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versión On-line ISSN 2521-9766versión impresa ISSN 1405-3195

Agrociencia vol.49 no.8 Texcoco nov./dic. 2015




Azospirillum lipoferum and nitrogen fertilization effect on chlorophyll content, nutrients uptake and biometric properties of Zea mays L.


Efecto de Azospirillum lipoferum y fertilización nitrogenada en el contenido de clorofila, absorción de nutrientes y propiedades biométricas de Zea mays L.


Elnaz Davaran-Hagh1, Bahram Mirshekari1*, Mohammad Reza-Ardakani2, Farhad Farahvash1, Farhad Rejali3


1 Department of Agronomy and Plant Breeding, Tabriz Branch, Islamic Azad University, Tabriz, Iran. *Author for correspondence. (email:

2 Department of Agronomy and Plant Breeding, Karaj Branch, Islamic Azad University, Karaj, Iran.

3 Soil and Water Research Institute, Karaj, Iran.


Received: October, 2014.
Approved: June, 2015.



Integrated nutrient management is a major agronomic practice which brings together all methods to support plants nutritional requirement. However, the interaction of these methods together is very complex and needs to be studied in different climatic conditions. So, the effect of nitrogen fertilizer and Azospirillum lipoferum inoculation on maize (Zea mays L.) growth, yield and nutrients uptake was determined in this study. The hypothesis was that the bacterial inoculation decreases the need for N fertilizer application without reducing plant yield. Therefore, we carried out a factorial experiment in a randomized complete block design with three replications. Nitrogen at 0, 25, 50, 75 and 100 % concentrations of the recommended dose, i.e. 150 kg N ha-1 (based on the soil sample analysis) were applied with or without A. lipoferum inoculation. There was a significant effect of N and A. lipoferum inoculation on most of the variables. In most cases, there was no significant difference between the two highest doses, i.e. 75 % and 100 % of N. Most variables showed their highest value in the interaction of A. lipoferum x 100 % N. But, the value of most variables was the same as in uninoculated x 100 % N or inoculated x 50 % N. Azospirillum lipoferum inoculation could be an alternative to high N fertilizer rates, increasing soil fertility and maize yield in sustainable farming. In conclusion, 75 % of the recommended N application rate along with A. lipoferum inoculation has the best effect on the variables increasing grain yield by 136.5 %.

Keywords: Biofertilizer, iron, nitrogen, phosphorus, zinc.



La gestión integrada de los nutrientes es una práctica agronómica importante que reúne todos los métodos para apoyar los requerimientos nutricionales de las plantas. Sin embargo, la interacción de estos métodos es muy compleja y debe estudiarse en condiciones climáticas diversas. Por tanto, el efecto de fertilizante nitrogenado y la inoculación de Azospirillum lipoferum en el crecimiento, rendimiento y absorción de nutrientes en maíz (Zea mays L.) fue evaluado en este estudio. La hipótesis fue que la inoculación bacteriana reduce la necesidad de aplicación de fertilizante N sin disminuir el rendimiento. Para ello realizamos un experimento factorial en un diseño de bloques al azar con tres repeticiones. Concentraciones de N de 0, 25, 50, 75 y 100 % de la dosis recomendada, es decir, 150 kg N ha-1 (basada en el análisis de muestras del suelo) fueron aplicadas con o sin inoculación de A. lipoferum. Hubo un efecto significativo del N y la inoculación de A. lipoferum en la mayoría de las variables. En la mayoría de los casos no hubo diferencia significativa entre las dos dosis más altas, 75 % y 100 % de N. La mayoría de las variables exhibieron sus valores mayores con la interacción de A. lipoferum x 100 % N. Pero, el valor de la mayoría de las variables fue el mismo que el no inoculado x 100 % N o inoculado x 50 % N. La inoculación con A. lipoferum podría ser una alternativa para los altos índices de fertilizante N, aumentando la fertilidad del suelo y el rendimiento de maíz en agricultura sostenible. La conclusión es que 75 % de la dosis recomendada de N junto con inoculación A. lipoferum tiene el mejor efecto en las variables y aumenta 136.5 % el rendimiento de grano.

Palabras clave: Biofertilizante, fierro, nitrógeno, fósforo, zinc.



