To stimulate growth of wheat seedlings and to form biofilms

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TO STIMULATE GROWTH OF WHEAT SEEDLINGS AND TO FORM BIOFILMS

V.S. Tverdokhlib, N.V. Limanska, K.D. Krylova, V.O. Ivanytsia

Odesa I. I. Mechnikov National University

Development of biological preparations for organic agriculture should include the study of interactions of microorganisms - the components of biopreparations, with representatives of natural microbiota of agrocoenoses. As a microorganism - typical representative of epiphytic and soil microbiota, species Bacillus megaterium has been selected. Aim. The study of effect of Lactobacillus plantarum ONU 12 and Bacillus megaterium ONU 484 on germination and growth of wheat seedlings. Materials and Methods. Seeds of wheat Triticum aestivum L. were inoculated with suspensions of bacteria B. megaterium OHy 484, L. plantarum OHy 12 and their mixture was germinated in hydroponics and soil under green house conditions. Germination of seeds, mean length of roots and height of seedlings were compared. Capability of these microorganisms to form biofilms was studied: inoculated seedlings were dyed with 0.1% acridin orange and observed with a magnification x600. Results. Simultaneous inoculation with bacteria L. plantarum ONU 12 and with the representatives of soil microbiota B. megaterium ONU 484 didn t decrease the stimulation effect of lactobacilli. Opposite, the highest stimulation effect on plants both in hydroponics and in soil caused the treatments with the mixture L. plantarum ONU 12 + B. megaterium ONU 484 and with the strain B. megaterium ONU 484 alone. In hydroponics mean length of roots increased in 8.0 - 16.9%, mean height of seedlings - in 8.8 - 24.3%. In soil germination of seeds increased in 7.0%, mean height of seedlings - in 7.6%, mean root length - in 13.1%. Lactobacilli and bacilli in the mixture were able to form biofilms with a developed matrix. Conclusions. In presence of representative of soil microbiota B. megaterium ONU 484 phytostimulation the activity of lactobacilli increased.

Strain B. megaterium ONU484 also showed stimulation activity when applied separately.

Key words: stimulation of plant growth, Lactobacillus plantarum, Bacillus megaterium.

The development of biological preparations for organic agriculture should include the study of interactions of microorganisms from biological preparations with representatives of natural microbiota of agrocenoses. Lactobacilli isolated from plants are known for their stimulatory activity on plant growth [9; 10], which is explained by the synthesis of auxin hormones or their precursors described in the literature [6]. It remains unclear whether the stimulatory effect of lactobacilli decreases when bacteria penetrate into the natural environment and interact with representatives of plant and soil microbiota. As a microorganism - a typical representative of the epiphytic and soil microbiota there were selected the species Bacillus megaterium in our study. The representatives of the genus Bacillus inhabit the surface and vessels of plants, as well as soil, and are known for their ability to stimulate plant growth [6; 8; 10; 13]. The phytostimulative activity of bacilli is explained by the possible synthesis of auxins, cytokinins, gibberellins and their precursors [8; 12; 13]. Gruneberg et al. (2006) indicate the successful application of the consortium of Bacillus subtilis and Lactobacillus spp. to improve the growth of ornamental plants. The previous studies described strains - representatives of the genera Bacillus and Lactobacillus with stimulatory activity for each strain separately [3; 9].

The aim of this work was to study the effect of Lactobacillus plantarum and Bacillus megaterium on germination and growth of wheat seedlings.

Materials and methods

Strains from the Collection of the Department of Microbiology, Virology and Biotechnology of Odesa National I. I. Mechnikov University L. plantarum ONU 12 i B. megaterium ONU 484 were used. Lactobacilli were grown in a liquid MRS medium [5] at 37 °C overnight and used in the experiments with a culture concentration of 10⁸ cells/ml. Bacilli were grown overnight in LB medium at 28 °C. To prepare the mixture, bacteria of both strains were grown to the concentration of 10⁸ CFU/ml, and mixed in a ratio of 1:1.

For simulating the conditions of hydroponics, we used the Aquasave S gel, prepared according to the instructions. As a test plant, Triticum aestivum L. (wheat variety - Kuialnik) was used. Seed surfaces were sterilized by 25% hydrogen peroxide for one minute. Then seeds were washed three times in sterile distilled water.

Overnight cultures of lactobacilli and bacilli at concentration of 10⁸ cells/ ml were used to prepare dilutions 1% (10⁷ cells/ml), 0.1% (10⁶ cells/ml), 0.01% (10⁵ cells/ml), 0.001% (10⁴ cells/ml), 0.0001% (10³ cells/ml), 0.00001% (10² cells/ ml) for each strain and for mixtures of strains. Seeds were soaked for one hour in each of the prepared dilutions. Subsequently, seeds were transferred in Aquasave S gel in glassware for germination. The control was seeds soaked for one hour in sterile distilled water. Germination was carried out in a greenhouse in Aquasave S gel for seven days.

