**2. Cultivation of soybean for a first time: In scope of inoculation view**

Cultural plants need considerable amount of macro and micro nutrients in mineral form to produce high quality of yield and these nutrients should be provided to correct yieldlimiting factors. Mineral and organic fertilizations are the pathways to enhance soil mineral nutrient budget. Due to the plant can only use mineral forms of nutrients, mineral fertilizers are readily available for the plants. Nutrient in organic fertilizers are in organic form that not readily available for the plants, thus the organic fertilizers have to be convert mineral form via the process called "mineralization".

Considerable amount of nitrogen is removed from soil when protein-rich grain or hay harvested, thus nitrogen is the most commonly deficient nutrient among macro and micro nutrients. Due to the nitrogen is the key component of healthy growing, all plants other than legumes should be fertilized by nitrogenous fertilizer. Legume plants are unique for their ability to fix nitrogen from atmosphere by symbiotic relationship with rhizobium bacteria. Rhizobia require a plant host therefore they cannot independently fix nitrogen. These bacteria located around root hair and fixing atmospheric nitrogen using particular enzyme called "nitrogenase". When this mutualistic symbiosis established, rhizobia use plant resources for their own reproduction whereas fix atmospheric nitrogen to meet nitrogen requirement of both itself and the host plants. Supply of nitrogen through biological nitrogen fixation has ecological and economical benefits. Farmers are not taking advantage of rhizobial inoculation to a number of reasons, thus they are passed up the potential of biological nitrogen fixation.

In many cases *Rhizobium spp.* might be existed in the soils, nevertheless, due to the insufficient number and activity (Gok and Onac, 1995), inoculation should be repeated. A number of studies indicate that no nodule formation appeared in the soybean roots if inoculated soybean isn't grown previously. Biren (2002) carried out the experiment to evaluate the effects of rhizobium inoculation in Turkish Republic of Northern Cyprus where soybean is not cultivated previously. He reported that there was no nodule formation in the non-inoculated control plants. Similarly, in Isparta where the soybean is not cultivated regularly there was no nodule occurrence (Coskan et al., 2009). In some circumstances it is possible to observe very limited number of infection even in first cultivation at non inoculated condition. Isler and Coskan (2009) reported that in the first cultivation in noninoculated condition there was a very few nodule formation in very light weight.

In scope of inoculation view, rhizobium inoculation should be realized with appropriate and healthy rhizobium strain in first cultivation of soybean plant and inoculation should be repeated every 2 to 3 year to sustain successful symbiotic nitrogen fixation. Depends on the rhizobium variety used, amount of nitrogen fixation greatly changed (Gok et al., 2001; Coskan et al., 2003). Thus, results obtained from local research should be considered in designating the effective strain.

inoculation of soybean by *Bradyrhizobium japonicum*, however, inoculation by irrigation water and seed bad inoculation methods are more effective according to nitrogen fixation parameters (Isler and Coskan, 2009). On the other hand organic compound such as fulvic and humic acids have stimulatory effect on soybean-rhizobium symbiosis (Coskan et al., 2010). Moreover, biological nitrogen fixation of soybean influenced by the number of factor such as pH, salinity, partial oxygen pressure, soil water content, ambient temperature as

Cultural plants need considerable amount of macro and micro nutrients in mineral form to produce high quality of yield and these nutrients should be provided to correct yieldlimiting factors. Mineral and organic fertilizations are the pathways to enhance soil mineral nutrient budget. Due to the plant can only use mineral forms of nutrients, mineral fertilizers are readily available for the plants. Nutrient in organic fertilizers are in organic form that not readily available for the plants, thus the organic fertilizers have to be convert mineral

Considerable amount of nitrogen is removed from soil when protein-rich grain or hay harvested, thus nitrogen is the most commonly deficient nutrient among macro and micro nutrients. Due to the nitrogen is the key component of healthy growing, all plants other than legumes should be fertilized by nitrogenous fertilizer. Legume plants are unique for their ability to fix nitrogen from atmosphere by symbiotic relationship with rhizobium bacteria. Rhizobia require a plant host therefore they cannot independently fix nitrogen. These bacteria located around root hair and fixing atmospheric nitrogen using particular enzyme called "nitrogenase". When this mutualistic symbiosis established, rhizobia use plant resources for their own reproduction whereas fix atmospheric nitrogen to meet nitrogen requirement of both itself and the host plants. Supply of nitrogen through biological nitrogen fixation has ecological and economical benefits. Farmers are not taking advantage of rhizobial inoculation to a number of reasons, thus they are passed up the potential of

