بررسی تأثیر سویه های باکتریایی گرم منفی و مثبت بر جوانه زنی و رشد گیاهچه های برخی غلات و گیاهان دانه روغنی

Introduction

A wide range of microorganisms ranging from pathogenic to beneficial, interact continuously with higher plants in the soil ecosystem (1). These microorganisms influence the growth, development and functions of plants (1, 2). Rhizobacteria are used as inoculants to enhance crop yield and control fungal pathogens biologically (12). 

Bacteria that colonize plant roots and promote plant growth are called plant growth-promoting rhizobacteria (PGPR) (3, 4). PGPRs isolated as free-living soil bacteria from plant rhizosphere, which can decrease the use of chemical fertilizer-N (4). The corresponding PGPR associated to plant roots and/or other plant parts could increase the growth and seed yield of plants (4).

PGPR could affect plant growth either indirectly or directly (8). The indirect promotion of plant growth occurs when PGPR lessens or prevents the deleterious effects of one or more phytopathogenic organisms (4). The direct promotion of plant growth by PGPR involves either providing the plants with certain bacterial-synthesized compounds or facilitating the uptake of certain nutrients from soil (4, 8). In general, microbial inoculation is considered as an important component for agricultural practice, due to loss of topsoil, soil infertility, decrease in plant growth and plant yield, and insufficient diversity of indigenous microbes (9).

PGRP could be classified into four groups: 1. Bio fertilizers (solubilisation of mineral phosphates, asymbiotic nitrogen fixation) 2. Phytostimulators (ability to produce phytohormones) 3. Rhizoremediators (degrading organic pollutants) and 4. Biopesticides (siderophores production and synthesis of antibiotics, enzymes and/or fungicidal compounds) (10).

Rhizobium meliloti, as a free-living microorganism in the soil, is grouped as Rhizobiaceae (1).It is considered as nitrogen-fixing bacteria, which have symbiotic relationships with legumes, especially alfalfa.

Some species of Pseudomonas bacteria like Azotobacter are useful due to their ability to make the unavailable nutrient elements accessible (13). For instance, inoculation with pseudomonas caused a significant increase in yield of wheat, sorghum and corn by 10 to 30% (5-12). Azadikhah et al. (14) showed that the Pseudomonas fluorescens strain had produced siderophore with promoting iron absorption by the plant and increasing the availability of iron in the surrounding soil of the root. Hamaoui et al. (15) reported that Azospirillum brasilense improved root and shoot development significantly, when compared with non-inoculated controls in Faba beans and chickpea. The Azotobacter strains had increased the disease tolerance in plants.

Bacillus sp.(asPGPR) could increase plant growth and yield when contacting the plant roots (4, 19). Bacillus species is inhibitory to several plant pathogens and improves the growth of many plants in streamed and natural soils (18). For example, inoculation of tea plants by Bacillus sp. reduced the disease incidence of blister blight for two seasons, which was almost comparable with the performance of chemical fungicide (16). Bacillus species could be considered as facultative anaerobes (17).

Bacillus subtilis is a rhizobacteria, which is attributed to synthesis of phytohormones, such as indole acetic acid, abscisic acid, gibberellins and cytokinins (20). This function of B. subtilis promotes root growth and increases the number of root hairs (20). The positive effect of B. subtilis has been documented in previous studies (5, 18). The cellular structure of B. pumilus is similar to other Bacillus species such as B. subtilis, B. megaterium, which generally shows high resistance to environmental stresses (21, 22). Kuan et al. (5) reported the significant effect of B. pumilus s1R1 on nitrogen fixation in maize. 

Stenotrophomonas sp. is a gram-negative obligate aerobe (23). The rod isolate is an environmental bacterium found in aqueous habitats, including plant rhizospheres, animals, foods, and water sources (23).

Several bacteria such as Klebsiella,  Azospirillum, Pseudomonas, Bacillus and Burkholderia are identified as PGPR through biological nitrogen fixation (BNF), phytohormone production (e.g., auxin, gibberellin and cytokinin) and biological control of soil pathogens in different plant species as maize (5,7). Kuan et al. (5) reported the positive effect of four bacterial strains including Klebsiella sp. Br1, Klebsiella pneumoniae Fr1, Bacillus pumilus S1r1، Acinetobacter sp. S3r2 on growth characteristics of maize. Safdarpour and Khodakaramian (31) reported the promoting effects of bacterial strains of Pseudomonas mosselli, Pseudomonas fuorescence and Stenotrophomonas maltophilia on germination and disease tolerance in tomato under in vitro conditions.

