BRM32110 and GTR + I for BRM32113. The BI analysis was carried out in MrBayes v.3.1.1 (Ronquist and Huelsenbeck 2003). The consensus tree was obtained after 10 million generations of a Markov chain were applied to two runs, four chains each, with a 15% burn- in. Likelihood log convergence was analyzed in TRACER v. 1.4.1 software (Rambaut et al. 2018). The tree was visualized and edited in ITol v.4 (Letunic and Bork 2019). Preparing the bacterial suspensions and obtaining the extracts The bacterial strains grown in Petri dishes were transferred to 5 different Erlenmeyers, each containing 100mL of the nutrient broth culture medium. The Erlenmeyers were kept under constant 150 rpm shaking at 28 °C for 6, 16, 24, 48, and 72 hours of growth. After the different growth periods, 50.0 mL of each bacterial suspension was transferred to a separation funnel, in which the same volume (50.0 mL) of ethyl acetate solvent was also added. The mixture was stirred slowly to avoid the formation of internal pressure in the funnel. The system was kept at rest until complete separation of the phases. The aqueous phase was separated from the organic phase and returned to the separation funnel. The aqueous phase was subjected to a further extraction step with the addition of another 50.0 mL of the same solvent. The aqueous layer was collected in an Erlenmeyer, and for one more time the process was repeated until three extraction steps were completed. The phase containing the ethyl acetate was concentrated at 50 °C in a rotary evaporator under reduced pressure. Thin-layer Chromatography The samples were analyzed via thin-layer chromatography (TLC) performed on an aluminum foil (4 x 5 cm) coated with a silica gel with fluorescent indicator F254 as the stationary phase. After applying acetone soluble samples on the plates, anethylacetate:hexane (9:1 v/v) mobile phase was eluted by capillarity. The chemical profile of the samples was visualized by ultraviolet light (254 and 365 nm) and vanillin solution (composed by135 mL of methanol, 3 mL of vanillin, 135mL of distillation water and 35 mL of sulfuric acid) as revealing, which forms colored spots, according to its interaction with the natural compounds classes. Extracts screening for antagonistic activity The isolates of M. oryzae and B. oryzae were previously grown in a Petri dish containing BDA (potato dextrose agar) culture medium during seven days of incubation at 24°C. Then, discs of culture medium, 5 mm in diameter, containing mycelia of the fungi were transferred to new Petri plates, 60 mm in diameter, containing BDA culture medium supplemented with 50 μl of the extracts of Bacillus sp. and Serratia sp solubilized in 1mL of DMSO (dimethylsulfoxide). The assay was conducted in an entirely randomized design with twelve treatments in four repetitions (tab....). The control treatment consisted of the medium supplemented with DMSO (negative control). The absolute control was considered a BDA plate containing only the mycelium discs of the two pathogens. The plates were incubated under a constant temperature of 24°C for 7 days. At the end of the incubation period, colony diameters were measured using a millimeter ruler. Antagonism was determined by comparing the percentage reduction of the mean colony areas in the different treatments (Filippi et al., 2011). The best treatment identified in the first assay antagonism assay, it was also quantified the B. oryzae conidial production by each colony, per Petri dish. Conida was removed with 3mL/plate of distilled and sterilized water and a brush. The concentration was estimated with a Neubauer chamber. Inhibition of conidial germination and appressorium formation of M. oryzae A suspension of M. oryzae conidia, prepared at a concentration of 2 x 10 5 conidia/mL was mixed with the different extracts at different concentrations. Five microliters of each extract were pipetted into an artficial hydrophobic plastic surface to induce germination and appressorium formation. The hydrophobic plastic surfaces containing the droplets were kept in a humid chamber in a growth chamber. Evaluations were performed after 4, 6 and 16 hours, under a 20x optical microscope. Germinated conidia and appressorium formed were quantified. The trial was composed of the treatments described in Table 2. Phytotoxicity of extracts on rice leaves Detached leaves of the rice cultivar BRS Primavera with 21 days after planting were placed in a humid chamber and sprayed with the extracts, selected in the test described in previous section. In an entirely randomized design, 4 treatments were evaluated, representing different extraction times and concentrations: T1: BRM 32113 6h of growth and sprayed at 25mg of exttract; BRM 32113 16h of growth, sprayed at 25mg extract; BRM 32110 6h of growth and sprayed at 100mg extract; BRM 16h of growth and sprayed at 50mg extract. The sprayed leaves were incubated at 25 ± 2 ° C under constant common light. Visual assessments of phytotoxicity, characterized by yellowish/whitish spots (chlorotic lesions), or any other changes on the leaf surface compared to the control, were performed daily for seven days, based on Sakthivel et al. (2002). The control of the experiment was represented by spraying leaves with sterilized distilled water. Assays in greenhouse Assays E1 and E2: Gray spot and blast leaf suppression It was composed of 8 treatments and three replicates, totaling 24 trays. Treatments in E1 and E2 were the same, but, E1 were spray inoculated with B. oryzae, and E2 with M. oryzae. the treatments of both assays are described in Table 3. Preparation of bacterial suspension: a cell suspension of the B. pyrrocinia and Bacillus sp. isolate was transferred to a 500 ml Erlenmeyer flask, containing 200 ml of nutrient broth culture medium, prepared according to Arriel-Elias et al. (2018). The bottles were under constant stirring at 150 rpm, for 48 h at 28°C. Then, each suspension was adjusted, with a spectrophotometer, to 0.5 absorbance, at a wavelength of 620 nm, corresponding to 1x10 8 CFU.ml -1 . Sowing in greenhouse Seeds of upland rice cultivar BRS Primavera were disinfectedwith 70% alcohol and 30% sodium hypochlorite before planting and sownin eight furrows in plastic trays (15 × 30 × 10 cm) in 3 kg of soil, that was fertilized with NPK (5 g of 5-30-15 + Zn and 3 g of ammonium sulphate). Besides, 5 g (NH 4 ) 2 SO 4 + Fe and Bo were applied per tray 19 days after sowing. A daily irrigation regime was adopted, corresponding to approximately 100% of the soil field capacity. Trays planted with rice seedswere maintained at greenhouse until therice plants get to V3 stage. B. oryzae suspension for spray inoculation (E1) B. oryzae isolate, was multiplied in Petri plates containing BDA and incubated at 25 ° C for seven days with continuous dark. Conidium was removed with sterilized distilled water and a brush, and the concentration was adjusted for 1x10 5 con.ml -1 . At the 35 st day (V4 stage), rice plants were transferred to cages coated with transparent plastic. Thirty ml of B. oryzae conidial was suspended. It was pulverized in each cage contained three trays with rice plants. The pulverization was performed with a pressure pump and a spray gun, at a pressure of 0.001 kg / cm 2 . The plants remained in the greenhouse under controlled environmental conditions, with temperatures ranging from 27 to 28 ° C and relative humidity above 80%, for 8 days. Leaf gray spot severity was evaluated using a severity scale (1.6%, 3.2%, 6.4%, 12.6, 23.1% and 38.6%), according to Lenz et al. (2010), and determining the percentage of affected leaf area by disease. M. oryzae suspension for spray inoculation (E2) M.oryzae isolates (BRM 31295), was multiplied in Petri plates containing agar, oatmeal and incubated at 25 ° C for seven days with continuous light. The aerial mycelium of M. oryzae colonies was removed with a sterile glass rod and the plates incubated in a growth chamber for 48 hours, under continuous fluorescent light. Conidium was removed with sterilized distilled water and a brush, and the concentration was adjusted for 3x10 5 con.ml -1 . At the 21 st day (V3 stage), rice plants were transferred to cages coated with transparent plastic. Thirty ml of M. oryzae conidial was suspended (Filippi et al. 2007). It was pulverized in each cage contained three trays with rice plants. The pulverization was performed with a pressure pump and a spray gun, at a pressure of 0.001 kg / cm2. The plants remained in the greenhouse under controlled environmental conditions, with temperatures ranging from 27 to 28 ° C and relative humidity above 80%, for 8 days. Leaf blast severity was evaluated using a severity scale (0%, 0.5%, 1%, 2%, 4%, 8%, 16%, 32%, 64%, and 82%), according to Notteghem (1981), and determining the percentage of affected leaf area by disease. Statistical analyses The averages of each test were calculated, the variances analyzed, and the proportions were compared by the Tukey test at 5% of significance, using the SPSS program, version 2.1. Results Bacterial isolates identification The 16S rRNA region from the genomes assembled, revealed that isolate BRM32113 pertains to the genus Serratia and species marcescens and isolate BRM32110 belongs to the genus Bacillus (Fig 1, A and B). Thin-Layer Chromatography The aspects considered were color and height after analysis with UV light and developer. The crude extract of isolates BRM32110 and BRM 32113 at the different extraction times of 6, 16, 24, 48 and 72 hours showed brown, yellow and blue spots on plate eluted with hexane anethylacetate:hexane (9:1 v/v) and revealed with vanillin solution, indicating the presence of terpenoids (yellow-brown) and fatty acids (blue), as described by Wagner & Bladt (1996). Stains with more marked brown coloration were detected at the same height in the extracts obtained after 6 and 16 hours of growth of both isolates. Antagonistic capacity of extracts Antagonism to B. oryzae The extracts obtained from Bacillus sp. (BRM 32110) and S. marcenses (BRM 32113), after 6 and 16 hours of growth, reduced up to 28% of the mycelial growth of B. oryzae (Table 4, Fig. 2, A). Quantification of conidia production of B. oryzae colonies grown in culture medium supplemented with extracts of Bacillus sp. (BRM 32110) and S. marcenses (BRM 32113) reduced conidia germination by up to 62%, statistically different from controls (Fig. 3, A). Antagonism to M. oryzae The extracts of Bacillus sp. (BRM 32110) and Serratia marcenses (BRM 32113) strains obtained after 6, 16, 24, 48 and 72 hours of growth reduced the growth of M. oryzae colonies. The extracts obtained after 6 and 16 hours, for both Bacillus sp. ( BRM 32110) and S. marcenses (BRM 32113) isolates reduced the mycelial growth of M. oryzae colonies by up to 94% (Table 4, Fig. 2, B). Conidium germination and appressorium formation After 6 hours of incubation: the extract of the isolate Bacillus sp. (BRM32110) (6h) at concentration of 100mg, significantly inhibited in 59.6% the germination of conidia, when compared to controls (Fig. 4-A), and in 85% the appressorium formation. The extract of the isolate S. marcenses (BRM32113) at concentrations of 50 and 100mg reduced by 45 and 52%, respectively, the germination of conidia (Fig. 5-A), and at the 25mg concentration reduced by 75% the number of appressorium formed. After 16 hours of incubation: the extract of the isolate Bacillus sp. (BRM32110 ) (16h) reduced in 45% and 38% the germinated conidia and 81% and 93% the appressorium formation in concentrations of 50 and 100mg (Fig. 4 - B and C). The extract of S. marcenses (BRM32113), at concentrations of 100mg and 50mg reduced conidia germination by 82% and 53% and appressorium formation by 91% and 92% respectively (Fig 5 - B and C). The negative control containing the solvent DMSO delayed conidia germination for both isolates, but after 6 hours of incubation it matched the control containing only water. The first appressorium were observed after 6 hours of incubation (Fig. 4 and 5) in the control treatment. Phytotoxicity Extracts individually tested showed negative for phytotoxic effect on detached rice leaves on a visual evaluation (Fig. 6 A and B). Different treatments did not alter the characteristics of leaves in a period of one week of evaluation, mainly in relation to the appearance of yellowish, whitish spots, necrosis or any other alteration of the leaf surface as compared to the control (Fig. 6 B, 1). In vivo tests under greenhouse conditions were also performed with the extracts. The results confirmed that extracts did not generate direct toxicity reactions in rice plants. Assays A1 (leaf brown) spot and A2 (leaf blast suppression) Brown spot suppression In A1, control treatments showed up to 23.5% of leaf area affected with brown spot at eight days after challenge inoculation. Treatments 3 (suspension of Bacillus sp. cells (BRM32110), with 7.8% of the leaf area affected, 5 (extract of Bacillus sp. BRM 32110, 6h) with 9. 8% affected leaf area, 6 ( Bacillus sp. extract BRM 32110, 16h) with 6.7% affected leaf area and 8 ( S. marcescens extract BRM32113, 16h) with 6.8% affected leaf area of (Fig. 7 A). Leaf blast suppression In A2, the treatments that stood out were 3 (suspension of Bacillus sp. (BRM32110) cells), with 10.4% affected leaf area, 4 (suspension of S. marcescens BRM32113 cells) with 11. 6%, 5 ( Bacillus sp. BRM 32110 extract 6h) with 8.8%, 6 ( Bacillus sp. BRM 32110 extract 16h) with 14.4%, 7 (BRM32113 extract 6h) with 10% and 8 (BRM32113 extract 16h) with 7.6%. All treatments differed statistically from controls that showed up to 37% leaf area affected with leaf blast (Fig 7 A). Discussion The results obtained in the present study showed that the use of Bacillus sp. and Serratia marcescens extracts were efficient and have potential for suppression of leaf brusone and brown spot in rice. The Bacillus sp. BRM 32110 and S. marcescens BRM32113 species under study, were isolated from the rhizosphere of upland rice by Filippi et al. (2011), who performed the phenotypic, biochemical and molecular characterization, through the amplification of the gene encoding the 16S rRNA region (Martins et al. 2020). Other works of the same research group revealed that these same isolates are growth promoters in rice, soybean and bean crops, antagonists to some phytopathogens such as M. oryzae and B. oryzae and resistance inducers. (Filippi et al 2011; Sousa et al. 2017; Côrtes et al. 2015; Nascente et al. 2017; Sperandio et al. 2017; Arriel-Elias et al, 2018; Arriel-Elias et al 2019; Silva et al. 2020; Rezende et al. 2021). However, complete genome characterization and chemical characterization of the metabolites of both bacterial isolates had not been performed so far. Bacteria have a simple genomic structure, comprising one or more chromosomes, and this has allowed the accumulation of a range of knowledge about these microorganisms. In recent years, the complete sequencing of genomes has advanced the knowledge of the phylogeny of these microorganisms (Marques 2012). Previously, phylogenetic identification was based on the sequencing of some regions of their genome, such as the 16S rRNA region. Sequencing of the ITS (Internal Transcribed Spacer) region separates the 16S rDNA gene and can be amplified with specific primers anchored to this region. This region is highly conserved intraspecifically but variable between different species, which makes distinction at the specific level possible. However, molecular identification of certain bacterial groups by 16S rRNA gene sequencing can lead to inaccurate results. Today a 16S rRNA identity greater than 98.7% is accepted as a minimum threshold to differentiate species because, in most cases, this similarity corresponds to a DNA-DNA identity greater than 70% (Fungaro 2000; Zanirati 2012). The bacterial isolate BRM32113 was initially identified by Martins et al. (2020), as Burkholderia pyrrocinia , analyzing the sequencing obtained from the amplification of the 16S rRNA gene, and the biochemical and morphological identification. But the analysis of the 16S rRNA gene extract from the complete genome sequencing of isolate BRM 32113 revealed that it is from the genus Serratia and species marcescens . The sequencing of isolate BRM 32110 also revealed that it is from the genus Bacillus and for specie identification we will continous the whole genome studies (Fig. 1). The aspects analyzed