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1. Res. on Crops 12 (2) : 471-478 (2011) With one figure Printed in India Effect of chemical and biological soil amendments on production and soil seed bank of annual medic (Medicago scutellata cv. Robinson) G. SHABANI*, M. R. CHAICHI1, M. R. ARDAKANI, J. K. FRIEDEL2, K. KHAVAZI3 AND H. R. ESHGHIZADEH4 Agriculture Research Center Karaj Branch, Islamic Azad University, Karaj , Iran *(e-mail : bb1379@yahoo.com)(Received : February, 2011) ABSTRACT In order to study the effect of different fertilizing systems consisting of control (no fertilizer), chemical, biological and its combination fertilizer on forage yield and conservation of soil seed bank of annual medic (Medicago scutellata cv. Robinson), an experiment was conducted based on a complete randomized block design with three replications in two locations under dry farming conditions in Kermanshah province, Iran, in 2009. The results showed that the highest amount of cumulative biomass of 4185 kg/ha was produced in urea fertilizer+phosphorus solubilizing bacteria+mycorrhiza treatment, and the lowest amount (3183 kg/ha) was observed in nitrogen fixing bacteria+mycorrhiza treatment, respectively. The number of pods in soil seed bank was significantly influenced by different fertilizing systems. The highest number of 314.6 pods per square metre was found in nitrogen fixing bacteria+phosphorus solubilizing bacteria treatment and the lowest of 139.8 pods per square metre in control treatment, respectively. Also the highest yield of pods (1147.2 kg/ha) was obtained in nitrogen fixing bacteria+phosphorus solubilizing bacteria application, while the lowest pod yield of 446 kg/ha was found in control treatment. Key words : Bio-fertilizers, medic, Medicago scutellata cv. Robinson, mycorrhiza, nitrogen fixation, solubilizing bacteria INTRODUCTION Efficient management and farming practices in the framework of a series of agricultural activities can increase agricultural production in the regions with limited rainfall (Ryan, 2008). Ley-farming system, in which a grain crop and annual medic pasture alternate each other in a short term rotation, is one of the popular agricultural systems to fulfill food production in dry regions (Oram, 1990). In rotation with other agricultural crops, annual medics have high water use efficiency and this characteristic makes them a suitable crop in rotation with wheat in dry climatic condition (Weston et al., 1996). In this experiment, application of nitrogen fixing bacteria+phosphorus solubilizing bacteria treatment led to sufficient seed production and rich soil seed bank ensuring natural regeneration. Plants in T , T , T and T treatments with 314.6, 278.8, 260 and 253.4 pods per square metre, respectively, were able to complete their physiological growth period soon after the first harvest at the beginning stage of flowering. This phenomenon could help the plant to produce more seed to support soil seed bank which makes these treatments more suitable for application in Ley-farming system. Similarly to this result, Wasule et al. (2002) in a field experiment on soybean indicated that there was a positive and synergistic interaction effect between Bradyrhizobium bacteria and phosphorus solubilizing bacteria (Pseudomonas putida). Pod Yield Pod yield was significantly affected by fertilizing systems but interaction with local condition was not significant (Table 3). The highest yield of pods (1147.2 kg/ha) was obtained in nitrogen fixing bacteria+ phosphorus solubilizing bacteria application, while the lowest pod yield of 446 kg/ha was found in control treatment (Table 5). De Freitas Table 6. Simple correlation coefficients between measured traits of annual medic as affected by fertilizing system bacteria+Mycorrhiza (T ). also indicate that if phosphorus solubilizing bacteria were applied with Glomus mossea, a significant increase in the growth of annual medic could be achieved (Piccini and Azcon, 1987). Soil Seed Bank Based on the results (Table 3), the number of pods in soil seed bank was significantly influenced by different fertilizing systems. The highest number of 314.6 pods per square metre was found in nitrogen fixing bacteria+phosphorus solubilizing bacteria treatment and the lowest of 139.8 pods per square metre in control treatment, respectively (Table 5). The superiority of combined inoculation of