In many cases *Rhizobium spp.* might be existed in the soils, nevertheless, due to the insufficient number and activity (Gok and Onac, 1995), inoculation should be repeated. A number of studies indicate that no nodule formation appeared in the soybean roots if inoculated soybean isn't grown previously. Biren (2002) carried out the experiment to evaluate the effects of rhizobium inoculation in Turkish Republic of Northern Cyprus where soybean is not cultivated previously. He reported that there was no nodule formation in the non-inoculated control plants. Similarly, in Isparta where the soybean is not cultivated regularly there was no nodule occurrence (Coskan et al., 2009). In some circumstances it is possible to observe very limited number of infection even in first cultivation at non inoculated condition. Isler and Coskan (2009) reported that in the first cultivation in non-

In scope of inoculation view, rhizobium inoculation should be realized with appropriate and healthy rhizobium strain in first cultivation of soybean plant and inoculation should be repeated every 2 to 3 year to sustain successful symbiotic nitrogen fixation. Depends on the rhizobium variety used, amount of nitrogen fixation greatly changed (Gok et al., 2001; Coskan et al., 2003). Thus, results obtained from local research should be considered in

inoculated condition there was a very few nodule formation in very light weight.

**2. Cultivation of soybean for a first time: In scope of inoculation view** 

well as soil mineral N content.

biological nitrogen fixation.

designating the effective strain.

form via the process called "mineralization".

#### **3. Effects of inoculation methods on fixation**

A number of inoculation methods are available, however, wetting the seeds by sugar, water and strain mixture or inoculation with peat culture are the most common methods in practise. Due to the rhizobium strain sensitive to the sunshine, inoculation and drying should be realized in indoor environment and seeds should also be protected from direct sunlight at sowing. Inoculation by wetting the seed methods has a number of disadvantages as follows: (1) Seeds are clinging to each other or to any surface of sowing equipment. Thus, farmers are abstained from inoculation to prevent time loss and extra workload. (2) The use of excessive water damages to the shell of the seed during inoculation therefore seeds become vulnerable to external conditions. Deaker et al. (2004) reported that seed inoculation method causes reduction of viable cell number when seed passes through machinery or lifting the seed coat out of the ground during germination. (3) Less amount of strain can be introduced to the seed especially in smaller seeds. Therefore, the higher amount of rhizobium bacteria per seed can be used in soil inoculation method compared to seed inoculation, especially for small seeded legumes (Brockwell, 1977).

Isler and Coskan (2009) tested the five different inoculation practises in pot experiment to evaluate the most effective method. They use the methods as follows: seed inoculation with sugar as an adhesive (SI), top inoculation with first irrigation (TI), two times top inoculation, one with first irrigation and one after germination (TTI), seed bad inoculation (SBI) and inoculation with peat culture-rhizobium suspension IWP). Result revealed that all practices other than control increased both number of nodule and nodule weight (Fig. 1). SI which commonly used inoculation technique was not effective as the other techniques tested. Observed nodule formation in TI proved that inoculants may reach rhizosphere area without any difficulties. Therefore inoculation with irrigation water may be used as an alternative inoculation technique considering the salt contents of the irrigation water. TTI application realized to compare with TI, however there wasn't statistical differences between TI and TTI.According to yield and the weight of seeds, SBI was the most effective inoculation techniques. Moreover SBI is the method that can easily adapt to sowing machinery with small changes.

Fig. 1. Effects of inoculation methods on nodule formation and nodule weight (SI: seed inoculation, TI: top inoculation, TTI: two times top inoculation, SBI: seed bad inoculation, IWP: inoculation with peat culture)

In this method seed is not directly contacted to the inoculants material, instead, seed located nearby or above the soil which rhizobia applied. Thus, the difficulties reported by Deaker et al. (2004) and Brockwell (1997) surmounted. Due to the inoculation material mostly stored in peat culture, inoculation with peat culture is another common inoculation method. But, if the peat culture dries out after inoculation, peat removed from seed and accumulate bottom of sowing machine (Gault 1978). Besides, when dry peat is wetted, great heat occurred which may reduce the number of viable rhizobia (Deaker 2004). Thus, in IWP application, water added to peat before adding the rhizobia to prevent high temperature occurrence, then this suspension applied to seed bed. Nodule count and nodule weight results revealed that the problem mentioned above is not realized and effective infection occurred in IWP. Results strongly indicate that, in the case of inoculants were not contact with seeds directly, the success of symbiotic relation increased. In general, SBI were the most effective methods among all methods tested. This method is also ripe for development of automated sowing machines.