in the CCD showed the most remarkable presence of brown coloration, indicating the presence of terpenoids, in the extracts obtained at 6 and 16 hours after bacterial growth (Wagner and Bladt 1996). Terpenes are one of the largest groups of natural products and comprise numerous substances with important roles in physiological and pathological processes, they are hydrocarbons formally divided into isoprenic units and present recognized antimicrobial activity (Felipe and Bicas 2017) that act directly in the process of infection of the fungus in the plant, inhibiting the growth of hyphae and the germination of conidia (Achotegui-Castells 2016). Some Serratia species produce volatile compounds such as terpenoids, which can influence the growth of other bacteria or fungi (Su et al. 2016). Also found in plants, they can be elicited by beneficial microorganisms and act as growth promoters, for example gibberellins, abscisic acid, carotenoids, and sterols, and also as toxins, inhibitors of phytopathogens and herbivores, such as limonoids, saponins, essential oils, among others (Hussein et al. 2019; Olanhewaju et al. 2017). The sum of all chemical reactions within a living organism is known as metabolism, classified as primary and secondary. In primary metabolism macromolecules are produced as essential functions for microorganisms, while in secondary metabolism we have the production of specific micromolecules that have adaptive functions to biological activity (Tortora 2012) and that also act as competitive weapons used against other microorganisms, nutrient transport agents, and sex hormones. We identified that the isolates BRM 21110 and BRM 32113 should grow for 6 and 16 hours in liquid nutrient broth medium for the production of molecules of primary and secondary metabolism, because the extracts obtained at these times were able to reduce the mycelial growth of M. oryzae and B. oryzae colonies up to 94 and 37% respectively (Fig. 2 A, Table 4). In addition we observed the change in the morphology of the fungal mycelium of both isolates (Fig. 2 B), which directly reflects in the pathogenicity of the fungus when in contact with the plant (Sangappillai and Nadarajah 2020) and also the reduction of up to 62% in the production of conidia of B oryzae . The process of penetration of these fungi into the plant cuticle is initiated when the conidia come in contact with the surface of rice leaves. It forms the germ tube that extends and differentiates into bulbous melanization, forming the appressorium, which is rich in melanin and contains cell wall degrading enzymes. Melanin is a secondary metabolite that protects conidia from UV damage and is also crucial in generating the turgor force generated by the apressorium to penetrate host tissue (Saha et al. 2020). We also evaluated morphological changes in conidia, germ tubes that did not form appressorium, non-melanized appressorium, and observed that all treatments differed from controls, which showed the formation of elongated hyaline conidia with thin apex and septate, as described in the literature (Prabhu and Filippi 2006). In the present study we observed that the LD 50 for M. orzae , which was 100mg for the extract of BRM 32110 (6h), 50mg for BRM32110 (16h), 25 for BRM 32113 (6 and 16h), through conidium germination and appressorium formation, of the extracts obtained from both bacterial isolates. These presented a reduction of up to 90% of the appressorium formation, when compared to the control. Therefore, even though all concentrations significantly reduced the aspects analyzed, the statistical and morphological evaluations led us to choose them. These same extracts did not show signs of fiotoxicity to rice leaves (Fig. 6). The reduction in the mycelial growth of B. oryzae and M. oryzae colonies, in the germination of conidia and appressorium formation by the metabolites represents a solid perspective for the development of a commercial bioproduct for rice culture because the insertion of a bioproduct in the management of rice culture can contribute to the reduction of doses and number of applications of synthetic chemical fungicides and fertilizers, known to cause problems to the microbiological balance of the soil and the environment. Therefore, aiming at the commercial use of the metabolites, it was necessary to define the LD 50, the ideal concentration and phytotoxicity of the extracts. The extracts obtained from the isolates BRM 32110 and BRM 32113 showed the same bioactivity of the bacterial cells of these isolates, as described by Filippi et al. 2011; Sousa et al. 2017; Sperandio et al. 2017; Arriel-Elias et al. 2017 and Arriel-Elias et al. 2019, who were successful in controlling diseases in rice culture when using the cells of these microorganisms after 48 hours of growth in culture medium. With 48 hours of culture we will have in the culture medium new cells and old cells, using extracts obtained after 6 and 16 hours of culture we will have a great advantage, which is uniformity by standardizing the growth time, extracting desirable characteristics of these PGPRs. In the greenhouse trials, to evaluate the suppression of diseases, we found that all treatments containing the extracts and bacterial suspension showed statistical differences from the controls. The solvent used did not harm the suppressive effect of bacteria, as previously proven in the antagonism test. When used alone, it did not show significant differences from the control. In A1 the best values for leaf brown spot suppression (LGS) were the treatments: 3 (BRM32110), presented 7.8% of affected leaf area, 4 (BRM32113) presented 17.1%, 5 (BRM32110 extract 6h) presented 9.8%, 6 (BRM32110 extract 16h) presented 6.7%, 7 (BRM32113 extract 6h) presented 11.4% and 8 (BRM32113 extract 16h) presented 6.8%. The controls treatments 1 (H2O), and 2 (DMSO) presented 21 and 23.5% of the leaf area affected by LGS, respectively (Fig. 6, 1. Treatments (3, 4, 5, 6, 7 and 8) presented small lesions and in a shorter amount, keeping most of the leaf area green (Fig. 6, 1 - C, D, E, F, G and H). In treatment 1 (H 2 O) and 2 (DMSO), we observed typical lesions of the disease, considered sporadic lesions, with a grayish center and brown edges that coalesce with time (Fig 7- 1 and 2). For leaf blast (LB) suppression, in A2, all treatments containing the extracts and bacterial suspension showed statistical differences from the controls and the best values for leaf blast suppression was: 3 (BRM32110), presented 10.4% of affected leaf area, 4 (BRM32113) presented 11.6%, 5 (BRM32110 extract 6h) presented 8.8%, 6 (BRM32110 extract 16h) presented 14.4%, 7 (BRM32113 extract 6h) presented 10% and 8 (BRM32113 extract 16h) presented 7.6%. The controls treatments 1 (H2O) and 2 (DMSO) presented 32 and 37% of the leaf area affected by LB respectively (Fig. 7 -2 ). Treatments (3, 4, 5, 6, 7 and 8) presented typical small brown open lesions, pinhead type and in a shorter amount; however, it was in a smaller size and number than the control treatment, and it did not coalesce with time, keeping most of the leaf area green (Fig. 6, 2 - C, D, E, F, G and H). In treatment 1 (H2O) and 2 (DMSO), we observed typical lesions of the disease, considered sporadic lesions that coalesce with time (Fig 7- 1 and 2). Similar effects were observed using cell-free extracts of B. subtilis and B. amyloliquefaciens , in inhibiting the mycelial growth of Fusarium up to 50.33%. Similarly, when using B. pumilus extracts obtained inhibitory activity for three soil pathogens Rhizoctonia solani, Phytium and Sclerotium rolfsii and furthermore identified one of the molecules responsible for the suppression of pathogens, pumilacidin (Mendes et al. 2018; Melo 2005), corroborating our results. Some authors have achieved similar results to ours when using extracts of Serratia sp. strains, as for example, Roberts et al. (2015) obtained the control of Phytium ultimum using extracts of Serratia marcescens, in the treatment of pumpkin seeds and Lenvenfors et al. (2004) when using extracts of Serratia plymuthica A 153 observed the inhibition of germination of conidia of species of Aspergillus and identified some molecules present in the extracts: pyrrolnitrin, and haterumalide NA, B and NE. These results show the efficiency of bacterial extracts on soil pathogens, and with our work, we achieved efficacy in suppressing leaf diseases in greenhouse. The most widely used control methods for brown spot and leaf blast, is the integration of genetic resistance and chemical control. But due to the high variability of the pathogen M. oryzae , the duration time of genetic resistance in improved rice cultivars is limited, thus increasing the number of fungicide applications which has caused high risk of resistance emergence in phytopathogen populations (Prabhu and Filippi 2006). Oliveira et al. (2015) found P. oryzae isolates with the presence of the G143A mutation in the cyt b gene that were 42 time more resistant to pyraclostrobin, due to the high selection pressure exerted by consecutive years of strobilurin applications. D'Ávila et al. (2021) examined the reaction of fungal field isolates, collected over a 26-year period, to the active ingredients of commercially relevant fungicides, and documented the gradual increase in the frequency of fungicide-resistant isolates in rice populations of P. oryzae on a long-term basis. The insertion of alternative measures in integrated disease management in rice can decrease the dependence on chemical control in the production system and safeguard the productive potential of genetically improved cultivars. Although biological control represents a strategy to increase the durability of resistance of rice cultivars to diseases and reduce toxic residues resulting from the indiscriminate use of chemicals (Filippi et al. 2011), there are still no biological products registered for rice cultivation (Agrofit 2021). To date, no other studies have been conducted using molecules of microbial metabolism of Bacillus sp. and Serratia marcescens in the control of brown spot and leaf blast in rice crop. The results obtained showed that the use of Bacillus sp. and S. marcescens bacteria extracts were efficient and have great potential for suppression of brown spot and leaf brusone in rice. Therefore, with this work we have the perspective of inserting a natural product in the agricultural market, aiming to favor producers with one more control tactic that can help the agroecological management to take off in Brazil. With this, more studies will be conducted in order to identify which are the molecules produced by these bacterial isolates and their production in large scale. We are at the forefront of developing bioproducts or biobased products for rice cultivation, and to help protect natural ecosystems while improving profitability and productivity in rice fields it is necessary to look for more components to integrated management. Conclusion The use of Bacillus sp. and Serratia marcescens extracts was efficient in reducing the severity of brown spot and leaf blast in rice culture. These results are significant because they demonstrate that it is possible to use bacterial extracts in the control of phytopathogens seeking the insertion of natural products in agriculture and is the first report in rice culture. Declarations Acknowledgments The authors would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for scholarship and Embrapa for the research financial support. Author contribution The original idea was suggested by VGPS and MCCF and the concept of this study was developed by MTAE. The experimental work was carried out by MTAE with guidance from GCTFA, GAB and PHDS. MTAE drafted the article. GCTFA and GAB were involved in the critical revision of the article for important intellectual content. All the authors read and approved the final manuscript. Funding CNPq (National Council for Scientific and Technological Development (CNPq) Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors not competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Agricultural Biologicals Market Size, Share & Trends Analysis Report By Crop Type (Cereals & Grains, Oilseeds & Pulses), By Product (Biopesticides, Biostimulants), By Application Method, And Segment Forecasts, 2020 – 2027 ( Global Agricultural Biologicals Market | Industry Report, 2020-2027 (grandviewresearch.com) ). https://www.grandviewresearch.com/industry-analysis/agricultural-biologicals-marke Accessed 20 April 2021 AGROFIT (2021) MAPA-Ministério da Agricultura Pecuária e Abastecimento. http://agrofit.agricultura.gov.br/agrofit_cons/principal_agrofit_cons. Accessed 20 april 2021 Ahmad P, Latef AAHA, Rasool S, Akram NA, Ashraf M, GUCEL S (2016) Role of Proteomics in Crop Stress Tolerance. Frontiers in Plant Science 7:1336. https://doi.org/10.3389/fpls.2016.01336 Arriel-Elias MT, Oliveira MIS, Silva-Lobo VL, Filippi MCC, Babana AH, Conceição EC, Cortes MVCB (2018) Shelf life enhancement of PGPRs using a simple formulation screening method. African Journal of Microbiology Research 12:115-126. https://doi.org/ 10.5897/AJMR2017.8787 Arriel-Elias MT, Côrtes, MVCB, de Sousa TP, Chaibub AA, Filippi MCC (2019) Induction of resistance in rice plants using bioproducts produced from Burkholderia pyrrocinia BRM 32113. Environ Sci Pullut Res 26:19705-19718 https://doi.org/10.1007/s11356-019-05238-3 Bach E, Cannavan FS, Duarte FRS, Taffarel JAS, Tsai SM, Brandelli A (2011) Characterization of feather degrading bacteria from Brazilian soils. Int Biodeterior Biodegrad 65:102-107. https://doi.org/ 10.1016/j.ibiod.2010.07.005 Bettiol W, Maffia LA, Castro MLMP (2014) Control biológico de enfermedades de plantas en Brasil. In: Bettiol W, Rivera MC, Mondino P, Montealegre JR, Colmenárez YC (eds) Control biológico de enfermedades de plantas em América Latina y el Caribe. Embrapa Meio Ambiente-Livro Científico (ALICE), pp 91-137 (in spanish) CONAB-Companhia Nacional de Abastecimento (2020). Séries históricas de safra. . Accessed 21 January 2021 CONAB-Companhia Nacional de Abastecimento (2021) Evolução dos custos de produção de arroz no Brasil. Compêndio de estudos, Conab 4:1-20 (in portuguese) Côrtes, ACAS, Sousa TP, Barros MV, Rodrigues FA, Silva GB, Filippi MCC (2015) Enzyme-Induced Defense Response in the Suppression of Rice Leaf Blast ( Magnaporthe Oryzae ) by Silicon Fertilization and Bioagents. International J of Res Studies in Biosciences 3: 22-32. ISSN 2349-0357 (Print) and ISSN 23490365 (Online) DEPEC-Departamento de Pesquisas e Estudos Econômicos (2019) Arroz e produtos 2017-Bradesco. Disponível em: www.economiaemdia.com.br. Accessed 22 de January 2021. EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA - EMBRAPA. Consumo Per capita de Arroz ( Oryza sativa L.) e de Feijão-comum ( Phaseolus vulgaris L.), no Brasil, de 1985 a 2019 (2020) https://www.cnpaf.embrapa.br/socioeconomia/docs/arroz/consumopercapitaarrozefeijao.ht m. Accessed 02 February 2021 (in portuguese) Felipe LO, Bicas JL (2017) Terpenos, aromas e a química dos compostos naturais. Química Nova na Escala 39:120-130. https://doi.org/10.21577/0104-8899.20160068 Filippi MCC, Silva GB, Silva-Lobo VL, Cortes MMCB, Moraes AJG, Prabhu AS (2011) Leaf blast ( Magnaporthe oryzae ) suppression and growth promotion by rhizobacteria on aerobic rice in Brazil. Biological Control 58:160-166. https://doi.org/10.1016/j.biocontrol.2011.04.016 Fungaro MHP (2000) PCR na micologia. Bioenol: Ciênc & Densenv, 3:12-16 (in portuguese) Guimarães RA (2014) Caracterização de Isolados de Sarocladium oryzae e seu Potencial na Supressão da Brusone em Arroz. Dissertation, Universidade Federal de Goiás (in portuguese) Lenz, G, Balardin RS, Corte GD, Marques LN, Debona (2010) Escala diagramática para avaliação de severidade de mancha-parda em arroz. Ciência Rural 40:752-758. https://doi.org/10.1590/S0103-84782010005000061 (in portuguese) Levenfors JJ, Hedmana R, Thaningb C, Gerhardsonb B, Welch CJ (2004) Broad-spectrum antifungal metabolites produced by the soil bacterium Serratia plymuthica A 153. Soil Biology & Biochemistry 36:677–685. https://doi.org/10.1016/j.soilbio.2003.12.008 Silva MA, Nascente AS, Filippi MCC, Lanna AC, Silva GB, Fernandes JPT, Arriel-Elias MT (2020) Screening of Beneficial Microorganisms to Improve Soybean Growth and Yield. Brazilian Archives of Biology and Technology 63:e20190463. https://doi.org/10.1590/1678-4324-2020190463 Marques MV (2012) Biologia Molecular e Genética Bacteriana (ed). SBG, Viçosa (in portuguese) Martins, BEM, Chaibub AA, Cortês MVCB, Silva-Lobo VL, Filippi MCC (2020) Characterization of bacterial isolates for sustainable rice blast control. Rev. Caatinga, Mossoró 33:702-712. http://doi.org/10.1590/1983-21252020v33n313rc Martins BEM (2015) Caracterização morfológica, bioquímica e molecular de isolados bacterianos antagonistas a Magnaporthe Oryzae . Dissertation, Universidade Federal de Goiás (in portuguese) Melo FMP (2005) Atividade antifúngica de metabólitos secundários produzidos pelo endófito de mandioca Bacillus pumilus MAIII4a. Dissertation, Universidade de São Paulo (in portuguese) Mendes HTA, Coutrim RL, Nolasco DSJ, Anjos DN, Barros BL, São José AR (2018). Metabólitos voláteis produzidos por Bacillus subtilis e Bacillus amyloliquefaciens sobre o crescimento micelial de Fusarium oxysporum f. sp. cubense . Cadernos de Agroecologia-Anais do VI CLAA, X CBA e V SEMDF, v. 13 ISSN 2236-7934 (in portuguese) Hussain M, Debnath B, Qasim M, Bamisile BS, Islam W, Hameed MS, Wang L, Qiu D (2019) Role of Saponins in Plant Defense Against Specialist Herbivores. Molecules 24:2067. https:?