ORIGINAL ARTICLE

Potentialities of Bacillus strains for promoting growth in maize (Zea mays L.)

Potencialidades de cepas de Bacillus para la promoción del crecimiento del maíz (Zea mays L.)

Marcia M. Rojas,^I B. Tejera,^I Diana M. Bosh,^I Y. Ríos,^II Janet Rodríguez,^II Mayra Heydrich,^I

ABSTRACT

The Bacillus genus has representatives promoting plant growth. The objective of this research was to isolate and select bacteria from Bacillus genus, associated to the maize cultivation with potential in promoting growth in this crop of great importance in human and animal feeding. Isolations of bacteria belonging to this genus were carried out, endophytes as rhizosphere, of maize (Zea mays L.) hybrid cultivar P-7928. The isolates were physiologically characterized, in terms of indolic compounds production, the phosphate solubilization capacity and qualitative determination of biological nitrogen fixation. Also, the ability to form biofilms was evaluated using the method crystal violet stain on polystyrene plates. Later, the isolates with better results were entirely selected through a cluster analysis. A total of 19 isolates, 9 rhizosphere and 10 endophytes were obtained. All produce indole-acetic acid in a concentration range between 2.92 and 17.02 μgmL^-1 and fix atmospheric nitrogen; nine solubilize inorganic phosphate in a range between 12.16 and 33.07 μgmL^-1. Furthermore, the ability to form biofilms of 78.94% of the isolates was showed, which gives advantages in the colonization of plants. The formation of in vitro biofilm of a good strain producing and a nonproducing was compared. Both were adhered to the polystyrene plate with different pattern, which was more consistent in the EAM4 strain, with high biofilm production. The obtained results allowed selecting the native isolates ERM1, RM5, EAM5 and RM1 as the most promising to promote plant growth and their future use in the sustainable agriculture.

RESUMEN

El género Bacillus tiene representantes que promueven el crecimiento vegetal. El objetivo de este trabajo fue aislar y seleccionar bacterias del género Bacillus, asociadas al cultivo del maíz con potencialidades para la promoción su crecimiento en este cultivo de gran importancia en la alimentación humana y animal. Se realizaron aislamientos de bacterias de este género, endófitos como rizosféricos, del cultivo del maíz (Zea mays L.) cultivar híbrido P-7928. Se caracterizaron fisiológicamente los aislados, en cuanto a la producción de compuestos indólicos, capacidad de solubilización de fosfatos y determinación cualitativa de la fijación biológica de nitrógeno. Además, se evaluó la capacidad de formar biopelículas mediante el método de tinción con violeta cristal en placas de poliestireno. Posteriormente, se seleccionaron los aislados con mejores resultados de manera integral mediante un análisis de conglomerado. Se obtuvieron 19 aislados, 9 rizosféricos y 10 endófitos. Todos producen ácido indolacético en un rango de concentración que oscila entre 2.92 y 17.02 μgmL^-1 y fijan nitrógeno atmosférico; nueve solubilizan fosfato inorgánico en un rango entre 12.16 y 33.07 μgmL^-1. Además, se demostró la capacidad de formar biopelículas de 78.94 % de los aislados, lo que le confiere ventajas en la colonización de las plantas. Se comparó la formación de biopelícula in vitro de una cepa buena productora y una no productora. Ambas se adhirieron a la placa de poliestireno con diferente patrón, que fue más consistente en la cepa EAM4, con elevada producción de biopelícula. Los resultados obtenidos permitieron seleccionar los aislados autóctonos ERM1, RM5, EAM5 y RM1 como los más promisorios para promover el crecimiento de  las plantas y su uso futuro en la agricultura sostenible.

INTRODUCTION

The maize (Zea mays L.) is an annual grass plant and one of the highest production cereals in the world jointly with wheat and rice. In most of the America countries, the maize historically forms the basis of the regional feeding and one of the central aspects of the Mesoamerican and Andean cultures. Human consumption worldwide is lower than other cereals, because maize is considered a staple food for animals (Paliwal et al. 2001).

