Influence of the application of Azospirillum lipoferum

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Pastos y Forrajes, Vol. 39, No. 1, January-March, xx-xx, 2016 / Lázaro J. Ojeda-Quintana

Scientific Paper

Influence of the application of Azospirillum lipoferum in Panicum maximum cv. guinea tobiatá on a Grayish Brown soil Lázaro J. Ojeda-Quintana1, Layda Toledo-Vazquez2, Consuelo Hernández-Rodríguez2, Yoandy MachadoDíaz2 y Eduardo Furrazola-Gómez3 1

CUM Cumanayagua, Universidad de Cienfuegos, Calle Los Filtros No. 18, Cumanayagua, Cienfuegos, Cuba 2 Estación Experimental de Suelos y Fertilizantes Escambray, Barajagua, Cumanayagua, Cienfuegos, Cuba 3 Instituto de Ecología y Sistemática. CITMA. E-mail: [email protected]

Abstract: A study was conducted during two years at the Soils and Fertilizers Research Station Escambray (located in the Cienfuegos province, Cuba), in order to evaluate the effect of the application of different doses of Azospirillum lipoferum in Panicum maximum cv. guinea tobiatá. For such purpose a randomized block design was used, with seven treatments and three replications. The treatments were: doses of application of the biofertilizer (L ha-1): 25, 50, 75, 100 and 125; a control with NPK at planting (45 kg ha-1 of nitrogen in alternate cuttings); and an absolute control. The plots measured 16 m2, with an evaluable area of 9 m2. The A. lipoferum strain used was INICA-8, with an average concentration of 108 cfu g-1 of seed. Four cuttings per year were made, with a frequency of 90 days, at a height above the soil of 25 cm, and in each cutting the dry matter yield was evaluated. The application of A. lipoferum had a favorable influence on the pasture establishment; the accumulated yield was 27,91; 28.34; 30,17; 30,23 and 31,92 t DM ha-1 for 25, 50, 75, l00 and 125 L ha-1, respectively, with significant difference from the control. The highest DM yield was found in the fertilized control (54,45 t DM ha-1), which differed significantly from the other treatments. It is concluded that this biopreparation can be an alternative to mineral fertilization. Keywords: organic fertilizers, yield

Introduction Agriculture, worldwide, constitutes a fundamental activity for the subsistence of the human population. Diverse factors have led to a process of deterioration of its scarce resources and an increasing difficulty to renew them. The soil as basis of the resources and the production is framed in a complex, heterogeneous and fragile environment, which shows a high susceptibility to erosion and a low natural fertility, with effects on the crop production, work productivity and feasibility of the establishment of sustainable productive systems. The recovery and maintenance of soil fertility on a sustainable basis constitutes a largely important factor in the development of the world’s agricultural production (Rueda-Puente et al., 2015). The objective of the nutrient management strategies is to achieve the required production in the crops, efficiently, economically and sustainably. There is consensus worldwide about the fact that the agriculture which depends exclusively on chemical inputs is not sustainable at long term, and

that only involving the combination of organic fertilizers, green manures and biofertilizers it will be possible to achieve sustainable food production, maintain the soil biodiversity and prevent environmental contamination (Aguirre et al., 2010). Biofertilizers are considered an alternative to substitute partially and totally mineral fertilizers, and the use of bacteria which interact with plants is a viable option in many countries. At present the development of biofertilizers is sought with the use of plant growth promoting bacteria, particularly of the Azospirillum genus, which fixes nitrogen and produces phytohormones (Ferlini, 2008). The first species of the Azospirillum genus was isolated by Beijerinck in 1925, in the Netherlands, from nitrogen-poor soils, and it was originally called Spirillum lipoferum. Since then, strains of this genus have been isolated from many species of wild and cultivated pastures, cereals and legumes in tropical and subtropical climates, according to the report by Rueda-Puente et al. (2015). This bacterium has progressed in the fields, leading to an

