CHARACTERIZATION OF ENDOPHYTIC BACTERIA OF THE GENUS BACILLUS AND THEIR INFLUENCE ON THE GROWTH OF MAIZE (ZEA MAYS) IN VIVO

Bacteria with positive properties on plant vitality are also called PGPB (Plant Growth Promoting Bacteria). Their presence can be observed not only in the root area but also in the above-ground parts of plants like endophytic bacteria. The aim of our study was to characterize promoting features of bacteria from Bacillus genus and compare them with Pseudomonas simiae WCS417 (plant growth promoting strain). The work was carried out in locality Kolíňany near Nitra (40°26 ́46 ́ ́N, 79°58 ́56 ́ ́W) and root samples were taken from 6 randomly selected plants of maize (Zea mays L.) in vegetative plant growth stage BBCH 14-15. Bacteria isolated from plant roots were identified and tested to biochemical parameters. From the biochemical features, we observed the detection of siderophores, determination of indole-3-acetic acid (IAA), monitoring the ability to dissolve phosphates and antifungal activity. Bacterial suspensions were applied to maize seeds and tested in vivo controlled conditions. Tested isolates were identified as Bacillus flexus, Bacillus megaterium and Bacillus subtilis. All 3 strains achieved the middle – class of phosphate solubilization index (2.00 ≤ SI ˂ 4.00), produced phytohormone IAA and showed positive production of siderophores and inhibited growth of Fusarium culmorum to more than 50%. All differences between tested strains and control strain P. simiae WCS417 were also statistically confirmed. All strains showed positive results in monitoring plant growth promoting properties. The effect of three Bacillus strains on maize seeds in vivo conditions showed significant differences in root length (P<0.0001) and weight of the young plant (P<0.001) compared to control.


Figure 1. Location of sampling point Isolation of bacterial strains
Root surface sterilization included the followings steps (Sun et al., 2008): to wash out the mechanical impurities roots were washed under distilled water and then surface sterilized in 99% ethanol for 1 minute and 3.125% sodium hypochloride solution for 6 minutes and followed by final wash in sterile distilled water for 3 times. Sterilized root samples were aseptically cat to small fragments which were placed on Luria-Bertani (LB) agar plates in 3 repetitions and incubated for 24 h at 30 °C. After incubation, distinct bacterial colonies were streaked on LB agar plates to get single colonies. Thus prepared bacterial isolates were used for further testing.

Molecular characterization
DNA was extracted from 1-day-old bacterial isolates prepared by the TSA agar cultivate method. Approximately 50 mg of each bacterial culture was placed in 200 μl of PrepMan solution (Life technologies) and homogenized with glass beads on BeadBug homogenizer (Benchmark scientific). DNA from all samples was use as a DNA template in PCR reaction. The bacterial 16S rRNA genes were amplified through PCR using the universal bacterial primers 27F (5'-AGAGTTTGATCATGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') (Nardi et al., 2004). Cycling conditions for PCR amplification (performed in thermocycler MJ Mini (Biorad, USA) were as follows: 95°C for 3 min. followed by 40 cycles of 95°C for 30 s, annealing at temperature 56°C for 30 s, 72°C for 90 s and a final elongation at 72°C for 10 min. Amplification products were sequenced and performed by Macrogen (South Korea). Acquired sequences were assembled and processed in MEGA 7 software (Kumar et al., 2018). Alignment was made in MUSCLE (Edgar, 2004). We used the reference sequences from Genbank database for phylogenetic analysis. A phylogenetic tree was constructed using method of Maximum likehood with substitute model Tamura-Nei (Tamura et al., 2007).

