Effect of inoculation with
on wheat yield and seed
Received for publication, March 7, 2011 Accepted, July 9, 2012 N.MILOŠEVIĆ1*, B.TINTOR1, R.PROTIĆ2, G. CVIJANOVIĆ3, T.DIMITRIJEVIĆ4 1Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia; 2Institute for Science Application in Agriculture, Bulevar despota Stefana 68b, Belgrade, Serbia;
Faculty of Biofarming, Maršala Tita 39, 24300 Bačka Topola, Serbia; 4AIC BEČEJ-AGRICULTURA A.D., Moše Pijade 2, 21220 Bečej, Serbia
*Corresponding author: email@example.com; Tel/fax: +381 21 4898 450
An investigation has been organized to study the effect of wheat seed inoculation (the cultivars Renesansa and Zlatka) with Azotobacter chroococcum, strain 86 (2-5x CFU 108 ml-1). Experiments were conducted under field conditions, on a chernozem soil. The effectiveness of inoculation on wheat seed quality (germination energy and percentage), yield and 1000-seed weight was shown to depend on the amount of applied urea (50, 100 and 200 kg ha-1 and nonfertilized control) and wheat cultivar. A. chroococcum treatment, depending on variety and fertilizer treatment, increased the energy of germination by 1 to 9% and seed viability by 2 to 8%. Negative effects on seed germination energy and percentage were found in the case of the cultivar Zlatka, in the variant with 100 kg urea ha-1. The largest increase in 1000-seed weight was obtained in the case of the cultivar Renesansa, in the variant without N application (16%). A. chroococcum inoculation increased the seed yield of both cultivars in the variant without urea application and with 50 kg ha-1 of urea. The highest yield increase (74%) was registered in the case of the cultivar Zlatka when inoculated and treated with 50 kg ha-1 of urea. All effects, except the yield, were low in the case of the cultivar Zlatka.
Key words: Azotobacter chroococcum,inoculation, seed, wheat
Nitrogen is the most limiting factor for high crop productivity. There has been an increased interest in biological nitrogen fixation (BNF) in the context of sustainable agriculture. The interest resulted from high cost of mineral fertilizers and their possible harmfulness to the environment. BNF plays an important role in maintaining soil fertility VANCE & GRAHAM . Non-symbiotic nitrogen-fixing bacteria that live in the rhizosphere DÖBEREINER  and/or endophytically HECHT-BUCHHOLZ  often increase yields of cereals and other crops. Free-living N-fixing bacteria play an important role in plant development on account of nitrogen fixation and supply of growth activators. Plant growth regulating substances may be produced the bacteria in the root zone, and in some cases there were indications of biological control of plant pathogens OKON .
Azotobacter is a free-living nitrogen-fixing bacterium MARTINEZ-TOLEDO  which is used as a biofertilizer in the cultivation of most crops. In the local soils, Azotobacter fixes annually about 60–90 kg N/ha and it may be used in crop production as a substitute for a portion of mineral nitrogen fertilizers GOVEDARICA & al. ; HAJNAL & al. . Inoculation with Azotobacter can increase the yield by 5-28% GOVEDARICA & al. ; KENNEDY & al. ; MILOŠEVIĆ & JARAK . The increase is a result of BNF, as well
as the production of antibacterial and antifungal compounds, growth regulators and siderophores PANDEY & KUMAR .
Among the field crops, wheat and sugarbeet are most responsive to nitrogen nutrition BOGDANOVIĆ & al. . Wheat is an important food crop on the global level. The aim of our investigation was to examine possibilities of successful wheat cultivation combined with bacterial inoculation. Inoculation with certain rhizobacteria clearly benefited growth and increased the seed yield and N content of field grown wheat. Effect of wheat inoculation with Azotobacter varies in dependence of the species and strain of N-fixing bacteria, physico-chemical soil properties, N fertilizers applied, climatic conditions and wheat cultivar MILOŠEVIĆ & GOVEDARICA ; MILOŠEVIĆ & JARAK ; MILOŠEVIĆ & al. . According to SOLIMAN & al.  inoculation with Azotobacter replaced up to 50% of urea-N for wheat grown in a greenhouse trial under aseptic conditions.
The objectives of this study were to assess the effects of inoculation with Azotobacter chroococcum and quantity of nitrogen mineral fertilizer on seed quality and seed yield of winter wheat.
Materials and Methods
This study was undertaken in order to assess the effects of inoculation with Azotobacter chroococcum and urea application on seed quality and seed yield of wheat cultivars Renesansa and Zlatka. Seed wheat yield performances were tested at AIC Bečej. Seed quality parameters (viability and germination energy) were analyzed under laboratory conditions, according to Rules of Seed Quality of Agricultural Crops SFRJ .
Experiments were conducted under field conditions, on a chernozem soil. The chemical soil properties were: pH (in H2O) - 7.47; CaCO3 - 15.27%; humus content - 3.81 %; available P and K contents - 32.70 mg/100g soil and 16.00 mg/100g soil, respectively. The field trial was established in a randomized block design with four replications. The size of experimental plots was 48x50 m.