Pastos y Forrajes, Vol. 39, No. 1, January-March, xx-xx, 2016 / Azospirillum lipoferum in Panicum maximum 25

increasingly higher and successful application in several regions of the world, especially in South America and Central America (Hartmann and Bashan, 2009). In this study the application of different doses of Azospirillum lipoferum in Panicum maximum cv. guinea tobiatá was evaluated, under field conditions, as an option to improve the dry matter yield of this pasture species. Materials and Methods The research was conducted during two years, on a Grayish Brown soil (Hernández et al., 1999), in 16-m2 plots plated with P. maximum cv. guinea tobiatá. The main soil fertility indicators showed the following values: pH (KCl): 5,4 (potentiometric method, National Normalization Office, 1999c); P2O5: 4,71 mg/100 g of soil (Oniani’s colorimetric method, National Normalization Office, 1999b); K2O: 13,1 mg/100 g of soil (by flame photometry according to Oniani’s colorimetric method, National Normalization Office, 1999a); organic matter: 1,24 % (according to the Walkley-Black method, NC-51, 1999); and total nitrogen: 0,185 %. A randomized block design was used with seven treatments and three replications. The evaluated treatments were: application doses of the biofertilizer (L ha-1): 25, 50, 75, 100 and 125; a control with NPK at planting (45 kg ha-1 of nitrogen in alternate cuttings); and an absolute control. The A. lipoferum strain used was INICA-8, with a population of 107 cfu g-1 of seed. Botanical seed was used for planting, with 96 % of germination. The biofertilizer was applied on the seed at the moment of planting; after the establishment cutting (cutting zero), and afterwards in alternate cuttings. Four cuttings were made per year, with a frequency of 90 days, at a height of 25 cm above the soil. NPK was applied after planting, at a rate of 45 kg N ha-1, 40 kg P2O5 and 120 kg K2O ha-1; N was applied later in similar doses, in alternate cuttings. The chemical carriers used were ammonium nitrate, simple superphosphate and potassium chloride. In each cutting the dry matter yield (t ha-1) was measured. The results were analyzed through an ANOVA, and when F was significantly different the means were compared according to Duncan’s (1955) multiple range test. Results and Discussion The influence of the application of Azospirillum on the pasture establishment is shown in figure 1.

The dry matter yield with regards to the absolute control was statistically higher in the treatments inoculated with the biopreparation, with growing increases according to the increase of the doses used (13,7; 21,8; 35,7; 35,2 and 44,0 %, respectively). The highest dry matter content was obtained with the initial application of NPK, which differed from all the treatments. This response in the establishment cannot necessarily be ascribed to the biopreparation, but to the adaptation process of Azospirillum in the soil, for which the synergic effect of the physiological relations that are shown in the pasture itself, the inoculated organism and the plant phenology should be taken into consideration. There was evidence of a more precocious germination in all the inoculated treatments with regards to the control. The capacity of Azospirillum to colonize the rhizosphere can depend on some properties of the bacterium, such as chemotaxis to root exudates, a versatile metabolism that includes nitrogen fixation, antagonism and competition with microorganisms. In that sense, its ability to join the plant roots and the soil particles is important, suggesting that the union of Azospirillum with the roots involves two distinct phases: adsorption and anchoring (De Bashan et al., 2010). The species A. lipoferum and Azospirillum brasilense are characterized by their motility and response to chemotaxic factors, and by remaining during a long period in the rhizosphere of crops. These characteristics enable them to compete with the native microflora; for such reason they can be found in a large number of tropical soils, even in tundras and semi-desert sites (Díaz-Franco and Ortegón, 2006; García-Olivares et al., 2012). These criteria should be taken into account to consider the stability of the microorganism in the soil and its influence on the establishment of P. maximum cv. guinea tobiatá. Most of the research aimed at improving the plant response has been based on the use of native nitrogen-fixing bacteria in cereals and forages, and recently other plants have been included, as well as the use of commercial biological products. Under certain circumstances, the quantity of nitrogen fixed by these microorganisms can be significant, but it does not explain in itself the increase of plant growth. In many essays it has been proven that Azospirillum produces a highly significant root increase in the initial stage of the plants under cultivation (Ramírez-Elías et al., 2014), and it can influence a better response in the establishment stage of pasture, as corroborated by the above-mentioned results.