/doi.org/10.3390/molecules24112067 Nascente AS, Filippi MCC, Lanna AC, Souza AC, Silva-Lobo VL, Silva GB (2017) Biomass, gas exchange, and nutrient contents in upland rice plants affected by application forms of microorganism growth promoters. Environmental Science and Pollution Research, 24:2956-2965. https://doi.org/10.1007/s11356-016-8013-2 Nascente AS, Filippi MCC, Sousa TP, Chaibub AA, Souza ACA, Lanna AC (2019) Upland rice gas exchange, nutrient uptake and grain yield as affected by potassium fertilization and inoculation of the diazotrophic bacteria Serratia spp. Southern Cross Publishing Group, Australia, Australian Journal of Crop Science 13:944-953. https://doi.org/10.21475/ajcs.19.13.06.p1689 Notteghem JL (1981) Cooperative experiment on horizontal resistance to rice blast. In: Blast and upland rice: report and recommendations from the meeting for international collaboration in upland rice improvement. Los Baños, IRRI, pp.43-51 Olanrewaju OS, Glick BR, Babalola OO (2017) Mechanisms of action of plant growth promoting bacteria. World J Microbiol Biotechnol 33:197. https://doi.org/10.1007/s11274-017-2364-9 Oliveira SC, Castroagudin VL, Maciel JLN, Pereira DAS, Ceresini PC (2015) Resistência cruzada aos fungicidas azoxistrobina e piraclostrobina no patógeno da brusone do trigo Pyricularia oryzae no Brasil. Summa Phytopathologica 41:298-304. https://doi.org/10.1590/0100-5405/2072 (in portuguese) Saha P, Ghosh S, Roy-Barmana S (2020) MoLAEA Regulates Secondary Metabolism in Magnaporthe oryzae . M Sphere 5:e00936-19. https://doi.org/10.1128/mSphere.00936-19 Prabhu AS (2006) Brusone em arroz: controle genético, progresso e perspectivas. Santo Antônio de Goiás, Embrapa Arroz e Feijão (in portuguese) Prabhu AS, Filippi MCC, Silva GB, Silva-Lobo VL, Morais OP (2009) An unprecedented outbreak of rice blast on a newly released cultivar BRS Colosso in Brazil. In: Wang GL, Valent B (ed) Advances in genetics, genomics, and control of rice blast. Springer Science, Netherlands, pp. 257–267 Dean RA, Talbot NJ, Ebbole DJ et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature Publishing Group 434:980-986. https://doi.org/10.1038/nature03449 Sansinenea E, Ortiz A (2011) Secondary metabolites of soil Bacillus spp. Biotechnol Lett 33:1523-1538. https://doi.org/10.1007/s10529-011-0617-5 Sperandio EM, Vale HMM, Reis MS, Cortes MVCBC, Lanna AC, Filippi MCC (2017) Evaluation of rhizobacteria in upland rice in Brazil: growth promotion and interaction of induced defense responses against leaf blast ( Magnaporthe oryzae ). Acta Physiologiae Plantarum 39:258-270. https://doi.org/10.1007/s11738-017-2547-x Rezende CC, Frasca LLM, Silva MA, Pires RAC, Lanna AC, Filippi MCC, Nascente AS (2021) Physiological and agronomic characteristics of the common bean as affected by multifunctional microorganisms. Semina: Ciências Agrárias 42:599-618. https://doi.org/10.5433/1679-0359.2021v42n2p599 Roberts DP, Lakshman DK, McKenna LF, Emche SE, Maul JE, Bauchan G (2016) Seed treatment with ethanol extract of Serratia marcescens is compatible with Trichoderma isolates for control of damping-off of cucumber caused by Pythium ultimum . Plant Dis. 100:1278-1287. https://doi.org/10.1094/PDIS-09-15-1039-RE Sakthivel N, Amudha R, Muthukrishnan S (2002). Production of phytotoxic metabolites by Sarocladium oryzae. Mycol. Res. 106:609-614. https://doi.org/10.1017/S0953756202005774 Achotegui-Castells A, Gianni DR , Llusi a J, Danti R, Barberini S, Bouneb M, Simoni S, Michelozzi M, Peñuelas J (2016) Terpene arms race in the Seiridium cardinale – Cupressus sempervirens pathosystem. Scientific Reports 6:18954. https://doi.org/ 10.1038/srep18954 D'Ávila LS , De Filippi MCC , Café-Filho AC (2021) Sensitivity of Pyricularia oryzae Populations to Fungicides Over a 26-Year Time Frame in Brazil. Plant Disease PDIS-08. https://doi.org/ 10.1094/pdis-08-20-1806-re Sharma A, Kumar V, Shahzad B et al. (2019) Worldwide pesticide usage and its impacts on ecosystem. SN Applied Sciences 1:1446. https://doi.org/10.1007/s42452-019-1485-1 Silva-lobo VL, Filippi MCC, Silva GB, Venancio WL, Prabhu AS (2012) Relação entre o teor de clorofila nas folhas e a severidade de brusone nas panículas em arroz de terras altas. Tropical Plant Pathology 37:83-87. https://doi.org/10.1590/S1982-56762012000100011 (in portuguese) SOSBAI-Sociedade Sul-Brasileira de Arroz Irrigado. Arroz irrigado: recomendações técnicas da pesquisa para o Sul do Brasil. Itajaí, SC: SOSBAI, 2012 (in portuguese) Sousa TP, Souza ACA, Cortes MVCB, Lanna AC, Pinheiro HA, Filippi MCC, Silva GB (2017) Bioagents and silicon promoting fast early upland rice growth. Environmental Sciences and Pollution Research 25:3657-3668. https://doi.org/10.1007/s11356-017-0753-0 Souza ACV (2012) Pesquisa com arroz irrigado ( Oryza sativa L.) no sistema pré-germinado: melhoramento genético, toxidez por ferro e brusone. Complete of course work, Universidade Federal de Santa Catarina (in portuguese) Sunder S, Singh R, Agarwal (2014). Brown spot of rice: an overview. Indian Phytopath., 67: 201-215. : https://www.researchgate.net/publication/296025966 Thomson NR, Crow MA, McGowan SJ, Cox A, Salmond GPC (2000) Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control. Molecular Microbiology 36:539-556. https://doi.org/10.1046/j.1365-2958.2000.01872.x Tortora GJ, Funke BR, Case CL et al. (2017) Microbiologia, 12 a ed. Artmed, Porto Alegre (in portuguese) Wagner H, Bladt S (1996) Plant drug analysis-A thin layer cromatography atlas. 2 a ed. Springer, Berlim Tremacoldi CR, Filho APSS (2006) Toxinas produzidas por fungos Fitopatógenos: Possibilidades de Uso no Controle de Plantas Daninhas. Embrapa Amazônia Oriental, Belém (in portuguese) Sangappillai V, Nadarajah K (2020) Fatty Acid Synthase Beta Dehydratase in the Lipid Biosynthesis Pathway Is Required for Conidiogenesis, Pigmentation and Appressorium Formation in Magnaporthe oryzae S6. Int. J. Mol. Sci. 21:7224. https://doi.org/doi:10.3390/ijms21197224 Zanirati DF (2012) Caracterização de bactérias láticas da microbiota de grãos de kefir cultivados em leite ou água com açúcar mascavo por metodologias dependentes e independentes de cultivo. Dissertation, Universidade Federal de Minas Gerais Tables Table 1 Collection details and GenBank accession numbers of isolates included in the present study. Serratia spp. Species Strain GenBank Accession number (16S rRNA ) Serratia plymuthica DSM 4540 NR 114579 S16.1.2 OK284921 Serratia ficaria NBRC 102596 NR 114155 DSM 4569 AJ233428 Serratia rubidaea DSM 4480 AJ233436 NBRC 103169 NR 114232 Serratia marcescens ZO1-A AJ296310 WO1-A AJ296309 BRM32113 In process Serratia nematodiphila PSI26 OK423573 B1 OK560052 Serratia ureilytica NiVa 51T AJ854062 NiVa 51 NR 042356 Bacillus spp. Bacillus cereus wg-3 EU624208 NVH 391-98 AM747234 Bacillus anthracis ATCC 14578 AB190217 LB5YE MW819989 Bacillus wiedmannii FSL W8-0169 NR 152692 Bacillus mycoides ATCC 6462 AB021192 2861 MT586023 Bacillus weihenstephanensis DSM 1182 AB021199 Bacillus sp. BRM32110 In process Bacillus toyonensis BCT-7112 NR121761 Bacillus cereus biovar toyoi NCIB 40112 AJ310100 Bacillus thuringiensis Bt11 MT292101 Bacillus pseudomycoides NRRL B-617 AF013121 B-617 NR11442 *Isolates and newly generated sequences are in bold. Table 2. Treatments description for Conidial germination and appressorium formation assays, combining growth period and extract concentration of BRM32110 and BRM32113 Isolate Treatments number Growth period (h) Concentration H 2 O 1 - - DMSO 2 - - BRM 32113 extration 3 6 100mg 4 6 50mg 5 6 25mg 6 6 10mg 7 6 5mg 8 16 100mg 9 16 50mg 10 16 25mg 11 16 10mg 12 16 5mg BRM 32110 13 6 100mg 14 6 50mg 15 6 25mg 16 6 10mg 17 6 5mg 18 16 100mg 19 16 50mg 20 16 25mg 21 16 10mg 22 16 5mg Table 3 Leaf gray spot and leaf blast suppression treatments, extraction time and extract concentration of BRM32110 and BRM32113, assays E1 and E3 Isolate Treatments number Extraction time (h) Concentration 1 H 2 O - - 2 DMSO - - BRM32110 3 - 10 8 CFU.mL -1 BRM32113 4 - 10 8 CFU.mL -1 BRM32110 5 6 100mg BRM32110 6 16 50mg BRM32113 7 6 25mg BRM32113 8 BRM32113 Extract 16 25mg Table 4. Reduction of mycelial growth of B. oryzae and M. oryzae by extracts of BRM32110 and BRM32113, after 7 days of growth in BDA. Means followed by the same letter do not differ statistically from each other by the Tukey’s test ( p <0.05) Treatments Extaction time (h) Reduction of colony area (%) B. oryzae Reduction of colony area (%) M. oryzae Control (H 2 O) - 0 c 0 h Control (DMSO) - 0 c 0 h BRM32110 - 6h 6 17.2 b 94,3 a BRM32110- 16h 16 28.9 a 93,2 a BRM32110 - 24h 24 0 c 80,0 bc BRM32110 - 48h 48 0 c 69,1 def BRM32110 - 72h 72 3.3 c 68,3 def BRM32113 - 6h 6 17.2 b 89,8 a BRM32113 - 16h 16 21.5 b 87,3 ab BRM32113 - 24h 24 3.3 c 76,6 cd BRM32113- 48h 48 1.4 c 74,4 cde BRM32113 - 72h 72 0.7 c 68,3 def Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1087379","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":67293027,"identity":"7291152a-ab6a-4651-ba7f-187d4e969dec","order_by":0,"name":"Marina Teixeira Arriel-Elias","email":"","orcid":"","institution":"UFG-EA: Universidade Federal de Goias Escola de Agronomia","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"Teixeira","lastName":"Arriel-Elias","suffix":""},{"id":67293028,"identity":"d7959139-a75b-4e3b-ae17-c0e1f618c77f","order_by":1,"name":"Gabriel Carlos Teixeira Freire Arriel","email":"","orcid":"","institution":"UniEVANGÉLICA Centro Universitário de Anápolis: UniEVANGELICA Centro Universitario de Anapolis","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"Carlos Teixeira Freire","lastName":"Arriel","suffix":""},{"id":67293029,"identity":"29b81f29-8bd4-4b6a-9fb1-96756b971632","order_by":2,"name":"Gustavo Andrade Bezerra","email":"","orcid":"","institution":"UFG-EA: Universidade Federal de Goias Escola de Agronomia","correspondingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"Andrade","lastName":"Bezerra","suffix":""},{"id":67293030,"identity":"1e500649-feb4-4d87-b9ef-9e33cc61b24c","order_by":3,"name":"Pedro Henrique Dias dos Santos","email":"","orcid":"","institution":"UFV: Universidade Federal de Vicosa","correspondingAuthor":false,"prefix":"","firstName":"Pedro","middleName":"Henrique Dias dos","lastName":"Santos","suffix":""},{"id":67293031,"identity":"1b4d3a99-1c36-4d49-b310-15bb06dc066d","order_by":4,"name":"Vanessa Gisele Pasqualotto Severino","email":"","orcid":"","institution":"UFG: Universidade Federal de Goias","correspondingAuthor":false,"prefix":"","firstName":"Vanessa","middleName":"Gisele Pasqualotto","lastName":"Severino","suffix":""},{"id":67293032,"identity":"e61c9a78-f596-4b3b-b834-a055615b4e58","order_by":5,"name":"Marta Cristina Filippi","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1676-8164","institution":"Embrapa Arroz e Feijão","correspondingAuthor":true,"prefix":"","firstName":"Marta","middleName":"Cristina","lastName":"Filippi","suffix":""}],"badges":[],"createdAt":"2021-11-17 01:35:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1087379/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1087379/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":16149341,"identity":"1d635b4f-c76f-4bce-a193-ed0b04ef8634","added_by":"auto","created_at":"2021-12-03 15:50:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149656,"visible":true,"origin":"","legend":"Phylogenetic trees based on the 16S gene recovered from the genomes of Bacillus sp. (A) and Serratia marcescens (B).","description":"","filename":"Fig.1copy.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1087379/v1/8bcfdfe956efbc25735a21d9.jpg"},{"id":16149512,"identity":"650d57e4-480d-4f2f-a9e3-6ed8c2f2bcc7","added_by":"auto","created_at":"2021-12-03 15:53:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":326521,"visible":true,"origin":"","legend":"(A) B. oryzae’s and (B) M. oryzae’s colony growth suppression in Petri plates containing PDA medium enriched with 50 μl of each extract (BRM32110 and BRM32113) solubilized in 1mL of DMSO (dimethylsulfoxide), seven days after incubation. A: first assay (a) control (PDA medium enriched with water), (b) PDA medium enriched with DMSO; BRM32113 extract collected at: 6h (c), 16h (d), 24h (e), 48h(f), 72h(g); BRM32110 extract collected at: 6h(h), 16h (i), 24h (j), 48h (l), 72h(m). B: second assay (a) control (PDA medium enriched with water), (b) PDA medium enriched with DMSO; BRM32113 extract collected at: 6h (c), 16h (d); BRM32110 extract collected at: 6h(e), 16h (f)","description":"","filename":"Fig.2copy.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1087379/v1/49259ff84109759b9d87f4bd.jpg"},{"id":16149340,"identity":"42e5919b-1e64-4256-a612-618692f8a78e","added_by":"auto","created_at":"2021-12-03 15:50:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68495,"visible":true,"origin":"","legend":"B. oryzae conidial concentration per Petri dish containing PDA culture medium, suplemented with 50 μl of each treatment solubilized in 1mL of DMSO (dimethylsulfoxide) seven days after incubation. Lowercase letters define the effect of treatmentsby the Tukey test (p ≤ 0.05). The bars indicate the mean standard error","description":"","filename":"Fig.3copy.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1087379/v1/3c76a629f86cb629d338e88c.jpg"},{"id":16149345,"identity":"5d4a9c5c-7930-4262-a652-c5eba9c75210","added_by":"auto","created_at":"2021-12-03 15:50:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":165135,"visible":true,"origin":"","legend":"Percentage of M. oryzae conidial germination (A and B) and appressorium (C and D) formation in drops containing conidal suspension and different BRM 32110’s extract concentration. The observations time were at 4, 6 and 16 hours after assay incubation. (A) BRM32110 extract obtained with 6h of bacterial cell growth in culture medium, (B) BRM32110 extract obtained with 16h of bacterial cell growth in culture medium, (C) BRM32110 extract 6h, (D) BRM32110 extract 16h. Lowercase letters define the effect of treatments in each incubation hour by the Tukey test (p ≤ 0.05). The bars indicate the mean standard error","description":"","filename":"Fig.4copy.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1087379/v1/05de1b9a8dca55be3ec4acd0.jpg"},{"id":16149542,"identity":"457b6b87-c881-4534-86cd-9560ca898e0d","added_by":"auto","created_at":"2021-12-03 15:56:33","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":150492,"visible":true,"origin":"","legend":"Percentage of M. oryzae conidial germination (A and B) and appressorium (C and D) formation in drops containing conidal suspension and different BRM 32113’s extract concentration. The observations time were at 4, 6 and 16 hours after assay incubation. (A) BRM32113 extract obtained with 6h of bacterial cell growth in culture medium, (B) BRM32113 extract obtained with 16h of bacterial cell growth in culture medium, (C) BRM32113 extract 6h, (D) BRM32113 extract 16h. Lowercase letters define the effect of treatments in each incubation hour by the Tukey test (p ≤ 0.05). The bars indicate the mean standard error","description":"","filename":"Fig.5copy.