The effect of bacterial isolates from rhizosphere soils on wheat and barley seed germination

Soil microorganisms, which are usually found in plant rhizosphere, have a wide spectrum of beneficial effects on the promotion of plant growth. The most comprehensively studied bacteria with these effects belong to the Bacillus genus. In this study, seven Bacillus isolates from Medicago sativa rhizosphere soils were isolated. Plant growth promoting characteristics of these isolates, such as production of indole-3aceticacid, siderophores and hydrogen cyanide were tested. The induction of wheat (Triticum aestivum) and barley (Hordeum jubatum) seed germination was evaluated in vitro. Isolate BMG2 produced the highest indole-3-aceticacid of 24.89 μg/mL. The length of roots of barley increased up to 60%, while the length of shoots of barley increased 2.23 times afterapplying isolatesPAZE-6 and BMG1, respectively. In addition, isolates BMG1, BMG2 and PAZE-6 improved germination of both types of seeds and showed ability to produce useful substances such as siderophores and indole-3-acetic acid. Further, these isolates could be used in the production of liquid crop additives that can improve the total yield of cultivated plants, especially barley.


INTRODUCTION
Plant growth promoting rhizobacteria (PGPR) are a heterogeneous group of bacteria that have the capability of synthesizing plant growth-promoting substances and decrease or prevent the deleterious effects of phytopathogenic microorganisms. They can be found in the rhizosphere, root surfaces and in association with roots, such as in symbiotic association inroot nodule formation. Soil microorganisms are important for agriculture as they promote the circulation of plant nutrients on the one side and reduce the need for chemical fertilizers on the other side 3 The ability of bacterial isolates to produce siderophores was tested by azurol S (CAS) agar plate assay, as described by Milagres (1999). A loopfull of bacterial culture, previously grown on a nutrient agar, was transferred to the borderline between CAS and NA medium. The inoculated Petri dishes were incubated for 7 days at 28C. The change of CAS agar color form blue to yellow was regarded as a positive reaction. The experiment was performed in 3 independent repetitions.
The production of indole-3-acetic acid was tested in a liquid nutrient broth enriched with 0.5mg/mL and 2mg/mL of tryptophan. Isolates were grown in liquid medium for 24h at 28C and centrifuged (13.000 rpm, 5min). After centrifugation, 1 mL of supernatant was mixed with 2mL of Salkowski reagent (0.5M FeCl 3 in 35% HCLO 4 solution) and placed in a dark place during 30 min. Optical density (OD) was recorded at 530 nm for each sample. The results were expressed as g/mL of IAA based on the comparison to the standard curve of auxin solution (0, 5, 10, 20, 50, 100 and 120 rpm).
The production of Hydrogen cyanide (HCN) was tested using Cyantesmo paper. Bacterial isolates were streaked on Petri dishes containing a nutrient agar, and pieces of Cyantesmo paper (approximately 2cm) were placed on the inner top of the Petri dish. Plates were incubated for 7 days at 28C. The color change of Cyantesmo paper from white to gray was considered asa positive result.
The API-ZYM system (bioMérieux, USA)was applied according to the manufacturer's recommendations. A pure overnight bacterial culture from the nutrient medium was centrifuged.
The suspension, with a turbidity of 5-6 McFarland in API Suspension Medium (2 mL), was prepared using precipitate. The inoculum (65 µL) was pipetted into each well of the kit, and then the API test strip was covered and incubated for 4.5 h at 30°C. After incubation, one drop of reagents "ZymA" and "ZymB" were added to stop the reaction, and the resulting color was developed over 5 min. The development of color was scored as positive reaction.
The ability of bacterial isolates to induce the germination of wheat and barley seeds was tested in vitro in Petri dishes. A sterile filter paper was moistened with 1 mL of sterile distilled water and placed with 40 wheat or 20 barley seeds in a Petri dish. The control sample consisted of wheat or barley seeds, without any bacterial treatment applied. The test treatment sample was prepared by immersing seeds in overnight bacterial culture. The experiment was performed in Effect of rhizosphere bacteria on seed germination, Buntic et al ZEMLJISTE I BILJKA, VOL 68, No 2, 1-11 Original paper doi: 10.5937/ZemBilj1902001B 4 triplicate. The Petri dishes were placed in a transparent sealed box at room temperature (22C) to provide humidity. The results were scored after seven days and expressed by the length of shoots and roots and the relative seed germination (%) index (RSGI) (Buntić et al., 2017). The percentage of RSGI is expressed by Eq.1: where SG S is seeds germination in samples and SG C is seeds germination in control.