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Several authors refer to the importance of the period after the inoculation for the survival of microorganisms in the soil, where a large number of microbial populations interact with the diverse substrata and plant roots. Research reports indicate that only those organisms capable of being transferred from the seeds to the roots and increase their biomass in the rhizosphere can be considered competitive root colonizers (De-Bashan et al., 2007). In order to find this “ideal” bacterium, it was considered important to study different associations between beneficial bacteria and plant types. Azospirillum showed the best results and, for such reason, it can serve as model for all the associative bacteria and other bacterial groups, such as cyanobacteria, phosphate-solubilizing bacteria and sulfur-oxidizing bacteria (Trejo et al., 2012). The consortia of microorganisms apparently work better when phosphate-solubilizing bacteria, Azotobacter, rhizobia, bacilli and arbuscular mycorrhizal (AM) fungi are included. This is explained by the synergic effects of each one of them on the increase of nutrient availability and the inactivation of inhibitory compounds which contribute to a better plant growth. The accumulated DM yield is shown in table 1. The response of the biofertilizer with regards to the absolute control in the evaluated seasons and in the accumulated total was evident; in the latter, the increase of the dry matter yield was 42,4; 44,6; 54,0; 54,3 and 62,9 % for 25, 50, 75, l00 and 125 L ha-1, respectively, without statistical differences among the doses. A preliminary economic appraisal presupposes the feasibility of using A. lipoferum as biofertilization alternative to the lack of mineral fertilization undergone by the pastures. Taking into consideration the significant increase of the dry matter yield with regards to the absolute control and

the increasing costs of synthetic mineral fertilizers, the co-inoculation of Azospirillum with other biofertilizers, such as mycorrhizae and efficient microorganisms, and also the application of low doses of mineral fertilizers, can be evaluated to increase the dry matter content. The results coincide with the findings by CuestaMuñoz (2005) in the region of the dry Colombian Caribbean, in a soil with a pH close to neutral and low organic matter content, where P. maximum reached a high dry matter productivity (around 12 to 18 t ha-1) due to the biological nitrogen fixation and to the effect of phytohormones, when Azospirillum strains were inoculated to supply the nitrogen sources of synthetic origin. In other crops, such as wheat, sorghum and corn (Díaz-Zurita and Fernández-Canigia, 2009) the inoculation of seeds with Azospirillum showed a positive effect on the ear length (7-30 %) and on the production of fertile spikelets (12-25 %). Huerta et al. (2008) stated that biofertilizers include those whose basis are microorganisms that normally live in the soil in low populations, and which, when increased through artificial inoculation, are capable of making available for the plants an important quantity of nutritional elements through their biological activity; for such reason, determining the presence of Azospirillum in the soil type where the research was conducted could corroborate the report by these authors. The favorable response of P. maximum cv. guinea tobiatá to inoculation was reached in a soil with pH 5,4 (moderately acid), which is not in correspondence with the report by Sánchez (cited by Rivera-Botía, 2008), who stated that the Azospirillum genus shows an optimum growth in pH range of 6,5-7,0, and claimed that for A. brasilense such range is 6,0-7,8; A. lipoferum, 5,7-6,8; Azospirillum

Pastos y Forrajes, Vol. 39, No. 1, January-March, xx-xx, 2016 / Azospirillum lipoferum in Panicum maximum 27

Table 1. Effect of the application of Azospirillum on the dry matter yield. Treatment 25 L ha

Dry season DM (t ha-1) 6,32

Rainy season

Increase (%) DM (t ha-1) Increase (%)

Accumulated total DM (t ha-1)

Increase (%)