In PGPR bacteria group, the deleterious rhizosphere bacteria (DRB) inhibits the growth of plants without causing symptoms of disease infection. One of the main mechanisms for growth inhibition by this undesirable group of rhizobacteria have been proposed as the production of phytotoxins such as cyanide and other volatile and non-volatile compounds (24). A certain concentration of indole-3-acetic acid (IAA), which is produced by DRB, has an inhibitory effect on root growth in sugar beet and blackcurrant (24). Tabatabaei et al. (25) reported the inhibitory effect of Pseudomonas, as a DRB strain, in seedling growth of durum wheat. Plant inoculation with PGPRs have different benefits in increasing different indices such as germination rate, higher mass production and better control of disease and microbial activities (26).

Identifying the native strains of PGPR as promoters of plant growth and/or inducers of plant defense response, may offer a practical way to achieve plant growth promotion and better management for plant diseases (11). In recent years, several studies have carried out to evaluate the effects of PGPRs on various plants, but there are few reports on the effects of some bacterial species asStenotrophomonas sp. Rhizobium meliloti and B. pumilus on seedling growth of different plant species. In addition, the PGPR function mechanisms are not fully understood in different plant species. This experiment was conducted to study the effect of seed inoculation with five different bacterial strains (Azotobacter chroococcum, Rhizobium meliloti, Bacillus subtilis, Bacillus pumilus and Stenotrophomonas sp.) on the germination and primary growth of mentioned plants under in vitro condition. This study reports the effect of B. Pumilus on germination and seedling growth of the studied plant species.

This experiment was conducted to study the effect of seed inoculation with five different bacterial strains (Azotobacter chroococcum, Rhizobium meliloti, Bacillus subtilis, Bacillus pumilus and Stenotrophomonas sp.) on germination and primary growth of mentioned plants under in vitro condition. This study reports, for the first time, the effect B. Pumilus on germination and seedling growth of the plants.

Materials and Methods

Bacterial Strains and Growth Conditions:Two Bacillus strains including B. subtilis (PTCC No:1204) and B. pumilus (PTCC No: 1733), Azotobacter chroococcum (PTCC No: 1658), Rhizobium meliloti (PTCC No:1684) and Stenotrophomonas sp. (Access No.: AB1616885.1) were obtained from the Department of Biology, University of Isfahan, Iran. The isolates were grown on nutrient agar (NA) (2 g yeast extract, 1 g meat extract, 5 g peptone, 5 g sodium chloride, 20 g agar-agar, 1000 mL distilled water) for routine use. The strains were maintained in NB with 20% glycerol at –80 °C for long-term storage. For preparing the bacterial cultures, single colony of each bacterial isolate was grown in 250 mL flasks containing 100 mL NB medium and incubated for 24 h at 28 ± 2 °C on a rotary shaker at 120 rpm. After incubation, the cell suspension was centrifuged at 5,000 (rpm) for 5 min at 4 °C and washed twice with sterile distilled water. The final pellet was re-suspended in sterilized distilled water and the bacterial cultures were standardized to 1×10⁸ colony-forming units (CFU)/mL and used, immediately, for seed germination experiments.

Effects of Bacterial Species on Seed Germination and Seedling Growth Traits: The effect of five different bacteria strains were evaluated in four different plant species. The examined species were both monocotyledon (wheat and barley) and dicotyledon (canola and sesame). The germination and seedling growth traits were evaluated at seeds of durum wheat (Triticum turgidum L. var durum), barley seeds (Hordeum vulgar L. cv. Valfajr), rape seeds (Brassica napus cv. Hayola) and sesame seeds (Sasamum indicum cv. Oltan). The experiment was conducted as a completely randomized design (CRD) with four replicates. Six treatments were made as follows: 1) seeds treated with Stenotrophomonas sp.; 2) seeds treated with B. subtilis; 3) seed treated with B. pumilus; 4) seeds treated with A. chroococcum; 5) seeds treated with R. meliloti; and 6) un-inoculated as control. Seeds were surface disinfected with 2% (v/v) solution of sodium hypochlorite for 15 min and were rinsed four times with sterile distilled water, and air-dried before being used in the germination experiments. All further manipulations were carried out under sterile conditions. The surface-sterilized seeds were immersed into individual bacterial suspensions for 30 min, shaking at 120 rpm. Twenty seeds were placed into sterile petri dishes (12 cm diameter), containing 1.5% (w/v) of distilled water agar. The Petri dishes were transferred into a growth chamber at 25 ± 2 °C for 7 days. Final percentage for germination and its rate were measured after 7 days from incubation. At the end of vegetative growth (30 days), different growth parameters viz., seedling fresh weight and seedling dry weight was measured from the base to the tip of the plant. Fresh weight was determined by uprooting the plant carefully, washing them thoroughly to remove remnants of soil particles. Dry weight was determined by drying the plants in an oven at 65 ºC until the weight remained constant.