In vitro tests for direct growth promotion traits
Bacterial isolates were tested for these direct growth promotion traits: production of phytohormone indole-3-acetic acid (IAA) and phosphate solubilisation. Pseudomonas simiae WCS417 (Netherlands) was used as a positive control. Production of phytohormone Indole-3-acetic acid (IAA) was evaluated according to Gordon and Weber (1951). Bacterial cultures were grown for 24 hours at 30 °C on Trypton Soya Agar (TSA) plates and than the concentration of each bacterial strain was standardized to 0.5 McFarland (10 8 CFU.ml -1 ). Bacterial suspension was incubated in Pikovskaya's broth with 0.2% L-tryptophan for 7 days at 30 °C. The supernatant was mixed with Salkowski reagent. The mixture was incubated in the dark for 30 minutes and then examined for the development of pink colour as an indication of IAA production. The colorimetric measurement of IAA was done spectrophotometrically at 530 nm. Activity of bacteria to dissolve phosphates was evaluated on Pikovskaya's agar with bromophenol blue (Gupta et al., 1994). Plates were inoculated with tested bacteria and incubated for 7 days at 30 °C. The yellow/clear zone around bacterial colonies showed positive solubilisation of phosphates and Solubilisation index (SI) was calculated according to Kumar and Narula (1999).

In vitro test for indirect growth promotion traits
Bacterial isolates were evaluated for these 2 traits associated with indirect growth promotion: antagonism against phytopatogenic fungi and siderophore production. In tested methods was used Pseudomonas simiae WCS417 as a positive control. Phytopathogenic fungi were inoculated on plates with Potato Dextrose agar (PDA) and incubated for 10 days at 25 °C. Tested bacteria were inoculated on TSA plates and incubated 24 hours at 30 °C. Bacterial suspensions were diluted to 0.5 McFarland (10 8 CFU.ml -1 ) and spreaded onto the surfaces of TSA plates using sterile bacterial cell spreaders. The centre of each plate was inoculated with 9 mm diameter fungal plug cut with a sterilized cork-borer from a Fusarium culmorum culture. Petri dishes were incubated for 10 days at 25 °C. Pseudomonas simiae WCS417 (Netherlands) was used as a positive control, sterile TSA plates as a negative control. The pathogenic fungus Fusarium culmorum 819. was obtained from Department of Plant Protection, Faculty of Agrobiology and Food Resources, SUA, Nitra.
Production of siderophores was tested according to Schwyn and Neilands (1987) using the Chrome Azurol S (CAS) medium. Each bacterial isolate was inoculated on agar plates and incubated for 7 days at 30 °C. The orange halo zone around a bacterial colony indicated siderophore production by bacteria.

Growth promotion effects in vivo
The potential PGPB strains isolated from maize roots were analysed for their ability to exhibit plant growth promotion. Firstly, the maize seeds were surface sterilized with 3.125% NaOCl for two min. and followed by three washes in sterile distilled water. The organically poor soil was sterilized in autoclave in followed conditions: pressure 0.1 MPa and temperature 121 °C for 20 minutes. Inoculants of the selected strains were prepared on TSA agar plates and incubated at 30 °C for 24 h. Concentration of bacteria was standardized to 0.5 McFarland (10 8 CFU.ml -1 ) for each strain. Sterilized seeds were immersed in the bacterial suspensions for 1 hour. After soaking, the seeds were sown in the soil, 1 cm below the surface. A growth chamber experiment lasted 4 weeks and was carried out under controlled conditions: changes in temperature depending on the light period -16 hours, 28 °C and dark period -8 hours, 22 °C and constant humidity in both phases was 80%. As a control, seeds were treated in distilled water. As a positive control we used the strain Pseudomonas simiae WCS417 (Netherlands). 30 days after sowing plants were removed from the soil and their roots were carefully washed. The root length (cm) and weight of the young plant (g) were evaluated.

STATISTICAL ANALYSIS
All results are the means of three independent replicates. Analysis was carried out using the GraphPad Prism where standard error was evaluated. We used One-way analysis of variance ANOVA to compare values from the analysis of plant growth promoting characteristics and analysis of the effect of these bacteria on maize growth performance (root length and weight of the young plant). A comparisons were done using Dunnett's test and the significant level was set at **** P < 0.0001 and *** P < 0.001.