In order to improve the use of the maize kernel in calf feeding there were developed experiences, in which which demonstrated that the processing effect is to increase starch digestibility in the digestive tract. However, as the processing method is more intense, decreases in daily liveweight gains or in the conversion efficiency are generated, associated with decreases in the dry matter intake.

Therefore, it would be possible to improve the efficiency of starch use with the choice of types of maize of high kernel size (Maresca et al. 2002). Lopez et al. (2003) showed that bran and defatted maize germ can substitute the kernel in the diet for growing pigs up to 40%, without affecting weight gains. However, the efficiency of the diet conversion is affected. In this research, the energy value estimated for growing pigs was 70% equivalent to maize, demonstrating the importance of maize kernel in these animals feeding.

Due to the economic importance of this kernel, it seeks to increase yields with the use of alternatives offered by the sustainable agriculture, in order to affect as little as possible to the environment. This includes the use of microorganisms present in the soil to produce biofertilizer that replace chemical fertilizers and thus, reduce soil damage. Among the most used microorganisms are the plant growth promoting bacteria (PGPB) (Saharan and Nehra 2011).

These microorganisms directly influence on the plants metabolism promoting the increase of water and nutrients intake and the root system development (Glick 2014). Also, they stimulate plant growth through the phytohormone synthesis, N2 fixation and inorganic phosphate solubilization. Indirectly, the effect is exercised by enhancing the function of other beneficial organisms, present in the rhizosphere (Gupta et al. 2015). The PGPB include the genus Pseudomonas, Azospirillum, Azotobacter, Bacillus and Gluconacetobacter (Ahemad and Kibret 2014).

The members of Bacillus genus are characterized by being Gram-positive bacteria, bacillary form, strict aerobes or facultative anaerobes, positive catalase and endospores forming. This genus has been extensively studied, as to their antagonistic capacity of several pathogens organisms to crops of economic importance, but is necessary to deepen in the promoter activity of plant growth, to combine both effects.

As de Araujo et al. (2011) stated, it is very important to obtain new strains of Bacillus and to study their potential to stimulate plant growth. Thus the objective of this study was to isolate and select bacteria from Bacillus genus, associated with the maize cultivation, with potentialities in promoting plant growth.

MATERIALS AND  METHODS

Asolations. The sampling area is located in the agricultural areas of the Instituto de Investigaciones en Agricultura Tropical "Alejandro de Humboldt", Santiago de las Vegas, La Habana. The soil of the area used in this study is classified as red ferrallitic (Hernández et al. 2015).

Isolations were performed from samples of aerial, root and rizhozphere of three plants of maize to obtain endophytes microorganisms.

For isolation, the plants were taken to separate the root of the aerial part. To isolate rhizobacteria, 1g of the root with rhizosphere soil was placed in a tube containing 9 mL of saline solution (NaCl 0.85%) and it was vigorously stirred using a (Genie) stirrer. To obtain endophytes, roots, stems and leaves were disinfected with sodium hypochlorite at 25% for 15 minutes. After this time, they were washed several times with sterile distilled water and macerated in sterile saline solution.

The samples were heated at 80 °C for 30 minutes and cultivated by dissemination in Tryptone Soy Agar (TSA, OXOID) medium. They were incubated at 30 °C for 24 h.

From the colonies with different characteristics, the microscopic observation with Gram stain was performed to check the purity and determine the micro-morphological and staining characteristics. Also, the presence of spores, mobility and catalase activity, recommended in Bergey's Manual for Bacillaceae family (Claus and Berkeley 1986) was determined. Pure strains with these characteristics were stored in tubes of Nutrient Agar and 20 % glycerol at -20 °C.