nitrogen fixing bacteria+phosphorus solubilizing bacteria on plant growth caused a significant increase in number of pods in soil seed bank. A proper fertilizing system is necessary for annual medic to ensure the capability of plant to produce adequate amount of seed in soil seed bank for a successful natural regeneration. Critical levels of soil seed bank for successful re-establishment of annual medic pasture in Ley-farming system is an average of 400 pods per square metre, although this critical level for various species of annual medic is different. For example, in different cultivars of M. scutellata and M. rigidula species around 600 and 250 pods per square metre are sufficient, respectively (Francis, 1988). Means with the same letter in each column are not significantly different at 5% probability level. Fertilizing systems : Control (without fertilizer) (T1), chemical, biological and integrated fertilizing systems of nitrogen and phosphorus as follows : Urea fertilizer+Triple superphosphate fertilizer (T ), Urea fertilizer+Phosphorus solubilizing bacteria (T3), Urea fertilizer+Mycorrhiza (T4), Urea fertilizer+Phosphorus solubilizing bacteria+Mycorrhiza (T ), Nitrogen fixing bacteria+Triple superphosphate fertilizer (T ), Nitrogen fixing bacteria+Phosphorus solubilizing 5 6 bacteria (T7), Nitrogen fixing bacteria+Mycorrhiza (T8) and Nitrogen fixing bacteria+Phosphorus solubilizing Trait RGB SSB Pod yield Forage yield CB et al. (1997), in canola, Cakmakc et al. (1999) Table 4. Mean comparison of annual medic traits as affected by different fertilizing systems in two experimental sites (Sararood and Mahidasht) under dry farming system 2 Sararood Mahidasht Sararood Mahidasht that sorghum seed was inoculated by B. cepucia, P. fluorecens or Enterobacter the plant growth was increased, however, the double and triple inoculation was not effective on plant growth (Chiarini et al., 1998). Ardakani et al. (2001) reported that combined application of mycorrhiza and Streptomyces on wheat yield, had a decreasing effect when compared with application of mycorrhiza alone. This is probably due to the sensitivity of mycorrhiza fungi to antibiotic substances which are secreted by Streptomyces. Research results have shown that seed inoculation with a mixture of several microorganism increased nutrient absorption and yield, compared with sole inoculation with each one of them (Perveen et al., 2002). Other research works Table 3. Degree of freedom and measuring F values of annual medic agronomic characteristics as affected by different fertilizing systems, grown in two experimental sites (Sararood and Mahidasht Experimental Stations) at dry farming conditions RGB : Re-growth biomass, AB : Accumulative biomass, SSB : Soil seed bank. CaCO (%) 30 28 Olsen phosphorus (mg/kg) 8.00 9.40 Available potassium (mg/kg) 530 430 DTPA extractable Zn (mg/kg) 0.38 1.56 DTPA extractable Cu (mg/kg) 0.70 1.40 DTPA extractable Fe (mg/kg) 2.00 4.76 DTPA extractable Mn (mg/kg) 2.42 3.78 (mm) (°C) Month Average precipitation Average temperature Table 2. Average precipitation and temperature during annual medic growing season in two experimental sites photosynthetic activities which lead to better in T treatment, respectively. In an experiment plant growth. In an experiment by Zaidi and Khan (2006), it was reported that inoculation of the vetch seed with a mixture of plant growth promoting bacteria, nitrogen fixing bacteria and mycorrhiza fungi, significantly improved dry matter. Adequate rainfall during re-growth Effect of fertilizing systems on the re- growth biomass was significant (Table 3). The highest re-growth biomass of 2581 kg/ha observed in nitrogen fixing bacteria+ phosphorus solubilizing bacteria (T ) followed by urea chemical fertilizer+solubilizing+ mycorrhiza treatment (2552 kg/ha) (Table 5). This result indicates the beneficial effect of integrated fertilizing system in nutrient supply stage in Mahidasht Station (Table 2) could explain 17.26% more dry matter production in this station (Table 4) compared to Sararood (Dryland Research Institute Station). Cumulative Biomass Production Different fertilizing systems had a significant effect on dry matter weight at early flowering stage (Table 3). The highest dry matter weight at early flowering stage was observed in urea fertilizer plus triple superphosphate treatment (T ). Using urea fertilizer+triple