غربال‏ گری باکتری‌های ریزوسفر یونجه از لحاظ صفات محرک رشدی گیاه

Introduction

Many microorganisms live in ‘rhizosphere’ as the part of soil modified or influenced by plant roots (1). Some of them such as Azospirillum, Azotobacter Pseudomonas and several gram positive Bacilli have positive effects on plants and therefore, are called plant growth promoting rhizobacteria (PGPR) (1). In various cropping systems, PGPR are directly or indirectly involved in increasing plant growth, as well as inducing resistance to pathogens and pests (2). The direct promotion of plant growth by PGPR can be either by providing the bacterial synthesized compound or by facilitating the uptake of certain nutrients from the environment. Some of direct effects include providing the host plant with i) fixed nitrogen, ii) soil phosphorus and iron solubilization iii) bacterial synthesized phytohormones such as auxins, cytokinins and gibberelins (3). The indirect promotion of plant growth occurs when PGPR lessen or prevent the deleterious effects of one or more phytopathogenic organisms. It was suggested that soil bacteria that indirectly stimulate plant growth be referred to as biocontrol- PGPB or biocontrol plant growth- promoting bacteria versus directly plant growth promoting bacteria as PGPB (4).In fact, there is a complex interaction between plant roots and soil microbes (5). So a part of photosynthesis products were released from root to soil, which were defined as rhizodeposition (6). It provides nutrients for soil microorganisms and is the main factor of rhizosphere creation and also plays a vital role in activity, population and the relative composition of species (7).

 Detection of bacteria other than rhizobia in root nodules was first reported by Beijerinck and Van Delden in the clover plants to be as Agrobacterium radiobacter (8). Manninger and Antal also detected rhizobia and other bacteria in the root nodules of legumes (9). Many studies have shown that simultaneous infection with rhizobia and the other rhizospheric bacteria increase nodulation and growth in a wide variety of legumes (10- 12). Co- inoculation of chickpea (Cicer arietinum) with some Pseudomonas and Bacillus strains along with effective Rhizobium spp. stimulated plantgrowth, nodulation and nitrogen fixation (13).

Production of plant growth regulators such as auxin, cytokinin and gibberellin by PGPR has also been suggested as a possible mechanism to promote plant growth and development (14). As some bacteria, such as Pseudomonas can stimulate plant growth by the production of indole- 3- acetic acid (IAA) (15- 17). In this regard, Patten and Glick showed that the production of IAA by Pseudomonas. putida was closely linked to optimal development of the root system in canola (Brassica napus L.) and mung bean (Vigna radiata) (18). As previous studies showed, rice is on of the most important stable crops in the developing world, and needs nitrogen as the most important essential nutrition (19). To make rice production sustainable and less dependent on nitrogen fertilizer, it is extremely important to know how to use PGPR fixing nitrogen and producing substances, as indole- 3- acetic acid and siderophores to improve rice growth (17).
To evaluate the efficacy of plant growth promoting bacteria (Azospirillum sp.) on canola crop, an experiment was conducted. Results indicated that Azospirillum sp. had positive effects on yield and yield components of oilseed rapes (20).

Azotobacter is a plant growth promoting rhizobacterium that can stimulate the growth of plants by different mechanisms. 10 isolates of Azotobacter chroococcum native to Iranian soils, were used for field experiments. In Azerbaijan Sharghi, 9 isolates increased the total wheat yield, in Khorasan Razavi only one isolate (T11) increased yield of straw and stubble. In Fars, T8 isolate had the highest seed yield and shoot dry weight. There was not significant effect of inoculation on growth indices of wheat in Kordestan and Azerbaijan gharbi provinces (21). Pseudomonas fluorescens is the most important plant growth promoting rhizobacteria. Thirty four Pseudomonas fluorescens isolates were isolated from pistachio orchards located in Rafsanjan. The PGP of all isolates were evaluated. Inoculation of pistachio seedlings with superior isolates increased the seedlings height, leaf area, shoot and root dry weights and concentrations of P, Fe, Zn, Mn and Cu in shoot significantly (22).

Enhancement of legume nitrogen- fixation by co- inoculation of rhizobia and plant growth promoting bacteria (PGPB) is important for improving nitrogen availability in sustainable agricultural systems. The aim of this study was to evaluate PGP activities of bacteria isolated from rhizospheric soil and plant root nodules of alfalfa (M. sativa) in Zanjan province.

Materials and methods

In order to isolate bacterial strains, rhizospheric soil and plant root nodules, samples were firstly collected from alfalfa (M. sativa) cultivated around Zanjan province. Then serial dilutions of samples were prepared. Dilutions were inoculated on plates which contain nutrient agar media. Inoculated plates were incubated at 28^oC for one week. Grown colonies were transfered to new plates containing Nutrient Agar (NA) media. After several subculturing, finally pure isolates were obtained. Isolates tested in the present study were grown on Nutrient Agar medium for their viability and maintained in Nutrient Broth (NB) medium with 15% glycerol at -80ºC for long- term storage. From the isolated bacterial strains, Bv1 and Bv2 were Pseudomonas and the other bacterial strains from Rm1 to Rm45 were Rhizobium. It should be noted that all measurements had 3 replications.