The excessive consumption of chemical fertilizers poses numerous adverse impacts on the environment and many life forms on earth. In contrast, biofertilizers are living microorganisms that colonize plant root and promote growth by increasing the supply or availability of nutrients to host plant (Lugtenberg et al., 2002). The application of biofertilizers is a cost effective necessity to obtain a high quality yield and avoid environmental pollution. Sylvia et al. (2005) report that there are about two million organisms in each gram of soil. They suggest that maintaining and improving the activity of this resource is required for sustained soil and crop management. Soil biological physical and chemical properties, have an important effect on soil fertility (Barea et al., 2005; Probst et al., 2008).

Azospirillum, a free—living rhizobacterium, is a beneficial microorganism for plants (Aguirre-Medina et al., 2013; Ramos et al., 2002). Zaied et al. (2007) state that the species Azospirillum provides a proper condition for maize (Zea mays L.) growth by producing phytohormones. The improvement of plant yield may result from the ability of bacteria to remove soil pathogens, produce plant growth regulators such as gibberellins, cytokines and auxin, increase plant access to nutrients, fix air N into the soil and facilitate the development of root system (Bashan et al., 2004; Rudresha et al., 2005; Bashan and de-Bashan, 2010). Zuberer (1987) report that maize inoculated with Azospirillum had higher dry weight than the uninoculated control, due to increased uptake of N, P, K, Mg, Mn and Zn. Ardakani and Mafakheri (2011) also point out that inoculating wheat seed with Azospirillum significantly affect plant growth factors. So, we carried out this experiment with the hypothesis that the bacterial inoculation reduces the need for chemical N fertilizer application without reducing plant yield. The aim was to determine the effect of inoculation with A. lipoferum on maize growth and yield and test replacement of chemical N fertilizer by this inoculation.



This study was carried out in 2011 at the Research Station of the Faculty of Agriculture, Islamic Azad University, Tabriz branch, Iran (46° 17' E, 38° 5' N). The experimental design was a randomized complete block with a factorial arrangement and three replications. Treatments of the experiment were N application rates: N0, control, N1 25 %, N2 50 %, N3 75 %, N4 100 % of the recommended dose, 150 kg N ha-1; and B0, without inoculation and B1, with inoculation of A. lipoferum. Treated seeds were inoculated with peat based inoculants of A. lipoferum, at 2X108 cell concentration g-1 of the carrier. Inoculated seeds were planted directly in the soil. Nitrogen fertilizer was top dressed at two stages of emergence and stamen appearance.

Maize seeds var. pop corn K.S.C. 600 were planted in order to obtain 66000 plants ha-1. Irrigation was conducted based on a 70 mm evaporation from evaporation pan. Prior to harvest, chlorophyll content was measured using a chlorophyll meter model Opti-Science (CCM-200). Then, 10 plants were harvested from the middle rows of each plot and grain yield was measured. To measure the absorption of nutrients, five other plants were harvested from each plot when grains were at dough stage and dried at 70 °C oven for 72 h. Then, samples were ground and analyzed. Nitrogen and protein were measured by Kjeldahl method and P was measured by spectrophotometry. Fe and Zn were measured by atomic absorption. Data were tested for normal distribution and subjected to statistical analysis using SAS. Duncan's multiple range test was used to compare means (p≤0.05).



Increasing N application rate from 0 % to 100 % of the recommended dose enhanced the value of variables except for the grain yield, 1000 seed weight and the number of seeds in ear. Their values were higher in 75 % rather than in 100 %, although the difference was not significant (Table 1 and 2).

Nitrogen is the main yield limiting nutrient because of its various roles in plants tissues. It is a structural component of amino acids, nucleic acids, enzymes and proteins, chlorophyll and cell wall (Fageria and Baligar, 2005; Wiedenhoeft, 2006; Fageria, 2009; Eftekhari et al., 2012). In an experiment on wheat, application of chemical N fertilizer significantly affected plant height, grain yield and biomass (Ardakani and Mafakheri, 2011). Njuguna et al. (2010) report the highest wheat grain yield, 1176.7 kg ha-1, against the lowest yield, 1022.9 kg ha-1 in the control, when 46 kg N ha-1 was applied; wheat root system development was also significantly improved by N fertilizer. Biesiada and Kus (2010) report the highest basil (Ocimum basilicum L.) yield upon applying 150-250 kg N ha-1; increasing N application from 50 to 150 kg ha-1 raised yield by 63.90 %, whereas increasing N dose to 250 kg ha-1 enhanced yield by 11.46 %. Results of our experiment also showed the improving effect of N application on maize growth variables (Table 2; Figure 1).