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1087379/v1/1a98e4460a1eb5deb60c857b.jpg"},{"id":16149344,"identity":"6816bcdb-572c-4c2a-8d62-2f91e97554e5","added_by":"auto","created_at":"2021-12-03 15:50:33","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":147687,"visible":true,"origin":"","legend":"Phytotoxicity of adjuvants used in the different formulations in rice leaves, evaluated during seven days. (A) day one, (B) day seven. Treatments: (1) water, (2) DMSO (dimetilsofoxide); BRM32110 extract at 6h (3), BRM32110 extract at 16h (4); BRM32113 extract at 6h (5); BRM32113 extract at 16h (6)","description":"","filename":"Fig.6copy.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1087379/v1/c80ec8a898eace01602c807c.jpg"},{"id":16149346,"identity":"b770e724-c4b4-4208-a761-22c280d6ad2c","added_by":"auto","created_at":"2021-12-03 15:50:33","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":125285,"visible":true,"origin":"","legend":"The severity of leaf gray spot (1) and leaf blast (2) according to the scale of notes. Uppercase letters indicate different symptoms in rice leaves of each treatment: (A) water; (B) DMSO; (C) BRM32110; (D) BRM32113; (E) BRM32110 extract 6h (100mg); (F) BRM32110 extract 16 h (50mg); (G)BRM32113 extract 6h (25mg), (H) BRM32113 extract 16h (25mg).Columns (lowercase letters) followed by the same letters were not significantly different from each other according to Tukey’s test (p \u003c 0.05). The bars indicate the mean standard error","description":"","filename":"Fig.7copy.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1087379/v1/a51550387ca9be204018c5d8.jpg"},{"id":16609673,"identity":"22197caa-b283-4ea6-8495-94d679e7b604","added_by":"auto","created_at":"2021-12-20 13:02:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1051383,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1087379/v1/6951f4d0-04c1-4233-b07c-e7c5ec090679.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eOptimization of the Extraction Process and Application of Bacterial Extracts in the Control of Brown Spot and Leaf Blast in Rice Culture\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe warning about the need to increase food production is recurrent, and rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) stands out for its strategic role in both social and economic development in Brazil and in the world (CONAB 2020). The average consumption in the world is 57 kg person-1 year-1 (Nascente et al. 2019), and in Brazil consumption is on average 45 kg of rice person-1 year-1 (Embrapa 2020). World rice production was approximately 495.23 million tons, and has the highest potential for increased production among other cereals, supplying 20% of calories and 15% of protein consumed in the diet (Sosbai 2012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The production of this cereal has been showing a drop in profitability, according to the increase in production costs, caused by the excessive number of fungicides and fertilizers, to ensure the production and quality of grains threatened by stresses (Guimar\u0026atilde;es et al. 2014; Ahmad et al. 2016), as the occurrence of brusone and grain spot. Brusone caused by the fungus \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e B. Couch ranks first in economic importance (Silva-Lobo et al. 2012; Dean et al. 2005), is difficult to control and can cause up to 100% losses in rice yield (Prabhu et al. 2009). Brown spot (\u003cem\u003eBipolaris oryzae\u003c/em\u003e) causes grain yield losses (90%), particularly when the leaf spot stage assumes epiphytic proportions (Sunder et al. 2014).\u003c/p\u003e\n\u003cp\u003eThe cost of pesticides in rice farming has increased by about 180% since the 2007/08 crop (CONAB 2021; Sharma et al. 2019) and fungicides account for up to 45% of production costs for both irrigated and upland systems (DEPEC 2019). In addition, the most commercially used molecules present specific site of action as triazoles and strobilurins, which facilitates the selection of isolates resistant to the most used molecules (Souza 2012; D\u0026apos;\u0026Aacute;vila et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, the market for biological inputs has been growing in recent years, The global agricultural biologicals market reached a value of US$ 8.1 Billion in 2020 and is estimated to register a compound annual growth rate of 9.4% from 2020 to 2027. worldwide due to the trend of an increasing demand in integrated pest and disease management (IPM) (Grand View Research 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite the fast growing market, the production of a bioproduct for plant health protection faces obstacles such as the development of an efficient and sustainable formulation. The main technical barriers to legalize and use these bioproducts are: lack of stability of the formulated product (low shelf life), low viability of microorganisms after exposure to ultraviolet rays of the sun, difficulty in the application of the product in the field, acceptance of the producers for presenting incompatibility with chemicals used in the management of various crops and species of microorganisms that are close to species pathogenic to humans (Bettiol et al. 2014).\u003c/p\u003e\n\u003cp\u003eIn previous studies conducted by our team, six bacterial isolates were shown to be potential biological agents in rice blast suppression. Among them we highlight those of the genus Bacillus and Serratia. (Filippi et al. 2011; Martins 2015; Martins et al. 2020). Bacteria of the genus Bacillus belong to the phylum Firmicutes, family Bacillaceae, can be found in soil, in association with plants, rivers, and estuaries and produce several metabolites of industrial interest such as enzymes, B-exotoxin, thuricin, petrobactim, bacillibactim (Bach et al. 2011; Sansinenea et al. 2011).\u003c/p\u003e\n\u003cp\u003eThe species of the genus Serratia stand out in their capacity to produce siderophores, indol acetic acid, phosphate solubilizers and production of antifungal metabolites, such as pyrrolnitrin, carbapenem, prodigiosin, haterumalide and siderophores (Levenfors et al. 2004; Thompson et al. 2002). These natural products, when compared to synthetic compounds, offer greater structural diversity, synergism between molecules and are considered exceptional sources of new agrochemicals (Tremacoldi and Filho 2006).\u003c/p\u003e\n\u003cp\u003eWe hypothesized that rhizobacteria produce primary and secondary metabolites that are responsible for their biological characteristics and activities, of potential importance, to be used in agriculture, because, they present a wide chemical diversity and can be a new integrant for the integrated management of diseases in rice culture. Therefore, the objective of this work was to optimize the extraction process and evaluate the efficiency of bacterial extracts of \u003cem\u003eBacillus\u003c/em\u003e sp. and \u003cem\u003eSerratia\u003c/em\u003e sp. in the control of leaf blast (\u003cem\u003eM. oryzae\u003c/em\u003e) and leaf brown spot (\u003cem\u003eB. oryzae\u003c/em\u003e) in rice culture.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eMicroorganisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolates \u003cem\u003eSerratia\u003c/em\u003e sp. (BRM 32113), \u003cem\u003eBacillus\u003c/em\u003e sp. (BRM 32110) \u003cem\u003eM. oryzae\u003c/em\u003e (BRM 31295) and \u003cem\u003eB. oryzae\u0026nbsp;\u003c/em\u003e(BRM46676)\u003cem\u003e\u0026nbsp;\u003c/em\u003ebelongs to Embrapa Microrganism Collection, preserved by the Castellani and deep freeze methods. Colonies of bacterial strains were transferred to Petri plates containing Nutrient Agar (AN-Extatro de carne, Peptona, Cloreto de s\u0026oacute;dio, \u0026Aacute;gar), and incubated during 48 hours at 28 \u0026deg;C. \u003cem\u003eM. oryzae\u003c/em\u003e isolate was transferred to Petri plates containing Potato Dextrose Agar (PDA), and incubated for seven days, at 24 \u0026deg;C, under light regime for seven days. \u003cem\u003eB. oryzae\u0026nbsp;\u003c/em\u003eisolate were transferred to Petri plates containing PDA, and incubated for seven days, at 24 \u0026deg;C, under dark conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial isolates identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe two bacterial isolates had their complete genome sequenced using the Illumina platform, where the Nextera XT kit (shotgun) is used, which contains the adaptor primers required for sequencing. Coverage: 100x (1 million reads). We extracted, for the BRM32110 and BRM32113, the 16S rRNA region from the genomes assembled, using the ContEst16S (Contamination Estimator by 16S) (Lee et al., 2017) in which 16S rRNA gene fragments from the query genome assemblies are screened. The database for both isolates was constructed by species from monophyletic clade of the S. marcescens and Bacillus thuringiensis (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSequences in the GenBank database were compared to check for molecular identification through the nucleotide BLAST algorithm\u0026nbsp;(Altschul et al. 1990). MUSCLE was used in the alignments\u0026nbsp;(Edgar 2004), implemented in MEGA v.7\u0026nbsp;(Kumar et al. 2016). Bayesian inference (BI) analysis was carried out, for each isolate reconstruction, based on the Monte Carlo Markov chain method (MCMC). The MrMODELTEST v. 3.04\u0026nbsp;(Nylander 2004)\u0026nbsp;was adopted to select the nucleotide substitution model for BI analysis. Likelihood values were calculated, and the model was selected according to the Akaike information criterion (AIC). Evolution models selected for each isolates reconstruction were HKY + I for BRM32110 and GTR + I for BRM32113. The BI analysis was carried out in MrBayes v.3.1.1\u0026nbsp;(Ronquist and Huelsenbeck 2003). The consensus tree was obtained after 10 million generations of a Markov chain were applied to two runs, four chains each, with a 15% burn- in. Likelihood log convergence was analyzed in TRACER v. 1.4.1 software\u0026nbsp;(Rambaut et al. 2018). The tree was visualized and edited in ITol v.4\u0026nbsp;(Letunic and Bork 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparing the bacterial suspensions and obtaining the extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacterial strains grown in Petri dishes were transferred to 5 different Erlenmeyers, each containing 100mL of the nutrient broth culture medium. The Erlenmeyers were kept under constant 150 rpm shaking at 28 \u0026deg;C for 6, 16, 24, 48, and 72 hours of growth.\u003c/p\u003e\n\u003cp\u003eAfter the different growth periods, 50.0 mL of each bacterial suspension was transferred to a separation funnel, in which the same volume (50.0 mL) of ethyl acetate solvent was also added. The mixture was stirred slowly to avoid the formation of internal pressure in the funnel. The system was kept at rest until complete separation of the phases. The aqueous phase was separated from the organic phase and returned to the separation funnel. The aqueous phase was subjected to a further extraction step with the addition of another 50.0 mL of the same solvent. The aqueous layer was collected in an Erlenmeyer, and for one more time the process was repeated until three extraction steps were completed. The phase containing the ethyl acetate was concentrated at 50 \u0026deg;C in a rotary evaporator under reduced pressure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThin-layer Chromatography\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples were analyzed via thin-layer chromatography (TLC) performed on an aluminum foil (4 x 5 cm) coated with a silica gel with fluorescent indicator F254 as the stationary phase. After applying acetone soluble samples on the plates, anethylacetate:hexane (9:1 v/v) mobile phase was eluted by capillarity. The chemical profile of the samples was visualized by ultraviolet light (254 and 365 nm) and vanillin solution (composed by135 mL of methanol, 3 mL of vanillin, 135mL of distillation water and 35 mL of sulfuric acid) as revealing, which forms colored spots, according to its interaction with the natural compounds classes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtracts screening for antagonistic activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolates of \u003cem\u003eM. oryzae\u003c/em\u003e and \u003cem\u003eB. oryzae\u003c/em\u003e were previously grown in a Petri dish containing BDA (potato dextrose agar) culture medium during seven days of incubation at 24\u0026deg;C. Then, discs of culture medium, 5 mm in diameter, containing mycelia of the fungi were transferred to new Petri plates, 60 mm in diameter, containing BDA culture medium supplemented with 50 \u0026mu;l of the extracts of \u003cem\u003eBacillus\u003c/em\u003e sp. and \u003cem\u003eSerratia\u003c/em\u003e sp solubilized in 1mL of DMSO (dimethylsulfoxide). The assay was conducted in an entirely randomized design with twelve treatments in four repetitions (tab....). The control treatment consisted of the medium supplemented with DMSO (negative control). The absolute control was considered a BDA plate containing only the mycelium discs of the two pathogens. The plates were incubated under a constant temperature of 24\u0026deg;C for 7 days. At the end of the incubation period, colony diameters were measured using a millimeter ruler. Antagonism was determined by comparing the percentage reduction of the mean colony areas in the different treatments (Filippi et al., 2011).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe best treatment identified in the first assay antagonism assay, it was also quantified the\u003cem\u003e\u0026nbsp;B. oryzae\u003c/em\u003e conidial production by each colony, per Petri dish. Conida was removed with 3mL/plate of distilled and sterilized water and a brush. The concentration was estimated with a Neubauer chamber. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of conidial germination and appressorium formation of \u003cem\u003eM. oryzae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA suspension of \u003cem\u003eM. oryzae\u003c/em\u003e conidia, prepared at a concentration of 2 x 10\u003csup\u003e5\u003c/sup\u003e conidia/mL was mixed with the different extracts at different concentrations. Five microliters of each extract were pipetted into an artficial hydrophobic plastic surface to induce germination and appressorium formation. The hydrophobic plastic surfaces containing the droplets were kept in a humid chamber in a growth chamber. Evaluations were performed after 4, 6 and 16 hours, under a 20x optical microscope. Germinated conidia and appressorium formed were quantified. The trial was composed of the treatments described in Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhytotoxicity of extracts on rice leaves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetached leaves of the rice cultivar BRS Primavera with 21 days after planting were placed in a humid chamber and sprayed with the extracts, selected in the test described in previous section. In an entirely randomized design, 4 treatments were evaluated, representing different extraction times and concentrations: T1: BRM 32113 6h of growth and sprayed at 25mg of exttract; BRM 32113 16h of growth, sprayed at 25mg extract; BRM 32110 6h of growth and sprayed at 100mg extract; BRM 16h of growth and sprayed at 50mg extract. The sprayed leaves were incubated at 25 \u0026plusmn; 2 \u0026deg; C under constant common light. Visual assessments of phytotoxicity, characterized by yellowish/whitish spots (chlorotic lesions), or any other changes on the leaf surface compared to the control, were performed daily for seven days, based on Sakthivel et al. (2002). The control of the experiment was represented by spraying leaves with sterilized distilled water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssays in greenhouse\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssays E1 and E2: Gray spot and blast leaf suppression\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was composed of 8 treatments and three replicates, totaling 24 trays. Treatments in E1 and E2 were the same, but, E1 were spray inoculated with \u003cem\u003eB. oryzae,\u0026nbsp;\u003c/em\u003eand E2 with \u003cem\u003eM. oryzae.\u0026nbsp;\u003c/em\u003ethe treatments of both assays are described in Table 3.\u003c/p\u003e\n\u003cp\u003ePreparation of bacterial suspension: a cell suspension of the\u0026nbsp;\u003cem\u003eB. pyrrocinia\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacillus\u0026nbsp;\u003c/em\u003esp.