40,1

21,59

43,1

27,91

42,4

50 L ha-1

7,21bc

59,8

21,13bc

40,1

28.34b

44,6

75 L ha-1

7,18bc

59,2

22,99bc

52,4

30,17b

54,0

bc

7,31

62,0

22,92

52,8

b

30,23

54,3

125 L ha-1

7,51bc

66,5

24,41b

61,8

31,92b

62,9

Absolute control

d

4,51

-

15,08

c

-

19,59

c

-

Fertilized control (NPK)

11,63ª

-

42,82ª

-

54,45ª

-

SE ±

0,3817**

-

1,9118**

-

-1

100 L ha

-1

c

bc

Values with different superscripts differ at p < 0,05(Duncan, 1955)

amazonense, 5,7-6,5; Azospirillum halopraeferens, 6,8-8,0; and Azospirillum doebereinerae, 6,0-7,0, which means that in spite of being out of the recommended range, Azospirillum found conditions for its establishment that allowed to influence favorably the inoculated pasture. The studies of more than twenty years indicate that the bacteria of the Azospirillum genus have a special affinity for the roots of grasses (Brasil et al., 2005), such as the case of pastures, which respond with increases in their growth when they are inoculated with Azospirillum spp. The secretion of plant growth promoting substances (such as auxins, gibberellins and cytokinins) by Azospirillum seems to be partially involved in this effect (Reis Junior et al.; Radwan et al; Kuss et al., cited by Cárdenas et al., 2010). In the dry Colombian Caribbean region, Cárdenas (2007) isolated Azospirillum strains in order to evaluate their effect on seeds of P. maximum cv. Tanzania, in simple inoculation and in co-inoculation with a phosphate-solubilizing strain of Enterobacter agglomerans (UV1), isolated from cotton production soils of the Cesar department. This inoculation increased crude protein up to 20 % and dry matter up to 45 %, with regards to plants fertilized with chemical nutritional sources under greenhouse conditions. In this study the dry matter yield exceeded 40 %, which endorses the favorable response of P. maximum to the inoculation with Azospirillum. Other authors report that the inoculation with A. brasilense is highly beneficial in such grasses as corn, sugarcane, pastures and sorghum, because it contributes from 30 to 50 % of nitrogen to those crops. Besides fixing nitrogen, this bacteria is capable of producing plant growth hormones such as indole acetic acid (IAA), which generates an important growth of

bc

b

1,9901**

-

** p < 0,01

the root system, with a higher absorption, even, of the applied mineral fertilizers (Aguilar et al., 2008). Microbial inoculants represent a new technology that contributes to improve the long-term productivity of the agricultural system. This can be considered a clean technology aligned with the principles of sustainable agriculture and opposed to the abusive increase of the utilization of pesticides and fertilizers in recent times. Several microorganisms are used in the habitual agricultural practice, and others have potential to be used in the future (Naiman et al., 2009). This indicates the need to continue studying the inoculation mechanisms and the advantages represented by these microorganisms in obtaining cleaner productions with a lower economic cost. It is concluded that the application of A. lipoferum influenced favorably the establishment of P. maximum cv. guinea tobiatá, as well as the increase of the accumulated dry matter yield. Likewise, the highest DM yield was found in the fertilized control, which differed significantly from the other treatments. The response of the dry matter yield to the inoculation with regards to the control indicates the possibility of using this biopreparation as alternative to mineral fertilization, and to test higher doses in later studies is recommended. BIBLIOGRAPHIC REFERENCES Aguilar-Piedras, J. J.; Luisa, Xiqui-Vásquez. Ma.; García-García, Silvia & Baca, Beatriz E. Producción del ácido indol 3- acético en Azospirillum brasilense. Revista Latinoamericana de Microbiología. 50 (1-2):29-37, 2008. Aguirre-Medina, J. F.; Irizar-Garza, Martha B. G.; Durán-Prado, A.; Grageda-Cabrera, O. A.; Peñadel-Río, Ma de los A.; Loredo-Osti, Catarina et

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Received: December 19, 2014 Accepted: December 1, 2015