**4. Effects of different tillage system**

Biological N2 fixation (BNF) was effected by different tillage system including agricultural practices, pesticides applications, addition of organic material, residue chopping. The ways in which these operations are implemented affect the physical and chemical properties of the soil, which in turn affect soil microorganisms as BNF bacteria.

The amount of nitrogen actually fixed by a legume depends not only on the genetics of the bacteria and host plant but also on the environment and agricultural practices. Among the common agricultural practices, fertilization with P and N has important effects in nitrogen fixation. It is a well-established fact that, when legumes are grown in soils high in available nitrogen, the nitrogen fixation rate is reduced.

According to different research, by definition, biological N2 fixation (BNF) is synonymous with sustainability. Advances in agricultural sustainability will require an increase in the utilization of BNF as a major source of nitrogen for plants. The process of BNF offers an economically attractive and ecologically sound means of reducing external nitrogen input and improving the quality and quantity of internal resources.

Soil tillage methods have complex effects on physical, chemical and biological properties of soil. Because of the changing physical and chemical properties of soil by soil tillage methods, the biological properties of soil may also change. Actually these changes are indirect results of tillage. Changed physical and chemical soil properties by soil tillage methods effect the parameters directly related with soil microbial activities such as organic matter, soil humidity, temperature and ventilation as well as the degrees of interaction between soil mineral and organic matter. As a result of these effects, significant differences can be observed in the population of microbial activities in soil (Kladivko, 2001; Lavelle, 2000; Wardle 1995; Saggar et al. 2001).

Plant and microorganism interactions in rhizosfer region are very important for plant growth. In the rhizosphere region, rhizobial activities occur as reciprocal and compulsory interactions (symbiosis) of plant-microorganism (Altieri, 2000; Garcia and Altieri, 2005). One of the important activities related to soil qualities is beneficial microorganism activities. The most important of these activities is a root nodule bacterium which provides to biological N2-fixation (Ferreira et al., 2000).

Microorganisms, that are important parts of the nature, are considerably affected by the environmental conditions. These organisms which rapidly reproduce and function in proper

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.

Biological N2 fixation (BNF) was effected by different tillage system including agricultural practices, pesticides applications, addition of organic material, residue chopping. The ways in which these operations are implemented affect the physical and chemical properties of

The amount of nitrogen actually fixed by a legume depends not only on the genetics of the bacteria and host plant but also on the environment and agricultural practices. Among the common agricultural practices, fertilization with P and N has important effects in nitrogen fixation. It is a well-established fact that, when legumes are grown in soils high in available

According to different research, by definition, biological N2 fixation (BNF) is synonymous with sustainability. Advances in agricultural sustainability will require an increase in the utilization of BNF as a major source of nitrogen for plants. The process of BNF offers an economically attractive and ecologically sound means of reducing external nitrogen input

Soil tillage methods have complex effects on physical, chemical and biological properties of soil. Because of the changing physical and chemical properties of soil by soil tillage methods, the biological properties of soil may also change. Actually these changes are indirect results of tillage. Changed physical and chemical soil properties by soil tillage methods effect the parameters directly related with soil microbial activities such as organic matter, soil humidity, temperature and ventilation as well as the degrees of interaction between soil mineral and organic matter. As a result of these effects, significant differences can be observed in the population of microbial activities in soil (Kladivko, 2001; Lavelle,

Plant and microorganism interactions in rhizosfer region are very important for plant growth. In the rhizosphere region, rhizobial activities occur as reciprocal and compulsory interactions (symbiosis) of plant-microorganism (Altieri, 2000; Garcia and Altieri, 2005). One of the important activities related to soil qualities is beneficial microorganism activities. The most important of these activities is a root nodule bacterium which provides to biological

Microorganisms, that are important parts of the nature, are considerably affected by the environmental conditions. These organisms which rapidly reproduce and function in proper

**4. Effects of different tillage system** 

nitrogen, the nitrogen fixation rate is reduced.

2000; Wardle 1995; Saggar et al. 2001).

N2-fixation (Ferreira et al., 2000).

the soil, which in turn affect soil microorganisms as BNF bacteria.

and improving the quality and quantity of internal resources.

environmental conditions, also struggle to continue their functions under poor conditions (Doğan et al., 2007).

