POLYPHASIC CHARACTERIZATION OF Bacillus STRAINS ISOLATED FROM MAIZE

The use of bioinoculants with plant growth-promoting bacteria (PGPB) is a sustainable agricultural practice that has been expanding worldwide. Thus, it is fundamental to characterize these bacteria molecularly to provide better security and traceability for bioinoculants production. This work aimed to identify, characterize and develop specific molecular markers for Bacillus strains related to plant-growing promotion. Five strains were identified as B. pumillus (B32), B. thuringiensis (B116), B. megaterium (B119) and B. subitilis (B2082 and B2084) using 16S rRNA gene sequencing and MALDI-TOF mass spectrometry. Repetitive element palindromic (Rep-PCR) and amplified ribosomal DNA restriction analysis (ARDRA) techniques allowed the discrimination of the strains, except B2082 and B2084 that presented identical genetic profiles, indicating that they are same or genetically close isolates. Moreover, specific molecular markers were developed for B116 and B119 strains and may be used for microbial inoculants production quality control, strains traceability and to detail PGPB colonization.

Maize crop is one of the main commodities of Brazilian agribusiness, given its different forms of use, ranging from animal feed and human food to the high-tech industry. To obtain high yield in maize crop, it is necessary to apply several farm inputs to the soil. Synthetic fertilizers comprise most of maize production cost and can cause several adverse environmental impacts, such as the eutrophication of water sources, soil degradation, micronutrient deficiency, toxicity to different beneficial organisms and reduction of microbiota biodiversity (Li et al., 2017). In this context, soil management practices and efficient genotypes, combined with bioinoculants, can be an efficient and sustainable alternative to mitigate these problems (Pereira et al., 2020;Santos et al., 2019). The use of bioinoculants, including the ones containing plant growth-promoting bacteria (PGPB), is a sustainable agricultural practice that has been expanding worldwide and shows positive results for several crops, including maize (Anzuay et al., 2017;Santos et al., 2019;Pereira et al., 2020). Maize can associate with several species of PGPB, including Bacillus (Velloso et al., 2019;Paiva et al., 2020). Bacillus is a heterogeneous group of gram-positive bacteria widely distributed in the environment, which comprises 293 species/ subspecies (Patel & Gupta, 2020). Some Bacillus species are capable to solubilize phosphate (Bahadir et al., 2018), produce indole-acetic acid (IAA) and other phytohormones (Mohite, 2013), siderophores (Bjelić et al., 2018), and control plant pathogens and pest insects (Shafi et al., 2017). Recently, the first commercial bioinoculant was released, named BiomaPhos ® , carrying a consortium of two P-solubilizing/ mineralizing Bacillus strains (B. subtilis and B. megaterium), which was developed by our group (Paiva et al., 2020). Bioinoculants containing Bacillus strains are stable in the environment because of their ability to form spores, allowing them to adapt to extreme abiotic conditions, such as temperatures, pH or exposure to pesticides (Bahadir et al., 2018). Despite isolating and characterizing strains regarding plant growingpromotion potential, it is necessary to have a fast and efficient technique to identify the bacterial strains in order to provide greater security and traceability for biological products (Van Elsas & Boersma, 2011;Reddypriya et al., 2018).
The most commonly employed target for molecular identification of bacteria is the 16S ribosomal RNA gene (16S rDNA) sequencing, followed by comparison with genomic databases (Banik et al., 2015;Fuks et al., 2018;Johnson et al., 2019). Mass spectrometry by matrix-assisted laser desorption/ionization with a time-of-flight analyzer (MALDI-TOF) is another technique that has been used to discriminate close related species, including members of the Bacillus genus PCR-based techniques using repetitive element palindromic PCR (Rep-PCR) (Versalovic et al., 1991) have been used for identification and taxonomy of prokaryotic microorganisms, 3 including Bacillus genus (Kumar et al., 2014).
The DNA sequences are amplified from highly conserved regions that present multiple copies in the genome of bacteria and the polymorphic DNA fragments are compared among the samples.
Several repetitive sequences, such as repetitive extragenetic palindromes (REP), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and repetitive DNA elements (BOX) were identified and used to study the genetic diversity of Bacillus in different environments, allowing phylogenetic studies and discrimination of genetically close species (Rai et al., 2015;Wang et al., 2020).
Thus, the objective of this work was to identify, characterize and develop specific molecular markers for genetic discrimination of Bacillus strains previously selected as P-efficient solubilizing and candidates to be part of maize bioinoculants.  (Oliveira et al., 2009;Gomes et al., 2014;Abreu et al., 2017), as well as their plant growthpromoting capacity (de Sousa et al., 2018;Ribeiro et al., 2018;.