RESULTS AND DISSCUSION
The results of CAS agar assay showed that all tested isolates, except B5 and PAZE-6,have the ability to synthesize siderophores (Table 1). Isolate B4 had the largest halo zone and showed the best potential in the production of siderophores. However, none of the tested isolates could produce HCN. A study conducted by Singh et al. (2015) showed that Bacillusstrains isolated from rhizosphere soil could not produce HCN and IAA, but they are potent producers of siderophores.
In addition, all tested isolates produced IAA in the presence of tryptophan concentration of 2 mg/mL (Table 1). However, the production of IAA was low in the presence of 0.5 mg/mL of tryptophan. Isolate BMG2 showed the highest IAA production of 24.89 g/mL, and was therefore characterized as the most potent producer. Isolates PAZE-6 and B4 also produced significant amounts of IAA, 8.67 g/mL and 6.00 g/mL, respectively. As all other tested Bacillus isolates showed lower IAA producing ability they could be characterized as weak producers. Research performed by Mohite (2013) also indicated that bacteria isolated from Effect of rhizosphere bacteria on seed germination, Buntic et al ZEMLJISTE I BILJKA, VOL 68, No 2, 1-11 Original paper doi: 10.5937/ZemBilj1902001B 5 rhizosphere soils could be very efficient in the production of plant growth stimulating substances, such as IAA. Furthermore, a research conducted by Djordjevic et al. (2017) indicated that the inoculation of maize seeds with indolacetic acid from Bacillus isolates has direct positive effects on root and shoot growth. Bacillus isolate used in their study had the ability to produce 8,86 μg/mL of IAA, which was similar as in the case of isolate PAZE-6 used in this research (Djordjevic et al., 2017).  According to the RSGI, all tested bacterial isolates showed goodeffect onT.aestivum and H.
jubatum seeds germination. However, the lowest seed germination was obtained after applying isolatesB3 and B4 on wheat and barley seeds, respectively. On the contrary, isolates BMG1, BMG2 and PAZE-6 improved the seed germination process (Table 3) in comparison to control.
In the literature, there are studies which also confirmed that Bacillus species were capable to improve seed germination including wheat and barley seeds (Hu et al., 2019;Abbasdokht and Gholami, 2010;Yadav et al., 2010;Canbolat et al. 2006).
In addition, all applied Bacillus isolates significantly improved the growth of shoots and roots of barley, recorded over seven days (Figs.1 and 2). The increase in the length of shoot of barley was between 1.39 (PAZE-6) to 1.99 (B3) times and 2.23 (BMG1) times in relation to control (Figs 1 and 2). While, the length of root of barley was increased by 31%, 39%, 54%, 55% and 60% with the applied PAZE-4, B5, BMG2, BMG1 and PAZE-6 respectively. The isolate BMG1 greatly increased the length of shoot and on the same time it contributed to the significant improvement of root length (Fig.1).
On the other hand, isolate B3 improved the growth of shoot and less growth of roots of barley seeds. Canbolat et al. (2006) also reported that Bacillus strains (RC01, RC02, RC03 and M-13) improved the growth both roots and shoots of barley seeds. The strain Bacillus RC01 showed the highest increase in root length of 8.88% (Canbolat et al. 2006). In addition, Ҫakmakҫi et al. (2007) (Ҫakmakҫi et al. 2007). However, applied Bacillusisolatesshowed low T.aestivum seeds germination. The highest increase in the length of shoot and root was given by BMG2 and it was 11.2% and 15.2 % in comparison to control, respectively (Fig.1). Taking into account the types of seeds, the isolate BMG2, with very good RSGI and good results of increasing the length of shoot and root for both seeds, can be considered as a good plant growth promoting bacteria forwheat and barley. In addition, this isolate showed ability for the production of IAA (24.89 µg/mL) and siderophores. Growth promotion may be due to other mechanisms such as production of plant growth promoting hormones in the rhizosphere and other PGP activities. These abilities depend on the availability of precursors for their production by microorganisms and their uptake by plant (Yadav et al., 2010).

CONCLUSION
Bacillus isolates BMG1, BMG2 and PAZE-6showed the strongest potential in the inductionof barley and wheat seed germination. All tested isolates improved the growth of shoots and roots of barely. None of the isolates has the ability to produce HCN. Isolates BMG2 and PAZE-6 produced the highest amount of IAA, while B4 showed the best potential in the production of siderophores. Based on the obtained results it can be considered that isolates BMG1, BMG2 and PAZE-6 could be used as PGP agents.