\u0026nbsp;isolate was transferred to a 500 ml Erlenmeyer flask, containing 200 ml of nutrient broth culture medium, prepared according to Arriel-Elias et al. (2018). The bottles were under constant stirring at 150 rpm, for 48 h at 28\u0026deg;C. Then, each suspension was adjusted, with a spectrophotometer, to 0.5 absorbance, at a wavelength of 620 nm, corresponding to 1x10\u003csup\u003e8\u003c/sup\u003e CFU.ml\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSowing in greenhouse\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeeds of upland rice cultivar BRS Primavera were disinfectedwith 70% alcohol and 30% sodium hypochlorite before planting and sownin eight furrows in plastic trays (15 \u0026times; 30 \u0026times; 10 cm) in 3 kg of soil, that was fertilized with NPK (5 g of 5-30-15 + Zn and 3 g of ammonium sulphate). Besides, 5 g (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e + Fe and Bo were applied per tray 19 days after sowing.\u0026nbsp;A daily irrigation regime was adopted, corresponding to approximately 100% of the soil field capacity. Trays planted with rice seedswere maintained at greenhouse until therice plants get to V3 stage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. oryzae suspension for spray inoculation (E1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eB. oryzae\u003c/em\u003e isolate, was multiplied in Petri plates containing BDA and incubated at 25 \u0026deg; C for seven days with continuous dark. Conidium was removed with sterilized distilled water and a brush, and the concentration was adjusted for 1x10\u003csup\u003e5\u003c/sup\u003e con.ml\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAt the 35\u003csup\u003est\u003c/sup\u003e day (V4 stage), rice plants were transferred to cages coated with transparent plastic. Thirty ml of \u003cem\u003eB. oryzae\u003c/em\u003e conidial was suspended. It was pulverized in each cage contained three trays with rice plants. The pulverization was performed with a pressure pump and a spray gun, at a pressure of 0.001 kg / cm\u003csup\u003e2\u003c/sup\u003e. The plants remained in the greenhouse under controlled environmental conditions, with temperatures ranging from 27 to 28 \u0026deg; C and relative humidity above 80%, for 8 days. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLeaf gray spot severity was evaluated using a severity scale (1.6%, 3.2%, 6.4%, 12.6, 23.1% and 38.6%), according to Lenz et al. (2010), and determining the percentage of affected leaf area by disease.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM. oryzae suspension for spray inoculation (E2)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM.oryzae\u003c/em\u003e isolates (BRM 31295), was multiplied in Petri plates containing agar, oatmeal and incubated at 25 \u0026deg; C for seven days with continuous light. The aerial mycelium of \u003cem\u003eM. oryzae\u003c/em\u003e colonies was removed with a sterile glass rod and the plates incubated in a growth chamber for 48 hours, under continuous fluorescent light. Conidium was removed with sterilized distilled water and a brush, and the concentration was adjusted for 3x10\u003csup\u003e5\u003c/sup\u003e con.ml\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAt the 21\u003csup\u003est\u003c/sup\u003e day (V3 stage), rice plants were transferred to cages coated with transparent plastic. Thirty ml of \u003cem\u003eM. oryzae\u003c/em\u003e conidial was suspended (Filippi et al. 2007). It was pulverized in each cage contained three trays with rice plants. The pulverization was performed with a pressure pump and a spray gun, at a pressure of 0.001 kg / cm2. The plants remained in the greenhouse under controlled environmental conditions, with temperatures ranging from 27 to 28 \u0026deg; C and relative humidity above 80%, for 8 days. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLeaf blast severity was evaluated using a severity scale (0%, 0.5%, 1%, 2%, 4%, 8%, 16%, 32%, 64%, and 82%), according to Notteghem (1981), and determining the percentage of affected leaf area by disease.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe averages of each test were calculated, the variances analyzed, and the proportions were compared by the Tukey test at 5% of significance, using the SPSS program, version 2.1.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eBacterial isolates identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA region from the genomes assembled, revealed that isolate BRM32113 pertains to the genus Serratia and species \u003cem\u003emarcescens\u003c/em\u003e and isolate BRM32110 belongs to the genus Bacillus (Fig 1, A and B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThin-Layer Chromatography\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe aspects considered were color and height after analysis with UV light and developer. The crude extract of isolates BRM32110 and BRM 32113 at the different extraction times of 6, 16, 24, 48 and 72 hours showed brown, yellow and blue spots on plate eluted with hexane anethylacetate:hexane (9:1 v/v) and revealed with vanillin solution, indicating the presence of terpenoids (yellow-brown) and fatty acids (blue), as described by Wagner \u0026amp; Bladt (1996). Stains with more marked brown coloration were detected at the same height in the extracts obtained after 6 and 16 hours of growth of both isolates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntagonistic capacity of extracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntagonism to \u003cem\u003eB. oryzae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extracts obtained from \u003cem\u003eBacillus sp.\u003c/em\u003e (BRM 32110) and \u003cem\u003eS. marcenses\u003c/em\u003e (BRM 32113), after 6 and 16 hours of growth, reduced up to 28% of the mycelial growth of \u003cem\u003eB. oryzae\u003c/em\u003e (Table 4, Fig. 2, A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuantification of conidia production of \u003cem\u003eB. oryzae\u003c/em\u003e colonies grown in culture medium supplemented with extracts of \u003cem\u003eBacillus sp.\u003c/em\u003e (BRM 32110) and \u003cem\u003eS. marcenses\u003c/em\u003e (BRM 32113) reduced conidia germination by up to 62%, statistically different from controls (Fig. 3, A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntagonism to \u003cem\u003eM. oryzae\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extracts of \u003cem\u003eBacillus sp.\u003c/em\u003e (BRM 32110) and \u003cem\u003eSerratia marcenses\u003c/em\u003e (BRM 32113) strains obtained after 6, 16, 24, 48 and 72 hours of growth reduced the growth of \u003cem\u003eM. oryzae\u003c/em\u003e colonies. The extracts obtained after 6 and 16 hours, for both \u003cem\u003eBacillus sp. (\u003c/em\u003eBRM 32110) and \u003cem\u003eS. marcenses\u003c/em\u003e (BRM 32113) isolates reduced the mycelial growth of \u003cem\u003eM. oryzae\u003c/em\u003e colonies by up to 94% (Table 4, Fig. 2, B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConidium germination and appressorium formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 6 hours of incubation: the extract of the isolate \u003cem\u003eBacillus sp.\u0026nbsp;\u003c/em\u003e(BRM32110) (6h) at concentration of 100mg, significantly inhibited in 59.6% the germination of conidia, when compared to controls (Fig. 4-A), and in 85% the appressorium formation. The extract of the isolate S. marcenses (BRM32113) at concentrations of 50 and 100mg reduced by 45 and 52%, respectively, the germination of conidia (Fig. 5-A), and at the 25mg concentration reduced by 75% the number of appressorium formed.\u003c/p\u003e\n\u003cp\u003eAfter 16 hours of incubation: the extract of the isolate Bacillus sp. (BRM32110 ) (16h) reduced in 45% and 38% the germinated conidia and 81% and 93% the appressorium formation in concentrations of 50 and 100mg (Fig. 4 - B and C). The extract of S. marcenses (BRM32113), at concentrations of 100mg and 50mg reduced conidia germination by 82% and 53% and appressorium formation by 91% and 92% respectively (Fig 5 - B and C).\u003c/p\u003e\n\u003cp\u003eThe negative control containing the solvent DMSO delayed conidia germination for both isolates, but after 6 hours of incubation it matched the control containing only water. The first appressorium were observed after 6 hours of incubation (Fig. 4 and 5) in the control treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhytotoxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Extracts individually tested showed negative for phytotoxic effect on detached rice leaves on a visual evaluation (Fig. 6 A and B). Different treatments did not alter the characteristics of leaves in a period of one week of evaluation, mainly in relation to the appearance of yellowish, whitish spots, necrosis or any other alteration of the leaf surface as compared to the control (Fig. 6 B, 1). In\u003cem\u003e\u0026nbsp;vivo\u003c/em\u003e tests under greenhouse conditions were also performed with the extracts. The results confirmed that extracts did not generate direct toxicity reactions in rice plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssays A1 (leaf brown) spot and A2 (leaf blast suppression)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrown spot\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;suppression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn A1, control treatments showed up to 23.5% of leaf area affected with brown spot at eight days after challenge inoculation. Treatments 3 (suspension of \u003cem\u003eBacillus sp.\u003c/em\u003e cells (BRM32110), with 7.8% of the leaf area affected, 5 (extract of \u003cem\u003eBacillus sp.\u003c/em\u003e BRM 32110, 6h) with 9. 8% affected leaf area, 6 (\u003cem\u003eBacillus sp.\u003c/em\u003e extract BRM 32110, 16h) with 6.7% affected leaf area and 8 (\u003cem\u003eS. marcescens\u003c/em\u003e extract BRM32113, 16h) with 6.8% affected leaf area of (Fig. 7 A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLeaf blast suppression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn A2, the treatments that stood out were 3 (suspension of \u003cem\u003eBacillus sp.\u003c/em\u003e (BRM32110) cells), with 10.4% affected leaf area, 4 (suspension of \u003cem\u003eS. marcescens\u003c/em\u003e BRM32113 cells) with 11. 6%, 5 (\u003cem\u003eBacillus sp.\u003c/em\u003e BRM 32110 extract 6h) with 8.8%, 6 (\u003cem\u003eBacillus sp.\u003c/em\u003e BRM 32110 extract 16h) with 14.4%, 7 (BRM32113 extract 6h) with 10% and 8 (BRM32113 extract 16h) with 7.6%. All treatments differed statistically from controls that showed up to 37% leaf area affected with leaf blast (Fig 7 A).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results obtained in the present study showed that the use of \u003cem\u003eBacillus sp.\u003c/em\u003e and \u003cem\u003eSerratia marcescens\u003c/em\u003e extracts were efficient and have potential for suppression of leaf brusone and brown spot in rice.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eBacillus sp.\u003c/em\u003e BRM 32110 and \u003cem\u003eS. marcescens\u003c/em\u003e BRM32113 species under study, were isolated from the rhizosphere of upland rice by Filippi et al. (2011), who performed the phenotypic, biochemical and molecular characterization, through the amplification of the gene encoding the 16S rRNA region (Martins et al. 2020). Other works of the same research group revealed that these same isolates are growth promoters in rice, soybean and bean crops, antagonists to some phytopathogens such as \u003cem\u003eM. oryzae\u003c/em\u003e and \u003cem\u003eB. oryzae\u003c/em\u003e and resistance inducers. (Filippi et al 2011; Sousa et al. 2017; C\u0026ocirc;rtes et al. 2015; Nascente et al. 2017; Sperandio et al. 2017; Arriel-Elias et al, 2018; Arriel-Elias et al 2019; Silva et al. 2020; Rezende et al. 2021). However, complete genome characterization and chemical characterization of the metabolites of both bacterial isolates had not been performed so far.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBacteria have a simple genomic structure, comprising one or more chromosomes, and this has allowed the accumulation of a range of knowledge about these microorganisms. In recent years, the complete sequencing of genomes has advanced the knowledge of the phylogeny of these microorganisms (Marques 2012). Previously, phylogenetic identification was based on the sequencing of some regions of their genome, such as the 16S rRNA region.\u003c/p\u003e\n\u003cp\u003eSequencing of the ITS (Internal Transcribed Spacer) region separates the 16S rDNA gene and can be amplified with specific primers anchored to this region. This region is highly conserved intraspecifically but variable between different species, which makes distinction at the specific level possible. However, molecular identification of certain bacterial groups by 16S rRNA gene sequencing can lead to inaccurate results. Today a 16S rRNA identity greater than 98.7% is accepted as a minimum threshold to differentiate species because, in most cases, this similarity corresponds to a DNA-DNA identity greater than 70% (Fungaro 2000; Zanirati 2012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe bacterial isolate BRM32113 was initially identified by Martins et al. (2020), as \u003cem\u003eBurkholderia pyrrocinia\u003c/em\u003e, analyzing the sequencing obtained from the amplification of the 16S rRNA gene, and the biochemical and morphological identification. But the analysis of the 16S rRNA gene extract from the complete genome sequencing of isolate BRM 32113 revealed that it is from the genus Serratia and species \u003cem\u003emarcescens\u003c/em\u003e. The sequencing of isolate BRM 32110 also revealed that it is from the genus Bacillus and for specie identification we will continous the whole genome studies (Fig. 1).\u003c/p\u003e\n\u003cp\u003eThe aspects analyzed in the CCD showed the most remarkable presence of brown coloration, indicating the presence of terpenoids, in the extracts obtained at 6 and 16 hours after bacterial growth (Wagner and Bladt 1996). \u0026nbsp;Terpenes are one of the largest groups of natural products and comprise numerous substances with important roles in physiological and pathological processes, they are hydrocarbons formally divided into isoprenic units and present recognized antimicrobial activity (Felipe and Bicas 2017) that act directly in the process of infection of the fungus in the plant, inhibiting the growth of hyphae and the germination of conidia (Achotegui-Castells 2016). Some \u003cem\u003eSerratia\u003c/em\u003e species produce volatile compounds such as terpenoids, which can influence the growth of other bacteria or fungi (Su et al. 2016). Also found in plants, they can be elicited by beneficial microorganisms and act as growth promoters, for example gibberellins, abscisic acid, carotenoids, and sterols, and also as toxins, inhibitors of phytopathogens and herbivores, such as limonoids, saponins, essential oils, among others (Hussein et al. 2019; Olanhewaju et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe sum of all chemical reactions within a living organism is known as metabolism, classified as primary and secondary. In primary metabolism macromolecules are produced as essential functions for microorganisms, while in secondary metabolism we have the production of specific micromolecules that have adaptive functions to biological activity (Tortora 2012) and that also act as competitive weapons used against other microorganisms, nutrient transport agents, and sex hormones.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe identified that the isolates BRM 21110 and BRM 32113 should grow for 6 and 16 hours in liquid nutrient broth medium for the production of molecules of primary and secondary metabolism, because the extracts obtained at these times were able to reduce the mycelial growth of \u003cem\u003eM. oryzae\u003c/em\u003e and \u003cem\u003eB. oryzae\u003c/em\u003e colonies up to 94 and 37% respectively (Fig. 2 A, Table 4). In addition we observed the change in the morphology of the fungal mycelium of both isolates (Fig. 2 B), which directly reflects in the pathogenicity of the fungus when in contact with the plant (Sangappillai and Nadarajah 2020) and also the reduction of up to 62% in the production of conidia of \u003cem\u003eB oryzae\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe process of penetration of these fungi into the plant cuticle is initiated when the conidia come in contact with the surface of rice leaves. It forms the germ tube that extends and differentiates into bulbous melanization, forming the appressorium, which is rich in melanin and contains cell wall degrading enzymes. Melanin is a secondary metabolite that protects conidia from UV damage and is also crucial in generating the turgor force generated by the apressorium to penetrate host tissue (Saha et al. 2020). We also evaluated morphological changes in conidia, germ tubes that did not form appressorium, non-melanized appressorium, and observed that all treatments differed from controls, which showed the formation of elongated hyaline conidia with thin apex and septate, as described in the literature (Prabhu and Filippi 2006).