EFFECT OF AZOTOBACTER AND ARBUSCULAR MYCORRHIZAL ON GROWTH OF SAFFLOWER (CARTHAMUS TINCTORIUS L.) AT DIFFERENT IRRIGATION REGIMES

Jamal Shariati¹, Weria Weisany², Shahram Torabian^3
1 Faculty of Agriculture, Mohaghegh Ardabili University, Ardabil, Iran
² Young Researchers and Elite Club, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran
³ Department of Plant Ecophysiology, Faculty of Agriculture, Tabriz University, Tabriz, Iran

ABSTRACT

In order to study the effect of Azotobacter and Arbuscular Mycorrhizal fungi (AMF) on the growth characteristics of safflower (Carthamus tinctorius L.) in different irrigation regimes, an experiment was conducted in 2010 at the greenhouse of Agricultural Faculty of Mohaggegh Ardebili University of Ardebil, Iran. The experimental design was factorial based on randomized complete block design with three replications. Treatments comprised of four irrigation regimes (field capacity, 20, 40 and 60% of drought stress) and four biological fertilizers (None-inoculated, inoculation with AMF and Azotobacter and their mixture as a mix-inoculated). Growth, yield and chlorophyll content of safflower decreased with increasing water stress. Seeds inoculation by combination of Azotobacter and AMF, increased leaf chlorophyll content, root and shoot length, shoot and root dry and fresh weight, grain yield at all irrigation levels. The highest safflower yield was for mix-inoculated at no stress treatment by 2.4 g per palnts. According to the results, seeds inoculation can alleviate the negative effects of drought stress on safflower plants, especially mix-inoculated treatment.

Key words: Arbuscular Mycorrhizal fungi, chlorophyll content, Irrigation, safflower, yield.

INTRODUCTION

Safflower (Carthamus tinctorius L.) is a plant adapted to moderate drought climates with rather low rates of available water. Safflower was primarily cultivated for its pharmaceutical usages but nowadays it is cultivated to produce edible oil and seed [21]. The main advantages of this plant are high percentage of seed oil (25–40%) and its high quality (due to the presence of oleic acid and linoleic acid), resistance to abiotic stresses such as salinity, drought and chilling [22]. There are limited researches around the world on safflower production under irrigated conditions that revealed it is a sensitive crop to water [7, 29] and moderately tolerant to salinity.

Drought is undoubtedly one of the most important environmental stresses that limit the productivity of crops around the world [8, 9]. In Iran, water is a scarce resource due to the high variability of rainfall. The effects of water stress depend on the timing, duration and magnitude of the deficits [25]. Water stress influences plant growth at various levels such as the quantity and quality of plant growth. Some investigations are reported the effect of water deficit on yield and seed components of safflower [19, 20, 23].

Fertilizer of nitrogen (N) and phosphor (P) are crucial to the growth of all plants. However, uncontrolled use of chemical and pesticides can destroy environment. In addition, the prices of these chemicals are high. Accordingly, application of plant growth promoting rhizobacteria (PGPR) and symbiotic Arbuscular Mycorrhizal fungi (AMF) are considered which can perform the goals of sustainable agriculture. The successful association between plants and AMF is a strategy to improve the nutritional status of both, which reduces the use of fertilizers specially P [4]. Azotobacteris a free-living nitrogen fixing rhizobacteria that can promote the growth of various crops by some mechanisms such as production of gibberellic acid, indole-3 acetic acid (IAA) and cytokinin [26]. Peng et al. [27] also reported the positive effect of Azotobacter application on maize biomass. The mechanisms of PGPRs can be divided into direct and indirect ones. Direct mechanisms include N2 fixation, soil mineral solubilization, production of phytohormones (auxins, cytokinins or gibberellins) and decrease of ethylene levels. Indirect mechanisms include favoring colonization by other favorable microorganisms of soil, such as AMF, and suppressing the growth of plant pathogenic microorganisms [10, 18, 27].

Seed inoculation and the foliar spray of PGPR have been used for boosting plant growth and reducing the negative impacts of stress conditions [34]. The alleviating effect of AMF symbiosis under drought stress generally relies on the positive effects of AMF fungi on the uptake and transport of water and on an improved uptake of nutrients, resulting in the hydration of plant tissues, a sustain-able physiology and a clear promotion of growth [6]. The Mycorrhiza take carbohydrates compounds from their plant host, while the plants benefit from the association by the increasing nutrients uptake, which improve tolerance to abiotic stress (drought or salinity), as well as enhanced plant disease control [38]. Mycorrhiza symbiosis protects host plants against the detrimental effects of drought stress through mechanisms of drought avoidance [6, 33]. The results in several studies on drought stress conditions indicated that the plant biomass, chlorophyll contents and rate of transpiration were greater in plants inoculated with AMF compared with plants without AMF infection [5, 6, 8]. Symbiosis of AMF delayed leaf senescence of Alfalfa under drought conditions [14]. Dashadi et al. [12] reported that co-inoculation of Rhizobiumand Azotobacterincreased total nitrogen content, nodulation, seed yield and biological yield of faba bean under water deficit condition.