As a result of symbiotic N2–fixation, legumes supply nitrogen to the soil not only with their nodules, but also by decomposition of their roots and shoots. Nitrogen might have formed by mixing the separated dead nodule tissues into the soil. This situation can be accelerated by cutting of the plant's shoots (Werner, 1987; Goormachting et al., 2004).

In a study of Dogan et al. (2011), the effects of six different soil tillage methods (Table 1) on some parameters related with nitrogen fixation have been investigated. According to the findings of the research in the No-Tillage with Direct Seeding (NTDS) plots, root weights (6.9 g/plant), number of nodules (96 number/plant), weight of nodules (0.318 g/plant) and root nitrogen content (% 0.71) are found to be statistically higher than with the other tillage applications. In the Reduced tillage with rotary tiller (RTR) plots, the values of up-root dry weight (51.3 g/plant), mean nodule weight (3.91 mg/nodule), root N content (2.38%), are found higher on the lands than in NTDS plots.


Table 1. Soil tillage methods in the major and secondary crop (soybean) production (Dogan et al., 2011)

Among the applications, in the plots of Reduced Tillage (RTHD and RTR) rhizobial nitrogen fixation parameters have been found considerably higher compared with the other applications (Fig. 2). However, some soil tillage methods used in this study negatively affected some soil parameters. For the Reduced Tillage with Rotary tiller (RTR) plots the dry root weight (4,8 g/plant), up-root weight (35,7 g/ plant) and root N content (% 0,68) values and for the Conventional Tillage with Burnt Residue (CTBR) plots, number of nodules and weight of nodule values were found to be lower than in the other tillage applications. The values of dry nodule weights, like in Conventional Tillage with Burnt Residue (CTBR) were low in the plots of Conventional Tillage with Residue (CTR) and Reduced Tillage with Heavy Disking (RTHD) with the values 0,071 and 0,088 g/plant, respectively. Besides, the lowest mean nodule weights (2,06 mg/nodule) have been observed in Conventional Tillage with Residue (CTR) plots and the lowest up root N content (%1,98) have been observed in Reduced Tillage with Heavy Disking (RTHD) plots. The results of the study have been showed that, parameters of nitrogen rhizobial fixation has been affected negatively by the conventional tillage methods in which 3-5 tillage operations are applied and soil is disturbed . There were differences among the tillage methods and these differences were found to be statistically significant. In general, the best results related with rhizobial activity have been obtained with No-Tillage with Direct Seeding (NTDS) and No-Tillage with Heavy Disking (NTHD). However, other soil tillage methods decreased the nitrogen fixation (Dogan et al., 2011). Similar studies have also showed that zero and reduced soil tillage methods have increased the soil microbial activity and population (Ferreria, 2000; Alvarez et al., 1995; Gassen and Gassen 1996).

Fig. 2. The effects of different soil tillage methods on number of nodule in secondary crop soybean plant (CTR: Conventional Tillage with Residue, CTBR: Conventional Tillage with Burnt Residue, RTHD: Reduced Tillage with Heavy Disking, RTR: Reduced tillage with rotary tiller, NTHD: No-Tillage with Heavy Disking, NTDS No-Tillage with Direct Seeding)

Generally, soil microbial activity is affected negatively by soil tillage (Jinbo et al., 2007; Kladivko, 2001; Hussain et al., 1999; Saggar et al., 2001). Therefore rhizobial activity is also be affected negatively by soil tillage (Hassen et al., 2007; Ferriera et al., 2000). Soil organic matter decreased by soil tillage operations is also important for the vital activities of soil microorganisms. The decrease of organic matter in the soil can also cause decreases in soil microbial activity (Saggar et al., 2001; Eliot el al., 1984). As it can be seen in the similar studies, the effects of soil tillage methods may differ depending on climate, regional, and