superphosphate treatment increased the yield by 7.6% compared to control (Table 5). This result could be explained by the ability of chemical fertilizer to supply readily nitrogen and phosphorus nutrients needed for plant growth. This characteristic of chemical fertilizer is more efficient, especially in the early period of plant growth which adequate moisture is available in dry land conditions. It seems that in this treatment due to the availability of nitrogen in the early stages of growth, faster growth is achieved with less need for weather heat units. This result is supported by the other study by Akbari et al. (2009) who reported that application of chemical fertilizer at different growth stages would need less heat units to promote plant growth. Re-growth Biomass Production Cumulative biomass was significantly affected by fertilizing systems (Table 3). The mean comparison showed that the highest amount of cumulative biomass of 4185 kg/ha especially in later stages of growth. It seems was produced in T treatment (Table 5), and that these treatments could provide with better the lowest amount of 3183 kg/ha was observed Urea fertilizer+mycorrhiza (T ), Urea fertilizer+phosphorus solubilizing bacteria+ mycorrhiza (T ), Nitrogen fixing bacteria+triple superphosphate fertilizer (T ), Nitrogen fixing bacteria+phosphorus solubilizing bacteria (T ), Nitrogen fixing bacteria+mycorrhiza (T ) and nitrogen fixing bacteria+phosphorus LSD (P=0.05) 149 428 699.0 152.2 40.8 RGB : Re-growth biomass, CB : Cumulative biomass, SSB : Soil seed bank. 1386ab 2581a 3576±448ab 1147.2a 314.6a T 8 1401ab 2184ab 3183±414b 976.7b 278.8ab 1397ab 2552a 4185±259a 740.2de 198.3d T 6 1369ab 2295ab 3727±72ab 799.8cde 210.8cd 1328b 2273ab 3502±138ab 910.2bc 260.0b T 4 1337b 2158ab 3496±62ab 865.7bcd 241.5bc solubilizing bacteria+Mycorrhiza (T ). Triple superphosphate and urea were applied according to soil test to fulfil the requirements of the crop in each site. Land preparation practices, including plowing, disking and furrowing were done before planting in early March. Medic seed was planted in both the locations in the second half of March by hand. Every experimental plot consisted of six planting rows of 25 cm distance with 5 m in length. The annual medic (cv. Robinson) seeds were planted at the rate of 20 kg of seed per hectare. Before sowing the seeds, based on soil analysis (Table 1), and according to fertilizer recommendation for annual medic, half of urea fertilizer and all phosphorus fertilizer (in treatments containing chemical fertilizer) were applied to the soil in bands by hand. The rest of the nitrogen fertilizer was broadcasted on the plots when plants reached to four-leaf stage. Phosphate solubilizing bacteria, nitrogen fixing bacteria and mycorrhiza solutions were prepared according to the Water and Soil Research Institute instructions. After calculating the amount of seed per treatment, the seeds were put inside a polyethylene bag (30 mg of each inoculation substance for 100 g of seed) alongwith 4% gum arabic solution. Then the seed and the adhesive substance were gently shaken for 30 sec. Then one gram of inoculation substance was added to the adhesive seeds and after 45 sec of shaking and making sure that the inoculation substance was uniformly distributed among the seeds, the inoculated seeds were spread on an aluminum sheet in shade to dry off. Then the seeds were promptly planted. The seeds were planted one centimeter deep within the sowing lines. At the early flowering stage and after elimination of border effects, random samples of one square metre were cut to the third node height from ground level in each experimental plot. The harvested samples were oven dried at 70°C for 24 h. The dried weight was measured as forage yield. The other measured traits were biomass in re-growth, pod and seed yield, cumulative biomass and soil seed bank. At the end of experimental period, soil samples were randomly taken from 0 to 5 cm in depth in each plot in both experimental locations using an auger to measure the soils seed bank. The soil samples were taken to the laboratory and after segregating the seeds from the soil, the samples were passed through sieve number 8 and 10; hence, all impurities were removed from the samples. Then the content of ground samples was passed through sieve number 8 and 10 which were placed on top of each other, respectively, in