Statistical Analysis:Analysis of variances (ANOVA) was conducted on the data when F values were significant (P ≤ 0.05). Mean comparisons were conducted using least significant differences (LSD, 0.05) procedure. Orthogonal independent comparisons were conducted for differences within and between the two crop groups, i.e. dicotyledonous versus monocotyledon and for their interaction with bacteria strain. The mean comparisons were conducted using Fisher’s least significant difference (LSD) at P ≤ 0.05.

Results

The bacterial strains, plant species and bacterial strains × plant species interaction showed significant effect on all the studied traits (data not shown). This result implied different responses of plant species to inoculation with bacterial strains. The highest germination percentage was related to sesame (85%) and wheat (86%) but the least one was associated to barley (36.4%) (Table1). All of the bacteria strains showed a significant increase in germination (%) of plant species, except B. pumilus (Table 1). The comparison effect of two Bacillus sp. (B. subtilis and B. pumilus) on wheat germination is presented in Figure 1.

Fig. 1- The Effects of B. Pumilus (a), and B. Subtilis (c) Wheat Blank (b) on Germination of Wheats

Table 1- Mean Comparisons of Plant Species and Bacteria Strains for Seed Germination (%), Root Number, Root Length, Shoot Length, Root Fresh Weight, Root Dry Weight, Shoot Fresh Weight and Shoot Dry Weight

Values within a column bearing different superscript are significantly different at P≤ 0.05. ¥: Abbreviations:  germination (G); root number (RN), root length (RL), shoot length (SL), root fresh weight (RFW), root dry weight (RDW), shoot fresh weight (SFW), shoot dry weight (SDW).

B. subtilis caused the highest germination (56.25%) in sesame (Fig. 2). B. pumilus had the most negative effect on the germination of barley and canola (0%) (Fig. 2). In canola, inoculation with all bacterial strains, except B. pumilus, showed significant increase on germination rather than the control (Fig. 2). Inoculation with bacterial strains had no significant effect on germination of wheat, barley and sesame (Fig. 2).

Fig. 2- The Plant Species ×Bacteria Species Interaction on Germination (%)

Root Number and Length: The highest number of root was observed under inoculation with R. meliloti that increased the number of roots by 2(%) in comparison with control (Table 1). The strains of B. subtilis, A. chroococcum and Stenotrophomonas sp. showed no significant effect on root number of the studied plant species, while the B. pumilus reduced the root number (83%) in comparison with control (Table 1). The non-significant effect of B. subtilis and A. chroococcum on all of the plant species could be explained by plant species × bacterial strains interaction. The negative effect of B. pumilus on root number, in comparison with control, was more obvious in barley (100%) rather than wheat (48.21%) (Fig. 3a).

The highest increase in root length was associated with Stenotrophomonas sp (7.07 cm) and A. chroococcum (6.45 cm), but the least one was related to B. pumilus (0.17 cm) (Table 1). Inoculation with all strains, except B. pumilus, showed an increase in the root length (Fig. 3b). B. pumilus reduced the root length by 95(%( in comparison with control (Fig. 3b). In comparison between different plant species, the highest root length was associated with wheat (9.4 cm), but the least ones were observed in the sesame (1.78 cm) and canola (1.41 cm) (Table 1). In barley, the root length increased significantly by 296 (%), 208 (%) and 210 (%) under inoculation with Stenotrophomonas sp, A. chroococcum and Rhizobium meliloti, respectively (Fig. 3b), but B. subtilis decreased the root length (100%) in comparison with control (Fig. 3b). 