RESULTS AND DISCUSSION
During last few years many studies focused on the isolation and characterization of bacterial strains isolated from soil or plant parts with plant growth promotion potential (Ramakrishna et (Nautiyal et al., 2013). Detailed data about taxonomic affiliation of tested strains and GenBank accession numbers are situated in Table 1. The phylogenetic tree of bacterial strains constructing by using their 16S rRNA sequences is shown on Figure 1.  Bacillus strains were examined for their morphological, physiological and biochemical properties. Relative results including cell morphology, Gram property and endospore formation, oxidase and catalase activity. All results are summarized in Table 2. In all properties we tested reference strain Pseudomonas simiae WCS417 (Netherlands). The aim of our work was to determine the effectiveness of bacteria by testing their ability to promote plant growth and apply them to seeds under in vivo conditions. For growth promotion, we tested the following: the ability to solubilize phosphates into a plant-accessible form, the production of phytohormone indole-3-acetic acid (IAA), the production of siderophores and antifungal activity. As a positive control we tested Pseudomonas simiae WCS 417 (Netherlands) strain. In the last years many publications described biological mechanisms involved in the ability of the PGPB strain P. simiae WCS417 to promote plant growth and health (Desrut et Table 3. Seed germination and plant growth are influenced by the nutrients available in the soil. Plants absorb phosphorus (P) from the soil through root transporters. Available forms of P are also limited (Bidondo et al., 2012). Bacillus spp. convert the complex form of essential nutrients, such as P to a simple available form that is used during uptake by plant roots. All tested strains solubilize phosphate. They are included in the group with middle phosphate solubilizaton index (2.00<SI>4.00) according to Kumar et al. (1999). A statistically significant difference (P<0.01) was observed between strain 239 (B. flexus) and P. simiae WCS417.
The presence of tryptophan and other bacterial food source compounds induces the synthesis of indole-3-acetic acid (IAA) and other hormones in bacterial populations (Glick, 2014). Plant-growth-promoting substance IAA is synthesized by Bacillus spp. and increase root and shoot cell division and elongation (Radhakrishnan and Lee, 2016). According to the IAA production assay results all 3 Bacillus isolates and Pseudomonas simiae WCS417 produce auxin from 2.07 to 5.60 µg.ml -1 . A statistically significant difference was observed between all Bacillus strains to the P. simiae WCS417. The P value was as follows 525-231 and 525-239 (P<0.001) and 525-246 (P=0,034). The iron-chelating properties of Bacillus spp. via siderophore production help to solubilize iron from minerals and organic compounds in rhizosphere (Nadeem et al., 2012). In the case of Pseudomonas simiae WCS417, its biological activity is mainly associated with the production of siderophores (Berendsen et al., 2015). In our study was production of siderophores positive in all tested isolates.
Maize is one of the crops that is attacked by a number of pests at all growth stages. Among the most common are microscopic fungi, especially representatives of the genus Fusarium (Javoreková et al., 2020). Genus Bacillus is well known for their antagonistic behavior by producing siderophores, HCN, hydrolytic enzymes and antibiotics. In the Srivastava's study nine of the soybean bacterial endophytes that belong to Bacillus sp. were reported to have antifungal activities against major soil-borne plant pathogens like Rhizoctonia, Fusarium and Sclerotinium (Srivastava et al., 2016). Bacillus spp. attaches to the mycelial cell walls and damage fungal mycelium (Akram et al., 2016). In our study was protection against pathogens more than 50% in all tested isolates. Statistically significant differences were observed between all tested strains to the control strain P. simiae WCS417. In our study, all strains produced IAA, although in different amounts. Pseudomonas simiae WCS417, which was used as positive control, promoted plant growth less than other tested bacteria. The length of the root system was extended by 12% and the amount of weight of young plant increased almost threefold compared to the control. Bacillus strains that produced lower levels (246 -Bacillus subtilis, 239 -Bacillus flexus), had bigger influence on root elongation and biomass, characteristics of great interest that provide greater surface area for the absorption of nutrients. According to Arshad and Frankenberger (1991) the effects of the auxin depend on its concentration, i.e. when it is low it can stimulate growth and when it is high can be inhibitory, same for seed germination. Microbial synthesis and secretion of IAA significantly increase the root length in all cases and play important role in the germination event of various plant species. Weight of the young plant increases in all tested samples, too.

Figure 2
Effects of Bacillus tested strains and reference strain Pseudomonas simiae WCS417 on maize growth (root length and weigth of the young plant) according to the control According to the all tested direct and indirect plant growth promoting traits and potential of bacteria to promote root length and weight of the young plant we evaluated as the most productive Bacillus flexus strain (239). Results of all tested strains are shown in Table 4.