\u003c/p\u003e\n\u003cp\u003eIn the present study we observed that the LD 50 for \u003cem\u003eM. orzae\u003c/em\u003e, which was 100mg for the extract of BRM 32110 (6h), 50mg for BRM32110 (16h), 25 for BRM 32113 (6 and 16h), through conidium germination and appressorium formation, of the extracts obtained from both bacterial isolates. These presented a reduction of up to 90% of the appressorium formation, when compared to the control. Therefore, even though all concentrations significantly reduced the aspects analyzed, the statistical and morphological evaluations led us to choose them. These same extracts did not show signs of fiotoxicity to rice leaves (Fig. 6).\u003c/p\u003e\n\u003cp\u003eThe reduction in the mycelial growth of \u003cem\u003eB. oryzae\u003c/em\u003e and \u003cem\u003eM. oryzae\u0026nbsp;\u003c/em\u003ecolonies, in the germination of conidia and appressorium formation by the metabolites represents a solid perspective for the development of a commercial bioproduct for rice culture because the insertion of a bioproduct in the management of rice culture can contribute to the reduction of doses and number of applications of synthetic chemical fungicides and fertilizers, known to cause problems to the microbiological balance of the soil and the environment. Therefore, aiming at the commercial use of the metabolites, it was necessary to define the LD 50, the ideal concentration and phytotoxicity of the extracts. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe extracts obtained from the isolates BRM 32110 and BRM 32113 showed the same bioactivity of the bacterial cells of these isolates, as described by Filippi et al. 2011; Sousa et al. 2017; Sperandio et al. 2017; Arriel-Elias et al. 2017 and Arriel-Elias et al. 2019, who were successful in controlling diseases in rice culture when using the cells of these microorganisms after 48 hours of growth in culture medium. With 48 hours of culture we will have in the culture medium new cells and old cells, using extracts obtained after 6 and 16 hours of culture we will have a great advantage, which is uniformity by standardizing the growth time, extracting desirable characteristics of these PGPRs.\u003c/p\u003e\n\u003cp\u003eIn the greenhouse trials, to evaluate the suppression of diseases, we found that all treatments containing the extracts and bacterial suspension showed statistical differences from the controls. The solvent used did not harm the suppressive effect of bacteria, as previously proven in the antagonism test. When used alone, it did not show significant differences from the control.\u003c/p\u003e\n\u003cp\u003eIn A1 the best values for leaf brown spot suppression (LGS) were the treatments: 3 (BRM32110), presented 7.8% of affected leaf area, 4 (BRM32113) presented 17.1%, 5 (BRM32110 extract 6h) presented 9.8%, 6 (BRM32110 extract 16h) presented 6.7%, 7 (BRM32113 extract 6h) presented 11.4% and 8 (BRM32113 extract 16h) presented 6.8%. The controls treatments 1 (H2O), and 2 (DMSO) presented 21 and 23.5% of the leaf area affected by LGS, respectively (Fig. 6, 1. Treatments (3, 4, 5, 6, 7 and 8) presented small lesions and in a shorter amount, keeping most of the leaf area green (Fig. 6, 1 - C, D, E, F, G and H). In treatment 1 (H\u003csub\u003e2\u003c/sub\u003eO) and 2 (DMSO), we observed typical lesions of the disease, considered sporadic lesions, with a grayish center and brown edges that coalesce with time (Fig 7- 1 and 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor leaf blast (LB) suppression, in A2, all treatments containing the extracts and bacterial suspension showed statistical differences from the controls and the best values for leaf blast suppression was: 3 (BRM32110), presented 10.4% of affected leaf area, 4 (BRM32113) presented 11.6%, 5 (BRM32110 extract 6h) presented 8.8%, 6 (BRM32110 extract 16h) presented 14.4%, 7 (BRM32113 extract 6h) presented 10% and 8 (BRM32113 extract 16h) presented 7.6%. The controls treatments 1 (H2O) and 2 (DMSO) presented 32 and 37% of the leaf area affected by LB respectively (Fig. 7 -2 ). Treatments (3, 4, 5, 6, 7 and 8) presented typical small brown open lesions, pinhead type and in a shorter amount; however, it was in a smaller size and number than the control treatment, and it did not coalesce with time, keeping most of the leaf area green (Fig. 6, 2 - C, D, E, F, G and H). In treatment 1 (H2O) and 2 (DMSO), we observed typical lesions of the disease, considered sporadic lesions that coalesce with time (Fig 7- 1 and 2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilar effects were observed using cell-free extracts of \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e, in inhibiting the mycelial growth of Fusarium up to 50.33%. Similarly, when using \u003cem\u003eB. pumilus\u003c/em\u003e extracts obtained inhibitory activity for three soil pathogens \u003cem\u003eRhizoctonia solani, Phytium\u003c/em\u003e and \u003cem\u003eSclerotium rolfsii\u003c/em\u003e and furthermore identified one of the molecules responsible for the suppression of pathogens, pumilacidin (Mendes et al. 2018; Melo 2005), corroborating our results.\u003c/p\u003e\n\u003cp\u003eSome authors have achieved similar results to ours when using extracts of \u003cem\u003eSerratia\u0026nbsp;\u003c/em\u003esp. strains, as for example, Roberts et al. (2015) obtained the control of Phytium ultimum using extracts of Serratia marcescens, in the treatment of pumpkin seeds and Lenvenfors et al. (2004) when using\u0026nbsp;extracts of \u003cem\u003eSerratia plymuthica\u003c/em\u003e A 153 observed the inhibition of germination of conidia of species of \u003cem\u003eAspergillus\u003c/em\u003e and identified some molecules present in the extracts: pyrrolnitrin, and haterumalide NA, B and NE. These results show the efficiency of bacterial extracts on soil pathogens, and with our work, we achieved efficacy in suppressing leaf diseases in greenhouse.\u003c/p\u003e\n\u003cp\u003eThe most widely used control methods for brown spot and leaf blast, is the integration of genetic resistance and chemical control. But due to the high variability of the pathogen \u003cem\u003eM. oryzae\u003c/em\u003e, the duration time of genetic resistance in improved rice cultivars is limited, thus increasing the number of fungicide applications which has caused high risk of resistance emergence in phytopathogen populations (Prabhu and Filippi 2006). Oliveira et al. (2015) found \u003cem\u003eP. oryzae\u003c/em\u003e isolates with the presence of the G143A mutation in the cyt b gene that were 42 time\u0026nbsp;more\u0026nbsp;resistant to pyraclostrobin, due to the high selection pressure exerted by consecutive years of strobilurin applications. D\u0026apos;\u0026Aacute;vila et al. (2021) examined the reaction of fungal field isolates, collected over a 26-year period, to the active ingredients of commercially relevant fungicides, and documented the gradual increase in the frequency of fungicide-resistant isolates in rice populations of\u003cem\u003e\u0026nbsp;P. oryzae\u003c/em\u003e on a long-term basis. The insertion of alternative measures in integrated disease management in rice can decrease the dependence on chemical control in the production system and safeguard the productive potential of genetically improved cultivars.\u003c/p\u003e\n\u003cp\u003eAlthough biological control represents a strategy to increase the durability of resistance of rice cultivars to diseases and reduce toxic residues resulting from the indiscriminate use of chemicals (Filippi et al. 2011), there are still no biological products registered for rice cultivation (Agrofit 2021).\u003c/p\u003e\n\u003cp\u003eTo date, no other studies have been conducted using molecules of microbial metabolism of \u003cem\u003eBacillus sp.\u003c/em\u003e and \u003cem\u003eSerratia marcescens\u003c/em\u003e in the control of brown spot and leaf blast in rice crop. The results obtained showed that the use of \u003cem\u003eBacillus sp.\u003c/em\u003e and \u003cem\u003eS. marcescens\u003c/em\u003e bacteria extracts were efficient and have great potential for suppression of brown spot and leaf brusone in rice. Therefore, with this work we have the perspective of inserting a natural product in the agricultural market, aiming to favor producers with one more control tactic that can help the agroecological management to take off in Brazil. With this, more studies will be conducted in order to identify which are the molecules produced by these bacterial isolates and their production in large scale.\u003c/p\u003e\n\u003cp\u003eWe are at the forefront of developing bioproducts or biobased products for rice cultivation, and to help protect natural ecosystems while improving profitability and productivity in rice fields it is necessary to look for more components to integrated management.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe use of \u003cem\u003eBacillus sp.\u003c/em\u003eand \u003cem\u003eSerratia marcescens\u003c/em\u003e extracts was efficient in reducing the severity of brown spot and leaf blast in rice culture. These results are significant because they demonstrate that it is possible to use bacterial extracts in the control of phytopathogens seeking the insertion of natural products in agriculture and is the first report in rice culture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for scholarship and Embrapa for the research financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003eThe original idea was suggested by VGPS and MCCF and the concept of this study was developed by MTAE. The experimental work was carried out by MTAE with guidance from GCTFA, GAB and PHDS. MTAE drafted the article. GCTFA and GAB were involved in the critical revision of the article for important intellectual content. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eCNPq (National Council for Scientific and Technological Development (CNPq)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors not competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u0026nbsp;\u003c/strong\u003eThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article\u0026apos;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026apos;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAgricultural Biologicals Market Size, Share \u0026amp; Trends Analysis Report By Crop Type (Cereals \u0026amp; Grains, Oilseeds \u0026amp; Pulses), By Product (Biopesticides, Biostimulants), By Application Method, And Segment Forecasts, 2020 \u0026ndash; 2027 (\u003ca href=\"https://www.grandviewresearch.com/industry-analysis/agricultural-biologicals-market\"\u003eGlobal Agricultural Biologicals Market | Industry Report, 2020-2027 (grandviewresearch.com)\u003c/a\u003e). https://www.grandviewresearch.com/industry-analysis/agricultural-biologicals-marke Accessed 20 April 2021\u003c/p\u003e\n\u003cp\u003eAGROFIT (2021) MAPA-Minist\u0026eacute;rio da Agricultura Pecu\u0026aacute;ria e Abastecimento. http://agrofit.agricultura.gov.br/agrofit_cons/principal_agrofit_cons. Accessed 20 april 2021\u003c/p\u003e\n\u003cp\u003eAhmad P, Latef AAHA, Rasool S, Akram NA, Ashraf M, GUCEL S (2016) Role of Proteomics in Crop Stress Tolerance. Frontiers in Plant Science 7:1336.\u0026nbsp;\u003ca href=\"https://doi.org/10.3389/fpls.2016.01336\"\u003ehttps://doi.org/10.3389/fpls.2016.01336\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eArriel-Elias MT, Oliveira MIS, Silva-Lobo VL, Filippi MCC, Babana AH, Concei\u0026ccedil;\u0026atilde;o EC, Cortes MVCB (2018) Shelf life enhancement of PGPRs using a simple formulation screening method. African Journal of Microbiology Research 12:115-126.\u0026nbsp;\u003ca href=\"https://doi.org/\"\u003ehttps://doi.org/\u003c/a\u003e\u003ca href=\"http://dx.doi.org/10.5897/AJMR2017.8787\" target=\"_blank\"\u003e10.5897/AJMR2017.8787\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eArriel-Elias MT, C\u0026ocirc;rtes, MVCB, de Sousa TP, Chaibub AA, Filippi MCC (2019) Induction of resistance in rice plants using bioproducts produced from \u003cem\u003eBurkholderia pyrrocinia\u0026nbsp;\u003c/em\u003eBRM 32113. Environ Sci Pullut Res 26:19705-19718\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s11356-019-05238-3\"\u003ehttps://doi.org/10.1007/s11356-019-05238-3\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eBach E, Cannavan FS, Duarte FRS, Taffarel JAS, Tsai SM, Brandelli A (2011) Characterization of feather degrading bacteria from Brazilian soils. Int Biodeterior Biodegrad 65:102-107.\u0026nbsp;\u003ca href=\"https://doi.org/\"\u003ehttps://doi.org/\u003c/a\u003e\u003ca href=\"http://dx.doi.org/10.1016/j.ibiod.2010.07.005\" target=\"_blank\"\u003e10.1016/j.ibiod.2010.07.005\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eBettiol W, Maffia LA, Castro MLMP (2014) Control biol\u0026oacute;gico de enfermedades de plantas en Brasil. In: Bettiol W, Rivera MC, Mondino P, Montealegre JR, Colmen\u0026aacute;rez YC (eds) Control biol\u0026oacute;gico de enfermedades de plantas em Am\u0026eacute;rica Latina y el Caribe. Embrapa Meio Ambiente-Livro Cient\u0026iacute;fico (ALICE), pp 91-137 (in spanish)\u003c/p\u003e\n\u003cp\u003eCONAB-Companhia Nacional de Abastecimento (2020). S\u0026eacute;ries hist\u0026oacute;ricas de safra. \u0026lt;https://www.conab.gov.br/info-agro/safras/serie-historica-das-safras\u0026gt;. Accessed 21 January 2021\u003c/p\u003e\n\u003cp\u003eCONAB-Companhia Nacional de Abastecimento (2021) Evolu\u0026ccedil;\u0026atilde;o dos custos de produ\u0026ccedil;\u0026atilde;o de arroz no Brasil. Comp\u0026ecirc;ndio de estudos, Conab 4:1-20 (in portuguese)\u003c/p\u003e\n\u003cp\u003eC\u0026ocirc;rtes, ACAS, Sousa TP, Barros MV, Rodrigues FA, Silva GB, Filippi MCC (2015) Enzyme-Induced Defense Response in the Suppression of Rice Leaf Blast (\u003cem\u003eMagnaporthe Oryzae\u003c/em\u003e) by Silicon Fertilization and Bioagents.\u0026nbsp;International J of Res Studies in Biosciences 3: 22-32. ISSN 2349-0357 (Print) and ISSN 23490365 (Online)\u003c/p\u003e\n\u003cp\u003eDEPEC-Departamento de Pesquisas e Estudos Econ\u0026ocirc;micos (2019) Arroz e produtos 2017-Bradesco. Dispon\u0026iacute;vel em: www.economiaemdia.com.br. Accessed 22 de January 2021.\u003c/p\u003e\n\u003cp\u003eEMPRESA BRASILEIRA DE PESQUISA AGROPECU\u0026Aacute;RIA - EMBRAPA. Consumo Per capita de Arroz (\u003cem\u003eOryza sativa\u003c/em\u003e L.) e de Feij\u0026atilde;o-comum (\u003cem\u003ePhaseolus vulgaris\u003c/em\u003e L.), no Brasil, de 1985 a 2019 (2020) https://www.cnpaf.embrapa.br/socioeconomia/docs/arroz/consumopercapitaarrozefeijao.ht m. Accessed 02 February 2021 (in portuguese)\u003c/p\u003e\n\u003cp\u003eFelipe LO, Bicas JL (2017) Terpenos, aromas e a qu\u0026iacute;mica dos compostos naturais. Qu\u0026iacute;mica Nova na Escala 39:120-130.\u0026nbsp;\u003ca href=\"https://doi.org/10.21577/0104-8899.20160068\"\u003ehttps://doi.org/10.21577/0104-8899.20160068\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFilippi MCC, Silva GB, Silva-Lobo VL, Cortes MMCB, Moraes AJG, Prabhu AS (2011) Leaf blast (\u003cem\u003eMagnaporthe oryzae\u003c/em\u003e) suppression and growth promotion by rhizobacteria on aerobic rice in Brazil. Biological Control 58:160-166.\u0026nbsp;\u003ca href=\"https://doi.org/10.1016/j.biocontrol.2011.04.016\"\u003ehttps://doi.org/10.1016/j.biocontrol.2011.04.016\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eFungaro MHP (2000) PCR na micologia. Bioenol: Ci\u0026ecirc;nc \u0026amp; Densenv, 3:12-16 (in portuguese)\u003c/p\u003e\n\u003cp\u003eGuimar\u0026atilde;es RA (2014) Caracteriza\u0026ccedil;\u0026atilde;o de Isolados de \u003cem\u003eSarocladium oryzae\u003c/em\u003e e seu Potencial na Supress\u0026atilde;o da Brusone em Arroz. Dissertation, Universidade Federal de Goi\u0026aacute;s (in portuguese)\u003c/p\u003e\n\u003cp\u003eLenz, G, Balardin RS, Corte GD, Marques LN, Debona (2010)\u0026nbsp;Escala diagram\u0026aacute;tica para avalia\u0026ccedil;\u0026atilde;o de severidade de mancha-parda em arroz. Ci\u0026ecirc;ncia Rural 40:752-758.