Test variants were: 1. control (without N fertilization); 2. 50 kg ha-1 urea; 3. 100 kg ha-1 urea; 4. 200 kg ha-1 urea, with and without inoculation with A. chroococcum.
Seeds were treated with bacterial suspension of Azotobacter chroococcum strain 86 (72 hrs old, density 2-5x108 CFU/ml) for 30 min before sowing. The strain, taken from a bacterial collection of Institute of Field and Vegetable Crops, was cultivated on a liquid medium of FEDOROV&SAVKINA . Biotests were conducted to monitor the effect of inoculation on seed quality (viability and germination energy) of wheat cultivars Renesansa and Zlatka. Subsequently, 1000-seed weight and seed yield were measured and shown per ha-1
of land. The presented results are 2-year averages (2003 and 2004).
ANOVA statistical method was used to determine lowest significant differences for average values and confidence interval of 95% (LSD 0.05%) STANKOVIĆ & al. .
Results and Discussion
The global annual volume of BNF is estimated at about 175 million tons of nitrogen, 79% of which are being fixed in plant-soil systems. Inoculation with diazotrophs may have a positive effect on plant growth parameters, which may but need not manifest itself on the yield of field crops. Namely, the effect of inoculation has been found to depend on cultural practicesapplied, plant species, hybrid, bacterial species and strain, inoculation technique and the method of application of bacterial preparations MILOŠEVIĆ & JARAK ; KENNEDY & al. .
Table 1. Effect of inoculation with A. chroococcum on germination energy of wheat seed (%) Urea (kg/ha)(C) Variety(A) Inoculation(B) 0 50 100 200 Average AB Average A Noninoculated 84.00 86.00 88.00 85.00 85.75 Renesansa Inoculated 86.00 88.00 88.00 88.00 87.50 86.63 Inoculated (%) in relation to noninoculated (100%) ＋2,38 ＋2.32 0.00 ＋3.53 Average AC 85,00 87.00 88.00 86.50 Noninoculated 82.00* 87.00 84.00 87.00 85.00 Zlatka Inoculated 90.00* 88.00 83.00 90.00 87.75 86.38 Inoculated (%) in relation to noninoculated (100%) ＋9,76 ＋1.15 －1.19 ＋3.45 Average AC 86,00 87.50 83.50 88.50 Average B 83,00 86.50 86.00 86.00 85.38 Average BC 88,00 88.00 85.50 89.00 87.63 Average C 85,50 87.25 85.75 87.50 A B C AB AC BC ABC LSD 5% 2.65 2.65 3.75 3.75 5.30 5.30 7.50
The analysis of variance of germination energy and viability based on LSD test showed that none of the major sources of variation (cultivar, inoculation and different doses of nitrogen fertilizer) exhibited a significant effect on the analyzed seed quality parameters. In the case of germination energy, statistical significance was recorded for interactions of all three variation sources. The effectiveness of inoculation and its effect on wheat seed quality (germination energy and percentage), yield and 1000-seed weight were shown to depend on the amount of applied urea (50, 100 and 200 kg ha-1 and nonfertilized control) and wheat cultivar. The inoculation with A. chroococcum increased the germination energy of both cultivars, with all fertilizer doses except the application of 100 kg of urea. The highest impact of inoculation was recorded in the cultivar Zlatka when not treated with mineral fertilizers (9.76%). The analysis of variance for the percentage of germination showed statistical significance only in the interaction inoculation x nitrogen fertilizer dose. A. chroococcum treatment, depending on the amount of applied urea and the cultivar, increased the energy of germination by 1-9% and seed viability by 2-8% (Tables 1 and 2). Negative and insignificant effects on seed germination percentage and energy were also found in the cultivar Zlatka treated with 100 kg urea ha-1. According to BURNS , inoculation with Azotobacter may significantly affect plant germination and growth, i.e., it may indirectly affect the yield. Table 2. Effect of inoculation with A. chroococcum on viability of wheat seed (%)
Urea (kg/ha)(C) Variety(A) Inoculation(B) 0 50 100 200 Average AB Average A Noninoculated 86.00 86.00 88.00 89.00 87,25 Renesansa Inoculated 92.00 88.00 88.00 91.00 89,75 88,50 Inoculated (%) in relation to noninoculated (100%) ＋6,98 ＋2.32 0.00 ＋2.25 Average AC 89,00 87.00 88.00 90.00 Zlatka Noninoculated 90.00 87.00 89.00 83.00 87,25 87,63
Inoculated 90.00 89.00 83.00 90.00 88,00 Inoculated (%) in relation to noninoculated (100%) 0,00 ＋2.3 －6.74 ＋8.43 Average AC 90.00 88.00 86.00 86.50 Average B 88.00 86.50 88.50 86.00 87.25 Average BC 91.00* 88.50 85.50* 90.50 88.88 Average C 89.50 87.50 87.00 88.25 A B C AB AC BC ABC LSD 5% 2.59 2.59 3.66 3.66 5.18 5.18 7.32
Average long-term results MILOŠEVIĆ & GOVEDARICA  showed mostly positive effects of A. chroococcum inoculation on wheat seed yield (up to 3%) and plant height (up to 6%). The latter parameter may be important in wheat breeding for resistance to lodging.