The objective of this study was to investigate the effect of Azotobacter and AMF symbiosis on the growth, chlorophyll and yield of safflower at different irrigation treatments.

MATERIALS AND METHODS

The experiment was conducted at the greenhouse of Agriculture Department of Mohagegh Ardebili University (latitude of 48°20′ N, and longitude of 38°05′ and 1350 m above sea level) in the period of May–July, 2010. This study was arranged as factorial, based on randomized complete block design with three replications. The certified seeds of safflower (cv. Goldasht) were obtained from Agricultural Research Center of Ardebil, Iran. Inoculation treatments included: Non-inoculated, inoculated with Arbuscular Mycorrhizal fungi (AMF) (Glomus hoi) and Azotobacter, and their mixture as a mix-inoculated. The G. hoi fungi were purchased as pure isolates from the Agricultural and Biotechnology Research Institute, Karaj, Iran. Before inoculation, the seeds surface was mixed with 15% sugar completely for more adhesion of inoculums. Seeds of safflower were washed with distilled water then inoculation was performed by a suspension of AM fungi and Azotobacter at the dose of 500 g per 100 kg^-1 seed in the darkness at 20–26°C. Finally, seeds were dried in the shade for 2 h. Details of soil properties are shown in Table 1. Ten seeds of safflower were sown in the plastic pot (25 cm diameter and 30 cm depth vol. 2.5 kg soil). Four seedlings were maintained in each pot one week after emergence. Plants in all treatments were left to grow for 7 weeks in a greenhouse at temperature 28±2°C and 60% relative humidity. Irrigation treatments were control (100% irrigation), 20% (80% of control), 40% (60% of control) and 60% (40% of control) water stress. The water content of the soil was kept at field capacity in the control treatment. Irrigation treatments were started 20 days after safflower planting. Leaf chlorophyll content was measured by Konica Minolta Sensing, Inc, SPAD- 502 every 15 days during the 5 stages. Plant roots were carefully removed from the soil and washed in tap water. Shoot and root samples were dried in oven at 75°C for 72 h and dry weights were measured. The yield of safflower was determined on the base of yield average of four plants and was expressed as gram per plant. The obtained data were subjected to an analysis of variance (ANOVA) with SAS 9.1 software (SAS Institute, Cary, NC, USA). Probabilities of significance (P≤0.01 or 0.05) were used to test the significance among the main treatment effects, treatment combinations and interactions. In addition, the Duncan’s multiple range test at a 0.05 probability level was used to compare the means.

RESULTS AND DISCUSSION

Data presented in Table 2 shown that length of stem and root, number of leaves, shoot and root fresh weight and dry weight, chlorophyll content and yield were significantly affected by water stress and inoculation and the their interaction effects (Tab. 2). Averaged over the inoculation, water stress significantly decreased growth parameters and yield of safflower. Additionally, safflower growth parameters and yield considerably improved by inoculation of seeds. The application of microorganisms increased the availability of nutrients, which had a positive impact on yield parameters [19]. Reddy et al. [31] evaluated the response of mulberry varieties to Azotobacter biofertilizers inoculation. The result revealed that the concentration of N, P, K and carbohydrate content increased in inoculated plants, which lead to improved yield. Performance of biofertilizers could be explained by the fixation of sufficient atmospheric nitrogen, production of plant growth promoters, decreasing the ethylene production in plants and solubilization of minerals such as phosphorus [18].

Length of stem
The result showed that stem length increased by inoculation with Azotobacter and AMF at different water regimes. Figure 1 showed that the highest of stem length was obtained to mix-inoculated at no stress; while the lowest was for none-inoculation at 60% stress. Increasing water stress considerably decreased positive effect of seeds inoculation (Fig. 1).

Length of root
According to result, the maximum length of root was observed in mix of Azotobacter and AMF at no water stress treatment (Fig. 1). The extent of root length increase by inoculation was at water stress treatments more than no water stress. These results were match with Al-Karaki et al. [3]. Improvement of growth parameters in inoculated seeds with mycorrhiza species under drought stress and no stress conditions can be related to ability of mycorrhiza in increasing the water absorption capacity of plant by increasing root hydraulic conductivity, the absorptive surface area of the root system, and access to small soil pores [6].