The colonies morphological characteristics and
Gram coloration were evaluated in a microscopy (Axioplan, Carl Zeiss, Germany) with digital zoom (Paiva et al., 2013). B116 strain was also grown in solid Luria-Bertani (LB) medium and incubated at 29 ºC for 72 hours to confirm its identity. The cells were suspended with 10 mL autoclaved deionized water and used for determination of protein crystal inclusions by phase contrast microscopy.

Amplification and sequencing of 16S rRNA gene
The 16S rRNA gene was amplified with 8F and 1492R primers (Turner et al., 1999). Nucleotide sequences were compared against the Genbank database using the BLASTN program (Altschul et al., 1997).

(ARDRA)
The 16S rRNA gene sequences from the five bacterial strains were analyzed in silico for restriction enzyme sites using NEBcutter V2.0 software (http://tools.neb.com/NEBcutter2/) and then the 16S rRNA amplified products were digested with Taq I enzyme (Thermo Fisher, USA) at 65 °C for 2 h and verified by electrophoresis on 1.0% (w/v) agarose gel stained with GelRed (Biotium Inc., USA).

Development of specific primers based on genome analysis
Specific primers were designed with the software Primer 3 Plus (https://primer3plus. com) based on B116 and B119 genomes previously available (JABXFG000000000.1 and JABXFF000000000.1, respectively). The specificity of the primers was checked using the BLAST N on GenBank (Altschul et al., 1997) and used for the amplification with 40 ng of DNA

Bacillus colony morphological characterization
The strains B32 and B116 showed predominantly circular beige colonies with convex elevation and smooth surface border.
B119 strain presented circular colonies with gray coloration, convex elevation and a border with slightly rough surface. The colonies of B2082 and B2084 isolates were similar, with a circular shape of beige coloration and a convex elevation border with smooth surface.
All strains are Gram positive and rod-shaped ( Figure 1).  Figure 2A). The amplification of the DNA with ERIC-PCR primers revealed between two and nine fragments with sizes ranging from 250 to 2,000 bp, whereas for REP primers, between two and six fragments with sizes ranging from 200 to 5,000 bp ( Figure 2B and 2C). These approaches allowed the genetic discrimination of the Bacillus strains, except B2082 and B2084 that showed identical profiles.

Development of specific molecular markers
Specific primers for detection of B116 (B.  *Values between 2.3 and 3.0 are equivalent to highly probable species identification; scores between 2.0 and 2.29 provide secure genus identification and probable species identification;and between 1.7 and 1.9, probable genus identification. ** Bacillus thuringiensis confirmed by protein crystal inclusions (data not shown).  increase food, animal feed and biofuel production.
Quality is one of the major concerns of bioinoculant technology which often leads to poor performance in the field and thereby loses the farmers' faith (Reddypriya et al., 2018). Strain authentication and detection of contaminants and pathogens for plant, animal, and human should be reinforced to ensure and control the quality of the bioinoculants produced (Soumare et al., 2020).
To address this issue, we explored different molecular techniques to characterize five Bacillus strains with plant growth-promotion capacity.
Initially, we uncovered significant phenotypic differences among colonies on PDA medium.  (Wang et al., 2007). In our work, long length 16S rRNA gene sequencing was used, which provided higher species-level resolution and accuracy and, when compared against sequences deposited in GenBank database, allowed Bacillus species identification. Our reported to be efficient in growth-promoting of several plant species, including maize, whose main mechanism is normally associated with phosphate solubilization, indole acetic acid production and/or nitrogen fixation (Kumar et al., 2014;Silva et al., 2016b;Bodhankar et al., 2017;Khati et al., 2018;. The results based on ARDRA, ERIC and  (Versalovic et al., 1991). These methods have been