With the concentrations of bacteria that exhibited the higher stimulatory activity we conducted the same experiment in soil conditions (Fig. 1). We used the soil "Poliskiy Universalniy" with a high content of peat, which was not sterilized before sowing.

In total in three independent experiments there were tested 300 seeds of each variants. After seven days, the growth characteristics of plants were measured: average lengths of the roots of seedlings and average height of the plant [2].

Statistical processing was performed using Excel package. The lengths of the roots and shoots as quantitative values were expressed as a mean and confidential interval at 95%, the germination as a qualitative value was expressed as a percentage and a standard error. Significant differences between control measurements and inoculated seedlings were detected in Student's t-test (P <0.05).

To study the ability of bacteria to form biofilms obtained wheat seedlings were washed from gel in distilled water. Biofilms were fixed in 96% ethanol for 15 min and stained with 0.1% solution of acridine orange for 10 minutes. The colored seedlings were dried on a slide and examined under a microscope. For observation, the microscope PrimoStar PC, Carl Zeiss with a total magnification x600 was used. The level of formation of biofilm was evaluated by the scale given in Table 1 [1].

Table 1

The evaluation criteria of the formation of biofilms [1]

We investigated 10 samples of each variant and evaluated the level of biofilm formation in 10 fields of vision. The biofilms were photographed using the camera Canon EOS 500D.

Results and Discussion

Inoculation of wheat seeds with bacteria of the strains L. plantarum ONU 12 and B. megaterium ONU 484 influenced germination and growth of seedlings in hydroponics conditions. Thus, treatment with bacilli at concentrations from 0,1% (10⁶ cells/ml) to 0.001% (10⁴ cells/ml) of the overnight cultures significantly increased the germination (Table 2).

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Table 2

Germination of wheat in gel (%) after the treatments with L. plantarum ONU 12 and B. megaterium ONU 484 in different concentrations, cells/ml

Note: the difference between the values is reliable at P = 0.05; * - compared with the corresponding indicator at concentration of 10⁷ cells/ml; ** - at concentration of 10⁶ cells/ ml; *** - at concentration of 10⁵ cells/ml; all values are significantly different from control.

In the control - seeds, soaked in water instead of the suspension of bacteria, the germination was 55.0 ± 2.6%. We can see that low concentrations of bacteria (10³ cells/ml) did not influence the germination. A high concentration (10⁷ cells/ml) did not influence or even suppress germination, as in the case with B. megaterium ONU 484. Application of the individual strains was more effective than treatment with the mixture of strains L. plantarum ONU 12 and B. megaterium ONU 484 (Table 1). Treatment with lactobacilli improved the germination in 5.0-25.0%, bacilli - in 8.3-35.0% (except for the concentration of 10⁷ cells/ml), and the mixture - in 1.7-5.0% (almost did not influence).

Measuring of the average length of the roots of seedlings showed that treatment of wheat seeds by suspensions of B. megaterium 484 increased root length in 8.0-14.8% (Fig. 1, A). In this case, the best concentrations were 0.1% (10⁶ cells/ml) and 0.0001% (10³ cells/ml) of B. megaterium 484 overnight cultures.

Fig. 1. Average length of the roots of wheat seedlings treated with bacterial
suspensions and germinated in gel:

A - B. megaterium ONU 484; B - L. plantarum ONU 12;

C - L. plantarum ONU 12 + B. megaterium ONU 484,

* values are significantly different from the control (P <0.05)

Treatment with lactobacilli increased the length of roots in 8.0% (Fig. 3, B). The average length of roots of seedlings after treatment with the mixture of lactobacilli and bacilli in gel increased in 8.0-17.0% (concentration of applied bacterial suspensions 10⁴-10⁶ cells/ml) (Fig. 5). This means, that interaction with representatives of soil and plant microbiota - bacilli, did not decrease the stimulatory activity of lactobacilli. Opposite - their activity increased due to stimulatory effect of bacilli.

Figure 2 shows the wheat seedlings in gels after the treatment by the mixture of lactobacilli and bacilli.

Fig. 2. Growth of root systems of wheat seedlings after the treatment with the mixture L. plantarum ONU 12 and B. megaterium ONU 484

Bacterial suspensions caused more significant effect on the height of seedlings. Thus, treatment with strain B. megaterium 484 increased the average height of seedlings in 8.8-21.4% (for concentrations of applied bacterial suspensions 10³10⁶ cells/ml) (Fig. 3, A).

Fig. 3. Average height of wheat seedlings treated with bacterial suspensions and germinated in gel:

A - B. megaterium 484, B - L. plantarum ONU 12;