\u0026nbsp;\u003ca href=\"https://doi.org/10.1590/S0103-84782010005000061\"\u003ehttps://doi.org/10.1590/S0103-84782010005000061\u003c/a\u003e (in portuguese)\u003c/p\u003e\n\u003cp\u003eLevenfors JJ, Hedmana R, Thaningb C, Gerhardsonb B, Welch CJ (2004) Broad-spectrum antifungal metabolites produced by the soil bacterium \u003cem\u003eSerratia plymuthica\u003c/em\u003e A 153. Soil Biology \u0026amp; Biochemistry 36:677\u0026ndash;685.\u0026nbsp;\u003ca href=\"https://doi.org/10.1016/j.soilbio.2003.12.008\"\u003ehttps://doi.org/10.1016/j.soilbio.2003.12.008\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eSilva MA, Nascente AS, Filippi MCC, Lanna AC, Silva GB, Fernandes JPT, Arriel-Elias MT (2020) Screening of Beneficial Microorganisms to Improve Soybean Growth and Yield. Brazilian Archives of Biology and Technology 63:e20190463.\u0026nbsp;\u003ca href=\"https://doi.org/10.1590/1678-4324-2020190463\"\u003ehttps://doi.org/10.1590/1678-4324-2020190463\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eMarques MV (2012) Biologia Molecular e Gen\u0026eacute;tica Bacteriana (ed). SBG, Vi\u0026ccedil;osa (in portuguese)\u003c/p\u003e\n\u003cp\u003eMartins, BEM, Chaibub AA, Cort\u0026ecirc;s MVCB, Silva-Lobo VL, Filippi MCC (2020) Characterization of bacterial isolates for sustainable rice blast control. Rev. Caatinga, Mossor\u0026oacute; 33:702-712.\u0026nbsp;\u003ca href=\"http://doi.org/10.1590/1983-21252020v33n313rc\"\u003ehttp://doi.org/10.1590/1983-21252020v33n313rc\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eMartins BEM (2015) Caracteriza\u0026ccedil;\u0026atilde;o morfol\u0026oacute;gica, bioqu\u0026iacute;mica e molecular de isolados bacterianos antagonistas a \u003cem\u003eMagnaporthe Oryzae\u003c/em\u003e. Dissertation, Universidade Federal de Goi\u0026aacute;s (in portuguese)\u003c/p\u003e\n\u003cp\u003eMelo FMP (2005) Atividade antif\u0026uacute;ngica de metab\u0026oacute;litos secund\u0026aacute;rios produzidos pelo end\u0026oacute;fito de mandioca \u003cem\u003eBacillus pumilus\u003c/em\u003e MAIII4a. Dissertation, Universidade de S\u0026atilde;o Paulo (in portuguese)\u003c/p\u003e\n\u003cp\u003eMendes HTA, Coutrim RL, Nolasco DSJ, Anjos DN, Barros BL, S\u0026atilde;o Jos\u0026eacute; AR (2018). Metab\u0026oacute;litos vol\u0026aacute;teis produzidos por \u003cem\u003eBacillus subtilis\u003c/em\u003e \u003cem\u003ee Bacillus amyloliquefaciens\u003c/em\u003e sobre o crescimento micelial de \u003cem\u003eFusarium oxysporum\u003c/em\u003e f. sp. \u003cem\u003ecubense\u003c/em\u003e. Cadernos de Agroecologia-Anais do VI CLAA, X CBA e V SEMDF, v. 13 ISSN 2236-7934 (in portuguese)\u003c/p\u003e\n\u003cp\u003e\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Hussain+M\u0026cauthor_id=31151268\"\u003eHussain\u003c/a\u003e M,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Debnath+B\u0026cauthor_id=31151268\"\u003eDebnath\u003c/a\u003e B,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Qasim+M\u0026cauthor_id=31151268\"\u003eQasim\u003c/a\u003e M,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Bamisile+BS\u0026cauthor_id=31151268\"\u003eBamisile\u003c/a\u003e BS,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Islam+W\u0026cauthor_id=31151268\"\u003eIslam\u003c/a\u003e W,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Hameed+MS\u0026cauthor_id=31151268\"\u003eHameed\u003c/a\u003e MS,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Wang+L\u0026cauthor_id=31151268\"\u003eWang\u003c/a\u003e L,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Qiu+D\u0026cauthor_id=31151268\"\u003eQiu\u003c/a\u003e D (2019) Role of Saponins in Plant Defense Against Specialist Herbivores. Molecules 24:2067. https:?/doi.org/10.3390/molecules24112067\u003c/p\u003e\n\u003cp\u003eNascente AS, Filippi MCC, Lanna AC, Souza AC, Silva-Lobo VL, Silva GB (2017) Biomass, gas exchange, and nutrient contents in upland rice plants affected by application forms of microorganism growth promoters. Environmental Science and Pollution Research, 24:2956-2965.\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s11356-016-8013-2\"\u003ehttps://doi.org/10.1007/s11356-016-8013-2\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNascente AS, Filippi MCC, Sousa TP, Chaibub AA, Souza ACA, Lanna AC (2019) Upland rice gas exchange, nutrient uptake and grain yield as affected by potassium fertilization and inoculation of the diazotrophic bacteria \u003cem\u003eSerratia\u003c/em\u003e spp. Southern Cross Publishing Group, Australia, Australian Journal of Crop Science 13:944-953.\u0026nbsp;\u003ca href=\"https://doi.org/10.21475/ajcs.19.13.06.p1689\"\u003ehttps://doi.org/10.21475/ajcs.19.13.06.p1689\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNotteghem JL (1981) Cooperative experiment on horizontal resistance to rice blast. In: Blast and upland rice: report and recommendations from the meeting for international collaboration in upland rice improvement. Los Ba\u0026ntilde;os, IRRI, pp.43-51\u003c/p\u003e\n\u003cp\u003eOlanrewaju OS, Glick BR, Babalola OO (2017) Mechanisms of action of plant growth promoting bacteria. World J Microbiol Biotechnol 33:197.\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s11274-017-2364-9\"\u003ehttps://doi.org/10.1007/s11274-017-2364-9\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOliveira SC, Castroagudin VL, Maciel JLN, Pereira DAS, Ceresini PC (2015) Resist\u0026ecirc;ncia cruzada aos fungicidas azoxistrobina e piraclostrobina no pat\u0026oacute;geno da brusone do trigo \u003cem\u003ePyricularia oryzae\u003c/em\u003e no Brasil. Summa Phytopathologica 41:298-304.\u0026nbsp;\u003ca href=\"https://doi.org/10.1590/0100-5405/2072\"\u003ehttps://doi.org/10.1590/0100-5405/2072\u003c/a\u003e (in portuguese)\u003c/p\u003e\n\u003cp\u003eSaha P, Ghosh S, Roy-Barmana S (2020) MoLAEA Regulates Secondary Metabolism in \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e. M Sphere 5:e00936-19.\u0026nbsp;\u003ca href=\"https://doi.org/10.1128/mSphere.00936-19\"\u003ehttps://doi.org/10.1128/mSphere.00936-19\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrabhu AS (2006) Brusone em arroz: controle gen\u0026eacute;tico, progresso e perspectivas. Santo Ant\u0026ocirc;nio de Goi\u0026aacute;s, Embrapa Arroz e Feij\u0026atilde;o (in portuguese)\u003c/p\u003e\n\u003cp\u003ePrabhu AS, Filippi MCC, Silva GB, Silva-Lobo VL, Morais OP (2009) An unprecedented outbreak of rice blast on a newly released cultivar BRS Colosso in Brazil. In: Wang GL, Valent B (ed) Advances in genetics, genomics, and control of rice blast. Springer Science, Netherlands, pp. 257\u0026ndash;267\u003c/p\u003e\n\u003cp\u003e\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Dean+RA\u0026cauthor_id=15846337\"\u003eDean\u003c/a\u003e RA,\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Talbot+NJ\u0026cauthor_id=15846337\"\u003e\u0026nbsp;Talbot\u003c/a\u003e NJ,\u0026nbsp;\u003ca href=\"https://pubmed.ncbi.nlm.nih.gov/?term=Ebbole+DJ\u0026cauthor_id=15846337\"\u003eEbbole\u003c/a\u003e DJ et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature Publishing Group 434:980-986.\u0026nbsp;\u003ca href=\"https://doi.org/10.1038/nature03449\"\u003ehttps://doi.org/10.1038/nature03449\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSansinenea E, Ortiz A (2011) Secondary metabolites of soil \u003cem\u003eBacillus\u003c/em\u003e spp. Biotechnol Lett 33:1523-1538.\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s10529-011-0617-5\"\u003ehttps://doi.org/10.1007/s10529-011-0617-5\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eSperandio EM, Vale HMM, Reis MS, Cortes MVCBC, Lanna AC, Filippi MCC (2017) Evaluation of rhizobacteria in upland rice in Brazil: growth promotion and interaction of induced defense responses against leaf blast (\u003cem\u003eMagnaporthe oryzae\u003c/em\u003e). Acta Physiologiae Plantarum 39:258-270.\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s11738-017-2547-x\"\u003ehttps://doi.org/10.1007/s11738-017-2547-x\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRezende CC, Frasca LLM, Silva MA, Pires RAC, Lanna AC, Filippi MCC, Nascente AS (2021) Physiological and agronomic characteristics of the common bean as affected by multifunctional microorganisms. Semina: Ci\u0026ecirc;ncias Agr\u0026aacute;rias 42:599-618.\u0026nbsp;\u003ca href=\"https://doi.org/10.5433/1679-0359.2021v42n2p599\"\u003ehttps://doi.org/10.5433/1679-0359.2021v42n2p599\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRoberts DP, Lakshman DK, McKenna LF, Emche SE, Maul JE, Bauchan G (2016) Seed treatment with ethanol extract of Serratia marcescens is compatible with Trichoderma isolates for control of damping-off of cucumber caused by \u003cem\u003ePythium ultimum\u003c/em\u003e. Plant Dis. 100:1278-1287.\u0026nbsp;\u003ca href=\"https://doi.org/10.1094/PDIS-09-15-1039-RE\"\u003ehttps://doi.org/10.1094/PDIS-09-15-1039-RE\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSakthivel N, Amudha R, Muthukrishnan S (2002). Production of phytotoxic metabolites by \u003cem\u003eSarocladium oryzae.\u003c/em\u003e Mycol. Res. 106:609-614.\u0026nbsp;\u003ca href=\"https://doi.org/10.1017/S0953756202005774\"\u003ehttps://doi.org/10.1017/S0953756202005774\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Achotegui-Castells%20A%5BAuthor%5D\u0026cauthor=true\u0026cauthor_uid=26796122\"\u003eAchotegui-Castells\u003c/a\u003e A,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Della%20Rocca%20G%5BAuthor%5D\u0026cauthor=true\u0026cauthor_uid=26796122\"\u003eGianni DR\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Llusi%26%23x000e0%3B%20J%5BAuthor%5D\u0026cauthor=true\u0026cauthor_uid=26796122\"\u003e\u0026nbsp;\u0026nbsp;Llusi\u003c/a\u003ea J,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Danti%20R%5BAuthor%5D\u0026cauthor=true\u0026cauthor_uid=26796122\"\u003eDanti\u003c/a\u003e R,\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Barberini%20S%5BAuthor%5D\u0026cauthor=true\u0026cauthor_uid=26796122\"\u003e\u0026nbsp;Barberini\u003c/a\u003e S,\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Bouneb%20M%5BAuthor%5D\u0026cauthor=true\u0026cauthor_uid=26796122\"\u003e\u0026nbsp;Bouneb\u003c/a\u003e M,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Simoni%20S%5BAuthor%5D\u0026cauthor=true\u0026cauthor_uid=26796122\"\u003eSimoni\u003c/a\u003e S,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Michelozzi%20M%5BAuthor%5D\u0026cauthor=true\u0026cauthor_uid=26796122\"\u003eMichelozzi\u003c/a\u003e M,\u0026nbsp;\u003ca href=\"https://www.ncbi.nlm.nih.gov/pubmed/?term=Pe%26%23x000f1%3Buelas%20J%5BAuthor%5D\u0026cauthor=true\u0026cauthor_uid=26796122\"\u003ePe\u0026ntilde;uelas\u003c/a\u003e J (2016) Terpene arms race in the \u003cem\u003eSeiridium cardinale\u003c/em\u003e \u0026ndash; \u003cem\u003eCupressus sempervirens\u003c/em\u003e pathosystem. Scientific Reports 6:18954. https://doi.org/\u003ca href=\"https://dx.doi.org/10.1038%2Fsrep18954\" target=\"_blank\"\u003e10.1038/srep18954\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e\u003ca href=\"https://europepmc.org/search?query=AUTH:%22Leilane%20S%20D%27%C3%81vila%22\"\u003eD\u0026apos;\u0026Aacute;vila LS\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://europepmc.org/authors/0000-0003-1676-8164\"\u003eDe Filippi MCC\u003c/a\u003e,\u0026nbsp;\u003ca href=\"https://europepmc.org/authors/0000-0002-5204-2961\"\u003eCaf\u0026eacute;-Filho AC\u003c/a\u003e (2021) Sensitivity of \u003cem\u003ePyricularia\u0026nbsp;oryzae\u003c/em\u003e Populations to Fungicides Over a 26-Year Time Frame in Brazil. Plant Disease PDIS-08. https://doi.org/\u003ca href=\"https://doi.org/10.1094/pdis-08-20-1806-re\"\u003e10.1094/pdis-08-20-1806-re\u003c/a\u003e\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSharma A, Kumar V, Shahzad B et al. (2019) Worldwide pesticide usage and its impacts on ecosystem. SN Applied Sciences 1:1446.\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s42452-019-1485-1\"\u003ehttps://doi.org/10.1007/s42452-019-1485-1\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSilva-lobo VL, Filippi MCC, Silva GB, Venancio WL, Prabhu AS (2012) Rela\u0026ccedil;\u0026atilde;o entre o teor de\u0026nbsp;clorofila nas folhas e a severidade de brusone nas pan\u0026iacute;culas em arroz de terras altas. Tropical Plant Pathology 37:83-87.\u0026nbsp;\u003ca href=\"https://doi.org/10.1590/S1982-56762012000100011\"\u003ehttps://doi.org/10.1590/S1982-56762012000100011\u003c/a\u003e (in portuguese)\u003c/p\u003e\n\u003cp\u003eSOSBAI-Sociedade Sul-Brasileira de Arroz Irrigado. Arroz irrigado: recomenda\u0026ccedil;\u0026otilde;es t\u0026eacute;cnicas da pesquisa para o Sul do Brasil. Itaja\u0026iacute;, SC: SOSBAI, 2012 (in portuguese)\u003c/p\u003e\n\u003cp\u003eSousa TP, Souza ACA, Cortes MVCB, Lanna AC, Pinheiro HA, Filippi MCC, Silva GB (2017) Bioagents and silicon promoting fast early upland rice growth. Environmental Sciences and Pollution Research 25:3657-3668.\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s11356-017-0753-0\"\u003ehttps://doi.org/10.1007/s11356-017-0753-0\u003c/a\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSouza ACV (2012) Pesquisa com arroz irrigado (\u003cem\u003eOryza sativa\u003c/em\u003e L.) no sistema pr\u0026eacute;-germinado: melhoramento gen\u0026eacute;tico, toxidez por ferro e brusone. Complete of course work, Universidade Federal de Santa Catarina (in portuguese)\u003c/p\u003e\n\u003cp\u003eSunder S, Singh R, Agarwal (2014). Brown spot of rice: an overview. Indian Phytopath., 67: 201-215. : https://www.researchgate.net/publication/296025966\u003c/p\u003e\n\u003cp\u003eThomson NR, Crow MA, McGowan SJ, Cox A, Salmond GPC (2000) Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control. Molecular Microbiology 36:539-556.\u0026nbsp;\u003ca href=\"https://doi.org/10.1046/j.1365-2958.2000.01872.x\"\u003ehttps://doi.org/10.1046/j.1365-2958.2000.01872.x\u003c/a\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTortora GJ, Funke BR, Case CL et al. (2017) Microbiologia, 12\u003csup\u003ea\u003c/sup\u003e ed. Artmed, Porto Alegre (in portuguese)\u003c/p\u003e\n\u003cp\u003eWagner H, Bladt S (1996) Plant drug analysis-A thin layer cromatography atlas. 2\u003csup\u003ea\u003c/sup\u003e ed. Springer, Berlim\u003c/p\u003e\n\u003cp\u003eTremacoldi CR, Filho APSS (2006) Toxinas produzidas por fungos Fitopat\u0026oacute;genos: Possibilidades de Uso no Controle de Plantas Daninhas. Embrapa Amaz\u0026ocirc;nia Oriental, Bel\u0026eacute;m (in portuguese)\u003c/p\u003e\n\u003cp\u003eSangappillai V, Nadarajah K (2020) Fatty Acid Synthase Beta Dehydratase in the Lipid Biosynthesis Pathway Is Required for Conidiogenesis, Pigmentation and Appressorium Formation in \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e S6. Int. J. Mol. Sci. 21:7224.\u0026nbsp;\u003ca href=\"https://doi.org/doi:10.3390/ijms21197224\"\u003ehttps://doi.org/doi:10.3390/ijms21197224\u003c/a\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZanirati DF (2012) Caracteriza\u0026ccedil;\u0026atilde;o de bact\u0026eacute;rias l\u0026aacute;ticas da microbiota de gr\u0026atilde;os de kefir cultivados em leite ou \u0026aacute;gua com a\u0026ccedil;\u0026uacute;car mascavo por metodologias dependentes e independentes de cultivo. Dissertation, Universidade Federal de Minas Gerais\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e Collection details and GenBank accession numbers of isolates included in the present study.