dry root weight (4,8 g/plant), up-root weight (35,7 g/ plant) and root N content (% 0,68) values and for the Conventional Tillage with Burnt Residue (CTBR) plots, number of nodules and weight of nodule values were found to be lower than in the other tillage applications. The values of dry nodule weights, like in Conventional Tillage with Burnt Residue (CTBR) were low in the plots of Conventional Tillage with Residue (CTR) and Reduced Tillage with Heavy Disking (RTHD) with the values 0,071 and 0,088 g/plant, respectively. Besides, the lowest mean nodule weights (2,06 mg/nodule) have been observed in Conventional Tillage with Residue (CTR) plots and the lowest up root N content (%1,98) have been observed in Reduced Tillage with Heavy Disking (RTHD) plots. The results of the study have been showed that, parameters of nitrogen rhizobial fixation has been affected negatively by the conventional tillage methods in which 3-5 tillage operations are applied and soil is disturbed . There were differences among the tillage methods and these differences were found to be statistically significant. In general, the best results related with rhizobial activity have been obtained with No-Tillage with Direct Seeding (NTDS) and No-Tillage with Heavy Disking (NTHD). However, other soil tillage methods decreased the nitrogen fixation (Dogan et al., 2011). Similar studies have also showed that zero and reduced soil tillage methods have increased the soil microbial activity

and population (Ferreria, 2000; Alvarez et al., 1995; Gassen and Gassen 1996).

Fig. 2. The effects of different soil tillage methods on number of nodule in secondary crop soybean plant (CTR: Conventional Tillage with Residue, CTBR: Conventional Tillage with Burnt Residue, RTHD: Reduced Tillage with Heavy Disking, RTR: Reduced tillage with rotary tiller, NTHD: No-Tillage with Heavy Disking, NTDS No-Tillage with Direct Seeding) Generally, soil microbial activity is affected negatively by soil tillage (Jinbo et al., 2007; Kladivko, 2001; Hussain et al., 1999; Saggar et al., 2001). Therefore rhizobial activity is also be affected negatively by soil tillage (Hassen et al., 2007; Ferriera et al., 2000). Soil organic matter decreased by soil tillage operations is also important for the vital activities of soil microorganisms. The decrease of organic matter in the soil can also cause decreases in soil microbial activity (Saggar et al., 2001; Eliot el al., 1984). As it can be seen in the similar studies, the effects of soil tillage methods may differ depending on climate, regional, and

CTR CTBR RTHD RTR NTHD NTDS

0

20

40

60

Number of nodule.plant-1

80

100

120

environmental factors. These factors must be taken into consideration before applying tillage methods. Otherwise, biological activity, fertility and sustainability of soil will be destroyed.

On high-input farms, microorganisms are generally thought to play a minor role in soil fertility because most nutrients in inorganic fertilizers are readily available for the plants and do not require degradation or mineralization (Smith et al., 2001).

Many studies have concluded that herbicides affect nitrogen fixation largely via indirect effects on plant growth and consequent availability of photosynthate to the root nodules (Wally et al., 2006; Abd-Alla et al. 2000); there is evidence that some pesticides might impair the ability of the rhizobia to recognize appropriate host plants. As a consequence, early nodulation events can be disrupted. However, according to their research, not all pesticides had a negative impact on nodulation and the degree to which nodulation was inhibited was dependant on pesticide concentrations. In some instances, results from various studies have been contradictory. For example, when examining the effects of chlorsulfuron under laboratory conditions, Anderson et al. (2004) observed that even at rates equivalent to two times field rates, chlorsulfuron did not influence rhizobial growth. However, although rhizobial growth was not influenced, the subsequent ability of these rhizobia to form nodules was reduced. Thus, they reported that when rhizobia were exposed to relatively high levels of chlorsulfuron, subsequent nodule size and total nitrogen fixation was reduced. In contrast, Martensson (1992) reported that nodulation ability was unaffected by previous exposure to chlorsulfuron. These contrasting results suggest that the impact of various herbicides on specific nodulation events may be highly dependent on specific environmental conditions, including different soil characteristics (i.e., pH, organic matter, moisture, etc.) and weather conditions. Martensson (1992) examined the impact of various herbicides on root hair formation. Rhizobia infect plant roots through root hairs and thus it was hypothesized that herbicides affecting root hair development might interfere with nodulation. Author reported that some herbicides, including glyphosate, caused root hair deformations that apparently resulted in fewer nodules being formed. It is important to note, however, that this was a laboratory study and consequently the herbicide rates used in these experiments were not necessarily similar to rates that would be encountered in soils under field conditions. Thus, although the research demonstrates the possibility for herbicides to affect nodulation via root hair deformations, it is not known if this phenomenon occurs under field conditions (Walley et al. 2006).