In wheat, Stenotrophomonas sp (183%), B. subtilis (193%) and A. chroococcum (159 %( were considered as the effective strains for the enhancement of root length, in spite of B. pumilus, which reduced the root length (86%( significantly (Fig. 3). In sesame, root length was reduced under inoculation with B. pumilus (51%) and B. subtilis (51%) (Fig. 3b), but A. chroococcum (109%), R. meliloti (59%) and Stenotrophomonas sp (47%) increased the root length in comparison with control (Fig. 3).

Fig. 3- The Plant Species × Bacteria Species Interaction on Root Number (a) and Root Length (b)

Shoot Length:Inoculation with all bacteria strains (except B. pumilus) showed a significant increase in shoot length (Table 1). Stenotrophomonas sp (105%), A. chroococcum (81%) and R. meliloti (73%) were considered as the most effective strains (Table 1). B. pumilus decreased the shoot length (90%), significantly. Inoculation with all strains, except B. pumilus, increased the shoot length in barley, wheat and canola, significantly (Fig. 4). In sesame B. subtilis (41%) and B. pumilus (100%) the shoot length was reduced (Fig. 4). B. pumilus showed the most increasing effect on shoot length (104%) of wheat in comparison with control (Fig.4). In barley (213%) and canola (180%), the highest increase in shoot length was caused by Stenotrophomonas sp., whereas in sesame the most one (14%) was observed under A. chroococcum inoculation (Fig.4).

Fig. 4- The Plant Species × Bacteria Species Interaction on Shoot Length

Root Fresh and Dry Weight: The highest fresh weight (0.034 g) and dry weight (0.005 g.) was associated with wheat, whereas the least was related to canola (0.008 g) and sesame (0.001 g), respectively (Table 1). The most effective strains for the increase of root fresh weight and root dry weight was Stenotrophomonas sp (166%) and A. chroococcum (267%), respectively (Table 1). Inoculation with B. pumilus reduced root fresh weight in all plant species, compared to control (Fig. 5a). The highest increase in root fresh weight of wheat (202%), barley (336%) and canola (180%) were related to Stenotrophomonas sp. (Fig. 6a). The highest increase in root fresh weight of sesame (27%) was associated with A. chroococcum (Fig. 6a). Al the bacterial strains, except A. chroococcum, reduced the root fresh weight in sesame (Fig. 6a). This inhibitory effect could be compromised by the significant effect for plant species × bacteria strain interaction.

The bacteria strains showed different root dry weight in the studied plants (Fig. 6b). The highest increase of dry root weight in wheat (142%) was achieved under inoculation with B. subtilis, whereas, in barley (262%) and canola (20%) the most increase was obtained under inoculation with Stenotrophomonas sp. (Fig. 6b). All the bacteria species reduced the dry root weight in sesame, in comparison with control (Fig. 6b). The differences in water absorption capacity between species could be a reason for this result.

Shoot fresh and dry weight: Bacterial inoculation increased the shoot fresh weight, significantly (Table 1), but the highest shoot fresh weight (0.086 g) was obtained in barley (Table 1). Among different bacterial strains, the highest increase in shoot fresh weight (150%) was related to Stenotrophomonas sp, whereas the least one (90%) was associated with B. subtilis (Fig. 7a). The effect of inoculation on increased shoot fresh and dry weight was more in wheat and barley rather than sesame and canola (Fig. 6a). In wheat and barley, all strains, except B. pumilus, showed an increasing effect on the shoot fresh weigh (Table 1). The highest shoot dry weight was observed in barley (0.012 g) and wheat (0.007 g) (Table 1). Inoculation with all strains of bacteria showed the beneficial role of these rhizobacteria on increasing shoot dry weight.

The highest increase in shoot dry weight (312%) was observed under inoculation with R. meliloti (Fig 6a). The highest shoot dry weight in sesame (56%) was observed under inoculation with R. meliloti (Fig. 6a). Shoot dry weight showed a significant increase in all of the plant species under inoculation with B. pumilus (Fig. 6b). The highest increase in shoot dry weight of wheat (163%), barley (608%), canola (523%) and sesame (8%) was observed under elicitation with B. subtilis, R. meliloti, Stenotrophomonas sp and A. chroococcum, respectively (Fig. 6a). The bacterial strains had the least positive effect on shoot dry weight of sesame.

Fig. 5 (a, b)- The plant species × bacteria species interaction on root fresh (a) and dry (b) weight

Fig. 6 (a, b)- The Plant Species × Bacteria Species Interaction on Shoot Fresh Weight (a) and Shoot Dry Weight (b)