Determination of potential as growth promoters. The determination  of indole acetic acid (IAA) production was performed according to the methodology which used Salkowski reagent(Glickmann and Dessaux 1995), in Tryptone Soy (TS) liquid medium. For this, the cultures were centrifuged at 5000 r.min^-1 for 15 minutes. Absorbance at wavelength of 535 nm was determined. In parallel, a pattern curve synthetic IAA was performed. This experiment was performed with three replications per strain. As a negative control the corresponding uninoculated medium was used.

To quantify phosphates solubilization, the strains which were positive in the qualitative assay were used. They were cultured in tubes with liquid medium NBRIP(Nautiyal 1999), which were incubated at 30 °C under stirring conditions in orbital shaker for 48 h.

This test was conducted according to the vanadomolybdate methodology proposed by Kundsen and Beegle (1988). The absorbance of samples at wavelength of 880 nm was determined in the spectrophotometer (Genesys 20). The reading of the results was performed at 48 h of incubation. In parallel, a pattern curve K2HPO4was performed. They were used to test three replications per isolates. As a negative control, the uninoculated culture medium was used.

Determination of the ability to form biofims. For this experiment the isolates were cultured on TS medium and incubated for 24 h at 37 °C under stirring conditions in orbital shaker. Then, the cell concentration to 10⁸ cells.mL^-1 was fitted, corresponding to 0.5 tube of McFarland (Jorgensen 1993) scale and were added 2 mL of each cultures in wells of polystyrene multiwell plates. These were incubated at 50 rpm and 30 °C for 8 h (Hsueh et al. 2006).

Subsequently, the contents of each well was removed, the plates were washed with distilled water and let to dry at room temperature. Then, they were stained for 10 minutes with 2 mL of crystal violet at 0.5 %. After, they were washed with distilled water and dried.

Later were added 2 mL of ethanol at 70%. Finally, the absorbance reading of the contents of each well at 590 nm was performed.

They were used for the experiment three replications per isolates and as negative control, the uninoculated medium.

After crystal violet staining, washing and drying of the plates, the biofilm formation in the bottom of the wells was observed in a trinocular biological microscope bright field, Zoel Model N-200M, China. Photographs were taken with a digital camera attached and Software Motic Images 2000 1.2 Micro-Optical Industrial Group Co.LT.

Statistical analysis. For data analysis, normality testing and variance homogeneity for all experiments variable were performed using the Statistica program version 8.0 (StatSoft 2007) they were performed. In the case where the normality and variance homogeneity was carried out, an ANOVA was performed and subsequently, Tukey test (Sigarroa 1985).

 A cluster analysis with the Euclidean distance application was performed for selecting strains that were completely better and proved to be promising for plant growth in maize cultivation. The results of the production of 3 indole-acetic acid and phosphate solubilization were used for this. They were taken as positive, the highest values obtained in each of the analyzed experiments and as negative, the lowers.

RESULTS AND DISCUSSION

Fontes et al. (2009), in the cultivation of Brazilian sweet corn, found more frequently the Bacillus genus among endophytes bacteria. When performing a molecular analysis of the isolated strains, concluded that the isolates found belonged to the species Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus and B. cereus.

In studies carried out in environments of importance to agriculture, this genus has been also found between rhizosphere microorganisms. The species B. badius, B. macerans, B. subtilis, B. licheniformis are highlighted (Wu et al. 2006), which showed potential in promoting plant growth.

Hernandez et al. (2003) found the Bacillus genus as one of the isolates when analyze the rhizosphere of Francisco Mejorado" cultivar in different types of soils in Cuba. This genus had the highest percentage of appearance after Pseudomonas, Azospirillum and Azotobacter.

Among the plant growth promoting hormones can be found auxins, gibberellins and cytokinins. Among the auxins, is the indole-3-acetic acid (IAA), whose production is widely distributed among plant growth promoting bacteria (PGPB) (Molina et al. 2015). It has been shown that the beneficial effects of rhizosphere microorganisms are related to auxins production, which may affect the initiation of lateral roots, their growth or both development processes. This brings about increase of the exploratory plant capacity and increases nutrient uptake (Ortiz et al. 2009).