Shoot fresh and dry weight
The highest shoot fresh and dry weight of safflower were obtained in mix-inoculated treatment (Azotobacter and AMF) at no water stress; in contrast, the lowest was observed in none-inoculated treatment at 60% water stress (Fig. 2). It seems that the promotion of water absorbing and nutrients from soil caused to positive effects on growth and performance of safflower. In other research, Sharma [35] showed that the inoculated with the Glomus increased the efficiency of nitrogen and phosphorus and plant growth. Mycorrhizal soybean plants had significantly higher root and shoot dry weights than non-mycorrhizal plants at all moisture levels [2]. Improvement of growth and yield by seed inoculation with N2-fixing bacteria may be attributed to the high nitrogen uptake by the inoculated plants and the ability of bacterial strains to produce growth promoting substances [15].

Root fresh and dry weight
The highest root fresh and dry weight of safflower were observed in mix-inoculated at no water stress; in contrast, the lowest were recorded in none-inoculated at 60% water stress (Fig. 3). AMF inoculation had a significant effect on various leaf and root-growth parameters of peanut, especially under drought stress conditions [28].

Number of leaves
The Figure 4 showed that the maximum number of leaves of safflower was recorded in mix-inoculated at no water stress, whereas, the minimum belonged to none-inoculated treatment at 60% water stress. In severe drought conditions many plant for the survival, reduce their leaves to decreases transpiration.

Chlorophyll content
At all levels of water stress, mix-inoculation had higher chlorophyll content than inoculation of AMF and Azotobacter individually (Fig. 5). The highest of chlorophyll content was for mix-inoculated at no stress treatment; however, the lowest was for none-inoculated treatment at 60% water stress. The plant leaves chlorophyll content values were decreased in drought stress comparing with same treatment in well irrigation condition, that indicate to plant photosynthesis decreased in drought, which lead to inhibit some essential material for protein synthesis [17]. Rawia et al [30] showed that the increase of leaf area and chlorophyll content in inoculated plants with bacteria are due to enhance uptake of nitrogen of Celosia argentea L. plant. Results of Sohrabi et al. [37] demonstrated that inoculation of root pea with various species of Mycorrhiza fungi significantly increased leaves chlorophyll content under drought stress. Chlorophyll content of Zea mays L. that inoculated with Glomus mosseae significantly increased compared with untreated plant in normal irrigation or drought treatment [1].

Yield per plant
The Figure 6 showed that the highest yield of safflower belonged to mix-inoculated at no stress treatment by 2.4 g, which had not significant difference with mix-inoculated at 20 and 40% stress. In contrast, the lowest yield of plant was recorded in none-inoculated under 60% water stress by 0.2 g. This result indicated that the main role of biological fertilizer on safflower yield. The use of different species of AMF under drought conditions may increase of the roots absorption by enhancement of surface root area, which causes to increase of safflower biomass and yield. Ortas et al. [24] revealed that the application of AMF can increase plant growth rate by improving allocation and transfer of assimilate. This result was according to other researches by Dey et al. [13] and Shehata and EL-Khawas [36]. An increased N2 fixation associated with increased leaf area due to AMF inoculation might increase total number of peanut pods [28]. Idris [16] indicated that the inoculation of wheat seeds with Azetobacter increased weight of 1000 seed. Auge [6] reported that AMF plants were found to be larger than non-AM plants in about 80% of the AM studies on plant growth during drought. The studies of Bryla and Duniway [11] and Ruiz-Lozano and Azcon [32] have suggested that, under drought conditions, any increase in water uptake by fungal hyphae would play a vital role in increasing plant drought resistance through improving leaf water potential, maintaining turgor pressure, and increasing the net photosynthetic rate and stomatal conductance. The higher photosynthetic rates associated with AMF colonization can result in higher concentrations of soluble sugars and other photosynthetic byproducts in the leaf symplasm, which can manifest itself as an increased cytoplast osmolality in AM plants as against non-AM plants [6]. Azotobacter plays an important role in yield attributed characters owing to the production of siderophores which regulate the availability of nutrients to the crop [10].

CONCLUSION

The results showed that water stress significantly reduced growth characteristics, chlorophyll content and yield of safflower. In addition, use of biological fertilizer improved nutrient uptake and growth of safflower under water stress. Mix-inoculated treatment with AMF and Azotobacter had considerably positive effect on all stages of plant growth and improved the number of leaves, roots and stem length, which can increase rate of photosynthesis. Use of AMF and Azotobacter as a mix-inoculated treatment simultaneously decreased the negative effects of drought more than other inoculated treatments, although inoculation of seed with AMF and Azotobacter singly was better than none-inoculated treatment in all water regimes.

Accepted for print: 3.10.2015

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