Assay for IAA production:IAA production was detected by the method described by Pattern and Glick (18). Each bacterial isolate was first grown in TSB (Trypton Soy Broth) medium (dextrose, 2.5 g; K₂HPO₄, 5 g; NaCl, 2.5 g; soybean peptone, 3 g; tryptone, 17 g; distilled water, 1 L; pH=7.2) for 48 h. Bacteria were then grown for 72 h on TSB medium amended with 25 µg/ml of L- tryptophane at about 28ºC. Fully grown cultures were centrifuged at 10000 g for 2 min. The culture supernatant (1 ml) was mixed with 4 ml of Salkowski reagent (concentrated sulfuric acid 150 g; distilled water 250 ml; 0.5 M FeCl₃.6H₂O 7.5 ml). Tubes containing the mixture were standed for 20 min, for color development. Optical density was read at 535 nm using spectrophotometer (Beckman DU 530). Concentration of IAA was measured with the help of standard graph of IAA obtained in the range of 0- 6 mg/ml. (18).

ACC- deaminase production: Production of ACC deaminase was determined as described by Amico et al. (23). Bacterial strains producing ACC- deaminase, use amino cyclopropane-1 carboxylic acid (ACC) as a nitrogen source. In this method 50 ml of fresh bacterial suspension was added to 20 ml DF (glucose, 2g; gluconic acid, 2g; citric acid, 2g; Na₂HPO₄, 6g; KH₂PO₄, 4g; ZnSO₄.7H₂O, 0.124mg; MnSO₄.7H₂O, 0.10mg; H₃BO₃, 0.10mg; FeSO₄.7H₂O, 1 mg; MgSO₄.7H₂O, 0.01mg; ; CuSO₄.7H₂O, 0.078mg; MoO₃, 0.01mg) medium containing 3 mM ACC, DF medium containing ammonium sulfate and DF medium without ACC and ammonium sulfate. After 48h, light absorbance was measured at 405 nm wave length by spectrophotometer in three mentioned media. The ratio of absorbance in ACC containing DF medium to absorbance of ammonium sulfate containing DF medium considered as ACC- deaminase production index.

HCN production: All bacterial strains were screened for the production of hydrogen cyanide (HCN) using the method described by Donate- correa et al. (24). At first each bacterial strain was cultured in TSA medium (peptone from casein 15 g; peptone from soymeal 5 g; sodium chloride 5 g; agar 15 g; distilled water 1000 ml) and glycine was added as precursor of hydrogen cyanide. Whatman filter papers no.1 soaked in sodium picrate solution (picric acid 0.5%; sodium carbonate 2%) was placed in the top of the plate and then plates were sealed with Parafilm and incubated at 28°C for 120 h. Development of light brown (low HCN production) to dark brown color (high HCN production) indicated production of hydrogen cyanide.

Siderophore production: Bacterial isolates were assayed for siderophore production on the Chrome azurol S agar medium as described by Alexander and Zuberer (15). For this medium, four distinct solutions (Fe- CAS [Chrome Azurol S] indicator solution; buffer solution; nutrient solution.

Casaamino acid solution: Casamino acids are a mixture of amino acids and some very small peptides obtained from acid hydrolysis of casein) were prepared separately, sterilized and then mixed with each other. Fresh cultures were plated onto blue- agar CAS plates, buffered with 0.1 M piperazin-1, 4-bisethanesulfonic acid (PIPES). In this media 10 mM FeCl₃ is complexed by 10 Mm chrome azurol S in the presence of 0.2 mM hexadecyltrimethylammonium bromide (HDTMA). Chrome azurol S agar plates were prepared and divided into equal sectors and spot inoculated with isolates and incubated at 28°C for 48 h. Development of orange halos around colonies was determined and considered as an ability of siderophore production.

.Chitinase production: Chitinase production was determined as described by Boller and Mauch (25). Bacterial suspension was spot inoculated on nutrient agar plates which amended with 0.1% colloidal chitin and zone of clearance was recorded after incubation at 28°C for 120 h.