Saggar et al. (2001) studied the effect of cultivation on soil organic C, functional chemical composition of SOM, and soil structure in soils of contrasting mineralogy. They found that soil susceptibility to structural degradation increased with years of cultivation, and from light textured to heavier textured soils. Because cultivation causes profound changes in the soil physical and chemical properties, and populations of microfauna and macrofauna, it is relevant to quantify its effects on soil microbial and microfaunal populations and on SOM dynamics.

#### **5. Mycorhiza-rhizobium interaction in light limited condition**

Vesicular Arbuscular Mycorrhiza (VAM) is symbiotically living organism with many crops and they enhance plant P uptake along with other micronutrients especially Zinc. Phosphorus efficiency in highly limy soil (in high pH) is considerably low whereas mycorrhiza assists plant to receive that immobile phosphorus by exudates and/or enhancing soil contact area. As mentioned previously, rhizobium is a microorganism, capable of fixing aerial nitrogen (N2) to soil/plant via symbiotic relations with legumes. Both organisms utilize the photosynthesis products that assimilated by host plants to survive. In non-limiting conditions those organism supports the plants for the most important macro and micro nutrients as N, P, Fe and Zn. On the other hand unsuitable soil or climatic conditions in growth season may result negative Rhizobium x Mycorrhiza interactions. Although both microorganisms use organic compounds formed by plants, they use trace amount of that compound compared to the plant biomass formation. Coskan et al. (2003) carried out the pot experiment to evaluate cross interactions of rhizobium and mycorrhiza in light limited condition. Results revealed that no nodule formation appeared in non-rhizobia-inoculated control variant whereas rhizobial inoculation increased number of nodule (Fig. 3) while decreased biomass weight (Fig. 4).

Fig. 3. Nodule formation (left) and mycorrhizal infection rate (right) in the light limited condition

Fig. 4. Effect of dual inoculation on root and shoot dry weight of soybean in light limited condition

capable of fixing aerial nitrogen (N2) to soil/plant via symbiotic relations with legumes. Both organisms utilize the photosynthesis products that assimilated by host plants to survive. In non-limiting conditions those organism supports the plants for the most important macro and micro nutrients as N, P, Fe and Zn. On the other hand unsuitable soil or climatic conditions in growth season may result negative Rhizobium x Mycorrhiza interactions. Although both microorganisms use organic compounds formed by plants, they use trace amount of that compound compared to the plant biomass formation. Coskan et al. (2003) carried out the pot experiment to evaluate cross interactions of rhizobium and mycorrhiza in light limited condition. Results revealed that no nodule formation appeared in non-rhizobia-inoculated control variant whereas rhizobial inoculation increased number

Fig. 3. Nodule formation (left) and mycorrhizal infection rate (right) in the light limited

%

Shoot dry matter Root dry matter


Infection rate

Fig. 4. Effect of dual inoculation on root and shoot dry weight of soybean in light limited



of nodule (Fig. 3) while decreased biomass weight (Fig. 4).

Number of nodule


0.0

0.5

1.0

1.5

g plant-1

2.0

2.5

3.0

condition

number of nodule per plant

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

condition

Although both of the rhizobium strains give rise to nodule formation, *B. japonicum* 1809 strain cause considerably higher nodule number. On the other hand, mycorrhiza inoculation increased the infection rate (Fig 3). Rhizobial inoculation decreased mycorrhizal infection rate in both with mycorrhiza and without mycorrhiza applications. Bacterial inoculation has no significant effect on plant growth except nodule formation. It is clearly seen that both rhizpbium and mycorrhiza applications reduced total plant dry weight. However, plant dry weight and phonological observations revealed that plant development is adversely effected due to mycorrhizal inoculation in light-limited growing session.