Azospirillum inoculation significantly affected the variables (Table 1). The inoculation increased grain yield by 19.38 %, biological yield by 5.23 %, chlorophyll content by 7.99 % and protein content by 10.25 %, compared with the uninoculated control (Table 2). The inoculation also increased N, P, Fe and Zn content by 11.87, 16.66, 7.39 and 5.95 %, respectively, compared with the control (Figure 2). The interaction of NX Azospirillum showed a significant effect only on the grain yield and the number of seeds in ear. Zaady et al. (1993) report 10 to 30 % of improvement in grain yield and biomass of maize and sorghum due to Azospirillum inoculation. Ribaudo et al. (2006) show that shoot and root fresh weights of inoculated tomatoes were 4 % and 30 %higher than the control plants, respectively; also, 20 % of improvement in shoot height was observed. Besides, Azospirillum brasilense inoculation increased growth variables of maize (El-Kholy et al., 2005).

One of the most important functions of Azospirillum is to fix atmospheric N to soil and making it available to plants (Bashan and de-Bashan, 2010). Our results indicate that grain yield was not statistically different in uninoculated treatment X 100 % N and inoculated treatmentX 50 % N. This means that A. lipoferum inoculation may be used as a substitute for high nitrogen fertilizer rates without reducing yield and shows the ability of the bacterium to fix atmospheric N. In an experiment with wheat, there were no significant differences in grain yield with 150 kg N ha-1 without Azospirillum inoculation and 50 kg N ha-1+ Azospirillum inoculation (Ardakani and Mafakheri, 2011). The combined application of Azospirillum + 60 kg N ha-1 caused a similar maize yield compared to the application of the recommended dose of 120 kg N ha-1 without inoculation (Yadav et al., 2011).

Biological N fixation is the primary mechanism by which Azospirillum bacteria increase plant growth and yield; besides the efficiency of water and nutrients absorption is improved by facilitating the development of root system and making higher soil volume available to plant root (Bashan and Holguin, 1997; Reis et al., 2000; Dalla Santa et al., 2004; Mehran et al., 2011). Ribaudo et al. (2006) show significant improvement in root fresh weight, main root hair length and root surface as the result of inoculating tomato plants with A. brasilense; the inoculation increased root fresh weight by 30 % over the control. Inoculation of plants with Azospirillum increased root length, root dry weight and shoot dry weight; these improvements are related to the presence of a well developed root system which facilitates water and nutrients absorption (Sprent and Sprent, 1990). According to Hasanabadi et al. (2010), inoculating barley plants with A. lipoferum significantly increase root growth parameters such as root dry weight and root length. Ardakani and Mafakheri (2011) also show that wheat inoculation with Azospirillum significantly increased root dry weight by 9 %, compared with the control.

Inoculation with Azospirillum changes root growth or morphology, resulting in better water and nutrient uptake, and consequently plant growth and yield (Lin et al., 1983; Bottini et al., 2004; Ribaudo et al., 2006). The result of our study showed that inoculation with Azospirillum increased N, P, Fe and Zn contents by 11.87 %, 16.66 %, 7.39 % and 5.95 %, as compared to the uninoculated control. This enhancement of the content of mineral nutrients may be due to increased nutrient absorption as the result of a more developed root system. Ardakani et al. (2011) report that inoculating wheat seeds with Azospirillum enhanced plant NPK content by 21 %, 17 % and 25 %. The inoculation with Azospirillum increased root growth and consequently nutrients and water uptake, which resulted in higher yield (Lin et al., 1983). According to Eid et al. (2006), inoculating Celosia argenta with Azospirillum increased N and P content in leaves.

Another mechanism by which Azospirillum inoculation improves plant growth is the production of plant growth promoting substances, polyamines and amino acids (Thuler et al., 2003; Tsavkelova et al., 2006). These phytohormones promote plant root growth, consequently increasing nutrients and water absorption (Reis et al., 2000; Steenhoudt and Vanderleyden, 2000; Dalla Santa et al., 2004; Chamangasht et al., 2012; Zakikhani et al., 2012). According to Ribaudo et al. (2006), Azospirillum inoculation increased indole-3-acetic acid (IAA) content in tomato shoots and roots by 7 and 19 fold; besides ethylene content was also increased.



Chemical nitrogen fertilizer and Azospirillum lipoferum inoculation significantly affected maize yield and nutrient uptake. Azospirillum lipoferum inoculation plus N rates higher than 75 % was not favorable due to the reduction in bacterial functioning. In most cases, 75 % of the recommended N application rate along with A. lipoferum inoculation had the best effect on the variables.



The authors highly acknowledge Islamic Azad University, Tabriz Branch-Iran, for their fields and laboratories support.



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