.Mineral phosphate solublization: Bacterial isolates previously grown in TSB medium were dropped into the basal Sperber (26) medium (glucose, 10 g; yeast extract, 0.5 g; MgSO₄.7H₂O, 0.32 g; CaCl₂, 0.14 g; Ca₃(PO₄)₂, 2.5 g; Agar, 15 g; distilled water, 1000 ml; pH=7.2) and incubated for 7 days at 28°C. Those isolates that formed visible clearing halos zone around their colonies were considered phosphate solubilizers, so their ratio of halo zone diameter to colony diameter was recorded.Quantitative analysis of mineral phosphate solubilization, in Sperber broth medium was made as described by Bano and Musarrat (3). The isolates were inoculated in 50 ml Sperber broth medium and shacked (125 rpm) for 120 h at 28°C. After this period, pH was measured and also bacterial suspensions were centrifuged at 10000 g for 10 min. 1ml of culture supernatant was placed into test tubes containing 3ml of distilled water, 1 ml ammonium molibdate- vanadate indicator and 1 ml of 1.5% ascorbic acid solution. The tubes containing the mixture were allowed to stand for 20min, for color development. The absorbance of yellow color was read at 470 nm using spectrophotometer (Beckman DU 530). The amount of soluble phosphate was measured from the standard curve of KH₂PO₄.
.Organic phosphate solubilization: Quantitative and qualitative methods which were used to evaluate bacterial isolates capability to solubilize organic phosphate were same as methods described for mineral phosphate, except that Inositol hexaphosphoric acid was used instead of Ca₃(PO₄)₂ (26).

Results

In this study, 47 bacterial isolates were studied for their PGP activities. On the basis of morphological and biochemical characteristics, two bacterial isolates were grouped into Pseudomonas fluorescent and they were shown as Bv1 and Bv2. A total of 45 nodule isolates of alfalfa were characterized as Sinorhizobium and they were shown as Rm1 to Rm45 (Table 1).

.Quantitative assay of IAA production: The results of this study showed that 91% of bacterial isolates (43 isolates) had the ability of IAA production. Mean amount of auxin production was 4.29 μg/ml and the range of production was between 4.04 μg/ml and 4.95 μg/ml. From the bacterial isolates, Rm4 produced the highest amount of IAA (4.95μg/ml) and Rm33, Rm41, Rm42, Bv2 and, Rm3 produced lowest amount (4.04 μg/ml), while Rm16, Rm20, Rm39 and Rm44 didn’t produce any IAA (Table 1).

Assay of siderophore production: Only 9 bacterial isolates of total 47 isolates (19%) produced visible orange halo zones around the colonies, showing that they were able to produce siderophore. The ratio of halo zone diameter to colony diameter ranged between 1.28 and 2. The lowest and highest ability of siderophore production (ratio of halo zone diameter to colony diameter) belonged to isolates Rm7and Rm43 respectively (Table 1).

.Assay of ACC- deaminase production: ACC- deaminase production was detected in 15 isolates and 32% out of total bacterial isolates were able to produce this enzyme. The range of production was 0.16- 1.05 μg/ml. Isolate Rm42 produced the lowest amount, while Rm40 produced the highest amount of ACC- deaminase (Table 1).

HCN production: The results of this study showed that HCN production ability in 24 isolates (51%) was weak, in 20 isolates (42%) was medium, in 2 isolates (4%) was high and in only one isolate (2%) (Rm42) was very high and the color filter paper changed to dark brown (Table 1).

Chitinase activity: The results of investigation showed that none of the bacterial isolates were able to produce chitinase, so all of them were known as chitinase negative isolates (Table 1).

Phosphate solubilization: The results of this study showed that 19 isolates (40%), were able to solubilize mineral phosphate. The highest amount of this ability belonged to Rm42 (96.41 μg/ml) and the lowest amount belonged to Rm15 (21.86 μg/ml). When a strain was active in solubilizing mineral phosphate, as a consequence of this effect, pH medium decreased more. Isolate Bv1 decreased the pH value of medium from 7.2 to 4.33 and was the most active strain in this way. Among the total bacterial isolates, 15 strains (32%) were able to solubilize organic phosphate (Table 2).

Thus it is possible that decreasing media pH might be due to mineral phosphate solublization activities (organic acids production) of bacterial isolates, and the relationship between Ca₃ (PO₄)₂ solublization (μg/ml) and pH can be shown as Fig. 1. Most of the isolates had the ability of auxin production (more than 90%) while none of them produced chitinase enzyme (Fig. 2).

 Fig. 1- Correlation between phosphate solublization (μg/ml) and pH decreasing

Fig. 2- Percentage (%) of tested isolates that had different PGP activities

Table 1- Production of IAA, siderophore, HCN, ACC- deaminase and chitinase by selected bacterial isolates*

* It should be noted that all measurements had 3 replications

** The ratio of orange halo zones diameter to colony diameter in CAS- agar medium

*** The color of filter paper in the test for 1, 2, 3, and 4 was yellow, cream, light brown, and dark brown respectively.

Table 2- Mineral and organic phosphate solubilization activities among selected bacterial isolates*

* It should be noted that all measurements had 3 replications.