The 19 obtained strains produced IAA in TS medium without addition of tryptophan, whose concentrations are between 2.92 and 17.01 μg.mL^-1 (Figure 1). There are no significant differences in IAA production between the isolates RM1, RM4, RM5, RM7, EAM3, EAM4, EAM5, EAM6, EAM7, ERM1, ERM3, which are the highest producers of this plant hormone.

These results agree from the quality and quantity point of view with those obtained by Swain et al. (2007) and Yu et al. (2016). Swain et al. (2007) showed that the inoculation of bacteria from this species to the yam plant (Dioscorea rotundata L.) increased the length of the stem and roots, as well as their fresh weight. This may be related to the production of such metabolite.

Other authors showed the ability to produce IAA for the genus in study, at concentrations higher than those obtained, but with the use of tryptophan as an inducer in the culture medium (Ali et al. 2009). It has also been shown that high concentrations of auxins have opposite effect, that is to say, inhibit plant growth (Arshad and Frankenberger 1997).

If the results of the production of IAA of isolates are compared with other microbial genera that produce this type of metabolite, it is observed that are similar to those of Rhizobium sp. and Azospirillum genera (de Souza et al. 2013).

The quantification of phosphate solubilization at 48 h showed that strains RM1, RM2, RM5, EAM5, ERM1, ERM2 and ERM5 there were not showed significant differences (figure 2), being RM2 that of  the highest solubilization value. The strains RM3 and RM9 were those of lower values. The strains that have high concentrations of soluble phosphate in the culture medium could potentially act as promoters of plant growth in soils with high levels of undissolved phosphates.

In this study the nitro-fixer ability of all isolates was qualitatively determined, when inoculated in semisolid nitrogen-free medium for five successive sweeps, as there was growth in the inoculation area in the semisolid nitrogen- free medium. Therefore, the microorganism was growing at the expense of atmospheric dinitrogen.

This would mean great contribution in the development of plant growth, since the dinitrogen is a limiting factor for its limited availability in the cultivated soil, in addition to the use of chemical fertilizers in large quantities it is an environmental danger (Bishnoi 2015).

The microorganisms able to carry out the biological nitrogen fixation are highly valued and used to increase the yields of different crops. Acetobacter, Azospirillum, Azotobacter, Bacillus, Enterobacter and Pseudomonas are among the microbial genera which have this ability. Bacillus is one of the genuses that are reported as dinitrogen fixer. It has been reported that the species Bacillus fusiformis shows high nitrogenase activity, so it is used in promoting the growth of plants, such as corn, wheat and rice (Różycki et al. 1999). This characteristic is interesting to use these strains in the subsequent development of a biofertilizer.

According to Arruda et al. (2013), the selection of PGPB with multiple potentialities for growth promotion suggests that they may have a better effect on plants at the level of greenhouse and in the yields of corn crop in the field. However, the physiological expression of bacteria under laboratory conditions does not guarantee their growth promoting activity in association with the plant (Fuentes and Caballero 2006). Therefore, it is essential to carry out studies plant- PGPB interaction to check the benefits of this interaction.

Biofilms are defined as communities of microorganisms which growth embedded in an exopolysaccharides matrix, adhering to an inert surface or a living tissue. Biofilms growth is the standard way of bacteria growth in nature. The ability of biofilms formation is not seemed to be restricted to any specific group of microorganisms. It is considered that, in appropriate environmental conditions, all microorganisms have the ability to form biofilms (Cairns et al. 2014).

The ability to form biofilms on the surface of the plant may be a competitive advantage. In this case, the ability of biofilms to adhere to abiotic surfaces like polystyrene plates was studied, which in vivo would allow a closer interaction of microorganisms with the plant, keeping interacting longer.