order to segregate the particles, in a way that stone, gravel and sand particles were left at the bottom of the container. The process of adding water to the container and passing through the sieves was repeated until the water added to the container became clear. In order to verify that no seeds were left among the gravel and sand in the container, the contents were placed in 25% salt water and the seeds floating on the surface of the salt water were collected. The soil seed bank population was measured by gathering, weighing and counting the seeds from the salt water surface. To analyze the experimental data and draw the charts, SAS and Excel software were used, respectively. Comparisons of all means were done on a 5% probability level based on a least significant difference (LSD) method. RESULTS AND DISCUSSION Forage Production at Early Flowering Stage a a RGB : Re-growth biomass, AB : Accumulative biomass, SSB : Soil seed bank. Means with the same letter in each column are not significantly different at 5% probability level. Table 5. Mean comparison of studied traits in different soil nutrient systems in annual medic (Medicago scutellata cv. Robinson) under dry farming culture m ) 1292b 1909b 3201±187b 446.7f 139.8e T 2 1486a 2108b 3285±164b 704.2e 242.2bc 1380 metres above the sea level, in 2009 growing season. The experiment was conducted in a randomized complete block design with three replications. Soil samples have been taken before the commencement of the experiment (Table 1). The experimental treatments consisted of : Control (without fertilizer) (T ), Urea fertilizer+triple superphosphate fertilizer (T ) * , Urea fertilizer+phosphorus solubilizing bacteria (T ), Table 1. Selected physical and chemical characteristics of soil (0-30 cm depth) in two experimental sites Characteristics Experimental stations 1327b 2268ab 3649±190ab 893.0bc 253.4b Sararood Mahidasht pH 7.68 7.93 Dissolved solids (EC 103) 30.0 55.0 Organic carbon (%) 0.31 0.62 Soil texture Loamy silt Loamy clay A set of seeds which stays intact and alive in the soil because of various reasons such as seed hardiness or lack of the right conditions for germination, is called the Soil Seed Bank. Medic soil seed bank changes with regard to both growth conditions in of different areas and the intensity and duration of grazing during the pasture period (Christiansen and Cocks, 1994). If the seed reserve in soil is less than 260 seed containing pods per square metre, the natural regeneration of annual medic will not be successful (Kassaim, 1979). However, this criterion varies with different 1Agronomy Department, College of Agriculture, University of Tehran, Karaj, Iran. 2Department of Sustainable Agricultural Systems, University of Natural Resources and Life Sciences, Vienna, Austria. 3Soil and Water Research Institute, Iran. 4College of Agriculture, Ferdowsi University of Mashhad, Iran. medic species and varieties, for instance in the case of Medicago rigidula about 600 pods, and for Medicago scutellata only 250 pods per square metre will suffice (Francis, 1988). Azizi (2003) reported that spring planting of annual medic and a maximum of one harvest in the beginning stage of flowering can supply soil seed bank and produce a considerable amount of dry foliage. Biological fertilizers are popular for their efficiency to supply phosphorus and other necessary nutrients for plants. The phosphorus solubilizing bacteria are the effective microorganisms in this process. These bacteria have a high efficiency to promote plant growth through providing P in its absorbable forms for plants. Mycorrhiza fungi are among the most important non-destructive microorganisms existing in most soils. According to the existing estimates, about 71% of the live biomass of soil microbial community contains mycelium of this fungus (Mukerji and Chamola, 2003). A symbiotic interaction between mycorrhiza and phosphate solubilizing bacteria can cause better moisture absorption from the soil by plants (Novella Legva et al., 2003). Application of Glomus macrocarpam and Rhizobium bacteria in Phaseolus mungo L. cultivation caused a significant increase in yield and the amount of phosphorus and nitrogen in the grain at the harvest time. Also the water uptake efficiency in fungi and bacteria treatment was higher compared to other treatments (Sheela and Sundaram, 2003). It has been reported