Table 3. Effect of inoculation with A. chroococcum on 1000-seed weight (g) Urea (kg/ha)(C) Variety(A) Inoculation(B) 0 50 100 200 Average AB Average A Noninoculated 31.00 33.00 36.00 34.00 33,50 Renesansa Inoculated 36.00 37.00 36.00 34.00 35,75 34,63 Inoculated (%) in relation to noninoculated (100%) ＋16,13 ＋12.12 0.00 0.00 Average AC 33,50 35.00 36.00* 34.00 Noninoculated 32.00 33.00 30.00 32.00 31,75 Zlatka Inoculated 33.00 33.00 31.00 34.00 32,75 32,25 Inoculated (%) in relation to noninoculated (100%) ＋3.12 0.00 ＋3.33 ＋6.25 Average AC 32.50 33.00 30.50* 33.00 Average B 31.50 33.00 33.00 33.00 32.63 Average BC 34.50 35.00 33.50 34.00 34.25 Average C 33.000 34.000 33.250 33.500 A B C AB AC BC ABC LSD 5% 2.33 2.33 3.29 3.29 4.66 4.66 6.59
The analysis of variance of the 1000-seed weight based on LSD test showed statistical significance only in the interaction variety x nitrogen fertilizer. When the optimal nitrogen fertilizer dose (100 kg urea) was applied, differences among the cultivars were largest. The highest increase in 1000-seed weight among the treatments (16%) was obtained with the cultivar Renesansa, in the variant without N application (Table 3). Yield and quality of wheat is directly related to the characteristics of variety and soil, crop management and characteristics of microbiological processes PROTIĆ & al. . According to GOVEDARICA & al.  inoculation of wheat seed with diazotrophs increased the 1000-seed weight from 2 to 14% under conditions of a greenhouse.
The analysis of variance of the yield showed that statistically significant differences existed for all major sources of variation and all interactions. The application of microorganisms increased the availability of nutrients, which had a positive impact on yield parameters MILOŠEVIĆ & al. . The enhancing effect of seed inoculation with N2-fixing
bacteria on the growth and yield of wheat was reported by many researchers BHATTARAI & HESS ; OZTURK & al. . This improvement may be attributed to the high nitrogen uptake by the inoculated plants and the ability of bacterial strains to produce growth-promoting substances HAAHTELA & al. . A. chroococcum inoculation increased the yield of seed in the variant without urea application and in the case with 50 kg ha-1 urea of both cultivars (Table 4). The highest yield increase (74%) was registered in the case of the cultivar Zlatka treated with 50 kg ha-1 of urea. According to KENNEDY & al. , inoculation with Azotobacter spp. increased the yield of wheat by 0.65 g per plant under conditions of a greenhouse.
Table 4. Effect of inoculation with A. chroococcum on seed yield (kg ha-1)
Urea (kg/ha)(C) Variety(A) Inoculation(B) 0 50 100 200 Average AB Average A Noninoculated 2292,00 2250,00 2333,00 3125,00 2500,00 Renesansa Inoculated 2667,00 2502,00 2667,00 2708,00 2636,00 2568,00 Inoculated (%) in relation to noninoculated (100%) ＋16,36 ＋11,2 ＋14,32 －13,34 Average AC 2479,50 2376,00 2500,00 2916,50 Noninoculated 3000,00 1667,00 2292,00 2542,00 2375,25 Zlatka Inoculated 3292,00 2917,00 2042,00 3292,00 2885,75 2630,50 Inoculated (%) in relation to noninoculated (100%) ＋9,73 ＋74,98 －10,91 ＋29,5 Average AC 3146,00 2292,00 2167,00 2917,00 Average B 2646,00 1958,50 2312,50 2833,50 2437,63 Average BC 2979,50 2709,50 2354,50 3000,00 2760,88 Average C 2812,75 2334,00 2333,50 2916,75 A B C AB AC BC ABC LSD 5% 13,47 13,47 19,05 19,05 26,94 26,94 38,09 In principle, bacterial inoculant biofertilizers can be used to supplement a portion of urea-N. The estimated amount of BNF by wheat–bacterial associations was between 10 and 30 kg N ha−1 for the crop or about 10% of the total N requirement KENNEDY & al. .
The effectiveness of bacterial inoculation on wheat seed quality (germination energy and percentage), yield and 1000-seed weight were shown to depend on the amount of applied urea and wheat cultivar. Wheat seed inoculation with A. chroococcum affected positively all parameters in the variants without urea application and the lowest dose of urea. Negative results were recorded in the variants with medium and high doses of urea in combination with inoculation. The results of this study clearly showed the beneficial role of rhizobacteria. Taking in consideration the ecological and economic importance of diazotroph application, the application of bifertilizers is a future imperative.
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