The isolates RM1, RM2, RM3, RM4, RM5, RM6, EAM 1 EAM 3 EAM4, EAM5, EAM7, ERM2, ERM3 and ERM5 have not significant differences and are the highest values (figure 3). For RM8 and RM9, which did not show significant differences, were the smaller values. The isolates RM7, EAM6 and ERM1 showed differences from to the rest of the mentioned isolates, with intermediate values between them. There are authors which delimit above 0.2 absorbance as a definition of biofilm-forming strains (Wakimoto et al. 2004).When considering this opinion, in this research 78.94 % of the strains can be considered positive.

It has been shown that the Bacillus genus is among the bacteria with ability to form biofilms which are used in agro- ecosystems managements.

Biofilms formation is also important, because it reduces the risk of susceptibility of bacteria that conform to adverse environmental conditions. It is also important because it increases the access to resources and niches that require critical mass and cannot be used effectively by isolated cells. The acquisition of new genetic traits, nutrients availability and metabolic cooperation are also suggested as a means that allowed optimizing the survival of the population in the biofilm (Anderson and O'Toole 2008). Another advantage of biofilms is that they protect the plant against pathogens and abiotic stress (Timmusk et al. 2009).

Bacterial interactions, growth and biofilms formation on the surface of the root involve complex mechanisms. The interaction between the roots and surrounding microorganisms form a single self-regulating complex system (Beauregard et al. 2013).

Biofilms formation is very beneficial for plants and to microorganisms associated with them, therefore it is a characteristic to consider if it were to produce a biofertilizer with the isolates obtained from this crop. Most of the plant-bacterium associations involve the physical interaction between bacteria and plant tissues. Direct observations of bacteria adhered to surfaces of plants have revealed variables multi-cellular associations, described as micro-colonies, aggregates and cell clusters (Beauregard et al. 2013). These multi-cellular structures exhibit many of the attributes that define the biofilms, groups of cells embedded in an exopolysaccharides matrix (EPS) on a solid surface.

In biocontrol agents, as Bacillus amyloliquefaciens SQR9, is associated the regulation of biofilms formation with the growth inhibition of Fusarium oxysporum in the cucumber rhizosphere (Xu et al. 2014).

The exopolysaccharides production is important in biofilms formation and also can contribute to the interaction of bacteria with roots and root accessories (Bogino et al. 2013).

Taking into account the values obtained in the experiments, a cluster analysis was carried out, which took as positive values the higher values of each of the capacities (in this case IAA production and phosphates solubilization) and as negative, the lowers. A cut at 20 % of the Euclidean distance (figure 4) was performed.

Jointly with the positive value, a group (I) was formed, in which RM1, EAM5, RM5 and ERM1 strains are included. These are strains that have the highest values in all analyzed capacities, so that may state that are the most promising for the promotion of crop growth. It is valid to note that RM5 exactly coincide with the positive value, which might suggest that it is the most promising.

There are three groups with intermediate values (II, III and IV). Group II includes EAM1 and RM8 strains. In group III, they are RM2, RM9, RM3, ERM2 and ERM5 and in group IV are grouped the strains RM4, EAM6, RM7, EAM3, EAM7, ERM3 and EAM4. In addition, a group (V) is obtained where only RM6 strain and the negative value are joining.

In this research 19 new Bacillus isolates were obtained from maize cultivation, root endophytes and of the aerial part, as rhizosphere. It was shown that all obtained strains have the ability to produce auxins and grow in nitrogen-free media. Nine of them solubilize phosphates, indicating their potential to promote plant growth. Fifteen strains form biofilms, which you may confer advantages in the plant colonization, important aspect for the future effectiveness of a bio-product. The RM1, EAM5, RM5 and ERM1 strains are the most promising for promoting plant growth in corn cultivation.

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Marcia M. Rojas, Departamento de Microbiología y Virología, Facultad de Biología, Universidad de La Habana, Cuba Calle 25 #455 e/ J e I Vedado CP 10400, La Habana, Cuba. Email: marcia@fbio.u.cu