\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" width=\"100%\"\u003e\n \u003cp\u003e\u003cem\u003eSerratia\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"22.8099173553719%\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"43.80165289256198%\"\u003e\n \u003cp\u003eGenBank Accession number (16S rRNA )\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eSerratia plymuthica\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eDSM 4540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eNR 114579\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003eS16.1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eOK284921\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eSerratia ficaria\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eNBRC 102596\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eNR 114155\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003eDSM 4569\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eAJ233428\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eSerratia rubidaea\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eDSM 4480\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eAJ233436\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003eNBRC 103169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eNR 114232\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eSerratia marcescens\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eZO1-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eAJ296310\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003eWO1-A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eAJ296309\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBRM32113\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eIn process\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eSerratia nematodiphila\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003ePSI26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eOK423573\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003eB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eOK560052\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eSerratia ureilytica\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eNiVa 51T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eAJ854062\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003eNiVa 51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eNR 042356\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" width=\"100%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus cereus\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003ewg-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eEU624208\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003eNVH 391-98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eAM747234\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus anthracis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eATCC 14578\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eAB190217\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003eLB5YE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eMW819989\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus wiedmannii\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eFSL W8-0169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eNR 152692\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus mycoides\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eATCC 6462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eAB021192\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003e2861\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eMT586023\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus weihenstephanensis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eDSM 1182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eAB021199\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBRM32110\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eIn process\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus toyonensis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eBCT-7112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eNR121761\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus cereus\u0026nbsp;\u003c/em\u003ebiovar toyoi\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eNCIB 40112\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eAJ310100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus thuringiensis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eBt11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eMT292101\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"33.388429752066116%\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus pseudomycoides\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.8099173553719%\"\u003e\n \u003cp\u003eNRRL B-617\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"43.80165289256198%\"\u003e\n \u003cp\u003eAF013121\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"34.24317617866005%\"\u003e\n \u003cp\u003eB-617\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"65.75682382133995%\"\u003e\n \u003cp\u003eNR11442\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Isolates and newly generated sequences are in bold.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eTreatments description for Conidial germination and appressorium formation assays, combining growth period and extract concentration of BRM32110 and BRM32113\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003eIsolate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003eTreatments number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003eGrowth period (h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003eDMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003eBRM 32113 extration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e100mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e50mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e25mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e10mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e5mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e100mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e50mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e25mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e10mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e5mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003eBRM 32110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e100mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e50mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e25mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e10mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e5mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e100mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e50mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e25mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e10mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.79858657243816%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"4.41696113074205%\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.381625441696112%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.73498233215548%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"17.6678445229682%\"\u003e\n \u003cp\u003e5mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eLeaf gray spot and leaf blast suppression\u0026nbsp;treatments, extraction time and extract concentration of BRM32110 and BRM32113, assays E1 and E3\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003eIsolate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003eTreatments number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.02826855123675%\"\u003e\n \u003cp\u003eExtraction time (h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.434628975265017%\"\u003e\n \u003cp\u003eConcentration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"3.0035335689045937%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"25.26501766784452%\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.02826855123675%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.434628975265017%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"3.0035335689045937%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"25.26501766784452%\"\u003e\n \u003cp\u003eDMSO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.02826855123675%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.434628975265017%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003eBRM32110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"3.0035335689045937%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"25.26501766784452%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.02826855123675%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.434628975265017%\"\u003e\n \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003eBRM32113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"3.0035335689045937%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"25.26501766784452%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.02826855123675%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.434628975265017%\"\u003e\n \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003eBRM32110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"3.0035335689045937%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"25.26501766784452%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.02826855123675%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.434628975265017%\"\u003e\n \u003cp\u003e100mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003eBRM32110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"3.0035335689045937%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"25.26501766784452%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.02826855123675%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.434628975265017%\"\u003e\n \u003cp\u003e50mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003eBRM32113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"3.0035335689045937%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"25.26501766784452%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.02826855123675%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.434628975265017%\"\u003e\n \u003cp\u003e25mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"28.268551236749115%\"\u003e\n \u003cp\u003eBRM32113\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"3.0035335689045937%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"25.26501766784452%\"\u003e\n \u003cp\u003eBRM32113 Extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"24.02826855123675%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.434628975265017%\"\u003e\n \u003cp\u003e25mg\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\n\u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Reduction of mycelial growth of \u003cem\u003eB. oryzae\u0026nbsp;\u003c/em\u003eand \u003cem\u003eM. oryzae\u0026nbsp;\u003c/em\u003eby extracts of BRM32110 and BRM32113, after 7 days of growth in BDA. Means followed by the same letter do not differ statistically from each other by the Tukey\u0026rsquo;s test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.34215167548501%\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.929453262786595%\"\u003e\n \u003cp\u003eExtaction time (h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003eReduction of colony area (%) \u003cem\u003eB. oryzae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.98236331569665%\"\u003e\n \u003cp\u003eReduction of colony area (%) \u003cem\u003eM. oryzae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eControl (H\u003csub\u003e2\u003c/sub\u003eO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e0 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e0 h\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eControl (DMSO)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e0 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e0 h\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32110 - 6h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e17.2 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e94,3 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32110- 16h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e28.9 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e93,2 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32110 - 24h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e0 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e80,0 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32110 - 48h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e0 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e69,1 def\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32110 - 72h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e3.3 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e68,3 def\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32113 - 6h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e17.2 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e89,8 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32113 - 16h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e21.5 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e87,3 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32113 - 24h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e3.3 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e76,6 cd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32113- 48h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e1.4 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e74,4 cde\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" width=\"18.34215167548501%\"\u003e\n \u003cp\u003eBRM32113 - 72h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"19.929453262786595%\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.746031746031747%\"\u003e\n \u003cp\u003e0.7 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" width=\"29.98236331569665%\"\u003e\n \u003cp\u003e68,3 def\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Metabolites, Natural products, Sustainability, Chemistry, Alternative control, Rice","lastPublishedDoi":"10.21203/rs.3.rs-1087379/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1087379/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this work was to optimize the extraction process and application of bacterial extracts of \u003cem\u003eBacillus\u003c/em\u003e sp. and \u003cem\u003eSerratia\u003c/em\u003e sp. in leaf blast control (\u003cem\u003eMagnapothe oryzae\u003c/em\u003e) and brown spot (\u003cem\u003eBipolaris oryzae\u003c/em\u003e) in rice culture. The work was divided into three stages: 1) Bacterial obtaining extracts through liquid-liquid extraction 2) Antagonistic capacity of bacterial extracts to \u003cem\u003eM. oryaze\u003c/em\u003e and \u003cem\u003eB. oryae \u003c/em\u003e3) Suppression of brown spot (A1) and leaf blast (A2) in greenhouse. The bacterial isolates in present study were identified as \u003cem\u003eBacillus sp. \u003c/em\u003e(BRM32110) and \u003cem\u003eSerratia marcescens\u003c/em\u003e (BRM32113).\u003cem\u003e \u003c/em\u003eThe crude extract of both isolates at different extraction times 6, 16, 24, 48 and 72 hours reduced the growth of colonies of \u003cem\u003eM. oryzae\u003c/em\u003e and \u003cem\u003eB. oryzae\u003c/em\u003e by up to 92% and 28%, respectively. The extracts that showed highest inhibition of colony growth were those obtained after 6 and 16h of incubation and were selected for subsequent assays. These, for both isolates were able to reduce conidia germination by up to 91% and apressorium formation of \u003cem\u003eM. oryzae\u003c/em\u003e by up to 93%. In green house, A1 the treatment that stood out was the extract of \u003cem\u003eBacillus sp.\u003c/em\u003e (16h) with 6.7% of leaf area affected and in A2 the treatment \u003cem\u003eS. marcescens\u003c/em\u003e extract (16h) stood out with only 7.6% of leaf area affected with brusone when compared to control. The use of extracts of \u003cem\u003eBacillus sp.\u003c/em\u003e and \u003cem\u003eSerratia marcescens\u003c/em\u003e was efficient in reducing the severity of brown spot and leaf blast in rice crop.\u003c/p\u003e","manuscriptTitle":"Optimization of the Extraction Process and Application of Bacterial Extracts in the Control of Brown Spot and Leaf Blast in Rice Culture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-12-03 15:50:31","doi":"10.21203/rs.3.rs-1087379/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bee60a16-4066-4e5c-a9f6-6312df80ed60","owner":[],"postedDate":"December 3rd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":8918317,"name":"Environmental Engineering"}],"tags":[],"updatedAt":"2021-12-20T13:02:03+00:00","versionOfRecord":[],"versionCreatedAt":"2021-12-03 15:50:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1087379","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1087379","identity":"rs-1087379","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}