that VAM fungi has a synergetic relation with other microorganisms such as phosphate solubilizing bacteria (PSB) and other plant growth promoting microorganisms (Azcon, 1989). Toro et al. (1988) studied annual medic response to combined inoculation of PSB, Enterobacter sp. and G. mossacee and found that mycorrhizal plants in every condition had a better growth. Neumann and George (2004) found that plant phosphorus content between mycorrhizal and non-mycorrhizal sorghum was not significantly different. The superiority of combined inoculation of Azotobacter sp. and Rhizobium sp. on bean yield and minerals uptake has been reported by Rodelas (1999). In an experiment by Piccini and Azcon (1987) on the effect of phosphate solubilizing bacteria and mycorrhiza fungi on annual medic, it was observed that using of phosphate solubilizing bacteria increased nutrient absorption of potassium, phosphorus and forage yield. Considering the importance of producing adequate forage alongwith maintaining an active seed bank in soil in ley-farming system and dry farming conditions, this experiment was conducted to investigate the effect of different fertilizing systems on the forage yield and seed bank reserves of annual medic (cv. Robinson) under dry farming conditions. MATERIALS AND METHODS This experiment was conducted in two locations : 1. Sararood Dryland Farming Research Station with the geographic longitude of 47′′, 20′ and latitude of 34′′, 20′ and elevation of 1351 metres above the sea level, and 2. Mahidasht Soil Fertility Research Station with the geographic longitude of 46′′, 50′ and latitude of 24′′, 16′ with elevation of T T T 3 9 5 7 9 3 4 8 9 2 5 experiment showed that by application of proper fertilizer on annual medic (M. scutellata cv. Robinson) and harvesting a forage yield at early flowering stage, not only a considerable amount of forage could be produced, but also an active soil seed bank could be achieved which was the main goal of this experiment. In urea chemical fertilizer+solubilizing+ mycorrhiza (T ) treatment application although the highest vegetative growth was achieved, but due to the lack of sufficient seed production, seed bank was poor. However, in a Ley-farming 480 in sugarbeet and De Freitas (2000) in wheat conducted experiments and all more or less concluded that seed inoculation with these bacteria significantly increased the grain yield, yield components and total dry matter production. There were positive significant correlations between cumulative biomass with re-growth biomass, as well as seed yield with pod yield and soil seed bank reserves (Table 6). Pod yield had a higher correlation (r=0.77**) with soil seed bank than other traits, also cumulative biomass with biomass in re-growth had a relatively high correlation. Positive correlation between seed yield and pod with the soil seed bank revealed a direct relationship of seed yield with available pod in soil seed bank. Application of proper fertilizing system can have a positive effect on the growth of annual medic. The results of this RGB : Re-growth biomass, AB : Accumulative biomass, SSB : Soil seed bank. *,**Significant at P=0.05 and P=0.01 levels, respectively. NS : Not Significant. 400 320 240 160 80 Sararood Station Mahidasht Station 6 Soil seed bank (pods/m2) 7 5 7 7 8 3 9 2 8 0.05 1 2 3 5 T 1 T LSD =57.7 0 1 2 3 4 T T T T T T T T 5 6 7 8 9 Fig. 1. Interaction effects of region and different fertilizer treatments on seed bank of annual medic at dryland conditions. system, when the regeneration and natural establishment of annual medic is the main goal, the application of nitrogen fixing bacteria+phosphorus solubilizing bacteria treatment is best recommended under the environment conditions of this experiment. ACKNOWLEDGEMENT The authors would like to thank the personnel of the Water and Soil Research Division of Kermanshah Agriculture and Natural Resources Center and the Dryland Farming Research Institute (Sararood) for their cooperation in carrying out this project. REFERENCES Akbari, P., Ghalavand, A. and Modarres Sanavi, S. A. M. (2009). Effect of different fertilizing systems and bacteria of incremental growth on phenology, yield and yield components of sunflower. Electron. J. Crop Produc. 2 : 134-19 (In Persian, Abstract in English). Ardakani, M. R, Mazaheri, D. and Noormohammadi, G. (2001). 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